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.     

Application of microwave irradiation to the synthesis of 14-helical beta-peptides

[structure: see text] We have evaluated the effects of microwave irradiation on the solid-phase synthesis of beta-peptides. Sequences designed to adopt the 14-helix, especially those containing the structure-promoting residue trans-2-aminocyclohexanecarboxylic acid (ACHC), suffer from poor synthetic efficiency under standard SPPS conditions. A comparison of microwave and conventional heating shows that both provide excellent synthetic results for shorter sequences; however, we identify a clear benefit from microwave irradiation for longer beta-peptides.     

Total synthesis of endothelin 1 by microwave-assisted solid phase method

<正>A microwave-assisted solid phase synthesis for endothelin 1 is presented.Reduced endothelin 1 was synthesized efficiently on Wang resin under microwave irradiation using Fmoc/tBu orthogonal protection strategy.The whole peptide was cleaved from the resin and two disulphide bridges were formed under air oxidation at room temperature.The purity and efficiency of synthesizing the peptide is much higher than other methods used before.     

Integrated SPPS on continuous-flow radial microfluidic chip

A novel integrated continuous-flow microfluidic system was designed and fabricated for solid phase peptide synthesis (SPPS) using conventional reactants. The microfluidic system was composed of a glass-based radial reaction chip, a diffluent chip, amino acid feeding reservoirs and continuous-flow reagent pathways. A tri-row cofferdam-fence structure was designed for solid phase supports trapping. Highly cross-linked, porous and high-loading 4-(hydroxymethyl)phenoxymethyl polystyrene (HMP) beads were prepared for microfluidic SPPS. The transfer losses, hazardous handling and time-consuming processes in traditional peptide cleavage steps were avoided by being replaced with the on-chip cleavage treatment. Six peptides from an antibody affinity peptide library against β-endorphin with different lengths and sequences were obtained simultaneously on the constructed continuous-flow microfluidic system within a short time. This microfluidic system is automatic, integrated, effective, low-cost, recyclable and environment-friendly for not only SPPS but also other solid phase chemical syntheses.     

Total synthesis of human urotension-Ⅱ by microwave-assisted solid phase method

Human urotension-Ⅱwas synthesized efficiently on Wang resin under microwave irradiation using Fmoc/tBu orthogonal protection strategy.Disulphide bridge was formed on solid phase with the irradiation of microwave,then the whole peptide was cleaved from the resin.The purity of crude peptide cyclized under microwave irradiation was higher than that under room temperature.     

Use of ionic liquid and microwave irradiation as a convenient, rapid and eco-friendly method for synthesis of novel optically active and thermally stable aromatic polyamides containing N-phthaloyl-l-alanine pendent group

An efficient, fast and easy method for synthesis of new optically active and thermally stable aromatic polyamides (PAs) containing pendent phthalimide group and l-alanine flexible side spacer using room temperature ionic liquid (RTIL) by microwave irradiation has been investigated. The results found that RTIL efficiently absorb microwave energy, thus leading to a very high heating rate. All the PAs showed excellent solubility and readily dissolved in various organic solvents. Thermogravimetric analysis (TGA) exhibited that polymers were stable, with 10% weight loss recorded above 373 and 418 °C in the nitrogen atmosphere. In order to see the efficiency of microwave irradiation, this method was compared with polycondensation of the same monomers in RTILs using conventional heating.     

Microwave synthesis of hybrid inorganic-organic porous materials: phase-selective and rapid crystallization

Microwave synthesis of two porous nickel glutarates was compared with conventional hydrothermal synthesis. The cubic nickel glutarate, [Ni20(C5H6O4)20(H2O)8] x 40 H2O (1), was synthesized by conventional electrical heating in several hours or days, depending on synthesis temperature. Crystallization was greatly accelerated by microwave irradiation, in which more stable, tetragonal nickel glutarate, [Ni22(C5H6O4)20(OH)4(H2O)10] x 38 H2O (2), was formed within a few minutes, suggesting the efficiency of the microwave technique in the synthesis of porous hybrid materials. The cubic phase 1 is formed preferentially at low pH, low temperature, and especially under conventional electrical heating. In contrast, the tetragonal phase 2 is obtained favorably at high pH, high temperature, and especially with microwave irradiation. This work demonstrates that the microwave method provides not only the very fast synthesis of a hybrid material, but also the possibility to discover a new porous hybrid material not yet identified by conventional hydrothermal synthesis. The hydrothermal formation of metal-organic hybrid materials in a matter of minutes is an important step towards developing commercially viable routes for producing this valuable class of materials.     

