Reversible proton-switchable fluorescence controlled by conjugation effect in an organically-functionalized polyoxometalate

A novel monosubstituted organoimido hexamolybdate containing 6-nitroquinoline moiety has been successfully synthesized. This organically-functionalized polyoxometalate exhibits proton-induced switchable fluorescence property in aqueous acetonitrile solution at room temperature. Experimental and theoretical investigations of this reversible “on” and “off” switching mechanism have been carried out, and it is found that the protonation and deprotonation at the heterocyclic nitrogen atom within quinoline fragment leads to the breaking and reformation of the conjugation through strong d-π interaction between the hexamolybdate anionic cluster and the quinoline moiety, resulting in “on” and “off” luminescence signal.

Molecular switches constitute a variety of reporter molecules able to show response to external stimuli by the reversible molecular structural change such as cis-trans isomerization, ring-closing/ring-opening, molecular proton-transfer processes, behavior of assembly and so on [1][2][3] . As an efficient and highly sensitive method, fluorescent molecular switches have attracted substantial research interest in the past decades [4][5][6][7][8][9] . In particular, proton sensors which can indicate the presence of protons are of great use in the field of detectors response to environmental changes, memories or logic gates in nanotechnology, and especially life sciences 10,11 . Up to date, though numerous fluorescent systems exhibiting reversible response to protons have been reported, the new challenge of analysis constantly needs to develop more proton-responsive fluorescent sensors. Therefore, the development of molecular switches having fluorescent response to external stimuli represents a continuous research topic.
In this sense, polyoxometalates (POMs) which are a rich class of metal oxide anionic clusters with diversity of structure and unique photonic, electronic, and magnetic properties [12][13][14][15][16] are good candidates for fabricating multifunctional fluorescent materials. Previously, one well understood synthetic method was to incorporate lanthanide ions, mainly Eu 3+ and Tb 3+ , into POMs [17][18][19][20][21][22] . Alternatively, the introduction of organic fluorophore may be an efficient synthetic strategy and have been attracted much attention in the past decade [23][24][25][26][27][28][29][30][31][32] . Especially, the organic functionalization of POMs via covalent linkage to form organic-inorganic hybrid fluorescent materials is a very attractive and challenging research area. Much pioneer work has been done by Neumann and Peng 27-30 , however it is found that POM clusters exhibit efficient fluorescence quenching when the inorganic clusters directly linked to the organic conjugated polymers or moieties through Mo-N triple bond [28][29][30] . Recently, Liu and Wei and co-workers reported that surfactant functionalized hexavanadates with proton or sodium ions as the counter-ions have fluorescent property 31 . Meanwhile, Liu and Hill and co-workers also found this photoluminescence in inorganic-organic hybrid hexavanadate clusters containing pyrene moiety 32 . However, to the best of our knowledge, POMs-based single-molecular switches displaying reversible "on" and "off " photoluminescence response to protons have not been reported yet.
In this paper, we report the synthesis of a novel organoimido hexamolybdate containing 6-nitroquinoline moiety, which exhibits reversible stimuli-response to protons. Furthermore, the mechanism of this reversible fluorescent behavior based on electron delocalization breaking and reforming is also investigated in detail. Furthermore, single-crystal X-ray diffraction investigation has also been performed. It is found that an asymmetric unit contains one independent cluster anion and two counter tetrabutylammonium cations as well as a half of acetonitrile molecule and one water molecule. The ORTEP drawings of the anionic cluster within compound Mo 6 -Q-NO 2 is presented in Fig. 1. As shown in Fig. 1, compound Mo 6 -Q-NO 2 displays the typical structural features as those of monofunctionalized hexamolybdates: one terminal oxo atom (O t ) of the hexamolybdate cluster has been replaced by an organoimido ligand. In addition, the formed Mo-N bond is found to be 1.730 Ǻ, and the nearly linear Mo-N-C angle is refined as 167.1° (Supplementary Tables S1 and S2), exhibiting the typical triple bond features 34 .

