N-Methylated Peptide Synthesis via Generation of an Acyl N-Methylimidazolium Cation Accelerated by a Brønsted Acid

The development of a robust amide-bond formation remains a critical aspect of N-methylated peptide synthesis. In this study, we synthesized a variety of dipeptides in high yields, without severe racemization, from equivalent amounts of amino acids. Highly reactive N-methylimidazolium cation species were generated in situ to accelerate the amidation. The key to success was the addition of a strong Brønsted acid. The developed amidation enabled the synthesis of a bulky peptide with a higher yield in a shorter amount of time compared with the results of conventional amidation. In addition, the amidation can be performed by using either a microflow reactor or a conventional flask. The first total synthesis of naturally occurring bulky N-methylated peptides, pterulamides I–IV, was achieved. Based on experimental results and theoretical calculations, we speculated that a Brønsted acid would accelerate the rate-limiting generation of acyl imidazolium cations from mixed carbonic anhydrides.     

N‐Methylated Peptide Synthesis via Generation of an Acyl N‐Methylimidazolium Cation Accelerated by a Brønsted Acid

The development of a robust amide‐bond formation remains a critical aspect of N‐methylated peptide synthesis. In this study, we synthesized a variety of dipeptides in high yields, without severe racemization, from equivalent amounts of amino acids. Highly reactive N‐methylimidazolium cation species were generated in situ to accelerate the amidation. The key to success was the addition of a strong Brønsted acid. The developed amidation enabled the synthesis of a bulky peptide with a higher yield in a shorter amount of time compared with the results of conventional amidation. In addition, the amidation can be performed by using either a microflow reactor or a conventional flask. The first total synthesis of naturally occurring bulky N‐methylated peptides, pterulamides I–IV, was achieved. Based on experimental results and theoretical calculations, we speculated that a Brønsted acid would accelerate the rate‐limiting generation of acyl imidazolium cations from mixed carbonic anhydrides.     

Application of serine- and threonine-derived cyclic sulfamidates for the preparation of S-linked glycosyl amino acids in solution- and solid-phase peptide synthesis

Cyclic sulfamidates were synthesized in 60% yield from L-serine and allo-L-threonine, respectively. These sulfamidates reacted with a variety of unprotected 1-thio sugars in aqueous bicarbonate buffer (pH 8) to afford the corresponding S-linked serine- and threonine-glycosyl amino acids with good diastereoselectivity (> or =97%) after hydrolysis of the N-sulfates. The serine-derived sulfamidate was incorporated into a simple dipeptide to generate a reactive dipeptide substrate that underwent chemoselective ligation with a 1-thio sugar to afford an S-linked glycopeptide. This sulfamidate was also incorporated into a peptide on a solid support in conjunction with solid-phase peptide synthesis. Chemoselective ligation of a 1-thio sugar with the cyclic sulfamidate was achieved on the solid support, followed by removal of the N-sulfate. Finally, the peptide chain of the resulting support-bound S-linked glycopeptide was extended using standard peptide synthesis procedures.     

Solid-phase synthesis of the cyclic lipononadepsipeptide [N-Mst(Ser1), d-Ser4, L-Thr6, L-Asp8, L-Thr9]syringotoxin

An optimized solid-phase strategy for the preparation of the cyclic lipononadepsipeptide [N-Mst(L-Ser1), D-Ser4, L-Thr6, L-Asp8, L-Thr9]syringotoxin is reported. The strategy is based on the use of a mild orthogonal protection scheme and the incorporation of the nonproteinogenic amino acid (Z)-Dhb into the peptide chain as the dipeptide Fmoc-Thr(tBu)-(Z)-Dhb-OH. The didehydrodipeptide was synthesized by a water-soluble carbodiimide-induced beta-elimination of a protected dipeptide containing a residue of Thr with its free hydroxy side chain unprotected.     

«Push-Pull»-Acetylene als Hilfsmittel zur Synthese von Peptiden

Peptide synthesis by Means of ‘Push-Pull’-Acetylenes ‘Push-pull’-acetylenes 1 are excellent reagents for peptide synthesis: Addition of ‘push-pull’-acetylenes to solutions of N-protected amino acids (or vice versa) gives enol esters 5 , which react selectively with the amino function of a second amino acid. In this way serine, tyrosine, 4-hydroxyproline, cysteine as well as histidine are linked to form dipeptide esters without protection of the second functional group (OH, SH or NH). The amount of racemization is very low. Similarly tri- as well as tetrapeptides are available. The versatility of the reaction is discussed.     

