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Molecular details for the timing and role of proton transfer in phosphoryl transfer reactions are poorly understood. Here, we have combined QM models, experimental NMR measurements, and X-ray structures to establish that the transition of an archetypal phosphoryl transfer enzyme, βPGM, from a very closed near-attack conformation to a fully closed transition state analogue (TSA) conformation triggers both partial proton transfer from the general acid-base residue to the leaving group oxygen and partial dissociation of the transferring phosphoryl group from the leaving group oxygen. Proton transfer continues but is not completed throughout the reaction path of the phosphoryl transfer with the enzyme in the TSA conformation. Moreover, using interacting quantum atoms (IQA) and relative energy gradient (REG) analysis approaches, we observed that the change in the position of the proton and the corresponding increased electrostatic repulsion between the proton and the phosphorus atom provide a stimulus for phosphoryl transfer in tandem with a reduction in the negative charge density on the leaving group oxygen atom. The agreement between solution-phase19F NMR measurements and equivalent QM models of βPGMWTand βPGMD10NTSA complexes confirms the protonation state of G6P in the two variants, validating the employed QM models. Furthermore, QM model predictions of an AlF4distortion in response to the proton position are confirmed using high resolution X-ray crystal structures, not only providing additional validation to the QM models but also further establishing metal fluorides as highly sensitive experimental predictors of active-site charge density distributions.

Isoelectronic metal fluoride transition state analogue (TSA) complexes, MgF3– and AlF4–, have proven to be immensely useful in understanding mechanisms of biological motors utilizing phosphoryl transfer. Here we report a previously unobserved octahedral TSA complex, MgF3(H2O)−, in a 1.5 Å resolution Zika virus NS3 helicase crystal structure. 19F NMR provided independent validation and also the direct observation of conformational tightening resulting from ssRNA binding in solution. The TSA stabilizes the two conformations of motif V of the helicase that link ATP hydrolysis with mechanical work. DFT analysis further validated the MgF3(H2O)− species, indicating the significance of this TSA for studies of biological motors.

Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β-lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β-lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β-lactamase inhibitors that work by mimicking a high energy ‘tetrahedral’ intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.

Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β-lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β-lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β-lactamase inhibitors that work by mimicking a high energy ‘tetrahedral’ intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.

Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF3 −) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO3 −), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the 1H, 15N and 13C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF3:ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the 1H–15N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex.

Azanucleosides, sugar-modified nucleoside analogues containing a 4′ nitrogen atom, have shown a lot of therapeutic potential, e.g. as anti-cancer and antiviral agents. We report the synthesis of a series of 2’-homoazanucleosides, in which the nucleobase is attached to the 2’-position of the pyrrolidine ring via a methylene linker. A suitable orthogonally protected iminosugar was synthesized by ring closing metathesis and dihydroxylation as key steps and further converted to a series of 8 nucleoside analogues through Mitsunobu reaction with suitably protected nucleobases. The 5′ position of the adenine analogue was then further derivatized with thiols to afford 2 additional compounds. The final compounds were evaluated for biological activity., Graphical abstract Image 1

β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two mechanistic groups: the serine β-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo β-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both β-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-β-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-β-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20–100 fold below that by which it inhibits its current clinical targets, the Class A serine β-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of β-lactamases and justify further work on the development of boronates as broad-spectrum β-lactamase inhibitors.\ud \ud

Graphical abstract, β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two mechanistic groups: the serine β-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo β-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both β-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-β-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-β-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20–100 fold below that by which it inhibits its current clinical targets, the Class A serine β-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of β-lactamases and justify further work on the development of boronates as broad-spectrum β-lactamase inhibitors.

BackgroundThe β-lactam antibiotics represent the most successful drug class for treatment of bacterial infections. Resistance to them, importantly via production of β-lactamases, which collectively are able to hydrolyse all classes of β-lactams, threatens their continued widespread use. Bicyclic boronates show potential as broad spectrum inhibitors of the mechanistically distinct serine- (SBL) and metallo- (MBL) β-lactamase families.MethodsUsing biophysical methods, including crystallographic analysis, we have investigated the binding mode of bicyclic boronates to clinically important β-lactamases. Induction experiments and agar-based MIC screening against MDR-Enterobacteriaceae (n = 132) were used to evaluate induction properties and the in vitro efficacy of a bicyclic boronate in combination with meropenem.ResultsCrystallographic analysis of a bicyclic boronate in complex with AmpC from Pseudomonas aeruginosa reveals it binds to form a tetrahedral boronate species. Microbiological studies on the clinical coverage (in combination with meropenem) and induction of β-lactamases by bicyclic boronates further support the promise of such compounds as broad spectrum β-lactamase inhibitors.ConclusionsTogether with reported studies on the structural basis of their inhibition of class A, B and D β-lactamases, biophysical studies, including crystallographic analysis, support the proposal that bicyclic boronates mimic tetrahedral intermediates common to SBL and MBL catalysis.General significanceBicyclic boronates are a new generation of broad spectrum inhibitors of both SBLs and MBLs.

