This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
DNA plays an import hole in biological activities and its study gradually grew during the years. Knowledge of DNA chemistry provided the basis for the development of synthesis methods of nucleic acids in the laboratory.1 This achievement is perhaps the most important in the area for understanding the functions of DNA,1 and nearly every molecular biology technique in use today employs chemically synthesized DNA’s and RNA’s for use in DNA sequencing, site directed mutagenesis, clone and express genes, etc.²
For most applications, very small quantities are required;³ however, a fast method with high yield and quality of product is necessary. Nevertheless, several years ago the synthesis was very laborious and time consuming. As an example, a 21 base paired DNA duplex could last the equivalent of four years of highly skilled and intense effort.4 In the 1950s to 1960s the solution technique for DNA synthesis developed by Khorana was used. It was known as phosphodiester method and it refers to the by-products: diester phosphate salts. The purification of products were extremely time consuming because of these salts, as the yield were not high and the product needed characterization in each step of the synthesis.¹
With the increasing of applications for DNA oligonucleotides, several laboratories focused on developing synthesis methodologies. The first improvement was the triester production instead of diester phosphate salts, which increased the yields and turned the purification easier.¹ The next advance was the shift from solution methods to solid support methods, which improved the purification step, virtually eliminated mechanical losses and allowed the use of large excess of reactants to drive the reaction to products.¹
By 1980’s, a breakthrough was achieved and a less than a day synthesis method of oligonucleotides was established.4 It is known as phosphoramidite method of DNA synthesis. A phosphoramidite monomer is very different synthesis unit compared with the previous ones. It is a normal nucleotide but with protection groups added to the reactive amine, hydroxyl and phosphate groups. (Figure 1). Also, the link to the support is made through the 3’ carbon, so the synthesis proceeds from 3’ to 5’ carbon,² rather than to 5’ – 3’ direction of DNA biosynthesis (replication). This synthesis method made it possible to make longer oligonucleotides, increasing the quality and yield and decreasing cost,² so this work will focus in the description of this chemical synthesis method of DNA.
Figure 1: Nucleoside phosphoramidite monomer for chemical synthesis of DNA.¹
CHEMICAL SOLID-PHASE DNA SYNTHESIS
- Solid supports
Solid-phase synthesis is really important and widely used in synthesis of peptides, oligonucleotides and oligosaccharides. The advantages are multiple, but the most important are: driving of reactions to completion by large use of reagents; cancellation of purification of products after each step due the washing of impurities and excess reagents before each reaction; and automation on computer-controlled solid-phase synthesizers.³
The solid-phase synthesis is carried out on a solid support held between filters in columns that enable all reagents and solvents to pass through freely. The solid support, also known as resins, are insoluble particles, can be of different compositions and typically 50-200 μmin diameter. In the DNA synthesis, the oligonucleotide is bounded during the synthesis.³ Two types of solid supports have proved to be the most useful for the phosphoramidite DNA synthesis method, the controlled pore glass (CPG) and polystyrene (PS).
- Synthesis of nucleoside phosphoramidite monomers
The adenine, guanine, cytosine and thymine phosphoramidites monomers used in the DNA synthesis are prepared on large scale from the free nucleosides, which are obtained from natural sources. The synthesis consists in the protection of the amines (except thymine, which does not need protection), followed by a tritylation of the 5’ carbon with the 4,4’-dimetroxytrityl chloride in pyridine at room temperature (protection of the 5’-hydroxyl group) and finally, after purification of the product, a phosphitylation at the 3’ carbon using 2-cyanoethyl diisopropylaminophosphorochloriditein the presence of the non-nucleophilic base diisopropylethylamine (DIPEA). After treatment in a silica gel column chromatography, precipitation into hexane and filtration through microfilter, the phosphoramidite monomers are ready to use in DNA synthesis. ³
- Protection of amides
The amides can be protected by two methods, a common benzoylation followed by adding a strong base to recover the alcohols groups (Figure 2) or a transient protection of the alcohol functions followed by benzoylation of the amino groups (Figure 3). The last has the advantage of being a one-pot synthesis, the isolation or purification of the intermediate silylated nucleoside is not necessary. ³
Figure 2: Benzoyl protection of adenine ³
Figure 3: Transient protection of adenine ³
There is another method for the protection of deoxycytidine: its amino group is sufficiently reactive to be functionalised with active esters that do not react with the hydroxyl functions. So, it can react with pentafluorophenylbenzoate affording a one-step synthesis of the protected N(4)-benzoyl dC (Figure 4). ³
Figure 4: Protection of cytosine using pentafluorophenyl benzoate ³
The protection of guanine follow same mechanisms than the protection of adenine, but using with an isobutyryl protecting group (Figure 5). Thymine, as said before, does not need protection.
