Iso-coenzyme A

Abstract

Iso-coenzyme A is an isomer of coenzyme A in which the monophosphate is attached to the 2′-carbon of the ribose ring. Although iso-CoA was first reported in 1959 (Moffatt, J. G., and Khorana, H. G. (1959) J. Am. Chem. Soc. 81, 1265–1265) to be a by-product of the chemical synthesis of CoA, relatively little attention has been focused on iso-CoA or on acyl-iso-CoA compounds in the literature. We now report structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and β-hydroxybutyryl-iso-CoA using mass spectrometry (MS), tandem MS, and homonuclear and heteronuclear NMR analyses. Although the 2′-phosphate isomer of malonyl-CoA was recently identified in commercial samples, previous characterizations of iso-CoA itself have been based on chromatographic analyses, which ultimately rest on comparisons with the degradation products of CoA and NADPH or have been based on assumptions regarding enzyme specificity. We describe a high performance liquid chromatography methodology for separating the isomers of several CoA-containing compounds. We also report here the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl transfer reactions. Finally, we describe a simple synthesis of iso-CoA from CoA, which utilizes β-cyclodextrin to produce iso-CoA with high regioselectivity, and we demonstrate a plausible mechanism that accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. We anticipate that these results will provide methodology and impetus for investigating iso-CoA compounds as potential pseudo-substrates or inhibitors of the >350 known CoA-utilizing enzymes. Iso-coenzyme A is an isomer of coenzyme A in which the monophosphate is attached to the 2′-carbon of the ribose ring. Although iso-CoA was first reported in 1959 (Moffatt, J. G., and Khorana, H. G. (1959) J. Am. Chem. Soc. 81, 1265–1265) to be a by-product of the chemical synthesis of CoA, relatively little attention has been focused on iso-CoA or on acyl-iso-CoA compounds in the literature. We now report structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and β-hydroxybutyryl-iso-CoA using mass spectrometry (MS), tandem MS, and homonuclear and heteronuclear NMR analyses. Although the 2′-phosphate isomer of malonyl-CoA was recently identified in commercial samples, previous characterizations of iso-CoA itself have been based on chromatographic analyses, which ultimately rest on comparisons with the degradation products of CoA and NADPH or have been based on assumptions regarding enzyme specificity. We describe a high performance liquid chromatography methodology for separating the isomers of several CoA-containing compounds. We also report here the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl transfer reactions. Finally, we describe a simple synthesis of iso-CoA from CoA, which utilizes β-cyclodextrin to produce iso-CoA with high regioselectivity, and we demonstrate a plausible mechanism that accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. We anticipate that these results will provide methodology and impetus for investigating iso-CoA compounds as potential pseudo-substrates or inhibitors of the >350 known CoA-utilizing enzymes. Iso-coenzyme A is an isomer of coenzyme A in which the monophosphate is attached to the 2′-carbon of the ribose ring. Iso-CoA was first reported in 1959 by Moffatt and Khorana (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar) to be a by-product of the chemical synthesis of CoA. The final step of this synthesis entailed acid-catalyzed hydrolysis of cyclic coenzyme A (Scheme 1) to produce a 50:50 mixture of two products that were separated using epicholorohydrin triethanolamine (ECTEOLA) cellulose ion exchange chromatography (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar). Direct structural analysis was not readily available in 1959; consequently, the authors deduced the structure of iso-CoA by establishing that, in contrast to CoA, iso-CoA was not a substrate for the enzyme phosphotransacetylase (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar). Moffatt and Khorana also used paper chromatography of phosphodiesterase hydrolysates to show that enzymatic hydrolysis of iso-CoA produces adenosine-2′,5′-diphosphate, exclusively (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar). Since