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Genetic deficiency of human purine nucleoside phosphorylase (PNP) causes T-cell immunodeficiency. The enzyme is therefore a target for autoimmunity disorders, tissue transplant rejection and T-cell malignancies. Transition state analysis of bovine PNP led to the development of immucillin-H (ImmH), a powerful inhibitor of bovine PNP but less effective for human PNP. The transition state of human PNP differs from that of the bovine enzyme and transition state analogues specific for the human enzyme were synthesized. Three first generation transition state analogues, ImmG (Kd = 42 pM), ImmH (Kd = 56 pM), and 8-aza-ImmH (Kd = 180 pM), are compared with three second generation DADMe compounds (4'-deaza-1'-aza-2'-deoxy-1'-(9-methylene)-immucillins) tailored to the transition state of human PNP. The second generation compounds, DADMe-ImmG (Kd = 7pM), DADMe-ImmH (Kd = 16 pM), and 8-aza-DADMe-ImmH (Kd = 2.0 nM), are superior for inhibition of human PNP by binding up to 6-fold tighter. The DADMe-immucillins are the most powerful PNP inhibitors yet described, with Km/Kd ratios up to 5,400,000. ImmH and DADMe-ImmH are orally available in mice; DADMe-ImmH is more efficient than ImmH. DADMe-ImmH achieves the ultimate goal in transition state inhibitor design in mice. A single oral dose causes inhibition of the target enzyme for the approximate lifetime of circulating erythrocytes.

The aza-C-nucleosides, Immucillin-H and Immucillin-G, are transition state analogue inhibitors of purine nucleoside phosphorylase, a therapeutic target for the control of T-cell proliferation. Immucillin analogues modified at the 2'-, 3'-, or 5'-positions of the azasugar moiety or at the 6-, 7-, or 8-positions of the deazapurine, as well as methylene-bridged analogues, have been synthesized and tested for their inhibition of human purine nucleoside phosphorylase. All analogues were poorer inhibitors, which reflects the superior capture of transition state features in the parent immucillins.

A polysaccharide rich in 4',6'-O-(1-carboxyethylidene)-substituted (i.e., pyruvylated) beta-carrageenan has been prepared by solvolytic desulfation of a polysaccharide containing predominantly pyruvylated alpha-carrageenan, which was extracted from the red seaweed, Callophycus tridentifer. The 13C and 1H NMR chemical shifts of pyruvylated beta-carrageenan have been fully assigned using 2D NMR spectroscopic techniques. The 4',6'-O-(1-methoxycarbonylethylidene) group, generated during chemical methylation of the polysaccharide, has been shown to survive under the conditions of acidic hydrolysis that cleave the 3,6-anhydro-alpha-D-galactosidic bonds in permethylated samples of both pyruvylated beta- and pyruvylated alpha-carrageenans. As a result, two novel pyruvylated carrabiitol derivatives have been prepared.

Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 A) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.

Purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyze N-ribosidic bond cleavage in purine nucleosides and nucleotides, with addition of phosphate or pyrophosphate to form phosphorylated alpha-D-ribose products. The transition states have oxacarbenium ion character with a positive charge near 1'-C and ionic stabilization from nearby phosphoryl anions. Immucillin-H (ImmH) and Immucillin-H 5'-PO(4) (ImmHP) resemble the transition state charge when protonated at 4'-N and bind tightly to these enzymes with K(d) values of 20 pM to 1 nM. It has been proposed that Immucillins bind as the 4'-N neutral form and are protonated in the slow-onset step. Solution and solid-state NMR spectra of ImmH, ImmHP, guanosine, and GMP in complexes with two PNPs and a HGPRTase have been used to characterize their ionization states. Results with PNP*ImmH*PO(4) and HGPRTase*ImmHP*MgPP(i) indicate protonation at N-4' for the tightly bound inhibitors. The 1'-(13)C and 1'-(1)H resonances of bound Immucillins showed large downfield shifts as compared to Michaelis complexes, suggesting distortion of 1'-C toward sp(2) geometry. The Immucillins act as transition state mimics by binding with neutral iminoribitol groups followed by 4'-N protonation during slow-onset inhibition to form carbocationic mimics of the transition states. The ability of the Immucillins to mimic both substrate and transition state features contributes to their capture of transition state binding energy. Enzyme-activated phosphoryl nucleophiles bound to PNP and HGPRTase suggest enhanced electrostatic stabilization of the cationic transition states. Distortion of the oxacarbenium ion mimic toward transition state geometry is a common feature of the three distinct enzymatic complexes analyzed here. Substrate complexes, even in catalytically cycling equilibrium mixtures, do not reveal similar distortions.

The 8-aza-immucillins (8-aza-9-deazapurines linked from C9 to C1 of 1,4-dideoxy-1,4-iminoribitol) have been designed as transition-state analogues of the reactions catalyzed by purine nucleoside phosphorylase and nucleoside hydrolases. Syntheses of the 8-aza-immucillin analogues of inosine and adenosine are described. They are powerful inhibitors of the target enzymes with equilibrium dissociation constants as low as 42 pM.

Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.

Readily available, crystalline penta-O-propanoyl-beta-D-glucofuranose is shown to be a suitable glycosylating agent for the acid-catalysed, direct synthesis of O-, S- and N-glucofuranosyl compounds. Beta-linked products are formed with good selectivity. Reaction with cyanotrimethylsilane gave the 1,2-O-(1-cyanopropylidene)acetal rather than the C-glycosyl cyanide. By selective acid-catalysed hydrolysis, the title compound was converted to the 1-hydroxy analogue from which the trichloroacetimidates were made as further potential glycosylating agents., (Copyright 2002 Elsevier Science Ltd.)

A chromogenic substrate, 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate was synthesized and used in combination with beta-N-acetylhexosaminidase for detection of the sulfatase, MdsA, by release of 4-nitrophenol. MdsA was originally isolated from the bacterium Prevotella strain RS2 and is believed to be involved in desulfation of sulfomucins, major components of the mucus barrier protecting the human colon surface. The exo nature of the MdsA sulfatase was indicated by its inability to de-esterify the disaccharide 4-nitrophenyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate. This latter compound was prepared from monosaccharide precursors by two different methods, the shorter requiring just six steps from 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and giving an overall yield of 26.4%. The syntheses of 4-nitrophenyl beta-D-galactopyranoside 3-triethylammonium sulfate and 6-triethylammonium sulfate and their use in combination with beta-galactosidase as chromogenic substrates for detecting Bacteroides fragilis sulfatases with different specificities was also demonstrated.

Plasmodium falciparum is responsible for the majority of life-threatening cases of malaria. Plasmodia species cannot synthesize purines de novo, whereas mammalian cells obtain purines from de novo synthesis or by purine salvage. Hypoxanthine is proposed to be the major source of purines for P. falciparum growth. It is produced from inosine phosphorolysis by purine nucleoside phosphorylase (PNP). Immucillins are powerful transition state analogue inhibitors of mammalian PNP and also inhibit P. falciparum PNP as illustrated in the accompanying article (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Kim, K., and Schramm, V. L. (2002) J. Biol. Chem. 277, 3219-3225). This work tests the hypothesis that erythrocyte and P. falciparum PNP are essential elements for growth and survival of the parasite in culture. Immucillin-H reduces the incorporation of inosine but not hypoxanthine into nucleic acids of P. falciparum and kills P. falciparum cultured in human erythrocytes with an IC(50) of 35 nm. Growth inhibition by Imm-H is reversed by the addition of hypoxanthine but not inosine, demonstrating the metabolic block at PNP. The concentration of Imm-H required for inhibition of parasite growth varies as a function of culture hematocrit, reflecting stoichiometric titration of human erythrocyte PNP by the inhibitor. Human and P. falciparum PNPs demonstrate different specificity for inhibition by immucillins, with the 2'-deoxy analogues showing marked preference for the human enzyme. The IC(50) values for immucillin analogue toxicity to P. falciparum cultures indicate that inhibition of PNP in both the erythrocytes and the parasite is necessary to induce a purine-less death.

Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226-3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K(i)* value of 0.6 nm. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nm. The K(m)/K(i)* value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K(m) for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5'-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.

Means have been developed for the synthesis and addition of 9-deaza-9-lithiopurine derivatives to the carbohydrate-derived cyclic imine 6 in facile convergent syntheses of biologically active aza-C-nucleosides.

A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) resembles the mammalian trimeric structure rather than the bacterial hexameric PNPs. The crystal structure of M. tuberculosis PNP in complex with the transition-state analogue immucillin-H (ImmH) and inorganic phosphate was solved at 1.75 A resolution and confirms the trimeric structure. Binding of the inhibitor occurs independently at the three catalytic sites, unlike mammalian PNPs which demonstrate negative cooperativity in ImmH binding. Reduced subunit interface contacts for TB-PNP, compared to the mammalian enzymes, correlate with the loss of the cooperative inhibitor binding. Mammalian and TB-PNPs both exhibit slow-onset inhibition and picomolar dissociation constants for ImmH. The structure supports a catalytic mechanism of reactant destabilization by neighboring group electrostatic interactions, transition-state stabilization, and leaving group activation. Despite an overall amino acid sequence identity of 33% between bovine and TB-PNPs and almost complete conservation in active site residues, one catalytic site difference suggests a strategy for the design of transition-state analogues with specificity for TB-PNP. The structure of TB-PNP was also solved to 2.0 A with 9-deazahypoxanthine (9dHX), iminoribitol (IR), and PO(4) to reconstruct the ImmH complex with its separate components. One subunit of the trimer has 9dHX, IR, and PO(4) bound, while the remaining two subunits contain only 9dHX. In the filled subunit, 9dHX retains the contacts found in the ImmH complex. However, the region of IR that corresponds to the oxocarbenium ion is translocated in the direction of the reaction coordinate, and the nucleophilic phosphate rotates away from the IR group. Loose packing of the pieces of ImmH in the catalytic site establishes that covalent connectivity in ImmH is required to achieve the tightly bound complex.

Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP). The gene was cloned, expressed, purified, and found to exhibit PNP activity. Purified M. tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Escherichia coli enzyme. Immucillin-H is a rationally designed analogue of the transition state that has been shown to be a potent inhibitor of mammalian PNP's. This inhibitor also exhibits slow-onset inhibition of M. tuberculosis PNP with a rapid, reversible inhibitor binding (K(i) of 2.2 nM) followed by an overall dissociation constant (K(i)) of 28 pM, yielding a K(m)/K(i) value of 10(6). Time-dependent tight binding of the inhibitor occurs with a rate of 0.1 s(-)(1), while relaxation of the complex is slower at 1.4 x 10(-)(3) s(-)(1). The pH dependence of the K(i) value of immucillin-H to the M. tuberculosis PNP suggests that the inhibitor binds as the neutral, unprotonated form that is subsequently protonated to generate the tight-binding species. The M. tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP. Analysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K(is) of 0.39 microM) while the 1,4-dideoxy-1,4-iminoribitol binds weakly (K(is) of 2.9 mM). Double-inhibition studies demonstrate antagonistic binding of 9-deazahypoxanthine and iminoribitol (beta = 13). However, the covalent attachment of these two components in immucillin-H increases equilibrium binding affinity by a factor of >14 000 (28 pM vs 0.39 microM) compared to 9-deazahypoxanthine alone, and by a factor of >10(8) compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements. The structural basis for M. tuberculosis PNP inhibition by immucillin-H and by its component parts is reported in the following paper [Shi, W., Basso, L. A., Santos, D. S., Tyler, P. C., Furneaux, R. H., Blanchard, J. S., Almo, S. C., and Schramm, V. L. (2001) Biochemistry 40, 8204-8215].

The accumulation of total water-soluble carbohydrate, and specifically sucrose and fructan, by excised leaves of Phormium tenax and P. cookianum (family Phormiaceae J. G. Agardh, order Asparagales) was investigated. Total water-soluble carbohydrate content of excised leaves of P. tenax and P. cookianum increased during 48 h of continuous illumination at an average rate of 1.3 and 0.9 mg g(-1) fresh weight leaf per hour, respectively. The sucrose content of excised leaves increased throughout the experimental period. The fructan content of excised leaves of P. tenax increased slightly throughout the experimental period, whilst that of P. cookianum was variable and showed no overall change. Chemical and spectroscopic analysis of the fructans obtained from the two Phormium species showed that they were similar to each other and contained mostly 1-linked and terminal fructofuranosyl (Fruf) residues, together with smaller amounts of 6-linked Fruf, 1,6-branched Fruf, terminal and 6-linked glucopyranosyl residues. Separation of the fructans by thin-layer and high-performance anion-exchange chromatography revealed the presence of a complex mixture of fructo-oligosaccharides and higher molecular weight fructan. The branched structure of the fructans isolated from excised leaves of Phormium resembles that of fructans and fructo-oligosaccharides isolated from some related species within the order Asparagales (Agave vera cruz, Cordyline australis and Urginea maritima), but is distinct from the linear structure of fructans from others (Allium cepa and Asparagus officinalis). The structural heterogeniety of fructans within both the order Asparagales and superorder Liliiflorae may be a useful chemotaxonomic aid.

Ricin toxin A-chain (RTA) is expressed by the castor bean plant and is among the most potent mammalian toxins. Upon activation in the cytosol, RTA depurinates a single adenine from position 4324 of rat 28S ribosomal RNA, causing inactivation of ribosomes by preventing the binding of elongation factors. Kinetic isotope effect studies have established that RTA operates via a D(N)*A(N) mechanism involving an oxacarbenium ion intermediate with bound adenine [Chen, X.-Y., Berti, P. J., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 1609-1617]. On the basis of this information, stem-loop RNA molecules were chemically synthesized, incorporating structural features of the oxacarbenium ion-like transition state. A 10-base RNA stem-loop incorporating (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds four times better (0.57 microM) than the 10-base RNA stem-loop with adenosine at the depurination site (2.2 microM). A 10-base RNA stem-loop with 1,2-dideoxyribitol [(2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] at the depurination site binds with a Kd of 3.2 microM and tightens to 0.75 microM in the presence of 9-deazaadenine. A similar RNA stem-loop with 1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds with a K(d) of 1.3 microM and improves to 0.65 micro;M with 9-deazaadenine added. When (3S,4R)-4-hydroxy-3-(hydroxymethyl)pyrrolidine was incorporated at the depurination site of a 14-base RNA stem-loop, the Kd was 0.48 microM. Addition of 9-deazaadenine tightens the binding to 0.10 microM whereas added adenine increases the affinity to 12 nM. The results of this study are consistent with the unusual dissociative D(N)*A(N) mechanism determined for RTA. Knowledge of this intermediate has led to the design and synthesis of the highest affinity inhibitor reported for the catalytic site of RTA.

