U.S. patent number RE37,194 [Application Number 09/567,618] was granted by the patent office on 2001-05-29 for high throughput solid phase chemical synthesis utilizing thin cylindrical reaction vessels useable for biological assay.
This patent grant is currently assigned to Pharmacopeia, Inc.. Invention is credited to Robert H. Grubbs, Gregory L. Kirk.
United States Patent |
RE37,194 |
Kirk , et al. |
May 29, 2001 |
High throughput solid phase chemical synthesis utilizing thin
cylindrical reaction vessels useable for biological assay
Abstract
A high throughput chemical synthesis system utilizing
cylindrical reaction vessels is disclosed. Reaction vessels are
utilized which include a tubular member adapted for placement of
electronically readable identifying indicia thereon. The
identifying indicia are representative of reaction conditions
within the tubular member and of one or more reagents utilized in a
reaction within the tubular members. A method of performing
chemical synthesis on solid phase reactive material within a
plurality of reaction vessels using a plurality of reaction stages
resulting in final products and employing identifying indicia
representing the reaction stages is also disclosed. The method
includes reading the identifying indicia located on the reaction
vessels, reacting one or more reagents within the reaction vessels
under particular reaction conditions which may be determined by
reading the identifying indicia, thereby synthesizing chemical
compounds within the reaction vessels. The method allows chemical
synthesis to occur according to a predetermined set of reactions
and also allows for combinatorial chemistry to be performed
utilizing random mix and split techniques. The final synthesized
products may be tested for chemical or biological activity. The
chemical structures of desired end products may be obtained by
reading recorded information wherein the reaction conditions and
reagents of reaction steps have been recorded, preferably in
conjunction with the identifying indicia.
Inventors: |
Kirk; Gregory L. (Winchester,
MA), Grubbs; Robert H. (Pasadena, CA) |
Assignee: |
Pharmacopeia, Inc. (Princeton,
NJ)
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Family
ID: |
24917046 |
Appl.
No.: |
09/567,618 |
Filed: |
May 9, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
726058 |
Oct 3, 1996 |
05798035 |
Aug 25, 1998 |
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Current U.S.
Class: |
205/335; 206/305;
206/459.5; 422/119; 422/129; 422/138; 422/232; 422/233; 422/236;
422/561; 422/63; 422/82; 436/165; 436/169; 436/47; 436/48; 436/49;
436/52; 436/55 |
Current CPC
Class: |
B01J
19/0046 (20130101); B82Y 30/00 (20130101); C07K
1/045 (20130101); G01N 30/46 (20130101); G01N
30/466 (20130101); B01J 2219/00286 (20130101); B01J
2219/00306 (20130101); B01J 2219/00308 (20130101); B01J
2219/00315 (20130101); B01J 2219/00355 (20130101); B01J
2219/00389 (20130101); B01J 2219/00418 (20130101); B01J
2219/00463 (20130101); B01J 2219/00495 (20130101); B01J
2219/005 (20130101); B01J 2219/00511 (20130101); B01J
2219/0052 (20130101); B01J 2219/00547 (20130101); B01J
2219/00585 (20130101); B01J 2219/0059 (20130101); B01J
2219/00592 (20130101); B01J 2219/00596 (20130101); B01J
2219/00605 (20130101); B01J 2219/0061 (20130101); B01J
2219/00612 (20130101); B01J 2219/00621 (20130101); B01J
2219/00637 (20130101); B01J 2219/00641 (20130101); B01J
2219/00644 (20130101); B01J 2219/00659 (20130101); B01J
2219/00689 (20130101); B01J 2219/00702 (20130101); B01J
2219/00711 (20130101); C40B 60/14 (20130101); C40B
70/00 (20130101); G01N 30/60 (20130101); G01N
30/6043 (20130101); G01N 2030/524 (20130101); G01N
30/60 (20130101); G01N 30/466 (20130101); Y10T
436/12 (20150115); Y10T 436/117497 (20150115); Y10T
436/114998 (20150115); Y10T 436/114165 (20150115); Y10T
436/113332 (20150115) |
Current International
Class: |
B01J
19/00 (20060101); C07K 1/00 (20060101); C07K
1/04 (20060101); G01N 30/00 (20060101); G01N
30/46 (20060101); G01N 30/60 (20060101); C25B
015/02 () |
Field of
Search: |
;205/335
;422/55,56,57,59,63,82,119,129,138,188,196,197,232,233,236
;436/52,55,47,48,49,165,169 ;206/305,459.5 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1312991 |
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Jan 1993 |
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CA |
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0 274 999 A2 |
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Dec 1987 |
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EP |
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0 355 266 A2 |
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Apr 1989 |
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EP |
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WO 88/05074 |
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Jul 1988 |
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WO |
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WO 92/00091 |
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Jan 1992 |
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WO |
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WO 92/22591 |
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Dec 1992 |
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WO |
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WO 93/02992 |
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Feb 1993 |
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WO |
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WO 93/05065 |
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Mar 1993 |
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WO |
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WO 93/12427 |
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Jun 1993 |
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WO |
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WO 93/17056 |
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Sep 1993 |
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WO |
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WO 94/05394 |
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Mar 1994 |
|
WO |
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WO 94/08051 |
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Apr 1994 |
|
WO |
|
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Primary Examiner: Bell; Bruce F.
Attorney, Agent or Firm: Goldstein; Ronald B. Heslin &
Rothenberg, P.C.
Claims
What is claimed is:
1. A method of performing chemical synthesis on solid phase
reactive material contained within a plurality of tubular reaction
vessels, using one or more reaction stages, resulting in a
plurality of final products and employing identifying indicia to
represent specific reactions and reagents utilized during said one
or more reaction stages, said method comprising:
reading said identifying indicia provided for each of a plurality
of tubular reaction vessels,
reacting a reagent with said reactive material by flowing a
reagent-containing solution through said tubular reaction vessels
under particular reaction conditions, wherein said reacting is
driven to completion within the reactive material by said flowing
of reagent-containing solution through said tubular reaction
vessels without agitation of said reactive material;
recording information concerning said reaction conditions and
reagents, said information corresponding to said identifying
indicia; and
repeating said reading and said reacting at least once using a
different reagent, thereby synthesizing chemical compounds within
said tubular reaction vessels.
2. The method of claim 1 wherein said identifying indicia comprise
electronically readable indicia located on said tubular reaction
vessels.
3. The method of claim 2 wherein said identifying indicia comprise
a bar code and said reading step is performed using a bar code
reader.
4. The method of claim 1 wherein said reactive material is coated
on an inside wall of said tubular reaction vessel.
5. The method of claim 1 wherein said reactive material comprises a
packing disposed within said tubular reaction vessels to allow
reagents to flow through said tubular reaction vessels.
6. The method of claim 1 wherein said tubular reaction vessels are
insertable into a reaction chamber wherein said reacting step is
performed within said reaction chamber.
7. The method of claim 6 wherein said identifying indicia comprises
the location of a tubular reaction vessel within said reaction
chamber.
8. The method of claim 1 wherein said tubular reaction vessels are
cylindrical in shape.
9. A method of performing chemical synthesis on solid phase
reactive material contained within a plurality of tubular reaction
vessels, using one or more reaction stages, resulting in a
plurality of final products and employing identifying indicia to
represent specific reactions and reagents utilized during said one
or more reaction stages said method comprising:
reading said identifying indicia provided for each of a plurality
of tubular reaction vessels,
reacting a reagent with said reactive material by flowing a
reagent-containing solution through said tubular reaction vessels
under particular reaction conditions;
recording information concerning said reaction conditions and
reagents, said information corresponding to said identifying
indicia;
repeating said reading and said reacting at least once, thereby
synthesizing chemical compounds within said tubular reaction
vessels; and
wherein said tubular reaction vessel comprises a central rod
axially oriented therein, said central rod being coated with said
reactive material.
10. The method of claim 9 wherein said reactive material comprises
a gel.
11. A method of performing chemical synthesis on solid phase
reactive material contained within a plurality of tubular reaction
vessels, using one or more reaction stages, resulting in a
plurality of final products and employing identifying indicia to
represent specific reactions and reagents utilized during said one
or more reaction stages, said method comprising:
reading said identifying indicia provided for each of a plurality
of tubular reaction vessels,
reacting a reagent with said reactive material by flowing a
reagent-containing solution through said tubular reaction vessels
under particular reaction conditions; recording information
concerning said reaction conditions and reagents, said information
corresponding to said identifying indicia;
repeating said reading and said reacting at least once, thereby
synthesizing chemical compounds within said tubular reaction
vessels;
wherein said tubular reaction vessels are insertable into a
reaction chamber, and wherein said reacting is performed within
said reaction chamber; and
removing said tubular reaction vessels from said reaction chamber
and sorting said tubular reaction vessels by reading said
identifying indicia thereon.
