The fluorescence regulation mechanism of the paramagnetic metal in a biological HNO sensor

Abstract

Paramagnetic metals are frequently used to regulate fluorescence emissions in chemical and biological probes. Accurate quantum calculations offer the first regulation theory that quenching is through the competitive nonradiative decay of the mixed fluorophore/metal ³ππ*/dd state isoenergetic to fluorophore-localized ¹ππ* state.

Graphical Abstract

A theoretical model was developed to reveal the origin of paramagnetic metal induced fluorescence quenching.

Paramagnetic metal ions, such as Cu²⁺, Co²⁺, Ni²⁺, and Fe³⁺, are frequently used to quench fluorescence to facilitate ON-OFF signaling of interesting chemical and biological systems.¹ For instance, [Cu^II(BOT1)Cl]⁺ (see Scheme 1), was recently reported as the first fluorescence probe² to directly detect nitroxyl (HNO), an important biological nitric oxides, in living cells. Among all the nitrogen oxides, HNO, the one-electron reduced and protonated analogue of the well-known signaling molecule nitric oxide (NO), is unique in its chemistry and biology.³ Investigations of HNO can be traced back to early studies of fundamental physical examinations and the elucidation of interactions in atmospheric, industrial and bacterial processes in the past century.³ The recent reports have indicated that HNO has important biological activity and pharmacological effects, such as vascular relaxation, enzyme activity regulation, neurological function regulation, enhanced cell oxidative stress, blood-brain barrier disruption, and neutrophil infiltration during renal ischemia/reperfusion.⁴ Although the original study of HNO first emerged in more than 100 years ago, the understanding of the chemistry and biochemistry of HNO and its detection in vivo have seriously lagged behind other redox nitrogen oxide congeners.³ To date, most HNO detection methods are indirect or inconvenient for in vivo uses.⁵

Scheme 1.

Chemical structure of [Cu^II(BOT1)Cl]⁺ with numbering and labelling schemes. Hydrogen atoms are omitted for clarity.

As an illustrative example of the direct HNO sensor, the metal's regulatory role is to quench the fluorescence of BOT1 upon Cu^II binding, and to regenerate fluorescence by reaction with the targeting molecule, HNO, leading to metal center reduction to diamagnetic Cu^I.² The basic idea of using Cu^II complex as the HNO receptor site and such redox reaction to couple with a fluorescence signaling site has been recently used to develop a few other metal-based HNO fluorescence probes.⁶ Although the quenching mechanisms of paramagnetic metals are usually hypothesized to result from photoinduced electron transfer (PET) from singlet fluorophore excited state to paramagnetic metal centers,^2,6a,7 a rigorous examination of this hypothesis and related fluorescence mechanistic details has not been reported. In addition, although derivatives of the central signaling unit, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), are widely used in chemical and biological studies,⁸ with relatively sharp fluorescence peaks, high quantum yields, and optical properties suited for cellular imaging,^2,6a,8 their fluorescence mechanisms are yet to be reported, despite early calculations of orbital energies.⁹ Here, we employed a multi-configurational quantum chemical study [see Electronic Supplementary Information (ESI) for computational details] that has been verified to be superior in investigations of excited states of various systems containing transition metals⁷ to investigate [Cu^II(BOT1)Cl]⁺ and related systems as the first example to elucidate a fluorescence regulation theory for paramagnetic metal systems and the associated mechanism-based design principle, based on excellent predictions of experimental absorption and fluorescence properties.

The central signalling unit of BOT1, 1,3,5,7,8-pentamethyl substituted BODIPY (1), was first studied. As shown in Fig.1, the ground state to excited state S₀→S_CT(1ππ*) transition is the lowest excitation with the largest oscillator strength (f=0.81). The calculated vertical excitation energy ($E_{\perp }$), 2.49 eV (498 nm) is in excellent accord with the experimental maximum absorption wavelength, λ_max,abs (493 nm).^8,10 The adiabatic excitation energy (E_0-0) was also shown in Table 1. A large photo-initiated charge translocation (PCT) with 0.481 e was found from the dimethylpyrro ring to the rest moiety (see Fig. 1 and ESI), leading to geometric inequality of this symmetrically substituted system.

Fig. 1.

Radiative relaxation pathway for 1 with S_CT(¹ ππ*) state energies (red star points) along the relaxation pathway.

