In recent years, breakthroughs in nucleic acid modification and delivery vectors have brought about a wave of innovative transformational therapies. Among them, nucleic acid drugs, which are considered to be the third generation of innovative drugs after small molecule drugs and antibody drugs, have experienced explosive growth. Its advantages lie in a wide range of druggable targets, strong specificity, high safety, lasting effects, high development success rate and low manufacturing costs. Oligonucleotide drugs are single-stranded or double-stranded structures composed of 20-60 Nucleotide units. They regulate the transcription and translation of disease genes and inhibit gene expression by acting on mRNA. They mainly include antisense oligonucleotides (ASOs), small interfering RNA (siRNA) and micro RNA (miRNA).
Currently, most oligonucleotide drugs are synthesized by solid-phase phosphoramidite chemistry. Chemical synthesis is carried out in the 3'-5' direction.
The commonly used solid phase carrier is controlled pore glass (CPG). CPG is covalently bound to the 3'-OH of the initial nucleotide ribose through a linker, while the 2'-OH of the ribose is protected with a protecting agent such as tert-butyldimethylsilyl (TBDMS), and the 5'-OH is protected with dimethoxytrityl (DMT). In addition, due to the presence of primary amino groups in adenine, guanine, and cytosine, they also need to be protected with acyl reagents (such as benzoyl).
Each cycle of solid phase synthesis mainly includes four steps: deprotection, coupling, oxidation, and capping.
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Use 2-3% trichloroacetic acid (TCA) or dichloroacetic acid (DCA) dissolved in dichloromethane/toluene to remove the DMT group of the ribose 5', exposing the 5'-OH for the next coupling. The deprotection time depends on the flow rate and column size. Insufficient reaction time/too weak acidity of the deprotection agent will produce N-1 impurities (only one nucleotide different from the full length N oligonucleotide). Too long reaction time/too strong acidity of the deprotection agent will lead to the production of depurine in RNA. After the reaction is completed, the residual deprotection agent is removed by washing with acetonitrile; the water content of acetonitrile in this step is typically less than 20 ppm, and the deprotection agent is not cleanly rinsed resulting in the production of N+1 impurities. Notably, trace trichloroacetaldehyde impurities in the DCA solution reacted with the free Hydroxyl Groups at the end of the oligonucleotide in the synthesis resulting in 147 Da more impurities than the full-length product (FLP).
The next nucleic acid phosphoramidite monomer reacts with tetrazole to form an active intermediate (this step is also called activation). The 5'-OH formed during deprotection attacks the active intermediate with nucleophilicity, forming a new phosphorus-oxygen bond and removing tetrazole, and the nucleotide chain is extended. In order to ensure a high overall yield, a high coupling efficiency is required in each cycle. N-1 impurities are the most common impurities in coupling, and they are the result of coupling efficiencies less than 100%. The N-1 impurity of the 25-mer oligonucleotide was tailed with dA, annealed and cloned with a plasmid with a dT tail, and 70 clones were randomly selected for sequencing. The results showed that all possible N-1 sequences were included, but the frequency of 3'-terminal truncated nucleotides was higher than that of 5′-terminal truncated nucleotides. It is speculated that this phenomenon is caused by steric hindrance, which makes the efficiency of the four-step reaction at the 3′-end low. The short extended chains at the 3′-end are close to each other, and DMTr also hinders the reaction. In addition, steric hindrance also reduces the efficiency of removing the DMTr group, making the protected chain unable to couple. Compared with FLP, higher molecular weight impurities are also present in the coupling step. The formation of long sequence impurities is attributed to the weak acidity of the activator tetrazole to remove a portion of the DMT group in the phosphoramidite solution. There are two mechanisms leading to the formation of this impurity. One is that the newly added phosphoramidite monomer forms a dimer by itself and then reacts with the deprotected 5'-OH; the other is that the newly added phosphoramidite monomer is coupled twice.
After the coupling reaction, the newly added nucleotide is connected to the oligonucleotide chain on CPG through a phosphite bond. The phosphite bond is unstable and easily hydrolyzed by acid and base. It is unstable in the acidic environment of the next cycle of deprotection, so it needs to be oxidized to a stable pentavalent phosphorus. The 2-cyanoethyl protecting group in the phosphodiester bond can make it more stable in subsequent synthesis. In addition, it can also be converted to pentavalent phosphorus by transferring a sulfur atom to trivalent phosphorus, thereby forming a thiophosphate bond. When synthesizing oligonucleotides containing both phosphodiester (PO) bonds and thiophosphate (PS) bonds, the oxidizing agent used in this step can oxidize the already formed PS bond to a PO bond, resulting in an impurity 16 Da less than FLP. The incomplete oxidation/sulfurization of the phosphite bond reacts with the DMT cation formed by the subsequent deprotection to produce two acid-stable impurities. One impurity is 366 Da more than N-1, and the other impurity is 286 Da more than FLP.