12. The method of claim 11 further comprising sorting said tubular
reaction vessels into two or more groups, said groups subsequently
being inserted into separate reaction chambers.
13. The method of claim 11 wherein said sorting comprises sorting
said tubular reaction vessels into at least a first and second
group, and further comprising reacting a first reagent within the
tubular reaction vessels of said first group and reacting a second
reagent within the tubular reaction vessels of said second
group.
14. The method of claim 13 further comprising testing the
synthesized chemical compounds for a particular chemical or
biological activity and identifying one or more of said compounds
based upon said identifying indicia and the recorded information
corresponding thereto.
15. The method of claim 14 further comprising transferring a
portion of each of said chemical compounds from said tubular
reaction vessels into one of a plurality of assay wells.
16. The method of claim 13 further comprising exposing said tubular
reaction vessels to a controlled intensity light source.
17. The method of claim 11 wherein said identifying indicia
comprises the location of a tubular reaction vessel within said
reaction chamber.
18. A method of performing chemical synthesis on solid phase
reactive material contained within a plurality of tubular reaction
vessels, using one or more reaction stages, resulting in a
plurality of final products and employing identifying indicia to
represent specific reactions and reagents utilized during said one
or more reaction stages, said method comprising:
reading said identifying indicia provided for each of a plurality
of tubular reaction vessels;
reacting a reagent with said reactive material by flowing a
reagent-containing solution through said tubular reaction vessels
under particular reaction conditions;
recording information concerning said reaction conditions and
reagents, said information corresponding to said identifying
indicia;
repeating said reading and said reacting at least once, thereby
synthesizing chemical compounds within said tubular reaction
vessels; and
removing said tubular reaction vessels from said reaction chamber
and randomly sorting said tubular reaction vessels into at least a
first and second group.
19. The method of claim 18 further comprising reacting a first
reagent within the tubular reaction vessels of said first group and
reacting a second reagent within the tubular reaction vessels of
said second group.
20. The method of claim 19 further comprising testing the
synthesized chemical compounds for a particular chemical or
biological activity and identifying one or more of said compounds
based upon said identifying indicia and the recorded information
corresponding thereto.
21. The method of claim 20 further comprising transferring a
portion of each of said chemical compounds from said tubular
reaction vessels into one of a plurality of assay wells.
22. The method of claim 19 further comprising exposing said tubular
reaction vessels to a controlled intensity light source.
23. A chemical synthesis reaction vessel for performing
combinatorial chemistry within a solid phase reactive material,
said reaction vessel comprising:
a tubular member, said tubular member allowing reagent-containing
solution to flow therethrough and having electronically readable
identifying indicia thereon, said identifying indicia representing
reaction conditions which have occurred or which will occur within
said tubular member and representing reagents reacted or to be
reacted within said tubular member; and
wherein said solid phase reactive material is disposed within said
tubular member such that axial flow of reagent-containing solution
through said tubular member causes the reagent-containing solution
to be in communication with all of said reactive material and
allows reactions to be driven to completion within the reactive
material without agitation of the reactive material.
24. The reaction vessel of claim 23 wherein said identifying
indicia comprise a bar code.
25. The reaction vessel of claim 24 wherein said bar code is
located at a first end of the tubular member.
26. The reaction vessel of claim 23 wherein said tubular member is
coated with said reactive material on an inside surface
thereof.
27. The reaction vessel of claim 26 wherein said coating of
reactive material on the inside surface of said tubular member is
25-100 microns thick.
28. The reaction vessel of claim 23 wherein said tubular member
comprises a photo-transparent material.
29. The reaction vessel of claim 23 wherein said reactive material
comprises a packing disposed within said tubular member and said
reactive vessel further comprises at least one frit located within
at least one end of the tubular member, said frit allowing fluid
reagents to flow therethrough while retaining said packing
therewithin.
30. The reaction vessel of claim 23 wherein said tubular member
comprises an elongate uniformly cylindrical vessel which can be
aligned and sorted in a rolling motion.
31. The apparatus of claim 30 further comprising means for
controlling flow rate through said plurality of tubular reaction
vessels when disposed within said one or more reaction
chambers.
32. A chemical synthesis reaction vessel for performing
combinatorial chemistry comprising:
a tubular member, said tubular member allowing reagent-containing
solution to flow therethrough and having electronically readable
identifying indicia thereon, said identifying indicia representing
reaction conditions which have occurred or which will occur within
said tubular member and representing reagents reacted or to be
reacted within said tubular member;
a solid phase reactive material disposed within said tubular member
to allow for axial flow of reagent-containing solution through said
tubular member in communication with said reactive material;
and
an elongate rod coaxially oriented within said tubular member, said
elongate rod being coated with said reactive material.
33. The reaction vessel of claim 26 or 32 wherein said reactive
material comprises a gel.
34. An apparatus for performing chemical synthesis by performing a
series of reactions on solid phase reactive material
comprising:
a plurality of tubular reaction vessels having electronically
readable identifying indicia disposed thereon, said tubular
reaction vessels containing a solid phase reactive material therein
and allowing reagent-containing solution to flow therethrough,
wherein said solid phase reactive material is disposed within each
tubular reaction vessel such that flow of reagent-containing
solution therethrough causes the reagent-containing solution to be
in communication with all of said reactive material and allows
reactions to be driven to completion within the reactive material
without agitation of the solid phase reactive material; and
one or more reaction chambers for receiving said tubular reaction
vessels therein, each of said reaction chambers having an inlet
disposed to allow said reagent-containing solution to flow into
said reaction chamber and axially through said tubular reaction
vessels disposed therein.
35. The apparatus of claim 34 further comprising one or more guide
arrays insertable into said one or more reaction chambers, each
guide array receiving said tubular reaction vessels therein.
36. The apparatus of claim 34 further comprising a
temperature-controlled enclosure surrounding said one or more
reaction chambers.
37. The apparatus of claim 34 wherein each of said tubular reaction
vessels is identically sized to allow substantially even flow of
reagent-containing solution therethrough.
38. The apparatus of claim 34 wherein each of said tubular reaction
vessels comprises an elongate uniformly cylindrical vessel which
can be aligned and sorted in a rolling motion.
39. An apparatus for performing chemical synthesis by performing a
series of reactions on solid phase reactive material
comprising:
a plurality of tubular reaction vessels having electronically
readable identifying indicia disposed thereon, said tubular
reaction vessels containing a solid phase reactive material therein
and allowing reagent-containing solution to flow therethrough;
one or more reaction chambers for receiving said tubular reaction
vessels therein, each of said reaction chambers having an inlet
disposed to allow said reagent-containing solution to flow into
said reaction chamber and axially through said tubular reaction
vessels disposed therein;
one or more guide arrays insertable into said one or more reaction
chambers, each guide array receiving said tubular reaction vessels
therein; and
wherein said inlet is located within a first reaction chamber
cover.
40. The apparatus of claim 39 further comprising a guide cap, said
guide cap comprising a plurality of apertures therein and being
located between said inlet and one of said guide arrays, wherein
said apertures are aligned with said tubular reaction vessels to
allow said reagent to flow from said inlet into, and axially
through, said tubular reaction vessels.
41. The apparatus of claim 40 further comprising a second guide
cap, said second guide cap having a plurality of apertures therein
and being located between one of said guide arrays and an outlet of
each of said reaction chambers, wherein said apertures are aligned
with said tubular reaction vessels to allow fluid reagents to flow
from said tubular reaction vessels to said outlet.
42. The apparatus of claim 41 wherein said outlet is located within
a second reaction chamber cover, said first and second reaction
chamber covers being located at opposite ends of a reaction
chamber.
43. An apparatus for performing chemical synthesis by performing a
series of reactions on solid phase reactive material
comprising:
a plurality of tubular reaction vessels having electronically
readable identifying indicia disposed thereon, said tubular
reaction vessels containing a solid phase reactive material therein
and allowing reagent-containing solution to flow therethrough;
one or more reaction chambers for receiving said tubular reaction
vessels therein, each of said reaction chambers having an inlet
disposed to allow said reagent-containing solution to flow into
said reaction chamber and axially through said tubular reaction
vessels disposed therein; and
wherein said one or more reaction chambers are in fluid flow
relationship to an array valve, said array valve being in fluid
flow relationship with a plurality of reservoirs wherein said array
valve distributes reagents from said reservoirs to the one or more
reaction chambers.
44. The apparatus of claim 43 further comprising one or more pumps
placed in fluid flow relationship between said array valve and said
one or more reaction chambers.
