SPOT-synthesis

Son ogashira reaction 182,279 SPOT synthesis 109 StUle reaction 159 Substitution reactions, electrophilic reagents 19... 

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). 

The most frequent application of SPOT-synthesis has been in the preparation of peptide arrays for the identification of linear B-cell epitopes. If the protein antigen is known, a set of overlapping peptides that encompass the entire sequence can be readily synthesized and assayed for binding of antibody (Reineke et al., 1999). The individual residues critical for binding can then be determined by SPOT-synthesis of peptides containing amino acid substitutions. 

Figure 7.5. Peptide array construction by SPOT-synthesis. fl-alanine groups (b-A) interact with the cellulose filter that serves as a planar support. Peptide synthesis then proceeds using Fmoc chemistries using the fl-alanine group as a starting point. The peptide is attached to the filter via its carboxy-terminus. In this case, lysine is added at the second position and various amino acids are present at the amino terminus of the peptide.

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

Reineke, U., Kramer, A., and Schneider-Mergener, J. (1999). Antigen sequence- and library-based mapping of linear and discontinuous protein-protein interaction sites by spot synthesis. Curr. Top. Microbiol. Immunol. 243, 23-36. 

In a recent study, this so-called SPOT synthesis was applied for the preparation of pyrimidines [45]. The group of Blackwell described primarily the appropriate support modification of commercially available cellulose sheets (Scheme 7.28). The initial introduction of the amine spacer was achieved within 15 min utilizing micro-wave irradiation, as compared to 6 h by conventional heating. The acid-cleavable Wang-type linker was attached by classical methods at ambient temperature. 

Scheme 7.28 Support modification for SPOT synthesis of pyrimidines.

The successfully generated chalcones could be cleaved by treatment with trifluoro-acetic acid or used for the subsequent synthesis of pyrimidines [45], Condensation of the polymer-bound chalcones with benzamidine hydrochloride under microwave irradiation for 30 min furnished the corresponding pyrimidines in good yields after TFA-induced cleavage. This new robust support/linker system for SPOT synthesis has been demonstrated to be compatible with a range of organic reactions and highly applicable for microwave conditions. 

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]. 

Aromax process, 1 665 25 171, 176 Aromizing-Aroformer process, 25 175 Arosolvan process, 3 606 P-Aroylacrylic acids, 15 486 2-Aroylbenzofurans, microwave-assisted synthesis of, 16 578 Array detector, 23 143 Array methodologies, 12 513-517 Arrays, large, 16 390 Array spotting/synthesis, in microarray fabrication, 16 385-387 Arrhenius equation, 10 85 14 622 ... 

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. 

Key Words Spot synthesis peptide array screening detection probing HRP POD biotin-labeling peptide-protein interaction. 

Protocols for spot synthesis using active esters (2)... 

Automated spot synthesis using HOBt/DIC activation (3)... 

Overview over spot synthesis of small organic molecules (7)... 

Detailed review regarding synthesis and application of spot synthesis (12)... 

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

Spot synthesis may be carried out manually, semi- or fully automated (see Fig. 1). Manual spot synthesis is convenient for the synthesis and screening of a relative small number of peptides (up to 100) and a large pipetting volume (>0.5 aL). For all other cases a semiautomated or fully automated synthesis is recommended. For synthesis of small spots with a pipetting volume of about 0.1 ptL, spot synthesizers from Intavis AG (Koln, Germany) are very useful (3). 

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). 

Fig. 1. Outline of spot synthesis (shown without washing steps).

Frank, R., Hoffmann, S., Overwin, H., Behn, C., and Gausepohl, H. (1996) Easy preparation of synthetic peptide repertoires for immunological studies utilizing SPOT synthesis. In Peptides in Immunology, ed. C.H. Schneider, pp. 197-204. New York John Wiley Sons, Ltd. 

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. 

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. 

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