(S)-4-Trimethylsilyl-3-butyn-2-ol as an auxiliary for stereocontrolled synthesis of salinosporamide analogs with modifications at positions C2 and C5

Analogs of salinosporamide A with variations of the C2 and C5 substituents are prepared in 8–10 steps using as the first and key transformation a diastereoselective Mukaiyama aldol reaction between the chiral 5-tert-butyldimethylsiloxy-3-methyl-1H-pyrrole-2-carboxylic ester depicted and various aldehyde substrates, promoted by tert-butyldimethylsilyl triflate. In this transformation, the 4-trimethylsilyl-3-butyn-2-ol ester functions to direct the formation of predominantly one of four possible diastereomeric aldol products. Introduction of the C2 appendage by a later-stage, stereocontrolled alkylation reaction permits the construction of analogs variant at this position. Results from in vitro and cell-based assays of proteasomal inhibition are reported. Mass spectrometric studies provide mechanistic details of proteasomal modification by salinosporamide A and analogs.     

Human glutathione transferase A1-1 demonstrates both half-of-the-sites and all-of-the-sites reactivity

A study of the kinetics of a heterodimeric variant of glutathione transferase (GST) A1-1 has led to the conclusion that, although the wild-type enzyme displays all-of-the-sites reactivity in nucleophilic aromatic substitution reactions, it demonstrates half-of-the-sites reactivity in addition reactions. The heterodimer, designed to be essentially catalytically inactive in one subunit due to a single point mutation (D101K), and the two parental homodimers were analyzed with seven different substrates, exemplifying three types of reactions catalyzed by glutathione transferases (nucleophilic aromatic substitution, addition, and double-bond isomerization reactions). Stopped-flow kinetic results suggested that the wild-type GST A1-1 behaved with half-of-the-sites reactivity in a nucleophilic aromatic substitution reaction, but steady-state kinetic analyses of the GST A1-D101K heterodimer revealed that this was presumably due to changes to the extinction coefficient of the enzyme-bound product. In contrast, steady-state kinetic analysis of the heterodimer with three different substrates of addition reactions provided evidence that the wild-type enzyme displayed half-of-the-sites reactivity in association with these reactions. The half-of-the-sites reactivity was shown not to be dependent on substrate size, the level of saturation of the enzyme with glutathione, or relative catalytic rate.     

Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress.

Sequence analysis of the vaccinia virus strain Western Reserve genome revealed the presence of an open reading frame (ORF), SalL4R, which has the potential to encode a transmembrane glycoprotein with homology to C-type animal lectins (G. L. Smith, Y. S. Chan, and S. T. Howard, J. Gen. Virol. 72:1349-1376, 1991). Here we show that the SalL4R gene is transcribed late during infection from a TAAATG motif at the beginning of the ORF. Antisera raised against a TrpE-SalL4R fusion protein identified three glycoprotein species of Mr 22,000 to 24,000 in infected cells. Immunogold electron microscopy demonstrated that SalL4R protein is present in purified extracellular enveloped virus particles but not in intracellular naked virus (INV). A mutant virus was constructed by placing a copy of the SalL4R ORF downstream of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible vaccinia virus promoter within the thymidine kinase locus and subsequently deleting the endogenous SalL4R gene. The growth kinetics of this virus demonstrated that SalL4R was nonessential for the production of infectious INV but was required for virus dissemination. Consistent with this finding, the formation of wild-type-size plaques by this mutant was dependent on the presence of IPTG. Electron microscopy showed that without SalL4R expression, the inability of the virus to spread is due to a lack of envelopment of INV virions by Golgi-derived membrane, a morphogenic event required for virus egress.     

Quantitative correlations relating the interaction of Zn(II)-tetraphenylporphine and horseradish peroxidase with amines

Reactions of nucleophilic substitution and enzymatic processes involving metalloporphirins (MP) are considered in terms of coordination of zinc(II)tetraphenylporphine (Zn-TPhP) with corresponding ligands/nucleophiles/substrates/bases. Linear correlations are performed between kinetic parameters of the Zn-TPhP coordination processes in chloroform (stability constants) and reactions of nucleophilic substitution both in aqueous and organic solvents involving pyridines, pyridine N-oxides, anilines, primary amines, as well as in reactions of oxidation of anilines with horseradish peroxidase in aqueous media (rate constants). Thermodynamic parameters of the complex formation and nucleophilic substitution linearly correlate with each other in the case of pyridines, anilines, and primary amines.     

