Reflections on second year

shallow focus photography of yellow star lanterns
Photo by 嘉淇 徐 on Pexels.com

This is my last week in the lab of 2018. It’s only really four days because we’re having our group’s Christmas party on Friday (laser quest and pub lunch) and then I’m at a conference on Monday before taking the rest of the week off before Christmas.

Towards the end of first year I wrote this post about how I thought first year had gone and I listed 5 things I wanted to change. In this post I’m going to see how I did with those goals and create 5 new goals for third year.

  1. Read more papers – I managed this one quite well. In first year I sporadically printed and read papers but this year I got organised and set up an RSS feed and have been pretty good at checking in with it most days – perhaps a little too often with my “inbox zero” tendencies. I use Mendeley to save anything I come across that might be useful for my project. I tried #365papers  and failed miserably though, partly due to me losing the spreadsheet I was keeping track of papers on in an IT nightmare but also me just not getting into a habit.
  2. Make more compounds – I certainly achieved this one. First year involved trying a lot of new chemistry and at the start of this year I optimised a lot of that chemistry making it far easier to get final compounds out. For example, one set of molecules I made last year took 8-9 separate reactions to get there and now with a small change to the chemical structure that I’ve learned doesn’t kill the activity of the drug in most cases, I can get to those compounds in 2-3 steps.
  3. Be more selective in the seminars I attend – In first year I felt I had to go to every single seminar to widen my knowledge but as you specialise you learn what interests you and what a good use of your time is. I still go to the odd seminar outside of my research area so I’m not in too much of a rabbit hole, but I certainly feel like I’ve been using my time a bit better when it comes to seminars.
  4. Attend more conferences – having only been to one conference in first year, I went to a few in 2018 – and still have one to go next week! I started the year by attending the Genome Stability Network Meeting in Cambridge in January, in March I went to the RSC-BMCS Mastering MedChem conference at University of Strathclyde in Glasgow, then RSC/SCI Kinase 2018 in May, again in Cambridge, and still have the RSC Biotechnology Group Chemical Tools in Systems Biology in London a week today.
  5. Use this blog more – this one I’ve technically achieved in the last month or so. Most of the year I found myself “procrasti-blogging” sporadically blogging if I was taking part in a science writing course where an assignment involved writing a blog or the Google Doodle of the day was linked to chemistry. Now I’m making a concerted effort to post regularly on here and also on my dedicated Instagram account.

I think I’ve done quite well in meeting all of those goals. They were fairly realistic goals without quantification. Now here are my goals for third year:

  1. Keep using this blog – weekly blog posts, a couple of Instagram stories/posts a week. Over Christmas I’m going to make a longer term plan for content and schedule as many posts as I can so it doesn’t take up too much of my time during term time. Let me know if there’s anything in particular you’d like to see.
  2. Get the desk/bench balance right – I continue to struggle with staying at my desk more often than being in the lab. Often I choose reading, agonising over lab book/write-up and writing off lab tasks to “tomorrow” that could be done today rather than making stuff in the lab. If anyone has any tips about this please let me know in the comments.
  3. Get something published – I have something to show for my research and I really want to get some of it published in a medicinal journal to show alongside my thesis at the end of the PhD. I’m waiting for some long-promised data from a collaborator which will help supplement my work but I’ve agreed with my supervisor that in February I need to start writing papers for publication without that research.
  4. Speak at a conference – similar to above, I have a sufficient story to tell that I would love to give a talk about just once about my research at a conference rather than just standing beside a research poster at said conferences where people may or may not come over to hear about it. I’d also like to go to a conference outside of the UK because travel is one of the perks of being in research.
  5. Finish the practical side of the project well – I plan to spend another year in the lab before writing up. I have until March 2020 technically but I’m leaving those three months as a “backstop” of sorts – #relevant. I have in my head I’d like to get to 100 final compounds for my thesis (I’m about two thirds of the way there so it seems tangible) and I’d also like to spend some time in the biology labs my group have testing some of those compounds.

Hopefully this time next year I’ll be writing a similar post about how well I did in achieving my third year goals. It’s crazy it’s got to my last year in lab already!

Did you make any goals/resolutions for 2018? Did you achieve them? If not, are you going to reattempt them in 2019? Let me know in the comments below.

My Placement Year in Switzerland

Increasingly, universities are offering integrated masters science degrees over bachelor degrees. These normally include an additional year of study and/or a placement year in industry or an academic research group. In this post I’ll tell you about the science I did on my placement in Switzerland and how it is linked to a major drug approval that happened a few months ago.

