Solid-phase approaches for labelling targeting peptides with far-red emitting coumarin fluorophores

Fluorophores based on organic molecules hold great potential for ligand-targeted imaging applications, particularly those operating in the optical window in biological tissues. In this work, we have developed three straightforward solid-phase approaches based on amide-bond formation or a Cu(I)-catalyzed azide-alkyne click (CuAAC) reaction for labeling an octreotide peptide with far-red emitting coumarin-based COUPY dyes. First, the conjugatable versions of COUPY fluorophores incorporating the required functional groups (e.g., carboxylic acid, azide, or alkyne) were synthesized and characterized. All of them were found fully compatible with Fmoc/ tBu solid-phase peptide synthesis, which allowed for the labeling of octreotide either through amide-bond formation or by CuAAC reaction. A near quantitative conversion was obtained after only 1 h of reaction at RT when using CuSO4 and sodium ascorbate independently of the click chemistry approach used (azido-COUPY/alkynyl-peptide resin or alkynyl-COUPY/azido-peptide resin). COUPY-octreotide conjugates were found stable in cell culture medium as well as noncytotoxic in HeLa cells, and their spectroscopic and photophysical properties were found similar to those of their parent coumarin dyes. Finally, the potential bioimaging applications of COUPY-octreotide conjugates were demonstrated by confocal microscopy through the visualization of living HeLa cells overexpressing the somatostatin subtype-2 receptor.


INTRODUCTION
Last few decades have witnessed an impressive growth in the development of novel therapeutic and diagnostic technologies against cancer. In this context, receptors overexpressed on cancer cells have been exploited to selectively deliver a large variety of cytotoxic drugs with the aim of minimizing toxic side-effects associated with chemotherapy.
Ligand-targeted imaging agents also offer great potential in the early detection of cancerous cells, as well as in fluorescence-guided surgery (FGS), which allows resection of solid tumours after illumination of malignancies directly in the operating room. 1 Recent advances in fluorophore chemistry and knowledge of targetable receptors have led to the clinical testing of several targeted fluorophores for intraoperative cancer detection and FGS. 1b,2 be tuned through the incorporation of CF 3 groups at the coumarin skeleton 6 or by replacement of N,N-dialkylamino groups at position 7 with the four membered ring azetidine, 6b the electron-withdrawing cyano group might also have a role in their photostability as previously demonstrated in some cyanine derivatives and in other dyes. 7 Considering the small size and the easy synthetic accessibility to COUPY dyes, these compounds are potential candidates for labelling targeting ligands such as receptor-binding peptides. In such a context, we envisaged that conjugatable versions of COUPY dyes (e.g., compounds 4 to 6 in Figure 1) could be obtained from scaffold 2 through N-alkylation with adequate reagents, thereby providing suitable functional groups (e.g., carboxylic acid, azide or alkyne) for conjugation via amidebond formation or copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC), respectively. 8 Herein, we report for the first time three straightforward solid-phase approaches for the conjugation of coumarin-based COUPY fluorophores to octreotide, a FDA-approved peptide that displays high affinity and selectivity for somatostatin receptors, mainly subtype-2 receptor (SSTR2) which is overexpressed on the membrane of various types of malignant cells. 9 This cyclic octapeptide is a promising candidate for developing novel targeted imaging agents since some derivatives, such as [ 111 In-DTPA]-and [ 90 Y-DOTA-Tyr3]-octreotide conjugates, are routinely used in the clinics for molecular imaging and therapy of neuroendocrine tumors, respectively, and several other SSTR2-targeted radiotherapeutics are currently under clinical evaluation. 1a Octreotide has also been conjugated successfully to

Synthesis and characterization of conjugatable COUPY dyes.
COUPY fluorophores 4-6 were easily synthesized through N-alkylation of 2 (Scheme 1), which was previously obtained by condensation of thiocoumarin 7 6,12 with 4pyridylacetonitrile. On the one hand, reaction of 2 with methyl bromoacetate afforded intermediate 8 in excellent yield after silica column chromatography (97%). This compound was transformed into coumarin 4 bearing the carboxylic acid function by acidic hydrolysis ( Figure S1). On the other hand, azido-(5) and alkynyl-(6) containing fluorophores were synthesized by reaction of 2 with N-alkyl bromoacetamide derivatives containing the appropriate functional groups for CuAAC (compounds 9 and 10, respectively). Compounds 5 and 6 were obtained as dark blue solids after purification (96% and 80% yields, respectively).
Full characterization was carried out by HR ESI-MS and 1D ( 1 H and 13 C) and 2D NMR, and the purity was assessed by reversed-phase HPLC ( Figure S1). Similarly to the parent COUPY dye 3, NOESY experiments revealed that the E rotamer was the major species in solution ( Figures S2-S4). 6 Scheme 1. Synthesis of conjugatable COUPY dyes 4-6.

