Chemical Synthesis of Oligonucleotides - Integrated DNA ...

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Here, we give a brief history of chemical synthesis, present the fundamentals of chemicalsynthesis of oligonucleotides as it is practiced today, and present some of the more basic issuesarising from syntheses including those affecting scale and yield.2. A History of Chemical SynthesisThe elucidation of the Genetic Code was accomplished by 1966 and is an example of bruteforce applied to a delicate problem. One of the main figures was Professor H. Gobind Khorana,a biochemist at the University of Wisconsin. It was understood then that, in nature, theformation of the phosphate linkages in DNA was catalyzed enzymatically in a reversiblereaction:dNTP + (dNMP) n ↔ (dNMP) n+1 + pp iThat is, a deoxynucleotide triphosphate was added to a growing deoxynucleotidemonophosphate polymer by a polymerase in the presence of Mg ++ and the forward reactionresulted in the n+1 polymer and a pyrophosphate. Based on this understanding, Khorana, andother investigators, began to try ways to accomplish the reaction synthetically. Many of theseinvestigators were already well versed in the chemistry involved in the synthesis ofpolypeptides. This very successful chemistry, shown graphically in Figure 1, utilized afoundation of a single amino acid bound to a solid support (in this case a resin bead), aprotected amino acid, and a condensing agent, dicyclohexylcarbodiimide (DCC) which catalyzedCH 3 H RCH 3 -C-O-C-N-C-COOHCH 3 O HRH 2 N-C-C-H ODCCResin BeadCondensing AgentBlocking GroupCH 3HRHCH 3 -C-O-C-N-C-C- N-C- + H 2 OC-CH 3 O H O R OGrowing PolypeptideFigure 1. The basic scheme of polypeptide synthesis from a solid support(resin bead). The final deprotection step removed the blocking group from thecoupled amino acid and permitted the next coupling reaction to proceed in thepresence of DCC.©2005, 2010, and 2011 Integrated DNA Technologies. All rights reserved. 2
the peptide linkage. The reaction would link the protected amino acid to the solid support inonly one way and the resulting di-peptide was then deprotected for the addition of the nextamino acid. In this way, the synthetic polypeptide grew at the direction of the synthesizingchemist one link at a time through a repeated cycle of steps.Khorana and his group at the Institute for Enzyme Research at the University of Wisconsinbegan experimenting with DCC on various solid supports and protecting groups on bothribonucleotides and deoxyribonucleotides. The scheme they developed involved threedifferent protecting groups surrounding the nucleotide (Figure 2). The integrity of the ringstructure of the base was protected by a benzoyl or an isobutyryl group, the oxygen on the 3’carbon of the sugar was prevented from forming a reactive hydroxyl until needed by theaddition of an acetyl group, and the 5’ carbon was blocked by a trityl group. In the synthesis ofthe trityl group Khorana chose to use a soluble polymeric support. In the case of polypeptidesynthesis the support was a resin bead but Khorana settled on a polystyrene bead for nucleicacid synthesis. Synthesis of the trityl began with the attachment of a benzene ring to thepolystyrene bead. Next, asecond ring was attached viaa Friedel-Crafts reactionwhich is the acetylation (oralkylation) of aromaticcompounds by aluminumchloride. This structure wasthen exposed to a Grignardreagent, an extremelyreactive class of compoundsused in the synthesis ofhydrocarbons. Finally, thenearly completed trityl groupwas treated with acetylchloride resulting in apolystyrenemethoxytritylchloride speciesthat could be easily attachedto the 5’ carbon of thepentose sugar (Figure 2).PolystyreneSolid SupportTritylGroupCoAcetyl GroupoAoB PBenzoyl orIsobutyryl GroupFigure 2. Starting point structure of an oligonucleotide synthesisby the Khorana group. Subsequent couplings would proceedthrough the 3’ carbon after removal of the acetyl group. Allnucleotides added from this point would have a reactive hydroxylin place of the trityl group but would retain the other twoprotecting groups.By placing the solid support on the trityl group and using the DCC condensing reagent,the synthesis of an oligodeoxynucleotide proceeded in the same 5’→3’ direction as in nature.The nucleotide to which the trityl was attached became the anchor of the synthesis. The oxygenon the 3’ carbon of the sugar was deprotected leaving the reactive hydroxyl group ready forcoupling. The protected nucleotide, without a trityl, was added via the condensing reaction ofDCC. This method was tedious and only modestly efficient but it led to two majorbreakthroughs. First, Khorana and his group were able to synthesize oligoribonucleotides thatwere used to confirm the Genetic Code. Second, in 1967, the Khorana-led team at Wisconsinannounced their intention to use this chemistry to synthesize a gene. Their attempt to do this©2005, 2010, and 2011 Integrated DNA Technologies. All rights reserved. 3
Page 1: Chemical Synthesis and Purification
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