Tug-of-war: molecular dynamometers against living cells for analyzing sub-piconewton interaction of a specific protein with the cell membrane

Protein–membrane interactions play important roles in signal transductions and functional regulation of membrane proteins. Here, we design a molecular dynamometer (MDM) for analyzing protein–membrane interaction on living cells. The MDM is constructed by assembling an artificial lipid bilayer and alkylated Cy3-DNA azide (azide-Cy3-Cx) on a silica bubble. After a functional aptamer is covalently anchored onto the corresponding target protein on a living cell through UV irradiation, azide-Cy3-Cx is conjugated with the aptamer through a click reaction to produce a “tug-of-war” between the MDM and the cell due to the buoyancy of the silica bubble. This induces the detachment of the protein from the cell membrane or the alkane terminal from the MDM enabling sub-piconewton embedding force measurement by changing the alkane chain length and simple fluorescence analysis. The successful analysis of embedding force variation of a protein on the cell membrane upon post-translational modifications demonstrates the practicability and expansibility of this method for mechanics-related research in biological systems.

A molecular dynamometer is designed to analyze the variation of sub-piconewton interaction between a specific protein and the membrane on living cells.     

An enzyme-mediated bioorthogonal labeling method for genome-wide mapping of 5-hydroxymethyluracil

DNA 5-hydroxymethyluracil (5hmU) is a thymine modification existing in the genomes of various organisms. The post-replicative formation of 5hmU occurs via hydroxylation of thymine by ten-eleven translocation (TET) dioxygenases in mammals and J-binding proteins (JBPs) in protozoans, respectively. In addition, 5hmU can also be generated through oxidation of thymine by reactive oxygen species or deamination of 5hmC by cytidine deaminase. While the biological roles of 5hmU have not yet been fully explored, determining its genomic location will highly assist in elucidating its functions. Herein, we report a novel enzyme-mediated bioorthogonal labeling method for selective enrichment of 5hmU in genomes. 5hmU DNA kinase (5hmUDK) was utilized to selectively install an azide (N₃) group or alkynyl group into the hydroxyl moiety of 5hmU followed by incorporation of the biotin linker through click chemistry, which enabled the capture of 5hmU-containing DNA fragments via streptavidin pull-down. The enriched fragments were applied to deep sequencing to determine the genomic distribution of 5hmU. With this established enzyme-mediated bioorthogonal labeling strategy, we achieved the genome-wide mapping of 5hmU in Trypanosoma brucei. The method described here will allow for a better understanding of the functional roles and dynamics of 5hmU in genomes.

We developed an enzyme-mediated bioorthogonal labeling strategy for the enrichment and genome-wide mapping of 5hmU. With this strategy, we provided the first map of 5hmU in the whole Trypanosoma brucei genome.     

Temperature triggered alternating copolymerization of epoxides and lactones via pre-sequenced spiroorthoester intermediates

We report the alternating copolymerization of caprolactone and epoxide through the in situ formation of pre-sequenced spiroorthoester monomer. The reaction is catalyzed by the temperature triggered, bifunctional cationic indium complex (±)-[(NN_iO_tBu)In(CH₂SiMe₃)][B(C₆F₅)₄] (1). 1 can catalyze the coupling of epoxide and lactone to form spiroorthoester at 60 °C and its double ring-opening polymerization at 110 °C to form poly(ether-alt-ester). The post-polymerization modification and degradation of the poly(ether-alt-ester) are further investigated.

We report the alternating copolymerization of caprolactone and epoxide through the in situ formation of pre-sequenced spiroorthoester monomer.     

