Bioconjugate Chemistry最新文献

{"title":"Thymidine Phosphodiester Chemiluminescent Probe for Sensitive and Selective Detection of Ectonucleotide Pyrophosphatase 1.","authors":"Omri Shelef, Sara Gutkin, Molhm Nassir, Anne Krinsky, Ronit Satchi-Fainaro, Phil S Baran, Doron Shabat","doi":"10.1021/acs.bioconjchem.4c00454","DOIUrl":"10.1021/acs.bioconjchem.4c00454","url":null,"abstract":"<p><p>ENPP-1 is a transmembrane enzyme involved in nucleotide metabolism, and its overexpression is associated with various cancers, making it a potential therapeutic target and biomarker for early tumor diagnosis. Current detection methods for ENPP-1 utilize a colorimetric probe, <b>TMP-</b><i><b>p</b></i><b>NP</b>, which has significant limitations in sensitivity. Here, we present probe <b>CL-ENPP-1</b>, the first nucleic acid-based chemiluminescent probe designed for rapid and highly sensitive detection of ENPP-1 activity. The design of probe <b>CL-ENPP-1</b> features a phenoxy-adamantyl-1,2-dioxetane luminophore linked to thymidine via a phosphodiesteric bond. Upon cleavage of the enzymatic substrate by ENPP-1, the probe undergoes an efficient chemiexcitation process to emit a green photon. Probe <b>CL-ENPP-1</b> demonstrates an exceptional signal-to-noise ratio of 15000 and a limit of detection value approximately 4500-fold lower than the widely used colorimetric probe <b>TMP-</b><i><b>p</b></i><b>NP</b>. A comparison of <b>TMP-</b><i><b>p</b></i><b>NP</b> activation by ENPP-1 versus alkaline phosphatase (ALP) reveals a complete lack of selectivity. Removal of the self-immolative spacer from probe <b>CL-ENPP-1</b> resulted in a new chemiluminescent probe, <b>CL-ENPP-2</b>, with an 18.4-fold increase in selectivity for ENPP-1 over ALP. The ability of probe <b>CL-ENPP-2</b> to detect ENPP-1 activity in mammalian cells was assessed using the human breast cancer cell line MDA-MB-231. This probe demonstrated a 19.5-fold improvement in the signal-to-noise ratio, highlighting its superior ability to detect ENPP-1 activity in a biological sample. As far as we know, to date, <b>CL-ENPP-1</b> and <b>CL-ENPP-2</b> are the most sensitive probes for the detection of ENPP-1 catalytic activity. We anticipate that our new chemiluminescent probes will be valuable for various applications requiring ENPP-1 detection, including enzyme inhibitor-based drug discovery assays. The insights gained from our probe design principles could advance the development of more selective probes for ENPP-1 and contribute to future innovations in chemiluminescence research.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"152-159"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11843602/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142941444","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Method for Screening Sodium Cyanoborohydride for Free Cyanide Content and Its Impact on Bioconjugation Chemistry.","authors":"Jarrod P Cohen, Adam DiCaprio, Jian He, Mikhail Reibarkh, James Small, Matthew Schombs","doi":"10.1021/acs.bioconjchem.4c00514","DOIUrl":"10.1021/acs.bioconjchem.4c00514","url":null,"abstract":"<p><p>Sodium cyanoborohydride (CBH) is commonly used as a mild reducing agent in the reductive amination of aldehydes and free amines. Within the pharmaceutical industry, this reaction is employed in the bioconjugation of proteins and peptides. Free cyanide species such as HCN and NaCN are known residual impurities in CBH that can contribute to the formation of undesired side products including cyanoamines and cyanohydrins. In commercial processes, the potential for bound cyanated species requires an analytical control strategy to monitor and mitigate any risk to human health. Given these concerns, minimization of cyanated side products is of utmost priority and can be achieved through a robust control strategy of quantitative screening of starting materials for free cyanide. Alternative risk mitigation strategies such as purification of bound cyanide containing species to pure species are less effective due to minor chemical differences between the expected product and bound cyanide species. Herein, we present a simple chromatographic assay for the quantitation of free cyanide in the raw material sodium cyanoborohydride. Method development, robustness evaluation, and scientific soundness assessment are reported with excellent linearity, accuracy, precision, and specificity. Additionally, this method was applied for the evaluation of raw material supplied from 10 commercial sources, none of which report a specification for free cyanide within their certificate of analysis. The measured free cyanide from these vendors ranged from 8 to 80 mM concentration, thereby confirming the value of screening these raw materials. Finally, we demonstrate the impact of free cyanide on a model bioconjugation reaction between ornithine and glyceraldehyde.