AU5141900A - Glycopeptide antibiotics, combinatorial libraries of glycopeptide antibiotics and methods of producing same - Google Patents

Description

WO 00/69892 1 PCT/US00/13679 GLYCOPEPTIDE ANTIBIOTICS, COMBINATORIAL LIBRARIES OF GLYCOPEPTIDE ANTIBIOTICS AND METHODS OF PRODUCING SAME 5 BACKGROUND OF THE INVENTION Related Applications 10 This application claims priority from U.S. Provisional Application No. 60/134,839 filed May 19, 1999. Field of the Invention The present invention relates to glycopeptide compounds and libraries of glycopeptide compounds structurally analogous to known glycopeptide antibiotics and methods of generating those libraries. 15 The compounds contain modified carbohydrate moieties. The libraries are generated using combinatorial chemical techniques that produce a diverse set of carbohydrate functionalities conjugated to an oligopeptide. Background of the Invention 20 Glycopeptide antibiotics are characterized by having at least one saccharide group chemically bonded to a rigid peptide structure having a cavity or cleft which acts as a binding site for the substrate used in bacterial cell wall synthesis. The glycopeptide antibiotics are further categorized into various subclasses depending on the identity and interconnections of the amino acids comprising the peptide backbone and the number and substitution pattern of the sugar residues in the molecule. The 25 glycopeptide antibiotics are generally active against Gram-positive bacteria but relatively ineffective against Gram-negative bacteria. Most notable among the glycopeptide antibiotics is vancomycin. Vancomycin is produced by Amycolatopsis orientalis, and is often referred to as "the drug of last resort" because it is effective 30 against most multi-drug-resistant gram positive bacteria. However, in recent years vancomycin resistant strains of some bacteria have emerged. [Cohen M., (1992); Neu H., (1992)]. It is estimated that 5-25% of enterococcal strains in hospitals are now resistant to vancomycin [Axelsen, P.H. et al. (1997)]. Most feared among the bacteria is Staphylococcus aureus, which can result in dangerous respiratory and blood infections. Vancomycin-resistant and vancomycin-insensitive strains of this 35 bacterium have also been recently reported [Milewski (1996)]. The structural formula of vancomycin is shown below and is characterized by a disaccharide moiety covalently linked to a heptapeptide structure. The structure of vancomycin places it in a class of molecules referred to as the "dalbaheptides." [Malabarba A., et al. (1997a)] Dalbaheptides in general WO 00/69892 2 PCT/US00/13679 are characterized by the presence of seven amino acids linked together by peptide bonds and held in a rigid conformation by cross-links through the aromatic substituent groups of at least five of the amino acid residues. In the heptapeptide structure of vancomycin, which is commonly referred to as the "aglycone" of vancomycin, the aromatic side-chains of amino acids 2, 4, and 6 are fused together 5 through ether linkages. The side-chains of amino acids 5 and 7 are joined via a carbon-carbon bond. Amino acids 1 and 3 are leucine and asparagine, respectively. Other naturally-occurring glycopeptide antibiotics are similar to vancomycin in that they have a glucose residue linked to the aromatic substituent on amino acid 4 through formation of a bond with a phenolic hydroxyl group. The glucose residue, in turn, is linked through its vicinal hydroxyl position to a unique amino sugar, either L 10 vancosamine. The sugars have been separately removed from glycopeptide antibiotics, and it has been found that the presence of both sugars enhances the pharmacokinetic properties of this class of antibiotics. [Nagarajan R. (1988), (1991), (1993] HO

NH

2 O OH SH OH 0 OO Cl O 0 H H 6,0M)4 \ H H 6 2 15 H O,,,, Cl /X ?". OH 0 N HN "NN ., NH CH3 HH 'H N 0 NH H NH O O N-H 0/ H HO /1". O O H 5

H

2 N OH HO OH 15 (I) The anti-microbial activity of vancomycin is known to be due to its ability to interfere with biosynthesis of the bacterial cell wall. [Nagarajan R. (1993)]. NMR evidence shows that the heptapeptide chain of 20 vancomycin forms a number of hydrogen bonds with the D-alanyl-D-alanine terminus of the disaccharide-pentapeptide precursors used to form the cell wall. [see, e.g., Prowse W., et al. (1995); WO 00/69892 3 PCT/US00/13679 Pierce C., et al. (1995); Williams D. et al. (1988)]. This interaction of vancomycin with cell wall precursors apparently inhibits or prevents the subsequent transglycosylation and/or transpeptidation steps of cell wall assembly. Supporting this mode of action is the fact that vancomycin-resistant strains of bacteria are found to produce a pentapeptide precursor terminating in a D-alanyl-D-lactate sequence. 5 It is hypothesized that the reduced effectiveness of vancomycin against resistant strains is due to reduced hydrogen bonding interactions between the drug and the D-alanyl-D-lactate substrate. The affinity of vancomycin for D-alanyl-D-lactate is estimated to be 2-3 orders of magnitude (4.1 kcal/mol) less than for D-alanyl-D-alanine. [Walsh C. (1993)]. 10 The sugar residues of the vancomycin and other glycopeptide antibiotics have been shown to affect binding activities. Structural changes in the sugar residues can produce significant changes in antibiotic activity. [Malabarba (1997), Nagarajan, R. (1993)] It has been proposed that the sugar residues on the glycopeptide antibiotics may enhance the avidity of these molecules for surface-bound peptide ligands. At least two different mechanisms for enhancing avidity have been proposed. [Kannan (1988), Gerhard 15 (1993), Allen (1997)] For example, it has been proposed that the biological activity of vancomycin, along with that of many other glycopeptide antibiotics, is enhanced by dimerization due to bonding interactions at the convex (non-ligand binding) face of the molecule. [Williams D., et al. (1993); Gerhard U., et al., (1993)] 20 Dimerization is believed to be facilitated by the disaccharide groups of the vancomycin molecule, and is thought to influence activity by increasing the avidity of vancomycin for surface-bound D-Ala-D-Ala peptide ligands. [Williams, (1998)] Structural evidence for dimerization has been obtained from both NMR and crystallographic studies, and it has been found that there are significant differences in the stability of the dimers formed in solution by different glycopeptide antibiotics. [MacKay (1994)] It is 25 proposed that differences in the dimerization constants may account at least partially for the remarkable differences in biological activity of different glycopeptide antibiotics which otherwise have very similar binding affinities for the natural d-Ala-d-Ala substrate. [Williams (1998)] A second mechanism for enhancing activity has also been proposed for the glycopeptide antibiotic 30 teicoplanin, which contains an N-alkyl chain on one of the sugars. It is suggested that this N-alkyl chain increases the effective avidity of teicoplanin for surface-bound D-Ala-D-Ala ligands by interacting with the membrane, thus "anchoring" the teicoplanin molecule at the membrane surface. [Beures (1995)] It should be noted that the attachment of hydrophobic substituents to the vancomycin carbohydrate moiety appears to enhance activity against vancomycin-resistant strains. For example, 35 attaching a hydrophobic group to the vancosamine sugar by alkylation on the amine nitrogen increases activity against vancomycin-resistant strains by two orders of magnitude. [Nagarajan (1991)] It is speculated that the lipophilic groups locate the antibiotic at the cell surface and make ligand binding an WO 00/69892 4 PCT/US00/13679 intramolecular process, which may partially overcome the decreased binding affinity for D-Ala-D-Lac. Hence, although the sugars on the glycopeptide antibiotics do not appear to interact substantially with the peptide substrates, they play a very important role in increasing the biological activity. Therefore, one potentially successful strategy for the design of new antibacterial agents based on the glycopeptide 5 class of antibiotics involves modifying the carbohydrate portions of the molecules. [Malabarba (1997a)] Related members of the vancomycin class of glycopeptide antibiotics include the ristocetins, the eremomycins, the avoparcins and teicoplanin. Several of these compounds are shown, together with 10 vancomycin in Figure 1. The chemical structures of all of these compounds include a dalbaheptide structure as the aglycone core, with minor differences in the amino acids and in cross-linking, but differ from each other most distinctively in terms of the nature of the sugar residues as well as the number and points of attachment of sugar residues to the aglycone core. It is known that biological activities of vancomycin-type antibiotics vary depending on the nature of the sugar residues. 15 One approach to obtaining new drug candidates derived from vancomycin and other glycopeptide antibiotics has involved chemical modification of one or more sugar residues of the naturally occurring glycopeptide. For instance, as noted previously, an alkyl chain can be attached to a sugar residue of the molecule, such as at the amino group of the amino sugar. [Cooper, R. et al. (1996)]. Other semi 20 synthetic approaches have involved traditional esterification and amidation methodologies applied to the peptide portion of the molecule. [Malabarba, A. et al. (1997b)] The attachment of lipophilic alkyl chains to the antibiotic has been proposed to afford better membrane anchoring, thereby increasing the effective activity of glycopeptide at the cell wall. [Felmingham, D. (1993)] The presence of an additional sugar has also produced compounds having enhanced activity, which may be due to their 25 improved dimerization ability. [Malabarba A., et al. (1997a); Allen N. et al., (1997]. Other semi synthetic approaches to modification of the vancomycin molecule have involved derivatization of the polypeptide binding pocket. [Pavlov A., et al. (1993)] Previous efforts in producing new compounds having increased activity against vancomycin-resistant 30 strains have typically involved a directed synthesis of a specific target derivative of a natural glycopeptide. This is a slow and relatively tedious process requiring a great deal of time and expense to obtain a suitable set of drug candidates for use in screening for activity. It is desirable to develop a combinatorial approach to the synthesis of new drug candidates based on the glycopeptide antibiotics. Recognizing this, Griffin and coworkers synthesized a combinatorial library of vancomycin derivatives 35 in which different peptide chains were appended to the carboxylate on amino acid 7. No candidates were identified which had significantly improved activity compared with the underivatized natural product for either vancomycin-sensitive or vancomycin-resistant strains. The failure of the effort WO 00/69892 5 PCT/US00/13679 highlights a key requirement for a strategy involving the synthesis of a library related to a natural product: it is imperative to introduce substituents at positions on the molecule where there is evidence that such substitutions will have an effect on activity. In the case of the glycopeptide antibiotics, changes to the carbohydrate portions of the molecules would seem to be warranted in light of the 5 relatively large role played by the sugar residues in increasing activity. The use of enzymes to generate glycosylated vancomycin derivatives wherein the saccharide residue carries a variety of functionalizations has been proposed and explored. [Solenberg (1997)] However, the range of compounds that can be prepared using enzymes in this manner is limited by the availability of enzymes specific to the desired functionalized saccharide residue. This has only been demonstrated for glucose .0 and xylose; vancosamine has never been attached using the enzyme method and no compounds displaying activity have been produced using the enzyme method. No other strategies for making libraries of glycopeptide antibiotics in which the carbohydrate moieties are combinatorially varied have been reported. 15 Comparison of the natural products have made it clear that the nature and placement of the sugars on the glycopeptide antibiotics play critical roles in antibiotic activity. Furthermore, there is some information from semi-synthetic efforts about positions on the carbohydrates that may be important in activity. For example, we have already noted that some vancomycin derivatives containing hydrophobic substituents on the vancosamine nitrogen show improved activity against vancomycin 20 resistant strains. However, there have been no reports of modifications on the glucose residue of vancomycin which have affected activity. In fact, for glycopeptide antibiotics containing two or more sugars attached to amino acid 4, there is no suggestion in the literature that the sugar directly attached to the aglycone can be modified to improve activity. It has even been argued that the glucose residue "has no independent contribution to binding, and it is likely that its role with respect to the binding constant 25 is merely to position the vancosamine optimally relative to the aglycon portion." [Kannan et al. (1988)] The structure-activity relationships among the vancomycin-like glycopeptide antibiotics show that the presence of an amino sugar at the residue 6 benzylic position and an N-alkyl or N-aryl substituted amino sugar at the amino acid-4 position increases antibiotic activity against both VRE and VSE. 30 However, these trends do not always hold against other gram-positive bacteria such as the Staphylococci and Streptococci. Furthermore, no studies have addressed the effects of introducing functionality on the sugar groups other than N-alkylation, N-acylation, formation of N-oxides or modification of ester groups at C-6. Because the nature and placement of the sugars on glycopeptide antibiotics play such critical roles in antibiotic activity, many more studies are needed to optimize the 35 sugar substituents. Such studies could not only lead to better antibiotics against vancomycin-resistant bacteria, but might provide more information about the mechanism of interaction at bacterial membranes. Preparation of derivatives with different sugar substituents will not only probe the sugar's WO 00/69892 6 PCT/US00/13679 role in currently proposed interactions, but may also lead to the discovery of new specific or non specific interactions of the glycopeptide antibiotics at the cell surface. For reviews regarding the structure activity relationships of natural and semisynthetic glycopeptide antibiotics see Malabarba et al. Med. Res. Rev., 1997, 17, 69; Nagarajan, Antimicrob. Agents Chemother., 1991, 35, 605; Nagarajan, 5 J. Antibiotics, 1993, 46, 1181; Cooper and Thompson, Ann. Rep. Med. Chem., 1996, 31, 131; Malabarba et al., Eur. J Med. Chem., 1997, 32, 459; Allen et al. J. Antibiotics, 1997, 50, 677. Combinatorial strategies have been successfully applied to the synthesis of peptide, nucleic acid, and various small molecule libraries, however, they have not been extensively employed to make 10 carbohydrate-based libraries. Most of the approaches to production of carbohydrate libraries have been conducted in solution. A solid phase approach to making diverse libraries of di- and tri-saccharide compounds has also been reported. [Liang et al. (1996)]. A solid phase method permits reactions to be driven to completion by using a large excess of reactants. The solid phase approach also permits spatial resolution of the product compounds. Glycopeptide libraries have been produced on the solid phase in 15 which amino acids were varied. However, no suggestion has been made that glycopeptide antibiotics can be made using a solid-phase method. SUMMARY OF THE INVENTION 20 This invention is directed to glycopeptide compositions which have the formula A,-A 2

-A

3

-A

4 -As-A 6 A 7 , in which each dash represents a covalent bond; wherein the group Al comprises a modified or unmodified a-amino acid residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl, 25 guanidinyl, carbamoyl, or xanthyl; wherein each of the groups A 2 to A 7 comprises a modified or unmodified a-amino acid residue, whereby (i) the group A 1 is linked to an amino group on the group

A

2 , (ii) each of the groups A 2 , A 4 and A 6 bears an aromatic side chain, which aromatic side chains are cross-linked together by two or more covalent bonds, and (iii) the group A 7 bears a terminal carboxyl, ester, amide, or N-substituted amide group. 30 It is further required that one or more of the groups A 1 to A 7 is linked via a glycosidic bond to one or more glycosidic groups each having one or more sugar residues; wherein at least one of said sugar residues bears one or more substituents of the formula YXR, N+(R 1

)=CR

2

R

3 , N=PRR 2

R

3 , N+RR 2

R

3 or

P+RIR

2

R

3 in which the group Y is a single bond, O, NR 1 or S; the group X is O, NR 1 , S, SO 2 , C(O)O, 35 C(S)O, C(S)S, C(NRI)O, C(O)NRI, or halo (in which case Y and R are absent); and R, R 1 , R 2 and R 3 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; and any WO 00/69892 7 PCT/US00/13679 pharmaceutically acceptable salts thereof; provided that: when Y is a single bond and X is O, NH or N alkyl, then R is not hydrogen; X and Y are not both O; X and Y are not S and O, or O and S, respectively; and if two or more of said substituents are present, they can be the same or different; and 5 provided that: when A 4 is linked to a glucose residue substituted at its 2-position by a group YXR in which Y is a single bond, X is NH and R is alkanoyl, then said glucose residue is further substituted by another sugar residue; when A4 is linked to a disaccharide in which a glucose residue bears an N substituted aminohexose residue, then said glucose residue bears at least one group YXR which is not alkanoyloxy; and when A 4 is linked to an acylaminoglucuronate residue, then said 10 acylaminoglucuronate residue is further substituted by a sugar residue. This invention is also directed to a chemical library comprising a plurality of glycopeptides, each having the formula described hereinabove. 15 This invention is further directed to a method of preparing a glycopeptide comprising: (a) selecting: (i) an aglycone that is soluble in one or more organic solvents, is derived from a glycopeptide antibiotic, and which aglycone has exactly one free phenolic hydroxyl group; and (ii) a protected first glycosyl donor; (b) allowing a non-enzymatic glycosylation reaction to proceed in an organic solvent such that a first glycosidic bond is formed, which links said free phenolic hydroxyl 20 group to the anomeric carbon of the first glycosyl donor to provide a pseudoaglycone having a protected first glycosyl residue; (c) selectively removing one protecting group from the first glycosyl residue to provide a pseudoaglycone bearing exactly one free hydroxyl group on the first glycosyl residue; (d) selecting a second protected glycosyl donor; and (e) allowing a lion-enzymatic glycosylation reaction to proceed in an organic solvent such that a second glycosidic bond is formed, 25 which links said free hydroxyl group on the pseudoaglycone to the anomeric carbon of the second glycosyl donor. This invention is further directed to a method of preparing a glycopeptide comprising: (a) selecting a glycopeptide antibiotic that is soluble in one or more organic solvents; 30 (b) contacting the glycopeptide antibiotic with a Lewis acid, and allowing a degradation reaction to proceed such that a sugar residue is removed, producing a pseudoaglycone having exactly one free hydroxyl group on a sugar residue of the pseudoaglycone; (c) selecting a protected glycosyl donor; and (d) allowing a non-enzymatic glycosylation reaction to proceed in an organic solvent such that a glycosidic bond is formed which links the free hydroxyl group 35 on the pseudoaglycone to the anomeric carbon of the glycosyl donor.

WO 00/69892 8 PCT/US00/13679 This invention is further directed to a method for preparing a glycopeptide comprising: (a) selecting a protected glycopeptide having a free primary hydroxyl group only at the 6-position of a hexose residue linked to A 4 ; (b) contacting the protected glycopeptide with a compound ArSO 2 G in which Ar is an aryl group and G is a leaving group under conditions effective to allow reaction of the free primary hydroxyl 5 group to form a glycopeptide sulfonate ester; (c) contacting the glycopeptide sulfonate ester with a nucleophile under conditions effective to allow displacement of a sulfonate group to produce a substituted glycopeptide. This invention is further directed to a method for producing a chemical library by performing at least 10 two steps in a combinatorial format to produce the chemical library, wherein each of the steps introduces a substituent on a glycopeptide. This invention is further directed to another method for producing a chemical library by performing at least two steps which are performed in a combinatorial format; wherein at least one of the steps 15 comprises a glycosylation reaction which introduces a substituted sugar residue. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 contains structure diagrams of vancomycin and related glycopeptide antibiotics. 20 Figure 2 illustrates a preparation of a protected aglycone of vancomycin suitable for glycosylation. Figure 3 illustrates another preparation of a protected aglycone of vancomycin suitable for glycosylation. 25 Figure 4 illustrates preparation of a sugar useful in glycosylation of an aglycone. Figure 5 illustrates glycosylation of a phenol which is a model compound for an aglycone. 30 Figure 6 illustrates glycosylation of a vancomycin aglycone. Figure 7 is a scheme for introduction of an amino substituent onto the glucose C6 position of vancomycin. 35 Figure 8 illustrates further functionalization of a glucose C6 amino substituent on vancomycin. Figure 9 illustrates substitution of both the glucose C6 position and the vancosamine nitrogen.

WO 00/69892 9 PCT/US00/13679 Figure 10 illustrates introduction of thio substituents at the glucose C6 position of vancomycin. Figure 11 illustrates removal of the A 1 amino acid of vancomycin and protection of the product to allow reaction at the A 2 terminal amino group. 5 Figure 12 illustrates reactions at the A 2 terminal amino group. Figure 13 illustrates preparation of a vancomycin dimer through the glucose C-6 position. 10 Figure 14 illustrates the substitution of a linker group on the vancosamine nitrogen. Figure 15 illustrates preparation of a suitably protected pseudoaglycone. Figure 16 is a graph of a time-kill study of N-decyl-C6-aminotriazole vancomycin against vancomycin 15 resistant enterococcusfaecium. Figure 17 is a graph of a time-kill study of N-4-(4-chlorophenyl)benzyl-C6-aminotriazole vancomycin against vancomycin-resistant enterococcusfaecium. 20 Figure 18 is a graph of a time-kill study of vancomycin against vancomycin-resistant enterococcus faecium. Figure 19 is a graph of a time-kill study of N-4-(4-chlorophenyl)benzyl vancomycin against vancomycin-resistant enterococcusfaecium. 25 DETAILED DESCRIPTION OF THE INVENTION Definitions 30 A "glycoconjugate" comprises any molecule linked to at least one carbohydrate of any size. The molecule can be a peptide or protein, a nucleic acid, a small molecule, a lipid, or another carbohydrate; it may be of natural or non-natural origin. A "glycopeptide" is a glycoconjugate comprising a peptide linked to at least one carbohydrate. A "glycopeptide antibiotic" is one of the naturally occurring glycopeptides with antibacterial activity, including, e.g., vancomycin, teicoplanin, ristocetin, 35 chloroeremomycin and avoparicin.

WO 00/69892 10 PCT/US00/13679 An "aglycone" is the result of removing the carbohydrate residues from a glycopeptide, leaving only a peptide core. A "pseudoaglycone" is the result of removing only one of two sugar residues of a disaccharide residue linked to residue A 4 of a glycopeptide. Thus, a pseudoaglycone comprises an aglycone in which A 4 is linked to a monosaccharide residue. 5 A "dalbaheptide" is a glycopeptide containing a heptapeptide moiety which is held in a rigid conformation by cross-links between the aromatic substituent groups of at least five of the seven a amino acid residues, including a cross-link comprising a direct carbon-carbon bond between the aryl substituents of amino acid residues 5 and 7, and aryl ether cross-links between the substituents of amino 10 acid residues 2 and 4, and 4 and 6. Amino acid residues 2 and 4-7 in different dalbaheptides are those found in the naturally occurring glycopeptide antibiotics. These amino acid residues differ only in that residues 2 and 6 do not always have a chlorine substituent on their aromatic rings, and in that substitution on free hydroxyl or amino groups may be present. Amino acid residues 1 and 3 may differ substantially in different dalbaheptides; if both bear aryl substituents, these may be cross-linked. 15 Molecules having a dalbaheptide structure include, e.g., the glycopeptide antibiotics mentioned above. The term "alkyl" refers to an acyclic or non-aromatic cyclic group having from one to twenty carbon atoms connected by single or multiple bonds. An alkyl group may be substituted by one or more of halo, hydroxyl, protected hydroxyl, amino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, COOH, aroyloxy, 20 alkylamino, dialkylamino, trialkylammonium, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl, heterocyclic, CONH 2 , CONH-alkyl, CON(alkyl) 2 , COO aralkyl, COO-aryl, COO-alkyl or phosphonium substituted by any combination of alkyl, aryl, aralkyl or heterocyclic. 25 The term "aryl" refers to a group derived from a non-heterocyclic aromatic compound having from six to twenty carbon atoms and from one to four rings which may be fused or connected by single bonds. An aryl group may be substituted by one or more of alkyl, aralkyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, halo, hydroxyl, protected hydroxyl, amino, hydrazino, alkylhydrazino, arylhydrazino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, 30 trialkylammonium, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl, COO-alkyl, COO-aralkyl, COO-aryl, CONH 2 , CONH-alkyl, CON(alkyl) 2 or phosphonium substituted by any combination of alkyl, aryl, aralkyl or heterocyclic. The term "aralkyl" refers to an alkyl group substituted by an aryl group. 35 The term "heterocyclic" refers to a group derived from a heterocyclic compound having from one to four rings, which may be fused or connected by single bonds; said compound having from three to twenty ring atoms which may be carbon, nitrogen, oxygen, sulfur or phosphorus. A heterocyclic group WO 00/69892 11 PCT/US00/13679 may be substituted by one or more of alkyl, aryl, aralkyl, halo, hydroxyl, protected hydroxyl, amino, hydrazino, alkylhydrazino, arylhydrazino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, trialkylammonium, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl, COO-alkyl, COO-aralkyl, COO-aryl, CONH 2 , CONH 5 alkyl, CON(alkyl) 2 or phosphonium substituted by any combination of alkyl, aryl, aralkyl or heterocyclic. The terms "alkoxy," "aryloxy" and "aralkyloxy" refer to groups derived from bonding an oxygen atom to an alkyl, aryl or aralkyl group, respectively. The terms "alkanoyl," "aroyl" and "aralkanoyl" refer to 10 groups derived from bonding a carbonyl to an alkyl, aryl or aralkyl group, respectively. The terms "heterocyclic-alkyl" and "heterocyclic-carbonyl" refer to groups derived from bonding a heterocyclic group to an alkyl or a carbonyl group, respectively. The term "heterocyclic-alkyl-carbonyl" refers to a group derived from bonding a heterocyclic-alkyl group to a carbonyl group. The term "protected hydroxyl" refers to a hydroxyl group bonded to a group which is easily removed to regenerate the free 15 hydroxyl group by treatment with acid or base, by reduction, or by exposure to light. The term "Lewis acid", as used herein, refers to any substance that can accept an electron pair from a base, with the exception of the mineral acids and organic carboxylic acids. The term "organic solvent", as used herein, refers to non-aqueous solvents, preferably to ketones, halogenated solvents, ethers, 20 esters and non-heterocyclic aromatic solvents. A "chemical library" is a synthesized set of compounds having different structures. The chemical library may be screened for biological activity to identify individual active: compounds of interest. 25 A "glycosyl donor" is a sugar or glycosidic residue that bears an anomeric leaving group, preferably a sulfoxide, which may be activated to render the anomeric carbon susceptible to reaction with a nucleophile to displace the activated group, thereby forming a glycosidic bond. The term "leaving group" as used herein is a group easily displaced from a sulfonyl group by a 30 nucleophile. Examples of leaving groups are halo, alkoxy, aryloxy, alkanoyloxy and arylsulfonyloxy. The term "DMF" refers to N,N-dimethylformamide; "THF" refers to tetrahydrofuran; "TFA" refers to trifluoroacetic acid; "EtOAc" refers to ethyl acetate; "MeOH" refers to methanol; "MeCN" refers to acetonitrile; "Tf" refers to the trifluoroacetyl group; "DMSO" refers to dimethyl sulfoxide; "DIEA" 35 refers to diisopropylethylamine; "All" in structural formulas refers to the allyl group; "Fmoc" refers to 9-fluorenylmethyloxycarbonyl; "HOBt" refers to 1-hydroxybenzotriazole and "OBt" to the 1 oxybenzotriazolyl group; "PyBOP" refers to benzotriazol- 1 -yl-oxytripyrrolidine-phosphonium WO 00/69892 12 PCT/US00/13679 hexafluorophosphate; "Su" refers to the succinimidyl group; "HBTU" refers to O-benzotriazol-1 -yl N,N,N',N'-tetramethyluronium hexafluorophosphate; "aloc" refers to allyloxycarbonyl; and "CBz" refers to benzyloxycarbonyloxy. 5 The glycopeptide compositions of this invention have the formula A 1

-A

2

-A

3

-A

4 -As-A 6

-A

7 , in which each dash represents a covalent bond; wherein the group A 1 comprises a modified or unmodified a amino acid residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl, guanidinyl, carbamoyl, or xanthyl; wherein each of the groups A 2 to A 7 comprises a modified or unmodified a-amino acid residue, 10 whereby (i) the group A 1 is linked to an amino group on the group A 2 , (ii) each of the groups A 2 , A 4 and

A

6 bears an aromatic side chain, which aromatic side chains are cross-linked together by two or more covalent bonds, and (iii) the group A 7 bears a terminal carboxyl, ester, amide, or N-substituted amide group. 15 It is further required that one or more of the groups A 1 to A 7 is linked via a glycosidic bond to one or more glycosidic groups each having one or more sugar residues; wherein at least one of said sugar residues bears one or more substituents of the formula YXR, N (R)=CR 2

R

3 , N=PRIR 2

R

3 , N RR 2

R

3 or P RiR 2

R

3 in which the group Y is a single bond, O, NRi or S; the group X is O, NRI, S, SO 2 , C(O)O, C(O)S, C(S)O, C(S)S, C(NR 1 )O, C(O)NRI, or halo (in which case Y and R are absent); and R, R 1 , R 2 20 and R 3 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; and any pharmaceutically acceptable salts thereof; provided that: when Y is a single bond and X is O, NH or N-alkyl, then R is not hydrogen; X and Y are not both O; X and Y are not S and O, or O and S, respectively; and if two or more of said substituents are present, they can be the same or different; and 25 provided that: when A 4 is linked to a glucose residue substituted at its 2-position by a group YXR in which Y is a single bond, X is NH and R is alkanoyl, then said glucose residue is further substituted by another sugar residue; when A 4 is linked to a disaccharide in which a glucose residue bears an N substituted aminohexose residue, then said glucose residue bears at least one group YXR which is not 30 alkanoyloxy; and when A 4 is linked to an acylaminoglucuronate residue, then said acylaminoglucuronate residue is further substituted by a sugar residue. Modified amino acid residues include amino acid residues whose aromatic groups have been substituted by halo, alkyl, alkoxy, alkanoyl, or other groups easily introduced by electrophilic substitution reactions 35 or by reaction of phenolic hydroxyl groups with alkylating or acylating agents; and amino acid residues which have protecting groups or other easily introduced substituents on their hydroxyl or amino groups, including, but not limited to alkyl, alkanoyl, aroyl, aralkyl, aralkanoyl, carbamoyl, alkyloxycarbonyl, WO 00/69892 13 PCT/US00/13679 aralkyloxycarbonyl, aryloxycarbonyl, alkylsulfonyl, arylsulfonyl, heterocyclic, heterocyclic-alkyl or heterocyclic-carbonyl substituents. Examples of preferred protecting groups include acetyl, allyloxycarbonyl (aloc), CBz, allyl, benzyl, p-methoxybenzyl and methyl. Modifications of hydroxyl groups occur on phenolic hydroxyl groups, benzylic hydroxyl groups, or aliphatic hydroxyl groups. 5 Other amino acid residues, in addition to A 2 , A 4 and A 6 , may be cross-linked through their aromatic substituent groups. Preferably, residues A 2 to A 7 of the glycopeptide are linked sequentially by peptide bonds and are cross-linked as in a dalbaheptide, as defined hereinabove. The preferred glycopeptides thus have a 10 peptide core in which the residues are linked as in the natural glycopeptide antibiotics, as shown in Figure 1. Substitution of different amino acids at A 3 is permitted, as are modified amino acid residues at all positions, as described hereinabove. In a preferred embodiment of this invention, residue A 1 is an a-amino acid, which may be substituted on the terminal amino group by alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, heterocyclic alkyl, alkylsulfonyl, arylsulfonyl, 15 guanidinyl, carbamoyl, or xanthyl, and the structures and interconnections of A] to A 7 are those of vancomycin, i.e., the glycopeptide has the heptapeptide core of vancomycin, subject to the amino acid modifications and substitutions on A, and A 7 described hereinabove. The glycopeptides of this invention contain at least one glycosidic group attached through a glycosidic 20 bond to the residues A 1 to A 7 . Preferably, a glycosidic group is attached to residue A 4 . In one preferred embodiment of the invention, a hexose residue is bonded directly to A 4 and is substituted by a group YXR. In the group YXR, when Y represents a single bond, XR is bonded directly to a carbon atom of the sugar residue. When X is halo, Y and R are absent and the halo group is attached directly to a carbon atom of the sugar residue, as in compounds LXX, CX, and others described hereinbelow. It is 25 not intended that YXR represent a peroxide, OOR, or the groups OSR or SOR. The hexose residue may be a monosaccharide residue or part of a disaccharide or oligosaccharide residue. Still more preferably, the group YXR is located at the C-6 position of the hexose. Most preferably, Y is a single bond and X is NRI or S, i.e., a substituted amino or thio group is attached to the C-6 position of the hexose. In one embodiment of the invention, a glycosidic group is also attached to residue A 6 . In 30 another preferred embodiment of the invention, a hexose residue linked to A 4 is substituted by an ylide group having the formula N=PR 1

R

2

R

3 , in which R 1 , R 2 and R 3 are preferably aryl. Although this invention includes all of the compounds described hereinabove, in a preferred embodiment of this invention, the glycopeptide composition is derived from vancomycin. Accordingly, 35 this invention includes methods for the selective derivatization of the C-6 position of the glucose residue of vancomycin. We have found that substituents at this position can have a dramatic effect on biological activity. For example, replacement of the glucose C-6 hydroxyl with the substituent WO 00/69892 14 PCT/US00/13679 N 0 H N N (2-thio-6-azathymine), attached to the glucose C-6 position through the sulfur, causes an increase in 5 activity against all strains tested. Furthermore, when this position is substituted and there is an additional substitution on the vancosamine sugar, the biological activity is affected in an unpredictable and non-additive way. For example, when the above 2-thio-6-azathymine substitution at C-6 of glucose is made concurrently with substitution of a 4-(4-chlorophenyl)benzyl group on the vancosamine nitrogen, which also increases activity against all strains, the activity against some strains of bacteria is 10 increased above the activity observed for either substitution alone, while against other strains, the activity is below that even of vancomycin. A strategy to introduce a suitable set of protecting groups and to differentiate the C-6 hydroxyl group from all other hydroxyl groups of a glycopeptide having a hexose residue at A 4 is illustrated below in 15 Scheme 1, showing functionalization of the glucose C-6 hydroxyl of vancomycin.

WO 00/69892 PCT/US00/13679 15 Scheme 1

HONHR

2 HO O H 0

OR

3 0 0 Cl 0 0 H / H HO,,, OH OH 0H H ,,H 0 H .O 0' N N N N NH CH 3 H H I,/H ' H_ NH O0 N-R2 H2 R0 HO H 2N N 0 0H HO OH OH Aloc-Su, (Vancomycin)

R=R

2

=R

3 = H NaHCO 3 NaHC03R2=Aloc ' R,=H, R3=H Allyl bromide,

R

2 =Aoc, R H, R 3 = NaHCO 3 - 3

R

2 =Aloc, R 1 =Allyl, R 3 =H 0 - C I - R2=Aloc, RI=Allyl, R3= -S O II 0 Protection of both amines by a similar group requires using excess acylation reagent while selective 5 protection of the N-methyl leucine residue is known, allowing selective functionalization of the vancosamine amine group. See Pavlov et al., J. Antibiotics, 1993, 46, 1731. Selectively introducing the mesitylenesulfonyl group at the glucose-6-position differentiates this position from the other hydroxyl groups and allows further reaction to displace the mesitylenesulfonyl group, affording many derivatives. A variety of functional groups are introduced at the glucose-6 position by using common 10 methods for nucleophilic displacement of primary arylsulfonyl groups directly, or by further synthetic modification of initial displacement products, including azido and iodo groups. For example, the iodo WO 00/69892 16 PCTUS00/13679 group is displaced by a variety of nucleophiles to produce additional C6-derivatives. A preferred nucleophile is a thiol compound, especially a heterocyclic thiol. Modification of an azido group at the 6-position is performed, e.g., by reducing the azido group to an amino group, which in turn is functionalized by means of reductive alkylation, nucleophilic substitution, or other amino-group 5 reactions well known to those skilled in the art. These approaches are illustrated in Figures 7-10, and in many of the Examples. In a preferred embodiment of the invention, an azido group is partially reduced by reaction with a phosphine compound to produce an iminophosphorane. Specific derivatives obtained by the aforementioned methods, and the antibiotic activities of these 10 derivatives, are presented hereinafter. For example, introduction of substituted diazines or substituted triazines, e.g., 2-thio-6-azathymine, at the glucose-6 position affords an increase in activity against all five strains of bacteria tested, including VRE. Large hydrophobic groups, especially those bearing a full or partial positive charge, e.g., N (R 1

)=CR

2

R

3 , N=PRiR 2

R

3 , NRIR 2

R

3 or P+RIR 2

R

3 in which one or more of R 1 , R 2 and R 3 are bulky groups, also increase activity. 15 Introduction of N-decyl and N-4-(4-chlorophenyl)benzyl groups at the vancosamine amine group of glucose-6 iodo derivatives effects further enhancement of activity beyond that observed from halide substitution alone. These two hydrophobic groups were previously shown to increase the activity of glycopeptide antibiotics against VSE and VRE when introduced onto the amine group of the 20 vancosamine or 4-epi-vancosamine residues of the amino acid-4 disaccharide. Since each of the hydrophobic and halide substitutions individually increases the antibiotic activity, it was anticipated that combining both changes into one structure would afford even better activity. Unexpectedly, the effects of these modifications are not additive and this result could not have been anticipated. While the individual changes increase activity, the combination of changes affords products that show 25 bacterial strain dependence affording either a combined increase in activity or a combined decrease in activity, not only below the individual changes but below vancomycin itself. Therefore, by introducing changes in the glucose residue, a glycopeptide is produced in which the changes made to the vancosamine result in activities that could not have been anticipated in this unnatural system. 30 Preferred glycopeptide compounds of this invention are: N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-mesitylenesulfonated vancomycin (compound XLII; see Examples). 35 Glucose-C6-2-thio-6-azathymine vancomycin (LXIV). Glucose-C6-2-thio-4-hydroxy-6-methylpyrimidine vancomycin (LXXVIII).

