U.S. patent application number 10/328625 was filed with the patent office on 2003-07-10 for saccharide compositions, methods and apparatus for their synthesis.
This patent application is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to Roth, Stephen A..
Application Number | 20030130175 10/328625 |
Document ID | / |
Family ID | 27496566 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030130175 |
Kind Code |
A1 |
Roth, Stephen A. |
July 10, 2003 |
Saccharide compositions, methods and apparatus for their
synthesis
Abstract
A method for preparing saccharide compositions is disclosed. The
method is reiterative and comprises the following three steps. (i)
A glycosyltransferase capable of transferring a preselected
saccharide unit to an acceptor moiety is isolated by contacting the
acceptor moiety with a mixture suspected of containing the
glycosyltransferase under conditions effective to bind the acceptor
moiety and the glycosyltransferase and thereby isolate the
glycosyltransferase. The acceptor moiety is a protein, a
glycoprotein, a lipid, a glycolipid, or a carbohydrate. (ii) The
isolated glycosyltransferase is then used to catalyze the bond
between the acceptor moiety and the preselected saccharide unit.
(iii) Steps (i) and (ii) are repeated a plurality of times with the
intermediate product obtained in the first iteration of the method
being used as the acceptor moiety of the second iteration.
Inventors: |
Roth, Stephen A.; (Gladwyne,
PA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP
1701 MARKET STREET
PHILADELPHIA
PA
19103-2921
US
|
Assignee: |
The Trustees of the University of
Pennsylvania
|
Family ID: |
27496566 |
Appl. No.: |
10/328625 |
Filed: |
December 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10328625 |
Dec 23, 2002 |
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09570236 |
May 12, 2000 |
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6518051 |
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09570236 |
May 12, 2000 |
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08480317 |
Jun 7, 1995 |
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6331418 |
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08480317 |
Jun 7, 1995 |
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08163534 |
Dec 9, 1993 |
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08163534 |
Dec 9, 1993 |
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07955687 |
Oct 2, 1992 |
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5288637 |
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07955687 |
Oct 2, 1992 |
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07683810 |
Apr 11, 1991 |
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5180674 |
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Current U.S.
Class: |
435/6.11 ;
435/101; 435/6.18; 435/68.1; 435/89; 514/19.3; 514/20.9; 514/5.5;
514/54 |
Current CPC
Class: |
C12P 19/26 20130101;
C12N 9/1051 20130101; C12N 11/00 20130101; C12P 19/18 20130101 |
Class at
Publication: |
514/8 ; 514/54;
435/68.1; 435/89; 435/101 |
International
Class: |
A61K 038/14; A61K
038/16; A61K 031/715; C12P 021/06; C12P 019/30; C12P 019/04 |
Goverment Interests
[0002] Portions of this invention were supported by National
Science Foundation Grant DCB8817883.
Claims
What is claimed as new and desired to be secured by Letters Patent
of the United States is:
1. A method for the glycosyltransferase-catalyzed preparation of a
saccharide composition by serially bonding preselected saccharide
units to an acceptor moiety, comprising the steps of: (i) preparing
a glycosyltransferase capable of transferring a preselected
saccharide unit to an acceptor moiety by contacting said acceptor
moiety with a mixture suspected to contain a plurality of
glycosyltransferases under conditions effective to bind said
acceptor moiety and said glycosyltransferase, and thereby isolating
said glycosyltransferase, wherein said acceptor moiety is one
member selected from the group consisting of proteins,
glycoproteins, lipids, glycolipids, and carbohydrates; (ii)
providing conditions and co-reagents sufficient to effect bonding
of said preselected saccharide unit to said acceptor moiety
catalyzed by said glycosyltransferase thereby obtaining a product;
and (iii) performing steps (i) and (ii) a plurality of times such
that the product obtained in step (ii) of a given iteration is used
as the acceptor moiety in step (i) of the following iteration until
said saccharide composition is obtained.
2. The method of claim 1 wherein said carbohydrate is one member
selected from the group consisting of monosaccharides,
disaccharides, oligosaccharides, and polysaccharides.
3. The method of claim 1 wherein the glycosyltransferase used in
step (ii) is immobilized to a solid support.
4. The method of claim 3 wherein said glycosyltransferase attached
to a solid support is obtained by protecting the active site of
said glycosyltransferase during the immobilization process.
5. The method of claim 1 wherein said co-reagents comprise
manganese cations.
6. The method of claim 1 wherein said saccharide used in step (ii)
is a saccharide nucleotide.
7. The method of claim 5 wherein said nucleotide is one member
selected from the group consisting of uridine, guanosine and
cytidine phosphates.
8. The method of claim 1 wherein said acceptor moiety used in said
first iteration is one member selected from the group consisting of
proteins, glycoproteins, lipids, and glycolipids.
9. The method of claim 1 wherein said acceptor moiety used in said
first iteration is one member selected from the group consisting of
monosaccharides, disaccharides, oligosaccharides, and
polysaccharides.
10. The method of claim 1 wherein said acceptor moiety used in said
first iteration is N-acetylglucosamine.
11. The method of claim 1 wherein said acceptor moiety used in said
first iteration is N-acetylglucosamine, said glycosyltransferase
used in said first iteration is galactosyltransferase, and said
glycosyltransferase used in said second iteration is
N-acetylneuraminyltransferase.
12. The method of claim 1 wherein said acceptor moiety used in said
second iteration is galactosyl .beta.1-4 N-acetylglucosamine.
13. The method of claim 1 wherein at least one of the donor
moieties used is cytidine monophosphate N-acetylneuraminic
acid.
14. The method of claim 1 wherein said mixture suspected to contain
a plurality of glycosyltransferases is a cell homogenate.
15. The method of claim 1 wherein said saccharide composition is a
compound of one of the following formulae: 12
16. A pharmaceutical composition comprising, in association with
pharmaceutically acceptable excipient or carrier, a saccharide
composition other than heparin prepared by a method for
glycosyltransferase-catalyzed serial bonding of preselected
saccharide units to an acceptor moiety comprising the steps of: (i)
preparing a glycosyltransferase capable of transferring a
preselected saccharide unit to an acceptor moiety by contacting
said acceptor moiety with a mixture suspected to contain said
glycosyltransferase under conditions effective to bind said
acceptor moiety and said glycosyltransferase, thereby isolating
said glycosyltransferase, wherein said acceptor moiety is one
member selected from the group consisting of proteins,
glycoproteins, lipids, glycolipids, and carbohydrates; (ii)
providing conditions and co-reagents sufficient to effect bonding
of said preselected saccharide unit to said acceptor moiety
catalyzed by said glycosyltransferase thereby obtaining a product;
and (iii) performing steps (i) and (ii) a plurality of times such
that the product obtained in step (ii) of a given iteration is used
as the acceptor moiety of step (i) of the following iteration.
17. The pharmaceutical composition of claim 16 comprising at least
100 mg of said saccharide composition.
18. The pharmaceutical composition of claim 16 comprising at least
1 gram of said saccharide composition.
19. The pharmaceutical composition of claim 16 wherein said
acceptor moiety used in said first iteration is a protein.
20. The pharmaceutical composition of claim 16 wherein said
acceptor moiety used in said first iteration is a glycoprotein.
21. The pharmaceutical composition of claim 16 wherein said
acceptor moiety used in said first iteration is a lipid.
22. The pharmaceutical composition of claim 16 wherein said
acceptor moiety used in said first iteration is a glycolipid.
23. The pharmaceutical composition of claim 16 wherein said
acceptor moiety used in said first iteration is a carbohydrate.