Polyvalent spherical aptamer engineered macrophages: X-ray-actuated phenotypic transformation for tumor immunotherapy

Spatiotemporally activatable immune cells are promising for tumor immunotherapy owing to their potential high specificity and low side effects. Herein, we developed an X-ray-induced phenotypic transformation (X-PT) strategy through macrophage engineering for safe and efficient tumor immunotherapy. Without complex genetic engineering, the cell membranes of M0-type macrophages were chemically engineered with AS1411 aptamer-based polyvalent spherical aptamer (PSA) via the combination of metabolic glycan labelling and bioorthogonal click reaction. Owing to the superior specificity, affinity and polyvalent binding effects of the high-density AS1411 aptamers, the engineered macrophages could easily recognize and adhere to tumor cells. With further X-ray irradiation, reactive oxygen species (ROS) generated by the Au-based PSA could efficiently transform the accumulated macrophages in situ from biocompatible M0 into antitumoral M1 phenotype via activating the nuclear factor κB signaling pathway, thereby achieving tumor-specific killing. In vitro and in vivo experiments confirmed the high tumor recognition and X-ray-induced polarization effect of the engineered macrophages. Compared to natural macrophages, our engineered macrophages significantly inhibited tumor growth in mice even if the radiation dose was reduced by three-fold. We believe this X-PT strategy will open a new avenue for clinical immune cell-based therapy.

An X-ray-induced phenotypic transformation strategy (X-PT) through macrophage engineering was developed for safe and effective immunotherapy.     

Cell–cell interactions via non-covalent click chemistry

Metabolic glycoengineering with unnatural sugars became a valuable tool for introducing recognition markers on the cell membranes via bioorthogonal chemistry. By using this strategy, we functionalized the surface of tumor and T cells using complementary artificial markers based on both β-cyclodextrins (β-CDs) and adamantyl trimers, respectively. Once tied on cell surfaces, the artificial markers induced cell–cell adhesion through non-covalent click chemistry. These unnatural interactions between A459 lung tumor cells and Jurkat T cells triggered the activation of natural killer (NK) cells thanks to the increased production of interleukin-2 (IL-2) in the vicinity of cancer cells, leading ultimately to their cytolysis. The ready-to-use surface markers designed in this study can be easily inserted on the membrane of a wide range of cells previously submitted to metabolic glycoengineering, thereby offering a simple way to investigate and manipulate intercellular interactions.

We designed complementary artificial markers that were introduced on the surface of cells previously modified by metabolic glycoengineering. These recognition markers enable unnatural cell–cell adhesion through non-covalent click chemistry.     

A bioinspired hollow g-C3N4–CuPc heterostructure with remarkable SERS enhancement and photosynthesis-mimicking properties for theranostic applications

Surface-enhanced Raman scattering (SERS) based on chemical mechanism (CM) has great potential for superior stability and selectivity. Moreover, a bioinspired CM-Raman substrate-Raman reporter system with charge separation and electron transport nature provides thylakoid-mimicking potential for multifunctional applications. Herein, hollow carbon nitride nanospheres hierarchically assembled with a well-oriented copper(ii) phthalocyanine layer and hyaluronic acid (HCNs@CuPc@HA) were designed as a light-harvesting nanocomposite and photosynthesis-mimicking nanoscaffold that enhance both CM-SERS and photoredox catalysis. Remarkable SERS enhancement was achieved due to the strengthened short-range substrate–molecule interaction, enriched CuPc molecule loading and enhanced light–mater interactions. Meanwhile, the uniform CuPc molecule film mimics a photo-pigment to accelerate the near infrared (NIR)-oxygen generation and photodynamic catalysis of photosynthetic membrane-like HCNs. The experimental findings were further validated by numerical theory analysis. The greatly enhanced SERS signal and photosynthetic-mimicking properties of the heterostructure (denoted as HCNCHs) were successfully employed for circulating tumor cell (CTC) diagnosis and SERS imaging-guided cancer catalytic therapy in tumor xenograft models.

Thylakoid-inspired HCNs@CuPc@HA is designed as a light-harvesting nanocomposite and photosynthesis-mimicking nanoscaffold to simultaneously enhance chemical mechanism-based SERS and photosynthesis-mimicking catalysis for theranostics application.     

