Rotaxane Formation Increases Squaraine Fluorescence Brightness beyond 900 nm by 25-fold

Abstract

A squaraine dye with two flanking benzo[cd]indoles absorbs strongly at 881 nm, but the fluorescence quantum yield is very low due to dye flexibility. Rigidification of the squaraine structure by encapsulation within a rotaxane produces a 25-fold increase in fluorescence brightness with an SWIR emission band that peaks at 915 nm and extends beyond 1100 nm. Excitation of the rotaxane’s anthrylene sidewalls with 375 nm light produces a very large pseudo-Stokes shift of 540 nm.

Graphical Abstract:

Fluorescence bioimaging is improved by using long wavelength light that can penetrate further through skin and tissue and produce sharper images due to reduced scattering. At present, there is great interest in developing next-generation fluorescent dyes that can absorb and emit light at near infrared (NIR) or short-wave infrared (SWIR) wavelengths (i.e., >900 nm).^1,2 Many of these long wavelength dyes are π-extended chemical structures with inherent spectral and physicochemical properties that limit bioimaging performance.³ A particularly important limitation is a very low fluorescence quantum yield.^4,5 In part, this is because the low-energy electronic excited state is coupled to vibronic transitions, which provides an efficient pathway for nonradiative relaxation of the excited state.⁶

One of the most common molecular design strategies to improve the fluorescence quantum yield is to rigidify the fluorochrome structure and eliminate dynamic processes, such as bond rotations, that favor nonradiative relaxation.⁷ This strategy is usually effective for dyes that emit visible light with wavelengths of <650 nm; however, systematic studies of longer wavelength dyes are rare, and clear trends have yet to be established. Recent work by Yang and co-workers has shown that structural rigidification of a bisbenzannulated carbon-rhodamine with a central spiro group leads to an increased NIR fluorescence quantum yield.⁸ Similarly, reports by Wang and co-workers and Si and co-workers have shown that intramolecular electrostatic locking can reduce bond rotations and increase SWIR emission intensity.^9,10 Conversely, the group of Schnermann and co-workers rigidified a set of homologous polymethine dyes by multiring annulation and observed a large increase in the fluorescence quantum yield for a deep-red pentamethine system (~650 nm)¹¹ but found no change in the quantum yield for a NIR heptamethine cyanine system (~780 nm).¹² A related problem with highly π-extended cyanine dyes is the tendency to favor nonsymmetrical π-bond isomers in polar solvents, which broadens absorption bands and greatly decreases fluorescence brightness.^13–15

For some time, we have developed synthetic methods to encapsulate deep-red squaraine dyes within a mechanically interlocked rotaxane structure.^16–18 Our efforts to develop squaraine rotaxanes with longer wavelength emission have been limited by the scarcity of suitable squaraine dye structures.^19–22 However, recent publications have shown that squaraines with flanking π-extended benzo[cd]indoles, such as SQ909 (Scheme 1), have SWIR absorption and emission bands, although they exhibit extremely low fluorescence quantum yields.^23,24 This is likely caused by photoisomerization and formation of a twisted intramolecular charge transfer state that promotes nonradiative relaxation.^25,26 The literature on homologous deep-red indole-based squaraines includes evidence that structural rigidification by internal hydrogen bonding, steric crowding, or macrocycle encapsulation can suppress photoisomerization and increase the fluorescence quantum yield.^27–30 We reasoned that encapsulation of benzo[cd]indole squaraine SQ909 within a rotaxane structure would restrict internal bond rotation and increase the fluorescence quantum yield. We have tested this hypothesis by preparing SR902 and SR915 as the first two examples of benzo[cd]indole squaraine rotaxanes with SWIR emission bands that are >900 nm. We find that the internal motion of the encapsulated dye within each rotaxane is greatly reduced, and there is a large increase in fluorescence brightness.

Scheme 1.

Chemical Structures with Atom Labels and Arrows Indicating Bond Rotations

Squaraine SQ909 was prepared by a published procedure and characterized by standard spectroscopic methods in organic solvents.²³ Shown in Figure 1 are partial ¹H NMR spectra at two different temperatures. Notably, the peaks for protons 6–8 exhibited splitting at lower temperatures. This dynamic NMR behavior has been reported previously,²⁵ and it is attributed to the bond rotations shown in Scheme 1 that produce stereoisomers. The six possible isomers of SQ909 are illustrated in Figure S2, and previous studies have concluded that the major isomer in solution is the “trans-anti-out” isomer that is depicted in Scheme 1.²⁵ Support for this assignment was provided by a 2D ¹H–¹H NOESY spectrum of SQ909 that indicated cross-relaxation between protons 7 and 8 but no cross-relaxation between protons 7 and 6 (Figure 2).

Figure 1.

