[2] R. G. Kirste, W A . Kruse, J . Schelten, Makromol. Chem. 162, 299 (1972). [3] a) A . I/: Tobolskg: Properties and Structure of Polymers. Wiley, New York 1960; b) Revised edition by M . Hofmann, Berliner Union, Stuttgart 1967, pp. 118f. [4] W W Grawsfey, Adv. Polym. Sci. 16, 1 (1974). [5] a) F . Btreche: Physical Properties of Polymers. Interscience, New York 1962, p. 61; h) J. Chem. Phys. 20, 1959 (1952); 25, 599 (1956). [6] M . Hofmann, Rheol. Acta 6. 92 (1967). [7] P. J . Flory: Principles of Polymer Chemistry. Cornell Univ. Press, New York 1953, p. 402. [8] H . A . Sruurr: Die Physik der Hochpolymeren. Springer, Berlin 1953, Vol. 2, p. 655. [9] M . Huj>nann, Rheol. Acta 6, 377 (1967). [lo] See ref. 171. p. 405; W Kuhn, F . Griin, Kolloid-Z. 101, 248 (1942): cf. P. J . Flory, Angew. Chem. 87, 787 (1975). [I 11 See ref. [3a], p. 94. [12] M . Hofmann, Kolloid-Z. Z . Polym. 250, 197 (1972). [I31 See ref. 171. p. 425; P . J . F l o r j , 7: G . Fox. J r . , J. Am. Chem. SOC. 73, 1904 (1951): J. Polym. Sci. 5 , 745 (1950). 1141 M . Huflmunn. H . Kriimer, R. Kuhn: Polymeranalytik. Thieme, Stuttgart 1977, Vol. 1 . [l5] M . Hofmann, K . Rother, Makromol. Chem. 80, 95 (1964). [I61 G. Dobrowofski, W Schnabef, Makromolekulares Colloquium, Freiburg, March 3, 1977.

[17] M . Hofmann, Makromol. Chem. 153,99 (1972). [18] M . Hoffmann. Rheol. Acta 6, 82 (1967). [!9] M . Mooney. J. Appl. Phys. 1 1 , 582 (1940); R . S. R i d i n , D . $4Saunders, Philos. Trans. R. SOC.London A243.251 (1951). [20] M . Hqffmann, Makromol. Chem. 174. 167 (1973). [21] a) A. S. Lodge, Rheol. Acta 7 , 379 (1968); b) R. Takserman-Krozer, A . Ziabicky, J. Polym. Sci. A2, 7, 2005 (1969); A2, 8, 321 (1970); c) F. S. Edwards, Proc. Phys. SOC.London 85, 613 (1965); 91, 513 (1967); 92, 9 (1967); Discuss. Faraday Soc. 49, 43 (1970); J. Phys. A 6 , 1169, 1186 (1973). 1221 See ref. 171, pp. 577, 512; P. J . Flory, J . Chem. Phys. 18, 108 (1950). 1231 M . L . Huggins, J. Phys. Chem. 46, 151 (1942). [24] M . Dauuil, J . P Cotton, €3. Farnoux, G . Janninck, G . Sarmu, H . Benoit, R. Dupfessiu, C . Picot, P. de Gennes, Macromolecules 8, 804 (1975). [25] P. Debye, J. Phys. Colloid Chem. 51, 18 (1947). 1261 G. K Sehufz, 2. Phys. Chem. 193, 168 (1944). 1271 See ref. [5a], p. 19; P. Debye, F. Bueche, J. Chem. Phys. 11, 470 (1943); A. Ishihora. J. Phys. SOC.Jpn. 5, 201 (1950). 1281 See ref. [7], p. 599. 1291 G. Gee, L . R. G. Peloar, Trans. Faraday SOC.38, 147 (1942); G. Gee, W I . C . Orr, ibid. 42, 507 (1946). 1301 C . E . H . Bawn, R . F . J . Freeman, A . R . Kamallidin, Trans. Faraday SOC.46, 677 (1950). 1311 J . Furukawu, H . Inaguki, Kautsch. Gummi Kunstst. 29.744 (1976). [32] U . Eisele, Lecture at the IISRP Conference Williamsburg (USA) 1976.

Flexible Drug Molecules and Dynamic Receptors[**] By R. J. P. Williams[*] When a small flexible drug molecule binds to its likewise mobile receptor (protein, membrane etc.) the shape and function of both can change. The study of the nature and extent of these changes by several independent methods gives an insight into the mode of action of drugs. The static lock and key model will most probably have to be revised or be replaced by the nebulous concept of dynamic states.

1. Introduction The basic problem of drug action in biological systems is easily formulated. In order to interact with a biological system any drug must first bind and may then react, whence a comprehensive minimal two-stage reaction path can be written

where D is the drug which undergoes a rapid reversible binding to a biological receptor L, i.e. a protein, DNA, RNA or a membrane etc., giving DL. This reaction has an equilibrium binding constant

[*] Prof. Dr. R. J . P. Williams Inorganic Chemistry Laboratory South Parks Road, Oxford OX1 3QR (England) [**] This article is based on the Merck, Sharp and Dohme Scientific Lecture 1976 given in London. It was also given in outline at the Belgian Chemical Society Meeting in Namur, 1976.

766

and there can be many successive steps of this kind before the 'final' binding condition is reached. The second step in the above simplified scheme [eq. (I)], which may or may not be a required part of action, is an irreversible chemical combination of D in the form D' with a part of the biological system L', where L' may be a protein, RNA, DNA, a membrane etc. which has incorporated D (or D'). L and L' could be the same receptor site of course. The reaction rate constant in its simplest form is a first order rate constant, kDL, but again many such steps could be involved. My analysis of drug action will be based upon the structural features of these reaction paths starting from the structures of D and L themselves. By structural features I imply the whole series of conformational states through which the two species D and L must go in order to reach some final state DL or D'L'. Recently the nature of such pathways has been stressed by Feeney, Roberts and Burgen"', who were motivated by their observations, using nuclear magnetic resonance (NMR) spectroscopy, on the mobility of certain drug and hormone molecules. My independent and parallel interest in the problem of conformational mobility has arisen through studies of both small and large molecules in solution also using sophisticated NMR methods for conformational analysis. In the past the solution structures of D and L (which Angrw. Chem. I n t . Ed. Engl. 16, 766-777 i1977 J

are the ones to be discussed in a drug/receptor interaction) have often been assumed to be known once X-ray crystal structures had been completed. This approach ignores the statistical occupancy and the time dependencies of many different conformations of a molecule in solution and may well have led to over-precise thinking about the general need for structural fitting, e.g. as in the notions of lock-and-key and induced fit. Examples illustrate the point. The study of different penicillins in the crystalline state by X-ray crystallography showed that there were two possible ring conformers and that different substituents on the fourmembered ring led to a switch from one conformation to the other (Fig. 1). We have studied the conformation of some of the same penicillins in aqueous solution by NMR probe

wriggling of the drug to a ‘best’ site. Drug design might have to be based on selected dynamic properties of a molecular frame as well as upon the possibility of providing a particular structure to fit a supposedly rigid receptor. Finally it will be shown in this article that the large molecules which form receptors are not necessarily more rigid than the small molecule drugs, and the same statistical fluctuations and timedependent changes must be analyzed for them. This motion also has an influence upon the constants in eq. (1). The obvious need for a careful analysis of the mobilities of drugs and receptors before indulging in a discussion of their interaction leads me to turn away from drug action for a while and to describe recently obtained knowledge of structures in solution and of the dynamics of these molecular conformations.

