固相同步多重肽合成

本文综述了近十年来在固相肽合成基础上发展的各种同步多重合成新技术。它们的应用可进一步提高肽合成的效率、降低成本,并将促进许多肽的结构-活性关系研究。因此,将对激素-受体间作用的机理研究、对分子免疫学的发展及研究新的肽类诊断试剂或疫苗均可创造较好的条件。     

Alpha-keto amide peptides: a synthetic strategy to resin-bound peptide isosteres for protease inhibitor screening on solid support

A synthetic strategy for the formation of resin-bound internal alpha-keto amide peptides suitable for protease inhibitor screening on solid support is presented. This general approach is based on the incorporation of alpha-keto amide building blocks during solid-phase peptide synthesis (SPPS). Such dipeptidyl building blocks were accessible using the acylcyanophosphorane methodology. The acid-labile alpha-keto carbonyl functionality was protected as a 1,3-dithiolane derivative. This protective group is fully compatible with standard SPPS reaction conditions and can be efficiently removed with N-bromosuccinimide in 10% aqueous acetone. The alpha-keto amide peptides were assembled on SPOCC-1500 resin and were characterized with high-resolution magic angle spinning (HR-MAS) NMR on bead. The methodology was evaluated and tested with a variety of building blocks containing natural and nonnatural amino acid moieties.     

Efficient Building Blocks for Solid-Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications

Specific spin labeling allows the site-selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the Fmoc-protection group, which makes them applicable for the use in solid-phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments employing these building-blocks in a TCE solution show enhancement factors of up to 26 for ¹H and ¹³C (¹H→¹³C cross-polarization). To proof the viability of the presented building-blocks for insertion of the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly was synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for ¹H and 13.9±0.5 for ¹³C (direct polarization).     

Towards a fully synthetic MUC1-based anticancer vaccine: efficient conjugation of glycopeptides with mono-, di-, and tetravalent lipopeptides using click chemistry

The membrane‐bound tumor‐associated glycoprotein MUC1 is aberrantly glycosylated in cancer cells compared with normal cells, and is therefore considered an attractive target for cancer immunotherapy. However, tumor‐associated glycopeptides from MUC1 do not elicit a sufficiently robust immune response. Therefore, antitumor vaccines were developed, which consist of MUC1 glycopeptides as the B epitopes and immune‐stimulating toll‐like receptor 2 (TLR 2) lipopeptide ligands. These fully synthetic vaccine candidates were prepared by solid‐phase synthesis of the MUC1 glycopeptides. The Pam₃Cys lipopeptide, also synthesized on solid‐phase, was C‐terminally coupled to oligovalent lysine cores, which N‐terminally incorporate O‐propargyl oligoethylene glycol acyl side chains. The MUC1 glycopeptides and lipopeptide lysine constructs were then conjugated by click chemistry to give oligovalent synthetic vaccines. Oligovalent glycopeptide–lipopeptide conjugates are considered more immunogenic than their monovalent analogues.     

Convergent solid phase peptide synthesis. v. synthesis of the 1-4, 32-34, and 53-59 protected segments of the toxin ii of androctonus australis hector.

Two protected peptide segments corresponding to the sequence 32-34 and 53-59 of toxin II of the north African scorpion Androctonus australis Hector have been synthesized on a photolabile Nbb-resin using the TFA-labile Boc -amino protection and HF-labile side chain protecting groups. A third protected peptide corresponding to segment 1-4 has been synthesized on the same resin but with a t-butyl group for β protection of aspartic acid and a Z group on the lysine side chain. For this last segment a combination of Boc and Fmoc groups for -amino protection has been used successfully on the Nbb-resin. After photolysis the crude peptides have been treated by solvent extraction and semi-preparative HPLC to yield highly purified segments. These syntheses show the flexibility of the convergent solid phase approach and how segments with different and independent protecting groups can be assembled by solid phase peptide procedure.     

Convergent chemo-enzymatic synthesis of mannosylated glycopeptides; targeting of putative vaccine candidates to antigen presenting cells

