U.S. patent application number 11/784253 was filed with the patent office on 2007-10-11 for processes for chemical synthesis of lipochitooligosaccharides.
Invention is credited to Subramaniam Sabesan.
Application Number | 20070238872 11/784253 |
Document ID | / |
Family ID | 38521085 |
Filed Date | 2007-10-11 |
United States Patent
Application |
20070238872 |
Kind Code |
A1 |
Sabesan; Subramaniam |
October 11, 2007 |
Processes for chemical synthesis of lipochitooligosaccharides
Abstract
Processes for the synthesis of lipochitooligosaccharides were
developed. A fully acylated oligoglucosamine precursor is prepared
and reacted with a glucosamine monomer that has an amine protecting
phthaloyl group. With removal of the phthaloyl group, a fatty acid
may be added on the terminal glucosamine unit, forming a
lipochitooligosaccharide. The processes can be adapted for use on a
commercial scale.
Inventors: |
Sabesan; Subramaniam;
(Wilmington, DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
38521085 |
Appl. No.: |
11/784253 |
Filed: |
April 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60790429 |
Apr 7, 2006 |
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Current U.S.
Class: |
536/53 ;
536/55.3 |
Current CPC
Class: |
C07H 3/06 20130101; C07H
13/06 20130101 |
Class at
Publication: |
536/053 ;
536/055.3 |
International
Class: |
C08B 37/00 20060101
C08B037/00 |
Claims
1. A process for synthesizing a lipochithooligosaccharide compound
having the structure: ##STR37## where individual groups R.sup.1,
R.sup.2 and R.sup.3 are independently selected from H and C.sub.1
to C.sub.20 alkyl, aryl, aralkyl, mono, di or polyalkenyl, mono, di
or polyalkynyl, groups; R.sup.4 is selected from a monosaccharide,
sulfate and phosphate; and n is from 0 to about 20; comprising: b)
combining a compound of structure C ##STR38## wherein R.sup.1 is
selected from H, C.sub.1 to C.sub.20 alkyl, aryl, and aralkyl
groups, with a compound of structure B ##STR39## where individual
groups R.sup.1 and R.sup.2 are independently selected from H and
C.sub.1 to C.sub.20 alkyl, aryl, and aralkyl groups; R.sup.4 is
selected from a monosaccharide, a sulfate and a phosphate; and n is
from 0 to about 19; in an aprotic solvent and agitating the
solution at a temperature between about 0.degree. C. to about
-78.degree. C. to form a first mixture; b) adding to the mixture of
a) a first activating agent selected from N-haloimides to form a
second mixture; c) adding a second activating agent selected from
perfluoroalkyl sulfonic acids, and optionally adding a reagent
selected from methyl perfluroalkyl sulfonates, to the second
mixture to form a third mixture; d) reacting the third mixture at a
temperature between about 0.degree. C. and about -78.degree. C. to
form a product comprising ester groups and an N-phthalimido group;
e) isolating the product of d); f) removing the ester groups and
the N-phthalimido group from the isolated product of e) forming a
de-esterified and de-N-phthalimido product; g) isolating the
product of f); h) selectively reacting the amino group of the
terminal sugar unit of the isolated product of g) with an acid or
acid halide of the formula R.sup.1COX: where X=OH or a halide, for
acids and acid halides, respectively, and R.sup.1 is selected from
H, C.sub.1 to C.sub.20 alkyl, aryl, aralkyl, mono, di or
polyalkenyl, mono, di or polyalkynyl groups; to form a
lipochitooligosaccharide; and i) isolating the
lipochitooligosaccharide.
2. The process of claim 1 wherein the first activating agent is an
N-halosuccinimide.
3. The process of claim 2 wherein the N-halosuccinimide is
N-iodosuccinimide.
4. The process of claim 1 wherein the perfluoroalkyl sulfonic acid
is in at least about a 0.25 molar equivalent amount to the compound
of formula B.
5. The process of claim 4 wherein the perfluoroalkyl sulfonic acid
is in about an equimolar equivalent amount to the compound of
formula B.
6. The process of claim 1 wherein the perfluoroalkyl sulfonic acid
is triflic acid.
7. The process of claim 1 wherein the methyl perfluroalkylsulfonate
is methyltriflate.
8. The process of claim 1 wherein the temperature of a) and d) is
about -20.degree. C. to about -70.degree. C.
9. The process of claim 1 wherein the temperature of a) and d) is
about -50.degree. C. to about -60.degree. C.
10. The process of claim 1 wherein the ester groups of the product
of e) are removed by transesterification with metal alkoxides in
alcohol and the N-phthalimido group of the product of e) is removed
by reacting with amines or diamines under refluxing conditions.
11. The process of claim 1 wherein the reacting of h) is carried
out using a base catalyst and an acid halide, or using a fatty acid
activated with a carbodiimide and N-hydroxybenztriazole.
12. A process for synthesizing a lipochithooligosaccharide compound
having the structure: ##STR40## where individual groups R.sup.1,
R.sup.2 and R.sup.3 are independently selected from: H, and C.sub.1
to C.sub.20 alkyl, aryl, aralkyl, mono, di or polyalkenyl, mono, di
or polyalkynyl, groups; R.sup.4 is selected from monosaccharides,
sulfates and phosphates; and n is from 0 to about 20; comprising:
a) providing a compound of structure D ##STR41## wherein R.sup.1 is
selected from H, and C.sub.1 to C.sub.20 alkyl, aryl, and aralkyl
groups; b) removing the ester groups and the internal N-phthalimido
groups of the compound of structure D; c) selectively reacting the
amino groups on the internal sugar units of the compound of
structure D with an acylating reagent to make an N-acyl derivative
product; d) removing the silyl group and the ester and the
N-phthalimido group on the terminal sugar unit of the N-acyl
derivative product of (c) by reacting the N-acyl derivative product
with tetra-N-alkyl ammonium fluoride followed by reacting with
amines or diamines under refluxing conditions to produce a
de-silylated and de-N-phthalimidated product; e) acylating the
terminal amino group of the de-N-phthalimidated product of (d) with
fatty acids activated with carbodiimide and N-hydroxylbenztriazole,
or an acid halide of the formula R.sup.1COX, in the presence of a
base catalyst, where X is a halide, and R.sup.1 is selected from H
and C.sub.1 to C.sub.20 alkyl, aryl, aralkyl, mono, di or
polyalkenyl, mono, di or polyalkynyl groups; to form a
lipochitooligosaccharide; and f) isolating the
lipochitooligosaccharide.
13. The process of claim 12 wherein the acylating agent of (c) is
acetic anhydride.
14. The process of claim 12 wherein the tetra-N-alkyl ammonium
fluoride of (d) is tetra-n-butyl ammonium fluoride and the diamine
is ethylene diamine attached to Merrifield resin.
15. A compound having the structure: ##STR42## where independent
groups R.sup.1 and R.sup.2 are independently selected from H and
C.sub.1 to C.sub.20 alkyl, aryl, and aralkyl groups; R.sup.4 is
selected from a monosaccharide, a sulfate and a phosphate; and n is
from 0 to about 19.
16. A composition comprising a chemically synthesized
lipochitooligosaccharide represented by the structure: ##STR43##
where individual groups R.sup.1, R.sup.2 and R.sup.3 are
independently selected from H and C.sub.1 to C.sub.20 alkyl, aryl,
aralkyl, mono, di or polyalkenyl, mono, di or polyalkynyl, groups;
R.sup.4 is selected from a monosaccharide, sulfate and phosphate;
and n is from 0 to about 20.
17. A composition comprising a chemically synthesized
lipochitooligosaccharide represented by the structure:
##STR44##
18. A composition comprising a chemically synthesized
lipochitooligosaccharide represented by the structure: ##STR45##
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to processes for chemical
synthesis of lipochitooligosaccharides, and the resulting
chemically synthesized lipochitooligosaccharides. The processes
disclosed herein allow the stepwise synthesis of low molecular
weight N-acylglucosamine oligomers having a fatty acid condensed on
the non-reducing end. The processes can be performed on a
commercial scale.
BACKGROUND
[0002] Lipochitooligosaccharides are naturally made in rhizobial
bacteria and function as nodulation factors. The nodulation factors
secreted from the bacteria elicit a response in the root cells of
legumes that leads to symbiotic nodule formation in the roots. In
these nodules nitrogen is fixed, and is provided as a nutrient to
the plant. The extent of legume root nodulation is directly linked
to plant growth and productivity.
[0003] The nodulation factor lipochitooligosaccharides have a
backbone of four or five 1,4-linked N-acylated glucosamine
residues, a structure also found in chitin
(poly-[1-4]-.beta.-N-acetyl-D-glucosamine). This backbone is
N-acylated and can carry diverse substitutions at both ends,
depending on the rhizobial species in which it is made. In some
rhizobia the N-acylation of the terminal unit is with fatty acids
of general lipid metabolism such as vaccenic acid (C18:1.DELTA.11Z)
and in other rhizobia the N-acylation is with polyunsaturated fatty
acids such as C20:3 and C18:2.
[0004] The nodulation factor lipochitooligosaccharides made in any
one species of bacteria are a mixture of compounds having different
substitutions that are not possible to completely separate. Some
nodulation factor lipochitooligosaccharides have been chemically
synthesized. There are various reported methods for making small
samples of lipochitooligosaccharides, for example as described in
Nicolaou et al., J. Am. Chem. Soc. 114: 8701-8702 (1992); Ikeshita
et al., Carbohydrate Research C1-C6 (1995); and Wang et al., J.
Chem. Soc. Perkin Trans. 1: 621-628 (1994).
[0005] There remains a need for a process to make the
lipochitooligosaccharide class of N-acylglucosamine oligomers in
larger quantities and economically. The present invention is
related to these and other ends.
