The length to diameter ratios of In2O3/Au NRs was periodically modulated in the range of 2.9–4.5 through controlling the initial content of indium salt and reaction time. Their gas sensing properties to volatile organic compounds (VOCs) were carefully studied and then applied in exhaled breath detection. The results demonstrate that In2O3/Au NRs gas sensor can effectively detect acetone at 250 °C and ethanol at 400 °C. The corresponding actual detection limit is as low as 0.1 ppm to acetone and 0.05 ppm to ethanol, respectively. Moreover, by using humidity compensation method, In2O3/Au NRs gas sensor can clearly distinguish the acetone and ethanol biomarkers in human breath. The main reason of the enhanced gas sensing properties was attributed to the “spillover effects” between Au and In2O3 NRs. The excellent sensing performance indicates that In2O3/Au NRs is a promising functional material to actual application in monitoring and detecting diabetes and safe driving area in a noninvasive and more accurate way.A series of In Significant interest has been generated in the field of selective detection of specific VOCs which also called biomarkers in exhaled breath from the metabolism process12345781011et al. fabricated Si doped WO3 sensors for quantitative analysis of acetone in dry and 90% relative humidity (RH) air sampleset al. prepared Pt nanoparticles (NPs) functionalized SnO2 fibers through electrospinning which could be a potential material for the detection of gaseous acetone and toluene, which are the biomarkers for the diabetes and lung cancer, respectivelyet al. synthesized In2O3 sensing film by a sparking process which can use to detect ethanol and acetoneRecently, nanomaterials based sensors are considered to be a promising clinical and laboratory diagnostic tool, because its large surface–to–volume ratio, controllable structure, easily tailored chemical and physical properties, which bring high sensitivity, fast dynamic process, and even the increasing specificity13142O3/Au NRs through co–precipitation method. By this way, the small Au NPs were effectively embedded in In2O3 NRs and acted as sensitizer. In particular, In2O3/Au gas sensor not only can detect acetone lower to 10 ppb at 250 °C, but also can detect ethanol lower to 50 ppb at 400 °C. Moreover, this sensor showed good anti–interference to humidity and could effectively detect gaseous acetone and ethanol in simulated exhale breath environment. And then, a humidity compensation method was used to increase the detection accuracy and the clinic test was also conducted.One–dimensional (1D) metal oxide nanostructures have been identified to be one of the most effective nanoarchitectures for chemiresistive VOCs detection18192021222323252O3/Au precursor sample  contained with 0.2 g In(NO3)3 and Au NRs (with reaction time of 120 min), and the as prepared precursor exhibits uniform NRs structures and small Au NRs are not observed elsewhere. Well, unlike with In2O3/Au precursor, the pure In2O3 without addition of Au NRs formed flower–like microspheres instead of NRs CO3 NPs aggregated due to the high surface energy of the NPs and finally self–assembled to flower like spheres− and CO32− ions tended to gather around Au NRs, and then induced the heterocoagulation process start from the surface of Au NRs and finally self–assembled to NR structure, that is to say, Au NRs played a role as template in forming the In2O3/Au NRs2O3/Au NRs after annealing have the average diameter of 75 nm and length of 310 nm, that is to say, the length to diameter ratio is about 4.1. Besides, the color of different samples as mentioned above is very different. As can be clearly seen in inset of 2O3/Au precursor is dark green, while the color of pure In2O3 is milk white to the naked eye, after annealing the color of In2O3/Au NRs is dark red, while the pure In2O3 is light yellow. This indicated that Au NRs were successfully introduced into In2O3/Au NRs.The morphologies and nanostructures of the precursor and annealing samples are first illuminated. The uniform Au NRs used in this work are as shown in d of NRs . As in a2O3/Au NRs samples with different initial amount of In(NO3)3 and reaction time were also synthesized. As shown in 3)3 and reaction time have a little influence on the morphologies. The length to diameter ratios are about 3.6, 3.8, and 4.5, respectively to 0.1g (3)3 at the reaction time of 120 min, which show a slight increase trend with the increasing of the In(NO3)3 amount. When the reaction time is different, the length to diameter ratios is changed. As can be seen from Besides, the In2O3/Au NRs sample was further carried out by TEM and STEM. It can be seen that the In2O3/Au NRs contains a lot of interspaces, indicating a large surface area as shown in 2O3. The corresponding SAED pattern . The same phenomenon can be observed in the characteristic spin–orbit split XPS data of trivalent indium as shown in 5/2 and In 3d3/2 are at 444.5 and 452.0 eV, respectively. Compared with the pure In2O3, clearly shift to the high binding energy also can be seen, those shifts may relate to the different local environment due to the introduced of Au. Moreover, typical XPS peaks of the core level region of Au4f can be observed in as–prepared In2O3/Au NRs. As can be seen in 0 state32332O3/Au NRs, which is consist with above analysis.In order to obtain a more detailed chemical composition of the as–prepared In2O3/Au NRs as well as pure In2O3 were carefully studied and compared, and then exhaled gas environment was simulated to further evaluate the performance of as prepared sensors towards to practical application. First, the responses of the In2O3/Au NRs with different initial amount of In(NO3)3 and reaction time compared with pure In2O3 sensors to 50 ppm acetone and ethanol gases as a function of operating temperature are exhibited in 2O3/Au NRs gas sensors are , , , and , respectively. Compared to pure In2O3 gas sensor , the response improves gradually when the atom ratios of Au/In increases (from S4 to S1), while the optimal working temperature shows a decreased trend only when the atom ratio of Au/In > 3.6. For ethanol gas, compared to pure In2O3 gas sensor , the optimal working temperature and corresponding responses for S1–S4 In2O3/Au NRs gas sensors are , , , and , respectively. As can be seen, the optimal responses mainly appear in the relatively higher temperature range and the changing trend of working temperature is similar with that of acetone gas. While the change of response value has only a little difference when the atom ratio of Au/In is larger than 3.6.The gas sensing properties of the In2O3, S4, and S3, respectively). However, when the Au amount further increases (S2 and S1), with the decrease of corresponding optimal working temperature, the response time and recovery time become longer rapidly. The changing trend of response time and recovery time for the ethanol gas is similar to that for acetone; however, the changing range is relatively small in the higher temperature range in the case of ethanol. The influence mechanism of the introduced Au on the sensing performance of the studied sensors is carefully discussed later in this article. It is worth mentioning that from the view of response, temperature difference, response and recovery time to this two gases, the as studied sensors, especially the S3 In2O3/Au NRs sensor, have dual mode sensing properties, which can quickly detect acetone gas at a relatively low working temperature (250 °C in our case) and detect ethanol at a higher temperature section (400 °C in our case), and the response of S3 In2O3/Au NRs sensor is ~2.2 and 3.3 times higher than that of pure In2O3 sensor under optimal working temperature, respectively.Furthermore, the response times and recovery times of different sensors working at corresponding optimal temperature were also evaluated in 2O3/Au NRs sensor toward to exhale breath, detail tests were conducted focused on a very low gas concentration which can meet the requirement of exhaled gas detection. First, the dynamic responses to different concentration of acetone at 250 °C and ethanol at 400 °C were investigated. Four response and recovery curves are shown in Generally, the biomarker gases in exhaled breath are low in concentrations. In order to obtain more information about the dual mode properties of In2O3/Au NRs and pure In2O3 gas sensors versus different acetone and ethanol concentrations at 250 °C and 400 °C respectively. As can be seen, the sensors show a good and wide linear relationship with the growth of the acetone and ethanol concentrations in all the studied range. Besides, the In2O3/Au NRs sensor exhibits good resolution to low concentration gas. For acetone detection, the In2O3/Au sensor response is below 2.5 for healthy humans (<0.9 ppm) and above 3.7 for diabetics (>1.8 ppm). This 48% response increase may allow reliable diagnosis of diabetic patients by breath acetone monitoring. For ethanol detection, the large linear range clearly covered the different alcohol levels encountered in breath  after drinking122O3/Au NRs sensor when Ra/Rg ≥ 1.2 was used as the criterion for reliable gas sensing, confirm that the as–fabricated gas sensor is a promising candidate for the detection low concentration of acetone and ethanol biomarkers in breath with a portable metal oxide detector.2O3/Au NRs compared with pure In2O3 gas sensors to 1 ppm acetone and 5 ppm ethanol were measured at 250 °C and 400 °C respectively as shown in 2O3/Au NRs sensor only decreased about 4% for acetone and 5% for ethanol in comparison to the original values, whereas the response of pure In2O3 sensor deceased about 12% and 13% to acetone and ethanol, respectively. This demonstrates that the sensor based on the In2O3/Au possesses excellent long time stability.In addition, the longtime stability of the Invs. different acetone and ethanol concentration of pure In2O3 and S3 In2O3/Au NRs gas sensors are listed in 2O3/Au NRs gas sensor are 10.8 s and 18.4 s for acetone gas and 9 s and 13.4 s for ethanol gas, respectively, which are systemically shorter than that of pure In2O3 gas sensor . This can be attributed to the introduction of the Au NPs which have catalytic properties. In addition, the dynamic processes of ethanol of both sensors are faster than that of acetone, this is because the higher the working temperature the faster the reaction of thermodynamics.Besides, the response times and recovery times τres) and recovery time (τrec) constants can be written as a function of temperature according to the well–known thermal activation function:τres(0) and τrec(0) are time coefficients, which depend on surface to volume ratio and reaction mechanism. KB is the Boltzmann’s constant and T is the absolute temperature. ΔEres and ΔErec present the forward and backward reaction barrier heights, respectively, while τres and τrec present the forward and backward reaction time. res and ΔErec are deduced to be 211 and 298 meV for acetone gas, and 251 and 355 meV for ethanol gas. As is calculated, the forward reaction barrier heights always lower than the backward reaction barrier heights, this indicated that to complete recovery processes need to overcome higher barrier height which may lead to the recovery times always longer than the response times in our case. Besides, the reaction barrier heights of ethanol gas are systematically higher than that of acetone, that is to say, more energy is needed to completed the dynamic processes in ethanol environment, this may have some relation with why the optimum operation temperature of the sensors to ethanol gas is higher than that to acetone gas.Actually, we have measured the response and recovery time constants at various temperatures to acetone and ethanol, respectively. As is known, the response and recovery is a reversible process. No matter in which process, a certain potential barrier should be overcame to complete the reaction. According to our previously work2, O2, some other VOCs and biomarker gases, and especially high humidity levels (>80% RH at 1 atm and 25 °C). Thus, the selectivity