24. The pharmaceutical composition of claim 16 wherein said
acceptor moiety used in said first iteration is a
monosaccharide.
25. The pharmaceutical composition of claim 16 wherein said
acceptor moiety used in said first iteration is a disaccharide.
26. The pharmaceutical composition of claim 16 wherein said
acceptor moiety used in said first iteration is an
oligosaccharide.
27. The pharmaceutical composition of claim 16 wherein said
acceptor moiety used in said first iteration is a
polysaccharide.
28. A pharmaceutical composition suitable for use in the therapy or
treatment of pneumonia comprising an effective amount of a compound
of the formula: 13in association with a pharmaceutically acceptable
carrier or excipient.
29. A pharmaceutical composition suitable for use in the therapy or
treatment of periodontal disease comprising an effective amount of
a compound of the formula: 14in association with a pharmaceutically
acceptable carrier or excipient.
30. A pharmaceutical composition suitable for use in the therapy or
treatment of solid tumors comprising an effective amount of a
compound of the formula: 15in association with a pharmaceutically
acceptable carrier or excipient.
31. A pharmaceutically composition suitable for use as a
contraceptive comprising an effective amount of a compound
comprising the formula: 16in association with a pharmaceutically
acceptable carrier or excipient.
32. A foodstuff comprising a saccharide composition other than gum
tragacanth or carrageenan prepared by a method for
glycosyltransferase-catalyzed serial bonding of preselected
saccharide units to an acceptor moiety comprising the steps of: (i)
preparing a glycosyltransferase capable of transferring a
preselected saccharide unit to an acceptor moiety by contacting
said acceptor moiety with a mixture suspected to contain a
plurality of glycosyltransferases under conditions effective to
bind said acceptor moiety and said glycosyltransferase, and thereby
isolating said glycosyltransferase, wherein said acceptor moiety is
one member selected from the group consisting of proteins,
glycoproteins, lipids, glycolipids, and carbohydrates; (ii)
providing conditions and co-reagents sufficient to effect bonding
of said preselected saccharide unit to said acceptor moiety
catalyzed by said glycosyltransferase thereby obtaining a product;
and (iii) performing steps (i) and (ii) a plurality of times such
that the product obtained in step (ii) of a given iteration is used
as the acceptor moiety in step (i) of the following iteration.
33. In infant foodstuff, the improvement comprising a compound of
the formula: 17present in an amount of from about 0.1 .mu.g
ml.sup.-1 to about 1000 .mu.g ml.sup.-1.
34. An apparatus for the glycosyltransferase-catalyzed synthesis of
a saccharide composition, said apparatus comprising: a reactor; at
least tour different glycosyltransferases in said reactor; inlet
means for introducing an acceptor moiety and a plurality of
preselected saccharide units into said reactor such that said
saccharide composition is synthesized; and outlet means for
discharging said saccharide composition from said reactor; wherein
said acceptor moiety is one member selected from the group
consisting of proteins, glycoproteins, lipids, glycolipids, and
carbohydrates.
35. The apparatus of claim 34 wherein said reactor comprises a
plurality of reaction zones serially connected so as to be in
sequential fluid communication with each other, wherein each
reaction zone comprises a glycosyltransferase specific to catalyze
bonding of a preselected saccharide unit onto the intermediate
product formed in the preceding reaction zone.
36. The apparatus of claim 34 wherein means for purifying the
intermediate products formed in each of said reaction zones from
the rest of the reaction mixture produced therein is situated in
fluid communication and between each of said reaction zones.
37. The apparatus of claim 34 wherein said glycosyltransferases are
immobilized onto a solid support.
38. The apparatus of claim 37 wherein the active sites of said
glycosyltransferases immobilized onto said solid support are
protected during the immobilization process.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 07/509,560, filed Apr. 16, 1990, and relates to subject
matter disclosed in copending U.S. patent application Ser. No.
07/241,012, filed Sep. 2, 1988, entitled "Carbohydrates and
Carbohydrate Complexes for Therapeutic and Preventative Treatment
of Mammals". Ser. No. 07/241,012 is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates to saccharide compositions such as,
for example, oligosaccharides, polysaccharides, glycolipids, and
glycoproteins. More specifically, this invention relates to
processes for preparing these and other saccharide compositions by
enzymatic techniques.
[0005] 2. Discussion of the Background
[0006] The term "carbohydrate" embraces a wide variety of chemical
compounds having the general formula (CH.sub.2O).sub.n, such as
monosaccharides, disaccharides, oligosaccharides and
polysaccharides. Oligosaccharides are chains composed of saccharide
units, which are alternatively known as sugars. These saccharide
units can be arranged in any order and the linkage between two
saccharide units can occur in any of approximately ten different
ways. As a result, the number of different possible stereoisomeric
oligosaccharide chains is enormous.
[0007] Of all the biological polymer families, oligosaccharides and
polysaccharides have been the least well studied, due in
considerable part to the difficulty of sequencing and synthesizing
their often complex sugar chains. Although the syntheses of
oligonucleotides and polypeptides are well developed, there is
currently no generally applicable synthetic technique for
synthesizing oligosaccharides.
[0008] Numerous classical techniques for the synthesis of
carbohydrates have been developed, but these techniques suffer the
difficulty of requiring selective protection and deprotection.
Organic synthesis of oligosaccharides is further hampered by the
lability of many glycosidic bonds, difficulties in achieving
regioselective sugar coupling, and generally low synthetic yields.
These difficulties, together with the difficulties of isolating and
purifying carbohydrates and of analyzing their structures, has made
this area of chemistry a most demanding one.
[0009] Much research effort has been devoted to carbohydrates and
molecules comprising carbohydrate fragments, such as glycolipids
and glycoproteins. Research interest in such moieties has been
largely due to the recognition that interactions between proteins
and carbohydrates are involved in a wide array of biological
recognition events, including fertilization, molecular targeting,
intercellular recognition, and viral, bacterial, and fungal
pathogenesis. It is now widely appreciated that the oligosaccharide
portions of glycoproteins and glycolipids mediate recognition
between cells and cells, between cells and ligands, between cells
and the extracellular matrix, and between cells and pathogens.
[0010] These recognition phenomena can likely be inhibited by
oligosaccharides having the same sugar sequence and stereochemistry
found on the active portion of a glycoprotein or glycolipid
involved in cell recognition. The oligosaccharides are believed to
compete with the glycoproteins and glycolipids for binding sites on
receptor proteins. For example, the disaccharide galactosyl
.beta.1-4 N-acetylglucosamine is believed to be one component of
the glycoproteins which interact with receptors in the plasma
membrane of liver cell. Thus, to the extent that they compete with
potentially harmful moieties for cellular binding sites,
oligosaccharides and other saccharide compositions have the
potential to open new horizons in pharmacology, diagnosis, and
therapeutics.
[0011] There has been relatively little effort to test
oligosaccharides as therapeutic agents for human or animal
diseases, however, as methods for the synthesis of oligosaccharides
have been unavailable as noted above. Limited types of small
oligosaccharides can be custom-synthesized by organic chemical
methods, but the cost for such compounds is typically very high. In
addition, it is very difficult to synthesize oligosaccharides
stereospecifically and the addition of some sugars, such as sialic
acid and fucose, has not been effectively accomplished because of
the extreme lability of their bonds. Improved, generally applicable
methods for oligosaccharide synthesis are desired for the
production of large amounts of widely varying oligosaccharides for
pharmacology and therapeutics.
