U.S. patent application number 14/036583 was filed with the patent office on 2014-04-03 for sugar chain-capturing substance and use thereof.
This patent application is currently assigned to NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY. The applicant listed for this patent is NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY, SUMITOMO BAKELITE COMPANY LIMITED. Invention is credited to Jun-ichi FURUKAWA, Yoshiaki MIURA, Shinichiro NISHIMURA, Hideyuki SHIMAOKA, Yasuro SHINOHARA.
Application Number | 20140094569 14/036583 |
Document ID | / |
Family ID | 39032711 |
Filed Date | 2014-04-03 |
United States Patent
Application |
20140094569 |
Kind Code |
A1 |
SHIMAOKA; Hideyuki ; et
al. |
April 3, 2014 |
SUGAR CHAIN-CAPTURING SUBSTANCE AND USE THEREOF
Abstract
The present invention provides a method for preparing a sample
characterized by binding a substance A containing a hydrazide group
to a sugar chain and/or a sugar derivative via hydrazone formation
between the hydrazide group of the substance A and the reducing end
of the sugar chain and/or the sugar derivative thereby to enable
the separation and purification of the sugar chain and/or the sugar
derivative for an analytical sample from a biological sample
containing the sugar chain and/or the sugar derivative by a simple
operation.
Inventors: |
SHIMAOKA; Hideyuki; (Tokyo,
JP) ; NISHIMURA; Shinichiro; (Hokkaido, JP) ;
SHINOHARA; Yasuro; (Hokkaido, JP) ; MIURA;
Yoshiaki; (Hokkaido, JP) ; FURUKAWA; Jun-ichi;
(Hokkaido, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY
SUMITOMO BAKELITE COMPANY LIMITED |
Sapporo-Shi
Tokyo |
|
JP
JP |
|
|
Assignee: |
NATIONAL UNIVERSITY CORPORATION
HOKKAIDO UNIVERSITY
Sapporo-Shi
JP
SUMITOMO BAKELITE COMPANY LIMITED
Tokyo
JP
|
Family ID: |
39032711 |
Appl. No.: |
14/036583 |
Filed: |
September 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12309945 |
Feb 4, 2009 |
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PCT/JP2007/000838 |
Aug 6, 2007 |
|
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14036583 |
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Current U.S.
Class: |
525/328.4 ;
526/301; 560/158 |
Current CPC
Class: |
C08F 222/102 20200201;
Y02P 20/55 20151101; C08J 3/24 20130101; C08F 122/38 20130101; G01N
2030/027 20130101; C07C 271/08 20130101; C08F 8/30 20130101; C07C
281/02 20130101; G01N 2030/8813 20130101; G01N 33/5308 20130101;
G01N 2030/8836 20130101; C07C 233/20 20130101; C08F 8/30 20130101;
Y10T 428/2982 20150115; C08F 220/60 20130101 |
Class at
Publication: |
525/328.4 ;
560/158; 526/301 |
International
Class: |
C08F 122/38 20060101
C08F122/38; C07C 271/08 20060101 C07C271/08 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 2006 |
JP |
2006-217165 |
Claims
1-54. (canceled)
55. A monomer having a structure represented by the following
formula (13), ##STR00042## wherein, in the formula, R.sub.2
represents H, CH.sub.3 or a hydrocarbon chain having 2 to 5 carbon
atoms; R.sub.6 represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--; and [P] represents a protective group.
56. A monomer according to claim 55, wherein the monomer has a
structure represented by the following formula (14),
##STR00043##
57. A polymer obtained by polymerizing the monomer according to
claim 55.
58. A polymer obtained by treating the polymer according to claim
57 with acid to deprotect the protective group to obtain the
polymer, wherein the obtained polymer is the polymer according
claim 3 having a hydrazide group at the side chain end.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for preparing a
sample using a prescribed sugar chain-capturing substance, and an
analytical sample obtained by the method. The present invention
relates to a method for preparing a sugar chain-capturing
substance, a compound used for the method and a polymer obtained by
polymerizing the compound. Further, the present invention relates
to the use of the sugar chain-capturing substance, for example, a
method for applying the sugar-chain capturing substance, a sugar
chain microarray, the use of the sugar chain microarray, sugar
chain affinity beads and the use of the sugar chain affinity
beads.
BACKGROUND ART
[0002] A biological polymer refers to a general term of a sugar
chain, glycoprotein, glycopeptide, peptide, oligopeptide, protein,
nucleic acid, lipid or the like.
[0003] Furthermore, these biological polymers play an importance
role in biotechnology fields such as medical science, cell
engineering, organs and medical engineering and the like. To
clarify the control mechanism of the biological reaction using
these substances is related to the development in the biotechnology
fields.
[0004] Of these biological polymers, a sugar chain is extremely
rich in its diversity, and is a substance participating in various
functions of an organism present in the nature. The sugar chain is
present as glycoconjugate bonded to protein, lipid or the like in
vivo in many cases, and is one of important components in vivo. It
has become clear that the sugar chain in vivo is deeply related to
information transfer between cells, regulation of functions or
interaction of protein, and the like.
[0005] Incidentally, the term "sugar chain" refers to a generic
term of a chain of molecules coupled with monosaccharide such as
glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine,
N-acetylgalactosamine, sialic acid or the like and a derivative
thereof by a glycosidic bond.
[0006] Examples of the biological polymer having a sugar chain
include proteoglycan of a cell wall of a plant cell contributing to
the stability of cells, glycolipid, affecting cell differentiation,
population growth, adhesion, migration or the like, glycoprotein
taking part in intercellular interaction or cellular recognition
and the like. A mechanism of the sugar chain contained in these
biological polymers of controlling a high-precision biological
reaction while acting for, helping, amplifying, regulating or
hindering functions mutually with the biological polymer has been
gradually made clear. Furthermore, when the relationship between
such a sugar chain and cell differentiation, population growth,
cell adhesion, immunity and a malignant change (cancer) in cells
becomes clear, a new development can be expected to be planned by
closely relating this sugar chain engineering to the medical
science, cell engineering or organs and medical engineering.
[0007] In Patent Document 1, there have been described a substance
capable of specifically reacting with such a sugar chain and a
method of separating a sugar chain by using the substance as well.
[0008] Patent Document 1: International Publication Pamphlet No
2004/058687
DISCLOSURE OF THE INVENTION
[0009] By the way, in Patent Document 1, there has been described
an example using an acid treatment employing trifluoroacetic acid,
acidic resin or the like in order to release (excise) the sugar
chain captured by using a sugar chain-capturing substance from the
sugar chain-capturing substance. To expose the sugar chain under
such severe conditions may cause degeneration of the sugar chain
such as separation of a sialic acid residue having a property of
being bonded to a terminal end of the sugar chain that is taken out
from a biological sample and a property of being easily separated
under acidic conditions. So, it has been demanded that excision of
the sugar chain is carried out under much milder conditions.
Incidentally, the existence of the sialic acid to be bonded to the
sugar chain and the binding site are related to diseases in many
cases so that it has been demanded that the sugar chain is analyzed
in the perfect state of the sialic acid. When even a part of the
sialic acid is separated at the pre-treatment step before analysis,
accurate information of the sugar chain cannot be obtained.
[0010] Then, the present invention is to provide a method for
preparing a sample which enables the separation and purification of
the sugar chain and/or the sugar derivative for an analytical
sample from a biological sample containing a sugar chain and/or a
sugar derivative by a simple operation, and an analytical sample
obtained by this method. Furthermore, the present invention is to
provide a method for preparing a sugar chain-capturing substance
which is used in the aforementioned method for preparing a sample,
a monomer which can be used for the preparation method, and a
polymer obtained by polymerizing this monomer. Further, the present
invention is to provide the use of the aforementioned method for
preparing a sample.
[0011] The present invention provides:
[0012] (1) a method for preparing a sample in which a substance A
containing a hydrazide group is bonded to a sugar chain and/or a
sugar derivative via hydrazone formation between the hydrazide
group of the substance A and the reducing end of the sugar chain
and/or the sugar derivative;
[0013] (2) the method for preparing a sample as set forth in (1),
in which the substance A includes a moiety containing chromophore
or fluorophore;
[0014] (3) the method for preparing a sample as set forth in (1),
in which the substance A is a substance selected from the following
substances or a salt thereof,
[0015] (substance A): 5-Dimethylaminonaphthalene-1-sulfonyl
hydrazine (Dansylhydrazine); 2-hydrazinopyridine; 9-fluorenylmethyl
carbazate (Fmoc hydrazine); benzylhydrazine;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionoc
acid, hydrazide;
2-(6,8-difluoro-7-hydroxy-4-methylcoumarin)acetohydrazide;
7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DOCH);
phenylhydrazine; 1-Naphthaleneacethydrazide; 2-hydrazinobenzoic
acid; biotin hydrazide; and phenylacetic hydrazide;
[0016] (4) the method for preparing a sample as set forth in (1),
in which the substance A contains a moiety consisting of at least
one of an arginine residue, a tryptophan residue, a phenylalanine
residue, a tyrosine residue, a cysteine residue and a derivative
thereof;
[0017] (5) the method for preparing a sample as set forth in (4),
in which the substance A is a compound having a structure of the
following formula (1),
##STR00001##
[0018] wherein, in the formula, R represents --CH.sub.3 or
--CD.sub.3;
[0019] (6) the method for preparing a sample as set forth in (1),
in which the substance A has a structure of the following formula
(2),
##STR00002##
[0020] wherein, in the formula, R represents any of H, --COCH.sub.3
gar --COCD.sub.3;
[0021] (7) the method for preparing a sample as set forth in (1),
in which the substance A is represented by the following formula
(3),
[Chemical Formula 3]
(Carrier)-R--NHNH.sub.2 (Formula 3)
[0022] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--;
[0023] (8) the method for preparing a sample as set forth in (7),
in which the substance A has a crosslinked polymer structure
represented by the following formula (4),
##STR00003##
[0024] wherein, in the formula, R.sub.1 and R.sub.2 represent a
hydrocarbon chain having 1 to 20 carbon atoms which may be
interrupted with --O--, --S--, --NH--, --CO-- or --CONH--; R.sub.3,
R.sub.4 and R.sub.5 represent H, CH.sub.3 or a hydrocarbon chain
having 2 to 5 carbon atoms; and m and n represent the number of
monomer units;
[0025] (9) the method for preparing a sample as set forth in (7),
in which the substance A has a crosslinked polymer structure
represented by the following formula (5),
##STR00004##
[0026] wherein, in the formula, m and n represent the number of
monomer units;
[0027] (10) the method for preparing a sample as set forth in any
one of (7) to (9), in which the substance A is a polymer particle
having an average particle diameter of equal to or more than 0.1
and equal to or less than 500 .mu.m;
[0028] 11) the method for preparing a sample as set forth in any
one of (7) to (10), in which the substance A is a polymer particle
having a hydrazide group of a dry weight of not less than 100 nmol
per 1 mg
[0029] (12) the method for preparing a sample as set forth in any
one of (7) to (10), in which the substance A is a polymer particle
having a hydrazide group of a dry weight of not less than 0.5
.mu.mol per 1 mg;
[0030] (13) the method for preparing a sample as set forth in any
one of (7) to (12), in which the substance A is stable at the pH of
3 to 8;
[0031] (14) the method for preparing a sample as set forth in any
one of (7) to (12), in which the substance A is stable under
pressure of at least not more than 1 MPa;
[0032] (15) the method for preparing a sample as set forth in (7),
in which the carrier in the above formula (3) is a substance to be
directly bonded to a solid phase substrate or a surface of the
solid phase substrate;
[0033] (16) a method for preparing a sample which involves the
sugar chain capture step including binding a substance A to a sugar
chain and/or a sugar derivative according to the method for
preparing a sample as set forth in any one of (1) to (15), and
[0034] the sugar chain release step including the action of a
substance B containing an aminooxy group or a hydrazide group on a
complex of the substance A and the sugar chain and/or the sugar
derivative, captured in the sugar chain capture step and binding
the sugar chain and/or the sugar derivative while cut off from the
substance A to the substance B in accordance with the
hydrazone-oxime exchange reaction or the hydrazone-hydrazone
exchange reaction occurred between the complex and the substance
B;
[0035] (17) the method for preparing a sample as set forth in (16),
in which the substance B includes a moiety containing chromophore
or fluorophore;
[0036] (18) the method for preparing a sample as set forth in (16),
in which the substance B is a substance selected from the group
consisting of the following substances containing a hydrazide group
or the following substances containing an aminooxy group or a
salt,
[0037] (substances containing a hydrazide group)
5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine);
2-hydrazinopyridine: 9-fluorenylmethyl carbazate (Fmoc hydrazine);
benzylhydrazine;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionoc
acid, hydrazide;
2-(6,8-difluoro-7-hydroxy-4-methylcoumarin)acetohydrazide;
7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH);
phenylhydrazine; 1-Naphthaleneacethydrazide; 2-hydrazinobenzoic
acid; and phenylacetic hydrazide; or
[0038] (substances containing an aminooxy group)
O-benzylhydroxylamine; O-phenylhydroxylamine;
O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine;
