Bioorthogonal reactions involving transition metals have diversified applications in imaging,drug development,chemical catalysis and other fields.Transition metals used to catalyze the bioorthogonal reaction mainly in...Bioorthogonal reactions involving transition metals have diversified applications in imaging,drug development,chemical catalysis and other fields.Transition metals used to catalyze the bioorthogonal reaction mainly include ruthenium,palladium,copper,and gold.However,the great potential for translational applications of bioorthogonal reaction needs to be further expanded and their reaction efficiency should be improved.Therefore,it is an urgent need for the development of this field to find more suitable catalysts to efficiently catalyze existing biological orthogonal reactions and expand the types of biological orthogonal reactions.Thus,this review not only summarizes those transition metal complexes-based catalysts participating in bioorthogonal reaction and some bioorthogonal reactions involving transition metals inside the cells,but also sheds light into the discovery of new transition metal complexes and their future development in applications.展开更多
Cell membrane camouflaged nanoparticles have been widely used in the field of drug leads discovery attribute to their unique biointerface targeting function.However,random orientation of cell membrane coating does not...Cell membrane camouflaged nanoparticles have been widely used in the field of drug leads discovery attribute to their unique biointerface targeting function.However,random orientation of cell membrane coating does not guarantee effective and appropriate binding of drugs to specific sites,especially when applied to intracellular regions of transmembrane proteins.Bioorthogonal reactions have been rapidly developed as a specific and reliable method for cell membrane functionalization without disturbing living biosystem.Herein,inside-out cell membrane camouflaged magnetic nanoparticles(IOCMMNPs)were accurately constructed via bioorthogonal reactions to screen small molecule inhibitors targeting intracellular tyrosine kinase domain of vascular endothelial growth factor recptor-2.Azide functionalized cell membrane acted as a platform for specific covalently coupling with alkynyl functionalized magnetic Fe_(3)O_(4)nanoparticles to prepare IOCMMNPs.The inside-out orientation of cell membrane was successfully verified by immunogold staining and sialic acid quantification assay.Ultimately,two compounds,senkyunolide A and ligustilidel,were successfully captured,and their potential antiproliferative activities were further testified by pharmacological experiments.It is anticipated that the proposed inside-out cell membrane coating strategy endows tremendous versatility for engineering cell membrane camouflaged nanoparticles and promotes the development of drug leads discovery platforms.展开更多
Mitochondria are essential for eukaryotic life as powerhouses for energy metabolism. Excessive mitochondrial hyperthermia and reactive oxygen species(ROS) production have been associated with aging, cancer,neurodegene...Mitochondria are essential for eukaryotic life as powerhouses for energy metabolism. Excessive mitochondrial hyperthermia and reactive oxygen species(ROS) production have been associated with aging, cancer,neurodegenerative diseases, and other disorders. Uncoupling protein 2(UCP2) is the effector responsible for regulating cellular thermogenesis and ROS production via dissipating protons in an electrochemical gradient. A UCP2 inhibitor named genipin(GNP) is being researched for its effect on mitochondrial temperature, but little is known about its mechanisms. This study developed several molecular probes to explore the interactions between GNP and UCP2. The result indicated that the hemiacetal structure in GNP could selectively react with the ?-amine of lysine on the UCP2 proton leakage channel through ringopening condensation at the mitochondrial, cellular, and animal levels. A notable feature of the reaction is its temperature sensitivity and ability to conjugate with UCP2 at high fever as lysine-specific covalent inhibitors that prevent mitochondrial thermogenesis. The result not only clarifies the existence of an antipyretic properties of GNP via its irreversible coupling to UCP2, but also reveals a bioorthogonal reaction of hemiacetal iridoid aglycone for selectively binding with the ?-amine of lysine on proteins.展开更多
Pretargeting is an innovative and promising approach in nuclear medicine for targeted-imaging/therapy through the following bioorthogonal reactions.It requires two reactive participants,one of which is a targeting vec...Pretargeting is an innovative and promising approach in nuclear medicine for targeted-imaging/therapy through the following bioorthogonal reactions.It requires two reactive participants,one of which is a targeting vector and the other is a small radiolabeled probe capable of specifically coupling through bioorthogonal reactions with the targeting vector accumulated in the disease site.Compared to the conventional direct targeting approach,such a two-step scheme conceptually can achieve a higher imaging contrast and an improved therapeutic effect owing to the suppressed non-specific targeting.In this review,we will first give a brief introduction on pretargeting systems and the history of bioorthogonal reactions,and then focus on some important works about radionuclide delivering through the bioorthogonal reaction based pretargeting strategy.Finally,we will discuss the steps forward in respect to the future clinical translation and truly hope that this methodology would continue to make contributions to nuclear medicines.展开更多
