In-Situ Observation of Photoswitching by NMR Spectroscopy: A

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In-Situ Observation of Photoswitching by NMR Spectroscopy: A Photochemical Analogue to the EXSY Experiment Eduard Stadler, Sebastian Tassoti, Pascal Lentes, Rainer Herges, Toma Glasnov, Klaus Zangger, and Georg Gescheidt Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02613 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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Analytical Chemistry

In-Situ Observation of Photoswitching by NMR Spectroscopy: A Photochemical Analogue to the EXSY Experiment Eduard Stadler*,‡,†, Sebastian Tassoti‡,§, Pascal Lentes⊥, Rainer Herges⊥, Toma Glasnov§, Klaus Zangger*,§, Georg Gescheidt*,† †Institute §Institute ⊥Otto

of Physical and Theoretical Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstraße 28, A-8010 Graz, Austria

Diels Institute for Organic Chemistry, University of Kiel, Otto-Hahn-Platz 4, DE-24118 Kiel, Germany

ABSTRACT: We present 1D and 2D NMR experiments providing in-situ insights into photoinduced isomerizations. Irradiation during the mixing period of an EXSY experiment leads to characteristic cross peaks in 2D spectra. The photo-triggered exchange of magnetization occurring in photoswitchable (Z) and (E) isomers of three selected azo compounds provides information on the dynamic E/Z equilibria. We report on the dependence of the diagonal-to-cross-peak ratio on concentration, light intensity and mixing time. In analogy to exchange spectroscopy, this ratio mirrors the efficiency of light induced molecular transformations. Furthermore, we present a time saving 1D version and a combined light/phase cycle scheme for enhanced detectability of photoinduced changes in the spectrum. This insight into light-induced structural changes is highly suited to study macromolecules in which photoswitchable units trigger conformational changes.

INTRODUCTION Photoswitchable molecules are utilized in many fields, where a straightforward on/off switching of molecular functionality by an external stimulus is desirable. In photochromic compounds, light triggers reversible, structural changes like ring closure or E/Z-isomerization with high spatial and temporal control. The synthetic accessibility, simple modification and large distance change between the (E) - and (Z)-isomer makes azobenzene one of the most frequently employed switchable systems. Azobenzenes are therefore used as actuators in chemical motors1–3, material science4–6, and in a number of biomedical applications.7,8 Proteins have been modified with azobenzene to block or open ion channels9 and change protein and peptide structures in a reversible fashion.10,11 Another promising field is the triggered release12 and development of smart, highly targeted drugs (photo pharmacology) where switching from an inactive to an active form can be achieved in vivo at specific positions.13,14 Recent findings suggest that an extremely high number of bioactive ligands could be made photoresponsive by incorporation of azobenzene units.15 NMR spectroscopy is an ideal tool for following photoinduced (reversible) transformations at molecular resolution. Photo-triggered reactions can be performed in-situ with optical fibers inserted into the sample tube.16,17 In addition to determining structure and concentrations, NMR is also a vital tool to study conformational changes or chemical exchange processes. These processes lead to line broadening effects in 1D spectra or change the appearance of cross peaks in 2D spectra.18 2D NMR has been used extensively to extract quantitative information, especially due to the limited resolution in 1D

spectra at low magnetic fields.19,20 Slow exchange processes become observable by following the fate of longitudinal magnetization. Experimentally, this is achieved by utilizing exchange spectroscopy techniques (EXSY)21 which are related to NOE type pulse sequences.22 Magnetization can also be transferred in chemical (photo)-reactions that are induced during the mixing time in modified NOE pulse sequences (SCOTCH experiment).23,24 The basic 2D-NOESY/EXSY and SCOTCH pulse sequences are shown in Scheme 1. During the first delay, t1, the spins are frequency labelled. After that, the magnetization is aligned with the z-axis by another radio-frequency-pulse. During the mixing time longitudinal magnetization may be transferred by the NOE or slow exchange. In the latter case, cross peaks correlate shifts of different conformations and allow quantification of the exchange processes.

