The 'O-acyl isopeptide method' for the synthesis of difficult sequence-containing peptides: application to the synthesis of Alzheimer's disease-related amyloid beta peptide (Abeta) 1-42.

An efficient 'O-acyl isopeptide method' for the synthesis of difficult sequence-containing peptides was applied successfully to the synthesis of amyloid beta peptide (Abeta) 1-42 via a water-soluble O-acyl isopeptide of Abeta1-42, i.e. '26-O-acyl isoAbeta1-42' (6). This paper describes the detailed synthesis of Abeta1-42 focusing on the importance of resin selection and the analysis of side reactions in the O-acyl isopeptide method. Protected '26-O-acyl isoAbeta1-42' peptide resin was synthesized using 2-chlorotrityl chloride resin with minimum side reactions in comparison with other resins and deprotected crude 26-O-acyl isoAbeta1-42 was easily purified by HPLC due to its relatively good purity and narrow elution with reasonable water solubility. This suggests that only one insertion of the isopeptide structure into the sequence of the 42-residue peptide can suppress the unfavourable nature of its difficult sequence. The migration of O-acyl isopeptide to intact Abeta1-42 under physiological conditions (pH 7.4) via O--N intramolecular acyl migration reaction was very rapid and no other by-product formation was observed while 6 was stable under storage conditions. These results concluded that our strategy not only overcomes the solubility problem in the synthesis of Abeta1-42 and can provide intact Abeta1-42 efficiently, but is also applicable in the synthesis of large difficult sequence-containing peptides at least up to 50 amino acids. This synthesis method would provide a biological evaluation system in Alzheimer's disease research, in which 26-O-acyl isoAbeta1-42 can be stored in a solubilized form before use and then rapidly produces intact Abeta1-42 in situ during biological experiments.     

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.     

Effect of the acyl groups on O-->N acyl migration in the water-soluble prodrugs of HIV-1 protease inhibitor

To improve the low water-solubility of HIV-1 protease inhibitors KNI-272, -279 and -727, we previously reported the water-soluble prodrugs of these inhibitors based on O-->N intramolecular acyl migration reaction. These prodrugs were rapidly converted to the corresponding parent drugs under physiological conditions. To understand the steric and electrostatic effects of O-acyl moiety on the migration rate, we examined several types of prodrug. A remarkably slow migration was observed in the benzoyl-type prodrugs, and Hammett plot of migration rate constants of p-substituted benzoyl-type prodrugs gave a linear free energy relationship.     

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.     

Development of first photoresponsive prodrug of paclitaxel

A prodrug of paclitaxel which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (O-acyl isoform of paclitaxel) has been synthesized. The prodrug was selectively converted to isotaxel by visible light irradiation (430 nm) with the cleavage of coumarin. Finally, paclitaxel was released by subsequent spontaneous O-N intramolecular acyl migration.     

Synthesis of oligopeptides through O-acyl isopeptide method

The O-acyl isopeptide method has recently gained attention as an efficient protocol for the synthesis of 'difficult sequence' peptides. Herein, synthesis of three oligopeptides of different length, a pentapeptide Gly-Leu-Leu-Ser-Val, a heptapeptide fragment 285-291 of transmembrane (M7-24-T40) Ala-Val-Leu-Ser-Leu-Pro-Leu and a decapeptide, Gly-Leu-Leu-Ser-Val-Leu-Gly-Ser-Val-Ala were demonstrated in solution phase by employing O-acyl isopeptide method. The peptides were established through an efficient pH triggered intramolecular O→N acyl migration under physiological conditions. The reactions were clean and complete in appreciable length of time.     

Water-soluble prodrugs of dipeptide HIV protease inhibitors based on O-->N intramolecular acyl migration: Design, synthesis and kinetic study

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-727, a potent small-sized dipeptide-type HIV-1 protease inhibitor consisting of an Apns-Dmt core (Apns; allophenylnorstatine, Dmt; (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid) as inhibitory machinery. These prodrugs contained an O-acyl peptidomimetic structure with an ionized amino group leading to an increase in water-solubility, and were designed to regenerate the corresponding parent drugs based on the O-->N intramolecular acyl migration reaction via a five-membered ring intermediate at the alpha-hydroxy-beta-amino acid residue, that is Apns. The synthetic prodrug 3a improved the water-solubility (13 mg/mL) more than 8000-fold in comparison with the parent compound, which is the practically acceptable value as water-soluble drug. Furthermore, to understand the structural effects of the O-acyl moiety on the migration rate, we evaluated several phenylacetyl-type and benzoyl-type prodrugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly under aqueous conditions from slightly acidic to basic pH at 37 degrees C.     

