Oligonucleotide on solid support

Fig. 2 Light-directed parallel chemical synthesis of peptides and oligonucleotides on solid supports of Affymetrix s DNA microarray [5, 6]. A photo-protected glass substrate is selectively exposed to UV light through a mask that contains the desired pattern (1). The deprotected areas are activated in step (2) followed by nucleoside incubation where chemical coupling occurs at activated positions (5). In step (4), a new mask pattern is applied. The coupling step is repeated in step (5). The deprotection and coupling process is repeated until the desired set of probes is obtained as shown in step (6)...

Synthesis (i) a hydrophobic polymer, terminally modified with a carboxylic acid moiety, was mixed with a coupling agent and reacted with a 50-amino-modified oligonucleotide on solid support (controlled pore glass, CPG) (ii) deprotection and cleavage of the resulting DNA polymer conjugate from solid support (iii) dialysis of cleaved DPA into deionized water to form a mixture... 

As the density of information derived from efforts to sequence, map and identify human genes increased, so did the demand for analytical tools capable of exploiting this information. DNA microarrays were developed in response to this demand. Southern(69) was the first to describe parallel, in situ ohgonucleotide synthesis as a means of generating oligonucleotide probe arrays on solid supports for highly parallel hybridization analysis. Southern s method uses standard nucleotide synthetic reactions to synthesize the oligonucleotides. The reactions are carried out in a movable chamber, which provides a physical barrier between the reaction chamber and the intended synthesis area. 

Schwyzer R, Felder ER, Failli P, The CAMET and CASET links for the synthesis of protected oligopeptides and oligonucleotides on solid and soluble supports, Helv. Chim. Acta., 67 1316-1327, 1984. 

The development of the synthesis of peptides on solid supports has also had a strong impact on other areas of organic synthesis. From the very beginning, some research groups tried, with some success, to export the idea from peptide synthesis to, for instance, the monoalkylation and monoacylation of carboxylic acids,the synthesis of aldehydes and ketones, or to the use of resin-bound dienes or dienophiles to trap reactive intermediates.PI This pioneering work has been fundamental to the much more recent explosion of combinatorial chemistry on solid supports. Synthesis on solid supports has also found application in the synthesis of oligonucleotides, PI oligosaccharides,PI peptide-DNA hybrids,P°l and peptide nucleic acids (PNAs).P l... 

In addition, fluorous tags have been developed for purification of oligonucleotides [18] and oilgosaccahrides [19] synthesized on solid support. 

During the development stages of combinatorial chemistry, it was beheved that efficient synthesis was only possible only by using solid-phase strategies. In part, this was influenced by the rapid and highly efficient synthesis of peptides and oligonucleotides by robots on solid support materials. One should bear in mind. 

Figure 23. Convertible triazolyl pyrimidine nucleoside. Postsynthetic reactions of the oligonucleotide (a) 10% thioacetic acid in acetonitrile on solid support, deprotection with 10% DBU then cone, ammonium hydroxide, 24 h, RT (b) 10% 1,1-dimethylhydrazine in acetonitrile on support, 3 h, RT 0.5 M aqueous sodium hydroxide, 16 h, RT (c) for R, = CH3,10% V/V DBU in anhydrous methanol 24 h, RT Ri = CH2CH3,10% V/V DBU in anhydrous ethanol, 48 h, RT (d) 0.5 M aqueous sodium hydroxide, 24 h, RT. Modified from [179],...

Figure 1 Oligonucleotide synthesis on solid support via the phosphoramidite approach.

Synthesis on solid supports was first developed by Merrifield [1] for the assembly of peptides. It has expanded to include many different applications including oligonucleotide, carbohydrate, and small-molecule assembly (see Chapters 11 and 14). The repetitive cycle of steps involved in the solid-phase synthesis of biopolymers can be performed manually using simple laboratory equipment or fully automated with sophisticated instrumentation. This chapter examines typical solid-phase reaction kinetics to identify factors that can improve the efficiency of both manual and automated synthesis. The hardware and software features of automated solid-phase instruments are also discussed. The focus of this discussion is not on particular commercial model synthesizers but on the basic principles of instrument operation. These considerations can assist in the design, purchase, or use of automated equipment for solid-phase synthesis. Most contrasting features have advantages and disadvantages and the proper choice of instrumentation depends on the synthetic needs of the user. 

Not surprisingly, combinatorial chemistry originated from linear assembly strategies developed in peptide and oligonucleotide chemistry (cf. Chapter 2), since both building blocks and coupling chemistry have been extensively studied in solution as well as on solid support. 

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