Stereoselective asymmetric synthesis of (+)‐sedamine and (+)‐allosedamine

A convenient method for the generation of (+)‐sedamine and (+)‐allosedamine in high optical purity has been elaborated. The key steps are the highly stereoselective 1,2‐nucleophilic addition to SAMP hydrazones allowing the installation of the stereogenic center at C2 and ring closing metathesis. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

New potent C2-symmetric malaria plasmepsin I and II inhibitors

A series of malaria plasmepsin (Plm) I and II inhibitors containing a C(2)-symmetric core structure have been synthesised and tested for protease inhibition activity. These compounds can be prepared using a straightforward synthesis involving a phenol nucleophilic ring opening of a diepoxide. Exemplar compounds synthesised exhibited remarkable inhibitory activity against both Plm I and II, notably 15c with K(i) values of 2.7nM and 0.25nM respectively, as well as showing >100-fold selectivity against Cathepsin D.     

Synthesis of (±)-Homosarkomycin and (±)-Rosaprostol

(±)-Homosarkomycin (2) and (±)-rosaprostol (3) were synthesized from (±)-methyl 2-oxo-bicyclo[3.1.0]hexane-1-carboxylate (1) by using the nucleophilic ring opening reaction on the double-activated cyclopropane ring as the key step.     

Synthesis, in vitro cytotoxicity and in vivo anti-inflammatory activity of long chain 3-amino-1,2-diols

The synthesis of long chain 3-amino-1,2-diols was carried out based on Sharpless asymmetric epoxidation of long chain allylic alcohols and regioselective nucleophilic ring opening by azido group. The in vitro cytotoxicity of the compounds prepared was evaluated against six solid tumor cell lines (A2780, H322, LL, WiDr, C26-10, UMSCC-22B). Free 3-amino-1,2-diols exhibited IC50 values between 1.45 microM and 32 microM. These compounds also presented interesting inhibition of carrageenin-induced paw edema in rats (85.3% - 79.6% at a concentration of 0.15 mmol/kg).     

Unexpected reaction of beta-cyclodextrin tosylate with pyrrolidinones

Substitution reactions of 6I-O-p-tolylsulfonylcyclomaltoheptaose with alkyl- and arylamines in 1-methyl-2-pyrrolidinone and various pyrrolidinones were investigated. An unexpected reaction of the tosyl group with pyrrolidinones was observed resulting in products deriving from nucleophilic attack by the lactam carbonyl oxygen and further opening of the heterocyclic ring. The new compounds have been fully characterized by ESIMS and NMR analyses.     

3-(2,5-Dihydro-1H-pyrrol-2-ylmethoxy)pyridines: synthesis and analgesic activity

We disclose an efficient procedure for the preparation of ethers of 2-substituted 2-hydroxymethylpyrroline and of 2-aminomethyl-3-pyrrolines, involving, as a key step, formation and nucleophilic ring opening of a cyclic sulfamidate. Several new analogs of epibatidine (1) and tebanicline (ABT-594, 2) were prepared and tested for analgesic activity in the mouse formalin model.     

Interactions of PAN's C‐termini with archaeal 20S proteasome and implications for the eukaryotic proteasome–ATPase interactions

Protein degradation in the 20S proteasome is regulated in eukaryotes by the 19S ATPase complex and in archaea by the homologous PAN ATPase ring complex. Subunits of these hexameric ATPases contain on their C‐termini a conserved hydrophobic‐tyrosine‐X (HbYX) motif that docks into pockets in the 20S to stimulate the opening of a gated substrate entry channel. Here, we report the crystal structure of the archaeal 20S proteasome in complex with the C‐terminus of the archaeal proteasome regulatory ATPase, PAN. This structure defines the detailed interactions between the critical C‐terminal HbYX motif and the 20S α‐subunits and indicates that the intersubunit pocket in the 20S undergoes an induced‐fit conformational change on binding of the HbYX motif. This structure together with related mutagenesis data suggest how in eukaryotes certain proteasomal ATPases bind to specific pockets in an asymmetrical manner to regulate gate opening.     

