Note: Descriptions are shown in the official language in which they were submitted.
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PURIFICATION DEVICE AND PURIFICATION METHOD
This application claims priority from provisional patent application
60/153,630
filed September 13, 1999, which is hereby incorporated by reference.
Background of the Invention
The invention relates to a purification device for use in chemical synthesis
and method of using the device.
Combinatorial chemistry and synthesis of chemical libraries have stimulated
i o the development of synthetic strategies to rapidly and efficiently purify,
isolate, and
manipulate compounds during each synthetic step of forming the library
members. For
example, peptide, DNA, and small organic molecules libraries can be
constructed using
either solid phase or solution phase synthetic methodologies. In principal,
solution
phase synthesis could produce higher purity and lower cost final product than
solid
15 phase synthesis. In practice, solution phase synthesis can lead to the
formation of an
emulsion which can lower the purity and yield of the final product and
variability of
results.
Summary of the Invention
a o In one aspect, the invention features a method of removing a residue from
a
target compound mixture. The method includes contacting a target compound
mixture
including a target compound and a residue with a quenching reagent, flowing
the target
compound mixture across the quenching reagent, immobilizing the residue on the
quenching reagent by forming a covalent bond between the residue and the
quenching
a s reagent, and removing the target compound from the quenching reagent. The
quenching reagent is coupled to an insoluble support.
In another aspect, the invention features a method of deprotecting a target
compound having a protecting group residue. The protecting group residue is an
acid
labile group or a base labile group. The method includes the steps of flowing
the target
3 o compound mixture across a quenching reagent coupled to an insoluble
support to allow
the covalent binding of the protecting group residue to the quenching reagent,
and
removing the target compound from the quenching reagent. The protecting group
residue can be a fluorenyl derivative.
In preferred embodiments the quencing reagent includes a piperazino, a
35 piperidino, or functionally equivalent moiety.
In a preferred embodiment the quenching reagent can be coupled to the
soluble support by a linker, e.g., a linear or aromatic linkers, e.g., a
linker described
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herein.
In a preferred embodiment the insoluble support can be an organic polymer
or an inorganic oxide such as silica.
The method can include the steps of introducing the target compound
mixture into a housing including the quenching reagent, and removing the
target
compound from the housing. Flowing can include moving a solvent through the
housing or cartridge.
In another aspect, the invention features a device for purifying a target
1 o compound. The device includes a first opening and a second opening. The
first
opening and the second opening are connected by a fluid flow path. The device
also
includes a quenching reagent immobilized on an insoluble support contained
within the
fluid flow path. The quenching reagent is capable of forming a covalent bond
with a
residue. The residue can be a protecting group residue and the protecting
group residue
15 can be an acid labile group or a base labile group. The device can also
include a second
quenching reagent immobilized on a second insoluble support.
The residue can be a protecting group or an excess reagent. The residue and
the target compound can be covalently bonded, such as when the residue is a
protecting
group. When the residue is a protecting group, the method can also include the
step of
a o releasing the residue from the target compound.
The insoluble support can be an organic polymer or an inorganic oxide such
as silica.
The housing can include a cartridge. In addition to the insoluble support,
the cartridge can include a solid phase support.
2 5 The quenching reagent can include a nucleophilic or an electrophilic group
or a base, such as an amine, an isocyanate, an aldehyde, or an acyl halide.
The
quenching reagent can include a polystyrene functionalized with the quenching
reagent
or a silane functionalized with the quenching reagent.
The target compound can be a polypeptide, a nucleotide, or an organic
3 o compound. A polypetide can include, for example, 2 or more natural or
unnatural
amino acids. Nucleotides include, but are not limited to, any sugar-phosphate-
based
moiety, as well as any derivatized sugar-phosphate-based moiety. Examples of
organic
compounds, include but are not limited to, organic molecules containing a
functional
group protected by an acid or base labile group which reacts with the
quenching agent
3 5 to form a covalent bond.
The polymer-supported quenching reagents are functionalized polymer
resins. The functional groups on the resins selectively bind to the residue
via a covalent
bond linkage.
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Solution phase synthesis can offer certain advantages over solid phase
synthesis. For example, a vast number of solution phase reactions have been
developed
in the synthesis literature, whereas relatively fewer reactions have been
optimized for
solid phase synthesis. The variety of protecting groups that can be used in
solution
phase synthesis overwhelms the relatively limited number of solid-phase
synthesis
resins. Solution phase synthesis can also lead to the elimination of resin
attachment
and cleavage steps during synthesis. In addition, solution phase synthesis is
amenable
to monitoring by ordinary chromatographic or spectroscopic techniques. Solid
phase
reactions can be more challenging to monitor.
