(+/-)-(1S,2R,5S)-5-Amino-2-fluorocyclohex-3-enecarboxylic acid. A potent GABA aminotransferase inactivator that irreversibly inhibits via an elimination-aromatization pathway

Inhibition of gamma-aminobutyric acid aminotransferase (GABA-AT) increases the concentration of GABA, an inhibitory neurotransmitter in human brain, which could have therapeutic applications for a variety of neurological diseases, including epilepsy. On the basis of studies of several previously synthesized conformationally restricted GABA-AT inhibitors, (+/-)-(1S,2R,5S)-5-amino-2-fluorocyclohex-3-enecarboxylic acid (12) was designed as a mechanism-based inactivator. This compound was shown to irreversibly inhibit GABA-AT; substrate protects the enzyme from inactivation. Mechanistic experiments demonstrated the loss of one fluoride ion per active site during inactivation and the formation of N-m-carboxyphenylpyridoxamine 5'-phosphate (26), the same product generated by inactivation of GABA-AT by gabaculine (8). An elimination-aromatization mechanism is proposed to account for these results.     

Inactivation of gamma-aminobutyric acid aminotransferase by (Z)-4-amino-2-fluorobut-2-enoic acid

(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.     

Synthesis of cyclopropane isosteres of the antiepilepsy drug vigabatrin and evaluation of their inhibition of GABA aminotransferase

The antiepilepsy drug vigabatrin (1; 4-aminohex-5-enoic acid; gamma-vinyl GABA) is a mechanism-based inactivator of the pyridoxal 5'-phosphate (PLP)-dependent enzyme gamma-aminobutyric acid aminotransferase (GABA-AT). Inactivation has been shown to proceed by two divergent mechanisms (Nanavati, S. M. and Silverman, R. B. (1991) J. Am. Chem. Soc. 113, 9341-9349), a Michael addition pathway (Scheme 2, pathway a) and an enamine pathway (Scheme 2, pathway b). Analogs of vigabatrin with a cyclopropyl or cyanocyclopropyl functionality in place of the vinyl group (2-5) were synthesized as potential inactivators of GABA-AT that can inactivate the enzyme only through a Michael addition pathway, but they were found to be only weak inhibitors of the enzyme.     

Synthesis of Cyclopropane Isosteres of the Antiepilepsy Drug Vigabatrin and Evaluation of their Inhibition of GABA Aminotransferase

The antiepilepsy drug vigabatrin (1; 4-aminohex-5-enoic acid; γ-vinyl GABA) is a mechanism-based inactivator of the pyridoxal 5'-phosphate (PLP)-dependent enzyme γ-aminobutyric acid aminotransferase (GABA-AT). Inactivation has been shown to proceed by two divergent mechanisms (Nanavati, S. M. and Silverman, R. B. (1991) J. Am. Chem. Soc. 113, 9341–9349), a Michael addition pathway (Scheme 2, pathway a) and an enamine pathway (Scheme 2, pathway b). Analogs of vigabatrin with a cyclopropyl or cyanocyclopropyl functionality in place of the vinyl group (2–5) were synthesized as potential inactivators of GABA-AT that can inactivate the enzyme only through a Michael addition pathway, but they were found to be only weak inhibitors of the enzyme.     

Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid

Evidence for an enamine mechanism of inactivation of pig brain gamma-aminobutyric acid (GABA) aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid is presented. apo-GABA aminotransferase reconstituted with [3H]pyridoxal 5'-phosphate is inactivated by (S,E)-4-amino-5-fluoropent-2-enoic acid and the pH is raised to 12. All of the radioactivity is released from the enzyme as an adduct of the cofactor; no [3H]pyridoxamine 5'-phosphate is generated.     

Enantiomers of 4-amino-3-fluorobutanoic acid as substrates for gamma-aminobutyric acid aminotransferase. Conformational probes for GABA binding

Gamma-aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of alpha-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches.     

