Shedding Light on the D 1 -Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D 1 and D 5 Receptors

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Introduction
[3] DA exerts its physiological functions via five G protein-coupled receptors (GPCRs), the dopamine D 1-5 receptors (D 1-5 R). [4] These five dopamine receptors are divided into two sub-families based on their ability to activate or inhibit the adenylyl cyclase and hence the production of the second messenger cAMP. [5]The D 1 R and D 5 R are Gα s -coupled and form the D 1 -like receptor family, whereas the D 2 R, D 3 R, and D 4 R are Gα i/o -coupled and members of the D 2like receptor family. [6,7]Due to the wide expression of dopamine and its receptors in the central and peripheral nervous system they have been the focus of research for many years.[16] The only approved drug targeting the D 1 -like receptors is the peripheral acting partial agonist fenoldopam for the intravenous treatment of hypertension. [17,18]he central active D 1 R selective full agonist dihydrexidine (Figure 1) was tested in clinical trials for the treatment of PD but failed due to severe side effects including flushing, hypotension, and tachycardia. [19,20]23][24] Fluorescent ligands represent attractive alternatives to radioligands, for example, to visualize receptors in cells and tissues or for studying the effect of drug-receptor-interactions with advanced biophysical technologies. [25,26][29] Furthermore fluorescence microscopy techniques such as confocal microscopy and total internal reflection fluorescence (TIRF) microscopy are often used for these purposes. [30,31]Traditionally, radiolabeling has been the method of choice to yield labeled ligands for the use as probes in receptor binding assays. [32]In the past decades, fluorescence-based binding assays have become increasingly important and are now almost as present as radiochemical binding assays. [28]The list of advantages and disadvantages of these two methods is relatively balanced.Especially in the areas of safety issues, accessibility, regulatory requirements and cost, fluorescence-based methods can be mentioned as more advantageous.Clear advantages are often seen in the simplicity of implementation and the fact that modern FRET/BRET-based assay systems allow kinetic measurements in real-time. [33,34]In contrast, radioassays are usually very robust, there is no need for receptor modification, access to autoradiography, and high sensitivity to name just a few. [32]For these reasons, both methods will continue to coexist and can be selected to characterize GPCRs and their ligands depending on the respective task.Although FRET/BRET-based assays provide valid results and bring several advantages over, e. g., radioassays, modifications must be made to the GPCR and cell protein of interest.[37] This allows studies under more physiological-like conditions, not least because cellular processes are not impaired by the addition of chemical substances that are often required for signal detection in conventional assays.Although not all processes from label-free readouts leading to the observed signal are yet fully understood and sometimes referred to as "black box" readouts, [38] these technique represents a highly interesting alternative to investigate ligand-receptor interactions.
We herein report the development and validation of a series of six novel fluorescent ligands for D 1 -like receptors and their use for binding studies in fluorescence microscopy.[41] The goal of the study is to find useful pharmacological tools that can further help to explore the complex functionalities and interactions of D 1 -like receptors.