Diverse substrate recognition and hydrolysis mechanisms of human NUDT5

Human NUDT5 (hNUDT5) hydrolyzes various modified nucleoside diphosphates including 8-oxo-dGDP, 8-oxo-dADP and ADP-ribose (ADPR). However, the structural basis of the broad substrate specificity remains unknown. Here, we report the crystal structures of hNUDT5 complexed with 8-oxo-dGDP and 8-oxo-dADP. These structures reveal an unusually different substrate-binding mode. In particular, the positions of two phosphates (α and β phosphates) of substrate in the 8-oxo-dGDP and 8-oxo-dADP complexes are completely inverted compared with those in the previously reported hNUDT5–ADPR complex structure. This result suggests that the nucleophilic substitution sites of the substrates involved in hydrolysis reactions differ despite the similarities in the chemical structures of the substrates and products. To clarify this hypothesis, we employed the isotope-labeling method and revealed that 8-oxo-dGDP is attacked by nucleophilic water at Pβ, whereas ADPR is attacked at Pα. This observation reveals that the broad substrate specificity of hNUDT5 is achieved by a diversity of not only substrate recognition, but also hydrolysis mechanisms and leads to a novel aspect that enzymes do not always catalyze the reaction of substrates with similar chemical structures by using the chemically equivalent reaction site.     

NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction.

The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate. The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product. This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group. No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate. No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP. Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates. The MutT protein is the first hydrolase shown to do so.     

Mechanism of the addition half of the O-acetylserine sulfhydrylase-A reaction

O-Acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants, substitution of the beta-acetoxy group of O-acetyl-l-serine (OAS) with inorganic bisulfide. The first half of the sulfhydrylase reaction, formation of the alpha-aminoacrylate intermediate, limits the overall reaction rate, while in the second half-reaction, with bisulfide as the substrate, chemistry is thought to be diffusion-limited. In order to characterize the second half-reaction, the pH dependence of the pseudo-first-order rate constant for disappearance of the alpha-aminoacrylate intermediate was measured over the pH range 6.0-9.5 using the natural substrate bisulfide, and a number of nucleophilic analogues. The rate is pH-dependent for substrates with a pK(a) > 7, while the rate constant is pH-independent for substrates with a pK(a) < 7 suggesting that the pK(a)s of the substrate and an enzyme group are important in this half of the reaction. In D(2)O, at low pD values, the amino acid external Schiff base is trapped, while in H(2)O the reaction proceeds through release of the amino acid product, which is likely rate-limiting for all nucleophilic reactants. A number of new beta-substituted amino acids were produced and characterized by (1)H NMR spectroscopy.     

Synthesis of cationic single-isomer cyclodextrins for the chiral separation of amino acids and anionic pharmaceuticals

We describe a protocol for the synthesis of mono-6(A)-(1-butyl-3-imidazolium)-6(A)-deoxy-beta-cyclodextrin chloride (BIMCD), a cationic, water-soluble cyclodextrin used in the chiral separation of amino acids and anionic pharmaceuticals by capillary electrophoresis. Starting from commercially available chemicals, BIMCD is synthesized in five steps. The first step involves a nucleophilic substitution between p-toluenesulfonyl chloride and imidazole to afford 1-(p-toluenesulfonyl)imidazole (A). In the second step, a nucleophilic substitution between beta-cyclodextrin and A affords mono-6(A)-(p-toluenesulfonyl)-6(A)-deoxy-beta-cyclodextrin (B). In the third step, a nucleophilic substitution between 1-bromobutane and imidazole affords 1-butylimidazole (C). In the fourth step, a nucleophilic addition between A and C affords BIMCD tosylate. In the final step, anion exchange using an ion-exchange resin yields BIMCD as a highly water-soluble solid. Each step takes up to 2 d, including the time required for product purification. The overall protocol requires approximately 6 d.     

Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies.