Figure 5: Protected guanine ³
- Protection of 5’-hydroxyl group – Tritylation
The protected dA, dG, dC and unprotected T nucleosides are tritylated selectively using 4,4’-dimethoxytrityl (DMT) at the 5’-hydroxyl group to protect it (Figure 6). ³
Figure 6: DMT nucleoside protection ³
The DMT-nucleosides are now phosphitylated at the 3’-position using 2-cyanoethyl diisopropylaminophosphorochloriditein the presence oof DIPEA to give the phosphoramidite monomers (Figure 7). ³ The reagent used for the phosphitilation is made by treating phosphorus trichloride with 2-cyanoethanol then with N,N-diisopropylamine in the presence of DIPEA (Figure 8). ³
Figure 7: Nucleoside phosphitylation ³
Figure 8: Synthesis of the phosphitylation reagent ³
- Resin Functionalization
After the synthesis of phosphoramidite monomers, it is necessary to treat the solid support to produce the functionalised resin for the synthesis of oligonucleotides. The functionalization occurs by the treatment of 5’-DMT nucleosides with succinic anhydride at room temperature in the presence of pyridine. Alarge excess of each nucleoside succinate is then added to a batch of the amino-functionalized resin along with a diimide coupling agent and an acidic alcohol such as 4-nitrophenol which forms an active ester in which it acts as a good leaving group. This is followed by a capping step to block any unreacted amino groups which would otherwise cause problems in oligonucleotide synthesis (Figure 9). ³
Figure 9: Resin functionalization ³
- The phosphoramidite method for chemical solid-phase DNA synthesis
The phosphoramidite method for DNA synthesis proceeds in the 3’ to 5’ direction and one nucleotide is added per synthesis cycle. It also consists of a series of steps, which are going to be outline below.
Figure 10: The phosphoramidite oligonucleotide synthesis cycle ³
- Detritylation of the support-bond at 3’ carbon of the nucleoside.
The first step consists in the removal of the protection group of the 5’ carbon. It is functionalised with DMT to prevent polymerization during the resin functionalization, but it is necessary to be removed so the synthesis can proceed.³ The mechanism of the detritylation is showed above (Figure 11), it uses trichloroacetic acid as catalyst.
Figure 11: Phosphoramidite nucleoside detritylation ³
- Activation and Coupling
After the detritylation, the support-bound nucleoside is ready to react with the next base. It is added in the form of nucleoside phosphoramidite monomer in large excess, mixed with an activator (tetrazole), both dissolved in acetonitrile. The mechanism of the activation of the nucleoside phosphoramidite monomer and the coupling is showed in Figure 11: the activation occurs by a protonation of the phosphoramidite monomer by the activator, turning the amino group of the phosphoramidite bond into a good leaving group, which is rapidly displaced by attack of the 5’-hydroxyl group of the support-bound phosphite trimester. The unbound base and by-products are washed out at the end of the reaction, and the tetrazole is reconstituted.³ The use of this activator increase coupling efficiency to greater than 99%.²
Figure 11: Phosphoramidite activation and coupling with support-bond nucleoside. ³
Even with a great coupling efficiency, the process still have a failure rate. In this case, the failure consists in the remain of unreacted support-bond nucleoside, it means, remain freely reactive and able to couple in the next cycle that would result in a missing base in the synthesis. Therefore, coupling failures must be removed from further participation in the synthesis by introducing a capping step to block the unreacted 5’-hydroxyl groups. It uses acetic anhydride and N-methyllimidazole (NMI) dissolved in tetrahydrofuran and a small quantity of pyridine (Figure 12).
Figure 12: Phosphoramidite capping ³
The oxidation step is a process necessary to stabilize the phosphite-triester (P(III)) formed during the coupling step, which is unstable in acid. The goal is to convert it into stable (P(V)) species, and it is achieved by iodine oxidation in presence of water and pyridine (Figure 13). The resultant phosphotriester is effectively a DNA backbone protected with a 2-cyanoethyl group that prevents undesirable reactions at phosphorous during subsequent cycles.
Figure 13: Phosphoramidite oxidation ³
After all these steps, it is necessary a new detritylation to remove the DMT protection group at the 5’-end of the resin-bound DNA chain so the hydroxyl can react with the next nucleotide phosphoramidite in the next cycle. It is the same process than the first step, but now using the support-bound DNA chain instead of the primarily support-bound nucleoside.
- Cleavage from the solid support
After the completion of all synthesis cycles to form the desired oligonucleoside, it is necessary to cleavage the DNA from the support. The linker used most frequently in DNA synthesis is the succinyl linker. It is stable in all reagents during the synthesis, but cleavable by treatment with concentrated ammonium hydroxide at room temperature for one hour (Figure 14).
Figure 14: oligonucleotide resin cleavage ³
- Oligonucleotide deprotection
The oligonucleotide dissolved in concentrated aqueous ammonia from the last step now is heated to remove the protection groups from the heterocyclic bases and phosphates (Figure 15).
Figure 15: Oligonucleotide deprotection ³
The last step of chemical synthesis of DNA is the purification of the products obtained. It mainly uses two methods for post-production purification: Polyacrylamide gel electrophoresis (PAGE) or High performance liquid chromatography (HPLC). ² The quality control of the purified product is also necessary and can be made by mass spectrometry (MS) or capillary electrophoresis (CE). ²
- FINAL CONSIDERATIONS
The first attempts of chemical DNA synthesis are from the same time of the discovery of DNA, due the importance of knowing this molecule. The method was developed, and the advent of phosphoramidite chemistry involving tetrazole intermediates in the 1980’s make it possible to make longer oligonucleotides, faster, and with higher yields and quality. Nowadays, this method is widely used for the chemical synthesis of DNA, and is very important for the multiple applications of synthetic DNA. This process also permits multiple alteration in the composition of the DNA, allowing different studies.
¹ Garrett Larson, Bruce E.Kaplan, John J. Ross, The American Biology Teacher, 1984, 46, 440-446.
² Chemical Synthesis and Purification of Oligonucleotides, Integrated DNA Technologies, https://eu.idtdna.com/Pages/docs/technical-reports/chemical-synthesis-of-oligonucleotides.pdf (Accessed December 2014).
³ Solid-phase Oligonucleotide synthesis, atdbio, http://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis (Accessed December 2014).
4 Marvin H. Caruthers, Acc. Chem. Res., 1991, 24, 278-284.