Solid-phase peptide synthesis Fmoc-based

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). 

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). 

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. 

Work in the Imperiali laboratory has also focused on exploring the ability of minimal peptide scaffolds to augment the rate of coenzyme-mediated transaminations [22-25]. To accomplish this, a strategy has been developed in which the core functionality of the coenzyme is incorporated as an integral constituent of an unnatural coenzyme amino acid chimera construct. Thus, non-cova-lent binding of the coenzyme to the peptide or protein scaffold is unnecessary. Both the pyridoxal and pyridoxamine analogs have been synthesized in a form competent for Fmoc-based solid phase peptide synthesis (SPPS) (Fig. 7) [23,24]. 

The development of chiral peptide-based metal catalysts has also been studied. The group of Gilbertson has synthesized several phosphine-modified amino adds and incorporated two of them into short peptide sequences.[45J,71 They demonstrated the formation of several metal complexes, in particular Rh complexes, and reported their structure as well as their ability to catalyze enantioselectively certain hydrogenation reactions.[481 While the enantioselectivities observed are modest so far, optimization through combinatorial synthesis will probably lead to useful catalysts. The synthesis of the sulfide protected form of both Fmoc- and Boc-dicyclohexylphosphinoserine 49 and -diphenylphosphinoserine 50 has been reported, in addition to diphenylphosphino-L-proline 51 (Scheme 14).[49 To show their compatibility with solid-phase peptide synthesis, they were incorporated into hydrophobic peptides, such as dodecapeptide 53, using the standard Fmoc protocol (Scheme 15).[451 For better results, the phosphine-modified amino acid 50 was coupled as a Fmoc-protected dipeptide 56, rather than the usual Fmoc derivative 52.[471 As an illustrative example, the synthesis of diphe-nylphosphinoserine 52 is depicted in Scheme 16J45 ... 

In addition to incorporating the 4-(2-aminoethyl)dibenzofuran-6-propanoic acid template into small peptides where a reverse turn is desired, we have also recently incorporated this template into a mini-protein called the PIN WW domain. WW domains have a three-stranded antiparallel p-sheet structure that mediates intracellular protein-protein interactions. 31 Substitution of this 3-turn mimetic into loop 1 of the PIN WW domain leads to a folded, three-stranded, antiparallel p-sheet structure with a stability indistinguishable from that of the all a-amino acid sequence. The template-incorporated PIN WW domain (11) was synthesized by an Fmoc-based solid-phase peptide synthesis strategy (Scheme 8), utilizing N-Fmoc-protected 4-(2-aminoethyl)dibenzofuran-6-propanoic acid 10. 11 The synthesis of 10, similar to that of 8, has been published.1 1 ... 

The two most commonly used types of allyl alcohol linker are 4-hydroxycrotonic acid derivatives (Entry 1, Table 3.7) and (Z)- or ( )-2-butene-l, 4-diol derivatives (Entries 2 and 3, Table 3.7). The former are well suited for solid-phase peptide synthesis using Boc methodology, but give poor results when using the Fmoc technique, probably because of Michael addition of piperidine to the a, 3-unsaturated carbonyl compound [167]. Butene-l,4-diol derivatives, however, are tolerant to acids, bases, and weak nucleophiles, and are therefore suitable linkers for a broad range of solid-phase chemistry. 

Support-bound triacylmethanes (e.g. 2-acetyldimedone) readily react with primary aliphatic amines to yield enamines. These are stable towards weak acids and bases, and can be used as linkers for solid-phase peptide synthesis using either the Boc or Fmoc methodologies, as well as for the solid-phase synthesis of oligosaccharides [456]. Cleavage of these enamines can be achieved by treatment with primary amines or hydrazine (Entries 2 and 3, Table 3.23 see also Section 10.1.10.4). 

Standard solid-phase peptide synthesis requires the first (C-terminal) amino acid to be esterified with a polymeric alcohol. Partial racemization can occur during the esterification of N-protected amino acids with Wang resin or hydroxymethyl polystyrene [200,201]. /V-Fmoc amino acids are particularly problematic because the bases required to catalyze the acylation of alcohols can also lead to deprotection. A comparative study of various esterification methods for the attachment of Fmoc amino acids to Wang resin [202] showed that the highest loadings with minimal racemization can be achieved under Mitsunobu conditions or by activation with 2,6-dichloroben-zoyl chloride (Experimental Procedure 13.5). iV-Fmoc amino acid fluorides in the presence of DMAP also proved suitable for the racemization-free esterification of Wang resin (Entry 1, Table 13.13). The most extensive racemization was observed when DMF or THF was used as solvent, whereas little or no racemization occurred in toluene or DCM [203]. 

A wide choice of peptide synthesizers is currently available, ranging from manual to fully automated. They are all based on solid-phase peptide synthesis methodologies in which either f-butoxy carbonyl (t-boc) (11), or 9-fluor-enylmethoxycarbonyl (Fmoc) (12) is the major protecting group during synthesis. A detailed description of peptide synthesis is clearly beyond the scope of this chapter, and further information on practical and theoretical approaches to this chemistry may be found elsewhere (13-15). However, a brief outline of solid-phase synthesis may prove useful. 

In solid phase peptide synthesis, it is important that the repetitive steps proceed rapidly, in high yield, and with minimal side reaction to prevent the accumulation of by-products (1). Solid phase peptide synthesis almost always employs either Boc or Fmoc chemistry. Boc chemistry requires acidic conditions for deblocking, and the potential side reactions have been extensively studied. However, in Fmoc chemistry (2) repetitive basic conditions are required for deblocking, and only a few of the base-catalyzed side reactions have been characterized. We present here a method used to demonstrate that a side reaction well known in Boc chemistry (3) but thought not to occur under the conditions of Fmoc, in fact occurs with both approaches aspardmide formation. 

There are numerous side reactions that can occur during solid-phase peptide synthesis, some of which are specific to the chemistries employed using Fmoc-based methodology. This section describes some of the prevalent reactions that will lead to heterogeneity in the resultant peptide. 

Montanari, V. and Kumar, K. (2006) A fluorous capping strategy for Fmoc-based automated and manual solid-phase peptide synthesis. European Journal of Organic Chemistry, (4), 874-877. 

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