and the response of the humidity environment are important in the actual breath gas measurement. Here, selectivity of In2O3/Au NRs sensor were evaluated at 250 °C and 400 °C respectively towards to some typical biomarkers  in exhale breath and compared with pure In2O3 sensor (acetone gas concentration was 1 ppm and ethanol was 5 ppm)12372O3/Au NRs sensor, which shows very low response or almost insensitive to the other typical interference gases at the same temperature.The constituent of people exhale breath gas is complicated, which composed by COa/Rwet response where Rwet is the resistance of sensor at a given RH environment versus RH ambience. Usually, Rwet is interpreted as the interaction between the surface adsorbed oxygen and the water molecules which induces a decrease in baseline resistance of the gas sensor. It can be seen that Ra/Rwet value of both In2O3/Au NRs and pure In2O3 sensors rise slightly in the as studied RH range (25%–94%), In the studied RH range, the Ra/Rwet value of In2O3/Au sensor only changed from 1.0 to 2.8 at 250 °C and 1.0 to 3.4 at 400 °C compared to pure In2O3 sensor that changed from 1.0 to 1.6 at 250 °C and 1.0 to 2.7 at 400 °C. The In2O3/Au sensor shows a little larger affected than pure In2O3 sensor due to the sensitized effect of Au.The interference of humidity is an important problem that must consider in breath sensing, because the VOCs signals could be screened by the high humidity levels in breath, and the small fluctuations in humidity also have big effect on the sensitivity of VOCs2O3/Au sensor exhibits good dual mode sensing properties towards to very low acetone and ethanol concentration as studied above, the response in simulated exhaled gas environment was further investigated and compared, as shown in 2, 17% for O2, 4% for CO2, and 1% for other gas, the simulated exhaled gas environment was built by mixed ambient atmosphere with a certain amount of CO2 and different concentration of acetone (250 °C) or ethanol gas (400 °C) in high RH surroundings, moreover, take into consideration the high humidity and individual difference in exhaled gas environment, 93.5 ± 1% RH (sim1) and 75.5 ± 1% RH (sim2) conditions were also provided.Since the as prepared Ina/Rg), the responses in simulated exhaled gas environment (Ra/Rsim) of S3 In2O3/Au NRs sensor shows gradually increased trend with the increasing of the RH value, because the sensor almost has no response to additional CO2, the main reason can be attributed to the effect of high RH conditions. Besides, all the Ra/Rsim responses in simulated exhaled gas environment of In2O3/Au NRs sensor show good linearity on the whole, and the slops of the three curves of acetone at 250 °C and ethanol at 400 °C are independent of the RH level, indicating the response to the VOCs is not effected by the background humidity. In this case, a humidity compensation method could use to increase the accuracy of the sensor response in the environment of high humidity levels or fluctuations in humidity, which has been proved to be effectiveAs depicted in 2O3/Au NRs sensor to the VOCs in human breath and the RH compensation method also was applied. For acetone detection, 1 diabetic and 6 healthy volunteers participated in the test. As shown in Furthermore, the clinic test were conducted to test the sensing properties of S3 In2O3/Au and In2O3 based sensors could be modeled using the surface–controlled model422O3 sensor is exposed to air, it interacts with oxygen by transferring electrons from the conduction band to the adsorbed oxygen atoms, forming reactive oxygen species . The reaction can be expressed as:Based on the above analysis of gas sensing characteristic, the sensing mechanism of both In2O3 sensor. This leads to the increase of the resistance in the In2O3 sensor. When the In2O3 sensor is placed into the reducing gas (acetone or ethanol in our case), the reactive oxygen ions will react with the analysis gas molecules and then the electrons are released back to the In2O3 sensor, resulting in the thinning of the depletion layer and the decrease of the resistance of the In2O3 sensorBased on the above reactions, a depletion layer is created due to the consumption of the electron in the surface region of the In2O3/Au NRs, exhibits good dual mode sensing properties both to acetone and ethanol gases in this work.In general, at low temperature, active sites at the semiconductor surface are rare and the amount of ion sorption of oxygen is deficient. These can lead to incompletely reaction of analysis gas. So the response is poor at low temperature. With increasing the working temperature, the ion sorption of oxygen on the sensor surface quickly increases, and the adsorbed oxygen gives rise to Schottky potential barriers at grain boundaries, and thus increases the resistance of the sensor surface. When reducing gases are introduced, the response will increase. While, further increasing the working temperature, the charge-carrier concentration and the conductivity increase and the Debye length decreases, which leads to the decrease of response. In fact, the response process of a semiconductor sensor on temperature is very complex. Its effect factors include the rates of adsorption and desorption of oxygen and reducing gases, the rate of surface decomposition of reducing gases, the charge carrier concentration, and the Debye length in the semiconductor2O3/Au NRs could promote the reactive oxygen species as chemical sensitizer and the most probable mechanism leading to improve sensor response is chemical sensitization via spillover effect23462O3 interaction. The shift of the In 3d peak to the higher binding energy is attributed to the interaction of In2O3 with Au and then leads to a modification of the surface electronic structure. This brings out the second role of Au as modulator of the surface electronic structure. So in our studies, the response value and response dynamic process are gradually increased with the increasing of the amount of Au at first . When Au amount further increases, the dynamic process slows down, this may attributed to the change of corresponding optimal working temperature.When Au are introduced, the uniform distributed Au in InGenerally, at low temperature most semiconductor metal oxides gas sensor materials are insulators rather than semiconductor, thus the gas sensing sensitivity is very poor. After the introduction of Au, ionsorption of oxygen ions can occur on the metal NPs surface at a low temperature (even at room temperature) due to the highly conductive nature, availability of free electrons, and chemical sensitization in AuHowever, despite the semiconductor oxide based gas sensors exhibited many advantage which meet the criteria of an ideal breath sensor, such as high response to low concentrations of VOCs, fast dynamic process, low-cost, simple and inexpensive to disposable2O3/Au NRs as well as pure In2O3 were fabricated using a facile co–precipitation technique. It is believed that the initial In(NO3)3 amount and reaction time have little effect on the morphology of In2O3/Au NR and the Au NPs were uniformly dispersed in In2O3/Au NRs. The dual mode gas sensing properties of different In2O3/Au NRs sensors compared with pure In2O3 were carefully studied, especially for very low concentration of acetone and ethanol. The results demonstrate that the optimal S3 In2O3/Au NRs sensor can effectively detect acetone at 250 °C and ethanol at 400 °C, which has response of 2.8 to 1 ppm acetone, and 9.8 to 5 ppm ethanol and low actual detection limit of 0.1 ppm to acetone and 0.05 ppm to ethanol. Besides, a humidity compensation method is applied to increase the accuracy of the sensor response in high humidity environment. Moreover, clinic tests were also performed, indicating that the In2O3/Au NRs sensor has the ability to actual detection acetone and ethanol biomarkers in exhaled breath. The present work suggests that the “spillover effects” of introduced Au NPs increased the activity oxygen species of In2O3 NRs and endowed In2O3/Au NRs gas sensor with enhanced dual mode gas sensing properties.In summary, In4 (1 mM aqueous) and 7.4 mL of cetyltrimethylammonium bromide  were mixed together, followed by the injection of 0.6 mL freshly prepared NaBH4 (10 mM aqueous) solution and magnetic stirring at 28 °C for 2 h. 2) Preparation of the Au NRs. In a flask, 13.2 mL of CTAB (0.1 M aqueous) was mixed with 12 mL of HAuCl4 (1 mM aqueous), and then 240 μL of AgNO3 (10 mM aqueous) and 220 μL of HCl (2 M aqueous) were added. After mixing, 192 μL of ascorbic acid  was added. While continuously stirring this mixture, 120 μL of the as–synthesized seed solution was added to initiate the growth of Au NRs. Then, these NRs were kept at 28 °C for 5 h to ensure full growth. Finally, after centrifuging at 13 000 rpm for 15 min, the upper liquid containing excess CTAB was removed, and NRs were left over in 40 mL deionized water.All the experiment reagents were used as received and without further purification. The Au NRs were prepared following the seed–mediated growth method reported by EI–Sayed and co–workers2O3/Au NRs were synthesized through co–precipitation method and annealed. First, various amounts  of In(NO3)3 were dissolved in 10 mL of deionized water which contained 1.8 g urea. After stirring, 20 mL of the as–synthesized Au NRs aqueous solution as motioned above was added. Then, the mixed solution heated to 80 °C and kept refluxing for 2 h in the oil bath. After self–cooling down to room temperature, the reaction product was separated by centrifugation and washing with deionized water for three times, then further dried at 60 °C for 6 h. At this time, the precursor NRs of [Au@In(OH)CO3] was obtained. Finally, the as–synthesized products were annealed in a tube furnace with a rising rate of 2.5 °C/min from room temperature to 600 °C and maintained for 3 h at 600 °C to obtain the In2O3/Au NRs samples. For comparison, pure In2O3 was also synthesized as follows, 0.2 g In(NO3)3 was dissolved in 30 mL of deionized water solution containing 1.8 g urea and no Au NRs were added, and the other processes were just the same with the above.The InThe morphology of the samples was inspected using JEOL JSM–7500F field emission SEM (Japan) with accelerating voltage of 15 kV and gold sputtering on surface. TEM and high resolution TEM (HRTEM) images were recorded on JEM–2010 transmission electron microscope under a working voltage of 200 kV. The phase structure of the samples were characterized by XRD; XRD patterns were conducted on Rigaku D/max 2550 using a monochromatized Cu target radiation resource (λ = 1.5045 Å) and the corresponding lattice constants of the samples were calculated by MDI Jade 5.0 software. XPS were conducted on an ESCAlab250 Analytical XPL Spectrometer with a monochromatic Al KR source. All the binding energies were referred to the C1s peak at 284.7 eV of the surface adventitious carbon. The fitted peaks in XPS spectra were deconvoluted using the XPS Peak 4.1 software.The as–synthesized samples were mixed with ethanol in the weight ratio of 5:1 to form a paste. The paste was coated on a ceramic tube on which a pair of gold electrodes was previously printed. A small spring–like Ni–Cr alloy was inserted into the ceramic tube to provide the operating temperature. After the solvent was evaporated, the ceramic tube with samples thin layer was sintered in an oven for 2 h at 400 °C. After sintering, the gas sensors were thermal aged with a heating voltage of 5 V at the ageing equipment for 6 days before the first measurement.a/Rg . The response and recovery times are defined as the time required reaching 80% of the final equilibrium value, and the details of the measurement are similar to those reported in the literatureThe gas–sensing properties were measured on a WS–30A system  and determined under laboratory conditions .The response is defined as S = RHow to cite this article: Xing, R. et al. Preparation and Gas Sensing Properties of In2O3/Au Nanorods for Detection of Volatile Organic Compounds in Exhaled Breath. Sci. Rep. 5, 10717; doi: 10.1038/srep10717 (2015).


Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures. The characterization of RNA–protein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X‐ray crystallography, or strategies based on chemical crosslinking, however, can be quite challenging. Herein, we have explored the use of an alternative method, native top‐down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single‐residue level of RNA. Our data show that the electrostatic interactions between HIV‐1 TAR RNA and a peptide comprising the arginine‐rich binding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNA by collisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TAR RNA by top‐down MS. Moreover, the MS data reveal time‐dependent 1:2 and 1:1 stoichiometries of the TAR–tat complexes and suggest structural rearrangements of TAR RNA induced by binding of tat peptide. Interactions between ribonucleic acids (RNA) and proteins are central to many fundamental biological processes, including gene expression and infection by RNA viruses. For a thorough understanding of such interactions, RNA–protein complexes are commonly investigated by nuclear magnetic resonance (NMR) spectroscopy or X‐ray crystallography, both of which require relatively large quantities of sample material. Moreover, NMR data interpretation can be complicated by unfavorable conformational dynamics,As an alternative to these methods, we explore the potential of native top‐down mass spectrometry (MS) using electrospray ionization (ESI)2N‐GRKKRRQRRRPP‐NH2) comprising the arginine‐rich binding region of tat (trans‐activating) protein from human immunodeficiency virus type 1 (HIV‐1). Previous studies showed that MS can be used to probe the effect of relative TAR and tat concentrations on complex stoichiometry.As a model system for our studies, we investigated complexes of 31 nt TAR (transactivation responsive) RNAn−, 42 % 1:1 complex, (TAR+tat−nH)n−, and 34 % 1:2 complex, (TAR+2tat−nH)n− , but ESI after approximately 30 min produced mostly the 1:1 complex 13− ions , y (12.0 %), a (1.8 %), w (2.4 %), and i  fragments from secondary backbone cleavage (0.3 %), along with 64.4 % undissociated ions and 9.0 % nucleobase loss from the latter. Nucleobase loss from fragments was also observed in 17.2 %, 5.1 %, 72.7 %, and 0.4 % of all c, y, a, and w ions, respectively; the substantially higher value for a ions is consistent with the dominant mechanism proposed for 3′ C−O cleavage that is initiated by nucleobase loss.c and y ions , on the other hand, does not involve nucleobase loss.c and y ions were generally higher on the 5′‐side of guanosine 3H13− ion6 %), y 1.0 %, a . This suggests that some of the net positive charge of the tat peptide is preserved in both the (TAR+tat−13H)13− ions and its c, y, a, and w fragments and corroborates the hypothesis that electrostatic interactions play a major role in stabilizing the gaseous TAR–tat complex. Importantly, noncovalent bonds between TAR and tat as well as the covalent bonds of tat can apparently be preserved at energies that are sufficiently high to cleave covalent RNA‐backbone bonds and disrupt intramolecular RNA interactions such as base pairing in stem regions /(Y(c or y with tat)+Y(c or y without tat)); within error limits, these were the same for ESI circa 5 and 30 min after preparation of the solution. Zero occupancy was found for c1–c5, c7–c9, y2–y9 (y1 was not detected) and 100 % occupancy for the complementary y30–y26, y24–y22, c30–c22 fragments, suggesting that TAR residues 1–5, 7–9, and 22–31 did not, or at least not strongly, interact with tat. Occupancy values other than 0 % or 100 % were found for all other sites, for example, from cleavage at site 18, 68.9±0.8 % for c18 and 49.4±1.0 % for its complement y13. This can be rationalized by the interactions of tat peptide with more than one residue of TAR RNA and the breaking by CAD of only some of them according to the strength and number of interactions with each fragment. In the above example, the higher fraction of c18 fragments with tat peptide attached compared to that of y13 suggests an overall higher stability of tat interactions with c18 than with y13, but both are sufficiently strong to prevent loss of tat during the backbone cleavage of TAR.Figure c and y fragments, however, generally exceeded or fell below the expected 100 %; for cleavage at site 18, the values for c18 and y13 add up to 118 %. The discrepancies of the added occupancy values from 100 % ranged from 2 % at site 10 to 37 % at site 11, with an average value of 17 %, and can be attributed to different stabilities of the c and y fragments, with and without peptide attached, against secondary backbone cleavage. To exemplify this point, yields of complementary c18(1 tat) and y13(0 tat) fragments (top) and c18(0 tat) and y13(1 tat) fragments (bottom) are illustrated in Figure c18 fragments undergo secondary backbone cleavage more readily than y13 fragments, and c18(1 tat) showed higher stability than c18(0 tat). Because secondary backbone cleavage of a c or y fragment produces both an internal i fragment and a smaller c or y fragment, it not only decreases the yield of larger c or y fragments .13− ions was obtained at energies greater than or equal to 111.8 eV, and the yields of the internal fragments were less than 1 % at energies below 130 eV, with 120.9 eV . We instead attribute this phenomenon to charge redistribution within fragment ions by proton transfer according to Coulombic repulsion,c and y fragments for tat. In this scenario, an electrostatic interaction of residue 6 of c6 with tat can be sufficiently strong to compete with the interactions between the complementary fragment y25 and tat, whereas in the c7, c8, and c9 ions, negative charge will move away from residue 6 to the terminal residues 7, 8, and 9, respectively,c6.The occupancy values of s Figure  b; howev12− ions  revealed the sites of tat binding to TAR in the 1:2 TAR–tat complex ,In conclusion, we have shown that native top‐down MS provides time‐resolved information on TAR–tat complex stoichiometry and reveals tat binding sites at the single‐residue level of TAR RNA. Apparently, the electrostatic interactions between TAR RNA and the basic tat peptide in the gas phase are sufficiently strong to survive at CAD energies that cleave phosphodiester backbone bonds of RNA. Native top‐down MS requires only small amounts of sample without the need for laborious preparation procedures, allows for the separate study of species that differ in Experiments were performed on a 7 T Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometer  equipped with an ESI source, a linear quadrupole for ion isolation, and a collision cell for CAD.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Our results indicate that the synthesis of a new class of compounds containing hypervalent nitrogen is within reach of current high-pressure experimental techniques.In all of its known connections nitrogen retains a valence shell electron count of eight therefore satisfying the golden rule of chemistry - the octet rule. Despite the diversity of nitrogen chemistry (with oxidation states ranging from + 5 to −3), and despite numerous efforts, compounds containing nitrogen with a higher electron count  remain elusive and are yet to be synthesized. One possible route leading to nitrogen’s hypervalency is the formation of a chemical moiety containing pentavalent nitrogen atoms coordinated by more than four substituents. Here, we present theoretical evidence that a salt containing hexacoordinated nitrogen(V), in the form of an NF Li3N exhibiting 8-fold N coordination)3PAu)5N]2+)Since the first synthesis of the NF5) in the gas phase9101112is a minimum on the potential energy surface (PES)11123 and F2 is highly exothermic (+1.82 eV per NF5)125 is a trigonal bipyramid with five covalent N–F bonds. Interestingly the energy of formation of the molecule is comparable with that of solid (NF4+)(F−)5 system an interesting example of the interplay between covalent and ionic bonding.Theoretical investigations into the properties of the nitrogen pentafluoride molecule . At present pressures up to 200 GPa are routinely achieved in diamond anvil cells (DACs), and the large influence of such high-pressure (HP) conditions on the properties and reactivity of the elements and chemical compounds is well documented151617In this communication we present results of a comprehensive computational investigation on the possibility of synthesizing solid NFincrease considerably at elevated pressures with calculations indicating that at such conditions F2 should oxidize Cs to CsF3212 to HgF32252627192930xHy structures should, and indeed does stabilize at HP conditions.Most importantly the oxidative strength of fluorine has been predicted to 5 were identified through the application of the USPEX evolutionary algorithm365 polymorphs exhibiting numerous and versatile bonding patterns. All thermodynamic and structural parameters reported here are obtained with the use of the HSE06 functional.Our solid-state calculations, performed within the Density Functional Theory, in the 0–300 GPa pressure range, utilized the hybrid HSE06 functional32335 are shown in P1 structure is a molecular crystal consisting of NF3 and F2 molecules, and is the only polymorph containing trivalent nitrogen. Similarly, the P–1 structure is also a molecular crystal, but in contrast it is composed of isolated NF5 units. Both R3m and I–4 structures exhibit ionic character and both contain NF4+ cations and F− anions. The I4/m and P4/n phases are also ionic, but apart from the NF4+ and/or F− ions they both contain the NF6− anion in which pentavalent nitrogen is bonded to six fluorine atoms. This anion was first proposed by Ewig and Van Wazer5 + F−. To our best knowledge there have been no prior reports on the stabilization of NF6− in the solid state.The lowest enthalpy structures of NFnm+ fragments is based not only on the fact that the optimized geometry of these fragments agrees well with that predicted by the VSEPR model3 – trigonal pyramid, NF4+ – tetrahedron, NF5 – trigonal bipyramid, NF6− – octahedron), but also on the excellent accordance between the bond lengths of these moieties at effectively 0 GPa and those obtained from gas-phase calculations . This is best exemplified by the fact that at 300 GPa the secondary N···F contacts of all structures are more than 25% longer than the intramolecular N–F bonds, while F···F distances are more than 30% longer compared to the genuine F–F bond in a F2 molecular crystal optimized at the same pressure. As expected all of the studied NF5 polymorphs are wide-gap insulators with the band gap exceeding 5 eV even at 300 GPa.The assignment of ionic/neutral NFulations . It’s noThe structures optimized with the PBE functional exhibit similar geometries to those described above (obtained with HSE06). Most importantly, we have compared the PBE and HSE06-optimized structure and found no evidence of a Peierls distortion ensuing after optimization of the PBE structures conducted with HSE06, in contrast to what was found for a polymeric phase of nitrogen5 unit (≈58 kJ/mol); see Surprisingly, despite the large diversity of bonding patterns exhibited by the structures