Note: Descriptions are shown in the official language in which they were submitted.
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Polymer supported reagents for natural products purification
Labeling of bio-molecules with a series of tags, such as biotinylating and/or
fluorescent
tags is a frequently used method in biochemical and biophysical laboratories.
These
compounds are useful for different applications, such as for example for
assays or
binding studies.
Since the purity of the desired product is important for the quality and the
reliability of
the subsequent applications, a series of purification steps, such as gel-
filtration, ion
exchange chromatography or reverse-phase RP-HPLC, must be done after the
labeling
of the bio-molecule. Nevertheless, during these steps a pure product with a
high
recovery cannot often be achieved.
In the field of the combinatorial chemistry the introduction of polymer-
supported
scavengers opened the possibility of a simple and efficient method to
facilitate the
purification of a desired product from un-reacted reagents and/or byproducts.
Once
reacted with the solid support, it can be removed from the reaction mixture,
e.g. by
filtration or extraction. A series of applications of scavengers bound on a
solid support
and their application in the practice of organic synthesis, combinatorial
synthesis and
automated organic synthesis have been developed. Examples of the solid support
are
polystyrene resins, Merrifield resins, polyamine resins, polystyrene-
divinylbenzene) /
poly(ethyleneglycol) grafted copolymer. These resins exhibit a significant
stability
2o towards organic solvents and temperature and pressure. Some examples are
described in
WO-A-9742230 and in the following references: Thompson L. A. Curr. Opin.
Chemical
Biol., 2000; 4: 324-337; Wentworth Jr., P. Trends Biotechnol., 1999, 17: 448-
452.
Protein purification by covalent chromatography is also known; the desired
product is
extracted from a crude reaction mixture by its selective reaction with a
polymeric
support, followed by filtration and rinsing. In a second step the protein can
be recovered
by cleavage from the resin. The polymeric support in the covalent
chromatography must
react with the desired product in a reaction mixture in the presence of other
(undesired)
compounds and the bound product has to be cleavable and retrieved from the
polymer.
The present invention provides a method for the purification of bio-molecules
after their
3o chemical modification using one or more insoluble polymer supported
reagents. Said
polymer supported reagents are put in contact with the mixture containing the
desired
chemically modified bio-molecule in order to react covalently with excess of
reagents
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and/or unwanted byproducts. Bio-molecules are substances originated by living
organisms. They can be proteins, peptides, DNA, RNA, lipids, small molecules,
all
originated by living organisms. The polymer supported reagent reacts with
byproducts
or reactants, leaving the desired pure product in solution.
The use of polymer supported reagents (also named scavenger resins) provides a
quick
and simple way of enhancing the purity of chemically modified bio-molecules.
The purification is carried out by incubating the polymer supported reagents
with the
mixture containing the desired bio-molecule and undesired byproducts and
educts and
removing the solid support. The solid support after incubation can be filtered
from the
1o solution or separated from the solution by aspiration.
These scavenger resins can be used packed in columns or in a batch system. The
incubation and the separation of the reaction mixture from the insoluble
polymeric
support can be repeated one or more times.
Preferably, the method of purification of this invention is performed under
non-
denaturing conditions. The bio-molecule maintains its three dimensional
structure
during the treatment with the scavenger resin. Therefore it does not have to
be re-
natured in a second step. The method of the present invention can be applied
as an
alternative and/or in combination with other purification steps, like ion
exchange
chromatography, affinity chromatography and gel filtration. Therefore, the
invention
2o also comprises the combination with other purification material such as ion-
exchange
resin; gel-filtration resin; NH2-reactive resins, (such as Actigel B, N-
hydroxy
succinimide esters activated columns, CNBr activated columns, Epoxy-activated
columns, Carbonyl diimidazole activated columns, but not limited to these
resins); SH-
reactive resins (such as Iodoacetyl, Bromoacetyl, maleimide, pyridyl disulfide
columns,
2s but not limited to it) chelating resins, desalting columns, reverse-phase
adsorbants. The
invention comprises the incubation of the reaction mixture with the scavenger
resins
combined with no, one or more of the other purification materials in one or in
more
subsequent steps.
A fiurther embodiment of this invention includes the automatic performance of
the
so process and the setup for a parallel purification array. Preferably, high
concentrations of
the reacting groups are present on the solid support, so an addition of a
small amount of
polymer is required.
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In particular, the present invention is relating to a method for the fast
purification of
labeled bio-molecules using an insoluble polymer supported quenching reagent.