微波场对固态氧离子导体上的甲烷氧化偶朕的影响

研究了微波场下甲烷在具有Bi2O3结构的固态氧离子导体上氧化偶联反应行为．与常规加热条件下的反应结果相比较,微波辐照下的反应有如下特点;（1）在达到相同甲烷转化率时,微波辐照下所需床层温度要远低于常规加热条件下所需床层温度;（2）微波辐照下,甲烷氧化偶联产物中C2烃的选择性普遍较高,在低温区尤为突出．微波场下甲烷偶联产物乙烷、乙烯的再氧化得到一定程度的抑制,致使微波场下的甲烷氧化偶联反应通常有较低的烯／烷比．     

Revealing protein structures in solid-phase peptide synthesis by 13C solid-state NMR: evidence of excessive misfolding for Alzheimer's β

Solid-phase peptide synthesis (SPPS) is a widely used technique in biology and chemistry. However, the synthesis yield in SPPS often drops drastically for longer amino acid sequences, presumably because of the occurrence of incomplete coupling reactions. The underlying cause for this problem is hypothesized to be a sequence-dependent propensity to form secondary structures through protein aggregation. However, few methods are available to study the site-specific structure of proteins or long peptides that are anchored to the solid support used in SPPS. This study presents a novel solid-state NMR (SSNMR) approach to examine protein structure in the course of SPPS. As a useful benchmark, we describe the site-specific SSNMR structural characterization of the 40-residue Alzheimer's β-amyloid (Aβ) peptide during SPPS. Our 2D (13)C/(13)C correlation SSNMR data on Aβ(1-40) bound to a resin support demonstrated that Aβ underwent excessive misfolding into a highly ordered β-strand structure across the entire amino acid sequence during SPPS. This approach is likely to be applicable to a wide range of peptides/proteins bound to the solid support that are synthesized through SPPS.     

Solid-phase synthesis of lysobactin (katanosin B): insights into structure and function

The solid phase synthesis of the cyclic depsipeptide antibiotic lysobactin is described. The natural product was synthesized via a linear approach using mostly an Fmoc-strategy solid phase peptide synthesis (SPPS) with a single purification. A lysobactin analog has also been synthesized displaying nanomolar membrane disruption activity not seen with the natural product.     

An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis

An amine-derivatized DOTA has been used to modify the surface of a polymeric support for conventional solid phase peptide synthesis (SPPS) following standard Fmoc chemistry methods. This methodology was used to synthesize a peptide-DOTA conjugate that was demonstrated to be a PARACEST MRI contrast agent. Therefore, this synthesis methodology can facilitate Fmoc SPPS of molecular imaging contrast agents.     

Mixed-phase synthesis of glycopeptides using a N-peptidyl-2,4-dinitrobenzenesulfonamide-thioacid ligation strategy

A strategy for the solid phase peptide synthesis (SPPS) and coupling of N-peptidyl and N-glycopeptidyl 2,4-dinitrobenzenesulfonamides (dNBS) with C-terminal peptidyl thioacids has been developed. The resulting N-dDNBS peptides were coupled to generate longer peptides. Ligation reactions were complete within 15 to 20 min.     

