摘要:

R. I .C. Reviews R.I.C. Reviews, published twice yearly, reviews areas of chemistry of interest to the chemist who has no specialist knowledge of the field under review, but who wishes to keep abreast of the growth of chemistry as a discipline. These reviews should prove useful to students in familiarizing themselves with a particular field. R.I.C. Reviews interprets the significance of chemistry in a wide context and publishes articles on the economic, social and historical aspects of chemistry, as well as on the reseaSch and applied sectors. Suggestions for future titles are welcomed. Prospective contributors should write to the Editor, enclosing a synopsis (of about 250 words) indicating the scope of their subject. The preferred length for reviews is 8000 words. Subscriptions from R.I.C. members are handled by the Royal Institute of Chemistry, 30 Russell Square, London WCl B 5DT. All other subscriptions are handled by The Chemical Society Publications Sales Office, Blackhorse Road, Letchworth, Herts. Annual Subsci-iption: f2 (R.I.C. members, f l 10s)

ISSN:0035-8940    

DOI:10.1039/RR97003FX001

出版商:RSC    

年代:1970

数据来源: RSC

摘要:

PRACTICAL ASPECTS QF PROGRAMME WRITING D. R. Hogg, B.Sc., Ph.D. Dept of Chemistry, The University, Aberdeen At39 2UE 29 . . .. .. .. .. .. .. . . 27 . . .. 39 Some aspects of programming in physical chemistry Choice of topic, 39 The objectives, background knowledge and testing of the pro- grammes, 41 Writing the programme, 42 . . * . * . Acknowledgements References and R. B. Moyes, B.Sc., Ph.D.,A.R.I.C. Dept of Chemistry, The University, Hull HU6 7RX Introduction The technique of programming . . .. .. .. .. . . Preliminary work, 29 Writing the programme, 32 Revision and rewriting, 38 INTRODUCTION . . . . . . . . * . .. .. . . . . .. 43 .. .. 43 Programmed learning is a teaching technique which has developed over the last decade into an important tool for education and training.It has found widespread use in such diverse fields as management training, systematic fault finding in electronic equipment, instruction in safety procedure for miners and in neuroanatomy. Its use is not however restricted to scientific and technological subjects ; a delightful programmed book1 has been written on the Battle of Waterloo and, in the same series,2 programmes have been published on the English sonnet, German grammar and Latin. In principle it appears that any material that has to be learnt can be taught by the pro- grammed technique. Probably the most obvious characteristics of a teaching programme are that the material is presented in a series of definite steps called ‘frames’ and that some form of active ‘response’ is required of the learner.In the ‘linear’ programme developed by Skinner3 each frame provides informat ion and poses a question or requires some other activity to complete the frame. The reader answers the question or completes the frame with the appropriate response before moving on to the next frame. The correct answer or response is supplied with this next frame so that immediate confirmation is given of the successful completion of the previous step. This is by far the most common type of programme and most programmed books, for example Skinner’s 0wn,4 are written in this way. 27 Hogg and Moyes The linear programme illustrates many of the essential properties of programmed material. The subject matter of the programme is presented in a carefully arranged series of steps of appropriate size by which the student proceeds in order to acquire the knowledge or skills that the programme is designed to teach.Active learning is maintained and comprehension is tested at each step by requiring the student to respond to each frame. In addition to ensuring that a given point has been understood before new material is introduced, the immediate provision of the correct answer was held3 to ‘reinforce’ the correct response, that is to increase the efficiency of the learning process, More recent work5 however has tended to cast doubt on the overriding importance of this effect. A low error rate in these responses is required so that the student can be made to feel that satisfactory progress is being maintained and the author be assured that he is communicating adequately with the student through the medium of the programme.In the linear programme, as with all programmed material, the student is able to learn at his own pace. In a linear programme all students follow the same path through the pro- gramme and no variation is allowed in the case of wrong answers. The alternative is the ‘branched’ programme and in this case there are many different paths through the programme. Several answers are provided to the question in each frame and the student chooses the answer he considers to be correct. In order to progress through the programme the student must choose the correct answer. If he chooses an incorrect answer he may be given more information and asked to choose again, be returned to an earlier frame, or be required to work through an alternative series of frames.In this way he is directed to learn material which, by his error, he has shown that he has not fully understood. Branched programmes can be produced in the form of a ‘scrambled book’, in which the reader does not progress from page one to page two, but only through those pages which are needed for him to understand the material to be learnt. Teaching machines generally use branched programmes. The programme is usually produced as a film-strip and each frame is projected on to a small screen. At the end of each frame the student is directed to press one of the buttons according to the answer chosen.This produces a fast film movement and the next frame the student requires is projected on the screen. Branched programmes are considered to favour the brighter pupil who does not have to waste time with the simpler steps, but machines are expensive and the programmes are more difficult to produce when compared with linear programmes. A more detailed account of programmed learning is to be found in the recent book by Kay, Dodd and Sime.6 An article on programmed learning in chemistry appeared in the first volume of Education in Chemistry7a and since then several further articles have appeared.7b Over the same period only 17 programmed books were reviewed in this magazines and although 80 titles are listed in Programmes in Printg many of these duplicate each other, or are available only with particu- lar teaching machines.These programmes cover a wide range of courses, some aimed at 0-level, others at undergraduate level. Some were written for the American college student and are not very suitable for British courses. R.I.C. Reviews 28 It is for this latter reason that the search has been limited to British sources. When all these books are examined, it is seen that the task of producing programmed material covering even the essentials of chemistry taught in the schools and at higher levels, has hardly been tackled. In 1966 the Royal Institute of Chemistry organized a scheme for the circula- tion of programmes at all levels of chemistry amongst interested teachers.It was hoped that this scheme would encourage the production of more programmes. The response has been disappointing and it was felt that some notes on the practical aspects of programme writing might be helpful. These were produced and distributed to the participants in the scheme. The res- ponse was generally favourable and the remainder of this review is based on a revision of these notes. It is hoped that they will stimulate an interest in programmed learning and programme writing in a wider audience. Programme writing, like all teaching-learning processes involves com- munication between the teacher, in this case the programme writer, and the pupil. There are of course certain basic principles10 which must be borne in mind but, within these limitations, the preparation of a programme and the style of presentation reflects to a large extent the preferences, personality and prejudices of the author.In this case it was considered desirable to present more than one approach to the subject. At the risk of some repetition, it is hoped that the necessary minimum of dogma has been introduced and the very personal nature of programme writing emphasized. THE TECHNIQUE OF PROGRAMMING D. R. H O W There are three main steps in writing a programme: the preliminary work, the writing of the programme and the testing, consequent revision and re- writing of the programme. It can be seen from these steps that writing a programme is in many ways analogous to preparing a new lesson or lecture.Programme writing is undoubtedly more time consuming, but the difference in the amount of time involved is not really so disproportionately large that it effectively prevents most teachers and lecturers from experimenting with the technique. Although this section concentrates on linear programmes, and the examples are chosen largely from the field of organic chemistry, much of the material also applies to branched programmes, and the examples could have been chosen equally well from other fields of chemistry. Preliminary work The obvious first step is the choice of a topic. It is useful to ask oneself three questions about this: Must the student be able to understand and apply this information in order to progress with the subject as a whole? At this stage in their development do students, in general, find this topic difficult? and Can the topic be sub-divided into smaller topics, each of which would give a viable programme ? The answers to the first two questions will indicate the usefulness of a programme on this topic, while the last question guards against tackling a project which may prove to be too ambitious.A large topic is probably best covered by a series of programmes which are prepared separately, rather than Hogg and Moyes 29 as a whole. Attempting too large a topic is probably one of the most common mistakes of aspiring programme writers. The next step is to decide what knowledge or skills the student should possess after working through the programme.This is probably best done by setting a series of questions that students would be expected to answer after completing the programme. It is desirable to set several questions on each of the final points in the topic, and also to set questions on any points which must be established in order to reach the final points in the topic. In setting these questions it is important to bear in mind that their purpose is to test comprehension and not necessarily to grade the students, although naturally some students will always do better than others. One of the most important properties of this set of questions is that it begins to divide the topic into objectives which are sufficiently small and precise to be useful. For a topic such as Boyle’s law the questions would probably encompass the statement of the law, the equation, a numerical problem the solution of which depends upon the application of the equation, a numerical problem which illustrates the experimental verification of Boyle’s law, possibly the identification of a statement as Boyle’s law and so on.If the ability to answer these questions represents the knowledge or skills that the student should possess after completing the programme then they collectively are the educa- tional objectives of the programme. It is much more useful when writing the programme to have as objectives ‘the ability to state and recognize Boyle’s law; the ability to express Boyle’s law as an equation; the ability, given the initial and final pressures and the initial volume, to calculate the final volume at the same temperature; the ability to solve a given problem involving pressures and volumes by applying Boyle’s law’, than an objective such as ‘to give a thorough understanding of Boyle’s law and its applications’.The latter will have to be converted, con- sciously, or subconsciously, into something resembling the former, before any real progress can be made with writing the programme. At this stage it is strongly recommended that the objectives of the pro- gramme be written down. It is also beneficial to revise any sentence or phrase in the objectives which contains one or more of the words, comprehend, knowledge, know, understand, understanding and appreciate. Their meaning is too dependent on the academic level of the programme for them to be useful in this context.Magerll suggests that when preparing objectives, one should ask the questions What will the student be doing when he is demonstrating proficiency? Under what conditions will this behaviour occur ? The answer to the first question must specify definite actions, e.g. stating Boyle’s law, writing P1V1 = P2V2. The second question refers to the informa- tion or equipment which must be given or be available in order to produce the required action. Having chosen a topic, decided precisely what is to be taught in the pro- gramme and produced a series of questions suitable for a terminal test (not necessarily in that order), the next step is to consider how much relevant knowledge the student can be assumed to possess.For a teacher writing a programme for his own students this does not pose too much of a problem, but if the programme is to be used by a wider audience then it is necessary to R. I. C. Reviews 30 state the knowledge which is assumed so that other teachers may decide whether it is suitable for their own classes. If too little previous knowledge is assumed the programme moves too slowly for the pupil and he tends to become bored. Boredom induces carelessness and he begins to make mistakes in his responses. If too much knowledge has been assumed then at some point the programme will move too rapidly for the student and again he will make mistakes. Although the assumption of too much knowledge and the assumption of too little knowledge both lead to mistakes in the responses, the latter is generally identified more readily as the mistakes are concentrated over a smaller section of the programme and involve a much higher percentage of the students.As the assumption of too much knowledge can be corrected by adding extra information, whereas the alternative error requires a much more extensive revision of the programme, it is preferable, in cases of doubt, to err on the side of assuming too much previous knowledge. In this way, after the pro- gramme has been revised, the students are given all the information they require and not all the information the programmer thinks they require. There is another way around this difficulty.If the amount of information is small and is not sufficiently familiar to be recalled by the student, then the problem can sometimes be resolved by incorporating the information in the frame (see later) or in a previous frame. Although this is not considered to be desirable by some programmer^,^^ in the opinion of the author the incorpora- tion of additional information is justified under the circumstances described or when it would obviously stimulate further interest. The tactic should not be used too frequently and it must be remembered that this information cannot be assumed to be known at a later stage of the programme. Having defined the initial and final levels of knowledge, the programme itself can be planned. Generally it is difficult, and by no means necessary, to avoid planning sections of the programme before this stage.It is imperative that the subject matter be developed in a logical orderly manner. A useful method is to use a paper with a line down the centre. On one side the points which are to be made are written in what appears to be the most appropriate order, leaving plenty of space; on the other side the type of frame considered suitable for this section of the subject matter is jotted down together with any ideas for actual frames which immediately come to mind and the approximate number of frames required to cover this point. These points are rearranged as required and the jottings amended and increased. This method is probably most useful when the logical order for the subject matter is fairly obvious or has been roughly determined.It is sometimes advantageous to produce a flowsheet for the development of the subject matter or to write on cards which can be rearranged more easily. The approxi- mate number of frames for each point is considered to ensure that a balance is maintained in the programme as a whole and that the programme will be approximately the required length. Naturally some experience is required before a confident estimate can be made of the average time taken by students to complete a programme. As a rough guide a collection of ‘long’ frames will take 2-3 min each, whereas if the programme consists of ‘very short’ frames the average falls to 20-30s per frame. In the experience of the author a 31 Hogg and Moyes 3 mixture of short and long frames seems to give an average of approximately 1 min/frame for university students.12 One of the much publicized advantages of programmed learning is that the student can work at his own pace, and there is invariably a spread from approximately one half of the average time to twice the average time.The average length of time required to complete a programme is obviously impor- tant if the programme is to be used within a lesson, where the timetable requirements are rigid, but the length is also important when the programme is intended for private study. Apart from the limitations imposed by the amount of time the student has available and the obvious proviso that the time required to complete the programme should not be disproportionate with respect to the importance of the subject matter, programmed learning is an active method of learning requiring a high degree of concentration and students tend to tire quite rapidly.It is doubtful whether a student can concentrate efficiently on a programme for more than 30-40 min and if the time taken to complete the programme considerably exceeds this limit then the content of the programme should be organized to provide a break after a suitable period of time. In this portion of the preparation the aim is to produce a set of notes or a plan which will assist in writing the programme. The aim is not to produce a written account of the topic which can be sliced up into convenient pieces, in order to provide small steps into which ‘active participation’ can be injected by the removal of one or more words.The programme should be written as a connected series of frames for each point, using the plan or notes merely as a guide: in the opinion of the author it is not desirable to give these notes any additional function. As in other forms of teaching the recall of certain types of information, or the practice of certain skills, e.g. recognition, requires frequent repetition in order that the material or skill be thoroughly learnt. There must be a reason- able time interval between the requests for repetition of the information or skill, to ensure that true retention has occurred and not brief temporary retention. In the final check through the plan it is worthwhile to look for situations in which earlier material can be repeated without ruining the logical development of the programme.The places at which definite revision loops can be inserted should also be indicated on the plan. When the objectives of the programme have been defined, the final test written, the amount of knowledge which can be assumed has been decided and a plan for the programme drawn up, then the actual writing of the programme can commence. Writing the programme In writing a programme the information is presented to the pupil in a series of small steps. This leaves unanswered the important question, How small must these steps be? A possible answer is that the step must be small enough to be negotiated without undue difficulty by the vast majority of the students for whom the programme is intended and yet pose a satisfactory challenge to them.From this it follows that the absolute size of step should increase as the average ability of the students increases. The size of step refers to the R.I.C. Reviews 32 degree of difficulty in comprehension not, of course, to the number of words used in the step. Each small piece of information presented to the student is referred to as a frame and in each frame the student must answer a question or make some other response. Consequently programme writing involves writing a connected series of frames requiring suitable responses. In writing frames three principles must be kept in mind. 1.The student must respond actively to each frame. This does not mean that the student must do something, however trivial, in each frame but that mentally he must process the entire frame to produce the required response. This principle is derived from the reasonable hypothesis that a student can only be assumed to have learnt the material he has been directed to read by the requirements of the response. 2. A frame must produce a minimal number of errors from the student. This principle does not require that all the students should produce the correct response but that most should. If a large number of erroneous res- ponses is produced it indicates that the frame is unsuitable either because it is ambiguous, or because the step is too large.It is not advisable to specify a minimal number and follow it rigidly. In practice there seems to be little difficulty with this point. 3. The student must be informed of the correct response immediately after completing the frame. This means that the correct response must be incor- porated& the programme adjacent to the frame, i.e. under the frame, on the back of the frame, at the side of the frame etc. A collection of correct responses at the end of the programme is not generally considered to be suitable. Within the limitations imposed by these three principles any type of frame and response may be used. Personally I consider it advantageous to use several types of frame and response in a programme as it introduces variety into the students’ work and presumably stimulates interest and reduces boredom.The second principle, minimal errors, leads to an important aspect of frame writing-methods for helping the student to produce the correct response. In this respect the orderly construction of the subject matter is probably of primary importance and gives the student a very powerful ‘prompt’ sometimes referred to as a ‘sequence prompt’. A prompt has been definedlob as ‘some- thing added to a frame to make the frame easier and which is not suficient of itself to produce a response, but depends on previous learning’. Under certain circumstancesframe l a could be called a ‘terminal frame’; that is, it contains an item which the student is expected to know after completing the pro- gramme.(Frame / a ) In a polar reaction the reacting centres are called The definition and use of the terms electrophile and nucleophile would be one of the objectives of this programme. If this is a terminal frame it should Hogg and Moyes 33 not contain a prompt and should be separated, as much as is practicable, from frames introducing this topic in order to reduce sequence prompting. Terminal frames may contain unavoidable hints arising from the grammatical structure. In frame la, for example, the response must be more than one noun, or a plural noun. If this type of hint is used intentionally it is sometimes called a ‘syntax prompt’. Frame l a can be made successively easier by the addition of prompts. First the response can be limited to two nouns, secondly the response can be further limited to two nouns one beginning with ‘n’ and the other with ‘e’, and finally the response is limited to two nouns one beginning with ‘e’ containing 12 letters in the word and the other beginning with ‘n’ containing 11 letters.This is illustrated in frames lb-ld. In a polar reaction the two reacting centres and the n (Frame Ib) In a polar reaction the two reacting centres are called the ~ _ _ . _ and the are called the . _ _ (frame Ic) - e __ - (Frame Id) are called the In a polar reaction the two reacting centres e - - - - - - - - - - - and the El - - - - - - - - - -. Another type of prompt which is used extensively is the choice of alterna- tives, This is illustrated in frame 2 where the response is limited to one of three words.