Here,we present a novel bioorthogonal platform that enables precise positioning of attached moieties in close proximity,thereby facilitating the discovery and optimization of biocompatible reactions.Using this platfor...Here,we present a novel bioorthogonal platform that enables precise positioning of attached moieties in close proximity,thereby facilitating the discovery and optimization of biocompatible reactions.Using this platform,we achieve a Horner-Wadsworth-Emmons(HWE)reaction under physiological conditions,generating a fluorophore in situ with a yield of up to 93%.This proximity platform should facilitate the discovery of various types of biocompatible reactions,making it a versatile tool for biomedical applica-tions.展开更多
Due to their excellent fluorescence properties and biological function,cyanine dyes have been widely applied in biological imaging.Heptamethine cyanine(Cy7)dyes,as a type of classic near-infrared(NIR)fluorescent dyes,...Due to their excellent fluorescence properties and biological function,cyanine dyes have been widely applied in biological imaging.Heptamethine cyanine(Cy7)dyes,as a type of classic near-infrared(NIR)fluorescent dyes,are considered as one of the effective fluorescent tools in the living organisms due to their good biocompatibility and very low background interference.Bioorthogonal reactions performed in living cells and tissues have developed by leaps and bounds in recent years.The NIR fluorescent labeling technique involving cyanine has attracted widespread attention.This review summarizes their recent application in the field of bioorthogonal imaging,mainly concluding Cy7-type dyes,labeling strategy,bioimaging application,etc.We expect this work can provide some helps for the studies of NIR bioorthogonal reaction in vivo.展开更多
Selective methods for modulating RNA epigenetic modifications within living cells and organisms represent attractive techniques for investigating biological functions and medicinal application.In contrast to enzymatic...Selective methods for modulating RNA epigenetic modifications within living cells and organisms represent attractive techniques for investigating biological functions and medicinal application.In contrast to enzymatic methods,abiotic chemical modulation offers access to diverse new-to-natural functionalities.We herein report a visible light-assisted bioorthogonal reaction involving flavin mononucleotide,sodium azide,and blue light irradiation.In concert with previous chemical approaches mimicking the demodification pathway,our system functions as a powerful and selective post-modification enzyme that targets N^(6)-prenyl adenosine i^(6) A in RNA and enables the efficient construction of an artificial N^(6)-cyanomethyl adenosine(cnm^(6) A).Notably,most endogenous biomacromolecular functions,including other common RNA epigenetics,remained unaffected.Consequently,we have successfully modified i^(6) A in tRNA to cnm^(6) A in human cells and establish this system as a bona fide bioorthogonal reaction with potential applications in chemical biology and in-cell modulation.展开更多
Benefiting from the advantage of taking place in biological environments without interfering with an innate biochemical process,the bioorthogonal reaction that commonly contains the“bond formation”and“bond cleavage...Benefiting from the advantage of taking place in biological environments without interfering with an innate biochemical process,the bioorthogonal reaction that commonly contains the“bond formation”and“bond cleavage”system has been widely used in targeted therapy for a variety of tumors.Herein,several prominent cases based on the bioorthogonal reaction that tailoring the metabolic glycoengineering tactics to modified cells for cancer immunotherapy,and the innovative tactics for reducing the metal ions’toxic and side effects with microneedle patches will be highlighted.Based on these applications,the complexities,potential pitfalls,and opportunities of bioorthogonal chemistry in future cancer therapy will be evaluated.展开更多
A dimesitylboryl-ended oligothiophene with tetrazine as core(BTz)was synthesized and its reactivity and spectral changes toward trans-cyclooctene((4E)-TCO-OH),cis-cyclooctene and bicyclo[6.1.0]non-4-yn-9-ylmethanol we...A dimesitylboryl-ended oligothiophene with tetrazine as core(BTz)was synthesized and its reactivity and spectral changes toward trans-cyclooctene((4E)-TCO-OH),cis-cyclooctene and bicyclo[6.1.0]non-4-yn-9-ylmethanol were comprehensively studied.The fluorescence intensity of BTz was enhanced up to more than 100 times upon bioorthogonal reaction with(4E)-TCO-OH.In addition,the first crystal structure of isolated product of tetrazine derivative with cyclooctene was determined,which clearly confirmed a dehydrogenation occurred after Diels-Alder reaction under ambient conditions.展开更多
