U.S. patent application number 09/795943 was filed with the patent office on 2001-07-12 for method for the production of sialylated oligosaccharides.
Invention is credited to Palcic, Monica Marija, Sujino, Keiko.
Application Number | 20010007760 09/795943 |
Document ID | / |
Family ID | 22516665 |
Filed Date | 2001-07-12 |
United States Patent
Application |
20010007760 |
Kind Code |
A1 |
Palcic, Monica Marija ; et
al. |
July 12, 2001 |
Method for the production of sialylated oligosaccharides
Abstract
Disclosed are methods for the enzymatic synthesis of
.alpha.-sialylated oligosaccharide glycosides. Specifically, in the
disclosed methods, .alpha.2,3-sialyltransferase is used to transfer
an analogue of sialic acid, employed as its CMP-nucleotide
derivative, to the non-reducing sugar terminus of an
oligosaccharide having a fucosyl group in the penultimate
saccharide unit to the non-reducing sugar terminus. The analogue of
sialic acid and the oligosacchairde employed in this method are
selected to be compatible with the sialyltransferase employed.
Inventors: |
Palcic, Monica Marija;
(Edmonton, CA) ; Sujino, Keiko; (Edmonton,
CA) |
Correspondence
Address: |
Gerald F. Swiss, Esq.
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
22516665 |
Appl. No.: |
09/795943 |
Filed: |
February 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09795943 |
Feb 27, 2001 |
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09146285 |
Sep 3, 1998 |
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6194178 |
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Current U.S.
Class: |
435/97 ; 435/74;
435/84; 435/85 |
Current CPC
Class: |
C12P 19/26 20130101;
C12N 9/1081 20130101; C12Q 1/48 20130101 |
Class at
Publication: |
435/97 ; 435/84;
435/85; 435/74 |
International
Class: |
C12P 019/18; C12P
019/26; C12P 019/44 |
Claims
1. A method for the enzymatic synthesis of an .alpha.-sialylated
fucosylated oligosaccharide containing a sialic acid or analogue
thereof which method comprises the steps of: a) selecting a
sialyltransferase compatible with a fucosylated oligosaccharide
having a fucose group in the non-reducing penultimate saccharide
position; b) selecting a CMP-sialic acid or an analogue thereof
which is compatible with the sialyltransferase selected in step
(a); c) contacting said CMP-sialic acid or an analogue thereof with
a fucosylated oligosaccharide of the formula 4wherein R.sub.1
represents a saccharide residue, R.sub.2 represents a saccharide
residue, and R.sub.1 and R.sub.2 together represent an acceptor for
the selected sialyltransferase; n is from 0 to about 10, Y is
selected from the group consisting of O, NH and S, and R.sub.3 is
selected from the group consisting of a protein, a lipid or an
aglycon moiety having at least one carbon atom, in the presence of
the sialyltransferase selected in (a) above under conditions
whereby the sialic acid or analogue thereof is transferred from the
CMP-sialic acid or analogue thereof to the non-reducing sugar
terminus of the fucosylated oligosaccharide so as to form an
.alpha.-sialylated fucosylated oligosaccharide containing a sialic
acid or analogue thereof.
2. The method of claim 1, wherein the aglycon moiety is selected
from the group consisting of --(A)--Z' wherein A represents a bond,
an alkylene group of from 2 to 10 carbon atoms, and a moiety of the
form --(CH.sub.2CR.sub.4R.sub.5G).sub.n(CH.sub.2CR.sub.4R.sub.5)--
wherein n is an integer equal to 0 to 5; R.sub.4 and R.sub.5 are
independently selected from the group consisting of hydrogen,
phenyl, phenyl substituted with 1 to 3 substituents selected from
the group consisting of amine, hydroxyl, halogen, alkyl of from 1
to 4 carbon atoms and alkoxy of from 1 to 4 carbon atoms, methyl,
or ethyl; and G is selected from the group consisting of a bond,
oxygen, sulphur, NH, and Z' is selected from the group consisting
of hydrogen, methyl, --OH, --SH, --NH.sub.2, --NHR.sub.6,
--N(R.sub.6).sub.2, --C(O)OH, --C(O)OR.sub.6, --C(O)NHNH.sub.2,
--C(O)NH.sub.2, --C(O)NHR.sub.6, --C(O)N(R.sub.6).sub.2, and
--OR.sub.7 wherein each R.sub.6 is independently alkyl of from 1 to
4 carbon atoms and R.sub.7 is an alkenyl group of from 3 to 10
carbon atoms.
3. A method for the enzymatic synthesis of a fucosylated and
.alpha.-sialylated oligosaccharide which method comprises the steps
of: a) selecting a sialyltransferase capable of sialylating an
oligosaccharide having a fucose group in the non-reducing
penultimate saccharide position; b) selecting a fucosyltransferase;
c) selecting a CMP-sialic acid or an analogue thereof which
compatible with the sialyltransferase selected in step (a); d)
selecting a GDP-fucose or an analogue thereof which is compatible
with the fucosyltransferase selected in step (b); e) contacting
said CMP-sialic acid or an analogue thereof and said GDP-fucose or
an analogue thereof with an oligosaccharide of the
formulaR.sub.1--R.sub.2-(saccharide).sub.n--Y--R.sub.3wherein
R.sub.1 represents a saccharide residue, R.sub.2 represents a
saccharide residue, and R.sub.1 and R.sub.2 together represent an
acceptor for the selected sialyltransferase and the selected
fucosyltransferase; n is from 0 to about 10, Y is selected from the
group consisting of O, NH and S, and R.sub.3 is selected from the
group consisting of a protein, a lipid or an aglycon moiety having
at least one carbon atom, in the presence of said sialyltransferase
and said fucosyltransferase selected in (a) and (b) above under
conditions whereby the sialic acid or analogue thereof and the
fucose or analogue thereof are transferred from the CMP-sialic acid
or analogue thereof and the GDP-fucose or analogue thereof,
respectively, to the non-reducing sugar terminus of the
oligosaccharide so as to form an .alpha.-sialylated fucosylated
oligosaccharide.
4. The method of claim 3, wherein the aglycon moiety is selected
from the group consisting of --(A)--Z' wherein A represents a bond,
an alkylene group of from 2 to 10 carbon atoms, and a moiety of the
form --(CH.sub.2CR.sub.4R.sub.5G).sub.n(CH.sub.2CR.sub.4R.sub.5)--
wherein n is an integer equal to 0 to 5; R.sub.4 and R.sub.5 are
independently selected from the group consisting of hydrogen,
phenyl, phenyl substituted with 1 to 3 substituents selected from
the group consisting of amine, hydroxyl, halogen, alkyl of from 1
to 4 carbon atoms and alkoxy of from 1 to 4 carbon atoms, methyl,
or ethyl; and G is selected from the group consisting of a bond,
oxygen, sulphur, NH, and Z' is selected from the group consisting
of hydrogen, methyl, --OH, --SH, --NH.sub.2, --NHR.sub.6,
--N(R.sub.6).sub.2, --C(O)OH, --C(O)OR.sub.6, --C(O)NHNH.sub.2,
--C(O)NH.sub.2, --C(O)NHR.sub.6, --C(O)N(R.sub.6).sub.2, and
--OR.sub.7 wherein each R.sub.6 is independently alkyl of from 1 to
4 carbon atoms and R.sub.7 is an alkenyl group of from 3 to 10
carbon atoms.
