Biomimetic Chemistry - ACS Publications - American Chemical Society

---

2 Models for Enzymic Systems An Introduction

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

FRANK H. WESTHEIMER Department of Chemistry, Harvard University, Cambridge, MA 02138

During the last two decades, the mechanisms of many enzymic processes have been established, and model systems have been developed that effectively mimic their action. In particular, the roles of thiamin, NAD, pyridoxal, flavins, B , ferridoxin, and metals in many enzymic processes now are understood. Model systems have been developed to imitate the action of decarboxylases and esterases, to imitate the action of enzymes in binding their substrates, and to approach the stereo-specificity of enzymes. Our laboratory recently has found phosphorylating agents that release monomeric methyl metaphosphate, which in turn carries out phosphorylation reactions, including some at carbonyl oxygen atoms, that suggest the actions of ATP. The ideas of biomimetic chemistry are illustrated briefly in terms of the processes mentioned above. 12

A

ll the chemical reactions in living systems are catalyzed by enzymes. Understanding the fabulous catalytic activity of enzymes and their remarkably precise specificity constitutes one of the great scientific problems of all time. Since Sumner crystallized urease in 1926, considerable progress has been made in developing the pathways, and in some instances rather detailed mechanisms for the action of enzymes. Theory has led to quantitative estimates of the catalytic activation that can be anticipated by a number of means: by bringing substrates into intimate contact with one another, by orienting molecules properly for reaction, by desolvating reactants, by internal acid and base catalysis, by special complexing of metal ions, by introducing steric strains, and by other means. Whereas two decades ago, no one 0-8412-0514-0/80/33-191-017$05.00/0 © 1980 American Chemical Society

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

18

BIOMIMETIC CHEMISTRY

knew how any enzyme worked, the more optimistic among the biochemical community now look at the problems of enzyme mechanisms as essentially solved. Such, however, is regrettably not the case. Although we like to believe that we "understand" some enzymes at a moderate level of sophistication, we cannot really claim to have solved the problem until we are able to design a structure, previously unknown, that will serve as catalyst for a specific reaction, and that will be selective for a particular substrate; we must then synthesize that structure and test the predictions. Further, we must be able to make at least an approximate estimate—within two orders of magnitude—of the expected rate and binding constant of the substrate for our "synthetic enzyme." If this sounds like a tall order—it is. But that is the challenge, that is the problem that chemists face in their attack on enzymology. Biomimetic chemistry is concerned with building models that imitate enzyme activity and specificity. The final objective is to convert the problem to one of molecular engineering, rather than one of answering fundamental questions. Even after someone has synthesized the first good enzyme-like catalyst, much will remain to be done as we explore the various ways in which the enormous catalytic activity of enzymes can be achieved. The leading synthetic organic chemists can now make any "small" molecule, but we are still interested in the ingenious ways in which such syntheses are achieved; similarly, even after we can synthesize appropriate catalysts, chemists will be interested in the actual accomplishment in specific cases. Although such catalysts eventually may have important industrial and medical uses, we are still far from the time when such catalysts can be designed and made. Biomimetic chemistry has been dominated by Japanese and American scientists; interestingly, a number of the joint publications from U.S. laboratories are from teams with both Japanese and American collaborators. A joint Japanese-American symposium on biomimetic chemistry is then especially appropriate. In this volume, Tagaki's chapter concerns the effects of metal ions in promoting a specific hydrolytic process. His work illustrates the effects of metal ions in numerous enzymic systems. Kaisers chapter is directed toward an understanding of the role of protein structure in enzymology. Since we cannot as yet predict how polypeptide chains will fold, he has adapted to his purposes a natural protein that has folded to a stable conformation, and is making an attempt to use its binding site to introduce a different catalytic activity than the one characteristic of the native enzyme; specifically, he is trying to convert a hydrolytic enzyme to one for oxidation-reduction. Yoshikawa has examined a system that promotes a highly stereoselective process; such systems lead us toward the objective of building catalysts with the complete

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

2.

