J. Phys. Ckem. 1985,89, 3 179-3 182 for C10, In conclusion, the measurements we made of C12, C14, and C16 TABS provide information about 4L,cmc, AHo/n, AH*/n, ACpo/n,and 4,. Generally speaking, the values we obtain are in good agreement with literature values, where (23) Anacker, E. W.; Rush, R. M.; Johnson, J. S. J. Phys. Chem. 1964,
68, 81. (24) Tartar. H.V. J. Colloid Sei. 1959. 14. 115. (25j Debye,'P. Ann. N . Y. Acad. Sci. 1949151, 575. (26) Debye, P. J. Phys. Chem. 1949, 53, 1. (27) Llanos, P.; Zana, R. J. Colloid Interface Sei. 1982, 88, 594. (28) Venable, R. L.; Nauman, R. V. J. Phys. Chem. 1964, 68 3498. ~K, J. "Critical ~ ~ ~ i l ~Concentrations ~~ l l , ~ of (29) Mukerjee, p.; ~
Aqueous Surfactant Systems", Natl. Stand. Ref: Data Ser. (US.Natl. Bur. Stand.) 1971, NSRDS-NBS 36.
3179
literature values are available. We have also shown that the model described above is applicable to our data and provides useful information about trends in the thermodynamic properties of these alkyltrimethylammonium bromide surfactants.
Acknowledgment. Acknowledgment is made to the donors of the Petroleum Research Fund. administered bv the American Chemical Society, for support 'of this research: Registry No. Decyltrimethylammonium bromide, 2082-84-0; dodecyltrimethylammonium bromide, 1 1 19-94-4; tetradecyltrimethylammonium bromide, 1 1 19-97-7; hexadecyltrimethylammonium bromide, 57-09-0.
Mechanism of Tritium-Atom Promoted Isotope Exchange in the Benzene Ring: 2. Substituent Effects and a Crossover in Labeling Mechanism M. F. Powellt and R. M. Lemmon* National Tritium Labeling Facility, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720 (Received: November 5, 1984)
Reaction of tritium atoms, generated by microwave activation of T2 gas, with substituted benzenes was studied at - 4 0 O C . The tritiations were usually carried out with a 1:l mole mixture of the desired phenyl compound and toluene. Both relative labeling yields (with benzene = 1 as a reference) and product isotope effects were determined; these were used to support a crossover in labeling mechanism from predominantly addition-elimination (as in benzene-?) to a hot-atom multiple-tritium addition (as takes place in xylene-t). The contribution of each labeling pathway could be controlled by varying the distance between the microwave discharge cavity and the sample; shorter distances gave "hotter" tritium atoms at the sample, resulting in less selectivity but higher labeling yields.
Introduction Tritium labeling of biological compounds by microwave activation of T2is one of the simplest of labeling procedures devised and, yet, is not used routinely because of the lack of general understanding of reaction mechanism(s) and conditions required for enhanced labeling selectivity. We have turned our attention to this and have recently delineated some of the factors for the For example, the effect of tritiation of aromatic sample temperature and isotopic substitution in the labeling of benzene demonstratd that the main labeling pathways are T. addition to give the cyclohexadienyl radical followed by either Ha elimination (yielding benzene-t, eq 1) or reaction of this radical
with additional T. atoms (resulting in saturated side products such as cyclohexadiene-t2 and cyclohexane-t,, eq 2). There was little indication from these previous studies of the reaction between T.* (translationally hot) and benzene, such as (i) H.abstraction by T.* to give HT followed by reaction of the phenyl radical with another T. atom (eq 3 and 4) or, (ii) a concerted substitution reaction (eq 5 ) . It may be possible, however, to identify hot T-* H (3)
Institute of Pharmaceutical Sciences, Syntex Research, 3401 Hillview Ave., Palo Alto, CA 94304.
