The kinetic parameters for activation of yeast triosephosphate isomerase (ScTIM), yeast orotidine monophosphate decarboxylase (ScOMPDC), and human liver glycerol 3-phosphate dehydrogenase (hlGPDH) for catalysis of reactions of their respective phosphodianion truncated substrates are reported for the following oxydianions: HPO32–, FPO32–, S2O32–, SO42– and HOPO32–. Oxydianions bind weakly to these unliganded enzymes and tightly to the transition state complex (E·S‡), with intrinsic oxydianion Gibbs binding free energies that range from −8.4 kcal/mol for activation of hlGPDH-catalyzed reduction of glycolaldehyde by FPO32– to −3.0 kcal/mol for activation of ScOMPDC-catalyzed decarboxylation of 1-β-d-erythrofuranosyl)orotic acid by HOPO32–. Small differences in the specificity of the different oxydianion binding domains are observed. We propose that the large −8.4 kcal/mol and small −3.8 kcal/mol intrinsic oxydianion binding energy for activation of hlGPDH by FPO32– and S2O32–, respectively, compared with activation of ScTIM and ScOMPDC reflect stabilizing and destabilizing interactions between the oxydianion −F and −S with the cationic side chain of R269 for hlGPDH. These results are consistent with a cryptic function for the similarly structured oxydianion binding domains of ScTIM, ScOMPDC and hlGPDH. Each enzyme utilizes the interactions with tetrahedral inorganic oxydianions to drive a conformational change that locks the substrate in a caged Michaelis complex that provides optimal stabilization of the different enzymatic transition states. The observation of dianion activation by stabilization of active caged Michaelis complexes may be generalized to the many other enzymes that utilize substrate binding energy to drive changes in enzyme conformation, which induce tight substrate fits.

Photoacoustic (PA) imaging is an emerging imaging modality that utilizes optical excitation and acoustic detection to enable high resolution at centimeter depths. The development of activatable PA probes can expand the utility of this technology to allow for detection of specific stimuli within live-animal models. Herein, we report the design, development, and evaluation of a series of Acoustogenic Probe(s) for Nitric Oxide (APNO) for the ratiometric, analyte-specific detection of nitric oxide (NO) in vivo. The best probe in the series, APNO-5, rapidly responds to NO to form an N-nitroso product with a concomitant 91 nm hypsochromic shift. This property enables ratiometric PA imaging upon selective irradiation of APNO-5 and the corresponding product, tAPNO-5. Moreover, APNO-5 displays the requisite photophysical characteristics for in vivo PA imaging (e.g., high absorptivity, low quantum yield) as well as high biocompatibility, stability, and selectivity for NO over a variety of biologically relevant analytes. APNO-5 was successfully applied to the detection of endogenous NO in a murine lipopolysaccharide-induced inflammation model. Our studies show a 1.9-fold increase in PA signal at 680 nm and a 1.3-fold ratiometric turn-on relative to a saline control.

Photoacoustic (PA) tomography is a noninvasive technology that utilizes near-infrared (NIR) excitation and ultrasonic detection to image biological tissue at centimeter depths. While several activatable small-molecule PA sensors have been developed for various analytes, the use of PA molecules for deep-tissue analyte delivery and monitoring remains an underexplored area of research. Herein, we describe the synthesis, characterization, and in vivo validation of photoNOD-1 and photoNOD-2, the first organic, NIR-photocontrolled nitric oxide (NO) donors that incorporate a PA readout of analyte release. These molecules consist of an aza-BODIPY dye appended with an aryl N-nitrosamine NO-donating moiety. The photoNODs exhibit chemostability to various biological stimuli, including redox-active metals and CYP450 enzymes, and demonstrate negligible cytotoxicity in the absence of irradiation. Upon single-photon NIR irradiation, photoNOD-1 and photoNOD-2 release NO as well as rNOD-1 or rNOD-2, PA-active products that enable ratiometric monitoring of NO release. Our in vitro studies show that, upon irradiation, photoNOD-1 and photoNOD-2 exhibit 46.6-fold and 21.5-fold ratiometric turn-ons, respectively. Moreover, unlike existing NIR NO donors, the photoNODs do not require encapsulation or multiphoton activation for use in live animals. In this study, we use PA tomography to monitor the local, irradiation-dependent release of NO from photoNOD-1 and photoNOD-2 in mice after subcutaneous treatment. In addition, we use a murine model for breast cancer to show that photoNOD-1 can selectively affect tumor growth rates in the presence of NIR light stimulation following systemic administration.

Photoacoustic (PA) imaging has emerged as a reliable in vivo technique for diverse biomedical applications ranging from disease screening to analyte sensing. Most contemporary PA imaging agents employ NIR-I light (650−900 nm) to generate an ultrasound signal; however, there is significant interference from endogenous biomolecules such as hemoglobin that are PA active in this window. Transitioning to longer excitation wavelengths (i.e., NIR-II) reduces the background and facilitates the detection of low abundance targets (e.g., nitric oxide, NO). In this study, we employed a two-phase tuning approach to develop APNO-1080, a NIR-II NO-responsive probe for deep-tissue PA imaging. First, we performed Hammett and Brønsted analyses to identify a highly reactive and selective aniline-based trigger that reacts with NO via N-nitrosation chemistry. Next, we screened a panel of NIR-II platforms to identify chemical structures that have a low propensity to aggregate since this can diminish the PA signal. In a head-tohead comparison with a NIR-I analogue, APNO-1080 was 17.7-fold more sensitive in an in vitro tissue phantom assay. To evaluate the deep-tissue imaging capabilities of APNO-1080 in vivo, we performed PA imaging in an orthotopic breast cancer model and a heterotopic lung cancer model. Relative to control mice not bearing tumors, the normalized turn-on response was 1.3 ± 0.12 and 1.65 ± 0.07, respectively.

We report pH rate profiles for k and K for the isomerization reaction of glyceraldehyde 3-phosphate catalyzed by wildtype triosephosphate isomerase (TIM) from three organisms and by ten mutants of TIM; and, for K for inhibition of this reaction by phosphoglycolate trianion (I). The pH profiles for K show that the binding of I to TIM (E) to form EH·I is accompanied by uptake of a proton by the carboxylate side-chain of E165, whose function is to abstract a proton from substrate. The complexes for several mutants exist mainly as E·I at high pH, in which cases the pH profiles define the p K for deprotonation of EH·I. The linear free energy correlation, with slope of 0.73 ( r = 0.96), between k/ K for TIM-catalyzed isomerization and the disassociation constant of PGA trianion for TIM shows that EH·I and the transition state are stabilized by similar interactions with the protein catalyst. Values of p K = 10-10.5 were estimated for deprotonation of EH·I for wildtype TIM. This p K decreases to as low as 6.3 for the severely crippled Y208F mutant. There is a correlation between the effect of several mutations on k/ K and on p K for EH·I. The results support a model where the strong basicity of E165 at the complex to the enediolate reaction intermediate is promoted by side-chains from Y208 and S211, which serve to clamp loop 6 over the substrate; I170, which assists in the creation of a hydrophobic environment for E165; and P166, which functions in driving the carboxylate side-chain of E165 toward enzyme-bound substrate.