Development of O-acyl isopeptide method

During over a decade of study on aspartic protease inhibitors and water-soluble prodrugs, in 2003, we discovered that the presence of an O-acyl instead of N-acyl residue within the peptide backbone significantly changed the secondary structure of the native peptide. In addition, the target peptide was subsequently generated by an O-N intramolecular acyl migration reaction. These findings led to the development of a novel method, called "O-acyl isopeptide method," for the synthesis of peptides containing difficult sequence. Further application of the method to Alzheimer's Abeta1-42 revealed that the O-acyl isopeptide of Abeta1-42 could be effectively synthesized and stored without spontaneous self-assembly. Intact monomer Abeta1-42 could then be obtained from the isopeptide under physiological experimental conditions. We named the O-acyl isopeptide as "Click Peptide," because of its "quick and easy one-way conversion" to the parent Abeta1- 42. Application of the click peptide has provided a new basis for the investigation of the biological functions of Abeta1-42 by inducible activation of its self-assembly. The O-acyl isopeptide method has further evolved as a general method for peptides synthesis with our recent developments of "O-acyl isodipeptide units" and "racemization-free segment condensation methodology." Isodipeptide units have enabled routine use of the O-acyl isopeptide method by avoiding the often difficult esterification reaction on resin. "Racemizationfree segment condensation methodology" has been achieved by employing N-segments possessing a C-terminal urethaneprotected O-acyl Ser/Thr residues. The synthesis of long peptides/proteins by racemization-free segment condensation has thus become possible at Ser/Thr residues instead of Cterminal Gly/Pro residues. As the O-acyl isopeptide method becomes more widely utilized, we have composed this review to facilitate its application for the production of peptides and proteins.     

Synthesis and functional studies of tuftsin analogs containing isopeptide bond

In the present paper a new approach is reported, to increase the resistance of tuftsin toward enzymatic cleavage by the introduction of an isopeptide bond into the molecule. The tetrapeptides H-Lys(Thr)-Pro-Arg-OH and H-Lys(Ala)-Pro-Arg-OH, the pentapeptides H-Thr-Lys(Ala)-Pro-Arg-OH, H-Thr-Lys(Thr)-Pro-Arg-OH and H-Ala-Lys(Ala)-Pro-Arg-OH and their For- and Boc-protected derivatives were built up by stepwise elongation of the chain, using conventional solution-phase methods. Preliminary experiments confirmed that from the Lys residue in position 2 of tuftsin the alpha-peptide bond between the Thr and Lys is cleaved with a significantly higher rate by leucine aminopeptidase than the epsilon-peptide bond. Several of the isopeptide derivatives increased to a higher extent the interleukin (IL-1) secretion by monocytes than tuftsin or [Ala1]-tuftsin.     

Synthesis and conformational analysis of AzAsx-containing oligopeptides

Summary For the sake of improving synthetic methods and evaluating the conformational perturbation induced by the substitution of AzAsx for the Asx residue in the cognate dipeptide R-CO-Asx-Pro-NHR, we prepared the AzAsx-dipeptide sequence by making use of the crystalline triphosgene. This reagent allows, in situ and under very mild experimental conditions, both the carbonylation and the activation of the properly substituted and N-protected hydrazine before coupling with the proline partner. With regard to conformational behaviour, the azadipeptide sequence displays a -fold, unlike the cognate dipeptide which adopts an Asx-turn.     

Synthesis and fungicidal activity of new N,O-acyl chitosan derivatives

Novel N,O-acyl chitosan (NOAC) derivatives were synthesized to examine their fungicidal activity against the gray mould fungus Botrytis cinerea (Leotiales: Sclerotiniaceae) and the rice leaf blast fungus Pyricularia oryzae (Teleomorph: Magnaporth grisea). The fungicidal activity was evaluated by the radial growth bioassay. NOAC derivatives were more active against the two plant pathogens than chitosan itself, and the effect was concentration dependent. Against B. cinerea, 4-chlorobutyryl chitosan (EC50=0.043%), decanoyl chitosan (EC50=0.044%), cinnamoyl chitosan (EC50=0.045%), and p-methoxybenzoyl chitosan (EC50=0.050%) were the most active (12-13-fold more active than chitosan). (Un)-substituted benzoyl chitosan derivatives were more active against B. cinerea than most of these with N,O-alkyl derivatives. Against P. oryzae chitosan derivatives with lauroyl, methoxy acetyl, methacryloyl and decanoyl were the most active.     

