Interactions of a Water-Soluble Diiron Hexacarbonyl Complex with Biologically Relevant Molecules and Their Promotion in CO-Release

JIANG Xiu⁃Juan XIAO Zhi⁃Yin LONG Li CHEN Li⁃Mei ZHANG Li⁃Qiu LIU Xiao⁃Ming

(College of Biological,Chemical Sciences and Engineering,Jiaxing University,Jiaxing,Zhejiang 314001,China)

Abstract:In this study,a water⁃soluble diiron carbonyl complex,[Fe2(μ⁃SCH2R)2(CO)6](R=CH(OH)CH2(OH),1),which has the potential as a CO⁃releasing molecule(CORM),was used to spectroscopically investigate its interac⁃tion with some biological molecules,such as hemoglobin(Hb),myoglobin(Mb),bovine serum albumin(BSA),gluta⁃thione(GSH),and DNA.The IR spectroscopic results showed that the proteins and GSH could promote the decom⁃position of complex 1 to release CO.All the CO⁃release progress followed the first⁃order kinetic model and GSH pos⁃sessed the highest efficiency in promoting CO⁃release.UV absorption spectral variations and fluorescent quench effect also indicated the interactions between these biologically relevant molecules and the diiron carbonyl complex.CD spectra of the mixture of the proteins and complex 1 indicated that no conformational changes in the proteins are induced.The interactions between pUC19 plasmid DNA and complex 1 suggested that the complex could not cause DNA damage.

Keywords:CO⁃releasing molecule;diiron hexacarbonyl complex;proteins;water⁃soluble

In the past decades,the therapeutic potentials of carbon monoxide(CO)have attracted considerable attention[1⁃4].This is because this gaseous molecule possesses cytoprotective, anti⁃inflammatory, anti⁃proliferative,and anti⁃apoptotic functions,which are of potential therapeutic applications[5⁃7].To explore the therapeutic potentials of CO,its controllable delivery is highly desired.Since there are several shortcomings in directly administering gaseous CO,CO⁃releasing mole⁃cules(CORMs)have been developed in the past decades aiming at precisely and controllably delivering CO to targeted tissues or organs.Metal carbonyl com⁃plexes are classic organometallic compounds of transi⁃tion metals with CO and release CO upon external stim⁃ulations such as solvolysis,ligand exchange,irradia⁃tions.Therefore,an increasing number of transition metal compounds have been investigated as CORMs[8⁃11].Among various CORMs,iron carbonyl complexes have attracted considerable attention[12⁃18],because not only there are tremendous iron carbonyl complexes available,but also both the complexes them⁃selves and the residues after CO⁃release are arguably less detrimental compared to other transition metal complexes since iron is one of the essential elements in the human body[18].

Of many iron carbonyl complexes,diiron carbonyl complexes have attracted particular attention in the last twenty years or so due to their structural resem⁃blance to the diiron subunit of[FeFe]⁃hydrogenase and thus,have intensively been investigated as the mimics of the diiron subunit[19⁃22].The high CO ⁃loading per diiron carbonyl complex molecule and capability of releasing CO upon ligand exchange reaction makes them extremely attractive as potential CORMs[18,23⁃24].Our previous reports have demonstrated that these diiron carbonyl complexes could release CO under the stimulation of nucleophilic substitution reactions[23⁃24].A water⁃soluble diiron hexacarbonyl complex,[Fe2{μ⁃SCH2CH(OH)CH2(OH)}2(CO)6](1,Scheme 1),is such a typical example that releases CO with the pres⁃ence of cysteamine(CysA)or some amino acids[23,25].

