We subsequently, in retrospect accidentally, encountered the elusive zero-CD product in a reaction mixture resulting from a nanogram-scale ouabain naphthoylation

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We subsequently, in retrospect accidentally, encountered the elusive zero-CD product in a reaction mixture resulting from a nanogram-scale ouabain naphthoylation. rise to the reported biological activities of hypothalamic inhibitory factor preparations remain to be clarified. Search for the endogenous ligands of Na+,K+-ATPase has been an enticing yet puzzling research subject for the last several decades (1C4). Putative endogenous sodium pump inhibitors have been detected many times in various mammalian tissues and plasma. However, purification, structural analysis, and physiological characterization of such compounds have been fraught with difficulties. Part of the reason for this problem is that the enzyme assays are susceptible to many nonspecific interferences, which sometimes lead to false-positive results (1). Recognition of this problem led to the employment of multiple assay systems, which greatly reduced the risk of falsely detecting putative physiologic Na+,K+-ATPase inhibitors. Nevertheless, progress is still hindered by the extreme paucity of material available from tissues. Among the putative endogenous inhibitors of Na+,K+-ATPase are a group of compounds that are considered to be related to ouabain, a plant-origin cardiac glycoside (5). These compounds have been extracted from various animal tissues, and in several cases sufficient material, albeit submicrogram-to-low microgram quantity, was purified to allow further chemical and physiological characterizations. The molecular mass appeared to be identical to ouabain by MS, and the retention time of reversed-phase HPLC (RP-HPLC) was the same as ouabain. Moreover, two compounds, ouabain-like compound (OLC) from human plasma (6) and adrexin C from bovine adrenal (7), were indistinguishable from herb ouabain by multiple biochemical and physiological criteria. On the other hand, a factor from bovine hypothalamus (hypothalamic inhibitory factor, HIF) showed different physiological profiles from ouabain in various assays, such as inhibition of ouabain-insensitive isoform (rat 1) of Na+,K+-ATPase (8), inhibition of sarcoplasmic reticulum Ca2+-ATPase, and inhibition of backdoor phosphorylation of purified Na+,K+-ATPase (9), binding kinetics to kidney epithelial cells and purified enzyme (10, 11), lipid bilayer permeability (11), and reversible inotropic effect on cardiac myocytes (12). For the structural characterization of HIF, further purification was carried out by using affinity chromatography combined with RP-HPLC (13). It turned out that this purified HIF was identical to ouabain by LC/MS. Furthermore, glycosidase treatment and acid hydrolysis showed HIF to be an -l-rhamnoside, as is usually ouabain; also, des-rhamnosyl HIF, the aglycone, was indistinguishable from ouabagenin by LC/MS. These findings led to attempts to differentiate JW74 HIF and herb ouabain by using nanogram-scale chemical derivatization. Naphthoylation of HIF (300 ng) followed by RP-HPLC showed that this major derivatization product from HIF eluted slightly earlier than ouabain pentanaphthoate, the major product from ouabain naphthoylation; moreover, CD spectroscopy revealed that this HIF derivative showed no distinct CD, whereas ouabain pentanaphthoate showed a clear positive CD couplet. Although the molecular ion peak of this HIF derivative was not clear, the product was described as HIF pentanaphthoate (13). Subsequent microscale derivatization of OLC (400 ng, gift from Upjohn Laboratories) also gave the same RP-HPLC and CD (zero-CD) profiles as those of HIF pentanaphthoate (14). This resulted in the final outcome how the ouabain-like sodium pump inhibitor can be an ouabain isomer, known as endogenous ouabain (2 frequently, 4, 5), despite the fact that the physiological discrepancies between HIF and additional substances still remained to become clarified (2): the word endogenous ouabain continues to be used to tell apart this substance from vegetable ouabain (2, 4, 5), although its accurate origin hasn’t however been clarified. Predicated on the JW74 JW74 spectroscopic and chromatographic info through the naphthoylation research on endogenous ouabain, a seek out the isomer of ouabain pentanaphthoate using the same HPLC and Compact disc (zero-CD) profile was initiated. Two lines of structural proof, the fact how the HIF aglycone was indistinguishable from ouabagenin by LC/MS which the sugars moiety of HIF was -l-rhamnoside, recommended that HIF could possibly be an -l-rhamnoside placement isomer of ouabain. This resulted in JW74 the computation of Compact disc spectra of most possible sugar placement isomers of ouabain pentanaphthoate and dedication from the validity Rabbit Polyclonal to ELOVL1 of computations by preparing as much synthetic isomers as you can (15). The group of assessment between theoretical.