Regio- and stereo-selective synthesis of carbohydrate isoxazolidines by 1,3-dipolar cycloaddition of nitrones to 5,6-dideoxy-1,2-O-isopropylidene- alpha-D-xylo-hex-5-enofuranose.

The synthesis of 2-phenyl-3-aryl and 2-phenyl-3-aroyl derivatives 5-(1,2-O-isopropylidene-alpha-D-xylo-tetrofuranos-4-yl)isoxazolidi ne (3) from nitrones and 5,6-dideoxy-1,2-O-isopropylidene-alpha-D-xylo-hex-5- enofuranose (1) is described. The 1,3-dipolar cycloaddition reactions given mainly anti adducts 3 and 4 (greater than or equal to 95% pi-facial stereoselectivity). The cycloadducts 3 with H-3,5 cis are formed either exclusively or preponderate over the trans diastereoisomers 4.     

First isolation of disubstituted cis-5,6-dihydro-1,10-phenanthrolines. Lipase-mediated resolution of cis- and trans-phenoxy alcohol isomers and assignment of absolute stereochemistry via CD and NMR spectroscopy

5,6-Dihydro-1,10-phenanthrolines can display axial and central chirality. In conjunction with the ligating properties of the diimino moiety, this class of compounds is of great interest to applications in supramolecular chemistry. We report the first preparation of cis-5,6-dihydro-1,10-phenanthroline derivatives by reacting triphenyl borate with the corresponding epoxide precursor. We found that solvent and temperature choice determined the stereoselectivity of the epoxide opening giving rise to the cis (14:1 dr) or trans (99:1 dr) product. Racemates of each stereoisomeric mixture, cis- and trans-phenoxy alcohol, were separated via highly enantioselective transesterifications with lipase PSCI from Burkholderia cepacia (97% ee, E > 200). Stereochemical assignments were carried out using CD and X-ray analyses in conjunction with NMR studies of α-methoxy-α-(trifluoromethyl)phenylacetic acid and α-methoxyphenylacetic acid esters.     

Stereoselectivity of the epoxidation of 7,8-dihydrobenzo[a]pyrene by prostaglandin H synthase and cytochrome P-450 determined by the identification of polyguanylic acid adducts

The stereoselectivity of the oxidation of 7,8-dihydrobenzo[a]pyrene (H2BP) to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (H4BP-epoxide) by prostaglandin H (PGH) synthase and cytochrome P-450 has been studied using microsomal preparations from ram seminal vesicles and rat liver. Incubations were performed in the presence of polyguanylic acid and the adducts formed between H4BP-epoxide and guanosine were isolated following the recovery and hydrolysis of the poly(G). When (+/-)-H4BP-epoxide was reacted with poly(G), four diastereomeric adducts were formed by the cis and trans addition of the exocyclic amino group of guanine to the benzylic carbon of the epoxide enantiomers. Each diastereomer was identified by a combination of ultraviolet, nuclear magnetic resonance, circular dichroism, and mass spectroscopy. Under comparable conditions, ram seminal vesicle microsomes in the presence of arachidonic acid triggered the binding of H2BP to poly(G) to a greater extent than rat liver microsomes from untreated and phenobarbital- and methylcholanthrene pretreated animals in the presence of NADPH. Quantitation of the (-)-cis- and (+)-cis-guanosine adducts revealed the degree of stereoselectivity of epoxidation. The ratio of (-)/(+) adducts was 54:46 for PGH synthase and 89:11 (control), 62:38 (phenobarbital), and 69:31 (methylcholanthrene) for cytochrome P-450-catalyzed reactions. PGH synthase catalyzed the epoxidation of H2BP with little or no stereoselectivity in contrast to cytochrome P-450. The utility of the poly(G) binding technique for the elucidation of the stereoselective generation of chiral electrophiles is discussed along with the mechanistic implications of the results.     

Paternò-Büchi reaction between aromatic carbonyl compounds and 1-(3-furyl)alkanols.

The photochemical reaction between aromatic carbonyl compounds and 3-furylmethanol derivatives occurs with high regioselectivity. In most of the experiments formation of oxetanes occurs at the hydroxyalkylated double bond. With chiral 1-(3-furyl)alkanols the reaction occurs with good-high stereoselectivity. In the case of 1-(3-furyl)-benzyl alcohol the stereoselectivity can be explained on the basis of the conformers of the reagent, assuming the formation of a complex between the carbonyl compound and the hydroxyl group.     

Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.     

