Drug Discovery Course - Ermondi PDF

L1 – ILT (Basic methods in drug discovery) – Prof. Giuseppe Ermondi – 12/12/21 INFORMATION ABOUT THE COURSE This is the chemistry module of the Integrated Laboratory Techniques course. The two sections of this course will be doing the same program (main topics and...

L1 – ILT (Basic methods in drug discovery) – Prof. Giuseppe Ermondi – 12/12/21 INFORMATION ABOUT THE COURSE This is the chemistry module of the Integrated Laboratory Techniques course. The two sections of this course will be doing the same program (main topics and main information are the same), but the Moodle pages are different. The course will be organized in 4 blocks – in every block there will be a PDF with the slides discussed during the lesson and some links to websites or other PDF slides useful. “Chemistry generalities”: we can find information about the course If you want to contact the prof, write an email using your official account (@edu.unito.it) Lessons organization These are all the 24 hours of the module (3 CFU): 12/12/2022 2 hours 13/12/2022 Sections joined 14/12/2022 2 hours 16/12/2022 mattino 2 hours 16/12/2022 pomeriggio Online asyncronous (to be confirmed) 19/12/2022 2 hours 20/12/2022 2 hours 21/12/2022 2 hours 22/12/2022 mattino 2 hours 22/12/2022 pomeriggio No lesson 23/12/2022 mattino Online asyncronous (to be confirmed) 23/12/2022 pomeriggio No lesson 09/01/2023 2 hours 10/01/2023 2 hours The last lesson in January will be about the exam preparation: we will provide you some examples of questions and we could discuss about some questions you might have. Exam The exam will be in written form. You will have 1 hour to answer 4 questions on all the program (even movies, software, exercises done in class). Every question is usually composed by some points. To reach at least 18/30 you have to reply to ALL questions. If there’s one answer missing, they consider it a penalty and so we have to pay attention to time. What’s more we have to answer following point by point. Moreover, it is important to know the basic chemistry and chemical structures: you will be asked to write them in the exam. If you do not write them correctly, the rest of the exam will not be taken into consideration. From the activity of a molecule, a particular group can be determinant to completely modify it in a biological system. YOU HAVE TO KNOW THE STRUCTURE OF THE 20 AMINO ACIDS THAT FORM PROTEINS. You have to be able to write a dipeptide structure: you have to write the mid bond and the structure of the 2 aa and the corresponding letter to indicate the aa. 1 During the course, it will be possible (not compulsory) to make a work on a given topic. If the homework is well done, also according to the deadline, you will have maximum 2 points of bonus for the exam. It’s up to you to do it or not but we will talk about this work when we reach the topic that you’ll do the work on. Program We will discuss both: 1. Experimental techniques 2. Computational techniques An important goal of the course joining the information that you find using experimental techniques and computational techniques. We have to put together this information to obtain further information to use in your research. Indeed, data can be obtained from experiments sensu stricto (in the lab) or from experiments sensu latu (using the computer). Either way, you should be aware of their origin, so if you are asked during the exam to answer for example “How to calculate the pKa?” you need to list and comment the methods that allow you to use the computer to calculate the pKa – I’m not asking you in this case to list the experimental methods to measure it (in the lab). SOFTWARES You will be asked to install some free software or to work on websites. There will be questions about these softwares We will be using: 1. Chemspider 2. Marvin Sketch 3. Chimera Link to Marvin Sketch: https://okta.chemaxon.com. It’s a software that allows you to draw molecules. You have to obtain a licence to use it , but the license expires after 2/3 months. You need to register to the Chemaxon site and download the Marvin Sketch software afte r [for next lessons would be better to have it]. On Marvin Sketch you can obtain the same result as searching from the 2D structure on the website. You can also change the structure. There’s the periodic table that you can use to change the structure. Notice that C are represented without hydrogen (they’re implicit) whereas polar groups are represented with H that is explicit. You can use the same method to write the structures in the exam but you have to be coherent: if you put them everywhere and then in another molecule you don’t put them, it’s an error. Once the structure is done, you can save it as a file. Click on File, and then Save. There are things that are common to all software that are based on Windows. To clear the display and re-initialize the system I click on File again. We always have to ask ourselves: What do I want to do with the software? Then, to pass from the 2D to the 3D structure, you have to go to “conformation < conformers”. Here it is described how the minimum energy conformer will be obtained: it is more complicated than on ChemSpider, but it allows you to really understand what you are doing. 3D structure requires some calculations. The 3D structure is related to conformers and so I can ask the software to calculate the conformer and I can tick the option “Give the lowest energy conformer that can be found”: it’s better if we select this option otherwise the software give all the possible conformations. In this case, you can move the molecule less, but you can save it as a file also in the 3D form. 2 Example: OKI/Ketoprofen OKI is the trade name for the anti-inflammatory drug whose active principle is Ketoprofen. We want to design Ketoprofen? How can I obtain this information? We can use Google by typing “ketoprofen”, but Google takes all information and put it on your display. This doesn’t always work. So, we can use other websites that are more specific. An example is ChemSpider. Link to ChemSpider: https://www.chemspider.com. It is a sort of mixture between Wikipedia and a curated database (database in which all the information are checked by specific people who have this job). This means that users put information on this database but most of them are corrected by communities of scientists. It is a database of chemical structures. It includes both drugs and not drugs. It does NOT include proteins. It provides the chemical structure and a huge number of information about it – e.g. on the side of the chemical structure, you can also have the molecular formula (in this case C 16H14O3), the average mass and monoisotopic mass. What I want you to remember is the structure: 1. Carboxyl group 2. Ketone 3. Aromatic rings (2) 4. Methyl group 5. Aliphatic chain It is important for you to know how to generate a 3D structure from a 2D one and to keep in mind that different software can have different functions and outputs. The code used generally almost everywhere is: 1. Grey = C 2. White = H 3. Red = O 4. Blue = N Marvin gives us some templates: small piece of molecules that are prepared by Marvin. An example of template is the aromatic group (you could also draw it by yourself using single and double bonds, but you can directly use this template). You can put the aromatic group in various positions. We have also a carboxylic group in ketoprofen. What I can do is asking the software to give you a molecule in 3D. This is a bidimensional (2D) structure, but you can also have the tridimensional (3D) one on ChemSpider. You are not expected to know how to draw these, but they are the real ones: indeed, there is only a bidimensional structure, but there are more tridimensional ones thanks to the flexibility of the molecule. Generally, we refer to the 3D structure that represents the minimum energy conformer. You can copy an image from Marvin to PowerPoint. Remember that to modify the structure you must work on Marvin and not on the picture on the PowerPoint. You can rotate, change dimensions, … 3 Link to Chimera: https://www.cgl.ucsf.edu/chimera/. This is to manipulate better the 3D structure saved on Marvin Sketch. There you can open the file you saved: finally there is the 3D structure in a minimum energy state. This will allow you to draw a lot of structures (more difficult than Ketoprofen) – we will eventually learn how to manage protein structures too, mostly to see how they move and interact with other molecules. We can use this other viewer to handle proteins but with it we can have also better views of our molecules to see how our molecules are oriented in the space. Link to a guide of Chimera: https://youtu.be/hQxKYSUdiD8. Other guides on these tools are on Internet and can be consulted in order to understand better how to draw the 2D and 3D structures on them. Pay attention on the different extensions in which you save the files: →.mvr stands for Marvin Sketch for example → [e.g. later on we will see.pdb, that stands for Protein Data Bank]. You can use a software like Chimera to put your molecules and to see how your drug interacts eith proteins and so obtain information about the mode of action of your drug.Marvin gives us some properties like pKa: carboxylic acids can be ionized and so we will see how important is obtaining information about ionization, pKa and now we can measure it. Example: Aspirin/Acetylsalicylic acid (done in Caron lesson, not in ours) The first thing you have to know is how to draw by hand the molecules. Then, on ChemSpider there is the 2D structure. 1. Aromatic ring 2. Carboxyl group 3. Ester You can work on this molecule in the same way we did before, on Marvin Sketch and Chimera. If you know how to draw the molecule by hand, you can also draw it on Marvin Sketch before, and then check on ChemSpider if it is correct. 4  L2 – ILT (Basic methods in drug discovery) – Prof. Ermondi (Sez. 1 + Sez. 2) – 13/12/2022 pH, IONIZATION AND BUFFERS Questa lezione è stata svolta dal Prof. Ermondi ad entrambe le sezioni. La spiegazione ha compreso la prima lezione (per intero) e la seconda lezione (in parte) delle sbobine dell’anno scorso, pertanto sono state integrate alla spiegazione di quest’anno le sbobine L1 ed L2 sia della sezione 1 (Caron) che della sezione 2 (Ermondi). PowerPoint di riferimento: “Block1”. Solubility Today our focus is on solutions: we need solutions when perfoming biological experiments (e.g. measure of protein or enzyme activity). The concept of solution is strictly related to solubility. There are two definitions of solubility: 1. Equilibrium (or Thermodynamic) Solubility (S) → classical inorganic chemistry definition: - the concentration of compound in a saturated solution when excess solid is present, and solution and solid are at equilibrium; - result is pH-dependent for ionisable drugs (SpH). 