This manuscript has been reproduced from the microfilm master. UMI films the

text directly from the original or copy submitted. Thus. some thesis and

dissertation copies are in typewriter face. M i l e others may be from any type of

cornputer printer.

The quality of this reproduction is dependent upon the qwlity of the copy

submitted. Broken or indistinct print, coiored or poor quality illustrations and

photographs, mnt bleedthrough, substandard margins, and irnproper alignment

can adversely affect reproduction.

In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages. these will be noted. Also, if unauthorized copyright

material had to be removed, a note will indicate the deletion.

Oversize materials (e-g.. maps, drawings, charts) are reproduœd by sedioning

the original. beginning at the upper lefthand corner and wntinuing from left to

right in equal sections with small overlaps. Each original is also photographed in

one exposure and is induded in reduced fom at the back of the book.

Photographs induded in the original manuscript have been reproduced

xerographically in this copy. Higher quality 6 x 9" black and white photographic

prints are available for any photographs or illustrations appearing in this copy for

an additional charge. Contact UMI directly to order.

BeH & Howell Infornation a d Leaming 300 NOM Zeeb Road, Ann Arbor, MI 481064346 USA

800-521-0600

NOTE TO USERS

Page(s) not included in the original manuscript are unavailable from the author or university. The

manuscript was microfilmed as received.

This reproduction is the best copy available.

UMI

Lanthanum(II1)-Promoted Cleavage of

RNA and Cydic Nucleotides

Phillip C. Hurst

A thesis submitted to the Faculty of Graduate Studies and Research of

McGill University in partial requirements for the degree of

Doctor of Philosophy

June 1997

Department of Chemis try

Mffiili University

Montreal, Quebec

Canada

O Phülip C . Hurst

National Library 1*1 of Canada Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibiiographic Services services bibliographiques

395 Wellington Street 395. rue WeUingtm Ottawa ON KI A ON4 OnawaON K l A W Canada Canada

Our nP Notre r w k r i l r > ~ ~

The author has granted a non- exclusive Licence allowing the National Library of Canada to reproduce, loan, distribute or seLl copies of this thesis in rnicrofom, paper or electronic formats.

L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction su. papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

Abstract

The RNA dimer ApA is rapidly deaved in the presence of lanthanum(III) chloride yielding the products of transesterification and hydrolysis, 2'-AMP, 3'-AMP and adenosine. The intermediate 2'3'-CAMP does not accumulate. Under pseudo-first-order conditions (approximately 100-fold excess lanthanum(III) chloride) the half-life for ApA deavage is only 13 seconds.

The potentiometric titration, pH-rate profile and dependence upon Ianthanum(III) chioride concentration support a penta-p-hydroxo dilanthanum species as the active species. The kinetic isotope effect of 1.9 and a one-proton inventory indicate that the 2'-hydroxyl deprotonation is not concerted with the

nudeophilic attadc. It is proposed that a rapid equilibrium proton transfer occurs between the 2'-hydroxyl and a bridging hydroxo prior to the nudeophilic attadc and rate-determining intramolecular general acid catalyzed breakdown of the phosphorane to products. It is proposed that the general acid is a bound water, formed from the protonation of the bridging hydroxo.

Lanthanum(II1) ion was found to be among the best of the trivalent lanthanides for promoting ApA deavage, contrary to literature reports. The proposed lanthanide dimer mechanism, which is consistent with the behavior of the O ther lanthanides, easil y explains this.

The Ianthanurn(III) dimer was also found to be highly reactive towards the DNA mode1 compow-d bis(pnitropheny1) phosphate and the cydic nudeo tides 3',5'-CAMP and 2'3'-CAMP# providing half-lives of 12s8 9min and 340ms respectively. The results were consistent with a bound p-hydoxo as the nudeophile.

The results for the ApA and cydic nucleotides reactions are faster than any reported for synthetic catalysts.

Résumé

Le dimere d'ARN ApA est rapidment coupé en présense de chlorure de lanthane(III). Ceci conduit à des produits de transestérification et d'hydroyse. le 2'-AMP, 3'-AMP et l'adenosine. L'intermédiaire 2'3'-CAMP ne s'accumule pas. Dans les conditions réactionelles de pseudo premier-ordre (exceès de 100 fois de lanthane(m)), la demie-vie pour le clivage de 1'ApA n'est que de 13 secondes.

Les titrages potentiométriques, les courbes pH-vitesse de réaction et la variation en fonction de la concentration en chlorure de lanthane(m) concordent pour indiquer que la forme active doit contenir Itesp&ce penta@-hydroxo)dilanthane. L'effet isotopique sur la cinétique ainsi que les résultats d'expériences d'inventaire de protons, qui montrent qu'un seul proton est échangé, indiquent que la déprotonation du groupment hydroxyle en position 2' n'est pas concertée avec l'attaque du nudéophile. il est proposé qu'un groupement hydroxo pontant agit comme base générale intramoléculaire pour deprotoner ce OH. La déprotonation est un équilibre rapide qui précède l'attque nudéophilique et l'étape déterminante de la décomposition i n t r a m o ~ ~ a i r e de l'intermédiaire phosphorane en produits. Cette dernière décomposition est catalysé par un acide général. Nous proposons que l'acid genérale est une molécule d'eau lié, formée par la protonation du groupement pontant. Des ions trivalents de la familie des lanthanides, et contrairement ce qui est connu dans la littérature, nous montrons que le lanthane(III) est parmi les meilleurs poun faciliter le clivage de 1'ApA.

Ceci s'explique aisément par le mécanisme proposé qui implique un dimere de lanthane(III) conformément à ce qui est connu du comportement des autres lanthanides. Nous trouvons également que le dimère de lanthane(III) est hautement réactif avec le compose modele de l'ADN, le bis@-nitrophény1)phosphate et les nucléotides cycliques 3I.5'-CAMP et 2','3'-CAMP, menant hdes demi-vies de 12s. Smin, et 340ms respectivement. Les résultats sont en accord avec le modele proposant un groupement v-hydroxo comme nudéophile. Les r&ultats pour les réactions avec 1'ApAet les nucléotides cycliques sont les plus rapides connus h ce jour pour un catalyseur synthétique.

1 would like to thank Dr. Jik Chin for his sound advice, guidance and words of encouragement throughout my studies in the chemistry department, and most certainly for the opporhlnity to work with hirn in the first place.

Thank you to Dr. Graham Darling, who shared s u p e ~ s o r y duties with Dr. Chin earlier in my studies and shared his fertile imagination with us.

During my stay in the Otto Maass building 1 had the pleasure of working in two different laboratories so 1 was fortunate to have made so many good friends. For their willingness to help, listen, share, speak their minds and be friends 1 must thank William Cheung, James Connolly, Rik Ganju, Lise Hubbard, Barry Linkletter, Erik Rubin, Jin Seog Seo, Brent Stranix, Bryan Takasaki, Petra Turkewitsch, Alex Vekselman, Daphne Wahnon, Mark Wall, Barbara Wandelt, Dan Williams, Ni& Williams and Mary Jane Young.

Very special thanks go to Nidc (for helping in so many ways and teaching me so much, about chemistry and how to handle fatherhood), Brent (also for being helpful, full of ideas and for sound advice on pregnant women and baby boys, and for al1 the coffee, cigarettes and snooker) and Petra (for listening to me cornplain, putting up with me in general and always k i n g a stalwart booster).

Thank you to Fred Kluck and Ridc Rossi for fixing the things I broke, and their good-natured helpfulness in general. Thank you to Renee Charron and Aline Gibson for making sure everything went smoothly. Everyone knows the chemistry department would most certainiy collapse without them. Thank you to Anne-Marie Lebuis for translating the abstract. Once 1 master English 1 may attempt French. Thank you for the beer Dr. Edward.

The most special thanks go to my immediate family. Thank you Rebecca for so many things. Montreal was a great big bore until you came dong. You are my sunshine ... Thank you to my son James, who makes m e laugh when 1 reaiiy need it. Thank you to my parents and brothers for ali their support, even if it wasn't monetary.

NOTE TO USERS

Page(s) not included in the original manuscript are unavailable from the author or university. The

manuscript was microfilmed as received.

This reproduction is the best copy available.

UMI

Glossary

2'3'-CAMr 2',3'-cCMP 2'5'-upu 2'-AMP

3'5'-ApA 3'3'-CAMP 3'3'-upu 3'-AMP S'-Ah@

AP A APUP BDNPP BNPP CHE5 cyden dApdA DNA

dpa EDTA EPPS

HEPES

HPLC HPNP 1s KIE

kobs

kW1

LFER neo

NPP

~ H 1 / 2

Pi

adenosine 2',3'-cydic monophosphate cytosine 2',3'-cydic monophosphate uridyl(2'+5')uridine adenosine 2'-monophosphate adenyl(3'+5')adenosine adenosine 3'$"-cydic monophosphate uridyl(3'+5')uridine adenosine 3'-monophosphate adenosine 5'-monophosphate 3'5'-ApA (above) adenyl(3'-t5')uridine 3'-phosphate bis(2.4dini trophenyl) phosphate bidpniîrophenyl) phosphate 2- [N-cycIohexylamino]ethanesulfonic acid 1 ,Q, 1 0-tetraazacydodododecane 2'-deoxyadenyl(3'+5')T-deoxyadenosine deoxyribonudeic acid 28P'-dipyridyiamllie ethylenediaminetetraacetic aad N-12-h ydroye thyl] piperazine-N'-[3-propammknk acid] N-[2-h ydroyethyll piperazine-NI-[2-e thanesulfonic acid] high performance liquid chromatography 2-hydroxypropyl pni trophen y1 phosphate intemal standard kinetic isotope effect observed rate cons tant relative rate

linear free energy rela tionship

pni trophenyl phosphate

half-neutralization value; midpoint of a titration ~ 0 ~ 3 - ; inorganic phosphate

poly(A) RDS RNA tacd

tacn

m ' Y TMEDA tren

polyadenylic aâd rate-detennining step ribonucieic acid 159- triazacydododecane 1,4,7-triazacyclononane 2,2':6',2-terp yridine te trame th yle th ylenediamine tris(2-aminoe thy1)amine tris(2-aminopropy1)amine

Table of Contents

Chapter I

Chapter II

Introduction

Fundamentai Cellular Functions Nucleic Acids Nucleotides Genetic Technologies

Mechanisrns of Phosphate Ester Cleavage Reactions Oxidative Mechanisms Nudeophilic Medianisms: DNA and Its Andogs Nudeophilic Mechanisms: RNA and Its Analogs Selected Enzyme Mechanisms

Advances in the Development of Phosphodiesterase Mimics

Mononuclear Transition-Metd Complexes The Dinudear Approach to Phosphate Ester Ueavage

Plan of Study

Rapid Cleavage of RNA by Lanthanum(111)

Introduction to Chapter II: Some Previous Reports of Nucleic Acid Cleavage by Lanthanide Ions and Other S ystems Results and Discussion

ApA 1s Rapidly Qeaved by Lanthanum Ion at Aikaline pHs Titration of LanthanumUII) Chloride A Mode1 for a Dimeric Lanthanum(II1) Species pH-Rate Psofile for Lanthanum-Promoted ApA Cleavage Second-Order Dependence upon Lanthanum(II1) Concentration Reaction Mechanisms

Summary and Conclusion to Chapter II

vii

2.4

2.5

2.6

2.7

Chapter III

3.1

3.2

3.2.1

3.2-2

3.2.3 3.2.4 3.2.5

3.3

3.4

3.5

3.5

Chapter IV

4.1

4.2

4.2.1 4.2.2

4.2.3

4.3

4.4

4.5

Experimental Appendix I: Derivation of Equation 2.3 Appendix 2 Mathematical illustration of Reaction Order versus pH for a Dimeric Active Species Appendix 3: Tabulated Kinetic Data

Kinetic Isotope Effects

Introduction to Chap ter III: Explorhg Transesterification Mechmisms in RNA Results and Discussion

LanthanumUII tPromoted Transesterif ication of HPNP Kinetic Isotope Effect for HPNP Kinetic Isotope Effect for ApA Proton Inventory Reaction Mechanisrns

Summary and Conclusion to Chapter III Experirnentai Appendix 1: Derivation of Equation 3.1 Appendix 2: Tabulated Kinetic Data

ApA and HPNP aeavage by Other Ln(1II)

Introduction to Chapter IV: Other Lanthanides in the Literature Results and Discussion

Sampling the Lanthanides: Titrations and Activities pH-Rate Profiles for Europium(II1) and Ihulium(II1) Second-Order Dependence upon Thulium(II1) Concentration

Conclusion to Chapter IV Experimental Avuendix: Tabulated Kinetic Data

viii

Chaptes V La(II1)-Promoted Hyàrolysis of DNA Models

Introduction to Chapter V: Metal Ion- and Metal Complex- Promoted Hydrolysis of DNA and DNA Models

Resuits and Discussion Lanthanum(II1)-Promoted Hydrolysis of BNPP: A Plateau in the pH-Rate Profile Rapid Ring-Opening of 3',S9-CAMP with Lanthanum(II1) The First Direct Measurement of 2*,3'-CAMP Ring-Opening with Lanthanum(II1)

Conclusion to Chapter V Experimental Appendix 1: Derivation of Equation 5.1 Appendix 2: Tabdated Kinetic Data

Chapter VI Conclusion to the Thesis

6.1.1 Contributions to Knowledge 6.1.2 Publications and Presentations

Literature Cited

Chapter 1 Introduction

1.1 Fundamental Cellular Functions

The chemistry of nudeotides and nudeic auds is basic to all cellular functions. Enzymes, which carry out multitudinous activities, owe their very existence to the nudeic aads that possess the code for their creation. Nudeotide hiphosp hates are vehides for energy capture, storage, transport and tram fer, so enabling many cellular processes. Cyclic purine nucleotides function as messengers, initiating and regulating the activities of complex systems. srne nudeotides are components of widely used coenzymes.

Many excellent tex ts describe these important activi ties and tha t material comprises a significant component of undergraduate and graduate level cwrïcula. Thus the material will receive only a cursory review here.

1.1.1 Nucleic Acids

Long term information storage is the domain of DNA (although viral genomes use RNA). From a set of ody four different base pairs (A, C, G, T) the genome is organized into codons, sets of three nudeotide base pairs that code for amino acids. The process of protein synthesis begins with the transcription of the genetic code. The codons are read and transcribed in sequence into a molecule of messenger RNA (rnRNA) which, in the case of eukaryotes, is transported out of the nucleus. Transfer RNA (tRNA) associates with the mRNA and an amino acid, enabling the translation of the genetic code into the proper sequence of amino auds. Each amino aad depends upon the function of a different tRNA molecule so that the tRNA specifically recognizes both the mRNA codon and the required amino aad. This process of translation occurs in the ribosomes. (De Robertis and De Robertis, 1980)

Another class of RNA can be involved in translation by specifically inhibiting the process. These RNAs were first proposed and termed translationai control RNA (tcRNA) by Heywood (Heywood et al., 1974; Bester et al., 197S), and are now broadly referred to as antisense RNA. An antisense RNA is complimentary to a specific mRNA and with it can f o m a stable duplex. When incorporated in the duplex the mRNA is mavailable for translation. Thus nudeic auds can also perform regdatory functions.

(transcription) protem

/ inactive duplex

Scheme 1.1 A simple cartoon overview of the involvement of nudeic acids in protein s ynthesis.

In the early 1980s the known repertoire of RNA was expanded to indude self-modifying reactions and tnily catalytic functions. T. R Cech found that the precursor ribosomal RNA (pre-rRNA) of Tetrahymena pigmentosa was capable of excising its own intervening sequence (IVS), the noncoding portion of the molecde, and splicing the remaining rRNA fragments together to produce the mature rRNA. (Cech et al., 1981; Kruger et al., 1982) Soon afterward S. Altman announced that the catalytic activity of ribonudease P (RNase P) was due to the RNA subunit of the enzyme and not at al1 to the protein subunit. RNase P catalyzes the deavage of short sequences from the 5' end of pre-tRNA to produce the mature tRNA with its 5'-phosphate and in vivo both the RNA and protein subunits are essential for its activity. However it was shown that under certain conditions in vitro the protein subunit was not the catalytic subunit and indeed was not even essential for the activity. (Guerrier-Takada et al., 1983; Guerrier- Takada and Altman, 1984)

The discovery that RNA could effect self-modifying and catalytic reactions

caused a paradigm shift in biodiemistry; previously such activities were believed to be the solely in the domain of protein enzymes. For this revolutionary work Altman and Cech shared the Nobel Prize for Chemistry in 1989. Since the discovery of the fïrst ribozymes many others have been reported. (Long and Uhlenbeck, 1993; Saldanha et al., 1993, and other articles in that journal issue; Yarus, 1993)

1-1.2 Nucleotides

Nucleotide triphosphates, in particular ATP, figure prominently in bioenergetics. The mitochondia harness redox reactions, exploiting the exothermiaty to produce ATP though a complex chain of events. The ATP may then be used elsewhere in the ce11 as an energy source; a cornmon cunency for a host of energy driven processes throughout the cell. (Rawn, 1983)

Adenosine-3'3'-cydic monophosphate (3',5'-CAMP) is an important second messenger in the regdation of many cellular processes. 3'3'-CAMP is synthesized from ATP by adenylate cydase. The activity of adenylate M a s e is controlled by hormones binding to cell surface receptors. in eukaryotic systems the 3'3'-CAMP produced binds to a protein kinase which catalyzes a phosphoryl transfer from ATP to a variety of proteins, leading ultimaiely to the required physiological effects. (Martin et al., 1985)

Adenine nudeotides are also employed in some coenzymes. Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are integral to the redox reactions and hydride transfers of a large number of dehydrogenases. The flavin coenzymes, flavin adenine dinucleotide

Figure 1.1 3',5'-CAMP, the ubiquitous second messenger, and ATP, the cell's energy currency.

(FAD) and flavin mononucleotide (FMN), are also redox coenzymes. Enzymes requiring FAD or FMN also employ hydride and one-electron transfers. Coenzyme A (CoA) is an acyl carrier involved in fatty acid oxidation and synthesis, acetylation reactions and oxidative decarboxylations. In none of the above coenzyme reactions does the adenine nudeotide participate directly (Le. in a chernical transformation event) but it is nonetheless important for aspects of molecular recognition. (Rawn, 1983)

1.1.3 Genetic Technologies

The last few decades have witnessed the developments of technologies that, while inspired by nature, have extended beyond nature's intended uses for nudeic acids. %me of these technologies are still in the stages of exploration and experimentation while others have been developed and refined to the point where they are widely applied and commeraalized.

Perhaps the mos t exotic of the recent novel technologies are catalytic DNA (Breaker and Joyce, 1994; Rawls, 1997) and molecular computation using DNA fragments. (Adleman, 1994) These are certainly weird and wonderful developments in saence today but rely heavily upon the design and construction of synthetic DNAs and automated sequencing. Nucleases, natural or synthetic, are not part of the game.

Polymerase Chain Reaction (PCR) and the various recombinant DNA

Scheme 1.2 Restriction endonudeases cut double stranded DNA site-speufically providing the molecular biologist with the desired fragments and the required precision for manipulating genes. These enzymes often recognize palindromic sequences and produce staggered cleavage sites. (Alberts et al., 1989)

methods are immensely successfd and powerfui genetic techn01ogies, and have been widely commeraalized. They too rely in part upon synthetic DNA and automated sequencing. However, they also rely heavily upon restriction endonudeases to excise targeted sequences in a genome and to cut DNA wherever a new oligonudeotide is to be inserted. (Alberts et al., 1989)

There is a limited repertoire of restriction endonudeases known and available to molecular biologis ts. The required si tespecif ic deavage c m of ten be a major hurdle in genetic research. Al-, when double stranded DNA is treated with one of these enzymes the result can be millions of fragments. Çorting through the fragments to isolate the target can be diffiicult and time consuming. (Alberts et al., 1989)

These frequent obstacles to molecular biology are major motivating factors in the research and develûpment of new, useful artificial systems for phosphate dies ter deavage. By synthesizing an an tisense oligonudeotide and tethering to i t a DNA cleaving agent one could in principle custom design one's own artifiuai endonudease, cut the target DNA site-specifically to produce only two fragments and then go about the business of conducting the research at hand.

%me researchers envision the day when artifiaal endonudeases will be used as therapeutic agents, to degrade the DNA or RNA of foreign pathogens. Perhaps they may one day be targeted against the host genome to regulate and modify at the mRNA level the effects of genetic diseases such as the progressive neurodegenerative trinucleotide repeat diseases (Huntington's Disease Collaborative Research Group, 1993) (e.g. Huntington's disease, fragile->< syndrome, myotonic muscular dystrophy) which are thought to be pathogenically the result of upregulation of their genes. (Housman, 1995; Bates, 1996)

The antisense component of artificial endonuclease research and development is sufficiently advanced. The remaining hurdle is to design, develop and exploit a simple artifiaal system capable of deaving the phosphate badcbone of DNA. Of course, the cleavage has to be fast enough to be practically useful and it must result in DNA fragments which can be readily Ligated.

1.2 Mechanisms of Phosphate Ester Qeavage Reactions

1.2.1 Oxida tive Mechanisms

DNA can be cleaved very rapidly by oxidative systems. The bis(1 ,l 0-phenan throline)-copper(I/ II) reagen t is one such example. When DNA is incubated with hydrogen peroxide and the copper complex for just a few minutes i t is extensively fragmented. (Sigman, 1986)

The bis(1,lO-phenan thro1ine)-copper(I/II) system shows no useful sequence specifiaty as is but Chen and Sigman have used this redox complex to develop a sequence-specific deaving reagent (Figure 1.2). The chemistry involves a diffusable hydroxyl radical which initiates the cleavage and so their "sequence-çpecific" system actually produced deavage over a fairly broad range of bases on the target oligodeoxynudeotide. (Chen and Sigman, 1988)

The mechanism of the cleavage reaction is an initial insertion of a

Figure 1.2 Three redox systems for deaving DNA.

Scheme 1.3 Sigman's reaction pathway for the oxidative deavage of DNA. (Sigman, 1986; Chen and Sigrnan, 1988)

hydroxyl radical at the C-1 position followed by loss of the base, converting the

deoxyribose ring into a lactone. Elimination of the 3'- and 5'-phosphates ensues producing a 5methylenefuranone. (Scheme 1.3) (Sigman, 1986; Chen and Sigman, 1988)

h o ther very effective redox sys tem is EDTA-iron(II/III). When tethered

to a DNA intercalator single strand and double strand DNA deavage are quite rapid (Figure 1.2). (Hertzberg and Dervan, 1982)

This too has been used to develop sequence-specific reagents. Dervan's group has constructed 01igodeoxynudeotideEDTA-iron(II/III) conjugates that bind in the minor groove of double strand DNA. PolypeptideEDTA-iron(II/m conjugates also bind in the minor groove and direct the deavage to a specific range of bases. (Wade et al., 1992; Szewczyk et al., 1996, as examples)

The redox systems usually involve a diffusable free radical that initiates the strand breaking so the deavage occurs over a rather broad range; they lads the precision required in artifiual restriction endonudeases. Regardless, the major drawback to al1 these redox systems is that they are destructive. As Scheme 1.3 shows, the nudeotide at the deavage site is destroyed in the process. Furthermore, the resul ting fragments are both terminated with phosphate groups and as such are not readily religated.

This is not to Say that these reagents have not been usefui; they are widdy

used as analytical tools. In footprinting experiments, the redox reagents are used to degrade DNA or RNA to which some ligand is bound. The portion of the nudeic acid which is tightly bound to a ligand is protected from the deavage. Thus analysis of the reaction products shows the binding site to be intact. It can also be applied to study three dimensional structure; the interior is shielded from the destructive radicals.

Tethering a redox system to a ligand can also provide information about binding phenornena. This is broadly referred to as affinity clemage, and artificial restriction endonucleases are just one application of the prinùple. (Wade et al., 1992; Szewuyk et ai., 1996, as examples)

Religa tion is possible for a manganese-persulfate sys tem (Figure 1.2). The cleavage occurs though the hydroxylation of specifically the s'-CH2 and produces a d'-phosphate terminus and a 5'-aldehyde. In a second step, sodium borohydride reduction of the aldehyde gives the natural5'-OH so that religation can be accomplished. (Pratviel et al., 1993)

1.2.2 Nudeophilic Mechanisms: DNA and Its Analogst

The phosphate diester badcbone of DNA (Figure 1.3) is a remarkably stable entity. It is es timated to have a hydrolytic half-life at neutral pH and 25°C on the order of two hundred mülion years. (Chin, 1991) That phosphate diester linkage has been entnisted by nature with the integrity of the genetic code.*

Although enzymes have evolved to hydrolyze that phosphate diester group with great facility it is too stable for mere chemists to study directly. Part of the resistance to hydrolysis arises from the negative charge on the nonbridging phosphoryl oxygens which repels incoming negatively charged nudeophiles. Another contributing factor is the high pKa of the conjugate a a d of the leaving group, a primary alkyl alcohol. (Westheimer, 1987) Mode1 compounds, wherein the leaving group is changed to one having a lower pK,, greatly facilitate the

Cytosine

Figure 1.3 The polymenc structure of DNA and its four bases.