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

Immucillin-H [ImmH; (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol] is a 23 pM inhibitor of bovine purine nucleoside phosphorylase (PNP) specifically designed as a transition state mimic [Miles, R. W., Tyler, P. C., Furneaux, R. H., Bagdassarian, C. K., and Schramm, V. L. (1998) Biochemistry 37, 8615-8621]. Cocrystals of PNP and the inhibitor are used to provide structural information for each step through the reaction coordinate of PNP. The X-ray crystal structure of free ImmH was solved at 0.9 A resolution, and a complex of PNP.ImmH.PO(4) was solved at 1.5 A resolution. These structures are compared to previously reported complexes of PNP with substrate and product analogues in the catalytic sites and with the experimentally determined transition state structure. Upon binding, ImmH is distorted to a conformation favoring ribosyl oxocarbenium ion formation. Ribosyl destabilization and transition state stabilization of the ribosyl oxocarbenium ion occur from neighboring group interactions with the phosphate anion and the 5'-hydroxyl of the ribosyl group. Leaving group activation of hypoxanthine involves hydrogen bonds to O6, N1, and N7 of the purine ring. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. Contacts between PNP and catalytic site ligands are shorter in the transition state analogue complex of PNP.ImmH.PO(4) than in the Michaelis complexes of PNP.inosine.SO(4) or PNP.hypoxanthine.ribose 1-PO(4). Reaction coordinate motion is dominated by translation of the carbon 1' of ribose between relatively fixed phosphate and purine groups. Purine and pyrimidine phosphoribosyltransferases and nucleoside N-ribosyl hydrolases appear to operate by a similar mechanism.

Ring-B derivatization of totarol (1) afforded the series of compounds 2-22 which were screened in vitro against: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and multiresistant Klebsiella pneumoniae. Several of the derivatives retained much of the antibacterial activity of totatol against the first three of these organisms (all gram-positive), but none was more active. The gram-negative Klebsiella was resistant to all compounds examined. Totarol (1) was shown to uncouple oxidative phosphorylation in isolated mitochondria at 50 microM.

Alterations of the C-12 and C-13 aromatic ring substituents of totarol (1) afforded the series of derivatives 2-14, and introduction of substituents at C-12 gave exclusively 2a-14a. The majority of these analogues were tested in vitro against the following organisms: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and multiresistant Klebsiella pneumoniae. The results were evaluated in terms of structure-activity relationship which reveals that: (a) the phenolic moiety at C-13, in general, is essential for antibacterial activity at < 32 microg/mL against gram-positive species, and (b) derivatization at C-12 has an undesirable effect on the antibacterial activity of this class of compounds, while (c) all compounds tested are ineffective against the gram-negative Klebsiella pneumoniae.

Giardia lamblia, the protozoan parasite responsible for giardiasis, requires purine salvage from its host for RNA and DNA synthesis. G. lamblia expresses an unusual purine phosphoribosyltransferase with a high specificity for guanine (GPRTase). The enzyme's sequence significantly diverges from those of related enzymes in other organisms. The transition state analogue immucillinGP is a powerful inhibitor of HGXPRTase from malaria [Li, C. M., et al. (1999) Nat. Struct. Biol. 6, 582-587] and is also a 10 nM inhibitor of G. lamblia GPRTase. Cocrystallization of GPRTase with immucillinGP led unexpectedly to a GPRTase.immucillinG binary complex with an open catalytic site loop. Diffusion of ligands into preformed crystals gave a GPRTase.immucillinGP.Mg(2+).pyrophosphate complex in which the open loop is stabilized by crystal contacts. G. lamblia GPRTase exhibits substantial structural differences from known purine phosphoribosyltransferases at positions remote from the catalytic site, but conserves most contacts to the bound inhibitor. The filled catalytic site with an open catalytic loop provides insight into ligand binding. One active site Mg(2+) ion is chelated to pyrophosphate, but the other is chelated to two conserved catalytic site carboxylates, suggesting a role for these amino acids. This arrangement of Mg(2+) and pyrophosphate has not been reported in purine phosphoribosyltransferases. ImmucillinG in the binary complex is anchored by its 9-deazaguanine group, and the iminoribitol is disordered. No Mg(2+) or pyrophosphate is detected; thus, the 5'-phosphoryl group is needed to immobilize the iminoribitol prior to magnesium pyrophosphate binding. Filling the catalytic site involves (1) binding the purine ring, (2) anchoring the 5'-phosphate to fix the ribosyl group, (3) binding the first Mg(2+) to Asp125 and Glu126 carboxyl groups and binding Mg(2+).pyrophosphate, and (4) closing the catalytic site loop and formation of bound (Mg(2+))(2). pyrophosphate prior to catalysis. Guanine specificity is provided by two peptide carbonyl oxygens hydrogen-bonded to the exocyclic amino group and a weak interaction to O6. Transition state formation involves N7 protonation by Asp129 acting as the general acid.

