Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci.,Vol. 8, Nos 1 & 2, August 1985, pp. 197–208. © Printed in India. Structural and mode of action studies on bio-active molecules

DUDLEY H. WILLIAMSUniversity Chemical Laboratory, Lensfield Road, Cambridge, CB2 1EW, England. Abstract. The technique of nuclear Overhauser effect difference spectroscopy allows the determination, from 1H nuclear magnetic resonance spectra, of those protons in a structure which are near in space to a selected, irradiated proton. The experiment is extremely powerful in the determination of structure in solution, and is sufficiently precise often to give stereochemical detail. The method was used in determination of the structures of the antibiotics of the teicoplanin complex (members of the vancomycin group), and the principles are briefly illustrated. Additionally, nuclear magnetic resonance pulse sequences can be used to edit 13C spectra (separate the spectrum into four spectra, containing C, CH, CH2, and CH3

carbons), and this technique also aided the structure elucidation of the teicoplanin complex. Finally, it is emphasised that nuclear Overhauser effect difference spectroscopy can be used to determine the molecular details of drug binding sites, and an example is given. Keywords. Nuclear Overhauser effect; actinomycin D; NMR spectra of oligonucleotides;teicoplanin antibiotics; DEPT edited 13C spectra.

Introduction Structural studies on organic molecules, especially those in the molecular weight range 1000–3000 Daltons, have recently been revolutionalised by the technique of nuclear Overhauser effect difference spectroscopy (nOeds).

The technique of nOeds allows the determination, from 1H NMR spectra, of those protons in a structure which are near in space to a selected, irradiated proton. Those protons which are near in space to the irradiated proton suffer a change in the intensity of their resonance signal. Since the rate of build-up of the intensity change is proportional to r –6 (where r is the distance between the irradiated proton and theproton whose change in resonance intensity is observed), the method is useful to determine approximate inter-nuclear proton-proton distances. Thus not only can structures of single components be determined, but also those of complexes, e.g. in which a drug interacts with its receptor. In this paper, work on single components is illustrated by reference to the structure elucidation of teicoplanin, and work on complexes by reference to the binding of actinomycin D to an oligonucleotide. Discussion Structure elucidation of the teicoplanin complex

The basic idea behind the use of the nOe in 1H spectra was outlined in the introductorysection. Since the technique is concerned with intensity differences (which may be only a Abbreviations used: nOeds, Nuclear Overhauser effect difference spectroscopy; NMR, nuclear magnetic resonance.

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198 Williams few per cent between irradiated and control spectra, the nOe effect is best observed by subtraction of irradiated and control spectra. After subtraction, the signals which remain [in the nOe difference spectrum (nOeds)] are due only to the irradiated proton, and those experiencing nOes.

The nOeds technique was employed quite extensively in the structure elucidation of the antibiotics of the teicoplanin complex. Teicoplanin is a mixture of similar antibiotics produced by a recently discovered species of actinomycete, Actinoplanes teichomyceticus (Parenti et al., 1978). The 5 main components of the mixture are designated A2-1, A2-2, A2-3, A24 and A2-5. The molecular weights of these separated components were determined by FAB MS. Important information on the numbers and nature of carbon atoms was then derived from DEPT 13C spectra. In this technique, (Doddrell et al., 1982; Bendali et al., 1982) the following pulse sequence is used to separate signals for CH, CH2, and CH3 carbons, spectra being recorded withpolarisation transfer from protons to carbon:

Additionally, the following pulse sequence yields 13C spectra of only quaternary (non-protonated) carbon atoms.

In the first given sequence, the carbon magnetisation fluctuates as a function of the length of the θ[H, y] pulse (where θ represents a flip-angle which is variable). The manner in which this fluctuation occurs is given in figure 1. It can be seen that the nature of the fluctuation depends on whether the carbon atom carries one, two or three attached hydrogens. Thus if θ = π/2, the carbon magnetisation associated with CH3 and CH2 groups is zero, and the resulting spectrum contains signals due to CH groups only. Combinations of spectra obtained for various values of θ can be arranged to provide spectra containing signals due to CH2 or CH3 groups only. The result ofcarrying out such experiments on A2-2 is given in figure 2 (Barna et al., 1984).

When the second given sequence was applied, the 13C spectrum shown in figure 3(a) was obtained; figure 3(b) gives the complete (normal) 13C spectrum. All these spectra were determined in CD3 CN solution, and thus only figure 3 show signals due to solvent carbon atoms.