Since it is impossible to achieve 100% coupling efficiency, there are still 5'-OH reactive groups that do not react after deprotection. If left untreated, these groups can still couple in the next cycle to produce N-1 impurities. Two reagents (capping reagent A, usually acetic anhydride; capping reagent B, usually N-methylimidazole) are usually used to acylate the 5'-OH. Reagents A and B are mixed and delivered to the system during the capping step. Acetic anhydride produces an impurity that is 41 Da more than expected by converting the protected guanine to acetylated diaminopurine.
Repeat the cycle until the desired length of oligonucleotide is synthesized. After synthesis, the oligonucleotide can be cleaved from the CPG and the bases can be deprotected by ammonia treatment. The cleaved oligonucleotide has a free 3'-OH at the end. When using the universal UnyLinker support, incomplete cleavage will result in an impurity with 261 or 275 Da more than FLP. Incubation at higher temperatures for longer times can convert the impurity to the final product.
When the 2'-OH in the oligonucleotide is deprotected from the TBDMS group, there is competition between the free 2'-OH and the hydroxyl group on the support to attack the phosphorus, resulting in an oligonucleotide with a 3' phosphate. Once this impurity is formed, it is difficult to remove. The isobutyl protecting group in Guanosine is one of the most difficult groups to remove when deprotecting the bases by ammonia treatment, requiring incubation at 55°C for several hours, and longer for guanosine-enriched sequences. Incomplete deprotection of guanosine produces an impurity 70 Da higher than FLP. Deprotection with AMA reduces the reaction time but increases the likelihood of an impurity 14 Da higher than FLP. In addition, methylamine can react with C-4 in cytidine to produce N-4-methylcytidine, which can be reduced by using an acetyl protecting group on cytosine. The more stringent conditions required for guanosine deprotection also result in the conversion of PS to PO, requiring optimization of incubation time to maintain a minimum ratio of PS to PO conversion while completely deprotecting guanosine. The alkaline conditions of C&D allow for deamination reactions that convert the exocyclic amines of cytidine and adenosine to carbonyl groups. Nonenzymatic deamination is catalyzed by acids and bases and accelerated at elevated temperatures, with a mass difference of only 1 Da before and after deamination.
Incomplete removal of TBDMS in the C&D stage produces an impurity 114 Da higher than FLP when the 2′-OH is protected with TBDMS. Deprotection is usually performed by treatment with fluoride ions in organic salts. The acidic properties of TEA-3HF neutralize the alkalinity of the C&D solution, otherwise exposure of the 2′-OH to alkaline conditions is likely to result in chain cleavage. This results in the formation of cyclic phosphates, 5′-2′ isomerization, and truncated sequences at the 3′ end.
In C&D of RNA using methylamine, methylamine reacts with uracil to produce impurities 52 Da and 95 Da smaller than FLP.
2′-F modified oligonucleotides will lose HF when subjected to harsh C&D conditions, and the subsequent addition of water molecules will eventually lead to the replacement of 2′-F with a hydroxyl group and stereochemical inversion at the 2′ position.
In the subsequent purification stage, ion exchange chromatography and reverse phase chromatography are often used to remove impurities in the product. The purified oligonucleotides can be stored for a long time or used directly for subsequent analysis.
Fig. 1 Oligonucleotide synthesis cycle. (Andrews, et al., 2021)
Unmodified oligonucleotides are susceptible to degradation by nucleases upon entering the human body and display unfavorable cellular uptake and biodistribution. Commonly modified positions on oligonucleotides include the phosphate backbone, ribose, pyrimidine bases, and the addition of targeting ligands.
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2' chemical modification of ribose can reduce the ability of nucleases to degrade small nucleic acid drugs. At the same time, it also significantly reduces the ability of TLR-3/7/8 to recognize small nucleic acid drugs as exogenous nucleic acids, and greatly improves the stability and overall half-life of nucleic acids.
Although PS modification slightly reduces the binding affinity of small nucleic acids to target sequences, it improves the nuclease resistance of oligonucleotides in blood and tissues. Since the replacement of the oxygen atom by a sulfur atom introduces an additional chiral center, there are two stereoisomers (Rp and Sp) on each PS bond. When one conformation can be degraded by nucleases, the other conformation often exhibits strong nuclease resistance. At the same time, the electron cloud of PS tends to be more dispersed than that of PO, resulting in more hydrophobicity. This property plays a role in promoting protein binding and extending plasma half-life, but non-specific protein binding ability also leads to toxicity, so the number of PS modifications is often strictly controlled in druggability studies.
LNA as well as cEt and tc-DNA modifications involve bridging of Sugar Rings, each of which promotes RNA-like structures, displays nuclease resistance and, most importantly, significantly increases binding affinity to target sequences.
Fig. 2 Therapeutic oligonucleotide modifications. (Baker, et al., 2022)
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| X22-09-ZQ075 | Adenosine 5'-triphosphate (ATP) tris solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ076 | Guanosine 5′-triphosphate (GTP) tris solution, 100 mM | >98% | Inquiry |
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