Bioenergetics and Thermodynamics: Model Systems
NATO ADVANCED STUDY INSTITUTES SERIES
Proceedings of the Advanced Study Institute Programme, which aims at the dissemination of advanced knowledge and the formation of contacts among scientists from different countries
The series is published by an international board of publishers in conjunction with NATO Scientific Mfairs Division
A Life Sciences Plenum Publishing Corporation B Physics London and New York
C Mathematical and D. Reidel Publishing Company Physical Sciences Dordrecht, Boston and London
D Behavioural and Sijthoff & Noordhoff International Social Sciences Publishers
E Applied Sciences Alphen aan den Rijn and Germantown U.S.A.
Series C - Mathematical and PhYSical Sciences
Volume 55 - Bioenergetics and Thermodynamics: Model Systems
Bioenergetics and Thennodynamics: Model Systems Synthetic and Natural Chelates and Macrocycles
as Models for Biological and Pharmaceutical Studies
Proceedings of the NATO Advanced Study Institute
held at Tabiano, Parma, Italy,
May 21-June 1, 1979
A. BRAIBANTI Faculty of Pharmacy, UniJlersity of Parma, Parma, Italy
D. Reidel Publishing Company Dordrecht : Holland I Boston: U.S.A. I London: England
Published in cooperation with NATO Scientific Affairs Division
library of Congress Cataloging in Publication Data
NATO Advanced Study Institute, Salsomaggiore, Italy,1979. Bioenergetics and thermodynamics.
(NATO advanced study institutes series: Series C, Mathematical and physical sciences; v. 55).
1. Complex compounds-Congresses. 2. Chelates-Congresses. 3. Thermodynamics-Congresses. 4. Bioenergetics-Congresses. I. Braibanti, A. II. Title. III. Series. QPSOl.C75N37 1979 574.19'2 80-15611 ISBN -13:97S-94-009-9037 -1 e-ISBN-13:97S-94-009-9035-7 DOl: 10.1007/978-94-009-9035-7
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TABLE OF CONTENTS
A. Braibanti BIOENERGETICS AND THERMODYNAMICS. AIMS AND METHODOLOGY OF THE SCHOOL 1
B. Sarkar DESIGN OF PEPTIDE MOLECULES TO MIMIC THE METAL BINDING SITES OF PROTEINS 7
J.M. Sturtevant CALORIMETRY AND BIOLOGY 17
B. Sarkar BIOINORGANIC CHEMISTRY OF NICKEL 23
J.P. Sauvage DESIGN AND SYNTHESIS OF LIGANDS 33
J.F. Stoddart HOLES, HANDEDNESS, HANDLES, AND HOPES: MEETING THE REQUIREMENTS OF PRIM~RY BINDING, CHIRALITY, SECONDARY INTERACTIONS AND FUNCTIONALITY IN ENZYME ANALOGUES 43
J.P. Sauvage TESTING OF LIGANDS 63
J.J. Christensen CALORIMETRIC TECHNIQUES TO STUDY PROTON-LIGAND AND METAL-LIGAND INTERACTIONS 75
P. Paoletti STRUCTURAL AND ENERGETIC ASPECTS OF METAL-LIGAND BINDING 93
J.J. Christensen TRANSPORT OF METAL IONS BY LIQUID MEMBRANES CONTAINING MACROCYCLIC CARRIERS III
vi TABLE OF CONTENTS
R.F. Jameson POTENTIOMETRIC AND SPECTROSCOPIC DETERMINATION OF EQUILIBRIUM CONSTANTS
A. Vacca EXPERIMENTAL DATA IN CHEMICAL AND BIOLOGICAL SYSTEMS AT EQUILIBRIUM
R.F. Jameson WHAT CONCENTRATION SCALE? WHAT STANDARD STATE? WHAT SPECIES ARE PRESENT?
A. Vacca TREATMENT AND ANALYSIS OF EQUILIBRIUM DATA BY COMPUTERS
G. Ostacoli MIXED COMPLEXES
T. Keleti KINETICS AND THERMODYNAMICS OF ENZYME ACTION AND REGULATION
P.M. May COMPUTER MODELS OF BIOLOGICAL SYSTEMS
D.E. Fenton MEMBRANES, ALKALI METALS AND TRANSFER
J. Jagur-Grodzinski SYNTHETIC SOLVENT-POLYMERIC MEMBRANES AND THEIR TRANSPORT CHARACTERISTICS
D.E. Fenton MACROCYCLES AND CATION SELECTIVITY
J. Jagur-Grodzinski SELECTIVITY OF MEMBRANES TOWARDS IONS AND MOLECULES
G. Scibona TRANSPORT PHENOMENA ACROSS SOLID AND LIQUID MEMBRANES
A. GHozzi CARRIERS AND CHANNELS IN ARTIFICIAL AND BIOLOGICAL MEMBRANES
127
145
157
165
175
181
207
221
229
253
275
297
313
339
G. Scibona LIPID BILAYER ELEeTROCHEMISTRY AND­ ROLE OF THE CARRIERS
A. Gliozzi THE LIPID BILAYER: A MODEL SYSTEM FOR BIOLOGICAL MEMBRANES
J .M. Sturtevant DIFFERENTIAL SCANNING CALORIMETRY. PROCESSES INVOLVING PROTEINS
J .M. Sturtevant RECENT ADVANCES IN BIOCHEMICAL CALORIMETRY
R. Lumry INTERPRETATION OF CALORIMETRIC DATA FROM COOPERATIVE SYSTEMS
J.P. Behr SELECTIVE COMPLEXATION OF AMMONIUM AND GUANIDINIUM SALTS BY SYNTHETIC RECEPTOR MOLECULES
R. Lumry DYNAMICAL ASPECTS OF SMALL-MOLECULE PROTEIN INTERACTION
J.M. Lehn MOLECULAR RECEPTORS, CARRIERS AND CATALYSTS: DESIGN, SCOPE AND PROSPECTS
R. Os terberg METAL IONS IN BIOLOGICAL SYSTEMS
R. Osterberg METAL ION - PROTEIN INTERACTIONS IN SOLUTIONS
J.M. Sturtevant FINAL COM1ENTS
vii
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PREFACE
This book reports the text of the lectures given at Tabiano, Sal so­ maggiore, Italy, during the Summer School on Bioenergetios and Thermodynamios: ModeZ Systems, in May 1979.
The aim of the School has been that of trying to employ the thermodynamic data on synthesised organic compounds with special reference to macrocylic ligands for the interpretation and predic­ tion of energetic processes involving small and large molecules.
A detailed description of the origin, scope and plan of the School can be found in the introductory lecture by A. Braibanti. In appendix to this lecture there are listed some introductory books recommended to achieve a sufficient background in the differ­ ent scientific fields contributing to the School.
The audience consisted of about a hundred scientists belong­ ing to different fields. Physical, organic, inorganic, pharmaceut­ ical, analytical and medicinal chemists, biochemists, biophysicists, pharmacologists interested in the problems of calorimetry, poten­ tiometry, spectroscopy, transport properties of synthetic and natu­ ral chelates and macromolecules spent two weeks to discuss topics ranging from thermodynamics to electrochemistry, from measurements on pure compounds to determinations on membranes. This picture of the audience is an indication of researchers which can be interested in this book.
I hope that the tremendous effort to put together scientists working in different fields for an interdisciplinary approach to the problem of the models for bioenergetics will be rewarded by a good appreciation of the book and by the flowering of the scien­ tific researches from the many seeds sown at the School.
ix
A. Braibanti
A. Braibanti
1. INTRODUCTION
The field of bioenergetics represents a link between the energetic phenomena in vivo and the thermodynamic interpretations of data from many chemical compounds designed and synthesised by organic chemists (1). Clearly, an interdisciplinary approach to the problem is necessary in order to compare different expertises and the lan­ guages of the physical, chemical, and life sciences.
The current state of knowledge in thermodynamics and in thermo­ chemistry is adequate and poised for such a union. The determination of the equilibrium constants for chemical reactions by potentiometry and other experimental methods (2),(3),(4),(5) has recently taken advantage of automatic measurements (6) and computerized treatment of data (7). In thermochemistry also the possibility of automatic monitoring equipment (8) has permitted the measurement of small amounts of heats, even in sophisticated biological systems (9).
Simultaneously, as precise thermodynamic and thermochemical data are collected (10), (11), (12), (13), (14), the possibility of using them to interpret chemical and biochemical processes be­ comes more realistic. However, the circulation of these data amongst thermochemists, organic chemists, biologists, biophysicists, and medicinal chemists is disencouraged by the difficulties of inter­ preting the data because of the different presentations and the incompleteness of our knowledge of the species present in the sys­ tems concerned. A common dialogue in bioenergetics (15) can only be achieved through an interdisciplinary discussion meeting. This is the primary objective of the Summer School.
A. Braibanti (ed.), Bioenergetics and Thermodynamics: Model Systems, 1-6. Copyright © 1980 by D. Reidel Publishing Company.
2 A. BRAIBANTI
2. GENERAL SCIENTIFIC. PLAN
The general plan for the School follows the usual sequence of con­ siderations involved in research concerning compounds that mimic the behaviour of naturally occurring species.
In the first instance, we try to combine our knowledge of biochemical and biological facts and our skills in synthesising organic ligands that may be expected to mimic the chelation and basicity properties of in vivo processes with respect to protons and cations (1), (16). In this context the macrocyclic ligands represent a very good example and a very promising field of study. Next, the problem of the choice of experimental methods for measur­ ing the equilibria between ligands and metal ions is to be tackled and, eventually, the structure of the solid complexes is determined using crystallographic analysis (17).The thermochemical analysis of the systems gives further insight into complexation processes, and in particular it enphasises the role of the solvent (18), (19).
Similarly, the complexing processes involved in a mixture of metal ions, protons, drugs, and proteins as measured from its endo-, or exothermicity is intimately related with changes in the con­ formation of the macromolecules, of the drugs, and of the solvent (20), (21). This aspect is usually investigated by employing anal­ ogous arguments based upon previous experience with the thermodyn­ amic investigations of simpler systems; these are essential in order to understand the selectivity of ligands towards metal ions and anions. This subject is considered also in connection with properties of membrane carriers and antibiotics (22), (23).
Both thermodynamic and kinetic aspects of the problem have to be discussed in order to get a realistic picture of the bio­ logical processes occurring (24).
This information, concepts, and data are the fundamental basis to discuss the important applications of calorimetry to biological systems (25) and the bioenergetics of drug-receptor interactions (26).
