First Step toward a Universal Fluorescent Probe: Unravelling the

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A First Step Towards a Universal Fluorescent Probe: Unravelling the Photodynamics of an Amino-Maleimide Fluorophore Michael Staniforth, Wen-Dong Quan, Tolga N. V. Karsili, Lewis A. Baker, Rachel K. O'Reilly, and Vasilios G. Stavros J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.7b04702 • Publication Date (Web): 04 Aug 2017 Downloaded from http://pubs.acs.org on August 5, 2017

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The Journal of Physical Chemistry

The First Step Towards a Universal Fluorescent Probe: Unravelling the Photodynamics of an AminoMaleimide Fluorophore Michael Staniforth,a,‡ Wen-Dong Quan,a,b,‡ Tolga N. V. Karsili,c,†,‡ Lewis A. Baker,a,b Rachel K. O’Reillya,* and Vasilios G. Stavrosa,* a.

Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. Email: [email protected], [email protected]

b.

Molecular Organization and Assembly of Cells Doctoral Training Centre, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom.

c.

Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.

ACS Paragon Plus Environment

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ABSTRACT: Continuous advancements in biophysics and medicine at the molecular level make the requirements to image structure-function processes in living cells ever more acute. While fluorophores such as the green fluorescent protein have proven instrumental towards such efforts, the advent of non-diffraction limited microscopy limits the utility of such fluorescent tags. Monoaminomaleimides are small, single molecule fluorophores that have been shown to possess stark variations in their emission spectra in different solvent environments, making them a potentially powerful tool for myriad applications. The ability to ‘autotune’ fluorescence according to different media allows for a probe capable of working in all regions of a cell, or accurately characterizing the purity of an environment. In this work, we present ultrafast pumpprobe studies of a model monoaminomaleimide, 1-methyl-3-(methylamino)-1H-pyrrole-2,5dione, and demonstrate how fluorescence quenching in polar protic solvents is caused by electron driven proton transfer from the solvent to the fluorophore. Armed with this knowledge, the present study acts as a first step for the rational design of future maleimides, potentially moving towards creating a universal fluorophore with tunable efficiency, dependent on environment.


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I.

INTRODUCTION

Fluorescent probes are amongst the most utilized tools in spectroscopy, ranging in applications from the macroscale, such as contamination-detection in water treatment,1-3 to the microscale, such as sub-cellular imaging.4-6 In particular, fluorescent dyes have been applied through immunostaining to visualize protein distribution in cells,7,8 a destructive technique that renders live cell imaging infeasible.9-11 Such limitations were in part negated by the discovery of green fluorescent proteins (GFPs),12 which utilize protein fusion,13,14 a non-destructive technique with unmatched specificity, making them by far the most applied fluorophores in bio-imaging applications.15-17 Indeed, tremendous progress has been made in the field of in vitro live cell imaging over the past two decades,18-20 with the use of fluorescent dyes being extended to more specialized fields such as visualization of ions, metabolites and niche assemblies in cells.21-23 There are, however, two inherent limitations in using GFPs in sub-cellular imaging applications: i) their relatively large size may alter the intracellular activities of smaller peptides, hence observations may not be representative of the ‘real world’ actions of the proteins under study;24,25 and ii) their emission profiles are usually insensitive to changes in the local environment.26 Furthermore, recent advances in light microscopy have resulted in non-diffraction limited techniques achieving spatial resolutions of up to 10-30 nm, potentially limiting the utility of such large fluorophores.27-30 It then follows that, along with the maturation of methods for incorporating non-canonical amino acids with highly modifiable functionalities into living organisms,31-33 small fluorophores will likely play a major role in the next generation of in vitro and in vivo intracellular imaging applications. It was recently reported that maleimides exhibit high fluorescence quantum yields upon amino or thiol functionalization at the 2/3- ring-centered positions.34,35 These maleimides already have


