Yuqi Go,Yuxing Lin,Tingting Liu,Xiomeng Zhng,Feng Xu,Pn Liu,Lupei Du,Minyong Li,b,*

a Department of Medicinal Chemistry,Key Laboratory of Chemical Biology(MOE),School of Pharmacy,Shandong University,Ji’nan 250012,China

b State Key Laboratory of Microbial Technology,Shandong University,Ji’nan 250100,China

Key words:Chemiluminescent imaging Chemiluminescence 1,2-Dioxetane Palladium(II)Bioassays

ABSTRACT As an important transition metal catalyst,palladium is extensively used in many areas including electronics industry,petroleum industry,automobile industry and fine chemicals engineering.However,it brings harm to the environment as well as people’s health.Herein,we managed to introduce a butynyl group to 1,2-dioxetane developing a reaction-based chemiluminescent probe as well as an imaging approach for monitoring palladium(II).Exhibiting enhanced total fl ux in Pd 2+enriched areas,palladium chemiluminescent probe(PCL)may afford potential utility for detecting Pd 2+in vitro,in cellulo and in vivo.?2018 Chinese Chemical Society and Institute of Materia Medica,Chinese Academy of Medical Sciences.

Palladium,an important transition metal,is w idely distributed in the environment and displays extraordinary catalytic performances in coupling reactions[1].In the past few decades,palladium has been extensively used in many areas including electronic industry[2],petroleum industry[3],automobile industry[3],glass manufacture[4]and fine chemicals engineering[1,4],which has done harm to the environment in the meantime.Moreover,palladium is hard to be biodegraded and easy to be enriched through the food chain[5].It is well know n that palladium can coordinate with macromolecules such as DNAand proteins leading to DNA degradation,allergic reaction as well as enzyme inhibition[6,7].Therefore,specific and selective detection of palladium in environmental and biological samples is urgently desirable.

There are many traditional methods for palladium detection,including X-ray,atomic emission spectroscopy(AES),inductively coupled plasma atomic emission spectrometry(ICP-AES),inductively coupled plasma mass spectroscopy(ICP-MS),solid-phase microextraction high-performance liquid chromatography(SPMEHPLC)and atomic absorption spectroscopy(AAS),by which accurate detection can be achieved rapidly and sensitively.However,these methods require complicated sample-pretreatment procedures,well-controlled experimental conditions,expensive facilities and highly-trained individuals.As a result of high selectivity,sensitivity,rapidity,low cost and operational simplicity,optical probes[8,9],such as colorimetric probes,fluorescent probes and luminescent probes,provide choices for determination of palladium species.

A highly sensitive readout can be provided by a triggered luminescence emission process,which is extremely favorable for bioimaging because of free interferences caused by light scattering and reduced background noise due to the absence of photonic excitation.For decades,bioluminescence has been w idely applied in the preclinical analysis using genetically modified cells or organisms.While bioluminescent enzymes,for example,fi re fl y luciferase and renilla luciferase,are necessary for bioimaging,chem iluminescence can be used with w ild-type cells,organisms,and even animals and offers opportunities for clinical imaging.

Therefore,we decided to develop chemiluminescent imaging(CLI)agents that can be utilized in luciferase-null systems[10–12].Currently,we chose 1,2-dioxetane,which can be sterically stabilized by spiroadamantane,as our chemiluminescent platform[13–20].Since the cleavage of a chemical bond triggers light emission of the chemiluminescence,it is signi fi cant to develop recognition moiety for detecting palladium ions sensitively and selectively.Bene fi tting from the capacity of palladium,“off-on”probes were explored,taking advantage of typical-metal binding[21],Pd-catalyzed Claisen rearrangement[22],Pd-catalyzed Tsuji-Trost reaction[23–25],Pdcatalyzed depropargylation[16,26]and so on.

Schem e 1.Structure and proposed chemiluminescent mechanism of palladium probe PCL.

On account of these considerations and expanding the techniques of chemiluminescent imaging,we managed to introduce butynyl group to 1,2-dioxetane moiety generating the palladium chemiluminescent probe(PCL)(Scheme 1).To be honest,our original plan was to design a mercury probe with alkynyl butylcarbonate as the recognition moiety[27,28].It was supposed that electronically excited m-oxybenzoate anion released once butynyl moiety is cleaved via metal-catalyzed reaction.Then,the excited intermediate undergoes an electron transfer process to return to the ground state,according to CIEEL(chemically initiated electron exchange luminescence)mechanism.Finally,the extra energy released in the form of light.However,according to the results of the preliminary experiment,our probe seems to be more sensitive to palladium cation than mercury at mild condition.Therefore,subsequent studies were centered on palladium(II)imaging using PCLin the current report.

The probe PCL was designed such that chemiluminescent emission w ould be initiated by the Pd-catalyzed cleavage of butynyl moiety and prepared conveniently according to the route illustrated in Scheme S1(Supporting information).In brief,the synthesis begins with the preparation of the intermediate compound 4 which can convert to 1,2-dioxetane by photocatalyzed oxidation.Assisted with N,N'-disuccinimidyl carbonate(DSC),but-3-yn-1-ol was connected to compound 4 to yield compound 6,the precursor of PCL.Finally,compound 6 was oxidized under visible light irradiation at 0?Cand the fi nal probes were obtained.

