==== Front
ACS Omega
ACS Omega
ao
acsodf
ACS Omega
2470-1343
American Chemical Society

10.1021/acsomega.3c01796
Article
Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging
Szepesi Kovács Dénes †‡§+
Chiovini Balázs ∥
Müller Dalma ⊥#∇
Tóth Estilla Zsófia §∇○
Fülöp Anna ◆
Ábrányi-Balogh Péter †‡§
Wittner Lucia §○
Várady György ¶
https://orcid.org/0000-0002-4217-0150
Farkas Ödön &
https://orcid.org/0000-0002-6753-6796
Turczel Gábor ●
Katona Gergely ∥
Győrffy Balázs §⊥#◊
Keserű György Miklós *†‡§
https://orcid.org/0000-0003-3224-8847
Mucsi Zoltán *▲□
Rózsa Balázs J. *∥▲^
https://orcid.org/0000-0002-3939-6925
Kovács Ervin *◆¢+
† Medicinal Chemistry Research Group, Research Centre for Natural Sciences, H-1117 Budapest, Hungary
‡ Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, H-1111 Budapest, Hungary
§ National Laboratory for Drug Research and Development, H-1117 Budapest, Hungary
∥ Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, H-1444 Budapest, Hungary
⊥ Oncology Biomarker Research Group, Research Centre for Natural Sciences, H-1117 Budapest, Hungary
# Department of Bioinformatics, Semmelweis University, H-1094 Budapest, Hungary
∇ Semmelweis University Doctoral School, H-1085 Budapest Hungary
○ Integrative Neuroscience Research Group, Research Centre for Natural Sciences, H-1117 Budapest, Hungary
◆ Femtonics Ltd., H-1094 Budapest, Hungary
¶ Molecular Cell Biology Research Group, Research Centre for Natural Sciences, H-1117 Budapest, Hungary
& Department of Organic Chemistry, Eötvös Loránd University, H-1117 Budapest, Hungary
● NMR Research Laboratory, Research Centre for Natural Sciences, H-1117 Budapest, Hungary
◊ Department of Pediatrics, Semmelweis University, H-1094 Budapest, Hungary
▲ Brain Vision Center, H-1094 Budapest, Hungary
□ Faculty of Materials and Chemical Sciences, University of Miskolc, Miskolc H-3515, Hungary
^ Laboratory of 3D Functional Network and Dendritic Imaging, Institute of Experimental Medicine, H-1083 Budapest, Hungary
¢ Polymer Chemistry and Physics Research Group, Research Centre for Natural Sciences, H-1117 Budapest, Hungary
* Email: keseru.gyorgy@ttk.hu. Tel.: +3613826911.
* Email: zmucsi@femtonics.eu.
* Email: rozsabal@brainvisioncenter.com.
* Email: kovacs.ervin@ttk.hu. Tel.: +3613826570.
14 06 2023
27 06 2023
8 25 2283622843
16 03 2023
01 06 2023
© 2023 The Authors. Published by American Chemical Society
2023
The Authors
https://creativecommons.org/licenses/by/4.0/ Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Magyar Tudományos Akadémia 10.13039/501100003825 BO/799/21/7 Innovációs és Technológiai Minisztérium 10.13039/501100015498 NKP-19-3-I-BME-408 Nemzeti Kutatási Fejlesztési és Innovációs Hivatal 10.13039/501100011019 TKP2021-NVA-14 Nemzeti Kutatási Fejlesztési és Innovációs Hivatal 10.13039/501100011019 TKP2021-EGA-42 Nemzeti Kutatási Fejlesztési és Innovációs Hivatal 10.13039/501100011019 PD124598 Nemzeti Kutatási Fejlesztési és Innovációs Hivatal 10.13039/501100011019 KFI-18-2018-00097 Nemzeti Kutatási Fejlesztési és Innovációs Hivatal 10.13039/501100011019 2020-1.1.5-GYORSTSV-2021-00004 Nemzeti Kutatási Fejlesztési és Innovációs Hivatal 10.13039/501100011019 2018-1.3.1-VKE-2018-00032 Magyar Tudományos Akadémia 10.13039/501100003825 NKP-22-ME3 document-id-old-9ao3c01796
document-id-new-14ao3c01796
ccc-price
==== Body
pmcIntroduction

Rapid and reliable identification of malignant tumor cells is crucial in cancer histological diagnosis. To achieve this goal, one of the most efficient and straightforward techniques is the use of specific antibodies that label exclusively the targeted tumor cells with high selectivity. This labeling usually visualizes the tumor cells for an imaging technique and distinguishes them from the healthy population; therefore, in vitro or in vivo methods based on antibody conjugation are a focus of research interest.1,2 Due to these reasons, there is a continuous demand for novel fluorescent dyes exhibiting specific emission maxima, higher photostability, better quantum yield, and significantly larger Stokes shift.3

