Effects of Eupalinilide E and UM171, alone and in combination on cytokine stimulated ex-vivo expansion of human cord blood hematopoietic stem cells.

Eupalinilide E was assessed for ex-vivo expansion activity on hematopoietic stem cells (HSCs) from human cord blood (CB) CD34+ cells in serum-free, SCF, TPO and FL stimulated 7 day cultures. Eupalinilide E ex-vivo enhanced phenotyped (p) HSCs and glycolysis of CD34+ cells isolated 7 days after culture as measured by extracellular acidification rate, but did not alone show enhanced NSG engrafting capability of HSCs as determined by chimerism and numbers of SCID Repopulating cells, a quantitative measure of functional human HSCs. This is another example of pHSCs not necessarily recapitulating functional activity of these cells. Lack of effect on engrafting HSCs may be due to a number of possibilities, including down regulation of CXCR4 or of the homing capacity of these treated cells. However, Eupalinilide did act in an additive to synergistic fashion with UM171 to enhance ex vivo expansion of both pHSCs, and functionally engrafting HSCs. While reasons for the disconnect between pHSC and function of HSCs with Eupalinilide E alone cultured CB CD34+ cells is yet to be determined, the data suggest possible future use of Eupalinilide and UM171 together to enhance ex vivo production of CB HSCs for clinical hematopoietic cell transplantation.

Employing modular polyketide synthase ketoreductases as biocatalysts in the preparative chemoenzymatic syntheses of diketide chiral building blocks.

Chiral building blocks are valuable intermediates in the syntheses of natural products and pharmaceuticals. A scalable chemoenzymatic route to chiral diketides has been developed that includes the general synthesis of α-substituted, ß-ketoacyl N-acetylcysteamine thioesters followed by a biocatalytic cycle in which a glucose-fueled NADPH-regeneration system drives reductions catalyzed by isolated modular polyketide synthase (PKS) ketoreductases (KRs). To identify KRs that operate as active, stereospecific biocatalysts, 11 isolated KRs were incubated with 5 diketides and their products were analyzed by chiral chromatography. KRs that naturally reduce small polyketide intermediates were the most active and stereospecific toward the panel of diketides. Several biocatalytic reactions were scaled up to yield more than 100 mg of product. These syntheses demonstrate the ability of PKS enzymes to economically and greenly generate diverse chiral building blocks on a preparative scale.

A robust platform for the synthesis of new tetracycline antibiotics.

Tetracyclines and tetracycline analogues are prepared by a convergent, single-step Michael-Claisen condensation of AB precursor 1 or 2 with D-ring precursors of wide structural variability, followed by removal of protective groups (typically in two steps). A number of procedural variants of the key C-ring-forming reaction are illustrated in multiple examples. These include stepwise deprotonation of a D-ring precursor followed by addition of 1 or 2, in situ deprotonation of a D-ring precursor in mixture with 1 or 2, and in situ lithium-halogen exchange of a benzylic bromide D-ring precursor in the presence of 1 or 2, followed by warming. The AB plus D strategy for tetracycline synthesis by C-ring construction is shown to be robust across a range of different carbocyclic and heterocyclic D-ring precursors, proceeding reliably and with a high degree of stereochemical control. Evidence suggests that Michael addition of the benzylic anion derived from a given D-ring precursor to enones 1 or 2 is quite rapid at -78 degrees C, while Claisen cyclization of the enolate produced is rate-determining, typically occurring upon warming to 0 degrees C. The AB plus D coupling strategy is also shown to be useful for the construction of tetracycline precursors that are diversifiable by latter-stage transformations, subsequent to cyclization to form the C ring. Results of antibacterial assays and preliminary data obtained from a murine septicemia model show that many of the novel tetracyclines synthesized have potent antibiotic activities, both in bacterial cell culture and in vivo. The platform for tetracycline synthesis described gives access to a broad range of molecules that would be inaccessible by semisynthetic methods (presently the only means of tetracycline production) and provides a powerful engine for the discovery and, perhaps, development of new tetracycline antibiotics.

Total synthesis of garsubellin A.

A concise approach to the laboratory synthesis of garsubellin A is described. Garsubellin A, an effective inducer of choline acetyltransferase (ChAT), has been shown to have potential as a therapeutic agent for the treatment of Alzheimer's disease. Starting from 3,5-dimethoxyphenol, the synthesis has provided garsubellin A in an 18-step sequence. Notable transformations include dearomative allylation, diastereoselective vinylogous lactonization, iodocarbocyclization, transannular Wurtz, and bridgehead functionalization reactions.

Studies directed toward the synthesis of terreulactone A: rapid construction of the A, B, C rings.

[structure: see text]. An efficient, rapid synthesis of the A, B, C rings of terreulactone A is described. Key steps in the synthesis include a diastereoselective benzylic acid rearrangement to create the desired quaternary center at C2 and a mild bromolactonization to assemble the lactonic ring A.

Synthesis of (-)-tetracycline.

We describe a convergent, enantioselective synthesis of (-)-tetracycline (1) from benzoic acid (17 steps, 1.1% yield). Benzoic acid was transformed into the AB precursor 2 in 10 steps (11% yield), as previously described, and the latter compound was activated toward Diels-Alder cycloaddition by the introduction of an alpha-phenylthio group (two steps, 66% yield). Heating of the resulting alpha-(phenylthio)enone (3) with the triethylsilyloxybenzocyclobutene derivative 4 at 85 degrees C gave the endo-Diels Alder adduct 5 in 64% yield. Deprotection and oxidation of the latter intermediate gave the 2-(phenylthio)-1,3-diketone 7, which was oxidized with m-chloroperoxybenzoic acid in the presence of trifluoroacetic acid. The sulfoxide intermediate(s) formed eliminated upon warming to 35 degrees C to give the anyhydrotetracycline derivative 8. Intermediate 8 underwent spontaneous autoxidation at 23 degrees C to form the hydroperoxide keto-9 stereoselectively. Without isolation, hydrogenolysis of 9 in the presence of palladium black gave (-)-tetracycline (42% yield from 7), indistinguishable from an authentic sample.

A convergent enantioselective route to structurally diverse 6-deoxytetracycline antibiotics.

Complex antibiotics based on natural products are almost invariably prepared by semisynthesis, or chemical transformation of the isolated natural products. This approach greatly limits the range of accessible structures that might be studied as new antibiotic candidates. Here we report a short and enantioselective synthetic route to a diverse range of 6-deoxytetracycline antibiotics. The common feature of this class is a scaffold of four linearly fused rings, labeled A through D. We targeted not a single compound but a group of structures with the D ring as a site of structural variability. A late-stage, diastereoselective C-ring construction was used to couple structurally varied D-ring precursors with an AB precursor containing much of the essential functionality for binding to the bacterial ribosome. Five derivatives were synthesized from benzoic acid in yields ranging from 5 to 7% over 14 to 15 steps, and a sixth, (-)-doxycycline, was synthesized in 8.3% yield over 18 steps.