Transition state analogue discrimination by related purine nucleoside phosphorylases

Transition state analogues of PNP, the Immucillins and DADMe-Immucillins, were designed to match transition state features of bovine and human PNPs, respectively. The inhibitors with or without the hydroxyl and hydroxymethyl groups of the substrate demonstrate that inhibitor geometry mimicking that of the transition state confers binding affinity discrimination. This finding is remarkable since crystallographic analysis indicates complete conservation of active site residues and contacts to ligands in human and bovine PNPs.     

L-Enantiomers of transition state analogue inhibitors bound to human purine nucleoside phosphorylase

Human purine nucleoside phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.     

Acyclic ribooxacarbenium ion mimics as transition state analogues of human and malarial purine nucleoside phosphorylases

Transition state analogues of PNP, the Immucillins and DADMe-Immucillins, were designed to match transition state features of bovine and human PNPs, respectively. A third generation of inhibitors has been designed that contain an acyclic iminoalcohol to replace the cyclic mimic of the ribooxacarbenium ion at the transition states of PNPs. The best third generation inhibitor is equivalent to the best inhibitors found in the previous transition state analogues.     

Synthesis of a transition state analogue inhibitor of purine nucleoside phosphorylase via the Mannich reaction

[reaction: see text] The expeditious convergent synthesis of the potent human purine nucleoside phosphorylase inhibitor DADMe-Immucillin-G (3) was achieved via the Mannich reaction. The Mannich chemistry of a series of deazapurines and amine hydrochlorides was also investigated.     

Synthesis and evaluation of macrocyclic transition state analogue inhibitors for alpha-chymotrypsin

Lactams 1 and 2 are readily formed from acyclic precursors in the presence of trypsin and chymotrypsin, respectively, identifying the macrocyclic ring system as a potential motif for constrained transition state analogue inhibitors of the serine peptidases. Ketone 3 was synthesized and shown to be a modest inhibitor of chymotrypsin (Ki = 220 microM), albeit 4-fold more potent than the acyclic hydroxy acid 25 (Ki = 1.5 mM as a mixture of epimers). A precursor (31) to the amino boronic acid 4 was also prepared; although this derivative was a potent inhibitor of chymotrypsin (Ki = 130 nM) by virtue of the boronic acid moiety, it showed no advantage over the des-amino analogue 32 (Ki = 120 nM), which is not capable of cyclizing.     

Novel UDP-glycal derivatives as transition state analogue inhibitors of UDP-GlcNAc 2-epimerase

The "epimerisation" of UDP-GlcNAc to ManNAc, the first step in the biosynthesis of sialic acids, is catalyzed by UDP-GlcNAc 2-epimerase. In this paper we report the synthesis of transition state based inhibitors of this enzyme. To mimic the assumed first transition state of this reaction (TS 1), we designed and synthesized the novel UDP-exo-glycal derivatives 1-4. We also report herein the synthesis of 5 and 6, the first C-glycosidic derivatives of 2-acetamidoglucal, and the synthesis of the ketosides 7 and 8, which were designed as bis-substrate analogue and bis- product analogue, respectively, to mimic the second step of the reaction via the assumed second transition state TS 2.     

Phosphate activation in the ground state of purine nucleoside phosphorylase

Phosphate and ribose 1-phosphate (R1P) bound to human purine nucleoside phosphorylase (PNP) have been studied by FTIR spectroscopy for comparison with phosphate bound with a transition state analogue. Bound phosphate is dianionic but exists in two distinct binding modes with similar binding affinities. The phosphate of bound R1P is also dianionic. Bound R1P slowly hydrolyzes to ribose and phosphate even in the absence of nucleobase. The C-OP bond is cleaved in bound R1P, the same as in the PNP-catalyzed reaction. Free R1P undergoes both C-OP and CO-P solvolysis. A hydrogen bond to one P-O group is stronger than those to the other two P-O groups in both the PNP.R1P complex and in one form of the PNP.PO4 complex. The average hydrogen bond strength to the PO bonds in the PNP.R1P complex is less than that in water but stronger than that in the PNP.PO4 complex. Hydrolysis of bound R1P may be initiated by distortion of the phosphate moiety in bound R1P. The unfavorable interactions on the phosphate moiety of bound R1P are relieved by dissociation of R1P from PNP or by hydrolysis to ribose and phosphate. The two forms of bound phosphate in the PNP.PO4 complex are interpreted to be phosphate positioned as the product in the nucleoside synthesis direction and as the reactant in the phosphorolysis reaction; their interconversion can occur by the transfer of a proton from one PO bond to another. The electronic structure of phosphate bound with a transition state analogue differs substantially from that in the Michaelis complexes.     

