Simultaneously Enhancing Aggregation-Induced Emission and Boosting Two-Photon Absorption Performance of Perylene Diimides through Regioisomerization

The integration of aggregation-induced emission (AIE) and two-photon absorption (2PA) into one molecule is very important because such combination would endow the material with synergetic effect for diverse and broader applications. Herein, two regioisomers based on perylene diimide and anthracene moieties are designed and synthesized. The as-prepared compounds exhibit distinct AIE and 2PA properties, where the ortho-substituted one not only exhibits enhanced and accelerated AIE performance, but also displays improved 2PA properties. This research would provide a general approach to explore ecient molecules by the marriage of AIE and 2PA that could improve their performance in various areas.


Introduction
Organic molecules exhibiting two-photon absorption (2PA) activity are of interest for a wide range of applications, including two-photon uorescence microscopy and biological imaging, 1-4 two-photon photodynamic therapy, 5-6 three-dimensional (3D) microfabrication, 7-8 3D optical data storage, [9][10] optical power limiting, [11][12] and up-converted lasing. 13 To facilitate these applications, the design and synthesis of e cient 2PA dyes are essential. Recently, this eld has been comprehensively reviewed. [14][15][16] However, 2PA dyes often show low emission e ciency ( uorescence quantum yields, Φ F ) when they are in high concentration or in the aggregated state, which is known as aggregation-caused quenching (ACQ).
These AIE molecules are designed as propeller-shaped molecules because the restriction of intramolecular rotations blocks the nonradiative pathway and opens up the radiative channel when aggregated. This design strategy of AIE molecule is opposite to that of 2PA molecule, where planar structure with elongated π-conjugation favors large 2PA cross-section. [23][24][25][26] Therefore, it is very important to nd the right balance between optical properties and structures for optimized 2PA and AIE molecules. A special molecular design for a 2PA molecule is required not only to ensure a large two-photon activity, but also to overcome uorescence quenching at a high concentration or in an aggregated state.
The key factor determining the AIE property is the moieties that can undergo active intramolecular motions to su ciently dissipate excited-state energy in the isolated state. 27 Thus, it is envisaged that the integration of the motional elements into the 2PA molecule may work as an effective rationale to eradicate the ACQ effect of the 2PA molecule and meanwhile retain the two-photon activity. 28 Tian, Hua and coworkers reported 2PA-active multibranched molecules incorporating triazine core and triarylamine branch, in which the rotational motions of the phenyl rings aid in dissipating excited-state energy in the solution state and assist in restricting intramolecular rotation (RIR) in the aggregated state. 29 Xu, Chi and coworkers reported D-π-D type 2PA molecules incorporating uorenyl-substituted triphenylamine as electron donor and distyrylbenzene/9,10-distyrylanthracene as π-conjugated bridge, in which Molecular Design Strategy, UV-Vis Absorption and Fluorescence Emission Properties In general, PDIs can be chemically modi ed in the imide-region, bay-region, or ortho-region. The substituents at the imide-region are primarily used to improve the solubility of PDIs in various solvents while minimally altering the optical and electronic properties. The modi cations in the bay-region result in a twist of the two naphthalene half units in PDIs, which can tune the optical and electronic properties. The functionalization in the ortho-region can tune the optical and electronic properties without affecting the planarity of the perylene core. 40 PDI is a typical electron acceptor. Thus, the attachment of an electron donor to the bay-or ortho-region of PDI core can effectively induce an intramolecular charge-transfer and nely tune the 2PA properties.
