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| United States Patent | 5324483 |
| Link to this page | http://www.wikipatents.com/5324483.html |
| Inventor(s) | Cody; Donna R. (Saline, MI);
DeWitt; Sheila H. H. (Dexter, MI);
Hodges; John C. (Ann Arbor, MI);
Kiely; John S. (Ann Arbor, MI);
Moos; Walter H. (Oakland, CA);
Pavia; Michael R. (Newton, MA);
Roth; Bruce D. (Ann Arbor, MI);
Schroeder; Mel C. (Dexter, MI);
Stankovic; Charles J. (Ann Arbor, MI) |
| Abstract | An apparatus and method which provides a suitable location for multiple,
simultaneous synthesis of compounds. The apparatus consists of: a
reservoir block having a plurality of wells; a plurality of reaction
tubes, usually gas dispersion tubes, having filters on their lower ends; a
holder block, having a plurality of apertures; and a manifold, which may
have ports to allow introduction/maintenance of a controlled environment.
The manifold top wall has apertures and a detachable plate with identical
apertures. The apparatus is constructed from materials which will
accommodate heating, cooling, agitation, or corrosive reagents. Gaskets
are placed between the components. Rods or clamps are provided for
fastening the components together. Apparatus operation involves placing
the filters on the lower ends of the reaction tubes in the reservoir block
wells, and the upper ends passing through the holder block apertures and
into the manifold. The apparatus provides in excess of 1 mg of each
product with structural knowledge and control over each compound. Using
the apparatus a series of building blocks are covalently attached to a
solid support. These building blocks are then modified by covalently
adding additional different building blocks or chemically modifying some
existing functionality until the penultimate structure is achieved. This
is then cleaved from the solid support by another chemical reaction into
the solution within the well yielding an array of newly synthesized
individual compounds, which after postreaction modification, if necessary,
are suitable for testing for activity. |
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Title Information  |
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Drawing from US Patent 5324483 |
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Apparatus for multiple simultaneous synthesis |
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| Publication Date |
June 28, 1994 |
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| Filing Date |
February 2, 1993 |
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| Parent Case |
This application is a continuation-in-part of application Ser. No.
07/958,383 filed Oct. 8, 1992, now abandoned. |
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Title Information |
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Description |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to an apparatus and method which are used for the
multiple, simultaneous synthesis of compounds, including but not limited
to organic compounds.
2. Description of Related Art
It is well known in the art that peptides or oligonucleotides may be
multiply and simultaneously synthesized. In a basic, single synthesis of a
peptide, amino acids are simultaneously coupled to a functionalized solid
support. Several methods have been developed by which peptides or
oligonucleotides may be multiply synthesized. One such methodology for
peptide synthesis was disclosed in Geysen, et al, International
Publication Number WO 90/09395. Geysen's method involves functionalizing
the termini of polymeric rods and sequentially immersing the termini in
solutions of individual amino acids. Geysen's approach has proven to be
impractical for commercial production of peptides since only very minute
quantities of polypeptides may be generated. In addition, this method is
extremely labor intensive. A second method of peptide or oligonucleotide
synthesis was developed by Affymax Technologies N.V. and disclosed in U.S.
Pat. No. 5,143,854. The Affymax method involves sequentially using light
for illuminating a plurality of polymer sequences on a substrate and
delivering reaction fluids to said substrate. This method of synthesis has
numerous drawbacks, including the fact that the products are noncleavable
and that the process produces large numbers, but only minute quantities of
products. A further method and device for producing peptides or
oligonucleotides is disclosed in Houghton, European Patent Number 196174.
Houghton's apparatus is a polypropylene mesh container, similar to a
tea-bag, which encloses reactive particles. The containers, however, are
not amenable to general organic synthesis techniques. Further apparatus
are disclosed in German Published Patent Application Number DE 4005518 and
European Patent Number 0355582, issued to Boehringer Ingelheim KG. Like
the earlier devices, these apparatus are not suitable for the synthesis of
general organic compounds and are directed toward peptide or
oligonucleotide synthesis.