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wide application in tissue imaging, being common handles for protein tagging through thiol-ene coupling.36-41 Further reports have also suggested that aggregation induced emission can be introduced via specific substitution at the nitrogen heteroatom.42-44 The former case is especially enticing for bio-imaging as it allows for single molecule resolved imaging. Such functionalization often involves simple synthetic techniques and relatively inexpensive reagents,36-41 making them highly accessible to most research groups. However, the applications referred to above were all achieved in hydrophobic environments, fluorescence in these maleimides being universally quenched upon solvation in protic solvents.36 While such sensitivity to environment has been utilized to identify disassembly of micelles via fluorescence lifetime imaging,37 such low emission in aqueous media limits their efficacy as fluorophores in biological applications. Creating bright, water-soluble, small fluorophore tags represents a major challenge in furthering biological imaging science. To begin to meet this challenge, we employ a combination of steady-state absorption and femtosecond transient electronic absorption spectroscopy (TEAS), and electronic structure calculations, in an attempt to unravel the nature of the fluorescence quenching in a model monoaminomaleimide (MAM), 1-methyl-3-(methylamino)-1H-pyrrole2,5-dione (NM-MAM, see Supporting Information for details on synthesis and structure), in a non-polar, polar aprotic and polar protic solvent. This provides us with a close to complete structure-dynamics-function description of this molecule, which can be used as a critical first step in a ‘guide-to-design’ to produce MAMs for a multitude of applications far beyond the field of bio-imaging.


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II. METHODS Materials Spectroscopic grade cyclohexane (c-hexane) and methanol (MeOH) were purchased from VWR. HPLC grade acetonitrile (MeCN) was purchased from Fisher Scientific. Water for synthesis and spectroscopy was purified to a resistivity of 18.2 MΩcm using a Millipore Simplicity Ultrapure water system. All other solvents and chemicals were purchased from Sigma, Aldrich, Fluka or Acros and used as received unless stated otherwise. Instrumentation 1

H and

13

C NMR spectra were recorded on a Bruker DPX-300, DPX-400, or AV-250

spectrometer at 25°C. Chemical shifts are given in ppm downfield from the internal standard tetramethylsilane (TMS). Infrared spectra were recorded on a Perkin Elma spectrum 100 FT-IR spectrometer. Fluorescence spectra were recorded using an Agilent Cary Eclipse Fluorescence spectrophotometer. High resolution mass spectrometry (HR-MS) was conducted on a Bruker UHR-Q-ToF

MaXis

with

electrospray

ionization.

Ultraviolet-visible

light

(UV-Vis)

spectroscopy was carried out on an Agilent Cary 60 UV-Vis spectrometer. Quartz cells with screw caps and four polished sides (Starna Scientific) were used for fluorescence and UV-Vis measurements. Time Resolved Transient Electronic Absorption Spectroscopy (TEAS) The detailed experimental procedures for TEAS can be found in previous reports.45-46 Briefly, a commercially available Ti:sapphire oscillator and amplifier system (Spectra-Physics) produces 3 mJ laser pulses of ~40 femtosecond (fs) duration centered around 800 nm with a repetition rate of 1 kHz. For TEAS, pump pulses (hνpu) were generated from a 1 mJ/pulse portion of the 800 nm fundamental using a Spectra-Physics TOPAS-C optical parametric amplifier, to produce hνpu of


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345, 360 or 365 nm with powers of 0.3-0.7 mW and a pulse duration of 80 fs. 5% of a second 1 mJ/pulse portion of the 800 nm laser beam (50 µJ/pulse) is focused onto a CaF2 window to generate the probe pulse: a white light continuum (330-725 nm). Pump-probe polarizations are held at the magic angle (54.7°) relative to one another. Changes in optical density (∆OD) of the sample are calculated from probe intensities, collected using a spectrometer (Avantes, AvaSpecULS1650F). The maximum pump-probe time delay (∆t) is 2 ns. All resultant transient absorption spectra (TAS) were chirp-corrected using the KOALA package.47 The delivery system for the samples is a flow-through cell (Demountable Liquid Cell by Harrick Scientific Products, Inc.). The sample is circulated using a peristaltic pump (Masterflex) with PTFE tubing, recirculating sample from a 50 mL reservoir (with minimum sample volume of ca. 20 mL), in order to provide each pump-probe pulse pair with fresh sample. Computational Methods Key reaction paths along the excited state potential energy surfaces (PES) were studied using state-of-the-art electronic structure theory. The ground state equilibrium geometry of NM-MAM in a cluster of three MeOH molecules (vide infra and Supporting Information) was optimized using the Møller-Plesset second-order perturbation theory (MP2)48 coupled to a cc-pVDZ basis set.49 The choice of three explicit methanol cluster molecules proximal to the NM-MAM chromophore was selected by recognizing that the NM-MAM solute contains two H-bond acceptors and one H-bond donor. Since methanol contains both H-bond acceptor/donor capabilities, the present minimum energy arrangement of the three proximal methanol molecules ensures a complete coverage of all important donor/acceptor sites of NM-MAM whilst reducing the computational expense. Indeed previous work on hydrogen bonded, solvated organic molecules have shown that small solvation shells, encapsulating only the key interacting sites,