Mechanism of inactivation of gamma-aminobutyrate aminotransferase by 4-amino-5-fluoropentanoic acid. First example of an enamine mechanism for a gamma-amino acid with a partition ratio of 0

The mechanism of inactivation of pig brain gamma-aminobutyric acid aminotransferase (GABA-T) by (S)-4-amino-5-fluoropentanoic acid (1, R = CH2CH2COOH, X = F) previously proposed [Silverman, R. B., & Levy, M. A. (1981) Biochemistry 20, 1197-1203] is revised. apo-GABA-T is reconstituted with [4-3H]pyridoxal 5'-phosphate and inactivated with 1 (R = CH2CH2COOH, X = F). Treatment of inactivated enzyme with base followed by acid denaturation leads to the complete release of radioactivity as 6-[2-hydroxy-3-methyl-6-(phosphonoxymethyl)-4-pyridinyl]-4-oxo-5-+ ++hexenoic acid (4, R = CH2CH2COOH). Alkaline phosphatase treatment of this compound produces dephosphorylated 4 (R = CH2CH2COOH). These results support a mechanism that was suggested by Metzler and co-workers [Likos, J. J., Ueno, H., Feldhaus, R. W., & Metzler, D. E. (1982) Biochemistry 21, 4377-4386] for the inactivation of glutamate decarboxylase by serine O-sulfate (Scheme I, pathway b, R = COOH, X = OSO3-).     

Structures of gamma-aminobutyric acid (GABA) aminotransferase, a pyridoxal 5'-phosphate, and [2Fe-2S] cluster-containing enzyme, complexed with gamma-ethynyl-GABA and with the antiepilepsy drug vigabatrin

Gamma-aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, gamma-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-A resolution) and in complex with vigabatrin as well as with the close analogue gamma-ethynyl-GABA (to 2.3 and 2.8 A, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5'-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radio-labeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.     

Syntheses of (Z)-and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and their inactivation of gamma-aminobutyric acid aminotransferase.

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respectively) were synthesized and investigated as potential mechanism-based inactivators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core alpha-trifluoromethyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxy-carbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from 4 or 5. This inactivating species is released from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for 4 and 5, respectively. The enzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A mechanism to account for these observations is proposed.     

Mechanism of inactivation of Escherichia coli aspartate aminotransferase by (S)-4-amino-4,5-dihydro-2-furancarboxylic acid

As a potential drug to treat neurological diseases, the mechanism-based inhibitor (S)-4-amino-4,5-dihydro-2-furancarboxylic acid (S-ADFA) has been found to inhibit the γ-aminobutyric acid aminotransferase (GABA-AT) reaction. To circumvent the difficulties in structural studies of a S-ADFA-enzyme complex using GABA-AT, l-aspartate aminotransferase (l-AspAT) from Escherichia coli was used as a model PLP-dependent enzyme. Crystal structures of the E. coli aspartate aminotransferase with S-ADFA bound to the active site were obtained via cocrystallization at pH 7.5 and 8. The complex structures suggest that S-ADFA inhibits the transamination reaction by forming adducts with the catalytic lysine 246 via a covalent bond while producing 1 equiv of pyridoxamine 5'-phosphate (PMP). Based on the structures, formation of the K246-S-ADFA adducts requires a specific initial binding configuration of S-ADFA in the l-AspAT active site, as well as deprotonation of the ε-amino group of lysine 246 after the formation of the quinonoid and/or ketimine intermediate in the overall inactivation reaction.     

Primary deuterium isotope effects for the 3-methylaspartase-catalyzed deamination of (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, and (2S,3S)-3-ethylaspartic acid