Protozoan parasites are unable to synthesize purines de novo and must rely on purine salvage pathways for their requirements. Nucleoside hydrolases, which are not found in mammals, function as key enzymes in purine salvage in protozoa. Inhibition of these enzymes may disrupt purine supply and specific inhibitors are potential therapeutic agents for the control of protozoan infections. A series of 1,4-dideoxy-1,4-imino-D-ribitols bearing C-bonded aromatic substituents at C-1 have been synthesized, following carbanion additions to the imine 2, and tested as potential nucleoside hydrolase inhibitors. Nucleoside analogues 8, 11, 14, 17, 20, 24-26, 28 exhibit Ki values in the range 0.2-22 microM against two representative isozymes of protozoan nucleoside hydrolases.

Nucleoside N-ribohydrolases from protozoan parasites are targets for inhibitor design in these purine-auxotrophic organisms. Purine-specific and purine/pyrimidine-nonspecific nucleoside hydrolases have been reported. Iminoribitols that are 1-substituted with meta- and para-derivatized phenyl groups [(1S)-substituted 1, 4-dideoxy-1,4-imino-D-ribitols] are powerful inhibitors for the nonspecific nucleoside N-ribohydrolases, but are weak inhibitiors for purine-specific isozymes [Parkin, D. W., Limberg, G., Tyler, P. C., Furneaux, R. H., Chen, X.-Y., and Schramm, V. L. (1997) Biochemisty 36, 3528-3534]. Binding of these inhibitors to nonspecific nucleoside hydrolase occurs primarily via interaction with the iminoribitol, a ribooxocarbenium ion analogue of the transition state. Weaker interactions arise from hydrophobic interactions between the phenyl group and the purine/pyrimidine site. In contrast, the purine-specific enzymes obtain equal catalytic potential from leaving group activation and ribooxocarbenium ion formation. Knowledge of the reaction mechanisms and transition states for these enzymes has guided the design of isozyme-specific transition state analogue inhibitors. New synthetic efforts have produced novel inhibitors that incorporate features of the leaving group hydrogen-bonding sites while retaining the iminoribitol group. These compounds provide the first transition state analogue inhibitors for purine-specific nucleoside hydrolase. The most inhibitory 1-substituted iminoribitol heterocycle is a sub-nanomolar inhibitor for the purine-specific nucleoside hydrolase from Trypanosoma brucei brucei. Novel nanomolar inhibitors are also described for the nonspecific nucleoside hydrolase from Crithidia fasciculata. The compounds reported here are the most powerful iminoribitol inhibitors yet described for the nucleoside hydrolases.

A series of analogues of, and potential pro-drugs derived from, the potent antibacterial diterpene totarol (1) were synthesized in order to elucidate the minimum structural requirements for antibacterial activity and to seek compounds with good bioavailability in vivo. These analogues varied in the structural features of their aromatic rings and the prodrugs were O-glycosylated derivatives. They were tested in vitro against three gram-positive bacteria: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA); and against the gram-negative multi-drug-resistant Klebsiella pneumoniae. None of the analogues was more potent than totarol itself, which is effective against these gram-positive bacteria at MIC values of 7 microM. The results were evaluated in terms of a structure-activity relationship and this showed that a phenolic moiety was essential for potent antibacterial activity. Amongst the pro-drugs, totaryl alpha-D-mannopyranoside (22) proved the most active in vitro (MIC 18 microM). The in vivo antibacterial activities of compounds 1, 22 and totarol beta-lactoside (23) were assessed in a mouse model of infection, but they were found to be ineffective. Compounds 1 and 22 were shown to be cytotoxic towards proliferating human cell cultures, CH 2983, HeLa, and MG 63, but only at concentrations of > 30 microM.

Malaria is a leading cause of worldwide mortality from infectious disease. Plasmodium falciparum proliferation in human erythrocytes requires purine salvage by hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase). The enzyme is a target for the development of novel antimalarials. Design and synthesis of transition-state analogue inhibitors permitted cocrystallization with the malarial enzyme and refinement of the complex to 2.0 A resolution. Catalytic site contacts in the malarial enzyme are similar to those of human hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) despite distinct substrate specificity. The crystal structure of malarial HGXPRTase with bound inhibitor, pyrophosphate, and two Mg(2+) ions reveals features unique to the transition-state analogue complex. Substrate-assisted catalysis occurs by ribooxocarbenium stabilization from the O5' lone pair and a pyrophosphate oxygen. A dissociative reaction coordinate path is implicated in which the primary reaction coordinate motion is the ribosyl C1' in motion between relatively immobile purine base and (Mg)(2)-pyrophosphate. Several short hydrogen bonds form in the complex of the enzyme and inhibitor. The proton NMR spectrum of the transition-state analogue complex of malarial HGXPRTase contains two downfield signals at 14.3 and 15.3 ppm. Despite the structural similarity to the human enzyme, the NMR spectra of the complexes reveal differences in hydrogen bonding between the transition-state analogue complexes of the human and malarial HG(X)PRTases. The X-ray crystal structures and NMR spectra reveal chemical and structural features that suggest a strategy for the design of malaria-specific transition-state inhibitors.