WO 00/69892 17 PCT/US00/13679 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C 6 -2-thio-5-amino-1,3,4-thiadiazole vancomycin (LXXXIII). N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-4-amino-3-hydrazino-1,2,4-triazole 5 vancomycin (LXXXIV). N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-4-hydroxy-6-methylpyrimidine vancomycin (LXXXV). 10 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-6-azathymine vancomycin (LXXXVI). Vancosamine-N-4-(4-chlorophenyl)benzyl-glucose-C 6 -iodo vancomycin (LXXIIa). Glucose-C6-N-2-quinoxalinyl-vancosamine-N-4-(4-chlorophenyl)benzyl vancomycin (LII). 15 Vancosamine-N-4-(4-chlorophenyl)benzyl-glucose-C6-S-3-amino-5-mercapto- ,2,4-triazole vancomycin (LXXIII). Glucose-C6-mesitylenesulfonyl vancomycin (XLI). 20 Glucose-C6-iodo vancomycin (LXX). Glucose-C6-azide vancomycin (XLVI). 25 Glucose-C6-bromo vancomycin (CX). Glucose-C6-amine vancomycin (XLVII). Glucose-C6-hydrazine vancomycin (XLIV). 30 Vancosamine-N-4-(4-chlorophenyl)benzyl-glucose-C6-iminotriphenylphosphorane vancomycin (CXXXVI). The chemical library of compounds of this invention is prepared to explore the effects of introducing a 35 large number of different substituents on glycopeptides on biological activity, especially substitutions on the sugar residues. In any preparation of a chemical library, at least two steps are performed, each of which introduces a substituent group on the glycopeptide. A combinatorial format is established in WO 00/69892 18 PCT/US00/13679 which many different predetermined substituent groups are introduced independently at each of at least two positions, resulting in a library containing a large number of glycopeptides, wherein each possible combination of the predetermined substituent groups is represented. For example, if three positions are to be substituted and 36 different substituent groups (3 sets of 12) are chosen, 1 of each set of 12 to be 5 substituted at each position, the total number of unique compounds (each of which bears 3 substituent groups) in the library will be 12x12x12=1,7 2 8. It is readily apparent that, when a combinatorial synthesis is performed in an automated system, a large number of related compounds may be prepared relatively quickly. Methods for performing combinatorial synthesis are well known and are described in several review articles. [Thompson (1996), Gallop (1994), Gordon (1994), Terrett (1995)] 10 Substituents are introduced on the glycopeptide library compounds of this invention through the use of two different reaction schemes. In one reaction scheme, glycosylation reactions are used to attach sugars bearing desired substituent groups to hydroxyl groups on various positions of glycopeptide antibiotics, aglycones, or pseudoaglycones, as described in detail hereinbelow. The other reaction 15 scheme is the method for derivatizing a hexose C-6 hydroxyl group, as shown in Scheme 1, and as described in the accompanying discussion and in several of the Examples. In construction of the library of this invention, at least two steps are carried out in a combinatorial format. These steps are selected independently from the two reaction schemes outlined hereinabove, such that a library is constructed using either scheme exclusively or a combination of the two. 20 One method suitable for preparing glycopeptide compounds, individually or as part of a chemical library, starts with the synthesis of a suitably protected aglycone. All reactive functional groups of the aglycone (amine, carboxylic acid, phenols, and benzylic alcohols) are suitably protected except for the hydroxyl group on which the sugars are to be attached, preferably the phenolic hydroxyl group on 25 residue 4. The carboxylic acid is protected with a group which is orthogonal to the other protecting groups used, i.e., the carboxylic acid protecting group is not removed under conditions suitable for removal of other protecting groups on the molecule. In addition, protecting groups are used that render the protected aglycone soluble in organic solvents. The protecting groups may either remain on the final glycopeptide compound or may be removed by exposure to acidic or basic conditions, catalytic 30 hydrogenation, or light. When the aglycone is derived from vancomycin, it is preferred that the protecting groups are as follows: carboxybenzyl (CBz) on the amine nitrogen, a benzyl ester group; benzyl, allyl or methyl phenolic ethers on the phenolic hydroxyls of A 5 and A 7 , and acetates on the aliphatic hydroxyls. Alternate methods for preparing aglycones of vancomycin are illustrated in Figures 2 and 3, and in the Examples. 35 This suitably protected aglycone is glycosylated via a non-enzymatic reaction in an organic solvent with a variety of glycosyl donors, thereby forming a glycosidic bond between the aglycone and the WO 00/69892 19 PCT/US00/13679 glycosyl donor. Preferably the glycosyl donors are activated monosaccharide anomeric sulfoxides which are functionalized at the 6 position or elsewhere. These sulfoxide donors are differentially protected so as to allow for selective deprotection of a single hydroxyl after formation of the glycosidic bond. Suitable protecting groups to allow for this selective deprotection are the 2,2-dimethyl 5 acetoacetate group, the 4-azidobutyryl group and any other groups which can be removed in the presence of other protecting groups. A modified sulfoxide glycosylation of the aglycone phenolic hydroxyl group may be accomplished using an acetate or other unhindered ester at C-2 of the sugar as a neighboring group. In this modified 10 glycosylation, as in the preferred glycosylation procedure utilizing activated monosaccharide anomeric sulfoxides, the leaving group at the anomeric center is a sulfoxide moiety which is activated by trifluoromethanesulfonic anhydride (Tf 2 0) in the presence of 2,6-di-t-butylmethylpyridine. The modification to the glycosylation procedure involves addition of BF 3 to the reaction. Without being bound to theory, it is believed that the presence of BF 3 prevents formation of the undesired ortho-ester 15 side product which is unstable in the presence of acid. Use of the modified procedure leads to the desired P3 glycosidic linkage. The use of BF 3 is an improvement because previously the presence of a very bulky ester at C-2 (e.g., pivalate) was required to prevent formation of the undesired ortho-ester during formation of a 13 glycosidic linkage by the sulfoxide method using neighboring group participation. These bulky esters can be very difficult to remove, except under strongly basic 20 conditions. It is preferred to perform the aforementioned glycosylation reactions on a polymeric resin, preferably after coupling the carboxylic acid functionality of these compounds to a suitable resin. In order to attach the carboxylic acid group to the resin, it must first be selectively deprotected. Use of a p 25 nitrobenzyl ester as a protecting group for the carboxylic acid is preferred to facilitate selective deprotection of the carboxylic acid in the presence of protected hydroxyl groups. A suitable resin is a cross-linked polymer insoluble in the reaction solvent which is suitably functionalized for attachment, e.g., SASRIN (Wang's resin). Once coupled to the resin, the differentially protected hydroxyl group on the attached sugar is deprotected. Alternatively, this hydroxyl group is freed before attachment to the 30 resin, since the hydroxyl group does not interfere with the coupling reaction. The free hydroxyl group then serves as the nucleophile in a second glycosylation reaction. In this second glycosylation, the hydroxyl is glycosylated, preferably in a solid phase reaction, with a variety of azido sugars. Following the glycosylation reaction, the azido groups are reduced and the resulting amino groups are then derivatized. The solid phase portion of the library construction can be carried out using a parallel 35 synthesis or a mix and split strategy. The carbohydrate-modified glycopeptide derivatives would then be deprotected and cleaved from the resin. This set of compounds would then be assayed for peptide binding and anti-bacterial activity.

WO 00/69892 20 PCT/US00/13679 When it is desired to remove protecting groups from any of the compounds of this invention, their removal is accomplished using methods well known to those skilled in the art. The preferred method for removal of protecting groups is as follows. Aloc groups on amines, and allyl esters or allyl ethers are removed by using Pd(0) mediated reactions, e.g., [Ph 3

P]

2 Pd(II)C 2 and Bu 3 SnH in 1:1 acetic 5 acid:DMF. Acetate protecting groups are removed using hydrazine in THF/methanol. An alternative method for construction of a library of glycopeptide compounds starts with the synthesis of a suitably protected pseudoaglycone. A protected glycopeptide antibiotic having a disaccharide at residue A 4 , i.e., a pseudoaglycone bearing an additional sugar residue, is treated with a Lewis acid in an 10 organic solvent to remove the additional sugar residue, as illustrated in Figure 15 and in the Examples. In a preferred embodiment of the invention, the Lewis acid is boron trifluoride, preferably as the complex with diethyl ether. When the glycopeptide antibiotic is vancomycin, it is preferred that allyloxycarbonyl (aloc) groups are present on the amines of A, and the vancosamine residue, acetates on the aliphatic hydroxyl groups, allyl phenyl ethers on the phenolic hydroxyls, and an allyl or o 15 nitrobenzyl ester on the A 7 terminal carboxyl; when solid-phase synthesis is employed, the o nitrobenzyl ester is preferred. A degradation reaction proceeds which removes the additional sugar residue, leaving a pseudoaglycone in which all reactive functional groups (amine, carboxylic acid, phenols, and benzylic alcohols) are suitably protected except for a hydroxyl group on the remaining residue A 4 sugar, which is where an additional sugar is to be attached. 20 This pseudoaglycone is glycosylated via a non-enzymatic reaction in an organic solvent, as described hereinabove for glycosylation of an aglycone to which one sugar residue has already been attached. In solid-phase synthesis of glycopeptide compounds from pseudoaglycones, the molecule is preferably 25 attached to the resin after removal of the o-nitrobenzyl group from the protected pseudoaglycone. The following examples are presented in order to illustrate various aspects of the present invention, but are not intended to limit it. 30 WO 00/69892 21 PCT/US00/13679 EXAMPLES General Procedures Unless specified otherwise, product purification by preparative reversed-phase HPLC is performed 5 using a PHENOMENEX LUNA C18 column (21.2 x 250 mm), 5 ipm particle size; and semi preparative reversed-phase HPLC is performed using a Vydac C18 column (10 X 250 mm), 5 pm particle size. Detection is by UV absorption measurement at 285 nm. Method A: Compounds are dissolved in DMF-water or DMF-methanol then diluted with water and 10 filtered (0.45 ptm). Multiple injections of 0.1 to 1 mL samples are required for most separations to avoid precipitation and overloading of the column. A gradient of acetonitrile in water containing 0.1% acetic acid at a flow rate of 7 to 8 mL/min. is used. Products purified by this method are treated with 1 butanol (approximately 1 to 1 with the anticipated water content) and evaporated to dryness under reduced pressure). The solid is then dissolved in methanol, diluted with toluene and evaporated under 15 reduced pressure. Method B: Compounds are dissolved in water or a water-methanol or water-DMF mixture and filtered (0.45 ptm). Multiple injections of 0.1 to 1 mL samples are required for most separations to avoid precipitation and overloading of the column. A gradient of acetonitrile in water containing 0.1% 20 trifluoroacetic acid at a flow rate of 7 to 8 mL/min. is used. Products purified by this method are evaporated under reduced pressure to remove the acetonitrile (bath temperature maintained at or below 25 'C to avoid loss of the vancosamine residue) and the remaining water solution froze and lyophilized. Purity of each aqueous sample is confirmed by analytical HPLC prior to I ,ophilizing. 25 Method C: Compounds are dissolved in water or a water-methanol or water-DMF mixture and filtered (0.45 pm). Multiple injections of 0.1 to 1 mL samples are required for most separations to avoid precipitation and overloading of the column. A gradient of acetonitrile in water (0.5% triethylamine adjusted to pH = 3 with phosphoric acid) is used. Products purified by this method are desalted by adsorption onto a polystyrene column (10 mm X 600 mm), followed by washing with 5 column 30 volumes of water, and eluted with 75 % methanol in water containing 0.1 % acetic acid. Fractions containing product are combined, the methanol removed under reduced pressure and the resulting water solution froze and lyophilized. Purity of each aqueous sample is confirmed by analytical HPLC prior to lyophilizing.

WO 00/69892 22 PCT/US00/13679 EXAMPLE 1: N,N'-dialoc Vancomycin Allyl Ester (III) a) N,N'-diallyloxycarbonyl Vancomycin (II) To a solution of vancomycin-HCI (13 g, 8.7 mmol) in 105 mL water is slowly added 80 mL acetone. A 5 30 mL aqueous solution of NaHCO3 (1.54 g, 18.3 mmol) is then added over 5 min. affording a thick white slurry. After stirring 10 min. the suspension is treated with a solution of N (allyloxycarbonyloxy)succinimide (18 g, 90 mmol) in 70 mL acetone. Within a few h the reaction became clear and stirred at room temperature for 36 h. TLC (6:4:1, chloroform-methanol-water) shows no vancomycin (baseline) remaining and one predominant glycopeptide product (Rf = 0.3). The crude 10 reaction mixture is treated with 1-butanol (100 mL) and evaporated to dryness under reduced pressure. The solid is dissolved in 50 mL methanol and precipitated by addition to 300 mL diethyl ether. Any chunks are crushed and the white suspension allowed to settle for 1 h at 4 0 C. Approximately 200 mL of the clear supernatant is decanted and the remaining suspension centrifuged and the supernatant decanted. The white solid is mixed vigorously with 240 mL acetone, the suspension centrifuged and 15 the supernatant decanted. The solid is dissolved in methanol, diluted with 300 mL toluene and evaporated under reduced pressure affording (II) (15.5 g, containing a trace of NHS impurity) which could be used without further purification. If desired removal of the NHS; The solid is dissolved in a minimum of methanol/DMF (1:1) and precipitated by addition to water. The suspension is mixed well, the suspension centrifuged and the supernatant decanted. The white solid is dissolved in methanol to 20 combine fractions, diluted with excess toluene, evaporated under reduced pressure, and dried en vacuo. Preparation of N-(allyloxycarbonyloxy)succinimide is reported in Int. J. Peptide Protein Res. 1991, 37, 556-564. b) N,N'-dialoc Vancomycin Allyl Ester (III) 25 Compound (II) (5 g, 3 mmol) is dissolved in 28 mL DMSO under an argon atmosphere (1 h with stirring). Powdered NaHCO 3 (2.5 g, 30 mmol) is added and the suspension stirred 10 min. followed by addition of allyl bromide (1.3 mL, 15 mmol). Stirring is continued for 7 h, at which time TLC shows the disappearance of (II) and one predominant product. The reaction is slowly diluted with acetone (ca. 25 mL) until the precipitate formed upon addition is just redissolved. This solution is vacuum filtered 30 (removing the insoluble NaHCO 3 ) into a flask containing 200 mL acetone and 450 mL diethyl ether. The flask is swirled occasionally during filtrate addition to disperse the mixture of white precipitate and oil that formed. The reaction flask and filter are rinsed with 10 mL acetone-methanol (1:1). The filtrate/suspension is stored at 4'C for 16 h with occasional swirling. The precipitate and oil coated the flask leaving a clear supernatant that is decanted. The solid mass is rinsed with acetone, dried under 35 high vacuum, and dissolved in 10 mL DMF-methanol (1:1). This solution is precipitated by addition to 180 mL water (6 x 30 mL in 6 centrifuge tubes). The suspension is mixed, chunks crushed, WO 00/69892 23 PCT/US00/13679 centrifuged, and the supernatant decanted. The solids are combined in methanol-acetone, diluted with toluene, evaporated under reduced pressure, and dried en vacuo affording (III) (4.5 g). TLC: Rf = 0.67; (chloroform-methanol-water; 6:4:1). An analytical sample is prepared by separation on HPLC; (Method A; 30 min. linear gradient of 25% to 60% acetonitrile; flow rate = 7.5 mL/min.) affording (III), 5 Ret. Time = 24 min.; LRESI-MS calc for C 7 7

H

87

N

9 0 28

CI

2 : 1655.5; [M+H]

+

= 1657; [M vancosamine+H] + = 1431 EXAMPLE 2: Allyl-dialoc-tri-OAll peracetate vancomycin pseudoaglycone (VI). 10 a) Allyl dialoc-tri-O-allyl vancomycin (IV). All-dialoc vancomycin (753 mg, 0.455 mmol) is taken in 5 mL DMF. Ground Cs 2

CO

3 (750mg, 2.30mmol) is added to the reaction solution. The suspension is stirred under high vacuum for 30 minutes. Then allyl bromide (400 pL, 2.36 mmol) is added. TLC at 6 hours shows completed reaction. 15 The suspension is precipitated in 100 mL water, centrifuged. The white solid is collected and loaded to a silica gel column (30mmxl2cm) and eluted with gradient from CHC1 3 to 5% MeOH/CHCl 3 to give 660mg (82%) of compound (IV) as white solid. Rf=0.6 (20% MeOH/CHCl 3 ). Mass Spec. [M+Na]+,1776; [M-V]

+

, 1550, [M-V-G], 1387. 20 b) Allyl-dialoc-tri-O-allyl peracetate vancomycin (V). Allyl dialoc-tri-O-allyl vancomycin (IV) (100 mg, 0.0563 mmol) is dissolved in 5 mL CH 2 C1 2 . Pyridine (164 gL, 2.027 mmol) is added followed by 2 mg DMAP. The reaction solution turns clear. Ac 2 0 (96 mL, 1.013 mmol) is added. After 5 hours, TLC shows completed reaction. The reaction is quenched with 1 mL methanol and then all solvents are removed. The residue is loaded to a silica gel 25 column (30mmxl2cm) and eluted with a gradient of 0% to 5% MeOH/CHCl 3 to give 104 mg (91%) of compound (V) as white solid. Rf=0.3 (5% MeOH/CHCI 3 ). Mass Spec. [M+Na] 2028. c) Allyl-dialoc-tri-OAll peracetate vancomycin pseudoaglycone (VI). Allyl-dialoc-tri-OAll peracetate vancomycin (V) (238 mg, 0.11 7mmol) is azeotroped with toluene 3 30 times and then dissolved in 8 mL CH 2 C1 2 . PhSH (120 pgL, 1.173 mmol) is added followed by BF 3 .Et 2 0( 431 pL, 3.5 Immol). TLC at 2 hours shows completed reaction. The reaction is quenched by 1 mL of DIEA and all solvents are removed. The residue is loaded to a silica gel column (30mmxl2cm) and eluted with a gradient of 0 to 5% MeOH/CHCI 3 to give 144mg (70%) of compound (VI) as white solid. Rf=0.3 (5% MeOH/CHCl 3 ). Mass Spec. [M+Na] + 2028. 35 EXAMPLE 3: Vancosamine N-CBz-C-6-O-acetyl sulfoxide (XI).

WO 00/69892 24 PCT/US00/13679 a) N,N'-bis-Cbz, Vancomycin (VII). To a solution of vancomycin-HCI (1.76 g, 1.19 mmol) dissolved in 8.5 mL water and diluted with 10 mL acetone is added 3 mL water containing NaHCO 3 (210 mg, 2.5 mmol). To the stirred suspension is 5 added 20 mL acetone, 15 mL water and N-(benzyloxycarbonyloxy)succinimide (1.2 g, 4.8 mmol) as a solution in 3 mL acetone. After 15 h. the clear solution is evaporated to dryness under reduced pressure with toluene azeotrope. The solid is dissolved in 15 mL DMF and precipitated by addition to 120 mL tetrahydrofuran. The suspension is centrifuged and the supernatant containing reagents decanted. The solid is then suspended in 120 mL acetone, mixed vigorously, centrifuged, and the supernatant 10 decanted. This acetone wash of the solid is performed 3 times to remove all reagents. The white solid is dried under reduced pressure affording (VII) (1.9 g, 95%) that is used without further manipulation. TLC: Rf = 0.33 (chloroform-methanol-water; 6:4:1). LRESI-MS calc for C 82

H

87

N

9 0 28

CI

2 1715.5; [M+Na] = 1739; [M-vancosamine+H] + = 1440; [M-disaccharide+H] = 1277 15 b) Vancosamine N-CBz methoxide (VIII). Crude vancomycin BisCBz (VII) (3.414g, 1.99 mmol) is dissolved in 18 mL methanol and 2.7 mL ION HCI aqueous solution is added. A white precipitate is formed during reaction. After 2 hours, TLC shows completed reaction. All the solvents are removed and the residue is precipitated in 300 mL acetone. The acetone layer is collected and concentrated to give a thick oil. This oil is loaded onto a 20 silica gel column (40mmxl4cm) and eluted with 60% ETOAc/PE to give 303 mg( 75%) of compound (VIII) as clear oil. (a:p =2:1) Rf=0.2 (40% ETOAc/PE) c) Vancosamine N-CBz C-4-O-acetyl methoxide (IX). The compound (VIII) (49 mg, 0.159 mmol) is dissolved in 2 mL CH 2 C1 2 . DMAP (0.2mg) is added to 25 the reaction followed by pyridine (13 gL, 12.6 mmol) and acetic anhydride (15 RL, 16.23mmol). After 12 hours, TLC shows completed reaction. The reaction is quenched by 0.5 mL methanol and all the solvents are removed. The residue is loaded to a silica gel column (20mmxl4cm) and eluted with 30% ETOAc/PE to give 53 mg (95%) of compound (IX) as clear oil. (a:=2:1). a anomer: Rf=0.4 (40% EtOAc/PE); 'H NMR (CDCl 3 , 300 MHz) 8 7.35 (m, 5 H), 5.25 - 4.90 (m, 3 H), 4.79 (d, J= 6.5 Hz, H-1, 30 1 H), 4.74 (bs, H-4, 1 H), 4.10 (m, H-5, 1 H), 3.34 (s, OCH 3 , 3 H), 2.10 (s, COCH 3 , 3 H), 2.00 - 1.88 (m, H-2, H-2', 2 H), 1.73 (s, CH 3 , 3 H), 1.14 (d, J = 6.4 Hz, CH 3 , 3 H). P anomer: Rf=0.3 (40% EtOAc/PE); 1 HNMR (CDCI 3 , 300 MHz) 8 7.35 (m, 5 H), 5.10 (d, J= 12.0 Hz, 1 H), 5.09 (s, 1 H), 4.95 (d, J = 12.0 Hz, 1 H), 4.73 (bs, H-4, 1 H), 4.55 (d, J = 12.0 Hz, H-1, 1 H), 3.84 (m, H-5, 1 H), 3.50 (s,

OCH

3 , 3 H), 2.07 (s, COCH 3 , 3 H), 2.00 - 1.70(m, H-2, H-2', 2 H), 1.64 (s, CH 3 , 3 H), 1.20 (d, J = 6.4 35 Hz, CH 3 , 3 H).

WO 00/69892 25 PCT/US00/13679 d) Vancosamine N-CBz C4-O-acetyl sulfide (X). The compound (IX) (144mg, 0.410 mmol) is azeotroped with toluene 3 times and then dissolved in 4 mL CH 2

CI

2 . PhSH (84 gL, 0.82 mmol) is added followed by BF 3 OEt 2 (100gL, 0.82mmol). TLC at 15 minutes shows completed reaction. The reaction is quenched by 20 mL saturated NaHCO 3 aqueous 5 solution. The CH 2 C1 2 layer is separated and the aqueous layer is further extracted with CH 2

C

2 (20 mLx3). The CH 2

CI

2 layers are combined and dried over anhydrous sodium sulfate, filtered, concentrated to give a clear oil. This oil is loaded to a silica gel column (30mmxl4cm) and eluted with 20% ETOAc/PE to give 125 mg (71%) compound (X) as white solid. Rf=0.7 (40% EtOAc/PE) (a:13=3:1) P3 anomer: H NMR (CDCl 3 , 500 Mhz) 8 7.47 - 7.24 (min, 10 H), 5.58 (dd, J = 2.8, 6.7 Hz, H-1, 10 1 H), 5.10 (d, J = 12.2 Hz, 1 H), 5.00- 4.97 (min, 3 H), 4.90 (s, H-4, 1 H), 4.51 (min, H-5, I H), 2.55 (dd, J = 6.7, 14.0 Hz, H-2, 1 H), 2.23 (d, J = 14.0 Hz, H-2', 1 H), 2.09 (s, COCH 3 , 3 H), 1.77 (s, CH3, 3 H), 1.16 (d, J = 6.4 Hz, CH3, 3 H); 13C NMR (CDCI 3 , 500 MHz) 8 170.94, 154.69, 136.60, 136.02, 131.20, 129.06, 128.71, 128.46, 128.35, 127.31, 83.12, 74.01, 66.61, 64.44, 53.66, 37.35, 24.11, 20.87, 17.13; a anomer: HNMR (CDCl 3 , 500 MHz) 8 7.47 - 7.24 (min, 10 H), 5.58 (dd, J= 2.8, 6.7 Hz, H-1, 1 H), 5.10 15 (d, J = 12.2 Hz, 1 H), 5.00 - 4.97 (min, 3 H), 4.90 (s, H-4, 1 H), 4.51 (min, H-5, 1 H), 2.55 (dd, J = 6.7, 14.0 Hz, H-2, 1 H), 2.23 (d, J = 14.0 Hz, H-2', 1 H), 2.09 (s, COCH 3 , 3 H), 1.77 (s, CH 3 , 3 H), 1.16 (d, J = 6.4 Hz, CH 3, 3 H); 1 3 C NMR (CDC1 3 , 500 MHz) 8 170.94, 154.69, 136.60, 136.02, 131.20, 129.06, 128.71, 128.46, 128.35, 127.31, 83.12, 74.01, 66.61, 64.44, 53.66, 37.35, 24.11, 20.87, 17.13. 20 e) Vancosamine N-CBz C4-O-acetyl sulfoxide (XI). The vancosamine sulfide (X) (18 mg, 0.0433mmol) is dissolved in 1.5 mL CH 2

CI

2 and cooled to -78 0 C. mCPBA is added and the reaction is slowly warmed up to -20 0 C in 1 hour. 'TLC shows completed reaction. The reaction is quenched by 100mL dimethyl sulfide. The reaction is extracted with 5 mL saturated NaHCO 3 aqueous solution. The aqueous layer is further extracted with CH 2 C1 2 (5 mLx3). The 25 CH 2

C

2 layers are combined and dried over anhydrous sodium sulfate, filtered, concentrated to a clear oil. This oil is loaded onto a silica gel column (20mmx8cm) and eluted with 60% ETOAc/PE to give 19 mg (95%) compound (XI) as white solid. Rf=0.15 (40% EtOAc/PE). 30 EXAMPLE 4: Regeneration of Vancomycin from (VI) a) Glycosylation of (VI) with (XI) to give (V). The compound (VI) (22.7 mg, 0.0127mmol) is azeotroped and dissolved in lmL CH 2

C

2 and cooled to 78 0 C. BF 3 .Et 2 0 (2pL, 0.0168mmol) is added followed by triflic anhydride (4gL, 0.0247mmol). Then 35 the sulfoxide (XI) (22mg, 0.0494mmol) in 0.5 mL CH 2

C

2 is added to the reaction vessel dropwise over 1 minute. TLC shows all sulfoxide is activated after addition. The reaction is slowly warmed up to - WO 00/69892 26 PCT/US00/13679 25 0 C in 1.5 hour and then quenched with 100mL methanol and 100mL DIEA. All the solvents are removed and the residue is loaded to a silica gel column (10mmx5cm) and eluted with a gradient of 0 to 5% MeOH/CHCl 3 to give 17mg of white solid. This white solid is purified by reverse-phase HPLC using a PHENOMENEX LUNA C18 column (21.2x250 mm), 5 pm particle, eluting with a 30 min. 5 linear gradient of 80% acetonitrile/0.1% acetic acid in water to 100% acetonitrile/0.1% acetic acid; flow rate of 8 mL/min. and UV detection at 285 nm. The fractions containing the pure products are combined and evaporated to give 11 mg (41%) of compound (V) as white solid. Rf=0.3 (5% MeOH/CHCl 3 ). Mass Spec. [M+Na] 2028. 10 b) Deprotection of compound (V) to give compound (IV). The glycosylation product (V) (9mg, 0.00443mmol) is dissolved in 0.4 mL methanol and 0.2 mL THF. Hydrazine (30pL) is added. The reaction is quenched with 0.2 mL of acetic acid after 4 hours. All solvents are removed and the residue is purified by reverse-phase HPLC using a PHENOMENEX LUNA C 18 column (21.2x250 mm), 5 gm particle, eluting with a 30 min. linear gradient of 80% 15 acetonitrile/0.1% acetic acid in water to 100% acetonitrile/0.1% acetic acid; flow rate of 8 mL/min. and UV detection at 285 nm. The fractions containing the pure products are combined and evaporated to give 5 mg (63%) of compound (IV) as white solid. Rf=0.3 (5% MeOH/CHCI 3 ). Mass Spec. [M+Na] 2028. 20 c) Deprotection of Compound (IV) to give Vancomycin. Compound (IV) (5 mg, 0.00281 mmol) is dissolved in 0.5 mL DMF/0.5 mL acetic acid. A catalytic amount of palladium dichloride-bis-triphenylphosphine is added and the reaction vessel is filled with nitrogen. To this mixture is added, with vigorous stirring, tributyltin hydride in 5 pL portions every 5 minutes until all starting materials and intermediates have disappeared by TLC. The crude reaction 25 mixture is precipitated with 20 mL diethyl ether in a 50 mL centrifuge tube. The mixture is centrifuged and decanted to give a white solid that is vortexed with 20 mL diethyl ether, centrifuged, decanted and dried. The resulting white solid is purified by reverse-phase HPLC using a PHENOMENEX LUNA C18 column (21.2x250mm), 5 pm particle, eluting with a 40 min. linear gradient of 0.1% trifluoroacetic acid in water to 20% acetonitrile/0.1% trifluoroacetic acid in water; flow rate of 7 mL/min. and 30 ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined, diluted with 10 mL water, organic solvents are evaporated and then the residue is lyophilized to give 3 mg (75%) of vancomycin TFA salt as white solid. R=0.05 (CHCl 3 :MeOH:H 2 0

=

3:4:2). Mass Spec. [M+Na

+

] 1471. 35 WO 00/69892 27 PCT/US00/13679 EXAMPLE 5: Benzyl N,N'-bis-Cbz Vancomycin (XII). To a solution of (VII) (1.49 g, 0.87 mmol) in 15 mL DMSO under an argon atmosphere is added NaHCO 3 (35 mg, 0.4 mmol), then benzyl bromide (0.3 mL, 2.5 mmol) and the mixture stirred for 3 h at 5 room temperature. The reaction is precipitated by addition to 400 mL 10% acetone in diethyl ether. The suspension is centrifuged, affording a thick sticky solid upon sitting, and the supernatant decanted. Combined supernatants are evaporated under reduced pressure to 10 mL volume and precipitated by addition to 200 mL diethyl ether. The suspension is centrifuged and the supernatant decanted. Solids are dissolved in methanol, combined, and evaporated under reduced pressure. Purification by HPLC 0 (Method A: 3 min. at 38% acetonitrile followed by a 40 min. linear gradient of 38% to 75% acetonitrile; flow rate = 8 mL/min.) affords (XII) (0.97 g, 61% from I). Ret. Time = 26 min.; TLC: Rf= 0.5 (chloroform-methanol-water, 50:21:4). LRESI-MS calc for C 8 9

H

93

N

9 0 2 8

CI

2 1805.6; [M+Na] += 1829; [M-vancosamine+H] = 1530; [M-disaccharide+H] = 1368 15 EXAMPLE 6: Benzyl CBZ-tri-O-methyl diacetate vancomycin aglycone (XV) a) Benzyl CBZ-tri-O-methyl vancomycin (XIII). To a stirring solution of crude benzyl bis-CBZ-vancomycin (XII) (1.0262 g, 0.5677 mmol, 100% from 20 vancomycin) in 20 mL of DMF is added CS 2

CO

3 (830 mg, 2.55 mmol) and Mel (530 pL, 8.52 mmol). The reaction is stirred for 3 hours and then lmL of acetic acid is added. The solution is filtered through a plug of silica gel with 15%MeOH/CH 2

CI

2 and concentrated. The residue is purified by flash chromatography (10-12.5% MeOH/CH 2

CI

2 ) to give 655.2 mg of semi-pure product. Rf 0.53 (15%MeOHI/CH2Cl2). 25 655.2 mg of the semipure product (0.354 mmol) is dissolved in 10.6 mL of acetic acid. Thiophenol (215 tL, 2.09 mmol) and 5.7 mL of 3% HBr in acetic acid are added. After 15 minutes, the reaction is poured into 150 mL of H 2 0 and the white precipitate is isolated by centrifuge. Purification of the precipitate by flash chromatography (5-7.5% MeOH/CH 2 C1 2 ) gives 313.5 mg (40% over 4 steps from 30 vancomycin) of (XIII). Rf. 0.26 (7.5% MeOH/CH2Cl2); MS (ESI) calc 1410.2 (C7 1

H

7 0N8019C12) found 1433.2 M+Na. b) Benzyl CBZ-tri-O-methyl p-methoxybenzyl diacetate vancomycin aglycone (XIV). To a solution of C (290.2 mg, .2058 mmol) in 10 mL of DMF is added Cs 2

CO

3 (162 mg, 0.497 mmol) 35 and p-methoxybenzyl chloride (PMBC1) (84 pL, 0.617 mmol). The reaction is stirred for 3.5 hours and then filtered through a plug of silica gel with 10%MeOH/CH 2

CI

2 and concentrated. The residue is WO 00/69892 28 PCT/US00/13679 purified by radial chromatography (0-6% MeOHICH 2 C1 2 ) to give 222.3 mg (71%) of purified intermediate product. Rf0.33 (7.5% MeOH/CH2Cl2); MS (ESI) calc 1530.3 (C79H78N8020C12) found 1530.3. 5 To a solution of the purified intermediate product (222.3 mg, 0.144 mmol) in 5 mL of pyridine is added 1.25 mL of acetic anhydride. The reaction is stirred for three hours and then concentrated in vacuo. The residue is purified by flash chromatography (0-4% MeOH/CH 2 C1 2 ) to give 228.3 mg (97%) of (XIV). Rf 0.29 (5% MeOH/CH2Cl2); MS (ESI) calc 1614.4 (C83H82N8022C12) found 1614.4. 10 c) Benzyl CBZ-tri-O-methyl diacetate vancomycin aglycone (XV). To a solution of (XIV) (241.8 mg, 0.150 mmol) in 10 mL of CH 2

CI

2 is added 1 mL of trifluoroacetic acid (TFA). After 5 minutes, 25 mL of toluene is added and the reaction is concentrated in vacuo. Purification by radial chromatography (0-6% MeOH/CH 2

CI

2 ) gives 206.5 mg (92%) of (XV). Rf=0.25 (5% MeOH/CH2Cl2); MS (ESI) calc 1494.2 (C 7 5H 7 4N8021C12) found 1517.2 M+Na. 15 EXAMPLE 7: N,N'-dialoc-glucose-C6-Amine-Vancomycin Allyl Ester (XVIII) a) N,N'-dialoc-glucose-C6-mesitylenesulfonyl-Vancomycin Allyl Ester (XVI). 20 To a stirred solution of compound (III) (370 mg, 0.22 mmol) in 2.5 mL anhydrous pyridine under an argon atmosphere at 4 oC is added 0.5 mL of a 1.4 M solution of mesitylenesulfonyl chloride in pyridine. The temperature is maintained at 4 oC for 24 h at which time the reaction is precipitated by addition to 50 mL diethyl ether (2 x 25 mL in two 50 mL centrifuge tubes). The suspension is centrifuged and the supernatant decanted. The solids are combined by dissolving in methanol and 25 evaporated under reduced pressure. Separation by HPLC (Method A; 40 min. linear gradient of 30% to 75% acetonitrile; flow rate = 7.5 mL/min.) affords starting material (III) (64 mg) and (XVI) (202 mg, 50%, 60% based on recovered III). Ret. Time = 28 min.; TLC: Rf= 0.7 (chloroform-methanol-water, 50:21:4). LRESI-MS calc for C 86

H

97

N

9 0 30 SIC1 2 : 1837.5; [M+H] += 1839; [M-vancosamine+H] = 1614; [M-disaccharide+H] = 1267. 30 b) N,N'-dialoc-glucose-C6-Azide-Vancomycin Allyl Ester (XVII). To a stirred solution of compound (XVI) (310 mg, 0.17 mmol) in 8 mL anhydrous DMF under an argon atmosphere is added sodium azide (112 mg, 1.72 mmol) and the suspension stirred at 85°C for 8 h. The mixture is cooled to room temperature and precipitated by addition to 80 mL diethyl ether. The white 35 solid is centrifuged and the supernatant decanted. The solid is dissolved in a minimum of methanol and precipitated by addition to 80 mL water. The suspension is mixed vigorously then stored at 4°C for 12 WO 00/69892 29 PCT/US00/13679 h. The suspension is centrifuged and the supernatant decanted. Separation by HPLC (Method A; 40 min. linear gradient of 25% to 50% acetonitrile; flow rate = 7.5 mL/min. affords (XVII) (172 mg, 60%). Ret. Time = 27 min.; TLC: Rf= 0.55 (chloroform-methanol-water; 50:21:4). LRESI-MS calc for