Variable-temperature ¹H NMR spectra of SQ909 (CDCl₃, 500 MHz).

Figure 2.

2D ¹H–¹H NOESY spectrum of SQ909 (CDCl₃, 500 MHz) with the red circle highlighting a cross-peak and the blue circle highlighting the absence of a cross-peak.

Squaraine SQ909 was converted into rotaxanes SR902 and SR915 by conducting Leigh-type clipping reactions as detailed in Scheme S1.¹⁷ Figure 3 shows a comparison of ¹H NMR chemical shifts for the three separate compounds. The spectra exhibit the expected loss in spectral symmetry and changes in chemical shift that are NMR signatures of squaraine rotaxanes. For example, the increased number of proton peaks for the sidewall components within the surrounding tetralactam macrocycles in both rotaxanes reflects decreased chemical symmetry due to anisotropic shielding by the encapsulated benzo[cd]indole squaraine and the loss of sidewall rotational freedom. Similarly, the peaks for squaraine protons 6–8, which are broad for free SQ909 at 298 K, are sharp for rotaxanes SR902 and SR915 (even when the sample is warmed to 333 K (Figure S3)), indicating the encapsulated dye is rigid and not undergoing significant bond rotation.

Figure 3.

¹H NMR (CDCl₃, 400 MHz, 298 K) comparison of SR902 (top), SQ909 (middle), and SR915 (bottom).

Theoretical support for increased squaraine rigidity within the rotaxanes was gained by conducting two sets of computational studies. The first used density functional theory (DFT) to estimate a rotational barrier for free SQ909 (Figure S4) that was commensurate with the dynamic NMR behavior. A subsequent set of molecular dynamics simulations quantified trajectories for SQ909, SR902, and SR915 at 400 K. As shown by the conformational distributions in Figure S5, the squaraine bond rotation was relatively restricted when the dye was encapsulated inside the rotaxane macrocycle.

Recrystallization of squaraine rotaxanes SR902 and SR915 produced single crystals that were suitable for analysis by X-ray diffraction. Each solid-state structure shows that the tetralactam macrocycle adopts a chair conformation and surrounds the core of the encapsulated squaraine that is in a “trans-anti-out” conformation (Figure 4). The phenylene sidewalls within the surrounding macrocycle for SR902 are located directly over the central ring of the encapsulated, planar squaraine dye, with bifurcated hydrogen bonds between the macrocycle NH residues and the squaraine oxygen atoms. The distance between the two phenylene sidewalls in SR902 (6.762 Å) is shorter than the distance between the two anthrylene sidewalls in SR915 (6.987 Å). This difference is attributed to the internal hydrogen bonding between the NH residues and the adjacent nitrogen atom in the two bridging 2,6-pyridinedicarboxamides within the surrounding macrocycle of SR902, which contracts the macrocyclic cavity. An interesting structural feature of both rotaxanes is the relative orientation of the two squaraine N-ethyl groups, which are directed in opposite directions away from the plane of the encapsulated squaraine. In the case of SR915, the steric crowding induced by these oriented N-ethyl groups forces the encapsulated squaraine to bend slightly at each end and the two anthrylene sidewalls to adopt a slipped–stacked orientation as shown in Figure 4b. These rotaxane structure distortion effects indicate that the anthrylene sidewalls within the surrounding macrocycle are held tightly against the encapsulated squaraine dye.

Figure 4.

X-ray crystal structures with side and top views of (a) SR902 and (b) SR915 (methyl groups shown as space filling, and red arrows indicate steric crowding with the anthrylene sidewalls of the surrounding macrocycle).

Listed in Table 1 are the spectral properties of the three dyes in dichloromethane (DCM). As shown by the absorption spectra in Figure 5, there was no evidence for dye self-aggregation, and the molar absorptivity decreased in the following order: SR915 > SQ909 > SR902 (Table 1). There was little difference in absorption and emission maximum wavelengths,³¹ and the Stokes shift in each case was close to 30 nm. There is a blue-shifted shoulder in each absorption spectrum and a symmetrical red-shifted shoulder in the corresponding emission spectrum, which has been noted before for this class of squaraine dyes and assigned to vibronic coupling.^10,25 As reflected by the values of the full width at half-maximum (fwhm) in Table 1, the absorption maximum peaks for the two rotaxanes are sharper than the peak for the free squaraine dye, reflecting a reduced number of vibrational transitions (i.e., increased squaraine rigidity). The rotaxane absorption spectral profiles hardly changed when the solvent was exchanged with the more polar methanol (Figure S6), and there was only a ~30% decrease in fluorescence brightness. This is a favorable SWIR fluorescence performance attribute, especially when compared to long wavelength π-extended cyanine dyes that exhibit extensive band broadening and quenched fluorescence in polar solvents such as methanol.^13–15

Table 1.

