Deuterium isotope effects on noncovalent interactions between molecules

Chemico-Biological Interactions 117 (1999) 191–217

Review article

Deuterium isotope effects on noncovalentinteractions between molecules

David Wade *

Faculty of Medicine, Department of Biochemistry, Kuwait Uni6ersity, PO Box 24923,13110 Safat, Kuwait

Accepted 22 October 1998

Abstract

The topic of deuterium isotope effects is usually concerned with the effects on chemicalreactions that are caused by the substitution of deuterium atoms for protium, or hydrogen,atoms in a molecule. These effects include changes in the rate of cleavage of covalent bondsto deuterium, or to an atom located adjacent to deuterium, in a reactant molecule.Deuterium isotope effects on other, noncovalent, interactions between molecules are knownto occur, but they are generally considered to be insignificant, especially in biologicalexperiments where deuterium substituted molecules are used as tracers. Noncovalent interac-tions between molecules include hydrogen bonding, and ionic and van der Waals interac-tions. This article reviews evidence for deuterium isotope effects on noncovalent interactions,with an emphasis on binding interactions between molecules of biological interest, but alsoincluding examples of nonbiological molecules in order to demonstrate the generality of theseeffects. The reality of this effect relies on the assumption that the only difference between theisotopomers considered is the presence of deuterium or hydrogen; there are no impuritiespresent. The physical basis of the effect may be due to differences in the polarities and/orsizes of deuterated versus nondeuterated isomers, and the extent of a deuterium isotope effecton a noncovalent interaction depends on the site of deuteration within a biomolecule. The

* Tel.: +965-5312300; fax: +965-5338908; e-mail: [email protected].

0009-2797/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.

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D. Wade / Chemico-Biological Interactions 117 (1999) 191–217192

presence of this effect requires careful interpretation of results obtained in experiments withdeuterium labeled compounds. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Deuterium; Isotope effects; Noncovalent interactions

1. Introduction

1.1. The isotopes of hydrogen, and their disco6ery

Hydrogen, the most abundant element in the universe and the first member of theperiodic table of elements, has three naturally occurring isotopes [1]: protium (1H),composed of a proton and an electron; deuterium or heavy hydrogen (D, 2H),composed of a proton, a neutron, and an electron, and which is stable; tritium (T;3H), composed of a proton, two neutrons, and an electron, and which is unstableand radioactive (b emitter; half-life=12.3 years). The relative abundance of thesethree isotopes on Earth are: protium, 99.985%; deuterium, 0.015%; tritium, traces.

According to the big bang model of the early universe, all of the deuterium inexistence was made within the first few minutes of the beginning of the universe,and any deuterium present now is a remnant of that event [2].

In 1932, Harold Urey and colleagues discovered deuterium, for which Urey wasawarded the Nobel Prize in Chemistry in 1934 [3]. This was done by collecting gasthat evaporated from liquid hydrogen at reduced pressure, slightly above the triplepoint of hydrogen (−259.2°C; 54 mmHg), and then examining the atomic spectraof the gas for the presence of spectral lines that had been previously predicted foreach of the hydrogen isotopes. Spectral lines were found for protium and deu-terium. Within the same year as Urey’s discovery, Cremer and Polyani predictedthat there would be differences in the rates of cleavage of carbon–hydrogen (C–H)and carbon–deuterium (C–D) bonds [4]. The following year, Eyring and Shermancalculated that the physical basis for Urey’s success in isolating deuterium was dueto the low temperature at which the procedure was done, a temperature at whichvan der Waals forces (i.e. long range polarizations) predominated. They alsopredicted that it would be possible to determine reactions involving hydrogen inbiological processes by differences in the rates of these reactions in the presence ofthe light and heavy isotopes [5].

1.2. Isotope effects (IEs) in6ol6ing co6alent bonds

The purpose of this paper is to discuss IEs on noncovalent interactions betweenbiological and other molecules, but it will be useful to briefly review the theory ofIEs on covalent interactions [6] in order to contrast these effects with those onnoncovalent interactions. Additional information about kinetic IEs on enzymecatalyzed reactions can be obtained from the reviews of Northrup [7] and Cleland[8], and information about their uses in the study of cytochrome P450 catalyzedreactions has been reviewed by Gillette and colleagues [9].

D. Wade / Chemico-Biological Interactions 117 (1999) 191–217 193

Kinetic isotope effects (KIEs) are changes in the reaction rate (k) that occurwhen a hydrogen (H) atom is replaced with a deuterium (D) or tritium atom in thereacting molecule, and they are categorized as either primary or secondary.

Reactant-(H/D)�k

Product-(H/D); kH"kD

A primary KIE is a change in the rate that occurs when the covalent bond to theisotopic atom is broken or formed in the rate determining step (RDS) of thereaction.

Reactant-(D)�k 1�k 2

(KDS)

�k 3

Product; k2H"k2

D

The physical basis of primary KIEs is the greater energy required to break or forma covalent bond to deuterium (or tritium) versus a covalent bond to hydrogen. Thedifference in bond energies is due to the greater mass of D than of H, which resultsin a lower vibrational frequency for bonds to D. The zero-point energy (ZPE), orthe vibrational energy remaining at 0 K, is lower for the C–D bond than for theC–H bond. It has been calculated that the rates of cleavage of C–H, N–H, andO–H bonds are 7, 8.5, and 10.6 times faster than the analogous rates for C–D,N–D, and O–D [1].

Approximately 79% of the elements that are essential for life exist in multiple,stable (nonradioactive) isotopic forms [10,11]. As mentioned above, the maximum

Table 1Selected results for the binding of phenobarbital, caffeine, theophylline, and deuterated isomers, tohuman serum albumin (HSA)

% Bound of 50Affinity constant [Ka (M−1)]Number of binding sitesCompoundmM doseon HSA (mean9S.D.)

Phenobarbitala 5249463 58Phenobarbital- 3 6089136 54

ethyl d5a

2 942990 46Phenobarbital-phenyl d5

a

1Caffeineb 486935 254059742 27Caffeine-1 CD3

b

2Caffeine-3 CD3b 362955 33

2Caffeine-1,7 284926 27(CD3)2

b

42Caffeine-3,7 2.5 267944(CD3)2

b

464609173Caffeine-1,3,7(CD3)3

b

Theophyllinec 492 8559101Theophylline-1 3.5 51486969

CD3c

a From Table II of [14].b From Table III of [14].c From Table III of [17].


Table 2Dimerization constants for caffeine isotopomersa

K (M−1) (mean9S.D. or range)Caffeine isotopomer

Caffeine 4.698 (S.D.=90.484)2.393 (SD=90.279)1 CD3

3.287 (SD=90.207)3 CD3

2.239 (SD=90.660)7 CD3

3.363 (R=90.030)1,3 (CD3)2

1,7 (CD3)2 3.705 (R=90.044)3.880 (R=90.514)3,7 (CD3)2

3.1961,3,7 (CD3)3

a From Part Four, Table I of [23].

primary KIE (kH/kD) for the cleavage of a bond to hydrogen has been calculatedto be 7, whereas those for carbon (kC12/kC13), nitrogen (kN14/kN15), oxygen (kO16/kO18), sulfur (kS32/kS34), and chlorine (kCl35/kCl37) range from 1.01 to 1.07 [6]. Thelarge difference between the effect for hydrogen and the effects for the otherbiologically important elements is due to the large differences in mass betweenhydrogen and deuterium, in contrast to the small differences in mass between theother elements and their isotopes.

A secondary KIE is a change in the overall reaction rate that occurs when thecovalent bond to the isotopic atom remains intact during the reaction. SecondaryKIEs may occur,

Reactant-(D)�k

Product-(D); kH"kD

for example, when D is located at bonds adjacent to the bond undergoing cleavageor formation in the reaction. Two subcategories of secondary KIEs are inductiveand steric KIEs. The slightly shorter length of the C–D versus C–H bond resultsin a slightly increased ability to donate electron density by an inductive effect, andthe reduced amplitude of C–D bending vibrations, versus those of C–H, results ina smaller size (i.e. a smaller steric requirement) of the group of atoms containing D.