C

77

H

86

N

12 0 2 7 C1 2 1680.5; [M+H] = 1682; [M-vancosamine+H] + = 1456; [M-disaccharide+H] + = 5 1267. c) N,N'-dialoc-glucose-C6-Amine-Vancomycin Allyl Ester (XVIII). A solution of azide (XVII) (172 mg, 0.1 mmol) and triphenylphosphine (180 mg, 0.7 mmol) in 25 mL THF containing 5 mL water is heated at 60 'C for 16 h. The reaction is cooled to room temperature, 10 diluted with 200 mL toluene and evaporated to dryness under reduced pressure. The white solid is dissolved in 5.5 mL methanol-DMF (10:1) and precipitated by addition to 75 mL diethyl ether (3 X 25 mL). The suspension is centrifuged and the supernatant containing triphenylphosphine decanted. The solid is dissolved in methanol, combined, and evaporated under reduced pressure. Separation by HPLC (Method A; 40 min. linear gradient of 15% to 50% acetonitrile; flow rate = 7.5 mL/min.) affords 15 (XVIII) (140 mg, 82%). Ret. Time = 24 min.; TLC: Rf= 0.3 (chloroform-methanol-water; 6:4:1). LRESI-MS calc for C 7 7 H88NioO27CI 2 : 1654.5; [M+H] = 1656; [M-vancosamine+H] + = 1429; [M disaccharide+H] + = 1267. 20 EXAMPLE 8: Allyl N,N'-Dialoc-Glucose-C6-N-4-(4-chlorophenyl)benzyl Vancomycin (XIX) To a stirred solution of (XVIII) (26 mg, 0.016 mmol) in 0.5 mL anhydrous DMF under an argon atmosphere is added 4-4-(4-chlorophenyl)benzylcarboxaldehyde (1.7 mg. (0.)008 mmol). After 10 min. sodium cyanoborohydride (2 mg, 0.03 mmol) is added and the mixture stirred an additional 4 h. The 25 reaction mixture is precipitated by addition to 8 mL diethyl ether. The suspension is centrifuged, the supernatant decanted, and the white solid then dried under reduced pressure to remove residual diethyl ether. Separation by HPLC (Method A; 30 min. linear gradient of 20% to 45% acetonitrile; flow rate = 7.5 mL/min.) affords (XIX) (9 mg, 61%, based on aldehyde) Retention time = 27 min., and 8 mg recovered (XVIII). TLC: Rf= 0.66 (chloroform-methanol-water; 6:4:1). LRESI-MS calc for 30 C 90

H

97

NIO

2 7 C1 3 1854.6; [M+H]

+

= 1856; [M-disaccharide+H] = 1267 The preparation of 4-(4-chlorophenyl)benzylcarboxaldehyde is given in J. Heterocyclic Chem. Vol. 22, 1985, pp. 873-878. 35 WO 00/69892 30 PCT/US00/13679 EXAMPLE 9: Allyl N,N'-Dialoc-Glucose-C6-N-5-(4-chlorophenyl)furan-1-methylene Vancomycin (XX) To a stirred solution of (XVIII) (63 mg, 0.036 mmol) in 0.9 mL anhydrous DMF under an argon 5 atmosphere is added DIEA (7.4 [tL, 0.04 mmol). After 5 min. 5-(4-chlorophenyl)furfal (7.3 mg, 0.035 mmol) is added and the solution heated at 70 °C for 100 min. Sodium cyanoborohydride (5 mg, 0.08 mmol) is then added and the mixture stirred an additional 2 h at 70 *C. The reaction mixture is cooled to room temperature and precipitated by addition to 25 mL diethyl ether. The suspension is centrifuged and the supernatant decanted. Residual diethyl ether is removed under a flow of argon. Separation by 10 HPLC (Method A; 40 min. linear gradient of 20% to 60% acetonitrile; flow rate = 7.5 mL/min.) affords (XX) (42 mg, 64%) Retention time = 23 min. TLC: Rf= 0.6 (chloroform-methanol-water; 6:4:1). LRESI-MS calc for C 88

H

9 5

N

0 0 2 8 C1 3 : 1844.5; [M+H] += 1846; [M-vancosamine+H] + = 1656; [M disaccharide+H] + = 1268. 15 EXAMPLE 10: Allyl N,N'-Dialoc-Glucose-C6-N-decyl Vancomycin (XXI) To a stirred solution of (XVIII) (11 mg, 0.007 mmol) in 0.45 mL anhydrous DMF under an argon atmosphere is added DIEA (1.3 pL, 0.0073 mmol). After 10 min. decylaldehyde (1.3 pL, 0.007 mmol) 20 is added and the solution stirred at room temperature for 45 min. Sodium cyanoborohydride (2 mg, 0.03 mmol) is then added and the mixture stirred an additional 5 h. The reaction mixture is precipitated by addition to 6 mL diethyl ether, the suspension centrifuged and the supernatant decanted. Residual diethyl ether is removed under a flow of argon. Separation by HPLC (Method A; 40 min. linear gradient of 20% to 75% acetonitrile; flow rate = 7.5 mL/min.) affords (XXI) (2 mg, 17%). Retention 25 time = 21 min. TLC: Rf= 0.68 (chloroform-methanol-water; 6:4:1). LRESI-MS calc for

C

87 Ho 108

N

10 0 27 C1 2 : 1794.7; [M+H]

+

= 1796; [M-disaccharide+H] + = 1267. EXAMPLE 11: Allyl N,N'-Dialoc-(6-N-thiocarbonyl methylamino glucose) Vancomycin (XXII) 30 A solution of amine (XVIII) (6 mg, 0.0036 mmol) in 0.2 mL anhydrous pyridine under an argon atmosphere is treated with methylisothiocyanate (0.8 mg, 0.01 mmol). After 10 min. TLC shows complete disappearance of starting material. The reaction mixture is added to 8 mL diethyl ether, the resulting suspension centrifuged and the supernatant decanted. The white solid is mixed vigorously 35 with 10 mL diethyl ether, centrifuged, supernatant decanted and solid dried under reduced pressure affording (XXII) (6 mg, 96%) TLC shows one compound; Rf= 0.8 (chloroform-methanol-water; WO 00/69892 31 PCT/US00/13679 6:4:1). This product is subjected to deprotection without further manipulation. LRESI-MS calc for

C

7 9

H

9 1

N

1 1 0 2 7

S

1 C1 2 : 1727.5; [M+H] += 1729; [M-vancosamine+H] + = 1502. 5 EXAMPLE 12: Allyl N,N'-Dialoc-glucose-C6-N-(thiophene-2-carboxamide) Vancomycin (XXIII) To a stirred solution of N-hydroxysuccinimide (NHS) (84 mg, 0.73 mmol) and triethylamine (92.5 pL, 0.66 mmol) in 1.3 mL acetonitrile-0.5 mL dichloromethane under an argon atmosphere at 4°C is added a solution of thiophene-2-carbonyl chloride (71 gL, 0.66 mmol) in 0.3 mL acetonitrile. After 30 min. 10 cooling is removed and the mixture stirred at room temperature for an additional 30 min. Stirring is stopped and triethylammonium chloride allowed to settle affording a 0.3 M solution of the NHS activated ester. To a stirred solution of amine (XVIII) (9 mg, 0.005 mmol) in 0.5 mL anhydrous DMF under an argon 15 atmosphere is added 50 giL of the 0.3 M NHS activated ester solution prepared above (0.015 mmol). After 48 h. the reaction mixture is precipitated by addition to 10 mL diethyl ether, the resulting suspension centrifuged and the supernatant decanted. Separation by HPLC (Method A; 40 min. linear gradient of 30% to 70% acetonitrile; flow rate = 7.5 mL/min.) affords (XXIII) (6.7 mg, 70%). Retention time = 19 min. TLC: Rf= 0.75 (chloroform-methanol-water; 6:4:1). LRESI-MS calc for 20 C 82

H

90 oN 10 0 28 SIC1 2 : 1764.5; [M+Na] += 1788; [M-vancosamine+Na] + = 1562; [M-disaccharide+H] = 1268. EXAMPLE 13: Allyl N,N'-Dialoc-glucose-C6-N-(glycine-carboxamide) Vancomycin (XXIV) 25 To a stirred solution of amine (XVIII) (20 mg, 0.012 mmol) in 1 mL anhydrous DMF under an argon atmosphere is added N-Fmoc-glycine pentafluorophenyl ester (13 mg, 0.028 mmol). After 1 h. the mixture is precipitated by addition to 15 mL diethyl ether, centrifuged and the supernatant containing excess reagents decanted. The white solid is taken up in methanol, diluted with 50 mL toluene and 30 evaporated under reduced pressure affording the Fmoc protected glycinamide product, one product by TLC: Rf= 0.73 (chloroform-methanol-water; 6:4:1). The dry solid is dissolved in 1 mL anhydrous DMF under an argon atmosphere and treated with 0.15 mL piperidine. After 30 min. the mixture is precipitated by addition to 25 mL diethyl ether, the suspension centrifuged and the supernatant decanted. Separation by HIPLC (Method A; 40 min. linear gradient of 10% to 45% acetonitrile; flow 35 rate = 8 mL/min.) affords (XXIV) (5 mg, 25%), Retention time = 25 min. which is used for analytical purposes and 14 mg of (XXIV) contaminated with an impurity. This material is subjected to deprotection without further manipulation. TLC: Rf = 0.4 (chloroform-methanol-water; 6:4:1).

WO 00/69892 32 PCT/US00/13679 LRESI-MS calc for C 79

H

91 NllO 28 C1 2 : 1711.5; [M+H]

+

= 1713; [M-vancosamine+H] = 1488; [M disaccharide+H] = 1268. 5 EXAMPLE 14: Allyl N,N'-Dialoc-glucose-C6-N-myristoyl Vancomycin (XXV) To a stirred solution of N-hydroxysuccinimide (NHS) (63 mg, 0.55 mmol) and triethylamine (69 [tL, 0.5 mmol) in 1 mL acetonitrile under an argon atmosphere at 4°C is added a solution of myristoyl chloride (135 pL, 0.5 mmol) in 1 mL acetonitrile-dichloromethane (1:1). After 30 min. cooling is 10 removed and the mixture stirred at room temperature for an additional 2 h. Stirring is stopped and the triethylammonium chloride precipitate allowed to settle affording a 0.23 M solution of the NHS activated ester. To a stirred solution of amine (XVIII) (15 mg, 0.009 mmol) in 0.6 mL anhydrous DMF under an argon 15 atmosphere is added 50 gL of the 0.23 M NHS activated ester solution prepared above (0.01 mmol). After 8 h the reaction mixture is precipitated by addition to 10 mL diethyl ether. The resulting suspension is centrifuged and the supernatant decanted. Separation by HPLC (Method A; 40 min. linear gradient of 50% to 100% acetonitrile; flow rate = 7.5 mL/min.) affords (XXV) (10 mg, 60%). Retention time = 26 min. TLC: Rf= 0.75 (chloroform-methanol-water; 6:4:1). LRESI-MS calc for 20 C 91

H

11 4

N

10 0 28 C1 2 : 1864.7; [M+Na]

+

= 1888; [M-vancosamine+H] = 1640; [M-disaccharide+H] + = 1268. EXAMPLE 15: Allyl N,N'-Dialoc-glucose-C6-N-2-iodo-benzoyl Vancomycin (XXVI) 25 To a stirred solution of N-hydroxysuccinimide (84 mg, 0.73 mmol) and triethylamine (92.5 [L, 0.66 mmol) in 1.5 mL acetonitrile under an argon atmosphere at 4*C is added a solution of 2-iodobenzoyl chloride (177 mg, 0.66 mmol) in 0.8 mL acetonitrile. After 30 minutes cooling is removed and the mixture stirred at room temperature for an additional 1 h. Stirring is stopped and the triethylammonium 30 chloride allowed to settle affording a 0.28 M solution of the NHS activated ester. To a stirred solution of amine (XVIII) (7 mg, 0.004 mmol) in 0.6 mL anhydrous DMF under an argon atmosphere is added 22 pL of the 0.28 M NHS activated ester solution prepared above (0.006 mmol). After 1 h an additional 30 pL of the 0.28 M NHS activated ester solution is added and the solution 35 stirred an additional 14 h. The reaction mixture is precipitated by addition to 14 mL diethyl ether. The suspension is centrifuged and the supernatant decanted. Separation by HPLC (Method A; 40 min. linear gradient of 20% to 70% acetonitrile; flow rate = 7.5 mL/min.) affords (XXVI) (5 mg, 66%) WO 00/69892 33 PCT/US00/13679 Retention time = 26 min. TLC: Rf= 0.6 (chloroform-methanol-water; 50:21:4). LRESI-MS calc for

C

84

H

91

N

0 0 28 1 1 C1 2 : 1884.4; [M+Na] += 1907; [M-vancosamine+Na] = 1681; [M-disaccharide+H] = 1268. 5 EXAMPLE 16: Allyl N,N'-Dialoc-glucose-C6-N-2-quinoxaloyl Vancomycin (XXVII) To a stirred solution of N-hydroxysuccinimide (NHS) (27 mg, 0.23 mmol) and triethylamine (29.7 pL, 0.21 mmol) in 0.6 mL acetonitrile under an argon atmosphere at 4°C is added a solution of 2 10 quinoxaloyl chloride (41 mg, 0.21 mmol) in 1.0 mL acetonitrile. After 10 minutes cooling is removed and the mixture stirred at room temperature for 30 min. The mixture is cooled to 4'C, stirring stopped, and the triethylammonium chloride allowed to settle affording a 0.12 M solution of the NHS activated ester. 15 To a stirred solution of amine (XVIII) (46 mg, 0.026 mmol) in 0.5 mL anhydrous DMF under an argon atmosphere is added 325 [iL of the 0.12 M NHS activated ester solution prepared above (0.04 mmol). After 45 minutes the reaction mixture is precipitated by addition to 25 mL diethyl ether, the suspension centrifuged and the supernatant decanted. Separation by HPLC (Method A; 40 min. linear gradient of 30% to 80% acetonitrile; flow rate = 7.5 mL/min.) affords (XXVII) (35 mg, 74%) Retention time = 24 20 min. TLC: Rf = 0.63 (50:21:4, chloroform-methanol-water). LRESI-MS calc for C 86

H

9 2

NI

2 0 2 8

CI

2 : 1810.6; [M+Na] += 1834; [M-vancosamine+H] = 1586; [M-disaccharide+H] = 1268. EXAMPLE 17: Allyl N,N'-Dialoc-Glucose-C6-N-4-(4-chlorophenyl)benzoyl Vancomycin (XXVIII) 25 4-(4-chlorophenvl)benzoic acid To a stirred solution of 4-(4-chlorophenyl)benzaldehyde (0.84 g, 3.9 mmol) in 30 mL acetonitrile acetone (2:1) is added 15 mL water and solid sodium bicarbonate (3.5 g, 41.7 mmol). After 5 min. a 25 mL solution of oxidation reagent (Oxone: 4.8 g, 7.8 mmol in 25 mL water containing 4 x 10 4 M 30 EDTA) is added dropwise over 15 min. then stirred for an additional 3.5 h. The reaction mixture is then treated with 18 mL aq. sodium bisulfite (9.5 g), stirred 2 h, then acidified with 10 mL 6 M HC1. The mixture is transferred to a separatory funnel and diluted with 300 mL dichloromethane and 400 mL water. The water layer is washed with dichloromethane (3 x 120 mL), organic layers combined, then washed with 500 mL water. The organic layer is dried over sodium sulfate, filtered, and evaporated 35 under reduced pressure. The desired acid is crystallized from acetone-water (5:2), filtered, washed with water, and evaporated under reduced pressure from toluene affording 0.6 g product. Remaining product WO 00/69892 34 PCT/US00/13679 could be isolated by chromatography but the amount obtained is satisfactory. TLC: Rf = 0.3 (chloroform-methanol; 10:1). The foregoing procedure is adapted from Webb et. al. Tetrahedron, 1998, 54, 401-410. 5 To a stirred solution of 4-(4-chlorophenyl)benzoic acid (5.3 mg, 0.023 mmol) in 0.4 mL anhydrous DMF under an argon atmosphere is added 1-hydroxybenzotriazole (HOBt) (4 mg, 0.03 mmol) then DIEA (10 1 tL, 0.06 mmol). After 10 min. the solution is treated with PyBOP (10 mg, 0.02 mmol) and stirred an additional 30 min. TLC shows a trace of starting acid and one new product (expected to be the HOBt activated ester) affording a reagent stock solution (ca. 57 mM in activated acid). .0 To a stirred solution of amine (XVIII) (21 mg, 0.012 mmol) in 0.25 mL anhydrous DMF under an argon atmosphere is added DIEA (2 ptL, 0.012 mmol). The solution is stirred 5 min. then treated with 0.3 mL of the 57 mM activated acid solution. After 20 min. the reaction is precipitated by addition to 20 mL diethyl ether, the resulting suspension is centrifuged, the supernatant decanted, and the residual 15 solvent removed under a flow of argon. Separation by HPLC (Method A; 40 min. linear gradient of 35% to 80% acetonitrile; flow rate = 7.5 mL/min.) affords (XXVIII) (18 mg, 82%); Retention time = 26 min. TLC: Rf= 0.7 (chloroform-methanol-water; 50:21:4). LRESI-MS calc for C 9 0

H

9 5

N

10 0 28

CI

3 : 1868.5; [M+Na] += 1892; [M-vancosamine+H] + = 1669; [M-disaccharide+H] + = 1268. 20 EXAMPLE 18: Deprotection of Compounds XIX - XXVIII General Procedure for Allyl/Aloc Removal. To a solution of glycopeptide in DMF-acetic acid (4:3 or 1:1) is added (Ph 3 P)2Pd(II)C12 (catalytic). 25 With vigorous stirring, Bu3SnH is added in 5 to 10 molar equivalent portions every 2 to 10 min. until TLC (chloroform-methanol-water; 6:4:1) shows all glycopeptide product is baseline. The biphasic mixture is precipitated by addition to diethyl ether, the suspension centrifuged and the supernatant decanted. The white solid is dried under reduced pressure or by a steam of argon to remove residual diethyl ether, dissolved in water and then filtered to remove any remaining catalyst or hydrophobic 30 salts. Separation by HPLC is performed as described for individual compounds. a) Glucose-C6-N-4-(4-chlorophenyl)benzyl Vancomycin (XXIX). Deprotection of compound (XIX) (8.5 mg, 0.0046 mmol) is performed as described in the general procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 5% to 35 50% acetonitrile; flow rate = 7.5 mL/min.) affords (XXIX) (7 mg, 95%). Retention time = 27 min. LRESI-MS calc for C 79

H

85

N

10 0 23 C1 3 : 1646.5; [M+H]

+=

1648; [M-disaccharide+H]

+

= 1143.

WO 00/69892 35 PCT/US00/13679 Note: see also preparation of (XLVIII) for formation of (XXIX). b) Glucose-C6-N-5-(4-chlorophenyl)furan-1-methylene Vancomycin (XXX). Deprotection of compound (XX) (31 mg, 0.017 mmol) is performed as described in the general 5 procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 5% to 60% acetonitrile; flow rate = 7.5 mL/min.) affords (XXX) (27 mg, 92%) Retention time = 26 min. LRESI-MS calc for C 77

H

83

N

10 0 2 4 C1 3 : 1636.5; [M+H] = 1638; [M-disaccharide+H] + = 1143. c) Glucose-C6-N-decyl Vancomycin (XXXI) 10 Deprotection of compound (XXI) (2 mg, 0.001 mmol) is performed as described in the general procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 0% to 60% acetonitrile; flow rate = 8 mL/min.) affords (XXXI) (2 mg, 95+%). Retention time = 25 min. LRESI-MS calc for C 76

H

96

N

10 0 2 3 C1 2 : 1586.6; [M+H] += 1588; [M-disaccharide+H] = 1143. 15 d) Glucose-C6-N-thiocarbonyl methylamino Vancomycin (XXXII). Deprotection of compound (XXII) (6 mg, 0.003 mmol) is performed as described in the general procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 0% to 50% acetonitrile; flow rate = 7.5 mL/min.) affords (XXXII) (1 mg, 17%). Retention time = 21 min. LRESI-MS calc for C 68

H

79

NIO

1 0 2 3

S

1 C1 2 : 1519.5; [M+H] += 1521; [M-vancosamine+H] + = 1379; [M 20 disaccharide+H] + = 1143. e) Glucose-C6-N-(thiophene-2-carboxamide) Vancomycin (XXXIII). Deprotection of compound (XXIII) (6.3 mg, 0.0034 mmol) is performed as described in the general procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 0% to 25 50% acetonitrile; flow rate = 7.5 mL/min.) affords (XXXIII) (5 mg, 97%). Retention time = 23 min. LRESI-MS calc for C 71

H

7 8

N

10 0 24

S

1 C1 2 : 1556.5; [M+H]

+

= 1558; [M-vancosamine+H] + = 1415; [M disaccharide+H] + = 1143. f) Glucose-C6-N-(glycine-carboxamide) Vancomycin (XXXIV). 30 Deprotection of compound (XXIV) (14 mg, containing impurity as described) is performed as described in the general procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 0% to 35% acetonitrile; flow rate = 7.5 mL/min.) affords (XXXIV) (8 mg, 57%). Retention time = 22 min. LRESI-MS calc for C 68

H

79 NllO 24 C1 2 : 1503.5; [M+H] += 1505; [M vancosamine+H] + = 1362; [M-disaccharide+H] + = 1143. 35 WO 00/69892 36 PCT/US00/13679 g) Glucose-C6-N-myristoyl Vancomycin (XXXV). Deprotection of compound (XXV) (10 mg, 0.005 mmol) is performed as described in the general procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 15% to 75% acetonitrile; flow rate = 7.5 mL/min.) affords (XXXV) (8 mg, 89%). Retention time = 27 min. 5 LRESI-MS calc for CsoH 102 No 10 0 24 C1 2 : 1656.6; [M+H] += 1658; [M-vancosamine+H] = 1517; [M disaccharide+H]* = 1143. h) Glucose-C6-N-2-iodobenzoyl Vancomycin (XXXVI). Deprotection of compound XXVI (4 mg, 0.002 mmol) is performed as described in the general 10 procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 0% to 50% acetonitrile; flow rate = 7.5 mL/min.) affords (XXXVI) (3 mg, 89%). Retention time = 23 min. LRESI-MS calc for C 73

H

7 9

N

1 0 0 24 1 1 C1 2 : 1676.4; [M+H] += 1678; [M-vancosamine+H] + = 1535; [M disaccharide+H] = 1143. 15 i) Glucose-C6-N-2-quinoxaloyl Vancomycin (XXXVII). Deprotection of compound (XXVII) (30 mg, 0.017 mmol) is performed as described in the general procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 5% to 60% acetonitrile; flow rate = 7.5 mL/min.) affords (XXXVII) (28 mg, 98%). Retention time = 22 min. LRESI-MS calc for C 7 5

H

80 soN 1 2 0 2 4

CI

2 : 1602.5; [M+H] += 1605; [M-vancosamine+H] + = 1460; [M 20 disaccharide+H] = 1143. j) Glucose-C6-N-4-(4-chlorophenyl)benzoyl Vancomycin (XXXVIII). Deprotection of compound (XXVIII) (10 mg, 0.005 mmol) is performed as described in the general procedure for allyl/aloc removal. Separation by HPLC (Method B; 40 min. linear gradient of 10% to 25 60% acetonitrile; flow rate = 7.5 mL/min.) affords (XXXVIII) (8 mg, 90%). Retention time = 24 min. LRESI-MS calc for C 79

H

83

N

10 0 2 4

CI

3 : 1660.5; [M+H] += 1663; [M-vancosamine+H] = 1520; [M disaccharide+H] = 1143.

WO 00/69892 37 PCT/US00/13679 EXAMPLE 19: Glucose-C6-5-chloro-2-hydroxy-benzylamine vancomycin (XXXIX) 0 o,1 N 0"H HO C O O H cl H H HO H N H

H

0 O OH OO NH 2 OH 0 H OH

C

84

H

93 Cl 3

N

10 0 28 Exact Mass: 1794.52 Mol. Wt.: 1797.05 C, 56.14; H, 5.22; CI, 5.92; N, 7.79; O, 24.93 (XXXIXa) 5 a) N,N'-Dialoc-allyl-glucose-C6-5-chloro-2-hydroxy-benzylamine vancomycin. N,N'-Dialoc-allyl-glucose-C6-amine vancomycin AcOH salt (XVIII, 193.7 mg, 0.113 mmol) is dissolved in dry DMF (5 mL) and DIEA (10.7 mL, 0.117 mmol) is added. The mixture is stirred at 70 oC under Ar. After 1.5 h 5-chlorosalicylaldehyde (11 mg, 0.0703 mmoL) is added then the solution 10 turns yellow. The mixture is stirred for 1 h then NaBH 3 CN (0.117 mL, 1M-THF, 0.117 mmoL) is added. The mixture is stirred for an additional 2 h then cooled down to room temperature. The mixture is evaporated and the residue is purified by ODS-HPLC (LUNA 5gm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% AcOH/H 2 0, B: 0.1% AcOH/MeCN, 20-60 % B 0-30 min., 8 mL/min, tr= 25 min.) to give the title compound as a white amorphous as AcOH salt (64.3 mg, 0.0346 mmol, 31%). 15 LRESI-MS 1796 (M+2H, for C 84

H

95 35 C1 3 N0oO 2 8)

+

, 1569 (M-alocvancosamine +2H)+. 1267 (M alocvancosamine-glucose+H)

+.

WO 00/69892 38 PCT/US00/13679

HNH

2 H/ 0 H

HO

~

, O HN

H

0 0 H Cl 7 3 H80Cl 3N 10024 Exact Mass: 1586.45 Mol. Wt.: 1588.85 C, 55.18; H, 5.14; CI, 6.69; N, 8.82; 0, 24.17 (XXXIX) b) Glucose-C6-5-chloro-2-hydroxy-benlzylaminle vancomycin (XXXIX). N,N'-Dialoc-allyl-glucose-C6-5-chloro-2-hydroxy-benlZYaminle vancomycin (64.3 mg, 0.0346 mmol) 5 is dissolved with dry DMF/AcOH (1/1) (2 mL). Pd(PPh 3

)

2 C1 2 (1.2 mg, 0.00171 mmol) is added, then the mixture is stirred at room temperature under Ar. Bu 3 SnH (10 mL) is added about every 5-20 min. After 5 h the reaction is done. Added ether then centrifuged three times. The residue is purified by ODS-HPLC (LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFAI- 2 0, B: MeCN, 0-50% B over 50 min., 8 mL/min, tr= 14 min.) to give a white amorphous as TFA salt (XXXIX, 15.2 mg, 10 0.00893 mmol, 26 %). LRESI-MS 1588 (M+2H, for C73H8 3 35 C1 3 N10024)

+

, 1143 (M-vancosamine glucose+H)

+

. 15 2 HO H H O OH HO H2NHMe HO OH O Cy3HaiClA10024 Exact Mass: 1586.45 Mol. Wt.: 1588.85 C, 55.18; H, 5.14; CI, 6.69; N, 8.82; O, 24.17 (XXXIX) b) Glucose-C6-5-chloro-2-hydroxy-benzylamine vancomycin (XXXIX). N,N'-Dialoc-allyl-glucose-C6-5-chloro-2-hydroxy-benzylamine vancomycin (64.3 mg, 0.0346 mmol) 5 is dissolved with dry DMF/AcOH (1/1) (2 mL). Pd(PPh3)2CI2 (1.2 mg, 0.00171 mmol) is added, then the mixture is stirred at room temperature under Ar. Bu3SnH (10 mL) is added about every 5-20 min. After 5 h the reaction is done. Added ether then centrifuged three times. The residue is purified by ODS-HPLC (LUNA 5Rm C 18(2), 21.2 x 250 mm, UV=285 nm, A: 0. 1% TFA/H-20, B: MeCN, 0-50% B over 50 min., 8 mL/min, tr= 14 min.) to give a white amorphous as TFA salt (XXXIX, 15.2 mg, 10 0.00893 mmol, 26 %). LRESI-MIS 1588 (M+2H, for C7zH83 35 3NIOO24)*, 1143 (M-vancosamnine glucose+H) . 15 20 WO 00/69892 39 PCT/US00/13679 EXAMPLE 20: Glucose-C6- trifluoroacetamide vancomycin (XL) 0 NH H H 0 H 0 0 CF0 H H O O HH OOH o NH2r.,N OH O HO OH O

C

7 9

H

8 7 CI2F 3 N10028 Exact Mass: 1750.50 Mol. Wt.: 1752.49 C, 54.14; H, 5.00; CI, 4.05; F, 3.25; N, 7.99; O, 25.56 5 (XLa) a) N, N'-Dialoc-allyl-glucose-C6-trifluoroacetamide vancomycin. N, N'-Dialoc-allyl-glucose-C6-amine vancomycin (AcOH salt of XVIII) (13.1 mg, 0.00768 mmol) is dissolved with dry pyridine (0.5 mL). The mixture is stirred at 0OC. Trifluoroacetic anhydride (1.7 10 mL, 0.012 mmol) is added. After 6 h an additional 10 mL of trifluoroacetic anhydride is added, and then the reaction is done. The mixture is passed through an ODS short column, then purified by ODS HPLC (LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 20-50-100% B 0-30-40 min., 8 mL/min, tr= 34 min.) to give the title compound as a white amorphous solid (5.6 mg, 0.00302 mmol, 39 %). LRESI-MS 1756 (M+6H, for C79H 9 3 35 Cl 2

F

3 N00o 2 8)

+

, 1507 (M 15 alocvancosamine-O+H) .

WO 00/69892 40 PCT/US00/13679 HN2 H HOH O H OH

C

6 8 H 7 5

CH

2

F

3

N

1 24 Exact Mass: 1542.43 Mol. Wt.: 1544.28 C, 52.89; H, 4.90; CI, 4.59; F, 3.69; N, 9.07; 0, 24.87 (XL) b) Glucose-C6- trifluoroacetamide vancomycin (XL). 5 N,N'-Dialoc-allyl-glucose-C6-trifluoroacetamide vancomycin (5.3 mg, 0.00302 mmol ) is dissolved with dry DMF/AcOH (1/1) (1 mL). Pd(PPh 3

)

2 C1 2 (1.0 mg, 0.00142 mmol) is added, then the mixture is stirred at room temperature under Ar. Bu 3 SnH (0.1 mL) is added about every 20 min. After 4 h the reaction is done. Diethyl ether is added, then the mixture is centrifuged twice. The residue is purified by ODS-HPLC (LUNA 5gi C18(2), 21.2 x 250 mm, UV=285 nim, A: 0.1% TFA/H 2 0, B: MeCN, 0 10 30% B over 30 min., 8 mL/min, tr = 14 min.) to give (XL) as a white amorphous TFA salt (13, 0.2 mg, H O OH HO NH2N HMe / OH HO0 OH 0.00012C681 mmol, 4 %). LRESI-MS 1543 (M+H, for C 68 H2 6 3 N10 2

F

3

N

10 4 2 , 1400 (M-vancosamine+H)4, Exact Mass: 1542.43 Mol. Wt.: 1544.28 C, 52.89; H, 4.90; CI, 4.59; F, 3.69; N, 9.07; O, 24.87 (XL) 1143 (M-vancosamine-glucose+H) . b)15 EXAMPLE 21: Glurcose-C6-mesitylenesulfonyl Vancomycin (XL).I) To5 NN'-Dialo-allyl-glucose-C6-trifluoroacetaminyl devancomyctive (XVI) (5.3 mg, 0.00028 mmol) in 3 mL anhydrouss dissolved with dry DMF is added 2 mL acetic acid then (Ph 3 )2Pd(II)CI2 (catalytic). This s00142 mmolution) is added, then the mixture is trstirred at room temper (20 additions at 5 minute intervals for 20 m.)added aot everywhich time TLC shows nearly 20 reaction is done. Diethyl ether the last addition, 0.45 mixture is centrifuged twiat nce. The reactsidue is purifiedon by ODS-H4PLC (LUNA 5gm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H20, B: MeCN, 0 10 30% B oveturns dark and TLC (Chloroform-methanol-water; 6:4:1(XL) shows all glycopeptide baseline. The biphasict (13, 0.2 mg, 0.000121mixture is diluted with 0.5 mL methanol and 5 mL diethyl ether and stirred 5 m-vanc. The solution ise+H), 1143 (M-vancosamine-glucose+H) . 15 EXAMPLE 21: Glucose-C6-mesitylenesulfonyl Vancomycin (XLI) To a stirred solution of mesitylenesulfonyl derivative (XVI) (52 mg, 0.028 mmol) in 3 mL anhydrous DMF is added 2 mL acetic acid then (Ph3P)2Pd(II)Cl2 (catalytic). This solution is treated with tributyltin hydride (20 RL additions at 5 minute intervals for 20 min.) at which time TLC shows nearly 20 so reaction. Five minutes after the last addition, 0.45 mL of Bu3SnH is added at once. The reaction turns dark and TLC (Chloroform-methanol-water; 6:4: 1) shows all glycopeptide baseline. The biphasic mixture is diluted with 0.5 mL methanol and 5 mL diethyl ether and stirred 5 min. The solution is WO 00/69892 41 PCT/US00/13679 precipitated by addition to 90 mL diethyl ether (3 x 30 mL in three centrifuge tubes). The resulting suspension is centrifuged, the supernatant decanted and the residual diethyl ether removed under a stream of argon. The solid is dissolved in water (ca. 5 mL per tube), stored at 4 0 C for 5 h and filtered to remove remaining catalyst or hydrophobic salts. The aqueous solutions are combined, volume reduced 5 under reduced pressure and separated by HPLC (Method B; 40 min. linear gradient of 0% to 60% acetonitrile; flow rate = 7.5 mL/min.) affords (XLI) (44 mg, 90%) Retention time = 24 min. LRESI MS calc for C 75

H

85

N

9 0 2 6 SIC1 2 : 1629.5; [M+H] += 1631; [M-vancosamine+H] + = 1488; [M disaccharide+H] = 1143. 10 EXAMPLE 22: N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-mesitylenesulfonated vancomycin (XLII) OOHO 0 H II I HH OH ci O/NH H H H H O O H00 HO 0NHMe 0O NH2 OH HO OH

C

88

H

94 C1 3

N

9 0 26 S Exact Mass: 1829.51 Mol. Wt.: 1832.16 C, 57.69; H, 5.17; CI, 5.81; N, 6.88; O, 22.70; S, 1.7 15 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (100.0 mg, 0.0573 mmol) is dissolved with dry DMF (2 mL) and wet DIEA (50 mL, 0.547 mmol) is added and the mixture is stabilized at 75 oC for 0.5 h. 4-(4-chlorophenyl)benzylcarboxylaldehyde (10.6 mg, 0.0490 mmol) is added and the reaction mixture is stirred at 75 oC for 2 h then NaBH 3 CN (0.3 mL, 1 M-THF, 0.3 20 mmoL) is added. The mixture is stirred for additional 2 h, cooled down to room temperature, filtered and purified by ODS-HPLC (LUNA 5tm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 10-60% B 0-30 min., 8 mL/min, tr= 27 min.) to give (XLI) as a white amorphous solid (37.7 WO 00/69892 42 PCT/US00/13679 mg, 0.0194 mmol, 35 %) and starting material (26.7 mg, 0.0153 mmol, 27 %) as TFA salts. LRESI MS 1831 (M+2H, for C 88

H

96 35 Cl 3

N

9 0 26

S)

+

, 1488 (M-N-4-(4-chlorophenyl)benzylvancosamine+2H) +. 1143 (M-N-4-(4-chlorophenyl)benzylvancosamine -glucose+H) +. 5 EXAMPLE 23: N-decylvancosamine-glucose-C6-2-mesitylenesulfonated vancomycin H o \ oo o Cl SI I H Cl / OH ci H H ONH

H

0 H H 0 0 0 0 HO NNHMe 0NH2 OH HO OH C8 5 H1001 2

N

9 0 2 6 S Exact Mass: 1769.63 Mol. Wt.: 1771.76 (XLIII) C, 57.62; H, 5.97; CI, 4.00; N, 7.11; O, 23.48; S, 1.81 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (101.5 mg, 0.0528 mmol) is dissolved 10 with wet DMF (5 mL) and DIEA (28 mL, 0.306 mmol) is added and the mixture is stabilized at 70 oC for 0.5 h. Decylaldehyde (9.30 mL, 0.0494 mmol) is added and the reaction mixture is stirred at 70 oC for 1.5 h then NaBH 3 CN (0.3 mL, 1M-THF, 0.3 mmoL) is added. The mixture is stirred for additional 2 h then cooled down to room temperature. The mixture is evaporated and the residue is purified by ODS-HPLC (LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 10-10 15 60-100 % B 0-5-30-40 min., 8 mL/min, trit.= 29 min.) to give white amorphous (XLIII) (18.9 mg, 0.010 mmol, 17 %) and the starting material (17.2 mg, 0.00985 mmol, 10 %) as TFA salts. LRESI-MS 1771 (M+2H, for C 85

H

10 7 3 5 Cl 2

N

9 0 2 6

S)

+

, 1488 (M-vancosamine+2H) , 1144 (M-N-decylvancosamine glucose+2H) +. 20 EXAMPLE 24: Glucose-C6-hydrazine vancomycin (XLIV) WO 00/69892 43 PCT/US00/13679 Oo ONH2 OH c2 0) H CI OO HH 0 HO NHMe 01 NH2 OO H OH H OH

C

6 6

H

77

CI

2

N

1 1023 Exact Mass: 1461.46 Mol. Wt.: 1463.30 C, 54.17; H, 5.30; CI, 4.85; N, 10.53; O, 25.15 Glucose-C6-2-mesitylenesulfonated vancomycin (XLI) (10.0 mg, 0.00527 mmol ) and hydrazine (50 5 mL, 0.00159 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 45oC. After 2.5 h. the solvent is removed in vacuo. Longer reaction time decomposes the compound. The residue is purified by ODS-HPLC (LUNA 5ptm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H20, B: MeCN, 0-30% B over 30 min., 8 mL/min, tr= 18 min.) to give a white amorphous TFA salt (XLIV) (1.2 mg, 0.000761 mmol, 14 %). LRESI-MS 1462 (M+H, for C 66

H

78

.

