High affinity threading of a new tetralactam macrocycle in water by fluorescent deep-red and near-infrared squaraine dyes

Abstract

A new tetralactam macrocycle was prepared and found to encapsulate deep-red and near-infrared squaraine and croconaine dyes in water with tunable threading kinetics. The new supramolecular paradigm of guest back-folding was used to increase macrocycle/squaraine affinity by 370-fold and achieve an association constant of 2.8 × 10⁹ M⁻¹.

Graphical Abstract

Guest back-folding increases squaraine affinity for a new tetralactam macrocycle 370-fold in water.

Over the last decade there has been an increasing effort to create fluorescent molecular probes for biological imaging.^1–8 While dyes that absorb in the UV-visible window can be used for microscopic imaging of transparent objects such as cells, it is well-recognized that fluorescence imaging of more opaque objects such as thick tissue samples or living subjects is best performed using dyes that absorb and emit deep-red or near-infrared wavelengths of light that penetrate more deeply through the sample with less scattering.⁹ To this end, our group has focused our efforts on improving the photophysical properties of deep-red and near-infrared squaraine dyes through encapsulation by tetralactam macrocycles.^10–11 Furthermore, we are developing a fundamentally new approach to the construction of targeted fluorescent probes for biological imaging by exploiting noncovalent assembly as a means to connect targeting ligands to a fluorescent squaraine dye.^12–13 Our previous biological imaging studies have employed targeted fluorescent probes that were preassembled by threading derivatives of the water-soluble tetralactam M1 (Scheme 1) with a deep-red squaraine dye that emitted ~700-nm light.^14–16 In the best cases, K_a values for squaraine threading of M1 in water were greater than 10⁹ M⁻¹. The primary noncovalent interactions that stabilize the threaded complex are strong hydrogen bonds between the tetralactam NH resides and the squaraine oxygens and stacking of the squaraine aromatic rings against the interior surfaces of the surrounding macrocycle 9,10-anthrylene sidewalls. Very recently we disclosed a new class of squaraine dyes that exhibit narrow absorption bands and emission near 800 nm, as well as the ability to thread M1 with high affinity.¹⁷ This breakthrough suggests a pathway towards a new family of next-generation preassembled threaded probes that emit at 700 or 800 nm for use as paired agents in multicolor imaging protocols. An important question at this decisive point in the research is the future choice of the tetralactam macrocycle. While M1 (and its derivatives) has several useful supramolecular properties that make it attractive for probe preassembly, we are concerned that the 9,10-anthrylene sidewalls in M1 may become problematic for certain types of biological imaging. For example, the flat and hydrophobic anthracene may promote poor solubility, low biocompatibility, or susceptibility to photoxidation.^18–19 In a search for alternative tetralactam macrocycles with different sidewalls, we recently reported that the organic-soluble tetralactam M2 with 2,3,5,6-tetramethylphenylene sidewalls adopts a highly preorganized structure that can be threaded by an organic-soluble squaraine dye in chloroform to give a stable fluorescent complex.²⁰ Here we describe the next step in this research, which is to disclose the synthesis and squaraine threading properties of M3, a new water-soluble analogue of M2. At the beginning of the project, it was not clear how the change in macrocycle sidewalls from 9,10-anthrylene in M1 to 2,3,5,6-tetramethylphenylene in M3 would affect the kinetics of squaraine threading in water, but we expected a decrease in association constant due to the lower sidewall hydrophobic surface area. As we suspected, initial studies with standard squaraine dyes noted a moderate drop in squaraine affinity; however, exploitation of guest back-folding as a new paradigm in supramolecular design (see below) enabled us to produce a specific squaraine guest for M3 with nanomolar affinity in water.

Scheme 1.

Structures of the compounds investigated in this study with relevant atom labels. The insert shows a cartoon picture of a macrocycle/squaraine complex.

