U.S. patent application number 10/317178 was filed with the patent office on 2003-07-31 for apparatus and method for synthesizing.
This patent application is currently assigned to Selecticide Corporation. Invention is credited to Baum, Stephen A., Flynn, Gary A., Patek, Marcel, Pavel, Safar, Smrcina, Martin, Strop, Peter, Wegrzyniak, Eric.
Application Number | 20030143630 10/317178 |
Document ID | / |
Family ID | 23783375 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030143630 |
Kind Code |
A1 |
Patek, Marcel ; et
al. |
July 31, 2003 |
Apparatus and method for synthesizing
Abstract
An apparatus and method for synthesizing a combinatorial library
comprising a plurality of chemical compounds such that the chemical
composition of each compound is easily tracked. The library
compounds are synthesized on solid-phase supports, which are
spatially arranged in frames during synthesis according to a
predetermined protocol, such that each solid-phase support passes
through a series of unique spatial 2D or 3D addresses by which the
chemical composition of each compound may be determined at any
point during synthesis. Solid-phase supports include hollow
tubular-shaped lanterns and gears.
Inventors: |
Patek, Marcel; (Tucson,
AZ) ; Pavel, Safar; (Tucson, AZ) ; Smrcina,
Martin; (Tucson, AZ) ; Wegrzyniak, Eric;
(Tucson, AZ) ; Strop, Peter; (Tucson, AZ) ;
Flynn, Gary A.; (Tucson, AZ) ; Baum, Stephen A.;
(Tucson, AZ) |
Correspondence
Address: |
SYNNESTVEDT & LECHNER, LLP
2600 ARAMARK TOWER
1101 MARKET STREET
PHILADELPHIA
PA
191072950
|
Assignee: |
Selecticide Corporation
Tucson
AZ
85711
|
Family ID: |
23783375 |
Appl. No.: |
10/317178 |
Filed: |
December 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10317178 |
Dec 11, 2002 |
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09449222 |
Nov 24, 1999 |
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6541211 |
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09449222 |
Nov 24, 1999 |
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09082038 |
May 20, 1998 |
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Current U.S.
Class: |
435/7.1 ;
422/129; 436/518 |
Current CPC
Class: |
B01J 2219/00675
20130101; B01J 2219/00459 20130101; B01J 2219/00286 20130101; C40B
60/14 20130101; B01J 2219/00722 20130101; B01J 2219/00322 20130101;
B01J 2219/00565 20130101; Y10T 436/24 20150115; B01J 2219/00328
20130101; B01J 2219/00502 20130101; B01J 2219/005 20130101; B01J
2219/00659 20130101; B01J 2219/00725 20130101; B01J 2219/00475
20130101; B82Y 30/00 20130101; B01J 2219/00472 20130101; B01J
2219/00461 20130101; B01J 2219/00527 20130101; B01J 2219/00317
20130101; B01J 2219/00319 20130101; Y10T 436/25 20150115; B01J
2219/00313 20130101; G01N 33/543 20130101; B01J 2219/00592
20130101; B01J 2219/00668 20130101; C40B 40/06 20130101; B01J
2219/0072 20130101; C40B 50/14 20130101; B01J 19/0046 20130101;
B01J 2219/0031 20130101; B01J 2219/0059 20130101; B01L 2300/0819
20130101; B01L 2300/0829 20130101; C40B 70/00 20130101; B01J
2219/00315 20130101; B01J 2219/00468 20130101; B01J 2219/00664
20130101; B01J 2219/00497 20130101; B01J 2219/00522 20130101; B01J
2219/00596 20130101; C40B 40/10 20130101; B01J 2219/00585 20130101;
B01J 2219/00689 20130101; Y10T 436/255 20150115 |
Class at
Publication: |
435/7.1 ;
422/129; 436/518 |
International
Class: |
G01N 033/53; B32B
005/02; G01N 033/543 |
Claims
What is claimed:
1. A system for synthesizing a combinatorial library comprising: a
containment device with a plurality of channels, each channel
fluidly connected to the outside of the containment device through
an opening; free solid supports, wherein the solid supports are
sized to stack inside the plurality of channels and thereby create
a columns of supports, wherein the spatial arrangement of the solid
supports in channels defines a 3-D array; and means for removing
the solid supports through said channel, retaining the spatial
relationship of the supports with each other.
2. The system as in claim 1 wherein the solid supports have a hole
in the middle and the means for removing the solid supports through
the channel is a rod with one stop-end larger than the hole in the
support, said rod used to insert through the holes of a vertical
stack of supports.
3. The system as in claim 1 wherein the solid supports are gears,
having a tubular structure with a plurality of outwardly projecting
fins.
4. The system of claim 3 wherein the gear is made of polypropylene
with a surface of polystyrene and having an outer diameter of from
about 4.0 mm to about 6.0 mm.
5. The system of claim 4 wherein the outer diameter of each gear is
about 5.0 mm.
6. The system as in claim 1 wherein the solid supports are rings,
having an inner borehole and an outer diameter.
7. The system of claim 6, wherein the rings are made of
polypropylene with a surface of polystyrene, with an outer diameter
of from about 7.8 mm, an inner bore diameter of about 6.9 mm, and a
height of about 3.1 mm.
8. The system as in claim 1 wherein the solid support are lanterns,
having holes in the middle.
9. The system of claim 8 wherein the lanterns are made of
polypropylene with a surface of polystyrene and having a tubular
structure with an outer diameter of from about 4.0 mm to about 6.0
mm and an inner bore diameter of from about 2.0 mm to about 3.0
mm.
10. The system of claim 9 wherein the outer diameter of each
lantern is about 5.0 mm, and the inner bore diameter is about 2.5
mm.
11. The system as in claim 1 wherein the means for removing the
solid supports through the channel is a vacuum that picks up X-Y
layers of solid-phase supports simultaneously.
12. The system as in claim 1 wherein the channels are cylindrical
wells.
13. The system as in claim 1 wherein the solid supports stacked in
one channel have mechanical means of linking supports, and the
means of removal of the entire stack of supports in one channel is
to grasp the first support closest to the channel opening, thereby
retaining the spatial relationship of the supports to the other
removed supports.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/449,222, filed Nov. 24, 1999, which is a
continuation-in-part of U.S. patent application Ser. No.
09/082,038, filed May 20, 1998, the entire disclosures of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of combinatorial
libraries. More specifically, the invention relates to methods of
synthesis utilizing arrays of solid-phase supports to produce a
combinatorial library of chemical compounds and, additionally, the
apparatuses used to carry out those methods.
BACKGROUND OF THE INVENTION
[0003] Citation or identification of any reference in section 2 or
any section of this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
[0004] A combinatorial library is a collection of multiple species
of chemical compounds comprised of smaller subunits or monomers.