Metabolism and hepatotoxicity of the naturally occurring benzo[b]pyran precocene I

The in vitro metabolism of precocene I by liver microsomes from control and treated rats and the effects of precocene I on the function and histology of the rat liver were examined. The major metabolites (80-90% of total metabolites) from all microsomal preparations were the cis and trans 3,4-diols of precocene I produced with a cis/trans isomer ratio of 1:2. These diols appear to arise mainly by spontaneous hydrolysis of precocene I 3,4-oxide. (+)-(3R,4R)-cis- and (-)-(3R,4S)-trans-precocene I 3,4-diols were the predominant enantiomers of the 3,4-diol formed. The enantiomeric excess of these diols (2-50%) is dependent on the microsomal preparation, with microsomes from control rats exhibiting the highest stereoselectivity and microsomes from phenobarbital-treated rats the least. 6-Hydroxyprecocene I was the next major metabolite and was formed to the extent of 5% (control), 10% and 17% (phenobarbital and 3-methylcholanthrene treatment, respectively) of total metabolites. Treatment of rats with a single i.p. dose of precocene I (300 mg/kg) resulted in extensive hepatic damage as evidenced by a marked increase of plasma glutamic pyruvic transaminase levels and histologic observation in liver sections of severe centrolobular necrosis. Although phenobarbital treatment of rats increased the rate of liver microsomal metabolism of precocene I by approximately 50% (nmol products/nmol cytochrome P-450/min) compared to liver microsomes from control rats, hepatic damage caused by precocene I was not significantly affected. Depletion of glutathione levels in the rats with diethyl maleate prior to precocene I treatment dramatically increased the severity of hepatic insult, whereas treatment of the rats with the mixed function oxidase inhibitor piperonyl butoxide prior to treatment with precocene I blocked hepatic damage. Treatment of rats with cysteamine prior to treatment with precocene I protected the animals against the toxic effects. Neither cis nor trans precocene I 3,4-diol nor 3,4-dihydroprecocene I elicited impaired liver function or cellular damage. The above results are consistent with the view that precocene I 3,4-oxide is the metabolite responsible for the hepatotoxic effects observed when precocene I is injected into rats.     

Stereoselective Degradation of alpha‐Cypermethrin and Its Enantiomers in Rat Liver Microsomes

Alpha‐cypermethrin (α‐CP), [(RS)‐a‐cyano‐3‐phenoxy benzyl (1RS)‐cis‐3‐(2, 2‐dichlorovinyl)‐2, 2‐dimethylcyclopropanecarboxylate], comprises a diastereoisomer pair of cypermethrin, which are (+)‐(1R‐cis‐αS)–CP (insecticidal) and (?)‐(1S‐cis‐αR)–CP (inactive). In this experiment, the stereoselective degradation of α‐CP was investigated in rat liver microsomes by high‐performance liquid chromatography (HPLC) with a cellulose‐tris‐ (3, 5‐dimethylphenylcarbamate)‐based chiral stationary phase. The results revealed that the degradation of (?)‐(1S‐cis‐αR)‐CP was much faster than (+)‐(1R‐cis‐αS)‐CP both in enantiomer monomers and rac‐α‐CP. As for the enzyme kinetic parameters, there were some variances between rac‐α‐CP and the enantiomer monomers. In rac‐α‐CP, the V_max and CL_int of (+)‐(1R‐cis‐αS)–CP (5105.22 ± 326.26 nM/min/mg protein and 189.64 mL/min/mg protein) were about one‐half of those of (?)‐(1S‐cis‐αR)–CP (9308.57 ± 772.24 nM/min/mg protein and 352.19 mL/min/mg protein), while the K_m of the two α‐CP enantiomers were similar. However, in the enantiomer monomers of α‐CP, the V_max and K_m of (+)‐(1R‐cis‐αS) ‐CP were 2‐fold and 5‐fold of (?)‐(1S‐cis‐αR)‐CP, respectively, which showed a significant difference with rac‐α‐CP. The CL_int of (+)‐(1R‐cis‐αS)–CP (140.97 mL/min/mg protein) was still about one‐half of (?)‐(1S‐cis‐αR)–CP (325.72 mL/min/mg protein) in enantiomer monomers. The interaction of enantiomers of α‐CP in rat liver microsomes was researched and the results showed that there were different interactions between the IC₅₀ of (?)‐ to (+)‐(1R‐cis‐αS)‐CP and (+)‐ to (?)‐(1S‐cis‐αR)‐CP(IC_50(?)/(+) / IC_50(+)/(?) = 0.61). Chirality 28:58–64, 2016. © 2015 Wiley Periodicals, Inc.     