Mitochondrial permeability transition involves dissociation of F1FO ATP synthase dimers and C‐ring conformation

The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F₁F_O ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F₁F_O ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F₁F_O ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT. Specific mutations in the F₁F_O ATP synthase c subunit that alter C‐ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F₁F_O ATP synthase dimers. Destabilizing F₁F_O ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT. The current study does not provide direct evidence that the C‐ring is the long‐sought pore‐forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F₁F_O ATP synthase dimers and involves the C‐ring.     

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.     

Complete synthesis of the bicyclic ring of a mutacin analog with orthogonally protected lanthionine via solid‐phase intracyclization

Mutacin 1140 (MU1140) is a naturally occurring lantibiotic derived from posttranslational modifications of a ribosomally synthesized peptide during the fermentation of a bacterium called Streptococcus mutans, the etiological agent of dental cavities. A practical approach for chemically synthesizing lantibiotics would be a valuable tool to expand the MU1140 library with additional semisynthetic analogs. In turn, an expanded library may prove useful to explore additional therapeutic indications for this pipeline of novel compounds. In this work, orthogonally protected lanthionine analogs were synthesized via an aziridine ring opening strategy. This lanthionine was utilized to synthesize a cysteamine (Cya) instead of the (S)‐aminovinyl‐D‐cysteine (AviCys) that is naturally found in MU1140. The Cya containing bicyclic C/D ring of MU1140 was synthesized by Fmoc solid‐phase peptide synthesis (SPPS). The linear peptides were synthesized using OPfp ester derivatives and using various common coupling reagents such as COMU and TCTU. The linear peptide was intracyclized with DEPBT to construct the so‐called bicyclic ring C/D. This is the first report on the complete chemical synthesis of the bicyclic C/D ring of a MU1140 analog using orthogonally protected lanthionines using SPPS.     

Synthesis and biological evaluation of isonucleosides derived from methyl 3,5-anhydro-2-O-(2-fluorobenzyl)-D-xylofuranosides

New isonucleosides [methyl 5-(1-pyrimidinyl)furanosides] are prepared by nucleophilic opening of the oxetane ring of methyl 3,5-anhydro-2-O-(2-fluorobenzyl)-D-xylofuranoside with silylated pyrimidine bases in the presence of trimethylsilyl triflate. Structures, configurations and conformations were determined by NMR techniques and several X-ray diffraction analyses, seven of the isonucleosides were tested for cytotoxicity and activity against HIV, HSV and several other viruses.     

2‐Azapurine Nucleosides: Synthesis,Properties, and Base Pairing of Oligonucleotides

This review deals with 2‐azapurine (imidazo[4,5‐d] [1,2,3]triazine) nucleosides and closely related analogs. Different routes are described to yield the desired target compounds, including a sequence of ring‐opening and ring‐closure reactions performed on purine nucleosides or direct glycosylation of a 2‐azapurine nucleobase with a sugar halide. Further, physical and spectroscopic properties of 2‐azapurine nucleosides are discussed, including fluorescence, ¹³C‐NMR data, single‐crystal X‐ray analyses, and conformation studies on selected compounds; new biological data are presented. The second part of this review is dedicated to oligonucleotides containing 2‐azapurines, including building‐block (phosphoramidite) preparation and their use in solid‐phase oligonucleotide synthesis. Base‐pairing properties of 2‐azapurine nucleosides as surrogates of canonical constituents of DNA were evaluated.     

