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

Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetery indicate that binding of a structurally rigid first-generation Immucillin (K d = 56 pM) is driven by large negative enthalpy values (ΔH = −21.2 kcal/mol) with a substantial entropic (-TΔS) penalty. The tightest-binding inhibitors (K d = 5 to 9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP·inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5'-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the seventeen Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. By introducing flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding.Human purine nucleoside phosphroylase (PNP) is required for the catabolism of 6-oxy-(and 2'-deoxy)-nucleosides to free nucleobases for recycling by phosphoribosyl transferases or oxidation to uric acid and excretion. Genetic deficiency of PNP in humans causes deoxyguanosine accumulation in the blood and its conversion to dGTP causes arrest of DNA synthesis and apoptosis specifically in activated T-cells. Human PNP is therefore a target for the treatment of autoimmune disorders and T-cell cancers (1-2).The transition state structure of human PNP has permitted the design of several transition state analogues with picomolar dissociation constants (3-7). The transition state is characterized by a fully-developed ribocation with a C1' -N9 distance of ≥ 3.0 Å and the phosphate nucleophile at a similar distance to C1' of the ribosyl cation (Figure 1) (3). Four structurally distinct generations of transition state analogues have been synthesized (Figure 2).

The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C- nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme.

The novel 6-lactone (1 ) of (3R,5S) -3hydroxy-5hydroxymethyltetrahydrofuran-3-carboxylic acid (2) has been isolated from tar produced by pyrolysis of cellulose doped with zinc chloride. Using Curie-point pyrolysis-g.1.c. analysis, the lactone was shown to be a significant product of the pyrolysis of cellulose in the presence of a wide variety of other Lewis acids, but not in the presence of protic acids or copper(ii) chloride. The lactone (1) was readily hydrolysed t o the acid (2) and yielded the methyl ester (3) on met ha n o I ysis.The pyrolysis of cellulose has been extensively studied because the tar produced is a potential chemical feedstock,' and a source of optically pure starting materials for At temperatures above 300 "C cellulose undergoes depolymerisation by intramolecular transglycosylation to yield levoglucosan ( 1,6-anhydro-~-~-glucopyranose) and minor amounts of other anhydrogluc~ses.~ The presence of acids in admixture with the cellulose profoundly influences the course of pyrolysis. In the presence of protic acids, pyrolytic dehydration, rearrangement and charring reactions are substantially accelerated. Thus the pyrolysis of cellulose doped with orthophosphoric acid gives levoglucosenone (1,6-anhydr0-3,4-dideoxy-P-~-glycero-hex-3enopyranos-2-ulose) as the predominant volatile carbohydrate product in yields of 2-12%.3*5 Pyrolysis in the presence of Lewis acids also results in considerable dehydration and charring, but levoglucosenone is produced in reduced yield (ca. 0.3%) and is no longer the major product.6Studies in this laboratory have shown that cellulose is converted into a liquid product, composed largely of oxygenated aromatics, by thermolysis in aqueous phenol in the presence of various catalysts7 Such liquefactions occur in the same temperature range that cellulose undergoes pyrolytic depolymerisation. A comparative study on the effects of a range of inorganic additives on the pyrolytic depolymerisation of cellulose was undertaken, in expectation that the results would help elucidate the mechanism by which cellulose is liquefied.For this comparative study, samples were subjected to analysis by Curie-point pyrolysis-gas-liquid chromatography (c.p.p.-g.l.c.).* In this method the sample is heated very rapidly to the desired temperature (358 "C) and, because the sample is suspended inside the head of the g.1.c. column, the volatile products are swept directly from the heated wire onto the column packing by a stream of inert gas, thus minimising the extent to which the primary pyrolysis products undergo secondary reactions.

The potent immucillin purine nucleoside phosphorylase (PNP) inhibitors F-DADMe-ImmH [(3S,4S)−3], and [(3R,4R)−3] are synthesized in seven steps. Cycloaddition to a fluoroalkene and an enzymic resolution are the key features of the construction of the fluoropyrrolidines 11, from which the immucillins are assembled by use of a three-component Mannich reaction. Slow-onset binding constants (Ki∗) for [(3S,4S)−3] and [(3R,4R)−3] with human PNP are 0.032 and 1.82 nM, respectively. F-DADMe-ImmH [(3S,4S)−3] exhibits oral availability in mice at doses as low as 0.2 mg/kg.