the original work of Moffatt and Khorana was done, four additional chemical syntheses of CoA have been reported (3Michelson A.M. Biochim. Biophys. Acta. 1961; 50: 605-607Crossref PubMed Scopus (9) Google Scholar, 4Michelson A.M. Biochim. Biophys. Acta. 1964; 93: 71-77Crossref PubMed Scopus (29) Google Scholar, 5Shimizu M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1965; 13: 1142-1144Crossref PubMed Scopus (6) Google Scholar, 6Shimizu M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1967; 15: 655-662Crossref PubMed Scopus (18) Google Scholar, 7Hashimoto M. Mukaiyama T. Chem. Lett. 1972; 7: 595-598Crossref Google Scholar, 8Taguchi Y. Nishimura N. Kakimoto T. Mushika Y. Bull. Chem. Soc. Jpn. 1976; 49: 1122-1125Crossref Google Scholar), all of which produce cyclic CoA as the penultimate product; cyclic CoA is then hydrolyzed either chemically to produce a mixture of CoA and iso-CoA (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar, 4Michelson A.M. Biochim. Biophys. Acta. 1964; 93: 71-77Crossref PubMed Scopus (29) Google Scholar, 5Shimizu M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1965; 13: 1142-1144Crossref PubMed Scopus (6) Google Scholar, 6Shimizu M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1967; 15: 655-662Crossref PubMed Scopus (18) Google Scholar, 8Taguchi Y. Nishimura N. Kakimoto T. Mushika Y. Bull. Chem. Soc. Jpn. 1976; 49: 1122-1125Crossref Google Scholar) or enzymatically with ribonuclease T2 to produce CoA regioselectively (3Michelson A.M. Biochim. Biophys. Acta. 1961; 50: 605-607Crossref PubMed Scopus (9) Google Scholar, 4Michelson A.M. Biochim. Biophys. Acta. 1964; 93: 71-77Crossref PubMed Scopus (29) Google Scholar, 5Shimizu M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1965; 13: 1142-1144Crossref PubMed Scopus (6) Google Scholar, 6Shimizu M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1967; 15: 655-662Crossref PubMed Scopus (18) Google Scholar, 7Hashimoto M. Mukaiyama T. Chem. Lett. 1972; 7: 595-598Crossref Google Scholar). Recently, Minkler et al. (9Minkler P.E. Anderson V.E. Maiti N.C. Kerner J. Hoppel C.L. Anal. Biochem. 2004; 328: 203-209Crossref PubMed Scopus (8) Google Scholar) observed the 2′-phosphate isomer of malonyl-CoA in commercial samples. These authors reported that HPLC 1The abbreviations used are: HPLC, high performance liquid chromatography; COSY, correlation spectroscopy; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; ESI, electrospray ionization; HMQC, heteronuclear multiple quantum correlation; LC, liquid chromatography; MES, 4-morpholineethanesulfonic acid; MRM, multiple reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PHB, poly-(β)-hydroxybutyric acid; TCEP, Tris(2-carboxyethyl)phosphine. -purified samples of each isomer exhibited identical UV, HPLC-MS, and HPLC-MS/MS properties, and the identity of the 2′-phosphate isomer was convincingly demonstrated by comparing the one-dimensional NMR spectra to that of 2′,5′-ADP. In related work, Retey and co-workers have synthesized several dethia-CoA analogs (10Michenfelder M. Retey J. Angew. Chem. Int. Ed. Engl. 1986; 25: 366-367Crossref Scopus (19) Google Scholar, 11Michenfelder M. Hull W.E. Retey J. Eur. J. Biochem. 1987; 168: 659-667Crossref PubMed Scopus (25) Google Scholar, 12Brendelberger V.G. Retey J. Ashworth D.M. Reynolds K. Willenbrock F. Robinson J.A. Angew. Chem. Int. Ed. Engl. 1988; 27: 1089-1090Crossref Scopus (23) Google Scholar, 13Brendelberger V.G. Retey J. Isr. J. Chem. 1989; 29: 195-200Crossref Scopus (3) Google Scholar, 14Wagner A.P. Retey J. Eur. J. Biochem. 1991; 195: 699-705Crossref PubMed Scopus (11) Google Scholar, 15Zhao Y. Michenfelder M. Retey J. Can. J. Chem. 1994; 72: 164-169Crossref Scopus (9) Google Scholar), in which the sulfur of CoA is replaced by a methylene, as mixtures of the 2′- and 3′-phosphate isomers; two of these analogs were purified to homogeneity and characterized by one-dimensional 1H-NMR, 31P-NMR, and, in one case, fast atom bombardment MS (13Brendelberger V.G. Retey J. Isr. J. Chem. 1989; 29: 195-200Crossref Scopus (3) Google Scholar, 14Wagner A.P. Retey J. Eur. J. Biochem. 