45. The apparatus of claim 43 further comprising extrusion means,
said extrusion means being insertable into said reaction chambers
and guide array to remove said tubular reaction vessels
therefrom.
46. The apparatus of claim 45 further comprising sorting means
configured to receive tubular reaction vessels extruded from said
reaction chambers.
47. The apparatus of claim 46 wherein said sorting means comprises
a reader for reading said identifying indicia on said tubular
reaction vessels.
48. The apparatus of claim 47 wherein said sorting means further
comprises means for moving extruded tubular reaction vessels from a
hopper.
49. The apparatus of claim 48 further comprising means for
receiving said extruded tubular reaction vessels therein from said
sorting means and for delivering said extruded reaction vessels to
said reaction chamber.
50. The apparatus of claim 48 further comprising transport and
fluid delivery means configured to receive reaction vessels therein
and to align said reaction vessels with a plurality of wells in an
assay plate.
51. The apparatus of claim 50 wherein said transport and fluid
delivery means comprise a robotic pipettor.
52. The apparatus of claim 45 wherein said means for moving
extruded reaction vessels comprises a ramp or a belt.
53. The apparatus of claim 52 wherein said means for moving
extruded reaction vessels further comprises a vessel director
disposed to move said tubular reaction vessels from said ramp or
belt into a vessel loading device.
54. The apparatus of claim 53 wherein said vessel loading device
receives a reaction chamber and guide array therein, said vessel
loading device being moveable along a first and second axis.
55. A method of chemically synthesizing compounds for direct
delivery to a biological assay comprising:
performing a series of reaction steps on a solid phase reactive
material contained within a plurality of tubular reaction vessels
having identifying indicia thereon, one or more of said reaction
steps being performed using reactive agents and under conditions
which may be determined by electronically reading said identifying
indicia;
sorting said tubular reaction vessels into an array based upon the
identifying indicia; and
transferring synthesized biological compounds from within said
array of vessels into an assay plate.
56. The method of claim 55 further comprising:
randomly sorting said tubular reaction vessels after one of said
reaction steps into at least a first group and second group of
reaction vessels;
reacting a first reagent within the reaction vessels of said first
group; and
reacting a second reagent within the reaction vessels of said
second group.
57. The method of claim 56 wherein said tubular reaction vessels
are sorted into more than one array and said compounds are
deposited into more than one assay plate.
58. The method of claim 55 wherein said identifying indicia
comprise a bar code.
59. The method of claim 55 wherein said reactive material is coated
on the inside of said tubular reaction vessels.
60. The method of claim 55 wherein said solid phase reactive
material comprises a packing retained within said reaction vessels
by frits disposed within the ends of said reaction vessels.
61. The method of claim 55 wherein said reaction steps are
performed in one or more reaction chambers configured to receive
one or more of said tubular reaction vessels therein, wherein each
tubular reaction vessel within a particular reaction chamber is
exposed to the same chemical reagent or reaction conditions during
a particular reaction step.
62. The method of claim 61 further comprising loading an elution
solvent into said tubular reaction vessels.
63. The method of claim 62 wherein said tubular reaction vessels
are exposed to ultraviolet radiation to release said compounds from
the tubular reaction vessels.
64. The method of claim 63 wherein said compounds are deposited
into said assay plate by dipping an end of a tubular reaction
vessel into the surface of said assay plate.
65. A method of performing chemical synthesis on solid phase
reactive material within a plurality of reaction vessels, said
method comprising:
separating tubular reaction vessels into a plurality of groups and
inserting each group into a different reaction chamber, said
tubular reaction vessels allowing reagent-containing solution to
flow therethrough and having said reactive material therein;
reacting a reagent with said reactive material within one or more
of said groups of tubular reaction vessels by flowing said reagent
through said reaction chambers;
recording data, said data being representative of reaction
conditions and reagents within a tubular reaction vessel during
said reacting step and being recorded in conjunction with
information about the location of the reaction vessel in a
particular chamber; and
repeating said separating, reacting and recording steps at least
once.
66. The method of claim 65 further comprising
testing final products synthesized within said tubular reaction
vessels; and
identifying one or more reaction vessels having a desired final
product synthesized therein.
67. The method of claim 66 further comprising evaluating said data
for the one or more reaction vessels having said desired final
product synthesized therein to determine the process by which said
product was synthesized, so as to thereby identify said
product.
68. The method of claim 66 wherein said reactive material comprises
a gel.
69. A method of identifying synthesized compounds having a desired
biological or chemical activity, said compounds having been
synthesized during multiple reaction stages on solid phase reactive
material contained within reaction vessels, said method
comprising:
transferring synthesized compounds from said reaction vessels to a
testing medium, said reaction vessels being tubular in shape and
comprising electronically readable identifying indicia thereon
representing reaction conditions which have occurred within said
tubular reaction vessels;
testing for a desired biological or chemical activity the
synthesized compounds transferred from said reaction vessels to
said testing medium; and
electronically reading the identifying indicia on the reaction
vessel wherein was synthesized a compound having said desired
biological or chemical activity.
70. The method of claim 69 further comprising reading recorded
information wherein specific identifying indicia correspond with
specific reaction histories.
71. The method of claim 70 wherein said recorded information is
used to determine the structure of said compound.
72. The method of claim 71 wherein said transferring synthesized
compounds from tubular reaction vessels to a testing medium
comprises depositing said compounds into an assay.
73. The method of claim 72 further comprising loading an elution
solvent into said reaction vessels.
74. The method of claim 73 further comprising exposing said
reaction vessels to ultraviolet radiation to release said compound
from said reaction vessels.
75. The method of claim 74 wherein said compounds are deposited
into said assay by dipping an end of a reaction vessel into said
assay.
76. The method of claim 75 wherein said identifying indicia
comprise a bar code and said reading step is performed using a bar
code reader.
77. The method of claim 71 or 72 wherein said reactive material
comprises a gel.
78. The method of claim 77 wherein said tubular reaction vessels
are configured for insertion into a reaction chamber and wherein
said reacting is performed within one or more reaction
chambers..Iadd.
79. A method of performing chemical synthesis utilizing a plurality
of reaction vessels, using one or more reaction stages and
resulting in a plurality of final products, each vessel comprising
solid phase reactive material and employing identifying indicia to
represent specific reactions and reagents utilized during the one
or more reaction stages, the method comprising:
(a) reading the identifying indicia provided for each of the
reaction vessels;
(b) sorting the reaction vessels into at least a first and a second
group, based on the indicia read in step (a);
(c) inserting the sorted groups into separate reaction
chambers;
(d) reacting a reagent with the reactive material by contacting a
reagent-containing solution with the reaction vessels in each
reaction chamber under particular reaction conditions, thereby
synthesizing chemical compounds on the reactive material;
(e) recording information concerning the reagent and reaction
conditions, the information corresponding to the identifying
indicia; and
(f) removing the reaction vessels from the reaction
chambers..Iaddend..Iadd.
80. The method of claim 79, further comprising:
(g) repeating steps (a) through (f) at least
once..Iaddend..Iadd.
81. The method of claim 79, further comprising reacting a first
reagent with the reaction vessels of the first group and reacting a
second reagent with the reaction vessels of the second
group..Iaddend.
Description
FIELD OF THE INVENTION
This invention relates to the field of combinatorial chemistry and,
more particularly, to a technique for performing combinatorial
chemistry using high throughput solid phase chemical synthesis
within a plurality of thin elongate reaction vessels.
DESCRIPTION OF RELATED ART
Combinatorial chemistry involves the synthesis of a large variety
of chemical compounds from a series of reactions or "chemical
recipes". Various combinatorial chemistry techniques have been used
to create a large number or library of compounds and these large
numbers of compounds can then be screened for various possible
biological activities for pharmaceutical, agricultural or other
purposes. Typically such a synthesis occurs in successive stages,
each of which involves a chemical modification of the then existing
molecules.
Geysen, in PCT Patent Appln. No., WO 90/09395 describes an approach
to high-throughput synthesis of peptide oligomers by synthesizing
these structures as they are attached to an array of "pins" that
are dipped into different reaction mixtures. However, using this
approach, quantities of synthesized compounds are limited. U.S. Pat
No. 5,143,854 to Fodor et al. discloses a similar array synthesis
method for peptides, oligonucleotides, and perhaps other oligomers
by using lithographic techniques derived from the semiconductor
industry, but applied to synthetic chemistry. Again, using these
techniques, yields are limited, typically to subpicomoles,
chemistries are also limited by the lithographic technique, and
biological activities must be assessed on the lithographic
array.
