High Throughput Technology PDF

High throughput technology  There are in principle two ways of generating an array of compounds (compound libraries) to find hits and leads and to optimize leads  Combinatorial synthesis (CS; produces mixtures of compounds within each reaction vessel)  Parallel synthesis (PS; produces a single co...

High throughput technology  There are in principle two ways of generating an array of compounds (compound libraries) to find hits and leads and to optimize leads  Combinatorial synthesis (CS; produces mixtures of compounds within each reaction vessel)  Parallel synthesis (PS; produces a single compound in each reaction vessel)  Advantage of PS: Easier to identify the synthesized structures  Advantage of CS: Generation of many more structures compared to PS (valuable for finding leads) 1  Both CS and PS are usually carried out using solid phase techniques (i.e., using beads)  First example of solid‐phase synthesis: Merrifield resin peptide synthesis Bruce Merrifield (Nobel Prize 1984) Resin: Divinylbenzene‐ crosslinked polystyrene Boc = t‐butyloxycarbonyl TFA = trifluoroacetic acid 2  The Merrifield resin is rarely used these days (because polystyrene is too hydrophobic – leads to peptide chain folding in on itself by H bonding)  A more modern resin used in peptide synthesis are the Wang and Rink resins Peptide synthesis using the Wang resin 3  Early solid‐phase synthesis efforts concentrated on peptides although they are not drug‐like (because of poor pharmacokinetic properties)  Jonathan Ellman (1992) showed that 1,4‐benzodiazepines could be generated on solid support via a cyclo‐ release strategy Valium* (diazepam) * Benzodiazepines such as valium are positive allosteric effectors of GABAA receptors (ligand‐gated Cl‐ channel) 4  Other multi‐component reaction types (designed to increase compound diversity in a single step) that have been used to generate large compound libraries  The number of commercially available screening compounds is about 20 million (as of 2019)  e.g., Pfizer held about 4 Mio compounds in 2019 5 Design of compound libraries  To achieve structural diversity of compounds, spider‐like scaffolds are often used  The scaffolds have a central body and arms bearing different functional groups (used to probe target binding)  Evenly spaced arms allow for higher success rates for finding a lead (better exploration of 3D space around the molecule) It is estimated that about half of all known drugs are derived from < 40 scaffolds. 6 Examples of scaffolds: tadpole‐like scaffold Not optimal for exploring the 3D space 7 Parallel synthesis (PS)  PS is most often used during lead optimization as it does not usually generate a very large number of compounds  Reactions are performed in a series of wells/vessels so that each contains a single (identifiable) product  For PS to work efficiently, certain standard organic synthesis procedures (e.g., extractions, solvent evaporation, purification) need to be eliminated or simplified  PS can be carried out using a solid‐phase approach or using solution phase organic synthesis (SPOS) Greenhouse parallel synthesizer (Radley) Parallel microwave synthesizer (CEM) 8 Useful techniques in PS  Solid phase extraction (SPE) can be used instead of liquid‐liquid extractions to remove acidic or basic compounds, or impurities  Columns can be run in sequence (e.g., following amide synthesis, an acidic SiO2 column can be used to remove basic impurities. This is followed by a basic column which removes acidic impurities)  A variant of SPE is fluorous SPE (F‐SPE) which uses highly fluorinated silica to remove fluorinated compounds: Example: 1. Reaction to form the urea derivative requires an excess of the amine 2. After the reaction, fluorinated isocyanate is added to consume all the excess amine. The fluorinated urea derivative is then removed by F‐SPE. 9  Scavenger resins can be used in PS work up to eliminate excess reagent and yield pure product  Catch‐and‐Release strategy: a reagent is attached to a resin. The reagent and can be easily removed by filtration after completion of the reaction. Amide synthesis using: carbodiimide urea 1. Carboxylic acid 2. Coupling reagent (resin‐bound) 3. Amine 10  Use of multiple resins  Example: Generation of a sulfonamide library using three polystyrene (PS) resins PS‐Morpholine: Base catalyst (used instead of the typical Et3N – smelly and volatile) PS‐Trisamine: Nucleophilic scavenger → removes excess RSO2Cl PS‐Isocyanate: Amine scavenger → removes unreacted amine 11  Microwave‐assisted organic synthesis (MAOS)  MAOS is used to accelerate synthesis (higher efficiency due to better energy transfer using microwaves)  MAOS is often “greener” than conventional synthesis (less energy, often solvent‐less, higher yields, less by‐products) Amide synthesis Suzuki coupling boronic acid Transition metal‐ mediated reductions (a) and aminations (b) 12  Microfluidics in parallel synthesis  Allows for syntheses to be carried out on microchips (microreactors) where the reactants continuously flow through the microfluidic channels  Some advantages: shorter reactions times, fewer by‐products, higher yield, good control of temperature, potential to carry out a vast number of chemical reactions on one chip Parallel synthesis of four products using four 2D microchips 13  Microfluidics in parallel synthesis Parallel synthesis of four products using a single 3D microchip 14 Facile High Throughput Wet-Chemical Synthesis Approach Using a Microfluidic-Based Composition and Temperature Controlling Platform Yang Hu 1, Bin Liu 2, Yating Wu 1, Ming Li 2, Xiaorui Liu 2, Jia Ding 2, Xiaopeng Han 3, Yida Deng 3, Wenbin Hu 2,3 and Cheng Zhong 2,3* Frontiers in Chemistry 2020 (8), 579828 15 Combinatorial synthesis (CS)  Useful to generate large amounts of compounds in a short time  Produces mixtures of compounds in each reaction vessel  The mixtures are not separated or purified, but are tested for biological activity as a whole  If there is no activity, there is no need to continue with the biological tests (in this case the mixtures are stored for future use in other drug design efforts)  Pitfall: A no‐activity result could also mean that the mixture does not contain all the expected structures or that some components of the mixture have conflicting activities 16 Mix‐and‐split method in CS  Example: The conventional synthesis of a dipeptide from 5 amino acids would require 25 individual syntheses  The mix‐and‐split method can be used to generate these compounds in less time 17 Mix‐and‐split method in CS 4. 