CA2209663A1 - Compositions and methods for inhibiting fungal cell wall formation - Google Patents

Abstract

Disclosed herein are antifungal compositions having an oligosaccharide that includes an N-acetylhexosamine residue linked .beta.(14) to a hexose and a biologically acceptable carrier. The isolation of the fungal enzyme chitin glucan .beta.(14) transferase (CG.beta.(14)T) is disclosed along with methods for using this enzyme in an assay for antifungal agents. CG.beta.(14)T
catalyses the formation of a .beta.(14) linkage between the terminal reducing N-acetylglucosamine residue of chitin and the non-reducing glucose residue of .beta.(13) linked glucans. Inhibitors of the enzyme, antifungals including such inhibitors, methods for the prevention and the treatment of fungal infections in animals and plants, and screening methods for identifying antifungals are also disclosed.

Description

WO 96/20717 . ' PCT/US96/00752 COMPOSITIONS AND METHODS FOR INHIBITING FUNGAL CE~L WALL
FORMATION

Field of the Invention This invention pertains to antifungal compositions and to methods for using such compositions in the prevention and treatment of fungal infections in plants and ~n~m~ls, The invention also encompasses the enzyme that catalyzes the formation of certain fungal cell wall oligosaccharides that are formed by a specific covalent linkage between chitin and glucans.

Background of the Invention Fungi are ubiquitous eukaryotic organisms of varying size and morphology. Fungi typically grow in two basic forms, yeasts and molds. Yeasts are single cell organisms that~are usually spherical or ellipsoidal in shape and that generally reproduce by budding. Molds are multicellular organisms that generally form filamentous colonies. Examples of fungi include monomorphic yeasts and yeast-like organisms, including Candida, Cryptococcus, and Saccharomyces; mo-nomorphic molds, such as Aspergillus and Coccidioides; and thermally dimorphic fungi, such as Blastomyces dermatitidis and Histoplasma capsulatum, which grow either in a yeast or a mold phase.
Fungal cell walls determine the shape of fungal cells and are essential for fungal integrity. The cell walls include three types of structural polysaccharides: glucans (polymers of glucose (Glc) containing ~(1~3) and ~(1 6) linkages~, mAnn~n~ (primarily glycoproteins with attached mannose ch~;n~), and chitin, Chitin is a linear polymer of N-acetylglucosamine (GlcNAc) residues joined by ~(1~4) linkages. It is scattered throughout the cell wall, although it is mainly concentrated at the septal region. Because chitin is concentrated at the S13BSllTUrE SHEET ~RULE 26) W096/20717 . PCT~S96tO0752 septal region, it is particularly instrllmPnt~l in the reproduction of fungi by budding. The amount of chitin in the dry weight of the cell wall varies according to the fungal species. For example in S. cerevisiae, chitin comprises only 1-2~ by weight, whereas in Sclerotium rolfsii, it may comprise up to 61~.
The architecture of the fungal cell wall is defined by the organization of these cell wall constituents as.well as by the constituents themselves. Several studies have suggested that different cell wall polysaccharides are covalently linked and particularly that chitin may be covalently linked to the glucan component of the yeast cell wall. Mol et al. (F.E.M.S.
Microbiol. ~etts., 41:95-99, 1987) disclose experiments in S.
cerevisiae in which treatment of alkali-insoluble glucan with chitinase rendered the material alkali-soluble, suggesting that covalent linkage of glucan to chitin was responsible for its initial alkali insolubility. Sietsma et al. (J. Gen.
Microbiol., 114:99-108, 1979) and Surarit et al (~. Gen.
Microbiol. 134:1723 1730, 1988) studied Schizophyllum co~m7 and~ Candida albicans, respectively, by digesting the cell walls with both ~(1~3) glucanase and chitinase. Sietsma et al.
suggest a model in which amino acids are involved in the linkages between chitin and glucan. Surarit et al. conclude that the linkage involves a $(1~6) linkage between carbon-6 of GlcNAc and carbon-1 of glucose.
Many fungi play an indi~pensable role in the cyclic transformation of organic matter, such as for example, in food and drug production. A broad range of fungi are frequent causes of diseases in plants, however. An historical example of a fungal plant disease that had extensive-adverse effects on man is the potato blight of the nineteenth century which led to the starvation of over one million people.
Fortunately, of the thousands of known species of fungi, only a relatively small number cause diseases in ~n;m~lS Most of these fungi are opportunistic pathogens, producing serious disease only in compromised individuals.
However, because of an aging population and an increase in the number of ;mmllnocompromised patients, such as those afflicted ~D;:~ I 1 1 ~TE S~T ~I~E 26~

-WO 96/20717 ~ ~ PCI~/US''''t~7:~2 with acquired ;mm~lno~eficiency syndrome (AIDS), patients undergoing cancer and corticosteroid therapy, and organ transplant recipients, fungal infections are increasing rapidly.
The major fungal ~n; m~ 1 pathogens in North America are Histoplasma capsulatum, Coccidioides innitis, Blastomyces dermatitidis, C~yptococcus neoformans, Candida species and Aspergillus species (Medically I~portant Fhngi, Second Edition, Davise H. Larone, Ed., American Society for Microbiology, W~sh;ngton, D.C.).
Development of effective methods and compositions for the prevention and the treatment of fungal infections is a critical goal of the agricultural and ~rm~ceutical industries. It has now been discovered that, in fungal cell walls, two saccharide-based constituents are cross-linked in a ~(1 4) covalent bond. The term; n~l reducing residue of a fungal chitin chain N-acetylglucosamine is covalently linked by a ~(1~4) linkage to the non-reducing end of a ~(1~3) glucan chain. Compositions incorporating these oligosaccharides or related compounds have antifungal properties in plants and ;3n;m~1 S.

Brief Description of the Drawin~s Figure 1 is an illustration of the elution profile from a Bio-Gel P-2 column of a yeast wall fraction solubilized by digestion with glucanase and chitinase.
Figure 2 is an illustration of the elution profile from a Bio-Gel P-2 column of compounds I, II, and III (Peaks A, B, and C of Figure 1, respecti~ely) after acid hydrolysis.
Figure 3a is an illustration of the mass spectrometry det~rm;n~tion of the molecular weight of compound I.
Figure 3b is an illustration of the mass spectrometry determ;n~tion of the molecular weight of compound II.
Figure 3c is an illustration of the mass spectrometry determ;n~tion of the molecular weight of compound III.
Figure 4a is an illustration of the elution profile from a Bio-Gel P-2 column of compound I after treatment with the 50 ~l of 100 mM citrate-phosphate buffer, pH 5Ø

~ TE S~EET (~LE 26~

WO 96/20717 ' ~ PCT/US96/00752 Figure 4b i8 an illustration of the elution profile from a Bio-Gel P-2 column of compound I after treatment with B-N-acetylglucos~m; n; dase.
Figure 4c is an illustration of the elution profile from a Bio-Gel P-2 column of compound I after treatment with ~-glucosidase.
Figure 5a i8 an illustration of the profile from paper chromatography of the products of a partial acid digestion of compound I.
Figure 5b is an illustration of the profile from paper chromatography of the products of enzymatic digestion of compound I.
Figure 6 is an illustration of the HPAEC profile of the products of periodate oxidation of the trisaccharide resulting from N-acetylglucosaminidase digestion of compound I.
Figure 7 is an illustration of the NMR spectra of compound I, l~m;n~ritriitol, and l~m;n~ribiitol.
Figure 8a is an illustration of the profile from paper chromatography of compound II after digestion with B-glucosidase.
Figure 8b is an illustration of the profile from paper chromatography of compound III after digestion with $-glucosidase.
Figure 9 is a graphic illustration of a'scheme for the generation of different oligosaccharides by chitinase digestion.
Figure 10 is an illustration of the profile from paper chromatography of a diacetylchitobiitol peak.
Figure lla is an illustration of the elution profile from a Bio-Gel P-2 column of material separated from diacetylchitobiitol by paper chromatography.
Figure llb is an illustration of the elution profile from a Bio-Gel P-2 column of the material separated from diacetylchitobiitol after treatment with B-N-acetylgluco~m; n; dase.

SlJBSm~E SHEI~ ULE 26) W096/tO717 . PCT~S96/00752 Figure llc is an illustration of the elution profile from a Bio-Gel P-2 column of the material separated from diacetylchitobiitol after treatment with ~-glucosidase.
Figure lld is an illustration of the elution profile from a Bio-Gel P-2 column of the material separated from diacetylchitobiitol after treatment with ~-N-acetylglucosaminidase and B-glucosidase.
Figure 12a is an illustration of the elution profile from a Bio-Gel P-2 column of a pentasaccharide which elutes with triacetylchitotriitol.
Figure 12b is an illustration of the elution profile from a Bio-Gel P-2 column of the pentasaccharide separated in Figure 12a after treatment with B-N-acetylgluco~m; n; dase.
Figure 12c is an illustration of the elution profile from a Bio-Gel P-2 column of the pentasaccharide separated in Figure 12a after treatment with B-glucosidase.
Figure 13a is an illustration of the elution profile from a Bio-Gel P-2 column of borotritide-reduced yeast cell walls after endoglucanase treatment and second borotritide reduction.
Figure 13b is an illustration of the elution profile from a Bio-Gel P-2 column of borotritide reduced yeast cell walls after endoglucanase treatment.
Figure 14a is an illustration of the elution profile from a Bio-Gel P-2 column of wild type strain D3C cell walls after treatment with endoglucanase, reduction, and incubation with chitinase.
Figure 14b is an illustration of the elution profile from a Bio-Gel P-2 column of strain ECY36-3C (chsl chs2::hEU2) cell walls after treatment with endoglucanase, reduction, and incubation with chitinase.
Figure 14c is an illustration of the elution profile from a Bio-Gel P-2 column of strain ECY36-3D (chsl call/csd2) cell walls after treatment with endoglucanase, reduction, and incubation with chitinase.
Figure 14d is an illustration of the elution profile from a Bio-Gel P-2 column of wild type strain ECY36-3D pHV9a S~ JTE SHEET (I~ULE 26) WO 96/20717 . PCT/US~ 2 cell walls after treatment with endoglucanase, reduction, and incubation with chitinase.
Summary of the Invention Compositions having antifungal properties in plants and ~n;m~l S are provided. These compositions comprise:
(a) an antifungal effective amount of (i) an oligosaccharide comprising an N-acetylhexosamine residue linked ~(1~4) to a ~lexose, (ii) an inhibitor of an enzyme having as a first substrate, the t~rm; n~ 1 reducing N-acetylglucosamine residue of chitin and having as a second substrate the non-reducing glucose residues of (B1 3) linked glucans, said enzyme catalyzing the formation of a (B1 4) linkage between said N-acetylglucosamine and said glucose, or (iii) any combination thereof; and (b) a biologically acceptable carrier.
Another aspect of the invention involves the isolation and identification of the fungal enzyme chitin glucan B(1 4) transferase (CGB(1~4)T). This enzyme catalyzes the formation of a B(1 4) linkage between the term;n~l reducing GlcNAc residue of chitin and the non-reducing glucose residue of B(1~3) linked glucans.
The invention also includes me~hods for the prevention and the treatment of fungal infections in ~n;m~l S
and plants. In such methods, prophylactically or therapeutically effective amounts of the antifungal compositions of the invention are ~min;~tered to the subject plant or ~n;m~l as a prophylactic measure or for the treatment of an existing fungal disease. High-throughput screening methods for identifying CG~(1~4)T inhibitors are also provided.