WESTHEIMER

19

Models for Enzymic Systems

stereospecificity that is characteristic of enzymes. Finally, Breslow will describe chemical modifications of cyclodextrins, modifications designed to allow an understanding of the specificity of binding, and the connection between binding and catalytic activity. These introductory remarks will have a twofold objective: first, to review the history of the field, in an attempt to put the following papers into prospective; second, to present some work from my own laboratory on a possible biomimetic system—the generation of monomeric metaphosphate, and its reactions that seem to parallel the action of pyruvate kinase and of amidotransferases. Pyridoxal Phosphate An accurate history of biomimetic reactions cannot easily be written. Emil Fischer's synthesis, in 1887, of fructose by the aldol condensation of a crude mixture of glyceraldehyde and dihydroxyacetone (I, 2) reasonably can be described as a biomimetic reaction; probably other, earlier syntheses could be cited with almost equal merit. The reactions mentioned here obviously have been selected somewhat arbitarily from the wealth of possibilities in the literature. Certainly one of the most elegant examples comes from the work of E . E . Snell and his collaborators. Although his work is modern by comparison with that of Fischer cited above, Snell reported in 1945 that he could transaminate glutamic acid and pyridoxal (3) by heating them together at about 120°C. Subsequently, in a series of papers with M . Ikawa, D . E . Metzler, and others, Snell reported (4-8) that the nonenzymic reactions of pyridoxal with amino acids in aqueous solutions, p H 4-10, are strongly promoted by polyvalent cations, such as A l , F e , and C u . Most amino acids tested under these conditions undergo reversible transamination; glycine is an exception. Here the equilibrium in transamination with pyridoxal lies far over the side of the amino acid; the reverse reaction, however, between pyridoxamine and glyoxylic acid, occurs readily in hot aqueous solutions in the presence of di- and trivalent cations. + + +

+ + +

+ +

CHO +

0 C—CH —CH —CH—C0 2

2

2

2

(1)

+

0 C—CH —CH —C—C0 " 2

2

2

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

2

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

20

BIOMIMETIC CHEMISTRY

Other reactions that mimic the enzymic processes that require pyridoxal phosphate also have been realized. Werle and Koch reported the nonenzymic decarboxylation of histine (9). The racemization of alanine occurs in preference to its transamination when aqueous solutions with polyvalent cations are maintained at p H 9.5. Other amino acids are likewise racemized; the order of rates is Phe, Met > Ala > Val > lieu. At lower p H , the dominant reaction is transamination, with p H maxima varying from 4.3-8 with the nature of the metal ion used as catalyst. When serine-3-phosphate or cysteine or threonine is heated with pyridoxal and an appropriate metal ion, the compounds are degraded to the corresponding ketoacids. Similarly, cystathionine is decomposed to yield a-ketobutyric acid (4-8). Further, serine reacts with indole in hot aqueous solution at p H 5 in the presence of metal ions to yield tryptophane, in strict imitation of the corresponding enzymic synthesis. The biomimetic reactions promoted by pyridoxal are remarkable in that so many different reactions of pyridoxal-promoted enzymes can be imitated with a simple metal ion serving as catalyst. Of course, these do not duplicate the enzymic processes (10, II). To begin with, the enzymes probably do not require metal ions. Furthermore, the metal-ion promoted reactions are not specific; many competing reactions of the same substrate occur concurrently, and of course, the nonenzymic processes are not stereospecific. Nevertheless, at a minimum, they have been important in helping to elucidate the chemical mechanism of reactions promoted by pyridoxal. In this connection, the work of Snell and his collaborators in finding substitutes for pyridoxal is of interest. For example, 4-nitrosalicylaldehyde proves an excellent substitute for the coenzyme in nonenzymic reactions; its electronic structure, and the possibilities for "electron-pushing" are similar to those pyridoxal phosphate. Still, research on the biomimetic reactions of pyridoxal will continue until we find out how to promote reactions with chemical and stereochemical selectivity, and at rates comparable with those that can be achieved with enzymes.

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

2.

