The Bis(enol triflate) Route to Dienediyne Models of the Biradical Forming and DNA-Cleaving Natural Product Neocarzinostatin Chromophore

Brückner, Reinhard; Suffert, J.

doi:10.1055/s-1999-2699

Cited by 31 publications

(9 citation statements)

References 26 publications

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“…[1][2][3] Neocarzinostatin (NCS), which was the first naturally occurring enediyne chromoprotein studied, [4,5] displays an interesting feature; its carrier protein is able to divert the enediyne cycloaromatization into a distinct pathway. How the protein directs the irreversible chemistry of its bound enediyne has remained a profound mystery.…”

Section: Introductionmentioning

confidence: 99%

“…Like path A, the protein-directed path B is also initiated by a thiolate attack at C12, but, unlike path A, it is followed by nucleophilic addition to the triple bond. [2,29] Whereas path B is not involved in the DNA-damaging reaction, [23] it might be an important inactivation route for self-protection of the microorganism (Streptomyces carzinostaticus) that produces NCS. [29] Abstract in Chinese: Scheme 1.…”

Section: Introductionmentioning

confidence: 99%

“…[2] Here, we account for the possible directing mechanism of the NCS protein (illustrated in Scheme 2). Once a thiol molecule approaches the holoNCS binding cleft, a nucleophilic attack occurs at the C12=C1 double bond to generate a C1 carbanion as a common precursor.…”

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confidence: 99%

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Role of Steric Effects in Protein‐Directed Enediyne Cycloaromatization of Neocarzinostatin

Chi

Chien

Liu

et al. 2010

Chemistry A European J

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The antibiotic neocarzinostatin comprises a carrier protein with a well-defined cavity for accommodating an active enediyne chromophore. The protein has two disulfides, one (Cys(37)-Cys(47)) lies on the cavity bottom and the other (Cys(88)-Cys(93)) in a constrained short loop. When the chromophore is not bound to the protein, a thiol-induced cycloaromatization of the enediyne into a tetrahydroindacene derivative is responsible for the potent antitumor activity. When it is protein-bound, the protein diverts the cycloaromatization pathway to form a distinct hydroxyisochromene-type product. How the protein directs the enediyne chemistry is an interesting puzzle, and various suggestions have been proposed in the past. We screened more than fifty thiols and manipulated conditions to locate reaction features and search for factors that could influence the protein directing strength. Thiol- and oxygen-concentration-dependence studies suggested that disulfides, which maintain the steric rigidity of the protein, could play a key role in diverting the cycloaromatization pathway. For direct proofs, we made mutations at each of the two disulfides by replacing sulfur atoms with oxygen. Circular dichroism and two-dimensional NMR spectroscopy studies suggested that the mutations changed neither the protein conformation nor the ligand interactions. Analyses of the thiol-induced cycloaromatization revealed that rupture of Cys(37)-Cys(47) made the protein almost completely lose its chemical directing ability, whereas rupture of Cys(88)-Cys(93) had only a minor influence. The results demonstrated that the steric rigidity of the binding cavity, but not necessary the whole protein, played an important role in the protein-directed mechanism.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Role of Steric Effects in Protein‐Directed Enediyne Cycloaromatization of Neocarzinostatin

Chi

Chien

Liu

et al. 2010

Chemistry A European J

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“…[10][11][12][13][14][15][16][17][18][19]). NCS is a 1:1 chromoprotein complex consisting of an apoprotein (apoNCS) and an enediyne chromophore (NCS-C [free form] or NCS-C(b) [protein-bound form]) (MW 659).…”