The synthesis of general organic compounds, poses many difficulties which
are absent in the synthesis of peptides or oligonucleotides. An approach
describing the synthesis of unnatural, oligomeric peptides is reported by
Simon, et al, in Proceedings of the National Academy of Sciences USA
1992;89:9367. Accordingly, none of the disclosed devices or methods for
the multiple, simultaneous synthesis of peptides or oligonucleotides are
useful for the synthesis of general organic compounds. Among the many
special problems found in the synthesis of general organic compounds, as
opposed to peptide or oligonucleotide synthesis, is the problem of
providing a device which will accommodate the wide range of synthetic
manipulations required for organic synthesis. The synthesis of general
organic compounds often requires such varied conditions as an inert
atmosphere, heating, cooling, agitation, and an environment to facilitate
reflux. Additionally, such synthesis requires chemical compatibility
between the materials used in the apparatus for multiple synthesis and the
reactants and solvents. Consequently, the apparatus must be constructed of
materials which are resistant to organic synthesis conditions and
techniques. Organic synthesis also often requires agitation. Such
agitation may be accomplished by magnetic stirring, sonicating, or
rotational shaking. None of the prior art devices are suitable for use
under these special conditions required for general organic synthesis.
While undeniably useful, peptides or oligonucleotides have significant
limitations in their application to pharmaceutical discovery programs. The
chemical leads discovered from these collections of compounds require
extensive modification due to the general unsuitability of peptides or
nucleotides as stable, orally active drugs. The building blocks utilized
are, in general, limited even allowing for the use of unnatural
enantiomers or artificial amino acids and modified nucleotides. The
peptides or oligonucleotides generated possess a repetitive linkage, amide
or phosphate moiety, which limits their structural diversity.
The principal object of the present invention, therefore, is to overcome
the limitations of the previous apparatus and methods which are limited to
peptide or oligonucleotide synthesis and to provide an apparatus and
method which will accommodate multiple, simultaneous synthesis of general
organic compounds including, but not limited to, nonpeptide or
nonnucleotide compounds.
SUMMARY OF THE INVENTION
The present invention is directed to an apparatus and method which will
provide a suitable environment for the synthesis of organic compounds.
Additionally, the synthesis of inorganic compounds, organometallic
compounds, and the like is entirely within the scope of the invention.
Central to the demonstration of this concept is the need to devise a
"general" method for multiple, simultaneous synthesis of organic
compounds. The synthesis method developed must satisfy the following
criteria. The compounds should be simultaneously synthesized in an array
format, which is compatible with the standard techniques of organic
synthesis. The final compounds should be produced individually (not as
mixtures) in soluble form. The quantity generated should be greater than 1
mg and in sufficiently pure form to allow direct biological testing.
Additionally, to the extent possible, sample handling should be carried
out using automated systems for speed, accuracy, and precision. A final
requirement is that the growing compounds must be readily separable from
by-products and reagents. Solid phase synthesis techniques commonly used
in peptide or oligonucleotide synthesis enable achievement of this
criteria. Typical solid supports (resins) include cross-linked
divinylbenzene-styrene (polystyrene), controlled pore glass (CPG),
polyacrylamides, poly(ethylene glycol) monomethyl ether and poly(ethylene
glycol) (PEG), silica gel, cellulose, acrylic acid grafted polypropylene,
and the like. Additionally, the solid support contains a reactive moiety.
Thus, a functionalized solid support is an insoluble material containing
an accessible reactive moiety such as, for example, a carboxylic acid,
alcohol, amine, halomethyl and the like which is used to covalently attach
an incoming building block. A further objective of the present invention
is to synthesize products with molecular weights of less than, but not
limited to, 1500 g/mol.
The approach described herein greatly increases the flexibility and
diversity of structures that can be produced by a parallel, solid phase
synthesis technology. Since neither the building blocks nor the methods
for connecting them are in any way limited, the chemistries compatible
with this apparatus and method are very broad, encompassing nearly all
organic reactions. The key feature which allows for the success of this
method is containment of a solid support within a gas dispersion tube.
This feature provides a unique means to segregate and manipulate the
growing compounds on a solid support. Equipment designed to simultaneously
manipulate the plurality of gas dispersion tubes and provide an
environment to perform standard organic synthesis techniques enables the
multiple, simultaneous synthesis of, for example, 8, 40, 100, or more
reactions at one time.