Scheme 1 Structure of complex 1

It is well known that proteins,one category of the essential biological molecules in living organisms,are full of nucleophilic residues.Therefore,the interaction between a CORM and proteins is inevitable.Indeed,the study of such an interaction has been one of the research interests and is of significance in drug design and understanding the pharmacology and biocompati⁃bility of CORMs as drugs.It is known that the concen⁃tration of glutathione(GSH)is about 5⁃fold(ca.10 mmol·L-1)higher in cancer cells than in normal cells(ca.2 mmol·L-1)[26].Therefore,it is of importance to study the interaction of iron⁃based CORMs prodrug with GSH since iron could cause Fenton reaction and the released CO can compete with O2to bind to cyto⁃chrome C in cells.These events in cells can play roles in suppressing cancer growth.In pharmacological investigations,how a drug interacts with biological mol⁃ecules such as proteins,GSH,DNA is of fundamental importance.Herein,we report our primary study in the interactions of hemoglobin (Hb),myoglobin (Mb),bovine serum albumin(BSA),GSH,and pUC19 plas⁃mid DNA with complex 1 to examine how these biologi⁃cally relevant molecules promote its CO⁃release.The kinetics of the CO⁃release of the complex upon the interaction with these proteins/GSH was examined using IR spectroscopy.Its interactions with these bio⁃logical molecules were further probed using other spec⁃troscopic techniques,such as UV⁃Vis,fluorescent,and circular dichroism(CD)spectroscopy.The interactions between the complex and pUC19 were monitored using gel electrophoresis.UV absorption spectra variation and fluorescent quench effect on the proteins in the presence of complex 1 showed interactions between the proteins and complex 1.But CD spectra showed that there was hardly any conformational change in the pro⁃teins in the interactions with complex 1.However,the DNA interaction test revealed that the complex could not cause any damage to the DNA.

1 Experimental

1.1 Materials and instrumentations

All operations were carried out under an argon atmosphere.Mb(equine heart),Hb(bovine),and BSA were purchased from Beijing BioDee Biotechnology Co.,Ltd.,and GSH,CysA,Na2HPO4,NaH2PO4were purchased from Aladdin.Complex 1 was synthesized using the method we reported before[23].FTIR spectra were recorded on Agilent 640 using a CaF2⁃cell with a spacer of 0.1 mm.UV⁃Vis spectra were measured on Evolution 201(Thermo ScientificTM).Fluorescent spec⁃tra were obtained on Varian(Cary Eclipse)with a 10 nm slit for both excitation and emission.Solid⁃state CD spectra were measured on a CD spectrometer(J⁃810,Jasco).

1.2 CO-release monitoring

A typical procedure for the monitoring was as follows:to a solution of complex 1(3.3 mg,0.006 6 mmol)in D2O(2.0 mL)was added a solution of Mb(50µL,4 mmol·L-1)in phosphate buffer solution(PBS,pH=7.4).The mixture was maintained at 37℃ and regu⁃larly monitored using IR spectroscopy.The CO⁃release initiated by Hb(50 µL,4 mmol·L-1),BSA(50 µL,4 mmol·L-1),GSH(50 µL,4 mmol·L-1),and CysA(50µL,4 mmol·L-1),was analogously performed,respec⁃tively.The above aqueous solutions were prepared in D2O since water molecule causes strong interference in a range from 2 000 to 1 900 cm-1.Owing to the limited solubility of complex 1 in D2O,a minimum DMSO(50µL)was added to assist the dissolution of complex 1.

1.3 Collection of UV-Vis and fluorescent spectra

To facilely compare the interactions of complex 1 with different biological molecules,the concentration of complex 1 and amounts of the biological molecules in the system(3 mL in total)should be kept at the same throughout UV⁃Vis and fluorescent spectroscopic stud⁃ies.A typical procedure was as follows:to a solution of Mb(3 mL,3.3 µmol·L-1)was added an appropriate amount of complex 1(33 µmol·L-1).Then the UV⁃Vis or fluorescent spectrum of the mixture was recorded.The monitoring for the interaction of complex 1 with the other two proteins was analogously performed.

1.4 Gel electrophoresis

The DNA interaction experiments were done using agarose gel electrophoresis at 37℃and a typical procedure was as follows:pUC19 plasmid DNA(0.01µg·µL-1)in 50 mmol·L-1Tris⁃HCl+18 mmol·L-1NaCl buffer(10µL,pH=7.2)was treated with complex 1 and CysA.The mixture was incubated for 4 h before load⁃ing buffer was added.Then the sample was electropho⁃resed for 2.5 h at 80 V on 0.8% agarose gel using 20 mmol·L-1Tris⁃boric acid⁃EDTA buffer.After the elec⁃trophoresis,DNA bands were visualized by UV light and photographed.