2. Intrinsic Solubility (S0) → same definition of the previous one, but it refers to the different species of the compound: - the equilibrium solubility (S) of the free acid or base form of an ionisable compound at a pH where it is fully un-ionised; - this solubility is pH-independent. Example: We want to prepare a saturated solution. At the beginning, the solution is transparent. If we add some reagents, we can observe the formation of the solid (we can see that because the solution is not transparent anymore). If we go on, we can see a dispersion of our solid, but if we perform a centrifugation we can observe the solid-phase at the bottom of the becher, above the transparent solution: this a saturated solution. This means that we obtain the equilibrium between the solid state and the solution: we’ve reached the solubility of our compound. Indeed, the concentration of the saturated solution that we can measure is the definition of the Thermodynamic Solubility (S) of the compound. It is a thermodynamic property in this case, because we wait until the equilibrium is reached. Example: What happens if we put an ester in an acidic environment? Nothing happens. And if we put in the acid a carboxylic acid? The carboxylic acid has a group that can be ionized, while the ester is not involved in any reaction when it is put in an acidic environment. If our drug is not ionizable, we have that the equilibrium solubility is the one that we measure when we are in a saturated solution (so, if compounds are not ionisable, the equilibrium solubility and the intrinsic solubility are equal). Otherwise, when we have an ionizable compound, this can be ionized (so, it loses a proton). When we put it in a basic environment (but in this case it’s sufficient that it is neutral), we have the compound changes with the pH. Therefore, if we want to solubilize in water the carboxylic acid in 1 the two forms (ionized and not), which is the most soluble between the two compounds? The ionized one, because probably it is more prone to move in water. So, when we define the solubility, we have that the solubility changes when pH changes: we have to define an Intrinsic Solubility (S0) - we measure it for the neutral form. Example: It is not strange that the Equilibrium Solubility (S) depends on pH – e.g. benzene (C6H6): it is not soluble in water, and there are not ionisable groups; e.g. benzoic acid (C7H6O2): it contains the acidic group that can be ionized, so the solubility depends on the ionization state of the molecule → if you want to solubilize the benzoic acid, would you use a basic or acidic solution? A basic one, because there is the ionization, so in principle the charged species are more soluble in water. In this case we introduce the Intrinsic Solubility (S0): this is the solubility of the neutral species. So, we start from the solubility of the neutral species, and then, when there is an ionization (presence of ionisable groups), we can change the solubility based on the pH we use. Obviously, depending on the presence of an acidic or basic group, if you want to increase the solubility you will have to choose the correct pH: - If there is an acidic group = you move to a basic pH; - If there is a basic group = you move to an acidic pH. Thermodynamic steps When we study the solubility, we can factorize the process in three substeps: 1. Dissociation of the molecule from the crystal: there is a transformation of the solid state into a sort of hypothetical gas state. The main force that governs this process is the interactions inside the solid: if they are strong (e.g. NaCl: sodium chloride is an ionic compound, so the interactions between the molecules are very strong because there are charges that are interacting with each other), it will be more difficult to have a solubilization. The ΔG here is positive, so the process is requiring energy; 2  2. Formation of a cavity in the solvent, that must be ready to accept the solute molecules: it depends on the strength of the interactions of the molecules of the solvent (e.g. water: there are some strong hydrogen bonds; e.g. alkanes: they have completely different properties). When the molecules move from the solid to the solvent, it is necessary that some spaces are formed: some solvent molecules are eliminated. Therefore, again there is a positive ΔG: this process requires energy; 3. Insertion of the molecules in the solvent cavity: having a good interaction between our molecules and the solvent is favorable for the solubility – ionized molecules in water interact very efficiently: solubility increases when we put an acid in a basic environment, because we have the formation of the un-ion, that interacts very well with water. This process produces energy, so there is a negative ΔG. Summing up, these are the three parameters that we have to consider when we have to understand the solubility of the compound (the first is the most difficult to predict) - The interactions in the solid state; - The interactions in the solvent; - The interactions between the solid and the solvent. Thermodynamic cycle These steps are not real physical steps, but ideal ones. Neverthelss, we need them to understand better what is going on, because otherwise it would be difficult to look up at the complete process. We can do a thermodynamic cycle in order to better understand which are the most important things that govern the solubility process. If I want to go from A to B I know that there is a chemical process governed by free energy G, that depends only on the initial state and the final state, so we can add the value of different ΔG (final energy minus initial energy) and then do the sum and subtraction – ΔG for state A is different from state B. 3 Now that we saw the physical-chemical basics of the solubility, we can move to something that is more practical during solubilization. Solutions and suspensions For any biological experiments, we need solutions, rather than suspensions. In a solution, the solute is completely solubilized, so no particles are visible in the becher: solutions are characterized by transparency (it does not mean uncolored). In a suspension, the compound is not solubilized: suspensions are characterized by opacity. Example: We want to measure the inhibition constant of an enzyme, so we prepare a solution. If it is effectively a solution, we are sure that the concentration inside our system is the concentration that we have prepared. If we have a suspension, it means that part of our compounds is not solubilized, but is in the solid part, so we don’t know what the concentration of our compound is. So, our test is not readable. To know the concentration, we have to solubilize all the compounds in the solution. Solvents Since we are discussing aqueous solutions, water is the most commonly used solvent in biological experiments. In particular, water is a polar solvent, and this can be shown by the constant of polarity (ε), which is 80. All the solvents that we can use are characterized by a specific value of polarity (e.g. alkanes are on the other side of the scale compared to water: they are apolar solvents – in the case of exane: ε is 2). Acetonitrile (ε = 37) and dimethylsulfoxide (DMSO ε = 46.7) are in the middle of the scale. Chloroform is not particularly suggested because it is very toxic, but for the preparation of nuclear magnetic resonance samples we need to dissolve our sample in the deuterated chloroform. There are different kinds of solvents: 1. Polar solvent = there is a partial charge on the atom (e.g. water, aceton → carbonylic group is a polar group); 2. Apolar solvent = no charge on the atoms (e.g. esan, benzene); 3. Aprotic solvent = no protons can dissociate (e.g. aceton); 4. Protic solvent = a proton or more can dissociate (e.g. isopropanol, acetic acid, methanol). 4 Scrivere la definizione in questa maniera, non in altre, dovesse uscire all’esame: A proto bound to a polar atom gives to the solvent a particular property, that is the protic property: a protic solvent is a solvent where there are hydrogens that are bound to polar molecules. Coulomb equation The dielectric constant is related to the interaction of charges that interact to each other. When it increases, the interaction between charges decreases. In water, the interaction between charges is lower respect to the situations where we have an apolar environment. We can measure all the ε and make a table like the one above. 5 It is true that a polar molecule is characterized by a higher difference in electronegativity and the structure of the molecule, but in general the polarity is related to the presence of polar moieties in our solvent. A polar solvent generally shows a dielectric constant that is greater respect to the other systems. It is possible to measure or make information about the polarity and the dielectric constant inside the cell membrane, that is quite similar to 4, and inside the cavity of proteins. Cosolvents (DMSO) If the compound we are interested in solubilizing is not soluble in water (e.g. benzene), DMSO (ε = 46.7) can be used, since it is able to solubilize compounds which are not soluble enough in water, because its polarity is not so different from water (its dielectric constant must be not too low). In this case, DMSO is used as a cosolvent. In fact, cells are not affected by DMSO, from 1% up to a 5% concentration in the total solution: we prepare a mother solution, that allows us to create different solutions with different concentration (the compounds solubilized in DMSO remain in solution after water addition) - for example, we can increase the concentration of our compounds in 10 solutions: the mother solution has a concentration C0, that is greater than all the concentrations that we want to obtain, and we perform dilution in series to reduce the errors. Normally, water is added to reach a concentration of solute at 5% in solution of DMSO. In some cases, the compound might reprecipitate after the addition of water and another solution must be prepared. Overall DMSO is sufficient in most cases. We use DMSO to be sure that our molecules are solubilized, because it is able to solubilize the greatest part of the compounds. DMSO seems less polar, so adding it to water we obtain a solvent that is less polar, because if our compounds are not so soluble in water this means that maybe they are not so polar. So, to increase solubility we have to decrease polarity by adding DMSO. Nonetheless, we must be aware that DMSO might modify the characteristics of our compound of interest. Actually, it is not trivial to predict when DMSO has an impact in some solutions, but since it is an organic solvent, it can modify the protein structure and the physico-chemical properties. For this reason, it is important to watch out for denaturation in the presence of DMSO. These are the main problems that DMSO alone can give us: 1. We cannot use it to inject a drug into patient because is a little bit toxic; 2. It modifies the physical chemical properties of our molecules - for example, if DMSO is used to study proteins: degradation of proteins increases if solubilized in DMSO and water; 3. The behavior of compounds in DMSO is different than in vivo. 6 The ionization is a problem for the drug discovery design, because when we have to present our report about our molecules to the agency to transform our molecule in a drug, we have to explain in our report what happens to our structure in every environment. So, when we have an ionize function, we need to explain what happen when our molecule is ionized, so it is an additional work, but a big amount of drug contains ionizable groups. pH: definition, calculation and measurement The professor remarked the importance of the proper writing of pH (not PH, Ph or ph). pH is defined as -log[H+] for acqueous solutions (in general, p means “-log”), where [H+] is the concentration of hydrogen ions in solution. The pH value depends on the components of the solution. pH scale starts from 0 and arrives to 14 (neutral pH is 7). It’s important to remember that the product of the concentration of [H+] and [OH-] is 10-14. This is the reason why, if there is the same concentration of H+ and OH-, the pH is 7. So, theoretically, the pH value assigned to pure water is 7. However, it is never 7, because of the presence of solubilized solutes. Some solutes can provide hydrolysis, others do not. In particular, acids or bases can be part of solutions. In the case of strong acids or strong bases, the pH is directly calculated from the concentration of the solution which is added in water. This is due to the fact that they are completely ionized. The main question to bear in mind, when we want to calculate the pH of a compound is: “Is the compound a strong acid/base or a weak acid/base?”. This is the equation that is the base of the ionization (for an acid): Example: HCl (strong acid) solution 10 -2M: pH = 2 NaOH (strong base) solution 10 -2M: pH = 12 It’s clear that if there is a strong acid, like a 10-2 M HCl solution, there will be a low pH (equal to 2). Contrariwise, if there is a strong base, like a 10-2 M NaOH solution in which the compound is 7 completely dissociated, the pH will be high (equal to 12). As said, the term “p” means “-Log”; so, in basic solution, it should be considered that there is a very small concentration of H +. The measurement of pH can be performed with different techniques: 1. Indicator papers (semi-quantitative measurement) → in order to understand if the reaction is going well, we need to check the pH. To measure the pH of a solution, it is necessary to take some drop and then put them on the paper. It’s important not to put the paper directly into the solution. The pH is measured through the color of the paper, compared with a legend that identifies the right pH for each color; for this reason, this method is called semi-quantitative. It’s impossible to precisely determine the exact pH because the comparison of the color of the paper with the legend is a subjective procedure. Anyway, this method is very used because it is a fast and immediate. 2. pH-meter (quantitative measurement) → The core of the instrument is an electrode that measures a potential (E) which is linearly related with pH. The potential can vary with temperature, the age of the electrode, etc. The pH-meter is more precise than the pH indicators papers; this because the first reports also some decimal digits, in which only the last is subject to errors, while the papers give only the full digit, without any decimals. Measurement at very low ionic strength aren’t reliable. It is important to consider that the measurement depends on the temperature, as well. Videos: Saggio ricerca cloruri et al. (by CaronErmondiVisentin) The video shows the procedure to measure the pH with the indicator paper. A piece of the indicator paper is broken, and a little bit of the solution is put on it; at this point, the change of colour occurs and it must be compared with the scale reported in the box of the paper box. REMEMBER: there are different types of indicators, with different scales; so, the scale to use must always be the one specific for the indicator paper we chose. On the other hand, the pH-meter provides a quantitative measurement. It is an analytical instrument, composed of an electrode connected to a box, which records the difference of potential. The pH value is given thanks to a calibration system. The equation of the calibration must be refreshed sometimes (at least once a week), by using 3 standards (standardization of pH-meter). Once this process has been done, then the pH value can be measured. pH-meter movie This video shows a pH measurement through the pH-meter. The electrode is in glass and it is fragile, so it must be handled carefully. The electrode, when not used, is immersed in a becker containing distilled water. Before the measurement it must be removed from the distilled water and it must be washed and dried (otherwise the concentration of the solution will be altered). Then it can be used in the solution of interest. The last part of the electrode must be completely immersed into the solution. It’s also important to pay attention when immersing the electrode into the 8 solution: it must not touch the bottom of the beaker. In the monitor it’s possible to read the pH value. It is important to wait for the pH value reported to be stable; it might take a couple of minutes. After that, it is important to wait for the pH value reported to be stable (it might take a couple of minutes). After the use, the electrode must be washed again and reinserted in the distilled water. REMEMBER: if the solute is not solubilized, the pH measurement is meaningless, because we are not dealing with a solution but a suspension. One of the things that should be done is the calibration of the electrode, but how? 1. Follow instruction provided by the manufacturer. 2. Use 3 buffers calibration and avoid single buffer calibration. A buffer is a pH-known solution. 3. Evaluate the parameters of the linear regression E = a x pH + b (where a and b are the parameters that are unknown). This linear equation correlates the potential to the pH. In general, all this calibration is made by the instrument, so we don’t have to take note of the pH, to measure the potential and to put these data in a file (for example on an excel paper) to do the correlation, but is sufficient to measure the three buffer and then put the correct balance of pH; after that, the instrument automatically calibrates and calculates the parameters in order to have the correct data. However, sometimes this calibration needs to be done to be sure that the instrument is going well. When we measure pH, in general we use a solution, in order to have a reliable measurement. Now we will define some important properties that are related to the ionization properties of the molecules. Ionization (pKa) 9 An acid (HA) in water is at the equilibrium with its dissociated form (H+ + A-), so HA (the neutral form) is in equilibrium with H+ (the ionization form/the proton) and A-: 𝑯𝑨 ↔ 𝑯+ + 𝑨− From this equilibrium, it is possible to obtain the dissociation constant Ka, that is expressed as the ratio between the product of the concentrations of the reactive species and the concentration of the undissociated species: [𝑯+ ][𝑨− ] 𝑲𝒂 = [𝑯𝑨] Then, if we want to consider the pKa, there should be a rearranging: [𝑯𝑨] 𝒑𝑲𝒂 = 𝒑𝑯 + 𝒍𝒐𝒈 [𝑨− ] If we are asked to think a base, usually it is NaOH. So, the reaction that represent NaOH is: 𝑵𝒂𝑶𝑯 → 𝑵𝒂+ + 𝑶𝑯− However, the reaction of equilibrium is different: when we study drugs, generally we don’t have a situation like NaOH, but we usually deal with drugs that contains nitrogen (N). If we take it with a pair of electrons, these can be protonated by a hydrogen (H): this is the kind of equilibrium we are interested in, and that’s the general equilibrium of the bases: 𝑩𝑯+ ↔ 𝑩 + 𝑯+ Also, the + is on the nitrogen because it shares four electrons: Then, the ionization capacity of bases must be expressed in the same way through pKa: this means that the dissociation at equilibrium is written in the same way as for acids: [𝑯+ ][𝑩] 𝑲𝒂 = [𝑩𝑯+ ] In this way, we have H+ on the right side of the equilibrium for bases, too. We have a specular view for both acids and bases. However, in this convention there is a problem: pKa alone is not sufficient to completely define this kind of equilibrium, but we need to know if the molecules are in an acidic or basic function: [𝑩𝑯+ ] 𝒑𝑲𝒂 = 𝒑𝑯 + 𝒍𝒐𝒈 [𝑩] By rearranging these formulas and introducing -log, we obtain the Henderson-Hasselbach equations. 10 Finally, when the concentrations of ionized and non-ionized species are equal: [𝑯𝑨] = [𝑨− ] or [𝑩𝑯+ ] = [𝑩] We have that: 𝒑𝑲𝒂 = 𝒑𝑯 Because the ration is equal to 1, and so the logarithm is 0. This works both for: 1. Acids = the lower the pKa, the stronger the acidity. 2. Bases = the higher the pKa, the stronger the basicity. Example: Here you can see an example of an acidic compound (pentanoic acid) and an example of a basic compound (pentylamine). For these two compounds, different Henderson-Hasselbach equations are used. IONIZATION PLOTS Acids and bases Ionization plots are used to verify which is the dominant species of your compounds at a given pH. This is important since the human body has compartments with different pH therefore, we have different species with different proprieties. To draw a plot, first you need to assume your pKa (let’s say pKa = 5). Then, it’s important to report: 1. On the axis of abscissa the pH value. 2. On the axis of ordinates the percentage of the species (HA, A-, B and BH+). The percentage of the species is the percentage of the species that is present as neutral form in the total concentration (from 0% to 100%, so the sum cannot be higher). Example: How can we understand the percentage? Let’s take into consideration an acid: when we have 100% acid, it means that all the acid is present in neutral form = HA. The other plot refers to the ionized species: 100% of ionized form means that the acid is totally ionized = A -. In the plot there’s the changing of the pH: when we look at the pH that is considered acid, we have that the acid is present as the neutral form. When we increase the pH there is the transformation from the neutral form to the ion. This transformation brings the plot to decrease, up to arrive where all HA is transformed in A-. As said before, when the percentage of species equals 50%, so: [𝑯𝑨] = [𝑨− ] 11 We have that: 𝒑𝑲𝒂 = 𝒑𝑯 This plot is almost always present in the exam, in different forms. Be sure to fully understand the concept of ionization. Example: How does each species vary in this plot? Let’s start with the BH+ species and analyze its trend. Before drawing the curve, what do we expect? Do we expect that the concentration of our base BH + is high at acid pH or basic pH? For a base, from the Henderson-Hasselbach equations, we know that BH+ concentration is higher at acidic pH. REMINDER: bases do not have pH value, they have pKa value. REMINDER: when the concentrations of ionized and non-ionized species are equal: [𝑯𝑨] = [𝑨− ] or [𝑩𝑯+ ] = [𝑩] We have that: 𝒑𝑲𝒂 = 𝒑𝑯 Because the ration is equal to 1, and so the logarithm is 0. This works both for: 1. Acids = the lower the pKa, the stronger the acidity. 