Detailed Description of the Invention The present invention includes several different types of antifungal compositions as well as methods, which may include the use of the enzyme CG~(1~4)T, for identifying such compositions. Preferred antifungal compositions comprise (a) an antifungal effective amoun~ of WO 96/20717 , PCT/US96tO07S2 (i) an oligosaccharide having an N-acetylh~osamine residue linked ~(1~4) to a hexose, (ii) an inhibitor of CG$(1~4)T, or (iii) any combination thereof; and ~ 5 (b) a biologically acceptable carrier.

N-Acetylhexosamine ~(1~4) Hexose Oligosaccharides An oligosaccharide is a carbohydrate that i8 made up of 2-10 monosaccharide units, any of which s y be the same or different. (Grant ~ Hackh's Chemical Dictionary, 5th Ed., McGraw Hill Book Co., (1987)). The present inventors have discovered that the cell wall of fungi, and particularly of S.
~ visiae yeast includes a specific covalent cross-linkage between chitin rh~;n~ and glucans. In the f~ngal cell wall, tPrm;nAl reducing N-acetylgluco~m;ne residues of chitin are linked ~(1~4) to the non-reducing glucose residues of ~(1~3) glucan rh~;n~. The oligosaccharides formed from N-acetylhexosamine residues linked ~(1~4) to a hexose are useful in antifungal compositions. Preferably, the hexose in either or each constituent of the oligosaccharide is glucose.

Chitin-Glucan $(1 4) Transferase (CG~(1~4)T) A particular enzyme or class of enzymes (CG~(l 4)Ts) catalyzes the formation of a B(1 4) linkage between an N-acetylhexosamine and a hexose and particularly between N-acetylglucosamine and glucose. The enzyme (whose identification is described below) can be used in the identification of antifungal agents and in the design of antifungal agents. The sugar moieties that are substrates of the enzyme(s) may be single residues or may be present a~ part of oligosaccharides as in, for example, chitin and glucan.
This enzyme is found in S. cerevisiae, and this enzyme or homologues thereof are found in other fungal species including, but not limited to, Candida, Aspergillus, Histoplasma, Cryptococcus, and Coccidioides specieC.
The enzyme(s) may be identified and isolated biochemically. An assay is devised to measure CG~(1 4)T
enzymatic activity in a quantitative manner. The assay JTE SHEEr (F~ULE 28) WO 96/20717 ~ . PCT/US96/00752 preferably includes a mixture of chitin (Sigma Chemical Co., St. Louis, MO) and in vivo-labeled glucan or l~m;n~rin (prepared by growing yeast cells in the presence of [3H] or [l~C] glucose and isolating alkali-soluble glucan according to Bowers, B. et al., J. Bacteriol. 119:564-575, 1974), that is exposed to the enzyme. The product of the reaction is then digested with Zymolyase 100 T (Seikagaku America, Inc.
Rockville, MD), precipitated, and quantified. The source of the CG~(1~4)T enzyme is a whole-cell extract of the fungal species studied, such as, for example, S. cerevisiae, produced by bead beating as described for glucan synthase (Kang et al.
Proc. Natl. Acad. Sci, USA, 83:5808-5812, 1986).
Using a whole-cell extract as starting material and the enzymatic assay described above, CGB(1 4)T is purified using methods that are well known in the art of protein chPm;~try. For example, the whole-cell extract may be fractionated by the sequential application of one or more of the following methods: ion-~ch~nge chromatography, molecular sieve chromatography, and hydrophobic chromatography. These methods may be combined with extraction in detergents and/or mixtures of organic solvents that are known to those of ordinary skill in the art.
In each case, chromatographic fractions and/or extracts are a~sayed for CGB(1~4)T activity and for total protein content. A balance sheet of purification i.e. total activity, total protein, and specific enzymatic activity~ is compiled at each step. Fractions showing peak CG~(1 4)T
activity are analyzed in parallel for their polypeptide profiles, using SDS-polyacrylamide gel electrophoresis. In this manner, fractions are obt~;ne~ cont~;n;ng progressively higher specific activity for CGB(1~4)T and fewer polypeptides.
The final, most purified CG~(1~4)T preparation is then sequenced, using the N-t~rm;n~l Edman degradation reaction. If the preparation contains only a single major polypeptide, the preparation itself is sequenced. If several polypeptides are present, they may be resolved on SDS-polyacrylamide gel electrophoresis and transferred to nylon or other suitable membranes or excised from the gel directly. The IT~ S~ ULE 26~

WO96/20717 L ' PCI~/'US9~ /a~

individual protein species may then be sequenced separately, using automated microseq~encing equipment such as, for ~mrle~
that available from Applied Biosystems (Foster, City, CA).
CG~ 4)T-related peptide sequences of about 10-20 amino acid residues are obtA;ne~.
Once a partial amino acid sequence of CG$(1 4)T is obt~;ne~, st~n~rd reverse-genetic methods may be used to identify, isolate, and sequence the complete CGB(1~4)T gene.
See, for example, Molecular Cloning, A Laboratory M~m~7 (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), or Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY, NY, 1992)~ A series of degenerate oligonucleotides are synthesized that collectively encode the partial peptide sequence identified as above. The m; Ye~
oligonucleotides are used to screen genomic and/or cDNA
libraries derived from the starting organism. Positive clones are re-screened, and the DNA inserts are excised, digested with restriction enzymes, re-cloned, and re-screened. Finally, DNA
fragments of 0.5-3 kb are sequenced, and open reading frames (i.e. a DNA sequencing capable of encoding a polypeptide) are determined.
The original peptide se~ence used to design screening probes, as well as deduced amino acid sequences derived from DNA clones, may also be used to design ;mmllnogenic peptides for the purpose of producing anti-CG$(1 4)T
antibodies. Peptides of up to 40 residues may be synthesized chemically, and used, in conjunction with appropriate carriers and adjuvants, as an ;mm~lnogen in rabbits or other ~n;m~l5 for the production of polyclonal and monoclonal antibodies. Such antibodies are conveniently made using the methods and compositions of Harlow and Lane, Antibodies~ A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
Anti-CGB(1~4)T antibodies are used to quantify and/or affinity purify the CG$(1~4)T enzyme, as well as to screen cDNA
expression libraries described above for the purpose of identifying and cloning CG$(1 4)T-related sequences.

S~c~ ~ E ~) WO 96120717 ' , PCI~/US~G~iu7:52 The CGB(1 4)T DNA sequence and ;he amino acid sequence of the CG$(1 4)T protein can be used as a basis for large-scale purification of the CGB(1 4)T protein and for the design and testing of CGB(1 4)T inhibitors. CG~(1~4)T
polypeptides, with or without additional sequence "tags", may be synthesized in large amounts in E. coli, using c~mmercially available vectors and bacterial hosts. An example of a suitable system is the Invitrogen Xpress~ system (San Diego, CA). The sequence n tags n enable the rapid affinity purification of the products, after which the "tags" may or may not be proteolytically removed to produce an authentic CG~(1~4)T polypeptide.
CGB(1~4)T polypeptides may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include, but are not limited to, radioisotopes, fluorescent compounds, and the like.
~abelled CG$(1~4)Ts can be used, for example, in assays for inhibitory compounds. According to the present invention, CGB(1~4)T may be a mo~o~er comprising a single polypeptide, may be a hom~ timer~ or may be a heteromultimeric molecule comprising different polypeptide ch~;n~. The polypeptide(s) may be modified by, for example, phosphorylation, sulfation, acylation, glycosylation, or other protein modifications.
Furth~rmore, the CGB(1 4)T may be isolated from its natural source or from heterologous org~n;s~ or cells, including, but not limited to, bacteria, yeast, insect cells, and ~mm~lian cells, into which the gene or genes encoding CGB(1~4)T
polypeptide(s) have been incorporated.
The present invention also contemplates derived proteins, and preferably fungal-derived proteins, with substantial sequence or functional homology tu the CGB(1~4)Ts described above. "Sequence homology~ describes the relatedness of CGB(1 4)Ts from different sources. Sequences are substantially homologous if at least about 70~, preferably at least about 80~, and most preferably at least about 90~ of the two sequences are identical. Functional homology describes the stringency of hybridization conditions under which two WO 96/20717 ~ ' PCI~/US~C~ 52 sequences effectively or substantially hybridize. "Stringent"
hybridization conditions are O.lX SSC at 65~C.
CGB(1~4)T may be derived from fungal sources such as Histoplasma capsulatum, Coeeidioides imnitis, Blastomyees dermatitidis, C~yptoeoeeus neoform~nR, Candida species, and Aspergillus species such as Aspergillus fumigates. Preferably the CG~(1~4)Ts are derived from other yeast-like org~n;~mc such as Candida, and most preferably, C. albicans. Non-S.
eerevisiae CG~(1~4)Ts may be identified and isolated by methods that are known in the art, such as, for example, antibody cross reactivity, PCR amplification from genomic DNA using degenerate oligonucleotide probes derived from the CG~(1~4)T sequence~
identified as described above, low-stringency hybridizations using similar S. cerevisiae probes, and finally, functional cloning, in which a cDNA expression library derived from another species is used to transform and to complement an absent or defective CGT function in S. cerevisiae.
The identification and analysis of CGB(1 4)T may also be carried out genetically. For example, one could select conditional mutants which are synthetically lethal with ehs2 mutants or osmotically r~me~;~l. Once yeast mutants that lack activity have been isolated, the CGB(1~4)T gene(s) may be identified by transforming the mutant strains with yeast genomic DNA libraries or yeast CDNA expression libraries.
Transformed clones are then screened for re-acquisition of CGB(1~4)T activity. Finally, the DNA clones are recovered from the yeast and are analyzed as described above for bacterial cloning systems~

Enzyme Inhibitors The present invention also encompasses agents that prevent the production of the enzyme CGB(1 4)T ~r that prevent the enzyme from catalyzing the formation of the N-acetylhexosamine B(1~4) hexose linkage described above. The inhibitory agents may comprise peptides, oligosaccharides, lipids, derivatives of any of the foregoing, or other small organic molecules. Preferably, the inhibitors comprise modified sugars or oligosaccharides that are also highly ~UBSIm~ S~EI~T ~RULE 26) WO96/20717 . PCT~S96/00752 specific in their inhibitory activity, i.e. inhibit pathogenic fungi without adversely affecting ~n;m~l or plant physiology.

