Chemoenzymatic Route for the Synthesis of (S)‐Moprolol,a Potential β‐Blocker

A biocatalytic route for the synthesis of a potential β‐blocker, (S)‐moprolol is reported here. Enantiopure synthesis of moprolol is mainly dependent on the chiral intermediate, 3‐(2‐methoxyphenoxy)‐propane‐1,2‐diol. Various commercial lipases were screened for the enantioselective resolution of (RS)‐3‐(2‐methoxyphenoxy)propane‐1,2‐diol to produce the desired enantiomer. Among them, Aspergillus niger lipase (ANL) was selected on the basis of both stereo‐ and regioselectivity. The optimized values of various reaction parameters were determined such as enzyme (15 mg/mL), substrate concentration (10 mM), organic solvent (toluene), reaction temperature (30 °C), and time (18 h).The optimized conditions led to achieving >49% yield with high enantiomeric excess of (S)‐3‐(2‐methoxyphenoxy)propane‐1,2‐diol. The lipase‐mediated catalysis showed regioselective acylation with dual stereoselectivity. Further, the enantiopure intermediate was used for the synthesis of (S)‐moprolol, which afforded the desired β‐blocker. Chirality 28:313–318, 2016. © 2016 Wiley Periodicals, Inc.     

High-throughput synthesis of conopeptides: a safety-catch linker approach enabling disulfide formation in 96-well format.

Conotoxins exhibit a high degree of selectivity and potency for a range of pharmacologically relevant targets. The rapid access to libraries of conotoxin analogues, containing multiple intramolecular disulfide bridges for use in drug development, can be a very labor intensive, multi-step task. This work describes a high-throughput method for the synthesis of cystine-bridged conopeptides.Peptides were assembled on a peptide synthesizer employing the Fmoc solid-phase strategy using a safety-catch amide linker (SCAL). Side-chain protecting groups were removed on solid phase before SCAL activation with ammonium iodide in TFA, finally releasing the peptide into the TFA solution. Disulfide bond formation was performed in the cleavage mixture employing DMSO.This improved method allows mixtures of oxidized peptides to be obtained in parallel directly from a peptide synthesizer. A single HPLC purification of the resulting crude oxidized material produced peptides of > 95% purity.     

Microwave‐assisted cleavage of Alloc and Allyl Ester protecting groups in solid phase peptide synthesis

Orthogonal protection of amino acid side chains in solid phase peptide synthesis allows for selective deprotection of side chains and the formation of cyclic peptides on resin. Cyclizations are useful as they may improve the activity of the peptide or improve the metabolic stability of peptides in vivo. One cyclization method often used is the formation of a lactam bridge between an amine and a carboxylic acid. It is desirable to perform the cyclization on resin as opposed to in solution to avoid unwanted side reactions; therefore, a common strategy is to use –Alloc and –OAllyl protecting groups as they are compatible with Fmoc solid phase peptide synthesis conditions. Alloc and –OAllyl may be removed using Pd(PPh₃)₄ and phenylsilane in DMF. This method can be problematic as the reaction is most often performed at room temperature under argon gas. It is not usually done at higher temperatures because of the fear of poisoning the palladium catalyst. As a result, the reaction is long and reagent–intensive. Herein, we report the development of a method in which the –Alloc/–OAllyl groups are removed using a microwave synthesizer under atmospheric conditions. The reaction is much faster, allowing for the removal of the protecting groups before the catalyst is oxidized, as well as being less reagent–intensive. This method of deprotection was tested using a variety of amino acid sequences and side chain protecting groups, and it was found that after two 5‐min deprotections at 38°C, all –Alloc and –OAllyl groups were removed with >98% purity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Resin comparison and fast automated stepwise conventional synthesis of human SDF‐1α

Human SDF‐1α contains 68 amino acids and is a member of the chemokine family of peptides. This long peptide was synthesized stepwise using classical conditions in 101 h. The reaction times were then reduced to deprotection times of 2 × 2 min and coupling times of 2 × 2.5 min, resulting in a total synthesis time of 22 h. The effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities was compared. A small portion of crude peptide was purified using an RP‐HPLC column and the mass of the final product was confirmed with MALDI‐TOF mass spectrometry. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Rapid solid-phase synthesis of a calmodulin-binding peptide using controlled microwave irradiation

A rapid and efficient microwave-assisted solid-phase synthesis method for the preparation of a nonapeptide using conventional Fmoc/Bu(t) orthogonal protection strategy is described. In this protocol, the coupling steps are performed within 5 min at 60 degrees C and the Fmoc-deprotection steps are completed within 3 min at 60 degrees C using a dedicated single-mode microwave peptide synthesizer utilizing temperature-controlled conditions. It is demonstrated that the model nonapeptide (containing the calmodulin-binding octapeptide sequence) is synthesized in a shorter time (approximately 3.5 h) and with high purity (>95%) under microwave irradiation conditions in comparison with a reference peptide that is obtained by standard methods at room temperature (within 11 h).     

