Preparation of 1-(3-C-(propa-1,2-dienyl)-D-ribo-pentofuranosyl)uracil, an allenic nucleoside.

The Crabbé reaction was extended to the preparation of C-3'-allenyl-uridine. The effects of solvent and protecting group on the reaction were studied. The conversion in refluxing dioxan of disilyl either 3 proceeds to the corresponding allenic nucleoside 7; whereas, in refluxing THF the Mannich base 5 was obtained. Fully deprotected Mannich base and allenic uridines 6 and 9 were tested for their antitumor activity.

Di-tert-butyl diethylphosphoramidite as the phosphitylating reagent in the preparation of 3-deoxy-3-C-methylene-D-ribo-hexose-6-phosphate and 3-deoxy-3-C-methylene-D-erythro-pentose-5-phosphate.

3-Deoxy-3-C-methylene-D-ribo-hexose-6-phosphate and 3-deoxy-3-C-methylene-D-erythro-pentose-5-phosphate were prepared from a common intermediate 3-deoxy-3-C-methylene-1,2-O-isopropylidene-alpha-D-ribo-hexofuranose. The preparation of the phosphorylated unsaturated sugars employed di-tert-butyl diethylphosphoramidite as the phosphitylating reagent. The removal of all the protecting groups was done under acidic conditions in the ultimate step. The unsaturated sugar phosphates were competitive inhibitors but neither substrates nor inactivators of glucose-6-phosphate and ribose-5-phosphate isomerases.

Synthesis of 8-(omega-Hydroxyalkyl)-, 8-(omega-hydroxyalk-1-enyl)-, and 8-(omega-hydroxyalk-1-ynyl)adenines using the tert-butyldimethylsilyloxymethyl group, a new and versatile protecting group of adenine.

The synthesis of 12 analogues of adenine substituted at C-8 by an omega-hydroxyalkyl, omega-hydroxyalk-1-enyl, or omega-hydroxyalk-1-ynyl chain of various length has been carried out in five or six steps starting from adenine. The analogues were obtained using a new protecting group of adenine, the tert-butyldimethylsilyloxymethyl group. 9-tert-Butyldimethylsilyloxymethyl-adenine is more soluble than adenine in organic solvents. It was prepared regiospecificaly in two steps from adenine and was amenable to C-8 iodination under basic conditions and to subsequent introduction of the various carbon chains at C-8 by palladium-catalyzed cross-coupling reactions (Stille or Sonogashira). The protecting group was removed under acidic conditions, thus demonstrating its versatility.

Evaluation of the antitumor activity of 1-(3-C-ethynyl-beta-D-ribofuranosyl) (PJ272), a recent ribonucleoside analogue.

The antiproliferative properties of a new ribonucleoside derivative, 1-(3'-C-ethynyl-beta-D-ribofuranosyl)uracil (PJ 272) that we synthesized a few years ago, were investigated in vitro on a variety of tumor cell lines from human and murine origins and in vivo, in tumor bearing mice. Using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we showed the ability of this compound to depress, at nanomolar concentrations, the growth of leukemia and lymphoma cultured cells. In 7 out of 8 tumor cell lines tested concentration of 50% inhibition (IC50) was found to be less than 25 nM. PJ 272 was also shown to present the same cytotoxicity against K562 Adriamycin-resistant cell line, which express a multi-drug resistance (MDR) phenotype, and its Adriamycin-sensitive parent cell line. Moreover, when injected intraperitoneally at 20 mg/kg every three days, PJ 272 was found to significantly increase the survival rate (T/C = 149%) of DBA/2 mice injected intraperitoneally with L1210 leukemic cells. Taken together, these results suggest that PJ 272 could be considered as a potentially very active drug against lymphoma and leukemia.

3'-Beta-ethynyl and 2'-deoxy-3'-beta-ethynyl adenosines: first 3'-beta-branched-adenosines substrates of adenosine deaminase.

The 3'-C-branched-adenosine and 2'-deoxyadenosine analogues 1-7 were tested as substrate of adenosine deaminase. The 9-(3'-C-ethynyl-beta-D-ribo-pentofuranosyl)adenine 1 and its 2'-deoxy analogue 7 were deaminated by the enzyme while the vinyl and ethyl derivatives 2 and 3 were not. The 9-(3'-C-branched-beta-D-xylo-pentofuranosyl)adenines 4-6 were deaminated by the deaminase.