O-(4-nitrobenzyl)hydroxylamine; 2-aminooxypyridine;
2-aminooxymethylpyridine; 4-[(aminooxyacetyl)amino]benzoic acid
methyl ester; 4-[(aminooxyacetyl)amino]benzoic acid ethyl ester;
and 4-[(aminooxyacetyl)amino]benzoic acid n-butyl ester;
[0039] (19) the method for preparing a sample as set forth in (16),
in which the substance B contains a moiety consisting of at least
one of an arginine residue, a tryptophan residue, a phenylalanine
residue, a tyrosine residue, a cysteine residue and a derivative
thereof;
[0040] (20) the method for preparing a sample as set forth in (16),
in which the substance B has a structure represented by the
following formula (7),
##STR00005##
[0041] wherein, in the structural formula, R represents --CH.sub.3
or --CD.sub.3;
[0042] (21) the method for preparing a sample as set forth in (16),
in which the substance B is a solid phase carrier;
[0043] (22) the method for preparing a sample as set forth in (21),
in which the substance B is a solid phase carrier containing an
aminooxy group;
[0044] (23) the method for preparing a sample as set forth in (22),
in which the substance B is represented by the following formula
(12),
[Chemical Formula 7]
(Carrier)-R--ONH.sub.2 (Formula 12)
[0045] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--;
[0046] (24) the method for preparing a sample as set forth in (23),
in which the substance B is a polymer particle having a structure
represented by the following formula (8),
##STR00006##
[0047] wherein, in the formula, R.sub.1 and R.sub.2 represent a
hydrocarbon chain having 1 to 20 carbon atoms which may be
interrupted with --O--, --S--, --NH--, --CO-- or --CONH--; R.sub.3,
R.sub.4 and R.sub.5 represent H, CH.sub.3 or a hydrocarbon chain
having 2 to 5 carbon atoms; and m and n represent the number of
monomer units;
[0048] (25) the method for preparing a sample as set forth in (23),
in which the substance B is a polymer particle having a structure
represented by the following formula (9),
##STR00007##
[0049] wherein, in the formula, m and n represent the number of
monomer units;
[0050] (26) the method for preparing a sample as set forth in (21),
in which the solid phase carrier is a substance to be directly
bonded to a solid phase substrate or a surface of the solid phase
substrate;
[0051] (27) the method for preparing a sample as set forth in any
one of (16) to (25), in which the hydrazone-oxime exchange reaction
or the hydrazone-hydrazone exchange reaction is carried out at
least one or more times after the completion of the sugar chain
release step;
[0052] (28) a method for preparing a sample in which a hydrazone
bond is dissociated and a sugar chain and/or a sugar derivative is
released by treating a substance A having a structure represented
by the following formula (3), (4) or (5) under acidic conditions to
which the sugar chain and/or the sugar derivative is bonded,
[Chemical Formula 10]
(Carrier)-R--NHNH.sub.2 (Formula 3)
[0053] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--,
##STR00008##
[0054] wherein, in the formula, R.sub.1 and R.sub.2 represent a
hydrocarbon chain having 1 to 20 carbon atoms which may be
interrupted with --O--, --S--, NH--, --CO-- or --CONH--; R.sub.3,
R.sub.4 and R.sub.5 represent H, CH.sub.3 or a hydrocarbon chain
having 2 to 5 carbon atoms; and m and n represent the number of
monomer units,
##STR00009##
[0055] wherein, in the formula, m and n represent the number of
monomer units;
[0056] (29) the method for preparing a sample as set forth in (28),
in which the treatment under acidic conditions is carried out using
a trifluoroacetic acid solution of 0.01 to 10 volume
percentage;
[0057] (30) the method for preparing a sample as set forth in (29),
in which the treatment under acidic conditions is carried out using
a trifluoroacetic acid solution of 0.01 to 1 volume percentage at
25 to 80 degree centigrade for 5 to 60 minutes;
[0058] (31) an analytical sample prepared by the method for
preparing a sample as set forth in any one of (1) to (30);
[0059] (32) a method for preparing a substance A represented by the
following formula (3), in which a polymer particle containing
carboxylic acid ester is obtained by polymerizing a raw material
containing a carboxylic acid ester monomer having a polymerizable
group in the presence of a crosslinking agent, and then the polymer
particle containing carboxylic acid ester is treated with a
hydrazine solution of a concentration of not less than 10 volume
percentage,
[Chemical Formula 13]
(Carrier)-R--NHNH.sub.2 (Formula 3)
[0060] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--;
[0061] (33) the method for preparing a substance A as set forth in
(32), in which the carboxylic acid ester monomer is carboxylic acid
methyl ester;
[0062] (34) a monomer having a structure represented by the
following formula (10),
##STR00010##
[0063] wherein, in the formula, R.sub.1 represents a hydrocarbon
chain having 1 to 20 carbon atoms which may be interrupted with
--O--, --S--, --NH--, --CO-- or --CONH--; and R.sub.2 represents H,
CH.sub.3 or a hydrocarbon chain having 2 to 5 carbon atoms;
[0064] (35) a polymer obtained by polymerizing the monomer as set
forth in (34);
[0065] (36) a monomer having a structure represented by the
following formula (11),
##STR00011##
[0066] (37) a polymer obtained by polymerizing the monomer as set
forth in (36);
[0067] (36) a monomer having a structure represented by the
following formula (13),
##STR00012##
[0068] wherein, in the formula, R.sub.2 represents H, CH.sub.3 or a
hydrocarbon chain having 2 to 5 carbon atoms; R.sub.6 represents a
hydrocarbon chain having 1 to 20 carbon atoms which may be
interrupted with --O--, --S--, --NH--, --CO-- or --CONH--; and [P]
represents a protective group;
[0069] (39) a polymer obtained by polymerizing the monomer as set
forth in (38);
[0070] (40) the polymer as set forth in (39), obtained by
subjecting the protective group [P] of the formula (13) to
deprotection by the acid treatment;
[0071] (41) a monomer having a structure represented by the
following formula (14),
##STR00013##
[0072] (42) a polymer obtained by polymerizing the monomer as set
forth in (41);
[0073] (43) a polymer obtained by subjecting a t-butoxycarbonyl
group of the polymer as set forth in (42) to deprotection by the
acid treatment;
[0074] (44) a method for preparing a substance A represented by the
following formula (4), in which a polymer particle containing
carboxylic acid ester is obtained by polymerizing a monomer having
a structure of the following formula (10) in the presence of a
crosslinking agent, and then the polymer particle containing
carboxylic acid ester is treated with a hydrazine solution of a
concentration of not less than 10 volume percentage,
##STR00014##
[0075] wherein, in the formula, R.sub.1 and R.sub.2 represent a
hydrocarbon chain having 1 to 20 carbon atoms which may be
interrupted with --O--, --S--, --NH--, --CO-- or --CONH--; R.sub.3,
R.sub.4 and R.sub.5 represent H, CH.sub.3 or a hydrocarbon chain
having 2 to 5 carbon atoms; and m and n represent the number of
monomer units,
##STR00015##
[0076] wherein, in the formula, R.sub.1 represents a hydrocarbon
chain having 1 to 20 carbon atoms which may be interrupted with
--O--, --S--, --NH--, --CO-- or --CONH--; and R.sub.2 represents H,
CH.sub.3 or a hydrocarbon chain having 2 to 5 carbon atoms;
[0077] (45) a method for preparing a substance A represented by the
following formula (5), in which a polymer particle containing
carboxylic acid ester is obtained by polymerizing a monomer having
a structure of the following formula (11) in the presence of a
crosslinking agent, and then the polymer particle containing
carboxylic acid ester is treated with a hydrazine solution of a
concentration of not less than 10 volume percentage,
##STR00016##
[0078] wherein, in the formula, m and n represent the number of
monomer units,
##STR00017##
[0079] (46) a method of applying a solid phase carrier onto which a
sugar chain and/or a sugar derivative obtained by subjecting a
mixture or a specific fraction from the mixture to the isolation
and purification is immobilized as a carrier for collecting a
substance having a bonding property or affinity to the sugar chain
and/or the sugar derivative;
[0080] (47) a method for preparing a sugar chain microarray, in
which a sugar chain and/or a sugar derivative is immobilized onto a
surface of a solid phase substrate composed of a solid phase
carrier including a substance B having a structure represented by
the following formula (3) or (12) according to the following steps
(1) to (4),
[0081] (step 1) a step of purifying and/or isolating the sugar
chain and/or the sugar derivative by a specific separation means by
binding the sugar chain and/or the sugar derivative to a compound
containing a soluble hydrazide group,
[0082] (step 2) a step of dispensing drops of a solution of the
compound obtained in the step (1) in a row onto the solid phase
substrate,
[0083] (step 3) a step of proceeding a reaction of exchanging a
sugar chain-substance A bond with a solid phase substrate-sugar
chain bond by incubating the solid phase substrate after the
completion of dispensing drops under prescribed conditions, and
immobilizing the sugar chain and/or the sugar derivative onto the
solid phase substrate, and
[0084] (step 4) a step of washing and removing the unreacted
substance on the solid phase substrate,
[Chemical Formula 22]
(Carrier)-R--NHNH.sub.2 (Formula 3)
[0085] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--,
[Chemical Formula 23]
(Carrier)-R--ONH.sub.2 (Formula 12)
[0086] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--;
[0087] (48) a sugar chain microarray produced by the method as set
forth in (47);
[0088] (49) a system for searching a sugar-binding substance for
specifically binding to or adsorbing the immobilized sugar chain
and/or the immobilized sugar derivative by bringing a solution
containing a specimen into contact with a surface of the sugar
chain mircoarray as set forth in (48), incubating and washing under
prescribed conditions, and then detecting the sugar-binding
substance collected at the sugar chain and/or the sugar derivative
of a drop-dispensed portion on the solid phase substrate;
[0089] (50) a system for evaluating the recognition specificity of
a binding protein by bringing a solution containing a specimen into
contact with a surface of the sugar chain mircoarray as set forth
in (48), collecting a sugar binding protein in the specimen at the
sugar chain and/or the sugar derivative of a drop-dispersed region
on the solid phase substrate, and quantitatively analyzing the
collected amount by a prescribed quantifying means;
[0090] (51) a method for preparing sugar chain affinity beads, in
which a sugar chain and/or a sugar derivative is immobilized onto a
surface of the polymer composed of a solid phase carrier including
a substance B having a structure represented by the following
formula (3) or (12),
[Chemical Formula 24]
(Carrier)-R--NHNH.sub.2 (Formula 3)
[0091] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--,
[Chemical Formula 25]
(Carrier)-R--ONH.sub.2 (Formula 12)
[0092] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--;
[0093] (52) the method for preparing sugar chain affinity beads as
set forth in (51), in which the sugar chain and/or the sugar
derivative is immobilized onto a surface of the polymer particle
according to the following steps (1) to (3),
[0094] (step 1) a step of purifying and/or isolating the sugar
chain and/or the sugar derivative by a specific separation means by
binding the sugar chain and/or the sugar derivative to a compound
containing a soluble hydrazide group,
[0095] (step 2) a step of immobilizing the sugar chain and/or the
sugar derivative onto the polymer particle by bringing the compound
obtained in the step (1) into contact with the polymer particle and
incubating under prescribed conditions for exchanging a sugar
chain-hydrazide group-containing compound bond with a sugar
chain-polymer particle bond, and
[0096] (step 3) a step of washing and removing the unreacted
substance on the polymer particle;
[0097] (53) sugar chain affinity beads prepared by the method as
set forth in (51) or (52); and
[0098] (54) a system for bringing a solution containing a specimen
into contact with the sugar chain affinity beads as set forth in
(53), incubating under prescribed conditions and washing, and then
isolating the captured sugar-binding substance,
[0099] According to the present invention, a sugar chain and/or a
sugar derivative for an analytical sample is enabled to be
separated and purified from a biological sample containing the
sugar chain and/or the sugar derivative by a simple operation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0100] The above and other objects, features and advantages will be
apparent from the following detailed description of the preferred
embodiments in conjunction with the accompanying drawings.
[0101] FIG. 1 illustrates a MALDI-TOF-MS chart of the reactant of a
sugar chain and 2-hydrazinopyridine. The horizontal axis represents
the molecular weight (m/z), while the vertical axis represents the
intensity. Furthermore, structures of the sugar chain expected at a
peak (a) in 2668.421 (=m/z), a peak (b) in 2978.005 (=m/z) and a
peak (c) in 3263.785 are schematically represented by : galactose,
.box-solid.: N-acetylglucosamine, .smallcircle.: mannose and
.diamond-solid.: sialic acid.
[0102] FIG. 2 illustrates a MAEDI-TOF-MS chart of the reactant of a
sugar chain and a substance A of the Example. The horizontal axis
represents the molecular weight (m/z), while the vertical axis
represents the intensity. Structures of the sugar chain expected at
each main peak are schematically represented by : galactose,
.box-solid.: N-acetylglucosamine, .smallcircle.: mannose,
.diamond-solid.: sialic acid and triangle figure: fucose.
[0103] FIG. 3 is a graph illustrating a relationship between the pH
and the exchange efficiency of the exchange reaction in the sugar
chain release step of the Example. The horizontal axis represents
the pH, while the vertical axis represents the exchange efficiency.
The exchange from oxime to oxime is indicated as .smallcircle., the
exchange from oxime to hydrazone is indicated as , the exchange
from hydrazone to oxime is indicated as .quadrature., and the
exchange from hydrazone to hydrazone is indicated as
.box-solid..
