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Karrikins (2H-furo[2,3-c]pyran-2-ones) are potent smoke-derived germination promoters for a diverse range of plant species but, to date, their mode of action remains unknown. This paper reports the structure-activity relationship of numerous karrikin analogues to increase understanding of the key structural features of the molecule that are required for biological activity. The results demonstrate that modification at the C5 position is preferred over modification at the C3, C4, or C7 positions for retaining the highest bioactivity.

The synthesis of some analogues of O -(2-acetamido-2-deoxy- d -glucopyranosylidene)amino N -phenylcarbamate, PUGNAc, an inhibitor of β- N -acetylglucosaminidases, is described. The analogues were tested against a range of β- N -acetylglucosaminidases to establish any biological activity. As well, the analogues were tested as inhibitors of a uridine diphosphate- N -acetyl- d -glucosamine: polypeptidyl transferase, OGT, a critical protein involved in the post-translational modification of nuclear and cytosolic proteins by N -acetyl- d -glucosamine.

Enzyme inhibition through mimicry of the transition state is a major area for the design of new therapeutic agents. Emerging evidence suggests that many retaining glycosidases that are active on alpha- or beta-mannosides harness unusual B2,5 (boat) transition states. Here we present the analysis of 25 putative beta-mannosidase inhibitors, whose Ki values range from nanomolar to millimolar, on the Bacteroides thetaiotaomicron beta-mannosidase BtMan2A. B2,5 or closely related conformations were observed for all tightly binding compounds. Subsequent linear free energy relationships that correlate log Ki with log Km/kcat for a series of active center variants highlight aryl-substituted mannoimidazoles as powerful transition state mimics in which the binding energy of the aryl group enhances both binding and the degree of transition state mimicry. Support for a B2,5 transition state during enzymatic beta-mannosidase hydrolysis should also facilitate the design and exploitation of transition state mimics for the inhibition of retaining alpha-mannosidases--an area that is emerging for anticancer therapeutics.

Four methods have been screened for the synthesis of some alpha-D-glucopyranosides, with the recently reported (Mukaiyama) combination of 2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl iodide and triphenylphosphine oxide being the most successful, especially in the diastereoselectivity exhibited. The alpha-D-glucopyranosides so obtained have been deprotected to yield 2-, 3-, 4- and 6-O-alpha-D-glucopyranosyl-D-galactopyranose. Only the last disaccharide showed any hydrolysis by alpha-glycosidases but this success was not emulated by mucosal extracts from the small intestine of the pig.

The inhibition of glycoside hydrolases, through transition-state mimicry, is important both as a probe of enzyme mechanism and in the continuing quest for new drugs, notably in the treatment of cancer, HIV, influenza, and diabetes. The high affinity with which these enzymes are known to bind the transition state provides a framework upon which to design potent inhibitors. Recent work [for example, Bülow, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568; Zechel, D. L. et al. J. Am. Chem. Soc. 2003, 125, 14313-14323] has revealed quite confusing and counter-intuitive patterns of inhibition for a number of glycosidase inhibitors. Here we describe a synergistic approach for analysis of inhibitors with a single enzyme 'model system', the Thermotoga maritima family 1 beta-glucosidase, TmGH1. The pH dependence of enzyme activity and inhibition has been determined, structures of inhibitor complexes have been solved by X-ray crystallography, with data up to 1.65 A resolution, and isothermal titration calorimetry was used to establish the thermodynamic signature. This has allowed the characterization of 18 compounds, all putative transition-state mimics, in order to build an 'inhibition profile' that provides an insight into what governs binding. In contrast to our preconceptions, there is little correlation of inhibitor chemistry with the calorimetric dissection of thermodynamics. The ensemble of inhibitors shows strong enthalpy-entropy compensation, and the random distribution of similar inhibitors across the plot of DeltaH degrees a vs TDeltaS degrees a likely reflects the enormous contribution of solvation and desolvation effects on ligand binding.

Gallium and indium complexes with pendant carbohydrates have been prepared and examined for their potential as radiopharmaceuticals. Carbohydrate-bearing 3-hydroxy-4-pyridinone ligand precursors and their tris(ligand)gallium(III) and -indium(III) complexes were synthesized and characterized by mass spectrometry, elemental analysis, and (1)H and (13)C NMR spectroscopy, and in the case of one intermediate, by X-ray crystallography. With three equivalents of ligand, neutral complexes formed with the bidentate hydroxypyridinone moiety complexing the gallium(III) and indium(III) metal centers.