Tetralactam M3 was prepared by the straightforward synthetic procedure described in the ESI (Scheme S1). The solubility of M3 (and its synthetic precursors) is high, which makes it an attractive synthetic host for detailed supramolecular studies. The methyl groups on the two aromatic sidewalls ensure that M3 adopts a highly preorganized structure in solution with all four NH residues directed into the macrocyclic cavity. A series of studies assessed the capabilities of five different squaraines and one croconaine dye to thread M3 in water. Each dye has a long polyethylene glycol (PEG) chain appended to the terminal nitrogen atom at each end of the structure which ensures good water solubility. Previous threading studies have shown that the threading kinetics and thermodynamics are not affected by the length of the two flanking PEG chains, but the rate of threading is quite sensitive to the steric size of the second N-alkyl substituent attached to each terminal nitrogen atom.²¹ Separate and independent proof that macrocycle threading occurred in each case was gained by observing diagnostic changes in ¹H NMR spectral patterns, squaraine absorption and fluorescence maxima bands, and electrophoresis migration behavior.¹⁰ An example of the changes in ¹H NMR spectral patterns is shown in Figure 1. Characteristic changes in chemical shift are observed when EtSQ700 is mixed with M3 at a 1:1 molar ratio in D₂O to produce M3⸧EtSQ700. As expected, the thiophene protons 1 and 2 for free EtSQ700 are broad due to fast exchange of the cis and trans squaraine conformers, but upon formation of M3⸧EtSQ700, they sharpen considerably because conformational exchange for the encapsulated squaraine is very slow.¹⁰ Additionally, the signal for interior macrocycle proton B is shifted substantially downfield and split into two unequal signals that reflect the conformation of the encapsulated squaraine. The major peak for proton B is a singlet corresponding to a squaraine conformation with its two thiophene units in a trans orientation, and the minor peak is a pair of singlets corresponding to the squaraine conformation with the thiophene units in a cis orientation. Similar ¹H NMR results were observed for threading of M3 by PrSQ700 and EtSQ800 (Figures S12 and S13).

Fig. 1.

¹H NMR (500 MHz, D₂O) of (a) EtSQ700 (1.0 mM), (b) M3⸧EtSQ700 (1.0 mM), and (c) M3 (1.0 mM). Atom labels are provided in Scheme 1. Asterisks indicate the minor complex where the thiophene units of EtSQ700 are in the cis conformation (trans preferred in 3:1 ratio by integration). The cartoons show EtSQ700 (colored blue) with the two thiophene units in a trans orientation. The small unlabeled peaks near proton 1 are residual NH signals that have not yet exchanged with the deuterated solvent.

As shown by the absorption and fluorescence data in Figures S7–S9 and Table S1, separate solutions of deep-red squaraines MeSQ700, EtSQ700, and EtSQ800 exhibited a red shift in absorption and fluorescence maxima bands, an increased Stokes’ shift, and an enhancement in fluorescence intensity when each squaraine solution was treated with one molar equivalent of water-soluble M3. In the case of near-infrared EtSQ800, a dye self-aggregation band at 700 nm in the absorption spectrum disappeared upon encapsulation by M3. The croconaine dye MeCR800 is not fluorescent, but dye encapsulation by M3 was indicated by a large red shift in the croconaine absorption maxima band (Figure S11 and Table S2). The one macrocycle threading system that unexpectedly did not produce a measurable change in dye absorption/emission wavelength was M3 threading by PrSQ700 (Figure S16), but the combination of ¹H NMR (Figure S13) and electrophoresis data (Figure S17) leaves no doubt that threading occurred to form M3⸧PrSQ700.

The changes in dye spectral properties (red shift in absorption and emission maxima bands and increase in fluorescence quantum yield) enabled absorption or fluorescence titration experiments, yielding titration isotherms (Figures S22–S27) that were fitted to 1:1 binding models to give the association constants in Table 1. Similarly, a standard fluorescence stopped flow method was used to acquire kinetic profiles and second-order rate constants for threading of M3. Inspection of the data in Table 1 shows that the dyes MeSQ700, EtSQ700, EtSQ800, and MeCR800 all threaded M3 with K_a values on the order of 10⁶ M⁻¹, approximately 100–1000 times lower than K_a for squaraine threading of M1.²¹ In each case, the values for k_on for dye threading of M3 are about 1000 times slower than threading of M1. This decrease in threading rate is most likely due to the reduced portal size, an effect that is analogous to that seen with cucurbiturils.²² There is a high sensitivity to the steric size of the N-alkyl substituent at each end of the squaraine structure, a trend seen with previous studies of M1.²¹ For example, changing N-methyl in MeSQ700 to N-ethyl in EtSQ700 produces a 1000-times slower rate constant for threading of M3.