(Frame 2)13 W e found that two similar charges may remain connected by the brass con- ductor without change, but were surprised that two (equal/different/ large) charges disappeared altogether when so connected. In frames 1 and 2 prompts were used to produce single word responses. With other responses, prompts must be incorporated in less obvious ways. In frame 3 the problem of incorporating a prompt has been solved by stating a rule and giving a worked example; the student is then required to respond by completing the incomplete example. Both the rule and the example are prompts and either of them may be removed in turn. (frame 3)f4 I n any reaction between two substances one is referred to as the REAGENT and the other as the SUBSTRATE.This nomenclature i s somewhat arbitrary but the inorganic substance, the ion, or the smaller organic molecule is usually referred to as the reagent, e.g. R. I . C. Reviews 34 + + reagent C6H6 substrate HzN03 -----+ CsHsNOz + H2O I- + (CH3)zCH’ + (CH3)zCHI reagent substrate Classify the following reactants as reagents or substrates. Clz + CH2=CH2 ____P etc. This is a particularly flexible type of frame which is suitable for the introduc- tion of many chemical concepts. After the material has been introduced it is sometimes useful to vary the order of presentation. That is, instead of using the rule as a prompt for the example, an example could be used as a prompt for the rule.Frames 4a-d illustrate this approach. (Frame 40) rule and example as prompts for an incomplete example. Markownikoff’s rule states ‘the negative part of the addendum adds predomi- nantly to the carbon atom carrying the smaller number.of hydrogen atoms’. In the examples below the halogen i s the negative part of the addendum, e.g. CH3CH==CH2 + HCI -----+ CH3CHCICH3 Complete the following reaction: (CH3)zC=CHz + HCI ------+ (Frame 46) rule as a prompt for an incomplete example. The addition of hydrogen iodide to 2-methyl but- I -ene obeys Markowni koff’s law (the negative part of the addendum adds predominantly to the carbon atom carrying the smaller number of hydrogen atoms). Complete the reaction: CH~CH~C=CHZ + HI -----+ I CH3 ____ (Frame 4c) example as a prompt for an incomplete example CHsCH=CHz + HCI ---+ CX3CHCICH3 Complete the following reactions: CH3CH=C(CH3)2 + HI ---+ etc.(Frame 4d) example as a prompt for the rule. The following additions obey Markowni koff’s law: CH3CH=CHz + HI ---+ (CH3)2C=CHz + HCI ---+ (CH3)2CCICH3 CH3CHICH3 State Markownikoff’s law: Hogg and Moyes 35 This type of frame is particularly suitable for programming descriptive work. In the programme, frame 5a (rule, example, incomplete example) is separated from frame 5b (example, incomplete example) by a series of frames on the mechanism of the reaction. (frame 50)15 R' \ / / \ \ / R \ R' R - c=c R Olefins can be prepared from alkyl halides and carbonyt compounds (alde- hydes and ketones) through the agency of triphenylphosphine (Ph3P) and a strong base such as sodium ethoxide (NaOEt) or n-butyl lithium (BuLi).The overall reaction can be written R' CHBr + O=C / Ph,P, BuLi R' R Met hylenecyclohexane Methylenecyclohexane can be prepared from two al kyl halide/carbonyl compound combinations. What are these? What other reagents (names and formulae) are needed? (Frame 5b) The whole sequence can be written: Ph,P EtBr -.+ f + - Ph3P=CHCH3 P h 3 P-C H C H3 Ph3PEt Br- _+ -+ Et,C=O EtzC=CHCH3 + Ph3PO Write your reaction between isopropyl bromide and acetone in this way. In frame 5a the last response merely requires the student to copy the statement given in the rule section of the frame.The amount of learning produced by this type of response is probably very small and frames in which this is the only, or the major response tend to irritate many students. The bask objection to this type of response, a copying response, is that the step is too small. Inframe 5a the copying response is only a minor part of the res- ponse, but it could have been avoided by rewording the statement defining the response. Frame 6a is undoubtedly just a copying frame of the type which tends to occur in introductory frames using single word responses. Another method of avoiding copying frames is illustrated in frame 6b which poses a series of simple direct questions. In the opinion of this author, copying frames should not be used.R.I.C. Reviews 36 (Frame 6a) Markownikoff (1869) studied the addition of hydrogen halides t o olefins and formulated the rule 'the negative part ofthe addendum adds predominantly to the carbon atom carrying the smaller number of hydrogen atoms'. With the hydrogen halides the halogen i s the negative portion of the addendum. The rule holds satisfactorily for hydrocarbons but there are some exceptions t o the rule when it i s applied to olefinic compounds which are not hydro- carbons. Markownikoff's rule states: the part of the addendum adds predominantly t o the carbon atom carrying-the smaller number of ~ ____-__-. atoms. (Frame 6b) CI I CH3--CH=CH2 + HCI + CH3-CH-CH3 CI CHrC=CH2 + HCI ---+ CH3-4-CHs CH3 or i s it polarized in the sense H8-- CI"? Hydrogen chloride readily donates a proton, Hf.Is hydrogen chloride polarized in the sense H*+-CI*-, CH3 I - CIS+. H*+-------C18-/H8- Would you refer t o chlorine, rather than t o hydrogen, as the negative portion of the molecule? yeslno In the reactions above does the negative portion of the molecule add to the carbon carrying the smaller number of hydrogen atoms? yes/no etc. In frame 6a the student only requires to read the words 'Markownikoff formulated the rule' and the portion in italics in order to respond correctly to this frame. The remainder of the first paragraph does not have to be considered in order to produce the response and thus, with respect to this information, the frame violates the principle of active response.If the two extra points introduced in frame 6a are necessary they should have been made the subject of additional frames. Although, as previously indicated, it does not seem necessary to follow this principle rigidly, it is advisable to check each frame to remove any unintentional deviations. At each step in the programme it is necessary to pose an adequate challenge to the student. With the more able students it is difficult to create a large enough learning step using frames which require only the addition of one or more words. This difficulty is aggravated further by the principle of active responding which restricts the length of the statement for each response. In my opinion this type of frame should be avoided as much as possible and its use limited to short frames having one or two responses.It would seem desirable to vary the type of frame used in a programme as much as possible, not only because this must surely make the programme more interesting to Hogg and Moyes 37 the student, although this would be reason enough, but also because it makes programmed learning more flexible with regard to the subject matter to which it can be applied. It also makes programme writing easier and more interesting. In this section examples have been given of various types of frame and these examples may be supplemented from a survey of recently published programmes.13-15 The only limitation on the format is that the frame should give a learning step of appropriate magnitude and that it should be capable of fulfilling the three basic principles.It is also desirable that the response be closely connected with the immediate aims of the teaching. If the programme teaches a practical skill the preferred response should be a practical response. Revision and rewriting It cannot be emphasized too strongly that, however carefully a programme is prepared and written, the first version can be regarded only as a rough draft. As programmed instruction is a learning method more than a teaching method, the document produced can be regarded as a programme only after it has been used by a reasonable number of students and suitably modified in the light of their comments, incorrect responses and general performance.The problem is how to obtain this information. From the literature on programmed learning it would appear that other authors have an ample supply of students and free periods in which they can test frame sequences written on talc for back-projection, or in which they can watch small batches of students work through the original manuscript and make the necessary corrections on the spot. If, as is generally the case, these facilities are not available, then this infor- mation can be obtained by incorporating an information sheet with the programme. The information sheet used by the author requests the numbers of the frames, including terminal frames to which incorrect responses were given, the incorrect response itself, any comments on this error, general comments on the programme and the time taken to complete the programme.The students hand in these sheets after completing the programme. Inevitably not all the students hand in sheets and, of those who do, not all of them fill in the sheets conscientiously, but from 80-100 students sufficient do so to make it worthwhile. These sheets produce encouragement and humour as by- products as well as honest criticism. All the incorrect responses and other details are then entered on to a copy of the programme. The results are generally clear cut. Certain frames and certain sections are unsatisfactory, either because they are badly expressed, or because examples have been badly selected, or because too much knowledge has been assumed.These frames and sections must then be rewritten and tested again until they are satis- factory. With a very much smaller number of students it takes longer to oollect enough information for a worthwhile revision. However, the teacher in this position will obtain more information per student than his colleague with a large group, where the relationship is generally more impersonal, and should obtain sufficient information from two or three classes. Assistance in testing R. I. C. Reviews 38 programmes can in any event be arranged through the Royal Institute of Chemistry’s Education Department. SOME ASPECTS OF PROGRAMMING IN PHYSICAL CHEMISTRY R. B. MOYES In the preceding part of this review Dr Hogg covered most of the practical matters related to programme writing; here I will confine myself to some personal views.I believe that the best programmes are those that the teacher has written for his own classes and which are related to their particular problems. Programmes written by other people always seem to contain difficulties of nomenclature or approach which make them inferior to those produced by the teacher himself. Further, once a teacher has written his own programmes, amendment and correction are often easy compared with the difficulties involved in making large numbers of minor corrections to others’ programmes in order to make them compatible with existing courses. The steps in writing a programme are dealt with in more detail below. They can be summarized as: (a) choice of topic; (b) definition of the aims and objec- tives of the programme; (c) consideration of the knowledge the student brings to the programme; (d) writing a draft; (e) testing the result; (f) revising the programme. Choice of topic In my view the choice of topic is crucial.Much of the early success of pro- grammed learning can be attributed to the students’ interest in a new teaching technique. When this motivation is removed and programmes are just a part of the course many of their advantages appear to be lost. Often, in order to deal with a topic in the programmed form, the student may be asked to spend much more time on it than he would normally. An example of this is the attention paid to the nomenclature of organic compounds by some programme writers.Some of these are short pithy accounts of the subject while others go to inordinate lengths because the subject lends itself to a logical approach. Organic nomenclature is not, however, a sensible subject for a programme at university level because most competent students can grasp the principles more quickly than the time involved in working through most of the published programmes. A further reason is that the student will want to refer to the prhciples of nomenclature when in doubt, something almost impossible within the usual programme form. This latter problem is often a difficulty with programmes since they seldom contain an easy method of reference to important sections. Some teachers would maintain that nomenclature is a matter which the student learns by practice and the artificial practice of the programme is not what is wanted.What then are the criteria for choosing a topic? First it must be one that the student$nds dzficult. Secondly, it must lend itself to the programmed form, and lastly it must justify the amount of time which the student will spend on it. Perhaps this could be amplified by an example from my own experience. I have to teach physical chemistry to students of biology whose mathematical abilities are modest. Nonetheless, I believe that much of the algebra of Hogg and Moyes 39 physical chemistry can be understood by these students and is worthwhile so long as each step is carefully explained. I feel that chemical lessons resulting from the mathematical logic are useful to the student and cannot be learned without working through the algebra.So my first programme deals with the commonly found algebra by which formulae for rate equations of various orders can be obtained, and their use in determining the order of reaction. The second programme shows how the steady-state approximation can be used to explain empirically-found rate equations. I think that these programmes fulfil the requirements given for choosing a programme. It is a topic found difficult by most of these students; it readily lends itself to the programmed form and the work involved is no greater than that normally expended by students on this subject. Example from kinetics I The time for half the reactant t o decompose will be for x = for x = a12 14 In the equation, kt = In a/a - x , so kt, = kt,= Inala- fa=In2= 15 16 so t, = 17 How does t, vary with initial concentration? 18 This constitutes another test of first-order kinetics. We look for a half-life time which is - - - - - - - - - - - of the initial concentration.In what units can k (the rate constant for a f i r s t order reaction) be expres- 19 sed? - _ _ _ _ ~ _ _ _ _ _ t, = 0.69/k i s independent of initial con- centration; ‘a’ does not appear in independent l a Time-1. As k = - In ___ the t a - x 2.30 x 0.3010 = 0.69 t, the formula. I units are - x a pure number. time The units of k must, then, be time-1 (i.e. s-1, min-1, years-”). You will remember logarithms are dimensionless.R.Z. C. Reviews 40 20 In what field do we find half-life times of years? Rad i oact i ve d is i n teg r a t i o n s. 21 This is a rate equation again. For example, if t, for radium i s 1590 years, what i s the value of k? - Example from kinetics / I ( i ) CH3CHO .-> CH3 + CHO ( i i ) CH3 + CH3CHO .kzc CH4 + CO + CH3 ( i i i ) 2CH3 ~-+ k3 k i C2Hs Then d[CH3]/dt = __. _ _ _ _ 12 Suppose the mechanism of the reaction is 13 So, by making the steady state approximation, we say d[CHs]/dt = 0 or rate of formation of methyl radicals = rate of loss of methyl radicals, d[CH3]/dt = 0 and we can calculate concentration of methyl radicals [CHs] a t this stage = 14 Now, rate of formation of methane is d[CHa]/dt = 15 Substitute for [CH3].d[CH4]/dt == k2(kl/k3)a[CH3CHO]g So this mechanism can explain why the reaction is three halves order in acetaldehyde. Reaction ( i ) produces CH3, reac- tion ( i i i ) destroys it, reaction ( i i ) has no effect on [CH3] as it is produced and destroyed, so d[CHsJ/dt = kl[CH&HO] - k3[CH3]2 [CH3] = (kl/k3)$[CH3CHO]a (nb .\/x = x i ) only reaction ( i i ) makes CH4 so d [ C H 4]/d t = kz[C H3C H 01 [ C H3] ___--__I_____ _____. The objectives, background knowledge, and testing of the programmes The objective of the first programme is to allow students successfully to complete the logical steps leading to formulae which will be of use to them, Hogg and Moyes 41 and of the second to afford them practice in applying a simple approximation which leads to interesting conclusions.Having completed these programmes, a student should be able to obtain the formulae mentioned in the first pro- gramme and to apply the steady-state approximation to simple reactions. Unlike Dr Hogg, I do not apply specific tests to gauge the success of the programmes but instead try to estimate the effect they have on the answers given in the usual class examinations. This is because I attempt to integrate programmes with the rest of the course in physical chemistry, of which they are only a small part, and I think that it is in this respect (assistance with the teaching of difficult areas of the subject as a whole) that they must be judged.In the development of programmes tests of their effectiveness are necessary, along with the answers to the frames. These make the revision of the pro- gramme possible but, in my view, give the programme unnecessary emphasis when it is used routinely. Again, because they are integrated parts of the course, I know what know- ledge students will have when they are given these programmes to work through. Specifically, they will have attended lectures in which most of the content of the first programme will have been discussed and the principles of the methods used in the second programme. For this reason, in the strictest sense, these programmes are useless to other teachers except where they follow lectures of the same kind. These programmes have been revised three times but there are still some errors which need correction.I have long regretted that I chose the HZ-Brz system as an example of a complicated chain reaction because it contains possibly too much algebra and too little chemistry. For this and other reasons, I do not consider these programmes to be ‘finished’. There is always a temptation to tinker with them but this may itself be an advantage as it presents one with the opportunity to bring material up to date. After all, how often does a good teacher teach exactly the same lesson every year? Writing the programme Far too much has been written on the topic of writing programmes, and far too much of it is still controversial for me to hazard more than a personal opinion.I feel most students are used to their teacher’s style and when written down it will still be familiar. Some obvious errors are worth remarking on, although personal preferences are difficult to forecast. In some programmes I have noticed a tendency to write long frames containing too much informa- tion and requiring too little response on the part of the student. It should be remembered that programmes are more than books with the occasional word .missing. Some authors spend too much time on definitions which are principally concerned with the technical use of words, for, while it is important to define these terms, they often occupy a disproportionately long and boring section of the programme and reduce the student’s concentration on the important work which follows it.The next comment, that concerning the minimal number of errors, involves R.I.C. Reviews 42 the achievement of a difficult balance between boring the student and con- fusing him, and it is here that the experience of the teacher in relation to his class is most valuable. Teachers attempting to write programmes should not be too anxious to avoid difficult steps since these encourage the brighter student who can sometimes be assisted by a hint written below the original quest ion. My final comment involves the careful placing of the correct response. Again there must be a judicious choice between a clear invitation to cheat and hiding the answer so far away that a frustrating search is required. For this reason I cannot recommend the scrambled book technique for dealing with branching programmes.My experience with the programmes I have written is that those students who cannot be bothered to work through the frames properly are unlikely to want to read the programme at all. Most students are quite willing to use the programme in the way intended and it seems pointless to include anti-cheating devices for the aberrant few. In any case there seems to be some evidence that simply reading the programme, answers and all, is still beneficial. In conclusion it must be said that programming is simply another method of teaching. It does not replace anything or anybody, but merely makes the task of learning simpler for some students. It is regrettable that the technique has been advertised to such an extent that unsuitable programmes which are readily available may be misused by students for whom they were not in- tended.It is for this reason that I repeat my belief that the teacher must write his own programmes for his own courses. ACKNOWLEDGMENTS The examples in frames 2, 3, 5a and 5b taken, respectively, from Ionic theory by R. B. Dunn, Electronic efjrects and their applications by D. R. Hogg, and Olefins and acetylenes by F. D. Gunstone are reproduced by permission of the English Universities Press. REFERENCES P. Thornhill, The Waterloo campaign. London : Methuen, 1965. Methuen’s Clearway Programmed Books. B. F. Skinner, ‘Teaching Machines’, Scient. Am., 1961, November, 90. J. G. Holland and B. F. Skinner, The anaZysis of behaviour. London: McGraw-Hill, 1961. (a) S. M. Markle, Good frames and bad, 21, 249. London: Wiley, 1964; (b) W. A. Hershberger and D. F. Terry, J. educ. Psychol., 1965, 56, 22; S. C. Lublin, ibid., 1965, 56, 295. H. Kay, B. Dodd and M. Sime, Teaching machines and programmed instruction. Harmondsworth: Penguin, 1968. (a) F. D. Gunstone and R. B. Moyes, Educ. Chem., 1964, 1, 189; (b) D. E. Hoare and G. R. Inglis, ibid., 1965,2,32; D. R. Hogg and H. P. R. Hodge, New Educ., 1965,l (12), 30; D. R. Hogg in Aspects of educational technology, ed. D. Unwin and J. Leedham. London: Methuen, 1967; R. B. Moyes, Educ. Chem., 1966, 3, 182; P. S. Adey, ibid., 1966, 3, 302; ibid., 1967, 4, 141 ; N. A. Coats, ibid., 1969,6,21; M. Collard, J. Griffith, H. Liddy, V. Shuk, and E. S. Swinbourne, ibid., 1969, 6, 130; G. M. Seddon, Chem. Brit., 1967,3, 160; (c) W. K. Richmond, Educ. Chem., 1968, 5, 109. Educ. Chem., 1964,1,163; 1966,3,44, 145; 1967,4,101,304; 1968,5,38,44, 129, 135, 177, 179; 1969,6, 30, 71, 189, 193, 194, 235. Hogg and Moyes 43 Learning and Educational Technology, 1969. 10 (a) P. Pipe, Practicalprogramming. New York: Holt, Rinehart and Winston, 1966; 9 P. Cavanagh and C. Jones, Yearbook of educational and instructional technology 1969-1970, incorporating Programmes in print. London : National Assn for Programmed (b) S. M. Markle, Good frames and bad. London: Wiley, 1964. 1969. 11 R. F. Mager, Preparing objectives for programmed instruction. San Francisco : Fearon, 1961. 12 In some very limited tests students from Further Education Colleges took an average of a little less than 1.5 min per frame. The error rate was similar to that of university students. 13 R. B. Dunn, Ionic theory, 13. London: English Univ. Press, 1965. 14 D. R. Hogg, Electronic efsects and their applications, 25. London: English Univ. Press, 15 F. D. Gunstone, Olefns and acetylenes, 22. London: English Univ. Press, 1966. 44 R.Z.C. Reviews