[0104] FIG. 4 illustrates a MALDI-TOF-MS chart of the reactant
obtained in the exchange reaction of FIG. 3. The horizontal axis
represents the molecular weight (m/z), while the vertical axis
represents the intensity. From the top of the chart, a functional
group of the beads is hydrazide when a releasing reagent is an
aminooxy compound (aoWR), a functional group of the beads is
hydrazide when a releasing reagent is a hydrazide compound (AcWRh),
a functional group of the beads is aminooxy when a releasing
reagent is an aminooxy compound (aoWR), and a functional group of
the beads is aminooxy, when a releasing reagent is a hydrazide
compound (AcWRh) respectively.
[0105] FIG. 5 illustrates a MALDI-TOF-MS chart of the sugar chain
recovered in the method of (1) of Experimental Example 7(B). The
horizontal axis represents the molecular weight (m/z), while the
vertical axis represents the intensity. The top of the chart
indicates control (serum sugar chain+400 .mu.M internal standard),
the middle indicates a flow through sample ((beads and unreacted
sugar chain)+400 .mu.M of internal standard), and the bottom
indicates a sample (serum sugar chain captured by using beads
containing a hydrazide group and then recovered by oxime
exchange+400 .mu.M of internal standard). When the intensity of the
sugar chain subjected to quantitative analysis of control was taken
as 100%, the flow through sample was 12% (88% bonded to beads),
while the sample was 56%.
[0106] FIG. 6 illustrates a MALDI-TOF-MS chart of the sugar chain
recovered in the method of (2) of Experimental Example 7(8). The
horizontal axis represents the molecular weight (m/z), while the
vertical axis represents the intensity. The chart indicates the
results when, from the top of the chart, 50 .mu.l (dry weight 16.5
mg) of Affi-Gel Hz, 100 .mu.l (dry weight 33 mg) thereof, 150 .mu.l
(dry weight 49.5 mg) thereof, and 2.5 mg of beads containing a
hydrazide group were used.
[0107] FIG. 7 illustrates a MALDI-TOF-MS chart of the sugar chain
recovered in the method of Experimental Example 8(1). The
horizontal axis represents the molecular weight (m/z), while the
vertical axis represents the intensity. The chart indicates the
results in cases of, from the top of the chart, before reaction,
after reaction with beads and without using beads (negative
control).
[0108] FIG. 8 is a graph illustrating the verification results of a
lectin capturing ability of Experimental Example 9(2). The vertical
axis indicates the absorbance at 450 nm. The lectin concentration
is 1 .mu.g/ml, and the mole number of lectin immobilized onto
monosaccharide is 2.5 mmol/g (saturated amount).
[0109] FIG. 9 is a graph illustrating the verification results of a
lectin capturing ability of Experimental Example 10(2). The
horizontal axis represents the molecular weight (m/z), while the
vertical axis represents the intensity.
BEST MODE FOR CARRYING OUT THE INVENTION
[0110] Embodiments of the present invention will be described
below.
[0111] From one of viewpoints, the present invention provides a
method for preparing a sample characterized by binding a substance
A containing a hydrazide group to a sugar chain and/or a sugar
derivative via hydrazone formation between the hydrazide group of
the substance A and the reducing end of the sugar chain and/or the
sugar derivative.
[0112] The substance A containing a hydrazide group used in the
method for preparing a sample is not particularly limited as long
as it contains a hydrazide group (--NHNH--) at the end part. This
hydrazide group is reacted with an aldehyde group for forming a
specific and stable bonding in an equilibrium between the cyclic
hemiacetal type and the non-cyclic aldehyde type formed by the
sugar chain in a solution such as an aqueous solution or the like,
thereby enabling to capture the sugar chain. Herein, the sugar
chain capture reaction refers to a reaction as illustrated
below,
##STR00018##
[0113] Herein, the substance A may be in the form of either a
low-molecular compound or a solid phase carrier.
[0114] When a low-molecular compound is used it is preferable that
the substance A contains a moiety consisting of at least one of an
arginine residue, a tryptophan residue, a phenylalanine residue, a
tyrosine residue, a cysteine residue and a derivative thereof from
the following viewpoints.
[0115] That is, when the substance A contains an arginine (R)
residue, there have been known that the ionization is accelerated
at the time of measurement of MALDI-TOF-MS, and the detection
sensitivity is improved. Furthermore, from the fact that tryptophan
(W) is a fluorescent amino acid and is hydrophobic, improvement in
the isolation by the reverse phase HPLC and improvement in the
fluorescence detection sensitivity can be attempted. Incidentally,
when phenylalanine (F) and tyrosine (Y) are used, such a sample is
suitable for detection by UV absorption.
[0116] Further, when cysteine is used, the substance A containing
cysteine can be bonded to other substance containing a --SH group
by an S--S bond using a thiol group in a side chain. For example,
such a substance A can be immobilized onto the solid phase carrier
containing a --SH group. Further, as other example, such a
substance A can be used for the ICAT method (Isotope-Coded Affinity
Tags).
[0117] As an example of such a substance A, a compound having a
structure of the following formula (1) can be cited,
##STR00019##
[0118] wherein, in the formula, R represents --CH.sub.3 or
--CD.sub.3.
[0119] The compound of the formula (1) can be produced in
accordance with the following Scheme 1,
##STR00020## ##STR00021##
[0120] In Scheme 1, the compound (b) can be obtained by subjecting
the compound (a) in which the amino group of a tryptophan moiety is
protected with the phenyl group or the like to deprotection by
hydrogenation in the presence of Pd/C. Herein, the tryptophan
moiety can also be substituted by phenylalanine, tyrosine, cysteine
or the like.
[0121] Subsequently, the amino group in a tryptophan side of the
compound (b) is acted on acetic acid (AcOH) in the presence of
water-soluble carbodiimide (WSC) and dimethylaminopyridine (DMAP),
in a solvent of dimethylformamide (DMF) for acetylation, whereby
the compound (c) is obtained.
[0122] Furthermore, hydrazine is acted on the compound (c) in a
solvent of methanol (MeOH) and a methoxy group of the C terminal
end in an arginine side is substituted with a hydrazide group,
whereby the compound (d) is obtained as a desired compound of the
formula (1).
[0123] When a deuterium (D) is introduced into the compound (d), in
Scheme 1, the compound (b) is acetylated in the presence of
water-soluble carbodiimide (WSC) and dimethylaminopyridine (DMAP),
in a solvent of dimethylformamide (DMF) using deuterated acetic
acid (CD3COOH) to obtain a compound in which the amino group in a
tryptophan side is acetylated using a deuterated acetyl group.
Furthermore, hydrazine is acted on the compound in a solvent of
methanol (MeOH) and a methoxy group of the C terminal end in an
arginine side is substituted with a hydrazide group, whereby the
compound (e) is obtained as a desired compound of the formula
(1).
[0124] Meanwhile, as other example of the substance A, a compound
having a structure of the following formula (2) can be cited,
##STR00022##
[0125] wherein, in the formula, R represents any of H, --COCH.sub.3
or --COCD.sub.3.
[0126] The compound of the formula (2) can be produced in
accordance with the following Scheme 2,
##STR00023## ##STR00024##
[0127] In Scheme 2, firstly, 2-aminobenzoic acid (compound (u)) is
acted on a cysteine dimer (compound (t)), whereby the compound (v)
is obtained.
[0128] Acetic anhydride is acted on this compound (v) for
acetylating the amino group of the 2-aminobenzoyl group, whereby
the compound (q) is obtained. At this time, a protinated compound
(q-1) is obtained by using the compound (o) that is acetic
anhydride. On the other hand, a deuterated compound (q-2) is
obtained by using the compound (p) that is deuterated acetic
anhydride.
[0129] Subsequently, the compound (q-1) or (q-2) is reacted with
hydrazine, whereby the compound (r) is obtained. Incidentally, when
R in the compound (r) is an acetyl group (--COCH3), a hydride (r-1)
is obtained. When R is a deuterated acetyl group (--COCD3) a
deuteride (r-2) is obtained.
[0130] Furthermore, there is obtained a compound containing a
hydrazide group of the formula (2) having a 2-aminobenzoyl group in
which the amino group is acetylated and cysteine by reducing the
compound (r) by use of a reducing agent such as OTT or the like for
cutting off the disulfide bond. Incidentally, when R in the
compound (r) is an acetyl group (--COCH3), a hydride (s-1) is
obtained. When R is a deuterated acetyl group (--COCD3), a
deuteride (s-2) is obtained.
[0131] As described above, by introducing an arginine residue, a
tryptophan residue, a phenylalanine residue, a tyrosine residue, a
cysteine residue and a derivative thereof into the substance A,
functions such as high sensitivity of mass spectrometry,
fluorescence.UV labeling or the like can be provided to the
captured sugar chain.
[0132] Further, as illustrated in the formula (2), chromophore or
fluorophore of the 2-aminobenzoyl group or the like may be
contained in a molecule. Examples of such a group include aromatic
residues having typical examples of a benzyl group, a naphthyl
group, an anthracenyl group, a pyridyl group and the like in
addition to the 2-aminobenzoyl group; and substituents containing a
Dansyl group or a Fmoc group. Such a substituent is a labeled
compound for providing fluorescence, and generally used for HPLC
analysis of the sugar chain. Accordingly, the sugar chain and/or
the sugar derivative captured by using a substance A into which
this substituent is introduced can be a labeled sample. By using
this labeled sample, the sugar chain and/or the sugar derivative
captured by using the substance A is enabled to be analyzed with
high resolution and high sensitivity by HPLC employing a reverse
phase column.
[0133] Furthermore, as explained in the formulae (1) and (2), a
deuterated substituent, for example, a deuterated acetyl group or
the like may be contained. A sample containing such a deuterated
functional group is enabled to perform qualitative and quantitative
analysis by enhancing the detection sensitivity by mass
spectrometry.
[0134] For example, a deuterated sample by using the substance A of
the formula (1) or (2) and a protinated sample are used in
combination, whereby qualitative and quantitative analysis of the
sugar chain contained, for example, in a sample containing an
unknown sugar chain (for example, those obtained by treating serum)
can be performed by mass spectrometry.
[0135] Accordingly, for example, when a sample with the composition
and the concentration already known from the past is
deuterium-labeled, an unknown sample is protium-labeled, and both
samples are mixed for carrying out mass spectrometry, it is
observed that each peak of the deuterated sample is shifted towards
the high molecular weight as much as the number of introduced
deuteriums rather than each peak corresponding to the protinated
sample. Then, the position (m/z value) and intensity of each peak
are analyzed so that the kind of the sugar chain illustrated by
each peak of the unknown sample and the concentration in the sample
are found. Such analysis can also be performed by making a known
sample to a protium and an unknown sample to a deuterium.
[0136] Furthermore, in this analysis, a sample extracted from a
healthy human is made to a deuterium, and a sample extracted from a
disease patient is made to a protium, or a sample of a healthy
human is made to a protium and a sample of a disease patient is
made to a deuterium, whereby the difference in the kind and amount
of the sugar chain contained in both samples can be analyzed.
Accordingly, such an analytical sample can be suitably used for
purposes of the pathological diagnosis on the basis of a biological
reaction participating in the sugar metabolism, medical treatment
by regulating such a biological reaction, and the like.
[0137] Regarding the substance A, the compound having a structure
of the formulae (1) and (2) will be described below, but the
substance A may be a substance selected from the following or a
salt thereof,
[0138] (substance A): 5-Dimethylaminonaphthalene-1-sulfonyl
hydrazine (Dansylhydrazine); 2-hydrazinopyridine; 9-fluorenylmethyl
carbazate (Fmoc hydrazine); benzylhydrazine;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionoc
acid, hydrazide;
2-(6,8-difluoro-7-hydroxy-4-methylcoumarin)acetohydrazide;
7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH);
phenylhydrazine; 1-Naphthaleneacethydrazide; 2-hydrazinobenzoic
acid; biotin hydrazide; and phenylacetic hydrazide.
[0139] Further, from the viewpoint that the captured sugar chain is
detected with high accuracy and high sensitivity, a moiety
containing chromophore, or fluorophore may be introduced or a
deuterium may be introduced into these compounds.
[0140] Herein, the sugar chain capture reaction, that is, a
reaction of the substance A with the sugar chain and/or the sugar
derivative, is carried out by introducing the substance A into a
sample containing the sugar chain and/or the sugar derivative. The
reaction is carried out in the reaction system under conditions of
pH of from 4 to 8, and the reaction temperature of from 4 to 90
degree centigrade, preferably from 25 to 90 degree centigrade and
more preferably from 40 to 90 degree centigrade for 10 minutes to
24 hours, preferably 10 minutes to 8 hours and more preferably 10
minutes to 2 hours.
[0141] Furthermore, when a solid phase carrier is used, the
substance
[0142] A is represented by the following formula (3),
[Chemical Formula 31]
(Carrier)-R--NHNH.sub.2 (Formula 3)
[0143] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --CO-- or
--CONH--.
[0144] In the formula (3), the carrier is a polymer matrix composed
of an inorganic substance or an organic polymer substance, and is
used in the form of a particle, or a solid phase substrate, or a
substance directly bonded to a surface of the solid phase
substrate.
[0145] Herein, as the inorganic substance which can be used as a
carrier, a substance in the form of a particle can be used, and
examples thereof include silica particles, alumina particles, glass
particles, metal particles and the like.