The selection of electron donor is based on two main points. Firstly, the intramolecular rotation (IMR) of donor "rotor" against the PDI core "stator" can deactivate the excited state of the molecule. For the largely planar PDI "stator", phenyl or biphenyl "rotors" are not enough to convert the emission behavior from ACQ to AIE due to the small volume. 39 Therefore, a donor "rotor" with a larger size should be considered. At the same time, to avoid a big rotational barrier, the molecular weight of "rotor" should not be too large. Secondly, it is recognized that a good conjugation between building blocks as well as conformational rigidity are favored for 2PA properties. So, the donor "rotor" should effectively prolong the molecular conjugation without introducing more rotatable aromatic rings. As one kind of polycyclic aromatic hydrocarbons (PAH), anthracene (ANT) is selected as the "rotor" because of the above reasons. Herein, and 550 nm with a series of well-resolved vibronic structures. This spectral feature indicates that the ortho-substitution by ANT has little in uence on the planarity of the PDI core. The above results are quite different from that observed for the PDI dye with a substituent at bay-position. Due to the steric hindrance, the perylene core is twisted in bay-substituted PDI dye. In comparison with the spectrum of O-PDI-ANT, the line-shape of the spectrum for the B-PDI-ANT is broader and displays less vibronic structures due to the loss of the planarity of the perylene core. According to the literature, the symmetrically forbidden S 0 -S 2 transition band of bay-substituted PDI is enhanced due to the core-twisting induced broken symmetry. 41 In this case, the spectral features of B-PDI-ANT are typical of PDI derivatives with modi cation at the bay-positions.
The uorescence emission spectra of PDI, B-PDI-ANT and O-PDI-ANT dyes in THF solution (10 −6 M) are shown in Figure 2(c). Upon excitation at 525 nm in THF, PDI displays uorescence emission with a peak and a pronounced shoulder at 530 nm and 571 nm, respectively. The emission and absorption spectra of PDI display a mirror image relationship, denoting that no aggregation occurs at this concentration in THF at room temperature. 42 The uorescence quantum yields (Φ F ) of PDI in THF solution is 70% using rhodamine B (Φ F = 97% in ethanol) as the calibration. At the same condition, B-PDI-ANT and O-PDI-ANT show signi cantly red-shifted emission bands and drastically reduced emission e ciencies (Φ F = 0.9% and 0.1%, respectively). The uorescence red-shift effect might be attributed to the expansion of the πconjugated system by introducing ANT to the PDI core (bay-or ortho-position). The uorescence quenching effect may be due to the intramolecular charge-transfer between ANT and PDI moieties, which is frequently observed in the D-π-A systems constructed by one electron-donating unit and one electronwithdrawing unit. Also, upon UV illumination, we noted that the wet spots of both B-PDI-ANT and O-PDI-ANT on TLC plates were nonemissive. However, they became emissive when they became dried spots. This result implies that both B-PDI-ANT and O-PDI-ANT would be AIE-active. Thus, the uorescence quenching of B-PDI-ANT and O-PDI-ANT in their dilute THF solutions can be tentatively associated with the intramolecular rotations of the ANT groups, which could exhaust the energy of the excited state.

AIE Properties
To verify the AIE property, uorescence emission spectra were tested to monitor the emission intensity uctuation of the molecules in THF-water mixtures at various water volume fractions (f w ). As shown in Figure 3a and 3c, the emission intensities are intensi ed for these two molecules when f w increases from 0 to 90%, exhibiting an AIE behavior. The comparison of their characteristic AIE properties discloses important information. Firstly, the evolution of the AIE process for their aggregates is different. To have a quantitative picture of their AIE process, we estimated their Φ F values in THF-water mixtures, using rhodamine B as the reference. As shown in Figure 3b and 3d, for B-PDI-ANT and O-PDI-ANT, the Φ F of the THF solution are very low (Φ F =0.93% and 0.13%) and are almost unchanged when water is added up to 50%, but start to increase swiftly upon addition of water to 80% and 50%, respectively. The trajectory of the Φ F change for B-PDI-ANT suggests that the molecules start to aggregate at a water fraction of >50%, but the highly twisted PDI core might provide enough room for the intramolecular rotation (IMR) of the ANT in B-PDI-ANT. Thus, the channel for consuming the excitation energy cannot effectively be switched off. As a result, B-PDI-ANT shows subdued emission at the f w of 60-80%. However, only when the molecules are packed much more tightly at the f w >80%, the aggregation-induced emission would be realized. A similar situation has also been reported by previous literature. 43 Unlike B-PDI-ANT, O-PDI-ANT exhibits accelerated AIE behavior. When the f w reaches 50%, aggregation occurs, resulting in a rapid increase in the Φ F based on the restriction of the intramolecular rotation (RIR) mechanism. The planar conformation of the PDI core could effectively diminish the molecular rotation to improve the e ciency of the radiative process. Accompanying the further increase of f w to 70%, the Φ F decreases, probably due to the transformation from ordered to random aggregates. Secondly, the degree of the AIE enhancement for their aggregates is different. Their aggregates formed in THF-water mixtures (f w = 60%) display increased emission with the Φ F values of 2.8 and 3.9%, respectively, resulting in the AIE ampli cation factors (α AIE = Φ F,aggr /Φ F,soln ) of 3 and 30, respectively. Their highest Φ F values are achieved at f w of 90%, with the α AIE of 18 and 34, respectively. These data suggest that O-PDI-ANT has a better AIE performance than B-PDI-ANT.