WESTHEIMER

Models for Enzymic Systems

21

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

Thiamin Another early success in biomimetic chemistry concerns reactions promoted by thiamin. In 1943, more than 35 years ago, Ukai, Tanaka, and Dokowa (12) reported that thiamin will catalyze a benzoin-type condensation of acetaldehyde to yield acetoin. This reaction parallels a similar enzymic reaction where pyruvate is decarboxylated to yield acetoin and acetolactic acid. Although the yields of the nonenzymic process are low, it is clearly a biomimetic process; further investigation by Breslow, stimulated by the early discovery of Ugai et al., led to an understanding of the mechanism of action of thiamin as a coenzyme. In 1957, Breslow (13) showed that the hydrogen atom in the 2-position of the thiazolium-ion portion of thiamin is ionized readily; the electronic structure of the anion imitates that of cyanide ion. The chemistry of thiamin can then be explained; the decarboxylation of pyruvate and the acetoin condensation are processes that follow conventional mechanisms; in modern language, thiamin allows an acyl group to become an anion equivalent. Subsequent to Breslow's initial discovery, he and McNelis (14) synthesized 3,4-dimethyl-2acetylthiazolium ion, and showed that in fact it is hydrolyzed rapidly. CH3 I

fi.

CH«*

^ 2

J\ 3

OCH Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

3

(10)

(fj)

2

OCH I

3

o—PO 2

I

3

o—PO 2

C—CH

3

NH —C H 2

6

5

Or

I C=CH

2

+ BW

JC—CH,

II

N—C H 6

S

+ CH3OPO3H+ BH

(11)

+

acetophenone. Apparently the attack of aniline at the carbonyl carbon atom is sufficiently rapid relative to the loss of a proton to the strong but sterically hindered base that is present, so that a Schiff base is formed preferentially to the enol phosphate. The second comment concerns control experiments. An alternative pathway for the formation of the Schiff base would involve the direct attack of aniline on acetophenone. Measurements of the rate of formation of the Schiff base suggested that, indeed, two separate processes occur, with the metaphosphatepromoted process being much the faster. This conclusion was supported strongly by the observation that, with 2-trifluoromethylaniline, the difference in rates is so large that, for practical purposes, the only reaction that occurs is the one promoted by metaphosphate. Finally, when methyl metaphosphate is generated in the presence of ethyl acetate and aniline, ethyl N-phenylbenzamidate is produced. Presumably, the reaction follows the course shown in Equation 12. These reactions suggest the corresponding biochemical ones (see Equation 13). In the presence of pyruvate kinase, A T P and pyruvate are equilibrated with phosphoenolpyruvate and A D P (74, 75, 76). Furthermore, in the presence of formylglycinamide ribonucleotide amidotransferase, A T P reacts with 5-phosphoribosyl-N-formylglycinamide and glutamine to yield 5-phosphoribosyl-N-formylglycinamidine according to Equation 14 (77).

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

30

BIOMIMETIC CHEMISTRY

O—CH

3

—PO 2

c

\

OC,H,

O C H + [CH OP0 ] 2

5

3

OCH

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

I

o—PO

2

'

HHNH

'• (12)

3

,NC H

2

B

I

C-NH C H \ OC H 2

6

\

5

+

2

C

S

+ CH OPO,H3

OC H 2

S

+ BH"

5

1

OPO. CH COCO,- +ATP 3

Pyruvate kinase

CH,=C

\

+ ADP

co

(13)

2

H N CH

2

\ : H O

0=C

A m idotran sf e rase

4- A T P + ghitamine

v

,

++

^ H (14)

Ribose phosphate H N / \ CH CHO 2

HN

A

NH Ribose phosphate

Of course, the mechanisms of these enzymic processes are at present unknown. G . Lowe and B. S. Sproat (78) have suggested that the action of pyruvate kinase involves monomeric metaphosphate as an intermediate. His evidence concerns the effect of the enzyme in scrambling the oxygen atoms of A T P in the absence of pyruvate, Equation 15. This scrambling process is enzyme catalyzed, and is interpreted most easily, but not exclusively, in terms of the reversible formation of monomeric metaphosphate.