Table 3Thermodynamic parameters for caffeine isotopomersa

Fusion temp.Caffeine isomer Phase transition Molar enthalpy ofMolar enthalpy offusion (kJ/mol)transition (kJ/mol)temp. (°C) (°C)

3.85 237.2 23.3Caffeine 142.119.6235.31 CD3 2.73138.121.23 CD3 135.6 2.67 236.6

236.57 CD3 139.4 3.04 21.3237.6 22.01,3 (CD3)2 2.81138.4236.61,7 (CD3)2 136.2 20.82.96

20.03,7 (CD3)2 138.7 3.48 238.6238.91,3,7 (CD3)3 135.0 21.82.40

a Adapted from Table LIX of [24].


Table 4Differences in the inhibition of RNase activity by ATA and deutero-ATAa

94 19 38Inhibitor conc. (mM)

% RNase acti6ityATA 5 3 38

19 1032Deutero-ATA 52

a Estimated from Fig. 1 of [26].

Another type of KIE is a change in the reaction rate due to a change in theisotopic composition of the reaction medium.

Reactant �Solvent-(H/D)

Product; kH"kD

The type of IEs considered in this review are sometimes classified as secondary IEs,although in almost all of the examples described, no breakage or formation of acovalent bond is involved.

Table 5Electroantennagram responses for isotopomers of (+)- and (−)-bornyl acetate, and (+)- and (−)-isobornyl acetatea

Response (mV)*Compoundb

(+)-Bornyl acetatec 1.71e

(+)-Bornyl-9,9,9-d3-acetate 1.66f

1.65f(+)-Bornyl-10,10,10-d3-acetate1.63e(+)-Bornyl-10-d1-acetate1.73e(+)-Bornylacetate-d3

1.71e(+)-Bornyl-2-d1-acetate(+)-Bornyl-2-d1-acetate-d3

d 1.76(−)Bornyl acetate 1.54g

1.53g(−)Bornyl-acetate-d3

1.17h(+)-Isobornyl-acetate(+)-Isobornyl-8,8,8-d3-acetate 1.16h

(+)-Isobornyl-2-d1-acetate 1.19I

1.18I(+)-Isobornyl-2-d1-acetate-d3

(−)-Isobornyl acetate 1.231.15j(−)-Isobornyl-8,8,8-d3-acetate1.12j(−)-Isobornyl-10,10,10-d3-acetate

a From Table I of [45].b Each experiment was repeated 180 times unless otherwise indicated.c 108 repetitions.d 36 repetitions.* Millivolt responses with the same superscript letter (e–j) are not significantly different at the 95%

confidence level.


Table 6Electroantennagram responses for isotopomers of trans-2-hexen-1-ala

RepetitionsCompound Response (mV)*

(1,3-d2)-Trans-2-hexen-1-al 2.179c602.122cd60(1,2,3-d3)-Trans-2-hexen-1-al2.121cd59(1-d1)-Trans-2-hexen-1-al

25 2.099cde(1,2,4,4-d4)-Trans-2-hexen-1-al(1,2-d2)-Trans-2-hexen-1-al 2.056de60

2.056de(1,2,3,4,4-d5)-Trans-2-hexen-1-al 592.027ef25(1,4,4-d3)-Trans-2-hexen-1-al2.013ef60(3-d1)-Trans-2-hexen-1-al2.011ef60(2-d1)-Trans-2-hexen-1-al

25 1.995ef(2,3-d2)-Trans-2-hexen-1-al1.951fg(3,4,4-d3)-Trans-2-hexen-1-al 591.922g382Trans-2-hexen-1-al

(2,4,4-d3)-Trans-2-hexen-1-al 1.899gh601.888gh(2,3,4,4-d4)-Trans-2-hexen-1-al 59

(4,4-d2)-Trans-2-hexen-1-al 60 1.832h

a From Tables I and 4.2 of [46].* Millivolt responses with the same superscript letter (c–h) are not significantly different at the 95%

confidence level.

2. Examples of DIEs on noncovalent interactions in biological processes

2.1. Binding studies

Some of the most convincing evidence for DIEs on noncovalent interactionsbetween biomolecules has been obtained from in vitro studies of bindinginteractions.

Table 7Differences in the relative retention timesa of chlorinated isotopomer pairs on GC with four differentstationary phasesb

Isotopomer pair D (mean9S.D.) (min)

1,4-Dimethylbenzene-d0/d10 0.01590.0060.05590.0062-Chloro-1,4-dimethylbenzene-d0/-d9

1-(Chloromethyl)-4-methylbenzene-d0/-d9 0.07590.0240.15590.0332,5-Dichloro-1,4-dimethylbenzene-d0/-d8

2,3-Dichloro-1,4-dimethylbenzene-d0/-d8 0.18090.0512-Chloro-1-(chloromethyl)-4-methylbenzene-d0/-d8 0.23090.090

0.22290.1143-Chloro-1-(chloromethyl)-4-methylbenzene-d0/-d8

0.59090.5021,4-Bis(chloromethyl)benzene-d0/-d8

2,3,5-Trichloro-1,4-dimethylbenzene-d0/-d7 0.92590.861

a Relative to retention time of 1,4-dimethylbenzene-d0/-d10.b Adapted from Table 1 of [66].


Table 8Differences in the boiling points and van der Waals (vdW) volumes of isotopomer pairsa

D boiling point (°C)Isotopomer pair D vdW volume (ml/mol)

2-Chloro-1,4-dimethylbenzene-d0/-d9 1.4 0.431-(Chloromethyl)-4-methylbenzene-d0/-d9 1.3 0.46

0.461.32,5-Dichloro-1,4-dimethylbenzene-d0/-d8

1.2 0.372,3-Dichloro-1,4-dimethylbenzene-d0/-d8

0.502-Chloro-1-(chloromethyl)-4-methylbenzene-d0/-d8 1.20.401.13-Chloro-1-(chloromethyl)-4-methylbenzene-d0/-d8

1.8 0.121,4-Bis(chloromethyl)benzene-d0/-d8

1.781.22,3,5-Trichloro-1,4-dimethylbenzene-d0/-d7

0.40Benzene-d0/-d6 1.00.411.5Chlorobenzene-d0/-d5

Bromobenzene-d0/-d5 – 0.480.111.6Toluene-d0/-d8

a Adapted from Tables V and VI of [66].

Thenot [12] examined the binding of phenytoin and phenytoin-d10, with bothphenyl rings perdeuterated, by plasma proteins of the dog and human. Theyindicated that the major phenytoin-binding protein of plasma is albumin, thatbinding between albumin and phenytoin reflected hydrophobic interactions betweenthe two molecules, and that they expected the extensive replacement of hydrogenatoms by deuterium atoms to affect the binding affinity. Protein binding wasdetermined by equilibrium dialysis, and phenytoin concentrations were measuredby mass spectrometry. The percent of free (unbound) phenytoin was higher for thedeuterated compound than for the unlabeled compound, in both the human (14.0vs. 11.8%, respectively) and dog (23.9 vs. 21.9%, respectively). The ratio ofequilibrium dissociation constants (KD; Kphenytoin/Kphenytoin-d10) was 1.195 for thehuman and 1.08 for the dog, indicating that deuteration had decreased the bindingaffinity of phenytoin for plasma proteins (by 20% in the human). In addition, the

Table 9Differences in the RP-HPLC retention times for isotopomers of aromatic hydrocarbonsa

Isotopomer pair D retention time (min)

2.0Benzene-d0/-d6

Toluene-d0/-d8 1.7Naphthalene-d0/-d8 2.1Anthracene-d0/-d10 0.9

0.9Phenanthrene-d0/-d10

1.0Durene-d0/-d14

Biphenyl-d0/-d14 1.6Nitrobenzene-d0/-d5 2.3

a Adapted from Table 1 of [67].