35 1

O

2 3 ) , 1321 (M 10 vancosamine+3H) , 1143 (M-vancosamine-glucose+H) . 15 20 WO 00/69892 44 PCT/US00/13679 EXAMPLE 25: Glucose-C6-1-pyrenesulfonated vancomycin (XLV) H H2 H 0 H 0 0ol 0 HOO NH III HCOy, \ Cl O OH ci a H H N /711 N H

H

0 H H 0 O O HO iH ' 1 NHMe OH NH2 OH H OH

HO

/ OH O

C

82 H83CI 2 N9026 S Exact Mass: 1711.45 Mol. Wt.: 1713.57 C, 57.48; H, 4.88; CI, 4.14; N, 7.36; O, 24.28; S, 1.87 5 N, N'-Dialoc-allyl-vancomycin (III) (25.0 mg, 0.0151 mmol) is dissolved dry pyridine (1 mL). The mixture is stirred at 4 oC in the refrigerator and 1-pyrenesulfonyl chloride (13.6 mg, 0.0452 mmol) with pyridine (0.5 mL) is added. Stirred at 4 oC in the dark. After 57 h additional 3 eq. of 2 mesitylenesulfonyl chloride with pyridine (5 mL) is added. After total 70 h quenched the reaction with MeOH (0.5 mL) then added ether (10 mL). Centrifuged and the layer is removed (x2). The residue 10 purified by ODS-HPLC (LUNA 5tm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% AcOH/H 2 0, B: 0.1% AcOH/MeCN, 40-80 % B 0-30 min., 8 mL/min, tr = 22 min.) to give a white amorphous. The product is dissolved with dry DMF/AcOH (1/1) (1 mL). Pd(PPh 3

)

2

CI

2 (cat.) is added, then the mixture is stirred at room temperature under Ar. Added Bu 3 SnH (0.2 mL) every about 5 min. After 3 h the reaction is done. Added ether then centrifuged twice. The mixture is filtered and purified by ODS 15 HPLC (LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 0-50-100 % B 0-30-50 min., 8 mL/min, trit. = 3 1min.) to give white amorphous (XLV) as a TFA salt (4.5 mg, 0.00246 mmol, 5.4%, 2 step). LRESI-MS 1713 (M+2H, for C 82

H

85 35 Cl 2

N

9 0 26

S)

+

, 1570 (M vancosamine+2H)

+

, 1144 (M-vancosamine-glucose+2H) . 20 WO 00/69892 45 PCT/US00/13679 EXAMPLE 26: Glucose-C6-azide vancomycin (XLVI) HO 0 0 N3 CII HH OH NH H H H H 0 0 H 00 HO H2'NHMe NH2 OH HO OH

C

66

H

74 C1 2

N

1 2023 Exact Mass: 1472.44 Mol. Wt.: 1474.27 C, 53.77; H, 5.06; CI, 4.81; N, 11.40; O, 24.96 5 N, N'-Dialoc-allyl-glucose-C6-azide vancomycin (XVII) (19.5 mg, 0.0116 mmol) is dissolved with dry DMF/AcOH (1/1) (1 mL). Pd(PPh 3

)

2

CI

2 (1.0 mg, 0.00142 mmol) is added, then the mixture is stirred at room temperature under Ar. Bu 3 SnH (0.1 mL) is added about every 20 min. After 4 h the reaction is done. Ether is added and the mixture is centrifuged twice. The residue is purified by ODS-HPLC (LUNA 5Rm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 0-30% B over 30 10 min., 8 mL/min, tr= 13 min.) to give a white amorphous (XLVI) as a TFA salt (6.3 mg, 0.0040 mmol, 34 %). LRESI-MS 1473 (M+H, for C66H 7 5 35 Cl120 2 3

)

+

, 1331 (M-vancosamine+2H)

+

, 1143 (M vancosamine-glucose+H) +. 15 EXAMPLE 27: Glucose-C6-amine Vancomycin (XLVII) To a solution of amine (XVIII) (18 mg, 0.01 mmol) in 1 mL anhydrous DMF containing 0.8 mL acetic acid is added (Ph 3 P)2Pd(II)C12 (catalytic). To the stirred solution Bu3SnH is added in 20 pL aliquots every 2 min. for 8 min. then 40 gL aliquots every 2 min. for 6 min., at which time addition of the 20 Bu3SnH affords a dark reaction mixture and TLC (chloroform-methanol-water; 6:4:1) shows all glycopeptide is baseline. The crude mixture is precipitated by addition to 20 mL diethyl ether, the suspension centrifuged and the supernatant decanted. The white solid is dried under reduced pressure to WO 00/69892 46 PCT/US00/13679 remove residual diethyl ether, dissolved in water (ca. 5 mL) and filtered to remove any remaining catalyst or hydrophobic salts. Separation by HPLC (Method B; 40 min. linear gradient of 0% to 40% acetonitrile; flow rate = 7.5 mL/min.) affords (XLVII) (15 mg, 96%) Retention time = 23 min. LRESI MS calc for C 6 6

H

76 NI0023C 2 : 1446.5; [M+H]

+

= 1448; [M-vancosamine+H] + = 1305; [M 5 disaccharide+H] = 1143. EXAMPLE 28: Glucose-C6-N-4-(4-chlorophenyl)benzyl Vancosamine-N4-4-(4-chlorophenyl)benzyl Vancomycin (XLVIII) 10 To a stirred solution of (XVIII) (10 mg, 0.007 mmol) in 0.4 mL anhydrous DMF under an argon atmosphere is added DIEA (6 pL, 0.035 mmol). After 5 min, 4-4-(4 chlorophenyl)benzylcarboxaldehyde (1.5 mg, 0.007 mmol) is added and the mixture stirred at 65°C for lh. Sodium cyanoborohydride (3 mg, 0.05 mmol) is then added and the mixture stirred an additional 5 15 h. at 65°C. The reaction is cooled to room temperature and precipitated by addition to 15 mL diethyl ether The resulting suspension is centrifuged and the supernatant decanted. Residual diethyl ether is removed under a stream of argon. Separation by HPLC (Method B; 40 min. linear gradient of 10% to 65% acetonitrile; flow rate = 7.5 mL/min.) affords (XLVIII) (2 mg, 15%); Retention time = 27 min. and (XXIX) (3 mg, 26%) which is identical to that prepared previously. LRESI-MS calc for 20 C 9 2

H

94

N

10 0 2 3 C1 4 : 1846.5; [M+H]

+

= 1848; [M-disaccharide+H] + = 1143. EXAMPLE 29: Glucose-C6-N-5-(4-chlorophenyl)furan- 1-methylene-Vancosamine-N-decyl Vancomycin (XLIX) 25 To a stirred solution of glucose-C6-N-5-(4-chlorophenyl)furan-1-methylene derivative (XXX) (8.8 mg, 0.005 mmol) in 0.5 mL anhydrous DMF under an argon atmosphere is added DIEA (4.4 p L, 0.025 mmol) then decylaldehyde (0.85 jtL 0.0045 mmol) and the solution stirred at 70 *C for 2 h. Sodium cyanoborohydride (2 mg, 0.03 mmol) is then added and the mixture stirred an additional 2 h. at 70'C. 30 The mixture is cooled to room temperature and precipitated by addition to 8 mL diethyl ether. The resulting suspension is centrifuged, the supernatant decanted and residual diethyl ether removed under a stream of argon. Separation by HPLC (Method B; 35 min. linear gradient of 20% to 80% acetonitrile; flow rate = 7.5 mL/min.) affords (XLIX) (1 mg, 12%). Retention time = 19 min. LRESI-MS calc for

C

87

H

103

N

10 0 24

CI

3 : 1776.6; [M+H] += 1778; [M-disaccharide+H] + = 1143. 35 WO 00/69892 47 PCT/US00/13679 EXAMPLE 30: Glucose-C6-N-5-(4-chlorophenyl)furan- 1-methylene-Vancosamine-N-4-(4 chlorophenyl)benzyl Vancomycin (L) To a stirred solution of glucose-C6-N-5-(4-chlorophenyl)furan-1-methylene derivative (XXX) (8.4 mg, 5 0.005 mmol) in 0.5 mL anhydrous DMF under an argon atmosphere is added DIEA (4.2 ptL, 0.024 mmol) then 4-4-(4-chlorophenyl)benzyl-carboxaldehyde (0.9 mg, 0.004 mmol) and the solution stirred at 70 'C for 2 h. Sodium cyanoborohydride (2.5 mg, 0.04 mmol) is then added and the mixture stirred an additional 2 h at 70'C. The mixture is cooled to room temperature and precipitated by addition to 8 mL diethyl ether. The resulting suspension is centrifuged, the supernatant decanted, and residual 10 diethyl ether removed under a stream of argon. Separation by HPLC (Method B; 40 min. linear gradient of 12% to 60% acetonitrile; flow rate = 7.5 mL/min.) affords (L) (2.5 mg, 28%) Retention time = 29 min. and 3.5 mg recovered starting material. LRESI-MS calc for C 9 0

H

9 2

N

10 0 24 C1 4 : 1836.5; [M+H] = 1838; [M-disaccharide+H] = 1143. 15 EXAMPLE 31: Glucose-C6-N-2-quinoxaloyl-Vancosamine-N-decyl Vancomycin (LI) To a stirred solution of glucose-C6-N-2-quinoxaloyl derivative (XXXVII) (11 mg, 0.007 mmol) in 0.5 mL anhydrous DMF under an argon atmosphere is added DIEA (6 [tL, 0.035 mmol) then 20 decylaldehyde (1.1 ItL, 0.006 mmol) and the solution stirred at 70 'C for 2 h. Sodium cyanoborohydride (3 mg, 0.05 mmol) is then added and the solution stirred an additional 2 h at 70 *C. The mixture is then cooled to room temperature and precipitated by addition to 15 mL diethyl ether. The resulting suspension is centrifuged, the supernatant decanted, and the residual diethyl ether removed under a stream of argon. Separation by HPLC (Method B; 40 min. linear gradient of 5% to 25 70% acetonitrile; flow rate = 7.5 mL/min.) affords (LI) (5 mg, 40%) Retention time = 32 min. LRESI MS calc for C 85 ssHo 10 oN120 24

CI

2 : 1742.6; [M+H] = 1744; [M-disaccharide+H] + = 1143. 30 35 WO 00/69892 48 PCT/US00/13679 EXAMPLE 32: Glucose-C6-N-2-quinoxaloyl-Vancosamine-N-4-(4-chlorophenyl)benzylVancomycin (LII) NH N HO o N N 0 0 H O I H HO,,, .. , OH 0 0 H 'N H N H N NN -. NH ~H H H 0 N HO H,, NH H3 H 0 ,I H2N00'5 HCH3 O H 2 N OH HO OH 5 To a stirred solution of glucose-C6-N-2-quinoxaloyl derivative (XXXVII) (10.3 mg, 0.006 mmol) in 0.4 mL anhydrous DMF under an argon atmosphere is added DIEA (5.2 gL, 0.03 mmol) then 4-4-(4 chlorophenyl)benzylcarboxaldehyde (1.2 mg, 0.0055 mmol) and the solution stirred at 70 'C for 100 10 min. Sodium cyanoborohydride (2.5 mg, 0.04 mmol) is then added and the mixture stirred an additional 2.5 h at 70'C. The mixture is cooled to room temperature and precipitated by addition to 15 mL diethyl ether. The resulting suspension is centrifuged, the supernatant decanted, and residual diethyl ether removed under a stream of argon. Separation by HPLC (Method B; 40 min. linear gradient of 5% to 70% acetonitrile; flow rate = 7.5 mL/min.) affords (LII) (3.3 mg, 30%, 60% based on 15 3 mg recovered starting material). Retention time = 29 min. LRESI-MS calc for C 88

H

89

N

12 0 24 C1 3 : 1802.5; [M+H]

+

= 1804; [M-disaccharide+H] = 1143. EXAMPLE 33: Glucose-C6-thiopropianato Vancomycin (LIII) 20 General Procedure for thiolate displacements on mesitylene sulfonyl derivative (XLI). To a stirred solution of mesitylenesulfonyl derivative (XLI) (10 to 100 mg) in 0.5 to 4 mL anhydrous DMF under an argon atmosphere is added powdered potassium carbonate (20 to 30 molar equivalents). 25 To the resulting suspension is added the thiol (10 to 20 molar equivalents) and the mixture is stirred at 60 to 65 'C until analytical HPLC shows disappearance of 9. The suspension is cooled to room WO 00/69892 PCT/US00/13679 49 temperature, diluted with 0.5 to 1 mL methanol, filtered (0.45 pim) to remove carbonate, and the filtrate is then evaporated under reduced pressure. Separation by HPLC is then performed. Mesitylenesulfonyl derivative (XLI) (13 mg, 0.008 mmol) is subjected to thiolate displacement with 2 5 propanethiol (30 pL, 0.32 mmol) as described in the general method. Separation by HPLC (Method B; 5 min. at 0% acetonitrile then 40 min. linear gradient of 0% to 45% acetonitrile; flow rate = 7.5 mL/min.) affords (LIII) (8 mg, 66%). Retention time = 37 min. LRESI-MS calc for C 69 Hs 1

N

9 0 23 SIC1 2 : 1505.5; [M+H]

+

= 1507; [M-vancosamine+H] + = 1364; [M-disaccharide+H] + = 1143. 10 EXAMPLE 34: Glucose-C6-thiophenyl Vancomycin (LIV) Mesitylenesulfonyl derivative (XLI) (5 mg, 0.003 mmol) is subjected to thiolate displacement with thiophenol (5 gL, 0.05 mmol) as described in the general method. Separation by HPLC (Method B; 40 15 min. linear gradient of 5% to 60% acetonitrile; flow rate = 7.5 mL/min.) affords (LIV) (3 mg, 67%). Retention time = 19 min. LRESI-MS calc for C 7 2 H79N 9 0 2 3

S

1 C1 2 : 1539.4; [M+H]

+

= 1540; [M vancosamine+H] + = 1397; [M-disaccharide+H] + = 1143. 20 EXAMPLE 35: Glucose-C6-3-chlorothiophenyl Vancomycin (LV) Mesitylenesulfonyl derivative (XLI) (8 mg, 0.005 mmol) is subjected to thiolate displacement with 3 chlorothiophenol (6 gL, 0.05 mmol) as described in the general method. Separation by HPLC (Method B; 40 min. linear gradient of 8% to 50% acetonitrile; flow rate = 7.5 mL/min.) affords (LV) (4 mg, 25 51%). Retention time = 30 min. LRESI-MS calc for C 72

H

78

N

9 0 23 SIC1 3 : 1573.4; [M+H]

+

= 1574; [M vancosamine+H] = 1433; [M-disaccharide+H] + = 1143. EXAMPLE 36: Glucose-C6-3-amino-5-mercapto-1,2,4-triazole Vancomycin (LVI) 30 Mesitylenesulfonyl derivative (XLI) (7 mg, 0.004 mmol) is subjected to thiolate displacement with 3 amino-5-mercapto-1,2,4-triazole (5 mg, 0.04 mmol) as described in the general method. Separation by HPLC (Method B; 40 min. linear gradient of 0% to 40% acetonitrile; flow rate = 7.5 mL/min.) affords (LVI) (4 mg, 65%). Retention time = 23 min. LRESI-MS calc for C 6 8

H

7 7

N

13 0 23

S

1 C1 2 : 1545.4; 35 [M+H]

+

= 1547; [M-vancosamine+H] + = 1404; [M-disaccharide+H] + = 1143.

WO 00/69892 50 PCT/US00/13679 EXAMPLE 37: Glucose-C6-Imidazole Vancomycin (LVII) Mesitylenesulfonyl derivative (XLI) (6 mg, 0.0034 mmol) and imidazole (18 mg, 0.26 mmol) are stirred in 0.7 mL anhydrous DMF under an argon atmosphere at 80 oC for 8 h. The mixture is cooled to 5 room temperature, diluted with water and separation by HPLC (Method B; 40 min. linear gradient of 0% to 50% acetonitrile; flow rate = 7.5 mL/min.) affords (LVII) (2.5 mg, 46%). Retention time = 24 min. LRESI-MS calc for C 6 9 H77NnO10 2 3

CI

2 : 1497.5; [M+H]

+

= 1498; [M-vancosamine+H] = 1357; [M disaccharide+H]* = 1143. 10 EXAMPLE 38: Glucose-C6-5-thio-1-methyl-tetrazole vancomycin (LVIII) H 0 N 0 0 o. H OH cl 0 H 0 HH H 0 H H 0 0 0 O C NH 2 I OH H OH

C

68

H

77

CI

2

N

1 3023 S Exact Mass: 1545.44 Mol. Wt.: 1547.40 C, 52.78; H, 5.02; CI, 4.58; N, 11.77; 0, 23.78; S, 2.07 15 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (10 mg, 0.00573 mmol), 5-mercapto 1-methyl-tetrazole (14 mg 0.121 mmol), and K 2

CO

3 (17 mg 0.123 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 70C. After 4 h. analytical HPLC indicates that the reaction is done. The mixture is filtered and purified by ODS-IPLC (LUNA 5Rtm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 O, B: MeCN, 0-50% B 0-30 min., 8 mL/min, tr 22 min.) to give 20 (LVIII) as a white amorphous TFA salt (3.4 mg, 0.00205 mmol, 36 %). LRESI-MS 1547 (M+2H, for

C

68 H79 35 C1 2

N

13 0 23

S)

+

, 1404 (M-vancosamine+2H) , 1144 (M-vancosamine-glucose+2H)

+.

WO 00/69892 51 PCT/US00/13679 EXAMPLE 39: Glucose-C6-1-thio-4-bromobenzene vancomycin (LIX) 20 H HO Br o o 0 0 I 11I H C OH C1 H H HO

H

0 H H 0 00 HO NNH2 OH HO OH

C

72

H

78 BrCl2 N 9

O

23 S Exact Mass: 1617.35 Mol. Wt.: 1620.32 C, 53.37; H, 4.85; Br, 4.93; CI, 4.38; N, 7.78; O, 22.71; S, 1.98 5 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (10 mg, 0.00573 mmol), 4 bromothiophenol (22.7 mg 0.12 mmol), and K 2

CO

3 (17 mg 0.123 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 70C. After I h. analytical HPLC indicates that the reaction is done. The mixture is filtered and purified by ODS-HPLC (LUNA 5iim C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 0-50% B 0-30 min., 8 mL/nmi. tr= 27 min.) to give (LIX) 10 as a white amorphous TFA salt (3.2 mg, 0.00185 mmol, 32%). LRESI-MS 1618 (M+H, for

C

72

H

79 79Br 35 Cl 2 N90 23 S), 1475 (M-vancosamine+H) , 1143 (M-vancosamine-glucose+H) . 15 20 WO 00/69892 52 PCT/US00/13679 EXAMPLE 40: Glucose-C6-2-thio-4-trifluoromethylpyridine vancomycin (LX) H H2 H F 10 H CI HO OHOO H, O HO N2NHMe OH HO OH

C

7 1

H

76

CI

2

F

3 N 1023S Exact Mass: 1609.42 Mol. Wt.: 1611.40 C, 52.92; H, 4.75; CI, 4.40; F, 3.54; N, 9.56; O, 22.84; S, 1.9 5 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (10 mg, 0.00573 mmol), 4 (trifluoromethyl)-2-pyridinethiol (21.6 mg 0.12 mmol), and K 2

CO

3 (17 mg 0.123 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 65 0 C. After 2 h. analytical HPLC indicates the reaction is done. The mixture is filtered and purified by ODS-HPLC (LUNA 5gm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 0-50-100 % B 0-30-40 min., 8 mL/min, tr= 35 min.) 10 to give (LX) as a white amorphous TFA salt (2.3 mg, 0.00133 mmol, 23 %). LRESI-MS 1613 (M+4H, for C 71

H

80 35 C1 2

F

3 N 1

O

2 3 S)', 1143 (M-vancosamine-glucose+H)

.

WO 00/69892 53 PCT/US00/13679 EXAMPLE 41: Glucose-C6-2-thio-4-aminopyrimidine vancomycin (LXI) HH os' OO ci 0 0 I II HI O OH H H Hl 0 0 HON OH .oo NH2 HO OH

C

70

H

78

CI

2

N

12 0 23 S Exact Mass: 1556.44 Mol. Wt.: 1558.41 C, 53.95; H, 5.04; CI, 4.55; N, 10.79; O, 23.61; S, 2.06 5 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (17.5 mg, 0.0100 mmol), 4-amino-2 mercaptopyrimidine (26.1 mg 0.12 mmol), and K 2

CO

3 (17 mg 0.123 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 80 0 C. After 1 h. analytical HPLC indicates the reaction is done. The mixture is filtered and purified by ODS-HPLC (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 0-70% B 0-60 min., 10 8 mL/min, tr= 30 min.) to give (LXI) as a white amorphous TFA salt (7.3 mg, 0.00436 mmol, 44 %). LRESI-MS 1557 (M+H, for C 7 oH 79 Cl 2 NI20 23

S)

+

, 1414 (M-vancosamine+H)

+

, 1143 (M-vancosamine glucose+H)

+

.

WO 00/69892 54 PCT/US00/13679 EXAMPLE 42: Glucose-C6-6-thio-2, 4-diaminopyrimidine vancomycin (LXII) 0 H NH 2 O S NH2 0 0 I I I H Q, Cl - OH H H NH HH H/ 0 HO NHMe 0NH2 H OH HO OH

C

7 0H 79 CI2N1 3 0 23 S Exact Mass: 1571.45 Mol. Wt.: 1573.42 C, 53.43; H, 5.06; CI, 4.51; N, 11.57; O, 23.39; S, 2.04 5 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (LXII) (13.3 mg, 0.00762 mmol), 4-amino 2-mercaptopyrimidine (31.2 mg 0.163 mmol), and K 2

CO

3 (22.1 mg 0.160 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 80 0 C. After 7 h. analytical HPLC indicates the reaction is done. The mixture is filtered and purified by ODS-HPLC (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5ptm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 0-70% B 0-60 min., 10 8 mL/min, tr= 36 min.) to give (LXII) as a white amorphous TFA salt (10 mg, 0.00593 mmol, 78 %). LRESI-MS 1573 (M+2H, for C 70

H

81 3 5 Cl 2

N

13 0 23

S)

+

, 1430 (M-vancosamine+2H) , 1143 (M vancosamine-glucose+H) .

WO 00/69892 55 PCT/US00/13679 EXAMPLE 43: Glucose-C6-2-thio-4-amino-6-hydroxypyrimidine vancomycin (LXIII) H NH2 0 0OH I II HC Cl / OH H H NH HOH H O O HO INHMe 01 N .... ,NH2 OH HO OH

C

7 0H78CI2N1 2 0 24 S Exact Mass: 1572.43 Mol. Wt.: 1574.41 C, 53.40; H, 4.99; CI, 4.50; N, 10.68; O, 24.39; S, 2.0 5 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (10 mg, 0.00573 mmol), 4, 5 diamino-6-hydroxy-2-mercaptopyrimidine (19.4 mg 0.122 mmol), and K 2

CO

3 (17 mg 0.123 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 65 0 C. After 5 h the mixture is filtered and purified by ODS-HPLC (LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 0-50-100 % B 0-30-40 min., 8 mL/min, tr= 29 min.) to give (LXIIT) as a white amorphous 10 TFA salt (1.0 mg, 0.000592 mmol, 10 %). LRESI-MS 1574 (M+2H, for C 7 0

H

80 35 C1 2

N

1 20 24

S)

+

, 1431 (M-vancosamine +2H) , 1143 (M-vancosamine-glucose+H) .

WO 00/69892 56 PCT/US00/13679 EXAMPLE 44: Glucose-C6-2-thio-6-azathymine vancomycin (LXIV) H H( . OH NH2H O H I H H ""Ni/"' NH HO H H O O H 0 0 HO I NHMe NH2 OH HO OH

C

7 oH 78 C1 2

N

1 2024 S Exact Mass: 1572.43 Mol. Wt.: 1574.42 C, 53.40; H, 4.99; CI, 4.50; N, 10.68; O, 24.39; S, 2.04 5 Glucose-C6-2-mesitylenesulfonated vancomycin (XLI) (175 mg, 0.1 mmol), 6-aza-2-thiothymine (307 mg 2.1 mmol), and K 2

CO

3 (304 mg 2.2 mmol), are dissolved with dry DMF (5 mL). The mixture is stirred at 80oC. After 5 h analytical HPLC indicates the reaction is done. The solvent is removed in vacuo. Water is added and the mixture is centrifuged. The residue is purified by ODS-HPLC (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5Rm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% 10 TFA/H 2 0, B: MeCN, 0-70% B 0-60 min., 8 mL/min, tr = 36 min.) to give (LXIV) as a white amorphous TFA salt (79 mg, 0.047 mmol, 47 %). LRESI-MS 1573 (M+H, for C 70

H

79 Cl 2 NI20 2 4

S)

+, 1430 (M-vancosamine+H) , 1143 (M-vancosamine-glucose+H)

.

1 H-NMR data in DMSO-d6 at 298 K: 8 0.84 (3H, d, J= 6.5 Hz, ld), 0.90 (3H, d, J= 6.5 Hz, ic), 1.09 (3H, d, J= 6.0 Hz, V6), 1.30 (3H, s, V7), 1.52 (2H, m, la), 1.70 (1H, br d, J= 8.5 Hz, V2e), 1.72 (1H, m, lb), 1.90 (1H, br d, J= 8.5 Hz, 15 V2a), 2.08 (3H, s, azathymine-6), 2.15 (1H, m, 3a), 2.41 (1H, m, 3a), 2.42 (3H, s, le), 3.17 (1H, br s, V4), 3.50-3.56 (5H, m, G2-6), 3.63 (1H, m, xl), 4.20 (1H, br s, x6), 4.37 (1H, m, x3), 4.44 (2H, br s, x5 and x7), 4.65 (1H, br d, J=5.0 Hz, V5), 4.92 (1H, br s, x2), 5.10 (1H, s, z6), 5.18 (1H, s, z2), 5.19 (1H, s, 4f), 5.24 (2H, s, VI and G1), 5.40 (2H, br s, G3OH and G4OH), 5.55 (1H, s, 4b), 5.73 (1H, br s, x4), 5.80 (1H, br s, Z2OH), 5.94 (1H, br s, Z6OH), 6.25 (1H, s, 7f), 6.40 (1H, s, 7d), 6.90 (1H, m, 20 w3 and w6), 6.72 (1H, d, J=9.0 Hz, 5e), 6.78 (1H, d, J=9.0 Hz, 5f), 7.19 (1H, s, 5b), 7.32 (1H, m, 2e), 7.39(1H, m, 2b), 7.46 (1H, d, J=8.5 Hz, 6e), 7.51 (1H, d, J=8.5 Hz, 6f), 7.60 (1H, m, 2f), 7.85 (1H, s, 6b), 8.49 (2H, br s, w5 and w7), 8.66 (1H, br s, w4), 9.10 (1H, br s, 7cOH), , 9.17 (1H, br s, 5dOH), 9.44 (1H, br s, 7eOH).

WO 00/69892 57 PCT/US00/13679 OH I NH 2

NH

2 V2a, e, 1.70,1.90 OH V4, 3.17 V OH G2-G6, 3.50-3.56 6, 2.08 G30H, G40H, V6, 1.09 V HO 5.40 V5, 4.651 V1, 5.2 G4 v7, 1.30 03 G5 G6 N G2 N O S 2" N N G1, 5.24 0 O CI 6e, 7.46 0 0 6f, 7.51 2b, 7.39 I 4 2 z20H, z6OH, 5.95 6 5.80 HO/ 4b, 5.55 z2, 5.18 OH / Cl 4f, 5.1 O 2e, 7.32 2f, 7.60 z6, 5.10 6b, 7.85 H H x2, 4.92 O / w6, 6.90 x5, 4.44 N x3, 4.37 N x6,4.20 N r 5, 8.49 x4, 575 N w3,6.90 NH w 2 , H H w4, 8.66 O HN w7, 8.49 5b, 7.19 O xl, 3.63 x7, 4.44 5f, 6.78 3a, 2.15, 2.45 //,,wl 0 HO 5 NHM le, I 2.42 5e, 6.72

NH

2 "b, 1.72 0 1 c, 0.90 la, 1.52 7f, 6.25 7 7c O, H 5dOH, 9.17 id, 0.84 HO 7e OH 7eOH, 9.44 7d, 6.40 7cOH, 9.10 WO 00/69892 58 PCT/US00/13679 EXAMPLE 45: Glucose-C6-2-thio-5-methoxybenzimidazole vancomycin (LXV) H(C 2 H 0 O H OMe o k o sZ H0 H 0 H HH / /NNH OH 0 74

H

81

CI

2

N

1 024H Exact Mass: 160)9.46 Mol. Wt.: 1611.47 C, 55.15; H, 5.07; CI, 4.40; N, 9.56; 0, 23.83; S, 1.9 5Glucose-C6-2-mesitylenlesulfonated vancomycin TFA salt (XLI) (5 mag, 000287 mmo|l), 5-methoxy 2-benzimidazole-thiol (11.0 mg 0.0610 mmol), and K 2

CO

3 (8.6 mg 0.0622 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 65°C. After 4 h. analytical HPLC indicates the reaction is H O OH H N2NHMe uNH HO OH C74H81Cl2N1 1024S Exact Mass: 1609.46 Mol. Wt.: 1611.47 C, 55.15; H, 5.07; CI, 4.40; N, 9.56; O, 23.83; S, 1.9 5 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (5 mg, 0.00287 mmol) 5-methoxy 2-benzimidazole-thiol (11.0 mg 0.0610 mmol), and K2CO3 (8.6 mg 0.0622 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 65oC. After 4 h. analytical HPLC indicates the reaction is done. The mixture is filtered and purified by ODS-HPLC (LUNA 5gm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% AcOH/H 2 0, B: 0.1% AcOH/MeCN, 0-50 % B 0-30 min., 8 mL/min, tr= 27 10 min.) to give (LXV) as a white amorphous TFA salt (1.2 mg, 0.000700 mmol, 24 %). LRESI-MS 1611 (M+2H, for C 74

H

83 35 C1 2

N

11 0 24 S), 1467 (M-vancosamine+2H), 1143 (M-vancosamine-glucose+H).

WO 00/69892 59 PCT/US00/13679 EXAMPLE 46: Glucose-C6-2-thio-5-chlorobenzothiazole vancomycin (LXVI) C I II HHH O N N//OH H H H c 0 O H " NHMe

SNH

2 OH HO OH

C

73

H

77

C

I

3 N10P23S2 Exact Mass: 1630.37 Mol. Wt.: 1632.94 C, 53.69; H, 4.75; CI, 6.51; N, 8.58; O, 22.54; S, 3. Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (103.9 mg, 0.0595 mmol), 5-chloro 5 2-mercapto-benzothiazole (256.9 mg 1.27 mmol), and K 2

CO

3 (176.1 mg 1.27 mmol), are dissolved with dry DMF (5 mL). The mixture is stirred at 75-80 oC. After 2 h. analytical HPLC indicates the reaction is done. The mixture is filtered and purified by ODS-HPLC (LUNA 5ptm C18(2), 50 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 20-100% B over 60 min., 20 mL/min, t,= 36 min.) to give (LXVI) as a white amorphous TFA salt (75.8 mg, 0.0434 mmol, 73 %). LRESI-MS 1632 10 (M+2H, for C 7 3 H79 3 5

C

3

N

1 i00 2 3

S

2

)

+

, 1488 (M-vancosamine+H)

+

, 1143 (M-vancosamine-glucose+H) +. 1 H-NMR data in DMSO-d6 at 298 K: 8 0.87 (3H, d, J= 5.5 Hz, ld), 0.92 (3H, d, J= 6.0 Hz, Ic), 1.08 (3H, d, J= 6.0 Hz, V6), 1.24 (3H, s, V7), 1.56 (1H, m, la), 1.65 (1H, m, lb), 1.66 (1H, m, la), 1.72 (1H, br d, J= 13 Hz, V2e), 1.90 (1H, br d, J= 12 Hz, V2a), 2.19 (1H, m, 3a), 2.62 (3H, s, le), 2.63 (1H, m, 3a), 3.15 (1H, br s, V4), 3.47-3.57 (3H, m, G2, G3, and G4), 3.59 (1H, br d, J= 11.0 Hz, G6), 3.73 15 (1H, m, G5), 3.83 (1H, br d, J= 11.0 Hz, G6), 3.96 (1H, m, xl), 4.20 (1H, br s, x6), 4.43 (1H, d, J= 5.5 Hz, x3), 4.45 (1H, br s, x7), 4.46 (1H, br s, x5), 4.67 (1H, br d, J=6.5 Hz, V5), 4.90 (1H, br s, x2), 5.10 (1H, s, z2), 5.16 (1H, s, z6), 5.21 (2H, br s, GI and VI), 5.26 (1H, d, J=5.5 Hz, 4f), 5.45-5.55 (2H, m, G3OH and G4OH), 5.67 (1H, s, 4b), 5.80 (1H, d, J= 7.5, x4), 5.90 (1H, br s, Z2OH), 5.95 (1H, d, J= 5.0, Z6OH), 6.25 (1H, s, 7f), 6.40 (1H, s, 7d), 6.57 (1H, m, w3), 6.74 (1H, s, Chlorobenzothiazole-4), 20 6.74 (1H, d, J=8.5 Hz, 5e), 6.78 (1H, d, J=8.5 Hz, 5f), 7.14 (1H, d, J=9.0 Hz, 2e), 7.16 (1H, s, 5b), 7.30 (1H, d, J=8.0 Hz, 6e), 7.45 (1H, d, J= 9.0 Hz, Chlorobenzothiazole-7), 7.46 (1H, s, 2b), 7.46 (1H, d, J=8.0 Hz, 6f), 7.53 (1H, d, J=9.0 Hz, 2f), 7.83 (1H, s, 6b), 8.05 (1H, d, J= 9.0 Hz, WO 00/69892 60 PCT/US00/13679 Chlorobenzothiazole-4), 8.51 (2H, br s, w5 and w7), 8.78 (1H, br s, w4), 9.08 (1H, br s, 7cOH), 9.17 (1H, br s, 5dOH), 9.44 (1H, br s, 7eOH). Cl OH

NH

2 V2a, e, 1.72, 1.90 4, 6.74 5 V4, 3.16 V,.16 V OH G2-G4, 3.47-3.57 S G3OH, G4OH, 6, 8.05 V6, 1.08 Vj 1 HO 3.45-3.55 V5, 4.671 V1, 5.21 G4 v,.4 G N* \ v7, 1.24 G GS, 3.73 G6, 3.59 7, 7.45 G2& 3.83 G1, 5.217 O S 2 S O Cl 6e, 7.30 0 0 6f, 7.46 2b, 7.46 6 4 2 z2OH, z60H, 5.95 1 I 25.90 HO// 4b, 5.67 z2, 5.10 OH CI 4f, 5.2 0 2e, 7.14 z6, 5.16 2f, 7.53 6b, 7.83 H H x2, 4.90 O w6 x5, 4.45 N x3, 4.44 N x6, 4.20 Nw5, 8.51 x4, 5.80 N w3, 6.57 NH w2, H H w4, 8.78 O HN w7, 8.51 5b, 7.16 O O xl, 3.96 x7 4.46 5f, 6.78 3a, 2.19, 2.63 i;. w1 O HO 5 INHMe le, I 12.62 5e, 6.73

NH

2 b, 1.65 O 1 5e 6 i c, 0.92 la, 1.56 & 1.66 7 .2 7OH 5dOH, 9.17 id, 0.87 HO 7e OH 7eOH, 9.44 7d, 6.40 7cOH, 9.08 WO 00/69892 61 PCT/US00/13679 61 EXAMPLE 47: Glucose-C6-2-thio-5-phenyl-1,3,4-oxadiazole vancomycin (LXVII) H

NH

2 H O H 0 Cl 0 0 H% I C OH Hl H H H H O O

H

0 0 HO NHMe OH HO OH

C

74

H

7 9gCI 2 N1 1 0 24 S Exact Mass: 1607.44 Mol. Wt.: 1609.46 C, 55.22; H, 4.95; CI, 4.41; N, 9.57; O, 23.86; S, 1.99 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (78.0 mg, 0.0447 mmol), 5-phenyl-1, 5 3, 4-oxadiazole-2-thiol (170.4 mg 0.956 mmol), and K 2

CO

3 (132.7 mg 0.960 mmol), are dissolved with dry DMF (2 mL). The mixture is stirred at 65 0 C. After 2 h. analytical HPLC indicates the reaction is done. The mixture is filtered and purified by ODS-HPLC (LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 10-60 % B 0-30 min., 8 mL/min, tr = 21 min.) to give (LXVII) as a white amorphous TFA salt (60.3 mg, 0.0350 mmol, 78 %). i RESI-MS 1608 (M+H, for 10 C 74

H

80 35 C1 2 NllO 24

S)

+

, 1466 (M-vancosamine+2H) . 1143 (M-vancosamine-glucose+H)

.