ISSN:0035-8940    

DOI:10.1039/RR9700300027

出版商:RSC    

年代:1970

数据来源: RSC

摘要:

E. J. Bourne, Ph.D., D.Sc., F.R.I.C., and P. Finch, BSc,, Ph.D. 45 46 49 52 .. 57 . . . . .. . . . . . . References . . . . . . . . .. . . . . . . . . 57 THE GLYCOSYL TRANSFER REACTION POLYSACCHARIDES-ENZYMIC SYNTHESIS AND DEGRADATION Dept of Chemistry, Royal Holloway College, Englefield Green, Surrey The glycosyl transfer reaction . . .. . . Polysaccharide biosynthesis . . . . . . . . . . . . . . . . * . * . Homopolysaccharides, 46 Branched and heteropolysaccharides, 48 Enzymic degradation of polysaccharides Mechanisms of action of glycosyl transfer enzymes . . Conclusions . . . . . . .. . . * . The wide occurrence and diversity of function of polysaccharides in nature is well documented,l as is the importance of this group of natural products in industry.2 These factors, coupled with the vast number of different structural examples so far encountered, are responsible for the considerable research effort which has been directed towards carbohydrate polymers.Perhaps the greatest problems to be overcome in any study of an enzyme- polysaccharide system involve purification of the two components. In addition to a variety of organic and inorganic contaminants of unrelated structure, each of the polymeric reactants is usually associated with other polymeric material of very similar structure. Ideally the reactants should be purified until constant physical and chemical properties (e.g. molecular weight, electrical charge, type of linkage, degree of branching) are achieved. During recent years greatly improved purifications have resulted from the wider application of procedures involving such techniques as ion-exchange chroma- tography, molecular-sieve chromatography, selective complex formation and electrophoresis.The successful elucidation of polysaccharide structures has led to con- sideration of the relations between chemical structure and physical and biological pr~pertiesla,~ and, in addition, has provided the basis for investiga- tions of the biological pathways of polysaccharide synthesis and degradation. This brief survey illustrates some general features of these pathways and indicates some topics of 'current interest by reference to examples selected from the many available. It is convenient to consider polysaccharide synthesis and degradation together for a number of reasons.The most important of these possibly is the fact that Bourne and Finch 45 both processes are examples of a single general reaction, termed the glycosyl transfer reaction : (1) G-OR + G-OR’ DONOR + H-OR’ ACCEPTOR The general applicability of this reaction to carbohydrate metabolism was first realized during the 1940s4 and, indeed, all subsequent observations may be discussed in terms of it. Thus, if the acceptor (H-OR’) is a polysaccharide chain the net result of the forward reaction is synthesis; if the acceptor is water (R’=H) the net result is hydrolysis; in each case the group transferred is the glycosyl group (G). POLYSACCHARIDE BIOSYNTHESIS One approach to a discussion of polysaccharide synthesis in terms of the transfer reaction is via an appraisal of the various possibilities for the nature of the glycosyl donor (substrate) G-OR, e.g.is G a single glycose residue or a number of residues previously linked by another process, and what possi- bilities exist for the structure of R? Clearly during synthesis the acceptor (H-OR’) in each successive step (except the first) is the growing poly- saccharide chain. Four main types of donor aglycone (OR) have been en- countered-phosphate, glycoses, nucleoside diphosphates and polyisoprenoid phosphates. 0 It (2) + HOR + GIC-O--(GIC), Homopolysaccharides The first examples of polysaccharide synthesis in vitro to be established con- clusively were the formation of a-l,4-linked amylose-type polymers by incuba- tion of a-D-glucose- 1 -phosphate with phosphorylases from muscle, yeast, liver, peas and potatoes (see ref.4 and refs therein): 0 ll Glc-0-P-0- + H-O-(Glc), I + HO-P-0- 0- A- The reactions are readily reversed by varying the relative proportions of inorganic phosphate and a-D-glucose- 1 -phosphate, and from these observa- tions the concept of transglycosylation was developed. It was later realized that the ratio of phosphate to a-D-glucose- 1 -phosphate in vivo is unfavourable to synthesis, and it is now postulated6 that in mammals the enzyme phos- phorylase is probably concerned with polysaccharide (glycogen) degradation only. However the stimulus provided by these observations resulted in the isolation of other cell-free extracts which would catalyse the incorporation of sugar units into polysaccharides.An early example was that of Hehre and Sugg,7 who demonstrated that an extract of the bacterium Leuconostoc mesenteroides catalysed the synthesis from sucrose (R = fructofuranosyl) of dextran, an a- 1,6-linked polyglucose. Further examples of polysaccharide synthesis by glycosyl transfer from donors containing carbohydrate ‘agly- cones’ are shown in Table 1. The last two examples in Table 1 refer to the action of enzymes which are R.I.C. Reviews 46 Table I. Glycosyl transfer from purely carbohydrate donors ~~ 10 Acceptor and linkage Dextran a- I ,6 Levan /3-2,6 Amylopectin a- I ,6 Glycogen 01- I ,6 Donor and linkage Sucrose a- I ,8-2 Sucrose a- I ,&2 Amylose a- I ,4 Glycogen a-1,4 ribofuranosyl or deoxyribofuranosyl Ref.7 8, 9 I 1 responsible for the synthesis of branch points in amylopectin and glycogen. Glycogen is generally held to possess a tree-like structure consisting of chains of a-174-linked glucose units connected by a-1,6-branch points.12 Glycogen branching enzyme catalyses the transfer of a chain of about six or seven glucose residues from an a-1,4-position to an a-l,(i-p~sition;~~ thus in this reaction glycogen is acting as both donor and acceptor. Amylopectin, the major energy reserve material in plants, is thought to possess a structure similar to that of glycogen, but one that is less highly branched and in fact glycogen branching enzyme can convert amylopectin into a glycogen-like molecule.14 The search for an alternative pathway to the glucose- 1 -phosphate/phos- phorylase system for the synthesis of the a-1,4-linked chains in glycogen was not rewarded until 1957, when Leloir and Cardin95 reported the isolation of an enzyme from liver tissue which catalysed the incorporation of glucose from uridine diphosphate glucose (UDPG) into glycogen.Uridine diphosphate glucose was the first example to be characterized16 of the general class of compounds known as the nucleoside diphosphate sugars. They possess the general formula : -5’-pyrophosphate- 1-sugar and act as glycosyl donors by cleavage of the linkage between the sugar anomeric carbon and the pyrophosphate oxygen, e.g.: UDPG + glycogen giycogen synthetase, G- I ,4-glycogen +- UDP Enzyme Dextransucrase Levansucrase Q-enzy me Branching enzyme purine or Bourne and Finch (3) In addition to their role as glycosyl donors nucleoside diphosphate sugars are important intermediates in metabolic interconversions of sugars, e.g. the formation of UDP-galactose from UDP-glucose and the formation of deoxy sugars.17 Some examples of polysaccharides believed to be synthesized by trans- glycosylation from nucleoside diphosphate sugars are given in Table 2. In the past few years evidence has been accumulating for the involvement, so far only in the synthesis of bacterial polysaccharides, of another class of sugar derivative in which the sugar aglycone (OR) takes the form of a poly- isoprenoid chain linked to the sugar through one26 or t ~ 0 ~ 7 + ~ * phosphate ester groups.The possible involvement of lipid intermediates in polysaccharide biosynthesis was perhaps first suggested by Colvin in 1961 in connection with the biosynthesis of cell~lose,~9 but it was not until 1965 that their importance was established by workers who were studying the cell-wall constituents of a number of Staphylococcus,30 Micrococcus3O and Salrn0nella~~9~1 species 47 4 -~ Ref. Rous chicken sarcoma Potato, pea, maize Acetobacter xylinum 19 20 Hyaluronic acid Starch Cellulose Mung beans 21 Table 2. Glycosyl transfer from nucleotide diphosphate sugars Source C, cytidine Donor UDP-N-acetyl-glucosamine ADP-gl u cose U DP-gl ucose G D P-g I ucose UDP-xylose TDP-rhamnose CDP-abequose A, adenosine Polysaccharide Xylan Lipopolysaccharide U, uridine Salmonella Hen oviduct typhimurium 1;: 22 G, guanosine T, thymidine Table 3.Proposed pathways of biosynthesis of some branched and heteropol ysaccharides Enzyme source Liver Potato Pathway iv (branching) iv (branching) iv (desu I phation) i , o r ii i , or ii, or iii and iv i, or ii Polysaccharide Glycogen Amylopectin Galactan Hyaluronic acid Chond roiti n Pneumococcus Type 111 capsule GI ycoprotei n Glycoprotein Lipopol ysaccharide Porphyra umbilicalis Staphylococcus hemolyticus Hen oviduct Pneumococcus Type 111 Colostrum Salmonella typhimurium Staphylococcus aureus ii iii iii 18 [24,25 Ref.35 10 36 37 38 39 40 25 32 of bacteria. Matsuhashi et al. have suggested32 that the formation of lipid intermediates may provide a means whereby intracellularly manufactured components can be transported through the hydrophobic cell membrane prior to synthesis of the outer wall. Branched and heteropolysaccharides Consideration of the biosynthesis of branched and heteropolysaccharides poses additional problems. Such polymers appear to be divisible into two types according to structure, namely those with regular, repeating structures and those with more random arrangements. A recent re-examination33 of Aerobacter aerogenes A3 (Sl) polysaccharide led to the formulation of a strict repeating tetrasaccharide unit, in conflict with earlier work, and it was suggested that other polysaccharides for which random structures have been proposed may merit re-examination.On the other hand, many polysaccharides definitely possess non-repeating structures, e.g. some dextrans are polymers of a-1 ,&linked glucose units having randomly arranged single unit branches.34 Four possible pathways may be envisaged for the formation of branched and heteropolysaccharides : ( i ) transfer of single units by a single enzyme having a number of specific binding sites; (ii) transfer of single units by a number of enzymes ; (iii) transfer of preformed oligosaccharides by one or more enzymes; and (iv) operation of transglycosylases, or of other enzymes, on a presynthesized chain of sugar units.Regular repeating structures would probably result from the operation of processes (i) and (iii) (where only one R.I.C. Reviews 48 enzyme is involved), while the regularity of polymers resulting from the other processes would depend on the specificities and possibly on the relative concentrations of the enzymes involved. All four types of system have been postulated; some examples are shown in Table 3. It is clear that this aspect of heteropolysaccharide biosynthesis is subject to considerable uncertainty, and that substantial developments can be expected in the near future by the use of the mild purification procedures now avail- able.A recent example is the isolation of Q-enzyme from potato juice by chromatography on DEAE-cellulose.41 A related aspect of polysaccharide biosynthesis is the question of direction of chain propagation, i.e. are units added to the reducing or to the non- reducing end of the growing polysaccharide chain? It has sometimes been assumed, by analogy with many other transglycosylation reactions, that the non-reducing chain terminus acts as the acceptor in polysaccharide biosyn- thesis, and this has been shown to be correct in the majority of cases which have been examined.42 However, application of the technique known as pulse labelling, in which incorporation of radioactively labelled substrate (the ‘pulse’) is followed by exposure of the synthetic system to a relatively large amount of unlabelled substrate (the ‘chase’), has revealed that in at least two i n ~ t a n c e s ~ ~ f ~ ~ polysaccharide elongation takes place other than at non-reducing chain ends.These observations are not exceptions to the gly- cosy1 transfer reaction; for example, the growing polysaccharide may be considered as the donor and the extra unit added as the acceptor in the elongation of SaZmoneZZa 0-antigen chains :42 M an-R ha-Gal-P-P-AC (Man-Rha-Gal),+r-P-P-ACL L -* (or P + P-ACL) (4) L + (Man-R ha-Gal),-P-P-AC + P-P-ACL In this example the bond cleaved is that between galactose (Gal) and phosphate (P) in the growing chain-antigen carrier lipid (ACL) complex. Gahan and Conrad43 succeeded in isolating a glycogen synthetase fraction from Aerobacter aerogenes which, although devoid of glycogen, retained the capacity to catalyse de novo glycogen synthesis from ADPG in the presence of an activator protein.The labelled glucose residues incorporated in the early stages of the reaction remained in external chains after an additional incorporation of five to six times as much glucose into the product. It appears that, at least in Aerobacter aerogenes, de novo glycogen synthesis may proceed via a different mechanism from the accepted glucosyl transferase-branching enzyme process. On storage the enzyme becomes more like a typical glycogen- dependent glycogen synthetase. ENZYMIC DEGRADATION OF POLYSACCHARIDES Enzymes which catalyse the hydrolysis of glycosidic linkages have attracted attention for more than 150 years.However, until recently their detailed investigation in mechanistic terms was somewhat neglected while extensive kinetic studies on the protein-hydrolysing enzymes, especially on chymotryp- sin by Bender and co-w~rkers,~~ were providing the basis for a molecular interpretation of enzyme action. Without doubt one of the major reasons Bourne and Finch 49 for this was the difficulty of purifying the glycosidases, particularly their reluctance to crystallize. In 1965 the picture changed when lysozyme, one of the few glycosidases to be crystallized, became the first enzyme to have its secondary and tertiary structure elucidated by x-ray cry~taIlography.~5 Furthermore, the detailed topography of the active site and the location of a substrate molecule therein was determined by x-ray diffraction analysis of the lysozyme-tri-N-acetyl- chitotriose complex.46 This brilliant work has stimulated some elegant experi- ments designed to elucidate the detailed mechanism of action of this enzyme, and has led to a resurgence of interest in glycosidases generally.One of the most obvious and intriguing properties of enzymes is their specificity, which may be defined as the range of compounds which a given enzyme will utilize successfully. Modern understanding of specificity stems from Fischer’s famous ‘lock and key’ hypothesis of 1894,47 but the original idea has been extended in three ways, two of which derive from the concept that enzyme molecules are flexible.The induced-fit theory48 postulates that an enzyme can adapt its conformation to that of a potential substrate, but that not all such induced conformations may be active. Secondly, there is the recognition of allosteric sites,49 which are distinct from the active site and catalytically inactive, but which may influence activity via conformational changes induced by the binding of small mo1ecules.49~50 The third additional factor is the recognition51 that most enzyme reactions are multi-step pro- cesses, and that selectivity or specificity may be exercised at any step including the initial (binding) one. Furthermore, selectivity may be due to electronic effects on the reactions involved as well as to steric effects.Thus the most quantitative approach to a discussion of specificity is in terms of the rate constants and energy parameters for the individual reaction steps, but since the nature of the steps is not known, except in a few cases, a more qualitative approach has often been used. A direct approach to the study of specificity is provided by x-ray crystallo- graphy, a difficult technique experimentally and one which may be applicable only to some enzymes (those which yield heavy-metal derivatives and form stable enzyme-substrate complexes). An alternative technique is to study the rates of interaction of an enzyme with a number of possible substrates of different structure. Carbohydrate substrates offer unusual opportunities for studying the factors involved since their structures may be varied between wide limits.The results obtained52 from studies of this type have been valuable in the classification of enzymes and in their application to the structural analysis of polysaccharides. Under natural conditions, water acts as the acceptor for degradative enzymes, except for some debranching enzymes and for phosphorylases. However if compounds which contain an alcohol group are added to the system they will often compete successfully with water for the role of acceptor; such compounds may for example be other carbohydrates or simple alcohols. One application of the use of simple alcohols is that the determination of anomeric configuration of the product glycoside reveals the stereochemistry of the enzyme reaction.53 If another sugar molecule is used as acceptor there will be a number of possible positions of linkage in the resultant disaccharide.R.I.C. Reviews 50 Wallenfels et aZ.s4 made the interesting observation that for transfer to glucose by ,B-galactosidase the disaccharide produced in greatest amount is that which is hydrolysed most readily, and it was concluded from this that the aglycone and the acceptor occupy the same position on the enzyme molecule. It has been rec0gnized~55~ for a long time that transglycosylation provides a means of preparing oligosaccharides which is often superior to available chemical methods of synthesis. Thus the action of Aspergillus niger a-glucosi- dase on glucose led to the first isolation of kojibiose (a-1,2-link).56 Further recent applications using different donor and acceptor molecules with extracts of yeast57 and of Tetrahymena pyriformi~~~ have afforded a number of unusual mixed disaccharides.A study of the transglycosylation reactions of fysozyme by Sharon and others59 has provided information on the mode and energies of binding of substrates to the active region of the enzyme. Examination of the mixture of higher oligosaccharides formed by the action of lysozyme on a