Double-Bond Requirement in a Fatty Acid Desaturase Mutant of Saccharomyces cerevisiae

A fatty acid Delta(9)-desaturase mutant of yeast was analyzed to establish the specificity of the fatty acid structural requirement. Several double-bond-containing and substituted fatty acids were tested. It was concluded that the presence of a Delta(9)cis double bond is necessary for growth. The need for a specific chain length or a specific number of double bonds, or for both, is more flexible. Tracer-containing 16:1Delta(9)cis, 18:1Delta(9)cis, 18:2Delta(9,12)cis,cis and 18:3Delta(9,12,15) all cis revealed that each of these growth-supporting components, once taken up, was not converted into other fatty acids. Concentration effects on doubling time were also considered.     

Defining a novel cis element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA: role in transforming growth factor-beta 1 induced mRNA stabilization.

Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 1 induced message stability. Using end-labeled RNA fragments in gel shift assays and UV cross-linking analyses, we detected in the 3'-UTR a novel 9 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis element effectively competed out the formation of the R2 3'-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, we were able to predict the consensus sequences, 5'-GAGUUUNNN-3' and 5'-NNNUUUGAG-3' for optimal protein binding. These results support a model in which the 9 nt region functions in cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is involved in TGF-beta 1-mediated effects.     

Biosynthesis of trans,trans- and cis,trans-farnesols by soluble enzymes from tissue cultures of Andrographis paniculata

A cell-free system obtained from tissue cultures of Andrographis paniculata produces 2-trans,6-trans-farnesol (trans,trans-farnesol) and 2-cis,6-trans-farnesol (cis,trans-farnesol) (5:1), incorporating 10% of the radioactivity from 3R-[2-(14)C]mevalonate. There is total loss of (3)H from 3RS-[2-(14)C,(4S)-4-(3)H(1)]mevalonate and total retention from the (4R) isomer in both the trans,trans-farnesol and cis,trans-farnesol formed. When 3RS-[2-(14)C,5-(3)H(2)]mevalonate is used as substrate, there is total retention of (3)H in the trans,trans-farnesol, but loss of one-sixth of the (3)H in the cis,trans-farnesol. With (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-trans,trans -farnesol and (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-cis, trans-farnesol as substrates, the label is lost from the (1R)-cis,trans and (1S)-trans,trans isomers but retained in the (1R)-trans,trans and (1S)-cis,trans isomers; this shows that the pro-1S hydrogen is exchanged in the conversion of trans,trans-farnesol into cis,trans-farnesol and the pro-1R hydrogen in the conversion of cis,trans-farnesol into trans,trans-farnesol. (1R)-[1-(3)H(1)]-trans,trans-Farnesol and (1R)-[1-(3)H(1)]-cis,trans-farnesol have been synthesized by asymmetric chemical synthesis and exchanged with liver alcohol dehydrogenase. Both the trans- and the cis-alcohol exchange the pro-1R hydrogen atom.     

Interaction of the antitumor agents cis,cis,trans-PtIV(NH3)2Cl2(OH)2 and cis,cis,trans-PtIV[(CH3)2CHNH2]2Cl2(OH)2 and their reduction products with PM2 DNA

The ability of two platinum(IV) antitumor agents, cis,cis,trans-PtIV[(CH3)2CHNH2]2Cl2(OH)2 (2) and cis,cis,trans-PtIV(NH3)2Cl2(OH)2 (4), to interact with PM2 DNA was examined. Analysis using gel electrophoresis showed that neither compound is able to alter the electrophoretic mobilities of the three forms of PM2 DNA in the gel. However, incubation of 2 and 4 with 2 equiv of Fe(ClO4)2 X 6H2O or 1 equiv of ascorbic acid results in reduction to yield the divalent complexes cis-PtII(NH3)2Cl2 (1) and cis-PtII-[(CH3)2CHNH2]2Cl2 (3). The structures of the reduction products were characterized by using elemental analysis as well as infrared and 195Pt NMR spectroscopies. Both 1 and 3 were found to bind to and unwind supercoiled form I PM2 DNA. The aforementioned observations support the suggestion that reduction is a means of activating the antitumor properties of 2 and 4.     

Stereoselectivity of Mucorales lipases toward triradylglycerols--a simple solution to a complex problem

The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn-2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O'-C1' ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N'-C1' amide bond or a phenyl ring in sn-2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn-1 and sn-3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn-1. To explain these experimental observations, computer-aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C-terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn-1 and sn-3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity.     