One of the most intriguing features of the 90 glycoside hydrolase families (GHs) is the range of specificities displayed by different members of the same family, whereas the catalytic apparatus and mechanism are often invariant. Family GH26 predominantly comprises β-1,4 mannanases; however, a bifunctional Clostridium thermocellum GH26 member (hereafter CtLic26A) displays a markedly different specificity. We show that CtLic26A is a lichenase, specific for mixed (Glcβ1,4Glcβ1,4Glcβ1,3)n oligo- and polysaccharides, and displays no activity on manno-configured substrates or β-1,4-linked homopolymers of glucose or xylose. The three-dimensional structure of the native form of CtLic26A has been solved at 1.50-A resolution, revealing a characteristic (β/α)8 barrel with Glu-109 and Glu-222 acting as the catalytic acid/base and nucleophile in a double-displacement mechanism. The complex with the competitive inhibitor, Glc-β-1,3-isofagomine (Ki 1 μm), at 1.60 A sheds light on substrate recognition in the –2 and –1 subsites and illuminates why the enzyme is specific for lichenan-based substrates. Hydrolysis of β-mannosides by GH26 members is thought to proceed through transition states in the B2,5 (boat) conformation in which structural distinction of glucosides versus mannosides reflects not the configuration at C2 but the recognition of the pseudoaxial O3 of the B2,5 conformation. We suggest a different conformational itinerary for the GH26 enzymes active on gluco-configured substrates.

Some attempts toward the synthesis of novel inhibitors of glycosyl transferases are described. More successfully, the synthesis of an activated cyclopropacyclohexene and an amide and an amine of a cyclopropa‐fused pyranose are described. None of these three novel compounds proved to be a significant inhibitor of a retaining α‐glucosidase from barley. † This paper is dedicated to the memory of Jacques H. van Boom.

The treatment of various glycosyl acceptors, each containing a reactive thiol group, with the appropriate glycosyl donor and a glycoside hydrolase or glycosynthase, failed to yield any thioglycosides––only the products of O-glycosylation were formed. However, thioglycosides were formed when a thioglycoligase was used to mediate the reaction between acceptor and donor. In fact, pyranose acceptors possessing a thiol group at C3, C4 or C6 (but not C2) were all capable of conversion into thioglycosides. Some comment is given regarding the mechanism of the various processes.

The understanding of transition state mimicry in glycoside hydrolysis is increasingly important both in the quest for novel specific therapeutic agents and for the deduction of enzyme function and mechanism. To aid comprehension, inhibitors can be characterized through kinetic, thermodynamic, and structural dissection to build an "inhibition profile." Here we dissect the binding of a tetrahydrooxazine inhibitor and its derivatives, which display Ki values around 500 nm. X-ray structures with both a beta-glucosidase, at 2 A resolution, and an endoglucanase at atomic (approximately 1 A) resolution reveal similar interactions between the tetrahydrooxazine inhibitor and both enzymes. Kinetic analyses reveal the pH dependence of kcat/Km and 1/Ki with both enzyme systems, and isothermal titration calorimetry unveils the enthalpic and entropic contributions to beta-glucosidase inhibition. The pH dependence of enzyme activity mirrored that of 1/Ki in both enzymes, unlike the cases of isofagomine and 1-deoxynojirimycin that have been characterized previously. Calorimetric dissection reveals a large favorable enthalpy that is partially offset by an unfavorable entropy upon binding. In terms of the similar profile for the pH dependence of 1/Ki and the pH dependence of kcat/Km, the significant enthalpy of binding when compared with other glycosidase inhibitors, and the tight binding at the optimal pH of the enzymes tested, tetrahydrooxazine and its derivatives are a significantly better class of glycosidase inhibitor than previously assumed.

