Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1968 Metal ion complex catalysis of amino acid ester hydrolysis Bruce Eugene Leach Iowa State University Follow this and additional works at:https://lib.dr.iastate.edu/rtd Part of theInorganic Chemistry Commons Recommended Citation Leach, Bruce Eugene, "Metal ion complex catalysis of amino acid ester hydrolysis " (1968).Retrospective Theses and Dissertations. 3487. https://lib.dr.iastate.edu/rtd/3487 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please [email protected]. I This dissertation has been microfilmed exactly as received 69-4254 LEACH, Bruce Eugene, 1942- METAL ION COMPLEX CATALYSIS OF AMINO ACID ESTER HYDROLYSIS. Iowa State University, Ph.D., 1968 Chemistry, Inorganic University Microfilms, Inc., Ann Arbor, Michigan METAL ION COMPLEX CATALYSIS OF AMINO ACID ESTER HYDROLYSIS by Bruce Eugene Leach A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILSOPHY Major Subject: Inorganic Chemistry Approved: Signature was redacted for privacy. Work Signature was redacted for privacy. Signature was redacted for privacy. of Graduate College Iowa State University Ames, Iowa 1968 ii TABLE OF CONTENTS Page INTRODUCTION 1 EXPERIMENTAL 20 Instrumentation 20 Materials 22 Preparation and Characterisation of Compounds 25 Equilibrium Measurements 4? Kinetic Measurements 46 RESULTS 52 Amino Acid Ester-N,N-diacetic Acids 52 Amino Acid Esters 81 DISCUSSION AND CONCLUSION 102 Amino Acid Estei-N,N-diacetic Acids 102 Amino Acid Esters 117 BIBLIOGRAPHY 132 ACKNOWLEDGMENTS 1?7 VITA 138 APPENDIX 139 Derivation of a Calculable Expression for the Formation Constant of Amino Acid Esters with [MZ]o 139 Derivation of a Calculable Expression for the Formation Constant of Amino Acids with [MZ]° 141 1 INTRODUCTION Metal ion catalysis of the hydrolysis of a-amino acid esters was discovered in 1952 by Kroll (1). Since that time those reactions have been studied by a number of research groups with hopes of elucidating the nature of the hydrolytic process and the role of metal ions in biological systems. Esters of a-amino acids are an example of a class of impor­ tant biological compounds which are energy-rich. An energy- rich compound has been defined as one whose reaction with a substance commonly present in the environment is accompanied by a large negative free energy change at physiological pH (2). Amino acid esters which are important intermediates in the biosynthesis of proteins, have a standard free energy of hydrolysis which is comparable to that of adenosine triphosphate, ATP. At physiological pH in the absence of metal ions the rate of hydrolysis of amino acid esters is very slow. In general, four reactions must be considered. They involve water and hydroxide ion attack upon the protonated and free base esters. EH + HgO products ^1 EH + OH" products ^2 E + HgO products *3 E + OH" products ^4 2 Conley and Martin (3) have investigated the hydrolysis of —9 — 1 glycine ethyl ester and found = 5 ± 3 x 10 sec. , kg = 24 M~^ sec."^, kg = immeasurably slow, and k^ = 0.58 sec. . In agreement with the greater nucleophilicity of hydroxide ion, most of the hydrolysis near pH 7 is catalyzed by hydroxide ion. Hay, Porter and Morris (4) have studied the basic hydrolysis of ethyl glycinate, methyl glycinate, ethyl betaine ethyl ester, ethyl leucinate, methyl leucinate, methyl cysteinate, methyl serinate, ethyl phenylalaninate and dimethyl glutamate at 25.0° and ionic strength 0.1 M and determined the ionization constants of the esters. They found that the ionization constants for amino acid esters were approximately two pK units lower than those for the corresponding amino acids. Protonation of the amino group of an amino acid ester results in a much greater reactivity of the protonated species in alkaline hydrolysis. The effect of charge on ester hydrolysis was demonstrated (4) by comparison of the rates of hydrolysis for ethyl glycinate, ethyl betaine ethyl ester and cysteine methyl ester. Positively charged betaine ethyl ester, [(C2Hg)gNCHgC00C2Hg]^, hydrolyzes 36 times faster than ethyl glycinate, NHgCHgCœCgHg, under similar conditions, whereas, the rate of cysteine methyl ester, ( 8CH2CH(NH2)C00CHg), hydrolysis was approximately one-eighth as fast as ethyl glycinate. 