[0146] Furthermore, examples of the organic polymer substance
include polysaccharide gels having typical examples of agarose and
sepharose, polymers of a vinyl compound in the form of a particle,
and substances immobilized onto a surface of the solid phase
substrate. Further, the surface of the solid phase substrate may be
formed by using these substances.
[0147] Meanwhile, the polymer particle is preferably in the shape
of a sphere, and is a polymer particle having its average particle
diameter of equal to or more than 0.1 and equal to or less than 500
.mu.m. The particle of the carrier having a particle diameter in
such a range is easily subjected to recovery by centrifugation,
filter or the like, and the reaction efficiency with the sugar
chain is also considered to be high because the particle has a
sufficient surface area. When the particle diameter is vastly
greater than the above range, the reaction efficiency with the
sugar chain is lowered in some cases since the surface area becomes
small. Furthermore, when the particle diameter is vastly smaller
than the above range, it is particularly difficult to recover the
particle by the filter in some cases. Further, when the particle is
filled in a column and the particle diameter is too small, the
pressure loss at the time of passing the fluid is high in some
cases.
[0148] Furthermore, examples of the solid phase substrate include a
microplate and a flat substrate. In this way, an analytical sample
can be prepared by applying the substance A to a substrate for a
sugar chain microarray.
[0149] R represents a hydrocarbon chain having 1 to 20 carbon atoms
which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--. For example, the following can be cited. In the following
formula, b and d represent an integer of 1 to 5, while c represents
an integer of 1 to 10,
##STR00025##
[0150] Herein, the sugar chain capture reaction may be carried out
by filling the aforementioned substance A in the form of a particle
in a column or the like and passing through the sample containing
the sugar chain and/or the sugar derivative (continuous), or may be
carried out by putting this particle into the sample and stirring
(batch). Furthermore, the reaction may be carried out by
continuously putting the sample into a reaction vessel filled with
particles in advance and stirring (semi-batch).
[0151] Furthermore, the substance A represented by the above
formula (3) can be obtained by polymerizing a raw material
containing a carboxylic acid ester monomer having a polymerizable
group in the presence of a crosslinking agent for obtaining a
polymer particle containing carboxylic acid ester, and then
treating the polymer particle containing carboxylic acid ester with
a hydrazine solution of a concentration of not less than 10 volume
percentage. Accordingly, from another viewpoint, the present
invention provides a method for preparing such a substance A
represented by the formula (3).
[0152] Herein, examples of the carboxylic acid ester monomer
include N-hydroxysuccinimide esters of carboxylic acids and
carboxylic acid methyl esters.
[0153] Meanwhile, as the crosslinking agent, a polyfunctional
compound, a compound subjected to copolymerization with a
carboxylic acid ester monomer, can be suitably used Examples
thereof include (1) di or tri(meth)acrylic esters of polyols, for
example, those ones in which the polyol is ethylene glycol,
propylene glycol, trimethylol propane, glycerin, polyoxyethylene
glycol, polyoxypropylene glycol, polyglycerin or the like, (2) in
the above (1), unsaturated acid esters of polyols in which the
unsaturated acid is other than (meth)acrylic acid, for example,
maleic acid, fumaric acid or the like, (3) bisacrylamides, for
example, N,N'-methylene bisacrylamide or the like, (4) di- or
tri(meth)acrylic esters obtainable by reacting polyepoxides with
(meth)acrylic acid, (5) di(meth)acrylic carbamyl esters obtainable
by reacting polyisocyanates with (meth)acrylic hydroxyesters, for
example, those ones in which the polyisocyanate is tolylene
diisocyanate, hexamethylene diisocyanate or the like, and (6)
polyvalent allyl compounds, for example, allylated starch,
allylated cellulose, diallyl phthalate, tetraallyloxyethane,
pentaerythritol triallyl ether, trimethylol propane triallyl ether,
diethylene glycol diallyl ether, triallyl trimellitate or the like.
Of these, ethylene glycol di(meth)acrylate, propylene glycol
di(meth)acrylate, N,N'-methylene bis(meth)acrylamide and the like
are preferable for use in the present invention.
[0154] Namely, the substance A represented by the formula (3)
having the following structure can be used.
[0155] --(Hydrazide Group-Containing Compound
Component)m-(Crosslinking Agent Component)n--
[0156] As the substance A having such a structure, those having a
crosslinked polymer structure represented by the following formula
(4) can be cited,
##STR00026##
[0157] wherein, in the formula, R.sub.1 and R.sub.2 represent a
hydrocarbon chain having 1 to 20 carbon atoms which may be
interrupted with --O--, --S--, --NH--, --CO-- or --CONH--; R.sub.3,
R.sub.4 and R.sub.5 represent: H, CH.sub.3 or a hydrocarbon chain
having 2 to 5 carbon atoms; and m and n represent the number of
monomer units.
[0158] R.sub.1 represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--, and examples thereof include the same as those cited in
the aforementioned R.
[0159] R.sub.2 represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--, and examples thereof include the following. In the
following formula, e and f represent an integer of 1 to 5, while g
represents an integer of 1 to 10,
##STR00027##
[0160] R.sub.3, R.sub.4 and R.sub.5 may each be the same or
different, and represent H, CH.sub.3 or a hydrocarbon chain having
2 to 5 carbon atoms. Examples thereof include the following. In the
following formula, h represents an integer of 1 to 4,
##STR00028##
[0161] Herein, as the carboxylic acid ester monomer that is a
precursor of the hydrazide group-containing compound component,
carboxylic acid methyl ester monomer represented by the following
formula (10) can be suitably used. Furthermore, as the crosslinking
agent component, the aforementioned crosslinking agents can be
used,
##STR00029##
[0162] wherein, in the formula, R.sub.1 represents a hydrocarbon
chain having 1 to 20 carbon atoms which may be interrupted with
--O--, --S--, --NH--, --CO-- or --CONH--; and R.sub.2 represents H,
CH.sub.3 or a hydrocarbon chain having 2 to 5 carbon atoms.
[0163] Concrete examples of R.sub.1 and R.sub.2 include the same as
those cited in the aforementioned formula (4).
[0164] That is, the substance A represented by the formula (4) can
be obtained by polymerizing a monomer having a structure of the
formula (10) in the presence of a crosslinking agent for obtaining
a polymer particle containing carboxylic acid ester, and then
treating the polymer particle containing carboxylic acid ester with
a hydrazine solution of a concentration of not less than 10 volume
percentage.
[0165] From a further viewpoint, the present invention provides a
preparation method for obtaining a substance A of the formula (4),
as described above, using a monomer represented by the formula
(10), a polymer obtained by polymerizing this monomer and this
formula (10).
[0166] Furthermore, in this preparation method, when the carboxylic
acid ester monomer is used as a starting material, this monomer is
polymerized in the presence of a crosslinking agent to obtain a
polymer particle, and then the polymer particle is treated with a
hydrazine solution to obtain a substance A of the formula (4).
However, for example, when the hydrazide group-containing monomer
of the following formula (13) is used as a starting material, this
monomer is polymerized in the presence of the aforementioned
crosslinking agent and then subjected to deprotection, whereby a
substance A of the formula (4) can also be obtained,
##STR00030##
[0167] wherein, in the formula, R.sub.2 represents H, CH.sub.3 or a
hydrocarbon chain having 2 to 5 carbon atoms; R.sub.6 represents a
hydrocarbon chain having 1 to 20 carbon atoms which may be
interrupted with --O--, --S--, --NH--, CO-- or --CONH--; and [P]
represents a protective group.
[0168] Herein, concrete examples of R.sub.2 include the same as
those cited in the aforementioned formula (4).
[0169] Furthermore, R.sub.6 represents a hydrocarbon chain having 1
to 20 carbon atoms which may be interrupted with --O--, --S--,
--NH--, --CO-- or --CONH--, and examples thereof include the
following. In the following formula, i and j represent an integer
of 1 to 5,
##STR00031##
[0170] Furthermore, examples of the protective group [P] in the
formula (13) include Boc, Fmoc, Tmoc and the like.
[0171] Meanwhile, as a method for deprotecting the protective group
[P], a usual acid treatment can be cited.
[0172] From a further viewpoint, the present invention provides a
preparation method for obtaining a substance A of the formula (4),
as described above, using a monomer represented by the formula
(13), a polymer obtained by polymerizing this monomer and, this
formula (13).
[0173] Furthermore, of the formula (4), as the particularly
suitable substance A, those having a crosslinked polymer structure
represented by the following formula (5) can be cited,
##STR00032##
[0174] wherein, in the formula, m and n represent the number of
monomer units.
[0175] Herein, as the carboxylic acid ester monomer that is a
precursor of the hydrazide group-containing compound component,
carboxylic acid methyl ester monomer represented by the following
formula (11) can be suitably used. Furthermore, as the crosslinking
agent component, ethylene glycol di(meth)acrylate can be used,
##STR00033##
[0176] That is, the Substance A represented by the formula (5) can
be obtained by polymerizing a monomer having a structure of the
formula (11) in the presence of a crosslinking agent for obtaining
a polymer particle containing carboxylic acid ester, and then
treating the polymer particle containing carboxylic acid ester with
a hydrazine solution of a concentration of not less than 10 volume
percentage.
[0177] From a further viewpoint, the present invention provides a
preparation method for obtaining a substance A of the formula (5),
as described above, using a monomer represented by the formula
(11), a polymer obtained by polymerizing this monomer and, this
formula (11).
[0178] Furthermore, in this preparation method, when the carboxylic
acid ester monomer of the formula (11) is used as a starting
material, this monomer is polymerized in the presence of a
crosslinking agent to obtain a polymer particle, and then the
polymer particle is treated with a hydrazine solution to obtain a
substance A of the formula (5). However, for example, when the
hydrazide group-containing monomer of the following formula (14) is
used as a starting material, this monomer is polymerized in the
presence of the aforementioned crosslinking agent and then
subjected to deprotection, whereby a substance A of the formula (5)
can also be obtained,
##STR00034##
[0179] Furthermore, as a method for deprotecting a t-butoxycarbonyl
(Boc) group that is a protective group, a usual acid treatment can
be cited.
[0180] From a further viewpoint, the present invention provides a
preparation method for obtaining a substance A of the formula (5),
as described above, using a monomer represented by the formula
(14), a polymer obtained by polymerizing this monomer and, this
formula (14).
[0181] Furthermore, the substance A represented by the formula (3),
(4) or (5) is a polymer particle having a hydrazide group of a dry
weight of not less than 100 nmol and preferably not less than 0.5
.mu.mol per 1 mg. It is preferable that the substance is stable at
the pH of 3 to 8, stable under pressure of at least not more than 1
MPa from the viewpoints that a shape of the particle is maintained
and the content of the active hydrazide group is not substantially
changed. Further, since the particle is copolymerized with a
crosslinking agent, the solubility into a solvent is lowered, and
the physical intensity is sufficiently obtained. Furthermore, there
is no moiety to be cut off at the pH of 3 to 8.
[0182] From another viewpoint, the present invention provides a
method for preparing a sample which involves the sugar chain
capture step including binding a substance A to a sugar chain
and/or a sugar derivative according to the aforementioned method
for preparing a sample, and the sugar chain release step including
the action of a substance B containing an aminooxy group or a
hydrazide group on a complex of the substance A and the sugar chain
and/or the sugar derivative captured in the sugar chain capture
step, and binding the aforementioned sugar chain and/or sugar
derivative while cut off from the aforementioned substance A to the
substance B according to the hydrazone-oxime exchange reaction or
the hydrazone-hydrazone exchange reaction occurred between the
complex and the substance B.
[0183] In the sugar chain capture step of this method, as described
above, the substance A and the sugar chain and/or the sugar
derivative are bonded via hydrazone formation between the hydrazide
group of the substance A and the reducing end of the sugar chain
and/or the sugar derivative, thereby capturing the sugar chain
and/or the sugar derivative using the substance A.
[0184] Subsequently, in the sugar chain release step, as shown in
the following Reaction Formula (1), the hydrazone-oxime exchange
reaction or the hydrazone-hydrazone exchange reaction is carried
out,
##STR00035##
[0185] Herein, substances as described above can be used for the
substance A.
[0186] Furthermore, the substance B may be in the form of either a
low-molecular compound or a solid phase carrier.
[0187] When a low-molecular compound is used, it is preferable that
the substance B contains a moiety consisting of at least one of an
arginine residue, a tryptophan residue, a phenylalanine residue, a
tyrosine residue, a cysteine residue and a derivative thereof from
the aforementioned viewpoints, similar to the substance A.
[0188] That is, by an arginine residue or the like contained in the
substance B, improvement in the detection sensitivity at the
measurement of MALDI-TOF-MS, improvement in the reverse phase HPLC
isolation, improvement in the fluorescence detection sensitivity
and the like can be designed for the complex of the substance B and
the sugar chain and/or the sugar derivative obtained through the
sugar chain release step.
[0189] Examples of such a substance B include a low-molecular
compound cited in the substance A and a compound having a structure
represented by the following formula (7). Furthermore, in the
formula (7), the tryptophan moiety can also be substituted by
phenylalanine, tyrosine, cysteine or the like,
##STR00036##
[0190] wherein, in the formula, R represents --CH.sub.3 or
--CD.sub.3.