SUMMARY OF THE INVENTION
[0006] One aspect of the present invention is a process for
synthesizing a lipochithooligosaccharide compound having the
structure: ##STR1## where individual groups R.sup.1, R.sup.2 and
R.sup.3 are independently selected from H and C.sub.1 to C.sub.20
alkyl, aryl, aralkyl, mono, di or polyalkenyl, mono, di or
polyealkynyl, groups; R.sup.4 is selected from a monosaccharide,
sulfate and phosphate; and n is from 0 to about 20; comprising:
[0007] a) combining a compound of structure C ##STR2## wherein
R.sup.1 is selected from H , C.sub.1 to C.sub.20 alkyl, aryl, and
aralkyl groups, with a compound of structure B ##STR3## where
individual groups R.sup.1 and R.sup.2 are independently selected
from H and C.sub.1 to C.sub.20 alkyl, aryl, and aralkyl groups;
R.sup.4 is selected from a monosaccharide, a sulfate and a
phosphate in a suitably protected form; and n is from 0 to about
19; in an aprotic solvent and agitating the solution at a
temperature between about 0.degree. C. to about -78.degree. C. to
form a first mixture; [0008] b) adding to the mixture of a) a first
activating agent selected from N-haloimides to form a second
mixture; [0009] c) adding a second activating agent selected from
perfluoroalkyl sulfonic acids, and optionally adding a reagent
selected from methyl perfluroalkyl sulfonates, to the second
mixture to form a third mixture; [0010] d) reacting the third
mixture at a temperature between about 0.degree. C. and about
-78.degree. C. to form a product comprising ester groups and an
N-phthalimido group; [0011] e) isolating the product of d); [0012]
f) removing the ester groups and the N-phthalimido group from the
isolated product of e) forming a de-esterified and de-N-phthalimido
product; [0013] g) isolating the product of f); [0014] h)
selectively reacting the amino group of the terminal sugar unit of
the isolated product of g) with an acid or acid halide of the
formula R.sup.1COX: where X=OH or a halide, for acids and acid
halides, respectively, and R.sup.1 is selected from H, C.sub.1 to
C.sub.20 alkyl, aryl, mono, di or polyalkenyl, mono, di or
polyalkynyl groups; to form a lipochitooligosaccharide; and [0015]
i) isolating the lipochitooligosaccharide.
[0016] Another aspect of the present invention is a process for
synthesizing a lipochithooligosaccharide compound having the
structure: ##STR4## where individual groups R.sup.1, R.sup.2 and
R.sup.3 are independently selected from: H, and C.sub.1 to C.sub.20
alkyl, aryl, aralkyl, mono, di or polyalkenyl, mono, di or
polyalkynyl, groups; R.sup.4 is selected from monosaccharides,
sulfates and phosphates; and n is from 0 to about 20; comprising:
[0017] a) providing a compound of structure D ##STR5## wherein
R.sup.1 is selected from H , and C.sub.1 to C.sub.20 alkyl, aryl,
and aralkyl groups; [0018] b) removing the ester groups and the
internal N-phthalimido groups of the compound of structure D;
[0019] c) selectively reacting the amino groups on the internal
sugar units of the compound of structure D with an acylating
reagent to make an N-acyl derivative product; [0020] d) removing
the silyl group and the ester and the N-phthalimido group on the
terminal sugar unit of the N-acyl derivative product of (c) by
reacting the N-acyl derivative product with tetra-N-alkyl ammonium
fluoride followed by reacting with amines or diamines under
refluxing conditions to produce a de-silylated and
de-N-phthalimidated product; [0021] e) acylating the terminal amino
group of the de-N-phthalimidated product of (d) with fatty acids
activated with carbodiimide and N-hydroxylbenztriazole, or an acid
halide of the formula R.sup.1COX, in the presence of a base
catalyst, where X is a halide, and R.sup.1 is selected from H and
C.sub.1 to C.sub.20 alkyl, aryl, aralkyl, mono, di or polyalkenyl,
mono, di or polyalkynyl groups; to form a lipochitooligosaccharide;
and [0022] f) isolating the lipochitooligosaccharide.
[0023] Another aspect of the present invention is a compound having
the structure: ##STR6## where individual groups R.sup.1 and R.sup.2
are independently selected from H and C.sub.1 to C.sub.20 alkyl,
aryl, groups; R.sup.4 is selected from a monosaccharide, a sulfate
and a phosphate; and n is from 0 to about 19.
[0024] A further aspect of the present invention is a composition
comprising a chemically synthesized lipochitooligosaccharide
represented by the structure: ##STR7##
DETAILED DESCRIPTION
[0025] The present invention provides processes for synthesizing
multigram to kilogram quantities of low molecular weight
N-acylglucosamine polymers (oligo N-acylglucosamines) having a
fatty acid condensed on the non-reducing end, called
lipochitooligosaccharides, that are scalable for commercial use.
The processes allow the use of simple purification procedures and
do not require cost prohibitive chromatographic separation
procedures. The oligo N-acylglucosamine portion of a
lipochitooligosaccharide is made by efficient coupling of monomers
that are stable to storage. Stepwise addition of a specific type of
monomer, described herein below, to a growing polymer chain results
in the synthesis of a defined chain length polymer, to which a
fatty acid is joined. The glucosamine monomer units are added to
each other one at a time, giving the opportunity to select each
glucosamine unit in an oligomer and allowing the incorporation of a
desired acyl group, including that of a fatty acid, to a
glucosamine unit of choice, thus enabling the synthesis of a large
array of analogs for biological evaluation.
[0026] Also provided are intermediates having the structure:
##STR8## where individual groups R.sup.1 and R.sup.2 are
independently selected from H and C.sub.1 to C.sub.20 alkyl, aryl,
and aralkyl groups; R.sup.4 is selected from a monosaccharide, a
suitably protected sulfate and a phosphate group, each of which is
in a suitably protected form; and n is from 0 to about 19. Since
each glucosamine unit is added to the chain separately, as
described herein below, the individual R.sup.1 or R.sup.2 group on
each glucosamine unit may be different. The intermediates are
useful in synthesizing the lipochitooligosaccharides.
[0027] When an amount, concentration, or other value or parameter
is recited herein as either a range, preferred range or a list of
upper preferable values and lower preferable values, the recited
amount, concentration, or other value or parameter is intended to
include all ranges formed from any pair of any upper range limit or
preferred value and any lower range limit or preferred value,
regardless of whether such ranges are separately disclosed. Where a
range of numerical values is recited herein, unless otherwise
stated, the range is intended to include the endpoints thereof, and
all integers and fractions within the range. It is not intended
that the scope of the invention be limited to the specific values
recited when defining a range.
[0028] Unless otherwise stated, the following terms, as used
herein, have the following meanings.
[0029] The term "shelf stable," as used herein, means that the
compound remains intact with storage at room temperature and when
exposed to moisture and air of laboratory storage conditions.
[0030] The term "large scale" refers to tens of grams to kilogram
quantities of material.
[0031] The term "low molecular weight polymer" refers to a chain of
monomer units that is greater than one unit and up to about 50
units in length. Oligomers are polymers with two to about 22 units.
Therefore an oligo-N-acylglucosamine, for example, is a type of low
molecular weight polymer.
[0032] The term "linkage position" means the position of the carbon
that is a part of the glycosyl bond. In 1,4-linkages, the linkage
position is 1on one glycoside and 4 on the linked glycoside.
[0033] The term "non-linkage position" means the position of a
carbon which is not a part of the glycosyl bond. For example, in a
1,4 linkage, the 2, 3 and 6 positions are non-linkage
positions.
[0034] The term "thioglycoside donor" means the glycosyl molecule
that participates at the C-1 position in the glycosyl bond.
[0035] The term "glycosyl acceptor" means the glycosyl molecule
that has a hydroxyl group at the position that will participate in
the glycosyl bond, and that connects through its oxygen to the C-1
glycosyl residue from the donor. In a .beta.1,4-linkage the
glycosyl acceptor has a hydroxyl group at the 4 position. The
glycosyl acceptor may be a single unit or a multiple unit chain
that is a low molecular weight polymer.
[0036] The term "suitably protected thioglycoside donor" means a
thioglycoside that has protecting groups at the positions that
become non-linkage positions following formation of the glycosidic
linkage. Protecting groups are used to prevent reaction at those
sites.
[0037] The term "suitably protected glycoside acceptor" means a
glycoside that has protecting groups at the positions that become
non-linkage positions following formation of the glycosidic
linkage. Protecting groups are used to prevent reaction at those
sites.
[0038] One embodiment of the present invention includes processes
for synthesizing compounds of Structure A: ##STR9## where
individual groups R.sup.1, R.sup.2 and R.sup.3 are independently
selected from H and C.sub.1 to C.sub.20 alkyl, aryl, aralkyl, mono,
di or polyalkenyl, mono, di or polyalkynyl, groups; R.sup.4 is
selected from a monosaccharide, sulfate and phosphate; and n is
from 0 to about 20. Since each glucosamine unit is added to the
chain separately, as described herein below, the individual
R.sup.1, R.sup.2 or R.sup.3 group on each glucosamine unit may be
different.
[0039] In preferred embodiments, the synthesis of compounds
disclosed herein, including those of Structure A, are synthesized
in sufficiently high yields and with adequate efficiency that
allows the processes to be carried out on a commercial scale.
[0040] In one process for synthesizing compounds of Structure A, an
oligo N-acylglucosamine precursor is synthesized, to which a fatty
acid is added forming the R.sup.3 group in Structure A. This
synthesis is made possible by preparing a fully acylated oligo
N-acylglucosamine of Structure B, then adding in .beta.1,4-linkage
an N-phthaloyl protected glucosamine monomer through glycosylation
with thioglycoside Compound C. The ester and N-phthaloyl groups are
removed from the glycosylated product, followed by the addition of
a fatty acid to the amino group of the terminal unit to obtain
compound A.