Synthesis of the first stable phosphonamide transition state analogue

Three methods were selected for the one-pot synthesis of the fully protected beta-fluoroaminophosphonic acids, using the readily accessible N-protected beta-fluoroaminals. These were activated by acylation leading, by beta-elimination, to a transient N-acylimine immediately trapped by reactive forms of dialkyl phosphites. Avoiding basic conditions, the complete or partial deprotection of these N-protected beta-fluoroaminophosphonic esters allowed the synthesis of the free amino acids, their esters, and a racemic beta-trifluorophosphonamidic acid. The latter, which represents a transition state analogue formed by the bacterial transpeptidase, is perfectly stable at pH 4.7, contrary to the nonfluorinated compounds.     

A three-component Mannich-type condensation leading to phosphinic dipeptides—extended transition state analogue inhibitors of aminopeptidases

N-Protected α-aminoalkylphosphinic acids bearing a P-H function were found to be novel practical building blocks in three-component condensations with formaldehyde and secondary amines (amino acids). Such Mannich-type N-phosphonomethylation is a common approach for phosphorus acid derived substrates and leads to multifunctional (phosphonic/amino/carboxylic) compounds of diverse relevance. The utility of this reaction was examined for construction, in a single synthetic step, of advanced phosphinic pseudodipeptides designed to act as extended transition state analogue inhibitors of selected aminopeptidases. Phosphinomethylation of primary amino acids was less efficient and yielded mixtures of products which were separated into individual components, and their structures identified.     

Structure-activity relationships for a collection of structurally diverse inhibitors of purine nucleoside phosphorylase

Values of inhibition constants, Ki, and concentrations required for 50% inhibition, IC50, for a collection of structurally diverse competitive inhibitors of calf spleen purine nucleoside phosphorylase have been determined employing inosine as substrate. These values have been employed to create predictive quantitative structure-activity relationships (QSAR) which link structure to values of Ki and IC50. These QSAR models have substantial power to predict values and the associated uncertainties for Ki and IC50 for unknown, structurally diverse inhibitors of purine nucleoside phosphorylase.     

Facile synthesis of 9-substituted 9-deazapurines as potential purine nucleoside phosphorylase inhibitors

A facile synthesis of 9-substituted 9-deazapurines as potential inhibitors of purine nucleoside phosphorylase has been achieved by the direct Friedel-Crafts aroylation or arylmethylation of 9-deazapurines using trifluoromethanesulfonic acid as catalyst. The aroylated 9-deazapurines could be transformed into the corresponding 9-aryimethyl derivatives by the Wolff-Kishner reaction. A novel synthesis of 9-deazahypoxanthine was also developed by treatment of 4-hydroxy-5-phenylazo-6-methylpyrimidin-2-thione with triethyl orthoformate in trifluoroacetic acid (TFA) to yield 8-oxo-7H-2-phenylpyrimido[5,4-c]pyridazin-6-thione followed by Raney nickel reduction.     

Syntheses of mycobactin analogs as potent and selective inhibitors of Mycobacterium tuberculosis

Three analogs of mycobactin T, the siderophore secreted by Mycobacterium tuberculosis (Mtb) were synthesized and screened for their antibiotic activity against Mtb H(37)Rv and a broad panel of Gram-positive and Gram-negative bacteria. The synthetic mycobactins were potent (MIC(90) 0.02-0.88 μM in 7H12 media) and selective Mtb inhibitors, with no inhibitory activity observed against any other of the microorganisms tested. The maleimide-containing analog 40 represents a versatile platform for the development of mycobactin-drug conjugates, as well as other applications.     