3. BACKGROUND INFORMATION AND FRONTIER TOPICS
The topics to be discussed at the Summer School are drawn from diverse disciplines and we shall assume that each participant possesses a good knowledge, not only of his or her own field, but also a reading knowledge of the other fields concerned. Such an acquaintance with the subject can be obtained by studying the in­ troductory books, of the list which had been compiled when the project of the School started. The list (s. Appendix) is by no means exhaustive but would serve as a guide for teachers when discussing with the students.
BIOENERGETICS AND THERMODYNAMICS 3
At the same time that elementary principles should be re­ called, the discussions during seminars should give the opportunity of enlighting even the up-to date and frontier topics of each chapter.
4. THE CONTENT OF THE SCHOOL
Does the content of the lectures correspond to the ideal outline of the scientific path from synthesis of compounds to application, from basic principles to frontier topics?
After a survey of the titles,summaries,and contents of the lectures and seminars, I think I may say that a large degree of success can be claimed.
The lectures and seminars will cover the main points of the model systems for the study of bioenergetic phenomena.
Some parts, however, will be only touched upon,almost inci­ dentally, and some left out. In particular non-equilibrium thermo­ dynamics and redox processes will not be presented with such an enphasis as they deserve in the field of bioenergetics. This has been a necessary choice because it would be counterproductive for the students if the sets of topics discussed here were enlarged.
Non-equilibrium thermodynamics and redox-processes would be the title of a possible school in the near future. Other points concerning structures either by quantum mechanics or by crystal­ lography will not be discussed in the School.
The first two days of the School will be devoted to the study of the project and synthesis of ligands; followed by calorimetric and potentiometric methods in connection with metal-ligand and ligand-protein equilibria. The distribution and role of the in­ organic metals in biological systems will also be presented.
Next the properties of membranes will come on the scene, with their pores and channels and with the mechanisms by which the carriers can influence the transport and electrical properties of the membranes.
The last part of the School will be reserved for the calor­ imetry of macromolecules and membranes, in relation also to the effects of the drugs on membranes and macromolecules.
4 A. BRAIBANTI
5. DISCUSSIONS, PROCEEDINGS, AND FUTURE RESEARCHES
The second step toward the final goal of the School will be on the shoulders of all the participants.
Students and lecturers are requested to give a continuos and active contribution of questions and discussions so as to keep alive the School. Theyare requested to point out also the mistakes and omissions of the organization.
The third step toward the success of the School will be the publication of the Proceedings. These could pass through the exam­ ination of the students at this School and then of the world of science and I hope that the opinions of our colleagues which are not present here will be favourable and encouraging. I recommend to the lecturers to help me in this hard work.
I think that I can claim a fourth step in that I am sure that this School will lead directly to many research projects that will come out of the School and after the School.
6. METHODOLOGY
The scheme of the work of the School consists of lectures in the morning with short discussions. In the afternoon the discussions, examples, and seminars should put forward the contribution of the students to the development of the topics presented by the lec­ turers in the morning.
I am sure that informal groups can meet here during these two weeks and discuss any point of interest not touched or sug­ gested by the official program.
(1) Lehn, J.M.: 1973, Structure and Bonding 16, 1. (2) Beck, M.T.D.: 1970, Chemistry of Complex Equilibria, Van
Nostrand, London. (3) Paoletti, P., Barbucci, R., Fabbrizzi L., Eds.: 1977,
Stability Constants Proc. of Summer School of Bivigliano (Florence), Edizioni Scuola Universitaria, Firenze.
(4) Nancollas, G.B.: 1970, Coord. Chern. Review 5, 379. (5) Jameson, R.F.: 1972, The~odynamias in Metal-complex Fo~ation
in: Skinner , H.A., Ed., Thermochemistry and Thermodynamics, Phys. Chem. Series One, Butterworths, London.
(6) Merciny, E., Gatez, J .M., Swennen, L., Duycaertz, G.: 1975, Annal. Chim. Acta 78, 159.
(7) Sabatini, A., Vacca, A., Gans, P.: 1974, Talanta, 21, 53. (8) Pilcher, G.: 1972, The~ochemistry of Chemical Compounds
BIOENERGETICS AND THERMODYNAMICS
in: Skinner, H.A., Ed., Thermochemistry and Thermodynamics, Phys. Chem. Series Two, Butterworths, London.
5
(9) Ria1di, G., Bi1tonen, R.: 1975, Thermodynamics and Thermo­ chemistry of Biologically Important Systems in: Skinner, H.A., Ed., Termochemistry and Thermodynamics, Phys. Chem. Series Two, Butterworths, London.
(10) 1964, Stability Constants of Metal-Ions Complexes 3 Chem. Soc. Special Publ. N.17, London.
(11) 1971, Stability Constants, Supplement N.1, Chem. Soc. Special Publ. N.25, London.
(12) Martell, A.E. and Smith, R.M.: 197 , Critical Stability Con­ stants, Vo1s.1,2,3,4 Plenum Press, London.
(13) Christensen, J.J., Hansen, L.D., and Izatt, R.M.: 1976, Hand­ book of Proton Ionization Heats and ReZated Thermodynamic Quantities, Wiley, New York.
(14) Christensen, J.J., Eatough, D.J., and Izatt, R.M.: 1975, Handbook of Metal Ligand Heats and ReZated Thermodynamic Quantities, Dekker, New York, II Ed ..
(15) Klotz, I.M.: 1967, Energy Changes in Biochemical Reactions, Academic Press, New York.
(16) Izatt, R.M., Nelson, D.P., Rytting, J.R., Haymore, B.L., and Christensen, J.J.: 1971, J. Am. Chem. Soc. 93, p. 1619.
(17) Truter, M.R.: 1971, Chem. Brit., p. 203. (18) Ria1di, G. and Bi1tonen, R.L.: 1975, Thermodynamics and
Thermochemistry of Biologically Important Systems in: Skinner, H.A., Ed., Thermochemistry and Thermodynamics, Phys. Chem. Series Two, Buttersworths, London.
(19) Klotz, I.M.: 1970, Water: Its fitness as a molecular environ­ ment in Bittar, Ed., Membranes and Ion Transport, Vol.1, Wi1ey-Interscience, London.
(20) Scheraga, H.A.: 1971, Theoretical and Experimental Studies of Conformations· of Polypeptides; Chemical Reviews 71, p. 195.
(21) Ivanov, V.T. and Ovchinnikov, Yu.A.: 1971, Conformation of membrane active cyclodepsipeptides in: Chiurdog1u, G., Ed., Conformational Analysis - Scope and Present Limitations, Academic Press, London.
(22) Fenton, D.E.: 1976, Alkali Metal Complexes as Probes for Membrane Transport. A Requirement for Multimethod Approach in Williams, D.R., Ed., Introduction to Bio-inorganic Che­ istry, Thomas, Springfield, U.S.A •.
(23) Shchori, E. and Jagur-Grodzinski, J.: 1977, 1st Symposium of Macrocyclic Compounds, Provo, Utah.
(24) Grell, E.: 1977, 1st Symposium of Macrocyclic Compounds, Provo, Utah. '
(25) Wadso, I.: 1972, Biochemical Thermochemistry in Skinner, H.A., Ed., Thermochemistry and Thermodynamics, Phys. Chem. Series One, Butterworths, London.
(26) Belleau, B. and Lavoie, J.: 1968, Canad. J. Biochem., pp. 1397- 1409.
6 A. BRAIBANTI
General introductory books
Allinger, N.L., Cava, M.P., Jongh, D.C., Johnson, C.R., Level, N.A., and Stevenson, C.L.: 1971, Organic Chemistry~ Worth.
Beck, M.T.D.: 1970, Chemistry of Complex Equilibria~ Van Nostrand, London.
Brown, E.G.: 1971, Introduction to Biochemistry~ Chem. Soc. Mono­ graph N. 17, London.
Fabiane, A.M. and Williams, D.R.:1977, The Principles of Bio­ inorganic Chemistry~ Chem. Soc. Monograph for Teacher N. 31, London.
Foye, W.O., Ed.: 1974, Principles of Medicinal Chemistry, Lea and Febiger, Philadelphia.
Hanzlik, R.P.: 1976, Inorganic Aspects of Biological and Organic Chemistry~ Academic Press, N.Y ..
Klotz, I.M.: 1967, Energy in Biochemical Reactions~ Academic Press, N.Y ..
Lehninger, A.L.: 1973, Bioenergetics~ Benjamin, MenloPark, Cal.
Martin, A.N., Swarbrick, J., and Cammarata, A.: 1969, Physical Pharmacy~ Lea and Febiger, Philadelphia.
Ovchinnikov, Yu.A., Ivanov, V.T., and Shkrob, A.M.: 1974, Membrane-Active Complexones, Elsevier, Amsterdam.
Paoletti, P., Barbucci, R., and Fabbrizzi, L., Eds: 1977, Stability Constants~ Proc. of Summer School~ Edizioni Scuola Universitaria.
Strier, L.: 1975, Biochemistry~ Freeman, San Francisco.
Sturtevant, J.M.: 1972, Calorimetry in Methods in Enzymology~ Ed. C. H. Hirs and S.N. Timasheff, Academic Press, N.Y.
DESIGN OF PEPTIDE MOLECULES TO MIMIC THE METAL BINDING SITES OF PROTEINS
Bibudhendra Sarkar
The Research Institute, The Hospital for Sick Children, Toronto, Ontario, and the Department of Biochemistry, The University of Toronto, Toronto, Ontario, Canada
The molecular design to m~m~c the metal-binding site of a protein molecule entails abstracting the minimum requirements which must be retained in a molecule in order to maintain parameters control­ ing the geometry, metal-binding ligands and microenvironment at the metal-binding site. A linear peptide may be designed to mimic a site which is located on a short linear amino acid sequence. However, when the metal-binding ligands originate from different parts of the polypeptide backbone, a cyclic peptide should be designed with amino acid residues having appropriate side chains. For the purpose of the design, building of suitable molecular models are essential. This can be supplemented by conformational calculations. Three examples are presented for the molecular de­ sign of the metal-binding sites: Copper(II)-transport site of hu­ man albumin, Zn(II)-binding sites of two metalloenzymes: carboxy­ peptidase and carbonic anhydrase.
1. INTRODUCTION
The three dimensional structures of several enzymes, proteins and hormones ~ave been elucidated at the atomic level. Some of them have a specific metal-binding site which on many occasions either imparts structural rigidity to the protein molecules .or contrib­ utes to the biological function of the molecule. The X-ray studies have provided a precise three dimensional atomic description which clearly reveals the molecular architecture, binding ligands, the associated geometry and the microenvironment at the binding site. Even though the tertiary structure of the protein and the nature of the metal binding site may be known, the functional aspects of the metal ion at the binding site may still remain very much unclear.