[0012] For certain applications, enzymes have been targeted for use
in organic synthesis as one alternative to more traditional
techniques. For example, enzymes have been used as catalysts in
organic synthesis; the value of synthetic enzymatic reactions in
such areas as rate acceleration and stereoselectivity has been
demonstrated. Additionally, techniques are now available for low
cost production of some enzymes and for alteration of their
properties.
[0013] The use of enzymes as catalysts for the synthesis of
carbohydrates has been proposed, but to date enzyme-based
techniques have not been found which are useful for the general
synthesis of oligosaccharides and other complex carbohydrates in
significant amounts. It has been recognized that a major limiting
factor to the use of enzymes as catalysts in carbohydrate synthesis
is the very limited current availability of the broad range of
enzymes required to accomplish carbohydrate synthesis. See Toone et
al, Tetrahedron Reports (1990) (45)17:5365-5422.
[0014] In mammalian systems, eight monosaccharides activated in the
form of nucleoside mono- and diphosphate sugars provide the
building blocks for most oligosaccharides: UDP-Glc, UDP-GlcUA,
UDP-GlcNAc, UDP-Gal, UDP-GalNAc, GGP-Man, GDP-Fuc and CMP-NeuAc.
These are the intermediates of the Leloir pathway. A much larger
number of sugars (e.g., xylose, arabinose) and oligosaccharides are
present in microorganisms and plants.
[0015] Two groups of enzymes are associated with the in vivo
synthesis of oligosaccharides. The enzymes of the Leloir pathway is
the largest group. These enzymes transfer sugars activated as sugar
nucleoside phosphates to a growing oligosaccharide chain.
Non-Leloir pathway enzymes transfer carbohydrate units activated as
sugar phosphates, but not as sugar nucleoside phosphates.
[0016] Two strategies have been proposed for the enzyme-catalyzed
in vitro synthesis of oligosaccharides. See Toone et al, supra. The
first strategy proposes to use glycosyltransferases. The second
proposes to use glycosidases or glycosyl hydrolases.
[0017] Glycosyltransferases catalyze the addition of activated
sugars, in a stepwise fashion, to a protein or lipid or to the
non-reducing end of a growing oligosaccharide. A very large number
of glycosyltransferases appear to be necessary to synthesize
carbohydrates. Each NDP-sugar residue requires a distinct class of
glycosyltransferases and each of the more than one hundred
glycosyltransferases identified to date appears to catalyze the
formation of a unique glycidic linkage. To date, the exact details
of the specificity of the glycosyltransterases are not known. It is
not clear, for example, what sequence of carbohydrates is
recognized by most of these enzymes.
[0018] Enzymes of the Leloir pathway have begun to find application
to the synthesis of oligosaccharides. Two elements are required for
the success of such an approach. The sugar nucleoside phosphate
must be available at practical cost and the glycosyltransferase
must be available. The first issue is resolved for most common
NDP-sugars, including those important in mammalian biosynthesis.
The problem in this technology however resides with the second
issue. To date, only a very small number of glycosyltransferases
are available. Access to these types of enzymes has been the single
limiting factor to this type of carbohydrate synthesis.
[0019] It has been reported that most glycosyltransferases are
difficult to isolate, particularly from mammalian source. This is
because these proteins are present in low concentrations and are
membrane-bound. Further, although a few glycosyltransferases have
been immobilized, these enzymes have been reported to be unstable.
To date only a very small number of glycosyltransferases are
available from commercial sources, and these materials are
expensive.
[0020] Much hope has therefore been put on future developments in
genetic engineering (i.e., cloning) of enzymes, particularly since
several glycosyltransferases have already been cloned, including
galacto-, fucosyl-, and sialyltransferases. It is hoped that future
advances in cloning techniques will speed the cloning of other
glycosyltransferases and enhance their stability.
[0021] Accordingly, in light of their potential uses and the
difficulty or impossibility to obtain them in sufficient
quantities, there exists a long-felt need for general synthetic
methods for the production of oligosaccharides, polysaccharides,
glycoproteins, glycolipids, and similar species in an efficient,
cost effective, stereospecific, and generally applicable
manner.
SUMMARY OF THE INVENTION
[0022] It is an object of the present invention to provide
saccharide compositions, particularly oligosaccharides,
polysaccharides and chemical moieties which comprise
oligosaccharide units.
[0023] It is another object of this invention to provide a wide
variety of saccharide compositions, including those not found in
nature.
[0024] It is a further object of this invention to provide
saccharide compositions useful in mitigating the effects of human
or animal diseases.
[0025] It is yet another object of this invention to provide
improved processes for preparing saccharide compositions.
[0026] It is a further object of this invention to provide
enzymatic processes for preparing saccharide compositions.
[0027] It is still another object of this invention to provide
processes for obtaining enzymes useful in synthesizing saccharide
compositions.
[0028] It is still another object of this invention to provide an
apparatus useful for the synthesis of saccharide compositions in
accordance with the present invention.
[0029] These and other objects are achieved by the present
invention, which provides enzymatic processes for preparing
oligosaccharides, polysaccharides, glycolipids, glycoproteins, and
other saccharide compositions. These processes involve the
enzyme-facilitated transfer of a preselected saccharide unit from a
donor moiety to an acceptor moiety. Saccharide compositions having
a plurality of saccharide units are preferably prepared by
appending the saccharide units in stepwise fashion to acceptor
moieties which are themselves saccharide compositions prepared in
accordance with this invention.
[0030] Accordingly, methods for preparing saccharide compositions
are provided comprising the steps of providing an acceptor moiety
and contacting the acceptor moiety with a glycosyltransferase. The
glycosyltransferase is prepared so as to be specific for the
acceptor moiety and capable of transferring a saccharide unit to
the acceptor moiety. This method of the present invention is
performed a plurality of times such that the product of the first
iteration becomes the acceptor moiety for a second iteration, and
so forth.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIGS. 1, 2 and 3 illustrate apparatuses suitable for use in
the glycosyltransferase catalyzed synthesis of saccharide
composition in accordance with the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] As employed herein, the term "saccharide composition" is
intended to include any chemical moiety having a saccharide unit
within its structure. Sugars, carbohydrates, saccharides,
monosaccharides, oligosaccharides, polysaccharides, glycoproteins,
and glycolipids provide examples of saccharide compositions.
Mixtures and solutions comprising such moieties are also saccharide
compositions.
[0033] Saccharide compositions are prepared according to this
invention by the enzyme facilitated transfer of saccharide units
from donor moieties to acceptor moieties. It will be appreciated
that such transfer occurs upon contacting the acceptor and donor
moieties with a glycosyltransferase, and typically results in
covalently bonding of the acceptor moiety and the saccharide unit
stereoselectively, that is, in but one stereoisomeric form.
[0034] The saccharide compositions prepared in accordance with this
invention are believed to find wide utility in diagnostics,
therapeutics, and pharmacological applications. Once the sugar
sequence of a desired target saccharide composition has been
determined by conventional methods, a retrosynthetic analysis is
generally performed to determine an appropriate synthetic scheme
for the saccharide composition. Such a synthetic scheme preferably
identifies the particular donor moieties, acceptor moieties, and
glycosyltransferases necessary to yield the desired saccharide
composition.
[0035] Instead of relying on future developments in genetic
engineering to provide the numerous glycosyltransferases required
for carbohydrate synthesis, the present invention relies on the
very different approach as follows. In the synthesis of a
saccharide composition in accordance with the invention, a
preselected saccharide unit is first enzymatically attached to an
initial acceptor moiety, i.e., a protein, a glycoprotein, a lipid,
a glycolipid, or a carbohydrate starting material. This is followed
by enzymatically attaching preselected saccharide units to the
product obtained in a stepwise fashion thereby forming the
saccharide composition.