A nitrone-modified 1,8-naphthalimide was desig ned as a novel bioorthog on alactivated turn-on probe based on strain-promoted alkyne-nitrone cycloadditio n(SPANC).The bioorthog onal cycloadducts were subseque ntly tra...A nitrone-modified 1,8-naphthalimide was desig ned as a novel bioorthog on alactivated turn-on probe based on strain-promoted alkyne-nitrone cycloadditio n(SPANC).The bioorthog onal cycloadducts were subseque ntly tran sformed into fluoresce nt rearra nge-ment products by photo-accelerati on,which exhibited sign ificant fluoresce nee enhan ceme nt,large stokes shift,and high fluores-cence qua ntum yield.DFT calculati ons were performed to elucidate the fluoresce nee OFF-ON mecha nism.This fluoroge nic strategy was successfully applied to labeling of proteins and visualizing mitochondria in live cells in real time.展开更多
Herein we presented a general strategy for in situ assembly of intramolecular charge-transfer(ICT)-based light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling reaction.By introducing iodo group at the ...Herein we presented a general strategy for in situ assembly of intramolecular charge-transfer(ICT)-based light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling reaction.By introducing iodo group at the appropriate position,five fluorophores with different scaffolds including naphthalimide,coumarin,naphthalene sulfonate,nitrobenzoxadiazole,and acetonaphthone,were designed as bioorthogonal multicolor fluorogenic probes,which could produce significant fluorescence enhancement and high fluorescence quantum yield after Suzuki-Miyaura reaction with aryl boronic acid or boronate.Manipulating the substituents andπscaffold in the fluorophores allows fine-tuning of their photophysical properties.With this strategy,we succeeded in peptide conjugation,no-wash fluorogenic protein labeling,and mitochondria-selective bioorthogonal imaging in live cells.展开更多
Bioorthogonal cleavage reaction-triggered prodrug activation by the pretargeted methods can achieve accurate cancer therapy.However,the click and release efficiency of these methods in vivo is limited by the space-tim...Bioorthogonal cleavage reaction-triggered prodrug activation by the pretargeted methods can achieve accurate cancer therapy.However,the click and release efficiency of these methods in vivo is limited by the space-time dislocation of bioorthogonal prodrug-trigger pairs within the tumor area,caused by their asynchronous administration and inconsistent accumulation for most delivery systems.We herein created a nanovoid-confinement and click-activated(NCCA)core–shell nanoreactor by incorporating prodrugs within zeolitic imidazolate framework-90(ZIF-90)as core and coating tetrazine-based covalent organic framework(COF)as shell.After surface modification of aptamer polymer,the NCCA nanoreactor enabled the sufficient delivery of photodynamic prodrugs within tumor.Notably,the core of ZIF-90 was decomposed by tumor acidic environment,inducing the high-efficiency activation of photodynamic prodrugs via nanoconfined bioorthogonal reaction with tetrazine-based COF shell.As a result,such photodynamic agents are efficiently and safely accumulated into tumor and specifically activated for precise photodynamic therapy of cancer cells and tumor bearing mice with minimizing toxic side effect.Taken together,such NCCA nanoreactor clearly demonstrates the critical feasibility to realize the synchronous delivery of both prodrugs and triggers for precise treatment,which most of delivery systems are not able to afford.展开更多
基金supported by the National Natural Science Foundation for Distinguished Young Scholars (82225025)the National Natural Science Foundation of China (21877049,22177038,32171296)+1 种基金Guangdong Natural Science Foundation (2020B1515120043,2022A1515012235)K.C.Wong Education Foundation。
文摘Bioorthogonal reactions involving transition metals have diversified applications in imaging,drug development,chemical catalysis and other fields.Transition metals used to catalyze the bioorthogonal reaction mainly include ruthenium,palladium,copper,and gold.However,the great potential for translational applications of bioorthogonal reaction needs to be further expanded and their reaction efficiency should be improved.Therefore,it is an urgent need for the development of this field to find more suitable catalysts to efficiently catalyze existing biological orthogonal reactions and expand the types of biological orthogonal reactions.Thus,this review not only summarizes those transition metal complexes-based catalysts participating in bioorthogonal reaction and some bioorthogonal reactions involving transition metals inside the cells,but also sheds light into the discovery of new transition metal complexes and their future development in applications.