5. A method for determining the non-reducing terminus structure of
an unknown oligosaccharide acceptor, which method comprises the
steps of: a) contacting the oligosaccharide acceptor with a
sialyltransferase which is not capable of sialylating a
non-reducing terminus of an oligosacchairde having a fucose group
in the non-reducing penultimate saccharide position and determining
whether the oligosaccharide was sialylated; b) contacting the
oligosaccharide with a sialyltransferase which is capable of
sialylating a non-reducing terminus of an oligosacchairde having a
fucose group in the non-reducing penultimate saccharide position
and determining whether the oligosaccharide was sialylated; and c)
comparing the results to determine whether the non-reducing
terminus was fucosylated.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This application is directed to methods for the enzymatic
synthesis of .alpha. -sialylated oligosaccharides. Specifically, in
the methods of this invention .alpha.2,3-sialyltransferase is
employed to transfer sialic acid or an analogue thereof, employed
as its CMP-nucleotide, to the non-reducing terminus of an
oligosaccharide which oligosaccharide has a fucosyl group in the
position penultimate to the non-reducing sugar terminus of the
oligosaccharide.
[0003] 2. State of the Art
[0004] Carbohydrates and/or oligosaccharides are present on a
variety of natural and pathological glycoconjugates.sup.1. Of
particular interest are carbohydrates and oligosaccharides
containing sialic acid residues particularly at the nonreducing
sugar terminus.sup.31. Such sialic acid terminated carbohydrates
and oligosaccharides are present in a number of products which have
been implicated in a wide range of biological phenomena based, in
part, on the concept of recognition signals carried by the
carbohydrate structures and by their binding to specific
ligands.
[0005] Specifically, such sialic acid terminated carbohydrates and
oligosaccharides are believed to be receptors for the binding of
toxins.sup.4, pathogenic agents such as viruses.sup.5, and are
believed to be recognition sites for a variety of lectins,
particularly those involved in cellular adhesion.sup.6,7, etc.
[0006] Similarly, certain oligosaccharides including sialic acid
terminated oligosaccharides have been identified as capable of
suppressing a cell-mediated immune response to an antigen. The
ability of such oligosaccharides to suppress a cell mediated immune
response to an antigen is described by Venot et al..sup.3
[0007] Additionally, the presence of certain sialyl terminated
oligosaccharides in tumor-related antigens is documented in the
art.sup.1 and, in general, the structures of the oligosaccharides
present on such antigens have been modified in some way from normal
oligosaccharides so as to lead to the expression of tumor related
antigens.sup.2. The prospect of passive immunotherapy with
monoclonal antibodies directed against some sialylated
tumor-associated antigens, such as the gangliosides GD.sub.2,
GD.sub.3 and GM.sub.2, in patients with melanoma has been
investigated.sup.8,9.
[0008] The synthesis of such oligosaccharides often involves
complex chemical reactions with corresponding low yields.
Accordingly, there has been much interest in using
glycosyltransferases in synthesizing at least a part of these
molecules.
[0009] Glycosyltransferases are a highly polymorphic group of
membrane-bound enzymes of endoplasmic reticulum and Golgi bodies
that catalyze the transfer of a single monosaccharide unit from a
nucleotide donor to the hydroxyl group of an acceptor saccharide in
the biosynthesis of N-glycan (Asn-GlcNAc N-glycosidic linkage;
GlcNAc, N-acetylglucosamine) and O-glycan (Ser/Thr-GalNAc,
O-glycosidic linkage; GalNAc, N-acetylgalactosamine) moieties of
glycoproteins and glycolipids.
[0010] The eukaryotic sialyltransferases comprise a family of
glycosyltransferases that catalyze the transfer of
N-acetylneuraminic acid (NeuAc), a sialic acid (SA), from CMP-SA to
the non-reducing terminus of oligosaccharide chains of
glycoconjugates. The addition of the sialic acid normally
terminates oligosaccharide chain elongation except for polysialic
chains found on neural cell adhesion molecule and gangliosides.
[0011] Known eukaryotic sialyltransferases involved in the
synthesis of N- and O-glycan derivatives of the glycoprotien and
glycolipid are summarized in Table 1, adapted from Palcic.sup.63.
In the table, the R represents the remainder of the acceptor
glycoprotein, glycolipid or oligosaccharide chain.
1TABLE 1 EC sialyltransferase (SL) Number Linkage Synthesized
Gal(2-6)-ST (ST6N) 2.4.99.1
NeuAc.alpha.2.fwdarw.6Gal.beta.1.fwdarw.4GlcNAc-R
GalNAc.alpha.(2-6)-ST 2.4.99.4
NeuAC.alpha.2.fwdarw.6GalNAc.alpha.-R (ST6OI) Gal(2-3)-ST (ST3O)
2.4.99.4 NeuAC.alpha.2.fwdarw.3Gal.be- ta.1.fwdarw.AGalNAc.alpha.-R
Gal(2-3)-ST (ST3N) 2.4.99.6
NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3/4GlcNAc-R
GalNAc.alpha.(2-6)-ST 2.4.99.7 NeuAc.alpha.2.fwdarw.6 (ST6OII)
.vertline. NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwda- rw.3GalNAc-R
N-Ac-neuramide 2.4.99.8 NeuAc.alpha.2.fwdarw.8NeuAc.a-
lpha.2.fwdarw.Gal.beta.-R .alpha.(2-8)- sialyltransferase
Gal.beta.1-3GlcNAc-ST
NeuAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GlcNA- c-R
[0012] .alpha.2,3-sialyltransferases are useful eukaryotic enzymes
for in vitro synthesis of N-linked and O-linked sialyl derivatives
of glycoproteins, for determinations of acceptors, and other
qualitative and quantitative research of glycoproteins. However, it
was previously reported that 2,3-sialyltransferases would not
synthesize N-linked and O-linked sialyl derivatives of
glycoproteins or glycolipids where the acceptor glycoprotein or
glycolipid possessed a fucosyl derivative in the penultimate
position to the non-reducing sugar terminus of the oligosaccharide
(U.S. Pat. No. 5,374,655.sup.67). This necessitated careful
planning in the synthesis of certain fucosylated and sialylated
oligosaccharides and in some cases required that certain steps be
completed using chemical synthesis, rather than enzymatic
synthesis.
[0013] In view of the above, it would be particularly advantageous
to develop methods for the facile preparation of .alpha.-sialylated
oligosaccharides from oligosaccharides having a fucosyl derivative
in the penultimate position to the non-reducing sugar terminus of
the oligosaccharide. The present invention accomplishes this by
using an .alpha.2,3-sialyltransferase to effect efficient coupling
of sialic acid activated as its CMP-nucleotide derivative (a donor
saccharide) to a saccharide or an oligosaccharide having a fucosyl
derivative in the penultimate position of the non-reducing end of
the sugar moiety (acceptor oligosaccharide).
SUMMARY OF THE INVENTION
[0014] The present invention is directed to methods for the
synthesis of oligosaccharides, glycoproteins and glycolipids
terminated in the non-reducing sugar end by an analogue of
N-acetylneuraminic acid. In particular, the methods of this
invention employ .alpha.2,3-sialyltransfe- rases to transfer a
sialic acid or analogue thereof, activated as their CMP-nucleotide
derivatives, to the non-reducing terminus of oligosaccharide
acceptors.