During my MChem placement year (2014/2015) at University of Strathclyde I worked as a Research and Development (R&D) intern for Corden Pharma Switzerland (CPS). They are a Contract Manufacturing Company of different Active Pharmaceutical Ingredients (APIs), Drug Products and Packaging Services. This means they produce various chemical products for pharmaceutical application for lots of different company clients.

CPSlogo
Picture caption: Corden Pharma logo with slogan “Experts taking care” Source: cordenpharma.com

The APIs produced at the CPS facility fall into a few categories: carbohydrates, lipids, peptides and other small molecules. At the start of my year there I worked on a short carbohydrate project where I produced a sugar-like compound for a big pharma client.

I spent the rest of my time working on a small molecule project for a slightly smaller company client. As part of a team of a few chemists I helped to make a large lipid-like molecule that helps deliver corrective pieces of nucleic acid into cells to treat genetically-linked diseases. This type of treatment is known as RNA interference (RNAi).

You may have heard of DNA, deoxyribose nucleic acid, a long double stranded molecule made up of individual nucleotides that contain the instruction manual for everything in our cells. There is another type of nucleic acid in the body called RNA, ribose nucleic acid. It possesses some structural differences to DNA such as the type of sugars and bases in the molecule and it exists as a single strand instead of a double strand.

DNA RNA
Picture caption: An partially unwound DNA molecule interacting with a single strand of mRNA. Source: Microsoft clipart

When a cell wants to carry out a particular function, the piece of DNA that codes for the protein that carries out that job is unwound by helicase enzymes. A temporary copy of that portion of DNA is made by RNA. This process is known as transcription.

This newly formed “messenger RNA” (mRNA) leaves the cell nucleus – the central region of the cell where DNA is stored – and travels to a ribosome that reads the instructions from the mRNA and builds the desired protein out of amino acids. Translation is the name given to this process.

protein translation
Picture caption: A green large green ribosome protein interacting with the RNA strand and building a new protein molecule. Blue tRNA molecules bring individual amino acids to the ribosome for building the protein. Source: clipart-library.com

But what if the protein being coded for is a harmful protein that leads to a disease symptom? The theory behind RNAi is that you can send foreign copies of RNA into cells to tell a ribosome to stop producing a particular protein.

These RNAs can tell the ribosome to stop making protein because they are complementary (similar in structure) to the original RNAs and so they bind together like a zip. This so-called interference could be used to treat diseases that are known to be caused by certain genes. Alnylam Pharmaceuticals have a great video that explains the science here.

RNAi
Picture caption: schematic summarising RNAi prcoess with natural blue mRNA and synthetic green small interfering RNA in moving around the cells. The siRNAs interact with the naturally produced mRNA leading to degradation of the mRNA. Source: the-scientist.com

RNAi won the 2006 Nobel Prize in Physiology/Medicine. It was awarded to Andrew Z. Fire and Craig C. Mello for successfully silencing a muscle gene in roundworms by injecting complementary RNA strands.

RNAi roundowrms
Picture caption: comparison of three green roundworms before and after treatment by RNA. Source: nobelprize.org

Their work showed that RNAi exists naturally in the body to regulate production of all proteins and can be done synthetically. The technique is now used by biologists around the world to shut down certain genes in cells and see the effect that it has. Researchers have also been trying to utilise it for medical use by giving patients complementary RNAs for disease-relevant proteins.

However, the body is usually very good at knowing where things should be in the body so it doesn’t expect to find bits of RNA outside of cells, e.g. in the bloodstream where most drugs end up.

The molecule I helped to make on my project allowed the attachment of these corrective RNAs to a big lipid. These big lipids then form nanoparticles (tiny bubbles) around the RNAs that help smuggle the RNAs across the cell membrane and into the cell where they can interact with ribosomes and shut down the production of harmful proteins.

I’m not allowed to tell you specifically how I made the molecule or what it looks like due to the confidentiality agreement I signed at the start of my placement year but it was wonderful to hear in August that the first RNAi therapy using this sort of chemistry was approved by the US Food and Drug Administration (FDA).

The treatment that was approved is called Patisiran (marketed as Onpattro by Alnylam Pharmaceuticals who developed it) and contains RNA strands that halt the formation of a protein called transthyretin. Transthyretin production leads to the symptoms of a genetic disorder called hereditary transthyretin (hATTR) amyloidosis. This is a rare disease where some 50,000 patients a year experience nerve damage due to the clumping of overproduced transthyretin.