1 o Polymer-supported quenching reagents have improved the purity and
reproducibility of solution phase synthesis. For example, polymer supported
reagents
can be used as a rapid purification technique in solution-phase parallel
synthesis.
Depending on functional groups the desired products, by-products or solution-
phase
reagents can be selectively trapped on a resin. Previously used purification
methods,
such as crystallization, extraction, and chromatography can be difficult to
apply when
synthesizing a diverse library of compounds. Moveover, separating a
structurally
diverse array of compounds based on physical properties such as solubility or
partition
coefficient can present a formidable challenge when preparing libraries of
compounds.
The use of polymer-supported quenching reagents as a purification platform
allows
a o rapid solution phase synthesis to be carried out by eliminating the
formation of
emulsion by avoiding the need to perform liquid-liquid extraction steps.
Flowing a reaction mixture across a supported quenching reagent can
overcome some of the difficulties encountered in solution phase method of
treating a
reaction mixture with a functionalized resin in a batch context. For example,
time
a s usage can be more efficient. The general procedure for removing the
protecting group
and purifying the solution phase product with polymer supported quenching
reagents
can requires between 14 hours and 4 days in a batch process. This reaction
duration is
long for combinatorial synthesis and purification situations. The reaction
speed is
significantly increased by flowing the target compound mixture across the
quenching
3 o reagent coupled to an insoluble support. This exposes the reaction mixture
to a higher
surface area of the resin. The mixture experiences a greater amount of
quenching
reagent of the surface, increasing the overall rate of the reaction.
In addition, a batch process can require the use of a large excess of polymer
resin. For example, typical solution phase methods require 1 mole of polymer
resin per
3 5 0.2 mole of blocked product, significantly increasing the cost of the
deblocking (e.g.,
deprotecting) step and the purification of the final product. By flowing the
target
compound mixture across the quenching reagent coupled to an insoluble support,
less
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resin can be used to achieve the same or improved purity of product.
Furthermore, batch processing can result in lower reaction yields due to
transfer losses and material losses during filtration of the resin. Multiple
transfers of
the product and filtration result in loss of product. This can be particularly
problematic
if the loss occurs near the end of a multiple step synthesis. Flowing the
target
compound mixture across the quenching reagent coupled to the insoluble support
increases the efficiency of the reaction and the purity of the product,
thereby increasing
reaction yields and purity.
Other features or advantages of the present invention will be apparent from
i o the following detailed description and also from the claims.
Brief Description of the Drawing
FIG. 1 is a schematic diagram depicting a device and method for purifying a
target compound mixture having an excess of a reagent.
FIG. 2 is a schematic diagram depicting a device and method for purifying a
compound.
FIG. 3 is a schematic diagram depicting a device and method for purifying a
target compound having a protecting group.
Fig. 4 is structural representation of several of reagents coupled to an
z o insoluble support.
Fig. S is a diagram of reactions involved in Bsmoc removal.
Detailed Description
In general, the purification device includes a quenching reagent coupled to
an insoluble support. For example, the quenching reagent can be covalently
bonded to
a polymeric support material. Alternatively, the quenching agent can be
covalently
bonded to an inorganic support material such as silica.
3 o A target compound, which is a product of a synthetic reaction, can be
separated from unwanted residues in a target compound mixture by selectively
forming
a covalent bond between the residue and the quenching reagent. For example,
the
residue can be excess starting material or a protecting group that has been
removed
from the target compound. The residue reacts with the quenching reagent to
form the
3 5 bond. This reaction immobilizes the residue on the insoluble support.
Removal of the
residue in this manner purifies the target compound once the target compound
is
removed from the quenching reagent coupled to the insoluble support.
By flowing the target compound mixture across the quenching reagent, the
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target compound mixture is exposed to a continual supply of unreacted
quenching
reagent. The effective increase concentration of the unreacted quenching
reagent
increases the kinetics of the reaction between the residue and the quenching
reagent. In
general, the contact time between the target compound mixture and quenching
reagent
is increased. This arrangement can allow the reaction to proceed more rapidly
and
more completely. Coupling of the quenching reagent to the insoluble support
permits
the supported quenching reagent to be contained within a device, such as a
disposable
column. The device allows the target compound mixture to be passed through the
column. Because the quenching reagent is coupled to the insoluble support
which in
to turn is contained within the device, the target compound mixture can flow
across the
quenching reagent. Accordingly, the purification of the target compound can
proceed
in higher yield in a shorter amount of time.
