WO 01/79267 PCT/US01/12630
2
A 6LYC0PEPTIDE
AND PREPARATION THEREOF
5 FIELD OF THE INVENTION
The present invention relates to the stereospecific
synthesis of a glycopeptide using a triply orthogonal
protection scheme, in particular, the synthesis of N-
acetylglucosaminyl-P- [1,4] -N-acetylmuramylmonopeptide and
10 derivatives thereof.
BACKGROUND OF THE INVENTION
U.S. 4,395,399 to Ovchinnikov et al. disclosed
glycopeptides of formula I

wherein Y' is a residue of an aminoacid or linear peptide of
2 to 5 amino acid residues. These glycopeptides are
prepared by coupling unblocked muramyl-containing N-
20 acetylamino-sugars of formula II

WO 01/79267 PCT/USOI/12630
3

with blocked aminoacids or peptides. The disaccharide acid
of formula II is obtained from large-scale fermentation of
5 the bacterium Micrococcus lysodeicticus. The peptide portion
is produced by conventional synthetic methods.
Compounds of formula I (hereinafter referred to as
"Ovchinnikov glycopeptides"), particularly N-
acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine
10 (GMDP) and N-acetylglucosaminyl-N-acetylmurainyl-L-alanyl-D-
glutamic acid (GMDP-A), are orally-active immunomodulators
for use in a number of indications. (see, e.g., Ivanov,
V.T., et al., Imrmxnologiya No. 2, 4-5 (1996); Adrianova,
I.E., et al., Radiobiologiia 32, 566-70 (1992); Paladie,
15 A.M., et al., Vaccine 14, 1327-30 (1996); and Khaitov, R.M..,
et al., "Immunotheraphy of Infections," Ed. Masihi, N. , 205-
211 (Marcel Dekker, Inc., 1994)). For example, compounds of
formula I possess adjuvant activity. Adjuvants are
compounds causing non-specific stimulation of the immune
20 system in human beings and animals, resulting in an
increased production of antibodies and enhancement of
protective reaction of the organism against infection.

WO 01/79267 PCT/US01/12630
4
Adjuvants are used in medicine for the manufacture of
vaccines and sera. In addition, U.S. Patent No. 5,506,204 to
R. Aston discloses the use of GMDP and GMDP-A for treatment
of septic shock.
5 The semi-synthetic approach for preparing glycopeptides
of formula I described above is utilized because the
disaccharide core, N-acetyl- (2-deoxy-2-aminoglucopyranosyl) -
P~[l,4]-N- acetylmuramic acid, is one of the most difficult
glucopyranosyl-glucopyranose disaccharides to synthesize.
10 For example, the order of glucopyranose hydroxyl acceptor
reactivity toward glycosyl cation donors, independent of
donor source, is water » ethanol > C(6)OH > C(2)OH > C(3)OH
> the required C(4)OH. In addition, 2-deoxy-2-
acylaminoglucopyranose C(4)OH acceptors are deactivated
15 electronically relative to glucose itself. Muramic acid
derivatives, in particular, suffer still further acceptor
reactivity disadvantage due to steric crowding around the
C(4) oxygen. Formation of the desired 3-[1,4]-glycosidic
bond requires a 2-deoxy-2-aminoglucopyranose glycosyl cation
20 donor with a nitrogen substituent that will favor equatorial
approach of the very modestly nucleophilic C(4)OH of a
muramic acid derivative.
Several approaches to this formidable glycosidation
problem have been documented, (see, e.g., Mercer, C, et
25 al., Tetrahedron Lett. 13, 1029 (1973); Durette, P.L., et

WO 01/79267 PCT/US«1/126J0
5
al., Carbohydr. Res., 77, Cl (1979); Kusumoto, D., et al.,
Bull. Chem. Soc. Jpn., 59, 1411 (1986); Kusumoto, D., et
al., Bull. Chem. Soc. Jpn., 59, 1419 (1986); Farkcis, J., et
al., Carbohydr. Res., 163, 63 (1987); Kinzy, W., et al.,
5 Liebigs Ann. Chem., 407 (1987); Termin, A., et al. , Liebigs
Ann. Chem., 789 (1989); Ledvina, M. , et al. , Collect. Czech.
Chem. Commun., 54, 2784 (1989) and Termin, A., et al.,
Liebigs Ann. Chem., 527 (1992). However, none of these
approaches provides a process for preparing the disaccharide
10 in sufficient amounts to be useful as an intermediate in the
preparation of glycopeptides of formula I.
SUMMARY OF THE INVENTION
The present invention provides a process for preparing
15 a protected glycopeptide of formula 1

1
by coupling a muramylamide compound of formula 2


WO 01/79267 PCT/US01/12630
6
with a glucopyranosyl compound of formula 3

3
5 to form the protected glycopeptide of formula 1,
wherein:
A is Br or Cl;
Pg0 is an acyl hydroxy-protecting group;
Pg1 is a hydroxy-protecting group which is not electron
10 withdrawing;
Pg2 is an amine-protecting group which does not lead to
oxazoline formation;
Pg5 is a hydroxy-protecting group;
Pg°, Pg1, Pg2, and Pg5 are mutually orthogonal
15 protecting groups; and
Y is a residue of an amino acid or peptide, wherein:
Y forms an amide linkage with the attached
carbonyl; and
Y comprises a protected terminal carboxy group.
20
The invention also provides compounds of formula III

WO 01/79267 PCT/US01/12630
7

wherein:
R° is Pgc or hydrogen;
5 R1 is Pg1, Pg3 or hydrogen;
R2 is Pg2 or acetyl;
RY is Y or Y';
Pg° is an acyl hydroxy-protecting group;
Pg1 is a hydroxy-protecting group which is not
10 electron- withdrawing;
Pg2 is a an amine-protecting group which does not lead
to oxazoline formation;
Pg3 is an acyl hydroxy-protecting group;
Pg5 is a hydroxy-protecting group;
15 Pg0/ Pg1, Pg2/ and Pg5 are mutually orthogonal
protecting groups;
Y' is a residue of an amino acid or peptide, wherein:
Y' forms an amide linkage with the attached
carbonyl; and
20 Y comprises an unprotected terminal carboxy group;
Y is a residue of an amino acid or peptide, wherein:

WO 01/79267 PCT/US01/1263O
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Y forms an amide linkage with the attached
carbonyl; and
*
Y comprises a protected terminal carboxy group.
5 Compounds of formula III' are useful intermediates in the
synthesis of the Ovchinnikov glycopeptides.
Detailed Description
Definitions
10 As used above, and throughout the description of the
invention, the following abbreviations, unless otherwise
indicated, shall be understood to have the following
abbrevi a tions:
Designation Reagent or Fragment
15 Ac -C(O)CH3
AcOH acetic acid
Ac2O acetic anhydride
BOC t-butyloxycarbonyl
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
20 THF tetrahydrofuran
TsOH p-toluenesulfonic acid
NMM N-methylmorpholine
TFA trifluoroacetic acid
Troc 2,2,2-trichloroethoxycarbonyl
25 min minutes
h hour(s)

WO 01/79267 PCT/US01/12630
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Cbz benzyloxycarbonyl
TLC thin layer chromatography
NMR nuclear magnetic resonance
ESI-MS electro-spray ionization mass
5. specturn
EtOAc ethyl acetate
IR infrared spectrum
MeOH methanol
NaOMe sodium methoxide
10 NHS N-hydroxysuccin imi de
EDCI 1 - [ 3 - (dimethylamino) propyl ] - 3 -
ethylcarbodiimide hydrochloride
As used above, and throughout the description of the
invention, the following terms, unless otherwise indicated,
15 shall be understood to have the following meanings:
"Antino acid" means an amino acid selected from the
group consisting of natural and unnatural amino acids as
defined herein. Amino acid is also meant to include -amino
acids having L or D stereochemistry at the cc-carbon.
20 Preferred amino acids are those possessing an cc-amino group.
The amino acids may be neutral, positive or negative
depending on the substituents in the side chain. "Neutral
amino acid" means an amino acid containing uncharged side
chain substituents. Exemplary neutral amino acids include
25 alanine, valine, leucine, isoleucine, proline,

