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).     

Enhancement of Chemical Reactions by Microwave Irradiation

Microwaves are low electromagnetic energy which induce molecular perturbation by stimulation of ionic diffusion and by enhancement of dipole rotation without causing rearrangement of molecular structures. Microwave energy irradiated on the samples in various liquid media is lost or absorbed to the samples by the following two mechanisms: ionic conduction and dipole rotation. These two effects take place simultaneously to account for the phenomenon of rapid heating associated with many practical applications of microwave irradiation. In contrast to conventional heating, the salient feat ure of “dipole rotation” constitutes one efficient form of “molecular agitation” or “molecular stirring” which can be explored for many aspects in chemical reactions. We will discuss some of the useful application of “molecular agitation” by means of microwave irradiation such as: 1. rapid hydrolysis of proteins and peptides, 2. selective hydrolysis of aspartyl peptide bond, 3. the racemization of amino acids, 4. rapid hydrolysis of sugars, 5. continuous-flow process of microwave reactions, and 6. the enhancement of coupling reactions in peptide synthesis.     

微波作用下大位阻氨基酸与H-Pro-CTC树脂的高效缩合

微波反应下, 运用新型固相肽合成反应器, 深入研究了五种大位阻氨基酸与H-Pro-CTC树脂(CTC树脂, 2-氯三苯甲基氯树脂)的缩合反应. 使用三次缩合的策略, 分别在DMF/NMP/THF (V∶V∶V＝1∶1∶1), NMP/DMSO/THF (V∶V∶V＝4∶1∶1), DMF/DMSO/THF (V∶V∶V＝4∶1∶1)混合溶剂中缩合一次, 每次缩合反应的最优条件为: 缩合试剂HBTU、氨基酸浓度7 mmol/L、微波辐射3 min、反应温度35 ℃、维持时间3 min, 与传统方法相比, 氨基酸的用量大大减少, 其过量倍数从5倍降低为2倍, 缩合反应速率提高了16倍以上. 五种大位阻氨基酸与H-Pro-CTC树脂的缩合率都提高到80%以上.     

Hot or not—the influence of elevated temperature and microwave irradiation on the solid phase synthesis of an affibody

Despite the advances of solid phase peptide synthesis (SPPS) the synthesis of long peptides is still challenging. Microwave irradiation and conventional heating are considered to improve the efficiency of SPPS. It has been shown that conventional heating and heating by microwave irradiation improves the efficiency of solid phase synthesis of peptides that are prone to aggregation as compared to the synthesis at room temperature. In this Letter, the influence of elevated temperature and microwave irradiation on the homogeneity of the synthesis product of a 58-mer peptide affibody has been compared. A detailed analysis by high resolution HPLC and LC-MS mass spectrometry using a high-mass resolution Orbitrap Exactive mass spectrometer was performed. This study revealed that neither thermal heating nor microwave heating improves the yield and purity of the crude product as compared to the synthesis at room temperature. In contrast, the formation of undesirable side products rather increased by microwave irradiation. These results indicate that neither heating nor microwave enhancement of solid phase synthesis does allow a significant improvement of peptide sequences with a low aggregation potential.     

Microwave-assisted, zinc-mediated peptide coupling of N-benzyl-α,α-disubstituted amino acids

Incorporation of the extremely hindered amino acid N-benzylaminoisobutyric acid into dipeptides under microwave irradiation with commercial zinc dust is described. A comparative survey of various methodologies for hindered peptide couplings was undertaken. The optimised coupling conditions were free from racemisation and applicable to other hindered amino acids.     