Guanine

Thymine

t For a very detailed and extensive review of nucleophilic substitution in phosphate esters see Thatcher and Kiuger. (1989) $ Two hundred million years rnay seem to be an unnecessarily long half-life but when one considers the size of the human genome, -6 x log nudeotides in our forty-six chromosomes, (Alberts et al., 1989) we realize that continua1 repair of our DNA is still required.

Scheme 1.4 Nudeophilic attack on a phosphate diester c m produce either P a bond or C-O bond fission.

study of phosphate diesters?

Very broadly, a cleavage mechanism might be associative (nudeophilic attadc upon the phosphate diester) or dissociative, whereby a unimolecular P-0 bond fission occurs to give a reactive metaphosphate species which combines with a nucleophile in a second step. The dissociative mechanism principally applies to phosphate monoester chemistry and so wilI not be discussed here.

In the assoaative mechanisms deavage of the phosphate diester group can occur by fission of the P-O bond or the C-O bond. (Scheme 1.4) In a study of

methyl-2,4-dinitrophenyl phosphate nucleophilic aromatic substitution was found to be the predominant pathway with alkoxide and phenolate nudeophiles.

Acetate and phosphate nucleophiles gave virtually exclusive P-O cleavage. When hydroxide was the sole nudeophile it attacked at both the phosphorus and the aromatic carbon to roughly equal extents. The third possibility, sN2 attack

upon the methyl carbon, did not occur to any significant extent under any of the conditions employed. (Kirby and Younas, 1970)

Westheimer detailed a set of guidelines which desaibe what may occur in the course of nudeophilic substitution at the phosphorus centre of phosphate

esters. Addition of hydroxide to a phosphate diester's phosphorus centre results in a pentacoordinate trigonal bipyramidal phosphorane species from which the

t A linear free energy relationship (LFER) has bcen shown to apply to the hydroxide-catalyzed hydrolysis of some phosphate diesters, the leaving group p&s of which span pH - 4 + 15.5. (Chin et al., 1989) A L E R with a similar P value also exists for the cerium(III/IV)-promoted cleavage of phosphate diesters suggesting that a "catalyst" may accelerate the cleavage of phosphate diesters with poor leaving groups to the same o r similar degree as for those with good leaving groups. (Takasaki, 1994) This may not always apply though and the researcher must be vigilant when studying the mode1 compounds and making inferences about the DNA chemistry.

Scheme 1.5 Pseudorotation in pentacoordinate phosphorus species.

leaving group departs. If the phosphorane is sufficiently stable (Le. it has an appreciable lifetime) it may undergo a process of bond angle and bond length rearrangement called pseudorotation (Berry, 1960) whereby ligand atoms may interchange axial and equatoriai positions. (Scheme 1.5) It was further proposed, as an extension of the principle of rnicroscopic reversibility, that nudeophiles attack at, and leaving groups depart from, axial positions. (Westheimer, 1968)

Whether this nudeophilic addition and elimination occurs in a concerted process or in sequence in a given reaction is a frequent question. In some reactions it would seem to be a two-step process whereby the phosphorane is a genuine, stable (albei t fleeting) in termedia te. Evidence for this is the iso topic exchange observed during the acidic hydrolysis reactions of cyclic phosphate esters in 180-labeled water; the scrambling would result from pseudorotation followed by unproductive breakdown to reactants. (Haake and Westheimer, 1961; Kluger et al., 1969)

Generally, in alkaline hydrolysis of phosphate diesters isotopic exchange is not observed, suggesting that pseudorotation is not occurring and probably that the phosphorane is in a transition state and not a true intermediate. A concerted addition-elimination mechanism for alkaline hydrolysis of some acyclic phosphate diesters is supported in Cleland's heavy atom isotope studies. (Hengge and Cleland, 1991; Cleland and Hengge, 1995; Hengge et al., 1995)

1.2.3 Nudeophilic Mechanisms: RNA and Its Analogs

Structurally, RNA is nearly identical to DNA. One difference is the occurrence of the base uraal in RNA instead of thymine. Chemicdy though the most significant difference is the RNA's ribose moiety; DNA possesses 2'-deoxyribose (Figure 1.4). The presence of the R N A 2'-hydroxyl means there is an efficient intramolecular nudeophile dose to the phosphate diester linkage.

The principal mode of cleavage of RNA is not hydrolysis but transesterification. Nudeophilic attadc of the 2'-oxygen on the neighbowing phosphate is facilitated by a Brensted base accepting the proton from the 2'-alcohol. The intramolecular transesterification cleaves the diester to give a 2',3'-cyclic intermediate and a leaving 5'-alcohol. Hydrolysis of the cyclic in termedia te gives the product monoes ter.

The reaction pathrvay in enzymatic systems is regiospecific; the phosphorane is in equilibrium rvith the 2',5'-isomer only and 3'-monoesters are the products of the ring-opening reaction.

In nonenzyrna tic sys tems there are additional possibili ties. According to

J Cyrosine Uracil

Figure 1.4 The polymeric structure of RNA and its four bases.

3'-monocsta terminus T-monoester terminus

Scheme 1.6 Reaction pathways for the cleavage of RNA. "NE" designates a nonenzymatic pathway.

Westheimer, (1968) the 2'-nudeophile WU initidy occupy an axial position in the phosphorane and it may depart from there as well, reverting to reactant. Pseudorotation of the transesterification's phosphorane though could place the 3'-oxygen in an axial position. Unproductive breakdown of the phosphorane would then yield the isomeric 2'-es ter.

Nonenzymatic ring-opening of the 2'3'-cydic intermediate produces both monoesters because the nucleophilic attack is indiscriminate, giving a phosphorane with either the 2'- or 3'-oxygen in the axial leaving position.

The presence of the 2'-hydroxyl not only provides a different mechanisrn for the diester deavage, relative to DNA, it confers upon the molecule a greatly decreased chemical stability. Estimates of RNA's stability to deavage can Vary somervhat. Extrapolating through pH, temperature and leaving group pK,, some

literature data (Davis et al., 1988a; Breslow et al., 1989; Jarvinen et al., 1991; Williams and Chin, 1996) would suggest a half-life (at pH=7 and 25OC) of from 17 + 69yr. Regardless of the estimate, it is undeniably more far prone to cleavage than DNA.

One might also gauge the increased reactivity of RNA by considering the effective molarity of the intramolecular nucleophile. Using data obtained by Usher et al. (1970) for the reaction of an RNA mode1 Kirby (1980) calculated that the effective molarity was on the order of 107. This makes intuitive sense; the estimated stabilities of DNA and RNA differ by about 107-fold.

Figure 1.5 Usher's RNA model, characterized by an effective molarity of 107. (Usher et ai., 1970; Kirby, 1980)

1.2.4 Selected Enzyme Mechanisms

Bovine pancreatic ribonudease A is among the most studied and best understood of enzymes. It catalyzes the deavage of RNA by transesterification, producing a 2'3'-cyclic intermediate which is ring-opened in a second slower step. It is also provides a classic example of a general acid - general base mechanism.

The bell-shaped pH-rate profile for the ribonudease reaction is explained by the action of two histidine residues at the active site- His 12 functions as a generd base, accepting a proton from the nudeophilic 2'-hydroxyl while His 119, the general aad, protonates the leaving 5'-alkoxide. (Fersht, 1985, and references therein) It has been proposed that His 119 also protonates a nonbridging phosphoryl oxygen rendering the phosphorus centre more susceptible to nucleophilic attack.+(Anslyn and Breslow, 1989a; Breslow et al., 1996, and work cited therein)

Lys 41

'3-

Figure 1.6 His 12 and His 119 of ribonuclease A function as general a a d and general base while Lys 41 provides electros tatic s tabiliza tion. (Fersh t 1985)

- --

t AIthough the necessity of this has been debated (HerschIag, 1994; Thompson and Raines, 1994) protonation of a nonbridging phosphoryl oxygen is implicated in acid-catalyzed cleavage and isomerization of diribonucleotides and models. ( J a ~ n c n et al., 1991; Oivanen et ai., 1991; Kuusela and Lonnberg, 1994a)

Figure 1.7 A proposed mechanism for the 3' - 5' exonuclease function of E. coli DNA polymerase 1 featuring nucleophile activation, double Lewis aud activation of the substrate and leaving group stabilization. (Beese and Steitz, 1991)

Lysine residues at the active site are important for the deavage. When the subshate is bound Lys 41 in particular iç in very dose proximity to the phosphate and believed to provide electrostatic stabilization for the phosphorane.

In the second s tep, the ring-opening reaction, the roles of the histidines are reversed; His 119, now the general base, activates a nucleophilic water by accepting a proton while His 12 delivers a proton to the leaving 2'-alkoxide. (Fersht, 1985, and references therein)

Many enzymes rely upon divalent metal ions to effect phosphoryl transfers and phosphate ester deavages. (Strater et al., 1996, review) A prime example of this is the Klenow fragment of E. coli DNA polymerase 1. It possesses 3' - 5' exonuclease activity and has been shown to contain two divalent metal ions at that active site. Based upon X-ray and kinetic analyses the metal centres are proposed to play a number of roles in the hydrolysis reaction (Figure 1.7). The phosphate bridges the two metal centres so the phosphate is doubly activated, and the phosphorane doubly stabilized, by these Lewis acids. One of the metal ions coordinates a water molecule (or hydroxide) providing an activated nudeophile. The other metai is within range of coordinating the leaving

3'-oxygen thus stabilizing the poor leaving group. (Beese and Steitz, 1991) It has b e n suggested that this double ion mechanism might also apply to

other systems, in particular, ribozymes. (Steitz and Steitz, 1993) Much is known about the functions and gross structure of many ribozymes but at the time of this writing the fine structural details and mechanisms are not fully ~nders tood.~ Inferences are made from kinetic studies and cornparison to well characterized systems. Ribonuclease P (Smith and Pace, 1993), self-splicing group 1 introns (Sjogren et al., 1997) and the protein enzyme ribonudease H (Davies et al., 1991) are examples where the Steitz medianism is invoked.

Bovine panaeatic deoxyribonuclease 1 (DNase I) is an endonudease that deaves double stranded DNA to produce 5'-phosphates and 3'-hydroxyls. X-ray analysis located a single calaum ion at the active site, coordinated to a nudeotide phosphate. The mechanism proposed (Suck et al., 1986) for the hydrolysis combines Lewis acid activation with nudeophilic attack of water, assisted by the tandem actions of His 131 and Glu 75. (Scheme 1.7)

end

end

Scheme 1.7 Lewis acid activation and general base catalysis are believed to be integral to the hydrolytic activity of DNase 1. (Suck et al., 1986)

t Mechanistically, ribozymes present fascinating challenges. Unlike the typical RNA transesterification which produces the 2'3'-cyclic intermediate and the 9-hydroxyl some ribozyme cleavage occurs by hydrolysis (Pace and Smith, 1990) or transestenfication to remote ribose moieties. (Saldanha et al., 1993) The nudeophiles in these reactions must be very well positioned and well activa ted indeed to compte wi th the 2'-hydroxyl adjacent to the phosphate.

1.3 Advances in the Development of Phosphodiesterase Mimics

1.3.1 Mononudear Transition-Metal Complexes

Meta1 complexes can accelerate the hydrolysis of phosphate diesters through various means. As Lewis acids the metai centres may coordinate a nonbridging phosphoryl oxygen, activating the phosphate towards nudeophilic attack and attenuating the developing negative charge in the transition state by drawing electron density away from the phosphorus centre. Lewis acid activation by a single cobalt(lIi) centre has been shown to provide approximately 400-fold rate acceleration in the hydrolysis of phosphate hiesters (Hendry and Sargeson, 1984) and two orders of magnitude is probably applicable to phosphate diesters as well. (Hendry and Sargeson, 1990)

A metal complex bearing a titratable aquo ligand may also serve as an increased supply of nucleophilic hydroxide (as hydroxo ligand) at the appropriate pH when hydroxide in solution is present in vanishingly small concentration (Chin, 1991) although the hydroxo is not as nucleophilic as a hydroxide. (Buckingham, 1976)

These two means of catalysis have been demonstrated separately producing modest rate accelerations. When both modes are operating in the same system though the acceleration can be several orders of magnitude greater; an additional advantage is had by replacing an intermolecular nucleophiiic pathway wi th an in tramolecular mechanism. In cis-aquohydroxo metal complexes the reaction benefits from the cooperation of Lewis acid activation and the intramolecular nucleophilic attadc of a hydroxo ligand. After a study of

OR' OR'

Scheme 1.8 Two principal modes of catalysis by metal ions and their complexes; Lewis acid activation of the substrate and nudeophile activation.

cis-aquohydroxo cobalt(II1) promoted hydrolysis of phosphate monoesters Sargeson suggested that the intramolecularity of the reaction could in some instances contribute up to 105-fold enhancement in rate. (Jones et al., 1983) The inhamolecular mechanism certainly can be efficient; Chin et al. (1989) demonstrated that the cis-aquohydroxo complex [( t rpn)Co(O~)(O~~)]*+ was capable of a 1010-fold enhancement in the rate of BNPP hydrolysis, nearly the same as that by a real enzyme from Enterobacter aerogenes.

The efficiency of the intramolecular hydroxo nucleophile is highly sensitive to the cornplex's geometry. When a set of cobalt(III) - tetraamine ligand complexes was tested for their ability to promote the hydrolysis of B N P P the reactivity was found to Vary among them by 300-fold (Figure 1.8). Examination of the X-ray aystal structures of their carbonato complexes provided a rational explanation for the order of reactivity; a larger N-Co-N angle opposite the OCo-O angle would better stabilize the four-membered ring that resulted from intramolecular attack on the bound substrate. (Chin et al., 1988; Chin et al., 1989; Chin, 1991)

Substantial differences in rate also arise when one metd ion is substituted for another. Even though the valence and the ligands were the same an

Figure 1.8 The N-Co-N angle opposite the O-Co4 angle is critical to the efficiency of the intramolecular nucleophile. The corresponding carbonato complexes showed that in (b) [(cyclen)Co(OH2) (OH)] *+ a n d (c) [(trpn)Co(OH2)(0H)]2+ the angles were much larger than the ideal octahedral angle of 90° whereas in (cl [( tren)Co(0~~)(0~)]2+ the angle was compressed.

(Chin et al., 1988; Chin et al., 1989; Chin, 1991)

iridium(III) complex was found to be approximately one thousand fold slower than the corresponding cobalt(XII) complex. The difference in Lewis acidity was known to be slight and the spread between the reactivities was ascribed to the larger ionic radius of iridium(III), manifesting itself in a less efficient intramolecular attack of the hydroxo. (Hendry and Sargeson, 1989)

1.3.2 The Dinuclear Approach to Phosphate Ester Cleavage

Inspired by natural nucleases which employ multiple metal ions, researchers have been developing simple synthetic mimics and discovering what makes them work. Dinudear metal ion complexes can provide multiple modes of activation simultaneously, some of which are unattainable in their mononudear counterparts. The literature abounds with interesting dinudear complexes offered as models of enzymatic systems but this discussion wiil be confined to a few well characterized, discrete dinuclear systems which exhibit significant reactivity and so serve as examples of the various means by which high reactivity can be achieved.

One of the earlier successes in dinuclear complex promoted hydrolysis was accomplished using two equivalents of [ ( t r p n ) ~ o ( ~ ~ z ) ( ~ ~ ) ] 2 + for the

hydrolysis of a phosphate monoester, hydroxyethyl phosphate (HEP). Solution phase 31P NMR indicated that one equivaient of the cobalt(III) complex chelated

the monoes ter and so no nudeophile was available for the hydrolysis. However when a second equivalent was added hydrolysis rapidly ensued. X-ray analysis

Figure 1.9 [ ( t r p n ) ~ o ( 0 ~ ~ ) ( 0 ~ ) ] 2 + can hydrolyze monoesters by a dinudear

mechanism. (Chin, 1991)

Figure 1.10 Structurally characterized by X-ray analysis, the dicopper0 complex provides double Lewis acid activation to bridging phosphates for rapid deavage of RNA models. (Wall et al., 1993)

of the product showed inorganic phosphate bridging two cobalt(III) centres by chelation to each. It was proposed that the second equivalent of the complex carried out the hydrolysis on the chelate by the Lewis aud-intramolecular nucleophile mechanism (Figure 1.9). It was found that other aquohydroxo complexes such as [(dpa)Cu(OH2)(0H)]+ could also hydrolyze the chelate. (Chin,

1991)

Dinuclear strategies have proven successful in the deavage of diesters as well. Wall et al. (1993) reported the application of a discrete dinudear complex in the transesterification reaction of the RNA analog HPNP (Figure 1.10). The dinuclear complex was 50-fold more effective than the corresponding mononuclear complex. Using X-ray crystallography they showed how diesters could easily bridge the two metal centres to provide the double Lewis aud activation of the substrate.

A novel dinudear copper(I1) complex was successfully employed in the transesterification reaction of the diribonucleotide, ApA. Coordinated by two

triamine macrocycles on a naphthalene spacer (Figure 1-11), the copper(I1) centres provided the double Lewis acid activation for the 105-fold rate acceleration. The cyclic intermediate, 2',3'-CAMP, did not accumulate in the reaction. In separate experiments it was learned that the dicopper(I1) complex

H p. \HW a ci-

"7 , Figure 1.11 Young's and Chin's (1995) dicopper(I1) complex deaves the

diribonucleotide ApA with a 105-fold rate acceleration wi thout accumulation of the cyclic intermediate (hydrolyzed 108-fold faster than the background reaction).

was responsible for a 108-fold acceleration in the ring-opening hydrolysis. (Young and Chin, 1995)

A remarkable rate enhancement was discovered through the novel application of a dinuclear cobalt(II1) complex. When the DNA analog methyl-p-nitrophenyl phosphate was bridged between the metal centres of a bis-p-hydroxo bridged complex (Figure 1.12) the hydrolysis reaction was 10"-fold faster than the background rate. Isotopic tracer experiments showed that the nucleophile was one of the bridging hydroxos. This system was demonstrating both double Lewis a a d activation and intramolecular attadc of an activated nucleophile. (Wahnon, 1995) This dinuclear cobalt(III) system also

Figure 1.12 Double Lewis acid activation and efficient intramolecular nucleophilic attack provide up to 10"-fold rate acceleration with the dimeric cobalt(III1 cornplex. (Wahnon, 1995)

Figure 1.13 Tsubouchi and Bruice propose their dinucleating phosphonate derives much of its reactivity from Lewis acid stabilization of the aikoxide leaving group. (Tsubouchi and Bruice, 1994; 1995)

enabled, for the first time, an accurate quantitative assessrnent of the effectiveness of double Lewis acid activation; it accounted for approximately 105-fold rate acceleration in the deavage of phosphate diesters. (Williams and Chin, 1996)

Tsubouchi and Bruice (1994; 1995) have reported an incredible 1013-fold rate acceleration in the hydrolysis of a phosphonate ester with a novel dinudear approach. The tactics centered around two &quinolino1 moieties tethered by the phosphonate (Figure 1.1 3). Upon the addition of two equivalents of lanthanum(II1) the phosphonate was promptly deaved. Based in part upon molecular modeling of the dinuclear system the authors propose that the meclianism involves the Lewis acid stabilization of the leaving group. It is a frequent component of proposed enzymatic mechanisms but had not been previously demonstrated in a synthetic system.

No discussion of dinudear artificiai nudeases would be complete without giving due attention to lanthanide chemishy. This special topic will be

introduced and discussed as the thesis develops.

Plan of Study

The li terature to da te sugges ts that lanthanides are quite reactive towards RNA but no dear consensus or coherent mechanistic theory about th& reactivity exists. It was suspected that the full potential of the lanthanides had not ken tapped. If lanthanides are to be employed in the development of antisense reagents with any utility a solid understanding of the origins of the reactivity and

reaction mechanism are required. The study had several objectives. In the past, lanthanide-RNA reactions

had always been conducted under seemingly arbitrary conditions. It was important to study the chemistry under rationally assigned constraints. Thus it would be possible further the understanding and make meaningful cornparisons among the lanthanides and to other systems.

A sound understanding of the origins of the reactivity and the reactions' mechanisms would be had by logical investigations employing kinetic analyses, product analyses and titrations.

Essential components of al1 RNA cleavage (by transesterification) reactions are deprotonation of the 2'-hydroxyl, intrarnolecular nudeophilic attack and protonation of the leaving group. As a complement to other data solvent isotope effects would be employed to delve into this sequence of events, to explore the possibilities of concerted versus sequential events and learn how lanthanides involve themselves in these events.

Also induded in the study was to be an examination of reactivity towards DNA and its analogs.

Chapter II Rapid Cleavage of RNA by LanthanumUII)

2.1 Introduction to Chapter II: Some Previous Reports of Nudeic Acid Cleavage by Lanthanide Ions and Other Systems

Credit for the discovery of lanthanide-promoted deavage of phosphate esters must go to Bamann and Meisenheimer who made the first reports back in the late 1930s (Bamann and Meisenheimer, 1938a; 1938b; 1938c; 1938d) although it was not until several years later that they and other researchers began to venture the first simple mechanistic explmations for the reactivities observed. Çhimomura and Egami were the first to apply lanthanides to the deavage of RNA. Their reaction conditions were quite severe, employing temperatures approaching 100°C to achieve nearly quantitative dephosphorylation in a twenty-four hour period. Based on a colorimetric assay of the produds they suggested that deavage had occurred to produce S'-phosphate groups and 2'- and 3'-hydroxyl groups, contrary to what we understand today about RNA transes teri fication. (Shimomura and Egami, 1953)

Soon afterward, Bamann's group turned their attention to biological substrates as well, examining lanthanum(III) and cerium(III) reactions with RNA, DNA and monoribonucleotides. They pondered the possible role of the lanthanides in the transesterification and speculated that the metal ion may interact with the 2'-hydroxyl. (Bamann et al., 1954)

Butcher and Westheimer used 1 8 0 tracer experiments to determine that the site of cleavage was the phosphorus-oxygen bond as opposed to the carbon-oxygen bond in the reaction of lanthanum(II1) hydroxide gels with phosphate monoesters. Based on the faster rates obsewed with monoesters bearing P-methoxy or amino groups they proposed that these substituents coordinated to the lanthanum but played no direct role the chernical step. In a l i

of the studies to this point the reactions were heterogeneous; the lanthanurn(m) hydroxide preupitated. The dosing paragraph of their paper sums up what they and the other researchers must have felt at the time:

The exact statement of mechanism for a heterogeneous reaction is extraordinarüy difficult at the present the; a more accurate determination of the kinetics must, in al1 probability, await a determination of the kinetics of metal-ion promoted hydrolysis of a phosphate ester in homogeneous solution. (Butcher and Westheirner, 1955)

It was not until the 1990s that researchers began in earnest to investigate lanthanide reactivities again. Some of the investigations were straightforward cornparisons of some lanthanides to various O ther metd ions under a single set of conditions. (Breslow and Huang, 1991; Morrow et al., 1992a) These showed that the lanthanides were indeed more effective than the others but the differences were modest and little mechanistic insight could be had from thaï limited data. The half-Me of the diribonucleotide UpU in the presence of ImM europium(m) was 31.5hr at pH = 7 and 80°C. (Breslow and Huang, 1991)

The studies by Kuusela and Lonnberg were much more revealing. Unsatisfied wi th the single pH studies of others they undertook to probe the pH dependence and [Ln] dependence of the cleavage reaction of UpU- The experiments were carried out well below neutraiity (approximately pH = 4 + 6)

and using gadolinium(III) as an example they demonstrated that the reactions differed in character from those of some transition-metal ions. The partial pH-rate profile for gadolinium(III) was nonlinear (upward curvature) as was the [Gd(III)] dependence. Zinc(I1) by contrast gave linear plots for the partial pH-rate profile (same pH range) and [Zn(II)I dependence. This implied that where zinc(II) was functioning as a mononuclear species, probably by a bifunctional Lewis acid-intramolecular-hydroxo-base mechanism, the gadolinium(III) was functioning as a polynuclear species. Despite the undetermined order of the polynudear species and that the mechanism was not dear given the data at hand this was the first bit of real insight into the lanthanide reactions. (Kuusela and Lonnberg, 1993)

The Komiyama group has made numerous reports of lanthanide-promoted cleavage of RNA, DNA and DNA mode1 compounds as well as ring opening reactions of 3',5'-CAMP. (Komiyama, 1995, review) For example, they exploited the increased reactivity of thulium(III) at a higher pH to achieve RNA half-iives of approximately lOmin (pH = 8, 30°C). (Komiyama et al., 1992) They have also applied mixed metal-ion systems to RNA and DNA cleavage showing significant conversion within minutes (rate constants were not provided) at neutral pH and 50°C. (Irisawa and Komiyama, 1995)

The Komiyama group has made contributions to the field by detennining the activities of their systems under fixed conditions and demonstrated some novelty with the mixed metal-ion systems but unfortunately reveal little regarding the mechanisms in their publications. The orders of the reactions with respect to [Ln] and the pH dependence are usually not discussed. The authors

Scheme 2.1 Proposed mechanism for the lanthanum(III)-hydrogen peroxide promo ted deavage of the DNA model compound BNPP. (Takasaki and Chin, 1993; 1995)

generally fall back on the stock bifunctional mechanism of Lewis aad activation and intrarnolecular general base without apparent investigation or discussion of its justification, or consideration of alternatives. Nonetheless, they have reported some very respectable rates.

