Biopolymers, automated synthesis

Automated synthesis of peptide and oligonucleotide libraries was initiated about 10 years ago [4], Within the last three years, there has been much attention focused on the generation of combinatorial libraries of small molecules. As with biopolymers, the use of solid resin support was central to the advance of this field. In solid-phase synthesis, one of the reactants is covalently bound to the solid support and an excess of the other reactants may be used in each step to drive reactions to completion. Purification of the intermediates and final product is easily achieved through extensive washing of the resin after each chemical step. For the purpose of high throughput synthesis, cleavage of the final... 

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. 

Although automated solid-phase instruments are increasingly being used to construct biopolymers, manual synthesis is still used extensively with methods that are used to create molecular diversity (see Chapter 15). The libraries of molecules produced by these methods are often used for screening against biological targets. 

Automated synthesis can ran unattended and is less prone to human error than manual synthesis. As the biopolymer assembly process was automated, there was a tendency to produce longer sequences. This, in turn, required better synthetic methods, which has led to continual improvements in solid-phase chemistries and instrument design. 

Of the three above-mentioned major classes of natural biopolymers, chemical synthesis of nucleic adds (in particular DNA) and proteins had already been achieved by the Merrifield solid-phase synthesis method a computer-controlled automated synthesizer became commerdally available more than 20 years ago. However, the chemical synthesis of polysaccharides like cellulose was far more difficult than that of the former two classes. Polysaccharide synthesis is the repetition of glycosylation, the most fimdamental reaction in carbohydrate chemistry (Scheme 2). 

In addition to being necessary for all forms of life, biopolymers, especially enzymes (proteins), have found commercial applications in various analytical techniques (see Automated instrumentation, clinical chemistry Automated instrumentation, hemtatology Biopolymers, analytical techniques Biosensors Immunoassay) in synthetic processes (see Enzyme applications, industrial Enzyme applications in organic synthesis) and in prescribed therapies (see Enzyme applications, THERAPEUTICS IMMUNOTHERAPEUTIC AGENTS Vitamins). Other naturally occurring biopolymers having significant commercial importance are the cellulose (qv) derivatives, eg, cotton (qv) and wood (qv), which are complex polysaccharides. 

The era of biotechnology was initiated by two major breakthroughs that paved the way for further developments in biochemical research. First, the sequencing of nucleic acids and proteins has been automated and allows for the composition of an unknown sample to be determined quickly and reliably [3]. Secondly, the synthesis of defined oligonucleotides [4] and peptides [5] has also been automated and even allows nonspecialists in this field to obtain rapidly larger-scale quantities of these important classes of biopolymers. The rational design of specific modifications has come within reach and is an important research tool in biomedicine, biotechnology, and pharmaceutics. 

Biopolymers such as proteins have a hierarchical structure (this can be compared to semicrystalline polymers. Section 2.7.2). The primary structure is simply the sequence of amino-acid residues in the polymer chain. This is created during the synthesis of each molecule, and is memorized , in other words it is not destroyed by thermal motion of the chain. Methods for determining primary structure have now been discovered, and even automated. The secondary structure of biopolymers results from hydrogen bonding between -CO and -HN groups. This leads to the so-called a and (3 structures of proteins. An a-helix and p-sheet are sketched in Fig. 6.13. The tertiary structure refers to the overall arrangement of the macromolecule, an example is shown in Fig. 6.14. 

Pedersen SL, Sorensen KK, Jensen KJ (2010) Semi-automated microwave-assisted SPPS optimization of protocols and synthesis of difficult sequences. Biopolymers 94(2) 206-212... 

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