1,3-Phenylene bis(ketoacid) derivatives as inhibitors of Escherichia coli dihydrodipicolinate synthase

doi:10.1016/j.bmc.2012.01.045

Bioorganic & Medicinal Chemistry

Volume 20, Issue 7, 1 April 2012, Pages 2419-2426

https://doi.org/10.1016/j.bmc.2012.01.045 Get rights and content

Abstract

Dihydrodipicolinate synthase is a key enzyme in the lysine biosynthesis pathway that catalyzes the condensation of pyruvate and aspartate semi-aldehyde. A series of phenolic ketoacid derivatives that mimic the proposed enzymatic intermediate were designed as potential inhibitors of this enzyme and were synthesized from simple precursors. The ketoacid derivatives were shown to act as slow and slow-tight binding inhibitors. Mass spectrometric experiments provided further evidence to support the proposed model of inhibition, demonstrating either an encounter complex or a condensation product for the slow and slow-tight binding inhibitors, respectively.

Graphical abstract

Introduction

Lysine and its immediate precursor meso-diaminopimelate (meso-DAP) are essential elements of bacterial peptidoglycan and proteins.1, 2 As the biosynthetic pathway to lysine is only found in plants and bacteria, it has attracted considerable attention as a target for the design and synthesis of novel herbicides and antibiotics.3, 4, 5, 6 The first committed step in the biosynthesis of lysine is the condensation of pyruvate 1 and (S)-aspartate semi-aldehyde (ASA, 2), catalyzed by the enzyme dihydrodipicolinate synthase (DHDPS) (Fig. 1).⁷

The DHDPS-catalyzed reaction is initiated by condensation of pyruvate 1 with an active site lysine residue (Lys161 in Escherichia coli DHDPS) forming a Schiff base. Subsequent tautomerization to the enamine 5 and aldol-type reaction with ASA 2 then generates the acyclic enzyme-bound intermediate 6 (Fig. 2). Transimination of the acyclic intermediate 6 yields the cyclic alcohol HTPA 3, with simultaneous release of the active site lysine residue.

We previously reported preliminary studies exploring a new class of inhibitor based on analogy to the acyclic enzyme-bound intermediate 6.8, 9 The crystal structure of DHDPS soaked with pyruvate and succinic semi-aldehyde (SAS) shows an enzyme-adduct closely related to 6, which lacks the amino group (Fig. 3A).¹⁰ The adduct exists in an extended conformation with dihedral angles ranging from 100 to 179°. We hypothesized that (1,3-phenylene)bisketoacid 7 and (2-hydroxy-1,3-phenylene)bisketoacid 8 would mimic the enzyme bound intermediate and would present the ketoacid functional groups in a constrained, extended conformation (Fig. 3B). Thus, we first synthesized the parent bis(ketoacid) 7 and derivatives 11–13 (Fig. 3C) and assayed these compounds for inhibition of DHDPS activity.⁸ The compounds showed moderate inhibitory activity against DHDPS (Table 1), and prompted further investigation into more functionalized analogues that more closely mimic the structure of the enzyme-bound intermediate 6; specifically, phenolic analogue 8 incorporating a hydroxyl group to mimic that at C4 of the enzymatic intermediate 6. Herein we report the synthesis of phenol-containing compounds 8, 19 and 20 and detailed analysis of the inhibitory activity and mechanism of action of these compounds and their simpler progenitors through kinetic and mass spectrometric analysis.

Section snippets

Synthesis

Our initial strategy toward synthesis of the phenolic bis(ketoacid) 8 was based on analogy to our previously developed synthesis of the model compound 7 in which the ketoacid groups were generated by oxidation of bis(acetyl)benzene 9 (Fig. 3C).⁸ Accordingly, we required synthesis of the corresponding 2,6-bis(acetyl)phenol 10. However, synthetic strategies towards this target were not successful and an alternate strategy was adopted. Li and Liu have reported the transformation of bromoacetylenes

Conclusions

In summary, new constrained bis(ketoacid) and bis(oximinoacid) derivatives have been synthesized and assayed for inhibition of DHDPS. These compounds display time-dependent inhibition and undergo condensation with the enzyme, generating an enzyme-bound adduct that closely resembles the enzymatic intermediate. Kinetic studies show these inhibitors act in a slow-tight binding manner that has not previously been observed for inhibitors of DHDPS. Inclusion of hydroxyl functionality in phenols 8 and

Materials and methods

Unless otherwise stated, all chemicals were purchased from Sigma–Aldrich or Novabiochem. (see Supplementary data for general experimental procedures).