Takasaki and Chin discovered that hydrogen peroxide can be employed with lanthanides to produce remarkable rate accelerations (- 108-fold over background) for the cleavage of the DNA model BNPP. These were important reports not just because of the reaction rates but because they were the first to communicate full and systematic studies of a lanthanide system. Based upon the pH-rate profiles, concentration dependencies, titrations and 1 8 0 tracer stuàies they concluded that the reactive species was a dinuclear structure composed of two lanthanide(II1) ions bridged by two nucleophilic peroxide dianions. (Takasaki and Chin, 1993; 1995)

II was also discovered that the activity previously asaibed to cerium(III) for DNA cleavage (Sumaoka et al., 1992b; Komiyama et al., 1993b; Komiyama et al., 1993c) was actually due to the redox couple of cerium(III) and molecular oxygen. The reactive species was proposed to be a dinuclear cerium(1V)-peroxide complex formed in situ. The mechanism involved nucleophilic attack of a peroxo ligand upon the phosphate diester followed by a rapid breakdown of the peroxyphosphate intermediate, in a fashion similar to the lanthanide(II1)-hydrogen peroxide system. Thus although the reaction involved redox chemistry the reaction products were the same as for hydrolytic cleavage. (Takasaki and Chin, 1994)

The peroxide mechanisms of Takasaki and Chin were novel yet they were well rooted in experirnental fact and were further substantiated by Barnes' and Blyth's X-ray structure of the bisy-peroxo dicerium(1V) complex (Barnes and Blyth, 1985) which was at the heart of the mechanisms.

The recent years have witnessed other notable advances in the science that employed lanthanide macrocyclic complexes and nonlanthanide systems. Morrow, et al. (199213) employed a hexadentate Schiff base maaocyde to make mononudear complexes with several of the lanthanides. It was found that the lan thanum(II1) and europium(III) complexes were quite stable to deligation and that the latter was quite reactive in RNA transesterification; under pseudo-first-order conditions (excess complex) the half-life of ApUp was five hours and the complex was also catalytic, capable of eight turnovers in fifty hours. Stability of the system and catalytic activity are desirable qualities for the developmen t of an tisense ar tificial nucleases.

Morrow has also developed other mononuclear lanthanide complexes from cyclen ligands bearing four pendant alcohols. (Chin and Morrow, 1994; Morrow et al., 1995) These complexes were remarkably stable to deligation yet still possessed significant reactivity; the half-life of poly(A) was just one hour under pseudo-first-order conditions. A europium(III) complex had a titratable proton at pH - 7 but it was not clear whether that was due to a bound water or the ionization of one of the pendant alcohols. The kinetics were first order in complex and the pH-rate profile showed a plateau beyond pH - 8. Consistent with the data, the researchers suggested the kinetics were due to the bifunctional Lewis acid-intramolecular-general-base mechanism aibeit the identity of the general base would be unknown: hydroxo ligand versus pendant alkoxo ligand.

Even faster RNA hansesterification was achieved with a simple copper(II) complex. The half-life for ApA (IOmM complex, O.lmM ApA, pH = 7, 20°C) was only three minutes. The kinetics showed a first-order dependence on complex concentration ruling out a dinuclear mechanism. Chelation of the phosphate moiety (so providing more than just simple single Lewis acid activation) to the copper(I1) centre was suggested in explanation of the unusually high reactivity. The chelate could not only activate the phosphorus centre to nucleophilic attack but could serve to attenuate the developing negative charge in the transition state. (Linkletter and Chin, 1995)

The dinuclear strategy (see Chapter 1) has proven to be quite successful (Wall et al., 1993; Wahnon et al., 1994) and using dinuclear cobalt(III) complexes

it has been shown that the double Lewis acid activation of bridging phosphate diesters can provide up to five orders of magnitude in the rate acceleration. (Williams and Chin, 1996) This was first successfully applied to RNA transesterification with a simple dinudear copper(I1) cornplex which provided - 105-fold acceleraîion over the ambient rate. The same complex ais0 gave - 108- fold rate acceleration for the intermediate 2',3'-CAMP suggesting that for that reaction it was using a hydroxo ligand as an efficient intramolecular nudeophiie. (Young and Chùi, 1995)

Some of the RNA cleaving systems discussed above and some other prominent examples from the literature are compiled in table 2.1 below. These might be considered benchmarks by which to judge the results that follow herein.

0.49mM E U L ~ pH=7.15,37"C ApUp 5.Ohr e

0.2m1M Zn(tacd) pH=7.6, M0C ApUp 8.lhr - - - - -

II lOrnM Cu(neo) pH=7.0,2S°C ApA 3.0min h

Il 2mM C U ~ L ~ 1 pH=6.0,50°C 1 ApA 53min i

1 (el (Morrow ct al., 1992b3 (f) (Chin and Morrow, 1994) (g) (Shelton and Morrow, 1991) (hl (Linkletter and Chin, 1995) (i) (Young and Chin, 1995)

Table 2.1 Prominent examples from the literature of RNA cleavage by artifiual sys tems.

2.2 Results and Discussion

2.2.1 ApA 1s Rapidly Ueaved by Lanthanurn Ion at Alkaline pHs

It was found that lanthanum(m) was more efficient at promoting RNA transesterification than was ever realized. Quenched aliquots from the reaction were analyzed by HPLC to obtain a time-resolved product profile. This showed that at 25OC and moderately alkaline pHs the RNA dimer ApA was rapidly deaved by 2mM lanthanum(IIi) yielding only the products of transesterification and hydrolysis, 2'-adenosine monophosphate, 3'-adenosine monophosphate and adenosine. The reaction was observed to go to completion and, provided the solutions were thoroughly degassed, preapitation did not occur (Figure 2.1).

Product analysis was detennined by separate HPLC analysis of authentic standards (Figure 2.2a) and by spiking reaction products with authentic

0 E 4 6 8

Retention time (min)

Figure 2.1 Typical stacked chromatograms from the lanthanum(II1)-promoted transesterification and hydrolysis reaction of ApA. [La] = 2.00mM. [ApA] - 0.025mM8 [CHES] = 20.00rnM, pH = 8.89, T = 25 OC. Chromatograms are (front to badc) of quenched aliquots taken at 15,30,45, 60, 90,180 and 720s. Peak are: 3'-AMP (3.6min), sodium p-nitrobenzenesulfonate (interna1 standard, 4.7min), 2'-AMP (8.0min), adenosine (SSrnin), ApA (1 1 Amin).

2 4 5 a Retention time (min)

Retention time (min)

1

I

J b

- 2 _ / 4 -1-i 6 8 ,L 1 6 - 12

Retention time (min)

e 4 7

6

5

I

Figure 2.2 (previous page) HPLC chromatograms. (a) An approximately equimolar mixture of (in order of appearance) 3'-AMP, 2',3'-CAMP, 2'-AMP, and adenosine. Relative integrated peak areas are 1.9, 1.0, 1.8 and 1.7 respectively. (b) Product profile for the lanthanum(III)-promoted ring-opening of 2'3'-CAMP. [La] = 2.00mMf [2',3'-CAMP] - 0.025mM, [CHES] = 20.00mMf pH = 8.95, T = 25 OC. Chromatogram is from a quenched aliquot taken at five seconds. (cl Adenine. Concentration is approximately the same as for those in Figure 2.2a and the relative integrated peak area is 1.5

standards (not shown here). The common RNA transesterification intermediate 2',3'-cydic adenosine

monophosphate did not accumulate but was most certainly involved as

evidenced by the production of both 2'-AMP and 3'-AMP in roughly equal amounts. Also when 2',3'-CAMP was employed as a substrate itself the ring-opened products were produced in the same ratio as from the ApA reaction (Figure 2.2b).

Oxidative deavage was not occurring as evidenced by the absence of the free base adenine in the product profile, an attribute of oxidative mechanisms (Figure 2.2~). This was not surprising as lanthanum(IIi) is not pa r t i dady redox active.

Between pH-9 and p H 4 0 the half-life for the lanthanum(III)-ApA reaction was only approximately 13 seconds. This result constituted the fastest nonenzymatic deavage of RNA reported at that time, not just for lanthanides but other metal ions, metal-ion complexes and metal-free systems as weil (see Table 2.1 earlier).

2.2.2 Titration of Lanthanum(II1) Chloride

One of the first ches as to why the lanthanum(III) reaction should be so fast came from the potentiometric titration of lanthanum(III) chloride. To mimic the reaction conditions as dosely as possible the titration was carried out upon 2mM lan thanum(II1) chioride as was used in the reaction.+

t This was not just a triviality; it was found that the titration curve would shift to lower values as lanthanum(I1I) concentration was increased.

32

A stock solution of 20mM lanthanum(Ei) chloride was standasdized by dilution and titration (NaOH) in the presence of an excess of standardized EDTA. The titration of 2mM lanthanum(l1I) revealed the consumption of a nons toichiometric 2.5 equivalen ts of hydroxide. Such nonstoichiometric titrations are common among the lanthanides. (Aksel'rud and Spivakovskii, 1960a; 1960b; 1960c; Aksel'rud and Emolenko, 1961)

The pH1/2 was approximately pH = 8.9 and preapitation did not occur

until after al1 the lanthanum(IIl) had been titrated (Figure 2.3). The abmpt curvature of the plot suggested that dimerization or higher ordered aggregation was occurring as had been observed in another study of a lanthanide system. (Takasaki and Chin, 1993)

2.2.3 A Mode1 for a Dimeric Lanthanum(II1) Species

The stoichiometry of the titration can easily be accommodated by invoking a simple dimerization reaction. (Scheme 2.2)

Scheme 2.2

The equations for the formation constant (Equation 2.1) and the mass balance (Equation 2.2) may be solved for the proposed dimer's concentration expressed as a function of hydrogen ion concentration. (Equation 2.3)

Equation 2.1

Equation 2.2

(Where &alT is the to ta1 lanthanum(III) added to the system).

Equation 2.3

(Where Kw is the auto-ionization constant of water. See Appendix 1 for the f d derivation of Equation 2.3).

Furthermore, the concentration of the dimer at a given pH during a titration can be related to the equivalents of hydroxide added through Equation 2.4.

Equation 2.4

Equation 2.4 was used to fit data from the titration providing the value for the formation constant of the dimeric speues, K = (2.9 t 0.3) x 1028 M-6 (Figure 2.3).

The reader may wish to point out that in the titrations of uncomplexed metal ions it is often viewed that the products are complex mixtures of metal hydroxides of varying order (monomeric, dimeric, trimeric, etc.). Indeed, efforts to characterize many such systems have further suggested that the composition of the systems can change throughout the titration. (Baes and Mesmer, 1976) However, without adopting a simple mode1 and hypothesis for the lanthanum(EI) titration the observations would be difficult to rationally explain and predictions difficult, if not impossible, to make.

It is entirely believable that a dimer is the first product to form from monomeric ions. A termolecular aggregation step might be conceived as occurring but there would not be any mechanis tic requirement for that; an initiai bimolecular aggregation would be, by far, the dominant process.

It was observed that precipitation was a rather slow process in these titrations. In fact, when solutions were thoroughly degassed and volumetric

equivalen ts NaOH

Figure 2.3 Titration of lanthanum(II1) chloride with sodium hydroxide.

[La] = 2.00mM, [NaOH] = IOOmM, [KCI] = IOOmM, vol = 50mL. The curve is

calculated from Equation 2.4

standard NaOH was used addition of 2.5 equivalents of base to the 2mM lanthanum(III) gave a homogeneous solution. In a stoppered via1 such a solution \vas often stable (no turbidity or precipitate) for more than a day. This would suggest that if higher ordered speues are present that they are in dynamic equilibrium with more soluble species of lower order (i.e. that the aggregation

processes are reversible).

Given the stability of the system and accepting the dimer as an initial product is appropriate to apply Ockham's Razor (the principle of parsimony) (Hoffmann et al., 1996) to the system to render it intelligible and permit the formulation of a good, simple scientific hypothesis, thereby enabling explanation and interpretation of the observations, and prediction. (Hempel, 1966) The

simplest model that can accommodate the nonstoichiometric requirement for hydroxide is the one based upon the dimer. Integer multiples of the dimer also fit the titration and ultimately these higher ordered aggregates are quite possibly formed. However, the dimer model is also easily incorporated into a kinetic model for the reaction. It is then, readily subject to experimental test.

2.2.4 pH-Rate Profile for Lanthanum-Promoted ApA Cleavage

The dimer model was used to develop the kinetic model for the system

time (s)

Figure 2.4 Typical kinetic data for the lanthanum(II1)-promoted transesterification of ApA. [La] = 2mM, [ApA] = 0.025mM, [CHES] = 20.00mM,

pH = 9.59, T = Z°C. ApA/IS is the ratio of ApA to the intemal standard, sodium nitrobenzene sulfonate (- 0.025mM). The cuve is a simple first-order decay fit to approximately the first two hdf-lives giving kOb, = 0.0559s-l.

Figure 2.5 pH-rate profile for the lanthanum(III)-promoted tramesterification of ApA. [LaIT=2.OOmM, [ApA]-0.025mM, [EPPS or CHES]=ZO.OOmM, T=25.0°C.

Data points are averaged from at least three determinations. Error bars are f three standard deviationç. k O b , the pseudo-first-order rate constant, is in units of reuprocal seconds. The curve is calculated from Equation 2.5.

according to Equation 2.5.

Equation 2.5

kobs = k[La2 (OH); I

(Where k is the second-order rate constant).

The pH dependence of the lanthanurn(III)-ApA reaction was detennined

by constructing the pH-rate profile in CHES and EPPS buffers. Excess lanthanum(II1) was employed providing pseudo-first-order conditions. Kinetic data was fit to a simple first-order decay oves the e s t two haif-lives (Figure 2.4).

The kinetic expression above was found to provide an excellent fit to the pH-rate profile data (Figure 2.5). This provided values of K = (2.8 f 0.6) x 1 0 ~ M - ~ and k = 57 IT 6 Lqmol-1s-1. Thus the K values from the titration and the pH-rate profile were in excellent agreement.

Aside from the close correspondence in the K values the pH-rate profile also exhibited a very steep slope = 5 prior to the pHl/* supporting the fifth-order dependence upon hydroxide for formation of the dimer as the active species. Also noteeworthy was the plateau, indicating that once the formation of the active species was quantitative there was no more requirement for hydroxide in the cleavage reaction, or at least in the rate-determining step. This was a signifiant feature of the reaction given that intermolecular base catalysis is a frequent component of mechanisrns for transesterification reactions of RNA and its model compounds. e.g. (Fersht, 1985; Breslow and Huang, 1991; Chandler and Kirby, 1992; Chin and Morrow, 1994; Breslow et al., 1996)

2.2.5 Second-Order Dependence upon Lanthanum(II1) Concentration

The data from the pH-rate profile fit the model quite well but it was of critical importance to establish the order of the reaction with respect to the lanthanum(1II). To do this the kinetics were run at varying concentrations of the lanthanum(II1).

Careful consideration was given to the effect pH would have upon the outcome of the experiment. If the active species was a dimer or some higher ordered species then the experiment would be most sensitive to the varying concentration if carried out below the pH1/2 of the lanthanum(EI). Returning to

Scheme 2.1 one can see that the experimental outcome is dependent upon the formation constant. If the experiment was to be carried out well above the pH1/2 then doubling [LaIT would simply double the concentration of the dimer or any other higher ordered species which may be active. The reaction would erroneously appear to be (or approach) first-order in lanthanum(III). (See Appendix 2 for a mathematical and graphical illustration of how the apparent reaction order will Vary with pH).

By carrying out the experiment below the pH1l2 the reaction will be

Figure 2.6 Dependence of the (logarithm of the) pseudo-first-order rate constant, kOb, , for the transesterification of ApA upon (the logarithm of') [La]=. [La]T = 1.00 + 2.00mM, [ApA] - 0.025mM, [EPPS] = 20.00mM, pH = 8.58 f 0.02,

T = 25OC. Data points are averages of ai least three determinations. Enor bars represent t three standard deviations. kOb, is in units of reciprocal seconds. [LaIT is in units of mol-L-1. Slope of the linear regression is 2.00 f 0.06.

sensitive to the varying concentration by virtue of the formation constant, K. Only then could the experimental outcome possibly refute the dimer hypothesis, revealing the active speues to be monomeric or of an order higher than two.

The experiment was carried out at pH = 8.58 f 0.02. Since the experiment was conducted upon the steep slope of the pH-rate profile it was necessary to ensure that for al1 concentrations of lanthanum(m) used the pHs were in very dose agreement.

Over the range of [La]T = 1.00 + 2.00 the reaction was found to have a

Figure 2.7 Proposed structure of the dimeric active speaes.

second-order dependence upon lanthanurn(m) (Figure 2.6). This was strong support for the proposed dimer-as-active-species model.

2.2.6 Reaction Mechanisms

The exact structure of the dimer is unknown. Deliberate preapitation with excess hydroxide produces only amorphous flocs and nothing suitabie for detailed X-ray analysis. A proposed structure is shown in Figure 2.7.

Recalling the titration, there is one pK, or plateau in the plot. This

indicates that the coordination of the hydroxides to the lanthanum is cooperative and that the hydroxo ligands bridge the two lanthanum centres. If some of the hydroxo ligands were not bridging then one would expect to see multiple pK,s

in the titration. For example, mer-triaquo cobalt(I1I) complexes can possess three discrete pK,s, one for each aquo ligand. The predominant speaes throughout the

titration are monomeric. Compare this to fac-triaquo cobalt(m) complexes which when titrated can display a single 1.5-equivalent pK, whereupon a

tris-CI-hydroxo dinuclear complex is formed quantitatively. (Schwarzenbach et al., 1971; Wieghardt et al., 1979)

The proposed structure of the penta-p-hydroxo lanthanum(III) dimer is also reminiscent of a crystal structure of a dinuclear cerium(1V) complex with two peroxo ligands bridging in a face-on marner such that each metal centre coordinated four oxygens. (Barnes and Blyth, 1985; Barnes et ai., 1987) This mode of peroxo coordination to lanthanides lies at the heart of Takasaki's and Chin's novel mechanisms for phosphate diester deavage by the cerium(III)-cerium(IV) redox couple and by the lanthanum(III)-hydrogen peroxide couple. (Takasaki

and Chin, 1993; 1994; 1995)

A phosphate moiety could bridge the two lanthanum(III) centres in the same fashion as has b e n shown by X-ray analysis for a bis-p-hydroxo dinudear cobal t(IJI) complex. (Wahnon, 1995) An X-ray structure of a dinudear cerium(III) complex has shown that acetates can bridge the two lanthanide centres in the same manner. (Panagiotopoulos et al., 1995) This mode of binding ApA to the

dimer would provide double Lewis aud activation of the phosphate toward nucleophilic attack which has been shown to provide a substantial component of the rate acceleration of phosphate diester hydrolysis. (Williams and Chin, 1996)

Based upon the results to this point there were two mechanisrns which could be discarded right away. The product analysis by HPLC showed the production of both the 2'-AMP and 3'-AMP. Thus intramolecular nucleophilic attack of a bridging hydroxo ligand was not occurring. (Scheme 2.3a) This was not expected to happen anyway given the presence of RNA's own efficient intramolecular nucleophile, the 2'-hydroxyl oxygen. Furthermore, if a bridging hydroxo ligand was the nucleophile the product profile would dictate that the resulting 3'-AMP be isomerized to give the observed ratio of monophosphates; metal ions are known to be ineffective in promoting the rapid interconversion of

a

Scheme 2.3

RNA 2'- and 3'-monophosphates. (Kuusela and Lonnberg, 1993) Scheme 2.3b describes a mechanism that is consistent with al1 the results

so far. It implies that by coordinating to a lanthanum(III) centre that the 2'-hydroxyl may spontaneously ionize and that the coordinated alkoxide is the thermodynamically more stable species. The possibility that coordination of the 2'-hydroxyl group to a metal may contribute to the rate acceleration of the transesterification has also been noted by other researchers. It was entertained as plausible although the researchers apparently favored the intramolecular base mechanism; no additional support codd be found in the experiments' results to justify its serious consideration over the popular alternative. (Breslow and Huang, 1991; Uebayasi et al., 1994; Chapman and Breslow, 1995; Kuusda et al., 1995; Sawata et al., 1995)

Intramolecular nudeophilic attack of alcohols has been shown to occur for copper(II) complexes (Young et al., 1995) and for lanthanide complexes as well. (Morrow et al., 1995) In these cases the alcohols were part of the ligands and the substrates were activated DNA mode1 compounds.

For the substrate bis(2,4-dinitrophenyl) phosphate (BDNPP) and a copper(I1) complex the ligand's pendant alcohol was suffiaently activated by the metal centre such that the usually efficient mechanism of intramolecular nucleophilic attack of a hydroxo was rendered ineffective; transesterification products dominated. However when the same copper(I1) complex was applied to

BNPP hydrolysis the product distribution changed dramatically; the pendant alcohol was no longer effective for the less activated substrate and the reaction products were predominantly those of nudeophilic attack by the metal-hydroxo.

This demons trated that the metai-alkoxo pathway would not be effective for substrates with poor leaving groups. In the case of the cornpetition between metal-hydroxo and metal-alkoxo nucleophiles it was proposed that the former dominated with the less activated substrate because the transition state was attained though a deprotonation of the attacking hydroxo; the p-nitrophenolate was then a better leaving group than the nucleophile. As for the coordinated alkoxide, it was proposed to be the better leaving group in the BNPP reaction so that its nudeophilic attack was not productive. (Young et al., 1995)

In the case of the lanthanurn(m)-ApA reaction the cleavage step is always transesterification yet the metal-alkoxo mechanism in Scheme 2.3b can be ruled out here as well based upon a similar leaving group argument; uniess the leaving 5'-alkoxide was stabilized to a comparable degree (by coordination) the

a

Scheme 2.4

coordinated 2'-alkoxide would be the better leaving group in the phosphorane transition state. Leaving group activation or assistance is proposed for a number of enzyme and ribozyme systems and is an attractive explanation (in part) for their reactivities. (Beese and Steitz, 1991; Steitz and Steitz, 1993) To date, only one synthetic system can be argued to provide Lewis acid stabilization for a poor leaving group and that is the Tsubouchi and Bruice (1994; 1995) bis(quinolino1) system (Figure 1.13, Chapter 1).

Scheme 2.4a is consistent with the plateau in the pH-rate profile; once the dimerization is quantitative there would be no further hydroxide requirement upon the part of the lanthanum(m) and so the generation of the intramolecular general base would be complete.

Although intramolecular general base catalysis is generally not regarded as being very effiaent in terms of effective molarity (Kirby, 1980) the concept of a hydroxo ligand acting as an intramolecular general base is nonetheless a reasonably attractive one. It has been drawn upon by several researchers in formulating mechanisms for RNA transesterification reactions. Some commoniy cited justifications for the bifunctional Lewis acid activation-inbamolecular general base mechanism are: (1) that the metal complexes' catalytic activities

show a first-order dependence upon hydroxide below the pK,; (2) that the pH-rate profiles exhibit plateaus above the pK,; (3) that organic bases of similar

pK, are less efficient general bases; (4) that metal hydroxo complexes with no

available coordination site are poor catalysts, and; (5) that the relative activities of various metal ions and their complexes correlate with the pKas of their aquo

forms.

This bifunctional motif has been applied in ewplanation of RNA (and RNA mode1 compound) kinetics involving uncomplexed transition-metai ions (Morrow et al., 1992a; Kuusela and Lonnberg, 1993; 1994b; Kuusela et al., 1995) and their complexes (Stem et al., 1990; Shelton and Morrow, 1991; Kuusela and Lonnberg, 1994b), cooperative catalysis by metal-ion mixtures (Irisawa et al., 19951, the magnesium requirements of some ribozymes (Uchimam et al., 1993; Uebayasi et al., 1994; Sawata et al., 1995), and RNA transesterification by lanthanide ions (Komiyama et al., 1992) and their maaocyclic complexes. (Chin and Morrotv, 1994)

The plateau in the pH-rate profile was a strong indication that in the rate-det ermining step depro tona tion of the 2'-hydroxyl by free hydroxide or a kinetic equivalent was not a requirement. (Scheme 2.4b) However, that

deprotonation could occur in a different chernical step; equilibrium deprotonation by an intermolecular base prior to the rate-determining step would be consistent with the results.

