Research advances of SERS analysis method based on silent region molecules for food safety detection.

Food safety is a critical issue that is closely related to people's health and safety. As a simple, rapid, and sensitive detection technique, surface-enhanced Raman scattering (SERS) technology has significant potential for food safety detection. Recently, researchers have shown a growing interest in utilizing silent region molecules for SERS analysis. These molecules exhibit significant Raman scattering peaks in the cellular Raman silent region between 1800 and 2800 cm-1 avoiding overlapping with the SERS spectrum of biological matrices in the range 600-1800 cm-1, which could effectively circumvent matrix effects and improve the SERS accuracy. In this review, the application of silent region molecules-based SERS analytical technique for food safety detection is introduced, detection strategies including label-free detection and labeled detection are discussed, and recent applications of SERS analysis technology based on molecules containing alkyne and nitrile groups, as well as Prussian blue (PB) in the detection of pesticides, mycotoxins, metal ions, and foodborne pathogens are highlighted. This review aims to draw the attention to the silent region molecules-based SERS analytical technique and to provide theoretical support for its further applications in food safety detection.

Phytochemical Analysis by FTIR of Zanthoxylum Acanthopodium, DC Fruit Ethanol Extract, N-hexan, Ethyl Acetate and Water Fraction.

Background: Andaliman fruit is used as spice in Batak cuisine, North Sumatera, Indonesia. The potency of andaliman fruit extract as herbal medicine is widely studied. Many studies elaborate the benefits of andaliman fruit extract as an antioxidant, antibacterial or anticancer. Objective: The aim of this study was to identify the phytochemical compounds of andaliman fruit extract and its fractions. Methods: The andaliman fruit was originated from Balige city, Tobasa Regency, North Sumatera. The extract was made by maceration within ethanol and followed by fractionation with n-hexan, ethyl acetate and water. The phytochemical screening by chemical reactions, thin layer chromatography, and Fourier Transform Infrared Spectrophotometer were performed. Results: This study found andaliman fruit ethanol extract consists of alkaloids, flavonoids, glycosides, tannins, triterpens, and steroids. The n-hexan fraction consists of triterpens and steroids, ethyl acetate fraction consists of flavonoids and glycosides, and water fraction consists of alkaloid, flavonoid, and glycosides. Andaliman fruit ethanol extract had eight color spots, n-hexan and ethyl acetate fraction had five color spots and water fraction had two color spots. Andaliman fruit ethanol extract had functional group of C-H alkanes, =C-H alkenes (aliphatic), O-H, C=C alkenes, C=C aromatics, C≡C alkynes, C-O, C=O, and NO2. The n-hexan fraction had C-H alkanes, =C-H alkenes (aliphatic), O-H, C=C alkenes, C-O, C=O, and NO2. The ethyl acetate fraction had C-H alkanes, =C-H alkenes (aliphatic), O-H, C=C alkenes, C=C aromatics, C-O, C=O, and NO2. The water fraction had C-H alkanes, =C-H alkenes (aliphatic), O-H, C=C aromatics, C≡C alkynes, and C-O. Conclusion: Phytochemical screening found that andaliman fruit ethanol extract, n-hexan, ethyl acetate and water fraction positive of phytochemical compounds. The FTIR of andaliman fruit ethanol extract, n-hexan fraction, ethyl acetate fraction, and water fraction showed the absorbance of C-H alkanes, O-H, C-O, and C=C alkenes indicating the presence of alkaloids, flavonoids, and triterpenoid saponin.

Design, synthesis, conformational analysis, and biological activity of Cα1-to-Cα6 1,4- and 4,1-disubstituted 1H-[1,2,3]triazol-1-yl-bridged oxytocin analogues.

