Long non‐coding RNA (lncRNA) lnc‐ISG20 has been found aberrantly up‐regulated in the glomerular in the patients with diabetic nephropathy (DN). We aimed to elucidate the function and regulatory mechanism of lncRNA lnc‐ISG20 on DN‐induced renal fibrosis. Expression patterns of lnc‐ISG20 in kidney tissues of DN patients were determined by RT‐qPCR. Mouse models of DN were constructed, while MCs were cultured under normal glucose (NG)/high glucose (HG) conditions. The expression patterns of fibrosis marker proteins collagen IV, fibronectin and TGF‐β1 were measured with Western blot assay. In addition, the relationship among lnc‐ISG20, miR‐486‐5p, NFAT5 and AKT were analysed using dual‐luciferase reporter assay and RNA immunoprecipitation. The effect of lnc‐ISG20 and miR‐486/NFAT5/p‐AKT axis on DN‐associated renal fibrosis was also verified by means of rescue experiments. The expression levels of lnc‐ISG20 were increased in DN patients, DN mouse kidney tissues and HG‐treated MCs. Lnc‐ISG20 silencing alleviated HG‐induced fibrosis in MCs and delayed renal fibrosis in DN mice. Mechanistically, miR‐486‐5p was found to be a downstream miRNA of lnc‐ISG20, while miR‐486‐5p inhibited the expression of NFAT5 by binding to its 3'UTR. NFAT5 overexpression aggravated HG‐induced fibrosis by stimulating AKT phosphorylation. However, NFAT5 silencing reversed the promotion of in vitro and in vivo fibrosis caused by lnc‐ISG20 overexpression. Our collective findings indicate that lnc‐ISG20 promotes the renal fibrosis process in DN by activating AKT through the miR‐486‐5p/NFAT5 axis. High‐expression levels of lnc‐ISG20 may be a useful indicator for DN. Here, these results provide evidence for that lnc‐ISG20 could promote the renal fibrosis in DN via miR‐486‐5p/NFAT5/AKT.Recently, the lncRNA lnc‐ISG20 was found to be up‐regulated in the glomerular in DN patients,22.1Signed informed consents were obtained from all participants or their families prior to the study, and each experimental procedure was approved by the Huaihe Hospital of Henan University Ethics Committee. All animal experiment procedures were strictly in accordance with the recommendations in the laboratory animal care and use guidelines published by the National Institutes of Health. Extensive efforts were made to minimize the suffering of the animals included in the study.2.2The kidney tissues were collected from 30 patients diagnosed as DN by renal biopsy at the Huaihe Hospital of Henan University from January 2018 to March 2019. The patients comprised of 15 males and 15 females, with a calculated mean age of 54.93 ± 10.17 years. In addition, adjacent normal kidney tissues taken from patients with kidney cancer served as controls, which included 15 males and 15 females, with a calculated mean age of 52.20 ± 8.24 years. The patients with renal cancer in the control group presented with normal renal function, blood glucose and urine protein before surgery, and no history of heart or liver disease.2.3C57BL/KsJ db/db male mice (aged 8‐weeks) and their age‐matched db/m mice were purchased from NBRI . Leptin receptor (db/db) gene‐deficient mice are widely used to establish type 2 diabetes models. Similarly, db/db mouse can be employed to construct a model of human DN, as they exhibit main common characteristics similar to humans such renal hypertrophy, glomerular enlargement, proteinuria and mesangial matrix expansion. Several studies have also confirmed that db/db mice exhibit hyperglycemia at 4 weeks of age, and the albumin excretion rate is 8‐62 times higher at 8 weeks of age.2.4The mice were assigned into control group, DN group, short hairpin RNA (sh)‐negative control (NC) group, sh‐lnc‐ISG20 group, overexpression (oe)‐NC group, oe‐lnc‐ISG20 group, oe‐NC + sh‐NC group, oe‐lnc‐ISG20 + sh‐NC group, oe‐NC + sh‐NFAT5 group, oe‐lnc‐ISG20 + sh‐NFAT5 group, 10 mice per group. Two weeks before HG treatment, mice were injected with adeno‐associated virus (AAV)2/2 vector expressing oe‐lnc‐ISG20, sh‐lnc‐ISG20, sh‐NFAT5 and AAV‐NC alone or in combination through tail vein.2.5A Bayer blood glucose metre was used to measure blood glucose, and the Coomassie brilliant blue method was applied to detect the 24‐hour urine albumin concentrations.2.6The collected kidney tissues were dehydrated with fixed gradient alcohol , paraffin‐embedded, and then sliced into 4 μm thick sections. The sections were then placed in an oven at 60°C with 1 hour, dewaxed with conventional xylene and dehydrated with gradient alcohol. The sections were stained with haematoxylin  for 3 minutes and eosin  for 1 minute. The sections were returned to blue with flow water for 5 minutes, dried, sealed with neutral gum, observed and imaged under an optical microscope . A total of 5 high‐magnification fields were randomly selected for each slice (× 200).2.7The collected kidney tissues were dehydrated with fixed concentrations of gradient alcohol , paraffin‐embedded, and then sliced into 4 μm thick sections. The obtained sections were then placed in an oven at 60°C with 1 hour, dewaxed with conventional xylene and dehydrated with gradient alcohol. After hydration, steps were performed according to the Masson staining kit . After Reguad staining for 5 minutes, and colour separation in picric acid ethanol solution, the sections were rinsed once with double distilled water. After dyeing with Ponceau red acid for 5 minutes, the sections were rinsed in double distilled water once, and immersed in l% phosphoaluminate for 5 minutes, aniline acetate blue solution for 7 minutes, and 1% acetic acid for 1 minute, in successive. After 95% ethanol dehydration, anhydrous ethanol dehydration and xylene clearing, the sections were sealed with neutral gum. Results of Masson staining were used to determine the collagen deposition in rat renal tubules and the proportion of total kidney collagen area. Digital images of kidney tissue were acquired using an optical microscope (Olympus) equipped with an image analysis software . Five fields were selected for each slice, and the area of fibrotic lesions in the cortical interstitium was calculated in any field and showed as a percentage of the area of fibrosis relative to the entire area.2.8The methods of fixing, dehydrating, slicing, baking, dewaxing and hydrating were as stated above. The sections were repaired with 0.1 M sodium citrate, heated and boiled for 20 min. After being allowed to cool down, the sections were washed with 0.2 mol/L PBS solution (pH 7.4) for 5 minutes × 3 times. Next, 3% catalase was added for 15 minutes reaction, and 0.2 mol/L PBS solution was used to rinse for 5 minutes × 3 times. With the addition of 5% BSA blocking solution dropwise, the sections were incubated at 37°C for 30 minutes. Primary antibodies from Abcam , rabbit anti‐collagen IV , rabbit anti‐fibronectin  and rabbit anti‐TGF‐β1  were added dropwise for incubation overnight at 4°C in a wet box. The sections were washed with 0.2 mol/L PBS solution for 5 minutes × 3 times. After drying the sections, the biotinylated goat anti‐rabbit IgG  was incubated with the sections at 37°C for 30 minutes. Following incubation with horseradish peroxidase‐labelled streptavidin protein working solution  at 37°C for 20 minutes, the sections were washed with 0.2 mol/L PBS for 5 minutes × 3 times. The sections were counterstained with haematoxylin, after which 1% ammonia was used to return to blue. After dehydration, transparency and sealing, tissue sections were observed under an optical microscope . A total of 5 high‐magnification fields were randomly selected for each section. 100 cells were counted in each field, where the number of positive cells <5% was considered as negative, and the number of positive cells ≥5% was positive. The results of immunohistochemistry were independently scored by two pathologists in a double‐blind manner.2.92 concentration and 95% saturation humidity. The MCs in the normal glucose (NG) group were cultured with L‐glucose 30 mmol/L, and those in the HG group were cultured with D‐glucose 30 mmol/L. HG stimulation simulated the growth environment of MCs under DN conditions, and NG stimulation simulated the normal growth environment.Human embryonic kidney cells HEK293T and mouse glomerular mesangial cells (MCs) were purchased from the Shanghai Academy of Sciences . The obtained HEK293T cells were cultured with HG DMEM , and MCs were cultured with low‐glucose DMEM . Next, 10% foetal bovine serum , 10 μg/mL streptomycin and 100 U/mL penicillin were cultured in an incubator  under the conditions of 37°C, 5% CO2.10TM 2000  was used to transfect sh‐NC (5′‐GGGUGAACUCACGUCAGAA‐3′ sequence), sh‐lnc‐ISG20‐1 (5′‐CCAGGUGUCGAUAAGUAAUTT‐3′ sequence), sh‐lnc‐ISG20‐2 (5′‐CUGUAAGUAGAUCCUGCAGTT‐3′ sequence), sh‐NFAT5‐1 (5′‐CGGACAACAAAGGCAACTCAA‐3′ sequence) or sh‐NFAT5‐2 (5′‐GCAGAGTAACTGGACGAAATA‐3′ sequence) into cells. After 48 hours of transfection, the transfection efficiency of sh‐lnc‐ISG20 and sh‐NFAT5 was determined by RT‐qPCR. AAV vectors expressing oe‐lnc‐ISG20, oe‐NFAT5, oe‐NC, shRNAs, sh‐NC and plasmids miRNA mimic, miRNA inhibitor and corresponding NCs were purchased from Shanghai GenePharma Co., Ltd .Cells were digested with trypsin in the logarithmic phase and inoculated with 6‐well plates at 1 × 102.112O 8 μL. Reaction conditions are as follows: 95°C for 10 minute, 95°C for 15 s, 60°C for 1 minute, 40 cycles in succession. Three replicate wells were set for each sample. The primers for lnc‐ISG20, miR‐486‐5p, GAPDH and U6 were synthesized by Shanghai Sangon Biotech Co., Ltd —. Each experiment was repeated three times.Total RNA content was extracted using Trizol kits , and RNA was reverse transcribed into cDNA using reverse transcription kit  with the volume of 20 μL. Reaction conditions were as follows: 37°C, 15 minutes; 85°C, 5 s. The SYBR Premix EX Taq kit  was used for loading. The samples were subjected to qPCR reaction in a real‐time fluorescence quantitative PCR instrument . Reaction system was as follows: SYBR Mix 9 μL, positive primer 0.5 μL, negative primer 0.5 μL, cDNA 2 μL and RNase Free dH2.12Cells in each group were collected following digestion with trypsin, and then lysed with enhanced radio‐immunoprecipitation assay lysis buffer  containing protease inhibitors. The protein concentration was detected by using bicinchoninic acid protein quantification kit (Boster). Proteins were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membrane. Afterwards, 5% bovine serum albumin was added for incubation at room temperature for 2 hours to block non‐specific binding. Diluted rabbit anti‐collagen IV , rabbit anti‐fibronectin , rabbit anti‐TGF‐β1 , rabbit anti‐NFAT5 , rabbit anti‐p‐AKT , rabbit anti‐AKT , rabbit anti‐p‐GSK3β , rabbit anti‐GSK3β , Rabbit anti‐GAPDH , all procured from Abcam, were incubated with the membrane overnight at 4°C. After washing 3 times with TBST for 5 minutes each time, HRP‐labelled goat anti‐rabbit secondary