微波辐射下L-氨基酸的快速消旋方法

微波辐射作用下的L-氨基酸消旋反应是一种新的氨基酸消旋方法，具有对环境友好的优点。本文报道了在微波辐射下，以1．Omol／L氢氧化钠水溶液替代有机酸作为反应溶剂，水杨醛为催化剂，水杨醛与L-氨基酸的摩尔比为0．1，L-氨基酸可以快速消旋；消旋反应随微波辐射功率的提高而加快，在600W时已接近最大反应速率。同时也讨论了微波作用下L-氨基酸的消旋反应机理。     

Use of N-trifluoroacetyl-protected amino acid chlorides in peptide coupling reactions with virtually complete preservation of stereochemistry

The use of protected amino acid chlorides for peptide coupling reactions has long been avoided due to the extensive racemization that commonly occurs during either the acid chloride formation or the coupling reaction itself. Conditions are described which allow N-trifluoroacetyl-protected amino acid chlorides to be generated in high purity and with high retention of stereochemical integrity. Control of temperature is the predominant factor in controlling racemization, and rapid formation of acid chlorides under low temperature can be conveniently achieved using Vilsmeier reagent. Stereochemical integrity is further maintained when coupling of N-trifluoroacetyl acid chlorides is carried out with amino acid esters under Schotten-Baumann conditions using specific controls on pH, temperature, and agitation. Second order rate constants for coupling and the azlactone formation associated with racemization were measured to be 4260 and 3.6 L/mol s, respectively. This high rate differential allows for the reaction to be run with a minimum excess of amine ester, and makes it suitable for continuous processing. The applicability of the preferred coupling conditions to a range of amino acid couplings is described.     

Peptide-Chain Elongation Using Unprotected Amino Acids in a Micro-Flow Reactor

Conventional peptide synthesis requires a deprotection step after each amidation step, which decreases synthetic efficiency. Therefore, peptide synthesis using unprotected amino acids is considered an ideal approach. Here, we report peptide chain elongation using unprotected amino acids via a mixed carbonic anhydride. Micro-flow technology enabled rapid mixing of an organic layer containing a protected amino acid or dipeptide and an aqueous layer containing an unprotected amino acid or dipeptide to accelerate the desired amidation, and this approach successfully suppressed undesired racemization/epimerization (≤0.4 %). Various di-, tri-, and tetra-peptides were obtained in good to high yields. This is the first report on peptide chain elongation that proceeds without severe racemization from unprotected amino acids using inexpensive, nonexplosive, less wasteful, and less toxic reagents.     

On-resin cyclization of a head-to-tail cyclopeptide using an allyldimethylsilyl polystyrene resin pre-loaded by metathesis

Here, we report the solid-phase synthesis of a 17-mer cyclopeptide which is expected to have anti-angiogenic properties. The peptidic synthesis is performed on an allyldimethylsilyl polystyrene support loaded by metathesis with a conveniently functionalized d-Tyrosine amino acid. The linear peptide was assembled by standard Fmoc chemistry and on-resin cyclization was enabled after selective deprotection of the C-terminal group with 2% hydrazine/DMF at room temperature. Final cleavage was realized under mild acidic conditions allowing to obtain a cyclopeptide under partially protected form.     

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.     

Aryldithioethyloxycarbonyl (Ardec): a new family of amine protecting groups removable under mild reducing conditions and their applications to peptide synthesis

The development of phenyldithioethyloxycarbonyl (Phdec) and 2-pyridyldithioethyloxycarbonyl (Pydec) protecting groups, which are thiol-labile urethanes, is described. These new disulfide-based protecting groups were introduced onto the epsilon-amino group of L-lysine; the resulting amino acid derivatives were easily converted into N alpha-Fmoc building blocks suitable for both solid- and solution-phase peptide synthesis. Model dipeptide(Ardec)s were prepared by using classical peptide couplings followed by standard deprotection protocols. They were used to optimize the conditions for complete thiolytic removal of the Ardec groups both in aqueous and organic media. Phdec and Pydec were found to be cleaved within 15 to 30 min under mild reducing conditions: i) by treatment with dithiothreitol or beta-mercaptoethanol in Tris.HCl buffer (pH 8.5-9.0) for deprotection in water and ii) by treatment with beta-mercaptoethanol and 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) in N-methylpyrrolidinone for deprotection in an organic medium. Successful solid-phase synthesis of hexapeptides Ac-Lys-Asp-Glu-Val-Asp-Lys(Ardec)-NH2 has clearly demonstrated the full orthogonality of these new amino protecting groups with Fmoc and Boc protections. The utility of the Ardec orthogonal deprotection strategy for site-specific chemical modification of peptides bearing several amino groups was illustrated firstly by the preparation of a fluorogenic substrate for caspase-3 protease containing the cyanine dyes Cy 3.0 and Cy 5.0 as FRET donor/acceptor pair, and by solid-phase synthesis of an hexapeptide bearing a single biotin reporter group.     