The steps necessary to perform a synthesis are 1) development of a
synthetic route that will be feasible on a solid support, 2) verification
of the resin-based synthesis using several representative examples, and 3)
execution of multiple, simultaneous synthesis within an array format to
generate, for example, 8, 20, 40 unit arrays and the like.
The method involves the sequential coupling of building blocks to form
resin-bound intermediates until the final or penultimate compound at each
location in the array is constructed, but still resin-bound. In addition
to coupling the building blocks directly, one may add, if required, a
coupling agent or reagent which is intended to chemically participate in
forming the covalent bond between the solid support and the building block
or between building blocks. Coupling reagents include catalysts, chemical
reagents, and the like. The sequential coupling reactions can be performed
as illustrated in the following procedures:
I. a functionalized solid support, a building block, a coupling reagent and
solvent are reacted together; or
II. a functionalized solid support, a reactive building block and solvent
are reacted together; or
III. a solid support with attached building block, a second building block,
a coupling reagent and a solvent are reacted together; or
IV. a solid support with attached building block, a second reactive
building block, and a solvent are reacted together.
Preferably, the sequential coupling reactions can be performed as
illustrated in the following procedures:
I. (a) charging the apparatus with a solid support with attached building
block wherein the building block has a reactive moiety protected by a
protecting group and a solvent;
(b) removing the protecting group from the reactive moiety with a
deprotection reagent;
(c) removing the deprotection reagent;
(d) sequentially adding additional reactive building blocks in solvents to
synthesize the compounds; and
(e) cleaving the compounds from the solid support within the apparatus to
afford the desired compounds; or
II. (a) charging the apparatus with a solid support with attached building
block wherein the building block has a reactive moiety protected by a
protecting group and a solvent;
(b) removing the protecting group from the reactive moiety with a
deprotection reagent;
(c) removing the deprotection reagent;
(d) adding a coupling reagent in a solvent;
(e) sequentially adding additional reactive building blocks and optionally
coupling reagents in solvents to synthesize the compounds; and
(f) cleaving the compounds from the solid support within the apparatus to
afford the desired compounds; or
III. (a) charging the apparatus with a solid support with attached building
block and a solvent;
(b) adding a reagent for changing the oxidation state of the reactive
moiety;
(c) sequentially adding additional reactive building blocks in solvents to
synthesize the compounds; and
(d) cleaving the compounds from the solid support within the apparatus to
afford the desired compounds; or
IV. (a) charging the apparatus with a solid support with attached building
block and a solvent;
(b) adding a reagent for changing the oxidation state of the reactive
moiety;
(c) adding a coupling reagent in a solvent;
(d) sequentially adding additional reactive building blocks and optionally
coupling reagents in solvents to synthesize the compounds; and
(e) cleaving the compounds from the solid support within the apparatus to
afford the desired compounds.
Other strategies for constructing the growing compounds on the solid
support are possible and are encompassed within the scope of the present
invention.
Cleavage of the final compound from the resin yields a product which can be
readily separated from the spent resin. Several options are available for
achieving this cleavage and these are illustrated in Scheme 1. A single,
invariant cleavage reagent can be employed to attack the resin-bound
product linkage to yield a final compound containing an invariant
functionality. Cleavage can be affected utilizing a variety of incoming
building blocks to attack the resin linkage and give a product with
variations in structure at the site of detachment. An alternative strategy
constructs a precursor compound (resin-bound) possessing a distal
functionality which, when activated or unmasked, will attack the
resin-linking bond resulting in ejection of the cyclized final compound
into solution. Since "unreacted" compounds remain attached to the resin,
the latter option provides a means to produce cleaner final products.