Scheme 1. (a): Basic NOESY and EXSY pulse sequence, consisting of three rf-pulses separated by the t1 evolution time for frequency labelling and a mixing period τm where longitudinal magnetization is transferred by NOE or chemical exchange. (b):

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Sequence for the SCOTCH experiment. During the mixing time magnetization is exchanged by a photochemical reaction.

Only recently, these sequences have been used with fiber coupled NMR techniques to study the photoinduced conversion of an azobenzene-linked peptide.25 In analogy to the SCOTCH acquisition scheme, the peptide was cycled between two distinct states during the NMR experiment. Irradiation of the sample during the mixing time induced E/Z-isomerization. The exchange of longitudinal magnetization between two isomers allowed correlating chemical shifts. After recording a single t1increment, the initial state was regenerated with light of a proper wavelength. The result of this experiment is a 2D correlation spectrum connecting equivalent resonances of the “on” and “off” state, a valuable tool for structure elucidation. While the structures of photoswitchable proteins, peptides or drugs are usually known, the efficiency of the isomerization, although being a critical parameter, is hardly addressed in current literature. In this work, we show an investigation of three azobenzene photoswitches depicted in Scheme 2. We have included parent azobenzene (1) representing a well-established reference system. In compound 2 ((E/Z)-1-(1-(4-(4-((4butylphenyl)diazenyl)phenyl)butanoyl)piperidin-4-yl)-1,3dihydro-2H-benzo[d]imidazole-2-one; OptoBI-1) the azobenzene is a part of an efficient photoswitchable system specially designed for the precise spatial and temporal optoregulation of TRPC3 – a Ca2+-transporter channel expressed in various human tissues.26,27 Successful in-vivo application of azobenzene based compounds requires to shift the switching wavelengths to the visible part of the spectrum. In this respect, substituting all ortho positions with methoxy groups11,28 or fluorine atoms29 proved to be successful. Another strategy is bridging of the aromatic rings, represented by compound 3 (5,6dihydrodibenzo[c,g][1,2]diazocine). This reverses the thermodynamic stability30 ((Z) being more stable than (E)) and increases the quantum yields of photoisomerization as compared to the parent azobenzene.31 The reversed stability has been used to switch the activity of neurotransmitters with light.32 N N

R2 N

N R2

R1

R1 (E) 1

R1 = R2 = H

2

R1 =

(Z) O R2 = O

N

N

NH

N N N N (Z)-3

2D correlation spectra. The quantification of slow exchange processes by exchange spectroscopy techniques is well established.21 However, to the best of our knowledge, this was not attempted for photoinduced isomerization reactions so far. In analogy to molecular rearrangements observed by EXSY experiments, photoinduced molecular movements become experimentally accessible in a selective way with the developed pulse sequences. To induce photoisomerization in-situ, we utilize a modified NOESY pulse sequence relying only on irradiation during the mixing time, a modification of the technique proposed by Nagashima et al.25 While previous work was based on fast, observable turnovers induced with light of high intensity during the NMR pulse sequence, we examine the exchange of magnetization in a steady state that is reached after prolonged irradiation. This removes the necessity of regenerating the initial state during the experiment and does not require high light intensities. In a first step, we explore the dependence of cros-peak intensity on irradiation intensity, mixing time and sample concentration. We include a quantitative description for the corresponding correlations in the NMR spectra. Then, we describe a 1D variant for these experiments, which allows rapid data collection. Furthermore, we exploit a novel dark/light phase cycling scheme to enhance spectral features. This approach suppresses diagonal and NOE peaks in 2D spectra and reveals photoinduced structural changes by their individual resonances.

