Showing papers by "George M. Whitesides published in 1987"

Regeneration of nicotinamide cofactors for use in organic synthesis.

Abstract: The high cost of nicotinamide cofactors requires that they be regeneratedin situ when used in preparative enzymatic synthesis. Numerous strategies have been tested forin situ regeneration of reduced and oxidized cofactors. Regeneration of reduced cofactors is relatively straightforward; regeneration of oxidized cofactors is more difficult. This review summarizes methods for preparation of the cofactors, factors influencing their stability and lifetime in solution, methods for theirin situ regeneration, and process considerations relevant to their use in synthesis.

Structure-Reactivity Relations for Thiol-Disulfide Interchange

Abstract: Equilibrium constants were determined for thiol-disulfide interchange between 36 di- and trithiols and the disulfides derived from either 2-mercaptoethanol or dithiothreitol. Reactions were conducted in methanol-d,/aqueous buffer (pH 7) or methanold, at 25 "C, using NMR spectroscopy to follow the reactions. These data were used to rank the dithiols in terms of reduction potential and to infer the structure of the disulfides formed from them on oxidation. There is a general correlation between the reducing ability of the dithiol and the size of the disulfide-containing ring formed on oxidation: dithiols that form six-membered rings are most strongly reducing (K = 103-105 M with respect to oxidized 2-mercaptoethanol); five- and seven-membered rings are approximately 1 order of magnitude less reducing. Compounds resembling 1,2-ethanedithiol form cyclic bis(disu1fide) dimers in relatively dilute solutions (- 1 mM) but polymerize at higher concentrations. Other classes of dithiols form polymers on oxidation.

Membrane-enclosed enzymic catalysis (MEEC): a useful, practical new method for the manipulation of enzymes in organic synthesis

Abstract: We wish to describe a useful technique for the efficient manipulation of enzymes in organic synthesis,3 in which the enzyme in soluble form is enclosed in commercially available dialysis membranes. We have tested this technique (membrane-enclosed enzymatic catalysis, MEEC) in a number of representative enzyme-c talyzed reactions (Scheme I)4-t2 and have found that it combines the simplicity of use of soluble enzymes with certain of the advantages of immobilizd enzymes. This technique may not be applicable to all enzymes, but it provides the simplest and most effective methods of using many of them in organic synthetic applications. Enzyme-catalyzed reactions can use either soluble or immobilized enzymes.l3'la Procedures based on soluble enzymes are, in general, more @nvenient than those using immobilized enzymes, but the enzymes are not easily recovered for reuse, and their lifetime may be shortened by shear or interfacial deactivation. Immobilization allows the separation and reuse of enzymes and often protects them from deactivation by organic cosolvents, shear, interfacial adsorption, and proteases,l5'16 but it is experimentally inconvenient and even under ideal conditions can qruse significant deactivation of sensitive proteins.

The Reactivity of Carboxylic Acid and Ester Groups in the Functionalized Interfacial Region of 'Polyethylene Carboxylic Acid' (PE-CO2H) and Derivatives: Differentiation of the Functional Groups into Shallow and Deep Subsets Based on a Comparison of Contact Angle and ATR-IR Measurements

Abstract: We have differentiated the carboxylic acid groups present in the functionalized interface of “polyethylene carboxylic acid” (PE4O2H, a material prepared from low-density polyethylene film by reaction with aqueous chromic acid) into two subsets: those sufficiently close to the surface of the polymer to influence its wettability by water and those too deep to do so. This differentiation was accomplished by taking advantage of differences in rates of esterification of carboxylic acid groups in different regions of the functionalized interface and of differences in rates of hydrolysis of ester groups derived from them. The subset of functional groups influencing wettability comprises <30% of the total groups present in the functionalized interface and appears to be homogeneous in its chemical reactivity. The remaining groups (-70% of the total) do not directly influence wettability and appear to become less reactive with increasing depth in the polymer. The surface and subsurface carboxylic acid and ester moieties are both less reactive in hydrolysis and formation reactions than are these groups in organic molecules in solution. Reactivities of the interfacial functional groups depend on structure in ways having no analogy in reactions in solution. For example, the rate of base-catalyzed hydrolysis of esters present in the functionalized polymer interface is a strong function of the length of the alcohol component of the ester: n-octyl esters are more than 20000 times less reactive than methyl esters. We have also explored the acidity of the interfacial carboxylic acid groups using ATR-IR spectroscopy and contact angle measurements as probes. Both the local polarity of the environment of individual carboxylic acid groups and charge-charge interactions between carboxylate anions appear to be important in determining acidity. Comparisons of wettability of samples containing different proportions of carboxylic acid, carboxylate anion, and ester groups indicate that wettability is particularly sensitive to low concentrations of carboxylate anion. We hypothesize that this sensitivity reflects a limited ability of the functionalized interfacial region to reconstruct-perhaps only by small-amplitude rotations of its constituent carboxylate ions-in a way that minimizes its free energy by maximizing the number of these hydrophilic moieties in direct contact with the polar, aqueous liquid phase.