The quenching reagent can be a functionality that can react to form a
covalent bond with an electrophile (e.g., an amine which can react with an
acyl halide
or other electrophilic residue). Alternatively, can be a functionality that
can react to
form a covalent bond with an nucleophile (e.g., an isocyante which can react
with an
amine or other nucleophilic residue). The reagent-coupled insoluble support
can be
mixed with other solid support material. The other solid support material can
be
ordinary chromatography column packing material, such as organic polymer
supports
z o or inorganic oxide supports. Suitable support materials can include cross-
linked
polystyrene beads, silica, or reverse-phase silica.
The quenching reagent can be coupled by reaction with the surface
functionality of the insoluble support. A commercially available an amine
quenching
reagent-coupled resin is Amberlyst i5 (Aldrich Chemical Co.). Quenching
reagents
z 5 (e.g., amines and isocyanates) can be coupled to a polymeric support as
described, for
example, in Booth and Hodges, J. Amer. Chem. Soc. 119:4882-4886 (1997) or in
Knapczyk et al. J. Org. Chem. 48:661-665 (1983), each of which is incorporated
herein
by reference. Quenching reagents can be coupled to inorganic supports, such as
silica,
as described in Carpino et al. J. Org. Chem. 48:666-669 (1983), each of which
is
3 o incorporated herein by reference.
The synthesis of a target compound can be facilitated by using reagents
coupled to an insoluble support. When the reagent is a quenching reagent that
can, for
example, form a covalent bond with a protecting group of the target compound,
the
protecting group can be immobilized on the insoluble support. The reaction can
be
3 5 facilitated by exposing the compound to an excess amount of the coupled
reagent. One
way to facilitate the reaction is to flow a solution of the compound over the
support
containing the immobilized reagent. In particular, a reagent coupled to an
insoluble
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support can be packed in a disposable cartridge and the target compound can be
exposed to the reagent by flowing a solution of the compound through the
cartridge.
The reaction can purify the target compound by removing the protecting group
residue
from the solution.
The insoluble support can be a resin or other chromatographic material, such
as silica. The solution containing the target compound is a liquid phase. The
insoluble
support is a solid or stationary phase. As the solution containing the target
compound
and the protecting group is exposed to the immobilized reagent, adsorption
occurs.
Adsorption is the equilibrium distribution of target compound or the
protecting group
1 o between stationary phase and the liquid phase. The adsorption capacity of
the insoluble
support or resin can be an important parameter to control to achieve adequate
purification.
An excess of a starting material can be used in a synthetic reaction to drive
the reaction to completion without fear of complicating isolation and
purification of the
i5 final products by using a column including a coupled quenching reagent.
Referring to
FIGS. 1 and 2, an excess of reagent A (e.g., an isocyanate compound or acyl
halide) is
combined with reagent B (e.g., an amine compound) to form a target compound
(e.g.,
an imide). The excess reagent is effectively removed by flowing the target
compound
mixture, which includes target compound 10 and excess residue 20, through
device 25.
a o Device 25 can be a cartridge or column used in chromatography.
Device 25 includes housing 30. Housing 30 has first opening 40 and second
opening 50, connected by fluid flow path 55. Fluid flow path 55, within
housing 30,
contains insoluble support 60. Insoluble support 60 is coupled to quenching
reagent 70
(e.g., an amine). Quenching reagent 70 is capable of forming a covalent bond
with
a s excess residue 20. Alternatively, the quenching reagent can be an
electrophile (e.g., an
isocyanate) which can form a covalent bond with an unreacted amine.
As the target compound mixture passes through fluid flow path 55, residue
20 passes across quenching reagent 70. Residue 20 reacts with quenching
reagent 70,
immobilizing residue 20 on the quenching reagent by forming a covalent bond
80. As a
3 o result, target compound 10 is obtained in high yield in a pure form. By
flowing the
target compound mixture through device 25, the quenching reaction occurs more
rapidly than in a batch process.
Refernng to FIG. 3, a target compound 10 bearing protecting group residue
20 can also be purified by the method. Protecting group residue 20 is base
labile (i.e.,
3 s is removed under basic conditions) and reacts to form a covalent bond with
an amine.