Table 10GLC behavior of n-alkanes and their perdeuterated isomers

D (DH) (cal/mol)b D (DS) (cal/K-mol)bD retention time (min) at temperaturen-Alkane pair(°C)a

160 180 200140120

C15/C15-d32 1.08 0.48 0.23 0.12 −152.6 −0.15342.691.844.86 0.78 0.36 0.18 −158.5 −0.1583C16/C16-d34

1.33 0.58 0.29 −177.4 −0.1827C17/C17-d36 3.309.14

a Adapted from Table I of [71].b Adapted from Table II of [71].

investigators were able to partially separate the two phenytoin isomers on a short(15 cm×4.6 mm), octadecyl-silica, reverse phase, high pressure liquid chro-matograph (RP-HPLC) column, using a linear gradient of 20–60% methanol inwater. The deuterated compound eluted first, indicating that it was lesshydrophobic.

Falconnet [13], Brazier [14], and Cherrah [15–17] studied the binding of thebarbiturate, phenobarbital, and the xanthines, caffeine and theophylline (Table 1),to human serum albumin (HSA) by equilibrium dialysis. The association or affinityconstants (Ka=1/KD) of HSA for phenobarbital and phenobarbital-ethyl d5 werenot significantly different. However, the Ka for phenobarbital-phenyl d5 wassignificantly higher, indicating that the phenyl d5 isotopomer had an increasedaffinity for HSA. Since perdeuteration of the ethyl group of phenobarbital had noeffect on binding to HSA, whereas perdeuteration of the phenyl ring increased itsability to interact with the protein, the investigators concluded that the phenyl ringwas involved in binding to the protein. They also noted that since perdeuteration ofthe ethyl group had no effect on binding, and no effect on the pharmacokinetics ofphenobarbital in man [18], the ethyl d5 compound could be used as a tracercompound in human metabolic studies. The Ka values of HSA for caffeine,

Table 11GC behavior of isotopic pairsa

Isotope pair Temperature (°C) D retention time (min)

Benzene-d0/-d6 25 0.0945 0.11Toluene-d0/-d3

50Toluene-d0/-d8 0.14Octane-d0/-d18 80 0.16

0.16110Naphthalene-d0/-d8

160Bibenzyl-d0/-d7 0.200.2560Ethylbenzene-d0/-d10

Ethylbenzene-(ethyl-d0/-d5) 0.186060Ethylbenzene-(ring-d0/-d5) 0.26

a Adapted from Figs. 1–3 of [73].


caffeine-1-CD3, and caffeine-1,3,7-(CD3)3 were not significantly different. However,the values for two other parameters, the number of binding sites on HSA and thepercent bound of a 50 mM dose, did differ for the deuterated isomers in comparisonwith caffeine. The Kas for caffeine-3-CD3, caffeine-1,7-(CD3)2, and caffeine-3,7-(CD3)2 were significantly lower than that for caffeine, indicating that HSA had alower affinity for the deuterated compounds. These three compounds also differedfrom caffeine with respect to the number of binding sites on HSA and the percentbound of a 50 mM dose. The Ka of HSA for theophylline-1-CD3 was substantiallyless than that for theophylline, indicating a decreased affinity for the deuteratedcompound, and the decrease in affinity for theophylline-1-CD3 was greater than thedecrease in affinity found for caffeine-1-CD3 versus caffeine. The investigatorscommented that substitution at the N-1 position of xanthines is known to berequired for the binding of xanthines to serum albumin [19], and that replacing theN-methyl group with a trideuteriomethyl group might strengthen a hydrogen bondto this nitrogen [20]. They also indicated that the site of isotopic modification of amolecule can effect its physicochemical properties (e.g. ionization, polarizablity,solubility, molecular geometry), and that changes in these properties can haveconsiderable influence on the molecule’s pharmacokinetics and pharmacodynamics(e.g. distribution, binding to transport proteins, enzymology). Earlier work byHorning [21] had shown that caffeine, caffeine-3-CD3, and caffeine-1,3,7-(CD3)3

could be partially separated from each other by RP-HPLC, and that the order ofelution was [first to last (or most polar to least polar)] caffeine-1,3,7-(CD3)3,caffeine-3-CD3, and caffeine, indicating that the polarity of the caffeine isomerincreased with an increasing content of deuterium. Similar results were obtained byCherrah [15] during gas chromatography–mass spectrometry (GC–MS) analyses ofcaffeine and caffeine-1,3,7-(CD3)3.

It is difficult to correlate the results of Brazier [14] and Cherrah [15–17] withthose of Horning [21], in terms of the HSA affinities for caffeine isotopomers beingrelated to deuterium content, because the affinities of HSA for caffeine andcaffeine-1,3,7-(CD3)3 (the least and most polar isomers according to HPLC andGC) were essentially the same, and were greater than that for caffeine-3-CD3 (ofintermediate polarity). However, a direct correlation can be made between thepolarities of these three compounds and two other parameters listed in Table 1, thenumber of binding sites on HSA and the percentage bound of a 50 mM dose. Bothof these parameters increased with increasing deuterium content.

Bechalany [22] also used RP-HPLC to investigate the effect of deuteration onlipophilicity of caffeine, and they also found that deuterated compounds elutedfaster than their protium isotopomers from an HPLC column containing apolyvinyl alcohol octadecanoyl stationary phase, using H2O/MeOH as the eluant.The order of elution was (first to last): caffeine-1,3,7-(CD3)3, caffeine-1,3-(CD3)2,caffeine-1,7-(CD3)2, caffeine-3,7-(CD3)2, caffeine-1-CD3, caffeine-3-CD3, caffeine-7-CD3, caffeine. They obtained the same results when the experiment was done atroom temperature (21–22°C) or at 30°C. These results led to the conclusion thatdeuteration of C–H groups is accompanied by a small decrease in the lipophilicityof caffeine isotopomers. Position specific effects of deuteration on the lipophilicity


of caffeine isotopomers were noted, as were found in the HSA binding studies.Replacement of protium with deuterium at position N-7 produced less of an IEthan at positions N-1 or N-3, for single substitutions, but the results obtained fordouble or triple position substitutions was more complex and not a simple additivephenomenon. The complexity was attributed to possible ‘second-order’ perturba-tion by substitution at the N-3 position, and/or to perturbation of the self-associa-tion of caffeine molecules.

Falconnet [23] used proton nuclear magnetic resonance spectroscopy to examinethe self association (dimerization) of caffeine, and the same seven deuteratedcaffeine isotopomers mentioned previously [caffeine-1-CD3, caffeine-3-CD3, caf-feine-7-CD3, caffeine-1,3-(CD3)2, caffeine-1,7-(CD3)2, caffeine-3,7-(CD3)2, caffeine-1,3,7-(CD3)3], over the concentration range of 5-150 mM caffeine or isotopomer.Chemical shifts for the N1-, N3-, and N7-methyl groups, and position 8 proton,were monitored over the concentration range of 5–150 mM, and a constant fordimerization was determined for each isotopomer. The results showed that deuter-ation reduced the degree of self association for all isotopomers examined (Table 2).The strength of self association was (strongest to weakest): caffeine, caffeine-3,7-(CD3)2, caffeine-1,7-(CD3)2, caffeine-1,3-(CD3)2, caffeine-3-CD3, caffeine-1,3,7-(CD3)3, caffeine-1-CD3, and caffeine-7-CD3.