Table 1.

Selected computational data for diamagnetic systems.

System	f	E⊥(eV)	E0-0	λFE(nm)
1	0.81	2.49	2.08	527
BOT1	1.03	2.37	2.16	565
[CuI(BOT1)]Cl	0.95	2.42	2.02	565

As shown in Fig. 1, following the initial excitation, 1 rapidly decays to its minimum, S_CT(1ππ*)-min, 0.41 eV below the Frank-Condon (FC) point through a flat relaxation path. A large oscillator strength (f_em=0.85) was found for the fluorescence emission, in excellent accord with the high experimental quantum yield (Φ=0.99).^8,10

The calculated fluorescence emission wavelength (λ_FE), 527 nm, is also close to experiment (519 nm).^8,10 This excellent fluorescence emission provides a strong theoretical basis of the experimental application of 1 as a useful signaling unit in [Cu^II(BOT1)Cl]⁺. When 1 is incorporated in BOT1, the calculated absorption peak of 523 nm is in good agreement with the experimental value of 518 nm.² The computed emission energy (corresponding to 565 nm) is only red shifted by 0.16 eV compared with experiment (526 nm).² Interestingly, these data are similar to those for 1, suggesting a minor effect of bridge and receptor units on absorption and emission wavelengths. However, they induce a significant decrease of PCT from 0.481 e in 1 to 0.210 e in BOT1, in correlation with a large Φ decrease from 0.99 to 0.12.^2,8,9b,10 This may be a result of the electron-donating triazole group to reduce the electron-accepting capability of part 2 in the fluorescence process. These results provide the first details of the fluorescence state of the signaling site and the importance of PCT.

To help understand the experimental fluorescence quenching effect of Cu^II binding to BOT1,² we first briefly examined a number of excitation mechanisms of [Cu^II(BOT1)Cl]⁺ shown in Table 2 with details in ESI. The lowest ground state to excited state excitation, ²D₀ →²D₁(dd), is localized in the metal center, involving an electron transition from ${{{d_x}^2}_{-y}}^2$ to ${d_Z}^2$, which confirms the conventional knowledge that d-d transition is a low energy excitation.^7a,11 In contrast, the second and third transitions are localized in the BODIPY part, with π→π* triplet ²D₀ →²D₂(³ππ*) and singlet ²D₀ →²D₃(¹ππ*) excitations, respectively. The singlet transition is predicted to be the strongest absorption due to the largest calculated oscillator strength (Table 2). The computed absorption peak (544 nm) is close to experiment (518 nm).² The similarity of calculated $E_{\perp }$ and f data to those of BOT1 suggests that ²D₃(¹ππ*) is responsible for the initial excitation and a reddish emission from the BODIPY unit might occur afterwards.

Table 2.

Selected computational data for [Cu^II(BOT1)Cl]⁺.

Transitions	Vertical		Adiabatic	Singly
	f	E ⊥	E 0-0	occupied orbitals
2D0		0		dz 2
2D0→2D1(dd)	<10−5	0.76	0.65	dx2-y2
2D0→2D2(3ππ*)	<10−8	1.56	1.16	π dz2 π*
2D0→2D3(1ππ*)	1.20	2.28	2.02	π dz2 π*
2D0→2D3′(3ππ*/dd)	<10−8	2.32	1.98	π dx2-y2 π*
2D0→2DLMCT(πd)	<10−5	--	1.51	π

However, there is nearly isoenergitic FC excitation (2.32 vs. 2.28eV), ²D₀→²D₃(³ππ*/dd)), where two unpaired electrons occupy the BODIPY π/π* orbits identical to that in ²D₂(³ππ*), and the third unpaired electron populates in the same metal dexcited state as in ²D₁(dd). This mixed excitation nature is further confirmed by $E_{\perp }’\mathrm{s}$ for these three FC transitions, since $E_{\perp }$ of ²D₀→²D_3’(³ππ*/dd) equals to the sum of $E_{\perp }$'s for ²D₀→²D₂(³ππ*) and ²D₀→²D₁(dd), see Table 2. In addition, this mixed excitation character is evident in the minimum structure of ²D_3’(³ππ*/dd) where bond parameters of BODIPY moiety resemble those of ²D₂(³ππ*) state minimum, and the elongated Cu-N/Cu-Cl bonds along d_Z² orbital direction bear similarity with the character of structural changes of d-d transition (see ESI).

