U.S. patent application number 09/863158 was filed with the patent office on 2001-10-11 for chemical synthesis apparatus employing a droplet generator.
This patent application is currently assigned to ISIS Pharmaceuticals, Inc.. Invention is credited to Acevedo, Oscar, Davis, Peter W., Ecker, David J., Hebert, Normand, Kiely, John S., Wyatt, Jacqueline R..
Application Number | 20010028866 09/863158 |
Document ID | / |
Family ID | 23200254 |
Filed Date | 2001-10-11 |
United States Patent
Application |
20010028866 |
Kind Code |
A1 |
Ecker, David J. ; et
al. |
October 11, 2001 |
Chemical synthesis apparatus employing a droplet generator
Abstract
Chemical reaction apparatus, materials and methods are provided
for the automatable, efficient synthesis of chemical species and
libraries. In accordance with preferred embodiments, chemical
droplet generation and direction techniques are employed to prepare
oligomers and libraries of chemical species. Reaction assemblies
adapted for efficient synthetic employment and for improved
collection are also disclosed.
Inventors: |
Ecker, David J.; (Leucadia,
CA) ; Acevedo, Oscar; (San Diego, CA) ;
Hebert, Normand; (Cardiff, CA) ; Davis, Peter W.;
(Carlsbad, CA) ; Wyatt, Jacqueline R.; (Carlsbad,
CA) ; Kiely, John S.; (San Diego, CA) |
Correspondence
Address: |
Woodcock Washburn Kurtz
Mackiewicz & Norris LLP
46th Floor
One Liberty Place
Philadelphia
PA
19103
US
|
Assignee: |
ISIS Pharmaceuticals, Inc.
|
Family ID: |
23200254 |
Appl. No.: |
09/863158 |
Filed: |
May 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09863158 |
May 23, 2001 |
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09313403 |
May 18, 1999 |
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09313403 |
May 18, 1999 |
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08778876 |
Jan 2, 1997 |
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5925732 |
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08778876 |
Jan 2, 1997 |
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08309925 |
Sep 21, 1994 |
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Current U.S.
Class: |
422/131 |
Current CPC
Class: |
B01J 2219/00585
20130101; B01J 2219/00378 20130101; B01J 2219/0059 20130101; B01J
2219/00729 20130101; C40B 40/06 20130101; B01J 2219/00617 20130101;
B01J 19/0046 20130101; B01J 2219/00641 20130101; B01J 2219/00612
20130101; B01J 2219/00626 20130101; B01J 2219/00351 20130101; B01J
2219/00621 20130101; B01J 2219/00527 20130101; C40B 60/14 20130101;
B01J 2219/0072 20130101; C07K 1/047 20130101; B01J 2219/0061
20130101; C12P 19/34 20130101; B01J 2219/0063 20130101; B01J
2219/00637 20130101; B01J 2219/00659 20130101; B01J 2219/00596
20130101; B01J 2219/00315 20130101; B01J 2219/00605 20130101; B01J
2219/00333 20130101; B01J 2219/00725 20130101; B01L 3/0268
20130101; C07H 21/00 20130101; C40B 40/10 20130101; B01J 2219/00722
20130101; C07B 2200/11 20130101 |
Class at
Publication: |
422/131 |
International
Class: |
B01J 019/00 |
Claims
What is claimed is:
1. A chemical reaction apparatus comprising a. a reaction support
having a plurality of reaction sites upon a first surface thereof;
b. a first droplet generator for jetting first reactant droplets
upon the first surface; c. a second droplet generator for jetting
second reactant droplets upon the first surface; and d. control
means for causing the droplets from each of the droplet generators,
to impact upon preselected sets of said reaction sites.
2. The apparatus of claim 1 wherein said reaction support is
porous.
3. The apparatus of claim 2 wherein the porous support comprises
controlled pore glass.
4. The apparatus of claim 2 wherein the porous support comprises a
porated solid.
5. The apparatus of claim 2 wherein the porous support comprises
fibers having a substantially common axis normal to the first
surface.
6. The apparatus of claim 2 wherein the porous support is an
anisotropic membrane.
7. The apparatus of claim 1 wherein the support comprises a second
surface substantially parallel with the first surface, the support
being capable of transporting fluid contacting the first surface to
the second surface of the support in a direction substantially
normal to the first surface.
8. The apparatus of claim 7 further comprising a collection plate
adjacent to the second surface.