Design rationale
Fluorescent ligands can be considered as an entity of three distinct parts: the ligand scaffold, the linker, and the fluorescent dye.44] The known D 1 R/D 5 R antagonist SCH-23390 (Figure 1) was chosen as the structural motif for D 1 -like receptors because it displays high affinity at the D 1 R and D 5 R and a high selectivity within the dopamine receptor family. [45]As a ligand scaffold, an antagonistic partial structure was preferred since agonists can induce receptor internalization and degradation, which could be disadvantageous for binding studies. [42]Structure-activity relationships (SAR) revealed that structural modifications and attachments at the para-position of the phenyl group are well tolerated and that a sulfonamide group may be beneficial for binding affinity. [46,47]In accordance with these SAR results, cryo-EM structures of D 1 R-G s complexes bound to an agonistic SCH-23390-derivative, SKF-83959, showed that this position points towards the extracellular space and is not involved in ligandreceptor interaction. [48,49]The solved cryo-EM structures with compound SKF-83959 and the D 1 R are shown in Figure S32 (Supporting Information (SI)) and underpin our design rationale.Therefore, this position and chemical moiety were chosen for the attachment of the linker.
Based on previously described D 1 R fluorescent ligands, which had the fluorescent dye directly attached to the D 1 -like scaffold, two ligands with a short alkylic linker were synthesized. [41]Additionally, fluorescent ligands with longer PEG-based linkers were prepared which should allow the fluorophore to reach outside the binding pocket and therefore, should have reduced impact on the ligand binding.PEG-based linkers are commonly used in fluorescent ligands because they usually don't interact with cell membranes, are chemically stable, and well soluble in water. [50]The connection of the ligand scaffold with the PEG-unit was performed in three steps.First, a short alkylic spacer was attached to the D 1 -like scaffold, which was then connected via a peptide coupling reaction to propargylamine.This product was coupled via a copper catalyzed azide alkyne click (CuAAC) reaction [51][52][53] to a PEG-2 or PEG-3 unit, which was orthogonally functionalized with a primary amine and an azide group (17 a, 17 b).The additional triazole moiety increases the solubility of the final fluorescent ligands.
The choice of the fluorescent dye is crucial and depends mainly on the intended use of the final fluorescent ligand.Important criteria to consider are spectral properties, quantum yield, solubility, solvatochromic effects, chemical stability, photostability, commercial availability, and an efficient coupling to the ligand scaffold. [42]The 5-TAMRA fluorescent dye was chosen to label the ligands because it is known to yield excellent results in fluorescence microscopy. [34,54,55]Additionally, DY549-P1 from Dyomics was used, as it possesses similar spectral properties as 5-TAMRA.Both dyes are hydrophilic, reducing the risk of unspecific interactions with cell membranes as compared to, for example, BODIPY fluorescent dyes, leading to lower non-specific binding. [56]