Photophysical Properties in DCM

	SQ909	SR902	SR915
ε (×105 M−1 cm−1)	1.5	1.0	2.2
λabs (nm)	881	871	882
Abs. fwhm (nm)	61.6	36.2	36.0
λem (nm)a	909	902	915
Stokes shift (nm)	28	31	33
Φf (%)	0.18b	0.91 ± 0.28c	3.0 ± 0.9c
brightness (×103 M−1 cm−1)	0.26	0.92 ± 0.28	6.6 ± 2.0

^aλ_ex = 790 nm; slit width = 29.4 nm.

^bFrom ref 22.

^cRelative to SQ909.

Figure 5.

Absorbance spectra of (a) SQ909, (b) SR902, and (c) SR915 in DCM and their (d) Beer–Lambert plots that give the molar absorptivities.

Most notably, conversion of SQ909 into a rotaxane produced a large increase in the fluorescence brightness. The reported fluorescence quantum yield for SQ909 is 0.18%, and we used it as reference dye to obtain the relative quantum yields of both rotaxanes.²³ From the ratio of emission peak areas, we determined fluorescence quantum yields of 0.91% and 3.0% for SR902 and SR915, respectively. Defining brightness as molar absorptivity multiplied by quantum yield, we find that rotaxane SR915 with anthrylene sidewalls is 25 times brighter than free dye SQ909. As shown in Figure 6a, the emission tail of SR915 continues beyond 1100 nm, suggesting great promise for future SWIR imaging applications.

Figure 6.

Normalized fluorescence emission of (a) SQ909, SR902, and SR915 (5 μM in DCM, λ_ex = 790 nm, slit width = 29.4 nm) and (b) SR915 at different excitation wavelengths (3 μM in DCM, slit width = 29.4 nm).

Another favorable fluorescence property exhibited by rotaxane SR915 is very efficient energy transfer from the excited anthrylene sidewalls in the surrounding macrocycle to the encapsulated squaraine. More specifically, excitation of the anthrylene sidewalls with blue light (375 nm) produces greatly reduced anthrylene fluorescence (94% decrease (Figure S8)) and relatively strong SWIR fluorescence with a very large pseudo-Stokes shift of 540 nm (Figure 6b). The capacity to excite a fluorescent SWIR dye at two distinctly different wavelengths is a useful attribute for in vivo imaging of living subjects. Often during fluorescence-guided surgery, there is a need to ascertain if a fluorescently labeled target is directly exposed to the imaging camera or buried beneath the tissue surface.³² This question can be readily answered by using an imaging protocol that switches between short and long excitation wavelengths. If the target is buried, there will be decreased imaging fluorescence due to poor tissue penetration of the shorter excitation light.^33,34

A common photochemical property of anthracene-containing dyes is facile reaction of the anthracene unit with in situ-photogenerated singlet oxygen to form a bleached 9,10-endoperoxide product.³⁵ However, a series of comparative experiments found that the surrounding anthrylene-containing macrocycle in rotaxane SR915 is completely resistant to reaction with photogenerated singlet oxygen (Table S1), which is a striking finding since 9,10-endoperoxide formation is rapid when the anthrylene macrocycle is a free compound.³⁶ We attribute this unusual decrease in reactivity to cross-component strain induced by the mechanical bond that interlocks the two components in rotaxane SR915.³⁷ As indicated by the X-ray structure in Figure 4, the anthrylene sidewalls within the surrounding macrocycle are held tightly against the encapsulated squaraine dye, which disfavors anthrylene reaction with singlet oxygen and formation of a nonplanar 9,10-anthrylene endoperoxide due to the large increase in bond angle strain (Scheme S3).

In summary, encapsulation of π-extended benzo[cd]indole squaraine dye SQ909 as rotaxane SR915 leads to a rigidified dye structure that exhibits 25-fold greater fluorescence brightness with an emission band that peaks at 915 nm and a tail that extends well beyond 1100 nm. Synthetic analogues of benzo[cd]indole squaraine dyes, with additional conjugation, are known to have emission maximum peaks that are >1000 nm,²⁵ which makes them very attractive for next-generation SWIR bioimaging. Our results suggest that rotaxane encapsulation of these π-hyperextended squaraine dyes will improve SWIR imaging performance by enhancing fluorescence brightness, increasing dye stability, enabling dual-wavelength excitation, and preventing undesired dye quenching upon self-aggregation.^3,16,38 Moreover, well-established synthetic methods can be used to append water solubilizing and biological targeting units and to generate new biomedically valuable molecular imaging agents.^29,33,39

Supplementary Material

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.orglett.5c02456.

Experimental procedures, compound characterization data, photophysical data, and computational results (PDF)

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.