Disaccharide Linkaqe Inhibitors Known inhibitors of disaccharide linkages, such as for example, allosamidin and related analogues (Tet. Letts., 35:4149, 1994); hydro~l;nones (Japanese Patent Publication No.
06-199649); moranoline (PCT Publication No. WO 94/04546);
galactosyl-beta-1,3-glycals (Scripps Res. Inst.); moenomycin 10 A (Tetrahedron, 50:2029, 1994); di-and tri-saccharides with intramolecular NH glycosidic linkages (Carbohyd. Res., 252:159, 1994); heteromethyl maltosides (J. Am. Chem. Soc., 116:1569, 1994); and thiol carbohydrates such as those disclosed in CArhQh. Res., 250:1, (1993), can be assayed for N-acetylh~yo~m;ne B(1~4) hexose linking inhibition. Suitable compounds can then be formulated into antifungal compositions.

Modified N-Acetylhexosamine and Hexose Other inhibitors of CGB(1 4)T include, but are not limited to, compounds comprising a t~rm;n~l GlcNAc residue in which the carbon at the 1 position of the ring (the 1-carbon) is modified. (Carbon atom ring numbers are illustrated in Figure 7). Non-limiting examples of suitable modifications at this position include, but are not limited to, (CH2)n-OH, wherein n is an integer from 1 to 12, C~-CI2 alkyl, unsubstituted aryl, aryl substituted preferably with C,-CI0 alkyl or alkenyl, or allosamizoline (Takehaski et al., Tet.
Letts., 32:5123, 1991). Also included as inhibitors are hexose and preferably glucose molecules in which the carbon at the 4 position of the ring (the 4-carbon) is modified to prevent formation of the (~1 4) GlcNAc linkage (see Figure 7).
Suitable modifications of glucose include, but are not limited to, (CH2)n-OH, wherein n is an integer from 1 to 12,) r Cl~CI~
alkyl, unsubstituted aryl, aryl subtituted preferably with C~-Cl0 alkyl or alkenyl, or allosamizoline (Takehaski et al., Tet.Letts., 32:5123, 1991). Either constituent of the oligosaccharide can be further modified at the other carbons.

~JBSmUrE SH~Er ~FtULE 2~) WO96/20717 PCT~S96/00752 Rational design of oligosaccharide-based inhibitors is based on an extensive enzymological analysis of CGB(1~4)T
activity.~ That is, the relative activity of different CGB(1-4)T substrates (including affinity and turnover time) is first tested for (~1 4)-linked GlcNAc polymers (chitin precursors) of different lengths, as well as for different configurations and lengths of (~1 3) and (~1~6)-linked glucose polymers (glucan precursors).
Both GlcNAc- and glucose-based inhibitory compounds may comprise one or more sugar units. These compounds may also contain other modifications to enhance their efficacy, including those that cause the compound to be ret~; n~ in the periplasmic space of the target organism. Preferably, inhibitors will be freely taken up across the fungal cell wall but will not cross the plasma membrane. The only limitation is that the modified compounds retain their capacity to bind CGB(1~4)T and to inhibit its enzyme activity.

Antifungals Antifungal compositions are prepared from the N-acetylhexosamine residue B(1~4) hexose oligosaccharides;
CGB(1~4)T inhibitors,including, but not limited to, modified N-acetylglucosamine or modified glucose; or any combination thereof as an active agent in a biologically acceptable carrier.
Suitable biologically acceptable carriers include, but are not limited to, phosphate-buffered saline~ saline, deionized water, or the like. Preferred biologically acceptable carriers are physiologically or phArmAcologically acceptable carriers.
The antifungal compositions include an antifungal effective amount of active agent. Antifungal effect amounts are those quantities of the antifungal agentc of the present invention that afford prophylactic protection against fungal infections in plants and An;mAls~ and which result in amelioration or cure of an existing fungal infection in plants or An;m~ls~ This antifungal effective amount will depend upon the fungus, the agent, and the host. The amount can be ~ ~--~ ~F~ULE ~ ' WO96/20717 PCT~S96/00752 det~rm;ne~ by experimentation known in the art, such as by establishing a matrix of dosages and frequencies and c~mp~ring a group of experimental units or subjects to each point in the matrix.
The antifungal compositions could act, for example, via inhibition of transglycosidation, by a competitive or non-competitive mech~n; ~m, These compositions could also inhibit the reaction by mass action end product inhibition.
Additionally, such compositions could inhibit the cleavage of the chitin-glucan linkage that may normally occur during growth or ~r~n~ion of the cell wall.
The antifungal active agents or compositions can be formed into dosage unit forms, such as for example, creams, ointments, lotions, powders, liquids, tablets, capsules, suppositories, sprays, or the like. If the antifungal composition is formulated into a dosage unit form, the dosage unit form may contain an antifungal effective amount of active agent. Alternatively, the dosage unit form may include less than such an amount if multiple dosage unit forms or multiple dosages are to be used to ~m; n; ster a total dosage of the active agent. Dosage unit forms can include, in addition, one or more excipient(s), diluent(s), disintegrant(s), lubricant(s), plasticizer(s), colorant(s), dosage vehicle(s), absorption enhancer(s), stabilizer(s), bactericide(s), or the like.
The antifungal agents and compositions of the present invention are useful for preventing or treating fungal infections in plants and ~n;m~lso Fungal infection prevention methods incorporate a prophylactically effective amount of an antifungal agent or composition. A prophylactically effective amount is an amount effective to prevent fungal infection and will depend upon the fungus, the agent, and the host. These amounts can be determined experimen~ally by methods known in the art and as described above. Fungal infection treatment 3S methods incorporate a therapeutically effective amount of an antifungal agent or composition. A therapeutically effective amount is an amount sufficient to stabilize or to ameliorate a fungal infection. Preferably, this amount will yield a S~RSmUlE S~~~ I (R~JLE 26) WO 96/20717 ~ ' PCT/U~GI'U~752 reduction to less than 10~ of the amount of fungus pre~ent at initiation of treatment. This amount also depends upon the fungus, the agent, and the host, and can be det~rm; n~ as explain above.
5Theprophylacticallyand/ortherapeuticallyeffective amounts can be ~m; n; stered in one ~m; n; ~tration or over repeated ~m; n; strations. Therapeutic ~m; n; stration can be followed by prophylactic ~m; n; ~tration, once the initial fungal infection has been resolved.
10The antifungal agents and compositions can be applied to plants topically or non-topically, i.e., systemically.
Topical application is preferably by spraying onto the plant.
Systemic ~m; n; stration is preferably by application to the soil and subsequent absorption by the roots of the plant. For example, the antifungal composition that includes an N-Acetylhexosamine (~ 4) hexose oligosaccharide can be A~m;n;~tered in an amount that effectively saturates the fungus and its environment, thereby inhibiting the CG~(1~4)T enzyme.
The antifungal agents and compositions can be 20 ~m; n; stered to An;m~ls topically or systemically. Systemic ~m;n;stration with respect to ~n;m~ls include both oral and parental routes. Parental routes include, without limitation, subcutaneous, intramuscular, intraperitoneal, intraduodenal, and intravenous ~m; n; stration.
Screeninq Assays Antifungal compounds may be identified using screening methods including, but not limited to, high-throughput screening methods that are based on a modified CG~(1 4)T assay. In addition, a screen for compounds that differentially affect the viability of chs2 versus chs3 mutant yeast strains (Gaughran et al., J. Bacterial, 176:5857-5860, 1994) would be expected to detect inhibitors of CG~ T.
Another screening method involves growing yeast cells in an osmotic remedial medium in the present of potential inhibitors, followed by an assay for alkali-soluble versus alkali-insoluble glucan. This ratio would increase upon inhibition of CG(1~4)To WO96/20717 PCT~S96/00752 In an alternate embodiment, compounds are screened for their ability to bind to CGB(1~4)T polypeptides purified as above.
Assays involve screening test inhibitory compounds from large libraries of synthetic or natural compounds.
Synthetic compound libraries are commerciall~ available from, for example, Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich Chemical C~r~ny~ Inc. (Milwaukee, WI).
Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and ~n;m~l extracts are available from, for example, Pan ~aboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
A preferred screening method includes the steps of (a) contacting a CG~(1~4)T enzyme as described above with an N-acetylhexosamine and a hexose in the presence of an antifungal candidate to form a test mixture; (~) contacting the enzyme with the same N-acetylhexose~m;ne and hexose in the absence of the candidates to form a control mixture; (c) detecting any formation of a ~(1 4) linkage between the N-acetylhexose~m;nP and the hexose in the test mixture and in the control mixture; (d) comparing the efficiency of formation of that linkage in the test mixture and in the control mixture;
and (e) selecting as an antifungal compound the test candidate compound that causes a decrease in the efficiency of formation of the linkage in the test mixture relative to the efficiency of formation of the linkage in the control mixture.
Once a particular test compound has been identified in a high-throughput screen, its inhibitory activity is then confirmed by measuring its effect on CG~(1~4)T activity in a st~n~rd (i.e. low-throughput) assay. Finally, the compound is tested for two properties: (1) the ability t._ inhibit fungal growth; and (2) a lack of effect on ~n;m~l and/or plant cells.
Fungal growth is measured by any method well-known in the art, for example, optical density of a liquid culture or colony WO96120717 ~ PCT~S96/007S2 formation on agar. The potential toxicity of an agent for m~mm~lian cells is measured by monitoring its effect in a typical m~mm~lian cell culture, such as, for example, L-cells.

Description of the Pre~erred Embs~;m~nts The following examples illustrate the invention without limitation.

Materials 0 Enzymes and Reagents ~-N-Acetylgluco~m;n;dases from jack beans or from Diplococcus~nP~m~n;~(OxfordGlycosystems,Inc.-Rosedale,NY).
~-N-Acetylgluco~m; n; ~A~e from beef kidney and $-glucosidase (Boehringer ~nnh~;m-Tn~;~n~polis~ IN).
B(l 3) endoglucanase.
Zymolyase 100 T (Seikagaku America, Inc.-Rockville, MD) Glusulase (Dupont Co.-Wilmington, DE).
Sodium [3H]borohydride (Na~3~) (American Radiolabeled Chemicals Inc.-St. Louis, MO) - 100-500 mCi/mmol, or ICN
Biomedicals, Inc.-Costa Mesa, CA - lO0 mCi/mmol).
[l-14C]glucose (American Radiolabeled Chemicals - 50-60 mCi/mmol).
Chitinase from Serratia marcescens was prepared as described in Roberts and Cabib, Anal. Biochem., 127 :402-412 (l982).