With the relative numbers of each kind of carbon atom known, it became evident that the structure was rather similar in the aglycone portion to that of ristocetin A (which contains no CH2 groups), but that in addition it contained some 3 sugars and a hydrocarbon sidechain (Barna et al., 1984). This last point was particularly evident from the presence of no less than 10 CH2 groups in A2-2.

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Figure 1. Fluctuation of the 13C magnetisation as a function of the angle θ in the DEPT sequence.

At this stage in the structure elucidation, with available chemical evidence for the structures of a number of sub-units of the antibiotics (from degradative experiments), the1H spectra were assigned and nOeds (among other techniques) used. The nOes were actually observed in a degradation product, A3-2, of A2-2. The A3-2 product was obtained by mild acid hydrolyses of A2-2; it corresponds to an A2-2 factor which has lost both D-mannose and an N-acyl-β-D-glucosamine unit by hydrolytic cleavage ofsugars (figure 4). The patterns of nOes observed (Barna et al., 1984) were very similar to those observed for ristocetin (Kalman and Williams, 1980), in particular amongs the nest of protons z6, 6b, x6, x5, 5b, w7 on the left of the molecule (figure 4). This evidence, added to that from δ,3 JH,H’ and Δδ / ΔTvalues, means that one can be confident that thestereochemistry is the same at all analogous asymmetric centres in the teicplanins andthe ristocetins.

200 Williams

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Structural studies on bio-active molecules 201

202 Williams

A specific example of the use of nOes is to be found in an aid to the determination of the stereochemistry and location of the sugar R of A3-2 (figure 4). On irradiation of Hl, nOes are seen to H3 and H5, as expected for the β-anomer of a glucose unit (with appropriate 1, 3-diaxial interactions, see scheme 1). Also in the same experiment an nOe is observed to z6, establishing the attachment of scheme 1 to the benzylic oxygen atom (figure 4).

Scheme 1 The interaction of drugs with DNA

Since the proton–proton nOe is strongly dependent on inter-proton distance, it can be used in the study of drug-receptor interactions. However, it is necessary that the receptor be studied as a compound of limited molecular weight. This restriction immediately suggested that drug-DNA interactions might be probed, since short, complementary sequences of DNA are now readily available through synthesis. We therefore decided to explore (i) the assignment of the proton nuclear magnetic resonance (NMR) spectra of short (4–8 residues) DNA duplexes (therefore containing 8–16 bases) using nOes and (ii) the use of inter-molecular nOes to determine the molecular basis for drug/DNA interactions.

When working with DNA duplexes, it is necessary to work in aqueous solution, since other solvents may disrupt the duplex. Our initial work (Reid et al., 1983a) was, therefore, carried out in 99.98 % enriched D2O. Although such studies provide a wealth of information, important information is lost due to the absence of NH resonances, which are frequently important in biological interactions. To overcome this problem, current work is being carried out in H2O with the use of multi-pulse sequences to suppress the water resonance efficiently.

It was found that nOes were very powerful in assigning, in D2O, the aromatic, anomeric (Η-1'), and C-2' protons (H-2' and H-2") in the 400 MHz proton spectrum of the self-complementary octanucleoside heptaphosphate d(G-G-T-A-T-A-C-C) (Reid et al., 1983a). The experiments were carried out under conditions in which duplex formation takes place (at 10°C). The duplex has a relatively high molecular weight (approaching 5000 Daltons), and, therefore, tumbles in solution relatively slowly. Under these circumstances, the energy provided by the pre-irradiation which induces the nOe passes inefficiently into other modes. Rather, it remains in the molecule and, by diffusing from proton to proton, perturbs the spin populations of protons at increasing distances from the originally excited proton as time passes. This process is known as spin diffusion (Kalk and Berendsen, 1976). Thus, for long pre-irradiation time, the nOe is no longer found simply for protons in close proximity. In order to re-establish this

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Figure 4. The structure of teicoplanin and its acid hydrolysis products, showing the protonnomenclature.

situation, it is necessary to use short pre-irradiation times (0·1–0·5 s), and follow the build-up of the nOe as a function of time. Those nOes which build-up most quickly denote the smallest inter-proton distances (Wagner and Wütherich, 1979).