WO 01/79267 PCT/US01/12630
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phenylalanine, tryptophan, methionine, glycine, serine,
threonine and cysteine. "Positive amino acid" means an
amino acid in which the side chain substituents are
positively charged at physiological pH. Exemplary positive
5 amino acids include lysine, arginine and histidine.
"Negative amino acid" means an amino acid in which the side
chain substituents bear a net negative charge at
physiological pH. Exemplary negative amino acids include
aspartic acid and glutamic acid. Preferred amino acids are
10 a-amino acids. Exemplary natural amino acids are
isoleucine, proline, phenylalanine, tryptophan, methionine,
glycine, serine, threonine, cysteine, tyrosine, asparagine,
glutamine, lysine, arginine, histidine, aspartic acid and
glutamic acid. Unnatural amino acid" means an amino acid
15 for which there is no nucleic acid codon. Examples of
unnatural amino acids include, for example, the D-isomers of
the natural a-amino acids as indicated above; Aib
(aminobutyric acid) , βAib (3-amino-isobutyric acid) , Nva
(norvaline) , fi-Ala, Aad (2-aminoadipic acid) , βAad (3-
20 aminoadipic acid) , Abu {2-aminobutyric acid) , Gaba (Y-
aminobutyric acid), Acp (6-aminocaproic acid), Dbu (2,4-
diaminobutryic acid) , α-aminopimelic acid, TMSA
(trimethylsilyl-Ala), alle (allo-isoleucine), Nle
(norleucine), tert-Leu, Cit (citrulline), Orn, Dpm (2,2'-
25 diarainopimelic acid), Dpr (2,3-diaminopropionic acid), α-or

WO 01/79267 PCMJS01/12630
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β-Nal, Cha (cyclohexyl-Ala), hydroxyproline, Sar
(sarcosine), and the like; cyclic amino acids; N°-alkylated
amino acids such as MeGly (Na-methylglycine) , EtGly (N5-
ethylglycine) and EtAsn (Na-ethylasparagine) ; and amino
5 acids in which the (X-carbon bears two side-chain
substituents. The names of natural and unnatural amino
acids and residues thereof used herein follow the naming
conventions suggested by the IUPAC Commission on the
Nomenclature of Organic Chemistry and the IUPAC-IUB
10 Commission on Biochemical Nomenclature as set out in
"Nomenclature of a-Amino Acids (Recommendations, 1974) "
Biochemistry, 14(2), (1975). To the extent that the names
and abbreviations of amino acids and residues thereof
employed in this specification and appended claims differ
15 from those noted, differing names and abbreviations will be
made clear.
"Amino acid protecting group" and "peptide—protecting
group* mean a group that protects an acid or amino moiety of
the amino acid/peptide or other reactive moiety on the side
20 chain of an amino acid/amino acid residue, e.g., hydroxy or
thiol. For examples of "corresponding protected
derivatives" of amino acid side chains, see T.W. Green and
P.G.M. Wuts in "Protective Groups in Organic Chemistry" John
Wiley and Sons, 1991. Protecting groups for an acid group
25 in an amino acid are described herein in the section

WO 01/79267 PCT/USO1/12630
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"carboxy-protecting group." Protecting groups for an amine
group in an amino acid are described in the section "amine-
protecting group."
"Amino acid residue" means the individual amino acid
5 units incorporated into a peptide, or peptide portion of a
molecule, through an amide linkage.
"Amine-protecting group" means an easily removable
group that is known in the art to protect an amino group
against undesirable reaction during synthetic procedures and
10 to be selectively removable. The use of amine-protecting
groups is well known in the art for protecting groups
against undesirable reactions during a synthetic procedure
and many such protecting groups are known, for example, T.H.
Greene and P.G.M. Wuts, Protective Groups in Organic
15 Synthesis, 2nd edition, John Wiley & Sons, New York (1991),
incorporated herein by reference. Amine protecting group
also includes acid-labile amine-protecting groups (e.g.,
BOC) and hydrogenation-labile amine-protecting groups (e.g.,
Cbz) . In the present invention, Pg2 is a group which does
20 not lead to the generation of undesirable oxazoline by-
products (i.e., Pg2 cannot be an acyl group). Suitable
amine-protecting groups include carbamate and imides groups.
Particular imide groups include phthalimide.
tetrachlorophthalimide and (Ac)2N-. Particular carbamate
25 groups include methoxy-carbonyl, 9-fluorenylmethoxycarbonyl,
2,2, 2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-

WO 01/79267 PCTYUS01/12630
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carbonyl, vinyloxycarbonyl, allyloxycarbonyl, t-
butyloxycarbonyl (BOC), 1,1-dimethyl-propynyloxycarbonyl,
ben zyloxy carbonyl (CBZ) , p-nitrobenzyloxycarbonyl, 2,4-
dichloro-benzyloxycarbonyl, trimethylsilyloxycarbonyl,
5 2,2,2-trichloroethoxycarbonyl, 1,1-dimethyl-2,2,2-
trichloroethoxycarbonyl, and the like. A preferred amine-
protecting group is 2,2,2-trichloroethoxycarbonyl.
"Carboxy-protecting group" means an easily removable
group that is known in the art to protect an acidic hydrogen
10 of a carboxyl group against undesirable reaction during
synthetic procedures, e.g., to block or protect the acid
functionality while the reactions involving other functional
sites of the compound are carried out, and to be selectively
removable. Such acid protecting groups are well known to
15 those skilled in the art, having been extensively used in
the protection of carboxyl groups, as described in U.S. Pat.
No. 3,840,556 and 3,719,667, the disclosures of which are
hereby incorporated herein by reference. For suitable acid
protecting groups, see T.W. Green and P.G.M. Wuts in
20 "Protective Groups in Organic Chemistry" John Wiley and
Sons, 1991. Acid protecting group also includes
hydrogenation labile acid protecting groups, such as benzyl.
Examples of acid protecting groups include esters such as
substituted and unsubstituted C1 to C8 alkyl, e.g., methyl,
25 ethyl, t-butyl, methoxymethyl, methylthiomethyl, 2,2,2-
trichloroethyl and the like, tetrahydropyranyl, substituted

WO 01/79267 PCT/US01/12630
and unsubstituted phenylalkyl such as benzyl and substituted
derivatives thereof such as alkoxybenzyl or nitrobenzyl
groups and the like, cinnamyl, dialkylaminoalkyl, e.g.,
dimethylaminoethyl and the like, trimethylsilyl, substituted
5 and unsubstituted amides and hydrazides, e.g., amides and
hydrazides of N,N-dimethylamine, 7-nitroindole, hydrazine,
N-phenylhydrazine and the like, acyloxyalkyl groups such as
pivaloyloxymethyl or propionyloxymethyl and the like,
aroyloxyalkyl such as benzoyloxyethyl and the like,
10 alkoxycarbonylalkyl such as methoxycarbonylmethyl,
cyclohexyloxycarbonylmethyl and the like,
alkoxycarbonyloxyalkyl such as t-butyloxycarbonyloxyroethyl
and the like, alkoxycarbonylaminoalkyl such as t-
butyloxycarbonylaminomethyl and the like,
15 alkylaminocarbonylaminoalkyl, such as
methylaminocarbonylaminomethyl and the like, acylaminoalkyl
such as acetylaminomethyl and the like,
heterocyclylcarbonyloxyalkyl such as 4-methylpiperazinyl-
carbonyloxymethyl and the like, dialkylaminocarbortylalkyl
20 such as dimethylaminocarbonyl-methyl and the like, (5-(lower
alkyl)-2-oxo-l,3-dioxolen-4-yl)alkyl such as (5-t-butyl-2-
oxo-1,3-dioxolen-4-yl)methyl and the like, and (5-phenyl-2-
oxo-1,3-dioxolen-4-yl)alkyl such as (5-phenyl-2-oxo-l,3-
dioxolen-4-yl)methyl and the like. Particular carboxy-
25 protecting groups include methyl, 9-fluorenylmethyl, 2-
(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-

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methylthioethyl, 1,3-dithianyl-2-methyl, 2- (p-
toluenesulfonyl) ethyl, 2-(p-nitrophenylsulf enyl) ethyl. 2-
(2 ' -pyridyl) ethyl, 2- (diphenylphosphino) ethyl, p-
(methylmercapto)phenyl, nitroethyl, allyl and the like.
5 Preferred carboxy-protecting groups are cyanoethyl, t-butyl
and -CH2CH2SO2Ph.
"Hydroxy-protecting group" means an easily removable
group that is known in the art to protect an hydroxyl group
against undesirable reaction during synthetic procedures and
10 to be selectively removable. The use of hydroxy-protecting
groups is well known in the art for protecting groups
against undesirable reactions during a synthetic procedure
and many such protecting groups are known, for example, T.H.
Greene and P.G.M. Wuts, Protective Groups in Organic
15 Synthesis, 2nd edition, John Wiley & Sons, New York (1991),
incorporated herein by reference. In the present invention,
the Pg°, Pg1, and Pg5 hydroxy-protecting groups are mutually
orthogonal, as described herein. Pg1 cannot be an electron-
withdrawing group, since such groups deactivate the coupling
20 reaction between the muramylamide compound of formula 2 and
glucosopyranosyl compound of formula 3. Suitable Pg1 groups
include aralkyl, aralkenly and silyl groups. Particular
aralkyl and alkenyl groups include benzyl and allyl,
respectively. Particular silyl groups include trialkylsilyl
25 groups, such as trimethylsilyl and (t-butyl) dimethylsilyl.
Preferred Pg1 groups are allyl and benzyl; a more preferred