Rhodamines, like tetramethylrhodamine (TAMRA, 1a) and fluorescein (1b), are widely used fluorophores (laser dyes, fluorescent probes, and chemosensors) because of their excellent photostability and photophysical properties (Figure 1A).4,5 The family of rhodols (2) is less prevailing and structurally similar based on the same xanthene scaffold. Rhodols usually have small Stokes shift (ca. 20–25 nm), which need to be increased for better detection. First-generation rhodols (2) were synthetized containing a diethylamino group on one side of the xanthene core (Figure 1A). However, there was still some room for improvement in fluorescence properties, and therefore, on the one hand, an electron-donating julolidine core6−10 was incorporated into next-generation compounds (3) that inhibits the internal rotation of the amino group, decreasing the twisted intramolecular charge transfer (TICT). On the other hand, in some cases, the π-system was extended (4) to fine-tune the emission.11−14 Furthermore, rhodols were also used as fluorescent probes for inorganic (H2O2 and HOCl) (5 and 6) or glutathione-detecting probes (7). The latter compound (7) was also applied in biological systems (Figure 1B).15−17

Figure 1 (A) Selected examples for previously published fluorophores (1a: TAMRA, R1 = NMe2, R2 = NMe2, R3 = COOH; 1b: fluorescein, R1 = OH, R2 = O, R3 = H). (B) Rhodol chemodosimeters used in in vitro studies. (C) Synthetic route and proposed application of the dyes synthesized in this work.

Specific fluorescently labeled proteins became effective tools to study the mechanism of complex biological systems.18,19 In tumor diagnostics, dye-labeled antibodies have great importance due to their combined unique selectivity and detectability.1,2,5,20,21 Nowadays, biorthogonal and click chemistry (e.g., azide-alkyne cycloaddition) offers a site-selective and simple labeling method; therefore, most of the fluorescent dyes are equipped with a functional group available for these reactions.22−25

Although the access to fluorescence or confocal microscopy is more general than that for two-photon fluorescence microscopy (2P microscopy), using 2P active fluorophores in imaging processes has many advantages. 2P excitation fluorescence imaging that provides thin optical sectioning has enabled a more precise quantification during analysis by restricting out-of-focus excitation (and thus emission). As out-of-focus fluorescence is never generated, no pinhole is required in the microscope detection path, resulting in an increase in the efficiency of fluorescence collection that makes a huge advantage and motivation to develop fluorescent dyes with 2P cross section. Moreover, excitation can be reached at longer wavelengths, the auto-fluorescence is negligible, and due to the lower energy of photons, less photobleaching is experienced.26,27 Finally, it is important to note that cancer cell behavior can be examined in vivo together with the surrounding tissue, e.g., non-tumorous, but tumor-associated stromal cells.28 Combining all of the advantages of 2P imaging, this technique is more and more widespread in cell imaging and the number of 2P studies in the diagnostic field is increasing, justifying the development of 2P active and specific fluorescent dyes.29−31

In light of these aspects, as a continuation of our interest in novel fluorescent dyes,32−36 two-photon active compounds,35,37 and antibody modification,25 we aimed to develop new rhodol derivatives with increased 2P cross section, large Stokes shift, and high photostability. After investigating the photophysical properties of the library, the best candidate of this set of dyes was cross-linked to the therapeutic antibody trastuzumab, and the dye-labeled antibody was investigated in vitro (Figure 1C) for the specific recognition of Her2+ cancer cells by confocal and 2P microscopy.

Results and Discussion

Synthesis of a Library of New Rhodol Fluorophores

The julolidine-decorated rhodols were synthesized in a two-step synthetic route. At first, the Friedel Craft acylation of 8-hydroxyjulolidine (10) using 1,2,4-benzenetricarboxylic anhydride (8) resulted in the mixture of ketones 14 and 15, which were separated successfully by preparative HPLC. Then, both intermediates 14 and 15 were transformed to the desired products 11a–11n using various resorcinol derivatives (9) in an acid-catalyzed condensation with up to 89% yields (Scheme 1).

Scheme 1 Two-Step Synthesis of Rhodol Derivatives (11) from Phthalic Anhydride Derivative (8), 8-Hydroxyjulolidine (10), and Various Phenol Derivatives (9) and the Structure of the Synthetized Compounds

Photophysical Development of the New Fluorophores

The spectroscopical properties of the rhodol carboxylic acids (11a–11n) have also been investigated in detail. Molar absorption coefficient, quantum yield, and brightness were determined (Table 1 and Table S1). We have also measured the 2P cross section and the emission intensity (Table 1). In most of the cases (11a–11h and 11k–11n), the excitation maxima (λexcmax) are located in the range of visible green light (542–551 nm) between the emission range of the common fluorescein and rhodamine dyes. It could be concluded that small changes in the structure did influence neither the excitation nor the emission significantly (571–606 nm). As expected, 11i and 11j with an extended aromatic skeleton showed higher absorbance maxima (λabsmax = 570 nm, Table S1). The emission maxima of 11i and 11j were detected in the range of yellow and orange light (603–606 nm), close to that of the rhodamine derivatives. The Stokes shifts for the members of the rhodol library are typically 30–41 nm, which is significantly larger than for that of the widely used rhodamines (the Stokes shift of 1a is only 23 nm; Table 1, entry 15). These advantages could be exploited in imaging experiments as the overlap of the absorption and emission spectra is negligible, allowing more comfortable selection of optical filters. Moreover, the recorded brightness is higher for 11a, 11d, and 11m (Table 1, entries 1, 4, and 13) than the brightness of the frequently used 5-TAMRA (1a, 31,554 M–1 cm–1 in Table 1, entry 15). In summary, the photophysical properties predict good applicability of these new dyes. In some cases (11c, 11e, 11h, 11i, 11j, and 11l in Table 1, entries 3, 5, 8–10, and 12), molar extinction coefficients and quantum yields were lower, decreasing the brightness of the compound. This could be explained by the well-known ring-chain tautomerism between the lactone and the free acid form of the rhodol along with the changes of pH38 and tuned by the substituents (Scheme 2). To explore the ring opening and closing equilibrium, we have investigated the pH dependence of the absorbance and fluorescence of 11d. The decreasing pH caused a significant drop in the fluorescence intensity; particularly, below pH = 6.4, the intensity decreased to 5% at 577 nm (Figure S49). This is in concordance with the presumption that under acidic conditions, the lactone form is more stable,39 while the anionic carboxylate supporting the electron gradient increasing the corresponding wavelengths is present under alkaline conditions. In the case of the excitation maxima using 2P techniques, the 2P properties are similar for all the members of the library. In particular, the excitation maxima could be achieved in the range of 830–850 nm; however, the intensity was generally in the range of 10–150 a.u. (Table 1). The 11d derivative was selected for further development based on its photospectroscopic properties.

