James F. Marecek

Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives.

Controlled modification of the carboxylic acid moieties of hyaluronic acid with mono- and polyfunctional hydrazides leads to biochemical probes, biopolymers with altered physical and chemical properties, tethered drugs for controlled release, and crosslinked hydrogels as biocompatible scaffoldings for tissue engineering. Methods for polyhydrazide synthesis, for prodrug preparation, for hydrogel crosslinking, and for monitoring biodegradation are described.

Kinetics of interfacial catalysis by phospholipase A2 in intravesicle scooting mode, and heterofusion of anionic and zwitterionic vesicles.

In this and the following three papers we examine the kinetics of action of pig pancreatic phospholipase A2 on vesicles of anionic phospholipids without any additives. The results provide the first unequivocal demonstration of interfacial catalysis in intravesicle scooting mode. In this paper we describe the conditions in which the action of pig pancreatic phospholipase A2 on DMPMe (ester) vesicles in the absence of any additive commences without a latency. Under these conditions the free monomer substrate concentration is insignificant; the bilayer enclosed vesicle organization remains intact even when all the substrate in the outer monolayer has been hydrolyzed; the rate of intervesicle exchange and the rate of transbilayer movement (flip-flop) of molecules is negligibly slow; and the rate of fusion of vesicles is insignificant. Thus an enzyme molecule bound to one vesicle hydrolyzes all the DMPMe molecules in the outer monolayer of the vesicle by a first-order process with a rate constant of 0.6 per min at 30 degrees C; or viewed another way, one enzyme molecule in a DMPMe vesicle can hydrolyze all the available substrate molecules at the rate of 3000 per min. At low anion concentrations excess substrate vesicles are not hydrolyzed unless the rate of intervesicle exchange of the bound enzyme is stimulated by anions in the aqueous phase. Higher calcium concentrations promote not only homofusion of DMPMe vesicles but also heterofusion of DMPMe and DMPC vesicles. It is proposed that calcium-induced isothermal lateral phase separation in DMPMe vesicles induces defects in the bilayer organization, and such defects are the sites for phospholipase A2 binding and for heterofusion with DMPC (ester) vesicles which do not have such sites.

Slow aggregation of buckminsterfullerene (C60) in benzene solution

Abstract Solution behavior of buckminsterfullerene (C60) in benzene was investigated by laser light scattering using an incident wavelength of 790 nm. C60 was found to aggregate slowly even in fairly dilute solution concentrations ranging from 1.39 to 2.11 mg/ml at room temperatures (23–26°C). The aggregation of C60 in benzene solution was reversible. The C60 aggregates could be dispersed simply by hand shaking the solution. The light-scattering intensity results indicated that the structure of C60 aggregates could be described as a fractal with dimension of 2.10 and the aggregation kinetics exhibited essentially an exponential behavior.

Hydrolysis of peptides in seawater and sediment

Abstract Protein hydrolysis and subsequent peptide production appear to be the links between degradation of protein and production of free amino acids in the marine environment. This model has not yet been fully demonstrated because neither presence of peptides nor peptide hydrolysis has been directly measured in seawater or sediments. Fluorescent Lucifer Yellow (LYA)-derivatives of several peptides were synthesized and tested for use as models in an investigation of peptide hydrolysis in seawater and sediment. We demonstrated that these LYA-derivatives behave to some degree as the natural peptides by showing that LYA-dialanine effectively competes with dialanine (ala 2 ) for the active sites of microbial hydrolytic enzymes found in seawater. LYA-derivatives of ala 2 , ala-leu and ala 4 hydrolyzed to smaller peptides or free amino acids in both seawater and sediments. In seawater, hydrolysis of the longer peptide, LYA-ala 4 was 90 × faster than hydrolysis of LYA-ala 2 and 30 × faster than LYA-ala-leu. In sediments, rapid disappearance of the initially-added substrate LYA-ala 4 from pore waters was followed by slower production of LYA-ala 3 , LYA-ala 2 and LYA-ala. Hydrolysis was not random; preferential cleavage of certain peptide bonds occurred.

Solution behavior of buckminsterfullerene (C60) in benzene

Solution characterization of buckminsterfullerene C60 in benzene was carried out by laser light scattering. Static and dynamic light scattering (SLS and DLS) experiments were accomplished by using an incident laser beam of 790 nm wavelength, and a fiber optic probe which was coupled to a RCA C31034 photomultiplier tube sensitive to the near ir wavelength. The effective hydrodynamic radius of C60 in benzene solution determined by using DLS was 6.4±0.1 A, in close agreement with the distance measured from the center of the solvated benzene to the center of the benzene‐solvated C60 (C60⋅4C6H6):6.7∼6.8 A as evaluated from the single crystal structure by x‐ray diffraction. C60 was found to aggregate slowly even in fairly dilute solution concentrations ranging from 0.78 to 1.39 mg/mL at temperatures 23∼27 °C. Based on the high specific refractive index increment of C60 in benzene, the molar mass of the C60 aggregates could be estimated. Together with the radius of gyration, the hydrodynamic radius, and its size...

Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2.