Cyclic amidine sugars as transition-state analogue inhibitors of glycosidases: potent competitive inhibitors of mannosidases

A series of monocyclic glycoamidines bearing different exocyclic amine, alcohol, or alkyl functionalities and bicyclic amidines derived from D-glucose and D-mannose were synthesized and tested as inhibitors of various glycosidases. All the prepared compounds demonstrated good to excellent inhibition toward glycosidases. In particular, the biscationic D-mannoamidine 9b bearing an exocyclic ethylamine moiety proved to be a selective competitive inhibitor of alpha- and beta-mannosidases (K(i) = 6 nM) making it the most potent inhibitor of these glycosidases reported to date. A favorable B(2,5) boat conformation might explain the selectivity of mannosidase inhibition compared to other glycosidases.     

Computer simulations of trypanosomal nucleoside hydrolase: determination of the protonation state of the bound transition-state analogue

Inosine-uridine nucleoside hydrolase (IU-NH) catalyzes the hydrolysis of nucleosides into base and ribose moieties via a ribooxocarbenium ion transition state, which has been characterized using kinetic isotope effects. Protozoan parasites lack de novo purine and pyrimidine biosynthesis and depend on the purine salvage from the host. Vern Schramm and co-workers characterized p-aminophenyliminoribitol (pAPIR) to be a potent inhibitor of IU-NH from Crithidia fasciculata with K(d) of 30 nM. The cyclic amine function of the iminoribitol ring can be either protonated (pAPIRH(+)) or unprotonated (pAPIR). pAPIRH(+) resembles the charge and geometry of the ribooxocarbenium ion transition state and can be looked upon as a transition-state analogue inhibitor; however, it is known that the pAPIR species is initially bound to the enzyme. We have characterized the pAPIRH(+) species as resident of the active site using ab initio calculations and molecular dynamics simulations. This is a novel use of molecular dynamics to investigate the protonation state of the bound ligand to the active site. Nanosecond molecular dynamics simulations reveal a short hydrogen-bonding network between pAPIRH(+)-O2'-Asp14-His241 triad, which is not seen in the crystal structure. Other features discussed are: hydrogen bonding between pAPIRH(+) and Asn168, unusual geometry of the iminoribitol ring, and hydrophobic interactions.     

Synthesis,characterization and biological evaluation of purine nucleoside analogues

We present a convenient route for the synthesis of C6-amino-C5′-N-cyclopropyl carboxamido-C2-alkynylated purine nucleoside analogues 11a–g via Sonogashira coupling reaction. The nine step synthesis is easy to perform, employing commercially available reagents. Compound 9 is used as key intermediate for the synthesis of analogues 11a–g. Synthetic intermediates and final products are appropriately characterized by IR, ¹H NMR, ¹³C NMR and Mass. The modified nucleoside analogues 11a–g is evaluated for in vitro anticancer activity against MDA-MB-231 and Caco-2 cell lines. Screening data reveals that compounds 11b and 11e displayed potent IC₅₀ value of 7.9, 6.8 µg/mL respectively against MDA-MB-231 and of 7.5, 8.3 µg/mL respectively against Caco-2 than the standard drug doxorubicin, thus establishing the potential anti-cancer properties of these newer derivatives.     

Synthesis of a novel diarylphosphinic acid: a distorted ground state mimic and transition state analogue for amide hydrolysis

We describe herein the synthesis of a new unsymmetrical diarylphosphinic acid, a hapten aimed to produce catalytic antibodies for the hydrolysis of heterocyclic amides. The phosphinate functionality was selected as a mimic both of the tetrahedral intermediate and the transition state of higher energy along the reaction profile. The phenyl and 2,4,6-(trimethyl)-phenyl groups flanking the phosphinate were chosen in order to impose rotation around the P–C bond, a choice supported by ab initio calculations. This new hapten should elicit catalytic antibodies whose binding site could affect the distortion at nitrogen as well as the twist along the N–C(O) bond for heterocyclic amides. This hapten along with a series of new sterically hindered unsymmetrical phosphinic acid derivatives was prepared by a key palladium-catalysed step.     