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"245-252"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11843608/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254206","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of Transiently Strainable Benzocycloheptenes for Catalyst-Free, Visible-Light-Mediated [3 + 2]-Cycloadditions.","authors":"Shivangi Kharbanda, Osaid Alkhamayseh, Georgia Eastham, Jimmie D Weaver","doi":"10.1021/acs.bioconjchem.4c00595","DOIUrl":"10.1021/acs.bioconjchem.4c00595","url":null,"abstract":"<p><p>Dynamic photogeneration of ephemeral and reactive species is enabling for chemical reactions, providing spatial and temporal control. A previous study from our group established the ability of 6,7-dihydro-5H-benzo[7]annulene, benzocycloheptene (<b>BC7</b>), to convert photochemical energy into ring strain, enabling the rapid cycloaddition of alkyl azides with the reversibly formed and transient <i>trans</i>-isomer, affording versatile nonaromatic triazolines. Despite the conceptual advances of the previous study, some challenges remained: the fragility of the triazoline products, the low regioselectivity for the cycloaddition, a need for an iridium-based photosensitizer and organic-based solvents, and a lack of convenient linchpin functional group handles. Herein, we communicate the development of a second generation of <b>BC7</b> molecules that overcome the issues of the first generation. A method to convert fragile triazoline products to stable triazoles was developed. The alkene component was polarized with a carbonyl group, dramatically improving the regioselectivity while simultaneously red-shifting the absorbance of the cycloalkene into the visible region, which was expected to facilitate direct excitation and eliminate the need for photocatalysts. However, experiments indicated that the cycloaddition involved passage through a triplet manifold, complicating the direct excitation strategy. This was successfully overcome by attaching a bromine atom directly to the alkene moiety, which accelerated singlet-to-triplet intersystem crossing by the heavy atom effect. Further exploration identified sites of substitution that can increase the water solubility and provide a handle for the loading of chemical tools and probes.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"302-308"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11843616/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143187549","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Unique Prodrug Targeting the Prostate-Specific Membrane Antigen for the Delivery of Monomethyl Auristatin E.","authors":"Hunter N Bomba, Melody D Fulton, Emily A Savoy, Beatrice Langton-Webster, Clifford E Berkman","doi":"10.1021/acs.bioconjchem.4c00297","DOIUrl":"10.1021/acs.bioconjchem.4c00297","url":null,"abstract":"<p><p>Monomethyl auristatin E (MMAE) is a promising treatment option for patients diagnosed with prostate cancer (PCa); however, toxicities prevent MMAE from being administered as free drug. No MMAE-based treatment is currently marketed for PCa. Herein, we describe a small-molecule-drug conjugate, CTT2274, for the selective delivery of MMAE. CTT2274 is composed of a prostate-specific membrane antigen (PSMA)-binding scaffold, a biphenyl motif, a pH-sensitive phosphoramidate linker, and MMAE payload. We demonstrate that CTT2274 shows selective binding to PSMA, which is overexpressed on PCa cells, and induces tumor cell death <i>in vitro</i>. In a patient-derived xenograft tumor model of PCa in mice, we show that weekly intravenous dosing of CTT2274 at 3.6 mg/kg for six weeks is superior to treatment with free MMAE at equivalent doses. Mice treated with CTT2274 experienced prolonged tumor suppression and significantly greater overall survival than mice treated with PBS. Additionally, the safety of CTT2274 compared to an equivalent dose of MMAE was assessed in healthy, non-tumor-bearing mice. Our results demonstrate that CTT2274 therapy is as efficacious as MMAE, results in superior overall survival, and has a more favorable safety profile. Together, these data indicate that CTT2274 is a candidate for clinical translation for the treatment of PCa.