Peptide Arrays

Peptide arrays were invented by Dr. Ronald Frank [1] who was the first to parallelize the Nobel prize awarded solid phase peptide synthesis [2; Bruce Merrifield, Nobel prize for chemistry in 1984]. In doing so, he first dissolved the 20 different amino acid building blocks for peptide synthesis in a solvent, and then spotted these 20 different solutions to defined spots on an amino-terminated solid support. If repeated for several layers, this procedure builds up many different peptides in a combinatorial synthesis, each on a different spot (Fig. 1). One drawback of this “SPOT synthesis” method is the low density of only ~25 peptides per cm². This is due to the difficulties to deposit very small droplets on a solid support that tend to mix with neighbouring droplets or evaporate before the coupling reaction is finished.

Solid-material-based-method: In order to circumvent these technical difficulties, we developed a prize awarded [3, 4] “solid-material-based-method”, that first embeds the amino acid building blocks within solid materials. The different amino acids are then consecutively addressed to very small areas on a two-dimensional support as solid “amino-acid-particles”, either by a “peptide-laser-printer“, [5] (more) or by the electrical fields of a computer chip’s pixel electrodes [6], or with the help of small cavities that randomly accommodate exactly one “amino-acid-particle”. [7] (more) In yet another variation of this theme, a nano3D printer uses single laser pulses to transfer tiny ”amino-acid-spots” from a donor to an acceptor. Simply by consecutively exchanging the first donor slide with additional donor slides the acceptor is structured with all the 20 different amino acid building blocks – each embedded within a solid material. [8] (more)

In all of these cases the coupling reaction is then induced simply by melting the solid-material-embedded layer of amino acid building blocks, which frees hitherto immobilized amino acid derivatives to diffuse to and react with amino groups on the surface of the support. [10] Technical advantages are (i) very small spot sizes due to structuring with solid materials, (ii) stability of reactive chemicals due to shielding them within a solid material, and (iii) the “ready-to-go-status” of polymer-embedded chemical reactants – once melted, they immediately start to diffuse towards their reaction partner. [9]

Fig. 1; Combinatorial synthesis. A limited number of monomeric amino acid building blocks yields a plethora of different peptides.

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

[2] Felgenhauer T, Schirwitz C und Breitling F. (2010) Peptide synthesis. Ullmann's Encyclopedia of Industrial Chemistry (Last updated: 29 Apr 2010) published online DOI: 10.1002/14356007.a19_157.pub2

[3] Stadler V, Bischoff FR, Felgenhauer T, Kring M und Breitling F. Winner of the business plan competition Science4Life (awarded with 30.000 €) with the project Fertigung und Vertrieb von Biochips zur Parallel-Synthese unterschiedlicher Peptide (Juni 2009).

[4] Breitling F, Bischoff FR, Stadler V, Felgenhauer T, Leibe K, Fernandez S (all from DKFZ or KIT) and Güttler S, Gröning M, Willems P, Biesinger B (Fraunhofer IPA). Winner of the „Wissenschaftspreis des Stifterverbandes“ (awarded with 50.000 € ) with the project Peptide laser printer (May 2008).

[5] Stadler V, Felgenhauer T, Beyer M, Fernandez S, Leibe K, Güttler S, Gröning M, Torralba G, Lindenstruth V, Nesterov A, Block I, Pipkorn R, Poustka A, Bischoff FR und Breitling F. (2008) Combinatorial synthesis of peptide arrays with a laser printer Angew. Chem. Int. Ed. 47, 7132–7135; DOI: 10.1002/anie.200801616.

[6] Beyer M, Nesterov A, Block I, König K, Felgenhauer T, Fernandez S, Leibe K, Torralba G, Hausmann M, Trunk U, Lindenstruth V, Bischoff FR, Stadler V und Breitling F. (2007) Combinatorial synthesis of peptide arrays onto a computer chip’s surface. Science 318, 1888; DOI: 10.1126/science.1149751.

[7] Popov R, Shankara GK, von Bojničić-Kninski C, Barua P, Mattes D, Breitling F, Nesterov-Müller A. (2019) Stochastic deposition of amino acids into microcavities via microparticles. Scientific Reports, 9: 16468; DOI: 10.1038/s41598-019-52994-w

[8] Loeffler FF, Foertsch TC, Popov R, Mattes DS, Schlageter M, Sedlmayr M, Ridder B, Dang FX, von Bojničić-Kninski C, Weber LK, Fischer A,Greifenstein J, Bykovskaya V, Buliev I, Bischoff FR, Hahn L, Meier MAR, Bräse S, Powell AK, Balaban TS, Breitling F, Nesterov-Mueller A. High-flexibility combinatorial peptide synthesis with laser-based transfer of monomers in matrix material. Nature Communications, 2016, DOI: 10.1038/NCOMMS11844

[9] Mattes DS, Jung N, Weber L, Bräse S, Breitling F. (2019) Miniaturized and automated synthesis of biomolecules – Overview and perspectives. Advanced Materials, first online published 29 April 2019; 31: 1806656; DOI: 10.1002/adma.201806656

[10] Stadler V, Kirmse R, Beyer M, Breitling F, Ludwig T, and Bischoff FR. (2008) PEGMA/MMA Copolymer Graftings: Generation, Protein Resistance, and a Hydrophobic Domain. Langmuir 24, 8151-8157