3-Methylaspartate ammonia-lyase catalyzes the deamination of (2S)-aspartic acid 137 times more slowly than the deamination of (2S,3S)-3-methylaspartic acid but catalyzes the amination of fumaric acid 1.8 times faster than the amination of mesaconic acid [Botting, N.P., Akhtar, M., Cohen, M. A., & Gani, D. (1988) Biochemistry (preceding paper in this issue)]. In order to understand the mechanistic basis for these observations, the deamination reaction was examined kinetically with (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, (2S,3S)-3-ethylaspartic acid, and the corresponding C-3-deuteriated isotopomers. Comparison of the double-reciprocal plots of the initial reaction velocities for each of the three pairs of substrates revealed that the magnitude of the primary isotope effect on both Vmax and V/K varied with the substituent at C-3 of the substrate. 3-Methylaspartic acid showed the largest isotope effect (1.7 on Vmax and V/K), 3-ethylaspartic acid showed a smaller isotope effect (1.2 on Vmax and V/K), and aspartic acid showed no primary isotope effect at all. These results, which are inconsistent with earlier reports that there is no primary isotope effect for 3-methylaspartic acid [Bright, H. J. (1964) J. Biol. Chem. 239, 2307], suggest that for both 3-methylaspartic acid and 3-ethylaspartic acid elimination occurs via a predominantly concerted mechanism whereas for aspartic acid an E1cb mechanism prevails.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resolution, absolute stereochemistry, and enantiopharmacology of the GluR1-4 and GluR5 antagonist 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid

The phosphono amino acid, (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propio nic acid (ATPO), is a structural hybrid between the NMDA antagonist (RS)-2-amino-7-phosphonoheptanoic acid (AP7) and the AMPA and GluR5 agonist, (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA). ATPO has been resolved into (S)-ATPO and (R)-ATPO using chiral HPLC, and the absolute stereochemistry of the two enantiomers was established by an X-ray crystallographic analysis of (R)-ATPO. (S)-ATPO and (R)-ATPO were characterized pharmacologically using rat brain membrane binding and electrophysiologically using the cortical wedge preparation as well as homo- or heteromeric GluR1-4, GluR5-6, and KA2 receptors expressed in Xenopus oocytes. (R)-ATPO was essentially inactive as an agonist or antagonist in all test systems. (S)-ATPO was an inhibitor of the binding of [(3)H]AMPA (IC(50) = 16 +/- 1 microM) and of [(3)H]-6-cyano-7-nitroquinoxaline-2,3-dione ([(3)H]CNQX) (IC(50) = 1.8 +/- 0.2 microM), but was inactive in the [(3)H]kainic acid and the [(3)H]-(RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([(3)H]CPP) binding assays. (S)-ATPO did not show detectable agonist effects at any of the receptors under study, but antagonized AMPA-induced depolarization in the cortical wedge preparation (IC(50) = 15 +/- 1 microM). (S)-ATPO also blocked kainic acid agonist effects at GluR1 (K(i) = 2.0 microM), GluR1+2 (K(i) = 3.6 microM), GluR3 (K(i) = 3.6 microM), GluR4 (K(i) = 6.7 microM), and GluR5 (K(i) = 23 microM), but was inactive at GluR6 and GluR6+KA2. Thus, although ATPO is a structural analog of AP7 neither (S)-ATPO nor (R)-ATPO are recognized by NMDA receptor sites.     

Paramyotonia congenita mutations reveal different roles for segments S3 and S4 of domain D4 in hSkM1 sodium channel gating

Mutations in the gene encoding the voltage-gated sodium channel of skeletal muscle (SkMl) have been identified in a group of autosomal dominant diseases, characterized by abnormalities of the sarcolemmal excitability, that include paramyotonia congenita (PC) and hyperkalemic periodic paralysis (HYPP). We previously reported that PC mutations cause in common a slowing of inactivation in the human SkMl sodium channel. In this investigation, we examined the molecular mechanisms responsible for the effects of L1433R, located in D4/S3, on channel gating by creating a series of additional mutations at the 1433 site. Unlike the R1448C mutation, found in D4/S4, which produces its effects largely due to the loss of the positive charge, change of the hydropathy of the side chain rather than charge is the primary factor mediating the effects of L1433R. These two mutations also differ in their effects on recovery from inactivation, conditioned inactivation, and steady state inactivation of the hSkMl channels. We constructed a double mutation containing both L1433R and R1448C. The double mutation closely resembled R1448C with respect to alterations in the kinetics of inactivation during depolarization and voltage dependence, but was indistinguishable from L1433R in the kinetics of recovery from inactivation and steady state inactivation. No additive effects were seen, suggesting that these two segments interact during gating. In addition, we found that these mutations have different effects on the delay of recovery from inactivation and the kinetics of the tail currents, raising a question whether this delay is a reflection of the deactivation process. These results suggest that the S3 and S4 segments play distinct roles in different processes of hSkM1 channel gating: D4/S4 is critical for the deactivation and inactivation of the open channel while D4/S3 has a dominant role in the recovery of inactivated channels. However, these two segments interact during the entry to, and exit from, inactivation states.     