It is not possible to distinguish between mechanisms 2.4a and 2.4b given

the results thus far. However, it is still appropriate to put those mechanisms

under the microscope and pose more questions. RNA transesterification requires

a deprotonation of the nudeophilic 2'-hydroxyl and a protonation of the leaving

5'-hydroxyl group. 1s the deprotonation concerted with the attack of the

2'-oxygen? Or does it occur in an earlier and separate step? What is the ra te-de termining s tep in the sequence of even ts leading to the deavage? Does the penta-p-hydroxo dimer really play a part in an intramolecular deprotonation of the 2'-hydroxyl? These questions will be considered further in the following chapter where the focus shall be upon the proton transfer from the 2'-hydroxyl

group.

2.3 Summary and Condusion to Chapter II

Lanthanum(II1) promotes the tramesterification of the RNA dimer ApA with a half-life of approximately 13 seconds at the maximal rate. This is the fastes t RNA cleavage repor ted to date. The potentiometric tihation of lanthanum(III) chioride, the pH-rate profile and the concentration dependence of the reaction al1 point to a penta-p-hydroxo lanthanum(III) dimer as the active speaes in the reaction. The pH-rate profile indicates that the moderately high pH for the reaction is simply a manifestation of the dimerization and that the transesterification reaction has no rate-determining requirement for free hydroxide.

Two reaction schemes can be conceived as being consistent with the results to this point. One features a p-hydroxo ligand functioning as an intramolecular general base for deprotonation of the 2'-hydroxyl group. The alternative is a preequilibrium intermolecular deprotonation followed by the nucleophilic attack.

For the sake of cornparison one can estimate the hydroxide-catalyzed background rate of ApA transesterification, based upon the linear free energy relationship that exists between the (logarithm of the) second-order rate constant for the hydroxide reaction and the pKa of the conjugate aud of the leaving group. (Davis et al., 1988a)+ If we take the pK, of the leaving 5'-hydroxyl as approximately pH = 16 and the pH of the reaction as 9 we arrive at an estimated half-life of approximately 5.3 x 106 s (-62 days). From the lanthanum(III) pH-rate profile at pH = 9 k O b , = 0.0419s-1 and so the half-life for the 2mM lanthanum(XII)-promoted reaction is approximately 16.5s. That would mean the lanthanum(II1) dimer is providing an approximately 320 000-fold acceleration over the ambient rate.

Hydroxide rates have been directiy measured for some diribonudeotides. Lonnberg's group have determined second-order rate constants for the hydroxide reaction with UpU at elevated temperatures. (Jarvinen et ai., 1991; Kuusela and Lonnberg, 1994a) Their values have k e n used to estimate* the second-order rate constant for hydroxide catalyzed transesterification of ApA, kZ - 3.2 x l W 3 ~ - m o l - ~ s - ~ at T = Z°C. (Williams and Chin, 1996) At pH = 9 then

t For aryl uridine-3'-phosphates at T = 25°C. $ Rate constant extrapolated from Lonnberg's data at T = 90°C and 60°C, and compensateci for inherent differences in reactivity between UpU and ApA.

the ambient reaction would have a half-life of about 2.2 x 107s (-250 days). Using the value derived from Lonnberg's work as the yardstick we arrive at a 1.3 x 106-fold acceleration over the background reaction.

These estimates of the background reactions differ owing to the methods and data employed but the difference is not great (-four-fold). The distinction is acadernic.

Consider though tha t given that the lanthanum(III)-promoted reac tion has no requirement for intermolecular base catalysis it might not be unreasonable to directly compare the half-life of this reaction to those of other systems at lower

pHs. The implication here is that if such a dimeric lanthanide speaes could be made to exist at lower pHs the half-life for the transesterification might be unchanged, or at least comparable. The acceleration over the ambient hydroxide

rate though would then be much greater. This point is addressed in Chapter IV.

2.4 Experimental

Water (deionized and distilled) was degassed under vacuum, and kept oxygen and carbon dioxide free under argon.

Other materials were as follows: lanthanum(III) chloride heptahydrate (ACS, Aldrich), CHES (ACS, Aldrich), EPPS (ACS, Aldrich), potassium chloride (ACS, BDH), sodium hydroxide (volumetric standard, 0.100M, "low carbonate*'? Anachernia), disodium EDTA (ACS, Fisher Saentific), sodium phosphate monobasic (HPLC grade, A&C), methanol (HPLC grade, BDH), ApA (98%, ICN), 2',3'-CAMP (98%, Aldrich), 2'-AMP (Sigma), 3'-AMP (99%, Aldrich), adenosine (99+%, Aldrich), adenine (99751, Aldrich), nitrobenzene sulfonic acid sodium sait (m- and p- isomers, Eastman-Kodak).

Titra tions

Titrations were carried out using a Mettler DL-21 titrator with a Mettler combination electrode. Data was collected electronically via an interfaced Packard Bell PC running TS1 titration software from McIntosh Analytical Systems. The data was transferred to a Macintosh computer, and processed and plotted using Kaleidagraph 3.0.1 from Abelbeck Software. Solutions were prepared in volumetric glassware and solutions were handled using volumetric pipettes and Eppendorf adjustable pipettors.

Primary standard EDTA was prepared by heating ACS grade disodium EDTA in an oven at -120°C overnight and cooling in a vacuum dessicator. A stock solution was prepared volumetrically from degassed water. This solution was standardized with volumetric standard NaOH and determined to be 0.126M.

A stock solution of lanthanum(III) chloride (prepared volumetrically in degassed water) was standardized by titration with excess EDTA. Cornparison of the NaOH consumption before and after addition of a measured aliquot of Ianthanum(1II) chloride allowed calculation of the lanthanum(II1) concentration, 20.0mM. Ionic strength was maintained with 0.100M potassium chloride.

5.000mL of the lanthanum(II1) solution and 5.000mL of 1M KCL were diluted to 50.000mL and acidified with concentrated HCL. This solution was then titrated with 0.100M NaOH to pH = 11. Consumption of NaOH by the

lanthanum(II1) was measured as that consumed between the inflections before and after the pH1/2 Data was corrected for the dilution that occurs during

titration and the water blank waç subtracted from the NaOH delivered during the titration. (Dick, 1973)

Kine tics

Stock solutions of lan thanumo chloride (20.0mM, standardized), CHES (100mM) and EPPS (100mM) were prepared volumetrically, degassed under vacuum and kept under argon. Buffers were adjusted with concentrateci NaOH to the approximate desired pHs for the reactions and degassed. Equd volumes of lOmmM ApA and lOmM nitrobenzene sulfonic acid sodium salt (intemal standard, 1s) were mixed to give a solution 5mM in each. Sodium hydroxide (volumetric standard, 0.100M) was degassed under vacuum and kept under argon.

In a typical kinetic experiment, measured (Eppendorf) volumes of the buffer, NaOH and lanthanum(II1) solutions were diluted with water to 2.000mL. The reaction would be carried out in a small glass via1 and magnetically stirred under a slow flow of argon. Temperature was maintained at 25OC with a jacketed beaker and thennostatted water circulator. The reaction was initiated with the injection of 10pL of the ApA/IS solution. pH was measured before and verified after the reaction with a Fisher Accumet 15 pH meter and an Orion 8013 Ross semimicro combination electrode.

Aliquots were removed from the reaction at measured intervals and rapidly quenched in 0.2M ammonium phosphate buffer (pH = 5.5) and then centrifuged (130 000 RPM, 5 minutes) to remove precipitates.

The supernatant was analyzed using a Hewlett-Packard 1090m HPLC with an autosampler unit and a Hewlett-Packard 2.1x100mm 5 p ODS Hypersil Cl8 column. Products were eluted by running O.SmL-min-l of 0.2M ammonium phosphate buffer (pH = 5.5) for five minutes followed by a linear gradient to 50% of a 60 /40 methanol/water solution over the next 10 minutes. Peaks were detected by UV absorbance at 254nrn. Column temperature was maintained at 40°C.

The chromatograms were integrated with a Hewlett-Packard 9153c computer and the data (ratio of ApA peak area to that of the intemal standard) was processed (plotted and curve-fitted) using Kaleidagraph 3.0.1 on a Macintosh computer.

2.5 Appendix 1: Derivation of Equation 2.3

Scheme 2.1 desaibes the equilibrium reaction of lanthanum(II1) ions with added hydroxide.

Scheme 2.1

Equation 2.1 is the expression for the formation constant of the dimer.

Equation 2.1

The total lanthanum in the system at any given point in the titration must obey the following mass balance.

Equation 2.2

(Where [La3+lT is the total lanthanum(III) added to the system).

Using the mass balance we may make a substitution for the monomeric ion in Equation 2.1.

Hydroxide ion may be substituted for hydronium ion using the expression for the auto-ionization of water to provide

where Kw is the auto-ionization constant of water. After expanding the squared term in parentheses and rearranging we arrive at

which is of the form ux* + bx + c = O . Applying the solution for quadratic equations and simplifying provides Equation 2.3.

Equation 2.3

2.6 Appendix 2: Mathematical Illustration of Reaction Order versus pH for a Dimeric Active Species

The following is meant to illustrate how dramatically the apparent reaction order with respect to lanthanum(III) concentration could Vary with pH according to the dimer-as-active-species model. Equation 2.3 c m be used to calculate dimer concentration (directly proportional to kobs) from total

lanthanum(III), pH, K and K w . Thus plots of loga~a3+lT) versus

log([La2 (OH); 1) at given pHs show the apparent order. The plot below shows

the calculated apparent order at pH = 8.35, 8.85 (the pH1/2) and 9.35 for the

concentration range 1 + 2mM.

Figure A2.1 Apparent reaction order versus pH for a pH112 = 8.85- pHs and

dopes for the hypothetical reactions are: (a) pH = 8.35 (m = 1.991, (b) pH = 8.85 (m = 1.39) and (c) pH = 9.35 (m=1.02). log([dimer]) is directly proportional to

ko bs -

Appendix 3: Tabulated Kinetic Data

Table A2.1 Complete data for the pH-rate profile of the lanthanum(III)-promoted transesterification reaction of ApA. [La] = 2.00mMf [ApA] - 0.025mMf [EPPS or CHES] = 20.00mM, T = 25°C. Values were nof rounded oJf until al1 calculations had been cornpleted.

[La 1, mol/ L IL

Table A2.2 Complete data for the lanthanum(1II) concentration dependence of the transesterification reaction of ApA. [ApA] - 0.025mM, [EPPS or CHES] = 20.00mM, pH = 8.58, T = 25OC. Values were not rounded off untii ali calczdations hnd been cornpleted.

Chapter III Kinetic Isotope Effects

3.1 Introduction to Chapter 1 II: Exploring Transesterification Mechanisms in RNA

At the end of the previous chapter were presented two kinetically indis tinguishable mechanistic schemes. By the defiition of kinetic equivalence they are both consistent with the data and would have identical rate laws; the stoichiornetric composition and charge of the transition state wouid be expected to be the same for each medianism. (Jendcs, 1987)

The RNA deavage reaction seems deceptively straightfonvard when one considers that the individual events are conceptually quite simple; deprotonation of the 2'-hydroxyl, nucleophilic attack on the phosphate, leaving group protonation, and hydrolysis of the cyclic intermediate. ALI investigations (and some times the ensuing deba tes) in to RNA transes terifica tion are concemed wi th when and how these events occur.

The situation becomes more complicated when one pauses to consider which event might be rate-determining and if some events occur in a sequential or a concerted fashion. Additionally the question usually arises as to whether or not in a given system the phosphorane species is in a transition state or is a genuine intermediate; it has never been directly observed in any instance.

Scheme 3.1 is an illustration of such a transesterification reaction reduced to its elementary, essential events. From these three events there are four conceivable combinations of concerted and sequential steps:

(1) al1 even ts occur sequentially (s tep-wise), [Il-[2]-[3]; (2) the first two events are concerted followed by the third, [l-21-[3]; (3) the first event is step-wise foliowed by two concerted events, [Il-[2-31,

and; (4) al1 even ts occur in a concerted fashion, El-2-31.

At this point consider what the term "concerted" implies. Jencks has illustrated how nudeophilic addition-elimination reactions vary from sequential general acid-base u ncatalyzed mechanisms through to general aud-base catalyzed, concerted mechanisms as a function of the stabilities of the reaction in termedia tes.

Scheme 3.1 The transesterification reaction of RNA reduced to its elementary even ts. Par tid structures shown for simpiici ty.

The former are characterized by very stable intermediates with Iifetimes long enough to permit diffusion through the solvent and abstraction (or donation) of a proton from (to) the solvent. General acid-base catalysis is not observed or is very weak.

The latter are characterized by "intermediates" of such high instability that the barrier for breakdown to reactants is insignificant. That being the case its lifetime will be shorter than a vibration frequency and the nudeophilic addition occurs in concert with the general acid-base catalysis. The "intermediate" would actually then be in the transition state and not really an intermediate at dl . (Jencks, 1980)

Applying this to RNA transesterification reactions w e can surmise whether or not a given reaction may be proceeding according to [l-2-31 or [ 11 - [2-31 (concer ted addi tion-elirnina tion) versus [Il-[2]-[3] or [l-21-(31 (sequential addition and elimination s teps). The intermedia te or transition state in question there is the phosphorane addition product. If the phosphorane is sufficiently stable then it may have a lifetime long enough to permit pseudorotation and so reversion to "reactant" could produce the isomerized 2',5'-diester. That would be indicative of sequential addition and elimination reactions, [Il-[2]-[3] or (1-21-[3]?

Such isomerization is known to occur in some specific instances. For example, the isomerization of 3',5'-UpU to the 2'5'-isomer (and vice versa) is acid-catalyzed below pH - 3 and above that proceeds at a pH-independent rate in the absence of buffers or other catalysts. (Jarvinen et al., 1991) In the presence

t This may be somewhat an oversimplification; although the pseudorotation involves no bond formation or fission it would not be an energy-free process. That is, there would be some small encrgy barrier associated with the interchange of axial and equatorial positions and in some instances this might contribute to the absence of the 3' + 2' migration.

of imidazole-imidazolium buffer the rate of isomerization appears to be accelerated. (Anslyn and Breslow, 1989a) Both of these observations were rationalized in terms of phosphorane intermediates in various states of pro tonation.

The isomerization is not observed to be accelerated in the presence of metal-ion catalysts or metal-complex catalysts. (Breslow et ai., 1989; Kuusela and Lonnberg, 1993; 1994b) This suggests that either coordination to the metal ion hinders pseudorotation or that in some way phosphorane stabilization by protonation differs from that by Lewis acids (i.e. that stabilization by protonation provides a stable phosphorane intermediate whereas stabilization by Lewis aads provides unstable phosphorane transition states or phosphorane intemediates w herein pseudoro ta tion is res tricted).

Bovine panaeatic ribonudease A ( N a s e A), perhaps the best understood RNase - enzymatic or otherwise - conducts the cleavage reaction in a concerted fashion. The deprotonation, nucleophilic attack and leaving group protonation are believed to occur simultaneously. RNase A does not catalyze the isomerization reaction. (Fersht, 1985) Whether or not the phosphorane is in the transition state or is a genuine intermediate is debatable. The enzyme imposes some stereochemical restraints on the reaction as well as the secure binding of the

subshate which could ensure a concerted reaction through an enforced catalysis mechanism. (Jencks, 1980)

Researchers have taken several different approaches to the ribonuclease puzzle (and that of other N a s e systems) over the years, making use of mode1

compound studies, computational methods and isotope effects. Davis et al. (1988a) have made considerable use of leaving group modifications in their studies of RNA transesterification. Studies of hydroxide- and imidazole-catalyzed deavage of a series of aryl uridine 3'-phosphates indicated that most of the charge buildup in the transition state resided on the attacking 2'-nucleophile. Leffler a values for the ArO-P fission and the 2'-0-P bond formation were 0.34 and 0.33 respectively (Figure 3.1), suggestive of an expanded or exploded transition state where bond breaking and forming are "only weakly advanced on a transition state that lies on a concerted pathway."

In the RNase A catalyzed reaction of the aryl uridine 3'-phosphates the charge buildup on the leaving oxygen was found to be much less (+0.55) than in the hydroxide and imidazole reactions (+0.15). (Davis et al., 1988b) This was taken as support for electrophilic catalysis by either Lys-41 (electrostatic

Figure 3.1 William's effective charges and Leffler a values for the nonenzymaüc transesterification of aryl uridine 3'-phosphates. (Davis et ai., 1988a)

interaction of the cationic amine with the phosphoryl group) or by His-119 (protonation of the nonbridging phosphoryl oxygens) in the enzyme as has been proposed by others. (Eftink and Biltonen, 1983b; Fersht, 1985; Breslow, 1991a; 1991b)

Clever design of model compounds and meticulous experimentation are the forte of Kirby's group, who are keenly interested in al1 manner of intramolecular reactions. A model for the ribonuclease reaction was used to show how modes of general acid catalysis can Vary according to reaction conditions (pH and identity of the general acid). Both the protonation of the nonbridging phosphoryl oxygens and general acid assistance for the leaving group were proposed as well as an electrostatic interaction or hydrogen bonding between the phosphorane and certain dicationic buffers such as the tetrarnethylethylene diammonium dication (Figure 3.2). That electrostatic stabilization is rerniniscent of the proposed function of Lys-41. In this model system pseudorotation products were not observed, perhaps for steric and electronic reasons it was suggested, but a phosphorane intermediate was proposed. (Daiby et al., 1992;

1993) Phosphoranes have also been studied with computational methods. Ab

ini tio calcula tions for the base-ca talyzed hydrol ysis of ethylene phosphate and for dimethyl phosphate seemed to indicate that dianionic phosphoranes can exist as genuine intermediates in alkaline solution. (Taira et al., 1993) This, say the authors, is supported by the observed production of a smail amount of methanol from the strongly alkaline reactions of methyl ethylene phosphate; pseudorotation of a stable phosphorane would be required. (Kluger et al., 1969)

Figure 3.2 Kirby's mode1 compound and the proposed transition state in the H ~ T M E D A + ~ catalyzed cleavage. Both general acid catalysis for the leaving

group and electrostatic (or H-bonding) stabilization of the phosphorane are implied. (Dalby et al., 1993)

Oddly, in the same publication Taira et al. later state that the general lack of evidence for pseudorotation in the alkaline reactions of RNA [and models thereof) "should be interpreted as a consequence of a high energy barrier to pseudorotation."t (Taira et al., 1993)

Kinetic isotope effects (KIE) have been used to probe RNase A reactions. Solvent isotope studies can provide information on proton transfer events in general base and general acid catalyzed reactions. In some cases one can make inferences about the number of proton transfer events occurring in the transition state of the rate-determining step. Eftink and Biltonen (1983a) found a solvent isotope effect of kcat(H20) /kcat(D20) - 4 for the RNase A catalyzed ring-opening of 2',3'-cCMP. The magnitude of the KIE was taken as evidence for the concerted general acid-general base catalysis of His-12 and His-119.

The proton inventory method was later applied to the very same reaction. Although the KIE was found to be slightly less in that study (3.07) it showed that

t The "high energy barrier" argument is perhaps unlikely. Pseudorotation involves only changing bond lengths and angles. Such processes have energy requirements satisfied in the infrared spcctrum. The methanol production they take as experimental support for their computations could be explained by SN2 attack of hydroxide on the methyl group resulting in C-O bond fission. No attack on the phosphorus and so no pseudorotation need be invoked. Thus if the methyl ethylene phosphate hydrolysis was to be carried out in 180 water the methanol produccd would be iabeled. Othcrwise the methanol would be unlabeled and the phosphate would exhibit isotopic scrambling from unproductivc breakdown of the pseudorotating phosphorane. (Haakc and Wcstheimer, 1961, for a related study wherein no isotopic saambling was observed in the alkaline hyàrolysis of ethylene phosphate)

the observed KIE in that reaction was due to (at least) two concerted proton transfer events in the rate-deterrnining step. (Matta and Vo, 1986)

Primary and secondary KIEs may also be obtained through the use of heavy atom isotopes such as 180 or I ~ N . (Cleland, 1995) Hydroxide-, imidazole, and acid-catalyzed transestenfication of uridine-3'-pnitrophenyl phosphate have been studied and the 180 and 1 5 ~ KIEs determined. The data, it was proposed, suggested that in al1 three reactions the nucleophilic addition and elimination events were concerted, although that may have been due in part to the low pK, (- 7) of the leaving group. (Cleland and Hengge, 1995)

Later, the 1 8 0 KIEs for the RNase A catalyzed transesterification of uridine 3'-tn-nitrobenzyl phosphate were determined. The primary (leaving-group oxygen) KIE of 1.6% was indicative of "considerable loss of bond order to the [leaving] oxygen atom in the transition state" but by itself wasn't diagnostic of the concerted or sequential mechanism. However, the authors argued that the secondary (nonbridging phosphoryl oxygen) KIE of 0.5% was more consistent with a concerted nucleophilic addition-elimination mechanism. (Sowa et al., 1997)

Although they really only skim the surface of the literature the several preceding examples represent some of the current leading thought in RNA transesterification reaction mechanisms. They also serve to illustrate how, even when conven tional kinetic data leads to equivocai mechanistic schemes, there are ways to further explore a mechanism, be it through rational thought or more elaborate experimentation.

It was decided that solvent isotope experiments might shed some light on the lanthanum(III)-promoted transes terification of ApA. If the deprotonation is part of the rate-determining step then it should show up in the KIE and, if it is concerted with general acid catalysis of the forward breakdown of an in termediate (or transition s ta te), a proton inven tory would also be revealing.

3.2 Results and Discussion

3.2.1 Lanthanwn(II1)-Promoted Transesterification of HPNP

Although the plateau in the pH-rate profile could be consistent with either (1) intramolecular general base ca ta1 ysis or (2) preequilibrium intermolecular deprotonation of the 2'-hydroxyl it was felt that one might be able to discriminate between the two by Iooking into the timing of the deprotonation. Scheme 2.4a implied that the intramolecular general base catalysis accomplishes the deprotonation for the nucleophilic attack. If that was to be the case then conducting the experiment in D20 might furnish the KIE for the general base

involvement. Scheme 2.4b illustrated intermolecular base catalysis. The plateau in the

pH-rate profile dictates that if this is occurring that it must occur in a rapid equilibrium prior to the rate-determining step. According to that model, if the experiment was to be conducted in D20, on the plateau, the deprotonation step

would not show up in the KIE. There was a potential pitfall or ambiguity in the ApA solvent isotope

Scheme 3.2 Transesterification of HPNP. (a) Intramolecular general base catalysis could furnish a one-proton KIE. (b) Preequilibrium intermolecular deprotonation would not exhibit a KIE in D20.

study; the leaving of the 5'-alcohol would quite possibly be subject to general acid catalysis. This mechanistic requirement could therefore confound the results. How would one interpret the occurrence and magnitude of the KIE while still not knowing with certainty how many proton transfers are occurring?

To circumvent this ambiguity it was deuded to employ the RNA mode1 compound Zhydroxypropyl p-nitrophenyl phosphate (HPNP). (Brown and Usher, 1965) p-Nitrophenol has its pK, at approxirnately neutral pH so under the

reaction conditions the leaving group is thermodynamically stable as the anion; no leaving group protonation need occur. That simplified the experimental outcome to either observing a one-proton KIE or not (Scheme 3.2).

time (s)

Figure 3.3 Typical kinetic data for the lanthanum(II1)-promoted transesterification of HPNP. [La] = 2.00mM, [HPNP] - O.lmM, [CHES] = 20.00mM, pH = 9.37, T = Z°C. The c u v e is a simple first-order growth fit to the first five half-lives giving kob, = 0.362~1. The inset shows the complete kinetic run.

Although it was not expect to differ the pH-dependence of the reaction was determined to es tablish that it did indeed exhibit the same plateau as for the ApA reaction.

The reactions were camed out by rapidly injecting buffered solutions of lanthanum(II1) chloride into buffered solutions of HPNP. Excellent pseudo-first-order kinetics were observed through to completion of the reaction (Figure 3.3).

Figure 3.4 Upper curve: pH-rate profile for the lanthanum(III)-promoted transesterification of HPNP. [La] = 2.00mM8 [HPNP] - O.lmM, [CHES] = 20.00mM, pH = 8.98 9.70, T = 2S°C. Data points are averaged from duplicate experiments except the point at 9.70 which is a single determination. Error bars are i three standard deviations. kobs8 the pseudo-first-order rate

constant, is in units of reciprocal seconds. The curve is calculated from Equation 2.5 (Chapter II). Lower curve: pH-rate profile for the lanthanum(III1-promoted transes terifica tion of ApA (from Chap ter II).