antibody  was added and incubated for 1 hours at room temperature. The membrane was reacted with enhanced chemiluminescence solution  for 1 minute at room temperature after 3 another 3 TBST rinses. Subsequently, the membranes were observed under a gel imager. GAPDH was used as an internal reference, and the ratio of the grey value of the target band to the internal reference band was used as the relative expression level of protein. Each experiment was repeated 3 times.2.13RIP assay was performed using EZ‐Magna RIP kits  according to the manufacturer's instructions. Cells were lysed with RIP lysis buffer containing a protease and phosphatase inhibitor cocktail (Sigma‐Aldrich). Magnetic beads were pre‐incubated (Invitrogen) with AGO2 antibody  or anti‐rabbit IgG  for 30 minutes, immunoprecipitated with the lysate, and spined at 4°C overnight. RNA was purified from the RNA‐protein complex bound to the magnetic beads and then analysed by RT‐qPCR.2.14+) was used as a calibration internal reference to calibrate the transfection efficiency between different samples. The ratio of RLU divided by the measured value of firefly luciferase RLU is used to evaluate the activation degree of the target reporter gene. Each experiment was repeated 3 times.Result of online website and sequence alignment indicated that lnc‐ISG20 was most likely to bind to miR‐486‐5p at two sites. The upstream and downstream sequences about 500 bp containing miR‐486‐5p potential binding site were synthesized. The wild‐type and mutant form of binding site were cloned into the psiCHECK‐2 vector R‐Luciferase (hRLuc) 3'UTR region, and then the mimics/NC of the tested miR were co‐transfected into 293T cells. The transfection method was the same as stated above. The cells were collected and lysed after 48 hours transfection. A dual‐luciferase reporter system  was used to detect luciferase activity. Firefly luciferase  each well at 4°C for 5 minutes. 200 μL of pre‐hybridization solution was added to each well and blocked at 37°C for 30 minutes. The cells were incubated with 250 μL of hybridization solution at 37°C overnight in the dark. The next steps should also be performed in dark conditions. The cells were rinsed at 42°C with Buffer I , Buffer II (2 × SSC), Buffer III (1 × SSC) and 1 × PBS in successively with 5 minutes each time. The cells were stained with DAPI (1:800) for 10 minutes, washed and sealed with nail polish. A total of 5 different fields of view were selected for observation and photographing under a fluorescence microscope .FISH technology was applied to identify the subcellular localization of lnc‐ISG20 and miR‐486‐5p. According to the instructions of RiboTM lncRNA FISH Probe Mix (Red) , a cell slide was plated in a 24‐well culture plate and inoculated with cells at 6 × 102.16P < .05 was considered statistically significant.Statistical analyses were performed using SPSS 21.0  statistical software. Measurement data were presented as mean ± standard deviation. Comparisons between two groups were analysed by the independent sample t test, and comparisons between multiple groups were analysed by the one‐way or repeated measurement analysis of variance followed by Tukey's post hoc test. Pearson correlation coefficient was used to observe the correlation of indicators. A value of 33.1Prior research indicated that lncRNA lnc‐ISG20 was up‐regulated in the glomeruli of DN patients.In order to further investigate the dysregulation of lnc‐ISG20 in DN, we established DN mouse models. Compared with the control group, it was found that the blood glucose and excretion rate of urinary albumin in the DN group were increased significantly Figure . HE staiExpression patterns of lnc‐ISG20 in the kidneys of DN mice were detected using RT‐qPCR, which revealed that the expression levels of lnc‐ISG20 in the DN group were significantly increased when compared with the control group Figure . Meanwhi3.2In order to explore the effect of lnc‐ISG20 on renal interstitial fibrosis, two sh‐lnc‐ISG20 sequences were designed and RT‐qPCR was performed to detect their efficiency in MCs. Compared with the sh‐NC group, the expression levels of lnc‐ISG20 in the sh‐lnc‐ISG20 group were found to be significantly reduced, and sh‐lnc‐ISG20‐1 exhibited the most obvious silencing effect, so sh‐lnc‐ISG20‐1 was selected for subsequent experimentation Figure . MCs wer3.3via orthotopic multi‐point injection to silence lnc‐ISG20. Compared with the sh‐NC group, the expression of lnc‐ISG20 in the sh‐lnc‐ISG20 group was significantly reduced  and oe‐NFAT5 were used at the same time under the HG condition. RT‐qPCR results validated the overexpression efficiency of oe‐NFAT5 Figure . Western3.7Our initial findings indicated that overexpression of lnc‐ISG20 could augment the expression of NFAT5, which could be reversed by miR‐486‐5p mimic. In order to explore whether lnc‐ISG20 exerts its biological function through the miR‐486‐5p/NFAT5/AKT axis, we designed a rescue experiment. The mRNA level of NFAT5 was significantly increased by lnc‐ISG20 overexpression but reduced by sh‐NFAT5 in the presence of lnc‐ISG20 Figure . ConsequMeanwhile, the expression levels of collagen IV, fibronectin and TGF‐β1 were found to be remarkably increased by enforced expression of lnc‐ISG20, but reduced by silencing of NFAT5. The increases of collagen IV, fibronectin and TGF‐β1 caused by oe‐lnc‐ISG20 were counteracted by sh‐NFAT5 Figure . Overall3.8In order to further verify the aforementioned results, multi‐point injection in situ of AAV2 vectors was performed in vivo. The results showed increased urinary albumin excretion rate of DN mice after lnc‐ISG20 overexpression and reduced urinary albumin excretion rate of DN mice after NFAT5 knockdown. Additionally, the urinary albumin excretion rate increased by oe‐lnc‐ISG20 was markedly reduced by sh‐NFAT5 Figure . RT‐qPCRTo further assess pathological condition of kidney tissues of DN mice after intervention, HE staining was conducted. The results demonstrated that the proliferation of mesangial cells and mesangial extracellular matrix was increased and the basement membrane became thickened after lnc‐ISG20 overexpression, accompanied with interstitial inflammatory cells and hyperplasia and infiltration in fibrous tissues of DN mice. The intact kidney base was observed without glomeruli, interstitial hyperplasia, and hyperplasia in fibrous tissues when NFAT5 was knocked down in DN mice. It was further indicated that inhibition of NFAT5 reversed the pathological conditions such as renal fibrosis caused by oe‐lnc‐ISG20 Figure . In addi4Currently, microalbuminuria evaluation is regarded as the gold‐standard for the diagnosis of early renal involvement, however, the unsatisfactory efficacy of urine testing exists as it can be precipitated by many common clinical factors, such as acute febrile disease, exercise and transient loss of glycemic control.et al firstly identified lnc‐ISG20 as long non‐coding RNA and characterized its positive regulatory effects on the protein levels of ISG20.Firstly, our findings revealed that lnc‐ISG20 was highly expressed in DN patients, the kidney tissues in DN mice, as well as the MCs under HG conditions. The ISG20 gene possesses the ability to encode a 20‐kDa protein, which is extensively involved in processes like oncogenesis and gene transcription.Additionally, the current study demonstrated that lnc‐ISG20 could promote cell fibrosis in MCs, while silencing of lnc‐ISG20 in DN mice brought about inhibitory effects on renal fibrosis. The progression of DN is commonly characterized by the loss of renal cells, and renal fibrosis represents the final pathological change in DN.via binding to miRNAs. For instance, lncRNA1700020l14Rik could interact with miR‐34a‐4p to alleviate cell proliferation and fibrosis in DN.Furthermore, the current study revealed that lnc‐ISG20 was capable of binding to miR‐486‐5p. Meanwhile, numerous studies have documented the tumour suppressive role of miR‐486‐5p in several kinds of malignancies, such as lung squamous cell carcinoma, non‐small cell lung cancer and breast cancer.via inducing AKT phosphorylation, which deepens our understanding of the molecular mechanisms underlying DN. Based on all above findings, our study indicates towards the existence of a regulatory axis in the pathophysiology of DN, wherein lnc‐ISG20 promotes renal fibrosis via the miR‐486‐5p/NFAT5/p‐AKT pathway. The newly proposed signalling axis in the present study contains many important regulatory factors, including lncRNAs, miRNAs, transcription factors as well as protein kinases, which enriches our understanding on both non‐coding RNAs and the pathogenesis of the disease.Expanding on our discoveries, we further uncovered that overexpression of NFAT5 could trigger cell fibrosis in MCs by activating the AKT protein. AKT, also known as protein kinase B or PKB, can be activated by multiple factors such as growth factors and the oncogenic mutations of upstream regulatory proteins.5From the present study, the results suggested that lnc‐ISG20 was aberrantly up‐regulated in DN. Hence, lnc‐ISG20 may be able to serve as a promising biomarker for the diagnosis of DN. Altogether, findings from the current study identified the unique miR‐486‐5p/NFAT5/p‐AKT signalling pathway in the progression of DN. Accordingly, we found that lnc‐ISG20 promoted renal fibrosis in DN through impairing miR‐486‐5p‐dependent inhibition of NFAT5 and activating AKT Figure . These hThe authors declare no conflict of interest.Yu‐Rui Duan: Conceptualization (lead); Data curation ; Formal analysis ; Investigation (lead); Methodology (lead); Writing‐original draft (lead); Writing‐review & editing . Bao‐Ping Chen: Conceptualization (supporting); Data curation ; Formal analysis ; Investigation (supporting); Methodology (supporting); Writing‐original draft (supporting); Writing‐review & editing . Fang Chen: Data curation ; Formal analysis ; Project administration (lead); Resources ; Software ; Writing‐review & editing . Su‐Xia Yang: Data curation ; Formal analysis ; Project administration (supporting); Software ; Validation ; Visualization ; Writing‐review & editing . Chao‐Yang Zhu: Data curation ; Formal analysis ; Software ; Validation ; Visualization ; Writing‐review & editing . Ya‐Li Ma: Data curation ; Formal analysis ; Software ; Validation ; Visualization ; Writing‐review & editing . Yang Li: Data curation ; Formal analysis ; Resources ; Software ; Supervision ; Validation ; Visualization ; Writing‐review & editing . Jun Shi: Data curation ; Formal analysis ; Funding acquisition (lead); Resources ; Software ; Validation ; Writing‐review & editing .YRD, BPC, FC, SXY, CYZ, YLM, YL and JS: Study design. YRD and BPC: Data collation; data analyses; and production of the initial draft of the manuscript. FC, SXY, CYZ, YLM, YL and JS: Contribution of manuscript drafting. All authors: Read and approval of the final submitted manuscript.