EXPERIMENTAL NMR spectroscopy. All experiments were performed on a Bruker Avance III 500 MHz spectrometer equipped with a 5 mm TXI probe with z-axis gradients at 300 K or a Bruker Avance III 700 MHz spectrometer using a 5 mm cryogenically cooled TCI probe with z-axis gradients. All NMR data were processed using TopSpin (Bruker) and MestreNova (Mestrelab Research S.L.) software. Sample irradiation. Light emitted from high power LEDs (Nichia, NVSU233B (385 and 405 nm), Lumitronix) was directly coupled into the blunt and polished end of a 2.8 mm thick optical fiber (ceramoptec). At the tip of the fiber the nylon jacket was removed and a pencil shaped tip created by sandblasting. The fiber tip was directly immersed in the sample solution or put into a flat bottomed NMR tube (OD=3.97 mm, ID=2.97 mm, Wilmad) filled with D2O for the field frequency lock. The fiber and inner tube were put into a screw capped NMR tube (ID=4.22 mm, Wilmad) usually containing 100 µL of sample solution. This geometry results in a layer thickness of 0.125 mm. The light intensity emitted from the fiber tip was measured with a spectrophotometer by immersing the tip in an integrating sphere (GL Optic). The high power LEDs were powered by a custom made power source allowing for precise modulation of intensity by pulse width modulation (Sahlmann Photochemical Solutions) and triggering by the NMR console. Azobenzene 1 was purchased from Sigma-Aldrich and used as received. The photoswitchable molecules 2 and 3 were synthesized according to published procedures.30,33

(E)-3

Scheme 2. Structures and photochemical equilibria of 1, 2, and 3. Note that the thermodynamically stable isomer is shown on the left. The aim of our investigation is to extract and employ quantitative information that is available in the cross peaks in


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Analytical Chemistry

Figure 1. PC-EXSY of azobenzene (1) in MeOD-d4 obtained by irradiation with light of λ = 390 nm during the mixing time. Cross-peaks assigned to photochemical exchange share the negative sign of the diagonal (red), NOEs are of the opposite sign (blue). 8 s mixing time, TD/TD1 = 4096/128, NS = 8; 4 h 2 min experimental time, fiber directly immersed in solution.

RESULTS AND DISCUSSION Light-coupled NOESY experiment.25 Irradiation of pure (E)- azobenzene (1) with UV-light at λ = 390 nm induces isomerization to (Z)-1. Since the absorption spectra of both isomers overlap in this spectral region, a photo stationary state containing 22 % (Z)-isomer is reached. Further irradiation does not lead to an observable concentration change because the forward and reverse reaction rates are identical. In this light induced “dynamic” equilibrium molecules are still exchanged between the two forms carrying magnetization. Therefore, we propose to name this type of experiments PhotoChemicalEXSY (PC-EXSY). This magnetization transfer can be monitored using a modified gradient-enhanced NOESY experiment (see Scheme 1b).22 The resulting spectrum is shown in Figure 1. Irradiation at 390 nm during the mixing time yields cross-peaks between protons of the (E) - and (Z)-isomers. Identical reaction rates in both directions result in a symmetrical spectrum with respect to the diagonal, i.e. the c,f and f,c peaks in Fig. 1 have the same intensitiy. Additionally, the spectrum shows NOE cross peaks (e.g. between protons c and b) with opposite sign, which is typical for fast tumbling molecules. Investigation of cross peak intensity. As a starting point we wished to examine the intensity dependence of the light induced cross peaks and correlate them with measured quantities and molecular parameters like molar absorption coefficients and quantum yields. Figure 2a correlates the relative light intensity emitted from a 390 nm LED with the cross peak intensity after a mixing time of 5 s. As expected, there is a clear relationship, since higher photon fluxes increase reaction rates and, hence, the amount of exchanged magnetization.

Quantitatively, the exchange between two sites A and B depends two rate constants 𝑘 + and 𝑘 ― for the forward and reverse reaction, respectively. A photochemical equilibrium under irradiation, may be described similarly: In this case, the two rates depend on molecular and experimental parameters. Analytic expressions for both rate constants (Equation S2 and S3) are given in the SI. When reaction rates are identical, prolonged irradiation will give a 50:50 mixture of the isomers. However, for most steady states of photochromic compounds the exchange will be asymmetric (𝑘 + ≠ 𝑘 ― ). Second, we were curious if a variation of mixing time allows for extracting the exchange rates in analogy with EXSY experiments. A set of experiments with different mixing times was performed (2b) and the evolution of cross and diagonal peak intensities was analyzed. We have fitted the cross/diagonal peak ratio (𝑎𝐴 𝑎𝐴𝐵) vs. the mixing time (𝜏𝑚) with an exponential function: 𝑎𝐴 𝑎𝐴𝐵 = 𝑎(1 ― exp ( ― 𝑏𝑡)). Accordingly, the rate constant 𝑘 + for the A → B reaction is dy/dt at 𝑡 = 0, i.e. 𝑘 + = 𝑎𝑏. For short mixing times or low exchange rates, a linear approximation can be used and 𝑘 + may be extracted from a linear fit (Equation 1): 𝑎𝐴 𝑎𝐴𝐵

≈ 𝑘 + 𝜏𝑚 .

