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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 t o the latter point, and is based
on the study' of the liquid system water-DMSO by means of the spin-lattice relaxa-
tion and chemical shift behavior of both the water and DMSO protons. The binary
system was investigated over a relatively large temperature range, and the relaxa-
tion times ( t l ) and chemical shifts were determined as a function of the concen-
tration of the two components and of their deuterated analogues.
The proton spin-lattice relaxation time is a measure of the energy decay of an ar-
ray of excited hydrogen nuclei. The predominant mechanism of the dissipation of
energy involves magnetic dipole-dipole interactions between the nuclei of neigh-
boring protons. Since this principal relaxation path is very sensitive t o the distance
that separates the interacting protons (a sixth-power relationship applies), the ex-
perimentally observed changes in the t l values reflect the dynamic changes that oc-
cur 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 t I
values, since the reciprocal of t l is directly proportional to the molecular correlation
time (rc),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 l / t l 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. I t is of in-
terest 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 ad-
vantage 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 t o an infinitely
dilute solution of the protonated species, dissolved in the magnetically relatively
inert counterpart, allows one to evaluate the intramolecular 1 , or 7,values.

which consists of six water molecules arranged in a chairlike conformation. the highly structured portion of liquid water.4”C). Naturally. maxima are discernible even at the highest temperature employed here (41.65. The replacement of water by deute- rium oxide has a relatively large effect on the relaxation of the DMSO protons. does not have an appreciable effect on the relaxation times of the water protons except at the lowest temperatures em- ployed (18. The relaxation behavior of each kind of proton was examined separately by means of the saturation recovery method. The replacement of DMSO by DMSO-d. is approximately 30% larger in the case of the protons of water. in which the DMSO molecules that are hydrogen-bonded to the same cluster (or aggregate of clusters) can bring their methyl groups into close . These results imply that a molar ratio of water and DMSO of approximately 2:l produces the greatest restriction on the rotational freedom of the components. ice-like water clusters. On the basis of these observations one may draw the conclusion that. and thus to increase their concentration above and beyond that present in neat water at a given temperature. DMSO-d. even though the lat- ter is presumably a more highly structured liquid than DMSO. and the presence of water is able to promote the latter. but it is noteworthy that the I / t . The addition of DMSO to water. and allows for a high degree of rotational freedom of the DMSO molecules. water and DMSO-d. mix- tures of DMSO. the relaxation mechanism of liquid DMSO involves intermolecular proton interac- tions to a relatively minor extent. apart from the intramolecular proton interactions. and then only in the water-rich mixtures. and finally. Szmant: Physical Properties and Biological Function 21 The results discussed here are based on work’ with four liquid systems: water and DMSO.. It appears that DMSO induces a more intensive structuring of water than vice versa.4”C.7”C and 9. regardless of whether the molecules are associated in the form of chains or large rings. The effect of the admixture of the other component. We shall turn our attention for a moment to those proton relaxation results that have a bearing on the liquid structure of DMSO. The structural implications of these observations can be stated as follows. X H ~ O = 0. at least in part. This ob- servation is consistent with the structural model described above.8-41. a modified Varian A60A NMR spectrometer was used. however. deuterium oxide and DMSO. even when they function to “lock in” the water clusters. and deuterium oxide. 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-recognizedz structural unit of ice I.3The introduction of water molecules disrupts this arrangement and finally brings about the proposed DMSO-stabilized.8”C). and this effect persists over the whole temperature range of 9. a decrease in the thermal agitation ofthe molecular system causes the I/tl maxima to develop to a higher degree. 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. although it is more pronounced in the water-rich mixtures.. and that such a restriction is more pronounced in the case of water. values of the water protons are about twice as high as the l / t l values of DMSO. even though apriori the absolute 111. and both protons exhibit maximum values at ca. since it places the burden of relaxation on interactions among the water protons. or vice versa. and again they are more so in the case of the water protons. These conclusions are consistent with the concept that DMSO has a pre- dominantly liquid structure in which pairs of methyl groups of neighboring DMSO molecules point away from each other. the above-mentioned results of the relaxation measurements suggest that DMSO molecules are able to “lock in” the hexameric water clusters. raises the I / t l values of both the water and DMSO protons. Since similar ice-like clusters of water are believed to represent.

