Design of Fluorescent Probes for Bioorthogonal Labeling of Carbonylation in Live Cells

With the rapid development of chemical biology, many diagnostic fluorophore-based tools were introduced to specific biomolecules by covalent binding. Bioorthogonal reactions have been widely utilized to manage challenges faced in clinical practice for early diagnosis and treatment of several tumor samples. Herein, we designed a small molecule fluorescent-based biosensor, 2Hydrazine-5nitrophenol (2Hzin5NP), which reacts with the carbonyl moiety of biomolecules through bioorthogonal reaction, therefore can be utilized for the detection of biomolecule carbonylation in various cancer cell lines. Our almost non-fluorescent chemical probe has a fast covalent binding with carbonyl moieties at neutral pH to form a stable fluorescent hydrazone product leading to a spectroscopic alteration in live cells. Microscopic and fluorometric analyses were used to distinguish the exogenous and endogenous ROS induced carbonylation profile in human dermal fibroblasts along with A498 primary site and ACHN metastatic site renal cell carcinoma (RRC) cell lines. Our results showed that carbonylation level that differs in response to exogenous and endogenous stress in healthy and cancer cells can be detected by the newly synthesized bioorthogonal fluorescent probe. Our results provide new insights into the development of novel bioorthogonal probes that can be utilized in site-specific carbonylation labeling to enhance new diagnostic approaches in cancer.

'Oxidative stress' is interrelated with an imbalance in the level of reactive oxygen species (ROS) and antioxidants metabolism in the cellular system. Low level of ROS not only induces the activation of transcription factors for oxidative stress adaptation 1 but also acts as a signaling molecule in cellular processes 2 , which is one of the critical research topics in the field of redox biology. The increased level of ROS and disturbed redox signaling results in the oxidative damage on biomolecules in living systems. Many disorders which can trigger cancer or metabolic diseases are closely correlated with oxidative stress 3,4 .
The dysregulation of ROS levels results in different oxidative modifications in biomolecules such as oxidation, nitration, hydroxylation and carbonylation. Protein carbonylation is the major modification affecting the activity and stability of proteins and this process consists of mainly primary and secondary protein carbonylation mechanisms 5 . Primary protein carbonylation mechanism comprises metal-catalyzed oxidation (MCO) and cleavage of the protein backbone by the α-amidation pathway. Fenton reaction initiates the catalysis of transition in the presence of H 2 O 2 to produce OH-radicals. Extremely reactive OHradical leads to direct oxidation of proline, arginine, lysine and threonine 6 . Secondary protein carbonylation is a consequence of covalent adduction either between advanced lipid peroxidation end products (ALEs) or advanced glycation end products (AGEs) with amino acid residues 7 .
Protein carbonylation is chemically irreversible. Decarbonylation process relies on the reduction of carbonyl moiety on proteins via enzymatic processes. Wong and co-workers demonstrated that an inhibitor of thioredoxin reductase can suppress decarbonylation while thioredoxin protein level increases during the decarbonylation process. They claimed that thiol-dependent reduction may support enzymatic decarbonylation processes 8 . In addition, alcohol dehydrogenases, carbonyl reductases and aldo-keto reductases are capable of reducing carbonyl moiety on proteins 9 . Another enzymatic decarbonylation mechanism based on activation of Lon protease, which is activated under stress conditions such as high level of H 2 O 2 and participates in the degradation of carbonylated proteins 10 . The non-enzymatic endogenous protection of carbonylation is provided by the presence of pyruvate www.nature.com/scientificreports www.nature.com/scientificreports/ In 2Hzin5NP, hydrazine formation was confirmed by the detection of NH peak in 1 H NMR spectrum. While NH protons had a broad peak at 9 ppm, proton of OH was indicated at 10 ppm. Deshielding of NH peak to 10.83 ppm and appearance of CH 3 peak at 1.31 ppm demonstrated the structural conformation of aliphatic hydrazone with the presence of CH peak at 7.78 ppm. However, impurities and solvent peaks were observed in the 1 H NMR and 13 C NMR spectra. Peak of NH 2 at 4.5 ppm showed the excess hydrazine in the product. Additionally, CH 3 peak of unreacted acetaldehyde was observed at 2 ppm. Furthermore, solvent peak of ethanol was observed at 1 ppm and presence of water was examined with a sharp peak at 3.5 ppm. The reaction between aromatic aldehyde and 2Hzin5NP resulted in deshielding effect of NH on 1 H spectrum. Presence of the two OH groups on the aromatic hydrazone led to two similar peaks at 10 ppm and 10.6 ppm. All products were also confirmed by their 13 C NMR.