2. General Comments about Rigidity of Small Molecules in Solution

Fig. I . General formula oi penicillins: spatial arrangement of the atoms in crystalline penicillin G and ampicillin.

methodsr2]but our results show clearly that there is little if any dependence of structure of the penicillins in solution on substitution of its rings (see Fig. 1). We also noted that the data suggested that although one conformation was dominant there was a smaller percentage of the other conformation in fast equilibrium with it. In principle the rate constants for conformational flipping of the molecules could have been determined too. We are forced to ask which particular structure if any is important for drug action and, has the nature and rate of conformational mobility an importance of its own? A rather general inspection of drug molecules shows in fact that only a limited number of drugs have rigid frames and obviously their conformations in the bound state are easily described from crystal or solution studies (but see below). In all other cases there are different conformations open to the drug molecule and these may well be in rapid equilibrium, as in the case of the penicillins. We need to consider the relative energies of these different conformations (open to the drug in the unbound state) since, presuming that only a limited set of the conformational forms are bound by the receptor, there is a population bias against the drug being bound in these forms in direct proportion to the probability of achieving the limited conformation set in the free state. Drug design might then be based partly on fitting, but if we are dealing with a conformationally mobile drug, partly on the restriction of conformational freedom of the (free) drug. However, this last approach could fail too for the very flexibility of the drug might also be an unavoidable requirement in order to achieve final binding or reaction, and in this case a knowledge of the conformational mobility of the drug would be essential in an understanding of action. In the above Scheme [eq. (1 )] certain steps, kf, would represent necessary movements on the way to a final binding site-a Angew. Chem. Int. E d . Engl. 16, 766-777 ( 1 9 7 7 )

Apart from the conformations of penicillins in solution we have examined the conformations of a series of amino-acids, some small peptides, some sugars, and a series of nucleotides, where there is obviously a much greater variety of possible mobilityr3~1‘. The general impression gained from these studies is that even though there must be mobility in the small molecules (rotation about single bonds with limited steric hindrance) certain “conformational families” or a very small set of families peculiar to each group of molecules in turn are overwhelmingly favored, possibly through the solvation by water. A family of conformations is to be thought of as a relatively small group of structures related to one another by minor changes in bond angles. We can illustrate the point by reference to the simple nucleotides but rather than making direct reference to the deduced nucleotide conformations, derived from NMR spectroscopic data, I shall use a comparison of the experimental NMR information so that experiment and interpretation are not confused. Our experimental procedure starts from an examination and an assignment of the NMR spectrum of a molecule in solution in as much detail as possible. This means that each ‘H, I3C, 31P,19F nucleus in the molecule is studied as far as is possible, but here I shall describe ‘H-NMR data in large part. These spectra in themselves contain coupling constant data derived from spectral fine structure and the coupling constants are related to the relative position in space of coupled protons. For example, in such a system of atoms as C ( H ’ F C(H’) the geometric relationship of H’ and H Zcan be uncovered. Restrictions upon free rotation about C-C bonds are then obvious in a qualitative and sometimes in a semi-quantitative way. Again proton relaxation times of the spectral signals can be used to calculate the non-bonded H-H distances and further data on small local regions of the conformation comes from the study of the nuclear Overhauser effect. All these methods have been used for some time by a large number of research workers. The considerable extension of the conformational analysis which we have made is to examine the NMR spectra of the small molecule in the presence of bound (in fast exchange) paramagnetic ions (see Fig. 2). The NMR spectra are found to be perturbed in two ways: 1) shifts of absorption lines ( 6 ) arise if the paramagnetic center has 767

*

nucleus

OH

Fig. 3. 5'-Adenosine monophosphate (5-AMP); bonds about which there is apparent free rotation are indicated by arrows.

Y

Fig. 2. The effect of a paramagnetic reagent placed at the origin of space (a) upon the line position (shift b-c) and line of width (broadening b-dj of an NMR spectrum (see ref. [3]). The broadening of lines is directly related t o TZm,and the shift is given as 6 for the most general rhomhic field case. In an axial field, R = 0.

Table 1. NMR shifts(6)of nucleotide sugar protons by lanthanoid(ir1j-containing probes given as the ratio R =6(i-H)/6(5'-H) [9] [see Fig. 2, Fig. 3 and

Eq.

(4.

Nucleotide

5'AMP

a fast electron relaxation time; 2) changes in relaxation time, e.g. broadening of lines ( B ) arise when the paramagnetic

5'-GMP

center has a long electron relaxation time. The equations

5'-CMP

5'-UMP

B=C

1

-

r6

(3)

show that 6 is a vector quantity depending upon the distance, r, of the nucleus under inspection from the paramagnetic probe atom, and upon the angle 6 between the principal axis of the paramagnetic probe and the direction from probe to the nucleus (see Fig. 2), while B is a scalar quantity. In the equations A and Care constants independent of the nucleus under study. (The above shift equation [eq. (2)] assumes that the bound paramagnetic ion generates an axially symmetric magnetic field. Details of the methods and measurements which allow the field to be so defined are given Let us assume that the site of binding of the paramagnetic ion to the molecule under study can be determined (which is the case), then these shift and relaxation measurements give directly a set of conformational parameters, 6 and B, just as coupling constants are conformational parameters and they can be used empirically as comparative molecular conformation data on very many different atoms which are separated by considerable distances. Of course all the different NMR methods add together to give the largest number of conformational parameters, and together should be used in the definition of structure"]. [*] All NMR methods provide data which refer t o the average conformation. It is a matter of convenience whether we search to describe the average in terms of a single conformation with non-Idealized bond-lengths and bond angles [2,3,5] or in terms of combinations of two, or three, or four idealized conformations etc. It has often been the case that averaged conformations have been analyzed usinysums oftwoor threeconformers where the contributing conformers introduced are those suggested by theoretical energy calculations in the gas phase. As shown by Feeney et d . [7] it is always possible t o fit NMR data by such a procedure. Even though it is certain that better fits of experimental parameters can be obtained by using such semi-theoretical approaches, since larger numbers of variables are introduced, than using single conformation fitt~ngthe fit must be suspect as the theory does not apply to solvated species. Thus it is essential that many indept.ildent methods are used to check structural representations of molecules in solution, e.9. coupling constants, relaxation data, shift data, and nuclear Overhauser effects, using first m e and only later combinations of two conformations.