The design and synthesis of transition-state mimics reflects the growing need both to understand enzymatic catalysis and to influence strategies for therapeutic intervention. Iminosugars are among the most potent inhibitors of glycosidases. Here, the binding of 1-deoxynojirimycin and (+)-isofagomine to the "family GH-1" beta-glucosidase of Thermotoga maritima is investigated by kinetic analysis, isothermal titration calorimetry, and X-ray crystallography. The binding of both of these iminosugar inhibitors is driven by a large and favorable enthalpy. The greater inhibitory power of isofagomine, relative to 1-deoxynojirimycin, however, resides in its significantly more favorable entropy; indeed the differing thermodynamic signatures of these inhibitors are further highlighted by the markedly different heat capacity values for binding. The pH dependence of catalysis and of inhibition suggests that the inhibitory species are protonated inhibitors bound to enzymes whose acid/base and nucleophile are ionized, while calorimetry indicates that one proton is released from the enzyme upon binding at the pH optimum of catalysis (pH 5.8). Given that these results contradict earlier proposals that the binding of racemic isofagomine to sweet almond beta-glucosidase was entropically driven (Bülow, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568), we reinvestigated the binding of 1-deoxynojirimycin and isofagomine to the sweet almond enzyme. Calorimetry confirms that the binding of isofagomine to sweet almond beta-glucosidases is, as observed for the T. maritima enzyme, driven by a large favorable enthalpy. The crystallographic structures of the native T. maritima beta-glucosidase, and its complexes with isofagomine and 1-deoxynojirimycin, all at approximately 2.1 A resolution, reveal that additional ordering of bound solvent may present an entropic penalty to 1-deoxynojirimycin binding that does not penalize isofagomine.

An endoglucanase from Humicola insolens has been used to glycosylate a range of alkenyl β- d -C- glucopyranosides with β-lactosyl fluoride. The resulting trisaccharides have been subjected to the action of a commercial β-galactosidase to form alkenyl β-C-cellobiosides. Oxidation of these has given a range of epoxyalkyl β-C-cellobiosides, putative inhibitors of cellobiohydrolases.

The treatment of derivatives of 1,6-epithio- and 1,6-episeleno-β- d -glucopyranose with various carbohydrate alcohols in the presence of N -iodosuccinimide/triflic acid gives rise to disulfides and diselenides, respectively, which may be transformed to the desired 6-deoxy-β- d -glucopyranosides.

‘β-Adiposin-2’, the 6″-hydroxylated derivative of ‘β-acarbose’, itself a diastereoisomer of the naturally occurring acarbose, has been prepared from a 1-epivalienamine derivative, 1,6:3,4-dianhydro-2-O-benzyl-β-d-galactose and benzyl 2,3,6,2′,3′,6′-hexa-O-benzyl-β-cellobioside.

A series of nine diethyl phosphonates were prepared and an analysis of their H-1 NMR spectra was undertaken. Some erroneous literature assignments of signals were revised.

Five trimethylarsonioribosides were prepared from naturally occurring and synthetic dimethylarsinylribosides (arsenosugars) by reducing them with 2,3-dimercaptopropanol and quaternizing the resultant arsine with methyl iodide. The trimethylarsonioribosides prepared in this manner were the four novel compounds methyl 5-deoxy-5-trimethylarsonio-β-D-riboside (as the iodide), (2′R)-2′, 3′-dihydroxypropyl 5-deoxy-5-trimethylarsonio-β-D-riboside (as the formate), 3′-[(2″, 3″ -dihydroxypropyl)hydroxyphosphinyloxy] - 2′ -hydroxypropyl 5-deoxy-5-trimethylarsonio-β-D-riboside and 3-(5′-deoxy-5′-trimethylarsonio-β-D-ribosyloxy)-(2S)-2-hydro xypropanesulfonate, and the known (2′S)-2′-hydroxy-3′-(sulfooxy)propyl 5-deoxy-5-trimethylarsonio-β-D-riboside. They were synthesized to serve as standards for chromatographic analyses of arsenic compounds in marine samples and for investigations into the biotransformation of arsenic in marine organisms. NMR spectral and chromatographic data for the five trimethylarsonioribosides are presented and compared with those of their dimethylarsinyl analogues.

Compounds (I) and (II), deoxyiminosugar equivalents of arabinobiose and arabinotriose, resp., are synthesized and evaluated for their arabinanase inhibitory activity.

