The Chemistry of a PhD

On the 2018 A-level results day I posted the first of two blogs about what learning outcomes of A-level Chemistry I still use in my day to day work as a medicinal chemistry PhD student. It was so many that I had to split it into two blog posts! Since it’s the first day of term for many English students today, here’s the second post where I further reflect on what aspects of Modules 2 and 3 of A-level Chemistry I need to remember to help me understand what I’m doing in the lab:

• Module 2: Inorganic chemistry
  • Electronegativity – knowing the relative affinity an element has for its electrons helps me to differentiate between different atoms when describing reaction mechanisms and when doing NMR analysis (e.g. the relative position of peaks associated with carbon atoms next to a nitrogen atom vs an oxygen atom)
  • Transtion metals – I use a lot of transition metal catalysts (mostly palladium) so my knowledge of metal complexes helps me to understand the structure of the catalysts with their various ligands attached and how they might work better because of those differences in complex structure and subsequent steric/electronic property changes.
  • Substitution reactions – knowing the exchanges that may take place between ionic species in my reaction mixture can help me know what side products may also be produced in my reaction mixture and how easy it will be to remove them.
  • Formation of coloured ions – a methylation reaction that I’ve carried out many times has a characteristic colour change from yellow to red, indicating the formation of the desired reaction intermediate. Knowledge of changes in the compound and the energies associated with that can be used to explain the colour change and can sometimes (but not always) indicate a reaction has worked.
  • Oxidation states in catalysis – understanding how the catalysts I use work at an oxidation level is important for the Suzuki reactions I’m running at the moment.

• Module 3: Organic chemistry
  • Organic chemistry nomenclature – I use different formula types to describe the compounds I make and the reagents I use. They come in handy for shorthand notation in my lab notebook (e.g. trethylamine = Et3N). Skeletal formula is the type of formula I use the most when describing the compounds I make – I’ve lost count of how many hexagons I’ve drawn.
  • Reaction mechanisms – as I’ve expressed above, I am expected to understand the mechanism of every reaction I’m carrying out and be able to draw curly arrows to show how electrons move to break and form the desired chemical bonds needed to make the product I want.
  • Isomerism – knowledge of the two types of stereoisomerism (E and Z) is important for knowing whether a reaction that forms one isomer over the other preferentially is going to make the product I want, or whether I’ve made a side product that I need to remove.
  • Organic functional groups and reactivities – knowledge of how alkanes, alkenes, alcohols, aldehydes, ketones, carboxylic acids, esters, acid anhydrides, acyl chlorides, amides, amines and many more functional groups react is central to organic chemistry.
  • Nucleophilic substitution and elimination reactions – understanding how these reactions work, not just regarding halogoalkanes and alcohols as taught in A-level, is important as I use these reactions a lot in my project.
  • Organic analysis – while I don’t use the qualitative colour change tests such as Fehling’s solution to detect ketones, I use mass spectrometry, infrared spectroscopy, NMR and optical isomerism as analytical tests to prove I’ve made the right molecule, as needed.
  • Aromatic chemistry – there are a lot of aromatic rings in drug discovery because our bodies uses these strong and stable ring systems to build lots of things in our bodies. I need to understand the properties of aromatic systems like benzene and how they react to make the molecules I need for my project. I am literally searching how to carry out electrophilic substitutions such as nitrations, chlorinations and acylations on a set of aromatic molecules right now!
  • Amino acids, proteins and DNA – while I’m not making amino acids in my project, the protein I am trying to shut down to kill cancer cells is made up of amino acids. I had a lot of fun learning to 20 fundamental ones in our bodies during my undergraduate degree. As eluded to previously, while I am learning to make specific kinds of compounds, I also need an understanding of the biochemical molecules and the processes they are associated with in order to combat disease.
  • Chromatography – another form of chemical analysis. I use thin layer chromatography daily in the lab to follow how a reaction is progressing and need to report the Rf of every new final compound I make. I also apply chromatography in a different way by carrying out column chromatography to purify my compounds when needed.
  • Organic synthesis – understanding that it often takes multiple reaction steps to get to a desired molecule and how I can design the safest, most cost effective and efficient process is a vital part of being a chemistry PhD student.

So as you can see, organic chemists genuinely do use a lot of the chemistry they use at school. If its a career you’re thinking about, it’s definitely worth putting the work in now and learning this stuff really well to put you in a good position for further study.