tritium- labelled tetrasaccharide revealed that transglycosylation to give higher oligosaccharides plays a vital part in the overall process of hydrolysis of tetrasaccharide to disaccharide. The tree-like structure of glycogen, in which non-reducing end groups comprise about 10 per cent of all units,60 was elucidated largely by the use of degradative enzymes.The major mode of degradation in vivo is the stepwise cleavage of glucosyl units from the non-reducing ends by phosphorolytic transglycosylation to give a-D-g~ucosyl phosphate. However this process is halted in the vicinity of the branch points, and a phosphorylase-resistant ‘limit dextrin’ results, which has residual chains of four glucose units attached at the branch points.61 Further degradation is made possible by the action of a debranching enzyme, first isolated from rabbit muscle,62 which (i) transfers an a-maltotriosyl fragment to another part of the molecule61 and (ii) catalyses the hydrolysis of the 1,6-linkage to the remaining residue.Attempts to separate the transferase and amylo- 1,6-glucosidase activities have so far been U ~ S U C C ~ S S ~ U ~ , ~ ~ ~ and it has been suggested that the two functions may be carried out by the same enzyme. However the discovery636 of a glycogen storage disease type IIID, in which transferase activity is missing but glucosi- dase activity is present, is in conflict with this conclusion. For the degradation of a polysaccharide three possible action patterns may be envisaged: (i) single chain, in which all the linkages are broken in one chain before the enzyme forms an active complex with another polymer molecule; (ii) multiple chain, in which only one link is broken per effective enzyme-substrate encounter ; and (iii) multiple atta~k,~4 a general case in which the enzyme catalyses the hydrolysis of several but not all bonds in a chain before dissociating from it.One may define the degree of multiple attack as the number of bonds broken per single enzyme-substrate encounter. The situation is complicated by the fact that some enzymes catalyse the cleavage of glycosidic linkages stepwise from the non-reducing end of a polysaccharide chain (exo-enzymes), while others do so at the interior of a chain (endo-enzymes). One of the first enzymes to be examined was the exo- enzyme p-amylase, and a number of workers proposed a single chain action pattern. This view was criticized by Bourne and Whelan65 who held that the Bourne and Finch 51 experimental evidence was more in accord with a multichain process.The question was resolved seven years later in favour of a multiple attack mecha- nism of degree 4.3 for a short chain amylose containing 44 units.66 For an amylose of higher molecular weight but of narrow molecular weight distribution Husemann and Pfannemuller67 observed a decrease in molecular weight during IS-amylolysis, thus confirming a multichain or multiple attack mode of action. French64 has pointed out that single and multichain patterns may be thought of as opposite extreme cases of multiple attack, and suggested that the degree of multiple attack depends on the relative values of the rate of cleavage of glycosidic linkages and the rate of dissociation of the enzyme- polysaccharide complex.The studies have been extended recently to endo- enzymes by Robyt and French68 who have examined the action of three a-amylases and of 1M sulphuric acid on recrystallized amylose of degree of polymerization 1000 & 50. The degrees of multiple attack for the three a-amylases were 7.0 (porcine pancreatic at pH 6.9), 3.0 (human salivary at pH 6.9), and 2.9 (Aspergillus oryzae at pH 5.5). An unexpected result was the value of 1.9 for 1M sulphuric acid, which was expected to give a value corresponding to purely random attack, i.e. 1 .O. The related problem of the ‘mode of action’ of enzymes which catalyse polysaccharide synthesis has been explored using starch phosphorylase,65~69 levansucrase70~71 and dextrans~crase~l-73 but the results obtained so far are conflicting, even for the same enzyme.This may be attributed, at least in part, to the experimental difficulties involved in the characterization of the synthesis products as reaction proceeds, and also to the possible controlling influence of external factors such as polymer solubility or crystallizability. MECHANISMS OF ACTION OF GLYCOSYL TRANSFER ENZYMES The ultimate goals of mechanistic interpretations of enzyme action must be to explain in chemical terms the enormous catalytic power and subtle selec- tivity of enzymes towards substrates and products. A particular requirement of any mechanism proposed for transglycosylation is that the stereochemistry of the enzyme-catalysed reaction must be accounted for.All transglycosylases which have been examined appear to operate with rigid stereochemistry (inversion or retention of configuration) with respect to the anomeric carbon of the donated sugar unit; some examples are given in Table 4. In common with other classes of enzymes, transglycosylases bring about a reduction of the free energy of activation for the reaction catalysed.79 This is generally thought to occur by the involvement of one or more functional groups at the enzyme active site which lowers the potential energy of activa- tion AH$. The concomitant increase in kinetic energy of activation --TASZ, due to participation of the extra groups, may be offset by the precise orienta- tion of bonds undergoing reaction with respect to the functional group(s).** However, opinions differ as to whether the rates of enzyme-catalysed reactions can be accounted for quantitatively in these t e r m ~ .~ ~ ~ ~ ~ ~ 8 ~ A frequently used qualitative approach is to identify functional groups at the enzyme active site, and then to postulate how these groups might be involved in the enzyme reaction. The postulations are usually made on the basis of the known chemistry of the types of substrate and reaction under consideration. Such a R.I.C. Reviews 52 j?-Amy lase a- Amy I ase Phosphor y i ase Inversion Retention Retention , ' f+rr Table 4. Stereochemistry of some transglycosylation reactions Enzyme I ,3-Giucanase Celiulase L y soz y m e Dextransucrase Levansucrase Q-enzyme UDPG/starch synthetase U DPG/cellulose synthetase 10 15 procedure can be quite successful, particularly if a stable enzyme-substrate intermediate is formed which can be characterized, as in the case of some protein hydrcllases.81 Unfortunately, this is not normally the case with glycolytic enzymes and mechanistic interpretations have so far been based on more indirect information, and in most cases must be regarded as specula- tive. Non-enzymic hydrolysis of glycosides is known to proceed by a variety of mechanisms depending on the nature of the substrate and on the reaction conditions. The hydrolysis and alcoholysis of glycosides derived from sugars and simple alcohols proceeds via an acid-catalysed mechanism, which is usually formulated as :83 20 Stereochemistry Inversion Retention Retention Retention Retention Retention Retention Inversion -H+ NH Ref. 74,75 75 76 77 78 79 7 8 ~ H, ' 0 'H Glycosides of phenols (which are often used as enzyme substrates, since the product phenol may be estimated spectrophotometrically) may also be hydrolysed via base-catalysed processes.Three types of mechanism have been proposed which involve ( i ) nucleophilic (anion) attack at the anomeric carbon OH Bourne aid Finch (5) 53 atom with the formation of anhydro-compounds ;84 (ii) nucleophilic substitu- tion at the aromatic carbon atom involved in the glycosidic linkage;85~~~ and (iii) ionic di~sociation.8~ The relative rates of acid- and alkali-catalysed hydrolyses of a series of substituted phenyl-a- and ,8-D-glucosides were measured by Rydon and co-workers,85Jj6 and under alkaline conditions both a- and ,8-glucosides exhibited positive Hammett p-values, while in acid zero (a-glucosides) and small negative (p-glucosides) values were obtained.When these studies were carried out using a ,8-glucosidase85 and an ~glucosidase~~ positive p-values were obtained in both cases when the substituent a-values were correlated with an enzyme-substrate affinity constant. Although the interpretation of substituent effects is hampered by the difficulty of assessing possible steric contributions, the results certainly suggest that basic or nucleophilic catalysis is involved in some way. The effect of the structure of the glycose residue on non-enzymic hydrolysis has been studied by a number of workers.A particularly pertinent case is that of the substituted phenyl-2-acetamido-2-deoxy-~-glucopyranosides which have been studied by Bruice and co-workers.88 Following the sugges- tions of Capon89 and of Inch and Fletcher,go the results were interpreted in terms of intramolecular general acid catalysis by a carboxyl substituent in the phenyl ring and intramolecular nucleophilic catalysis by a neutral 2-acetamido group. Lowe et aE.91 measured the rates of lysozyme-catalysed hydrolysis of several p-aryf-N-acetyl-chitobiosides and analysed the tesults in terms of the Michaelis-Menteng2 equation. It was found that the Michaelis constant Km was independent of phenyl substituents but that the catalytic constant kcat showed a marked dependence, with p equal to + 1.2.It would seem that the action of lysozyme involves nucleophilic or basic participation in some way. With few exceptions,85@ all the mechanisms proposed for non-enzymic hydrolysis of glycosides involve glycosyl-oxygen fission. This has been observed experimentally for reactions catalysed by acid,93 and also by the enzymes sucrose phosphorylase, 94 yeast a-glucosidase,g5 almond ,8-gluco- sidase,95 invertase, 96 takamaltase, 97 ,&amylase, 98-100 a-amylase,100-101 fl-glucuronidase102 and lys0zyrne.7~ The acid-catalysed hydrolysis of methyl a-D-glucopyranoside is about twice as fast in deuterium oxide as in water, and this can be explained in terms of the mechanism shown in (5).lo3 However several enzymic hydrolyses proceed more slowly in deuterium oxide,104 and it has been argued105 that a different mechanism must be in operation.An alternative explanation106 is that heavy water may adversely affect the conformation of enzymes in solution. Consideration of the various mechanisms proposed for glycoside hydrolysis reveals that the rate determining step is that which involves the loss of the aglycone moiety. Thus one may write a general reaction sequence for an enzyme-catalysed hydrolysis which embodies all the features of the mecha- nisms proposed : E + S + ES 4 E.Glycose + Aglycone a__). H*O fast slow E + Glycose + Aglycone (6) R.I.C. Reviews 54 Some or all of the individual steps may be catalysed by acidic, basic, or nucleophilic groups at the enzyme active site and the enzyme complex E.Glycose could be ionic or covalent in nature.One would not expect to be able to isolate the enzyme substrate complex in the presence of acceptor, and as far as the authors are aware there have been only two reports of the isolation of glycolytic enzyme-substrate intermediates. Silverstein et aZ.107 have submitted evidence for the formation of a complex of sucrose phos- phorylase with glucose on reaction of sucrose with this enzyme, and the kinetic data were consistent with a double displacement mechanism originally proposed by Doudoroff et a1.108 Leglerl O9 has reported the isolation of a radioactive p-glucosidase complex after reaction with 14C-labelled conduritol B epoxide (2,3-anhydro-myo- inositol). The radioactivity could be released as (+)-inositol by treatment with 0.05M hydroxylamine, but not by treatment with sodium carbonate/hydrogen carbonate buffer of the same pH.It was suggested that complex formation occurred by acid-catalysed trans opening of the epoxide ring via attack of an enzyme carboxylate anion to give an ester of (+)-inositol, which released inositol when treated with hydroxylamine. However the operation of the same process during the hydrolysis of p-glucosides would presumably lead to a-glucose, which is in conflict with experimental evidence that the action of /I-glucosidase proceeds with retention of configuration.l1° It is possible that the stability of the inositol-enzyme intermediate may be due to the presence of a cyclohexane rather than a pyranosyl ring in the substrate, and it will be interesting to see if this type of experiment can be extended to other glycosi- dases. It has been postulated that transglycosylation proceeding with inversion occurs by a single-step nucleophilic .displacement. Products with retained configuration are supposed to arise by two successive displacements, the first of donor aglycone by an enzyme functional group, the second of the enzyme functional group by the acceptor.ll1 This latter explanation was proposed as the mechanism of sucrose phosphorolysis, and strong evidence was pro- vided by the observation of an exchange reaction between a-D-glUCOSyl phosphate and H32POz- catalysed by sucrose phosphorylase in the absence of fructose :I08 sucrose phosphorylase -..A a-D-glucose-1 -phosphate , postulated glucosyl enzyme + phosphate (7) This double inversion hypothesis has been extended to cover retention during polysaccharide synthesis.lf2 However, phosphate exchange does not occur in the case of polysaccharide phosphorylase unless the acceptor (glycogen or starch) is present.113 Lack of exchange may be explained in terms of Kosh- land’s induced fit theory by postulating that the enzyme active site adopts the correct configuration only in the presence of acceptor.48 Retention of configuration has been rationalized in two other ways: ( i ) formation of a carbonium ion followed by an addition whose stereo- specificity is controlled by the enzyme;ll4 or (ii) the operation of an SNi process.115 However it is difficult to explain lack of exchange in terms of Bourne and Finch 55 E + P E + S HOCHZ H \ ke Fig.I . Proposed mechanism of action of lysozyme. these last two theories. Present research in the field of polysaccharide bio- synthesis is largely concentrated on the isolation of donors and enzymes from a wide variety of sources, but it appears that substantial progress would result from the detailed examination of one or two systems. The x-ray crystallographic studies of lysozyme and of its complex with N-acetyl-chitotriose45s46 have provided a basis for further investigations of the mechanism of action of this enzyme.Under this stimulus, detailed studies of the enzymic and non-enzymic hydrolysis of glycosides of various mono- and oligosaccharides structurally related to the natural substrates have been carried out. As a result the possible mechanisms have been narrowed down to a small number of alternatives, although within these limitations some uncertainties and anomalies still remain. A large amount of experimental data45~46@~919116 supports the assignment of an enzyme-catalysed pathway involving general acid catalysis by the carboxyl group of glutamic acid 35 and intramolecular nucleophilic catalysis by the substrate 2-deoxy-2-acetamido group as represented in Fig. 1. The mechanism proposed postulates a double inversion at the substrate anomeric carbon atom, leading to retention of con- figuration, as has been 0bserved.7~ However the mechanism is not yet established with the certainty which this brief description might suggest, since the importance of intramolecular acetamido group participation is challenged by other experimental evidence.Raftery and Rand-Meir reported117 that, in the presence of the tetra- saccharide chitotetraose, lysozyme will catalyse the release of p-nitrophenol from p-nitrophenyl-/3-glycosides of N-acetyl-D-glucosamine, D-glUCOSe, and 2-deoxy-~-glucose. The relative rates of release of p-nitrophenol were 2 : 1 : 16 respectively, and it was shown that release ofp-nitrophenol occurred via synthesis of p-nitrophenyl oligosaccharides by transglycosylation.Thus it was proposed that acetamido group participation is not necessary to explain catalysis, and that the alternative pathways involve (i) a carbonium ion possibly stabilized by an enzyme basic group, e.g. aspartic acid 52,469118 or (ii) a covalent intermediate resulting from participation by an enzyme basic group. The steric distortion of the substrate proposed by Phillips and co- workerslls would presumably facilitate the production of a carbonium ion but not necessarily that of a covalent intermediate. It may be noted that the rate of acid-catalysed hydrolysis of methyl-2-deoxy-~-~-glucoside is ca 103 R. I. C. Reviews 56 times that of methyl-~-~-glucoside,~~~ and this has been ascribed to the greater ease of production of a carbonium ion (due to both electronic and steric effects).CONCLUSIONS In this brief survey we have attempted to demonstrate the extreme variety which has been encountered in the biochemical pathways of polysaccharide synthesis and degradation, while emphasizing how the majority of these pathways are unified by the concept of glycosyl transfer. It may be concluded that further progress towards the elucidation of the details of such pathways will be facilitated by the multitude of mild separation and purification procedures now available, coupled with the use of radioactively labelled enzyme substrates. As far as the mechanisms of the enzymically-catafysed reactions are concerned the situation is rather more uncertain. It would appear that further significant progress will require the detection and study of individual reaction steps and intermediates, perhaps by application of the rapid reaction techniques which have already been used with considerable success with a number of enzymes.12* Another area in which further informa- tion is desirable is in the non-enzymic catalysis of glycosyl cleavage reactions.A number of functional groups have been proposed as participants in enzy- mically catalysed transglycosylations but, apart from hydrogen ion, experi- mental data concerning the reactivity of such groups towards glycosides is extremely fragmentary. REFERENCES 1 See, for example, (a) D. A. Rees, The shapes of molecules. London: Oliver and Boyd, 2 See, for example, Industrialgums, ed.R. L. Whistler. New York and London: Academic 3 (a) D. A. Rees and J. Skerrett, Carbohydrate Res., 1968,7,334; (b) G. N. Ramachandran 1967; (b) M. Stacey, Chem. Brit., 1970, 6, 113 Press, 1959. 87. 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ISSN:0035-8940    

