If Biomolecule Synthesis were like LEGOs :: Bio-Orthogonal Click Chemistry

 

Introduction

After exploring a few topics in "Molecular Biology" in my previous blogs, such as gene editing, nucleic acids such as DNA and RNA, lets explore a bit of "Chemical Biology" :: specifically Bio-Orthogonal Chemistry.

Our body is much like a railway network, with multiple "junctions" where these pathways meet. The path followed by the train (the product) depends on its final destination. But if there are too many trains on that route, it can cause congestion and delays. Too few trains would result in lack of connectivity between the two places.

Let me introduce you to a few terminologies Pharmacology (adding external chemical to stop a "bad process, like a run-away train / cancer cell) Metabolism managing the body's internal chemistry when the natural "conductors" (enzymes) aren't working) and Side-effects.

Metabolism

The lack or excess production of a particular metabolic product in our body can potentially interfere with other reactions in our body. For example, the accumulation of phenylalanine in the brain (due to the lack of an enzyme called phenylalanine hydroxylase, which breaks down phenylalanine into tyrosine) causes developmental and neurological issues. 

Pharmacology

Drugs like Azithromycin (bacterial infection), Paracetamol (for fever), Doxorubicin (cancer treatment through damaging the cancer cell DNA and by inhibiting enzymes crucial for DNA replication) inhibit some or the other pathway which functions in our body when our body has been invaded by some or the other organism, to bring the net product of our metabolism back to normal. 

Side Effects

Some of these drugs may affect other pathways too, much like cars jumping signals at a crossroad causing trouble for both lanes, causing what we commonly call 'side effects'. For example, aspirin (acetylsalicylic acid) is often used as a blood thinner, for relieving headaches or improving blood flow in the body, but the very same mechanisms that relieve our problem cause other problems such as stomach ulcers (since aspirin affects the cells of the blood vessels along the stomach lining, causing a break in the lining, exposing bare tissue to stomach acid). 

 

The main question to be answered is, How can we develop new compounds with similar effects and very high yields, without any side effects i.e. without interfering in other pathways?" 

For this to occur, one most effective way is to synthesise the compound inside the living organism itself, in a way that does not affect other reactions. This is where a relatively new field of biochemistry, called "Bio-orthogonal chemistry", shines through. 

BIO-ORTHOGONAL CHEMISTRY

Bio-orthogonal chemistry is that field of bio-chemistry which involves performing synthetic chemical reactions inside of a living organism, without interfering with the other reactions occurring inside the cells of that organism. 

While Pharmacology (adding external chemical to stop a "bad process, like a run-away train / cancer cell) and Metabolism managing the body's internal chemistry when the natural "conductors" (enzymes) aren't working) are deeply intertwined tracks that often affect one another, Bio-orthogonal chemistry is like a ghost train: it runs on the same network but never hits another train, never stops at a standard station, and doesn't cause any "metabolic congestion." 

These reactions are aimed at the modification of certain biomolecules within our body, inside our body itself, at particular parts of our body, which are reached through transportation vehicles such as lipid nanoparticles (hats off to whoever thought of effectively producing an empty cell, and replacing its living matter with a lot of fats (lipids) to stabilise drugs). These type of reactions usually involve two blocks :: the 'handle' and the 'probe'. This is similar to the International Space Station and the Space Shuttle (the US space program that ran between  1981 - 2011). The probe 'scans' for a suitable binding site and when it finds such a site, it 'docks' to the site, much like how the Space Shuttle docks to the International Space Station. 

However, there are some conditions that must be fulfilled for this methodology to be successful

1. It must occur at the same conditions as our body functions at i.e. at a pH of around 7 or the pH of that region of the body in case we are trying to perform that reaction in one particular region or organ (if not, then the reaction will not occur at all). 
2. It must occur fast enough to prevent other reactions from interfering with it in any way, while providing only a single type of product. 
• We need only a single type of product because our body is designed in a way that it can operate only on one particular type of a given compound, due to the highly choosy nature of protein-based structures called enzymes, which select particular forms of a compound very strictly, to ensure that only the right product is formed. 
3. Of course, it must be non-toxic and yield the product at high efficiency. (A chemist's dream; iykyk) 
4. Most importantly, it must not involve any component or group of atoms (functional groups) which involve in natural metabolic reactions in our body. 

Recently, significant research has been done investigating the different methods of synthesis of these materials inside our body. 