7
8 B.SARKAR
If one can design a small molecule which approximates the natural site on a protein molecule, it will allow a variety of studies which often cannot be done with a large protein molecule. Apart from allowing a study of the functional aspects of the metal-bind­ ing site, such type of molecules will possess the natural speci­ ficity and other properties which could have potential practical applications in therapeutics and medicine.
2. CRITERIA INVOLVED IN THE DESIGN
The designing of a small molecule to mlm~c the metal-binding site of a protein molecule entails abstracting the minimum requirements which must be retained in a molecule in order to maintain par­ ameters controlling the geometry, the metal-binding ligands and microenvironment at the metal-binding site. The binding site may be located at the a-NH2 terminus of a protein in a linear sequence; alternatively, the binding site may be a complex one where the ligands are composed of amino acid residues originating from dif­ ferent parts of the polypeptide chain.
Figure 1. Design of a peptide model to mimic the metal­ -binding site of a protein located on a short linear sequence . Metal (M) is bound to four nitrogens. The carboxyl terminal (C) is blocked.
PROTEIN-MIMICKING PEPTIDES 9
The secondary and tertiary structures of the protein molecule bring together and maintain the necessary residues in the required ge­ ometry. In the former case, a linear peptide can be designed by incorporating the essential binding residues interspaced by amino acid residues which do not possess liganding type side chains (Fig. 1). In this type of binding site, it is quite possible that the peptide nitrogens or carbonyl oxygens are also involved in the binding in addition to the other liganding groups. The C-terminal and the N-terminal of such a linear peptide should be protected if not involved in the metal-binding . The essential binding residues must be the same amino acids that are binding metal in the native mol­ ecule. For the latter type of binding ·site, a short linear peptide would have little chance of forming a stable matrix. The large number of possible conformational states available to a linear peptide chain is substantially reduced by cyclization. Thus, a cyclic peptide should be designed , incorporating liganding amino acid residues in strategic positions to provide the metal-binding site (Fig. 2).
Figure 2 . Design of a cyclic peptide model to m1m1C the metal-binding site of a protein where ligand residues originate from different parts of the p'olypeptide chain. Metal (M) is shown bound to ligands originating from the cyclic peptide backbone structure.
10 B. SARKAR
Several features should be considered for the cyclic peptide design. A proline residue can give a suitable bend which may help orient the peptide backbone for cyclization (1). One can gain further knowledge from peptide antibiotics such as enniatin and valinomycin, both of which have cyclic structures with repeating sequence containing LD or DL pairs. It has been shown that this type of conformation has the property of assuming special types of folding in which the three peptide units linked together prod­ uce a reversal in chain direction. It is also known that with alternating L- and D-amino acids, it produces a conformation which has low energy and is further stabilized by a hydrogen bond be­ tween the NH group of the third peptide unit and the carbonyl oxygen of the first peptide unit (2).· The occurrence of a reversal of chain direction is a key feature for the closure of the ring in cyclic structures.
As is well known that the disulfide linkage can render rigid­ ity to the backbone structure also, inclusion of n,S-unsaturated amino acid residues in the backbone structure may be of advantage. Recent crystallographic data (3) provide the eviderice for a degree of rigidity in the dehydro amino acid system.
The native metal binding site may have a hydrophobic environ­ ment. One could provide hydrophobic environment by introducing in appropriate geometry aromatic residues, such as tryptophan, phenyl­ alanine, etc.
For the purpose of molecular design, building of appropriate molecular models seems to be essential. This can be advantageously supplemented by conformational calculations. However, the lack of an adequate representation of solvent and the problem of intro­ ducing metal in the calculation still pose a major challenge. Never­ theless, with a computer and a set of potential functions, one can systematically explore many more conformers than is feasible with a set of molecular models.
3. EXAMPLES
3.1. Copper(II)-transport site of human albumin
Copper(II)-transport site of human albumin is located at the NH2- -terminal segment of the albumin molecule on a short linear se­ quence: Asp-Ala-His .... This Cu(II)-binding site has been proposed to be a composite site involving n-amino nitrogen, two intervening peptide nitrogens and the imidazole nitrogen of the histidine resi­ due in the third position (4). It is known that albumins from bov­ ine, human and rat show a characteristic of a specific first bind­ ing site for Cu(II), but dog albumin does not have a similar site for Cu(II) (5). When sequence determination at the NH2-terminal
PROTEIN-MIMICKING PEPTIDES 11
region of dog albumin showed that the histidine in the third pos­ ition is replaced by a tyrosine (6), it became quite clear that the histidine residue in that position is obligatory to produce a specific site for Cu(II).
The design scheme produced a simple peptide having the native sequence; Asp-Ala-His-NHCH3 and another peptide which was further simplified from the native sequence, namely, GlyGly-L-His-NHCH3. According to the design criteria discussed above, the carboxyl group was derivatized to N-methyl amide in order to resemble more closely the protein molecule. The a-NHz group was left unprotected since this group is required for the Cu(II)-binding. Both these pep tides showed a square planar geometry of the nitrogen ligands around Cu(II). Molecular models the simplified peptide, GlyGly-L­ -His-NHCH3 are shown in Fig. 3.
Figure 3. Space-filling and Kendrew models of the designed peptide, GlyGly-L-His-NHCH3 , showing the square planar geometry of the nitrogen ligands for Cu(II)-binding and the peptide backbone.
12 B. SARKAR
In order to gain further insight into the design, theoretical conformational analyses were undertaken with the peptides. Inthese calculations, the conformational energy U is expressed as a sum of the energies due to various interactions as follows:
U=U b dd+Ul . +U . non- on e e ectrostat1c tors10n
A large number of folded conformations of both peptides were found to occur from the calculations, possessing the conformational prop­ erties for forming a square planar complex, utilizing terminal N­ -atom, two peptide N-atoms and the imidazole N-atom. However, one interesting observation was that the conformation of the native sequence peptide was far more restricted than the designed peptide, due to the presence of side chains.
The peptides were synthesized and a detailed study of their Cu(II)-binding properties revealed the fact that the molecules were successfully designed to mimic the Cu(II)-transport site of human albumin (7-10) (Table 1). Furthermore, X-ray crystal struc­ ture of Cu(II)-GlyGly-L-His-NHCH3 shows Cu(II) is tetradentately chelated by the amino terminal nitrogen, the next two peptide nitrogens and the histidyl nitrogen of a single tripeptide in a slightly distorted square planar arrangement (11).
Table 1. Comparison of Cu(II)-binding properties
Cu(II)-Complex A £ Kn logS max max (l017 M)
Human albumin 525 101 6.61
GlyGly-L-His-NHCH3 525 103 2.07 -0.479
L-Asp-L-Ala-L-His- NHCH3 525 103 1.04 -0.55
3.2. Zinc(II)-binding site of carboxypeptidase A
Carboxypeptidase A is a Zn(II)-containing metalloenzyme which catalyzes the hydrolysis of free carboxyl C-terminal of pep tides and esters.
The ligands involved in the Zn(II)-binding have been ident­ ified as His (69) , Glu(72) and His(196) (12).
PROTEIN-MIMICKING PEPTIDES
A critical survey of several metalloenzymes, whose crystal structure has been elucidated reveals the natural occurence of one to three amino acid residues in between two ligands for the metal at the active site. Other ligands originate from a distant part of the molecule. Taking into account these features as well as the geometry of the Zn(II)-binding according to the criteria discussed above, a cyclic octapeptide of the sequence:
Gly-L-Glu-Gly-Gly-L-His-Gly-L-His-Gly
13
was designed. Model building showed that the peptide provided the satisfactory requirements for the Zn(II)-binding geometry (Fig. 4).
Figure 4. Space filling and Kendrew models of the de­ signed cyclic octapeptide to mimic the Zn(II)­ -binding segment of the active site of carboxy­ peptidase.
The designed molecule possess the native Zn(II)-ligands inter­ spaced by glycine residues. The y-carboxyl moiety of the glutamate and the imidazole residues of the two histidines in the model seemed to interact well with the tetrahedral Zn(II). The space in between the ligand amino acid residues were filled with glycylresi­ dues. Glycyl residue was chosen mainly because of its simple structure.
Theoretical conformational analysis of the peptide was under~
14 B. SARKAR
taken. First, the minimum energy conformations of the linear octa­ peptide were obtained. Ring closure was then attempted by system­ atically incrementing the backbone angles, ~ and ~ until the two loose ends were within a reasonable bonding distance to each other. Selected conformations satisfying the ring closure constraints were minimized. Several minimum energy conformations were obtained and at least one such conformation suggested that Zn(II) could bind two histidine and one glutamic acid residue in a somewhat distorted tetrahedral coordination geometry.
The peptide has been synthesized and Zn(II)-binding studies suggest that the peptide can compete with carboxypeptidase for the Zn(II). Furthermore there is evidence for the involvement of both the histidines and glutamyl residues in the binding. Detailed studies are currently underway to characterize fully the Zn(II)­ -binding site of the cyclic octapeptide.
3.3. Zinc(II)-binding site of carbonic anhydrase
Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide. Zinc(II) in carbonic anhydrase is bound to protein through three histidyl residues: His (94), His (96), and His (119) (13). It has been pointed out before that several metalloenzymes have been found to contain one to three amino acid residues between two metal-binding ligands while other metal-binding sites originate from distant parts of the polypeptide chain. Consideration of all the above information, coupled with examination of molecular models of a variety of peptides, resulted in the choice of a cyclic hepta­ peptide containing the three native Zn(II)-ligands with the se­ quence:
Gly-L-His-Gly-L-His-Gly-L-His-Gly
Again, cyclization of the peptide was used to introduce some degree of rigidity in the backbone of the molecule. Model building re­ vealed that the imidazole residues of the three histidines in the peptide possess the ability to interact with Zn(II) to form a tetrahedral complex (Fig. 5).
The peptide was synthesized and preliminary data suggest that at low pH there is a 1:1 Zn(II)-peptide complex. However, at higher pH values, species of higher stoichiometry became dominant.
