Spot synthesis on cellulose

Oligonucleotide and peptide microarrays can be prepared in situ with light-directed synthesis on a glass surface in conjunction with either a photolithographic method or using a micromirror device (2,11-15). However, these methods are not useful for most organic synthesis. Furthermore, such approaches require equipment that is not readily available. Spot synthesis on cellulose membrane is another in situ synthesis method, but the resulting microarrays are low density (16). 

Bowman Matthew D, Jeske Ryan C, Blackwell Helen E (2004) Microwave-accelerated SPOT-synthesis on cellulose supports. Org Lett 6 2019-2022... 

The SPOT-synthesis method also employs Fmoc chemistry but uses hydroxyl groups present on cellulose filter paper to derivatize and thereby immobilize (3-alanine groups onto the paper. After deprotection, the 13-alanine groups can be used as platforms for the synthesis of peptide arrays (Fig. 7.5) (Frank, 1992 Gausepohl et al., 1992). This method has been widely used for mapping antigen-antibody interactions as well as protein-DNA, protein-metal and other protein-protein interactions (Reineke et al., 2001). 

An 8000-member library of trisamino- and aminooxy-l,3,5-triazines has been prepared by use of highly effective, microwave-assisted nucleophilic substitution of polypropylene (PP) or cellulose membrane-bound monochlorotriazines. The key step relied on the microwave-promoted substitution of the chlorine atom in monochlorotriazines (Scheme 12.7) [35]. Whereas the conventional procedure required relatively harsh conditions such as 80 °C for 5 h or very long reaction times (4 days), all substitution reactions were found to proceed within 6 min, with both amines and solutions of cesium salts of phenols, and use of microwave irradiation in a domestic oven under atmospheric reaction conditions. The reactions were conducted by applying a SPOT-synthesis technique [36] on 18 x 26 cm cellulose membranes leading to a spatially addressed parallel assembly of the desired triazines after cleavage with TFA vapor. This concept was later also extended to other halogenated heterocycles, such as 2,4,6-trichloropyrimidine, 4,6-dichloro-5-nitropyrimidine, and 2,6,8-trichloro-7-methylpurine, and applied to the synthesis of macrocyclic peptidomimetics [37]. 

The Spot Technique Synthesis and Screening of Peptide Macroarrays on Cellulose Membranes... 

Hilpert, K., Winkler, D.F.H., and Hancock, R.E.W. (2007) Peptide arrays on cellulose support SPOT synthesis - a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nature Protocols 2, 1333-1349. 

Frank, R. and Overwin, H. (1996) SPOT synthesis. Epitope analysis with arrays of synthetic peptides prepared on cellulose membranes. In Epitope Mapping Protocols, ed. G.E. Morris, pp. 149-169. Totowa, NJ Humana Press. 

A peptide library can be synthesized using the SPOT synthesis technique to form a low-density peptide spot array (e.g., 25 spots/cm ). In this method, different peptides are synthesized in situ as low-density arrays on cellulose membrane or paper (8). The volume of Fmoc-amino acids and coupling reagents dispensed creates a specific SPOT size that determines both the scale of reaction and the absolute number of peptides that can be arranged on an area of a membrane. Cotton (another form of cellulose) and polystyrene-grafted polyethylene film segments also have been used as solid supports. Recently, polymeric membranes that are chemically, mechanically, and thermally more stable have been developed, which include hydroxy-functionalized PEG acrylate polypropylene membranes and an amino-functionalized ester-free PEG... 

Peptide arrays on paper or cellulose membranes generated by SPOT synthesis generally are low density, even with the commercially available automatic SPOT synthesizer. Such a low-density peptide chip (several thousand peptides) now is available commercially, for example, PepChip microarray from Mimotopes. Foder et al. (4) first described the... 

Spot synthesis Solid-phase synthesis at certain points (spots) on u (wo-dimensionul (2D) surface (e.g.. a cellulose membrane). Recently, (he techniques of ink-jet printing have been applied to spot synthesis, yielding very den.se arrays of compounds. 

A different way to produce chemical microarrays in situ is spot synthesis of combinatorial libraries on cellulose sheets [56]. Spot synthesis is configured as an open system to be operated at room temperature. Despite attempts to replace cellulose with polypropylene as a synthesis support [57], cellulose is still the support of choice for spot synthesis, and reaction conditions have to be compatible with the restricted chemical stability of cellulose. Due to the acid labihty of such membranes, the diversity content of these arrays was initially restricted to the synthesis of peptides. Recently, a method was described that could widen the scope of spot synthesis arrays. Germeroth and coworkers [57] succeeded in the assembly of a library of 8000 cellulose-bound 1,3,5-triazines under mild reaction conditions. They employed a strategy that took advantage of a temperature-dependent, successive displacement of cyanuric chlorides by different nucleophiles in a first report of the synthesis of smaU organic compounds on ceUulose sheets. 

Thereafter, Frank developed a novel technical concept for the cellulose paper sheet-supported simultaneous parallel synthesis utilizing conventional Fmoc/tBu chemistry [78]. As he carried out different coupling reactions simultaneously on distinct areas of the same sheet of paper instead of using separate pieces of paper for individual peptides, this approach is named SPOT synthesis. Recent advances in this field have expanded the SPOT technology beyond peptidic systems into... 

A novel concept for the synthesis of macrocyclic peptidomimetics 262, which incorporate heteroaromatic units, was reported (Scheme 39). This method involves sequential SNAr reactions of orthogonally protected amino groups of peptides and other linear oligomers on halogenated heterocycles such as 2,4,6-trichloro[l,3,5]triazines. The scope of this novel solid-phase approach was evaluated systematically by means of the SPOT-synthesis methodology on planar cellulose membranes <2000JC0361>. 

On-chip synthesis provides a further improvement of efficiency, but has practical limitations in terms of flexibility and the quality of the cm the spot synthesized compounds. Moreover, the homogeneity and the quality of these microarrays are difficult to control. Chemical libraries, applying compoimds with similar fimctional groups and reactivity (like in the case of amino acids), were successfully used in making macroarrays on cellulose membranes using the SPOT synthesis concept (Frank, 2002). 

Paper as a solid support fascinated Frank, and he later developed another unique and ingenious technique, the spot synthesis (SPOT). In SPOT synthesis, peptides are synthesized on functionalized membrane sheets as spots that could be as large as 10 mm or as small as 1 mm. The spot synthesis technique became a popular tool for peptide synthesis and was soon automated." The first semiautomated SPOT synthesizer, the ASP222, was launched by ABIMED Analysen-Technik of Germany in 1993." The automated method provided an economical way to synthesize very large numbers of peptides. The Intavis synthesizer Auto-Spot (www.intavis.com) allowed the simultaneous synthesis of 1600 peptides. The chemical and physical problems associated with the use of cellulose sheets as synthesis support were, for the most part, overcome by developing synthetic membranes from polypropylene and Teflon. ... 

In the late 1980s, Frank and coworkers developed a method for parallel solid phase synthesis where the molecules were linked to a membrane rather than to conventional polystyrene beads [26,27]. Libraries were prepared by adding the different reagents at each of the spots on the membrane. This SPOT-synthesis method can be carried out on cellulose and polypropylene sheets (loading lOO-lOOOOnM/cm or lO-lOOnM/cm, respectively) and requires no specialized infrastructure to obtain libraries. The mean spot size... 

Heine N, Germeroth L, Schneider-Mergener J, Wenschuh H (2001) A modular approach to the SPOT-synthesis of 1,3,5-trisubstituted hydantoins on cellulose membranes. Tetrahedron Lett 42 227-230... 

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