Sc Cerevisiae Yeast Strains and Yeast Growth D3C (M~T~ ura3) - wild type.
ECY36-3C (M~Ta chs1-23 chs2::LEU2 trpl-l ura3-52 leu2-2) - CHsl- and Chs2-deficient.
ECY36-3D pHV9A - contains plasmid pHV9A which carrie~
the CALl/CSD2 gene that restores Chs3 activity.
Strains D3C and ECY36-3D were grown in YEPD (l~ yeast extract, 2~ peptone, 2~ glucose), and strains ECY36-3C and ECY36-3D pHV9A were grown in m;n;m~l medium (2~ glucose, 0.7~
Difco yeast nitrogen base without amino acids) plus nutritional requirements. In all cases growth was at 30~C.

SUBSTITUTE SHEET (RULE 26) WO96/20717 PCT~3~'~C~75 Procedures Preparation of Cell Walls and Digestion with Gl~cAn~-~e and Chitinase.
In a typical preparation, 16.3 g (wet weight) of cells (Strains D3C or ECY36-3C which has a higher chitin content). (See, Shaw et al., ~. Cell. Biol., 14~ 123, 1991) were harvested in the exponential phase of growth. The harvested cells were suspended in about 45 ml of 50 mM Tris chloride, pH 7.5, and were added to 75 grams of glass beads (0.5 mm diameter, Braun-Melsungen, Germany) in the intermP~;~te size vessel of a Bead-Beater (Biospec Products, Bartlesville, Ohio). After cooling the suspension to 5~ C, the Bead-Beater was operated for two 3 minute periods, with a 10 minutes cooling period inbetween. The extract was aspirated from the glass beads, and the aspirated extract was washed several times with small portions of Tris buffer.
Cell walls were s~ nted by centrifugation at 4,000 x g for 10 minutes, and the pellets were washed five times with Tris buffer. The washed cell walls were suspended in the same buffer to a final volume of 495 ml.
~ (1~3) endoglucanase (Zymolyase 100 T) (4.3 ml of a 7.5 mg/ml solution in 50 mM sodium phosphate, pH 6.3) was added. The suspension was incubated at 37~ C with shaking, and the absorbance at 660 nm was monitored. After about one hour, the absorbance had decreased to about 5~ of the original value.

The suspension was centrifuged for 10 minutes at 16,000 g. The pellet was washed twice with Tris buffer and twice with 1~ SDS. The suspension was placed for 5 minutes in a boiling water bath in the second SDS washing. The pellet was then washed three more times with water and was suspended in water to a concentration of 10 mg chitin/ml (about one ml in the preparation described.) The ratio of total N-acetylglucosamine to total glucose at this step was 1:0.7~
600 ~l of the resultant suspension was treated with 600 ~l of NaB 3H4 (6 mCi) in dimethylformamide at room temperature for 5 hours. The reaction was t~rm;n~ted by the addition of 300 ~l of 1 M acetic acid, and the insoluble WO96/20717 PCT~S96/00752 material was washed by repeated centrifugation, followed by suspension in 50 mM potassium phosphate at pH 6.3. The washed, reduced chitin was digested overnight at 30~C with 600 ~l (184 mU) of S. marcescens chitinase. Any insoluble residue was - 5 removed by centrifugation.
The supernatant fluid was used directly for Bio-Gel P-2 chromatography. The compounds isolated from the eluate of the Bio-Gel column were again reduced with an excess of unlabeled sodium borohydride and repurified by Bio-Gel chromatography prior to further analysis.

High Performance Anionic ~ h~nge Chromatography Samples were analyzed on a Dionex high performAncp amino P~ch~nge chromatography (HPAEC) instrument equipped with a pulsed amperometric detector Model PAD2, and pellicular anion-~X~h~nge columns (PA-1 or MA-1, 4 x 250 mm). The Dionex eluent degas module was used to sparge and pressurize the eluents with the helium set at 100 p.s.i. The flow rate was maintained at 0.8 ml/min. The applied pulse potential was 0.05 V, and detector sensitivity was set at 300 nA. The system was used at ambient temperature. Samples were applied via a Dionex microinjection valve with a 50-~1 loop. Areas under the curve were recorded and integrated with a Spectra-Physics integrator.
The eluent cont~;ne~ 100-500 mM NaOH.
Paper Chromatography The solvent used for paper chromatography was butanol/pyridine/water 6:4:3, and the paper was Whatman NoO 1 or No. 3 MM.
Carbohydrate Det~rmin~tions Total carbohydrate was measured with the anthrone reagent (See Trevelyan et al., Biochem. J., 50:298-303 (1952)) and glucose was measured by glucose oxidase (Glucose Trinder Kit - Sigma Chemical Co. - St. ~ouis, MO). Free GlcNAc was ~uantified by the method of Reissig et al., J. Biol. Chem., 217:959-966, 1955) and combined GlcNAc by the same method after digestion of 60 ~1 of sample (0.3-0.5 ~mol of combined GlcNAc) with 60 ~1 Glusulase, 60 ~1 chitinase and 5 ~1 1 M sodium phosphate, pH 6.3, for 45 minutes at 30~C.

SJJ~~ ITES~

WO 96120717 ' . PCT/USS~'0~52 Mass spec~ t~y Chemical iodization mass spectra were obt~;ne~ with a Finigan 1015D spectrometer, using ~mmon;~ as the reactive gas.
A~ Spe~L~ -etry 1H and 13C NMR spectra were measured at ambient temperature with a Varian FX 300 or a Varian Gemini spectrometer, operating at 300 MEz for protons and 75 MHZ for 13C Chemical shifts found in the spectra recorded for solutions in CDCl3 and D2O are reported, respectively, with Me~Si and meth~nol (~ MeOH vs. ~Me4Si 49.0) as internal st~nA~rds. Proton-signal assignments were done by COSY or hnmonllclear decoupling experiments. The non-equivalent g~rm;n~l proton resonating at lower field is denoted Ha and the one at higher field Hb. Carbon signal assignments were based on heteronuclear shift-correlated 2D experiments (HECTOR).

General Synthesis of ~-D-GlcNAc (1 6)-D-Glc Optical rotations were measured at 25~ C with a Perkin Elmer Model 241 MC automatic polarimeter. All reactions were monitored by thin-layer chromatography on pre-coated slides of silica gel G F254 (Analtech). Detection was effected by charring with 5~ sulfuric acid in ethanol or, when applicable, with W light. Preparative chromatography was performed by gradient elution from columns of Silica Gel 60 (Merck, No. 9385)~ Reactions requiring anhydrous conditions were performed under dry nitrogen usin~ common laboratory glassware and reagents and solvents were handled with gas-tight syringes. Solutions in organic solvents were dried with anhydrous sodium sulfate and concentrated under a vacuum at 40~Cr 1~ 2l3,4-Tetra-O-acetyl-$-D-glucopyranose (SigmaChemical Co.) and bromo-2-deoxy-2-N-phtalimido-3, 4~ 6-tri-O-acetyl-~, $-D-glucopyranose (Toronto Research Chemicals Inc.) were used as suppl ied.

t~ULE ~;~

:

WO 96120717 . ~ PCI~/US96/00752 1, 2, 3, 4-Tetra-O-acetyl -6-0- (3, 4, 6-tri -O-acetyl -2-deoxy-2 -N-phtalimido -~ -D-glucopyranosyl -B-D-glucopyranose 1, 2, 3,4-Tetra-O-acetyl-~-D-glucopyranose (0.7 g, 2 mmol) was dissol~ed in dry nitromethane (30 ml), and 4A
molecular sieve (1 gram) was added. After cooling to 30~C, the reaction mixture was stirred for 30 minutes, and sym-collidine (0.28 ml, 2 mmol) and silver triflate (0.54 gram, 2.1 mmol) were added. Finally, bromo-2-deoxy-2-N-phtalimido-3,4,6-tri-O-acetyl-a,B-D-glucopyranose (1 gram, 2 mmol) dissolved in nitromethane ( 5 ml) was added dropwise. After 1 hour, additional portions of sym-collidine (0.056 ml, 0.4 mmol), silver triflate (0.1 gram, 0.4 mmol), and bromo-2-deoxy-2-N-phtalimido-3,4,6-tri-O-acetyl-a,B-D-glucopyranose and (0.2 gram, 0.4 mmol) were AAA~A. The reaction mixture was stirred for 2 hours at -30~ C. At this point, 1,2,3, 4- Tetra-O-acetyl-B-D-glucopyranose was no longer detected by thin layer chromatography (TLC-toluene/acetone 6:1) After filtration through Celite, the filtrate was washed with saturated sodium bicarbonate and with water, dried with sodium sulfate, concentrated, and purified on a silica gel column (toluene/acetone 10:1) to yield 1,2,3, 4- tetra-O-acetyl-6-O-(3, 4, 6-tri-O-acetyl-2-deoxy-2-N-phtalimido-B-D-glucopyranosyl-B-D-glucopyranose.

SUBSm~ SH~EI (RULE 26) WO96/20717 PCT/U~3G~752 Properties are summarized below.
[~]D + 18.47~ (c. 7.82, chloroform); lH NMR (CDCl3) ~; 7.89-7.3 (m, 6H, Ph), 5.75 (dd, lH, J3.,4, 9.1 Hz, J2.,3. 10.6 Hz, H-3'), 5.59 (d, 1 H, Jl2 7.9 Hz, H-l), 5.44 (d, 1 H, Jl~.8.2 Hz, H-l'), 5.16 (dd, 1 H J4.~ 9.4 Hz, H-4'), 5.13 (dd, 1 H, J2. 9.9 Hz, H-3), 5.01 (dd, lH,H-2), 4.90 (dd, lH, J4~ 9.8 Hz, H-4), 4.34 (m, 2 H, H-2', H-5',), 4.17 (dd, 1 H, J5.6~ 2.4 Hz, H-6b), 3.88 (m, lH, H-5'), 3.72 (m, 1 H, H-5), 3.62 (m, 2H, H-6" H-6b), 2.14 (q, 3 H, COCH3), 2.03 (8, 3 H, COCH3); 1.99 (s 3 H, COCH3), 10 1.93 (s, 3H, COCH3, 1.93 (s, 3 H, COCH3), 1.89 (s, 3 H, COCH3 (s, 3 H, COCH3), 1.85 (s, 3 H, COCH3), l3C-NMR (CDCl3) ~:170.86, 170.27, 170.16, 169.60, 169.32, 169.21, 168,86 (COCH3), 97.94 (C-l'), 91.51 (C-l), 73.63 (C-5), 72-64 (C-2), 71.87 (C-5'), 70.29 (C-4), 68.82 (C-4'), 68.27 (C-3), 67.33 (C-6), 61.88 (C-15 6'), 54.22 (C-2'), 20.63, 20.58, 20.48, 20.48, 20.37, 20.26, 20.20 (COCH3).