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group is benzyl. In addition, Pg° and Pg3 must be removable
by saponification (i.e. Pg° and Pg3 must be acyl groups).
Particular acyl groups include formyl, acetyl, chloroacetyl,
trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxy-acetyl,
5 trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl,
o-nitrocinnamoyl, picolinoyl, acylisothiocyanate,
aminocaproyl, benzoyl, and the like. Preferred groups are
chloroacetyl and acetyl; a more preferred group is acetyl.
Finally, since Pg5 is orthogonal to the other hydroxy-
10 protecting groups, it must be stable under saponification
conditions (i.e., Pg5 cannot an acyl group) and some
conditions suitable for removal of Pg1. Suitable Pg5 groups
aralkyl and alkenyl groups. Preferred Pg3 groups include
allyl, n-pentenyl, and benzyl; a more preferred group is
15 benzyl.
"Leaving group" of an activated ester means a
substituent having sufficient lability such that it can be
substituted by a good nucleophile (i.e., an amino group of a
peptide unit) . The lability of a particular substituent
20 will vary depending upon substituents on the same and/or
adjacent carbon atoms and the nature of the leaving group.
Those skilled in the art will appreciate the types of
leaving groups capable of substitution by an amino
nucleophile. For suitable leaving groups, see M. Bodanszky
25 and A. Bodanszky in "The Practice of Peptide Synthesis"
Springer-Verlag, 1984; and M. Bodanszky in "Principles of

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Peptide Synthesis" Springer-Verlag, 1984. In the present
invention, the leaving group activates the attached carbonyl
such that the terminal amino acid group acts as a linker for
linking the disaccharide with the peptide unit. Particular
5 leaving groups include pentafluorophenoxy, N-oxysuccimide,
N-oxyphthalimide, and N-oxybenzotriazole. A preferred
leaving group is N-oxysuccinimide.
"Orthogonal protecting groups" means protecting groups
for which there exists a set of conditions wherein one of
10 the groups can be removed without removing the other(s) . The
term encompasses protecting groups for different moieties
(e.g., orthogonal amine and hydroxy protecting groups) as
well as the same moiety (e.g., orthogonal hydroxy-protecting
groups) . It is not a requirement that orthogonal protecting
15 groups necessarily be different. For example, when the term
is used to describe protecting groups for the same moiety,
the groups may be different (e.g., orthogonal acetyl and
benzyl hydroxy-protecting groups) or the same (e.g.,
orthogonal benzyl protecting groups).
20 "Electron-withdrawing group" means a group which is a
more powerful electron attractor than hydrogen. Electron
withdrawing groups exhibit negative inductive effects,
whereas groups which are poorer electron attractors than
hydrogen exhibit positive inductive effects, (see, e.g., E.
25 S. Gould, Mechanism and Structure in Organic Chemistry,

WO 01/79267 PCT/US01/12630
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Holt, Rinehart and Winston, New York (1959), incorporated
herein by reference).
"Acyl" means an R-C(O)- group, wherein R is bonded to
i the CO group through a carbon-carbon bond.
5 "Alkyl" means an aliphatic hydrocarbon group which may
be straight or branched having about 1 to about 20 carbon
atoms in the chain. Preferred alkyl groups have 1 to about
12 carbon atoms in the chain, more preferred is lower alkyl
as defined herein. Branched means that one or more lower
10 alkyl groups such as methyl, ethyl or propyl are attached to
a linear alkyl chain. "Lower alkyl" means about 1 to about
4 carbon atoms in the chain that may be straight or
branched.
"Alkenyl" means an aliphatic hydrocarbon group
15 containing a carbon-carbon double bond and which may be
straight or branched having about 2 to about 15 carbon atoms
in the chain. Preferred alkenyl groups have 2 to about 12
carbon atoms in the chain; and more preferably about 2 to
about 4 carbon atoms in the chain. Branched meant; that one
20 or more lower alkyl groups such as methyl, ethyl or propyl
are attached to a linear alkenyl chain. "Lower alkenyl"
means about 2 to about 4 carbon atoms in the chain that may
be straight or branched. Exemplary alkenyl groups include
ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl,
25 n-pentenyl, heptenyl, octenyl, cyclohexylbutenyl amd
decenyl.

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"Aryl" means an aromatic monocyclic or multicyclic ring
system of about 6 to about 14 carbon atoms, preferably of
about 6 to about 10 carbon atoms. Exemplary aryl groups
include phenyl or naphthyl, or phenyl substituted or
5 naphthyl substituted.
"Carboxy" means an HO(O)C- (carboxylic acid) group.
"N-oxysuccinimide" means a moiety of the following

"Peptide" means a polymer encompassing amino acid
10 residues joined together through amide bonds.
"GMDP" refers to N-acetylglucosaminyl-N—acetylmuramyl-
L-alanyl-D-isoglutamine, which has the following structure:

"GMDP-A" refers to the N-acetylglucosaminyl-N-
15 acetylmuramyl-L-alanyl-D-glutamic acid, which has the
following structure:

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Embodiments
With reference to formulas 1-19, as described herein,
particular and preferred embodiments are as follows:
5 In a first particular embodiment of the invention, the
muramylamide compound of formula 2, as described herein, and
glucopyranosyl compound of formula 3, as described herein,
are reacted under scrupulously anhydrous conditions.
In a second particular embodiment of the invention, Pg0
10 is acetyl.
In a third particular embodiment of the invention, the
Pg° group of a compound of formula 8, as described herein,
is removed to form a compound of formula 7, as described
herein.
15 In a fourth particular embodiment of the invention, th e
Pg° group of a compound of formula 13, as described herein,
is removed to form a compound of formula 12, as described
herein.
In a preferred embodiment, the Pg° group is removed in
20 the presence of aqueous sodium hydroxide.

WO 01/79267 PCT/US01/12630
21
In a fifth particular embodiment of the invention, Pg5
is benzyl, allyl or n-pentenyl.
In a preferred embodiment, Pg5 is benzyl.
In a sixth particular embodiment of the invention, the
5 Pg5 group of a compound of formula 12, as described herein,
is removed to form a compound of formula 11, as described
herein.
In a seventh particular embodiment of the invention,
the Pg5 group of a compound of formula 7, as described
10 herein, is removed to form a compound of formula I, as
described herein.
In a preferred embodiment, the Pg? group is removed in
the presence of hydrogen and a palladiam/carbon catalyst.
In an eighth particular embodiment of the invention,
15 Pg1 is a benzyl, allyl or silyl hydroxy-protecting group;
In a preferred embodiment, Pg1 is benzyl.
In a preferred embodiment, a murainylarnide compound of
formula 3a, as described herein, is prepared by reductively
opening the 1,3-dioxane ring of a muramylamide of formula 6,
20 as described herein.
In a ninth particular embodiment of the invention, the
Pg1 group of a compound of formula 1, as described herein,
is exchanged with a Pg3 group to form a, compound of formula
10, as described herein.
25 In a tenth particular embodiment of the invention, the
Pg1 group of a compound of formula la, as described herein.

WO 01/79267 PCT/USO1/1263O
22
is exchanged with a Pg3 group to form a compound of formula
19, as described herein.
In a preferred embodiment, Pg3 is acetyl.
In a more preferred embodiment, the exchanging is
5 carried out in the presence of acetic anhydride, acetic
acid, and zinc chloride.
In a eleventh particular embodiment of the invention,
Pg2 is a carbamate or imide amine-protecting group;
In a preferred embodiment, Pg2 is 2,2,2-
10 trichloroethoxycarbonyl.
In an twelfth particular embodiment of the invention,
the Pg2 group of a compound of formula 19, as described
herein, is exchanged with an acetyl group to form a compound
of formula 18, as described herein.
15 In a thirteenth particular embodiment of the invention,
the Pg2 group of a compound of formula 10, as described
herein, is exchanged with an acetyl group to form a compound
of formula 9, as described herein.
In a preferred embodiment, the exchanging is carried
20 out in the presence of acetic anhydride, acetic acid, and
zinc dust.
In a fourteenth particular embodiment of the invention,
the Pg3 group of a compound of formula 8, as described
herein, is removed to form a compound of formula 7, as
25 described herein.

