Huihuan Qin,Lingling Li,Kun Li*,Xiaoqi Yu*

Key Laboratory of Green Chemistry and Technology,Ministry of Education,College of Chemistry,Sichuan University,Chengdu 610064,China

Key words:Monoamine oxidase-B Fluorescent probe Covalent-assembly Coumarin Cell imaging

ABSTRACT To detect monoamine oxidase B(MAO-B),the level of which is one of the most important indicators of neurodegenerative diseases such as Parkinson's disease,a new type of cascade reaction based on the formation of coumarin was applied in this research.After the reaction with MAO-B,the protecting group of hydroxyl group in probes(DEAN-MA and DEAB-MA)was removed,and the fluorescence intensity significantly increased(l em=456 nm)as the structure of coumarin wasformed subsequently.The probes showed excellent sensitivity and selectivity to MAO-B.The detection limit of DEAN-MA and DEAB-MA were 0.6ng/m Land 0.2 ng/m L,respectively.We succeeded in detecting MAO-B[23_TD DIFF]in vitro and imaging it in human astrocyte(U[24_TD DIFF]87).

Monoamine oxidase B(MAO-B)belongs to the fl avin monoamine oxidase family,which catalyzes the oxidation of biogenic and xenobiotic amines and plays an important role in the catabolism of neuroactive and vasoactive amines in the central nervous system and peripheral tissues.MAO dysfunction is thought to be responsible for several psychiatric and neurological disorders.Especially,the level of MAO-B is related to Alzheimer’s disease and Parkinson’s disease[1,2].Therefore,detecting MAO-B in human nerve cells is of great signi fi cance.Various technologies have been developed for the detection of MAO-B.Compared with technologies like positron emission tomography(PET)[3,4],magnetic resonance imaging(MRI)[5,6],and enzyme-linked immunosorbent assay(ELISA)that exploits bio-reducible agents,fluorescence imaging as a powerful non-invasive method with high sensitivity is regarded particularly suitable for monoamine oxidase detection[7–14].

In recent years,many fluorescent probes have been reported for MAO-B,most of which explored the fluorescence-enhancement strategy by removing the reducible group and releasing the fluorescence signal of the fluorophore based on MAO-B’s redox reaction[10](Scheme 1a).However,one shortage of this design strategy is that the original fluorescence intensity of the probes is hard to form a zero background before reacting with MAO,which w ould influence the sensitivity of the detection.Therefore,developing “off-on”fluorescent probe from a zero background is highly desirable.Probe[25_TD DIFF]via “covalent-assembly”strategy(raised by Yang group)could provide an approach to get “off-on”or ratiometric fluorescent probe,since a new fluorophore can be formed via covalent reaction of tw o fragments [15].The fundamental feature of “covalent-assembly”probe is that it w arrants both a colorimetric change and a turn-on fluorescent signal from a zero background.Moreover,the conjugative covalent-assembly of the backbone may cause a “red shift”of the fluorescence spectra,since a new fluorescent conjugated structure is developed.

In fact,several“covalent-assembly”probes have been successfully applied in the detection of small molecules including sulfurcontaining species(SCS)and reactive oxygen species(ROS)[16–24].However,such strategy is hardly applied in the detection of MAO[12,13].Sames group[13]reported a coumarin sw itch for MAO-B,based on a sequence of enzymatic oxidation and intramolecular cyclization(Scheme 1b).In this work,a different design strategy was applied in the “covalent-assembly”to detect MAO-B.Instead of expanding the conjugate planes of the fluorophore,we new ly built a fluorescent skeleton.As indicated in Scheme 1c,after reacting with MAO-B,the re-exposed oxygen anion w ill attack the nearest carbonyl carbon atom,leading to the formation of the fluorophore and the off-on fluorescence effect.

Schem e 1.(a)The reaction process of probes with MAO-B in most previous research.(b)Previously reported “covalent assembly”MAO-B probe.(c)The“covalent assembly”strategy applied in this work.

As a kind of important fluorophore,coumarin has large conjugate system,strong molecular electron transfer ability,good thermodynamic and photochemical stability.As a result,a large number of coumarin based fluorescent probes have been reported for anions,metal ions and small molecules[25–28].As an important reactant of coumarin in synthesis,salicylaldehyde compound has a hydroxyl moiety which is the key reaction group.If the hydroxyl group was protected,the formation of coumarin w ill be restricted because of the lack of nucleophilic group.Once the protecting group was removed,coumarin skeleton w ould be formed.In the light of“covalent-assembly”strategy,we want to develop a novel approach for MAO-B detectionvia this cascade reaction.In this work,tw o salicylaldehyde derivatives decorated with the 3-aminopropoxy group were designed for the detection of MAO-B.With the sequence of enzymatic oxidation by MAO-B and intramolecular cyclization,fluorescence signal of coumarin w ould be turned on.Furthermore,the development of inhibitors of MAOB is an important item in the treatment of neurodegenerative diseases[29,30].We therefore detected the inhibition of pargyline(N-benzyl-N-methylprop-2-yn-1-amine)to MAO-B using our probes.It was proved that both DEAN-MA and DEAB-MA were capable of screening MAO-B inhibitors.Importantly,it has been suggested that the expression level of MAO-B in nerve cells is higher than non-nerve cells[31],we therefore applied our probes to the imaging of MAO-Bin both HL-7702 cells(human liver cells)and U 87 cells(human astrocyte).