To verify the response mechanism of hydrazine probe, 2Hzin5NP towards carbonyl groups, compounds were analyzed by LC-MS. Compound 2Hzin5NP indicated a molecular ion peak of [2Hzin5NP + H] + at m/z = 170.05 in PBS buffer solution. LC-MS spectra of aliphatic and aromatic hydrazone demonstrated that peak of [2Hzin5NP + H] + was absent and new peaks at m/z = 196.07 and at m/z = 274.08 appeared in PBS buffer solution.
Hydrazine HCl salt is a stable compound in aqueous solutions. We have designed a small molecule-based cell-permeable fluorescent sensor 2Hzin5NP which achieved successful labeling of carbonyls on biomolecules inside live cells. Acetaldehyde and salicylaldehyde were used as mimic molecules of carbonyl groups which correspond to carbonylated biomolecules in cells. The fluorescent probe, 2Hzin5NP, has an absorption maximum at 354 nm and fluorescence emission maximum at 469 nm with the intensity of 2 × 10 5 CPS. The absorption and emission spectra of 2Hzin5NP (Fig. 1) undergoes a red Stoke shift on reaction with an aliphatic and an aromatic aldehyde on Table 1. While aliphatic hydrazone, provided a 12-fold increase in fluorescence intensity compared to its hydrazine aromatic hydrazone, demonstrated a 3-fold fluorescence intensity increase in MeOH. Fluorescence intensity and color shift changes from hydrazine turn off to hydrazone turn-on transformation were visualized  www.nature.com/scientificreports www.nature.com/scientificreports/ under normal and long wavelength fluorescence light (Fig. 1A). These data also assisted to demonstrate the large difference in fluorescence intensity between an aliphatic and aromatic hydrazone formation. Due to fluorescence quenching of amine derivatives, Hydrazine probe, 2Hzin5NP only showed extremely weak fluorescence as expected at 469 nm (ɸ = 0.001) and addition of aliphatic aldehyde lead to a 'turn on' fluorescence emission (ɸ = 0.085) with a bathochromic shift to 502 nm (Fig. 1B, Table 1). Hydrazone formation delocalized the electron distribution on the amide bond increase on the quantum yield of aliphatic and aromatic hydrazone 21 . Since it is highly expected to see a larger emission shift and fluorescence increase on aromatic hydrazone than aliphatic hydrazone due to its conjugation of electron donating electrons through the aromatic ring, cellular systems contain only aliphatic aldehydes.

Bioorthogonal labeling of carbonyls in live cells.
Carbonylation of biomolecules is one of the consequences of ROS damage in live cells, which occurs with the addition of aldehyde, ketone or lactam moieties giving rise to the lipid peroxidation, glycation and protein carbonylation. Carbonyl group on biomolecules is also utilized as a chemical reporter for the bioorthogonal labeling. We reasoned that hydrazine-based fluorescent probe can undergo a spectroscopic change upon hydrazone formation as a fluorescent sensor and hence used in the detection of carbonyl functional group on biomolecules in living systems 22,23 . In order to clarify the relative contribution of carbonylation, A-498 primary site and ACHN metastatic site cancer cells along with the normal human dermal fibroblasts, were examined in this study. A-498 cell line is a VHL-mutated while ACHN cell line is a c-MET-overactive cell model of Renal Cell Carcinoma (RCC). VHL protein (pVHL) is responsible for the regulation of Hypoxia-induced factor-1α (HIF-1α) via ubiquitin-mediated degradation by 26 S proteasome 24 . Loss of VHL and increased hypoxia results in overexpression of c-MET 25,26 , which was accompanied by increased metastatic potential and poor prognosis in RCC 27 . Phosphorylated MET activation is suppressed by wild-type VHL gene, hence VHL mutation induces the phosphorylation of MET protein resulting in overactive c-MET pathway. Given that overactive c-MET pathway plays an important role in protection against ROS-induced oxidative stress in renal carcinoma, HDF, A-498 and ACHN cells will possess different systemic levels of oxidative stress hence serve as excellent models to measure the efficiency of 2Hzin5NP probe to monitor relative protein carbonylation levels in a cellular microenvironment. Cytotoxicity assay of H 2 O 2 on HDF, A-498 and ACHN cell lines demonstrated that HDF cells endured higher concentrations of H 2 O 2 , which can be explained by the entrance of HDF into the transition shock state. Shock state is defined as