'O-Acyl isopeptide method' for peptide synthesis: Solvent effects in the synthesis of Abeta1-42 isopeptide using 'O-acyl isodipeptide unit'.

'O-Acyl isopeptide method' is an efficient synthetic method for peptides. We designed 'O-acyl isodipeptide units', Boc-Ser/Thr(Fmoc-Xaa)-OH, as important building blocks to enable routine use of the O-acyl isopeptide method. In the synthesis of an Abeta1-42 isopeptide using O-acyl isodipeptide unit Boc-Ser(Fmoc-Gly)-OH, a side reaction, resulting in the deletion of Ser(26) in the O-acyl isopeptide structure, was noticed during coupling of the unit. We observed that the side reaction occurred during the activation step and was solvent-dependent. In DMF or NMP, an intramolecular side reaction, originating from the activated species of the unit, occurred during the activation step. In non-polar solvents such as CHCl(3) or CH(2)Cl(2), the side reaction was less likely to occur. Using CH(2)Cl(2) as solvent in coupling the unit, the target Abeta1-42 isopeptide was synthesized with almost no major side reaction.     

O‐Acyl isopeptide method: development of an O‐acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aβ1‐42 isopeptide

The O‐acyl isopeptide method was developed for the efficient preparation of difficult sequence‐containing peptide. Furthermore, development of the O‐acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid‐phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Chemistry of alpha-hydroxymethylserine: problems and solutions

Further improvements related to the synthesis of peptides containing HmS are presented. Efficient synthetic protocols have been developed to synthesize "difficult" sequences containing a C-terminal HmS residue, MeA-HmS or consecutive HmS. Preparative methods for orthogonal N- and/or C-protected HmS(Ipr) derivatives are described. Their compatibility with standard solution or solid-phase peptide chemistry protocols allows synthetic flexibility toward HmS-containing peptides. In the synthesis of the sterically hindered dipeptides with the C-terminal HmS(Ipr) residue, HATU proves the highest efficiency, as compared with the fluoride and PyBroP/DMAP coupling methods. The HATU method also outperforms the fluoride activation in the solid-phase assembly of HmS homosequence. Specific protocols are described to overcome an undesired cyclization to diketopiperazines that occurs during the removal of Fmoc from dipeptides with the C-terminal HmS(Ipr) or HmS residues, thus precluding their C-->N elongation. The successful protocols involve: (i) the 2+1 condensation using mixed anhydride activation yielding the desired product with the highest optical integrity or (ii) use of the 2-chlorotrityl resin as a solid support sterically suppressing the undesired cleavage due to diketopiperazine formation. The latter approach allows the mild conditions of peptide cleavage from solid support, preserving the isopropylidene protection and minimizing the undesired N-->O-acyl migration that was observed under prolonged acid treatment used for cleaving the HmS peptide from the Wang resin.     

The depsipeptide method for solid‐phase synthesis of difficult peptides

After about one century of peptide chemistry, the main limitation to the accessibility of peptides and proteins via chemosynthesis is the arising of folding and aggregation phenomena. This is true not only for sequences above a critical length but also for several biologically relevant substrates that are relatively short yet form either highly folded structures (e.g. WW domains) or fibrils and aggregates after final deprotection (β‐amyloid peptide). Such so‐called difficult sequences may be more easily obtained via their corresponding depsipeptides (O‐acyl isopeptides), ester isomers that are often easier to assemble and purify, and are smoothly converted to the parent amides under mild conditions. The depsipeptide method is the most recent technique to improve the outcome of difficult syntheses, applicable to sequences containing residues of serine or threonine. A brief overview is presented about chemical aspects of the method, the steps that have been undertaken for its optimization, and the evaluation of its efficiency. Further applications of analogous principles to other critical topics in peptide synthesis such as condensation of peptide segments and solid‐phase synthesis of naturally occurring cyclodepsipeptides are addressed as well. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A biomimetic strategy in the synthesis and fragmentation of cyclic protein.