A-level chemistry isn’t just used by chemists though. Doctors, pharmacists, chemical engineers, pharmacologists, product designers and many more roles rely on an understanding of chemical matter. Do you use chemistry in your work?

Today is A-level results day. The Independent reported that about 46,800 students took A-level chemistry in 2017 – the fourth most popular subject after mathematics, biology and psychology. That’s a lot of people learning about the ingredients of the universe!

While deep in the exam period, you might think the things you are revising are topics you’ll never need to remember in real life circumstances. I’m aware my job as a chemist means I’m much more likely to apply these concepts than the average person but for those of you thinking about pursuing a chemistry career, it’s useful to know what you should learn now to save you re-learning it later in life.

Having gone through the A-level Chemistry syllabus, here’s a list of things from the first module that I use in my day-to-day life as a chemistry PhD student:

MODULE 1 PHYSICAL CHEMISTRY

• Atomic structure – the study of chemistry is based on developing knowledge about the particles that the universe is made up of. In medicinal chemistry we specifically look at the molecules that make up our bodily functions an how we can design molecules that alter these functions for therapeutic benefit.
• Mass spectroscopy – an analytical technique that tells you the atomic mass of your molecule. I use this type of analysis on a daily basis in my lab to make sure I’ve made the right product – assuming the mass number I’m given matches the one I’m expecting.
• Relative atomic mass, relative molecular mass – when looking at mass spec data I have to take relative molecular masses into account if e.g. I have a chlorine atom in my molecule (35-Cl and 37-Cl isotopes give me two different product mass peaks). Also, another analytical technique I use called Nuclear Magnetic Resonance relies on the existence of 13-C in 1.1% of all carbon atoms.
• The mole and Avogrado’s constant – Moles are the typical unit used to describe a quanitity of a chemical substance, like a dozen eggs, you would talk about needing X moles of a chemical. Luckily, I use an electronic lab book that calculates the majority of mole calculations I need to do for all my experiments to work out how much of each chemical I need. I still need to be able to calculate the number of moles of a chemical I need for my reaction to work from time to time if transferring a protocol from a scientific paper to the lab book software, and also to be able to engage with the software and check its calculating the correct number of moles for my reaction.
• Empirical and Molecular formula – I often express my compounds in empirical formula (just the relative number of carbon, hydrogen. Nitrogen, oxygen etc.) as a way of keeping the chemical structures confidential. When writing up the reagents I use I often use molecular formula shorthand in my lab notebook (e.g. Chloroform = CH3Cl).
• Percentage yield calculations – again, my lab book software does a lot of these calculations for me. We use % yields a lot to express how efficient a reaction is and is a very quick way of communicating to fellow chemists how well my chemistry is working (a reaction with yield >85% are pretty good, whereas a 5% yielding reaction needs optimising!). Similarly, I still try to check whether the calculated percentage makes sense for the mass of product I have.
• Balanced equations – while I don’t write out balanced equations in the same format as I did in high school, I need to make sure the ratio of different reagents I use makes sense for the reaction I’m doing.
• Volume and concentration calculations – sometimes the prescribed concentration of, say ammonia solution, in my protocol doesn’t match what we have in the cupboard. While I can often use the lab book software to calculate how much 2 M solution I need to use in absence of 13 M solution, a solution concentration is sometimes expressed in different units (%, g/L, M etc.) so I like to do these calculations manually to make sure I can still convert between units and have the right amount of reagent.
• Knowledge of bonding – the different reagents I use in the lab have different types of bonding properties (covalent, ionic, metallic, polar covalent) which affects how they interact. When working out the mechanism of a reaction (i.e. a detailed step by step rationale for how it works) I need this knowledge to explain why a catalyst might co-ordinate to a starting material, or why a particularly strong covalent bond won’t break.
• Bonding and non-bonding electrons – again, using this knowledge when working through the mechanisms of the reactions I’m carrying out, it’s important I know which pairs of electrons will get involved in my reaction and which won’t.
• Intermolecular forces – the forces that hold molecules together or repel them (permanent dipole-dipole forces, Van der Waals, dispersion, London forces and hydrogen bonding) are really important for understanding how a drug molecule fits into a protein to shut down a biological function. When I design molecules for my specific project, it’s usually to increase my understanding of these forces and how to utilise them to make a drug that mimics an ATP molecule in the way it binds to a kinase enzyme.
• Kinetics and thermodynamics – these fundamental concepts are applied to explain why reactions go a certain way depending on different factors. It explains why my reaction needs to be a particular temperature, or why one chemical product is preferentially formed over another.
• Chemical equilibria – The equilibrium (balance) involved in a reaction can be used to explain why a reaction hasn’t gone particularly well in converting starting material to product and how I might push that reaction to go to completion (i.e. 100% product in an ideal world) by changing the concentration of certain reagents, temperature etc. (applying Le Chatelier’s principle).
• Oxidation, reduction and redox equations – Oxidation and reduction reactions are some of the transformations I apply to my molecules so being able to write reactions showing which chemical species are being oxidised/reduced is important to remember.
• Acids and bases – I frequently use acids and bases in various ways during my reactions e.g. one of my reactions involved acetic acid catalysing the formation of a benzimidazole ring system. I also alter the pH of my reaction mixture sometimes in the purification process  – e.g. if a solid dissolves at a certain pH environment but crashes out as a precipitate at a different pH.
• pKa – the acidity of protons involved in my molecules is a very important aspect of predicting and explaining reactivity. I learned a bunch of pKa values during my undergraduate degree to explain why one region of a molecule is more reactive than another.
• Buffers – while I don’t personally use buffer solutions in the chemistry I’m currently doing, a lot of chemical reactions require a buffer to maintain the pH of the reaction at a certain level. The biologists I work with run different biochemical assays (tests involving enzymes and cells etc.) use buffers a lot to mimic cellular conditions when testing molecules I make for them.