6-0- (2-aceta~rudo-2-deoxy-13-D-glucopyranosyl) -o~,$-D-glucopyranose .
1,2,3,4-Tetra-O-acetyl-6-O-(3,4,6-tri-O-acetyl-2-deoxy-2-N-phtalimido-~-D-glucopyranosyl-~-D-glucopyranose (0.055 gram, 0.072 mmol) was dissolved in anhydrous methanol (10 ml), and sodium methoxide in methanol (1 M, 0.0 ml) was added. The reaction mixture was stirred at room temperature 25 for 4 hours. At this time, 1,2,3,4-Tetra-O-acetyl-6-O-(3,4,6-tri-O-acetyl-2-deoxy-2-N-phtalimido-~-D-glucopyranosyl-~-D-glucopyranose was no longer detected by TLC (propanol/ethyl acetate/water 4:2:1). ~fter neutralization with Amberlite 120 (H+) and filtration, the filtrate was concentrated and dried under a vacuum. The crude product was dissolved in methanol (5 ml), and anhydrous hydrazine (14 ~1, 0.45 mmol) was added. The reaction mixture was kept under reflux (65~ C) for 2 hours, at which point starting material was consumed as monitored by TLC
(ethyl acetate/ethanol/water, 8:4:2). After cooling to room temperature, acetic anhydride (0.5 gram, 0.46 ml, 4.9 mmol) was added, and the mixture was stirred for 20 minutes. When the starting material was no longer detected by TLC

~UBSm~ S~tE I (RULE 26) WO 96/20717 PCr/US96/007S2 (propanol/ethylacetate/water 2:1:1), the reaction mixture was concentrated and purified on a Bio-Gel P-2 extra-fine, 2 x 90 cm column to yield 6-O- (2-acetamido-2-deoxy-$-D-glucopyranosyl ) - ~, B - D - glucopyranose .
Properties are su~unarized below.
[~X] D + 3.82- (c 17.00, water); 1H N~ (D2O) ~ : a: 5.21 (d, lH, Jl,2, 3,7 Hz, H-1), 4.55 (d, 1 H, J1,2 8.3 Hz, H-11), 4.11 (dd, 1 H, J5,6, 2Hz, J6"6~,11.4 Hz, H-6.), 3.94 (m, 2H, H-6'.7 H-5), 3.81 (dd, lH, J5,6 4.3 Hz, H-6b), 3.73 (m, H-6'b), 3.71 (m, H-2', H-3), 3.68-3.46 (m, 4 H, H-3', H-2, H-5', H-4) 3.41 (dd, 1 H, J,.,4. 6.1 Hz, J4.5. 9.6 Hz, H-4' ), 2.07 (s, 3 H, COCH~);
1~: 4.63 (d, 1 H, J1,2 7.9 Hz, H-1), -4.57 (d, 1 H, J1.,2., 8.4 Hz, H-1' ), 4.17 (dd, 1 H, J5,6. 1.9 Hz, J6.b 11.5 Hz, H-6a), 3.75 (m, 15 2 H, H-2', H-6b), 3.73 (m, H-6'b), 3.68-3,46 (m, 4 H, H-3', H-5, H-5', H-3) 3.41 (ddl, 1 H, J34, 6.1 Hz, J4,,5 9.6 Hz, H-4'), 3.38 (dd, 1 H, J 5.6 Hz, J 9.2 Hz, H-4), 3.24 (dd, 1 H, J2,3 9.2 H-2), 2.07 (~, 3 H, COCHa);
13C NMR (D20) ~: ~X: 175.11 (COCH3), 101.98 (C-1' ), 92.45 (C-1), 76.11 (C-5' ), 73.97 (C-3' ), 73.02 (C-3), 71.69 (C-2), 70.57 (C-5), 70.18 (C-4), 69.83 (C-4'), 68.71 (C-6), 55.73 C-2'), 22.40 ( CO~H3);
~:: 175.11 (COCH3), 102.06 (C-1~ ), 96.31 C-1), 76.11 (C-5' ), 76.05 (C-5), 75.06 (C-3), 74.35 (C-2), 73.97 (C0-3' ), 70.18 (C-4), 69.83 (C-4' ), 68.95 (C-6), 55.73 (C-2' ), 22.40 (COCH3) .

Isolation and Identification of the ~ (1 4) Linkage Example 1 - Between Chitin and ~ (1 3) Glucan in S.
30 cerevisiae To isolate the linkage region between chitin and ~-glucan~ yeast cell walls were digested with a ~ (1 - 3) endoglucanase ( zymolyase ), with the expectation of f orming 35 short glucose oligosaccharide stubs attached to the chitin.
After removal of all the solubilized material, the insoluble fraction was reduced with sodium borotritide, to label the reducing ends of the stubs. This treatment also reduced and labeled GlcNAc residues at the reducing end of chitin ch~3; n!::

~IBSrlTUlE S~EET (F~ULE 26~

CA 02209663 l997-07-04 WO96/20717 . PCT~S96/00752 not bound to glucan. The labeled material w-s then digested with S. marcescens exo-chitinase, an enzyme that se~uentially cleaves diacetylchitobiose residues from chitin, starting from the non-reducing end (See, Roberts et al., Anal. Biochem ., 127:402-412, 1982).

The chitinase-solubilized fraction from 6 mg of glucanase-resistant insoluble residue was applied to an extra-fine Bio-Gel P-2 column (2 X 90 cm) and was eluted with 0.1 M
acetic acid. (In Bio-Gel P-2 column chromatography, each GlcNAc residue, whether free or combined, counts as two hexose residues in det~rm; n; ng relative elution positions (See, Y~m~h;ta et al., Meth. Enzymol., 83:105-106, 1982). Thus, diacetychitobiose elutes in the same volume as a glucose tetrasaccharide. This is not true of paper chromatography, where the mobility of each monosaccharide depends on the solvent used, and, within the same series, is inversely proportional to molecular weight). 1. 8 ml fractions were collected, and a 20-~1 portion of each sample was counted.
Results are illustrated in Figure 1. ~1" indicates the void volume position, and ~2-8~ indicate the positions of the following st~n~rds: 2, triacetylchitotriitol (or l~m; n~ r;hp~;tol);3~l~m;n~ripentaitol; 4, diacetylchitobiitol (l~m~ n~ ritetraitol); 5, l~m;n~ritriitol; 6, GlcNAc-ol (or l~m;n~ribiitol); 7, glucitol; 8, Glc ([14c]) glucose was added as internal st~n~Ard).
Two of the major radioactive peaks correspond to diacetylchitobiitol (Figure 1, peak 4) and triacetylchitotriitol (Figure 1, peak 2), which originate from free chitin ch~;n~ cont~;n;ng an even and an odd number of GlcNAc residues, respectively (See Kang et al., ~. Biol. Chem., 25g:14966-14972, 1984). A large peak in the void volume and some additional minor peaks were also detected. The latter appeared to be candidates for the linkage region and were named Peaks A (Compound I), B (Compound II), and C (Compound III).
Peak A contained both N-acetylglucosamine and glucose, whereas Peaks B and C cont~;ne~ only glucose.

Structure of Compound I (Peak A. Fiqure 1 SUBSTITUTE SHEET (RULE 26~

WO 96/20717 ' PCT/US96/00752 Acid Hydrolysis/EPAEC

5 nmol each of compounds I, II or III were evaporated to dryness under nitrogen, and were hydrolyzed with 25 ~l of 2M TFA at 100~C for 2 hours, evaporated under nitrogen and - 5 diluted to 200 ~l of water. A 50 ~l sample was analyzed by HPAEC in a PA-1 column as described abo~e. Results are illustrated in Figure 2. Acid hydrolysis of Eeak A gave rise to glucosamine, glucose and glucitol, ln the ratio 1.1:2:0.9 as illustrated in Figure 2.
Mas~ Spectrometry 100 nmol of each of compounds I, II, and III were evaporated to dryness under nitrogen. The residue was dissolved in 200 ~l of pyridine, and 200 ~l of acetic anhydride were added. After overnight incubation at room temperature, a few drops of toluene and of methanol were added, and the solution was evaporated to dryness. The dissolution-evaporation was repeated several times, first with toluene, then with methanol. Finally, the sample was dissolved in 20 ~l of dichloromethane and was analyzed by mass spectrometry.
Because ~mmo~;~ was used as the reactive gas, the ~mmo~;um ion weight was added to the calculated molecular weight. Results are illustrated in Figures 3a-c.
The molecular weight of the acetylated compound, as measured by mass spectrometry, was 1316, compared to a value of 1315 for an acetylated tetrasaccharide consi~ting of a residue each of GlcNAc and glucitol and two gluco~e residues.
(This includes the weight of ~mmon; um ion). This composition is also reflected in the elution of volume of Peak A (Figure 1) which corresponds to a reduced glucose pentasaccharide stA n~l~3 rd .

~-N-acetylglucos~minidase or ~-glucosidase Trea~ment Compound I (Peak A - Figure 1) was treated with N-acetyl-~-glucosaminidase and ~-glucosidase to determine the manner and position at which the GlcNAc was attached. Aliquots (~13 0 pmol, 13 o,000 cpm) of compound I were evaporated to dryness and were redissolved in 50 ~l of lO0 mM citrate-phosphate buffer, pH 5.0 (Figure 4a); in the same buffer plus SUBS~IT~ SHEET (RVLE 26) W O 96/20717 . PC~rrUS96/00752 5 ~1 (135 mU) of B-N-acetylglucosaminidase from jack beans (Figure 4b); and in 60 ~ l of O.lM acetate buffer pH 4.5, cont~;n;ng 0.1 mg of sweet ~l m o~l B-glucosidase (Figure 4c).
All mixtures were incubated 16 hours at 37~C, then diluted with 300 ~1 water and subjected to Bio-Gel P-2 chromatography.
Standards were: 1, triacetylchitotriitol; 2 , diacetylchitobiitol; 3, l~m;n~ritriitol; 4, l~m~n~ribiitol; 5, glucitol; 6, glucose ([14C]) glucose was the internal st~ni~rd).
Results are illustrated in Figure 4. The positions of peaks A, B and C are also indicated.
Whereas B-glucosidase treatment had no effect on the elution position of compound I (Figure 4c), incubation with B-N-acetylglucosaminidase moved it to the position of l~m;n~ritriitol (Figure 4b). This result showed that the GlcNAc in compound I was to be at the non-re~llc;ng end and was attached to a trisaccharide by a B-linkage.