Convenient preparation of 3,5-anhydro- and 2,5-anhydropentofuranosides, and 5,6-anhydro-D-glucofuranose by use of the Mitsunobu reaction

Methyl 3,5-anhydro-alpha-D-xylofuranosides are obtained by use of the Mitsunobu reaction from 2-O-protected methyl alpha-D-xylofuranosides, which are easily prepared from D-xylose. The Mitsunobu reaction of methyl 3-N-benzylamino-3-deoxy- and 3-azido-3-deoxyarabinofuranosides, which are prepared from the conveniently available methyl 2,3-anhydro-alpha-D- and 2,3-anhydro-alpha-l-lyxofuranosides by nucleophilic ring opening, yields the corresponding methyl 2,5-anhydro-alpha-D- and 2,5-anhydro-alpha-l-arabinofuranosides. Ring opening of 3,5-anhydro-1,2-O-isopropylidene-alpha-D-xylofuranose with azide yields the corresponding 5-azido derivative. The structure and configuration of the products is confirmed by NMR spectroscopy. 5,6-Anhydro-1,2-O-isopropylidene-alpha-D-glucofuranose is formed by the Mitsunobu reaction of 1,2-O-isopropylidene-alpha-D-glucofuranose. Its structure is verified by single-crystal X-ray diffraction analysis.     

Synthesis of 4-Deoxy-4-C-hydroxymethyl-α-L-Lyxo-Pyranosyl Thymine

Abstract

The synthesis of 1-[4-deoxy-4-C-hydroxymethyl-α-L-lyxopyranosyl]thymine has been accomplished by two synthetic routes both starting from methyl 2, 3-O-isopropylidene-β-D-ribopyranoside. The first route makes use of a ring opening, ring closure reaction sequence to increase the proportion of the desired L-isomers. The second route utilizes the soft nucleophilic character of malonyl anions and ozonolytic cleavage of enol ether to introduce the branched chain. The newly obtained pyranosyl nucleoside obtains a ⁴C₁ conformation with an equatorially oriented thymine moiety.     

Metal ion complexes of macrocyclic polyamines enhance both the phosphate hydrolysis and imidazole ring opening of RNA 5'-cap structure

The cleavage of P1-(7-methylguanosyl-5') P3-(guanosyl-5') triphosphate, a RNA 5'-cap model, by 2-hydroxyethyl- (6a-6c) and 2-aminoethyl- (7a-7c) substituted macrocycles in the presence and absence of Zn2+ and Cu2+ ions has been studied at pH 7.2 and 60 degrees. In the presence of the metal ions, hydrolysis of the phosphate group is enhanced. The mono- and dinuclear Zn2+ complexes promote solely the phosphate hydrolysis, whereas the corresponding Cu2+ complexes accelerate both the phosphate hydrolysis and the imidazole ring opening of the 7-methylguanine base. In the absence of the metal ions, the macrocycles mainly promote breakdown of the 7-methylguanine base, most probably by enhancing the nucleophilic attack of hydroxide ion on the C(8)-atom by shielding the repulsive negative charge on the phosphate moiety. The 2-hydroxyethyl and 2-aminoethyl side arms exhibit a two- to three-fold rate acceleration. Opening of the imidazole ring eventually results in cleavage of the triphosphate bridge.     

Exploring the S4 and S1 prime subsite specificities in caspase-3 with aza-peptide epoxide inhibitors

Caspase-3 is a prototypic executioner caspase that plays a central role in apoptosis. Aza-peptide epoxides are a novel class of irreversible inhibitors that are highly specific for clan CD cysteine proteases. The five crystal structures of caspase-3-aza-peptide epoxide inhibitor complexes reported here reveal the structural basis for the mechanism of inhibition and the specificities at the S1' and the S4 subsites. Unlike the clan CA cysteine proteases, the catalytic histidine in caspase-3 plays a critical role during protonation and subsequent ring opening of the epoxide moiety and facilitates the nucleophilic attack by the active site cysteine. The nucleophilic attack takes place on the C3 carbon atom of the epoxide and results in an irreversible alkylation of the active site cysteine residue. A favorable network of hydrogen bonds involving the oxyanion hole, catalytic histidine, and the atoms in the prime site of the inhibitor enhance the binding affinity and specificity of the aza-peptide epoxide inhibitors toward caspase-3. The studies also reveal that subtle movements of the N-terminal loop of the beta-subunit occur when the P4 Asp is replaced by a P4 Ile, whereas the N-terminal loop and the safety catch Asp179 are completely disordered when the P4 Asp is replaced by P4 Cbz group.     

