Gene synthesis machines - DNA chemistry and its uses V. Amaranth and A. D. Broom, Chem. Rev. 77, 183 (1977) G. J. Powers et al., J. Am. Chem. Soc. 97, 875 (1975) M. D. Matteucci and M. H. Caruthers, Tetrahedron Lett. 21, 719 (1980) J. Am. Chem. Soc. 103, 3185 (1981) S. L. Beaucage and M. H. Caruthers, Tetrahedron Lett. 22, 1859 (1981) Over the past 30 years, three methods for synthesizing DNA have been successfully developed; these are the: (12, 13, 14) 1) phosphate diester 2) phosphate triester (16) 3) phosphite triester (15) 12. S. A. Narang, Tetrahedron 39, 3 (1983) 13. J. E. Davies and H. G. Gassen, Angew. Chem. Int. Ed. Engl. 22, 13 (1983). 14. K. Itakura, J. J. Rossi, R. B. Wallace, Annu. Rev. Biochem. 53, 323 (1984). 15. R. L. Letsinger and W. B. Lunsford, J. Am. Chem. Soc. 98, 3655 (1976). 16. R. L. Letsinger and V. Mahadevan, ibid. 88, 5319 (1966) lab of R. L. Letsinger R. L. Letsinger ------------- Novel Methods for Synthesis of High Quality Oligonucleotides Solid-phase oligonucleotide synthesis The development of oligonucleotide synthesis methods started from the syn- thesis of thymidilyl-(3´ 5´)-thymidine [12] after the structure of DNA was revealed by Watson and Crick [13]. Initially, three different approaches has been used to create phosphate linkages: phosphodiester [14, 15], H- phosphonate [16-18] and phosphotriester [19-21]. The former two ap- proaches did not require internucleotide linkage protection (Scheme1) and were thus favored over the phosphotriester method (Scheme 2). It became obvious that rapid and nearly quantitative coupling reactions are needed to build long oligonucleotides with satisfactory overall yields. As a logical result phosphite triester approach [22] emerged later. More reactive 3´-O-phosphorochloridites reacted rapidly with the corresponding 3´- protected nucleoside yielding elongated oligonucleotides after subsequent oxidation of the resulting phosphite (Scheme 3). Unfortunately, due to their high reactivity, phosphorochloridites could neither be isolated nor stored, so they were obtained in situ from 5´-O-protected nucleosides by phosphityla- tion with protected phosphorodichloridate. As a result symmetrical by- products with 3´ 3´ and 5´ 5´ internucleotide linkages were also formed. In order to meet the requirements for automation, the phosphoramidite method of nucleotide coupling was developed [23] as an elegant modifica- tion of the phosphite triester approach (Scheme 3). This new class of inter- mediates gave rise to the traditional solid-phase oligosynthesis method (Fig.1), as phosphoramidites are suitable for both storage and handling under normal conditions and react rapidly with the corresponding nucleophile (e.g. the 5´ hydroxyl group of the oligonucleotide linked to the solid-phase sup- port) upon activation with 1H-tetrazole [24]. Usually, phosphoramidites are obtained in the reaction of a protected nucleoside with either chloro- phosphine [25] or diamidophosphite [26] and can be isolated and stored as solids at –20 C. [12] synthesis of thymidilyl-(3´ 5´)-thymidine [14,15] phosphodiester [16-18] H-phosphonate [19-21] phosphotriester [22] phosphite triester approach [23] phosphoramidite method of nucleotide coupling (as a modification of the phosphite triester approach) [25] chlorophosphine - phosphoramidites are obtained in the reaction of a protected nucleoside with chlorophosphine [26] diamidophosphite - phosphoramidites are obtained in the reaction of a protected nucleoside with diamidophosphite The growing oligonucleotide chain has to be isolated from the synthesis reagents after each reaction. The concept of using an organic polymer as a support for oligonucleotide synthesis was adapted from Merrifield’s ap- proach of peptide synthesis [27], which gave the basic principle employed in the modern solid-phase oligosynthesis. Currently CPG [28, 29] and highly cross-linked polystyrene [30], containing the 3´-terminal nucleoside attached via an ester linkage, are used as solid supports for oligosynthesis. [27] Merrifield's approach to peptide synthesis - organic polymer as a support for oligonucleotide synthesis [28-29] CPG as a solid support for oligosynthesis [30] highly cross-linked polystyrene containing the 3´-terminal nucleoside attached via an ester linkage, as a solid support for oligosynthesis [31-37] studies on optimal phosphoramidite