Scheme 2 Protonation and Ring Closure Equilibria of Compounds 16a–16n for Computational Study

Computed at the B3LYP/6-31G(d,p)//PCM(water) level of theory. Below is the most possible distribution of the rhodol derivatives.

Table 1 Single-Photon Characterization of the Novel Rhodols (11a–11n) and the Extensively Used 5-TAMRA in HEPES (10 μM, pH = 7.4)

 	 	single-photon properties	 	two-photon properties	
#	compound	λexcmax [nm]	λemmax [nm]	Δλ [nm]	B [M–1 cm–1]	 	λexcmax [nm]a	intensity [a.u.]+	
1	11a	546	585	37	32,678	 	845	46.0	
2	11b	547	589	40	19,021	 	845	96.4	
3	11c	542	571	28	18,834	 	845	128.5	
4	11d	542	577	34	35,663	 	845	151.7	
5	11e	547	582	34	18,712	 	845	59.4	
6	11f	547	586	38	28,912	 	845	51.4	
7	11g	551	580	27	19,153	 	855	69.0	
8	11h	550	584	32	7955	 	845	2.0	
9	11i	568	606	36	526	 	855	4.8	
10	11j	568	603	33	673	 	855	7.0	
11	11k	551	592	40	22,142	 	845	23.2	
12	11l	551	594	41	18,574	 	845	28.8	
13	11m	540	573	30	36,473	 	835	31.0	
14	11n	543	579	35	31,055	 	845	11.8	
15	5-TAMRA	551	574	24	31,554	 	855	104	
a λexcmax: wavelength of the maximum of excitation spectrum. λemmax: wavelength of the maximum of emission spectrum. Δλ: Stokes shift calculated by λemmax – λexcmax. B is brightness (B = ε·Φ). For more details, see Table S1 and experimental section in the Supporting Information.

Theoretical Calculations to Explain the Differences in Fluorescence Properties

In the course of the computational study, the fluorophore was modeled as the R,1R2-functionalized methyl amide derivative (16a–16n, corresponding to 11a–11n, and in addition, the non-substituted derivatives as R,1R2 = H in 16o and 16p (see Scheme 2 and Table S3a), mimicking the antibody–fluorophore conjugate (13). Both 4- and 5-carboxamide isomers (series derived from 14 and 15, respectively) were considered. The rhodol scaffold, bearing a positive charge, participates in complex deprotonation and isomerization equilibria (Scheme 2). The various species (forms I, II, and III) exhibit different fluorescence properties, and their appearance in the solution depends on their thermodynamic stability, indicated by the computed ΔG values (Table S2). Here, we studied only the substituent effect on this equilibrium to estimate the relationship between the distribution of the tautomeric forms and fluorescence intensities. Form IIb is responsible for the high fluorescence intensity with overall neutral charge, so in our scope, we looked for a derivative, where form IIb has the lowest ΔG value, suggesting that it is the most preferred form.

Interestingly, the position of the carboxamide functional group on the phenyl ring has no significant effect on the computed result (ΔΔG is less than 2 kJ mol–1), so we focused on the functional groups decorating ring A of the xanthene. Considering a protonation equilibrium with water molecules (Figure S55), in general, at neutral pH, form IIb is more stable than form I (ranges from +5 to +42 kJ mol–1) and, except 16k–16n, form IIb is more stable than form IIIb (ranges from +4 to +20 kJ mol–1, Table S2). For R1 = R2 = F (16g and 16h), form IIb and form IIIb are quite close to each other (ΔΔH less than 4 kJ mol–1). In the cases of 16k and 16l (R = napht2) and 16m and 16n, (R = −C2H4COOH), form IIIb is the most stable. Since we are looking for neutral structures, these compounds are not suggested for conjugation based on theoretical assumptions.

In the next section, we compare only the different form II species. Among the substituents, the OH functional group (16a and 16b) prefers mostly form IIb by ca. 40 kJ mol–1, which is due to the strong and stabilizing hydrogen bond between O– and the neighboring OH group. This also refers to the increased acidity of the rhodol OH. Compounds 16i and 16j (R = napht1) favor the ring closed forms IIc and IIIb, which are not fluorescent in accordance with the experimental findings, so these are also excluded from the selection. The ethyl (16c and 16d), carboxyethyl (16m and 16n), and methoxy (16e and 16f) substituents, however, exhibit favorable distribution for the fluorescent form IIb. In conclusion, both theory and experiments suggest that the last six derivatives can be promising candidates for antibody conjugation.