[0191] The compound of the formula (7) can be prepared in according
with the following Scheme 3. Incidentally, R is CH.sub.3,
##STR00037##
[0192] In Scheme 3, the compound (b) (WR--OMe) is obtained by
subjecting the compound in which the amino group of the tryptophan
moiety is protected by the phenyl group or the like to
deprotection. Herein, the tryptophan moiety can also be substituted
by phenylalanine, tyrosine or the like. Subsequently, the compound
(m) is synthesized by the condensation reaction such as the mixed
acid anhydride method or the like of the compound (b) and
Boc-protected hydroxylamine (compound (w)). A protective group of
this hydroxylamine is not restricted to Boc such as the compound
(w), but may be Fmoc, Troc or the like. Subsequently, by subjecting
the compound (m) to a deprotection procedure, the compound (n) is
obtained as the compound of the formula (7), a desired product. As
this deprotection procedure, for example, when a protective group
is Boc, a procedure by trifluoroacetic acid (TFA) can be cited.
[0193] As described above, by introducing an arginine residue, a
tryptophan residue, a phenylalanine residue, a tyrosine residue, a
cysteine residue and a derivative thereof to the substance B, the
captured sugar chain is enabled to be detected with high accuracy
and high sensitivity.
[0194] Furthermore, as shown in the formula (7), chromophore or
fluorophore of the 2-aminobenzoyl group or the like may be
contained in a molecule. Examples of such a group include aromatic
residues having typical examples of a benzyl group, a naphthyl
group, an anthracenyl group, a pyridyl group and the like in
addition to the 2-aminobenzoyl group; and substituents containing a
Dansyl group or a Fmoc group. Such a substituent is a labeled
compound for providing fluorescence, and is generally used for HPLC
analysis of the sugar chain. Accordingly, the sugar chain and/or
the sugar derivative captured by using the substance A into which
this substituent is introduced can be made into a labeled sample.
This labeled sample is used, whereby the sugar chain and/or the
sugar derivative captured by using the substance A can be analyzed
with high resolution and high sensitivity by HPLC employing a
reverse phase column.
[0195] Furthermore, as described in the formula (7), a deuterated
substituent, for example, a deuterated methoxy ester group or the
like may be contained. Such a deuterated functional group contained
therein enables to perform quantitative analysis using a difference
in the mass numbers measured, for example, by mixing a standard
substance with a sample at a state of the standard substance with
heavy labeling and the sample with light labeling, in addition to
an effect of enhancing the detection, sensitivity by mass
spectrometry.
[0196] According to this, a complex of the substance B and the
sugar chain and/or the sugar derivative obtained through the sugar
chain release step can also be suitably used for a complex of the
aforementioned substance A and the sugar chain and/or the sugar
derivative.
[0197] As for the substance B, a compound having a structure
represented by the formula (7) is described, but the substance B
may be a substance selected from the group consisting of the
following substances containing a hydrazide group or the following
substances containing an aminooxy group or a salt,
[0198] (substances containing a hydrazide group)
5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine);
2-hydrazinopyridine; 9-fluorenylmethyl carbazate (Fmoc hydrazine);
benzylhydrazine;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionoc
acid, hydrazide;
2-(6,8-difluoro-7-hydroxy-4-methylcoumarin)acetohydrazide;
7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH);
phenylhydrazine; 1-Naphthaleneacethydrazide; 2-hydrazinobenzoic
acid; and phenylacetic hydrazide, or
[0199] (substances containing an aminooxy group)
O-benzylhydroxylamine; O-phenylhydroxylamine;
O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine;
O-(4-nitrobenzyl)hydroxylamine; 2-aminooxypyridine;
2-aminooxymethylpyridine; 4-[(aminooxyacetyl)amino]benzoic acid
methyl ester; 4-[(aminooxyacetyl)amino]benzoic acid ethyl ester;
and 4-[(aminooxyacetyl)amino]benzoic acid n-butyl ester.
[0200] Further, from the viewpoint that the captured sugar chain is
detected with high accuracy and high sensitivity, a moiety
containing chromophore or fluorophore may be introduced or a
deuterium may be introduced into these compounds.
[0201] Herein, a reaction in the sugar chain release step is
carried out by introducing the substance B into a sample containing
the complex of the substance A and the sugar chain and/or the sugar
derivative. The reaction is carried out by heating in the pH
adjusted to 3 to 8 and preferably adjusted to 4 to 6 at a
temperature of 4 to 90 degree centigrade and preferably 60 to 90
degree centigrade for 15 minutes to 16 hours, preferably 15 minutes
to 5 hours and more preferably 30 minutes to 3 hours. As a solvent,
preferably used is any of water, volatile organic solvents having
typical examples of acetonitrile, methanol and the like, or a mixed
solvent thereof. More preferably used is a mixed solvent of water
and acetonitrile, and the most preferably used is a mixed solvent
of water and acetonitrile (1:9) containing 2% acetic acid as a pH
adjusting agent.
[0202] Meanwhile, when a solid phase carrier is used, substances
cited in the aforementioned substance A, and substances containing
an aminooxy group, for example, represented by the following
formula (12) can be suitably used for the substance B,
[Chemical Formula 45]
(Carrier)-R--ONH.sub.2 (Formula 12)
[0203] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--.
[0204] In the formula (12), the carrier is a polymer matrix
composed of an inorganic substance or an organic polymer substance,
similar to the carrier described in the formula (3), and is used in
the form of a particle, or a solid phase substrate, or a substance
directly bonded to a surface of the solid phase substrate.
Furthermore, concrete examples of R include the same as those cited
in the formula (3).
[0205] Herein, the exchange reaction of the sugar chain release
step may be carried out by adding a solution of the substance B of
a low-molecular compound to the substance A in the form of a
particle capturing the sugar chain and/or the sugar derivative
introduced into a vessel or the like. Furthermore, the exchange
reaction may also be carried out by introducing the aforementioned
substance B in the form of a particle into a vessel or the like,
and adding a solution of the substance A of a low-molecular
compound capturing the sugar chain and/or the sugar derivative. Or,
the exchange reaction may be carried out such that the solution of
the substance B of a low-molecular compound may be passing through
the substance A in the form of a particle capturing the sugar chain
and/or the sugar derivative filled in a column or the like.
Further, the exchange reaction may also be carried out such that
the aforementioned substance B in the form of a particle is filled
in a column or the like through which the solution of the substance
A of a low-molecular compound capturing the sugar chain and/or the
sugar derivative is passed. Also, the exchange reaction may also be
carried out such that the substance B of a low-molecular compound
is introduced into the solution of the substance A of a
low-molecular compound capturing the sugar chain and/or the sugar
derivative.
[0206] In the complex of the substance B and the sugar chain and/or
the sugar derivative obtained by this exchange reaction, the
substance B and the sugar chain and/or the sugar derivative are
bonded by a hydrazone bond when a substance having a hydrazide
group is used as a substance B, and by an oxime bond when a
substance having an aminooxy group is used as the substance B.
[0207] As the substance B represented by the formula (12), a
polymer particle having a structure represented by the following
formula (8) can be suitably used,
##STR00038##
[0208] wherein, in the formula, R.sub.1 and R.sub.2 represent a
hydrocarbon chain having 1 to 20 carbon atoms which may be
interrupted with --O--, --S--, --NH--, --CO-- or --CONH--; R.sub.3,
R.sub.4 and R.sub.5 represent H, CH.sub.3 or a hydrocarbon chain
having 2 to 5 carbon atoms; and m and n represent the number of
monomer units. Furthermore, concrete examples of R1 to R5 include
the same as those cited in the formula (4).
[0209] Furthermore, as such a polymer particle represented by the
formula (8) a polymer particle having a structure represented by
the following formula (9) can be suitably used,
##STR00039##
[0210] wherein, in the formula, m and n represent the number of
monomer units.
[0211] Furthermore, it is preferable that the substance B
represented by the formula (12), (8) or (9) is stable at the pH of
3 to 8 and stable under pressure of at least not more than 1 MPa
from the viewpoints of the use of a sugar chain-capturing carrier
and the use of a solid phase substrate.
[0212] Further, after the completion of the aforementioned sugar
chain release step, the hydrazone-oxime exchange reaction or the
hydrazone-hydrazone exchange reaction may be carried out at least
one or more times. According to this, a labeled sample can be
obtained by the action of the substance B composed of any optional
labeled compound on the released sugar chain and/or the released
sugar derivative so that the sample can be applied to the planned
analysis method.
[0213] Herein, the method for obtaining a labeled sample by
releasing the captured sugar chain using a sugar chain-capturing
substance. However, according to the following method, a
non-labeled sample can be obtained, and the present invention
provides such a method for preparing a sample as well.
[0214] Such a method for preparing a sample is characterized in
that the hydrazone bond is dissociated and the sugar chain and/or
the sugar derivative is released by treating the substance A having
a structure represented by the above formula (3), (4) or (5) under
acidic conditions to which the sugar chain and/or the sugar
derivative is bonded.
[0215] The treatment under acidic conditions at this time is
carried out using a trifluoroacetic acid solution of 0.01 to 10
volume percentage and preferably a trifluoroacetic acid solution of
0.01 to 1 volume percentage at 25 to 80 degree centigrade for 5 to
60 minutes.
[0216] The thus-obtained analytical sample composed of the sugar
chain sample is not labeled, and is useful for the application of
those which may not be labeled.
[0217] In this way, it is possible to perform the separation and
purification of the sugar chain and/or the sugar derivative for an
analytical sample from a biological sample containing the sugar
chain and/or the sugar derivative by a simple operation. From a
further viewpoint, the present invention provides an analytical
sample obtained by this method for preparing an analytical
sample.
[0218] Furthermore, from another viewpoint, the present invention
provides the use of the method for preparing a sample including the
step of capturing the sugar chain by using the substance A. That
is, the present invention provides a method of applying a solid
phase carrier onto which a sugar chain and/or a sugar derivative
obtained by subjecting a mixture or a specific fraction from the
mixture to the isolation and purification is immobilized as a
carrier for collecting a substance having a bonding property or
affinity to the sugar chain and/or the sugar derivative.
[0219] In this application method, firstly, the sample containing
the sugar chain and/or the sugar derivative may be used by
subjecting a mixture or a specific fraction from the mixture to the
isolation and purification. Furthermore, using a solid phase
carrier as the substance A, the sample solution is passing through
this solid phase carrier so that the sugar chain and/or the sugar
derivative in this sample is bonded to the hydrazide group or the
aminooxy group of the solid phase carrier and immobilized
thereunto.
[0220] Subsequently, by the action of the sample (hereinafter
referred to as a specimen sample) collected from a specimen for
performing diagnosis or examination on this solid phase carrier, a
substance having a bonding property or affinity to the sugar chain
and/or the sugar derivative contained in this specimen sample, for
example, a sugar binding protein such as lectin or the like is
collected. In this way, the solid phase carrier onto which the
sugar chain and/or the sugar derivative in the specimen sample is
immobilized, and can be used as a carrier for collecting lectin or
the like in the specimen sample.
[0221] Further, the collected substance is detected and
quantitatively analyzed, whereby it is possible to perform the more
advanced analysis of the relationship between the sugar chain and
those of specimen cell differentiation, population growth, cell
adhesion, immunity and a malignant change (cancer) of cells.
[0222] Furthermore, from another viewpoint, the present invention
provides the use of the method for preparing a sample including the
sugar chain capture step and the sugar chain release step.
[0223] From one viewpoint, the present invention provides a method
for preparing a sugar chain microarray.
[0224] That is, in this method for preparing a sugar chain
microarray, the sugar chain and/or the sugar derivative is
immobilized onto a surface of the solid phase substrate composed of
a solid phase carrier including a substance B having a structure
represented by the following formula (3) or (12) according to the
following steps (1) to (4),
[Chemical Formula 48]
(carrier)-R--NHNH.sub.2 (Formula 3)
[0225] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--,
[Chemical Formula 49]
(Carrier)-R--ONH.sub.2 (Formula 12)
[0226] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--.
[0227] Incidentally, concrete examples of these substances include
the same as those cited above.
[0228] (step 1) a step of purifying and/or isolating the sugar
chain and/or the sugar derivative by a specific separation means by
binding the sugar chain and/or the sugar derivative to a compound
containing a soluble hydrazide group,
[0229] (step 2) a step of dispensing drops of a solution of the
compound obtained in the step (1) in a row onto the aforementioned
solid phase substrate,
[0230] (step 3) a step of proceeding a reaction of exchanging a
sugar chain-substance A bond with a solid phase substrate-sugar
chain bond by incubating the solid phase substrate after the
completion of dispensing drops under prescribed conditions, and
immobilizing the sugar chain and/or the sugar derivative onto the
solid phase substrate, and
[0231] (step 4) a step of washing and removing the unreacted
substance on the solid phase substrate.
[0232] In the step (1), by reacting the aforementioned sugar chain
and/or the sugar derivative with the substance A for capturing the
sugar chain and/or the sugar derivative using the substance A, the
sugar chain and/or the sugar derivative is purified and/or isolated
by using a specific separation means, for example, a technique such
as chromatography or the like. In the step (2), drops of the sugar
chain and/or the sugar derivative obtained in the step (1) are
dispersed onto the solid phase substrate in a row. In the step (3),
the exchange reaction illustrated in the aforementioned. Reaction
Formula (1) is carried out by incubating the solid phase substrate
under conditions of, for example, pH of 5, a temperature of 60 to
90 degree centigrade and a reaction time of 1 to 16 hours. As a
result, the sugar chain and/or the sugar derivative is immobilized
onto the solid phase substrate. In the step (4), the unreacted
substance in the step (3) is washed and removed by a usual method
using a buffer or the like.