Effect of cholesterol on the bond ordering in hydrated unsaturated lipid bilayers

Molecular dynamics computer simulations of hydrated bilayers of unsaturated phosphatidylcholines in which double bonds are in the states: 18:0/18:1(n-9)cis (PC), 18:0/18:2(n-6)cis (PC), 18:0/18:3(n-3)cis (PC), 18:0/20:4(n-6)cis (PC), and 18:0/22:6(n-3)cis in the presence of cholesterol (40 mol%) and its absence have been performed. The simulation have been performed at 303 K and 1 atm, under the conditions corresponding to the experimentally observed liquid-crystalline state of the bilayer from phosphatidylcholine. The C-C and C-H bond order parameter profiles with respect to the bilayer normal and the C-C bond orientation distribution functions have been calculated. The widths of the functions and positions of their maxima have been determined. The dependence of these characteristics on the type of the bond, the degree of unsaturation of the chain, the presence of cholesterol in the bilayer, and the bond order parameters have been analyzed.     

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).     

Isomerization increases the postprandial oxidation of linoleic acid but not alpha-linolenic acid in men

Human lipid intake contains various amounts of trans fatty acids. Refined vegetable and frying oils, rich in linoleic acid and/or alpha-linolenic acid, are the main dietary sources of trans-18:2 and trans-18:3 fatty acids. The aim of the present study was to compare the oxidation of linoleic acid, alpha-linolenic acid, and their major trans isomers in human volunteers. For that purpose, TG, each containing two molecules of [1-(13)C]linoleic acid, alpha-[1-(13)C]linolenic acid, [1-(13)C]-9cis,12trans-18:2, or [1-(13)C]-9cis,12cis,15trans-18:3, were synthesized. Eight healthy young men ingested labeled TG mixed with 30 g of olive oil. Total CO(2) production and (13)CO(2) excretion were determined over 48 h. The pattern of oxidation was similar for the four fatty acids, with a peak at 8 h and a return to baseline at 24 h. Cumulative oxidation over 8 h of linoleic acid, 9cis,12trans-18:2, alpha-linolenic acid, and 9cis,12cis,15trans-18:3 were, respectively, 14.0 +/- 4.1%, 24.7 +/- 6.7%, 23.6 +/- 3.3%, and 23.4 +/- 3.7% of the oral load, showing that isomerization increases the postprandial oxidation of linoleic acid but not alpha-linolenic acid in men.     

Thermodynamic origin of cis/trans isomers of a proline-containing beta-turn model dipeptide in aqueous solution: a combined variable temperature 1H-NMR, two-dimensional 1H,1H gradient enhanced nuclear Overhauser effect spectroscopy (NOESY), one-dimensional steady-state intermolecular 13C,1H NOE, and molecular dynamics study

The cis/trans conformational equilibrium of the two Ac-Pro isomers of the beta-turn model dipeptide [13C]-Ac-L-Pro-D-Ala-NHMe, 98% 13C enriched at the acetyl carbonyl atom, was investigated by the use of variable temperature gradient enhanced 1H-nmr, two-dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY), 13C,1H one-dimensional steady-state intermolecular NOE, and molecular dynamics calculations. The temperature dependence of the cis/trans Ala(NH) protons are in the region expected for random-coil peptides in H2O (delta delta/delta T = -9.0 and -8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (delta delta/delta T approximately -4.8 ppb) than that of the cis isomer (-7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProH alpha-AlaNH and AlaNH-NH(R) for the trans isomer. The experimental NOE data, coupled with computational analysis, can be interpreted by assuming that the trans isomer most likely adopts an ensemble of folded conformations. The C-CONH(CH3) fragment exhibits significant conformational flexibility; however, a low-energy conformer resembles closely the beta II-turn folded conformations of the x-ray structure of the related model peptide trans-BuCO-L-Pro-Me-D-Ala-NHMe. On the contrary, the cis isomer adopts open conformations. Steady-state intermolecular solute-solvent (H2O) 13C,1H NOE indicates that the water accessibility of the acetyl carbonyl carbons is nearly the same for both isomers. This is consistent with rapid fluctuations of the conformational ensemble and the absence of a highly shielded acetyl oxygen from the bulk solvent. Variable temperature 1H-nmr studies of the cis/trans conformational equilibrium indicate that the trans form is enthalpically favored (delta H degree = -5.14 kJ mole-1) and entropically (delta S degree = -5.47 J.K-1.mole-1) disfavored relative to the cis form. This demonstrates that, in the absence of strongly stabilizing sequence-specific interresidue interactions involving side chains and/or charged terminal groups, the thermodynamic difference of the cis/trans isomers is due to the combined effect of intramolecular and intermolecular (hydration) induced conformational changes.     