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23
In a fifteenth particular embodiment of the invention,
the Pg3 group of a compound of formula 13, as described
herein, is removed to form a compound of formula 12, as
described herein.
5 In a preferred embodiment, the Pg3 group is removed in
the presence of aqueous sodium hydroxide.
In a sixteenth particular embodiment of the invention,
LOH is N-hydroxysuccinimide.
In a seventeenth particular embodiment of the
10 invention, A is Br.
In an eighteenth particular embodiment of the
invention, Y is a peptide comprising 2 to 5 amino acid
residues.
In a preferred embodiment, Y is a linear peptide.
15 In an nineteenth particular embodiment of the
invention, each of X' and W is a residue of an amino acid
or peptide comprising 2 to 4 amino acid residues, provided
that the total number of amino acid residues in X' and W is
2 to 5;
20 In a preferred embodiment, each of X' and W is a
linear peptide.
In a more preferred embodiment, the -X'-W is a linear
peptide;
With reference to formula III above, particular and
25 preferred embodiments are as follows:

WO 01/79267 PCT/US01/12630
In a twentieth particular embodiment of the invention,
R° is Pg°.
In a preferred embodiment, Pg° is acetyl.
In a twenty-first particular embodiment of the
5 invention, R° is hydrogen.
In a twenty-second particular embodiment of the
invention, R1 is Pg1.
In a preferred embodiment, Pg1 is benzyl, allyl or
silyl.
10 In a more preferred embodiment, Pg1 is benzyl.
In a twenty-third particular embodiment of the
invention, R1 is Pg3.
In a preferred embodiment, Pg3 is acetyl.
In a twenty-fourth particular embodiment of the
15 invention, R1 is hydrogen.
In a twenty-fifth particular embodiment of the
invention, R2 is Pg2.
In a preferred embodiment, Pg2 a carbamate or imide
amine-protecting group;
20 In more preferred embodiment, Pg2 is 2,2,2-
trichloroethoxycarbonyl.
In a twenty-sixth particular embodiment of the
invention, R2 is acetyl.
In a twenty-seventh particular embodiment of the
25 invention, Pg5 is benzyl, allyl or n-pentenyl.
In a preferred embodiment, Pg5 is benzyl.

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25
In a twenty-eighth particular embodiment of the
invention, Y is a peptide comprising 2 to 5 amino acid
residues.
In a preferred embodiment, Y' is a linear peptide.
5 In a twenty-ninth particular embodiment of the
invention, Y' is a peptide comprising 2 to 5 amino acid
residues.
In a preferred embodiment, Y' is a linear peptide.
This invention also includes all combinations of.
10 particular and preferred embodiments described herein.
Prsparatian of Compounds of Formula 1
Synthesis of a compound of formula 1, the central
disaccharide core, in orthogonally protected form presents a
15 significant synthetic challenge. For example,
identification of a protective scheme hairing triple
orthogonality is highly desirable to accomplish selective
unmasking of the three types of pendant hydroxyl gxoups
(i.e., anomeric OH, peripheral OH, and carboxyl OH). In
20 addition, the stereoselective construction of the β-[1,4]
glycosidic linkage is expected to be difficult irrespective
of the method used to generate a reactive glycosyl cation
donor. For example, with respect to the glycosyl cation,
each of the following inherent properties contribute to a
25 loss of reactivity of the glycosyl cation acceptor: (i) the
intrinsic lack of nucleophilicity of the C(4)-hydroxyl group

WO 01/79267 PCT/US01/12630
26
of glucopyranose-based acceptors, (ii) additional steric
crowding around the C(4)-hydroxyl of muratnic acid-based
acceptors, and (iii) additional electronic deactivation of
2-deoxy-2-acylaminoglucopyranose acceptors relative to their
5 glucopyranose-based counterparts. With respect to the
glycosyl cation donor, an activation method with ei
predisposition toward formation of a p-[l,4] glycosidic
linkage is needed. The reaction conditions for glycosyl
cation generation also needs to be compatible with
10 functionality resident in both the donor and acceptor.
A compound of formula 1, wherein the variables are as
described herein, may be prepared by coupling a murainylamide
compound of formula 2, wherein the variables are as

described herein, with a glucopyranosyl compound of formula
3, wherein the variables are as described herein, under
appropriate conditions. Particular conditions include
carrying out the coupling reaction under scrupulously
20 anhydrous Konigs-Knorr conditions (e.g., in a silver
triflate/dichloromethane solution including molecular
sieves), or the like.

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A compound of formula 2 is prepared according to the
procedures described in Imoto, M., Bull. Chem. Soc. Jpn.,
60, 2205 (1987) .
A compound of formula 3, wherein the variables are as
5 described herein, may be prepared by coupling an acid of
formula 4, wherein the variables are as described herein,

with a protected amino acid/peptide compound of formula 5,
10 wherein Y is as described herein, under appropriate
conditions. Particular conditions encompass carrying out
the coupling reaction in a solution of NMM (or the like) and
2-chloro-4, 6-dimethoxy-l, 3,5'triazine (or the like) in
CH2CI2 (or the like) , wherein the compound of formula 5 is
15 added as the tosylate salt, or the like.
A compound of formula 3a, wherein Pg1 is benzyl and the
other variables are as described herein, may be prepared by
treating a compound of formula 6, wherein the variables are


WO 01/79267 PCMJS01/12630
28
as defined herein, with a reducing agent under appropriate
conditions. A particular reducing agent is triethylsilane,
or the like. Particular conditions include carrying out the
reduction in CH2C12 (or the like) and TFA (or the like) at
5 about 0 °C. This reaction provides an efficient means for
regioselective introduction of the benzyl
protection/activation at C(6)OH of the muramic acid
derivative.
It is known that when an ester derivative of a compound
10 of formula 6 is treated with trifluoroacetic acid euid
triethylsilane as described in DeNinno, M.P., et al.,
Tetrahedron Lett., 36, 669 (1995), only a small amount of
the analogous compound of formula 3a is observed. The major
product formed is a lactone, as shown in scheme I.

Scheme .1
The acid-catalyzed lactonization proceeds at a rate
competitive with the reductive ring opening, thus leading to
the undesired lactone. In the present invention, however,
20 introduction of an amide bond in place of the ester bond
eliminates the conversion to the lactone, thus allowing the
desired product (compound 3a) to be isolated in much higher
yields.

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A compound of formula 6, wherein the variables are as
described herein, may be prepared by coupling an acid of
formula 4a, wherein the variables are as described herein.

with a protected amino acid/peptide compound of formula 5,
wherein Y is as described herein, under appropriate
conditions. Particular conditions encompass carrying out
10 the coupling reaction in a solution of NMM (or the like) and
2-chloro-4, 6-dimethoxy-l, 3, 5-triazine (or the like) in
CH2CI2 (or the like) , wherein the compound of formula 5 is
added as the tosylate salt, or the like.
The synthesis of the core disaccharide outlined above
15 provides a high throughput access to intermediates in the
total synthesis of the Ovchinnikov glycopeptides. Due to the
convergent approach, protecting group economy, and
crystalline nature of the intermediates, one can easily
scale the process to substantial or commercial volumes.
20
Preparation of Compounds of Formula I

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I. Direct Attachment of the Amino Acid/Peptide Portion
A Compounds of formula I, wherein Y' is the unprotected
form of Y, may be prepared by removing the Pg5 group of a
compound of formula 7, wherein the variables are as
5 described herein, in the presence of a hydroxy-deprotecting
agent and under appropriate conditions.