[0233] From a further viewpoint, the present invention provides a
sugar chain microarray obtained by such a method. This sugar chain
microarray can be used as a carrier for collecting a substance
which interacts, for example, with the sugar chain and/or the sugar
derivative contained in the specimen sample. Accordingly,
identification, quantitative analysis or the like of the collected
substance can be performed.
[0234] From a further viewpoint, the present invention provides the
use of the sugar chain microarray.
[0235] For example, a solution containing the specimen is brought
into contact with a surface of this sugar chain microarray for
incubating under prescribed conditions and washing, and then a
sugar-binding substance collected at the sugar chain and/or the
sugar derivative of a drop-dispersed region on the solid phase
substrate is detected, whereby a system for searching the
sugar-binding substance for specifically binding to or adsorbing
the immobilized sugar chain and/or the immobilized sugar derivative
is provided.
[0236] In this search system, in accordance with the aforementioned
method, the solution containing a specimen (sample) is introduced
into the solid phase substrate onto which the sugar chain and/or
the sugar derivative is immobilized from an inlet tube or the like,
and the specimen sample is brought into contact with the sugar
chain and/or the sugar derivative.
[0237] Furthermore, in a state that the specimen sample is brought
into contact with the sugar chain and/or the sugar derivative, the
solid phase substrate is incubated. Incubation at this time is
carried out under conditions of, for example, pH of 4 to 10, a
temperature of 37 degree centigrade and a reaction time of 1 to 16
hours.
[0238] As a result of incubation, the sugar-binding substance
collected at the sugar chain and/or the sugar derivative, for
example, protein such as lectin or the like is detected. As the
detection method and means at this time, when the sugar-binding
substance can be directly fluorescent dyed, fluorescence is
measured by using a fluorescent scanner after the completion of
dyeing, whereby the substance is detected; and when a sugar-binding
substance is already known and is bonded to a specific antibody,
the antibody is bonded, whereby the substance is detected. Further,
when a sugar-binding substance is protein, the method is not
restricted thereto, and other general methods for detecting protein
can be applied.
[0239] According to this system, the collected sugar-binding
substance is detected, whereby the sugar-binding substance for
specifically binding to or adsorbing the immobilized sugar chain
and/or the immobilized sugar derivative can be searched. According
to this, it is possible to perform the more advanced analysis of
the relationship between the sugar chain and specimen cell
differentiation, population growth, cell adhesion, immunity and a
malignant change (cancer) in cells.
[0240] Further, there is provided a system for evaluating the
recognition specificity of a binding protein, for example, as the
use of the aforementioned sugar chain microarray, by bringing a
solution containing a specimen into contact with a surface of the
sugar chain mircoarray, collecting a sugar binding protein in the
specimen at the sugar chain and/or the sugar derivative of a
drop-dispersed region on the solid phase substrate, and
quantitatively analyzing the collected amount by a prescribed
means, for example, fluorescence, color development or the
like.
[0241] As described above, the sugar binding protein in the
specimen sample is collected at the sugar chain microarray composed
of the solid phase substrate onto which the sugar chain and/or the
sugar derivative is immobilized, and the collected amount is
quantitatively analyzed.
[0242] According to this system, based on the result, the
recognition specificity of the binding protein can be
evaluated.
[0243] Furthermore, from another viewpoint, the present invention
provides a method for preparing sugar chain affinity beads as the
use of the method for preparing a sample including the sugar chain
capture step and the sugar chain release step.
[0244] That is, the method for preparing sugar chain affinity beads
includes immobilizing the sugar chain and/or the sugar derivative
onto the surface of the polymer composed of the solid phase carrier
consisting of the substance B having a structure represented by the
following formula (3) or (12),
[Chemical Formula 50]
(Carrier)-R--NHNH.sub.2 (Formula 3)
[0245] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --CO-- or
--CONH--,
[Chemical Formula 51]
(Carrier)-R--ONH.sub.2 (Formula 12)
[0246] wherein, in the formula, the carrier represents a polymer
matrix; and R represents a hydrocarbon chain having 1 to 20 carbon
atoms which may be interrupted with --O--, --S--, --NH--, --CO-- or
--CONH--.
[0247] Incidentally, concrete examples of these substances include
the same as those described above.
[0248] Furthermore, in the method for preparing the sugar chain
affinity, beads, the sugar chain and/or the sugar derivative may be
immobilized onto the surface of the polymer particle according to
the following steps (1) to (3),
[0249] (step 1) a step of purifying and/or isolating the sugar
chain and/or the sugar derivative by a specific separation means by
binding the sugar chain and/or the sugar derivative to a compound
containing a soluble hydrazide group,
[0250] (step 2) a step of immobilizing the sugar chain and/or the
sugar derivative onto the polymer particle by bringing the compound
obtained in the step (1) into contact with the polymer particle and
incubating under prescribed conditions for exchanging a sugar
chain-hydrazide group-containing compound bond with a sugar
chain-polymer particle bond, and
[0251] (step 3) a step of washing and removing the unreacted
substance on the polymer particle.
[0252] In the step (1), by reacting the aforementioned sugar chain
and/or the sugar derivative with the substance A for capturing the
sugar chain and/or the sugar derivative using the substance A, the
sugar chain and/or the sugar derivative is purified and/or isolated
by using a specific separation means, for example, a technique such
as chromatography or the like. Incidentally, concrete examples of
the conditions of the sugar chain capture reaction at this time
include conditions of pH of 4 to 7, a reaction temperature at 25 to
90 degree centigrade and a reaction time for 1 to 16 hours,
specifically pH of 5, a reaction temperature at 80 degree
centigrade and a reaction time for 1 hour.
[0253] In the step (2), the exchange reaction illustrated in the
aforementioned Reaction Formula (1) is carried out by brining the
sugar chain and/or the sugar derivative obtained in the step (1)
into contact with the polymer particle and incubating the solid
phase substrate under specific conditions of, for example, pH of 4
to 7, a temperature at 25 to 90 degree centigrade and a reaction
time for 1 to 16 hours, specifically pH of 5, a temperature at 80
degree centigrade and a reaction time for 1 hour. As a result, the
sugar chain and/or the sugar derivative is immobilized onto the
polymer particle. In the step (3), the unreacted substance in the
step (2) is washed and removed by a usual method using a buffer or
the like.
[0254] Incidentally, the sugar chain and/or the sugar derivative is
captured by using the substance A having a hydrazide group in
advance and the exchange reaction is carried out by using the
substance B, whereby sugar chain affinity beads are prepared. The
procedure for preparing sugar chain affinity beads is not
restricted thereto. The sample containing the sugar chain and/or
the sugar derivative is directly brought into contact with the
polymer particle prepared using the solid phase carrier consisting
of the substance B, whereby the sugar chain affinity beads may be
prepared.
[0255] From a further viewpoint, the present invention provides
sugar chain affinity beads obtained in accordance with such a
method. Further, the present invention provides a system for
isolating the sugar-binding substance for the use of the sugar
chain affinity beads.
[0256] That is, in the system for isolating the sugar-binding
substance, a solution containing the specimen (sample) is brought
into contact with the aforementioned sugar chain affinity beads for
incubating under prescribed conditions and washing, and then the
captured sugar-binding substance is isolated.
[0257] In this system, firstly, a solution containing the specimen
sample is brought into contact with the sugar chain affinity beads
for incubating in a buffer solution containing Ca.sup.2+ and
Mg.sup.2+ under specific conditions of, for example, pH of 4 to 10,
a temperature at 37 degree centigrade and a reaction time for 1 to
16 hours, whereby a sugar-binding substance is collected at the
sugar chain and/or the sugar derivative which is immobilized onto a
surface of the sugar chain affinity beads. Furthermore, the surface
of the sugar chain affinity beads at which the sugar-binding
substance is collected is washed by the usual washing procedure
using a buffer or the like.
[0258] Furthermore, the collected sugar-binding substance is
isolated from the sugar chain affinity beads. At this time, as a
method for releasing the sugar-binding substance from the sugar
chain affinity beads, there can be exemplified, for example, a
method including adding an organic solvent such as methanol or the
like and separating a sugar and a sugar-binding substance; a method
including adding a solution of hapten sugar of a high concentration
(0.1 to 1M) and separating a sugar-binding substance, i.e.,
transferring lectin to the added sugar of a high concentration; and
the like.
[0259] As descried above, the method for preparing a sample of the
present invention can be applied to various purposes.
EXAMPLES
[0260] The present invention is now illustrated in detail below
with reference to the following Experimental Examples. However, the
present invention is not restricted to these Experimental
Examples.
Experimental Example 1
(A) Preparation of Hydrazide Group-Containing Compound
[0261] Low-Molecular Hydrazide Compound
[0262] 1. Commercial Products were Used for the Following Hydrazide
Compounds,
[0263] 5-Dimethylaminonaphthalene-1-sulfonyl hydrazine
(Dansylhydrazine); 2-hydrazinopyridine; 9-fluorenylmethyl carbazate
(Fmoc hydrazine); benzylhydrazine;
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionoc
acid, hydrazide (BODIPY.TM. FL hydrazide);
2-(6,8-difluoro-7-hydroxy-4-methylcoumarin)acetohydrazide (Marina
Blue.TM. hydrazide); 7-diethylaminocoumarin-3-carboxylic acid,
hydrazide (DCCH); phenylhydrazine; 1-Naphthaleneacethydrazide; and
2-hydrazinobenzoic acid.
[0264] 2. Synthesis of AcWRh (Substance A)
[0265] AcWRh was synthesized through the route illustrated in the
aforementioned Scheme 1 (incidentally, Ac represents an acetyl
group, W represents a tryptophan residue, R represents an arginine
residue, and h represents a hydrazide group).
[0266] (1) Synthesis of WR-OMe (Compound (b))
[0267] Methanol (5 ml) was added to Z--WR-OMe (a) (10 mg, 20 mmol)
and 10% Pd/C (10 mg), and the resulting mixture was stirred in a
hydrogen has atmosphere at room temperature for 2 hours. The
reaction solution was filtered using an aqueous membrane filter to
remove Pd/C and the filtrate was concentrated under a reduced
pressure, whereby a desired compound (h) (WR-OMe) was obtained. By
the analysis according to MALDI-TOF-MS, a desired product, the
[M+H]+ ion was observed at m/z: 376.
[0268] (2) Synthesis of AcWROMe (c)
[0269] WR-OMe (b) (53 mg, 0.14 mmol) was dissolved in 1 ml of DMF,
and WSC (40 mg, 0.20 mmol), DMAP (5 mg, 0.041 mmol) and acetic acid
(200 .mu.l) were added thereto. The resulting mixture was stirred
at room temperature for 3 hours. Furthermore, WSC (50 mg, 0.26
mmol) was added and stirred all night. The reaction solution was
concentrated, and the obtained residue was purified by silica gel
column chromatography (chloroform:methanol=1:1), whereby a desired
AcWROMe was obtained.
[0270] 1H NMR (500 MHz, CD3OD) .delta. 7.70-7.00 (m, 5H, indole),
3.62 (s, 3H, OMe), 1.93 (s, 3H, Ac)
[0271] (3) Synthesis of AcWRh (d)
[0272] 10 mg of AcWROMe was dissolved in 10% hydrazine/methanol (5
ml), and the resulting solution was reacted at room temperature for
12 hours and then concentrated, whereby AcWRh (d) was prepared. By
the analysis according to MALDI-TOF MS, a desired product, the
[M+H]+ ion was observed at m/z: 416.89.
[0273] On the other hand, according to the above procedure, d-AcWRh
(e) in which an acetyl group was substituted by a deuterium was
prepared.
[0274] d-AcWROMe: 1H NMR (500 MHz, CD3OD) .delta.7.59-6.99 (m, 5H,
indole), 3.62 (s, 3H, OMe), 1.93 (s, 3H, Ac), d-AcWRh: MALDI-TOF-MS
[M+H]+ m/z: 419.94
[0275] Synthesis of Hydrazide Group-Containing Polymer Beads
[0276] 1. Synthesis of Methyl Ester-Containing Monomer
[0277] A methyl ester-containing monomer was synthesized through
the route illustrated in the following Scheme 4,
##STR00040##
[0278] (1) Synthesis of Compound (h)
[0279] A solution prepared by dissolving methacrylic anhydride
(MAH: 5 g, 0.03 mol) (f) in 100 ml of chloroform was put dropwise,
on an ice bath, to a solution prepared by dissolving 25 g of
(ethylenedioxy)bis(ethylamine) (EDBEA: 25 g, 0.17 mol) (g) in 100
ml of chloroform. The resulting material was filled with nitrogen
and the content was stirred all night. The residue obtained by
evaporating a solvent from the obtained reaction solution was
applied to silica gel column chromatography (development solvent:
mixed solvent of 90 volume % chloroform/10 volume % methanol) for
taking out a prescribed fraction, and the solvent was evaporated
from this fraction, whereby a compound (h) was obtained.
