Synthesis of fluorinated curcumin derivatives for detecting amyloid plaques by 19F-MRI

Organic & Biomolecular Chemistry PAPER Cite this: Org. Biomol. Chem., 2024, 22, 5948 Received 6th May 2024, Accepted 2nd July 2024 DOI: 10.1039/d4ob00730a rsc.li/obc Synthesis of fluorinated curcumin derivatives for detecting amyloid plaques by 19 F-MRI† Sebastiano Micocci,‡ a Rachele Stefania, *‡ b Francesca Garello, a Umberto Fasoglio, a Ivan Hawala, a Lorenzo Tei, b Simonetta Geninatti Crich a and Giuseppe Digilio b The most prominent pathophysiological hallmark of Alzheimer’s disease is the aggregation of amyloid-β (Aβ) peptides into senile plaques. Curcumin and its derivatives exhibit a high affinity for binding to Aβ fibrils, effectively inhibiting their growth. This property holds promise for both therapeutic applications and diagnostic molecular imaging. In this study, curcumin was functionalized with perfluoro-tert-butyl groups to create candidate molecular probes specifically targeted to Aβfibrils for use in 19 F-magnetic resonance imaging. Two types of fluorinated derivatives were considered: mono-substituted (containing nine fluorine atoms per molecule) and disubstituted (containing eighteen fluorine atoms). The linker connecting the perfluoro moiety with the curcumin scaffold was evaluated for its impact on binding affinity and water solubility. All mono-substituted compounds and one disubstituted compound exhibited a binding affinity toward Aβfibrils on the same order of magnitude as reference curcumin. The insertion of a charged carboxylate group into the linker enhanced the water solubility of the probes. Compound Curc-Glu-F9 (with one L-glutamyl moiety and a perfluoro-tert-butyl group), showed the best properties in terms of binding affinity towards Aβfibrils, water solubility, and intensity of the 19 F-NMR signal in the Aβ oligomer bound form. Introduction Alzheimer’s disease (AD) is characterized by a progression from episodic memory problems to a slow global decline in cognitive function. The scientific research in the field is focused on the investigation of a simple and accurate way to detect Alzheimer’s before these devastating symptoms begin. Cerebral amyloid-beta (Aβ) accumulation and aggregation is the primary event in AD pathogenesis. 1a,b It has been proposed that the rest of the disease process, including the formation of neurofibrillary tangles containing tau protein, results from an imbalance between Aβproduction and Aβclearance. 2a,b Accordingly, estimating the level of Aβdeposition in the brain would be informative for early diagnosis of AD and for evaluating AD progression. Among the various aggregates that Aβcan generate, oligomers gather during the early phases of Aβaggregation and have the most neurotoxic effects. 2a,3 Larger aggregates, such as protofibrils and fibrils, can fragmentate, releasing them and acting as sinks. In order to achieve early detection of Aβoligomers and protofibrils, many researchers have tried to develop chemical probes that have a specific affinity for Aβaggregates. Compounds able to bind selectively with high affinity the Aβaggregates in vitro and in vivo are derivatives based on highly conjugated aromatic systems, such as thioflavin T, Congo red, chrysamine G, benzoxazoles, curcumin, and stilbenes. 4 Among them, curcumin is of great interest because it is food-derived and shows a superior safety profile. Curcumin is a low molecular weight yellow-orange pigment derived from the turmeric plant with numerous pharmacological properties including anti-tumor, anti-oxidative, anti-inflammatory, hepatoprotective, nephroprotective, and anti-amyloid effects. Several studies have reported that curcumin has a high binding affinity to Aβaggregates and inhibits the aggregation. 5 Structurally, curcumin contains two methoxyphenol rings linked by a conjugated dieneβ-dicarbonyl backbone. Curcumin is a potentially great scaffold to develop probes for Aβdiagnostic imaging and/or therapy because of its blood–brain barrier (BBB) permeability, high-affinity binding to senile plaques, and low toxicity. 6 Curcumin is also reported to reduce Aβaggregates in Alzheimer’s transgenic mice. 7 Considerable progress in Aβ †Electronic supplementary information (ESI) available. See DOI: https://doi.org/ 10.1039/d4ob00730a ‡These authors contributed equally. a Department of Molecular Biotechnology and Health Sciences, University of Turin, Via Nizza 52, 10126 Torino, Italy b Department of Science and Technological Innovation, University of Eastern Piedmont “Amedeo Avogadro”, Viale Teresa Michel 11, 15120 Alessandria, Italy. E-mail: r[email protected] 5948 |Org. Biomol. Chem.,2024,22,5948–5959 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 03 July 2024. Downloaded on 7/26/2024 4:53:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue imaging has been achieved in recent years. 