Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome.Methyltransferases (MTases) 3 play key roles in normal physiology and human diseases through methylating DNA, RNA, and proteins. Almost all MTases use S-adenosyl-L-methionine (SAM) as a methyl donor and generate S-adenosyl-Lhomocysteine (SAH) as a by-product. Pharmacological modulation of MTases by small molecules represents a novel approach to therapeutic intervention in cancer and other diseases (1). However, because the core domains of various MTases are conserved, designing inhibitors that specifically block the disease-related MTase without affecting other MTases, has been challenging. The ability to rationally design and generate selective inhibitors would have profound implications for development of new medicines for many methyltransferase-mediated diseases.Dengue virus (DENV), from genus Flavivirus in the family Flaviviridae, is the most prevalent mosquito-borne viral pathogen that infects humans. The four serotypes of DENV (DENV-1 to -4) pose a public health threat to 2.5 billion people worldwide, and cause 50 -100 million human infections each year. Neither vaccine nor antiviral therapy is currently available for DENV. The flavivirus MTase methylates the guanine N7 and ribose 2Ј-O positions of the viral RNA cap in a sequential manner (i.e. GpppA-RNA 3 m7GpppA-RNA 3 m7GpppAm-RNA) (2, 3). Recent studies have shown that flavivirus MTase is critical for viral replication and, therefore, represents a valid target for antiviral therapeutics (4 -6). We therefore examined the feasibility to design inhibitors that specifically modulate flavivirus MTase. EXPERIMENTAL PROCEDURESPreparation of DENV-3 MTases-The DNA fragment representing the MTase domain of DENV-3 was cloned into expression vector pGEX4T1 (Amersham Biosciences). Ala-substitution mutant MTases were prepared using a standard overlapping PCR procedure. Recombinant MTases, containing an N-terminal GST, were expressed in Escherichia coli. BL21 cells and purified through a GSTPrep TM FF 16/10 column (GE Healthcare). The GST tag was then cleaved by thrombin and removed from the MTases using the GST column. The MTases were further purified through gel filtration to ensure protein purity was Ͼ95%. The p...

Dedicated to Professor Y. Kishi on the occasion of his 70th birthday.Herein we address the total synthesis of the natural product oasomycin A by assembly of the C1-C12, C13-C28, and C29-C46 subunits, whose syntheses have been described in the preceding Communications.[1]The synthesis plan (Scheme 1) incorporates a speculative late-stage macrolactonization of the linear seco acid precursor to form a 42-membered lactone that upon global deprotection would provide the natural product. Since oasomycin A is known to rearrange to the oasomycins D and E under basic conditions, [2] an acid-mediated global deprotection was obligatory. It was our intention to assemble the requisite seco acid by using an aldol addition of the C1-C28 ketone I to the C29-C46 aldehyde II with a concomitant installation of the C29 stereocenter, followed by a stereoselective reduction of the C27 ketone.The assembly of ketone I through a Kocienski-Julia olefination [3] of the C13-C28 aldehyde III with C1-C12 fragment IV was undertaken first (Scheme 2). Sulfone 1 was selectively deprotonated with KHMDS and treated with aldehyde 2[3] to afford the coupling product 3 a as a 7:1 mixture of E/Z isomers (57 % yield). In addition, a significant amount of a by-product was consistently formed in 15-25 % yield in this and related olefinations. This by-product with the general structure 3 b (Scheme 2) may be rationalized by a Brook rearrangement of the Julia intermediate followed by alkoxide attack on the sulfur center. All efforts to suppress this side reaction were unsuccessful. [4] With both the C1-C28 and C29-C46 subunits in hand, we addressed the aldol coupling which would provide the oasomycin A skeleton. The logic behind the selection of an aldol addition to form the C28 À C29 bond was based on the fact that the diastereoselectivity of this reaction should be reinforced by resident chirality in both reaction partners: the C25 stereocenter on the enolate [Eq. (1)], [5] and the C31 stereocenter on the aldehyde fragment [Eq. (2)].[6] Although Scheme 1. Assembly of oasomycin A subunits.

Putting the pieces together: The total synthesis of the natural macrolide oasomycin A has been realized. Key fragment couplings include an anti‐Felkin selective aldol addition (green), Kociensky–Julia olefinations (red), and competitive Weinreb amide acylation reaction (blue). The utility of the 4,5‐diphenyloxazole as a carboxy surrogate and the late‐stage macrolactonization affording the 42‐membered macrocycle of oasomycin A are also described.