DOI:10.1039/RR9700300045

出版商:RSC    

年代:1970

数据来源: RSC

摘要:

ELECTRON RESONANCE IN ANISOTROPIC SOLVENTS . . . . . . . . 63 . . . . . . 66 * . . . . . 68 . . . . 75 Meldola medal lecture G. R. Luckhurst, B.Sc., Ph.D. , . . . * . Dept of Chemistry, Tbe University, Southampton SO9 5NH * . Liquid crystals The anisotropic spectrum . . . . . . . . Examples . . .. . . . . .. . . The sign of the isotropic coupling constant, 68 The sign of the spin density, 71 Radical geometry, 72 The g-factor shift . . Radicals with more than one unpaired electron Conclusion . . Acknowledgments . . . . .. . . 74 * . . I . . .. . . 83 . . . . 83 .. . . .. . . .. . . . . . . . . . . . . . . 1 . References . . . . . . . . . . . . . . .. . . 83 Any filled molecular or atomic orbital contains two electrons which have exactly the same energy, but opposite spins.When there is only one electron in an orbital, this electron can be in either of the two spin states which are equal in energy. States of equal energy are said to be degenerate. In the presence of a magnetic field, the degeneracy of the two spin states of an unpaired electron is removed-the state in which the electron spin is anti- parallel to the applied magnetic field decreases in energy and becomes favoured over the state in which the electron spin is parallel to the field. The simplest electron resonance experiment consists of inducing transitions between these two spin levels, the resulting absorption of energy being observed as a single line in the spectrum. When the unpaired electron interacts with nuclei which also possess spin the electron resonance spectrum contains more than one line.This splitting of the single line is produced by smaller magnetic fields generated by the nuclei either adding to or subtracting from the applied field. If there is just one nucleus, with spin I, then the spectrum has 21 4- 1 equally spaced lines with the same intensity. The spacing between adjacent lines is called the nuclear hyperfine coupling constant. There are two mechanisms responsible for the electron-nuclear coupling. The first is the Fermi-contact term which occurs when the electron has a finite probability of being found at the nucleus. This is only true for electrons in orbitals with some s-character and the interaction is therefore isotropic- the same in all directions.The dipolar interaction between the electron and nuclear spins is responsible for the other coupling which is anisotropic- direction dependent. The Fermi-contact term has no classical analogue while the dipolar coupling is comparable to the interaction between two bar magnets. 1 Measurement of both components of the interaction is important because it leads to the determination of certain features of the molecular structure. Luckhurst 61 The isotropic coupling constant gives the s-character of the molecular orbital containing the unpaired electron, while the anisotropic hyperfine coupling is determined by the average electron-nuclear separation. Provided the radical can be incorporated in a crystalline lattice both components of the hyperfine coupling may be determined.Experimentally the crystal is mounted in the spectrometer and the spacing between the hyperfine lines measured as a function of the relative orientation of the crystal and the magnetic field. But for the majority of organic radicals, it is difficult to grow single crystals which are magnetically dilute and therefore they have only been studied in fluid solution. In a typical solvent, such as benzene, the dipolar or anisotropic hyperfine splitting is averaged to zero by the rapid rotational diffusion of the solute. Analysis of the line positions in a solution electron resonance spectrum yields only the isotropic coupling constants. Indeed, even the sign of this coupling cannot be determined directly. Solution studies therefore result in a consider- able loss of structural information, especially as other magnetic interactions within a radical are also anisotropic.When the molecular motion is slow, details of the anisotropic interactions can be gleaned from the widths of the hyperfine lines.2~3 This technique is not generally applicable and a method of obtaining the anisotropic interactions from the line positions is desirable. A liquid differs from a crystal in two important respects. In a crystal the molecular motion is usually completely quenched, and the molecules are macroscopically ordered. In a liquid the motion is rapid and there is no molecular ordering. Clearly, a series of intermediate states exists between these two extremes.For example, in a polycrystalline sample there is no molecular motion to average the anisotropic interactions, but the molecules are still distributed isotropically. As a result the lines in the electron resonance spectrum are very broad and the components of the hyperfine interaction can only be determined when they are large.4 The technique is not generally applicable. Another state is obtained by retaining the rapid molecular motion which is responsible for the narrow hyperfine lines and removing the isotropic character of the motion. The result of partially aligning the radical is to retain some dependence of the line positions on the anisotropic interactions. An early study of a partially aligned radical in a liquid was made by Ohnishi and McConnell.5 They measured the spectrum of the chloroproma- zine cation bound to DNA while flowing the solution through a narrow tube.The spectrum was found to depend on the orientation of the tube with respect to the magnetic field because the large size of DNA, together with viscous forces, results in partial alignment of the radical. Although the experiment is intriguing the technique is of limited applicability. The problem of partial alignment is encountered in nuclear magnetic reson- ance, where the anisotropic interactions include the nuclear dipolar coupling and the quadrupole coupling.6 If the molecule possesses an electric dipole moment then it can be aligned in an electric field. The extent of the alignment is extremely small, even with the maximum fields attainable, and it is only because the anisotropic interactions are enormous in comparison with their isotropic components that the effects of alignment can be discerned in the ~pectrum.~~ In electron resonance the anisotropic interactions are comparable R.I.C. Reviews 62 to their isotropic components and the alignment resulting from the applica- tion of an electric field would be quite incapable of altering the positions of the lines in the electron resonance spectrum. Fortunately, the use of liquid- crystalline solventsg has provided a more satisfactory and general solution to the problem of aligning solute molecules in magnetic resonance spectroscopy.lOJ1 LIQUID CRYSTALS The tendency for the planes of benzene molecules to lie parallel to one another in the bulk fluid only extends over several molecules.This situation occurs in the majority of liquids and can be described by saying that the local order or angular correlation is small. There is a class of compounds, known as liquid crystals, in which the angular correlation extends over many thousands of molecules. Liquid crystals are usually solid at room temperature and on heating they melt sharply to give the liquid-crystalline phase. Further increase in the temperature reduces the degree of local order until there is a second first-order phase transition to an isotropic fluid. There are three distinct types of thermotropic liquid crystal each characterized by a different mole- cular arrangement in the liquid-crystalline mesophase. In certain cases a compound may adopt more than one molecular arrangement before finally passing to the isotropic phase.The order-disorder phase transition in liquid-crystalline systems is a result of the anisotropy in the intermolecular potential function.l2 Indeed the temperature at which the transition to the isotropic phase occurs increases directly with this anisotropy. Any compound whose molecules deviate from spherical symmetry should exhibit liquid-crystalline behaviour. Obviously, most compounds are not liquid crystals; this is because, on cooling, the isotropic fluid freezes before the transition to the ordered fluid can be reached. Of the three liquid-crystalline forms the nematic mesophase is most commonly used as a solvent in magnetic resonance experiments.Compounds which give a nematic mesophase have essentially rod-like molecules and hence a highly anisotropic intermolecular potential. For example, 4,4'-dimethoxy- azoxybenzene is a yellow solid which melts at 118 "C and has a nematic- isotropic transition point at 135 "C. In the nematic mesophase, between 118 "C and 135 "C, the long axes of the molecules tend to be arranged parallel to one another as in Fig. 1. Even though the mesophase is highly ordered the high angular correlation in a given region has only a transitory existence and the rate of molecular motion is comparable to that in a normal liquid. Providing no constraint is applied to the mesophase the regions of high local order are randomly oriented with respect to one another, and the system is still isotropic in the macro- scopic sense. Application of a magnetic field greater than about one kilogauss13 aligns the molecules with their long axes parallel to the direction of the magnetic field.This ordering is a result of the anisotropy in the magnetic susceptibility, enhanced by the high local order, interacting with the magnetic field. A magnetic field is, of course, an integral part of a magnetic resonance experiment, and it is natural to use this to align the liquid-crystalline solvent. Luckhurst 63 5 Fig. I (above). The molecular arrange- ment in a nematic mesophase, e.g. 4,4’- dimethoxyazoxybenzene. Fig. 2 (right). Possible local molecular arrangements in a smectic mesophase, e.g.ethyl 4-azoxy- benzoate. This procedure has slight limitations because the direction of the alignment cannot be varied. However, electric fields can also align the molecules in the mesophase with their long axes parallel to the electric field. When the two fields are in competition an electric field of about 5 kV cm-1 is sufficient to overcome the magnetic field of 3 kG typically found in an X-band electron resonance spectrometer.14J5 The use of an electric field to align the sample does not have the inflexibility of a magnetic field alone. When the attraction between the sides of neighbouring molecules is large the high degree of local order extends in two dimensions;g two possible molecular arrangements are shown in Fig.2. The resulting mesophase is called smectic and is again formed by rod-like molecules. The crystal-Iike structure of the smectic mesophase implies a high viscosity and the molecular motion is very inhibited. As a result, molecules in the mesophase are not aligned by magnetic fields, although an ordered state is said to be obtained on cooling the isotropic phase below the smectic-isotropic transition point in the presence of a magnetic field.16J7 Alternatively, if the compound, for example 4,4’-di-n-heptyloxyazoxybenzene, passes through a nematic phase before reaching the smectic phase, the nematic phase can be aligned by a magnetic field and this alignment is frozen into the sample on passing into the smectic mesophase.18 The technique is valuable because the direction of R.I.C.Reviews 64 Me Me I Me Me 0 Pr \C-0 // s Fig. 3. The helical structure of the cholesteric mesophase, e.g. cholesteryl propionate. alignment, with respect to the magnetic field, can then be altered simply by rotating the sample tube. The third type of thermotropic liquid crystal, known as cholesteric, is formed by esters of cholesterol but not by cholesterol itself, which does not exhi bit any liquid-crystalline properties. The molecules in the cholesteric phase are arranged with their long axes parallel as in a nematic mesophase. However, on passing from one molecular layer to the next there is a contin- uous and constant change in the direction of alignment.The resulting helical arrangement (Fig. 3) is responsible for the unusual and technologically important optical properties of the cholesteric mesophase. The factors respon- sible for the helix are the asymmetric centres in the cholesteryl esters. Indeed, if the nematogen has an asymmetric centre, both the d- and the I-enantio- morphs are found to be cholesteric,lg while the racemate is nematic. The helical arrangement in the ordered regions reduces the anisotropy in the diamagnet- Luckhurst 65 50 ism and so a magnetic field of several kilogauss is unable to align the choles- teric mesophase. However, at high magnetic fields the helix is unwound to form a nematic mesophase which is then aligned. If two cholesteric liquid crystals with opposite rotations are combined to form an optically inactive mixture, then this is nematic and aligned by a magnetic field.20 Such mixtures have been used to orient solute molecules in nuclear magnetic resonance, but not in electron resonance.The addition of a solute to a nematic liquid crystal must affect the properties of the mesophase. In fact, the addition depresses the nematic-isotropic transition point, often below the melting point because the entropy changes at the two transition points are quite different. Although the range of the mesophase may be reduced by the presence of the solute the ordering proper- ties of the solution are comparable to those of the pure solvent.21 The solute will also be aligned on application of a magnetic field, not because of direct interaction of its anisotropic diamagnetic susceptibility with the magnetic field but because of interaction with the anisotropic potential generated by the macroscopically ordered solvent.The sense of the solute-solvent inter- action can often be inferred from the shape of the solute molecule and its preferred orientation can then be deduced. For example, a rod-like solvent will orient a planar solute with its plane parallel to the long axis of the rod, and hence to the direction of the magnetic field. THE ANISOTROPIC SPECTRUM The electron resonance spectrum of a partially aligned radical, which will be called the anisotropic spectrum, can only be interpreted fully if the spin hamiltonian is known. The most elegant derivation of this spin hamiltonian is obtained by writing the hamiltonian in terms of irreducible tensor operators and transforming it under rotation with Wigner rotation r n a t r i ~ e s .