0022-3654/85/2089-3 179$01.50/0
T
@+ T-6 ,H
(4) T
atom labeling in substituted benzenes because labeling by the addition-elimination pathway of substituted benzenes (eq 1) should decrease due to the higher stability of the intermediate cyclohexadienyl radical3 formed. Since most substituents on benzene are thought to stabilize aryl radicals (vide i ~ ~ f r a almost ) , ~ , ~ all substituted phenyl compounds should exhibit lower tritium labeling yields than in benzene (due to eq 2 predominating over eq 1) with concomitant increases in the amounts of saturated products formed. We have demonstrated this herein and found that by changing the geometry of the labeling apparatus-specifically, varying the distance between the microwave cavity and the sample holder-we were able to show evidence for a change in the major labeling mechanism from addition-elimination (eq 1 and 2) to a translationally "hot-atom" reaction ( eq 3,4, and/or 5) in microwave-T. labeling of aromatic compounds. Experimental Section and Results All phenyl compounds were spectral grade or chromatography standards and were used without further purification. Deuterated (1) Powell, M. F.; Morimoto, H.; Erwin, W.R.; Gordon, B. E.; Lemmon, R. M. J. Phys. Chem. 1985, 88, 6266-71. (2) Gordon, B. E.; Peng, C. T.; Erwin, W. R.; Lemmon, R. M. Int. J. Appl. Radiar. Isot. 1982, 33, 721-24. ( 3 ) Nelson, R. F.; Adams, R.N. J. Am. Chem. Sor. 1968, 90, 3925-30. (4) Nelsen, S. F.; Landis, R. T.; Kiehle, L. H.; Leung, T. H. J. Am. Chem. Sor. 1972, 94, 1610-14.
0 1985 American Chemical Society
3180
The Journal of Physical Chemistry, Vol. 89, No. 14, 1985
TABLE I: Relative Labeling Yields and Product Isotope Effects ( Y H/ Y D) for Various Substituted Benzenes“
compound benzene benzene-d6 fluorobenzene fluorobenzene-d, toluene toluene-d8 m-xylene o-xylene
o-xylene-dl0 4-fluorotoluene ethylbenzene n-propylbenzene tert-butylbenzene 4-tert-butyltoluene anisole
re1 yield 1.Ob
0.17 f 0.01 0.27 f 0.04 0.11 f 0.02 0.24 f 0.02 0.24 f 0.03 0.18 f 0.01 0.052 f 0.009 0.045 f 0.003 0.082 f 0.014 0.048 f 0.005 0.0086 f 0.0009 0.010 f 0.006 0.019 f 0.002 0.043 f 0.004
yu I Yn 5.80 f 0.34
Powell and Lemmon TABLE II: Correlation of Relative Labeling Yields and Product Isotope Effects ( YH/Yo) with the Mstance between the Microwave Cavity and Light Trap
distance, cm 2
2.45 f 0.57 .OO f 0.15
. I 5 f 0.21
“The microwave cavity was 8 cm from the sample holder for these experiments. *Reference. compounds were obtained commercially (Aldrich) and were usually >98% atom D; no correction was made for the small isotopic impurity. In order to minimize side reactions, only those phenyl compounds with the least reactive (toward T-) substituents were chosen. For example, fluorobenzene is better than bromobenzene for these microwave studies because fluorine is fairly stable to halogen substitution, whereas bromine is not.s Tritium gas was generated by heating uranium tritide as described earlier.z A schematic diagram of the labeling apparatus and experimental details are shown in an earlier pub1ication.l In all experiments the visible flow discharge extended -0.5-1.0 cm beyond the edges of the microwave cavity. Inherent difficulties in strictly controlling the microwave plasma from run-to-run often resulted in different product yields and so the relative labeling yields (see Table I) were obtained from experiments carried out competitively. These were done by reacting a stream of T- in H e (0.5 torr of Tz; 4.5 torr of He1) with a 1:l mole mixture of toluene and the phenyl compound of study for 1 min at ---SO O C . The relative yields of Table I are referenced to the most reactive compound, benzene (at l,O), but could have just as easily been given in terms of toluene (by multiplying the relative yields in Table I by 1.0/0.24 or 4.17). The sample workup and GLRC analyses were carried out as described previously.1*6To prevent relative yield errors caused by the selective evaporation of the lighter compound from the liquid sample mixture during the 1-min run, the composition of the 1:l mole mixture was checked both before and after the exposure. In the event that some fractional evaporation had occurred (Le,, a few percent difference), the radioactive peak counts were corrected by measuring the mass peaks with an HP 3390 A integrator. No attempt was made to identify many of the other radioactive compounds formed during the microwave labeling; however, recent advances in retention index assignments that facilitate this task are soon forthcoming.’ (To illustrate the complexity of this problem, we note that exposure of only m-fluorotoluene to a microwave activated stream of Tz gas can give more than 30 different cycl+c6 compounds of varying degrees of substitution and saturation.) In almost all experiments herein, the total yield of labeled saturation products was greater than the yield of labeled parent compounds. Total sample activities were comparable with our earlier study on benzene and biphenyl.’ All isotope effect measurements using deuterated phenyl compounds were carried out similarly except for the reaction of toluene-d8. In this case the sample was made of a 1:l mole mixture of benzene and toluene-& (5) Peng, C. T.; Gordon, B. E.; Erwin, W. R.; Lemmon, R. M. Inr. J . Appl. Radiat. Isot. 1982, 33, 419-27. ( 6 ) Gordon, B . E.; Erwin,W. R.; Press, M.; Lemmon, R. M. Anal. Chem. 1978, 50, 179-82. ( 7 ) Buchman, 0.;Cao, G.-Y.; Peng, C. T. J . Chromatogr. 1984, 312, 75-90.