The concept of bead-based split synthesis to make large collections
of oligomers was first disclosed by Furka et al. in the 14th Int'l
Congress of Biochemistry, Prague, Czechoslovakia, Jul. 15, 1988. A
mix and split methodology is used whereby solid phase reactive
materials are randomly mixed and separated between reaction stages.
The reactive materials are then exposed to reactive agents and
conditions which are tracked. Dower et al. in PCT Patent Appln. No.
WO 93/06121, also disclose a "split synthesis" methodology for
synthesizing large numbers of different oligomers while they are
attached to bead-based solid phase supports as disclosed by
Merrifield in Science, vol. 236, Apr. 18, 1986, pgs 341-347. The
oligomers on each bead can be identified directly or indirectly
using an oligonucleotide "tag" that tracks each synthetic step.
However, the quantity available on each bead is generally less than
1 nanomole and the process of identifying compounds involves PCR or
some other tedious chemical method.
Still et al. in PCT Patent International Publ. No. WO 94/08051 have
disclosed an improved bead-based split synthesis technique which
includes a wide variety of chemical reactions not limited to
oligomers. The beads disclosed by Still et al. are also "tagged,"
but with a binary code utilizing independent chemical entities that
are more readily detected and identified using Gas Chromatography
techniques. Nonetheless, the quantities of synthetic compound
yielded from this technique are still subnanomole and their
identification is still based on chemical methods.
Houghten in European Patent Appln. No. 196174, describes yet
another method applicable to the synthesis of peptides or
oligonucleotides where solid support resin beads are partitioned
using inert plastic mesh bags or "tea-bags" where they stay
together during synthesis. Many "tea-bags" can be processed using
split synthesis to generate sufficient quantities of an array of
compounds. The methods as described by Houghten are specific to
oligomeric reactions. However, others, like Cody et al., in U.S.
Pat. No. 5,324,483, have generalized the basic "tea-bag" principle
to include segregating beads during reactions in systems that are
compatible with a wide range of chemical reactions. However, these
systems are somewhat awkward to use, require that the beads be
agitated, require many fluid-tight seals that must allow removal
and insertion and may require physically large layout areas for a
reasonable number of distinct compounds. In addition, detachment of
compounds and delivery to bioassays would be cumbersome for large
numbers of compounds.
Others such as Pavia et al., as disclosed in Bioorganic &
Medicinal Chem. Letters. (1993), vol. 3, pgs. 387-396 have used
robotic or automated systems for the synthesis of compounds in
liquid or solid phase (e.g., on beads) with milligram quantities
possible and some economies gained. All of these robotic systems
allow reasonably flexible programming of parallel synthesis of an
adequate quantity of compounds in separate reaction wells. However,
these systems are essentially serial in nature and do not enjoy the
enormous advantages of the split synthesis methods described above.
Operation is generally limited to hundreds or thousands of
compounds, and the automation can add extra constraints on the
choices or efficiencies of the synthetic reaction steps used.
Moreover, the solid phase bead-based systems require agitation as
mentioned above.
Beattie et al., in U.S. Pat. No. 5,175,209 and Frank et al., in
U.S. Pat. No. 4,689,405 describe a method of synthesizing oligomers
in large quantities using mix and split techniques similar to those
described above where solid support structures in the form of disks
are sorted into reaction vessels and resorted after each step. The
identity of the oligomer on each disk is defined by the reaction
chamber in which such disk was located during each reaction step.
Since the disks are large and can be marked, the sorting is
deliberate and direct. Moreover, non-chemical (e.g., mechanical)
methods may be used to mark and identify the disks as they go
through each synthesis step and thereby identify the structures
thereon. The quantities achievable using this method are greater
than 1 mg. However, the methods described by Beattie et al. and
Frank et al. are specific to oligomers, and the disk support
structures do not provide a convenient interface to standard
bioassays such as 96-well-plate-based tests. Compounds in solutions
extracted from these disks are not easily loaded into standard
96-well plates. The sorting and loading methods for the disks may
also be inadequate if the numbers of different structures desired
reaches 100,000 or more. Moreover, the mesh-like solid supports
described by Beattie et al. and Frank et al. may not be applicable
to non-oligomeric chemistries.
Therefore, none of these methods is ideal for synthesizing greater
than milligram quantities of non-oligomeric chemical compounds in
combinatorial or otherwise large libraries in such a way that
sampling such libraries into 96-well plates or other standard
bio-assay formats is easily achieved.
It is therefore desirable to obtain a combinatorial chemical
library synthesis system which is capable of a wide variety of
synthetic operations creating medicinally relevant molecules as
well as biopolymers.
It is also desirable to obtain a combinatorial chemical library
synthesis system which provides a yield in excess of 1 milligram
for each compound in the library with reasonable purity.
It is further desirable to obtain a system of executing
combinatorial "mix and split" synthetic strategies for up to
100,000 distinct elements. Such a system should provide for random
as well as deliberate mixing, which is important when one desires
to reduce the library from the set of all combinations because of
chemical or other constraints.
It is further desirable to obtain a combinatorial chemical library
synthesis system which conveniently prepares samples for biological
screening while allowing for quantitative partial extraction and
compatibility with 96-well or high density screening formats.
It is also desirable to provide a combinatorial chemical library
synthesis system which facilitates the deliberate sorting and
selecting of library members.
SUMMARY OF THE INVENTION
The present invention provides all of the above desirable aspects,
and a goal of the present invention is to provide a method of
synthesizing a large variety of potentially novel organic compounds
from a systematic scheme of "chemical recipes"(i.e., to perform
combinatorial chemistry) with sufficient yield (>1 .mu.mol or
>1 mg) and purity to allow a wide range of tests that would
enable discovery of drugs, agrochemicals, or specialty chemicals
from this set of organic structures. In the case of drug discovery,
these tests would include standard assay formats in the areas of
microbiology, enzymology, biological receptor binding studies,
toxicology, etc.
The present invention also has the advantage of providing a
convenient means to facilitate delivery of chemical samples to
typical testing formats (e.g. 96-well or higher density arrays of
vessels used in bio-tech laboratories).
The present invention also provides convenient and reliable means
to track the identity of each compound during the synthesis,
sampling and testing process to facilitate detailed studies of
chemical structure vs. biological activity relationships (QSAR).
Improved tracking and control of samples during synthesis also
increases the flexibility in designing such "combinatorial
libraries."
The aforementioned advantages may be achieved by use of a high
throughput solid phase chemical synthesis system using thin
tubular, and preferably cylindrical, reaction vessels.
In the present invention, a single reaction vessel may include a
straight, coaxial tubular member. The tubular member may be adapted
for placement of electronically readable identifying indicia
thereon so as to represent reaction conditions which have occurred
or which will occur within the tubular member and reagents utilized
in such reactions. As used herein, the term "electronically
readable" includes optically readable.
The identifying indicia may comprise a bar code which may be formed
by a series of readable bars which extend around the outer
circumference at a first end of the tubular member. The tubular
member may be coated with a defined thickness of a solid phase
reactive material on an inside surface thereof. Alternatively, the
reaction vessel may contain an elongated rod, coaxially oriented
within the tubular member, which is coated with such a reactive
material. The solid phase materials may be in the form of a gel
that is swellable in a variety of solvents and which has reactive
sites distributed throughout the volume thereof. As yet another
alternative, the reaction vessel may be filled with a plurality of
solid phase reactive beads which are retained therein by frits
located at the ends of the tubular member.
The present invention further includes a system which allows
chemical synthesis to be performed on solid phase reactive material
disposed within one or more reaction vessels, using one or more
reaction stages, resulting in one or more final products and
employing identifying indicia for each reaction vessel to represent
the specific reactions and reagents utilized during each reaction
stage in each vessel. The method employed in such system may
include reading the identifying indicia of a tubular reaction
vessel having the reactive material therein, reacting a reagent
within the reaction vessel under particular reaction conditions,
and subsequently determining such reagent and reaction conditions
by reading the identifying indicia, so as to identify the compound
so synthesized. The aforementioned steps may be repeated at least
once, thereby synthesizing and identifying chemical compounds
within the reaction vessel. Information concerning the reagent
and/or reaction conditions may be recorded in conjunction with
information concerning the identifying indicia. When recorded,
information concerning the reagent or reaction conditions may
correspond to particular indicia so that when particular indicia
are read, the corresponding information concerning the reagent or
reaction conditions used in a particular reaction vessel may be
readily determined.
