Linear Free Energy Relationship Analysis of Transition State Mimicry by 3‑Deoxy‑d-<i>arabino-</i>heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic

3-Deoxy-d-<i>arabino-</i>heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with <i>K</i>_i = 1.5 μM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, <i>m</i> = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log­(<i>K</i>_M,Mn<i>K</i>_M,PEP<i>K</i>_M,E4P/<i>k</i>_cat) versus log­(<i>K</i>_i), revealed a slope, <i>m</i>, of 0.34, with <i>r</i>² = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups

Transition State Analysis of Enolpyruvylshikimate 3‑Phosphate (EPSP) Synthase (AroA)-Catalyzed EPSP Hydrolysis

Proton transfer to carbon atoms is a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. The main catalytic challenge for enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) is protonating the methylene carbon atom of phosphoenolpyruvate, or EPSP, in the reverse reaction. We performed transition state analysis using kinetic isotope effects (KIEs) on AroA-catalyzed EPSP hydrolysis, which also begins with a methylene carbon (C3) protonation, as an analog of AroA’s reverse reaction. As part of this analysis, an inorganic phosphate scavenging system was developed to remove phosphate which, though present in microscopic amounts in solution, is ubiquitous. The reaction was stepwise, with irreversible C3 protonation to form an EPSP cation intermediate; that is, an A_H^‡*A_N mechanism. The large experimental 3-¹⁴C KIE, 1.032 ± 0.005, indicated strong coupling of C3 with the motion of the transferring proton. Calculated 3-¹⁴C KIEs for computational transition state models revealed that the transition state occurs early during C3–H⁺ bond formation, with a C3–H⁺ bond order of ≈0.24. The observed solvent deuterium KIE, 0.97 ± 0.04, was the lowest observed to date for this type of reaction, but consistent with a very early transition state. The large 2-¹⁴C KIE reflected an “electrostatic sandwich” formed by Asp313 and Glu341 to stabilize the positive charge at C2. In shifting the transition state earlier than the acid-catalyzed reaction, AroA effected a large Hammond shift, indicating that a significant part of AroA’s catalytic strategy is to stabilize the positive charge in the EPSP cation. A computational model containing all the charged amino acid residues in the AroA active site close to the reactive center showed a similar Hammond shift relative to the small transition state models

Potent Inhibition of 3‑Deoxy‑d‑arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic

3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate pathway. It catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP. The kinetic mechanism was rapid equilibrium sequential ordered ter ter, with the essential divalent metal ion, Mn²⁺, binding first, followed by PEP and E4P. DAHP oxime, in which an oxime group replaces the keto oxygen, was a potent inhibitor, with <i>K</i>_i = 1.5 ± 0.4 μM, though with residual activity at high inhibitor concentrations. It displayed slow-binding inhibition with a residence time, <i>t</i>_R, of 83 min. The crystal structure revealed that the oxime functional group, combined with two crystallographic waters, bound at the same location in the catalytic center as the phosphate group of the tetrahedral intermediate. DAHP synthase has a dimer-of-dimers homotetrameric structure, and DAHP oxime bound to only one subunit of each tight dimer. Inhibitor binding was competitive with respect to all three substrates in the subunits to which it bound. DAHP oxime did not overlap with the metal binding site, so the cause of their mutually exclusive binding was not clear. Similarly, there was no obvious structural reason for inhibitor binding in only two subunits; however, changes in global hydrogen/deuterium exchange showed large scale changes in protein dynamics upon inhibitor binding. The <i>k</i>_cat value for the residual activity at high inhibitor concentrations was 3-fold lower, and the apparent <i>K</i>_M,E4P value decreased at least 10-fold. This positive cooperativity of binding between DAHP oxime in subunits B and C, and E4P in subunits A and D appears to be the dominant cause for incomplete inhibition at high inhibitor concentrations. In spite of its lack of obvious structural similarity to phosphate, the oxime and crystallographic waters acted as a small, neutral phosphate mimic

Transition State Analysis of Acid-Catalyzed Hydrolysis of an Enol Ether, Enolpyruvylshikimate 3‑Phosphate (EPSP)