Finally, to demonstrate the ability of the selected rhodol 11d for biological application, we have investigated the photostability in HEPES buffer by exciting continuously using a 520 nm LED light source (5 and 10 W). The original fluorescence intensity decreased to 50% after 15 min in the case of 5 W and after 10 min in the case of 10 W irradiation (Figure S50). The rates of bleaching were acceptable, considering an imaging process that usually requires sequential excitation for just a couple of minutes and lower than the commonly used fluorescein. Furthermore, investigating the solvent effect on the photophysical properties of 11d (Figure S51) in a series of solvents, we have observed decreased absorbance and fluorescence intensity for apolar-aprotic solvents (toluene, dioxane, and tetrahydrofuran). In the case of slightly polar and protic ethanol, we detected a significant increase in the absorbance and two times higher fluorescence intensity compared to HEPES buffer. Also, there is a slight hypsochromic effect in EtOH compared to buffer, DCM, and acetonitrile. Considering the emission wavelength, the aqueous buffer seems to be an appropriate medium, confirming the usability in biological investigations. In aqueous medium, suppressed aggregation quenching is often relevant;3,26,40 however, in the case of 11d, there is no significant loss of intensity with and without SDS in HEPES buffer (Figure S52). Furthermore, chemical stability tests over 24 h in aqueous solutions using various buffers (pH 3 to 11; Figure S53) also proved the excellent applicability of the selected candidate 11d.

Antibody Conjugation and Microscopy Imaging

After having these promising results in hand, we transformed 11d to 17 azide, enabling azide-alkyne click reaction. Knowing that antibodies target specific and unique cancer cells and deliver their fluorescent1 or cytotoxic payloads41 with high accuracy, we have conjugated 17 to the human IgG trastuzumab having four cyclooctynyl harbors (created after literature procedures, shown in Scheme 3),42 resulting in a potential diagnostical tracer 13 for imaging of Her2+ cells.43

Scheme 3 Transformation of 11d Acid with a Simple and Fast Method to NHS active ester Followed by the Introduction of the Azide Functional Group and the Use of 17 in Click Reaction to Produce the Antibody–Fluorophore Conjugate 13

At first, an activated44 NHS ester of 11d was prepared with TSTU (18), followed by the smooth acylation of 3-azidopropane-1-amine (19), resulting in 17. Then, we were able to label the antibody (12) with the fluorescent dye in a copper-free strain-promoted azide-alkyne click reaction at room temperature. The antibody–fluorophore conjugate 13 was characterized by UV/Vis spectroscopy to determine the fluorophore–antibody ratio (FAR). With the use of the Lambert–Beer equation, the ideal FAR = 4 value was confirmed (Table S2). The homogeneity of the conjugate was determined by SDS-PAGE that showed high (95%) homogeneity after the modification steps (Figure S53). Investigating the gel under UV light (366 nm), the fluorescent spot of the antibody–fluorophore conjugate could be seen by the naked eye (Figure S54).

Having the antibody–fluorophore conjugate (13) in hand, first, we examined the selectivity of the conjugates in flow cytometry using living cells. We treated NCI-N87 cells overexpressing Her2 receptor and MCF7 Her2-negative cells with 13 conjugate and observed the same selectivity as the native antibody45,46 and its pyridazinone conjugates,42 indicating that the conjugates might be useful tools also on living cells (Figure S55). Second, cell sections of the same cell lines were treated with 13 (Figure 2). Confocal microscopy showed no membrane labeling for Her2-negative MCF-7 cells, while in the case of the Her2+ cell line, the membrane labeling (red, Figure 2B) was significant next to the signal of DAPI (blue, Figure 2A).

Figure 2 Confocal microscopy images of NCI-N87 (Her2+) (A–C) and MCF-7 (Her2−) (D–F) cells treated with 13 fluorophore–antibody conjugate. (A) DAPI cell nucleus labeling of NCI-N87 cells. (B) 13 conjugate labeling of NCI-N87 cells. (C) Merged image of DAPI and 13 conjugate labeling of NCI-N87 cells. (D) DAPI cell nucleus labeling of MCF-7 cells. (E) 13 conjugate labeling of MCF-7 cells. (F) Merged image of DAPI and 13 conjugate labeling of MCF-7 cells. Scale bars: 10 μm. (A, C, D) Excitation: 405 nm; emission: 456 nm. (B, E, F) Excitation: 543 nm; emission: 687 nm.

We examined the cell section with 2P microscopy as well, observing the analogue labeling pattern (Figure 3). Membrane labeling is clearly visible on the merged picture (Figure 3C) using the red fluorescence signal (Figure 3A) on the surface of NCI-N87 cells (Her2+) and GFP fluorescence images (Figure 3B) of the same (Her2+) cells similar to using confocal techniques (Figure 2C). In the case of MCF-7 (Her2−) cells, only autofluorescence was detected on the same wavelength (Figure 2D–F). Therefore, we confirmed that novel rhodol 11d is also an effective tool for 2P microscopy imaging and the antibody–fluorophore conjugate retained its selectivity examined on two cell lines.