The use of a solid support to multiply and simultaneously synthesize a
subset of related, individual compounds requires a means of preparing the
compounds in an array format. The method for constructing a compound array
is illustrated with the following two examples: In one variant, the final
compound, prior to detachment from the solid support, can be constructed
from two building blocks/portions/halves utilizing a single coupling
reaction to join the two smaller parts. One starts by selecting the
congeners of building block #1 (for example, 3; A, B, and C) to be
directly attached to the solid support and the number of congeners of the
second half (building block #2) of the final compound that will be
attached to the first building block (for example, 3; X, Y, and Z). The
number of congeners of building block #1 multiplied by the number of
congeners of building block #2 gives the number of locations contained in
the array, in this example 3.times.3=9. Each of the congeners of #1 is
covalently attached to the solid support a number of times equal to the
number of congeners of #2, herein each of the first building blocks (A, B,
and C) is coupled to the support in three locations each (9 couplings
total). The covalent joining of the second building block to the first
building block is now carried out with each of the congeners of building
block #2 (i.e., A, B, and C are each coupled once with Z giving AZ, BZ,
and CZ). Completion of the progression of couplings yields all nine
expected permutations. This is illustrated in Scheme A.
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SCHEME A.
Construction of an Array Involving One
Coupling Reaction
Building Building Block #1
Block #2 A B C
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X AX BX CX
Y AY BY CY
Z AZ BZ Cz
______________________________________
To achieve additional structural variation, one can utilize the addition of
a third set of building blocks (for example, 3; 1, 2, and 3) and thus a
second coupling reaction to provide 27 elements from an array of
3.times.3.times.3=27. To accomplish this in the array synthesis will
require that the first array (building block #1 coupled to building block
#2 which is nine elements) be replicated three times so that when the
third building block is added, the final elements are produced separately.
This would be carried out as is illustrated in Scheme B. The expansion of
the number of congeners within each building block set and the expansion
of the number of discrete building blocks can be carried to whatever level
desired to prepare arrays of any desired size or structural variability.
An alternative array construction can be carried out using a large number
of congeners of several building blocks but choosing not to prepare every
permutation possible (for example, two building blocks each with 30
congeners leads to 900 possible compounds). In this instance quantitative
structure activity relationship techniques and statistical methods can be
used to select the most desired subsets of congeners to employ in
preparing a smaller array.
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SCHEME B.
Construction of an Array Involving Two
Coupling Reactions
Build-
ing
Block Building Block #1 (A, B, C)
#2 A B C A B C A B C
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X AX1 BX1 CX1 AX2 BX2 CX2 AX3 BX3 CX3
Y AY1 BY1 CY1 AY2 BY2 CY2 AY3 BY3 CY3
Z AZ1 BZ1 CZ1 AZ2 BZ2 CZ2 AZ3 BZ3 CZ3
1 2 3
Building Block #3 (1, 2, 3)
______________________________________
To achieve the foregoing objects and in accordance with the purpose of the
invention, as embodied and broadly described herein, the invention
provides an apparatus for the multiple, simultaneous synthesis of
compounds comprising: (a) a plurality of reaction tubes, with each
reaction tube having a filter device on its lower end; (b) a reservoir
block which has a means for containing a plurality of reaction wells and
receiving the filter devices on the lower ends of the plurality of
reaction tubes; (c) a holder block with a plurality of apertures which
correspond to the location of the plurality of reaction wells in the
reservoir block and the plurality of reaction tubes; (d) a manifold
located adjacent to the holder block, with the lower end of the manifold
open such that the manifold may be placed on the holder block and surround
the upper ends of the reaction tubes which are protruding upward through
the apertures in the holder block; (e) a means for providing a sealed
connection which is impermeable to gases and liquids between the holder
block and the manifold and the holder block and the reservoir block; and
(f) a means for fastening together the components of the apparatus. FIGS.
1-10 illustrate the components and embodiments of the apparatus.
In a second embodiment, the upper end of the manifold has a plurality of
apertures which correspond in location to the apertures in the holder
block. In this second embodiment, a plate which has a plurality of
apertures which correspond in location to the apertures in the upper end
of the manifold, and a means for providing a seal between the plate and
the upper end of the manifold is provided. FIGS. 1 and 6 illustrate the
components of this particular embodiment.
A further advantage of the present invention is that the apparatus provides
the ability to monitor the reaction process by removal of a filtrate
aliquot from the reaction well and analyze the solution by common
chromatographic methods, such as Gas Chromatography/Internal Standard
(GC/ISTD), High Pressure Liquid Chromatography/Internal Standard
(HPLC/ISTD) or Thin Layer Chromatography (TLC), titration, colorimetry,
spectroscopic methods, and the like. Additionally, by providing a separate
reaction vessel for each reaction, the apparatus allows for the integrity
of the filtrates, intermediates, and compounds which are generated.