Partial Acid Digestion A sample of compound I (~25 pmol, 25,000 cpm) was 2 0 evaporated to dryness under nitrogen and was hydrolyzed with 50 ~1 of 0.05 M trifluoracetic acid for 2 hours at 100~C. The hydrolyzate was subjected to paper chromatography. Segments (1-cm) of the paper were counted. St~n~rds: 1,gluco~e ([l4C]
glucose internal st~n~rd); 2, glucitol; 3, l~m;n~ribiitol; 4, l~m;n~ritriitol or gentiobiitol; 5, l~m;n~ritetraitol; 6, gentiotriitol; 7, l~m;n~ripentaiol.
Results are illustrated in Figure 5a. Only products that still retained the labeled sorbitol were detected by the radiometric assay. After partial acid hydrolysis, three additional peaks were detected, which migrated as Glc(B1-3)Glc(B1-3)Glc-ol (or Glc(1-6)Glc-ol), Glc(B1-3)Glc-ol and Glc-ol. All of these compounds would be expected if GlcNAc were attached tc a reduced 1~ m; n;:lrit rios e.

Partial Enzyrnatic Digestion Aliquots of compound 1 (10 pmol, 10,000 cpm each) were evaporated to dryness and were dissolved in 30 ~1 of 100 mM citrate-phosphate buffer, pH 6Ø Both aliquots were HE~T tR~J~E 2~!

WO 96t20717 , ' PCT/US96/00752 incubated 16 hours at 37~C, one with 7.5 mU of ~-N-acetylgluco~Am~n;dase from Diplococcus pnel~mnn i~ and the other with the same enzyme plus 0.02 mg of ~-glucos dase from sweet Almon~. Both samples were subjected to paper chromatography as above. StAn~Ards: 1, glucose; 2, l~m~nAribiitol; 3, sophoritol; 4, cellobiitol; 5, lAm;n~ritriitol or gentiobiitol;

6, gentiotriitol.
Results are illustrated in Figure 5b. (-) -incubated with N-acetylglucoYAm;n;dase; (o) - incubated with both enzymes. The tentative structure of compound I and of the hydrolysis products are shown, where an open square stands for GlcNAc, an open circle for Glc and a filled circle for glucitol.
Partial hydrolysis with B-N-acetylglucos~m; n; ~ARe gave rise to a peak in the position of the original material and another one moving as Glc(~1-3)Glc($1-3)Glc-ol or Glc)B1-6)Glc-ol. This indicates that the glucose trisaccharide cannot be gentiotriitol, since the latter (stAn~Ard 6 in Figure 5b) moves much more slowly than the product of the reaction. When both B-N-acetylhexosaminidase and B-glucosidase were allowed to act on compound I, the product comigrated with Glc(B1-3)Glc-ol and was clearly different from the 1-2, 1-4 and 1-6 isomers (Figure 5b).
In separate experiments it was found that lAm;nAribiitol is resistant to B-glucosidase. Taken together, the above results are consistent with a structure in which GlcNAc is B-linked to reduced lAm;nAritriose.

Trisaccharide Periodate Oxidation A portion of compound I (60 nmol) was digested with jack bean B-N-acetylglucosaminidase and was subjected to Bio-Gel P-2 chromatography essentially as described above. The recovered trisaccharide (50 ~1) was oxidized .~ith 700 nmol of sodium metaperiodate for 70 hours at 4~C in the dark.
Ethyleneglycol (1~, 23 ~l) was added. After 2 hours at room temperature, 40 ~l of 0.1 NaOH and 50 ~l of sodium borohydride in 0.01 M NaOH were added. Incubation was continued for 3 additional hours. The sample was evaporated to dryness under TE ~EET ~iJ~

7 PCT~S96/00752 nitrogen, dissolved in 100 ~l of 2 M trifluoracetic acid, and heated at 100~C for 2 hours. After evaporation to dryness, the residue was dissolved in 350 ~l water and a 50-~l portion was subjected to HPAEC on a PA-1 column with 0.2 M NaOH as solvent.
T~m;n~ribiitol and l~m;n~ritriitol (50 nmol of each) were subjected to the same treatment and chromatographed.
Results are illustrated in Figure 6. The large peak at 2.34-2.36 minutes is ethyleneglycol. Glucose (retention time 5.15-5.25 min) was present in the samples resulting from oxidation of l~m;n~ritriitol or of the trisaccharide from compound I, but not in the one from l~m;n~ribiitol.
Any configuration of the glucose to glucose linkage other than 1~3 would have resulted in the destruction of both ~lc residues. However glucose was recovered after oxidation, indicating that the term; n~l and penultimate glucose residue~
were bound in a 1 3 linkage.

H-A~ Spectrum From1H NMR studies of reduced l~m;n~ritriose, it was concluded that the ~n~meric proton of the internal residues (H'-1 from ring C~, see Figure 7 top) resonates at 4.65 ppm.
Only two doublets were found, with chemical shifts of 4.58 ppm and 4.48 ppm, in the IH-NMR spectrum of Compound I (data not shown). Therefore, it may be inferred that the 4.58 ppm doublet corresponds to the ~nom~ric protons H~-1 and H~-1 of I. Because the doublet is partially overlapped by the HO~
signal, the signal cannot be quantified by integration to confirm that it is originated in two protons. The corresponding coupling constant Jl'2'=Jl"2" iS 7.9 Hz, as expected for a $ linkage.
Given this attribution of the 4.58 ppm signal, the doublet at 4.48 ppm must represent the ~nsm~ric proton (H'''-1) of the GlcNAc unit. This chemical shift is in good agreement with known shifts for branched penta- and hexasaccharides bearing GlcNAc at the nonreducing end (4.45-~.58 ppm). See, S118S~lllrrE SHEET (~ULE 26) WO 96/20717 PCT/US9~ IIC752 Kahn et al., Carbohyd. Res. 262:283-295, 1994. The coupling constant J1...2... ~f the 4.48 ppm doublet is 8.3 Hz, a value typical of ~-linked units.

~ 5 Analoqous Compound Cnmr~rison GlcNAc(B1-6)Glc was synthesized. This compound ~ eliminated the 1 6 linkage as a possibility for the chitin/N-acetylglucoc~m;ne linkage because the synthetic compound was decomposed by beef kidney ~-N-acetylglucosaminidase whereas compound I was resistant tdata not shown). The possibility that GlcNAc was attached to Glc by a 1-2, 1-3 or 1-4 linkage still rem~;ne~. Since the a-m~ount of material available was insufficient for methylation analysis, NMR =pectroscopy was employed.
l3C-M!IR Spectrum Approximately 1 ~mol of compound I was evaporated to dryness several times with D2O, and then was dissolved in 600 ~l of D2O. The spectrum was measured continuously for 5 days, as described above. Interpretation of the spectra was based on identification of the signals in two-~;m~n~ional COSY and HECTOR spectra of st~n~rd l~m;n~ribiitol, lAm;n~ritriitol and GlcNAc(Bl-6)Glc st~n~rds.
Results are illustrated in Figure 7. Attribution of peaks to different carbons are shown for the spectrum of Compound I. For each bracketed group of peaks, the carbon listed on top refers to the first peak from left under the bracket. The other carbons follow from top to bottom and from left to right, respectively. The position a~- which the C4"
peak was expected is shown, together with that where it wa~
actually found (arrow).
Model oligosaccharides l~m;n~ribiitol and l~m;n~ritriitol and GlcNAc(~1-6)Glc were studied by 2D NMR
spectroscopy (COSY, ~ECTOR). The groups of signals belonging to the different carbons of D-sorbitol (C), D-glucopyranosyl units (C' and C'') and 2-acetamido-2-deoxy-D-glucopyranosyl (C~) were identified. The largest deviation of chemical shifts of the C'' unit of compound I compared to the corresponding unit of reduced l~m; n~ ritriose was expected at Sl~ 111 IJTE SHEET (~IJLE 26) CA 02209663 l997-07-04 WO 96/20717 PCT/U~3C/01~752 the carbon involved in the glycosidic bond. C''-2 and C''-6 were eliminated as participants in the bond, because their chemical shifts, 73.55 and 73.35 ppm for C''-2 and 60.65 and 60.86 ppm for C''-6, are the same for compound I and for reduced l~m;n~ritriose. If the glycosidic linkage were at position C''-3, one of the signals in the region 75.41-75.97 ppm would move to lower field in the spectrum of compound I, because this is the area in which carbons C'-5, C~-5 and C''-3 are located. This shift did not occur. Also, in the spectrum of compound I, signalc for five carbons should appear in the region 68.35-70.83 ppm, representing C-5 and all four C-4 carbons (C-4, C'-4, C''-4, C~-4 ? . However, only four signals were found in thi~ area, and they were assigned to carbons C-4, C-5, C'''-4 and C'-4. This meant that carbon C''-4 signal was moved to lower field, where it can be found at 78.68 ppm (Figure 7), due to the large positive ~-effect of the glycosidic linkage at that position (18). Therefore, the glycosidic linkage between GlcNAc and Glc in compound I was $(1~4). The complete structure of the substance was GlcNAc(B1~4)Glc($1 3)Glc($1 3)Glc-ol.

Structure of Compound in Peak B - Fiqure 1 Acid Hydrolysis Complete acid hydrolysis of Compound II (Peak B, Figure 1) gave rise to glucose and sorbitol in a 2.0:1.0 ratio (See Figure 2).

Mass Spectrometry The molecular weight, as measured by mass spectrometry was in agreement with this result (calculated 1028; found 1028). See Figure 3b.

~-Glucosidase Digestion A portion (5000 cpm) of compound II was evaporated to dryness and dissolved in 30 ~l of acetate buffer at pH 4.51 cont~;n;ng 10 ~g of sweet ~lm~n~ $-glucosidase. After 16 hours of incubation at 37~C, the sample was analyzed by paper chromatography.

V~ TE S~EE r (~ULE 2~) WO 96/20717 PCI/US~ 7 Results are illustrated in Figure 8a. (O)-digested sample; (-) incubated control.
Digestion of compound II with B-glucosidase followed by paper chromatography showed that the labeled material had moved to the position of l~m;n~ribiitol. The NMR spectrum of compound II is identical to that of reduced l~m; n~ritriose (Table 1). Therefore, compound II was identified as 1 ;Im; n~ ritriitol.

C. . ' Ring C-l C-2 C-3 C-4 C-S C-6 NHCOCH3 T~ l C 62.11 72.89 78.71 70.88 70.29 62.91 C' 103.10 73.35 84.35 68.45 76.13 60.86 C" 102.87 73.59 75.69 69.73 75.46 60.86 GlcNAc(Bl-6)Glc C a 92.45 71.96 73.02 70.18 70.57 68.71 B 96.31 74.35 75.06 70.18 76.05 68.95 C'a 101.98 55.73 73.97 69.83 76.11 175.11(C0) B 102.06 22.40(CH3) I C 62.03 72.83 79.37 70.83 70.25 62.83 C' 103.09 73.33 84.17 68.35 75.97 60.83 C" 102.54 73.55 75.68 78.68 75.41 60.65 C"' 101.55 55.69 74.62 69.83 74.35 60.21 23.35(CH3) n C 62.11 72.85 78.75 70.33 62.11 C' 103.14 73.33 84.49 68.46 76.15 60.86 C" 102.91 73.60 75.72 69.73 75.46 SVElSrl~UrE S~EET t~ULE 2~) WO96/20717 PCT~S96/00752 Structure of Compound III (Peak C - Fiqure l) Acid Hydrolysis Acid hydrolysis of compound III (Peak C - Figure l) liberated glucose and sorbitol in l.l:l.0 ratio. See Figure 2. The molecular weight of the compound was as expected from the analysis (calculated 739.4; found 740). See Figure 3c.