The protected compound can be purified by passing the target compound mixture,
which includes protecting group residue 20 covalently bonded to target
compound 10,
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though device 25, including housing 30, and first opening 40 and second
opening 50,
connected by fluid flow path 55. As described above, fluid flow path 55,
within
housing 30, contains insoluble support 60. Insoluble support 60 is coupled to
quenching reagent 70 (e.g., an amine). Device 25 can also include solid phase
support
s 65 (e.g., other chromatography beads). Quenching reagent 70 is capable of
forming
covalent bond 80 with protecting group residue 20.
For example, a fluorenylmethoxycarbonyl (Fmoc)-protected amino acid was
deprotected and purified using the device including a piperazine coupled to a
silica
support prepared as described Carpino et al. J. Org. Chem. 48:666-669 (1983).
200 mg
to of Fmoc-Ile-DCPM in 1 mL of DMF was injected into a Flash 12 cartridge
(Biotage)
packed with the silica-supported piperazine. The cartridge was washed with 10
mL of
DMF at a flow rate of 0.4 mL per minute for an elapsed wash time of 250
minutes.
HPLC analysis of the eluent showed nearly complete deprotection and no
detectable
dibenzofulvene residue. By comparison, 15% dibenzofulvene remains in solution
when
15 the protected reagent is stirred with the bulk reagent for five days. The
reaction is more
complete and proceeds more rapidly using the cartridge method.
In another example, an alkylating reagent (e.g., an epoxide) can be used to
synthesize a tertiary amine by alkylating a secondary amine. If the secondary
amine is
used in excess amount, an electrophilic quenching reagent, such as an
isocyanate,
z o which is coupled to an insoluble support in the device, can be used to
purify the product
by immobilizing the excess secondary amine on the quenching reagent.
In order to purify a target compound in a target compound mixture that can
include a nucleophilic residue and an electrophilic residue (i.e., that have
not reacted
with each other), an electrophilic scavenger device and a nucleophilic
scavenger device
25 Can be used sequentially. Alternatively, a mixed bed device can be used. In
a mixed
bed device, two or more layers of quenching reagent functionalized insoluble
support
can be used. For example, the device can have two layers. One layer can
include an
insoluble support having a quenching reagent capable of forming a covalent
bond with
a nucleophile. A second layer can be placed above or below the first layer.
The second
3 0 layer can include a coupled quenching reagent that is capable of forming a
covalent
bond with an electrophile. A solid phase support layer that is not
functionalized can be
placed in between the first layer and the second layer to prevent adverse
interactions
between the layers. The order of exposure each layer to the target compound
mixture
can depend on the nature of the residues in the target compound mixture. The
order of
3 s exposure that provides the target compound in highest purity can be more
efficient to
use in the purification method, although this must be determined for each type
of target
compound mixture produced.
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Scheme I depicts a series of synthetic reactions that can be carried out under
excess reagent conditions to provide a target compound mixture that can be
purified by
the method using an electrophilic quenching reagent device, a nucleophilic
quenching
reagent device, or a mixed layer device.
_ g _
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Scheme I
O
R OCOCI
R~~ ~ // 'NCO ~--~
v
OR2
-NCO y~-- NH2
R2SO2C1
Ri~N NS02Rz
R2R3C0, NaCNBH ; ~ ~ ; ~-N~
p RI N N-~ -1
R,
R2
R1~N N~ ~-NCO ?;, NEi2
R2C02H o
Excess reagent ~ R2
o~~ O
-NCQ
R~~N N R
OH
R2NC0 0 ~ o 'i~Co
R,~N N--~
~2
g _
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Example
Four non-swelling silica-base reagents were prepared and compared for carrying
out
de-blocking/scavenging reactions by continuous methods, e.g., column, and
batch
technique. Silica reagents bearing the piperazino and piperidino function were
prepared. These organic bases were attached to silica using linear and
aromatic linkers.
Fig. 4 shows the four linker/reagent combinations.
Piperazino and piperidino functionalized silica gel were synthesized and
evaluated in
to batch and on-column procedures. For on-column use, disposable columns
(Biotage
Flash Samplets) were prepared. These products were designed to: (1) de-block
the
amino protecting group 9-fluorenylmethyloxycarbonyl (Fmoc); and (2) scavenge
the
dibenzofulyene liberated in the de-blocking process. The application of these
functionalized silica reagents was also shown for 1,1-Dioxobenzo[b]thiophene-2-
ylmethyloxycarbonyl (Bsmoc) amine protecting group.