Femtosecond Transient Absorption and Theoretical Calculation
In order to study the excited state dynamics and especially the AIE mechanism of B-PDI-ANT and O-PDI-ANT, the femtosecond transient absorption (fs-TA) spectroscopy studied on PDI dyads were performed. The pump wavelength was 525 nm. Figure S5-S6 and Figure 4a show the fs-TA spectra of B-PDI-ANT. As shown in Figure S5, at the earlier delay time from 0 to 0.39 ps, the intensity of the broad band (465-625 nm) with centered wavelength at 528 nm increases, which is attributed to the ground-state bleaching (GSB). Two bands at short wavelength (centered at 350 and 420 nm, respectively) and one band at long wavelength (centered at 680 nm) also increase at this delay time range, which are attributed to the excited-state absorption (ESA) associated with the S 0 →S 1 . As shown in Figure S6, at later delay time from 0.39 to 6.58 ps, the ESA bands show apparent hypsochromic shift with the center wavelength emerging at 325 nm, 396 nm, and 675 nm, respectively. This process is mainly associated with the twisted intramolecular charge-transfer (TICT) from ANT to PDI groups. As shown in Figure 4a, at the last delay time from 6.58 to 925 ps, the GSB and ESA bands gradually drop to zero. The rotation of the ANT group in B-PDI-ANT is the primary channel to relax its excited state energy, which completely overwhelms the radiative process. As shown in Figure S7, global tting analysis of the decay kinetics at all wavelengths yields three time constants: 0.43 (τ 1 ), 1.92 (τ 2 ) and 250 ps (τ 3 ), which is consistent with the three excited-state decay processes observed in Figure S5-S6 and Figure 4a. O-PDI-ANT is a positional isomer to B-PDI-ANT. Nevertheless, the fs-TA spectra results show that the excited state of O-PDI-ANT undergoes a process apparently different from that of B-PDI-ANT. As shown in Figure S9, at the rst stage (0-0.36 ps), the GSB band (450-550 nm) is not a broad peak but multiple peaks with ne structure (457, 488, and 528 nm), which is in agreement with the steady-state absorption spectrum. At the same time, a stimulated emission (SE) band is observed at 572 nm, which corresponds to the steady-state uorescence emission. As shown in Figure S10, at the second stage (0.36-2.87 ps), the decrease of the SE peak at 572 nm is seen, accompanied by the blue-shift of the ESA peaks at 335, 402 and 678 nm. This reveals the conversion from uorescence emission to ICT. Therefore, the excited state of O-PDI-ANT is mainly consumed by the nonradiative process rather than the radiative process. As shown in Figure 4b, in the third stage (2.87-7510 ps), the GSB and ESA bands completely disappears, which is attributed to the rotation of the ANT group in O-PDI-ANT. As shown in Figure S11, global tting of the kinetics for O-PDI-ANT also obtains three-time constants: 0.36 (τ 1 ), 3.00 (τ 2 ) and 39 ps (τ 3 ). These values denote the procedures of excitation, TICT and rotation. The τ 3 for O-PDI-ANT is signi cantly shorter than that of B-PDI-ANT, indicating the rotation process dramatically accelerates in O-PDI-ANT. It is worthy to note that two new positive peaks at 470 and 504 nm begin to increase at this stage, suggesting an alternative relaxed pathway for O-PDI-ANT. An intersystem crossing may take place to generate a triplet state. The formation of the triplet state of O-PDI-ANT was con rmed with nanosecond transient absorption (ns-TA) spectroscopy ( Figure S13-S14). The results and discussion are available in the ESI †.