Proton transfer to carbon represents a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. Multiple kinetic isotope effects (KIEs) were measured for acid-catalyzed hydrolysis of the enol ether functionality of enolpyruvylshikimate 3-phosphate (EPSP) as a nonenzymatic analog of the EPSP synthase (AroA) reaction. The large solvent deuterium KIE demonstrated that protonating C3 was the rate-limiting step, and the lack of solvent hydron exchange into EPSP demonstrated that protonation was irreversible. The reaction mechanism was stepwise, with C3, the methylene carbon, being protonated to form a discrete oxacarbenium ion intermediate before water attack at the cationic center, that is, an A_H^‡*A_N (or A_H^‡ + A_N) mechanism. The calculated 3-¹⁴C and 3,3-²H₂ KIEs varied as a function of the extent of proton transfer at the transition state, as reflected in the C3–H⁺ bond order, <i>n</i>_C3–H+. The calculated 3-¹⁴C KIE was a function primarily of C3 coupling with the movement of the transferring proton, as reflected in the reaction coordinate contribution (^lightν^‡/^heavyν^‡), rather than of changes in bonding. Coupling was strongest in early and late transition states, where the reaction coordinate frequency was lower. The other calculated ¹⁴C and ¹⁸O KIEs were more sensitive to interactions with counterions and solvation in the model structures than <i>n</i>_C3–H+. The KIEs revealed a moderately late transition state with significant oxacarbenium ion character and with a C3–H⁺ bond order ≈0.6

A ^99mTc-Labelled Tetrazine for Bioorthogonal Chemistry. Synthesis and Biodistribution Studies with Small Molecule <i>trans</i>-Cyclooctene Derivatives

<div><p>A convenient strategy to radiolabel a hydrazinonicotonic acid (HYNIC)-derived tetrazine with ^99mTc was developed, and its utility for creating probes to image bone metabolism and bacterial infection using both active and pretargeting strategies was demonstrated. The ^99mTc-labelled HYNIC-tetrazine was synthesized in 75% yield and exhibited high stability <i>in vitro</i> and <i>in vivo</i>. A <i>trans</i>-cyclooctene (TCO)-labelled bisphosphonate (TCO-BP) that binds to regions of active calcium metabolism was used to evaluate the utility of the labelled tetrazine for bioorthogonal chemistry. The pretargeting approach, with ^99mTc-HYNIC-tetrazine administered to mice one hour after TCO-BP, showed significant uptake of radioactivity in regions of active bone metabolism (knees and shoulders) at 6 hours post-injection. For comparison, TCO-BP was reacted with ^99mTc-HYNIC-tetrazine before injection and this active targeting also showed high specific uptake in the knees and shoulders, whereas control ^99mTc-HYNIC-tetrazine alone did not. A TCO-vancomycin derivative was similarly employed for targeting <i>Staphylococcus aureus</i> infection <i>in vitro</i> and <i>in vivo</i>. Pretargeting and active targeting strategies showed 2.5- and 3-fold uptake, respectively, at the sites of a calf-muscle infection in a murine model, compared to the contralateral control muscle. These results demonstrate the utility of the ^99mTc-HYNIC-tetrazine for preparing new technetium radiopharmaceuticals, including those based on small molecule targeting constructs containing TCO, using either active or pretargeting strategies.</p></div

Synthesis scheme for the preparation of 3.

<p>A protected form of HYNIC (<b>1</b>) was coupled to a commercially available tetrazine to form <b>2</b>. The Boc group was removed prior to labelling by treatment with TFA in DCM to produce <b>3</b>. Tz* = (4-(1,2,4,5-tetrazin-3-yl)phenyl) methanamine.</p

Synthesis scheme for ^99mTc-HYNIC-tetrazine-TCO-vancomycin (7) from TCO-vancomycin (6) and 4.

<p>Synthesis scheme for ^99mTc-HYNIC-tetrazine-TCO-vancomycin (7) from TCO-vancomycin (6) and 4.</p

Biodistribution data for 4.

<p>Data are presented as the mean (± SEM) percent injected dose per gram (%ID/g) for selected tissues and fluids from CD1 mice at 0.5, 1, 2 and 6 h post injection (n = 3 per time point). Approximately 0.88 MBq were administered per mouse. Full biodistribution data can be found in the supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167425#pone.0167425.s004" target="_blank">S4 File</a>).</p

Radiolabelling scheme for the HYNIC-tetrazine ligand 3 and HPLC chromatogram of the isolated product 4.

<p>^99mTc labelling of the HYNIC-tetrazine <b>3</b> (A). A γ-HPLC chromatogram of the purified final product <b>4</b> (B).</p

Biodistribution data for active targeting of <i>S</i>. <i>aureus</i> infection using ^99mTc-HYNIC-tetrazine-TCO-vancomycin (7).

<p>Compounds <b>4</b> and <b>6</b> were combined prior to i.v. injection of Balb/c mice (n = 3 per time point). Select fluids and tissues were collected at 1 (gray bars) and 6 h (black bars) post injection, including the infected calf muscle (right), and the non-infected calf muscle (left). Data are expressed as the mean percent injected dose per gram (%ID/g) ± SEM. Tabulated biodistribution data can be found in the supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167425#pone.0167425.s004" target="_blank">S4 File</a>).</p