[0036] With the attachment of each preselected saccharide unit one
obtains an intermediate product. The present invention is based on
the inventor's discovery that the starting material of the
synthesis (i.e., the protein, glycoprotein, lipid, glycolipid or
carbohydrate) and each intermediate product formed in the synthesis
can be advantageously used to obtain, for each corresponding step
of the synthesis, a glycosyltransferase specific to catalyze the
attachment of the next intermediate product in the synthesis of the
target saccharide composition.
[0037] Thus, in accordance with the invention, the
glycosyltransferase needed for any given step is isolated with the
intermediate product (the acceptor moiety) and used to attach to
the acceptor moiety the next saccharide unit necessary for
construction of the target carbohydrate molecule. In accordance
with the present invention, this process is repeated, with each
iteration (time) yielding the particular glycosyltransferase
required to attach the next saccharide unit onto the growing
molecule being isolated, until the target carbohydrate molecule is
obtained.
[0038] Also provided by the invention are reaction conditions and
co-reagents as may be necessary and sufficient to effect the
covalent bonding of the saccharide unit to the acceptor moiety.
[0039] In accordance with preferred embodiments, the acceptor
moiety may be a protein, glycoprotein, lipid, glycolipid, or
carbohydrate, such as a monosaccharide, disaccharide,
oligosaccharide, or polysaccharide. In accordance with other
preferred embodiments, the glycosyltransferase is attached to a
solid support.
[0040] The present methods are capable of stereospecific attachment
of the saccharide unit to the acceptor moiety. In general, it is
preferred to employ saccharide nucleotides as donor moieties.
Uridine, guanosine, and cytidine phosphate materials terminated by
the saccharide units to be donated preferably comprise the donor
moieties.
[0041] The present invention thus also provides means for preparing
a glycosyltransferase specific for a particular acceptor moiety and
capable of transferring a preselected saccharide unit to the
acceptor moiety. Such methods comprise contacting the acceptor
moiety with a mixture suspected to contain a plurality of
glycosyltransferases under conditions effective to bind the
acceptor moiety and the glycosyltransferase specific for the
acceptor moiety. The resulting, bound glycosyltransferase is
subsequently isolated. It is preferred that the glycosyltransferase
be sequenced and that the glycosyltransferase be produced in
enhanced quantities by genetic engineering techniques.
[0042] The mixture suspected to contain a glycosyltransferase of
interest may be identified as follows. For the most common
glycosidic linkages, the glycosyltransferase activities have been
described in publications. This is largely true for compounds like
milk oligosaccharides, or the carbohydrate moieties of typical
(i.e., prevalent) glycoproteins and glycolipids. For less well
described linkages, one may first look to the tissue, organ,
foodstuff organism, in which the linkage is found. Generally, if
the linkage is found in a particular source, the enzyme that made
the linkage is also present in the source.
[0043] If one is presented only with a saccharide structure, and
not a source, one can then test examples of organisms that are
likely to contain such a saccharide structure using the most
sensitive screening assay available. For example, if the compound
contained iduronic acid, N-acetylgalactosamine and
N-acetylglucosamine, one would test vertebrate connective tissue.
If the target compound contain abequose, one would test bacteria
and plants for the presence of the appropriate
glycosyltransferase.
[0044] Various assays for detecting glycosyltransferases which can
be used in accordance with the invention have been published. The
following are illustrative. Furukawa et al, Biochem. J., (1985)
227:573-582 describe a borate-impregnated paper electrophoresis
assay and a fluorescence assay (FIG. 6) developed by the inventor.
Roth et al, Exp'l Cell Research (1983) 143:217-225 describe
application of the borate assay to glucuronyl transferases,
previously assayed colorimetrically. Benau et al, J. Histochem.
Cytochem. (1990) 38(1):23-30 describe a histochemical assay based
on the reduction, by NADH, of diazonium salts.
[0045] Once a source for the glycosyltransferase of interest has
been found, the source is homogenized. The enzyme is purified from
homogenate by affinity chromatography using the acceptor moiety as
the affinity ligand. That is, the homogenate is passed over a solid
matrix having immobilized thereon the acceptor moiety under
conditions which cause the glycosyltransferase to bind to the
acceptor moiety. The solid support matrix having the
glycosyltransferase bound thereto is then washed. This is followed
by an elution step in which the glycosyltransferase is desorbed
from the solid support matrix and collected. As known, the absorbed
glycosyltransferase may be eluted, for example, by passing an
aqueous salt (e.g. NaCl) solution over the solid support
matrix.
[0046] In actual practice of the invention, the "enzyme" purified
from the homogenate by affinity chromatography and which is used to
attach a preselected saccharide unit onto the acceptor moiety
comprises a mixture of various glycosyltransferases which have been
purified away from other extraneous biological material present in
the homogenate which includes enzymes which can interfere with the
desired activity of the purified glycosyltransferases. Thus, the
glycosyltransferases used in accordance with the present invention
are frequently a mixture of various "glycosyltransferase". If
desired, this material may be further purified with a single
purified glycosyltransferase being isolated and used in the process
of the present invention, but such further purification is
generally not necessary.
[0047] In accordance with the present invention, an acceptor moiety
is provided which is capable of being covalently bound to a
preselected saccharide unit. Representative acceptor moieties
include proteins, glycoproteins, lipids, glycolipids and
carbohydrates. It will be appreciated that acceptor moieties are
preferred to the extent that they are present as a structural
component of a saccharide composition of interest. For example, in
preparing a saccharide composition such as N-acetylneuraminyl
.alpha.2-3 galactosyl .beta.1-4 N-acetylglucosamine, preferred
acceptor moieties would be N-acetylglucosamine and galactosyl
.beta.1-4 N-acetylglucosamine. It will likewise be appreciated that
where an acceptor moiety is terminated by a saccharide unit,
subsequent saccharide units will typically be covalently bound to
the nonreducing terminus of the terminal saccharide.
[0048] The saccharide unit to be transferred to an acceptor moiety
is provided by a donor moiety for the saccharide unit. A donor
moiety according to this invention includes the saccharide unit to
be transferred and is capable of providing that saccharide unit to
the acceptor moiety when contacted by the acceptor moiety and the
appropriate glycosyltransferase. Preferred donor moieties are
saccharide nucleotides, such as saccharide-terminated uridine
phosphates, saccharide-terminated guanosine phosphates, and
saccharide-terminated cytidine phosphates.
[0049] It will be appreciated that donor moieties are preferred to
be capable of readily providing their component saccharide unit to
an acceptor moiety when placed in contact therewith and with a
glycosyltransferase. For example, uridine diphosphate galactose is
preferred for transferring galactose to N-acetylglucosamine, while
cytidine monophosphate N-acetylneuraminic acid is preferred for
transferring N-acetylneuraminic acid, a sialic acid, to galactosyl
.beta.1-4 N-acetylglucosamine.
[0050] Upon identification of acceptor moieties and donor moieties
necessary for the preparation of a saccharide composition, a
glycosyltransferase for each acceptor/donor pair should be
prepared. Those skilled in the art will appreciate that a
glycosyltransferase may be broadly defined as an enzyme which
facilitates the transfer of a saccharide unit from one chemical
moiety (here defined as a donor) to another (here defined as an
acceptor) and which is named phenomenologically according to the
saccharide unit it transfers. Thus, galactosyltransferase transfers
galactose, while fucosyltransferase transfers fucose.