containing pentavalent nitrogen their relative enthalpies at low pressure fall in a narrow range of 0.6 eV per NFP1 phase (Z = 1), containing molecular NIIIF3 mixed with F2, which is the lowest enthalpy structure. Interestingly upon compression of the P1 structure the F–F bond in F2 lengthens from 1.39 Å at 0 GPa to 1.44 Å at 39 GPa, while the shortest secondary N···F contact contracts from 3.14 Å to 1.89 Å. This points to a significant pressure-induced enhancement of the donor-acceptor interactions between the HOMO of NF3 and the antibonding LUMO of F2. In fact, upon compression to 40 GPa this interaction leads to heterolytic dissociation of the F2 molecules and formation of an ionic structure containing NF4+ cations separated by F− anions. At 40 GPa the shortest F···F contact in P1 is 2.17 Å and the four N–F bonds have a length of 1.29 Å. Those changes clearly illustrate that compression of the P1 phase up to 40 GPa leads to spontaneous oxidation of NIIIF3 by F2 and subsequent formation of NVF4+ and F−.At pressures lower than 11 GPa it is the P1 phase turns out to be identical in terms of geometry with another NF5 polymorph, R3m (F−) structure of the I–4 symmetry (Z = 2), which is even more densely packed than the R3m (F− anions surrounded by 12 F atoms originating from 8 NF4+ cations). This structure is noteworthy since the I–4 phase becomes more stable than the R3m polymorph at 37 GPa. Nevertheless, already above 33 GPa both I–4 and R3m have a higher enthalpy than a more complex I4/m phase (Z = 6), which remains the lowest-enthalpy structure up to 151 GPa (F−) and (NF4+)2 layers leading to a general formula of (NF4+)2(NF6−)(F−) = 3NF5. This structure bears many similarities to the HP phase of PCl5 (I2/m symmetry) which is best formulated as (PCl4+)2(PCl6−)(Cl−)I2/m polymorph is also layered, but in contrast to the I4/m structure of NF5 it exhibits tilting of the complex ions about an axis lying in the plane of the layers. Geometry optimization of a NF5 polymorph isostructural with I2/m indicated that such a structure is not competitive with I4/m in terms of enthalpy.The 6− anion in I4/m exhibits a slight tetragonal distortion at the low pressure limit (effectively 0 GPa) with two axial N–F bonds shorter by 1.3% than the four equatorial ones. Still all the bond lengths are very close to those obtained in molecular calculation (I4/m remains nearly constant with increasing pressure and does not exceed 1% at 300 GPa. Upon compression of I4/m the N–F bonds in NF4+ shorten by 5% (to 1.25 Å at 300 GPa), while in case of the axial/equatorial bonds of NF6− the contraction is approximately 8%.The NFculation . The difI4/m is predicted to become thermodynamically less stable than a P4/n structure (Z = 4) containing a 1:1 ratio of NF4+ and NF6−, and no F− anions. This polymorph is isostructural with the ambient pressure phase of PCl5 = (PCl4+)(PCl6−)P4/n the NF6− anions are more distorted compared to I4/m with two unequal axial N–F bonds  is correct requires more elaborate calculations which are beyond the scope of this communication.Above 151 GPa –F bonds . High prnion see . Unequiv5 molecules would be the most stable polymorph of NF5. The P–1 structure, which contains such units, becomes more stable than R3m/I–4 at 78/243 GPa, but still in the whole pressure region studied it remains less stable than the NF6-containing polymorphs (I4/m and P4/n). The coordination of NF5 molecules in the P1 phase changes from trigonal bipyramidal to square pyramidal above 40 GPa 2(NF6−)(F−) (I4/m polymorph). Furthermore, we found NF5 to be stable against decomposition into NF4, another possible fluorine-rich phase of the N/F phase diagram (see Section III of the SI). Also, we emphasize that the calculated phonons remain positive within the whole Brillouin zone; a proof that the I4/m phase is dynamically stable both at the pressure of synthesis (40 GPa) as well as at higher pressures (see section IV in SI).The enthalpy change associated with the reaction: NF3 by F2 is accompanied by the ionization of the reactants, that is formation of NF4+, NF6− and F− ions in place of neutral NF5 molecules. The tendency for heterolytic, rather than homolytic splitting of the F–F bond in F2 reacting with NF3 is further exemplified by the molecular-ionic transition observed in the P1 polymorph of NF5.Our results hint that the high-pressure oxidation of NFet al. also predict formation of ionic species at high pressure and at large hydrogen contentsnormal valent species , in contrast to the hypervalent NF5, NF6− species reported in this study. Another difference is that for the N/H system the NH5 composition is metastable with respect to decomposition into stable NH4 and H2 while we find NF4 to be unstable at both ambient and high pressure. The instability of hypervalent N/H molecules can be traced back to the large steric crowding around the nitrogen atom in NHn (n > 4) species. In fact, as calculated by Ewig and van Wazer, the NH5 molecule is not only thermodynamically but also dynamically unstable in the gas phase, in contrast to NF5 which is dynamically stableOn a side note, we here point that for the related N/H system Qian 3, N2O)2529L) of ionic phases which leads to additional stabilization of such structures with respect to molecular, van der Waals bonded polymorphs. The increase in HL is a consequence of the volume reduction upon compression, as the lattice enthalpy is proportional to the inverse cube root of the molecular volume 43The propensity for the formation of ionic phases at HP, observed also for other nitrogen compounds (F−) stoichiometryInterestingly in the case of the NF3 and F2. A newly formed salt, of (NF4+)2(NF6−)(F−) stoichiometry, would constitute the first example of a compound containing hypervalent nitrogen atoms. Most interestingly, due to relatively low pressures involved and due to the involvement of strong ionic interactions this new species might be stable even upon decompression to ambient pressure, particularly at low temperatures. Due to the computer-intensive nature of phonon calculations a detailed study of the dynamic stability of various NF5 phases at low pressure is beyond the scope of this study.In summary our calculations indicate that a compound containing nitrogen(V) covalently bound by six fluorine atoms can be synthesized via a HP reaction between NF5 bear many similarities to those exhibited by PCl5, which serves as good example on the rule of thumb that at HP elements tend to resemble their heavier congenersFinally, it is also worth to remark that the phase transitions of NF−6 eV. Valence electrons were treated explicitly, while standard VASP pseudopotentials were used for the description of core electrons. The k-point mesh was set at 2π × 0.06 Å−1. All structures were optimized using a conjugate gradient algorithm until the forces acting on the atoms were smaller than 10 meV/Å. The abovementioned parameters ensured convergence of the calculated enthalpy within 2 meV per atom.Periodic DFT calculations utilized the HSE06 hybrid potential323347483, F2, NF5, as well as NF4  using the HSE06 functional. Beside the best structures identified at PBE level, we also used a number of enthalpically low lying meta-stable structures in the HSE06 re-optimization. For NF3 our structure search identified the Pnma, Pnma (2), and P212121 molecular phases proposed in an earlier study3(s)  + F2(s) → NF5(s) was calculated taking (at each pressure) the lowest enthalpy polymorph of NF5 and NF3, as well as the ambient-pressure α polymorph of F22 (C2/c space group) symmetrizes spontaneously to a Cmca structure which is analogous to the high-pressure polymorph of Cl22 remains in the form of a molecular crystal.The candidate structures of NFl as NF4  were ide5 polymorphs we have calculated dispersion corrections (in the form of D3 correction proposed by Grimme and co-workers53545 polymorphs (changes of transition pressures do not exceed 2 GPa upon inclusion of D3 corrections).In order to determine the influence of dispersion-type interactions on the relative stability of NFStructure visualization was performed with the VESTA 3.1 softwareHow to cite this article: Kurzydłowski, D. and Zaleski-Ejgierd, P. Hexacoordinated nitrogen(V) stabilized by high pressure. Sci. Rep.6, 36049; doi: 10.1038/srep36049 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


To understand the K+ selectivity in the presence of a high concentration of competing Na+ ions requires consideration of more than just ion binding affinities. Here, equilibrium‐based calculations of the extracellular occupation of the Na+,K+‐ATPase transport sites by Na+ and K+ are compared to fluxes through Na+ and K+ transport pathways. The results show that, under physiological conditions, there is a 332‐fold selectivity for pumping of K+ from the extracellular medium into the cytoplasm relative to Na+, whereas equilibrium calculations alone predict only a 7.5‐fold selectivity for K+. Thus, kinetic effects make a major contribution to the determination of extracellular K+ selectivity.The sodium potassium pump (Na Similarly, K+ binds to K+ channels from the cytoplasm where the K+ concentration is already high and the concentration of competing Na+ ions is low.Selectivity for Na+,K+‐ATPase, K+ ions must bind from the extracellular medium, where the concentration of competing Na+ ions is much higher than that of K+. Therefore, the ion selectivity of the Na+,K+‐ATPase needs to be much more stringent than that of ion channels. In principle, there are two contributions to ion‐transporting selectivity: firstly, the equilibrium binding affinities of the enzyme for either Na+ or K+, and secondly, the kinetics of Na+‐ and K+‐ transporting pathways.In contrast, in the mechanism of the Na+,K+‐ATPase for Na+ and K+ can be gained from the structure of the enzyme and theoretical free energy calculations. Recent crystal structures showed the Na+,K+‐ATPase in a transition state preceding the E1P.2Na+ conformation and a sodium occluded (Na3). E1P‐ADP state + to the E1 state, especially to the third ion binding site, site III + channels for Na+ over K+ ions within the channel's selectivity filter +,K+‐ATPase have shown the E2 conformation of the enzyme preferentially binds two Rb+ ions  or two K+ ions et al. +,K+‐ATPase is essential for K+ selectivity on the extracellular face of the protein. Structure‐based energy simulations can in principle explain differences in equilibrium ion binding affinities. However, as Yu et al. +,K+‐ATPase is not in equilibrium and its ion pumping activity occurs on the millisecond–second timescale, a timeframe not currently accessible to molecular dynamics simulations, and that equilibrium‐based calculations of binding affinities to particular enzyme conformations are based on assumptions of slow subsequent reactions, that is, slow occlusion or dephosphorylation. Therefore, to fully understand the basis for the selective inward pumping of K+ by the Na+,K+‐ATPase, it is necessary to consider both the thermodynamics and the kinetics of the enzyme.Information on the affinity of the Nacluded Na. E1P‐ADP+,K+‐ATPase activity, the E2P conformation of the enzyme releases 3Na+ ions to the extracellular medium per ATP hydrolysed