4 8c
compound benzene
I .04
re1 yield
benzene-d6 toluene
0.69 f 0.06 0.85 f 0.05
1.45 f 0.13
benzene-d6
b
benzene-d6
0.17 f 0.01
2.53 f 0.10 5.80 f 0.34
YUI Yn
“ Reference. From ref 1. The relative yield is not given because YH/YD was obtained by comparison with 1,4-~yclohexadieneyields. From Table I. The competitive method was also checked by reacting T. with m-xylene and toluene or benzene in separate experiments. When benzene was used as the reference compound, the ratio of mxy1ene:benzene was 0.18 f 0.01. When toluene was used and then corrected for the ratio of to1uene:benzene (0.24), the calculated m-xy1ene:benzene ratio was reasonably close (0.14 f 0.03). Most experiments were carried out with the microwave cavity 8 cm from the light trap in contrast to our earlier work where the distance was -4 cm. The relative labeling yields for toluene and benzene-d6 were also measured with the microwave cavity moved as close as possible (-2 cm) to the light trap without direct UV irradiation from the microwave source on the sample. In these cases (seeTable 11), the relative yield for toluene was significantly higher (0.85 f 0.05) and the product isotope effect (YH/YD) for benzene-d6 was reduced (1.45 f 0.13).
Discussion Inspection of Table I shows that the relative labeling yields of substituted benzenes drop quite dramatically with the number or size of substituents. Both electron-donating and electron-withdrawing substituents cause a decrease in the labeling yields, as compared with benzene. In certain other cases, however, the relative amounts of exchange-labeled product are different even though the substituents are electronically similar. For example, both toluene and propylbenzene have similar alkyl groups, but the former gives approximately 30 times more exchange-labeled product. Since these experiments were carried out on a 1:l mole mixture, one might expect (based on the number of hydrogen atoms per molecule) that the yield of labeled propylbenzene-t should be higher than toluene-t. The data for the alkyl substituted benzenes in Table I refute this, however. Comment on this is deferred to the next subsection. Another comparison of relative labeling yields warrants attention. Approximately 3 to 4 times more labeled m-xylene than o-xylene was formed under like reaction conditions. In that the highly exothermic rates of T. attack on the isomers should be similar as should the Ha elimination rates (both compounds have the same number of methyl-stabilized radical resonance forms), some other factor(s) may account for the difference in labeling yields. We also note that the product isotope effects (YH/YD)decrease for the series benzene-& fluorobenzene-ds, toluene-& and oxylene-dlo. The “extra” deuterium in the substituent groups contribute little to the overall isotope effect because isotope exchange in the methyl group(s) (if it occurs) probably follows a “hot-atom” reaction in which small (Le., 1) isotope effects are expected (for example, eq 6 and 7). Thus YH/YD is primarily
-
CH,
T:+@-6+
tH9
HT
determined by the isotopic substitution in the aryl ring. (Recent observations in our laboratory have shown a small amount of labeling in hexamethylbenzene; this indicates that the methyl
T Atom Isotope Exchange in Benzene
groups of toluene or xylene may also be tritiated by a minor with increasing substituent pathwaye8) The decrease in Y H / Y D size is strongly indicative of a change in labeling mechanism and is the subject of the following paragraphs. The electronic effect of the substituent is one of the factors that changes the labeling mechanism from additioq-elimination (eq 1 and 2) to a hot-atom type of reaction (eq 3-7). This can be illustrated in the following example: If most of the tritium atoms react with the phenyl compound by addition and the remainder by a hot-atom reaction (eq 3-7), then the primary labeling path will be addition-elimination providing the elimination step (eq 1) is faster than subsequent T. addition (eq 2). Only unstable cyclohexadienyl radicals of short lifetime will exhibit fast Helimination; otherwise the labeling will occur by a hot-atom reaction path. Phenyl substituents, which stabilize the intermediate radical (resulting in longer