[0280] (2) Synthesis of Compound (j)
[0281] To a solution prepared by dissolving 5 g of the compound (h)
(0.023 mol) in 100 ml of chloroform were added 1.5 equivalents of
monomethyl succinate (i) and 1.5 equivalents of a water-soluble
carbodiimide compound (WSC), i.e.,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The reaction vessel
was tightly sealed and filled with nitrogen, and the content was
stirred all night. The residue obtained by evaporating a solvent
from the obtained reaction solution was applied to silica gel
column chromatography (development solvent: mixed solvent of 90
volume % chloroform/10 volume % methanol) for taking out a
prescribed fraction, and the solvent was evaporated from this
fraction, whereby a compound (j) was obtained.
[0282] Furthermore, the obtained substance was confirmed to be the
compound (j) by NMR and a matrix-assisted laser-desorption ionized
time-of-flight mass spectrometer (MALDI-TOF-MS).
[0283] 2. Synthesis of Methyl Ester-Containing Polymer
[0284] A polymer particle containing methyl ester was synthesized
through the route illustrated in the following Scheme 5,
##STR00041##
[0285] Into a three-necked flask was introduced 25 ml of a 5%
aqueous solution of polyvinyl alcohol (PVA) and purged with
nitrogen. A mixture composed of 1 g (2.6 mmol) of the compound (j),
ethylene glycol dimethacrylate (k) (EGDMA: 5 mol % with respect to
(j)) and 1 ml of chloroform was introduced into said three-necked
flask, and the reaction solution was stirred while the temperature
was maintained at 60 degree centigrade to disperse the compound (j)
and EGDMA, which were monomers contained in the mixture, in the
aqueous PVA solution. Subsequently, to start polymerization,
azobisisobutyronitrile (AIBN) was added as a polymerization
initiator in an amount of 3 weight % with respect to the monomer.
The reaction was carried out at 60 degree centigrade for 16 hours,
and then the resultant polymer particles were, collected by
centrifugation and washed with methanol and water.
[0286] 3. Introduction of Hydrazide Group
[0287] 400 mg of a polymer particle was taken in a vessel, 4 ml of
hydrazine monohydrate was added, and the resulting mixture was
stirred and allowed to stand at room temperature for 2 hours. After
the reaction, hydrazine monohydrate was removed, and the resulting
reactant was washed with methanol, and then rinsed with 1M
hydrochloric acid. Thereafter, the resulting reactant was further
washed with pure water.
[0288] 4. Quantitative Analysis of Amount of Functional Group
[0289] 1 mg of a polymer particle was taken in a vessel. 1 .mu.mol
of N-acetyl-D-lactosamine (LacNAc) was added thereto, and 180 .mu.l
of acetonitrile containing 2% acetic acid was further added. The
resulting material was heated at 80 degree centigrade for 45
minutes, whereby LacNAc and a hydrazide group on beads were
reacted. Beads were rinsed with pure water so that the unreacted
sugar chain was recovered and quantitatively analyzed by the
measurement of MALDI-TOF-MS (internal standard method) to obtain
the bonding amount of LacNAc with beads. It was found that 0.86
.mu.mol (860 nmol) of LacNAc was bonded per 1 mg of beads.
[0290] Commercial Hydrazide Group-Containing Beads
[0291] Affi-Gel Hz manufactured by Bio-Rad Co., Ltd, was used as it
was.
[0292] 1. Quantitative Analysis of Amount of Functional Group
[0293] 50 .mu.l (16.5 mg as a dry weight of beads) of a dispersion
of Affi-Gel Hz was taken in a vessel. 1 .mu.mol of LacNAc was added
thereto, and 180 .mu.l of acetonitrile containing 2% acetic acid
was further added. The resulting material was heated at 80 degree
centigrade for 45 minutes, whereby LacNAc and a hydrazide group on
beads were reacted. Beads were rinsed with pure water so that the
unreacted sugar chain was recovered and quantitatively analyzed by
the measurement of MALDI-TOF-MS (internal standard method) to
obtain the bonding amount of LacNAc with beads. It was found that
about 8 nmol of LacNAc was bonded per 1 mg of beads (dry
weight).
[0294] (B) Synthesis of Aminooxy Group-Containing Compound
[0295] Synthesis of Aminooxy Group-Containing Low-Molecular
Compound aoWR
[0296] aoWR (compound (n)) was synthesized according to the
aforementioned Scheme 3 (ao represents an aminooxy group).
[0297] (1) Synthesis of Boc-NHOCH2CO--W--R--OMe (Compound (m))
[0298] A THF (6 ml) solution of Boo aminooxyacetic acid (1) (2.5
mmol) was cooled down to -20 degree centigrade. Next,
N-methylmorpholine (3.0 mmol) and isobutyl formate (3.0 mmol) were
added thereto, and the resulting mixture was stirred for 15
minutes, whereby a mixed acid anhydride was prepared. The reaction
solution was at a temperature of 0 degree centigrade, the compound
(b) (WR-OMe (3.0 mmol)) was dissolved in water (3 ml), and sodium
hydrogen carbonate (3.0 mmol) was added to prepare a WR--OMe
solution. The WR--OMe solution was mixed with another reaction
solution and the resulting mixture was stirred for 1 hour. The
reaction solution was concentrated under a reduced pressure, and
the obtained residue was purified by silica gel column
chromatography, whereby a desired compound (m)
(Boc-NHOCH2CO--W--R--OMe) was obtained. By the analysis according
to MALDI-TOF-MS, a desired product, the [M+H]+ ion was observed at
m/z: 547.
[0299] (2) Synthesis of NH2OCH2CO--W--R--OMe (Compound (n))
[0300] To the compound (m) was added trifluoroacetic acid (TFA) (2
ml), and the resulting mixture was stirred at -20 degree centigrade
for 2 hours. The reaction solution was concentrated under a reduced
pressure, toluene was added and the reaction solution was
repeatedly azeotroped to remove TFA, whereby a desired compound (n)
was obtained. By the analysis according to MALDI-TOF-MS, a desired
product, the [M+H]+ ion was observed at m/z: 448.
[0301] On the other hand, in accordance with the above procedure,
aoWR (H) in which hydrogen of a methyl group was protium and aoWR
(D) in which hydrogen was deuterium were prepared.
Synthesis of Aminooxy Group-Containing Polymer Beads
[0302] A polymer particle containing an aminooxy group was prepared
according to the method as described in Patent Document:
International Publication Pamphlet No. 2005/097844.
Experimental Example 2
Preparation of Sugar Chain Sample
[0303] (1) Preparation of Glycoprotein Sugar Chain
[0304] Fetuin was used as glycoprotein or ribonuclease B was used
as a sample. 10 mg of glycoprotein was taken in a vessel and
dissolved in a 50 mM ammonium bicarbonate solution. A small amount
of a surface active agent was added thereto and the reaction
solution was incubated at 60 degree centigrade for 30 minutes, and
then 10 units of N-glycosidase F (a product of Roche) were added,
and the resulting solution was incubated at 37 degree centigrade
for 16 hours, whereby a sugar chain was released.
[0305] (2) Preparation of Glycoprotein Sugar Chain Contained in
Serum
[0306] 5 mL of normal serum was taken in a vessel and dissolved in
a 50 mM ammonium bicarbonate solution. A small amount of a surface
active agent was added thereto and the reaction solution was
incubated at 60 degree centigrade for 30 minutes, and then 5 units
of N-glycosidase F (a product of Roche) were added, and the
resulting solution was incubated at 37 degree centigrade for 16
hours, whereby a sugar chain was released.
Experimental Example 3
Reaction of Low-Molecular Hydrazide Compound with Sugar Chain
[0307] (1) Reaction of Commercial Hydrazide Compound with Sugar
Chain
[0308] The hydrazide compound of Experimental Example 1 (A) with a
concentration of 10 mM was dissolved in methanol or acetonitrile to
obtain a hydrazide compound solution. To the fetuin sugar chain
solution of Experimental Example 2(1) (corresponding to 500
.mu.mol) were added 1 .mu.l of a hydrazide compound solution and
further 100 .mu.l of acetonitrile. The reaction solution was heated
at 80 degree centigrade for 45 minutes, whereby the sugar chain and
the hydrazide compound were reacted. The product after the
completion of the reaction was measured by MALDI-TOF-MS.
[0309] FIG. 1 illustrates a MALDI-TOF-MS chart when
2-hydrazinopyridine was typically used, whereas a peak was observed
at the position indicating the molecular weight of the sugar chain
(in the figure, a structure represented in a schematic view) and
2-hydrazinopyridine bonded to each other. The same results were
also obtained when other compounds were used.
[0310] (2) Reaction of AcWRh with Sugar Chain
[0311] AcWRh of Experimental Example 1(A) was dissolved to the
concentration of 10 mM in methanol or acetonitrile to obtain a
hydrazide compound solution. The serum sugar chain solution
(corresponding to 5 .mu.l of serum) of Experimental Example 2(2)
was taken in a vessel. 1 .mu.l of the hydrazide compound solution
was added thereto and 100 .mu.l of acetonitrile was further added.
The resulting material was heated at 80 degree centigrade for 45
minutes, whereby the sugar chain and the hydrazide compound (AcWRh)
were reacted. The product after the completion of the reaction was
measured by MALDI-TOF-MS.
[0312] FIG. 2 illustrates a MALDI-TOF-MS chart, whereas a peak was
observed at the position indicating the molecular weight of the
sugar chain (in the figure, a structure represented in a schematic
view) and AcWRh bonded to each other.
Experimental Example 4
Reaction of Hydrazide Group-Containing Polymer Beads with Sugar
Chain
[0313] 2.5 mg of the polymer beads containing a hydrazide group of
Experimental Example 1(A) was measured in a vessel. The serum sugar
chain solution (corresponding to 5 .mu.l of serum) of Experimental
Example 2(2) was added, and 180 .mu.l of acetonitrile containing 2%
acetic acid was further added. The resulting material was heated at
80 degree centigrade for 45 minutes, whereby the sugar chain and a
hydrazide group on beads were reacted. Beads were rinsed with a
small amount of pure water so that the unreacted sugar chain was
recovered and quantitatively analyzed by the measurement of
MALDI-TOF-MS (internal standard method). As a result, it was found
that 80 to 90% sugar chain was bonded to beads. Thereafter, beads
were washed with a 0.5% aqueous solution of sodium dodecyl sulfate
(SOS), 50% methanol, an aqueous solution of 4M guanidine and pure
water, and supplied to the experiment of re-releasing of the sugar
chain to be described below.
Experimental Example 5
Preparation of Labeled Sugar Chain
[0314] (1) Bonding of AcWRh (d), AcWRh (e) and Sugar Chain
[0315] To an aqueous chitotriose solution was added 10 equivalents
of AcWRh (d) or d-AcWRh (e) of Experimental Example 1(A), and the
pH was adjusted to 5 with acetic acid. The reaction solution was
heated at 90 degree centigrade for 1 hour, whereby a labeled
chitotriose-AcWRh (d) and a labeled chitotriose-d-AcWRh (e) were
obtained.
[0316] (2) Bonding of aoWR(H), aoWR(D) and Sugar Chain
[0317] To an aqueous chitotriose solution was added 10 equivalents
of aoWR (H) or aoWR (D) of Experimental Example 1(B), and the pH
was adjusted to 5 with acetic acid. The reaction solution was
heated at 90 degree centigrade for 1 hour, whereby a labeled
chitotriose-aoWR was obtained.
Experimental Example 6
Liquid Phase Exchange Reaction of Functional Groups
[0318] In the following exchange reaction, the sugar chain labeled
with a hydrazide group-containing compound was reacted with an
aminooxy group-containing compound (or a hydrazide group-containing
compound), whereby label was transferred by the hydrazone-oxime
exchange (or hydrazone-hydrazone exchange). By comparison, as for
the sugar chain labeled with an aminooxy group-containing compound,
label was also attempted to be transferred. The progress of the
reaction was confirmed by MALDI-TOF-MS.
[0319] (A) Exchange Reaction of Functional Groups
[0320] (1) Hydrazone-Hydrazone Exchange Reaction
[0321] To a solution of the labeled chitotriose-AcWRh (d) of
Experimental Example 5(1) was added 10 equivalents of d-AcWRh (e)
of Experimental Example 1(A), and the pH was adjusted to 5 with
acetic acid. The reaction solution was heated at 90 degree
centigrade for 1 hour, and then a part of the reaction solution was
taken out and mixed with the labeled chitotriose-aoWR (H) of
Experimental Example 5(2) with a known concentration as an internal
standard. This resulting mixture was analyzed by MALDI-TOF-MS, and
a ratio of the labeled chitotriose-AcWRh (d) contained in the
reaction solution to the labeled chitotriose-d-AcWRh (e) obtained
in this reaction was calculated.
[0322] (2) Hydrazone-Oxime Exchange Reaction
[0323] To a solution of the labeled chitotriose-AcWRh (d) of
Experimental Example 5(1) was added 10 equivalents of aoWR (H) of
Experimental Example 1(B), and the pH was adjusted to 5 with acetic
acid. The reaction solution was heated at 90 degree centigrade for
1 hour, and then a part of the reaction solution was taken out and
mixed with the labeled chitotriose-aoWR (D) of Experimental Example
5(2) with a known concentration as an internal standard. This
resulting mixture was analyzed by MALDI-TOF-MS, and a ratio of the
labeled chitotriose-AcWRh (d) contained in the reaction solution to
the labeled chitotriose-aoWR (H) obtained in this reaction was
calculated.