Combinatorial libraries come in a variety of sizes, ranging from a
few hundred to several thousand species of chemical compounds.
There are also a variety of library types, including oligomeric and
polymeric libraries comprised of compounds such as peptides,
carbohydrates, oligonucleotides, and small organic molecules, etc.
Such libraries have a variety of uses, such as identifying and
organic molecules, etc. Such libraries have a variety of uses, such
as identifying and characterizing ligands capable of binding an
acceptor molecule or mediating a biological activity of
interest.
[0005] The library compounds may comprise any type of molecule of
any type of subunits or monomers, including polymers wherein the
monomers are chemically connected by any sort of chemical bond such
as covalent, ionic, coordination, chelation bonding, etc., which
those skilled in the art will recognize can be synthesized on a
solid-phase support. The term polymer as used herein includes those
compounds conventionally called heteropolymers, i.e., arbitrarily
large molecules composed of varying monomers, wherein the monomers
are linked by means of a repeating chemical bond or structure. The
polymers of the invention of this types are composed of subunits or
monomers that can include any bi-functional organic or
herteronuclear molecule including, but not limited to amino acids,
amino hydroxyls, amino isocyanates, diamines, hydroxycarboxylic
acids, oxycarbonylcarboxylic acids, aminoaldehydes, nitroamines,
thioalkyls, and haloalkyls. In the disclosure of the present
invention, the terms "monomer," "subunits" and "building blocks"
will be used interchangeably to mean any type of chemical building
block of molecule that may be formed upon a solid-phase
support.
[0006] Various techniques for synthesizing libraries of compounds
on solid-phase supports are known in the art. Solid-phase supports
are typically polymeric objects with surfaces that are
functionalized to bind with subunits or monomers to form the
compounds of the library. Synthesis of one library typically
involves a large number of solid-phase supports. Solid-phase
supports known in the art include, among others, polystyrene resin
beads, cotton threads, and membrane sheets of
polytetrafluoroethylene ("PTFE").
[0007] To make a combinatorial library, solid-phase supports are
reacted with a one or more subunits of the compounds and with one
or more numbers of reagents in a carefully controlled,
predetermined sequence of chemical reactions. In other words, the
library subunits are "grown" on the solid-phase supports. The
larger the library, the greater the number of reactions required,
complicating the task of keeping track of the chemical composition
of the multiple species of compounds that make up the library.
Thus, it is important to have methods and apparatuses which
facilitate the efficient production of large numbers of chemical
compounds, yet allow convenient tracking of the compounds over a
number of reaction steps necessary to make the compounds.
[0008] One method of making combinatorial libraries is described in
U.S. Pat. No. 5,510,240 to Lam et al. ("Lam '240 patent"), the
disclosure of which is incorporated herein by reference in its
entirety. More specifically, the Lam '240 patent discloses a split
and mix method of synthesizing combinatorial libraries of
bio-oligomers on resin beads, in certain embodiments of which the
library contains all possible combinations of monomer subunits of
which the bio-oligomers are composed. Although there may be several
resin beads containing the same species of bio-oligomer, each resin
bead contains only one species of bio-oligomer.
[0009] Another example of a method of making combinatorial
libraries on divisible solid-phase supports is described in U.S.
Pat. No. 5,688,696 to Lebl ("Lebl '696 patent"), the disclosure of
which is incorporated herein by reference in its entirety. In the
method disclosed in the Lebl '696, each of a set of predetermined
species of test compounds is present on a predetermined number of
solid-phase supports--preferably on only one--and each solid-phase
support has only a single species of test compound.
[0010] The use of radio-frequency identification ("RFID") chips to
record the steps of library synthesis is also known. See, for
example, U.S. Pat. Nos. 5,741,462, 5,770,455, and 5,751,629, as
well as WO 98/15826.
[0011] A method and apparatus for synthesis of a combinatorial
library using a 3-D array of reaction zones is provided in Glaxo's
WO 99/32219 ("Glaxo Application"). This application discloses
stackable frames having a plurality of holes. Membranes, which act
as the solid supports, are trapped between stacked frames, and
these membranes are exposed at the frame holes. In an alternative
embodiment, solid support beads are placed on flow-through sieves
that allow flow-through of reagents around the support beads.
Reagents are pumped in from the top and vacated at the bottom or,
alternatively, pumped in from the bottom and vacated at the top.
The apparatus disclosed allows reagents to be delivered to groups
of supports in the X-Z planes or in the Y-Z planes during synthesis
steps.
[0012] The Glaxo Application also employs a 3-D (X-Y-Z) array of
supports. However, instead of using a containment apparatus having
true wells in which solid supports are stacked, the Glaxo method
employs stackable 2-D (X-Y) frames. The Glaxo Application discloses
two distinct embodiments of stackable frame structures. One
embodiment sandwiches a membrane between stacked frames, the frames
having a plurality of holes. The membranes are solid-phase supports
which are held between the frames. The frame holes expose the
membranes. The membranes also have holes to allow reagents to pass
through the layers of membranes and contact other membranes in the
vertical "column" of the array. Another embodiment has sieves in
place of the membranes, and free solid supports are placed on each
sieve between the frames. The sieves allow reagents to flow
vertically from top to the bottom of the stacked 3-D array
contacting a vertical column of solid-phase supports resting on
sieves.
[0013] A major disadvantage with Glaxo's apparatus and method,
however, is that after the synthesis is completed, the solid
supports, whether as the membrane or the solid-phase support beads
suspended on the sieve, are not easily freed from the stacked array
while retaining their spatial identities. The frames must be taken
apart one by one to gain access to the supports and to provide some
means to retain the identities of each support. This requires a
burdensome additional step that makes the apparatuses disclosed
less attractive for commercial production of libraries.
[0014] While methods exist in the art that can be used to produce a
library of compounds, there is still a need for methods and
apparatuses effective for commercial use to build a large library
of compounds quickly and with a minimum of cost. Thus, there is
still a need for alternative methods of synthesis that use 2-D or
3-D arrays of solid-phase support as part of the synthesis process
for the purpose of commercially making large libraries of compounds
efficiently.
[0015] Moreover, there is still a need for apparatuses and methods
for efficiently synthesizing extremely large libraries, e.g.,
greater than 100,000 compounds, using 2-D or 3-D arrays as tools in
the synthesis.
SUMMARY OF THE INVENTION
[0016] The present invention provides methods and apparatuses that
use 2-D or 3-D array of solid-phase supports and that may be used
to commercially synthesize a library of compounds. In particular a
method is provided which may be commercially used to produce large
libraries having between about 100,000 to 200,000 compounds. A
number of embodiments of methods and apparatuses for synthesizing
libraries of compounds are provided herein in accordance with the
present invention.