Oxytocin (OT) is a neurohypophyseal peptide hormone containing a disulphide-bridged pseudocyclic conformation. The biomedical use of OT peptides is limited amongst others by disadvantageous pharmacokinetic parameters. To increase the stability of OT by replacing the disulphide bridge with the stable and more rigid [1,2,3]triazol-1-yl moiety, we employed the Cu2+-catalysed side chain-to-side chain azide-alkyne 1,3-cycloaddition. Here we report the design, synthesis, conformational analysis, and in vitro pharmacological activity of a homologous series of Cα1-to-Cα6 side chain-to-side chain [1,2,3]triazol-1-yl-containing OT analogues differing in the length of the bridge, location, and orientation of the linking moiety. Exploiting this macrocyclisation approach, it was possible to generate a systematic series of compounds providing interesting insight into the structure-conformation-function relationship of OT. Most analogues were able to adopt similar conformation to endogenous OT in water, namely, a type I ß-turn. This approach may in the future generate stabilised pharmacological peptide tools to advance understanding of OT physiology.

SNAP-tagging live cells via chelation-assisted copper-catalyzed azide-alkyne cycloaddition.

SNAP-tag is a single-turnover enzyme that has become a powerful tool, hence a popular choice, of targeted cellular protein labeling. Three SNAP-tag substrates that carry the copper-chelating 2-picolyl azide moiety are prepared, one of which has an unconventional 5-pyridylmethyl-substituted guanine structure, rather than the usual benzylguanine that is optimized to be accepted by SNAP-tag. All three substrates are effective in transferring a 2-picolyl azide moiety to SNAP-tag in live cells under conventional labeling conditions (30-minute incubation of cells with labeling reagents at 37 °C under 5% CO2). Live cells that are decorated with chelating azido groups on the extracellular side of membranes undergo copper-catalyzed azide-alkyne cycloaddition (CuAAC) with an ethynyl-functionalized fluorophore to accomplish membrane protein labeling by a fluorescent dye. The chelation-assisted CuAAC labeling step is rapid (<1 minute) with a relatively low dose of the copper catalyst (20 µM), and consequently exerts no ill effect on the labeled cells. A SNAP-tag substrate that carries a non-chelating azide moiety, on the other hand, fails to produce satisfactory labeling under the same constraints. The rapid, live cell-compatible SNAP-tag/chelation-assisted CuAAC two-step method expands the utility of SNAP-tag in protein labeling applications.

2'-Modified Thymidines with Bioorthogonal Cyclopropene or Sydnone as Building Blocks for Copper-Free Postsynthetic Functionalization of Chemically Synthesized Oligonucleotides.

The development of facile methods for conjugating relevant probes, ligands, or delivery agents onto oligonucleotides (ONs) is highly desirable both for fundamental studies in chemical biology and for improving the pharmacology of ONs in medicinal chemistry. Numerous efforts have been focused on the introduction of bioorthogonal groups onto phosphoramidite building blocks, allowing the controlled chemical synthesis of reactive ONs for postsynthetic modifications. Among these building blocks, alkyne, cyclooctynes, trans-cyclooctene, and norbornene have been proved to be compatible with automated solid-phase chemistry. Herein, we present the development of novel 2'-functionalized nucleoside phosphoramidite monomers comprising bioorthogonal methylcyclopropene or sydnone moieties and their introduction for the first time to ON solid-phase synthesis. Traceless ON postsynthetic modifications with reactive complementary probes were successfully achieved through either inverse electron-demand Diels-Alder (iEDDA) reactions or strain-promoted sydnone-alkyne cycloaddition (SPSAC). These results expand the set of bioorthogonal phosphoramidite building blocks to generate ONs for postsynthetic labeling.

Specific N-terminal attachment of TMTHSI linkers to native peptides and proteins for strain-promoted azide alkyne cycloaddition.

The site specific attachment of the reactive TMTHSI-click handle to the N-terminus of peptides and proteins is described. The resulting molecular constructs can be used in strain-promoted azide alkyne cycloaddition (SPAAC) for reaction with azide containing proteins e.g., antibodies, peptides, nanoparticles, fluorescent dyes, chelators for radioactive isotopes and SPR-chips etc.