[0015] Accordingly, in one of its method aspects, the present
invention is directed to a method for the enzymatic synthesis of an
.alpha.-sialylated and fucosylated oligosaccharide containing a
sialic acid or analogue thereof which method comprises the steps
of:
[0016] a) selecting a sialyltransferase compatible with a
fucosylated oligosaccharide having a fucose group in the
non-reducing penultimate saccharide position;
[0017] b) selecting a CMP-sialic acid or an analogue thereof which
is compatible with the sialyltransferase selected in step (a);
[0018] c) contacting said CMP-sialic acid or an analogue thereof
with a fucosylated oligosaccharide of the formula 1
[0019] wherein R.sub.1 represents a saccharide residue, R.sub.2
represents a saccharide residue, and R.sub.1 and R.sub.2 together
represent an acceptor for the selected sialyltransferase; n is from
0 to about 10, Y is selected from the group consisting of O, NH and
S, and R.sub.3 is selected from the group consisting of a protein,
a lipid or an aglycon moiety having at least one carbon atom, in
the presence of the sialyltransferase selected in step (a) above
under conditions whereby the sialic acid or analogue thereof is
transferred from the CMP-sialic acid or analogue thereof to the
non-reducing sugar terminus of the fucosylated oligosaccharide so
as to form an .alpha.-sialylated fucosylated oligosaccharide
containing a sialic acid or analogue thereof.
[0020] This invention is also directed to a method for the
enzymatic synthesis of a fucosylated and .alpha.-sialylated
oligosaccharide which method comprises the steps of:
[0021] a) selecting a sialyltransferase capable of sialylating an
oligosaccharide having a fucose group in the non-reducing
penultimate saccharide position;
[0022] b) selecting a fucosyltransferase;
[0023] c) selecting a CMP-sialic acid or an analogue thereof which
is compatible with the sialyltransferase selected in step (a);
[0024] d) selecting a GDP-fucose or an analogue thereof which is
compatible with the fucosyltransferase selected in step (b);
[0025] e) contacting said CMP-sialic acid or an analogue thereof
and said GDP-fucose or an analogue thereof with an oligosaccharide
of the formula
R.sub.1--R.sub.2-(saccharide).sub.n--Y--R.sub.3
[0026] wherein R.sub.1 represents a saccharide residue, R.sub.2
represents a saccharide residue, and R.sub.1 and R.sub.2 together
represent an acceptor for the selected sialyltransferase and the
selected fucosyltransferase; n is from 0 to about 10, Y is selected
from the group consisting of O, NH and S, and R.sub.3 is selected
from the group consisting of a protein, a lipid or an aglycon
moiety having at least one carbon atom, in the presence of said
sialyltransferase and said fucosyltransferase selected in (a) and
(b) above, under conditions whereby the sialic acid or analogue
thereof and the fucose or analogue thereof are transferred from the
CMP-sialic acid or analogue thereof and the GDP-fucose or analogue
thereof, respectively, to the non-reducing sugar terminus of the
oligosaccharide so as to form an .alpha.-sialylated fucosylated
oligosaccharide.
[0027] This invention is also directed to a method for determining
the non-reducing terminus structure of an unknown oligosaccharide
acceptor, which method comprises the steps of:
[0028] a) contacting the oligosaccharide acceptor with a
sialyltransferase which is not capable of sialylating a
non-reducing terminus of an oligosacchairde having a fucose group
in the non-reducing penultimate saccharide position and determining
whether the oligosaccharide was sialylated;
[0029] b) contacting the oligosaccharide with a sialyltransferase
which is capable of sialylating a non-reducing terminus of an
oligosacchairde having a fucose group in the non-reducing
penultimate saccharide position and determining whether the
oligosaccharide was sialylated; and
[0030] c) comparing the results to determine whether the
non-reducing terminus was fucosylated.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0031] The present invention is directed to the discovery that
certain .alpha.2,3-sialyltransferases will transfer compatible
analogues of sialic acid to certain oligosaccharides,
glycoproteins, and glycolipids having a fucosyl group in the
penultimate position to the non-reducing end of the sugar. This
discovery permits the synthesis of oligosaccharides
.alpha.-sialylated at the non-reducing terminus from
oligosaccharides having a fucosyl group in the penultimate position
to the non-reducing end of the sugar. This method also permits the
transfer of compatible analogues of sialic acid to the fucosylated
oligosaccharide. This invention also permits the determination of
the structure of acceptors and other qualitative and quantitive
research of glycoproteins and glycolipids.
[0032] However, prior to discussing this invention in further
detail, the following terms will first be defined.
[0033] A. Definitions
[0034] As used herein, the following terms have the definitions
given below:
[0035] The term "sialic acid" means all of the naturally occurring
structures of sialic acid including
5-acetoamido-3,5-dideoxy-D-glycero-D-- galacto-nonulopyranosylonic
acid ("Neu5Ac") and the naturally occurring analogues of Neu5Ac,
including N-glycolyl neuraminic acid (Neu5Gc) and 9-O-acetyl
neuraminic acid (Neu5,9Ac.sub.2), which are compatible with the
selected sialyltransferase. A complete list of naturally occurring
sialic acids known to date are provided by Schauer.sup.31.
[0036] Naturally occurring sialic acids which are recognized by a
particular .alpha.2,3-sialyltransferase so as to bind to the enzyme
and are then available for transfer to an appropriate acceptor
oligosaccharide structure are said to be compatible with the
sialyltransferase and are sometimes referred to herein as a
"compatible naturally occurring sialic acid".
[0037] The term "analogues of sialic acid" refers to analogues of
naturally occurring structures of sialic acid including those
wherein the sialic acid unit has been chemically modified so as to
introduce, modify and/or remove one or more functionalities from
such structures. For example, such modification can result in the
removal of an OH functionality, the introduction of an amine
functionality, the introduction of a halo functionality, and the
like. In so far as the sialic acid analogues are compatible with
the sialyltransferase, they are sometimes referred to herein as a
"compatible sialic acid analogues".
[0038] Certain analogues of sialic acid are known in the art and
include, by way of example, 9-azido-Neu5Ac, 9-amino-Neu5Ac,
9-deoxy-Neu5Ac, 9-fluoro-Neu5Ac, 9-bromo-Neu5Ac, 8-deoxy-Neu5Ac,
8-epi-Neu5Ac, 7-deoxy-Neu5Ac, 7-epi-Neu5Ac, 7,8-bis-epi-Neu5Ac,
4-O-methyl-Neu5Ac, 4-N-acetyl-Neu5Ac, 4,7-di-deoxy-Neu5Ac,
4-oxo-Neu5Ac, 3-hydroxy-Neu5Ac, 3-fluoro-Neu5Ac acid as well as the
6-thio analogues of Neu5Ac. The nomenclature employed herein in
describing analogues of sialic acid is as set forth by Reuter et
al..sup.20
[0039] Insofar as sialyltransferases are designed to transfer or
donate compatible naturally occurring sialic acids, analogues of
Neu5Ac are sometimes referred to herein as "artificial donors"
whereas the compatible naturally occurring sialic acids are
sometimes referred to herein as the "natural donors".