The above finding provides an interesting insight for molecu­ lar design. Recent studies have shown that Zn(II) greatly facili­ tates the deprotonation of both bound water molecules and pyrrole
PROTEIN-MIMICKING PEPTlDES 15
proton of the bound imidazole (14),(15). The formation of com­ plexes of higher stoichiometry may be the result of polymeriz­ ation via Zn(II) induced deprotonation. Such polymerization would not occur in the native enzyme due to steric restrictions. It would appear that Zn(II) in the peptide-Zn(II) complex remains exposed and therefore may form complex chain of Zn(II)-peptide polymers. Design of subsequent molecules should include the intro­ duction of bulky side chain residues which would result in steric restrictions which in turn may inhibit the formation of polymers .
Figure 5. Space-filling and Keridrew models of the de­ signed cyclic heptapeptide to mimic the Zn­ -binding segment of the active site of car­ bonic anhydrase.
16 B. SARKAR
(1) Deber, C.M., Madison, V., and B1out, E.R.: 1976, Aaaounts Chem. Res. 9, pp. 106-113.
(2) Ramachandran, G.N. and Chandrasekharan, V.: 1972, Progress in Peptide Research, Ed. S. Lande, Gordon and Beach Science Publishers Inc., New York, Vol. 2, pp. 195-215.
(3) Pieroni, 0., Montagnoti, G., Fissi, A., Merlino, S., and Ciarde11i, F.: 1975, J. Amer. Chem. Soa. 97, pp. 6820-6826.
(4) Peters, T., Jr. and Blumenstock, F.A.: 1967, J. Biol. Chem. 242, pp. 1574-1578.
(5) Appleton, D.W. and Sarkar, B.: 1971, J. Biol. Chem. 246, pp. 5040-5046.
(6) Dixon, J.W. and Sarkar, B.: 1965, J. Biol. Chem. 240, pp. 5972-5977.
(7) Lau, S., Kruck, T.P.A., and Sarkar, B.: 1974, J. Biol. Chem. 249, pp. 5878-5884.
(8) Lau, S. and Sarkar, B.: 1975, Can. J. Chem. 53, pp. 710-715. (9) Kruck, T.P.A., Lau, S., and Sarkar, B.: 1976, Can. J. Chem.
54, pp. 1300-1308. (10) Iyer, K.S.N., Lau, S., Laurie", S.H., and Sarkar, B.: 1978,
Biochem. J. 169, pp. 61-69. (11) Camerman, N., Camerman, A., and Sarkar, B.: 1976, Can. J.
Chem. 54, pp. 1309-1316. (12) Lipscomb, W.N., Hartsuck, J.A., Quiocho, F.A., and Reeke, G.N.,
Jr.: 1969, Proc. Nat. Aaad. Sai. USA 64, pp. 28-35. (13) Kannan, K.K., Notstrand, B., Fridborg, K., Lovgren, S.,
Ohlsson, A., and Petef, M.: 1975, Proc. Nat. Aaad. Sai. USA 72, pp. 51-55.
(14) Appleton, D.W. and Sarkar, B.: 1974, Proc. Nat. Acad. Sai. USA 71, pp. 1686-1690".
(15) Demoulin, D., Pullman, A., and Sarkar, B.: 1977, J. Amer. Chem. Soa. 99, pp. 8498-8500.
APPENDIX
(1) Sarkar, B.: 1977, Conaept of moleaular design in relation to the metal-binding sites of proteins and enzymes in Metal­ -Ligand Interaation in Organic Chemistry and Biochemistry, Ed. B. Pullman and N. Goldblum, Part 1, pp. 193-228 by D. Reidel Publishing Company, Dordrecht-Ho11and.
(2) Freeman, H.C.: 1967, Crystal structures of metal-peptide aom­ plexes in Adv. Protein Chem. 22, pp. 258-424.
(3) Pullman, B. and Pullman, A.: 1974, Moleaular orbital aalcu­ lations on the aonformation of amino acid residues of protein in Adv. Protein Chem. 28, pp. 348-526.
(4) Anfinsen, C.B. and Scheraga, H.A.: 1975, Experimental and theoretiaal aspects of protein folding in Adv. Protein Chem. 29, pp. 205-300.
CALORIMETRY AND BIOLOGY
Department of Chemistry, Yale University, New Haven, Connecticut 06520, U.S.A.
Professor Braibanti has suggested that it might be useful to give some attention at the start of this School to the historical as­ pects of the application of thermochemistry to biology. Although I guess it is safe to assume that I have been interested in this general area for a longer period that anyone else in this School, such longevity certainly does not qualify me as as historian, and I have found this to be a difficult assignment.
It is actually quite appropriate to consider historical mat­ ters at this particular time since we are now at approximately the bicentennial anniversary of the first application of calorimetry to biology. In 1780 Lavoisier and de Laplace described to the Academie des Sciences in Paris (1) a simple ice calorimeter and its application in showing that the heat evolved in forming car­ bon dioxide by animal respiration is the same as that evolved in a combustion process.
Apparently, although much happened in calorimetvy and in its application to chemistry and physics during the next century, not much of significance transpired in our field until 1910. In that year J. Barcroft, a Fellow at King's College, and A.V. Hill, a Scholar at Trinity College, both in Cambridge, published a paper (2) which seems to me to be of the greatest importance, showing its authors to be decades ahead of their time. I will quote the introduction to this paper.
In the present paper we propose (1) to set forth some ad­ ditionaZ evidence for supposing the union of oxygen with hemogZobin is a chemicaZ one, (2) to press this supposition to its ZogicaZ concZusion and on thermodynamicaZ principZes deduce the heat
17
18 J. M. STURTEVANT
generated when one moz.ecuz.ar weight of hemogz.obin unites with cxcygen, (3) by actual. determinations of the heat produced by the union of one gram of hemoglobin, to calculate the molecuz.ar weight of hemogz.obin. In a footnote to this introduction it is stated that the responsi­ biz.ity for the mathematical portion of the work rests with Hill. and for the oxygen estimates with Barcroft.
This was written at a time when protein chemistry was laboring under the view that proteins are colloids in the older sense - that is that they are dispersed as lump of a wide range of sizes, lumps of glue in the case of proteins, probab ly of indef ini te and variable composition. True, in the case of hemoglobin there was a rather accurately known and reproducible minimum molecular weight of about 16,500 based on the known iron content, but was an exceptional case. It was also widely, though vaguely, felt that reactions in­ volving such apparently ill-defined entities as proteins were of a different character from the reactions of small molecules - almost as though there were a vital. principle involved. In view of these prejudices, the work of Barcroft and Hill stands out as particularly daring and significant.
Henri in 1904 had suggested that the temperature variation of the binding of oxygen to hemoglobin could be employed to evaluate the heat of the reaction, but Barcroft and Hill appear to be the first to have clearly distinguished between van't Hoff and calorimetric enthalpies and to have made good use of the distinction.
Barcroft and Hill used a calorimeter composed of a Dewar flask and a Beckmann thermometer, with a four capillary gas inlet tube. The reduced hemoglobin was protected from air oxidation by a layer of olive oil. Their calorimetric experiments gave a heat of 1.85 cal per g of hemoglobin at oxygen saturation, and their equilibrium measuremepts at constant oxygen partial pressure over the tempera­ ture range 16-49° gave van't Hoff enthalpy of 28,000 cal per mole. Since the ratio, l5,100,agreed within experimental uncertainty with the molecular weight of 16,700 deduced from iron determi­ nations, they concluded that in the equation
( 1)
n = 1. They used hemoglobin extensively dialyzed against distilled water, which was presumably monomeric.
In a later paper in 1923 (3), Brown and Hill, the latter now F.R.S. and located at Manchester, worked with blood and with un­ dialyzed hemoglobin. They noted that the mass action equation
........:L- = Kxn (2) 1 - y
CALORIMETRY AND BIOLOGY 19
based on equation (1) would predict a linear plot when log ~, y being the fractional saturation of the hemoglobin, .is plotted against log x, x being the oxygen partial pressure. The slope of the line would equal n, which we now call the Hill coefficient, and the value of y/(l - y) at log x = 0 would equal K. Brown and Hill found n to be around 2.9. From the variation of K with tem­ perature they obtained a van't Hoff enthalpy wich was about 2.8 times their redetermined calorimetric enthalpy expressed in calories per mole of oxygen bound. They took this agreement in the values of n obtained in two totally independent ways to be a strong ar­ gument in support of the view that the binding of oxygen to hemo­ globin is truly a chemical reaction subject to the law mass action.
I find these papers to be most impressive considering their dates. This sort of application of calorimetry has been redis­ covered numerous times since 1910. I myself was guitly of such rediscovery when I studied calorimetrically the mutarotation of a- and B-glucose in 1941 (4). In that work, a small difference between the calorimetric and van't Hoff enthalpies, the latter calculated from calorimetrically evaluated equilibrium constants, was taken to indicate the probable participation of more than two forms in the mutarotational equilibrium. Forward and reverse rate constants for the reaction, as well as the heats of solution of a- and B-glucose, were also obtained from the calorimetric exper­ iments.
In another pioneering paper (5), this one in 1911, A.V. Hill, by then a Fellow of Trinity College, described a differential calor­ imeter composed of two Dewar flasks with a differential thermo­ couple. With this device he measured the heat produced in the souring of milk, and by living, resting frogs, by resting muscles, by the action of yeast cells on cane sugar, and by the action of saliva on starch. This work was the precursor of Hill's famous extensive work on the energetics of muscle action.
There is another early development in the applications of calor­ imetry which has been frequently rediscovered - the calorimetric determination of the rates of chemical reactions. The first examples of this application that I know of are the works of Duclaux in 1908 (6), Chelintzev in 1912 (7), Barry in 1920 (8), Tian in 1923 (9) and Hartridge and Roughton in 1925 (10). Barry, in what was the first such work having any biochemical significance, studied the inversion of sucrose, obtaining both heats and rates of excellent accuracy.
If I may be permitted a personal anecdote, I had in 1934 started work on the kinetics of various organic reactions using dilatometry, when I was struck by what I thought was a nice idea - why not construct the dilatometer in the form of a Dewar flask and observe the rise or fall of the meniscus due to the expansion
20 1. M. STURTEVANT
or contraction of the solution resulting from the heat of the re­ action? This led by easy steps to direct calorimetry for rate de­ terminations, and I then began to be more interested in the ther­ mal data than in the kinetic data, and so I became a calorimetrist.