The apparatus of the present invention has the additional advantages of
being constructed of materials which are chemically compatible with
organic reagents, such as corrosive acids and organic solvents, required
for organic reactions. The present invention has the further advantage of
having the ability to provide a suitable means for the manipulations, such
as agitation, heating, cooling, refluxing, and an inert atmosphere, common
to organic synthesis.
Additional objectives and advantages of the invention will be set forth in
part in the description that follows, and in part will be obvious from
this description, or may be learned by practice of the invention. The
objects and advantages of the invention may be realized and attained by
means of the instrumentalities and combinations particularly pointed out
in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
While this specification concludes with claims particularly pointing out
and distinctly claiming that which is regarded as the present invention,
the objects and advantages of this invention may be more readily
ascertained from the following description of a preferred embodiment when
read in conjunction with the accompanying drawings.
FIG. 1 is an illustration of the components of the preferred embodiment of
the apparatus of the invention.
FIG. 2 is a perspective view of the components of the preferred embodiment
of the apparatus of the invention.
FIG. 3 is an exploded perspective view showing each of the components of
the preferred embodiment of the apparatus of the invention.
FIG. 4 is a cross-sectional view FIG. 3.
FIG. 5 is an enlarged partial cross-section of FIG. 4.
FIG. 6 is an exploded perspective view of the preferred embodiment of the
invention.
FIG. 7 is an alternative embodiment for pressure equalization by a jacketed
gas dispersion tube.
FIG. 8 is an alternative embodiment for pressure equalization by a
capillary tube.
FIG. 9 is an alternative embodiment of the components for providing a
gas-tight seal.
FIG. 10 is an illustration of the solid-phase extraction equipment for
postcleavage manipulations.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Reference will now be made in detail to the presently preferred embodiments
of the invention.
The apparatus of the present invention comprises a plurality of reaction
vessels. For the sake of illustration only, the accompanying drawings and
description describe a device containing eight such reaction vessels,
unless otherwise described. A device having a greater or lesser number of
reaction vessels is entirely within the scope of the invention. Further,
the apparatus is described in the accompanying drawings as having a
horizontal cross-section which is rectangular in shape. An apparatus
having a square or circular horizontal cross section is also entirely
within the scope of the present invention. Additionally, the apparatus may
be adapted to be fully automated or semiautomated, and such adaptations
are also within the scope of invention.
An initial element of the apparatus (10) as shown in FIGS. 1-6 is reaction
vessels which can be any vessels capable of containing resin and organic
reactants. In the preferred embodiment of the invention, the reaction
vessels are a combination of reaction tubes and reaction wells. The
reaction tubes are most preferably gas dispersion tubes (11). The lower
ends of the reaction tubes have filters (14) and one or more pressure
equalization holes (40) pierced in the reaction tubes (11) above the
filters (14). The total length of each reaction tube can be from 50 to 300
mm with a preferred length of 250 mm. The upper ends of the reaction tubes
may have an outside diameter of from 5 to 25 mm, with a preferred outside
diameter of 8 mm. The inside diameter of the upper ends of the reaction
tubes can be from 1 to 24 mm, with a preferred inside diameter of 5 mm. To
allow for the materials in the reaction tube to mix with reactants, a
filter (14) should be located in the lower end of the reaction tube (11).
The length of the filters (14) on the lower ends of the reaction tubes can
be from 1 to 300 mm, with a preferred length of 25 mm. The filters (14) on
the lower ends of the reaction tubes may have an outside diameter of from
5 to 30 mm, with a preferred outside diameter of 12 mm. The filters (14)
on the lower ends of the reaction tubes may have an inside diameter of
from 1 to 24 mm, with a preferred inside diameter of 5 mm. Preferably, in
order to allow the maximum reaction between material placed in the
reaction tube and surrounding reactant, this filter (14) is preferably
constructed of fritted glass. The porosity of the frit may be modified or
selected to accommodate v | | |