~-Glucosidase Digestion A portion (l0 pmol, l0,000 cpm) of compound III was evaporated to dryness and dissolved in 30 ~l of acetate buffer at pH 4.5, contA;n;ng l0 ~g of sweet ~lmon~ B-glucosidase.
After 16 hours of ;nCl~h~tion at 37~ C, the sample was analyzed by paper chromatography. St~n~rds: Glc; 2, l~m;n~ribiitol;
3, sophoritol; 4, cellobiitol; 5, gentiobiitol.
The material behaved on paper chromatography (Figure 8~) or HPAEC (results not shown) like l~m;n~ribiitol and could be distinguished from the 1~2, l 4 and 1~6 isomers. On the basis of these data and by analogy with compounds I and II, compound III was identified as l~m;n~ribiitol.
Other Mixed Oliqosaccharides Containinq N-Acetylqlucosamine and Glucose Figure 9 illustrates a scheme for the generation of different oligosaccharides by chitinase digestion. Chitinase is able to cut between a GlcNAc and a Glc residue, if the linkage between the two sugars is $(1 4). Therefore, compounds I and II would be derived from chitin ch~;n~ with an odd or even number of GlcNAc residues, respectively, both attached to a reduced l~m;n~ritriose. Similarly, Compound III would result from hydrolysis of an even-numbered chain linked to l~m;n~ribiitol. The corresponding oligosaccharide from an odd-numbered chain (Figure 9, compound IV) should behave in the P-2 column either as a reduced tetrasaccharide of glucose or as reduced diacetylchitobiose. Thus, it may have been hidden under the large peak of diacetylchitobiitol in Figure l.
Accordingly, material from that peak was subjected to paper chromatography. Results are illustrated in Figure l0.

CA 02209663 l997-07-04 WO 96/20717 ' ~ 6/00752 This peak cont~;ne~ in addition to the reduced disaccharide, some slowly moving labeled material.
The slow-moving labeled band was excised and eluted with water.
The st~n~rds were~ m;nAritriitol; 2, l~m;n~ribiitol; 3, diacetylchitobiitol; 4, glucitol.

Treatment of material separated from di a ce tyl chi tobi i tol wi th 1~ -N-acetylglucoff~minidaseand/orB-glucosidase The slow-moving band in the paper chromatogram of Figure 10 was eluted with water, concentrated, and rechromatogr~phe~ on a Bio-Gel P-2 column (lx90cm). Results are illustrated in Figure lla.
Aliquots of the original material were subjected to incubation with B-N-acetylglucos~m;n;dase~ ~-glucosidase, and both ~-N-acetylglucosaminidase and ~-glucosidase. Results are illustrated in Figure llb, c, and d, respectively. The standards were: 1, triacetylchitotriitol; 2, diacetychitobiitol; 3, l~m; n~ritriitol; 4, l~m; n~ribiitol; 5, glucitol; 6, glucose.
Treatment of the material with ~-N-acetylglucosaminidase caused displacement of a large portion of the label to the elution position of either l~m; n~ribiit or N-acetylglucosaminitol, whereas incubation with B-glucosidase had the same effect on a minor portion of the radioactive material. Finally, with a mixture of both enzymes, all of the radioactivity moved to the new position. The results are consistent with the hypothesis that the material eluted from paper cont~;ne~ a mixture of GlcNAc-~-Glc-~-Glc-ol (compound IV) and Glc-~-Glc-~-Glc-~-Glc-ol (compound V - Figure 9) .
The isolation of compound V suggested ~hat the corresponding compound VI (Figure 9) should also have been formed. According to its composition, this compound would be eluted in the P-2 column together with reduced triacetylchitotriose. Therefore, material from the reduced trisaccharide peak was analyzed by paper chromatography and found to contain a small amount of slower-moving labeled material (See Figure 12a).

~ SHEET(~U~26) WO96120717 PCT~S96/00752 The slow moving radioactive material was eluted with water from paper, concentrated and treated with $-N-acetylglucos~m; n; dase followed by Bio-Gel P-2 chromatography as described above. Results are illustrated in Figure 12b.
The pentasaccharide was also incubated with B-glucosidase followed by Bio-Gel P-2 chromatography as described above.
Results are illustrated in Figure 12c. The st~n~rds were:
l, triacetychitobiitol or l~m; n~ r; heX~ itol; 2, l~m;n~ripentaitol; 3, diacetychitobiitol orl~m;n~ritetraitol;

10 4, 1 ~m; n~ritriitol; 5, compound III or l~m;n~ribiitol; 6, glucose.
This substance was resistant to B-glucosidase, but was digested by B-N-acetylgluco~m;n;~se, with co~cnm;tant displacement to the l~m;n~ritetraitol position in the P-2 column. The substance had the expected proper~ies of compound VI (GlcNAc-$-Glc-B-Glc-B-Glc-B-Glc-ol).

Glucose Oligosaccharides Do not Preexist in the Intact Cell Wall The possibility r~m~; ne~ that the glucose oligosaccharides attached to chitin preexisted as such in the intact cell wall before glucanase digestion, rather than being part of a larger chain. If the glucose oligosaccharides did pre-exist, they would be labeled if the borotritide reduction were performed before, rather than after treatment with glucanase. Therefore, walls were reduced twice, before and after B(l~3) endoglucanase treatment and were chromatographed on a Bio-Gel P-2 column. Results are illustrated in Figure 13a. The st~n~rds were: l, void volume; 2, triacetylchitotriitol; 3, laminaripentaitol; 4, diacetychitobiitol; 5, l~;n~ritriitol; 6, l~m;n~ribiitol; 7, glucitol; 8, glucose. ~ell walls were also reduced before $(1~3) endoglucanase treatment as described above~ Results are illustrated in Figure 13b.
Only the reduced chitooligosaccharide peaksl resulting from free chitin ch~;nc~ were labeled (Figure 13b).
Accordingly, the oligosaccharides were part of an extended ~l~lITU~ SHEEr (FffJLE 2B) -WO 96/20717 . PCT/US96/00752 glucan chain and can only be exposed to reduction by degradation of the chain with glucanase.

Synthesis of the Glucan-linked Chitin RequireB Chitin Synthetase 3 Three different chitin synthetases (Chsl, Chs2 and Chs3) participate in different aspects of chitin synthesis in yeast (Shaw et al., J. Cell . Biol ., 114~ 123, 1991; Cabib et al., J. Cell. Biol., lQ8:1665-1672, 1989). Mutant~ in each of the three synthetases are available.
Wild type strain D3C cell walls were treated with endoglucanase, reduced, ;ncllh~ted with chitinase, and chromatographed on Bio-Gel P-2 columns as described above.
Results are illustrated in Figure 14a. The st~n~rds were: 1, void volume; 2, triacetylchitotriitol; 3, diacetylchitobiitol;
4, diacetylchitobiose; 5, l~m; n~ritriitol; 6, l~m; n~ribiitol;
7, GlcNAc; 8, Glc.
The procedure was repeated with Strain ECY 36-3C
(chsl chs2:LEU2), Strain ECY36-3D (chsl call/csd2) (this strain is deficient in Chs3), and Strain ECY36-3D pHV9a (the plasmid contains the CAL1/CSD2 gene and restores Chs3 activity).
Results are illustrated in Figures 14b, c, and d, respectively.
Strain ECY36-3C, with mutations in Chsl and Chs2/
still chowed a full array of oligosaccharides (Figure 14b), i.e. it is not impaired in the formation of chitin-glucan ch~inc. However, strain ECY36-3D, which lacked Chs 1 and Chs3, showed only the two reduced chitooligosaccharide peaks, derived from free chitin (Figure 14c). Transformation of this strain with a plasmid carrying the CAL1/CSD2 gene, required for Chs3 activity, restored a full complement of oligosaccharides (Figure 14d). Therefore, Chs3 is the synthetase involved in the formation of the chitin that becomes glucan-linked.

Tritium- labeled Void Volume Peak The material solubilized by glucanase and chitinase digestion was fractionated on P-2 columns. A fairly large amount of radioactivity emerged at the void volume (Figure 1).

S~lBSllTUTE SH~ I (RULE 26) WO 96/20717 . PCT/US96/00752 This material was rechromotographed on Sephacryl S-200 and Sephacryl S-300 columns. Results indicated that the material was heterogeneous and of high molecular weight, in the 200,000-300,000 dalton range. NMR spectra were s;m;l~r to those of pustulan, a $(1 6)-linked glucan, although other components appeared to be present. Acid hydrolysis released glucose and some mannose.
The void volume material was barely detectable in the Chs3 mutant (Figure 14c) and was restored by the CA~l/CSD2plasmid (Figure 14d), which indicated that it was originally bound to chitin whose synthesis depends on the presence of Chs3. The void volume labeled material was also somewhat reduced in the chsl- chs2 mutant (Figure 14b) as well as in the wild-type fraction resulting from cell walls reduced with borotritide before glucanase digestion (Figure 13b).
Example 1 illustrates that the oligosaccharides contA;n;ng GlcNAc linked ~ 4) to glucose were not solubilized until cell walls were digested with both $-glucanase and chitinase. This indicates that the oligosaccharides originate in the linkage region of glucan and chitin. The presence of both N-acetylglucos~m;ne and glucose in some of the compounds confirmed this. The short glucose cha;ns were originally part of the glucan, because they are protected from reduction when the polysaccharide is intact.
The structure of compound I corresponds to an original oligosaccharide (before reduction) cont~;n;ng one N-acetylglucosaminyl group linked in $(1~4) to l~m;n~ritriose.
Compound I and the other five compounds studied can be arranged in two homologous serie~, one cont~;n;ng 2, 3, or 4 $(1-3)-linked glucose units and the other with the same units plu~ an N-acetylglucosaminyl group at the non-reducing end. The different lengths of the glucose moieties was due to some variability in the position of the $(1~3) linkage hydrolyzed by the zymolyase preparation. The main activity in zymolyase appeared to be a $(1~3)endoglucanase that gives rise to l~m;n~ripentaose as the major product (Kitamura et al., J. Gen.
Appl. Microbiol., 20:323-344, 1974). Therefore, the r~m~;n;ng stubs attached to chitin should not be much more than five ~ISSllTU~ S~lE~ ~ E 26~

