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The porphyrin dianion unit is planar with perpendicular aromatic rings (Supplementary Fig. S31). The structures of lanthanide double-decker porphyrins have been previously discussed in the literature25, 26. The schematic structure of the double-decker complexes is depicted in Fig. 1a. The skeleton structure, without DEG sidechains and with or without a negative charge, was optimized by calculation (refer to Section 5, SI) because the total charge depends upon the pH and solvent. The structure optimization of YbDD and [YbDD]- using MOPAC27 in the LUMPAC 1.3.028, 29 suite of programs is shown in Supplementary Fig. S32a, b, and is similar to that using the ORCA30 program (Supplementary Fig. S33) and Firefly QC31 package (Supplementary Figs. S35 and S37), which is partially based upon the GAMESS (US)32 source code. The porphyrin ring system is no longer planar due to (i) the cation-π attractive forces and (ii) π-π repulsive forces. The N-N distance from the bottom to the top of the double decker is comparable with the distances within each sandwich layer. The structure was also optimized for the AlDD system (Supplementary Fig. S36) and shows six short Al-N bonds and two longer bonds. These bonds give rise to a distorted structure. The calculated highest occupied molecular orbital/lowest unoccupied molecular orbital (HOMO/LUMO) are also given for LnDD in Fig. 1b and Supplementary Fig. S34 (DEG chains are omitted for clarity).
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Scanning tunneling microscopy (STM) is an advanced technique that can be used for probing molecular assemblies on an individual molecule basis. The study of porphyrins assembly and structure at the vacuum and liquid interface on surfaces is relatively advanced33. In particular, several studies have been undertaken on double-decker structured molecules34, 35. YbDD was deposited on a clean highly oriented pyrolytic graphite (HOPG) (0001) surface by placing a drop of dilute solution and evaporating at room temperature. The molecules formed self-assembled motifs without further treatment through surface adsorption and diffusion33. The STM topographic image in Fig. 1c shows a high-magnification image of a region of a drop-cast surface with additional features decorating the step edges. As shown in the zoom inset, these form a ~4 nm periodic row of separation, and an apparent height of ~1 nm (line profile Fig. 1g) is present. This height, which was recorded at -1.5 V filled state, is strongly influenced by the electronic effects of both the tip apex and molecular surface junctions36, 37. In a different trial with YbDD, a close-packed arrangement was observed and is shown in Fig. 1d, e. This ordered arrangement is long-ranged ~100 nm and aligned parallel to the HOPG step edge direction. The features also show a separation of ~4 nm in the direction perpendicular to the step direction (parallel unresolved) as indicated by the height line profile in Fig. 1e inset. Therefore, a templating effect originating at the step is suggested. The overall behavior of the drop-cast double-decker YbDD on HOPG is in line with previous studies of double-decker motifs and porphyrin ligands, with a favorable interaction and ability to spontaneously form a periodic assembly. The large ~4 nm spacing between resolvable features is consistent with literature accounts of similarly structured molecules with the spacing correlated to alkyl chain length34, 38 with individual molecules packing face-on with the oxy-alkyl chain R groups having a favorable arrangement on the HOPG surface, leading to the observed spacing. We attribute the observed features, rows and protrusions (Fig. 1c, e) to single molecules with further work underway to resolve the exact inner-molecular structure.
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The synthesis and characterization of the double-decker porphyrinate lanthanide complexes with Ln = La, Er, Gd, and Yb trivalent ions are shown in Supplementary Scheme S1, Supplementary Figs. S1-S10 and Supplementary Table S1. Due to the paramagnetic properties of the latter three lanthanide ion complexes, LaDD was synthesized as the analogue for nuclear magnetic resonance (NMR) analysis. Upon the addition of hydrazine hydrate, a well-resolved LaDD NMR spectrum could be obtained (Fig. 2) because hydrazine hydrate served as a reducing agent and assisted the formation of monoanionic diamagnetic complexes9. The protons of the single ligand Por(2DEG) can be categorized into peripheral and internal. The peripheral aromatic protons are typically located approximately at 6.5-10.0 ppm, while the DEG sidechain aliphatic protons normally lie within the range of 1.5-4.0 ppm. The peak of the hydrazine hydrate mixed with DMSO-d6 is observed at 2.6 ppm. The ring current effect strongly shifts the two internal protons on the porphyrin upfield to -3.2 ppm. The disappearance of internal N-H peaks and the proton shifting can then serve as an indication of metallization with the lanthanide ion. No signal is observed in the negative range (equated to internal N-H protons) in the spectrum of LaDD, while all peaks are subjected to upfield shifting due to the anisotropy of the f-metal ion as well as the impact of lanthanide-induced shifts10. It is noted that the theoretically most possible supramolecular trimers or even multiple aggregate structures can also give rise to similar NMR spectra, but the high-resolution mass spectra (HRMS) and STM images corroborate the double-decker structure of LaDD (and thus the LnDD series) unambiguously (Supplementary Fig. S6).