As Figure 3.4 illustrates, comparison to the pH-rate profile for ApA showed the same plateau above a kinetic pH1i2 near pH = 9. Fitting the data in

the same fashion as for the ApA data provided the formation constant for the lanthanum dimer, K = 3.1 x 1027 Md, and the second-order rate constant, k = 367 ~ * r n o l - k ~ . The pH corresponding to half-maximal rate can be calculateci according to Equation 3.1. This yields a value of pH = 9.04 from the HPNP pH-rate profile which corresponds well with the midpoint of the lanthanurn(m) titration (Figure 2.3, Chapter II), pH = 8.85. The half-Me for HPNP on the plateau is only 1.9 seconds.

Equation 3.1

Having established that the pH-rate profile of HPNP exhibits a plateau as does ApA it is reasonable to assume that the same deprotonation step occurs for this substrate. Thus the kinetic isotope experiment should be a sound way to proceed.

3.2.2 Kinetic Isotope Effect for HPNP

The kinetic isotope experiment was carned out in the same fashion as for the pH-rate experiments with the exception that the CHES and sodium hydroxide (to adjust buffer pH) were lyophilized from 4 0 and then used to prepare solutions in D20. The HPNP was simply dissolved in HzO. The stock lanthanum(II1) solution (H20) used was the sarne as used for the pH-rate

experiments thus providing reaction media of 90% deuterium fraction. The reactions were well behaved as before, going to completion and

providing good pseudo-first-order fits. The results (Table 3.1) indicate there is no significant KIE in the HPNP

reaction; collecting and averaging the data from al1 the pHs (pDs) provides an overall result of kobs H/kobs D = 1.01 + 0.06.t t Since the data was collccted on the plateau of the pH-rate profile it is valid to treat the data as if collected at a single pH. Note that if in the D20 reaction there was no plateau (Le. the= was a first-order dependencc on hydroxide) the rates would have approximately doubled over the range of pD us&.

Table 3.1 Kinetic isotope results for the lanthanum(II1)-promoted transesterification of HPNP. [La] = 2.00mM, [HPNP] - O.lmM, [CHES] = 20.00mM, pH (PD) - 9.3 + 9.6, T = 25°C. Results at each pH (PD) are the averages of three replicate experiments. In the D 2 0 reactions D20 /H20 = 9/1.

The absence of the KIE would seem to indicate that in the rate-determining step there is no proton transfer occurring. It is unlikely that binding of the HPNP to the lanthanum(II1) dimer or the departure of the p-nitrophenolate ion are rate-determining. Thus we have to consider that the deprotonation of the nucleophilic alcohol occurs independent of, and prior to, the rate-determining step. In other words, the deprotonation appears to be occurring in a sequeritial fashion, not concerted with the later steps which would be ra te-de termining.

3.2.3 Kinetic Isotope Effect for ApA

The kinetic isotope result for the HPNP reaction is interesting in its own right as intermolecular or intramolecular general base catalysis are frequently invoked in the mechanisms for reactions of RNA and its models. However the reaction of ApA itself is also of interest. The kinetic isotope experiments for the ApA reaction were carried out in similar fashion to earlier ApA experiments; quenched aliquots were analyzed by HPLC. The solutions were prepared as for the HPNP experiments providing reaction media with 90% D20 content.

As with the HPNP reaction, the ApA-D20 data rests squarely on a

pH-rate plateau. (Table 3.2) In contrast to the HPNP kinetic isotope result though the ApA reaction shows a significant KIE. If again the data are pooled from

Table 3.2 Kinetic isotope results for the lanthanum(II1)-promoted transesterification of ApA. [La] = 2.00mM, [ApA] - 0.025mM, [CHES] = 20.00mM, pH (PD) - 9.1 + 9.6, T = 2S°C. Resdts at each pH (PD) are the averages of two to four replicate experiments. In the D20 reactions D20/H20 = 9/1.

across the pH (PD) ranges the overall result is kOb, H/kobs, D = 1.73 I O . = . If one may extrapolate to 100% D20 the KIE is approximately 1.9.

From the two kinetic isotope experiments some of the arnbiguity seems to be dissipating. The absence of a KIE for the HPNP reaction implies that the KIE observed for the ApA reaction is a manifestation of general acid catalysis in the rate-determining breakdown of the phosphorane to products.

The general acid assistance is certainly unavoidable for RNA transesterification given the leaving group concerned. Furthemore, it also appears to be an integral part of the rate-determining steps in enzymatic and nonenzymatic systems; the fission of the 5'-O-P bond has been implicated in the transition states in RNase A catalysis (Sowa et al., 1997), hydroxide catalysis (Davis et al., 1988a; Davis et al., 1988b), and buffer catalysis of RNA and its models (Anslyn and Breslow, 1989a; Dalby et al., 1993; Thompson and Raines, 1994).

The magnitude of the KIE found here compares well with some related studies in the literature. Usher et al. carried out solvent isotope studies for an RNA model, cis-tetrahydrofuran-4-01 3-phenyl phosphate (see Figure 1.5, Chapter 1) from approximately pH (PD) = 7.5 + 9. If one plots their kob, data and interpolates, a KIE - 4.25 is the result (Usher et al., 1970) This is most likely due to the concerted actions of acid and base catalysis and, if the contributions are equal, KIEs of approximately 2.06 eadi.

The KIE of 3.07 for the RNase A cataiyzed ring-opening of +,3'-cAMP was best explained by the combined action of general aad and general base catalysis, His-12 and His-119 contributing 1.75 each to the overall KIE. (Matta and Vo, 1986) A proton inventory for a nonenzyrnatic model of the same reaction yielded similar values of 2.12 and 1-90 for the general acid and general base components of its KIE. (Anslyn and Breslow, 1989b)

Kirby's RNA model compound (mentioned earlier, Figure 3.2) is reported to be subject only to general acid catalysis. The KIEs for this compound in several buffer systems range from 1.76 to 1.92.

The KIE observed for the ApA reaction at hand is consistent with general acid catalysis as the sole proton transfer step but it is not inconceivable that it is the resul t of combined general acid and general base catalysis. Concerted proton transfers are multipticative; theory dictateç a quadratic relationship for two such events. (Schowen, 1977) Thus, using the extrapolated value as an approximation, the KIE cortld be the result of two separate KIEs of roughly 1.3 to 1.4. Proton inven tory experimen ts are useful tools for inves tigating such possibili ties.

3.2.4 Proton Inventory

The proton inventory experiment involves measuring reaction rates in reaction media of varying H20/D20 ratios. The rate constants from each reaction (k,) are normalized relative to the rate constant in pure H20 (k,) and k,/ko is

plotted against the mole fraction of deuterium (n). Linear data is indicative of a single proton transfer in the rate-determining step. Curvature in the plot can appear in various forms; shallow, downward curvature may be indicative of a two-proton mechanism and a good fit to mathematical schemes for such models is generally regarded as good experimental support. (Schowen, 1977) Alternatively, if the data is consistent with a two-proton mechanism, a plot of (kn/ko) ' /2 versus deuterium fraction will be linear. (Matta and Vo, 1986; Anslyn and Breslow, 1989b)

The results of the lanthanum(III)-ApA proton inventory are illustrated in Figure 3.5. The kJk0 versus n plot is linear supporting the argument that there is only one proton transfer in the rate-determining step. A plot of (k,/k,)lj2

versus n shows a very slight upward deviation from linearity as would occur with a linear model.

It was hoped that the upward curvature obtained from transforming the

Figure 3.5 Proton inventory for the lanthanum(m)-promoted transesterification of ApA. [La] = 2.00mMf [ApA] - 0.025mMf [CHES] = 20.00mM, pH (PD) - 9.7 t, 9.9, T = 2S°C. The solid line is the k,,/k, plot. The dashed line is the ( k , / k , ) i / z plot. The point at n = 1 was obtained by extrapolation of the k , /k , data. Data

points are the averages of three to five determinations. Error bars are f one standard deviation. The lower line is a linear regression of the data; the upper line is simply a straight line co~ec t ing the data points at each extreme.

linear data would be much more pronounced providing a dearer distinction between the alternative one- and two-proton hypotheses in the square root plot. This small degree of curvature though is a consequence of the relatively small slope of the kJk0 versus n plot.+ It was unfortunate that the

expenmental error spanned the straight 1ine.S Although the proton inventory result did not clearly define the KIE as a

one-proton effect (it has not refuted the argument either) it is consistent with the proposition. The magnitude of the KIE abo compares quite weIi with literature values for related reactions. Together these results, dong with the absence of a KIE for the HPNP reaction, are strong indicators of a single proton transfer in the rate-determining step - general aad catalysis for the phosphorane's breakdown to products.

3.2.5 Reaction Mechanisms

Had the solvent isotope experiment showed a significant KIE for the HPNP reaction and had the proton inventory suggested two proton transfers for the ApA reaction choosing a mechanism would at this point be a simple matter. However, we m u t now apply careful, rational thought to reconcile the results rvith just one of the two alternative mechanisms.

The HPNP KIE and proton inventory demonstrated that the

deprotonation of the 2'-hydroxyl occurs prior to the rate determining step. Thus the correct sequence of events is a sequential deprotonation followed by the rate-deterrnining step. The results though could be consistent with either of the depro tonation mechanisms.

To resolve this dilemma we momentarily turn Our attention to the acid catalyzed departure of the leaving group. Two alternatives here are intermolecular and intramolecular acid catalysis. Buffer catalysis was not observed for the lanthanum(II1)-ApA reaction. In addition, this system is sufficiently fast that the combination of double Lewis-acid activation and intermolecular acid catalysis would be an inadequate explanation of its reactivity. It is proposed then that the mechanism involves intramolecular

t The rcader may satisfy himself-hcrself about this point by constructing linear plots of various slopes and then constructing the corrcsponding square root plots. The linear plots with srnaIler slopcç lcad to square root plots with Iess pronounced curvature. $ The crrors givcn are actually quite reasonable for the method(-4% or les).

general acid catalysis of the leaving group departure and a rational, detailed mechanism can be proposed.

The preequilibrium deprotonation occurs via equilibrium proton transfer between the 2'-hydroxyl and a bridging hydroxo, rigidly held and properly

positioned. (The kinetic equivalent would be deprotonation by a base in solution

followed by proton transfer to a bridging hydroxo though it is a more elaborate

scheme). The transition state (or intermediate) phosphorane breaks down to products assisted by the intramolecular general acid catalysis of an aquo ligand, that which resulted from the deprotonation step. Hydrolysis of the cyclic intermediate is rapid given its failure to accumulate and this step is considered in Chapter V. (Scheme 3.3)

2'- and 3'-monophosphates (fast) P

Scheme 3.3 The proposed mechanism for the lanthanum(1II)-promoted transesterification of ApA; preequilibrium proton transfer to a bridging hydroxo followed by 2'-alkoxide attadc at the phosphorus and intramolecular general acid catalysis o f the leaving group departure.

3.3 Summary and Conclusion to Chapter III

Kinetic isotope effects have been determined for the lanthanum(II1)-promoted transesterification of the RNA analog HPNP and for ApA. The former shows no difference in rates for the H20 and D20 reactions;

general acid catalysis is not a requirement for this compound so the la& of a KIE indicates generai base catalysis is not implicated in the rate-determining step.

ApA exhibited a KIE of 1.9 (extrapolated from the result in 90% D20). Together with the HPNP and proton inventory results this is interpreted as

arising from a single pro ton tr ans fer; general acid ca ta1 ysis of the phosp horane's breakdown to products.

A detailed mechanism for the lanthanum(El)-prornoted transesterification reaction has been proposed. The deprotonation of the 2'-hydroxyl occurs by a rapid equilibrium proton transfer to a bridging hydroxo ligand prior to the rate-determining step. Intramolecular general acid catalysis by an aquo ligand (formed in the deprotonation step) occurs in the rate-determining breakdown of the phosphorane to products. The results do not allow one to determine if the phosphorane is in a transition state or is a true intermediate. Though it is speculative, a careful study of the literature (see introduction to this chapter) suggests that it may be in the transition state, implying that the nucleophilic attack of the Falkoxide and the general aud catalyzed leaving of the 5'-hydroxyl are concerted. Hydrolysis of the cyclic intermediate is rapid (it is not observed to accumulate) and this step is examined in Chapter V.

3.4 Experimental

Ma tenals

Water (deionized and distilled) and deuterium oxide (Cambridge Isotopes) were degassed under vacuum, and kept oxygen and carbon dioxide free under argon.

Other materials were as follows: lanthanum(1II) chloride heptahydrate (ACS, Aldrich), CHES (ACS, Aldrich), sodium hydroxide (volumetric standard, O.ZOOM, "low carbonate", Anachernia), sodium phosphate monobasic (HPLC orade, A&C), methanol (HPLC grade, BDH), ApA (98%, ICN). nitrobenzene 0

sulfonic acid sodium sait (m- and p- isomers, Eastman-Kodak). 2-Hydroxypropyl pnitrophenyl phosphate was synthesized according to

the li terature. (Usher et al., 1970)

Spectrophotometer Kinetic Isotope Experiments

Volumetric standard NaOH was stripped of its water on a rotary evaporator and redissolved in D20. This was repeated twice more to prepare a solution of NaOD in DzO. CHES and EPPS were lyophilized once from D20 solutions prior to preparing solutions volumetrically (100mM) in D20 and

adjusting pD with NaOD. The stock solution of lanthanum(III) chloride (2O.OmM) was prepared volumetrically in H 2 0 and standardized (as described in

Chapter II). Al1 these solutions were degassed under vacuum and kept under argon. HPNP was dissolved in D20 or H20 as required (-1Om.M).

In a typical kinetic experiment, measured (EppendorD volumes of buffer, NaOD and HPNP solutions were diluted with D20 to 1.000mL. A second 2.000mL solution was prepared from measured volumes of D20, buffer, NaOD,

and lanthanum(II1) solutions. Concentrations of the two solutions were such that when 1.000mL of the second was added to the first solution the final concentrations were: [La] = 2.00mM8 [HPNPJ - O.lmM, and [buffer] = 20.00mM- The reaction would be carried out in a 2mL cuvette maintained at 2S°C. With the firs t solution in the cuvette the spectrophotometer would begin to collect data for a few seconds and then the second solution would be rapidly injected. The reaction was monitored by the increase in absorbance at 400nm using a Hervlett-Packard 8452 diode array spectrophotometer equipped with a jacketed

cuvette cornpartment and an RMS Lauda water circulator. pD was measured at the end of the reaction and compared to that of an identicai (iess HPNP) solution using a Fisher Accumet 15 pH meter and an Orion 8013 Ross semimicro combination electrode. The values given for pD are the meter readings, uncorrected.

Data was processed (plot ted and cuve-fitted) using Kaleidagaph 3.0.1 on a Macintosh computer.

HPLC Kinetic Isotope Expenments

Stock solutions of lan thanurn(II1) chIoride (20.0mM, s tandardized), and CHES (100mM) were prepared volumetricaily, degassed under vacuum and kept under argon. Buffers were adjusted with concentrated NaOH to the approximate desired pHs for the reactions and degassed. Equal volumes of lOmM ApA and lOmM nitrobenzene sulfonic acid sodium salt (interna1 standard, IS) were mixed

to give a solution 5rnM in each. Sodium hydroxide (volumetric standard, 0.100M) was degassed under vacuum and kept under argon.

Solutions in D20 were prepared as for the 'W-Vis" kinetics (above).

In a typical kinetic experiment measured volumes of buffer, base and lan thanum(II1) were dilu ted wi th wa ter to 2.000mL. By varying the volumes of D 2 0 solutions and H20 solutions used the deuterium fraction could be varied

from O to 0.9. Final concentrations in the reaction mixture were: [La] = 2.00mM,

[ApA] - 0.025mM, and [buffer] = 20.00rnM. The reaction would be carried out in a small glass via1 and magnetically

stirred under a slow flow of argon. Temperature was maintained at 25OC with a jacketed beaker and an RMS Lauda water circulator. The reaction was initiated with the injection of 10pL of the ApA/IS solution. pH (PD) was measured before and verified after the reaction with a Fisher Accumet 15 pH meter and an Orion 801 3 Ross semimicro combination electrode.

Aliquots were removed from the reaction at measured intervals and rapidly quenched in 0.2M ammonium phosphate buffer (pH = 5.5). The quenched samples were centrifuged (130 000 RPM, 5 minutes) to remove precipitates.

The supernatant was analyzed using a Hewlett-Packard 1090m HPLC with an autosampler unit and a Hewlett-Packard 2.1x100mm 5 p ODS Hypersil Cl8 column. Products were eluted by running 05mL-min-1 of 0.2M ammonium

phosphate buffer (pH = 5.5) for five minutes followed by a linear gradient to 50% of a 60/40 methanol/water solution over the next 10 minutes. Peaks were detected by UV absorbante at 254nm. Column temperature was maintaineci at 40°C.

The chromatograms were integrated with a Hewlett-Packard 9153c computer and the data (ratio of the ApA peak area to that of the intemal standard) was processed (plotted and curve-fitted) using Kaleidagraph 3.0.1 on a Macintosh computer.

3.5 Appendix 1: Denvation of Equation 3.1

At the midpoint of the titration (pH1 12) the mass balance dictates

This may be used to substitute [LaIT into the expression for the equilibrium formation constant, K. Substituting [Hl and Kw for [OH] and rearranging provides

*

Taking the negative logarithm of that equation provides Equation 3.1

Appendix 2: Tabulated Kinetic Data

Table A3.1 Complete data for the pH-rate profile of the lanthanum(III)-promoted transesterification reaction of HPNP. [La] = 2.00mM, [HPNP] - O.lmM, [CHES] = 20.00mh4, T = 2S°C. Values were not rozinded off until al1 calculations hnd becri corn pleted.

1 kobs, I-1# S-'

0333

Q

0.0158 'obs, S-'

Q KIE a

0330 0.0109 1.01 0.06

Tables A3.2 (Previous page) Complete data for the kinetic isotope experiment for HPNP. [La] = 2.00mM, [HPNP] - 0-lmM, [CHES] = 20.00mM, T = 25OC. Deuterium fraction = 09.Valites were not rounded off until al1 calculaiions had been cornple fed.

Tables A3.3 Complete data for the kinetic isotope experiment for ApA. [La] = 2.00mM, [ApA] - O.lmM, [CHES] = 20.00mM, T = 2S°C. Deuterium fraction = 0.9. kOb,, H and kobs, D are calculated from pooled data. The KIE in parenthesis is from extrapolation to n = 1.Values were not rounded off until al1 cnlc~ilntiorrs had been completed.

Table A3.4 Complete data for the proton inventory experiment for ApA. [La] = 2.00mM, [ApA] - 0-lmM, [CHES] = 20.00mM, T = 25"C.VaIues were not rort rideci off ztntil al1 calculations had been completed.

Chapter IV ApA and HPNP Cleavage by Other Ln(II1)

4.1 Introduction to Chapter IV: Other Lanthanides in the Literahve

The few reports that appeared in from the 1960s to the 1980s contained few real insights into lanthanide-promoted cleavage of RNA. The researchers were primarily interested in the development of new tools for molecular biology paying particular attention to the stoichiometry of the polyribonudeotide reactions and the patterns of deavage in relation to the secondary and tertiary structures of the polymers. (Eichhorn and Butzow, 1965; Rordorf and Kearns, 1976; Ciesiolka et al., 1989)

With the resurgent interest in lanthanide-promoted reactions of nudeic acids researchers have been exploring the chemistry from a number of perspectives; lanthanide ion reactions, lanthanide complexes, comparative studies and, RNA and DNA chemistry have been investigated.

Breslow's group, long interested in RNA cleavage chemistry, briefly studied lanthanide(II1)-promoted cleavage of the RNA dimer UpU. The three lanthanides considered (EuC13, TbC13, and YbC13) provided virtually the same

rate accelerations for the RNA analog HPNP; half-lives were in the vicinity of 40min ([Ln] = 0.5mM, pH = 7, T = 37OC). Europium(iII) was shown to be capable of cleaving the RNA dimer UpU under more rigorous conditions ([Eu] = ImM, pH = 7, T = 80°C) with a half-life of approximately 32hr. (Breslow and Huang,

1991)

Shortly thereafter Morrow investigated the ability of a larger set of divalent metal and lanthanide(III) ions to promote the cleavage of HPNP under similar conditions (pH = 6.85, T = 37OC). Expressing their results as second-order rate constants, their results contained no particular revelations; al1 the lanthanides they tested had comparable reactivities (k2 - 0.1 + 0.6~-mol-~s-l) and

were rouglily one to two orders of magnitude faster than the transition metals studied. They attempted to correlate the lanthanides' relative reactivities to ionic radius but no clear trend was apparent. They did make note of the wide variation in titrations of the lanthanides (expressing surprise that the reactivities should be so similar given the widely differing pH1i2s) but did not report any pH-rate

studies. (Morrow et al., 1992a) In later work on mononuclear lanthanide(II1) macrocyclic complexes

Morrow and Chin considered the reactivities of lanthanum(III), europium(ID)

and lutetium(II1) complexes for the deavage reactions of HPNP and poly(A). The relative reactivities did not correlate with the complexes' pK,s as had been

expected but it was suggested that differences in the coordination spheres were the con trolling factor. (Chin and Morrow, 1994)

At about the same time it was reported that the RNA dimers ApA and UpU could be cleaved by thulium(II1) (IOmM) under quite mild conditions (pH = 8, T = 30°C) with half-lives of approximately ten minutes. The authors

seemed satisfied to state that the mechanism involved intramolecdar general

base catalysis by a thulium(II1)-bound hydroxo ligand although their report

contained no detailed studies. They did however present an order-of-reactivity

series for the lanthanides in the RNA reaction:

Tm, Lu > Nd, Eu, Sm > Ce, Gd, Tb > Pr, Dy z Ho, Er > Yb > La

It is to be assumed by the reader that those studies were carried out under the same conditions as for thulium(XII); the authors were not explicit. (Komiyama et al., 1992; Matsumura et al., 1992)

After we had realized the previously unrecognized reactivity of

lanthanum(II1) and established the chemisü-y of the ApA reaction it was deaded

that an investigation of some other lanthanides was in order. There were a few

motivating concerns. Which lanthanide could provide the shortest half-life?

There was noiv sound justification for reconsidering the conclusions of others;

the previous studies made cornparisons under arbitrarily fixed conditions. The titrations of the lanthanides are known to differ widely with respect to their pH1/2s and, as was shown earlier, the lanthanurn(III) reactivity correlated with

the titration. This provides a working mode1 for assessing the full potentials of

the other lanthanides.

The reactivi ty of the lanthanum(III) can only realiy be tapped a t fairly high pH. To some this is a concern simply because it is far removed from so-called physiological conditions. Others who read this work and are interested in exploiting metal-ion chemistry in general for molecular biology may have

applications in mind that strictly require other ranges of pH. So if comparable reactivity can be demonstrated with other lanthanides at lower pHs the lanthanide chemistry may be given further considerations by others.

It is also of interest to see if other lanthanides, regardless of reactivity,

function in the same manner as lanthanum(m), providing a general modd.

4.2 Results and Discussion

4.2.1 Sampling the Lanthanides: Titrations and Activities

Several lanthanides were sampled for titration and activity studies. Carrying out the titrations with standardized solutions and under the same conditions as for lanthanum(1II) showed that, within approximately five percent, they al1 exhibited the same 2.5-equivalent consumption of hydroxide.+ The trend was for decreasing pHi 12 across the row with the differences becoming smaller

near the end of the row (Figure 4.1).

O O 5 1 1.5 2 2 5 3

eq NaOH

t Cerium(II1) was intended to be included in the survey as well. Cerium had k e n dcmonstrated to possess great rcactivity for the cleavage of DNA (and its analogues) and it was establishcd that the ccrium(III/IV) rcdox equilibrium was integral to the reactions. It was of intercst to sec if, by excluding molecular oxygen, the reactivity of cerium(II1) could be cleanly asscsscd. Howevcr, even whcn precautions were taken to exdude oxygen, the titration was not rcvcrsiblc suggesting it would not fit the same mode1 as lanthanum(II1).

Figure 4.1 (Previous page) Titrations of several trivalent lanthanides. (a) Lanthanum, (b) praseodymium, (c) neodyrnium, (d) europium, (e) holmium, (0 thulium and (g) lutetium. [Ln(m)] = 2mM, [KU] = 100mM, [NaOH] = 100rnM,

T = 25OC. Al1 lanthanides were chloride sai ts.

The reactivity of the series was then assessed using the RNA analog HPNP. To avoid any complication of the data due to possible involvement of buffers the reactions were carried out simply using 2.5 equivalents of sodium hydroxide to ensure in each case the metal ion was fully titrated. The trend in reactivity was found to follorv a similar pattern as for the titrations although the

. Lanthanum - - - Praseodymium

timc (s)

Figure 4.2 Typical time traces for lanthanide(III1-promoted reactions of HPNP. [LnUII)] = 2mM, 2.5 equivalents of NaOH, [HPNP] - 0.05mM or O.lmM, T = 2S°C, h = 400nm.