Mapping blood biochemistry by Raman spectroscopy at the cellular level

We report how Raman difference imaging provides insight on cellular biochemistry in vivo as a function of sub-cellular dimensions and the cellular environment. We show that this approach offers a sensitive diagnostic to address blood biochemistry at the cellular level. We examine Raman microscopic images of the distribution of the different hemoglobins in both healthy (discocyte) and unhealthy (echinocyte) blood cells and interpret these images using pre-calculated, accurate pre-resonant Raman tensors for scattering intensities specific to hemoglobins. These tensors are developed from theoretical calculations of models of the oxy, deoxy and met forms of heme benchmarked against the experimental visible spectra of the corresponding hemoglobins. The calculations also enable assignments of the electronic transitions responsible for the colour of blood: these are mainly ligand to metal charge transfer transitions.

We assign the electronic transitions responsible for the colour of blood and present a Raman imaging diagnostic approach for individual blood cells.     

Support stabilized PtCu single-atom alloys for propane dehydrogenation

PtCu single-atom alloys (SAAs) open an extensive prospect for heterogeneous catalysis. However, as the host of SAAs, Cu suffers from severe sintering at elevated temperature, resulting in poor stability of catalysts. This paper describes the suppression of the agglomeration of Cu nanoparticles under high temperature conditions using copper phyllosilicate (CuSiO₃) as the support of PtCu SAAs. Based on quasi in situ XPS, in situ CO-DRIFTS, in situ Raman spectroscopy and in situ XRD, we demonstrated that the interfacial Cu⁺–O–Si formed upon reduction at 680 °C serves as the adhesive between Cu nanoparticles and the silicon dioxide matrix, strengthening the metal–support interaction. Consequently, the resistance to sintering of PtCu SAAs was improved, leading to high catalytic stability during propane dehydrogenation without sacrificing conversion and selectivity. The optimized PtCu SAA catalyst achieved more than 42% propane conversion and 93% propylene selectivity at 580 °C for at least 30 hours. It paves a way for the design and development of highly active supported single-atom alloy catalysts with excellent thermal stability.

This paper describes PtCu single-atom alloys supported on copper phyllosilicate via Cu⁺–O–Si. The catalyst exhibits sintering resistance in propane dehydrogenation reaction without sacrificing activity and selectivity.     

Surface-enhanced Raman spectroscopy: benefits,trade-offs and future developments

Surface-enhanced Raman spectroscopy (SERS) is a vibrational spectroscopy technique with sensitivity down to the single molecule level that provides fine molecular fingerprints, allowing for direct identification of target analytes. Extensive theoretical and experimental research, together with continuous development of nanotechnology, has significantly broadened the scope of SERS and made it a hot research field in chemistry, physics, materials, biomedicine, and so on. However, SERS has not been developed into a routine analytical technique, and continuous efforts have been made to address the problems preventing its real-world application. The present minireview focuses on analyzing current and potential strategies to tackle problems and realize the SERS performance necessary for translation to practical applications.

Surface-enhanced Raman spectroscopy (SERS) is a vibrational spectroscopy technique with sensitivity down to the single molecule level that provides fine molecular fingerprints, allowing for direct identification of target analytes.     

Fluorescent probes for visualizing ROS-associated proteins in disease

Abnormal expression of proteins, including catalytic and expression dysfunction, is directly related to the development of various diseases in living organisms. Reactive oxygen species (ROS) could regulate protein expression by redox modification or cellular signal pathway and thus influence the development of disease. Determining the expression level and activity of these ROS-associated proteins is of considerable importance in early-stage disease diagnosis and the identification of new drug targets. Fluorescence imaging technology has emerged as a powerful tool for specific in situ imaging of target proteins by virtue of its non-invasiveness, high sensitivity and good spatiotemporal resolution. In this review, we summarize advances made in the past decade for the design of fluorescent probes that have contributed to tracking ROS-associated proteins in disease. We envision that this review will attract significant attention from a wide range of researchers in their utilization of fluorescent probes for in situ investigation of pathological processes synergistically regulated by both ROS and proteins.

Abnormal proteins, influenced by reactive oxygen species (ROS), are directly related to the development of various diseases.     