Firstly,the enzyme activity of MAO-Bwas tested to be 4.8 U/mg[30_TD DIFF]via UV?vis absorption spectra(Fig.S1 in Supporting information)[32].Then the reaction conditions for probe DEAN-MA and DEABMA with MAO-Bwere studied,including the effects of p H and the concentration of probes.The enzymatic reaction was carried out in the p H range of 5.3 ?9.0.And It was discovered that the fluorescence barely changed under acidic conditions(p H ?7.0),while a signi fi cant enhancement of the probe could be observed in biological conditions(p H around 7.4)after incubation with MAO-B for 1h.This indicated that DEAN-MA and DEAB-MAshould be able to serve as sensitive MAO-Bprobes under physiological conditions(Fig.S2 in Supporting information).Follow ing that,the optimum concentration of the probes to react with MAO-B(10 m g/m L)were studied.As indicated in Fig.S3(Supporting information),the fluorescence intensity reached a plateau w hen the concentration of the probes reached 10 m mol/L.The follow ing experiments were therefore all carried out using 10 m mol/L as the concentrations of the probes.

As seen from Fig.1,fluorescence studies revealed that probe DEAB-MA and DEAN-AM had very weak fluorescence in HEPES buffer.Reaction of probe DEAN-MA and DEAB-AM with MAO-B leaded to the change of fluorescence signal at 456 nm from “off”state to “on”state.These results indicated that both fluorescence probes DEAN-MA and DEAB-MA were MAO-B reactive.

Fig.1.(a)The fluorescence response to MAO-B(10 m g/m L)and the image of fluorescence response of probe DEAN-MA(10 m mol/L)to different concentration of MAO-B.(b)The fluorescence response of probe DEAB-MA(10 m mol/L)to MAO-B(10 m g/m L)and the image of fluorescence response of probe DEAN-MA(10 m mol/L)to different concentration of MAO-B.Spectra collected 1h after MAO-B addition.l ex:360 nm(DEAB-MA),375 nm(DEAN-MA).Solvent:HEPESbuffer(100 mmol/L,p H 7.4,1%DMSO and 5%glycerol included)[14].

Fig.2.The signal-to-background ratio of probe DEAN-MA(a)and DEAB-MA(b)(10 m mol/L)to several distractors.Spectra was collected 1 h after substance addition.The concentration of enzymes except MAOs was 1 mg/m L,and the concentration of h2O2 and amino acids was 1 mmol/L.Insert:1.horse radish peroxidase,2.glucoseoxidase,3.H2O2,4.sparsely cotton like thermophilic lipase,5.[42_TD DIFF]L-GSH,6.L-cysteine,7.L-serine,8.pancrelipase,9.trypsin,10.L-high cysteine,11.D-glutamic acid,12.acyl transferase,13.MAO-A(10 m g/m L),14.MAO-B(10 m g/m L).

We then studied the specificity and selectivity of the probes towards MAO-B.We examined various potential interfering species in parallel under the same conditions,including H2O2,serine,glutamic acid,cysteine,glutathione,GSH,high-cysteine,some oxidoreductase and other related enzymes.As depicted in Fig.2,probe DEAN-MA exhibited high selectivity to MAO-B over the species tested,which may be ascribed to the specific oxidation of the substrate by the enzyme.Probe DEAB-MA also showed high signal to background ratio after reacting with MAO-B,however,interferences fromL-GSH and L-cysteine were also hard to omit.Therefore,only DEAN-MA was applied in the living cell imaging experiments.Furthermore,both DEAN-MA and DEAB-MA showed selectivity tow ards MAO-B to MAO-A under the same concentration,which indicates that probes are more suitable for detecting MAO-B than MAO-A.

Fig.3 show s the fluorescence change of probes with MAO-B at various concentrations.As is seen,the fluorescence intensity of the probes increased gradually with increasing concentration of MAOB.A good linearity between the fluorescence intensity and the concentration of MAO-Brange of 0–10m g/m Lwith an equation of[I456=13.82209?[MAO-B]+38.11( R2=0.960)was observed.The detection limit(k[38_TD DIFF]=3)of DEAN-MA and DEAB-MAwas respectively determined to be 0.6ng/m L and 0.2 ng/m L[33],which was better or comparable to previously published MAO probes.

