Peptide synthesis Fmoc

The Fmoc group protection is common in solid-phase peptide synthesis. Fmoc is resistant to acidic conditions and easily deprotected by weak bases, particularly secondary amines. Deprotection occurs through base-catalyzed abstraction of the (3-proton of the protecting group with elimination leading to formation of dibenzofulvene (1.83) (Scheme 1.36). 

Besides performing multiple syntheses in parallel, the accelerating of reaction rates may also achieve the same result— production of multiple peptides in the same time period. CEM (http //www.cem.com) has employed microwave irradiation to shorten both coupling and deprotection times. Their synthesizer, Odyssey (Fig. 7), achieves one cycle of peptide synthesis (Fmoc-based) in less than 10 min and is theoretically capable of the synthesis of 12 peptides in a row. However, for each new peptide the reaction vessel has to be cleaned and new batch of resin has to be transferred into it. This step raises concerns with the authors of this review about the possibility of cross-contamination. 

The aim of release and deprotection is to separate the peptide from the solid support as well as from the side-chain protecting groups. To avoid side reactions, the peptide should only be exposed to the cleavage mixture for the minimum time it takes to release and deprotect it. The peptide is then removed from the support by filtration and from the protecting groups by precipitation, centrifugation, and decantation. The methods described herein are for release of peptides assembled via Fmoc-based solid-phase peptide synthesis (Fmoc-SPPS). 

The carboxamidomethyl ester was prepared for use in peptide synthesis. It is formed from the cesium salt of an A-protected amino acid and a-chloroacetamide (60-85% yield). It is cleaved with 0.5 M NaOH or NaHCOa in DMF/H2O. It is stable to the conditions required to remove BOC, Cbz, Fmoc, and r-butyl esters. It cannot be selectively cleaved in the presence of a benzyl ester of aspartic acid. ... 

Thermitase, pH 7.5, 55°, 50% DMSO, 3-140 min. This method was used to avoid the degradation of base-sensitive side chains during peptide synthesis. The method is compatible with the Fmoc group. ... 

This tertiary ester was developed to reduce aspartimide and piperidide formation during the Fmoc-based peptide synthesis by increasing the steric bulk around the carboxyl carbon. A twofold improvement was achieved over the the standard Fbutyl ester. The Mpe ester is prepared from the acid chloride and the alcohol and can be cleaved under conditions similar to those used for the r-butyl ester. ... 

Cleavage is effected with acid. The following table compares the acidolysis rates with Bn and cyclohexyl esters in TFA/phenol at 43°. The Dmp group reduces aspartimide formation during Fmoc-based peptide synthesis. 

This active ester was used for carboxyl protection of Fmoc-serine and Fmoc-threonine during glycosylation. The esters are then used as active esters in peptide synthesis. 

The Dmab group was developed for glutamic acid protection during Fmoc/r-Bu based peptide synthesis. The group shows excellent acid stability and stability toward 20% piperidine in DMF. It is formed from the alcohol using the DCC protocol for ester formation and is cleaved with 2% hydrazine in DMF at rt. ... 

The Phacm group is stable to the following conditions DIEA-CH2CI2, TFA-CH2CI2, piperidine-DMF, 0.1 M TBAF-DMF, and DBU-DMF for 24 h at It to HF-anisole or / -cresol (9 1) at 0° for 1 h and to TFA-scavengers (phenol, HSCH2CH2SH, p-cresol, anisole) for 2 h at 25°. It is partially stable (>80%) to TFMSA-TFA-/ -cresol for 2 h at 25°. These stability characteristics make the group compatible with BOC- or Fmoc-based peptide synthesis. ... 

For a review of the use of Fmoc protection in peptide synthesis, see E. Atherton and R. C. Sheppard, The Fluorenylmethoxycarbonyl Amino Protecting Group, in The... 

When the Ac group is removed (20% piperidine/DMF or 5% hydrazine/DMF), it becomes the Hmb group that is used to improve solubility and prevent aspar-tamide formation and is readily cleaved with TFA. The related 2-Fmoc-4-methoxybenzyl group has also been prepared and used in peptide synthesis. ... 

These derivatives provide effective protection for phosphotyrosine in Fmoc-based peptide synthesis. They are cleaved with 95% TFA. ... 