This mixture pattern was also found in another Cu^II-based sensor,^7a suggesting that it may be general for fluorescence unit-Cu^II complexes. An important consequence is to provide a bypass channel to quench fluorescence emission (vide infra). In addition, a π→d transition, ²D₀→²D_LMCT(πd), between the BODIPY moiety and the Cu²⁺ center was found to be ~0.8 eV lower than the above isoenergetic transitions. This makes the electron communication between metal and fluorescence unit energetically favorable to regulate the nonradiative relaxation (vide infra). It also supports the significance of PET from the fluorescence site to the metal center repeatedly implied/speculated in experimental studies,^1,12 which, however, was found here to be only part of the full mechanism.

The key decay processes from the joint FC region of isoenergetic ²D_3’ (³ππ*/dd) and ²D₃(¹ππ*) are shown in Fig. 2 with more details in ESI. Although the fluorescent ²D₃(¹ππ*) state is most possibly to be initially populated due to its largest oscillator strength (Table 2), luminous relaxation is slow with a ns timescale (0.15 ns),² which cannot compete with the ultrafast nonradiative decay of the isoenergetic ²D_3’(³ππ*/dd) state (vide infra). Therefore, the ²D_3'(³ππ*/dd) state governs the dominant decay channel through direct initial population to this state or an effective internal conversion of ²D₃(¹ππ*)→²D_3’(³ππ*/dd) in the isoenergetic FC region. This explains the considerably low experimental Φ (0.01).²

Fig. 2.

Decay pathways from ²D₃ (¹ππ*) vs. ²D_3’ (³ππ*/dd) of [Cu^II(BOT1)Cl]⁺.

As shown in Fig. 2 and ESI, the initial decay of ²D_3’(³ππ*/dd) is associated with structural changes in both BODIPY and Cu^II center, resembling those for ²D_3’(³ππ*/dd)-min with the characteristics of mixed ³ππ*/dd excitation. It then rapidly decays to the conical intersection, CI(²D_3’/²D₂), between ²D_3’(³ππ*/dd) and ²D₂(³ππ*) with a 0.17 eV energy decrease. The energy gap between two excited states at CI(²D_3’/²D₂) is calculated to be only 0.082 eV. Such strong non-adiabatic coupling allows a fast interconversion to occur effectively from ²D_3’(³ππ*/dd) to ²D₂(³ππ*) state. Meanwhile, axial Cu-Cl and Cu-N bonds are elongated to be 2.363 and 2.253 Å in CI(²D_3’/²D₂) compared with those (2.324/2.199 Å) in the ²D₀ ground state, but are shorter than those in the ²D_3’(³ππ*/dd)-min excited state (see ESI), suggesting that CI(²D_3’/²D₂) is an intermediate to approach ground state structure for the moiety of Cu^II center. It also functions as an effective nonadiabatic relay to repopulate the unpaired electron of Cu^II from ${{{d_x}^2}_{-y}}^2$ to ${d_Z}^2$ to help recover its ground state electronic structure, allowing an ultrafast decay to ²D₂(³ππ*) state, in which the excited state pattern now becomes centered on BODIPY only. The evolution of ²D₂(³ππ*) along a slightly downhill energy path then reaches the singlet triplet crossing between ²D₂(³ππ*) and ²D_LMCT(πd), referred as STC(²D_LMCT/²D₂) in Fig. 2. The small energy difference (0.144 eV) and strong spin-orbit coupling at STC(²D_LMCT/²D₂) enable the ²D₂→²D_LMCT transition with high efficiency in the subpicosecond timescale,¹³ facilitated by the electron transfer from the high energy BODIPY π* orbital to the low energy Cu^II d_Z² orbital. This type of π→d electron communication induces the weakened axial Cu-Cl and Cu-N bonds (~2.50 Å) around the Cu²⁺ center, besides the retention of the excited BODIPY triplet state structural character (see ESI). Consistently, dipole moment increases significantly to ca. 30 Debye in STC(²D_LMCT/²D₂) from ca. 15 Debye in ²D₂(³ππ*). This triggers the electron repopulation to recover the BODIPY ground state structure by a reverse electron transfer from ${{{d_x}^2}_{-y}}^2$ to the π orbital leading to the double occupancy of the BODIPY π orbital, which is much more stable than ²D_LMCT(πd). This generates the ²D₁(dd) state, e.g. the axial Cu-Cl and Cu-N bonds are further shortened to 2.320 and 2.298Å from ~2.50 Å in ²D_LMCT(πd)-min. CI[²D₁(dd)/²D₀] was found to seam the surface between ²D₁(dd) and the ground state. It is 0.73 eV lower than ²D_LMCT(πd)-min, acting as an effective relay by switching the unpaired electron from ${{{d_x}^2}_{-y}}^2$ to ${d_Z}^2$. The closeness of the energy levels (0.095 eV) ensures CI[²D₁(dd)/²D₀] to function as an effective non-adiabatic funnel by the enhanced non-adiabatic coupling between dd and ground states. With the electron repopulation, the ground state recovery is achieved by the changes of axial Cu-Cl and Cu-N bonds in a similar downhill pathway to its hot ground state (²D₀*) with high efficiency. In this way, this non-radiative decay competitively shuts off the fluorescence emission channel.