Synthesis and characterization of COUPY-octreotide conjugates.
Having at hand the conjugatable COUPY derivatives (4-6), we focused on labelling octreotide following a stepwise solid-phase strategy since it allows the regioselective attachment of the fluorescent dye at the N-terminal end of the peptide sequence, either through amide-bond formation or by Cu(I)-catalyzed click chemistry. First, the linear octapeptide sequence incorporating a short polyethylenglycol spacer (11) was assembled manually on a Rink amide resin-p-MBHA using standard Fmoc-tBu methodology (Scheme 2). After coupling of coumarin 4 with HATU in the presence of DIPEA, side-chain deprotection and cleavage from the resin (TFA/TIS/H 2 O/EDT 94:2.5:2.5:1, 2.5 h RT) and cyclization via disulfide bond formation in an aqueous buffer (pH 7-8) were carried out. Analysis by HPLC-ESI MS showed a main peak ( Figure S5) that was isolated and characterized as the expected COUPYoctreotide conjugate (12). Finally, after purification by semipreparative HPLC and lyophilization, the formate salt of 12 (overall yield 12%) was obtained as a pink solid and fully characterized by HR ESI-MS and NMR. As shown in Figure S6  Once demonstrated the compatibility of COUPY dyes with solid-phase peptide synthesis (SPPS), we investigated the potential applications of coumarin derivatives bearing azide (5) and alkynyl (6) functional groups for labelling octreotide via CuAAC reaction on solid-phase, since click chemistry is routinely employed for modifying peptides, oligonucleotides, small molecules and polymers. 13 First, the required functional groups were incorporated at the Nterminal end of peptide-bound resin 11 by coupling 4-pentynoic acid or 2-azidoacetic acid, which provided alkynyl-(13) and azido- (14) peptide-bound resins, respectively, (Figures S9-S10). Click chemistry was investigated by reacting 13 and 14 resins with 5 and 6, respectively, in the presence of CuSO 4 (3 mol equiv.) and sodium ascorbate (3 mol equiv.) in DMF/H 2 O 20:1 for 18 h (Scheme 2). To our delight, after cleavage and deprotection, HPLC-MS analysis revealed the formation of the expected linear COUPY-octreotide conjugates.
Since no significant side reactions derived of the presence of sodium ascorbate and Cu(I) were detected, we decided not to explore the use of Cu-stabilizing ligands. Although most click chemistry procedures reported in the literature describe the use of long reaction times (12-48 h) and even microwave irradiation for labelling peptides with organic fluorophores, 13a we obtained near quantitative conversions after only 1 h of reaction at RT (see Figures S11 and S14 for the HPLC-MS analyses after 1 h, 4 h and overnight reaction times), independently of the click chemistry approach used (azido-COUPY/alkynyl-peptide resin or alkynyl-COUPY/azido-peptide resin). Finally, after cyclization and purification, clicked COUPY-octreotide conjugates (15 and 16) were obtained as pink solids (Figures S13-S14 and S15-S16).

Photophysical characterization of COUPY-octreotide conjugates.
The photophysical properties (absorption and emission spectra, molar absorption coefficients (), and fluorescence quantum yields ( F )) of COUPY-octreotide conjugates were studied in water and in PBS buffer, and compared with those of their respective coumarin precursors (see Table 1 and Figure 3 and Figures S17-S20). All the compounds showed an intense absorption band in the yellow-red part of the visible spectrum, being the wavelength absorption maximum slightly red-shifted with respect the parent dye 3 (e.g.,  abs = 543 nm for 3 vs  em = 555 nm for 5-6 and 8 in H 2 O) because of the additional electron-withdrawing effect of ester and amide functions. Similarly, the emission maximum was red-shifted by ca 10 nm (e.g.,  em = 605 nm for 3 vs  em = 615 nm for 5-6 and 8 in H 2 O) and, consequently, the Stokes's shifts remained around 60-62 nm. An additional red-shift in absorption (ca 4-6 nm) and emission (ca 3-5 nm) maxima occurred after conjugation to octreotide. As shown in Table 1, the  F of the conjugatable coumarin derivatives (8 and 5-6) in aqueous media was reduced when compared with 3 (e.g.,  F = 0.066 for 5 vs  F = 0.15 for 3 in H 2 O). However, a clear improvement in the fluorescent quantum yield of these fluorophores was achieved when conjugated to the peptide (e.g.,  F = 0.066 for 5 vs  F = 0.19 for conjugate 15 in H 2 O), independently of the chemical conjugation approach used.

