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

Abstract

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.

Thiols Screened by the Neocarzinostatin Protein for Preserving or Detoxifying its Bound Enediyne Antibiotic

“…The temperature at which half of the protein is unfolded (T m ) was found to be 67.8 8C for WT apoNCS (Figure 1c). This value is reasonably close to those reported previously (63.5-68 8C) [48,51,53,54,56,57] taking moderate variations in experimental methods and conditions into account. The thermal stability measurements (T m ) of mutants F76A, Figure 1.…”

“…In natural apoNCS produced by S. carzinostaticus, two disulfide bonds (C37ÀC47 and C88ÀC93) are present, both of which are important for maintaining the stability of the folded conformation. [49][50][51] To confirm the formation of both disulfide linkages in the recombinant apoNCSs, we performed a disulfide assay that was specifically developed for apoNCS. [48,51] As shown by mass spectrometry, no free Cys residues were observed in any of the purified proteins used in this study.…”

“…The far-UV CD spectrum of native apoNCS shows a typical negative band at 212 nm for the b-sheet-dominant structure and an atypical positive signal that is centered at 224 nm. [48,[51][52][53][54][55][56] The near-UV CD spectrum, attributed to the characteristic motion of the aromatic side-chains of Y32, W39, and W83 in the tertiary environment of apoNCS, exhibits a weak but broad negative band at around 271 nm. [48,51,[54][55][56] The far-and near-UV CD spectra of mutants F76A, L77A, and S98A apoNCSs (data not shown) are in good agreement with our previous results [48] and closely resemble those of native apoNCS.…”

Substituent‐Dependent, Iron‐Mediated Tandem Cyclization of Diynes with Benzaldehyde Acetals to Form Highly Functionalized Indene Derivatives

“…[1] Diynes have been used as versatile building blocks for the preparation of carbo-and heterocycles through transition-metal catalyzed carbocyclization, cycloaddition, and cycloisomerization reactions. [7] Although enediynes have been documented for the synthesis of indenes, [8] diynes have seldom been applied for this purpose. [7] Although enediynes have been documented for the synthesis of indenes, [8] diynes have seldom been applied for this purpose.…”

“…[2] The indene backbone is frequently found in natural products, [3] pharmaceuticals, [4] functional materials, [5] and metallocene complexes, [6] and a variety of synthetic methods have been developed for their construction. [7] Although enediynes have been documented for the synthesis of indenes, [8] diynes have seldom been applied for this purpose. [9] Recently, iron catalysis has emerged as a promising, environmentally benign alternative to traditional transition-metal catalysis due to the many advantages of utilizing iron-catalyst precursors, such as low cost, nontoxicity, good stability, and straightforward methods of handling.…”