The tubular reaction vessels may be marked with the identifying
indicia thereon and any of the reaction vessel embodiments
previously discussed supra may be used within this method. In
addition, the tubular reaction vessels may be adapted for
insertion, individually or in a group, into a reaction chamber
wherein the reacting steps are performed, in parallel, in each such
reaction vessel in the group. The reaction steps may include
flowing a reagent-containing solution through groups of such
tubular reaction vessels within one or more reaction chambers so as
to communicate with the solid phase reactive material therein,
effectively accessing all binding sites within such reactive
material. The tubular reaction vessels in the group may be removed
from the reaction chamber and sorted by reading the identifying
indicia, such as a bar code, thereon. The reaction vessels may be
pooled and/or sorted into two or more new groups, and each group
then inserted into a separate reaction chamber.
In addition, the system allows for the random mixing and separating
of the reaction vessels after one or more reaction steps, which may
be performed when creating combinatorial libraries. For example, a
group of reaction vessels may be removed from several reaction
chambers, mixed and randomly separated into two or more groups for
delivery to different reaction chambers for different subsequent
reactions. After these reactions, additional random mixing and
separating, as well as additional reactions may occur. Data
relating to the reactions. e.g., reagents and reaction conditions,
may be reflected by the reaction vessels' identifying indicia,
e.g., bar codes. These identifying indicia may be read, e.g.,
electronically, immediately prior to or after a reaction step has
occurred. Such indicia, along with the specific reaction step
information corresponding thereto, may be recorded and stored,
e.g., in a database. The reaction history of each individual
reaction vessel can therefore be tracked, and the chemical recipe
determined for the compounds synthesized, respectively, within each
vessel.
In the random synthesis just described, unless multiple copies of
each reaction product are made, there is a likelihood that not all
members of the set of potential final products will be synthesized.
Therefore, in accordance with the invention, solid phase chemical
synthesis also may be performed according to a pre-selected
chemical recipe performed on reactive material within a plurality
of tubular reaction vessels, resulting in the synthesis of either
the complete set of all possible combinations of a set of reagents
in a series of reactions or a predetermined subset thereof in a
predetermined number of copies, e.g., one copy. The method includes
reading coded indicia located on the reaction vessels, reacting one
or more reagents within the reaction vessels under particular
reaction conditions where the reagents and reaction conditions
subsequently are determined by the reading of the coded indicia,
and repeating the reading and reacting steps until the desired
chemical compounds have been synthesized within the reaction
vessels, the identities of such compounds being readily
ascertainable from reading the coded indicia. When the reacting
step is repeated, in accordance with this "pre-selected recipe
method," the reaction vessels may be deliberately sorted into
reaction chambers to undergo specific reactions represented by the
indicia. The chemical recipe of each chemical compound corresponds
to particular pre- or post-coded indicia on the reaction vessel
wherein its synthesis occurred.
The present system also facilitates synthesizing chemical or
biological compounds for direct delivery to an assay. The
synthesized compounds may be attached to the solid phase reactive
material by photo-labile covalent linking techniques, in which case
the tubular reaction vessels may be exposed to a controlled
intensity light source to release synthesized compounds from the
reactive material for elution with appropriate solvents. The
resulting eluent (i.e., compound dissolved in solvent) remains in
the reaction vessel and is available for delivery to a bioassay.
Such synthesized chemical compounds may be transferred directly
from the reaction vessels into a plurality of assay wells.
Alternative chemical methods can be used to detach compounds, e.g.,
by adding, neutralizing, and removing detachment reagents. The
synthesized chemical compounds may be screened for biological,
chemical, agricultural or other purposes. The method may be
performed in parallel on multiple reaction vessels having different
solid phase reactive materials therein. Optionally, before
detachment of a synthesized chemical compound, a reaction vessel
may be separated, e.g., "cut-up," into multiple pieces for use in
multiple assays.
The method employed in such direct delivery system includes
performing a plurality of reaction steps on the reactive material
within each of a plurality of tubular reaction vessels. After one
or more such reaction steps, the reaction vessels may be mixed and
separated into one or more groups. Each group of reaction vessels
may then be placed into a different reaction chamber where the
reactive materials therein will be exposed to different reactions.
Data relating to the reactions, e.g., reaction conditions and
reagents, may be recorded in such a manner as to be identified with
the particular reaction vessel. For example, for each of the
reaction vessels, data relating to each reaction may be recorded
and stored on in a database along with the reaction vessel's bar
code, identifying the chemical recipe for the synthesized compound
within. (It also is possible to track the reactions by recording
the location of each reaction vessel in a particular reaction
chamber for each reaction step and subsequently corresponding this
data to other recorded data relating to the reaction conditions for
each reaction chamber.) After all the reaction stages are
completed, the reaction vessels are sorted into a transfer array
based upon the identifying indicia, and a portion of the compound
within each of the sorted reaction vessels may be removed therefrom
for delivery to an assay plate or to a lawn-type assay. The
compounds subsequently can be tested and the chemical recipe for
desired (i.e., active) compounds obtained by matching their
respective positions in the assay plate with the identifying
indicia on the reaction vessels in the corresponding positions in
the transfer array.
The present invention also facilitates the testing of synthesized
compounds for biological or chemical activity. The tested compounds
are synthesized during multiple reaction stages on solid phase
reactive material contained within the reaction vessel prior to any
testing. The testing may include transferring synthesized compounds
from the tubular reaction vessels into a testing medium, the
reaction vessels comprising electronically readable identifying
indicia thereon representing reaction conditions which have
occurred within the tubular reaction vessels. The synthesized
compounds are then tested for a desired biological or chemical
activity. By electronically reading the identifying indicia on the
reaction vessel wherein was synthesized a compound having the
desired biological or chemical activity, the method of synthesis of
such compound (and, consequently, the structure of such compound)
may be determined by looking up recorded information wherein
specific identifying indicia correspond with specific reaction
histories.
The present invention also encompasses an apparatus for performing
chemical synthesis by performing a series of reactions on solid
phase reactive material within a plurality of tubular reaction
vessels, and one or more reaction chambers adapted to receive,
individually or in a group, the tubular reaction vessels therein.
Each of the reaction chambers has an inlet disposed to allow
reagents to flow into the reaction chamber and into the reaction
vessels disposed within the reaction chamber.
One or more guide arrays may be adapted for insertion into the
reaction chambers and also adapted to receive the tubular reaction
vessels therein. Each reaction chamber may have an inlet and an
outlet located within a first and second reaction chamber cover,
respectively. A guide cap having a plurality of apertures therein
may be located between the inlet and the guide array so that the
apertures are aligned with the reaction vessels to allow fluid
reagents to flow, in parallel fashion, from the inlet to the
respective reaction vessels within the reaction chamber. A second
guide cap having a plurality of apertures therein may be located
between the guide array and the outlet of the reaction chamber so
that the apertures are aligned with the reaction vessels to allow
fluid reagents to flow, in parallel fashion, from the respective
reaction vessels to the outlet of the reaction chamber.
One or more reaction chambers may be placed in fluid flow
relationship to an array valve which is in fluid flow relationship
with a plurality of reservoirs each having reagents therein. The
array valve is capable of distributing the reagents within the
reservoirs to the plurality of the reaction chambers in various
combinations. One or more pumps may be placed in fluid flow
relationship between the array valve and each of the reaction
chambers. Temperature control enclosures may be used to surround
each of the reaction chambers to allow reactions to occur within
specific temperature ranges.
An extrusion means such as a tool or other device may be used to
remove the reaction vessels from the reaction chambers. The
extrusion means may be adapted for insertion into the reaction
chambers and for contact with individual reaction vessels therein.