Mapengo [24] used differential scanning calorimetry to determine thermodynamicparameters of caffeine and the same seven caffeine isotopomers studied by Falcon-net. The purities of the compounds were 99.97% for caffeine, and 99.63–99.97%[mean (9S.D.)=99.77 (90.08)%] for the deuterated caffeines. The temperaturesof the phase transition and fusion (i.e. melting; 238°C) were measured, and themolar enthalpies of these processes were calculated (Table 3). The transitiontemperatures for the deuterated isomers (135.0–139.4°C) were 1.019–1.053-fold lessthan that for caffeine (142.1°C), indicating that all of the deuterated isomers wereless ordered. The fusion temperatures for caffeine-1-CD3, caffeine-3-CD3, caffeine-7-CD3, and caffeine-1,7-(CD3)2 were less than that of caffeine, indicating less selfassociation, probably via hydrogen bonding, between molecules in the crystallineforms of these compounds. However, the fusion temperatures for caffeine-1,3-(CD3)2, caffeine-3,7-(CD3)2, caffeine-1,3,7-(CD3)3 were 0.4–1.7°C higher than forcaffeine, indicating a higher degree of hydrogen bonding in the crystalline forms ofthese compounds.

In a different experiment, LaReau [25] examined the binding of NAD+ to theenzyme, lactate dehydrogenase (LDH), a preliminary step in the conversion oflactate to pyruvate. They used NAD+ that was labeled with a single deuterium atposition 4 of the nicotinamide ring, and found a significant DIE on the binding ofthe coenzyme to LDH. The investigators also noted that a common assumption ofenzymologists is that the binding of isotopically labeled molecules to enzyme activesites is not affected by the presence of the label (in contrast to their results forNAD+), but that this assumption contradicted another generally accepted conceptof enzymology, that enzymes bind more tightly to the transition state than to thesubstrates or products of a reaction.


2.2. Inhibition studies

DIEs on noncovalent interactions are often observed in studies of the mechanismof action of enzymes or enzyme inhibitors. For example, Gonzalez [26] encounteredsuch an IE during studies of the mechanism of action of aurintricarboxylic acid(ATA), a potent inhibitor of interactions between proteins and nucleic acids, and ofapoptosis, or programmed cell death. ATA is a dye that consists of a mixture ofpolyanionic polymers, and it is capable of competing with DNA or RNA in theirinteractions with nucleic acid binding proteins, such as ribonuclease (RNase),probably by interfering with electrostatic interactions between the nucleic acid andthe protein. The investigators synthesized totally deuterated ATA, isolated a highmolecular weight fraction (MW\3500) of the synthetic material, and used it toinvestigate the interaction of ATA with bovine pancreatic RNase by protonmagnetic resonance. They indicated that the deuterated ATA fraction possesed aultraviolet–visible absorption spectra that was nearly identical to that obtainedwith the same high MW fraction isolated from commercially available, undeuter-ated ATA, and that both the undeuterated and deuterated ATA fractions gave verysimilar electron spin resonance (ESR) spectra (i.e. they are both stable freeradicals). They also demonstrated that the synthetic deutero–ATA fraction wascapable of inhibiting the degradation of yeast RNA by bovine pancreatic RNase, aproperty possessed by the same fraction of commercially available ATA. Althoughthe authors did not comment on it, their published data also shows that thedeuterated form of this ATA fraction was a less potent inhibitor of RNase activityover the entire concentration range tested (about 4–38 mM), and that the differencein inhibitory activity increased with decreasing ATA, or deutero–ATA, concentra-tion (Table 4). Assuming that the undeuterated and deuterated forms of this highMW ATA fraction are chemically identical, it is apparent that deuteration substan-tially reduced its potency (3–6-fold) as a competitive inhibitor of RNase activity,and that this effect was on its ability to noncovalently bind to the protein.

2.3. Effects on hydrophobicity

Luan and Urry [27] used differential scanning calorimetry to study the effect ofreplacing H2O with D2O, as a solvent, on the apparent hydrophobicity of a bovineelastin-based polypentapeptide, (L-Val1-L-Pro2-Gly3-L-Val4-Gly5)n. In water, thishydrophobic peptide is known to undergo an inverse temperature transitionwhereby an increase in temperature causes an increase in intra- and intermolecularorder (i.e. a phase separation resulting in a more ordered, dense viscoelastic phase).The inverse temperature transition is due to the fact that the amino acids compris-ing this polymer are hydrophobic and do not interact strongly with the solvent,water [28]. Consequently, the water molecules surrounding the protein form anordered, internally hydrogen-bonded, lattice structure that acts as a force to preventchanges in the structure of the polymer. When the temperature of this system isincreased, the order within the water lattice breaks down, and the protein is able toassume an energetically more favorable conformation. Therefore, the proteincondenses due to an increase in its internal hydrophobic interactions.


Replacement of the solvent, H2O, with D2O caused a 2°C decrease in thetransition temperature, and a 10% increase in the heat of transition. The effectcould be viewed as an increase in the hydrophobicity of the peptide in D2O, withrespect to peptide hydrophobicity in H2O. The investigators noted that the appar-ent increase in hydrophobicity that occurs when D2O is substituted for H2O is notdue to a lower degree of internal order in D2O, which is considered to be morestructured than H2O. Rather, the effect is due to differences between the structuresof the protein hydration shell, as opposed to bulk solvent, in D2O versus H2O.

It should be noted that, in addition to a demonstration of solvent DIEs onnoncovalent interactions, this experiment is also an elegant demonstration of therole of the solvent in the origin of another very important biological phenomenon,hydrophobicity [29].

2.4. Metabolic studies

Gately [30] investigated the biodistribution of glucose (Glc) and perdeuteratedGlc in mice, and found that the deuterated compound was cleared more slowlyfrom the blood. The kinetics of clearance was similar for both compounds duringthe first 15 min following intravenous injection, with most (96%) of both com-pounds being cleared during the first minute. However, during the next 100 min therate of clearance of residual perdeuterated Glc was substantially less than that forthe undeuterated form (e.g. only 22% of the rate of clearance for Glc at 60 min).Hepatocytes are freely permeable to Glc, but its entry into other cell types requirestransport systems [31], and the interaction of Glc with these systems is noncovalent[32]. The investigators also found an IE on the separation of Glc and perdeuteratedGlc by silica gel TLC using an ethyl acetate/ethanol (1:1, v/v) mobile phase(Rfs=0.5 and 0.45, respectively), indicating differences in the polarities of the twoisomers (i.e. Glc more polar than the perdeuterated isomer). The author of thisreview (D. Wade) would like to speculate that if the potential influence of hormonalfactors (e.g. insulin) is excluded, the clearance data combined with the TLC resultscould be interpreted as indicating a possible DIE on the interaction of Glc with itstransport systems, and that this effect was reflected in a reduction in the clearancerate of perdeuterated Glc versus Glc from the blood.

Stable isotopes are used as tracers in human biomedical experimentation anddiagnosis, and at all stages of the life cycle [33]. No harmful effects have beendetected in mammals at levels of deuterium in body water less than 15%, and theoverall effect of using deuterated molecules as tracers appears to be a depression oftissue metabolism due to the slower reaction rates of deuterated compounds in vivo.Deuterium labeled amino acids are commonly used in studies of amino acidmetabolism. In a study of the role of the splanchnic vascular bed in gastrointestinal(GI) absorption, Krempf [34] found higher rates of flux between the GI tract andblood for L-phenylalanine (L-Phe), with all five of its phenyl ring hydrogensreplaced by deuteriums (L-Phe-ring d5), than for a carbon isotopic version of Phe[L-(1-13C)-Phe]. They suggested that the difference in flux might be due to protonexchange during amino acid transport, however, the transport model to which they


referred [35] deals only with proton exchange of a-amino group hydrogens, notphenyl ring hydrogens. In addition, it is highly unlikely that phenyl hydrogen, ordeuterium, exchange was involved because the pKa for a closely related modelsystem, benzylic hydrogen, is approximately 43 [36]. These investigators alsoindicated that their results did not exclude the possibility that amino acid transport,from the intestinal lumen into the blood, discriminated against L-Phe-ring d5 (seealso the previous discussion regarding L-Phe and L-Phe-ring d5 in Section 2.1).