One of the older forms of calorimetry, and certainly the most highly developed form, is combustion calorimetry. Berthelot built his first bomb calorimeter in 1885, and he and Thomsen and Stohmann and others vigorously pursued the task of determining heats of com­ bustion from which heats of reaction could be calculated, in the mistaken idea that chemical affinities could be inferred from such data. Even after it became clear that the change in free energy rather than in enthalpy determines the spontaneity of a chemical reaction, the development of combustion calorimetry continued until at present the accuracy of combustion data is frequently limited by the purity of the substances burned. It may be difficult for those interested in biochemistry to appreciate it now, but there was a time not so long ago when it was generally assumed that the heat of a chemical reaction could best be obtained by the addition and substraction of combustion heats, or of the heats of formation derived from them, and almost no attention was given to the direct calorimetric determination of reaction enthalpies. However, even with combustion heats accurate to 0.01%, the reaction heats de­ rived from them are of little or no use in the biochemical field. Consider again the mutarotation of glucose. I found (4) for the process
a-Glucose (solid) = B-glucose (solid) (3)
the value 6H298 = 1165 cal mol-I. Combustion data of Huffman and Fox (11) lead to the value ~H298 = 1501 cal mol-I, in very poor agreement. And, of course, in biochemical contexts the reaction involving solid species, which is all that one can obtain from combustion calorimetry, is of no interest at all. Unfortunately, then, the most highly developed area of calorimetry is of no rel­ evance in biochemistry. But fortunately there have been notable improvements during the past quarter century in calorimeters suit­ able for the study of reactions in solution, and perhaps of even greater importance, several excellent instruments have become commercially available. The newer developments in reaction calor­ imetry will be considered at other sessions in this School.
A major purpose of combwstion calorimetry has traditionally been the accumulation of enthalpies of formation which could be combined with entropies of formation to give free energies of for­ mation. This application led to the development of highly precise heat capacity calorimeters for the evaluation of Third Law en­ tropies. There was in the early sixties a short-lived program di­ rected toward the utilization of combustion data and Third Law en­ tropies,together with additional necessary data such as solubilities, for amino acids, peptides and proteins to attempt to obtain some
CALORIMETRY AND BIOLOGY 21
thermodynamic understanding of protein biosynthesis. This very ambitious program appears to have been dropped. I think the most useful data to come from this effort were the low temperature heat capacities for several proteins determined by Hutchens et al. (12).
I mentioned earlier the 1911 work of A.V. Hill on the heat effects produced by frogs, muscles, yeast cells, and so on. His calorimeter was a more or less adiabatic device, with the evacuated walls of the Dewar flasks minimizing heat exchanges between the calorimeter and its surroundings. Many reaction calorimeters have been described and employed in biochemical work which are adiabatic, or nearly so, in design. A radically different approach was adopted by A. Tian in 1923 (13), which was further developed by CalveL Tian placed a multijunction thermopile between a calorimetric cell and a relatively massive, essentially isothermal, heat sink sur­ rounding the cell, the thermopile being distributed in such a manner that its output was accurately proportional to the rate of heat transfer between the cell and the heat sink. The magnitude of a heat effect was then evaluated by integration over time of the thermopile output, the value of the proportionality constant being obtained from suitable calibration experiments with reactions of known heat effects. I mention the Tian calorimeter primarly because several of the calorimeters which are currently being employed in biochemical or biological studies, including some commercially available instruments, are of the heat conduction type. The Tian-Calvet calorimeter has been used in a very wide variety of work, including studies of single cell and higher living organisms.
I have attempted here to point out what seem to me to be some of the earlier milestones in the development of biological calor­ imetry. And now I· expect that, as should be the case, our further attention here will be turned to recent progress in various branches of biothermodynamics.
(1) Lavoisier, A.L. and de Laplace P.S.: 1784, 1780 Mern. Acad. Sci ..
(2) Barcroft, J. and Hill, A.V.: 1910, J. Physiol. 39, p. 411. (3) Brown, W.E.L. and Hill, A.V.: 1923, Froc. Roy. Soc. London
94B, p. 297. (4) Sturtevant, J.M.: 1941, J. Phys. Chern. 45, p. 127. (5) Hill, A.V.: 1911, J. Physiol. 43, p. 261.. (6) Duclaux, J.: 1908, Camp. Rend. 146, p. 120. (7) Chelintzev, V.: 1912, J. Russ. Phys.-Chern. Soc. 44, p~ 865. (8) Barry,F.: 1920, J. Am. Chern. Soc. 42, p. 1295, p. 1911. (9) Tian, A.: 1923, BuU. Soc. Chim. 33, p. 427 .•
(10) Hartridge, H. and Roughton, F.J.W.: 1925, Proc. Cambro Phil. Soc. 22, p. 426.
22 J. M. STURTEVANT
(11) Huffman, H. and Fox, S.: 1938, J. Am. Chern. Soc. 60, p. 1400. (12) Hutchens, J.O., Cole, A.G. and Stout, J.W.: 1964, J. Biol.
Chern. 239, p. 4194. (13) Tian, A.: 1923, Bull. Soc. Chim. 33, p. 427.
BIOINORGANIC CHEMISTRY OF NICKEL
Bibudhendra Sarkar
The Research Institute, The Hospital for Sick Children, Toronto, Ontario, and the Department of Biochemistry, The University of Toronto, Toronto, Ontario, Canada
The toxicity caused by excessive intake of metals due to occu­ pational or environmental pollution is a health problem throughout the world. In our programme of studying the biological transport of metals and its removal, a detailed investigation was carried out with Ni(II). The major Ni(II)-binding substances in human blood were found to be albumin and L-histidine. The Ni(II)-binding site of human albumin was shown to involve a-NHZ nitrogen, two inter­ vening peptide nitrogens, imidazole nitrogen of the third histi­ dine residue and the carboxyl side chain of the aspartic residue. Triethylenetetramine and D-penicillamine were found to be most efficient antidotal agents against Ni(II)-toxicity. A detailed Ni(II)-binding studies were undertaken with these chelating agents with a view to understand the reason for their efficiency in the removal of Ni(II).
1. INTRODUCTION
Metal toxicity caused by excessive intake of metal due to occu­ pational exposure or environmental pollution is of much concern in recent years. There are reports of high level of Ni(II) in the blood of individuals in the mining town of Sudbury, Ontario, the site of the largest open-pit nickel mines in North America (1). The results showed that the serum nickel in the healthy inhabitants of Sudbury was double that of healthy inhabitants of Hartford, Connecticut, a city with relatively low environmental concentration of nickel. A recent investigation of workers at a nickel refinery in Norway found that the degree of epithelial keratinization. was more pronounced in the nickel exposed group than in controls (2). Little is known, however, as to how the metal is handled by the
23
24 B. SARKAR
body or how they are transported . In our programme of studying the biological transport of metals and its removal we have undertaken a detailed investigation with Ni(II).
2 . STATE OF NICKEL(II) IN HUMAN BLOOD
Three Ni(II)-binding fractions were obtained when 63NiC1 2 was added to the native serum (Fig . 1).
1.0
0.8
I 10.7 ..... -I z
FRACTION NUMBER (4ml)
Figure 1 . Fractionat i on on Sephadex G-150 of native human serum with Ni(II)----, 0.0 . at 280 nm, ---, Ni(II) concentration .
Of the total Ni(II), 95.7% were associated with albumin, 4.2% were bound to low molecular weight components and a small fraction, usually less than 0.1%, was associated with a high molecular weight protein which eluted in the void volume of Sephadex G-150.
The low molecular weight Ni(II)-binding component was detected
BIOINORGANIC CHEMISTRY OF NICKEL 25
by ultracentrifugation technique (3). It was identified as amino acid and L-histidine alone could account for all the low molecular Ni(II)-binding substances in blood serum (Fig. 2).
12-
+. /0 \~%0 0 0 +
-
-
-
-
-
-
Figure 2. Distribution of supernatant Ni(II) as percent­ age of total Ni(II) after ultracentrifugation at various Ni(II): albumin ratio. Native serum, 0---0; Dialyzed serum + amino acids, + --- +; Dialyzed serum, ,~. ; Albumin + amino acids, ---..
Also L-histidine was shown to possess a greater affinity for Ni(II) than albumin does. The typical U shaped curve seen in Fig . 3 is the result of a competition between L-histidine and albumin for Ni(II)-binding. The albumin binding to Ni(II) becomes evident only when no more L-histidine is available for Ni(II)­ -binding. No other amino acid did show any significant Ni(II)- -binding in serum.
26
, 2.0
Figure 3. Effect of L-histidine on the supernatant dis­ tribution of Ni(II) in dialyzed serum at various Ni(II): albumin ratio. Dialyzed serum + L­ -histidine, e---e; Dialyzed serum 0---0.
The equilibrium existing between Ni(II)-L-histidine and Ni(II)­ -albumin may have important biological significance. The Ni(II)-L- - histidine complex, which is a much lower order of molecular size than Ni(II)-albumin complex, may mediate transport through biologi­ cal membrane by virtue of the equilibrium between these two mol­ ecular species of Ni(II).
3. NICKEL TRASPORT SITE OF HUMAN ALBUMIN
Figure 4, presents the results of an equilibrium dialysis experiment of increasing molar equivalent of 63NiC12 against albumin. It was shown that there is a 1:1 correspondence between the number of
BIOINORGANIC CHEMISTRY OF NICKEL 27
• « 0.8 V)
:::::> V)
Ni/ HSA
Figure 4. Stoichiometry of Ni(II)-binding to human albu­ min as a function of Ni(II): albumin molar ratio.
moles of Ni(II) bound per mole of albumin. It was concluded that there is a first, stronger binding site for Ni(II) on albumin. When Ni(II)-albumin dialysis described above is performed in the presence of one molar equivalent of Cu(II), the specific Ni(II)­ -binding is essentially abolished indicating the exclusion of Ni(II) by Cu(II). This finding confirms that the specific Ni(II)­ -binding site involves the NH2-terminal region of the protein.
Proton displacement analysis indicates that between pH 6.5 and 7.5 there is an increase in the number of protons released, followed by a decrease from pH 7.5 to 8.0. Since the pKa for the a-amino group of albumin NH2-terminus is known to be approximately
28 B.SARKAR
>- :!::
400 450 500 Wavelength (nm)
Figure 5. Visible absorption spectra of 1:1 Ni(II)-albu­ min complex at various pH values.
At neutral pH an absorptioq maximum at 420 nm was observed. Interestingly, when one molar equivalent of Ni(II) is added to a 1:1 Cu(II)-albumin solution at pH 7.5, no absorption peak with a maximum at 420 nm is developed in 12 hours, again demonstrating that the primary binding site for both Ni(II) and Cu(II) is the NH2-terminal region of albumin.
In order to delineate clearly the nature of the Ni(II)-binding
BIOINORGANIC CHEMISTRY OF NICKEL 29
site of albumin, a detailed Ni(II)-binding studies were carried out with a peptide: L-aspartyl-L-alanyl-L-histidine-N-methyl amide (AAHMA) representing the native amino acid sequence tripeptide of the amino terminus of human albumin.
