Peptide synthesizers, continuous-flow

There are a couple of polyethylene-based composites that have shown some success in solid-phase organic synthesis. Merrifield and co-workers [32] introduced a polystyrene-polyethylene composite support in the form of a sheet for solid-phase peptide synthesis, which exhibited an amine loading level of 1.0 mmol amine/g. This material has since been molded into various shapes, including tubing for a continuous-flow peptide synthesizer (reported loading=0.21 mmol amine/m tube or 0.67 mmol amine/g) [33], Geysen and coworkers [34,35] synthesized a poly ethylene-polymethaciy late copolymer which was molded... 

Kearney, T. and Giles, J. (1989) Fmoc peptide synthesis with a continuous flow synthesizer. Amer. Biotechnol. Lab. 7, 34-44. 

Krchnak, V., Cabel, D., and Lebl, M. (1996) MARS Multiple automated robotic synthesizer for continuous flow of peptides. Peptide Res. 9, 45-49. 

Insolubility is, of course, the most fundamental property of a polymer support for it to be useful in peptide synthesis, but it is not the only relevant one. Particle size and shape as well as mechanical stability are important in order to permit easy manipulation and rapid filtration from liquids. Mechanical stability is especially critical when the solid support is to be used in a continuous-flow peptide synthesizer. 

A branched peptide 11 [prothrombin(l-9)-Lys(Leu-enkephalin)-NH2] has been synthesized on a PAL-PEG-PSty resin in a continuous flow peptide synthesizert l by combining A -Fmoc for temporary protection, A -Aloc for semipermanent lysine protection, and Boc for permanent protection of the terminal amino group of prothrombin, as outlined in Scheme 6. Deprotection of Aloe in the continuous flow synthesizer is achieved by use of the Pd(PPh3)4/ NMM/ACOH/CHCI3 system. 

Conventional batch and continuous-flow peptide synthesizers typically produce one to three peptides at a time in quantities from 0.025 to 2 mmol. To satisfy the demand for a greater number of sequences, robotic instruments have been adapted for peptide assembly which are able to construct 8 to 144 peptides simultaneously. Production scale synthesizers have also been designed that are able to prepare up to 5 moles of a peptide. 

The first connmercially available continuous-flow synthesizers were manual instruments the PEPSYNthesiser I from Cambridge Research Biochemicals (CRB) and the Model 4175 from LKB Biochrom. In these instruments, one manual valve switches between the flow and recirculate modes and another manual valve selects the solvent, reagent or the annino acid injection port. CRB later released the semiautomatic PEPSYNthesiser II before selling the rights to the instrument to MilhGen, who ultimately released the fully automated 9050 PepSynthesizer. Pharmada-LKB Biochrom also developed an automated continuous-flow synthesizer, the Biolynx Model 4170. Several years later, the rights to this instrument were obtained by Novabiochem. Most of the automated, semiautomated, and manual continuous-flow peptide synthesizers are fisted in Table 3. 

Similarly to the tea bag approach, pieces of any solid-phase support suitable for solid-phase synthesis can be used for parallel peptide preparation. One of the first materials used in this manner was paper. Frank used paper disks l (Whatman 3MM, 1.5-cm diameter) packed into the columns of a multicolumn continuous-flow synthesizer. Prior to the synthesis, the paper was derivatized with 4-alkoxybenzyl (Pab) linkage to allow for the cleavage with trifluoroacetic acid. The disks can be easily labeled with a pencil, sorted, and combined depending on the common amino acid to be coupled at the next step. 

Figure 2.—Continued. Flow chart of the protocol used to synthesize tetrameric bundles of defined amino acid sequence. (F), (G) Single amino acids are added to sequentially assemble the peptide modules of the synthetic proteins. Continued...

Peptide synthesis A solution of 20% in DMF (v/v) is most commonly employed for both batch and continuous-flow syntheses, although concentrations as high as 50% in DMF (v/v) have also been reported [26,57]. For some difficult syntheses, DBU (see next) has been added to the piperidine solution to accelerate the rate of Fmoc group removal [58]. Solvents other than DMF may be used successfully. These include DMA and NMP or mixtures of these together with DCM. 

The authors noted that with a typical commercial continuous flow peptide synthesizer, it should be possible to accomplish six different amino acid couplings per day on 50 disks resulting in 300 peptide couplings per day. This rather classical but very straightforward and efficient approach combines the continuous flow technique with traditional solid-phase supported chemistry. 

In this connection another utility of the Ddz-group should be mentioned briefly. In dioxane and tetrahydrofurane solutions of Ddz-amino acids and peptides the masking group can be cleaved continuously and quantitatively in a flow procedure by photochem-ically induced autoheterolysis with light irradiation [93]. This possibility of deprotection is particularly useful in peptide syntheses with Ddz-amino acid active esters insolubilized on polymer phase [104] By aminolysis the Ddz-amino acid is transferred from its support onto an amino component in solution. The dissolved Ddz-peptide so formed can be depro-... 

Glycopeptide 31 was synthesized in a custom-made, fully automatic continuous-flow peptide synthesizer constructed essentially as described elsewhere (19). A resin consisting of a cross-linked polystyrene backbone grafted with poly(ethylene glycol) chains (TentaGel , Rapp Polymer, Germany) and functionalized with the Rink amide linker (p-[a-(fluoren-9-ylmethoxyformamido)-2,4-dimethoxybenzyl]phenoxyacetic acid, Nova-biochem, Switzerland) was used for the syntheses. DMF was distilled before use. 

Naphthalenequinone methides (382) and (384) were photochemically synthesized from 1- and 2-adamantylnaphthol derivatives (381) and (383), respectively (Scheme 82). Photorearrangement of allqrl-aryl nitrones (385) via oxaziridines (386) afforded amide derivatives (387) as peptide fragments via continuous flow process (Scheme 83). ... 

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