4660
J . Am. Chem. SOC.1994,116, 4660-4668
Synthesis of Novel Analogs of Acetyl Coenzyme A: Mimics of Enzyme Reaction Intermediates David P. Martin, Richard T. Bibart, and Dale G. Drueckhammer' Contribution from the Department of Chemistry, Stanford University, Stanford, California 94305 Received September 13, 1993"
Abstract: An improved method for the synthesis of analogs of coenzyme A (CoA) and its thioesters, which are modified in the thiol or thioester moiety, has been developed using a combination of chemical and enzymatic reactions. The enzymes catalyzing the last two steps of CoA biosynthesis were used to prepare a CoA analog (lc) in which an amide bond is replaced by a thioester bond and the thiol group is replaced by a methyl group. Reaction of ICwith a primary amine in aqueous solution results in aminolysis of the thioester linkage to form the desired CoA analog. Reaction with different amines permits the introduction of a variety of functional groups in place of the normal thiol or thioester group. This methodology has been used in the synthesis of five new analogs of acetyl-coA in which the thioester sulfur is replaced by a methylene group and the acetyl group is replaced by carboxylate (14a), nitro (14b), carboxamide (14c), methyl sulfoxide (14d), and methyl sulfone (14e) groups. 14a-c were designed to mimic the possible enolate or enol intermediate in the reaction of citrate synthase and related enzymes. 14a and 14c are potent inhibitors of citrate synthase, with Ki values 1000- and 570-fold lower than the K,,,for acetyl-coA, respectively. CD titrations indicate that 14a and 14c have low affinity for citrate synthase in the absence of oxaloacetate, consistent with their recognition as enol or enolate analogs. 14b is a poor inhibitor of citrate synthase, with affinity slightly lower than that for acetylCoA. These results are consistent with generation of the enol form of acetyl-coA as the nucleophilic intermediate in the reaction of citrate synthase. 14d and 14e were designed to mimic the tetrahedral intermediate or transition state in the reaction of chloramphenicol acetyltransferase and related acetyl-CoA-dependent acetyltransferases. Both compounds are poor inhibitors of chloramphenicol acetyltransferase, with affinities slightly lower than that of acetylCoA, indicating that these compounds are not good mimics of the enzyme-bound tetrahedral intermediate or transition state.
Cozenzyme A (CoA, la) and its thioesters are involved in a variety of metabolic functions including glycolysis, fatty acid &oxidation, and numerous biosynthetic pathways.' It has been estimated that 4% of the known enzymes require CoA or a CoA ester as substrate.* These include enzymes of pharmaceutical importance such as HMG-CoA r e d ~ c t a s eacyl-CoA:cholesterol ,~ acyltransferase? myristoy1CoA:protein N-myristoyltran~ferase,~ and ATP citrate lyase.6 We recently reported a combined chemical and enzymatic synthesis of CoA analog^.^ In this approach, the enzymes catalyzing the last two steps in CoA biosynthesis were used to prepare a coenzyme A analog lb, having an amide bond replaced by a thioester (Scheme 1). Aminolysis of the thioester bond of compound l b with the amine 5 formed the dethio (carba) analog of acetyl-coA (acetonyldethio-CoA, 4), as shown in Scheme 2. It was envisioned that by aminolysis with a variety of amines, l b could serve as a versatile synthon for a wide range of CoA and CoA ester analogs modified in the thiol/thioester portion of the molecule. Since the specific functionality of interest is introduced in a final chemical step, each analog would not be subject to substrate specificity limitations of the CoA biosynthetic enzymes. Abstract published in Advance ACS Abstracts, May 1, 1994. (1) McCormick, D. B.; Wright, L. D., Eds. Methods in Enzymology; Academic Press: New York, 1970; Vol. 18A, Chapter IV. (2) Lee, C.-H.; Chen, A. F. Immobilized Coenzymes and Derivatives. In The Pyridine Nucleotide Coenzymes; Everse, J., Anderson, B.; You, K., Eds.; Academic Press: New York, 1982; p 189. (3) Grundy, S . M. N . Engl. J . Med. 1988, 319, 24. (4) Sliskovic, D. R.; White, A. D. Trends Pharmacol. Sci. 1991, 12, 194. Largis, E. E.; Katocs. A. S. In Drugs Affecting Lipid Metabolism X; Gotto, A. M., Smith, L. C., Eds.; Elsevier Science Publishers: New York, 1990. ( 5 ) Shaw, W.V.; Leslie, A. G. W. Annu. Reo. Biophys. Biophys. Chem. 1991, 20, 363. (6) Elshourbagy, N. A.; Near, J. C.; Kmetz, P. J.; Sathe, G. M.; Southan, C.; Strickler, J. E.; Gross, M.; Young, J. F.;Wells, T. N. C.; Groot, P. H. E. J. Biol. Chem. 1990, 265, 1430. (7) Martin, D. P.; Drueckhammer, D. G. J. Am. Chem. SOC.1992, 114, 7287.