Enantioselective total synthesis of the potent anti-HIV nucleoside EFdA

A concise enantioselective total synthesis of 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), an extremely potent anti-HIV agent, has been accomplished from (R)-glyceraldehyde acetonide in 18% overall yield by a 12-step sequence involving a highly diastereoselective ethynylation of an α-alkoxy ketone intermediate.     

Exploring nucleoside hydrolase catalysis in silico: molecular dynamics study of enzyme-bound substrate and transition state

The mechanism of action of inosine-uridine nucleoside hydrolase has been investigated by long-term molecular dynamics (MD) simulation in TIP3P water using stochastic boundary conditions. Five MD studies have been performed with enzyme substrate complex (E.S), enzyme substrate complex with protonated His241 (EH.S), enzyme transition state complex (E.TS), enzyme transition state complex with protonated His241 (EH.TS), and His241Ala transition state complex E(H241A).TS. Special attention has been given to the role of His241, which has been considered as the general acid catalyst to assist departure of the leaving nucleobase on the basis of its location in the active site in the X-ray crystal structure (). Yet on the basis of the location in the active site, Tyr229 is closer to the aniline ring of pAPIR as compared to His241. On initiation of MD simulations, His241 does not approach the nucleobase in the structures of EH.S, E.S, EH.TS, and E.TS. In the solvated enzyme, Tyr229, which is a member of the hydrogen bonding network inosine O2'.Asp14.His241.Tyr229.inosine N7, serves as a proton source to the leaving nucleobase. The loss of significant activity of His241Ala mutant is shown to be related to the disruption of the above hydrogen bonded network and the distancing of Tyr229 from inosine N7. The structures of the enzyme complexes with substrate or TS are not visibly altered on protonation of His241, a most unusual outcome. The bell-shaped pH dependence upon pK(app)'s of 7.1 and 9.1 may be attributed to the necessity of the dissociation of Asp10 or Asp15 and the acid form of Tyr229, respectively. In TS, the residue Ile81 migrated closer, whereas Arg233 moved away from the nucleobase. The probability of ribooxocarbenium ion stabilization by Asn168 and Asp14 is discussed. The Asp14-CO(2)(-) is hydrogen bonded to the ribose 2'-OH for 96% of the MD simulation time. Nucleophilic addition of water138 to ribooxocarbenium ion is suggested to be assisted by the proton shuttle from water138 --> Asp10 --> Asp15 --> water pool. An anticorrelation motion between Tyr229-OH and Asn168-OD1 in EH.S and E.S is observed. The relationship of this anticorrelated motion to mechanism, if any, deserves further exploration, perhaps the formation of a near attack conformation.     

Synthesis and structure-activity relationships of human renin inhibitors designed from angiotensinogen transition state

The synthesis and the structure-activity relationships of renin inhibitors designed from the angiotensinogen transition state are described. These inhibitors contained residues modified at P1-P1', P2, and P4-P3. Decrease in the size of side chain alkyl group in norstatine analog at P1 diminished the inhibitory activities of the compounds. Compound 5j, which contained valine residue instead of histidine residue at P2, inhibited potently cathepsin D (IC50 = 6.0 x 10(-9) M) and pepsin (IC50 = 3.5 x 10(-7) M) to the same extent as renin (IC50 = 8.5 x 10(-10) M), and thus was not specific for renin. The reduction of the beta-carbonyl group to methylene group in beta-carbonylpropionyl residue at P4-P3 decreased the potency about 2 orders against human renin (5i: IC50 = 1.1 x 10(-7) M vs. 1: IC50 = 2.4 x 10(-9) M). These results confirmed the rationality of our analysis of the interaction between an orally potent human renin inhibitor 1 and the active site of human renin using modeling techniques, showing that 1 fits the active site of renin favorably. The experimental details of the synthesis are presented.