Racemisation of N‐Fmoc phenylglycine under mild microwave‐SPPS and conventional stepwise SPPS conditions: attempts to develop strategies for overcoming this

We have been engaged in the microwave‐solid phase peptide synthesis (SPPS) synthesis of the phenylglycine (Phg)‐containing pentapeptide H‐Ala‐Val‐Pro‐Phg‐Tyr‐NH₂ (1) previously demonstrated to bind to the so‐called BIR3 domain of the anti‐apoptotic protein XIAP. Analysis of the target peptide by a combination of RP‐HPLC, ESI‐MS, and NMR revealed the presence of two diastereoisomers arising out of the racemisation of the Phg residue, with the percentage of the LLLDL component assessed as 49%. We performed the synthesis of peptide (1) using different microwave and conventional stepwise SPPS conditions in attempts to reduce the level of racemisation of the Phg residue and to determine at which part of the synthetic cycle the epimerization had occurred. We determined that racemisation occurred mainly during the Fmoc‐group removal and, to a much lesser extent, during activation/coupling of the Fmoc‐Phg‐OH residue. We were able to obtain the desired peptide with a 71% diastereomeric purity (29% LLLDL as impurity) by utilizing microwave‐assisted SPPS at 50 °C and power 22 Watts, when the triazine‐derived coupling reagent DMTMM‐BF₄ was used, together with NMM as an activator base, for the incorporation of this residue and 20% piperidine as an Fmoc‐deprotection base. In contrast, the phenylalanine analogue of the above peptide, H‐Ala‐Val‐Pro‐Phe‐Tyr‐NH₂ (2), was always obtained as a single diastereoisomer by using a range of standard coupling and deprotection conditions. Our findings suggest that the racemisation of Fmoc‐Phg‐OH, under both microwave‐SPPS and stepwise conventional SPPS syntheses conditions, is very facile but can be limited through the use of the above stated conditions. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

蛋白质体积对其亚细胞定位的影响

为了探索N(2)-L-丙氨酰-L-谷氨酰胺的合成工艺,确定最佳工艺以符合工业化生产的需求,以L-谷氨酰胺为原料,通过氨解反应合成L-丙氨酰-L-谷氨酰胺,并对反应条件及精制工艺进行优化.实验结果显示,当设定氨解反应温度为60℃,反应压力为0.5 MPa,反应时间为5.5h,以及氨水体积与α-D-氯丙酰-L-谷氨酰胺质量之比(V∶m)为1.5∶1时,将所得粗品用75％乙醇溶液进行精制,合成得到的N(2)-L-丙氨酰-L-谷氨酰胺经检测含量为99.65％,总收率约为55.77％.结果表明,经优化后的工艺简便安全、条件温和易控、生产周期短,适合工业化大量生产,且产品纯度、收率较高.     

Soluble polymer-supported synthesis of benzodiazepinones

Soluble polymer-supported synthetic method provides a highly efficient route for the construction of biologically important 1,5-benzodiazepin-2-ones. A library of N-substituted benzodiazepinones can be readily assembled utilizing S(N)Ar reaction, reduction of nitro group and one-pot cyclization following N-alkylation as the key step in the synthesis. All reactions in the sequence were performed at room temperature to facilitate the generation of libraries in a parallel fashion. The crude products were obtained in 80-95% yield with 60-96% HPLC purity.     

An HPLC‐ESMS study on the solid‐phase assembly of C‐terminal proline peptides

DKP formation is a serious side reaction during the solid‐phase synthesis of peptide acids containing either Pro or Gly at the C‐terminus. This side reaction not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides lacking the first amino acids. For the preparation of protected peptides using the Fmoc/tBu strategy, the use of a ClTrt‐Cl‐resin with a limited incorporation of the C‐terminal amino acid is the method of choice. The use of resins with higher loading levels leads to more impure peptide crudes. The use of HPLC‐ESMS is a useful method for analysing complex samples, such as those formed when C‐terminal Pro peptides are prepared by non‐optimized solid‐phase strategies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Peptide synthesis ‘in water’ by a solution‐phase method using water‐dispersible nanoparticle Boc‐amino acid