微通道连续流动高效绿色合成亮丙瑞林

开发了一种高效绿色的连续流动多肽合成方法,并成功应用于一种含有9个氨基酸的促性腺激素释放激素类似物(亮丙瑞林)的合成.该方法采用苄氧羰基(Cbz)保护氨基酸,在微通道反应器中实现高效偶联与水洗萃取除杂,并通过钯碳催化剂填充柱氢解反应实现快速洁净地脱除Cbz保护基,使原料和溶剂的消耗量大大减少,原子经济性大幅提高.该高效绿色合成方法将在多肽制药工业中得到更多应用.     

Application of DEPBT on the Synthesis of the Protected Dipeptides Containing Histidine with Unprotected Imidazole Group by Solution Method

3- (Diethoxyphosphoryloxy)- 1,2,3-benzotriazln-4 (3H)-one (DE-PBT) was an organophosphorus coupling reagent developed by our group. It was an effective coupling reagent for the synthesis of protected peptides containing Tyr, Ser and Thr with unprotected hydroxy group on their side chain. The further study of the synthesis of a series of protected dipeptides containing hisfidine with unprotected imidazole group using DEPBT is reported. During the synthetic procedure, the imidazole group of histidine did not need to be protected. When the carboxyl components were N-protected aromatic amino acids or basic amino acids, the yields were relatively high (63%--81%). However,when the carboxyl components were N-protected acidic amino acids, the yields were relatively low (47%--48%). The results expanded the application of DEPBT on the synthesis of bioactive peptides containing histidine.     

Enzyme-labile protecting groups in peptide synthesis: development of glucose- and galactose-derived urethanes

The development of the tetra-O-acetyl-D-glucopyranosyloxycarbonyl (AGlOC) and tetra-O-acetyl-beta-D-galactopyranosyloxycarbonyl (AGalOC) protecting groups, which are fully enzyme-labile, carbohydrate-derived urethanes, is described. The protected amino acids were easily synthesized and subsequently converted into a series of model dipeptides through classical peptide couplings. Cleavage of an alpha/beta-anomeric mixture of a model AGlOC dipeptide was achieved with a "one-pot" procedure in good yield. To gain a better understanding of the enzymatic deprotection reaction, the AGalOC group was removed through a two step biotransformation (lipase catalyzed deacetylation, followed by beta-galactosidase catalyzed glycosidic bond fragmentation). Under these very mild reaction conditions (aq. buffer pH7.0, 37 degrees C), the desired N-terminal, unprotected dipeptide conjugates were obtained. The methodology was further utilized for the synthesis of an advanced tetrapeptide model system.     

γ-Valerolactone (GVL): An eco-friendly anchoring solvent for solid-phase peptide synthesis

Due to the hazardous nature of CH₂Cl₂, regulatory authorities have imposed restrictions to minimize or even stop its use. It has therefore become imperative to identify environmentally benign solvents to replace it. Here we report on a bio derived solvent, γ-valerolactone, for the incorporation of the first amino acid onto p-alkoxybenzyl alcohol resin in solid-phase peptide synthesis. Satisfactory loading values (by a spectrophotometric method) were achieved. Furthermore, racemization and dipeptide formation were also checked and found to be acceptable.     

Chiral analysis of biogenic DL-amino acids derivatized by urethane - protected alpha-amino acid N-carboxyanhydride using capillary zone electrophoresis and micellar electrokinetic chromatography

A new analytical method for enantioselective separation of DL-amino acids derivatized by N-fluorenylmethoxycarbonyl-L-alanyl N-carboxyanhydride (FMOC-L-Ala-NCA) using capillary electrophoresis was developed. Separation parameters, such as composition and pH of the background electrolyte, and concentration of gamma-cyclodextrin (in capillary zone electrophoresis) and sodium dodecyl sulfate (in micellar electrokinetic chromatography) were optimized. The separation method was validated and it suits well for purity analysis. Detection limit of the method was 0.2% of the minor enantiomer in the major one. The level of racemization in coupling during solid-phase peptide synthesis was studied using capillary electrophoresis with gamma-cyclodextrin as a chiral selector. The anchorage of the first (C-terminal) amino acid derivative to the solid supports bearing the hydroxylic groups is the key step of the synthesis affecting the extent of its racemization. FMOC-L-phenylalanine was chosen as the suitable model amino acid derivative making it possible to study the degree of racemization of N-fluorenylmethoxycarbonyl-L-alanine-L-phenylalanine synthesized on different polymer resins, using the different condensation agents.     