Determination of enantiomeric purity of polar substrates with chiral lanthanide NMR shift reagents in polar solvents

Abstract: The proton NMR spectra of 19 1,2and l,3-dioxygenated compounds were studied in deuteriated acetonitrile, acetone, and chloroform in the presence of chiral lanthanide NMR shift reagents tris(ll-((heptafluoropropyl)hydroxymethylene)-d-camphorato)europium(III) (1) and tris(((trifluoromethyl)hvdroxymethylene)-d-camphorato)europium(III) (2). Enantiotopic OH, CH, and CH3 NMR resonances were best resolved in acetonitrile; line broadening obscured the scalar coupling. Enantiomeric excesses as high as 98Vo can be determined for 16 of these compounds in this polar solvent.

Measurement of thiol-disulfide interchange reactions and thiol pKa values.

Abstract: Publisher Summary Thiol-disulfide interchange (SH/S2 interchange) reactions involving proteins are important in a number of biochemical processes including formation and cleavage of structural cystines, control of enzyme activities by reversible redox reactions of enzyme thiols and disulfides, and redox processes requiring thiols. The reaction is mechanistically simple: it involves initial ionization of thiol to thiolate anion, followed by nucleophilic attack of thiolate anion on the sulfur-sulfur bond of the disulfide. There are three types of parameters that must be determined to characterize fully a SH/S2 interchange reaction: (1) the rates at which the displacement steps occur, (2) the values of pKa of the participating thiols, and (3) the positions of the equilibria between the thiol (thiolate) and disulfide species. The chapter discusses the characteristics of each of these parameters and describes methods of determining them. The methods are useful for biologically relevant thiols and cysteine groups in proteins.

[25] Enzymatic regeneration of adenosine 5′-triphosphate: Acetyl phosphate, phosphoenolpyruvate, methoxycarbonyl phosphate, dihydroxyacetone phosphate, 5-phospho-α-d-ribosyl pyrophosphate, uridine-5′-diphosphoglucose

Abstract: Publisher Summary This chapter describes the best methods available for regeneration of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and details several applications of these methods in enzyme-catalyzed syntheses In particular, it give procedures for synthesis of three phosphate donors—acetyl phosphate, phosphoenolpyruvate, and methoxycarbonyl phosphates—and apply these reagents to syntheses of sn -glycerol 3-phosphate, dihydroxyacetone phosphate, glucose 6-phosphate, 5-phospho-α-D-ribosyl pyrophosphate, and uridine-5'-diphosphoglucose Three procedures for the enzymatic regeneration of ATP are presently available, which are useful in practical-scale organic synthesis One is based on acetyl phosphate (AcP) as the phosphorylating agent and acetate kinase as the catalyst; the second uses phosphoenolpyruvate (PEP) and pyruvate kinase; and the third uses methoxycarbonyl phosphate [CH3OC(O)OPO3 2-, MCP] and acetate kinase The advantages and disadvantages of each method are summarized in the chapter Both pyruvate kinase and acetate kinase have high specific activity and show excellent stability in immobilized form Pyruvate kinase is currently the less expensive enzyme Further, it is effective for regeneration of ATP from ADP at lower concentrations of ADP than is acetate kinase, because the Michaelis constant for pyruvate kinase is lower than that for acetate kinase In practice, for most synthetic applications, either acetyl phosphate/acetate kinase or phosphoenolpyruvate/pyruvate kinase is used for the regeneration of ATP

Fumarase-catalyzed synthesis of L-threo-chloromalic acid and its conversion to 2-deoxy-D-ribose and D-erythro-sphingosine