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"169-178"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062212","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantum Dot Erythropoietin Bioconjugates Enhance EPO-Receptor Clustering on Transfected Human Embryonic Kidney Cells.","authors":"Ryan N Porell, Okhil K Nag, Michael H Stewart, Kimihiro Susumu, Eunkeu Oh, James B Delehanty","doi":"10.1021/acs.bioconjchem.4c00521","DOIUrl":"10.1021/acs.bioconjchem.4c00521","url":null,"abstract":"<p><p>Erythropoietin (EPO)-induced cellular signaling through the EPO receptor (EPOR) is a fundamental pathway for the modulation of cellular behavior and activity. In our previous work, we showed in primary human astrocytes that the multivalent display of EPO on the surface of semiconductor quantum dots (QDs) mediates augmented JAK/STAT signaling, a concomitant 1.8-fold increase in the expression of aquaporin-4 (AQPN-4) channel proteins, and a 2-fold increase in the AQPN-4-mediated water transport activity. Our hypothesis is that this enhanced signaling involves the simultaneous ligation and clustering of EPOR by QD-EPO conjugates. Here, we utilized a human embryonic kidney (HEK 293T/17) cell line transfected with EPOR fused to enhanced green fluorescent protein (eGFP) to visualize EPOR clustering. We demonstrate that QDs displaying five copies of EPO (bearing a C-terminal 6-histidine tract) on the nanoparticle surface induce a 1.8-fold increase in EPOR clustering compared to monomeric EPO at the same concentration. Our findings confirm the critical role played by the multivalent display of EPO in mediating clustering of the EPOR. More generally, these results illustrate the capability of nanoparticle-growth factor bioconjugates to control the activity of cognate receptors and the important role played by multivalent display in the modulation of selective cellular delivery and signaling.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"160-168"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142996195","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis and Biological Evaluation of Bile Acid-Triclosan Conjugates: A Study on Antibacterial, Antibiofilm, and Molecular Docking.","authors":"Neha V Rathod, Satyendra Mishra","doi":"10.1021/acs.bioconjchem.4c00539","DOIUrl":"10.1021/acs.bioconjchem.4c00539","url":null,"abstract":"<p><p>This work describes the synthesis, characterization, and antibacterial properties of four bile acid-triclosan conjugates. The in vitro antibacterial activity of synthetic bile acid-triclosan conjugates was investigated against a panel of Gram-positive and Gram-negative bacteria. Conjugates <b>3</b> and <b>4</b> show high activity against <i>Escherichia coli</i> (ATCC25922), with IC₅₀ values of 2.94 ± 0.7 and 1.51 ± 0.05 μM, respectively. Conjugate <b>4</b> demonstrated 9 times the activity of triclosan (6.77 μM) and 18 times the potency of kanamycin, a well-known antibiotic. Compound <b>3</b> showed higher potential activity against all evaluated strains, including <i>Bacillus megaterium</i> (IC₅₀: 3.05 ± 0.02), <i>Bacillus amyloquefaciens</i> (IC₅₀: 8.79 ± 0.01), <i>Serratia marcescens</i> (IC₅₀: 6.77 ± 0.4), and <i>E. coli</i> (IC₅₀: 1.51 ± 0.05 μM). These findings indicate that it has broad-spectrum antibacterial activity. Bile acid-triclosan conjugates prevent biofilms by up to 99% at low doses (conjugates <b>4</b>; 4.16 ± 0.8 μM), compared to triclosan. Conjugate <b>5</b> was most potent against <i>B. amyloquefaciens</i> (IC₅₀ = 5.23 ± 0.2 μM), while conjugate <b>4</b> was most effective against <i>B. megaterium</i> (IC₅₀ = 4.16 ± 0.8 μM) in biofilm formation. These conjugates inhibit biofilm formation by limiting the extracellular polymeric substance generation. The in vitro antibacterial study revealed that bile acid-triclosan conjugates were more effective than the parent molecule triclosan at inhibiting bacterial growth and biofilm formation against both Gram-positive and Gram-negative bacteria.