Pyrimidine nucleotides were tested as substrates for pure yeast orotidylate decarboxylase in an attempt to gain insight into the nature of the catalytic mechanism of the enzyme. Substitutions of the 5-position in the pyrimidine ring of the orotidylate substrate resulted in compounds that are either excellent inhibitors or substrates of the enzyme. The 5-bromo- and 5-chloroorotidylates are potent inhibitors while the 5-fluoro derivative is a good substrate with a turnover number 30 times that observed with orotidylate. When carbon 5 of the pyrimidine ring is replaced by nitrogen in 5-azaorotidylate, the resulting compound is unstable in solution with a half-life of 25 min at pH 6. However, studies with freshly generated 5-azaorotidylate show that an enzyme-dependent reaction occurs, presumably decarboxylation. This enzyme reaction follows simple Michaelis-Menten kinetics. Because the 5-aza group is not electrophilic, an enzyme mechanism utilizing a nucleophilic addition of the enzyme at the 5-position is ruled out. We also present studies that are not compatible with a mechanism requiring the formation of a Schiff's base prior to decarboxylation. The enzyme is tolerant of modest substitution at the 4-position, for the 4-keto group can be replaced with a thioketone. However, no catalysis is observed when the same substitution is made at the 2-position. Similarities in the substrate specificity of orotate phosphoribosyltransferase and orotidylate decarboxylase led us to compare the amino acid sequences of the two enzymes; significant (20%) sequence homology was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a bacterial dehalogenase with activity toward halogenated alkanes, alcohols and ethers

An enzyme that is capable of hydrolytic conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols was purified from a 1,6-dichlorohexane-degrading bacterium. The dehalogenase was found to be a monomeric protein of relative molecular mass 28,000. The affinity for its substrates was relatively low with Km values for short-chain haloalkanes in the range 0.1-0.9 mM. The aliphatic dehalogenase showed a much broader substrate range than has been reported for halidohydrolases so far. Novel classes of substrates include dihalomethanes, C5-C9 1-halo-n-alkanes, secondary alkylhalides, halogenated alcohols and chlorinated ethers. Several of these compounds are important environmental pollutants, e.g. methylbromide, dibromomethane, 1,2-dibromoethane, 1,3-dichloropropene, and bis(2-chloroethyl)ether. The degradation of chiral 2-bromoalkanes appeared to proceed without stereochemical preference. Optically active 2-bromobutane was converted with inversion of configuration at the chiral carbon atom, suggesting that the dehalogenase reaction proceeds by a nucleophilic substitution involving a carboxyl group or base catalysis.     

Synthesis of structurally identical fluorine-18 and iodine isotope labeling compounds for comparative imaging

The synthesis of a benzophenone-based labeling compound designed for comparative imaging studies with both in vivo positron emission tomograph (PET) and single-photon computed tomography (SPECT) and ex vivo autoradiography is described. The new compound can be labeled with either F-18 or iodine radioisotopes to give two different radioisotopmers: N-[2-fluoro-5-(3-[I-131]iodobenzoyl)benzyl]-2-bromoacetamide (1) and N-[2-[F-18]fluoro-5-(3-iodobenzoyl)benzyl]-2-bromoacetamide (2). Compound 1 and 2 have a 2-bromoacetyl group, which can be used to conjugate with biomolecules through a nucleophilic substitution reaction. Compound 1 was synthesized from the corresponding tributyltin derivatives via an oxidative destannylation reaction, and compound 2 was prepared via a four-step radiosynthesis (nucleophilic aromatic substitution, reduction, oxidation, and alkylation) starting from 4-(N,N,N-trimethylammonio)-3-cyano-3'-iodobenzophenone triflate. A remarkably high radiochemical yield (>90%) was achieved for the F-18 nucleophilic aromatic substitution under mild conditions (room temperature in less than 10 min), indicating the structural advantage of the designed molecule to facilitate the F-18 for trimethylammonium substitution in the presence of two electron-withdrawing groups (nitrile and carbonyl). The overall radiosynthesis time for compound 2 is less than 3 h after end of bombardment (EOB) with an unoptimized radiochemical yield of about 2% (not decay corrected) and specific activity of 0.8 Ci/micromol at EOB. The radiolabeling precursors for compound 1 and 2 were synthesized via a carbon-carbon bond-forming reaction between 2-substituted-5-lithiobenzonitrile and 3-substituted benzaldehyde derivatives. Compounds 1 and 2 should allow us to label biomolecules with F-18 or iodine isotopes and gives structurally identical products, which are expected to have identical biological properties and should be useful for comparative imaging studies.     

