Synthesis and Analysis of Biologics 3900-BT1-6
Course schedule:
Week 1
Day 1:
Introduction: course structure, an overview of the planned syntheses
Safety course: an overview of the laboratory techniques used during the course (working under anhydrous conditions, working with moisture-sensitive reagents, solution synthesis, solid-phase synthesis, equipment operating under reduced pressure – rotavaps, pumps, lyophylizer), working with organic reagents, safety precautions
Synthesis and purification of modified building block for solid-phase RNA synthesis:
modification of a commercial adenosine phosphoamidate under PTC conditions; workup of the reaction mixture.
Day 2 and 3
Synthesis of mRNA cap analogue:
Purification of the modified building block
Synthesis and purification of dinucleotide part of the cap analogue
Week 2
Synthesis of mRNA cap analogue (continuation)
Day1
Activation of the dinucleotide (reaction setup, reaction monitoring, precipitation of the product)
Day 2 and 3
Final cap assembly (reaction setup, reaction monitoring, initial workup)
Week 3
Synthesis of mRNA cap analogue (continuation), purification of final cap (ion exchange chromatography, preparative HPLC), characterization (MS, NMR), quality control
Week 4
Automated synthesis of modified oligonucleotide
Day 1
Synthesizer set-up
Preparation of the proper solutions and reagents
reaction monitoring
Day 2
Reaction workup, oligonucleotide deprotection
Day 3
purification of the oligonucleotide (precipitation and/or preparative HPLC)
Week 5
Bioconjugate reactions and final seminar
Day 1 and 2
selected bioconjugation reactions, modification of oligonucleotides (amide coupling, “click chemistry”),
Working in microscale, reaction monitoring (HPLC),
Day 3
Group seminar, presentation of results, discussion of the results
Course coordinators
Type of course
Mode
Prerequisites (description)
Learning outcomes
Knowledge and understanding
1. Explains the basic strategies used in the synthesis of modified nucleotides, mRNA cap analogues and modified oligonucleotides.
2. Describes the principles of solution-phase and solid-phase synthesis in the context of nucleotide and oligonucleotide chemistry.
3. Understands the role of protecting groups, and activated building blocks in oligonucleotide synthesis.
4. Explains the principles of automated solid-phase oligonucleotide synthesis.
5. Understands of the main purification techniques used for biomolecules, including ion-exchange chromatography, reversed-phase chromatography and preparative HPLC.
6. Understands the purpose and limitations of analytical methods used for nucleotide and oligonucleotide characterisation, including HPLC, NMR and MS.
7. Explains the basic principles of post-synthetic oligonucleotide modification using bioconjugation chemistry.
Practical and analytical skills
1. Carry out selected multistep synthetic procedures involving nucleotide derivatives and oligonucleotide-based compounds.
2. Works safely with organic solvents, moisture-sensitive reagents, fluorescent dyes and chromatographic systems.
3. Performs basic reaction monitoring using TLC and HPLC.
4. Applies suitable purification techniques to intermediates, final products and modified oligonucleotides.
5. Prepares samples for NMR, MS and HPLC analysis.
6. Interprets basic analytical data to confirm product identity and assess purity.
7. Identifies common problems in synthesis and purification, such as incomplete conversion, product degradation, low recovery or insufficient purity.
8. Prepares detailed laboratory reports containing experimental procedures, analytical data and discussion of results.
Competences
1. Plans and organizes laboratory work in a multistep synthetic project.
2. Works effectively and safely in a laboratory team.
3. Critically discusses experimental results and relate them to the applied synthetic strategy.
4. Presents laboratory results in a clear and structured way during a group seminar.
5. Evaluate the reliability of analytical evidence supporting the identity and quality of synthesised compounds.
Assessment criteria
The exam takes the form of a multiple-choice test covering the material presented during the lectures. It lasts up to 90 minutes and consists of up to 30 questions or tasks, each scored individually, for a maximum of 100 points.
The final grade is based on the percentage of points earned out of the maximum possible score:
up to 50% – fail (2.0)
51–60% – satisfactory (3.0)
61–70% – satisfactory plus (3.5)
71–80% – good (4.0)
81–90% – good plus (4.5)
91–100% – very good (5.0)
A minimum of 51% of the total points is required to pass the exam.
A maximum of two absences is allowed. For multi-stage experiments conducted in pairs, at least one member of the team must be present to continue the work.
Bibliography
Modification of phosphoamidates and synthesis of cap analogs:
1) Kamil Ziemkiewicz, Marcin Warminski, Radoslaw Wojcik, Joanna Kowalska, Jacek Jemielity; Quick Access to Nucleobase-Modified Phosphoramidites for the Synthesis of Oligoribonucleotides Containing Post-Transcriptional Modifications and Epitranscriptomic Marks. Journal of Organic Chemistry 5 August 2022; 87 (15): 10333–10348. https://doi.org/10.1021/acs.joc.2c01390
2) Marcin Warminski et. al. ; Trinucleotide mRNA Cap Analogue N6‑Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo. Journal American Chemical Society 27 March 2024; 146 (12): 8149–8163. https://doi.org/10.1021/jacs.3c12629
Solid phase synthesis and nucleic acid chemistry:
https://atdbio.com/nucleic-acids-book
chapter 5
Bioconjugate reactions:
1) Hermanson, Greg. (2013). Bioconjugate Techniques: Third Edition. Bioconjugate Techniques: Third Edition. 1-1146. 10.1016/B978-0-12-382239-0.00005-4.
Selected publications and books will be recommended for individual task.