Enzyme-activated near-infrared fluorogenic probe with high-efficiency intrahepatic targeting ability for visualization of drug-induced liver injury

Hepatotoxicity is a serious problem faced by thousands of clinical drugs, and drug-induced liver injury (DILI) caused by chronic administration or overdose has become a major biosafety issue. However, the near-infrared (NIR) fluorescent probes currently used for liver injury detection still suffer from poor liver targeting ability and low sensitivity. Enzyme-activated fluorogenic probes with powerful in situ targeting ability are the key to improving the imaging effect of liver injury. Herein, we rationally designed a leucine aminopeptidase (LAP) activated fluorogenic probe hCy-CA-LAP, which greatly improved the hepatocyte-targeting capability by introducing a cholic acid group. The probe hCy-CA-LAP is converted into a high-emission hCy-CA fluorophore in the presence of LAP, showing high selectivity, high sensitivity and low detection limit (0.0067 U mL⁻¹) for LAP, and successfully realizes the sensitive detection of small fluctuations of LAP in living cells. Moreover, the probe can achieve effective in situ accumulation in the liver, thereby achieving precise imaging and evaluation of two different types of drug-induced hepatotoxicity in vivo. Therefore, the probe hCy-CA-LAP may be a potential tool for exploring the roles of LAP and evaluating the degree of DILI.

We rationally designed a leucine aminopeptidase (LAP) activated fluorogenic probe hCy-CA-LAP with high hepatocyte-targeting ability for accurate and sensitive imaging of DILI.     

Molecular planting of a single organothiol into a “gap-site” of a 2D patterned adlayer in an electrochemical environment

The self-assembled inclusion of molecules into two-dimensional (2D) porous networks on surfaces has been extensively studied because 2D functional materials consisting of organic molecules have become an important research topic. However, the isolation of a single molecular thiol remains a challenging goal. Here, we report a method of planting and isolating organothiols onto a 2D patterned organic adlayer at an electrochemical interface. In situ scanning tunneling microscopy revealed that the phase transition of an ovalene adlayer is electrochemically induced and that the gap site created by three ovalene molecules serves as a 2D molecular template to isolate thiol molecules and to standardize the distance between them via the formation of precise selective open spaces, suggesting that electrochemical “molecular planting” opens applications for 2D patterns of isolated single organothiol molecules.

Gap sites electrochemically created in the ovalene adlayer can accept a single thiol.     

Expanding the functionality of proteins with genetically encoded dibenzo[b,f][1,4,5]thiadiazepine: a photo-transducer for photo-click decoration

Genetic incorporation of novel noncanonical amino acids (ncAAs) that are specialized for the photo-click reaction allows the precisely orthogonal and site-specific functionalization of proteins in living cells under photo-control. However, the development of a r̲ing-strain i̲n situ l̲oadable d̲ipolarophile (RILD) as a genetically encodable reporter for photo-click bioconjugation with spatiotemporal controllability is quite rare. Herein, we report the design and synthesis of a photo-switchable d̲ib̲enzo[b,f][1,4,5]t̲hiad̲iazepine-based a̲lanine (DBTDA) ncAA, together with the directed evolution of a pyrrolysyl-tRNA synthetase/tRNA_CUA pair (PylRS/tRNA_CUA), to encode the DBTDA into recombinant proteins as a RILD in living E. coli cells. The fast-responsive photo-isomerization of the DBTDA residue can be utilized as a converter of photon energy into ring-strain energy to oscillate the conformational changes of the parent proteins. Due to the photo-activation of RILD, the photo-switching of the DBTDA residue on sfGFP and OmpC is capable of promoting the photo-click ligation with diarylsydnone (DASyd) derived probes with high efficiency and selectivity. We demonstrate that the genetic code expansion (GCE) with DBTDA benefits the studies on the distribution of decorated OmpC-DBTD on specific E. coli cells under a spatiotemporal resolved photo-stimulation. The GCE for encoding DBTDA enables further functional diversity of artificial proteins in living systems.