The
labeling of the bio-molecules can be, for example, biotinylation or the
introduction of
fluorescent tags.
Therefore, in a preferred embodiment, the present invention provides a method
of
removing biotinylating and/or fluorescent agent from a reaction mixture
containing
labeled bio-molecules, such as for example proteins, protein mixtures and/or
peptides,
which are either obtained by cellular expression or by chemical or enzymatic
cleavage
from proteins.
As stated above, this invention is addressed to the improvement of the purity
of a
modified bio-molecule by trapping unwanted byproducts and/or un-reacted
starting
material. Bio-molecules can be proteins, peptides obtained by enzymatic and/or
chemical cleavage from proteins and/or produced by cellular systems. These
proteins
and peptides can contain post-translational modifications, such as
phosphorylations,
glycosylations, myristolations and palmitoylations. The bio-molecules can also
be
DNA, RNA, oligonucleotides, lipids, nucleotides, nucleosides, phospholipids,
carbohydrates, small molecules (like NAD, NADH, ATP, ADP, AMP, GTP, GDP,
GMP, but not limited to it) originated from living organisms.
The chemical modification of these bio-molecules can be the tagging with
biotinylating
or fluorescent agents. 'The biotinylating and fluorescent agents contain three
parts:
biotin and/or the fluorescent tag; no, one or more linkers and a chemical
group, which
permits the binding to the bio-molecule. The linker is expected to be
chemically robust
at the conditions the binding and quenching step is carried out. It is
generally composed
from CHz-CHz; CHz-CH; CH-CHz; CH-CH; CH=CH; CH-N; CHz-N; CH-O; CHz-O;
CH-S; CHz-S; CDz-CDz; CDz-CD; CD-CDz; CD-CD; CD=CD; CD-N; CDz-N; CD-O;
CDz-O; CD-S; CDz-S C(=O)-N; N-C(=0); C(=O)-O; O-C(=O); O-C(=O)-N; N-C(=O)-
O; N-C(=O)-N; O-C(=O)-O and combinations thereof. (D is equal deuterium). The
group reacting with the bio-molecule can be iodoalkyl, bromoalkyl, maleimido,
dithiopyridine, disulphides, isothiocyanat, succiilimidyl esters,
sulphosuccinimidyl
3o esters, aldehydes, ketones, dichlorotriazines, diazoles carboxylic acids,
sulphonyl
chlorides, acyl azides, acyl nitrites, acid chlorides, amino groups,
hydrazines,
hydrazides, hydroxylamines, alcohol, carbodiimides and combinations thereof.
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The fluorescent tags include all molecules, which are able to emit photons
after having
been excited by light or heat. Examples are fluorescein, eosin, dinitrophenyl,
naphthalene and substituted naphtalenes (such as dansyl), coumarin,
[Ru(bipy)3]2+,
BODIPY~ fluorophores, rhodamine, Texas Reds, Indocarbocyanine (Cy3) or
indodicarbocyanine (Cy5), Alexa Fluor~, Nile Red, allophycocyanine, Oregon
Green~,
indotricarbocyanine (CY7), Europium trisbipyridine cryptate, N-[7-nitrobenz-2-
oxa-1,3-
diazol-4-yl].
The method of the present invention is particularly attractive for the
nanotechnological
approach, owing to two characteristics. It is a simple one step approach, in
which the
to desired product does not have to bind or interact with a solid support.
Second, high
concentrations of the quenching groups on the solid support allow the addition
of a
small amount of quenching polymer.