基金the National Natural Science Foundation of China(No.82073807)。
文摘Cell membrane camouflaged nanoparticles have been widely used in the field of drug leads discovery attribute to their unique biointerface targeting function.However,random orientation of cell membrane coating does not guarantee effective and appropriate binding of drugs to specific sites,especially when applied to intracellular regions of transmembrane proteins.Bioorthogonal reactions have been rapidly developed as a specific and reliable method for cell membrane functionalization without disturbing living biosystem.Herein,inside-out cell membrane camouflaged magnetic nanoparticles(IOCMMNPs)were accurately constructed via bioorthogonal reactions to screen small molecule inhibitors targeting intracellular tyrosine kinase domain of vascular endothelial growth factor recptor-2.Azide functionalized cell membrane acted as a platform for specific covalently coupling with alkynyl functionalized magnetic Fe_(3)O_(4)nanoparticles to prepare IOCMMNPs.The inside-out orientation of cell membrane was successfully verified by immunogold staining and sialic acid quantification assay.Ultimately,two compounds,senkyunolide A and ligustilidel,were successfully captured,and their potential antiproliferative activities were further testified by pharmacological experiments.It is anticipated that the proposed inside-out cell membrane coating strategy endows tremendous versatility for engineering cell membrane camouflaged nanoparticles and promotes the development of drug leads discovery platforms.
基金supported by the National Natural Science Foundation of China(No.81973449)the National Key Research and Development Program of China(Nos.2018YFC1704800 and 2018YFC1704805)。
文摘Mitochondria are essential for eukaryotic life as powerhouses for energy metabolism. Excessive mitochondrial hyperthermia and reactive oxygen species(ROS) production have been associated with aging, cancer,neurodegenerative diseases, and other disorders. Uncoupling protein 2(UCP2) is the effector responsible for regulating cellular thermogenesis and ROS production via dissipating protons in an electrochemical gradient. A UCP2 inhibitor named genipin(GNP) is being researched for its effect on mitochondrial temperature, but little is known about its mechanisms. This study developed several molecular probes to explore the interactions between GNP and UCP2. The result indicated that the hemiacetal structure in GNP could selectively react with the ?-amine of lysine on the UCP2 proton leakage channel through ringopening condensation at the mitochondrial, cellular, and animal levels. A notable feature of the reaction is its temperature sensitivity and ability to conjugate with UCP2 at high fever as lysine-specific covalent inhibitors that prevent mitochondrial thermogenesis. The result not only clarifies the existence of an antipyretic properties of GNP via its irreversible coupling to UCP2, but also reveals a bioorthogonal reaction of hemiacetal iridoid aglycone for selectively binding with the ?-amine of lysine on proteins.
基金This work was supported by the National Key Research and Development Program of China(No.2018YFA0208800)the National Natural Science Foundation of China(No.81720108024)+3 种基金the Natural Science Foundation of Jiangsu Higher Education Institutions,China(No.20KJA150006)the Natural Science Foundation of Jiangsu Province,China(No.BK20191418)the Suzhou Key Industry Technology Innovation Projects,China(No.SYG202036)the Priority Academic Program Development(PAPD)of Jiangsu Higher Education Institutions,China.
文摘Pretargeting is an innovative and promising approach in nuclear medicine for targeted-imaging/therapy through the following bioorthogonal reactions.It requires two reactive participants,one of which is a targeting vector and the other is a small radiolabeled probe capable of specifically coupling through bioorthogonal reactions with the targeting vector accumulated in the disease site.Compared to the conventional direct targeting approach,such a two-step scheme conceptually can achieve a higher imaging contrast and an improved therapeutic effect owing to the suppressed non-specific targeting.In this review,we will first give a brief introduction on pretargeting systems and the history of bioorthogonal reactions,and then focus on some important works about radionuclide delivering through the bioorthogonal reaction based pretargeting strategy.Finally,we will discuss the steps forward in respect to the future clinical translation and truly hope that this methodology would continue to make contributions to nuclear medicines.