There are three complex species MHA, MAZ and MH_ZA (M=Ni(II), A=ligand and H=hydrogen) detected in the pH range 5.4-9.Z in the metal-peptide system. The MHA species existing at pH 5.4-6.6 rep­ resents Ni(II) bound to the peptide molecule with one of its three titrable groups still protonated. Both the MAZ and MH_ZA species represent fully deprotonated peptide bound to Ni(II) ion. It is known that with small peptides, several metals form bis-ligand complexes. However, it is unlikely that such a complex will form with protein at physiological pH because of the steric restric­ tions imposed by the large size of the protein molecule. The physio­ logically relevant species seems to be MH_ZA. The MH_ZA species has lost two extra protons not normally titrated in the absence of Ni (II). Around pH 9.0 this species is the only one present. Hence the MH_ZA species requires not only the involvement of the a-amino and the imidazole nitrogens but also two other coordination sites from where protons can be prematurely released. The stoichiometry of the species MH_ZA is such that a total of four protons must be released from the peptide provided that carboxyl group is already deprotonated. Two of the MH_ZA protons can be accounted for by the deprotonation of the a-amino and imidazole groups of the peptide. For the other two protons, there are three possible sources. One source is from the water molecules resulting in the formation of hydroxyl species. A second source is the pyrrole group of the imidazole and the third source is the proton on the peptide bound nitrogen of AAHMA. Nickel(II) complexes in which the peptide groups are deprotonated have been demonstrated by infra-red spectroscopy (4) and by crystallographic studies (5). The Ni(II)-triglycine system, for example, shows Ni(II) binding to peptide nitrogen and it has in fact~ been shown that 'the second peptide proton is lost more readily than the first. This is due to a favoured change from octahedral NiA (A=triglycine) to square planar, NiH_ZA coor­ dination. The coordination of a single peptide nitrogen is not sufficient to cause the change in geometry, however, accessibility of the more favourable square planar configuration in the Ni(II)­ -triglycine system promotes the dissociation of additional peptide protons so as to make these ligand donor available. In view of this it seems possible that in the present system, the extra protons displaced by Ni(II) may come from the peptide nitrogens. In this way, Ni(II) would be coordinated to four nitrogens in the MH_ZA species.
At pH 9.Z, the visible absorption spectrum is that of pure MH_ZA complex since it is the only species in the pH range. The spectra shows one absorption peak with Amax=4Z0 nm and £max=IZ8. The spectral results reported here is consistent with the results
30 B. SARKAR
of Ni(II)-albumin complex. Peters and Blumenstock (6) also noted that a solution of Ni(II) and human serum albumin was yellow in colour. On the basis of the results presented here it seems that the modes of binding of Ni(II) to the peptide and to albumin are very similar.
Further structural elucidation was made by l3C_ and lH-NMR investigation of the Ni(II)-peptide system. The l3C-spectrum showed that the Ni(II) complex is in slow exchange on the NMR time scale and resonances for bound and unbound complexes were observed in the pH range 6.4 to 9.0. The aspartic carboxyl carbon is the most affected by Ni(II)-binding (6 = 10.85 ppm at pH = 6.4) which is consistent with carboxylate-Ni(II) coordination. This is the first evidence of carboxylate participation in such a complex. The carbonyl carbon resonances (Aps-CO and Ala-CO) are also affec­ ted by Ni(II)-binding. The shifts provide strong supportive evi­ dence that the a-amino group is a metal-binding group. The imidaz­ ole ring system also has a large variation. The introduction of Ni(II) ion into the solution containing peptide causes changes in the lH-IDm. spectra. Most interest"ing in DMSO-d6 solution there is constant disappearance of Ala-NH and His-NH protons, which confirm the coordination of the peptide nitrogens. The overall lH-NMR re­ sults are consistent with 13C-NMR results.
Thus, it appears that Ni(II) transport site of human albumin is a penta-coordinated structure involving a-NHZ nitrogen, two in­ tervening peptide nitrogens, imidazole nitrogen of the third his­ tidine residue and the carboxyl side chain of the aspartic residue.
4. ANTIDOTAL EFFICACY OF CHELATING DRUGS UPON ACUTE TOXICITY OF Ni(II) IN ANIMALS
Several chelating drugs were administered to rats by im injection at equimolar dosages in order to compare their relative effective­ ness in prevention of death after a single parenteral injection of NiClZ' Among them, triethylenetetramine (Trien) and D-penicillam­ ine (Pen) were the most effective antidotes for acute Ni(II)-tox­ icity (7). In order to understand the reason for their effective­ ness, a detailed Ni(II)-binding studies were undertaken with these two chelating agents.
4.1. Nickel(II)-triethylenetetramine system
Studies show that MA is the only major species in the pH 5.0-6.5 region. The species MHA is only evident as a possible species be­ low pH 5, but must be insignificant since most of the Ni(II) is in the form of the aquo complex below pH 5. Trien forms the fol­ lowing complexes, with Ni(II): MA, MAZ' MHZA2 • MH2A3' M2HA3 • and MZH2A3 with overall log stability constants: 14.34. 20.64. 37.28.
BIOINORGANIC CHEMISTRY OF NICKEL 31
40.05, 49.Z0, and 55.0Z, respectively. The resolved visible absorp­ tion spectra indicate all the species to be octahedral.
The cis isomers are likely to be more important structures: 1. The Trien is less strained than in the planar configuration required for the trans isomer; Z. Two available cis coordination sites are necessary for the formation of the species MHZAZ' MAZ and MZA3. The species M2A3 makes maximum use for the available metal coordination sites and the available ligand donor atoms. Molecular models show that a Trien molecule can bridge the two pairs of cis sites on adjacent (Ni(trien)(H20)2)2+ ions resulting in the central ~N-CH2-CH2-N: moiety being elongated and the two methylene groups having a staggered conformation with respect to each other. Formation of the species M2HA3 from MZA3 must involve protonation at a coordination site with consequent breaking of the Ni-N+ bond.
4.2. Nickel(II)-D-penicillamine system
This system is exceedingly simple, only two species being formed over the pH range 4-10. The first species MA is only a minor one at lower pH, while above pH 5.8 all the Ni(II) is in the form of the species MAZ' The species and their stability constants (log SlOl = 11.ZZ and log SlOZ = ZZ.71) are in good agreement wi th previous reports. An interes ting feature of this sys tem is the finding of the K2 > Kl , a reverse of the usual order. The spec­ troscopic measurements show that this can be attributed to the en­ hancement of stability gained by the formation of a square planar MAZ species, in which the Pen ligands act as bidentate S, N-donor ligands rather than in the expected tridentate fashion. A similar conclusion regarding the square planar configuration of MA2 for both Pen and the much studied L-cysteine, has been reached by others (8),(9). L-cysteine, which is closely related to Pen (peni­ cillamine = S,S-dimethylcysteine) is a naturally occurring amino acid and an important metal-binding agent in blood serum. A com­ parison of the binding properties of Pen and L-cysteine reveals that both form MA and MA2 species in aqueous solution, the latter in both cases being a red, square planar complex. The stability constants for MA and MA2 species are greater, by a factor of ap­ proximately 10 in each case for the Pen system, presumably re­ flecting the influence of the two methyl substituents adjacent to the thiol group in Pen. The methyl group also seems to exert a steric influence in that no polynuclear species are observed with Pen, in constrast to the finding of polynuclear species with L-cysteine of the type M2A3 and M3A4'
4.3. Reason for the efficiency of triethylenetetramine and D-peni­ cillamine as therapeutic agents
The results presented above do show that these ligands are very
32 B.SARKAR
effective chelating agents for Ni(II), but number of additional features are also shown to be important. For example, at ratios of Trien/Ni(II»1 and at the physiological pH of 7.4, Trien che­ lates Ni(II) in a number of ways, i.e. as the species M2HA3' MH2A2 , MA, plus the minor species M2A3' MA2 and M2H2A3. The ef­ ficiency of Trien as a therapeutic agent therefore lies in the flexibility in being able to adopt a number of configurations. In contrast, Pen at high Pen/Ni(II) exclusively forms Ni(Pen)2-, this complex gains additional stability by adopting a square planar configuration. Complexes of Ni(II) involving Pen are also more stable than those formed with the related, naturally occurring amino acid, L-cysteine.
(1) McNeely, M.D., Neckay, M.W., and Sunderman, F.W., Jr.: 1972, Clin. Chern. 18, pp. 992-995.
(2) Torjussen, W. and Solberg, L.A.: 1976, Acta Otolaryngol 82, pp. 266-267.
(3) Sarkar, B.: 1970, Can J. Biochem. 48, pp. 1339-1350. (4) Billo, E.J. and Mergerum, D.W.: 1970, J. Amer. Chern. Soc.
92, pp. 6811-6818. (5) Freeman, H.C.: 1973, in Inorganic Biochemistry, Ed. G.L.
Eichorn, Vol. 1, pp. 121-166, Elsevier Publisher, New York. (6) Peters, T., Jr. and Blumenstock, F.A.: 1967, J. Biol. Chern.
242, pp. 1574-1578. (7) Horak, E., Sunderman, F.W., Jr., and Sarkar, B.: 1976, Res.
Corm. Chern. Path. & Pharmaco l. 14·, pp. 153-165. (8) Letter, J.E. and Jordan, R.B.: 1975, J. Amer. Chern. Soc. 97,
pp. 2381-2390. (9) Natusch, D.F.S. and Porter, L.J.: 1971, J. Chern. Soc. A,
p. 2527 and references therein.
APPENDIX
(1) Blomberg, M., Hellsten, E., Henriksson-Enfl0, A., Sundbom, M., and Vokal, H.: 1977, A Report on Nickel, Published by the University of Stockholm, Institute of Physics.
(2) National Academy of Sciences: 1975, Medical and Biological Effects on Environmental Pollutants: Nickel, Washington, D.C.
(3) Sunderman, F.W., Jr.: 1973, The Current Status of Nickel Carcinogenes. Ann. Clin. Lab. Sci. 3(3), pp. 156-180.
(4) Underwood, E.J.: 1971, Trace Elements in Human and Animal Nutrition, p. 170, 3rd ed., Academic Press, New York.
