Synthesis Methods and Specific Drugs

• Combinatorial synthesis: the automated synthesis of a large numbers of compounds in a short time period using a defined reaction route and a large variety of reactants.
• In solid phase synthesis, reactants are bound to a solid support and remain attached to the bead during the synthetic sequence.

  • Excess reagents can be used to drive reactions to completion
  • Excess reagents and by-products are easily removed
  • Polymeric support can be regenerated and re-used after cleaving the product if used
  • Automation is possible
• Solid phase combinatorial synthesis: beads can be mixed and reacted in the same reaction vessel. Products formed are distinctive for each bead and physically distinct.
• Parallel synthesis: automatic synthesis of a number of compounds in a shot time using a defined reaction route and a variety of reactants. Single products are formed in each reaction vessel.
• Solid phase synthesis requires a resin bead/functionalised surface to act as a solid support, as well as:

  • Anchor/linker
  • Bond linking to the anchor/linker
  • Ability to cleave the product
  • Protecting groups

  
• Linker: a molecule covalently attached to the support which contains a reactive functional group, allowing attachment of the first reactant.
• Linkers must be stable to the reaction conditions in the synthesis. Different linkers are choice dependant on the group to be attached and the desired functionality on the product and amount.
• Solid supports can be controlled pore glass (CPG) and microporous polystyrene (MPPS) for nucleic acids. These are non-swellable/low-swellable solid supports. Resins are the insoluble particles of 50 - 200 microns in diameter.
• Resins functionalised with DNA bases are available commercially to start oligonucleotide synthesis.

  • 5'-DMT nucleosides are treated with succinic anhydride in pyridine at room temperature to form nucleoside succinate
  • Large excess of nucleoside succinate coupled with aminoalkyl resin
  • Capping step then blocks any unreacted amino groups
  • Loading of nucleoside on resin is determined by trityl analysis

  
• Controlled pore glass (CPG):

  • Rigid, non-swelling
  • Deep pores in which synthesis takes place
  • Suitable for oligonucleotides up to 150 bases or longer
  • 500 angstrom pores
  • Yields fall of for oligonucleotides > 40 bases
• RNA deprotection strategy uses aqueous ammonia and can cleave oligonucleotides from the solid support and remove protecting groups from the bases. t-butyldimethylsilyl (TBS) needs a different reagent, fluoride.
• Common protecting groups for bases:

  • N(6)-benzoyl dA
  • N(2)-isobutyryl dG
  • N(4)-benzoyl dC
• For chemically modified oligonucleotides (sensitive to ammonia), protecting groups used:

  • N(6)-phenoxacetyl dA
  • N(2)-isopropyl phenoxyacetyl dG
  • N(2)-acetyl dC
• The teabag method: a method of parallel synthesis with a cheaper economy. It is not suitable for producing large quantities of different products.

  
• Tagging: the method of identifying the structure present on a bead. The molecule is constructed on the same bead as the target molecule.
• Tagging requires a linker with two functional groups, one to release the tag and one to release the target molecule.
• Hypertension: high blood pressure.
• High cholesterol: build-up of fatty deposits inside the arteries and an increased risk of blood clots.
• Coronary heart disease: when the blood supply to the heart becomes restricted.
• Angina: chest pain caused by reduced blood flow to the heart muscles.
• Heart attack: when the supply of blood to the heart is suddenly blocked.
• Stroke: when the supply of blood to the brain becomes blocked.
• Targets and medications for cardiovascular disease:

  • Calcium channel blockers (CCBs) to relax the arteries, increasing blood supply to the heart muscle
  • Beta blockers (BBs) to make the heart beat slower with less force
  • Angiotensin converting enzyme (ACE) inhibitors to reduce blood pressure
  • Diuretics, decreasing the amount of fluid through the arteries to reduce blood pressure
  • Cholesterol-lowering drugs such as statins (small molecule) and Inclisiran (siRNA)
• Hypercholesterolaemia:

  • Consequence of high-fat diet in combination with genetic risk factors
  • High levels of low-density lipoprotein (LDL)
  • Accumulation of LDL accelerates the progress of atherosclerosis
• Statins: block the pathways for synthesising cholesterol in the liver by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme of cholesterol synthesis.
• Antibodies can be used as inhibitors to proprotein convertase subtilisin kexin type 9 (PCSK9) which leads to degradation of the LDL receptor.
• Severe hypocholesterolaemia cannot be controlled by statins as it is caused by mutations in the hepatic LDL receptor on liver cells. This is by mutation of apolipoprotein B, the main protein component of LDL particles which facilitates binding.
• A cause of familial hypercholesterolaemia is gain-of-function mutations in the PCSK9 gene.

  • PCSK9 binding to LDL loaded LDLR is directed to the lysosome for degradation
  • Without PCSK9 binding the LDL particle is returned to the cell surface
  • PCSK9 causes LDLR degradation
• Pharmacophore: molecular features that determine target specificity.
• Dianophore: molecular features that determine tissue distribution and metabolism.
• RNA interference: sequence-specific suppression of gene expression by dsRNA through translational or transcriptional repression.
• Types of vaccines:

  • Inactivated
  • Live-attenuated
  • Viral vector
  • Subunit, recombinant, polysaccharide or conjugate
  • Toxoid
  • DNA and mRNA
• Inactivated vaccines: a dead version of a germ that causes disease e.g., hepatitis A, flu, polio, rabies.
• Live-attenuated vaccines: live vaccines with a weakened form of the germ e.g., MMR, rotavirus, smallpox, chickenpox, yellow fever.
• Viral vector vaccines: use vector to deliver genetic material for protein or antigen to elicit an immune response e.g., Ebola, zika, flu, HIV, COVID-19.
• Subunit, recombinant, polysaccharide and conjugate vaccines: specific pieces of the germ e.g., haemophilus influenzae type B (HIB), hepatitis B, whooping cough, pneumococcal and meningococcal, shingles.
• Toxoid vaccine: toxin made by the germ that causes disease e.g., diphtheria, tetanus.
• DNA and mRNA vaccines: make proteins to trigger an immune response e.g., COVID-19.
• Antigen: a foreign particulate matter that can bind to a specific antibody or T-cell receptor. Can trigger an immune response.
• Synthetic mRNA:

  • 5' cap for ribosome initiation, translation and stability
  • 5' untranslated region (UTR) for translation and stability
  • Coding sequence region, encoding gene of interest
  • 3' UTR for translation and stability
  • Polyadenylated (polyA) tail for ribosome initiation, translation and stability