I was quite surprised by quite how much physical chemistry feeds into my organic chemistry project. It just shows how much chemistry overlaps. Hopefully this shows you how useful this stuff is to know when thinking about becoming a chemist. Check back for a future post that will list the parts of A-level modules 2 and 3 that I still use as part of my PhD.

What was your favourite thing that you learned during A-level chemistry? Is there anything from your A-level days that you still use in your job today? 

Today’s Google Doodle celebrates the Danish biochemist S.P.L. Sørensen (1868-1939) (Figure 1). He came up with the famous pH scale that is taught to high school chemistry students and is an important property to consider when using chemicals. The Doodle is a game which gets you to guess how acidic or basic some common household items are.

The pH scale varies from 1-14 (Figure 2). A neutral solution (e.g. water) is considered to have pH 7 and any pH lower than 7 is deemed to be an acidic solution and anything more than 7 a basic/alkaline solution. The pH scale is logarithmic so something with a pH of 6 is 10 times more acidic than something with a pH of 7.

The average pH of our body is slightly alkaline at pH 7.4, “physiological pH” but it actually varies across different parts of the body:

• Mouth: pH 6.5-7.5
• Stomach: pH 1.5-4
• Intestines: pH 4-7
• Blood: pH 7.4

This is important in drug design because as the pH environment changes, a drug molecule may undergo chemical changes because of the acidic/basic solution it is in.

If an environment is too acidic, a drug may become “protonated”. Chemists use the term proton/hydrogen interchangeably because a hydrogen ion is by definition a proton as it has lost its single electron to form H⁺.

An acid is defined as a proton/hydrogen donor, it loses hydrogen atoms to other species. This means any acid present in an environment, say gastric acid in our stomach, may react with the drug, adding a proton to the molecule (Figure 3). This forms a drug salt.

Similarly, if an environment is too basic, a drug may become “deprotonated”. Bases react with acidic protons present on the drug molecule and remove them from the drug molecule, again causing it to become a salt (Figure 4). A drug salt will behave differently to the neutral drug and might not get to where it needs to be in the body.

Have you ever tried mixing oil and water? They don’t tend to like mixing together very well (Figure 5). This phenomenon is important for how a drug behaves in the body. The neutral form of the drug will typically be happier dissolved in the oil over the water, whereas the protonated/deprotonated salt form of a drug would be happier dissolved in the water.

Our body is made up of a mixture of oily and watery (“aqueous”) environments. For example, the outside of our cells is an oily phospholipid membrane whereas the inside and outside of our cells are water-based. A drug needs to be sufficiently oil soluble to be able to cross the cell membrane to get inside the cell (Figure 6).

If the drug has been chemically altered by the acidic/basic environment around it and is in its salt form, it is highly unlikely to cross the cell membrane to get into cells and have the desired effect. This is because salts are much happier dissolved in water. The non-salt version of the drug may well dissolve in water too but it is more likely to also dissolve in oily substances to cross a cellular membrane.