[0041] The oligo N-acylglucosamine of Structure B to which the
terminal phthaloyl-protected glucosamine monomer is added may
consist of from 2 to about 21 glucosamine units that are joined by
.beta.1,4-linkage. ##STR10## where individual groups R.sup.1 and
R.sup.2 are independently selected from H and C.sub.1 to C.sub.20
alkyl, aryl, and aralkyl groups; R.sup.4 is selected from a
monosaccharide, a sulfate group and a phosphate group, each of
which is in a suitably protected form; and n is from 0 to about 19.
Since each glucosamine unit is added to the chain separately, as
described herein below, the individual R.sup.1 or R.sup.2 group on
each glucosamine unit may be different.
[0042] The N-phthaloyl protected glucosamine monomer that is to be
joined to the oligo N-acylglucosamine is shown as Structure C.
##STR11## where the R.sup.1 groups are independently selected from
H, C.sub.1 to C.sub.20 alkyl, aryl, and aralkyl groups.
[0043] The oligoglucosamine that is needed for the synthesis of
Structure B, is prepared using the process for forming glycosidic
linkages between hexoses that is described in copending U.S.
application Ser. No. 11/154457 (attorney docket no. CL2695), which
is herein incorporated by reference. The oligoglucosamine is
synthesized as follows.
[0044] A thioglycoside monomer, represented by Structure (I), is
very efficiently coupled to a position 4 glycosyl acceptor
represented by Structure (II) by using activating agents generated
from N-haloimides and an approximately equimolar amount of a strong
protic acid. ##STR12## where R.sup.1 and R.sup.2 are each
independently selected from H and C.sub.1 to C.sub.20 alkyl, aryl,
and aralkyl groups; [0045] R.sup.3 and R.sup.4 are each
independently selected from monofunctional acyl, bifunctional acyl,
phthaloyl, trichloroacetyl, and tetrachlorophthaloyl groups; [0046]
and R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are each independently
selected from C.sub.1 to C.sub.20 alkyl, aryl, and aralkyl groups.
[0047] Preferably R.sup.1 and R.sup.2 are phenyl groups. [0048]
Preferably R.sup.3 and R.sup.4 are acyl groups derived from a
phthaloyl unit. [0049] Preferably R.sup.5 is a p-toluyl group.
[0050] Preferably R.sup.6 and R.sup.7 are methyl groups. [0051]
Preferably R.sup.8 is a tertiary butyl group
[0052] The position 4 glycosyl acceptor is represented by Structure
(II): ##STR13## where R.sup.1 is selected from an acyl group and
protected glycosyl units; [0053] R.sup.2 is selected from H and
C.sub.1 to C.sub.20 alkyl, aryl, and aralkyl groups; [0054] R.sup.3
and R.sup.4 are each independently selected from monofunctional
acyl, bifunctional acyl, phthaloyl, trichloroacetyl, and
tetrachlorophthaloyl groups; and R.sup.5 is selected from C.sub.1
to C.sub.20 alkyl, aryl, and aralkyl groups. [0055] Preferably
R.sup.1 and R.sup.2 are phenyl groups. [0056] Preferably R.sup.3
and R.sup.4 are acyl groups derived from a phthaloyl unit. [0057]
Preferably R.sup.5 is a methyl group.
[0058] The glycosylation process used in the synthesis of an
oligoglucosamine precursor is illustrated as follows. A unit, e.g.,
molecule, of monomer (II), the glycosyl acceptor, provides an
initial unit onto which units of a monomer (I), the thioglycoside
donor, are added to extend the polymer chain. Monomers (I) and (II)
can be made from D-glucosamine hydrochloride, which is commercially
available. To synthesize monomer (I), for synthesis of 1,4-linked
glucosamines, the D-glucosamine hydrochloride is derivatized with a
phthaloyl group using phthalic anhydride to protect the amine
(product 2 in Example 1). The hydroxyl groups are then protected by
acetylation (product 3 in Example 2), and the product is purified
by crystallization. Next, a benzenethiol group is added to the 1
position (product 4 in Example 2) and the product is purified by
washing with protic solvents. The resulting product is deacetylated
(product 5 in Example 2), benzoyl protecting groups are added at
the 3 and 6 hydroxyl positions (product 6 in Example 2) and the
product is purified by crystallization. Finally a silicon
protecting group, referred to as a t-butyldimethylsilyl (tBDMS)
group, is added as a temporary protecting group at the 4-hydroxyl
group of product 6, creating the compound shown as monomer (I) in
Reaction 1 below (S-(p-toluyl) 4-O-(dimethyl-t-butyl
silyl)-2-deoxy-3,6-di-O-benzoyl-2-phthalimido-1-thio-.beta.-D-glucopyraos-
ide). This compound represents one example of a monomer (I), which
is a suitably protected thioglycoside donor. Each of the individual
reactions used in the preparation of monomer (I) is known to one
skilled in the art. The combination of reactions and purifications
is amenable to large scale preparation of monomer (I), which is
used in the process of the instant invention. The resulting monomer
(I) provides a building block for the synthesis of
oligoglucosamines.
[0059] One skilled in the art will know that other protecting
groups can be used in the preparation of intermediates to
glucosamine-monomer (I). For example, the amine can be protected
with monofunctional acyl, bifunctional acyl, trichloroacetyl or
tetrachlorophthaloyl groups and the hydroxyl groups can be
protected with C.sub.1 to C.sub.20 alkyl, aryl, or aralkyl groups
as a part of an ester group. Similarly, the silyl group can be any
tri-substituted silicon, substituted with, for example, C.sub.1 to
C.sub.20 alkyl, aryl, and aralkyl groups.
[0060] To synthesize monomer (II), for synthesis of 1,4-linked
glucosamine, a similar sequence of reactions as used for monomer
(I) is used. The phthaloyl derivative of D-glucosamine
hydrochloride (product 3 in Example 2) is acetylated and methylated
at the 1 position hydroxyl (product 7 in example 3), then
deacetylated at the other hydroxyls (product 8 in example 3).
Benzoyl groups are added to product 8 at the 3 and 6 positions
creating monomer (II). Alternatively, a hydroxyl-protected
monosaccharide may be added to the 6 position. Each of these
individual steps is carried out using reaction conditions well
known to one skilled in the art. The resulting monomer (II)
provides the initial unit onto which molecules prepared as for
monomer (I) are added for the synthesis of a low molecular weight
glucosamine. The compound shown in Reaction 1 below (methyl
2-deoxy-3,6-di-O-benzoyl-2-phthalimido-.beta.-D-glucopyraoside)
represents one example of a monomer (II) type compound, which is a
suitably protected glycosyl acceptor containing a hydroxyl group at
the 4-position.
[0061] One skilled in the art will know that other protecting
groups can be used in the preparation of intermediates to monomer
(II). For example, the amine may be protected with monofunctional
acyl, bifunctional acyl, trichloroacetyl or tetrachlorophthaloyl
groups and the hydroxyl groups may be protected with with C.sub.1
to C.sub.20 alkyl, aryl, or aralkyl groups as a part of an ester
group.
[0062] Through iterative glycosylation and silicon protecting group
removal from the product polysaccharide, glycosyl units can be
added to the desired length. Coupling of monomers (I) and (II), as
well as coupling of an oligoglucosamine chain+monomer (I), is
carried out using thioglycoside activating agents under saturating
substrate concentration in the reaction. The thioglycoside
activating agents are generated from N-haloimides and strong protic
acids. For example, N-halosuccinimides such as N-iodosuccinimide
and N-bromosuccinimide can be used as activating agents in
combination with strong protic acids such as triflic acid
(trifluoromethanesulfonic acid) and other perfluroalkylsulfonic
acids. Though triflic acid alone is sufficient to activate the
thioglycoside, the combined use of triflic acid and methyltriflate
(methyltrifluoromethanesulfonate) facilitates the removal of
by-products that may be detrimental to the glycosylation reaction.
Thus, while methyltriflate is not sufficient to activate the
monomer (I), the combination of triflic acid/methyltriflate
provides optimal efficiency for reaction and purification
conditions.
[0063] Use of N-halosuccinimide at 1 to 1.8 molar equivalent to
monomer (I) and approximately a molar equivalent (to monomer (II))
amount of any perfluoroalkyl sulfonic acid, of which triflic acid
is an example, together with a molar equivalent (to monomer (II))
of methyltriflate provides efficient glycosylation. Use of triflic
acid in amounts of about 0.25 to about 1.0 molar equivalent amount
can be employed for effective glycosylation. The coupling
efficiency is directly related to the ease of purification of the
desired product from starting material. Thus of particular use is
approximately a molar equivalent amount each of triflic acid and
methyltriflate, for forming a readily purifiable product. Such high
concentrations of triflic acid do not cleave the sugar molecule,
especially when the reaction is carried out at low
temperatures.
[0064] Using the above described activating agents, the coupling
reaction can be driven to quantitation, forming the glycosidic
linkage, as shown in Reaction 1. Shown is an example reaction of
glucosamine-monomer (I) and glucosamine-monomer (II) forming a
dimer low molecular weight polyglucosamine. The coupling of monomer
(I) to the glycosyl acceptor (in this case monomer (II)) is step A.
##STR14##
[0065] In addition, using a minimum amount of reaction solvent
keeps the reactants at saturation levels and at high effective
concentration, and results in more efficient glycosylation. The
activating agents are added to the glycosides and the coupling
reaction is carried out at a low temperature. Temperatures from
about 0.degree. C. to about -78.degree. C. are suitable for the
reaction. It is preferred that the temperature for the reaction be
between about -20.degree. C. and about -70.degree. C. More
preferred is that the temperature be between about -50.degree. C.
and about -60.degree. C. The reaction time is from about 15 minutes
to about 8 hours. The reaction is desirably allowed to run for a
time sufficient for all potential glycosidic linkages to be formed.
Preferred is a reaction time between about 4 and about 6 hours.