WO 00/69892 62 PCT/US00/13679 EXAMPLE 48: Glucose-C6-5-thio-1-(4-hydroxyphenyl)-1H-tetrazole vancomycin (LXVIII) H 2 H H N OS,, NN I I HQ~, CIOH H H NH H O O H 0 00 1 OH HO OH

C

73

H

79 C1 2 N1 3 024S Exact Mass: 1623.45 Mol. Wt.: 1625.47 C, 53.94; H, 4.90; CI, 4.36; N, 11.20; O, 23.62; S, 1.97 5 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (10 mg, 0.00573 mmol), 1-(4 hydroxyphenyl)-1H-tetrazole-5-thiol (23.3 mg 0.12 mmol), and K 2

CO

3 (17 mg 0.123 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 65oC. After 14 h. analytical HPLC indicates the reaction is done. The mixture is filtered and purified by ODS-HPLC (LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 0-50-100 % B 0-30-40 min., 8 10 mL/min, tr = 35 min.) to give (LXVIII) as a white amorphous TFA salt (1.8 mg, 0.00103 mmol, 18 %). LRESI-MS 1625 (M+2H, for C 73

H

81 35 Cl 2 NI30 24

S)

+

, 1480 (M-vancosamine +2H)

+

, 1143 (M vancosamine-glucose+H)

.

WO 00/69892 PCT/US00/13679 63 EXAMPLE 49: Glucose-C6-2-thio-4,5-diphenyloxazole vancomycin (LXIX) 0 H OI )OS N c0 1~ 11 H O /0/ O H H H SHNH H O HO NH H 0 NH 2 H 0OH HO OH

C

82

H

86

CI

2

N

1 0023S Exact Mass: 1680.50 Mol. Wt.: 1682.60 C, 58.53; H, 5.15; CI, 4.21; N, 8.32; O, 21.87; S, 1.9 Glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLI) (10 mg, 0.00573 mmol), 4, 5 5 diphenyl-2-oxazole thiol (30.4 mg 0.12 mmol), and K 2

CO

3 (17 mg 0.123 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 65oC. After 2 h. analytical HPLC indicates the reaction is done. The mixture is filtered and purified by ODS-HPLC (LUNA 5tm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 10-10-60 % B 0-5-30 min., 8 mL/min, tr= 28 min.) to give (LXIX) as a white amorphous TFA salt (3.4 mg, 0.00133 mmol, 33 %). LRESI-MS 1683 (M+3H, for 10 C 82

H

89 35 Cl 2

N

10 0 23

S)

+

, 1540 (M-vancosamine +3H)

+

, 1143 (M-vancosamine-glucose+H) +. EXAMPLE 50: Glucose-C6-Iodo Vancomycin (LXX) 15 a) N,N'-dialoc-glucose-C6-Iodo-Vancomycin Allyl Ester. To a stirred solution of mesitylenesulfonyl derivative (XVI) (500 mg, 0.27 mmol) in 12 mL anhydrous dimethylacetamide (DMA) under an argon atmosphere is added powdered potassium iodide (0.9 g, 5.4 mmol) and the mixture stirred at 85°C for 12 h. The reaction is cooled to room temperature and precipitated by addition to 120 mL diethyl ether (4 x 30 mL), the suspension centrifuged, the 20 supernatant decanted, and the remaining diethyl ether removed under argon flow. The white solid in each tube is dissolved in 3 mL methanol/0.8 mL DMF, precipitated by the addition of 30 mL water, and the suspension stored at 4 'C for 12 h. Each suspension is then centrifuged and the supernatant is decanted. The solids are dissolved in methanol, combined, diluted with 250 mL toluene and evaporated WO 00/69892 64 PCT/US00/13679 to dryness under reduced pressure. The solid is dissolved in a minimum of methanol and diluted with as much dichloromethane as possible without precipitating, loaded onto a silica column packed in dichloromethane and eluted; first with 2 column volumes of dichloromethane-methanol-water (100:15:1), then dichloromethane-methanol-water (100:16:11). Fractions containing pure product are 5 combined and evaporated affording (350 mg, 73%). Fractions containing impure product are combined, evaporated and separated by HPLC (Method A; 40 min. linear gradient of 25% to 70% acetonitrile; flow rate = 7.5 mL/min.) affording additional pure product (30 mg). Ret. Time = 25 min. Combined yield of title compound = 380 mg, 80%. TLC: Rf= 0.6 (chloroform-methanol-water; 50:21:4). LRESI-MS calc for C 77

H

86

N

9 0 2 7 1 1 C1 2 : 1765.4; [M+Na] = 1788; [M-vancosamine+Na] + = 10 1562; [M-disaccharide+H] + = 1289. b) Glucose-C6-Iodo Vancomycin (LXX). The iodo derivative from step (a) (109 mg, 0.062 mmol) is dissolved in 8 mL anhydrous DMF and divided into two separate 4 mL reactions. Acetic acid (3 mL) is then added to each flask followed by 15 (Ph 3 P)2Pd(II)C12 (catalytic). Bu3SnH is added to the vigorously stirred solution in 30 tL portions every min. for 4 min. After the forth addition, waited 5 min., then added 60 RL Bu3SnH. The mixture turns dark and TLC (chloroform-methanol-water; 6:4:1) shows all glycopeptide is baseline. The crude mixture is diluted with 0.5 mL methanol and precipitated by addition to 80 mL diethyl ether. The suspension is centrifuged and the supernatant decanted. The white solid is suspended in diethyl ether 20 and mixed vigorously. The suspension is centrifuged and the supernatant decanted. The white solid is dried under reduced pressure to remove residual diethyl ether, dissolved in water (ca. 10 mL) stored at 4* C for 12 h then filtered to remove any remaining catalyst or hydrophobic salts. Separation by HPLC (Method B; 40 min. linear gradient of 5% to 60% acetonitrile; flow rate = 7.5 mL/min.) affords (LXX) (89 mg, 86%). Retention time = 22 min. LRESI-MS calc for C 66

H

74

N

9 0 2 3 1C1 2 : 1557.3; [M+H]

+

= 25 1558; [M-vancosamine+H] = 1415; [M-disaccharide+H] + = 1143. EXAMPLE 51: Glucose-C6-thioacetato Vancomycin (LXXI) 30 To a stirred solution of iodide (LXX) (2.5 mg, 0.0016 mmol) in 0.2 mL anhydrous DMF under an argon atmosphere is added powdered potassium carbonate (10 mg, 0.07 mmol). To the resulting suspension is added mercaptoacetic acid, monosodium salt, (8 mg, 0.07 mmol) and the mixture stirred at 60 oC for 40 min. The suspension is cooled to room temperature, diluted with 1 mL methanol and filtered (0.45 pm) to remove carbonate. The filtrate is evaporated under reduced pressure to remove methanol then 35 diluted with water (0.3 mL) and separation by HPLC (Method B; 40 min. linear gradient of 0% to 45% acetonitrile; flow rate = 7.5 mL/min.) affords (LXXI) (1.5 mg, 62%). Retention time = 22 min.

WO 00/69892 65 PCT/US00/13679 LRESI-MS calc for C 6 8

H

77

N

9 0 25

S

1 C1 2 : 1521.4; [M+H]*= 1523; [M-vancosamine+H] = 1380; [M disaccharide+H]* = 1143. 5 EXAMPLE 52: Vancosamine-N-decyl-Glucose-C6-S-3-amino-5-mercapto-1,2,4-triazole Vancomycin (LXXII) a) Vancosamine-N-decyl-Glucose-C6-odo-Vancomycin (LXXIIa). To a stirred solution of (LXX) (32 mg, 0.019 mmol) in 0.6 mL anhydrous DMF under an argon 10 atmosphere is added DIEA (17 ptL, 0.1 mmol). After 10 min., decyl aldehyde (2.86 pL, 0.015 mmol) is added and the solution heated at 70 'C for 2 h. Sodium cyanoborohydride (3 mg, 0.05 mmol) is then added and heating continued for an additional 2 h. The reaction mixture is cooled to room temperature and precipitated by addition to 20 mL diethyl ether. The suspension is centrifuged and the supernatant decanted. The white solid is dried under reduced pressure to remove residual diethyl ether. Separation 15 by HPLC (Method B; 40 min. linear gradient of 5% to 80% acetonitrile; flow rate = 8 mL/min.) affords the iodo product (LXXIIa) (10 mg, 30%). (Retention time = 28 min.) and 6 mg recovered (LXX). LRESI-MS for (LXXIIa) calc for C76H 94

N

9 0 23 IIC1 2 : 1697.5; [M+H] += 1699; [M-vancosamine+H] = 1415; [M-disaccharide+H] + = 1143. 20 b) Vancosamine-N-decyl-Glucose-C6-S-3-amino-5-mercapto-1,2,4-triazole Vancomycin (LXXII). To a stirred solution of the iodo product from step (a) (5 mg, 0.003 mmol) in 0.5 mL anhydrous DMF under an argon atmosphere is added potassium carbonate (10 mg, 0.07 mmoil). After 5 min. 3-amino-5 mercapto-1,2,4-triazole (4.2 mg, 0.036 mmol) is added and the mixture stirred at 55 oC for 30 min. The 25 mixture is cooled to room temperature, filtered (0.45 pm) to remove carbonate, and diluted with 8 mL water. Separation by HPLC (Method B; 40 min. linear gradient of 5% to 70% acetonitrile; flow rate = 7.5 mL/min.) affords (LXXII) (4.8 mg, 95%). Retention time = 28 min. LRESI-MS calc for

C

78

H

9 7 NI3023SIC1 2 : 1685.6; [M+H]1 = 1687; [M-vancosamine+H] + = 1404; [M-disaccharide+H] + = 1143. 30 WO 00/69892 66 PCT/US00/13679 EXAMPLE 53: Vancosamine-N-4-(4-chlorophenyl)benzyl,Glucose-C6-S-3-amino-5-mercapto-1,2,4 triazole Vancomycin (LXXIII) HQHN \ \/CI HO 0 HO OH 0 HN-N SS N

NH

2 0 0 0 O C H C I .H HO,,, COH 0 H 0 ;'" "H H H ""N N N NH H H ,H HO, NH O HO NCH I 0 H O

H

2 N / I OH HO OH 5 (LXXIII) a) Vancosamine-N-4-(4-chlorophenyl)benzyl Glucose-C6-Iodo-Vancomycin (LXXIIIa). To a stirred solution of (LXX) (21 mg, 0.013 mmol) in 0.5 mL anhydrous DMF under an argon atmosphere is added DIEA (11 IL, 0.06 mmol). After 10 min., 4-4-(4-chlorophenyl)benzyl 10 benzaldehyde (2.5 mg, 0.11 mmol) is added and the solution heated at 70°C for 90 min.. Sodium cyanoborohydride (3 mg, 0.05 mmol) is then added and the mixture stirred at 70°C for an additional 2 h. The reaction mixture is cooled to room temperature and precipitated by addition to 25 mL diethyl ether. The suspension is centrifuged and the supernatant decanted. The white solid is dried under reduced pressure to remove residual diethyl ether. Separation by HPLC (Method B; 40 min. linear 15 gradient of 5% to 60% acetonitrile; flow rate = 8 mL/min.) affords the iodo product (LXXIIIa) (11 mg, 46%); retention time = 32 min.; and 3 mg recovered (LXX). LRESI-MS for (LXXIIIa) calc for

C

79

H

83

N

9 0 23 1IC1 3 : 1757.4; [M+H]

+

= 1759; [M-vancosamine+H] + = 1415; [M-disaccharide+H] + = 1143. 20 b) Vancosamine-N-4-(4-chlorophenyl)benzyl,Glucose-C6-S-3-amino-5-mercapto-1,2,4-triazole Vancomycin (LXXIII). To a stirred solution of the iodo product (LXXIIIa) (5.4 mg, 0.003 mmol) in 0.5 mL anhydrous DMF under an argon atmosphere is added potassium carbonate (10 mg, 0.07 mmol). After 5 min. 3-amino-5 mercapto-1,2,4-triazole (4.2 mg, 0.037 mmol) is added and the stirred mixture heated at 55 'C for 50 25 min. The mixture is cooled to room temperature, filtered (0.45 pm) to remove carbonate, and the filtrate diluted with 6 mL water. Separation by HPLC (Method B; 40 min. linear gradient of 10% to WO 00/69892 67 PCT/US00/13679 67 65% acetonitrile; flow rate = 8 mL/min.) affords (LXXIII) (4.5 mg, 90%). Retention time = 28 min. LRESI-MS calc for C 8 1

H

86

N

13 0 2 3 SIC1 3 : 1745.5; [M+H] += 1747; [M-vancosamine+H] + = 1405; [M disaccharide+H] + = 1143. 5 EXAMPLE 54: N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-azide vancomycin (LXXIV) Cl H H o o -0

N

3 0 0 I I I

HC,

' 1 OH ci H H ,, NNH2 H 0 H H 0 0 o0 H jH NHMe 1 OH HO OH

C

79 H83Cl3N12023 Exact Mass: 1672.48 Mol. Wt.: 1674.93 C, 56.65; H, 4.99; CI, 6.35; N, 10.04; O, 21.9 10 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-iodo vancomycin TFA salt (LXXIIIa) (10.9 mg, 0.00582 mmol) and NaN 3 (7.6 mg 0.116 mmol) are dissolved with dry DMF (1 mL). The mixture is stirred at 45 oC. After 4 h. analytical HPLC indicates the reaction is done. the mixture is filtered then purified by ODS-HPLC (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 20-70% B 0-60 min., 8 mL/min, tr= 33 min.) to give 15 the white amorphous title compound (8.2 mg, 0.00458 mmol, 79 %) as TFA salt. LRESI-MS 1673 (M+H, for C 7 9

H

8 4 3 5 C1 3 NI2023), 1330 (M-N-4-(4-chlorophenyl)benzylvancosamine-glucose+H)

+

. 1143 (M-N-4-(4-chlorophenyl)benzylvancosamine-glucose+H) . 20 WO 00/69892 68 PCT/US00/13679 EXAMPLE 55: N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-amine vancomycin (LXXV) cl H QCI 0 6 H NH2 "' NH H O O H 0o HHO 'NHMe OOH I H OH

C

7 9 HasCI 3 N1 0023 Exact Mass: 1646.49 Mol. Wt.: 1648.93 C, 57.54; H, 5.20; CI, 6.45; N, 8.49; O, 22.32 5 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-azide vancomycin (LXXIV) (7.7 mg, 0.00430 mmol) and PPh 3 are suspended with THF (0.8 mL) and the mixture is stirred at room temperature under Ar for 0.5 h. Added H 2 0 (0.4 mL) and the mixture is stirred at 75 oC. After 9 h the mixture is filtered then purified by ODS-HPLC (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5ptm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 20-70% B 0-60 min., 8 mL/min, tr= 30 10 min.) to give the white amorphous title compound (3.2 mg, 0.00182 mmol, 42 %) as TFA salt. LRESI MS 1648 (M+2H, for C79H8 6 3 5 Cl 3 N100 23

)

+

, ? (M-N-4-(4-chlorophenyl)benzylvancosamine glucose+H)

+

. 1143 (M-N-4-(4-chlorophenyl)benzylvancosamine-glucose+H)

+

.

WO 00/69892 PCT/US00/13679 69 EXAMPLE 56: Glucose-C6-2-thio-5-amino-1,3,4-thiadiazole vancomycin (LXXVI) H H 2NH 2 H OH ON NH Cl o HO OH H H HO NHMe aNH H / OH

C

6 8

H

76

CI

2 N 1 2 0 23

S

2 Exact Mass: 1562.40 Mol. Wt.: 1564.44 C, 52.21; H, 4.90; CI, 4.53; N, 10.74; O, 23.52; S, 4.10 5 Glucose-C6-iodo vancomycin TFA salt (LXX) (14 mg, 0.00837 mmol), 5-amino-I, 3, 4-thiadiazole-2 thiol (24 mg 0.18 mmol), and K 2

CO

3 (25 mg 0.181 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 40-50oC. After 0.5 h. analytical HPLC indicates the reaction is done. the mixture is filtered then purified by ODS-HPLC (COSMOSIL 5C 18-AR, 20 x 250 mm, and LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 10-70% B 0-60 min., 8 mL/min, 10 tr = 27 min.) to give the white amorphous title compound as the TFA salt (6.3 mg, 0.0375 mmol, 45 %). LRESI-MS 1565 (M+H, for C 68

H

77 35 C1 2

N

1 20 23

S

2

)

+

, 1143 (M-vancosamine-glucose+H) .

WO 00/69892 70 PCT/US00/13679 EXAMPLE 57: Glucose-C6-5-thio-4-amino-3-hydrazino-1,2,4-triazole vancomycin (LXXVII) OH NHNH2 H QO Cl OH H H HO H H 0 H H 0 O HO NHMe 0 NH2 OO K OH HO OH C68H 79 C1 2

N

1 5 0 23 S Exact Mass: 1575.46 Mol. Wt.: 1577.41 C, 51.78; H, 5.05; CI, 4.50; N, 13.32; O, 23.33; S, 2.03 5 Glucose-C6-iodo vancomycin TFA salt (LXX) (20 mg, 0.0120 mmol ), 4-amino-3-hydrazino-5 mercapto-1, 2, 4-triazole (Purpald@, 37.5 mg 0.257 mmol), and K 2

CO

3 (35.4 mg 0.256 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 45oC. After 2 h. analytical HPLC indicates the reaction is done. The mixture is filtered then purified by ODS-HPLC (COSMOSIL 5C18-AR, 20 x250 mm, and LUNA 5gm C18(2), 21.2 x250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: 10 MeCN, 0-70% B 0-60 min., 8 mL/min, tr= 32 min.) to give the white amorphous title compound as the TFA salt (8.3 mg, 0.0491 mmol, 41 %). LRESI-MS 1569 (M-NHNH 2 +H+Na, for

C

68

H

81 35 Cl 2

N

1 50 23 SNa)

+

, 1143 (M-vancosamine-glucose+H)

+

.

1 H-NMR data in DMSO-d6 at 298 K: 6 0.86 (3H, d, J= 6.0 Hz, Id), 0.90 (3H, d, J= 6.0 Hz, Ic), 1.07 (3H, d, J= 6.5 Hz, V6), 1.26 (3H, s, V7), 1.41 (1H, m, la), 1.48 (IH, m, la), 1.70 (1H, br d, J= 12 Hz, V2e), 1.73 (1H, m, lb), 1.89 (1H, br d, J= 15 12 Hz, V2a), 2.14 (1H, m, 3a), 2.32 (3H, s, le), 2.36 (1H, m, 3a), 3.05 (1H, m, xl), 3.15 (1H, br s, V4), 3.46 (1H, br d, J= 12.5 Hz, G6), 3.51 (1H, br d, J= 12.5 Hz, G6), 3.52 (3H, m, G2, G3, and G4), 3.72 (1H, m, G5), 4.20 (1H, br s, x6), 4.37 (1H, m, x3), 4.43 (1H, s, x7), 4.44 (1H, br s, x5), 4.69 (1H, br d, J=6.5 Hz, V5), 4.88 (1H, br s, x2), 5.10 (1H, s, z6), 5.16 (1H, s, z2), 5.20 (1H, s, 4f), 5.20 (1H, br s, G4OH), 5.22 (1H, s, V1), 5.29 (1H, br s, GI), 5.43 (1H, br s, G3OH), 5.54 (1H, s, 4b), 5.74 (1H, br s, 20 Z2OH), 5.75 (1H, br s, x4), 5.94 (1H, br s, Z6OH), 6.25 (1H, s, 7f), 6.40 (1H, s, 7d), 6.64 (1H, m, w3), 6.72 (1H, d, J=8.5 Hz, 5e), 6.77 (1H, d, J=8.5 Hz, 5f), 6.89 (1H, m, w6), 7.19 (1H, s, 5b), 7.33 (1H, m, 2e), 7.34-7.57 (5H, m, NH 2 and NHNH 2 of 5-thio-4-amino-3-hydrazino-1, 2, 4-triazole), 7.37 (1H, m, WO 00/69892 71 PCT/US00/13679 2b), 7.47 (1H, d, J=8.5 Hz, 6e), 7.51 (1H, d, J=8.5 Hz, 6f), 7.57 (1H, m, 2f), 7.85 (1H, s, 6b), 8.46 (2H, br s, w5 and w7), 8.66 (1H, br s, w4), 9.09 (1H, br s, 7cOH), 9.41 (1H, br s, 7eOH). OH 7.34-7.57

NH

2 V2a, e, 1.70,1.89 V4, 3.15 v OH G3OH, 5.42 NHNH 2 V \ 15, s o 0 G40H, 5.20 V6, 1.07 HO H 2 N V5, 4.69 Vi, 5.21 G4, 3.52 N v7, 1.26 G3 , 52 3.72 G6,3 N G5,, 53.72, .85 H6, 3.462 4.N G23.52 & 3..51 . G N Gl, 5.29 O S 0 CI 6e, 7.47 0 0 6f, 7.51 2b, 7.37 66 4 2 z20H, z60H, 5.94 6 5.70 HO/ 4b, 5.54 z2, 5.16 OH CI 4f, 5.2 O 2e, 7.33 z6, 5.10 2f, 7.57 6b, 7.85 H H x2, 4.88 O / w6, 6.89 x5 4.4 N x3, 4.37 N >-w5 8.4 x, .7 x6, 4.20 N 5, 8.46 x4, 5.75 N w3, 6.63 NH w2, H H w4, 8.66 0 HN W 7 , 8

.

4 6 5b, 719 O O 0 xl, 3.05 S, 7.19 x7, 4.43 5f, 6.77 3a, 2.14, 2.36 //,,wl 0 HO 5 NHMe le, 2.32 Se, 6.72

NH

2 lb, 1.73 O 6.2 7c ic, 0.90 la, 1.41 & 1.48 7f,6.2y5 7 7c OH id, 0.86 HO 7e OH 7eOH, 9.41 7d, 6.40 7cOH, 9.09 5 WO 00/69892 72 PCT/US00/13679 EXAMPLE 58: Glucose-C6-2-thio-4-hydroxy-6-methylpyrimidine vancomycin (LXXVIII) ~H OH OH 0 0 0 H4 I I O H%,, \ ClO OH ci 0 H H HO H H 0 O H 0 00 ,0 HO 0 4"NHMe 0NH 2 /OH HO OH

C

7 1

H

79 C1 2

N

11 0 24 S Exact Mass: 1571.44 Mol. Wt.: 1573.42 C, 54.20; H, 5.06; CI, 4.51; N, 9.79; O, 24.40; S, 2.04 5 Glucose-C6-iodo vancomycin TFA salt (LXX) (15 mg, 0.00897mmol ), 4-hydroxy-2-mercapto-6 methylpyrimidine (27.4 mg 0.193 mmol), and K 2

CO

3 (26.5 mg 0.192 mmol), are dissolved with dry DMF (1 mL). The mixture is stirred at 45oC. After 0.5 h. analytical HPLC indicates the reaction is done, the mixture is filtered then purified by ODS-HIPLC (COSMOSIL 5C 18-AR, 20 x 250 mm, and LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 0-70% B 0-60 min., 10 8 mL/min, tr= 36 min.) to give the white amorphous title compound as the TFA salt (3.0 mg, 0.00178 mmol, 20 %). LRESI-MS 1572 (M+H, for C71H 8 0 35 Cl2N 1 1 0 24

S)

+

, 1430 (M-vancosamine +2H) , 1143 (M-vancosamine-glucose+H)*. 15 20 WO 00/69892 73 PCT/US00/13679 EXAMPLE 59: N-decylvancosamine-glucose-C6-2-thio-6-azathymine vancomycin (LXXIX) HN OH OS oHQ . ,O 0 H O 0 SHNH

H

0 H H OO H NHMe

NH

2 OH HO OH

C

80

H

98

CI

2 N 2024S Exact Mass: 1712.59 Mol. Wt.: 1714.67 C, 56.04; H, 5.76; CI, 4.14; N, 9.80; O, 22.39; S, 1.8 5 N-decylvancosamine-glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (Ex. 52 a) (10 mg, 0.00530 mmol), 6-aza-2-thiothymine (16.0 mg 0.112 mmol), and K 2

CO

3 (31.0 mg 0.224 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 75 0 C. After 8.5 h analytical HPLC indicates the reaction is done. The mixture is filtered and the residue is purified by ODS-HPLC (LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, I: MeCN, 10-60% B 0-30 10 min., 8 mL/min, tr= 26 min.) to give the white amorphous title compound as a TFA salt (4.8 mg, 0.00262 mmol, 50 %). LRESI-MS 1714 (M+2H, for CsoHoo 100 35 Cl 2

N

1 2 0 24

S)

+

, 1143 (M-N decylvancosamine-glucose+H).

WO 00/69892 74 PCT/US00/13679 EXAMPLE 60: N-decylvancosamine-glucose-C6-2-thio-5-chlorobenzothiazole vancomycin (LXXX) C' H\ I H O H II OS S HII I HQ, C OH ci H H HO H H 0 H H 0 0 HO 1 H2NHMe O NH 2 OH HO OH

C

8 3

H

97

CI

3

N

10 0 2 3

S

2 Exact Mass: 1770.52 Mol. Wt.: 1773.20 C, 56.22; H, 5.51; CI, 6.00; N, 7.90; O, 20.75; S, 3.62 5 N-decylvancosamine-glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (Ex. 52 a) (6.5 mg, 0.00345 mmol), 5-chloro-2-mercapto-benzothiazole (14.8 mg 0.0734 mmol), and K 2

CO

3 (10.1 mg 0.0731 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 75oC. After 2.5 h. analytical HPLC indicates the reaction is done. The mixture is filtered and the residue is purified by ODS-HPLC (LUNA 5gm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 10 10 60% B 0-30 min., 8 mL/min, tr = 28 min.) to give the white amorphous title compound as a TFA salt (1.4 mg, 0.000742 mmol, 22%). LRESI-MS 1771 (M+H, for C 83

H

98 3 5

CI

3

N

10 0 23

S

2

)

+

.

WO 00/69892 75 PCT/US00/13679 EXAMPLE 61: N-decylvancosamine-glucose-C6-2-thio-5-phenyl- 1,3,4-oxadiazole vancomycin (LXXXI) HH H H O I K HO ci H H H H 0 H H 00 HO NHNHMe ~ N NH 2 0 OH HO OH

C

84

H

9 9 C 12 N 11 0 2 4 S Exact Mass: 1747.60 Mol. Wt.: 1749.72 C, 57.66; H, 5.70; CI, 4.05; N, 8.81; O, 21.95; S, 1.83 5 Glucose-C6-2-thio-5-phenyl-1,3,4-oxadiazole vancomycin TFA salt (LXVII) (50.0 mg, 0.0290 mmol) is dissolved with wet DMF (2 mL) and DIEA (14.2 mL, 0.155 mmol) is added and the mixture is stabilized at 70 oC for 20 min. Decylaldehyde (4.70 mL, 0.0250 mmoL) is added and the reaction mixture is stirred at 70 oC for 1.5 h then NaBH 3 CN (0.1 mL, 1M-THF, 0.1 mmol) is added. The mixture is stirred for additional 2 h then cooled down to room temperature. The mixture is evaporated 10 and the residue is purified by ODS-HPLC (LUNA 5tm C 18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1%

TFA/H

2 0, B: MeCN, 10-60 % B 0-30 min., 8 mL/min, trit.= 22 min.) to give the white amorphous title compound (10.8 mg, 0.00579 mmol, 20 %) and the starting material (12.1 mg, 0.00702 mmol, 24 %) as TFA salts. LRESI-MS 1749 (M+2H, for C 84

H

101 35 Cl 2 NO10 24

S)

+

, 1144 (M-N-decylvancosamine glucose+2H) +. 15 20 WO 00/69892 PCT/US00/13679 76 EXAMPLE 62: N-decylvancosamine-glucose-C6-2-thio-4, 5-diphenyloxazole vancomycin (LXXXII) HH oo i HH NHNH

H

0 H H 0 HO NHMe O NH2 HO OH

C

9 1

H

1 0 4 C 2 N10024S Exact Mass: 1822.63 Mol. Wt.: 1824.82 C, 59.89; H, 5.74; CI, 3.89; N, 7.68; O, 21.04; S, 1.76 5 N-decylvancosamine-glucose-C6-2-mesitylenesulfonated vancomycin TFA salt (XLIII) (5.6 mg, 0.00321 mmol), 4, 5-diphenyl-2-oxazole thiol (16.0 mg 0.0632 mmol), and K 2

CO

3 (8.8 mg 0.0637 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 65oC. After 2 h. analytical HPLC indicates the reaction is done. The mixture is filtered and the residue is purified by ODS-HPLC (LUNA 5lm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 20-60% B 0-30 10 min., 8 mL/min, tr= 29 min.) to give the title compound as a white amorphous TFA salt (2.1 mg, 0.00108 mmol, 34 %). LRESI-MS 1824 (M+H, for C 91

H

1 o 6 35 Cl 2

N

10

O

24

S)

+

, 1143 (M-N decylvancosamine-glucose+H)

+

.

WO 00/69892 77 PCT/US00/13679 EXAMPLE 63: N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-5-amino-1,3,4-thiadiazole vancomycin (LXXXIII) H io O NH H -- 0 -. H ~H2 0 0 H 4// Cl O OH ci OH H H HH

H

0 H H o HO NH Me NHeNH2 OH H OH HO OH

C

8 lH 85

CI

3 N12023S2 Exact Mass: 1762.44 Mol. Wt.: 1765.10 C, 55.12; H, 4.85; CI, 6.03; N, 9.52; O, 20.85; S, 3.63 5 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C 6 -iodo vancomycin TFA salt (Ex. 53 a) (13.0 mg, 0.00694 mmol), 5-amino-1, 3, 4-thiadiazole-2-thiol (19.7 mg, 0.148 mmol), and K 2

CO

3 (20.5 mg 0.148 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirrd at 40-50oC. After 0.5 h analytical HPLC indicates the reaction is done. The mixture is filtered then purified by ODS-HPLC 10 (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5gm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1%

TFA/H

2 0, B: MeCN, 20-70% B 0-60 min., 8 mL/min, tr = 33 min.) to give the white amorphous title compound (3.0 mg, 0.00160 mmol, 23 %) as TFA salt. LRESI-MS 1763 (M+H, for

C

81

H

86 35 Cl 3

N

1 20 23

S

2

)

+

, 1420 (M-N-4-(4-chlorophenyl)benzylvancosamine-glucose+H) . 1143 (M-N-4 (4-chlorophenyl)benzylvancosamine-glucose+H) . 15 20 WO 00/69892 78 PCT/US00/13679 EXAMPLE 64: N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-5-thio-4-amino-3-hydrazino 1,2,4-triazole vancomycin (LXXXIV) cl OHHC O OH

NHNH

2 0 CI o 0 HO0H Cl1O OH H H OV N H N NH 0 Ni H OOO HO "NHMe HOI H0 I ~NH 2 OH HO OH

C

81

H

88 C1 3

N

15 023S Exact Mass: 1775.50 Mol. Wt.: 1778.08 C, 54.71; H, 4.99; CI, 5.98; N, 11.82; O, 20.70; S, 1.80 5 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-iodo vancomycin TFA salt (Ex. 53a) (12.0 mg, 0.0639 mmol ), 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole (20.0 mg 0.137 mmol), and K 2 CO3 (18.8 mg 0.136 mmol), are dissolved with dry DMF (1 mL). The mixture is stirred at 45oC. After 3 h analytical HPLC indicates the reaction is done. The mixture is filtered then purified by ODS-HPLC (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5tm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% 10 TFA/H 2 0, B: MeCN, 0-70% B 0-60 min., 8 mL/min, tr= 43 min.) to give the title compound as a white amorphous TFA salt (43, 5.1 mg, 0.0491 mmol, 41 %). LRESI-MS 1748 (M-NHNH2+H, for

C

81

H

86 35 C1 3

N

1 3 0 23

S)

+

, 1403 (M-NHNH 2 -N-4-(4-chlorophenyl)benzylvancosamine +H)+. 1143 (M-N-4 (4-chlorophenyl)benzylvancosamine-glucose+H) . 15 20 WO 00/69892 79 PCT/US00/13679 EXAMPLE 65: N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-4-hydroxy-6 methylpyrimidine vancomycin (LXXXV) H H 0 0 H H H

H

0 0H HN CI H H HO H H O OH HO 0N2*NHMe uNH HO OH C 84

H

88

CI

3

N

11 0 24 S Exact Mass: 1771.48 Mol. Wt.: 1774.08 C, 56.87; H, 5.00; CI, 6.00; N, 8.68; O, 21.64; S, 1.81 5 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C 6 -iodo vancomycin TFA salt (Ex. 53 a) (10.0 mg, 0.00534 mmol), 4-hydroxy-2-mercapto-6-methylpyrimidine (16.2 mg 0.114 mmol), and K 2

CO

3 (15.8 mg 0.114 mmol), are dissolved with dry DMF (1 mL). The mixture is stirred at 45oC. After 1 h analytical HPLC indicates the reaction is done. The mixture is filtered then purified by ODS-HPLC 10 (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5pum C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1%

TFA/H

2 0, B: MeCN, 0-70% B 0-60 min., 8 mL/min, tr= 46 min.) to give the title compound as a white amorphous TFA salt (6.0 mg, 0.00318 mmol, 60 %). LRESI-MS 1773 (M+2H, for

C

84 Ho 90 35 Cl 3

N

11 0 24

S)

+

, 1429 (M- N-4-(4-chlorophenyl)benzylvancosamine

+H)

+

. 1143 (M-N-4-(4 chlorophenyl)benzylvancosamine-glucose+H) +. 15 20 WO 00/69892 80 PCT/US00/13679 EXAMPLE 66: N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-6-azathymine vancomycin (LXXXVI) H .C H H 0 HO 0 I O.. N 0 s N cl II IK S H OH CI H N HO H H O O0 o HO NNHMe OH HO OH

C

8 3

H

8 7 CI3N 1 2 0 24 S Exact Mass: 1772.47 Mol. Wt.: 1775.07 C, 56.16; H, 4.94; CI, 5.99; N, 9.47; O, 21.63; S, 1.81 5 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-iodo vancomycin TFA salt (Ex. 53 a) (10.5 mg, 0.00560 mmol), 6-aza-2-thiothymine (18.0 mg 0.126 mmol), and K 2

CO

3 (17.4 mg 0.126 mmol), are dissolved with dry DMF (1 mL). The mixture is stirred at 45oC. After 1 h. analytical HPLC indicates the reaction is done. The mixture is filtered and the residue is purified by ODS-HPLC (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5pm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H20, B: 10 MeCN, 0-70% B 0-60 min., 8 mL/min, tr= 44 min.) to give the title compound as a white amorphous TFA salt (4.4 mg, 0.00233 mmol, 42 %). LRESI-MS 1774 (M+2H, for C 83

H

89 35 Cl 3

N

1 2 0 24

S)

+

, 1432 (M vancosamine+2H)

+

, 1143 (M-vancosamine-glucose+H)

+.