In an experiment designed to evaluate the use of urine, instead of blood, as a testmaterial to monitor amino acid metabolism, Zello [37] obtained evidence for a DIEon the reabsorption of L-Phe-ring d5 by the kidney. Amino acids that enter thearterial blood will eventually enter the nephrons of the kidney, where they are freelyfiltered through the glomerulus and into the kidney tubules. They are thenreabsorbed to the extent of 100% by highly specific, active-transport processes (i.e.processes that require energy to move a molecule against a concentration gradient[32]) within the proximal tubules. These investigators found that L-Phe-ring d5 waspresent at a significantly greater concentration in the urine than the plasma, incontrast to the results obtained with L-(1-13C)-Phe, which was present at equalconcentrations in the urine and plasma. This indicated that the process of filtrationand reabsorption was not effected by L-(1-13C)-Phe, but that there was an effect onthe reabsorption of L-Phe-ring d5. To reiterate, any possible carbon isotope effectcould be expected to be so small as to be negligible. After eliminating potentialsources of error from their experimental methods, including the possibility ofD-to-H exchange of phenyl ring hydrogens, they concluded that L-Phe-ring d5 wasnot metabolized in the same manner as the unlabeled compound. It is important tonote that the authors use the word ‘metabolized’ to refer simply to active-transportprocesses; processes which do not involve the breaking of C–D (or C–H) bonds inthe phenyl ring of Phe [32,38].

2.5. Olfaction (in general)

The sense of smell is thought to be a receptor mediated phenomenon in which thebinding, a noncovalent interaction, of a stimulant molecule to a membrane receptorinduces an electrophysiological response. One theory of olfaction suggests thatdifferences in the odors of molecules are related to differences in the molecularvibrations of odorant molecules. A good test of this theory would involve acomparison of the odors of molecules that differ only in their vibrational frequen-cies, such as deuterated and nondeuterated isotopomers. For air-breathing animals,odorant molecules are usually volatile, nonionic, hydrophobic compounds, withmolecular weights less than 300, and which contain a limited number of functionalgroups. Alternatively, the best odorants for aquatic animals are water solublecompounds, such as amino acids [39].

2.5.1. Olfaction in fishHara [40] investigated the ability of the whitefish, Coregonus clupeaformis, to


distinguish between the odor of glycine (Gly) and fully deuterated glycine (Gly-d5).Over the concentration range of 10−8–10−4 M, these fish avoided solutions ofGly-d5 and preferred solutions of Gly. Both compounds were shown to be able tostimulate similar electrical responses in the olfactory bulbs of these fish, and thepreference reactions could be eliminated by cauterization of the fish nares. Haranoted that previous work had demonstrated that stimulatory effectiveness dependedon the interaction of an amino acid with a receptor of definite shape, size, andcharge distribution, and that the receptor should contain two charged subsites forinteractions with the ionized a-amino and a-carboxyl groups of the amino acid. Itwas also noted that deuteration of a molecule should not effect the molecular shapeor molecular properties associated with electronic structure and force fields, butthat molecular motions would be effected, and differences in these motions mightbe responsible for the reaction of fish to these olfactory stimuli. It is important toemphasize that if the different responses of fish to these olfactory stimuli were, infact, mediated via an initial interaction between the stimulant molecule and anolfactory receptor, that the nature of this interaction was almost certainlynoncovalent.

2.5.2. Olfaction in insectsMeloan and colleagues [41] used molecules that are olfactory stimulants for

insects to study the effects of deuterating these molecules on their abilities tomodify insect behavior, and also to stimulate electrical responses in isolated insectantennae. Acetaldehyde and acetone are aggregation stimulants, and methanol and2-butanone are oviposition stimulants, for the red flour beetle, Tribolium castaneum.The bornyl acetates, (+ )-, and to a lesser extent, (− )-bornyl acetate, are syntheticsex attractants for the American cockroach, Periplaneta americana, whereas theisomeric (+ )- and (− )-isobornyl acetates are nonstimulatory.

Wang [42] showed that perdeuteration of acetaldehyde (-d0 vs. -d4) and acetone(-d0 vs. -d6), and partial deuteration of methanol (-d0 vs. -d1) and 2-butanone (-d0

vs. -d5), eliminated the aggregation and oviposition responses of red flour beetles toall of these compounds, except methanol-d1. These results suggested that insectswere unable to distinguish the deuterated isomers from the nondeuterated com-pounds, except for methanol-d1.

Kuo [43] found that deuteration of (− )-bornyl acetate affected the sex attractionresponse of the cockroach. The insects responded to all compounds tested, includingthe parent compound and isomers: 3,3-d2; 2-d1; acetate-d3; 2,3,3-d3; 3,3-d2, acetate-d3;2-d1, acetate-d3; 2,3,3-d3, acetate-d3. However, the responses induced by the deuter-ated isomers were significantly different from those induced by the parent compound.In addition, there were significant differences between the responses induced by thedeuterated isomers themselves. The order of attractiveness was: undeuterated\CD3\CD2\CD\ (CD3+CD2)\ (CD3+CD)\ (CD2+CD)\ (CD3+CD2+CD). A single substitution at the 2-position had a greater effect than two deuteriumsat the 3-position or three deuteriums on the acetate methyl group. The combinedresults showed that the attractiveness of a labeled compound was related more tothe position of substitution than to differences in mass.


Scriven [44], Havens [45], and DeCou [46] used a biophysical approach, theelectroantennagram assay, to investigate differences in the abilities of deuteratedisotopomers of (+ )- and (− )-bornyl acetates, (+ )- and (− )-isobornyl acetates,and the olfactory stimulant, trans-2-hexen-1-al to stimulate electrical responses inisolated antennae, the olfactory organ, of cockroaches. The data showed that allcompounds tested were capable of eliciting electrical responses in the antennae, thatthe magnitude of the response was proportional to the attractiveness of thecompound to the insect, that deuteration of the compound could either reduce orenhance the electrical response, and that the position of deuteration within amolecule was important (Tables 5 and 6). If it is assumed that the responses elicitedby these molecules are mediated by their interaction with a receptor, as is generallyassumed for olfactory stimulants, then studies such as these can begin to provideinformation about potential features of the receptor molecule or site.

2.5.3. Olfaction in humansTurin [47] and colleagues found striking differences in the odors of acetophenone

and acetophenone-d8. Turin criticized Hara’s experiment, on olfaction in fish, onthe basis that glycine contains only exchangeable protons, however, the methylenehydrogens/deuteriums of glycine cannot exchange with solvent protons at physio-logical pH.