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are sufficient to explain observed photodynamics.50,51 Furthermore, these studies have shown the precise orientation and number of the solvent molecules to have only minimal effect on the energetics of the molecules under study.50-52 As such, the cluster geometry presented here can be considered an adequate representation of the solvated system while minimizing computational expense. Following on from the above argument, only two solvent molecules were used in calculations involving NM-MAM in MeCN and c-hexane. In the former case, this method was chosen as there are only two dominant interactions (hydrogen bonding to the NH site on NM-MAM, and π-stacking between the solvent and solute) and addition of further molecules proved unnecessary in adequately describing the system. In the case of c-hexane, once again, two discrete solvent molecules were deemed necessary, a third being consistently localized away from the cluster, when the cluster geometry was relaxed. Further details can be found in the Supporting Information. First excited state singlet and triplet relaxed potential energy scans were undertaken at the second-order algebraic diagrammatic construction (ADC(2))53,54 using a bond stretch (OH/NH) dimension as the driving coordinate (vide infra). The corresponding ground state (S0) energies at a particular S1/T1 relaxed geometry were computed using MP2/cc-pVDZ. Vertical, or Franck-Condon, excitation and emission energies were calculated for NM-MAM in MeOH with three discrete solvent molecules as described above and in MeCN and c-hexane with two solvent molecules (vide supra and Supporting Information). The ground state was optimized as described above, then vertical excitation energies were calculated using ADC(2) with a cc/pVDZ basis set. The S1 excited state was then optimized using ADC(2) with a cc/pVDZ basis set and Franck-Condon emission energies (i.e. emission assuming a static, S1


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minimum, geometry) extracted. All MP2 and ADC(2) calculations were undertaken in TURBOMOLE.55,56 III. RESULTS Steady state absorption and emission spectroscopy The absorption and emission spectra of NM-MAM in c-hexane, MeCN, MeOH and MeO-d4, were first studied. From the steady-state absorption spectra in Figure 1a (extinction coefficients, ε, given in Table 1), there are no qualitative changes in the shape of the absorption spectra, with a modest spectral shift of the peak maximum depending on solvent. It is very likely therefore that, in each case, the same electronic state transition is responsible for the absorption observed with the relative stability of the excited state changing depending on the solvent used. As such, we have opted to excite with a different wavelength for each solvent such that the same region of the excited state manifold is accessed in each case, to achieve the most comparable results between solvent environments.57-60 A similar spectral shift to that seen in the absorption spectra was observed in the emission spectra, shown in Figure 1, recorded at the absorption maximum for NM-MAM in each solvent studied. The decrease in energy of the emission maximum, however, exceeds that of the absorption maximum when moving from non-polar to polar aprotic to polar protic solvent, resulting in an increase in Stokes shift as solvent-solute interaction increases. This shift coincides with the notable drop in quantum yield (φf) common to these fluorophores, as already discussed above and presented in Table 1.61 The above results, taken together, may be indicative of a state change being accessed in polar protic solvents, proceeding photoexcitation and leading to fluorescence quenching. This is explored in more detail in the following sections.