Table 1.

Association constants (K_a) and rate constants (k_on and k_off) for threading of M1 and M3 in H₂O at 25 °C.^a

Complex	Ka (M−1)	kon (M−1s−1)	koff (s−1) b
M3⸧MeSQ700	(7.6 ± 1.1) × 106	(8.4 ± 1.5) × 105	0.11
M1⸧MeSQ700	(1.0 ± 0.2) × 109	(7.2 ± 0.3) × 108	0.72
M3⸧EtSQ700	(9.0 ± 5.1) × 106	(6.6 ± 0.9) × 103	0.00073
M1⸧EtSQ700	(1.1 ± 0.4) × 109	(4.3 ± 0.3) × 106	0.0039
M3⸧EtSQ800	(1.8 ± 1.4) × 106	(9.4 ± 0.2) × 103	0.0052
M1⸧EtSQ800	(6.3 ± 3.4) × 108	(7.3 ± 1.6) × 106	0.012
M3⸧MeCR800	(1.4 ± 0.3) × 106	-	-
M1⸧MeCR800	(1.4 ± 0.2) × 109	-	-
M3⸧BzSQ700	(2.8 ± 2.4) × 109	(5.8 ± 0.6) × 105	0.00023
M1⸧BzSQ700	(8.5 ± 1.7) × 109	(7.4 ± 2.2) × 108	0.087

^aData for M1 taken from references 10, 17, 21, and 23.

^bk_off calculated from K_a and k_on.

The values of K_a and k_on for threading of M3 by EtSQ700 are high enough to ensure quantitative formation of M3⸧EtSQ700 within a few minutes when both components are mixed at high micromolar concentrations at 25 °C. Equally important is the low value of k_off indicating that the threaded complex has high kinetic stability. Thus, appropriate derivatives of M3 and EtSQ700 will likely be quite useful for preassembly of long-lived fluorescent probes for future biological imaging studies. In the case of in situ capture applications, however, it will be much easier to develop useful protocols if K_a for the squaraine/macrocycle pair was at least 10⁹ M⁻¹.^12–13 With this goal in mind, we were drawn to the new supramolecular paradigm of guest back-folding as a potential way to improve K_a for squaraine threading of M3.²³ We investigated threading using squaraine BzSQ700, which has a flanking N-benzyl ring at each end of its structure. As indicated by the cartoon in Scheme 2, each flanking N-benzyl can stabilize a threaded complex by folding back and interacting with the exterior surface of the surrounding macrocycle. Recently we observed that K_a for threading of M1 with BzSQ700 was 8-fold higher than K_a for threading with MeSQ700, a control squaraine whose structure lacks the flanking N-benzyl rings.²³ In this present study, we sought to determine if a guest back-folding effect would improve squaraine affinity for M3. Macrocycle threading to form M3⸧BzSQ700 was confirmed by observing the expected diagnostic changes in ¹H NMR chemical shifts (Figure S14) and absorption/emission spectra (Figure S10). The complexation-induced enhancement in fluorescence emission permitted fluorescence titration experiments that measured association constant and threading rate constant. Analysis of the data in Figure S25 and S31 gave values of K_a = (2.8 ± 2.4) × 10⁹ M⁻¹ and k_on = (5.8 ± 0.6) × 10⁵ M⁻¹s⁻¹. Inspection of the data in Table 1 shows that K_a to form M3⸧BzSQ700 is a remarkable 370 times higher than the K_a to form M3⸧MeSQ700, suggesting a much stronger guest back-folding effect than previously observed with M1. Independent verification of K_a for M3⸧BzSQ700 was gained by conducting a competitive NMR macrocycle threading experiment. A solution of M1 was mixed with an equimolar amount of M3⸧BzSQ700 and the mixture was analyzed by ¹H NMR (see spectra in Figure S19). Integration of the diagnostic signals for macrocycle protons B in M1⸧BzSQ700 and M3⸧BzSQ700 indicated an equilibrium ratio of 1:0.34, within error of the ratio predicted by the respective K_a values in Table 1. Strong experimental evidence for guest back-folding in the threaded M3⸧BzSQ700 complex was provided by a NOESY experiment at 700 MHz which showed that the benzyl protons 3 and 4 of BzSQ700 undergo through-space cross-relaxation with macrocycle protons C (Figure 2 and S15). Additional computational support for guest back-folding was gained by conducting a 100 ns molecular dynamic (MD) simulation of a model of M3⸧BzSQ700 utilizing explicit water solvation (a movie of the MD simulation can be found in the ESI). Trajectories suggest that most of the complex’s co-conformational states allow stacking of one or both of the flanking N-benzyl rings from encapsulated BzSQ700 against the isophthalamide bridging units of the surrounding M3.²⁴ The combination of NOESY and MD data suggests that a likely reason for the enhanced guest back-folding effect exhibited by M3 is the additional stacking interactions provided by the triazole group attached to each isophthalamide bridging unit (these attached triazole groups are absent in M1).²⁵