Synthesis of functionalized guanidino amino acids

We report the synthesis of guanidino amino acids (GuAA), which are structurally related to Arg and resemble a dipeptide consisting of alpha- and gamma-amino acid with a guanidinium group in the main chain. The compounds are available with different protecting groups in gram amounts and are intended as synthetic building blocks for the construction of synthetic oxoanion or peptide receptors. Tyr, Trp or dansyl-functionalized Lys can be introduced as the alpha-amino acid part, which leads to luminescent GuAAs. The compounds signal carboxylate binding in MeOH, DMSO and buffered water by change of the emission intensity. The property may find use in the construction of chemosensors.     

1-Ethoxyethylidene, a new group for the stepwise SPPS of aminooxyacetic acid containing peptides

For more than a decade, the oxime ether ligation has proven to be one of the most efficient technique for the preparation of various peptide conjugates. However, despite numerous reports, the preparation of aminooxy-containing peptides is still hampered by N-overacylation of the NH-O function either during its incorporation or through the peptide-chain elongation. This restricts the introduction of protected-NH-O function at the last acylation step and prevents the use of standard solid-phase peptide synthesis (SPPS) procedures for the preparation of more complex aminooxy-peptides. We have studied the coupling of modified Fmoc-lysine containing either N-Boc- or N,N'-bis-Boc-protected aminooxyacetic acids (Aoa) during the elongation of the peptide chain and found that none of them is adequate. To circumvent this limitation, we propose to protect the Aoa moiety with a 1-ethoxyethylidene group (Eei) to provide 2-(1-ethoxyethylideneaminooxy)acetic acid building block. We showed that the Eei group is fully compatible with standard SPPS conditions and safely allows the multiple incorporation of the aminooxy functionality into the growing peptide. Since Eei-protected Aoa remains as flexible as normal amino acids in peptide synthesis, it may become the rule for the straightforward preparation of aminooxy peptides.     

Aziridine-mediated ligation and site-specific modification of unprotected peptides

A synthesis of aziridine-containing peptides via the Cu(II)-promoted coupling of unprotected peptide thioacids and N-H aziridine-2-carbonyl peptides is reported. The unique reactivity of the resulting N-acylated aziridine-2-carbonyl peptides facilitates their subsequent regioselective and stereoselective nucleophilic ring-opening to give unprotected peptides that are specifically modified at the ligation site. The aziridine-mediated peptide ligation concept is exemplified using H(2)O as the nucleophile, producing a Xaa-Thr linkage (where Xaa can be an epimerizable and hindered amino acid). The overall process is compatible with a variety of unprotected amino acid functionality, most notably the N-terminal and Lys side chain amines.     

Optimized ‘inverse activation’ methodology for esterification of hydroxyl-functionalized resins

Since the advent of the solid-phase method for peptide synthesis, the esterification of protected amino acid derivatives onto hydroxyl-functionalized resins has been problematic on many levels. Most methods for this reaction are attended by unacceptable levels of racemization and/or dipeptide formation, or require the use of expensive reagents with difficult handling properties. Herein, we describe a straightforward, generally-applicable method for the esterification of hydroxyl-functionalized resins, in high yield and complete stereochemical integrity.     

Visible light photoredox catalysed amidation of carboxylic acids with amines

A visible-light promoted photoredox catalysed, green one-pot approach for the amidation of carboxylic acids with amines has been developed for the synthesis of diverse aliphatic and aromatic amides. The proposed strategy is extendable also to biologically active amides and could represent a low-cost alternative to the common synthetic pathways. The developed strategy may hold great potential for a comprehensive display of biologically interesting peptide synthesis and amino acid modification.     