[0066] A general description of a process for coupling of monomer
(I), a suitably protected thioglycoside donor, and monomer (II), a
position 4 glycosyl acceptor, is as follows. Monomer (II) (about
1.0 eq.) and monomer (I) (at least 1 and up to about 3 eq., with
about 1-2 eq. being preferred) are dissolved in a minimum of an
aprotic solvent, such as methylenechloride, diethylether,
acetonitrile, and benzotrifluoride. The most preferred solvent is
methylenechloride. The solution is cooled to about -55.degree. C.
to -60.degree. C. under nitrogen atmosphere with agitation.
Agitation may be by any method which thoroughly mixes the
components of the solution, such as shaking or stirring. Typically,
vigorous stirring is used. Powdered N-iodosuccinimide (NIS) is
added to the cold solution. After about 15 min, a solution of a
perfluoroalkyl sulfonic acid, such as triflic acid (about 1.0 eq.)
and methyltrifluromethanesulfonate (about 1.0 eq.), dissolved in
minimum of aprotic solvent, e.g., methylenechloride, is added in
drops, while maintaining the reaction temperature under about
-60.degree. C. After the addition, the reaction mixture is
maintained at the same temperature with stirring, for about 6 hours
and then poured directly over a 1:1 mixture of saturated sodium
thiosulfate and saturated sodium bicarbonate solution. Additional
solvent such as methylenechloride is employed to dilute the
reaction mixture and provide washing of the reaction flask. The
solution is thoroughly mixed and the organic layer separated. The
organic layer is then washed sequentially with 1% to 6% bleach
solution, preferably 0.6% to 3% bleach solution, then water, and
finally with saturated sodium bicarbonate solution. The product is
recovered by concentration of the solution at reduced pressure. The
impurities are removed by dissolving the material in diethylether
or ethylacetate, followed by precipitation with n-hexane. It is to
be understood that variations known to one skilled in the art can
be introduced into the process, without departing from the scope of
the invention.
[0067] The efficiency of the described coupling reaction reduces
the level of undesired by-products and starting materials in the
reaction mixture following coupling, thereby facilitating the
removal of the existing minor impurities through selective solvent
extraction methods. There is no need for the commonly used and
expensive purification methods of silica gel chromatography,
although these methods may be used. Selective washing with organic
solvents provides a simplified purification method that is useful
for large-scale production. Solvents useful for the washing during
purification include diethylether and hexane-ethylacetate mixture.
Any combination of solvents in which the product is insoluble, but
the impurities and the by-products are soluble, may be used. This
selective extraction of impurities derived from excess monomer (I),
using solvents in which the desired product is insoluble, is a
highly preferred method for isolation of the product.
[0068] Following coupling and optional purification, chain
extension is carried out. Prior to extension of the disaccharide
product, the silicon blocking group is removed from the
polyglucosamine linkage position as shown in Reaction 2 below, step
B. The silicon group can be removed, for example, by dissolving in
minimum anhydrous tetrahydrofuran (THF), then reacting with acetic
acid (2-3 eq.) and n-tetrabutylammonium fluoride solution in THF (1
M, 2-3 eq.). The reaction progress may be monitored either by TLC
or NMR of the reaction mixture. Additional methods for removing
silicon protecting groups are well known to one skilled in the
art.
[0069] Upon completion, the reaction mixture is concentrated to
dryness, the residue dissolved in solvent such as methylenechloride
and washed with water, 1M aqueous HCl solution, 0.6%-3% bleach
solution (to remove the dark brown color), and aqueous saturated
sodium bicarbonate solution. The remaining organic layer is dried
over anhydrous magnesium sulfate and concentrated to dryness.
Purification of the product is typically accomplished by
precipitation with, for example, diethylether or an n-hexane-ethyl
acetate mixture, which ensures the removal of residual monomer from
the previous step as well as the silicon impurity. Any combination
of solvents in which the product is insoluble, but the impurities
and the by-products are soluble may be used for precipitation.
[0070] Additional monomer (I), a suitably protected thioglycoside
donor, is then added through a glycosyl bond to the unblocked
disaccharide using activating agents as described above. The
disaccharide is used in place of monomer (II), as shown in Reaction
2, according to the general coupling procedure described above.
Shown is an example reaction of a glucosamine dimer with removal of
the silicon blocking group in step B and addition of a
glucosamine-monomer (I) forming a trimer low molecular weight
polyglucosamine. The coupling of monomer (I) to the glycosyl
acceptor (in the example reaction, the glucosamine dimer) is step
A. ##STR15##
[0071] Purification by organic solvent washing is also as described
above. Further rounds of chain extension are accomplished by
silicon blocking group removal and addition of monomer (I). The
process is repeated in a stepwise manner such that the
thioglycoside and the polyglucosamine form a beta linked
polyglucosamine that has a length of x+1, wherein x is the length
of the starting polyglucosamine and 1 is one monomer unit. Since
the reaction at each step is nearly quantitative, the completion of
each step results in a product that contains more than about 80% of
molecules having a single chain length. Thus the product is
enriched in a single anomer of beta linkage oligoglucosamine
molecules.
[0072] The steps are repeated until a polyglucosamine chain is made
that is of unit length appropriate to form the precursor
oligoglucosamine used in synthesis of a lipochitooligosaccharide.
The polyglucosamine chain length may be between 2 and about 21
units. Chain lengths of between about three and about seven units
are particularly suitable for use in the present process.
[0073] The benzoyl and phthalimido protecting groups on the
precursor oligoglucosamine are then converted to their acetates.
The protecting groups are removed by methods well known by one
skilled in the art. For a polymer containing 2-5 residues, this is
carried out in a two step procedure. First, de-O-benzoylation can
be accomplished by Zemplens' method, which is well known to those
skilled in the art, using sodium methoxide in methanol. The
phthaloyl group can be removed by using an
ethylenediamine-derivatized Merrifield resin (P. Stangier, O.
Hindsgaul, Synlett. 1996, 2: 179-181), as well known to one skilled
in the art. Alternatively, removal of the benzoyl and the
phthalimido groups can be accomplished in a single step by treating
the protected product at refluxing temperature with hydrazine or
hydrazine in n-butanol, followed by selective extraction of the
product polyhexosamine with water. The single step method is
preferred for polymers of length greater than 4, due to their
incomplete de-benzoylation under Zemplens' condition and their lack
of solubility in methanol and n-butanol. The silyl protecting group
remains on the terminal 4-hydroxyl group at the chain extension
end.
[0074] The hydroxyl and the amino groups of the resulting compound
are then acylated using procedures well known to those skilled in
the art. For simple acyl groups such as an acetyl group, acylation
can be carried out by addition of pyridine and acetic anhydride,
with addition of a small amount of 4-N,N-dimethylamino pyridine, as
is well known to one skilled in the art. Anhydrides of simple acyl
groups, such as acetyl or propionyl groups, are commercially
available and are readily used. For other types of acyl groups
where anhydrides are not available, the corresponding acid
chlorides are used. It is desired that the acyl groups at the amino
groups stay permanently, as seen in the lipochitoligosaccharide
molecule, whereas the acyl groups at the hydroxyl function are
removed. Also there may be differential introduction of acyl groups
at the amino and hydroxyl functions if desired, by acylating the
highly reactive amino groups first, followed by acylation of the
hydroxyl groups by methods well known to one skilled in the
art.
[0075] The silicon blocking group is then removed from the
resulting compound as described previously in the polyglucosamine
chain extension reaction. The resulting N- and O-acyl
oligoglucosamine compound is shown as Structure B above. The
compound of Structure B is then reacted with the compound of
Structure C (which has a protecting phthaloyl group; structure
shown above). The compound of Structure C is an intermediate in the
synthesis of Monomer (I), and its preparation is as for Product 4
in Example 2, which is the same compound as shown in Structure C.
The reaction of a compound of Structure B and a compound of
Structure C is carried out as described above for coupling of
Monomers (I) and (II), as well as coupling of an oligoglucosamine
chain+Monomer (1). The resulting coupled Structures B+C product is
isolated as described above for the coupled Monomers (I)+(II)
product.
[0076] The protecting N-phthalimido group and the ester groups of
the coupled Structures B+C product are removed in a two-step
reaction, using conditions well known by one skilled in the art.
The ester groups are first removed by transesterification with
metal alkoxides in alcohol, specifically by treating the ester with
sodium methoxide in methanol. The N-phthaloyl group is then removed
by reacting with amines or diamines under refluxing conditions,
specifically by treating the de-esterified product with hydrazine
in alcoholic solvents such as methanol and ethanol, or by treating
the de-esterified product with ethylenediamine derivatized
Merrifield resin. The de-esterified product with phthalimido group
removed is isolated by extracting with water, and removing the
impurities by washing the aqueous layer with solvents capable of
extracting the impurities, such as methylene chloride. The
resulting compound has a free amino group on the terminal sugar
unit, while all other nitrogens are acylated.
[0077] Another process for making a compound having a free amino
group on the terminal sugar unit, while all other nitrogens are
acylated, may be carried out starting with a compound of structure
D: ##STR16## wherein R.sup.1 is selected from H and C.sub.1 to
C.sub.20 alkyl, aryl, and aralkyl groups.
[0078] The compound represented by structure D can be synthesized
according to the process described in the Examples herein for
synthesizing Product 13. The ester groups are removed under
transesterification conditions using metal alkoxides in alcohols
under refluxing conditions. The internal N-phthalimido groups are
removed by reacting with ethylenediamine resins. Acylation of
internal amino groups are carried out by methods well known to one
skilled in the art, followed by removing the silyl group and the
ester and the N-phthalimido group on the terminal sugar unit by
reacting with tetra-N-alkyl ammonium fluoride, followed by reacting
with amines or diamines under refluxing conditions to produce a
de-silylated and de-N-phthalimidated product containing a free
amino group on the terminal sugar unit.
[0079] The free amino group is selectively reacted with an acid or
acid halide of the formula R.sup.1COX: [0080] where X=OH or a
halide, for acids and acid halides, respectively, and [0081]
R.sup.1 is selected from H , C.sub.1 to C.sub.20 alkyl, aryl,
aralkyl, alkenyl, dienyl, and trienyl groups.