The combination of solid phase peptide synthesis and endo-β-N-acetylglucosaminidase (ENGase) catalysed glycosylation is a powerful convergent synthetic method allowing access to glycopeptides bearing full-length N-glycan structures. Mannose-terminated N-glycan oligosaccharides, produced by either total or semi-synthesis, were converted into oxazoline donor substrates. A peptide from the human cytomegalovirus (CMV) tegument protein pp65 that incorporates a well-characterised T cell epitope, containing N-acetylglucosamine at specific Asn residues, was accessed by solid phase peptide synthesis, and used as an acceptor substrate. High-yielding enzymatic glycosylation afforded glycopeptides bearing defined homogeneous high-mannose N-glycan structures. These high-mannose containing glycopeptides were tested for enhanced targeting to human antigen presenting cells (APCs), putatively mediated via the mannose receptor, and for processing by the APCs for presentation to human CD8+ T cells specific for a 9-mer epitope within the peptide. Binding assays showed increased binding of glycopeptides to APCs compared to the non-glycosylated control. Glycopeptides bearing high-mannose N-glycan structures at a single site outside the T cell epitope were processed and presented by the APCs to allow activation of a T cell clone. However, the addition of a second glycan within the T cell epitope resulted in ablation of T cell activation. We conclude that chemo-enzymatic synthesis of mannosylated glycopeptides enhances uptake by human APCs while preserving the immunogenicity of peptide epitopes within the glycopeptides, provided those epitopes are not themselves glycosylated.     

Silica precipitation with synthetic silaffin peptides

Silaffins are highly charged proteins which are one of the major contributing compounds that are thought to be responsible for the formation of the hierarchically structured silica-based cell walls of diatoms. Here we describe the synthesis of an oligo-propyleneamine substituted lysine derivative and its incorporation into the KXXK peptide motif occurring repeatedly in silaffins. N(ε)-alkylation of lysine was achieved by a Mitsunobu reaction to obtain a protected lysine derivative which is convenient for solid phase peptide synthesis. Quantitative silica precipitation experiments together with structural information about the precipitated silica structures gained by scanning electron microscopy revealed a dependence of the amount and form of the silica precipitates on the peptide structure.     

Quaternary β2,2‐Amino Acids: Catalytic Asymmetric Synthesis and Incorporation into Peptides by Fmoc‐Based Solid‐Phase Peptide Synthesis

β‐Amino acid incorporation has emerged as a promising approach to enhance the stability of parent peptides and to improve their biological activity. Owing to the lack of reliable access to β^2,2‐amino acids in a setting suitable for peptide synthesis, most contemporary research efforts focus on the use of β³‐ and certain β^2,3‐amino acids. Herein, we report the catalytic asymmetric synthesis of β^2,2‐amino acids and their incorporation into peptides by Fmoc‐based solid‐phase peptide synthesis (Fmoc‐SPPS). A quaternary carbon center was constructed by the palladium‐catalyzed decarboxylative allylation of 4‐substituted isoxazolidin‐5‐ones. The N?O bond in the products not only acts as a traceless protecting group for β‐amino acids but also undergoes amide formation with α‐ketoacids derived from Fmoc‐protected α‐amino acids, thus providing expeditious access to α‐β^2,2‐dipeptides ready for Fmoc‐SPPS.     

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.     

Sustainable Peptide Synthesis Enabled by a Transient Protecting Group

The growing interest in synthetic peptides has prompted the development of viable methods for their sustainable production. Currently, large amounts of toxic solvents are required for peptide assembly from protected building blocks, and switching to water as a reaction medium remains a major hurdle in peptide chemistry. We report an aqueous solid‐phase peptide synthesis strategy that is based on a water‐compatible 2,7‐disulfo‐9‐fluorenylmethoxycarbonyl (Smoc) protecting group. This approach enables peptide assembly under aqueous conditions, real‐time monitoring of building block coupling, and efficient postsynthetic purification. The procedure for the synthesis of all natural and several non‐natural Smoc‐protected amino acids is described, as well as the assembly of 22 peptide sequences and the fundamental issues of SPPS, including the protecting group strategy, coupling and cleavage efficiency, stability under aqueous conditions, and crucial side reactions.     

A Tandem In Situ Peptide Cyclization through Trifluoroacetic Acid Cleavage

We present a new approach for peptide cyclization during solid phase synthesis under highly acidic conditions. Our approach involves simultaneous in situ deprotection, cyclization and trifluoroacetic acid (TFA) cleavage of the peptide, which is achieved by forming an amide bond between a lysine side chain and a succinic acid linker at the peptide N‐terminus. The reaction proceeds via a highly active succinimide intermediate, which was isolated and characterized. The structure of a model cyclic peptide was solved by NMR spectroscopy. Theoretical calculations support the proposed mechanism of cyclization. Our new methodology is applicable for the formation of macrocycles in solid‐phase synthesis of peptides and organic molecules.     

N-Alkyl cysteine-assisted thioesterification of peptides

A new method for the preparation of peptide thioester by the post-solid phase peptide synthesis (SPPS) approach was developed. A series of N-alkyl cysteine derivatives were prepared and used as the C-terminus residue of the peptides prepared by the Fmoc SPPS. The synthetic peptides released from resin by TFA were readily converted to the peptide thioester in aqueous 3-mercaptopropionic acid (MPA) without significant side reactions.     