1991; 195: 699-705Crossref PubMed Scopus (11) Google Scholar). Dethia-CoA analogs were also synthesized and characterized by Stewart and co-workers (16Stewart C.J. Wieland T. Liebigs Ann. Chem. 1978; 1: 57-65Crossref Scopus (21) Google Scholar, 17Ciardelli T.L. Stewart C.J. Seeliger A. Wieland T. Liebigs Ann. Chem. 1981; 5: 828-841Crossref Scopus (26) Google Scholar, 18Thorpe C. Ciardelli T.L. Stewart C.J. Wieland T. Eur. J. Biochem. 1981; 118: 279-282Crossref PubMed Scopus (31) Google Scholar); unfortunately, they were unable to separate the 2′- and 3′-phosphate isomers, and the NMR data could only be obtained on isomeric mixtures (17Ciardelli T.L. Stewart C.J. Seeliger A. Wieland T. Liebigs Ann. Chem. 1981; 5: 828-841Crossref Scopus (26) Google Scholar). However, to the best of our knowledge no full structural characterization has ever been reported in the literature for iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, or β-hydroxybutyryl-iso-CoA. The issue of whether or not enzymes that act upon CoA-containing substrates discriminate between the 2′- and 3′-phosphate isomers is of obvious relevance to the design of new classes of inhibitors and pseudosubstrates. As mentioned above, in the early work of Moffatt and Khorana the enzyme phosphotransacetylase was reported to be unreactive toward iso-CoA (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar). Similarly, Retey and co-workers have reported that FAD-dependent isobutanoyl-CoA dehydrogenase is reactive with isobutanoyl-dethia-CoA but not with the iso-CoA analog (13Brendelberger V.G. Retey J. Isr. J. Chem. 1989; 29: 195-200Crossref Scopus (3) Google Scholar) and that methylmalonyl-CoA pyruvate carboxylase also distinguishes between the isomers, reacting exclusively with propionyl-dethia-CoA (10Michenfelder M. Retey J. Angew. Chem. Int. Ed. Engl. 1986; 25: 366-367Crossref Scopus (19) Google Scholar). In contrast, Thorpe et al. (18Thorpe C. Ciardelli T.L. Stewart C.J. Wieland T. Eur. J. Biochem. 1981; 118: 279-282Crossref PubMed Scopus (31) Google Scholar) reported that substrate mixtures containing both dethia-iso-CoA and dethia-CoA derivatives are capable of reducing enzyme-bound FAD. Likewise, Rossier J. Biochem. J. PubMed Scopus Google Scholar) reported that is by both and and et al. A.P. Retey J. Eur. J. Biochem. 1991; 195: 699-705Crossref PubMed Scopus (11) Google Scholar) have reported that is by both and in both the iso-CoA analogs are In the of and (17Ciardelli T.L. Stewart C.J. Seeliger A. Wieland T. Liebigs Ann. Chem. 1981; 5: 828-841Crossref Scopus (26) Google Scholar), is to a from the data the were with mixtures containing both the 2′- and this issue to be for CoA-utilizing enzymes using purified and characterized iso-CoA substrate we report structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and β-hydroxybutyryl-iso-CoA. characterizations of the structure of iso-CoA have been based on chromatographic (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar) that rest on comparisons with the degradation products of CoA and NADPH J. Am. Chem. Soc. Scopus Google Scholar, J. Chem. PubMed Google Scholar, W.E. J. Am. Chem. Soc. Scopus (19) Google Scholar) or have been based on assumptions regarding enzyme M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1965; 13: 1142-1144Crossref PubMed Scopus (6) Google Scholar, 6Shimizu M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1967; 15: 655-662Crossref PubMed Scopus (18) Google Scholar, 8Taguchi Y. Nishimura N. Kakimoto T. Mushika Y. Bull. Chem. Soc. Jpn. 1976; 49: 1122-1125Crossref Google Scholar). We describe HPLC methodology to separate the isomers of several CoA-containing compounds and the characterization of iso-CoA using MS, MS/MS, and NMR analyses. We also report here the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl transfer reactions. Finally, we describe a simple synthesis of iso-CoA from CoA that utilizes β-cyclodextrin to produce iso-CoA with high regioselectivity, and we demonstrate a plausible mechanism that accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. of CoA purified from the of 1-ethyl-3-(3-dimethylaminopropyl) and β-cyclodextrin were from was from HPLC or and all compounds were obtained from HPLC CoA and were with a tandem mass with an to an HPLC of the isomers was with an attached to a using an of and a of mass spectra were obtained in the ion