[0082] Typically the acid halide is a chloride reagent, but
bromides and iodides may also be used.
[0083] The reaction of the free amino group on the terminal sugar
of the coupled Structures B+C product, with the protecting
N-phthalimido group and the ester groups removed, and R.sup.1COX
may be performed by methods well known by one skilled in the art
(some of which are described in WO2005063784A1). For example,
reactants may be dissolved in a DMF-water mixture, or water and
methanol or ethanol mixture. When an acid halide is employed in the
reaction, base catalysts such as sodium carbonate, potassium
carbonate, bicarbonate, triethylamine, or hydroxides of alkali or
alkaline earth metals are used. When acids are employed in the
amidation reaction, it is carried out in the presence of a
carbodiimide such as ethyl-(N,N-dimethylaminopropyl)-carbodiimide
hydrochloride, and N-hydroxybenztriazole may be added to promote
the reaction. The product may be isolated by methods known to those
skilled in the art such as by filtering through an acidic resin
column, followed by drying. The reaction results in an
N-acylglucosamine compound having a fatty acid condensate linked at
the amino group of the terminal residue, having a Structure A
(shown above), which is called a lipochitooligosaccharide.
Compounds made in the present process may have one or more fatty
acid groups on internal residues as well. During the synthesis of
compounds of Structure B, the glucosamine monomer units are added
to each other one at a time, giving the opportunity to select each
glucosamine unit in an oligomer and allowing the incorporation of a
desired acyl group, including that of a fatty acid, to a
glucosamine unit of choice. Thus a fatty acid may be incorporated
with a glucosamine unit at an internal position, in addition to
adding a fatty acid to the terminal glucosamine unit.
[0084] Lipochitooligosaccharides include natural nod factors that
are signaling factors involved in nodulation of legume roots by
nitrogen fixing bacteria. Through increasing nodulation, thereby
increasing the nitrogen supply to the plant,
lipochitooligosaccharide nod factors enhance plant growth and
yield. Lipochitooligosaccharides may be used to treat the roots,
leaves, or seeds of plants. The compounds may be applied in the
soil, to plant foliage, or as a seed coating. Both legume and
non-legume plants may benefit from these treatments.
[0085] Individual lipochitooligosaccharides prepared using
processes disclosed herein may be readily tested for effects on
legume root nodulation by one skilled in the art, for example as
described in Demont-Caulet et al. (Plant Physiology 120:83-92
(1999)). Also the effectiveness of individual
lipochitooligosaccharides, prepared using processes disclosed
herein, in promoting plant growth enhancement and yield improvement
of legume and non-legume plants may be readily tested, as is well
known to one skilled in the art. Thus compounds of Structure A
which do not correspond to known natural nodulation factors, but
which have nodulation stimulating activity, plant growth enhancing
activity, or yield enhancing activity may be prepared using
processes disclosed herein and readily identified by testing for
these applications.
EXAMPLES
General Methods and Materials
[0086] Unless specified, all the reagents were purchased from
Aldrich Chemical Co (St. Louis, Mo.). Thin layer chromatography was
performed on pre-coated plates of Silica Gel 60 F.sub.254 (EM
Science) and the spots were visualized with a spray containing 5%
sulfuric acid in ethanol, followed by heating. Column
chromatography was done on silica gel 60 (230-400 mesh, EM
Science). .sup.1H NMR spectra were recorded at 500 MHz. The
hydrogen chemical shifts in organic solvents are expressed relative
to deuterated methylenechloride, with a reference chemical shift of
5.36 ppm. For solutions of compounds in deuterium oxide or
deuterated methanol, the hydrogen chemical shift values are
expressed relative to the HOD signal (4.75 ppm at 296.degree.
K.).
Example 1
Synthesis of
2-deoxy-1,3,4,6-tetra-O-acetyl-2-phthalimido-D-glucopyranose
[0087] ##STR17##
[0088] D-Glucosamine hydrochloride (compound 1, 1.0 Kg) was
suspended in methanol (5.0 L) and vigorously stirred. NaOH (184.8
g) was dissolved in minimum deionized water and added to the
D-Glucosamine/Methanol suspension. The suspension was stirred for
15 min and the insoluble material (sodium chloride) was filtered
off by vacuum filtration. The theoretical amount of NaCl formed
should be about 270 g.
[0089] To the filtrate, phthalic anhydride (342 g) was added and
the solution was stirred until most of the solid dissolved (about
30 min). This was then followed by the addition of triethylamine
(468 g) and stirred for 10 to 15 min. To the resulting clear
solution, another portion of phthalic anhydride (342 g) was added
and the mixture was allowed to stir overnight at room temperature.
Product usually began to precipitate out after two hours.
[0090] The precipitated product was filtered and the residue was
washed with minimum ice cold methanol so as to remove the yellow
color from the product. The residue was then washed three times
with acetonitrile, with enough solvent added to the filter to
completely immerse the solid, and dried at room temperature under
high vacuum. The weight of the white solid, product 2, was 954 g.
.sup.1H-NMR (D.sub.2O): 7.74-7.56 (phthalimido hydrogens), 5.42
(H-1.alpha.), 4.94 (H-1.beta.), 4.17 and 4.01 (H-6), 3.27 (CH.sub.2
of N-ethyl group), 1.35 (CH.sub.3 of N-ethyl group).
[0091] The product 2 from above (1.01 Kg, made from two batches)
was placed in a 10 liter 3 neck round bottom flask set up with an
overhead electric stirrer, an N.sub.2 inlet and an addition funnel.
Acetic anhydride (3 L) and N,N-dimethylaminopyridine (1.0 g) were
added to the flask and stirred vigorously. Pyridine (2.8 L) was
added slowly and the reaction mixture was stirred for 2 days at
room temperature. The reaction mixture was quenched with ice-water
(4 L) and the product was extracted with methylenechloride. The
organic layer was repeatedly washed with aqueous hydrochloric acid
solution, and then with saturated sodium bicarbonate solution. The
organic layer was dried over anhydrous magnesium sulfate, filtered,
and concentrated to dryness. The product was recrystallized from
hot ethanol. Weight of the recrystallized product 3 was 701 g.
.sup.1H-NMR (CD.sub.2Cl.sub.2) .delta.: 7.91-7.80 (phthalimido
hydrogens), 6.62 (H-1), 5.59 (H-3), 5.21 (H-4), 4.47 (H-2), 4.36
and 4.16 (H-6), 4.06 (H-5), 2,12, 2.06, 2.02, 1.88 (acetyl methyl
groups). Thus the above NMR chemical shift data verified the
structure of product
3,2-deoxy-1,3,4,6-tetra-O-acetyl-2-phthalimido-D-glucopyranose,
which is shown below in Example 2.
Example 2
Synthesis of Monomer (I)
[0092] Preparation of Intermediate Product 4: ##STR18##
[0093] Product 3 (464 g) was dissolved in toluene and the solvent
was evaporated. This was repeated and the remaining solid was
placed on a high vacuum line overnight.
[0094] The dried solid was dissolved in minimum methylenechloride
(ca. 600 ml), and stirred well. To this, 4-methylbenzenethiol (181
g, 1.45 mol, 1.5 eq.) was added followed by the dropwise addition
of boron trifluoride diethyl etherate (BF3-etherate; 165 g, 1.16
mol, 1.2 equivalent, over 180 min). The reaction mixture was
stirred overnight. White crystals formed in the morning when
stirring was stopped. The crystals were filtered, giving product
4A. The filtrate was diluted with methylenechloride, washed
sequentially with saturated NaHCO3 solution, water, then
bicarbonate solution, and dried giving product 4B. Both 4A and 4B
products were extensively washed with anhydrous methanol and dried
under vacuum. Since the NMR spectrums of 4A and 4B products were
identical, these two were combined (Product 4, 426.3 g).
[0095] .sup.1H-NMR (CD.sub.2Cl.sub.2) .delta.: 7.96-7.80
(phthalimido hydrogens), 7.36 & 7.13 (S-aromatic hydrogens),
5.78 (H-3), 5.69 (H-1), 5.13 (H-4), 4.33 (H-2), 4.30 & 4.12
(H-6), 3.93 (H-5), 2.36 (S--Ph--Me group), 2.13, 2.04, 1.85
(methyls of acetyl groups). Thus the NMR spectrum verified the
structure of product 4, as shown above. Preparation of Intermediate
Product 5 ##STR19##
[0096] Product 4 (350 g) was suspended in nearly 4 L of dry
methanol. To this, 35 ml of 0.5 M sodium methoxide solution was
added and the solution immediately turned basic. The suspension was
left stirring at room temperature overnight. The solid deposited
was filtered and washed with dichloromethane, giving pure Product 5
(232 g). The filtrate was neutralized with sulfonic acid resin and
concentrated to dryness. The dry solid was washed with
methylenechloride and dried, giving impure compound 5 (43.8 g).
.sup.1H-NMR (CD.sub.3OD) of pure 5 .delta.: 7.87-7.76 (phthalimido
hydrogens), 7.22 & 6.99 (S-aromatic hydrogens), 5.46 (H-1),
4.18 (H-2), 4.03 (H-3), 3.89 & 3.70 (H-6), 3.39 (H-5), 3.37
(H-4), 2.22 (S--Ph--Me group). Thus the NMR spectrum verified the
structure of product 5, as shown above. Preparation of Intermediate
Product 6 ##STR20##
[0097] Product 5 (295 g; 638 mmol) was suspended in dry toluene (1
L) and evaporated under vacuum. This procedure was repeated once
more to ensure the removal of methanol contaminant that is
detrimental to the reaction. 265 grams total was recovered. The
residue after toluene evaporation was suspended in
methylenechloride (3 L) in a 3-neck flask fitted with an overhead
stirrer and the suspension was stirred under dry nitrogen
atmosphere. The flask was cooled in an ice bath and the following
reagents were added: Pyridine=126 g, N,N-Dimethylaminopyridine=500
mg; and Benzoyl Chloride: 171 g (added by means of an addition
funnel slowly in drops over 60 min). The reaction mixture was milky
white, but began to clear when all benzoyl chloride was added. The
reaction was allowed to stir for 18 h at room temperature. The
reaction was diluted with methylenechloride and was washed with
water (2.times.), 1 M aqueous HCl (2.times.), then saturated
NaHCO.sub.3 and dried with MgSO.sub.4.