768

OH

5'-d-TMP

PH

R = 6(i-Hj/6(5'-H) i=2' i=3'

i=4'

2.0 7.6 9.0

0.09 0.09 0.1 1

0.24 0.26 0.27

0.40 0.38 0.38

0.32 0.34 0.33

2.0 7.6 2.0 7.6

0.10 0.08 0.07 0.08

0.20 0.2 1 0.20 0.25

0.40

0.38 0.38 0.37

0.32 0.31 0.32 0.32

2.3

0.08

0.30

0.37

0.35

Table 1 gives shift data for the protons of some nucleotides (Fig. 3). The observed shift 6(i-H) of the NMR signal of a proton i-H due to presence of a bound lanthanide(rI1) ion is expressed by the ratio R of this observed shift relative to the shift of a chosen proton, here 6(5'-H) of the 5'-H proton (see Fig. 2). The paramagnetic lanthanide ions have been used to generate the shifts but a wide variety of reagents can be employed. A striking result is that, almost within experimental error limits, all the nucleotides give the same set of conformation parameters. Broadening (relaxation) and coupling constant data are also almost invariant which can only mean that independent of the base (pyrimidine or purine) and of the sugar (oxy- or deoxyribose), the general conformational outline of nucleotide (including mobility averaging) is fixed, and it is surprisingly closely related to the single conformation observed in crystals. (As an aside we observed that although the crystal structures of deoxy- and oxy-uracil monophosphate were different from that of deoxy- and oxycytidine monophosphate this difference was not seen in solution and it may well be that the difference seen is an "artefact" of crystallization as in the case of the penicillin.) These results were deduced independently from coupling constant data alone by Sundara/ingam[6~who has gone so far as to state that the nucleotides must be relatively rigid in solution. As there is clearly a high degree of constraint in the mobility of these molecules the relative stability of the observed (averaged) set of conformations seen in these nucleotides must be due in part to the nature of water as a solvent. If we change solvent we observe a different stable conformational family. In dimethyl sulfoxide the (average) conformation of the nucleotide monomers is different from that found in water, and in some aqueous systems we find that even salt and denaturants such as urea also affect the conformations of small molecules, as indicated by probe measurementsr3I. We Angrw. Chem. lnt. Ed. Enqi. 16, 766 777 (1977)

note that receptor sites may be very unlike water in their “solvation” properties and may prefer energetically a quite different conformational family from that found in aqueous solution. All these data tell us nothing about the nature of other non-preferred conformations or the rate of flipping to lowly populated conformational states, although we may presume this flipping is fast since we see no separate signals for different states (but of course we do not know that such states of low probability exist). In fact many conformations widely different from those determined by NMR methods of analysis could be reached and for 1 % of the time almost any conformation X could be present and it would go undetected by these solution methods. However, if X is the desired conformation for action there is then a factor of 100 against its formation for this particular molecule and it must be possible to build a better molecule, drug, for binding alone where this mobility factor is considerably reduced. The implications of these findings for the study of drugs is simple but has not been clearly stated before: 1. Only for those drugs which have rigid frames (Table 2), are we able to neglect mobility, but even here we must consider the aqueous (or other) solvation environment. For example our study of cyclic 3’,5’-adenosine monophosphate (3’,5’-CAMP)gave the same structure for the two rings containing the phosphate and the sugar (X-ray structure analysis), and we may assume that this frame is relatively rigid[’]. The Table 2. Rigid and flexible drug molecules [a]. Relatively rigid

Flexible

Moderately flexible

Quinine (Many alkaloids)

Straight-chain alcohols

Penicillins Chloramphenicol

Nitrogen mustards (N-methyl bis(2-chloroethyl)amine) and derivatives Sulfonamides

Streptomycin Linear polypeptides

Steroids Tetracyclines Morphine Cocaine

Mepacrine Phenothiazine Nicotine

[a] It is of great interest t o make molecular models of drugs and attempt to visualize the number of different static and dynamic modes of action which they could have.

receptor could bind exactly this frame, but just because the hydration remains unknown it is conceivable that it is the hydrated form of the molecule which generates action when the stereochemistry of the receptor site can not be deduced from the solution or crystal structure studies of the small molecule. 2. Other drug molecules may be much more mobile (Table 2); for example, we have been unable to determine a “structure” for 2’,3’-CAMP by our methods, a fact which suggests that the NMR datacan not be represented by a single conformation family[91.For such a molecule (drug) which has considerable mobility between two o r more rather different conformational families knowledge of the conformations observed in crystals or in solution is of much more limited value for we do not know not only the hydration but which or how many conformationally different steps are required in each of the steps Anguw. Chem. I n t . Ed. Engl. 16. 766 777 ( 1 9 7 7 )

of equation (l), and we d o not know which are the important conformations in the final condition. The problem becomes even more serious as we turn to molecules of increasing size, and we note that it is not just a problem of drug binding alone but is perhaps a general feature of biological activity. 3. From our studies, the orientation of the base in 3’3’CAMP is clearly not fixed relative to the two rings of the cyclic system to the same degree as was found in the simple nucleotides 5‘- or 3’-AMPr81. Thus it may be easy for this part of 3‘,5’-CAMPto wriggle or corkscrew its way to a required binding site, receptor. Thus, whether a molecule binds or not can depend upon whether it can reach a certain site or not, for the receptor site need not be on an open surface[”*]. Impressions as to the importance of molecular dynamics for drug action are strongly supported by the inspection of hormones.

2.1. Hormones and Peptide Hormones O n first inspection it is surprising that small molecules such as adrenaline, 5-hydroxytryptamine, and acetylcholine should be used as hormones and transmitters, for these molecules contain only a very small rigid unit-if they contain a rigid unit at all-and rather few sites (e.y. -OH or -NH2 groups) by which they can be recognized selectively. The molecules also contain some bonds about which rotation is relatively easy. Given the requirement for virtually specific action of a hormone a much more rigid molecule would seem to be appropriate. However, if we consider that a molecule has to “wriggle” to reach its site of action then rapid on/off reactions using a series of different conformations could be required[’? Thus as above a compromise may be reached between a good match of hormone and binding surface in some final state and a mobility required in order to reach the site. This compromise will include a loss of some binding capability which is governed by the adverse statistical weighting of conformers in free and bound forms. It is not easy to work out an optimum system for such fast and selective binding but, as it may be that the actual binding site is closely related to the heavily weighted conformation in solution, it will still pay in designing drugs which compete with hormones or transmitters to try first conformational matching with these states. It is remarkable too how many larger hormones are not like 3,5’-cAMP or sterols, which must have very limited conformational mobilities. Instead many of them are apparently disordered linear polymers ranging from tripeptides to higher peptides such as glucagon, which have molecular weights

[**I A change of attitude may now be required on the part of chemists who arc engaged in the design of drug molecules. The outline “shape” of a molecule, at 0 K, when examined by instantaneous methods (time constant < 10- ” s ) u~III conform to bond angle and bond length predictions based upon our knowledge of simple molecules such as methane. Houever for a mobile molecule.;mobile receptor interaction it could well be that a rather different space-filling model would be more appropriate: for example. methane would become a simple ball and an indole ring would be represented by a flattened spinning disc. Dynamics in these cases involves rotational mocement about various axes, but no translation. However. translation could also be important and perhaps we should seek for tunnels and channels in receptors which may have gates at which “wriggling” motions may be involved in sitc interactions. The shape of these channels must be such that given wriggling the uhole molecule can pass through, but such a shape is clearly not closely related to any single molecular structure. 7 69

of > 10000. These molecules must be flexible to a very high degree, and in fact this mobility has been confirmed by NMR measurements. The longer the chain of these hormones the smaller the probability of a correct matching between the hormone and receptor assuming that the receptor recognizes all the hormone. It would seem that fast action should then belong to small or rigid hormone molecules while slower action, but perhaps very highly selective control, should belong to longer flexible molecules, all static and dynamic ways of achieving selectivity having then been found (Table 3). Table 3. Naturally occurring rigid and flexible small molecules and hormones

[.I. Relatively rigid

Flexible

Moderately flexible

Alkaloids Sterols Auxins Thyroxine Trypsin inhibitor Heme Coenzyme BIZ

Spermine Glutathione Corticotropins (ACTH) Releasing hormone Many lipids Glucagon Melanocyte-stimulating hormones

Vassopressin Oxytocin Nucleotides Vitamins E and K Bacetracin Folk acid Polysaccharides ATP

[a] I t is assumed that steric hindrance as in thyroxine, ring formation as in vasopressin, and cross-linking as in the trypsin inhibitor make a part of a molecule effectively rigid.