The dissolution of CC in NaOH/ZnO aqueous solutions using the freezing–thawing method was an exothermic process, and the lower temperature was favorable for the dissolution of CC. ZnO dissolved in NaOH aqueous solutions with the formation of Zn(OH)42−, and no free Zn2+ ions existed in the solvents. NaOH/Na2Zn(OH)4 system formed strong interactions with the hydroxyl groups of CC to improve its solubility and the stability of CC solution. The results indicate that 7 wt% NaOH/1.6 wt% ZnO aqueous solution was the most appropriate solvent for the dissolution of CC. This work revealed the dissolution interaction of CC-NaOH/ZnO solutions, which is beneficial for the industrialization of the CarbaCell process.The dissolution and molecular interactions of cellulose carbamate (CC) in NaOH/ZnO aqueous solutions were studied using optical microscopy, differential scanning calorimetry (DSC),  The CC and solvent mixtures were taken out of the refrigerator at different freezing times. Subsequently, an appropriate amount of mixture was directly pressed between the two glass slides, then sealed with paraffin, observed, and photographed. The solubility of CC was determined by observing the CC solutions with the polarized mode. After a certain concentration of CC was obtained, the solution was subjected to polarized microscope observation at room temperature. The area observed in the polarized microscope was a 20 mm diameter circular field. When no fibers were observed, CC was considered to be dissolved at that concentration. Each solubility was tested at least 3 times to confirm the accuracy and repeatability.2 atmosphere. 1H NMR spectra were measured on a Varian INOVA-600 spectrometer in the proton noise-decoupling mode at 25 °C. The chemical shifts of protons were referenced to the signals of D2O and tetramethylsilane (TMS). The CC concentrations for DSC and 1H NMR measurements were 5 wt% and 0.3 wt%, respectively.Differential scanning calorimetry (DSC) measurements were made using a TA Q20 instrument . CC was dispersed in NaOH (5–10 wt%)/ZnO (0–1.6 wt%) hydrates and sealed in a stainless pan. The temperature was programmed from 20 to −40 °C and then from −40 to 20 °C at a rate of 1 °C/min under Nh) distributions of CC in NaOH/ZnO aqueous solutions were characterized by dynamic light scattering (DLS) on a multi-angle light scattering spectrometer  equipped with an ALV-5000/E multi-digital time correlator. The scattering angle was 90°. The solutions were kept for 10 min at each condition before measurement. All of the CC solutions (c = 0.3 g/L) were optically cleaned through 0.45 and 0.22 µm Millipore filters .The hydrodynamic radius  images were measured with an AFM  in ac mode at 25 °C. Silicon probes with a spring constant of 2 N/m and resonance frequency of 70 kHz  were employed. All data from the images were analyzed using AFM accessory software, and the images presented were flattened only when necessary. The CC solution was purified by a 0.22 µm Millipore filter , deposited onto the wafer, and air-dried overnight before imaging. TEM images were obtained using a JEM-2010(HT) transmission electron microscope . Before TEM observation, the CC solution was cast onto a perforated carbon film, which was supported on a copper grid, and air-dried overnight. The concentration of CC for AFM and TEM measurements was 1.0 × 102+, Na+, OH− and H2O were introduced into the simulation process. The composition of the force field in the simulation included covalent bonds, angles, and twists, in addition to the interaction of electrostatic and hydrogen bonds ,551H NMRct of CC . Therefoh distributions. h distributions of CC in various solvent systems. Only one symmetrical peak existed in the CC solutions. As the NaOH content increased from 5 to 10 wt%, the Rh distributions of CC gradually narrowed, suggesting that the higher concentration of OH− was beneficial to the dissolution of CC 2 crystals gradually precipitated out of the CC solution when the solvent was 7 wt% NaOH/2.0 wt% ZnO hydrates. An appropriate amount of ZnO added to the 7 wt% NaOH solution could dissolve a greater quantity of CC. However, excessive ZnO added to the 7 wt% NaOH solution would worsen the dissolution effect. h values of CC in NaOH/ZnO aqueous solutions with various NaOH and ZnO contents. When the ZnO content was 1.6 wt%, the Rh values of CC decreased from 37.7 ± 2.2 to 18.0 ± 1.4 nm as the NaOH content increased from 5 to 10 wt%. When the NaOH content was fixed at 7 wt%, the Rh values of CC decreased from 43.7 ± 2.8 to 21.4 ± 1.6 nm as the ZnO content increased from 0 to 1.6 wt%. When the ZnO content attained 2.0 wt%, the Rh of CC chains increased to 29.0 ± 2.1 nm due to the instability of the solution. Rh reflects the solvatization of CC [The dissolved state of CC in NaOH/ZnO aqueous solutions was also verified by DLS, which was evidenced by its Ron of CC a. As dison of CC . The str42− hydrates played the auxiliary role in the dissolution of CC.Under strictly controlled and identical drying conditions, the CC solutions lose water and the CC shrink during the drying process, resulting in the aggregation of nanofibers ,58. To i2O 42− hydrates, and CC-NaOH/ZnO interactions could be formed in the solution 4 systems significantly improved the solubility of CC and the stability of CC solution, due to the strong interactions between the Zn(OH)42− hydrates and the hydroxyl groups of CC. Therefore, 7 wt% NaOH/1.6 wt% ZnO aqueous solution was proven to be the most appropriate solvent for CC, and this work provides a theoretical basis for the application of the CarbaCell process.CC can be dissolved in NaOH/ZnO aqueous systems via a freezing–thawing method to form transparent solutions. This was found to be a typical enthalpy-driven process, and low temperature was favorable for the dissolution of CC. With the increase of NaOH and ZnO contents, the amount of hydrates increased; the interaction of CC-NaOH and CC-NaOH/ZnO systems also increased. Thus, the melting enthalpies of the frozen mass increased. ZnO existed as Zn(OH)


L-benzyl tyrosine thiol carboxylic acid analogues (2a–2k) were synthesized after the study of computational simulation by adding of methyl, chloro, bromo and nitro groups to the benzyl ring for investigation of SAR analysis. Although the synthesized molecules 2a–k shows the potent inhibitory effects against metallo-β-lactamase (IMP-1) with the range of icK values of 1.04–4.77 µM, they are not as potent as the candidate inhibitor.The emergence of drug-resistant pathogenic bacteria is occurring due to the global overuse and misuse of β-lactam antibiotics. Infections caused by some bacteria which secrete metallo-β-lactamases (enzymes that inactivate β-lactam antibiotics) are increasingly prevalent and have become a major worldwide threat to human health. These bacteria are resistant to β-lactam antibiotics and MBL-inhibitor/β-lactam antibiotic combination therapy can be a strategy to overcome this problem. So far, no clinically available inhibitors of metallo-β-lactamases (MBLs) have been reported. In this study,   The β-lactam antibiotics are the most frequently prescribed drugs used in the treatment of bacterial infections since the 1940s. The β-lactam antibiotic resistance crisis has been attributed to the global overuse and misuse of these medications. The study of classifications β-lactam antibiotics and mechanisms of their inactivation by resistant bacteria can assist scientists to design a strategy to overcome this problem. So far, the β-lactamases have been classified to serine-β-lactamases  and metallo-β-lactamases  based on their mechanisms of action. Moreover, MBLs are further divided into three subclasses, B1, B2 and B3, which differ in their amino acid sequences and metal occupancies.In total, all MBLs are characterised by αββα quaternary structural fold and zinc ions of the active sites are located at the edge of the ββ sheets at a distance measurement range nearly 3.4–4.4 ÅPseudomonas aeruginosa and Serratia marcescens. In the active site of this enzyme, Zinc 1 is coordinated by a water molecule and three identical amino acids  with tetrahedral arrangement while Zinc 2 is coordinated by two water molecules and three different amino acids  in distorted trigonal bipyramidal geometry. The proposed mechanism in In this research, only IMP-1, a subclass B1 enzyme, was only investigated. IMP-1 is secreted by bacteria such as One strategy to overcome this problem is to find a potent inhibitor that can attach to the active site of IMP-1 and prevent the hydrolysis of the β-lactam rings of antibiotics. Although numerous MBLs inhibitors have been reported in the literature, there is no clinically available inhibitor. Moreover, none of the clinically useful serine-β-lactamases inhibitors, such as clavulanic acid, which is co-administered with penicillin is not effective against MBLs.Ki values in a range of 7–19 µM against NDM-1. The co-crystallised enzyme/inhibitors of these compounds demonstrated that the sulphur atom of the mercaptomethyl group is positioned nearly equidistant between the two zinc ions, displacing the bridging water/hydroxide molecule that is proposed to be the attacking nucleophile, but with little impact on the Zn1 − Zn2 distance.Ki value of 41 µM against GOB-18.Ki values in a range of 6–14 µM. Arjomandi et al.Kic value of 86 nM against IMP-1. In this study, this inhibitor was used as a candidate molecule for further modification and development with the aim of improving inhibition activity.Thiol carboxylic acid compounds have been reported as the most common inhibitors of IMP-1.1 can be exerted and investigated for the aim the study of improving inhibition activity. The study of computational modelling predicts the thiol group of the candidate inhibitor is coordinated to both Zinc ions of the active site of the enzyme which is probably one of the key interactions for strong chelating. Therefore, the substitution of the carboxylic acid group with a thiol