A sorting means receives the reaction vessels extruded from the
reaction chambers and sorts the reaction vessels into one or more
groups, to be determined based on the particular reaction or
reactions to be performed within the reaction vessel. The sorting
means may include a reader adapted to read identifying indicia,
such as a bar code, on the reaction vessel. The sorting means may
also include means for removing the extruded reaction vessels from
a hopper which collects the reaction vessels after extrusion from
the reaction chamber. A ramp or belt may be used to move the
extruded reaction vessels to a vessel director disposed to load one
or more reaction vessels into a vessel loading device. The vessel
loading device is adapted to receive a reaction chamber and guide
array therein and is moveable along a first and second axis to
allow insertion of any reaction vessel in a particular location
within the reaction chamber. A means for receiving one or more
extruded reaction vessels from the sorting means, such as a robotic
pipettor adapted to a vessel transfer array (described above), may
be used to communicate with the reaction vessels and deposit
synthesized compounds eluted therefrom into a plurality of wells
of, e.g., a 96-well plate, or onto a lawn bioassay plate.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A depicts a top sectional view of a preferred embodiment of a
disposable reaction vessel adapted for synthesis of a single
compound therein;
FIG. 1B depicts a cut-away sectional view from the side of the
reaction vessel of FIG. 1A;
FIG. 2A depicts a schematic representation of a preferred
embodiment of a reaction chamber containing a plurality of reaction
vessels;
FIG. 2B depicts a schematic representation of the reaction chamber
shown with reaction fluids moving therethrough;
FIG. 2C depicts a sectional view from the side of a portion of the
interface between an inlet guide cap and guide array insert,
located within a reaction chamber, having a reaction vessel
therein;
FIG. 3 depicts a schematic representation of an embodiment of a
reaction system, including reagent bottles, pumps, reaction
chambers and reagent switching valve-arrays;
FIG. 4 depicts a schematic representation of an embodiment of a
sorting means useable to perform solid phase chemical synthesis in
a plurality of reaction vessels where reaction vessels are
identified and moved into one or more appropriate reaction chambers
for a subsequent synthetic step;
FIG. 5 depicts a schematic representation of an embodiment of a
bioassay sampling system where reaction vessels are sorted onto
special fixtures for photoelution of compounds and for pipetting
eluent into 96-well plates using robotic pipettors;
FIG. 6 depicts a schematic representation of an embodiment of
another bioassay sampling system where reaction vessels are bundled
directly from a reaction chamber in a dense format, dipped into an
elution solvent, photoeluted with end-on illumination, and dipped
into a lawn type assay plate where small eluent volumes are
transferred by capillary action in the same dense pattern;
FIG. 7 depicts an isometric view of an alternative embodiment of
the tubular reaction vessel having reactive polymer beads and glass
beads therein; and
FIG. 8 depicts a sectional view of yet another embodiment of the
tubular reaction vessel having a rod coaxially oriented therein
coated with reactive material.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring to FIGS. 1A and 1B, a preferred reaction vessel 1 is
shown. The reaction vessel 1, which is preferably disposable, is
one component of a system for performing solid phase chemical
synthesis in accordance with the principles of the present
invention. Each vessel contains an inner reactive coating 2 (or
inner reactive packing--see discussion of FIG. 7 below) to which
molecules of a synthesized compound are attached, throughout the
volume thereof, during such synthesis. Typically, the coatings or
packings are chemical polymers such as cellulose, pore-glass,
silica gels, polystyrene optionally cross-linked with
divinylbenzene and optionally grafted with polyethylene glycol and
optionally functionalized with amino, hydroxy, carboxy, or halo
groups, grafted co-poly beads, polyacrylamide beads, latex,
dimethylacrylamide optionally cross-linked with N,N'-bis-acryloyl
ethylene diamine, glass coated with hydrophobic polymers, etc.
Preferably, the coating or packing is divinylbenzene-cross-linked,
polyethyleneglycol-grafted polystyrene optionally functionalized
with amino groups (for example, TentaGel.RTM. S NH.sub.2, Rapp
Polymere).
The outer shell 3 of the reaction vessel 1 is a structural element
shaped such as a capillary tube, that provides an attachment
surface, protects the polymer coating 2 and provides a rigid shape
and defined size to the vessel 1. Although the tubular reaction
vessel 1 is preferably thin and cylindrical in shape, other shapes
and configurations may be used. For example, instead of a cylinder
shape, other polygonally shaped tubular structures may be used. The
shell 3 is preferably clear to allow penetration by UV radiation to
effect the photocleavable release of compounds for bioassay. The
shell 3 should also be inert and stable against a variety of
organic solvents, strong acids and bases, heat, and other
conditions typical of organic synthesis. The polymer coating 2 is
preferably attached to the inner surface of the rigid shell 3 so
that the polymer remains attached throughout the various synthesis
steps. Such steps may involve the use of high temperatures (up to
100.degree. C.), organic solvents (MEK, chloroform, etc.), strong
acids and bases, and oxidizing reagents. Preferred materials for
the reaction vessel shell 3 include glass, quartz, or optically and
chemically compatible plastics (e.g. Teflon that is transparent to
light at 300-400 nm wavelength). The polymer coating may be
deposited onto the inner surface of the shell by methods
conventional in the art. An example of such a method is disclosed
by Garner et al. in U.S. Pat. No. 5,387,526, the disclosure of
which is incorporated herein by reference.
Nominal dimensions for a preferred reaction vessel 1 may be as
follows:
inner diameter of shell, 3 1 mm outer diameter of shell, 3 2 mm
length of shell, 3 100 mm thickness of coating, 2 0.05 mm
Use of the tubular reaction vessel 1 having the aforementioned
nominal dimensions in solid phase chemical synthesis allows for a
high output yield. For example, using TentaGel.RTM. S NH.sub.2,
mentioned supra as a coating, which has a vendor-published value of
active sites at 0.3 .mu.mol/mg and a density of 1.2 g/cm.sup.3 for
the coating, then the vessel should support roughly 2.4 .mu.mol of
compound during synthesis. This represents an ideal yield of
roughly 1.2 mg based on a molecular weight of 500 g/mol.
As shown in FIG. 7, an alternative embodiment of a thin tubular
reaction vessel is shown. Each of the ends of the reaction vessel
is capped with a permeable frit 50, typically glass, that allows
fluid to flow therethrough. In this embodiment, the reaction vessel
shell 3 is filled with a mixture of glass beads 51 (300-700 .mu.m)
and reactive polymer beads 53 (100-200 .mu.m). The glass beads 51
are disposed to allow reagents containing solvents to flow rapidly
over the polymer beads 53, and through the vessel shell without
causing the polymer beads to collapse into a flow-restricting
aggregation. This type of reaction vessel may be constructed from
commercially available materials. However, the flow and diffusion
properties near any polymer bead may not be as desirable as those
attainable with the coated tube depicted in FIG. 1.
As shown in FIG. 8, another alternative embodiment of a reaction
vessel is shown, incorporating a small tube or rod 55 coated with
the reactive polymer-gel 2. The smaller tube or rod is coaxially
oriented inside the larger reaction vessel shell 3 using alignment
fixtures 58 located at each end of the vessel 1 to position the
small tube or rod concentrically within the larger tube. Each
alignment fixture 58 may be constructed of a glass frit. The use of
the smaller tube or rod 55 allows for coating of its exterior
surface, which may be more easily accomplished than coating the
interior of the shell 3.
Referring again to FIGS. 1A and 1B, electronically readable
indicia, preferably an optically readable bar-code 4, is preferably
etched or marked in rings around the outside of the vessel to
establish unique identifying indicia for each vessel. The markings
or etching should be resistant to all the solvents and synthesis
conditions to which the vessel will be exposed and should not
contribute to any chemical contamination during synthesis. A
typical bar-code covering integers from 1-1,000,000 can be
accommodated within a 1 inch segment of the length of the reaction
vessel 1. Since the bar-code 4 is marked in bands, its readout will
be independent of the rotational orientation of the vessel 1 with
respect to its central, longitudinal axis. Specific methods of
rapid bar-code 4 readout are disclosed herein with reference to
FIGS. 4 & 5.
Referring to FIG. 2A and 2B, an exploded view and cutaway side view
of a preferred reaction chamber assembly 5 are shown with a
plurality of reaction vessels 1 formed in an array inserted within
the chamber. The reaction chamber 5 is designed to provide a
uniform, efficient reaction environment for the reaction vessels
therein. Referring to FIG. 2B, the bold arrows indicate the reagent
flow entering through the chamber inlet cover 11 and eventually out
the chamber outlet cover 9. The flow is made even throughout the
internal cross sectional area of the chamber by use of flow
diffuser 12 disposed within the chamber inlet cover 11. Each
reaction vessel 1 is aligned in parallel with the flow direction
when inserted into the guide array insert 6. The inlet guide cap 10
and exit guide cap 8 provide further alignment and immobilization
for the reaction vessels when the chamber covers 11, 9 are fastened
on the chamber body 7. A detailed view, such as that of FIG. 2C,
indicates how the inlet guide cap 10 can direct reagent flow
efficiently through the reaction vessel 1. The reagent flow may
contain a variety of corrosive and volatile solvents that can
attack most plastics and metals. Furthermore, reaction temperatures
within the chamber may reach as high as 100.degree. C. The chamber
body 7 and chamber covers 9, 11 are constructed from structurally
rigid materials with sufficient solvent resistance properties;
borosilcate glass and highly corrosion resistant plastics are
preferred materials. However, thermoformable resins such as PVDF,
polyethylene, or polypropylene may also be used in some
applications. Also, the guide array insert 6 and the guide caps 8,
10 may be molded or extruded using very solvent-resistant resins
such as PTFE, TFE, or PVDF. The chamber covers 9, 11 are preferably
sealed to the chamber body 7 through a pair of O-rings 31, 33 (FIG.