2. Bases = the higher the pKa, the stronger the basicity. By pKa definition = pKa is the pH at which a substance is 50% ionized: since we have pKa = 5, we will have that BH+ species is 50% present at pH 5. So, the concentration of BH+ increases towards the acid pH and decreases towards the basic pH. This is a simple semi-quantitative resume based on the Henderson-Hasselbach equations - if we apply numerically Henderson-Hasselbach equation, we can determine how much species is present at each pH. Once I have calculated the amount of BH+ at each pH, I can join the points and have a sigmoid curve: we have obtained the ionization plot of the species BH+. We can build an ionization plot for any monoprotic acid or base, once pKa is provided or assumed, by applying the same principles we did with the BH + specie. 12 How to draw a plot: Equilibrium of the acids: 1. Represent on the abscissa the pH value (from 0 to 14) and on the ordinate the percentage of species at equilibrium: 2. Indicate what we are talking about (e.g. in case of acids: indicate the neutral form on the left side of the graph and the ionized form on the right side): 3. It’s important to indicate the pKa, because we know that when pH equals pKa, the percentage of AH and the percentage of A- is 50%, so the two lines will intersect in this point: 4. We have to focus on HA: when we have a pH that is acid, the percentage is about 100%. With a very basic pH, all the AH is transformed, and here the percentage is about 0%. In the middle part we know that AH is transforming in A-. There are two simple rules that can help: A. When pH = pKa – 2, the percentage of AH is about 100%. B. When pH = pKa + 2, the percentage of AH is about 0%. 5. Now, we focus on A-. when we have a pH that is very basic, the percentage is 100%, while when the pH is acid, the percentage is 0%. The rules are similar to AH but: A. When pH = pKa + 2, the percentage of A- is about 100%. B. When pH = pKa + 2, the percentage of A- is about 0%. 13 REMEMBER: the reason why in the rules that we used there is the number 2 depends on the equation: [𝑯𝑨] 𝒑𝑲𝒂 = 𝒑𝑯 + 𝒍𝒐𝒈 [𝑨− ] When the Log = 2, the concentration of HA is about 2 order of magnitude bigger than A - Equilibrium of the bases: the principle is exactly the same of acids, so when the pH is very acid the BH+ form is the most prevalent, while when the pH is very basic the neural form B is about 100%. The difference is that when pH is less than pK a in acids we have a neutral form, while in bases we have the neutral form when pKa is less than pH. Zwitterions Zwitterions are species that have an acidic and a basic function. They are important because amino acids belong to this family (you should know the chemical structure of all the natural amino acids). At which pH zwitterions are present and at which pH zwitterions are not dominant? In order to determine this, we have to draw the ionization plot of the amino acid (more complex than acids and bases. Example: We take in consideration alanine: We have two ionic centers: 1. Basic amino group → H2N (pKa is about 9). 2. Acidic carboxylic group → COOH (pKa is about 3) When there is a big difference between the two different pK a it’s a very good situation, because they can be treated separately: there are two ionizable groups and two widely separated pKas, so the two equilibria do not interfere one with the other. In practice, we have to put together the acidic and basic ionization plots (we consider two equilibria). Indeed, in the plot we can see three curves because we have three species (we need to follow the color, not the position: zwitterion in blue, anion in red and cation in orange) with three pKa: 14  1. pKa = 2,34 in case of the carboxylic acid. 2. pKa = 9,69 in case of the amino group. 3. pKa = 6,02 in case of neutral form. The resulting ionization plot comes of course from Henderson-Hasselbach equation, because the two equilibria don’t interfere one with another. We can see in the plot that our three curves cross in two points that correspond to the pKas. At acidic pH we have the protonated species dominating. Then we have a decrease of the basic species since there is an increase of the zwitterion species due to the protonation of the acidic center. There is a wide region comprised between pH 4 and 8 in which we have almost 100% zwitterion. After pH 8 there is a decrease of the zwitterion and an increase of the anionic specie. In particular, at pH = 6,02 there is the isoelectric point, in which the net charge is 0, so if you calculate the isoelectric pH is about 6, in the middle of the range where the zwitterion is almost 100%. In details: 1. In condition of very acid pH (for instance, pH = 2), so in an acid solution, there is this situation: The equilibrium is: 𝑩𝑯+ ↔ 𝑩 + 𝑯+ 2. When we increase the pH up to, for example, pH = 5 (always in acidic situation), the pKa = 3 is involved. So, the equilibrium involves the acidic function. At this point the first equilibrium that we have to consider is: 15  3. Then, if we move to a basic pH, like pH = 11, the part that is involved is the one with pKa = 9. So, the new species is: The new equilibrium will be: SUMMARY: when the pH is acid the most prevalent form is A. When the pH begins to increase, the form A decreases up to 0. Following the blue line, so the percentage of B, it is in equilibrium with the carboxylic part, because the pKa is 3. In this case its concentration increases. Then, it begins the dissociation of the ammonium group and so B begins to decrease, while C increases. Zwitterions are very important because all of them show these properties. Examples of questions in the exam: 1. If you are asked to draw the ionization profile of the zwitterionic species of a given amino acid, you should draw the blue curve, not the red or orange ones). 2. If you are asked to draw the ionization profile of the charged species of the anionic drug, you draw the A- curve (one curve = one species). pKa VALUES pKa values of common acidic substructures: 16 Phenols are interesting because their typical pKa is in the range between 7 and 10.5. Sometimes it’s possible that they can mislead. For example, if we see a pKa = 9 we think that is a basic pKa, and it is true. But the phenols have a basic pK a despite they are acids. So, it’s not sufficient to have the value of the pKa but we need to know what type of group is present in our molecule. pKa values of common basic substructures: Indole (C8H7N) is an aromatic heterocyclic organic compound. The amino acid tryptophan is an indole derivative. Exercise: Some drugs: which are their pKa? The functional groups? The ionizable centers? Ketoprofen is an acid, because there is the carboxylic group. The pKa can be deduced from the tables we saw before (page 16), so it ranges from 3 to 5. It is an aliphatic carboxylic group, because the aromatic cycle is not near enough. Benzoicaine is an aromatic base instead: in principle NH can have acidic behaviour in principle, but these H2 cannot be ionized. Atenolol is a little bit more complex: when you have to decide if the compound is a base or an acid, you can consider three groups. 17 Tetracaine will be see next lesson. Solutions (from last year lessons): There are some databases where we can find some collections of data that can be used to obtain the informations that we need. On Moodle we can find a database, called “pK a database”, where some researchers tried to search on the literature various informations about pK a and so they collected these resources and made them free to all the researchers. The most important part of the informations that it provides is the reference, because it’s important to know how the pK a was measured, in order to evaluate if the value is correct or not. The professor suggests to practice finding the pKa of these molecules through the database. 18  Lezione 03 del 14/12/2022 – Prof. Ermondi. In the previous lesson we have focused on tetracaine. And if we take a look to this molecule, we can see a basic center (an aliphatic ammine) that is the most important ionization center of this molecule. Then we have seen some tables with range of the pKa of some of the most common ionization moieties. Now we move to another point that is how we can measure the pKa because we have seen that it is an important property of molecules, and it is a descriptor related to the ionization properties. Depending on the value of the pKa, you have that your molecule can be ionized or not in a certain environment (the typical environment is the physiological pH). Looking to the value of the pKa you can know the most relevant form present in a particular environment. METHODS TO MEASURE pKa pKa is a sperimental property. We have a lot of systems that we can use to measure the pKa. In general, every technique shows some advantages and some disadvantages as we can see in the table below. Another point to considerate is the expertise. The table shows how strong (++) or weak (--) are the different methods, referring to amount, restrictions, pKa range, cost/time, precision and the different effects of temperature, ionic strength, and organic modifier. 1  1) The potentiometric method In the instrument there are the electrodes and some acids and bases containers that are useful for titration. The compound is dissolved in water and titrated with an acidic or basic buffer of known molarity. In the classical potentiometric method, the pH of the test solution changes as the titrant is added. This change is monitored using a pH electrode. The first phase is the Blank Titration, that is a titration without an analyte being present, only the solvent used in the analyte solution. After this, firstly we add acid, in order to arrive to the pH that is acid, and then add the titration solution. In this case we don’t have the same curve, because now it is influenced by the compounds. When it arrives to basic pH, the compounds are involved in the modification of pH, because when we add the KOH titration solution, we have that these compounds are involved in the equilibrium of the solution. The difference in the plot depends on the compound, in particular in its pKa. So, the difference between the blank titration and the blank + sample curve makes a plot that depends on the pKa of the compounds. Then, the equation of the curve permits to obtain the value of pKa. The most important thing is that we can obtain information of pKa measuring the pH. It is possible to make this kind of titration also in the lab, just using the pHmeter; the interesting thing is that this system does all these steps completely autonomously. On Moodle there is a video that shows this system (T3 (potentiometric) pKa determination). 