New substrates and inhibitors of gamma-aminobutyric acid aminotransferase containing bioisosteres of the carboxylic acid group: design, synthesis, and biological activity

A series of potential substrates of gamma-aminobutyric acid aminotransferase (GABA-AT) with lipophilic bioisosteres of the carboxylic acid group (2-7) were synthesized and tested. Most of the synthesized compounds showed substrate activities with GABA-AT; 1H-tetrazole-5-propanamine (6) was the best of those tested. The potential time-dependent inhibitor of GABA-AT, 1H-tetrazole-5-(alpha-vinyl-propanamine) (8), was designed based on the structures of 6 and the antiepilepsy drug vigabatrin (4-aminohex-5-enoic acid, 1). The synthesized compound 8 showed time-dependent inhibition of GABA-AT, but its potency is lower than that of vigabatrin. Methylation of the tetrazole group in 8 resulted in loss of time-dependent activity, suggesting that the tetrazole ring, the carboxylate bioisostere, exists in its deprotonated form in the enzyme active site.     

Evidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitase

Beef heart aconitase, as isolated under aerobic conditions, is inactive and contains a [3Fe-4S]1+ cluster. On incubation at pH greater than 9.5 (or treatment with 4-8 M urea) the color of the protein changes from brown to purple. This purple form is stable and can be converted back in good yield to the active [4Fe-4S]2+ form by reduction in the presence of iron. Active aconitase is converted to the purple form at alkaline pH only after oxidative inactivation. The Fe/S2- ratio of purple aconitase is 0.8, indicating the presence of [3Fe-4S] clusters. The number of SH groups readily reacting with 5,5'-dithiobis(2-nitrobenzoic acid) is increased from approximately 1 in the enzyme as isolated to 7-8 in the purple form, indicating a partial unfolding of the protein. On conversion of inactive aconitase to the purple form, the EPR signal at g = 2.01 (S = 1/2) is replaced by signals at g = 4.3 and 9.6 (S = 5/2). M?ssbauer spectroscopy shows that purple aconitase has high-spin ferric ions, each residing in a tetrahedral environment of sulfur atoms. The three iron sites are exchange-coupled to yield a ground state with S = 5/2. Analysis of the data within a spin coupling model shows that J13 congruent to J23 and 2 J12 less than J13, where the Jik describe the antiferromagnetic (J greater than 0) exchange interactions among the three iron pairs. Comparison of our data with those reported for synthetic Fe-S clusters (Hagen, K. S., Watson, A. D., and Holm, R. H., (1983) J. Am. Chem. Soc. 105, 3905-3913) shows that purple aconitase contains a linear [3Fe-4S]1+ cluster, a structural isomer of the S = 1/2 cluster of inactive aconitase. Our studies also show that protein-bound [2Fe-2S] clusters can be generated under conditions where partial unfolding of the protein occurs.     

Inactivation of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue in Corynebacterium glutamicum, by (R)- and (S)-oxirane-2-carboxylate: analysis and implications

( R)- and ( S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the ( R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the ( R) and ( S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by ( R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the ( R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.     