2,6-Bis((trimethylsilyl)ethynyl)anisole (15)

A 3-neck r.b.f. equipped with a nitrogen inlet and seals was flame dried and flushed with nitrogen, then charged with 2,6-diiodoanisole 14 (1.00 g, 2.78 mmol), copper iodide (57.0 mg, 0.29 mmol, 10 mol %), trans-dichloro-bis(triphenylphosphine) palladium (101 mg, 0.13 mmol, 4.8 mol %) and triethylamine (25 mL). The reaction flask was flushed with nitrogen

Acknowledgments

This work was supported by the Defense Threat Reduction Agency (Project ID AB07CBT004) (C.A.H.) and the Royal Society of New Zealand Marsden Fund (J.A.G. and C.A.H.). The authors thank R.C.J. Dobson, S.R.A. Devenish, F.G. Pearce, S. Dommaraju and M.A. Perugini for providing samples of DHDPS and DHDPR used in enzyme assays, and for useful discussions.

References and notes (26)

R.J. Cox et al.
Bioorg. Med. Chem.
(2000)
J.G. Shedlarski et al.
J. Biol. Chem.
(1970)
B.A. Boughton et al.
Bioorg. Med. Chem. Lett.
(2008)
B.A. Boughton et al.
Bioorg. Med. Chem.
(2008)
L.-S. Li et al.
Tetrahedron Lett.
(2002)
G.I. Feutrill et al.
Tetrahedron Lett.
(1970)
A.I. Bukhari et al.
J. Bacteriol.
(1971)
M.S. Pavelka et al.
J. Bacteriol.
(1996)
C.A. Hutton et al.
Mol. BioSys.
(2007)
C.A. Hutton et al.
Mini-Rev. Med. Chem.
(2003)

R.J. Cox

Nat. Prod. Rep.

(1996)

S. Blickling et al.

Biochemistry

(1997)

Cited by (26)