WO 00/69892 81 PCT/US00/13679 EXAMPLE 67: N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-4,5-diphenyloxazole vancomycin (LXXXVII) C1 H NH 0 0 O Cl HO C1 0 OH H H N NH H OH H 0 0 HO NH 2 NHMe OH HO OH

C

94

H

93

CI

3

N

10 0 24 S Exact Mass: 1882.52 Mol. Wt.: 1885.22 C, 59.89; H, 4.97; CI, 5.64; N, 7.43; O, 20.37; S, 1.70 5 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-mesitylenesulfonated vancomycin (Ex. 53 a) (11.6 mg, 0.0060 mmol ), 4, 5-diphenyl-2-oxazole thiol (31.9 mg 0.126 mmol), and K 2

CO

3 (17.5 mg 0.127 mmol), are dissolved with dry DMF (0.5 mL). The mixture is stirred at 75 0 C. After 3 hours, analytical HPLC indicates the reaction is done. The mixture is filtered and purified by ODS-HPLC (LUNA 5ptm C18(2), 21.2 x 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 20-60% B 0-30 10 min., 8 mL/min, tr= 19 min.) to give the white amorphous title compound as the TFA salt (3.5 mg, 0.00175 mmol, 29 %). LRESI-MS 943 (M+4H, for C 94

H

97 35 Cl 3

N

10 0 24

S)

2+. EXAMPLE 68: N,N'-Dialoc, Methyl glycine Vancomycin (LXXXVIII) 15 a) Methyl glycine vancomycin. To a stirred solution of vancomycin-HCI (3.16 g, 2.13 mmol) in 21 mL of DMSO-DMF (16:5) under an argon atmosphere is added glycine methyl ester-HCl (0.53 g, 4.26 mmol) and diisopropylethylamine (1.13 mL, 6.5 mmol). The solution is cooled with an ice bath and 7 mL of a 0.45 M solution of 20 HOBT/HBTU in DMF is added. After 1 h the ice bath is removed and stirring continued for 6h. The reaction mixture is precipitated by addition to 400 mL acetone-ethanol (3:1), stored at 4 oC for 16 h, WO 00/69892 82 PCT/US00/13679 clear supernatant decanted, and the remaining suspension centrifuged. The white solid is suspended in 100 mL ethanol, centrifuged, and supernatant decanted. The ethanol wash is repeated twice, at which time TLC shows complete removal of reagents. The white solid is dried en vacuo affording 3.6 g of crude product. This product is used in the next step without further purification. 5 b) N,N'-Dialoc, Methyl glycine vancomycin (LXXXVIII). To a stirred solution of the crude product (2.1 g, 1.4 mmol) in 18 mL DMSO-DMF (5:4) under an argon atmosphere with ice bath cooling is added allyl 1-benzotriazolyl carbonate (0.76 g, 3.5 mmol) and triethylamine (0.4 mL, 2.83 mmol). After 1.5 h the reaction is warmed to room temperature and stirring 10 continued for an additional 1.5 h. The reaction mixture is precipitated by addition to 200 mL of acetone-diethyl ether (1:1) affording a white precipitate that is centrifuged and the supernatant decanted. The solid is suspended in 200 mL diethyl ether, centrifuged and the supernatant decanted. The solid is dissolved in methanol and evaporated under reduced pressure affording a tan foam. Separation by HPLC (Method A; 25 min. linear gradient of 30% to 44% acetonitrile; flow rate = 7 15 mL/min.) affords (LXXXVIII) (1g, 48%) Ret. Time = 21.5 min. TLC: Rf = 0.7 (chloroform-methanol water, 6:4:1). LRESI-MS calc for C 7 7

H

88

N

10 0 29

CI

2 : 1686.5; [M+Na]

+

= 171. EXAMPLE 69: Glucose-C6-mesitylenesulfonyl-N,N'-Dialoc, Methyl glycine Vancomycin (LXXXIX). 20 A solution of 2-mesitylenesulfonyl chloride (160 mg, 0.73 mmol) in 0.5 mL anhydrous pyridine is stirred at 4 oC for 30 min. This solution is added to compound (LXXXVIII) (310 mg, 0.18 mmol) and stirred in 2.5 mL anhydrous pyridine under an argon atmosphere at 4 'C. The stirred mixture is maintained at 4 oC for 12 hours, precipitated by addition to 30 mL diethyl ether-acetone (3:2), 25 centrifuged and the supernatant decanted. The white solid is taken up in methanol and evaporated under reduced pressure. Separation by HPLC (Method A; 40 min. linear gradient of 30% to 75% acetonitrile; flow rate = 7.5 mL/min.) affords starting material (60 mg) and the title compound (225 mg, 65%, 81% based on recovered starting material) Ret. Time = 30.7 min. TLC: Rf= 0.7 (chloroform methanol-water, 50:21:4). LRESI-MS calc for C 86

H

98

N

1 0 0 3 1 SIC1 2 : 1868.6; [M+H] += 1870; [M 30 vancosamine+H] + = 1645; [M-disaccharide+H] + = 1299. EXAMPLE 70: Glucose-C6-Azide-N,N'-Dialoc, Methyl glycine Vancomycin (XC) 35 To a stirred solution of mesitylenesulfonyl derivative (LXXXIX) (54 mg, 0.03 mmol) in 2 mL anhydrous DMF under an argon atmosphere is added sodium azide (50 mg, 0.8 mmol). The suspension is stirred at 85 oC for 6 h then cooled to room temperature. The mixture is diluted with a minimum of WO 00/69892 83 PCT/US00/13679 methanol (ca 0.5 mL) to dissolve the sodium azide then diluted with chloroform until precipitate formed. Methanol is then added dropwise to dissolve the precipitate. The mixture is subjected to a short Silica gel column (3 X 15 cm) eluting with chloroform-methanol-water (50:21:4). Fractions containing product are combined and evaporated under reduced pressure. Separation by HPLC 5 (Method A; 40 min. linear gradient of 25% to 50% acetonitrile; flow rate = 7 mL/min.) affords (XC) (35 mg, 70%); Ret. Time = 29.1 min. TLC: Rf= 0.5 (chloroform-methanol-water, 50:21:4). LRESI MS calc for C 7 7

H

8 7 N130 2 8

CI

2 : 1711.5; [M+Na] += 1735; [M-vancosamine+H] + = 1486; [M disaccharide+H] = 1299. 10 EXAMPLE 71: Glucose-C6-Amine-N,N'-Dialoc, Methyl glycine Vancomycin (XCI) To a stirred solution of azide (XC) (59 mg, 0.035 mmol) in 1 mL DMF under an argon atmosphere is added trimethylphosphine (100 jtL of 1 M THF solution). After 1.5 h, 0.2 mL water is added and the 15 mixture stirred at room temperature for 17 h and then at 45 oC for an additional 6 h. The mixture is cooled to room temperature, evaporated to 0.5 mL and precipitated by addition to 16 mL diethyl ether. The resulting suspension is centrifuged, the supernatant is decanted, and the solid is dried under reduced pressure. Separation by HPLC (Method A; 40 min. linear gradient of 15% to 50% acetonitrile; flow rate = 7 mL/min.) affords (XCI) (28 mg, 64%); Ret. Time = 18.4 min. TLC: Rf= 0.2 20 (chloroform-methanol-water, 6:4:1). LRESI-MS calc for C 77

H

8 9 NI 1 0O 2 8 C1 2 : 1685.5; [M+H] += 1687; [M-vancosamine+H] = 1460; [M-disaccharide+H] = 1298. EXAMPLE 72: Glucose-C6-N,N-bis-Cbz-guanidinyl, N,N'-Dialoc, Methyl glycine Vancomycin 25 (XCII) and Glucose-C6-N,N-bis-Cbz-guanidinyl, Methyl glycine Vancomycin (XCIII) To a stirred solution of amine (XCI) (12 mg, 0.007 mmol) in 0.3 mL anhydrous DMF is added N,N' bis-Cbz-methylpsuedothiourea (25 mg, 0.07 mmol) and stirring continued for 12 h. The reaction mixture is precipitated by addition to 10 mL diethyl ether, centrifuge and decanted. The white solid is 30 suspended in 20 mL diethyl ether, suspension centrifuged, supernatant decanted and solid dried under reduced pressure affording (XCII) (13 mg, 93%). TLC: Rf = 0.8 (chloroform-methanol-water, 6:4:1). This product is used in the next step without further purification. An analytical sample of (XCII) is similarly prepared followed by separation using HPLC (Method A; 40 min. linear gradient of 40% to 75% acetonitrile; flow rate = 7 mL/min.) 7 Ret. Time = 23.3 min. LRESI-MS calc for 35 C 94 HIo 03

N

13 0 32 C1 2 : 1995.6; [M+H]

+=

1997; [M-disaccharide+Na]

+

= 1321.

WO 00/69892 84 PCT/US00/13679 To a stirred solution of guanidine derivative (XCII) in 0.5 mL anhydrous DMF is added 0.15 mL acetic acid and a catalytic amount of (Ph 3

P)

2 PdCl 2 . The mixture is treated with tributyltin hydride (5 ptL every 10 min. for 2 h) until TLC shows all glycopeptide is baseline (chloroform-methanol-water, 6:4:1). The reaction mixture is precipitated by addition to 10 mL diethyl ether, suspension centrifuged, supernatant 5 decanted and the solid dried under reduced pressure. Separation by HPLC (Method A; 40 min. linear gradient of 10% to 60% acetonitrile; flow rate = 7 mL/min.) affords (XCIII) (11 mg, 86% from XCI) Ret. Time = 23.5 min. LRESI-MS calc for C 86

H

95

NI

3 0 28

CI

2 : 1827.6; [M+H]

+

= 1830; [M disaccharide+H] + = 1216. 10 The preparation of N,N'-bis-Cbz-methylpsuedothiourea is given in Int. J. Pep. Prot. Res. Vol. 40, 1992, pp. 119-126. EXAMPLE 73: Glucose-C6-mesitylenesulfonyl, Methyl glycine Vancomycin (XCIV) 15 To a stirred solution of mesitylenesulfonyl derivative (LXXXIX) (10 mg, 0.005 mmol) in 0.1 mL anhydrous DMF containing 2 1 tL formic acid is added triphenylphosphine (0.5 mg, 0.002 mmol) and a catalytic amount of tetrakis(triphenylphosphine)Pd(0). After 72 h the reaction mixture is precipitated by addition to 6 mL diethyl ether, suspension centrifuged, supernatant decanted, and the solid dried 20 under reduced pressure. Separation by HPLC (Method C; semi-prep column; 40 min. linear gradient of 5% to 75% acetonitrile; flow rate = 4 mL/min.) affords (XCIV) (4 mg, 40%) Ret. Time = 17.4 min. LRESI-MS calc for C 78

H

90

NI

0 0 27

S

1 C1 2 : 1700.5; [M+H] + = 1703; [M-vancosamine+H] + = 1561; [M disaccharide+H] + = 1215. 25 EXAMPLE 74: Glucose-C6-amine, Methyl glycine Vancomycin (XCV) To a stirred solution of amine (XCI) (6 mg, 0.004 mmol) in 0.5 mL anhydrous DMF containing 0.35 mL acetic acid is added a catalytic amount of (Ph 3

P)

2 PdCl 2 . This mixture is treated with tributyltin 30 hydride (10 ptL every 10 min. for 1 h) until TLC shows all glycopeptide is baseline (chloroform methanol-water, 6:4:1). The reaction mixture is precipitated by addition to 20 mL diethyl ether, the suspension centrifuged, the supernatant decanted and the remaining diethyl ether removed under reduced pressure. The solid is separated by HPLC (Method B; 40 min. linear gradient of 0% to 40% acetonitrile; flow rate = 7.5 mL/min.) affording (XCV) (5 mg, 92%) Ret. Time = 19.7 min. LRESI-MS 35 calc for C 69

H

81 Nl]O 24

CI

2 : 1517.5; [M+H] += 1519; [M-vancosamine+H] + = 1378; [M-disaccharide+H] = 1216.

WO 00/69892 PCT/US00/13679 85 EXAMPLE 75: Glucose-C6-guanidine, Methyl glycine Vancomycin (XCVI) 5 Guanidine derivative (XCIII) (6 mg, 0.003 mmol) is dissolved in 0.4 mL water-methanol (1:1) and hydrogenated under balloon pressure with catalytic 10% Pd/C for 3.5 h. The reaction mixture is filtered and the methanol removed under reduced pressure. Separation by HPLC (Method B, semi-prep column; 40 min. linear gradient of 5% to 25% acetonitrile; flow rate = 4 mL/min.) affords (XCVI) (1 mg, 15%) Ret. Time = 19.3 min. LRESI-MS calc for C 70

H

83

N

13 0 24

CI

2 : 1559.5; [M+H] += 1561; [M 10 vancosamine+H] + = 1418; [M-disaccharide+Na] + = 1239. EXAMPLE 76: Glucose-C6-Iodo-N,N'-Dialoc, Methyl glycine Vancomycin (XCVII) 15 To a stirred solution of mesitylenesulfonyl derivative (LXXXIX) (26 mg, 0.014 mmol) in 0.7 mL anhydrous dimethylacetamide (DMA) is added potassium iodide (50 mg, 0.3 mmol). The mixture is stirred at 85 oC for 16 hours then cooled to room temperature. The solution is diluted with water and separation by HPLC (Method A; 40 min. linear gradient of 30% to 60% acetonitrile; flow rate = 7 mL/min.) affords (XCVII) (19 mg, 75%) Ret. Time = 23.9 min. TLC: Rf = 0.55 (chloroform-methanol 20 water; 50:21:4). LRESI-MS calc for C 77

H

87

N

1 0 0 2 8 1IC1 2 : 1796.4; [M+H]+ = 1798; [M-vancosamine+H] + = 1571; [M-disaccharide+Na] + = 1323. EXAMPLE 77: Glucose-C6-Iodo, Methyl glycine Vancomycin (XCVIII) and Glucose-C6-deoxy, 25 Methyl glycine Vancomycin (XCIX) a) Preparation of (XCVIII) only. A catalytic amount of (Ph 3

P)

2 PdCl 2 is added to a stirred solution of (XCVII) (4 mg, 0.002 mmol) dissolved in 0.2 mL anhydrous DMF containing 0.1 mL acetic acid. This mixture is treated with 30 tributyltin hydride (5 pL every 10 min. for 50 min.) until TLC shows all glycopeptide is baseline (chloroform-methanol-water; 6,4,1). The reaction mixture is precipitated by addition to 6 mL diethyl ether, the suspension centrifuged, the supernatant decanted and the remaining diethyl ether removed under reduced pressure. Separation by HPLC (Method C; 2% acetonitrile for 5 min. then 30 min. linear gradient of 2% to 30% acetonitrile; flow rate = 4 mL/min.) affords (XCVIII) (3 mg, 75%) Ret. Time = 35 23.1 min. LRESI-MS calc for C 6 9

H

7 9

NI

0 0 24 1 1 C1 2 : 1628.4; [M+H]1 = 1630; [M-vancosamine+H] + = 1487; [M-disaccharide+H]

+

= 1215.

WO 00/69892 86 PCT/US00/13679 b) Preparation of (XCVIII) and (XCIX). A catalytic amount of (Ph 3

P)

2 PdCl 2 is added to a stirred solution of (XCVII) (12 mg, 0.007 mmol) dissolved in 0.5 mL anhydrous DMF containing 0.35 mL acetic acid. This mixture is treated with tributyltin hydride (10 jtL every 5 min. for 30 min.) at which time TLC shows all product is baseline 5 (chloroform-methanol-water; 6:4:1). The reaction mixture is precipitated by addition to 25 mL diethyl ether, the suspension centrifuged, the supernatant decanted and the remaining diethyl ether removed under reduced pressure. Separation by HPLC (Method A; linear gradient of 0% to 40% acetonitrile at a flow rate of 7.5 mL/min.) affords (XCVIII) (4 mg, 33%) Ret. Time = 26.7 min. and (XCIX) (2.5 mg, 21%) Ret. Time = 22.3 min. 10 EXAMPLE 78: Glucose-C6-deoxy, Methyl glycine Vancomycin (XCIX) A stirred solution of (XCVIII) (1 mg) and 10% Pd/C (catalytic) in 0.4 mL 50% aq. methanol is 15 hydrogenated under balloon pressure for 3 h. The reaction mixture is filtered through a 0.2 pm syringe filter and separated by HPLC (Method B, semi-prep column; linear gradient of 2% to 30% acetonitrile; flow rate = 4 mL/min.) affording (XCIX) (1 mg) Ret. Time = 23.4 min. LRESI-MS calc for

C

6 9

H

80 soN 10 0 2 4 C1 2 : 1502.5; [M+H] += 1504; [M-vancosamine+H] + = 1360; [M-disaccharide+H] + = 1215. 20 EXAMPLE 79: N,N'-bis-Cbz, Vancomycin (VII) To a solution of vancomycin-HCI (1.76 g, 1.19 mmol) dissolved in 8.5 mL water and diluted with 10 mL acetone is added 3 mL water containing NaHCO 3 (210 mg, 2.5 mmol). To the stirred suspension is 25 added 20 mL acetone, 15 mL water and N-(benzyloxycarbonyloxy)succinimide (1.2 g, 4.8 mmol) as a solution in 3 mL acetone. After 15 h the clear solution is evaporated to dryness under reduced pressure with toluene azeotrope. The solid is dissolved in 15 mL DMF and precipitated by addition to 120 mL tetrahydrofuran. The suspension is centrifuged and the supernatant containing reagents decanted. The solid is then suspended in 120 mL acetone, mixed vigorously, centrifuged, and the supernatant 30 decanted. This acetone wash of the solid is performed 3 times to remove all reagents. The white solid is dried under reduced pressure affording the title compound (1.9 g, 95%) that is used without further manipulation. TLC: Rf = 0.33 (chloroform-methanol-water; 6:4:1). 35 WO 00/69892 87 PCT/US00/13679 EXAMPLE 80: N,N'-bis-Cbz Benzyl Vancomycin (XII) To a solution of(C) (1.49 g, 0.87 mmol) in 15 mL DMSO under an argon atmosphere is added NaHCO 3 (35 mg, 0.4 mmol), then benzyl bromide (0.3 mL, 2.5 mmol) and the mixture stirred for 3 h at 5 room temperature. The reaction is precipitated by addition to 400 mL 10% acetone in diethyl ether. The suspension is centrifuged, affording a thick sticky solid upon sitting, and the supernatant decanted. Combined supernatants are evaporated under reduced pressure to 10 mL volume and precipitated by addition to 200 mL diethyl ether. The suspension is centrifuged and the supernatant decanted. Solids are dissolved in methanol, combined, and evaporated under reduced pressure. Purification by HPLC 10 (Method A; 3 min. at 38% acetonitrile followed by a 40 min. linear gradient of 38% to 75% acetonitrile; flow rate = 8 mL/min.) affords (XII) (0.97 g, 61% from 1). Ret. Time = 26 min.; TLC: Rf = 0.5 (chloroform-methanol-water, 50:21:4). 15 EXAMPLE 81: Glucose-C6-mesitylenesulfonyl-N,N'-bis-Cbz Benzyl Vancomycin (CII) To a stirred solution of compound (CI) (250 mg, 0.138 mmol) in 1.8 mL anhydrous pyridine under an argon atmosphere at 4 'C is added 0.25 mL of a 1.12 M solution of mesitylenesulfonyl chloride in pyridine. The temperature is maintained at 4 oC for 18 h at which time 0.1 mL of 1.12 M 20 mesitylenesulfonyl chloride in pyridine is added. After an additional 8 h the mixture is precipitated by addition to 50 mL diethyl ether, centrifuged, supernatant decanted and the white solid dried en vacuo. Separation by HPLC (Method A; 40 min. linear gradient of 35% to 95% acetonitrile; flow rate = 7.8 mL/min.) affords starting material (33 mg) and (CII) (154 mg, 56%, 64% iased on recovered 3). Ret. Time = 27 min.; TLC: Rf= 0.53 (chloroform-methanol-water, 45:10:1). LRESI-MS calc for 25 C 98 HIo 03

N

9 0 30 SIC1 2 : 1987.6; [M+H]

+

= 1989; [M-vancosamine+H] = 1711; [M-disaccharide+Na] = 1390. EXAMPLE 82: Glucose-C6-Azide-N,N'-bis-Cbz Benzyl Vancomycin (CIII) 30 To a stirred solution of mesitylenesulfonyl derivative (CII) (80 mg, 0.04 mmol) in 1 mL anhydrous DMF under an argon atmosphere is added sodium azide (26 mg, 0.4 mmol). The suspension is heated at 85 'C for 7.5 h then cooled to room temperature. The mixture is precipitated by addition to 20 mL diethyl ether, centrifuged, and the supernatant decanted. The tan solid is dissolve in methanol (ca 1 35 mL) and precipitated by addition to 20 mL water. The suspension is centrifuged and the supernatant decanted. Separation by HPLC (Method A; 40 min. linear gradient of 35% to 80% acetonitrile; flow rate = 7.8 mL/min.) affords (CIII) (38 mg, 52%). Ret. Time = 24 min.; TLC: Rf= 0.45 (chloroform- WO 00/69892 88 PCT/US00/13679 methanol-water, 45:10:1). LRESI-MS calc for C 89

H

92

N

12 0 2 7 C1 2 : 1830.6; [M+Na]

+

= 1854; [M vancosamine+H] = 1556; [M-disaccharide+Na] + = 1389. 5 EXAMPLE 83: Glucose-C6-Amine-N,N'-bis-Cbz, Benzyl Vancomycin (CIV) A solution of triphenylphosphine (32 mg, 0.12 mmol) and azide (CIII) (25 mg, 0.014 mmol) in 3 mL THF containing 1 mL water is heated at 55 oC for 5 h. After cooling to room temperature, the mixture is diluted with 40 mL toluene and evaporated to dryness under reduced pressure. The solid is dissolved 10 in methanol (ca. 1 mL) and precipitated by addition to 25 mL diethyl ether. The resulting suspension is centrifuged, supernatant decanted, and solid dried under reduced pressure. Separation by HPLC (Method A; 40 min. linear gradient of 20% to 75% acetonitrile; flow rate = 7.5 mL/min.) affords (CIV) (18 mg, 73%). Ret. Time = 21 min.; TLC: Rf = 0.15 (chloroform-methanol-water, 50:21:4). LRESI MS calc for C 89

H

94 No 10 0 27 C1 2 : 1804.6; [M+H]

+

= 1806; [M-disaccharide+H] + = 1369. 15 EXAMPLE 84: Glucose-C6-N-Acetyl-N,N'-bis-Cbz Benzyl Vancomycin (CV) and Glucose-C6-N Acetyl Vancomycin (CVI) 20 To a solution of amine (CIV) (15 mg, 0.008 mmol) in 0.3 mL anhydrous DMF under an argon atmosphere at 4 oC is added acetic anhydride (0.1 mL, 0.01 mmol). After 30 min. 8 mL toluene is added and the mixture evaporated to dryness under reduced pressure affording 15 mg (CV) (one spot by TLC: Rf= 0.7 (chloroform-methanol-water, 50:21:4)). This product is subjected to deprotection without further purification. 25 N-acetyl derivative (CV) (11 mg) is dissolved in 0.8 mL DMF-methanol-water (1:2:1) and hydrogenated under balloon pressure with a catalytic 10 % Pd/C. After 70 min. the reaction mixture is filtered to remove catalyst and diluted with 0.6 mL water. Separation by HPLC (Method B; 50 min. linear gradient of 0% to 30% acetonitrile; flow rate = 7.5 mL/min.) affords (CVI) (6 mg, 67% from 30 CIV). Ret. Time = 24 min. LRESI-MS calc for C 6 8

H

7 8

N

10 0 24

CI

2 : 1488.5; [M+H] += 1490; [M vancosamine+H] + = 1346; [M-disaccharide+H] + = 1143. EXAMPLE 85: N,N'-Di-Fmoc Vancomycin (CVII) 35 To a solution of vancomycin-HCl (178 mg, 0.012 mmol) in 2 mL water is added 21 mg NaHCO 3 . The resulting suspension is diluted with 3 mL acetone and stirred for 10 min. The clear solution is then WO 00/69892 89 PCT/US00/13679 treated with N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-succinimide ) (90 mg, 0.26 mmol) and 1 mL DMSO and stirred for 24 h. An additional 80 mg Fmoc-succinimide is then added and the mixture stirred for an additional 16 h. The mixture is precipitated by addition to 6 mL diethyl ether acetone (5:2), the suspension centrifuged and the supernatant decanted. The white solid is suspended in 5 diethyl ether, the suspension centrifuged, the supernatant decanted and the solid dried under reduced pressure. Purification by HPLC (Method A; 75 min. linear gradient of 20% to 100% acetonitrile; flow rate = 7 mL/minute) affords (CVII) (167 mg) Ret. Time = 73 min. TLC: Rf= 0.6 (chloroform methanol-water; 6:4:1). LRESI-MS calc for C 9 6

H

9 5

N

9 0 28 C1 2 : 1891.6; [M+H]

+

= 1893. 10 EXAMPLE 86: N,N'-Di-Fmoc Allyl Vancomycin (CVIII) To a solution of Fmoc protected derivative (CVII) (35 mg, 0.018 mmol) in 0.6 mL DMSO is added NaHCO 3 (13 mg, 0.15 mmol) and the mixture is stirred 10 min. Allyl bromide (10 pL, 0.12 mmol) is 15 then added and stirring continued for 24 h. The reaction mixture is precipitated by addition to 10 mL THF-ethyl acetate (9:1), the suspension centrifuged, the supernatant decanted and the solid dried under reduced pressure. Separation by HPLC (Method A; 45 min. linear gradient of 30% to 80% acetonitrile; flow rate = 7 mL/min.) affords (CVIII) (24 mg, 68%) Ret. Time = 37 min. TLC: Rf= 0.8 (chloroform methanol-water; 6:4:1). LRESI-MS calc for C 9 9

H

9 9

N

9 0 2 8 C1 2 : 1931.6; [M+Na]*= 1955. 20 EXAMPLE 87: N,N'-dialoc-glucose-C6-Bromo-Vancomycin Allyl Ester (CIX) To a stirred solution of mesitylenesulfonyl derivative (XVI) (10 mg, 0.005 mmol) in 0.3 mL anhydrous 25 DMF under an argon atmosphere is added lithium bromide (10 mg, 0.11 mmol) and the mixture stirred at 80'C for 7.5 h. The reaction is cooled to room temperature and evaporated to dryness under reduced pressure. Separation by HPLC (Method A; 40 min. linear gradient of 30% to 55% acetonitrile; flow rate = 7.5 mL/min.) affords (CIX) (10 mg, containing a small amount of apparent mesitylenesulfonate salt) Ret. Time = 25 min.; TLC: Rf= 0.6 (chloroform-methanol-water; 50:21:4). LRESI-MS calc for 30 C 77

H

86

N

9 0 2 7 7 9 BrIC1 2 : 1717.4; [M+Na]

+

= 1741; [M-vancosamine+H] = 1493; [M-disaccharide+Na] = 1290. This intermediate is subjected to deprotection without further purification. 35 WO 00/69892 90 PCT/US00/13679 EXAMPLE 88: Glucose-C6-bromo Vancomycin (CX) To a stirred solution of bromide (CIX) (10 mg, 0.005 mmol, containing impurity as described) in 0.4 mL anhydrous DMF containing 0.3 mL acetic acid is added (Ph 3 P)2Pd(II)C12 (catalytic). With 5 vigorous stirring, Bu3SnH is added in 10 pL aliquots every 10 to 20 min. for 2 h (110 j1L total added), at which time TLC (chloroform-methanol-water; 6:4:1) showed all glycopeptide baseline. The biphasic mixture is diluted with 150 gL methanol and precipitated by addition to 15 mL diethyl ether. The suspension is centrifuged and the supernatant decanted. The white solid is dried under a stream of argon to remove residual diethyl ether, dissolved in DMF-water (1:2, ca. 2 mL) and filtered to remove any 10 remaining catalyst or hydrophobic salts. Separation by HPLC (Method B; 40 min. linear gradient of 0% to 45% acetonitrile; flow rate = 7.5 mL/min.) affords (CX) (7 mg, 85% from 4). Retention time = 24 min. LRESI-MS calc for C 66

H

74 N90237 9 BrIC1 2 : 1509.3; [M+H]

+

= 1511; [M-vancosamine+H] = 1369; [M-disaccharide+H] = 1143. 15 EXAMPLE 89: 2-(2,2-dimethylacetoacetyl)-3,4,6-tri-O-benzyl- -D-glucose phenyl sulfoxide (CXVI) Compound (CXI) (3,4,6-tri-O-benzyl-D-glucose) is prepared on a multi-gram scale from commercially available p-D-glucose pentaacetate in 5 steps with an overall yield of 50%. 20 [V. Betaneli et al., Carbohydrate Research, 1982, Vol. 107, page 285] a) 2-acetyl-3,4,6-tri-O-benzyl-D-glucose phenyl sulfide (CXII). To a solution of (CXI) (5.1 g, 11.3 mmol) in 200 mL of dry CH2Cl2 is added pyridine (9.2 mL, 113 mmol), acetic anhydride (Ac 2 0) (5.3mL, 56.7 mmol), and 4-dimethylaminopyridine (DMAP) (100 mg, 25 0.82 mmol). The reaction is stirred for 1.5 hours and then concentrated in vacuo. The residue is dissolved in 500 mL of EtOAc and washed with 1 N HCI (2 X 100 mL), saturated NaHCO3 (2 X 100 mL), H20 (100 mL) and saturated NaCl (100 mL). The organic layer is dried over Na2SO4 and concentrated in vacuo to give 6.1 grams of crude diacetate. This material is dissolved in 200 mL of dry CH2Cl2 and the solution is cooled to -40 0 C. Thiophenol (1.2 mL, 11.7 mmol) is added followed by 30 BF3'Et20 (2.9 mL, 22.6 mmol). The reaction is allowed to warm slowly to room temperature and then stirred at room temperature for 1.5 hours. The reaction is then poured into 200 mL of saturated NaHCO3 and stirred for 30 minutes. The product is extracted with CH2Cl2 (3 X 200 mL). The organic layers are combined, dried over Na2SO4 and concentrated. The residue is purified by flash chromatography (10-15% EtOAc/petroleum ether) to give 5.6 g (85%) of (CXII) as a white solid (14:1 35 ratio of 3:a sulfides). Rf=.41 (15% EtOAc/petroleum ether); 1 H NMR (CDCl3, 500 MHz) 8 7.22-7.54 WO 00/69892 91 PCT/US00/13679 (min, 20H), 5.05 (appt, J = 9.0 Hz, 1H, H2), 4.80-4.83 (min, 2H), 4.55-4.70 (min, 5H), 3.68-3.82 (min, 4H), 3.55-3.58 (min, 1H), 2.02 (s, 3H, CH3 on acetate); 13C NMR (CDCl3, 500 MHz) 8 170.2, 138.9, 138.8, 138.6, 133.7, 133.0, 129.6, 129.2, 129.1, 128.7, 128.6, 128.5, 128.4, 128.3, 86.7, 85.2, 80.1, 78.5, 76.0, 75.8, 74.2, 72.6, 69.7, 21.8. 5 b) 3,4,6-tri-O-benzyl-D-glucose phenyl sulfide (CXIII). To a solution of (CXII) (802 mg, 1.37 mmol) in 10 mL of THF is added MeOH (20 mL) and 12 drops of a saturated methanolic solution of NaOH. The reaction is stirred overnight then diluted with 150 mL of MeOH. Amberlite acidic resin is added and the reaction is stirred for 10 minutes. Litmus paper 10 indicates that the pH is neutral and the resin is filtered off. The filtrate is concentrated in vacuo and the residue is purified by flash chromatography (20% EtOAc/petroleum ether to give 654 mg (93%) of the P3 sulfide (CXIII), and 49 mg (7%) of the a sulfide as white solids. Rf(3)= .20 (15% EtOAc/petroleum ether) Rf(a) =.13 (15% EtOAc/petroleum ether); 1 H NMR (P3) (CDCl3, 500 MHz) 8 7.25-7.63 (inm, 20H), 4.87-4.98 (min, 3H), 4.55-4.68 (min, 4H), 3.85 (dd, J = 11.0, 1.5 Hz, 1H), 3.79 (dd, J = 10.5, 4.5 Hz, 15 1H), 3.63-3.68 (min, 2H), 3.54-3.60 (min, 2H), 2.50 (s, 1H, free OH); 13C NMR (P) (CDCI3, 500 MHz) 8 139.2, 139.0, 138.8, 133.6, 132.6, 129.7, 129.3, 129.2, 129.1, 128.8, 128.7, 128.5, 128.4, 128.3. c) 2-(2-methyl)-acetoacetyl-3,4,6-tri-O-benzyl- -D-glucose phenyl sulfide (CXIV). To a 2-neck 100 mL round bottom flask outfitted with a condenser is added (CXIII) (1.06 g, 1.96 20 mmol), dry toluene (35 mL), DMAP (240 mg, 1.96 mmol), and ethyl-2-methyl acetoacetate (1.5 mL, 9.8 mmol). The reaction is heated at reflux for 48 hours then cooled and concentrated in vacuo. Purification of the residue by flash chromatography (15-20% EtOAc/petroleum ether) gives 1.07 g (86%) of (CXIV) as a white solid along with 95 mg (9%) of recovered (CXII). Rf=.20 (15% EtOAc/petroleum ether) (mixture of isomers). 25 d) 2-(2,2-dimethylacetoacetyl)-3,4,6-tri-O-benzyl- 3 -D-glucose phenyl sulfide (CXV). A solution of (CXIV) (189.5 mg, 0.296 mmol) in 12 mL of THIF is cooled to 0OC and potassium-t butoxide (66.5 mg, .592 mmol) is added. The solution is stirred at 0OC for 10 minutes and then methyl iodide (37 RL, .592 mmol) is added. The reaction is stirred at 0OC for 45 minutes and then poured into 30 20 mL of saturated NH4Cl and extracted with CH2Cl2 (3 X 20 mL). The organic layers are combined, dried over Na2SO4 and concentrated in vacuo. Purification by flash chromatography (20% EtOAc/petroleum ether) gives 181 mg (94%) of (CXV) as an oil. Rf.25 (15% EtOAc/petroleum ether); 'HNMR (CDCl3, 500 MHz) 8 7.17-7.54 (min, 20H), 5.15 (appt, J 10Hz, 1H, H2), 4.86 (d, J = 11 Hz, 1H), 4.74 (d, J = 10.5 Hz), 4.56-4.69 (min, 5H), 3.70-3.82 (min, 1H), 3.55-3.58 (min, 1H), 2.22 (s, 3H), 1.44 35 (s, 3H), 1.37 (s, 3H); 13 C NMR (CDCl3, 500 MHz) 8 206.2, 172.6, 138.8, 138.6, 138.4, 133.6, 132.9, WO 00/69892 92 PCT/US00/13679 129.7, 129.1, 128.7, 128.6, 128.4, 128.3, 127.6, 86.7, 84.8, 80.0, 78.6, 75.7, 75.5, 74.2, 73.0, 69.5, 56.5, 27.1, 22.8, 22.6. e) 2-(2,2-dimethylacetoacetyl)-3,4,6-tri-O-benzyl- -D-glucose phenyl sulfoxide (CXVI). 5 A solution of (CXV) (189.5 mg, .290 mmol) in 15 mL of CH2C12 is cooled to -60 0 C and m chloroperoxybenzoic acid (mCPBA) (64% purity, 85 mg, .315 mmol) is added. The reaction is allowed to warm slowly to -5oC and quenched with 50 gL of Me2S. The reaction is poured into 20 mL of saturated NaHCO3 and extracted with CH2Cl2 (3 X 20 mL). The organic layers are combined, dried over Na2SO4, and concentrated. The residue is purified by flash chromatography 10 (40%EtOAc/petroleum ether) to give 186.1 mg (96%) of (CXVI) as a 1:1 mixture of sulfoxide isomers. Rf (less polar isomer)=.29 (40% EtOAc/petroleum ether); Rf (more polar isomer)=.23 (40% EtOAc/petroleum ether); 1 H NMR (less polar isomer) (CDCI3, 500 MHz) 8 7.15-7.61 (m, 20H), 5.51 (appt, J = 9.5 Hz, 1H, H2), 4.19-4.86 (m, 6H, 3 X CH2 on Bns), 4.17 (d, J = 9.5 Hz, 1H, Hi1), 3.78 (appt J = 8.5, 1H), 3.51-3.69 (m, 3H), 3.45-3.48 (m, 1H), 2.23 (s, 3H), 1.47 (s, 3H), 1.40 (s, 3H); H NMR 15 (more polar isomer) (CDCI3, 500 MHz) 8 7.14-7.82 (m, 20H), 5.29 (appt, J = 8.5 Hz, 1H, H2), 4.54 4.85 (m, 4H, 2 X CH2 on Bns), 4.30-4.37 (m, 3H), 3.78 (appt, J = 8.5 Hz, 1H), 3.64-3.74 (m, 3H), 3.50 3.52 (m, 1H), 2.22 (s, 3H), 1.46 (s, 3H), 1.42 (s, 3H); 13 C NMR (less polar isomer) (CDCl3, 500 MHz) 8 206.4, 172.1, 139.7, 138.7, 138.4, 138.2, 132.1, 129.6, 129.2, 129.1, 128.6, 128.4, 127.7, 126.3, 91.3, 84.5, 81.0, 75.6, 75.4, 74.2, 70.0, 69.2, 56.7, 26.9, 22.8, 22.6; 13C NMR (more polar isomer) (CDC13, 20 500 MHz) 8 206.2, 173.1, 139.8, 138.7, 138.3, 129.4, 129.1, 129.0, 128.6, 128.5, 128.4, 128.3, 128.0, 127.1, 93.7, 83.7, 80.2, 75.5, 75.4, 74.2, 71.4, 68.8, 56.6, 26.8, 22.8, 22.7. EXAMPLE 90: Glycosylation of a model phenol. Preparation of 3,4,6-tri-O-benzyl-f3-D 25 glucopyranosyl 2,6-dimethoxy phenol (CXVIII) a) 2-(2,2-dimethylacetoacetyl)-3,4,6-tri-O-benzyl- p-D-glucopyranosyl-2,6-dimethoxy phenol (CXVII). 2,6-dimethoxyphenol (48.6 mg, 0.315 mmol) is dissolved in 5 mL of benzene and bis(tributyltin)oxide (88.3 gL, 0.173 mmol) is added. The solution is refluxed overnight with a Dean Stark trap which 30 contains 4 angstrom molecular sieves in the side arm. The reaction is cooled and concentrated in vacuo to give tributyltin-2,6-dimethoxy phenoxide which is dissolved in 1 ml of dry methylene chloride to give a stock solution. In a separate flask, (CXVI) (62.4 mg, .0931 mmol) and 2,6 di-t-butyl-4-methyl pyridine (40.6 mg, 0.198 mmol) are azeotroped 3 times with toluene. Flame dried 4 angstrom sieves and a stir bar are added to the flask followed by 4 ml of EtOAc. The solution is stirred for 1 hour and 35 cooled to -78'. 157 RL of a stock solution containing 100 gL of Tf 2 0 and 900 gL of CH 2 Cl 2 is added WO 00/69892 PCT/US00/13679 93 (.093 mmol of Tf 2 0). The reaction is allowed to warm to -60 0 C. The temperature is maintained at 60 0 C for 10 minutes and then the reaction is cooled back to -78oC. 170.5 pL (0.0473 mmol) of the stock solution of tributyltin 2,6-dimethoxy phenoxide is added dropwise by syringe. After 5 minutes, 40 pL of pyridine is added and then the reaction is diluted with 50 ml of EtOAc and poured into 25 ml 5 of saturated NaHCO 3 . The EtOAc layer is washed with 25 mL of saturated NaCl, dried over Na 2