9. The apparatus of claim 8, wherein said collection plate has a
plurality of wells for receiving fluid transported through said
support.
10. The apparatus of claim 1 further comprising additional droplet
generators for jetting chemical reactant upon said first
surface.
11. The apparatus of claim 1 wherein said control means is a
digital computer.
12. The apparatus of claim 1 wherein at least one droplet generator
is adapted for traversing over the first surface of the reaction
support.
13. A chemical reaction apparatus comprising a. a reaction support
having a plurality of preselected reaction sites upon a first
surface thereof; b. a droplet generator for jetting droplets of
chemical reactant upon the first surface; c. a plurality of
reactant reservoirs in fluid communication with said droplet
generator; d. control means for causing chemical reactants from
selected ones of said plurality of reactant reservoirs to be jetted
by said droplet generator upon preselected sets of said reaction
sites.
14. The apparatus of claim 13 further comprising valve means for
directing chemical reactants from said reactant reservoirs to the
droplet generator in response to control signals from said control
means.
15. The apparatus of claim 1 wherein said reaction support is
porous.
16. The apparatus of claim 15 wherein the porous support comprises
controlled pore glass.
17. The apparatus of claim 15 wherein the porous support comprises
a porated solid.
18. The apparatus of claim 15 wherein the porous support comprises
fibers having a substantially common axis normal to the first
surface.
19. The apparatus of claim 15 wherein the porous support is an
anisotropic membrane.
20. The apparatus of claim 13 wherein the support comprises a
second surface substantially parallel with the first surface, the
support being capable of transporting fluid contacting the first
surface to the second surface of the support in a direction
substantially normal to the first surface.
21. The apparatus of claim 20 further comprising a collection plate
adjacent to the second surface.
22. The apparatus of claim 21 wherein said collection plate has a
plurality of wells for receiving fluid transported through said
support.
23. The apparatus of claim 13 further comprising additional droplet
generators for jetting chemical reactant upon said first
surface.
24. The apparatus of claim 13 wherein said control means is a
digital computer.
25. The apparatus of claim 1 wherein the droplet generator is
adapted for traversing over the first surface of the reaction
support.
26. A method for synthesizing a chemical species comprising a.
identifying a plurality of reaction sites upon a first surface of a
reaction support; b. jetting upon a first set of said reaction
sites, drops of fluid comprising a first chemical reactant species;
and c. jetting upon a second set of said reaction sites, drops of
fluid comprising a second chemical reactant species.
27. The method of claim 26 wherein said reaction support is
porous.
28. The method of claim 27 wherein the porous support comprises
controlled pore glass.
29. The method of claim 27 wherein the porous support comprises a
porated solid.
30. The method of claim 27 wherein the porous support comprises
fibers having a substantially common axis normal to the first
surface.
31. The method of claim 27 wherein the porous support is an
anisotropic membrane.
32. The method of claim 26 wherein the support comprises a second
surface substantially parallel with the first surface, the support
being capable of transporting fluid contacting the first surface to
the second surface of the support in a direction substantially
normal to the first surface.
33. The method of claim 26 further comprising collecting fluid from
the first surface on a collection plate adjacent to the second
surface.
34. The method of claim 33, wherein said collection plate has a
plurality of wells for receiving said fluid.
35. The method of claim 26 under control of a digital control
means.
36. The method of claim 26 wherein said first and second sets are
substantial identical.
37. The method of claim 26 wherein said synthesis is of an
oligonucleotide.
38. A method for synthesizing a chemical species comprising a.
bonding an initial reaction fragment to a first surface of a
reaction support, said first surface having a plurality of
preselected reaction sites; b. jetting upon a first set of said
reaction sites a first chemical reactant species to effect a
chemical reaction with the initial reaction fragment at the first
set of reaction sites; and c. jetting upon a second set of said
reaction sites a second chemical reactant species to effect a
chemical reaction with either i. the initial reaction fragment at
sites not in common with said first set of reaction sites, or ii.
the reaction product of the initial reaction fragment and the first
chemical reactant at those sites which are in common with said
first set of reaction sites.
39. The method of claim 38 further comprising d. jetting upon a
further set of said reaction sites a further chemical reactant
species, which may be the same as or different from any prior
chemical reactant species, to effect a chemical reaction with
either i. the initial reaction fragment at sites not in common with
any prior set of reaction sites, or ii. the reaction product of the
initial reaction fragment and the additional chemical reactants
delivered to sites of said further set.