A particular Pg5 group is benzyl, or the like. A particular
10 hydroxy-deprotecting agent is H2/(Pd/C), or the like.
Particular hydroxy-deprotecting conditions encompass
carrying out the deprotection in an alcohol solvent (e.g.,
methanol, ethanol or the like) at about room temperature.
A compound of formula 7, wherein the variables are as
15 described herein, may be prepared by saponifying the Pg° and
Pg3 groups of a compound of formula 8,

8 7
wherein Pg3 is an acyl hydroxy-protecting group and the
20 other variables are as described herein, in the presence of

WO 01/79267 PCT/US01/1263O
31
a saponifying agent and under appropriate conditions. A
particular Pg° and Pg3 group is acetyl, or the like. A
particular saponifying agent is aqueous sodium hydroxide, or
the like. Particular saponifying conditions encompass
5 carrying out the saponification in an alcohol solvent (e.g.,
methanol, ethanol or the like) at about room temperature.
A compound of formula 8, wherein the variables are as
defined herein, may be prepared by depro tec ting the Y group
a compound of formula 9, wherein the variables are as .

described herein, under appropriate conditions. The
peptide-deprotecting is carried out using an appropriate
deprotecting agent that depends on the nature of the
15 peptide-protecting group, i.e., whether it is removable
(labile) under acid, base, or hydrogenation conditions, and
other reactive moieties in the compound undergoing
deprotection, i.e., a deprotecting agent is chosen to carry
out the deprotection without affecting the other reactive
20 moieties unless a concomitant reaction is desired. A
particular peptide-protecting group for a carboxylic acid
moiety is C1 to C8 alkyl; more particularly t-butyl, or the
like. A particular peptide-deprotecting agent for such a

WO 01/79267 PCT/USOl/12630
32
group is an inorganic acid; more particularly HC1, or the
like. Another particular peptide-protecting group for a
carboxylic acid moiety is -CH2CH2SO2Ph, or the like. A
particular peptide-deprotecting agent for such a group is
5 DBU (or the like), wherein particular conditions encompass
dissolving the compound of formula 9 in THF (or the like)
and adding DBU (or the like) dropwise as a THF solution (or
the like) .
A compound of formula 9, wherein the variables are as
10 described herein, may be prepared by exchanging the Pg2
group of compound of formula 10, wherein the variables are
as described herein, with an acetyl group in the presence of

15 an amine-deprotecting agent and acylating agent under
appropriate conditions. The amine-deprotecting is carried
out using an appropriate deprotecting agent that depends on
the nature of the amine-protecting group, i.e., whether it
is removable (labile) under acid, base, or hydrogenation
20 conditions, and other reactive moieties in the compound
undergoing deprotection, i.e., a deprotecting agent is
chosen to carry out the deprotection without affecting the

WO 01/79267 PCT/USO1/126JO
33
other reactive moieties unless a concomitant reaction is
desired. A particular amine-protecting group is β,β,β-
trichloroethoxycarbonyl, or the like. A particular
deprotecting agent is Zn dust (or the like) in the presence
5 of a proton source (e.g., acetic acid or the like). A
particular acylating agent is acetic anhydride, or the like.
Particular conditions include adding Zn dust (or the like)
and about a 3:2:1 mixture of THF:Ac2O:AcOH (or the like) to
a solution of the compound of formula 10 in about a 2:1
10 mixture of Ac20:Ac0H, or the like.
A compound of formula 10, wherein the variables are as
described herein, may be prepared by exchanging the Pg1
group a compound of formula 1, wherein the other variables
are as described herein, with a Pg3 group in the presence

of a solvolyzing agent and acylating agent under appropriate
conditions. The solvolysis is carried out using an
appropriate solvolyzing agent that depends on the nature of
20 the hydroxy-protecting group, i.e., a solvolyzing agent is
chosen to carry out the solvolysis without affecting the
other reactive moieties unless a concomitant reaction is

WO 01/79267 PCT/US01/1263O
34
desired. A particular hydroxy-protecting group is benzyl,
or the like. A particular solvolyzing agent is ZnCl2, or
the like. A particular acylating agent is acetic anhydride,
or the like. Particular conditions include carrying out the
5 solvolysis/acylation in about a 2:1 mixture of AC2O:AcOH, or
the like.
II. Attachment of the Amino Acid/Peptide Portion in Stages
A compound of formula I, wherein -Y' is -X'-W, X' is a
10 residue of an amino acid or peptide which forms an amide
linkage with the attached carbonyl, and W is a residue of
an andno acid or peptide, may be prepared by deprotecting
the W group of a co:npound of formula 11 in the presence of a

peptide-deprotecting agent under appropriate conditions.
The peptide-deprotecting is carried out using an appropriate
deprotecting agent that depends on the nature of the
peptide-protecting group, i.e., whether it is removable
20 (labile) under acid, base, or hydrogenation conditions, and
other reactive moieties in the compound undergoing
deprotection, i.e., a deprotecting agent is chosen to carry
out the deprotection without affecting the other reactive

WO 01/79267 PCT/US01/12630
35
moieties unless a concomitant reaction is desired. A
particular peptide-protecting group for a carboxylic acid
moiety is C1 to CB alkyl; more particularly t-butyl, or the
like. A particular peptide-deprotecting agent for such a
5 group is an inorganic acid; more particularly HC1, or the
like. Another particular peptide-protecting group for a
carboxylic acid moiety is -CH2CH2SO2Ph, or the like. A
particular peptide-deprotecting agent for such a group is
DBU (or the like), wherein particular conditions encompass
10 dissolving the compound of formula 11 in THF (or the like)
and adding DBU (or the like) dropwise as a THF solution, or
the like.
A compounds of formula 11 may be prepared by removing
the Pg5 group of a compound of formula 12, wherein the
15 variables are as described herein, in the presence of a
hydroxy-deprotecting agent and under appropriate conditions.

A particular protecting group is benzyl, or the like. A
20 particular hydroxy-deprotecting agent is H2/(Pd/C carbon),
or the like. Particular hydroxy-deprotecting conditions
encompass carrying out the deprotection in an alcohol

WO 01/79267 PCT/US01/1263O
36
solvent (e.g., methanol, ethanol or the like) at aibout room
temperature.
A compound of formula 12, wherein the variables are as
described herein, may be prepared by saponifying the Pg° and
5 Pg3 groups of a compound of formula 13,

wherein the variables are as described herein, in the
presence of a saponifying agent under appropriate
10 conditions. A particular saponifying agent aqueous sodium
hydroxide, or the like. Particular conditions encompass
carrying out the saponification in an alcohol solvent (e.g.,
methanol, ethanol or the like) at about room temperature.
A compound of formula 13, wherein the variables are as
15 described herein, may be prepared by coupling a compound of
formula 15, wherein -X"C(O)OL is the activated ester of -X',
-0L is a leaving group capable of substitution by an amino
nucleophile, and the other variables area as desciribed
herein, with a protected amino acid/peptide of formula 14,
20 wherein W is as defined herein, and under appropriate
conditions.

WO 01/79267 PCT/USOl/12630
37

A particular protected amino acid is γ-Obut-iso-Gln, or the
like. Particular conditions encompass adding dropwise a
5 solution of y-Obu'-iso-Gln or the like (in about a 2:1
mixture of acetonitrilerDMF, or the like) to a solution of
compound 15 (in acetonitrile or the like), followed
immediately by diisopropylethyl amine, or the like.
A compound of formula 15, wherein the variables are as
10 described herein, may be prepared by esterifying an acid of
formula 17, wherein the variables are as defined herein,
with a compound of formula 16, wherein the variables are as
described herein, under appropriate conditions.

A particular compound of formula 16 is N-hydroxysuccinimide,
or the like. Particular conditions encompass forming a

WO 01/79267 PCT/USO1/1263O
38
slurry of the compound of formula 17 in acetonitrile (or the
like) , and adding EDCI (or the like) and N-
hydroxysuccinimide (or the like) to the slurry at about room
temperature.
5 A compound of formula 17, wherein the variables are as
described herein, may be prepared by deprotecting the
terminal carboxy moiety of the X group of a compound of
formula 18, wherein the variables are as described herein,
with an appropriate amino acid/peptide deprotecting agent

under appropriate conditions. The peptide-deprotecting is
carried out using an appropriate deprotecting agent that
depends on the nature of the carboxy-protecting group, i.e.,
15 whether it is removable (labile) under acid, base, or
hydrogenation conditions, and other reactive moieties in the
compound undergoing deprotection, i.e., a deprotecting agent
is chosen to carry out the deprotection without affecting
the other reactive moieties unless a concomitant reaction is
20 desired. A particular peptide-protecting group for a
carboxylic acid moiety is C1 to C8 alkyl; more particularly
t-butyl, or the like. A particular peptide-deprotecting

WO 01/79267 PCT/US01/1263Q
39
agent for such a group is an inorganic acid; more
particularly HC1, or the like. Another particular peptide-
protecting group for a carboxylic acid moiety is
-CH2CH2SO2Ph, or the like. A particular peptide-deprotecting
5 agent for such a group is DBU (or the like) , wherein
particular conditions encompass dissolving the compound of
formula 18 in THF (or the like) and adding DBU (or the like)
dropwise as a THF solution, or the like.
A compound of formula 18, wherein the variables are as
10 described herein, may be prepared by exchanging the Pg2
group of a compound of formula 19, wherein the variables are
as described herein, with an acetyl group in the presence of

15 an amine-deprotecting agent and an acylating agent under the
appropriate conditions. The amine-deprotecting is carried
out using an appropriate deprotecting agent that depends on
the nature of the amine-protecting group, i.e., whether it
is removable (labile) under acid, base, or hydrogenation
20 conditions, and other reactive moieties in the compound
undergoing deprotection, i.e., a deprotecting agent is
chosen to carry out the deprotection without affecting the

