Difficult peptides

Importantly, it was demonstrated that no significant racemization occurred in the course of peptide formation. Furthermore, the complete coupling of difficult peptide sequences could be accomplished within a few minutes, and it was determined that peptide fragments have higher reactivity than single amino acid derivatives under microwave irradiation conditions. However, the exact reaction temperature during the irradiation period was not determined. 

Peptides with up to 166 amino acids (aglycon of erythropoietin [34]) have been prepared using the Fmoc strategy on cross-linked polystyrene. The preparation of peptides containing several /V-methylamino acids (e.g. cyclosporine [35,36]) has also been successfully performed using Fmoc protection. Flowever, such difficult peptides require careful planning of the synthesis and often the use of special coupling techniques. 

Scheme 6. The Hnb-protecting group serves as an activated O- N acyl transfer auxiliary for the efficient synthesis of difficult peptides.

In 2004, Kumar and Montanari introduced a fluorous tagged trivalent iodonium compound that can be used as a tag for /-Boc based solid phase peptide synthesis by tagging free amines with a perfluoroheptyl (W-C7F15) group.It is an efficient fluorous tagging reagent that has the potential to help in the synthesis of both routine and difficult peptide and protein sequences. The... 

Very difficult peptide syntheses are those which contain sterically hindered C -dialkylannino adds such as Aib residues, especially if these residues are found next to each other. Due to their influence on the stabilization of helical stmcturest l in small peptides, the introduction of such amino acids is of interest. Various protocols for the coupling of hindered systems using UNCAs,t l symmetric anhydrides,t HATU,b CIP/HOAt, and PyBroPl l activation have been reported. In a comparative analysis of activation methods for the synthesis of H-Aib-Aib-Aib-Aib-OH as a model peptide which included the symmetric anhydride, PyBroP, acid fluoride, and UNCA methods, the acid fluoride technique was shown to be exceptionally well suited.t 1... 

So far, pseudoprolines have found applications in providing solubilizing, secondary structure-disrupting building blocks for the synthesis of difficult peptide sequences and for the reversible induction of cis-amide bonds into peptide backbones (Fig. 11.15). 

For difficult peptides, incomplete Fmoc deprotection can be a problem and use of the stronger tertiary base, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) has been shown to increase reaction efficiency. Typically, small amounts of piperidine are... 

Recently, Anteunis and Sharma [58] reported a study devoted to the lactam cyclization. Cyclization of the virginiamycin Si linear precursor into the natural product offered a special challenge. Creation of a peptidic bond between the two N-alkylamino acid residues (N-methylphenylalanine and 4-oxopipecolinic acid) - which is the most difficult peptidic bond to form - led to extensive racemiz-ation of the N-methylphenylalanine residue as the carboxy group has to be strongly activated (BOP-Cl in different conditions) during the cyclization step. 

Tickler AK, Wade JD (2007) Overview of Solid Phase S5mthesis of "Difficult Peptide" Sequences. In Coligan, JE, editor. Current protocols in protein science. Chapter 18. 

During the early 1970s it was suggested that the occurrence of difficult peptides possibly arose due to an incompatibility between the apolar polystyrene resins, polar peptide chains, and apolar reaction media, such as dichloro-methane, used for SPPS as depicted in Figure Iboxlc (13). This prompted the investigation of alternative polymer supports and reaction media to the... 

Following the introduction of polyamide supports, Sheppard and co-workers advocated a milder orthogonal Fmoc/r-butyl chemistry for SPPS in place of the somewhat harsh Boc/benzyl protocols of the original Merrifield scheme (18,19). This new chemistry, along with the advent of a flow stable physically supported polyamide gel, led to the widely adopted continuous flow Fmoc-polyamide technique for SPPS (20-22). This proved to be a pivotal contribution in the understanding of the difficult peptide phenomenon through the real-time spectrophotometric monitoring of a fluorene derived chromo-phore, released at each A/ -Fmoc deprotection cycle, into the solvent stream. 

The unhindered release of fluorene derivatives from a fully solvated peptide-polymer matrix results in a characteristic bell-shaped elution profile (20) Figure 2a). Syntheses that exhibit such profiles throughout their assembly are generally of an excellent crude quality. In the case of difficult peptide sequences, the slowing of reaction kinetics at the onset of aggregation is readily observed as a broadening of the release of fluorene derivatives from the reaction matrix Figure 2b). In severe instances, the monitored release of... 

Numerous modified SPPS protocols have been suggested to improve the quality of difficult peptide syntheses. A common theme among the majority of these protocols is an attempt to limit the extent of p-sheet secondary structure formation by altering the solvation properties of the polymer, the polymer-bound protected peptide, and/or the surrounding reaction media. 

In order to effectively assembly difficult peptides, more complex protocols aimed at the disruption of secondary structure formation need to be introduced prior to the onset of synthetic difficulties. Ideally, this could be achieved if a predictive method describing where and when -structure formation will occur, based simply on examination of the primary sequence, was available. Unfortunately, there is no complete solution available to date, although a number of groups have developed reasonably successful guidelines. Current predictive methods are derived from four main investigations ... 

The effects of host-guest substitutions within a given difficult peptide framework (50,51). 

Figure 3. Deprotection peak heights as a function of host residue (-X-X-) and chain length in the SPPS of the known difficult peptide test sequence H- Ala) -X-X-(Ala)3-Val-poly-dimethylacrYlamide (I). (a) General hydrophobic residues, (b) Eff of side-chain protection of cysteine, (c) Effect of trityl protection of side-chain carboxamide of glutamine and asparagine, (d) Effect of arginine and tertiary amino acids proline and sarcosine. (Reproduced from ref. 51 with permission from Munksgaard International Publishers Ltd., Copenhagen.)...

The host-guest results, along with the segment solubility results of Narita et al. (48, 49) and Blackmeer et al. (52), suggested that the difficult peptide problem might be eliminated by the introduction of a reversibly A -alkylated amino acid residue prior to aggregation. 

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