moiety may improve the potency of the inhibitory activity as there will be two thiol groups for metal chelating. On the other hand, computational simulation is only a predicted model and does not reveal the real configuration of the ligand and its interactions with the amino acids of the active site, thus, the replacement of the thiol group with the carboxylic acid can also be investigated when the modified molecule would have two carboxylic acid groups. The alternative modification is the substitution of hydrogen(s) of the methylene groups of the skeleton with the electron-donating and electron-withdrawing groups or hydrogen acceptor and donor moieties and examination of their interactions with the amino acids of the active site for the study of a better fixing in the active site. The subsequent modification is the substituting of the central phenyl ring with heteroaromatic rings which may lead to a better fixing of the molecule into the active site due to the creation of further interactions like hydrogen bond(s). Moreover, removing either one or two methylene groups of the skeleton reduce the size of the molecule that may create a better conformation and configuration with the creation of stronger interactions with Zinc ions or amino acids of the active site. The other modification is the study of addition of hydrogen acceptor and donor groups or electron withdrawing or electron donating moieties on either central phenyl ring or the tale benzyl ring. In this study, the latest modification was investigated by the addition of methyl, chloro, bromo and nitro groups to the benzyl ring of the candidate molecule  of all analogues were higher and better than the lead compound. On the other hand, the predicted distance of the coordination of the thiol group of most analogues to Zinc 2 is shorter than Inhibitor 1 which can be considered as one of the key factors for stronger chelation. The predicted distance between Zinc 1 and the thiol group of target molecules and the reference ligand is nearly unaffected  was downloaded from Protein Data Bank (PDB) and Chain B was deleted as the topologies of the active sites of both chains A and B are identical. Further elimination for chain A was carried out by removing water molecules (solvents), single Zinc ion and the coordinated ligand of the active site. The cavity of the active site with two zinc ions was set by MolDock for the investigation of docking study. Compounds ractions . The calaffected . The docaffected . Altogetin vitro assays of MBLs inhibitors. CENTA,The IMP-1 enzyme, lacking the first 21 signal peptide amino acid residues, was expressed and purified using the protocol of Vella et al.50 values) of the inhibitors 2a–k were measured by a screening assay which evaluated the residual activity of the enzyme. The assay results showed that all compounds 2a–k exhibited approximately 50% inhibition at the range of concentration of 1–5 µM. Further kinetic analyses then allowed the determination of icK values of these compounds based on the procedure reported by Arjomandi et al.λ = 235 nm.50 values) was performed in duplicate, with benzyl penicillin as the substrate and HEPESX  as the buffer at 25 °C. The final concentrations of enzyme and benzyl penicillin were 10 nM and 500 µM, respectively. Bovine serum albumin  was added to the enzyme for stability. For each well, 196 µL substrate and 4 µL inhibitor were added followed by the addition of 200 µL of enzyme. The rate of the hydrolysis of substrate was recorded for 5 min. To measure icK value, four solutions with different concentrations for each molecule were prepared and then were assayed based on the aforementioned procedure while the concentrations of substrate in wells were 200, 400, 600, 800 and 1000 µM. The raw kinetic data were analysed by non-linear regression using the Win Curve Fit program (Kevin Raner Software) and the icK value was calculated for each molecule.icK values of molecules 2a–k are in the range of 1.04–4.77 µM which was unexpected based on the computational modelling results. Although the introduced compounds are still potent enzyme-based inhibitors, they are nearly at least ten times less as potent as the lead inhibitor is. Due to the less inhibition activities of the synthesized molecules in compare to the reference compound, their cell-based assays have not carried out.Benzyl penicillin solutions were prepared fresh at the time of assay as their activities will change and stop after 24 h even with keeping in the fridge.2a–c are better enzyme-based inhibitors when compared compounds 2d–k. In particular ortho and para substituted compounds 2a and 2c are similar and better than meta-substituted compound 2b which indicates the influence of electron donating group. Although the docking poses failed to show any interaction of the benzyl group in the receptor site, apparently a monosubstituted electron donating group on benzyl ring can lead to a better and stronger affinity of the inhibitor in the active site while electron withdrawing one decrease that affinity.Among the reported molecules, compounds with methyl groups 2a–k are potent inhibitors against IMP-1 and the ortho-substituted compounds on benzyl ring show the highest inhibitory activities than meta- and para-substituted ones . Consequently, the addition of either electron donor or electron withdrawing group to the benzyl ring can decrease the potency of inhibitors against IMP-1.To compare the information of the computational modelling and the achieved experimental data, the inc ions . Althoug2a–2k was similar to what had been reported by Arjomandi et al.L-tyrosine methyl ester 3 was used as the precursor to reduce the number of steps in the synthesis  was synthesized by the procedure that was reported by Arjomandi et al.L-amino acid methyl esters 6a–k and acetylthio acetic acid 7 was carried out by HBTU as a coupling reagent and DIPEA as a base to produce compounds 8a–k which then purified by flash column chromatography.1H NMR signal appears at 2.7 ppm. To remove this impurity, running a mixture of 30–70% petroleum ether–ethyl acetate solvent in flash column chromatography can take out tetra methyl urea as well as other impurities. The monitoring of the fraction samples by TLC indicated that no product(s) is available. The final products then were collected with increase the percentage of the polar component of the solvent. Intermediates 8a–k are new compounds, and their 1H NMR and 13 C-NMR spectra (as observed by the appearance of the characteristic thioacetyl methyl group) as well as LRMS and HRMS (positive mode) were recorded and confirmed the constitutions of molecules. The final hydrolysed compounds 2a–k were prepared by the treatment of intermediates 8a–k with sodium hydroxide solution under nitrogen to give the final thiol carboxylic acid products , as well as LRMS and HRMS (negative mode), were recorded and confirmed the constitutions of molecules.The synthesis pathway of compounds ynthesis . CompounL-benzyl tyrosine thiol carboxylic acid 1. These compounds demonstrated potent enzyme-based in vitro inhibitory activities against IMP-1 with icK values of ranging 1.04 ± 0.28–4.77 ± 2.81 µM but they were less potent than the lead compound. The compounds with electron donating group to ortho, meta and para of the benzyl ring of candidate molecule show better inhibition than electron withdrawing one. The addition of substitution to the benzyl ring of the potent cell-based inhibitor 1 decreases its activity against IMP-1.We described a straightforward and efficient synthesis of analogues of δ scale and referenced to the residual solvent peak . Coupling constants (J) are reported in Hertz. Multiplicities of the peaks are abbreviated as follows: s for singlet, bs for broad singlet, d for doublet, t for triplet and q for quartet. Low- and high-resolution EI-MS were measured on a Finnigan MAT 900 XL-Trap mass spectrometer in the positive and the negative ionisation mode. LR-ESI were recorded on a Bruker HCT 3D Ion Trap and HR-ESI were performed on a BrukerMicrOTof-Q with the DIONEX Ultimate 3000 LC in the positive and the negative electrospray ionisation mode, with CH3OH as a solvent. For IR analyses, samples were measured neat on a PerkinElmer Frontier FT-IR spectrometer .All chemicals were purchased from Sigma-Aldrich , Merck  and Fluka Chemical Companies . All NMR experiments were recorded on Bruker AVANCE 500, 400 or 300 MHz spectrometers . Chemical shifts are reported in parts per million (ppm) on a 5a–k. A mixture of N-Boc-L-tyrosine methyl ester , benzyl derivative 4a–k (13.5 mmol), potassium carbonate  and sodium iodide  in acetone (50 mL) was heated under reflux, under nitrogen, for 24 h. The resulting mixture was then filtrated and evaporated under the vacuum to give the crude oil which was purified by flash chromatography (30–100% EtOAc in petroleum ether).The general procedure reported by Wolf et al.6a–k. A mixture of L-Boc protected compounds 5a–k (5 mmol), chloroform (30 mL) and TFA  was stirred at room temperature, and the progress of the reaction was monitored by TLC. Then the organic solution was washed with saturated sodium bicarbonate (30 mL), dried over sodium sulphate and evaporated under the vacuum to give the products 6a–k which was used for the next step without further purification.The general procedure reported by Benthem et al.8a–k. A mixture of deprotected Boc compounds 6a–k (4.0 mmol), 2-(acetylthio)acetic acid 7 , HBTU  and DIPEA  in THF (30 mL) was stirred at room temperature for 24 h. Then the organic solvent was evaporated under the vacuum, and the residue was dissolved in DCM (50 mL). The organic phase was then washed with saturated sodium bicarbonate (2 × 50 mL) and evaporated under the vacuum to give the crude oil which was purified by flash chromatography (30–100% EtOAc in petroleum ether) to give the pure products 8a–k.The general method reported by Knorr et al.2a–k were synthesized using the following general procedure: compounds 8a–k (3.0 mmol) were dissolved in methanol (10 mL), then sodium hydroxide solution  was added and the solution was stirred under nitrogen for an hour. The organic solvent (methanol) was evaporated under the vacuum, then water (50 mL) was added and the combined water phase was washed with ethyl acetate (50 mL) and DCM (50 mL) under nitrogen. Then pH was adjusted to 1 using concentrated HCl. The product in the water phase was extracted with ethyl acetate (50 mL) and washed with water (5 × 50 mL)2a–k. All 1H NMR, 13 C NMR, LRMS and HRMS (negative mode) spectra were recorded.Compounds 1H NMR : δ 7.34 , 7.26–7.16 , 7.03 , 6.89 , 4.98 , 4.96 , 4.54 , 3.70 , 3.04 , 2.99 , 2.35 , 1.40 3). 