6,7,8,9-Tetrahydro-3-methyl-1H-pyrano-[4,3-b]quinolin-1-one.

The condensation reaction of 4-amino-6-methyl-2-pyrone with 1-cyclohexenecarboxaldehyde and a catalytic amount of (S)-(+)-10-camphorsulfonic acid in toluene at 358 K gave a 1:2.5 ratio of the title compound, (1) (C13H13NO2), and 7,8,9,10-tetrahydro-1H-pyrano[4,3-c]isoquinoline-1-one, (2). The formation of (2) presumably proceeds through an intermediate imine. Both (1) and (2) show inhibitory activities against acetylcholinesterase and human aldose reductase. Of the three linear-fused rings of (1), both ring A and ring B are planar and the angle between these planes is 0.46 (13) degrees. While the two C atoms of cyclohexane ring C attached to its common atoms with ring B are in the plane of the latter, as expected, the remaining two C atoms of ring C are out of this plane, by 0.342 (4) and -0.402 (3) A, respectively.

Binding of aldose reductase inhibitors: correlation of crystallographic and mass spectrometric studies.

Aldose reductase is a NADP(H)-dependent enzyme, believed to be strongly implicated in the development of degenerative complications of Diabetes Mellitus. The search for specific inhibitors of this enzyme has thus become a major pharmaceutic challenge. In this study, we applied both X-ray crystallography and mass spectrometry to characterize the interactions between aldose reductase and four representative inhibitors: AminoSNM, Imirestat, LCB3071, and IDD384. If crystallography remains obviously the only way to get an extensive description of the contacts between an inhibitor and the enzymatic site, the duration of the crystallographic analysis makes this technique incompatible with high throughput screenings of inhibitors. On the other hand, dissociation experiments monitored by mass spectrometry permitted us to evaluate rapidly the relative gas-phase stabilities of the aldose reductase-inhibitor noncovalent complexes. In our experiments, dissociation in the gas-phase was provoked by increasing the accelerating voltage of the ions (Vc) in the source-analyzer interface region: the Vc value needed to dissociate 50% of the noncovalent complex initially present (Vc50) was taken as a gas-phase stability parameter of the enzyme-inhibitor complex. Interestingly, the Vc50 were found to correlate with the energy of the electrostatic and H-bond interactions involved in the contact aldose reductase/inhibitor (Eel-H), computed from the crystallographic model. This finding may be specially interesting in a context of drug development. Actually, during a drug design optimization phase, the binding of the drug to the target enzyme is often optimized by modifying its interatomic electrostatic and H-bond contacts; because they usually depend on a single atom change on the drug, and are easier to introduce than the hydrophobic interactions. Therefore, the Vc50 may help to monitor the chemical modifications introduced in new inhibitors. X-ray crystallography is clearly needed to get the details of the contacts and to rationalize the design. Nevertheless, once the cycle of chemical modification is engaged, mass spectrometry can be used to select a priori the drug candidates which are worthy of further crystallographic investigation. We thus propose to use the two techniques in a complementary way, to improve the screening of large collections of inhibitors.

A 'specificity' pocket inferred from the crystal structures of the complexes of aldose reductase with the pharmaceutically important inhibitors tolrestat and sorbinil.

BACKGROUND: Aldose reductase (AR) is an NADPH-dependent enzyme implicated in long-term diabetic complications. Buried at the bottom of a deep hydrophobic cleft, the NADPH coenzyme is surrounded by the conserved hydrophilic residues of the AR active site. The existence of an anionic binding site near the NADP+ has been determined from the structures of the complexes of AR with citrate, cacodylate and glucose-6-phosphate. The inhibitor zopolrestat binds to this anionic site, and in the hydrophobic cleft, after a change of conformation which opens a 'specificity' pocket. RESULTS: The crystal structures of the porcine AR holoenzyme and its complexes with the inhibitors tolrestat and sorbinil have been solved; these structures are important as tolrestat and sorbinil are, pharmaceutically, the most well-studied AR inhibitors. The active site of the holoenzyme was analyzed, and binding of the inhibitors was found to involve two contact zones in the active site: first, a recognition region for hydrogen-bond acceptors near the coenzyme, with three centers, including the anionic site; and second, a hydrophobic contact zone in the active-site cleft, which in the case of tolrestat includes the specificity pocket. The conformational change leading to the opening of the specificity pocket upon tolrestat binding is different to the one seen upon zopolrestat binding; this pocket binds inhibitors that are more effective against AR than against aldehyde reductase. CONCLUSIONS: The active site of AR adapts itself to bind tightly to different inhibitors; this happens both upon binding to the inhibitor's hydrophilic heads, and at the hydrophobic and specificity pockets of AR, which can change their shape through different conformational changes of the same residues. This flexibility could explain the large variety of possible substrates of AR.