The probe can also be used for the screening of potential MAO-B inhibitors.As show n in Fig.4,with increasing concentrations of pargyline(N-benzyl-N-methylprop-2-yn-1-amine),a typical inhibitor of MAO-B,the emission intensity gradually decreased.The IC50value of pargyline obtained by using the logistic fi t of the experimental data was 9.3 m mol/L for DEAN-MA and 2.6 m mol/L for DEAB-MA.This result suggests that DEAN-MA and DEAB-MA have the potential of being applied to the screening of MAO-B inhibitors.

Fig.3.The fluorescence intensity of DEAN-MA(a)and DEAB-MA(10 m mol/L)(c)after reacting with MAO-B(the concentration of MAO-Brange is 0[13_TD DIFF]?10 m g/m L)for 1 h and their linear graphs for DEAN-MA(b)and DEAB-MA(d).

Fig.4.The fluorescence intensity of DEAN-MA(a)and DEAB-MA(b)(10 m mol/L)reacting with MAO-B(10 m g/m L)for one more hour after deferent concentration of inhibitor incubated with MAO-B 1h.

The reaction mechanism was proved by ESI-MSof the reacting product and the fluorescence spectra.The peak at m/z 255.0655[M-Na+]of fluorescent molecule EMCCwas found in the reaction mixture with DEAB-MAand MAO-B,and the peaks at m/z 269.0802[M-H+]and 291.0616[M-Na+]of EBCCwere found in the reaction mixture with DEAN-MA(Fig.S4 in Supporting information).The results of fluorescence spectra showed that the emission spectra of the theoretical product EMCCwas the same to the reaction mixture of DEAB-MA and MAO-B,and DEAN-MA showed the same result.These results proved the proposed reaction mechanism between the probes and MAO-B(Scheme 1c,Fig.S5 in Supporting information).

Fig.5.The fluorescence image of control of U87 cells(a,b,c)and incubated with DEAN-MA(d,e,f),control of HL-7702 cells(g,h,i)and incubated with DEAN-MA(j,k,l).a,d,g,j for blue channel;b,e,h,k for overlay;c,f,i,l for bright.

Follow ing that,we found the DEAN-MAcould response to MAOBat lex[39_TD DIFF]=405 nm as well(Fig.S6 in Supporting information),so the cytotoxicity of probe DEAN-MA to U87 and HL-7702 cells were studied(Fig.S7 in Supporting information).The results showed that the cells viability was still more than 80%w hen the concentration of DEAN-MA reached 10 m mol/L.Therefore,it was safe to say that DEAN-MApossessed relatively low cytotoxicity and the follow ing fluorescence imaging experiments could be carried out with 10 m mol/L DEAN-MA.

Fluorescence imaging of HL-7702 cellsand U87 cellswasshow n in Fig.5.The fluorescence in U87 cells was higher than that in HL-7702 cells,through which we successfully demonstrated that the level of MAO-Bin nerve cells is higher than somatic cells including liver epithelial cells.This result is in line with the previous report[28].We then studied the distribution of the probe inside cells.As show n in Fig.6,there was no obvious colocation effect between probe DEAN-AM and mitochondria tracker red,lysosome tracker red,or endoplasmic reticulum tracker red in U87 cells.The result suggested that probe DEAN-AM was distributed in various organelles after diffusion into cells.

In summary,the tandem reaction-based probes were used to detect MAO-B selectively[40_TD DIFF]in vitro under the suitable p H value(p H 7.4)and temperature(37?C).The 5.8-fold signal enhancement for DEAN-AM and 6.4-fold signal enhancement for DEAB-AM were detected and both of them showed selectivity between MAO-Band MAO-A.Importantly,the novel design strategy of utilizing targeted enzyme to build a fluorophore in the reaction process could be of great importance for the future design of fluorescent probes.Meanwhile,probe DEAB-MA showed stronger fluorescence response and lessselectivity,which may be caused by the different substituent on the benzene ring from DEAN-MA.Moreover,the inhibition assay suggested the potential use of DEAB-MA and DEAN-MA on the screening of MAO-B inhibitors.Furthermore,fluorescence imaging results corresponded with previous published results about the higher level of MAO-B in nerve cells.Altogether,these results proved the great value and potential of DEAB-MA and DEAN-MA in the future study of MAO-B.

Fig.6.The fluorescence image of U87 cells incubated with DEAN-MA and mitotracker(a,b,c,d),lyso-tracker(e,f,g,h),ER-tracker(i,j,k,l).a,e,ifor red channel of tracker;b,f,j for blue channel of DEAN-MA;c,g,k for overlay;d,h,l for bright.

Acknow ledgments

This work was fi nancially supported by the National Natural Science Foundation of China(Nos.21572147 and 21472131).We also thank the Comprehensive Training Platform of Specialized Laboratory,College of Chemistry,Sichuan University for sample analysis.

Appendix A.Supp lementary data

Supplementary material related to this article can be found,in the online version,at doi:10.1016/j.cclet.2018.05.018.