The structure of human HGPRT bound to the transition-state analog immucillinGP and Mg2+-pyrophosphate has been determined to 2.0 A resolution. ImmucillinGP was designed as a stable analog with the stereoelectronic features of the transition state. Bound inhibitor at the catalytic site indicates that the oxocarbenium ion of the transition state is stabilized by neighboring-group participation from MgPPi and O5'. A short hydrogen bond forms between Asp 137 and the purine ring analog. Two Mg2+ ions sandwich the pyrophosphate and contact both hydroxyls of the ribosyl analog. The transition-state analog is shielded from bulk solvent by a catalytic loop that moves approximately 25 A to cover the active site and becomes an ordered antiparallel beta-sheet.

The proposed transition state for hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) has been used to design and synthesize powerful inhibitors that contain features of the transition state. The iminoribitols (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinHP) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinGP) are the most powerful inhibitors yet reported for both human and malarial HGPRTs. Equilibrium binding constants are >1,000-fold tighter than the binding of the nucleotide substrate. The NMR spectrum of malaria HGXPRT in the Michaelis complex reveals downfield hydrogen-bonded protons. The chemical shifts move farther downfield with bound inhibitor. The inhibitors are lead compounds for species-specific antibiotics against parasitic protozoa. The high-resolution crystal structure of human HGPRT with immucillinGP is reported in the companion paper.

8-(Methoxycarbonyl)octyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (10) was synthesized in 54% yield by regioselective diglycosylation of unprotected mannoside 4, employing the trichloroacetimidate donor 1, followed by debenzoylation. Derivatives of compounds 4 and 10 were used to prepare conjugates containing fluorochromes for the study of carbohydrate-lectin interactions, as well as conjugates with phospholipids for the preparation of liposomes.

A new and simple colorimetric method has been developed for determining activity in vitro against Giardia intestinalis. The microtitre plate assay is based upon the nucleoside hydrolase activity released from G. intestinalis by lysis. Action of the nucleoside hydrolase on the substrate analogue, 4-nitrophenyl beta-D-ribofuranoside (NPR), gives rise to a coloured product which may be determined directly by the change in absorbance. A number of other such nucleoside analogues can be similarly used, but NPR is the preferred substrate, since it gives high enzymic activity at a relatively low substrate concentration. The IC50 values determined using this method for the known anti-giardials metronidazole, tinidazole and furazolidone were consistent with previously published values. The method is simple, does not involve radioisotopes or complex instrumentation, and thus provides a convenient method for screening potential anti-giardial agents.

Genetic defects in human purine nucleoside phosphorylase cause T-cell deficiency as the major phenotype. It has been proposed that efficient inhibitors of the enzyme might intervene in disorders of T-cell function. Compounds with features of the transition-state structure of purine nucleoside phosphorylase were synthesized and tested as inhibitors. The transition-state structure for purine nucleoside phosphorylase is characterized by (1) an elevated pKa at N7 of the purine ring for protonation or favorable H-bond interaction with the enzyme and (2) oxocarbenium ion formation in the ribosyl ring (Kline, P. C., and Schramm, V. L. (1995) Biochemistry 34, 1153-1162). Both features have been incorporated into the stable transition-state analogues, (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol (immucillin-H) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1, 4-imino-D-ribitol (immucillin-G). Both inhibitors exhibit slow-onset tight-binding inhibition of calf spleen and human erythrocyte purine nucleoside phosphorylase. The inhibitors exhibit equilibrium dissociation constants (Ki) from 23 to 72 pM and are the most powerful inhibitors reported for the enzyme. Complete inhibition of the homotrimeric enzyme occurs at one mole of inhibitor per mole of enzymic trimer. Binding of the transition-state inhibitor at one site per trimer prevents inhibitor binding at the remaining two sites of the homotrimer. A mechanism of sequential catalysis at each subunit, similar to that of F1 ATPase, is supported by these results. Slow inhibitor dissociation (e.g., t1/2 of 4.8 h) suggests that these compounds will have favorable pharmacologic properties. Interaction of transition-state inhibitors with purine nucleoside phosphorylase is different from reactant-state (substrate and product analogue) inhibitors of the enzyme which bind equally to all subunits of the homotrimer.