Preparation of a core-shell type MBHA resin and its application for solid-phase peptide synthesis

MBHA (4-methylbenzhydrylamine) resin has been extensively used as a solid support for the synthesis of peptide amides. Herein, we prepared the core-shell-type MBHA resin by benzotriazole-catalyzed amidoalkylation and partial hydrolysis. The core-shell structure of the MBHA resin was confirmed by two-photon microscopy and its synthetic performance in solid-phase peptide synthesis (SPPS) was evaluated.     

EPR investigation of the influence of side chain protecting groups on peptide-resin solvation of the Asx and Glx model containing peptides

In spite of all progressive efforts aiming to optimize SPPS, serious problems mainly affecting the assembly of aggregating sequences have persisted. Following the study intended to unravel the complex solvation phenomenon of peptide-resin beads, the XING and XAAAA model aggregating segments were labeled with a paramagnetic probe and studied via EPR spectroscopy. Low and high substituted resins were also comparatively used, with the X residue being Asx or Glx containing the main protecting groups used in the SPPS. Notably, the cyclo-hexyl group used for Asp and Glu residues in Boc-chemistry induced greater chain immobilization than its tert-butyl partner-protecting group of the Fmoc strategy. Otherwise, the most impressive peptide chain immobilization occurred when the large trytil group was used for Asn and Gln protection in Fmoc-chemistry. These surprising results thus seem to stress the possibility of the relevant influence of the amino-acid side chain protecting groups in the overall peptide synthesis yield.     

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.     

Synthesis of an anion-binding amino acid

We achieved the synthesis of a derivative of phenylalanine with a diazamacrocycle on its side chain by macrocyclization of a dichloride on l-DOPA. We also report its incorporation into peptide structures by solid phase peptide synthesis which will lead to the development of artificial anion channels.     

Site-selective conjugation of thiols with aziridine-2-carboxylic acid-containing peptides

The synthesis and convergent site-selective conjugation of aziridine-2-carboxylic acid-containing peptides with thiols, both in solution and on solid support, are described. The synthesis and use of FmocAzyOH in this capacity demonstrate both the efficient incorporation and tolerance of the Azy moiety in multistep Fmoc solid-phase peptide synthesis (SPPS), as well as the competence of solution and on-bead ligation through a highly regioselective base-promoted aziridine ring-opening process.     

Pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction

In one of the peptide condensation methods termed thioester method, an amino protecting group is required in the lysine side chain. In this study, to investigate the efficiency of the pyruvoyl group as an amino protecting group, we synthesized N^α-fluorenylmethoxycarbonyl (Fmoc)-N^ε-pyruvoyl-lysine and introduced it into peptides and glycopeptides by the ordinary Fmoc-based solid phase peptide synthesis. The pyruvoyl peptide could be condensed with a peptide thioester by the thioester method, and this protecting group was easily removed by o-phenylenediamine treatment without significant side reactions.     

Synthesis of linear tuftsin analogues modified at the ε-amino group of lysine

In this Letter, eight tuftsin analogues, seven of which are novel, are presented. All the linear tuftsin analogues contain an isopeptide bond. Modification of the tuftsin chain was based on the introduction of simple amino acids such as valine, glycine, alanine and β-alanine into the peptide chain at the ε-amino group of lysine. The peptides were synthesized by a solid-phase method using the standard Fmoc procedure. Simultaneous deprotection of the peptide side chain and liberation from the resin was achieved using TFA, and the free novel tuftsin analogues were purified and characterized.     

Aqueous synthesis of sialylglycopeptide-grafted glycopolymers with high affinity for the lectin and the influenza virus hemagglutinin

Glycopolymers with pendant complex-type sialyl N-glycans containing heptapeptides, that is, sialylglycopeptides (SGPs), were synthesized using a water soluble polymer backbone bearing N-hydroxysulfosuccinimidyl esters by post-polymerization modification in water. Although SGP has three amino groups on the peptide chain, the substitution reaction occurs preferentially at the N-terminus α-amino group in the lysine residue onto the polymer side chain because the reactivity of such α-amino group is higher than that of the ε-amino group in the lysine residue under mild acidic aqueous condition. The resulting SGP-grafted glycopolymers exhibited strong interaction with the lectin Sambucus sieboldiana agglutinin and the human influenza A virus hemagglutinin, with higher binding associate constant values than those of free saccharide according to quartz crystal microbalance analysis. © 2020 Wiley Periodicals, Inc. J. Polym. Sci. 2020 , 58, 548–556