A comparison of column-based use with pre-packed samplets and batch technique
is
shown in this work. The mechanical simplicity and efficiency of the column-
based
approach make possible the rapid, parallel synthesis and purification of
solution-phase
2 o syntheses. These cartridges offer an alternative to solid-phase organic
synthesis in the
practice of combinatorial chemistry.
De-blocking studies were performed with 9-fluorenylmethyl p-chlorocarbanilate
(Fmoc-PCA) as a test probe. Chromatography conditions were as follows: column,
C4
z5 Vydac, 4.6X100mn; Eluant, A, HZO+ CAN (95+5), B, ACN + H20 (95+5);
Gradient,
B(30-100%) in 15 minutes; Detection, UV @ 240 nm; Flow, 2 ml/minutes.
1g of piperazino-benzyl-functionalized silica (Reagent 2) was packed into
Biotage
samplets. 30 mg, 60 mg, or 90 mg of Fmoc-PCA was dissolved in 2 ml of DMSO and
3 o deposited on samplet. The solution was left in the samplet for 30 minutes.
The amount
of de-blocking was dependent of sample load. The 30 mg and 60 mg trial gave
100%
de-blocking 100%. At a 90 mg load 97% de-blocking was achieved. Scavenging was
dependent on, time, sample load, and solvent composition. The 30 mg, 60 mg,
and 90
mg trials gave, respectively, 90% 76% and 60% scavenging.
Batch and column technique were compared for de-blocking. In a batch trial, 5
g of
reagent 2 + Sml of DMSO + 300 mg of Fmoc-PCA were stirred at room temperature
for 30 minutes. In a column samplet/on-column trial, 5 g of reagent was packed
in a
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samplet and 300 mg of Fmoc-PCA was dissolved in SmL of DMSO and added to
samplet. After 30 minutes the samplet was washed with 10 ml of ACN and
collected
mixture tested by HPLC. Higher de-blocking and scavenging was achieved using
column-based Samplet technology. The batch process gave 66% scavenging and 98%
de-blocking. The samplet/On-column gave 87% scavenging and 100% de-blocking.
The samplet method minimized amount of solvent used to recover.
1, 1-Dioxobenzo[b]thiophene-2-ylmethyloxcarbonyl (Bsmoc) is a common base
sensitive amino group protecting group. See Fig. 5. Bsmoc undergoes Michael-
like
to addition by base to generate the free amine and the reactive intermediate
(2). The
intermediate (2) decays over 8-10 minutes to give the stable de-blocking
product (3).
Bsmoc undergoes simultaneous de-blocking and scavenging steps (scheme 2)
Scavenging adduct (2) doesn=t leave the column. Only two equivalents of base
are
needed for complete liberation of free amine (product).
Bsmoc-p-chlorocarbanilate (Bsmoc-PCA) was used as test probe. 1g of
piprazinobenzyl-functionalized silica (reagent 2) was packed in the samplet.
30mg,
60mg, 100 mg, or 130 mg of Bsmoc-PCA dissolved in 2 ml of DMSO placed on
samplet. The solution was left in the samplet for 30 minutes. De-blocking was
100%
z o complete for the 30 and 60 mg of samplet. 98% de-blocking was achieved
with the 100
mg of sample. Bsmoc-de-blocking is dependent on, time, sample load, and
solvent
composition.
Piperazino-benzyl-functionalized silica (Reagent 2) was introduced for on-
column de
blocking and scavenging of Fmoc and Bsmoc groups. This reagent is very
successful
in Biotage samplet format and allows the automation of parallel solution phase
synthesis using Fmoc-chemistry. Base functionalized silica gel can be used in
different
applications including: synthesis of peptide using Fmoc- and Bsmoc- chemistry;
solid phase organic synthesis; solid phase Knoevenagel catalyst. Unreacted
excess
3 o starting material is selectively removed from reaction mixture.
Advantages of solid supported de-blocking reagents include: no loss of product
due to
formation of emulsion; eliminate extractions; decrease the quantity of
solvents utilized;
decrease the number of steps in the process including no filtration of solid
support; and
s 5 allow automation of parallel solution phase chemistry.
Other Embodiments
It is to be understood that while the invention has been described in
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conjunction with the detailed description thereof, the foregoing description
is intended
to illustrate and not limit the scope of the claimed invention. For example,
other
reactive functionalities, such as activated esters, alkyl halides, alkyl
triflates, malonate
derivatives, dimes, or dienophiles, can react with a residue of the
appropriate reactivity
s in a target compound mixture to form a covalent bond.
What is claimed is:
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