Via directed evolution of the tRNA synthetase, genetic encoding of a unique DBTD derived ncAA into proteins is realized. The DBTD residue is capable of transducing photon energy into ring-strain energy in situ for photo-clicking with diarylsydnone.     

A redox-enabled strategy for intramolecular hydroamination

Metal- or acid-catalyzed intramolecular hydroamination and Cope-type intramolecular hydroamination, a distinct concerted approach using hydroxylamines, typically suffer from significant synthetic limitations. Herein we report a process for intramolecular hydroamination that uses a redox-enabled strategy relying on efficient in situ generation of hydroxylamines by oxidation, followed by Cope-type hydroamination, then reduction of the resulting pyrrolidine N-oxide. The steps are performed sequentially in a single pot, no catalyst is required, the conditions are mild, the process is highly functional group tolerant, and no chromatography is generally required for isolation. A robustness screen and a gram-scale example further support the practicality of this approach.

A redox strategy enables hydroaminations: mild conditions allows efficient hydroxylamine formation & cyclization, then B₂(OH)₄ as reductant also facilitates isolation!     

HSA-Lys-161 covalent bound fluorescent dye for in vivo blood drug dynamic imaging and tumor mapping

The specific combination of human serum albumin and fluorescent dye will endow superior performance to a coupled fluorescent platform for in vivo fluorescence labeling. In this study, we found that lysine-161 in human serum albumin is a covalent binding site and could spontaneously bind a ketone skeleton quinoxaline–coumarin fluorescent dye with a specific turn-on fluorescence signal for the first time. Supported by the abundant drug binding domains in human serum albumin, drugs such as ibuprofen, warfarin and clopidogrel could interact with the fluorescent dye labeled human serum albumin to feature a substantial enhancement in fluorescence intensity (6.6-fold for ibuprofen, 4.5-fold for warfarin and 5-fold for clopidogrel). The drug concentration dependent fluorescence intensity amplification realized real-time, in situ blood drug concentration monitoring in mice, utilizing ibuprofen as a model drug. The non-invasive method avoided continuous blood sample collection, which fundamentally causes suffering and consumption of experimental animals in the study of pharmacokinetics. At the same time, the coupled fluorescent probe can be efficiently enriched in tumors in mice which could map a tumor with a high-contrast red fluorescence signal and could hold great potential in clinical tumor marking and surgical resection.

HSA lysine-161 covalent bound quinoxaline–coumarin based fluorescent dye realized in situ blood drug concentration monitoring and tumor visualization.     

Enantioselective synthesis of α-amino ketones through palladium-catalyzed asymmetric arylation of α-keto imines

Chiral α-amino ketones are common structural motifs in natural products and pharmaceuticals, as well as important synthons in organic synthesis. Thus, establishing efficient methods for preparing compounds with these privileged scaffolds is an important endeavor in synthetic chemistry. Herein we disclose a new catalytic asymmetric approach for the synthesis of chiral α-amino ketones through a chiral palladium-catalyzed arylation reaction of in situ generated challenging α-keto imines from previously unreported C-acyl N-sulfonyl-N,O-aminals, with arylboronic acids. The current reaction offers a straightforward approach to the asymmetric synthesis of acyclic α-amino ketones in a practical and highly stereocontrolled manner. Meanwhile, the multiple roles of the chiral Pd(ii) complex catalyst in the reaction were also reported.

Chiral α-amino ketones are common structural motifs in natural products and pharmaceuticals, as well as important synthons in organic synthesis.     