~ ~ ~ ~ The resulting equations can be rationalized using arguments which emphasize the physics of the problem. Consideration of a specific example simplifies the arguments still further. The electron resonance spectrum of di-t-butylnitroxide, whose structure is shown in Fig. 4, consists of three equally-intense lines caused by the interaction of the unpaired electron with the nitrogen nucleus (I = 1). Provided the radical tumbles rapidly in solution the separation between adjacent hyperfine lines is the isotropic nitrogen coupling constant a ( N ) .If the radical is fixed in a crystal with the magnetic field along the 3 axis, shown in Fig. 4, the spacing between the lines is now the sum of the isotropic splitting and the anisotropic coupfing, splitting is found to be a ( N ) plus A’(:) and an identical spacing of a ( N ) + A’\:) appropriate for this axis. Similarly, when the field is along axis 2 the is found when the field is along axis I (i.e. A’(;) = A’(:)). Such a situation is R. I. C. Reviews 66 described by saying that the anisotropic hyperfine interaction has cylindrical symmetry about axis 3. In a nematic mesophase di-t-butylnitroxide will tend to be aligned with the 1-2 molecular plane parallel to the direction of the would be a ( N ) + A'(:). However, the molecular motion prevents the align- magnetic field.The anisotropic coupling constant for complete alignment ment from ever reaching completion and the observed spacing is a(N) plus some fraction of the anisotropic coupling A'(:). The value of this weighting fraction is readily determined. If the magnetic field makes an arbitrary angle with the molecular axis system, the spacing between the hyperfine lines is where l3 is the cosine of the angle between the symmetry axis 3 and the magnetic field. When the radical moves rapidly from one orientation to another the observed splitting is obtained by averaging equation 1 over all orienta- - tions. In a nematic mesophase the anisotropic nitrogen coupling constant, a ( N ) , is where the ensemble average is denoted by the bar.It is convenient to denote the weighting fraction by the symbol 03, where 3 occurs twice in the subscript because it is found in both direction cosines, I,. Although the average p3 is a measure of the alignment of the 3 molecular axis with respect to the mag- netic field, @,, is a more logical indication of the degree of alignment. For example, when the motion is isotropic all values of l3 are equally probable and so 03, is zero. If axis 3 is parallel to the magnetic field 033 is unity. The values of Oil and 02, are given by expressions analogous to that for C33. It is also possible to define quantities which involve the direction cosines of different axes and in general The 0 values can be arranged in a square array which, not unexpectedly, is called the ordering matrix.24 The properties of the direction cosines mean that the sum of the diagonal elements of the matrix (its trace) is zero.The ordering matrix notation may be included in equation 2 to give This can then be written in a more symmetric form25 by using the traceless property of the anisotropic hyperfine interaction, Luckhurst 67 Fig. 5. The structure and axis system for d i pheny Initroxide. Equation 5 has been derived for a cylindrically symmetric hyperfine tensor. In general the shift in the coupling constant 6 - a is given by (7) i,i 6a = 3 C Aij 8ij where i, j , . . . represent molecular axes.25 When the radical is cylindrically symmetric, as for example perinaphthenyl, the alignment of any axes set in the molecular plane will be equal.The elements of the ordering matrix el1 and 8,, will also be equal and because the matrix is traceless 011 == 822 = - 83312 (8) Substitution of this result into equation 7 gives an equation of the same form as 5, (9) EXAMPLES 6a = A& 833 The coupling constant shift, 6a, is obtained experimentally by measuring the spectrum of the radical dissolved in the liquid crystal both above and below the nematic-isotropic transition point. As we shall see this shift can then be used to provide information about the anisotropic hyperfine ten~or.269~7 Alternatively, if the components of A’ are known then the elements of the ordering matrix can be determined from the shift. This is important because the form of the anisotropic solute-solvent intermolecular poten- tia1,28>’9 which determines the degree of alignment, can be obtained from 0.Indeed, it is also possible to investigate the properties of the pure liquid crystal given the ordering matrix for the solute.29y30 We now illustrate the structural information which can be obtained from the coupling constant shifts with three examples. Tlie sign of the isotropic coupling constant The first example is diphenylnitroxide, whose structure and molecular axis system are shown in Fig. 5. The isotropic spectrum of the radical dissolved in 4,4’-dimethoxyazoxybenzene has three groups of lines : the spacing between these is the nitrogen coupling constant of 28.17 MHz. Below 135°C the solvent exists in its nematic mesophase and there is a dramatic change23 in the electron resonance spectrum (Fig.6). The nitrogen coupling constant decreases to 18.18 MHz, demonstrating quite clearly the partial alignment of the radical. The anisotropic spectrum also exhibits a linewidth variation, as shown by the digerent heights of the first derivative line shapes illustrated R.I.C. Reviews 68 10 gauss Fig. 6. The isotropic and anisotropic electron resonance spectra of diphenylnitroxide dissolved in 4,4'-d imet hoxyazoxybenzene. in Fig. 6. The differences in the linewidth contain important information concerning the dynamic structure of the mesophase, but we shall not explore this aspect here.31~32 Although the separation between the hyperfine lines gives the magnitude of the nitrogen coupling constant it does not yield its sign.This information can be obtained from the nitrogen shift adN) but Luckhurst 69 6 Fig. 7. The axis system for the X-Y fragment. Y before this can be done we must look into the theoretical expressions for the anisotropic hyperfine tensor. We shall only discuss n-radicals in this article and expressions for the anisotropic hyperfine tensors in such systems have been derived by McConnell and Strathdee.33 They calculated the hyperfine tensor resulting from the dipolar interaction between a nucleus Y and an electron contained in a Slater 2pz orbital on a nucleus X. The axis system for the fragment X-Y is given in Fig. 7. The principal components of the tensor are where p is the spin density on atom X.The quantities P and Q are related to the internuclear separation Y and the effective nuclear charge Z for atom X by In these equations yy is the magnetogyric ratio for nucleus Y and a is Zr/1.058. These results can also be used to estimate the hyperfine tensor for Y when the electron is in a 2pz orbital on Y by taking the limit of equation 10 as r tends to zero. The resulting components of the tensor are The spin density on the nitrogen atom in diphenylnitroxide is predicted to be both large and positive by all molecular orbital calculations. According to equation 13 the nitrogen hyperfine tensor will approximate closely to cylindrical symmetry about the’3 axis normal to the molecular plane and so the nitrogen shift will be given by equation 5: The tensor component A’$;) is positive because of the positive spin density on the nitrogen and so to calculate the sign of the shift we need to know the sign of 033.Since diphenylnitroxide is essentially planar the 3 axis will tend R.I.C. Reviews 70 to be aligned perpendicular to the direction of the magnetic field. The average value of 1; will tend to be small, O,, will be negative, and the nitrogen shift constant the must therefore is found be to negative. be larger Experimentally than the anisotropic isotropic coupling; nitrogen ldN)I > coupling v[. Since both splittings have the same sign the experimental shift, a ( N ) - a(N) can only be negative if the isotropic splitting is positive.The liquid crystal experiment therefore confirms the theoretical prediction of a positive nitrogen coupling constant. Before leaving this example it is appropriate to comment on the assertion that the isotropic and anisotropic coupling constants have the same sign. The experimental values of a and are usually found to be comparable and in order for the coupling to have changed sign the shift would need to be twice the isotropic splitting. Theoretically the shift is C A;j oij and even for a i, i completely aligned radical this sum is unlikely to be 2a. For the remainder of the article the coupling constant is assumed to retain its sign on partial alignment. The sign of the spin density The isotropic proton coupling constants in an aromatic radical are propor- tional to the mpin density on the adjacent carbon atom: (14) The proportionality constant, Q, in the McConnell relationship, which provides a useful method for testing r-molecular wave-functions, is equal to about -80 MHz for a neutral radical.Because molecular orbital theory can calculate the absolute magnitude of the spin density it is important to be able to measure this quantity in order to provide a complete test. However, as the absolute magnitudes of the coupling constants are not obtained from the isotropic spectrum, equation 14 cannot be used to determine the signs of the spin densities. As we shall see measurement of the anisotropic spectrum can lead to a sign determination. a(i) = Qp(t) The first such determination was made for the perinaphthenyl radical whose spectrum was measured above and below the nematic-isotropic transition point of 4,4’-dimethoxyazoxybenzene.35 The structure of the radical and its axis system are shown in Fig.8. Both spectra are readily analysed in terms of a large septet splitting from the six equivalent /3-protons, and a smaller quartet splitting from the three equivalent a-protons. The isotropic coupling constants are 5.10 MHz for the a-protons and 17.6 MHz for the protons whereas the corresponding anisotropic values are 5.75 MHz and 17.6 MHz. Only Fig. 8. The structure of the perinaphthenyl radical. P a Luckhurst 71 the a-protons exhibit a significant shift, 6 a ( a ) , on alignment. The ordering matrix for perinaphthenyl must be cylindrically symmetric because of the circular shape of the radical.The proton shifts are therefore given by equation 9 6a = A;, O,, where 3, the symmetry axis, is perpendicular to the molecular plane. The sign determination is based on calculating values for both sides of this equation. On the one hand 6a can be measured from the isotropic and anisotropic spectra and on the other hand we can calculate the hyperfine tensor A’ and estimate the degree of alignment 8. In order to determine the sign of the spin density both sides of the equation are calculated for either choice of sign. We then seek to eliminate one of these by seeing if the signs obtained for 6a and Aj.3 03, are opposite. Clearly, if both 6a and A;, 0 3 3 have the same dependence on the spin density this procedure must fail.Fortunately, the sign of the isotropic proton coupling constant, and hence 6a, depends only on the spin density at the adjacent carbon atom, whereas the anisotropic proton hyperfine tensor is determined by a sum of contri- butions from all the spin density in the radical. In practice the technique works in the following way. If p(a) is positive the isotropic coupling constant for the a-protons will be negative, and so the shift will also be negative since ~p[> la(a)l. The /?-proton splitting of 17.6 MHz ensures that the spin density p(P) must be positive. Calculation of the hyperfine tensor element, A;:), with positive spin density at both a and /3 positions gives a value of -2.76 MHz.As for diphenylnitroxide the element 0,, of the ordering matrix will be negative and so the term A’s“) 03, is found to be positive. This result is inconsistent with the sign of 8a@) and so the a spin density cannot be positive. Clearly the choice of a negative sign should be consistent with the a shift. If p ( ~ ) is negative then the isotropic splitting is positive and the shift is also positive. The tensor element A’%\ calculated with p(a) negative and p(B) positive is still negative, although equal to -2.17 MHz. The term A’$) 8,, is positive and agrees with the sign of 8 a ( a ) . The a spin density is quite clearly negative in agreement with self-consistent-field molecular-orbital calcula- tions.36 in 4,4‘-dimethoxyazoxybenzene,23 Radical geometry The use of liquid-crystalline solvents in nuclear magnetic resonance has been valuable for measuring the bond lengths and bond angles of the solute.6,7 The method is capable of high accuracy because the anisotropic nuclear dipolar coupling can be calculated exactly from the internuclear separation.In principle a similar analysis should be possible in electron resonance. In practice the inaccuracy of the 2pz wave-function used in calculating the hyper- fine tensor limits the use of the coupling constant shift in determining the geometry of the radical. However, an indication of the potential of liquid- crystal experiments is given by a study of the triphenylmethyl radical dissolved As usual the spectrum was measured in both phases but in this case the R.I . C. Reviews 72 Table. Temperature variation of the coupling constants in the triphenyl- methyl radical Coupling constant (MHz) Temperature ("C) meta ortho para I25 (isotropic) I20 3.187 7.18 7.78 3.310 7.10 7.66 3.335 7.08 7.64 3.372 7.08 7.64 3.391 7.09 7.65 3.422 7.11 7.67 3.488 7.13 7.69 I I 5 I10 I05 I 00 95 anisotropic spectrum and hence the proton shifts were determined as a func- tion of temperature. The radical possesses cylindrical symmetry so the anisotropic proton coupling constants are given by equation 9 : ii = a -+ A;, O,, where axis 3 is perpendicular to the molecular plane. The anisotropic coupling will either increase or decrease with decreasing temperature, depending on the relative signs of a and A;, 03,, because the degree of alignment increases at lower temperatures.For the majority of solutes the temperature dependence of 0 is found to be in qualitative but not quantitative agreement with theory.29 The results for triphenylmethyl, given in the Table, are not in complete agreement with those of other solutes. The anisotropic meta splitting does increase with decreasing temperature, whereas both the ortho and para splittings first decrease, pass through a minimum and then increase. The structure of triphenylmethyl is thought to be like a symmetrically pitched propeller with the phenyl groups inclined at an angle of about 25" to the horizontal plane. In the nematic mesophase the radical is subjected to a highly anisotropic potential whose magnitude increases with decreasing temperat~re.~g As the anisotropy increases we might expect the radical to be forced into a more planar configuration.This squashing of the molecule is thought to be responsible for the unusual temperature dependence of the anisotropic proton coupling constants. Small distortions of tetramethylsilane and neopentane dissolved in 4,4'-di-n-hexyloxyazoxybenzene have already been observed by nuclear magnetic resonance. The increasing planarity of the radical will effect a" by changing both the isotropic coupling a and the tensor component A;,. According to molecular orbital theory the spin density at the ortho and para positions will increase while that at the meta position becomes more negative as the radical tends to a planar conformation.23 The three isotropic coupling constants will therefore increase in magnitude. The change in both the spin distribution and the geometry will also be reflected in Aj303, because of modifications in A j 3 .Although quantitative calculation of the hyperfine tensor is unreliable22J3 i t is certain that 4, is negative for all three protons. Since the element 03, of the ordering matrix is also negative the anisotropic contribution to ii is Luckhurst 73 positive. In contrast the isotropic contribution a is negative for the ortho and para protons but positive for the meta. Thus, the anisotropic meta coupling constant is a sum of two positive terms both of which should increase with decreasing temperature.On the other hand, a' for the ortho and para protons is a sum of two terms with opposite signs. As the magnitude of both terms increase with decreasing temperature, the anisotropic coupling constant might well pass through a minimum. The observed trends are therefore in accord with a model based on molecular deformation by the anisotropic potential of the liquid crystal. THE G-FACTOR SHIFT The value of the magnetic field HO at which the centre of an electron resonance spectrum occurs, is determined by the g-factor for the radical. If the frequency of the microwaves produced by the klystron in the spectrometer is V , gP (16) hv ffo = -- where h is Planck's constant, and ,8 is the electron Bohr magneton.For a free electron which has no orbital angular momentum the g-factor is 2.0023 19 and isotropic. The orbital angular momentum is largely quenched in the majority of organic radicals, although some is retained because of spin-orbit coupling. As a result the g-factor exhibits a small departure from the free- spin value. Furthermore, different molecular axes in the radical have different g-factors. The components of this anisotropic g-tensor constitute another source of structural information, but, like the hyperfine tensor, cannot usually be determined for organic radicals. The use of liquid-crystalline solvents changes this s i t ~ a t i o n . ~ ~ ~ 2 3 ~ ~ 8 W e n radicals are aligned, the centre of the anisotropic spectrum will depend on the total g-tensor. The value of g determined from equation 15 is now the average g which is related to the components of the anisotropic tensor g' by g = g + C g,>Bij i,i This relationship is analogous to the equation for the hyperfine shift and similarly reduces to Sg = g;3 033 (17) when either the g-tensor or the ordering matrix possesses cylindrical symmetry about the 3 axis.The sign of the g-shift can be determined directly from experi- ment because the g-factor is always positive. Provided the components of the g-tensor are known then the sign of the ordering matrix can be derived without making any assumptions. This derivation is important because intuitive ideas about the direction of the preferred orientation for the solute can be tested.g-Shifts have been determined for a number of radicals dissolved in various liquid crystals. In general, analysis of the shifts yields information about the anisotropic g-tensor and often all the components of the tensor can be determined. For example,22 the isotropic g-factor of perinaphthenyl dissolved R. I.C. Reviews 74 in 4,4’-dimethoxyazoxybenzene is 2.00261 and at 100°C the g-shift is 10.9 x 10-5. Both g and 0 are cylindrically symmetric because of the circular shape of the radical. The component gj, of the anisotropic g-tensor could be determined from the g-shift if the degree of alignment O,, was known. Although 03$ can be obtained from the shift for the a-protons the possible inaccuracies in the theoretical proton hyperfine tensor makes the procedure unreliable.Fortunately the 13C hyperfine tensors can be calculated more precisely; measurement of the 13C shifts for perinaphthenyl22 gives a value for 03, of -0.31 at 100°C. The value of g;3 is therefore -35.3 + 10-5 and the component of the g-tensor perpendicular to the molecular plane is 2.00226. Theoretically the in-plane components of the g-tensor are expected to vary from one radical to the next, whereas the component perpendicular to the plane is predicted to be independent of the radical and to have the free-spin val~e.