8 Currently, positron emission tomography (PET) is the most efficient imaging modality to detect Aβdeposition because of its high sensitivity and the ability to quantify the accumulated probe. Pittsburgh compound B ([ 11 C]PiB), which is a derivative of thioflavin (ThT), was the first successful Aβ-selective PET radioligand; 9 then, different 18 F-labeled Aβ-targeting derivatives emerged to overcome the 20 min radioactive decay half-life limitation of 11 C-PiB. 10,11a,b Magnetic resonance imaging (MRI) is another important and widely clinically used diagnostic technique that provides detailed anatomical information with excellent soft tissue contrast, and it is amenable to molecular imaging applications provided that suitable MRI probes are available. MRI probes are typically based on paramagnetic contrast agents, but diamagnetic probes carrying the MR active 19 F nuclei are attracting a growing interest because of several advantages: first, the 19 F nucleus has a high gyromagnetic ratio and a natural isotopic abundance of 100%, hence an MR sensitivity approaching that of 1 H. Second, biological tissues contain essentially no 19 F: only negligible amounts of endogenous fluorine are embedded in the teeth and bone matrix of the human body. Therefore, the introduction of exogenous 19 F signals in vivo will yield background-free images. Third, 19 F MRI exhibits relatively high spatial resolution. In addition, it is worth noting that 19 F is a naturally occurring halogen and a stable, non-radioactive isotope of fluorine. Thus, unlike the radioactive isotope 18 F commonly used in PET imaging, incorporating fluorine into a probe is synthetically smoother. Yanagisawa et al. developed the first perfluoro curcumin analogue, FMeC1, 12 for 19 F MRI to facilitate visualization of Aβ in vivo. FMeC1, named then Shiga-Y5, containing two trifluoromethoxy groups in place of the methoxy ones and a methylpropanoate moiety on the C4 position (Fig. 1), could cross the blood–brain barrier and bind to Aβplaques in a transgenic mouse model of AD after injection via the tail vein. They also developed and investigated several 19 F-containing curcumin analogue, called the Shiga-Y series, with different moieties at the C4 position; 13 among them, Shiga-Y25 (Fig. 1) with a short PEG chain ending with a trifluoromethoxy group successfully detected Aβdepositions in the brain of a living mouse. 14 All developed probes contained a limited number of 19 F atoms/ molecule, typically six/molecule, except the case of Shiga-Y25 which contains nine 19 F atoms. This work aims at synthesizing a series of novel 19 F-containing curcumins with a high number of equivalent 19 F nuclei, suitably spaced from the aromatic part of the molecule. Furthermore, we present a novel synthetic approach that involves direct modification of the OH group attached to the phenol moiety of curcumin to produce novel 19 F curcumin derivatives (Curc-C 3 -F9,Curc-C 6 -F9,Curc-Glu-F9,Curc-C 6 -F18, Curc-Glu-F18). The affinity of the novel derivatives for Aβ fibrils was evaluated in vitro by a fluorescence-based assay, and the 19 F-NMR properties in the fibril-bound state were investigated in vitro. Results and discussion Design of the 19 F-curcumin imaging probes Several aspects must be kept into account when designing curcumin-based 19 F-MRI probes targeted to Aβfibrils. The enol form of these compounds must be preserved as it shows preference for binding to Aβfibrils, 15 whereas the keto form favours the binding to Aβoligomers. 16 Another important parameter to consider is the hydrophilicity/hydrophobicity balance of the fluorinated probe for an efficient crossing of the BBB. 17 Finally, the highest achievable sensitivity to 19 F-MRI detection must be pursued by introducing in the molecule as many as possible magnetically equivalent fluorine atoms. A common drawback of fluorinated molecular probes is that their 19 F-NMR linewidth can be significantly affected by binding interactions. If the re-orientational motions of the perfluoroalkyl moieties are restricted in the bound state, a significant line broadening of the 19 F-NMR signal would arise, leading ultimately to a dramatic loss of the 19 F-MRI signal. Based on these observations, we have designed and synthesized a range of novel curcumin derivatives that contain nine or eighteen equivalent 19 F atoms. To counteract potentially detrimental line-broadening effects, the perfluorinated alkyl groups were linked to the curcumin structure through flexible linkers, such to preserve local re-orientational freedom also in the bound-state. These linkers varied in length and hydrophilicity. Specifically, we have developed three monosubstituted curcumin derivatives, one with a short aliphatic chain (Curc-C 3 -F9), one with a longer aliphatic chain (Curc-C 6 -F9), and one with a spacer containing a carboxylic group suitable to improve the solubility (Curc-Glu-F9, Fig. 2). The conjugation reactions occur on the hydroxyl groups (4-OH) attached to the phenyl rings of natural product of curcumin. The monosubstituted compounds maintain one curcumin phenolic group, which is known to be important for interaction with fibrils. 18 We also synthesized bis-functionalized derivatives to maximize the number of 19 F atoms per molecule. These probes were assessed for the best compromise between water solubility, fibril targeting ability, and sensitivity to 19 F-MRI detection.Fig. 1 Structure of curcumin, Shiga-Y5, Shiga Y51, Shiga-Y25. Organic & Biomolecular Chemistry Paper This journal is © The Royal Society of Chemistry 2024 Org. Biomol. Chem.,2024,22,5948–5959 | 5949 Open Access Article. Published on 03 July 2024. Downloaded on 7/26/2024 4:53:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Synthesis of the fluorinated curcumin imaging probes The synthesis of the probes started by preparing the perfluorinated amines, characterized by the presence of the nonafluoro-tert-butyloxy tail, using the condensation under Mitsunobu conditions 19 of the readily available 6-(Boc-amino)- 1-hexanol or 3-(Boc-amino)-1-propanol with perfluoro-tertbutanol (Scheme 1). The corresponding perfluoro-tert-butyl ethers were obtained, from which the Boc group was removed in acidic conditions, using a 1 : 1 mixture