Chemistry
The syntheses of the D 1 -like scaffold and linkers were performed separately followed by the connection of both parts.The last step in the synthesis of the different fluorescent ligands was always the labeling with a fluorescent dye.
The ligand scaffold was synthesized following the general synthetic route for benzazepines used by Neumeyer et al. and Shen et al. with modifications (Scheme 1). [57,58]In a three-step synthesis 4-nitroacetophenone was transformed into the corresponding epoxide 3 with good yields.Commercially available 4methoxybenzaldehyde was first chlorinated using sulfurylchloride in acetic acid to afford 3-chloro-4-methoxybenzaldehyde (4).A Henry-reaction with nitromethane in acetic acid, followed by a reduction with LiAlH 4 in THF afforded the primary amine 6.The secondary amine 8 was prepared via the introduction of a Boc-protecting group (7) and subsequent reduction with LiAlH 4 .This two-step synthesis provided selectively the mono-methylated amine in high yields.A nucleophilic substitution of the secondary amine 8 with epoxide 3 in acetonitrile generated product 9 in excellent yields, which was converted to the benzazepine 10 by an acid catalyzed cyclization reaction using Eaton's reagent at room temperature for 72 h.After deprotection with boron tribromide in DCM, the resulting phenol 11 was protected with acetyl chloride or triisopropylsilyl chloride, respectively (12 a, 12 b).A reduction of the nitro group with hydrogen and catalytical amount of palladium on charcoal in a mixture of MeOH and THF afforded the anilines 13 a and 13 b.
For the introduction of the sulfonylamide moiety two different sulfonyl chlorides (14 a, 14 b) were required.They were synthesized starting from aliphatic bromides using thiourea and N-chlorosuccinimide (Scheme 2A) following the procedure of Yang and Xu et al. [59] The PEG-linkers (22 a, 22 b) were synthesized starting from the corresponding polyethylene glycols following the reaction protocol published by Iyer et al. [34,60] Coupling of the sulfonylchlorides 14 a and 14 b with the anilines 13 a and 13 b in the presence of pyridine afforded the structures 18 and 20 (Scheme 2B) in moderate yields.18 was converted to the primary amine 19 using hydrazine hydrate in ethanol, which simultaneously cleaved the acetyl group affording the free phenol.Purification by preparative HPLC afforded 19 in high purity as the final precursor for coupling to the fluorescent dyes.Ester hydrolysis of 20 under basic conditions and subsequent amide coupling with propargylamide using HATU as a coupling reagent obtained the terminal alkyne 21 in high yields.The triisopropylsilyl protecting group was cleaved as well during this reaction affording the free phenol, which is essential for ligand binding to the D 1 -like receptors.By using a copper catalyzed alkyne azide click reaction protocol with CuSO 4 pentahydrate and ascorbic acid as catalysts [53] 21 was coupled to the PEG linkers.Due to complex purification by preparative HPLC the products (22 a, 22 b) were isolated in low but sufficient yields for the coupling with the fluorescent dyes.
All three precursors were labelled with both fluorescent dyes (5-TAMRA-NHS ester and DY549-P1-NHS ester) in DMF with an excess of NEt 3 as a base (Scheme 3). [61]Purification by preparative HPLC afforded the final fluorescent ligands (23-28)  in moderate to good yields, great purity, and stability (Figures S1-S6; SI).
Since the PEG-containing compounds 25-28 were able to show significantly better properties in terms of solubility, further competition experiments were performed with the same compounds at the D 5 R (Figure 2B).In all cases, reduced affinity values of about 0.4-0.8logarithmic units were found.Again, the best results were obtained with 25 (pK i (D 5 R) = 7.62, Table 1)  and 27 (pK i (D 5 R) = 7.65, Table 1).Both compounds were subsequently tested at the D 2long R, D 3 R, and D 4 R, and only weak binding to the D 2 -like receptors was detected, representing a great selectivity profile over D 2 -like receptors for 25 and 27 (at least 1,000-fold for D 1 R and at least 500-fold for D 5 R).Because of the highest affinity and selectivity, 25 seemed to be the most promising ligand to be used as a fluorescent tracer in microscopy studies.For this purpose, knowing the ligand's mode of action is of great importance since agonists can alter the results of binding studies by inducing receptor internalization and degradation.Therefore, we confirmed the antagonistic behavior of 25 in a BRET-based G s heterotrimer dissociation assay (G s -CASE) (Figure 3; Table 2). [62]In the agonist mode, only at high concentrations of 1 μM a slight decrease could be observed, which is most likely due to optical interference of 25 with the BRET components of G s -CASE as observed in an earlier study with a 5-TAMRA-labeled histamine H 3 receptor ligand (Figure 3A). [34]In contrast, 25 reduced already at lower concentration the 1 μM dopamine-induced BRET response (pK b (D 1 R) = 7.29, pK b (D 5 R) = 9.48; Table 2), indicating that it acts as a neutral antagonist at both, D 1 and D 5 receptors.Concentration-response curves of dopamine-induced G s activation at the D 1 R and D 5 R are shown in Figure S7 (SI).

Fluorescence Properties
The fluorescence properties are usually not heavily affected by the addition of a ligand scaffold, if the fluorophore is chemically not changed, as it's the case for the 5-TAMRA and DY549-P1 dye.Nevertheless, the absorption and emission spectra and the quantum yield should be determined for new fluorescent ligands.The knowledge of the excitation and emission spectra and a high quantum yield are essential for their use in pharmacological assays and fluorescence microscopy.The emission and excitation spectra of 25-28 were recorded in PBS buffer containing 1 % of BSA (Figure 4).The excitation and emission maxima are presented in Table 3.
The quantum yield of the compounds was determined in PBS buffer containing 1 % BSA for all four compounds and additionally in PBS buffer for compounds 25 and 27 (Table 3) following a previously published protocol. [63]The 5-TAMRAlabeled compounds 25 and 27 have a higher quantum yield of approximately 36 % in PBS buffer compared to the DY549-P1labeled ligands (26 and 28; approx.19 %).Furthermore, it was observed that the addition of 1 % BSA led to a decrease in the quantum yield of the 5-TAMRA-labeled fluorescent ligands, but still with a satisfactory quantum yield of approx.30 %.Based on these results all four ligands, but especially 25 and 27, should be suitable for the use in fluorescence microscopy.