and then takes up 2K+ ions, pumping them into the cell +,K+‐ATPase under physiological conditions based on the Albers–Post model was published + ions for 2Na+ ions, that is, net one Na+ ion leaving the cell, in addition to the more well‐known 3Na+/2K+ exchange. Based on this model, kinetic calculations can be performed to predict the relative frequencies of 3Na+/2K+ versus 3Na+/2Na+ pumping, that is, to determine the overall extracellular ion selectivity of the Na+,K+‐ATPase. Here, the aim is to dissect the relative contributions of pure ion binding affinities  and kinetically induced perturbations of ion binding affinities due to subsequent reactions to the overall extracellular ion selectivity of the protein.In the conventional Albers–Post scheme of Na+,K+‐ATPase turnover were performed using the commercially available program berkeley‐madonna 8.0  and the variable step‐size Rosenbrock integration method for stiff systems of differential equations. The simulations yielded the time course of the concentration of each enzyme intermediate involved and the steady‐state flux through each of the enzyme's parallel pathways. For the purposes of simulations, each enzyme intermediate was normalized to a unitary concentration and the enzyme was assumed arbitrarily to be initially totally in the E1 state. To determine the steady‐state flux through each pathway, each simulation was carried out until the distribution between the different enzyme states no longer changed and the fluxes reached constant values. The entire kinetic model is shown in Fig. Computer simulations of the steady‐state Na+/2K+ pumping accounts for 99.7% of all ion exchanges carried out by the Na+,K+‐ATPase, whereas 3Na+/2Na+ pumping only accounts for 0.3%. The kinetic simulations do not take into account dephosphorylation by the back transfer of phosphate to ADP, which has only been demonstrated + and no external K+. Taking the ratio of the two percentages given above amounts to a K+ inward transport preference over Na+ of 332 : 1. In the absence of any preference for either ion, that is, simply based on the extracellular ion concentrations m K+; 140 mm Na+). Thus, equilibrium and kinetic factors together in fact produce an enhancement of the relative frequencies of K+ inward transport over Na+ by a factor of 11 620. The kinetic simulations show that the 3Na+/2K+ pathway is almost exclusively favoured under physiological conditions. The question is whether binding affinities alone can account for the high preference for the 3Na+/2K+ pathway or whether kinetic factors play an important role.Kinetic simulations based on the model shown in Fig. +,K+‐ATPase, the degrees of occupancy of all of the E2P forms of the enzyme need to be determined, because this state of the enzyme has the ion binding sites facing the extracellular medium  equation +,K+‐ATPase derived from kidney tissue, which occurs as the α1β1 isoform of the enzyme. The values of the dissociation constants used were KN1o = 13.4 mm, KNo = 128 mm and KKo = 0.424 mm. These values were calculated from the corresponding values at Vm = 0 mV = KV=0exp(aFVm/RT). The dielectric coefficients, a, for KN1o, KNo and KKo were taken to be 0.65, 0.37 and 0.37, respectively Based on Eqns E2PK2+ conformation, that is, the state immediately prior to occlusion of 2K+ and dephosphorylation of the enzyme  predict a 332‐fold preference for K+ transport over Na+.From the results shown in Table yme Fig. , relativ6% Table  of the t+ selectivity to the level of virtual exclusive inward K+ pumping. These kinetic factors include the following: (a) occlusion of Na+ or K+ from the E2PNa2+ or E2PK2+ states, respectively, (b) much more rapid extracellular K+ occlusion than Na+, and (c) subsequent dephosphorylation, which continually perturb the extracellular ion binding equilibria and effectively siphon virtually all of the enzyme through the 3Na+/2K+ pathway.Thus, kinetic factors are crucial to enhancing extracellular K+ and K+ occlusion pathways on the perturbation of the E2P binding equilibria can be assessed by simulating the enzyme turnover using the kinetic model +/2K+ exchange and only 19.3% 3Na+/2Na+ exchange. The preference for 3Na+/2K+ exchange can be explained in part due to the fact that only the E2PNa2+ and E2PK2+ states are considered to undergo ion occlusion. Both of these reactions cause a shift away from the E2PNa3+ state, hence facilitating K+ binding. Assuming the mixed state E2PK+Na+ is incapable of ion occlusion, its occupation would also be depleted by both the Na+ and K+ occlusion pathways. The further increase in the preference for 3Na+/2K+ exchange from 80.7% to 99.7% is achieved by a much more rapid ion occlusion by E2PK2+ than E2PNa2+. Thus, the ion binding equilibria are shifted even further in the direction of E2PK2+.The effect of the extracellular Na+,K+‐ATPase does bind K+ ions extracellularly much more strongly than Na+ ions, because of the high degree of competition from the much higher concentration of Na+ ions in the extracellular medium relative to K+, the stronger K+ binding affinity alone is insufficient to explain the virtual exclusive pumping of K+ ions from the extracellular medium into the cytoplasm under physiological conditions. As recently pointed out by Rui et al. +,K+‐ATPase overcomes this problem by having a much higher activation energy barrier for the occlusion of Na+ than K+. Rui et al. +. After K+ ions bind to the E2P conformation, a conformational change of the protein occurs, occluding the ions within the protein matrix so that they have no access to either the extracellular medium or the cytoplasm. Although this conformational change can also occur when Na+ binds, presumably because of the larger size of the K+ ion and its ability to coordinate with surrounding amino acid side chains, the reaction is much faster with K+ ions present in the binding sites. The rate constant for K+ occlusion by the E2P conformation has been experimentally determined to be 342 s−1+ occlusion occurs with a rate constant of only around 4 s−1+ ions draws virtually all of the enzyme cycles through the K+ pumping pathway in spite of an expected equilibrium occupation of the E2P conformation by K+ of only 20%.In summary, although the E2P conformation of the Na+,K+‐ATPase for K+ is also consistent with experimental measurements in which the enzyme was treated with palytoxin (PTX) +,K+‐ATPase and turns it from a pump into a channel by stabilizing an open ion transport pathway across the entire membrane +,K+‐ATPase with PTX bound still hydrolyses ATP, Harmel and Apell Km of the protein for external Na+ and K+ in the PTX bound and free states. If one uses 1/Km as an approximation of binding affinity, their results indicated that treatment with PTX causes a 22‐fold reduction in the selectivity of the E2P state for K+ over Na+. This could be explained by the inability of the PTX‐treated enzyme to preferentially occlude K+ ions, thus in qualitative agreement with the results reported here, stressing the importance of occlusion in determining ion selectivity.It is worth mentioning that the finding here that kinetics plays a major role in determining the extracellular selectivity of the NaRJC designed the project, developed the theory and wrote the paper. EV performed simulations.


GDM) is a glucose intolerance disorder which occurs during pregnancy as a result of insulin insensitivity; it usually disappears after delivery. However, some women with GDM can develop type 2 diabetes (T2D) after delivery, and the mechanisms by which this occurs remain unknown. This study compared the levels of sirtuins (NAD‐dependent deacetylases) and antioxidative enzymes in postpartum women with previous GDM (pGDM) or T2D and in postpartum women with a previous healthy pregnancy (controls). Women with pGDM showed upregulated levels of sirtuin 1 (SIRT1) mRNA and protein, with reduced expression levels of sirtuin 3 (SIRT3) and superoxide dismutase 2 (SOD2), relative to the controls. Women with T2D similarly showed a lower level of SIRT3 mRNA than the controls. Lipid peroxidation  was higher in women with pGDM than in the controls. These data show that in women with pGDM, the reduced level of SIRT3 may play a role in the reduced SOD2 level, possibly leading to oxidative stress, which, in turn, upregulates the level of SIRT1. These results might confer the risk of future diabetes development in women with pGDM, as a similar reduction in SIRT3 was found in women with T2D.Gestational diabetes mellitus ( BMIbody mass indexF.Insfasting insulinFPGfasting plasma glucoseGDMgestational diabetes mellitusHbA1cglycosylated hemoglobinMDAmalondialdehydeNFkBnuclear factor kappa BPARPpoly (ADP‐ribose) polymerasepGDMprevious GDMROSreactive oxygen speciesRPGrandom plasma glucoseSIRTsirtuinSODsuperoxide dismutaseT2Dtype 2 diabetesDiabetes mellitus, a metabolic disease characterized by chronic hyperglycemia, with disturbances in the metabolism of carbohydrates, proteins, and fats, is due to defects in insulin action or insulin secretion or both. The number of people with diabetes stood at around 171 million worldwide in 2000, and this is predicted to increase to about 366 million cases by 2030 m) reduces SIRT1 at the mRNA and protein levels and hence its activity m) for 1 week followed by normal glucose conditions for 2 weeks, the cells showed persistent gene and protein expression of the inflammatory and apoptotic nuclear factor kappa B (NFkB), poly (ADP‐ribose) polymerase (PARP), and Bcl‐2‐associated X protein (Bax), which are involved in diabetic vascular complications Sirtuins are NAD‐dependent deacetylases that have a wide spectrum of metabolic and stress‐tolerance properties. Among them are the well‐characterized antioxidative and cytoprotective sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3). Mammalian SIRT1 can activate or suppress the expression of target genes involved in aging, cellular metabolism, and stress response by deacetylating substrates, including histones, coregulators, and transcription factors SIRT3 gene is deleted SIRT3 is the downstream target gene of peroxisome proliferator‐activated receptor‐gamma coactivator‐1 alpha and regulates mitochondrial biogenesis SIRT3 gene knockdown increased apoptosis and cellular ROS in pancreatic islet beta cells isolated from patients with T2D Recent studies have reported that SIRT3 regulates high‐glucose‐induced oxidative stress through the deacetylation and activation of superoxide dismutase 2 (SOD2) The aim of this study was to test the alterations in the levels of circulating sirtuins and SOD in postpartum women with pGDM or T2D in relation to hyperglycemia‐induced oxidative stress.