基金supported by the National Natural Science Foundation of China(Nos.21977075,22271200)the National Key R&D Program of China(Nos.2022YFC2009902,2022YFC2009900)+1 种基金the Science and Technology Plan Project of Sichuan Province(No.2023YFS0121)the 1·3·5 Project for Disciplines of Excellence at West China Hospital(No.ZYYC23003),Sichuan University。
文摘Here,we present a novel bioorthogonal platform that enables precise positioning of attached moieties in close proximity,thereby facilitating the discovery and optimization of biocompatible reactions.Using this platform,we achieve a Horner-Wadsworth-Emmons(HWE)reaction under physiological conditions,generating a fluorophore in situ with a yield of up to 93%.This proximity platform should facilitate the discovery of various types of biocompatible reactions,making it a versatile tool for biomedical applica-tions.
基金supported by the National Key R&D Program of China(No.2022YFA1207400)National Natural Science Foundation of China(No.22274061)+1 种基金the 111 Project(No.B17019)Fundamental Research Funds for the Central Universities(No.CCNU22QN007).
文摘Due to their excellent fluorescence properties and biological function,cyanine dyes have been widely applied in biological imaging.Heptamethine cyanine(Cy7)dyes,as a type of classic near-infrared(NIR)fluorescent dyes,are considered as one of the effective fluorescent tools in the living organisms due to their good biocompatibility and very low background interference.Bioorthogonal reactions performed in living cells and tissues have developed by leaps and bounds in recent years.The NIR fluorescent labeling technique involving cyanine has attracted widespread attention.This review summarizes their recent application in the field of bioorthogonal imaging,mainly concluding Cy7-type dyes,labeling strategy,bioimaging application,etc.We expect this work can provide some helps for the studies of NIR bioorthogonal reaction in vivo.
基金supported by the National Natural Science Foundation of China(grant nos.22022704,21977097,and 22271291)Chinese Academy of Sciencessupported by the Postdoctoral Innovative Talents Support Program(grant no.BX20200337).
文摘Selective methods for modulating RNA epigenetic modifications within living cells and organisms represent attractive techniques for investigating biological functions and medicinal application.In contrast to enzymatic methods,abiotic chemical modulation offers access to diverse new-to-natural functionalities.We herein report a visible light-assisted bioorthogonal reaction involving flavin mononucleotide,sodium azide,and blue light irradiation.In concert with previous chemical approaches mimicking the demodification pathway,our system functions as a powerful and selective post-modification enzyme that targets N^(6)-prenyl adenosine i^(6) A in RNA and enables the efficient construction of an artificial N^(6)-cyanomethyl adenosine(cnm^(6) A).Notably,most endogenous biomacromolecular functions,including other common RNA epigenetics,remained unaffected.Consequently,we have successfully modified i^(6) A in tRNA to cnm^(6) A in human cells and establish this system as a bona fide bioorthogonal reaction with potential applications in chemical biology and in-cell modulation.
基金supported by the Major State Basic Research Development Program of China (Grant No.2017YFA0205201)the National Natural Science Foundation of China (Grant Nos.81925019 and U1705281).
文摘Benefiting from the advantage of taking place in biological environments without interfering with an innate biochemical process,the bioorthogonal reaction that commonly contains the“bond formation”and“bond cleavage”system has been widely used in targeted therapy for a variety of tumors.Herein,several prominent cases based on the bioorthogonal reaction that tailoring the metabolic glycoengineering tactics to modified cells for cancer immunotherapy,and the innovative tactics for reducing the metal ions’toxic and side effects with microneedle patches will be highlighted.Based on these applications,the complexities,potential pitfalls,and opportunities of bioorthogonal chemistry in future cancer therapy will be evaluated.
基金National Natural Science Foundation of China(Nos.52150222,21672130 and 52073163)the Shenzhen Science and Technology Research and Development Funds(No.JCYJ20190806155409104)the State Key Laboratory of Crystal Materials.We acknowledge Prof.Dr.Cuihua Zhao of Shandong University for her valuable suggestions.