of trifluoroacetic acid (TFA) in dichloromethane (CH 2 Cl 2 ). In order to improve the solubility of the final perfluoro curcumin derivative, we also designed a perfluorinated amine containing a carboxylate moiety: thus, compound F9-C 3 -NH 2 with the shorter chain was conjugated to Boc-L-glutamic acid 5-tert-butyl ester (Boc-Glu (OtBu)-OH) using DCC/DMAP approach. Then, compound F9Glu-NH 2 was obtained as a trifluoroacetate salt after the deprotection of the Boc group with TFA. While curcumin is naturally derived, its derivatives like those containing 19 F in its structure are generally produced by a chemical reaction between by acetylacetone and its derivatives with appropriate aryl-aldehydes. This assembly method can yield multiple chemical analogues, such as compounds with trifluoromethoxy groups on the benzene ring and alkyl substituents on the middle carbon of the linker (C4 position). 15,20 The functionalization at C4 can be performed by a Michael addition starting from curcumin. 21 Here, the synthesis of perfluoro curcumin derivatives MRI probes was carried out in four steps starting from commercial Curcuma longa powder (C1386, Merck), which is composed of curcumin (77%), demethoxycurcumin (17%), and bisdemethoxycurcumin (3%). Conjugation reactions take place on one or two hydroxyl groups of the phenyl rings of curcumin to synthesize monoor bifunctional nonafluoro derivatives in a facile synthetic route. The reaction of curcumin with 0.5 equivalents of t-butyl bromoacetate in the presence of potassium carbonate as base led to the formation of mono-tBu ester derivative and bis-tBu ester derivative as by-product (compound 1a and compound 1b, Scheme 2). The monofunctionalized curcumin was isolated, after purification by column chromatography on silica gel, with about 30% yield. The bis-functionalized derivative was also collected (10% yield) and used for further functionalization to evaluate its binding to the Aβaggregates and to compare with the mono-functionalized derivatives. Then, the t-Bu esters were deprotected with TFA and the mono and bis-carboxylic acid curcumin derivatives (compound 2a and 2b, respectively) were reacted with EDC/NHS in order to obtain the mono and bis-N-hydroxysuccinimide esters (Curcmono-NHS and Curc-bis-NHS, compounds 3a and 3b). The final 19 F-MRI probes were obtained by amide coupling reaction between the NHS-activated esters of curcumin and the perfluorinated amine (F9-C 3 -NH 2 ,F9-C 6 -NH 2 and F9-Glu-NH 2 )in a mixture of CH 3 CN and a phosphate buffer at pH 7.5, at room temperature (Scheme 2). The bis-NHS curcumin 3b was also conjugated to F9-C 6 -NH 2 and F9-Glu-NH 2 to afford final bisperfluorinated derivatives with a higher number of 19 F nuclei (Curc-C 6 -F18 and Curc-Glu-F18) (Scheme 2). The compounds were then purified by RP-HPLC and characterized by UPLC-UV-MS(ESI+) and NMR spectroscopy. The characteristic 1 H, 19 F, 2D 1 H, 1 H-COSY, 2D 1 H, 13 C-HSQC, 2D 1 H, 13 C-HMBC NMR (600 MHz, DMSO-d 6 , 300 K) and 19 F NMR (500 MHz, ethanol, 300 K) of all 19 F-curcumin imaging probes reported are presented as ESI,†as well as the UPLC-UV-MS(ESI+) chromatogram. The complete 1 H NMR chemical shift assignment of Curc-C 3 -F9 dissolved in DMSO-d 6 is shown in Fig. 3. The peak at 6.13 ppm corresponds to the proton on the α-carbon in the keto–enol tautomer (H1 in Fig. 3). Moreover, the methoxy, the aromatic and the conjugated methyne protons of the two sides of the molecule are not magnetically equivalent confirming the asymmetry of the structure. For all compounds, the purity was found to be between 96 and 98% as measured by UPLC at λ= 220 nm and λ= 413 nm. Binding activity of curcumin derivatives to the amyloid-beta (Aβ) fibrils Aβaggregates in protofibrillar and fibrillar state were prepared by incubating Aβ1–42 peptide (50 μM) in phosphate buffer 10 mM (pH 7.4) containing 11 mM NaCl, for 4 days at 37 °C under stirring. 22 The formation of Aβaggregates was checked by measuring Thioflavin-T (ThT) fluorescence enhancement during fibril formation at 37 °C under stirring at 600 rpm (see Fig. 2 Structure of 19 F MRI curcumin-based probes synthesized in this work. Scheme 1 Synthesis of perfluoroamine derivatives: (i) nonafluoro-tertbutyl alcohol, PPh 3 , DIAD, Et 2 O, (ii) TFA, CH 2 Cl 2 , (iii) Boc-Glu(OtBu)-OH, DCC, DMAP, CH 2 Cl 2 , (iv) TFA, CH 2 Cl 2 (1 : 1). Paper Organic & Biomolecular Chemistry 5950 |Org. Biomol. Chem.,2024,22,5948–5959 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 03 July 2024. Downloaded on 7/26/2024 4:53:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Fig. 4A). ThT exhibits a significant shift in the excitation maximum (from 385 nm to 450 nm) and the emission maximum (from 445 nm to 482 nm) due to its binding to Aβ. 23 ThT is an effective indicator of fibrillization, as confirmed by morphological analysis using field emission scanning electron microscopy (FESEM) (Fig. 4B). These experiments showed that aggregation and maturity of the fibrils was optimal after 5 days incubation under our experimental conditions. Curcumin and its fluorinated derivatives, at the concentrations used, generate negligible fluorescence emission in the range 488–497 nm. In the absence of fibrils (Fig. 5, dashed line, λ ex = 450 nm). The binding of curcumin and its Scheme 2 Synthesis of mono and bis-perfluorinated curcumin derivatives: (i) t-butyl bromoacetate, K 2 CO 3 in CH 3 CN; (ii) TFA/CH 2 Cl 2 (1 : 1); (iii) EDC/NHS, 5 mol% DMAP in NMP; (iv) F9-C 3 -NH 2 or F 9 -C 6 -NH 2 or F9-Glu-NH 2 ,buffer phosphate (0.1 M, pH = 7.5), CH 3 CN. Fig. 3 1 H-NMR spectrum of compound Curc-C 3 -F9 (DMSO-d 6 , 300 K). Organic & Biomolecular Chemistry Paper This journal is © The Royal Society of Chemistry 2024 Org. Biomol. Chem.,2024,22,5948–5959 | 5951 Open Access Article. Published on 03 July 2024. Downloaded on 7/26/2024 4:53:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online derivatives to mature Aβfibrils is known to be characterised by a steep increase of their fluorescence emission 24a,b and also by a red shift of the absorption maximum. 