−1 and postload plasma glucose concentration at 2 h of > 11.1 mmol·L−1 for a 75 g oral glucose tolerance test performed during pregnancy −1 were considered as type 2 diabetic according to the American Diabetes Association For this study, 15 pregnant women with GDM (GDM group), 13 pregnant women without diabetes (control group), and five pregnant women with T2D (T2D group) were recruited from the Obstetrics and Gynecology Department of the King Abdulaziz University Hospital, Jeddah, Saudi Arabia. All the donors were informed about the objectives of the research, and informed consent was obtained from each study participant prior to sample collection. This study was approved by the Ethics Committee of the King Abdulaziz University Hospital. Exclusion criteria for the control group were as follows: subjects on medication or who have a family history of diabetes and/or high blood pressure and/or are smokers. Inclusion criteria for the GDM group were as follows: a history of GDM in the second or third trimester of gestation, and absence of infection, hypertension, and/or any other concomitant systemic diseases. Subjects enlisted had a previous fasting plasma glucose (FPG) concentration of > 5.8 mmol·LA venous blood sample was collected for glycosylated hemoglobin (HbA1c) and random plasma glucose (RPG) analyses on the day of delivery. The level of circulating HbA1c, an indicator of blood glucose control, was measured with a Hitachi 911 autoanalyzer . Normal values of HbA1c as reported previously g for 15 min, and the plasma samples were stored at −80 °C until assayed. The fasting blood glucose concentration and insulin levels were measured by an automated enzymatic method . Another 3 mL of blood was collected directly into plain tubes for the ELISA and MDA experiments. The obtained serum samples were stored at −80 °C until processing. Serum concentrations of SIRT1 and MDA were measured with an ELISA system  and a colorimetric assay kit , respectively, following the manufacturers’ instructions.At 1 day postpartum, blood samples were collected again from all three study groups for FPG, fasting insulin, ELISA, and lipid peroxidation (MDA) analyses. The 5 mL of fasting venous blood sample obtained was centrifuged at 3000 rest 2009 software version 2.0.13 For the genetic test, 2.5 mL of blood was collected into PAXgene blood RNA tubes at 1 day postpartum and stored at −80 °C until processing. Total cellular RNA was isolated from the samples with the PAXgene blood RNA kit  as per the manufacturer's instructions. The RNA concentration and purity were assessed spectrophotometrically, and the integrity was evaluated using an Agilent 2100 Bioanalyzer . cDNA was synthesized from 1 μg RNA using an ImProm‐II Reverse Transcription System kit  following the manufacturer's protocols. The SIRT1 primer was designed using the Primer3Primer design tool, whereas the primers for β‐actin (reference gene), SIRT3, and SOD2 were taken from earlier studies t‐test was applied for unpaired data. Results are represented as mean ± SEM of a minimum of four independent values, and differences among means were considered statistically significant when P < 0.05.Student's P = 0.1, n = 10). However, there were no significant changes in MDA levels in the T2D group relative to the control group  in the patients with a history of GDM than in the healthy controls when compared with the housekeeping gene β‐actin     . However, the expression level of SOD2 mRNA did not differ between the T2D and control groups   Fig. A than diWomen with GDM can develop T2D after delivery, the mechanisms of which are unclear.SIRT3 and SOD2 were decreased; this may be accompanied by pathological damage. On the other hand, women with T2D showed significant change in SIRT3 level only, supporting the notion that the glycemic programming of gene expression in women with pGDM could result from a recent exposure to hyperglycemia induced by GDM. It also shows that these altered genes can potentially serve as diabetes risk factors, although this needs to be further investigated in a large cohort study. SIRT1 localized in the nucleus is implicated in mitochondrial biogenesis et al. SIRT1 expression in GDM patient‐derived human umbilical vein endothelial cells cultured under normal glucose conditions. It can be inferred from that study that the increased SIRT1 expression postdelivery seen in our model may be due to a recent history of GDM. The underlying mechanism remains to be elucidated. Although not significant, we also observed a slight increase in the expression of SIRT1 in the women with T2D. Contradictory results were reported by Balestrieri et al. In this study, we found that the expression of circulating SIRT1 was increased at the mRNA and protein levels in women with pGDM, whereas the expression levels of in vivo study showed that SIRT1 expression increased to fourfold in response to oxidative stress (paraquat injection) in adult mouse and monkey hearts SIRT1 expression in women with pGDM could be also due to the mild oxidative stress observed in the present study.High level of MDA has been reported in patients with T2D as a result of hyperglycemia‐induced ROS production and lipid peroxidation SIRT3 expression was decreased in both groups, which can lead to altered mitochondrial function through increased ROS generation and oxidative stress development. Reduced SIRT3 expression has been verified in other models of T2D SIRT3‐knockout mice exhibit metabolic changes, including increased oxidative stress SIRT3 deletion plays a role in impairment of beta‐cell function in T2D through accelerated development of the metabolic syndrome SIRT3, located in mitochondria SOD2, the key superoxide scavenger, was markedly decreased in women with pGDM relative to that in the healthy women, which was in line with studies that have documented reduced antioxidative enzyme levels in diabetic animal models SIRT3 may account for the low SOD2 expression in our model. However, the level of SOD2 mRNA did not differ between the T2D and control groups. Nonetheless, SIRT3 expression was reduced in the T2D group, which might be implicated in the pathogenesis of T2D.Antioxidative enzymes, such as SOD2, glutathione peroxidase, and catalase, exert defense mechanisms to alleviate the excess ROS and prevent oxidative stress We recognize that this study is limited by the low number of study subjects; nevertheless, the statistical significance values obtained strongly confirm the validity of the results. However, further study with a larger number of cases is recommended to explore the mechanisms behind the results observed in the current study. There were no significant differences in characteristics of the subjects except for the significant increases in HbA1c and random glucose levels and higher age of the women with pGDM. In addition, elevated random glucose and postpartum fasting glucose levels were noted in the women with T2D.SIRT1, SIRT3, and SOD2 in women with pGDM. These alterations can confer an increased risk for the development of T2D in women with pGDM as a similar reduction in SIRT3 was also found in women with T2D. Therefore, we believe that measures to enhance SIRT3 expression and its activity would be useful to protect women against the development of diabetes.The results of the current study showed alterations in the levels of SS conceived and designed the project. SS and NA acquired the data. SS, NA and KS analyzed and interpreted the data. SS wrote the paper.


There is an error in the third paragraph of the Introduction. Specifically, the following sentence is incorrect: "The karyotype is represented by a diploid number of A chromosomes (2n = 24) and a variable number (2–10) of additional B chromosomes [12]". This sentence should read: "The karyotype is represented by a diploid number of A chromosomes (2n = 48) and a variable number (2–10) of additional B chromosomes [12]".


Frustrated Lewis pair (FLP) chemistry enables a rare example of alkyne 1,2‐hydrocarbation with N‐methylacridinium salts as the carbon Lewis acid. This 1,2‐hydrocarbation process does not proceed through a concerted mechanism as in alkyne syn‐hydroboration, or through an intramolecular 1,3‐hydride migration as operates in the only other reported alkyne 1,2‐hydrocarbation reaction. Instead, in this study, alkyne 1,2‐hydrocarbation proceeds by a novel mechanism involving alkyne dehydrocarbation with a carbon Lewis acid based FLP to form the new C−C bond. Subsequently, intermolecular hydride transfer occurs, with the Lewis acid component of the FLP acting as a hydride shuttle that enables alkyne 1,2‐hydrocarbation. Scheme 3C][BF4] in a FLP with (o‐tolyl)3P that resulted in either intramolecular Friedel–Crafts reactivity or 1,2‐addition to the alkyne (Scheme N‐methylacridinium ([1]+) with alkynes, with and without additional Lewis bases. These experiments led to the discovery of a new FLP‐mediated route for the 1,2‐hydrocarbation of alkynes. The mechanism of this reaction is distinct from that reported by Mayr and co‐workers and those established for alkyne hydroboration reactions.One approach to access novel products from alkynes and Lewis acids is to treat alkynes with Lewis acid/Lewis base mixtures that form frustrated Lewis pairs (FLPs).2]+) has to be more stable than the vinyl cation initially formed from the interaction of the carbon Lewis acid and the alkyne. Calculations at the M06‐2X/6‐311G level (with PCM (CH2Cl2)) confirmed that this relationship was true for the combination of the N‐methylacridinium cation ([1]+) with 4‐ethynylanisole, with the 1,2‐hydrocarbation product, [2]+, being energetically favored over the vinyl cation 4]−; this anion was chosen as it was an effective counterion in previous FLP chemistry with [1]+)1]+  and the lower nucleophilicity of 4‐ethynylanisole (relative to ynamides), thus resulting in a larger kinetic barrier, which prevents hydride migration in this case despite the favorable thermodynamics.trans‐hydrocarbation with mixtures of [1]+, N‐methylacridane (as a hydride source), and 4‐ethynylanisole ,1][BArCl] had occurred; nevertheless, crystallization (by layering the sample with pentane) and X‐ray diffraction studies revealed formation of the Z 1,2‐hydrocarbation product [3][BArCl] . The formation of the E 1,2‐hydrocarbation product [2][BArCl] was confirmed by NMR spectroscopy, mass spectrometry, and X‐ray diffraction studies. Identical outcomes were observed when the reactions were performed in the dark, and during these reactions N‐methylacridane (1‐H) and a new acridinium species were observed as intermediates.However, the combination of [BArCl]  and a stepwise mechanism  are precluded. The FLP of [1]+/2,6‐lutidine could conceivably react with alkynes by a number of pathways, including dehydrocarbation or 1,2‐addition. To rule out the 1,2‐addition pathway, a non‐nucleophilic Lewis base was used instead of 2,6‐lutidine. On replacing 2,6‐lutidine with the hindered base 2,4,6‐tri‐tert‐butylpyridine (TBP), [2][BArCl] was again formed in situ as the major product from [1][BArCl]/4‐ethynylanisole (after 72 h at 60 °C). The formation of [2][BArCl] with TBP indicates that a deprotonation (dehydrocarbation) pathway is most likely, as alkyne 1,2‐addition products are precluded with this extremely hindered base. The most probable proton source is the Lewis acid activated alkyne .As no hydrocarbation reaction proceeded in the absence of 2,6‐lutidine, a concerted mechanism . The latter variable was explored as the FLP‐mediated production of [2]+/[3]+ is slow; therefore, any intermediates formed during alkyne hydrocarbation (such as 4) will exist in solution in the presence of [1]+. Upon mixing 4, [TBP−H][BArCl], and [1][BArCl] (10 mol %), an immediate color change was noted, with [1][BArCl] completely