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

2.

WESTHEIMER

31

Models for Enzymic Systems

®—P—o—PO AMP—®

3

=

^ @—P—o + [ P 0 ] 3

AMP—®

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

(15)

AMP—® Although these data suggest a monomeric metaphosphate mechanism, they are far from conclusive. Furthermore, while the reactions shown for methyl metaphosphate apparently mimic the enzymic processes, the mechanisms are not necessarily parallel. In particular, Kuo and Rose (79) have shown that the action of pyruvate kinase probably involves the energetically unfavorable enolization of pyruvate, followed by phosphorylation of the enol. (More precisely, they studied the reaction of Equation 13 in the direction from right to left.) By contrast, the reaction of methyl metaphosphate on acetophenone almost certainly proceeds by initial attack of the monomeric metaphosphate on the carbonyl oxygen atom of the ketone. Nevertheless, an interesting speculation concerns the possibility that, in some instances at least, phosphorylation proceeds by pathways such as those shown in Equations 11 and 12; if this proves to be the case, then A T P is not only thermodynamically, but also kinetically important. F. Lipmann (80), in an early and classical advance in biochemistry, showed that thermodynamically unfavorable processes could be carried forward if they were coupled to the hydrolysis of ATP, or to that of another "highenergy phosphate". The net free energy for the overall process, for instance, of the amidation of 5-phosphoribosyl-N-formylglycinamide is negative, provided that in the process A T P is degraded to A D P and inorganic phosphate. Now it appears that A T P may serve a second important function. It could activate a carbonyl group by phosphorylating it, much in the fashion that a superacid might activate a carbonyl group by protonating it, converting the polar carbonyl compound to an even more polar intermediate with a positively charged oxygen atom. The molecule would then be primed for rapid reaction, even with a relatively mild nucleophile. The kinetic activation of carbonyl compounds by methyl metaphosphate may prove to be a biomimetic process, and to illuminate the kinetic importance of ATP. Literature Cited 1. Fischer, E.; Tafel, J. Ber. Dtsch. Chem. Ges. 1887, 20, 3384. 2. Fischer, E. Ber. Dtsch. Chem. Ges. 1890, 23, 387. 3. Snell, E. E. J. Am. Chem. Soc. 1945, 67, 194.

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

32

BIOMIMETIC CHEMISTRY

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

4. 5. 6. 7. 8. 9. 10.