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"276-290"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143021299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unlocking the Potential of Antimicrobial Peptides: Cutting-Edge Advances and Therapeutic Potential in Combating Bacterial Keratitis","authors":"Bingru Xiao,&nbsp;Jie Wang,&nbsp;Jie Xing,&nbsp;Lulu He,&nbsp;Chen Xu*,&nbsp;Aiguo Wu* and Juan Li*,&nbsp;","doi":"10.1021/acs.bioconjchem.4c0059410.1021/acs.bioconjchem.4c00594","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.4c00594https://doi.org/10.1021/acs.bioconjchem.4c00594","url":null,"abstract":"<p >Bacterial keratitis is a prevalent, and severe corneal illness resulting from bacterial pathogens. Failure to administer a timely and suitable therapy may lead to corneal opacity, ulceration, significant vision impairment, or potential blindness. Current clinical interventions for bacterial keratitis involve the administration of topical antimicrobial agents and systemic antibiotics. However, the misuse and overuse of antibiotics have led to the rapid emergence of antibiotic-resistant bacteria. Additionally, the restricted antibacterial spectrum and possible adverse effects of antibiotics have provided considerable obstacles to traditional therapies. This highlights the urgent need for novel and highly effective antimicrobial agents. Antimicrobial peptides (AMPs) are a class of naturally occurring or synthetically designed small molecules that have gained significant attention due to their unique antimicrobial mechanisms and low risk of resistance development. AMPs exhibit promising potential in treating bacterial keratitis through direct antibacterial mechanisms, such as inhibiting cell wall synthesis, disrupting cell membranes, and interfering with nucleic acid metabolism, as well as indirect mechanisms, including modulation of the host immune response. This review provides a comprehensive overview of the antibacterial mechanisms of AMPs and their advancements in the treatment of bacterial keratitis. It emphasizes the role of various modification strategies and artificial-intelligence-assisted design in enhancing the antibacterial efficacy, stability, and biocompatibility of AMPs. Furthermore, this review discusses the latest progress in combining AMPs with delivery systems for improved therapeutic outcomes. Finally, the review highlights the current challenges and future perspectives of AMPs in bacterial keratitis treatment, providing valuable insights for developing novel AMPs with high antibacterial efficacy, stability, and safety for bacterial keratitis therapies.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":"36 3","pages":"311–331 311–331"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143641438","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Targeted NIR Fluorescent Mechanically Interlocked Molecules-Peptide Bioconjugate for Live Cancer Cells Submitochondrial Stimulated Emission Depletion Super-Resolution Microscopy.","authors":"Samiran Kar, Rabi Sankar Das, Tapas Bera, Shreya Das, Ayan Mukherjee, Aniruddha Mondal, Arunima Sengupta, Samit Guha","doi":"10.1021/acs.bioconjchem.4c00476","DOIUrl":"10.1021/acs.bioconjchem.4c00476","url":null,"abstract":"<p><p>Herein, a water-soluble, ultrabright, near-infrared (NIR) fluorescent, mechanically interlocked molecules (MIMs)-peptide bioconjugate is designed with dual targeting capabilities. Cancer cell surface overexpressed α_Vβ₃ integrin targeting two RGDS tetrapeptide residues is tethered at the macrocycle of MIMs-peptide bioconjugate via Cu(I)-catalyzed click chemistry on the Wang resin, and mitochondria targeting lipophilic cationic TPP⁺ functionality is conjugated at the axle dye. Living carcinoma cell selective active targeting, subsequently cell penetration, mitochondrial imaging, including the ultrastructure of cristae, and real-time tracking of malignant mitochondria by MIMs-peptide bioconjugate (RGDS)₂-Mito-MIMs-TPP⁺ are established by stimulated emission depletion (STED) super-resolved fluorescence microscopy. Water-soluble NIR (RGDS)₂-Mito-MIMs-TPP⁺ is an effective class of MIMs-peptide bioconjugate with promising photophysics; for instance, remarkable photostability and thermal stability, strong and narrow NIR abs/em bands with high quantum yield, ultrabrightness, decent fluorescence lifetime, reasonable stability against cellular nucleophiles, biocompatibility, noncytotoxicity, and dual-targeted living cancer cell submitochondrial imaging ability are all indispensable criteria for targeted super-resolved STED microscopy.