with used as the and of and The was to and the to using multiple reaction was on the of the isomers of CoA and using as the a of for the of the The commercial were the CoA, and and and and and MS of mass and were using samples of CoA and the isomer on an mass The was in the ion using a of MS analysis was a mass of and spectra of the ion were obtained the mass of NMR spectra of iso-CoA were obtained in in a on a for and for using an K. The was using the with the on the data in the and in The data was to a of and a was The for the heteronuclear multiple quantum correlation were the data in in an and The chemical for the CoA and iso-CoA are in the The were either to the iso-CoA and analysis in the of reducing or to an were obtained and the were then obtained by the spectra using the S. J. Am. Chem. Soc. Google Scholar) and Scopus Google Scholar) of CoA, and the isomers were on a HPLC with of samples using the chromatography A of and was used with a containing a were a of with a containing and a of HPLC was on an with a containing a using with a of and for each CoA-containing were observed with four HPLC and HPLC using both and MS samples of CoA were in and iso-CoA was not in or not of Iso-CoA and enzymes and poly-(β)-hydroxybutyric not all of which CoA-containing were for the to with the iso-CoA-containing were in a of isomers, and in with a final of The first reaction was by of and and the reaction was with were with by to HPLC analysis as CoA of CoA were in of and then to produce CoA with by The reaction was HPLC using the HPLC above, that the CoA CoA was in of of 1-ethyl-3-(3-dimethylaminopropyl) was and the was was by HPLC reducing an of the to the with for the reaction was the mixture was The was in and the was to a that was with the and the by-product were from the with the and the cyclic CoA was in of and The cyclic CoA-containing were and of Iso-CoA cyclic CoA was in of containing and and the was The was with the for or the reaction was by HPLC, which the was to to the a final of iso-CoA with by and to of the and β-cyclodextrin from the iso-CoA-containing and by to The final was then in the was and the was by a The isomeric mixture of iso-CoA and CoA was as and the mixture was of iso-CoA was by the in a of in to the CoA to the the mixture a and with by a final using HPLC, as of Iso-CoA CoA was in of and the reaction was by HPLC was a mixture of CoA and The identity of iso-CoA in this was by NMR analysis HPLC and as of CoA and and HPLC-MS/MS obtained for commercial CoA samples are in A of The of A in an of a commercial CoA that two with identical that commercial CoA samples two compounds with identical HPLC-MS/MS using were to structural is from the of A in that the CoA samples two for the and these and HPLC-MS/MS results the of isomeric compounds with identical these analyses, each in the commercial CoA was purified by HPLC, and high mass and were on each As in the the two samples exhibited identical of with an of with to the the ion spectra for the two samples identical that only in the of and These results the of isomeric of CoA in the commercial and the results that these isomers identical that these isomers have and HPLC-MS/MS were also with commercial and in the of this these also the of two isomeric compounds with identical that exhibited identical by HPLC-MS/MS using the observed of Iso-CoA by spectra for the two isomers of CoA are in A and of The in for the isomer is to several CoA spectra J. Chem. PubMed Google Scholar, J. Am. Chem. Soc. PubMed Scopus Google Scholar, J. Am. Chem. Soc. PubMed Scopus Google Scholar, Anal. Biochem. 1988; PubMed Scopus Google Scholar, Anderson V.E. Chem. Scopus Google Scholar, Chem. PubMed Scopus (9) Google Scholar), and the chemical were to et al. Anderson V.E. Chem. Scopus Google Scholar). In contrast, the in for the isomer is in the were obtained for this isomer in to the chemical The chemical and are in is that for the isomer the is by the and are by and and the two have that the of the on the ribose to these were to the of each As in the spectra for the that the monophosphate is attached the this as The HPLC in that commercial samples of CoA, and as and and of the isomers, of The of iso-CoA the chemical of the ribose ring. The the chemical of the was used to