an increase in BCL-2 levels that causes delayed apoptosis to provide enough time for the reversal of cell damage 28   www.nature.com/scientificreports www.nature.com/scientificreports/ differences in their carbonylation responses to oxidative stress 16 . In redox homeostasis, healthy cells maintain a low level of ROS production and possess antioxidant defenses which are necessary to prevent the oxidative damage. Cancer cells, however are continuously exposed to elevated levels of oxidative stress, which cause the steady-state upregulation of antioxidant defense in order to prevent ROS induced apoptosis 29 . Under oxidative stress conditions, the detoxification process is triggered in order to prevent oxidative damage in healthy cells by neutralizing the high level of ROS, explaining why HDF can endure higher H 2 O 2 concentrations when compared to the RCC cell lines. On the other hand, A-498 primary site ccRCC cell line could tolerate higher concentrations of H 2 O 2 compared to the ACHN metastatic site ccRCC cell line. Since A-498 cell line has VHL gene mutation which causes HIF-1α accumulation and activation of several survival pathways including transforming growth factor, epidermal growth factor, insulin-like growth factor and vascular endothelial growth factor, enhanced cell survival against H 2 O 2 treatment could be possible for A-498 cells 30 .

Determination of cytotoxic effects of H
In our study, HDF, A-498 and ACHN cells were treated with six different concentrations of 2Hzin5NP for 30 minutes to detect the cytotoxic effect of 2Hzin5NP in live cells as shown in Fig. 2B. The labeling of HDF cells with 2Hzin5NP resulted in a 15% decrease of cell viability at the concentrations of 5 µM and 10 µM. Exposure to 2Hzin5NP at 15-35 µM led to a 25% reduction in HDF cell viability, while 50 µM 2Hzin5NP incubation resulted in a 35% decrease in the cell viability. In addition, we examined the effect of sodium pyruvate in detection of H 2 O 2 -induced carbonylation, since sodium pyruvate is a natural scavenger which reacts with H 2 O 2 to yield sodium acetate, carbon dioxide and water as byproducts 31 . Physiological concentrations of sodium pyruvate present in cell culture medium also in serum, could alter the effective lifetime of exogenously added H 2 O 2 and inhibits both oxidative stress damage and H 2 O 2 -induced carbonylation of biomolecules. To verify that, cells were incubated with increased concentrations of sodium pyruvate in FBS containing DMEM and labeled with 2Hzin5NP. As expected, in the absence of pyruvate, cells displayed maximum fluorescence labeling, while increased sodium pyruvate concentration decreased the fluorescence labeling (Fig. 3). Otherwise, detection of the basal level of oxidative stress by 2Hzin5NP labeling demonstrated a visible but faint fluorescent signal for all cells, suggesting that 2Hzin5NP can be used to detect the basal level of protein carbonylation (Fig. 3, top panels). In the absence of pyruvate, H 2 O 2 treated groups showed dot-like fluorescence staining in cytoplasmic and pre-nuclear regions, indicating the presence of carbonylated protein aggregates. Protein carbonylation is necessary for the recycling of inactivated and misfolded proteins in proteasomal degradation system. Proteasome-dependent degradation of carbonylated proteins depends on the level of carbonylation. While mildly carbonylated proteins can be recognized by proteasomes, highly carbonylated proteins have relatively less ubiquitination site so they cannot be degraded by proteolysis 32 . 20S and 26S proteasomes are responsible for the recognition of carbonyl moieties on proteins and their degradation. While 26S proteasome activity is inhibited by oxidative stress byproducts, the 20 S proteasome is more effective to degrade carbonylated proteins 33 . As a high level of carbonyl moiety on proteins increases hydrophobicity 34 , dysfunctional proteins are accumulated and form protein aggregates called aggresomes 35 , which may induce autophagy 36 and apoptosis due to their high cytotoxicity in cellular systems 37 . In agreement with this notion, confocal microscopy images suggested the presence of carbonylated protein aggregates that appeared around the nucleus with an even distribution in the cellular cytoplasm (Fig. 3). This result presumably indicates that 2Hzin5NP is membrane-permeable but not nuclear membrane-permeable.