(1)

The isomeric ratio of the steady state provides 𝑘 ― and the sum of both rate constants, 𝑘 = 𝑘 ― + 𝑘 + (see SI). The data shown in Figure 2b were acquired with the fiber tip directly immersed in a 0.182 M azobenzene solution. For each data point a 2D spectrum was recorded and the cross/diagonal peak ratio extracted from a single slice along the indirect dimension containing the peaks “c” and “c,f” as indicated by the dotted line in Fig. 1. For mixing times between 0 and 20 s the relative



cross peak intensity is in the range of 1-7 %. With a light power of approximately 50 mW with a peak wavelength at 390 nm we obtained a value for 𝑘 + of 0.0029 s-1. This range is compatible with the calculated value (𝑘𝑐𝑎𝑙𝑐. = + 0.002 s-1) based on literature values for azobenzene isomerization34,35 at 405 nm (estimated irradiation volume of 100 µL). The (Z) content of 22% allows to extract the rate constant for the reverse (Z → E) process. This yields 0.0029/0.22 = 𝑘 ― = 0.0132 s-1 and hence, 𝑘 = 0.0161. The exchange rate k directly reflects the switching frequency induced in the NMR active volume, indicating that, on average, an azobenzene molecule undergoes isomerization every 62 s (= 1/0.0161 s-1). In general, rate constants well below 1 s-1 are expected, because the number of absorbed photons per second is much lower than the concentration of the photochromic compound. Applying this methodology for characterizing photodynamic equilibria is possible but values should be handled with care. The light distribution and reaction rates may not be distributed uniformly in the NMR active volume. A scheme of the irradiation setup is shown in the supplementary information (Figure S7). For instance, a vertical mismatch of the fiber tip and the NMR active volume leaves a fraction of sample in the dark. As a result, molecules in this sample volume do not undergo isomerization and contribute only to the diagonal peak intensity.

Figure 2. (a): Cross peak intensity as a function of light intensity. The insert shows slices of 2D spectra showing the light induced

cross peak (390 nm, 5s mixing time) correlating protons in para position of (E) and (Z)-1. (b): Correlation of the diagonal/cross peak ratio with the employed mixing time. A linear fit allowing to estimate the light induced isomerization rate according to Equation 1 is also shown. (c): Cross/diagonal peak ratio as a function of concentration. Data obtained with irradiation with 405 nm and 2 s mixing time. Data measured with the proposed 1D sequence.