Scheme 1 NHZ
0 0-P-0
o w
0-7.0
? O.
ATP
0
HO OH
El
\PPI
2. a. X = NH, Y = SH b. X = S,Y = SH C. X = S, Y = CH,
E, = Phosphopantetheine adenylyltransferase E2 = Dephospho-CoA kinase E, = Inorganic pyrophosphatase 0
0
1. a. X = NH, Y = SH (CoASH) b. X = S,Y = SH C. X = S,Y = CH,
CoA esters are readily prepared by chemical8 or enzymatic9 acylation of CoA. Unnatural CoA esters have been useful in mechanistic studies of CoA ester utilizing enzymes.I0J Thioether analogs of CoA esters have been prepared by alkylation of CoA.12 (8) Ouyang, T.; Walt, D. R. J . Org. Chem. 1991,56,3752 and references therein. (9) Billhardt, U. M.; Stein, P.; Whitesides, G. M. Bioorg. Chem. 1989,17, 1.
0002-7863/94/1516-4660$04.50/00 1994 American Chemical Society
Synthesis of Novel Analogs of Acetyl Coenzyme A
J. Am. Chem. SOC.,Vol. 116, No. 11, 1994 4661 Scheme 3 OH
0
OH
1 . (CH~)~AI-S-(CH,)Z-CH~
0
::
CoA-CHz-C-CH,
2. (CH,O),P(O)CI,
pyridine 0
0 9. R = H 10, R = (CH3O)*P(O)-
4
Such compounds have shown some utility in mechanistic studies but generally have lower binding affinities for the target enzyme than the natural substrate. A more interesting example of a CoA thioester is carboxymethyl-CoA, which is a potent inhibitor of citrate synthase.12 This compound has been viewed as an analog of the enolate or enol form of acetyl-coA 6,despite its nonisosteric nature relative to acetyl-CoA.IZ-ls Another very interesting class of CoA ester analogs is thedithioesters.16J7 Thedithioester analog of acetyl-coA has greatly enhanced acidity of the acetyl methyl hydrogens (pK, 12.5) and has been used to study the mechanism of citrate synthase.16J7 The thioester is intimately involved in most of the enzymecatalyzed reactions of CoA esters. Functionalization of natural CoA provides access to a limited number of analogs modified in the thioester portion. Many other potentially valuable CoA ester analogs cannot be prepared directly from CoA and thus require assembly of the CoA moiety. A few examples of ketone analogs of CoA esters, having the thioester sulfur atom replaced with a methylene g r o ~ p , l have ~ * ' ~been prepared, as has an amide analog of propionyl-CoA.20 These analogs have been synthesized using chemistry analogous to the original chemical synthesis of CoA.21 These compounds have received very limited attention, probably due to the difficulty of their synthesis. The combined chemical and enzymatic CoA analog synthesis developed in this lab7permits for the first time the routine synthesis of CoA ester analogs modified in the thioester functionality. In our initially reported synthetic strategy, the ethanedithiol thioester intermediates l b and 2b were not isolable as they existed as a mixture of interconverting species due to disulfide bond formation and thioester thiolysis reactions.' We report here the improvement of our synthesis by deletion of the terminal thiol group. We also report the use of this methodology in the synthesis of five new analogs of acetyl CoA (14a-e) and studies of these compounds as inhibitors and mechanistic probes of acetyl-coA utilizing enzymes. Results
Improved Synthesis of Coenzyme A Analogs. To overcome problems associated with the terminal thiol group of 2b, the (10) Palmer, M. A. J.;Differding, E.;Gamboni, R.; Williams,S. F.; Peoples, 0. P.; Walsh, C. T.; Sinskey, A. J.; Masamune, S.J. Eiol. Chem. 1991, 266, 8369. (1 1) Lau, S.-M.; Brantley, R. K.;Thorpe, C. Biochemistry 1988,27,5089. (12) Eggerer, H.Eur. J. Eiochem. 1990, 198, 767. (13) Kurz, L. C.;Shah,S.; Crane, B. R.;Donald, L. J.;Duckworth,H. W.; Drysdale, G. R. Biochemistry 1992, 31, 7899. (14) Karpusas, M.; Branchaud, B.; Remington, S . J. Biochemistry 1990, 29, 2213. (15) Bayer, E.; Bayer, B.; Eggerer, H. Eur. J. Eiochem. 1981, 120, 155. (16) Wlassics, I. D.; Stille, C.; Anderson, V. E. Eiochim. Eiophys. Acta 1988, 952, 269. (17) Wlassics, I. D.;Anderson, V. E. Biochemistry 1989, 28, 1627. (18) Nikawa, J.; Numa, S.;Shiba, T.; Stewart, C. J.; Wieland, T. FEES Lett. 1978, 91, 144. Stewart, C. J.; Wieland, T. Liebigs Ann. Chem. 1978, 57. (19) Michenfelder, M.; Retey, J. Angew. Chem. 1986, 98, 337. (20) Martini, H.; Retey, J. Angew. Chem., Inr. Ed. Engl. 1993, 32, 278. (21) Moffat, J. G.; Khorana, H. G. J. Am. Chem. SOC.1959, 81, 1265. Moffatt, J. G.; Khorana, H. G. J. Am. Chem. SOC.1961, 83, 663.