3. Examples of DIEs on other noncovalent interactions between molecules

3.1. Mixtures

Jancso [48,49] reviewed the experimental evidence for nonideal behavior inmixtures of isotopomers, in terms of the differences in the vapor pressures ofdeuterated and nondeuterated molecules and mixtures of the two. Ideal mixturesare solutions of two or more components in which the presence of one componentdoes not effect the properties of the other component. Traditionally, mixtures ofisotopomers had been assumed to be ideal mixtures, but careful measurements havenot supported this hypothesis. Liquid methane (CH4) has a lower vapor pressurethan perdeuterated liquid methane (CD4), and Fuks [50] investigated the basis forthis difference by determining the molar volumes of these isotopomers between 98and 112 K. They found that the molar volume of CH4 was about 1% larger thanthat of CD4 over the entire temperature range, and interpreted this difference asbeing due to two factors: a translational quantum effect, and small differences inthe intermolecular forces of the two liquids. Simultaneous measurements by Rebelo[48,49] of the vapor pressure differences of methane, deuteromethane, and mixturesof the two, showed small but significant differences. These vapor pressure isotopeeffects (VPIEs) were consistent with other data for molar volume isotope effects(MVIEs) of nonpolar molecules which showed that the nature of MVIEs wastemperature dependent. Below 170 K, the MVIE was normal, with the molarvolume of CH4 being greater than that of CD4, whereas above 170 K, the situation


was reversed. Similar results were obtained for measurements of the vapor pressuresof benzene (C6H6) and perdeuterobenzene (C6D6). The vapor pressure of C6D6 isalmost 3% higher than that of C6H6, an inverse VPIE, but the molar volume ofC6H6 is almost 0.3% larger than that of C6D6, a normal MVIE. The investigatorsfound evidence indicating that when C6H6 was dissolved in C6D6, it underwent aslight compression.

Bates and others [51–53] investigated the effect of deuteration on the mixing ofisotopomers of 1,4-polybutadiene (PBD), polyethylene (PE), poly(ethylethylene)(PEE), polystyrene (PS), and poly(vinylethylene) (PVE), using the technique ofsmall-angle neutron scattering. They found that binary mixtures of a polymer andits perdeuterated isomer did not form ideal solutions, and, under the appropriateconditions, would separate into two phases. They indicated that the effect wascaused by small, but measurable differences in the repeat unit volumes of normaland perdeuterated isomers, and that these volume differences were, in turn, causedby the difference between the C–H (longer) and C–D (shorter) bond lengths.

3.2. Mimicking the effect of pressure

Investigators have found that deuteration of a molecule can have the same effecton its physical properties as applying pressure on it. For example, it was found thatperdeuteration of nitromethane (H3C–NO2) corresponded to the application of 400bar, or about 360 atmospheres, of pressure on the molecule [54,55].

Sinzger [56] studied the effects of deuteration on copper salts of the organicmetal, 2,5-dimethyl-N,N %-dicyano-p-benzoquinone diimine, (Me2-DCNQI)2Cu, andfound that substitution of methyl group and ring hydrogens, with deuteriums,produced large changes in the solid-state properties and crystal structure of thecompound. The parent compound is a conductor of electrical current at tempera-tures from ambient to as low as 0.4 K, but when the methyl group and ringhydrogens were substituted with an increasing number of deuteriums (from CH3/CH3/H2 to CH3/CD3/H2, CD3/CD3/H2, and CD3/CD3/D2) there was a progressiveincrease in the temperature at which the compound underwent transition from theconductive to the insulative states (0.4 to 58, 73, and 82 K, respectively). Substitu-tion with deuterium also produced significant changes in ESR signals, magneticproperties, and the crystal structure, as determined by X-ray analysis. The investi-gators indicated that this very strong secondary DIE was due to an effect ofdeuteration on the crystal structure of (Me2-DCNQI)2Cu which mimicked the effectof pressure.

The equivalence of selective deuteration and pressure, on the physical propertiesof (Me2-DCNQI)2Cu, was confirmed by Aonuma [57]. They synthesized 15 of thepossible 35 deuterated isomers of this compound, measured their electrical resistiv-ities, correlated the results to effective pressures, and determined the crystalstructures of the compounds. They were able to determine that the deuteriumsubstitution pattern within the methyl groups was not a significant factor, but thatdeuteration of the methyl groups was more effective than deuteration of thebenzoquinone ring in producing the metal-to-insulator transitions. They also found


that the effective pressure versus temperature (Peff–T) phase diagram for thesecompounds reproduced the P–T phase diagram. They also found that deuterationproduced contraction of the unit cell volume in the crystal structures, and this wascorrelated with the Peff.

Both groups of investigators described the crystal structure of (Me2-DCNQI)2Cuand its isotopomers as consisting of stacks of ligand and copper ions that areorganized into infinite superhelices, resulting in a diamonoid structure. Theysuggested that the shorter length of the C–D bond caused each Me2-DCNQImolecule to become less bulky, and resulted in each of the molecules in the crystalcoming closer to one another, similar to the effect caused by increasing pressure.They also hypothesized that the resulting distortion of the tetrahedral coordinationgeometry around copper ions was the basis for the observed changes in theconductivity properties of the compound.

3.3. Polymer chain conformation

Gu [58] and Okamoto [59] found that solutions of poly ((R)-1- and -2-deuterio-n-hexyl isocyanate)s gave extraordinarily large optical rotations, and these wereattributed to the amplification, by the polymer, of very small energy differencesbetween the deuterated monomer units in their left- and right-handed helicalconformations. Polyisocyanates contain backbone amide groups that are forced toadopt helical conformations due to steric reasons. The polymer chain consists of analternating sequence of left- or right-handed helices with reversal points joining thehelical segments. The replacement of hydrogen by deuterium at only one positionin a hexyl side chain (e.g. positions 1 or 2) makes the monomeric unit chiral, and,therefore, optically active. Nonbonded interactions between the chiral side chain ofa given monomeric unit and the main chain atoms of the preceeding units causes asymmetry break, in the helix sense, leading to a predominance in the content of theleft-handed helix in the polymer. This is due to a small energetic preferrence, in themonomer, for one particular helix handedness, and the resultant amplification, bycooperativity of multiple monomer units, of this preferrence in the polymer. Thenonbonded interactions are stronger in the polymer with deuterium substituted atposition 1, rather than position 2, in the hexyl side chain because the chiral centeris located closer to the main chain than in the latter situation, and the magnitudeof the free energy difference between right- and left-handed helices is about twice asgreat for the position 1-substituted polymer as for the position 2-substitutedpolymer. The investigators found that the right-handed helix predominated in theposition 2-substituted polymer, whereas the left-handed helix predominated in theposition-1 substituted polymer.

3.4. Adsorption/complex formation

Although the main emphasis of this review is to present examples of deuteriumIEs on interactions between molecules in which there is no covalent bond breakinginvolved, the following example of a DIE on the binding of an alkene to a


transition metal complex involves the cleavage of one of the two bonds in a doublebond between two carbon atoms in the alkene, but no cleavage of a bond todeuterium. Consequently, it is an example of a secondary DIE. Bender [60] notedthat transition metal complexes are known to bind alkenes reversibly, and thatsecondary deuterium equilibrium isotope effects (EIEs) for alkene complexes areusually inverse, with the deuterated isomer binding better. Bender used infraredabsorbance spectroscopy to measure differences in the reversible binding ofethylene (C2H4) and ethylene-d4 (C2D4) to the transition metal complex, diosmacy-clobutane [(OC)4Os–Os(CO)4], and found that C2D4 bound better to the complexthan did C2H4. Differences in the binding abilities were attributed to differences inthe zero point energies of a new vibrational mode that was present in bound, butnot free, C2H4/C2D4, and Bender suggested that this hypothesis would also apply tothe coordination of other small molecules to transition metal complexes.

Schultz [61] used flash photolysis in the liquified rare gas solvents, krypton (Kr)and xenon (Xe), to investigate activation of the carbon–hydrogen bonds incyclohexane by the transition metal complex, [C5(CH3)5]Rh(CO)2. They found thatbond activation was preceded by an equilibration step where cyclohexane wasweakly bound to rhodium in the metal complex, and also that cyclohexane-d12 wasbound 12-fold more tightly than the undeuterated isomer. They speculated that theactivation mechanism involved the initial formation of a weak s-complex betweenthe alkane and the metal, and that this step was followed by C–H/C–D bondactivation prior to further reaction. They calculated that the bond activation stephad energy requirements of 4.2 kcal/mol for C–H, and 5.3 kcal/mol for C–D.Bengali [62] performed a similar experiment using the same transition metalcomplex, liquid Kr as the solvent, and the alkane isotopomers, neopentane andneopentane-d12. They obtained the same type of results as Schultz [61] with theperdeuterated isomer being bound more tightly to the metal than the undeuteratedisomer. In addition, the use of a high resolution probe laser enabled them to obtaindirect evidence for the existence of the postulated weak alkane–metal complex thatpreceded bond activation.