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Transient electronic absorption spectroscopy TEAS experiments were performed on NM-MAM in c-hexane, MeCN and MeOH, following photoexcitation at the lowest energy absorption peak in each case (see Figure 1a for steady-state absorption spectra). Figures 2a and 2d show the measured TAS of NM-MAM in c-hexane. In the false color maps maps, as well as in the selected TAS, positive signal (red) corresponds to an increase in absorption following excitation. Negative signal (blue) corresponds to a decrease in absorption or an increase in emission. In the aforementioned TAS, two positive signals are present, which we assign to excited state absorption (ESA). The first is a relatively narrow and weak feature centered at ~400 nm, whilst the second, a broader and more intense feature, spans the wavelength range ~600-725 nm. Between these ESA features, a negative signal is also evident. Such negative signals, red-shifted from the absorption maximum, are plausibly assigned to stimulated emission (SE). This is supported by the emission spectrum (see Figure 1b). A weak negative signal appearing close to the pump wavelength (>2 ns and their exact value cannot be determined. The extracted lifetime is in accord with the high φf measured of 38%, as expected from S1 state emission. To investigate the effect of polarity on its photodynamics, NM-MAM was dissolved in MeCN, an aprotic polar solvent. It should be noted that a higher concentration of NM-MAM was used in MeCN and MeOH (8 mM and 10 mM respectively) than in c-hexane (>2 ns. The latter accords with the φf returned from the fluorescence emission studies (Table 1). The DAS associated with the two lifetimes extracted from the global fit are shown in Figure 2h. The first of these (5.1 ps) shows negative components below 400 nm, and between 450 and 570 nm, with positive components elsewhere. This likely represents a flow of population between two states. The second DAS (>>2 ns) then represents the long-lived feature, which extends beyond our temporal window. Finally, TAS were obtained for NM-MAM in MeOH (Figure 2c and 2f). These TAS demonstrate a marked difference to those of c-hexane. In contrast, the TAS in MeCN and MeOH are near identical at early time delays: ESA features at ~450 nm and ~640 nm superimposed with SE at ~530 nm and GSB at ~365 nm. At longer times, however, the similarities are tenuous, notably the spectral features of the TAS for NM-MAM in MeOH tend to baseline while those of NM-MAM in MeCN extend beyond the temporal window of our experiment. Global fitting the TAS reveals three time constants, with three corresponding DAS (see Figure 2i). The first DAS, with a lifetime of ~0.7 ps, has (unsurprisingly) a spectral profile similar to the MeCN DAS with the lifetime of ~5 ps; the second and third DAS (11 ps and 850 ps) comprise of both positive and negative features indicative, once again, of population flow between states. Importantly, the third DAS reflects a near complete baseline recovery by 2 ns, consistent with the measured φf of 1.2%. Electronic structure calculations To gain mechanistic insight into the excited state dynamics of NM-MAM in MeOH, the solvent which leads to near baseline recovery in the TAS of NM-MAM within 2 ns and with almost complete quenching of its fluorescence, relaxed potential energy cuts (PECs) were


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constructed along the RO–H stretch coordinate of MeOH (the path along which represents intermolecular proton transfer from MeOH to NM-MAM). These cuts included the ground and first excited (1ππ*) state of the locally excited (LE) NM-MAM molecule, as well as the corresponding ground and excited states for a charge transfer (CT) configuration. CT states were located by increasing RO–H and allowing all other coordinates to relax. The CT state then becomes the lowest energy configuration. By stepping the RO–H coordinate back, the PEC for the CT state was recovered. We comment here, and return to discuss below, that similar calculations were carried out for associated triplet states. We briefly consider here other competitive pathways. The long excited state lifetimes of NMMAM in c-hexane and MeCN, coupled with their high quantum yield of fluorescence, suggest that the classic ultrafast decay pathways observed in analogous systems (e.g. deformations, or N– H dissociation)64-66 are likely not competitive in these solvents, as this would result in reduced lifetimes and likely fluorescence quenching. It can also be concluded that the increased polarity of MeCN is not sufficient to change the electronic landscape enough to make these processes competitive. As such, we propose that the protic nature of MeOH leads to the significant changes observed in the photodynamical behavior of NM-MAM, and so this makes up the focus of the rest of this work. While other proton transfer mechanisms than that presented in Figure 3 are possible, PECs following transfer to the opposite oxygen atom on NM-MAM or from the NH group to methanol demonstrated less favorable energetic landscapes and so are not considered further (see Supporting Information for PECs and further details). The returned cuts given in Figure 3 confirm that the relaxed S1(CT) state contains a labile path with respect to proton transfer from MeOH to NM-MAM (blue dashed arrow, Figure 3 and inset). This stability in the relaxed S1(CT) profile can be understood on electronic grounds: the