25a,b In our conditions, the maximum fluorescence emission of curcumin derivatives in the presence of Aβfibrils was found using an excitation wavelength of 450 nm. This excitation wavelength was used in binding titrations, where a fixed amount of a curcumin derivative was incubated with increasing amounts of Aβfibrils. The fluorescence emission at around 490 nm was plotted as a function of the fibril concentration to obtain binding isotherms (Fig. 6), from which the binding affinity K a for Aβcould be extracted by computer aided best fitting to eqn (1) (Experimental section). The obtained thermodynamic association constants K a , assuming a 1 : 1 interaction with Aβ,are listed in Table 1. The affinity constant K a for curcumin obtained by this assay is in line with that reported in the literature. 26 All fluorinated compounds showed a good affinity for fibrils, with K a in the range (1.1–3.9 × 10 5 M −1 ), except Cur-C 6 -F18. The latter compound showed a very low fluorescence increase which prevented the calculation of its affinity constant. This could be likely due to the very poor solubility of the compound in aqueous medium (see below). While the extension of the spacer between curcumin and the perfluoro-tert-butyl ether from C 3 to C 6 does not affect the affinity, the presence of a carboxylic group, which is negatively charged at neutral pH, slightly reduces the K a of Curc-Glu-F9 derivative. Although it is known that at least one phenolic group in the aromatic portion of the structure is needed to preserve binding to the fibril beta-sheets layer, we found that the disubstituted Curc-Glu-F18 (having no such phenolic group) had a binding affinity in the same order of magnitude of the monosubstituted Curc-Glu-F9 counterpart. We might speculate that the two amide groups within each of the linkers of Curc-GluFig. 4 (A) ThT fluorescence enhancement at different times of incubation (37 °C, under stirring, [Aβ] 300 nM, [ThT] 50 nM, ex. 450 nm, em. 476 nm). (B) FESEM images of Aβaggregates (5 days incubation) at 15k× and 100k× magnification, probe set at 100 pA and the electron beam energy at 5 keV. Fig. 5 Fluorescence emission spectra (λ ex = 450 nm) as a function of aggregated Aβ: (A) Curc-C 3 -F9, 22.0 nM; (B) Curc-C 6 -F9, 25.6 nM; (C) Curc-Glu-F9, 20.8 nM; (D) Curc-Glu-F18, 28.3 nM; (E) Curc-C 6 -F18, 25.0 nM; (F) curcumin, 21.0 nM. Fig. 6 Plot of the fluorescence emission (λ ex = 450 nm) of curcumin and its derivatives: (A) Curc-C 3 -F9,λ em = 497 nm, R 2 = 0.99957; (B) Curc-C 6 -F9,λ em = 495 nm, R 2 = 0.99836; (C) Curc-Glu-F9,λ ex = 495 nm, R 2 = 0.99334; (D) Curc-Glu-F18,λ em = 488 nm, R 2 = 0.992; (E) Curc-C 6 -F18,λ ex = 488 nm; (F) curcumin, λ em = 495 nm, R 2 = 0.91. Titrations were done in triplicate. Error bars correspond to ±SD of the mean values. Paper Organic & Biomolecular Chemistry 5952 |Org. Biomol. Chem.,2024,22,5948–5959 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 03 July 2024. Downloaded on 7/26/2024 4:53:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online F18 may provide suitable hydrogen bonding capabilities to form a suitable hydrogen bond network with the fibril. Binding to human serum albumin (HSA) For an initial assessment of the binding selectivity, we chose serum albumin as a model protein offering multiple potential binding sites for the fluorinated compounds, each site having different binding properties. It has been reported that the curcumin fluorescence emission in the 450–490 nm range (with λ ex at 430 nm) strongly increases when bound to the hydrophobic pockets of HSA. 26 To this purpose the fluorescence emission at 485 nm was plotted as a function of the HSA concentration, assuming a 1 : 1 interaction (see ESI, section 3†). As expected, reference curcumin showed specific binding to HSA with an affinity constant K a of 5.9 × 10 5 M −1 , in line with the value reported in the literature. 27 The monosubstituted CurcC 6 -F9 and Curc-Glu-F9 showed a very low binding to HSA as demonstrated by the low fluorescence increase compared to the autofluorescence of HSA alone at the same concentrations. The only compound showing a binding affinity for HSA was Curc-Glu-F18, with a binding affinity constant higher than that of reference curcumin (K a = 4.2 × 10 6 M −1 ). Solubility of fluorinated curcumin derivatives All fluorinated curcumin derivatives exhibit pronounced hydrophobicity, especially the bis-functionalized Curc-C 6 -F18 derivative, which does not dissolve in any of the potentially injectable formulations prepared for in vivo use. The very low solubility in aqueous medium is due to the presence of the central curcuminoid body, consisting of a conjugated alkenyl and aromatic components, as well as the transformation of the curcumin –OH groups into alkyl ethers terminating with three bulky and hydrophobic –CF 3 groups. To achieve a suitable solubility and stability of the solutions in water, addition of CREMOPHOR EL, a non-ionic surfactant derived from castor oil, was necessary. The solubility properties of the compounds at 10 mg mL −1 in DMSO, MeOH, and HEPES buffer with the addition of 10% and 20% CREMOPHOR are shown in Table 2. The curcumin derivatives that gain a negative charge at physiological pH (Curc-Glu-F9 and Curc-Glu-F18) have been shown to effectively enhance water solubility. 