[0040] The term "sialyltransferase" refers to those enzymes which
transfer a compatible naturally occurring sialic acid, activated as
its cytidine monophosphate (CMP) derivative, to the terminal
oligosaccharide structures of glycolipids or glycoproteins
(collectively glycoconjugates) and include enzymes produced from
microorganisms genetically modified so as to incorporate and
express all or part of the sialyltransferase gene obtained from
another source, including mammalian sources. Numerous
sialyltransferases have been identified in the literature with the
different sialyltransferases generally being distinguished from
each other by the terminal saccharide units on the glycoconjugates
which accept the transferase..sup.64 For example,
sialyltransferases, which build the following terminal
oligosaccharide structures on glycoconjugates have been
characterized:
[0041]
.alpha.Neu5Ac(2-3).beta.Gal(1.fwdarw.3/4).beta.GlcNAc-.sup.21
[0042] .alpha.Neu5Ac(2-6).beta.Gal(1-4).beta.GlcNAc-.sup.21, 22
[0043] .alpha.Neu5Ac(2-3).beta.Gal(1-3).alpha.GalNAc-.sup.23-25
[0044] .alpha.Neu5Ac(2-6).alpha.GalNAc-.sup.26-28
[0045] .alpha.Neu5Ac(2-6).beta.GlcNAc-.sup.29, 30.
[0046] Other sialyltransferases with a variety of specificities
have been isolated from a variety of sources.
[0047] A "sialyltransferase compatible with a fucosylated
oligosaccharide having a fucose group in the non-reducing
penultimate saccharide position" means that the sialyltransferase
is capable of sialylating an oligosaccharide having a fucose group
or analogue thereof in the non-reducing penultimate saccharide
position. It has been found that the myxoma virus
.alpha.2,3-sialyltransferase, as disclosed in International Patent
Application Publication No. WO97/18302.sup.65, has this
capability.
[0048] It is contemplated that related sialyltransferases also
encoded by other genera of the sub-families of Chorodopoxvirinae,
Entomopoxvirinae and the unclassified viruses of the family of
Poxviridae will be compatible with a fucosylated
oligosaccharide.
[0049] Analogues of sialic acid activated as their
cytidinemonophosphate derivative which are recognized by a
particular sialyltransferase so as to bind to the enzyme and are
then available for transfer to an appropriate acceptor
oligosaccharide structure are said to be compatible with the
sialyltransferase and are sometimes referred to herein as a
"compatible analogue of sialic acid". Because the transfer reaction
employs a sialyltransferase, it goes without saying that an
analogue of sialic acid employed in such a reaction must be a
compatible analogue of sialic acid.
[0050] CMP-nucleotide derivative of Neu5Ac refers to the compound:
2
[0051] CMP-derivatives of analogues of sialic acid refer to those
compounds having structures similar to that above with the
exception that the Neu5Ac residue is replace with an analogue of
sialic acid.
[0052] The term "fucosyltransferase" refers to those enzymes which
transfer a compatible naturally occurring fucose, activated as its
guanosine diphosphate (GDP) derivative, to the terminal
oligosaccharide structures of glycolipids or glycoproteins
(collectively glycoconjugates) and include enzymes produced from
microorganisms genetically modified so as to incorporate and
express all or part of the fucosyltransferase gene obtained from
another source, including mammalian sources. Numerous
fucosyltransferases have been identified in the literature.
[0053] The term "analogues of fucose" refers to analogues of
naturally occurring structures of fucose including those wherein
the fucose unit has been chemically modified so as to introduce,
modify and/or remove one or more functionalities from such
structures. For example, such modification can result in the
removal of an OH functionality, the introduction of an amine
functionality, the introduction of a halo functionality, the
introduction of a sulfate or phosphate moiety, and the like.
Certain analogues of fucose are known in the art and include, by
way of example, 3-deoxy-fucose.sup.68, arabinose, C-6 modified
fucoses.sup.69 (i.e. 6-O-propyl fucose) and 3,6
dideoxy-L-galactose.sup.7- 0.
[0054] It is also contemplated that the fucose or analogues of
fucose may be transferred from other purine diphosphates including,
adenosine-5'-diphosphofucose, xanthosine-5'-diphospho-fucose,
inosine-5'-diphospho-fucose, etc..sup.71
[0055] Analogues of fucose activated as their diphosphate
derivative which are recognized by a particular fucosyltransferase
so as to bind to the enzyme are then available for transfer to an
appropriate acceptor oligosaccharide structure are said to be
compatible with the fucosyltransferase. In so far as the fucose
analogues are compatible with the fucosyltransferase, they are
sometimes referred to herein as a "compatible fucose
analogues".
[0056] The term "oligosaccharide" refers to compounds of the
formula
R.sub.1--R.sub.2-(saccharide).sub.n--Y--R.sub.3
[0057] wherein R.sub.1 represents a saccharide residue, R.sub.2
represents a saccharide residue, and R.sub.1 and R.sub.2 together
represent an acceptor for the selected sialyltransferase and the
selected fucosyltransferase; n is from 0 to about 10, Y is selected
from the group consisting of O, NH and S, and R.sub.3 is selected
from the group consisting of a protein, a lipid or an aglycon
moiety having at least one carbon atom.
[0058] The term "fucosylated oligosaccharide" refers to compounds
of the formula 3
[0059] wherein R.sub.1 represents a saccharide residue, R.sub.2
represents a saccharide residue, and R.sub.1 and R.sub.2 together
represent an acceptor for the selected sialyltransferase and the
selected fucosyltransferase; n is from 0 to about 10, Y is selected
from the group consisting of O, NH and S, and R.sub.3 is selected
from the group consisting of a protein, a lipid or an aglycon
moiety having at least one carbon atom.
[0060] Since naturally occurring oligosaccharides and fucosylated
oligosaccharides are acceptors for certain
.alpha.2,3-sialyltransferases, and are believed to be acceptors of
certain sialyltransferases in vivo, these oligosaccharides and
fucosylated oligosaccharides are sometimes referred to herein as
"natural acceptors". Contrarily, since the oligosaccharides and
fucosylated oligosaccharides employed in this invention are
sometimes different from such "natural acceptors", they are
sometimes referred to herein as "artificial acceptors". That is to
say that artificial acceptors are those oligosaccharides and
fucosylated oligosaccharides which contain a substituent at the
anomeric carbon atom of the reducing sugar which substituent is
other than hydroxyl, a protein, or a lipid capable of forming a
micelle or other large molecular weight aggregate. Accordingly, a
protein linked to the anomeric carbon atom of the reducing sugar of
the oligosaccharide or fucosylated oligosaccharide through its
aglycon moiety would be an artificial acceptor since this acceptor
contains an "artificial" unit, i.e., the aglycon linking group.
[0061] The fucosylated oligosaccharides of this invention may be
further distinguished from natural acceptors by virtue of chemical
modification(s) to one or more of the saccharide units of the
oligosaccharide. Such chemical modification could involve the
introduction and/or removal of one or more functionalities in one
or more of the saccharide unit(s). For example, such modification
can result in the removal of an OH functionality, the removal of
saccharide unit(s), the introduction of an amine functionality, the
introduction of a halo functionality, the introduction of one or
more saccharide unit(s), and the like.