The Studies of Microwave Effects on the Chemical Reactions

Microwave heating involves direct absorption of energy by functional groups that bear ionic conductivity or a dipole rotational-effect, and this energy is then released into the surrounding solution. This absorption of energy causes the functional groups involved to have higher reactivity to other surrounding reactants than when they are simply incubated with the reactants at the same temperature. In other word the enhanced rate of the reaction can be due to the reactant stirred by the molecular dipole rotation and molecules themselves acting as a stirring bar. In contrast to conventional heating, the salient feature of “dipole rotation” constitutes one efficient form of “molecular agitation” or “molecular stirring” many aspects of which can be explore in chemical reactions. We will discuss some of the useful applications of this “molecular agitation” by means of microwave irradiation. Using this unique technology, we have developed: 1) a method to control the cleavage sites of peptide bonds, especially those bonds connected to aspartic acid residues inside the native peptides and proteins, 2) a method to increase coupling efficiency in solid-phase peptide synthesis using a common microwave oven, 3) a novel procedure that increases the rate of alcalase-catalyzed reactions using microwave irradiation in peptide-bond formation with proline as a nucleophile and selective benzoylation of a pyranoside derivative, 4) a procedure to solubilize and hydrolyze retrograded starch, 5) a novel procedure to enhance the rate of saponification in a serum sample for very long chain fatty acid analysis.     

Microwave heating in solid-phase peptide synthesis

The highly refined organic chemistry in solid-phase synthesis has made it the method of choice not only to assemble peptides but also small proteins - mainly on a laboratory scale but increasingly also on an industrial scale. While conductive heating occasionally has been applied to peptide synthesis, precise microwave irradiation to heat the reaction mixture during coupling and N(α)-deprotection has become increasingly popular. It has often provided dramatic reductions in synthesis times, accompanied by an increase in the crude peptide purity. Microwave heating has been proven especially relevant for sequences which might form β-sheet type structures and for sterically difficult couplings. The beneficial effect of microwave heating appears so far to be due to the precise nature of this type of heating, rather than a peptide-specific microwave effect. However, microwave heating as such is not a panacea for all difficulties in peptide syntheses and the conditions may need to be adjusted for the incorporation of Cys, His and Asp in peptides, and for the synthesis of, for example, phosphopeptides, glycopeptides, and N-methylated peptides. Here we provide a comprehensive overview of the advances in microwave heating for peptide synthesis, with a focus on systematic studies and general protocols, as well as important applications. The assembly of β-peptides, peptoids and pseudopeptides are also evaluated in this critical review (254 references).     

A Diaminodiacid (DADA) Strategy for the Development of Disulfide Surrogate Peptides

Disulfide bond‐containing peptides are useful molecular scaffolds with diagnostic and therapeutic applications due to their good biological activity and good target selectivity, but their utility is sometimes limited by the lability of the disulfide moiety under reducing conditions and in the presence of disulfide bond isomerase. The development of disulfide surrogates with improved redox stability has been an area of ongoing research; and one possible strategy is based on a diaminodiacid (DADA) moiety, which can be used to synthesize the disulfide bond replacement peptides with precise structures and enhanced stability through automated solid‐phase peptide synthesis (SPPS). This review summarizes recent developments in the DADA‐based SPPS of peptide disulfide surrogates. Some representative applications and structural studies on the DADA‐based disulfide surrogates are described.     

Biomimetic studies on the mechanism of stereoselective lanthionine formation

Selenocysteine derivatives are useful precursors for the synthesis of peptide conjugates and selenopeptides. Several diastereomers of Fmoc-3-methyl-Se-phenylselenocysteine (FmocMeSec(Ph)) were prepared and used in solid phase peptide synthesis (SPPS). Once incorporated into peptides, the phenylselenide functionality provides a useful handle for the site and stereospecific introduction of E- or Z-dehydrobutyrine residues into peptide chains via oxidative elimination. The oxidation conditions are mild, can be performed on a solid support, and tolerate functionalities commonly found in peptides, including variously protected cysteine residues. Dehydropeptides containing unprotected cysteine residues undergo intramolecular stereoselective conjugate addition to afford cyclic lanthionines and methyllanthionines, which have the same stereochemistry as found in lantibiotics, a family of ribosomally synthesized and post-translationally modified peptide antibiotics. The observed stereoselectivity is shown to originate from a kinetic rather than a thermodynamic preference.     