Microscopy
In order to test the suitability of 25 for laser scanning confocal microscopy (LSCM) and to visualize the binding of 25 to the D 1 R, HEK-293T cells were transiently transfected with the D 1 R Cterminally tagged with YFP.48 hours after transfection confocal microscopy images were recorded (Figure S8, SI).After addition of 50 nM of 25 a rapid accumulation of fluorescence at the cell surface was observed.This is caused by the fast association of 25 to the D 1 R expressed on the cell membrane reaching saturation binding in less than one minute.Based on the fluorescence intensity of individual cells, a time-dependent association curve could be obtained illustrating the fast binding of 25 to the D 1 R followed by a constant dissociation of 25 from the receptor after addition of 50 μM SCH-23390 (1,000-fold excess) (Figure 5).Based on the association and dissociation   18.00 � 0.33 [a] Data shown are mean values � SEM of three different slit adjustments (exc./em.):5/5 nm, 5/10 nm, 10/10 nm.
rates a kinetic pK d value of 8.07 was calculated (Table 4).These results demonstrate the suitability of 25 for the use in fluorescence microscopy as a labeling agent to visualize the D 1 R in live cells.

Molecular Brightness
To further characterize the ligand's binding affinity in intact living cells we employed molecular brightness to calculate the number of receptors decorated by the ligand on the basal membrane of cells, for varying ligand concentrations. [64]In order to reference the fraction of ligand-receptor complexes to the total amount of receptors expressed, we transfected HEK293-AD cells with D 1 R C-terminally tagged with the photostable fluorescent protein mNeonGreen (D 1 R-mNeonGreen).Cells were incubated with increasing concentrations of 25 and after reaching equilibrium, the ligand was washed out and the cell's basal membranes were imaged over time in the two spectral channels (GFP /receptor and TAMRA/ligand) using a confocal microscope (Figure 6A).From the obtained movies the average number of emitters within the confocal excitation volume was calculated based on the fluctuation of the fluorescence photon counts within each pixel (see methods) and plotted against each other.A linear regression was fitted to the data points and the slope calculated (Figure 6B).A slope of 1 signifies a 1 : 1 ratio in the number of GFPs and TAMRAs and with this a full occupancy of the receptor by the ligand.Slopes between 0 and 1 represent partial occupancy.Plotting obtained slopes versus logarithmic expression of ligand concentration returns a sigmoid concentration response curve and yields a pK d of 8.92 � 0.13, that matches well the results obtained by radioligand binding (Figure 6C).  a] 17 cells from four independent experiments were analyzed.Data represent mean values � SEM.Experiments were performed at HEK293T cells transiently expressing the hD 1 R. [b] Data represent mean values � CI (95 %).c] Five cells from one experiment were analyzed.Data represent mean values � SEM.Experiments were performed at HEK293T cells transiently expressing the hD 1 R. [d] Data represent mean values � CI (95 %).e] Association rate constant: k on = (k obs -k off )/c (25).f] K d (kin) = k off /k on ; pK d (kin) = À log K d (kin).Indicated errors were calculated according to the Gaussian law of error propagation.

tert-Butyl (3-Chloro-4-methoxyphenethyl)carbamate (7)
Di-tert-butyldicarbonate (3.23 g, 14.82 mmol, 1.1 eq.) was dissolved in DCM and added slowly at room temperature to a solution of 6 (2.50 g, 13.47 mmol, 1 eq.) in DCM.The reaction was stirred at room temperature overnight.The solvent was removed under reduced pressure and the crude product was dried in vacuo.A slightly yellow solid was obtained (3.79 g, 98 %).7 (6.2 g, 21.70 mmol, 1 eq.) was dissolved in THF and added slowly at room temperature to a suspension of LiAlH 4 (2.47 g, 65.10 mmol, 3 eq.) in THF.The reaction was heated to reflux for 3 h.After cooling to room temperature water (10 ml) and 20 % aq.KOHsolution was added carefully at 0 °C and the reaction was stirred for 30 min at room temperature.The white solid was filtered off and the filtrate was dried under reduced pressure.The crude product was dried in vacuo.A yellow oil was obtained (3.88 g, 90 %).R f = 0.16 (DCM/MeOH + 1 % NH 3 95 : 5  [58] 8 (2.44 g, 12.22 mmol, 1 eq.) and 3 (2.02g, 12.22 mmol, 1 eq.) were dissolved in acetonitrile and heated to reflux overnight.The solvent was removed under reduced pressure and the crude product was purified by column chromatography (DCM/MeOH + 1 % NH 3 98 : 2).A brown oil was obtained (4.00 g, 90 %).R f = 0.