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Photophysical properties of LnDD (Ln = Yb, Er, Gd, La) have been measured (Supplementary Figs. S20-S24) and summarized in Supplementary Table S3. Upon photoexcitation at 425 nm (representing the strongest absorption band, the B or Soret band, Fig. 3a, black experimental spectrum), YbDD showcases superior photophysical performance compared with the monoporphyrinato counterpart/analogue YbN (serving as the control) under various solvent systems (Supplementary Figs. S11-14 and Supplementary Table S2). The NIR quantum yields of YbDD were measured by comparison with the standard YbTPP(Tp), which was reported as 3.2% in dichloromethane with the same excitation wavelength of 425 nm39. The NIR emission quantum yields of YbDD in toluene (water) were recorded as 3.5% (2.8%), while those of YbN in these solvents were 2.8% (2.7%), shown in Table 1. To explain these results, firstly, YbDD, which has two antenna ligand groups, should transcend YbN, which has only one. As shown in Fig. 3b, the emission spectrum of YbDD comprises several parts: the porphyrin ligand visible-NIR emission and the Yb3+ (2F5/2 → 2F7/2) NIR emission, which has an equal peak height in this figure. The peaks at 647 and 699 nm represent the porphyrin fluorescence from the Q-band singlet nominally labeled S1. The S2 singlet (B-band) emission is also observed at a much weaker intensity and at shorter wavelengths (not shown). From the comparison with the low temperature 77 K emission spectrum of YbDD (Supplementary Fig. S19), the hot emission bands 1, 2, and 3 in Fig. 3b may correspond to the transitions from the three excited states of 2F5/2, and the energy intervals between band 3 (975 nm; 10, 260 cm-1) and bands 4-6 in Supplementary Fig. S19 can identify the three levels above the ground state energy of 2F7/2. The energy transfer from the porphyrin ligands to the Yb3+ ion is observed to be efficient because the metal ion is not excited by 425 nm radiation in the absence of an antenna. However, the presence of both ligand fluorescence and lanthanide emission at room temperature suggests that the energy transfer rate from the porphyrin to Yb3+ is similar to the nanosecond regime. The lower emission quantum yield of YbDD in water than that in toluene is attributable to the quenching by high-frequency O-H vibrations. The trivalent lanthanide ions belong to the hard Lewis acid category with the coordination number of up to 8-12 so that under saturation of the lanthanides' inner coordination sphere by ligands offers vacancies for solvent molecule coordination35. The YbN system was confirmed to have unsaturated seven-coordinated Yb3+: four N from the porphyrin ring and three O from the Kläui [(η5-C5H5) Co{(MeO)2P = O}3]- anion capped oxygen atoms. To shield the Yb3+ ion in an aqueous environment and suppress luminescence quenching, the double-decker complexation strategy in YbDD fulfills the eight-coordination number requirement.
Fig. 3 Photophysical properties of the Yb(Ⅲ) complexes.