An efficient synthetic route to glycoamino acid building blocks for glycopeptide synthesis

[reaction: see text] Chemical glycopeptide synthesis requires access to gram quantities of glycosylated amino acid building blocks. Hence, the efficiency of synthesis of such building blocks is of great importance. Here, we report a fast and highly efficient synthetic route to Fmoc-protected asparaginyl glycosides from unprotected sugars in three steps with high yields. The glycosylated amino acids were successfully incorporated into target glycopeptides 7 and 8 by standard Fmoc solid-phase peptide synthesis.     

Extensive conformational searches of 13 representative dipeptides and an efficient method for dipeptide structure determinations based on amino acid conformers

Conformations of peptides are the basis for their property studies and the predictions of peptide structures are highly important in life science but very complex in practice. Here, thorough searches on the potential energy surfaces of 13 representative dipeptides by considering all possible combinations of the bond rotational degrees of freedom are performed using the density functional theory based methods. Careful analyses of the conformers of the 13 dipeptides and the corresponding amino acids reveal the connections between the structures of dipeptide and amino acids. A method for finding all important dipeptide conformers by optimizing a small number of trial structures generated by suitable superposition of the parent amino acid conformations is thus proposed. Applying the method to another eight dipeptides carefully examined by others shows that the new approach is both highly efficient and reliable by providing the most complete ensembles of dipeptide conformers and much improved agreements between the theoretical and experimental IR spectra. The method opens the door for the determination of the stable structures of all dipeptides with a manageable amount of effort. Preliminary result on the applicability of the method to the tripeptide structure determination is also presented. The results are the first step towards proving Anfinsen's hypothesis by revealing the relationships between the structures of the simplest peptide and its constituting amino acids. It implies that the structures of peptides are not only determined by their amino acid sequences, but also closely linked with the amino acid conformations. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009     

Chemical synthesis of a glycoprotein having an intact human complex-type sialyloligosaccharide under the Boc and Fmoc synthetic strategies

The chemical synthesis of complex glycoproteins is an ongoing challenge in protein chemistry. We have examined the synthesis of a single glycoform of monocyte chemotactic protein-3 (MCP-3), a CC-chemokine that consists of 76 amino acids and one N-glycosylation site. A three-segment native chemical ligation strategy was employed using unprotected peptides and glycopeptide. Importantly, the synthesis required the development of methods for the generation of sialylglycopeptide-alphathioesters. For the sialylglycopeptide-alphathioester segment, we examined and successfully implemented approaches using Fmoc-SPPS and Boc-SPPS. To avoid use of hydrogen fluoride, the Boc approach utilized minimal side chain protection and direct thiolysis of the resin bound peptide. Using these strategies, we successfully synthesized a glycoprotein having an intact and homogeneous complex-type sialyloligosaccharide.     

Solid-phase synthesis of difficult peptide sequences at elevated temperatures: a critical comparison of microwave and conventional heating technologies

The Fmoc/t-Bu solid-phase synthesis of three difficult peptide sequences (a 9-mer, 15-mer, and 24-mer) was performed using N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole as coupling reagent on polystyrene, Tentagel, and ChemMatrix resins. In order to obtain an insight into the specific role of the elevated temperature and/or the electromagnetic field for peptide syntheses carried out using microwave irradiation, peptide couplings and Fmoc-deprotection steps were studied under microwave and conventionally heated conditions at the same temperature. While room temperature couplings/deprotections generally produced the difficult peptides in rather poor quality, excellent peptide purities were obtained using microwave heating at a temperature of 86 degrees C for both the coupling and deprotection steps in only 10 and 2.5 min reaction time, respectively. While for most amino acids no significant racemization was observed, the high coupling temperatures led to considerable levels of racemization for the sensitive amino acids His and Cys. It was demonstrated for all three peptide sequences that when performing the coupling/deprotection steps at the same reaction temperature using conventional heating, nearly identical results in terms of both peptide purity and racemization levels were obtained. It therefore appears that the main effect of microwave irradiation applied to solid-phase peptide synthesis is a purely thermal effect not related to the electromagnetic field.     

Random peptide synthesis using immobilized enzymes in high concentrations of organic solvents

Trypsin, leucine aminopeptidase, and carboxypeptidase B were separately immobilized on controlled pore glass and reacted with a dipeptide substrate in high concentrations of either acetone or 1-propanol. Hydrolytic activity was demonstrated and evidence for the possible synthesis of peptide polymer is presented. Directed synthesis using amino acids and blocked amino acids as substrates was not successful.