In this blog, I shall focus only on the MVP : Azide-Alkyne Cycloaddition (the original Click Chemistry reaction, whose variant with copper won two-time Nobel laureate, Karl Barry Sharpless, the 2022 Nobel Prize in Chemistry, his second in the same field). 

AZIDE-ALKYNE CYCLOADDITION (AAC) : THE OG 

Among every single reaction in the field, this one stands out, particularly for its efficiency and high yield.  Azides in its linear (-N=N=N), straight simplicity and the short kid, the alkyne (R-C≡C-R, where R can be any functional group). As the name suggests, the reaction involves the formation of a 'cyclic' closed compound, which goes by the name "1,4 (or 1,5 depending on the method of reaction as we will see shortly)- disubstituted triazole", which is formed by the reaction of the terminal nitrogen atoms of the azide and the carbon atoms involved in the triple bond.

This reaction was originally discovered in the 1930s by Huisgen, who discovered this reaction under high temperature and pressures. But one would now be thinking, how can two stable compounds, like these, react, even at high temperature and pressure? So now, it is time for us to understand how this reaction truly works, but there are multiple ways in which this reaction can occur, depending on the use of a catalyst. 

The main concept behind this reaction, though it is universal (nearly) for all reactions, is the energy difference between the two reactants. For that, let us briefly understand how electrons are contained in an atom. 

THE APPLICATIONS OF CLICK CHEMISTRY

Since these reactions are fast, selective and compatible with living systems, they have become powerful tools in targeted drug delivery systems and in-vivo (inside the body) drug synthesis. Some of the most important applications of this reaction is in the design of lipid nanoparticles that can accurately target organs or particular cells, by using the low-energy barrier of this reaction which allows these nanoparticles to 'couple', very specifically, to the targeted cell. 

This actually solves a long-standing issue with in-vivo drug delivery, where we know how to transport these drugs yet do not know if it will ever reach the target organ before it degrades or is attacked by the immune system since it is still recognised as a foreign body. Solving this problem has also led to the possibility of potential cures for cancer, where research continues to progress.

This technique, coupled with the use of resistant polymers such as PEG (polyethylene glycol) and click-synthesised PEG-BLOCK polymers (PEG + any polymeric unit containing a functional azide or alkyne-substituted unit) allows for the formation of highly resistant (since it evades the immune system by preventing markers known as opsonins from binding to the particle and by increasing its hydrophilicity , increasing particle circulation time in a largely aqueous blood plasma) pH-sensitive transport systems which can effectively bind to the target cells with high efficiency. 

Recently, in May 2025, a group of scientists, from the Northeastern University in Boston, USA, managed to produce a drug delivery system that involves the transportation of the precursor azide and alkyne molecules using RNA 'probes', small strands of RNA to which the alkyne group was fixed, along with azide molecules marked with fluorescent chemicals to indicate the occurrence of the reaction and also act as a visible measure of the quantity of product formed (this would likely be denoted by the fluorescence being detected close to the RNA probe, indicating that the azide has reacted with the alkyne molecule attached to the RNA). They utilised CuAAC, the copper-catalysed version of the click reaction, while managing to prevent the spreading of copper throughout the entire cell through the use of a specialised DNA ligand, simply a molecule attached to the DNA, which managed to confine the copper ions to a particular region in the cell.  This is just a small sample of the possibilities that arise in the field of 'in-vivo' drug synthesis and delivery!

This also allows us to build a huge collection of possible azide-alkyne drug precursors, through computer simulations mimicking the conditions of our body, and helps to greatly speed up the process of drug discovery, by targeting the particular cells that are impacted by the problem and delivering the cure.  Currently, multiple such drugs are being tested by the FDA, for their approval. 

APPENDIX - For the Every Curious Minds

A Deep-Dive into the AAC MECHANISM - DOWN TO THE GRASSROOTS

Think about it, just like how two people with similar interests, who share the same vibe and energy, get together quite easily, similarly two reactants that share the same energy bind together quite fast and very easily. 

As we would be familiar, 'orbitals' are regions in the space of an atom that possess a particular amount of energy and are almost guaranteed to contain some of the fundamental particles of  matter, electrons. When these orbitals involve in the formation of a chemical bond, the orbitals of similar energy levels always choose each other to form a strong, stable bond and, usually, this occurs between the highest occupied molecular orbitals (HOMO), the highest-energy orbital containing electrons, and lowest unoccupied molecular orbitals (LUMO), the lowest-energy orbital which is left empty. 