22 Annals New York Academy of Sciences proximity. statistical contributions of both proton environments in accord with the com- position of the mixture.~ and is probably of little interest in biological systems. while the relaxation results suggest a molecular ratio of 6:3. On the basis of chemical shifts. because of the very rapid hydrogen-deuterium exchange in mix- tures of water and deuterium oxide. Another deviation in chemical shifts was found.O=0. and it exhibited a maximum when the water and DMSO molecules were at a molar ratio of 3: 1. it was impossible to separate the intra. however. and that this tendency becomes more intense at the lower temperatures. . the intramolecular interactions contribute nearly twice as much as the intermolecular interactions between DMSO molecules. the water molecules tend to become doubly hy- drogen-bonded to DMSO. The latter molecular ratio happens to coincide with the composition of a compound that melts at -62"C. and thus it is not surprising that the chemical shifts also in- crease as the temperature is lowered. ice-like water. and DzO allows one to separate the intra. Unfortunately. The determination of the DMSO proton relaxation times in mixtures of DMSO. At low concentrations of water. It is found that in the temperature range of 29. the DMSO-induced structuring of water comes to a climax when water and DMSO are at a molecular ratio of 6:2. and the presence of D 2 0 has relatively little effect on these contributions. and the direction of a given deviation indicates the type of liquid structure that is preferred. it was rather sensitive to the temperature of the liquid system. The formation of 1:2 water-DMSO complexes has been reported previ~usly.and intermolecular contributions. Since the chemical shifts of the water protons at the other extreme condition of a water molecule in a DMSO environment are also known. The chemical shifts of the water protons in the presence of DMSO were de- termined over a temperature range from -54. DMSO-d. the buildup of the structure that gives rise to this deviation begins as soon as DMSO is introduced into water. The direction of this deviation was toward the chemical shift values of neat water. This dis- crepancy 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). there is a deviation of the chemical shifts in the direction of the DMSO-rich values.6.8-18.2"C to 41.7"C.9-41.4"C. it is relatively temperature-insensitive. 10% water. in the lower temperature range of 9. This observation again agrees with the previously proposed effect of DMSO on the struc- ture of water. in the range of high concentrations of water.and in- termolecular contributions to the relaxation of the water protons. Any deviation of the observed chemical shifts from the cal- culated shifts indicates preferential structuring of the liquid. Even though the maximum deviation in the chemical shift corre- sponds to a mixture that contains only ca. which has been detected' in the phase diagram of the water-DMSO system. it is possible to calculate the chemical shifts that would result from simple. Thus it seems that even low concentrations of DMSO are able to promote the accu- mulation of ice-like water clusters. The ap- plication of such a treatment to the chemical shifts of water protons in the presence of DMSO reveals the existence of two such deviations. and reaches a maximum when the water and DMSO molecules are at a molar ratio of 1:2. The relatively greater chemical shift (downfield) of the water protons observed in a water-rich environ- ment suggests that this circumstance is conducive to the formation of linear hydrogen bonds of polar character. and this happens to the greatest extent when XD. because it represents a mixture that contains only ca. On the other hand. This result reveals that in relatively DMSO-rich mixtures.. the magnitude of the intermolecular interactions approaches that of the intramolecular ones.4"C. 41% water. The latter are associated with the highly struc- tured.

5. 3. Phys. WHIPPLE. RASMUSSEN. in favor of the latter. EISENBERG. Oxford University Press. H. 1968. Y . TOKUHIRO. T. New York.. 1974. W. 125(2): 249. N. the changes induced in its structure by the presence of different solutes. Since the hydration of cell constituents and the activity of water in general are not necessarily the same in the different states of water. Szmant: Physical Properties and Biological Function 23 Eight years ago The New York Academy of Sciences sponsored a conference on the subject of forms of water in biological system^. D. 2. S. Ed.Acad. In press. P. Jacob. H. Inc. 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. The Structure and Properties of Water. Eds. Chem.^ and the structure of water. H. E. E. and that it may therefore be capable of displacing the equilibrium between the less and more highly structured water. MACKENZIE. The evidence presented here suggests that DMSO stabilizes ice-like water clusters. Nature 220: 1315. 1965. C. Wood. Among the more important biological consequences of this indirect effect of DMSO. & W. H. without a profound change in its chemical identity. E. 1971. and the bio- logical implications of the different states of water were of great concern to the par- ticipants. In Dimethyl Sulfoxide. Sci. SZMANT. N . Rosenbaum Kc D. REFERENCES 1 ..Y. one can mention changes in the conformations and associations of proteins and other molecules. H. SZMANT. More direct biological effects caused by DMSO. N . Marcel Dekker Inc. 1969. L. New York. may include changes in ion-pairing equili- bria and in the specific solvation of hydrogen-bond donors. . & A . Y . D. MENAFRA & H. J. KAUZMANN. Ann. 4.