[0098] The crude product was recrystallized in 8 liters of hot
EtOH, crystals were filtered, and washed in EtOH giving Crop 6A
(225 g). The filtrate was concentrated to dryness giving Crop 6B
(131 g). A second recrystallization of Crop 6A was done to give
pure product 6 (172g). The residue (40 g) from the filtrate of the
second recrystallization had product 6 of purity greater than 95%,
as determined by NMR. Crop 6B was not further processed as NMR
analysis showed that it had a significant amount of undesired
products and was therefore recycled back to compound 5.
[0099] .sup.1H-NMR (CD.sub.2Cl.sub.2) .delta.: 8.14, 7.88, 7.69,
7.57, 7.41 (benzoate hydrogens), 7.80-7.72 (phthalimido hydrogens),
7.34 & 7.00 (S-aromatic hydrogens), 5.93 (H-3), 5.79 (H-1),
4.77 & 3.99 (H-6), 4.47 (H-2), 4.03-3.99 (H-5), 3.91 (H-4),
3.25 (OH), 2.31 (S--Ph--Me group). Thus the NMR spectrum verified
the structure of product 6, as shown above. Preparation of Monomer
(I) ##STR21##
[0100] Product 6 (171.9 g; 275.6 mmol) was dissolved in minimum
methylenechloride (350 mL) containing collidine (41.7 g; 344.5
mmol; 1.25 eq.). t-BDMS-Triflate (80.0 g; 303.1 mmol; 1.1 eq.) was
added drop-wise by addition funnel (over 50 minutes). The reaction
mixture was allowed to stir overnight. The reaction mixture was
diluted with methylenechloride and washed sequentially with
ice-cold water, 0.5 M aqueous HCL (ice cold), then aqueous
saturated NaHCO.sub.3. It was then dried with MgSO.sub.4, filtered
and concentrated to give monomer (I) as a white solid (207 g). The
product was dissolved in dry toluene and concentrated to dryness
before use in a glycosylation reaction. The 207 g of monomer (I)
product recovered was essentially equal to the theoretical yield,
calculated to be 203.4 g.
[0101] .sup.1H-NMR (CD.sub.2Cl.sub.2) .delta.: 8.16-7.41 (benzoate
hydrogens, phthalimido hydrogens), 7.30 & 6.95 (S-aromatic
hydrogens), 5.97 (H-3), 5.82 (H-1), 4.89 & 4.49 (H-6), 4.40
(H-2), 4.14 (H-4), 4.01 (H-5), 2.30 (S--Ph--Me group), 0.80
(t-butyl group on silicon), 0.09 & -0.16 (methyl groups of
silicon). Thus the NMR spectrum verified the structure of Monomer
(I), as shown above.
Example 3
Synthesis of Monomer (II)
[0102] Preparation of Intermediate Compound 7 ##STR22##
[0103] To ensure that the starting glycoside was free of EtOH
traces, compound 3 (60.0 g; 126 mmol) was dissolved in toluene and
evaporated. It was then dissolved in anhydrous CH.sub.2Cl.sub.2
(500 ml) containing MeOH (6.5 g; 202 mmol; 1.6 eq.). Tin
tetrachloride (SnCl.sub.4; 18.4 g; 70.5 mmol; 0.56 eq.) was diluted
with CH.sub.2Cl.sub.2 (25 ml) and added drop-wise. The reaction
mixture was poured over ice water and shaken well. This was
repeated once more and then the organic layer was washed twice with
aqueous saturated NaHCO.sub.3, dried with MgSO.sub.4, filtered, and
concentrated. The crude product was recrystallized from hot EtOH,
giving crystals of product 7 (43.1 g). The crude yield of 49.8 g of
product 7 was 88% of the theoretical yield, calculated to be 56.6
g, while the recrystallized product 7 yield of 43.1 g was 76%.
[0104] .sup.1H-NMR (CD.sub.2Cl.sub.2) .delta.: 7.86-7.74
(phthalimido hydrogens), 5.78 (H-3), 5.31 (H-1), 5.18 (H-4), 4.31
(H-2), 4.34 & 4.20 (H-6), 3.88 (H-5), 2.20, 2.03, 1.86 (methyls
of acetyl groups). Thus the NMR spectrum verified the structure of
product 7, as shown above. Preparation of Intermediate Product 8
##STR23##
[0105] Product 7 (141.0 g; 314 mmol) was suspended in MeOH (1000
ml), and NaOMe (0.5 M, 10 ml) was added. The methyl glycoside
product 7 did not readily dissolve in MeOH. The solution was tested
to ensure basicity. The reaction was stirred overnight. The
solution became clear. Examination of the reaction mixture by TLC
(EtOAc-Hexane-EtOH=10:20:1) indicated the disappearance of the
starting material and the formation of a polar product (near the
origin). The solution was neutralized with sulfonic acid resin,
filtered, and concentrated to dryness. Weight of the residue,
called product 8, was 105.3 g, which probably includes some
methanol.
[0106] The crude yield of 105.3 g of product 8 was essentially
equal to the theoretical yield, calculated to be 101.3 g.
.sup.1H-NMR (CD.sub.3OD) .delta.: 7.85-7.80 (phthalimido
hydrogens), 5.07 (H-1), 4.21 (H-2), 3.94 (H-3), 3.92 & 3.74
(H-6), 3.40 (H-5), 3.40 (OCH.sub.3), 3.38 (H-4). Thus the NMR
spectrum verified the structure of product 8, as shown above.
Preparation of Monomer (II) ##STR24##
[0107] Product 8 (crude; 105.3), after being evaporated with
toluene-DMF, was suspended in CH.sub.2Cl.sub.2 (500 ml). Pyridine
(61.8 g; 782 mmol; 2.5 eq.) was added first, followed by the
drop-wise addition of benzoyl chloride (88 g; 626 mmol; 2.0 eq.) to
the mixture. The reaction mixture was allowed to stir at room
temperature for 24 h. It was then diluted with CH.sub.2Cl.sub.2 and
washed sequentially with H.sub.2O, 1 M HCl (2.times.), then aqueous
saturated sodium bicarbonate solution, dried with MgSO.sub.4,
filtered, and concentrated. The product was purified by
chromatography on silica gel, using EtOAc-Hexane=3:8 as eluant. The
weight of the purified product was 116.1 g. The product was about
90% pure as determined by NMR. A portion (21.1 g) of this product
was crystallized from diethylether-hexane to obtain pure
crystalline material (13.8 g) of monomer (II).
[0108] .sup.1H-NMR (CD.sub.2Cl.sub.2) .delta.: 8.15, 7.92, 7.67,
7.56, 7.42 (benzoate hydrogens), 7.83-7.74 (phthalimido hydrogens),
5.93 (H-3), 5.40 (H-1), 4.82 & 4.72 (H-6), 4.43 (H-2),
4.03-3.92 (H-5, H-4), 3.50 (OCH.sub.3), 3.33 (OH). Thus the NMR
spectrum verified the structure of monomer (II), as shown
above.
Example 4
Synthesis of Derivatized Glucosamine Disaccharide
Structural Characterization of Oligoglucosamine Derivatives:
[0109] The structures of the coupled products described below were
confirmed by proton NMR and mass spectrometry as follows. The
chemical shifts of hydrogens H-3 and H-1 of the phthalimido
glucosamine unit appeared in proton NMR spectrum at chemical shifts
between 5 and 6.5 ppm. The hydrogen H-3 appeared as a doublet of a
doublet with a coupling constant of about 8-10 Hz. By counting the
number of these hydrogen signals, the length of the
oligoglucosamine can easily be determined, for the disaccharide to
the pentasaccharide. For oligoglucosamine derivatives of 6 and
above, the signals for these hydrogens started to overlap. However,
a sufficient number of these signals could be identified to confirm
the structure. A similar observation was seen for the anomeric
hydrogens, which appeared as a doublet with a coupling constant of
about 8-8.5 Hz, thereby confirming the .beta.-glycosidic
configuration. Furthermore, the chemical shift of H-4 in the
terminal glucosamine unit appeared around 3.5 ppm, when the
corresponding carbon carried a hydroxyl group. This was shifted to
3.7 ppm upon glycosylation at this site. Thus, H-4 could be used as
a reporter group for establishing the success of the glycosylation
reaction. Further proof of structure was obtained by MALDI and
electrospray mass spectral data of the product, which are indicated
for each compound. Synthesis of Dimer Product 9 ##STR25##
[0110] Monomer (II) (80.6 g, 109.3 mmol, 1.2 eq.) and monomer (II)
(48.4 g, 91.1 mmol), both previously evaporated with toluene once,
were dissolved in CH.sub.2Cl.sub.2 (150 mL) in a 3-necked, 500 ml
flask. 4A Molecular sieve was added (5 g). The mixture was cooled
to -60.degree. C. under nitrogen atmosphere with vigorous stirring.
After 10 min, N-lodosuccinimide (NIS; 44.3 g; 196.7 mmol; 2.2 eq.)
was added as a dry powder, followed by the drop-wise addition of a
solution of triflic acid (TfOH; 13.7 g, 91.1 mmol, 1.0 eq.) and
methyltriflate (14.9 g, 54.8 mmol, 1.0 eq.) in methylenechloride.
