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In chemistry, an azide is the functional group N3, that is particularly useful for dipolar cycloaddition reactions, such as click chemistry reactions. Organic azides are readily formed by reacting sodium azide with halogenated alkanes. They can also be used as antibacterial agents.

Resonance Structures

The azide group is a linear molecule, and the most stable electron resonance structure is 1, depicted below, which contains four nitrogen-nitrogen bonds and low charge density. Two other resonance forms, 2 and 3, also contain four nitrogen-nitrogen bonds, but these structures are less stable due to the high charge density, 2^-, on one of the nitrogen atoms. Two additional resonance forms, 4 and 5, are also less stable because they contain only three nitrogen-nitrogen bonds.

Click Chemistry

Azides are frequently used to form cycloaddition products with alkynes under click chemistry reactions. Such chemistry is often used to synthesize triazole and tetrazole-based drugs.

DNA synthesis (chemically)

DNA is often chemically synthesized in laboratories, or by commercial entities, for many reasons. For example, DNA sequencing often relies on short DNA primers which must be synthesized. DNA-based aptamers and thioaptamers, which are nucleic acid-based protein-binding affinity reagents that can be used in place of an antibody in assays, are a more recent use. Another use is the creation of small DNA strands, coding for a particular protein, that can be ligated into a much larger bacterial plasmid for protein over-expression. Directed mutagenesis of over-expressed proteins also relies on synthesized DNA. The DNA is synthesized on an automated machine by passing a variety of solvents and reagents through a solid support under anhydrous conditions.

Conventional DNA synthesis

During chemical DNA synthesis, as opposed to biological DNA synthesis, using phosphoramidite reagents, four steps are typically used for each DNA base addition: 1) deprotection of the growing DNA strand, 2) coupling of the new base, 3) capping (termination) of failed strands, and 4) oxidation. Finally, once the entire DNA strand has been synthesized, the DNA is subjected to ammoniolysis (concentrated ammonium hydroxide) to cleave away the support and deprotect the DNA bases. Each of these steps is discussed in more detail below.

DNA synthesis begins with a single, protected DNA base attached (on the 3'-side) to a solid support, typically ~ 70 micron beads made of polystyrene/divinylbenzene, although a variety of support materials and sizes are now available, including non-cleavable beads and heat-labile beads. Because the nucleophilic amine groups of natural DNA bases can undergo coupling chemistry, the base attached to the bead, and the phosphoramidites later added to the bead, have protecting groups on the bases to ensure only phopho-based coupling reactions occur. These protected base forms have tradionally been N2-isobutyryl-dG, N4-benzoyl-dC, and N6-benzoyl-dA, which require about 16-24 hours of deprotection in concentrated ammonium hydroxide at 55C. More recent protected forms such as phenoxyacetyl-dA (PAC-dA), dimethylformadinyl-dG (DMF-dG), and acyl-dC can be deprotected quickly in milder conditions(Reddy, M. et al., 1994). Most phosphoramidites and the initial base are also protected at the 5'-oxygen atom by a dimethoxytrityl group (DMT), although some specialty chemical linkers and dyes are sometimes protected with a monomethoxytrityl group (MMT). Once the DNA beads are loaded onto a DNA synthesizer, typically in a column packed between two frits, the first step, DMT removal, begins.

Deprotection of the 5'-DMT group The 5'-dimethoxytrityl group on the bead support is acid labile and it is removed quickly (~ 30 seconds) by addition of dichloroacetic acid in methylene chloride. Subsequent washing of the beads with acetonitrile minimizes loss of DNA base protecting groups on the bead and on subsequent phosphoramidites. The resulting dimethoxytrityl cation flushed away is bright orange and provides a spectrometric estimate of coupling efficiency at the end of each base addition. However, due to differing kinetics (ie reaction rates) of detritylation, the dG bases always provide a smaller signal than do the other DNA bases.

Elongation of the DNA is then promoted by the addition of an "activator", typically tetrazole, a weak acid, and a phosphoramidite. The weak acid activates the phosphorus atom of the phosphoramidite, thereby increasing the rate of successful nucleophilic addition of the unprotected 5'-oxygen atom of the DNA strand to the P atom. An excess of phosphoramidite (compared to the bead support) ensures near complete coupling (99+%). Although this reaction takes about 30 seconds, longer coupling times, or double coupling steps, can be used to ensure more complete coupling. Although 99% success at each coupling stage seems excellent, when longer DNA strands are synthesized, ie 200 bases, (0.99)^200 is a catastrophic yield. After the coupling period, the excess reagents are flushed away with acetonitrile.