converted into 1‐H along with approximately 10 mol % formation of a new N‐methylacridinium species, proposed to be [5]+  of [5]+ (relative to Et3B) was computationally determined  level (DCM solvation (PCM)). The HIA of [5]+ at the β position  is lower than that of [1]+ , thus confirming that intermolecular hydride transfer from 4 to [1]+ is favored, consistent with the formation of 1‐H observed during the FLP reaction. More notably, the HIA of [5]+ at the δ position is higher than that at the β position and higher than that of [1]+. This result indicates that the hydride may be transferred from 4 to the δ position of 5 . An analogous transformation was reported for the acid‐catalyzed rearrangement of tertiary propargyl alcohols to allenenols (known as the Meyer–Schuster rearrangement).6 would then lead to the observed product, [2]+.Having identified m Scheme , the deu5]+, we heated equimolar amounts of [5][I] and N‐methylacridane (1‐H), both independently synthesized, in solution at 60 °C. Interestingly, no formation of the expected product 6 from hydride transfer was detected. Instead, after 30 h, the major product observed was the alkene [7]+ , which was presumably formed by the reaction of allene 6 with the by‐product [1]+ from hydride transfer . The lower electrophilicity in the δ position of [2]+ relative to that in [5]+ is attributed to the reduced conjugation between the γ,δ system and the acridinyl moiety . Combined, the data indicate that this 1,2‐hydrocarbation reaction is the result of an initial FLP‐type process: Lewis acid activation of the alkyne/deprotonation (dehydrocarbation) to form 4, followed by stepwise intermolecular hydride transfer/protonation steps to yield the final product [2]+ or [3]+, with [1]+ acting as an exogenous Lewis acid to facilitate hydride shuttling . Similar reactivity has been observed for the protonation of allenolates, with the Z isomer being the kinetic product and the E isomer the thermodynamic product.The hydrocarbation product + (10 mol %) to facilitate hydride transfer, the hydrocarbation product [9]+ was observed as the major product. Notably, the conversion of 8 into [9]+ was significantly quicker when [1]+ was used as an exogenous hydridophilic Lewis acid to mediate the 1,3‐hydride migration than in the absence of [1]+, as observed in the formation of [2]+ from 4.The rate‐determining step of the overall reaction appears to be the dehydrocarbation of the alkyne with the Lewis acid/Lewis base FLP. This hypothesis was supported by the observation of minimal reactivity of the carbon Lewis acid based FLP combination after 72 h at 60 °C when 4‐ethynylanisole was replaced with the less nucleophilic alkyne 4‐ethynyltoluene Scheme , left. H2CH]+ to an alkyne/olefin reacting with an external nucleophile and not undergoing hydrocarbation.In summary, the first example of alkyne 1,2‐hydrocarbation enabled by FLP chemistry has been presented. It is based on extending an established FLP reaction, alkyne activation/deprotonation, to carbon Lewis acid based FLPs, which in this case results in dehydrocarbation. This step is then followed by intermolecular hydride transfer enabled by the Lewis acidic component of the FLP, which acts as a hydride shuttle. Finally, protonation delivers the hydrocarbation product. A series of control experiments disfavored a mechanism based on intramolecular 1,3‐hydride migration; instead, a new alkyne 1,2‐hydrocarbation mechanism was identified that requires a Lewis base and a free Lewis acid. The ability to circumvent the intramolecular 1,3‐hydride migration step in this hydrocarbation mechanism is particularly important, since 1,3‐hydride migration is a slow process (even when highly exothermic), as highlighted by Mayr and co‐workers.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


In this work, we illustrate the potential of using 19F‐containing ligands as future magnetic resonance imaging (MRI) contrast agents and as probes in magnetic resonance spectroscopy studies by significantly increasing their magnetic resonance detectability through the signal amplification by reversible exchange (SABRE) hyperpolarization method. We achieve 19F SABRE polarization in a wide range of molecules, including those essential to medication, and analyze how their steric bulk, the substrate loading, polarization transfer field, pH, and rate of ligand exchange impact the efficiency of SABRE. We conclude by presenting 19F MRI results in phantoms, which demonstrate that many of these agents show great promise as future 19F MRI contrast agents for diagnostic investigations.Fluorinated ligands have a variety of uses in chemistry and industry, but it is their medical applications as  Its detection has already been shown to give remarkable results in traditional  MRS applications.18F radio‐labelled positron emission tomography (PET) agents are highly successful in the clinic as diagnostic probes.Hyperpolarized magnetic resonance spectroscopy (MRS) methods have been successfully used in conjunction with 19F presents several significant advantages over 13C and 15 n detection, as it is spin‐1/2, 100 % abundant, and has 83 % of the sensitivity of 1H detection. Moreover, it has a very wide and, hence, diagnostic chemical shift range (much larger than those of 13C and 31P and comparable to that of 15 n) and it is not present endogenously in biological tissue, so there is no competing background signal to obscure the corresponding in vivo data. The large chemical shift range exhibited by hyperpolarized 129Xe nuclei reflects one of the reasons this alternative probe is attracting so much attention. So far, 19F MRS has been used to aid the probing of catabolic and anabolic drug conversions of anticancer agents such as 5‐fluorouracilIn the context of MRS applications, 19F MRI experiments are presented in the literature, such as increasing the number of 19F atoms per molecule. Variations in transverse relaxation times have also been exploited to provide molecular environment data.19F signal compared to conventional methods. So far, the 19F nuclear magnetic resonance (NMR) response of various compounds has been successfully enhanced using both dynamic nuclear polarizationpara‐hydrogen‐induced polarization).19F nucleus of 3‐fluoropyridine using signal amplification by reversible exchange (SABRE), which is also a para‐hydrogen (p‐H2)‐based technique, was reported in 2009 by Adams et al.19F SABRE MRI results using the same molecule and achieved signal enhancements of 100‐fold at 9.4 T by using SABRE‐SHEATH .1H and 19F spectra using SABRE.bis(trifluoromethyl)pyridine were investigated. In addition, a further report described an attempt to utilize continuous hyperpolarization to hyperpolarize pentafluropyridine,19F hyperpolarization by SABRE.Various approaches to improve the typically low signal‐to‐noise ratio (SNR) of p‐H2,2. As a nuclear singlet, it has no net spin and, so, it is unobservable from an NMR perspective. However, once p‐H2 has come into contact with a metal center, thereby forming p‐H2‐derived hydride ligands, this previously NMR invisible singlet state can be accessed. Now, polarization is transferred from the p‐H2‐derived nuclei to spin‐1/2nuclei that are located in ligands attached to the same metal center through the J‐coupling network.trans to the p‐H2‐derived hydrides receive polarization optimally, as they exhibit the largest difference in cis and trans couplings. The reversible nature of both H2 addition and analyte ligation at the metal center enables hyperpolarization to buildup in non‐ligated analyte molecules in a process that has been described as being catalytic in the transfer of polarization. The hyperpolarization that is created in this way can be readily read‐out in an NMR experiment and, hence, the signal intensity of the resulting NMR spectrum or magnetic resonance (MR) image is suitably enhanced  condition is met.19F nuclei; in 2010, it was calculated that about 20 % of administered drugs contained fluorine atoms or fluoroalkyl groups.19F‐containing substrates and drug molecules with a pyridyl arrangement imidazole‐2‐ylidene), we probed the ability of 1L–6L to displace the chloride ligand of 1 to give [Ir(IMes)(COD)L]+. This step has been shown to be important during SABRE activation and its absence would suggest a substrate that is unlikely to polarize.3L.HCl as the free base were unsuccessful under the conditions that we needed to employ the ligand. This is attributed to the fact that 4‐fluoropyridine reacts with itself to give N‐(4′‐pyridyl)‐4‐fluoropyridinium fluoride, which hydrolyzes to N‐(4′‐pyridyl)‐4‐pyridone (see Section 2.1 of the Supporting Information).1L, 2L, 4L, 5L, and 6L were successfully employed.Prior to investigating the polarization transfer to the 6 Scheme  using th19F NMR spectrum of 20 equivalents of 1L or 5L in the presence of 1 (5 mm) yielded only a single set of peaks, that of free 1L or 5L. A second set of peaks, owing to the formation of [Ir(IMes)(COD)L]+, was not observed. This contrasts strongly with the data obtained for 2L under the same conditions, in which two peaks are observed in a ratio of 0.84:19.16 that correspond to [Ir(IMes)(COD)2L]+ and free 2L, respectively. These two peaks are separated by 362 Hz in a 1.4 T NMR field. The same observation was made when 2L was exchanged for 4L; in this case, the two peaks were separated by 335 Hz in a 1.4 T NMR field, although the ratio was smaller (0.25:19.75 for 4L/[Ir(IMes)(COD)4L]+). Conversely, when 5L was used, the 19F NMR spectrum comprised of only three peaks, representative of the three different 19F NMR environments that 5L possesses. Just like 1L, there were no other observable peaks for bound 5L. As both 1L and 5L possess ortho‐fluorine nuclei, there could be a possibility that steric hindrance around the nitrogen used to ligate to the metal center of 1 is preventing effective ligation. Compared to hydrogen, 19F has a greater van der Waals radius by 0.27 Å, but is still smaller than a methyl group .ortho‐position yielded no detectable 15 n hyperpolarization through SABRE‐SHEATH.pKa of 1L (−0.44) is such that it may not be an effective ligand in comparison to pyridine (pKa=5.23) or 3‐fluoropyridine (pKa=2.97). To the best of our knowledge, no reported pKa data are available for 4L, although 3,5‐dichloropyridine has a reported pKa of 0.51.4L would be expected to have a similar pKa and, thus, is more basic than 1L but less so than 2L. Wang et al. indicated that the pKa of 4L was <2, as the hydrolysis rate of their ruthenium complexes increased relative to when pyridine or 3‐picoline were employed.5L is reported to not react with hot aqueous hydrogen iodide or hydrochloric acid, owing to the base‐weakening effect of the two ortho‐fluorines.1L and 5L acting as efficient ligands to 51. L has previously been reported as not being SABRE active.A 256‐scan 2.21 to form efficient SABRE polarization‐transfer catalysts with the ligands shown in Scheme 2(IMes)(L)3]Cl. 1L, when examined under SABRE, produced a small enhancement (below unity) for both ligand loadings tested here . Furthermore, a strong hydride signal for the corresponding complex [Ir(H)2(IMes)(1L)3]Cl was not observed. Attempts to increase the binding