The mechanism investigation in substitution of 21-bromo-3alpha-hydroxyl-3beta-methoxymethyl-5alpha-pregnan-20-one with nucleophiles

A mechanistic study on the nucleophilic substitution of a strictly geometric 21-bromo-3alpha-hydroxyl-3beta-methoxymethyl-5alpha-pregnan-20-one was described. Reaction of the alpha-bromoketone with excess lithium imidazole followed by the addition of extra bases including n-butyllithium, methyllithium, lithium piperidine, and lithium pyrrolidine provided unexpected alpha-nucleophilic carbonyl adducts that derived from strong base. Data from HPLC and proton NMR suggested an epoxide as the intermediate. Two possible reaction pathways were proposed for the nucleophilic substitution reaction. One pathway is the normal SN2 substitution reaction, directly provided the imidazoly product without the formation of the unexpected alpha-substituted products. The other pathway went through an epoxide intermediate, in which imidazole anion or the strong bases added would attack from the less hindered site of the epoxide to give the substitution product.     

Nucleophilic substitution at the anomeric position of 1,2-O-isopropylidenefuranose derivatives. A novel stereoselective synthesis of cyclic phosphates analogous to cAMP

1,2-O-Isopropylidenefuranose derivatives were treated with various nucleophiles in the presence of either BF(3).OEt(2) or trimethylsilyl trifluoromethanesulfonate (TMSOTf) leading to substitution products in a regio- and stereoselective manner. In particular, nucleophilic substitution of 1,2-O-isopropylidenefuranose derivatives when treated with allyltrimethylsilane was controlled by steric and electronic factors (similar to Woerpel's stereoelectronic model). On the other hand, when 1,2-O-isopropylidenefuranose derivatives were treated with trimethylsilane, in the presence of bis-O-trimethylsilyl-5-iodouracil or bis-O-trimethylsilyl-thymidine, substitution products were generated in high regio- and stereoselectivities via an unusual nucleophilic substitution with opening of the furanose ring. Based on these results, a stereoselective method for the synthesis of neutral cyclic phosphates analogous to cAMP was developed.     

Mechanism of nucleophilic substitution of thiamine and its analogs: Methanol and water solvents

4-Amino-1,2-dimethyl-5-(substituted methyl)pyrimidinium ions undergo lyate ion-catalyzed nucleophilic substitution in methanol and in water. The substituents at position 5 of these thiamine analogs include phenols and pyridinium ions. The observation of separate rate- and product-determining steps indicates a multistep mechanism of substitution. Linear free energy relationships suggest that addition of lyate ion to the pyrimidinium ring is rate-limiting when phenoxide ions act as leaving groups and that a change occurs when pyridines depart. Elimination of the pyridine becomes the rate-limiting step following rapid addition of the lyate ion. Reactivities of phenol substrates in methanol and in water are similar but, when pyridine departs, changing from water to methanol gives rise to a large rate acceleration. An increase in ground state electrostatic destabilization of the dicationic substrate in the less polar alcohol provides the rate enhancement.     

Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols

The relationship between the structure and activity of meta- and para-hydroxylated monophenols was studied during their tyrosinase-catalysed hydroxylation and the rate-limiting steps of the reaction mechanism were identified. The para-hydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a -OCH3 group on C-1, as p-hydroxyanisol (4HA) and m-hydroxyanisol (3HA), or with a -CH2OH group, as p-hydroxybenzylalcohol (4HBA) and m-hydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electron-donating effect of the -OCH3 group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (para-hydroxylated substrates) or for electrophilic attack (meta-hydroxylated substrates). The electron-attracting effect of the -CH2OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steady-state rate (V(M)Max) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K(M)m), with the meta-hydroxylated substrates having very large values. The catalytic efficiency k(M)cat/K(M)m is much greater for thepara-hydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the meta-hydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K(M)m of the meta-hydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the para-hydroxylated substrates, the binding constant decreases, leading to an increase in K(M)m. The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V(M)max. The small difference between the absolute values of V(M)max suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.     

Synthesis and antihyperglycemic activity of suitably functionalized 3H-quinazolin-4-ones

A series of 2-sec-amino-3H-quinazolin-4-ones (4a-p) and 4-sec-amino-2-chloroquinazolines (5a-b) have been synthesized by nucleophilic substitution reaction of 2-chloro-4(3H)-quinazolones (3) and 2,4-dichloroquinazolines (2) with amines, respectively. Most of the synthesized compounds were evaluated for antihyperglycemic activity but only 4a,b,d,j,o displayed significant reduction in blood glucose level in streptozotocin and sucrose loaded rat models.     