19 F-NMR study Compounds Curc-C 6 -F9,Curc-Glu-F9, and Curc-Glu-F18, which showed an acceptable solubility in the hepes/cremophor biocompatible medium, were selected for 19 F-MRI sensitivity assessment. All these compounds show a single 19 F-NMR singlet falling 4–5 ppm upfield relative to TFA (see ESI section 2.14†). In MRI signal acquisition, the relaxation times T 1 and T 2 of 19 F nuclei play a crucial role, as they determine both the signal-to-noise ratio achievable in the image and the acquisition times of the imaging experiment. Indeed, high values of T 1 (greater than 1 s) require long acquisition times, while too small values of T 2 (less than 100 ms) result in signal loss due to both line-broadening and dead times of the acquisition sequence. The T 1 and T 2 values for the single 19 F resonance of the synthesized products are reported in Table 3. The relaxation times are within the suitable range for 19 F-MRI. Indeed, T 1 values around 500 ms allow for the acquisition of a reasonable number of images in a short time without saturating the signal, while T 2 values slightly above 100 ms are enough to avoid appreciable signal loss during spin-echo trains. Accordingly, 19 F MR-images show a good signal-to-noise and the expected proportionality of the 19 F-MRI signal intensity to the number of fluorine atoms present in each compound (Table 3 and Fig. 7). Next, we tried to acquire the 19 F-NMR signal in the presence of amyloid fibrils. The critical issue in this kind of measurement is that our model of amyloid fibrils is incompatible with the surfactant needed to solubilize the probe. Therefore, experiments were carried out without the surfactant with compound Curc-Glu-F9, which showed the best compromise between binding affinity to fibrils, selectivity and water solubility. In phosphate buffer (without any surfactants) Curc-Glu-F9 (50 μM) yielded a barely detectable signal (Fig. 8A), because the compound aggregates into supramolecular structures having a heterogeneous distribution of molecular sizes and leading to a substantial line-broadening. In the presence of monomers or oligomers at 50 μM concentration, a 19 F-NMR Table 1 Molar fluorescence intensities of the Aβbound compounds (F b ) and association constants (K a ) Sample F b ± SD (CPS mA −1 M −1 )K a ±SD(M −1 ) Curc-C 3 -F9 5.5 × 10 13 ± 1.1 × 10 13 3.8 × 10 5 ± 0.9 × 10 5 Curc-C 6 -F9 3.2 × 10 13 ± 3.2 × 10 13 3.9 × 10 5 ± 0.8 × 10 5 Curc-Glu-F9 1.7 × 10 14 ± 0.4 × 10 14 1.1 × 10 5 ± 0.9 × 10 5 Curc-Glu-F18 3.5 × 10 13 ± 2.9 × 10 13 2.1 × 10 5 ± 0.2 × 10 5 Curc-C 6 -F18 n.d. n.d. Curcumin 9.7 × 10 14 ± 7.4 × 10 14 2.6 × 10 5 ± 1.3 × 10 5 Table 2 Solubility of the synthesized compounds in DMSO, methanol and HEPES + cremophor a at 10 mg mL −1 Sample DMSO MEOH HEPES + cremophor 10% HEPES + cremophor 20% Curc-C 6 -F9 ++—+ Curc-C 6 -F18 ++—— Curc-Glu-F9 +++ + Curc-Glu-F18 +++ + a The LD 50 of cremophor surfactant was verified in the literature 28 and a significantly smaller amount was used in the tests. Table 3 19 F-NMR relaxation times and 19 F-MRI signal-to-noise ratio (SNR) in phantoms (at 7 T and 25 °C) of fluorinated curcumin derivatives at a concentration of 4.5 mM T 1 (ms) T 2 (ms) SNR Curc-C 6 -F9 HEPES + cremophor 20% 610 126 13.7 Curc-Glu-F9 HEPES + cremophor 10% 558 139 11.4 Curc-Glu-F18 HEPES + cremophor 10% 523 102 22.0 Organic & Biomolecular Chemistry Paper This journal is © The Royal Society of Chemistry 2024 Org. Biomol. Chem.,2024,22,5948–5959 | 5953 Open Access Article. Published on 03 July 2024. Downloaded on 7/26/2024 4:53:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online signal at −70.6 ppm (linewidth about 25 Hz) became clearly detectable (Fig. 8B and C). In such experimental conditions, the theoretically expected fraction of fibril-bound probe would be 80%. In the presence of mature fibrils, the signal disappears (Fig. 8D). The increase of 19 F-NMR signal intensity in the presence of monomers/oligomers is explained in terms of the establishment of a binding equilibrium between the aggregated, NMR invisible form of the fluorinated compound and an NMR detectable form where Curc-Glu-F9 is bound to amyloid monomers/oligomers. In the presence of mature fibrils, such adducts have a large molecular size, leading to massive 19 F-NMR line broadening which in turn hampers the detection of the 19 F-NMR signal. These results indicate that, under the in vitro assay conditions, Curc-Glu-F9 can detect Aβmonomers/oligomers rather than mature fibrils. Soluble Aβoligomers are toxic to neurons and are believed to be major contributors to synaptic dysfunction and neuronal death observed in Alzheimer’s disease. Detection and quantification of soluble Aβand tau oligomers in cerebrospinal fluid (CSF) may serve as pre-symptomatic biomarkers. 29a,b This approach to study the binding interaction has limitations, as the in vitro conditions may not mimic properly those likely met in a physiological environment. For instance, fibril-bound forms may exist in equilibrium with forms bound to the hydrophobic components of the ECM rather than in equilibrium with self-aggregated forms. In vivo studies with murine models of brain deposition of amyloid fibrils are awaited to gain further insights about the NMR properties of the 19 F-NMR signal, and to assess whether the high number of fluorine atoms is paralleled by a proportional increase of signal intensity. As a matter of fact, such kind of studies for the compounds of the Shiga-family were carried out directly by in vivo measurements. 