DESIGN AND SYNTHESIS OF LIGANDS
Jean-Pierre Sauvage
Institut Le Bel, Universite Louis Pasteur, 4 Rue Blaise Pascal, Strasbourg, France
Macropolicyclic molecules contain intramolecular cavities enabling these molecules to form inclusion complexes with given substrates. In addition to selective complexation of substrates, these macro­ policycles may perform chemical activation and/or transport of the bound substrate. The field of synthetic receptor molecules capable of molecular recognition and catalysis has rapidly devel­ oped over the past decade (1-6). In this article we shall mainly focus our attention on the design and synthesis of ligands capable of displaying strong and selective complexation of small charged species, such as alkali and alkali-earth cations, transition metal cations and inorganig anions.
The nature of the charged substrate to be complexed will determine the nature of the chemical groups contained in the ligand. In general, eiectron pair donating groups will be required for the complexation of cations while positively charged, hydrogen bonding groups will be required for the complexation of anions. The ge­ ometry of the substrate to be complexed will determine the topology of the ligand. The ligands we have prepared can be roughly divided into 4 topological classes:
1. Macrobicyclic compounds, dispalying strong complexation of spherical cations (type B).
2. Cylindrical macrotricyclic ligands presenting two complexation sites for cationic species (type C).
3. Spherical macrotricyclic receptors containing a large sphe­ roidal internal cavity and leading to the complexation of large cations and of spherical anions (type D).
33
34 J.P. SAUVAGE
4. Acyclic or monocyclic molecules conta1n1ng positively charged functional groups displaying complexing ability towards anionic substrates of different geometries (type A).
The various topologies of some of the ligands to be discussed are depicted in Figure 1 .
...---- ..... ,.- -Z Z ....,
A B
c FIGURE 1
1. MACROBICYCLIC COMPOUNDS
The monocyclic crown ether prepared by Pedersen (7),(8) twelve years ago display interesting complexation properties towards spherical cations although the internal cavity of these ligands is far from having spherical symmetry. The cavity of a macrobi­ cyclic molecule was expected to be more adapted for the complex­ ation of a spherical cation. The general scheme (9) for the syn­ thesis of macrobicyclic compounds is presented in Figure 2.
zl\ or
or z
I: (Z
36 J. P. SAUVAGE
This synthetic route and chemical steps leading to macrobi­ cyclic compounds have been widely used for preparing other related compounds (10 - 12). Len~hening the bridges of the macrobicyc1ic systems from [! .1.:iJ to []. 3.1I leads to a gradual change in size of the intramolecular cavity, from about 1.2 to 4.8 ! diameter. The lipophilicity of the ligand can be adjusted at will by the introduction of aromatic rings or of aliphatic chains (13),(14) in the organic skeleton surrounding the complexation site. A few cryptands presenting a lipophilic character are presented in Figure 3.
ro~o~ rr'-o~ ro/\.~ ~ N"\.,.~lC""\...N MeN Mole N""v0 ""vo'"\..-... N~"'"\..-O~N \.JO~ 'v°,--/V ~ '0°'00 x = CHz ~ !J ~ !J
2.2.CS 2.2. (NMe) 2 2.2.28 2.28 .28
FIGURE 3
The replacement of oxygen binding sites in macrobicyclic cryptands by nitrogen (12) or sulfur (10) ahs been carried out by the use of appropriate protecting groups. Although the topology of the ligands and the size of" the internal cavity are only little mod­ ified, the complexing properties were expected to be different than those of the ligands 1-8 since the internal complexation site presents a "softer" donor character (Figure 4). An extension of these macrobicyclic cryptands is presented by the ligand bis­ tren (15) whose synthesis is depicted in Figure 5. Depending on the conformation of the bridges the large internal cavity is able to adjust itself to different types of substrates. The tri­ podal subunit "tren" is known as a good complexing agent towards transition metal cations, therefore, bis-tren should lead to binuclear complexes. Furthermore, the hexa-protonated form of bis-tren should be a good receptor of anionic species of various shapes and in particular, it should recognise linear cylindrical inorganic anions of suitable size (16).
DESIGN AND SYNTHESIS OF LIGANDS
2.2.2. (NMe)
40.2S 4S.20 6S.
FIGURE 4
MeOOC COOMe
I ACID Ts /",cYj Ts rN N\
31
CIOC COCI
CIOC COCI
MIG" DILUllOtl
'NH HN\-0",,- ..J 0
z~z
FIGURE 5
~O~ ! DIBORANE
~O~
38 J. P. SAUVAGE
2. CYLINDRICAL MACROTRICYCLIC LIGANDS
The first macrotricyclic ligand was obtained in the course of the synthesis of macrobicyclic cryptands (17) ,(18).
tC)~o + (0 0') _
VJ
FIGURE 6
Later, a series of compounds presenting the same general topology and nitrogen and oxygen atoms as binding sites were prepared (19); the size of the two monocyclic subunits and the length of the linking bridges can be varied at will. In order to complex two transition metals in the same molecule, oxygen was replaced by sulfur (20). The synthesis of sulfur containing macrotricyclic ligands is presented in Figure 7.
FIGURE ?
DESIGN AND SYNTHESIS OF LIGANDS 39
The synthetic route can be choosen in order to introduce either different macrocyclic subunits, the two bridges being the same (as exemplified in Figure 7) or different linking bridges, the two monocyclic subunits being identical. These cylindrical macro­ tricyclic ligands define three cavities: two lateral circular cavities located inside the macrocyclic subunits and a central cavity.
3. SPHERICAL MACROTRICYCLIC RECEPTORS
The first spherical macrotricyclic compound to be synthesised (21) is represented in Figure 8. It contains four nitrogen sites and six oxygen sites located respectively at the corners of a tetra­ hedron and an octahedron.
rY'\
FIGURE 8
o This ligand contains a large spherical cavity (about 1.7 A radius) which should be ideal for spherical recognition. Furthermore, the four nitrogen sites are ideally located for the recognition of a tetrahedron of the appropriate size such as NH4+ (see Part II).
The synthetic route involves three high dilution reactions and protecting-deprotecting steps for the successive contruction of a macrocyclic, a macrobicyclic and the final macrotricyclic molecule.
In its tetraprotonated form, the positively charged cavity might be occupied by an anionic species (X-) held in a tetrahedral array of ~-H ... X- hydrogen bonds. Indeed, these tetraprotonated macrotricyclic ligands may be considered as the topologically optimal receptors for spherical anions.
40 J. P. SAUVAGE
4. ANION RECEPTOR MOLECULES
The protonated forms of some of the polycyclic ligands mentioned above have been revealed as efficient receptors for various anionic substrates (Part II). In addition to ammonium as an anionic binding site, one may envisage other positively charged functional groups susceptible of forming hydrogen bonds providing the binding strength necessary for the complexation of anions. In particular, the guani­ dinium group presents interesting properties: it may form zwitter­ ionic hydrogen bond N-H+ ... X-, it has a very high pKa and is there­ fore little affected by pH changes, it is of importance in the maintenance of protein conformations (arginyl residues) and in the binding of anionic substrated by enzymes. In addition, the guani­ dinium group is relatively easily introduced into organic skeletons providing the desired architecture for anion complexation. Some of the cationic guanidinium macrocycles sinthetized (22) are rep­ resented in Figure 9.
FIGURE 9
The synthetic scheme leading to such a macrocyclic molecule is depicted in Figure 10.
B r
N a
C N
M eO
H ,H
42 J. P. SAUVAGE
(1) Lehn, J.M.: 1973, Structure and Bonding 16, p. 1. (2) Lehn, J.M.: 1977, Pure Applied Chern. 49, p. 857. (3) Lehn, J.M.: 1978, Pure Applied Chern. 50, p. 871. (4) Lehn, J.M.: 1978, Accounts Chern. Res. 11, p. 49. (5) Cram, D.J., Hegelson, R.C., Sousa, L.R., Timko, J.M., Newcomb,
W., Moreau, P., De Jong, F., Gokel, G.W., Hoffman, D.H., Domeier, L.A., Peacock, S.C., Madan, K., and Kaplan, L.: 1975, Pure Applied Chern. 43, pp. 327-349.
(6) Laidler, D.A., and Stoddart, J.F.: 1977, J.C.S. Chern. Comrn., p. 481 and references cited therein.
(7) Pedersen, C.J.: 1967, J. Arn. Chern. Soc. 89, p. 2495, 7017. (8) Pedersen, C.J., and Frensdorff, H.K.: 1972, Angew. Chern. 84,
p. 16 and references cited therein. (9) Dietrich, B., Lehn, J.M., Sauvage, J.P., and Blanzat, J.:
1973, Tetrahedron 29, p. 1629. (10) Dietrich, B., Lehn, J.M., and Sauvage, J.P.: 1970, J.C.S.
Chern. Comm., p. 1055. (11) Dietrich, B., Lehn, J.M., and Sauvage, J.P.: 1973, J.C.S.
Chern. Comm., p. 15. (12) Lehn, J.M., and Montavon, F.: 1976, Helv. Chirn. Acta 59,
p. 1566. (13) Cinquini, M., Montanari, F., and Tundo, P.: 1975, J.C.S. Chern.
Comm., p. 393. (14) Clement, D., Damm, F., and Lehn, J.M.: 1976, Heterocycles 5,
p. 477. (15) Lehn, J .M., Pine, S.H., Watanabe, E., and Willard, A.K.: 1977,
J. Am. Chern. Soc. 99, p. 6766. (16) Lehn, J.M., Sonveaux, E., and Willard, A.K.: 1978, J. Am.
Chern. Soc. 100, p. 4914. (17) Cheney, J., and Lehn, J.M.: 1972, J.C.S. Chern. Comm., p. 487. (18) Cheney, J., Lehn, J.M., Sauvage, J.P., and Stubbs, M.: 1972,
J.C.S. Chern. Comm., p. 1100. (19) Lehn, J.M., Simon, J., and Wagner, J.: 1973, Angew. Chern. 85,
P •. 621,622; 1977, Nouveau J. Chimie 1, p. 77. (20) Alberts, A.H., Annunziata, R., and Lehn, J.M.: 1977, J. Arn.
Chern. Soc. 99, p. 8502. (21) Graf, E., and Lehn, J .M.: 1975, J. Arn. Chern. Soc. 97, p. 5022. (22) Dietrich, B., Fyles, T., Lehn, J.M., Pease, L.G., and Fyles,
D.L.: 1978, J.C.S. Chern. Comm., p. 934.
HOLES, HANDEDNESS, HANDLES, ~D HOPES : MEETING THE REQUIREMENTS OF PRIMARY BINDING, CHIRALITY, SECONDARY INTERACTIONS AND FUNCTIONALITY IN ENZYME ANALOGUES
J. Fraser Stoddart
Department of Chemistry, The University, Sheffield S3 7HF and Corporate Laboratory, Imperial Chemical Industries Ltd., P.O. Box 11, The Heath, Runcorn, Cheshire WA7 4QE.