~9740 Clearly the result for perinaphthenyl is in good but not complete agreement with theory. RADICALS WITH MORE THAN ONE UNPAIRED ELECTRON Until now we have been concerned with radicals possessing a single unpaired electron. The presence of a second electron introduces another important magnetic interaction.As well as coupling with the spins of magnetic nuclei the two electron spins can couple with each 0ther.l The discussion of this electron-electron interaction is simplified when the biradical does not contain magnetic nuclei. The interaction has both an isotropic and anisotropic component, like most forms of magnetic coupling. The isotropic coupling, known as the exchange interaction J , separates the four electron-spin orienta- tions into three degenerate levels: which form the triplet state and the singlet state The triplet and singlet states are separated in energy by an amount J. When J is positive the ground state of the system is a diamagnetic singlet, but if J is negative the ground state is a paramagnetic triplet.We shall ignore the singlet state for the moment. The degeneracy of the triplet levels is removed by a magnetic field and electron resonance transitions can be induced between the spin levels.1 The two allowed transitions (Fig. 9) occur at the same field value, and so the electron resonance spectrum contains a single line. In an isotropic solvent the only interactions not averaged to zero by rapid tumbling are the isotropic couplings and so the spectrum of a biradical is identical to that of the com- parable monoradical. In organic biradicals the anisotropic electron-electron coupling results from the dipolar interaction between the electron spins.This anisotropic interaction is known as the zero-field splitting tensor D Luckhurs f 75 Fig. 9. The energy levels and allowed electron resonance transitions for a triplet state dissolved in the isotropic and anisotropic phase. because it partially removes the degeneracy of the triplet levels even in zero magnetic field. For example, when a biradical is aligned in a nematic meso- phase the relative energies of the triplet levels are: and The effect of a magnetic field on these new energy levels is also shown in Fig. 9. The presence of the unaveraged zero-field splitting has removed the degeneracy of the two allowed transitions and the anisotropic spectrum contains two lines separated by 4 C Dgj Oij.The quite different forms of the i , i anisotropic spectra for monoradicals and biradicals should provide a simple means of distinguishing between the two species. The biradical bi~galvanoxyl~~1 whose structure is shown in Fig. 10, is an excellent example. Bisgalvanoxyl is prepared by oxidation of the parent diphenol using lead dioxide ; the oxidation rarely proceeds to completion and the product is contaminated with the monoradical. The isotropic spec- trum (Fig. 10) of the product dissolved in 4,4’-dimethoxyazoxybenzene at 139°C consists of an intense signal from the monoradical superimposed on a barely discernible broad line from the biradical. However, on alignment in R. I. C. Reviews 76 c 0” Vk& 0 T= 109°C m loo gauss I f( Fig.10. The isotropic and anisotropic spectra of bisgalvanoxyl dissolved in 4,4’-dimethoxy- azoxybenzene. the nematic mesophase, the monoradical spectrum is essentially unaltered whereas the biradical line is split into a doublet of separation 395 M H z . ~ ~ The experiment clearly demonstrates the presence of two unpaired electrons in one of the oxidation products. The description of the electron resonance spectra of biradicals is more complicated when they contain magnetic nuclei. The nitroxide biradicals of the form: are well characterized43 and we shall limit the discussion to these species. The unpaired electrons interact predominantly with the nitrogen nuclear spins, and the system may be regarded as containing two electrons and just two nuclei.Because the electrons are coupled to the nuclei as well as to each other, there is a competition for the spin of a particular electron and the form of the isotropic electron resonance spectrum depends critically on the result of this competition. When the electrons are coupled more strongly to each other than to either Luckhurst 77 * 10 gauss gl utarate. Fig. I I . The isotropic and anisotropic spectra of bis(2,2,6,6,-tetramethylpiperidin-4-yloxyl-l) nitrogen nucleus, that is when J>> a ( N ) , the spectrum contains five lines, J : separated > a ( N ) the by spectrum a(N)/2 with is characteristic intensities 1 of : 2 two 3 : equivalent 2 : 1. In other nitrogen words, nuclei. if The only effect of alignment in a nematic mesophase is to split each of the five lines into a doublet separated by 4 C Dij oij, as before.44 Bis(2,2,6,6- i,j L-/ tetrarnethylpiperidin-4-yloxyl-l)glutarate, that iswithX equal to OC(CH&CO, provides an example of this behaviour.The isotropic spectrum at 142°C (Fig. 11) consists of five equally spaced lines. The heights of the lines are not in the expected ratio of 1 : 2 : 3 : 2 : 1 because the intramolecular motion modulates the value of J which produces an alternating linewidth effect.45 On alignment in the nematic mesophase of 4,4'-dimethoxyazoxybenzene at 98"C, each line is split into a doubleP4 but, because of overlap, only nine of the expected 10 lines can be seen (Fig. 11). At the other extreme, when J < a(N), each electron is unaware of the other's presence in the biradical.The electron resonance spectrum is therefore indistinguishable from that of the appropriate monoradical and contains three lines separated by a ( N ) . For example, the isotropic spectrum (at 141 "C) of the terephthalate (X = OC-(--\--CO) has only three lines, and gives 78 R. I . C. Reviews & 10 gauss Fig. 12. The spectra of bis(2,2,6,6-tetrarnethylpiperidin-Cyloxyl- I ) terephthalate dissolved in the isotropic and nematic mesophase. no indication that the radical has two unpaired electrons;44 but in the nematic mesophase of 4,4'-dimethoxyazoxybenzene at 95°C each of the three lines is apparently split into a doublet44 (Fig. 12). The successful synthesis of a biradical is demonstrated by this additional splitting in the anisotropic spectrum.In principle the anisotropic spectrum should be more complex but the large linewidth results in a deceptively simple spectrum.46 i,i The isotropic electron resonance spectrum for an intermediate coupling scheme is complicated because the triplet and singlet levels are mixed by the hyperfine term. However, the complexity is valuable since electron-electron coupling J can be obtained by analysing the spectrum. In contrast analysis of the simpler spectra for the two extremes only allows a h i t to be placed on J . The spectral analysis does not yield the sign of J , but this can be deter- mined from the anisotropic spectrum. In the nematic mesophase certain lines are split into doublets separated by 4 C Dij oij whereas the spacing between other lines now depends on both J and C Dzj 023.The signs of the i , j components of D can be obtained from a point-charge model for the electrons and the sign of 8 can be inferred from the shape of the biradical. Thus the sign of the sum C Dij Oij can be calculated and that of J obtained. For the carbonate i,i Luckhurst 79 (X = CO) J is positive and so its ground state is a singlet, but the separation from the triplet is small and the latter is thermally pop~lated.47y4~ The use of liquid-crystalline solvents is important when the number of unpaired electrons in the paramagnetic species is not known with cer- taint~.*~$5O The formula for certain conjugated binitrones can be written with diamagnetic or paramagnetic structures, for example 0- -0 can be reformulated as the biradical:5* 0 I* 0 * I 0 The intensities of the electron resonance spectra increase with increasing temperature, suggesting that this triplet state is thermally accessible ;51 but the isotropic and anisotropic spectra in 4,4'-dimethoxyazoxybenzene (Fig.13) failed to confirm the existence of a biradical.50 The only effect on passing into the mesophase is to decrease the nitrogen splitting. The observation of the nitrogen shift of 2.8 MHz is important since it shows that the radical is aligned. Clearly, the absence of any splitting must be because the radical contains just one unpaired electron. The paramagnetic species is now thought to be the mononitroxide : OH formed from the binitrone by the abstraction of a hydrogen atom, most probably from the solvent.50 The chemist's ingenuity in linking any number of monoradicals together to form a polyradical makes it important to have a procedure for testing the success of the synthesis.The use of liquid-crystalline solvents in electron resonance spectroscopy provides such a technique. Although the form of the R.I.C. Reviews 80 - 5 gauss Fig. 13. The isotropic and anisotropic spectra of the radical derived from the binitrone. anisotropic spectrum of a polyradical is complicated by any magnetic nuclei certain lines will behave as if the nuclei were absent. If the polyradical contains n unpaired electrons, then on alignment the single line in the iso- tropic spectrum will be split into an n-line multiplet with a binomial distri- bution of intensities. This behaviour is completely analogous to the nuclear magnetic resonance experiment with a solute containing n equivalent nuclei of spin 4.6 The triradical: 52 illustrates the technique.The widths of the hyperfine lines for the triradical are rather large and in order to resolve the triplet splitting expected on align- Luckhurst 81 189°C of I M°C Fig. 14. The spectrum the triradical dissolved in the isotropic and aniso- tropic phase of C(p-meth- oxy benzy 1 id en e) am i noazo- benzene. ment, 4-(pmethoxybenzylidene) aminoazobenzene was used as solvent because it had a high nematic-isotropic transition point (180 0C).53 The isotropic spectrum exhibits the alternating linewidth effect 54 found in nitrox- ide biradicals but we need only be concerned with the three intense lines shown in Fig.14. In the anisotropic spectra these lines are each split into a triplet whose separation increases at lower temperatures because of the increased alignment. The paramagnetic species must contain three unpaired electrons. v 82 20 gauss R.1. C. Reviews CONCLUSION The use of isotropic solvents in electron resonance spectroscopy is restrictive because it results in the loss of all knowledge of any anisotropic interaction. This limitation is removed by using liquid-crystalline solvents which can partially align the solute.Analysis of the anisotropic spectrum can then yield the signs of coupling constants, spin densities and the exchange interaction in biradicals as well as the geometry of the radical, the magnitude of the g-tensor and the number of unpaired electrons in the radical. The way is now open for the investigation of anisotropic interactions, even in the most complicated radicals. There is, of course, another side to the story. The aniso- tropic couplings are already known for certain species and so the form of the anisotropic spectrum can be used to study the fascinating and important properties of the liquid-crystalline state. ACKNOWLEDGEMENTS My work has undoubtedly benefited from collaboration with my friends Drs David Chen, Howard Falle, Andy Hudson, Janet Ockwell and Mr Peter James: it is a pleasure to acknowledge their collaboration. I owe my greatest debt to Professor Alan Carrington for his continuing inspiration and guidance.REFERENCES 1 A. Carrington and A. D. McLachlan, Introduction to magnetic resonance. New York: Harper and Row, 1967. 2 A. Hudson, A. Carrington and G. R. Luckhurst, Proc. R. SOC., 1965, A284,582. 3 A. Hudson and G. R. Luckhurst, Chem. Rev., 1969, 69, 191. 4 P. W. Atkins and M. C. R. Symons, The structure of inorganic radicals. Amsterdam: Elsevier, 1967. 5 S. Ohnishi and H. M. McConnell, J. Am. chem. SOC., 1965,87,2293. 6 G. R. Luckhurst, Q. Rev. chem. SOC., 1968, 22, 179. 7 A. D. Buckingham and K. A. McLauchlan, Prog. NMR Spectrosc., 1967,2,64. 8 C.W. Hilbers and C. Maclean, Molec. Phys., 1969, 16, 275. 9 G. W. Gray, Molecular structure and the properties of liquid crystals. London: Academic, 1962. 10 A. Saupe and G. Englert, Phys. Rev. Lett., 1963, 11,462. 11 A. Carrington and G. R. Luckhurst, Molec. Phys., 1964,8,401. 12 W. Maier and A. Saupe, 2. Naturf., 1959,14a, 882. 13 W. Maier and A. Saupe, 2. phys. Chem., 1956,6,327. 14 D. H. Chen and G. R. Luckhurst, Molec. Phys., 1969,16,91. 15 C. F. Schwerdtfeger and P. Diehl, Molec. Phys., 1969,17,417. 16 G. Foex, Trans. Faraday SOC., 1933, 29, 958. 17 C. S. Yannoni, J. Am. chem. Soc., 1969,91,4611. 18 P. D. Francis and G. R. Luckhurst, Chem. Phys. Lett., 1969, 3, 213. 19 G. W. Gray, Molec. Crystals, 1969, 7, 127. 20 E. Sackmann, S. Meiboom and L. C. Snyder, J. Am. chem. SOC., 1967,89,5981. 21 D. H. Chen and G. R. Luckhurst, Trans. Faraday SOC., 1969, 65, 656. 22 S. H. Glarum and J. H. Marshall, J. chem. Phys., 1966, 44,2884. 23 H. R. Falle and G. R. Luckhurst, to be published. 26 G. R. Luckhurst, Molec. Phys., 1966, 11, 205. 50,258. 24 A. Saupe, 2. Naturf., 1964, 19a, 161. 25 G. R. Luckhurst, Molec. Crystals, 1967, 2, 363. 27 H. R. Falle, G. R. Luckhurst, A. Horsfield and M. Ballester, J. chem. Phys., 1969, 28 A. Saupe, Molec. Crystals, 1966, 1, 527. 29 D. H. Chen, P. G. James and G. R. Luckhurst, Molec. Crystals, 1969, 8, 71. 30 H. C. Longuet-Higgins and G. R. Luckhurst, Molec. Phys., 1964, 8, 613. 31 H. R. Falle and G. R. Luckhurst, Molec. Phys., 1967, 12,493. Luckhurst 83 32 S. H. Glarum and J. H. Marshall, J. chem. Phys., 1967,46,55. 33 H. M. McConnell and J. Strathdee, Molec. Phys., 1959, 2, 129. 34 H. M. McConnell, J. chem. Phys., 1956, 24, 764. 35 H. R. Falle and G. R. Luckhurst, Molec. Phys., 1966,11,299. 36 L. C. Snyder and T. Amos, J. chem. Phys., 1965,42,3670. 37 L. C. Snyder and S . Meiboom, J. chem. Phys., 1966,44,4057. 38 K. Mobius, H. Haustein and M. Plato, 2. Naturf., 1968, 23a, 1626. 39 G. G. Hall and A. Hardisson, Proc. R . Soc., 1964, A278, 129. 40 A. J. Stone, Molec. Phys., 1964, 7, 31 1. 41 E. A. Chandross, J. Am. chem. Soc., 1964, 86, 1263. 42 H. R. Falle and G. R. Luckhurst, unpublished results. 43 A. R. Forrester, J. M. Hay and R. H. Thomson, Organic chemistry of stable free radicals. London : Academic, 1968. 44 H. R. Falle, G. R. Luckhurst, H. Lemaire, Y . Marechal, A. Rassat and P. Rey, Molec. Phys., 1966,11,49. 45 G. R. Luckhurst, Molec. Phys., 1966, 10, 543. 46 H. Lemaire, A, Rassat, P. Rey and G. R. Luckhurst, Molec Phys., 1968, 14,441. 47 H. Lemaire, J. chim. Phys., 1967, 64, 559. 48 S. H. Glarum and J. H. Marshall, J . chem. Phys., 1967,47, 1374. 49 I. Agranat, M. Rabinovitz, H. R. Falle, G. R. Luckhurst and J. N. Ockwell, J. chem. SOC. (B), 1970, 294. 50 A. R. Forrester, R. H. Thomson and G. R. Luckhurst, J . chem. Soc, (B), 1968, 1311. 51 M. Colonna and P. Bruni, Gazz. chim. Ital., 1964, 94, 1448. 52 A. L. Butschatschenko, V. A. Golubev, M. B. Neiman and E. G. Rosantsev, Dokl. Akad. Nauk SSSR, 1965, 163, 1416. 53 G. R. Luckhurst and E. G. Rosantsev, Izv. Akad. Nauk SSR Ser. Khim., 1968, 8, 1708. 54 A. Hudson and G. R. Luckhurst, Molec. Phys., 1967,13,409. 84 R. I. C. Reviews