[0017] In a first embodiment, in accordance with the present
invention, a 3-D array of solid-phase supports is used to provide
parallel synthesis. One embodiment of the apparatus which provides
this 3-D array is a containment device which has a plurality of
wells wherein discrete solid-phase supports can be placed into and
stacked in a column. In another embodiment of the apparatus, a 3-D
array is formed by stacking a plurality of 2-D frames which have
solid-phase supports arranged in an orderly X-Y array. The frames
have a plurality of holes arranged in an orderly X-Y array and
solid-phase supports can be friction fitted or interlocked into
these holes to temporarily hold the supports to the frame during
synthesis. Alternatively, the supports can be physically attached
to the frames in a manner in which, when desired, they can be
easily cut from the frame. Associated with this 3-D array, specific
embodiments of the apparatuses are disclosed, in accordance with
the present invention, including a 3-D containment plate which has
double-drilled holes, a gear-shaped solid-phase supports ("gear")
designed to be friction fitted or interlocked into 2-D frame holes,
a lantern-shaped solid-phase supports ("lantern"), and ring
supports used in conjunction with a containment device having a
plurality of wells.
[0018] A specific synthesis method is provided, which can be used
with an apparatus having a 3-D arrangement of solid-phase supports,
in accordance with the present invention. A preferred method
provides a monomer or subunit diversity to the library compounds on
the solid-phase supports between the X-Y layers in the Z direction.
The method comprises: providing reagents to react with solid-phase
supports in the X-Z layers, providing reagents to react with
solid-phase supports in Y-Z layers, and retrieving columns of
solid-phase supports, while retaining their spatial
relationships.
[0019] A defining characteristic of this first method embodiment
using a 3-D array of support is once the array is formed, the
supports are generally not moved during the subsequent synthesis
steps. Reagents for reacting with the supports are brought to the
array and usually a particular reagent is delivered only to a
subset of the supports in the 3D array. Additionally, the size of
the library of compound will be limited by the size of the 3-D
array.
[0020] In a second method embodiment, in accordance with the
present invention, the same stackable frames are used as in the
first embodiment. Frames having X-Y arrays of solid-phase supports
are stacked into 3-D arrays ("stacks"). Instead of a single 3-D
array, in this second embodiment a multiple N number of stacks are
formed in preparation for making a library of compounds.
[0021] In the first synthesis step, each stack numbered 1 to N is
completely immersed into separate reactors 1 to N respectively,
each reactor having a distinct reagent and a subunit is attached to
each support in the stack. After each stack is removed from its
reactor, a first randomization occurs by taking one and only one
layer (frame) of each original stack, combining these layers to
form a new stack. Thus, the first layer or frame from each original
stack is grouped to create a first new stack, the second layer or
frame from each original stack is grouped to create a second new
stack. This reshuffling process is repeated until all the original
frames of each old stack are transferred to a set of N number of
new stacks. Then, in the second synthesis step, each new stack from
1 to N is immersed in a set of reactors, each reactor having a
different reagent.
[0022] In the second randomization step, one vertical column of
solid-phase supports is removed from each new stack keeping the
spatial identification of the supports intact and then reassembled
to make a new grouping of 3-D supports. Another vertical column of
supports is removed from each new stack and regrouped to another
grouping of 3-D supports. This process is repeated until all the
supports in the new stack arrays have been regrouped into a N
number of new 3-D arrays. In this regrouping, randomization step,
only one vertical column of supports is taken from each new stack
to make a new grouping of supports. In the third and final
synthesis step, the new groupings of supports are each put into
separate 1 to N reactors, each reactor having a different
reagent.
[0023] The apparatuses used with this second embodiment are the
same as used in the first embodiment. A preferred embodiment of the
frame and solid-phase support is a 2-D frame having a plurality of
holes arranged in an X-Y rectangular order. A preferred apparatus
comprises gears or lanterns friction fitted or interlocked into the
plurality of holes. Additionally, reactors having a capacity large
enough for immersion of 3-D stacks are needed.
[0024] The defining characteristics of this second embodiment are:
(a) many N number of 3-D original stacks are formed; (b) the
original stacks do not have solid supports which have a subunit
attached in contrast to embodiment one; (c) the solid supports are
disturbed from the original 3-D arrays because the supports are
moved during the synthesis process when the frames are reshuffled
and vertical columns of supports are regrouped; and (d) every solid
support in each 3-D stack is completely immersed in the reagent
during a synthesis step because the stack is brought to the
reagents/reactors. The second embodiment lends itself to large
scale production of libraries of compounds because the final number
of unique compounds is based on the number N of original stacks
made.
[0025] The third embodiment, in accordance with the present
invention, uses 2-D frames in a "sort and combine" method of
synthesis. There is no stacking of the frames into a 3-D array.
Instead, the 2-D frames are split during synthesis of the
combinatorial library. The method of this third embodiment can be
implemented by automation since no rods are required and may be
used to generate large libraries, having between about 100,000 to
200,000 compounds.
[0026] In this method, a Q number of 2-D frames is chosen. The 2-D
frames have rows and columns. Solid supports are placed into
reagents for a first synthesis step. Solid supports thus reacted
with a single subunit are placed into the frame holes such that the
frame has columns of supports which have the same subunit, but
between columns, there is a diversity of subunits. This placement
provides the first randomization. Each Q number of frames is
initially identically prepared. Next, in a second synthesis step,
the Q frames are placed into 1 to Q reactors, each having a
different reagent. After removal from the reactors, the Q number of
frames are split up into subframes to provide the second
randomization. M new groups of subframes are regrouped by taking
one and only one subframe from each original frame. M represents
the number of subframes a frame has been split into. The M new
groups of subframes, each are immersed into 1 to M reactors, each
reactor having a different reagent. After final synthesis the
supports are detached from the subframes and placed into a labeled
cleavage plate.
[0027] The total number of unique compounds in the library is
Q.times.M.times.N, where N is the number of columns present in the
original 2-D frames, and Q is arbitrarily chosen. The size of the
library will be controlled by choice of three variables Q, M and
N.
[0028] The preferred apparatuses used in this embodiment are 2-D
frames. Solid-phase supports such as gears are friction fitted or
interlocked into the plurality of holes in the frame. The
additional feature of the frame is that it must be easily
splittable into subframes. Reactors are need which have capacity
for accepting groups of subframes. Additionally, in accordance with
a preferred embodiment of the present invention, a 2 row subframe
having a RIFD chip is disclosed.
[0029] The defining characteristics of this third method embodiment
are: (a) user choice of the number of frames Q to use in the
synthesis; (b) the solid supports are disturbed from the original
2-D arrays because the supports are moved during the synthesis
process when the frames are split and regrouped; and (c) every
solid support in each 2-D frame or 2-D subframe is completely
immersed in the reagent during a synthesis step because the frame
or group of frames is brought to the reagents/reactors. The third
embodiment lends itself to large scale production of libraries of
compounds because the final number of unique compounds is based on
the number Q of original frames used.