The in situ study of surface species and structures of oxide-derived copper catalysts for electrochemical CO2 reduction

Oxide-derived copper (OD-Cu) has been discovered to be an effective catalyst for the electroreduction of CO₂ to C2+ products. The structure of OD-Cu and its surface species during the reaction process are interesting topics, which have not yet been clearly discussed. Herein, in situ surface-enhanced Raman spectroscopy (SERS), operando X-ray absorption spectroscopy (XAS), and ¹⁸O isotope labeling experiments were employed to investigate the surface species and structures of OD-Cu catalysts during CO₂ electroreduction. It was found that the OD-Cu catalysts were reduced to metallic Cu(0) in the reaction. CuO_x species existed on the catalyst surfaces during the CO₂RR, which resulted from the adsorption of preliminary intermediates (such as *CO₂ and *OCO⁻) on Cu instead of on the active sites of the catalyst. It was also found that abundant interfaces can be produced on OD-Cu, which can provide heterogeneous CO adsorption sites (strong binding sites and weak binding sites), leading to outstanding performance for obtaining C2+ products. The Faradaic efficiency (FE) for C2+ products reached as high as 83.8% with a current density of 341.5 mA cm⁻² at −0.9 V vs. RHE.

CuO_x species were shown to exist on OD-Cu during the CO₂RR, which resulted from the adsorption of preliminary intermediates (such as *CO₂ and *OCO⁻) on Cu instead of on the active sites of the catalyst.     

Tumor suppression via diverting intracellular sialylation with multifunctional nanoparticles

Sialylation plays an important role in tumor-related physiological processes. Therefore, intervention of sialylation has great potential to explore new paths for tumor therapy. In view of the immune modulation of sialic acid (SA) on tumors, this work designs a multifunctional mesoporous silica nanoparticle (MFMSN) to divert intracellular sialylation for tumor suppression. The galactose groups covered on MFMSN act as sialylation substrates to bind intracellular SAs competitively, which inhibits the SA expression on the tumor cell surface. The diverted intracellular sialylation can be visualized on living cells and in vivo by specifically binding the sialylated galactose with a phenylboronic acid labeled ssDNA probe released from the pore of MFMSN to induce DNA strand displacement, which recovers the fluorescence of the dsDNA probe covered on MFMSN surface. The diverting of sialylation efficiently suppresses tumor growth in mice, demonstrating the great potential of the designed strategy for revealing SA-related biological processes and clinical cancer therapy.

Multifunctional nanoparticles are designed to divert intracellular sialylation, which can suppress tumor growth and be visualized.     

All on size-coded single bead set: a modular enrich-amplify-amplify strategy for attomolar level multi-immunoassay

Ultrasensitive protein analysis is of great significance for early diagnosis and biological studies. The core challenge is that many critical protein markers at extremely low aM to fM levels are difficult to accurately quantify because the target-induced weak signal may be easily masked by the surrounding background. Hence, we propose herein an ultrasensitive immunoassay based on a modular Single Bead Enrich-Amplify-Amplify (SBEAA) strategy. The highly efficient enrichment of targets on only a single bead (enrich) could confine the target-responsive signal output within a limited tiny space. Furthermore, a cascade tyramide signal amplification design enables remarkable in situ signal enhancement just affixed to the target. As a result, the efficient but space-confined fluorescence deposition on a single bead will significantly exceed the background and provide a wide dynamic range. Importantly, the SBEAA system can be modularly combined to meet different levels of clinical need regarding the detection sensitivity from aM to nM. Finally, a size-coded SBEAA set (SC-SBEAA) is also designed that allows ultrasensitive multi-immunoassay for rare samples in a single tube.

A modular single bead enrich-amplify-amplify strategy is proposed for simultaneous detection of multiple proteins at the aM level.     

Reusable Co-nanoparticles for general and selective N-alkylation of amines and ammonia with alcohols

A general cobalt-catalyzed N-alkylation of amines with alcohols by borrowing hydrogen methodology to prepare different kinds of amines is reported. The optimal catalyst for this transformation is prepared by pyrolysis of a specific templated material, which is generated in situ by mixing cobalt salts, nitrogen ligands and colloidal silica, and subsequent removal of silica. Applying this novel Co-nanoparticle-based material, >100 primary, secondary, and tertiary amines including N-methylamines and selected drug molecules were conveniently prepared starting from inexpensive and easily accessible alcohols and amines or ammonia.

A general cobalt-catalyzed N-alkylation of amines with alcohols by borrowing hydrogen methodology to prepare different kinds of amines is reported.