12,14,16 Conclusions The use of 19 F-MRI to detect amyloid fibrils in the brain presents several challenges. The main hurdle is the limited sensitivity of the 19 F-MRI technique, eventually requiring a high concentration of fluorine-19 nuclei in the imaging voxel (in the order of millimoles per litre), under a realistic clinical scenario, 30 and a suitably small signal linewidth. We successfully synthesized new curcumin derivatives substituted with one or two perfluoro-tert-butyl groups, containing 9 or 18 equivalent fluorine atoms respectively. Amongst these compounds, the monosubstituted Curc-Glu-F9 (i) retained a high binding affinity towards Aβfibrils; (ii) had an acceptable water solubility in formulation with surfactants; and (iii) showed a detectable 19 F-NMR signal in the form bound to Aβoligomers in a preliminary in vitro assay. In vivo studies are needed to determine if the large number of equivalent fluorine atoms per molecule will result in a corresponding increase in 19 F-MRI signal intensity in a more realistic brain imaging setting. Experimental General synthetic methods All reagents were purchased by Sigma Aldrich (Darmstad, Germany) and solvents by VWR International (Radnor, USA) and were used without further purifications. Column chromatographic separations were performed using silica gel (VWR International) with a particle size of 0.040–0.063 mm. Preparative HPLC-MS were carried out on a Waters AutoPurification system (3100 Mass Detector, 2545 Pump Gradient Module, 2767 Sample Manager, and 2998 PDA detector). UPLC analysis was performed using a UPLC Acquity H-Class coupled with the QDa and TUV detectors, using Kinetex® F5 column, 1.7 μm, 2.1 × 100 mm, applying a gradient of CH 3 CN (0.05% TFA) in H 2 O (0.05% TFA) from 50% to 100% in 8 min and 100% of B in 4 min (0.2 mL min −1 ), peak area revealed at 210 nm and 430 nm (method 1). All compounds are >95% pure by HPLC. NMR spectra were recorded at 310 K on a Bruker AVANCE 600 MHz and a Bruker Avance Neo 500 MHz spectrometer. Fig. 7 1 H, 19 F, and merged MRI of (a) Curc-C 6 -F9, (b) Curc-Glu-F9, (c) and Curc-Glu-F18 solubilized at 4.5 mM in HEPES/NaCl buffer + cremophor. The normalized 19 F signal intensity scale is reported in the calibration bar. The central cone is to provide an appropriate volume for shimming. Fig. 8 19 F-NMR spectrum of Curc-Glu-F9: (A) without Aβ, (B) with Aβ monomers, (C) with soluble Aβoligomers and (D) with mature fibrils. Asterisks denote TFA (*) and hexafluoro-2-propanol (HFIP, **) as contaminants. Paper Organic & Biomolecular Chemistry 5954 |Org. Biomol. Chem.,2024,22,5948–5959 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 03 July 2024. Downloaded on 7/26/2024 4:53:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Synthesis of mono-NHS and bis-NHS esters of curcumin Synthesis of 1a and 1b. 1.0 g of curcumin (2.7 mmol) was dissolved in 100 mL of acetonitrile and 0.2 g (1.4 mmol) of sodium sulfate and 0.7 g (5 mmol) of potassium carbonate were added to the solution. Then, 0.2 mL (1.3 mmol) of t-butyl bromoacetate in 5 mL CH 3 CN were added slowly dropwise to the mixture which was stirred under reflux and argon atmosphere for 2 h. The reaction was cooled to room temperature, all salts were filtered and the solvent was evaporated. The crude product was purified by flash chromatography (silica gel column, petroleum ether/ethyl acetate 8 : 2 to 1 : 1) to give 1a (0.34 g, yield 30%) and 1b (0.53 g, yield 19%) as a yellow oil. Compound 1a: 1 H NMR (DMSO-d 6 ), δ(ppm): 9.70 (s, 1H, phenolic-OH), 7.60 (d, 2H, J= 15.8 Hz, H4/H4′), 7.41 (s, 1H, H6), 7.41 (s, 1H, H6′), 7.27 (d, 1H, J= 8.0 Hz, H10), 7.20 (d, 1H, J= 8.2 Hz, H10′), 6.93 (d, 1H, J= 8.3 Hz, H9), 6.88 (d, 1H, J= 15.8 Hz, H3), 6.86 (d, 1H, J= 8.3 Hz, H9′), 6.80 (d, 1H, J= 15.9 Hz, H3′), 6.13 (s, 1H, H1), 4.76 (s, 2H, H12), 3.89 (s, 3H, H20), 3.88 (s, 3H, H20′), 1.47 (s, 9H, tBu) (see ESI section 2.1†). Direct infusion mass analysis with methanol/water 9 : 1 v : v at 0.2 mg mL −1 : ESI-MS (m/z) calcd: for C 27 H 30 O 8 [M + H] + 483.19 found: 483.35. Compound 1b: 1 H NMR (DMSO-d 6 ), δ(ppm): 7.61 (d, 2H, J= 15.9 Hz, H4/H4′), 7.30 (s, 2H, H6/H6′), 7.27 (d, 2H, J= 8.4 Hz, H10/H10′), 6.94 (d, 2H, J= 8.4 Hz, H9, H9′), 6.87 (d, 2H, J= 15.9 Hz, H3/H3′), 6.16 (s, 1H, H1), 4.72 (s, 4H, H12/ H12′), 3.88 (s, 6H, H20/H20′), 1.46 (s, 18H, tBu). Direct infusion mass analysis with methanol/water 9 : 1 v : v at 0.2 mg mL −1 : ESI-MS (m/z): calcd: for C 33 H 40 O 10 [M + H] + 597.26 found: 597.37. Synthesis of 2a and 2b. 10 mL of TFA were added at room temperature to a solution of 0.28 g (0.58 mmol) of 1a in 3 mL CH 2 Cl 2 . After 4 h, the solution was concentrated in vacuo and the product was precipitated adding Et 2 O (20 mL); the resulting yellow solid was centrifuged (6000 rpm, 30 min) and then washed with Et 2 O (20 mL × 3), collected and dried in vacuo to give 2a (0.22 g, yield 88%) as a yellow powder. Compound 2b was obtained by performing the same procedure starting from 0.3 g (0.5 mmol) of 1b (0.37 g, yield 84%). Compound 2a: UPLC-UV (λ= 220 nm, 430 nm): t R 2.05 min, 98% purity. 1 H-NMR (DMSO-d 6 ), δ(ppm): 16.33 (exch br s, 1H, H11), 13.06 (exch br s, 1H, COOH), 9.67 (s, 1H, phenolic-OH), 7.60 (d, 2H, J= 15.9 Hz, H4/H4′), 7.41 (s, 1H, H6), 7.36 (s, 1H, H6′), 7.26 (d, 1H, J= 8.1 Hz, H10), 7.20 (d, 1H, J= 8.3 Hz, H10′), 6.94 (d, 1H, J= 8.1 Hz, H9), 6.87 (d, 1H, J= 15.9 Hz, H3), 6.86 (d, 1H, J= 8.3 Hz, H9′), 6.80 (d, 1H, J= 15.9 Hz, H3′), 6.13 (s, 1H, H1), 4.78 (s, 2H, H12), 3.88 (s, 3H, H20), 3.87 (s, 3H, H20′). 