The insoluble polymer supported reagents to be used in the present
purification method
are consisting of a polymer bearing at least one reagent residue. Preferably,
the reagent
15 residue is selected from the group consisting of thiol (-SH), hydroxyl (-
OH), carboxyl,
formyl, keto (COOH, CH=O, C=O), guanidino, amino groups and derivative thereof
(NR1R2 wherein Rl and R2 independently represent hydrogen atom, C1-C12 alkyl
or aryl
group, NRIRa, OH), azlactone. Preferably, at least 0.01 mmoles of reagent
residues,
more preferably at least 0.05 mmoles, are covalently bonded or immobilized on
1 gram
20 of resin. In general, there are two synthetic strategies by which the
preferred high
loading of quenching functionality on polymeric supports is achieved. In the
first
strategy, a polymer with existing functionalities of greater than 0.01 mmol
per gram of
polymer is chemically modified to give a novel polymer-supported quenching
reagent
which has greater than 0.01 mmol of quenching functionalities per gram of
polymer. In
25 the second strategy, polyfimctional or dendritic molecules bearing
connecting functional
groups and tvvo or more quenching functional groups axe attached to polymers
with less
than 2 mmol of attachment sites per gram of polymer. In this manner, the
munber of
quenching sites is amplified compared to the number of attachment sites. One
required
characteristic of the polymers to be used according to the present invention
is the
3o insolubility. The term insolubility means that the polymer does not
dissolve in the
solvent, wherein the purification reaction is carried out, such as water, but
also water/
organic solvent mixtures and organic solvents. For example, the polymeric
support can
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be built up by cross-linked polystyrenes, polyacrylamides and polyalcrylates,
polystyrene-divinylbenzene), poly(styrene-divinylbenzene/poly-(ethyleneglycol)
copolymer (known also as Tentagel~ resin). ). Of course, also other polymeric
supports
known in the art can be conveniently used in the present invention, such as
for example
5 polyamide, polyamide/polyethylene glycol copolymer, polysulfone, latex,
polyester,
paper, polypropylene, polyethylene, nitrocellulose.
TentaGel is a trade mark of Rapp Polymere GmbH. TentaGel resins are grafted
copolymers consisting of a low cross linked polystyrene matrix on which
polyethylene
glycol (PEG or POE) is grafted.
Another classes of polymeric supports are polysaccharide resins like
Sephaxose, agarose
(non cross-linked and cross-linked), cellulose, polysaccharides copolymerized
with
acrylamide (for example N,N'-methylene-bis(acrylamide), N-acryloyl-2-amino-2-
hydroxymethyl-1,3-propane diol, but not limited to these two compounds). Even
though
these last compounds do not show such a high stability as those above, they
keep being
the mostly used resins in the purification procedures for proteins.
The reagent residue may be linked to the polymeric supports by means of a
spacer. The
spacer is chemically resistant at the conditions in which the purification or
quenching
step is carned out, and is generally of the formula -CH2-CH2-; -CH2-CH-; -CH-
CH2;
CH-CH-; -CH=CH-; =CH-N=; -CH2-N; -CH-O-; -CH2-O-; -CH-S-; -CH2-S-; -C(=0)-
2o N-; -N-C(=O)-; -C(=O)-O-; -O-C(=O)-; -O-C(=O)-N-; -N-C(=O)-O-; -N-C(=O)-N-;
-O-C(=O)-O or combinations thereof, and the like. A spacer can be linked to
one or
more reagent residues, and can also be linked to another spacer, of the same
formula or
different. The insoluble polymer supported reagents to be used in the method
of this
invention may be prepared by converting a polymeric starting material into a
polymer
2s supported reagent in one to four synthetic steps, rinsing thoroughly with
one or more
solvents after each synthetic step, for example as described in WO-A-9742230.
The
preparation of the polymer supported reagents starts from known polymers.
Polymer
supported reagents axe made in one to four synthetic steps from readily
available
starting materials, such as for example, insoluble polymers or derivatives
thereof which
3o contain convenient linker functionality, and one or more polyfunctional
reagents which
bear a compatible connecting functionality and one or more functionalities
used in the
purification or quenching reaction process.
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The preferred polymeric starting materials are well-known to those skilled in
the art of
solid-phase peptide or solid-phase organic synthesis.
The present invention also provides polymer supported reagent of the formula
I:
Rs-Sp-Re I
wherein Rs is insoluble polymer selected from polysaccharide resins,
cellulose,
polysaccharides copolymerized with acrylamide; Re is one or more reagent
residues that
are capable of selective covalent reaction with unwanted byproducts, or excess
reagents;
and Sp is one or more chemically robust spacer that join Rs and Re.
Preferably, the
reagent residue Re is selected from the group consisting of thiol, hydroxyl ,
carboxyl,
1o formyl, keto, guanidine, amino groups and derivative thereof as defined
above,
azlactone and the spacer Sp is as defined above. It is a further object of the
invention a
process of preparing a compound of the formula I as defined above, which
comprises
conversion of a polymeric starting material Rs as above defined into a
compound of
Formula I in one to four synthetic steps, rinsing thoroughly with one or more
solvents
I5 after each synthetic step.
The starting materials are well-known to or those skilled in the art of
purification of bio-
molecules. They are commercially available or are known in the scientific
literature.