a Room temperature absorption spectrum of YbDD (black solid line) and calculated spectra using half-height widths of 1500 cm-1 using LUMPAC (blue dashed line) and ORCA (red dashed line) programs (refer to Supplymental Information Section 4). b Comparison of the emission bands of YbN (black) and YbDD (red) at 298 K in aqueous solution (λex = 425 nm). c Decay curve of Yb3+ emission in Fig. 3b (blue points) and fitted curve (red dashed line) using a monoexponential functionτ (µs)a τ (μs)a Φ (%)b Φ (%)b BRc Toluene H2O Toluene H2O H2O YbDD 28.19 23.62 3.5 2.8 2540 YbN 23.02 12.04 2.8 2.7 1850 aDetermined from the emission decay curve monitored at λem = 978 nm (2F5/2 → 2F7/2) with λex = 425 nm (Conc.: 1 μM)
bThe relative quantum yields of the Yb3+ emission (λex = 425 nm) from the two Yb3+ complexes in toluene and H2O were obtained by comparison with the standard YbTPP(Tp)
cCalculated from BR = attenuation coefficient × quantum yield; molar attenuation coefficients were obtained from the absorption spectra in water at 425 nm by applying the Beer–Lambert LawTable 1. Luminescence lifetime (τ), quantum yield (Φ) and brightness (BR) of YbDD and YbN in toluene or water
Brightness is the product of quantum yield and molar attenuation coefficient40, 41, and demonstrates the radiant energy emitted per frequency interval unit area per solid angle. For bioimaging purposes where low dosage is preferred because of adverse effects, the brightness is a more superior indicator of applicability than quantum yield, since, with higher brightness, low-abundance fluorescent compounds are detected more easily. The brightness of YbDD exceeds that of YbN by a factor of 1.37 (Table 1). The NIR 2F5/2 → 2F7/2 emission lifetimes of YbDD and YbN were determined to be 23.6 μs in water (Fig. 3c and Supplementary Fig. S17, S18) and 28.2 μs in toluene, which are both higher than the values for YbN (Table 1). YbDD shows a longer 2F5/2 → 2F7/2 lifetime, which mainly results from its higher symmetry than YbN. With a more symmetric structure, the f-f transition mechanism is of less forced electric dipole character and more vibronic, and the lifetime for a specific transition is longer42. This trend is consistent with the measured NIR emission quantum yields in water and toluene. It is worth noting that most porphyrin-based NIR dyes for biological applications have little emission in the NIR-II biological window because no metal ion is coordinated. Furthermore, the maximal absorption peaks of these dyes are usually located only from 650 to 800 nm43. Both of these reasons limit the application prospects. One commercially available NIR-II dye (NIR-II dye #900883, Sigma-Aldrich) has a similar emission peak located at 1050 nm, which is the same as that of YbDD, but its NIR emission quantum yield is ~2%, which is lower than that of YbDD. The impressive NIR emission quantum yields and long NIR emission lifetime of YbDD in aqueous solution, together with its hydrophilic property, hold tremendous promise as a (NIR) bioimaging probe.
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As a cross-system validation, the singlet oxygen quantum yield of GdDD was also examined in chloroform by comparison with the spectrum of the reference compound H2TPP (ΦΔ = 55% in CHCl3). A new-generation anticancer agent GdN, which consists of only one porphyrin ring, with high-singlet oxygen quantum yield was selected to serve as a comparison. The near-infrared 1O2 phosphorescence spectra of GdDD, GdN and the reference are shown in Fig. 4a. From these spectra, the singlet oxygen quantum yields of GdDD and GdN were measured at 66% and 51%, respectively. The singlet-oxygen quantum yield was also evaluated in aqueous solution with a PBS buffer using rose bengal (RB) as the standard by absorption changes of the decomposition of 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ABDA) at 402 nm (Supplementary Figs. S15 and S16). The values of ΦΔ were determined as 46% for GdDD and 42% for GdN. Hence GdDD displayed superior singlet oxygen generation in both organic and aqueous media. The comparison with two U.S. Food & Drug Administration approved PDT agents, porfimer sodium (Photofrin®) and 5-aminolevulinic acid (Levulan®) was made. Although GdDD shows lower-singlet oxygen quantum yield (46% in aqueous solution, Photofrin®: 89%; Levulan®: 56%), it has a much higher maximal absorptivity (GdDD: 223, 872 M-1 cm-1 @412 nm, and 52480 M-1 cm-1 @580 nm) than these two commercial photosensitizers (Photofrin®: 3000 M-1 cm-1 @632 nm; Levulan®: 5000 M-1 cm-1 @632 nm)44. With a double-decker porphyrinato structure and the resulting high molar extinction coefficient values, GdDD shows great applicability in photodynamic effects, which is also consistent with the high brightness of YbDD. In vitro experiments have also been performed to practically compare photodynamic therapeutic efficiency in different cell lines, which also suggest GdDD as a potential PDT agent (Supplementary Figs. S25-S30 and Supplementary Table S4).