These orbitals are usually found far away from the centre of the atom i.e. the nucleus, since electrons in an atom are filled up in the orbitals in ascending order of their energies, which is determined by the Aufbau principle (i.e. electrons fill orbitals in order of lowest to highest energies; the relative energy of these orbitals is determined by the sum of their principal and angular quantum numbers, as per the s-p-d-f (these letters are an indication of their angular orientation in 3-dimensions) system (eg:- 1s :- 1 + 0(s) =1 ; 2p :- 2 + 1(p) = 3; 3d:- 3 + 2(d) =5 and so on).  

The way in which electrons are filled can be graphically represented through the Molecular Orbital Diagram, which represents the formation of new orbitals during the formation of a bond between any two atoms or more through the overlap of atomic orbitals.  These also help us to determine the HOMO and LUMO during bond formation. 

Based on the overlap of the HOMO and LUMO, a theory called 'Frontier Molecular Orbital (FMO)' theory, was proposed by Japanese scientist, Kenichi Fukui. This theory proposes that during a chemical reaction, the product formed depends only on the nature of the HOMO and LUMO orbitals, the last occupied and first unoccupied orbitals. This makes sense since the HOMO of one atom and the LUMO of the other atom will have relatively lower energy gaps, usually the lowest, allowing for an almost-perfect orbital overlap, provided their orientations are correct, like two LEGO blocks of similar, but not the same, type being fitted together. Also, when we combine the basic concepts of electrostatics, the study of static charges, we see that the nucleus, which is just a collection of positively charged protons and neutrally charged electrons, 

This, along with the fact that each orbital has a distinct orientation in a 3-D space, allows us to predict the type of product, along with the particular atoms that actually react together (called 'regioselectivity' i.e. the reaction occurs only at a particular atom and a particular orientation of the reactant molecules and forms only a particular form of the same compound). 

This reaction obeys a similar pathway; the azide and alkyne share similar energies between their HOMO and LUMO and 'click' together like the two LEGO blocks mentioned before, under the right conditions. However, the high temperature and pressure required for the original reaction was almost unfeasible. 

This is where the work of Sharpless and his fellow scientists breaks the ice, by introducing metal catalysis into the reaction, particularly copper and ruthenium, or by simply forcing the alkyne into bonding with the azide by increasing its frontier orbital energy level.  The copper-based reaction works only with terminal alkynes (alkynes of the form R-C≡CH), since it involves the formation of a charged intermediate (R-C≡C Cu), which is further stabilized by the another copper atom, forming a metal-coordination complex, which interacts with the terminal negatively charged nitrogen atom of the azide group,  Then, following a bond shift, that occurs due to overloading of the nitrogen atom with electrons, the other terminal, negatively charged nitrogen atom attacks the other carbon atom in the bond, forming a cyclic product, particularly the 1,4-triazole ring (don't worry, '1,4' depends on a specific numbering system for such compounds, the IUPAC system). 

The Mechanism of CuAAC

       

The ruthenium-catalysed reaction, on the other hand, does not involve the ionic intermediate, rather it involves the formation of a cyclic complex compound, called a 'ruthenacycle', which is a 6-sided cyclic intermediate containing both the azide and alkyne. For this, a ruthenium complex, stabilised by heavy, electron-donating ligands such as pentamethylcyclopentadienyl (Cp*) anion, is used. The electron-donating nature promotes the formation of high oxidation states of Ruthenium atom, which is required to maintain the stability of the cycle. This also contributes to steric crowding, which allows the reaction to occur only at a particular region, hence leading to the formation of only a single type of product. The alkyne which, unlike in the copper-catalysed reaction,  and the azide then bind to the ruthenium atom, where a similar process like that of the copper-coordinated intermediate takes place, but the region of occurrence of bonding slightly varies which, after the formation of a closed ring between the alkyne and azide, results in the formation of a 1,5-triazole instead of the 1,4-triazole. An important point to note is that in either reaction, only the respective isomers mentioned, also called regioisomers, are synthesised, not both. This ensures the formation of only a selective isomer, in line with the principle of bio-orthogonal chemistry. 

The mechanism of RuAAC

In the end, there is only one way to put it clearly:- In many ways, AAC in bio-orthogonal chemistry is one of the most elegant yet efficient tools of modern chemical science: a set of reactions so selective and reliable that they have seamlessly integrated themselves into the modern world of drug discovery. 

With that note, I take your leave. I hope you enjoyed my blog, and that I have managed to at least break the ice over the field that is Bio-Orthogonal Chemistry. 

ADIOS!! TAKE CARE!