13 C NMR (DMSO-d 6 ), δ(ppm): 184.4, 183.2, 170.5, 150.0, 149.8, 149.7, 148.6, 141.6, 140.7, 128.8, 126.9, 123.8, 123.0, 122.9, 121.7, 116.3, 113.5, 112.0, 111.6, 101.5, 65.5, 56.3. ESI-MS (m/z): calcd: for C 23 H 22 O 8 [M + H] + 427.13 found: 427.33; [M + Na] + 449.12 found: 449.34 (see ESI section 2.2†). Compound 2b: UPLC-UV (λ= 220 nm, 254 nm): t R 2.50 min, 93% purity. 1 HNMR (DMSO-d 6 ), δ(ppm): 7.62 (d, 2H, J= 15.9 Hz, H4/H4′), 7.41 (s, 2H, H6/H6′), 7.27 (d, 2H, J= 8.4 Hz, H10/ H10′), 6.94 (d, 2H, J= 8.4 Hz, H9, H9′), 6.88 (d, 2H, J= 15.9 Hz, H3/H3′), 6.16 (s, 1H, H1), 4.77 (s, 4H, H12/H12′), 3.88 (s, 6H, H20/H20′). 13 C NMR (DMSO), δ(ppm): 184.9, 172.6, 151.5, 151.2, 141.8, 131.2, 123.9, 123.8, 115.4, 112.5, 67.0, 56.8. ESI-MS (m/z): calcd: For C 25 H 24 O 10 [M + H] + 485.14 found: 485.26 (see ESI section 2.3†). Synthesis of 3a and 3b. 0.36 g of 2a (0.84 mmol) were dissolved in 1 mL of N-methyl pyrrolidone (NMP). EDC (0.24 g, 1.26 mmol), NHS (0.14 g, 1.26 mmol) and 5 mol% DMAP were added to the solution. The reaction mixture was stirred for 2 h at room temperature. The product was precipitated adding Et 2 O (30 mL), the resulting yellow solid was centrifuged (6000 rpm, 30 min) and washed with Et 2 O (20 mL × 3) and EtOAc (20 mL × 3) and then collected and dried. For compound 2b:3 equivalents of EDC and NHS were used starting from 0.2 g (0.4 mmol) of compound 2b. The products were used directly for the next step without further purification. Direct infusion mass analysis with methanol/water 9 : 1 v : v at 0.2 mg mL −1 : Compound 3a: ESI-MS (m/z): calcd: for C 27 H 25 NO 10 [M + H] + 524.15 found: 524.26; [M + Na] + 546.49; Compound 3b: ESI-MS (m/z): calcd: for C 33 H 30 N 2 O 14 [M + H] + 679.17 found: 679.37; [M + Na] + 701.61. Synthesis of F9-C 3 -NH 2 and F9-C 6 -NH 2 3-(Boc-amino)-1-propanol (0.37 g, 2.1 mmol) or 6-(Boc-amino)- 1-hexanol (0.46 g, 2.1 mmol) and triphenylphosphine (0.66 g, 2.52 mmol) in Et 2 O (16 mL) were added to an ice cooled and stirred solution of nonafluoro-tert-butyl alcohol (0.5 g, 2.1 mmol) in Et 2 O (8 mL). After 5 min, a solution of diisopropyl azodicarboxylate (DIAD, 0.55 g, 2.73 mmol) in Et 2 O (5 mL) was added during 15 min. Then, the ice bath was removed and the mixture stirred at RT for 24 h. The solid precipitate was removed by filtration and the filtrate evaporated. The crude product was purified by chromatography (silica gel column, DCM/MeOH 98 : 2) to afford Boc-HN-C 3 -F9 or Boc-HN-C 6 -F9 as a yellow oil (52% and 32%, respectively). For the deprotection of the Boc-group, 0.3 g of Boc-perfluoroamine were dissolved in CH 2 Cl 2 (2 mL) and cooled to 0 °C. 2 mL of TFA were added and the solution was allowed to warm to room temperature. After stirring at room temperature until starting material was consumed (TLC monitoring) the solution was concentrated in vacuo (ca. 80%). Boc-NH-C 3 -F9: 1 H-NMR (CDCl 3 , 600 MHz) δ 1.48 (s, 9H), 1.94 (t, 2H, J= 5.92), 3.29 (m, 2H), 4.13 (t, 2H, J= 5.96). ESI-MS (m/z): calcd: for C 12 H 16 F 9 NO 3 [M + H] + 394.10 found: 394.14. F9-C 3 -NH 2 : 1 H-NMR (MeOD, 600 MHz) δ2.12 (m, 2H), 3.10 (m, 2H), 4.28 (t, 2H, J= 5.93), 6.87 (m, 3H) (see ESI section 2.4†). ESI-MS (m/z): calcd: for C 7 H 8 F 9 NO [M + H] + 294.05 found: 294.13. Boc-NH-C 6 -F9: 1 H-NMR (CDCl3, 600 MHz) δ1.39 (m, 2H), 1.46 (m, 2H), 1.50 (s, 9H), 1.54 (m, 2H), 1.73 (m, 2H), 3.16 (t, 2H), 4.05 (t, 2H). 13 C-NMR (CDCl 3 , 600 MHz) δ24.32, 25.60, 27.67, 28.88, 29.22, 39.71, 68.99, 116.76, 118.72, 120.66, 122.58, 155.26. ESI-MS (m/z): calcd: for C 15 H 22 F 9 NO 3 [M + H] + 436.15 found: 436.15. F9-C 6 -NH 2 : 1 H-NMR (MeOD 600 MHz) δ1.51(m, 4H), 1.71 (m, 2H), 1.79 (m, 2H), 2.97 (m, 2H), 4.15 (t, 2H, J= 5.94) (see ESI section 2.5†). 13 C-NMR (MeOD, 600 MHz) δ23.91, 24.92, 26.30, 28.43, Organic & Biomolecular Chemistry Paper This journal is © The Royal Society of Chemistry 2024 Org. Biomol. Chem.,2024,22,5948–5959 | 5955 Open Access Article. Published on 03 July 2024. Downloaded on 7/26/2024 4:53:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 38.50, 69.17, 116.80, 118.72, 120.67, 122.62, 159.03. ESI-MS (m/ z): calcd: for C 10 H 14 F 9 NO [M + H] + 336.09 found: 336.27. Synthesis of F9-Glu-NH 2 Boc-L-glutamic acid 5-tert-butyl ester (0.18 g, 0.6 mmol) was dissolved in 10 mL of CH 3 CN and DIPEA (0.18 g, 1.4 mmol) and HATU (0.23 g, 0.7 mmol) were added. After 5 min, F9-C 3 - NH 2 (0.2 g, 0.7 mmol) dissolved in CH 3 CN (1 mL) was added dropwise and then the mixture was stirred at room temperature for 6 h under N 2 atmosphere. The solvent was then removed under vacuum and the residue was dissolved in CH 2 Cl 2 (10 mL) and washed three times with brine (2 × 10 mL) and water (2 × 10 mL) and the separated organic phases were dried with anhydrous Na 2 SO 4 . Then, the crude product obtained by evaporation of the solvent was purified by chromatography (silica gel column, petroleum ether/ethyl acetate 7 : 3) to give F9-C 3 -Glu(OtBu)-NH-Boc as a pale yellow oil (55%). 1 H-NMR (CDCl 3 , 600 MHz) δ1.49 (s, 9H), 1.51 (s, 9H), 1.88 (m, 1H), 1.98 (m, 2H), 2.19 (m, 1H), 2.30 (m, 2H), 3.43 (m, 2H), 4.16 (m, 3H), 5.31 (s, 1H), 6.63 (s, 1H). 