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CT state contains a charge-separation that is neutralized by proton transfer. However, it should be noted that the relaxed S1(CT) profile contains a local minimum at short RO–H which could trap population at near threshold excitations (green curved arrow, Figure 3), and that the minimum energy geometry of the S1(LE) state (see Supporting Information) lies near degenerate in energy to that of the S1(CT) state, again potentially trapping population at low RO–H distances. These calculations imply that electron driven proton transfer (EDPT) is a viable path by which NM-MAM in MeOH non-radiatively decays, thus quenching fluorescence (vide infra). Although PECs of NM-MAM in MeCN were not calculated, the electron withdrawing CN group in MeCN is likely to stabilize a CT state containing the electronic configuration π(CN)1 π*(NM-MAM)1, analogous to that of MeOH, though this is unlikely to undergo charge recombination due to the absence of a labile proton donor on MeCN. IV. DISCUSSION Consolidating the experimental and theoretical data, we can see that the marked difference in the observed TAS when moving from non-polar c-hexane to polar MeCN or MeOH solvents, can be understood in terms of a population transfer to a CT state in the latter two cases. The short time (∆t 2 ns; ii) a charge transfer (CT) pathway is introduced by polar aprotic solvents, MeCN in this case, which is accessed in ~5 ps. Some population, trapped in the locally excited (LE) minimum energy geometry returns to S0 via fluorescence in competition with internal conversion from both the LE and CT states in >>2 ns; iii) in protic MeOH, CT occurs in ∼0.7 ps, thus quenching fluorescence. This CT state then undergoes electron driven proton transfer (EDPT, ∼11 ps). Either of these first two process may be convoluted with ultrafast ISC to the triplet manifold. Finally, back H-atom transfer from NM-MAM to the MeO• radical occurs, mediated via ISC (∼850 ps). Through synergized experiment and theory, we have demonstrated that fluorescence quenching in MAM systems in protic solvents is a result of EDPT. While the present work is far


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removed from the final goal of a true ‘universal’ small-molecule fluorophore, the fundamental understanding developed herein on the photodynamical processes involved in MAMs represents an essential first step towards realizing this goal. Armed with this knowledge, future rational design of MAMs which retain charge transfer character in protic solvents but prevent EDPT, and thus fluorescence quenching, would yield an extremely powerful fluorescent probe. Furthermore, proton loss and proton transfer mechanisms have been shown to be crucial in vital processes such as radical scavenging in vitamin E.76 Small, single molecule MAM fluorophores such as the one discussed here could prove instrumental in probing such mechanisms within living cells. We thus consider the present work as a platform for exploration into the, thus far, unexploited and far-reaching potential of MAM chromophores, launching exciting research opportunities into such fields as; two-photon absorption fluorescence imaging with MAMs; modification of MAMs to reduce absorption onset energy; and proton transfer mechanisms as a biological probe.


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Figure 1. a) Absorption spectra for 3 µM NM-MAM in c-hexane, MeCN and MeOH. Note: The feature at ~375 nm is caused by a lamp change in the spectrometer. b) Normalized excitation spectra (Exc) alongside normalized emission spectra (Em) for ease of comparison. MeO-d4 was chosen for this comparison data as it is qualitatively identical to that for MeOH, but gives a slightly higher quantum yield of fluorescence.