Binding selectivity of conformationally restricted analogues of (-)-indolactam-V to the C1 domains of protein kinase C isozymes

Two conformationally restricted analogues of (-)-indolactam-V (1) (cis and trans amides) were examined for their binding selectivity to the synthetic C1 peptides of all protein kinase C (PKC) isozymes. Although the binding constants of the cis amide-restricted analogue (2) were equal to those of 1, the trans amide-restricted analogue (3) bound significantly only to the novel PKC (delta, epsilon, eta, theta) isozymes.     

Stereoselectivity of rat liver glutathione transferase isoenzymes for alpha-bromoisovaleric acid and alpha-bromoisovalerylurea enantiomers.

The stereoselectivity of purified rat GSH transferases towards alpha-bromoisovaleric acid (BI) and its amide derivative alpha-bromoisovalerylurea (BIU) was investigated. GSH transferase 2-2 was the only enzyme to catalyse the conjugation of BI and was selective for the (S)-enantiomer. The conjugation of (R)- and (S)-BIU was catalysed by the isoenzymes 2-2, 3-3 and 4-4. Transferase 1-1 was less active, and no catalytic activity was observed with transferase 7-7. Isoenzymes 1-1 and 2-2 of the Alpha multigene family preferentially catalysed the conjugation of the (S)-enantiomer of BIU (and BI), whereas isoenzymes 3-3 and 4-4 of the Mu multigene family preferred (R)-BIU. The opposite stereoselectivity of conjugation of BI and BIU previously observed in isolated rat hepatocytes and the summation of activities of enzymes known to be present in hepatocytes on the basis of present data are in accord.     

Characterization of secondary amide peptide bond isomerization: thermodynamics and kinetics from 2D NMR spectroscopy

Secondary amide cis peptide bonds are of even lower abundance than the cis tertiary amide bonds of prolines, yet they are of biochemical importance. Using 2D NMR exchange spectroscopy (EXSY) we investigated the formation of cis peptide bonds in several oligopeptides: Ac-G-G-G-NH(2) , Ac-I-G-G-NH(2) , Ac-I-G-G-N-NH(2) and its cyclic form: I-G-G-N in dimethylsulfoxide (DMSO). From the NMR studies, using the amide protons as monitors, an occurrence of 0.13-0.23% of cis bonds was obtained at 296 K. The rate constants for the trans to cis conversion determined from 2D EXSY spectroscopy were 4-9 × 10(-3) s(-1) . Multiple minor conformations were detected for most peptide bonds. From their thermodynamic and kinetic properties the cis isomers are distinguished from minor trans isomers that appear because of an adjacent cis peptide bond. Solvent and sequence effects were investigated utilizing N-methylacetamide (NMA) and various peptides, which revealed a unique enthalpy profile in DMSO. The cyclization of a tetrapeptide resulted in greatly lowered cis populations and slower isomerization rates compared to its linear counterpart, further highlighting the impact of structural constraints.     

Changes in cis-zeatin and cis-zeatin riboside levels and biological activity during potato tuber dormancy

The effects of postharvest storage duration and temperature on endogenous cis -zeatin ( cis -Z) and cis -zeatin riboside ( cis -ZR) levels in potato ( Solanum tuberosum L.) tubers were determined in relation to tuber bud dormancy. The tubers used in these studies were completely dormant for at least 81 days of storage. Thereafter, tuber bud dormancy diminished gradually and after 165 days of postharvest storage, the tubers were completely non-dormant. Immediately after harvest, endogenous levels of cis- Z and cis -ZR were approximately 25 pmol (g fresh weight)⁻¹ and 8 pmol (g fresh weight)⁻¹, respectively. In tubers exiting dormancy but stored at a growth-inhibiting temperature (3°C), endogenous levels of cis -Z rose over threefold after 25 days of storage and remained elevated for the duration of the study. Levels of cis -ZR remained essentially constant during this same period. In tubers transferred to a growth permissive temperature (20°C) prior to use, the rise in endogenous cis -Z was less dramatic and more protracted; increasing twofold after 53 days of storage. No change in cis -Z riboside content was observed in these tubers during this period. Dose-response studies using either cis -Z or trans -Z demonstrated a time-dependent increase in cytokinin sensitivity during postharvest storage. Immediately after harvest, dormant tubers were insensitive to both zeatin isomers. Thereafter, tubers exhibited a dose-dependent increase in premature sprouting following injection with either cytokinin isomer. After injection into dormant tubers, cis -[8-¹⁴C]-zeatin was metabolized primarily to adenine/adenosine and cis -Z riboside. Seven days after injection, less than 10% of the recovered radioactivity was associated with trans -ZR. These results are consistent with a role for endogenous cis -Z (and its derivatives) in the regulation of potato tuber dormancy.