40. The method of claim 39 performed iteratively.
41. The method of claim 38 further comprising recovering the
chemical synthesized.
42. The method of claim 38 wherein said synthesis is of
oligonucleotide.
43. A chemical reaction assembly comprising a reaction support
having first and second surfaces and being capable of transporting
fluid contacting the first surface to the second surface of the
support in a direction substantially normal to the first surface,
and a collection plate adjacent to the second surface having a
plurality of wells for receiving fluid transported through said
support.
44. A chemical reaction apparatus comprising a. a shaped body
having first and second surfaces; b. said body having an array of
reaction wells therein in fluid communication with each of said
first and second surfaces; and c. porous reaction support in said
reaction wells.
45. The apparatus of claim 44 further comprising d. a collection
plate adjacent the second surface of the shaped body, said
collection plate having a plurality of collection wells in an
cooperative array with the array of said reaction wells.
46. The apparatus of claim 44 wherein said reaction support has a
plurality of reaction sites defined thereupon.
47. The apparatus of claim 44 wherein the reaction support is
porous.
48. The apparatus of claim 44 wherein the porous support comprises
controlled pore glass.
49. The apparatus of claim 44 wherein the porous support comprises
a porated solid.
50. The apparatus of claim 44 wherein the porous support comprises
fibers having a substantially common axis normal to the first
surface.
51. The apparatus of claim 44 wherein the porous support is an
anisotropic membrane.
52. A method for chemical synthesis comprising a. providing a
reaction support having first and second surfaces; b. transporting
to selected sites on the first surface of said reaction support a
plurality of chemical reagents, the chemical nature, amount and
sequence of which is predetermined and under the control of a
control means to effect synthesis of a chemical species; c.
transporting said chemical species through the reaction support to
the second surface thereof; d. providing a collection plate having
a plurality of collection wells adjacent to said second surface
such that the chemical species is recovered in a well or wells.
53. The method of claim 52 further comprising challenging said
chemical species in an assay.
Description
FIELD OF THE INVENTION
[0001] This invention is concerned with novel apparatus, materials
and methodologies for synthesizing chemical compounds, especially
oligomers. Chemical reactions are accomplished on surfaces in a
fashion which is both easy and economical and which is amenable to
the attainment of high yields. Automation of chemical reaction
processes is facilitated in the present invention. Synthesis of
oligomers, especially oligonucleotides and polypeptides, is
especially benefitted by the employment of the present invention. A
wide variety of other chemical reactions can be achieved in
accordance with the present invention, however. The present
invention is also suited for the preparation of chemical libraries
which are useful per se, inter alia for screening purposes and
otherwise.
BACKGROUND OF THE INVENTION
[0002] It has been proposed heretofore to employ apparatus commonly
called an "ink jet" for the delivery of deblocking reagents in
solid state oligomeric reactions. Such proposal, however, is crude,
is limited in scope, and generally requires non-automatable
procedures for its employment. Thus, it has been proposed to use an
"ink jet" apparatus to place droplets of a deblocking reagent, zinc
bromide, upon specified locations of a reaction surface to deblock
a growing oligonucleotide chain to render it amenable to chain
elongation. This has been proposed for use, for example, on a
microscope slide. Following the delivery of deblocking reagent, it
was proposed to manipulate the microscope slide such as by dipping
it into a quantity of further reagent to accomplish a chain
elongation. Further application of the "ink jet" delivery of
chemical reagent was then proposed, however realignment of the
microscope slide would be necessitated by that proposed
methodology. In addition to the cumbersome nature of the prior
proposal and its lack of suitability to full automation, only
relatively small harvests of oligomeric product were anticipated
using the proposed scheme.
[0003] There is a great need for chemical reaction apparatus,
materials and attendant methodologies which permit the automated,
high yield, relatively large scale synthesis of chemical species,
especially oligomers. The apparatus and methodologies provided by
the present invention now enable the use of chemical "jetting"
technology for the practical synthesis of chemical and biochemical
products in high yield and with ease of synthesis. There is also
provided a need for synthetic systems which deliver complex
synthesized products to receiving vessels with ease and in high
yield.
[0004] The apparatus and methods of the present invention also
address a long-felt need by permitting the preparation of libraries
of chemical compounds having predictable diversity among the
functional moieties thereupon.