Advancements in Enacyloxins Total Synthesis: Access to the Chlorinated Polyunsaturated Chain Peculiar to this Promising Family of Antibiotics.

The first synthesis of the protected chain specific to the enacyloxin antibiotic family is reported. The noticeable features are (a) the construction of the chlorinated undecapentaenoic moiety implementing the sequence Tsuji's alkyne syn allyl-chlorination, E-selective Pd/Cu-catalyzed allene-alkyne coupling, Horner-Wadsworth-Emmons olefination, dehydration; (b) control of the C18 chlorinated stereogenic center by organo-catalyzed aldehyde α-chlorination; and (c) the assemblage of this aldehyde with the C1-C16 ketone using a highly diastereoselective Mukaiyama aldol.

Enzymatic Halogenation of Terminal Alkynes.

The biosynthetic installation of halogen atoms is largely performed by oxidative halogenases that target a wide array of electron-rich substrates, including aromatic compounds and conjugated systems. Halogenated alkyne-containing molecules are known to occur in Nature; however, halogen atom installation on the terminus of an alkyne has not been demonstrated in enzyme catalysis. Herein, we report the discovery and characterization of an alkynyl halogenase in natural product biosynthesis. We show that the flavin-dependent halogenase from the jamaicamide biosynthetic pathway, JamD, is not only capable of terminal alkyne halogenation on a late-stage intermediate en route to the final natural product but also has broad substrate tolerance for simple to complex alkynes. Furthermore, JamD is specific for terminal alkynes over other electron-rich aromatic substrates and belongs to a newly identified family of halogenases from marine cyanobacteria, indicating its potential as a chemoselective biocatalyst for the formation of haloalkynes.

Alkyne as a Latent Warhead to Covalently Target SARS-CoV-2 Main Protease.

There is an urgent need for improved therapy to better control the ongoing COVID-19 pandemic. The main protease Mpro plays a pivotal role in SARS-CoV-2 replications, thereby representing an attractive target for antiviral development. We seek to identify novel electrophilic warheads for efficient, covalent inhibition of Mpro. By comparing the efficacy of a panel of warheads installed on a common scaffold against Mpro, we discovered that the terminal alkyne could covalently modify Mpro as a latent warhead. Our biochemical and X-ray structural analyses revealed the irreversible formation of the vinyl-sulfide linkage between the alkyne and the catalytic cysteine of Mpro. Clickable probes based on the alkyne inhibitors were developed to measure target engagement, drug residence time, and off-target effects. The best alkyne-containing inhibitors potently inhibited SARS-CoV-2 infection in cell infection models. Our findings highlight great potentials of alkyne as a latent warhead to target cystine proteases in viruses and beyond.

7-[18F]Fluoro-8-azaisatoic Anhydrides: Versatile Prosthetic Groups for the Preparation of PET Tracers.

18F-Fluorination of sensitive molecules is often challenging, but can be accomplished under suitably mild conditions using radiofluorinated prosthetic groups (PGs). Herein, 1-alkylamino-7-[18F]fluoro-8-azaisatoic anhydrides ([18F]AFAs) are introduced as versatile 18F-labeled building blocks that can be used as amine-reactive or "click chemistry" PGs. [18F]AFAs were efficiently prepared within 15 min by "on cartridge" radiolabeling of readily accessible trimethylammonium precursors. Conjugation with a range of amines afforded the corresponding 2-alkylamino-6-[18F]fluoronicotinamides in radiochemical conversions (RCCs) of 15-98%. In addition, radiolabeling of alkyne- or azide-functionalized precursors with azidopropyl- or propargyl-substituted [18F]AFAs using Cu-catalyzed click cycloaddition afforded the corresponding conjugates in RCCs of 44-88%. The practical utility of the PGs was confirmed by the preparation of three 18F-labeled PSMA ligands in radiochemical yields of 28-42%. Biological evaluation in rats demonstrated excellent in vivo stability of all three conjugates. In addition, one conjugate ([18F]JK-PSMA-15) showed favorable imaging properties for high-contrast visualization of small PSMA-positive lesions.

