Fmoc chemistry

Although use of Boc has enabled successful syntheses, its application has been limited, because of the use of HF - a major factor in the current preference for Fmoc chemistry [6]. Little known work has been performed with Boc chemistry using microwave energy. For this reason, this chapter focuses on the application of microwave energy for Fmoc SPPS. 

Although the Fmoc method does not require use of HF, it does suffer from inherent difficulties during stepwise synthesis. These can occur because of intermo-lecular aggregation, j8-sheet formation, steric hindrance from protecting groups, and premature termination of the sequence. As a result, standard amino acid cycle times range from 30 min to 2 h and incomplete deprotection and coupling reactions are still common. 

Carboxylic acids and amines will react to give an ammonium salt (acid-base equilibrium) instead of a carboxamide. Activation of the carboxylic acid to form an ester species enables peptide bond formation to occur (Fig. 20.3). 

This reduces the electron density on the C=0 group, which favors nucleophilic attack by the amine group (Fig. 20.4). The improved nucleofugicity of the leaving group also increases the reaction rate. 

The phosphonium method was originally developed from derivatives of tris-dimethylaminophosphonium salts for the activation of carboxylic acids [10-13]. The first developed phosphonium salt-type reagents were //-oxo-bis-[tris(dimethyl-amino)phosphonium]-bis-tetrafiuoroborate (Bates Reagent) and benzotriazol-l-yl-N-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). Although 

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Fmoc chemistry is compatible with methyl phosphates when methanolic... 

The investigation of minor groove-binding polyamides was greatly accelerated by the implementation of solid-phase synthesis [48]. Originally demonstrated on Boc-y9-Ala-PAM resin with Boc-protected monomers, it was also shown that Fmoc chemistry could be employed with suitably protected monomers and Fmoc-y9-Ala-Wang resin (Fig. 3.8) [49]. Recently, Pessi and coworkers used a sulfonamide-based safety-catch resin to prepare derivatives of hairpin polyamides [50]. Upon activation of the linker, resin-bound polyamides were readily cleaved with stoichiometric quantities of nucleophile to provide thioesters or peptide conjugates. 

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). 

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. 

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. 

A Trzeciak, W Bannwarth. Synthesis of head-to-tail cyclic peptides on solid support by FMOC chemistry. Tetrahedron Lett 33, 4557, 1992. 

Common protectors of hydroxyls are benzyl and 2-bromobenzyl for Boc chemistry and tert-butyl for Fmoc chemistry. Trityl provides a third level of selectivity for both chemistries because it can be removed by mild acid (1% CF3C02H in CH2C12), which does not affect tert-butyl based protectors. O-Allyl is not removable by palladium-catalyzed allyl transfer, so it is not appropriate. Protection by acyl such as benzyloxycarbonyl is possible, but 0 -acyl protectors can be problematic because of their tendency to shift to adjacent amino groups (see Section 6.6) and... 

The main considerations in protecting the hydroxyl of tyrosine are the stability to acid of the protector and the protector s tendency to alkylate the ortho-position of the ring when it is removed (Figure 6.7 see Section 3.7). The standard protector for Fmoc chemistry is tert-butyl, but the substituent is sensitive to acid. Preferable... 

Thus, the best compromises for Boc and Fmoc chemistries seem to be cyclohexyl and 2,4-dimethylpent-3-yl (Dmpn), which is of intermediate stability, and the removal of which by trifluoromethanesulfonic acid with the aid of thioanisole (see Section 6.22) leads to minimal imide formation (see Section 6.13). Points to note are that acidolysis of esters by hydrogen fluoride can lead to fission at the oxy-car-bonyl bond instead of the alkyl-oxy bond, thus generating acylium ions that can react with nucleophiles (see Sections 6.16 and 6.22), and that benzyl esters may undergo transesterification if left in methanol. The side reactions of cyclization (see Section 6.16) and acylation of anisole (see Section 6.22) caused by acylium ion formation do not occur at the side chain of aspartic acid.47-51... 

Efficient, though not total, suppression of the reaction is achieved in a synthesis using Fmoc chemistry by including an acid such as 1-hydroxybenzotriazole or... 

R Dolling, M Beyermann, J Haenel, F Kemchen, E Krause, P Franke, M Brudel, M Bienert. Base-mediated side reactions on Asp(OtBu)-X sequences in Fmoc-chemistry (piperazine), in HLS Maia, ed. Peptides 1994. Proceedings of the 23rd European Peptide Symposium. Escom, Leiden, 1995, pp 244-245. 

Double lipidated peptides incorporating a C(GerGer)XC(GerGer)-OMe 33 motif that is found in several Rah and homologous proteins were also synthesized in solution via Fmoc chemistry following cysteine deprotection and geranylgeranylation. ° ... 

The PNA chain was linked to the peptide spacer glutamic acid-(y-tert-butyl ester)-(fi-aminohexanoic acid)-(fi-aminohexanoic acid) (Glu [OtBuj-fiAhx-fiAhx) via an enzymatically cleavable Glu-Lys handle. The Glu [OtBuj-fiAhx-fiAhx spacer was coupled to the amino-functionalized membrane by standard Fmoc-Chemistry. Then the membranes were mounted in an ASP 222 Automated SPOT Robot and a grid of the desired format was dispensed at each position. The free amino groups outside the spotted areas were capped and further chain elongation was performed with Fmoc-protected PNA monomers to synthesize the desired PNA oligomers (18). After completion of the synthesis, the PNA oligomers were cleaved from the solid support by incubation with bovine trypsin solution in ammonium bicarbonate at 37 °C for 3 h. 

AM-beads (22) leaves the majority of the peptide attachment sites in the interior uncleaved to afford (23) ( shaving methology). The first residue is attached using orthogonal FMOC-chemistry to provide (24). Coding is achieved by using standard BOC-chemistry in the interior of the bead to yield (25). Repetition of this process furnishes a surface bound peptide, which is encoded internally (26). 

A general procedure for the iodine reaction in the solid phase is shown in Scheme 9. Both Boc and Fmoc chemistry can be used to assemble the linear S-protected bis-cysteine peptides and for thiol protection the well-established Acm and Trt groups are usually used. Suitable solvents for the thiol-deprotection/oxidation step by iodine to form the disulfide are CH2C12, DMF, or aqueous AcOH. The final deblocking and cleavage from the resin is carried out under standard conditions. Modification at sensitive amino acid residues caused... 

The monomeric peptides [Cys-I], [Cys,Cys(Acm-II], [Cys(Acm),Cys-III], and [Cys-IV] were synthesized by Fmoc chemistry on Rink-amide resin as 5-Mob derivatives and cleaved/deprotected with 1M TMSBr/thioanisole in TFA in the presence of m-cresol and 1,2-ethanedithiol as scavengers. Following gel filtration on Sephadex G-10 with 1M AcOH as solvent and HPLC purification the peptides were obtained in 30—40% yield. Each product was characterized by LSIMS, HPLC, and amino acid analysis. 

Caged peptides (NPY and angiotensin II) containing Tyr[Bzl(2-N02) were synthesized on an automated solid-phase peptide synthesizer using Fmoc chemistry and cleaved from the resin by treatment with TFA. Crude peptides were purified by semipreparative HPLC with a gradient phase of 0.1% TFA and MeCN. For fractions containing peptides, the eluant was not passed through the UV monitor to avoid potential photolysis. The collected peptide was examined by analytical HPLC and MS. 

Resin Rink amide MBHA (Fmoc chemistry) TentaGel-OH (Fmoc chemistry) Trt resin (Fmoc chemistry)... 

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