Fluorescence imaging of COUPY-octreotide conjugates in living cells
Finally, we investigated the potential bioimaging applications of COUPY-octreotide conjugates. As a representative compound, we selected conjugate 12 and studied its cellular uptake by confocal microscopy in SSTR2-overexpressing HeLa cells after irradiation with a yellow light laser (λ ex = 561 nm). Interestingly, fluorescent vesicles, mostly-like endosomes, were visible in the cytoplasm of all of the examined cells after 30 min of incubation with 12, thereby confirming the internalization and accumulation of COUPY-octreotide conjugate in the cells (Figures 4 and S23). This pattern of staining contrasts with that of coumarin 3 ( Figure S23), which accumulates preferentially in mitochondria and nucleoli, 6 and indicates that the internalization of COUPY-octreotide conjugate is driven exclusively by the peptide moiety and not by the coumarin tag. In order to get more insights into the cellular uptake of the conjugate, we incubated HeLa cells with 12 at 4 o C for 30 min. As shown in Figure 5, no staining was observed inside the cytoplasm, which confirms that 12 enters the cells only through an energy-dependent pathway. By contrast, the pattern of staining observed with 3 was not modified at low temperature ( Figure 5), thereby suggesting internalization through simple passive diffusion. Next, we decided to compare the visualization ability of COUPY dyes when conjugated to octreotide with that of two common commercially available fluorophores: 5(6)carboxyfluorescein, which is one of the most popular fluorescent tags for labelling peptides and is typically excited at 488 nm, and Atto-Rho12, a rhodamine dye that can be excited with the same yellow light laser than our COUPY fluorophore. Atto-Rho12-octreotide (17) was synthesized by reaction of the corresponding succinimidyl ester derivative with octreotide and carboxyfluorescein-octreotide (18) was prepared by SPPS. 10b As shown in Figure 4, the performance of the COUPY dye when conjugated to octreotide was comparable to that of the rhodamine dye when exciting at 561 nm under similar conditions. By contrast, COUPY fluorophore allowed a much better visualization of HeLa cells than carboxyfluorescein, even at much lower concentrations and with a more convenient excitation wavelength (561 nm vs 488 nm). On the other hand, it is worth noting that conjugates involving carboxyfluorescein and Atto-Rho12 dyes (17 and 18) were obtained as regiomeric mixtures since both commercially available fluorophores are supplied as mixtures of isomers. However, COUPYoctreotide conjugate 12 was easily obtained as a single product, which represents a suitable alternative to many conventional fluorophores when labeled biomolecules with a well-defined structure are required for biological applications. Moreover, it is important to note that the photostability of COUPY-octreotide conjugate (12) in PBS buffer under green light irradiation (505 ± 15 nm; Figure 6) was found similar to that of the parent coumarin 3, 6 which indicates that peptide derivatization through the pyridine heterocycle does not significantly modify the photostability of COUPY dyes.

CONCLUSIONS
In conclusion, we have synthesized three conjugatable versions of COUPY dyes (compounds 4 to 6) incorporating suitable functional groups for conjugation via amide-bond formation (e.g., carboxylic acid) or copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction (e.g., azide or alkyne). The compounds were easily obtained from a cheap, commercially available precursor, 7-N,N-diethylaminocoumarin, in only three or four linear steps, being the N-alkylation of the pyridine ring the key step. All the conjugatable coumarin derivatives were found fully compatible with Fmoc/tBu solid-phase peptide synthesis, which allowed the straightforward labeling of octreotide peptide with a far-red emitting fluorophore. On the one hand, attachment of the coumarin 4 bearing a carboxylic acid function to the N-terminal end of the linear peptide-bound resin followed by acidic cleavage and deprotection and cyclization led to the expected COUPY-octreotide conjugate (12). This conjugate was found stable in cell culture medium as well as non-cytotoxic in HeLa cells. On the other hand, clicked COUPYoctreotide conjugates (15 and 16) were efficiently obtained by CuAAC reaction on solidphase between azide-(5) and alkynyl- (6) containing COUPY dyes and peptide-bound resins containing the complementary functional groups.
The spectroscopic and photophysical properties of COUPY-octreotide conjugates were found similar to those of their parent coumarin dyes, being the wavelength absorption maximum located in the yellow-red part of the visible spectrum, while the emission ranged from the far red to the NIR region. Importantly, the fluorescent quantum yields of COUPY-octreotide conjugates in water were found higher than those of their coumarin precursors. Finally, the potential bioimaging applications of COUPY-octreotide conjugates were demonstrated by confocal microscopy in SSTR2-overexpressing living HeLa cells. Both the pattern of staining and the inhibition of the cellular uptake at low temperature indicate that the internalization of the conjugates is driven by the peptide moiety and not by the coumarin tag. Moreover, the fact that the visualization ability of COUPY dyes when conjugated to octreotide was similar to that of a commercially available rhodamine fluorophore, Atto-Rho12, and much better than that of the 5(6)-carboxyfluorescein, makes them as a suitable alternative when labeled biomolecules with a well-defined structure are required for biological applications. Work is in progress in our laboratory to increase the red-shifted properties of COUPY dyes with the aim of using them in ligand-targeted imaging applications such as fluorescence-guided surgery.