[0005] This invention also diminishes waste stream pollution
associate with many prior synthetic technologies through the
precision application of reagent moieties in synthetic schemes.
[0006] Precisely arrayed pluralities of defined chemical compounds
are also possible through employment of this invention. Binding,
reaction, degradation, chemical and biological interaction and
other testing protocols may, thus, be performed with unparalleled
convenience through practice of embodiments of this invention.
[0007] The invention minimizes reagent usage such that the impact
of toxic, explosive, radioactive, or expensive sensitive materials
on syntheses is reduced.
SUMMARY OF THE INVENTION
[0008] In accordance with the present invention, there are provided
chemical reaction apparatuses. These apparatuses comprise a
reaction support having a first surface (alternatively called a
reaction surface) thereupon. The reaction surface is considered to
have a plurality of preselected reaction sites upon it. These are
generally in a regular geometric array such as a grid, but may be
in other patterns as well. The apparatus further comprises a first
droplet generator for jetting reactant droplets upon the first
surface. The apparatus further comprises a second droplet generator
for jetting droplets of a second reactant species upon the first
surface. The droplets of each of the first and second droplet
generators are under control of a control means such as a general
or special purpose digital computer, with attendant switches and
actuators, for causing the droplets from each of the droplet
generators to impact upon definable sets of the preselected
reaction sites on the reaction surface of the reaction support. The
impacting of the droplets upon the sets of reaction sites is
preferably in accordance with a preselected pattern or patterns. In
this way, chemical reactants can be directed to particular reaction
sites upon the reaction surface in any desired order so as to
achieve desired chemical reactions at such reaction site without
the need for removing, manipulating, and redeploying the reaction
support.
[0009] In accordance with preferred embodiments, three, four, or
more droplet generators are provided, each connected to one or more
chemical reactant reservoirs, in order to provide diversity among
the chemical reactions possible at the reaction surface. The
control means is preferably programmed to deliver different
sequences of reactants at different reaction sites so as to
synthesize differing chemical compounds at such sites. Preferably,
the methods of the invention are performed iteratively such that
relative complex molecules, such as oligomers, can be
synthesized.
[0010] Synthesis has been found to be benefitted by employment of
porous reaction supports, especially those having the capability of
transporting fluid from the first (or reaction) surface to a second
surface thereof. Improvement of yield through exploitation of the
internal pore volume of the support and amenability for improved
product harvest is also now possible.
[0011] Once the different chemical moieties have been synthesized
at the plurality of reaction sites, they may be employed in a
variety of highly useful ways. Thus, the different chemical
moieties may be exposed to test solutions, such as bodily fluids of
a test animal, to diagnose or ascertain bodily states in such
animal. A wide variety of assays may thus be formulated using the
apparatus and methodologies of the present invention. In accordance
with other utilities, the chemical species thus formed may be used
as probes in biological systems or as primers or substrates for
polymerase chain reaction amplification or the like. Hybridization
studies may also be conducted using apparatus and methods in
accordance with the present invention, where the products of the
methodologies are oligonucleotides, polypeptides or other
hybridizable species. All of the foregoing utilities are known per
se to those skilled in the art.
[0012] In accordance with other preferred embodiments of the
invention, reaction apparatuses are provided where one or more
droplet generators, each in fluid communication with pluralities of
reactant reservoirs, are employed to perform synthesis. Control
means are caused to effect the operation of valving moieties to
select reactants in appropriate orders to achieve the desired
reactions at individual reaction sites.
[0013] In accordance with the invention, methods for synthesizing
chemical species comprise identifying a plurality of reaction sites
upon a reaction surface and jetting upon a first set of such
reaction sites, droplets of fluid comprising a first chemical
reactant. The methods also comprise jetting upon a second set of
such reaction sites, droplets of fluid comprising a second chemical
reactant species. It will be appreciated that, through practice of
the present invention, the first jetted reactant species and the
second jetted reactant species may be jetted to the same or
different reaction sites on the reaction surface. Control means for
effectuating the jetting of such fluids upon the selected sets of
reaction sites is also invoked to attain the ends of the invention.
Iterative jetting of reactant species permits the elaboration of
wide varieties of chemical moieties as the reaction sites and
permits the generation of libraries of diverse species.