Reaction of 1-amino-2-methylenecyclopropane-1-carboxylate with 1-aminocyclopropane-1-carboxylate deaminase: analysis and mechanistic implications

1-aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyzes the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia. In an early study of this unusual C(alpha)-C(beta) ring cleavage reaction, 1-amino-2-methylenecyclopropane-1-carboxylic acid (2-methylene-ACC) was shown to be an irreversible inhibitor of ACC deaminase. The sole turnover product was identified as 3-methyl-2-oxobutenoic acid. These results provided strong evidence supporting the ring cleavage of ACC via a nucleophilic addition initiated process, thus establishing an unprecedented mechanism of coenzyme B(6) dependent catalysis. To gain further insight into this inactivation, tritiated 2-methylene-ACC was prepared and used to trap the critical enzyme nucleophiles. Our results revealed that inactivation resulted in the modification of an active site residue, Ser-78. However, an additional 5 equiv of inhibitor was also found to be incorporated into the inactivated enzyme after prolonged incubation. In addition to Ser-78, other nucleophilic residues modified include Lys-26, Cys-41, Cys-162, and Lys-245. The location of the remaining unidentified nucleophile has been narrowed down to be one of the residues between 150 and 180. Labeling at sites outside of the active site is not enzyme catalyzed and may be a consequence of the inherent reactivity of 2-methylene-ACC. Further experiments showed that Ser-78 is responsible for abstracting the alpha-H from d-vinylglycine and may serve as the base to remove the beta-H in the catalysis of ACC. However, it is also likely that Ser-78 serves as the active site nucleophile that attacks the cyclopropane ring and initiates the fragmentation of ACC, while the conserved Lys-51 is the base required for beta-H abstraction. Clearly, the cleavage of ACC to alpha-ketobutyrate by ACC deaminase represents an intriguing conversion beyond the common scope entailed by coenzyme B(6) dependent catalysts.     

Mechanistic studies of 1-aminocyclopropane-1-carboxylate deaminase: characterization of an unusual pyridoxal 5'-phosphate-dependent reaction

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give α-ketobutyric acid and ammonia as products. The cleavage of the C(α)-C(β) bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile- or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK(a) of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and (13)C KIE studies of the wild type enzyme suggest that the C(α)-C(β) bond cleavage step in the chemical mechanism is at least partially rate-limiting under k(cat)/K(m) conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294.     

Crystal structure of phenylacetic acid degradation protein PaaG from Thermus thermophilus HB8

Microbial degradation of phenylacetic acid proceeds via the hybrid pathway that includes formation of a coenzyme A thioester, ring hydroxylation, non‐oxygenolytic ring opening, and β‐oxidation‐like reactions. A phenylacetic acid degradation protein PaaG is a member of the crotonase superfamily, and is a candidate non‐oxygenolytic ring‐opening enzyme. The crystal structure of PaaG from Thermus thermophilus HB8 was determined at a resolution of 1.85 Å. PaaG consists of three identical subunits related by local three‐fold symmetry. The monomer is comprised of a spiral and a helical domain with a fold characteristic of the crotonase superfamily. A putative active site residue, Asp136, is situated in an active site cavity and surrounded by several hydrophobic and hydrophilic residues. The active site cavity is sufficiently large to accommodate a ring substrate. Two conformations are observed for helix H2 located adjacent to the active site. Helix H2 is kinked at Asn81 in two subunits, whereas it is kinked at Leu77 in the other subunit, and the side chain of Tyr80 is closer to Asp136. This indicates that catalytic reaction of PaaG may proceed with large conformational changes at the active site. Asp136 is the only conserved polar residue in the active site. It is located at the same position as those of 4‐chlorobenzoyl‐CoA dehalogenase and peroxisomal Δ³,Δ²‐enoyl‐CoA isomerase, indicating that PaaG may undergo isomerization or a ring‐opening reaction via a Δ³,Δ²‐enoyl‐CoA isomerase‐like mechanism. Proteins 2009. © 2009 Wiley‐Liss, Inc.