Terminal Alkyne-Modified DNA Aptamers with Enhanced Protein Binding Affinities.

Nucleic acid-based receptors, known as aptamers, are relatively fast to discover and manufacture but lack the diverse functional groups of protein receptors (e.g., antibodies). The binding properties of DNA aptamers can be enhanced by attaching abiotic functional groups; for example, aromatic groups such as naphthalene slow dissociation from proteins. Although the terminal alkyne is a π-electron-rich functional group that has been used in small molecule drugs to enhance binding to proteins through noncovalent interactions, it remains unexplored for enhancing DNA aptamer binding affinity. Here, we demonstrate the utility of the terminal alkyne for improving the binding of DNA to proteins. We prepared a library of 256 terminal-alkyne-bearing variants of HD22, a DNA aptamer that binds the protein thrombin with nanomolar affinity. After a one-step thrombin-binding selection, a high-affinity aptamer containing two alkynes was discovered, exhibiting 3.2-fold tighter thrombin binding than the corresponding unmodified sequence. The tighter binding was attributable to a slower rate of dissociation from thrombin (5.2-fold slower than HD22). Molecular dynamics simulations with enhanced sampling by Replica Exchange with Solute Tempering (REST2) suggest that the π-electron-rich alkyne interacts with an asparagine side chain N-H group on thrombin, forming a noncovalent interaction that stabilizes the aptamer-protein interface. Overall, this work represents the first case of terminal alkynes enhancing the binding properties of an aptamer and underscores the utility of the terminal alkyne as an atom economical π-electron-rich functional group to enhance binding affinity with minimal steric perturbation.

Global Assessment of Drug Target Engagement and Selectivity of Covalent Cysteine-Reactive Inhibitors Using Alkyne-Functionalized Probes.

Covalent inhibitors are emerging as a promising therapeutic means for efficient and sustained targeting of key disease-driving proteins. As for classic non-covalent inhibitors, understanding target engagement and selectivity is essential for determining optimal dosing and limiting potential on- or off-target toxicity. Here, we present a complementary activity-based protein profiling (ABPP) strategy for unbiased proteome-wide profiling of cysteine-reactive inhibitors based on two orthogonal approaches. We illustrate the use of clickable alkyne probes for in-gel fluorescence and mass spectrometry studies using a series of therapeutic XPO1 inhibitors as an example.

Bioconjugation of Functionalized Oligodeoxynucleotides with Fluorescence Reporters for Nanoparticle Assembly.

In the field of nucleic acid nanotechnology and therapeutics, there is an imperative need to improve the oligodeoxynucleotides' (ODNs) properties by either chemical modification of the oligonucleotides' structure or to covalently link them to a reporter or therapeutic moieties that possess biologically relevant properties. The chemical conjugation can thus significantly improve the intrinsic properties not only of ODNs but also reporter/therapeutic molecules. Bioconjugation of nucleic acids to small molecules also serves as a nano-delivery facility to transport various functionalities to specific targets. Herein, we describe a generalized methodology that deploys azide-alkyne cycloaddition, a click reaction to conjugate a cyanine-3 alkyne moiety to an azide-functionalized ODN 12-mer, as well as 3-azido 7-hydroxycoumarin to an alkyne functionalized ODN 12-mer.

Anti-inflammatory acetylenic meroterpenoids from the ascidian-derived fungus Amphichorda felina SYSU-MS7908.