The reaction mixture was left at -55.degree. C. for an additional 4
hr. An additional 100 ml of the triflic acid/methyltriflate
solution was added to the reaction mixture dropwise to reduce of
the viscosity. The reaction mixture was filtered cold over a celite
pad into a filter flask containing 1:1 saturated sodium
thiosulfate-sodium bicarbonate solution that was stirred thoroughly
during the filtration. The flask and the residue on the filter were
rinsed with methylenechloride and the combined filtrate was worked
up as follows. The filtrate was poured into a separatory funnel.
The contents were thoroughly mixed, the aqueous solution separated,
and the organic layer washed one more time with saturated aqueous
sodium thiosulfate solution, followed by water, and aqueous
saturated sodium bicarbonate solution. The solution was then dried
with magnesium sulfate, filtered and concentrated. Weight of the
crude product was 111.1 g. Analytically pure sample was prepared by
subjecting the crude product to separation by silica gel
chromatography, using ethyl acetate-hexane as eluant. 1H-NMR
(CD.sub.2Cl.sub.2) .delta.: 8.17-7.19 (phthalimido and benzoate
hydrogens), 6.11 and 5.76 (2.times.H-3), 5.74 and 5.31
(2.times.H-1), 4.36 and 4.32 (2.times.H-2), 4.32 and 3.93
(2.times.H-4), 3.90 and 3.53 (2.times.H-5), 4.65, 4.38, 4.12, and
3.63 (4.times.H-6), 3.38 (OCH.sub.3), 0.68 (t-butyl), -0.12, -0.40
(2.times.CH.sub.3). Mass spec.: M. wt. Calc. 1144.37; Obs.
M+Na=1167.5. Thus the NMR spectrum verified the structure of
product 9, as shown above. The crude product as such was used in
the next step, where complete removal of the tBDMS was
accomplished.
Example 5
Removal of the Silicon Group from Disaccharide Product 9 for Chain
Extension
[0111] Preparation of Intermediate Product 10 ##STR26##
[0112] Product 9 (111.1 g) was dissolved in THF (350 ml). To this
solution, a 1 M solution of acetic acid (110 ml) and a 1 M solution
of n-tetrabutylammonium fluoride in THF (110 ml) were added and the
reaction mixture was stirred at room temperature for 3 days.
Completion of the reaction was ascertained by TLC using
EtOAC:Hex:EtOH=4:8:1 as a solvent, which indicated that the
reaction was complete. The solvent of the reaction was evaporated
on high vacuum (without heat) and the residue was dissolved in
CH.sub.2Cl.sub.2, washed sequentially with water, 1 M aqueous HCl,
10% sodium thiosulfate aqueous solution, and finally, with
saturated aqueous NaHCO.sub.3. The solution was then dried with
MgSO.sub.4, filtered and concentrated. The resulting solid was
treated with diethylether which resulted in a gluey material. The
supernatant was filtered and the gluey material was repeatedly
washed with diethylether. To the filtrate, hexane was added to
precipitate any ether soluble product and this was filtered
(Fraction B, 5.9 g). The final filtrate from ether-hexane was
concentrated to dryness (Fraction C).
[0113] The NMR spectrum indicated that Fraction B product had about
5% silicon impurity (peak around 0 ppm) along with the major
desired disaccharide. Fraction A was contaminated about 10% with
tBDMS impurities and a tetrabutylammonium derivative. Therefore,
Fraction A was resuspended in 600 ml of ether, mixed for about 10
minutes, filtered and the process was repeated once more (weight of
the solid recovered was 77.3 g). This solid was purified once more
by dissolving the product in ethyl acetate and precipitating the
product with the aid of hexane (weight of the product recovered was
71.7 g). The filtrates were combined, hexane was added to
precipitate the remaining product and additional 10.8 g of the
product was recovered. .sup.1H-NMR (CD.sub.2Cl.sub.2) .delta.:
8.12- 7.14 (phthalimido and benzoate hydrogens), 6.14 and 5.73
(2.times.H-3), 5.72 and 5.34 (2.times.H-1), 4.37 and 4.34
(2.times.H-2), 4.10 and 3.69 (2.times.H-4), 3.97 and 3.44
(2.times.H-5), 4.66, 4.18, 4.12-4.06 (4.times.H-6), 3.38
(OCH.sub.3), 3.35 (OH). Mass spec.: M. wt. Calc. 1030.98; Obs.
M+Na=1053.1. Thus the NMR spectrum verified the structure of
product 10, as shown above.
Example 6
Synthesis of Derivatized Glucosamine Trisaccharide
[0114] Synthesis of Trimer Product 11 ##STR27##
[0115] Monomer (I) (88.6 g; 120 mmol; 1.5 eq.) and product 10 (82.5
G; 80.0 mmol) were dissolved in CH.sub.2Cl.sub.2 (100 ml) in a
flask. Molecular sieve (4A, 5.0 g) was added. The flask was placed
in a -55.degree. C. water bath and stirred for 15 min. NIS (48.6 g;
216 mmol) was added as a powder to the cold solution, while
maintaining vigorous stirring. A solution of methyl triflate (13.1
g; 80 mmol; 1.0 eq.) and TfOH (12 g; 80 mmol; 1.9 eq.), both
dissolved together in CH.sub.2Cl.sub.2 (5 ml), was added to the
cold solution in drops by means of an addition funnel (over 60
min). After 6 h at -60.degree. C. to -50.degree. C., the reaction
mixture was poured over saturated sodium bicarbonate and saturated
sodium thiosulfate aqueous solution (1:1, 400 ml) contained in an
Erlenmeyer flask and thoroughly stirred. Additional
methylenechloride (200 ml) was added and the contents were
thoroughly mixed for 10 min, the aqueous solution separated, and
the organic layer washed with 0.6% aqueous bleach solution,
de-ionized water, and aqueous saturated sodium bicarbonate
solution. The solution was then dried with MgSO.sub.4, filtered and
concentrated.
[0116] To remove the excess monomer impurity from the
trisaccharide, the crude product was suspended in diethylether (600
ml), the solid thoroughly mixed and the supernatant filtered. This
process was repeated three times and the residue finally dissolved
in methylenechloride, then concentrated to dryness giving 93.5 g of
product 11. To the filtrate, about 40% volume of hexane was added
and the precipitated material filtered, redissolved in
methylenechloride and concentrated to dryness under vacuum to
obtain an additional amount of compound 11 (26.0 g). .sup.1H-NMR
(CD.sub.2Cl.sub.2) .delta. (only select hydrogen chemical shifts
are reported): 8.13-7.12 (phthalimido and benzoate hydrogens),
6.03, 5.88, and 5.62 (3.times.H-3), 5.64, 5.48, and 5.29
(3.times.H-1), 3.77 (H-4 of the terminal glucosamine unit), 3.90
(H-5 of the terminal glucosamine unit), 4.63 (H-6 of the terminal
glucosamine unit), 3.35 (OCH.sub.3), 0.64 (t-butyl), -0.18, -0.33
(2.times.CH.sub.3 of the silicon unit). Mass spec.: Exact m. wt.
Calc. 1643.49 ; Obs. M+Na=1666.3. Thus the NMR spectrum verified
the structure of product 11, as shown above.
Example 7
Removal of the Silicon Group from Trisaccharide Product 11 for
Further Chain Extension
[0117] Preparation of Intermediate 12 ##STR28##
[0118] Product 11 was dissolved in minimum THF (500 ml). To this
solution, 1 M solution of acetic acid (150 ml) and a 1 M solution
of n-tetrabutylammonium fluoride in THF (150 ml) were added and the
reaction mixture was stirred at room temperature for 3 days. The
reaction mixture was evaporated to dryness, the residue redissolved
in methylenechloride, washed sequentially with deionized water, 1M
HCl, 1% aqueous bleach solution (to remove the dark brown color),
and saturated sodium bicarbonate solution, then concentrated to
dryness.
[0119] In order to remove the nonpolar silicon and other
impurities, the solid was dissolved in minimum ethyl acetate.
Hexane was added in drops (the final solvent ratio EtOAc-Hexane was
17:14). This resulted in a gluey material. The liquid was filtered
and the gluey material redissolved in EtOAc (200 ml) and
precipitated with hexane (100 ml) as described above. Finally,
diethylether was added to solidify the gluey material and the solid
was filtered. The solid was redissolved in methylenechloride and
concentrated to dryness giving 81.4 g of product 12.
[0120] The filtrate EtOAc-Hexane-ether was concentrated to dryness.
The residue was suspended in diethylether, shaken well and
filtered. This process was repeated twice. Finally, the precipitate
was dissolved in methylenechloride and concentrated to dryness to
obtain additional product 12 (16.5 g).). .sup.1H-NMR
(CD.sub.2Cl.sub.2) .delta. (only select hydrogen chemical shifts
are reported): 8.08-7.16 (phthalimido and benzoate hydrogens),
6.03, 5.92, and 5.59 (3.times.H-3), 5.67, 5.48, and 5.29
(3.times.H-1), 3.56 (H-4 of the terminal glucosamine unit), 3.91
(H-5 of the terminal glucosamine unit), 4.63 (H-6 of the terminal
glucosamine unit), 3.35 (OCH.sub.3), 3.01 (OH), 0.64. Mass spec:
Exact m. wt. Calc. 1529.41; Obs. M+Na=1553.4. Thus the NMR spectrum
verified the structure of product 12, as shown above.