[0051] Glycosyltransferases according to this invention are those
able to effect the transfer of a predetermined saccharide unit to
an acceptor moiety. Glycosyltransferases are preferably specific
for an acceptor moiety or at least some significant, active, or
exposed portion thereof. Specificity is manifested for a
glycosyltransferase by its tendency to bind with a particularly
sequenced portion of an acceptor moiety when placed in contact or
close proximity therewith and to effect the transfer of a
particular saccharide unit to that acceptor moiety.
[0052] Currently, glycosyltransferases are available only from
natural sources and, as a result, are somewhat limited in number.
It will be appreciated that known glycosyltransferases are only
capable of effecting saccharide unit transfers which are highly
specific, both in terms of the chemical identity of the saccharide
unit transferred and the stereochemistry of its subsequent
attachment to the acceptor moiety. For example, it is known that
one N-acetylneuraminyltransferase can effect the transfer of
N-acetylneuraminic acid to an acceptor moiety bearing only a
galactose unit to produce a saccharide composition having an
.alpha.2-3 linkage between the N-acetylneuraminic acid unit and the
galactose unit.
[0053] Thus, the invention permits construction of sugar linkages
found in nature. For example, the linkage of galactose .alpha.1-2
to N-acetylneuraminic acid, which has not been found in nature,
cannot presently be effected. The methods disclosed herein are,
however, applicable to any type of glycosyltransferase which may
become available.
[0054] While the behavior of a number of glycosyltransferases is
known, most glycosyltransferases are currently not fully
characterized. The present invention, however, provides methods by
which all glycosyltransferases amenable to its practice may be
identified and prepared. It has now been found that an acceptor
moiety can be used as an affinity chromatographic tool to isolate
enzymes that can be used to transfer particular saccharide units
and, thus, synthesize other glycosides.
[0055] In a preferred embodiment, an acceptor moiety is immobilized
as, for example, on a solid support. It will be appreciated that
the term "solid support" includes semi-solid supports as well. Once
immobilized, the acceptor moiety is contacted with a mixture
suspected to contain glycosyltransferases, such as one comprising
naturally-occurring cell homogenate. Since an immobilized acceptor
moiety will bind an enzyme specific for it, this system is then
monitored for acceptor-bound enzyme.
[0056] Monitoring for acceptor-bound enzyme may be carried out as
follows. The cell homogenate is passed over the immobilized
acceptor moiety. This may be achieved, for example, by passing the
cell homogenate over a column charged with immobilized acceptor
moiety. The column is then washed and the amount of protein which
passes through the column charged with immobilized acceptor moiety
is monitored. When no more protein is detected, an aqueous salt
solution eluant is passed through the column to elute the enzyme.
The eluant obtained is then assayed for the presence of
glycosyltransferase(s). The assays which can be used are noted
above, i.e., the methods described by Furukawa et al, Roth et al
and Benau et al.
[0057] If no binding of the enzyme to the acceptor moiety occurs
(i.e., the assay of the eluate fails to reveal the presence of
glycosyltransferase(s) therein), then it can be concluded that the
mixture did not contain an enzyme specific for the particular
acceptor. Other mixtures of, for example, animal and/or plant cell
homogenates are then contacted with the acceptor moiety until
enzyme binding is observed.
[0058] When the acceptor moiety is bound by an enzyme, the species
are separated and further studied. In a preferred embodiment, the
acceptor and the candidate enzyme are again contacted, this time in
the presence of a donor moiety which comprises the saccharide unit
desired to be transferred to the exceptor moiety. If such
contacting results in the transfer of the saccharide unit to the
acceptor, the enzyme is a glycosyltransferase useful in the
practice of this invention.
[0059] It will be appreciated that once the glycosyltransferase is
identified, it can be sequenced and/or replicated by techniques
well-known to those skilled in the art. For example, replication
might be accomplished by recombinant techniques involving the
isolation of genetic material coding for the glycosyltransferase
and the preparation of an immortal cell line capable of producing
the glycosyltransferase. Replication will likely prove desirable
for commercial scale production of saccharide compositions in
accordance with this invention.
[0060] After the glycosyltransferase is identified, it is contacted
with the acceptor moiety and donor moiety under conditions
sufficient to effect transfer and covalently bonding of the
saccharide unit to the acceptor moiety. It will be appreciated that
the conditions of, for example, time, temperature, and pH
appropriate and optimal for a particular saccharide unit transfer
can be determined by one of skill in the art through routine
experimentation. Certain co-reagents may also prove useful in
effecting such transfer. For example, it is preferred that the
acceptor and donor moieties be contacted with the
glycosyltransferase in the presence of divalent cations, especially
manganese cations such as may be provided by MnCl.sub.2.
[0061] In a preferred embodiment, the glycosyltransferase is
immobilized by attachment to a solid support and the acceptor and
donor moieties to be contacted therewith are added thereto. As
discussed above, the glycosyltransferase used in accordance with
the present invention is frequently a mixture of
glycosyltransferases containing at least one glycosyltransferase
possessing the desired activity, but purified single
glycosyltransferases may also be used in accordance with the
present invention. In this preferred embodiment, either the mixture
of glycosyltransferases or the purified single glycosyltransferase
may be immobilized. Alternatively, the glycosyltransferase, donor
and acceptor are each provided in solution and contacted as
solutes.
[0062] A preferred procedure for immobilization of
glycosyltransferases--a- nd of acceptor moieties, where
necessary--is based on the copolymerization in a neutral buffer of
a water soluble prepolymer such as
poly(acrylamide-co-N-acryloxysuccinimide (PAN), a cross-linking
diamine such as triethylenetetramine, and the glycosyltransferase,
as disclosed by Pollack et al., J. Am. Chem. Soc. (1980)
102:6324-36. The immobilization of the enzymes on PAN is useful
because small amounts of enzyme can be used, high yields of enzyme
activity are obtained, and the bond between enzyme and polymer is
stable.
[0063] More preferred methods of immobilization include
immobilization of the glycosyltransferase amino groups onto solid
support oxirane groups (see, e.g., Chun et al, Enzyme Eng. (1980)
5:457-460) or onto cyanogen bromide activated "SEPHADEX" or
"SEPHAROSE" (Axen et al, Nature (1967) 214:1302-1304).
[0064] In a preferred embodiment, the glycosyltransferase is
immobilized from a moderately purified composition containing the
glycosyltransferase. Extremely pure enzyme preparations (ie, with
specific activities in the range of 1 nMole transferred per .mu.g
protein per minute of incubation) are less efficiently immobilized
covalently to solid supports, in that the percent derivatization is
lower, compared to 10 or 100 times less pure preparations.
[0065] It will be appreciated that impairment of the active sites
of the glycosyltransferase due to immobilization should be avoided.
The inventor observed that contaminating enzyme activities tend to
disappear during the immobilization process as compared to the
activity of the glycosyltransferase of interest which is
specifically protected during the immobilization process. During
the immobilization process the glycosyltransferase may be protected
by the cation required by the enzyme, the nucleotide recognized by
the enzyme, and the acceptor recognized by the enzyme. For example,
a galactosyl transferase may be protected with Mn.sup.2+,
N-acetylglucosamine and UDP during the immobilization, regardless
of which immobilization method is used. In this way, contaminating
proteases are not protected in any way during the immobilization
process.
[0066] Because only the desired glycosyltransferase is protected
during the immobilization process, enzymes that interfere with the
synthesis of the target saccharide composition tend to be lost.
Examples of interfering enzymes are proteases, which would
otherwise attack the desired glycosyltransferase, and glycosidases,
which would otherwise attack the product saccharide.