[0030] All three method embodiments use 2-D or 3-D arrays of
supports held in frames to facilitate parallel synthesis on
solid-phase supports and to provide spatial identification and thus
the synthesis history of the compound produced on a particular
support.
[0031] There is interchangeability of apparatuses used in the
various embodiments described above, in accordance with the present
invention. For example, the supports, frames, rods and devices for
removing the supports from the frames are interchangeable. A gear
design of solid support for use with 2-D frames is provided in
accordance with the present invention. A new embodiment of a 3-D
containment plate having double-drilled holes and RFID chip is
provided in accordance with the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Reference is next made to a brief description of the
drawings, which are intended to illustrate a number of embodiments
of the apparatus and method of making a combinatorial library
according to the present invention. The drawings and detailed
descriptions which follow are intended to be merely illustrative,
and are not intended to limit the scope of the invention as set
forth in the appended claims.
[0033] FIG. 1a illustrates six flasks 20a-20f, having six different
reagents, and 96 solid supports used in the first embodiment of the
present invention;
[0034] FIG. 1b provides a cutaway side view of a containment well
showing the first layer of solid-phase supports after being
distributed from the first flask;
[0035] FIG. 1c shows a top view of the 96 well plate;
[0036] FIG. 1d shows another cutaway side view of the containment
apparatus with wells containing all six layers of solid-phase
supports, wherein each layer has a different subunit or building
block;
[0037] FIG. 2 is a combined diagram and top view of the 96 well
containment apparatus, wherein R2(A) through R2(H) represent
different reagents delivered into the rows of wells (X direction of
the array) of the apparatus in the second synthesis step;
[0038] FIG. 3 is a combined diagram and top view of the 96 well
containment apparatus, wherein R3(A) through R3(L) represent
different reagents delivered into the columns of wells (Y direction
of the array) of the apparatus in the third and final synthesis
step;
[0039] FIG. 4 is a mixed side view and perspective view
illustrating the final distribution of the solid-phase supports, in
which each solid support now provides a unique compound and each
layer is distributed into one-layer cleavage plates, 38a-f;
[0040] FIG. 5 is a perspective view of a single frame, showing the
friction fitted or interlocked gears in a plurality of holes, a
stack of frames providing a 3-D array, and a complete set of
original "stacks" consisting of 24 total stacks;
[0041] FIG. 6 is a perspective view illustrating the first
randomization step involving taking one layer from each original
stack to create a new stack and a set of new stacks;
[0042] FIG. 7 is a perspective view illustrating the second
randomization step of the present method, wherein a single column
of supports is taken from each new stack and regrouped to form a
new group of supports in a 3-D array and each column is then
treated with reagents in a third randomization step;
[0043] FIG. 8 is a top view of a gear-shaped solid support ("gear")
made by Chiron;
[0044] FIG. 9 is a top view of a gear frame that may be used in all
embodiments of the present invention;
[0045] FIG. 10 is a perspective view of a solid-phase support
shaped as a ring;
[0046] FIG. 11 is a top view of a 3-D containment plate having
double-drilled holes for wells;
[0047] FIG. 12 is a schematic diagram illustrating an embodiment of
the present invention used to synthesize a library having 27
subunits;
[0048] FIG. 13 is a perspective view of a Chiron lantern solid
support;
[0049] FIG. 14a is a perspective view of a frame used to contain
solid-phase supports, the frame having an RFID chip; and
[0050] FIG. 14b is a top view of the frame shown in FIG. 14a.
DETAILED DESCRIPTION OF THE INVENTION
[0051] A detailed explanation of the methods and apparatuses in
accordance with the present invention with reference to the
drawings is provided as follows:
[0052] A. Synthesis of Compounds Using Frames Stacked to Provide a
3-D Array
[0053] One method of synthesizing solid supports was disclosed in
co-pending Baum et. al. U.S. patent application Ser. No. 09/082,038
the disclosure of which is incorporated by reference in its
entirety. ("Baum Application") (See Baum Application, p. 6, para. 1
and p. 10, para. 2, for discussion of the method utilizing a 3-D
array.) The method uses a containment apparatus having a plurality
of vertical wells. Free solid-phase supports are "stacked" into
each well, each support physically contacting adjacent supports
within a well. (See Baum Application, pp. 4-6, in particular, top
of p. 5, lines 4-5, discussing a "plurality of discrete supports
arranged in a plurality of columns in one or more wells." See also
FIGS. 12-17, 23-29 which describe various embodiments of 3-D
apparatuses containing a plurality of wells, wherein supports are
stacked, and pp. 6-7 which provide Figure captions and discussions
of those Figures in pp. 8-41.) The stacking of supports in the
containment structure thereby provides an overall 3-D spatial
arrangement of supports within the containment apparatus. Thus,
after stacking, each support in the 3-D array may be identified by
its X-Y-Z position in the array. Once the supports are placed
inside the wells, reagents may be directed into sets of wells to
react with the supports during steps of the synthesis process.
Importantly, because of the open well structure of the containment
apparatus, when the synthesis steps are completed, the supports can
be easily retrieved from the wells, while retaining the spatial
identification of each support.
[0054] An example implementing the specific steps of the method is
illustrated FIG. 1. The solid support 10 must be of a type which is
free and can be stacked vertically in the containment wells. The
solid support may be of various types including, but not limited
to, those disclosed in co-pending U.S. application Ser. No.
09/082,038, as well as those known in the art. (See Baum
Application, FIGS. 12-17, 25-28, which describe various embodiments
of solid supports contained in wells wherein "discrete" or free
supports are stacked; pp. 6-7 which provide Figure captions and
discussion of those Figures in pp. 8-41 and ; p. 21 at para. 1-2
discussing possible shapes of supports.) The solid supports used in
the following example is a commercially available Chiron lantern,
as shown in FIG. 13.
[0055] As shown in FIG. 1a, there are six flasks, 20a-f, each
containing a different reagent, R1. As a first synthesis step 96
lanterns 10 are placed into each flask to provide a total of 576
lanterns reacted. The reagents in the flasks attaches to the
functionalized surface of the lanterns, thereby forming first
synthesis intermediates. It can be seen that six different types of
synthesis intermediates are formed by placement in the six flasks,
having different reagents.
[0056] Next, the 96 lanterns are taken from the first flask 20a and
distributed into the 96 vertical wells 45 as the first X-Y layer
(Z=1) of supports in the containment apparatus 35, as shown in FIG.