Figure 3 Artificially recolored two-photon images of 13 antibody conjugate-treated Her2+ and Her2– cells. (A) DAPI labeling of NCI-N87 cells excited at 750 nm (PMT filter: 490–550 nm). (B) Fluorescence of 13 antibody conjugate on NCI-N87 cells excited at 840 nm (PMT filter: 570–640 nm). (C) Merged image of NCI-N87 cells. (D) DAPI labeling of MCF-7 cells excited at 750 nm (PMT filter: 490–550 nm). (E) Image of 13 conjugate-treated MCF-7 cells excited at 840 nm (PMT filter: 570–640 nm). (F) Merged image of MCF-7. Scale bars: 100 μm. For camera images, see Figures S56–S58.

Conclusions

In conclusion, we have synthesized a library of next-generation rhodol fluorophores with improved fluorescence properties compared to 5-TAMRA, particularly better Stokes shift, and increased brightness. The new dyes exhibited specific emission wavelength, excellent brightness, applicability in confocal as well as in 2P microscopy, and good photostability for imaging in aqueous buffer. In the case of some derivatives, the decreased fluorescence could be explained by ring-chain tautomerism supported by theoretical calculations. The dye with the best properties was equipped with an azide functional group and linked to cyclooctyne-derived trastuzumab. Finally, this fluorophore–antibody conjugate was proven to be efficient to selectively label Her2-positive NCI-N87 cells in both confocal and 2P microscopy imaging of cells designated as trastuzumab targets.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c01796.Experimental procedures and compound characterization (Figures S1–S55), 2P measurements with camera images (Figures S56–S58), structure and data of computed water clusters (Figure S59), FAR determination (Table S2), and theoretical data (Tables S3 and S4) (PDF)

Supplementary Material

ao3c01796_si_001.pdf

Author Contributions

+ D.S.K. and E.K. contributed equally to this work.

Author Contributions

The synthesis of fluorescent dyes was carried out by E.K. and A.F. The spectroscopic characterization was made by D.S.K., G.T., and A.F. The conjugation was prepared by D.S.K. Theoretical calculations and data curation were accomplished by Z.M. and Ö.F. Biological experiments were designed by B.G. and carried out by D.M., confocal microscopy was performed by E.Z.T. and L.W., and two-photon imaging was implemented by B.C. Acquisition of reagents and resources for biological measurements was carried out by B.G., B.R., and K.G. Flow cytometry was performed by G.V. Validation was accomplished by P.A.B., E.K., Z.M., and G.M.K. G.M.K. and R.J.B. were responsible for resources, funding acquisition was accomplished by G.M.K., G.K., R.J.B., and Z.M., project administration was implemented by P.A.B. and E.K., and E.K., Z.M., and G.M.K. supervised the project. The visualization was carried out by E.K., D.S.K., and M.Z. The manuscript was written through contributions of E.K., D.S.K., P.A.B., and M.Z. All authors have given approval to the final version of the manuscript.

The authors declare the following competing financial interest(s): Gergely Katona and Balzs J. Rzsa are founder of Femtonics and members of its scientific advisory board. The other authors declare that no conflict of interest exists. However, two of my co-author have, as they are founder of Femtonics, the company which employed researchers also.

Acknowledgments

The research was supported by the 2018-1.3.1-VKE-2018-00032, KFI-18-2018-00097, TKP2021-EGA-42, 2020-1.1.5-GYORSÍTÓSÁV-2021-00004, and TKP2021-NVA-14 grants of the National Office of Science, Innovation and Technology (NKFIH). We are grateful for the support of NKFIH PD124598 and ÚNKP-19-3-I-BME-408 New National Excellence Program of the Ministry for Innovation and Technology and János Bolyai Research Scholarship (BO/799/21/7, ÚNKP-22-ME3). The authors acknowledge the supportive work of Krisztina Németh. We are grateful for the possibility to use ELKH Cloud,47 which helped us achieve the results published in this paper.
==== Refs
References