There are a number of different ways of administering a drug to a patient (topical creams, intravenous injections, slow-release patches, inhalers etc.) but the preferred method is oral dosing, that is, a tablet you swallow with water. Each delivery method has its own set of advantages and disadvantages. Regarding oral tablets, the drug then needs to bypass the gastrointestinal tract (GIT) (Figure 7) to get to where it needs to be in the body to have its desired biological effect.

The GIT is made up of the mouth, throat, stomach and upper and lower intestines. When the drug first enters the body by mouth, it is in a neutral environment (pH 6.5-7.4). Some of the drug may be absorbed into the lining of our mouths but most of it travels down our throats to our stomach which is relatively much more acidic (pH 1-4).

If we don’t want the drug to be absorbed into the stomach lining, we design our drug such that it contains groups of atoms that will be protonated to form the salt. This means the drug will be happier in the stomach acid than the oily stomach lining. Similarly, it is important to make sure a drug is sufficiently stable to survive this acidic environment and not be broken down by our body’s metabolism processes.

The drug then moves on to the intestines which are relatively more basic than the stomach (pH 4-7). Usually any protonated parts of the drug will be deprotonated again to give the neutral form of the drug and this is where many drugs travel through the oily intestine lining and into the blood (pH 7.4).

As you can see, knowing about pH and how it can affect molecules is very important in drug design. Sørensen’s pH scale is a helpful way of quantitatively measuring how acidic or basic a chemical environment is. Chemists use this to predict how a drug might behave in that environment.

1. Google homepage, accessed 29^th May 2018
2. Image used under creative commons license using Bing Image Search software

During my undergraduate degree, people would often ask what course I was studying. Usually the response to “Chemistry with Drug Discovery” was “Chemistry with what?”. No, it doesn’t involve hunting around with a magnifying glass looking for paracetamol. The drug discovery process is a huge chain of events involving numerous people that gets a medicine from initial idea to market. This process can take up to 20 years and many projects fail at various points of the journey. Below is a diagram that I will talk through to take you through the drug discovery process (the blue portion).

1. Identify target – Scientists (mainly biologists) work out how they might target a disease. We have many tiny molecular machines in our bodies called proteins which do various jobs and keep us alive. Sometimes those proteins are missing or go rogue and that often causes a disease, so it becomes the drug target.
2. Identify compounds – Often, the way to treat a disease is to shut down the protein that is causing the symptoms. These are called inhibitor molecules and they do so by binding to the protein, rendering it useless. Think of it like putting a key in a lock that means a door can’t be unlocked from the other side. These compounds can be found using a number of techniques: by doing a screen (chucking hundreds to millions of potential molecules at the desired protein to see what sticks), working on an existing molecule someone else has identified, or using educated guesses based on the shape of the protein. Chemists will then make dozens, if not hundreds, of these molecules and also slight variants of them based on the same general shape to find a brand new drug.
3. Establish activity – It’s now back to team biology who test the drug compounds the chemists have made to see if they have the desired effect. This is done using a test called an assay where the individual proteins/cells are monitored to see if adding the drug stops the protein from doing its job. The results of these assays can be relayed back to the chemists who can then make more molecules to ask further questions about how the protein is inhibited and improve on their compounds. Many rounds of this exchange can take place (which is what my PhD project largely involves).
4. Select clinical candidates – Once scientists are concretely sure about how their drug binds to the protein and have found compounds that shut down the protein with high potency (that is, requiring very little drug to carry out this effect), a shortlist of clinical candidate compounds are selected and made ready for testing in live subjects instead of cells.
5. Test safety – This is the most controversial aspect of the drug discovery process. Before clinical trials, where drugs are tested in increasingly larger groups of humans, they are first tested in other animal models to see what effect the drug has on an entire organism’s system. Such models include rat, mice, dog and primates, with increasing similarity to human anatomy. I am not involved with this work as a chemist but from what I’ve learned such testing is heavily regulated and I personally think it is a vital step before putting drug candidates into humans. I may discuss this further in a future post.

In the rare occurrence that a drug compound makes it through all of the above stages, it is ready to test in humans. This first part of the process can take years. As I said above, my PhD comes in at stages 2/3 of this diagram and that is 3-4 years worth of work on my part in addition to the work done before me by biologists in identifying the protein I am targeting for treating cancer.