[0067] As noted above, in accordance with the invention, a
saccharide composition prepared by contacting an acceptor moiety
with a donor moiety and a glycosyltransferase can, in turn, serve
as an acceptor moiety for isolating further enzymes and as an
acceptor moiety to which subsequent saccharide units may be
transferred. The addition of saccharide units to saccharide
compositions prepared by such contact is preferred for the
synthesis of carbohydrates and saccharide chains having greater
than about three saccharide units.
[0068] For example, in preparing the trisaccharide
N-acetylneuraminyl .alpha.2-3 galactosyl .beta.1-4
N-acetylglucosamine, the disaccharide galactosyl .beta.1-4
N-acetylglucosamine is prepared according to this invention and
then employed as an acceptor moiety to which a subsequent unit is
added. Those skilled in the art will appreciate that the saccharide
units attached to the saccharide compositions of this invention can
be the same or different.
[0069] The saccharide compositions of this invention find use in an
exceedingly wide variety of applications and may be used in the
same manner as saccharide compositions available from known
sources. It is preferred that the saccharide compositions be
employed in therapeutic and preventative treatments for mammals,
such as disclosed in U.S. Ser. No. 07/241,012.
[0070] The saccharide compositions of this invention are expected
to find use as blocking agents for cell surface receptors in the
treatment of numerous diseases of viral, bacterial, or fungal
origins, such as pneumonia, candidiasis, urinary tract infections,
periodontal disease, and diarrhea. For example, oligosaccharides
prepared according to this invention may inhibit the attachment of
pathogens such as pneumonia-causing bacteria to mammalian membrane
molecules. Such pathogens might be incubated with cellular
glycoproteins and glycolipids that have been separated by
chromatography or electrophoresis. After detecting specific
adherence patterns, the target compound could be analyzed and
inhibitory saccharide composition prepared. If either of the
complimentary molecules functions through its saccharide component,
then specific saccharide compositions should inhibit
attachment.
[0071] The saccharide compositions which can be prepared in
accordance with the invention can be used in the following
applications:
[0072] 1. Nutritional Supplements:
[0073] infant formulas 1
[0074] geriatric formulas
[0075] special care formulas
[0076] 2. Antibacterials:
[0077] pneumonia (e.g., 2
[0078] urinary tract infection 3
[0079] dental carries 4
[0080] periodontal disease 5
[0081] diarrhea 6
[0082] surgical (nosocomial) infections
[0083] catheter-associated infections
[0084] 3. Antitumor:
[0085] solid tumor metastases 7
[0086] 4. Anti-inflammatory:
[0087] neutrophil-platelet interactions
[0088] WBC-endothelium interactions
[0089] 5. Naval Drag-reduction:
[0090] ship hulls
[0091] 6. Contraceptives 8
[0092] foam and jelly components
[0093] 7. Antivirals:
[0094] Herpes
[0095] influenza
[0096] HIV
[0097] 8. Antifungals and Yeasts
[0098] oral and vaginal candidiasis (e.g., glucomannan complex,
.alpha.-D-MAN(1-6).sub.n branched .alpha.-(1-2) with L-RHAM, D-Gal,
9
[0099] actinomycetes
[0100] 9. Food Additives: 10
[0101] emulsifiers
[0102] thickeners (e.g., carrageenan 11
[0103] 10. Veterinary:
[0104] antibacterial
[0105] antiviral
[0106] antifungal
[0107] anti-inflammatory
[0108] The present invention thus also provides pharmaceutical and
other compositions, such as foodstuff compositions, containing
saccharide compositions prepared in accordance with the present
invention. In both the pharmaceutical compositions and the
foodstuff compositions provided by the invention, the saccharide
composition of the invention may be present in an amount of from
10.sup.-3 .mu.g ml.sup.-1 to 100 mg ml.sup.-1.
[0109] The concentration of the saccharide composition of the
present invention in any given particular pharmaceutical
composition or foodstuff composition will vary in terms of the
activity of the saccharide being used. For pharmaceutical
compositions the concentration of saccharide present in the
composition will depend on the in vitro activity measured for any
given compound. For foodstuff compositions, the concentration of
the saccharide composition of the present invention may be
determined measuring the activity of the compound being added.
[0110] For example, mother's milk contains the saccharide
composition set forth above where it is indicated as being useful
both in infant formula and as an antibacterial for fighting urinary
tract infections. As such, the present invention provides an
improvement in commercial infant formulas by permitting the
addition to these commercial infant formulas the saccharide
composition illustrated above. The particular saccharide
composition illustrated above may be present in the commercial
infant formula in an amount of 0.1 .mu.g per ml to 1000 .mu.g per
ml. It is present in mother's milk at ca. 10 .mu.g per ml.
[0111] The pharmaceutical compositions should be pyrogen free.
Pharmaceutical compositions in accordance to the present invention
may be prepared as is known in the art so as to be suitable for
oral, intravenous, intramuscular, rectal, transdermal or nasal
(e.g., nasal spray) administration. It may also be prepared for
topical administration in the form of creams, ointments,
suspensions, etc.
[0112] A few saccharides have been noted as being important both as
commodity chemicals in the food, textile, and petroleum industries,
and as specialty chemicals, primarily in the medical field. To
date, the absence of an efficient process for preparing saccharide
compositions has made it impossible to obtain commercial
compositions containing, as an active ingredient, a saccharide
composition.
[0113] The present invention makes such saccharide compositions
readily available in large quantity for the first time. With the
method of the present invention, saccharide compositions heretofore
available only in miniscule quantities, and saccharide compositions
heretofore unavailable, are readily made in gram and kilogram
quantities. The purity of the saccharide compositions provided in
accordance to the present invention exceeds 95 wt. %. In some
applications requiring a high level of purity, the method of the
present invention can be used to obtain saccharide compositions
containing purity levels of from 98 wt. % to essentially 100 wt.
%.
[0114] The present invention thus now provides for the first time
pharmaceutical compositions and other compositions containing
saccharide compositions present invention present in an effective
amount. The present invention provides compositions containing the
saccharide compositions obtained in accordance with the present
invention present in the amount of at least 100 mg, preferably at
least 500 mg, and up to 95 wt. % of the composition.
[0115] In another embodiment, the present invention provides an
apparatus suitable for use in accordance with the present invention
for the glycosyltransferase catalyzed synthesis of a saccharide
composition. Illustrative configurations for such apparatus are
provided in FIGS. 1, 2 and 3.
[0116] In a very basic embodiment the apparatus of the present
invention contains one reaction chamber in which all of the
glycosyltransferases, all the preselected saccharide units and the
initial acceptor moiety are combined. Due to the specificity of the
glycosyltransferases, this mixture, given sufficient time, will
produce the saccharide composition of the present invention.
[0117] FIGS. 1, 2 and 3 illustrate more efficiently designed
apparatuses which may be used in accordance with the present
invention. The apparatuses illustrated in the figures, comprise, as
their basic elements, a reactor equipped with an inlet and an
outlet. The reactor is suitable,for carrying out the sequential
covalent bonding of a plurality of preselected saccharide units
onto an acceptor moiety, catalyzed by a plurality of corresponding
glycosyltransferases specific to each covalent bonding. It contains
at least three, preferably four, and even more preferably a number
greater than four, such as five, six, seven, or more, different,
glycoltransferases which are preferably immobilized.
[0118] The inlet means is suitable for introducing the acceptor
moiety and the plurality of preselected saccharide units into the
reactor such that the saccharide composition is synthesized.
Preferably, the inlet means is suitable for also introducing into
the reactor the glycosyltransferases which are themselves
preferably immobilized. The outlet means permits discharging the
saccharide composition from the reactor.
