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Lecture 12

CHEM564 Lecture 12: Sec 3 (1)

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University of Alberta
Christopher Cairo

BioconjugateChemistry Cairo 3 Nucleic acids 3.1 Structure and synthesis of Nucleic acids 3.1.1 Structure and function of nucleic acids The structure of oligonucleotides consists of individual nucleotides linked by a phosphodiester. DNA/RNA synthesis is now done routinely using established protocols, and can often be obtained from commercial sources. The versatility of oligonucleotide synthesis is a central technology for molecular biology, as it is used to generate specific primers and other reagents necessary for these methods. 3.1.2 Chemical Synthesis of Nucleic acids Several strategies have been developed for the synthesis of oligonucleotides. The central problem to be solved in oligonucleotide synthesis is the efficient generation of the phosphodiester. A brief summary in approximate chronological order is given below. Phosphate coupling Coupling using a phosphate was one of the earliest strategies applied. In the scheme below, where X is a good leaving group. 139 BioconjugateChemistry Cairo R Base R Base O O Nomenclature of O O phosphorous derivatives 1 OH + O Base O P O X O- O Base O P O O O OH R O R Base R O O O + O P X Base OH HO O O R An example would be the use of TsCl to form the tosylated phosphate ester, which is then reactive with the 3’-OH of the other residue. Coupling reagents, such as carbodiimides may also be used to accelerate this type of coupling. Khorana received the Nobel prize for work on this problem in 1968 (Chemistry). Phosphite coupling Phosphites have been used to generate phosphodiester bonds. Letsinger, at Northwestern, identified that phosphites under acidic conditions react very rapidly with nucleophiles, and this principle was developed to generate phosphites that could be oxidized to the phosphate diester linkage. Phosphoramidites 140 BioconjugateChemistry Cairo The phosphite strategy has been expanded to phosphoramidites. This is the most commonly applied system for oligonucleotide synthesis. The phosphonium species are much more reactive with nucleophiles than the corresponding phosphite or phosphoramidite, which is desirable for coupling reactions. One rationale for this is that the phosphite has fewer O lp, which would donate electron density to the empty orbitals on P. However, the phosphonium which results from protonation of a phosphite is much more reactive with water, making this pathway difficult to implement. The phosphoramidites are less susceptible to reaction with water, and retain the electrophilic character needed for coupling. The identity of the alkyl groups (R) can be selected to allow easy conversion of the resulting pentavalent phosphorous species to the desired phosphate linkage. Typical modern strategy for oligonucleotide synthesis General reviews of the synthesis of oligonucleotides are available. Abriefsummaryofthe key features used in modern oligonucleotide synthesis is given below. Solid support Analogous to the iterative strategy used in peptide synthesis (discussed in Sec 2), solid support has advantages for the generation of oligonucleotides. Although polystyrene supports have been used for oligonucleotide synthesis, it is more typical to use controlled pore glass (CPG). 3 141 BioconjugateChemistry Cairo H2N OH OH OH Si Si Si O O OH Si O O O OH Si NH2 Si Si Si O O O O R Base O O O O O2N Base R O O O O Base O OH OH O R O O cleavage synthesis O O O HN conc. N3 HN Si OH OH O O OH Si O O OH Si Si Si O O Si Si Si O O 5’-OH Protection The 5’-OH protecting group is an important feature of the strategy, as it will need to be removed at every coupling step. Therefore, it should have relatively mild conditions for removal. The Trityl group (Trt) is the most commonly employed protecting group for this purpose. 142 BioconjugateChemistry Cairo Coupling The general coupling scheme relies on use of a phosphoramidite: The specific implementation is shown below, where the 3’-OH is R1, and 5’-OH is R2. 1H Tetrazole is used as an additive which generates a reactive P-tetrazolyl intermediate. The tetrazole has a remarkably low pKa (4.9). 143 BioconjugateChemistry Cairo Note that the resulting phosphite product after each coupling step is very reactive. To avoid cleavage, the nucleotide chain must be capped and oxidized shortly after coupling. Capping Capping is used to prevent any unreacted 5’-OH groups from continuing to be used later in the synthesis. Having a capping step should reduce the number of side products formed from deletion of an individual residue. Typical capping conditions are acetic anhydride and N,N- dimethyl aminopyridine. Oxidation Oxidation of the phosphite to a phosphate is performed with iodine and water in the presence of a strong base, lutidine. 144 BioconjugateChemistry Cairo Cleavage and deprotection The final cleavage step is performed using concentrated ammonia, to cleave the ester linkage to the CPG, and simultaneously cleave the cyanoethylester. The elimination reaction can be ix referred to as A N +E , oN an E cb 1E conj1gate base) mechanism. Summary of iterative synthesis A summary of the scheme for modern synthesis of a DNA sequence is shown below. This scheme uses the convention that individual nucleotide residues are represented as a vertical line, with the 5'-OH shown on an upward diagonal line, and the 3'-OH on a downward diagonal line. ixE1cb mechanism is discussed on pg 1488-1489, March 6 Ed. 145 BioconjugateChemistry Cairo 3' 1. Solid support Base HO OH O CPG O HO DMT O 5' OH TCA 2. 5' deprotection O CPG 5. cleavage HO 1) TCA 2) NH (aq) N 3 N O N 3. coupling O O H P N DMT O O P O R O HO OH n O CPG O P O DMT O R O 1) Ac O, DMAP 2 4. Capping and 2) 2 ,2 O, Oxidation lutidine O O CPG O P O DMT O R O 3.2 Modification & reactivity of nucleic acids 3.2.1 Synthesis of modified oligonucleotides Oligonucleotides have been dissected with a variety of strategies which allow for modification of the nucleotide base, the ribose backbone, and the phosphodiester linkage. Excellent reviews are available with additional examples. Modified bases Many modified bases have been generated, too many in fact to summarize in much detail here., 5A few examples to illustrate the versatility available to nucleotide chemists are given below. 146 BioconjugateChemistry Cairo 5’-OH Modificiation The most common strategy is to introduce modifications at the 5' end, this can most easily be done using a modified phorphoramidate. Alternatively, a thiol nucleophile can be installed in place of the5'OH,allowingselective coupling at this position. Oligonucleotides with a 5’-SH or 5’-NH have b2en synthesized and 8, 9 can be easily used for conjugation chemistry. A number of useful reagents are available for introduction of other reactive handles. 147 BioconjugateChemistry Cairo 3’-OH Modification To modify the 3' end of an oligonucleotide, the linkage to solid support can be exploited. Commercially available reagents are available that allow simple incorporation of a thiol at the 3' end. DMT O Base O S H S O O N CPG S
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