13 C NMR : δ 172.4 (CO2CH3), 158.0 (CO2Boc), 155.1 (Ar-C), 136.7 (Ar-C), 134.7 (Ar-C), 130.4 (Ar-C), 130.2 (Ar-C), 128.6 (Ar-C), 128.2 (Ar-C), 128.2 (Ar-C), 126.0 (Ar-C), 114.8 (Ar-C), 79.9 (C(CH3)3), 68.6 (CH2O), 54.5 (CH2CHNH), 52.1 (CH3OCO), 37.5 (CH2CHNH), 28.3 (C(CH3)3), 18.8 (CH3Ph). HRMS calculated for C23H29NNaO5 [M + Na]+ 422.1938, found 422.1939.Pale yellow viscous oil, 2.17 g (80%). 1H NMR : δ 7.39 , 7.27–7.17 , 7.11 , 6.93 , 5.00 , 3.71 , 3.70 , 3.04 , 2.84 , 2.36 , 1.97 . 13 C NMR : δ 175.3 (CO), 157.9 (Ar-C), 136.6 (Ar-C), 134.7 (Ar-C), 130.3 (Ar-C), 130.3 (Ar-C), 129.2 (Ar-C), 128.6 (Ar-C), 128.2 (Ar-C), 126.0 (Ar-C), 114.8 (Ar-C), 68.6 (CH2O), 55.8 (CH2CHNH), 52.0 (CH3O), 39.9 (CH2CHNH), 18.8 (CH3Ph). HRMS calculated for C18H22NO3 [M + H]+ 300.1594, found 300.1595.Orange solid, 1.47 g (98%). 1H NMR : δ 7.37 , 7.27–7.17 , 6.99 , 6.88 , 6.60 , 4.98 , 4.77 , 3.71 , 3.52 , 3.08 , 3.00 , 2.35 , 2.32 . 13 C NMR : δ 195.2 (COS), 171.6 (CO2), 167.6 (CONH), 158.0 (Ar-C), 136.6 (Ar-C), 134.7 (Ar-C), 130.4 (Ar-C), 130.3 (Ar-C), 128.6 (Ar-C), 128.3 (Ar-C), 127.8 (Ar-C), 126.0 (Ar-C), 114.8 (Ar-C), 68.5 (CH2O), 53.5 (CH2CHNH), 52.3 (CH3O), 36.7 (CH2CHNH), 32.7 (CH2S), 30.1 (CH3CO), 18.8 (CH3Ph). HRMS calculated for C22H25NNaO5S [M + Na]+ 438.1346, found 438.1349.Pale yellow viscous oil, 1.33 g (80%). 1H NMR : δ 7.34 , 7.24–7.10 , 6.99 , 5.00 , 4.62 , 3.14 , 313 , 2.95 , 2.33 . 13 C NMR : δ 174.4 (CO2H), 172.9 (CONH), 159.3 (Ar-C), 138.0 (Ar-C), 136.4 (Ar-C), 131.5 (Ar-C), 131.3 (Ar-C), 130.3 (Ar-C), 129.7 (Ar-C), 129.2 (Ar-C), 126.9 (Ar-C), 115.8 (Ar-C), 69.7 (PhCH2O), 55.3 (CH2CHNH), 37.5 (CH2CHNH), 28.0 (CH2SH), 18.9 (CH3). HRMS calculated for C19H20NO4S [M-H]− 358.1119, found 358.1125.Off white solid, 0.66 g (61%). 1H NMR : δ 7.28–7.17 , 7.12 , 7.02 , 6.88 , 4.98 , 4.94 , 4.33 , 3.69 , 3.07–2.93 , 2.35 , 1.40 3). 13 C NMR : δ 172.4 (CO2CH3), 158.0 (CO2Boc), 155.1 (Ar-C), 138.3 (Ar-C), 136.9 (Ar-C), 130.3 (Ar-C), 128.7 (Ar-C), 128.5 (Ar-C), 128.2 (Ar-C), 128.2 (Ar-C), 124.6 (Ar-C), 114.9 (Ar-C), 79.9 (C(CH3)3), 70.1 (CH2O), 54.5 (CH2CHNH), 52.2 (CH3OCO), 37.5 (CH2CHNH), 28.3 (C(CH3)3), 21.4 (CH3Ph). HRMS calculated for C23H29NNaO5 [M + Na]+ 422.1938, found 422.1935.Pale yellow viscous oil, 2.17 g (80%). 1H NMR : δ 7.91 , 7.24 , 7.20 , 7.17 , 7.11 , 7.07 , 6.91 , 4.94 , 4.23 , 3.73 , 3.23 , 3.13 , 2.34 . 13 C NMR : δ 169.2 (CO), 158.8 (Ar-C), 138.3 (Ar-C), 136.5 (Ar-C), 130.3 (Ar-C), 128.9 (Ar-C), 128.5 (Ar-C), 128.3 (Ar-C), 124.6 (Ar-C), 124.6 (Ar-C), 115.6 (Ar-C), 70.1 (CH2O), 54.5 (CH2CHNH), 53.4 (CH3O), 35.2 (CH2CHNH), 21.4 (CH3Ph). HRMS calculated for C18H22NO3 [M + H]+ 300.1594, found 300.1592.Olive green solid, 1.47 g (98%). 1H NMR : δ 7.29–7.16 , 7.12 , 6.98 , 6.86 , 6.62 , 4.97 , 4.75 , 3.71 , 3.52 , 3.07 , 2.99 , 2.35 , 2.31 . 13 C NMR : δ 195.2 (COS), 171.6 (CO2), 167.7 (CONH), 158.0 (Ar-C), 138.3 (Ar-C), 136.8 (Ar-C), 130.3 (Ar-C), 128.7 (Ar-C), 128.5 (Ar-C), 128.2 (Ar-C), 127.7 (Ar-C), 124.6 (Ar-C), 114.9 (Ar-C), 70.0 (CH2O), 53.5 (CH2CHNH), 52.4 (CH3O), 36.7 (CH2CHNH), 32.7 (CH2S), 30.1 (CH3CO), 21.4 (CH3Ph). HRMS calculated for C22H25NNaO5S [M + Na]+ 438.1346, found 438.1343.Viscous yellow oil, 1.33 g (80%). 1H NMR : δ 7.25–7.16 , 7.15–7.08 , 6.89 , 4.99 , 4.61 , 3.14 , 3.12 , 2.93 , 2.33 . 13 C NMR : δ 174.4 (CO2H), 172.9 (CONH), 159.3 (Ar-C), 139.3 (Ar-C), 138.7 (Ar-C), 131.4 (Ar-C), 130.3 (Ar-C), 129.5 (Ar-C), 129.4 (Ar-C), 129.1 (Ar-C), 125.6 (Ar-C), 115.9 (Ar-C), 71.0 (PhCH2O), 55.3 (CH2CHNH), 37.5 (CH2CHNH), 28.0 (CH2SH), 21.4 (CH3). HRMS calculated for C19H20NO4S [M-H]− 358.1119, found 358.1129.Off white solid, 0.70 g (65%). 1H NMR : δ 7.29 , 7.17 , 7.01 , 6.87 , 4.97 , 4.94 , 4.61–4.44 , 3.68 , 3.02–2.92 , 2.34 , 1.40 3). 13 C NMR : δ 172.4 (CO2CH3), 158.0 (CO2Boc), 155.1 (Ar-C), 137.7 (Ar-C), 133.9 (Ar-C), 130.3 (Ar-C), 129.3 (Ar-C), 128.1 (Ar-C), 127.6 (Ar-C), 114.9 (Ar-C), 79.9 (C(CH3)3), 69.9 (CH2O), 54.5 (CH2CHNH), 52.2 (CH3OCO), 37.5 (CH2CHNH), 28.3 (C(CH3)3), 21.2 (CH3Ph). HRMS calculated for C23H29NNaO5 [M + Na]+ 422.1938, found 422.1936.Off white solid, 2.44 g (90%). 1H NMR : δ 7.29 , 7.17 , 7.07 , 6.89 , 4.97 , 3.74 , 3.70 , 3.03 , 2.84 , 2.68 , 2.34 . 13 C NMR : δ 174.5 (CO), 158.0 (Ar-C), 137.7 (Ar-C), 133.9 (Ar-C), 130.3 (Ar-C), 129.3 (Ar-C), 128.6 (Ar-C), 127.6 (Ar-C), 115.0 (Ar-C), 69.9 (CH2O), 55.6 (CH2CHNH), 52.2 (CH3O), 39.4 (CH2CHNH), 21.2 (CH3Ph). HRMS calculated for C18H22NO3 [M + H]+ 300.1594, found 300.1591.Pale yellow viscous oil, 1.47 g (98%). 1H NMR : δ 7.28 , 7.17 , 6.97 , 6.86 , 6.57 , 4.97 , 4.75 , 3.70 , 3.51 , 3.06 , 2.99 , 2.34 , 2.31 . 13 C NMR : δ 195.2 (COS), 171.6 (CO2), 167.6 (CONH), 158.0 (Ar-C), 137.8 (Ar-C), 133.9 (Ar-C), 130.3 (Ar-C), 129.3 (Ar-C), 127.7 (Ar-C), 127.6 (Ar-C), 114.9 (Ar-C), 69.9 (CH2O), 53.5 (CH2CHNH), 52.3 (CH3O), 36.8 (CH2CHNH), 32.8 (CH2S), 30.1 (CH3CO), 21.2 (CH3Ph). HRMS calculated for C22H25NNaO5S [M + Na]+ 438.1346, found 438.1342.Off white solid, 1.36 g (82%). 1H NMR : δ 7.26 , 7.19–7.06 , 6.88 , 4.96 , 4.61 , 3.13 , 3.11 , 2.93 , 2.31 . 13 C NMR : δ 174.4 (CO2H), 172.9 (CONH), 159.2 (Ar-C), 138.7 (Ar-C), 135.7 (Ar-C), 131.4 (Ar-C), 130.2 (Ar-C), 130.1 (Ar-C), 128.7 (Ar-C), 115.9 (Ar-C), 70.9 (PhCH2O), 55.3 (CH2CHNH), 37.5 (CH2CHNH), 28.0 (CH2SH), 21.2 (CH3), 1.90 . HRMS calculated for C19H20NO4S [M-H]- 358.1191, found 358.1192.Off white solid, 0.73 g (68%). 1H NMR : δ 7.53 , 7.38 , 7.30–7.20 , 7.03 , 6.88 , 5.12 , 4.96 , 4.53 , 3.69 , 3.04 , 2.97 , 1.40 3). 13 C NMR : δ 172.4 (CO2CH3), 157.6 (NHCO2), 155.1 (Ar-C), 134.7 (Ar-C), 132.5 (Ar-C), 130.4 (Ar-C), 129.3 (Ar-C), 128.9 (Ar-C), 128.7 (Ar-C), 128.5 (Ar-C), 126.9 (Ar-C), 114.9 (Ar-C), 79.9 (C(CH3)3), 67.1 (CH2O), 54.5 (CH2CHNH), 52.2 (CH3O), 37.5 (CH2CHNH), 28.3 (C(CH3)3). HRMS calculated for C22H26ClNNaO5 [M + Na]+ 442.1392, found 442.1397.Off white solid, 2.34 g (82%). 1H NMR : δ 7.53 , 7.37 , 7.28–7.20 , 7.10 , 6.90 , 5.12 , 3.69 , 3.01 , 2.81 , 1.67 . 13 C NMR : δ 175.3 (CO), 157.4 (Ar-C), 134.7 (Ar-C), 132.5 (Ar-C), 130.3 (Ar-C), 129.6 (Ar-C), 129.3 (Ar-C), 128.9 (Ar-C), 128.7 (Ar-C), 126.9 (Ar-C), 114.9 (Ar-C), 67.1 (CH2O), 55.8 (CH2CHNH), 51.9 (CH3O), 40.0 (CH2CHNH). HRMS calculated for C17H19ClNO3 [M + H]+ 320.1048, found 320.1049.Green viscous oil, 1.57 g (98%). 1H NMR : δ 7.53 , 7.37 , 7.28–7.20 , 7.00 , 6.88 , 6.60 , 5.12 , 4.75 , 3.70 , 3.51 , 3.07 , 2.99 , 2.32 . 13 C NMR : δ 195.1 (COS), 171.6 (CO2), 167.6 (CONH), 157.6 (Ar-C), 134.6 (Ar-C), 132.5 (Ar-C), 130.3 (Ar-C), 129.3 (Ar-C), 129.0 (Ar-C), 128.7 (Ar-C), 128.1 (Ar-C), 126.9 (Ar-C), 114.8 (Ar-C), 67.1 (CH2O), 53.5 (CH2CHNH), 52.3 (CH3O), 36.7 (CH2CHNH), 32.7 (CH2S), 30.1 (CH3CO). HRMS calculated for C21H22ClNNaO5S [M + Na]+ 458.0799, found 458.0803.Yellow viscous oil, 1.43 g (82%). 1H NMR : δ 7.52 , 7.40 , 7.31–7.25 , 7.14 , 6.90 , 5.10 , 4.62 , 3.14 , 3.12 , 2.95 . 13 C NMR : δ 174.3 (CO2H), 172.9 (CONH), 159.0 (Ar-C), 136.1 (Ar-C), 134.0 (Ar-C), 131.5 (Ar-C), 130.6 (Ar-C), 130.4 (Ar-C), 130.4 (Ar-C), 130.3 (Ar-C), 128.1 (Ar-C), 115.9 (Ar-C), 68.3 (PhCH2O), 55.3 (CH2CHNH), 37.5 (CH2CHNH), 28.0 (CH2SH). HRMS calculated for C18H17ClNO4S [M-H]− 378.0572, found 378.0566.Tan solid, 0.79 g (69%). 1H NMR : δ 7.41 , 7.28 , 7.02 , 6.86 , 4.99 , 4.96 , 4.53 , 3.69 , 3.03 , 2.98 , 1.40 3). 13 C NMR : δ 172.4 (CO2CH3), 157.5 (NHCO2), 155.1 (Ar-C), 139.1 (Ar-C), 134.5 (Ar-C), 130.3 (Ar-C), 129.8 (Ar-C), 128.6 (Ar-C), 128.0 (Ar-C), 127.4 (Ar-C), 125.3 (Ar-C), 114.9 (Ar-C), 79.9 (C(CH3)3), 69.1 (CH2O), 54.5 (CH2CHNH), 52.2 (CH3O), 37.5 (CH2CHNH), 28.3 (C(CH3)3). HRMS calculated for C22H26ClNNaO5 [M + Na]+ 442.1392, found 442.1394.Pale yellow oil, 2.28 g (80%). 1H NMR : δ 7.40 , 7.26 , 7.08 , 6.87 , 4.97 , 3.68 , 3.66 , 3.00 , 2.80 , 1.65 . 13 C NMR : δ 175.3 (CO), 157.3 (Ar-C), 139.0 (Ar-C), 134.4 (Ar-C), 130.2 (Ar-C), 129.7 (Ar-C), 129.6 (Ar-C), 127.9 (Ar-C), 127.3 (Ar-C), 125.2 (Ar-C), 114.8 (Ar-C), 69.0 (CH2O), 55.7 (CH2CHNH), 51.8 (CH3O), 40.0 (CH2CHNH). HRMS calculated for C17H19ClNO3 [M + H]+ 320.1048, found 320.1047.Yellow solid, 1.57 g (98%). 1H NMR : δ 7.40 , 7.29 , 6.99 , 6.85 , 6.59 , 4.99 , 4.76 , 3.70 , 3.51 , 3.07 , 2.99 , 2.32 . 13 C NMR : δ 195.2 (COS), 171.5 (CO2), 167.6 (CONH), 157.6 (Ar-C), 139.0 (Ar-C), 134.5 (Ar-C), 130.4 (Ar-C), 129.9 (Ar-C), 128.2 (Ar-C), 128.1 (Ar-C), 127.4 (Ar-C), 125.3 (Ar-C), 114.8 (Ar-C), 69.1 (CH2O), 53.5 (CH2CHNH), 52.4 (CH3O), 36.8 (CH2CHNH), 32.8 (CH2S), 30.1 (CH3CO). HRMS calculated for C21H22ClNNaO5S [M + Na]+ 458.0799, found 458.0800.Brown viscous oil, 1.45 g (83%). 1H NMR : δ 7.42 , 7.33–7.26 , 7.13 , 6.89 , 5.02 , 4.62 , 3.14 , 3.12 , 2.94 . 13 C NMR : δ 174.3 (CO2H), 172.9 (CONH), 158.9 (Ar-C), 141.2 (Ar-C), 135.4 (Ar-C), 131.5 (Ar-C), 131.0 (Ar-C), 130.6 (Ar-C), 128.8 (Ar-C), 128.3 (Ar-C), 126.6 (Ar-C), 115.9 (Ar-C), 70.0 (PhCH2O), 55.3 (CH2CHNH), 37.5 (CH2CHNH), 28.0 (CH2SH). HRMS calculated for C18H17ClNO4S [M-H]− 378.0572, found 378.0569.Tan solid, 0.77 g (68%). 1H NMR : δ 7.33 , 7.02 , 6.85 , 4.98 , 4.94 , 4.59–4.46 , 3.69 , 3.05 , 2.95 , 1.40 3). 13 C NMR : δ 172.4 (CO2CH3), 157.6 (NHCO2), 155.1 (Ar-C), 135.5 (Ar-C), 133.7 (Ar-C), 130.3 (Ar-C), 128.7 (Ar-C), 128.5 (Ar-C), 128.2 (Ar-C), 114.9 (Ar-C), 79.9 (C(CH3)3), 69.2 (CH2O), 54.5 (CH2CHNH), 52.2 (CH3O), 37.5 (CH2CHNH), 28.3 (C(CH3)3). HRMS calculated for C22H26ClNNaO5 [M + Na]+ 442.1392, found 442.1395.Yellow solid, 2.28 g (82%). 