Warram J. M. ; de Boer E. ; Sorace A. G. ; Chung T. K. ; Kim H. ; Pleijhuis R. G. ; van Dam G. M. ; Rosenthal E. L. Antibody-Based Imaging Strategies for Cancer. Cancer Metastasis Rev. 2014, 33 , 809–822. 10.1007/s10555-014-9505-5.24913898
Pal R. ; Kang H. ; Choi H. S. ; Kumar A. T. N. Fluorescence Lifetime-Based Tumor Contrast Enhancement Using an EGFR Antibody–Labeled Near-Infrared Fluorophore. Clin. Cancer Res. 2019, 25 , 6653–6661. 10.1158/1078-0432.CCR-19-1686.31481509
Liang X. ; Zhang Q. Recent Progress on Intramolecular Charge-Transfer Compounds as Photoelectric Active Materials. Sci. China Mater. 2017, 60 , 1093–1101. 10.1007/s40843-016-5170-2.
Beija M. ; Afonso C. A. M. ; Martinho J. M. G. Synthesis and Applications of Rhodamine Derivatives as Fluorescent Probes. Chem. Soc. Rev. 2009, 38 , 2410 10.1039/b901612k.19623358
Ariztia J. ; Solmont K. ; Moïse N. P. ; Specklin S. ; Heck M. P. ; Lamandé-Langle S. ; Kuhnast B. PET/Fluorescence Imaging: An Overview of the Chemical Strategies to Build Dual Imaging Tools. Bioconjugate Chem. 2022, 33 , 24–52. 10.1021/acs.bioconjchem.1c00503.
Gao M. ; Lee S. H. ; Das R. K. ; Kwon H.-Y. ; Kim H. S. ; Chang Y.-T. A SLC35C2 Transporter-Targeting Fluorescent Probe for the Selective Detection of B Lymphocytes Identified by SLC-CRISPRi and Unbiased Fluorescence Library Screening. Angew. Chem., Int. Ed. 2022, 61 , e202202095 10.1002/anie.202202095.
Ming W. ; Hu X. ; Zhang Z. ; Chang S. ; Chen R. ; Tian B. ; Zhang J. Synthesis and Properties of Seminaphthorhodafluor Red Laser Dyes. Res. Chem. Intermed. 2020, 46 , 1991–2002. 10.1007/s11164-019-04076-9.
Hanaoka K. ; Iwaki S. ; Yagi K. ; Myochin T. ; Ikeno T. ; Ohno H. ; Sasaki E. ; Komatsu T. ; Ueno T. ; Uchigashima M. ; Mikuni T. ; Tainaka K. ; Tahara S. ; Takeuchi S. ; Tahara T. ; Uchiyama M. ; Nagano T. ; Urano Y. General Design Strategy to Precisely Control the Emission of Fluorophores via a Twisted Intramolecular Charge Transfer (TICT) Process. J. Am. Chem. Soc. 2022, 144 , 19778–19790. 10.1021/jacs.2c06397.36191139
Homma M. ; Takei Y. ; Murata A. ; Inoue T. ; Takeoka S. A Ratiometric Fluorescent Molecular Probe for Visualization of Mitochondrial Temperature in Living Cells. Chem. Commun. 2015, 51 , 6194–6197. 10.1039/C4CC10349A.
Liang Q. ; Chen W. ; Ren X. ; Song X. Red-Emitting Rhodamine-Based Probe with Large Stokes Shift for ClO– Detection. Tetrahedron 2022, 127 , 133020 10.1016/j.tet.2022.133020.
Poronik Y. M. ; Vygranenko K. V. ; Gryko D. ; Gryko D. T. Rhodols – Synthesis, Photophysical Properties and Applications as Fluorescent Probes. Chem. Soc. Rev. 2019, 48 , 5242–5265. 10.1039/C9CS00166B.31549709
Xu S. ; Knight J. R. ; Brummett B. J. ; Shieh M. ; Cui Q. ; Wang Y. ; Ramush G. ; Xian M. Benzothiazole-Derived Sulfones and Sulfoxides as Reactive Templates for Biothiols and Sulfane Sulfurs. Org. Lett. 2022, 24 , 2546–2550. 10.1021/acs.orglett.2c00734.35352985
Wang L. G. ; Munhenzva I. ; Sibrian-Vazquez M. ; Escobedo J. O. ; Kitts C. H. ; Fronczek F. R. ; Strongin R. M. Altering Fundamental Trends in the Emission of Xanthene Dyes. J. Org. Chem. 2019, 84 , 2585–2595. 10.1021/acs.joc.8b03030.30719911
Tian J. ; Shi D. ; Zhang Y. ; Li X. ; Li X. ; Teng H. ; James T. D. ; Li J. ; Guo Y. Stress Response Decay with Aging Visualized Using a Dual-Channel Logic-Based Fluorescent Probe. Chem. Sci. 2021, 12 , 13483–13491. 10.1039/D1SC04162B.34777768
Ren M. ; Wang L. ; Lv X. ; Sun Y. ; Chen H. ; Zhang K. ; Wu Q. ; Bai Y. ; Guo W. A Rhodol-Hemicyanine Based Ratiometric Fluorescent Probe for Real-Time Monitoring of Glutathione Dynamics in Living Cells. Analyst 2019, 144 , 7457–7462. 10.1039/c9an01852b.31710053
Dickinson B. C. ; Huynh C. ; Chang C. J. A Palette of Fluorescent Probes with Varying Emission Colors for Imaging Hydrogen Peroxide Signaling in Living Cells. J. Am. Chem. Soc. 2010, 132 , 5906–5915. 10.1021/ja1014103.20361787