Another competing cyclisation during peptide synthesis is the formation of aspartimides from aspartic acid residues [15]. This problem is common with the aspartic acid-glycine sequence in the peptide backbone and can take place under both acidic and basic conditions (Fig. 9). In the acid-catalysed aspartimide formation, subsequent hydrolysis of the imide-containing peptide leads to a mixture of the desired peptide and a (3-peptide. The side-chain carboxyl group of this (3-peptide will become a part of the new peptide backbone. In the base-catalysed aspartimide formation, the presence of piperidine used during Fmoc group deprotection results in the formation of peptide piperidines. 

Chan WC, White PD (2000) Fmoc solid phase peptide synthesis, a practical approach. Oxford University Press, New York... 

Scheme 13.76. Fmoc Protocol for Solid Phase Peptide Synthesis...

Akaji K, Kiso Y, Carpino LA. Fmoc-based solid-phase peptide synthesis using a new t-alcohol type 4-(l, l -dimethyl-l -hydroxypropyl)phenoxyace-tyl handle (DHPP)-resin (Fmoc = 9-fluorenyloxycarbonyl). J Chem Soc Chem Comm 1990 584-586. 

Breipohl G, Knolle J, Stuber W. Synthesis and application of acid labile anchor groups for the synthesis of peptide amides by Fmoc-solid-phase peptide synthesis. Int J Peptide Protein Res 1989 34 262-267. 

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.

Aurora Biomolecules dedicates to peptide synthesis (and polyclonal antibody production) for any small quantity purpose. FMOC chemistry (on Perceptive Biosystems Pioneer instruments) is used for peptides synthesis Online monitoring of the coupling efficiencies and HATU activation helps insure that the major component of the synthesis is the correct oligopeptide. Purification is firstly carried out by size exclusion chromatography, and then by HPLC on a PE vision purification workstation. Typically, 20 mg of pure peptide are obtained. The molecular weight of the purified peptide is determined as a final confirmation of quality. 

More recently, Somfai and coworkers have reported on the efficient coupling of a set of carboxylic acids suitable as potential scaffolds for peptide synthesis to a polymer-bound hydrazide linker [24]. Indole-like scaffolds were selected for this small library synthesis as these structures are found in numerous natural products showing interesting activities. The best results were obtained using 2-(7-aza-l H-benzo-triazol-l-yl)-l,l,3,3-tetramethyluronium hexafluoride (HATU) and N,N-diisopropyl-ethylamine (DIEA) in N,N-dimethylformamide as a solvent. Heating the reaction mixtures at 180 °C for 10 min furnished the desired products in high yields (Scheme 7.4). In this application, no Fmoc protection of the indole nitrogen is required. 

With the C-terminal residue introduced as part of the BAL anchor and the penultimate residue incorporated successfully by the optimized acylation conditions just described, further stepwise chain elongation by addition of Fmoc-amino acids generally proceeded normally by any of a variety of peptide synthesis protocols. 

Atherton, E. and Sheppard, R.C. (1989) Solid Phase Peptide Synthesis, a Practical Approach. 203pp. IPR Press, UK. A working handbook focussing on polyacrylamide resins and Fmoc-chemistry. 

Chan, W.C. and White, P.D. (Eds.) (2000) Fmoc Solid Phase Peptide Synthesis. A Practical Approach. 368pp. Oxford University Press. Essential procedures and advanced techniques. 

DS King, CG Fields, GB Fields. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Ini J Pept Prot Res 36, 255, 1990. 

RESINS AND LINKERS FOR SYNTHESIS OF PEPTIDES USING FMOC/TBU CHEMISTRY... 

FIGURE 5.17 Resins and linkers for synthesis of peptides using Fmoc/tBu chemistry. The linkers are secured to supports by reaction with aminomethyl resins. A protected amino acid is anchored to the support as an ester by reaction with a hydroxyl or chloro group (italicized). The alkoxy and phenyl substituents render the benzyl esters sensitive to the cleavage reagents. 

FIGURE 5.20 Resins for the synthesis of protected peptides using Fmoc/tBu chemistry. The first residue is esterified to the handle by reaction with the italicized functional group. The protected peptide is detached by cleavage of the ester bond with 1% CF3C02H for (A) and (B) and 10% CF3C02H for (C). 

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