Next, fs-TA spectra ( Figure S16-S27) of B-PDI-ANT and O-PDI-ANT were recorded on their aggregated states in THF and H 2 O of different proportions. An increase in the proportion of H 2 O leads to the eventual aggregation of molecules, and hence, a corresponding change is observed in the fs-TA spectra. The study above shows that the Φ F of B-PDI-ANT and O-PDI-ANT do not show an obvious change until the proportion of H 2 O increase to 60%. Thus, the measurements were conducted at the f w = 60 %. For B-PDI-ANT, the measurement is also conducted at the f w = 90 % because a further signi cant increase in the Φ F of B-PDI-ANT is observed at the f w = 90 %. It can be easily seen that the GSB and ESA bands show an apparent redshift upon aggregation. To quantitatively describe the difference, the decay dynamics are tted at the selected wavelength. In comparison to the global tting results, the similar time constants of B-PDI-ANT and O-PDI-ANT can be obtained by using the wavelength tting at 430 nm. Therefore, these two wavelengths are selected. As shown in Figure S28, for non-aggregated B-PDI-ANT in THF, the rotation amplitude is 38.5%, but this value shows a slight decrease to 31. These results provide evidence of rapid Φ F growth for O-PDI-ANT within the aggregates as the rotation can be constrained much quicker than that of B-PDI-ANT.
To obtain a deep insight into the different ground-and excited-state properties between B-PDI-ANT and O-PDI-ANT, TD-DFT calculation using a CAM-B3LYP/6-31G(d) method based on Gaussian 16 is performed. [44][45] As the crystal structures were rather challenging to obtain, the optimized molecular structures are examined at the ground state. As shown in Figure S29 and S31, the perylene core of O-PDI-ANT is more planar than that of B-PDI-ANT, as also indicated by their UV/Vis spectra. Similar results are observed in the optimized molecular structures at the excited state ( Figure S30 and S32). The more twisted conformation of perylene core in B-PDI-ANT should be ascribed to the introduction of the ANT group into the bay-position of PDI. We also calculated the rotational barriers (potential energy surface, PES) of ANT group in the ground state and excited state for isolated B-PDI-ANT and O-PDI-ANT. As shown in Figure 4c and 4d, the PESs of O-PDI-ANT exhibit "single-well", but those of B-PDI-ANT display "doublewell". Not surprisingly, the intramolecular rotation of the ANT group in O-PDI-ANT is free because no energy barrier exists. In contrast, the intramolecular rotation of the ANT group in B-PDI-ANT should overcome an energy barrier of 1.1-1.8 kcal/mol at the ground state and 4.1-6.4 kcal/mol at the rst excited state. Overcoming these energy barriers will cause a longer lifetime for the ANT group in B-PDI-ANT to rotate. Therefore, the PES calculations provide some evidence to explain the dynamic differences of the solution-phase results. As shown in Figure S33, B-PDI-ANT and O-PDI-ANT exhibit the chargetransfer nature for the spatially separated HOMOs and LUMOs. Their HOMOs are restricted on the ANT groups, while LUMOs are distributed to the PDI core. The TICT process can be suppressed by the physical constraint in the packing mode, thus the amplitude of TICT shows obvious decrease for B-PDI-ANT (from 27.6 to 9.08%) and O-PDI-ANT (from 28.6 to 10.8%) upon aggregation in THF/H 2 O (f w = 60%).