Bai X. ; Yang B. ; Chen H. ; Shen J. ; Yang D. HKOCl-4: A Rhodol-Based Yellow Fluorescent Probe for the Detection of Hypochlorous Acid in Living Cells and Tissues. Org. Chem. Front. 2020, 7 , 993–996. 10.1039/D0QO00081G.
Toseland C. P. Fluorescent Labeling and Modification of Proteins. J. Chem. Biol. 2013, 6 , 85–95. 10.1007/s12154-013-0094-5.24432126
Hansen R. A. ; Märcher A. ; Gothelf K. V. One-Step Conversion of NHS Esters to Reagents for Site-Directed Labeling of IgG Antibodies. Bioconjugate Chem. 2022, 33 , 1811–1817. 10.1021/acs.bioconjchem.2c00392.
Grus T. ; Lahnif H. ; Klasen B. ; Moon E.-S. ; Greifenstein L. ; Roesch F. Squaric Acid-Based Radiopharmaceuticals for Tumor Imaging and Therapy. Bioconjugate Chem. 2021, 32 , 1223–1231. 10.1021/acs.bioconjchem.1c00305.
Poplinger D. ; Bokan M. ; Hesin A. ; Thankarajan E. ; Tuchinsky H. ; Gellerman G. ; Patsenker L. Ratiometric Fluorescence Monitoring of Antibody-Guided Drug Delivery to Cancer Cells. Bioconjugate Chem. 2021, 32 , 1641–1651. 10.1021/acs.bioconjchem.1c00205.
Bird R. E. ; Lemmel S. A. ; Yu X. ; Zhou Q. A. Bioorthogonal Chemistry and Its Applications. Bioconjugate Chem. 2021, 32 , 2457–2479. 10.1021/acs.bioconjchem.1c00461.
Peplow M. Click Chemistry Targets Antibody-Drug Conjugates for the Clinic. Nat. Biotechnol. 2019, 37 , 835–837. 10.1038/d41587-019-00017-4.31375794
Vatansever E. C. ; Kang J. ; Tuley A. ; Ward E. S. ; Liu W. R. An Optimal “Click” Formulation Strategy for Antibody-Drug Conjugate Synthesis. Bioorg. Med. Chem. 2020, 28 , 115808 10.1016/j.bmc.2020.115808.33071032
Petri L. ; Szijj P. A. ; Kelemen Á. ; Imre T. ; Gömöry Á. ; Lee M. T. W. ; Hegedűs K. ; Ábrányi-Balogh P. ; Chudasama V. ; Keserű G. M. Cysteine Specific Bioconjugation with Benzyl Isothiocyanates. RSC Adv. 2020, 10 , 14928–14936. 10.1039/D0RA02934C.35497170
Xu L. ; Zhang J. ; Yin L. ; Long X. ; Zhang W. ; Zhang Q. Recent Progress in Efficient Organic Two-Photon Dyes for Fluorescence Imaging and Photodynamic Therapy. J. Mater. Chem. C 2020, 8 , 6342–6349. 10.1039/D0TC00563K.
Xu L. ; Lin W. ; Huang B. ; Zhang J. ; Long X. ; Zhang W. ; Zhang Q. The Design Strategies and Applications for Organic Multi-Branched Two-Photon Absorption Chromophores with Novel Cores and Branches: A Recent Review. J. Mater. Chem. C 2021, 9 , 1520–1536. 10.1039/D0TC05910B.
Bussard K. M. ; Mutkus L. ; Stumpf K. ; Gomez-Manzano C. ; Marini F. C. Tumor-Associated Stromal Cells as Key Contributors to the Tumor Microenvironment. Breast Cancer Res. 2016, 18 , 84 10.1186/s13058-016-0740-2.27515302
Lee M. ; Downes A. ; Chau Y.-Y. ; Serrels B. ; Hastie N. ; Elfick A. ; Brunton V. ; Frame M. ; Serrels A. In Vivo Imaging of the Tumor and Its Associated Microenvironment Using Combined CARS / 2-Photon Microscopy. IntraVital 2015, 4 , e1055430 10.1080/21659087.2015.1055430.28243514
Benninger R. K. P. ; Piston D. W. Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues. Curr. Protoc. Cell Biol. 2013, 59 , 4–11. 10.1002/0471143030.cb0411s59.
Shaya J. ; Corridon P. R. ; Al-Omari B. ; Aoudi A. ; Shunnar A. ; Mohideen M. I. H. ; Qurashi A. ; Michel B. Y. ; Burger A. Design, Photophysical Properties, and Applications of Fluorene-Based Fluorophores in Two-Photon Fluorescence Bioimaging: A Review. J. Photochem. Photobiol., C 2022, 52 , 100529 10.1016/j.jphotochemrev.2022.100529.
Sóvári D. ; Kormos A. ; Demeter O. ; Dancsó A. ; Keserű G. M. ; Milen M. ; Ábrányi-Balogh P. Synthesis and Fluorescent Properties of Boroisoquinolines, a New Family of Fluorophores. RSC Adv. 2018, 8 , 38598–38605. 10.1039/C8RA08241C.35559080
Szepesi Kovács D. ; Hajdu I. ; Mészáros G. ; Wittner L. ; Meszéna D. ; Tóth E. Z. ; Hegedűs Z. ; Ranđelović I. ; Tóvári J. ; Szabó T. ; Szilágyi B. ; Milen M. ; Keserű G. M. ; Ábrányi-Balogh P. Synthesis and Characterization of New Fluorescent Boro-β-Carboline Dyes. RSC Adv. 2021, 11 , 12802–12807. 10.1039/D1RA02132J.35423835