The synthesis and evaluation of a molecularly imprinted polymer has been undertaken using an oxazine-based tryptophanamide transition state analogue (TSA) as template. An efficient route to the synthesis of oxazine-based TSAs for the reaction of pyridoxamine and indole-3-pyruvic acid has been established, with yields of up to 80%. NMR titration studies were performed to examine the interactions between the functional monomer, methacrylic acid and the template. Complexation of the template by functional monomer in the presence of crosslinker showed an apparent KD of 0.63-0.79 ± 0.04 M (293 K, acetonitrile-d3) based upon the chemical shift of the template amide protons. TSA-imprinted and non-imprinted reference polymers were synthesized by free radical polymerization in acetonitrile. Polymer monoliths were ground and fractionated into a 25-63 μm size range. Polymer-ligand recognition studies were conducted using the polymers as HPLC stationary phases. An imprinting factor (IF) of 2.93 was observed for the TSA, indicating the selectivity of the imprinted sites for the template. Studies using the D- and L-enantiomers of the phenylalaninamide analogue of the template showed enantioselectivity in the case of the imprinted polymer, α = 1.10, though not in the case of the non-imprinted reference polymer (1.00). Using UV-spectroscopy based polymer-ligand binding studies, a maximum theoretical capacity (Bmax) of 0.059 ± 0.004 mmol·g-1 was observed for the imprinted polymer. Conclusively, an imprinted polymer with binding sites selective for the TSA was successfully prepared and shall subsequently be studied with respect to its capacity to catalyse the transamination reaction between pyridoxamine and indole-3-pyruvic acid to yield pyridoxal and tryptophan.

The development of novel and efficient biomimetic materials able to exhibit high catalytic activity is a massive challenge in synthetic chemistry. Natural enzymes have ability to catalyze chemical reactions with high specificity, stereoselectivity, and regioselectivity under moderate conditions (i.e., lower temperature and pressure and near-neutral pH). However, they have some drawbacks, such as the high cost of enzyme isolation and purification, and low operational stability owing to denaturation under harsh reaction conditions. Molecularly imprinted polymer (MIP)-based biomimetic catalysts can overcome these drawbacks of natural enzymes.This chapter provides an overview of recent developments in the design and synthesis of biomimetic catalysts based on MIPs. It begins with a short summary of the transition state theory. This is followed by a description approaches to the construction of biomimetic catalysts. Then, several studies reported in the literature regarding the use of MIP-based biomimetic catalyst systems in hydrolysis, elimination, and C-C bond formation reactions are discussed

Background: Small organic molecules coupled to a carrier protein elicit an antibody response on immunisation. The diversity of this response has been found to be very narrow in several cases. Some antibodies also catalyse chemical reactions. Such catalytic antibodies are usually identified among those that bind tightly to an analogue of the transition state (TSA) of the relevant reaction; therefore, catalytic antibodies are also thought to have restricted diversity. To further characterise this diversity, we investigated the structure and biochemistry of the catalytic antibody 7C8, one of the most efficient of those which enhance the hydrolysis of chloramphenicol esters, and compared it to the other catalytic antibodies elicited in the same immunisation. Results: The structure of a complex of the 7C8 antibody Fab fragment with the hapten TSA used to elicit it was determined at 2.2 A resolution. Structural comparison with another catalytic antibody (6D9) raised against the same hapten revealed that the two antibodies use different binding modes. Furthermore, whereas 6D9 catalyses hydrolysis solely by transition-state stabilisation, data on 7C8 show that the two antibodies use mechanisms where the catalytic residue, substrate specificity and rate-limiting step differ. Conclusions: Our results demonstrate that substantial diversity may be present among antibodies catalysing the same reaction. Therefore, some of these antibodies represent different starting points for mutagenesis aimed at boosting their activity. This increases the chance of obtaining more proficient catalysts and provides opportunities for tailoring catalysts with different specificities.