Metzler, D. E.; Snell, E. E. J. Am. Chem. Soc. 1952, 74, 979. Olivard, J.; Metzler, D. E.; Snell, E. E. J. Biol. Chem. 1952, 199, 669. Metzler, D. E.; Ikawa, M.; Snell, E. E. J. Am. Chem. Soc. 1954, 76, 648. Ikawa, M.; Snell, E. E. J. Am. Chem. Soc. 1954, 76, 653. Longnecker, J. B.; Snell, E. E. J. Am. Chem. Soc. 1957, 79, 142. Werle, E.; Koch, W. Biochem. Z 1949, 319, 305. Braunstein, A. E. "The Enzymes"; Boyer, P. D., Lardy, H.,Myrbäch,K., Eds.; Academic: New York, 1960; Vol. II, 113. 11. Snell, E. E. Vitam. Horm. (N.Y.) 1958, 16, 77. 12. Ukai, T.; Tanaka, R.; Dokowa, T. J. Pharm. Soc. Jpn. 1943, 63, 269. 13. Breslow, R. J. Am. Chem. Soc. 1957, 79, 1762. 14. Breslow, R.; McNelis, E. J. Am. Chem. Soc. 1960, 82, 2394. 15. Carlson, G. L.; Brown, G. M. J. Biol. Chem. 1960, 235, PC3. 16. Holzer, H.; Beaucamp, K. Biochim. Biophys. Acta 1961, 46, 225. 17. Krampitz, L. O.; Suzuki, I.; Greull, G. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1961, 20, 971. 18. Lienhard, G. E. J. Am. Chem. Soc. 1966, 88, 5642. 19. Crosby, J.; Lienhard, G.E.J. Am. Chem. Soc. 1970, 92, 5707. 20. Westheimer, F. H. Proc. Chem.Soc.,London 1963, 253. 21. Westheimer, F. H. Robert A. Welch Foundation Conference on Chemical Research, 15th, 1971, 7. 22. Hamilton, G.; Westheimer, F. H. J. Am. Chem. Soc. 1959, 81, 6332. 23. Fridovich, I.; Westheimer, F. H. J. Am. Chem. Soc. 1962, 84, 3208. 24. Warren, S.; Zerner, B.; Westheimer, F. H. Biochemistry 1966, 5, 817. 25. Tagaki, W.; Westheimer, F. H. Biochemistry 1968, 7, 891, 895, 901. 26. Frey, P. A.; Kokesh, F. C.; Westheimer, F. H. J. Am. Chem. Soc. 1971, 93, 7266. 27. Kokesh, F. C.; Westheimer, F. H. J. Am. Chem. Soc. 1971, 93, 7270. 28. Guthrie, J. P.; Westheimer, F. H. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1967, 26, 562. 29. Guthrie, J. P. J. Am. Chem. Soc. 1972, 94, 7024. 30. Guthrie, J. P.; Jordan, F. J. Am. Chem. Soc. 1972, 94, 9132, 9136. 31. Taguchi, K.; Westheimer, F. H. J. Am. Chem. Soc. 1973, 95, 7413. 32. Westheimer, F. H.; Fisher, H. F.; Conn, E. E.; Vennesland, B.J.Am. Chem. Soc. 1951, 73, 2403. 33. Vennesland, B.; Westheimer, F. H. "The Mechanism of Enzymes"; McElroy, W. D., Glass, B., Eds.; The Johns Hopkins Press: Baltimore, 1954; 357. 34. Abeles, R. H.; Hutton, R. F.; Westheimer, F. H. J. Am. Chem. Soc. 1957, 79, 712. 35. Norcross, B. E.; Kleindinst, P. E., Jr.; Westheimer, F. H.J.Am. Chem. Soc. 1962, 84, 797. 36. Dittmer, D. C.; Fouty, R. A. J. Am. Chem. Soc. 1964, 86, 91. 37. Creighton, D. J.; Sigman, D. S. J. Am. Chem. Soc. 1971, 93, 6314. 38. Bränden, C. I.; Jörnvall, H.; Eklund, H.; Furugren, B. Enzymes 1975, 105. 39. Holbrook, J.; Liljas, A.; Steindel, S. J.; Rossmann, M. G. Enzymes 1975, 191. 40. Smith, E. L.; Austen, B. M.; Blumenthal, K. M.; Nye, J. F. Enzymes 1975, 294. 41. Banaszak, L. J.; Bradshaw, R. A. Enzymes 1975, 369. 42. Shinkai, S.; Bruice, T. C. Biochemistry 1973, 12, 1750. 43. Ohnishi, Y.; Kagami, M.; Ohno, A. J. Am. Chem. Soc. 1975, 97, 4766. 44. Van Bergen, T. J.; Kellogg, R.M.J.Am. Chem. Soc. 1977, 99, 3882. 45. Abeles, R. H. "Biological Aspects of Inorganic Chemistry," Dolphin, D., Ed.; John Wiley: New York, 1978. 46. Dowd, P.; Shapiro, M.; Kang, K. J. Am. Chem. Soc. 1975, 97, 4754. 47. Dowd, P.; Trivedi, B. K.; Shapiro, M.; Marwaha, L. K. J. Am. Chem. Soc. 1976, 98, 7875. 48. Dowd, P.; Shapiro, M. J. Am. Chem. Soc. 1976, 98, 3724.

