N10-Formyltetrahydrofolate is the formyl donor for glycinamide ribotide transformylase in Escherichia coli.

Glycinamide ribotide transformylase from Escherichia coli was obtained free of N5,N10-methenyltetrahydrofolate cyclohydrolase activity by DEAE-cellulose chromatography. In reaction mixtures containing this enzyme preparation in potassium maleate buffer, pH 7.2, no detectable interconversion of N5,N10-methenyltetrahydrofolate occurred. Upon addition of glycinamide ribotide, N-formylglycinamide ribotide was formed when N10-formyltetrahydrofolate was present; no formylation occurred in the presence of N5,N10-methenyltetrahydrofolate. A method for the synthesis and purification of glycinamide ribotide is presented.     

The accumulation of glycinamide ribotide by ade3 and ade8 mutants of Saccharomyces cerevisiae

The purine intermediate GAR is present in cell free extracts of $\text{ade3}$ and $\text{ade8}$ mutants of yeast. It is also detectable following acid hydrolysis of extracts of $\text{ade6}$ and $\text{ade7}$ which accumulate FGAR and FGAM respectively. GAR accumulation is repressed by growing cells in high levels of adenine. Neither $\text{ade4}$ nor $\text{ade5}$ accumulate GAR and both prevent accumulation of GAR in $\text{ade3}$ and $\text{ade8}$ and FGAR in $\text{ade6}$. Since $\text{ade3}$ is known to be defective in folate metabolism these results indicate that $\text{ade8}$ is blocked in the conversion of GAR→FGAR.     

Down-regulation of melanin synthesis by a biphenyl derivative and its mechanism

Down-regulation of melanin synthesis is required for recovery of pigmentary disorders and it is known that direct inhibitors of tyrosinase, the key enzyme in melanin synthesis, such as hydroquinone with a phenol structure, suppress melanin synthesis. We screened several phenolic derivatives using B16 melanoma cells and found that a biphenyl derivative, 2,2'-dihydroxy-5,5'-dipropyl-biphenyl (DDB), down-regulated melanin synthesis effectively. Although DDB has a phenol structure, it did not inhibit tyrosinase in vitro, thus we examined its mechanism in detail. Western blotting revealed that the amount of tyrosinase was decreased by DDB, and pulse-chase labeling and immunoprecipitation analysis showed a decrease of mature tyrosinase and acceleration of tyrosinase degradation in its presence. These results suggest that DDB down-regulates melanin synthesis by inhibiting the maturation of tyrosinase, leading to acceleration of tyrosinase degradation.     

One-step synthesis of hypoxanthine from glycinamide and diformylurea

Because of their easy availability and their relative chemical stability, urea, formic acid, and glycine might have played a role in the assembly process of nucleobases. In this paper, a short reaction path is described to prepare hypoxanthine starting from the above mentioned precursors. The formation of hypoxanthine has been verified by high-resolution mass spectrometry with the 15N-labelled urea as starting material, and HPLC analysis. The yield of this condensation reaction has been determined spectrophotometrically.     

On the structural and functional modularity of glycinamide ribonucleotide formyltransferases

Glycinamide ribonucleotide formyltransferases (GARTs) are part of the de novo purine biosynthetic pathway, catalyzing the direct transfer of a formyl group from the tetrahydrofolate cofactor to the glycinamide ribonucleotide substrate. Despite the low amino acid-sequence identity between the GARTs from Escherichia coli and human, their tertiary structures are superimposable. As part of our functional studies of these enzymes, we have investigated the interchangeability of individual protein fragments or modules between the two enzymes and the functional properties of the resulting hybrids. The modular nature of GART facilitated the creation of combinatorial libraries of chimeras between the Escherichia coli and human enzymes, which were functionally selected through complementation of an auxotrophic Escherichia coli strain. From a pool of several dozen sequence distinct hybrids, six in vivo-functional fusion genes were selected, overexpressed, and purified to homogeneity. The kinetic analysis of these constructs and the comparison of their k(cat) and K(M) values to the parental enzymes suggest that the characteristic kinetic properties from the two parents are "modular encoded" and can be exchanged by domain swapping. The chimeras in general, however, are subject to temperature instability and misfolding; thus, they serve primarily as useful candidates for further rounds of optimization.     

Penicilin acylase mediated synthesis of formyl cefamandole

Summary Penicillin acylase-catalyzed synthesis from D,L-methyl mandelate (1c) and the cefalosporin nucleus (2) at pH 6 gives rise preferentially to the L-diastereoisomer (3a) of cefamandole, whereas D,L-O-formyl methyl mandelate (1f) affords exclusively O-formyl cefamandole (cefamandole nafate) (4), in 30–35% yields.     

On the mechanism of CFTR inhibition by a thiazolidinone derivative

The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels inactivate despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.     

Convenient synthesis of human calcitonin and its methionine sulfoxide derivative

The human calcitonin peptide chain was assembled using Fmoc solid-phase peptide synthesis chemistry. The combinations of cleavage Reagent H with trans-[Pt(en)(2)Cl(2)](2+) and Reagents B, K, and R with trans-[Pt(CN)(4)Cl(2)](2-) provide convenient methods for the synthesis of human calcitonin and its methionine sulfoxide derivative; the formation of intramolecular disulfide bonds by the above Pt(IV) oxidants is essentially quantitative.     

A DFT study on the mechanism of the organocatalytic synthesis of a benzoxazine-substituted indolizine derivative

A series of theoretical computations were conducted via density functional theory at the B3LYP/6–31 + G(d,p) level to study the mechanism of the organocatalytic synthesis of a benzoxazine-substituted indolizine derivative. Four possible alternative pathways were considered in this work. The calculated results show that the formation of an N-ylide precursor from 4-dimethylaminopyridine (DMAP) is a key step as it provides the necessary nucleophilic centre for the subsequent H-migration and H-elimination processes. The precursor N-ylide and Schiff base isomers with the most favourable activities in the preliminary work were identified theoretically by analysing the reaction mechanism. The synthetic mechanism to obtain the indolizine derivative was found to be a two-step reaction, with the rate-determining step being the first H migration to form a transition state with a four-membered ring. The catalytic activity of DMAP in the first H-migration step in the overall synthetic process greatly reduces the reaction barrier height. The chiral selectivity of the synthesis is dominated by the spatial geometry of the Schiff base functional group.