1. TMS-CI, LiBr
2. aq. NaHCO,
2c
Table 1. Kinetic Comparison of 2n, 3a vs 212, 3c with CoA Biosynthetic Enzymes
phosphopantetheine adenylyltransferase
K, (mM) 2a 2c
0.19 0.38
dephosphocoenzyme A kinase
relative V , 1.o 0.45
Km (mM) 3a 3c
0.12 0.13
relative , V 1.o 0.90
pantetheine phosphate analog 2c, having the thiol group replaced with a methyl group, was prepared as shown in Scheme 3. The sodium salt of pantothenic acid 7 was reacted with methyl tosylate to form the methyl ester 8.22 8 was converted to the propyl thioester 9using a dimethyl(propy1thio)aluminum reagent.Z3The aluminum reagent was prepared by reaction of trimethylaluminum with propanethiol. When this chemistry was performed with ethanethiol instead of propanethiol, separation of the ethyl thioester product from unreacted methyl ester 8 was difficult. This separation was much simpler with the propyl thioester 9, thus this compound was used in further work. The primary hydroxyl group was selectively phosphorylated with dimethyl phosphoro~hloridate~~ to give the dimethyl phosphate 10, which was demethylated using trimethylsilyl chloride and lithium bromide to form 2c.25 The enzymes used in this work were obtained from Brevibacterium ammoniagenes in a one-step purification (DEAE Sepharose chromatography) of the crude cell extract.26~2~In kinetic analysis of phosphopantetheine adenylyltransferase in a coupled assay with pyrophosphate-dependent phosphofructokinase,28 2c gave a V,,, near half that obtained with the natural substrate 2a and a 2-fold higher K,, as indicated in Table 1. The intermediate dephospho-CoA analog 3c was isolated from a preparative phosphopantetheine adenylyltransferasereaction and found to be almost as good as natural dephospho-CoA 3a as a substrate for dephospho-CoA kinase, as also shown in Table 1, For preparative work, fractions from the DEAE Sepharose chromatography containing the two desired enzyme activities were combined and immobilized in polyacrylamide gel.29 2c was efficiently converted to the corresponding CoA analog IC in a preparative reaction using the enzymes of Scheme 1 with 1 equiv ofATP. Phosphoenolpyruvateand pyruvate kinase wereincluded (22) Ueda, K. Bull. Chem. SOC.Jpn. 1979, 53, 1879. (23) Hatch, R. P.; Weinreb, S.M, J. Org. Chem. 1977, 42, 3960. (24) Shemin, D., Ed. Biochemical Preparations; John Wiley and Sons, Inc.: New York, 1957; Vol. 5 , p 1. (25) McKenna, C. E.; Higa, M. T.; Cheung, N. H.;McKenna, M. Tetrahedron Lett. 1977, 155.
(26) Martin, D. P.; Drueckhammer, D. G. Eiochem.Eiophvs. . . Res. Commun.
1993, 192, 1155.
(27) Shimizu, S.;Komaki, R.; Tani. Y.; Yamada. H.FEES Lett. 1983. 151, 303. (28) O'Brien, W .E. Anal. Eiochem. 1976, 76, 423. (29) Pollak, A.; Blumenfeld, H.;Wax, M.; Baughn, R. L.;Whitesides, G. M. J. Am. Chem. SOC.1980, 102,6324.
Martin et al.