After preparation,chemiluminescence intensity was measured with an IVIS kinetic imaging system(Caliper Life Sciences,Hopkinton,Massachusetts,U.S.A.)equipped with a cooled charge-coupled device(CCD)camera.More details about synthetic experiments and chemiluminescence imaging assays are well described in Supporting information.

Stability of PCL in an aqueous system with different p H was examined initially,since acid-base status in the environment may influence the carbonate moiety in PCL.The probe(100 m mol/L)was incubated in Tris-HCl with a w ide p H range from 3 to 10 for approximately 60 min.After incubation,the chemiluminescence intensity was recorded with a living imaging system as depicted in Fig.1.These results suggested that the palladium probe is stable in the aqueous system within a p H range of 3–7,although luminescence augmented remarkably in the system with p H more than 9.

Fig.1.influence of p H on the stability of PCL.(A)Chemiluminescence imaging of PCL in Tris-HCl with various p H ranging from 3 to 10.(B)Quanti fi cation of chemiluminescence intensity of PCL for each p H.

Fig.2.Sensitivity assay of PCL in vitro.(A)Time scans of the chemiluminescence intensity of PCL(50 m mol/L)reacted with Pd Cl2(12.5 m mol/L)in PBS(1?,p H 7.4)containing 10%(v/v)Enhancer Emerald II.(B)Quanti fi cation of chemiluminescence intensity of PCL(50 m mol/L)with Pd 2+(0–2.5 mmol/L)in PBS(1?,p H 7.4)containing 10%(v/v)Enhancer Emerald II.(C)Quanti fi cation of chemiluminescence intensity of PCLwith Pd Cl2(100 m mol/L)and EDTA(0 to 2 mmol/L)in PBS(1?,p H 7.4)containing 10%(v/v)Enhancer Emerald II.

Sensitivity of PCL(50 m mol/L)for palladium was evaluated in an aqueous system.As illustrated inFig.2 and Fig.S1(Supporting information),optical signals of the probe fl uctuated with time,w hen reacted with palladium species(25 m mol/L).As 60 min passed,a plateau occurred,and the luminescence intensity was almost invariant although declined slightly.Subsequently,after incubation with Pd Cl2in PBS(1?,p H 7.4)of variousconcentrations(0 to 2.5 mmol/L)for 75 min,the probe’s luminous fl ux was measured.It is indicated that PCL is sensitive to Pd2+within a concentration range from 3.125 m mol/Lto 500 m mol/L.Meanwhile,limit of detection(LOD)of PCLto Pd2+can be calculated aslessthan 6.25 m mol/L.As for the decrease of photon emission w hen the concentration of palladium species reached a high level,we speculated that heavy metal species in higher concentration might depress the effect of Enhancer Emerald II.

It is also speculated that only free palladium ion can catalyze the cleavage of the butynyl moiety in PCL.Therefore,we commenced another experiment to con fi rm our conjecture.Previously,palladium chloride(100 m mol/L)was incubated with EDTAsolution at variousconcentrations(0 to 2 mmol/L)at 37?Cfor 60 min.Follow ing the addition of PCL(50 m mol/L),the samples were incubated for an extra 60 min.Finally,the optical signals of PCLwere measured by an IVISkinetic imaging system to fi nd that chemiluminescent intensity declined by the increasing amount of EDTA( Fig.2 and Fig.S1).Accordingly,the probe displays an immediate concentration-dependent photon emission,which isan ideal property for imaging of free palladium ion in biological environment.

Fig.3.Selectivity assay of PCL in vitro.(A)Chemiluminescence imaging of selectivity of PCL tow ards various metal cations.(B)Quanti fi cation of the chemiluminescent intensity of PCL for each condition.

Selectivity of PCL(50 m mol/L)for Pd2+(50 m mol/L)against a range of metal species,including heavy metals as well as microelements existing in the body,was assessed in an aqueous system.As illustrated in Fig.3 and Fig.S2(Supporting information),an evident enhancement in chemiluminescence intensity displayed w hen Pd2+existing,compared with other metal cations.These response and selectivity data suggested that PCL with fine sensitivity and selectivity,combined with bene fi cial characteristics of the chemiluminescence technique,affording potential utility for detection of Pd2+in cellulo and in vivo.

Experiments mainly focusing on proving the hypothesis of the probe’s mechanism were also carried out.To evaluate the liability of PCL to palladium ion,the probe in an aqueous solution was reacted with palladium chloride at room temperature for 3 h.After microporous fi ltration,the sample was analyzed with HPLC by comparing the sample with PCL or methyl 3-hydroxybenzoate.According to the HPLC spectra exhibited in Fig.4,w hen existing palladium ion,PCL decomposed,making absorbance at 4.3 min(the retention time of PCL)declined.At13.0 min,which is the retention time of methyl 3-hydroxybenzoate,a new peak appeared simultaneously.The proposed mechanism of the palladium probe was con fi rmed by HPLC(Fig.4).