3 An^ïolici and Hopgood (5) have measured the rates of hydrolysis of butyl glycinate, ethyl alaninate, ethyl P- alaninate, and ethyl valinate at 25.0® and ionic strength 0.060 M. They concluded that the steric effect of the R group of ethyl esters of a-amino acids, NHgCHRCOOCgH^, is a major factor determining the relative rates. The results indicate that the rates fall into an order predicted by the Newman "rule of six" (6). This rule states that those atoms which are effective in providing steric hindrance are separated from the attacking atom in the transition state by a chain of four atoms, i.e., numbering the oxygen atom of the hydroxyl group attacking the carbonyl atom 1, then the greatest steric hinderance will result from atoms in position 6. For example the rate of hydrolysis of ethyl valinate which has six atoms ip position 6 would be expected to be quite slow. NH„CHCOOC„H NH„CHCOO 3 3 3 3 Ester hydrolysis has been observed to be catalyzed by both general base catalysis and nucleophilic catalysis, depending upon the type of ester. Esters activated in the alcohol portion have been found to undergo nucleophilic catalysis (7, 8) whereas esters activated by electron- withdrawing substituents in the acyl portion have been sliown l.o hydro I yx.o wi l.h a median ism ol general base catalysis (9). Tht' nuclc'ophilic catalysis mechanism dopicLod below in the reactions written for the base hydrolysis of esters is 2 one in which the reagent B directly attacks the sp hybridized carbonyl carbon atom to form an intermediate which is subsequently hydrolyzed by water in a fast step to result in the products of the reaction. In this case, k <k' - k • ^HgO^OH ' ^HgO • O II H RCOR' B: RGB + OR n O II ^OH ' O^ " RCOR" OH (H„0) 2^ V RCOH + OR O 11 ^*nH~ H o^ RGB + OH , (H„0) 2ÎLJ RGOH + B: For general base catalysis the proton transfer from the general base must occur in the rate determining step which is illustrated for the abstraction of a proton by HgO from the tetrahedral intermediate formed by hydration of the ester carbonyl f^roup. 0 - Ô I I R-G OR' 1 I /\ ^ H ^H 0H„ 5 In general, evidence for the intermediate in the nucleophilic catalysis mechanism is not obtainable since the subsequent hydrolysis is usually faster than its forma­ tion. Sometimes the deuterium oxide solvent isotope effect may be used as a criterion to distirguish between general base- and nucleophile-catalyzed reactions (10) but the results may also be ambiguous. . One method which has been used is to compare the catalytic effects of two bases of similar pK^ but widely different nucleophilicity (11). Bruice and Lapinski (8) found for example that whereas imidazole and phosphate dianion, HPO^ , have similar pK^ values the coefficients of catalytic activity are 4000 times greater for imidazole toward p-nitrophenyl acetate than for phosphate dianion but similar coefficients are found toward ethyl dichloroacetate. They concluded that p-nitrophenyl acetate was hydrolyzed by nucleophilic catalysis. Ethyl glycinate was studied (9) and the mechanism was stated to be general base catalysis on the basis of the reaction in the presence of the buffer tris-(hydroxymethyl)- aminomethane. The evidence certainly cannot be considered conclusive in the absence of more data. There is also a problem of definitions as to the function of a general base catalyst (12). If the function of the general base catalyst is not only a rate determining proton transfer but also the 6 i M Lt'oduc (. ir>ii o 1 a base into the substrate to lorm an unstable intermediate then little difference exists between the two cases. Glycine ethyl ester (13) and ethyl acetate (14) hydrolyses exhibit nearly identical activation energies of 10.9 and 11.0 kcaL/mole respectively. The entropy of activa­ tion for the basic hydrolysis of ethyl glycinate was -21.7 e.u. (13). The much larger value of 18.1 kcal./mole for the activation energy for ethyl glycinate basic hydrolysis reported by Connor, Jones, and Tuleen (15) is probably due to the contribution of protonated ethyl glycinate to the rate of hydrolysis to give a much larger rate than that reported by a number of other investigators (3,4,13,16,17). Even at pH 9.5 - 11.0 care must be taken that the rate observed does not include contributions from the hydrolysis of small concentrations of the protonated ester. At lower pH values due to the lower concentrations of hydroxide ion the rate observed decreases and at pH 7 the rate observed is too slow Lo measure by pH stat techniques compared to the metal ion catalyzed hydrolysis (3). Kroll (1) found that heavy metal ions accelerated the hydrolysis of amino acid esters. It was observed that a maximum ia to of hydrolysis occurred when the metal ion ; ester ratio approached unity. This was interpreted to mean that the velocity of the hydrolysis at a constant pH main­ tained by tris-(hydroxymethyl)-aminomethane, tris, buffer