Sagun Tandel

Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride.

Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparentK i of the inhibition was 125 μm (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k 2) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 Å resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.

The crystal structure of palmitoyl protein thioesterase-2 (PPT2) reveals the basis for divergent substrate specificities of the two lysosomal thioesterases, PPT1 and PPT2.

Mutations in palmitoyl protein thioesterase-1 (PPT1) have been found to cause the infantile form of neuronal ceroid lipofuscinosis, which is a lysosomal storage disorder characterized by impaired degradation of fatty acid-modified proteins with accumulation of amorphous granular deposits in cortical neurons, leading to mental retardation and death. Palmitoyl protein thioesterase-2 (PPT2) is a second lysosomal hydrolase that shares a 26% identity with PPT1. A previous study had suggested that palmitoyl-CoA was the preferred substrate of PPT2. Furthermore, PPT2 did not hydrolyze palmitate from the several S-palmitoylated protein substrates. Interestingly, PPT2 deficiency in a recent transgenic mouse model is associated with a form of neuronal ceroid lipofuscinosis, suggesting that PPT1 and -2 perform non-redundant roles in lysosomal thioester catabolism. In the current paper, we present the crystal structure of PPT2 at a resolution of 2.7 Å. Comparisons of the structures of PPT1 and -2 show very similar architectural features; however, conformational differences in helix α4 lead to a solvent-exposed lipid-binding groove in PPT1. The limited space between two parallel loops (β3-αA and β8-αF) located immediately above the lipid-binding groove in PPT2 restricts the binding of fatty acids with bulky head groups, and this binding groove is significantly larger in PPT1. This structural difference accounts for the ability of PPT2 to hydrolyze an unbranched structure such as palmitoyl-CoA but not palmitoylcysteine or palmitoylated proteins. Furthermore, differences in fatty acid chain length specificity of PPT1 and -2, also reported here, are explained by the structure and may provide a biochemical basis for their non-redundant roles.

Facile acid catalyzed ring cleavage of N-acylated lactams

Abstract N-Alkoxycarbonyl and N-arylsulfonyl lactams ( 1a - e ) were prepared and converted to the corresponding acyclic products ( 2a - h ) by acid catalyzed cleavage of the lactam ring in good yields and excellent regioselectivity.

Preparation of 7-Arylmethyl-1H-pyrrolo[3,4-c]pyridine-1,3-(2H)-diones and α-aryl-3-hydroxy-5-pyridylacetonitriles using arynic methodology

Abstract 7-Arylmethyl-1H-pyrrolo[3,4-c]pyridine-1,3-(2H)-diones and α-aryl-3-hydroxy-5-pyridylacetonitriles can be prepared in modest yields from the respective reactions of 5-bromonicotinamide and 5-chloro-3-pyridinol with arylacetonitriles and LDA.

Conversion of thiocarbamates to carbamates

Abstract Treatment of methyl N-methylthionocarbamate (2a) with a catalytic amount of iodine or conc. H2SO4 results in the unexpected formation of the isomer, methyl N-methylthiolcarbamate (3a) in 90% yield. This has subsequently been transformed into methyl N-methylcarbamate (4a), by sodium methoxide. A curious transformation of methyl N-methyldithiocarbamate (1a) to (4a) on prolonged treatment with sodium methoxide is also discussed.

The reaction of 2-chloro-4-nitrophenol and the isomeric chloronitrobenzenes with LDA under aryne-forming conditions

Abstract The unprecedented base-initiated generation of a nitrobenzyne and subsequent addition of preformed arylacetonitrile anion nucleophiles is reported. In all cases, 2-amino-5-nitro-3-benzo[b]furans are obtained as major product with small amounts of 3-arylmethyl-2-cyano-4-nitrophenols. A mechanism involving ring closure of phenoxide and nitrile groups of the initial aryne-nitrile anion adduct is proposed to account for the formation of the benzofurans. The three isomeric chloronitrobenzynes, however, do not give aryne products when treated with LDA, but rather are reduced to the corresponding bis-dichloroazoxybenzenes.

One-step synthesis of 1-hydroxymethyl-6-aryl-2H-anthra[9,1-bc]furans, 6-benzyl-2-hydroxymethylbenzonitriles, and 3-arylmethyl-4-methylphthalimides

Abstract Titled compounds were prepared by the reaction of 2-chlorobenzyl alcohol or 2-bromobenzamide with arylacetonitriles in the presence of LDA. A mechanism in terms of an initial tandem rearrangement pathway followed by appropriate cyclization processes is proposed.

The synthesis of 2-arylmethyl-6-hydroxybenzenecarbonitriles from the base-mediated aryne reaction of bromophenols with arylacetonitriles

A facile one-step synthesis of 2-arylmethyl-6-hydroxybenzenecarbonitriles from readily available arylacetonitriles is described. The substitution pattern was verified by X-ray diffractometric analysis of 2-benzyl-6-hydroxybenzonitrile. An explanation of the addition to and reactivity of the 2,3-didehydrophenoxide intermediate, which is supported by DFT calculations, is presented.