General procedure D
The alkyne (1 eq.) and the linker (1.5 eq.) were dissolved in DCM/ MeOH (4 : 1).CuSO 4 *5 H 2 O (0.1 eq.) and ascorbic acid (0.3 eq.) were added and the reaction was stirred at room temperature for 72 h.The solvents were removed under reduced pressure and the crude product was purified by HPLC.

General procedure E
The primary amine (1.5 eq) was dissolved in DMF (30 μL).NEt 3 (10 eq.) and the fluorescent dye NHS-ester (1 eq.) in DMF (60 μL) were added, and the reaction was shaken for 2.5 h in the dark at room temperature.The reaction was quenched with 10 % aqueous TFA (20 μL), and the crude product was purified by preparative HPLC.

Radioligand competition binding experiments at the dopamine receptors
Cell homogenates containing the D 2long R, D 3 R, and D 4.4 R were kindly provided by Dr. Lisa Forster, University of Regensburg.Homogenates containing the D 1 R and D 5 R were prepared and radioligand binding experiments with cell homogenates were performed as previously described with minor modifications. [77,78]For radioligand competition binding assays homogenates were incubated in BB at a final concentration of 0.
R) at room temperature, bound radioligand was separated from free radioligand through PEI-coated GF/C filters using a Brandel harvester (Brandel Inc., Unterföhring, Germany), filters were transferred to (flexible) 1450-401 96-well sample plates (PerkinElmer, Rodgau, Germany) and after incubation with scintillation cocktail (Rotiszint eco plus, Carl Roth, Karlsruhe, Germany) for at least 3 h, radioactivity was measured using a MicroBeta2 plate counter (PerkinElmer, Waltham, MA, USA).Competition binding curves were fitted using a four-parameter fit ("log(agonist) vs. response-variable slope").Calculations of pKi values with SEM and graphical presentations were conducted with GraphPad Prism 9 software (San Diego, CA, USA).

G s heterotrimer dissociation assay
HEK293 A cells (Thermo Fisher) were transiently transfected with the G s BRET sensor, G s -CASE (http://www.addgene.org/168124/), [62]long with either D 1 R or D 5 R using polyethyleneimine (PEI).Per well of a 96-well plate, 100 μl of freshly resuspended cells were incubated with 100 ng total DNA mixed with 0.3 μl PEI solution (1 mg/ml) in 10 μl Opti-MEM (Thermo Fisher), seeded onto poly-Dlysine (PDL)-pre-coated white, F-bottom 96-well plates (Brand GmbH) and cultivated at 37 °C, 5 % CO 2 in penicillin (100 U/ml)/ streptomycin (0.1 mg/ml)-, 2 mM L-glutamin-and 10 % fetal calf serum (FCS)-supplemented Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher).48 hours after transfection, cells were washed with Hank's Buffered Salt Solution (HBSS) and incubated with a 1/1000 furimazine (Promega) dilution in HBSS for 2 minutes.Next, the baseline BRET ratio was recorded in three consecutive reads, cells were stimulated with serial dilutions of 25 or vehicle control, and BRET was recorded for another 15 reads.For experiments in antagonist mode, serial dilutions of 25 were added together with furimazine before the experiment and 1 μM dopamine or vehicle control was added after the first three baseline recordings.All experiments were conducted using a ClarioStar Plus Plate reader (BMG Labtech) with a cycle time of 120 seconds, 0.3 seconds integration time and a focal height of 10 mm.Monochromators were used to collect the NanoLuc emission intensity between 430 and 510 nm and cpVenus emission between 500 and 560 nm.BRET ratios were defined as acceptor emission/ donor emission.The basal BRET ratio before ligand stimulation (Ratio basal ) was defined as the average of all three baselaine BRET values.Ligand-induced ΔBRET was calculated for each well as a percent over basal ([(Ratio stim À Ratio basal )/Ratio basal ]×100).To correct for non-pharmacological effects, the average ΔBRET of vehicle control was subtracted.