Particularly interesting in this respect is the recent work on insulin, which illustrates conformational mobility of at least one tenth of the peptide“’! Here it is the cooperative effect of metal ion and anion binding, at different sites, which causes a rearrangement of the peptide. Using NMR methods we have confirmed that a corresponding conformational switch of insulin occurs in solution with addition of the same reagents[’’]. The changes are anion-, cation- (i. e. salt) and temperature-dependent. This result throws doubt again on the detailed use of crystal structures in the discussion of the action of such hormones-except for the regions of immobilized (rigid) surface (see the trypsin inhibitor in Section 2.2). The observations can also be used to describe the synergism of two small-molecule “drugs”, here X i (ZnZf) and Y - (C1or I-), on a receptor surface (insulin) when it is seen that Ala

there can be large conformational effects in some regions of the large molecule and that also general changes may occur to a smaller degree over larger regions of the molecule (see Section 2.2).

2.2. Small Molecules: Summary Table 3 lists a variety of molecules found in biological systems. Apart from peptides and polynucle~tides[~~ 12] we have looked at such molecules as adenosine triphosphate (ATP)[13] and vitamin B12[’41by the same conformational methods using paramagnetic probes. In the last two cases the structures found in solution are again relatively closely related to those in the solid state and we class these molecules as largely rigid. Other molecules such as polyamines and long chain fatty acids must have a much greater mobility and Table 3 puts them into a different group. Not all control peptidemolecules of higher molecular weight are of the mobile kind such as is glucagon. The nature of the trypsin inhibitors which are quite large peptides but not simple linear polymers and which must act rapidly, suggest that they are rather rigid, for their -S-S cross-links and internal P-pleated sheet restrict the conformation very greatly[15. l6]. The rigidity of the trypsin inhibitor is shown too by the fact that even some of the aromatic residues of amino-acid side chains do not flip freely despite the fact that this is a small molecule[161. We stress that the possible combinations of rigid and flexible elements can lead to all kinds of intermediate behavior and different parts of large molecules can behave differently.

3. The Receptor Site: Introduction The approach in the discussion of the structure of the receptor must be the same as that used in the study of small molecules. We must go from an examination of constrained molecules in crystals or free molecules in the solution phase to a semi-speculative knowledge of a receptor geometry and dynamics. Now we suspect that receptor sites are particular regions of proteins, DNA, RNA, membranes or polysacchar-

42

h Met 12

I

Val 109

Met 105

I

T

Ala

I

1

31

Leu 8

I

0

Fig. 4. Part of the ‘H-NMR spectrum of lysozyme with assignments (methyl groups in the given amino acids).

I10

Angew. Chem. Int. Ed. Engl. 16, 766-777 ( 1 9 7 7 )

ides. What can we say about the structure of such large molecules in biological systems? First we turn to the examination of the nature of these large molecules in solution.

3.1. Proteins as Flexible Molecules In the last few years, 1974-77, a change away from rigid pictures of the conformation of macromolecules has developed. We pioneered these studies in so far as the use of high resolution 'H-NMR spectroscopy on proteins was concerned["! (Fig. 4), while parallel work by Sykes and by Wiitkvick[161was underway on peptides, and many authors were carrying out I3C-NMR studies. In other work a variety of techniques" 8. have been employed which also demonstrate motion, such as oxygen quenching of fluorescence and hydrogen exchange, but this work is restricted to the general discussion of the protein and it has not been possible to locate the mobility within the protein sequence. Our work shows clearly the nature of the motions and how they are distributed within proteins. The types of motion observed are: 1. Rotation and libration (sometimes restricted) about certain single bonds internal to the protein. 2. High mobility of protein side-chains on the surface. 3. General mobility of (hydrophobic) groups internal to proteins (breathing). 4. Occasional segmental movements of the main chain. 5. Some proteins are quite generally mobile i. e. close to random coil polymers. Table 4 gives some details of these motions. Table 4. Protein motion. Type of Motion

Observation

Expansion (Vibration)

Temperature dependence of ring cnrrent shifts relaxation times Insulin: transitions Heme: resonances (hernoglobin) Lysine: correlation times Tryptophan: oscillation Phenylalanine and tyrosine: flip Temperature dependence of resonance positions Relaxation times

Fast segmental motion Surface motion Slow motion of side chains

Random coil motions

3.2. Segmental Motion: Summary In some proteins we must suppose that a part of the protein may be mobile while another part is not and that these parts can be of quite diverse sizes for different proteins[20-22! Vallee et al. have stressed the mobility of the active site region of carboxypeptidase in solution[231and it would appear that the effect is seen even in the crystals. This mobility effects a relatively small back-bone change, comparable with the type of change which occurs on the binding of oxygen to hemoglobin when the FG region undergoes perturbation. In the meantime quite a number of proteins have been examined and they would appear to fall into very different classes of behavior. Lysozyme, peroxidases, peptidases, carbonic anhydrase and cytochrome c show least conformational change of the main frame; kinases, concanavalin A, colicin Ia, and hemoglobin show rather larger changes; histones, chromaAngew. Chrm. Int. Ed. Engl. 16, 766-777 (1977)

granin A, and phosvitin are very adaptable, flexible, chains (Table 5). Thus it seems that large molecules (receptors) show the same range of flexibility as small molecules. There must be some very rigid systems and some very mobile ones and all intermediate behavior patterns will emerge. Table 5. Rigid and flexible large molecules [a] Relatively rigid

Flexible

Moderately flexible

Cytochrome c Neurotoxins [b] Lysozyme Peptidases Nucledses

Phosvitin Chromogranin A Histones Glucagon Myelin protein A. 1 . Vesicnlin

Insulin DNA-binding proteins t-RNA Kinases (?) Antibodies (?) Lipases

[a] Generally, extracellular proteins are rigid and are often crosslinked by S-S bridges. lntracellular proteins are more mobile especially if they are required to equilibrate between bound and unbound states involving DNA, RNA or membrane surfaces. [b] Personal communication from Prof. C.Petsku.

The case of cytochrome c is of great interestrz1! Although it is known that the two oxidation states of cytochrome c have different physical properties e. g. solubility and chromatographic mobility we have been unable to detect differences in the interior of the molecule in the two oxidation states in solution. A similar result has been found in crystals. However we do observe that the binding of various anionic and cationic reagents on the surface of cytochrome c is different in the two oxidation states which proves that their surface energies are changed by the change in charge on the iron. Change in surface energy will be reflected by change in charge, conformation, and hydration, but present methods do not detect the surface states readily. Even so it is these surface states which will be important in protein/protein and at least initially in drug/protein interactions.