Figure 4.3 Trends in pHl/p (circles) and kob, (diarnonds) of the HPNP reactions

for several trivalent lanthanides. Conditions for the titrations and reactions are as described previously. kobs values are from at least triplicate determinations. The

lines drawn are simply smooth curves; no mathematical relationships are implied.

first several lanthanides provided nearly the same rates for the reaction in spite of having very different pHti2s (Figure 4.2).

Figure 4.3 shows how the kOb,s correlate with the titrations. Reasonably

assuming the remainder of the trivalent lanthanides behave similarly

interpolation of this body of results will provide sound estimates of any lanthanide(II1) ion's reactivity.

It would also appear that by using lanthanides which titrate at lower pHs that comparable reactivity toward authentic RNA might be had. Also the order-of-reactivity series described by Komiyama requires amendment; it would only apply at a fixed pH.

Figure 1.4 pH-Rate profile for the europium(IIl)-promoted tramesterification of ApA. [Eu] = 2.00m.M, [ApA] - 0.025mM, [EPPS or CHES] = 20.00mM, T = 2S°C. Data points are averaged from duplicate determinations. Error bars are f one standard deviation. kOb,, the pseudo-first-order rate constant, is in units of reciprocal seconds. The curve is calculated from Equation 2.5 (Chapter II).

4.2.2 pH-Rate Profiles for Eucopium(II1) and Thulium(I11)

HPNP is in some instances a good preliminary indicator of reactivity to RNA and as such is widely used. However the reactivity may not aiways exactly parallel that of RNA; it has been observed, although not without exception (Chin et al., 1989; Takasaki, 1994), that greater acceleration may be obtained for substrates with better leaving groups. For that reason the more detailed studies were conducted for europium(II1) and thulium(III) using ApA as the substrate in

Figure 4.5 pH-Rate profile for the thulium(II1)-promoted transesterification of A p A [Tm] = 2.00mM, [ApA] - 0.02SmM, [EPPS] = 20.00mM, T = 2S°C. Data

points are averaged from duplicate determinations. Error bars are + three standard deviations. kobs, the pseudo-first-order rate constant, is in units of

reciprocal seconds. The cuve is calculated from Equation 2.5 (Chapter II).

buffered systems. The pH-rate profiles for the europium(II1)- and thulium(II1)-promoted

reactions were constructed in the same manner as for the lanthanum(II1)-promoted reaction, analyzing quenched aliquots from the reaction by HPLC to obtain the time-resolved product profile. Replicate determinations afforded the pH-rate profiles shown in Figure 4.4 and in Figure 4.5.

The product analyses from the europium(II1) and thulium(III) reactions

rvere identical to that from the lanthanum reaction. Only hydrolytic products rvere obtained and the intermediate 2'3'-CAMP was not observed to accumulate (not shown).

Both europium(III) and thulium(III) furnished pH-rate profiles exhibiting the very steep dope prior to a plateau. The half-maximal rates were found to occur in the near vicinity of the pHi/2. Given these commonalities, the resulting

pH-rate profiles were fit to the same equation as for the lanthanum(III) pH-rate profile, providing values for the formation constants, K, and the secondsrder rate constants, k.

Table 4.1 sumarizes the results for the lanthanum(m), europium(III) and thulium(II1) reactions with ApA. The relative reactivities toward ApA compare well with the HPNP results as was expected. What stands out though is the acceleration the three lanthanides provide over the ambient (hydroxide) rate. Although the three lanthanides provide comparable rates for the reaction, as Table 4.1 demonstrates the acceleration over the ambient hydroxide rate differs greatly as the pH decreases; at pH = 7.5 thulium accomplishes a 6.9 x 106-fold acceleration over the hydroxide reactiont whereas those for europium(IID and

Table 4.1 Comparison of the lanthanum(II1)-, europium(II1)- and thulium(II1)-promoted transesterification reactions of ApA. koH, the

pseudo-first-order rate constant for the hydroxide reaction, was estimated according to Williams and Chin (1996).

t Komiyama et al. report 6.8 x 106-fold acceleration for the thulium(I1l)-ApA reaction at pH = 8. If their value for the ambient hydroxide reaction (tlIZ = 130yr) is used for this study then wc have found a 5 5 x 207-fold accclcration.

lanthanum(1m are 1.5 x 107-fold (pH = 8) and 1.3 x le-fold (pH = 9) respectiveiy.

4.2.3 Second-Order Dependence upon Thulium(II1) Concentration

As for the lanthanurn(m)-ApA reaction, it was important here to establish the order of the reaction with respect to the lanthanide concentration. The concentration dependence for the thulium reaction was found to be the same as

Figure 4.6 Dependence of the (logarithm of the) pseudo-first-order rate constant, kObs, for the transesterification of ApA upon (the logarithm of) [TrnlT. [Tm]T = 1.00 + 2.00mM, [ApA] - 0.025mM. [HEPES] = 20.00mM,

pH = 7.06 + 0.02, T = 25OC. Data points are averages of three to four determinations (except the point at log([Tm]) = -2.9 which is a single determination). kobs is in units of reciprocal seconds. [?mlT is in units of rnol*~-l.

Slope of the linear regression is 2.17 + 0.26.

for the lanthanum reaction; second-order in the 1 + 2mM range (Figure 4.6).

Thus it appears that the reactive species for the thulium(II1) reaction is a thulium(II1)-hydroxo dimer of a two-to-five stoichiometry as for the

lanthanum(III) reaction and the ApA reaction foliows the same mechanism. Regrettably, a convincing europium(III) concentration plot could not be

ob tained. Al though the data (not shown) indicated an order greater than unity, it was widely scattered. This may be a manifestation of carrying out the experiment

on the steep slope of the pH-rate profile where even small variances in pH c m

result in comparatively large differences in rate. Nonetheless, the other data for

europium fits the dimenc mode1 quite well and it is not unreasonable to suppose

that it applies. Even if the order with respect to europium(III) was greater than two the titration and pH-rate profile suggest that the mechanism of the ApA transesterification reaction is the same as for the others.

4.3 Conclusion to Chapter IV

Titra tions and reactivi ties for several lanthanides have been determineci. That body of results indicates that the reactivities of the trivalent lanthanides correlates with their pHl/p. The fastest absolute rates are provided by the earlier lanthanides. There the pHi/2s rapidly drop in value yet they provide very comparable rates. Later in the series the pHi/2s Vary little but the reactivities continue to dedine slightly. Interpolation of the results confidently predicts the reactivities for the entire lanthanide(III) series.

The pH-rate profiles have been obtained for thulium(II1) and europium(1II). Like lanthanum(II1) they show a strong (fifth-order) dependence upon hydroxide prior to a plateau, indicating that they too show no requirement for hydroxide in the rate-determining step once fully titrated. The dependence upon thulium(III) concentration was detennined to be second order. Given these common features and the stoichiometry of the titrations it is proposed that the penta-p-hydroxo lanthanide(II1) dimer mode1 and the ApA transesterification mechanism are generally applicable to the lanthanide series.

It is norv quite clear that dinuclear lanthanide species provide a tremendous advantage over mononudear systems. Further advances in this area must be directed towards finding dinudeating ligands which can stabilize the reactive dimer and perhaps even promote its formation in a cooperative fashion at even lower pHs. The pH-rate profiles clearly demonstrate that once the dimerization is complete the reactivity will be maintained over a wide range of pH. Selecting one lanthanide over another for preparing complexes will not likely confer any great advantages in terms of rates so choices could be made based on the desired coordination properties.

Ma terials

Water (deionized and distilled) was degassed under vacuum, and kept oxygen and carbon dioxide free under argon.

Other materials were as folbws: neodymium(III) chloride hexahydrate (99.9%, Aldrich), praseodymium(II1) chloride hexahydrate (99.9%, Aldrich), europium(II1) chloride hexahydrate (99.9%, Aldrich), terbium(III) chloride hexahydrate (99.9%, Aldrich), holmium(I1I) chloride hexahydrate (99.9%, Aldrich), thulium(II1) chloride hexahydrate (99.99%, Aldrich), lutetium(m) chloride hexahydrate (99.9%, Aldrich), HEPES (ACS, Aldrich), EPPS (ACS, Aldrich), potassium chloride (ACS, BDH), sodium hydroxide (volumetric standard, O.IOOM, "low carbonate", Anachernia), disodium EDTA (ACS, Fisher Scientific), sodium phosphate monobasic (HPLC grade, A&C), methanol (HPLC grade, BDH), ApA (98%, EN), and nitrobenzene sulfonic acid sodium sait (m- and y- isomers, Eastman-Kodak).

Titra tions

Titrations were carried out using a Radiometer PHM63 pH meter, ' I1T80

titration controller, ABU80 automatic burette , RTSS22 automatic titrator and REC chart recorder (with the REAI60 titration module). pH was measured with K-4010 calomel reference and G-2040 g l a s electrodes. Solutions were prepared in volumetric glassware and solutions were handled using volumetric pipettes and Eppendorf adjus table pipettors.

Standardization and titration of the lanthanides was carried out in a fashion similar to that described in Chapter II.

The piotted (chart recorder) titration was scanned using an Apple Onescanner and Macintosh PC running Ofoto 1.0 software. The image file was digi tized, conver ting the ti tration to numeric Cartesian data, using Flexi trace 1 .O2 and replo tted using Kaleidagraph 3.0.1.

Spectrop ho tome ter Kine tic Experimen ts

Stock solutions of the lanthanides were the same, or prepared in identical fashion, as described for the titrations (above).

The experiments were carried out using the same apparatus and instrumentation as desaibed in Chapter III.

In a typical kinetic experiment a measured volume of a stock lanthanide solution (20.00mM, standardized) and 2.5 equivalents of sodium hydroxide

solution (100mM) were diluted to 2-000mM Ln(III). With 5pL or 10pL of HPNP solution (IOrnM) placed in an empty cuvette data collection (400nm) was begun. The alkaline lanthanide solution was injected as rapidly possible into the cuvette to initia te the reaction.

Data was processed (plotted and curve-fitted) using Kaleidagraph 3.0.1 on a Macintosh cornputer.

HPLC Kinetic Experiments

Stock solutions of the lanthanides were the same as or prepared in identical fashion as described above. Stock solutions of buffers (HEPES and EPPS) and a stock solution of ApA with internal standard was prepared as described in Chapter II.

Kinetics for the pH-rate and concentration profiles were carried out in the same manner as described in Chapter II using the same apparatus and instrumentation.

Appendix: Tabulated Kinetic Data

Table A4.1 pHl 1 2 values and pseudo-first-order rate constants for lan thanide(II1)-promo ted transes terification of HPNP. For the ti trations [Ln] = 2.00mM, [NaOH] = 0.100mM and T = 25°C. For the kinetics [Ln] = 2.00m.M, 2.5 equivalents of sodium hydroxide, [HPNP] - 0.05 + O.lmM, T = 23°C

Table A4.2 Complete data for the pH-rate profile of the thulium(m)-promoted transesterification reaction of ApA. [Tm] = 2.00mM, [ApA] - 0.025mM, [HEPES or EPPS] = 20.00mM, T = 25OC. Values were not rounded off until al1 calczrla tions had been completed.

11 pH 1 k,, s-' 1 1 avg 1

Table A4.3 Complete data for the pH-rate profile of the europium(III)-promoted transesterification reaction of ApA. [Eu] = 2.00mM, [ApA] - 0.025mM, [HEPES or EPPS] = 20.00mM, T = 25OC. Values were mot rounded off until al1 cnlciilntions had been cotnpleted.

Table A4.4 Complete data for the thulium(III) concentration dependence of the transesterification reaction of ApA. [ApA] - 0.025mM, [HEPES] = 20.00mM. pH = 7.06 f 0.02, T = 25OC. Valites wme not rounded off until al1 calculations had been coin pleted.

Chapter V La(1II)-Promoted Hydrolysis of DNA Models

5.1 Introduction to Chapter V: Metal Ion- and Metai Complex- Promoted Hydrolysis of DNA and DNA Models

Creation of artificial systems for the cleavage of DNA has been a pursuit of rnany research teams for a number of years, motivated by the potential for the development of new powerful and versatile tools for molecular biology and perhaps even therapeutic agents. In the process much has been learned about the nature of DNA's remarkably stable phosphate backbone and what is, or will be, necessary to accomplish its cataiytic hydrolysis.

The first generation of metal complexes developed were mononuclear. They provided important mechanistic information and showed how variations in a complex's structure can affect reactivity. A series of cobalt(III) complexes was shown to be the most effective for the hydrolysis of DNA analogs, the fastest of rvhich, [Co( trpn) ] +3, was capable of 1010-fold acceleration in the hydrolysis of BNPP. This kvas the result of combined Lewis acid activation and efficient

intramolecular attack of a bound hydroxo ligand. It was learned that slight changes in ligand structure (N-Co-N angle opposite the binding site) impart significant differences in rates. (Chin and Zou, 1988)

The next generation of artificial DNases makes use of a dinuclear approach to provide double Lewis acid activation of a bridging phosphate. Inspiration and insight for this strategy stems from studies of enzymes that employ two metal ions in the hydrolysis and polymerization of nudeic acids. X-ray and kinetic studies have shown that two or more metal ions lie at the active sites of a number of enzymes (Strater et al., 1996, for a review) including DNA polymerase 1 (which can carry out exonudease reactions) (Beese and Steitz, 1991; Han et al., 1991), T4 DNA polymerase (Wang et al., 19961, alkaline phosphatase (Kim and Wyckoff, 1991) and ribonudease H from HIV-I reverse transcriptase- (Davies et al., 1991)

Multiple metal ion reqirements are also known for the ribozyme RNase P (Altman et al., 1993; Smith and Pace, 1993) and two-metai-ion mechanisms have been proposed for it and other ribozymes. (Steitz and Steitz, 1993)

Clever use was made of a dinuclear cobalt(IlI) complex from the literature (Figure 5.1). Bridging of a phosphate diester was proven with X-ray analysis. This complex provided a remarkable 1011-fold acceleration for the hydrolysis of

Figure 5.1 A dinuclear cobalt(IKI) complex capable of up to 10"-fold acceleration in the cleavage of ac tivated phosphate dies ters. (Wahnon, 1995)

methyl p-nitrophenyl phosphate. With '$0 tracer studies it was shown that the nucleophile was a F-hydroxo ligand as opposed to hydroxide in solution. (Wahnon, 1995)

As discussed previously and was shown in Chapters II and IV, lanthanides owe their great reactivity to reactive dinuclear species. The cerium(ILI/IV) system was actually capable of cleaving authentic DNA with a half-life of roughly 100min, approximately 1012-fold faster than the ambient (hydroxide) reaction. (Takasaki and Chin, 1994 ) The dinuclear lanthanum(II1)-hydrogen peroxide couple cleaved the DNA mode1 BNPP 10"fold fas ter than the ambient reaction (Scheme 2.1, Chapter II). (Takasaki and Chin, 1993; 1995)

Schneider's group has also expiored lanthanide systems for DNA cleavage. They have recently reported on europium(1II)-promoted cleavage of

Figure 5.2 Schneider's dinucleating ligand for lanthanides enables 106-fold acceleration for nicking supercoiled DNA. (Rammo et ai., 1996)

supercoiled plasmid DNA. This substrate is more prone to cleavage than h e a r DNA but nonetheless the reported acceleration was 106-fold. (Rammo et al., 1996)

In another report they explored the use of maaocyclic ligands (Figure 5.2) designed to form dinuclear complexes with lanthanides. With BNPP and supercoiled plasmid DNA results they suggest that the active speaes were the dinuclear complexes. Differences in rates between europium(111) and praesod ymium(III) complexes were ascribed to dif feren t distances between the metal centres in the dinuclear complexes although structural data was not presented. Accelerations of 106-fold were reported for the dinuclear complexes. (Ragunathan and Schneider, 1996)

Komiyama's group has made numerous announcements of hydrolytic cleavage of DNA and related compounds using lanthanides and their complexes. (Some of these articles concern the use of cerium(III) and it is quite possible that in some cases the activity may have been due to cerium(1V) formed in situ). Thuliurn(II1) was reported to be able to hydrolytically cleave the DNA dimer dTpdT. Chrornatographic evidence was presented for the appearance of thymidine after seven days at 50°C and pH = 8 but no rate constant was offered. (iMa tsumo to and Komiyama, 1992)

Macrocyclic lanthanide complexes, used effectively in RNA cleavage, were also reported to promote the hydrolysis of a linear 35-mer of DNA under quite mild conditions. Degradation of the DNA was illustrated with electrophoresis results but regrettably no rate constants were provided. The results are nonetheless intriguing. (Shiiba et al., 1993)

This group has also boldly taken the chemistry up to the next level, cons truc ting a an an tisense-cerium(1V) complex conjuga te and applied it to the scission of a specific DNA 50-mer. After 12hr deavage at the targeted site was detectable by autoradiography. (Komiyama et al., 1993a)

3',5'-cAVP is another subshate that may be employed as a DNA model. In the ring-opening reaction the leaving groups are the very same as for DNA (Figure 5.3). The six membered ring though imparts some strain on the phosphate diester group which accounts for its estimated haif-life of 500 OOOyr (Chin and Zou, 1987) compared to that for DNA, 200 million years. (Chin and Zou, 1988)

A mononuclear cobalt(1II) complex is known to accelerate 3'3'-CAMP'S ring-opening reaction by approximately 108-fold. (Chin and Zou, 1987) An

Figure 5.3 The leaving groups for DNA hydrolysis are the same as for 3*,5'-CAMP ring-opening.

acceleration of 101'-fold is cla imed for a cerium(II1) hydroxide "cluster". (Contribution to the reactivity by cerium(IV) might be a factor here). The authors present a partial pH-rate profile although the purported plateau is not fully defined. Chelation of the phosphate to a mononuclear cerium(III) centre and intramolecular attack of a hydroxo ligand is the proposed mechanism. In addition they specuiate that bound aquo ligands provide general acid catalysis for the leaving group; the data does not necessarily lead us to this conclusion. (Sumaoka et al., 1992b; 1992a)

Our realization of the full potential of lanthanum(II1) and other trivalent lanthanides in RNA transesterification provided motivation to reconsider its reactivity with DNA and its analogs (BNPP and the cydic phosphate diesters).

Enzymatic and nonenzymatic catalysis of the 2',3*-CAMP ring-opening reaction have been studied. RNase A deaves RNA to produce the 2'3'-cyclic intermediate, which is hydrolyzed more slorvly in a second step. (Thompson et al., 1991) In contrast, the hydroxide catalyzed reaction of RNA produces the 2'- and 3'-monoesters without accumulation of the cydic intermediate.

2',3'-CAMP production is directly observed in some artificial RNase systems (metal ion complexes) and not in others. (Morrow et al., 199213; Young and Chin, 1995, as examples) This is a curious fact, that some systems might be effective for both of the consecutive reactions whereas other systems are effective (relatively so) for only one, and suggests that the contrasting systems exhibit

different rate-determining steps or that they are employing some different mechanis tic fea tures al together.

The lack of accumulation of 2' 3'-CAMP in the lanthanum(IU)-promoted Ap A transesterifica tion (Figure 2.1, Chapter ID suggested that there Nght be significant reactivity towards this family of substrates. The HPLC tedinique was inadequate for this investigation; after just a few seconds into the reaction it had reached completion. However, it was possible to make an estimate of the lower limit of the kobs; if the reaction had just reached ten half-lives by five seconds (essen tiall y a comple ted reaction) then the kob, was a t leas t -1.4~~. Stop-flow

investigations would refïne this estima te. Aside from assa ying the reactivi ty for phosphate dies ter hydrolysis, a

mechanistic question had come to mind. From where would the nudeophilic hydroxide come? Two possibilities were conceivable. Perhaps the reactive dimer would only provide the double Lewis acid activation and the nucleophilic hydroxide would come from the surrounding solution. Aitematively, the dimer might behave as the dinuclear cobalt(II1) complex (above) did, utilizing a p-hydroxo ligand as an efficient nucleophile.

5.2 Results and Discussion

5.2.1 Lanthanum(II1)-Promoted Hydrolysis of BNPP: A Plateau in the pH-Rate Profile

BNPP \vas used as a convenient substrate for the construction of the pH-rate profile. The conditions used were the same as for the ApA and HPNP studies. The time course of the reaction, monitored by the appearance of the p-nitrophenolate anion, showed biphasic kinetics; the lanthanum(IIT) dimer was cleaving the diester and then the product monoester in a slower second step (Figure 5.4).

O 10 20 30 40 50 60 70

time (s)

Figure 5.4 Typical kinetic data for the lanthanum(1II)-promoted cleavage of BNPP. [La] = 2.00mM, [BNPP] - 0.05mM, [CHES] = 20.00mM, pH = 9.79,

T = 25OC. The cuve (see Equation 5.1) is fit to the first five half-lives of the diester cleavage giving k = 0.0647s-1. The inset shows complete kinetic run.

The kinetics were fit to a double exponential equation, providing the pseudo-first-order rate constants for both the diester hydrolysis and that of the monoester (Equation 5.1).

Equation 5.1

[p - ni trophenolate] = 2[BNPP], + 'BNPP1O [Xl(e + e-12') - Zkze-'~'] k2 - kl

Figure 5.5 pH-rate profile for the lanthanum(III)-promoted cleavage of BNPP. [La] = 2mM, [BNPP] - 0.05mM, [CHES] = 20.00mM, T = 2S°C. Data points are averaged from three determinations. Error bars are + three standard deviations. kobs, the pseudo-first-order rate constant for the cleavage of the diester (kl in

Equation 5.1) is in units of reciprocal seconds. The curve is calculated from Equation 2.5 (Chap ter II).

The pH-rate profile from pH - 9 + 10 exhibited a plateau just as for the ApA and HPNP reactions (Figure 5.5). This indicated that the nudeophile was not hydroxide from the surrounding solution; that would have manifested itself as a first-order dependence upon the rising pH. Thus the nudeophile is dearly a p-hydroxo iigand as was shown for the dinudear cobalt(III) complex discussed earlier.

Fitting the pH-rate data to the equation for equilibriurn dimer formation (Equations 2.3 and 2.5, Chapter II) provided the value for its formation constant, K = (3.6 + 0.8) x 1027 M-6, and the second-order rate constant for the diester hydrolysis reaction, k = 59 + 7 L-mol-1s-1.

On the plateau of the lanthanum(III) pH-rate profile the half-life for BNPP is only ttvelve seconds, corresponding to an estimated acceleration of 2.2 x 107-fold over the hydroxide reaction at pH = 9. (Chin et ai., 1989)

5.2.2 Rapid Ring-Opening of 3:s'-CAMP with Lanthanum(II1)

2 6 e

Retention time (min)

Figure 5.6 Typical stacked chromatograms from the lanthanum(III)-promoted ring-opening reaction of 3',S1-CAMP. [La] = 2.00mM8 [3',5'-CAMP] - 0.025mM, [CHESI = 20.00mM, pH = 9.00, T = Z°C. Chromatograms are (front to back) of quenched aliquots taken at 15,60,120,180,300,600,900,1800 and 3600s. Peaks are: 5'-AMP (1.7min), 3'-AMP (3.4min) and ApA (8.8 min).

2 4 6 8 1 8

Retcntion time (min)

Figure 5.7 An approximately equimolar mixture of (in order of appearance) 5'-AMP and 3'-AMP. Relative integrated peak areas are 1 and 1.09 respectively.

The ability of the lanthanum(III) dimer to promote the ring-opening

reaction of 3',5'-CAMP was tested on the plateau of the pH-rate profile,

monitoring the kinetics by HPLC as before. The reaction was followed for two

half-lives (Figure 5.6). The identity of the products, 3'- and 5'-AMP, was

confirmed by analysis of an equirnolar solution of authentic materials (Figure 5.7) and by spiking the reaction products (not shown).

The disappearance of the substrate was fitted to a simple first-order decay (Figure 5.8). The half-life for the reaction at pH = 9.82 was approximately five minutes based upon triplicate determinations (kOb, = 0.00218 + 0.00021 SI). The

reaction was also carried out at pH = 9.00, where the half-life was approximately nine minutes (kOb,= 0.00130 f 0.00023 s-1, for triplicate determinations). The

lanthanum(II1) dimer provides 2 x 109-fold rate acceleration at pH = 9 and

5.5 x 108-fold acceleration at pH = 9.8. The ratio of 5 ' - A m to 3'-AMP was approximately 1 to 2.4. This unequal

partitioning of the products reflects the relative rates of 3'-0-P and 5'-O-P cleavage respectively in the phosphorane transition state (or intermediate). A 5'-hydroxyl is thought to have a higher pK, than a 3'-hydroxyl and so would be

Figure 5.8 Typical kinetic data for the lanthanum(III)-promoted ring-opening of 3',5'-CAMP. [La] = 2.00mM, [3',5'-CAMP] - 0.025mM, [CHES] = 20.00mM,

pH = 9.00, T = 25°C. 3'5'-cAMP/IS is the ratio of 3'3'-CAMP peak area to that of the interna1 standard, sodium nitrobenzene sulfonate (-0.025mM). The c u v e is a simple first order decay fit to approximately the first two half-lives giving kob, = 0.00227s-1-

expected to be the poorer leaving group. The observed ratio of product monophosphates is opposite to what one would expect based solely on the leaving group argument but may be a consequence of some interaction of the 2'-hydroxyl or inherently unequal bond orders in the cydic phosphate.