WO96120717 PCT~S96/007S2 glucose units long. The existence of chitin ~h~in~ with an odd or e~en number of GlcNAc residues and the ability of Serratia exochitinase to hydrolyze a GlcNAc(B1 4)Glc residue P~pl~;n the presence or absence of GlcNAc.
5The sum of reduced diacetylchitobiose and triacetylchitobiose is equivalent to the number of free chitin ~ ch~ ~ n~ . The gum of oligosaccharides should be equivalent to the number of glucan-linked chA; n~ . This analysis suggests that between 40 and 50~ of the chitin ~h~; n~ are engaged in linkage with glucan. The chitin to glucan ratio in the cell wall is about 1:10 in strain D3C. The effect of small amounts of chitin on the solubilization in hot alkali of about 70~ of the glucan (Mol et al., F.E.M.S. Microbiol. Lett., 41:95-99, 1987) may be ~xrl~;neA by the different chain lengths of chitin and glucan. The reported values are ~100 for the former (Kang et al., ~. Biol. Chem., 259:14966-14972, 1984) and ~1500 for the latter (Manners et al., Biochem ~., 13$:19-30, 1973; Fleet et al., J. Gen. Microbiol., 94:180-192, 1976). Thus, a relatively small number of chitin molecules may suffice to affect the properties of a 15-fold higher amount of glucan.
The results above with chitin synthetase mutants indicate that Chs3 is the enzyme responsible for the formation of the chitin that is incorporated into the chitin-$(1~4) linked-hexose oligosaccharide. This is consistent because it is known that Chs3 i9 involved in the synthesis of 80-90~ of the cell wall chitin, including that present in a ring at the base of an emerging bud and that dispersed throughout the wall (Shaw et al., ~. Cell. Biol., 114:111-123, 1991; Bulawa et al., P.N.A.S., USA, 87:7424-7428, 1990). This chitin i8 incorporated into the cell wall late in the cell cycle, after cytokinesis and during bud maturation (Shaw et al.).
Therefore, the glucan of the bud cell wall f~rmed until that m~m~nt could not be bound to chitin and must represent an alkali-soluble glucan. This is supported by the finding that soluble glucan is the precursor of insoluble glucan and that bud walls disappear after prolonged alkali extraction. Thus, chitin is attached to preexisting glucan.

Sl~mllrE SHE~T (RULE 26) WO96/20717 ~ PCT~S96/00752 It has been suggested that the chitin glucan bond may be formed in the periplasmic space by transglycosylation from a newly-formed chitin chain (Cabib et al., Microbiol . Sci ., S:370-375, 1988). According to this hypothesis, a portion of the chitin chain would be released in the reaction. An alternative merh~n;~m is possible if chitin ch~;nc grow from reducing end, as does the O-antigen of Gram-negative bacteria (Robbins et al., Science, 158:1536-1542, 1967). In that case, the GlcNAc residue at the reducing end would remain activated during synthesis, and the whole nascent chain could be transferred directly to glucan.

Example 2 - An Enzymatic Assay for Chitin-Glucan Linkage 50 ~l of chitin (10 mg/ml in PBS), 50 ~l of 3H-glucan (prepared as in Example 1) (10~ cpm/mg, 10 mg/ml in PBS), and 10 ~l of CG$(1 4)T source are m;~e~. The mixture is incubated at 30~C for 1 hour. 5 ~l of a solution of zymolyase lOOT (7.5 mg/ml, prepared as in Example 1) are added, and the incubation is continued for 1 hour at 37~C. Subsequently, 200 ~l of ice-cold 20~ trichloroacetic acid is added, and the resultant mixture is incubated on ice for 5 minutes. Finally, the reaction mixture is filtered through Whatman GF/C filters. The radioactivity associated with the filters is then ~uantified by liquid scintillation counting.
Controls are prepared either omitting the enzyme source or with 2 mg unlabelled glucan.
For a given sample, a dose-dependent increase in acid-precipitable radioactivity indicates the formation of a chitin-glucan linkage.
Example 3 - Hiqh-Throughput Screening for Inhibitors of Chitin-Glucan Linkage The assay described in Example 2 is adapted for high-throughput screening as follows:
A known source of the enzyme is used, such that 50 of the 3H-glucan (i.e. 2500 cpm) is converted from an acid-soluble to acid-insoluble form during the reaction. Reaction mixtures are formed in 96-well microliter disnes according to 111 UTE SHE~ (F~ULE 2~;) WO 96/20717 ' PCI~/US96tO0752 the procedure of Example 2, with the addition of 15 ~1 of a solution contA;n~ng test inhibitory compounds. After ;ncl~hAtion at 30~C for 1 hour, zymolyase lOOT i8 added according to the procedure of Example 2 and ;nCllhAtion is continued for an additional hour.
Using automated equipment, the contents of each well are transferred to a sheet of Whatman 3MM filter paper. The paper is immersed in ice-cold 10~ trichloroacetic acid for 10 minutes and is then w~Rhe~ in 5~ trichloroacetic acid. Areas of the paper corresponding to each well are excised and counted. A reduction in the number of cpm detected in a given well indicates a candidate inhibitory compound.

Example 4 - Test of Antifungal Properties A candidate antifungal agent is dissol~ed in a biologically acceptable solvent such as saline. Serial 10-fold dilutions of the agent are prepared in yeast growth medium.
10 ml aliquots of each dilution are inoculated with 10~ yeast cells, followed by incubation at 30~C. At hourly intervals, the growth of the cultures is ascertA;ne~ by measuring the absorbance at 600 nm.
An effecti~e antifungal agent is one that suppresses the growth of yeast cells by ~90~ at concentrations that are practical for agricultural or medicinal applications.
Example 5 - Antifungal Formulation for Agricultural Use An antifungal formulation for agricultural use is prepared by m; ~; ng N-acetylglucos~m;n~-~(1~4)-glucose with deionized water. The resultant composition is sprayed on a fungally infected plant.

Example 6 - Antifungal Formulation for Medicinal Use An antifungal formulation suitable for An;m~l use is prepared by m;~;ng N-acetylgluco~Am;ne $(1~4)glucose with saline. The resultant solution is A~m;n;stered systemically to a m~mmAl suffering from a fungal infection.

WO 96/20717 ~ r PCT/U~r ~tC7~2 All patents, applications, articles, publications, and test methods mentioned above are hereby incorporated by reference.
Many variations of the present invention will suggest themselves to those skilled in the art in light of the above detailed description. Such obvious variations are within the full intended scope of the appended cl~; m~ .

SUBSTITUTE SHEET (RULE 26)

Claims (21)

IN THE CLAIMS:

1. An antifungal composition comprising:
(a) an antifungal effective amount of (i) an oligosaccharide comprising an N-acetylhexosamine residue linked .beta.(1->4) to a hexose, (ii) an inhibitor of an enzyme having as a first substrate, the terminal reducing N-acetylglucosamine residue of chitin and having as a second substrate the non-reducing glucose residues of (.beta.1->3) linked glucans, said enzyme catalyzing the formation of a (.beta.1->4) linkage between said N-acetylglucosamine and said glucose, or (iii) any combination thereof; and (b) a biologically acceptable carrier.

2. An antifungal composition as defined in claim 1, wherein said biologically acceptable carrier comprises a pharmaceutically acceptable carrier.

3. An antifungal composition as defined in claim 1, wherein said N-acetylhexosamine of said oligosaccharise comprises N-acetylglucosamine and said hexose of said oligosaccharide comprises glucose.

4. An antifungal composition as defined in claim 1, wherein said oligosaccharide comprises a disaccharide.

5. An antifungal composition as defined in claim 1, wherein said enzyme inhibitor comprises N-acetylglucosamine modified at the 1-carbon by a member selected from the group consisting of (CH2)n-OH wherein n is an integer from 1-12, C1-12 alkyl, aryl, aryl substituted with C1-C10 alkyl or C1-C10 alkenyl, and allosamizoline.

6. An antifungal composition as defined in claim 1, wherein said enzyme inhibitor comprises glucose modified at the 4-carbon by a member selected from the group consisting of (CH2)n-OH wherein n is an integer from 1-12, C1-12 alkyl, aryl, aryl substituted with C1-C10 alkyl or C1-C10 alkenyl, and allosamizoline.

7. An antifungal composition as defined in claim 5, wherein said 1-carbon modified N-acetylglucosamine is further modified at an additional carbon.

8. An isolated fungal enzyme, said enzyme having as a first substrate the terminal reducing N-acetylglucosamine residue of chitin and having as a second substrate the non-reducing glucose residues of .beta.(1->3) linked glucans, said enzyme catalyzing the formation of a .beta.(1->4) linkage between said N-acetylglucosamine and said glucose.

9. An enzyme as defined in claim 8, wherein said fungal species is selected from the group consisting of Saccharomyces cerevisiae, Candida albicans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, and Aspergillus fumigatus.

10. An enzyme as defined in claim 9, wherein said fungal species is Saccharomyces cerevisiae.

11. An enzyme as defined in claim 9, wherein said fungal species is Candida albicans.

12. A composition comprising an inhibitor of an enzyme as defined in claim 8.

13. An antifungal composition as defined in claim 1, wherein said biologically acceptable carrier comprises a pharmaceutically acceptable carrier.

14. A method of preventing fungal infection of a plant, said method comprising applying to said plant, a prophylactically effective amount of an antifungal composition as defined in claim 1.

15. A method of preventing fungal infection in an animal, said method comprising administering to said animal, a prophylactically effective amount of an antifungal composition as defined in claim 1.

16. A method as defined in claim 15, wherein said administration is performed by a mode selected from the group consisting of topical administration and systemic administration.

17. A method of treating a fungal infection in a plant in need of such treatment, said method comprising applying to said plant, a therapeutically effective amount of a composition as defined in claim 1.

18. A method of treating a fungal infection in an animal in need of such treatment, said method comprising administering to said mammal, a therapeutically effective amount of a composition as defined in claim 1.

19. A method as defined in claim 18, wherein said administration is performed by a mode selected from the group consisting of topical administration and systemic administration.

20. A method for screening for antifungal compounds, said method comprising (a) contacting said compound with an oligosaccharide comprising an N-acetylhexosamine residue linked .beta.(1->4) to a hexose, and (b) detecting any free N-acetylhexose or free hexose.

21. A screening method for identifying antifungal compounds, said method comprising (a) contacting an enzyme as defined in claim 8 with an N-acetylhexosamine and a hexose in the presence of said compound, to form a test mixture;

(b) contacting said enzyme with said N-acetylhexosamine and said hexose in the absence of said compound, to form a control mixture;
(c) detecting any formation of a .beta.(1->4) linkage between said N-acetylhexosamine and said hexose in said test mixture and in said control mixture;
(d) comparing efficiency of said formation of said linkage in said test mixture and in said control mixture;
and (e) selecting as an antifungal compound a test compound that causes a decrease in the efficiency of formation of said linkage in said test mixture relative to the efficiency of formation of said linkage in control said mixture.