Multipin peptide synthesis

Valerio, R. M. Bray, A. M. Campbell, R. A. DiPasquale, A. Margellis, C. Rodda, S. J. Geysen, H. M. Maeji, N. J. Multipin Peptide Synthesis at the Micromole Scale using 2-Hydroxyethyl Methacrylate Grafted Polyethylene Supports, hit. J. Pept. Protein Res. 1993, 42, 1. 

NJMaeji, AM Bray, RM Valerio, W Wang. Larger scale multipin peptide synthesis. Peptide Res 8 33-38, 1995. 

RM Valerio, AM Bray, RA Campbell, A Dipasquale, C Margellis, S. J. Rodda, FtM Geysen, NJ Maeji. Multipin peptide synthesis at the micromole scale using 2-hydroxyethyl methacrylate grafted polyethylene supports. Int J Peptide. Protein Res 42 1-9, 1993. 

Originally, Multipin peptide synthesis was performed using a Boc protocol (39) however, due to more streamlined handling (required for efficient multiple synthesis), Fmoc/tBu synthetic protocols are now used. The TFA-mediated cleavage method used with Fmoc/tBu synthesis is far more easily adapted for large numbers than the HF required with Boc synthesis. 

Combinatorial libraries are prepared by the (1) parallel synthesis of arrays, (2) split-pool method, (3) biological method, or (4) spatially addressable parallel synthesis [74,78-80]. Parallel synthesis is carried out by the simultaneous synthesis of an array of different compounds. Several methods are available. In the multipin method, the peptide synthesis is carried out on polyethylene rods that have attached protected amino acids [81]. The amino acid sequence of a synthesized peptide on a particular pin depends on the order in which the amino acids are added. The number of products synthesized is the same as the number of pins. Another version of parallel synthesis, known as the teabag method, uses resin-filled bags in place of pins [74]. By pooling the resin portions from the appropriate bags, followed by redistribution and further coupling with a specific amino acid, a peptide library can be synthesized. The SPOT method uses a cellulose paper membrane as a solid support, which acts as an open reactor. Respective reagent solutions are pipetted onto several spots to synthesize as many peptides as the spots chosen [74,82]. 

J. L. Aubaganac, C. Enjalbal, et al.. Application of time-of-flight secondary ion mass spectrometry to in situ monitoring of solid-phase peptide synthesis on the multipin system, J. Mass Spectrom. 33, 1094-1103 (1998). 

The Multipin method is an effective, low cost, simultaneous multiple peptide synthesis technology which gives researchers ready access to large... 

The pin consists of a radiation-grafted polypropylene crown fitted to an inert polypropylene stem . Graft polymers used with the Multipin system include polystyrene, a methacrylamide copolymer (38), and poly(hydroxyethyl methacrylate) (34) (HEMA). We have found that the HEMA surface is best suited to peptide synthesis. Historically, peptides were prepared in a non-cleavable format on the crown surface for epitope mapping applications (39). Over the past decade, however, most peptides prepared by the Multipin method have been synthesized on cleavable linkers. The linkers used for peptide synthesis are outlined below. 

Figure 1 summarizes the linkers used for peptide synthesis by the Multipin method. Most carboxylic acids are prepared on the 4-(hydroxymethyl)-phenoxyacetic acid handle 1 (40). To avoid diketopiperazine formation during the synthesis of peptides containing a C-terminal Pro residue, the sterically hindered linker 2 is used (41). To minimize the risk of racemization dining linker derivatization. Asp and Glu are linked via their side-chain functionality, as shown in 3. Similarly, Asn and Gin are prepared by coupling Fmoc-Asp-OrBu and Fmoc-Glu-OiBu, respectively, to the Rink linker 4 (42). 

Multipin multiple peptide synthesis (MPS) kit (Chiron Technologies). The k rt includes crowns and stems, plastic baths and reaction trays, software (for IBM or Macintosh), and a manual... 

If multiple sets of peptides are being synthesized, it is advisable to refer to the manual provided with the Multipin Multiple Peptide Synthesis (MPS) kit. Use the software to generate the set of peptide sequences to be synthesized. 

Linear polystyrene can be generated on insoluble polymers by /-irradiation of the latter in a solution of styrene [110]. Polystyrene grafted onto polytetrafluoroethylene [111-116], polyethylene [2,110,117], or polypropylene [15] can be functionalized in the same way as cross-linked polystyrene, and loadings of up to 1.0 mmol/g can be attained. These supports, which are also available as crown-shaped pins (Multipin, 2-3 mm diameter, 8-10 pmol per crown), have been used for the synthesis of peptides [2,110,111,118], oligonucleotides [112-115,117,119], and small molecules [120-122]. 

Polyethylene has been grafted with acrylic acid, 2-hydroxyethyl methacrylate//V,/V-dimethylacrylamide, or methacrylic acid//V,/V-dimethylacrylamide to yield supports (e.g. Figure 2.10) suitable for the synthesis of peptides [2,222-224] and other compounds [216,225,226]. A similar support, also suitable for the synthesis of peptides, is polypropylene grafted with hydroxypropyl acrylate [227]. These supports can be used as membranes or as crown-shaped pinheads (Multipin 2-4 mm diameter) with loadings of 1.2-2.2 pmol per crown. 

Bray AM, Jhingran AG, Valerio RM, Maeji NJ, Simultaneous multiple synthesis of peptide amides by multipin method. Application of vapor-phase ammonolysis, J. Org. Chem., 59 2197-2203, 1994. 

A useful vapor-phase ammonolysis has been developed for simultaneous multiple synthesis of peptide amides by both the resin and multipin methods. Pins or peptide-resins are deprotected and washed with CHjCb, DMF, and CH2CI2, and are then air-dried. A bottle of NH3/THF (3 7 v/v, 50-100 mL) was cooled to —78 °C and placed in a well-greased desiccator together with the multipin pin holders (1-4) or the open vials containing resins (130mg/vial). The desiccator was clamped shut, partially evacuated (10 s), and then left to stand at 20°C for 24 h. After this time, the multipin pin holders and the resins were removed from the dessicator and allowed to stand for 30 min. Qeaved peptides were eluted from the pins by immersing them in MeCN/H20 (4 6) for 3 h, or from the resins by washing with AcOH/MeCN/HjO (3 4 3, 5 x ImL). 

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