Studies on the activity and activation of rat liver microsomal glutathione transferase, in particular with a substrate analogue series

A number of potential substrates for the microsomal glutathione transferase have been investigated. Out of 11 epoxides tested, only two, i.e. androstenoxide and benzo(a)pyrene-4,5-oxide, were found to be substrates. Upon treatment of the enzyme with N-ethylmaleimide, its activity toward only certain substrates is increased. It appeared upon inspection of the bimolecular rate constants from the corresponding nonenzymatic reactions that the substrates for which the activity is increased are the more reactive ones. This hypothesis was investigated further using a series of para-substituted 1-chloro-2-nitrobenzene derivatives as substrates. Activation was seen only with the more reactive nitro-, aldehyde-, and acetaldehyde-substituted compounds and not with the amide and chloroanalogues, thus demonstrating the predicted effect with a related series of compounds. Interestingly, kcat values are increased 7-20-fold by N-ethylmaleimide treatment, whereas the corresponding kcat/Km value is increased only for the p-nitro derivative. Effective molarity and rate enhancement values were found to increase with decreasing reactivity of the substrate, attaining maximal values of 10(5) M and 10(8), respectively. It is concluded that the glutathione transferases are quite effective catalysts with their less reactive substrates. Hammett rho values for the kcat values of unactivated and activated enzyme were 0.49 and 2.0, respectively. The latter value is close to those found for cytosolic glutathione transferases, indicating that activation changes the catalytic mechanism so that it more closely resembles that of the soluble enzymes. The rho values for kcat/Km values were 3 and 3.5 for the unactivated and activated enzyme, respectively, values close to those observed for the nonenzymatic bimolecular rate constants and thereby demonstrating that these reactions have similar properties. The high coefficients of correlation between resonance sigma- values and all of these parameters demonstrate a strong dependence on substrate electrophilicity, as expected for nucleophilic aromatic substitution.     

Unmasking Anticooperative DNA-binding interactions of vaccinia DNA topoisomerase I

Vaccinia DNA topoisomerase (vTopo) catalyzes highly specific nucleophilic substitution at a single phosphodiester linkage in the pentapyrimidine recognition sequence 5'-(C/T)+5C4+C3+T+2T+1p \N-1 using an active-site tyrosine nucleophile, thereby expelling a 5' hydroxyl leaving group of the DNA. Here, we report the energetic effects of subtle modifications to the major-groove hydrogen-bond donor and acceptor groups of the 3'-GGGAA-5' consensus sequence of the nonscissile strand in the context of duplexes in which the scissile strand length was progressively shortened. We find that the major-groove substitutions become energetically more damaging as the scissile strand is shortened from 32 to 24 and 18 nucleotides, indicating that enzyme interactions with the duplex region present in the 32-mer but not the 24- or 18-mer weaken specific interactions with the DNA major groove. Regardless of strand length, the destabilizing effects of the major-groove substitutions increase as the reaction proceeds from the Michaelis complex to the transition state for DNA cleavage and, finally, to the phosphotyrosine-DNA covalent complex. These length-dependent anticooperative interactions involving the DNA major groove and duplex regions 3' to the cleavage site indicate that the major-groove binding energy is fully realized late during the reaction for full-length substrates but that smaller more flexible duplex substrates feel these interactions earlier along the reaction coordinate. Such anticooperative binding interactions may play a role in strand exchange and supercoil unwinding activities of the enzyme.     

A new synthetic route to 2- and 4-methoxyestradiols by nucleophilic substitution

A new snythetic route to 2- and 4-methoxyestradiols is described. Benzo-15-crown-5 with CuI catalyzes the specific nucleophilic substitution at the carbon atom carrying a non-activated halogen on ring A of the estradiol.     

Short and efficient syntheses of analogues of WAY-100635: new and potent 5-HT1A receptor antagonists

Simple syntheses of four new and potent analogues of the 5-HT1A receptor ligand, WAY-100635 are described, namely the 6-(pyridinyl)-bromo-, the 6-(pyridinyl)-fluoro-, the pyrimidine- and the 5-(pyridinyl)-bromo-analogues. The first three analogues were obtained by aromatic nucleophilic substitution of the 2,6-dihalogenopyridine (activated or not as an N-oxide) or of the 2-chloropyrimidine with the corresponding amine nucleophile as a key step. The fourth analogue, the 5-(pyridinyl)-bromo-analogue, was synthesized from the 2-amino-5-bromopyridine via a progressive elongation of the skeleton. The four compounds described are all full antagonists and show good in vitro binding affinities (Ki).