the of each in the ribose and chemical and chemical chemical in a new of The for the isomer that the to the of the ribose this isomer is as the two of the with the on the and no are of four commercial CoA-containing samples. The HPLC of commercial CoA, and two for each and have the and CoA, and iso-CoA, and and and and and and and β-hydroxybutyryl-iso-CoA and for and reaction in for CoA, and and iso-CoA isomers demonstrate that the and with either These data the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl transfer reactions. In the in two of to which is to by the In a has been that the to is from the in both that in all both the CoA and iso-CoA isomers are with In we that and iso-CoA isomer with in the reaction to produce the not of iso-CoA and acyl-iso-CoA compounds be as potential inhibitors or a synthesis capable of high of the 2′-phosphate isomer is to this we report a simple synthesis of iso-CoA from CoA. As in CoA was to the and the was then used to the CoA in a reaction to the synthesis of J. 1976; 27: PubMed Scopus Google Scholar). We that the first of the synthesis are using the CoA to of the the is to CoA in step Finally, hydrolysis is in the of β-cyclodextrin in high to produce iso-CoA with a the acid-catalyzed of the CoA monophosphate in an to a plausible mechanism for the existence of the 2′-phosphate isomers in commercial preparations of CoA-containing compounds. In these CoA was in the of and the of CoA and iso-CoA were by HPLC as a of As in a final mixture of CoA and iso-CoA was We that iso-CoA is only the iso-CoA of commercial preparations be in this the of the The of coenzyme A in is by the of enzymes A. C. M. C. G. 2004; PubMed Google Scholar) The that with CoA-containing we of the known enzymes to be of this The 2′-phosphate isomer of CoA, first iso-CoA in the 1959 report of Moffatt and Khorana (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar), has been an by-product or has been a of the literature that only of the CoA-utilizing enzymes The have been for the to discriminate between the 2′- and 3′-phosphate isomers (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar, M. Retey J. Angew. Chem. Int. Ed. Engl. 1986; 25: 366-367Crossref Scopus (19) Google Scholar, 13Brendelberger V.G. Retey J. Isr. J. Chem. 1989; 29: 195-200Crossref Scopus (3) Google Scholar, 14Wagner A.P. Retey J. Eur. J. Biochem. 1991; 195: 699-705Crossref PubMed Scopus (11) Google Scholar, 17Ciardelli T.L. Stewart C.J. Seeliger A. Wieland T. Liebigs Ann. Chem. 1981; 5: 828-841Crossref Scopus (26) Google Scholar, 18Thorpe C. Ciardelli T.L. Stewart C.J. Wieland T. Eur. J. Biochem. 1981; 118: 279-282Crossref PubMed Scopus (31) Google Scholar, J. Biochem. J. PubMed Scopus Google Scholar, T.L. C.J. J. Am. Chem. Soc. Scopus Google Scholar), and in two of these the enzymes have been to purified 2′-phosphate isomers as substrates or inhibitors A.P. Retey J. Eur. J. Biochem. 1991; 195: 699-705Crossref PubMed Scopus (11) Google Scholar, J. Biochem. J. PubMed Scopus Google Scholar). the of the compounds in these are analogs of CoA, as the or and not iso-CoA In the results CoA and iso-CoA were purified by HPLC, and high mass MS and that the compounds are isomers with Direct structural of the two isomers was then using 1H-NMR, COSY, and HMQC, and the results that the monophosphate of the iso-CoA isomer is attached to the 2′-carbon of the ribose ring. of acetyl-iso-CoA, acetoacetyl-iso-CoA, and β-hydroxybutyryl-iso-CoA was also using and HPLC-MS/MS analyses. We report here the first of iso-CoA-containing compounds acting as substrates in acyl transfer reactions. and with the of and the reaction that all enzymes with either isomer with These results that enzymes be for toward the 2′-phosphate isomers of CoA-containing We that enzymes have been for to with In the of and J.G. J. PubMed Scopus (19) Google Scholar) have identified a the of this enzyme that they have a for CoA a between an and the 3′-phosphate of CoA has also been reported for H.G. S. A. 1981; PubMed Scopus Google Scholar, J. Chem. PubMed Scopus Google Scholar). of be a of enzymes that with CoA-containing to be whether this be by the of a the the of the ribose ring. The for isomers of CoA-containing ion exchange chromatography (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar, 14Wagner A.P. Retey J. Eur. J. Biochem. 