Rapid Protein Hydrolysis by Microwave Irradiation Using Heat-Resistant Teflon-Pyrex Tubes

A rapid peptide-bond hydrolysis by means of microwave irradiation is introduced for the facile preparation of protein hydrolysates used for amino acid analysis. The optimal hydrolysis condition has been determined using several enzymes with known amino acid compositions. The effects of hydrolysis time on the recovery of various labile and hydrophobic amino acids are also exemplified in the microwave heating of standard amino acids. The method has been applied to the complete amino acid analysis with a single nonvolatile solvent of methanesulfonic acid with good recovery of tryptophan and half-cystine. It provides a radical expedition of protein and peptide hydrolysis via commercial microwave ovens and specially-designed Teflon-Pyrex tubes, circumventing the tedious procedures using vacuum-sealed pyrex lubes heating at 110°C for more than 24 h. This novel type of microwave chemistry associated with rapid peptide-bond cleavage is of great potential in the automation of the complete process of amino acid analysis starting from the preparation of protein hydrolysates.     

An activated O --> N acyl transfer auxiliary: efficient amide-backbone substitution of hindered "difficult" peptides

Overcoming the phenomenon known as "difficult" synthetic sequences has been a major goal in solid-phase peptide synthesis for over 30 years. In this work the advantages of amide backbone-substitution in the solid-phase synthesis of "difficult" peptides are augmented by developing an activated N(alpha)()-acyl transfer auxiliary. Apart from disrupting troublesome intermolecular hydrogen-bonding networks, the primary function of the activated N(alpha)()-auxiliary was to facilitate clean and efficient acyl capture of large or beta-branched amino acids and improve acyl transfer yields to the secondary N(alpha)()-amine. We found o-hydroxyl-substituted nitrobenzyl (Hnb) groups were suitable N(alpha)()-auxiliaries for this purpose. The relative acyl transfer efficiency of the Hnb auxiliary was superior to the 2-hydroxy-4-methoxybenzyl (Hmb) auxiliary with protected amino acids of varying size. Significantly, this difference in efficiency was more pronounced between more sterically demanding amino acids. The Hnb auxiliary is readily incorporated at the N(alpha)()-amine during SPPS by reductive alkylation of its corresponding benzaldehyde derivative and conveniently removed by mild photolysis at 366 nm. The usefulness of the Hnb auxiliary for the improvement of coupling efficiencies in the chain-assembly of difficult peptides was demonstrated by the efficient Hnb-assisted Fmoc solid-phase synthesis of a known hindered difficult peptide sequence, STAT-91. This work suggests the Hnb auxiliary will significantly enhance our ability to synthesize difficult polypeptides and increases the applicability of amide-backbone substitution.     

Solid phase synthesis of aromatic oligoamides: application to helical water-soluble foldamers

Synthetic helical aromatic amide foldamers and in particular those based on quinolines have recently attracted much interest due to their capacity to adopt bioinspired folded conformations that are highly stable and predictable. Additionally, the introduction of water-solubilizing side chains has allowed to evidence promising biological activities. It has also created the need for methods that may allow the parallel synthesis and screening of oligomers. Here, we describe the application of solid phase synthesis to speed up oligomer preparation and allow the introduction of various side chains. The synthesis of quinoline-based monomers bearing protected side chains is described along with conditions for activation, coupling, and deprotection on solid phase, followed by resin cleavage, side-chain deprotection, and HPLC purification. Oligomers having up to 8 units were thus synthesized. We found that solid phase synthesis is notably improved upon reducing resin loading and by applying microwave irradiation. We also demonstrate that the introduction of monomers bearing benzylic amines such as 6-aminomethyl-2-pyridinecarboxylic acid within the sequences of oligoquinolines make it possible to achieve couplings using a standard peptide coupling agent and constitute an interesting alternative to the use of acid chloride activation required by quinoline residues. The synthesis of a tetradecameric sequence was thus smoothly carried out. NMR solution structural studies show that these alternate aminomethyl-pyridine residues do not perturb the canonical helix folding of quinoline monomers in protic solvents, contrary to what was previously observed in nonprotic solvents.     

