CsF and CuI do the trick: Stille coupling reactions of aryl/vinyl iodides, triflates, and bromides with aryl/vinyl stannanes are greatly enhanced by the inclusion of CsF and CuI in the reaction mixture (see scheme; Conditions A: [Pd(PPh3)4]/CsF/CuI/DMF/45 °C; Conditions B: PdCl2/PtBu3/CsF/CuI/DMF/45 °C).

Escherichia coli 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH).These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9 -91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues. 5Ј-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN)1 functions at two steps in bacterial pathways related to polyamine biosynthesis, quorum sensing, methylation, and purine and methionine salvage reactions ( Fig. 1; see Refs. 1-4). It catalyzes the physiologically irreversible hydrolysis of 5Ј-methylthioadenosine (MTA) to adenine and 5-methylthio-D-ribose (MTR). The product adenine is subsequently recycled into the adenine nucleotide pool by the widely distributed adenine phosphoribosyltransferase (5), and the 5-methylthio-D-ribose is subsequently phosphorylated to 5-methylthio-␣-D-ribose 1-phosphate and converted into methionine (6). MTA is a by-product of the reactions involving sequential transfers of the aminopropyl group from decarboxylated S-adenosylmethionine to form spermidine and spermine (although spermine is absent in Escherichia coli). Polyamine synthesis is sensitive to product inhibition by MTA, with inhibition constants reported to be 0.3 M for bovine spermine synthase (7) and 50 M for rat spermidine synthase (8). Inhibition of MTAN is therefore expected to inhibit polyamine biosynthesis and the salvage pathways for adenine and methionine. Another function of MTAN in bacteria is generation of S-ribosylhomocysteine (SRH) from SAH. SRH is a precursor for synthesis of tetrahydrofurans, quorum-sensing molecules involved in expression of the enzymes for biofilm formation, exotoxin synthesis, and antibiotic resistance factors (9 -14). Previously characterized nucleoside and nucleotide N-ribosyl hydrolases proceed through transition state structures in which the N-ribosidic

Streptococcus pneumoniae 5′-methylthioadenosine/S-adenosylhomocysteine hydrolase (MTAN) catalyzes the hydrolytic deadenylation of its substrates to form adenine and 5-methylthioribose or S-ribosylhomocysteine (SRH). MTAN is not found in mammals but is involved in bacterial quorum sensing. MTAN gene disruption affects growth and pathogenicity of bacteria, making it a target for antibiotic design. Kinetic isotope effects and computational studies have established a dissociative SN1 transition state for E. coli MTAN and transition state analogues resembling the transition state are powerful inhibitors of the enzyme (Singh, V., Lee, J. L., Núñez, S., Howell, P. L. and Schramm, V. L. (2005) Biochemistry 44, 11647-11659). The MTAN from S. pneumoniae has 40% sequence identity to E. coli MTAN, but exhibits remarkably distinct kinetic and inhibitory properties. 5′-Methylthio-Immucillin-A (MT-ImmA) is a transition state analogue resembling an early SN1 transition state. It is a weak inhibitor of S. pneumoniae MTAN with a K i of 1.0 μM. The X-ray structure of S. pneumoniae MTAN with MT-ImmA indicates a dimer with the methylthio group in a flexible hydrophobic pocket. Replacing the methyl group with phenyl (PhT-ImmA), tolyl (p-TolTImmA) or ethyl (EtT-ImmA) groups increases the affinity to give K i values of 335 nM, 60 nM and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fullydissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a K i value of 24 nM and replacing the 5′-methyl group with p-Cl-phenyl (p-Cl-PhTDADMe-ImmA) gave a K i * value of 0.36 nM. The inhibitory potential of DADMe-Immucillins relative to the Immucillins supports a fully dissociated transition state structure for S. pneumoniae MTAN. Comparison of active site contacts in the X-ray crystal structures of E. coli and S. pneumoniae MTAN with MT-ImmA would predict equal binding, yet most analogues bind 10 3 to 10 4 fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since k cat /K m for S. pneumoniae MTAN is <10 -2 that of E. coli MTAN. Keywords5′-methylthioadenosine; 5′-methylthioadenosine nucleosidase; quorum sensing; nucleoside hydrolase; transition state; transition state analogue inhibitors; polyamines; MTAN structure; catalytic efficiency *Corresponding author: telephone (718) 430-2813, Fax (718) Email vern@aecom.yu.edu. 3 Immucillin-H is (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol and has been shown to have a pK a > 10 at N7 (47). MT-ImmA is chemically similar in the 9-deazaadenine ring and is expected to have similar pK a . NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN 1 ) is a bacterial enzyme encoded by the pfs gene and catalyzes hydrolytic depurination of 5′-methylthioadenosine (MTA) to form 5-methylthioribose (MTR) and S-adenosylhomocysteine (SAH) to S-ribosylh...

Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum expresses a distinct PNP (PfPNP) with a unique substrate specificity that includes 5-methylthioinosine. The PfPNP functions both in purine salvage and in recycling purine groups from the polyamine synthetic pathway. Immucillin-H is an inhibitor of both huPNP and PfPNPs. It kills activated human T-cells and induces purine-less death in P. falciparum. Immucillin-H is a transition state analogue designed to mimic the early transition state of bovine PNP. The DADMeImmucillins are second generation transition state analogues designed to match the fully dissociated transition states of huPNP and PfPNP. Immucillins, DADMe-Immucillins and related analogues are compared for their energetic interactions with human and P. falciparum PNPs. Immucillin-H and DADMe-Immucillin-H are 860 and 500 pM inhibitors against P. falciparum PNP but bind human PNP 15-35 times more tightly. This common pattern is a result of k cat for huPNP being 18-fold greater than k cat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the k chem /k cat binding argument for transition state analogues. Preferential PfPNP inhibition is gained in the Immucillins by 5-methylthio substitution which exploits the unique substrate specificity of PfPNP. Human PNP achieves part of its catalytic potential from 5-OH neighboring group participation. When PfPNP acts on 5-methylthioinosine, this interaction is not possible. Compensation for the 5-OH effect in the P. falciparum enzyme is provided by improved leaving group interactions with Asp 206 as a general acid compared with Asn at this position in huPNP. Specific atomic modifications in the transition state analogues cause disproportionate binding differences between huPNP and PfPNPs and pinpoint energetic binding differences despite similar transition states.Inhibition of human purine nucleoside phosphorylase (huPNP) 1 by transition state analogue inhibitors shows promise for the control of T-cell cancers and autoimmune diseases (1-3). The combined inhibition of human and P. falciparum PNPs kills parasites cultured in human erythrocytes by purine-less death (4, 5). Immucillin-H (ImmH) is the first transition state analogue inhibitor developed in this class and shows a 56 pM K d for huPNP (6). It has recently entered phase II clinical trials against T-and B-cell cancers (7). ImmH was designed from the early ribooxacarbenium ion transition state structure of bovine PNP (8). More recently, the transition state structures have been solved for huPNP and PfPNP, which are both characterized by symmetric near fully dissociated ribooxacarbenium ion transition states (9).The second generation transition state analogue inhibitors were synthesized to mimic these dissociated transition states and include DADMe-Immucillin-H and DADMe-Immucillin-G. These are extraordinary inhibitors of huPNP with K d values of 16 and 7 pM, respectively. DA...

The combination of copper(I) iodide and cesium fluoride significantly enhances the Stille reaction. After extensive optimisation, a variety of electronically unfavourable and sterically hindered substrates were coupled in very high yields under mild conditions.