The principle of the technique may be illustrated by taking one simple example from the assignment (Reid et al., 1983b) of the proton spectrum of the self-complementary tetranucleoside triphosphate d(A-G-C-T). At 0°C, this exists primarily as a duplex. The protons of the bases can be assigned largely on the basis of chemical shifts, coupling constants, and T1 values, but assignment of the anomeric protons (Η-1' protons, see scheme 2) is not possible in this way. Experience shows that in a right-handed helix, irradiation of an anomeric proton gives an nOe to the proton anti to it in the base of the same nucleotide residue, i.e. CH-1' → CH-6, GH-1' → GH-8 , AH-1' → AH-8 in scheme 2; andalso to the corresponding proton in the adjacent base moving along the nucleotide in [

204 Williams the 3'-direction, i.e. AH-1'

→ GH-8, GH-1' → CH-6 in scheme 2. Thus, as can be seen from theschematic illustration of the fragment scheme 2, irradiation of the AH-8 proton gives an nOe to only one anomeric proton which must be that of its own residue. When AH-1' is inturn irradiated, nOes are observed to both AH-8 and GH-8, while irradiation of GH-8 gives nOes to both AH-1' and GH-1'. The double headed arrows in scheme 2 indicate theobservation of mutual nOes, and thus all the anomeric protons can be assigned.

Scheme 2

In no reasonable right-handed helical conformation does any Η-1' approach closer than 4·0 A to a 2'-proton (see the C residue in scheme 2) on any other deoxyribosyl residue. Thus, nOes from Η-1' signals identify the vicinal methylene pair exclusively. Additionally, their relative build-up rates make distinction between the two geminal methylene protons possible. In general., a faster rate of build-up of the nOe to H"-2' from H-l' than to H'-2' from H-l', is observed (Reid et al., 1983a). Although the methylene region of d(G-G-T-A–T-A-C-C) gives only seven resolved signals at 10°C, it is possible to assign specifically the eight H'-2' and the eight H"-2' protons among these seven resolved signals.

With techniques for the secure assignment of most of the protons in a short duplex established, we were then in a position to analyze the DNA binding site of an antibiotic. We chose the antibiotic actinomycin D (figure 5), to bind to the duplex d(A-G-C-T) since (i) the interaction of this antibiotic with nucleic acids is one of the most studied (Remers, 1978) (and, therefore, provides a reasonable test of the methods), (ii) despite all this study, it had never before proved possible to probe, by a high resolution method,

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Structural studies on bio-active molecules 205

Figure 5. Structure of actinomycin D. the interaction in solution of actinomycin D with a DNA-duplex, and (iii) the antibiotic binds by intercalation and its preference for G-C rich DNA gave a good probability that it would intercalate between the two G-C base pairs of d(A-G-C-T). The model for the binding of actinomycin D to double helical DNA is derived from an X-ray crystal structure of the antibiotic complexed to deoxyguanosine (Sobell and Jain, 1972; Sobell et al., 1977).

Since we had the 1H spectrum of the d(A-G-C-T) duplex adequately assigned, we next required the assignment of the bound nucleotide and bound actinomycin D resonances. This was achieved by analysis of the 1H spectrum of a mixture of the helix and antibiotic in the ratio 2:1 (Reid et al., 1983b). The intercalated antibiotic stabilizes the helix, so that at 30°C, one set of signals can be seen due to the complex (1:1), and a second set of sharper signals due to 1 part of free helix. Evidently, the free and bound helix are in slow exchange on the NMR timescale. The signals due to the bound helix can then be assigned by saturation transfer. In this experiment, a resonance of the free helix is irradiated, and the signal is, therefore, effectively removed from the spectrum by [[[[

206 Williams removing the population differences that exist at equilibrium. Before this equilibrium is re-established, the helix passes from the free to the bound state, with the result that the corresponding resonance in the bound state is reduced in intensity. Thus, the free and bound resonances are correlated. Once this has been done, the helix: antibiotic ratio is decreased to 1:1 by addition of more actinomycin D, and nOes determined in the complex.