Example 8
Synthesis of Derivatized Glucosamine Tetrasaccharide
[0121] Synthesis of the Tetramer Product 13 ##STR29##
[0122] Thioglycoside monomer (I) (37.4 g; 50.7 mmol) and
trisaccharide product 12 (45.6 g; 29.8 mmol) were dissolved in
CH.sub.2Cl.sub.2 (150 ml) in a flask. Molecular sieve (4A, 10.0 g)
was added. The flask was placed in a -55.degree. C. bath and
stirred for 15 min. NIS (20.5 g; 91.25 mmol) was added as a powder
to the cold solution, while maintaining vigorous stirring. A
solution of methyl triflate (4.9 g; 29.8 mmol) and TfOH (4.5 g;
29.8 mmol), both dissolved together in CH.sub.2Cl.sub.2 (20 ml),
was added to the cold solution in drops by means of an addition
funnel (over 60 min). After 6 h, at -60.degree. C., the reaction
mixture was poured over saturated sodium bicarbonate and saturated
sodium thiosulfate aqueous solution (1:1, 400 mL) contained in an
Erlenmeyer flask and thoroughly stirred. Additional
methylenechloride (200 ml) was added and the contents were
thoroughly mixed for 10 min, the aqueous solution separated, and
the organic layer washed sequentially with 10% aqueous sodium
thiosulfate solution, 1% aqueous bleach solution, and aqueous
saturated sodium bicarbonate solution. The solution was then dried
with MgSO.sub.4, filtered and concentrated (75.1 g).
[0123] To remove the excess monomer impurity from the
tetrasaccharide, the crude product was suspended in diethylether
(600 ml), the solid thoroughly mixed and the supernatant filtered.
This process was repeated three times, and the residue finally
dissolved in methylenechloride and concentrated to dryness (13 A,
54.2 g).
[0124] To the filtrate, about 40% volume of hexane was added and
the precipitated material filtered, redissolved in
methylenechloride and concentrated to dryness giving 5.8 g of
product 13 B. NMR analysis of 13 A and 13 B indicated that these
were nearly the same and they were combined. .sup.1H-NMR
(CD.sub.2Cl.sub.2) .delta. (only select hydrogen chemical shifts
are reported): 8.09-7.03 (phthalimido and benzoate hydrogens),
6.00, 5.83, 5.76, and 5.62 (4.times.H-3), 5.62, 5.42, 5.41, and
5.27 (4.times.H-1), 3.74 (H-4 of the terminal glucosamine unit),
3.88 (H-5 of the terminal glucosamine unit), 4.60 (H-6 of the
terminal glucosamine unit), 3.33 (OCH.sub.3), 0.63 (t-butyl),
-0.19, -0.34 (2.times.CH.sub.3 of the silicon unit). Mass spec.:
Exact m. wt. Calc. 2142.62 ; Obs. M+Na=2166.4. Thus the NMR
spectrum verified the structure of product 13, as shown above.
Example 9
Conversion of Benzoyl and Phthalimido Protecting Groups to their
Acetates Synthesis of Acetylated Product 15 from 13
[0125] ##STR30##
[0126] Product 13 is dissolved in hydrazine and heated to
105.degree. C. After 20 h, the reaction mixture is concentrated to
dryness. The residue is then extensively washed with
methylenechloride to remove the by-products and 10 to give product
14.
[0127] Product 14 is dissolved in minimum amount of anhydrous
pyridine containing equal volume of acetic anhydride. A small
amount of 4-N,N-dimethylamino pyridine is added and the reaction is
stirred at room temperature for 24 h. It is then poured over
ice-water and is extracted with methylenechloride. The
methylenechloride layer is washed with ice-cold 1M aqueous
hydrochloric acid, and then saturated sodium bicarbonate solution.
It is then dried over anhydrous magnesium sulfate and concentrated
under reduced pressure to obtain product 15.
Example 10
Desilylation and Addition of Terminal Phthalimido-Glucosamine
Unit
[0128] Synthesis of De-silylated Product 16 ##STR31##
[0129] Tetrasaccharide 15 is dissolved in minimum THF followed by
the addition of 1 M solution of acetic acid in THF and 1 M solution
of tetrabutylammoniumfluoride in THF and stirred at room
temperature. Reaction progress is checked after 18 h by NMR for
completion of the reaction. The reaction mixture is evaporated to
dryness, redissolved in methylenechloride, washed sequentially with
saturated sodium thiosulfate solution, 1M HCl, and saturated sodium
bicarbonate solution, then concentrated to dryness.
[0130] To remove nonpolar silicon impurities, the solid is
dissolved in ethyl acetate (400 ml). Hexane (400 ml) is added in
drops with stirring of the precipitated material. The precipitate
is filtered and the process is repeated once more, followed by a
final washing of the solid with 1:1 EtOAc-Hexane and then is dried
to get product 16.
Glycosylation of Tetrasaccharide
[0131] Synthesis of Tetrasaccharide Product 17 ##STR32##
[0132] Thioglycoside monomer (Product 4 from Example 2; 2 mole
equivalent to product 16) and tetrasaccharide product 16 are
dissolved in minimum CH.sub.2Cl.sub.2 containing 4A Molecular
sieves. The solution is cooled to -60.degree. C. and is stirred
well. After ten minutes at -60.degree. C., NIS (3.5 mole equivalent
to pentamer 16) is added quickly. After five minutes, a solution of
triflic acid (1 equivalent) and methyl triflate (1 equivalent),
dissolved together in CH.sub.2Cl.sub.2 (20 ml), is added in drops.
The reaction mixture is left at -60.degree. C. for an additional 5
hr. The reaction mixture is poured over saturated sodium
bicarbonate and saturated sodium thiosulfate aqueous solution (1:1,
500 ml) contained in an Erlenmeyer flask and is thoroughly stirred.
Additional methylenechloride is added and the contents are
thoroughly mixed for 10 min, the aqueous solution is separated, and
the organic layer is washed sequentially with 1% aqueous bleach
solution, 10% aqueous sodium thiosulfate solution, and aqueous
saturated sodium bicarbonate solution. The solution is then dried
with MgSO.sub.4, filtered and concentrated. The residual solid is
dissolved in minimum EtOAc, and is followed by dropwise addition of
hexane. The liquid portion is filtered, and the insoluble material
is redissolved in EtOAc, then precipitated again in hexane.
Finally, diethylether is added to solidify the gluey material, and
the residue is washed with ether and dried to get product 17.
Example 11
Removal of O-acetyl and N-phthalimido Groups and Conversion to
Lipochitooligosaccharide
[0133] Synthesis of Product 18 ##STR33##
[0134] Product 17 is suspended in MeOH, then NaOMe (0.5 M) is added
and is stirred at room temperature for 2 days. The reaction is
neutralized with acidic resin, and is concentrated to dryness. The
product is then suspended in n-butanol containing
MR-ethylenediamine resin and heated to 100.degree. C. for 24 h. The
hot solution is filtered over a celite pad and is washed with 1:1
methanol-water. The combined filtrate is concentrated to dryness to
obtain product 18. Synthesis of Lipochitooligosaccharide 19:
##STR34##
[0135] Product 18 is dissolved in minimum water containing the
2E,9Z-hexadecadienoic acid for amidation of the amine group of the
terminal glucosamine unit.
Ethyl-(N,N-dimethylaminopropyl)-carbodiimide hydrochloride (1
equivalent) and N-hydroxybenztriazole (1 equivalent) are added and
stirred at room temperature overnight. The reaction mixture is
passed through a column of acidic resin and the filtrate is
concentrated to dryness to get product 19.
Example 12
Synthesis of a Lipochitooligosaccharide Tetramer
[0136] ##STR35## ##STR36##
[0137] Product tetrasaccharide 13 of Example 8 (25 g) was suspended
in anhydrous methanol (900 ml). Sodium methoxide solution (0.5 M,
20 ml) was added and the reaction mixture was stirred at room
temperature for 1 day, forming a thick white precipitate. The
reaction mixture was then heated to reflux causing all of the solid
to dissolve. After 72 h at reflux, lots of precipitate was again
formed in the reaction flask. The heating was stopped, the flask
was cooled, and the precipitated material was filtered and washed
with methanol. Weight of the precipitated product was 11.2 g. This
was identified as product 20 by proton NMR.
[0138] Product 20 (11.2 g) was refluxed in methanol (1 L)
containing ethylenediamine Merrifield resin (152 g) for 5 days. The
warm reaction mixture was then filtered and washed with methanol.
Unreacted starting material remained as solid, whereas the
methanolic filtrate contained product. This was concentrated to
dryness and the solid was suspended in methanol (175 ml) containing
acetic anhydride (6 ml) and triethylamine (6 ml), and stirred at
room temperature for 2 h. A white precipitate formed in the flask,
which was filtered. The filtrate was treated with H+resin (10 g),
filtered, and concentrated to dryness to get product 21 (6.7 g).
This was identified as product 21 by proton NMR.
[0139] Product 21 was suspended in tetrahydrofuran (100 ml), and 1M
solutions of acetic acid and tetrabutylammonium fluoride (5 ml
each) were added. After 24 h of stirring at room temperature the
mixture remained cloudy. N,N-dimethylformamide (10 ml) was added to
assist in dissolving the product, and the reaction was stirred at
65.degree. C. for 3 days and then concentrated to dryness. The
resulting product was then suspended in methanol (100 ml) and
ethylenediamine Merrifield resin (25 g) was added. The reaction was
heated to 75.degree. C. and stirred for 44 h. The reaction was
allowed to cool to room temperature and filtered. The filtrate
which contained product 22 was concentrated to dryness (2.2 g).
This was identified as product 22 by proton NMR.
[0140] A solution of the fatty acid C18:1 or C16:1 (0.37 g) in
N,N-dimethylformamide (10 ml) containing EDCI (0.28 g) and
HOBt-H.sub.2O (0.20 g) was added to a suspension of product 16 (1.0
g) in DMF. Additional N,N-dimethylformamide (10 ml) was added to
completely dissolve the oligomer. The reaction mixture was stirred
for 18 h at room temperature and then heated to 80.degree. C. for 2
h, resulting in the formation of a thick gel. The gel was diluted
with methanol and the gelatinous material containing the product
(product 23 from C18:1 and product 24 from C16:1) was filtered. The
residue was repeatedly washed with methanol, followed by washing
with water. The residue on the filter paper was collected and dried
to obtain product 23 (520 mg) or product 24 (552 mg). This was
identified as product 23 and 24 by proton NMR.
* * * * *