Materials and Methods
Unless otherwise stated, common chemicals and solvents (HPLC grade or reagent grade quality) were purchased from commercial sources and used without further purification.
Fmoc-protected amino acids, resins, and coupling reagents for solid-phase synthesis were obtained from Novabiochem, Bachem, or Iris Biotech. Fmoc-L-threoninol p-carboxyacetal was synthesized following previously reported procedures. 14 Solid-phase peptide synthesis  (9). The published method with some modifications was followed to synthesize compound 9. 15 3-Azido-1-propanamine (250 mg, 2.5 mmol) was dissolved in 10 mL of DCM and then 10 mL of saturated aqueous NaHCO 3 were added. The mixture was vigorously stirred at -10 ºC and bromoacetyl bromide (1.01 g, 5.0 mmol) was slowly added. The reaction mixture was slowly allowed to warm to room temperature. After stirring for 3 h, the reaction mixture was partially concentrated to remove the organic solvent and then poured into 100 mL of water. The aqueous phase was extracted with AcOEt (2 x 100 mL), and the combined organic phases were washed with saturated NaHCO 3 (100 mL), 5 % aqueous HCl solution (100 mL) and saturated NaCl (100 mL (10). The published method with some modifications was followed to synthesize compound 10. 16 Propargylamine (500 mg, 9.1 mmol) was dissolved in 20 mL of DCM and then 20 mL of saturated aqueous NaHCO 3 were added. The mixture was vigorously stirred at -10 ºC and bromoacetyl bromide (3.66 g, 18.2 mmol) was slowly added. The reaction mixture was slowly allowed to warm to room temperature. After stirring for 3 h, the reaction mixture was partially concentrated to remove the organic solvent and then poured into 100 mL of water. The aqueous phase was extracted with AcOEt (2 x 100 mL), and the combined organic phases were washed with saturated NaHCO 3 (100 mL), 5 % aqueous HCl solution (100 mL) and saturated NaCl (100 mL
COUPY-octreotide conjugate (12). After removal of the Fmoc protecting group from peptidebound resin 11 with 20 % piperidine in DMF (2 x 10 min), coumarin 4 (4 mol equiv.) was coupled by using HATU (3.9 mol equiv.) and DIPEA (2+2 mol equiv.) in anhydrous DMF for 3 h in the dark by using the following procedure: DIPEA (2 mol equiv.) was first added to a solution of coumarin 4 and HATU in anhydrous DMF and, after stirring for 5 min a RT, the mixture was added to DMF-swollen peptide-bound resin 11; then, DIPEA (2 mol equiv.

Photophysical characterization of the compounds.
Absorption spectra were recorded in a Varian Cary 500 UV/Vis/NIR spectrophotometer at room temperature. Molar absorption coefficients () were determined by direct application of the Beer-Lambert law, using solutions of the compounds in each solvent with concentrations ranging from 10 −6 to 10 −5 M. Emission spectra were registered in a Photon Technology International (PTI) fluorimeter. Fluorescence quantum yields (Φ F ) were measured by comparative method using cresyl violet in ethanol (CV;  F;Ref = 0.54 ± 0.03) as reference. 17 Then, optically-matched solutions of the compounds and CV were excited and the fluorescence spectra was recorded. The absorbance of sample and reference solutions was set below 0.1 at the excitation wavelength and  F were calculated using the following equation

Fluorescence imaging
All microscopy observations were performed using a Zeiss LSM 880 confocal microscope equipped with a 405 nm laser diode, an Argon-ion laser, a 561 nm laser and a 633 nm laser.
The microscope was also equipped with a full enclosure imaging chamber (XLmulti S1, Pecon) connected to a 37 ºC heater and a 5% CO 2 providing system. Cells were observed using a 63X 1.2 multi immersion objective. Compounds 3, 12 and 17 were excited using the 561 nm laser and detected from 570 to 670 nm. Compound 18 was observed using the 488 nm laser line of the Argon-ion laser. Image analysis was performed using Fiji. 18 Unless otherwise stated images are colorized using Fire lookup table.

Supporting Information
Copies of HPLC traces and UV−vis absorption and fluorescence emission spectra of the compounds; additional fluorescence imaging studies; 1D NMR ( 1 H, 13 C, and 19 F), MS, and selected 2D NMR spectra.
This material is available free of charge via the Internet at http://pubs.acs.org.