Study of non-covalent enzyme-inhibitor complexes of aldose reductase by electrospray mass spectrometry.

Specific non-covalent interactions between aldose reductase (AR), its NADP+ cofactor and five inhibitors have been characterized by electrospray mass spectrometry (ES-MS). These results indicated that the protein could be desorbed and maintained in the gas phase in a form very close to its native conformation. Collisionally induced dissociation (CID)-MS and CID-MS-MS showed that the adenosine diphosphate part of the cofactor interacts strongly with AR. The relative stability of the ternary AR x NADP+ x inhibitor complexes was established and successfully correlated with the IC50 values. All inhibitors were shown to only bind to AR holoenzyme. These results are important for the field of drug development insofar as ES-MS might provide a rapid and very sensitive method for the screening of potential drugs or for the identification of compounds displaying high binding affinity to a target biomolecule.

[Change of cholesterol oxidase of Brevibacterium sterolicum and substrate specificity]. / Modification de la cholestérol oxydase de Brevibacterium sterolicum et spécificité de substrat.

Cholesterol oxidase modified by hydrogen peroxide is inactive with cholesterol solubilized in buffer with surfactants. Pregn-5-en-3 beta-ol when solubilized in the same conditions and substrates soluble in buffer, like 3 beta-hydroxy-androst-5-en-17-one or 3 beta-hydroxy-androst-5-en-17 beta-carboxylic acid are substrates of the modified enzyme. The observed loss of activity on cholesterol could be due to the inability of the oxidized cholesterol oxidase to extract cholesterol from mixed cholesterol/surfactant aggregates. Cholesterol oxidase on storage undergoes modifications close to those with hydrogen peroxide and care should be taken for the use of cholesterol oxidase as cholesterol probe.

Sequence of pig lens aldose reductase and electrospray mass spectrometry of non-covalent and covalent complexes.

The complete sequence of pig lens aldose reductase (EC 1.1.1.21), a member of the nicotinamide coenzyme-dependent aldo-keto reductase super family, was determined by the combined use of data obtained from Edman degradation, fast-atom-bombardment mass spectrometry and electrospray mass spectrometry. The N-terminal residue of human and pig aldose reductase was shown to be acetylated. The assignment of a disulfide bridge (Cys298-Cys303) was obtained by mass spectrometry. Electrospray mass spectrometry has been used for molecular mass measurement of human muscle (35758 +/- 7 Da) and pig lens (35778 +/- 3Da) aldose reductase; using mild ionization conditions, it has also been used to study the reversible interaction involved in a non-covalent complex with NADP+ (36527 +/- 4Da). An alkylating analog of NADP+ (3-chloroacetylpyridine-adenine dinucleotide phosphate) was used as an irreversible inhibitor to investigate the NADP binding site and the mass of the covalent complex was measured (36521 +/- 3 Da).

Mechanism-based inhibition of proline dehydrogenase by proline analogues.

The inactivation of proline dehydrogenase by several L-Pro analogues was investigated with the aim to block the essential metabolic pathway of tsetse flies allowing the degradation of L-Pro to L-Glu. In vitro studies on rat liver mitochondria showed that only 4-methylene-L-proline was able to inactivate proline dehydrogenase. The inactivation kinetics agreed with a mechanism-based inhibition. The other tested analogues E- and Z-4-fluoromethylene-L-proline, and cis and trans-5-ethynyl-D,L-proline were neither substrate nor inactivator of the enzyme. In vivo 4-methylene-L-proline showed no toxicity against Drosophila flies, but was lethal for Glossina pallidipes flies. This result allows the consideration of 4-methylene-L-proline as an attractive compound molecule in the struggle against tsetse flies.