13 C-NMR (CDCl 3 , 600 MHz) δ27.16, 27.56, 28.85, 29.21, 31.85, 35.78, 52.64, 67.62, 79.05, 81.67, 116.33, 118.58, 120.64, 122.51, 155.43, 170.70, 171.73. ESI-MS (m/z): calcd: for C 21 H 31 F 9 N 2 O 6 [M + H] + 579.20 found: 579.36. For the deprotection of the Boc and tertbutyl groups, 0.2 g of F9-C 3 -Glu(OtBu)-NH-Boc were dissolved in CH 2 Cl 2 (2 mL) and cooled to 0 °C; then, 2 mL of TFA were added and the solution was allowed to warm to room temperature. After stirring at room temperature overnight the solution was concentrated in vacuo to obtain F9-C 3 -Glu-NH 2 in 92% yield. 1 H-NMR (DMSO-d 6 , 600 MHz) δ1.85 (m, 2H), 2.02 (m, 2H), 2.32 (m, 2H), 3.18 (m, 2H), 3.97 (m, 1H), 4.13 (t, 2H, J= 6.41 Hz), 8.03 (t. 1H, J= 5.33 Hz), 8.27 (s, 2 H) (see ESI section 2.6†). 13 C-NMR (CDCl 3 , 600 MHz) δ28.05, 30.36, 32.86, 36.58, 54.74, 64.52, 116.24, 118.42, 172.40, 174.83. ESI-MS (m/z): calcd: for C 12 H 15 F 9 N 2 O 4 [M + H] + 423.09 found: 423.18. Synthesis of monosubstituted curcumin derivatives (Curc-C 3 - F9, Curc-C 6 -F9, Curc-Glu-F9) A solution of mono-NHS ester of curcumin (compound 3a, 0.38 mmol) in acetonitrile (4 mL) was slowly added at room temperature to a solution of perfluoroamine (F9-C 3 -NH 2 ,F9C 6 -NH 2 ,F9-Glu-NH 2 ) (0.38 mmol) dissolved in sodium phosphate buffer (0.1 M, pH 7.5, 4 mL) and CH 3 CN (4 mL). The biphasic mixture was allowed to stir vigorously for 2 h. Then the acetonitrile was evaporated under reduced pressure and the aqueous phase was washed with dichloromethane (3 × 100 mL). The organic phase was dried with anhydrous Na 2 SO 4 , and, after filtration, the solvent was evaporated to give yellow solids. The solids were then purified by preparative HPLC by using a Water XTerra™Prep RPdC8 19/100 column, applying a gradient of CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) from 50% to 100% in 15 min (20 mL min −1 ). The pure products were obtained as yellow powders. The purity of the compounds was determined by UPLC using method 1. Curc-C 3 -F9: (0.17 g). Yield: 62%, t R 5.80 min, 98% purity. 1 H-NMR (600 MHz, DMSO-d 6 , 300 K), δppm: 9.69 (s, 1H, phenolic-OH), 8.08 (t, J= 5.6 Hz, 1H, H14), 7.61 (d, J= 15.9 Hz, 2H, overlapping H4/H4′), 7.43 (d, J= 1.9 Hz, 1H, H6), 7.37 (d, J = 1.9 Hz, 1H, H6′), 7.27 (dd, J= 8.3 and 1.9 Hz, 1H, H10), 7.20 (dd, J= 8.2 and 1.9 Hz, 1H, H10′), 6.97 (d, J= 8.3 Hz, 1H, H9), 6.88 (d, 15.9 Hz, 1H, H3), 6.87 (d, J= 8.2 Hz, 1H, H9′), 6.81 (d, J= 15.9 Hz, 1H, H3′), 6.13 (s, 1H, H1), 4.57 (s, 2H, H12), 4.13 (t, J= 6.2 Hz, 2H, H17), 3.90 (s, 3H, H20), 3.88 (s, 3H, H20′), 3.27 (q, J= 6.2 Hz, 2H, H15), 1.88 (m, J= 6.2 Hz, 2H, H16) (see ESI section 2.7†for details and NMR assignment). 19 F-NMR (470 MHz, ethanol/DMSO-d 6 550 : 50, 298 K), δppm (relative to TFA −76.55 ppm): −71.71 (s). 13 C-NMR (150 MHz, δppm from 2D HSQC and 2D HMBC, DMSO-d 6 , 300 K): 184.4 (C2′), 182.8 (C2), 168.0 (C13), 150.0 (overlapping C7, C8, C8′), 148.5 (C7′), 141.4 (C4′), 140.3 (C4), 129.2 (C5), 126.7 (C5′), 123.7 (C10′), 123.0 (C3), 122.7 (C10), 121.6 (C3′), 116.3 (C9′), 114.3 (C9), 111.8 (C6′), 111.5 (C6), 101.3 (C1), 68.8 (C17), 68.6 (C12), 56.2 (overlapping C20, C20′), 35.2 (C15), 29.8 (C16). ESI-MS (m/z): calcd: for C 30 H 28 F 9 NO 8 [M + H] + 702.17 found: 702.19; [M + Na] + 724.33. Curc-C 6 -F9: (0.15 g). Yield: 52%, t R 6.58 min, 98% purity. 1 H-NMR (600 MHz, DMSO-d 6 , 300 K), δppm: 9.70 (s, 1H, phenolic-OH), 7.95 (t, J= 5.7 Hz, 1H, H14), 7.60 (d, J= 15.8 Hz, 2H, overlapping H4/H4′), 7.43 (d, J= 1.9 Hz, 1H, H6), 7.36 (d, J = 1.9 Hz, 1H, H6′), 7.27 (dd, J= 8.4 and 1.9 Hz, 1H, H10), 7.20 (dd, J= 8.4 and 1.9 Hz, 1H, H10′), 6.97 (d, J= 8.4 Hz, 1H, H9), 6.88 (d, J= 15.8 Hz, 1H, H3), 6.86 (d, J= 8.4 Hz, 1H, H9′), 6.80 (d, J= 15.8 Hz, 1H, H3′), 6.12 (s, 1H, H1), 4.56 (s, 2H, H12), 4.09 (t, J= 6.2 Hz, 2H, H20), 3.90 (s, 3H, H23), 3.88 (s, 3H, H23′), 3.15 (q, J= 6.2 Hz, 2H, H15), 1.66 (m, J= 6.4 Hz, 2H, H19), 1.46 (m, J= 7.4 Hz, 2H, H16), 1.37 (m, J= 7.4 Hz, 2H, H18), 1.295 (m, J= 7.4 Hz, 2H, H17) (see ESI section 2.8†for details and NMR assignment). 19 F-NMR (470 MHz, ethanol/ DMSO-d 6 550 : 50, 298 K), δppm (relative to TFA −76.55 ppm): −71.79 (s). 13 C-NMR (150 MHz, δppm from 2D HSQC and 2D HMBC, DMSO-d 6 , 300 K): 184.4 (C2′), 182.7 (C2), 167.7 (C13), 149.8 (overlapping C7, C8, C8′), 148.7 (C7′), 141.5 (C4′), 140.2 (C4), 129.2 (C5), 126.7 (C5′), 123.7 (C10′), 123.0 (C3), 122.9 (C10), 121.6 (C3′), 116.2 (C9′), 114.4 (C9), 111.8 (C6′), 111.5 (C6), 101.4 (C1), 70.7 (C20), 68.5 (C12), 56.15 (overlapping C23, C23′), 38.6 (C15), 29.55 (C19), 29.3 (C16), 26.3 (C17), 24.9 (C16). ESI-MS (m/z): calcd: for C 33 H 34 F 9 NO 8 [M + H] + 744.21 found: 744.28; [M + Na] + 766.25. Curc-Glu-F9: (0.08 g). Yield: 34%, t R 4.13 min, 97% purity. 1 H-NMR (600 MHz, DMSO-d 6 , 300 K), δppm: 9.70 (s, 1H, phenolic-OH), 8.24 (br, 1H, H14), 7.92 (t, J= 5.6 Hz, 1H, H19), 7.60 (d, J= 15.8 Hz, 2H, overlapping H4/H4′), 7.43 (d, J= 1.6 Hz, 1H, H6), 7.36 (d, J= 1.6 Hz, 1H, H6′), 7.26 (dd, J= 8.4 and 1.6 Hz, 1H, H10), 7.20 (dd, J= 8.4 and 1.6 Hz, 1H, H10′), 7.01 (d, J = 8.3 Hz, 1H, H9), 6.88 (d, J= 15.8 Hz, 1H, H3), 6.86 (d, J= 8.4 Hz, 1H, H9′), 6.80 (d, J= 15.8 Hz, 1H, H3′), 6.13 (s, 1H, H1), 4.64 (AB system, 2H, H12), 4.28 (m, br, 1H, H15), 4.11 (t, J= 6.2 Hz, 2H, H22), 3.90 (s, 3H, H26), 3.88 (s, 3H, H26′), 3.15 (q, J= 6.6 Hz, 2H, H20), 2.16 (m, 2H, H17), 2.06 (m, 1H, H16a), 1.87 (m, 1H, H16b), 1.81 (m, J= 6.5 Hz, 2H, H21) (see ESI section 2.9†for details and NMR assignment). 19 F-NMR (470 MHz, ethanol/DMSO-d 6 550 : 50, 298 K), δppm (relative to Paper Organic & Biomolecular Chemistry 5956 |Org. Biomol. Chem.,2024,22,5948–5959 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 03 July 2024. Downloaded on 7/26/2024 4:53:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online