Fmoc removal

For the addition of the first amino acid to supports derivatized with peptide amide-forming linkers, such as trialkoxybenzhydrylamine 9, trialkoxybenzyl-amine 10, and aminoxanthenyl 11 resins, any of the standard peptide coupling methods described in Section 7 can be used. Since basic reagents such as 4-dimethylaminopyridine are not required to effect this transformation, there is no risk of enantiomerization or dipeptide formation. Many resins of this type are supplied Fmoc-protected and these should be treated with piperidine prior to the addition of the first amino acid. With automated peptide synthesizers, Fmoc removal and loading with the C-terminal residue can be programmed to occur as part of the normal coupling cycle. 

The addition of the first residue to dimethoxybenzhydrylamine-based resins can be sluggish, particularly the 3-branched amino acids, such as valine and isoleucine. Repeating the coupling after 1 h with fresh reagents generally leads to complete resin derivatization. 

Removal of the Fmoc group from the N-terminus of the resin-bound peptide chain is normally achieved by treating the peptidyl resin with 20-50% piperidine in DMF. The reaction is typically complete within 4-10 min, depending on the nature of the peptide being synthesized. With peptides containing aspartic acid and asparagine, inclusion of 0.1 M HOBt in the deprotection mixture has been found to be partially effective in suppressing aspartimide formation (18). 

In the case of sequences which have become aggregated the standard treatment with piperidine in DMF may not always be effective. Even extending the time of deprotection reaction considerably does not always result in com- 

Protocol 6. Manual removal of Fmoc groups Reagents 

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Alternatively to using prelipidated building blocks palmitoylation on resin is possible with the hydrazine linker. In Scheme 27 the synthesis route for the palmitoylated and farnesylated N-Ras peptide 78 is shown. Here the initial loading of trityl-protected cysteine to the hydrazine linker was mediated by A,A-diisopropylcarbodiimide (DIG) and HOBt. After Fmoc removal the proline was coupled using HBTU and HOBt. The trityl-protected dipeptide 75 was subsequently S-deprotected using TFA with triethylsilane (TES) as a scavenger. Farnesylation of the free thiol was achieved with an excess of farnesyl bromide. 

More recently, Aitken and coworkers described a short and convergent formal total synthesis of cyclotheonamide C using a process that involves a Passerini reaction, amine deprotection, and an acyl migration (PADAM sequence. Scheme 22) [90]. The key linear pentapeptide 22e is obtained by a Passerini reaction of isocyanide a, Fmoc-amino aldehyde b, and Boc-dipeptide acid e followed by Fmoc removal and consequently 0,N-acyl migration [91]. The macrocyclization was achieved with TBTU and HOBt after Boc and fBu removal in good yield (52%) to furnish intermediate f. 

Fmoc-Rink-Nle-pMeBHA-resin (1 g, 0.55 mmol g ) was swollen for 1.5 h in NMP in a reaction vessel equipped with a sintered glass bottom, and placed on a shaker. The Fmoc group was removed with 20% piperidine in NMP (8mL, 2 x 15 min) and after washing with NMP (8mL, 5 x 2 min), the Fmoc removal was monitored by the ninhydrin Kaiser test. Coupling of building blocks such as iV -Fmoc-/Va>-carb-oxyalkyl(OAl)Xaa-OH or /V -Fmoc-/V" -aminoalkyl(Aloc)Xaa-OH, or N -alkylated amino acids such as... 

Removal of the temporary Fmoc protecting group from the N-terminus of the peptidyl-resin is normally achieved by short treatment with 20% piperidine in DMF (Protocol 7). The reaction is generally complete within 10 min, but can be longer in some cases. For safe removal a 20-min deprotection time is recommended. The deprotection results in formation of a dibenzofulvene-piperidine adduct that strongly absorbs in the UV range (see Note 3). Fmoc removal can be monitored by UV spectroscopy. This is a common procedure with automatic... 

The cyclization of aspartic acid residues to form aspartimide is the most likely side-reaction observed in routine SPPS (Fig. 10). This is a sequence-dependent side-reaction that occurs either during chain elongation or during final TFA cleavage when Asp(OtBu)-AA sequence (AA = Ala, Gly, Ser, Asn(Trt)) is present in the peptide. Hydrolysis of the aspartimide ring leads to a mixture of both a- and P-peptides. Its reaction with piperidine used for Fmoc removal also leads to the formation of a- and p-piperidides. Normally, in Fmoc-based SPPS, Asp (OtBu) provides sufficient protection. However, for particular sequences such as Asp(OtBu)-Asn(Trt) particularly sensitive to aspartimide formation, addition of HOBt to the piperidine solution or protection of the aspartyl amide bond with the 2-hydroxy-4-methoxybenzyl (Hmb) group should be considered (36). 

Protocols used for standard Fmoc cleavage in SPPS and related spectroscopic monitoring techniques are reported in Section 4.S.2.2, while protocols used for Fmoc removal in solution- and solid-phase synthesis of glyco- and phosphopeptides, and O-sulfated peptides are reported in Sections 6.3, 6.5, and 6.6, respectively. 

Although aspartimide formation can occur during base-catalyzed acylation in Boc-based chemistry, the reaction is more pronounced during Fmoc removal with piperidine.The reaction, shown in Scheme 6, is sequence dependent, but aspartimide formation can be up to 0.3% per N-terminal deprotection cycle when the required peptide contains -Asp-Gly-. When the resultant peptide is cleaved from the support the aspartimide ring may remain intact, but can also open to form the P-branched peptide. 

Multiple addition of amino acids can occur when the acylation conditions are basic enough to lead to premature Fmoc removal during the coupling. This has been observed when using TBTU/DIPEA-based activationf and can be overcome by addition of excess HOBt or changing the conditions for peptide bond formation. 

Racemization of C-terminal Cys bound to the solid support as an ester is promoted by repetitive Fmoc removal.Cys is often introduced at the C-terminal of a peptide to provide an anchoring point, via the thiol, for subsequent conjugation so in some circumstances racemization at this point may not be important. Where it is essential to retain the chiral integrity, preparation of the peptide amide using a Rink-type linkage agent will overcome the problem. 

The cycle of alternating amino acid coupling and amide alkylation consisted of the following steps, which are further described below (i) coupling of an Fmoc-protected amino acid, (ii) Fmoc removal followed by tritylation of the N-terminal amino group, (iii) amide alkylation, and (iv) removal of the trityl group. 

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