4. Small Molecule/Protein (Drug/Receptor) Combination This article .has stressed dynamic features of both drugs and receptors where drugs have been likened to many types of small molecules and receptors to many types of large polymers or assemblies. In discussing the combination of drug with receptor it is the dynamic features which we wish to keep to the forefront, although we realize that static matching has a very important and proven role to play. The discussion will proceed through examples, but before proceeding to these examples the states of the receptor and small molecule must be visualized as they approach one another. All the evidence provided above indicates that the small molecule will be fluctuating very rapidly between at best a small number of similar conformations and at worst a complete spectrum of very different states. The receptor surface may be equally mobileespecially in the case of sugars and lysines-but in other regions, pockets and grooves, may show limited mobility. An agonist drug might bind and then pass through these mobile regions, in some cases going to a site much deeper in the receptor while other, antagonist, molecules may just block access of the agonist to the deep-seated site. From here on we examine the bound drug/receptor sites. 771

4.1. LysozymeSaccharide Reactions The “receptor site” of lysozyme is the enzyme binding groove for substrate. It is lined with readily recognizable (by NMR) tryptophan residues (Fig. 5), and it is these groups which we can follow most readily during the binding of the saccharide[24,25]. The NMR method is fast enough to follow steps which occur with time constants of 5 1 0-4 s, and its specificity allows a precise statement as to which groups move and, if sufficient care is taken, the movement itself can be defined with some precision (Fig. 6). 0

Fig. 5. The lysozyme pocket showing the binding of a trisaccharide (alter

1251).

I

I

I

I

6.4

6.2

6.0

5.8

Fig. 6. Aromatic ‘H-NMR spectral region of hen lysozyme (3mmol/l) at pH 5.3 in the presence of tris(N-acetylglucosamine) (4 mmol/l) showing the effect of temperature on the tryptophan > N H resonances at a) 3 7 T , b) 45T,c) 55°C.

In the initial state there is present say the disaccharide bis(N-acetylglucosamine), which is a semi-mobile molecule but which may well be largely constrained by interaction with water, so that perhaps 95 % of the molecules belong to a small family of conformers. The protein lysozyme has a groove which is undergoing some dynamic motion including fast motion of side chains such as valines and the slow movement of the tryptophans (see Fig. 10). In other words the 772

two molecules generate a set of geometries, some compatible and some incompatible, and on binding they settle down into the final energetically most favorable bound state, which-as we have shown-has rather less mobility. The overall binding reaction path is then a succession of conformational states Ef S-r z E S ( l ) + xES(2)+ final state

(1 final conformations)

where the sum sign, X, covers all the dynamic states of a species. It is the dynamic nature of the initial and intermediate states of the reaction sequence which allows all the steps to the final condition to be rapid[241.The observed greater rigidity in the last step (or stages) means that the reverse process, the first dissociation step, may have a slow step as its initial step. Ignoring the consequences of this description as far as enzyme action is concerned we note here that an inhibitor of the enzyme could be a molecule which reaches states CES(1) or ZES(2) but cannot go through to the final state for reaction, when the inhibitor need not be a very good match for a substrate so long as it binds. As an almost trivial example CN- is a very strong poison (drug) when it replaces O2 in heme proteins. Sulfonamides do not match C 0 2 or HCOy very well but they bind in the pocket of carbonic anhydrase in place of CO,. Thus the match of enzyme and substrate is not highly restricted and we visualize a series of poor, moderate and good matches to different states of the enzyme groove. A very interesting test of this situation has been observed by accident.

We wished to study the surface of lysozyme using the spin-labeled substrate 3-[4-(2-deoxy-2-acetylamino-l-glucosyloxy)-phenylcarbamoyl]-2,2,5,5-tetramethyl-3-pyrrolin-l-oxyl ( I )[261. However, we found that the binding was not through the N-acetylglucosamine moiety, which is a portion of the natural substrate, but was directly to the spin-label itself which binds in site C. The fit can not be perfect but the binding of the spin-label molecule (2) by itself, which was tested as a result of the above finding, showed that it is bound better than N-acetylglucosamine ! Thus by accident a potential inhibitor (drug) has been uncovered which is chemically unlike the substrate but binds better than the substrate analog. No examination of the spin-label molecule (2) and the N-acetylglucosamine would have suggested the parallel. Detailed inspeG tion ofpossible protein surfaces and the geometry of a molecule such as N-acetylglucosamine or even more so a steroid reveals how difficult it is for an enzyme to match precisely the spacefilling and bonding capabilities of a substrate or a drug. Restricted mobility of the surface then becomes a very great advantage in assisting good (not excellent) matching. In fact I believe the matching has been rather over-stressed, for all that is required is good, relatively specific, binding and that the kinetics of this binding should be under control. In both respects mobility is highly valuable (Fig. 7). Angew. Chem. I n t . Ed. Engl. 16. 766-777 (1977)

ring is a rigid frame and we have examined such frames extensively in simple complexes, showing for example how they form plane-to-plane stacked molecular complexes with porphyrinscZ9].We then used these methods in the study of peroxidase complexes‘301. Peroxidases are very large glycoproteins which have some obvious mobile features such as the saccharide side chains. In the native state they are high-spin Fe”’ enzymes. Reaction with cyanide converts them into the low-spin state, whereas reaction with azide gives a mixture of the two forms (low-spins high-spin) in equilibrium. On the NM’R time-scale all these states

High Temperat ure

Fig. 7. Active center of lysoryme [25]. Overall survey of the conformational states of lysozyme in solution. Each block represents a conformational state and the linewidths referred to are those of the ‘H-NMR spectrum.

A somewhat more extensive rearrangement is seen on binding saccharides to concanavalin A[”]. Here binding results in such changes to the protein structure that two complete crystal structure analyses (protein with sugar and protein without sugar) were required. No evidence is available about the dynamics of the protein in this case. Note that here too a carefully labeled reagent, 1-(o-iodopheny1)-P-D-glucopyranoside, designed to find the sugar binding site, actually became bound to an abnormal site. Another example of mobility at a receptor site has been uncovered by Burgen et ~ l . [ They ~ ~ l observed . that the absorption spectrum of carbonic anhydrase (as the c o b a l t ( ~enzyme) ) was changed by the addition of sulfonamides. The change is consistent with the reaction

Native (largely high-spin)

+ Azide (high-spin)+ Azide (low-spin)

are rapidly in equilibrium so that both the on/off reactions of azide and the spin-changes are very fast. In fact there is probably present some 5 % of low-spin state in the native enzyme, as is the case in myoglobin and hemoglobin. As shown in Figure 8, high-spin and low-spin Fe”’ heme complexes have rather different structures. We must suppose that iron(ri1) oscillates rapidly between the different states and that the protein chain linked directly to the iron through the proximal histidine also undergoes rapid changes‘”, 311.

(b) Low-spin Fern

Co(r1)(base-) + sulfonamide --t Co(ir)(sulfonamide-) distorted geometry tetrahedral geometry

N

for the 3- and 4-substituted sulfonamides; but the 2-substituted sulfonamide complex has an absorption spectrum lying between these extremes and thus indicating an “intermediate” geometry at the cobalt. There is general agreement that the metal site in carbonic anhydrases can flick between different coordination states. Although only a small change is apparent this metalloenzyme is comparable with such enzymes as peroxidase and cytochrome P-450 in that the coordination sphere of the metal is sensitive to drug binding (see also Table 6).