Abstract: These enzyrnes are indisputably valuable but have certain disadvantages, among which is often imperfect enantioselectivity. The standards in enantioselective synthesis are now very high: for most applications, an enantiomeric excess (ee) in an enantioselective step of less than 90% is essentially useless, and values of ee >99% are very desirable.a It is often difficult to achieve broad substrate selectivity and high enantioselectivity simultaneously. This difficulty poses a strategic question for the synthetic chemist: Is it better to use a structurally \"efficient\" intermediate with modest enantiomeric purity in a synthetic

Enzymatic routes to enantiomerically enriched 1-butene oxide

Abstract: This paper compares several routes to enantiomerically enriched l-butene oxide (1) in which resolution is achieved by using an enzymatic reaction (Scheme I). This research had two objectives: to enumerate several general strategies that can be followed in practical, enzvme-based routes to enantiomerically enriched epoxides and to compare the utility of these routes in the particular case of compound 1. In general, we restrict our account to reactions that proceed with high values of enantiomeric excess (ee) and that have the potential for preparing 50-g quantities of product. Enantiomerically enriched epoxides are useful synthons in chiral synthesis. General routes are now available to only epoxy alcohols and analogues of these substances.2'3 All reactions were conducted on scales that generated 1-5 g of I as product. The chemical yields reported in Scheme I are oueroll yields for conversion of the substrate for the enzymatic reaction to 1; these yields are not optimized. We established the optical purity of I by 1H NMR analysis in the presence of Eu(hfc)r.a With careful calibration, this method can detect a 1To enantiomeric impuri ty ( i .e. , 98% ee). Acylase I (EC 3.5.1.14) is commercially available, inexpensive, and stable. It hydrolyzes a range of Nacyl-a-amino acids with high (>99% ee) enantioselectivity.s Both enantiomers are easily recovered, and the reaction can be run virtually to completion. In resolutions of 2aminobutanoic acid, the reaction stops spontaneously after hydrolysis of the S enantiomer is complete, and losses of material occur entirely in the recovery and purification of product. Conversion of the 2-amino acid via the corresponding 2-chloro acid and chlorohydrin to the epoxide preserves chirality well.6

Combined microbial/chemical synthesis of (+)-(R)-methyloxirane having high enantiomeric excess

Abstract: Synthese par fermentation du glucopyranose par Clostridium thermosaccharolytycum suivie de transformation du (−)-(R)-propanediol-1,3 obtenu par HBr dans l'acide acetique puis par le pentanolate-1 de potassium dans le pentanol-1

Enzyme-catalyzed synthesis of nucleoside triphosphates from nucleoside monophosphates

Abstract: Syntheses of adenosine 5′-triphosphate (ATP) from adenosine 5′-monophosphate (AMP) and ribavirin 5′-triphosphate (RTP) from ribavirin 5′-monophosphate (RMP) (1) were performed using enzymes as catalysts. Synthesis of ATP is based on acetyl phosphate as the phosphate donor, and acetate kinase (Bacillus stearothermophilus, EC 2.7.2.1), adenylate kinase (porcine muscle, EC 2.7.4.3), and inorganic pyrophosphatase (yeast, EC 2.6.1.1) as the catalysts. Three reactions on a 150-mmol scale provided ATP as its barium salt in 82% yield and 67% purity. Synthesis of RTP used phosphoenol pyruvate (PEP) as the phosphate donor, and pyruvate kinase (rabbit muscle, EC 2.7.1.40) and adenylate kinase (rabbit muscle) as the catalysts. A gram-scale reaction provided RTP as its barium salt in 93% yield and 97% purity. This work demonstrates the utility of the autoxidationresistant acetate kinase fromB. stearothermophilus, the value of pyrophosphatase in controlling the level of pyrophosphate in the reactions and the ability of adenylate kinase to accept at least one substrate other than a derivative of adenosine.

Limited nerve impulse blockade by "leashed" local anesthetics.