[0062] In a preferred embodiment, the aglycon moiety has from 1-20
carbon atoms and, more preferably, is selected from the group
consisting of --(A)--Z' wherein A represents a bond, an alkylene
group of from 2 to 10 carbon atoms, and a moiety of the form
--(CH.sub.2CR.sub.4R.sub.5G).sub.n- (CH.sub.2CR.sub.4R.sub.5)--
wherein n is an integer equal to 0 to 5; R.sub.4 and R.sub.5 are
independently selected from the group consisting of hydrogen,
phenyl, phenyl substituted with 1 to 3 substituents selected from
the group consisting of amine, hydroxyl, halogen, alkyl of from 1
to 4 carbon atoms and alkoxy of from 1 to 4 carbon atoms, methyl,
or ethyl; and G is selected from the group consisting of a bond,
oxygen, sulphur, NH, and Z' is selected from the group consisting
of hydrogen, methyl, --OH, --SH, --NH.sub.2, --NHR.sub.6,
--N(R.sub.6).sub.2, --C(O)OH, --C(O)OR.sub.6, --C(O)NHNH.sub.2,
--C(O)NH.sub.2, --C(O)NHR.sub.6, --C(O)N(R.sub.6).sub.2, and
--OR.sub.7 wherein each R.sub.6 is independently alkyl of from 1 to
4 carbon atoms and R.sub.7 is an alkenyl group of from 3 to 10
carbon atoms. Preferably, the --(A)--Z' group defines a group
capable of being linked to a carrier or a group capable of being
derivatized to a group which is capable of being linked to a
carrier.
[0063] Preferably, the aglycon group is a hydrophobic group of at
least 2 carbon atoms and more preferably at least 4 carbon atoms.
Most preferably the aglycon group is --(CH.sub.2).sub.8COOMe.
[0064] When the aglycon group is one which is capable of being
linked to a carrier such as an antigenic carrier, the methods of
this invention are useful in preparing artificial conjugates such
as artificial antigens having one or more .alpha. -sialylated
oligosaccharide groups containing an analogue of sialic acid which
groups are pendant to the antigen.
[0065] The carrier is a low or high molecular weight,
nonimmunogenic or antigenic carrier including the linking to a
fluorescent label, a radioactive label, biotin, or a photolabile
linking arm or a moiety to be targeted. Preferably, the carrier is
an antigenic carrier and accordingly, the artificial conjugate is
an artificial antigen. In some cases it may be advantageous to
employ a non-immunogenic carrier.
[0066] On the other hand, the carrier can be a low molecular weight
carrier such as ethylene diamine, hexamethylene diamine,
tris(2-aminoethyl)amine, L lysilysine, poly-L-lysine, and polymers
of various molecular weights.
[0067] Saccharide units (i.e., sugars) useful in the
oligosaccharides described above include by way of example, all
natural and synthetic derivatives of glucose, galactose,
N-acetyl-glucosamine, N-acetyl-galactosamine, fucose, sialic acid,
3-deoxy-D,L-octulosonic acid and the like. In addition to being in
their pyranose form, all saccharide units in the oligosaccharides
are in their D form except for fucose which is in its L form.
[0068] As noted above, oligosaccharides useful in the processes
disclosed herein contain terminal units which are compatible with
the selected sialyltransferase. That is to say that such compatible
terminal units permit recognition of the oligosaccharide by a
particular sialyltransferase so that the sialyltransferase binds to
the oligosaccharide and further permits transfer of the compatible
analogue of sialic acid onto the oligosaccharide.
[0069] B. Synthesis and Methodology
[0070] Preparation of Oligosaccharides
[0071] Oligosaccharides to which the sialic acid analogue is to be
enzymatically coupled are readily prepared either by complete
chemical synthesis or by chemical/enzymatic synthesis wherein
glycosyltransferases (other than sialyltransferases) are employed
to effect the sequential addition of one or more sugar units onto a
saccharide or an oligosaccharide. Such methods are well known in
the art. For example, chemical synthesis is a convenient method for
preparing either the complete oligosaccharide glycoside; for
chemically modifying a saccharide unit which can then be chemically
or enzymatically coupled to an oligosaccharide glycoside; or for
chemically preparing an oligosaccharide glycoside to which can be
enzymatically coupled one or more saccharide units.
[0072] Chemical modifications of saccharide units are well known in
the art which methods are generally adapted and optimized for each
individual structure to be synthesized. In general, the chemical
synthesis of all or part of the oligosaccharide first involves
formation of a glycosidic linkage on the anomeric carbon atom of
the reducing sugar. Specifically, an appropriately protected form
of a naturally occurring or of a chemically modified saccharide
structure (the glycosyl donor) is selectively modified at the
anomeric center of the reducing unit so as to introduce a leaving
group comprising halides, trichloroacetimidate, thioglycoside, etc.
The donor is then reacted under catalytic conditions (e.g., a
soluble silver salt such as silver trifluoromethanesulfonate, a
Lewis acid such as boron trifluoride etherate or
trimethylsilyltrifluorom- ethanesulfonate, or thioglycoside
promoters such as methyl trifluoromethanesulfonate or
dimethyl(methylthio)sulfonium trifluoromethanesulfonate) with an
aglycon or an appropriate form of a carbohydrate acceptor which
possess one free hydroxyl group at the position where the
glycosidic linkage is to be established. A large variety of aglycon
moieties are known in the art and can be attached with the proper
configuration to the anomeric center of the reducing unit.
Appropriate use of compatible blocking groups, well known in the
art of carbohydrate synthesis, will allow selective modification of
the synthesized structures or the further attachment of additional
sugar units or sugar blocks to the acceptor structures.
[0073] After formation of the glycosidic linkage, the
oligosaccharide can be used to effect coupling of additional
saccharide unit(s) or chemically modified at selected positions or,
after conventional deprotection, used in an enzymatic synthesis. In
general, chemical coupling of a naturally occurring or chemically
modified saccharide unit to the saccharide glycoside is
accomplished by employing established chemistry well documented in
the literature. See, for example, Okamoto et al..sup.32, Ratcliffe
et al..sup.33, Abbas et al .sup.34, Paulsen.sup.35, Schmid.sup.36,
Fugedi et al..sup.37, and Kameyama et al..sup.38. The disclosures
of each of these references are incorporated herein by reference in
their entirety.
[0074] On the other hand, enzymatic coupling is accomplished by the
use of glycosyl transferases which transfer sugar units, activated
as their appropriate nucleotide donors, to specific saccharide or
oligosaccharide acceptors, generally at the non-reducing sugar
portion of the saccharide or oligosaccharide. See, for example,
Toone et al..sup.62 and U.S. Pat. No. 5,374,655.sup.67. Moreover,
it is possible to effect selected chemical modifications of the
saccharide or oligosaccharide acceptor, of the sugar donor or the
product of the enzymatic reaction so as to introduce modifications
or further modifications into the structure.
[0075] Preparation of Analogues of Sialic Acid
[0076] Certain analogues of sialic acid are well known in the art
and are prepared by chemical modification of sialic acid using
procedures well documented in the art. For example, chemically
modified Neu5Ac derivatives including 9-azido-Neu5Ac..sup.39,
various 9-amino-Neu5Ac derivatives.sup.40, 9-deoxy-Neu5Ac.sup.41,
9-fluoro-Neu5Ac.sup.42, 9-bromo-Neu5Ac.sup.43,
8-deoxy-Neu5Ac.sup.41, 8-epi-Neu5Ac.sup.44,
7-deoxy-Neu5Ac.sup.47-epi-Neu5Ac.sup.45, 7,8-bis-epi-Neu5Ac.sup.45,
4-O-methyl-Neu5Ac.sup.53, 4-N-acetyl-Neu5Ac.sup.48,
4-epi-Neu5Ac.sup.47, 4,7-di-deoxy-Neu5Ac.sup.41,
4-oxo-Neu5Ac.sup.49, 4-deoxy-Neu5Ac.sup.52,
3-hydroxy-Neu5Ac.sup.50, 3-fluoro-Neu5Ac.sup.51 acid, the product
of cleavage of the side chain at C-8 or at C-7.sup.46 as well as
the 6-thio analogues of Neu5Ac.sup.54 are reported in the
literature. Other sialic acid analogues are disclosed in U.S. Pat.