2-Phenyl- 1,3-benzodithiolylium trifluoromethanesulfonate, easily prepared from 1,2-benzenedithiol, converts alcohols into dithioorthoesters and, ultimately, benzyl ethers (Bu3SnH) and benzoates (HgO/HBF4).

A variety of approaches towards the synthesis of lincosamine, a derived portion of the antibiotic lincomycin, are reported. Initial approaches involved the intramolecular delivery of a nitrogen atom (trichloroacetimidate, trichloroacetylcarbamate, carbamate, 2-amino-2phenylacetate) attached to 0 4 onto C6 of a 6,7-anhydrooctoside. Later approaches, albeit more direct but again largely unsuccessful, involved the Sharpless titanium(1v)-mediated nucleophilic opening of a suitable 6,7-anhydrooctose, and the Sharpless oxyamination and the aziridination of suitable octenoses. As an aid to the structure elucidation of several compounds encountered in this work, single-crystal X-ray structure determinations are reported for methyl 6,7-anhydro-2,3-diO-benzyl-8-deoxy-oc-~-threo-~-galacto-octopyranoside, methyl 6,7-anhydro-2,3-di-0-benzyl-8deoxy-a-D-threo-D-gluco-octopyranoside and 7-azido-7-deoxy-l,2:3,4-d1-0-isopropylidene-/3-~erythro-D-galacto-octose. In the preceding paper we outlined the syntheses of various octenoses (1)-(4) as potential substrates for the synthesis of lincosamine (3, a derived portion of the antibiotic lincomycin.' This paper describes firstly our attempts to convert the alcohol (I), via an intermediate such as (6), into lincosamine ( 9 , and concludes with approaches from the alkenes (2)-(4) to the same target molecule. For the synthesis of an intermediate such as (6) we needed to prepare initially the epoxide (7), and two routes were available. Epoxidation of the alkene (8) followed by removal of the protecting group at 0 4 could formally lead to Stick, R. V., and Tilbrook, D. M. G., Aust. J. Chem., 1990, 43, 1643. 0004-9425/90/101657$03.00 L. M. Engelhardt et aL (7), whereas epoxidation of the alkene (I) , taking advantage of the possible directing effect of the hydroxy group at ~ 4 , ~ could lead directly to the same epoxide (7). Thus, treatment of the alkene (8) with m-chloroperbenzoic acid gave a mixture of two epoxides which were separable by flash chromatography. The major product, formed in 57% yield, was the unwanted P-L-threo epoxide (9); the required a-D-threo epoxide (10) was obtained in only 17% yield. The assignment of structure to (9) and (10) was based on 13c n.m.r. spectroscopy and comparison with related epoxides (see below): for (9), C6 resonated at 6 54 .39 and C7 at 57.28, whereas the corresponding resonances in (10) were at 51.18 and 5 8 . 7 0 . ~ The same oxidation (m-chloroperbenzoic acid) of the alkene (1) yielded an inseparable mixture of the two possible epoxides (1 1) and (7), in 58% yield and a ratio of 7 : 3. The 13c n.m.r. spectrum was consistent with the structures for (1 1) (6 54 .0 , C 6 ; 57.2, C 7) and (7) (6 51 . 3 , C 6 ; 58.6

Treatment of methyl 2,3-anhydro-4,6-O-benzylidene-α-D-alloside with the anion derived from trimethyl(prop-1-ynyl)silane, allylmagnesium chloride or isobutenylmagnesium chloride introduces a three- or four- carbon substituent at C2 of the sugar. In each case, a hydroboration-oxidation sequence helps convert the centre of unsaturation in the new substituent into a carboxylic acid residue. Subsequent manipulations allow the introduction ofanother carbon at C6 of the sugar to give a highly functionalized precursor to the C1-C9 fragment of tylonolide, the aglycon of the antibiotic tylosin. Attempts at the introduction of an axial methyl group at C4 of the extended sugar also described.

