No abstract

HAJDUCH, M.; VYDRA, D.; HUSTOVA, A.; KVASNICA, M.; BIEDERMANN, D.; MARKOVA, L.; URBAN, M.; SAREK*, J.; Nat. Prod. Rep. 23 (2006) 3, 394-411; Dep. Org. Nucl. Chem., Charles Univ., CZ-128 40 Prague 2, Czech Republic; Eng.) -Lindner 37-244

Soluble adenylate cyclase [EC 4.6.1.1] accumulates in the culture medium of exponentially growing Bordetella pertussis (300-900 pmol of cAMP formed/min per ml of 24 hr culture supernatant). In addition, there is an extracytoplasmic adenylate cyclase which enables the intact organisms to form [32P] cAMP (adenosine 3':5'-cyclic monophosphate) from exogenous [alpha-32P] ATP (200-1200 nmol of cAMP formed/min per g wet weight of cells) and which comprises 20-45% of the total adenylate cyclase activity. In contrast, only 1.7 and 2.4% of the total cell malate dehydrogenase [EC 1.1.1.37] and alkaline phosphatase [EC 3.1.3.1], respectively, are detectable in the intact cell. Trypsin treatment of intact organisms destroys 96% of the extracytoplasmic adenylate cyclase, but does not reduce the total cell malate dehydrogenase or a small pool of intracellular adenylate cyclase. Four compartments of adenylate cyclase in B. pertussis are proposed; (A) soluble enzyme in the culture supernatant (up to 20% of the total activity); (B) enzyme associated with intact cells and measurable without cell disruption (20-45%); (C) extracytoplasmic enzyme sensitive to trypsin, but not measurable in intact cells at standard substrate concentrations (40-60%); and (D) intracellular enzyme (7-9%). In comparison with previously studied bacterial adenylate cyclases, the extracytoplasmic location appears to be unique to the B. pertussis enzyme.

We studied the incorporation of the fluorescent cytidine analogues 1, 3-diaza-2-oxo-phenothiazine (tC) and 1, 3-diaza-2-oxo-phenoxazine (tCo) by human DNA polymerase α and Klenow fragment of DNA polymerase I (E. coli). These tricyclic nucleobases possess the regular hydrogen bonding interface of cytosine but are significantly size expanded toward the major groove. Despite the size alteration both DNA polymerases insert dtCTP and dtCoTP with remarkable catalytic efficiency. Polymerization opposite guanine is comparable to the insertion of dCTP, while the insertion opposite adenine is only ∼4-11 times less efficient than the formation of a T-A base pair. Both enzymes readily extend the formed tC(o)-G and tC(o)-A base pairs, and can incorporate at least 4 consecutive nucleotide analogues. Consistent with these results, both DNA polymerases efficiently polymerize dGTP and dATP when tC and tCo are in the template strand. KF inserts dGTP with a 4-to 9-fold higher probability than dATP, while pol α favors dGTP over dATP by a factor of 30-65. Overall, the properties of tC(o) as templating base and as incoming nucleotide are surprisingly symmetrical and may be universal for A and B family DNA polymerases. This finding suggests that the aptitude for ambivalent base pairing is a consequence of the electronic properties of tC(o).

Twelve lupane, 18alpha-oleanane, and des-E-lupane derivatives (1a-5b) were either extracted from natural sources or synthesized from betulinic acid (1a) and betulin (2). Compounds 1b, 1c, 3b, 3c, 4b, 4c, 5a, and 5b were then used as starting materials for further synthesis of a series of pyrazines and benzopyrazines (6a-18); 20 of them are new (6a-6e, 7a-7d, and 10a-18). Activity of pyrazine 6a against the T-lymphoblastic leukemia cell line CEM encouraged us to synthesize several new esters (6b-6d) to study structure-activity relationships with respect to substitution of the carboxyl group at position 28. The synthesized compounds were tested for cytotoxicity against a variety of cancer cell lines of different histogenetic origin, and the results were compared with cytotoxicity of the known starting compounds. Significant cytotoxic activity against A 549, K 562, and multidrug-resistant K 562-tax cell lines was found in pyrazines 6a, 6d, and 6e.