Fig. 4 Emission spectra of the Gd(Ⅲ) complexes.
a The near-infrared 1O2 phosphorescence spectrum sensitized by GdDD, GdN, and the standard tetraphenylporphyrin H2TPP (in CHCl3. Absorbance = 0.05 at the excitation wavelength of 425 nm). b The 77 K phosphorescence spectrum of GdDD and GdN in MeOH (Conc.: 10 μM, λex = 425 nm)The energy gap between the antenna donor state and the lanthanide ion plays a crucial role in the energy transfer efficiency. The lowest triplet state of the lanthanide double-decker complex was determined experimentally from phosphorescence. The 77 K phosphorescence spectra of GdDD and GdN are shown in Fig. 4b. The zero-phonon lines are at a very similar wavelength (~ 745 nm: 13405 cm-1), and the prominent vibrational progression in the ring carbon-nitrogen stretching mode of 1410 cm-1 is at a lower energy. The triplet energy level is therefore located at 2610 cm-1 above the highest 2F5/2 level of Yb3+ in YbDD. The optimum energy gap has been given as between 2000 and 5000 cm-1 to eradicate back energy transfer45, 46. The weak features marked 1 and 2 in Fig. 4b correspond to the singlet fluorescence bands S1(0, 0) and S1(0, 1), as in Fig. 3b for YbDD at 298 K. The triplet state lifetimes of GdDD and GdN at 77 K were measured as 0.21 ± 0.03 and 0.14 ± 0.02 ms, respectively.
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Previous calculations of the energy levels of double-decker complexes have shown poor agreement with experiments47, 48. Herein, the absorption spectrum was modeled from the optimized structure by two programs. First, an excited states calculation was performed using the RM1 semiempirical quantum chemistry method using the LUMPAC suite of programs28, 29, and the calculated result is shown as the dashed blue line in Fig. 3a. The strong singlet-singlet transition is located at 346 nm. In the alternative calculation using ORCA30, this feature is shifted to lower energy at 530 nm (red dashed line, Fig. 3a).
Transient absorption (TA) spectroscopy, as two-dimensional spectroscopy, was used to investigate both the spectral and temporal properties of the samples. The femtosecond (fs) TA spectra at different delay times for YbDD in chloroform at low laser fluence are displayed in Fig. 5a. The S0 → S2 Soret absorption band is shown in orange color, and its stimulated emission band has a small red shift with respect to the ground-state bleach and gives a negative signal49. The triplet-triplet (T1 → Tn) absorption bands are observed at longer wavelengths (440-530 nm)50, with maximum intensity at 451 nm, corresponding to the terminal state energy of 35, 578 cm-1. The lifetimes of the bleach and the excited state transients for YbDD were determined by monitoring at wavelengths of 424 and 451 nm, respectively. (Fig. 5b). The two results are effectively the same and are in the picosecond scale, denoting a rapid singlet-to-triplet intersystem crossing. The femtosecond TA absorption spectra were also obtained using a higher pump fluence (Fig. 5c). The pulsed laser with high fluence produces a thermal effect of the YbDD, which causes distortions of the porphyrin structures and results in significant redshifts in the electronic absorption spectra51. In contrast to the lower fluence, a redshift of the Soret band (Δλ = + 36 nm) and the T1 → Tn absorption bands were observed. It is worth noting that the structural change was detected instantly by ultrafast TA spectroscopy: the peak at 501 nm started to shift to 527 nm after 100 ps, and the whole conformation changing process was completed in nanoseconds (Fig. 1d). Furthermore, the formation of the triplet state from the singlet excited state is clearly observed from the kinetics at 527 nm in Fig. 5d. In contrast to the 527 nm, the excited singlet state at 501 nm de-excited exponentially to the ground states. However, when using nanosecond (ns) TA spectroscopy, the detailed kinetics of the triplet-triplet absorption peak at approximately 526 nm could not be resolved since the conformation was changing too quickly (Fig. 5e). The ground state bleach recovery lifetime was extracted from the nanosecond TA spectra (0.69 μs), which is consistent with the decay lifetime by monitoring deactivation of the triplet state signal at 526 nm (0.69 μs) (Fig. 5f). Isosbestic points were found in all TA spectra (Fig. 5a, c, e), which suggest that only one single photoexcited species was formed in each case.
Fig. 5 Transient absorption spectra of YbDD.
a fs-TA spectra at different time delays at a low pump fluence (15 µJ cm-2), b ground state bleach recovery dynamics at 424 nm and excited state decay at 451 nm of YbDD in chloroform following 395 nm laser excitation. c Spectral evolution in fs-TA at 395 nm laser excitation having 45 µJ cm-2 fluence. d Normalized excited state decay kinetics at 501 and 527 nm. e ns-TA spectra at different time delays with a 45 µJ cm-2 pump fluence. f Ground state bleach recovery dynamics at 460 nm and the excited state decay at 526 nm of YbDD in chloroform following 395 nm laser excitation. The UV-vis spectra of YbDD are added in the upper panels to guide the ground state bleach