1991; 195: 699-705Crossref PubMed Scopus (11) Google Scholar, T.L. C.J. J. Am. Chem. Soc. Scopus Google Scholar), is not and have isomers of CoA J. Biochem. J. PubMed Scopus Google Scholar). We report here an HPLC methodology that of CoA and which upon work by et al. J. Scopus Google Scholar), is the first HPLC to separate a of iso-CoA and we were to separate four iso-CoA isomers with using We that of HPLC of CoA-containing compounds several that to iso-CoA compounds that were not identified as by the authors T. Anal. Biochem. PubMed Scopus (36) Google Scholar, H. Biochem. Biophys. 1991; PubMed Scopus (36) Google Scholar, A. J. A. Scopus (6) Google Scholar, A. Y. S. M. J. Scopus (3) Google Scholar, M. Anal. Biochem. 2004; 328: PubMed Scopus Google Scholar). iso-CoA have been by previous authors the isomers will HPLC We also report here the first chemical synthesis of iso-CoA from CoA. reported that produce iso-CoA compounds as a by-product of cyclic CoA hydrolysis mixtures containing both the 2′- and 3′-phosphate isomers in of and (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar, 5Shimizu M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1965; 13: 1142-1144Crossref PubMed Scopus (6) Google Scholar, 6Shimizu M. Nagase O. Okada S. Hosokawa Y. Tagawa H. Chem. Pharm. Bull. 1967; 15: 655-662Crossref PubMed Scopus (18) Google Scholar, 8Taguchi Y. Nishimura N. Kakimoto T. Mushika Y. Bull. Chem. Soc. Jpn. 1976; 49: 1122-1125Crossref Google Scholar). In our simple the cyclic CoA is from CoA, and then hydrolysis in the of β-cyclodextrin in high produces iso-CoA regioselectively in high results are in with of and co-workers S. M. J. Chem. 5: Scopus Google Scholar, M. S. Y. J. Am. Chem. Soc. Scopus Google Scholar), reported that of and compounds as with enzymatic hydrolysis by These authors that the an with which that hydrolysis toward the the 2′-phosphate We as a plausible mechanism to for the existence of the 2′-phosphate isomers in commercial preparations of CoA-containing compounds. In and co-workers the acid-catalyzed synthesis of iso-CoA from CoA J. Chem. PubMed Google Scholar, J. Chem. PubMed Google Scholar); these authors not iso-CoA but only demonstrated that CoA to the acid-catalyzed of the 2′- and 3′-phosphate of NADPH J. Chem. PubMed Google Scholar) and D.M. J. Chem. Soc. Scholar, M. H. J. Chem. 1989; Scopus Google Scholar, M. H. J. Chem. 1991; Scopus Google Scholar, M. H. J. Chem. Scopus (21) Google Scholar) is in the literature. In our of CoA with an mixture of iso-CoA and CoA and of the isomer could be These results that is a mechanism for iso-CoA from CoA, that are used in several CoA F. J. Chem. PubMed Google Scholar, D.M. H. J. Chem. PubMed Google Scholar, H. H. J. Am. Chem. Soc. Scopus (3) Google Scholar, A. J. Chem. PubMed Google Scholar, H. H. J. Chem. PubMed Google Scholar). The of iso-CoA compounds is The original report of Moffatt and Khorana (1Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1959; 81: 1265Crossref Scopus (36) Google Scholar, 2Moffatt J.G. Khorana H.G. J. Am. Chem. Soc. 1961; 83: 663-675Crossref Scopus (114) Google Scholar) that phosphotransacetylase not with iso-CoA has to the that the are compounds. In contrast, et al. S. K. Y. Chem. Pharm. Bull. 1967; 15: PubMed Scopus (6) Google Scholar) that iso-CoA be from of in the of Similarly, (3Michelson A.M. Biochim. Biophys. Acta. 1961; 50: 605-607Crossref PubMed Scopus (9) Google Scholar) reported that an purified enzyme from iso-CoA from cyclic CoA in enzyme was which produces 2′-phosphate from as from J. 1976; 27: PubMed Scopus Google Scholar). Although this enzyme of all to the in substrate for this has not been G. J. M. P.E. K. J. Chem. PubMed Scopus Google Scholar, J. M. P.E. J. Chem. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). These that iso-CoA could be enzymatically in from either or no for this Although the of iso-CoA is the potential for this of isomeric compounds is Iso-CoA have potential as and inhibitors and as for investigating CoA to the of CoA-utilizing enzymes. We anticipate that our in to the first of enzymes acyl transfer with iso-CoA isomers, will provide the methodology for iso-CoA and acyl-iso-CoA compounds. with