This paper describes a simple biomimetic strategy to prepare small cyclic proteins containing multiple disulfide bonds. Our strategy involves intramolecular acyl transfer reactions to assist both the synthesis and fragmentation of these highly constrained cyclic structures in aqueous solution. To illustrate our strategy, we synthesized the naturally occurring circulin B and cyclopsychotride (CPT), both consisting of 31 amino acid residues tightly packed in a cystine-knot motif with three disulfide bonds and an end-to-end cyclic form. The synthesis of these small cyclic proteins can be achieved by orthogonal ligation of free peptide thioester via the thia zip reaction, which involves a series of reversible thiol-thiolactone exchanges to arrive at an alpha-amino thiolactone, which then undergoes an irreversible, spontaneous ring contraction through an S,N-acyl migration to form the cyclic protein. A two-step disulfide formation strategy is employed for obtaining the desired disulfide-paired products. Partial acid hydrolysis through intramolecular acyl transfer of X-Ser, X-Thr, Asp-X, and Glu-X sequences is used to obtain the assignment of the circulins disulfide bond connectives. Both synthetic circulin B and CPT are identical to the natural products and, thus, the total synthesis confirms the disulfide connectivity of circulin B and CPT contain a cystine-knot motif of 1-4, 2-5, and 3-6. In general, our strategy, based on the convergence of chemical proteolysis and aminolysis of peptide bonds through acyl transfer, is biomimetic and provides a useful approach for the synthesis and characterization of large end-to-end cyclic peptides and small proteins.     

Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs.

The amyloid A4 or beta peptide is a major component of extracellular amyloid deposits that are a characteristic feature of Alzheimer's disease. We synthesized a series of peptide analogs of the A4/beta peptide which are progressively longer at their carboxyl termini, including 42- and 39-residue peptides which represent the major forms of the A4/beta peptide in senile plaque and the hereditary cerebral hemorrhage with amyloidosis form, respectively. All peptides tested, beta 1-28 through beta 1-42, formed amyloid-like fibrils and previously unreported thin sheet-like structures which stained with thioflavin T and Congo Red. The solubility of beta 1-42 and shorter peptides was pH and concentration dependent, with a broad insolubility profile in the pH range of 3.5-6.5 and at concentrations above 0.75 mg/ml. Only peptides of 42 residues or longer were significantly insoluble at pH 7.4. beta 1-47 and beta 1-52 peptides are highly insoluble in aqueous media but are soluble at 40 mg/ml in the alpha helix-promoting solvent, 1,1,1,3,3,3-hexafluoro-2-propanol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the beta 1-42 peptide migrates as a series of higher molecular mass aggregates whereas shorter peptides migrate as monomers. Aggregation is also dependent on pH, peptide concentration, and time of incubation in aqueous medium. These results indicate that the length of the hydrophobic carboxyl terminus of the A4/beta peptide is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition.     

Synthesis of an O‐acyl isopeptide by using native chemical ligation in an aqueous solvent system

O‐Acyl isopeptides, in which the N‐acyl linkage on the hydroxyamino acid residue (e.g. Ser and Thr) is replaced by an O‐acyl linkage, generally suppress unfavorable aggregation properties derived from the corresponding parent peptides. Here, we report the synthesis of an O‐acyl isopeptide of 34‐mer pyroGlu‐ADan (2), a component of amyloid deposits in hereditary familial Danish dementia, by using native chemical ligation. Native chemical ligation of pyroGlu¹‐ADan(1‐21)‐SCH₂CH₂SO₃^?Na⁺ (3) and Cys²²‐O‐acyl isopeptide (4), in which the amino group of the Ser²⁹ residue at the isopeptide moiety was protected by an allyloxycarbonyl group, proceeded well in an aqueous solvent to yield a ligated O‐acyl isopeptide (5). Subsequent disulfide bond formation and deprotection of the allyloxycarbonyl group followed by HPLC purification gave 2 with a reasonable overall yield. 2 was converted to the parent peptide 1 via an O‐to‐N acyl migration reaction. The sequential method, namely (i) native chemical ligation of the O‐acyl isopeptide, (ii) HPLC purification as the O‐acyl isopeptide form, and (iii) O‐to‐N acyl migration into the desired polypeptide, would be helpful to solve problems with HPLC purification of hydrophobic polypeptides in the process of chemical protein synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A switchable stapled peptide

The O‐N acyl transfer reaction has gained significant popularity in peptide and medicinal chemistry. This reaction has been successfully applied to the synthesis of difficult sequence‐containing peptides, cyclic peptides, epimerization‐free fragment coupling and more recently, to switchable peptide polymers. Herein, we describe a related strategy to facilitate the synthesis and purification of a hydrophobic stapled peptide. The staple consists of a serine linked through an amide bond formed from its carboxylic acid function and the side chain amino group of diaminopropionic acid and through an ester bond formed from its amino group and the side chain carboxylic acid function of aspartic acid. The α‐amino group of serine was protonated during purification. Interestingly, when the peptide was placed at physiological pH, the free amino group initiated the O‐N shift reducing the staple length by one atom, leading to a more hydrophobic stapled peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Polymerization site in the beta chain of fibrin: mapping of the B beta 1-55 sequence