Methyl (E)-2,3-di-O-benzyl-6,7,8-trideoxy-α-D-galacto-oct-6-enopyranoside, a potential precursor to lincosamine, has been prepared from both methyl α-D-galactopyranoside and methyl α-D-glucopyranoside, with the required inversion at C4 in the latter sequence being effected by a conventional oxidation-reduction. As well, (E)-6,7-dideoxy-1,2: 3,4-di-O-isopropylidene-α-D-galacto-oct-6-enose and (E)- and (Z)-6,7,8- trideoxy-1,2:3,4-di-O-isopropylidene-α-D-galacto-oct-6-enose have been prepared from 1,2: 3,4-di-O-isopropylidene-α-D-galactose.

The kidney of the giant clam Tridacna maxima contains algal arsenic compounds, probably as a consequence of the clam's symbiotic relationship with unicellular algae (zooxanthallae). Extraction of Tridacna kidneys yielded three novel arsenic compounds, N-(5′-deoxy-5′-dimethylarsinoyl-β-D-ribosyloxycarbonyl)glycine 5, (2S)-3-(5′-deoxy-5′-dimethylarsinoyl-β-D-ribosyloxy)-2-hydroxypropanoic acid 4a, and (2R)-3-(5′-deoxy-5′-dimethylarsinoyl-β-D-ribosyloxy)-2-hydroxypropanoic acid 4b, in addition to four previously reported dimethylarsinoylribosides. The structures of the three new Compounds were assigned chiefly from NMR data, and those for compounds 4a and 4b were confirmed by synthesis. Extraction of a second batch of Tridacna kidneys gave, in addition to compounds identified in the first extraction, (2S)-3-[(5′-deoxy-5′-trimethylarsonio-β-D-ribosyloxy)-2-hydroxypropyl]sulfate 18 and two novel compounds: an arsenic-containing nucleoside, 9-(5′-deoxy-5′-dimethylarsinoyl)-9H-adenosine 16 and N-[4-(dimethylarsinoyl)butanoyl]taurine 21. Syntheses of compounds 16 and 21 are reported. The presence of the nucleoside 16 in Tridacna, as a consequence of algal metabolism, supports a proposed pathway for the biogenesis of arsenic-containing ribosides by algae involving methylation and adenosylation by S-adenosylmethionine. A biogenetic pathway for compound 21 involving donation of the 3-amino-3-carboxypropyl moiety of S-adenosylmethionine is also proposed. The presence of both compounds 16 and 21 in Tridacna may represent the first example of donation, by S-adenosylmethionine, of all three of its alkyl groups to a single acceptor (arsenic) within one organism.

Procedures are described for the attempted conversion of the pyrrolidine (2) into a bicyclic system that would ultimately lead to retronecine (3). The only molecule to be isolated from these attempts was the epimeric ketone (6), and AM1 and MM2 calculations were used to probe this apparently favoured isomerization. Alternative procedures were then investigated that sought to convert the pyrrolidine (1) into precursors to retronecine, and notable successes were obtained with the addition of the amine (21) to both nitroethene and ethyl 2-diethylphosphonoacrylate. However, not only were these precursors resistant to further transformations into retronecine , but also the problem of epimerization again arose. Consequently, AM1 calculations were again employed, and an X-ray structure determination of ethyl (7R,8R)-7-benzyloxy-1-benzyloxymethyl-5,6,7,8-tetrahydro-3H-pyrrolizine-2-carboxylate (39) is reported. Attempts to convert (39) into a diastereoisomer of retronecine were unsuccessful.

Methyl β-D-galactopyranoside was converted into methyl 4-azido-3-O-benzyl-6-O-t-butyldiphenylsilyl-2,4-dideoxy-β-D-arabino-hexoside, but subsequent transformation of the anomeric centre into a dithioacetal was unsatisfactory. Alternatively, methyl β-D- galactopyranoside easily gave methyl 4-azido-3,6-di-O-benzyl-2,4-dideoxy-β-D-arabino-hexoside, and this could be transformed by a reductive amination into two pyrrolidines as potential precursors to retronecine. Related chemistry gave (4S,5S,6R)-6-benzyloxy-4-benzyloxymethyl-3-oxa-1-azabicyclo[3.3.0]octan-2-one, the structure of which was confirmed by a single-crystal X-ray structure determination. Finally, the synthesis of a potential precursor to heliotridine (an epimer of retronecine ), namely methyl 4-azido-3-O-benzyl-6-O-t-butyldiphenylsilyl-2,4-dideoxy-α-D-ribo-hexoside, from methyl α-D- mannopyranoside is reported.