2. 3. 1. Reactive beads are split into 5 vials 2. A (different) amino acid is added to each vial giving 5 different beads (1 in each vial) 1. 3. The beads for all 5 vials are mixed together and split into 5 new vials (i.e., each vial is identical and has 5 different types of beads) 4. A (different) amino acid is added to each of the 5 vials to make dipeptides Overall, the 5 vials will contain all 25 possible dipeptides 18 Structure Identification in CS (Tagging Approach)  The structural identification of compounds in a mixture of many compounds can be achieved by performing the syntheses with beads that harbour bifunctional linkers (such as SCAL) SCAL = Safety catch acid‐labile linker The idea behind these linkers is that they have two reactive sites (red circles) one that is involved in making the target molecules, the other being used for tagging/coding (after each synthetic step). The Trp residue is the site for target molecule synthesis. The Lys residue is the site for tagging/coding. 19 Tag Compound Tag Simplified example of a 3‐step synthesis using SCAL The order of synthetic steps: synthesis 1 → tag 1 → synthesis 2 → tag 2 → synthesis 3 → tag 3 Tag 1 codes for synthesis 1, tag 2 for synthesis 2, … Compound Tag At the end of the 3‐step synthesis, a triple tag (tripeptide) with a specific sequence (code) is obtained. Compound 20  After the last synthetic step (synthesis + tag), the target molecule on the Trp residue can be released in two steps:  1. Reduction of both sulfoxide groups  2. Treatment with an acid  Following treatment, the tags (code) still remain on the Lys residue (and are therefore still attached to the bead)  The code (attached to the bead) can then be determined by peptide microsequencing (a typical 0.1 mm bead contains about 100 pmoles of peptide) Because the target molecule is released with an acid SCAL = Safety catch Because the target molecule is safe on the linker until a special step (i.e., the reduction) is performed acid‐labile linker Because the linker catches the reactants that make up the target molecule 21 The use of photolithography in CS  In photolithography, specific products can be synthesized and detected on a plate (solid support) with very high resolution (up to 250,000 compounds per cm2)  For the synthesis of peptides (see below) the surface carries a photolabile protecting group (X)  A mask is used to expose only certain parts of the surface with light, causing deprotection  These deprotected sites are now available for coupling with an amino acid (which is also protected with the photolabile group) 22  After washing of the plate, the process can be repeated with different masks (leading to peptide chains with different sequences)  The sequences at all locations on the plate are known because they are related to the order and type of masks used  In the example below, bioactive compounds are detected with a fluorescently‐labeled receptor 23 Dynamic CS  Dynamic CS (DCS) is an emerging alternative to the typical mix and split CS  In DCS, all compounds are synthesized in one flask at the same time, and are (in‐situ) screened as they are being formed Three important aspects of DCS:  The target (enzyme/receptor) is in the reaction vessel along with the building blocks  The reactions should be reversible (compounds are being formed and are breaking back down constantly)  Because the target is present, (only) active compounds bind to it, effectively removing the compound from solution. This shifts the reversible equilibrium to generate more of the active compound (Amplification!)  Identification of the active compound(s) is achieved by freezing the equilibrium reactions  By performing an irreversible reaction that converts all equilibrium products to stable compounds 24 Example: DCS of 12 imines from 3 aldehydes and 4 primary amines in the presence of carbonic anhydrase (target) reversible irreversible  After a certain time, cyanoborohydride was added to reduce all imines in the mixture to secondary amines  The mixture was then separated by HPLC and products were identified by mass spectrometry  The same reactions were carried out also in the absence of carbonic anhydrase to see which product is amplified  It was found that the sulfonamide (below) was significantly amplified → hence, this is the active compound! 25 High throughput screening (HTS)  In drug development, HTS is used to test a vast number of compounds for biological activity  HTS is a heavily automated process using large (but compact) compound storage facilities, computer‐controlled sample‐ and liquid‐handling robots, and analyzers (often optical) a) Compound storage facility and handling robot (Boehringer–Ingelheim) b) Robot systems for screening 10,000 samples per day (Boehringer–Ingelheim) c) Design‐synthesis‐test‐analyze facility for automated drug discovery (AstraZeneca) 26 UltraHTS system at Bristol‐Myers Squibb 27  HTS assays are mostly optical assays (fluorescence, luminescence, absorbance)  The are performed in multi‐well plates (these have either 96, 192, 384, 1536, 3456 or 6144 wells) Standard 96‐well plate 384‐well plate 1536‐well plate  Since the plate dimensions are all equal (127.71 x 85.43 mm), the well size decreases with increasing number of wells on the plate  Running a 1536‐well plate compared to a 96‐well plate translates to significant cost savings (much less sample per assay!) 28