Live cell confocal microscopy at the D 1 R
Confocal images were recorded with kind assistance from Manel Bosch (Universitat de Barcelona).HEK293T cells were transfected based on previously described procedures with the D 1 R-YFP (Cterminally tagged) with minor modifications. [80,81,22]Cells were seeded in 35 mm wells containing 1.5 cover slips.48 h after transfection medium was changed to OptiMem media (Gibco) supplemented with 10 mM HEPES.Imaging was performed using a Zeiss LSM880 Laser Scanning Confocal Microscope equipped with a "Plan-Apochromat" 40x/1,3 Oil DIC M27 objective and a photomultiplier tube (PTM) detector.For excitation of 25 an DPSS laser with a wavelength of 561 nm was used.Fluorescence was detected within an emission window of 569-669 nm.Image size was set to 512×512 pixels.After adjusting the focus, time-lapse images were recorded in intervals between 0.32 and 1 s. 25 was added in a final concentration of 50 nM.Dissociation was induced by the addition of SCH-23390 (50 μM, 1,000-fold excess).Time-lapse confocal images were processed using the ImageJ software.Contrast was adjusted for each file to facilitate the visualization of the fluorescence signal.Total fluorescence was plotted as a function of time using GraphPad Prism9 software (GraphPad, San Diego, USA).The time of addition of 25 was set as 0 min.Data was fitted using the "Association-one conc. of hot ligand" or "association then dissociation" functions from Prism9.Kon, Koff, and kinetic Kd values were calculated by Prism9.

Molecular Brightness
HEK-293AD cells were seeded in 8-well Ibidi μ-slides with a density of 25,000 cells per well and transfected with 2 μg hD 1 R-mNeon-Green after 24 h using JetPrime transfection reagent according to manufacturer's protocol.After further 24 h cells were washed and imaged in FRET-buffer (144 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM HEPES) on a confocal laser scanning microscope, Leica SP8, with a white-light laser at wavelengths of 488 and 552 nm, and laser power of 5 %.All measurements were conducted with an HC PLAP CS2 40×1.3 numerical aperture (NA) oil immersion objective (Leica).Movies were acquired 1.3 seconds per frame for 100 frames with two hybrid detectors in the range of 498 to 547 nm and 562 to 612 nm respectively, in a line sequential, counting mode.Molecular brightness ɛ and number of molecules N are calculated from the average (k) of the photon counts collected in a pixel and its variance (σ) according to the formulas ɛ = σ 2 /k-1 and N = k 2 /σ 2 .ImageJ was used to extract molecular brightness and fluorescence intensity values, number of emitters was calculated with Word Excel and obtained values were plotted and fitted with Prism v. 9.5.1.

Figure 2 .
Figure 2. Competition binding curves from radioligand binding experiments at the D 1 R (A) and D 5 R (B) homogenates performed with compounds 23-28 and the respective radioligands (cf.Table 1 footnotes).Competition binding curves of 25 (C) and 27 (D) at the D 1-5 R homogenates.Graphs represent the means from three independent experiments each performed in triplicates.Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration-response curves.

Figure 3 .
Figure 3. Concentration-response curves (CRCs) for G s activation of 25 in the absence (A) and presence (B) of 1 μM dopamine in HEK293 A cells transiently expressing the G s BRET sensor along with the wild-type D 1 R or D 5 R. Graphs represent the means of three independent experiments each performed in duplicates.Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration-response curves.

[ a ]
Competition binding assay at HEK293T_CRE Luc 2P D 1 R homogenates, HEK293T_CRE Luc 2P D 2long R homogenates, HEK293T_CRE Luc 2P D 3 R homogenates, HEK293T_CRE Luc 2P D 4 R homogenates, or HEK293T_CRE Luc 2P D 5 R homogenates. [b] Displacement of 1 nM [ 3 H]SCH-23390 (K d = 0.4 nM). [c] Displacement of 0.05 nM [ 3 H]N-Methylspiperone (K d = 0.015 nM). [d] Displacement of 0.05 nM [ 3 H]N-Methylspiperone (K d = 0.026 nM). [e] Displacement of 0.1 nM [ 3 H]N-Methylspiperone (K d = 0.078 nM). [f] Displacement of 1 nM [ 3 H]SCH-23390 (K d = 0.4 nM).Data shown are mean values � SEM of N experiments, each performed in triplicates.Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration-response curves.Competition binding curves are shown in Figure 2. Table 2. Functional data of 25 at the D 1 R and D 5 Competition binding experiment at HEK293 cells expressing the G s BRET sensor with the wild type D 1 R or D 5 R. [b] Inhibition of dopamine-induced (c = 1 μM, EC 50 = 393 nM, Figure S7, SI) G s activation. [c] Inhibition of dopamine-induced (c = 1 μM, EC 50 = 26 nM, Figure S7, SI) G s activation.Data shown are mean values � SEM of N experiments, each performed in duplicates.Data were analyzed by nonlinear regression and were best fitted to sigmoidal concentration-response curves.Competition binding curves are shown in Figure 3.