No. 5,352,6703. Chemical modification leading to other sialic acid
analogues would follow such established procedures.
[0077] Activation of Analogues of Sialic Acid to Their
CMP--Nucleotide Derivatives
[0078] The enzymatic transfer of analogues of sialic acid require
the prior synthesis (i.e., activation) of their nucleotide (CMP)
derivatives. Activation of the analogues of sialic acid is usually
done by using the enzyme CMP-sialic acid synthase which is readily
available and the literature provides examples of the activation of
various analogues of sialic acid such as 9-substituted
Neu5Ac.sup.28,39,40,55-57, 7-epiNeu5Ac.sup.58,
7,8-bis-epi-Neu5Ac.sup.58, 4-O-methyl-Neu5Ac.sup.59,
4-deoxy-Neu5Ac.sup.60, 4-acetamido-Neu5Ac.sup.48,
7-deoxy-Neu5Ac.sup.56, 4,7-dideoxy-Neu5Ac.sup.56, the 6-thio
derivatives of Neu5Ac.sup.61 and Neu5OH (KDN). Still other examples
of activated sialic acid analogues are disclosed in U.S. Pat. No.
5,352,670.sup.3.
[0079] Transfer of the Analogues of Sialic Acid to the
Oligosaccharide Acceptor
[0080] The nucleotide derivative of a compatible analogue of sialic
acid and the compatible acceptor (i.e., a fucosylated
oligosaccharide or an oligosaccharide having terminal saccharide
unit(s) on the non-reducing end which are recognized by the
selected sialyltransferase) are combined with each other in the
presence of the selected sialyltransferase compatible with a
fucosylated oligosaccharide under conditions wherein the sialic
acid or analogue thereof is transferred to the acceptor. As is
apparent, the saccharide or oligosaccharide acceptor employed must
be one which functions as a substrate of the particular
sialyltransferase employed.
[0081] In this regard, the art recognizes that artificial acceptors
are tolerated in some cases by sialyltransferases especially where
modification is in the aglycon part of the structure.
[0082] Likewise, when an analogue of sialic acid (i.e., an
artificial donor) is to be enzymatically transferred, it is
necessary that the CMP derivative of the analogue also be
recognized by the sialyltransferase. In this regard, the art
recognizes that certain sialyltransferases can tolerate some
modifications to naturally occurring sialic acids and still
transfer these analogues of sialic acid to glycoproteins or
glycolipids possessing a suitable terminal acceptor structure.
[0083] It has been found that sialyltransferases possess sufficient
recognition flexibility so as to transfer an artificial donor to an
artificial acceptor.sup.3. Such flexibility permits the facile
synthesis of a panel of oligosaccharides containing different
analogues of sialic acid at the non-reducing sugar terminus of the
oligosaccharide.
[0084] As noted above, a nucleotide derivative of a compatible
sialic acid or a compatible analogue thereof is combined with a
compatible acceptor (i.e., a saccharide or an oligosaccharide
having terminal saccharide unit(s) on the nonreducing end which are
recognized by the selected sialyltransferase) in the presence of
the sialyltransferase under conditions wherein the sialic acid or
analogue thereof is transferred to the acceptor. Suitable
conditions, known in the art, include the addition of the
appropriate sialyltransferase to a mixture of the compatible
acceptor and of the CMP-derivative of the compatible sialic acid
analogue in a appropriate buffer such as 0.1M sodium cacodylate in
appropriate conditions of pH and temperature such as at a pH of 6.5
to 7.5 and a temperature between 25.degree. and 45.degree. C.,
preferably 35.degree.-40.degree. C. for 12 hours to 4 days. The
resulting oligosaccharide can be isolated and purified using
conventional methodology comprising HPLC, ion exchange-, gel-,
reverse-phase- or adsorption chromatography.
[0085] Once formed, the .alpha.-sialylated oligosaccharide
glycoside can be further modified by chemical and/or enzymatic
means to further derivatize this compound. For example, other
glycosyltransferases can be used to add a glycosyl group to an
.alpha.-sialylated oligosaccharide recognized by the transferase.
This latter aspect is important insofar as the modifications made
to the oligosaccharide must be compatible with the desired
enzymatic transfers.
[0086] Additionally, the .alpha. sialylated oligosaccharide can be
chemically modified to provide further derivatization of these
compounds. Such chemical modification includes reduction of a
9-azido group on an analogue of sialic acid to an amine group which
can be still further functionalized to another derivative such as
the 9-acetamido derivative. Similarly, the carboxyl group found on
analogues of sialic acid can be selectively transformed on a
sialylated oligosaccharide glycosides via lactonization, reduction
or transformation into an amide.
[0087] In one or more of the enzymatic steps recited above, the
enzyme can be bound to a solid support so as to facilitate the
reaction of the reagents and the recovery of the product from the
enzyme.
[0088] C. Utility
[0089] The methods of this invention are useful in preparing
oligosaccharides containing sialic acid or an analogue thereof
bound via an .alpha.-linkage to the non-reducing sugar terminus of
the oligosaccharide. Such oligosaccharides are recognized in the
art as being useful as pharmaceuticals, as well as in the
generation of antibodies to these structures, which antibodies are
useful in diagnostic assays.
[0090] Additionally, methods of this invention are useful in
preparing oligosaccharides containing an analogue of sialic acid
bound via an .alpha.-linkage to the non-reducing sugar terminus of
the oligosaccharide which can be coupled to an antigenic carrier so
as to produce artificial antigens. Accordingly, such
oligosaccharides act as intermediates in the preparation of
artificial antigens.
[0091] Additionally, methods of this invention are useful in the
determination of the non-reducing terminus of an unknown
oligosaccharide.
EXAMPLES
[0092] The following examples are offered to illustrate this
invention and are not to be construed in any way as limiting the
scope of this invention.
[0093] In these examples, unless otherwise defined below, the
abbreviations employed have their generally accepted meaning:
2 PBS = phosphate buffered saline MES = morpholine ethane sulfonic
acid PMSF = .alpha.-toluenesulfonyl fluoride Le.sup.x-gr =
8-methoxycarbonyloctyl .alpha.-L-fucopyranosyl-
(1.fwdarw.3)-[.beta.-D-galactopyranosyl-(1.fwdarw.4)]-.beta.-D-2-
acetamide-2-deoxy-glucopyranoside Le.sup.a-gr =
8-methoxycarbonyloctyl .beta.-D- galactopyranosyl-(1.fwdarw.3)
[.alpha.-L-fucopyranosyl- (1.fwdarw.4)]-.beta.-D-2-acetamide-2-d-
eoxy- glucopyranoside CMP-NANA = cytidine
5'-monophospho-N-acetyl-neuraminic acid CMP-.sup.3H-NANA = cytidine
5'-monophospho-N-acetyl-neuraminic acid [sialic-9-.sup.3H] BSA =
bovine serum albumin d = doublet dd = doublet of doublets s =
singlet t = triplet GDP-Fuc = guanosine-5'-diphospho-L-fucose
UDP-galactose = uridine-5'-diphospho-galactose TMR =
tetramethylrhodamine
[0094] Commercially avaliable components are listed by
manufacturer. Some of the recited manufacturers are as follows:
[0095] Millipore=Millipore Corp., Bedford Mass.
