Proteomics of rat liver Golgi complex: minor proteins are identified through sequential fractionation

The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.     

The preparation of quinolines and related fused-ring heterocycles from the dianions of benzoylacetone,certain cyclic ketone oximes,or certain substituted hydrazones

C(α),O-Dilithiooximes, C(α),N-dilithiobenzoylhydrazones, or C(α),N-dilithiocarboalkoxyhydrazones were prepared in an excess of lithium diisopropylamide (LDA) and condensed with 2-aminobenzophenones, or isatoic anhydrides to give intermediates that were treated with aqueous acid, which caused their hydrolysis, cyclodehydration and/or linear dehydration to give products which were substituted quinolines or related fused-ring heterocycles (e.g., cycloheptaquinolines). Dilithiobenzoylacetone was condensed with 2-aminobenzophenones, which was followed by acid cyclodehydration to substituted 2-phenacylquinolines.     

The effect of pressure on order destruction and order creation in linear or branched alkane mixtures

The pressure dependence of the excess enthalpy H ^E , dH ^E /dP, has been calculated from experimental excess volumes V ^E and dV ^E /dT using dH ^E /dP=V ^E –TdV ^E /dT. dH ^E /dP at zero pressure are reported at 25°C and equimolar concentration for the mixtures: cyclohexane with the series of normal alkanes (n-C _n , where n=6,8,10,12,14 and 16) and with the series of highly branched alkanes (br-C _n , where n=6,8,12 and 16), benzene, toluene and p-xylene +n-C _n and 1-chloronaphthalene +n-C _n and br-C _n . Experimental and Flory theory dH ^E /dP values are in good agreement for the whole cyclohexane +br-C _n series. For the n-C _n series, dH ^E /dP becomes increasingly positive deviating from the Flory predictions. This discrepancy is due to the presence of short-range orientational order in the higher n-C _n pure liquids which makes dH/dP more negative and which, upon mixing, is destroyed producing a positive contribution to dH ^E /dP not accounted for by the theory. The discrepancy between theoretical and experimental dH ^E /dP is large for benzene, but progressively smaller for toluene, p-xylene and 1-chloronaphthalene. These results are consistent with creation of order between the aromatic plate-like molecule and the long n-C _n in solution. For 1-chloronaphthalene +n-C _n , this order creation process produces a negative contribution to dH ^E /dP which balances the positive order-destruction contribution originated by the rupture, upon mixing, of short-range orientational order in pure n-C _n .     

A convergent solution-phase synthesis of the macrocycle Ac-Phe-[Orn-Pro-D-Cha-Trp-Arg], a potent new antiinflammatory drug

Relatively few cyclic peptides have reached the pharmaceutical marketplace during the past decade, most produced through fermentation rather than made synthetically. Generally, this class of compounds is synthesized for research purposes on milligram scales by solid-phase methods, but if the potential of macrocyclic peptidomimetics is to be realized, low-cost larger scale solution-phase syntheses need to be devised and optimized to provide sufficient quantities for preclinical, clinical, and commercial uses. Here, we describe a cheap, medium-scale, solution-phase synthesis of the first reported highly potent, selective, and orally active antagonist of the human C5a receptor. This compound, Ac-Phe[Orn-Pro-d-Cha-Trp-Arg], known as 3D53, is a macrocyclic peptidomimetic of the human plasma protein C5a and displays excellent antiinflammatory activity in numerous animal models of human disease. In a convergent approach, two tripeptide fragments Ac-Phe-Orn(Boc)-Pro-OH and H-d-Cha-Trp(For)-Arg-OEt were first prepared by high-yielding solution-phase couplings using a mixed anhydride method before coupling them to give a linear hexapeptide which, after deprotection, was obtained in 38% overall yield from the commercially available amino acids. Cyclization in solution using BOP reagent gave the antagonist in 33% yield (13% overall) after HPLC purification. Significant features of the synthesis were that the Arg side chain was left unprotected throughout, the component Boc-d-Cha-OH was obtained very efficiently via hydrogenation of d-Phe with PtO(2) in TFA/water, the tripeptides were coupled at the Pro-Cha junction to minimize racemization via the oxazolone pathway, and the entire synthesis was carried out without purification of any intermediates. The target cyclic product was purified (>97%) by reversed-phase HPLC. This convergent synthesis with minimal use of protecting groups allowed batches of 50-100 g to be prepared efficiently in high yield using standard laboratory equipment. This type of procedure should be useful for making even larger quantities of this and other macrocyclic peptidomimetic drugs.     

A polymer-supported rhodium catalyst that functions in polar protic solvents

Highly cross-linked macroporous polymers are excellent supports for heterogenizing rhodium alkene hydrogenation and hydroboration catalysts. The permanent pore structure of the support enables high conversions and excellent yields with minimal workup (filtering). These heterogenized catalysts can be reused, and due to the permanent pore structure, they function in a broad range of solvents including polar protic. Control experiments reveal that catalysis occurs exclusively within the polymer matrix, and not due to leached catalyst.     

The Chemistry of Some Isopolar Analogues of Phosphoric and Pyrophosphoric Acids

Abstract

We have previously established that the α-fluorination of alkanephosphonates provides analogues of phosphate esters which have improved ‘isopolarity’ relative to simple alkanephosphonates.¹ This property is manifest, inter alia, in enhanced acidity and in the upfield shift for the ³¹P n.m.r. resonance. Indeed, for a range of halomethanephosphonic acids we have found the relationship “δ_P=9.61 (pK_a2 - 4.59) ppm” gives an excellent correlation between these parameters. In this context, the properties of CF₂CIPO(OR)₂ species, derived from the Michaelis-Becker reaction of dialkyl phosphonates with Freon 22, CF₂Cl₂, will be described.