Purification and electrospray mass spectrometry of aldose reductase from pig lens.

Aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) has been purified from pig lens to homogeneity by a rapid and efficient three-step procedure involving poly(ethylene glycol) fractionation, ion-exchange chromatography and chromatofocusing. The homogeneity of the purified enzyme was examined by polyacrylamide gel electrophoresis under native and denaturing conditions, by isoelectric focusing and by high-performance liquid chromatography on a size-exclusion column. The highly purified enzyme is a monomeric protein with a molecular mass of 35,775 +/- 3 Da as determined by electrospray mass spectrometry (ESMS). This purification procedure is particularly suited for the preparation of triclinic single crystals of pig lens aldose reductase, which are currently used in X-ray studies of this enzyme.

Novel NADPH-binding domain revealed by the crystal structure of aldose reductase.

Aldose reductase is the first enzyme in the polyol pathway and catalyses the NADPH-dependent reduction of D-glucose to D-sorbitol. Under normal physiological conditions aldose reductase participates in osmoregulation, but under hyperglycaemic conditions it contributes to the onset and development of severe complications in diabetes. Here we present the crystal structure of pig lens aldose reductase refined to an R-factor of 0.232 at 2.5-A resolution. It exhibits a single domain folded in an eight-stranded parallel alpha/beta barrel, similar to that in triose phosphate isomerase and a score of other enzymes. Hence, aldose reductase does not possess the expected canonical dinucleotide-binding domain. Crystallographic analysis of the binding of 2'-monophospho-adenosine-5'-diphosphoribose, which competitively inhibits NADPH binding reveals that it binds into a cleft located at the C-terminal end of the strands of the alpha/beta barrel. This represents a new type of binding for nicotinamide adenine dinucleotide coenzymes.

Modification of alpha-amylase from Bacillus licheniformis by the polyaldehyde derived from beta-cyclodextrine and alpha-amylase thermostability.

The cleavage of beta-cyclodextrine by sodium periodate at the seven 2-3 diols of the glucose unit gives rise to the polyaldehyde 1, used to modify alpha-amylase. The reductive modification of alpha-amylase from Bacillus licheniformis reduced the number of reactive lysine groups from 8 to 3.5 per mol of enzyme with an activity loss of 25% and increased the half-life at 80 degrees C from 4.7 to 7.0 minutes.

Beta-hydroxysteroid dehydrogenase: activity in microemulsion and extraction from Pseudomonas testosteroni cells with microemulsion.

The stability of purified beta-hydroxysteroid dehydrogenase activity measured as a function of time was good in buffered cationic and non-ionic microemulsions. The use of 1-pentanol and 1-hexanol in place of 1-butanol as cosurfactant gave increased activity and stability. The NAD+ Michaelis constant was 0.22 mM in buffer and 3.5 mM in waterpool concentration in microemulsion. Proteins, among them beta-hydroxysteroid dehydrogenase, were extracted from Pseudomonas testosteroni with cationic microemulsion, thus indicating that microemulsions may be utilized in protein release from cells.

X-ray analysis of D-xylose isomerase at 1.9 A: native enzyme in complex with substrate and with a mechanism-designed inactivator.

The structures of crystalline D-xylose isomerase (D-xylose ketol-isomerase; EC 5.3.1.5) from Streptomyces rubiginosus and of its complexes with substrate and with an active-site-directed inhibitor have been determined by x-ray diffraction techniques and refined to 1.9-A resolution. This study identifies the active site, as well as two metal-binding sites. The metal ions are important in maintaining the structure of the active-site region and one of them binds C3-O and C5-O of the substrate forming a six-membered ring. This study has revealed a very close contact between histidine and C1 of a substrate, suggesting that this is the active-site base that abstracts a proton from substrate. The mechanism-based inhibitor is a substrate analog and is turned over by the enzyme to give a product that alkylates this same histidine, reinforcing our interpretation. The changes in structure of the native enzyme, the enzyme with bound substrate, and the alkylated enzyme indicate that the mechanism involves an "open-chain" conformation of substrate and that the intermediate in the isomerization reaction is probably a cis-ene diol because the active-site histidine is correctly placed to abstract a proton from C1 or C2 of the substrate. A water molecule binds to C1O and C2O of the substrate and so may act as a proton donor or acceptor in the enolization of a ring-opened substrate.