Treatment of 1,3,4,6-tetra-O-acetyl-2-(o-carboxybenzoylamino )-2-deoxy-β-D-glucose with ethyl chloroformate , followed by workup and treatment with methanol, did not give 1,3,4,6-tetra O-acetyl-2-deoxy-2-phthalimido-β-D-glucose as reported in the literature, but rather the methyl ester 1,3,4,6-tetra-O-acetyl-2-deoxy-2-(o- methoxycarbonylbenzoylamino )-β-D-glucose. The formation of this methyl ester is believed to proceed via 1,3,4,6-tetra-O-acetyl-2-deoxy-2- [(3′-oxo-1′,3′-dihydroisobenzofuran-1′-ylidene)amino]-β-D-glucose which was also formed from the amido carboxylic acid by treatment with dicyclohexylcarbodiimide . Optimal procedures are given for the preparation of the title compound 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucosyl trichloroacetimidate.

Five trimethylarsonioribosides were prepared from naturally occurring and synthetic dimethylarsinylribosides (arsenosugars) by reducing them with 2,3-dimercaptopropanol and quaternizing the resultant arsine with methyl iodide. The trimethylarsonioribosides prepared in this manner were the four novel compounds methyl 5-deoxy-5-trimethylarsonio-β-D-riboside (as the iodide), (2′R)-2′, 3′-dihydroxypropyl 5-deoxy-5-trimethylarsonio-β-D-riboside (as the formate), 3′-[(2″, 3″ -dihydroxypropyl)hydroxyphosphinyloxy] - 2′ -hydroxypropyl 5-deoxy-5-trimethylarsonio-β-D-riboside and 3-(5′-deoxy-5′-trimethylarsonio-β-D-ribosyloxy)-(2S)-2-hydro xypropanesulfonate, and the known (2′S)-2′-hydroxy-3′-(sulfooxy)propyl 5-deoxy-5-trimethylarsonio-β-D-riboside. They were synthesized to serve as standards for chromatographic analyses of arsenic compounds in marine samples and for investigations into the biotransformation of arsenic in marine organisms. NMR spectral and chromatographic data for the five trimethylarsonioribosides are presented and compared with those of their dimethylarsinyl analogues.

The treatment of hexopyranosyl bromides, also activated at C6 (Br, OTs, OMs), with H2S/HCONMe2 under basic conditions gives rise to 1,6-dideoxy 1,6-epithio sugars. One such sugar has been further transformed into the synthetically useful 3,4-anhydro-1,6-dideoxy-1,6-epithio-β-D-galactose. The treatment of this epoxide with sodium azide and with cyclohexylamine is described. An analogous treatment of one doubly activated hexopyranosyl bromide with sodium hydrogen selenide has led to a novel 1,6-dideoxy 1,6-episeleno sugar which displayed interesting n.m.r. spectra. Finally, in an attempt to prepare 1,6-dideoxy 1,6-epidithio sugars, a tetraalkylammonium tetrathiomolybdate reagent was found to be the reagent of choice for converting doubly activated hexopyranosyl bromides into 1,6-dideoxy 1,6-epithio sugars.

A fluorescent analogue of cholesteryl oleate , namely 22-[ethyl(2'-naphthyl)amino]-23,24-dinorchol-5-en-3β-yl oleate, has been prepared from a steroidal carboxylic acid, 2-naphthylamine and oleic acid.

β-D-Glucosidases and β-D-glucan hydrolases are two important classes of enzymes that are responsible for various processes that occur in plants and bacteria. Here we describe our efforts, over the past decade, towards the design and synthesis of potential inhibitors of these enzymes. In particular, approaches are described to epoxyalkyl glycosides, deoxy halo epoxides, aziridines, thiirans and epoxyalkyl C-glycosides, all potentially irreversible, mechanism-based inhibitors of the above enzymes. Our efforts towards tetrahydro-l,2-oxazines are also included. Some time is devoted to the concept of “β-acarbose”, a diastereoisomer of the naturally occurring acarbose, as a novel, competitive inhibitor of the above enzymes. Throughout this work, attention was continually directed to the synthesis of potential inhibitors in optically-pure form.