As you can see, the drug discovery process is a rigorous and lengthy one. In a future post I will talk about the various phases of clinical trials that take place to make sure a drug is safe before going on the market.

Are there aspects of the drug discovery process that surprise you? Is this how you expected new drugs to come about? Let me know in the comments.

Everyone is different. Everyone chooses to do a PhD for a variety of reasons. Everyone wants to get different things out of a PhD project. Below is how I decided which projects to apply for.

Why a PhD?

Before even thinking of applying, you need to be sure you know exactly why you want to apply for a PhD as it is something you will be asked in application forms and interviews. Is it the next natural step in your chosen field? Are you excited to be part of cutting edge research and contributing something new to the scientific narrative? Are you up for a few more years of studying? Can you afford/do you want to live on a stipend for the next few years for the sake of research? Or do you just not know what you want to do next? Speak to people who have done/dropped out of PhDs to find out their motivation and see if you’re on the same wavelength as them.

While I don’t think I want to stay in academia forever, I knew I wanted to contribute something to research into treating cancer and a PhD is a very easy way to do that. I had spent the final year of my MChem in a med chem group and saw what doing a PhD was like and could see myself doing that in the future.

My absolutes

First and foremost, once deciding to do a PhD, I knew I wanted to do a cool project in an interesting place. This took the pressure off the overwhelming number of options to take into account. With those two criteria met, other factors can then be weighed up, such as location, discipline, university credibility etc. After answering yes to the two questions “Is it a cool project? Is it in an interesting place?”, I settled on the following checklist:

• Medicinal chemistry with a bit of biology
• Oncology-related project
• Good university, reputable research group
• Interesting location – Scotland (not Glasgow, needed a change of scene) or UK (if England, close to London for easy access to theatre etc. and friends who had moved south)
• Applying to a project, not a Centre for Doctoral Teaching (CDT, will explain below)

Finding PhDs

Now the all important where do I find the things to apply to:

• FindaPhD.com is where I found most of the PhD projects that I applied for. It has a very useful search function where you can search for projects based on subject, institution, funding grant and even by supervisor.
• Ask staff at your current institution if they’re looking or can recommend people for the work you’re interested in. Academia is a smaller world than you think.
• Good old Google. If a particular institution takes your fancy, look up what research is going on, by whom, and contact them directly for any opportunities.
• Look up industry options – there are a number of industry-based PhD options popping up for chemists (GSK/Strathclyde, AstraZeneca/Cambridge etc.). If you’re not wanting to spend a few more years entirely in an academic environment, looking for PhDs with placements/mostly based in industry might be a good alternative.
• Attend postgrad fairs – to be honest, I didn’t find this massively helpful as it was geared towards grad schemes but there are universities present. I had to inform the GSK recruitment staff there that they offered a PhD scheme with Strathclyde.
• Attend Open Days – I only attended one open day (St Andrews) but it was very worthwhile. After the generic morning being shown around the university, my coursemates and I that attended got a chance to speak to academics one-to-one about potential projects. Sadly the funding for a project I agreed to fell through but it was a useful day.

PhD or CDT?

In the UK there are two approaches to offering PhD scholarships. There is the traditional route where you apply to individual projects with specific supervisors at a specific institution. Increasingly, due to the way funding is being offered, universities are setting up “Centres for Doctoral Training” which focus on offering a number of PhD scholarships dedicated to a specific topic across a number of groups/institutions. Some examples I have come across are listed below.

• GSK Stevenage/Strathclyde – med chem, organic synthetic chemistry either academically/industrially based with a placement at the other site within the CDT.
• AstraZeneca/Cambridge – similar to above
• CRITICAT – catalysis-based projects based at St Andrews, Edinburgh and Heriot Watt universities.
• Synthesis for Biology and Medicine based at Oxford University

CDTs typically offer a first year of rotation around different research groups and projects and then you decide on your PhD project and supervisor after that period of training. This strategy is useful for candidates who know which institution or what field they’d like to work in but not neccessarily which project and allows an informed decision. Personally I decided to apply for a specific project and supervisor as there wasn’t a guarantee I’d get a project I was after once joining a CDT but I have ex-coursemates who are largely enjoying the CDT option.

I could say much more about my personal process of finding a PhD as there’s lots to take into account. Please comment below or tweet me (@fi0n0) any other questions you might have if you’re looking into studying for a doctorate.