ISSN:0035-8940    

DOI:10.1039/RR9700300061

出版商:RSC    

年代:1970

数据来源: RSC

摘要:

* . Cu m u 1 a t i ve 1 nd ex Volume 1, 1968 ; Volume 2, 1969; Volume 3, 1970. Air pollution . . .. * . . . .. . . I . 3, 119 Barrett, C. F., Air pollution . . . . .. . . .. 0 . 3, 119 0 . Betteridge, D., The teaching of chemistry in Victorian and Edward- ian times Bond, G. C., Catalysis in the context of chemistry Bourne, E. J. and P. Finch, Polysaccharides-enzymic synthesis and Briggs, G. G.--see .. .. .. .. . . . . .. . . 3, 161 . . 3, I Graham-Bryce, I. J. degradation . . .. . . .. .. . . .. .. 3, 45 Cairns, A. C. H., Chemicals and the world economy Catalysis in the context of chemistry Chemical applications of ultrasonic absorption measurements in .. 1, 1 . . 2, 41 .. 2, 143 .. 2, 1 .. 2, 117 .. 1, 135 .. 2, 41 . , .. .. .. .. 3, 1 .. .. .. .. . . . . 2, 59 . . .. .. 1,205 the liquid-state, Some Chemical education : problems of innovation Chemicals and the world economy . . Chemistry and nutrition Chemistry and the consumer . . .. .. .. .. Chemistry and the origin of life Chemistry and physics of enzyme catalysis, The , . .. Chemistry of tribology, The . . .. . . .. .. Chemistry in Victorian and Edwardian times, The teaching of .. 3, 161 CurrelZ, B. R. and M. J. Frazer, Inorganic polymers .. . . .. .. .. .. . . .. . . . . . . .. . . .. 2, 13 .. 2, 117 .. 2, 41 .. 2, 87 .. . . * . .. Doonan, S., The chemistry and physics of enzyme catalysis .. Economy, Chemicals and the world . . .. ,. .. Electrochemistry, Organic Electron resonance in anisotropic solvents .. . . .. Environment, Pollution of the Enzyme catalysis, The chemistry and physics of . . .. . . .. .. 3, 61 . * 3, 85 .. 2, 117 .. 3, 135 .. 3, 105 . . 2, 87 .. 2, 143 Frazer, M. J.-see Currell, B. R. . . .. .. .. . * Fermentation-the last ten years and the next ten years Finch, P.-see Bourne, E. J. Fish, H., Water pollution Fleischmann, M. and D. Pletcher, Organic electrochemistry Frazer, Alastair, Chemistry and nutrition , . .. * . . . .. * . Frost, B. R. T., Nuclear fuels . . .. .. .. .. Fuels, Nuclear . . .. . f .. .. . . * . .. 2, 163 .. 2, 163 177 Gowenlock, B. G. and C. A. F. Johnson, Techniques of physical measurement: vacuum technique . . .. . . .. .. 1, 107 Graham-Bryce, 1.J. and G. G. Briggs, Pollution of soils . . . . 3, 87 Hallam, H. E., Infrared and Raman spectra of inorganic compounds Halliwell, H. F., Chemical education: problems of innovation . . 1, 205 Hogg, D. R . and R. B. Moyes, Practical aspects of programme writ- ing 1, 39 . . . . .. .. .. .. . . .. . . .. * . Infrared and Raman spectra of inorganic compounds Inorganic polymers Ives, D. J. G. and T. H. Lemon, Structure and properties of water . . Johnson, C. A. F.-see Gowenlock, B. G. Lemon, T. H.-see Ives, D. J. G. Luckhurst, G. R., Electron resonance in anisotropic solvents Life, Chemistry and the origin of Moyes, R. B.-see Hogg, D. R. Nutrition, Chemistry and Nuclear fuels . . . . . . . . .. . . . . . . .. . . Oparin, A .I., Chemistry and the origin of life Organic electrochemistry I . . . * . , . . . .. . I Pollution of soils Pollution of the environment Pollution, Water Polymers, Inorganic . . .. . . .. * . Polysaccharides-enzymic synthesis and degradation Practical aspects of programme writing Programme writing, Practical aspects of . . . . . . * . Soils, Pollution of Structure and properties of water * . Pletcher, D.-see Fleischmann, M . Pollution, Air . . . . . . . . .. . . .. . . .. . . .. I . 178 .. .. . . .. .. 3, 27 . . 3, 61 .. .. . . . . 2, 1 Miall, L . M., Fermentation-the last ten years and the next ten years 3, 135 . . 2, 163 . . 2, 143 . . 2, 1 .. 2, 87 . . 3, 119 . . 3, 105 . . . . 1, 39 . . . . 2, 13 1, 62 . . . . . . 3, 87 . . 3, 85 . . 2, 13 .. 3, 45 3, 27 . . 3, 27 * . 1, 135 . . .. . . . . Roberts, Eirlys, Chemistry and the consumer Rowe, Geoflrey W., The chemistry of tribology .. . . .. . . . . . I . . .. .. Teaching of chemistry in Victorian and Edwardian times, The Transition metal ions in biological processes, Role of .. .. . . .. .. . . . . .. .. . . . . . . * . ,. 1, 1 * . . . .. 3, 87 . . 1, 62 .. 3, 161 . . . . 1, 13 * . 2, 59 Ultrasonic absorption measurements in the liquid states, Some chemical applications of . . . . * . Vacuum technique . . . . . . . . .. I . . . .. 3, 105 . . . . . . .. . . .. . . .. 1, 13 . . . I . . . . . . .. . . . . 1, 107 Water pollution Williams, R. J. P., Role of transition metal ions in biological processes Wyn-Jones, E., Some chemical applications of ultrasonic absorption meawrements in the liquid state . . .. .. . . .. 2, 59 179

ISSN:0035-8940    

DOI:10.1039/RR9700300177

出版商:RSC    

年代:1970

数据来源: RSC