A combination strategy of 13C NMR and bioinformatics was established to expedite the discovery of acetylenic meroterpenoids from the ascidian-derived fungus Amphichorda felina SYSU-MS7908. This approach led to the identification of 13 acetylenic meroterpenoids (1-13) and four biogenic analogs (14-17), including five new ones named felinoids A-E (1-4 and 15). Their structures and absolute configurations were elucidated using extensive spectroscopy, ECD quantum chemical calculations, and single-crystal X-ray diffraction analysis. Compound 1 possessed a rare cyclic carbonate in natural acetylenic meroterpenoids. The plausible shikimate-terpenoid biosynthetic pathways of 1-4 were also postulated. Five of these isolates exhibited anti-inflammatory activity by inhibiting NO production in LPS-induced RAW264.7 cells (IC50 = 11.6-19.5 µM). Moreover, oxirapentyn E diacetate showed a dose-dependent inhibition of pro-inflammatory cytokines IL-6 and TNF-α. Structural modification of oxirapentyn B yielded 29 new derivatives, among which seven showed improved activity (IC50 < 3 µM) and higher selectivity index (SI > 22). The structure-activity relationship study indicated that 7, 8-epoxy, and 6-acylation were crucial for the activity. These findings may provide a powerful tool to accelerate the discovery of new fungal acetylenic meroterpenoids for future anti-inflammatory drug development.

Investigating the Effects of Donors and Alkyne Spacer on the Properties of Donor-Acceptor-Donor Xanthene-Based Dyes.

NIR dyes have become popular for many applications, including biosensing and imaging. For this reason, the molecular switch mechanism of the xanthene dyes makes them useful for in vivo detection and imaging of bioanalytes. Our group has been designing NIR xanthene-based dyes by the donor-acceptor-donor approach; however, the equilibrium between their opened and closed forms varies depending on the donors and spacer. We synthesized donor-acceptor-donor NIR xanthene-based dyes with an alkyne spacer via the Sonogashira coupling reaction to investigate the effects of the alkyne spacer and the donors on the maximum absorption wavelength and the molecular switching (ring opening) process of the dyes. We evaluated the strength and nature of the donors and the presence and absence of the alkyne spacer on the properties of the dyes. It was shown that the alkyne spacer extended the conjugation of the dyes, leading to absorption wavelengths of longer values compared with the dyes without the alkyne group. In addition, strong charge transfer donors shifted the absorption wavelength towards the NIR region, while donors with strong π-donation resulted in xanthene dyes with a smaller equilibrium constant. DFT/TDDFT calculations corroborated the experimental data in most of the cases. Dye 2 containing the N,N-dimethylaniline group gave contrary results and is being further investigated.

A Simple and Practical Bis-N-Heterocyclic Carbene as an Efficient Ligand in Cu-Catalyzed Glaser Reaction.

Conjugated diyne derivatives are important scaffolds in modern organic synthetic chemistry. Using the Glaser reaction involves the coupling of terminal alkynes which can efficiently produce conjugated diyne derivatives, while the use of a stoichiometric amount of copper salts, strong inorganic base, and excess oxidants is generally needed. Developing an environmentally friendly and effective method for the construction of symmetrical 1,3-diynes compounds by Glaser coupling is still highly desirable. In this study, we present an economical method for the production of symmetric diynes starting from various terminal acetylenes in a Glaser reaction. A simple and practical bis-N-heterocyclic carbene ligand has been introduced as efficient ligands for the Cu-catalyzed Glaser reaction. High product yields were obtained at 100 °C for a variety of substrates including aliphatic and aromatic terminal alkynes and differently substituted terminal alkynes including the highly sterically hindered substrate 2-methoxy ethynylbenzene or 2-trifluoromethyl ethynylbenzene and a series of functional groups, such as trifluoromethyl group, ester group, carboxyl group, and nitrile group. The established protocol is carried out in air under base-free condition and is operationally simple. These research work suggest that bis-N-heterocyclic carbene could also an appealing ligand for Glaser reaction and provide a reference for the preparation of symmetric 1,3-diynes in industrial filed.

Enolate-Azide [3 + 2]-Cycloaddition Reaction Suitable for DNA-Encoded Library Synthesis.

