De novo generation of a bright blue fluorophore from 2-oxoglutarate in biological samples

We discovered the generation of a new bright blue fluorophore from a particular type of amine and 2-oxoglutarate (2-OG) under mild conditions without any chemical additives. Two β-aminoethylamine molecules and three 2-OG molecules form an unprecedented 2-pyridone structure with a fused γ-lactam ring (DTPP) via complex reactions including double decarboxylation and quintuple dehydration. The DTPP fluorophore shows a high quantum yield (80%) and photostability. The great potential of the present DTPP generation in the quantitative analysis of 2-OG in biosamples is demonstrated.


General methods
All reactions were performed in oven-dried glassware. 2 were purchased from Sigma-Aldrich (USA). Propylamine was purchased from Junsei chemical (Japan). Triethylamine (TEA), acetonitrile (ACN) and dichloromethane (DCM) were purchased from Samchun (Korea). CDCl 3 , D 2 O and CD 3 OD were purchased from BK instruments (Korea). Dulbecco's modified Eagle's medium (DMEM), Dulbecco's phosphatebuffered saline (DPBS), and trypsin-EDTA were purchased from Welgene (Gyeongsan, Korea). All chemical reagents and solvents were used without further purification. Thin layer chromatography was performed on glass coated with Silica gel 60 F254 and Silica gel 60 RP-18 F254s (Merck). Flash column chromatography was performed using silica gel (0.040-0.063 mm, Merck). Anion exchange column was performed using DOWEX ® 1×8 chloride form (100-200 mesh, Sigma-Aldrich). Cation exchange column was performed using CM Sephadex ® C-25 (Sigma-Aldrich). Proton NMR spectra were recorded at 300 MHz, 500 MHz or 850 MHz and chemical shifts were reported relative to the residual solvent peaks (for spectra recorded in CDCl 3 ; 7.26 ppm, D 2 O; 4.79 ppm, CD 3 OD; 3.31 ppm). Multiplicity was indicated as follow: s (singlet), d (doublet), t (triplet), q (quartet), h (hextet) and m (multiplet). Carbon NMR spectra were recorded at 75 MHz, 126 MHz or 214 MHz. Two-dimensional NMR spectra were recorded at 500 MHz or 850 MHz. NMR data were processed with MestReNova (Version 6.0.2) or Topspin (Version 4.0.6). High resolution MS data was recorded on the AB SCIEX Q-TOF 5600 LC/MSMS spectrometer using electrospray and atmospheric pressure chemical ionization. High performance liquid chromatography (HPLC) was performed on the Shimadzu HPLC system equipped with an LC-20AD pump and an SPD-20A UV detector. Agilent ZORBAX 300SB-C18 5 µm 9.4 × 250 mm column was used for purification. For the mobile phase, Solution A (water with 0.1% v/v TFA) and Solution B (ACN with 0.1% v/v TFA) were used as eluents. FTIR spectra was recorded on the Thermo Scientific NICOLET iS10 spectrometer. UV absorption was recorded on the JASCO V-650 spectrophotometer and fluorescent emission was recorded on the JASCO FP-8300 spectrofluorometer.

3. Quantum yield measurement
The relative quantum yields (φ F ) of each fluorophore was measured in reference to quinine sulfate (φ F = 58% at 354 nm excitation in 0.1 M H 2 SO 4 ). The formula used for measurements is shown below: where η is the refractive index of the solvent, and PL area and OD are the fluorescence area and absorbance value, respectively.

5. The cell culture
HeLa (human cervical cancer) cells were grown in DMEM supplemented with 10% FBS at 37℃. The cells were seeded in a 100 mm culture dish at a density of 300,000 cells per dish.

6. Fluorometic analysis of 2-OG in cell extracts
Cell extracts were prepared according to the previous study [1]. Dishes with HeLa cells were taken out of the 37℃ incubator and immediately placed on ice. Then, the growth medium was

7. Two-photon microscopy (TPM) analysis of 2-OG in fixed cells
HeLa (human cervical cancer) cells were grown in DMEM supplemented with 10% FBS at 37℃. The cells were seeded on 10 mm coverglasses in 6-well plates at a density of 150,000 cells per well. After incubating for 48 h, the growth medium was changed with fresh DMEM supplemented with 10% FBS containing 0 mM, 3.75 mM, or 7.50 mM of DM 2-OG. After incubation for 24 h at 37℃, the medium was removed and the cells were washed with 10 mL of PBS. Then, 10 mM DEEDA in isopropyl alcohol (5 mL) was added to each well. The DTPP fluorescence was developed by a further incubation for 24 h at 37℃. After washing the cells with DPBS twice, image acquisition was performed using a two-photon laser scanning microscope (FVMPE-RS; Olympus, Japan) with a 25× objective (XLPLN25XSVMP2; Olympus, Japan) and the Olympus Fluoview FV31S-SW Software. Fluorescence images of the DTTP distribution were acquired in the blue channel (λ ex = 720 nm and λ em = 410-455 nm).
All images were analyzed using the ImageJ software (National Institutes of Health, http://rsb.info.nih.gov/ij/). The total blue fluorescence intensity was integrated in each cell. The fluorescence intensity of more than 80 cells were analyzed per each group (n > 80).

Crystallization of 6.
Because DTPP is difficult to be crystallized, the compound 6 with the same core structure was crystallized instead. The NMR patterns of compound 6 (Figure S19-S23) are nearly identical to those of the DTPP core structure. Vapor diffusion method was used to crystallize 6. In an inner vial, 5.4 mg of 6 was dissolved in 0.5 mL chloroform. In an outer vial, 2 mL of cyclohexane was added and sealed with paraffin film. After 24 h-incubation at room temperature, crystal was obtained.

X-ray crystallographic analysis
A suitable crystal was selected and recorded on a SuperNova, Dual, Cu at home/near, AtlasS2 diffractometer. The crystal was kept at 292.6(3) K during the data collection. Using Olex2 [2], the structure was solved with the ShelXT [3] structure solution program using Intrinsic Phasing and refined with the ShelXL [4] refinement package using Least Squares minimization. Figure S27. The X-ray crystal structure and structure refinement for compound 6