H. Harry Szmant
Department of Chemistry • University of Detroit • Detroit, Michigan 48221

The unique capability of dimethyl sulfoxide (DMSO) to penetrate living tissues without causing significant damage is most probably related to its relatively polar nature, its capacity to accept hydrogen bonds, and its relatively small and compact structure. This combination of properties results in the ability of DMSO to associate with water, proteins, carbohydrates, nucleic acid, ionic substances, and other constituents of living systems. Of foremost importance to our understanding of the possible functions of DMSO in biological systems is its ability to replace some of the water molecules associated with the cellular constituents, or to affect the structure of the omnipresent water.

The work discussed in this paper addresses itself to the latter point, and is based on the study¹ of the liquid system water-DMSO by means of the spin-lattice relaxation and chemical shift behavior of both the water and DMSO protons. The binary system was investigated over a relatively large temperature range, and the relaxation times (t₁) and chemical shifts were determined as a function of the concentration of the two components and of their deuterated analogues.

The proton spin-lattice relaxation time is a measure of the energy decay of an array of excited hydrogen nuclei. The predominant mechanism of the dissipation of energy involves magnetic dipole-dipole interactions between the nuclei of neighboring protons. Since this principal relaxation path is very sensitive to the distance that separates the interacting protons (a sixth-power relationship applies), the experimentally observed changes in the t₁ values reflect the dynamic changes that occur in the stereochemical environment of the protons under consideration as one varies the composition and the temperature of the system. Changes in the structure of the liquid can be inferred to have occurred from the experimentally determined t₁ values, since the reciprocal of t₁ is directly proportional to the molecular correlation time (_c), which in turn represents the time required for a complete rotation of the molecular moiety that contains the protons under consideration. Thus maxima in the plots of 1/t₁ versus composition, for example, reveal an increased structuring of the system, which may involve attractions between the molecular moieties that contain the interacting protons, hence prolonging their correlation times. It is of interest to note that the relaxation of deuteron nuclei results from the interaction not of nuclear dipoles, but rather of nuclear quadrupoles, and consequently the deuteron relaxation mechanism involves only intramolecular interactions. One can take advantage of this difference in the behavior of protons and deuterons by examining mixtures of the analogous isotopic substances; in this way one can separate the intra- and intermolecular contributions to the relaxation process. It stands to reason that a decrease in the concentration of the protonated species dissolved in its deuterated counterpart gradually eliminates the intermolecular contributions to the total relaxation time, and that extrapolation of the relaxation times to an infinitely dilute solution of the protonated species, dissolved in the magnetically relatively inert counterpart, allows one to evaluate the intramolecular t₁ or _c values.

The results discussed here are based on work1 with four liquid systems: water and DMSO; water and DMSO-d₆; deuterium oxide and DMSO; and finally, mixtures of DMSO, DMSO-d₆ and deuterium oxide. The relaxation behavior of each kind of proton was examined separately by means of the saturation recovery method; a modified Varian A60A NMR spectrometer was used.

The addition of DMSO to water, or vice versa, raises the 1/t₁ values of both the water and DMSO protons, and both protons exhibit maximum values at ca. XH2O=0.65. The effect of the admixture of the other component, however, is approximately 30% larger in the case of the protons of water, even though a priori the absolute 1/t₁ values of the water protons are about twice as high as the 1/t₁ values of DMSO. These results imply that a molar ratio of water and DMSO of approximately 2:1 produces the greatest restriction on the rotational freedom of the components, and that such a restriction is more pronounced in the case of water, even though the latter is presumably a more highly structured liquid than DMSO. Naturally, a decrease in the thermal agitation of the molecular system causes the 1/t₁ maxima to develop to a higher degree, but it is noteworthy that the 1/t₁ maxima are discernible even at the highest temperature employed here (41.4° C), and again they are more so in the case of the water protons. The structural implications of these observations can be stated as follows. It appears that DMSO induces a more intensive structuring of water than vice versa, and the concentration dependence of this phenomenon suggests that three DMSO molecules are very effective in producing a highly structured cluster of six water molecules. The decision to translate the 2:1 molar ratio to structural units that involve six water and three DMSO molecules is based on the well-recognized structural unit of ice I, which consists of six water molecules arranged in a chair-like conformation. Since similar ice-like clusters of water are believed to represent, at least in part, the highly structured portion of liquid water, the above-mentioned results of the relaxation measurements suggest that DMSO molecules are able to "lock in" the hexameric water clusters, and thus to increase their concentration above and beyond that present in neat water at a given temperature.

The replacement of DMSO by DMSO-d₆ does not have an appreciable effect on the relaxation times of the water protons except at the lowest temperatures employed (18.7° C and 9.8° C), and then only in the water-rich mixtures. This observation is consistent with the structural model described above, since it places the burden of relaxation on interactions among the water protons, and allows for a high degree of rotational freedom of the DMSO molecules, even when they function to "lock in" the water clusters.

