Phosphinotripeptidic Inhibitors of Leucylaminopeptidases

Abstract

Phosphinate pseudopeptide are analogs of peptides containing phosphinate moiety in a place of the amide bond. Due to this, the organophosphorus fragment resembles the tetrahedral transition state of the amide bond hydrolysis. Additionally, it is also capable of coordinating metal ions, for example, zinc or magnesium ions. These two properties of phosphinate pseudopeptides make them an ideal candidate for metal-related protease inhibitors. This research investigates the influence of additional residue in the P2 position on the inhibitory properties of phosphinopeptides. The synthetic strategy is proposed, based on retrosynthetic analysis. The N-C-P bond formation in the desired compounds is conveniently available from the three-component condensation of appropriate amino components, aldehydes, and hypophosphorous acid. One of the crucial synthetic steps is the careful selection of the protecting groups for all the functionals. Determination of the inhibitor activity of the obtained compounds has been done using UV-Vis spectroscopy and standard substrate _L-Leu-p-nitroanilide toward the enzymes isolated from the porcine kidney (SsLAP, Sus scrofa Leucine aminopeptidase) and barley seeds (HvLAP, Hordeum vulgare Leucine aminopeptidase). An efficient procedure for the preparation of phosphinotripeptides has been performed. Activity test shown that introduction of additional residue into P2 position obtains the micromolar range inhibitors of SsLAP and HvLAP. Moreover, careful selection of the residue in the P2 position should improve its selectivity toward mammalian and plant leucyl aminopeptidases.

Keywords: LAP inhibitors; barley aminopeptidase inhibitor; ligand-enzyme interaction; molecular modeling; organophosphorus compound; phosphinate pseudopeptide.

Figure 1

Schematic structure of phosphinic peptide with marked position P2-P2’.

Figure 2

Putative phosphinic pseudotripeptides derived from leucine and homophenylalanine containing modified P2 substituents as inhibitors of leucylaminopeptidase.

Scheme 1

Retrosynthetic analysis of phosphinic pseudotripeptides derived from leucine and homophenylalanine.

Scheme 2

Synthesis of the key building blocks 16 and 17 bearing the side chains of pseudo-HPhe and -Leu, respectively, and required for preparing the target phosphinopeptides 1–9. Reagents and conditions: (a) HMDS, 110 °C; (b) H₂C=CHCO₂Me, 90 °C, 66–74%; (c) 1-AdBr, Ag₂O, CHCl₃, 65 °C, 87–90%.

Scheme 3

Synthesis of phosphinic pseudotripeptide analogs 1–3 and their free acids 4 and 5. Reagents and conditions: (a) HCO₂NH₄/Pd-C, MeOH, reflux or rt; (b) H₂/Pd-C, MeOH; (c) Boc-_L-Met-OH (R¹ = CH₂SMe) or Boc-_L-Hse(TBDMS)-OH (R¹ = CH₂OTBDMS), HOBt, EDC, TEA, DMF, 51–70%; (d) TBAF, THF, 0 °C to rt, 70%; (e) TFA/CH₂Cl₂ (1:1), 85–99%; (f) NaOH/MeOH (0.4 M) them 0.5 M HCl, pH 1, 98–99%.

Scheme 4

Synthesis of phosphinic pseudotripeptide analogs 6–9. Reagents and conditions: (a) H₂/Pd-C, MeOH; (b) Boc-_L-Bip-OH (R¹ = Ph) or Boc_-L-Bpa-OH (R¹ = COPh), HOBt, EDC, TEA, DMF, 50–76%; (c) TFA/CH₂Cl₂ (1:1), 78–99%.

Figure 3

The designation of type inhibition and the inhibition constant K_I by Dixon method for compound 3 in the presence of SsLAP.

Figure 4

The designation of type inhibition and the inhibition constant K_I by Dixon method for compound 1 in the presence of HvLAP.

Figure 5

Alignment of (a) 1-SR isomer and (b) 2-SR isomer in the active site of SlLAP (PDB: 4KSI), and (c) 1-SR isomer and (d) 2-SR isomer in the active site of BtLAP (PDB: 1LCP). Magnesium and zinc ions are shown, respectively, as the green and gray spheres. The surface of the active site is colored according to hydrophobicity.

Figure 6

Alignment of (a) 2-SR isomer, (b) 2-SS isomer, (c) 3-SR isomer, (d) 3-SS isomer in the active site of SlAP (PDB: 4KSI). Magnesium ions are shown as a green sphere. Green lines represent intermolecular hydrogen bonds; blue lines represent ligand–metal interactions. The surface of the active site is colored according to hydrophobicity.

Figure 7

Modeled binding mode of 3-SS isomer in the active center of bovine leucyl aminopeptidase (PDB: 1LCP). Amino acid residues of the inhibitor and enzyme are shown as sticks, while the zinc ions are shown as a gray sphere. Hydrogen bonds are shown as thin green lines, metal ion—phosphinic moiety interaction is shown as a thin blue line, while the surface of the active site is colored according to hydrophobicity.

Figure 8

Modeled binding mode of (a) 7-SR isomer and (b) 7-SS isomer in the active center of bovine leucyl aminopeptidase (PDB: 1LCP). Amino acid residues of the inhibitor and enzyme are shown as sticks, while the zinc ions are shown as a gray sphere. Hydrogen bonds are shown as thin green lines, metal ion—coordinating moieties interaction shown as a thin blue line, aromatic π-electrons—aliphatic group, and sulfur atom shown as dashed lines. The surface of the active site is colored according to hydrophobicity.