Alnylam’s website says 51% of patients treated with Onpattro “experienced improvement in quality of life at 18 months” compared to 10% of patients who were treated with a placebo. This first-in-line treatment means that now one disease can be safely treated this way it might be possible to treat other genetically-linked diseases using RNAi.

It was really cool to see the science my project being approved as a new type of treatment which will hopefully pave the way for future RNAi therapies. While at times I was frustrated with the tricky chemistry I was doing on placement, I’ve been reminded by this approval that sometimes the goal of helping patients is met, which makes it worthwhile.

fiona matterhorn
Picture caption: Fiona in winter clothing standing in front of the famous triangular shaped Matterhorn mountain in snowy Zermatt, Switzerland.

Outside of the lab I enjoyed sampling different Swiss cheese and chocolate and travelling around the country on their first-class public transport system as well as further afield. While the country is very expensive, it’s a beautiful place to live with the Alps and picturesque old towns. I enjoyed being back last weekend and showing my friend Emily around – check out my Instagram for a post about her job as a consultant process engineer.

I learned a lot of chemistry (and Swiss German) on my placement and would thoroughly recommend taking any opportunity to gain industrial experience during your degree to give a taste of what life as a scientist is like.

Have you come across RNAi? Does it seem like a viable way to treat diseases? Have you visited Switzerland? Let me know in the comments below.

I kept a photo-a-day blog on blipfoto during my time in Switzerland and wrote a few blog posts about living en Suisse, one of which was reposted on globalgraduates.com.

 

How do I know I’ve made the right molecule?

I spend my days in a chemistry lab making drug-like molecules. A lot of these end up being small quantities (less than 0.1 g!) and usually have the appearance of a white/off-white powder. Occasionally I get a colour which is very exciting.

Picture caption: stacked boxes of sample bottles of ca. 40 different chemical products I’ve made in the last few months

The question a non-chemist might ask is “how do you know you’ve made the product”? Lots of different analytical techniques have been developed over decades to help chemists determine the chemical structure of the products they’ve made.

In this post I will give you an introduction to the seven analytical techniques I carry out on my samples to prove I have made the right molecule. These various bits of data go in my experimental write up which will make up a large chunk of my thesis.

1) LCMS

LCMS stands for Liquid Chromatography Mass Spectrometry. This combines two techniques: liquid chromatography allows to you separate a mixture into its components while mass spectrometry will tell you the molecular weight of each of those components.

Picture caption: A large grey boxed machine on a lab bench. This is our LCMS instrument in the lab.

Ideally the read off of a pure sample will just show that there’s one component in your mixture and the molecular weight from the mass spec will match the calculated weight of your product (i.e. the number you get when you add up the molecular weights of the individual nitrogen, carbon, oxygen, hydrogen etc. atoms).

2) 1H NMR

NMR stands for nuclear magnetic resonance. There are many different “flavours” of NMR. Proton NMR (written as “1H”) looks at the different hydrogen atoms present in a molecule and the different environments they’re in.

Picture caption: The blue door that leads to the NMR lab with safety signs warning about strong magnetic fields. 

For example, some protons will be attached to carbon atoms, while others will be attached to nitrogen atoms. This means they will have a different “magnetic environment” due to the different number of electrons in different atoms and how they’re distributed between different atoms and chemical bonds.

Below is an NMR spectrum for ethanol, the type of alcohol found in alcoholic beverages. There are three different proton environments in the molecule of ethanol: a hydrogen attached to an oxygen (blue); a hydrogen attached to a carbon that’s attached to a carbon and an oxygen (green); and a hydrogen that’s attached to a carbon that’s attached to another carbon (red).

ethanolnmr
Picture caption: a graph with three sets of peaks/spikes corresponding to different hydrogen atoms found in ethanol (chemical structure of ethanol also shown).

The appearance of the peaks is affected by the number of nearby protons e.g. the CH3 peak is split into a triplet because there are two protons on the adjacent carbon atom. Undergraduate chemistry degrees have entire modules dedicated to interpreting NMR spectra, so I won’t go into too much detail here.

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LCMS and 1H NMR are usually the minimum pieces of data you need to be sure you’ve made the right compound to move on with other chemistry. The two pieces of data combined give sufficient evidence that you’ve made something that weighs the same as the expected molecular weight; has the expected number of protons in the predicted magnetic environments to be the right products; and gives an indication of the purity of the compound.