Regulatory pressure has compelled the chemical manufacturing industry to reduce the use of organic solvents in synthetic chemistry, and there is currently a strong focus on replacing these solvents with water. Here, we describe an efficient in‐water solution‐phase peptide synthesis method using Boc‐amino acids. It is based on a coupling reaction utilizing suspended water‐dispersible nanoparticle reactants. Using this method, peptides were obtained in good yield and with high purity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational analysis of sea cucumber (Caudina arenicola) C globin 94-97 fragment,Pro-Glu-Leu-Leu

The tetrapeptide Pro-Glu-Leu-Leu forms the 94-97 fragment of C globin in sea cucumber. 2% Butanediol dimethacrylate-cross linked polystyrene (2% BDDMA-PS), which had been optimized, was used for the synthesis of the tetrapeptide Pro-Glu-Leu-Leu. The peptide was synthesized by using Boc-amino acid strategy. The peptide purity was checked by RP-HPLC and the peptide was characterized by (1)H NMR spectroscopy and amino acid analysis. Conformation of the peptide was studied by 1D- and 2D- homonuclear (1)H NMR, in DMSO-d6 at 300K. The conformation of the synthetic tetrapeptide (extended backbone conformation) is not in agreement with that in C globin.     

Peptide purification by affinity chromatography based on α‐ketoacyl group chemistry

Significant advances have been achieved in the fields of peptide/protein synthesis, permitting the preparation of large, complex molecules. Shortcomings, however, continue to exist in the area of peptide purification. This paper details some studies we undertook to develop a new strategy for peptide purification based on a reactivity of α‐ketoacyl groups in peptides. The α‐ketoacyl peptide was generated from N^ε‐acyl‐lysyl‐peptide in the solid phase via a transamination reaction using glyoxylic acid and nickel(II) ion. Cleavage of the α‐ketoacyl group with o‐phenylenediamine gave the target peptide in an acceptable yield and purity. We first carried out a careful step‐by‐step optimization of the purification conditions using a model peptide. The strategy was then used in the purification of a transmembrane peptide that could not be effectively purified using a conventional RP‐HPLC system due to the strong hydrophobicity of the peptide and its high tendency to aggregate. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of biologically active analogs of the dodecapeptide a-factor mating pheromone of Saccharomyces cerevisiae

A number of dodecapeptides with the sequence YIIKGVFWDPAC were synthesized using solid phase peptide synthesis. The purity of the crude cleavage product was found to be directly related to the cysteine protecting group and the conditions employed for cleavage of the peptide from the resin. When 4-methyl-benzyl cysteine was used, complete deprotection was only achieved with low-high HF conditions at temperatures of 10 degrees-25 degrees, whereas milder conditions could be used for dodecapeptides containing ethyl cysteine or acetamidomethyl cysteine. In several syntheses the biological activity of the crude cleavage product greatly exceeded the biological activity of a purified major peptide component. The high activity found in the crude cleavage peptide was probably due to minor peptide side products in which the cysteine sulfur was alkylated by hydrophobic species during HF treatment. Two dodecapeptides, YIIKGVFWDPAC and YIIKGFWDPAC(Ethyl), had significant a-factor activity against MAT alpha strains of Saccharomyces cerevisiae. These peptides represent the first synthetic analogs with a-factor activity.     

Synthesis of peptide thioesters via an N–S acyl shift reaction under mild acidic conditions on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl auxiliary group

An efficient method of peptide thioester synthesis is described. The reaction is based on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl (Dmmb) auxiliary‐assisted N‐S acyl shift reaction after assembling a peptide chain by Fmoc‐solid phase peptide synthesis. The Dmmb‐assisted N‐S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S‐peptide with sodium 2‐mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on‐resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41‐mer peptide thioester, a phosphorylated peptide thioester and a 33‐mer peptide thioester containing a trimethylated lysine residue. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

High‐molecular‐weight poly(Gly‐Val‐Gly‐Val‐Pro) synthesis through microwave irradiation