Scheme 2.

Schematic representation of increased squaraine/macrocycle affinity due to guest back-folding. After macrocycle threading, the N-benzyl ring at each end of squaraine BzSQ700 can fold back and stabilize the complex by interacting with the macrocycle exterior.

Fig. 2.

Partial 2D NOESY (700 MHz, D₂O) of M3⸧BzSQ700 showing cross-peaks between proton C of M3 and benzyl protons 3 and 4 of BzSQ700 indicating guest back-folding.

To demonstrate the potential utility of this guest back-folding effect, a pair of guest-selection experiments were conducted that measured the capability of M3 to selectively bind a squaraine dye (MeSQ700 or BzSQ700) in the presence of a structurally similar croconaine MeCR800. The experiment was enabled by the narrow near-infrared absorption band of MeCR800 at 780 nm which is easily distinguished from the narrow squaraine absorption band at 670 nm. Tetralactam macrocycles are known to have similar affinities for croconaine and squaraine dyes,²⁶ and the measured K_a of (1.4 ± 0.3) × 10⁶ M⁻¹ for threading of M3 with MeCR800 (Figure S11, S26, and S27) was expected for a croconaine structure which did not allow guest back-folding. The two related guest-selection experiments are illustrated in Scheme S2. In the first experiment, aliquots of M3 were sequentially added to a binary equimolar mixture of high affinity BzSQ700 (due to guest back-folding) and relatively low affinity MeCR800 in water. The absorption maxima bands for the two dyes were monitored simultaneously, with an expectation that M3 would preferentially encapsulate BzSQ700 and induce a red shift in its absorption maxima. As shown by the spectral data and speciation plots in Figure S20, the selectivity of M3 for BzSQ700 over MeCR800 was observed to be very high. This contrasts with the outcome of the second experiment in Scheme S2; a control squaraine competition experiment that found M3 only has a very small preference for MeSQ700 over MeCR800 (Figure S21). Both dyes in this second experiment have similar affinities for M3 because they do not have the flanking N-benzyl rings needed to provide a strong guest back-folding effect. The results of these two experiments illustrate how guest back-folding can be exploited as a powerful new molecular design paradigm for various high affinity and high selectivity in situ capture applications. In the current case, macrocycle selectivity for a specific squaraine guest was greatly increased not by the traditional approach of synthesizing a more complementary squaraine core structure but by the novel design strategy of attaching peripheral flanking N-benzyl units that promoted guest back-folding after squaraine capture.

From the perspective of preassembly, the new water-soluble M3 is a welcome addition to the portfolio of tetralactam macrocycles that can be used to encapsulate squaraine and croconaine dyes and make preassembled deep-red and near-infrared fluorescent probes for various biological imaging and phototherapeutic studies. The systematic collection of thermodynamic and kinetic data produced by this study allows us to choose the best squaraine or croconaine dyes for preassembly of 700-nm or 800-nm probes for a range of different theranostic applications. We are grateful for funding support from the NIH (R01GM059078), Dr. J. Peng for NMR advice, and helpful comments from anonymous reviewers.