Two classes of phosphatidylinositol manno-oligosaccharides (PIMs) were isolated from each of Mycobacterium bovis AN5 and WAg201 and Mycobacterium smegmatis mc2 155. The deacylated PIMs (dPIMs), were identified as hexasaccharide (dPIM-6) and disaccharide (dPIM-2) species composed of mannose, myo-inositol, glycerol and phosphate residues in the proportions of 6:1:1:1 and 2:1:1:1, respectively. Structural analysis, employing a combination of microanalytical methods, nuclear magnetic resonance spectroscopy, and mass spectrometric techniques established that the sequence of residues within dPIM-6, as shown below, was identical in the three mycobacterial strains investigated.

The synthesis of spiroacetals [3R,5S,6S]-3-benzyloxy-5-methoxy-1,7-dioaxaspiro[5.5]undecane 3 and [3R,5S,6S]-3-methoxy-5-benzyloxy-1,7-dioaxaspiro[5.5]undecane4, where the substituents on the spiroacetal assembly are in a 1,3-diaxial orientation, is described. Epoxidation of unsaturated spiroacetal 5 using dimethyldioxirane showed greater preference for the alpha-epoxide 11 over the beta-epoxide 12. Treatment of the alpha-epoxide 11 with lithium diethylamide in tetrahydrofuran afforded both the allylic alcohol 7 and the homoallylic alcohol 13 in approximately equal amounts. Use of a nonpolar solvent (hexane) suppressed the isomerization of the allylic alcohol 7 to the homoallylic alcohol 13, affording a 21:1 ratio of 7:13. Coordination of an oxygen lone pair with the electron-deficient lithium center of the reagent sets up a transition state in which abstraction of a syn-proton and opening of the epoxide leads to stereoselective formation of allylic alcohol 7 with a pseudoaxial hydroxyl group at C-5. The hydroxyl group liberated in the epoxide rearrangement step was then used to direct a second epoxidation to the lower face of the alkene. Thus, treatment of alcohol 7 with m-CPBA buffered with sodium acetate afforded alpha-syn-epoxy alcohol 9. Subsequent epoxide opening using lithium aluminum hydride proceeded smoothly, affording syn-3,5-diaxial diol 10 and 4,5-diol 22. Epoxy alcohol 9 was then treated with sodium hydride and methyl iodide or benzyl bromide, affording methyl ether 23 or benzyl ether 24, respectively. Reduction of methoxy epoxide 23 with lithium aluminum hydride then afforded alcohol 25 together with the regioisomeric alcohol 27. Benzyloxy epoxide 24 afforded alcohols 29 and 31. Finally benzylation of alcohol 25 afforded bis-ether 3 whereas methylation of alcohol 29 afforded bis-ether 4. Spiroacetals 3 and 4were screened for herbicidal activity and exhibited significant activity against the weeds Avena fatua, Setaria viridis, Amaranthus retroflexus, and Chenopodium album when applied preemergence. Bis-ethers 3 and 4, which contain alkoxy groups anchored in a 1,3-diaxial orientation on a spiroacetal ring, represent the first examples of herbicides based on a spiroacetal ring system.

Protozoan parasites lack de novo purine biosynthesis and require purine salvage from the host. Nucleoside hydrolases are involved in nucleoside salvage and are not found in mammals, making them protozoan-specific targets for inhibitor design. Several protozoan nucleoside hydrolase isozymes with distinct substrate specificities have been characterized. Novel substituted iminoribitols have been synthesized to resemble the transition state structure of the nonspecific inosine-uridine nucleoside hydrolase from Crithidia fasciculata (IU-nucleoside hydrolase). These inhibitors have been characterized for this enzyme and for a purine-specific nucleoside hydrolase (IAG-nucleoside hydrolase) from Trypanosoma brucei brucei. Inhibitors which provide nanomolar inhibition constants for IU-nucleoside hydrolase exhibit micromolar inhibition constants for the IAG-enzyme. For example, p-bromophenyliminoribitol inhibits the IU- and IAG-enzymes with dissociation constants of 28 nM and 190 microM, respectively. Substrate specificity, the action of transition state inhibitors and the pH-dependence of the kinetic constants establish that the catalytic mechanisms and transition state structures are fundamentally different for the IU- and IAG-isozymes. The finding is remarkable since these isozymes share significant homology at the catalytic sites and both use inosine as a preferred substrate. The specificity of the transition state analogues indicates that logically-designed transition state inhibitors are isozyme-specific, with (Km/Ki IU-nucleoside hydrolase)/(Km/Ki IAG-nucleoside hydrolase) values up to 39,000. The mechanism of the differential inhibition is based on the relative leaving group activation and ribosyl-oxocarbenium-forming abilities of these enzymes. In addition to providing isozyme-specific inhibitors, the novel molecules described here have diagnostic value for the nature of the transition states for N-ribohydrolase enzymes.

Two new isomers of allosamidin and allosamizoline, and two dimeric allosamidin analogues have been synthesized. The new compounds have been tested for inhibition of insect chitinase and for toxicity to insect larvae.