Figure 5 .
Figure 5. Binding kinetics of 25 at the hD 1 R in whole HEK-293T cells using LSCM.The graph shows association of 25 (c = 50 nM) to the receptor and dissociation of 25 induced by addition of SCH-23390 (c = 50 μM, 1,000-fold excess, after 15 seconds) from a representative experiment.Data represent mean � SEM of four independent cells of one experiment.

ConclusionsA
set of six different fluorescent ligands containing linkers of different lengths and chemical compositions and two different fluorescent dyes were designed and synthesized.The known D 1 R/D 5 R antagonistic SCH-23390 was used as a D 1 -like scaffold for the fluorescent ligands.Two fluorescent dyes, 5-TAMRA and DY549-P1, were chosen because of their proposed suitability as a fluorescent tracer for microscopy studies.After the successful synthesis and purification of the ligands, their fluorescent and pharmacological properties were determined.Radioligand binding studies were performed revealing 25 and 27 as D 1 -like receptor selective fluorescent ligands with binding affinity in the low nanomolar range.Compound 25, equipped with the medium-length hydrophilic PEG linker and 5-TAMRA as dye, showed the best overall results regarding affinity, selectivity, quantum yield and was used as an exemplary compound to determine its mode of action.A G protein biosensor assay confirmed the expected neutral antagonism of 25.Furthermore, 25 was used successfully to label D 1 Rs in live cells for LSCM demonstrating its suitability for fluorescence microscopy.In molecular brightness studies, the ligand's binding affinity could be determined in a range that was in good agreement with radioligand binding data and a full occupancy of the receptor by the ligand.Overall, the set of fluorescent ligands, especially 25, represent a versatile tool for different experimental setups for further investigations at the D 1 -like receptors.

Figure 6 .
Figure 6.Association of 25 to the hD 1 R using molecular brightness analysis.Basolateral membranes of HEK-293AD cells transiently expressing hD 1 R-mNeonGreen and preincubated with indicated amount of 25 (A); calculated numbers of emitters for 5-TAMRA and mNeonGreen channels, and the corresponding linear regression fit (mean � SEM; n = 23 cells from 3 independent experiments) (B), slopes obtained from linear fits plotted against log concentrations of 25 with the corresponding non-linear fit and pK d value (mean � SEM; n = at least 20 cells from 3 independent experiments for each datapoint) (C).

Table 4 .
Kinetic binding constants of 25 at the hD 1 R in confocal microscopy.
C NMR) spectra were purchased from Deutero GmbH (Kastellaun, Germany).All reactions carried out with dry solvents were accomplished in dry flasks under nitrogen or argon atmosphere.For the preparation of buffers, HPLC eluents, and stock solutions millipore water was used.Column chromatography was accomplished using Merck silica gel Geduran 60 (0.063-0.200 mm).
35mmol, 1 eq.) and thiourea (1.08 g, 14.35 mmol) were dissolved in EtOH and heated to reflux overnight.The solvent was removed under reduced pressure and the obtained solid was added at 5 °C to a suspension of NCS (9.58 g, 71.75 mmol, 5 eq.) in acetonitrile and 2 N aq.HCl (5 ml).The reaction was stirred for 20 min below 10 °C and poured onto water.The aqueous phase was extracted with diethyl ether.The combined organic phases were washed with brine, dried over Na 2 SO 4 and the solvent was removed under reduced pressure.