1b. The lanterns from the next flask 20b are then distributed in
the same manner forming the second layer of supports within the
containment apparatus. This process is repeated until all lanterns
from each remaining flask 20c-f are distributed by layers into the
containment apparatus.
[0057] The containment apparatus 35 must be made of a material
which is inert to reagents and can provide proper structural
rigidity. A standard 96-well plate, each well approximately 2 ml
deep, can be used as 3-D containment apparatus with the proper
choice of stackable solid supports. As provided in FIG. 1c, which
shows a top view of the containment apparatus, the apparatus has 96
total wells placed in an eight by twelve arrangement in the X-Y
plane. In the Z vertical direction of the array, the well must have
a depth to accommodate the total number of different reagents as
shown in FIG. 1d. Because there are six flasks, having six
different reagents in the example depicted, the depth of the well
must accommodate at least six lanterns. Ultimately, there will be a
vertical stack of six lanterns in each well, and each ring will
have attached a different subunit, monomer or building block. The
configuration or construction of any stackable solid support 10,
including the example lanterns, should be designed with the
dimensions to prevent relative movement of the supports within the
wells 45.
[0058] With the supports placed in 3-D array arrangement, the
second synthesis step takes place. As shown in FIG. 2, reagent
R2(A) is directed to the first row of wells in the X direction of
the array, the row consisting of 12 wells. The reagents bond to the
particular monomer of each lantern to create second synthesis
intermediates. Continuing the process, a different reagent R2(B)
through R2(H), as shown in FIG. 3, is directed to successive rows
of the containment apparatus. At the conclusion of this step, 48
distinct compounds are formed in the array.
[0059] The third synthesis step repeats the previous steps by
taking a different set of reagents, R3(A) to R3(L), and directing
the reagents successively into row groupings of eight wells pointed
in the Y direction of the array. The reagents react with the second
synthesis intermediates to create the third and final synthesis
product. At the conclusion of this step, there are a total of 576
distinct compounds representing each element of the X-Y-Z
combinatorial array.
[0060] The last step, as shown in FIG. 4, is the transfer of the
solid supports (lanterns) within the containment apparatus 35 into
to six separate 96-well plates 38a-f. Each plate will accept only a
single X-Y layer of the original 3-D array of supports. Thus, the
top X-Y layer of supports is transferred to plate 38a. The next
underlying X-Y layer of supports is transferred to plate 39b. The
process is repeated until all layers have been transferred. The
transfer should be performed in a manner which retains the spatial
relationships of the supports. The new plates 38a-f must be
properly labeled to identify which X-Y layer is contained, and
thus, each transferred lantern may be identified by its original
location in the X-Y-Z array of supports.
[0061] After the lanterns are transferred to single-layer 96 well
plates, the compounds still attached to the lanterns may be stored
within these plates. Alternatively, the compounds may be cleaved
from the lanterns using a cleavage solution. After cleavage, the
compounds may be extracted onto another plate, dried and prepared
for biological screening or other purposes for which they may be
suited in a manner known in the art.
[0062] In sum the synthesis system comprises: (a) a 3-D array of
supports; (b) free solid supports; (c) a containment apparatus with
a plurality of open wells in X-Y arrangement; and (d) means for
removing vertical Z column array of supports from the well from the
top or bottom of the 3-D array, once the rounds of synthesis are
completed.
[0063] The synthesis method comprises: (a) providing free solid
supports; (b) providing a containment apparatus having a plurality
of open wells; (c) stacking free solid supports into the wells to
create a 3-D array of supports; (d) delivering reagents to portions
of the 3-D array and; and (e) removing the supports in vertical Z
columns.
[0064] A particular method of synthesis using the system above
comprises: (a) providing an X-Y layer of supports all having one
building block attached and diversity of building blocks between
X-Y layers in the Z vertical direction of the 3-D array; (b)
providing randomization and synthesis by providing reagents first
in the X-Z layers and then Y-Z layers of the 3-D array; and (c)
removing vertical columns of supports all at once through the well
opening, thereby preserving the spatial information of the
supports.
[0065] In accordance with one embodiment of the present invention,
a variation of the above described method of combinatorial
synthesis using a single, 3-D array of supports is provided. The
formation of the 3-D array of supports is different in this
embodiment. In contrast to stacking free solid supports into a
separate well containment apparatus, in this variation frames of
supports are stacked together to provide a 3-D array of supports.
Each 2-D frame defines a single X-Y layer of supports. When
stacked, the frames form their own solid support reagent
containment compartments, and therefore a separate containment
apparatus is not needed.
[0066] The supports are either attached temporarily by some
mechanical means, such as friction fitting or interlocking into
holes of the frames, or the supports come physically attached to
the frames but in a manner whereby the supports may be easily cut
from the frame.
[0067] Referring to FIG. 5, an example of a single, 3-D stacked
frame 76 is shown at frames having gears for solid-phase supports
fitted into holes by friction fit or interlocking. Only a single
3-D stack 76 is used in this synthesis embodiment. The method of
synthesis is nearly identical to the process used with the open
well 3-D containment apparatus. In the first synthesis step, all
supports in a single layer or frame are reacted with one type of
reagent creating layer diversity. There are several ways to have a
frame having all supports attach a single building block. Assuming
that free solid supports are used with frames with holes, in which
the supports are friction fitted or interlocked, a first way is to
have free solid supports such as a lantern or a gear reacted in a
reactor such as a flask. The solid supports are then inserted into
the holes in a frame and held in place by friction fit or some
other means. A second way is to insert the solid supports into the
frame first, and immerse the entire frame in a reactor. Assuming
that the frame has integral supports attached, immersion of the
entire frame into a reactor is the only alternative. Each frame
must be immersed in its own reagent. Stacking the layers of frames
thereby provides a diversity of monomers or building blocks between
layers in the Z direction of the support array.
[0068] Once the frames have been stacked, the steps of synthesis
and randomization are identical as with the 3-D array using free
solid supports and a well containment apparatus. The only
difference may be in the last step of removing vertical columns of
solid supports from the array. If the solid supports are attached
to the frame by friction fit or interlocking, a means must be used
to remove individual columns of supports in the Z direction of the
3-D array. If lanterns are used as the solid supports, a rod may be
inserted through the holes of the lanterns to capture a single
vertical column of rings. As shown in FIG. 7 the rod 82 has a
stop-end 90 on one end. The other end of the rod is inserted
through the holes of the lanterns and then pulled to free the
vertical column of lanterns from the stacked frames. The lanterns
thus captured on the rod are spatially intact and may be labeled
and stored. Additionally, each ring may be taken out and placed
into a single layer cleavage plate and further labeled. If the
solid supports are integral to the frame, then there must be an
intervening step of cutting the supports from the frame with some
cutting device.