1H NMR : δ 7.33 , 7.08 , 6.87 , 4.98 , 3.69 ), 3.01 , 2.80 , 1.71 . 13 C NMR : δ 175.3 (CO), 157.5 (Ar-C), 135.5 (Ar-C), 133.7 (Ar-C), 130.3 (Ar-C), 129.6 (Ar-C), 128.9 (Ar-C), 128.7 (Ar-C), 114.9 (Ar-C), 69.2 (CH2O), 55.8 (CH2CHNH), 52.0 (CH3O), 40.0 (CH2CHNH). HRMS calculated for C17H19ClNO3 [M + H]+ 320.1048, found 320.1046.Brown solid, 1.57 g (98%). 1H NMR : δ 7.33 , 6.98 , 6.84 , 6.59 , 4.98 , 4.75 , 3.70 , 3.51 , 3.07 , 2.98 , 2.32 . 13 C NMR : δ 195.2 (COS), 171.6 (CO2), 167.7 (CONH), 157.7 (Ar-C), 135.4 (Ar-C), 133.8 (Ar-C), 130.4 (Ar-C), 130.3 (Ar-C), 128.7 (Ar-C), 128.1 (Ar-C), 114.9 (Ar-C), 69.2 (CH2O), 53.5 (CH2CHNH), 52.4 (CH3O), 36.8 (CH2CHNH), 32.7 (CH2S), 30.1 (CH3CO). HRMS calculated for C21H22ClNNaO5S [M + Na]+ 458.0799, found 458.0806.Brown viscous oil, 1.39 g (80%). 1H NMR : δ 7.39 , 7.34 , 7.13 , 6.89 , 5.02 , 4.61 , 3.14 , 3.13 , 2.94 . 13 C NMR : δ 174.3 (CO2H), 172.9 (CONH), 159.0 (Ar-C), 137.6 (Ar-C), 134.6 (Ar-C), 131.5 (Ar-C), 130.5 (Ar-C), 130.1 (Ar-C), 129.6 (Ar-C), 115.9 (Ar-C), 70.1 (PhCH2O), 55.3 (CH2CHNH), 37.5 (CH2CHNH), 28.00 (CH2SH). HRMS calculated for C18H17ClNO4S [M-H]− 378.0572, found 378.0557.Off white soild. 0.80 g (70%). 1H NMR : δ 8.13 , 7.85 , 7.65 , 7.44 , 7.03 , 6.88 , 5.44 , 4.97 , 4.52 , 3.69 , 3.04 , 2.97 , 1.39 3). 13 C NMR : δ 172.3 (CO2CH3), 157.2 (NHCO2), 155.0 (Ar-C), 146.8 (Ar-C), 134.0 (Ar-C), 133.9 (Ar-C), 130.4 (Ar-C), 128.9 (Ar-C), 128.5 (Ar-C), 128.3 (Ar-C), 124.9 (Ar-C), 114.9 (Ar-C), 79.9 (C(CH3)3), 66.8 (CH2O), 54.5 (CH2CHNH), 52.2 (CH3O), 37.4 (CH2CHNH), 28.3 (C(CH3)3). HRMS calculated for C22H26N2NaO7 [M + Na]+ 453.1632, found 453.1635.Pale yellow viscous oil, 2.78 g (95%). 1H NMR : δ 8.13 , 7.86 , 7.65 , 7.46 , 7.10 , 6.90 , 5.44 , 3.69 , 3.01 , 2.82 , 1.95 . 13 C NMR : δ 175.2 (CO), 157.1 (Ar-C), 146.9 (Ar-C), 134.0 (Ar-C), 133.9 (Ar-C), 130.4 (Ar-C), 129.9 (Ar-C), 128.5 (Ar-C), 128.3 (Ar-C), 124.9 (Ar-C), 115.0 (Ar-C), 66.8 (CH2O), 55.7 (CH2CHNH), 52.0 (CH3O), 39.9 (CH2CHNH). HRMS calculated for C17H19N2O5 [M + H]+ 331.1288, found 331.1290.Tan yellow viscous oil, 1.62 g (98%). 1H NMR : δ 8.13 , 7.86 , 7.66 , 7.47 , 7.00 , 6.88 , 6.59 , 5.43 , 4.76 , 3.70 , 3.51 , 3.08 , 2.99 , 2.33 . 13 C NMR : δ 195.2 (COS), 171.5 (CO2), 167.6 (CONH), 157.3 (Ar-C), 146.9 (Ar-C), 134.0 (Ar-C), 133.8 (Ar-C), 130.5 (Ar-C), 128.5 (Ar-C), 128.5 (Ar-C), 128.3 (Ar-C), 125.0 (Ar-C), 114.9 (Ar-C), 66.8 (CH2O), 53.5 (CH2CHNH), 52.4 (CH3O), 36.8 (CH2CHNH), 32.8 (CH2S), 30.2 (CH3CO). HRMS calculated for C21H22N2NaO7S [M + Na]+ 469.1040, found 469.1045.Pale yellow solid, 1.48 g (83%). 1H NMR : δ 8.22 , 8.12 , 7.84 , 7.64 , 7.45 , 7.13 , 7.00 , 6.89 , 5.41 , 4.80 , 3.20 , 3.16 , 3.07 , 1.83 . 13 C NMR : δ 177.0 (CO2H), 170.0 (CONH), 157.4 (Ar-C), 146.9 (Ar-C), 134.0 (Ar-C), 133.7 (Ar-C), 130.6 (Ar-C), 128.5 (Ar-C), 128.3 (Ar-C), 128.3 (Ar-C), 124.9 (Ar-C), 115.1 (Ar-C), 66.8 (PhCH2O), 53.5 (CH2CHNH), 36.4 (CH2CHNH), 28.1 (CH2SH). HRMS calculated for C18H17N2O6S [M-H]− 389.0813, found 389.0809.Pale yellow solid, 0.79 g (67%). 1H NMR : δ 8.27 , 8.15 , 7.72 , 7.52 , 7.03 , 6.86 , 5.09 , 4.98 , 4.51 , 3.67 , 3.03 , 2.96 , 1.38 3). 13 C NMR : δ 172.3 (CO2CH3), 157.2 (NHCO2), 155.0 (Ar-C), 148.3 (Ar-C), 139.2 (Ar-C), 133.0 (Ar-C), 130.4 (Ar-C), 129.5 (Ar-C), 128.9 (Ar-C), 122.8 (Ar-C), 122.0 (Ar-C), 114.8 (Ar-C), 79.8 (C(CH3)3), 68.5 (CH2O), 54.4 (CH2CHNH), 52.1 (CH3O), 37.4 (CH2CHNH), 28.2 (C(CH3)3). HRMS calculated for C22H26N2NaO7 [M + Na]+ 453.1632, found 453.1639.Yellow viscous oil, 2.63 g (90%). 1H NMR : δ 8.26 , 8.12 , 7.72 , 7.52 , 7.09 , 6.87 , 5.08 , 3.72 , 3.67 , 3.02 , 2.84 , 2.75 . 13 C NMR : δ 174.5 (CO), 157.1 (Ar-C), 148.3 (Ar-C), 139.1 (Ar-C), 133.0 (Ar-C), 130.4 (Ar-C), 129.5 (Ar-C), 122.8 (Ar-C), 122.0 (Ar-C), 121.2 (Ar-C), 114.8 (Ar-C), 68.5 (CH2O), 55.4 (CH2CHNH), 52.0 (CH3O), 39.3 (CH2CHNH). HRMS calculated for C17H19N2O5 [M + H]+ 331.1288, found 331.1286.Brown solid, 1.62 g (98%). 1H NMR : δ 8.28 , 8.15 , 7.73 , 7.54 , 7.00 , 6.88 , 6.62 , 5.10 , 4.74 , 3.69 , 3.51 , 3.07 , 2.98 , 2.33 . 13 C NMR : δ 195.2 (COS), 171.5 (CO2), 167.6 (CONH), 157.3 (Ar-C), 148.4 (Ar-C), 139.1 (Ar-C), 133.1 (Ar-C), 130.5 (Ar-C), 129.5 (Ar-C), 128.6 (Ar-C), 122.9 (Ar-C), 122.1 (Ar-C), 114.8 (Ar-C), 68.6 (CH2O), 53.5 (CH2CHNH), 52.4 (CH3O), 36.8 (CH2CHNH), 32.7 (CH2S), 30.1 (CH3CO). HRMS calculated for C21H22N2NaO7S [M + Na]+ 469.1040, found 469.1045.Yellow solid, 1.45 g (81%). 1H NMR : δ 10.56 , 8.25 , 8.13 , 7.71 , 7.52 , 7.20 , 7.09 , 6.88 , 5.07 , 4.80 , 3.20 , 3.16 , 3.07 , 1.84 . 13 C NMR : δ 174.8 (CO2H), 170.3 (CONH), 157.4 (Ar-C), 148.3 (Ar-C), 139.0 (Ar-C), 133.1 (Ar-C), 130.5 (Ar-C), 129.5 (Ar-C), 128.2 (Ar-C), 122.9 (Ar-C), 122.1 (Ar-C), 114.9 (Ar-C), 68.5 (PhCH2O), 53.5 (CH2CHNH), 36.3 (CH2CHNH), 28.0 (CH2SH). HRMS calculated for C18H17N2O6S [M-H]− 389.0813, found 389.0805.Pale yellow solid, 0.82 g (70%). 1H NMR : δ 8.21 , 7.56 , 7.03 , 6.86 , 5.12 , 4.96 , 4.50 , 3.68 , 3.03 , 2.97 , 1.38 3). 13 C NMR : δ 172.3 (CO2CH3), 157.2 (NHCO2), 155.0 (Ar-C), 147.5 (Ar-C), 144.5 (Ar-C), 130.4 (Ar-C), 129.0 (Ar-C), 127.5 (Ar-C), 123.8 (Ar-C), 114.8 (Ar-C), 79.9 (C(CH3)3), 68.6 (CH2O), 54.5 (CH2CHNH), 52.2 (CH3O), 37.4 (CH2CHNH), 28.2 (C(CH3)3). HRMS calculated for C22H26N2NaO7 [M + Na]+ 453.1632, found 453.1636.Brown viscous oil, 2.69 g (92%). 1H NMR : δ 8.16 , 7.80 , 7.53 , 7.08 , 6.87 , 5.05 , 4.28 , 3.77 , 3.26 , 3.12 . 13 C NMR : δ 169.5 (CO), 158.1 (Ar-C), 147.4 (Ar-C), 144.3 (Ar-C), 130.4 (Ar-C), 127.5 (Ar-C), 125.1 (Ar-C), 123.8 (Ar-C), 115.5 (Ar-C), 68.5 (CH2O), 54.7 (CH2CHNH), 53.6 (CH3O), 35.2 (CH2CHNH). HRMS calculated for C17H19N2O5 [M + H]+ 331.1288, found 331.1287.Dark brown solid, 1.62 g (98%). 1H NMR : δ 8.21 , 7.57 , 7.00 , 6.85 , 6.60 , 5.11 , 4.75 , 3.69 , 3.51 , 3.07 , 2.97 , 2.33 . 13 C NMR : δ 195.1 (COS), 171.5 (CO2), 167.5 (CONH), 157.2 (Ar-C), 147.5 (Ar-C), 144.4 (Ar-C), 130.4 (Ar-C), 128.6 (Ar-C), 127.5 (Ar-C), 123.8 (Ar-C), 114.8 (Ar-C), 68.6 (CH2O), 53.5 (CH2CHNH), 52.3 (CH3O), 36.8 (CH2CHNH), 32.7 (CH2S), 30.1 (CH3CO). HRMS calculated for C21H22N2NaO7S [M + Na]+ 469.1040, found 469.1042.Brown viscous oil, 1.48 g (83%). 1H NMR : δ 9.43 , 8.21 , 7.57 , 7.09 , 6.88 , 5.12 , 4.81 , 3.21 , 3.17 , 3.08 , 1.81 . 13 C NMR : δ 175.4 (CO2H), 169.9 (CONH), 157.4 (Ar-C), 147.6 (Ar-C), 144.3 (Ar-C), 130.6 (Ar-C), 128.3 (Ar-C), 127.6 (Ar-C), 123.8 (Ar-C), 115.0 (Ar-C), 68.6 (PhCH2O), 53.5 (CH2CHNH), 36.4 (CH2CHNH), 28.1 (CH2SH). HRMS calculated for C18H17N2O6S [M-H]− 389.0813, found 389.0809.Brown solid, 0.84 g (72%). 1H NMR : δ 7.56 , 7.43 , 7.32 , 7.22 , 7.02 , 6.86 , 4.98 , 4.94 , 4.53 , 3.68 , 3.03 , 2.97 , 1.40 3). 13 C NMR : δ 172.4 (CO2CH3), 157.5 (NHCO2), 155.1 (Ar-C), 139.3 (Ar-C), 131.0 (Ar-C), 130.4 (Ar-C), 130.3 (Ar-C), 130.1 (Ar-C), 128.6 (Ar-C), 125.8 (Ar-C), 122.6 (Ar-C), 114.9 (Ar-C), 79.9 (C(CH3)3), 69.1 (CH2O), 54.5 (CH2CHNH), 52.2 (CH3O), 37.5 (CH2CHNH), 28.3 (C(CH3)3). HRMS calculated for C22H26BrNNaO5 [M + Na]+ 486.0887, found 486.0893.Tan viscous oil, 2.84 g (90%). 1H NMR : δ 7.56 , 7.42 , 7.32 , 7.22 ,7.09 , 6.87 , 4.98 , 3.69 (4H: 3H-singlet for CH3O and 1H-multiplet for CH2CHNH), 3.01 , 2.82 , 1.90 . 13 C NMR : δ 175.2 (CO), 157.4 (Ar-C), 139.3 (Ar-C), 130.9 (Ar-C), 130.3 (Ar-C), 130.3 (Ar-C), 130.1 (Ar-C), 129.6 (Ar-C), 125.8 (Ar-C), 122.6 (Ar-C), 114.9 (Ar-C), 69.0 (CH2O), 55.7 (CH2CHNH), 52.0 (CH3O), 39.9 (CH2CHNH). HRMS calculated for C17H19BrNO3 [M + H]+ 364.0543, found 364.0547.Brown solid, 1.78 g (98%). 1H NMR : δ 7.56 , 7.42 , 7.31 , 7.22 , 6.98 , 6.84 , 6.61 , 4.97 , 4.75 , 3.69 , 3.51 , 3.07 , 2.98 , 2.32 . 13 C NMR : δ 195.2 (COS), 171.5 (CO2), 167.6 (CONH), 157.5 (Ar-C), 139.3 (Ar-C), 131.0 (Ar-C), 130.3 (Ar-C), 130.3 (Ar-C), 130.1 (Ar-C), 128.2 (Ar-C), 125.8 (Ar-C), 122.6 (Ar-C), 114.8 (Ar-C), 69.0 (CH2O), 53.5 (CH2CHNH), 52.3 (CH3O), 36.7 (CH2CHNH), 32.7 (CH2S), 30.1 (CH3CO). HRMS calculated for C21H22BrNNaO5S [M + Na]+ 502.0294, found 502.0291.Tan viscous oil, 1.63 g (85%). 1H NMR : δ 7.57 , 7.43 , 7.37 , 7.25 ,7.13 , 6.89 , 5.01 , 4.61 , 3.14 , 3.12 , 2.94 . 13 C NMR : δ 174.3 (CO2H), 172.9 (CONH), 158.9 (Ar-C), 141.5 (Ar-C), 131.8 (Ar-C), 131.5 (Ar-C), 131.3 (Ar-C), 131.3 (Ar-C), 130.6 (Ar-C), 127.1 (Ar-C), 123.4 (Ar-C), 115.9 (Ar-C), 69.9 (PhCH2O), 55.3 (CH2CHNH), 37.5 (CH2CHNH), 28.0 (CH2SH). HRMS calculated for C18H17BrNO4S [M-H]− 422.0067, found 422.0071.Off white solid, 0.89 g (70%). 1H NMR : δ 7.48 , 7.27 , 7.01 , 6.85 , 4.96 , 4.94 , 4.52 , 3.68 , 3.03 , 2.97 , 1.39 3). 13 C NMR : δ 172.4 (CO2CH3), 157.6 (NHCO2), 155.1 (Ar-C), 136.0 (Ar-C), 131.7 (Ar-C), 130.3 (Ar-C), 129.0 (Ar-C), 128.5 (Ar-C), 121.8 (Ar-C), 114.9 (Ar-C), 79.9 (C(CH3)3), 69.2 (CH2O), 54.5 (CH2CHNH), 52.2 (CH3O), 37.5 (CH2CHNH), 28.3 (C(CH3)3). HRMS calculated for C22H26BrNNaO5 [M + Na]+ 486.0887, found 486.0896.Off white solid, 2.90 g (92%). 1H NMR : δ 7.50 , 7.30 , 7.10 , 6.88 , 4.96 , 3.69 , 3.67 , 3.01 , 2.81 . 13 C NMR : δ 175.6 (CO), 157.6 (Ar-C), 136.2 (Ar-C), 131.8 (Ar-C), 130.5 (Ar-C), 129.8 (Ar-C), 129.2 (Ar-C), 122.0 (Ar-C), 115.1 (Ar-C), 69.4 (CH2O), 56.0 (CH2CHNH), 52.1 (CH3O), 40.2 (CH2CHNH). HRMS calculated for C17H19BrNO3 [M + H]+ 364.0543, found 364.0546.Brown solid, 1.78 g (98%). 1H NMR : δ 7.48 , 7.27 , 6.97 , 6.83 , 6.60 , 4.96 , 4.75 , 3.70 , 3.51 , 3.06 , 2.97 , 2.31 . 13 C NMR : δ 195.2 (COS), 171.5 (CO2), 167.6 (CONH), 157.6 (Ar-C), 135.9 (Ar-C), 131.7 (Ar-C), 130.3 (Ar-C), 129.0 (Ar-C), 128.1 (Ar-C), 121.8 (Ar-C), 114.8 (Ar-C), 69.2 (CH2O), 53.5 (CH2CHNH), 52.4 (CH3O), 36.7 (CH2CHNH), 32.7 (CH2S), 30.1 (CH3CO). HRMS calculated for C21H22BrNNaO5S [M + Na]+ 502.0294, found 502.0289.Tan viscous oil, 1.59 g (83%). 1H NMR : δ 7.50 , 7.33 , 7.13 , 6.89 , 5.01 , 4.61 , 3.14 , 3.12 , 2.94 . 13 C NMR : δ 174.4 (CO2H), 172.9 (CONH), 159.0 (Ar-C), 138.2 (Ar-C), 132.6 (Ar-C), 131.5 (Ar-C), 130.6 (Ar-C), 130.4 (Ar-C), 122.5 (Ar-C), 116.0 (Ar-C), 70.2 (PhCH2O), 55.3 (CH2CHNH), 37.5 (CH2CHNH), 28.0 (CH2SH). HRMS calculated for C18H17BrNO4S [M-H]− 422.0067, found 422.0073.Off white solid, 0.87 g (68%). Click here for additional data file.Click here for additional data file.