[0096] Waters=Waters Corp., Milford, Mass.
[0097] Boehringer Mannheim=Boehringer Mannheim, Laval, Quebec,
Canada
Example 1
Preparation of viral .alpha.2,3-sialyltransferase cell lysates
[0098] The myxoma viral .alpha.2,3-sialyl transferase cell lysate
was prepared by a method similar to that set forth in International
Patent Application Publication No. WO97/18302.sup.65, which is
incorporated herein by reference.
[0099] Ten T180 flasks of confluent layers of European rabbit
kidney cell (RK13) cells were infected with Brazilian myxoma virus
strain, Lausanne (Lu) (ATCC VR-115) isolated Campinas, Brazil, 1949
and Uriarra (Ur) isolated Australian Capital Territory, 1953 (a
derivative of Moses strain (ATCC VR-116)). The cells were kept at
37.degree. C. for 24 hours. Twenty-four hours post infection, the
cells were detached by scraping and washed with PBS. Cell lysates
were prepared by suspension in 20 mL of extraction buffer (50 mM
MES, pH 6.1, 0.5% Triton-X100, 100 mM NaCl, 1.5 mM MgCl.sub.2, 0.1
mM PMSF, 10 mg/ml aprotinin) at 4.degree. C., for 45 minutes. The
lysate was clarified by centrifugation at 2,000 g at 4.degree. C.
for 15 minutes.
[0100] The supernatant was recovered and applied to a 5 mL HiTrap
Blue Affinity chromatography column (Pharmacia, Piscataway N.J.) in
loading buffer (50 mM MES, pH 6,1, 0.1% Triton-CF54, 100 mM NaCl,
25% glycerol). The .alpha.2,3-sialyltransferase was eluted from the
column with a step NaCl elution (0.5 M, 1.0 M, 1.5 M, and 2.0 M
NaCl). The .alpha.2,3-sialyltransferase was desalted by passing the
eluant through a PD-10 column (BioRad, Hercules, Calif.) in column
buffer (50 mM MES, pH 6,1, 0.1% Triton-CF54, 25% glycerol).
[0101] Total protein concentrations were measured using Bradford
Bio-Rad and following the manufacturers instructions with IgG as a
protein standard.
Example 2
Transfer of sialic acid to Lewis.sup.a and Lewis.sup.x
oligosaccharide acceptors
[0102] Acceptor (54 nmol), CMP-NANA (40 nmol), and CMP-.sup.3H-NANA
(150,000-180,000 dpm) were added to a mixture of cell lysate (16
.mu.L), water (3 .mu.L) and 1 .mu.L of assay buffer (250 mM MES,
0.5% Triton CF54, pH 7.0) in a 0.5 mL microfuge tube. Reaction
mixtures were incubated at 37.degree. C., diluted with water to 200
.mu.L and loaded onto a C.sub.18 Sep-Pak reverse-phase cartridge
which had been pre-equilibrated with 20 mL of MeOH and 20 mL of
water. The cartridge was washed with 50 mL of water and the product
eluted with 4 mL of MeOH into a scintillation vial. The
radioactivity of the MeOH eluates were quantitated by liquid
scintillation in 10 mL of EcoLite (+) scintillation cocktail (ICN,
Montreal, Quebec, Canada) in a Beckman liquid scintillation counter
(LS1801).
[0103] Results of the radioactive transfer to the different
acceptors in duplicate experiments were as follows:
3 Acceptor total CMP-NANA Incubation time dpm Le.sup.x-gr 152644 90
min. 1330/1130 Le.sup.a-gr 176174 170 min. 4161/4045
Example 3
Transfer of sialic acid to various oligosaccharide acceptors
[0104] The ability of the isolated viral a2,3-sialyltransferase to
transfer a sialic acid to various acceptors was tested.
[0105] Acceptor (54 nmol), CMP-NANA (40 nmol), and CMP-.sup.3H-NANA
(150,000-180,000 dpm) were added to a mixture of cell lysate (16
.mu.L), water (3 .mu.L) and assay buffer (250 mM MES, 0.5% Triton
CF54, pH 6.1) in a 0.5 mL microfuge tube. Reaction mixtures were
incubated at 37.degree. C., diluted with water and measured by the
method set forth in Example 2 above to obtain relative rates of
transfer. Kinetics were carried out in an analogous method by
varying the acceptor concentration from about 0.2.times.Km to
3.times.Km.
4 Relative Rate Relative rVmax/ Acceptor (2.7 mM) Km (.mu.M) Vmax
Km LacNAc-O-gr 100 112 .+-. 11 100 (1.5 0.896 nmol/mL/min)
6'-dimethyl-lacNAc-)-gr 17 Lactose -O-gr 90 211 .+-. 40 112 0.531
3'-methyl-lactose-O- 4.1 octyl 4'-Methyl-lactose-O- 20 octyl
Le.sup.c -O-gr 79 202 .+-. 10 90 0.446 T-disaccharide-O-gr 64 427
.+-. 110 51 0.119 Gal.alpha.(1.fwdarw.3)-lacNAc-O-gr 18
Gal.alpha.(1.fwdarw.4)-lacto- se-O-gr 12 9270 .+-. 1848 17 0.00183
GlcNAc-O-gr <1 Glc-O-octyl <1 Gal-O-phenyl <1
Fuc-Gal-O-octyl 1.5 LacNAc-OH 171 Lactose-OH 121 97 .+-. 43 120
1.23 Le.sup.a-O-gr 50 1578 .+-. 105 42 0.0266 Le.sup.x-O-gr 21 9490
.+-. 1930 30 0.00316 CMP-NANA 244 .+-. 36 gr =
(CH.sub.2).sub.8COOMe CMP-NANA = cytidine 5'-monophospho-N-acetyl-
-neuraminic acid
[0106] This indicates that the viral .alpha.2,3-sialyltransferase
is able to use a number of different oligosaccharide structures as
acceptors for the transfer of a sialic acid.