lifetimes), increase the possibility of subsequent T. addition with proportionately less likelihood for elimination of H- and addition-elimination labeling. In that most aryl substituents stabilize radicals more than H. (support for this comes from both spin density studies of CH3-CH-X9 and from selectivity ratios of aryl radicalsI0J’), the contribution to exchange labeling by the reactions of eq 1 and 2 is less for the more substituted benzenes. The isotope effect data support this conclusion. In benzene, where the relative labeling yield is high (primarily by the addiis much greater than unity since tion-elimination pathway) YH/YD the major exchange mechanism involves product-forming H transfer in the elimination step (eq 1). Fluorobenzene exhibits a moderate labeling yield (0.27 f 0.04) and a smaller isotope effect (2.45 f 0.57), presumably because both labeling mechanisms (eq 1, 2, and 3-7) are almost equally operative. Toluene and especially xylene, however, show lower labeling yields and smaller isotope effects caused by a crossover in the exchange-labeling mechanism to primarily “hot atom” labeling. This is not unexpected in that the alkyl groups in toluene and propylbenzene readily stabilize the cyclohexadienyl radical formed by T. addition to the ring. We are presently setting up tritium NMR facilities in order to determine the labeling patterns in these compounds. The electronic effect on radical stability is not the only factor governing labeling yields, otherwise 0-and m-xylene would have nearly identical labeling yields as would toluene and propylbenzene. The diffusion rates of the target compounds also play an important role in determining relative labeling yields. The larger aryl compounds-which cannot diffuse rapidly away from the sample surface after accepting a single T.-will react with more than one tritium atom becoming multiply labeled. Thus the major exchange-labeling route for molecules of low diffusional ability will be mainly through a “hot-atom” type of reaction. On the other hand, smaller compounds that diffuse rapidly in the liquid sample matrix (Le., radicals of benzene or fluorobenzene) quickly become buried below the sample surface. This allows (i) sufficient time for the elimination of H. to occur and (ii) covering up of labeled species to prevent multiple labeling by additional T.. This molecular ”size dependence” of substituted phenyl compounds on tritium labeling is illustrated in Figure 1. The importance of molecular diffusion in microwave labeling is demonstrated vividly by comparing the relative labeling yields for toluene (0.24 f 0.02) and propylbenzene (0.0086 f 0.0009). The electronic substituent effects are similarly radical stabilizing (8) Tang, Y.-S., Morimoto, H., unpublished results. (9) Norman, R. 0. C.; Gilbert, B. C . Ado. Phys. Qrg. Chem. 1967.5.53. (10) Riichardt, C.; Herwig, K.; Eichler, S.Tetrahedron Lett. 1969, 421. (11) Riichardt, C . Agnew. Chem., Int. Edit. Engl. 1970, 9, 830-843. (12) Mnelander, L. C . S.;Saunders, W. H. Jr. “Reaction Rates of Isotopic Molecules ; Wiley: New York, 1980.
The Journal of Physical Chemistry, Vol. 89, No. 14, 1985 3181
I
-2
1 -1 6
i
-1 2
I -4
i
-8
i i
log (re1 yield)
Figure 1. Dependence of relative labeling yield (shown in logarithmic form for convenience) with the molecular weight of the labeled compound. The dashed line is included to show the trend.
and so one might expect that both compounds should show limited but like labeling yields. This is not the case, however, when saturation is a strongly competing reactiqn and the tritium exchange step is a hot atom type of reaction giving small Y H / Y D as expected for the mechanism in eq 8. CH3
T The yield of toluene-t should be relatively high for an overall exchange scheme (eq 9) where a diffusion step, kdiff, removes
‘
Saturated Products
J
labeled toluene at the sample interface at a rate competitive with saturation k,,[T.]. However, in the case of propylbenzene where k,,,[T.] is greater than kdifr[T.] (as demonstrated experimentally by the higher yields of saturated products formed), the yield of propylbenzene-t is greatly reduced because k,,,[T.] removes the labeled product much faster than kdiff [not shown in eq lo].