[0119] FIG. 1 illustrates a column-type reactor charged with a
solid support matrix. The various glycosyltransferases (enzymes 1,
2, 3) used in the process may be either randomly distributed
throughout the solid support matrix or they may be arranged in
zones as illustrated in FIG. 1. The initial acceptor moiety (shown
as A in the figures) and the preselected saccharide units (shown as
B, C and D in the figures) are charged into the reactor via the
inlet means and passed through the solid support matrix whereupon
the saccharide composition is produced due to the action of the
specific glycosyltransferases and recovered via the outlet means as
molecule A-B-C-D.
[0120] In the embodiment illustrated in FIG. 2, the initial
acceptor moiety and the preselected saccharide unit to be attached
to the initial acceptor moiety are charged at the top of the solid
support matrix, with the glycosyltransferases specific to the
addition of each preselected saccharide units being arranged in
corresponding zones along the direction of flow of the reaction
mixture. The various preselected saccharide units are then
individually added at correspondingly appropriate locations along
the flow of the reaction mixture as shown in the figure.
[0121] In another preferred embodiment, illustrated in FIG. 3, the
reactor comprises a plurality of (n) reaction zones serially
connected so as to be in sequential fluid communication with each
other where (n) roughly corresponds to not more than the number of
saccharide units being attached. Each reaction zone contains at
least one glycosyltransferase specific to catalyze the bonding of a
particular preselected saccharide unit onto the intermediate
product formed in the preceding reaction zone.
[0122] In accordance with this embodiment the initial acceptor
moiety (A) and the first preselected saccharide unit (B) to be
attached to the acceptor moiety are passed through the first
reaction zone which comprises a glycosyltransferase specific to
catalyze the bonding of the first preselected saccharide unit onto
the initial acceptor moiety thus producing a first intermediate
product. This first intermediate product is then transferred to the
second reaction zone (n=1) where it is combined with the second
preselected saccharide unit (X.sub.n) and the glycosyltransferase
(E.sub.1+n) specific to catalyze the bonding of the second
preselected saccharide unit with the first intermediate product
formed. This process is repeated in a corresponding number of
reaction zones until the target saccharide composition provided by
the invention and illustrated as A--B--(X)--.sub.nZ, wherein each X
moiety is independently selected and n is an integer of from 1 to
500 or more, is obtained.
[0123] In another preferred embodiment, also illustrated in FIG. 3,
means for purifying 4 each intermediate product formed from the
reaction mixture emanating from any given reaction zone are
situated in fluid communication and between each of the reaction
zones. The means for purifying, which may comprise, e.g., an ion
exchange resin, remove contaminants in the reaction mixtures which
inhibit the efficiency of the bonding of the next preselected
saccharide unit onto the intermediate product formed.
[0124] Additional objects, advantages, and novel features of this
invention will become apparent to those skilled in the art upon
examination of the following examples thereof, which are not
intended to be limiting.
EXAMPLE 1
Preparation of the Trisaccharide N-Acetylneuraminyl .alpha.2-3
Galactosyl .beta.1-4 N-Acetylglucosamine
[0125] To each of five test tubes was added 10 .mu.l of pH 7.4
potassium phosphate buffer, 10 .mu.l of 50 MM MnCl.sub.2, 17,000
CPM of cytidine monophosphate-[.sup.14C]-N-acetyineuraminic acid,
25 .mu.l of galactosyltransferase, and 25 .mu.l of
N-acetyineuraminyltransferase. The glycosyltransferases were
purified from bovine colostrum by Sephadex G-100 gel
chromatography.
[0126] To test tube 1 was also added 10 .mu.l of 40 mm uridine
diphosphate galactose and 10 .mu.l of 40 mM N-acetylglucosamine.
Test tube 1 was incubated in ice for one hour.
[0127] To test tube 2 was also added 10 .mu.l of 40 mM uridine
diphosphate galactose. Test tube 2 was incubated at 37.degree. C.
for one hour.
[0128] To test tube 3 was also added 10 .mu.l of 40 mM
N-acetyllactosamine. Test tube 3 was incubated at 37.degree. C. for
one hour.
[0129] To test tubes 4 and 5 were also added 10 .mu.l of 40 mM
uridine diphosphate galactose and 10 .mu.l of 40 mM
N-acetylglucosamine. Test tubes 4 and 5 were incubated at
37.degree. C. for one hour.
[0130] After incubation, the contents of the test tubes were each
subjected to high voltage electrophoresis on paper saturated with
sodium tetraborate. Isotopically labeled trisaccharide product was
identified by its mobility, as demonstrated by the product formed
in test tube 3.
1 Test Tube Trisaccharide (cpm) 1 0 2 0 3 3375 4 670 5 954
[0131] As can be seen, the presence of suitable acceptor moieties,
donor moieties, and glycosyltransferases in test tubes 4 and 5
yielded the expected trisaccharide product from monosaccharide
starting materials. Typically, the sialic acid N-acetylneuraminate
presents special problems for synthetic organic chemists seeking to
incorporate it into saccharide compositions, due to the acid
lability of its glycosidic bond. Synthesizing a trisaccharide from
cytidine monophosphate N-acetylineuraminic acid enzymatically
eliminates the synthetic problems associated with removing
protecting groups under strong acidic condition.
[0132] It is believed that an acceptor moiety (N-acetylglucosamine)
initially contacts a donor moiety (uridine diphosphate galactose)
and a glycosyltransferase (galactosyltransterase) to produce a
saccharide composition (galactosyl .beta.1-4 N-acetylglucosamine) ,
which then acts as an acceptor moiety upon contacting a second
donor moiety (cytidine monophosphate N-acetylneuraminic acid) and a
second glycosyltransferase (N-acetylneuraminyltransferase).
[0133] The synthesis of the trisaccharide product in test tubes 4
and 5 from monosaccharide starting materials is confirmed by
comparison with the product of test tube 3, in which the
trisaccharide is formed by contacting a disaccharide acceptor
moiety (N-acetyllactosamine) with cytidine monophosphate
N-acetylneuraminic acid and N-acetylneuraminyltransferase.
[0134] The absence of trisaccharide in test tube 2 illustrates that
a suitable acceptor moiety is necessary for trisaccharide
formation. The absence of trisaccharide in test tube 1 indicates
that the synthesis of the trisaccharide is, indeed, dependent upon
the action of any enzyme (the glycosyltransferase) that is inactive
at low temperatures.
[0135] It is expected that the oligosaccharides
N-acetylgalactosaminyl .alpha.1-3 (fucosyl .alpha.1-2) galactosyl
.beta.1-4 N-acetylglucosaminyl .beta.1-3 galactose (a target for
diarrhea-causing bacteria) and N-acetylgalactosaminyl .beta.1-4
galactosyl .beta.1-4 glucose (a target for pneumonia-causing
bacteria) can likewise be prepared by the processes of the present
invention.
EXAMPLE 2
Tetrasaccharide Biosynthesis Protocol
[0136] Enzymes:
[0137] N-acetylglucosaminyltransferase:
[0138] Human colostrum is centrifuged for one hour at
70,000.times.G. A 25% saturated ammonium sulfate cut yields a
supernatant that is dialyzed to remove the ammonium sulfate. The
retentate is applied to a Sephadex G-200 column (2.5.times.83 cm).
The protein profile is determined spectrophotometrically at 280 nm,
and a radioactive assay is performed to locate the fractions with
transferase activity. The fractions containing the single enzyme
peak are pooled and concentrated 10-fold by Amicon filtration. The
pooled enzyme preparation is again assayed, and the protein
concentration is determined using a BioRad assay. The specific
activity of the preparation is 5.3 pMoles per .mu.g
protein-min.