The DNA-encoded chemical library (DEL) is a powerful hit selection technique in either basic science or innovative drug discovery. With the aim to circumvent the issue concerning DNA barcode damage in a conventional on-DNA copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC), we have successfully developed the first DNA-compatible enolate-azide [3 + 2] cycloaddition reaction. The merits of this DEL chemistry include metal-free reaction and high DNA fidelity, high conversions and easy operation, broad substrate scope, and ready access to the highly substituted 1,4,5-trisubstituted triazoles. Thus, it will not only further enrich the DEL chemistry toolbox but also will have great potential in practical DEL synthesis.

Total Synthesis of Phenanthroindolizidines Using Strained Azacyclic Alkynes.

We report a concise approach to phenanthroindolizidine alkaloids, wherein strained azacyclic alkynes are intercepted in Pd-catalyzed annulations. Two types of strained intermediates were evaluated: a functionalized piperidyne and a new strained intermediate, an indolizidyne. We show that each can be employed, ultimately allowing access to three natural products: tylophorine, tylocrebine, and isotylocrebine. These efforts demonstrate the successful merger of strained azacyclic alkyne chemistry with transition-metal catalysis for the construction of complex heterocycles.

Development of an Effective Functional Lipid Anchor for Membranes (FLAME) for the Bioorthogonal Modification of the Lipid Bilayer of Mesenchymal Stromal Cells.

The glycosylation of cellular membranes is crucial for the survival and communication of cells. As our target is the engineering of the glycocalyx, we designed a functionalized lipid anchor for the introduction into cellular membranes called Functional Lipid Anchor for MEmbranes (FLAME). Since cholesterol incorporates very effectively into membranes, we developed a twice cholesterol-substituted anchor in a total synthesis by applying protecting group chemistry. We labeled the compound with a fluorescent dye, which allows cell visualization. FLAME was successfully incorporated in the membranes of living human mesenchymal stromal cells (hMSC), acting as a temporary, nontoxic marker. The availability of an azido function─a bioorthogonal reacting group within the compound─enables the convenient coupling of alkyne-functionalized molecules, such as fluorophores or saccharides. After the incorporation of FLAME into the plasma membrane of living hMSC, we were able to successfully couple our molecule with an alkyne-tagged fluorophore via click reaction. This suggests that FLAME is useful for the modification of the membrane surface. Coupling FLAME with a galactosamine derivative yielded FLAME-GalNAc, which was incorporated into U2OS cells as well as in giant unilamellar vesicles (GUVs) and cell-derived giant plasma membrane vesicles (GPMVs). With this, we have shown that FLAME-GalNAc is a useful tool for studying the partitioning in the liquid-ordered (Lo) and the liquid-disordered (Ld) phases. The molecular tool can also be used to analyze the diffusion behavior in the model and the cell membranes by fluorescence correlation spectroscopy (FCS).

Regio- and Stereoselective Switchable Synthesis of (E)- and (Z)-N-Carbonylvinylated Pyrazoles.

Regio- and stereoselective switchable synthesis of (E)- and (Z)-N-carbonylvinylated pyrazoles is first developed by using the Michael addition reaction of pyrazoles and conjugated carbonyl alkynes. Ag2CO3 plays a key role in the switchable synthesis of (E)- and (Z)-N-carbonylvinylated pyrazoles. Ag2CO3-free reactions lead to thermodynamically stable (E)-N-carbonylvinylated pyrazoles in excellent yields whereas reactions with Ag2CO3 give (Z)-N-carbonylvinylated pyrazoles in good yields. It is noteworthy that (E)- or (Z)-N1-carbonylvinylated pyrazoles are obtained with high regioselectivity when asymmetrically substituted pyrazoles react with conjugated carbonyl alkynes. The method can also extend to the gram scale. A plausible mechanism is proposed on the basis of the detailed studies, wherein Ag+ acts as coordination guidance.