Synthesis and characterization.
Photo-physical property. The UV-Vis spectrum of compound Mo 6 -Q-NO 2 is shown in Fig. 2. The lowest-energy electronic transition (LET) absorption band in 50% aqueous acetonitrile solution locates at 423 nm, which exhibits obviously bathochromic shift compared to the parent hexamolybdate due to the quinoline aromatic ring conjugated with the hexamolybdate framework through the strong d-π interaction 35 . To assess the effect of acid and base, absorption changes of Mo 6 -Q-NO 2 in the presence of 10 equivalents of H + and OH − in 50% aqueous acetonitrile solution have been determined respectively. It is found that the LET absorption band  has no obviously change in the presence of 10 equivalents of OH − , while the LET absorption band has significant hypsochromic shift from 423 nm to 383 nm in the presence of 10 equivalents of H + . Interestingly, upon further addition of 0.5 mL dilute NaOH solution (10 equivalents) into the acidic solution to neutralize the HCl, the LET absorption band migrates back to 423 nm. Note that the decreased intensity of absorption peaks attributes to the dilution effect of the addition of 0.5 mL dilute NaOH solution. This reversible change of LET absorption band not only indicates that Mo 6 -Q-NO 2 is stable after these treatments but also implies that a corresponding reversible change of conjugate delocalization system within compound Mo 6 -Q-NO 2 takes place.
Theoretical investigation of the protonated structure. To explore the cause of reversible change of LET absorption band, we firstly need to determine the protonation site. Previous studies have pointed out that bridge oxygen (O b ) is a preferable binding site to proton as compared to terminal oxygen [36][37][38][39] . Therefore, within  Fig. 3, it was found that the structure corresponding to the protonation at the heterocyclic nitrogen atom is the most stable isomer. Based on this result, we can explain the reversible change of absorption band from the corresponding change of structure. The protonation at the heterocyclic nitrogen atom leads to the Mulliken charge of the hydrogen bound to the 4 site carbon in quinoline moiety increased from 0.362 to 0.555, resulting in much stronger hydrogen bonding between 4-H and the bridge atom O15. Combination of the effect of hindrance of 6-nitro, in addition, the angle of Mo-N-C decreased to 150.5°. Simultaneously, the bond length of Mo-N increased to 1.851 Ǻ while that of N-C decreased to 1.315 Ǻ (Supplementary Fig. S3). All these changes of structure indicate that the Mo-N bond exhibits much more like single bond, largely decreasing the d-π interaction between inorganic hexamolybdate cluster and organic quinoline aromatic ring. Therefore, the absorption band in UV-Vis spectrum shifts to 383 nm after protonation.
Reversible proton-switchable fluorescence. In previous investigations, it was found that the Mo-N triple bond linking POM clusters with conjugated aromatic moiety displayed significant fluorescence quenching because of the photo-induced electron transfer from the fluorophore to the clusters [27][28][29][30] . Likewise, compound Mo 6 -Q-NO 2 also exhibits no fluorescence. However, compound Mo 6 -Q-NO 2 clearly displays proton responsive "on" fluorescence at 560 nm with excitation wavelength of 377 nm. As shown in Fig. 4a, upon gradual addition of dilute HCl (0~20 equimolar) to the 50% aqueous acetonitrile solution of Mo 6 -Q-NO 2 , the intensity of the emission at 560 nm increased gradually. While upon further gradual addition of dilute NaOH (0~16 equimolar) to the acidic solution, the emission intensity exhibited gradual decreasing until the fluorescence almost "off ", (presented in Fig. 4b). To verify this reversible "on" and "off " fluorescence originated from the presence and absence of H + , further gradual addition of 8 and 16 equimolar dilute HCl have been performed, and the recovered fluorescence is observed. A control experiment confirmed that this proton-switchable fluorescence was due to Mo 6 -Q-NO 2 , as the organic ligand 5-amino-6-nitroquinoline showed almost no fluorescence with excitation wavelength of 377 nm (Supplementary Fig. S4). It is noteworthy that this is a first example of organoimido derivative of POMs with significant reversible proton-switchable fluorescent property.