2  Another important thing is that this technique can be used to obtain informations about pKa from more than one sample. This is important because in drug discovery process, at the beginning, we need to make measurement of more than one compound. 2) The spectrophotometric method Spectrophotometry is an experimental technique that is used to measure the concentration of solutes in a specific solution by calculating the amount of light absorbed by those solutes. This technique is powerful because certain compounds will absorb different wavelengths of light at different intensities. By analyzing the light that passes through the solution, you can identify dissolved substances in solution and how concentrated those substances are. It's important the presence of a chromophore close to the ionization site in the molecule. In principle any wavelength can be used for the determination of pKa: - the best choice: a wavelength at which the molar absorbivities are as different as possible. - the worst choice: the isosbestic point at which wavelength of both forms have the same molar absorptivity. Taking into consideration the compound, in an acidic pH it is in its neutral form. Then, moving to a pH that is basic, the spectra of the neutral form decrease, while the spectra of the ionized species increase. So, if the pH change, also the species change. Now, we have to remember this equation (1): It can be expressed also in term of mole fractions (X). The mole fraction is the amount of a constituent in moles divided by the total amount of all constituents in the mixture in moles (2) We should also consider the formula of absorbance. It is proportional to the concentration. (3) For example, if we have a pH that is basic, we have that all the compounds are present as ionized form; in this case, X will be 1 and A = AA-. Contrariwise, if the pH is acidic, we have that X = 0 and A = AHA. At this point, after had measured the pH, we can calculate the three different absorbances. Then, we can put there absorbances in the equation (4) and determinate the mole fraction. Now, we know X and the pH so, we can obtain pKa with (2). PKA MEASUREMENT: WATER VS SOLVENTS It is possible to measure the pKa in solvents different from water. For instance, sometimes, a pKa measurement in organic solvents (miscible with water) is needed. This happens for two main reasons: 3 - by creating a solution, usually the solubility increases. - It has to be taken in account the environment in which molecules work in real situation. The solvent can be non-polar, even without water (es. phospholipidic layer). If organic solvents are added to water, the parameter to look at to monitor the solution is the polarity. By changing the polarity of the system, also the electrostatic interactions between charges change. By the way, it is not very easy to predict the effect of the solvent added to water. In general, when an acid dissociates, it starts from a neutral form and dissociates into charged species. If the polarity of the solvent is reduced by adding an organic solvent to water, the equilibrium tent to maintain the neutral from (shift to the left). In the table we can see the changing in the pKa in a mixture of water and 2-propanol according to the percentage of organic solvent content in weight. When % increase, the pKa increase, too. Various pKa from proteins and small molecules have already been measured and can be found on databases (links are on Moodle, section 2). Anyway, there are software available to calculate pKa: it is possible to develop models that allow to predict the value starting from chemical structures. Different tools use different algorithm. It is advised to use more then one tool to compare the result and asses the veracity of the prediction. Also, those tools don’t consider the impact of the cosolvent. 4  EXERCISE – MARVIN SKETCH MARVIN SKETCH It is necessary to install the free trial of the software. Once opened Marvin Sketch, the page that appears let you draw molecules and do some calculations on them. In the lower part you can see that there are templates to help in drawing molecules. Click on the shape and then on the blank page to automatically draw the template. On the left, by selecting the line button, you can draw bonds. Click on the button and then on the position you want to create the bond. It is possible to change the CH3 with other groups by clicking on a group on the right of the page and then click on the methyl you want to change. Here we drew a phenol. For simple molecules it is easily possible to draw them manually directly on MarvinSketch. When you need to draw complex or unusual molecule, to be sure to draw it correctly and rapidly a good advice is to use the web resource ChemSpider: go to http://www.chemspider.com/ and write on the searchbar the name of the molecule. 5 The website brings you to a page dedicated to the molecule, when you can visualize either the 2D and 3D structure, the formula, properties, and other features. By clicking on more details, you can find the SMILES code, which is an alphanumeric string that represent the molecule. This code can be copied and pasted on MarvinSketch, that will recognize the string as the molecule and will automatically reproduce the drawing. All you have to do is to copy the aplhanumeric code from ChemSpider, then go to Marvin, click on Edit → Paste, and the molecule will automatically appear on the worksheet. Back to MarvinSketch features. It is possible to use the drown molecules in your own works. Using the same program for all the drawing will gave homogeneity and integrity. Make sure to work with the circled option selected (Rectangle Selection), then select your molecule, click on Edit → Copy as MRV. Now the molecule is copied and can be pasted on other program like word, excel… 6 Some calculation can be made starting from the molecule. To predict the pKa, as before, it is necessary to select the molecule with the rectangle selection. Then click on Calculation → Protonation → pKa. The program gives you a window with some parameters that you can change. For simple calculation, the standard parameters are good. Now click on Ok. Now the software will show another window. In the first frame the molecule is shown with its pKa value or values written near to the group(s) that can be protonated and change the pH (in red acidic pKas, in blue basic pKas). On the left we have a graph showing the ionization profile. On the y axis there are the percentages of microspecies distribution, while on the x axis there is the pH. In the middle there are the molecule represented in two ways: neutral form and ionized form. A colour code indicates which plot represent which form of the molecule. If you point one of the two forms, the calculated values will appear on the plot. In this case, the values aren’t taken from experimental data in some database. Those are calculated on the spot by algorithms. In fact, using the software doesn’t substitute for experimental measurement. I want to show you a couple of things that you can find on Moodle. Here there is a couple of paper that are devoted on the analysis of the influenced of the ionizations in Drug Discovery process. 7 PAPER 1: A Chemogenomic Analysis of Ionization Constants— Implications for Drug Discovery PAPER 2: Acidic and Basic Drugs in Medicinal Chemistry: A Perspective Medicinal Chemistry is a branch of the chemistry that is used to synthetize new drugs. Many of the drugs that are presents in the market were discovered in medicinal chemistry projects. In general, medicinal chemistry is part of the more general process that is the drug discovery process. The drug discovery process is the process that starting from the problem that in general is the cure of a particular disease and arrived to the discover of the new drugs. Is a long process, there are a lot of steps and one of these steps is the medicinal chemistry. (E’ il pdf “Acid and bases as drugs”). Here we have a couple of links in Moodle: pKa predictor, pKa database… There are other software that basically do the same work of MarvinSketch. Link on Moodle: https://xundrug.cn/molgpka This resource is aimed at predicting pKa starting from a molecule. As in Marvin, it is possible to draw manually a molecule with an easy editor program or copypaste the SMILES code to draw it automatically. Once the molecule has been represented, click on predict pKa by Graph Neural Network. The site provides a page showing the pKa and other information about the molecule. pKa database: is a tool that you use to search pKa that are measured and not calculated. Is a different type of information that you can obtain. The problem here maybe is the molecules that you search are not present in database because simply no one has measured the pKa. EXERCISE 1: pKa This is the table. Let’s focus on BENZOIC ACID 1) Build the 2D structure with Marvin Sketch and the 3D structure 8 2) Calculate aqueous pKa with MarvinSketch And then whit pKa predictor 3) Check on pKa database 9  4) Build the plot pKa (y) vs % isopropanol (x) using the data reported in the table (but not the one with 0% cosolvent), extrapolate the pKa value in pure water and compare with those you obtained in the previous steps. To do the assignment it is necessary to use a math editor, like Excel. On the program, write the values in two column and then build the plot. It can be seen that, from about 70% on, the linearity is progressively lost, so the pKa values tent to change a lot. From the graph, it is possible to extrapolate the pKa value in pure water (0% cosolvent) by taking from the program the curve equation and calculating the point in which the curve meets the y axis (x,%isopropanol=0). To do this we need a linear equation. Change the graph from “dispersione con linee rette” to “dispersione”. Then, click on the “+” and select “linea di tendenza” which will be our straight line. To obtain the equation, check more options of linea di tendenza. Select “visualizza l’equazione sul grafico” and “visualizza il valore R quadro sul grafico”, then close the window. This is what you will obtain. We can obtain the pK a in water by replace y with 0 in the equation. 10  R2 is the correlation factor. The more the values in near to 1, the more accurate is the correlation. It is basically an index that assure correlation. We can see that, by reducing the graph excluding the 100% isopropanol value, the correlation factor increases because the linearity is much more conservated. Excluding the last value is also important to calculate pKa because the equation will be more realistic and so the value. By doing so, we can see that the pKa at 0% cosolvent is: y = 0,0452·(0)+3,904 y = 3,904 which can be compared to the value in the table 4,21 which comes from experimental data. The R2 is 0,964. 