Efficient synthesis of complex glycopeptides based on unprotected oligosaccharides

N-Glycopeptides containing 1 to 4 trisaccharide chains, with the carbohydrates vicinal to each other in the multivalent glycopeptides, were efficiently synthesized by using the glycosylated Fmoc-asparagine as a key building block. While the couplings of amino acids with glycopeptides could be achieved in the homogeneous solutions in N-methylpyrrolidinone (NMP) to give excellent yields, all products were conveniently isolated from the reaction mixtures through a precipitation method by using the free carbohydrate chains as phase tags. Commercially available pentafluorophenyl (Pfp) esters of amino acids were employed for the glycopeptide elongation. Longer glycopeptides were constructed by means of a highly convergent synthetic design that is based on the coupling of glycopeptide/peptide fragments. Hydrogen bond interactions between free oligosaccharides were proposed to explain the exceptionally high efficiency of the couplings between two glycosylated building blocks.     

Microwave-assisted traceless synthesis of thiohydantoin

An efficient, microwave-assisted method for the liquid-phase combinatorial synthesis of 3,5-disubstituted-thiohydantoin has been developed. Fmoc-protected amino acids were coupled with HO-PEG-OH and after deprotection, reacted with various isothiocyanates in microwave cavity. The PEG bound thiourea compounds underwent base mediated cyclization/cleavage step by microwave flash heating. The desired products were then liberated from the soluble matrix in good yield and purity under microwave exposure.     

Efficient,Optimized Applications of p-Nitrophenyl Active Ester Temporarily Protecting Groups Together with Simultaneous Activation in Synthesis of Special Glu and Lys Peptides

Benzyloxycarbonyl-glutamylpeptide p-nitrophenylesters were prepared from protected amino acids, e.g.: p-nitrophenyl-glutamate as carboxyl component and aminoacid or peptide p-nitrophenylesters as amino components by different kinds of peptide coupling methods. Mixed carbonic anhydride and azide methods gave good results. The p-nitrophenylesters existed as temporary protecting groups, so the peptide couplings proceeded together with simultaneous activation. The conditions and applications of the procedure are discussed. The peptides having one or more active ester groups were used to form amides by their aminolysis with derivatives of ethylamine or were polycondensated (after deprotection) to obtain polypeptides.     

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.     

Application of microwave method to the solid phase synthesis of pseudopeptides containing ester bond

A new procedure was developed for reducing the reaction time and improving the yield of esterification reaction in solid phase synthesis of pseudopeptides containing an ester bond by utilizing microwave irradiation. We selected a pseudodipeptide (Fmoc-LysΨ[COO]Leu-NH₂) and optimized the microwave-assisted esterification reaction in solid phase synthesis using Fmoc chemistry. For this, microwave-assisted esterification reactions with different reaction time, temperature, and solvents were performed using 1,3-diisopropylcarbodiimide (DIC) as the coupling reagent. We synthesized several pseudodipeptides containing an ester bond by using the optimized microwave irradiation method. The purity and yield of the pseudodipeptides synthesized in this way were better than those obtained without microwave irradiation. Furthermore, we applied this methodology for synthesizing pseudopeptides (6- and 12-mer) corresponding to the α helical peptide. The microwave-assisted esterification reaction afforded the target pseudopeptides with high yield (∼80%) and purity within 12 min, whereas the reaction without microwave irradiation afforded the target compound with poor yield (∼45%) and low purity.