Example 4
Confirmation of 2,3 linkage of sialic acid to Lewis.sub.a and
Lewis.sub.x oligosaccharide acceptors
[0107] Le.sup.a-TMR (35 nmol) and CMP-NANA (200 nmol) were
incubated with viral cell lysate (4.9 .mu.L) and alkaline
phosphatase solution (0.1 .mu.L) (5 .mu.L of alkaline phosphatase
(Boehringer Mannheim) 1000 U/ml and 1 .mu.L BSA solution (100
mg/mL)). After gentle rotation at room temperature (25.degree. C.)
for 42 hours, additional alkaline phosphatase solution (0.2 .mu.L)
and CMP-NANA solution (100 mM, 0.2 .mu.L) were added to the mixture
which was reacted for 2 more days at room temperature. The reaction
mixture was elevated and maintained at 37.degree. C. for 48 hours,
then the mixture was loaded onto a C.sub.18-Sep-Pak cartridge
(Waters) which had been pre-equilibrated with 10 mL of MeOH and 10
mL of water. The cartridge was washed with 5 mL of water then
TMR-labeled compounds were eluted with 5 mL of MeOH. This solution
was dried under vacuum, passed through a filter (Milliex-GV filter,
0.22 .mu.m, Millipore Corp.) and lyophilized. Water was added to
dry material to make 100 .mu.M TMR concentration. This solution
(0.5 .mu.L) was mixed with capillary electrophoresis running buffer
(10 mM phosphate, 10 mM sodium borate, 10 mM sodium dodecyl
sulfate, 10 mM phenyl boronic acid pH 9.0, 499.5 .mu.L). This
solution was used for separation and analysis by capillary
electrophoresis with laser induced fluorescence detection by the
method set forth in Le et al..sup.66 A new product peak produced in
the enzyme reaction had the same migration time as authentic
sialylated Le.sup.x-TMR and the new product peak was converted back
to Le.sup.x-TMR by treatment with neuraminidase.
Example 5
Preparative Synthesis of sialylLe.sup.x-gr
[0108] Cell lysates (14 mL) were mixed with BSA solution (5 .mu.L,
100 mg/mL). This solution was concentrated in a Slide-A lyzer
(Pierce Chemical Company, Rockford, Ill.) to 1.8 mL. Le.sup.x-gr
(4.7 mg, 6.7 .mu.mol) and CMP-NANA (6.8 mg, 10.3 .mu.mol) were
added to 200 .mu.L of concentrated cell lysate Alkaline phosphatase
(Boehringer Mannheim, 1000 U/mL, 10 .mu.L) was added. This reaction
mixture was turned at room temperature for 24 days. During this
incubation, CMP-NANA was added (6 times, after 3 days, 7 days, 11
days, 3.0 mg each, after 14 days, 18 days, 21 days, 2.0 mg each).
This mixture was loaded onto a C.sub.18-Sep-Pak cartridge (Waters)
which had been pre-equilibrated with 10 mL of MeOH and 10 mL of
water. The cartridge was washed with 40 mL of water, then the
product was eluted with 50 mL of 10% MeOH. This eluate was dried
under vacuum and again loaded onto a C.sub.18-Sep-Pak cartridge
which had been pre-equilibrated with 10 mL of MeOH and 10 mL of
water. After washing with water (10 mL); 1 % MeOH (10 mL), and then
5 % MeOH (10 mL), the desired product was eluted with 10% MeOH (17
mL). This solution was dried under vacuum, passed through a filter
(Milliex-GV filter, 0.22 .mu.m, Millipore Corp.) and lyophilized to
give sialylated Le.sup.x-gr (2.19 mg, 33%). The structure of the
product sialyl Le.sup.x-gr was confirmed by both NMR spectroscopy
and mass spectrometry.
[0109] NMR (300 Hz, only typical peaks are shown, D.sub.2O) .delta.
5.10 (d, H, J=3.9 Hz, H-1(Fuc)), 4.51 (d, 2H, J=8.0 Hz, H-1(Gal,
GlcNAc)), 3.69 (s, 3H, CO.sub.2Me), 2.76 (dd, H, J=4.7, 12.6 Hz,
H-3, (NANA)), 2.38 (t, 2H, J=11.4 Hz, CH.sub.2CO.sub.2Me), 2.04
(s,3H, Ac), 2.02 (s, 3H, Ac), 1.79 (t, H, J=12.2, 1.8 Hz, H-3
(NANA)), 1.17 (d, 3H, J=6.6 Hz, H-6 (Fuc)).
[0110] Mass calculated for C.sub.41H.sub.69N.sub.2O.sub.25=989.4;
found 989.0
[0111] Sialylated Le.sup.a-gr was also synthesized from Le.sup.a-gr
in a similar manner. Its structure was confirmed by both NMR
spectroscopy and mass spectrometry.
[0112] NMR (300 Hz, only typical peaks are shown, D.sub.2O) .delta.
5.00 (d, H, J=3.9 Hz, H-1(Fuc)), 4.52 (d, 2H, J=7.7 Hz, H-1(Gal,
GlcNAc)), 3.69 (s, 3H, CO.sub.2Me), 2.76 (dd, H, J=4.7, 12.5 Hz,
H-3, (NANA)), 2.39 (t, 2H, J=7.3 Hz, CH.sub.2CO.sub.2Me), 2.02 (s,
6H, Ac), 1.76 (t, H, J=12.3, H-3 (NANA)), 1.16 (d, 3H, J=6.4 Hz,
H-6 (Fuc)).
[0113] Mass calculated for C.sub.41H.sub.69N.sub.2O.sub.25=989.4;
found 989.0
Example 6
Synthesis of sialylLe.sup.x-TMR from GlcNAc-TMR
[0114] GlcNAc-TMR (35 nmol), UDP-galactose (70 nmol), 0.5 .mu.L
isolated bovine milk .beta.-1,4 galactosyltransferase (0.3 mU),
GDP-fucose (70 nmol), 0.5 .mu.L isolated human milk
.alpha.1,3,4-fucosyltransferase (0.03 mU), CMP-NANA (100 nmol), 0.1
mU) were incubated with 5.7 .mu.L of concentrated viral cell lysate
solution. After gentle rotation at room temperature for 24 hours,
0.2 .mu.L aliquots were removed and spotted onto a silica gel
60F.sub.254 thin layer chromatography plate (Merck, Darmstadt
Germany). The plate was developed with isopropanol:H.sub.2O:NH.-
sub.4OH (7:2:1). Le.sup.x-TMR (Rf=0.18) and sialyl Le.sup.x-TMR
(Rf=0.30) were visible due to the pink chromophore TMR. These Rfs
correspond to those of authentic material and the sialyl
Le.sup.x-TMR formed in the enzyme reaction mixture co-migrated with
sialyl Le.sup.x-TMR. The starting material GlcNAc-TMR (Rf=0.39) and
the reaction intermediate LacNAc-TMR (Rf=0.25) were not
detected.
Example 7
Synthesis of sialylLe.sup.a-TMR from Le.sup.c-TMR
[0115] Le.sup.c-TMR (15 nmol), GDP-fucose (70 nmol), 0.5 .mu.L
isolated human milk, .alpha.-1,3,4-fucosyltransferase (0.03 mU),
CMP-NANA (100 nmol), 0.1 .mu.L of 1 M MnCl.sub.2 and 0.1 .mu.L
alkaline phosphatase (Boehringer Mannheim, 10 mU) were incubated
with 5.1 .mu.L of concentrated viral cell lysate solution. After
gentle rotation at room temperature for 72 hours, 0.2 .mu.L
aliquots were removed and spotted onto a silica gel 60F.sub.254
thin layer chromatography plate (Merck, Darmstadt Germany). The
plate was developed with isopropanol:H.sub.2O:NH.- sub.4OH (7:2:1).
Le.sup.a-TMR (Rf=0.18) and sialyl Le.sup.a-TMR (Rf=0.25) were
visible due to the pink chromophore TMR. These Rfs correspond to
those of authentic material and the sialyl Le.sup.a-TMR formed in
the enzyme reaction mixture co-migrated with sialyl Le.sup.a-TMR.
The starting material Le.sup.c-TMR (Rf=0.31) was not detected.
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