The results show that interaction of the actinomycin aromatic chromophores (figure 5) does indeed Occur between the G-C pairs. This increases the perpendicular distance between adjacent G and C residues, which is evidenced by the abolition of nOes between these two residues in the complex, although they are seen in the free nucleotide. Positive evidence to support the same conclusion is evident from figure 6. In this figure, the methyl groups attached to the actinomycin chromophore (figure 5) are coded by the letters b and c. NOes are found between these methyl groups and the guanine H-8 protons (g and f), and the cytosine H-6 protons (d and h). Additionally, H-7 (a) of the phenoxazone ring (figure 5) is involved in nOes with H-5 and H-6 of cytosine (e and d) and H-8 of guanosine (f).

Figure 6. View of the antibiotic-duplex complex into the major groove. The antibioticprotons are identified by diagonal hatching. Arrows connect antibiotic and nucleotide protonsbetween which nOes are observed.

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The orientation of the peptide portion of the antibiotic in the minor groove is also evident (figure 7). Irradiation of H-2 of adenine (see i) gives a large nOe to the N-methyl group (k) of N-methyl-L-valine. and a smaller nOe to the side chain methyl group (j) of the same amino acid. Irradiation of the anomeric proton Η-1' (1) of the thymine gives an nOe to the N-methyl group (m) of sarcosine. These, and a number of other nOes, are sufficient to define the molecular details of the interaction in solution, and give further support to the model proposed by Sobell and co-workers (Sobell and Jain, 1972; Sobell et al., 1977).

Figure 7. View of the antibiotic-duplex complex into the minor groove. The antibioticprotons are identified by diagonal hatching. Arrows connect antibiotic and nucleotide protonsbetween which nOes are observed.

In the study of biological interactions, an ability to identify readily the NH resonances of amide groups is important. Since DNA duplexes must be examined in aqueous solution, and as the NH-resonances are lost in D2O solution, it is important to be able to work in H2O. As an alternative to pulse techniques which selectively suppress the H2O resonance, using its characterized chemical shift or relaxation time, we have developed a pulse sequence for selective observation only of those protons directly bonded to 15N (Doddrell et al., 1983; Reid et al., 1983c). When molecules of interest can

208 Williams be readily biosynthesized enriched with 15N, the technique may be useful for probing inter-molecular interactions in H2O.

There was considerable justification for the view that for complex biological structures, in the 1970’s NMR spectroscopy in many cases merely gave support, in solution, for structures previously established by X-ray methods. This is certainly no longer the case; NMR is now making extremely important contributions in its own right, and appears likely to do so to an even larger extent in the future. Acknowledgements The author wishes to thank past and present members of his research group, names in the references to the work carried out in Cambridge, for their contributions to the research. SERC (UK) and the Royal Society (London) are thanked for financial support. References Barna, J. C. T., Williams, D. H., Stone, D. J. Μ., Leung, C. T-W. and Doddrell, D. M. (1984) J. Am. Chem. Soc.

(in press). Bendall, M. R., Pegg, D. T., Doddrell, D. M., Johns, S. R. and Willing, I. (1982) J. Chem. Soc. Chem. Commun.,

1138. Doddrell, D. M., Pegg, D. T. and Bendall, M. R. (1982), J. Mag. Res., 48, 323. Doddrell, D. M., Williams, D. H., Reid, D. G., Fox, K. and Waring, M. (1983) J. Chem. Soc. Chem. Commun.,

218. Kalk, A. and Berendsen, H. J. C. (1976) J. Mag. Res., 24, 343. Kalman, J. R. and Williams, D. H. (1980) J. Am. Chem. Soc., 102, 897. Parenti, F., Beretta, G., Berti, Μ. and Arioli, V. (1978) J. Antibiot., 31, 276. Reid, D. G., Salisbury, S. Α., Bellard, S., Shakked, Z. and Williams, D. H. (1983a) Biochemistry, 22, 2019.Reid, D. G., Salisbury, S. A. and Williams, D. H. (1983b) Biochemistry, 22, 1377. Reid, D. G., Doddrell, D. M., Fox, K. R., Salisbury, S. A. and Williams, D. H. (1983c) J. Am. Chem. Soc., 105,

5945. Remers, W. A. (1979) in The Chemistry of Antitumor Antibiotics, (New York: Wiley) Vol. 1. Sobell, H. M. and Jain, S. C. (1972) J. Mol. Biol., 68, 21. Sobell, Η. Μ. and Tsai, C-C., Jain, S. C. and Gilbert, S. G. (1977) J. Mol. Biol., 114, 333. Wagner, G. and Wutherich, J. (1979) J. Mag. Res., 33, 675.