Membrane spot synthesis

Frank, R. (1992). Spot synthesis an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48, 9217-9232. 

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]. 

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). 

Frank, R. (2002) The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports—principles and applications. J. Immunol. Methods 267, 13-26. 

Detection of weak binding by electrochemical blotting to PVDF membranes (13) Spot synthesis and screening on living cells (14)... 

If not noted elsewhere, all data correspond to the preparation of a 10 x 12 cm cellulose membrane (96-well plate size). For larger membranes preparation of related amounts of reagents is required. If we write about the use of amino acids, it should always include the use of other organic building blocks (e.g., PNA monomers, peptoidic elements, heterocycles) (22,23), which can be used under spot synthesis conditions. Here we describe only the basic procedures for spot synthesis of linear peptides. For the synthesis of modified peptides, such as cyclization or side-chain modifications, see ref. (24). 

Frank, R. and Schneider-Mergener, J. (2002) SPOT synthesis—scope of applications. In Peptide Arrays on Membrane Support, eds. J. Koch and M. Mahler, pp 1-22. Berlin Heidelberg Springer-Verlag. 

Martens, W., Greiser-Wilke, I., Harder, T.C., et al. (1995) Spot synthesis of overlapping peptides on paper membrane supports enables the identification of linear monoclonal antibody binding determinants on morbillivirus phosphoproteins. Vet. Microbiol. 44, 289-298. 

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. 

Volkmer-Engert, R., Hoffmann, B., and Schneider-Mergener, J. (1997) Stable attachment of the HMB-linker to continuous cellulose membranes for parallel solid phase spot synthesis. Tetrahedron Lett. 38, 1029-1032. 

Besides classical resin beads, other polymeric carriers were also used for the synthesis ofpeptide libraries in various formats. Poly aery late-grafted polypropylene pins were used for the synthesis of the first combinatorial chemical library [1,2], This type of support continues to be heavily used in multiple peptide [27] and non-peptide [28] library synthesis. Cellulose paper, originally used by Frank et al. as a solid-phase support for oligodeoxy-ribonucleotide synthesis [29], has also been used as the support for multiple SPOT synthesis of peptide libraries [30,31], Polystyrene-grafted polyethylene film (PS-PE) may also be used in combinatorial peptide library synthesis [32], The specific feature of the membrane type of carrier is its dividability. This feature has been used for the synthesis of libraries with a nonstatistical distribution of library members, where no compound is missing and none is represented more than once [33],... 

SPOT synthesis Structure determination not needed convenient on-paper assay moderately expensive spot synthesis equipment and custom-spot peptide membrane are commercially available. Peptide library is relatively small limited to binding and simple functional assay if bound peptides are used for the assay releasable peptides possible, but each in small quantity and peptide spot membrane generally not recyclable for subsequent use. 

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... 

Scheme 4 Cleavable Linkers for Spot Synthesis and Membrane Sheetsl l...

The spot synthesis technique is simple and easily accessible for practical use. There are some limitations to the scope of chemistries that can be used with cellulose as a solid support. To eliminate some of these shortcomings, Gao and EsnoufP° 4 proposed using a different membrane, Immobilon AV-1, for spot synthesis. Before the synthesis of peptide arrays can be carried out, this polyvinylidene fluoride based material is derivatized with ethane-1,2-diamine, followed by coupling of Fmoc-(3Ala as a spacer. In most other aspects, this peptide array synthesis is very similar to the spot synthesis proposed by Frank.t l... 

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. 

---