Facile Synthesis of Internal and C‐Terminal Peptide α‐Ketoamides with Fmoc‐Solid Phase Peptide Synthesis

We report a general and operationally simple method for the solid phase synthesis of α‐ketoamide peptides using standard Fmoc solid phase peptide synthesis. The method delivers deprotected peptide α‐ketoamides directly upon resin cleavage without any additional steps, and tolerates all side chain functional groups. A small collection of C‐terminal and internal α‐ketoamide peptides – including two reported protease inhibitors (HCV and SUB1) – were prepared in good yields. In addition, we demonstrate that our method serves as versatile platform for the convenient preparation of cyclic α‐ketoamide peptides, photocagged peptide α‐ketoamides, and fluorescently labeled peptides.     

Synthesis of an olefin-containing cyclic peptide using the solid-phase Horner-Emmons reaction

Linear and cyclic olefin peptides containing the substrate sequence for human T-cell leukemia virus type-1 (HTLV-1) were efficiently synthesized on a solid support using the Horner-Emmons reaction. The precursor peptide aldehyde was prepared by oxidation of the corresponding peptide alcohol with Dess-Martin periodinane. The oxidation reaction proceeded quantitatively on a cross-linked ethoxylate acrylate resin (CLEAR) support instead of a polystyrene-based support. Cyclization on the solid support was achieved via an amide bond formation mediated by EDC/HOAt to yield a single major product. The linear olefin peptide was cleaved by HTLV-1 protease at the scissile site, whereas the cyclic olefin peptide functions as a competitive inhibitor rather than a substrate.     

A method for rapid protease substrate evaluation and optimization

We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.     

DNA-Barcoded Plasmonic Nanostructures for Activity-Based Protease Sensing

We report the development of a new class of protease activity sensors called DNA-barcoded plasmonic nanostructures. These probes are comprised of gold nanoparticles functionalized with peptide-DNA conjugates (GPDs), where the peptide is a substrate of the protease of interest. The DNA acts as a barcode identifying the peptide and facilitates signal amplification. Protease-mediated peptide cleavage frees the DNA from the nanoparticle surface, which is subsequently measured via a CRISPR/Cas12a-based assay as a proxy for protease activity. As proof-of-concept, we show activity-based, multiplexed detection of the SARS-CoV-2-associated protease, 3CL, and the apoptosis marker, caspase 3, with high sensitivity and selectivity. GPDs yield >25-fold turn-on signals, 100-fold improved response compared to commercial probes, and detection limits as low as 58 pM at room temperature. Moreover, nanomolar concentrations of proteases can be detected visually by leveraging the aggregation-dependent color change of the gold nanoparticles. We showcase the clinical potential of GPDs by detecting a colorectal cancer-associated protease, cathepsin B, in three different patient-derived cell lines. Taken together, GPDs detect physiologically relevant concentrations of active proteases in challenging biological samples, require minimal sample processing, and offer unmatched multiplexing capabilities (mediated by DNA), making them powerful chemical tools for biosensing and disease diagnostics.     

A high-complexity, multiplexed solution-phase assay for profiling protease activity on microarrays

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.     

Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles

We report a simple staining-free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au-NPs) dual-functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining-free gel electrophoresis. The use of Au-NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide-mediated approach facilitates the synthesis of DNA/peptide conjugated Au-NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.     

Differential isotopic mass splitting as a mass spectrometric tool for identifying protease substrates

A method is described whereby stable isotopic signatures were partially incorporated into both termini of a peptide sequence giving rise to a characteristic cluster of four peaks in the mass spectral analysis. Cleavage of this peptide by a protease between the labeled positions generates two fragments both displaying their own individual signature peaks. The event of protease cleavage of the peptide was monitored by the changes in clusters within the spectrum. We believe that this technique could be used to aid the discovery of new cleavage substrates for proteases. Additionally, the analysis can be automated with dedicated software designed to select and interpret the data since all peaks of interest contain predefined signatures and can be easily distinguished from background noise.     

Studies on the insolubility of a transmembrane peptide from signal peptide peptidase

We have undertaken fundamental studies on the solubility properties of a peptide derived from the fourth transmembrane (TM) domain of signal peptide peptidase, a 7-TM intramembrane-cleaving protease. We have found that by disfavoring secondary structure formation we are able to greatly improve the solubility, handling, and purification properties of this peptide. Our findings suggest that preventing secondary structure formation by reversible modification of the polypeptide backbone of hydrophobic transmembrane peptides may be a useful strategy for the total chemical protein synthesis of integral membrane proteins.     

Comparative investigation of the cleavage step in the synthesis of model peptide resins: implications for Nalpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nalpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nalpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.     

Inhibition of hepatitis C virus serine protease in living cells by RNA aptamers detected using fluorescent protein substrates

Hepatitis C virus is one of the causative agents of non-A non-B hepatitis. Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation. NS3 serine protease is considered to be a suitable target for anti-HCV reagents. We report an assay of HCV NS3 protease in living cells. We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP. Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease. The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed. As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection. We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro. We studied the effect of RNA aptamers introduced into HeLa cells. The addition of G9-II RNA in the medium at a concentration of 2.5 micro g/ml reduced cleavage by one-third that of control.     