A method which affords novel polymer supported reagents are described in the
following schemes, wherein Rs and Sp are as defined above:
2o Rs-Br + NH3 or H2N-Sp-NHZ -> Rs-NH2 or Rs-NH-Sp-NH2
Rs-Br + HS-Sp-SH -> Rs-S-Sp-SH
Specific methods which afford selected examples of most preferred polymer-
supported
quenching reagents are illustrated in Examples 1 and 2.
The following examples illustrate the invention without limiting it:
25 Description of Actigel B resin:
Specifications
...............................................................................
..........:;~%.....................................:............::.::::::::.:::
::::::::::::::::::::::::::::::
se bead conc:
exclusion limit: 4 million Daltons
size: y60-160 N,m
acer: 7 atoms, hydrophilic
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Example 1
Preparation of the Actigel B-NHz resin
Matrix-O-spacer-O-CHz-CH(OH)-CHBr + HzN-CHz-CHz-NHz -->
Matrix-O-spacer-O-CHz-CH(OH)-CHz-NH-CHz-CHz-NHz
Actigel B resin (1 ml, 150 mmoles/1) was washed for 3 times with 0.1 M K2C03
and
incubated with 333 ~,1 of ethylenediamin (300 mg, 5 mmoles) in 4.5 ml of 0.1 M
K2C03 overnight at 4°C.
The resin was washed with 0.1 M K2C03 (3 x 5 ml), PBS (saline phosphate
buffer,
l0 3 x 5 ml) and finally with PBS/20% ethanol (3 x 5 ml). For storage the
resin was kept in
a solution of PBS/ethanol (4:1, v:v)
Example 2
Preparation of the Actigel B-SH resin
Matrix-O-spacer-O-CHz-CH(OH)-CHBr + HS-CHz-CHz-CHz-SH -->
Matrix-O-spacer-O-CHz-CH(OH)-CHz-S-CHz-CHz-CHz-SH
Actigel B resin (1 ml, 150 mmoles/1) was washed for 3 times with 0.1 M K2CO3
and
incubated with 500 ~,1 propanedithiol (540 mg, 5 mmoles) in 4.5 ml 0.1 M KZC03
overnight at 4°C.
The resin was washed Wlth O.1 M K2CO3 (3 x 5 ml), PBS (3 x 5 ml) and for
storage
2o with PBS /20% ethanol (3 x 5 ml).
Example 3
Validation of the Actigel B-NHz resin
60 ~,g of fluorescein-5(6)-carboxyamido-caproic acid-N-hydroxy succinimide
ester
(0.10 .moles) were dissolved in 6 ~,1 DMF, diluted with 100 ~,l PBS and
incubated with
2, 10 and 20 p,l Actigel B-NHz resin for 90 minutes to overnight at 4
°C. Finally the
solid support is filtered and washed with PBS. The amount of the not bound
fluorescein
can be established by fluorescence spectroscopy.
Example 4
Validation of the Actigel B-SH resin
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70 p,g of fluorescein maleimide (0.16 p,moles) were dissolved in 7 ~,1 DMF,
diluted with
100 p,l PBS, pH=7.2, and incubated with 5, 20 and 50 ~,l Actigel B-SH resin
for 90
minutes to overnight at 4 °C. Finally the solid support was filtered
and washed with
PBS. The amount of the not bound fluorescein can be established by
fluorescence
s spectroscopy.
Example 5
Usage of the resins for purification:
40 ~,g of a mixture of casein proteins, dissolved in 40 ~1 NH4HC03 SOmM, pH=8,
were
incubated with 20 ~,l of a solution containing 20 ~,g of EZ-Link PEO Biotin
(Pierce) in
NH4HC03 SOmM, pH=8, for 2 hours at 37C. After that, 10 ~1 Actigel B-SH resin
were
added to the reaction mixture and the incubation carried out for another 30-60
minutes.
The resin was removed by filtration or spinning down. The resin was washed
twice with
10 Nl NH4HC03 SOmM, pH=8 and the washing solution was added to the protein
solution. In order to validate this approach and to check the efficiency of
the quenching,
the supernatant solution was injected in aHP1090 separation system using a RP-
C4
column (Vydac 4.6x250 mm, 300A pore size, 7.5 ~,). The efficiency was
monitored by
comparing the absorption of the traces at wavelengths of 220 and 280 nm. A
reduction
of 90% of EZ Link PEO-Biotin could be observed, whereas the recovery of the
protein
was >90%.