I

N 0

Tdble 6 Changes in metalloenzymes due t o drug binding

________

-~

~

Enzyme

~~

Metal

Drug

Change

Cytochrome P-450

Iron(I1l)

Sterol

Peroxiddse

Iron(ll1)

(Myoglobin

Iron(l11)

3-Indolepropionic acid HgI;

Low-spin H high-spin g-value changes

(Hemoglobin

lron(ll1)

Carbonic anhydrase

Cobalt(I1)

-

~

-~

Xe 1.3-diphosphoglycerate Sulfonamides

zinc(^)]

Low-spin tt high-spin) Low-spin t - i high-spin) Distortion of tetrahedral coordination

4.2. Peroxidase-Substrate Complexes

The complexes of peroxidase with substrates can be readily studied since some of the substrates-indoles and phenolshave NMR spectra which are easy to interpret. The indole Angew. Chem. I n t . Ed. Engl. 16, 766 777 ( 1 9 7 7 )

Fig. 8. Stereochemistry of the heme iron spin-states

When an aromatic substrate binds to the protein it does not form a plane-to-plane stacked molecular complex like in the model reactions with porphyrins. Taking peroxidase complexes of 3-indolepropionic acid as an example we find that the indole system lies as is shown in Figure 9I3O1. The indole derivative is bound only by weak Van der Waals interactions in the pocket of the enzyme. Binding of other substrates is in the same pocket but the relationship between these substrates and their binding sites and between the indoles and their binding sites differ considerably. We conclude that the site of binding is more like a small mobile oily pool than a lock and key fit. Thus it is anticipated that a large number of organic chemicals can act as inhibitors of these 773

enzymes. Maybe enzymes for detoxification behave in this rather general way, for similar features can be seen in the binding sites of cytochrome P-450 and of alcohol dehydrogenases.

The effect of a drug can occur (i) at its immediate site which may be a long way from the iron, i. e. 1,3-diphosphoglycerate, (ii) at the iron by way of the spin-state change-a considerable relay, (iii) at the -SH group which is an even greater distance away. Antagonists could block these processes while agonists could potentiate them in a quite different way. Moreover, there are several very different reagents which can act on the same process, i.e. O2 uptake by hemoglobin, by binding at totally different sites, e.g. 1,3-diphosphoglycerate, xenon, HgI:, CO, and sulfydryl reagents. The very different chemical behavior of these reagents as compared with 02, the substrate which the drug challenges, shows that drug design can be quite impossibly difficult. Note too that it is the mobility of the receptor, here hemoglobin, which is responsible for all these different “drug” effects. 4.3. Wriggling and Gating Another illustration of this “wriggling” principle can be given by examining again the mobility exhibited by the binding groove of lysozyme (Fig. 10).This groove contains two aminodicarboxylate residues, Glu 35 and Asp 52, of which Glu

Fig. 9. The peroxidase complex of indolepropionic acid showing the disposition of heme and substrate, (a) and (b) are orthogonal views.

Now these oily pools in the proteins for taking up substrates need not be immediately accessible to the small molecules and it would be very interesting to observe exactly the time course of approach to the final state of binding. An inhibitor or a drug could act at any of the approach stages, or it could act indirectly by preventing the conversion of one form of a protein into another. Again it does not appear that the mobility within the oily drop is much altered by the insertion of the substrate even though the substrate binding affects the iron(m) site somewhat. A good example of the wriggling of a group to reach such an “oily-drop’’ binding site is the uptake of the hydrophobic anion HgI; into myoglobin. The binding site as observed by X-ray crystallography is at the back of the heme. There are gross restrictions to movement in this region of the protein and even quite small ligands cannot bind to the iron itself for this region of space is designed to accept oxygen only. We have studied the effect of the uptake of Hgl; on the properties of the Fe(r1r) form of myoglobin and have shown that there is a small change in the spin-state equilibrium (high-spin G low-spin) on binding. Thus the binding group (drug), here HgI;, has to wriggle through the protein to its binding center where it alters the conformation of the protein slightly. Uptake of xenon (a very large atom) must take place by similar wriggling. Again the effects of oxygen on the fluorescence of proteins demand a similar conformational mobility allowing access by wriggling. It is of interest to see how such binding could adjust the chemistry of the receptor. In the P-chains of hemoglobin[321 there is a sulfydryl group F G (p-93) which is on the helix which carries the Fe-linked histidine (proximal). Perutz has shown that the degree to which this sulfydryl is exposed depends upon the spin-state of the iron, for change of spin-state causes rotation of the helix. Thus we have the following dependencies: a) drug bound-more low-spin Fe, exposed -SH; b) drug absent -more high-spin Fe, hidden -SH. I74

Irp 6 2

A s p 101

,g

~, , I r p I08

2-

Ala 107

V a l 109

4 Fig. 10. The active site region of lysozyme; section of the crystal structure. A l l the amino acid residues can he detected in the NMR spectrum.

35 is held in the protonated form, against Trp 108. If a cation is bound in this region of the groove it may interact with sufficient strength that Glu 35 is ionized, and the resulting carboxylate group moves away from Trp 108 (configuration 2 in Scheme 1). In the case of weakly interacting cations this movement does not occur (configuration 1). We can therefore distinguish strongly interacting and weakly interacting cations. Now let us suppose that exactly the same structural relationship of two carboxylate groups occurs, not in lysozyme, but in a region of space behind which there is a second binding site for the cation. In the case of strong interaction (configuration 2) the actual binding at the two carboxylate centers would prevent (gate) access to this site. In the case of weak interaction (configuration 1) binding at the rear site would occur as the gate would remain open. Clearly the cations could act in very different ways. Gated reactions will always be associated with wriggling paths. Of course the same pattern can be imagined for the action of a cation such as acetylcholine. Angew. Chem. Int. Ed. Engl. 16,766777 (1977)

T r p 108

T r p 108

. . .CH2-COZ-H

. . .CH, \cop

Glu 3 5

Glu 35 t__

..

M@

M20

..

.CH,-COp A s p 52

.CH2 A s p 52

Configuration 1

Configuration 2

Scheme 1

Another example concerns the passage of an ion, e.g. Li+, through a membrane using an ionophore. There is a binding on/off reaction of metal and ionophore followed by a release. There is also an outward directed ion pump and we may suppose a site of action for Li’ say in competition with C a 2 + or Mg2+. It must be seen that the ionophore-governed steps are very important in the overall effect of lithium. As we have stated above the mobility of the surfaces of proteins is much greater than that of the internal regions. Our measurements show that surface lysine and sugar side chains (in glycoproteins) have motions with correlation times of I 0-9 s, i. e. as fast as collision diffusion rates. In an assembly of proteins there must be a pathway between the protein molecules which is diffusion restricted by the constraints imposed by the packing of the mobile side chains. The general point can be visualized from an examination of packing of proteins in crystals. Examination of these crystals shows that between protein molecules there are large channels of “disordered” solvent and protein side chains. Inhibitors and other quite large groups such as heavy atom labels diffuse down these channels quite readily to binding sites. But quite clearly there are restrictions on this diffusion which can be of almost any degree of selectivity. Thus only certain molecules will be able to reach particular regions of proteins in an assembly. It is possible to describe this as a “gating” of (drug) action, and the actual motion as “wriggling” to a site. The importance of this mobility of proteins extends to the binding reaction when an antibody combines with an antigen. Huber et u ~ . [ ‘ ~ I have suggested that the Fc fragment ofthe antibody is relatively mobile so that it changes conformation on receiving the antigen.

4.4. More Complicated Receptors A protein or a group of proteins may or may not be the receptor site of a drug. Rather a drug may act on a similar type of large molecule, e.g. RNA or DNA, or it could act at a membrane or similar condensed phase. The effect of small molecules on a condensed phase should now be examined in the same way as we have examined their effect on large molecules. The parallels between these problems has been described in a recent review on phases in biological We use lipid phases, membranes, as an example.