Jancsó A. ; Kovács E. ; Cseri L. ; Rózsa B. J. ; Galbács G. ; Csizmadia I. G. ; Mucsi Z. Synthesis and Spectroscopic Characterization of Novel GFP Chromophore Analogues Based on Aminoimidazolone Derivatives. Spectrochim. Acta, Part A 2019, 218 , 161–170. 10.1016/j.saa.2019.03.111.
Kovács E. ; Cseri L. ; Jancsó A. ; Terényi F. ; Fülöp A. ; Rózsa B. ; Galbács G. ; Mucsi Z. Synthesis and Fluorescence Mechanism of the Aminoimidazolone Analogues of the Green Fluorescent Protein: Towards Advanced Dyes with Enhanced Stokes Shift, Quantum Yield and Two-Photon Absorption. Eur. J. Org. Chem. 2021, 2021 , 5649–5660. 10.1002/ejoc.202101173.
Csomos A. ; Kontra B. ; Jancsó A. ; Galbács G. ; Deme R. ; Kele Z. ; Rózsa B. J. ; Kovács E. ; Mucsi Z. A Comprehensive Study of the Ca2+ Ion Binding of Fluorescently Labelled BAPTA Analogues. Eur. J. Org. Chem. 2021, 2021 , 5248–5261. 10.1002/ejoc.202100948.
Chiovini B. ; Pálfi D. ; Majoros M. ; Juhász G. ; Szalay G. ; Katona G. ; Szőri M. ; Frigyesi O. ; Lukácsné Haveland C. ; Szabó G. ; Erdélyi F. ; Máté Z. ; Szadai Z. ; Madarász M. ; Dékány M. ; Csizmadia I. G. ; Kovács E. ; Rózsa B. ; Mucsi Z. Theoretical Design, Synthesis, and In Vitro Neurobiological Applications of a Highly Efficient Two-Photon Caged GABA Validated on an Epileptic Case. ACS Omega 2021, 6 , 15029–15045. 10.1021/acsomega.1c01164.34151084
Yuan L. ; Lin W. ; Yang Y. ; Chen H. A Unique Class of Near-Infrared Functional Fluorescent Dyes with Carboxylic-Acid-Modulated Fluorescence ON/OFF Switching: Rational Design, Synthesis, Optical Properties, Theoretical Calculations, and Applications for Fluorescence Imaging in Living Animals. J. Am. Chem. Soc. 2012, 134 , 1200–1211. 10.1021/ja209292b.22176300
Obukhova O. M. ; Mchedlov-Petrossyan N. O. ; Vodolazkaya N. A. ; Patsenker L. D. ; Doroshenko A. O. Stability of Rhodamine Lactone Cycle in Solutions: Chain–Ring Tautomerism, Acid–Base Equilibria, Interaction with Lewis Acids, and Fluorescence. Colorants 2022, 1 , 58–90. 10.3390/colorants1010006.
Xu L. ; Long X. ; He J. ; Liu L. ; Kang F. ; Deng Z. ; Wu J. ; Jiang X. F. ; Wang J. ; Zhang Q. Aggregation Effects on the One- and Two-Photon Excited Fluorescence Performance of Regioisomeric Anthraquinone-Substituted Perylenediimide. J. Mater. Chem. C 2023, 125 , 10.1039/d2tc03928a.
Khongorzul P. ; Ling C. J. ; Khan F. U. ; Ihsan A. U. ; Zhang J. Antibody–Drug Conjugates: A Comprehensive Review. Mol. Cancer Res. 2020, 18 , 3–19. 10.1158/1541-7786.MCR-19-0582.31659006
Bahou C. ; Richards D. A. ; Maruani A. ; Love E. A. ; Javaid F. ; Caddick S. ; Baker J. R. ; Chudasama V. Highly Homogeneous Antibody Modification through Optimisation of the Synthesis and Conjugation of Functionalised Dibromopyridazinediones. Org. Biomol. Chem. 2018, 16 , 1359–1366. 10.1039/C7OB03138F.29405223
Smith T. A. D. Towards Detecting the HER-2 Receptor and Metabolic Changes Induced by HER-2-Targeted Therapies Using Medical Imaging. Br. J. Radiol. 2010, 83 , 638–644. 10.1259/bjr/31053812.20675463
Kovács E. ; Rózsa B. ; Csomos A. ; Csizmadia I. G. ; Mucsi Z. Amide Activation in Ground and Excited States. Molecules 2018, 23 , 2859 10.3390/molecules23112859.30400217
Hudis C. A. Trastuzumab — Mechanism of Action and Use in Clinical Practice. N. Engl. J. Med. 2007, 357 , 39–51. 10.1056/nejmra043186.17611206
Yamashita K. ; Iwatsuki M. ; Yasuda-Yoshihara N. ; Morinaga T. ; Nakao Y. ; Harada K. ; Eto K. ; Kurashige J. ; Hiyoshi Y. ; Ishimoto T. ; Nagai Y. ; Iwagami S. ; Baba Y. ; Miyamoto Y. ; Yoshida N. ; Ajani J. A. ; Baba H. Trastuzumab Upregulates Programmed Death Ligand-1 Expression through Interaction with NK Cells in Gastric Cancer. Br. J. Cancer 2021, 124 , 595–603. 10.1038/s41416-020-01138-3.33100329
Héder M. ; Rigó E. ; Medgyesi D. ; Lovas R. ; Tenczer S. ; Török F. ; Farkas A. ; Emődi M. ; Kadlecsik J. ; Mező G. ; Pintér Á. ; Kacsuk P. The Past, Present and Future of the ELKH Cloud. Információs Társadalom 2022, 22 , 128 10.22503/inftars.xxii.2022.2.8.