4662 J . Am. Chem. SOC.,Vol. 116, No. 11, I994 Scheme 6
Scheme 4 1. (CH3O)ZCH
C H 3 S Na'
CHZOH
cat. Tos-OH
C H ~ OO C H ~ Ha Pd/C
-
x
2. NaN3, DMSO
d
-5
Na104
17
MeOH/H,O
Scheme 5 17
Oxone
-
MeOH/H20 H 2 N m R
Phth=N-(CH2)3-S-CH3
DMSO, 25' C
17
CHBOH
12. x = CI 13. X = N3
11
Phth=N-(CH2)3--Br 16
-
0 Phth=N--(CH2)3-S-CHz 18
0
Phth=N-(CH2)3-S-CH3 19 0
HZN-NHZ EtOH, 25' C
-
HzN-NH, EtOH, 25' C
-
15d
15e
Scheme 7
1c
15
0 CoA-S-C-CHI
-
0
0
. 02C-C-CH2-C02-
14 (COA-R)
B
a, R =CH,-C-O
FI
b, R =CHa-,N-OC,
B
a
d, R =CH,--S-CH3
e,
FI
p
a
R =CHp-C-NHz
for regeneration of ATP from the ADP formed in the kinase step.30 Inorganic pyrophosphatase was included to hydrolyze pyrophosphate, thus making the adenylyltransferase step irreversible. Upon completion of the reaction, the immobilized enzymes were recovered and reused. After three uses, the kinase activity was too low for further use of the immobilized enzyme. A total of about 1 g of IC was obtained in three preparative reactions using the enzymes obtained from a 12-L culture of B. ammoniagenes. The product ICwas purified by anion-exchange chromatography followed by reverse-phase HPLC. Synthesis of Analogs of Acetyl-coA. The previously reported dethia analog of acetyl-coA 4 was prepared from ICas shown in Scheme 2. The amine 5 was prepared as shown in Scheme 4.l This involved carbonyl protection of 5-chloro-2-pentanone (1 l), nucleophilic displacement of chloride by azide, and reduction of the azide to the primary amine 5. The aminolysis reaction (Scheme 2) was performed with a 1.9 M concentration of the amine 5 at pH 10.2 at room temperature for 20 h. About 50% deprotection of the carbonyl occurred during the course of the aminolysis reaction. Carbonyl deprotection was completed upon acidification of the reaction mixture of p H 4.5. 4 was purified by reverse-phase HPLC and isolated in 50% yield. Five additional new analogs of acetyl-coA 14a-e were prepared by reaction of ICwith the amines 15a-e as shown in Scheme 5. The nitro and amido amines 15b,c were prepared according to literature meth0ds.3~3~2The sulfoxide and sulfone 15d,e were prepared via a common intermediate 17, as shown in Scheme 6. 17 was prepared by reaction of 16 with thiomethoxide anion and converted to the sulfoxide 18 and sulfone 19 according to standard oxidation method^.^^,^^ Deprotection by reaction with hydrazine in ethanol gave the amines 15dand 1 5 . 3 5 Theaminolysis reactions of Scheme 5 were all carried out near pH 10 with an amine concentration near 1.0 M. Reaction times were typically about (30) Hirschbein, B. L.; Mazenod, G. M.; Whitesides, G. M. J . Org. Chem.
1982,47, 3765.
(31) Alston, T. A.; Porter, D. J. T.; Bright, H. J. J . Enzyme Inhib. 1987,
1, 215.
(32) Kleeman, A.; Leuchtenberger, W.;Martens, J.; Weigel, H. Angew. Chem., Int. Ed. Engl. 1980, 19, 621. (33) Johnson, C. R.;Leonard, N. J. J . Org. Chem. 1962, 27, 282. (34) Trost, B. M.; Curran, D. P. Tetrahedron Lett. 1981, 14, 1287. (35) Bickel, H.; Fechtig, B.; Hall, G. E.; Keller-Schierlein, W.; Prelog, V.; Vischer, E. Helu. Chim. Acta 1960, 43, 901.
7H2
2
cop.
R =CHz-S-CH,
cop-
Table 2. Affinity of Citrate Synthase for Acetyl-coA Analogs
6L 4b 14a 14b 14c a
1.6 x 10-5 ( K ~ ) 1.6 x 10-5 1.6 X 10-8 1.6x 10-5
2.8 X 10-8
1.0 x 10-4
1.4 X 10-4 1.3
X
10-4
References 40 and 15. Reference 18.
24 h a t room temperature. Analytical HPLC consistently showed >90% of the desired aminolysis products 14, with the only other detectable product being the acid resulting from thioester hydrolysis, which represented