4.5. Membranes Fast tumbling of groups such as we have described in proteins is observed in artificial vesicle membranes of most lipids. For example lecithin vesicles give a high resolution Aiigrw. C h ~ w Inf. Ed. Engl. 16, 7 6 6 7 7 7 ( 1 9 7 7 )

NMR spectrum which shows that motion must be fast for the -N(CH,); group on the head and the methyl group on the tail of the fatty acid chains but is somewhat slower for the region around the glycerol group. When the size of the vesicle is changed or other molecules incorporated into it, or the temperature changed, or ions bound to the surface, these mobilities can undergo quite considerable changes. Turning to biological membranes we and others have observed that the ‘H-NMR spectrum of almost all cell membranes is very broad, even for membranes which contain a high percentage of lecithin, and it is not possible to detect in the NMR spectrum either the choline group on the head or the methyl group on the tail of the fatty acid chains. It is readily shown that inclusion of such molecules as cholesterol in the bilipid layer will cause a general reduction of the motion of the lipid sections of the molecule but the headgroup remains free. We are therefore left with the problem that there are gross restrictions on mobility in real biological membranes which are not seen in vesicles. This restriction could be related to size, curvature of membrane surface, or to the binding of the head-groups. The membrane lipids are also in rapid lateral motion, i. e. in the plane of the membrane. Inversion of lipids is however relatively slow; hence the membrane retains its asymmetry. The time scale for lateral movement is not vastly different from diffusion and it has frequently been supposed that drugs can influence this motion. The inversion has a time constant in the range of minutes. Thus although inversion is not important in any steady-state change and return, which takes place within seconds, the steady-state itself can change in response to ambient conditions. We proposed some years ago that it would be interesting to study this asymmetry with the paramagnetic binding probes, but the first actual study was made 351. A major problem-as in the independently in study of proteins-was (and still is) the question of the structure and the structural dynamics of the membrane and what chemical factors control the dynamics. To this end we looked at the binding of probe ions to a variety of phosphate esters including phosphatidylcholines. The first feature of binding is that the metal ions do not bind to the phosphate of a membrane phosphatidylcholine in the same way as they bind to phosphate esters such as phosphoglycerolcholine in water. If we use our standard procedures for the study of biological molecules we obtain the structure shown in Figure 11, in which the choline head-group stretches away from the bilipid membrane to a large degree. The result need not be analyzed in great detail, for there is surely much motion, but it is important to observe that the head-group of a lipid in a membrane does not behave in the same way as the dissociated free head-group, say phosphoglycerolcholine, behaves in aqueous solution. Secondly, the binding strength of cations at the two membrane surfaces (inside and outside) is not equal. Thirdly, as the cations bind selectively to different phospholipids they generate asymmetry in the membrane if their concentrations are different on the two sides of the The effect of the metal binding on the mobility of the membrane has not yet been assessed. However, it will be recognized immediately that the action of a chemical (drug) on such a system is not likely to be dependent upon any one simple factor. Thus as with protein receptors we anticipate that an extremely complicated story 715

\

drug action, for this description covers the whole of solid state and solution chemistry with its wide variety of interactions which are expressed quantitatively only in terms of free energy changes. Recently we have been using NMR to examine whole organs and have found a remarkable variety of mobilities in different phases[371.However, these features of cells cannot be discussed at length here; but in conclusion it should be pointed out that we must contend with many new problems when dealing with added chemicals (drugs) which can affect mobility, and static solid state models of fitting the geometry and chemistry of one surface to another may have to be supplanted by the more nebulous concepts of dynamic (solution) states The work described in this article was made possible by grants from the Science and Medical Research Councils, England. The author wishes to acknowledge the great help he has received from fellow members of The Oxford Eizqme Group and from his students.

Fig. 1 1 . The structure of phosphatidylcholine. Note that this structure has considerable mobility, and is for a cation-bound condition.

lies behind the drug action. Though it may be possible to follow drug-protein binding atom by atom, this possibility does not really exist in a membrane phase and we may have to be content with a more phenomonological functional thermodynamic approach. This would mean that drug design would remain very empirical. For example, fluidity in a cooperative liquid is open to a phase change of the solpgel kind and it could be that a small quantity of an impurity could shift the sol e gel equilibrium and thus change the dynamics of the phase dramatically. 4.6. Mobility of Bound States

The conclusion seems inevitable that just as all degrees ofmolecular motion are to be found in drugs (small molecules) and in receptors (proteins, RNA, membranes) so in their combination we shall find all kinds of mobilities (Fig. 12), for the binding site of a drug can vary from a condition in which the best model is a molecule held on a rigid crystalline material to one in which the drug has partitioned from water into an organic medium. There cannot be a general theory of

Fig. 12. Schematic diagram for a mobile protein (linked by ari-ows)-mobile drug interaction.

716

Received: November 8, 1976 [Z 187 IE] German version: Angew. Chem. 89, 805 (1977)

!I Burgen, G. C. K . Roherrs, J. Feeriey, Nature 253. 753 (1975): and references cited therein. [2] C . M . Dohson. L. 0 . Ford, S . E. Summers. R. J . P. Williums. J. Chem. Soc. Faraday Trans. 11 71, 1 I45 (1975). 131 B. A . Lrrine, R. J . P. Willirm~s,Proc. Roy. Soc. (London) 345, 5 (1975): B. A . L e i w e , R . J . P. Williums in B. Pullman: Environmental Effects on Molecular Structure and Properties. Reidel, Dordrecht 1976, p. 95 rr. [4] See r. g. E. Bergmanri, B. Pullmuif: Molecular and Quantum Pharmacology. Reidel. Dordrecht 1974. [5] C . D. Burry, J. A. Glo.sel. R. J . P. Williams, A. K Xur-ier. J . Mol. Biol. 84, 471 (1974). J. Am. Chem. Soc. 95. 2333 (1973). [6] C . .4lronu, M. Sutidurulin~pn~, 171 B. Birrlsull, N. J . M . Birdsull, J. Feeney. J. Thornrun, J. Am. Chem. Soc. Y7, 2845 (1975). [ 8 ] C . D. Burr)., D. R. Murrin, R. J . P. Williums, 4 . K Xrriirr. J. Mol. Biol. 84, 49 1 ( I 974). [9] C. Geruldes, D. Phil. Thesis, Oxford 1976. [lo] G. B e d r y , E. D o d m l . G. Dodson, D. Hodgkin. D. Merc~olo.Nature -261, 166 (1976). . J . P. William,$.K . Williumson. unpublished. J. A . Gfascl, A. C. 7: Norrh, R. J. P. Williums. A. K Xorirr. Blochim. Biophys. Acta 262, 101 (1972). [I31 P. Trunswll, J. M . Thornron, A. T.: Kordu. R . J. P. U’illiani.~, Eur. J . Biochem. 57, 135 (1975). [I41 0. D. Hensens, H . A. 0. Hill, J. Thorriron, R. J. P. Willianis, Phil. Trans. Roy. Soc. (London) 8 2 7 3 , 353 (1976). 11 51 R . H u h , D. Kirkla, M! Sriegrfnanri, J . Drirenhqfer, A . Jone.s: Bayer Symposium V. Proteinase Inhibitors. Springer, Berlin 1973. 1161 K . Ct’iirhrich. G. Wugner, FEBS Lett. 50. 265 (1975). [I71 1. D. Curuphell. C. M . Dohaon. R J . P. Wdliums, Proc. Roy. Soc. (London) 345. 23. 41 (1975). [ I S ] K . Lindrrafrum-Lung, J. A . S~hrllmunriin P. D. Boyrr, H. Lorfly, K . M ) r h u c k . The Enzymes. Vol. 1. Academic Press. Nor York 1959. p. 443 ff. [Is] J . R . Lahowirz, G. Weher, Biochemistry 12. 4161 (1973) [20] 4 . Care. C . M. Dohson, J . Porrllo, R. J . P. Williums, FEBS Lett. 65. 190 ( 1976). [?1] G. R. M o o r e . R . J . P. Willrcrms, Coord. Chem. Re>. 18. 125 (1956). [ZZ] K. J. P. Williams, Cold Spring Harbor Symp. Quant. Biol. 36. 53 (19711. [23] J . T Johunseri. B. L . W i l l e e ~Biochemistry 14, 649 11975) [24] C. M. Dohsoii. R. J. P. CtElliun~s.FEBS Lett. 56. 362 (1975). [25] 7: Inioro. L. N . Johnsor~. 4 C. T N o r t h , D. C. Pkiiiips. J. -1. Ruplr!, in P D. B o w : The Enrymcs. Vol. 7. Academic Press, New York 1972, p. 665. [26] J . P o ~ ~ l s e C. n . M . Dohsori. R J . P. Williunis, unpublished. 1271 J. IV U e i ~ h r r G. , N . Reeke. U. 4. Cuiininghurn, G. ,At. Erlchnun, Nature 259. 407 (1976). [2X] R. M! King. A . S. K Bnrgt~n.Proc. Roy. SOC. (London) B193, 107 (1976). [I]