Abstract: To measure the depth of the local anesthetic binding site within the neuronal membrane, biotin-containing polyethylene glycols having zero, three, and six ethylene glycol subunits were added to the p-amino termini of tetracaine and procaine, thereby interposing a pharmacologically inert "spacer" molecule between the local anesthetic and the biotin moiety. These biotinyl-local anesthetic derivatives produced "tonic" inhibition of the compound action potential of split, desheathed frog sciatic nerves in a concentration-dependent, reversible manner. However, no inhibition of the action potential occurred when sufficient avidin, a 66,000-MW protein that binds four biotins, was present to bind and anchor the biotin-containing end of each derivative outside the plasma membrane. Increasing the "leashed" anesthetic derivative's concentration to 4 times that which reduced impulse height by 50% in the absence of avidin still produced no detectable block when equimolar avidin was present. Apparently, the "spacer" in the derivative compound was too short to permit the avidin-complexed anesthetic to reach its site of action on the sodium channel. In a similar fashion, the local anesthetic derivatives produced "use-dependent" block when drug-treated nerves were stimulated at 40 Hz in the absence of equimolar avidin, but failed to produce "use-dependent" block when equimolar avidin was present. In common with others, we assume that tertiary amine local anesthetics may reach their binding site via hydrophobic (transmembrane) pathways without necessarily entering the cytoplasm. Thus, since our longest local anesthetic derivative, that containing six ethylene glycol subunits, placed the local anesthetic group a maximum of 15-18 A from the surface of the avidin moiety, we conclude that the local anesthetic binding site for block of sodium channels of amphibian nerve must be greater than or equal to 15 A from the outer surface of the plasma membrane.

Protein synthesis in cell-free reticulocyte lysates on multi-hour incubation.

Abstract: Cell-free protein synthesis in rabbit reticulocyte lysate translation mixtures has been studied during multi-hour incubations. In an impaired lysate obtained from cells stored at 0 degrees C before lysis, and showing a low initial rate of synthesis, translation could be stimulated by a factor of 4 by including RNase inhibitor and additional ATP and GTP. In translation mixtures prepared from normal lysates, protein synthesis could be improved by approximately 50% by the addition of excess GTP. The observed increases in protein synthesis were obtained by improved maintenance of the initial rate of synthesis.

What, if Anything, Can Chemistry Offer to Fracture Mechanics?

Abstract: Chemistry is primarily concerned with the structures and properties of bonds between atoms and with relating these bonds and their properties to the structure, reactivity, and other properties of molecules. The principle concerns of chemistry have thus been individual molecules and molecular reactivity. Attention is now slowly turning to the structure, reactivity, and properties of solids: that is, to collections of molecules and to very large molecules. Why slowly? Certainly not because the problems in the solid state, or more broadly in materials science, are unimportant. The objectives of rationalizing properties such as tensile modulus, fracture strength, corrosion resistance, electrical conductivity using atomic-level structural information, dielectric constant, and thermal conductivity are clearly simultaneously immensely interesting scientifically and important technologically.1,2 They are also, unfortunately, very difficult to attain. Moreover, the difficulty often is of an annoying sort--that is, it is associated with defects. The properties of large, regular ensembles of atoms and bonds can often be calculated with a high degree of precision using standard techniques of statistical mechanics.3 What cannot be readily calculated is the often critical influence of unknown impurities or defects of unknown composition and structure on the properties of interest.

Aldolase-Catalyzed Synthesis of Complex C8 and C9 Monosaccharides.

Abstract: Fructose-1,6-diphosphate aldolase (E.C. 4.1.2.13) from rabbit muscle catalyzes the stereospecific aldol condensation of dihydroxyacetone phosphate with a variety of pentose-5-phosphates and hexose-6-phosphates. The aldol products can be dephosphorylated with acid phosphatase (E.C. 3.1.3.1) and the resulting free carbohydrates are peracetylated. These C8 and C9 sugars can serve as precursors to and analogs of KDO, sialic acids and related substances.

cis-(Bis(dicyclohexylphosphino)ethane)platinum(0) Reacts with Unactivated Carbon-Hydrogen Bonds.

Abstract: Although platinum(0) is centrally important in heterogeneous catalytic reforming of petroleum, the only soluble platinum complexes that react with saturated hydrocarbons are platinum chlorides and acetates. In particular, and by contrast with iridium, rhodium, and the other transition metals that have provided the basis for the recent major advances in carbon-hydrogen bond activation, no phosphine-stabilized platinum species has been reported that reacts intermolecularly with unactivated C-H bonds, although intramolecular reaction is facile. Here, the report that thermal reductive elimination of neopentane from cis-hydrido-neopentyl(bis(dicyclohexylphosphino)ethane)platinum(II) (1) produces the reactive intermediate (bis(dicyclohexylphosphino)-ethane)platinum(0) (2) and that 2 reacts with C-H bonds in saturated and unsaturated hydrocarbons.