[0139] Galactosyltransferase:
[0140] Human colostrum is centrifuged at 8700.times.G for 15
minutes. The supernatant is poured through cheesecloth and 10 ml is
applied to a Sephadex G-100 column (2.5.times.90 cm). The protein
profile is determined spectrophotometrically at 280 nm, and a
radioactive assay is performed to locate the fractions with enzyme
activity. The fractions with the highest activity are pooled and
concentrated 10-fold by Amicon filtration. The pooled enzyme
preparation is again assayed, and the protein concentration is
determined as above. The specific activity of the preparation is
15.4 pMoles per .mu.g protein-min.
[0141] Enzyme Immobilization:
[0142] N-acetylglucosaminyltransferase:
[0143] 300 mgs of Eupergit beads (1.2 ml) are washed three times
with deionized water, and then three times with aseptic
Hepes-buffered water. One ml of the enzyme preparation is combined
aseptically with the beads along with UDP, lactose, MnCl.sub.2,
(final concentrations: 10, 25, and 10 mM, respectively) and a drop
of chloroform in a Hepes-buffered solution. The beads are gently
agitated at 4.degree. C. for 21/2 days. Aliquots are taken and
assayed periodically. To stop the derivatization, the beads are
washed three times with an aseptic buffer, and stored in buffer, in
the cold, with UDP, lactose, MnCl.sub.2, and chloroform.
[0144] Galactosyltransferase:
[0145] 3.75 grams of beads are washed three times with deionized
water, and then three times with aseptic Hepes-buffered water. The
beads are added to 3-mls of the enzyme preparation (in both cases,
optimum derivatization occurs at about 1 mg protein per 200 mgs
beads) along with UDP, GlcNAc, MnCl.sub.2, (final concentrations
are all 10 mM) and a drop of chloroform in a Hepes-buffered
solution. Derivatization and storage are as described above, except
that the GlcNac is used with the galactosyltransferase in place of
lactose, which is the acceptor for the
N-acetylglucosaminyltransferase.
[0146] Tetrasaccharide Production:
[0147] Derivatized N-acetylglucosaminyltransferase (0.5 ml beads)
is incubated under constant stirring with lactose (25 mM),
UDPGlcNAc (80 .mu.M), and MnCl.sub.2 (10 mM) for 21 hours. This
incubation is carried out in duplicate-the supernatant of one
incubation is used to measure the amount of trisaccharide produced
(14 .mu.g), and the supernatant from the other incubation is added
to 0.5 ml beads derivatized with the galactosyltransferase. The
galactosyltransferase incubation contains, therefore, 14 .mu.g of
trisaccharide, 25 .mu.M UDPgal, and 10 mM MnCl.sub.2. After 24
hours at room temperature, the second enzyme preparation produces
about 1.6 .mu.g of tetrasaccharide. After 31 hours, 2.2 .mu.g of
tetrasaccharide were produced.
EXAMPLE 3
[0148] The following schemes will be used for synthesizing three,
relatively complex oligosaccharides: the A- and B-type milk
oligosaccharides (I and II), and gum tragacanth (III), a plant
oligosaccharide used by the ton as a food additive.
galNAC.alpha.1,.fwdarw.(fuc.alpha.1,2.fwdarw.)gal.beta.1,3.fwdarw.(fuc.alp-
ha.1,4.fwdarw.)GlcNAc.beta.1,3.fwdarw.gal.beta.1,4.fwdarw.glc
(I)
[0149] First, the hexanolamine glycoside
(glc-O--(CH.sub.2).sub.6--NH.sub.- 2) of glucose that will be
affixed to CNBr-activated supports, e.g., Sepharose, via the amino
group of the hexanolamine will be synthesized. Then the
glucose-recognizing galactosyltransferase will be purified from
human milk or colostrum using this affinity ligand. The enzyme,
once partly purified, will be used to galactosylate glucose, making
lactose.
[0150] Alternatively, the hexanolamine glycoside of lactose, which
is an inexpensive and readily available disaccharide, will be
synthesized. The lactose so produced will be attached to Sepharose
and used as an affinity ligand to purify in part the
N-acetylglucosaminyltransferase from human colostrum, or from human
plasma.
[0151] Next, this second transferase will be used to add
N-acetylglucosamine to lactose, making the trisaccharide, which
will again be attached to Sepharose. This bound trisaccharide will
be used to obtain the .beta.1,3 galactosyltransferase (from porcine
submaxillary gland), which will, in turn, yield the substrate for
purifying the next enzyme--the .alpha.1,4 fucosyltransferase (from
porcine liver). The .alpha.1,2 fucosyltransferase (from porcine
submaxillary gland), and, finally, the .alpha.1,3
N-acetylgalactosaminyltransferase (from porcine submaxillary
glands) that terminates the synthesis of the A-type milk
oligosaccharide will be affinity purified in this step-wise
fashion. Each transferase so obtained will be immobilized to a
solid matrix by any of several means, and the matrices will be
poured in column configurations.
[0152] The enzyme-containing columns will be used sequentially, in
the same order that the smaller amounts of derivatized substrates
were synthesized, to synthesize large amounts each soluble
oligosaccharide.
[0153] The order of attachment of the sugars is critical. The
proximal fucose (that attached .alpha.1,4 to glcNAc) must be
attached to the completed core tetrasaccharide before the addition
of the second fucose (that attached .alpha.1,2 to the galactose.
Finally, the terminal galNAc (.alpha.1,3) is added to complete the
seven-sugar oligosaccharide. This order is required by the
specificities of the glycosyltransferases.
gal.alpha.1,3.fwdarw.(fuc.alpha.1,2.fwdarw.)gal.beta.1,3.fwdarw.(fuc.alpha-
.1,4.fwdarw.)GlcNAc.beta.1,3.fwdarw.gal.beta.1,4.fwdarw.glc
(II)
[0154] Having synthesized I, II will be synthesizes in precisely
the same fashion, except that the hexasaccharide will be used,
first, to purify an .alpha.1,3 galactosyltransferase that will be
derivitized with protective groups for a galactosyl-, and not an
N-acetylgalactosaminyltransferase. This enzyme will then be used to
synthesize the B-type oligosaccharide.
[ . . .
(fuc.alpha.1,3.fwdarw.xy1.beta.1,3.fwdarw.)galA.alpha.1,4.fwdarw.(-
gal,.beta.1,4.fwdarw.xy1.beta.1,3.fwdarw.)galA . . . ] (III)
[0155] To isolate the enzyme that synthesizes the .alpha.1,4
galacturonic acid backbone of gum tragacanth, which currently is
available only from the bark of a tree species indigenous to the
Middle East, hexagalacturonans will be prepared from pectin, a
common constituent of citrus rinds, and used as an affinity
ligand.
[0156] The same affinity ligand can next be used to isolate from
tree tissues the xylosyltransferase that synthesizes the proximal
.beta.1,3 xylosides. The xylosylated galacturonans, once
derivatized, will be used to isolate both the fucosyl- and
galactosyltransferases that, respectively, fucosylate and
galactosylate the xylosylated galacturonan. In the case of this
oligosaccharide, the degree of xylosylation, fucosylation, and
galactosylation will be controlled empirically by the number of
passes of the compounds through the appropriate enzyme-containing
columns. The number of repeat units produced will depend on the
number of galacturonic acid residues used initially; this number
will vary in length from four to twenty monosaccharide units.
[0157] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
* * * * *