Fmoc solid-phase synthesis of peptide thioesters using an intramolecularn,S-acyl shift

[reaction: see text] We describe the Fmoc solid-phase synthesis of peptide thioesters based on the alkylation of the safety-catch sulfonamide linker with a protected 2-mercaptoethanol derivative. The thioester is generated on the solid phase after the peptide chain assembly as a consequence of an intramolecular N,S-acyl shift. Depending on the stability of the spacer separating the sulfonamide linker from the resin toward TFA, treatment of the peptidyl resin with TFA led to a soluble or supported deprotected thioester.     

Synthesis of bifunctional peptide derivatives based on a β-cyclodextrin core with drug delivery potential

A selective, versatile, robust methodology for bifunctionalization of β-cyclodextrin is achieved allowing the attachment of peptides in varying C- and/or N-terminal combinations on resin using Fmoc SPPS. Two linkers are attached to cyclodextrin enabling selective binding to the resin (or a peptide attached to the resin). Continuation of peptide growth and/or cleavage from the resin follows, thus various combinations of peptide-cyclodextrin species are achieved. A model peptide (Gly-Ala) is used in this study to illustrate the potential of this system for attaching one or more bioactive peptides for drug transport and release purposes.     

Carbon Nanotube‐Enhanced Polarization of Fluorescent Peptides: A Novel Amplification Strategy for Homogeneous Detection of Proteases

A new fluorescence polarization (FP) amplification strategy based on the use of multiwalled carbon nanotubes (MWCNTs) as the FP enhancer was developed for the simple, sensitive, and universal monitoring of protease activity in homogeneous solution. A fluorophore‐labeled peptide that includes a protease‐cleavable element and ten histidine residues for binding MWCNTs is adsorbed on MWCNTs through strong π–π stacking and electrostatic interactions. When the fluorophore‐labeled peptide/MWCNT complexes are exposed to a protease target, specific peptide cleavage by the protease target occurs, thus releasing fragments carrying the fluorophore from the surface of MWCNTs, which in turn results in a significant decrease in the FP value. The detection limits of this assay for two proteases, thrombin and chymotrypsin (CTP), were estimated to be 0.5 pM and 0.3 pM , respectively. In addition, it is also demonstrated that this MWCNT‐enhanced FP assay is suitable for protease inhibitor screening.     

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.     

A new method for the preparation of 2-chlorotrityl resin and its application to solid-phase peptide synthesis

2-Chlorotritylchloride (2-CTC) resin was prepared efficiently from 1% DVB-crosslinked polystyrene resin and 1-chloro-2-(dichloro(phenyl)methyl)benzene, which was easily obtained from 2-chlorobenzophenone. This 2-CTC resin showed excellent properties as a support for solid-phase peptide synthesis. Four peptide fragments were obtained in high purity using the resin.     

Solid-phase synthesis of peptide thioacids through hydrothiolysis of resin-bound peptide thioesters

We report that solid-phase hydrothiolysis is an efficient method to convert resin-bound peptide thioesters to thioacids in aqueous buffer by using a total PEG-based resin. Also demonstrated is the use of the so-prepared peptide thioacids in chemoselective amide bond formation reactions.     

Solid-phase synthesis of metal-complex containing peptides

We report the synthesis of Fmoc protected single amino acid chelates (SAAC) and their metal complexes. The modified amino acids are suitable for solid-phase peptide synthesis. The use of 4-hydroxymethylbenzoic acid AM (HMBA-AM) resin allows the nucleophilic cleavage of the peptide-metal complexes from the resin without decomplexation.     

一种新的玉米抗氧化肽的制备与结构表征

用酶法在受控条件下降解玉米醇溶蛋白,对酶解产物进行分离纯化,获得一种新的抗氧化肽;对其一级结构进行了表征,氨基酸组成和顺序为Leu-Asp-Tyr-Glu;从结构上分析了其可能的抗氧化机制.     

Rapid identification of substrates for novel proteases using a combinatorial peptide library

Fluorogenic substrates for assaying novel proteolytic enzymes could be rapidly identified using an easy, solid-phase combinatorial assay technology. The methodology was validated with leader peptidase of Escherichia coli using a subset of an intramolecularly quenched fluorogenic peptide library. The technique was extended toward the discovery of substrates for a new aspartic protease of pharmaceutical relevance (human napsin A). We demonstrated for the first time known to us that potent fluorogenic substrates can be discovered using extracts of cells expressing recombinant enzyme to screen the peptide library. The straightforward and rapid optimization of protease substrates greatly facilitates the drug discovery process by speeding up the development of high throughput screening assays and thus helps more effective exploitation of the enormous body of information and chemical structures emerging from genomics and combinatorial chemistry technologies.     

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.