Optically dense samples lead to a similar situation. The exponential decrease in light intensity (Beer-Lambert law) leads to hardly irradiated regions in the sample. From an experimental point of view, this implies that highest exchange rates should be obtained in dilute samples in thin layers with a fiber tip matched well with the radiofrequency coil. For a verification of this assumption we increased the concentration of azobenzene samples in a stepwise fashion and extracted the cross/diagonal peak ratio with a 1D-experiment (description see later section and Figure 2c) with a 405 nm LED. Additionally, we decreased the layer thickness drastically by placing the fiber inside a reference tube with an outer diameter of 3.97 mm, creating a uniform, thin (ca. 0.125 mm) layer. As shown in previous work, this reduction of the NMR active volume effectively increases the photon flux.36Due to the quantitative nature of NMR, the diagonal peak intensity increases linearly with concentration. At the same time, the cross peaks show saturation behavior, due to an increasing optical densities of the sample. As a result, the cross/diagonal peak ratio drops with increasing concentration. This may also be shown by consulting the corresponding photochemical rate law in the formed stationary state (Equation S5). In contrast to the EXSY experiment the herein obtained values for rate constants depend crucially on concentration, irradiation intensity and geometry. However, preparing samples with alike optical densities ensures comparability of the obtained data. As a further test for the reproducibility of our experiment, we determined exchange rates of an azobenzene sample at five different temperatures ranging from 283 to 303 K. The temperature dependence of the isomerization quantum yields of azobenzene has been reported.37 Within the narrow range of temperatures attainable in our experiment, however it is not observable in our data, (see Figure S8 and Table S1). The extracted exchange rates have a small standard deviation of 6 % of the average value. 1D sequence for efficient data collection. The above presented 2D experiments map all light induced correlations in a photochromic system. However, acquisition times for a series of 2D experiments with incrementally longer mixing times are exceedingly time consuming. If exchange rates are the only required information a pulse sequence analogous to a selective NOESY experiment can be used.38–44 In this experiment, the selective excitation of a single resonance replaces the t1 frequency labelling necessary for the 2D experiment. This provides a fast lane to selectively determine the exchange rate. For instance, 128 t1-increments can be replaced with a single 1D experiment. This is especially important for photoinduced exchange, since only small amounts of transferred magnetization may require averaging of many transients. This 1D experiment may be repeated with different mixing times which allows to extract the desired exchange rates. A pulse sequence diagram is provided in the Supporting Information. Photoswitching efficiency of three different compounds. This 1D variant of the experiment allows efficient data collection and is a useful tool for investigating the switching capabilities of different compounds. To indicate the scope of our method we have also investigated photoswitch 2, a photo


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Analytical Chemistry pharmacological substance enabling photo-control of a cation channel.27 Compound 3 was developed to achieve a reverted stability of (E) and (Z)-isomers. Especially in the field of photo pharmacology a stable and inactive state is highly desirable.45 Bridging the two aromatic rings moves the n-π*-bands utilized in the switching process further to the red part of the spectrum.30 Additionally, this modification increases the quantum yields of isomerization considerably.31 The results show how substitution/and modification of the azobenzene core alters the switching behavior. For the experiment we prepared solutions with equal optical densities at the irradiation wavelength. Absorbance spectra of these samples after prolonged irradiation with 405 nm recorded at a sample thickness of 1 mm are presented in Figure S9. The photoinduced isomerization rate was measured with the proposed 1D sequence, starting with the selective excitation of a resonance of the (E)-isomer. An exponential fit (see Equation S6 and Table S2) yields the rate constant in the E → Z direction. Thermodynamically, this direction is “uphill” for compounds 1 and 2 (hence we define it as 𝑘 + ) and “downhill” for compound 3 (i.e. 𝑘 ― ). The resulting data are presented in Figure 3 and compiled in Table 1. The rate constants for E → Z (numbers carrying superscript “a” in Table 1) are clearly in the order 3 > 2 > 1. The investigated compounds have similar content of (Z)isomer. In the case of compounds 1 and 2 light at 405 nm induces incomplete Z → E conversion, whereas for comp. 3 the equilibrium is pushed from the more stable (Z)-isomer far to the E-side ( 𝜒𝐸 = 0.75). The slope for 2 in Fig. 3 is slightly higher (0.06 s-1 vs. 0.09 s-1 for 𝑘 + ). The ethylene bridge in compound 3 further increases the observed switching frequency (𝑘 ― = 0.12 s-1). The rate constants 𝑘 ― and k are calculated using the isomeric ratio, which is conveniently read from 1D spectra. Due to the similar Z content the total exchange rate basically follows the same trend 3 > 2 > 1. These numbers suggest that the modifications in para position of compound 2 had no negative effect on the quantum yields, whereas higher quantum yields in compound 3 almost doubles the light induced switching frequency in the NMR active volume. Table 1. Exchange rates and isomeric ratios determined on samples having equal optical densities at the irradiation wavelength (405 nm). comp.

𝑘 + (s1)

𝑘 ― (s-

𝜒𝐸

𝜒𝑍

k (s-1)

1)

1

0.06a

0.21b

0.78

0.22

0.26

2

0.09a

0.36b

0.80

0.20

0.44

3

0.37b

0.12a

0.75

0.25

0.49

avalues

obtained by the exponential fit shown in Figure 3. bvalues calculated using the isomeric ratio.