WO 0 1/79267 PCT/USO1/126J0
40
other reactive moieties unless a concomitant reaction is
desired. A particular amine-protecting group is β,β,β-
trichloroethoxycarbonyl, or the like. A particular
deprotecting agent is Zn dust (or the like) in the presence
5 of a proton source (e.g., acetic acid or the like). A
particular acylating agent is acetic anhydride, or the like.
Particular conditions include adding Zn dust (or the like)
and about a 3:2:1 mixture of THF:Ac2O:AcOH (or the like) to
a solution of the compound of formula 8 in about a 2:1
10 mixture of Ac2O:AcOH, or the like.
A compound of formula 19, wherein the variables are as
described herein, may be prepared by exchanging the Pgr1
group of a compound of formula la, wherein the variables are
as described herein, with a Pg3 group in the presence of a

solvolyzing agent and acylating agent under appropriate
conditions. The solvolysis is carried out using an
appropriate solvolyzing agent that depends on the nature of
20 the hydroxy-protecting group, i.e., a solvolyzing agent is
chosen to carry out the solvolysis without affecting the
other reactive moieties unless a concomitant reaction is

WO 01/79267 PCT/US01/12630
41
desired. A particular hydroxy-protecting group is benzyl,
or the like. A particular solvolyzing agent is ZnCl2 or
the like. A particular acylating agent is acetic anhydride,
or the like. Particular conditions include carrying out the
5 solvolysis/acylation in about a 2:1 mixture of AC20:Ac0H, or
the like.
It is understood that the process described above can
be modified so that the peptide portion can attached in
three or more stages.
10
EXAMPLES
General
Reactions are carried out with continuous stirring
under a positive pressure of nitrogen except where noted.
5 Dilutions/solutions of liquids are shown as volume:volume.
Reagents and solvents are purchased and used without further
purification. TLC is performed with 0.25 mm silica gel 60
plates with a 254 nm fluorescent indicator from E. Merck.
Plates are developed in a covered chamber and visualized by
20 ultraviolet light or by treatment with 5% phosphomolybdic
acid in ethanol followed by heating. Flash chromatography
is carried out with silica gel 60, 230-400 mesh (0.040-0.063
mm particle size) purchased from EM Science. HPLC analyses
and purifications are performed using Dynamax C8 columns
25 with the specified solvent system and flow rate. NMR
spectra are reported as chemical shifts in parts-per-million

WO 01/79267 PCT/US01/12630
42
(ppm) downfield from a tetramethylsilane internal standard
(0 ppm) . 1H NMR spectra are recorded in the solvent
indicated on either a Bruker Avance spectrometer at 500.18
MHz, a Varian Mercury spectrometer at 400.21 MHz, or a GE
5 QE-300 spectrometer at 300.15 MHz. 13C NMR spectra are
recorded in the solvents indicated on the previously
mentioned spectrometers at 125.78 MHz, 100.15 MHz, and 75.48
MHz, respectively. IR spectra are recorded on a Nicolet
510P FT-IR spectrometer; electrospray mass spectra are
10 recorded on a Micromass Platform LCZ spectrometer. High
resolution mass spectra are recorded on a Micromass QTOF
mass spectrometer.
The synthetic process for preparation of a protected
disaccharide, compound vi, is outlined in scheme II. and
15 exemplified in Example 1, both shown below.

WO 01/79267 PCT/USOI/12630
43

Example 1
5 J. Regioselective Installation of Benzyl Protection &
Attachment of Peptide Linker:

WO 01/79267 PCT/US01/12630
44

A mixture of (L)-alanine (15.0 g, 168 mmol),
phenylsulfonyl ethanol (37.6 g, 202 mmol) , and TsOH*H2O
(35.2 g, 185 mmol) in benzene (750 mL) is refluxed using a
5 Dean-Stark apparatus. After 16 h, additional phenylsulfonyl
ethanol (25 g, 135 mmol) and TsOH*H2O (25 g, 134 mmol) is
added along with benzene (180 mL), and the reaction mixture
is refluxed overnight. Concentration in vacuo gives the
product, compound i, in quantitative yield as a white solid.
10 Analytical (compound i) : 1H NMR(DMSO-d6, 300 MHz) δ
8.25(br s, impurities, TsOH), 7.94-7.88(m, 2H) , 7.81-7.74(q,
J = 6.2 Hz, 1H), 7.71-7.62(m, 2H), 7.49(d, J= 8.1 Hz, 1H),
7.12(d, J = 8.1 Hz, 1H), 5.39(br s, 2H), 4.52-4.44(m, 1H),
4.41-4.33(m, 1H) , 3.90-3.82 (m, 1H), 3.78(t, J = 5.5 Hz, 2H),
15 3.67(t, J = 6.2 Hz, 1H), 3.44(t, J = 6.6 Hz, 1H), 2.29(s, 3H
+ impurities, TsOH), 1.20(d, J = 7.3 Hz, 3H) 13C NMR (DMSO-
d6, 75 MHz) 6 169.5, 145.5, 139.3, 137.7, 134.1, 133.6,
129.5, 129.3, 128.0, 127.7, 127.6, 125.5, 58.9, 57.5, 54.9,
53.6, 47.7, 20.7, 15.2: MS(ESI) m/z 258.1 (100%, M-TsOH-H);
20 IR KBr) vmax 3424(br), 2927(br), 1745 (m) , 1309(m), 1224 (m) ,
1195 (m), 1147(s), 1124(m), 1087 (m), 1007(m) cm"1; Anal.
l

WO 01/79267 PCI7US01/12630
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Calcd for C18H25NO4S: C, 50.10; H, 5.84; N, 3.25; S, 14.86.
Found: C, 48.49; H, 5.31; N, 2.56; S, 14.72.
To a slurry of benzyl W-acetyl-4,6-benzylidine muramic
acid (20.0 g, 42.5 mmol) in CH2C12 (300 mL) at 0° C is added
5 N-methylmorpholine (NMM) (4.67 mL, 42.5 mmol) and 2-chloro-
4,6-dimethoxy-l,3/5-triazine (8.94 g, 51.0 mmol). After
stirring for 45 min at 0° C, CH2C12 (300 mL) followed by NMM
(9.34 mL, 83.0 mmol) and L-alanine(phenylsulfonylethyl
ester, tosylate salt) (15.4 g, 51.0 mmol) (i.e., compound i)
10 are added to the above reaction mixture. The resulting
solution is slowly warmed to room temperature and stirred
for 3 days. The reaction mixture is then filtered. The
filtrate is washed first with IN HCl then with brine, and
dried (MgSO4) . The filtrate is then concentrated under
15 reduced pressure, evaporated with toluene(x2), and vacuum
dried overnight to afford the product, compound ii, (23.5 g,
95%) as a white solid.
Analytical (compound ii) : 1H NMR (CDC13, 400 MH2:) δ
7.44(d, J = 3.0 Hz, 2H), 7.35(m, 8H) , 6.95(d, J = 6Hz, 1H),
20 6.15(d, J = 6.0 Hz, 1H), 5.85(m, 1H), 5.47(s, 1H), 5.21{dd,
J = 3.0, 12.0 Hz, 1H), 5.30(dd, J = 3.0, 15.0 Hz, 1H),
4.90(d, J = 3.0 Hz, 1H), 4.72(d, J = 12.0 Hz, 1H), 4.60 (m,
2H), 4.42 (m, 2H), 4.30-4.20(m, 2H), 4.15(q, J= 3.0 Hz, 1H),
4.00(q, J = 3.0 Hz, 1H) , 3.82(m, 1H). , 3.75(d, J = 9.0 Hz,
25 1H), 3.65(m, 1H), 1.93(s, 3H), 1.43(d, J= 3.0, 9.0 Hz, 3H),