2 Results and discussion

2.1 CO-release promoted by the proteins

Hb,BSA,and Mb are common proteins existing in blood or muscle in organisms.GSH is a short⁃chain peptide composed of three amino acids which is an important antioxidant and radical scavenger in vivo.Therefore,these proteins or peptides were selected in the study to demonstrate how complex 1 interacts with these biologically relevant substances in PBS.In the investigations,the molar ratio of complex 1 over a pro⁃tein was kept at 10∶1.For comparison,the CO⁃release initiated by CysA was also carried out under the same conditions.As shown in Fig.1,the characteristic IR absorption peaks of complex 1(2 068,2 032,and 1 989 cm-1)decreased continuously with reaction time in the presence of Hb in PBS.The spectroscopic monitoring results for the CO⁃release initiated by other substances(Mb,BSA,and GSH)and the stability of complex 1 in PBS are shown in Fig.S1⁃S4(Supporting information).For CysA,the spectral variation with reaction time is shown in Fig.S5.For comparison,the CO⁃release was also examined in the presence of both CysA and BSA(Fig.S6).The results indicate clearly that the presence of these biologically relevant substances can promote CO⁃release from the diiron carbonyl complex.Since these molecules possess no other capabilities but nucleophilicity,the ligand exchange mechanism for the CO⁃release initiated by these substances should also apply[23⁃25].The reaction initiated by Hb leads to decom⁃pose of complex 1 to completely release CO since no other intermediate bearing CO was detected at the spec⁃troscopic time scale.CysA can react with complex 1 slowly to promote CO⁃release[23],and its co⁃existence with BSA could synergistically accelerate the decompo⁃sition of complex 1(Fig.S6).

Fig.1 IR spectral variation of complex 1 in the presence of Hb in PBS/D2O at 37℃under an open atmosphere

Kinetic analysis of the decomposition of complex 1 initiated by an appropriate nucleophilic agent was performed.The linear plots are shown in Fig.2,which suggest that the reactions between complex 1 and the proteins,GSH or CysA follow a first⁃order kinetic model.All the kinetic data are summarized in Table 1.For the proteins,Mb showed the most effective interac⁃tion in promoting the decomposition of complex 1 whereas the reaction initiated by BSA was the most sluggish one.Compared to the reaction rate constant with BSA,the reaction rates for Mb and Hb were about two and one times faster,respectively.GSH turned out the most effective accelerator to trigger CO release from complex 1(Table 1).The reason may be attribut⁃ed to its two carboxylic groups and one thiol group,of which the thiol group is of strong affinity towards iron carbonyl complexes.Further,in PBS(pH=7.4),the car⁃boxylic groups of GSH with an isoelectric point of 5.93 would exist mostly in carboxylate form which would be more nucleophilic.The concentration of GSH was about 5⁃fold higher than in normal cells.The recent report of our collaborator also revealed that endogenous GSH in cancer cells can trigger the CO release of com⁃plex 1 and the releasing rate of CO in cancer cells was faster than that in normal cells[26].Therefore,such a CO⁃releasing system can be of potential as anticancer drugs.Interestingly,both BSA and CysA could jointly accelerate the decomposition of complex 1 demonstrat⁃ing a synergistic effect(Table 1).

Fig.2 Plots of the logarithm of IR absorbance of complex 1 vs reaction time in the presence of various nucleophiles in PBS/D2O

Table 1 Kinetic data of the decomposition of complex 1 in the presence of different nucleophiles in PBS/D2O at 37℃under an open atmospherea

2.2 Spectroscopically probing the interaction between the proteins and complex 1

UV⁃Vis spectroscopy is the most adopted method for studying conformation change of proteins.The UV⁃Vis spectral variations of different proteins and control in PBS(pH=7.4)in the presence of complex 1 are shown in Fig.3⁃5 and Fig.S7.The UV⁃Vis spectrum of complex 1 in PBS showed a strong absorption at 333 nm and a weak shoulder absorption band at 460 nm(Fig.S7),which should be attributed to the electron transition bands of LMCT andd⁃dtransition,respec⁃tively.In addition,the UV⁃Vis absorption of complex 1 decreased steadily with time(Fig.S7),which indicates the slow decomposition of complex 1 in PBS.