Two-photon Absorption Properties
As shown in Figure 5a, the 2PA spectrum of PDI parent shows a weak absorption with a maximum 2PA cross-section of 95 GM at 800 nm, which is also consistent with previously reported 2PA data for unsubstituted PDI parents. 46 In the measuring range of 800-900 nm located at the first near-infrared (NIR-I) window, B-PDI-ANT exhibits the 2PA cross-sections ranging from 404 GM at 800 nm to 332 GM at 900 nm, which are 4 and 12 folds that of parent PDI, respectively. Within the same scanned wavelength range, O-PDI-ANT exhibits a quick increase in the δ values from 117 GM at 800 nm to 2159 GM at 900 nm, which are 1 and 77 folds of parent PDI, respectively. Particularly, in the measuring range of 900-1100 nm located at the second near-infrared (NIR-II) window, B-PDI-ANT exhibits an increase in the δ values from 332 GM at 800 nm to 969 GM at 1100 nm, which is similar to the most cases of small molecules based on bay-substituted PDI. In sharp contrast to B-PDI-ANT, O-PDI-ANT shows very large δ values (2159-4787 GM) across the same measuring wavelength range of 900-1100 nm. And the maximum δ is 4787 GM at 1100 nm. The signi cant enhancement of the δ values for O-PDI-ANT could be attributed to its structural features: that is a more extensive conjugated system because of the planar perylene core. This structural feature of O-PDI-ANT can be bene cial to π-electronic delocalization and afford more effective coupling channels to increase δ values. We also summarized the previously reported 2PA data for small organic molecules containing one or two PDI unit in recent ten years from 2011-2021 (Figure 5b and Table S3). [47][48][49][50][51][52][53][54][55][56][57][58] Herein, to make the δ values (measured under different experimental conditions) comparable, all of the δ values presented here are measured in the femtosecond regime, which can prevent the contribution from the excited-state absorption known to lead to arti cially enhanced δ values when conducted in the nanosecond regime. 59 It is worth mentioning that there are very few reports on ortho-substituted PDI and our result is among the highest δ values reported for small organic molecules containing one or two PDI units. And inspired by the excellent 2PA performance, this work would provide critical guidelines for the design of highly effective 2PA dyes.

Discussion
In summary, we have developed a practical strategy to enhance the aggregation-induced emission (AIE) by altering the substitution position of perylene diimides (PDI) from bay to ortho area. On one hand, such a regioisomerization enhances the AIE performance. For bay-substituted product, the uorescence quantum e ciency of the aggregates formed in THF-water mixtures (f w = 90%) is 18 folds that of the dilute THF solution. While, for ortho-substituted product, the uorescence quantum e ciency of the aggregates formed in THF-water mixtures (f w = 90%) is 34 folds that of the dilute THF solution. Besides, the AIE process is accelerated in the case of the ortho-substituted product. The threshold of the dramatic uorescence quantum e ciency increase appears at a water fraction of 80% for bay-substituted product, but only 50% for ortho-substituted product. The enhancement of the AIE performance is probably because of the restriction of the fast rotation process of the anthracene group in the ortho-substituted product. The acceleration of the AIE process is probably because of the rapid drop of the amplitude for the rotation process. The reasons for the enhanced and accelerated AIE are supported by the femtosecond transient absorption spectroscopy study of the bay-and ortho-substituted product in their isolated and aggregated state. Meanwhile, the theoretical calculation also proves that the rotation of anthracene group for bay-substituted product needs to overcome a small energy barrier; in contrast, the rotation of anthracene group for ortho-substituted product is free because no energy barrier exists. On the other hand, such a regioisomerization signi cantly improves the two-photon absorption (2PA) properties. For bay-substituted product, the maximum 2PA cross-section is 969 GM at 1100 nm, while that for orthosubstituted product is 4787 GM at the same wavelength. The large increase in the 2PA cross-section is ascribed to the improved planarity of the perylene core in the ortho-substituted product. These results demonstrate the feasibility to enhance the AIE and boost the 2PA performance by simply changing the substitution position of PDI from bay to ortho area. It is believed that, inspired by these preliminary results, many other ortho-substituted PDI with better uorescence property and 2PA performance could be further developed.

