Structure-function Relationships in Human Hypoxanthine-guanine Phosphoribosyltransferase （HGPRT） by Random Mutagenesis

Introduction Hypoxanthine guaninephosphoribosyltransferase(HGPRT,EC2.4.2.8)isakeyenzymeofthepurine salvagepathway,whichallowsrecyclingofpurinebases intoDNAandRNA.Itiswidelydistributedinnature andhasbeenstudiedbothinprokaryotesandeu karyotes.Inhumans,acomp…     

Concise synthesis of the core bicyclo[2.2.2]diazaoctane ring common to asperparaline, paraherquamide, and stephacidin alkaloids

A versatile synthesis of the bicyclo[2.2.2]diazaoctane core structure of asperparaline, brevianamide, paraherquamide, and stephacidin natural products is demonstrated. This convergent synthesis relies on an intramolecular hetero Diels-Alder reaction to construct the key tetracycle from a diketopiperazine derived azadiene; which in turn was formed from prolinamide and a pyruvic acid derivative. The stereochemical outcome of the Diels-Alder reaction was found to favor the brevianamide stereochemistry.     

Belamide A, a new antimitotic tetrapeptide from a Panamanian marine cyanobacterium

The isolation and structure elucidation of belamide A from the marine cyanobacterium Symploca sp. is described. Belamide A is a highly methylated linear tetrapeptide with structural analogy to the important linear peptides dolastatins 10 and 15. Disruption of the microtubule network in A-10 cells was observed at 20 μM and displayed classic tubulin destabilizing antimitotic characteristics. The moderate cytotoxicity of belamide A (IC₅₀ 0.74 μM vs HCT-116 colon cancer line) provides new insights into structure-activity relationships for this drug class.     

Spectrophotometric determination of niobium in tantalum metal

A new method for the determination of traces of niobium in tantalum metal has been developed. The niobium is separated from tantalum by solvent extraction with hexone from hydrofluoric acid-hydrochloric acid solution, and from molybdenum and tungsten by solvent extraction with oxine-chloroform solution from ammoniacal citrate solution. The niobium is then determined by the spectrophotometric thiocyanate method.     

Synchronism in the Diels-Alder reaction

Defined as the formation of two new bonds of equal length at the same time, synchronism has been shown to be not possible for the Diels-Alder reaction when the products are not of strict C_s symmetry. This has been done in two steps. First, the SCF -MO transition-state structures for the cyclization reactions of ethylene with cyclopentadiene and furan have been calculated. The activation energies (and heat of reaction) with the STO -3G, 3-21G, and 4-31G basis sets are as follows: with cyclopentadiene 35.0 (?80.7), 30.0 (?32.0), 38.4 (?19.9) kcal/mol; with furan 33.4 (?66.4), 34.6 (?16.5), 42.1 (?8.8) kcal/mol. Then, using a bond-order-bond-length relationship, a value for the tendency toward asynchronism has been calculated for substituted reactants where the two new bonds are arbitrarily held equal. This tendency has been shown in all cases which break strict C_s symmetry. Further, inspection of the atomic-orbital coefficients showed that the substituents cannot be considered as mere perturbations on the pi system in many cases.     

Hetero-tripodal hydroxypyridonate gadolinium complexes: syntheses, relaxometric properties, water exchange dynamics, and human serum albumin binding

The synthesis and relaxometric properties of hetero-tripodal hydroxypyridonate-terephthalamide gadolinium (Gd(3+)) chelates with differing structural features for probing human serum albumin (HSA) interactions are reported. The Gd(3+) complexes are divided into two series. The first series (3-5) features a benzyl derivative connected to the hydroxypyridonate (HOPO) moiety. The second series of complexes (6-10) has the common feature of a poly(ethylene glycol) (PEG) attached to the terephthalamide (TAM) moiety and is nonbenzylated. The water exchange of the complexes is in the fast exchange regime with rates (k(ex)) in the range 0.45-1.11 x 10(8) s(-1). The complexes have a moderate interaction with HSA with association constants (K(A)'s) in the range 0.7-8.6 x 10(3) M(-1). Protein binding results in an enhancement in proton relaxivity from 7.7-10.4 mM(-1) s(-1) (r(1p)) to 15-29 mM(-1) s(-1) (r(1p)(b)). It is concluded that the interaction of the complexes with HSA (i) is enhanced by the presence of benzyl groups, (ii) is entropically driven, and (iii) results in a lower hydration number (q).     

On-chip cell lysis by local hydroxide generation

We present a novel method for on-chip cell lysis based on local hydroxide electro-generation. Hydroxide ions porate the cell membrane, leading to cell lysis. After lysis occurs, hydrogen ions, also generated on chip, react with excess hydroxide, creating a neutral pH lysate and eliminating the need for a wash step. Three different cell types are shown to be effectively lysed by this method: red blood cells, HeLa (human tumor line) and Chinese Hamster Ovary (CHO) cell lines. The release of cytoplasmic molecules from HeLa and CHO cells is demonstrated by monitoring the escape of a membrane impermeant dye from the cytoplasm. In the vicinity of the cathode, the hydroxide concentration is predicted by finite element simulations and shown to fit the lysis rates at different distances from the generating cathode. For flow-through experiments, a second device integrating a mechanical filter with hydroxide generation is fabricated and tested. The purpose of the filter is to trap whole cells and only allow lysate to pass through. The flow rate dependence of hydroxide concentration at the lysis filter is modeled and lysis efficiency is experimentally determined to be proportional to the hydroxide concentration for flow rates from 15 to 30 microl min(-1).