2B) which should be made of solvent-resistant materials such as
KALREZ.RTM.. This type of material is manufactured by E.I. Dupont
de Nemours and Company of Wilmington. Del., and is capable of
withstanding a variety of solvent, high temperature and high
sealing forces while maintaining thermal stability. Other sealing
means, however, may also suffice. The outlet and inlet covers 9, 11
may be clamped to the chamber body 7 by any appropriate clamping
mechanism (not shown). A tab structure 35, or other mark, on an
outer corner of the chamber body 7 may be used as a reference for
the specific locations of the respective reaction vessels 1 as
arrayed in the reaction chamber 5. This reference is important for
the sorting process prior to or following various reaction steps
and in the sampling process for bio-assays described herein.
Referring to FIG. 3, a reaction system is shown with four reaction
chambers 5 installed therein. Reagent reservoirs such as bottles 26
are prepared to contain the desired quantity of solvent with the
desired concentration of reagents. The reagent bottles 26 are shown
as sealed with purge/sparge lines 37 inserted to allow removal of
oxygen and/or equilibration with specific dissolved gases. Syringe
lines 39 are shown as means for adding reagents. However, other
means for adding reagents may be used. For example, solid reagents
may be added and dissolved through a resealable tube in the reagent
bottles 26.
The reaction system of FIG. 3 allows for a parallel method of
synthesizing a large collection of compounds, the respective
compounds retained in the gel phase interiors of the respective
reaction vessels 1 that are contained inside each reaction chamber
5. All reaction vessels inside a given reaction chamber 5 will
experience the same chemical conditions for a particular step in
the synthetic process. The array valve 22 in fluid flow
relationship with each reagent bottle 26 can direct a different
reagent solution to each reaction chamber from any of the reagent
bottles 26 shown. The array valve 22 can also facilitate a sequence
of reagent exposures for a given synthetic step.
Temperature-controlled enclosures 24 may surround each of the
reaction chambers 5, allowing each reaction step to occur at a
prescribed temperature in addition to the controlled
reagent/solvent exposure. The reagent/solvent exposure is further
controlled by the independent peristaltic pumps 23 each placed
downstream of the array valve 22. The peristaltic pumps can control
the flow rates to the corresponding reaction chambers downstream
therefrom. Hence, the volumetric rate of fresh reagent/solvent
communicating with the gel phase inside the reaction vessels 1,
necessary for specific reactions, may be controlled by the pumps
23.
The peristaltic pumps 23 can be programmed to exchange fresh
reagent/solvent into the reaction chambers and subsequently halt
flow during a prescribed equilibration or "incubation" period. This
allows fluid reagents adequate time to fully react with the
intermediate compounds retained in the gel phase inside the
reaction vessels 1. After equilibration, flow can be resumed to
remove unwanted dissolved byproducts and to introduce fresh
reagents. Reactions can be optimized and reagents can be used in a
more efficient manner with such a programmed flow. A waste manifold
25 accepts the post-reaction effluent and maintains a closed fluid
system to prevent unwanted air exposure to the
reagent/solvents.
Alternatively, to further conserve on reagents while maintaining a
flowing reaction environment, a recirculation fluid path 41 can be
arranged for each reaction chamber. However, when using a
recirculation path, care should be taken to avoid clogging of
valves by any gel particles that may dislodge or precipitate from
the reaction vessels. Moreover, the extent of recirculation may
have to be limited due to the potential increasing concentration of
reaction byproducts in the recirculating fluid. Another alternative
is to provide a means of sealing the entrance and exit ports of the
reaction chambers 5 so that they may be removed from the reaction
system for equilibration "off-line." While such equilibration is
occurring, different reaction chambers may be installed in the
reaction system.
In a preferred embodiment, each reaction chamber 5 may accommodate
100 reaction vessels 1. The reaction system shown in FIG. 3 can be
replicated so that groups of reaction chambers can share a set of
reagent bottles. An arrangement involving as many as 100 reaction
chambers is practically feasible for the construction of a large
collection of compounds. Such an arrangement would utilize 10,000
reaction vessels, wherein each vessel can be involved in one of
potentially 100 different synthesis steps, resulting in a high
potential for diversity of the resulting chemical library. However,
even with the convenient fluidics shown in FIG. 3, the task of
sorting and tracking the reaction vessels in between each synthetic
step could be onerous for a collection of 10,000 vessels or
more.
Referring to FIG. 4, a sorting means is shown, including means for
tracking and transferring reaction vessels 1 from one reaction
chamber to another reaction chamber in preparation for the next
synthetic step. After completion of a synthetic step, the reaction
chamber 5 is equilibrated with an inert solvent and carefully
drained so as to avoid damage to the reactive material in the
reaction vessels 1. The chamber covers and inlet guide cap are
removed to allow the reaction vessels 1 to exit the chamber using
an extrusion means such as an extrusion tool 13 having an array of
long pins that are guided through the holes in the exit guide cap
by a plunger 14. Although an extrusion tool 13 is depicted in FIG.
4, any mechanism or technique which may extrude the reaction
vessels 1 from the reaction chamber may be used. The reaction
vessels are deposited in a sorting hopper 15 with all the tubes
oriented in the same direction. For instance, the barcoded end of
the tubes is oriented towards the reaction vessel after extrusion
therefrom into the hopper 15. A hopper feed 16 randomly accepts the
tubular reaction vessels 1 from the hopper and transfers them to a
conveyor belt 17 in fixed intervals. Alternatively, a ramp (not
shown) may be used to allow the reaction vessels to roll thereon.
The belt 17 may have rib structures 43 that maintain the reaction
vessels 1 in fixed spacings. The reaction vessels may be released
in intervals ranging from 0.1 to 1 second so that each chamber
containing 100 tubes can be sorted in under 2 minutes.
Sorting of 10,000 reaction vessels 1 can be accomplished in from 20
minutes to 3.3 hours. All subsequent sorting steps including bar
code reading and ejection from the belt into the appropriate new
chamber may be timed to correspond with this feed rate. The bar
code reader 18 is adapted to, and placed in relationship with, the
belt 17 in order to read each bar coded reaction vessel 1 placed
thereon. The bar code reader 18 identifies each reaction vessel as
it travels within scanning distance. The bar code reader 18 is
interfaced with a computer (not shown) which controls and monitors
the reaction-chamber-location and identification of each reaction
vessel 1. Conventional software may be used to decode the barcode
for each reaction vessel and determine the destination, e.g., the
location within any reaction chamber 5 for the next reaction step
for the particular reaction vessel.
One of a series of mechanical vessel directors 19, each disposed on
the opposite side of belt 17 from the respective reaction chambers
5 and in alignment therewith, may then be electronically actuated
to push any vessel 1 into the appropriate reaction chamber 5 when
the vessel moves into the proper position. The bar code reader 18,
or another optical reader, may be used to determine the location of
such vessel on the belt 17 and actuate the vessel director 19 when
appropriate. The vessel director 19 contains a punch 45 which, when
activated, contacts the end of a reaction vessel and directs the
vessel 1 into a vessel loading device 20. Vessel loading device 20
may be used within the reaction chamber 5 to carefully move any
vessel 1 into the reaction chamber 5 after such vessel has been
ejected from belt 17 by the vessel director 19. The reaction
chambers 5 are mounted on a dual axis adjustable receiver 21 which
moves along both an X and Y axis. The adjustable receiver 21 moves
the reaction chamber to the appropriate location for insertion of
one reaction vessel 1 at a time therein. The adjustable receiver 21
is controlled by a computer which also monitors the position of
each reaction vessel within the chamber 5 mounted on the receiver
21. The reaction chambers 5 are indexed after each vessel 1 is
loaded so that each chamber can be filled with reaction vessels in
a deliberate fashion using the grid of holes defined by the guide
array insert 6. The software and hardware required for these
sorting operations may be similar to that used by manufacturing
engineering groups or consultants in the clinical or diagnostic
reagents and disposables industries, which is readily commercially
available. After all the reaction vessels have been sorted and
loaded into the reaction chambers 5, the reaction chambers 5 can be
reassembled with their guide caps and chamber covers and installed
in a reaction system, such as that of FIG. 3, for the next step in
the combinatorial synthesis.
The present invention provides flexibility with regard to the
"mix-and-split" operations performed in combinatorial synthesis.