[0069] Thus, the system comprises: (a) 3-D stackable frames; (b)
means for temporarily attaching the supports to the 2-D frame; (c)
means for removing the solid supports without disassembly of the
3-D stack, retaining the 3-D spatial relationship of the solid
supports; and (d) a channel means to allow reagents in a vertical
column in the Z direction to allow supports to contact and react
with the reagent directed into the channel.
[0070] The method of synthesis is the same as described herein
above. The only difference is the addition of an optional cutting
step if supports are integral to the frames. A defining feature of
this method is that the reagent is brought to the stacked 3-D
array. The final compounds formed are identified by their 3-D
spatial locations.
[0071] B. Split-Mix Synthesis Using Stacked Frames and Rods
[0072] In accordance with another embodiment of the present
invention, a method is disclosed which uses multiple stacks of
frames as shown in FIG. 5. The method involves (a) stacking of
frames having a plurality of supports attached to the frames,
forming a plurality of identical stacked frames; (b) providing a
first synthesis step comprising immersing each stack in a separate
reactor to attach a building block to all of the solid supports in
the stack of frames; (c) reshuffling the original stacks, for
example, such that each first layer of each original stack of
frames is grouped in a new stack of frames, each second layer of
each original stack of frames is grouped in a new stack of frames,
and this process is repeated until all the original stack of frames
are reshuffled into new stack of frames; (d) providing a second
synthesis step immersing these new stacks each into its own reactor
to provide the third step of synthesis; (e) reshuffling the stacks
a second time by liberating the columns of supports from each 3-D
stack, in a manner that retains the spatial relationship of the
supports with the other supports of each column in the Z direction
and grouping corresponding columns of supports from the first
re-shuffled stacks to form new final stacks; and (f) providing a
third synthesis step by immersing each new final stack into its own
reactor.
[0073] FIG. 5 provides a specific example of the method using
particular embodiments of the apparatus. Frames having 48 holes are
shown. The solid supports depicted are shaped as gears which may be
placed inside the holes by friction fit or interlocking. As shown
in the particular example, a complex library having 27,648
compounds is synthesized on solid-phase supports, wherein the
compounds are ultimately arranged in a 3-D array, and wherein each
compound has a unique 3-D spatial address. In this example, the
solid-phase supports comprise gears, which will discussed in more
detail below.
[0074] As shown in FIG. 5, gear-shaped solid supports 70 ("gears")
are placed in plastic gear frames 72. Each frame has a six-by-eight
arrangement of holes, which holes have 48 gears inserted. The 24
total frames 72 are stacked together to provide a 3-D stack 76. In
this example, 24 identical 3-D set of stacks 74 are created. Given
that there are 24 total stacks, 24 frames in each stack, and 48
gears in each frame, the total number of gears in the twenty-four
stacks is 27,648. Each stack has 1152 gears.
[0075] After the total of 24 stacks are formed, each of these
stacks is immersed in its own reactor for the first round of
synthesis. Because there are 24 stacks, there are 24 corresponding
reactors, each reactor containing a unique subunit, monomer or
building block to be attached to the gears. After completion of the
first synthesis, each of these 1152 gears in a stack has attached a
single building block.
[0076] After the first round of synthesis is completed, a first
randomization step follows by reshuffling the 24 original stacks
into a new stacks. As illustrated in FIG. 6, frames 72 in the first
set of 24 original frame stacks 74 are rearranged in a
predetermined pattern into a second set of frame stacks 78. In the
second set of stacks 78, because all the frames must be accounted
for, there are again 24 frame stacks, and each stack consists of 24
frames.
[0077] In a particular example of a predetermined pattern, as shown
in FIG. 6, the top-most frame of each of the frame stacks in the
original frame stack set 74 is arranged in a new frame stack,
identified by the label (r1, c1) depicted in newly reshuffled frame
stack 78. Similarly, the second layer frames of original frame
stacks 74 are arranged in a new frame stack, identified by the
label (r1, c2). In this way, all gear frames in original stack 74
are rearranged such that each frame stack in the second set of
frame stacks 78 includes one and only one gear frame 72 from each
frame stack in the original set of frame stacks 74.
[0078] After reshuffling of the frame stacks is completed, each new
stack 80, in the set of new stacks 78, is placed in its reactor for
the second round of synthesis. Similar to the first round of
synthesis, there are 24 reactors, each containing a reagent, with
no reagent repeated among the second set of 24 reactors.
[0079] As shown in FIG. 7, gears 70 are then liberated from the
gear frames 72 and frame stacks 80 and placed on rods 82, thereby
forming a column of gears 84. Each of frame stacks 80 yields 48
columns of gears 84 placed on rod 82.
[0080] After a second randomization step illustrated in FIG. 7
column of gears 84 are arranged into a group of gear columns 86.
Each group of gear columns 86 includes one and only one column of
gears from each of the twenty-four set, once-reshuffled frame
stacks 80. This re-arrangement results in 24 new groups, each group
consisting of 48 gear columns. The liberation of columns of gears
may be done manually using rods 82 that have one end having a
stop-end 90. The other rod end may be inserted through the holes in
each gear.
[0081] Each of the group of gear columns 86 are then reacted with a
third reagent in a third and final round of synthesis. The method
repeats the previous synthesis steps i.e. each group of the newly
formed set of twenty-four groups is placed into its own reactor,
wherein none of the twenty-four reactors has the same reagent.
[0082] After the third round of synthesis has been completed, the
gears are stored on their respective rods 82 or removed from their
rods and placed in a single layer X-Y plate for future processing,
such as cleavage and extraction. One can determine the chemical
composition of the compounds on each gear by the 2D spatial address
of the gear. Because more than one plate is required to store the
entire library of compounds in this example, a label must provide a
third component to provide a 3-D spatial identification.
[0083] In a preferred embodiment of the apparatuses, Chiron
lanterns or gears or other similar supports are placed into holes
in frames and held in place by friction fit or interlocking. The
means for removing the supports from the frames can be provided in
a number of ways depending on whether the supports are attached to
the frames by friction fit or whether the supports are physically
attached. If the supports are in the frames by friction fit or
interlocking, the supports must be taken out from the frames, while
preserving the spatial relationship of the supports relative to the
other supports. If the supports are physically attached to the
frames, the supports must first be cut and then liberated from the
frames.
[0084] The gears may be pushed or pulled out from the holes of the
frames using a variety of tools. One such tool already discussed is
a rod 82 having a stop-end 90 as shown in FIG. 7. The support,
whether a gear, lantern or another shape, is designed with a hole
through the middle. The rod is placed through a vertical line of
support holes using one rod end. The stop-end of the rod cannot go
through the small hole of the supports and thus a vertical column
of supports is caught on the rod and can be liberated from the
frames by pulling the tip of the rod. The supports may be
conveniently stored on the rods or the supports may be labeled and
stored for later cleaving of each unique compound from the
supports.