文摘A dimesitylboryl-ended oligothiophene with tetrazine as core(BTz)was synthesized and its reactivity and spectral changes toward trans-cyclooctene((4E)-TCO-OH),cis-cyclooctene and bicyclo[6.1.0]non-4-yn-9-ylmethanol were comprehensively studied.The fluorescence intensity of BTz was enhanced up to more than 100 times upon bioorthogonal reaction with(4E)-TCO-OH.In addition,the first crystal structure of isolated product of tetrazine derivative with cyclooctene was determined,which clearly confirmed a dehydrogenation occurred after Diels-Alder reaction under ambient conditions.
基金This work was supported by the Beijing Nova Program(Z201100006820049)the National Natural Science Foundation of China(No.21907109).
文摘A nitrone-modified 1,8-naphthalimide was desig ned as a novel bioorthog on alactivated turn-on probe based on strain-promoted alkyne-nitrone cycloadditio n(SPANC).The bioorthog onal cycloadducts were subseque ntly tran sformed into fluoresce nt rearra nge-ment products by photo-accelerati on,which exhibited sign ificant fluoresce nee enhan ceme nt,large stokes shift,and high fluores-cence qua ntum yield.DFT calculati ons were performed to elucidate the fluoresce nee OFF-ON mecha nism.This fluoroge nic strategy was successfully applied to labeling of proteins and visualizing mitochondria in live cells in real time.
基金supported by the Beijing Nova Program(No.Z201100006820049)the National Natural Science Foundation of China(No.21907109)the CAMS Innovation Fund for Graduate Students(No.2019–1007–03)
文摘Herein we presented a general strategy for in situ assembly of intramolecular charge-transfer(ICT)-based light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling reaction.By introducing iodo group at the appropriate position,five fluorophores with different scaffolds including naphthalimide,coumarin,naphthalene sulfonate,nitrobenzoxadiazole,and acetonaphthone,were designed as bioorthogonal multicolor fluorogenic probes,which could produce significant fluorescence enhancement and high fluorescence quantum yield after Suzuki-Miyaura reaction with aryl boronic acid or boronate.Manipulating the substituents andπscaffold in the fluorophores allows fine-tuning of their photophysical properties.With this strategy,we succeeded in peptide conjugation,no-wash fluorogenic protein labeling,and mitochondria-selective bioorthogonal imaging in live cells.
基金supported by the National Key R&D Program of China(No.2019YFA0210103)the National Natural Science Foundation of China(No.21974039)+3 种基金China Postdoctoral Science Foundation(Nos.2020TQ0092 and 2020M682537)the Science and Technology Innovation Program of Hunan Province(No.2020RC2019)the Special Funds for the Construction of Innovative Provinces in Hunan Province(No.2019RS1031)the Opening Fund of Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research(Hunan Normal University),the Ministry of Education.
文摘Bioorthogonal cleavage reaction-triggered prodrug activation by the pretargeted methods can achieve accurate cancer therapy.However,the click and release efficiency of these methods in vivo is limited by the space-time dislocation of bioorthogonal prodrug-trigger pairs within the tumor area,caused by their asynchronous administration and inconsistent accumulation for most delivery systems.We herein created a nanovoid-confinement and click-activated(NCCA)core–shell nanoreactor by incorporating prodrugs within zeolitic imidazolate framework-90(ZIF-90)as core and coating tetrazine-based covalent organic framework(COF)as shell.After surface modification of aptamer polymer,the NCCA nanoreactor enabled the sufficient delivery of photodynamic prodrugs within tumor.Notably,the core of ZIF-90 was decomposed by tumor acidic environment,inducing the high-efficiency activation of photodynamic prodrugs via nanoconfined bioorthogonal reaction with tetrazine-based COF shell.As a result,such photodynamic agents are efficiently and safely accumulated into tumor and specifically activated for precise photodynamic therapy of cancer cells and tumor bearing mice with minimizing toxic side effect.Taken together,such NCCA nanoreactor clearly demonstrates the critical feasibility to realize the synchronous delivery of both prodrugs and triggers for precise treatment,which most of delivery systems are not able to afford.