We shall turn our attention for a moment to those proton relaxation results that have a bearing on the liquid structure of DMSO. The replacement of water by deuterium oxide has a relatively large effect on the relaxation of the DMSO protons, and this effect persists over the whole temperature range of 9.8-41.4° C, although it is more pronounced in the water-rich mixtures. On the basis of these observations one may draw the conclusion that, apart from the intramolecular proton interactions, the relaxation mechanism of liquid DMSO involves intermolecular proton interactions to a relatively minor extent, and the presence of water is able to promote the latter. These conclusions are consistent with the concept that DMSO has a predominantly liquid structure in which pairs of methyl groups of neighboring DMSO molecules point away from each other, regardless of whether the molecules are associated in the form of chains or large rings.³ The introduction of water molecules disrupts this arrangement and finally brings about the proposed DMSO-stabilized, ice-like water clusters, in which the DMSO molecules that are hydrogen-bonded to the same cluster (or aggregate of clusters) can bring their methyl groups into close proximity. The determination of the DMSO proton relaxation times in mixtures of DMSO, DMSOd₆ and D2O allows one to separate the intra- and intermolecular contributions. It is found that in the temperature range of 29.9-41.4° C, the intramolecular interactions contribute nearly twice as much as the intermolecular interactions between DMSO molecules, and the presence of D 2O has relatively little effect on these contributions. On the other hand, in the lower temperature range of 9.8-18.7° C, the magnitude of the intermolecular interactions approaches that of the intramolecular ones, and this happens to the greatest extent when XD2O=0.6.

Unfortunately, because of the very rapid hydrogen-deuterium exchange in mixtures of water and deuterium oxide, it was impossible to separate the intra- and intermolecular contributions to the relaxation of the water protons. The chemical shifts of the water protons in the presence of DMSO were determined over a temperature range from -54.2 C to 41.4° C. The relatively greater chemical shift (downfield) of the water protons observed in a water-rich environment suggests that this circumstance is conducive to the formation of linear hydrogen bonds of polar character. The latter are associated with the highly structured, ice-like water, and thus it is not surprising that the chemical shifts also increase as the temperature is lowered. Since the chemical shifts of the water protons at the other extreme condition of a water molecule in a DMSO environment arc also known, it is possible to calculate the chemical shifts that would result from simple, statistical contributions of both proton environments in accord with the composition of the mixture. Any deviation of the observed chemical shifts from the calculated shifts indicates preferential structuring of the liquid, and the direction of a given deviation indicates the type of liquid structure that is preferred. The application of such a treatment to the chemical shifts of water protons in the presence of DMSO reveals the existence of two such deviations. At low concentrations of water, there is a deviation of the chemical shifts in the direction of the DMSO-rich values; it is relatively temperature-insensitive, and reaches a maximum when the water and DMSO molecules are at a molar ratio of 1:2. This result reveals that in relatively DMSO-rich mixtures, the water molecules tend to become doubly hydrogen-bonded to DMSO. The formation of 1:2 water-DMSO complexes has been reported previously,³ and is probably of little interest in biological systems, because it represents a mixture that contains only ca. 10% water. Another deviation in chemical shifts was found, however, in the range of high concentrations of water. The direction of this deviation was toward the chemical shift values of neat water; it was rather sensitive to the temperature of the liquid system, and it exhibited a maximum when the water and DMSO molecules were at a molar ratio of 3:1. This observation again agrees with the previously proposed effect of DMSO on the structure of water. Even though the maximum deviation in the chemical shift corresponds to a mixture that contains only ca. 41% water, the buildup of the structure that gives rise to this deviation begins as soon as DMSO is introduced into water. Thus it seems that even low concentrations of DMSO are able to promote the accumulation of ice-like water clusters, and that this tendency becomes more intense at the lower temperatures. On the basis of chemical shifts, the DMSO-induced structuring of water comes to a climax when water and DMSO are at a molecular ratio of 6:2, while the relaxation results suggest a molecular ratio of 6:3. This discrepancy can be resolved by considering the accuracy of the basic data and the sensitivity of both criteria to structural changes: the preference falls on the molecular ratio of 6:2 (this is deduced from the chemical shift measurements). The latter molecular ratio happens to coincide with the composition of a compound that melts at -62° C, which has been detected4 in the phase diagram of the water-DMSO system.

Eight years ago The New York Academy of Sciences sponsored a conference on the subject of forms of water in biological systcms,⁵ and the structure of water, the changes induced in its structure by the presence of different solutes, and the biological implications of the different states of water were of great concern to the participants. The evidence presented here suggests that DMSO stabilizes ice-like water clusters, and that it may therefore be capable of displacing the equilibrium between the less and more highly structured water, in favor of the latter. Since the hydration of cell constituents and the activity of water in general are not necessarily the same in the different states of water, it follows that DMSO may exert an indirect effect on biological systems by virtue of the changes that it causes in the liquid structure of water. Among the more important biological consequences of this indirect effect of DMSO, one can mention changes in the conformations and associations of proteins and other molecules. More direct biological effects caused by DMSO, without a profound change in its chemical identity, may include changes in ion-pairing equilibria and in the specific solvation of hydrogen-bond donors.

REFERENCES

1. Tokuhiro, T., L, Memefra &, H. H. Szmant. 1974. J. Chem. Phys. In press.
   
2. Eisenberg, D. & W. Kauzmann. 1969. The Structure and Properties of Water. Oxford University Press, Inc. New York, N.Y.
   
3. Szmant, H. H. 1971. In Dimethyl Sulfoxide. S. W. Jacob, E. E. Rosenbaum & D. C. Wood, Eds. Marcel Dekker Inc. New York, N.Y.
   
4. Rasmussen, D. H. & A. P. MacKenzie. 1968. Nature 220: 1315.
   
5. Whipple, H. E., Ed. 1965. Ann. N.Y. Acad. Sci. 125(2): 249.