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"223-232"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142941442","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Site-Specific Molecular Engineering of Nanobody-Glucoside Conjugates for Enhanced Brain Tumor Targeting.","authors":"Siyu Zhou, Xiaofeng Fang, Yunhe Luo, Yicheng Yang, Weijun Wei, Gang Huang, Xuanjun Zhang, Changfeng Wu","doi":"10.1021/acs.bioconjchem.4c00555","DOIUrl":"10.1021/acs.bioconjchem.4c00555","url":null,"abstract":"<p><p>Nanobodies play an increasingly prominent role in cancer imaging and therapy. However, their <i>in vivo</i> efficacy is often constrained by inadequate tumor penetration and rapid clearance from the bloodstream, particularly in brain tumors due to the intractable blood-brain barrier (BBB). Glycosylation is a favorable strategy for modulating the biological functions of nanobodies, including permeability and pharmacokinetics, but it also leads to heterogeneous glycan structures, which affect the targeting ability, stability, and quality of nanobodies. Here, we describe a post-translational modification strategy to produce precisely engineered and homogeneous nanobody-glucoside conjugates for effective BBB penetration and brain tumor targeting. Specifically, we employ an enzymatic method and click chemistry to functionalize nanobodies with glucoside and poly(ethylene glycol) (PEG), facilitating efficient transcytosis into the brain via glucose transporter-1 (GLUT1). Furthermore, we rationally select a near-infrared (NIR) fluorophore for labeling to maintain the metabolic pathway and biodistribution of nanobodies and assess their potency in two tumor models. The resulting nanobody-glucoside conjugates demonstrate a remarkable increase in BBB penetration and brain tumor accumulation, which are ∼2.9-fold higher in the transgenic mouse model and ∼5.7-fold higher in the orthotopic glioma model compared to unmodified nanobodies. This study provides a promising approach for the production of nanobody therapeutic agents for central nervous system (CNS) delivery.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"291-301"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142941441","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"5'-Amino-Formyl-Thieno[3,2-<i>b</i>]thiophene End-Label for On-Strand Synthesis of Far-Red Fluorescent Molecular Rotors and pH-Responsive Probes.","authors":"Ryan E Johnson, Keenan T Regan, Richard A Manderville","doi":"10.1021/acs.bioconjchem.4c00457","DOIUrl":"10.1021/acs.bioconjchem.4c00457","url":null,"abstract":"<p><p>The ability to label synthetic oligonucleotides with fluorescent probes has greatly expanded their nanotechnological applications. To continue this expansion, it is essential to develop approachable, modular, and tunable fluorescent platforms. In this study, we present the synthesis and incorporation of an amino-formyl-thieno[3,2-<i>b</i>]thiophene (AFTh₂) handle at the 5'-position of DNA oligonucleotides. The 5'-AFTh₂ end-label participates in both on-strand Knoevenagel and heterocyclization reactions, yielding far-red hemicyanines and pH-responsive probes with p<i>K</i>_a values in the biological regime. The Knoevenagel products, designated 5'-ATh₂Btz and 5'-ATh₂Ind, demonstrate excitation maxima beyond 640 nm with brightness up to ∼50,000 M^-1 cm^-1. Notably, 5'-ATh₂Btz demonstrates strong topology sensitivity, allowing it to probe transitions from duplex- to single-strand (SS)/G-quadruplex (GQ) topologies with an ∼9-fold increase in fluorescence in the absence of quenchers. In contrast, the heterocyclization product, 5'-ATh₂BIM, displays visible excitation and emission and is weakly fluorescent in basic solution. Upon lowering the pH from ∼8 to 5, this probe undergoes an unprecedented 400-fold light-up. Additionally, attaching 5'-ATh₂BIM to a polymorphic GQ allows for a shift in p<i>K</i>_a by ∼1.5 pH units simply by changing topology. The performance of the probes has been demonstrated in various contexts, including GQs, i-motifs, duplexes, and SS oligonucleotides. Their performance should facilitate the development of new DNA-based sensing platforms.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"216-222"},"PeriodicalIF":4.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142996101","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}