SO

4 and concentrated. The residue is purified by flash chromatography (30% EtOAc/petroleum ether) to give 30.8 mg (93%) of (CXVII). Rf=0.30 (25% EtOAc/petroleum ether); 'H NMR (CDCl3, 270 MHz) 8 7.17-7.33 (min, 15H), 7.04 (t, J = 8.6 Hz, 1H, Ha of phenol), 6.565 (d, 2H, J = 8.6 Hz, Hb of phenol), 5.44 (appt, J = 8.4 Hz, 1H, H-2), 5.075 (d, J = 7.9 Hz, 1H, H-1), 4.46-4.88 (min, 6H, 3 X CH2 on Bns), 10 3.69-3.88 (min, 2H), 3.79 (s, 6H, 2 X OMe on phenol), 3.39-3.46 (min, 1H), 2.12 (s, 3H), 1.37 (s, 3H), 1.32 (s, 3H); 13 C NMR (CDCl3, 270 MHz)5 206.1, 172.4, 153.7, 138.6, 138.3, 138.0, 133.7, 128.5, 128.0, 127.6, 127.2, 124.8, 105.4, 100.5, 83.1, 78.1, 76.1, 75.0, 74.7, 74.1, 73.9, 68.7, 56.2, 55.9, 26.0, 22.3, 21.5; MS (ESI) calc 698.8 (C41H46010) found 721.8 M+Na. 15 b) 3,4,6-tri-O-benzyl-f3-D-glucopyranosyl 2,6-dimethoxy phenol (CXVIII). To a solution of (CXVII) (53.2 mg, 0.0761 mmol) in 650 gL of THF is added 1.3 ml of MeOH followed by hydrazine (40gL, 1.3 mmol). The reaction is stirred for 3 hours and then 1 00pil of acetic acid is added. The reaction is poured into 40 mL of saturated aqueous ammonium chloride and extracted with methylene chloride (3 X 25 mL). The organic extracts are combined, dried over Na 2

SO

4 20 and concentrated. Purification by flash chromatography (20%EtOAc/petroleum ether) gives 35.1 mg (79%) of (CXVIII). Rf=0.24 (25% EtOAc/petroleum ether); 'H NMR (CDCl3, 270 MHz) 8 7.17-7.44 (min, 15H), 7.06 (t, J = 8.6 Hz, 1H, Ha of phenol), 6.61 (d, J = 8.6 Hz, 2H, Hb of phenol), 5.07 (d, J = 11.2 Hz, 1H), 4.84 (appt, J = 11.7 Hz, 2H), 4.54-4.60 (min, 4H), 3.88-3.94 (min, 1H), 3.85 (s, 6H, 2 X OCH 3 on phenol), 3.49-3.78 (min, 5H); " 3 C NMR (CDCl3, 270 MHz) 25 8 153.2, 139.0, 138.6, 138.3, 136.0, 127.2-129.0 (multiplet, aromatics), 125.0, 106.5, 105.6, 85.0, 77.4, 76.1, 75.8, 75.2, 75.1, 73.8, 69.6, 56.5. EXAMPLE 91: CBz-tetra-O-benzyl diacetate vancomycin aglycone (CXXIII) 30 a) CBZ-Bn-vancomycin aglycone (CXIX). Trifluoroacetic acid (6.4 mL) is added to bis-CBz-Bn-vancomycin (XII) (250.2 mg; 0.138 mmol; >80% pure by HPLC). The reaction mixture turns black and is stirred at room temperature for 11 hours and then precipitated in 80 mL of H20. The precipitate is collected by centrifugation and subjected to silica 35 gel flash chromatography (15% MeOH/CH2Cl2). Fractions containing the desired product are WO 00/69892 94 PCT/US00/13679 combined and concentrated. This material is purified by reverse phase preparatory HPLC (C 18, 40 80% CH3CN/H20 with 0.1% HOAc over 40 min) to give (CXIX) (60.1 mg; 32%) as a white solid. Rf = 0.17 (15% MeOH/CH2Cl2); MS (ESI) calc 1368.1 (C68H64N8019C12) found 1369.1 M+H. 5 b) CBZ-Bn-O-allyl vancomycin aglycone (CXX). 4A molecular sieves are added to (CXIX) (171.2 mg; 0.125 mmol) and then DMF (7.5 mL) is added. The solution is stirred for 30 minutes and then Cs2CO3 (52.6 mg, 0.162 mmol) is added and the mixture is stirred for 30 minutes. The solution is cooled to 0 oC and allyl bromide (75.6 piL, 0.625 mmol) is added. After 50 minutes the reaction is quenched by the addition of HOAC (100 gtL). The 10 reaction mixture is filtered through a plug of silica gel with 15% MeOH/ CH2Cl2 and the filtrate is concentrated. Purification by reverse phase preparatory HPLC (C 18, 40-80% CH 3 CN/H20 with 0.1% HOAc over 45 min) gives (CXX) (77.4 mg; 44%) as a white solid along with recovered (CXIX) (23.8 mg; 14%). Rf = 0.28 (15% MeOH/CH2CI2); MS (ESI) calc 1408.2 (C7 1

H

6 8 N8019CI2) found 1409.2 M+H. 15 c) CBZ-tetra-O-benzyl-O-allyl vancomycin aglycone (CXXI). 4A molecular sieves are added to (CXX) (26.3 mg; 0.0187 mmol) and then DMF (1.5 mL) is added. The solution is stirred for 30 minutes and then Cs2CO3 (29.0 mg, 0.089 mmol) is added and the mixture is stirred for 30 minutes. The solution is cooled to 0 oC and benzyl bromide (44.4 pL, 0.3736 20 mmol) is added. The reaction is stirred for 2.5 hours at 0 oC and then warmed to room temperature and stirred at room temperature for 5 hours. The reaction is then quenched with HOAc (40 pL) and filtered through a plug of silica gel with 15% MeOH/CH2Cl2. The filtrate is concentrated and the residue is purified by radial chromatography (5% MeOH/CH2Cl2) to give (CXXI) (22.7 mg; 73%) as a white solid. Rf = 0.125 (5%MeOH/CH2Cl2); MS (ESI) calc 1678.5 (C92H86N8019C12) found 1701.5 25 MNa. d) CBZ-tetra-O-benzyl-O-allyl diacetate vancomycin aglycone (CXXII). Compound (CXXI) (47.8 mg; 0.0285 mmol) is dissolved in pyridine (4mL) and Ac20 (1 mL) is added. The reaction is stirred at room temperature for 2.5 hours and then concentrated. The residue is filtered 30 through a plug of silica gel with 10% MeOH/CH2Cl2 and the filtrate is concentrated. The residue is purified by radial chromatography (4% MeOH/CH2Cl2) to give (CXXII) (47.8 mg; 95%) as a white solid. Rf = 0.30 (5% MeOH/CH2Cl2); MS (ESI) calc 1762.6 (C 96

H

9 0 N8021C12) found 1785.6 M+Na.

WO 00/69892 PCT/US00/13679 95 e) CBZ-tetra-O-benzyl diacetate vancomycin aglycone (CXXIII). To (CXXII) (44.8 mg; 0.0254 mmol) is added CHCl3 (4.5 mL), HOAc (0.59 mL), and N-methyl morpholine (0.29 mL). The solution is degassed for 5 minutes and then Pd(PPh3)4 (11.1 mg; 9.6 x 10 3 mmol) is added. The reaction is stirred for 45 minutes and then an additional amount of Pd(PPh3)4 5 (3.5 mg; 3 x 10 - 3 mmol) is added. The reaction is stirred for another 15 minutes and then filtered through a plug of silica gel with 10% MeOH/CH2Cl2. The filtrate is concentrated and the residue is purified by radial chromatography (5% MeOH/CH2Cl2) to give (CXXIII) (41.9 mg; 96%). Rf = 0.25 (5% MeOH/CH2Cl2); MS (ESI) calc 1722.5 (C93H86N8021C12) found 1723.5 M+H. 10 EXAMPLE 92: [2-(2,2-dimethylacetoacetyl)-3,4,6-tri-O-benzyl-3-D-glucopyranoside]-N-CBZ-tetra-O benzyl-diacetato vancomycin aglycone (CXXIV). Sulfoxide (CXVI) (101.3 mg, 0.151 mmol) is combined with 2,6-di-t-butyl-4-methyl pyridine (62.7 mg, 15 0.303 mmol) and 5 mL of dry CH2CI2 is added. The solution is cooled to -70 oC and Tf20 (25.5 pL, 0.151 mmol) is added. The reaction is warmed to -60 oC and maintained at this temperature for 30 minutes. Then (CXXIII) (39.6 mg; 0.023 mmol) is added dropwise in 1 mL of CH2CI2. The reaction is allowed to warm slowly to -50 'C and then the temperature is maintained between -50 oC and -55 oC for 30 minutes. The reaction is quenched by the addition of thiophenol (15 ptL) followed by DIEA 20 (100 pL). The cold reaction mixture is filtered through silica gel with 10% MeOH/CH2Cl2 (100 mL). The filtrate is concentrated and subjected to radial chromatography (4% MI:OH/CH2Cl2). Fractions containing the desired product are combined and repurified by radial chromatography (3.5% MeOH/CH2Cl2) to give (CXXIV) (8.7 mg;17%). Rf = 0.23 (3.5% MeOH/CH2Cl2); MS (FAB) calc 2,267.1 (C 12 6H122N8028Cl2) found 2268.2 M+H. 25 EXAMPLE 93: N, N'-Diallyloxycarbonyl-methoxy-glycine-deleucine aspartatic acid Vancomycin (CXXVIII). 30 a) Deleucine-vancomycin (CXXV). Vancomycin-HCI (497 mg, 0.335 mmol) is dissolved in 4 mL water to which is added 4 mL distilled pyridine with stirring in a 40 0 C oil bath. To this solution is added phenyl isothiocyanate (50 mg, 0.368 mmol). After stirring for 30 minutes the clear solution is evaporated of organic solvent under reduced pressure and then added 100 mL water is added, which is frozen and lyophilized to dryness. To the WO 00/69892 96 PCT/US00/13679 powder is added 4 mL of CH2Cl2 and 4 mL of trifluoroacetic acid. This clear solution is stirred at room temperature for 3 minutes and then evaporated under reduced pressure to dryness. The brown oil is partitioned between 100 mL of ethyl acetate (EtOAc) and 100 mL H20. The aqueous layer is collected and the organic layer is extracted twice with water (40 mL each). The aqueous layers are 5 combined and evaporated under reduced pressure to dryness. The white solid is dissolved in methanol, loaded to a C18 reverse phase column (50mm x 12cm, particle size 40tm, pore size 60 A (J. T. Baker) and eluted with 10% acetonitrile/0.1% acetic acid in water. The fractions containing the pure products are combined and evaporated to give 325 mg of (CXXV) as a white powder, 73.5%. Rf=0.1 (CHCl 3:MeOH:H20 = 3 :5 : 1.5). Mass Spec. [M+H]

+

, 1322; [M-V]

+

, 1178. 10 b) Methoxy-glycine-deleucine vancomycin (CXXVI). Compound (CXXV) (162 mg, 0.117 mmol) and glycine methyl ester hydrochloride (74 mg, 0.585 mmol) are dissolved in 0.8 mL DMSO and 0.8 mL DMF and stirred at 0 0 C. Diisopropylethylamine (204 il, 0.585 mmol) is added to the reaction vessel via syringe followed by HOBt/HBTU (1.17 mL 15 0.45M DMF solution, 0.526 mmol). The ice bath is removed after addition. After 10 minutes, the reaction is completed and the reaction solution is directly loaded to a poly(divinylbenzene) column (30mm x 8cm, 50-100 micron particle size) and eluted with methanol/water (0, 10%, 20%, 30%, 40%, 50% of 100 mL each). The fractions containing the pure products are combined and evaporated to give 160 mg of (CXXVI) as a white powder, 95%. Rf-0.1 (CHCl3:MeOH:H20=3:3:1). Mass Spec. 20 [M+H]

+

, 1393; [M-V]

+

, 1249. c) N-allyloxycarbonyl-methoxy-glycine-deleucine vancomycin (CXXVII). Compound (CXXVI) (647 mg, 0.465 mmol) is dissolved in 10 mL water and 10 mL dioxane mixture. Fmoc-succinimide (172 mg, 0.511 mmol) in 5 mL dioxane is added to the solution over 10 hours via 25 syringe pump. The reaction mixture is stirred for an additional 5 hours after addition. Then the solution is rotary evaporated to dryness under reduced pressure. The crude oil obtained is dissolved in 10 mL DMF. To this clear solution is added diisopropylethylamine (406 gL, 2.32 mmol) followed by Aloc-OBt (102 mg, 0.465 mmol) in 1 mL DMF. The reaction is stirred at room temperature for 30 minutes. Piperidine (2 mL) is added to the reaction flask at this time. After stirring for another 5 30 minutes, the solution is suspended into 160 mL of acetone and stirred, centrifuged, and decanted. The white precipitate obtained is collected, loaded to a C18 reverse phase column (50mm x 12cm, particle size 40im, pore size 60 A (J. T. Baker) and eluted with isopropanol/water (0, 10%, 20%, 30%, 40%, 50%, 60% of 100 mL each). The fractions containing the pure products are combined and evaporated to give 309 mg of (CXXVII) as a white powder, 58% over 3 steps. Rf-0.4 35 (CHCl3:MeOH:H20=3:2:0.5). Mass Spec. [M+2H]+,1478; [M-V+H]

+

, 1250.

WO 00/69892 97 PCT/US00/13679 d) N, N'-diallyloxycarbonyl-methoxy-glycine-deleucine aspartatic acid vancomycin (CXXVIII). Compound (CXXVII) (102 mg, 0.0691 mmol) and Aloc-Asp(OFm)-OH (55 mg, 0.138 mmol) are premixed and azeotroped with toluene three times, dissolved in 1.5 mL DMF and then cooled to 0oC. Diisopropylethylamine (48 [tL, 0.276 mmol) is added to the reaction vessel followed by HOBt (19 mg, 5 0.138 mmol) and PyBOP (72 mg, 0.138 mmol). After stirring for 15 minutes, 200 1 IL piperidine is added to the reaction. The ice bath is removed and the reaction is stirred at room temperature for 5 minutes. The clear solution is suspended in 45 mL acetone and stirred, centrifuged, and decanted. The solid is dried under reduced pressure and purified by reverse-phase HPLC using a PHENOMENEX LUNA C18 column (21.2 x 250mm), 5 micron particle, eluting with a 30 min. linear gradient of 0.1% 10 acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 71 mg of compound (CXXVIII), 62% over 2 steps. Rf = 0.5 (CHC13:MeOH:H20=3:2:0.5). Mass Spec. [M+Na] , 1698; [M-V+Na] , 1472. 15 EXAMPLE 94: N-Allyloxycarbonyl-N'-methoxyglycine [N-acetato-vancosamino] Vancomycin (CXXXI) a) Methoxy-glycine vancomycin (CXXIX). 20 Vancomycin hydrochloride (317 mg, 0.213 mmol) and glycine methyl ester hydrochloride (54 mg, 0.426 mmol) are dissolved in 2 mL DMSO and 2 mL DMF and stirred at 0OC. Diisopropylethylamine (186 pL, 0.3195 mmol) is added to the reaction vessel via syringe followed by HOBt/HBTU (710 pL 0.45M DMF solution, 0.319 mmol). The ice bath is removed after addition. After 10 minutes, the reaction is completed and the reaction solution is directly loaded to a poly(divinylbenzene) column 25 (30mm x 8cm, 50-100 micron particle size) and eluted with methanol/water (0, 10%, 20%, 30%, 40%, 50% of 100 mL each). The fractions containing the pure products are combined and evaporated to give 249 mg of (CXXIX) as a white powder, 77%. Rf = 0.15 (CHCl3:MeOH:H20 = 3:2:0.5). Mass Spec.

[M+H]

+

, 1521; [M-V]

+

, 1377. 30 b) N-allyloxycarbonyl-N'-methoxyglycine vancomycin (CXXX). Compound (CXXIX) (110 mg,0.0723 mmol) is dissolved in 3 mL DMF. Aloc-OBt (17 mg, 0.0795 mmol) in 0.5 mL DMF is added to the solution over 10 hours via syringe pump. The reaction is stirred for additional 5 hours after addition. The solution is then suspended into 160 mL of acetone and stirred, centrifuged, and decanted. The white solid is directly loaded to a poly(divinylbenzene) column (30mm 35 x 8cm, 50-100 micron particle size) and eluted with methanol/water (0, 10%, 20%, 30%, 40%, 50% of 100 mL each). The fractions containing the pure product are combined and evaporated to give 115 mg WO 00/69892 PCT/US00/13679 98 of (CXXX) as a white powder, 62%. Rf = 0.4 (CHCl3:MeOH:H20 = 3:2:0.5). Mass Spec. [M+H] +, 1605; [M-V] 4 ,1461. c) N-allyloxycarbonyl-N'-methoxyglycine [N-acetato-vancosamino] vancomycin (CXXXI). 5 Compound (CXXX) (32 mg, 0.0202 mmol) and glyoxylic acid monohydrate (2 mg, 0.0222 mmol) are dissolved in 400 pL methanol and stirred at 40 0 C for 2 hours. A white precipitate is generated and the suspension is cooled back to room temperature and 100 tL DMF is added followed by 61 PL of NaCNBH3 in THF (1M solution). After 20 minutes, the resulting clear solution is directly purified by reverse-phase HPLC using a PHENOMENEX LUNA C 18 column (21.2 x 250mm), 5 ptm particle, 10 eluting with a 30 min. linear gradient of 20% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 18 mg of product (CXXXI), 54%. Rf= 0.4 (CHCl3:MeOH:H20 = 3:2:0.5). Mass Spec.: [M+H] , 1662; [M-V] , 1460. 15 EXAMPLE 95: 2-(4-Azidobutyryl)-3,4,6-triacetyl glucose sulfoxide (CXXXII) a) 2-(4-azidobutyryl)- 1,3,4,6-tetraacetyl-D-glucose. 1,3,4,6 tetraacetyl D-glucose (W.E. Dick, Carbohyd. Res., 21, 255-268 (1972)) is dissolved in CH 2

C

2 20 to make a 0.1M solution. 6 eqivalents of pyridine and 3 equivalents of 4-azidobutyryl chloride (S. Kusumoto et. al., Bull. Chem. Soc. Jpn., 59, 1289-1298 (1986)) are added. After several hours, the reaction is poured into saturated NaHCO 3 , extracted with CH 2 Cl 2 , dried over Na 2

SO

4 , and concentrated. The residue is purified by flash chromatography to give the title compound. 25 b) 2-(4-azidobutyryl)-3,4,6-triacetyl-D-glucose sulfide. The product of step a) is dissolved in CH 2

C

2 to make a 0.1M solution. 5 equivalents of BF 3 Et 2 0 and 1.25 equivalents of thiophenol are added. After several hours, the reaction is poured into saturated NaHCO 3 , extracted with CH 2 Cl 2 , dried over Na 2

SO

4 , and concentrated. The residue is purified by flash chromatography to give the title compound. 30 c) 2-(4-azidobutyryl)-3,4,6-triacetyl-D-glucose sulfoxide. The product of step b) is dissolved in CH 2 C1 2 to make a 0.1M solution. The solution is cooled to -780 and 1.1 equivalents of mCPBA is added. The reaction is slowly warmed until conversion to sulfoxide is complete. The reaction is quenched with 1 equivalent of Me 2 S, poured into saturated NaHCO 3 , 35 extracted with CH 2 Cl 2 , dried over Na 2

SO

4 , and concentrated. The residue is purified by flash chromatography to give the title compound (CXXXII).

WO 00/69892 99 PCT/US00/13679 EXAMPLE 96: Modified Sulfoxide Glycosylation Procedure. CBZ-Bn-tri-O-Me Vancomycin pseudoaglycone (CXXXIII) a) CBZ-Bn-tri-O-Me-hexaacetyl Vancomycin pseudoaglycone. 5 Peracetylated glucose sulfoxide (47.3 mg, 0.1036 mmol) and 2,6-di-t-butyl-4-methyl pyridine (43.3 mg, 0.2108 mmol) are azeotroped 3 times with toluene. Flame dried 4 angstrom molecular sieves and a stir bar are added followed by 3 ml of CH 2

C

2 . The solution is stirred for 45 minutes and then cooled to 780. 174 pL of a stock solution containing 100 pgL of Tf 2 0 and 900 gL of CH 2

CI

2 is added (0.1036 mmol of Tf 2 0). The reaction is warmed to -600, maintained at that temperature for 20 minutes, and 10 then cooled back to -780. CBZ-Bn-tri-O-Me-diacetyl vancomycin aglycone (XV) (49.0 mg, 0.0328 mmol) is dissolved in 1 ml of CH 2

CI

2 and BF 3 -Et 2 0 (83 pL, 0.656 mmol) is added. This solution is added to the activated sulfoxide and the reaction is warmed to -150 over 1.5 hours. The reaction is then filtered through a plug of silica gel with 7.5% MeOH/CH 2 C1 2 into a flask containing 200 gL of pyridine. The filtrate is concentrated. Purification by radial chromatography gave 28.3 mg (47%) of 15 the title compound. Rf 0.21 (50% EtOAc/petroleum ether then 5% MeOH/CH 2

CI

2 ); MS (ESI) calc 1824.5 (C 89

H

9 2

N

8 0 30 C1 2 ) found 1847.5 MNa. b) CBZ-Bn-tri-O-Me Vancomycin pseudoaglycone. The product of step a) (7.2 mg, 0.0039 mmol) is dissolved in 250 pL of THF and 500 pL of MeOH are 20 added. 20 pgL of H 2

NNH

2 are added and the reaction is allowed to stir for 10 hours. The reaction is then quenched with 60 pL of acetic acid (HOAc) and filtered through a plug of silica gel with 20% MeOH/CH 2 C1 2 . The filtrate is concentrated and purified by reverse-phase HPLC using a PHENOMENEX LUNA C 1 8 column (21.2x250mm), 5 pm particle, elutin, '~ith a 35 minute linear gradient of 35% - 80% acetonitrile/0.1% acetic acid in water, flow rate 7ml/min. 2.0 mg (32%) of the 25 title compound (CXXXIII) is isolated as a white solid. Retention time on HPLC is 24.8 minutes; MS (ESI) calc 1572.3 (C 7 7 Hs 80 NsO 8 24

CI

2 ) found 1595.3 MNa. EXAMPLE 97: Modified Sulfoxide Glycosylation Procedure. Preparation of Aloc-tetra-O-allyl 30 pentaacetyl vancomycin pseudoaglycone (VI). a) Aloc-tetra-O-allyl-pentaacetyl-2(4-azidobutyryl)-glucose vancomycin pseudoaglycone. 2-(4-azidobutyryl)-3,4,6-triacetyl-D-glucose sulfoxide (CXXXII) (3 equivalents) and 2,6-di-t-butyl-4 methyl pyridine (6 equivalents) are azeotroped 3 times with toluene. Flame dried 4 angstrom molecular 35 sieves and a stir bar are added followed by 3 ml of CH 2

C

2 . The solution is stirred for 45 minutes and then cooled to -78o. Tf 2 0 (3 equivalents) is added. The reaction is warmed to -60', maintained at that temperature for 20 minutes, and then cooled back to -780. Aloc-tetra-O-allyl diacetate vancomycin WO 00/69892 100 PCT/US00/13679 aglycone (1 equivalent, prepared analogously to the preparation of XV, using allyl bromide in place of benzyl bromide; and methyl iodide and aloc-succinimide in place of N (benzyloxycarbonyloxy)succinimide) is dissolved in 1 ml of CH 2 C1 2 and BF 3 Et 2 0 (20 equivalents) is added. This solution is added to the activated sulfoxide and the reaction is warmed to -150 over 1.5 5 hours. The reaction is then filtered through a plug of silica gel with 7.5% MeOH/CH 2 Cl 2 into a flask containing 200 IL of pyridine. The filtrate is concentrated. Purification by radial chromatography gives alloc-tetra-O-allyl-pentaacetyl- 2 (4-azidobutyryl)- glucose vancomycin pseudoaglycone. b) Aloc-tetra-O-allyl-pentaacetyl vancomycin pseudoaglycone (VI). 10 Aloc-tetra-O-allyl-pentaacetyl-2-(4-azidobutyryl)-glucose vancomycin pseudoaglycone is dissolved in 5:1 THF/H 2 0 to make a 0.1 M solution. 5 equivalents of Ph 3 P are added and the reaction is heated to 600. The reaction is maintained at this temperature until TLC indicates that the reaction is complete. Then the reaction is cooled to room temperature and filtered through silica gel with 10% MeOH/CH 2

CI

2 . Purification by radial chromatography gives (VI). 15 EXAMPLE 98: Glycosylation of a Model Phenol. Preparation of 2,3,4,6-Tetra-O-benzyl-P3-D glucopyranosyl-2,6-dimethoxy phenol. 20 Peracetylated glucose sulfoxide (50.1 mg, 0.1098 mmol) and 2,6-di-t-butyl-4-methyl pyridine (47.4 mg, 0.231 mmol) were azeotroped 3 times with toluene. Flame dried 4 angstrom sieves and a stir bar were added to the flask, followed by 3 ml of CH 2 Cl 2 . This solution is stirred for 45 minutes and then cooled to -780. 185 gL of a stock solution containing 100 gL of Tf 2 0 and 900 tL of CH 2

CI

2 is added (0.1098 mmol of Tf 2 0). The reaction is warmed to -600, maintained at this temperature for 20 minutes, and 25 then cooled back to -780. 2,6-dimethoxy phenol (8.4 mg, .0545 mmol) is dissolved in Iml of CH 2 Cl 2 and BF3-Et 2 0 (140 RL, 1.098 mmol) is added. This solution is added to the activated sulfoxide by syringe. The reaction is allowed to warm to 00 and then filtered through a plug of silica gel with ethyl acetate into a flask containing 200 gL of pyridine. This filtrate is concentrated and purified by flash chromatography (45% EtOAc/petroleum ether) to give 14.9 mg (56%) of the title compound. Rf 0.27 30 (50%EtOAc/petroleum ether); 1 H NMR (CDCl 3 , 500 MHz) 8 7.05 (t, J = 8.5 Hz, 1H, Ha of phenol), 6.59 (d, J = 8 Hz, 2H, Hb of phenol), 5.25-5.36 (min, 3H, H2, H3, and H4), 5.10 (d, J= 7.5 Hz, 1H, H1), 4.28 (dd, J = 12.3 Hz, J = 5 Hz, 1H, H6), 4.15 (dd, J = 12 Hz, J = 2.5 Hz, H6') 3.86 (s, 6H, 2 X Me on phenol), 3.70-3.73 (min, 1H, H5), 2.05-2.06 (min, 12H, 4 acetates). 35 WO 00/69892 101 PCT/US00/13679 101 EXAMPLE 99: Di- {N,N'-diallyloxycarbonyl-O-allyl-6-glucosamino vancomycin}-C(O)CH 2 - (0) (CXXXV) a) Allyl N,N'-diallyloxycarbonyl [6-N-acetato-glucosamino] vancomycin (CXXXIV). 5 Compound (III) (17 mg, 0.0103 mmol) and glyoxylic acid monohydrate (0.95 mg, 0.103 mmol) are dissolved in 1 mL methanol and stirred at 40 oC for 2 hours to generate a white precipitate. The suspension is cooled back to room temperature and 250 1 tL DMF is added followed by 200 tL of NaCNBH3 in THF (IM solution). After 20 minutes, the resulting clear solution is directly purified by reverse-phase HPLC using a PHENOMENEX LUNA C18 column (21.2 x 250mm), 5 1 tL particle, 10 eluting with a 30 min. linear gradient of 20% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product were combined and evaporated to give 6 mg of product (CXXXIV), 33%. Rf=0.28 (CHCl3:MeOH:H20 = 3:2:0.5). Mass Spec. [M+H]

+

, 1716; [M-V]

+

, 1488. 15 b) Di- {N,N'-diallyloxycarbonyl-O-allyl-6-glucosamino vancomycin}-C(O)CH 2 - (0) (CXXXV). As shown in Fig. 13, compound (CXXXIV) (5 mg, 0.00292 mmol) is dissolved in 1 mL methanol and 300 tL DIEA is added. This solution is stirred for 10 minutes and then loaded to a 5mm x 30mm polystyrene column and eluted with methanol/water/1%DIEA (0%, 10%, 20%, 30%, 40%, 50% of 10 mL each). The fractions containing compound (CXXXIV) are combined and concentrated to give a 20 white solid. This white solid is mixed with C-6 amine (III) (10 mg, 0.00582 mmol, purified from silica gel column as free base), azeotroped with toluene 3 times and dissolved in 100 ptL DMF. The reaction solution is stirred at 0 oC and DIEA (5 ptL, 0.0283 mmol) is added followed by HOBt (2mg, 0.0148mmol) and pyBOP (5 mg, 0.00962 mmol). After 10 minutes, the :.action is directly loaded to a 10mm x 12cm silica gel column and eluted with 30% methanol/CHCl3 to give a crude product. The 25 crude product is purified by reverse-phase HPLC using a PHENOMENEX LUNA C18 column (21.2 x 250mm), 5 micron particle, eluting with a 40 min. linear gradient of 20% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 2 mg of dimer (CXXXV), 20%. Rf=0.7 (30% CHCl3/MeOH). Mass Spec. [M+2Na]

+

, 3396. 30 35 WO 00/69892 102 PCT/US00/13679 EXAMPLE 100: N-4-(4-Chlorophenyl)benzyl Vancosamine-glucose-C6-iminotriphenylphosphorane Vancomycin (CXXXVI). H a PPh3 H HH

H

0 HOP.,,, O Hc 0 HO e O NH2 OH HO OH

C

97

H

98 C1 3

N

10 0 23 P Exact Mass: 1906.56 Mol. Wt.: 1909.20 C, 61.02; H, 5.17; CI, 5.57; N, 7.34; O, 19.27; P, 1.62 5 N-chlorobiphenylvancosamine-glucose-C6-azide vancomycin (LXXIV) (15 mg, 0.00838 mmol) and PPh 3 (44.0 mg, 0.168 mmol) are suspended with THF/ H 2 0 (lmL, 4/1) and the mixture is stirred at 45 oC. After 6 hours, 10 eq. of PPh 3 and 1 mL of THF are added. After 18 hours, the mixture is filtered then purified by ODS-HPLC (COSMOSIL 5C18-AR, 20 x 250 mm, and LUNA 5tm C18(2), 21.2 x 10 250 mm, UV=285 nm, A: 0.1% TFA/H 2 0, B: MeCN, 20-70% B 0-60 min., 8 mL/min, tr= 49 min.) to give the white amorphous solid product (CXXXVI) (5.5 mg, 0.00182 mmol, 42 %) as a TFA salt. LRESI-MS 1908 (M+2H, for C 99

H

98 35 Cl 3 No 10

O

23

P)

+

, 1708 (M- N-4-(4-chlorophenyl)benzyl+2H)

+

, 1564 (M- N-4-(4-chlorophenyl)benzylvancosamine+2H) . 1143 (M- N-4-(4 chlorophenyl)benzylvancosamine-glucose+H) +. 15 EXAMPLE 101: N, N'-diallyloxycarbonyl-methoxy-glycine-deleucine glucosamino-aspartate vancomycin (Fig. 12, I). 20 As shown in Fig. 12, compound (CXXVII) (20 mg, 0.0119 mmol) and glucosamine.HCI (8 mg, 0.0358 mmol) are premixed and azeotroped with toluene 3 times, dissolved in 240 tL DMF and then cooled to WO 00/69892 PCT/US00/13679 103 0 oC. Diisopropylethylamine (21 iL, 0.119 mmol) is added to the reaction vessel followed by HOBt (4.8 mg, 0.0357 mmol) and pyBOP (18 mg, 0.0358 mmol). After stirring 15 minutes, the clear solution is suspended in 45 mL acetone and stirred, centrifuged, and decanted. The solid is dried under reduced pressure and purified by reverse-phase HPLC using a PHENOMENEX LUNA C 18 column (21.2 x 5 250mm), 5 [im particle, eluting with a 40 min. linear gradient of 0.1% acetic acid in water to 40% acetonitrile/0.1% acetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 13 mg of the title compound, 60% over 2 steps. RfO0.15 (CHCl3:MeOH:H20=3:2:0.5). Mass Spec. [M+Na]+,1859; [M-V+Na] +, 1632. 10 EXAMPLE 102: Evaluation of Vancomycin Analogs for Anti-Microbial Activity Evaluation of vancomycin analogs is performed using in vitro susceptibility tests and a time-kill assay. 15 [NCCL Standard, 1993] In susceptibility tests, five strains of bacteria, two of the most important: Staphylococcus aureus and Enterococcusfaecalis (both susceptible and resistant strains), and Bacillus cereus are chosen and bacteria viability remaining in each well is evaluated using a colorimetric assay based on the tetrazolium salt 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) and minimum inhibitory concentration (MIC) values are determined as gg/mL. [Mosmann, T. 20 (1983); Damour, O., et al. (1992); Mikami, Y., et al. (1994)] This assay gives susceptibility information quickly, efficiently, and clearly. Promising analogs that pass this first screening are studied to evaluate their activity against resistant strains in greater detail using a time-kill assay. [Pankuch G., et al., (1994); Zelinitsky, S. et al. (1997); 25 Mercier, R-C., et al. (1997)] This study gives the information regarding their bactericidal ability or mode of action. Bacteria All strains [Bacillus cereus (ATCC® 11778), Staphylococcus aureus (ATCC® 29213), Methicillin 30 resistant Staphylococcus aureus (ATCC® 33591), Enterococcusfaecalis (ATCC® 29212), and gentamicin, streptmycin, vancomycin resistant Enterococcusfaecalis (ATCC® 51299), are purchased from REMEL (Lenexa, KS). Susceptibility tests 35 MICs are determined by the microdilution method using 96-well microplates. Samples are suspended with Cation Adjusted (20 to 25 mg of Ca 2 +/mL and 10 to 12.5 mg/mL of Mg 2+ ) Mueller-Hinton broth WO 00/69892 104 PCT/US00/13679 104 (Difco Laboratories, Detroit, MI) and are two-fold diluted from 5 p.g/mL to 0.0025 mg/mL on microplates (12 step dilution). To each well, which contained 100 pL of cell suspension (106 CFU/mL), 100 p.L of antibiotic solution is added and the plates are incubated at 37 oC for 24 h. 50 pL of MTT solution (1 mg/mL) is added to each well, then the plates are incubated under the same 5 conditions (incubation time; 30 min for Bacillus cereus, Staphylococcus aureus, and Methicillin resistant Staphylococcus aureus ; 2 hours for Enterococcusfaecalis and Gentamicin, Streptmycin, Vancomycin Enterococcusfaecalis ). MTT is a yellow tetrazolium salt that is reduced by mitochondrial enzymes in viable cells to an insoluble blue formazan product. MIC values are evaluated by observing the lowest drug concentration to inhibit bacteria growth. Results for the anti-bacterial 10 activities of the compounds tested are given in the Tables provided hereinbelow. Time-kill Assays For time-kill studies, a 24-well microplate containing 1 mL of Cation-Adjusted Mueller-Hinton broth plus 5% lysed horse blood with doubling antibiotic concentrations are used. Antibiotic concentrations 15 are chosen to be 7 doubling dilutions above the microdilution MIC. A drug-free control well is included with each run. Lysed horse blood is prepared by freezing and thawing horse blood REMEL (Lenexa, KS) six times. Equal volumes of lysed blood and sterile deionized water are then mixed and centrifuged at 12000 x g for 20 min. Appropriate amounts of 50% lysed blood are then added to the Cation Adjusted Mueller-Hinton broth to yield a final concentration of 5% lysed horse blood. To each 20 well, which contained 0.5 mL of cell suspension (106 CFU/mL), 0.5 mL of antibiotic solution is added and the plates are incubated at 37 'C in a shaking incubator. Viability counts of antibiotic containing suspensions are performed at 0, 1, 2, 4, 6, 12, 24, 36, and 48 h by plating of 10-fold dilutions of 0.1 mL aliquots from each well in Cation Adjusted Mueller-Hinton broth onto Trypticase soy agar-5% sheep blood agar plates. Recovery plates are incubated for up to 48 h. The lower limit of sensitivity of 25 colony counts is 300 CFU/mL. [Pankuch G., et al. (1994)] The results are illustrated in Figs. 16-19, where the amount of antibiotic is given in gtg/mL and is indicated by the respective symbol.