Im Rahmen dieser Arbeit gelang die Darstellung neuer 6,5 bizyklischer Lactame durch die Kondensationsreaktion einer Zuckerkomponente und eines vicinalen Aminothiol oder –alkohols. Die Zuckerkomponente bestimmt dabei die Konfiguration des sechsgliedrigen Lactamrings, wohingegen durch die Wahl des Aminothiols bzw. Alkohols der Fünfring variiert wird. Ausgehend von den 6,5-bizyklischen Lactamen wurden zwei Eliminierungsstrategien zur Darstellung verschiedener 3,2-Hydroxypyridinonderivate angewendet, deren Synthese die Konfiguration der verwendeten Arabinuronsäure ausnutzte. Die cis-trans-cis Anordnung der Hydroxylgruppen sowie des Heteroatoms am Brückenkopf ermöglicht dabei die selektive Darstellung der 3,2-Hydroxypyridinone. Das Redoxverhalten der so erhaltenen Hydroxypyridinone wurde mittels Zyklovoltammetrie untersucht und mit bereits in der Arbeitsgruppe vorhandenen Pyridinonderivaten verglichen. Die Messungen der Hydroxypyridinone zeigten eine reversible Oxidation. Für N-substituierte 2-Pyridinone wurde dabei verglichen mit den Hydroxypyridinonen ein erhöhtes Potenzial gefunden. Des Weiteren wurden erste Studien bezüglich des Komplexierungsverhaltens der erhaltenen Hydroxy¬pyridinone durchgeführt und die Bildung eines 3:1 Komplexes mit Eisen(III) anhand einer spektrophotometrischen Titration nachgewiesen. Im Rahmen dieser Arbeit konnte zudem gezeigt werden, dass das Oxo-Hydroxypyridinon im Gegensatz zu den isosteren Thio Hydroxypyridinonen über eine diastereoselektive Reduktion des Pyridinonrings zum Aufbau von Hydroxypiperidinonen geeignet ist. Der Substitutionsgrad der Hydroxypyridinone konnte im Weiteren durch das Anbringen eines zusätzlichen Substituenten an Position 8 erhöht werden. Die triflierten Acetonide, die in der Synthese der Hydroxypyridinone als Eliminierungsvorläufer dienen, lassen sich bei ausreichender Stabilität auch über eine nukleophile Substitutionsreaktion zur Darstellung von Dipeptidbausteinen nutzen. Es konnte gezeigt werden, dass die Neigung des Triflats zur Eliminierung mit der Acidität des Protons am Brückenkopf korreliert. Aus diesem Grund ist die Einführung des Stickstoffs an Position 8 nur für das Oxazolidinlactam möglich. Der so erhaltene Dipeptidbaustein Tra=Oap wurde auf seine konformationsinduzierende Wirkung hin untersucht. Es konnte anhand von NMR-spektroskopischen Untersuchungen gezeigt werden, dass es sich bei dem Baustein nicht um ein aktives Turnmimetikum, sondern um ein gestrecktes Derivat handelt., Mimicking the transition states of enzymes plays an important role in medicinal chemistry. I have developed a synthesis of a potential transition state mimic, starting from a bicyclic oxazolidine lactam. The dipeptide building block, which itself has an intact peptide backbone with one C-O-double bond substituted by an C-O sigma bond in order to mimic the transition state found in proteases, was subsequently incorporated into peptide fragments in order to investigate their inhibitory potential. Bicyclic oxa- or thiazolidine lactams can further serve as precursors for Hydroxypyridinons (HOPOs). The HOPO moiety is the chelating structural feature in several ligand systems already used in a broad range of applications and is therefore an interesting synthetic target. Due to the large number of functional groups present, our synthetic route offers an easy entry for further modifications of the HOPO scaffold. I have developed the synthesis of a variety of hydroxypyridinons with different substitution patterns and investigated their metal affinities, for iron for example a 3:1 complex was observed by UV/vis titration. The reduction of the HOPO moiety to the corresponding piperidin¬ons proceeds diastereoselectively.

The crystal structure of the complex of transketolase and thiamine thiazolone diphosphate has been determined at 2.3 Å resolution. The complex has a structure which closely resembles that of this enzyme with the cofactor ThDP. This is consistent with the observation that the binding of the analogue to transketolase involves ground state rather than transition state interactions. Since thiamine thiazolone diphosphate resembles an expected intermediate in the catalytic pathway, the structure of the intermediate was modelled from the crystal structure. Based on this model, enzymic groups responsible for binding of the intermediate and proton transfer during catalysis are suggested.

We have recently shown that alpha-MAPI, a peptidic aldehyde of microbial origin, inhibits the HIV protease with a potency comparable to pepstatin, having, differently from pepstatin, no activity on other aspartic proteases. In this study different peptide derivatives containing a C-terminal aldehyde have been tested to assess the potential of this function for the inhibition of HIV protease. The results of our analysis correspond with the recently published subsite preferences of the viral enzyme, indicating that aldehydes bind to the active site of the HIV protease. Our data suggest that peptide aldehydes can act in their hydrated forms as transition state analogues with the most potent inhibitor having an IC50 of 0.9 microM.

Succinylphosphonate (SP) is a powerful inhibitor or α-ketoglutarate dehydrogenase (KGD). Methylation of the phosphonate reduces its inhibitory effect. The complex of KGD with SP undergoes a kinetically slow transition similar to the process observed during catalysis. α-Ketoglutarate binds to the enzyme—inhibitor complex, preventing its isomerisation.