[0324] (3) Oxime-Hydrazone Exchange Reaction
[0325] To a solution of the labeled chitotriose-aoWR (H) of
Experimental Example 5(2) was added 10 equivalents of AcWRh (d) of
Experimental Example 1(A), and the pH was adjusted to 5 with acetic
acid. The reaction solution was heated at 90 degree centigrade for
1 hour, and then a part of the reaction solution was taken out and
mixed with the labeled chitotriose-d-AcWRh (e) of Experimental
Example 5(1) with a known concentration as an internal standard.
This resulting mixture was analyzed by MALDI-TOF-MS, and a ratio of
the labeled chitotriose-aoWR (H) contained in the reaction solution
to the chitotriose-AcWRh (d) obtained in this reaction was
calculated.
[0326] (4) Oxime-Oxime Exchange Reaction
[0327] To a solution of the labeled chitotriose-aoWR (H) of
Experimental Example 5(2) was added 10 equivalents of aoWR (D) of
Experimental Example 1(B), and the pH was adjusted to 5 with acetic
acid. The reaction solution was heated at 90 degree centigrade for
1 hour, and then a part of the reaction solution was taken out and
mixed with the labeled chitotriose-AcWRh (d) of Experimental
Example 5(1) with a known concentration as an internal standard.
This resulting mixture was analyzed by MALDI-TOF-MS, and a ratio of
the chitotriose-aoWR (H) contained in the reaction solution to the
chitotriose-aoWR (D) obtained in this reaction was calculated.
[0328] (B) Study of Exchange Reaction of Functional Groups
[0329] FIG. 3 is a graph illustrating the yields of the exchange
reactions of the above 4 patterns.
[0330] It was found that the efficiency of the hydrazone-hydrazone
exchange reaction and the hydrazone-oxime exchange reaction was
higher than that of the oxime-hydrazone exchange reaction and the
oxime-oxime exchange reaction. That is the hydrazone bond was
easily subjected to the exchange reaction rather than the oxime
bond. Furthermore, it was also found that the hydrazone-oxime
exchange proceeded with good efficiency rather than the
hydrazone-hydrazone exchange.
Experimental Example 7
Exchange Reaction from Solid Phase to Liquid Phase
[0331] (A) Comparison of Exchange Reaction Efficiency
[0332] From the results of liquid phase exchange reaction in
Experimental Example 6, it was found that the hydrazone-oxime
exchange proceeded the most efficiently. As shown below, the
efficiency of the exchange reaction from solid phase to liquid
phase was also compared in the same manner.
[0333] Beads containing a hydrazide group capturing the serum sugar
chain in Experimental Example 4 and beads containing an aminooxy
group capturing the serum sugar chain in the same manner as in
Experimental. Example 4 were used for the comparison. To each of
sugar chain capturing beads was added 20 .mu.l of aoWR(H) solution
(20 mM) or AcWRh (H) solution (20 mM) and further 180 .mu.l of 2%
acetic acid/acetonitrile solution, and the resulting mixture was
heated at 80 degree centigrade for 45 minutes. After the completion
of the reaction, the supernatant was recovered and the MALDI-TOF-MS
measurement was carried out
[0334] FIG. 4 is a MALDI-TOF-MS chart.
[0335] As clear from the S/N ratio of the chart, it was found that
the efficiency was the best when beads containing a hydrazide group
captured the sugar chain and an aminooxy compound released it. This
result is consistent with the result from the comparison of the
reaction efficiency in the liquid phase in Experimental Example
6.
[0336] (B) Re-Release of Sugar Chain from Beads by Exchange
Reaction
[0337] (1) Re-Release of Sugar Chain from Polymer Beads Containing
Hydrazide Group
[0338] To polymer beads containing a hydrazide group bonded to the
sugar chain in Experimental Example 4 were added 20 .mu.l of aoWR
(H) solution (20 mM) and 180 .mu.l of 2% acetic acid/acetonitrile
solution, and the resulting mixture was heated at 80 degree
centigrade for 45 minutes. After the completion of the reaction,
the supernatant was recovered and an internal standard substance
(chitotetraose) was added thereto for carrying out the MALDI-TOF-MS
measurement, whereby the amount of the recovered sugar chain was
calculated.
[0339] (2) Re-Release of Sugar Chain from Commercial Beads
(Affi-Gel Hz)
[0340] With respect to Affi-Gel. Hz capturing the serum sugar chain
in the same manner as in Experimental Example 4, the sugar chain
was re-released in the same manner as in (1) of Experimental
Example 7(B), whereby the amount of the recovered sugar chain was
calculated.
[0341] FIG. 5 illustrates a MALDI-TOF-MS chart of the sugar chain
recovered in the method of (1) of Experimental Example 7(B).
[0342] As a result of the calculation from the signal amount of the
internal standard substance, it was found that 56% of the amount of
the sugar chain used could be recovered.
[0343] FIG. 6 illustrates a MALDI-TOF-MS chart of the sugar chain
recovered in the method of (2) of Experimental Example 7(B). At the
same time, the chart of the sugar chain recovered in (1) of
Experimental Example 7(B) was also illustrated.
[0344] From the S/N ratio of the mass spectrum, it was obviously
found that the amount of the recovered sugar chain was small when
commercial beads were used. It was found that, even when the amount
of beads used was increased by 3 times, it failed to reach the
polymer beads containing a hydrazide group by the signal amount.
This is considered, as described above, because the amount of the
functional group of commercial beads is small (about 1/100) as
compared to beads of the present invention.
Experimental Example 8
[0345] Exchange Reaction from Liquid Phase to Solid Phase
[0346] As illustrated below, the sugar chain labeled with a
hydrazide compound (hydrazone bond) was brought into contact with
polymer beads containing an aminooxy group, whereby the
hydrazone-oxime exchange reaction from liquid phase to solid phase
was carried out. This method could be applied such that, for
example, the sugar chain was labeled with a fluorescent hydrazide
compound, and then the sugar chain was isolated by HPLC or the like
and reacted with beads, whereby the isolated sugar chain was
immobilized onto beads. In short, this method can be used as a
method for randomly selecting a sugar chain from sugar chain
mixtures (for example, sugar chain recovered from glycoprotein) and
presenting the selected sugar chain onto a surface of the beads.
Further, as an application example, a solid phase substrate can
also be used instead of beads.
[0347] (1) Bonding of Hydrazine Compound (AcWRh (d)) with Sugar
Chain
[0348] AcWRh (d) of Experimental Example 1(A) was dissolved in
methanol with a concentration of 10 mM. 10 pmol of the sugar chain
released from fetuin that was glycoprotein was taken in a vessel. 1
.mu.l of a hydrazide compound solution was added and 100 .mu.l of
acetonitrile containing 2% acetic acid was further added. The
resulting material was heated at 80 degree centigrade for 45
minutes, whereby the sugar chain and AcWRh (d) were reacted.
[0349] (2) Bonding of Hydrazine Compound (d-AcWRh (e)) with Sugar
Chain
[0350] The sugar chain and d-AcWRh (e) were reacted in the same
manner as in Experimental Example 8(1).
[0351] (3) Exchange Reaction
[0352] 5 mg of polymer beads containing an aminooxy group was taken
in a vessel, and the sugar chain bonded to AcWRh (d) was put
thereinto. The pH was adjusted to 4 with an acetic acid buffer
solution, and the resulting solution was reacted at 80 degree
centigrade for 1 hour. After the completion of the reaction, the
supernatant was recovered, the labeled d-AcWRh (e) sugar chain with
a known concentration was added as an internal standard, and the
MALDI-TOF-MS measurement was carried out, whereby the amount of the
sugar chain (NA3: Asialo, galactosylated triantennary glycan
typically paid attention to) was obtained. As the negative control,
the system free from polymer beads containing an aminooxy group was
treated in the same manner.
[0353] FIG. 7 illustrates a MALDI-TOF-MS chart.
[0354] In the system in which polymer beads containing an aminooxy
group was reacted, about 20% of the amount of the sugar chain was
observed. On the other hand, in the negative control, the amount of
the sugar chain was hardly changed. From this fact, it was found
that about 80% of the amount of the sugar chain was bonded on
polymer beads containing an aminooxy group.
Experimental Example 9
Monosaccharide Immobilized Beads
[0355] Preparation of Monosaccharide Immobilized Beads
[0356] 10 mg of polymer beads containing an aminooxy group of
Experimental Example 1(B) was taken in a vessel, and 10 .mu.mol of
galactose (Gal) or N-acetylglucosamine (GlcNAc) was added thereto.
200 .mu.l of acetonitrile containing 2% acetic acid was added
thereto, and the resulting material was heated at 80 degree
centigrade for 1 hour, whereby the sugar was immobilized onto
beads. Thereafter, beads were sequentially washed with a 0.5% SDS
solution, methanol and pure water, whereby foreign matters were
removed.
[0357] (2) Verification of Lectin Capturing Ability
[0358] Three kinds of labeled HRP (horse radish peroxidase)
lectins: HRP-Concanavalin A (Con A), HRP-Wheat germ agglutinin
(WGA) and HRP-Ricinus communis agglutinin (RCA120) (all lectins
manufactured by Seikagaku Corporation) were respectively dissolved
to the concentration of 1 .mu.g/ml in a binding buffer (50 mM
Tris/HCl, 100 mM NaCl, 10 mM CaCl2, 10 mM MgCl2, pH7.6). 1 mg of
beads onto which monosaccharide was immobilized in Experimental
Example 9(1) was taken in a vessel, and 100 .mu.l of any of lectin
solutions was added thereto, and the resulting mixture was mildly
stirred at 37 degree centigrade for 16 hours. Thereafter, beads
were washed with a binding buffer containing 0.05% of Tween-20 and
a binding buffer respectively for 1 hour. Labeled HRP lectin bonded
on beads was the development of the color with a peroxidase
coloring kit (a product of Sumitomo Bakelite Co., Ltd.) for
measuring the absorbance of the solution at 450 nm, whereby the
binding amount of lectin was estimated.
[0359] As shown in FIG. 8, it was found that lots of WGA was bonded
to GlcNAc immobilized beads, while lots of RCA120 was bonded to Gal
immobilized beads. It has been known that WGA mainly recognized
GlcNAc, while RCA120 mainly recognized Gal. Monosaccharide
immobilized beads obtained by applying the method of the present
invention could surely recognize and capture corresponding
lectin.
Experimental Example 10
Oligosaccharide Immobilized Beads
[0360] Preparation of Oligosaccharide Immobilized Beads
[0361] 10 mg of polymer beads containing an aminooxy group of
Experimental Example 1(B) was taken in a vessel, 1 .mu.mol of the
fetuin sugar chain of Experimental Example 2(1) or a ribonuclease B
sugar chain solution was added in terms of the amount of the sugar
chain. 200 .mu.l of acetonitrile containing 2% acetic acid was
added and the resulting material was heated at 80 degree centigrade
for hour, whereby the sugar chain was immobilized onto beads.
Thereafter, beads were sequentially washed with a 0.5% SDS
solution, methanol and pure water, whereby foreign matters were
removed.
[0362] (2) Verification of Lectin Capturing Ability
[0363] Capture of Concanavalin A (Con A)
[0364] 1 mg of beads with Ribonuclease B sugar chain immobilized
thereon in Experimental. Example 10(1) was taken in a vessel. 1
.mu.l of the Con A solution (10 .mu.g/ml) and 100 .mu.l of the
binding buffer were added thereto, and the resulting material was
mildly stirred at 37 degree centigrade for 16 hours, whereby Con A
was bonded to the sugar chain on beads. Thereafter, beads were
washed with a binding buffer containing 0.05% of Tween-20 and a
binding buffer respectively for 1 hour. The cleaning solution was
removed, 20 .mu.l of 0.5 M methyl-.alpha.-mannopyranoside was added
as hapten, and the resulting mixture was stirred at 37 degree
centigrade for 2 hours, whereby Con A bonded to the sugar chain on
beads was released. The supernatant was recovered, a trypsin
solution (sequence grade trypsin, a product of Promega) was added,
the reaction mixture was allowed to stand at 37 degree centigrade
for 16 hours, and the released Con A was fragmented into peptides.
A part of the reaction solution was taken and the MALDI-TOF-MS
measurement was carried out. As shown below, data was compared with
already known amino acid sequence of Con A.
[0365] (1) The trypsin cut position was presumed from the amino
acid sequence of Con A (already known) for estimating the mass
number of a peptide fragment. Further, it was compared with the
measured mass spectrum data so that it was confirmed that they were
consistent. At this time, the mass number of the peptide fragment
was estimated using PeptideMass (a tool published on web).
[0366] (2) MS/MS analysis of the representative spectrum of the
obtained spectra was carried out. As a result of the analysis using
MASCOT MS/MS Ion Search (a tool for estimating original protein
from MS/MS data of the peptide fragment), it was confirmed that the
peptide was surely derived from Con A.
[0367] As shown in FIG. 9, lots of peaks were detected, in which
the Con A-derived peptide and the mass number were consistent. The
main sugar chain contained in Ribonuclease B was a high
mannose-type sugar chain (lots of mannose residues contained),
while Con A has a property of recognizing mannose and binding to
it. From these facts, Con A could be recognized and captured by the
sugar chain immobilized onto beads.
* * * * *