Figure 3. Switching frequency determined with a 1D version of the experiment, utilizing a 405 nm LED and solutions of photoswitchable compounds 1-3. The fitted curves (dotted lines) used to extract the exchange rates are also shown.

Phase cycle for enhancing spectral features. Despite the investigation of very efficient switchable molecules, the achievable reaction rates with reasonable light intensities are small. Intense cross peaks may require a combination of long mixing times, high light intensities, low concentrations and special irradiation geometries. Thus, spectra are usually dominated by large diagonal peaks; a problem also encountered in SCOTCH experiment.46 Also, in more complex systems spectra can become overcrowded by NOE cross peaks. As discussed, exchange only takes place if triggered by irradiation. To highlight photoinduced processes, a spectrum recorded in the dark (no exchange) can be subtracted from a spectrum recorded with irradiation (exchange). This is achieved by combining “dark” and “light” scans with opposite receiver phases. In the light scan, diagonal peaks, NOE and exchange cross peaks are observed. From these signals, the diagonal peaks and NOE peaks acquired in the dark scan are subtracted due to the opposite receiver phase, leaving a spectrum with no observable NOEs and 99 % diagonal peak suppression. As a small tradeoff, the cross peak intensity in these phase-cycled spectra is also reduced by ca. 30 %. However, the resulting spectra are much simpler due to the absence of NOEs and the low intensity of diagonal peaks (Figure 4). This intensity change allows easy extraction of the correlation information at shorter mixing times.



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AUTHOR INFORMATION Corresponding Author *G.G.: E-mail: [email protected] E.S.: E-mail: [email protected] K.Z.: E-mail: [email protected]

Author Contributions ‡E.S.

and S.T. contributed equally to the work. *G.G. and K.Z. contributed equally to the work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Figure 4. Phase-cycled PC-EXSY of azobenzene in MeOD-d4. Negative diagonal peaks (red) are of the opposite sign of photochemical exchange peaks (blue). 2 s mixing time, TD/TD1 = 4096/128, NS = 16; 2 h 36 min experimental time.

CONCLUSION In conclusion, we showed how a simple setup with a glass fiber and a single high-power LED allows for facile and selective monitoring of photoinduced exchange processes. Instead of observing concentration changes we show that correlation experiments may also be performed in a steady state of photochromic molecules. This removes the necessity to regenerate the initial state by irradiating the sample with a second wavelength. Most photoswitchable compounds offer a suitable wavelength where both isomers absorb for this type of experiments. In the presented 2D experiments cross peaks indicate the correlation between two isomers and, when compared to diagonal peaks at various mixing times, are a measure for photochemical exchange rates. A time saving 1D variant can be used when photoinduced rates are of prior interest. If structural information is needed an experiment employing a “light” and “dark” phase cycle provides valuable information. This experiment may become a viable tool for mapping light induced structural changes in natural47 or modified lightresponsive proteins.48 High switching frequencies of photochromic units attached to macromolecules could be utilized to probe changes in their secondary structure. Using this method, we compared the efficiency of three azobenzene based photoswitches. Up to date, light-coupled NMR spectroscopy was mainly used to detect concentration changes in photoreactions49,50 and photochromic 51,52 compounds. However, the results demonstrate how NMR is also capable of capturing light induced molecular dynamics. This paves the way to assess the efficiency of photoswitches in a facile manner, using a setup that matches well with intended in-vivo application.

ASSOCIATED CONTENT

Financial support to K.Z. by the Austrian Science Foundation (FWF) under project number P30230 and through the Doktoratskolleg Molecular Enzymology (W901) is gratefully acknowledged. We also thank the interuniversity program in natural sciences, NAWI Graz, for financial support. We thank Christian Holly for assembling the high power LEDs.

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Pulse sequence diagrams of PC-EXSY, PC-EXSY-PC, selective PC-EXSY; Bruker code for the pulse sequences; (PDF)

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TOC Figure: For Table of Contents Only


In-Situ Observation of Photoswitching by NMR Spectroscopy: A

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