Non-pretreated O-acyl isopeptide of amyloid β peptide 1–42 is monomeric with a random coil structure but starts to aggregate in a concentration-dependent manner

An isopeptide of amyloid β peptide 1–42 (isoAβ42) was considered as a non-aggregative precursor molecule for the highly aggregative Aβ42. It has been applied to biological studies after several pretreatments. Here we report that isoAβ42 is monomeric with a random coil structure at 40 μM without any pretreatment. But we also found that isoAβ42 retains a slight aggregative nature, which is significantly weaker than that of the native Aβ42.     

Synthesis and optical studies of L-methionine oligopeptides in solution

A series of L -methionine oligomers [BOC-(Met)_n-OMe] (n = 2–7) and the corresponding diastereomeric di- and tripeptides were synthesized using the mixed anhydride method. Oligomers prepared in this manner were optically pure and were obtained in reasonable yield. Preliminary optical examination of the peptides suggests that secondary structures may begin forming in the pentamer or hexamer in trifluoroethanol. BOC-(Met)₄-OMe and BOC-(Met)₅-OMe were also synthesized using an insoluble resin containing BOC-L -methionine as the nitrophenol active ester.     

Alpha-glutamyl oligopeptides: preparation by the solid-phase method

The synthesis of the oligomeric peptides (Glu)_n–Phe(NO₂)–Phe (up to n = 4) by the solid-phase method is reported. Fractionation by ion-exchange column chromatography of the crude materials cleaved from the resin and subsequent amino acid analysis revealed that the desired peptides were obtained, although contaminated by several by-products whose number depends on the length of peptide chain and on the experimental conditions.     

Sequential oligopeptides. Synthesis and characterization of the oligopeptides and a polypeptide with the repeating sequence L-norvalyl-glycyl-L-proline

The synthesis and characterization of a series of oligopeptides (from the tripeptide to the octadecapeptide) with the repeating sequence L -norvalyl-glycyl-L -proline and a polytripeptide with this sequence are reported. The oligomers were synthesized step by step using the mixed anhydride method. All the products were chemically and optically pure. The polymer was prepared by the active ester method, using the p-nitrophenyl ester as the polymerizable tripeptide derivative. Good yield of relatively high average molecular-weight polymer was obtained. In the accompanying paper conformational investigations, both in solution and in the solid state, on the oligomers and the polymer are described.     

Synthesis and fungicidal activity of lipophylic N- and O-acyl derivatives of beta-hydroxy DL-alpha-amino acids

Synthesis of N- and O-acyl derivatives of DL-serine and threo-DL-phenylserine was accomplished by a regioselective acylation of the corresponding amino acid. The residues introduced into amino acid structure contain hydrophobic long chain or aromatic, namely lauroyl, myristoyl and phenylacetyl moieties. The fungicidal activity against six strains of fungi was studied. Several compounds were found to be effective against growth of fungi, and O-myristoyl-DL-serine 2 and N-phenylacetyl-threo-DL-phenylserine 8 completely inhibited the growth of the mycelium of the fungus Verticillium dahliae.     

Synthesis of sequential oligopeptides and polypeptide having the sequence of L-alanyl-L-leucylglycine

A series of sequential oligopeptides having simple nonpolar side chains, Nps-(L -Ala-L -Leu-Gly)_n- OEt has been prepared by a stepwise fragment-condensation method using Nps-L -alanyl-L -leucylglycine N-hydroxysuccinimide ester, which was prepared by the Nps-N-carboxy α-amino-acid-anhydride method. The success of the synthesis of the peptide having a high-molecular weight, such as octadecapeptide, results from the highest solubility of the tripeptide unit, L -alanyl-L -leucylglycine. The sequential polypeptide having the same tripeptide sequence was also prepared by polycondensation of the tripeptide N-hydroxy-succinimide ester.     

Phenylalanine oligopeptides: Synthesis,polarimetric and ultraviolet absorption studies

A series of l-phenylalanine oligomers was synthesized by the dicyclohexyl-carbodiimide and acylazide methods. The compounds prepared in this manner were chemically and optically pure and were obtained in good yield. A preliminary stereo-chemical analysis of these oligopeptides by means of polarimetry and ultraviolet absorption spectroscopy is reported. The vibrational fine structure observed in the ultraviolet spectra is also discussed.     