Modern chemical and spectroscopic techniques have been used to characterise the polysaccharides extracted from the tetrasporic life stages of Gigartina clavifera and Gigartina alveata. Both are predominantly xi-carrageenans. About one in six of the 3-linked residues in both polysaccharides also have a pyruvate acetal group at the 4- and 6-positions. In addition, a similar proportion of the 4-linked units of each polysaccharide are devoid of sulfate groups, whilst more have sulfate esters on both O-2 and O-6. Some of the 3-linked units contain a sulfate at the 6-position in addition to that at O-2. The polysaccharide from tetrasporophytic G. alveata also contains a small but significant number of 3,6-anhydrogalactosyl units, most of which are naturally sulfated at the 2-position.

The highly complex polysaccharide extracted from the New Zealand red alga Pachymenia lusoria (Grev.) J. Ag. has been characterised and certain structural features defined. A reductive hydrolysis procedure was used for constituent sugar and linkage analyses, with trideuteriomethylation being employed to enable the location of natural methyl ether groups to be determined. A reductive partial-hydrolysis procedure allowed agarobiosyl constituent residues to be identified. The analytical results are consistent with the polymer having a linear backbone of 3-linked D-galactopyranosyl alternating with 4-linked D- or L-galactopyranosyl residues. The 3-linked residues are nearly all 2-sulfated, with 1 in 3 also being 6-O-methylated and 1 in 5 also bearing a 4,6-pyruvic acetal residue. About one-third of the polymer is comprised of blocks of agarobiosyl repeat units that are 2-sulfated on the beta-D-galactopyranosyl and one-third 2-O-methylated on the 3,6-anhydro-L-galactosyl constituents. Of the remaining 4-linked residues, half are 2-O-methyl-D-galactopyranosyl residues and half are galactopyranosyl residues, of which approximately half are in the L configuration.

The structure of the polysaccharide isolated from tetrasporophytic plants of the New Zealand red alga Gigartina decipiens has been determined by chemical and spectroscopic techniques. It is a linear polymer composed primarily of alternating 3-linked beta-D-galactopyranosyl 2-sulphate and 4-linked alpha-D-galactopyranosyl 2,6-disulphate residues. About 15% of the 3-linked residues have an additional sulphate ester group at the 6-position. Aside from this small extra sulphate substitution, the structure is that of the idealised lambda-carrageenan. Good quality solution-state 13C NMR spectra were recorded and interpreted for this carrageenan and for the carrageenans produced from it by solvolytic desulphation and alkali modification.

Commercially available lactase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) enzymes produced from Kluyveromyces fragilis and Kluyveromyces lactis were accessed as catalysts for use in the production of beta-galactopyranosides of various alcohols using lactose as galactosyl donor. The yield of galactoside was enhanced by using the highest practical concentrations of both lactose and alcohol acceptor. The concentrations and thus yield, were limited by the solubility of the substrates. The increase in galactoside yield with increasing lactose concentration appeared to be specific to the lactose substrate and not due to water activity alterations, because addition of maltose to a fixed concentration of lactose had no effect. During the course of the reaction, the yield of galactoside peaked after around 70% to 80% of the lactose was consumed, due to hydrolysis of the product by the enzyme. A wide variety of compounds with primary or secondary hydroxyl groups could act as acceptors, the essential requirement being at least some water solubility. Addition of organic cosolvents had little effect on galactoside yield except when it increased the water solubility of sparingly soluble alcohols. Some galactosides were synthesized on a gram scale to determine practical product recoveries and improve purification methods for large-scale synthesis. Initial purification by hydrophobic chromatography (for galactosides of hydrophobic alcohols) or strong anion-exchange chromatography (for galactosides of hydrophilic alcohols) separated galactosides, galactobiosides, and higher oligomers from reducing sugars. A facile separation of the galactoside and galactobioside could then be effected by flash chromatography on silica gel.

Methods are reported that facilitate the structural characterization of complex sulphated galactans of the red algae. Two procedures have been developed for the production of alditol acetates from carrageenans and agaroids. Both procedures generate 3,6-anhydrogalactitol acetate from the easily destroyed 3,6-anhydrogalactosyl residues in near quantitative yield. The "double hydrolysis-reduction" method involves preliminary hydrolysis under conditions sufficient to cleave all of the 3,6-anhydrogalactosidic bonds, but mild enough to avoid significant further degradation. The "reductive hydrolysis" method uses the acid-stable 4-methylmorpholine-borane to reduce the 3,6-anhydrogalactose end groups as they are released during acid hydrolysis. An alditol acetate sample can be prepared from a polysaccharide in a single tube, ready for g.l.c. analysis, in less than 2.5 h, i.e. more quickly than by any previous procedure. Problems associated with incomplete methylation of sulphated carrageenans and agaroids by the Hakomori procedure have been overcome by first converting the sulphated polysaccharide into its triethylammonium salt form. The reductive hydrolysis method is effective for the production of partially methylated alditol acetates from the methylated polysaccharides, enabling the rapid determination of the substitution pattern of these polysaccharides. These improved analytical methods have been applied successfully to kappa-, iota-, and lambda-carrageenans, as well as some agars.