11 ILT – Drug Discovery – 16/12/2022 prof. Ermondi In the previous lesson we discussed the problem of ionization for drugs and how this property is important because depending on the pH of the environment your molecules can be neutral or ionized, if ionized it's in general charged (obviously it's not true for zwitterions but that's another problem). We saw how we can handle the problem of zwitterions when the pkas are very different. We've seen how we can calculate the pka, always keep in mind the difference between calculation and measure (when you use a calculator you are not measuring anything, the calculator simply predicts the pka). Some calculators are very precise whereas others are not so precise. The complexity is not always related to the increase of the size of the molecules. Prediction is an important point because you can predict the pka of a molecule without having the molecule and in drug discovery when you are designing the potential molecules it could be useful to have an idea of the properties of the molecule of interest without synthetizing it, because when you synthetize a molecule you have to spend time, solvents, reagents and so it's very useful if you can predict it without all this work. PROTEIN PKA In this lesson we will focus our attention on protein pKa. We already know that there are some amino acids with a chain with ionizable groups; we can mention: arginine, lysin, histidine, aspartate, and glutamine. We can make an example of a peptide: Depending on the charge of aminoacids’ side chains, it changes the way a protein interacts with a drug. In the proteins, some ionizable groups are located on the surface, whereas other can be located in the proteins interior. Why there is a difference between these two different areas of the protein? Because when your amino acids face the solvent, in general you have that the solvent is water and so you have a polar environment. When the amino acid is present, for example, in a pocket of the protein, you have that for water molecules is very difficult to reach this amino acid, so you have an environment very low in water. So, you have an environment which is defined not polar. We can observe the two groups: the amine (blue circle) and the carboxylic group (red circle) that are ionizable. In the case of big proteins, is not interesting the N-terminal or the C-terminal ionizable groups but the lateral chains, because they have an influence in the behaviour of the protein. We will focus on the lateral chain, the most important thing that we have to consider when we are talking about pKa. The pKa is influenced by the environment: the lateral chain can be located on the protein surface (polar region) or can be buried in the interior of the protein (non -polar region). Now, we can consider two tools where we can find information about proteins: The most important things are experimental values and computational values and is important to distinguish if the value that we are observing is experimental or computational. collected in a database (the experimental values of PKa are not very easy to measure), for a limited number of proteins, usually the proteins are not very big because increasing the dimensions of the proteins is more difficult to calculate the experimental value of PKa. For the computational value, we can predict in principle the PKa to all the structure, but we have to calculate carefully the results because it is necessary to understand for every value if the computational prediction is good or not. We know that chancing the co-solvent changes the pka, because you change the polarity of the environment, in particular the polarity of your solvent. So, when you add an organic solvent, you have a lower dielectric constant, so the environment is less polar and this mimics the situation of the proteins’ pockets. Unfortunately, it’s not so easy to characterize the dielectric constant in a pocket, so you have to develop some tools that try to predict the pka so try to predict how the environment present in the pocket changes the pka of your aminoacids. A couple of tools: experimental values collected in a database => for a limited number of proteins computational prediction => in principle it can be applied to all the structures PKAD DATABASE Link of the database: http://compbio.clemson.edu/lab/software/5/ It is a database in which some experimentally measured pKa values of ionizable groups in proteins are stored. It reports: The record number, which is not so much relevant; The protein PDB identification code which allows the identification of the protein because it’s also used in another database (e.g. 1A2P); The residue name (for 1A2P – record 2 it’s the aspartate); The type of the chain (for 1A2P – record 2 it’s C); The position of the residues in the protein, the “RES ID” (for 1A2P – record 2 it’s 8); The experimental pKa value (for 1A2P – record 2 it’s 3.8); The experimental uncertainty of the experimental pKa value (for 1A2P – record 2 it’s ± 0.1); The SASA% (for now we don’t analyse it); The experimental method that has been used to measure the pKa (for the 1A2P – record 2 it’s 2D – 1H – NMR so, it has been used a nuclear magnetic resonance); The experimental condition that has been used (Expt. pH and Expt. Temp); The link to the paper in which it’s reported that specific pKa value. CODE is used to collect experimental structure from a database which is called PDB, protein database. PDB = it is an important web resource, in the web site there is this RCSB that is the organization that cure the PDB database. The PDB is a collection of protein structure that are obtained from some experimental techniques. It is an important resource because it gives you structures of proteins. Main drugs interact with proteins and so, you can try to use this tool to predict how a drug interact with proteins. The structures present in PDB are characterized by a label that is formed by G4 characters that can be numbers or letters. So, when you use the CODE you can download the structure of your PDB of your proteins, and then you can submit the structure to the toll of prediction of pKa. So, you can check your protein by knowing the PDB code and then search the pKa values of its ionizable amino acids This is a paper that introduce to a database of an experimental measured PKa values where there are some remarks about the importance of this problem. In particular that the ionizable side chains in proteins play a key role in various functionalities of the corresponding proteins and protein complexes. The ionization is important, not only for protein interaction, but also for a lot of other function, in particular, for example they can influence the protein directions, the influence of stability, the structure of molecules, the solubility of the molecule, the structure of the proteins. Ionization play a key role in protein folding, and the protein stability depends on the PH and the conformation can explain this dependance by the change of ionization. Another important point is that the ionizable point can be present in the active site of the molecule and again it’s very important because, for example, a drug can interact with the protein in a particular cavity and in the cavity there are ionizable residues; this is very important because the ionization state strongly influence the interaction between the protein and the drug or the legant. It is possible to identify the active site of the protein on the basis o f predicting perturbed PKa, It is important to have an idea of the values of PKa, it is important to predict the PKa values of ionizable groups of proteins and which are the factors contributing to the corresponding PKa shifts. The measure of PKa of proteins is not very easy as in the case of small molecules, because you have to select them. In this paper are reported some data and their related plots. In the following table it’s reported, for each residue ID, the number of available data in the database ( No. of measurements), the average pKa, the lowest pKa and the highest pKa. For instance, if you look at tyrosine (which has phenol as a lateral chain), the database includes 47 measurements of the pKa of tyrosine and the average value is approximately 11. (This is rather normal if you think that the pKa for phenol is about 10). This 10.98 comes both from 12.5 (the tyrosine having this value is significantly less acidic than expected) and 6.08 (the tyrosine having this value is much more acidic than expected). We can say that the 12.5 value is likely coming from the fact that the related tyrosine is located on the interior of the membrane, whereas the 6.08 from the fact that the OH is embedded in an electrostatic interaction, in particular, a hydrogen bond which in some way can force the ionization of the H linked to the oxygen. The same reasoning can be applied for the remaining ionizable residues reported in the database This is a database of experimental values that we can measure with different techniques, we can see that we have some measure that are performed on a small percentage of molecules, and we can also see how changes the PKa for the various ionizable amino acids. The number of measure is not huge because it is difficult to measure PKa for pr oteins and in general you can measure the protein only for small molecules because when the size of the molecule increase, the NMR determinations increases difficulty, so it is very difficult to obtain information for very big molecules. We can see how for the values of amino acids, PKa changes in a very wide range. The istograms show us how change the various PKa in the various environment where you can find the amino acids. You can see also that the pKa extremities are significantly less distant for th e C-terminal (COH), in which the pKa ranges from 2.4 to 4.03, while in the N-terminal (NH2) from 6.91 to 9.14. Since the two extremities (C-term and N-term) are always exposed to the solvent, they have a more limited variation in their pKa and they are often not involved in the formation of intramolecular interactions. Similarly, the authors of this paper plotted the distribution of the pKa value for the six different ionizable amino acids using a histogram. You can see that there is a huge difference in t he distribution of the pKa values for the different ionizable amino acids. PROPKA Another tool is the PropKa calculator (https://www.ddl.unimi.it/vegaol/propka.htm). Here you can insert the PDB code of your protein of interest. The calculator tries to take in consideration the local environment of each side chain and predict pKa. [By going back to the PKAD database, clicking with the right mouse button on your protein (e.g. 1A2P) and selecting “apri in un’altra scheda”, you will be moved to the PDB database and see all the information about your protein.] Let’s say you put 1A2P as your PDB code, and then click “Run PropKa”: you will see the predicted value of the pKa at the different positions of a specific residue (following table; the remaining amino acids are not reported). In this case, the predicted value of the pKa for the aspartate in position 8 is 3.16 (the experimental value was 