Prostate cancer (PCa) is the second leading cause of cancer-related death among men in the United States. Androgen receptor (AR) signaling is the dominant oncogenic pathway in PCa and the main strategy of PCa treatment is to control the AR activity. A large number of patients acquire resistance to Androgen deprivation therapy (ADT) due to AR aberrant activation, resulting in castration-resistant prostate cancer (CRPC). Understanding the molecular mechanisms underlying AR signaling in the PCa is critical to identify new therapeutic targets for PCa patients. The recent advances in high-throughput RNA sequencing (RNA-seq) techniques identified an increasing number of non-coding RNAs (ncRNAs) that play critical roles through various mechanisms in different diseases. Some ncRNAs have shown great potentials as biomarkers and therapeutic targets. Many ncRNAs have been investigated to regulate PCa through direct association with AR. In this review, we aim to comprehensively summarize recent findings of the functional roles and molecular mechanisms of AR-related ncRNAs as AR regulators or targets in the progression of PCa. Prostate cancer (PCa) is the most frequently diagnosed cancer and the second-highest cause of cancer death among men in the United States, with an estimated 248,530 new cases and 34,130 deaths expected in 2021 in the United States . The groNext-generation RNA sequencing (RNA-seq) advances have become overly convenient to measure gene expression levels and explore new transcriptional units across the transcriptome. 2% of the human genome encodes approximately 20,000 protein-coding genes  and up tEZH2 expression by binding to EZH2 3′ untranslated region (UTR), and has a strong negative correlation with PCa progression from benign to localized disease to metastasis , and an exceptionally long 3′ UTR with a length of approximately 6.8 kb , including the miR-135, miR-185, miR-34, miR-421, and miR-9 families reported by Several groups have systemically explored AR modulatory miRNAs through different strategies . Ostlingeting AR . Similar AR mRNA . The rol AR mRNA . Among 2AR 3′ UTR activity by miR-346, miR-361-3p, and miR-197 was also confirmed by AR 3′ UTR reporter assay combined with miRNA mimics. Mimics of miR-346, miR-361-3p, and miR-197 also prevented Actinomycin D-induced loss of AR transcript. Previous studies demonstrated that miR-346 binds to 3′ UTR of AGO2 and hTERT. Upon miR-346 binding to the 3′ UTR of AGO2 and hTERT, the middle sequence motif (CCGCAU) of miR-346 recruits G-rich RNA sequence binding factor 1 (GRSF1) to form a “bulge loop,” thus facilitating the recruitment of AGO2 mRNA and hTERT mRNA to ribosomes to promote translation (AR mRNA stability and promote AR expression through a similar mechanism. In AR-V7) expression  and altering histone H3K27 methylation   and splicing factor U2AF65. The PCGEM1-hnRNP A1 interaction could suppress hnRNP A1 interaction with AR pre-mRNA, while PCGEM1-U2AF65 interaction could promote U2AF65 interaction with AR pre-mRNA, indicating that PCGEM1 may participate in the AR signaling by regulation of AR mRNA splicing  , which ima (RCC) . LncRNA  targets , 2017. Ppatients . Howeversplicing . The relGAS5) is a lncRNA firstly identified in growth-arrested mammalian cells , mineralocorticoid receptor (MR), progesterone receptor (PR), and AR, at their DNA binding domains through GAS5 contained hairpin RNA glucocorticoid receptor response element (GRE)-mimic and, thereby, inhibit the association of these receptors with their DNA recognition sequence and thus repress the transcriptional activity of these steroid receptors (SRA) could selectively enhance the transactivation of steroid receptors, such as PR, GR, estrogen receptor (ER), and AR, through interaction with their N-terminal, regulatory domain (NTD) (SLNCR1), recruits AR to MMP9 and EGR1-bound genomic loci to regulate melanoma invasion and proliferation. Interestingly, several confirmed AR bound lncRNAs, such as HOTAIR, SRA, SLNCR1, and even PCGEM1, all include a conserved region with a similar sequence (SLNCR1609–637) which is required for AR-lncRNA interaction  . Schmidtin (NTD) , 2019 reeraction . Furtherh AR NTD . These sKLK3 eRNA (KLK3e) is an eRNA produced from the upstream enhancer regions of Kallikrein-related peptidase 3 (KLK3)  are derived from super-enhancers and have been proven to control mRNA transcription through facilitating enhancer-promoter interaction . KLK3 eR3 (KLK3) , a well-ARLNC1 was reported to directly bind to AR mRNA 3′ UTR, stabilize AR mRNA, and increase the cytoplasmic fraction of AR mRNA, thus regulating PCa cell growth and apoptosis , one of the most studied lncRNAs, was previously reported to bind to EZH2 and enhance EZH2-mediated repression of Polycomb-dependent target genes  which are highly expressed in CRPC tissues, were also reported to participate in AR mRNA splicing through association with splicing factors -binding micropeptide, YY1BM. YY1BM could suppress the interaction between YY1 and AR and thus downregulate eEF2K expression and induce apoptosis in human esophageal squamous cell carcinoma -on-chip, ChIP-seq assays . AR direMUC1 3′ UTR  patients and may serve as biomarkers of high-risk primary PCa  (NCT00085228) and Clusterin mRNA (Custirsen- OGX011) (NCT01188187) had been evaluated in PCa human patients in Phase II and III clinical trials, but both failed due to major toxic events or no significant survival improvement (Hsp27 mRNA (Apatorsen- OGX-427) (NCT01120470) and AR mRNA (ARRx- AZD5312) (NCT03300505) are currently under Phase I clinical trials. In addition to targeting the protein-coding mRNAs, ASO targeting lncRNA MALAT1 dramatically prevented lung cancer metastasis in a pulmonary metastatic mouse model (Antisense oligonucleotides (ASOs) as an RNA-based therapeutic approach can induce gene silencing through RNase H-mediated degradation of target RNAs. It has shown improved target specificity and stability as well as tolerated toxicity after significant advancements in the design, chemical modifications, and delivery . Severalrovement . Additiose model , showingin vitro study until to date, and several lncRNAs are proven to be necessary for life and brain development through the studies in the knockout mouse model  or siRNA without appropriate rescue assays. Silencing nuclear lncRNAs requires the nuclear enrichment of AGO2 and RNA interference (RNAi) factors Dicer, TRBP, and TRNC6A/GW182 (cis mechanisms to regulate their neighboring genes. The effect of destructing these regulatory elements located in the lncRNA genome loci and neighboring genes prevails against the effect of lncRNA loss (MALAT1 in MMTV-PyMT mouse which is a transgenic model of metastatic breast cancer, induced significantly increased lung metastasis of breast cancer cells (MALAT1 in mouse with normal physiological condition showed no apparent phenotype (MALAT1 is dispensable for development but plays important roles in suppressing breast cancer metastasis. Taken together, it is critical to choose the proper method to generate lncRNA depleting cells and mouse model based on lncRNA’s cellular distribution, genome localization and its function mechanism, and it is also important to take rescue experiments into consideration when investigating lncRNAs’ function through loss of function methods (There are several possible reasons to explain why some 6A/GW182 , while A6A/GW182 , and thu6A/GW182 . ThirdlyRNA loss . LncRNA RNA loss . Fourthler cells , while ihenotype , suggest methods .A growing number of novel discovered ncRNAs and various research has revealed the crucial roles of ncRNAs in different disease processes. Many ncRNAs have been verified to participate in PCa initiation or progression by regulating or mediating AR signaling. These ncRNAs hold great potentials as diagnostic biomarkers and therapeutic targets. Given that ncRNAs consist of the majority of the human transcriptome, a long journey in the understanding of ncRNAs, especially lncRNAs, circRNAs, and other ncRNAs, is yet to be achieved. Further investigations based on high-throughput sequencing technology and integrative bioinformatics analysis will enable the discovering of new functional ncRNAs and their regulation mechanisms, and these works will further promote the development of effective therapeutic strategies for PCa.YY and QC developed the concept of the review. YY and KL collected the related publications and drafted this manuscript. QL and QC helped to revise this manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the effectiveness of tele-rehabilitation for children diagnosed with unilateral cerebral palsy.The design of this study is a narrative review. An electronic search was conducted for studies that related to tele-rehabilitation using the following databases: CINAHL, PubMed, MEDLINE, OTSeeker, and PEDro. The data extracted were analyzed by evaluating them according to the key results, limitations, suitability of the methods used to the initial hypothesis, interpretation of the results, and impact of the conclusions in the field.Out of 139 studies, 3 studies met the inclusion criteria. Further, manual searches of the references of included studies identified 2 more relevant studies. The interventions applied in those studies were web-based multi-modal therapy program using Move-it-to-improve-it (Mitii™), home-based hand-arm bimanual intensive therapy (H-HABIT), and lower-extremity functional training (LIFT). The outcomes were executive functions, occupational performance, activity capacity, dexterity, quality of bimanual hand-use, functional goals, gait capacity, and performance.Tele-rehabilitation is effective in improving the functions of the upper and lower extremities in daily living activities for children with unilateral cerebral palsy (UCP), aged between 2 to 18 years old, classified to levels I and II in GMFCS and levels I, II, and III in MACS. Webcam and good internet connection are essential requirements to conduct tele-rehabilitation. Children need to be contacted weekly via phone or e-mail for further follow-ups. Additionally, tele-rehabilitation may be considered one of the intervention strategies for patients who live in rural areas. Cerebral palsy (CP) is a neurodevelopmental disorder that affects children’s development and limits their functions and activities. It occurs in 1.5 to 2.5 per 1000 live births  dyskinetThe symptoms of CP include muscle tone abnormalities which are considered to be the most common symptoms among CP children. Hypertonicity, hyperreflexia, clonus, and poor coordination are also the symptoms of children diagnosed with CP. Other symptoms include feeding difficulties, drooling, hip dislocation, scoliosis, seizures, intellectual impairment, communication difficulties, and impaired vision and hearing; those symptoms cause many impairments for the child .The most common impairment that CP children would have is upper limb impairment . AnotherCP treatment depends on symptoms and impairments. The treatments have been divided into pharmacologic treatment , which targets muscle tone abnormalities, and non-pharmacologic treatment, including surgical or rehabilitation treatment. Surgical treatment aims to reduce spasticity and improve gait kinematics, while rehabilitation programs may enhance motor development and help to prevent secondary deformities . The outEarly intervention is essential for infants diagnosed with CP because infants who are not actively using their motor cortex may lose cortical connections and functions. An infant’s motor behavior increases by discovering and interacting with the environment. Hence, controlling and generating the growth, development of muscles, ligaments, and bones and development of the neuromotor system lead to improving the neuroplasticity . Early iDuring pandemics and crises such as coronavirus (COVID-19), rehabilitation services would be almost impossible and difficult for children and their caregivers in the rehabilitation centers. One of the solutions that might be effective for children with disabilities for getting rehabilitation services is tele-rehabilitation .Tele-rehabilitation has become an interest for many physical therapy professions especially during the crisis of COVID-19. World Confederation for Physical Therapy (WCPT) provided a global hub of resources to support the physiotherapy profession in its response to COVID-19, and advise the rapid, measured, and responsive way the global profession has responded, whereas tele-rehabilitation became a viable solution for offering rehabilitation services .Tele-rehabilitation defines as a creative way to deliver rehabilitation services remotely using tele-communication technologies . CommuniRecent studies have proven that tele-rehabilitation reinforces the early intervention for children diagnosed with CP . ProvidiTele-rehabilitation might have many advantages such