A. S.

Aiigew Chem. l n t . Ed. Engl. 16, 766-777 ( 1 9 7 7 )

1291 C. D . B u r r ] , H . A. 0.Hill,P. J . Sudler, R. J . P. Williams, Proc. Roy. SOC.(London) A 334,493 (1973). [30] P. S . Burns. R. J . P. Williums,P. E. Wright,J. Chem. SOC.Chem. Commun. 1975. 795. [31] This problem has been studied in much detail recently by Prof. H . Frauevifelder (Hamburg Int. Biochemistry Meeting 1976). [32] M . F . Perirrz, L. F . Teneyck, Cold Spring Harbor Symp. Quant. Biol. 36. 295 (1971).

[33] [34]

R. J . P. Williams, Biochim. Biophys. Acta 416, 237 (1975). See H . Hauser, M . C . Phillipx. B. A. Leririr, R. J . P. Williums, Nature 261, 390 (1976).

1351 L. I . Bursirkor. Y. E. Sliayiro, A. V Viktoroc, A . F . Bystror. A. Bergelson, Akad. Nauk USSR 208, 717 (1973).

D.

1361 R. J . P. Williums, Physiol. Chem. Phys. 4, 427 (1972). 1371 A . Daniels. R. J . P. Williorm, P. E . Wrighr,Nature 261, 321 (1976).

C 0 M M U N I C AT1 0N S Racemic proline ( 4 )

Simple Synthesis of Racemic Proline By Ulrich Schmidt and Huns Poise/[*] Dedicated to Professor Engelbert Broda on the occasion of his 65th birthday Racemic proline has so far only been accessible by time-consuming multi-step syntheses"! We have now found a simple synthetic pathway starting from pyrrolidine: N-Chlorination, preferably with tert-butyl hypochlorite, leads to N-chloropyrrolidine ( 1 ) which is not isolated but instead reacted directly with sodium methoxide to give l-pyrroline (2). This product trimerizes even in solution within a few hours and cannot be isolated[']. However, we found it to be sufficiently stable in dilute solution t o add reactive compounds. Hydrogen cyanide adds smoothly to 1-pyrroline to form the nitrile (3) whose hydrolysis affords racemic proline

12)

ill

131

141

( 4 ) in about 457, yield (based on pyrrolidine). Hydrogen chloride must be eliminated from the N-chloropyrrolidine ( I ) prior to addition of hydrogen cyanide; direct reaction of ( 1 ) with cyanide in alkaline solution yields N-pyrrolidinecarbonitrile. For large scale preparations it is recommended that chlorination of the pyrrolidine be carried out in the two-phase system: ether (or toluene)/aqueous hypochlorite solution ~~

~~

[*] Prof. Dr. U. Schmidt and Dr. H. Poise1 Organisch-Chemisches Institut der Universitit A-1090 Wien, Wiihringerstrazse 38 (Austria)

Pyrrolidine (7.1 g, 0.10 mol) and tert-butyl hypochlorite (12.0 g, 0.11 mol) are simultaneously added dropwise to stirred, ice-cooled ether (100 ml). After 5 min the colorless solution is extracted once with dilute HCI and twice with a small volume of water. The mixture is dried with sodium sulfate, treated with sodium (2.76g, 0.12 mol) in methanol (70ml) and refluxed for 25 min, whereupon NaCl is precipitated. After cooling, a solution of anhydrous hydrogen cyanide (10 ml, 0.26mol) in ether (10ml) is added and reaction allowed to continue overnight at room temperature. The solvent is removed in a rotary evaporator and the residue dissolved in methylene chloride, washed twice with water, and dried with sodium sulfate. Vacuum distillation yields 5.6g (58 %) of ( 3 ) , b.p. 77"C/12 torr.-Compound (3) (5.5g) is heated with 19%aqueoushydrochloricacid(t00ml)for l 4 h a t 100°C in a bomb tube. After evaporation of the acid the residue is dissolved in water and decolorized with animal charcoal. After concentration the product is dried over sulfuric acid. Esterification of the salt mixture by Fischer's method with HCl/ethanol affords 6.6g (81 %) of D,L-proline ethyl ester which is allowed to stand overnight with 20ml of water. Removal of solvent leaves 5.0g [76 'i:based on ( 3 ) ] of D.L-prOline ( 4 ) . Received: September 22, 1975 [ Z 316 IE] German version: Angew. Chem. 89. $ 2 1 ( 1 977) Publication delayed at authors' request CAS Registry numbers: ( I ) , 19733-68-7; (2). 5724-81-2; (3). 57015-08-4: ( 4 ) . 609-36-9 See, e.g., K . Hasse and A. Wirlund. Chem. Ber. 93. 1686 11960); K . H . Buecliel and F . Korte, ihid. 95, 2453 (1962); R A . Srrojiii.. H . C . Whitr, and E . Sirojnj, J. Org. Chem. 27, 1241 (1962). [2] D. W Fulh~igeand C . A . liilii iler We~rf,J. Am. Chem. Soc. XO. 6249 (1958). [I]

Photochemical [2 + 21 Cycloaddition between Parallel CC and NN Double Bonds[**] By Wilfr.ied Berning and Siegfried Hiinig[*] Rigid polycyclic compounds containing two parallel C=C bonds are well known and their chemistry, particularly photochemical [2 +2] cycloaddition to cage compounds, has been ~~

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[*] Prof Dr. S. Hunig. Dr. W. Berning Institut fur Organische Chemie der Universitht Am Hubland. D-8700 Wiiriburg (Germany)

[**I From the dissertation by W Beriii!ig. Universitiit Wiirrburg 1977. This work was supported by the Fonds der Chemischeii Industrie and the BASF AG. Ludwigshafen. 177