[0085] If the supports are attached to the frame, the supports must
first be cut from the frame before removal. There are many
conceivable variations for liberating the supports from the frames
in the last step dependent on the specific design of the frames and
supports. Some have been described in co-pending application Ser.
No. 09/082,038. (See Baum Application at FIGS. 25-28 for various
embodiments for removing the supports from the wells and the
accompanying discussions pp. 35-40.)
[0086] Gears 70, like lanterns 10, are made of polypropylene with a
thin layer of polystyrene on its surface that has been
functionalized to react with reagents used in synthesizing the
compound libraries. As shown in FIG. 8 gear 70 comprises a tubular
structure with 10 short outwardly projecting fins evenly spaced
around the circumference of gear 70. The outer diameter of gear 70
is approximately 4.0 mm to 6.0 mm, preferably around 5.0 mm.
Library subunits are synthesized on all surfaces of the gears
70.
[0087] One type of gear frame suitable for use with gears 70 is
shown in FIG. 9. Gear frame 92 is made of high density polyethylene
or polypropylene. Gear frame 92 includes 96 apertures arranged in a
8.times.12 array. The apertures extend through the thickness of
gear frame 92 such that rods may be passed through both gears 70
and gear frame 92. Of course, gear frames with greater than or less
than 96 apertures may be manufactured. Gears 70 are maintained in
the apertures of gear frame 92 by a friction fit or interlocking.
Gear frame 92 also may be fitted with one or more radio-frequency
identification (RFID) chips to confirm the identification of gear
frame 92 and gears 70 within gear frame 92.
[0088] In yet another illustrative example, manipulation of
solid-phase supports is minimized through use of a plate that
functions both as a container to maintain the solid-phase supports
in a 3D array and as a reactor for the various reaction steps. In
this example, a combinatorial library having 576 compounds is
synthesized on solid-phase supports, which comprise tubes cut into
individual solid-phase support rings. As discussed above and shown
in FIG. 10, tube rings are structurally similar to lanterns, having
a tubular structure with an outer diameter of approximately 7.8 mm,
an inner bore diameter of approximately 6.9 mm, and a height of
approximately 3.1 mm. Each tube ring supports approximately 15
.mu.mols of compound, which is approximately 6 mg of compound at an
average molecular weight of 400.
[0089] FIG. 11 illustrates a 96 wells or holes, 3-D containment
plate 200 which can be used in the first embodiment of this present
invention. Typically such a plate will have 96 holes or more. Note
the double-drilled first hole 130 and second hole 140. The two
holes intersect and connect the two holes. The first hole is
intended to be a channel or well for which the solid supports may
be inserted and stacked inside. The second hole is intended to
provide a separate channel for reagents to flow through and contact
each stacked solid support in the first channel. Since the holes
intersect, there is an opening between the first and second
channels where the reagent may pass through. Later, when the solid
supports need to be retrieved, a rod having a stop-end at one end
may be used to pull the stacked column of supports out of the 3-D
array by inserting a first end of the rod into the second channel
and a bend in second end of the rod is used to catch the end of the
column of supports stacked in the first channel. The first end of
the rod can be pulled to free the friction fitted or interlocked
gears from the frames.
[0090] C. "Sort and Combine" Synthesis Using Frames
[0091] The third embodiment, in accordance with the present
invention, comprises a "sort and combine" synthesis using 2-D
frames having N row by M column of solid supports. This method is
suitable for large scale production of combinatorial libraries
wherein the numbers of unique compounds exceed 100,000. A frame is
prepared by placing supports having the same monomer or building
block into the first column, filling all N places. The second
column of the frame is filled with another set of supports all
having the same monomer but different from the monomer in the first
column. Each column is thus filled with supports having different
monomers attached to the supports.
[0092] If Q numbers of identical frames are used, prepared as
described above, there should be Q reactors, each having a
different reagent. Each frame numbered 1 through Q is immersed in
its own reactor to allow the supports on the frames to react with a
reagent. After this step, each frame 1 through Q is then taken from
the reactors and physically split into subframes of rows of the
original frame. Next, all of the subframes are reassembled in
groups such that all of the same numbered rows 1 of each original
N.times.M frame are assembled into one group of subframes, all rows
2 of each original frame are assembled into another group of
subframes and so on until the last, Nth row of each original
N.times.M frame is assembled into a group of subframes. After
reassembly there are N groups of subframes. In the second synthesis
step, each of these groups in turn is immersed into N number of
different reagents to provide M.times.N.times.Q diversity. Q, which
represents the number of original frames and also the number of
reactors, is independently chosen.
[0093] FIG. 12 provides an example of the preferred implementation
of the method in accordance with the present invention. While the
method may be used to synthesize highly complex libraries, i.e.,
greater than about 100,000 compounds per library, the following
example illustrates synthesis on a much smaller scale in order to
provide a simplified, yet complete, explanation of the method.
[0094] In this example, a library of only 27 different compounds
will be synthesized on solid-phase supports. Each final compounds
is composed only of three subunits or building blocks: A, B, and C.
The 27 compounds are ultimately arranged in a 2D spatial array,
wherein the chemical composition of the compound may be determined
by its unique 2D spatial address.
[0095] Many known types of free, solid supports may be used with
this method. We have already described Chiron lanterns and gears
which may be friction-fitted into the holes in frames. We will
assume in this example that Chiron lanterns 10, as depicted in FIG.
13, are used.
[0096] Referring to FIG. 12, in the first round of synthesis, 27
identical lanterns 10 are reacted with a first reagent A, B, or C,
in a manner known in the art, e.g., as described in the Lebl '696
patent. The 27 solid-phase supports 10 are evenly distributed into
three reaction flasks 20a, 20b, and 20c. The flasks are essentially
reactors except that flasks have smaller volumes. After the first
synthesis step is completed, nine lanterns in flask 20a will have
attached the A subunit, nine lanterns in flask 20b will have
attached the B subunit, and nine lanterns in flasks 20c will have
attached the C subunit.
[0097] The groups of nine lanterns from each flask are then
rearranged into the holes of three lantern frames 30a, 30b, and 30c
by friction-fitting the lanterns. It is necessary that each lantern
frame be provided equal numbers of lanterns from each flask in an
orderly arrangement. In this case each flask A, B and C contributes
three lanterns. Note that in this example, each frame 30a, 30b and
30c has the identical 2-D spatial arrangement of lanterns. For each
lantern frame 30a-c, lanterns from first flask 20a are placed in
the first column (c1), lanterns from second flask 20b are placed in
column (c2), and lanterns from third flask 20c are placed in the
column (c3) farthest to the right. Note that this is but one
example of a workable orderly arrangement. Other arrangements can
serve equally well as long as each frame is provided lanterns in
equal numbers from each different flask available, and the
arrangement is orderly and known.