building blocks A successful synthesis of oligonucleotides, besides amidite reactivity, de- pends on the integrity of other groups of the molecule. Protecting groups have to be introduced at several places of the nucleoside phosphoramidite molecule: 5´ hydroxyl; exocyclic amino groups of cytidine, adenosine and guanosine; phosphoramidite moiety; and 2´ hydroxyl of ribonucleosides (Fig.2). Some studies reported the necessity of guanosine lactam group pro- tection [38-40], but replacing DMAP by NMI in the capping mixture [38], and introduction of a capping step before oxidation in the oligosynthesis procedure allowed this group to be left unprotected [41]. sections: - 5-prime hydroxyl protection - phosphate protection - 2-prime hydroxyl protection - acyl and sulfonyl protecting groups - acid-labile protecting groups - protecting groups cleavable by oxidation, reduction, or photolytic hydrolysis - fluoride-labile protecting groups - A strategy of using protected protecting groups and its perspectives - purification of oligonucleotides - Functionalized solid support as a purification aid - Oligonucleotide purification using RPC [24] .. phosphoramidites are suitable for both storage and handling under normal conditions and react rapidly with the corresponding nucleophile (e.g. the 5´ hydroxyl group of the oligonucleotide linked to the solid-phase support) upon activation with 1H-tetrazole -------------------------------------------- -------------------------------------------- -------------------------------------------- -------------------------------------------- -------------------------------------------- A short history of oligonucleotide synthesis (2006) However, general accessibility to the chemistry would require many advancements before reliable, automated oligonucleotide synthesis was a reality. The beginning of the era of investigating the human genome can be dated to the collaboration between Professor Marvin Caruthers of the University of Colorado, and Professor Leroy Hood of CalTech, when they set out to automate Caruthers' new phosphoramidite oligonucleotide synthesis chemistry. This collaboration, which formed Applied Biosystems, Incorporated (ABI), commercialized the first phosphoramidite DNA synthesizer in the early 1980's. Many labs now had routine access to oligonucleotides, which was critical in advancing the overall understanding of biological systems. The first published account of the directed chemical synthesis of an oligonucleotide occurred in 1955 when Michelson and Todd reported the preparation of a dithymidinyl nucleotide (Michelson and Todd, 1955). In their report, the phosphate link between two thymidine nucleosides was made by first preparing the 3' phosphoryl chloride of a 5' benzoyl protected thymidine, using phenylphosphoryl dichloride. This was then reacted with the 5' hydroxyl of a 3' protected thymidine. The chemistry worked reasonably well, albeit slowly. Additionally, the phosphoryl chloride intermediate was not stable, being susceptible to hydrolysis. Figure 1 shows the basic scheme. Khorana contribution In the late 1950's a creative and forward-thinking researcher at the University of Chicago by the name of H. Gobind Khorana became interested in the synthesis of oligonucleotides. He introduced two concepts to the field that made possible the convenient synthesis of oligonucleotides more than just a few bases long. One concept, the on-off protection scheme necessary for sequential oligonucleotide synthesis, is still widely used today by oligonucleotide chemists, virtually unmodified from Khorana's initial publications (Schaller, e.t al., 1963; Smith, et. al., 1961). The other was the first use of a stable phosphorylated nucleoside that coupled to the desired nucleoside when activated (Khorana, et. al., 1956). This protocol, called the phosphodiester method of oligonucleotide synthesis (Figure 2), is the same cyclic scheme used today with the exception of the addition of one step, oxidation. In place of the hydrolysable phosphoryl chloride, he prepared 3' phosphates of the 5' protected nucleoside using phosphorochloridates that then hydrolyzed to the phosphomonoester. These 5' protected nucleoside 3' phosphates were subsequently activated using a condensation reagent, such as dicyclohexyl carbodiimide (DCC), to couple to the 5' hydroxyl of another 3'-protected