Ultrasonication Improves Solid Phase Synthesis of Peptides Specific for Fibroblast Growth Factor Receptor and for the Protein-Protein Interface RANK-TRAF6

Considering our interest in the use of peptides as potential target-specific drugs or as delivery vectors of metallodrugs for various biomedical applications, it is crucial to explore improved synthetic methodologies to accomplish the highest peptide crude purity in the shortest time possible. Therefore, we compared “classical” fluorenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) with ultrasound(US)-assisted SPPS based on the preparation of three peptides, namely the fibroblast growth factor receptor 3(FGFR3)-specific peptide Pep1 (VSPPLTLGQLLS-NH₂) and the novel peptides Pep2 (RQMATADEA-NH₂) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH₂), which are being developed aimed at interfering with the intracellular protein-protein interaction(PPI) RANK-TRAF6. Our results demonstrated that US-assisted SPPS led to a 14-fold (Pep1) and 4-fold time reduction (Pep2) in peptide assembly compared to the “classical” method. Interestingly, US-assisted SPPS yielded Pep1 in higher purity (82%) than the “classical” SPPS (73%). The significant time reduction combined with high crude peptide purity attained prompted use to apply US-assisted SPPS to the large peptide Pep3, which displays a high number of hydrophobic amino acids and homooligo-sequences. Remarkably, the synthesis of this 25-mer peptide was attained during a “working day” (347 min) in moderate purity (approx. 49%). In conclusion, we have reinforced the importance of using US-SPPS towards facilitating the production of peptides in shorter time with increased efficacy in moderate to high crude purity. This is of special importance for long peptides such as the case of Pep3.     

Total synthesis of microcin B17 via a fragment condensation approach

The total synthesis of the 43 amino acid antibacterial peptide Microcin B17 (MccB17) is described. The natural product was synthesized via a convergent approach from a heterocycle-derived peptide and peptide thioester fragments prepared via Fmoc-strategy solid phase peptide synthesis (SPPS). Final assembly was achieved in an efficient manner using two Ag(I)-assisted peptide ligation reactions to afford MccB17 in excellent overall yield.     

Low-temperature synthesis of allyl dimethylamine by selective heating under microwave irradiation used for water treatment

Low-temperature synthesis of allyl dimethylamine (ADA) by selective heating under microwave irradiation (MI) used for water treatment is investigated. The effect of MI, ultrasound irradiation (UI) and conventional heating on yield of ADA, reaction time and the flocculation efficiency of polydiallyl dimethylammunion chloride (PDADMAC) prepared form ADA were studied. The results show that by selective heating at low temperature, MI not only increases yield of ADA and reduces reaction time, but also greatly enhances the flocculation efficiency of PDADMAC.     

Synthesis of fluorescein-labelled O-mannosylated peptides as components for synthetic vaccines: comparison of two synthetic strategies

Mannose-binding proteins on the surface of antigen-presenting cells (APCs) are capable of recognizing and internalizing foreign agents in the early stages of immune response. These receptors offer a potential target for synthetic vaccines, especially vaccines designed to stimulate T cells. We set out to synthesize a series of fluorescein-labelled O-mannosylated peptides using manual solid phase peptide synthesis (SPPS) on pre-loaded Wang resin, in order to test their ability to bind mannose receptors on human APCs in vitro. A flexible and reliable method for the synthesis of fluorescein-labelled O-mannosylated glycopeptides was desired in order to study their lectin-binding properties using flow cell cytometry. Two synthetic strategies were investigated: incorporation of a fluorescein label into the peptide chain via a lysine side chain epsilon-amino group at the final stage of standard Fmoc solid phase peptide synthesis or attachment of the fluorescein label to the N(alpha)-amino group of a lysine with further incorporation of a mannosylated peptide unit through the side chain N(epsilon)-amino group. The latter strategy proved more effective in that it facilitated SPPS by positioning the growing mannosylated peptide chain further removed from the fluorescein label.