In this study, we synthesized a polypeptide from its pentapeptide unit using microwave irradiation. Effective methods for polypeptide synthesis from unit peptides have not been reported. Here, we used a key elastin peptide, H‐GlyValGlyValPro‐OH (GVGVP), as the monomer peptide. It is difficult to obtain poly(Gly‐Val‐Gly‐Val‐Pro) (poly(GVGVP)) from the pentapeptide unit of elastin, GVGVP, via polycondensation. Poly(GVGVP) prepared from genetically recombinant Escherichia coli is a well‐known temperature‐sensitive polypeptide, and this temperature sensitivity is known as the lower critical solution temperature. When microwave irradiation was performed in the presence of various additives, the pentapeptide (GVGVP) polycondensation reaction proceeded smoothly, resulting in a product with a high molecular weight in a relatively good yield. The reaction conditions, like microwave irradiation, coupling agents, and solvents, were optimized to increase the reaction efficiency. The product exhibited a molecular weight greater than Mr 7000. Further, the product could be synthesized on a gram scale. The synthesized polypeptide exhibited a temperature sensitivity that was similar to that of poly(GVGVP) prepared from genetically recombinant E. coli. Therefore, this technique offers a facile and quick approach to prepare polypeptides in large amounts. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of Aβ[1‐42] and its derivatives with improved efficiency

It has been proved that the principal component of senile plaques is aggregates of β‐amyloid peptide (Aβ) in cases of one of the most common forms of age‐related neurodegenerative disorders, Alzheimer's disease (AD). Although the synthetic methods for the synthesis of Aβ peptides have been developed since their first syntheses, Aβ[1‐42] is still problematic to prepare. The highly hydrophobic composition of Aβ[1‐42] results in aggregation between resin‐bound peptide chains or intrachain aggregation which leads to a decrease in the rates of deprotection and repetitive incomplete coupling reactions during 9‐flurenylmethoxycarbonyl (Fmoc) synthesis. In order to avoid aggregation and/or disrupt internal aggregation during stepwise Fmoc solid phase synthesis and to improve the quality of crude products, several attempts have been made. Since highly pure Aβ peptides in large quantities are used in biological experiments, we wanted to develop a method for a rational synthesis of human Aβ[1‐42] with high purity and adequate yield. This paper reports a convenient methodology with a novel solvent system for the synthesis of Aβ[1‐42], its N‐terminally truncated derivatives Aβ[4‐42] and Aβ[5‐42], and Aβ[1‐42] labeled with 7‐amino‐4‐methyl‐3‐coumarinylacetic acid (AMCA) at the N‐terminus using Fmoc strategy. The use of 10% anisole in Dimethylformamide/Dichloromethane (DMF/DCM) can substantially improve the purity and yield of crude Aβ[1‐42] and has been shown to be an optimal coupling condition for the synthesis of Aβ[1‐42]. Anisole is a cheap and simple aid in the synthesis of ‘difficult sequences’ where other solvents are less successful in the prevention of aggregation during the synthesis. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.     

An optimized Boc synthesis of indolicidin

Indolicidin, an antimicrobial peptide from bovine neutrophils containing five tryptophan out of a total of 13 residues, has the highest molar proportion of tryptophan of any known peptide sequence and is thus considered a difficult synthetic target. Conventional Boc chemistry can be applied to the synthesis of indolicidin with an appropriate choice of scavenger mixtures, reaction times and temperatures at the crucial acidolytic cleavage and deprotection step. In particular, treatment with HF/p-cresol/p-thiocresol (90:7:3) for 40 min at –8 °C results in a crude product containing ca. 90% indolicidin, from which the target compound can be isolated in satisfactory yields and purity after reverse-phase purification. The main byproducts arising during the synthesis and cleavage steps have been identified by HPLC with on-line electrospray mass spectrometric detection.     

Hydrophobic benzyl amines as supports for liquid‐phase C‐terminal amidated peptide synthesis: application to the preparation of ABT‐510

C‐terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C‐terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C‐terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work‐up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag‐assisted liquid‐phase peptide synthesis as supports, leading to the total synthesis of ABT‐510 ( 2 ). Although an ethyl amide‐forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C‐terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Investigation of peptide thioester formation via N→Se acyl transfer

Native chemical ligation is widely used for the convergent synthesis of proteins. The peptide thioesters required for this process can be challenging to produce, particularly when using Fmoc‐based solid‐phase peptide synthesis. We have previously reported a route to peptide thioesters, following Fmoc solid‐phase peptide synthesis, via an N→S acyl shift that is initiated by the presence of a C‐terminal cysteine residue, under mildly acidic conditions. Under typical reaction conditions, we occasionally observed significant thioester hydrolysis as a consequence of long reaction times (~48 h) and sought to accelerate the reaction. Here, we present a faster route to peptide thioesters, by replacing the C‐terminal cysteine residue with selenocysteine and initiating thioester formation via an N→Se acyl shift. This modification allows thioester formation to take place at lower temperatures and on shorter time scales. We also demonstrate how application of this strategy also accelerates peptide cyclization, when a linear precursor is furnished with an N‐terminal cysteine and C‐terminal selenocysteine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.