Acid‐Resistant BODIPY Amino Acids for Peptide‐Based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets

Abstract The G protein‐coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid‐resistant BODIPY‐based amino acid (Trp‐BODIPY PLUS), and its implementation for solid‐phase synthesis of fluorescent bioactive peptides. Trp‐BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn‐on fluorescence emission upon target binding for wash‐free imaging. Finally, we employed Trp‐BODIPY PLUS to prepare some of the first fluorogenic kisspeptin‐based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.


Materials and Methods
Chemicals were obtained from commercial sources and were used without further purification.Fmoc-Trp-BODIPY-OH was synthesized as described elsewhere. [2]actions were monitored by HPLC-MS analysis using a HPLC Agilent 1200 System comprising a Kinetex C 18 column (5 µm, 100

Fmoc-Trp-BODIPY PLUS
Fmoc-Trp-BODIPY-OH (15.0 mg, 0.020 mmol) was weighted in a 10 mL round bottom flask and the system was purged with N 2 .The amino acid was dissolved in 4.0 mL dry DCM followed by the addition of BF 3 OEt 2 (24.7 µL, 0.20 mmol, 10 eq) and trimethylsilyl cyanide (52.3 µL, 0.40 mmol, 20 eq).The reaction was stirred at room temperature for S19 reaction was then quenched with H 2 O (7.5 mL), followed by DCM removal with N 2 .
The crude was extracted with AcOEt (10 mL) and the organic layer was washed with brine (x3).The combined organic layers were dried over anhydrous magnesium sulfate, filtrated, and concentrated under rotatory evaporator.Purification was conducted by semi-preparative HPLC using a 50-95% gradient over 17 min with detection at 254 and 500 nm.Pure fractions were collected and lyophilized to afford pure compound as a red solid (8.9 mg, 58%).General procedures for SPPS [3] Amino acids were obtained from Sigma-Merck, Cambridge Bioscience and Iris Biotech.DIC was obtained from Sigma-Aldrich.Rink Amide and chlorotrityl polystyrene resins were obtained from Rapp Polymer GmbH and Novabiochem, respectively.Unless otherwise indicated, peptides were manually synthesized in 2-10 mL polystyrene syringes fitted with porous polyethylene discs using common Fmoc-S20 SPPS protocols.Solvents, excess of reagents and soluble by-products were removed by suction.The Fmoc group was removed with piperidine/DMF (1:4) (1×1 min, 2×5 min), followed by DMF (×5), and DCM (×5) washes.All syntheses were carried out at r.t.Peptides bearing fluorescent moieties were always protected from light.
Resin loading for 2-chlorotrityl polystyrene resin.The first amino acid was loaded onto the resin using DIPEA (3.0 eq.) in DCM for 10 min followed by additional DIPEA (7.0 eq.) for extra 40 mins.MeOH (0.8 μL mg -1 resin) was added to cap remaining trityl groups.The resin was then filtered and washed using DCM (5×1 min) and DMF (5×1 min).The loading of the resin was determined by measuring the absorbance of piperidine-dibenzofulvene adduct at 290 nm.
Cleavage from resin for peptides 4-5 and 7-8.The peptide was cleaved from the resin using 95% TFA, 2.5% TIS 2.5% H 2 O for 1 h and washed with DCM (4×1 min).The combined filtrates were collected into a round bottom flask and concentrated under reduced pressure.

H-Trp(BODIPYplus)-Lys-Tyr-Arg-Ala-Glu-NH2 (peptide 4)
The synthesis was performed on 70 mg of Rink Amide resin (0.18 mmol g -1 ).Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH and Fmoc-Glu(tBu)-OH were used as side-chain protected building blocks and their incorporation was carried on a Liberty Blue microwave peptide synthesizer (CEM).For that, DIC and OxymaPure reagents were used for each amide coupling and 20% piperidine in DMF was employed for the removal of Fmoc protecting groups.After cleavage as described above, the crude peptide was precipitated by adding cold Et 2 O (dropwise) and the resulting precipitate was decanted and dried (×2).Purification was conducted by semi-Preparative HPLC using a 5-50% gradient over 17 min, with detection at 254 and 500 nm.Pure fractions were collected and lyophilized to afford peptide 4 as a red solid (3.4 mg, 23% yield).

cyclo(Gly-Arg-Lys-Lys-Trp-Phe-Trp(BODIPYplus)) (peptide 6)
The synthesis was performed on 40 mg of 2-chlorotrityl polystyrene resin (0.46 mmol g -1 ).Fmoc-Gly-OH, Fmoc-Arg(Boc) 2 -OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp-OH and Fmoc-Phe-OH were used as building blocks.After cleavage as described above, the peptide crude was precipitated by adding cold Et 2 O (dropwise) and the resulting solid was decanted and dried to afford 24 mg of protected peptide (75% yield).23 mg of cleaved peptide (1.0 eq) were then dissolved in DMF (0.026 M) with COMU (1.1 eq) and DIEA (2.5 eq) and the resulting mixture was stirred for 1 h at r.t.The peptide crude was purified by semi-Preparative HPLC using a 60-100% gradient over 25 min, with detection at 254 and 500 nm.The peptide was dissolved in TFA:DCM:DTT (95:2.5:2.5) for 1 h to remove the side-chain protecting groups, followed by precipitation in cold Et 2 O (dropwise) and lyophilization to afford pure peptide 6 as an orange solid (2.2 mg, 13% yield from cyclization step).