Fig.3 UV⁃Vis spectra of complex 1(33 µmol·L-1)and Hb(3.3 µmol·L-1)in PBS(pH=7.4)in the presence of complex 1 with various concentrations

Fig.4 UV⁃Vis spectra of complex 1(33 µmol·L-1)and Mb(3.3 µmol·L-1)in PBS(pH=7.4)in the presence of complex 1 with various concentrations

Both Mb and Hb showed two absorption peaks at 280 and 405 nm,which are ascribed to the residue of amino group and Soret peak,respectively(Fig.3 and 4).BSA just comprised the residue of the amino group and presents one peak at 280 nm(Fig.5).When complex 1 was gradually added,considerable interactions of com⁃plex 1 with these proteins were demonstrated as indi⁃cated by the increasing band at 280 nm(Fig.3⁃5).How⁃ever,there was almost no change for the band at 405 nm,the peak belonging to Mb and Hb,which implies that the moiety and coordination sphere of complex 1 is not affected.This is attributed to the protection of the moiety offered by the protein domains.Additionally,a new band at 333 nm appeared and increased gradually,which may be contributed to the absorption band of complex 1.The highly similar spectral change suggests a similar interaction mechanism.These results suggest that the products of complex 1 with these proteins pos⁃sess a rather similar coordination atmosphere.

Fig.5 UV⁃Vis spectra of complex 1(33 µmol·L-1)and BSA(3.3 µmol·L-1)in PBS(pH=7.4)in the presence of complex 1 with various concentrations

Proteins are always fluorescent owing to the exis⁃tence of those aromatic amino residues,such as trypto⁃phan,tyrosine,and phenylalanine.As shown in Fig.6,the fluorescence of the proteins was significantly affect⁃ed when complex 1 was present.Upon excitation at 280 nm,Hb,Mb,and BSA exhibited considerable fluo⁃rescence at 327,328,and 348 nm,respectively.With the addition of complex 1,the fluorescent intensity of these proteins decreased instantly while band position and spectral profiles did not change.The fluorescent quench effect of the complex on the proteins could be attributed to the interaction of the proteins with com⁃plex 1,which agrees with the UV⁃Vis spectral changes upon the interactions with the proteins as discussed above.

Fig.6 Fluorescent spectra of different proteins in the absence and presence of complex 1 in PBS

As discussed above,there are strong interactions between the proteins and the iron carbonyl complex,particularly Hb and Mb.To examine whether the inter⁃action changes significantly the conformation of the proteins,CD spectroscopy was employed to check the reactions and the results are shown in Fig.7.The CD spectra indicate that the interaction does not lead to significant variations in their conformations.But there were subtle differences among the three proteins.For BSA,the two spectra were nearly superimposable whereas,for both Hb and Mb,there were observable changes in peak intensities.These observations are in good agreement with the interactions of the proteins with the iron carbonyl complex.

Fig.7 CD spectra of different proteins(a:Hb,b:BSA,c:Mb)in the presence and absence of complex 1

2.3 Interaction of CORM with DNA

To examine whether the CO⁃release and the resul⁃tant residues would lead to any DNA degradation,the mixture of CysA and complex 1 was incubated with pUC19 plasmid DNA for 4 h.Analysis of electrophore⁃sis of the DNA suggests no observable DNA degrada⁃tion during the incubation(Fig.8).

Fig.8 Interaction of the CO⁃releasing system(CysA+1)with pUC19 plasmid DNA

3 Conclusions

In summary,the interactions of a diiron hexacar⁃bonyl complex 1 with Hb,Mb,BSA proteins,and GSH,and their promotion to the CO⁃release from the com⁃plex were studied.UV⁃Vis and fluorescent spectroscop⁃ic investigations suggest that among the examined three proteins,Hb,Mb,and BSA,the interaction of BSA with the complex is the weakest one,which echoes rightly the trend of CO⁃release promoted by these proteins.All the CO⁃release follows the first⁃order kinetic model.In the promotion of CO⁃release,GSH shows the best efficiency,which may be of significance in exploring anticancer drugs since,in cancer cells,the level of GSH is higher than normal cells.Despite the strong interactions between the proteins and the iron complex,the CO ⁃releasing process and relevant resi⁃dues do not either significantly alter the conformations of the proteins or degrade pUC19 plasmid DNA.

Supporting information is available at http://www.wjhxxb.cn