Next, we demonstrated the quantitative fluorescence response of 2Hzin5NP towards carbonylated proteins by fluorescence spectrophotometry. A significant increase of the fluorescence intensity at 506 nm was obtained upon following H 2 O 2 treatment in the absence of sodium pyruvate in all cell lysates. In contrast, a negligible difference in fluorescence intensities was noticed among the treatments in the presence of 2 mM sodium pyruvate in the medium. When HDF cells were incubated with 2 mM pyruvate containing DMEM with the presence of H 2 O 2 , the cells demonstrated 37% fluorescence response as the indication of H 2 O 2 -induced carbonylation level in HDF cell line. While lack of pyruvate led to a 63% increase in fluorescence emission, in HDF cells treated with H 2 O 2 (Fig. 4A), a 10% difference in fluorescent intensity was detected in the basal redox statue of HDF in the absence of pyruvate. As expected, the fluorescence intensity of 2Hzin5NP labeled cells was decreased by increasing the concentration of pyruvate in the medium. The result is consistent with the findings by Salahudeen et al. showing the pyruvate protection of H 2 O 2 induced damages in renal tissue in vivo and in vitro 38 . Analysis of carbonylated protein level in A-498 cell line demonstrated that H 2 O 2 treated A-498 cells generated 42% fluorescent response when cultured in 2 mM pyruvate DMEM (Fig. 4B). There was a 3-fold increase in basal protein-carbonylation levels in response to pyruvate in A-498 cells. 2Hzin5NP labeling of H 2 O 2 treated ACHN cells exhibited 73% fluorescent labeling in the presence of pyruvate when compared to H 2 O 2 treated cells grown in the absence of pyruvate, which www.nature.com/scientificreports www.nature.com/scientificreports/ represents 100% (Fig. 4C). Without H 2 O 2, 2Hzin5NP labeled ACHN cells showed 39% fluorescence intensity, which was brought down to 29% with 2 mM pyruvate pre-incubation. When H 2 O 2 -induced carbonylation levels were compared among HDF, A-498 and ACHN cells (Fig. 4D), HDF cells demonstrated 2.6-fold and 3.9-fold higher fluorescent staining than A-498 ACHN, respectively. Comparison of primary site versus metastatic site RCC cell lines showed that A-498 cells had a 1.4-fold higher level of H 2 O 2 induced carbonylated protein when compared to ACHN. This difference could be due to the VHL mutation in A-498 resulting in HIF1α-induced upregulation of glucose transporters GLUT1-GLUT4 39 as GLUT-mediated glucose influx stimulates oxidative stress via disruption of cellular energy homeostasis and redox status 40 .

Fluorescence Labeling of Serum Starvation Induced Carbonyls. Serum starvation is a classical
method for endogenous ROS generation, which was introduced by Troppmair and co-workers 41 . Serum starvation causes an alteration in redox homeostasis, which initiates intrinsic cell death pathways 42 . In order to detect the effects of endogenous ROS generation on carbonylation of biomolecules, HDF, A-498 and ACHN cells were serum starved and 2Hzin5NP labeled. In Fig. 5A, confocal images on the right panel, demonstrated an increased carbonylation level with fluorescence staining under the condition of serum deprivation. In 2011, Kuznetsov et al. reported that serum starvation caused up to a 3.5 -fold increase in ROS generation in 32D myeloid cells, NIH3T3 mouse embryonic fibroblast cells and in HL-1 cardiac muscle cells 41 . Likewise, in our study, serum starvation led to an increased protein carbonylation levels in healthy cells with respect to cancer cells. In order to verify quantitative results of serum starvation induced carbonylation on live cells, spectrofluorometric analysis was done. RFU obtained for cells that were serum starved and 2Hzin5NP labeled was normalized to 100% labeling yield. When cells were incubated with 10% FBS containing DMEM, fluorescence labeling of HDF and A-498 cell lines was decreased to 41%, while a 58% fluorescence response was detected for ACHN cell line (Fig. 5B). The relative fluorescence level of carbonylated proteins was detected in all cell lines for comparison. Carbonylated protein level of serum starved ACHN cell line was 1.7 times higher than the carbonylation level of serum starved A-498 cell line. While serum starved HDF cells demonstrated a respective 3.6-fold and 1.9-fold higher fluorescent intensity than A-498 and ACHN cells (Fig. 5C) This difference can be explained by the c-MET mutation in ACHN cells or loss of VHL function in A-498 cells 43 both of which results in direct or indirect activation of the c-MET pathway 44 . www.nature.com/scientificreports www.nature.com/scientificreports/ Pal and coworkers confirmed that renal tumors overactivate c-MET to cope with ROS-induced oxidative stress 45 . Overexpressed c-Met mediates PI3K/Akt activation which is involved in endogenous ROS generation and oxidative stress 46 . Activation of PI3K/Akt pathway stimulates expression of the redox-sensitive transcription factor nuclear factor-κB (NF-κB), which regulates anti-apoptotic target genes 47 . Therefore, cancer cells evolve to eliminate carbonylated proteins to evade apoptosis. In agreement, we found that A-498 and ACHN cells possess lower levels of carbonylated proteins in response to oxidative stress induced by serum starvation and H 2 O 2 treatment.