These results for the first time show that quenching is primarily based on the efficient conversion from the fluorescent ²D₃(¹ππ*) state to the mixed state of ²D_3’(³ππ*/dd) due to close energy proximity and the subsequent ultrafast downhill decay of this mixed state, not the widely reported experimental hypothesis⁴ of only electron transfer from fluorophore singlet excited state to Cu^II. In fact, as shown in Fig. 2, both the forward and backward electron transfers from fluorophore to metal are important for the non-radiative decay, along with other localized changes. The close energy proximity of the fluorescent state and non-fluorescent state was also found to be an important feature of quenching effect in non-metal systems, e.g. fluorescent ¹ππ* state vs. non-fluorescent ¹nπ* state.^7a This suggests that close energy proximity facilitates the conversion from the fluorescent state to the non-fluorescent state via energy resonance. Our results thus offer the first mechanism-based design principle for such systems: the metal d-d transition energy shall be close to the singlet-triplet splitting of the fluorophore to result in almost isoenergetic ¹ππ* state and the mixed ³ππ*/dd state.

Experimental results show that upon the introduction of HNO to the solution of [Cu^II(BOT1)Cl]⁺, the copper center is reduced to Cu(I) with the concomitant release of NO.² Since the HNO/NO conversion mechanism via the reduction of [Cu^II(BOT1)Cl]⁺ has been investigated in our previous work,¹⁴ herein we focus to understand how the reduced metal complex restores the fluorescence of the BODIPY unit that is quenched in the oxidized Cu(II) system. In [Cu^I(BOT1)]Cl, the Cu⁺ atom with a completely filled d¹⁰ electronic configuration is penta-coordinated by four nitrogen atoms from the triazole bridge and tripodal dipicolylamine, and Cl⁻, see ESI. There are four weak Cu-N bonds with bond lengths of 2.22–2.48Å in the ground state of the [Cu^I(BOT1)]Cl complex. Upon photo-excitation of S₀→S_CT(¹ππ*), these Cu-centered bond lengths remain almost unchanged, while significant structural changes mainly take place in the BODIPY moiety. This suggests that the perturbation to the excited state properties of BOT1 from Cu⁺(d¹⁰) binding is negligible. Indeed, as shown in Table 1, the fluorescence emission wavelength is predicted to be the same as in BOT1. As a result, the reddish fluorescence emission is re-generated from the BODIPY moiety when [Cu^II(BOT1)Cl]⁺→[Cu^I(BOT1)]Cl conversion occurs in the presence of HNO, which is in excellent agreement with experiment.²

The solvent effect has also been examined to inspect how solvent polarity or microenvironment influences the radiative/non-radiative mechanism (see ESI). It was found that these factors may slightly alter the energy level but unlikely modifies the basic mechanism of the competitive non-radiative relaxation through energy resonance, although solvent polarity may accelerate the PET from the fluorescence site to the metal center, a part of the full mechanism described in this work.

In summary, the most important discovery from this work is the fluorescence regulation theory for paramagnetic metal systems due to the competitive nonradiative decay of the mixed fluorophore/metal ³ππ*/dd state isoenergetic to fluorophore-localized ¹ππ* state, which will help understand other similar sensors and facilitate mechanism-based design for chemical and biological applications.