Action of pig pancreatic phospholipase A2 on vesicles of over 50 synthetic 1,2-diacylglycerol-3-phosphate derivatives and analogs is examined in the absence of any additives. In general, shorter acyl chains and small substituents on the phosphate make a better substrate, while phospholipids with large apolar substituents are not hydrolyzed. The interfacial turnover rate constant for scooting kinetics, ki, for the various phospholipids were from less than 0.1 to 1 per min. Intervesicle exchange of the bound enzyme is faster in vesicles of phospholipids with larger polar substituents, and it is promoted in the presence of anions like chloride, sulfate and thiocyanate. These factors lower the residence time of the enzyme on the bilayer and therefore effectively decrease the rate of hydrolysis. The apparent Km for the enzyme in the interface of anionic phospholipids in the presence of salts is in the 40 to 100 microM range which is 3- to 7-times larger than the dissociation constants for the bound enzyme measured by fluorescence enhancement of Trp-3. The quantum yield of the bound enzyme in vesicles of the various lipids is found to be up to 4-fold different. It is suggested that this difference is due to the E* + S to E*S equilibrium, where E*S has higher fluorescence intensity. The role of calcium in generating the enzyme binding site at the anionic interface, the role of anion anchoring site on the enzyme, and the relationship between the catalytic efficiency and the fluorescence quantum yields are discussed.

Non-redox-active lipoate derivates disrupt cancer cell mitochondrial metabolism and are potent anticancer agents in vivo

We report the analysis of CPI-613, the first member of a large set of analogs of lipoic acid (lipoate) we have investigated as potential anticancer agents. CPI-613 strongly disrupts mitochondrial metabolism, with selectivity for tumor cells in culture. This mitochondrial disruption includes activation of the well-characterized, lipoate-responsive regulatory phosphorylation of the E1α pyruvate dehydrogenase (PDH) subunit. This phosphorylation inactivates flux of glycolysis-derived carbon through this enzyme complex and implicates the PDH regulatory kinases (PDKs) as a possible drug target. Supporting this hypothesis, RNAi knockdown of the PDK protein levels substantially attenuates CPI-613 cancer cell killing. In both cell culture and in vivo tumor environments, the observed strong mitochondrial metabolic disruption is expected to significantly compromise cell survival. Consistent with this prediction, CPI-613 disruption of tumor mitochondrial metabolism is followed by efficient commitment to cell death by multiple, apparently redundant pathways, including apoptosis, in all tested cancer cell lines. Further, CPI-613 shows strong antitumor activity in vivo against human non-small cell lung and pancreatic cancers in xenograft models with low side-effect toxicity.

Coordination of magnesium with adenosine 5′-diphosphate and triphosphate

Abstract 31 P NMR chemical shifts of salts of adenosine 5′-triphosphate and diphosphate: ATPH 2− 2 2(Me 4 N + ) · H 2 O, ATPH 2− 2 2 Na + · 3.5 H 2 O, ATPH 2− 2 Mg 2+ · 4 H 2 O, ATPH 2− 2 Ca 2+ · 2 H 2 O, ADPH 2− 2(Me 4 N + ) · H 2 O and ADPH 2− Mg 2+ · 4 H 2 O have been measured in 0.02 M 2 H 2 O solutions at 145.7 MHz (22° C) at constant p 2 H values (8.20 and 6.20). The results are compared with those obtained from salts of adenosine 5′-monophosphate and other simpler phosphomonoesters, e.g. AMP 2− 2(Me 4 N + ), AMP 2− Mg 2+ , AMPH − Me 4 N + and (AMPH − ) 2 Mg 2+ . It is concluded that the effects exerted by Mg 2+ and Ca 2+ on the 31 P NMR shifts of dipoly- and tripolyphosphates relative to monovalent cations are due mainly to changes in conformation of the polyphosphate chain rather than to purely electronic factors associated with the binding of divalent cations to the phospho-oxyanions. The data are consistent with the existence of the following complexes at p 2 H 8.20: (MgP α P β )ADP − and (MgP α P γ )ATP 2− af (MgP α P β )ATP 2− af (MgP β P γ )ATP 2− with the latter equilibrium relatively fast in the NMR time scale. Monoprotonation of the terminal phosphate appears to weaken the Mg 2+ -polyphosphate binding, particularly at P β of MgADPH and at P β and P γ of MgATPH − . The Mg 2+ -polyphosphate binding weakens further at p 2 H 3.70, i.e. in MgATPH 2 . Possible implications of the results in the mechanism of actomyosin Mg 2+ -ATPase in muscle contraction are discussed.

The affinity of phospholipase A2 for the interface of the substrate and analogs

In the intravesicle scooting mode of interfacial catalysis, the interfacial complex E*S is formed by the interaction of the membrane bound phospholipase A2 (E*) with the substrate monomer (S) in the interface. In the presence of nonhydrolyzable substrate analogs (I) the kinetics of interfacial catalysis is modified. If phospholipase A2 is added to a mixture of the vesicles of L-DMPMe ester and of DTPMe ether or D-DMPMe ester, the extent of hydrolysis, A, decreases and the interfacial scooting rate constant, ki, remains unchanged. On the other hand, when the enzyme is added to the vesicles prepared from premixed L-DMPMe ester with D-DMPMe ester or L-DTPMe ether, ki decreases but A remains constant. Qualitatively, these results are in excellent accord with the Scheme I for interfacial catalysis. However, a quantitative departure has been noted, which suggests that the interfacial dissociation constant for E*S is larger than that for E*I. These results are interpreted to suggest that the catalytic rate constant for decomposition of E*S to E* + P is larger than the rate constant for decomposition of E*S to E* + S. Broader implications of the scooting mode of interfacial catalysis are discussed.

Synthesis of all optically active spermine macrocycle, (S)-6-(hydroxymethyl)-1,5,10,14-tetraazacyclooctadecane, and its complexation to ATP

The title compound was prepared in high yield from L-ornithine via a Richman-Atkins type macrocyclization. 31P-NMR binding studies indicate formation of a 1:1 complex of the protonated macrocycle with ATP.