[0098] In the second synthesis step, each frame 30a, 30b, and 30c
is then reacted with a second set of reagents, also having subunits
A, B, and C, by immersing each frame into its respective reactors,
40a, 40b and 40c. Note that subunits may be the same subunits in
the first synthesis step as the example provided. However, within a
synthesis step, each subunit provided in each reactor should be
unique.
[0099] As a result of the second round of synthesis, the lantern
frames 30a, 30b, 30c contain nine different two-subunit synthesis
intermediates. The lanterns in frame 30a will have three different
synthesis intermediates as follows: in column c1, three lanterns
having the intermediates AA; in column c2, three lanterns having
the intermediates AB; and in column c3, three lanterns having the
intermediates AC. The lanterns in frame 30b will have three
different synthesis intermediates as follows: in column c1, three
lanterns having the intermediates BA; in column c2, three lanterns
having the intermediates BB; and in column c3, three lanterns
having the intermediates BC. Similarly, the lanterns in frame 30c
will have three different synthesis intermediates as follows: in
column c1, three lanterns having the intermediates CA; in column
c2, three lanterns having the intermediates CB; and in column c3,
three lanterns having the intermediates CC.
[0100] The next step provides a randomization. Each row of the
frame 30a is then broken into smaller subframes of rows as
indicated by subframes 50a, 50b, and 50c. Each split subframe has
three supports. Frame 30b is broken into smaller subframes 52a, 52b
and 52c. And similarly, frame 30c is broken into subframes 54a, 54b
and 54c. The subframes are regrouped such that all split frames
from the same rows are grouped together. For example, subframes
from the first rows, 50a, 52a and 54a are grouped into new group of
subframes 60a. Subframes from the second rows, 50b, 52b and 54b are
grouped into a new group of subframes 60b. Similarly, subframes
from the third rows, 50c, 52c, and 54c are grouped into new group
of subframes 60c.
[0101] In the third synthesis step, each new group of subframes,
60a, 60b and 60c, is immersed into reactors 40a, 40b, and 40c,
respectively. Note that in this example, the same reactors that
were used in the second round of synthesis are used again in this
third round of synthesis. Alternatively, other reactors (not shown)
having different subunits, e.g., H, I, and J may be used. After the
third round of synthesis, all 27 of the possible three unit
combinations of building blocks A, B, and C will have been
synthesized. Each compound will be attached to and located on one
and only one lantern.
[0102] In an alternative embodiment, smaller frames may be used in
lieu of a breakable larger frame. Specifically, in this example,
rather than using the three lantern frames 30a-c, which are adapted
to contain 9 lanterns apiece, one can use nine smaller frames,
which are adapted to contain 3 lanterns apiece.
[0103] All 27 lanterns 10 are then removed from their respective
subframes, and transferred to plate 65 comprising a 3 row by 9
column (3.times.9) array of wells. The compounds are then removed
from the lanterns, such that there is one unique compound per well.
Therefore, each compound has a unique location or spatial 2D
address within the plate, i.e., row (1-3), column (1-9), and may
therefore be identified by its unique spatial 2D address. For
example, compounds located in well at r2, c5 will have a chemical
composition comprising BBB. According to the spatial 2D address,
one can therefore determine the chemical composition of the
compound.
[0104] As explained above, each lantern 10 moves through a given
pattern throughout synthesis such that its ultimate location or
spatial address reveals the chemical composition of the compound
attached to each lantern. In addition, the spatial address of each
compound and its associated chemical composition will also reveal
the history of the synthesis, including the various rounds or
reactions of synthesis. For example, by its spatial address within
the 3.times.9 plate, it may be determined which flask the compound
originated from in the first round of synthesis. The spatial
address therefore contains a wealth of useful information about the
compound.
[0105] In addition, the present method is not limited to any
particular pattern or grouping of solid-phase supports. Any
ordered, nonrandom pattern or grouping may be incorporated into the
present method as long as the relationship between the pattern and
the ultimate spatial address of the library compounds is
determinable. For example, in an alternative embodiment of the
present method, the lanterns may first be arranged such that the 9
lanterns from the first flask comprise the first row (rather than
column) of each 3.times.3 lantern frame.
[0106] Lantern 10 is known in the art and is commercially available
from Chiron. Lantern 10 is made of polypropylene with a thin layer
of polystyrene on its surface similar to other solid-phase supports
known in the art. This polystyrene surface is functionalized to
react with reagents used in synthesizing the compound libraries. As
shown in FIG. 13, a lantern comprises four tubular substructures,
121, 122, 123, and 124, that are attached to each other, creating
an overall tubular structure. Lantern 10 has an outer diameter of
approximately 5.0 mm to approximately 6.0 mm, preferably around 5.0
mm, and an inner bore with a diameter of approximately 2.0 mm to
approximately 3.0 mm, preferably around 2.5 mm. In addition, the
height of each lantern is approximately 5 mm. In addition, each
lantern 10 supports approximately 15 .mu.mols of compound. Library
subunits are synthesized on all surfaces of the lanterns, including
both the outer and inner surface. Although lanterns are used in the
preferred embodiment of the present method, it will be appreciated
by those of ordinary skill in the art that any physically
manipulable solid-phase support may be incorporated into the
present method. Tubes cut into individual solid-phase support rings
may also be used.
[0107] A preferred type of lantern frame that may be used in the
present method is shown in FIGS. 14a and 14b. Frame 32 comprises a
high density polyethylene, polypropylene, or other chemically
resistant material and has dimensions of approximately 18 mm by 81
mm. Frame 32 includes 16 wells 33 arranged in a 2.times.8 array,
and wells 33 are dimensioned to contain lanterns 10. In addition,
frame 32 includes knife cut grooves (not shown) that allow it to be
divided or broken apart into subframes having at least two wells
apiece. Optionally, frame 32 may be fitted with one radio-frequency
identification (RFID) chips 34 known in the art to record the
identity of frame 32 and lanterns 10 within frame 32. RFID chip 34
is approximately 11 mm long and is positioned at one end of frame
32. In addition, a variety of alternative structural supports may
be incorporated into the present invention.
[0108] In sum, the apparatus comprises: (a) single frames which can
be broken into subframes; (b) reactors; and (c) means for holding
solid supports on the frames temporarily or means to allow cutting
of the supports.
[0109] The present invention may be embodied in other forms without
departing from its spirit or essential characteristics. The
described embodiments are to be considered only as illustrative and
not as restrictive. For example, in each of the examples described
above, each synthesis comprises three rounds of reactions. However,
depending on the combinatorial library desired, one may need fewer
than three rounds or less than three rounds. The scope of the
invention is, therefore, indicated by the appended claims.
* * * * *