Methods
Synthesis and characterization of the AIE molecules. All the AIE molecules were synthesized using Suzuki-coupling protocols as detailed in Supplementary Information. Chemical structures were con rmed by 1 H NMR and 13 C NMR spectroscopy using a Brucker-Biosipin Avance 400 MHz spectrometer in CDCl 3 at room temperature.
Optical property spectroscopy. UV-Vis absorption spectra and uorescence emission spectra were determined with a Shimadzu UV-Vis-2600 spectrophotometer and a Shimadzu RF-6000 spectrophotometer.
Transient absorption spectroscopy. The fs-TA measurements were performed based on a femtosecond Ti:sapphire regenerative ampli ed Ti:sapphire laser system (Coherent, Astrella) and an automated data acquisition system (Ultra-fast Systems, HeliosFire). The half pulse width of output from ampli er is 80 fs. The probe pulse was obtained by using approximately 1 W of the ampli ed 800 nm output from the Spit re to generate a white-light continuum (320-780 nm) in a sapphire plate or CaF 2 crystal. The maximum extent of the temporal delay was 8000 ps. The instrument response function was determined to be 200 fs. At each temporal delay, data were averaged for 2 s and collected by the acquisition system.
The probe beam was split into two before passing through the sample. One probe beam traveled through the sample, the other was sent directly to the reference spectrometer that monitored the uctuations in the probe beam intensity. Fiber optics was coupled to a multichannel spectrometer with a CMOS sensor that had a 1.5 nm intrinsic resolution. For the experiments described in this study, the sample solution was excited by a 525 nm pump beam which is obtained by using femtosecond TOPAS ampli er. The solutions were excited in a 2 mm path-length cuvette with a sample concentration of 0.2 mM throughout the data acquisition. The data were stored as three-dimensional (3D) wavelength-time-absorbance matrices that were exported for use with the tting software. Therefore, in order to remove the contributions of the solvent, the raw spectra of the sample were subtracted by the spectra of the solvent under the same conditions. Kinetics tting method. Equation 1 shows the exponential function for wavelength kinetics tting. The wavelength kinetics tting for B-PDI-ANT and O-PDI-ANT are both tted by three exponential functions and result in three lifetimes at 430 nm.
Potential energy surface scanning method. The potential surface energy scanning (PES) was obtained from DFT and TD-DFT calculation using Gaussian 16, employing M062X method and 6-311g (d,p) basis set with an SMD solvent model of water. The scanning mainly investigates the energy change with different dihedral between perylene ring and the anthracene ring. The step size was set to be 10 degree from -10° to -170° (The situation in 0° was excluded because the two rings were totally overlapped). The energy of the optimized geometry at ground state was set as 0, and all energy in PES used the relative energy.
Two-photon excited uorescence spectroscopy. A homemade spectro uorometer was used while a femtosecond pulsed laser (Chameleon Discovery NX, Coherent), with pulse duration, ≈140 fs (FWHM), 80 MHz, repetition rate, tuning range 800-1100 nm served as the excitation source.2PA measurements were performed in 10 mm spectro uorometric quartz cuvettes with the dye concentrations of 5 × 10 −5 M.Possible reabsorption of uorescence emission was analyzed and taken into account. The quadratic dependence of two-photon induced uorescence intensity on the excitation power was checked for each excitation wavelength. The reference is Rhodamine B with a concentration of 5 × 10 −5 M solution in spectroscopic grade methanol. The samples are measured in spectroscopic grade THF. In the measuring range of excitation wavelengths, from 800 to 1100 nm, linear absorption could be neglected for all of the solutions. For comparison, the 2PA spectrum of PDI parent is also measured at the same condition. Figure 1 The enhancement of AIE performance for PDIs in previous reports and this work Page 18/21   Fs-TA spectra of (a) B-PDI-ANT and (b)O-PDI-ANT in THF. The potential energy curves of (c) B-PDI-ANT and (d) O-PDI-ANT in S0 and S1 as a function of dihedral angle between the perylene and ANT planes.