Pr
‘
Saturated Products The important role of molecular diffusion is also demonstrated by comparing the labeling yields of m- and o-xylene. Both isomers should be tritiated nearly equally if the labeling pathway(s) is determined solely by substituent electronic effects; this, however, is not found experimentally. The higher labeling yield for m-xylene may be explained by -a difference in diffusional rates, providing m-xylene diffuses somewhat faster than o-xylene. Speculation of which isomer should diffuse faster can be made from the viscosities (at --50 bC) for m-xylene (- 1.3 cP) and for o-xylene ( N 1.9 cP); the lower value for m-xylene indicates this compound
3182 The Journal of Physical Chemistry, Vol. 89, No. 14, 1985
probably undergoes molecular diffusion more rapidly than its ortho isomer and so the yield of labeled m-xylene is higher. (A similar correlation was noted between the labeling yields of other substituted benzenes and their respective viscositites at 0 OC;generally, the higher the viscosity, the lower was the relative labeling yield.) A change in mechanism from addition-elimination (eq 1 and 2) to hot-atom tritium labeling (q3-7) is predicted if IT.*] can be increased and [T-] reduced. One way of bringing about this increase is to move the microwave discharge cavity closer to the sample holder (see Figure 1 in ref 1) such that the T-* atoms formed have less time (before reaching the sample) to lose their excess translational energy by collision with the He carrier gas and glass walls. In such experiments, caution must be used so that the microwave cavity is not close enough to permit direct UV irradiation of the sample. The data of Table I1 show that by moving the microwave cavity closer to the light trap (to -2 cm), the product isotope effect for reaction of benzene and T.* is smaller (YH/YD = 1.45 f 0.13) and the competitive labeling reaction between benzene and toluene is less selective (relative labeling yield of toluene = 0.85 f 0.05). Both of these observations are consistent with a mechanistic crossover to a hot-atom type of reaction (i.e,, eq 3-7). The exergonic reaction of translationally hot T.* atoms with aromatic compounds is expected to proceed / with little selectivity (Hammond postulate) and smaller Y H YD (Bigeleisen, Melander, and Westheimer theory of isotope effects”), as was observed herein. Thus, moving the microwave cavity closer for the labeling of substituted phenyl compounds increases the relative labeling yields (as the fraction of hot-atom labeling be. the labeling of toluene comes greater) and reduces Y H / Y D In and o-xylene where near unity isotope effects were observed (see Table I), almost all of the exchange labeling occurred by a hot-atom pathway and not by addition-limination. Thermalized T- atoms react with alkyl substituted compounds and give fairly stable alkyl cyclohexadienyl radicals; such radicals resist rapid
Powell and Lemmon
H- expulsion (or bimolecular H. transfer to another molecule) and instead react with the excess T- impinging on the sample surface to give various saturated compounds.
Conclusions Since most bioorganic aryl molecules have radical-stabilizing substituents, which favor saturated side product formation upon T. addition, it is imperative that the microwave labeling be carried out under conditions in which hot atom labeling is greatest. This may be done by minimizing the distance between the microwave cavity and the sample or possibly reducing the total pressure of T2 (or T2 and He); in these cases the T. atoms formed should remain “hot” for a longer time because of fewer deactivating collisions before reaching the sample. Similarly, pumping the gas mixture more rapidly from the microwave cavity to the sample should also increase the labeling yields (given that P and T remain the same). This may be achieved by bleeding a T2/He mixture from an upstream bulb, and vacuum pumping away the T,/He gas mixture after a single pass by the microwave cavity and the sample. Experiments of this nature are currently in progress. Acknowledgment. This research has been supported by the Biotechnology Resources Program, Division of Research Resources, National Institutes of Health under Grant 5 P41 RR01237-02, and by the Department of Energy under Contract DE-AC03-76SF00098, Acknowledgment is also made of the helpful comments of R. G. Aune, W. R. Erwin, H. Morimoto, C. T. Peng, Y . 4 . Tang, and especially B. E. Gordon. Registry No. Hydrogen, 1333-74-0; deuterium, 7782-39-0; benzene, 71-43-2; benzene-d6, 1076-43-3; toluene, 108-88-3; toluene-& 2037-26-5; fluorobenzene, 462-06-6; fluorobenzene-d5, 1423-10-5; m-xylene, 10838-3; o-xylene, 95-47-6;o-xylene-d,o, 56004-61-6; 4-fluorotoluene, 35232-9; ethylbenzene, 100-41-4; n-propylbenzene, 103-65-1; terr-butylbenzene, 98-06-6; 4-tert-benzyltoluene, 98-51-1; anisole, 100-66-3.