Synthesis and structure-activity relationship studies of 2,4-thiazolidinediones and analogous heterocycles as inhibitors of dihydrodipicolinate synthase
2021, Bioorganic and Medicinal Chemistry
Citation Excerpt :
Furthermore, as mammals do not require meso-DAP and lysine is obtained nutritionally, the DAP pathway is absent in humans, thus increasing the potential of strategically developing selective inhibitors.14 All inhibitors of the DAP pathway identified to date mimic the natural substrates present including ASA,7,15 pyruvate,15–19 lysine15,20 and the heterocyclic intermediates, DHDP and THDP.7,21–25 More recently, Skovpen et al. designed and synthesised R,R-bislysine with a Ki of 140 nM against Campylobacter jejuni DHDPS.26
Dihydrodipicolinate synthase (DHDPS), responsible for the first committed step of the diaminopimelate pathway for lysine biosynthesis, has become an attractive target for the development of new antibacterial and herbicidal agents. Herein, we report the discovery and exploration of the first inhibitors of E. coli DHDPS which have been identified from screening lead and are not based on substrates from the lysine biosynthesis pathway. Over 50 thiazolidinediones and related analogues have been prepared in order to thoroughly evaluate the structure-activity relationships against this enzyme of significant interest.
Base-mediated Benzannulation Reactions for the Synthesis of Densely Functionalized Aryl α-Keto Esters
2021, Asian Journal of Organic Chemistry
A novel and straightforward strategy for the construction of versatile densely functionalized aryl α‐keto esters is disclosed through a one‐pot and efficient [4+2] benzannulation reaction of α‐cyano‐β‐methylenones and ynediones, which could be synthesized easily from the corresponding starting materials. This protocol features high yield, mild metal‐free reaction condition, high atom economy, high functional‐group tolerance, easy handing and gram‐scale synthesis.
Kinetic, spectral, and structural studies of the slow-binding inhibition of the Escherichia coli dihydrodipicolinate synthase by 2, 4-oxo-pentanoic acid
2021, Archives of Biochemistry and Biophysics
Citation Excerpt :
However acyclic compounds, intended to mimic the enzyme bound intermediate in the reaction, such as diethyl (E)-4-oxo-2-heptadienedioate irreversibly inhibit DHDPS [22,23]. Diketopimelic acid and phenolic keto acid analogs also intended to mimic the enzyme intermediate typically irreversibly inactivated the enzyme, but bound relatively weakly to enzyme with binding constants generally greater than 1 mM [24,25]. However, a potent inhibitor of DHDPS that binds to the allosteric lysine binding site has been developed.
Dihydrodipicolinate synthase (DHDPS) catalyzes the first step in the biosynthetic pathway for production of l-lysine in bacteria and plants. The enzyme has received interest as a potential drug target owing to the absence of the enzyme in mammals. The DHDPS reaction is the rate limiting step in lysine biosynthesis and involves the condensation of l-aspartate-β-semialdehyde and pyruvate to form 2, 3-dihydrodipicolinate. 2, 4-oxo-pentanoic acid (acetopyruvate) is a slow-binding inhibitor of DHDPS that is competitive versus pyruvate with an initial K_i of about 20 μM and a final inhibition constant of about 1.4 μM. The enzyme:acetopyruvate complex displays an absorbance spectrum with a λ_max at 304 nm and a longer wavelength shoulder. The rate constant for formation of the complex is 86 M⁻¹ s⁻¹. The enzyme forms a covalent enamine complex with the first substrate pyruvate and can be observed spectrally with a λ_max at 271 nm. The spectra of the enzyme in the presence of pyruvate and acetopyruvate shows the initial formation of the pyruvate enamine intermediate followed by the slower appearance of the E:acetopyruvate spectra with a rate constant of about 0.013 s⁻¹. The spectral studies suggest the formation of a Schiff base between acetopyruvate and K161 on enzyme that subsequently deprotonates to form a resonance stabilized anion similar to the enamine intermediate formed with pyruvate. The crystal structure of the E:acetopyruvate complex confirms the formation of the Schiff base between acetopyruvate and K161.
A Facile Approach to α-Keto Esters via Oxidative Esterification of α-Amino Carbonyl Compounds
2019, Asian Journal of Organic Chemistry
A direct oxidative esterification of α‐amino carbonyl compounds by two kinds of hydroperoxides, for the synthesis of α‐keto esters has been developed. The reaction shows broad substrates scope and good functional group tolerance, even for the synthesis of bulkyl esters, and is free of metal catalysts and external additives. Notably, the inhibitor of protein tyrosine phosphatases (PTPases) could be successfully synthesized with our protocol.
A Short Covalent Synthesis of an All-Carbon-Ring [2]Rotaxane
2017, Organic Letters
While the current supramolecular syntheses of [2]rotaxanes are generally efficient, the final product always retains the functional groups required for non-covalent preorganization. A short and high-yielding covalent-template-assisted approach is reported for the synthesis of a [2]rotaxane. A terephthalic acid template core preorganizes the covalently connected ring precursor fragments to induce a clipping-type cyclization over the thread moiety. Cleavage of the temporary ester bonds that connect the ring and thread fragments liberates the [2]rotaxane.
TEMP and copper cocatalyzed oxygenation of ketones with molecular oxygen: Chemoselective synthesis of α-ketoesters
2015, Organic Chemistry Frontiers
A novel and practical method for the synthesis of α-ketoesters through TEMP and copper cocatalyzed chemoselective oxidative coupling of commercially available methyl ketones with alcohols is developed. Mild conditions and broad substrate scope make this chemistry an efficient tool for the late-stage modification of bioactive compounds. Detailed mechanistic studies demonstrate a novel mechanism involving an organocatalytic cycle and SET process. The oxygenation process with molecular oxygen enables this transformation.

View all citing articles on Scopus

View full text

1,3-Phenylene bis(ketoacid) derivatives as inhibitors of Escherichia coli dihydrodipicolinate synthase

Abstract

Graphical abstract

Introduction

Section snippets

Synthesis

Conclusions

Materials and methods

2,6-Bis((trimethylsilyl)ethynyl)anisole (15)

Acknowledgments

Bioorg. Med. Chem.

J. Biol. Chem.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem.

Tetrahedron Lett.

Tetrahedron Lett.

J. Bacteriol.

J. Bacteriol.

Mol. BioSys.

Mini-Rev. Med. Chem.

Nat. Prod. Rep.

Biochemistry