Intercalation of the (1S,2R,3S,4R)-N6-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence

The (1S,2R,3S,4R)-N(6)-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, results from trans opening of (1R,2S,3S,4R)-1,2-epoxy-1,2,3, 4-tetrahydrobenz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Two conformations of this adduct exist, in slow exchange on the NMR time scale. A structure for the major conformation, which represents approximately 80% of the population, is presented. In this conformation, an anti glycosidic torsion angle is observed for all nucleotides, including S,R,S,RA6. The refined structure is a right-handed duplex, with the benz[a]anthracene moiety intercalated on the 3'-face of the modified base pair, from the major groove. It is located between S,R,S,RA6.T17 and A7.T16. Intercalation is on the opposite face of the modified S,R,S,RA6.T17 base pair as compared to the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2, 3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct, which intercalated 5' to the modified R,S,R,SA6.T17 base pair [Li, Z. , Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981]. The spectroscopic data do not allow refinement of the minor conformation, but suggest that the adenyl moiety in the modified nucleoti111S,R, S,RA6 adopts a syn glycosidic torsion angle. Thus, the minor conformation may create greater distortion of the DNA duplex. The results are discussed in the context of site-specific mutagenesis studies which reveal that the S,R,S,RA6 lesion is less mutagenic than the R,S,R,SA6 lesion.     

First enantiospecific synthesis of a 3,4-dihydroxy-L-glutamic acid [(3S,4S)-DHGA], a new mGluR1 agonist

The first synthesis of one of the 4 possible stereoisomers of 3,4-dihydroxy-L-glutamic acid ((3S,4S)-DHGA 3), a natural product of unknown configuration, is described. The synthesis is based on the Lewis acid catalyzed reaction of benzyl alcohol with a D-ribose-derived 2,3-aziridino-gamma-lactone 4-benzyl carboxylate (6). Preliminary pharmacological studies showed that (3S,4S)-3 is an agonist of metabotropic glutamate receptors of type 1 (mGluR1) and a weak antagonist of mGluR4 but has no discernible activity with respect to mGluR2. This activity profile can be rationalized by fitting extended conformations of (3S,4S)-3 in proposed models of each of these receptor subtypes.     

Inactivation of gamma-aminobutyric acid aminotransferase by (S)-4-amino-4,5-dihydro-2-furancarboxylic acid does not proceed by the expected aromatization mechanism

Inactivation of pyridoxal 5'-phosphate (PLP)-dependent gamma-aminobutryic acid aminotransferase by (S)-4-amino-4,5-dihydro-2-furancarboxylic acid (SADFA) gives pyridoxamine 5'-phosphate, not the expected SADFA-PLP aromatization product. Inactivation appears to proceed by a Michael addition/hydrolysis mechanism instead.     

Intercalation of the (-)-(1R,2S,3R, 4S)-N6-[1-benz[a]anthracenyl]-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence

The solution structure of the (-)-(1R,2S,3R,4S)-N6-[1-(1,2,3, 4-tetrahydroxy-benz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61(italic), and 62 of the human N-ras protooncogene, was determined. This adduct results from the trans opening of 1S,2R,3R,4S-1, 2-epoxy-1,2,3,4-tetrahydro-benz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Molecular dynamics simulations were restrained by 509 NOEs from 1H NMR. The precision of the refined structures was monitored by pairwise root-mean-square deviations which were <1.2 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 9.1 x 10(-)2 at 250 ms. The refined structure was a right-handed duplex, in which the benz[a]anthracene moiety intercalated from the major groove between C5.G18 and R,S,R,SA6.T17. In this orientation, the saturated ring of BA was oriented in the major groove of the duplex, with the aromatic rings inserted into the duplex such that the terminal ring of BA threaded the duplex and faced toward the minor groove direction. The duplex suffered localized distortion at and immediately adjacent to the adduct site, evidenced by the increased rise of 8.8 A as compared to the value of 3.5 A normally observed for B-DNA between base pairs C5.G18 and R,S,R,SA6.T17. These two base pairs also buckled in opposite directions away from the intercalated BA moiety. The refined structure was similar to the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9, 10)-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct of corresponding stereochemistry at X6 of the same oligodeoxynucleotide [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224]. Both adducts intercalated toward the 5'-direction from the site of adduction. The similarities in solution structures were reflected in similar biological responses, when repair-deficient AB2480 Escherichia coli were transformed with M13mp7L2 DNA site-specifically modified with these two adducts.