The Fhst Direct Measurement of 2',3'-CAMP Ring-Opening with LanthanumUII)

The ring-opening reaction of 2',3'-CAMP was monitored by a stop-flow technique using a pH sensitive d ye, thymol ph thalein+. Ring-opening converts the cyclic diester monoanion to a monoester which, under alkaline reaction conditions, rvould be a dianion. Thus, the reaction produces a stoichiometric amount of hydronium ion which is observed by the quenching of thymolphthalein's absorbante in the visible spectrum.

The thymolphthalein was added to the buffered lanthanum(IXI) solution to

O 0.5 1 1.5 2 2.5

time (s)

Figure 5.9 Typical kinetic data for the lanthanurn(m)-promoted ring-opening of 2',3'-CAMP. [La] = 1.00mM, [2',3'-CAMP] - O.lmM, [thymolphthalein] - O.lmM, [CHES] = 5.00mM, pH = 9.82, T = 2S°C. The cuve is a simple first-order decay fit to approximately the first seven half-lives giving kob, = 1.99s-1. The inset shows

the complete kinetic run.

t Colorlcsç to blue (598nm) transition at pK, = 10.

104

ensure that absorbance changes were not due to any interaction between them. When the buffered lanthanum(m)-thymolphthalein solution was diluted with an equal volume of buffer (pHs were carefuiIy matched) no absorbance change was observed. When substrate was induded in the diluent the ring-opening reaction was readily observed (Figure 5.9).

The stop-flow data provided good fits to a simple first-order decay over the first five to ten half-lives. Table 5.1 summarizes the results of this set of experiments at three pHs on the pH-rate plateau. When the entire set of results (pH = 9.67 + 9.96) are pooled the average is kOb, = 2-02 f 0.13 s-1. In the

introduction to this chapter the upper limit for the half-life was estimated to be roughly 500ms; the achial result, 340ms, is quite dose to that.

The second-order rate constant for the hydroxide reaction is 1.5 x 10-3 L-mol-1s-1 (Abrash et al., 1967). At pH =9.8 the ambient half-life of

Zf,3'-CAMP would be roughly 7.3 x 106 s (-84days). The lanthanum(III) dimer accomplishes a 2 x 107-fold aiceleration.

At the time of this writing this is the fastest reported result for this

ring-opening reaction and it has never been determined for the

Ianthanide-promoted reactions. It is adcnowledged that due to the high reaction

pH herein (and so a faster background reaction) the acceleration is not as great as in other reports at lower pHs. (Young and Chin, 1995, for example) However,

otlier lanthanides provide practically the same absolute rates for RNA (and HPNP) transesterification at lower pHs and 2',3'-CAMP did not accumulate in any of

Table 5.1 Results for the lanthanum(II1)-promoted ring-opening reaction of 2',3'-CAMP on the pH-rate plateau. [La] = l.OOmM, [2',3'-CAMP] - O.lmM,

[thymolphthalein] - 0-lmM, [CHES] = 5.00mM, T = Z°C. Results at each pH are the averages of four to eight replicate determinations.

those cases. Thus, other lanthanides, operating at lower pHs, wiU provide simiiar aDsolz<te rates yet greater accelerations of 109 to 10lo as the background (ambient hydroxide) rate is deaeased.

5.3 Conclusion to Chapter V

The pH-rate profile of the lanthanum(III1-promoted deavage of BNPP exhibits a plateau above the of the lanthanum(II1). The half-maximal rate for this reaction coincides with the pHi/> These observations establish that as

with the ApA and HPNP reactions the reactive species is the penta-p-hydroxo lanthanum(II1) dimer. The plateau also demonstrates that the nudeophile is a hydroxo ligand as opposed to hydroxide from solution. On the plateau the lanthanum(II1) dimer is responsible for up to a ten million-fold acceleration for the BNPP cleavage.

The lanthanum(II1) dimer is also very reactive towardç the cyclic phosphates. At pH = 9 a billion-fold acceleration for 3',5'-CAMP was observed over the ambient hydroxide reaction. For the ring-opening of 2',3'-CAMP, which had never been directly measured in lanthanide-promoted RNA cleavage, the reaction was observed to proceed ten million times faster than the background reac tion.

These results demonstrate that lanthanum(III) (and likely lanthanides in general) are much more efficient for phosphate diester hydrolysis than had been observed by anyone before. The identity of the reactive species has also been establislied and a mechanism has been proposed, grounded in experimentai resul ts.

It was hoped that it would be possible to observe the hydrolysis of actual DiLTA with this system but the accelerations provided are not yet great enough to make this possible in a practical time frame. Under reaction conditions typically employed in this study no hydrolysis of the DNA dimer dApdA could be observed after eight hours, after which the lanthanum ultimately precipitated. Several more orders of magnitude in rate acceleration are required to achieve this and so far only the cerium(III/IV) system (Takasaki and Chin, 1994) can accomplish this nonenzymatically and give hydrolytic products. Although slower than the cerium(III/IV) system the other lanthanides still warrant consideration for future research; the redox active cerium system could conceivably present complications in practical applications wherein some components of a system other than the target phosphate diester may be vulnerable to oxidation or reduction.

Research in these lanthanide-promoted reactions will undoubtably be continued by others. Although the rates attained in this work are remarkable a

major advance must be made in ligand chemistry; to be useful as toolç for genetic research ligands for the lanthanides are required to make site-specific cleavage possible and practicai. %me advances have been reported in the literature but both speed and specificity require improvment. Furthemore, the reported ligand systems rvere not developed with a dinudear mode1 in mind such as is proposed in this work.

5.4 Experimental

Ma terials

Water (deionized and distilled) was degassed under vacuum, and kept

oxygen and carbon dioxide free under argon. Other materials were as follows: lanthanum(III) chloride heptahydrate

(ACS, Aldrich), CHES (ACS, Aldrich), sodium hydroxide (volumehic standard, 0.1 OOM, "10 w carbonate", Anachernia), sodium phosphate monobasic (HPLC arade, A&C), methanol (HPLC grade, BDH), BNPP (Sigma), 2',3'-CAMP (98%, u

Aldrich), 3',5'-CAMP (9895, Aldrich), 3'-AMP (99%, Aldrich), 5'-AMP (99%,

Aldrich), nitrobenzene sulfonic acid sodium salt (m- and p isomers,

Eas tman-Kodak), thymolphthalein (analytical grade, Fisher Scientific).

Spectrophotometer Kinetic Experimen ts

Stock solutions employed were prepared in the same fashion as described in Chapter IV.

The experiments were carried out using the same apparatus and

instrumentation as described in Chapter III. In a typical kinetic experiment measured volumes of stock lanthanum(m)

(20.00mM, standardized), CHES (100mM) and sodium hydroxide (100mM)

solutions were diluted to 2.000mM La(II1) and 20mM CHES. 5pL or lOpL of

BNPP solution (IOmM) kvas injected, rapidly mixed and data collection (400nm)

\vas begun. Data was processed (plotted and curve-fitted) using Kaleidagraph 3.0.1 on

a .Macintosh cornputer.

HPLC Kinetic Experiments

Stock solutions were the same as or prepared in identical fashion as above.

A stock solution of 3',5'-CAMP with interna1 standard was prepared as desaibed

for ApA in Chapter II. Kinetics were carried out in the same manner as described in Chapter II

using the same apparatus and instrumentation. Integration and data processing were carried out also as described earlier.

Stopflow Kinetic Experiments

Measured volumes of stock lanthanum(III) (20.00m.M), CHES (100mM) and thymolphthaiein (IûmM in absolute ethanol) were diluted with water to give final concentrations of 2.00mM La(III), 5.000mM CHES and 0.2mM thymol phthalein.

A second solution was prepared as 5.000mM CHES and 0.2mM 23'-CAMP in water.

The pHs of the two solutions were carefuily matched to within 0.02 pH units using a Fisher Accumet 15 pH meter and an Orion 8013 Ross semimicro combination eiectrode.

The two solutions were placed in the syringes of a HI-TECH Saentific SFA-12 Rapid Kinetia Accessory, used with a Hewlett-Packard 8452 diode array spectrophotometer equipped with a jacketed cuvette cornpartment and an RMÇ Lauda water circulator. Several stop-flow "shots" would be carried out while continuously monitoring the absorbance at 594nm.

Data was processed (plotteci and curve-fitted) using Kaleidagraph 3.0.1 on a Macintosh cornputer.

5.5 Appendix 1: Derivation of Equation 5.1

The lanthanum(II1)-promoted deavage of BNPP proceeds according to

Scheme A5.1.

Scheme A5.1

BNPP '' > NPP + p - nitrophenolate

NPP '' > P i + p-nitrophenolate

The rate of change of p-nitrophenolate obeys Equation A 5 1 and the concentration of BNPP at a given time is given by Equation A5.2. The rate of chance of NPP is described by A5.3.

Equation A51

d -[y - ni trophenola te] = kl [BNPP] + k2[NPP] dl

Equation A5.2

Equation A5.3

Equation A5.3 is not readily integrable but rnay be rendered so by rearranging and multiplying both sides by an integrating factor. This is a function, 1, such that the left-hand side of the equation

will be the deriuat ive of the product I(t)[NPP]. (Stewart, 1989) The integrating

factor in this case is ekzf . Appl ying this integrating factor then gives

Applying the limiting condition t = O ([NPP] = 0) one can solve for the

integra tion cons tant,

Substituting for C and rearranging provides the expression for NPP concentration a t time r, Equation A5.5.

Equation A5.5

Norv Equation A 5 1 can be re-expressed as

d - [ p - ni trophenola te] = kl [BNPP], e-'lt + klk2 [ ~ ~ p p ] , (e-k~' - e-k2') dr k2 - kl

~vhicli is rearranged, integrated and simplified to give the double exponential

Equa tion A56

Equation A5.6

[p - ni trophenola te] = 7[BNPP], + [BNPPIo [4 (e-hf + ehk2') - 2k2e~11] k2 - kl

5.6 Appendix 2: Tabulated Kinetic Data

Table A5.l Complete data for the pH-rate profile of the lanthanum(III)-promoted

cleavage of BNPP. [La] = 2.00mM, [BNPP] - 0.05mM, [CHESJ = 20.00mM, T = 25°C. Values uere riof roztnded off trrrtil al1 calmlations had been completed.

Table A5.2 Complete data for the lanthanum(II1)-promoted ring-opening reactions of 3',5'-CAMP. [La] = 2.00mM, [3',5'-CAMP] - O.OlmM, [CHEÇI = 20.00mM, T = 25OC. Valzres iuere not rounded off until al1 calculations had lireri coiri pletrd

Table A5.3 Complete data for the lanthanum(II1)-promoted ring-opening of 2',3'-CAMP. [La] = l.OOmiM, [2',3'-CAMP] - 0-lmM, [thymolphthaiein] - O.lmM,

[CHESI = 5.00miM, T = 25OC. Vnlltes were not rourided off until al1 calculntions had lieen cowpleted.

Chapter VI Conclusion to the Thesis

6.1.1 Contributions to Knowledge

Lanthanum(m) was discovered to provide the greatest absolute rates for RNA transes terification of al1 the lanthanides. With a half-life of approximately 13s it is also fas ter than any nodanthanide system.

For the first time a coherent theory for the lanthanide reactivity is available and is well rooted in experimental fact. The reactive species was found to be a penta-p-hydroxo dilanthanum(III) species. This dinudear system is proposed to operate by double Lewis-aud activation and the intramolecular attack of the 2'-alkoxide. The kinetic isotope effect of 1.9 and a one-proton inventory indicate that the 2'-hydroxyl deprotonation is nof concerted with the nudeophilk attack. It is proposed that a rapid equilibrium proton transfer occurs between the 2'-hydroxyl and a bridging hydroxo prior to the nudeophilic attack and rate-determining intramolecular-generai-acid catalyzed breakdown of the phosphorane to products. It is proposed that the general aad is a bound water, formed from the protonation of the bridging hydroxo.

Other lanthanides have b e n shown to function by the same mechanism. Maximal reactivities are al1 quite comparable in spite of having pHi/* values

spanning neariy two pH units. The mie order of reactivity of the lanthanides in RNA deavage is the reverse of what has k e n reported in the literature.

The reactivity of lanthanum(III) towards the cydic nucleotides 2',3'-CAMP and 3',5'-CAMP has been determined. The half-lives of 34Oms and 9min respectively show that lanthanum(XII) is the most reactive system, lanthanide or otherwise, reported for these substrates. Kinetic and product analysis indicate that the reactive speaes is the same penta-p-hydroxo dilanthanum(IlI) complex whereas the nudeophile is a p-hyàroxo.

6.1.2 Publications and Presentations

Publications and Patents

Hurst, P.C.; Takasaki, B.K.; Chin, J.; Rapid Cleavage of RNA with a kÀz(IZ1) Dimer (J.Am.Chem.Çoc. 1996,118,9982)

Abbas, KA., Hurst, P.C., Edward, J.T.; Re-examination of the Kirkwood- Westheimer Theory of Electrostatic Effects. V . Effect of Churged Substituents on the Rates of Alknline Hydrolysis of SubstitutPd Strychnines (Can J-Chem. 1997,75,44)

stranix, B.R.; Hurst, P.C.; Chin, J.; Darling, G.D.; Functional Polyrners pom (Viny1)polystyrene. Short Routes to Binding Carbon Centered Functionnl Groups to Polys f yrene Resin through a Dimethylene Spacer (patent ap plied for)

Presen ta tions

June 1996, Canadian Society for Chemistry 79th Conference and Exhibition; Hurst, P.C.; Takasaki, B.K.; Chin, J.; Rapid Cleavage of RNA by Lanthanum Chloride (presenter, PCH)

References

Abrash, H. I., Cheung, C.-CI S., and Davis, J. C. (1967) The Nonenrymatic Hydrolysis of Nudeoside 2 '3 '-Phosphates. Biochemistrv, 6,1298-1 303.

Adleman, L. M. (1994) Molecular Cmputation of Soluiions to Cmbinatorjal Problems. Mence, 266, 1021-1024.

Aksel'rud, N. V., and Ermolenko, V. 1. (1961) Hydroxides and Basic Chlodes of Europium, Terbium, and Holmium. Russ. 1. Inow. - Chem., 6,397-399.

Aksel'rud, N. V., and Spivakovskii, V. B. (1960a) Hydroxides and Basic Chlorides of Yttrium and Lanthanum. Russ. 1. Inom. Chem.,5,158-163.

Aksel'rud, N. V., and Spivakovskii, V. B. (1960b) Busiç Chloride and Hydroxide of Smnmim. Fusse 1. in or^. - Chem., 5,163-167.

Aksel'rud, N. V., and Spivakovskii, V. B. ( 1 9 6 0 ~ ) Basic Chlorides and Hydroxide of Dysprosium. Russ. 1. Inore. - Chem., 5,168-171.

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D. (1989) The Molecular Biolow of the Cell. New York: Garland Publishing, Inc.

Altman, S., Kirsebom, L., and Talbot, S. (1993) Recent Studies of Ribonuclevzse P. FASEB T., 7,7-14.

Anslyn, E., and Breslow, R. (1989a) On the Mechanism of Catalysis by Ribonuclease- Claaaage and Isomeriurtion of the Dinuckotide UpU Gztalyzed by Imidazole Buffes. 1. Am. Chem. Soc., 11 1,4473- 4482.

Anslyn, E., and Breslow, R. (1989b) Proton lnventory of a Bifunctional Ribonuclease Model. J. Am. Chem. Soc., 1 1 1,8931-8932.

Baes, C. F., Jr., and Mesmer (1976) The Hvdrolvsis of Cations. Toronto: John Wiley & Sons

Bamam, E-, and Meisenheimer, M. (1938a) Spaltung von Estem der Phosphorsaure in Gegenwart von Lanthanhydroryd (1. Mit teil. über "phosphatische" Wirkungett von Hydrogelen). çhem- Ber., 71,171 1- 1720.

Bamann, E., and Meisenheimer, M. (1938b) Über dos Verhalten von Metthydroxyden gegeniiber Phosphorsaure-ester (11. Mitteil. über "phosphatische" Wirkungen von Hydrogelen). Chem. Ber.,71, 1980-1983.

Bamann, E., and Meisenheimer, M. (1938~) Umwandlung von Metaphosphat in Orthophosphat un ter d m EinfluJ? von Metalloxyden und Hydroxyden (III. Mitteil. über "phosphatische" Wirkrtngen von Hydrogelen). Chem. Ber., 72,2086-2089.

Bamann, E., and Meisenheimer, M. (19386) Katalytische Aufspaltrt ng m n Pyro- und Polyphosphaten (IV. Mitteil. über "phospfuztische" Wirkungen uon Hydrogelen). Chem. Ber., 77,2233-2236.

Eiamann, E., Traprnann, H., and Fischler, F. ( 1 9 s ) Verhallen und Spe#taf von Cm und Lanthan als Phosphatase-Modelle gegen über Nucleinsüuren und Monon ucleotiden (Behaim and Speci@ity of Cetium and Lanthanum as Phosphatase Models Towards Nucleic Acids and Mononuclwtides). Biochem. Z., 326, 89-96.

Barnes, J. C., and Blyth, C. S. (1985) Structure and Thermal Decomposition of Potassium Bis-p-pe~oxo Hexacmbonatodicerate(N) Doddydrate . Inow. - Chim. Acta, 2 10,133-1 37.

Barnes, J. C., Blyth, C. S., and Knowles, D. (1987) Sodium Salts of the Bis-p-peroxo- hexncnrbonatodicerate(IV) Anion. Cystal structure of NaglCe(OZ)IC03)3~2-18H20. ïnorg. Chim. AC@ 126, L3-L6.

Bates, G. (1996) Exp~nded Glutamines and Neurodegenmtion-A Gain of I m @ . BioEssavs, 18,175- 178.

Ekese, L. S., and Steitz, T. A. (1991) Structural Bais for the 3 '-5 ' Exonuclease Adiwity of Eschhh ia coli DNA Polymerase Ir A Two Meta1 Ion Mechanism. EMBO l., 10,25-33.

Berry, R. S. (1960) Correlation of Rates of Intramolecuhr Tunneling Processe, with Application tu Some Group V Compounds. J. Chem. Phvs., 32,933-938.

Bester, A., Kennedy, D., and Heywood, S. (1975) Two Classes of Translational Control RNA: Their Role in the Reguhtiun of Protein Synthesis. P m . N a t Acad. Sci. USA, 72,1523-1528.

Breaker, R. R., and Joyce, G. F. (1994) A DNA Enzyme that C l m a RNA. Chemistrv 1 , 223-229.

Breslow, R. (1991a) Bifunctional Acid-Base Catalysis by Imidazole Groups in Enzyme Mimics. L, Molecular Cat-, 91,161-174.

Breslow, R. (1991b) How Do lmidawle Groups Cotalyre the Cleavage of RNA in Enzyme Models and in Enzymes ? Evidence fiom "Negative Gztalysis ". Acc. Chem. Res., 24,317-324.

Breslow, R., Dong, S. D., Webb, Y., and Xu, R. (1996) Further Studies on the Buffer-Catdyzed Cleavage and Isornerization of Uridyluridine. Medium and lonic Strength Effecfs in Catalysis by Morpholine, Irnidazole, and Acetafe Buffers Help Clarify the Mechanisms Used by a Ribonuclease Mimic. J. Am. Chem. Soc., 118,6588-660.

Breslow, R., and Huang, D.-L. (1991) Effects of Meta1 Ions, Including ~ 2 + and Lanthanides, on the Cleavage of Ribonucleotides and RNA Mode1 Compounds. Proc. Natl. Acad. çci. USA, 88,4080-4083.

Breslow, R., Huang, D.-L., and Anslyn, E. (1989) On the Mechanism of Action of Ribonuclerrses: Dinucleotide Clemage Catalyzed by Imidozole and 2n2+. Proc. Natl. Acad. Sci. USA, 86,1746-lm.

Brown, D. M., and Usher, D. A. (1965) Hydrolysis of Hydroxyalkyl Phosphate Esters: Effect of Changing Ester Group. J. Chem. Soc., ,6558-6564.

Buckingham, D. A. (1977) Mefal-OH and Its Ability fo Hydrolyse (or Hydrate) Substrates of Biologi~l Interest. Biolopical Asriects of Inor~ranic Chemistry. Eds. Addison, A.W., et al. New York: Wiley InterScience,

Butcher, W. W., and Westheimer, F. X. (1955) iunthanum Hydroxide Gel Promoted Hydrolysis of Phosphate Esters. J. Am. Chem. Soc., 77,2420-2424.

Cech, T. R., Zaug, A. J., and Grabowski, P. J. (1981) In vitro Spicing of the Ribasmal RNA Precursor of Tetrahymena: Involvement of a Guanosine Nucleotide in the Exrision of the Intemening Sequence. Cell, 27,487-496.

Chandler, A. J., and KYby, A. J. (1992) Inhamolecular Attack by the Hydroq Group on Phosphate Diesters is both General Acid and General &Ise Cafalysed. J. Chem. Soc. Chem. Commun., ,1769-1770.

Chapman, W. H., Jr., and Breslow, R. (1995) Selective Hydrolysis of Phosphte Esters, Nitrophenyl Phosphates and UpU, &y Dirneric Zinc Compléxes Depends on the S p e e r Length. 1. Am- Chem- Soc, 1 17,5462-5469.

Chen, C.-h., and Sigman, D. (1988) S q ~ ~ c e - S p e c i f ü Scission of RNA by 1 ,IO-PhenanthrolinFCupper Linked to Dwxyoligonuclwtides. 1. Am. Chem. Soc., 11 O, 65706572.

Chin, J. (1991) Dmeloping A r t i f i d Hydrolytic Mefalloenzyma by a Unified Mechanistic Approach. Acc. Chem. Res., 24,145-152.

Chin, J., Banaszayk, M., and Jubian, V. (1988) N d Ligand Effect on Cobdt (U~ Cornplex Prmted Hydrolysis ofa Phosphate Diesta. J. Chem- Soc. Chem. Commun., ,735-736.

Chin, J., Banaszczyk, M., Jubian, V., and Zou, X, (1989) Co(liI) Complex Promoted Hydrolysis of Phosphate Diesters: Cornpariçon in Reactiwity of Rigid cis-Diaquotefraaracobalt(III) Complexes. J- Am- Chem. Soc., 11 1,18&190.

Chin, J., and Zou, X. (1987) Catalytic Hydrolysis of CAMP. Çan. 1. Chem., 65,1882-1884.

Chin, J., and Zou, X. (1988) Cobalt(lll) Complex Promoted Hydrolysis of Phosphate Diesters: Change in Rate-Deferrnining Step With change in Phosphate Diester Reactizn'ty. J. Am. Chem. Soc., 110,223-225.

Chin, K. O. A., and Mono w, J. R. (1994) RNA Cltnvage and Phosphate Diester Transesterr@xtion by Enurpçulated Lanthanide Ions: Trmersing the Lanthanide Series with Lanthanurn(lll), Europiurn(LII) and LutetiundIiI) Complexes of 1,4,7,l O-Tetrakis(2-hydroxyalky1)-1 ,4,7,l O-tehaazacyclododerane. Inore. Chem., 33,5036-5041.

Ciesiolka, J., Marciniec, T., and Krzyzosiak, W. 1- (1989) Probing the Environment of hnthanide Binding Sifes in Yerist by Specific Metal-Ion Promoted Clavages. Eur. 1. Biochem., 182,445- 450.

Cleland, W. E., and Hengge, A. C. (1995) Mechanimis of Phosphoryf and Acyl Tramfer. FASEB T,9, 1585-1594.

Cleland, W. W. (1995) Isotope Effects: Detemination of Enzyme Trann*tion Sfate Structure. Enz-me Kinetics and Mechanism. Ed. Purich, D.L. Mcthods in Enzymology. Toronto: Acadernic Press, 249: 341-373.

Dalby, K. N., Hollfelder, F., and Kitby, A. J. (1992) Elecfrostatic Cat~!ysis of the Hydrolysis of a Phosphate Diester in Water. J . Chem, Soc. Chem. Commun., , 1770.

Dalby, K. N., Kirby, A. J., and Hollfelder, F. (1993) Models for Nuclease Catalysis: Mechanism for General Acid Catalysis of the Rapid Inhamolecular Displacement of Mefhoxidefiorn a Phosphate Dk te t . J. Chem. Soc. Perkin Trans. 2,, 1269-1281.

Davies, J. F., Hostomska, Z., Hostomsky, Z., Jordan, S. R., and Matthews, D. A. (1991) Crystal Structure of the Ribonuclaase H Dommn of MV-1 Reverse Transcriptase. Science, 252,88-94.