strength by using a smaller carbene (IMe) were unsuccessful (see Section 2.1 of the Supporting Information). Hence, 1L is poorly suited to SABRE.We then sought to evaluate the ability of 2L proved to provide a substantial 1H SABRE response. When the substrate loading is 1:4, 1H SABRE NMR spectra recorded after adding 3 bar of p‐H2 and shaking the sample for 10 s in a field of approximately 65 G show that the protons H‐2 and H‐6 of 2L exhibit a 2103‐fold summed signal gain, which compares to an enhancement of −2397 reported for pyridine by Lloyd et al. under analogous conditions.2L, a value approximately six times higher than the one reported by Shchepin et al. for the same sample at 9.4 T.p‐H2 gas used for sample polarization (H2)(2L)3] is formed and is the dominant SABRE polarization‐transfer catalyst in solution, with a diagnostic hydride signal at δ−23.11 ppm. A second complex, indicated by a pair of hydride resonances δ at 23.87 and −25.15 ppm, was found to be [Ir(IMes)(H2)(2L)2(MeOH)] (see the Supporting Information for further details and characterization data). The possibility of Cl rather than methanol binding was excluded by acquiring spectra of the same reaction in dichloromethane and comparing the results. As exemplified elsewhere,H resonance. Although evidence for methanol binding was obtained, no OH or residual CD2HOD signal enhancement was observed. Shchepin et al. presented 1H NMR spectra in their Supporting Information, which possess unattributed signals that may reflect the observation of species of this type.2L buildup rate at 300 K was extrapolated through Eyring analysis and was found to be 65.1 s−1, that is, approximately 2.8 times larger than the corresponding rate of pyridine measured using the same conditions.−1 suggests that the binding energy is slightly higher than that of pyridine (for which the site trans to hydride has a ΔH≠(build‐up) value of 95 kJ mol−1).The activation parameters corresponding to the process of free ligand buildup in solution following dissociation from the dominant complex were calculated by using data obtained from a series of variable‐temperature exchange spectroscopy (EXSY) measurements (see Section 2.4 of the Supporting Information). The 4L, 6L, and 7L, very good 1H SABRE polarization (of the order of hundreds up to thousands) was obtained in all cases. The SABRE polarization‐transfer catalysts formed with 1 in solution are the tris‐substituted species, [Ir(IMes)(H)2(L)3]+, and the analogous methanol complex, [Ir(IMes)(H)2(L)2(MeOH)]+ .When considering the related molecules 1H NMR spectra were collected on samples containing one‐fold and 17‐fold excesses of 7L in the presence of 5 mm of 1 in MeOD solution. In the case of the sample containing a one‐fold excess, substantial signal enhancements were observed for all three of the non‐exchangeable resonances of the free substrate, with the largest 1H NMR signal enhancement being observed for H‐2 (−272±24), followed by H‐4 and H‐6 . The corresponding values for the solution containing a 17‐fold excess of ligand were 59±2, 50±2, and 55±2. These values are much lower than those seen for 2L and reflect the fact that the protonated form of the free ligand (7L a) dominates in solution.A series of one‐shot 1H NMR spectra, which, in the case of 7L, contains a resonance at δ−23.45 ppm, corresponding to the tris‐substituted complex. A pair of resonances, located at δ−23.84 and −24.04 ppm, arise from a complex where the equatorial sites are occupied by one substrate molecule, one methanol molecule, and two hydrides.Evidence for methanol binding to the iridium center was again seen in the hydride region of the associated 1H SABRE NMR experiments now result in a polarized OH resonance, exhibiting average enhancement values of 6.65±0.5 (one‐fold excess) and 51±2 (17‐fold excess). We have shown, in our previous work,1, can be achieved by adding a mild base to the solution. When analyzing samples containing 20 and 100 mm of 7L after adding Cs2CO3 in equal amounts to the substrate , the enhancement of the free‐ligand resonances increases considerably in both cases, as a result of the molecule being deprotonated (7L b) and increasing the ligand's probability of binding to the catalyst. We note, however, that in the case of low ligand loading, the presence of base promotes and accelerates H–D exchange, a phenomenon that occurs immediately after activation and on the timescale of the experiments, resulting in a progressive decrease of the total enhancement with each addition of fresh p‐H2. Data shows that at least 40 % deuteration of the three sites takes place in the first 15 minutes (see Section 2.2 of the Supporting Information).The corresponding For the sample prepared using a one‐fold excess of ligand and a one‐fold excess of base, relative to the catalyst, the total signal enhancement obtained across the three sites was 2.5‐times higher than that seen for a similar sample prepared without base; the corresponding difference increased to 4.5 times for the 17‐fold loading , and the anti‐fungal drug Vorincazole (9L), both of which are listed on the World Health Organisation's essential medicines list. As 8L is present in solution in its fully protonated form (thus preventing the N centers from binding to 1), we adopted a strategy similar to the one used for 7L to promote the formation of the SABRE polarization‐transfer catalyst and to improve the signal enhancements. In the hydride region of this 1H NMR spectrum, we initially see two signals at δ−15.4 and −18.1 ppm, which are in emission and absorption, respectively, owing to the formation under ALTADENA H resonances belonging to a bound COE (cyclooctene) ligand at δ 3.57 and 3.35 ppm, which is consistent with a prior report,We have also analyzed the fluorine‐substituted nucleobase used widely used in cancer therapy, 5‐fluorouracil (2(IMes)(COE)(8L)2]Cl, exhibits significant SABRE activity. The signal enhancement reduces over the next 5 minutes, as this complex is converted into multiple species (see the Supporting Information for detailed information). Under these conditions, when the excess of 8L is fourfold and 0.5‐fold excess of Cs2CO3 is present, we see a signal at δ 7.56 ppm, corresponding to the free resonance of H‐5. This signal possesses an initial enhancement of 53‐fold and a second peak at δ 5.97 ppm, corresponding to the H‐5 resonance of the ligand when bound to the Ir center, shows an initial 26‐fold enhancement. A set of optimization experiments were undertaken to identify the best base and ligand concentrations, and ultimately a 100‐fold 1H NMR signal enhancement was obtained for the free H‐5 resonance of 8L and approximately a 57‐fold enhancement was observed for the peak corresponding to the bound ligand. Unfortunately, these values decrease rapidly with time, owing to a process of deactivation that quenches the activity of the catalyst. Attempts to block this cyclometalation process using a smaller carbene were unsuccessful under the conditions employed. We also note that rapid H/D exchange leads to the formation of CD3OH alongside the deuteration of the iridium hydride.Hence, the initial product,  , the total enhancement obtained for each molecule is considerably lower than for pyridine. However, when moving to high ligand loadings (17‐fold excess of substrate), the performance of pyridine is surpassed by 2L and 6L (with enhancements which are 3.2 and 1.2 higher), whereas the enhancements obtained for 4L and 7L b become comparable to the ones of pyridine. We show that this can be explained by considering the effect of relaxation in these fluorinated ligands, which relax slower, on average, than pyridine under the same conditions. We note that, in the case of 7L, deprotonation using Cs2CO3 leads to a remarkable increase in T1 of the proton located between the N center and the carboxylic group, from 14.5 to 46.1 s. Our work demonstrates that, by varying the ligand loading and p‐H2 concentration, it is possible to dramatically exceed the previously reported 1H hyperpolarization levels. 19F NMR hyperpolarized spectra were successfully recorded for 2L, 4L, 6L, 7L a, 7L b, 8L, and 9L, and the average enhancements of the free fluorine resonance, with the exception of 8L and 9L, were quantified. The highest values were obtained for 2L and 7L b , although we note that the enhancement values reported by Shchepin et al.p‐H2, the enhancement of the 19F resonance can be significantly improved.When comparing the 1H and 19F can be explained by taking into account the short longitudinal relaxation times of 19F, which range between 3 and 5 s at 11.74 T for all of the ligands investigated here. Although this is apparently an obstacle in the way of 19F SABRE MRI applications, we show that, in some cases, one can circumvent this situation by exploiting the long relaxation times of the protons in INEPT polarization‐transfer experiments or by working at relatively low detection fields.The two orders of magnitude difference between the enhancements obtained for 19F MRI images have been recorded by using a similar polarization‐transfer procedure to that used in NMR spectroscopy experiments. When comparing the results with their Boltzmann equilibrium counterparts, we find that hyperpolarization facilitates an SNR gain of over one order of magnitude for most of the substrates investigated, particularly 2L and 7L b, for which the maximum SNR gains obtained were approximately 104 and 76, respectively, representing a very promising for future diagnostic MRI applications.Furthermore, hyperpolarized 2D 19F‐substituted ligands, including drugs included on the WHO list of essential medicines. We present various methods, through which the polarization of 19F nuclei can be optimized and we show, using NMR and MRI results, that some of the substrates investigated in this work exhibit tremendous potential for future applications such as clinical MRI investigations, molecular imaging, and in vivo pH assessment.This work demonstrates the feasibility of using SABRE to hyperpolarize a wide range of 1H and 13C NMR measurements were recorded on a Bruker Avance III series 400 MHz spectrometer, whereas 19F NMR measurements as well as T1 inversion recovery and characterization experiments were performed on a Bruker Avance III series 500 MHz system. Samples concerning 9L and the T1 measurements at 1.4 T were collected by using an Oxford Instruments Pulsar equipped with a 1H and 19F probe. NMR samples were prepared containing 5 mm catalyst precursor in 0.6 mL of [D4]MeOH. NMR measurements were collected using either 4 or 20 equivalents of substrate to 5 mm of iridium in 0.6 mL MeOD . p‐H2 was prepared by cooling hydrogen gas over charcoal at 20 K for all samples, except those of 9L and measurements made at 1.4 T; these utilized hydrogen gas over charcoal at 77 K. After adding p‐H2 at 3 bar pressure, 1H NMR spectra were recorded by using π/2 excitation pulses immediately after shaking the sample in a magnetic field of 65 G. A similar procedure was used for the polarization of heteronuclei. For more details about the sample preparation and experimental procedures, please see Section 1 of the Supporting Information.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.