Random mix-and-split methods as disclosed in PCT Patent
International Publ. No. WO 94/08051 to Still et al. are easily
achieved by pushing all the vessels 1 using the extrusion tool 13
into a large hopper 15 where the reaction vessels 1 can be mixed by
rotation thereof in a direction parallel to the longitudinal axis
of the reaction vessels 1. Bundles of vessels 1 can be removed,
randomly separated and loaded into the next reaction chamber 5
using the system of FIG. 4 or by hand. Alternatively, complex
mix-and-split algorithms that systematically eliminate certain
combinations or provide multiple (i.e., extra) copies of other
combinations can be implemented through a preprogrammed
computer-controlled function that determines which reaction vessel
director 19 is activated for each reaction vessel 1. Random mix and
split operations performed in combinatorial chemistry can be
facilitated in large quantities by recording data, immediately
prior to the placement of each reaction vessel 1 into a reaction
chamber 5 or immediately subsequent to its removal therefrom,
relating to the reactions performed in such reaction vessel. For
example, data relating to a particular reaction, such as the
reagents and/or the reaction conditions, may be recorded and stored
in a database along with identifying data from an electronically
readable medium on the reaction vessels, such as the bar code. In
this manner, the reaction vessels may be tracked by the
electronically readable indicia and the chemical recipe of
compounds synthesized therein deciphered based upon reading the
indicia and corresponding the same to the relevant data relating to
the reactions performed. Alternatively, data relating to the
reactions undergone within a particular reaction vessel may be
recorded by a recording means, such as a computer. The reaction
vessels then may be tracked by their particular locations within
reaction chambers at every reaction stage, e.g., by placing each
reaction vessel in predetermined locations within such reaction
chambers at each reaction stage, and recording reaction conditions
for each reaction chamber at every reaction stage. Using this
technique, the chemical recipe for the compound synthesized within
any reaction vessel may be determined based solely upon the
location of such reaction vessel within a particular reaction
chamber after the final reaction stage.
FIG. 5 depicts a schematic of a system which shows how the
compounds in the reaction vessels 1 are released and sampled into
standard 96-well microtiter plates after combinatorial synthesis
within the reaction vessels 1 is complete. Reaction vessels 1 are
loaded into a hopper 15 using the extrusion tool 13 and plunger 14.
The reaction vessels 1 may be removed from the hopper via feed
mechanism 16 and loaded in order of feeding onto an alignment
fixture 27, which may be designed specifically for use with the
96-well plate format, using a short ramp 28 or conveyor belt (not
shown) and by mechanically moving the alignment fixture 27,
preferably in increments, under the short ramp 28. A bar-code
reader 18 reads the codes on the reaction vessels 1 as they roll
down the ramp 28 and a computer (not shown), interfaced with the
bar code reader, tracks the vessels as they are loaded onto each
alignment fixture 27. One of ordinary skill in the art can readily
utilize software to determine which vessels 1 are loaded in what
order onto each alignment fixture 27 for screening.
The alignment fixtures contain indentations 47, preferably
V-shaped, wherein the reaction vessels 1 are seated. The alignment
fixtures 27 may also contain an electronically readable and/or
writable indicia such as a barcode, allowing additional
traceability and monitoring of each reaction vessel 1 with respect
to each alignment fixture 27 and sample well 31. The reaction
vessels 1 may be filled with an elution solvent before they are
loaded onto the alignment fixtures. This is preferably accomplished
by flushing the reaction chamber 5 with elution solvent before
removing the vessels 1 therefrom and loading them into the hopper
15. However, the elution step can be performed in other ways
conventional in the art.
The extrusion means such as extrusion tool 13, which is also
interfaced with the computer, may be programmed to extrude only
certain reaction vessels from any given reaction chamber. This
controlled extrusion may be performed either after the completion
of all combinatorial synthesis steps, as shown in FIG. 5, or
between reaction stages during the combinatorial synthesis, as
shown in the system of FIG. 4. Such controlled extrusion could
facilitate sorting of the reaction vessels into the proper reaction
chambers for the next reaction step.
Once the alignment fixtures 27 are loaded with reaction vessels 1,
if the compounds synthesized therein were attached to the reactive
polymer coating by a photo-labile covalent linker, the vessels are
placed under a controlled intensity ultraviolet source 29 to detach
the synthesized compound from the polymer within each such reaction
vessel 1. Such photo-labile covalent linking techniques used in
solid phase synthesis are described by Still et al. in PCT Patent
International Publ. No. WO 94/08051. A portion of each of the bound
compounds can be detached and eluted as a linear function of the
ultraviolet power multiplied by exposure time when the fractional
release is small. Alternatively, a portion can be detached by
exposing a fractional section of a tube that has been cut up. Since
microtiter plate well volumes are typically about 100 .mu.l and
desired test concentrations are usually about 1 .mu.M, only about
0.01% release of the estimated 1-2 .mu.mol total quantity of each
compound is needed. It may be desirable to expose the entire
surface of the reaction vessels to the ultraviolet light in order
to optimize the release of compounds therefrom. To accomplish this,
the alignment fixtures 27 may be made of a photo-transparent
material and the reaction vessels may be held in sandwich fashion
between two alignment fixtures. After exposing one side of each of
the reaction vessels to the light, the mated alignment fixtures
could then be easily flipped to allow the opposite sides of these
reaction vessels to be exposed to the light. Other conventional
techniques, however, for optimizing exposure may be used.
After exposure to ultraviolet light, each alignment fixture 27 may
be rotated so that the reaction vessels 1 are vertically oriented.
The alignment fixture 27 nominally holds the vessels in rows of 12
or columns of 8. Means for transport and fluid delivery, such as
robotic pipettor 30, moves over to an alignment fixture and
captures the vessels using a septum or seal-based capture mechanism
(not shown), which engages the ends of the reaction vessels 1 to
form a seal therewith. The arm of the robotic pipettor 30 then can
move the vertically oriented reaction vessels into alignment with
the wells of a target plate 31, and the elution solvent is
dispensed from the vessels 1 into said wells by pressuring the
vessels via the pipettor 30. The pipettor may wash out any
remaining compound that has been released by the UV light by
delivering additional wash solvent from a reservoir. After all the
desired rows (or columns) of the 96-well plate 31 are filled using,
if desired, different sets of vessels 1 from different alignment
fixtures 27, plate 31 is removed and the elution solvent dried off,
leaving just the sample compounds from the vessels 1 therein.
Multiple compounds may be loaded into each well if desired, using
additional alignment fixtures 27 and by computer-controlling the
robotic pipettor 30 appropriately. Plate 31 may also contain
electronically readable and/or writable indicia such as a barcode
to allow for computerized tracking of compound identity for each
plate and well location.
FIG. 6 depicts a schematic of an alternative, high throughput
technique of sampling the synthesized compounds from the reaction
vessels 1 and performing a bioassay. Rather than arranging the
reaction vessels with the typical 9 mm spacing corresponding to the
rows or columns of a 96-well microtiter plate as described with
reference to FIG. 5, the vessels 1 may be arranged in a more dense
grouping. This type of system may be used when precise volume
control of the synthesized compounds is not necessary, or when
moving small volume fluid samples of compounds is desired. The
reaction vessels may be extracted from a reaction chamber 5 by
using one of the inlet or exit guide caps 8 as a means of holding
onto one end of the group of tubular reaction vessels 1. The other
end of the group of reaction vessels 1 may be loaded with elution
solvent by dipping into a solvent tank 32. The amount of solvent
loaded, typically about 2 to about 20 .mu.l, may be determined by
capillary action.
The ends of the vessels 1 dipped into the solvent may then be
exposed to ultraviolet source 33. The exposure may be adjusted to
release a pre-determined portion of each compound, typically about
0.1% of that bound to the solvent-exposed polymer. The elution
solvent from each reaction vessel 1 may then be transferred to a
lawn bioassay plate 34 by dipping the ends of the vessels 1 therein
and relying on capillary forces to deliver the compound-containing
solvent. Alternatively, the small volume of solvent from each
vessel 1 may be transferred by aligning the group of vessels 1 with
guide cap 8, over the lawn bioassay plate 34 in a fixture, placing
the entire assembly in a centrifuge (not shown) and spinning the
solvent out of the tubes into the bioassay plate. The bioassay
plate 34 may be adapted to have small individual wells to
efficiently receive the solvent.
Use of the tubular reaction vessels 1 may also allow for the use of
liquid chromatography techniques and systems for purification of
the synthesized compounds prior to deposition into plates or
bioassays. Compounds extracted into solvent from the vessels 1 can
be easily transferred to a chromatographic system through simple
tubing connectors as in FIG. 5. Purified compounds obtained from
liquid chromatographs may be deposited directly into a plate or
bioassay.
Although the invention has been disclosed with reference to the
embodiments depicted herein, it will be apparent to one of ordinary
skill in the art that various modifications and substitutions may
be made to such embodiments. For example, different indicia other
than bar codes may be used on the reaction vessels; various
configurations for the reaction chambers may be used; various
configurations may be appropriate for reaction systems using
multiple reaction chambers, solvents and reagents; and various
different extrusion means and sorting means may also be used. Any
such modifications and/or substitutions are intended to be within
the scope of the invention as defined by the following claims.
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