The studies demonstrating an IE on the binding of molecules to transition metalcomplexes may have relevance to the reactions of enzymes containing transitionmetals at their active sites, such as the cytochromes P450.

Ramasamy and Hurtubsie [63] examined the solid-matrix luminescence propertiesof perdeuterated and undeuterated phenanthrene adsorbed on five different solidmatrices: sodium acetate (NaOAc), 1% a-cyclodextrin/sodium chloride (1% a-CD/NaCl), 80% a-CD/NaCl, 30% b-CD/NaCl, and filter paper. The results showedthat substitution of D for H atoms in phenanthrene had a considerable effect on thesolid matrix phosphorescence. When phenanthrene was added to NaOAc, it wasnot fluorescent or phosphorescent, either at 296 K (room temperature) or 96 K,indicating that it did not interact very strongly with the salt. However, phenan-threne-d10 was both fluorescent and phosphorescent when in contact with NaOAc,which indicated that it interacted more strongly with NaOAc than did phenan-threne. Also, the fluorescence increased 2.3-fold, and the phosphorescence increased12.3-fold, when the temperature was decreased from 296 to 96 K. This result


indicated that processes within the solid matrix, probably thermally inducedvibrations, were causing a loss of energy from excited phenanthrene-d10 at roomtemperature, but that these processes were reduced at the lower temperaturethereby enabling a stronger interaction with the matrix. Also, at the lower temper-ature, the normalized phosphorescence spectral areas for phenanthrene-d10, on theother four solid matrices, were an average of 3-fold greater than the same areas forphenanthrene.

3.5. Additional chromatographic separations

The topic of isotope fractionation is concerned with the phenomenon that isobserved when an isotopically labeled compound is subjected to a separationprocedure, such as chromatography. It is frequently possible to resolve molecules ofthe compound that contain the isotope label from those molecules of the compoundwhich do not contain label. This is different than the separation of the molecule ofinterest from impurities, labeled or unlabeled. Klein [64] discussed common miscon-ceptions about this phenomenon including: the observation of isotope fractionationin large molecules (200–500 MW) is rare and unusual, that it only occurs in systemsof extremely high fractionation efficiencies, and that the majority of the observa-tions can be explained on the basis of labeled impurities. Klein noted that the lastmisconception had been a considerable psychological barrier to progress in thisfield of research.

The lipophilicity or hydrophobicity of isotopomers can easily be assessed byobserving their chromatographic behaviors during GC or liquid chromatography(LC), and, in most cases, the replacement of carbon-bound hydrogen with deu-terium makes a molecule less polar, lipophilic, or hydrophobic. This effect has beentermed, ‘inverse isotope effect’. A few examples involving RP-HPLC have alreadybeen mentioned (see Section 2.1). However, the effect can sometimes be compli-cated by the presence of heteroatoms, as noted above for the studies of xanthines,and in one of the following examples. RP-HPLC has also been used to modelligand–receptor interactions [65]. It may, therefore, be possible to utilize the resultsof RP-HPLC (or other chromatographic method) analyses to demonstrate that theeffect of deuteration on the noncovalent interactions of molecules may be related tothe effects of deuteration on lipophilicity or polarity.

Bermejo [66] chlorinated mixtures of 1,4-dimethylbenzene and 1,4-dimethylben-zene-d10, and then separated the products by capillary GC on four differentstationary phases of different polarities (Table 7). They obtained eight differentpairs of isotopomeric products: 2-chloro-1,4-dimethylbenzene-d0/-d9; 1-(chloromethyl)-4-methylbenzene-d0/-d9; 2,5-dichloro-1,4-dimethylbenzene-d0/-d8;2,3-dichloro-1,4-dimethylbenzene-d0/-d8; 2-chloro-1-(chloromethyl)-4-methylben-zene-d0/-d8; 3-chloro-1-(chloromethyl)-4-methylbenzene-d0/-d8; 1,4-bis-(chloromethyl)benzene-d0/-d8; 2,3,5-trichloro-1,4-dimethylbenzene-d0/-d7. In allcases, the deuterated isomer eluted before its nondeuterated counterpart, indicatingthat there was less interaction between the stationary phase and the deuteratedcompound than there was between the stationary phase and the nondeuterated


compound. This ‘inverse IE’ was not affected by the polarity of the stationaryphase. These investigators also calculated the boiling points and van der Waalsvolumes for the pairs of isomers (Table 8), and compared the results with theliterature values of these parameters for similar compounds. The values of bothparameters were, in all cases, less for the deuterated compounds than for theundeuterated isomers. The decreased values of the bulk properties for deuteratedcompounds were attributed to the slightly shorter internuclear distance in the C–Dversus C–H bond, resulting in a more compact electron distribution and a decreasein electronic polarizability.

Baweja [67] separated eight isotopomer pairs of aromatic hydrocarbons usingRP-HPLC on a C18 column with mobile phases consisting of mixtures of water andacetonitrile in various combinations of 30–85% H2O with 15–70% CH3CN (notethat except for benzene and toluene, the author did not specify the deuteriumcontent of the compounds, and it is assumed that each was perdeuterated). For allcompounds tested, the deuterated compound eluted first (Table 9), and substitutionof D2O for H2O in the mobile phases used for anthracene and phenanthreneseparations did not change the order of elution. The author speculated that van derWaals forces were responsible for the order of elution, and noted that thechromatographic behavior depends on the interaction between the C–H or C–Dbonds, and the stationary phase. It was noted that the oscillation frequency of theC–H or C–D bond, creates an electromagnetic field which induces a field ofopposite charge in surrounding molecules. Since the C–H bond has a higheroscillation frequency than the C–D bond (3300 vs. 2334 cm−1, respectively), itinduces greater forces of attraction between itself and the stationary phase, and itselution from the column is slowed with respect to the deuterated compound. It isinteresting to note the differing effects of deuterium substitution in the experimentsof Bermejo [66] and Baweja [67]. In the former case, deuterium substitution resultedin an apparent decrease in the polarity of the compound whereas in the latter caseit resulted in an increase in polarity.

Dluzneski and Jorgensen [68] utilized fluorescein thiocarbamyl methylamine(FTCM) and FTCM-d3 to test the efficiency of a new method for use in opentubular liquid chromatography, and they were able to achieve near baselineseparation of these two large isotopomers, in spite of the very small differences intheir sizes due to the presence of three deuteriums. FTCM-d3 eluted ahead ofFTCM, indicating that it was less polar, lipophilic, or hydrophobic.

Bushey and Jorgenson [69] used dansylated methylamine (DNS-NHCH3) anddeuterated DNS-NHCH3 (DNS-NHCD3) to demonstrate that micellar electroki-netic capillary chromatography is a powerful separation technique. As in theFTCM/FTCM-d3 experiment, it was possible to achieve near baseline separation ofthese isotopomers, which were only slightly different in size due to the replacementof three hydrogens by deuteriums in the methyl group attached to the primaryamine nitrogen. In their chromatographic conditions, DNS-NHCD3 migrated fasterthan DNS-NHCH3 toward the positive electrode, indicating that the deuteratedcompound was more polar than the undeuterated isomer.


Matucha [70] investigated the separation of C15–C17 n-alkanes and theirperdeuterated isomers by GC on a fused silica capillary column, with a cross linked5% phenyl methylsilicone stationary phase. The order of elution from the columnwas (first to last) n-C15D32, n-C15H32, n-C16D34, n-C16H34, n-C17D36, n-C17H36. Thisresult indicated that the shorter alkanes were less lipophilic than the longer ones,and that the deuterated compounds were less lipophilic than their nondeuteratedisomers.