3.1), while for the same amino acid in position 12 is 3.29 (the experimental value was 3.8). So, you can see that there are some differences between the predicted and the experimental values. In the PropKa you also have other indications related to a specific pKa value. The PropKa tool is based on an empirical algorithm. With the permission of the authors, the PropKa executable is included in the VEGA ZZ package and now is accessible though the Vega On-line service. APPLICATION Insert the following code: 6Y3C (you can click on this code through the slides and you will be moved to the PDB database) Run PropKa Download the output (you can download the text file and open it in Excel for instance) / you can work directly on the output online Draw the chemical structure of lysine with Marvin (to understand which is the ionizable side chain of lysin) Show the variation of predicted lysine pKa values in the same graphical way (histogram) shown in page 2 in the box in red (you have to extract of the pKa values of the lysin residue) [We did an exercise in class together using the tool, use the link on moodle.] We can see the graphic interface and you can choose a file or you can use the PDB; because in principle you can obtain the protein structure using other methods, so in principle you can use the file or directly your label. Then the software is able to retrieve the structure from PDB so you don’t see this step because it’s automatically done. Then you can see the results. You have the name of the amino acid and a number, which is the position in your chain. This number can go from 1 to the length of your protein. You can of course have other information like the predicted pka, which is the goal of this exercise, or the location of the aminoacid (some on the surface, whereas other are bonded). You have other parameters that are used for the prediction, like the solvation of the solvent that is due to the exposure of your aas in a water environment. You have other informatio n related to the H bond: this is important because if the H is involved in a H bond, this changes the availability of protons, so the pka changes. The H bond requires a donor and an acceptor. So, the aminoacid can be involved in a H bond network inside the pocket and the pka can change because of this H bond network. Your side chain can be ionized and then you have a strong interaction of coulombic nature (that involves charges). So, the software analyses all the ionizable chains, the environment and on the base on this analysis uses a model to help the software to predict the pka. We can also see the pka range. Then we want to compare these values to the predicted value. We can see the structure of lysin. (you have to learn it). The pka is not influenced by the stereochemistry. At the end of the exercise, you can see that you have your predicted values and you can report the values obtained by propka and study the frequency of the values to study the distribution. This prediction is made considering the lysin in water: the pka changes if the lysin on the surface or in the pocket. This is how you can obtain this information. I look on this ionizable centre but obviously when you are on marvin you have two ionization centres because you have the aa not involved in proteins. You are interested only in the side chains because they are the parts that get ionized in a protein. BUFFERS Definition and generalities A buffer is a solution that can maintain a nearly constant pH if it is diluted, or if relatively s mall amount of strong acids or bases are added. In this definition there are some basic point: the buffer is a solution so if in the buffer there are some solids, it doesn’t work; the buffer has to maintain a nearly constant pH: there are a lot of situation in which we want to maintain a specific constant pH, for example to use a UV procedure to determine a pKa value; There are a lot of biological buffer because in the physiological state we need a constant pH range. For example human plasma is buffered at pH 7.4 by a carbonic acid/bicarbonate buffer system. The buffer capacity is defined as “maximum amount of either strong acid or strong base that can be added before a significant change in the pH will occur”. This is one of the main features of buffer, so if we add acid or bases the pH doesn’t change. The largest buffer capacity is for pH = pKa: in this particular conditions the concentration of the two species are equal [ A- ] = [ AH ]. In general, the buffer capacity works well in a specific range of pH: pKa-1 < buffer capacity < pKa+1. It means that when you create a buffer, you probably use some molecules with a certain pKa. In principle buffers can be prepared from any weak acid or base (if you have strong acid or base, you don’t consider the pKa). How does the buffer work? A weak acid and a weak base are required to obtain a buffer. We have also some biological buffers to control the pH, because if you have, for example, changes in the pH of the plasma, you can have problems in the functioning of the body because some proteins require a specific pH. For example if we have a weak acid we have that in the solution we have the neutral form [HA] and the ionized form [A- ]. When we put together the two systems, we obtain the equilibrium and then we have to prepare a buffer. It is important that we have a buffer, when we have the presence both of the neutral and ionic part, we need both the compounds. In the buffer solution there are the presence of neutral and ionic species. If in the system we add a strong acid (for example HCl that in solution dissociates completely), the weak bases will react with the H+ from the strong acid to form the weak acid HA: [H+ + A- -> HA]. The H+ gets absorbed by the A- instead of reacting with water to form H3O+ (H+ ). The pH changes only slightly. The protons (hydrogens) react with the ionic part, and adding HCl to a solution, all of these protons produce a variation in the pH; but if some of these protons react with the ionic part of our weak acid, they cannot contribute to the decrease of the pH. The pH does not change. This system works only if we have also a weak acid. If in the system we add a strong base (for example NaOH), the weak acid will give up its H+ in order to transform the base (OH- ) into water (H2O) and the conjugate base: HA + OH- →A- + H2O. Since the added OH is consumed by this reaction, the pH will change only slightly. When we add NaOH, the hydroxyl ion can increase the pH, but a part of hydroxyl can react with the acid and part of this ions is buffered by the acid. In this case the neutral part. Or ag ain we can always think that we have the same equilibrium, in the equilibrium of dissociation of the acid, when we add OH the equilibrium moves to the right. When do the buffers work in the best way? When the concentrations of these two compounds are the same, because in this condition there is the maximum power. The maximum power is present when the pH is the same of the pKa. This is the reason why you choose a molecule with a pKa near the pHtaht you want to use. If for example, only the neutral part is present and it is assumed that only HA is present in the solution, it is obvious that HA tend to form HA; but if the concentration of HA prevails, the buffer work well when we add OH- and it does not work if we add H+. The same is true for the other conditions. It is important that the concentration of the buffer has to be related with the concentration of the strong acid or of the strong bases that probably we add to the solution. If the concentration of the buffer is low, it doesn’t work. For example, consider the addition of HCl. If the concentration of [HA]= 0.01 and [A-]= 0.01, adding [HCl]= 1 M, all A- react completely with H+. But when there is a big amount of strong acid, it is able to interact with all the system and we have 1M-0.01= 0.99M that cannot react with A-, because it disappears and the buffer doesn’t work. COMMON BUFFERS 1) Acetic acid (CH3COOH). It is a classical system and an educational one because in general it is not useful in lab. CH3COOH ↔ CH3COO- + H+ , pKa = 4.5. The region with the maximum buffer power is illustrated in the graph below. 2) Ammonia (NH4). Another classical system. NH4 ↔ NH3 + H+ , pKa = 9.0. The region with the maximum buffer power is illustrated in the graph below. It is possible to choose between acid acetic or ammonium depending on the pH that we want to buffer, and the pH depends on the experiment that we have perform. There are two others system interesting for two reasons: 1. from a practical point of view, phosphoric acid and carbonic acid are two physiological buffers, so a system that is present in biological system; 2. using a multiprotic acid, it is able to buffer at different pH depending on the di fferent pKa; 3) Phosphoric acid (H3PO4): There are various equilibrium: H3PO4 ↔ H2PO4- + H+ for pKa1 H2PO4 -1 ↔ HPO4 2- + H+ for pKa2 HPO4 2- ↔ PO4 3+ + H+ for pKa3 For each of this pKa values, three different buffers can be created: H3PO4/N2H2PO4 for pKa1 NaH2PO4/Na2HPO4 for pKa2 Na2HPO4/ Na3PO4 for pKa3 4) Carbonic acid. The first equilibrium (H2CO3 ↔ HCO3- + H+ ), with pKa1= 6-7, can be used to obtain a buffer at about 7, that is the physiological pH. It is the buffer present in the blood. When you consider multiprotic acid you have to keep in mind the different pkas of these important buffers and you have to consider that for every pka you have a different equilibrium. The carbonic acid is mainly used in the body, it’s not as used in the lab, whereas phosphoric acid is used in many experiments because it’s a excellent buffer. How can I get a buffer solution? Buffer solution could be commercially available, so it is possible to directly buy the buffer. Buffer preparation is reported in literature protocols. More often standard buffer solutions are routinely prepared in the lab by students: for instance, TRIS buffer. On Moodle you can see the commercial source of this important buffer (TRIS); the main group is a base. You can order these compounds. Depending on your use it could be required to have a more or less pure compound. So, another point you have to check is the purity of your molecules. Another tool that you can find online is the buffer calculator that allows you to obtai n a recipe to prepare your buffer. The tool gives you the substances and their corresponding pka. You can select various kind of buffers and you have to choose the better one. The most used pH is neutral pH, but you can choose what pH to get. The buffer calculator gives you all these choices because depending on what kind of experiment you want to perform, there are some buffers that work better than others. Another problem, we will see when we will study spectroscopy, it’s based on the absorption on the UV spectrum of your molecules and sometimes the solvent absorbs. So, you can choose a buffer that does not absorb in the range that you are using. So, the choice of