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

2. WESTHEIMER

Models for Enzymic Systems

49. Carter, C. W., Jr.; "Iron-Sulfur Proteins"; Lovenberg, W., Ed.; Academic: 1977; Vol.III,p. 157. 50. Wolff, T. E.; Berg, J. M.; Warrick, C.; Hodgson, K. O.; Holm, R. H.; Frankel, R. B. J. Am. Chem. Soc. 1978, 100, 4630. 51. Reynolds, J. G.; Laskowski, E. J.; Holm, R. H. J. Am. Chem. Soc. 1978, 100, 5315. 52. Laskowski, E. J.; Frankel, R. B.; Gillum, W. O.; Papaefthymiou, G. C.; Renaud, J.; Ibers, J. A.; Holm, R. H. J. Am. Chem. Soc. 1978, 100, 5322. 53. Johnson, R. W.; Holm, R. H. J. Am. Chem. Soc. 1978, 100, 5338. 54. Kurtz, D. M., Jr.; Wong, G. B. Holm, R. H.J.Am. Chem. Soc.1978,100, 6777. 55. McKenna, C. E. "Molybdenum and Molybdenum-Containing Enzymes;" Coughlan, M. P., Ed.; Pergamon: Oxford, 1980; 14. 56. Lloyd, G. J.; Hsu, C. M.; Cooperman, B. S. J. Am. Chem. Soc. 1971, 93, 4889. 57. Hsu, C. M.; Cooperman, B.S.J.Am. Chem. Soc. 1976, 98, 5652, 5657. 58. Buckingham, D. A.; Marzilli, L. G.; Sargeson, A. M. J. Am. Chem. Soc. 1967, 89, 2772, 4539. 59. Anderson, B.; Milburn, R. M.; Harrowfield, J. MacB.; Robertson, G. B.; Sargeson, A.M.J.Am. Chem. Soc. 1977, 99, 2652. 60. Butler, L. G. Enzymes 1971, 529. 61. Reid, T.W.;Wilson, I. B. Enzymes 1971, 373. 62. Hartsuck, J. A.; Lipscomb, W. N. Enzymes 1971, 1. 63. Delange, R. J.; Smith, E. L. Enzymes 1971, 81. 64. Bernard, P. W. C.; Bunton, C. A.; Llewellyn, D.R.;Oldham, K.G.;Silver, B. L.; Vernon, C. A. Chem. Ind. (London) 1955, 760. 65. Butcher, W. W.; Westheimer, F. H. J. Am. Chem. Soc. 1955, 77, 2420. 66. DiSabato, G.; Jencks, W. P.J.Am. Chem. Soc. 1961, 83, 4400. 67. Brown, D.; Hamer, N. J. Chem. Soc. (London) 1960, 1155. 68. Samuel, D.; Silver, B. J. Chem. Soc. (London) 1961, 4321. 69. Miller, D. L.; Ukena, T. J. Am. Chem. Soc. 1969, 91, 3050. 70. Rebek, J.; Gaviña, F. J. Am. Chem. Soc. 1975, 97, 1591, 3221. 71. Clapp, C. H.; Westheimer, F. H. J. Am. Chem. Soc. 1974, 96, 6710. 72. Clapp, C. H.; Satterthwait, A . ; Westheimer, F.H.J.Am. Chem. Soc. 1975, 97, 6873. 73. Satterthwait, A. C.; Westheimer, F. H. J. Am. Chem. Soc. 1978, 100, 3197. 74. Rose, I. A. J. Biol. Chem. 1960, 235, 1170. 75. Robinson, J. L.; Rose, I. A. J. Biol. Chem. 1972, 247, 1096. 76. Levitzki, A.; Koshland, D. E., Jr. Biochemistry 1971, 10, 3365. 77. Buchanan, J. Adv. in Enzymol. 1973, 39, 91. 78. Lowe, G.; Sproat, B. S. Chem. Commun. 1978, 783. 79. Kuo, D. J.; Rose, I.A.J.Am. Chem. Soc. 1978, 100, 6288. 80. Lipmann, F. Adv. in Enzymol. 1941, 1, 99. ;

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 3, 2015 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch002

33

RECEIVED May 21,

1979.

In Biomimetic Chemistry; Dolphin, D., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.