WO 01/79267 PCT/US01/12630
46
1.38 (d, J = 3.0, 9.0 Hz, 3H) ; 13C NMR (CDC13, 75 MHz) δ
173.2, 172.2, 170.6, 131.6, 129.0, 128.9, 128.4, 128.3,
125.9, 101.4, 97.5, 81.7, 78.3, 76.6, 75.6, 75.1, 70.1,
68.9, 65.8, 64.1, 63.2, 55.3, 53.2, 48.1, 23.0, 17..4, 17.8.
5 MS (ESI) m/z 583.2 (86%, M+H) , 581.3(100%, M-H) ; IR Vmax
(CHC13) 3010(m), 1740(m), 1681(s), 1616(m), 1569(s),
1523(m), 1470(m), 1377(s), 1333 (m) , 1119 (m) , 1090 (m) cm-1,
Anal. Calcd for C31H38N2O9: C, 63.90; H, 6.57; N, 4.81.
Found: C, 63.78; H, 6.55; N, 4.89.
10 Triethylsilane (16.4 mL, 103 mmol) is added to a
solution of compound ii (12.0 g, 20.6 mmol) in CH2C12 (150
mL) at 0° C, followed by dropwise addition of TFA (8.1 mL,
103 mmol). The mixture is allowed to stir for 5 h, after
which an additional 3 equivalents of TFA (5.0 mL) is added
15 dropwise, and stirred at 0° C overnight. Upon completion of
the reaction, as evidenced by TLC (EtOAc), the reaction
mixture is diluted with CH2C12. then NaHCCO3 is added slowly
to neutralize the TFA. The aqueous layer is extracted with
CH2Cl2. The organic layer is washed with brine (x2) , then
20 dried (MgSO4) , and concentrated in vacuo. Purification by
prep-LC (eluting with 70:30 EtOAc:hexane to EtOAc), followed
by recrystallization from CH2Cl2 and isopropyl ether gives
the product, compound iii, (7.4 g, 61%) as a white solid.
Analytical (compound iii) : 1H NMR (CDC13, 300 MHz) δ
25 7.38-7.26(m, 10H), 6.99 d, J = 7.3 Hz, 1H), 6.16(d, J = 8.8

WO 01/79267 PCT/US01/12630
47
Hz, 1H), 5.94-5.81(m, 1H) , 5.30(dd, J= 1.1, 17.2 Hz, 1H),
5.22(dd, J = 1.1, 10.6 Hz, 1H) , 4.92{d, J = 3.7 Hz, 2H) ,
4.68(t, J = 11.7 Hz, 1H), 4.59(d, J = 7.7 Hz, 4H), 4.49(q, J
= 6.2 Hz, 1H), 4.46(dd, J = 2.2, 11.7 Hz, 2H), 4.21(dq, J=
5 3.7, 9.9 Hz, 1H), 4.17(q, J= 7.0 Hz, 1H), 3.83-3.75(m, 1H),
3.71(t, J = 5.1 Hz, 1H) , 3.68-3.65(m, 1H) , 3.54(t, J = 10.2
Hz, 1H) , 1.89(s, 3H) , 1.44(d, J = 7.0 Hz, 3H) , 1.40 (d, J =
7.0 Hz, 3H) ; 13C NMR(CDCl3, 75 MHz) 5 173.0, 172.3, 170.3,
167.7, 137.8, 137.1, 131.7, 128.6, 128.5, 128.1, 127.8,
10 127.7, 118.5, 97.1, 80.5, 77.7, 73.7, 71.6, 70.5, 70.2,
69.8, 65.8, 55.1, 52.5, 48.0, 24.5, 23.3, 19.2, 17.7;
MS(ESI) m/z 585.2 (100%, M+H), 583.2 (100%, M-H); IR
Vmax(CHCl3) 3433(m), 3010(m) , 1741(m), 1677(s), 1522(m),
1454 (m), 1124 (m), 1058 (m) cm-1; Anal. Calcd for C32H4oN209:
15 C, 63.68; H, 6.90; N, 4.79; Found: C, 63.67; H, 6.58; N,
4.83.
II. Glycosidation


WO 01/79267 PCTAJS01/12630
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Compound iv is prepared using the procedures described
in Imoto, M., Bull. Chem. Soc. Jpn., 60, 2205 (1987).
To a solution of compound iii (4.59 g, 6.43 mmol) in
CH2CI2 (30 mL) are added 4A molecular sieves (10 g) and
5 silver triflate (5.12 g, 20.0 nunol) . To this mixture is
added a solution of freshly prepared compound iv (10.8 g,
20.0 mmol) in CH2CI2 (9.5 mL) in four portions over a 1 h
period. Each of the starting materials is dried prior to
use, and the reaction is performed under controlled
10 anhydrous conditions. After stirring at room temperature
for 24 h, the reaction mixture is filtered through Celite
and washed with CH2Cl2. The organic layer is washed with
NaHCO3, brine, dried (Na2SO4) , and concentrated in vacuo.
Purification by column chromatography on silica (Flash Elute
15 system) utilizing a solvent gradient of 50% hexane in EtOAc,
15% hexane in EtOAc, EtOAc, and 5% MeOH in EtOAc yields the
product, compound v, (5.73 g, 76%) as a white solid, along
with unreacted starting material, compound iii, (630 mg,
14%) .
20 Analytical (compound v): 1H NMR(CDC13, 300 MHz)
67.91(d, J = 7.0 Hz, 2H), 7.66(t, J = 7.3 Hz, 1H), 7.58-
7.50(m, 4H), 7.45(t, J= 7.3 Hz, 2H), 7.33-7.26(m, 6H),
6.83{d, J = 7.3 Hz, 1H), 6.52(d, J = 7.0 Hz, 1H), 5.09(d, J
= 2.9 Hz, 1H), 4.97(t, J = 9.5 Hz, 1H), 4.87(d, J = 12.1 Hz,
25 1H) , 4.79-4.73(m, 2H), 4.60(dd, J=7.3, 12.1 Hz, 2H), 4.53-

WO 01/79267 PCT/US01/126J0
49
4.29(m, 5H), 4.26-4.04(m, 7H), 4.00-3.88(m, 2H) , 3.70-
3.50(m, 4H) , 3.42(t, J= 10.6 Hz, 4H) , 2.03(s, 3H) , 1.98(s,
6H) , 1.89(8. 3H), 1.34(d, J = 6.6 Hz, 3H), 1.24(d, J = 7.3
Hz, 3H) ; 13C NMR(CDC13, 75 MHz) 5173.4, 171.8, 170.6, 170.3,
5 169.4, 154.1, 137.3, 134.0, 129.4, 129.1, 128.5, 128.1,
100.0, 97.1, 96.9, 77.4, 77.0, 76.6, 75.7, 74.5, 73.8, 72.2,
71.2, 70.4, 70.3, 68.3, 67.2, 61.5, 58.1, 26.2, 54.9, 53.6,
47.7, 23.2, 20.6, 18.3, 17.5; MS (FAB) m/z 1176.3 (73%,
M+H) , (ESI) m/z 1174.5 (62%, M-H) IR (KBr) V^ 3385 (br) ,
10 3067(w), 2939(w), 1753(s), 1669m), 1537(m), 1233(s),
1145 (m), 1045 (s) cm-1; UV-vis (95% EtOH) λmax 264
(1223.11)nm; Anal. Calcd for C51H64Cl3N302oS: C, 52.02; H,
5.48; N, 3.57; S, 2.72; Cl, 9.03. Found: C, 51.72; H, 5.40;
N, 3.64; S, 2.72; Cl, 9.07.
15
III. Protective Group Interchange

20 To a solution of compound v (1.9 g, 1.57 mmol) in
Ac2O:AcOH (2:1, 11 mL) is added a solution of ZnCl2 (2.1 g,

WO 01/79267 PCT/US01/12630
50
15.7 mmol) in Ac2O:AcOH (2:1, 5 mL) in one portion. Upon
completion of the reaction (24 h) as judged by TLC (EtOAc) ,
Troc is removed by adding Zn dust (4.1 g, 62.8 mmol) and a
mixture of THF:AC2O:AcOH (3:2:1, 25 mL) to the above
5 reaction mixture and stirring until no starting material is
evidenced by TLC (EtOAc). The reaction mixture is filtered
through Celite, washed with EtOAc, and then concentrated
under reduced pressure. The residue is repeatedly
evaporated with toluene to remove any remaining AC2O and
10 AcOH, and then diluted with EtOAc. The organic layer is
washed with NaHCO3 (x2), H2O (x2) , and brine. The organic
layer is then dried (Na2SO4) and concentrated in vacuo.
Purification via column chromatography on silica (Flash
Elute system) eluting with 2% MeOH in EtOAc affords the
15 product, compound vi,(1.0 g, 67%) as a white solid.
Analytical (compound vi) : 1H NMR (CDCl3, 300 MHz) δ
7.89(d, J = 7.0 Hz, 2H) , 7.66(t, J = 7.3 Hz, 1H) , 7..56(t, J
= 7.7 Hz, 2H), 7.34-7.23(m, 6H) , 7.16(d, J = 7.7 Hz, 1H),
6.88(d, J = 7.0 Hz, 1H), 6.12(d, J = 9-5 Hz, 1H), 5.12-
20 5.07(m, 3H), 4.56 (dd, J = 12.1, 40.0 Hz, 2H), 4.45(d, J =
9.0 Hz, 1H), 4.39(d, J= 8.4 Hz, 1H), 4.35-4.23(m, 4H),
4.17(d, J- = 12.0 Hz, 2H) , 4.09-3.95{m, 3H) , 3.78(d, J = 5.5
Hz, 2H), 3.60-3.48(m, 3H), 3.41-3.30(m, 2H), 2.12(s, 3H),
2.01(s, 3H), 2.00(s, 3H), 1.99(s, 3H) , 1.94(s, 3H), 1.92(s,
25 3H),1.38 (d, J = 6.6 Hz, 3H) , 1.28(d, J = 7.3 Hz, 3H) ; 13C