The formation of a fibrin clot occurs through binding of putative complementary sites, called fibrin polymerization sites, located in the NH2- and COOH-terminal domains of fibrin monomer molecules. In this study, we have investigated the structure of the NH2-terminal fibrin polymerization site by using fibrinogen-derived peptides and fragments. Fibrinogen was digested with Crotalus atrox protease III, to two major molecular species: a Mr 325,000 derivative (Fg325) and a peptide of Mr 5000. The peptide and its thrombin-cleavage product were purified by ion-exchange and reverse-phase HPLC; the authenticity of the B beta 1-42 and beta 15-42 peptides, respectively, was confirmed by amino acid sequencing. Since Fg325 had decreased thrombin coagulability, we addressed the question of whether the peptide B beta 1-42 contained a fibrin polymerization site. In order to identify and map the site, the peptides B beta 1-42 and beta 15-42 were tested for their ability to inhibit fibrin monomer polymerization. In addition the following peptides prepared by chemical synthesis were also tested: beta 15-18, beta 15-26, beta 24-42, beta 40-54, beta 50-55, and alpha 17-19-Pro. While B beta 1-42 had no inhibitory activity, the peptide devoid of fibrinopeptide B, beta 15-42, was a strong inhibitor. The peptides beta 15-18, beta 15-26, and beta 15-42 decreased the rate of fibrin polymerization by 50% at a molar excess of the peptide to fibrin monomer of 500, 430, and 50, respectively. The peptides beta 24-42, beta 40-54, and beta 50-55 were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and conformation of an analog of the helix‐loop‐helix domain of the Id1 protein containing the O‐acyl iso‐prolyl‐seryl switch motif

Synthetic peptides reproducing the helix‐loop‐helix (HLH) domains of the Id proteins fold into highly stable helix bundles upon self‐association. Recently, we have shown that the replacement of the dipeptide Val‐Ser at the loop–helix‐2 junction with the corresponding O‐acyl iso‐dipeptide leads to a completely unfolded state that only refolds after intramolecular O → N acyl migration. Herein, we report on an Id HLH analog based on the substitution of the Pro‐Ser motif at the helix‐1–loop junction with the corresponding O‐acyl iso‐dipeptide. This analog has been successfully synthesized by solid‐phase Fmoc chemistry upon suppression of DKP formation. No secondary structure could be detected for the O‐acyl iso‐peptide before its conversion into the native form by O → N acyl shift. These results show that the loop–helix junctions are determinant for the folded/unfolded state of the Id HLH domain. Further, despite the high risk of DKP formation, peptides containing O‐acyl iso‐Pro‐Ser/Thr units are synthetically accessible by Fmoc chemistry. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Neurotoxicity and oxidative stress in D1M-substituted Alzheimer's A beta(1-42): relevance to N-terminal methionine chemistry in small model peptides

Small model peptides containing N-terminal methionine are reported to form sulfur-centered-free radicals that are stabilized by the terminal N atom. To test whether a similar chemistry would apply to a disease-relevant longer peptide, Alzheimer's disease (AD)-associated amyloid beta-peptide 1-42 was employed. Methionine at residue 35 of this 42-mer has been shown to be a key amino acid residue involved in amyloid beta-peptide 1-42 [A beta1-42]-mediated toxicity and therefore, the pathogenesis of AD. Previous studies have shown that mutation of the methionine residue to norleucine abrogates the oxidative stress and neurotoxic properties of A beta(1-42). In the current study, we examined if the position of methionine at residue 35 is a criterion for toxicity. In doing so, we tested the effects of moving methionine to the N-terminus of the peptide in a synthetic peptide, A beta(1-42)D1M, in which methionine was substituted for aspartic acid at the N-terminus of the peptide and all subsequent residues from D1 to L34 were shifted one position towards the carboxy-terminus. A beta(1-42)D1M exhibited oxidative stress and neurotoxicity properties similar to those of the native peptide, A beta(1-42), all of which are inhibited by the free radical scavenger Vitamin E, suggesting that reactive oxygen species may play a role in the A beta-mediated toxicity. Additionally, substitution of methionine at the N-terminus by norleucine, A beta(1-42)D1Nle, completely abrogated the oxidative stress and neurotoxicity associated with the A beta(1-42)D1M peptide. The results of this study validate the chemistry reported for short peptides with N-terminal methionines in a disease-relevant peptide.