Another palladium chemiluminescent probe with Pd-catalyzed depropargylation mechanism was designed developed previously[16].It is con fi rmed that the chemiluminescence could be triggered via the catalytic depropargylation of Pd(0).Although light emission of the probe occurred w hen treated with Pd(II),the responsibility still can be attributed to the reduction of Pd(II)to Pd(0)by PPh3actually.While the selectivity data of PCL manifested that this palladium probe is sensitive to Pd(II)with specificity.Thus,PCLisa novel chemiluminescent probe for Pd(II)detection despite it may not be the fi rst reported one among its analogues.

Fig.4.HPLCspectra of PCL(100 m mol/L),PCL(100 m mol/L)incubated with Pd Cl2(50 m mol/L)for 3 h and methyl 3-hydroxybenzoate(100 m mol/L).

Fig.5.Palladium imaging in HeLa cells using PCL.(A)Variation of chemiluminescence intensity in 60 min(400 m mol/L of Pd Cl2).(B)Quanti fi cation of the chemiluminescent intensity of PCL for each condition.

Afterw ards,the ability of PCL to imaging palladium in living cells was investigated.To import palladium ion into living cells,human cervical cancer cell lines HeLa wascultivated with the Pd Cl2solution in PBS(1?,p H 7.4)for 1 h before the test.As illustrated in Fig.5 and Fig.S3(Supporting information),chemiluminescence intensity continually enhanced along with the increasing concentrations(0–400 m mol/L)of palladium chloride.After 45-minreaction,luminescent fl ux reached the peak value,w hen the relative chemiluminescence intensity at 400 m mol/Lof Pd Cl2came up to approximately 130%.The results exposed that PCL was capable of palladium imaging at the cellular level with a series of dynamic changes.According to the results of cytotoxicity test accomplished by SRB cytotoxicity assay,the hypoxia chemiluminescent probe is low-toxic and fine-biocompatible(Fig.S4 in Supporting information).

Additionally,bioactivity of PCL tow ards palladium ion in commercial rabbit plasma(20 folds diluted with saline)was investigated.For a 1-hour-incubation,as depicted in ig.6 and Fig.S5(Supporting information),the chemiluminescence intensity augmented with the concentrations of Pd Cl2from 25 m mol/L to 100 m mol/L.These results indicated that PCL could detect palladium quantitatively even in such complex biological samples.Unfortunately,20-fold diluted rabbit plasma treated only with PCL released a chemiluminescence signal over 2.5 times stronger than PCL treated with saline,which drove dow n the LOD of PCL.The disadvantage may also be attributed to the instability of carbonate structure in complex biological samples.However,this study can still provide some valuable data and experience for establishing a chemiluminescent system for the animal model.

Fig.6.Palladium imaging in 20-fold diluted rabbit plasma using PCL.(A)Variation of chemiluminescence intensity in 60 min(100 m mol/Lof Pd Cl2).(B)Quanti fi cation of the chemiluminescent intensity of PCL for each condition.

Fig.7.Palladium imaging in vivo using PCL.(A)Chemiluminescence imaging of PCL tow ards Pd2+with various concentrations(0–100 m mol/L).(B)Quanti fi cation of chemiluminescence intensity from the subcutaneous injection areas of the rude mice of triplicates(n=4).(C)Variation of chemiluminescence intensity within 35 min.

The capability of PCLfor palladium(II)imaging in living animals was interrogated.We attempted to subcutaneous inject the 50 m L of saline or palladium chloride(20 m mol/L,50 m mol/L or 100 m mol/L)in saline.Follow ing,50 m L of PCL(1 mmol/L)in saline containing 10%(v/v)Enhancer Emerald IIwas subcutaneous injected in situ respectively.Thereafter,subsequent chemiluminescence intensity was recorded with living imaging system.As show n in Fig.7,after injection of PCL,obviously enhancing emission signals from injection regions in rude mice were observed,reaching peak values at about 30 min.The enhanced chemiluminescence intensity lasted for about 5 min.As was expected,distinct enhancement of luminescence intensity can be observed visually by living imaging,as injection amount of Pd Cl2grew from 0 to 100 m mol/L.

In summary,herein we designed and prepared a palladium chemiluminescent probe(PCL)by using 1,2-dioxetane as the chemiluminescence platform and butynyl moiety as the recognition unit.Exhibited desirable properties,including no requirement for excitation light,low background noise,high sensitivity,high selectivity and the ability to function well in aqueous media,PCLis capable of monitoring palladium ion visually in vitro,in cellulo and in vivo.Although the instability of carbonate structure in PCLmay drive dow n the LODs in the basic system or in complex biological samples,the exploration brought experience and expand the palladium imaging toolkit and applications of chemiluminescence technology.

Acknow ledgments

The present work was supported by grants from the Taishan Scholar Program at Shandong Province,the Qilu/Tang Scholar Program at Shandong University,the Key Research and Development Project of Shandong Province(No.2017CXGC1401)and the Major Project of Science and Technology of Shandong Province(No.2015ZDJS04001).

Appendix A.Supp lementary data

Supplementary data associated with this article can be found,in the online version,at https://doi.org/10.1016/j.cclet.2018.03.028.