In summary, our results suggest that our novel compound, 2-Hydrazine-5-nitrophenol (2Hzin5NP) is a carbonyl moiety specific bioorthogonal sensor, which is applicable for fluorescent labeling and detection of ROS induced carbonylation in live cells.

conclusion
Carbonylation is an irreversible post-translational modification on biomolecules, which can also serve as an oxidative stress biomarker. Determination of carbonylation level of biomolecules may provide information about the distortion of redox biology in cancer cells. Bioorthogonal chemistry provides labeling strategies with the designing of site-specific fluorescent probes to target biomolecules carbonylation via click reaction that gives quantitative and qualitative measurements. Aldehyde -hydrazine reactions are well suited for the detection of carbonylation in live cells. 2-Amino-5-nitrophenol is reduced to 2-Hydrazine-5-nitrophenol by diazotization reaction. 2Hzin5NP is specific for carbonyl groups on aldehydes, ketones and lactams. In this study, 2Hzin5NP was used as a site-specific fluorescent probe for the detection of oxidative stress induced carbonylation in HDF, A498 and ACHN. Primary site and metastatic site of RCC demonstrated different carbonylation levels due to exogenous or endogenous variability in ROS generation.
This site-specific bioorthogonal labeling may be announced as a potentially useful strategy that can be used as a small molecule-based diagnostics for the molecular detection of oxidative damage in biological systems. In future investigations, bioorthogonal near-IR fluorescent probes would be the next key molecules to synthesize for the detection of carbonylation due to their minimal cellular or tissue components autofluorescence production in the near-IR region. Near-IR dyes have the potential to offer highly specific and sensitive fluorescence detection in complex biological systems. It is therefore essential that future probes should have the specificity and selectivity to define oxidative stress induced carbonylation of biomolecules in vitro and in vivo. Bioorthogonal labeling of carbonylation would allow us to determine the dynamics of oxidative stress induced carbonylation which have vital roles in the diagnosis and determination of therapeutic targets for future cancer therapies. 1 H-NMR and 13 C-NMR spectra were measured with an AVANCE III 500 MHz spectrometer (Bruker) (TMS as internal standard). The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet (denotes complex pattern), dd = doublet of doublets and dt = doublet of triplets. Absorption spectra were obtained by an Agilent/HP 8453 UV-Visible Spectrophotometer using a Starnacell Hellma quartz back wall cuvette with a 1 cm path length. Emission spectra were recorded at room temperature using a Jobin Yvon Horiba FluoroMax-4 spectrofluorometer using a Starnacell Hellma 2 × 10 mm fluorescence cuvette, oriented such that the light passes through the shorter path. In cell culture, live cell imaging was performed by Zeiss Lsm 800 Confocal Microscope and emission spectra of cell lysate was recorded by Thermo Fisher Varioskan Lux Multimode Microplate Reader using a black 96-well microplate.
Quantum yields were calculated using the following equation: where F denotes the area under the fluorescence band, A denotes the absorbance at the excitation wavelength, and ƞ denotes the refractive index of the solvent. The integration of the emission bands was performed using GraphPad Prism 6.
Cell culture. HDF, A-498 and ACHN cells were grown in DMEM containing 4.5 g/L glucose, 1 mmol/L sodium pyruvate and 200 mM L-glutamine which was supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin in a humidified incubator containing 5% CO 2 at 37 °C. Once the cells reached to 80% confluent cells, they were sub-cultivated with trypsin-EDTA solution (0.05%). www.nature.com/scientificreports www.nature.com/scientificreports/ Statistical analysis. All data were obtained from three independent experiments and presented as the mean ± SD (error bars). Quantification of carbonylation in cell lysates were analyzed by two-tailed Student t-test. P value less than 0.05 was considered as a statistically significant.