Peptide-resin

Peptide- resin with the designated numbfer of units of(leucyl-alanylglycylvalyloxymethylphenylacetyl) attached. Peptide loading wis 0.2 mmole/g PS (1 phenyl in 50 substituted). 

The similarity of results for peptide resins in both solvents fits nicely with the estimate that this resin sample with about 50 weight % peptide should have similar swelling characteristics in the two solvents (3). In these samples we have measured the integrals of the aromatic region relative to an unenriched peptide peak, and the results are consistent with the observation of all the carbon nuclei, and hence the results are typical of all portions of the cross-linked matrix. 

Preparation of BAL-anchored peptide-resins by on-resin reductive amination followed by stepwise chain elongation... 

PREPARATION OF BAL-ANCHORED PEPTIDE RESINS BY ON-RESIN REDUCTIVE AMINATION, FOLLOWED BY STEPWISE CHAIN ELONGATION (SCHEME B)... 

Final products are cleaved with TFA-H20 (19 1) (1 mL/50mg of resin) at 25°C for 1 h. The filtrate from the cleavage reaction is collected, combined with TFA washes (1 mL/50mg of resin) of the cleaved peptide-resin, and dried. Cleavage yields (>85%) are calculated by amino acid analysis. 

Peptide resin samples are hydrolyzed in 12 N aqueous HC1-propionic acid (1 1) at 155°C for 3h. 

FIGURE 5.8 Relative volumes of a polystyrene resin and a peptide-resin adduct in the presence or absence of solvent molecules. The values are for a peptide of 40 residues (molecular weight = 5957 daltons), the first residue loaded at 0.95 mequiv/g of resin, giving a peptide to resin ratio of 4 1 by weight after assembly.15... 

The symmetrical anhydride is prepared using dicyclohexylcarbodiimide in dichloromethane, the urea and solvent are removed, and the anhydride is dissolved in dimethylformamide and added to the peptide-resin (see Section 2.5). The anhydride is a more selective acylating agent than the 0-acylisourea and, thus, gives cleaner reactions than do carbodiimides, but twice as much amino-acid derivative is required, so the method is wasteful. It avoids the acid-catalyzed cyclization of terminal glutaminyl to the pyroglutamate (see Section 6.16) and is particularly effective for acylating secondary amines (see Section 8.15). 

Activated esters (see Section 2.9) with 1-hydroxybenzotriazole as a catalyst are employed — pentafluorophenyl or 4-oxo-3,4-dihydrobenzotriazin-3-yl esters in particular for continuous-flow systems and special cases such as dicarboxylic amino acids. Other activated esters are not reactive enough. An alternative is preparation of benzotriazolyl esters using a carbodiimide followed by addition of the solution to the peptide-resin. 

RA Houghten, MK Bray, ST Degraw, CJ Kirby. Simplified procedure for carrying out simultaneous multiple hydrogen fluoride cleavages of protected peptide resins. Ini J Pept Prot Res 27, 673, 1986. 

Sequence inversion and racemization have been associated with uncatalyzed formation of the cyclic dipeptides and has been shown to greatly complicate the kinetics of formation. Cyclic dipeptide formation, by uncatalyzed processes, is rapid enough to pose an apparent threat to the stability of proteins and a possible rationale for the posttranslational N-acetylation of proteins that have been observed in higher organisms. The rate of DKP formation will also depend on the carbonyl ester protecting groups or the structures of the peptide-resin linkage in the solid-phase mode. Furthermore, cyclization is a concentration-independent reaction and demands the use of dilute solutions. ... 

The ruthenium catalysts have also been attached to various solid supports, including nolyethy-leneglycol (PEG), " mesoporous molecular sieves, " and Merrifield s peptide resin. " ... 

When solid-phase peptide synthesis was initially being developed, the question of whether or not a separate neutralization step is necessary was considered. Since it was known from the work of others that the chloride ion promotes racemization during the coupling step in classical peptide synthesis, and since we were deprotecting the Boc group with HC1, it seemed advisable to neutralize the hydrochloride by treatment with TEA and to remove chloride by filtration and washing. This short, additional step was simple and convenient and became the standard protocol. Subsequently, we became aware of three other reasons why neutralization was desirable (1) to avoid weak acid catalysis of piperazine-2,5-dione formation, 49 (2) to avoid acid-catalyzed formation of pyroglutamic acid (5-oxopyr-rolidine-2-carboxylic acid), 50 and (3) to avoid amidine formation between DCC and pro-tonated peptide-resin. The latter does not occur with the free amine. 

The insolubility of peptides is not limited to the solution phase and can occur during peptide synthesis on the solid phase where it manifests itself as the difficult sequence problem. On-resin peptide aggregation and poor peptide segment solubility are due to the formation of (3-sheet-like structures. Difficult sequences contain predominantly hydrophobic residues that consistently show incomplete acylation and a characteristic shrinkage of the peptide-resin during synthesis.111 Reversible backbone protection was introduced into Fmoc/ tBu SPPS after the study of synthesis failure during the assembly of difficult sequences1121 and is now routinely used to circumvent synthesis failure in SPPS. 

The peptide segment to be ligated to the polymer supported bound peptide was dissolved in 6M Gn-Cl, 0.1 M Na3P04 (pH 7.0), 0.1 M Met, and 1% PhSH (10-50 mM peptide), added to the N-terminal Cys-peptide-resin, and allowed to react at rt. Ligation reactions were complete in 2-24 h. 

The peptide-resin was washed with 8 M urea and 0.1 M Na3P04 (pH 7.0), and treated with 0.25 M NaOH in the same urea buffer (pH 12-14) for 2 min, washed with an equal volume of 0.25 M HC1 in urea buffer... 

The peptide resin Boc-Cys(Acm)-Arg(Tos)-Gly-Asp(OCy)-Phe-Cys(Acm)-MBHA-resin (0.5 mmol) was swollen with CH2a2 (10 mL) and suspended in DMF (10 mL). A soln of I2 (381 mg, 1.5 mmol) in MeOH (10 mL) was added and the mixture was stirred for 3h at rt. The resin was washed repeatedly with DMF and CH2Cl2/MeOH (1 1) and dried. It was then treated with HF/anisole (95 5) at 0°C for 1 h. After removal of the HF, the residue was washed repeatedly with Et20 and extracted with 5% AcOH, followed by lyophilization. The product was purified by HPLC and converted into its hydrochloride by repeated lyophilization from HCI-containing H20 yield 51 mg (16%) [a]D28 —68.8 (c 1.0, H20) the product was characterized by amino acid and MS analysis. 

When H-Cys(Acm)-Arg-Gly-Asp-Phe-Cys(Acm)-NH2, prepared from the same protected peptide resin by HF treatment, was subjected to I2 oxidation in soln, the peptide and other reagents could not be separated, and the desired product was not obtained. 

The peptide resin 1 was treated for 1 h with 50% TFA in CH2C12 containing 2% anisole, washed with CH2C12, neutralized with 5% DIPEA in CH2C12, washed with CH2C12, and then DMF. The product 2 was treated with a 5-fold molar excess of HOBt in DMF, followed after 20 min with a 5-fold molar excess of DCC in CH2C12. The mixture was adjusted with DIPEA to pH 7.5. After 24 h, the resin was drained and after a neutralization step with DIPEA, the cyclization was repeated for another 24 h. The extent of the cyclization to 3 was estimated to be about 95% by the ninhydrin test. 

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