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confidence: 99%

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Jayachithra¹,

Hariharan²,

Kumar³

et al. 2008

Analytical Biochemistry

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Most conjugate proteins undergo both conformational and stability changes on ligand removal. When architecture remains unchanged in the protein holo and apo forms, it is uncertain whether the protein stability also remains unaltered in both of the forms. Neocarzinostatin (NCS), a chromoprotein possessing a potent enediyne chromophore stands for such an instance. Protein-chromophore interaction has not been thoroughly explored previously due to a lack of strategies to independently and simultaneously monitor changes in the NCS conjugates. Here we report a method by which one can detect the signal exclusively from only one of the NCS conjugates without the spectral interference from the other. Stability of the NCS protein is significantly correlated to the proteinbound chromophore, irrespective of denaturation by heat, pH, urea, or ethanol. Despite the similarity in protein backbone conformation, protein stability of the NCS holo form diminishes and equalizes to that of the apo form when the chromophore is released and degraded. Although the enediyne chromophore is highly unstable, it intriguingly protects the protein by which it is protected. Significant mutual reliance between the carrier protein and its naturally associated ligand unveils important information on the NCS drug stability. KeywordsEnediyne; Neocarzinostatin; Chromophore; Ligand binding; Holoprotein; Protein stability Recently, much attention has been focused on the protein-ligand conjugation. Most conjugate proteins undergo changes in their conformation and subsequently, in stability on ligand removal [1][2][3]. For example, binding of heme to cytochrome changes the protein conformation and consequently, increases the protein stability [4]. It is rather uncommon for conjugate proteins to adopt a like conformation with or without the ligand. This includes azurin (β protein with a copper ion), flavodoxin (α/β protein with a flavin mononucleotide) [5], neocarzinostatin (NCS, 1 β protein with an enediyne chromophore) [6,7] and so on. In spite of structural similarity, the holo form of azurin is more stable than its apo form [8]. The flavodoxin holo form, in contrast, has little changes in either conformation or stability on its cofactor removal *Corresponding author. Fax: +886 4 22862547. E-mail address: chdhchin@dragon.nchu.edu.tw (D.-H. Chin). 1 Abbreviations used: NCS, neocarzinostatin; holoNCS, neocarzinostatin chromoprotein conjugate complex; apoNCS, apoprotein of neocarzinostatin; NCS-C, neocarzinostatin chromophore; NCS-C(b), protein-bound neocarzinostatin chromophore; CD, circular dichroism; HPLC, high-performance liquid chromatography; EtBr, ethidium bromide; NOESY, nuclear Overhauser effect spectroscopy; NMR, nuclear magnetic resonance; T m , temperature at which half of the protein is unfolded; T r , temperature at which half of the NCS-C(b) is released from NCS complex; C m , concentration of denaturant at which half of the protein is unfolded; C r , concentration of denaturant at which half of the NCS-C(b) is released from NCS com...

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“…Das hier beschriebene, einfache Eintopfverfahren eröffnet in Verbindung mit einer großen Auswahl an käuflichen Carbonylverbindungen einen flexiblen, konvergenten Zugang zu einer Reihe konjugierter Enine. Dieses Verfahren sollte neue Synthesemöglichkeiten für Zielstrukturen bieten, die konjugierte Enin‐21 oder Endien‐Fragmente enthalten und vielversprechende antibiotische, Antitumor‐ und andere biologische Aktivität aufweisen 2d. 22…”

Section: Methodsunclassified

Eine allgemeine und vielseitige Methode zur C‐C‐Kreuzkupplungssynthese konjugierter Enine: eine Eintopfsynthese, ausgehend von Carbonylverbindungen

Lyapkalo

Vogel

2006

Angewandte Chemie

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Alles in einem Topf: Die erste allgemeine Methode für die konvergente Kreuzkupplungssynthese von konjugierten Eninen aus leicht verfügbaren enolisierbaren Aldehyden und Ketonen wurde entwickelt (siehe Schema; NfF=Nonafluorbutan‐1‐sulfonylfluorid). Die Syntheseroute besteht aus wenigstens vier operativen Schritten, die allesamt in einer Eintopfreaktion ausgeführt werden, wobei gebräuchliche Reagentien, Katalysatoren und Additive Verwendung finden.

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The Bis(enol triflate) Route to Dienediyne Models of the Biradical Forming and DNA-Cleaving Natural Product Neocarzinostatin Chromophore

Cited by 31 publications

References 26 publications

Role of Steric Effects in Protein‐Directed Enediyne Cycloaromatization of Neocarzinostatin

Role of Steric Effects in Protein‐Directed Enediyne Cycloaromatization of Neocarzinostatin

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Eine allgemeine und vielseitige Methode zur C‐C‐Kreuzkupplungssynthese konjugierter Enine: eine Eintopfsynthese, ausgehend von Carbonylverbindungen

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