For a PhD thesis you usually need to provide “full characterisation” for compounds which involves more analysis than those two techniques. I agreed with my supervisor that I would only get full analysis for final compounds that are to be tested by a biologist or compounds that don’t appear to have been made before by anyone else.

I determine if a compound is new by doing a literature database search and if zero hits come up in the search, I can assume no one has published the synthesis of this molecule before. I need to provide as much information as possible to prove it is the right compound. Below are 5 additional types of analysis I get on samples that fall into the final/unknown category.

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3) HRMS

High resolution mass spectrometry is just a slightly higher quality version of LCMS. Having two mass spectrometry experiments that match up gives more concrete evidence about what the molecular weight of my chemical product is.

Picture caption: blue lab door leading to the mass spectrometry lab, covered in warning signs about high voltage and magnetic fields.

4) 13C NMR

13C NMR is like 1H NMR but it instead looks at the different environments of carbon atoms in a molecule. There are lots of different types of NMR experiment that are used depending on atoms are in your compounds e.g. fluorine, phosphorus. Going back to the ethanol example, I would expect to see two peaks in a 13C NMR spectrum of ethanol because there are two types of carbon atom present in the molecule.

Picture caption: a grey space ship like instrument in the centre of a room. This is one of the university’s NMR instruments that I run 1H and 13C NMR experiments on.

There are also different types of NMR experiment that combine different types of NMR e.g. COSY, NOESY, HSQC, HMBC etc. which I may talk about in another post some time.

5) IR spectroscopy

IR spectroscopy involves firing infrared radiation (IR) at your sample to determine what types of chemical bonds are present. Different chemical bonds have different energies and absorb and emit different levels of IR. An IR spectrum (see below) will tell me if I have C-H/C=O/C-N bonds present in my product, but not how many there are.

6) Melting Point

We know that ice melts at 0 °C. Similarly, different products will have characteristic temperature at which they change phase. Recording the melting point of a sample will allow a chemist making the same compound in the future to compare their melting point with yours. The melting point also gives an idea of how volatile a compound is i.e. how easily might it boil off into the atmosphere.

7) Rf value

The final type of analysis I get is an Rf value, which stands for retention factor. You may have carried out chromatography at school or a science fair, whereby you separate a mixture into its components by running a liquid through a medium such as filter paper.

I use a slightly more sophisticated version of this technique called thin layer chromatography (TLC) where I run a spot of my product up a silica plate. The Rf value is a ratio of the distance travelled by the spot of product divided by the distance travelled by the liquid.

Picture caption: small white silica plates with run TLC experiments on them. Lines and spots have been drawn on in pencil for samples that are not visible under normal light.

This gives an indication of how clean the product is (I’ll see multiple spots if there are any impurities). It also tells you how well the product dissolves in that particular liquid – it will travel further up the plate if it dissolves really well in the e.g. water that I run my TLC plate in.

There are other types of analysis beyond this set that I could also use but they are either excessive, time consuming or unnecessary for the type of molecules I make. I think of full characterisation as compiling as much evidence as possible to be sure I’ve made the right molecule, fitting various jigsaw puzzle pieces together to build up a concrete picture of what my product is.

Some of the techniques are qualitative rather than quantitative and aren’t necessarily as sophisticated as others e.g. IR just tells me I have a C-N bond present while NMR will tell me if there are protons attached to that nitrogen, how many, and what other protons/carbons they are close to.

Picture caption: a big pile of printed data to analyse on my desk, and an IR spectrum on the computer screen.

These seven types of data I get are the standard ones expected in most theses and journals, but it wasn’t always that way: in the 1960s chemists only had IR and MP techniques available whereas now 1H NMR and mass spectrometry are the standard minimal analytical techniques, with MP and IR as added extras.

Last month a new analytical technique, called micro-electron diffraction, was published and generated a lot of excitement amongst chemists – on my Twitter feed at least! MicroED might save chemists a lot of time analysing compounds in the future by rapidly generating a 3D “X-ray skeleton” of the molecule using electron beams. This would save having to do all these other analyses.

For more information about different types of spectroscopy and analytical techniques used in chemistry, check out the resources below:

Does it surprise you how much analysis is done on one sample? What types of analyses do you use in your work to make sure a job has been done properly? Let me know in the comments below.