as reducing the cost for both therapists and patients and providing healthcare services for patients who live in rural areas and who have difficulties with transportation . On the Therefore, the purpose of this narrative review is to assess and synthesize the studies that used tele-rehabilitation to improve functions in daily activities. This will include investigating the effectiveness of tele-rehabilitation performed for children diagnosed with CP and investigate the psychometric properties of the outcome measures involved in the selected studies.The study design is a narrative review.An electronic search was conducted for studies that were published in English which is related to tele-rehabilitation using the following databases: CINAHL, PubMed, MEDLINE, OTSeeker, and PEDro from 2010 to September 2021, using the medical subject heading (MeSH) or keywords such as “tele-rehabilitation,” “tele-health,” “tele-medicine,” and “remote rehabilitation,” and every set search included MeSH terms which was combined by AND cerebral palsy. Table The included studies were selected according to the following inclusion criteria:  childrenOn the other hand, the exclusion criteria were  childrenThe data extracted from each study were retrieved and include the citation details, participants’ characteristics, the interventions applied, the concluded outcomes, and the outcome measures that were performed to assess the improvement.The data were analyzed through the critical assessment process by evaluating them according to the following:  key resuThe process of the search strategy and screening of articles that were followed in the current review is shown in Fig. The main findings are summarized in Table The interventions were applied remotely including a web-based multi-model therapy program (Mitii), home-based hand-arm bimanual hand function intensive therapy (H-HABIT), and lower-extremity intensive functional training (LIFT). The main outcomes were functional outcomes including executive function, occupational performance, activity capacity, dexterity, bimanual hand function, and lower extremity function. The selected studies were assessed critically using the critical assessment process; Table The studies , the lower extremities muscle group was targeted to be strengthened using body weight and thera-band such as bridging and clamshells. Regarding balance, children performed activities such as single-leg standing, and standing on unsteady surfaces, ball kicking, tandem walking, jumping jacks, and galloping/skipping to improve coordination. The program was performed daily, 2 h/day.They concluded that intensive intervention for lower extremity applied at home environment given by caregivers has a significant improvement in gait capacity and performance. Tele-rehabilitation program using LIFT with remote supervision resulted in an improvement in ambulation distance and overall walking ability.The outcome measures used in the included studies were briefly described and investigated according to the population (condition and age) and psychometric properties . Table This narrative review aims to evaluate and synthesize studies that used tele-rehabilitation to improve functions in daily activities for children with CP. This involved investigating the effectiveness of tele-rehabilitation for children with CP.During pandemics and crises, tele-rehabilitation is the best solution that seems to be an effective treatment for children with disabilities during the pandemic; World Confederation for Physical Therapy (WCPT) provided a global hub of resources to support the physiotherapy profession to continue the rehabilitation programs during the pandemic . The stuThe effectiveness of tele-rehabilitation might have the same outcomes as compared to interventions that are applied face-to-face . HoweverThis narrative review showed that tele-rehabilitation has significant effectiveness for children with UCP to improve functions ; the age range was between 2 and 18 years old, levels I and II in GMFCS, and levels I, II and III in MACS. However, children with younger age were not included. Also, children with levels III, IV, and V in GMFCS and levels IV and V in MACS were not included as there is no evidence to support this hypothesis, which indicates certain barriers to be encountered in such cases. We have also noticed that all the selected studies included children diagnosed with UCP, which reflects that the result of our study can be applied to children with UCP only and not for all children with CP. In contrast, tele-rehabilitation has a significant improvement for other cases in other studies that were not included in our study as we focused only on CP cases.From our point of view, treating children with levels III, IV, and V in GMFCS and levels IV and V in MACS might need special equipment and special techniques that the caregivers are unable to perform them as they do not have enough experience, which corresponds to the study , that teDuring investigating the outcome measures, we found that some of the included studies used invalid measurement tools for children aged from 2 to 18 years old. For example, BBT is a tool designed to assess gross manual dexterity for children aged between 6 and 19 years old . Thus, tGiven the above limitations, we are unable to generalize that tele-rehabilitation is effective for all children with CP just because the selected studies specified children with levels I and II GMFCS and levels I, II, and III on MACS.This study is a narrative review; a risk of bias or quality assessment of the included studies was not performed in this review. Additionally, the included studies were all RCTs because they are given the highest level of evidence in the hierarchies rank studies. Furthermore, there were clear methodological limitations in the included articles which could have influenced the findings of this review, for example, the selected studies did not include children with moderate or severe disability given that all children were levels I and II on GMFCS and levels I, II, and III on MACS, which affect on the results of the treatment for children with moderate or severe disabilities. Also, the included studies did not mention any risk of injury that may be associated with tele-rehabilitation, which may influence the treatment and cause complications and difficulties for the patients.Further studies are recommended by performing a quality assessment for the selected studies in form of a systematic review. Additionally, it is recommended to include other studies rather than RCTs for further investigation regarding the effectiveness of tele-rehabilitation. Risk of injury associated with tele-rehabilitation should also be investigated. All levels of disability children are suggested being included.The findings of this narrative review showed that tele-rehabilitation is effective in improving the functions of the upper and lower extremities in daily living activities  for children diagnosed with UCP, aged between 2 and 18 years old, classified to levels I and II in GMFCS and levels I, II, and III in MACS.Children should be monitored via a webcam and good internet connection. In addition, they should be supervised by therapists for at least once weekly via a phone, e-mail, or video conference to promote an effective tele-rehabilitation. The suggested duration and intensity are 30 min for 5 days weekly regarding the Mitii and 2 h for 5–6 days regarding performing LIFT or H-HABIT. Additionally, tele-rehabilitation may be considered one of the intervention strategies for patients who live in rural areas. Tele-rehabilitation should be performed with caution; the children should be monitored and followed up frequently to prevent the risks of injury that may occur during performing the suggested intervention that is performed remotely.


Although home-based pulmonary rehabilitation programs have been shown in some studies to be an alternative and effective model, there is a lack of consensus in the medical literature due to different study designs and lack of standardization among procedures. Therefore, the purpose of this study was to compare the efficacy of a home-based versus outpatient pulmonary rehabilitation program for patients with chronic obstructive pulmonary disease (COPD).Five electronic databases including Embase, PubMed, Scopus, Science Direct, and Cochrane Library will be searched in May 2021 by 2 independent reviewers. The reference lists of the included studies will be also checked for additional studies that are not identified with the database search. There is no restriction on the dates of publication or language in the search. The randomized controlled trials focusing on comparing home-based and outpatient pulmonary rehabilitation for COPD patients will be included in our meta-analysis. The following outcomes should have been measured: functional exercise capacity, disease-specific health-related quality of life, and cost-effectiveness measures. Risk ratio with a 95% confidence interval or standardized mean difference with 95% CI is assessed for dichotomous outcomes or continuous outcomes, respectively.It was hypothesized that these 2 methods would provide similar therapeutic benefits.10.17605/OSF.IO/5CV48. Patients with severe airflow restriction and those who experience repeated acute exacerbations often suffer from reduced quality of life, impaired exercise ability, and an increased risk of readmission to hospital. Intervention designed to accelerate post-hospitalization recovery and improve symptoms may lead to reduced use of healthcare in the future and to real improvements in quality of life and functional capacity in patients with shortness of breath and vulnerable COPD. However, the use of pulmonary rehabilitation therapy is generally low although strong scientific advice for its routine use in the treatment of COPD.,4Pulmonary rehabilitation is a multidisciplinary intervention that combines nutritional treatment, psychological support, physical exercise, and patient education.,6 In addition, since most studies have been conducted under remote supervision, such as home visits or phone calls from physiotherapists or doctors, there is limited data on unsupervised rehabilitation programs.–9 Therefore, the purpose of this study was to compare the efficacy of home-based versus outpatient pulmonary rehabilitation programs for patients with COPD. It was hypothesized that these 2 methods would provide similar therapeutic benefits.Home-based unsupervised pulmonary rehabilitation therapy has been proposed as an alternative model that can increase access and absorption while curbing the rising health care costs associated with COPD. Although home-based pulmonary rehabilitation programs have been shown in some studies to be an alternative and effective model, there is a lack of consensus in the medical literature due to different study designs and lack of standardization among procedures.22.1https://osf.io/5cv48), and the registration number is 10.17605/OSF.IO/5CV48. The protocol was written following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols statement guidelines.The prospective registration has been approved by the Open Science Framework (OSF) registries  is used for the meta-analysis. Extracted data are entered into Review Manager by the first independent author and checked by the second independent author. Risk ratio with a 95% confidence interval or standardized mean difference with 95% CI is assessed for dichotomous outcomes or continuous outcomes, respectively. The heterogeneity is assessed using the 3,6 In addition, since most studies have been conducted under remote supervision, such as home visits or phone calls from physiotherapists or doctors, there is limited data on unsupervised rehabilitation programs.–9 Therefore, the purpose of this study was to compare the efficacy of home-based versus outpatient pulmonary rehabilitation programs for patients with COPD. It was hypothesized that these two methods would provide similar therapeutic benefits.Home-based unsupervised pulmonary rehabilitation therapy has been proposed as an alternative model that can increase access and absorption while curbing the rising health care costs associated with COPD. Although home-based pulmonary rehabilitation programs have been shown in some studies to be an alternative and effective model, there is a lack of consensus in the medical literature due to different study designs and lack of standardization among procedures.Conceptualization: Guiyun Shi.Data curation: Guiyun Shi, Chuanjun Chen.Formal analysis: Guiyun Shi, Chuanjun Chen.Investigation: Guiyun Shi, Chuanjun Chen.Methodology: Chuanjun Chen, Guiyun Shi.Resources: Chuanjun Chen.Software: Guiyun Shi.Supervision: Chuanjun Chen, Guiyun Shi.Validation: Chuanjun Chen.Writing – original draft: Guiyun Shi.Writing – review & editing: Chuanjun Chen.