H-Tyr-Asn-Trp(BODIPYplus)-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH2 (peptide 7)
The synthesis was performed on 56 mg of Rink Amide resin (0.18 mmol g -1 ).Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Leu-OH and Fmoc-Arg(Pbf)-OH were used as side-chain protected building blocks.Incorporation of the residues prior to Trp(BODIPY) PLUS were carried on a Liberty Blue microwave peptide synthesizer (CEM).For that, DIC and OxymaPure reagents were used for each amide coupling and 20% piperidine in DMF was employed for the removal of Fmoc protecting groups.After cleavage as described above, the crude peptide was precipitated by adding cold Et 2 O (dropwise) and the resulting precipitate was decanted and dried (x2).Purification was conducted by semi-Preparative HPLC using a 5-60% gradient over 17 min, with detection at 254 and 500 nm.Pure fractions were collected and lyophilized to afford peptide 7 as an orange solid (3.3 mg, 20% yield).

HRMS (ESI+
DIPEA in NMP (2 M) were added to the resin, with subsequent reaction for 40 min at r.t.Coupling of Fmoc-NH-PEG 4 -OH (2 eq.) was carried out using HBTU (2 eq.) and DIPEA (4 eq.) in DMF for 1.5 h.Coupling of Trp-BODIPY PLUS (1.5 eq.) was carried with HBTU (1.5 eq.) and DIPEA (4 eq.) in DMF for 1.5 h.After cleavage as described above, the crude peptide was precipitated by adding cold Et 2 O (dropwise) and the resulting precipitate was decanted and dried (x3).Purification was conducted by semi-Preparative HPLC using a 0-100% gradient over 25 min, with detection at 254 nm.
Pure fractions were collected and lyophilized to afford peptide 8 as a red solid (1.0 mg, 2% yield).Primary islet isolation.Animals were euthanised in accordance with a schedule 1 procedure and bile ducts injected with Serva NB8 1 mg mL -1 collagenase.The pancreas was dissected, islets isolated using a histopaque gradient and cultured at 37°C, 5% CO 2 in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin and 1% glutamine.
Islets were then transferred to a well with 100 µL complete RPMI with 10 µM peptide 7 and incubated for 1 h at 37°C, 5% CO 2 .For control, non-competition assay wells, islets were incubated for 1 h in 100 µL complete RPMI with: a) 10 µM peptide 7, b) 10 µM KP-10, c) no probes.Islets were washed with complete RPMI prior to transfer to a glass, flat-bottomed 96-well imaging plate for imaging in complete RPMI.
Islet labeling with LUXendins.LUXendins are GLP1R markers [4] used to identify GLP1R+ cells.12-15 medium to large WT islets were picked 24-72 h after isolation into a plastic, round-bottomed 96-well plate into wells containing complete RPMI with: Representative images had linear adjustments applied to brightness and contrast and to enable cross-comparison, intensity values were maintained between samples.
Fluorescence intensity was measured using a ROI and mean intensity in ImageJ.

3 .
Determination of the extinction coefficient of Trp-BODIPY PLUS in EtOH.Values presented as means±SEM (n=6).

Table 2 .
Analysis of the chemical stability of Trp-BODIPY and Trp-BODIPY PLUS under conventional SPPS conditions.Amino acids were diluted with different reagent mixtures (70 µM, final volume 100 uL) and stirred at r.t. for 1 h (conditions A-B and E-H) or 5 min (conditions C and D).Mixtures were analyzed by HPLC-MS.
: t R : 4.7 min (90% purity).Optical properties were measured in a Synergy H1 BioTek spectrophotometer at the indicated concentrations on 96 or 384-well plates.
MS(ESI+) m/z calcd.forC114 H 199 BN 39 O 29 [M+H] + : 2071.0,found:2071.3.Spectroscopy measurements.Flow cytometry.Peptide 6 was reconstituted at 5 mM in DMSO and used at 300 nM in HEPES-NaCl buffer containing 2 mM CaCl 2 , with or without 2.5 mM EDTA, and were incubated with the cells for 10 min at r.t.before flow cytometry analysis, unless otherwise stated.AF647-Annexin V was used at the indicated concentration and added to cells 10 min prior to analysis.Fluorescence emission was measured on a 5 L LSR flow cytometer (BD).Excitation sources/emission filters used: peptide 6