Davis, A. M., Hall, A. D., and Williams, A. (1988a) Charge Description of Buse-Catalyzed Alcoholysis ofAry1 Phosphodiesters: A Ribonuclease Model. J. Am- Chem. Soc-, 1 IO, 5105-5108.

Davis, A. M., Regan, A. C., and Williams, A. (1988b) Experimental Charge Measurement at b v i n g Oxygen in the Buvine Ribonuclease A Catalyzed Cyclization of Uridine 3'-Phosphate Aryl Esters. Bioc hemistrv, 27,9042-9047.

De Robertis, E. D. P., and De Robertis, E. M. F. (1980) 0. Seventh ed. Philadelphia: Saunders ColIege/Holt, Rinehart and Winston

Dick, J. C. (1973) Analvtical Chemistrv. Montreal: McGraw-Hill Book Company

Eftink, M. R., and Biltonen, R L. (1983a) Energetics of Ribanuclease A Catalysis. 1. pH, Ionic Sfrength, and Soloent lsotope Dependence of the Hydrolysis of Cyfidine Cyclic 2',3'-Phosphate. Biochernism 22,5123-5134.

Eftink, M. R., and Biltonen, R. L. (1983b) Energetics of Ribonuclease A Catalysk 2. Nonenzymatic Hydrolysis of Cyfidine Cyclic 2 '3 '-Phosphate. Biochemistw, 22,5134-5140.

Eichhorn, G. L., and Butzow, J. J. (1965) Interactions of Mefal Ions with Polynucleotides and Related Compo unds. III. Degradatiun of Polyribonuclwtides by Lanthanurn Ions. Biomlvmers, 3,79-94.

Fersht, A. (1985) Enzvrne Structure and Mechanism. Second ed. New York: W. H. Freeman and Company

Guerrier-Takada, C., and Altman, S. (1984) Catalytic Activity of an RNA Molecule Prepared by Transcription in vitro. Mencc 223,285-286.

Guemer-Takada, C., Gardiner, K., Marsh, T., Pace, N., and Altman, S. (1983) The RNA Moiety of Ribonuclase P Is the Catalytic Subunit of the Enzyme. (Zell. 35,849-857.

Haake, P. C., and Westheimer, F. H. (1961) Hydrolysis and Exchange in Esters of Phosphoric Acid. Ir Am. Chem. Soc., 83,1102-1 109.

Han, H., Rifkind, J. M., and Mildvan, A. S. (1991) Role of Divalent Cations in the 3 '5'-Exonucltxse Reaction of DNA Polyrnerase 1. Biochemistw, 30,11104-1 1108.

Hempel, C. G. (1966) Philoso hv of Naturd Science. Foundations of Philosophy Series. Toronto: Pren tice-Hall

Hendry, P., and Sargeson, A. M. (1984) Base Hydrolysis of the Pentu-arnrnine(trimethy1 phosphate)indiurn(lll) Ion. J. Chem. Soc. Chem. Commun., ,164-165.

Hendry, P., and Sargeson, A. M. (1989) Meta1 Ion Promoted Phosphate Ester Hydrolysis. Intrarnolecular Attackof Coordinated Hydroxide Ion. J. Am. Chem. Soc., 111,2521-2527.

Hendry, P., and Sargeson, A. M. (1990) Reactivity of Coordinated Phosphate Esters: Pen taamminecobalt (III) Complexes. Inore. Chem., 29,92-97.

Hengge, A. C., and Cleland, W. W. (1991) Phosphoryl-Transfer Reactions of Phosphodiesters: Characterization of Transition States by Heauy-Atom Isotope Effects. J. Am. Chem. Soc., 113,5835- 5841.

Hengge, A. C., Tobin, A. E., and Cieland, W. W. (1995) Studies of Transition-State Structures In Phosphoryl Tram fer Raactionç of Phosphodiaters of p-Mtrophenol. J . Am. Chem. Soc., 2 17,5919-5929.

Herschiag, D. (1994) Ribanuclease Revisited: Catalysis aia the Classical General And-Base Mechunisrn or a Trigter-ZikeMechanisrn? J. Am. Chem. Soc., 116,11631-11635.

Hertzberg, R P., and Dervan, P. B. (1982) Claaage of Double Helical D N A by (Methidiump'opyl- EDTA)iron(ii). J. Am. Chem. Soc., 104,313-315-

Heywood, S. M., Kennedy, D. S., and Bester, A. J. (1974) Separation of SpecaFc Initiation Fuctors lnvol~ed in the Translation of Myosin and Myoglobin Messenger RNAs and the Isolation ofa New RNA Inuolved in Translation. Roc. Natl. Acad. Sci. USA, 71,2428-2431.

Hoffmann, R., Minkin, V. I., and Carpenter, B. K. (1996) Ockharn's Razor and Chemistry. Bull. Soc- Chim. Fr., 133,117-130.

Housman, D. (1995) Gain of Glutamines, Gain of Function? Nature Genetics, 20,344.

Huntington's Discase Collabora tive Research Grou p (1993) A Nmel Gene Containing a Trinuclwtide Repeat That Is Expanded and Unstable cm HUntirigt~n'~ Disease Chromosomes. CZelt, 72,971-983.

Irisawa, M., and Komiyama, M. (1995) Hydrolysis of DNA and RNA Through Cooperation of Two Metal Ions: A Nwel Mimic of Phosphdiesteruses. J. Biochem. (To kvQZ 1 17,465466.

Irisawa, M., Takeda, N., and Komiyama, M. (1995) Synergetic Cafalysis by Two Nonlanthanide Metal ions for Hydrolysis of Din'banucleorides. 1. Chem. Soc. Chem. Commun., ,1221-1222.

Jarvinen, P., Oivanen, M., and Lonnberg, H. (1991) Intmconuersion and Phosphoester Hydrolysis of 2 '5',- and 3 '5 '-Dinuclwside Monophosphates Kinefics and Mechanisms. J. Orp. Chem., 56,5396-5401.

Jencks, W. P. (1980) M e n 1s an lntemrediate Not an Intermediate? Enforced Mechanisms of General Acid-Base Catalyzed, Carbocation, Carbanion and Ligand Exchange Reactions. Acc. Chem. Res., 13, 161-169.

Jencks, W. P. (1987) Catalvsis in Chemistrv and Enzvmology. - Toronto: General Publishing Company

Jones, D. R., Lindoy, L. F., and Çargeson, A. M. (1983) Hydrolysis of Phosphate Esters Bound to Cobalt(I1I1. Kinetics and Mechanism of Intrarnokcukr Attack of Hydroxide on Coordinated 4-Nitrophenyl Phosphate. J . Am. Chem. Soc., 105,7327-7336.

Kim, E. E., and Wyckoff, H. W. (1991) h c t i o n Mechanism of Alkaline Phosphatase &rred on Crystal Shuctures. Two-Meta1 Ion Catalysiç. J . Mol. Biol., 218,449-464.

Kirby, A. J. (1980) E f l e t i ~ e Molarities for Intrarnolemlar Reactions. Advances in Phvsical Owanic Chemistry. Eds. Gold, V., et al. Toronto: Academic Press, 17: 183278.

Kirby, A. J., and Younas, M. (1970) The Reactimty of Phosphate Esters. Reactions of Diesters with Nucleuphiles. Journal of the Chernical Societv. Part B., ,1165-1 172.

Kluger, R., Covitz, F., Demis, E., Williams, L. D., and Weçtheimer, F. H. (1969) pH-Product and pH-Rate Profiles for the Hydrolysis of Methyl Ethylene Phosphate. Rate Limiting Pseudorotation. 1. Am. Chem. Soc., 91,6066-6072.

Komiyama, M. (1995) Sequence-Specijîc and Hydrolytic Scission of DNA a d RNA by hanthanide Cornplex-OligoDNA Hybrids. J. Biochem. (Tokvo), 1 18,665470.

Komiyama, M., hokawa, T., Shiiba, T., Takeda, N., Yoshinari, K., and Yashiro, M. (19931) Molecular Design oJArtiFcia1 Hydrolytic nu cl^ and Ribonuclases. Nucleic Acids Svmp. Sm., 29, 197-1 98.

Komiyama, M., Matsumoto, Y., Hayashi, N., Matsumura, K., Takeda, N., and Watanabe, K. (1993b) Hydrofysis of OfigoDNAs by lanthanide Metal(III) Chluride. Pol-m. l., 35,1211-1214.

Komiyama, M., Matsumura, K., and Matsumoto, Y. (1992) Unptecedentedly Fasf Hydrolysis cf the RNA Dinuclwsïde Monophosphates ApA and UpU by Rare Earth Meta1 Ions. 1. Chem. Soc. Chem. Commun., ,640-641.

Komiyam, M., Matsumura, K., Yonezawa, K., and Matsumoto, Y. (1993~) C O ~ ~ S B C L I ~ ~ ~ ~ Cafaiys& 6y Cmiurn(1II) Ion for Complefe HydrolysLr of Phosphodiester Linkage in DNA. Chem. Emress, 8,8588.

Komiyama, M., Takeda, N., Takahashi, Y., Uchida, H., Shiiba, T., Kodama, T., and Yashiro, M. (1995) Efficient and Oxygen-independent Hydrolysk of Single-strand DNA by Ceriurn(IV) Ion. j. Chem. Çoc. Perkin Trans. 2, ,269-274.

Kruger, K., Grabowski, P. J., Zaug, A. J., Sands, J., Gottschling, D. E., and Cech, T. R. (1982) Self- Splicing RNA: Autoexcision and Au tocyclization of the Ribosomal RNA Intmening Sequence of Tetrahymena. Cell, 3I,l47-157.

Kuusela, S., and Liinnberg, H. (1993) Metal Ions that Promote the Hydrolysis of Nucfeotide Phosphoesters Do Not Enhance Intramofecular Phosphate Migration. J- Phys. &P. Chem., 6,347-356-

Kuusela, S., and Lonnberg, H. (1994a) Hydrolysis and Isomerization of the Internucleosidic Phosphodiester Bonds of Polyuridylic Acid: Kinetics and Mechanism. 1. Chem. Soc. Perkin Trans. 2, , 2109-21 12.

Kuusela, S., and Lonnberg, H. (1994b) Metal-Ion-Promoted Hydrolysis of Polyrtridyfic An'd. JI Chem- Soc. Perkin Trans. 2, ,2301-23û6.

Kuusela, S., Rantanen, M., and Lonnberg, H. (1995) Metal-Ion-Promoted Hydrolysis of Uridylyl(3'S')Uddine. Interna1 as. Exferml General Base Catalysis. J. Chem. Soc. Perkin Trans- 2,, 2269-2273.

Linkletter, B., and Chin, 1. (1995) Rupid Hydrolysis of RNA with a CU' Cornplex. Angew. Chern. Int- Ed. En~l. , 34,472-474.

Long, D. M., and Uhlenbeck, O. C. (1993) Self-Clean'ng Catalytic RNA. FASEB L., 7,25-30.

Martin, D. W., Ir., Mayes, P. A., Rodwell, V. W., and Granner, D. K. (1985) Hamr ' s Review of Biochemistry. Twentieth ed. East Norwalk, CT: Appleton-Century-Crofts

Matsumoto, Y., and Komiyama, M. (1992) DNA Hydrolysis 6y Rare Earth Metal Ions. Chem. Express, 7,785-788.

Matsumura, K., Matsumoto, Y., and Komiyama, M. ( 1992 ) Rare Earth Metal Ions for Unprecedentedly Fast RNA Hydrolysis. Nucleic Acids Syrn~. Ser., , 11-12.

Matta, M. S., and Vo, D. T. (1986) Proton Inuentory of the Second Step of Ribonucle~se Catolyçis. L Am. Chem. Socy 108,5316-5318.

Morrow, J. A., Buttrey, L. A., and Berback, K. A. (1992a) TransestenenI;r,tion of a Phosphate Diester by Divalent and Tn'mlent Metal Ions. Inore. Chem., 3l,W2O.

Morrow, J. R., Aures, K., and Epstein, D. (1995) Metal Ion Promoted Attack of an Akohol on a Phosphate Diester: Modelling the Role of Meta1 Ions in RNA Self-splicing Reactions. J. Chem. Soc. Chem. Commun., ,2431-2432.

Morrow, J. R., But trey, L. A., Shel ton, V. M., and Berback, K. A. (1992b) EfFcient Catalytic Cleavage of RNA by lanthaniddLii) Macrocyciic Complexes: Toward Synthetic Nucleases fm in V i m Applications. 1. Am. Chem. Soc., 124,1903-1905.

Oivanen, M., Schnell, R., Pfleiderer, W., and k ~ b e r g , H- (1991) Interconversian and Hydroiysis of Monomethyl and Monoisopropyl Esters of Adenosine 2'- and 3'-Monophosphates: Kinetics und Mechunisms. J. Orp. Chem, 56,3623-3628.

Pace, N. R., and Smith, D. (1990) Ribonuclerzse P: Function and Variation. J. Biol. Chem., 265,3587- 3590.

Panagiotopoulos, A., Zafiropoulos, T. F., Perlepes, S. P., Bakalbassis, E., Masson-Ramade, I., Khan, O., Terzis, A., and Raptopoulou, C. P. (1995) Molecular Structure and Magnetic Properties of Acetato-Bridged iiznthanide(1II) Dimers. Inora. Chem., 34,491 8-4920.

Pratviel, G., Duarte, V., Bernadou, /., and Meunier, B. (1993) Nonenzymatic Cleavage and Ligation of D N A at a Three A-T Base Pair Site. A Two-Step "Pseudohydrolysis" of DNA. 1. Am. Chem. Soc, 115, 7939-7943.

Ragunathan, K. G., and Schneider, H.-J. (1996) Binuclear Lanthanide Complexes as Catalysts For the Hydrolysis of Bis(p-nihopheny1)phospbfe and Double-Shanded DNA. A n ~ e w . Chem. Int. Ed. Enpl, 35,1219-1221.

Rammo, J., Hettich, R., Roigk, A., and Schneider, H.-J. (1996) Cafalysis of DNA Clenuuge by Lanthanide Complexes with Intercalating Ligands and their Kinetic Characteriaztion. Chem. Commun, , 105-107.

Rawls, R. (1997) Catalytic DNAs. Chernical & EnPineri News February 3: 33-35.

Rawn, J. D. (1983) Biochemistrv. New York: Harper & Row

Rordorf, B. F., and Kearns, D. R. (1976) Effect of Europium(If1) on the T h m a l Denaturation and Cleavage of Transfer Ribonucleic Acids. Biowlvrners, 15,1491-1504.

Saldanha, R., Mohr, G., Belfort, M., and Lambowitz, A. (1993) Group f and Group II Inhons. FASEB 1,7,15-24.

Sawata, S., Komiyarna, M., and Taira, K. (1995) Kinetic Eoidence Based on Solvent Isotope Effecis for the Nonexistence of a Proton-Transfer Process in Reactions Catalyzed by a Harnrnerhaad Ribozyme: Implication to the Double-Metal-Ion Mechanism of Catalysis. 1. Am. Chem. Soc., 117,2357-2358.

Schowen, R. L. (1977) Soluent Isotope Effcts on Enrymatic Reactions. @tom Effects on Enzvme- Catalvzed Reactions. Eds. Cleland, W.W., et al. Baltimore: University Park Press,

Schwarzenbach, G., Boesch, J., and Egli, H. (1971) The Formation of Dinuclem p-Hydroxo-cobalt(LII) Complexes. J . Inorp. Nucl. Chem., 34,2141-2156.

Shelton, V. M., and Morrow, J. R. (1991) Catalytic Transesten~uation and Hydrolysis of RNA by Zinc(II) Complexes. Inorp. - Chem., 30,4295-4299.

Shiiba, T., Yonezawa, K., Takeda, N., Matsumoto, Y., Yashiro, M., and Komiyama, M. (1993) hnthanz.de Meta1 Complexes for the Hydrolysis of L i m r DNAs. J. Molecular Ca t., 84, L21-U5.

Shimomura, M., and Egami, F. (1953) Dephosphorylation of Nucleic Acids, Mononuclsotides and Casein by iiznthanurn and Cerium Hydroride Gels. Bull. Chem. Soc. Jpn., 26,263-267.

Sigman, D. S. (1986) Nuclease Actiuity of 1.10-Phenanthroline-Cupper Ion. Acc. Chem. Res., 19,180- 186.

Sjogren, A. S., Pettersson, E., Sjoberg, B. M., and Stfimberg, R (1997) Metaf Ion interaction with Cosubshafe in Self-Splicing of Croup 1 Introns. Nucleic Acids Res, 25,648-653.

Smith, D., and Pace, N. R. (1993) Multiple Magnesium Ions in the Ribonuclease P Reaction Mechunism. Biochemistrv, 32,5273-5281.

%wa, G. A., Hengge, A. C., and Cleland, W. W. (1997) Isotope EMls Support a Concertrd Mechanism for Ribonuclease A. J. Am. Chem. Soc., 119,2319-2320.

Steitz, T. A., and Steitz, J. A. (1993) A General Two-Metal-Ion Mechanism for Catalytic RNA. Proc. Na tI. Acad. Sci. USA, 90,64984502.

Stem, M. K., Bashkin, J. K., and Sall, E. D. (1990) Hydrolysis of RNA by Transition-Metal Complexes. J . Am. Chem. Soc., 11 2,5357-5359.

Stewart, J. (1989) Multivariable Calculus. Pacific Grove, CA: Brooks/Cole Publishing Company and Rand McNalIy & Company

Strater, N., Lipscomb, W. N., Klabunde, T., and Krebs, B. (1996) Two-Meta1 Ion Catalysis in Enzymatic Acyl- and Phosphuryl-Transe Reactions. A n ~ e w . Chem. I n t Ed. En& 35,2024-2055.

Suck, D., Lahm, A., and Oefner, C. (1986) Structure of DNme I at 2.0A Resolution Suggests a Mechnnism for Binding and Cutting DNA. Nature (London), 321.62û-625.

Sumaoka, J., Yashiro, M., and Komiyama, M. (1992a) Enormous Catalysis of Lanthanide Meta1 Ions for Unprecedentedly Fast Hydrolysis of 3 '5'-Cyclic Adenosine Monophosphate. Nucleic Acids S-yn~ . Ser., ,37-38.

Sumaoka, J., Yashiro, M., and Komiyama, M. (1992b) Remarkably Fast Hydrolysis of 3 '5'-Cyclic Adenosine Monophosphate by Cerium (III) Hydronde Cluster. J. Chem. Soc. Chem. Commun., ,1707- 1708.

Szewczyk, J. W., Baird, E. E., and Dervan, P. B. (1996) Couperatbe Triple-Helix Formation vi4z a Minor Grome Dimerizatwn Domain. J. Am. Chem. Soc., 1 18,67784779.

Taira, K., Uchimaru, T., Storer, J. W., Yliniemela, A., Uebayasi, M., and Tanabe, K. (1993) Properties of Dianionic Oqphosphorane In termediates: Implication to the React ion Profile for Base- Catalyzd RNA Hydrolysis. J. Orn. Chem., 58, XMl9-3OOl7.

Takasaki, B. (1994) Lanthanide Ions in the Development of Artificial Nucleases. Ph-Dg Mail1 University

Takasaki, B. K., and Chin, J. (1993) Synergistu Effect between La(Iïl) and Hydrogen Peroxide in Phosphate Diester Claurage. J. Am. Chem. Soc., 115,9337-9338.

Takasaki, B. K., and Chin, J. (1994) Cleavage of the Phosphate Diester Buckbone of DNA with Cerium(I11) and Moleculat Oqgen. J. Am. Chem. Soc., 116,1121-1122.

Takasaki, B. K., and Chin, J. (1995) La(Iii)-Hydrogen Peroxide Cooperativity in Phosphate Diester Cleavagc A Mechanistic Shcdy. J. Am. Chem. Soc, lV,8582+5ûS.

Thatcher, G. R. J., and Kluger, R. (1989) Mechanism and Catalysis of Nuclmphilic Substitution in Phasphaie Esters. Advances in Phvsical Otnanic Chemistrv. Ed. Bethell, D. Toronto: Academic Press, 25: 99-265.

Thompson, J. E., and Raines, R. T. (1994) Vdue of General Acid-Base Catalysis to Ribonuclease A. L Am. Chem. Çoc., 1 1 6,5467-5468.

Thompson, J. E., Venegas, F. D., and Raines, R. T. (1994) Energebis of Catalysis by Ribonucleases: Fate of the 2 '3 '-Cyclic Phosphodiester In temediate. Biochemistrv, 33,7408-7414.

Tsubouchi, A., and Bruice, T. C. (1994) Remarhblc (-10'~) Rate Enfuncement in Phosphonate Ester Hydrolysis Catalyzed by Two Metal Ions. J. Am. Chem. Soc., 11 6,11614-1 1615.

Tsubouchi, A., and Bruice, T. C. (1995) Phosphonate Ester Hydrolysis Catalyzed by Two hnthanum Ions. Inhamolecular Attack of Coordinated Hydroxide and Lewis Acid Actiuation. J. Am. Chem. Soc., 7 17,7399-7411.

Uchirnaru, T., Uebayosi, M., Tanabe, K., and Taira, K. (1993) Theoretical Analyses on the Role of M~'+ Ions in Ribozyme Rmctions. FASEB l., 7,137-139.

Uebayasi, M., Uchimaru, T., Koguma, T., Sawata, S., Shimayama, T., and Taira, K. (1994) Theoretical and Experimental Considerations on the Hammerheaà Ribozyme Reactions: Divalent Magnesium Ion Mediated Clmage of Phosphorus-Oqgen Bonds. 1. Orp. Chem., 59,7414-7420.

Usher, O. A., Richardson, D. I., and Oakenfull, D. G. (1970) Models of Ribonuclease Action. II. Specific A d , Spmpc Base, and Neutra1 Pathways for Hydrolysis ofa Nucleotide Diester Analog. J. Am- Chem. Soc., 92,4699-471 2.

Wade, W. S., Mrksich, M., and Dervan, P. B. (1992) Design of Peptides That Bind in the Minor G r m e of DNA at 5'-(A,T)G(A,T)C(A,T)-3' Sequences by a Dimerù Side-by-Side Motif. J. Am. Chem. Soc, 114,8783-8794.

Wahnon, D., Hynes, R. C., and Chin, J. (1994) Dramatic Ligand Effect in Coppmtll) Complex Romoted Transesteri/ication of a Phosphate Diester. j. Chem. Soc. Chem. Commun., ,1441-1442.

Wahnon, D. C (1995) A Dinuclear Approach to Phos~hate Diester Hvdrolvsis. Ph-D. MG11 University

Wall, M., Hynes, R. C., and Chin, J. (1993) Double LaLtis Acid Activation in Phosphate Diester Cleavage. Anpew. Chem. Int. Ed. End, 32,1633-1635.

Wang, J-, Yu, P., Lin, T. C., Konigsberg, W. H., and Steitz, T. A. (1996) Crystal Structures of an NH2- Terminal Fragment of 7 4 D N A Polymeruse and Ifs Complexes with Single-Shanded DNA und &h Divalent Meta1 Ions. Biochemistry, 35,8110-8119.

Westheimer, F. H. (1968) Pseudo-rotatwn in the Hydrolysis of Phosphate Esters. Acc. Chem. Res., 1, 70-78.

Wes theimer, F. H. (1987) Why Nature Chose Phosphates. Science, 235,1173-1 178.

Wieghardt, K., Schmidt, W., Nuber, B., and Weiss, J. (1979) Neue p-Hydroxo- U b e r g a n g m e t e l l p l e , 1. Dars tellung und Shukt UT des tram-Dinqua-di-p-hydroxo-bisl(l,4,7- triazacyclononan)co6alt(~)l-Kah'ons; Kinetik und Mechanismus seiner Bt7dung. Chem. Ber, 112,2220- 2230.

Williams, N. H., and Chin, J. (1996) Mefal-Ion Catalysed Phosphafe Diester T~unsesterijïc(1tion: Q u m tifying Double Lauis-Acid Activation. Chem. Commun., ,131 -1 32.

Yarus, M. (1993) How Many Catalyfic R N A ? Ions and the Cheshire Cat Conjecture. FASEB J., 7,31- 39.

Yashiro, M., Ishikubo, A., and Komiyama, M. (1997) Efficient and Unique Cooperation of Three ZindII) Ions in the Hydrolysis of Dirr'bonuclwtides by a Trinuclear Zt'nc(ïI1 Cornplex. Chem. Commun., ,8344.

Young, M. J., and Chin, J. (1995) Dinuclear Copper(I1) Complex That Hydrolyzes RNA. J . Am. Chem. Soc., 717,10577-10578.

Young, M. J., Wahnon, D., Hynes, R. C., and Chin, J. (1995) Reactim-fy of CoppedII) Hydroxides and Coppedll) Alkoxtiies for Cleaaing an Acfivuted Phosphate Diester. J. Am. Chem. Soc., 1 1 7,9441 -9447.