Synthesis and nmr conformational studies of polyoxyethylene-bound, alpha-deuterated glutamate oligopeptides

The syntheses of three series of glutamate oligopeptides attached to a macromolecular solubilizing polyoxyethylene (POE) group Boc-[Glu(OMe)]_n-OPOE, Ac-[Glu(OMe)]_n-OPOE, pGlu-[Glu(OMe)]_n?1-OPOE (n ? 1–7) and their various analogs specifically deuterated at individual α-CH positions using the liquid-phase method of peptide synthesis are described. It was shown that stepwise synthesis using the symmetrical anhydride gave homo-oligopeptides that are analytically pure. Fragment condensation methods using DCC-HOBt yield POE-peptides with POE-HOBt impurities but the peptide synthesis may be carried stoichiometrically with smaller quantities of amino acid derivatives. 360 MHz ¹H-nmr conformational studies of these homo-oligopeptides in DMSO-d₆ are presented. The α-deuterated peptides are shown to allow unequivocal homoligopeptide backbone NH assignments.     

A new method for the histochemical demonstration of O-acyl sugars in human colonic epithelial glycoproteins

Summary A new general method has been developed for the specific histochemical identification ofO-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose andN-acetylhexosamine with an ester substituenent(s) located on a potentialvicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA-Bh*-Bh-PAS) method] the initial PA-Bh treatment rendersvicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from bothO-acyl sugars andO-acyl sialic acids by saponification (KOH), sialic acidvicinal diols are selectively removed by the PA^*-Bh sequence andO-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections.Using the PA-Bh-KOH-PA^*-Bh-PAS method we have demonstrated, for the first time, thatO-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence ofO-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA^*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars andO-acyl sialic acids are selected.     

Synthesis of an actinomycin-related peptide lactone from the corresponding cyclic pentapeptide by N,O-acyl shift

The N,O-acyl shift was investigated as a method for the synthesis of an O-peptide or peptide lactone from a linear or cyclic peptide respectively. Protected derivatives of glycyl-L-threonine could be converted to O-peptides by the action of HCl/dioxane at room temperature and N-acylated under conditions which precluded a reverse O,N-acyl shift. For effecting N,O-acyl shift in cyclo(Thr-D-Val-Pro-Sar-MeAla) this reagent was unsatisfactory and p-toluenesulfonic acid in dioxane at 80 degrees was used instead. The resulting crystalline peptide lactone p-toluenesulfonate salt was N-acylated with 3-benzyloxy-4-methyl-2-nitrobenzoyl chloride to afford a known intermediate in the synthesis of 5,5'-MeAla actinomycin D. This approach constitutes a novel synthetic route to actinomycins and potentially to other peptide lactone antibiotics.     

Synthesis and interaction with metal ions of cyclic oligopeptides bearing carboxyl groups

Cyclic di- and tetrapeptides bearing carboxyl or carboxylate groups, cyclo[Glu(OBzl)-Glu(OMe)], cyclo[Glu-Glu(OMe)], cyclo(Glu-Glu), cyclo[Glu(OMe)-Pro)2, and cyclo(Glu-Pro)2, were synthesized and investigated on the intramolecular interaction of carboxyl side chains in the complexation with metal ions in relation with the conformation. The three kinds of cyclic dipeptides were found to take a flagpole boat conformation. Folded conformation of side chains was predominant for cyclo[Glu(OBzl)-Glu(OMe)] and cyclo[Glu-Glu(OMe)]. However, cyclo(Glu-Glu) took an unfolded conformation. Intramolecular interaction of carboxyl groups was observed neither in free state nor in complexation with metal ions. The intramolecular interaction of carboxyl groups was observed in the case of cyclo(Glu-Pro)2 in the absence of metal ions added. Cyclo[Glu(OMe)-Pro]2 and cyclo(Glu-Pro)2 formed a complex with Ca2+ and Ba2+ without participation of side chains.     

Isopeptide method: development of S‐acyl isopeptide method for the synthesis of difficult sequence‐containing peptides

A novel strategy for a more efficient synthesis of difficult sequence‐containing peptides, the S‐acyl isopeptide method, was developed and successfully applied. A model pentapeptide Ac–Val–Val–Cys–Val–Val–NH₂ was synthesized via its water‐soluble S‐acyl isopeptide using an S‐acyl isodipeptide unit, Boc–Cys(Fmoc–Val)–OH. An S‐acyl isopeptide possessing excellent water solubility could be readily and quantitatively converted to the native peptide via an S N intramolecular acyl migration reaction at pH 7.4. Thus, the S‐acyl isopeptide method provides a useful tool in peptide chemistry. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.