Discussion
In light of the structural analysis as above, fluorescence enhancement upon gradual addition of dilute HCl into the 50% aqueous acetonitrile solution of Mo 6 -Q-NO 2 is due to the protonation at the heterocyclic nitrogen atom leading to the Mo-N changing from triple bond to single bond, resulting in the effective channel of photo-induced electron transfer from the organic 6-nitroquinoline moiety to the hexamolybdate cluster that is blocked. Contrarily, the quenching of fluorescence upon addition of NaOH into this system attributes to subsequent deprotonation of the heterocyclic nitrogen atom, regenerating the effective photo-induced electron transfer channel, illustrated in Fig. 5.
In summary, we have successfully synthesized a novel mono-substituted organoimido hexamolybdate containing isoquinoline moiety which, as a first example of organoimido-POMs-based fluorescent single-molecule switch, exhibits proton-induced switchable fluorescence property in aqueous acetonitrile solution at room temperature. Moreover, experimental and theoretical investigations of reversible "on" and "off " switching mechanism have been carried out, and it is found that the protonation and deprotonation at the heterocyclic nitrogen atom within quinoline fragment leads to the breaking and reformation of the conjugation through strong d-π interaction between the hexamolybdate anionic cluster and the quinoline moiety, resulting in "on" and "off " luminescence signal. This proton-responsive switchable fluorescent activity provides a potential application in sensors using organic functionalized polyoxometalates. More importantly, this work paves a new way to design and synthesize POM-based organic-inorganic hybrid fluorescent single-molecule switches. Spectroscopic Characterization. Infrared spectra were measured using the method of KBr pellets and recorded on a Perkin-Elmer FTIR spectrometer. The electrospray mass spectra (ESI-MS) were measured on a Bruker Apex IV FTMS Plus ion-trap mass spectrometer, and all experiments were carried out in the negative-ion mode using CH 3 CN as solvent. UV-Vis absorption spectra of Mo 6 -Q-NO 2 in a mixed solution of acetonitrile and water were recorded on a UN-2100s spectrometer. Fluorescence measurements were recorded in 3 mL quartz cuvettes at room temperature using a Hitachi F-7000 FL Spectrophotometer equipped with a xenon lamp excitation source. All fluorescence spectra were measured at an excitation wavelength of 377 nm.

Crystallographic structural determinations.
A suitable crystal of the compound Mo 6 -Q-NO 2 was covered with petroleum jelly, mounted onto glass fibers, and transferred directly to a Rigaku RAXIS-SPIDER IP diffractometer. Data collection, data reduction, cell refinement, and experimental absorption correction were performed with the software package of Rigaku RAPID AUTO (Rigaku, 1998, Ver2.30). Structures were solved by direct methods and refined against F 2 by full matrix least squares. All non-hydrogen atoms, except disordered atoms, were refined anisotropically. Hydrogen atoms were generated geometrically. All calculations were performed using the SHELXTL V. 5.10 program 43 .
Theoretical calculations. All the calculations presented in this work were carried out with the Gaussian-09 program package 44 . The structures of each stationary point were fully optimized using the B3LYP method, in combination with the LANL2DZ basis set for molybdate atoms and the 6-31G(d) basis set for rest atoms. Vibrational frequencies of each stationary point were calculated at the same level of theory to characterize the nature of the stationary points and give the thermal correction to Gibbs free energy. In addition, to consider the solvent effect, a single point energy calculation was performed by using the integral equation formulism within the polarized continuum model (IEF-PCM) 45 with the Bondi atomic radii for the united atom topological model at the B3LYP level based on each optimized geometry, with the LANL2DZ basis set for molybdate atoms and the 6-31 + G(d, p) basis set for rest atoms denoted as IEFPCM-B3LYP/BS1. The dielectric constant of acetonitrile is the default value of the G09 program.