WO 01/792*7 PCT/US01/J2d30
51
NMR(CDG13 75 MHz) 8173.8, 171.9, 171.2, 170.9, 170.8,
170.6, 169.3, 139.2, 137.3, 134.1, 129.4, 128.9, 128.5,
128.1, 128.0, 127.8, 100.2. 96.9, 77.1, 76.6, 75.9, 75.6,
72.5, 71.8, 70.2, 69.5, 68.2, 62.3, 61.6, 58.0, 54.9, 54.6,
5 53.6, 47.8, 23.2, 23.1, 20.9, 20.6, 18.4, 17.3; MS (ESI)
m/2 994.7 (100%, M-H); IR (KBr) Vmax 3384 (br), 3301 (br),
3068(w), 2939 (w), 1748 (s), 1670(s), 1540(m), 1372 (m) ,
1236(s), 1144{m), 1041(s) cm-1; Anal. Calcd for C45H61N302oS:
C, 54.26; H, 6.17; N, 4.22; S, 3.22. Found: C, 53.96; H,
10 5.78; N, 4.17; S, 3.09.
Scheme III and Example III, both shown below,
illustrate the synthesis of GMDP from compound vi.

WO 01/79267 PCT/USO1/12630
52

5 Example 2
I. Preparation of compound vii

WO 01/79267 PCT/US01/12630
53

5 The phenylsulfonyl ester, compound vi, (549 mgr, 0.52
mMol) is dissolved in THF (15 mL). After stirring
commences, DBU (90 \iL, 0.6 mMol) is added dropwise as, a THF
solution (5 mL). After 1.5 h, TLC analysis (10% MeOH in
chloroform) indicates complete conversion to a new product
10 at lower Rf. The reaction mixture is partitioned between
EtOAc and 1 N HC1. The organic phase is layered with water
and, with vigorous stirring, the pH is adjusted to 8.8 with
2 N NaOH (meter) . In a similar manner, the basic aqueous
phase is layered with chloroform, the system is stirred
15 vigorously while the pH is adjusted to 1.5 with concentrated
HC1. The acidic aqueous phase is extracted again with
chloroform. The combined chloroform solutions are dried
(MgSO4) and concentrated to give the desired disaccharide
acid, compound vii, as a colorless solid (412 mg, 96%) .
20 ESI-MS (negative ion) = 824.8.
II. Preparation of compound ix

WO 01/79267 PCT/US01/12630
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The disaccharide acid, compound vii, (1.02 g, 1.24
5 mMol) is slurried in acetonitrile. N-hydroxysuccinimide
(156 mg, 1.36 mMol) and EDCI (261 ing, 1.36 mMol) are then
added. The system becomes homogeneous'immediately. After 4
h, TLC analysis (10% MeOH in chloroform) indicates complete
formation of the NHS active ester intermediate, i.e.,
10 compound viii. A solution of γ-Obut-iso-Gln (275 mg, 1.36
mMol) in 2:1 = acetonitirile:DMF (5 mL) is added dropwise,
followed immediately by diisopropylethyl amine (237 |1L, 1.36
mMol). After 3.5 h, TLC analysis (10% MeOH in chloroform)
indicates complete conversion of the active ester to the
15 desired glycodipeptide at Rf = 0.32. A small amount of a
very slightly higher Rf product, possibly the diastereomer
at Ala, is also observed. The reaction mixture is
partitioned between EtOAc and N HC1. The organic phase is
dried (MgSO4) and concentrated to a solid. The crude
20 product is adsorbed on silica gel (10 g) , and
chromatographed over silica gel (10 g) using an elution

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55
gradient of chloroform to 10% MeOH in chloroform. The
product thus obtained, tetraacetyl glycodipeptide (compound
ix), (colorless solid, 1.10 g, 88%) is diastereomerically
pure. ESI-MS (positive ion) = 1010.4, 1032.4
5
III. Preparation of GMPD

The tetraacetyl glycodipeptide (compound ix) (1.02 g,
10 1.0 mMol) is dissolved in dry MeOH (25 mL) . A solution of
0.5 M NaOMe in MeOH (2.0 mL, 1 mMol) is added with stirring.
The reaction mixture is stirred at room temperature until
ESI-MS analysis indicates that all four acetyl groups have
been removed, thereby forming the tetrahydroxy plycopeptide
15 (compound x) (positive ion, M+H = 843) . Dowex resin 50WX8-
400 is added portionwise with stirring until the apparent pH
(paper) reaches 4-5. The resin is removed by filtration and
the solution concentrated to a thick oil. The oil is taken
up in 0.25 M HCl in ethanol and stirred at room temperature
20 for 3 hr. Pd/C (0.5 g) is added to the reaction mixture,

WO 01/79267 PCMJS01/12630
56
and the system is brought under a hydrogen atmosphere.
After 2.5 h, the catalyst is removed by filtration and the
filtrate concentrated in vacuo. The concentrate is
lyophilized twice from saturated aqueous NH4HCO3 to afford
5 GMDP as an off-white solid (598 mg, 86%). ESI-MS (positive
ion, M+14 = 709.4; negative ion, M-H = 693.9).

X-13169
57
1. A compound of formula III

wherein:
5 R° is Pg° or hydrogen;
R1 is Pg1, Pg3, or hydrogen;
R2 is Pg2 or acetyl;
RY is Y or Y';
Pg° is an acyl hydroxy-protecting group;
10 Pg1 is a hydroxy-protecting group which is not
electron-withdrawing;
Pg2 is a an amine-protecting group which does not lead
to oxazoline formation;
Pg3 is an acyl hydroxy-protecting group;
15 Pg5 is a hydroxy-protecting group;
Pg°, Pg1, Pg2, and Pg5 are mutually orthogonal
protecting groups;
Y is a residue of an amino acid or peptide, wherein:
Y forms an amide linkage with the attached
20 carbonyl; and
Y comprises a protected terminal carboxy group;
and

X-13169
58
Y' is a residue of an amino acid or peptide, where Y'
forms an amide linkage with the attached carbonyl.
2. The compound of Claim 1, wherein R° is Pg0.
5
3. The compound of Claim 2, wherein Pg° is acetyl.
4. The compound of claim 1, wherein R° is hydrogen.
10 5. The compound of Claim 1, wherein R1 is Pg1.
6. The compound of Claim 5, wherein Pg1 is a benzyl,
allyl or silyl hydroxy-protecting group.
15 7. The compound of Claim 6, wherein Pg1 is a benzyl.
8. The compound of Claim 1, wherein R1 is Pg3.
9. The compound of Claim 8, wherein Pg3 is acetyl.
20
10. The compound of Claim 1, wherein R1 is hydrogen.
11. The compound of Claim 1, wherein R2 is Pg2.
25 12. The compound of Claim 11, wherein Pg2 is a
carbamate or imide amine-protecting group.

X-13169
59
13. The compound of Claim 12, wherein Pg2 is 2,2,2-
trichloroethoxycarbonyl.
5 14. The compound of Claim 1, wherein R2 is acetyl.
15. The compound of Claim 1, wherein Pg5 is a benzyl,
allyl, or n-pentenyl hydroxy-protecting group.
10 16. The compound of Claim 15, wherein Pg5 is benzyl.
17. The compound of Claim 1, Y wherein is a peptide
comprising 2-5 amino acid residues.
15 18. The compound of Claim 17, wherein Y is a linear
peptide.
19. A compound substantially as herein described with
reference to the given examples.
20
Dated this 30th day of March, 2007.

The stereospecific synthesis of a glycopeptide using a triply orthogonal protection
scheme is described, in particular, the synthesis of N-acetylglucosaminyl- beta -[1,4]-
N-acetylmuramylmonopeptide and derivatives thereof. The glycopeptide is useful for
the preparation of GMDP and related compounds having a glucosaminyl- beta -[1,4]-
N-acetylmuramic acid disaccharide core.