WO 00/69892 105 PCT/US00/13679 MIC values of glucose-C6 modified vancomycin derivatives (ptg/mL) X= BC SA MRSA EF VREF CL5137 CL5244 OH (vancomycin) 1 1.0 1.0 1.3 1.3 5.0< 1600 25 I LXX 0.63 0.63 1.3 0.63 1.9 50< 6.3

N

3 XLVI 1.0 0.63 1.3 1.3 2.5 50< 25

NH

2 XLVII 1.0 0.63 0.32 1.9 1 50< 25

NHNH

2 XLIV 1.3 0.63 1.3 1.3 2.5 50< 50 0 XL 1.3 0.48 1.0 1.3 5.0 50< 25 HN CF3 0 XXXIV 5.0 1.9 2.5 2.5 5.0< nd nd NH2 H S XXXII 1.9 1.9 2.5 1.3 5.0 nd nd HNNHMe s- LIII 1.3 2.5 2.5 2.5 5.0< nd nd SLXXI 5.0< 5.0 5.0< 5.0< 5.0< nd nd S OH - XXXI 1.3 1.3 1.3 0.32 2.5 nd nd -- XXXV 3.8 __2.5 1.3 0.015 0.16 nd nd .,N LVII 2.5 1.3 1.9 2.5 5.0 50< 50 e 'N LVIII 1.3 1.9 2.5 2.5 5.0 50< 50 FNN.NH, LVI 1.0 0.48 0.63 0.63 0.63 50< 1.6 S N" 1NK LXXVI 0.63 0.63 0.63 1.3 5.0< 50< 19 S S ._ N-- LXXVII 0.63 1.3 1.3 0.63 1.3 63 3.2 S NH 0 0 XXXIII 1.3 1.3 2.5 1.3 5.0 nd nd LIV 1.3 1.0 0.63 1.3 2.5 50< 13 LIX 1.3 0.63 1.0 1.0 0.32 50< 6.3 Br Ik LV 0.63 0.48 0.32 0.63 0.63 50< 3.2 Sl F LX 5.0 5.0 5.0 2.5 2.5 50< 50< s LXI 0.63 0.63 1.3 0.63 0.63 50< 3.2 I LXII 0.63 1.9 2.5 1.3 1.3 50< 6.3

NHI

WO 00/69892 106 PCT/US00/13679 S -rOH LXIII 5.0 5.0 5.0< 2.5 5.0< 50< 6.3 N" O SOH LXXVIII 0.63 1.0 1.3 0.63 2.5 50< 6.3 &N OH Ny LXIV 0.08 0.24 0.32 0.12 0.16 500< 1.2 NI

S

XXXIX 1.3 1.3 1.3 2.5 5.0< 50< 25 0 HA XXXVI 1.3 1.3 2.5 1.3 2.5 nd nd O XLI 1.3 0.63 1.3 1.3 1.3 50< 6.3 II LXV 0.63 1.9 2.5 0.63 2.5 50< 13 LXVI 0.16 0.63 1.3 0.24 0.32 50 0.4 XXXVII 0.32 0.32 0.63 0.32 0.32 50< 1.6 N XXIX 0.63 0.63 0.63 0.63 0.63 50 0.6 XXXVIII 0.32 1.0 0.63 0.32 0.16 50< 0.4 XXX 0.63 0.32 0.32 0.63 0.63 50< 1.6 oi LXVII 0.32 0.48 1.3 0.63 0.63 50< 3.2 LXVIII 1.3 2.5 2.5 2.5 5.0< 50< 13 N-N r LXIX 1.3 1.9 2.5 0.24 0.32 50 0.6 XLV 1.3 2.5 1.3 0.63 0.48 50< 3.2 BC; Bacillus ceres ATCC1 1778 SA; Staphylococcus aureus ATCC29213 MRSA; Staphylococcus aureus ATCC3359, Methicillin resistance 5 EF; Enterococcus faecalis ATCC29212 VREF; Enterococcus faecalis ATCC51299, vancomycin resistance CL51 37; Enterococcus faeciumCL5137, VanA CL5244; Enterococcus faecalis CL5244, VanB WO 00/69892 107 PCT/US00/13679 MIC values of glucose-C6 modified N-4-(4-chlorophenyl)benzyl vancomycin derivatives (ptg/mL) X= BC SA MRSA EF VREF CL5137 CL5244 OH 0.08 0.16 0.08 0.08 0.08 13 0.4 I LXXIIa 0.32 0.48 0.63 0.16 0.32 13 0.3

N

3 LXXIV 0.32 1.0 0.48 0.16 0.16 13 0.3

NH

2 LXXV 0.16 1.3 0.63 0.16 0.63 25 0.4 N=PPh 3 CXXXVI 1.3 1.3 -- 0.32 - 0.32-- 0.16 5.0 0.32 N N LXXIII 0.48 1.9 1.0 0.16 0.08 3.2 0.2 S NIN N LXXXIII 0.32 0.48 0.48 0.16 0.32 3.2 0.2 S .N N-tHK N N, LXXXIV 0.08 0.63 0.48 0.12 0.16 6.3 0.2 N S

NI

Sy .OH OH LXXXV 1.3 1.9 1.3 0.32 0.32 6.3 0.4 I.N. LXXXVI 0.32 1.0 1.0 0.12 0.16 3.2 0.4 0 XLII 1.9 5.0< 5.0< 1.0 1.9 6.3 0.8 H N LII 1.9 5.0 5.0 0.08 0.16 3.2 0.2 L 3.8 5.0 2.5 1.3 1.0 6.3 0.8 ,o LXXXVII 5.0< 5.0< 5.0< 5.0 5.0 50 10

NW

WO 00/69892 108 PCT/US00/13679 MIC values of glucose-C6 modified N-decyl vancosamine vancomycin derivatives ([tg/mL) X= BC SA MRSA EF VREF CL5137 CL5244 OH 0.32 0.63 0.48 0.16 0.32 25 3.2 ............................................................................ 1 1.0 1.3 0.32 0.08 0.12 6.3 0.4< NyN, LXXII 2.5 3.8 1.9 0.32 0.32 6.3 0.3 S SN OH NI LXXIX 0.63 2.5 1.3 0.32 0.63 25 0.6 NN S LXXX 5.0 5.0< 5.0 1.3 2.5 50 2.4 LI 2.5 5.0< 5.0< 0.63 1.3 6.3 0.2 N XLIX 3.8 5.0 2.5 1.9 1.3 13 0.8 , LXXXI 2.5 5.0< 2.5 0.32 0.63 16 0.8 N-. S( zLXXXII 5.0< 5.0< 5.0< 5.0< 5.0< 50< 13 N1 5 BC; Bacillus ceres ATCC11778 SA; Staphylococcus aureus ATCC29213 MRSA; Staphylococcus aureus ATCC3359, Methicillin resistance EF; Enterococcus faecalis ATCC29212 VREF; Enterococcus faecalis ATCC51299, vancomycin resistance 10 CL5137; Enterococcus faeciumCL5137, VanA CL5244; Enterococcus faecalis CL5244, VanB WO 00/69892 109 PCT/US00/13679 The preceding Examples are intended to describe certain preferred embodiments of the present invention. It should be appreciated, however, that obvious additions and modifications of the invention will be apparent to one skilled in the art. The invention is not limited except as set forth in the claims. 5 REFERENCES CITED Cohen M. (1992), Science, 257: 1050 10 Neu H. (1992), Science, 257: 1064. Axelsen, P.H. et al. (1997), J. Am. Chem. Soc. (JACS), 119:1516. Westwell et al. (1995), J. Antibiotics, 48: 1292. Walsh C. (1993), Science, 261:308. Malabarba A., et al. (1997a), "Structural Modifications of Glycopeptide Antibiotics," Med. Res. 15 Rev., 17(1):69-137. Nagarajan R., et al. (1988), "Selective cleavage of vancosamine, glucose, and N-methyl-leucine from vancomycin and related antibiotics," J.Chem.Soc.Chem.Comm., 1306-1307. Nagarajan R. (1991), "Antibacterial Activities and Modes of Action of Vancomycin and Related Glycopeptides," Antimicr. Agents Chemother., 35:605-609. 20 Nagarajan R. (1993), "Structure-activity relationships of vancomycin-type glycopeptide antibiotics," J Antibiotics, 46:1181-1195. Yan L., et al. (1994), JACS, 116: 6953. Prowse W., et al. (1995), Biochemistry, 34:9632-9644. Pierce C., et al. (1995), J.Chem.Soc. Perkin Trans., 2:153-157. 25 Williams D., et al. (1988), "Molecular Basis of the Activity of Antibiotics of the Vancomycin Group," Biochem. Pharm., 37:133-141. Kannan R., et al. (1988), "Function of the Amino Sugar and N-terminal Amino Acid of the Antibiotic Vancomycin in its Complexation with Cell Wall Peptides," JACS, 110: 2946-2953. Mackay J., et al. (1994), "Dissection of the contributions toward Dimerization of Glycopeptide 30 Antibiotics," JACS, 116:4573. Williams D. et al., (1993), "Toward an estimation of binding constants in aqueous solution: Studies of associations of vancomycin group antibiotics," PNAS USA, 90:1172-1178. Gerhard U., et al. (1993), "The role of the sugar and chlorine substituents in the dimerization of vancomycin antibiotics," JACS, 115:232-237. 35 Beauregard D., et al. (1995), "Dimerization and Membrane Anchors in Extracellular Targeting of Vancomycin Group Antibiotics," Antimicr.Agents & Chemo., 39: 781-785.

WO 00/69892 110 PCT/US00/13679 Loll P., et al. (1997), "Simultaneous Recognition of a Carboxylate-containing Ligand and an Inramolecular Surrogate Ligand in the Crystal Structure of an Asymmetric Vancomycin Dimer," JACS, 119:1516-1522. Felmingham, D. (1993), "Towards the ideal glycopeptide," J.Antimicrob. Chemother., 32, 663 5 666. Cooper, R. et al. (1996), "Chapter 14. Semisynthetic Glycopeptide Antibiotics," in Ann. Rept. In Med. Chem.- 31. Academic Press, Inc., 131-140. Malabarba, A. et al. (1997b), "Glycopeptide resistance in multiple antibiotic-resistant Gram positive bacteria: a current challenge for novel semi-synthetic glycopeptide derivatives," Eur. J. Med. 10 Chem., 32:459-478. Allen N. et al., (1997), "The Role of Hydrophobic Side Chains as Determinants of Antibacterial Activity of Semisynthetic Glycopeptide Antibiotics," JAntibiot., 50: 677-684. Pavlov A., et al. (1993), "Synthesis and Biological Activity of Derivatives of Glycopeptide Antibiotics Eremomycin and Vancomycin Nitrosated, Acylated or Carbamoylated at the N-terminal," 15 JAntibiot., 46:1731-1739. National Committee for Clinical Laboratory (NCCL) Standard. 1993. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow Aerobically-Third Edition; Approved Standard. NCCLS document M7-A3. National Committee for Clinical Laboratory Standard, Villanova, PA. 20 Mosmann, T. (1983), "Rapid colorimetric assay for cellular growth and survival; application to proliferation and cytotoxicity assays," J. Immunol. Methods. 65: 55-63. Damour, O., et al. (1992) "Cytotoxicity evalution of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes," Burns 18:479-485. Mikami, Y., et al. (1994) "Comparsion of in vitro antifungal activity of itraconazole and 25 hydroxy-itraconazole by colorimetric MTT assay," MYCOSES 37:27-33. Pankuch, G., et al. (1994) "Study of comparative anti-pneumococcal activities of penicillin G, RP 59500, erythromycin, sparfloxacin, and vancomycin by using time-kill methodology," Antimicrob. Agents Chemother. 38: 2065-2072. Zelenitsky, S., et al. (1997) "Time-kill curves for a semisynthetic glycopeptide, LY333328, 30 against vancomycin-susceptible and vancomycin-resistant Enterococcusfaecium strains," Antimicrob. Agents Chemother. 41:1407-1408. Mercier, R-C., et al. (1997) "Pharmacodynamic evalution of a new glycopeptide, LY333328, and in vitro activity against Staphylococcus aureus and Enterococcusfaecium," Antimicrob. Agents Chemother. 41:1307-1312. 35 Gerhard, U., et al. (1993) "The role of the sugar and chlorine substituents in the dimerization of vancomycin antibiotics," J. Am. Chem. Soc. 115: 232-237.

WO 00/69892 111 PCT/US00/13679 Solenberg, P.J. et al. (1997) "Production of hybrid glycopeptide antibiotics in vitro and in Streptomyces toyocaensis," Chem. Biol. 4:195-202. Milewski, W.M. et al. (1996) "Overproduction of a 37-Kilodalton Cytoplasmic Protein Homologous to NAD+-Linked D-Lactate Dehydrogenase Associated with Vancomycin Resistance in 5 Staphylococcus aureus," Antimicrobial Agents and Chemotherapy 40:166-172 Thompson, L.A. and Ellman, J.A. (1996) "Synthesis and Applications of Small Molecule Libraries," Chem. Rev. 96:555-600. Gallop. M.A. et al. (1994) "Applications of Combinatorial Technologies to Drug Discovery. 1. Background and Peptide Combinatorial Libraries," J. Med. Chem. 37:1233-1251. 10 Gordon, E.M. et al. (1994) "Applications of Combinatorial Technologies to Drug Discovery. 2. Combinatorial Organic Synthesis, Library Screening Strategies, and Future Directions," J. Med. Chem. 37:1385-1401. Terrett, N.K. et al. (1995) "Combinatorial Synthesis - The Design of Compound Libraries and their Application to Drug Discovery," Tetrahedron 51:8135-8173. 15 Betaneli, V.I. et al. (1982) "A Convenient Synthesis of 1,2-O-Ethylidene Derivatives of Carbohydrates," Carbohydrate Research 107:285-291. Williams, D.H. et al. (1998) "An Analysis of the Origins of a Cooperative Binding Energy of Dimerization," Science 280:711-714.

Claims (116)

1. A glycopeptide of the formula A _A 2 _ A 3 _ A 4 _ A 5 _ A 6 _ A 7 , in which each dash represents a covalent bond; wherein the group A 1 comprises a modified or unmodified a-amino acid residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, 5 heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl, guanidinyl, carbamoyl, or xanthyl; where each of the groups A 2 to A 7 comprises a modified or unmodified a-amino acid residue, whereby (i) the group A 1 is linked to an amino group on the group A 2 , (ii) each of the groups A

2 , A 4 and A 6 bears an aromatic side chain, which aromatic side chains are cross-linked together by two or more covalent bonds, and (iii) the group A 7 bears a terminal carboxyl, ester, amide, or N-substituted 10 amide group; and wherein one or more of the groups Aj to A 7 is linked via a glycosidic bond to one or more glycosidic groups each having one or more sugar residues; wherein at least one of said sugar residues is a disaccharide modified to bear one or more substituents of the formula YXR, N+(R 1 )=CR 2 R 3 , N=PR 1 R 2 R 3 , N+R 1 R 2 R 3 or P+R 1 R 2 R 3 in which the group Y is a single bond, O, NR 1 or S; the group X is 15 O, NR 1 , S, SO2, C(0)O, C(0)S, C(S)O, C(S)S, C(NR 1 )O, C(O)NR 1 , or halo (in which case Y and R are absent); and R, R 1 , R 2 , and R 3 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; and any pharmaceutically acceptable salts thereof; provided that at least one of the substituents of the formula YXR is not hydroxyl; X and Y are not both O; X and Y are not S 20 and O, or O and S, respectively; and if two or more of said substituents are present, they can be the same or different; and provided that when A 4 is linked to a disaccharide having a glucose residue that bears an N-substituted aminohexose residue, then said glucose residue is modified to bear at least one of said substituents YXR, N+(R1)=CR 2 R 3 , N=PR 1 R 2 R 3 , N+R 1 R 2 R 3 or P+R 1 R 2 R 3 . 25 2. The glycopeptide of claim 1 in which said disaccharide comprises two hexose residues linked to A 4 and wherein at least the hexose residue linked directly to A 4 is modified to bear at least one of said substituents YXR, N+(R1)=CR 2 R 3 , N=PR 1 R 2 R, N+R 1 R 2 R 3 or P+R 1 R 2 R 3 .

3. The glycopeptide of claim 2 in which said substituent is attached to the C6 position of said hexose residue linked directly to A 4 . 30

4. The glycopeptide of claim 3 in which said hexose residue linked directly to A 4 is glucose.

5. The glycopeptide of claim 4 in which at least one of said substituents is YXR wherein Y is a single bond and X is O, NR 1 , S or SO 2 . WO 00/69892 PCT/US00/13679 113

6. The glycopeptide of claim 5 wherein X is NR 1 .

7. The glycopeptide of claim 5 wherein X is S.

8. The glycopeptide of claim 5 wherein X is SO 2 .

9. The glycopeptide of claim 5 wherein X is 0 and R is not H. 5

10. The glycopeptide of claim 4 wherein at least one of said substituents YXR is halogen.

11. The glycopeptide of claim 2 wherein A 1 -A 2 -A 3 -A 4 -A 5 -A 6 -A 7 form a dalbaheptide.

12. The glycopeptide of claim 3 wherein A I -A 2 -A 3 -A 4 -A 5 -A 6 -A 7 form a dalbaheptide.

13. The glycopeptide of claim 4 wherein Al-A 2 -A 3 -A 4 -A 5 -A 6 -A 7 form a dalbaheptide.

14. The glycopeptide of claim 5 wherein Al-A 2 -A 3 -A 4 -A 5 -A 6 -A 7 form a dalbaheptide. 10

15. The glycopeptide of claim 6 wherein AI-A 2 -A 3 -A 4 -A 5 -A 6 -A 7 form a dalbaheptide.

16. The glycopeptide of claim 7 wherein A 1 -A 2 -A 3 -A 4 -A 5 -A 6 -A 7 form a dalbaheptide.

17. The glycopeptide of claim 8 wherein Al-A 2 -A 3 -A 4 -A 5 -A 6 -A 7 form a dalbaheptide.

18. The glycopeptide of claim 9 wherein A 1 -A 2 -A 3 -A 4 -A 5 -A 6 -A 7 form a dalbaheptide.

19. The glycopeptide of claim 10 wherein A I A 2 -A 3 -A 4 -A 5 -A 6 -A 7 form a dalbaheptide. 15

20. The glycopeptide of claim 11, wherein A 6 in said dalbaheptide is linked via a glycosidic bond to one or more sugar residues.

21. The glycopeptide of claim 11 wherein the amino acids in said dalbaheptide are those in vancomycin.

22. The glycopeptide of claim 20 wherein A 1 , which is N-methyl leucine, has been 20 selectively removed and replaced with another of said groups A 1 .

23. The glycopeptide of claim 2 in which the other hexose residue bears at least one of said substituents.

24. The glycopeptide of claim 3 in which the other hexose residue bears at least one of said substituents.

25 25. The glycopeptide of claim 4 in which the other hexose residue bears at least one of said substituents.

26. The glycopeptide of claim 5 in which the other hexose residue bears at least one of said substituents.

27. The glycopeptide of claim 6 in which the other hexose residue bears at least one of said 30 substituents.

28. The glycopeptide of claim 7 in which the other hexose residue bears at least one of said substituents.

29. The glycopeptide of claim 8 in which the other hexose residue bears at least one of said substituents. WO 00/69892 PCT/US00/13679 114

30. The glycopeptide of claim 9 in which the other hexose residue bears at least one of said substituents.

31. The glycopeptide of claim 10 in which the other hexose residue bears at least one of said substituents. 5

32. The glycopeptide of claim 11 in which the other hexose residue bears at least one of said substituents.

33. The glycopeptide of claim 12 in which the other hexose residue bears at least one of said substituents.

34. The glycopeptide of claim 13 in which the other hexose residue bears at least one of 10 said substituents.

35. The glycopeptide of claim 14 in which the other hexose residue bears at least one of said substituents.

36. The glycopeptide of claim 23 wherein at least one of said substituents is YXR wherein Y is a single bond and X is O, NR 1 , S or SO2. 15

37. The glycopeptide of claim 36 wherein X is NR 1 .

38. The glycopeptide of claim 37 wherein said substituent is attached to C3 of said other hexose residue.

39. A chemical library comprising a plurality of glycopeptides, each of said glycopeptides having the formula AIA 2 _A 3 _A 4 _A 5 _A 6 _A 7 , in which each dash represents a covalent bond; wherein 20 the group A 1 comprises a modified or unmodified a-amino acid residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl, guanidinyl, carbamoyl, or xanthyl; where each of the groups A 2 to A 7 comprises a modified or unmodified a-amino acid residue, whereby (i) the group A 1 is linked to an amino group on the group A 2 , (ii) each of the groups A 2 , A 4 and A 6 bears an aromatic side chain, 25 which aromatic side chains are cross-linked together by two or more covalent bonds, and (iii) the group A 7 bears a terminal carboxyl, ester, amide, or N-substituted amide group; and wherein one or more of the groups A 1 to A 7 is linked via a glycosidic bond to one or more glycosidic groups each having one or more sugar residues; wherein at least one of said sugar residues is a disaccharide modified to bear one or more substituents of the formula YXR, N+(R 1 )=CR 2 R 3 , N=PR 1 30 R 2 R 3 , N+R 1 R 2 R 3 or P+R 1 R 2 R 3 in which the group Y is a single bond, O, NR 1 or S; the group X is O, NR 1 , S, SO 2 , C(O)O, C(O)S, C(S)O, C(S)S, C(NR 1 )O, C(O)NR 1 , or halo (in which case Y and R are absent); and R, R 1 , R 2 , and R 3 are independently hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl or arylsulfonyl; and any pharmaceutically acceptable salts thereof; provided that at least WO 00/69892 115 PCT/US00/13679 one of the substituents of the formula YXR is not hydroxyl; X and Y are not both O; X and Y are not S and O, or O and S, respectively; and if two or more of said substituents are present, they can be the same or different; and provided that when A 4 is linked to a disaccharide having a glucose residue that bears an 5 N-substituted aminohexose residue, then said glucose residue is modified to bear at least one of said substituents YXR, N+(R 1 )=CR 2 R 3 , N=PR 1 R 2 R 3 , N+R 1 R 2 R 3 or P+R 1 R 2 R 3

40. The chemical library of claim 39 wherein AlA 2 _A 3 _A 4 _A 5 _A 6 _A 7 form a dalbaheptide and wherein said disaccharide comprises two hexose residues linked to A 4 and wherein at least the hexose residue linked directly to A 4 is modified to bear said substituent at the C6 position. 10

41. The chemical library of claim 40 wherein the other hexose residue bears a group YXR in which Y is a single bond and X is NR 1 .

42. A method for preparing a glycopeptide comprising the steps of: (a) selecting a protected glycopeptide of the formula AlA 2 _A-3_A 4 _A 5 _A 6 _A 7 , in which each dash represents a covalent bond; wherein the group A 1 comprises a modified or unmodified 15 ca-amino acid residue, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl, guanidinyl, carbamoyl, or xanthyl; where each of the groups A 2 to A 7 comprises a modified or unmodified a-amino acid residue, whereby (i) the group A 1 is linked to an amino group on the group A 2, (ii) each of the groups A 2 , A 4 and A 6 bears an aromatic side chain, which aromatic side chains are 20 cross-linked together by two or more covalent bonds, and (iii) the group A 7 bears a terminal carboxyl, ester, amide, or N-substituted amide group; at least A 4 is linked to a glycosidic group which has a hexose residue linked to A 4 ; and said protected glycopeptide has no free amino or carboxyl groups and has a free primary hydroxyl group only at the 6-position of said hexose residue; 25 (b) contacting said protected glycopeptide with a compound ArSO 2 G in which Ar is an aryl group and G is a leaving group under conditions effective to allow reaction of said free primary hydroxyl group to form a glycopeptide sulfonate ester; (c) contacting said glycopeptide sulfonate ester with a nucleophile under conditions effective to allow displacement of a sulfonate group to produce a substituted glycopeptide. 30

43. The method of claim 42 in which said nucleophile is a thiol compound.

44. The method of claim 42 in which said nucleophile is a halide.

45. The method of claim 44 in which said halide-substituted glycopeptide is contacted with a second nucleophile under conditions effective to allow displacement of said halide to produce a second substituted glycopeptide. WO 00/69892 116 PCT/US00/13679

46. The method of claim 45 in which said second nucleophile is a thiol compound.

47. The method of claim 42 in which the nucleophile is an azide ion, and further comprising reduction of an azido group at the 6-position of the substituted glycopeptide to an amino group. 5

48. The method of claim 47 further comprising the step of introducing a substituent onto said amino group.

49. The method of claim 42 in which the nucleophile is an azide ion, and further comprising a step of contacting said substituted glycopeptide with a phosphine compound under conditions effective to allow formation of an iminophosphorane. 10

50. A method for producing the chemical library of claim 39, said method comprising at least two steps in each of which a substituent is introduced on a glycopeptide.

51. The method of claim 50 wherein at least one of said two steps comprises introducing a substituent on the 6-position of a hexose residue directly linked to A 4 .

52. The method of claim 51 wherein the other of said at least two steps comprises 15 . introducing an N-substituent on an aminohexose residue bonded to said hexose residue directly linked to A 4 .

53. The method of claim 52 wherein said hexose residue directly linked to A 4 is a glucose residue.

54. The method of claim 51 wherein AIA 2 _A 3 _A 4 _A 5 _A 6 _A 7 form a dalbaheptide. 20

55. The method of claim 54 wherein the amino acids in said dalbaheptide are those in vancomycin.

56. The method of claim 55 wherein A 1 , which is N-methyl leucine, has been selectively removed and replaced with another of said groups A 1

57. A method of preparing a glycopeptide comprising: 25 (a) selecting: (i) an aglycone that is soluble in one or more organic solvents, is derived from a glycopeptide antibiotic, and which aglycone has exactly one free phenolic hydroxyl group; and (ii) a protected first glycosyl donor; (b) allowing a first non-enzymatic glycosylation reaction to proceed in an organic solvent such that a first glycosidic bond is formed, which links said free phenolic hydroxyl group to the 30 anomeric carbon of the first glycosyl donor to provide a pseudoaglycone having a protected first glycosyl residue; (c) selectively removing one protecting group from the first glycosyl residue to provide a pseudoaglycone bearing exactly one free hydroxyl group on the first glycosyl residue; (d) selecting a second protected glycosyl donor; and WO 00/69892 117 PCT/US00/13679 (e) allowing a second non-enzymatic glycosylation reaction to proceed in an organic solvent such that a second glycosidic bond is formed, which links said free hydroxyl group on the pseudoaglycone to the anomeric carbon of the second glycosyl donor.

58. A method of preparing a glycopeptide comprising: 5 (a) selecting a protected glycopeptide antibiotic that is soluble in one or more organic solvents, (b) contacting the glycopeptide antibiotic with a Lewis acid, and allowing a degradation reaction to proceed such that a sugar residue is removed, producing a pseudoaglycone having exactly one free hydroxyl group on a sugar residue of the pseudoaglycone; 10 (c) selecting a protected glycosyl donor; and (d) allowing a non-enzymatic glycosylation reaction to proceed in an organic solvent such that a glycosidic bond is formed which links the free hydroxyl group on the pseudoaglycone to the anomeric carbon of the glycosyl donor.

59. The method of claim 57 in which each of the first glycoside and the second glycosyl 15 donor is a monosaccharide bearing an activated anomeric sulfoxide group.

60. The method of claim 58 in which the glycosyl donor is a monosaccharide bearing an activated anomeric sulfoxide group.

61. The method of claim 59 in which BF 3 is present in the first non-enzymatic glycosylation reaction. 20

62. The method of claim 61 in which the first glycosyl donor is a glucose.

63. The method of claim 60 in which the glycopeptide antibiotic is vancomycin.

64. The method of claim 61 in which the glycopeptide antibiotic is vancomycin.

65. The method of claim 63 in which the Lewis acid is boron trifluoride.

66. The method of claim 63 in which the glycopeptide antibiotic is rendered soluble in 25 organic solvents by substitution with protecting groups.

67. The method of claim 66, further comprising removal of said protecting groups subsequent to step (d).

68. The method of claim 67 in which said protecting groups comprise: aloe groups substituted on amine nitrogens, an allyl ester group, allyl phenolic ether groups, and acetates of 30 aliphatic hydroxyls.

69. The method of claim 57 in which the aglycone is rendered soluble in organic solvents by substitution with protecting groups.

70. The method of claim 69, further comprising removal of said protecting groups and protecting groups on the glycosides following step (e). 35

71. The method of claim 70 in which said protecting groups comprise: a CBz group on the amine nitrogen, a benzyl ester group, methyl phenolic ether groups on residues 5 and 7, and acetates of aliphatic hydroxyls. WO 00/69892 PCT/US00/13679 118

72. The method of claim 57 in which the glycopeptide is attached to a polymeric support.

73. The method of claim 58 in which the glycopeptide is attached to a polymeric support.

74. A method for producing the chemical library of claim 39; said method comprising at least two steps, wherein at least one of said at least two steps comprises a glycosylation reaction which 5 introduces a substituted sugar residue.

75. The method of claim 74 in which A 1 to A 7 are linked sequentially by peptide bonds and cross-linked as in a dalbaheptide.

76. The method of claim 75 in which said glycosylation reaction links said substituted sugar residue to an A 4 residue of an aglycone. 10

77. The method of claim 76 in which said glycosylation reaction links said substituted sugar residue to a sugar residue of a pseudoaglycone, wherein said sugar residue of a pseudoaglycone is linked to an A 4 residue of the pseudoaglycone.

78. The method of claim 76 in which a second glycosylation reaction links a second substituted sugar residue to said substituted sugar residue. 15

79. The method of claim 77 in which Al 1 is a modified or unmodified ca-amino acid residue, and in which A 1 to A 7 are linked sequentially by peptide bonds and cross-linked so as to have the structure of a dalbaheptide.

80. The method of claim 78 in which A 1 is a modified or unmodified ca-amino acid residue, and in which A 1 to A 7 are linked sequentially by peptide bonds and cross-linked so as to have 20 the structure of a dalbaheptide.

81. The method of claim 77 in which the structures and interconnections of A 1 to A 7 are those found in vancomycin.

82. The method of claim 81 in which a glycosyl donor bearing an activated anomeric sulfoxide group is employed in each glycosylation reaction. 25

83. The compound of claim 1 which is N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-mesitylenesulfonated vancomycin.

84. The compound of claim 1 which is glucose-C6-2-thio-6-azathymine vancomycin.

85. The compound of claim 1 which is glucose-C6-2-thio-4-hydroxy-6-methylpyrimidine vancomycin. 30

86. The compound of claim 1 which is N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-5-amino-1,3,4-thiadiazole vancomycin.

87. The compound of claim 1 which in N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-4-amino-3-hydrazineo- 1,2 ,4-triazole vancomycin. WO 00/69892 119 PCT/US00/13679

88. The compound of claim 1 which is N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-4-hydoxy-6 methylpyrimidine vancomycin.

89. The compound of claim 1 which is 5 N-4-(4-chlorophenyl)benzylvancosamine-glucose-C6-2-thio-4-hydoxy-6-azathymine vancomycin.

90. The compound of claim 1 which is N-4-(4-chlorophenyl)benzylvancosamine-glucose-C 6 -ido vancomycin.

91. The compound of claim 1 which is glucose 0 -C6-N-2-quinoxalinyl-vancosamine-N-4-(4-chlorophenyl)benzyl vancomycin.

92. The compound of claim 1 which is vancosamine-N-4-(4-chlorophenyl)benzyl-glucose-C6- S-3-amino- 5-mercapto-1,2,4 triazole vancomycin.

93. The compound of claim 1 which is glucose-C6-mesitylenesulfonyl vancomycin. 15

94. The compound of claim 1 which is glucose-C6-iodo vancomycin.

95. The compound of claim 1 which is glucose-C6-azide vancomycin.

96. The compound of claim 1 which is glucose-C6-bromo vancomycin.

97. The compound of claim 1 which is glucose-C6-amine vancomycin. 20

98. The compound of claim 1 which is glucose-C6-hydrazine vancomycin.

99. The compound of claim 1 which is N-4-(4-chlorophenyl)benzyl vancosamine-glucose-C6-iminotriphenylphosphorane vancomycin.

100. The compound of claim 1 which is glucose-C6-N-decyl vancomycin.

101. The compound of claim 1 which is N-4-(4-chlorophenyl)benzyl 25 vancosamine-glucose-C6-amine vancomycin.

102. A glycopeptide antibiotic bearing at least one disaccharide group, said disaccharide group comprising two saccharide groups, a first of said saccharide groups bearing at least one amino or substituted amino group, and a second of said saccharide groups modified to bear at least one substituent which is not hydroxyl, or a pharmaceutically acceptable salt thereof. 30

103. The glycopeptide antibiotic of claim 102 wherein the second of said saccharide groups is glucose modified to bear at least one substituent which is not hydroxyl at the C6 position of said glucose.

104. The glycopeptide antibiotic of claim 103 which is vancomycin modified to bear at least one substituent which is not hydroxyl at the C6 position of said glucose. 35

105. The glycopeptide antibiotic of claim 104 wherein said at least one substituent which is not hydroxyl at the C6 position of said glucose is amino. WO 00/69892 120 PCT/US00/13679 120

106. The glycopeptide antibiotic of claim 105 wherein the first of said saccharide groups bears at least one substituted amino group.

107. The glycopeptide antibiotic of claim 106 wherein said substituted amino group is -NR 1 H wherein R 1 bears one or more alkyl, substituted alkyl, aryl, substituted aryl, heterocyclic or 5 substituted heterocyclic groups.

108. The glycopeptide antibiotic of claim 107 wherein at least one of said substituted alkyl groups is aralkyl.

109. The glycopeptide antibiotic of claim 107 wherein at least one of said substituted aryl groups is aralkyloxy substituted aryl. 10

110. The glycopeptide antibiotic of claim 107 wherein at least one of said substituted aryl groups is halo-substituted aryl.

111. The glycopeptide antibiotic of claim 102 wherein the first of said saccharide groups bears at least one substituted amino group. 15

112. The glycopeptide antibiotic of claim 111 wherein said substituted amino group is -NR 1 H wherein R 1 bears one or more alkyl, substituted alkyl, aryl, substituted aryl, heterocyclic or substituted heterocyclic groups.

113. The glycopeptide antibiotic of claim 112 wherein at least one of said substituted alkyl groups is aralkyl. 20

114. The glycopeptide antibiotic of claim 112 wherein at least one of said substituted aryl groups is aralkyloxy substituted aryl.

115. The glycopeptide antibiotic of claim 112 wherein at least one of said substituted aryl groups is halo-substituted aryl.

116. The glycopeptide antibiotic of claim 112 wherein said at least one substituent 25 which is not hydroxyl is amino.