Matucha [71] also investigated the temperature dependence of the DIE on thegas–liquid chromatographic (GLC) behavior of alkane isotopomers, on a FSOTcolumn coated with PS-255 cross-linked polydimethylsiloxane. The IE on theretention times of these isotopomer pairs increased with increasing carbon chainlength or increasing content of deuterium, and decreased with increasing tempera-ture (Table 10). The investigators also calculated differences in the enthalpy andentropy changes for the pairs of isotopomers, and the absolute values of bothparameters increased with increasing deuterium content. They interpreted theirresults as showing that the heavier isotopomers, with lower molar volumes, elutedearlier than the lighter forms because van der Waals dispersion forces dominatedthe solute-stationary phase interaction.

Mraz [72] studied the DIEs on the retention behaviors of N,N-dimethylfor-mamide (DMF), N-methylformamide (MF), and formamide (F) using GC andvarious stationary phases (methyl and phenylmethyl polysiloxane, and polyethyleneglycol). They found that methyl-deuterated DMF (DMF-d6 or -d3) eluted earlierthan unlabeled DMF, indicating that the deurated compounds were less lipophilicthan the unlabeled compounds. Alternatively, formyl deuterated DMF, MF, and Feluted later than their unlabeled isotopomers, indicating that the deuterated com-pound was more lipophilic than the unlabeled compound. The polarity of thestationary phase did not effect the elution order of these isotopomers.

Shi and Davis [73] were able to completely separate nine isotopomeric pairs ofmolecules, within 5–21 min, by GC on a DB-5 column (fused silica with a liquidphase of 5% diphenyl, 95% dimethylsilicone) (Table 11). In each case, the deuter-ated species eluted first, an ‘inverse IE’, indicating that each deuterated compoundwas less polar than its nondeuterated isomer. They examined the effect of tempera-ture on the differences in GC retention times, and also calculated differences in theenthalpy, entropy, and free energy changes for the isotope pairs. The resultsindicated that the IEs were additive, and that deuterium on the aliphatic part of amolecule was more important than deuterium on an aromatic ring in determiningthe inverse IE. The investigators noted that the GC process consists of twosubprocesses: mixing of the eluting component with the liquid partitioning material,which they indicated was relatively isotope insensitive, and condensation–vaporiza-tion of the eluting component. They suggested that the latter process was mostimportant for the observed inverse IE because intermolecular van der Waals forcesare operative in the condensed phase, and result in a shift in the isotope-sensitiveZPE when a molecule is transferred from the gas to the condensed phase. Theseinvestigators later demonstrated that it was possible to completely separate theisotopomers of decane-d0/-d22, and to partially separate the isotopomers of octane-


d0/-d18, by RP-HPLC on a C-18 column using a methanol–water mixture as themobile phase [74]. Again, the deuterated compounds eluted first, indicating thatthey were more polar than the undeuterated isomers.

Yang [75] studied the effect of deuterium substitution on the lipophilicity ofN-nitrosodimethylamine (NDMA), a structural analog of DMF, by subjectingNDMA and its perdeuterated isotopomer, NDMA-d6, to RP-HPLC, withmethanol or water as the elution solvent and at different pH values. As was foundin the previous example for the DMF/ DMF-d6 pair, perdeuterated NDMA elutedbefore the undeuterated compound, indicating that NDMA-d6 was less lipophilic orhydrophobic than NDMA. These same investigators had previously shown thatboth demethylation and denitrosation of NDMA, by the hepatic microsomalenzyme, cytochrome P450 2E1, was accompanied by a substantial DIE on the Km

for these reactions (i.e. 5-fold greater for NDMA-d6 than for NDMA) whereas theVmax was unaffected [76]. In addition, more recent work has shown that the activesite of this P450 enzyme has a preference for small, hydrophobic substrates [77].These combined results could be interpreted as indicating that the reduction inlipophilicity that accompanies perdeuteration of NDMA effects its ability to bindto the active site of the enzyme. The examples cited above for FTCM and forDNS-NHCH3 illustrate that even very small changes in the sizes of large moleculesupon deuteration can be detected. Therefore, it is not unreasonable to postulatethat relatively larger changes in the sizes of smaller molecules, such as thedifferences in the sizes of NDMA and NDMA-d6, would have relatively largereffects on the lipophilicities of such molecules, and that these differences would bedetectable by the active site of an enzyme. In a potentially relevant study, Evans[78] used host/guest chemistry to show that an alkane, heptane, can coordinate toa metal center, iron(II), in a crystalline porphyrin complex. They noted that an sp3

C–H bond can act as an electron pair donor in a three-center (metal C–H), twoelectron bond, designated as an agostic interaction, that such s-complexes areobligatory intermediates in a number of important C–H activation reactions (e.g.cytochrome P-450 catalyzed reactions), and that they are sufficiently weak to beunstable under ambient conditions unless the alkane is anchored to an adjacent,more strongly bound ligand, or trapped in a molecular cavity near the metal center.Since alkane isotopomers of a similar size (octane-d0/-d18) are separable by gaschromatography, NDMA isotopomers are separable by RP-HPLC, and deuteratedalkenes have been shown to bind more strongly to transition metal complexes [60],it seems possible that alkane isotopomers (and possibly NDMA isotopomers) mightexhibit different binding affinities to Fe–porphyrin complexes. An alternativehypothesis was recently proposed by Bell and Guengerich [79], who studied theoxidation kinetics of ethanol, versus deuterated ethanol (1,1-2H ethanol), to ac-etaldehyde by human cytochrome P450 2E1. These investigators found the releaseof product, acetaldehyde, from the enzyme was the rate limiting step of catalysis,and that this could account for the effects of isotopic hydrogen substitution on theKm for the conversion of ethanol to acetaldehyde. Their results demonstrated thatdeuterated acetaldehyde binds more tightly to the enzyme, and is released moreslowly from it, than nondeuterated acetaldehyde. They suggested that rate-limiting


product release might account for all of the P450 2E1 catalyzed reactions in whicha kinetic hydrogen IE on Km has been observed (e.g. NDMA/NDMA-d6)metabolism. In either case, substrate binding to or product release from theenzyme, it is likely that deuterium substitution in the substrate or product effectsthe noncovalent interaction of the molecule with the enzyme.

4. Summary and conclusions

The purpose of this review was to provide evidence for DIEs on noncovalentinteractions between molecules, in order to demonstrate the potential importance ofsuch effects for the interpretation of data from experiments involving deuteriumsubstituted compounds. Both biological and nonbiological examples were cited inorder to illustrate the generality of the phenomenon, and, in many cases, thenonbiological examples can be used to help understand noncovalent deuterium IEsobserved in biological experiments. The origin of the IEs on noncovalent interac-tions has been attributed primarily to differences in the lengths of the C–D versusC–H covalent bonds, and secondarily to the changes in the physical properties ofa molecule that are a consequence of deuteration, such as changes in polarity,polarizability, and molecular volume. There is currently enough information avail-able in the literature to begin to formulate some generalizations about DIEs onnoncovalent interactions. For example, deuteration of hydrocarbons will generallyresult in a more polar, or less lipophilic, molecule and this effect will become morepronounced as the content of deuterium in the molecule increases. If the deuteriumsubstituted molecule also contains heteroatoms (e.g. N, O, S), then the effect ofdeuteration upon polarity or lipophilicity, either an increase or a decrease, willdepend on the position of deuterium substitution with respect to the location ofheteroatoms in the molecule. DIEs on noncovalent interactions between moleculescan be substantial, and it would be useful for biologists to be aware of thisphenomenon when planning experiments or interpreting the results of experimentsinvolving the use of deuterium labeled compounds.

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