The basic requirements of an enzyme analogue are recognised as binding, chirality, functionality, and catalysis. The attrac­ tions of molecular receptors of the crown ether type are dis­ cussed and the attributes of carbohydrates as sources of chirality and functionality are listed. The influence of consti­ tution and stereochemistry upon complex stabilities between l8-crown-6 derivatives and primary alkyl ammonium ions are examined in considerable detail. It is concluded that all aspects of structure - namely, constitution, configuration, and conformation - associated with the primary binding site of a crown ether receptor define the natures and strengths of the complexes they form. Sources of secondary interactions between appropriate substrates and the carbohydrate moieties of the receptors are identified as providing a further means of structuring complexes. The prospect of designing more sophisti­ cated molecular receptors of the crown ether type is raised and discussed as a prerequisite to achieving high selectivity in binding - and ultimately also in catalysis - using this brand of enzyme analogue.
1. HOLES AND PRIMARY BINDING
The notion of binding organic species be they neutral, cationic, anionic, or even radical in nature by synthetic receptor molecules is a relatively recent development in
43
44 J. F. STODDART
chemistry (1-5). The realisation of this notion during the last few years owes much to the pioneering spirit of Cram (1) and Lehn (2,3) following the initial observation by Pedersen (6) in 1967 that crown ethers in general, and dibenzo-18-crown-6 (!) in particular, will form stable complexes with substituted
1
ammonium chlorides such as those derived from Me3CNH2, Me2CHCH2NH2, Me2CHCH2CH2NH2, C6HllNH2 (cyclohexylamine), HONH2, H2NNH2, and H~CCH2NH2. This information, coupled with the close inspection of Corey-Pauling-Koltun (C.P.K.) space-filling molecular models, led Cram (7) to propose a three-point binding model for the face-to-face complex formed between l8-crown-6 (2) and a RNH3+ cation. This proposal can be illustrated by refer~ ence to the cationic complex (~)-Me3CNH3+ formed between (~) and
Me
\ .... ... H ...
~O~'o o
the Me3CNH3+ ion. It is en~isaged that the three hydrogens on the positively charged nitrogen of the Me3CNH3+ ion form hydrogen bonds to alternate oxygens in (~) leaving the other three oxygens to contribute some additional ion-dipole stabil­ isation to the complex. More recently, attention has been drawn (4,5,8) to the fact that (2) prefers to adopt an "all­ gauche-OCH2CH20" conformation in-crystalline complexes of
x
HOLES, HANDEDNESS, HANDLES, AND HOPES 45
lS-crown-6 derivatives with RNH3+ ions and that the hydrogen bonds are associated invariably (to date at least in the published literature) with the three oxygens (e) syn to the R group. Thus, holes built around the crown ether constitution would appear to have shape and size as two synthetically adjustable parameters in their design. Appreciation of these factors leads one to recognise in the crown ether hole, the potential for a lot of structure in complexes without resort to a vast expenditure of synthetic effort in building a receptor molecule from first principles.
One long term goal of the present research effort on molecular receptors of the crown ether type is the building of enzyme analogues. The basic requirements for an enzyme analogue in relation to an appropriate substrate are binding, chirality, functionality, and catalysis. Crown compounds with the lS­ crown-6 constitution not only form strong complexes in organic solvents with RNH3+ ions (vide s~ra) but they also complex albeit weakly with neutral molecules (e.g. H2NCONH2, H2NCSNH2, MeCN, MeN02' and Me02CCiCC02Me) that contain acidic hydrogens (9-12). Crown compounds with the 12-crown-4 constitution form strong face-to-face complexes with R2NH2+ ions as well as with RNH3+ ions in organic solvents (13). Here, a two-point binding model involving hydrogen bonding of the ion to diagonally disposed heteroatoms in the 12-crown-4 constitution applies. Other crown constitutions (e.p. 15-crown-5 and macroheterocycles containing more than an lS-membered ring) form complexes with appropriate organic cations (1-3). The fact that strong non­ covalent bonds (e.g. hydrogen bonds and ion-dipole interactions) are formed at these primary binding sites means that it is possible to build highly structured molecular complexes around relatively low molecular weight receptor molecules. A further advantage of building enzyme analogues around the crown ether constitution is the ease with which crown compounds can be synthesised in high yields from readily available precursors.
2. CONSTITUTION AND COMPLEX STABILITY
The dependence of complex stabilities of Me3CNH3+ ions with crown compounds upon constitutional changes in a number of lS-crown-6 derivatives has been investigated (14) in considerable detail by a two-phase equilibration procedure between CDC13 and D20. An lH n.m.r. spectroscopic method may be used to measure the extent of transfer of Me3CNH3+SCN- from the D20 layer into the CDC13 layer containing the crown compound and so obtain a stability constant (Ka) in CDC13 for the equilibrium:
46 J. F. STODDART
HMH (4180000 ; -9'01 ) 7.. X = .. , 0
!!. X = M (5800000; -9·20)
HOLES, HANDEDNESS, HANDLES, AND HOPES 47
In the remainder of this article, the stability constants for complex formation_yith Me3CNH3+SCN- will be quoted in parentheses as Ka values in M together with the derived free energies of complexation (~G) in kcal mol- l after each crown compound for which values have been reported. The format (Ka , ~G) will be adopted for the presentation of this data. The Ka values associated with the various 18-membered ring macrocycles (l)-(~) demonstrate how constitutional modification of lB-crown-6 (~) can be used to influence the complexing abilities of this kind of molecular receptor. Replacement of one oxygen by a CH2 group as in (l) results in loss of half the complexing ability of (~). The m-xylyl unit in (i) also provides considerable destabilisation of the complex which can be compensated for, in part at least, by providing an additional binding site as in (5) in the form of a C02Me group. A furanyl oxygen is consid­ erably less basic than an ethereal oxygen and so (~) forms a weaker complex than does (~). However, a tetrahydrofuranyl oxygen, which is more basic than an "ordinary" ethereal oxygen provides a source of increased complexation in (~) compared with that observed for (2). It is also possible that the cooperativity characteri~ing binding by (7) to the Me3CNH3+ ion is enhanced in a stereochemical sense (vide infra). In (~), the stronger hydrogen bonding potential of a pyridyl nitrogen is demonstrated by its high Ka value relative to that for (~).
3 . HANDEDNESS AND SYMMETRY
Chirality can be introduced into synthetic receptor molecules in one of two ways. Resolution of a racemic modification of an appropriate synthon provides one approach to the problem. The other approach - which will be illustrated here - is to incor­ porate natural products into the syntheses of the receptor molecules. The most obvious way to render the crown ether binding sites chiral is to employ readily available enantio­ merically pure carbohydrates with known chiroptical properties in their syntheses. The abundance of substituted bismethylenedioxy units in carbohydrate derivatives for incorporation into the crown ether constitution is an immediately obvious attribute of this class of natural product. In addition, carbohydrates contain numerous functional groups (most commonly OH groups) which can be used to form derivatives that are often conforma­ tionally biased and hence rigid. This rigidity lends itself to the elaboration of side arms which can be either convergent or divergent with respect to the primary crown ether binding site and can also become sources of either secondary binding sites or catalytic sites. The presence of good 1H and 13C n.m.r. probes in carbohydrate derivatives also renders them attractive from the analytical and structural viewpoints.
48 J. F, STODDART
Q-0-13 R' =OMe; R2= H _. P-O-!!. R'= H ; R2= OMe
Q-0-12 R' = OMe; R2 = H -'
A-0-12 R' = H R2 = OMe t' _,
HO
Q-0-15
Q-0-14
)(e o Me
)(e O· Me
0 o Me
0y ~V XMe
HOLES, HANDEDNESS, HANDLES, AND HOPES 51
The numerous carbohydrate precursors to chiral crown ethers fall (5) into two categories: there are those with C2 symmetry, and those which are asymmetric. Examples of the first category are provided by 1,4-di-O-benzyl-L-threitol L-(9), 1,2:5,6-di-O­ isopropylidene-D-mannitol D-(lO)~ and 2,5-~ydro-D-mannitol - D-(ll). The 4,6-0-benzyliaene-derivatives of methyl a- and lr-O-glucopyranosides a-0-(12) and 8-0-(12), methyl a- and 8-~galactopyranosides ~-o:(13) and lr-D-(13), methyl a-D-manno pyranoside 8-D-(14), and methyl a-o-altropyranoside a-0=(15) are examples in the S;cond category. All contain substituted-Chiral ethylene glycol units and D-(ll) also has a substituted chiral diethylene glycol unit as wel~ The nature of the substituted bismethylenedioxy units ranges from being formally flexible in L-(9)and 0-(10) through a situtation of greatly reduced flexi­ Eility in=o-(Il) to being more or less rigid in a-0-(12) - a-O­ (~) where=diequatorial, axial-equatorial, and diaxia~orientations for the conformations of the two OH groups are all represented.
Symmetry introduces welcome economies into syntheses of chiral crown ethers incorporating more than one carbohydrate residue. For example, in 18-crown-6 derivatives containing two carbohydrates with a constitutionally diagonal relationship, at least four different situations can be identified. They are (i) the incorporation of two identical residues with C2 symmetry to give (15) one 18-crown-6 derivative with D2 symmetry, e.g. 00-(16), (ii) the incorporation of two non-identical residues each~aving C2 symmetry resulting in one 18-crown-6 derivative with C2 symmetry, e.g. OL-(17), (iii) the incorporation of two non-identical residues,-one with C2 symmetry and the other asymmetric, to give (16) one asymmetric 18-crown-6 derivative, e.g. a-00-(18), and (iv) the incorporation of two identical asymmetric residues to give (17) two asymmetric 18-crown-6 derivatives which are constitutional isomers of each other. An example of this situation is summarized in Figure 1. Base­ promoted condensation of the "half-crown" diol 0-(19) with its derived bistosylate D-(20) yielded (17) two 18-~rown-6 deriva­ tives 2,3:2' ,3'-aa-0~(21) and 2,3:3',2'-aa-00-(22) with different chromatographic properties, melting points, specific rQtations etc. Constitutional assignments were made on the basis of dynamic lH n.m.r. spectroscopy in C02C12 of 1:1 complexes formed with RNH3+X- salts. The isomer with m.p. 233-2340 was assigned to 2,3:2' ,3'-aa -00-(21) with heterotopic faces because unequally populated anisometric--(18) complexes were identified at low tempe