a particular buffer is related to the experiment you want to perform. You can fix the volume of the buffer that you need, you can fix the pH, the concentration. We’ve seen that if our buffer is not concentrated correctly, there’s the risk that when you add a reagent the buffer won’t work. The solution could be directly prepared: the buffer arrives in a bottle in the correct solution. But the buffer can also arrive with a concentration that is not the correct one, so it arrives in a si ring where there’s the buffer but to prepare the right concentration it is necessary to add some distilled water, to obtain the right concentration. Buffer can be produced directly in laboratory and its preparation is reported in literature protocols. Using the link on Moodle we have an example of buffer calculator where we can choose: different type of buffer (different acid and corresponding sault depending on pKa), so there are different compound that we can choose to create our buffer; the quantity; the concentration, that depends on the experiment that we are performing; if during the experiment the concentration vary with the introduction of H+ or OH, we need to use a buffer where the concentration is bigger. Fix the ionic strength: in general we use the concentration of the system, but if it is not correct, we have to take into account the ionic strengths, that depends on the concentration of the ions. We should maintain constant the ionic strengths, because if it is constant we can use c oncentrations values. In some cases we need to add some ions, in order to maintain also the ionic strength. If we want to fix the ionic strength, there are different choice. All this system are corresponding at strong bases, and are not involved in the acid/bases equilibrium of the buffers. For example: There the ionic strength depend on the presence of CH3COO- and H+. If we want to maintain constant the ions, we have to add a big amount of different ions. So if we add NaCl ↔ Na+ + Cl- where Na+ and Cl- do not interfere with the equilibrium. Fix the temperature (in general we use 25°) Then we calculate the recipe, and we see instruction to produce the solution. You can also get different recipes and you can follow the one you want. Some rules that you have to follow: The buffer concentration is generally ten times sample concentration, otherwise the risk is that the sample interacts with all the buffer and it doesn’t work. KCl 0.15 M could be add or not: it depends on if in the experiment it is important to fix the ionic strengths. Buffer solution has to be properly conserved and buffer pH has to be checked daily: in general is prepare a big amount of buffer because otherwise a lot of time is spend to prepare the buffer every day and because working with big amount of quantity, the weight is more accurate. Do not prepare buffers without knowing their composition: a lot of salts are sodium salt, but if we look to the system immediately, we know if a sault is possible or not; so if we think to the system that we will have we are able to avoid errors. How to select the best buffer for our experience? ❖ Largest buffer capacity: depending on the pH we want to prepare the buffer we have to select the correct buffer solution; ❖ Organic buffers often give interferences in UV analysis, because there is the absorption of the buffer and not of the sample; ❖ Phosphate buffers can block HPLC instruments, because they are sault and give problems to the instrument; ❖ It depends on the applications It must be clear that if you want to prepare a buffer at 2-3 pH, you the acid and the conjugated base. To produce this one way is to add the acid and the salt, but another way is to produce the acid and then, add, for example, sodium nitrogen in order to transform the acid in the conjugated base. This is the reason why there are two recipes and not only one. You can watch the buffer preparation movie. At the end it’s very important to measure the pH with pHmeter and you can correct it using acid/base until you get the pH that you need. 19/12/22 - ILT Drug Discovery - L05 - prof. Ermondi YOUTUBE VIDEO https://www.youtube.com/watch?v=S6bgIeM5wSQ In the video is showed how to prepare a buffer, it can be useful both for the life in laboratory and for the examination. It is not sufficient to follow the recipe, but it is important to check the pH of the buffer. At the end you want to fix the pH with a pH meter, because the paper method is not sufficient. PAPER learn.unito.it/pluginfile.php/106088/mod_resource/content/3/jmc_acids_bases_review.pdf The topic of the paper is the acidic and basic properties of drugs in drug discovery. It is important in the market of drugs. There are some graphs about the pKa and the pH and you can see how to determine the percentage of ionised species in different conditions. In this graph there are the pH of some tis- sues presents in the body. It is important to know the ionization properties of your mole- cule, because different tissues have differ- ent pH and so the properties of your mole- cule can change. This means that the phar- macodynamics can change, but also the pharmacokinetics (how the molecule reaches the target). Important aspect of the drug discovery process that are influenced by the ionization: Solubility, because it governs the pharmacokinetics Permeability, that is related to how the molecule moves across the cellular membrane. For ex- ample, if your molecule is an oral drug, it must cross the gastrointestinal tract and to move from the stomach to the blood. To do that, it is important the permeation to cellular membrane. There are some molecules that governs this type of transport, for example PGP moves the molecules away from the cell. The charge of the molecule is also important because the cell membrane prefers to select non-polar molecules. Bioavailability Clearance There are some proteins present in the blood, such as albumin, that interact with your drug and are important in the distribution of the drug. In the paper has been done an analysis based on properties that are regraded by using some data- bases, such as ChEMBL, Pubchem, and Drugbank. These are free databases accessible using the website, in which you can find information: Drugbank gives us information about the activity, the phasmacokinetics properties of the drug ChEMBL is more devoted to biological properties, for example the interaction with other mole- cules PubChem, in which you can find similar information but form a chemistry point of view 1 19/12/22 - ILT Drug Discovery - L05 - prof. Ermondi Now we can move to the new topic. UV-VIS AND FLUORESCENCE SPECTROSCOPY: SPECTROSCOPY: is the study of radiation between matter and electromagnetic radiation. We can start this lesson with some information about electromagnetic spectrum. You know that the electromagnetic spectrum is wide, and you can find different kind of radiation, from the most energetics X-rays until the microwaves, radio waves, gamma radiation and so on. From our point of view, it is interesting how these radiations interacts with molecules and how to use these interaction to obtain information about the molecule. In particular, the ultraviolet spectrum is a small part of the total spectrum. https://www.youtube.com/watch?v=S6bgIeM5wSQ We can see the classification of the different type of radiations presented in the electromagnetic spectrum, moreover, we have the frequency and the wavelength. One of the most important things is that the energy of the radiation is proportional to the frequency: when the energy incre ases, the frequency will become higher than before. https://www.youtube.com/watch?v=dkARLSQWHH8which makes a brief introduction to spectroscopy (This is the link of the video on Moodle, the professor shows it during the class) Now, we can focus our attention on some basic concepts: In electromagnetic radiation we have two perpendicular fields that have to move together to from the radiation: they are the electric and the magnetic field. This is why it’s named electromagnetic radiation. In the videos linked in Moodle, you can find information about the magnetic and electric fields. In particular we have an interaction with the molecule only if the energy of the wavelength is exactly equal to the difference of energy between two energetic levels. Depending on the energy used, you can have different kinds of energetic levels and you will have a different kind of spectroscopy. The molecule is not a rigid body, in fact it can have vibration or rotation, depending on the energy used to excite the molecule. 2 19/12/22 - ILT Drug Discovery - L05 - prof. Ermondi The spectroscopy regards the interaction of electromagnetic radiation with the matter, this interaction can produce two different phenomena: the first one is the absorption, in which there is a movement from one energetic level to another that is higher; An electron can be promoted from its ground state (low energetic level) to its excited state (Higher energetic level) thanks to the absorption of energy. The energy absorbed is equal to the difference between the two levels. The spectroscopy is related to the quantum property of the matter. At the subatomic level, the system cannot have all the energy values and thus only some states are allowed. The electron can stay only in the lower energy state (ground level) or can stay, for a brief amount of time, at the higher energy state. At the same time, an electron cannot go from n=1 to n=2 because there are some rules that don’t allow this kind of configuration. The quantum origin of the subatomic particle is responsible for spectroscopy. Then, considering interactions, we have a first step when we have the absorption, that we have already described and the second one is the emission, in which we have the release of photons due to the shift from excited state back to ground state. In other words, there is the release of energy in the form of photons. What is the main important difference between these two phenomena? The absorption is an independent phenomenon. The emission is a process that depend on excitation. (We have two steps: excite the system by radiation and then we have the emission that is a phenomenon that is subsequent to the absorption) There are advantages in spectroscopy: It is very easy to use It is fast to record: the spectra require 2 seconds The technique is very sensible, so it requires a very small amount of sample, and this could be important because sometime if you want to record the spectra of a protein, to purify your protein, it could be a very complex and expensive process with too much material. It is not very expensive because is a very simple instrument, this is the advantage of this technique. You can measure the absorption using different kinds of descriptors. UV spectrum has on the X axes the wavelengths [nm] and on the Y axes you can have various options: - Transmittance, (T = I/I0) often in %. I= emerging radiation I0= initial radiation - Absorba