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Figure 2. a)-c) TAS false color maps for NM-MAM in a) cyclohexane (c-hexane, > 2000

-

-

4.7 ± 0.7 (345)

MeCN

30.4 ± 3 %

5.1±2.6

>> 2000

-

5.3 ± 0.8 (360)

MeOH

1.2 ± 0.2 %

0.7±0.3

11±1.7

850±110

5.5 ± 0.9 (365)

MeO-d4

2.5 ± 0.3 %

0.95

11.8±2

1400±170


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ASSOCIATED CONTENT Supporting Information. Details on synthesis; steady-state UV-VIS absorption and fluorescence data; global fitting analysis; computational trajectory data; and potential energy cuts for various decay pathways. AUTHOR INFORMATION Corresponding Author *[email protected], [email protected] Present Addresses †T.N.V.K. is now at the Department of Chemistry (Beury Hall), Temple University, 13th and Norris Streets, Philadelphia PA 19122, United States of America. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. ACKNOWLEDGMENTS The authors would like to thank Dr. Kay Doncom for helpful discussions and Yujie Xie for assistance with the fluorescence quantum yield measurements. M.S. thanks the EPSRC for postdoctoral funding (grant EP/N010825/1). T.N.V.K thanks TUM for the award of a postdoctoral fellowship and the EPSRC for funding (EP/L005913). W.D.Q. and L.A.B. thank EPSRC for studentship grants EP/F500378/1. R.K.O’R thanks the ERC (615142) for funding. V.G.S thanks the EPSRC for an equipment grant (EP/J007145/1) and the Royal Society and


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Leverhulme Trust for a Royal Society Leverhulme Trust Senior Research Fellowship. We thank the Scientific Computing Research Technology Platform at the University of Warwick for providing computational resources and the Warwick Centre for Ultrafast Spectroscopy for the use of experimental apparatus. REFERENCES (1) Baghoth S. A.; Sharma S. K.; Amy G. L., Tracking Natural Organic Matter (NOM) in a Drinking Water Treatment Plant Using Fluorescence Excitation–Emission Matrices and PARAFAC, Water Research, 2011, 45, 797–809. (2) Matilainen A.; Gjessing E. T.; Lahtinen T.; Bhatnagar A.; Sillanpää M., An Overview of the Methods Used in the Characterisation of Natural Organic Matter (NOM) in Relation to Drinking Water Treatment, Chemosphere, 2011, 83, 1431–1442. (3) Bridgeman J.; Bieroza M.; Baker A., The Application of Fluorescence Spectroscopy to Organic Matter Characterisation in Drinking Water Treatment, Rev. Environ. Sci. Biotechnol., 2011, 10, 277–290. (4) Zhang J.; Campbell R. E.; Ting A. Y.; Tsien R. Y., Creating New Fluorescent Probes for Cell Biology, Nature Rev., 2002, 3, 906–918. (5) Guo Z.; Park S.; Yoon J.; Shin I., Recent Progress in the Development of Near-Infrared Fluorescent Probes for Bioimaging Applications Chem. Soc. Rev., 2014, 43, 16–29. (6) Giepmans B. N. G.; Adams S. R.; Ellsinman M. H.; Tsien R. Y., The Fluorescent Toolbox for Assessing Protein Location and Function, Science, 2006, 312, 217–224. (7) Hervieu G. J.; Cluderay J. E.; Harrison D. C.; Roberts J. C.; Leslie R. A., Gene Expression and Protein Distribution of the Orexin-1 Receptor in the Rat Brain and Spinal Cord, Neurosci., 2001, 103, 777–797.


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TOC Graphic:


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Figure 1. a) Absorption spectra for 3 µM NM-MAM in c-hexane, MeCN and MeOH. Note: The feature at ~375 nm is caused by a lamp change in the spectrometer. b) Normalized excitation spectra (Exc) alongside normalized emission spectra (Em) for ease of comparison. MeO-d4 was chosen for this comparison data as it is qualitatively identical to that for MeOH, but gives a slightly higher quantum yield of fluorescence. 135x54mm (300 x 300 DPI)



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Figure 2. a)-c) TAS false color maps for NM-MAM in a) cyclohexane (c-hexane,

First Step toward a Universal Fluorescent Probe: Unravelling the

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