[0014] It is also possible to employ the present invention in a
hybrid fashion by combining it with other chemical reaction
schemes. Thus, for example, a support for reaction may be coated
with an initial reactant species and then reacted through the
jetting of chemical reactants in preselected locations on the
surface. Subsequent reaction at the selected locations on the
surface in accordance with the invention may ensue whereby a
plurality of reactant species are delivered to such locations.
Thus, by pretreating the support where the reaction is to occur
with a first reactant moiety, certain economies of scale may be
attained. The employment of chemical jetting technology to deliver
often expensive reagents for at least some of the subsequent steps,
is, however, preferred.
[0015] At any reaction site, either or both of the first and second
reactants may be delivered thereto. Thus, at the reaction site,
either the first reactant, the second reactant, or the first
reactant followed by the second reactant can be so delivered. The
ensuing chemical reactions will depend upon the identity and order
of chemical reactants delivered to any particular reactant
site.
[0016] This is also true where an initial reaction species is
applied to the support prior to the delivery of jetted reactants.
In such case, at reaction site R.sub.n, one of four situations will
prevail after jetting of two reactants has occurred. First, it may
be that neither the first nor the second reactant are directed to a
given reaction site. In this case, only the initial reaction
species will have been delivered to this site. In two additional
cases, either the first reactant or the second reactant, but not
both is jetted to site. In this case, two different combinations of
two reagents are delivered to the site. Finally, it may be seen
that both the first and the second reactant can be jetted to the
site whereupon three reaction species will have been so delivered.
Persons of ordinary skill in the art will readily appreciate how a
complex series of reactants can be delivered to particular sites on
a reaction surface to achieve complex and varied chemical
reactions. The present invention is suited to the delivery of a
large variety of chemical species including reagents,
intermediates, blocking and deblocking agents, monomers, dimers,
oligomers, solvents, washing agents, cleaving agents and the
like.
[0017] In accordance with preferred embodiments, the methodologies
of the present invention are performed iteratively. Thus, three,
four, five, and more reactants can be delivered to a reaction
surface in varying combinations at different reaction sites on the
surface. The number of different chemical moieties which may, thus,
be elaborated is extraordinarily numerous and varied. It is, thus,
possible to generate, isolate and recover a wide variety of
different chemical species in a highly automated fashion on small
reaction surfaces. The present invention also provides reaction
assemblies wherein a reaction support surmounts a collection plate,
preferably one having a plurality of collection is wells. Transport
of chemical species through the reaction support enables their
collection in the collection wells where they may be easily
recovered, analyzed, tested, hybridized, screened, assayed and
otherwise utilized. The efficient deposition of product species
into such collection vessels is a particularly advantageous aspect
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1 and 2 are generalized drawings of droplet generating
apparatus useful in the invention depicting salient features.
[0019] FIG. 3 depicts a single head droplet generating apparatus,
in schematic, wherein a plurality of reagent reservoirs is in fluid
communication with the droplet generating head. Delivery of
chemical species to a reaction surface having a plurality of
reaction sites is depicted.
[0020] FIG. 4 is a chemical jetting apparatus in schematic, wherein
a plurality of chemical droplet generating heads is employed to
direct different chemical reagents to sites on a reaction
surface.
[0021] FIG. 5 shows the transitting of a chemical droplet generator
over a reaction surface to deposit droplets of reagent at
preselected sites on the surface.
[0022] FIG. 6 shows a reaction support surmounting a collection
plate and collection wells in the collection plate. The transport
of liquid species from the reaction surface to a second surface of
the reaction support and its collection into a collection well is
shown.
[0023] FIG. 7A depicts one embodiment of the invention where
reaction wells are formed in a shaped body for holding a reaction
support. The wells funnel liquid species transitting the reaction
support such that the same may be collected by collection wells of
collection plate.
[0024] FIG. 7B is a plan view of a portion of a shaped body
containing reaction wells and reaction support, the whole suited
for surmounting a collection plate.
[0025] FIG. 8 is a depiction of a preferred apparatus having a
transitting droplet generating head together with preferred
reactive supports within reaction wells of a shaped body.
Collection wells in a collection plate are shown.
[0026] FIG. 8A shows reaction wells in a shaped body while
[0027] FIG. 8B shows a reaction support within a well.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0028] The present invention provides significant improvements in
chemical synthesis and recovery technology. The apparatuses and
methods of the present invention are advantageously employed in the
preparation of oligomers, especially oligonucleotides and
polypeptides, and in the preparation of libraries of compositions
having diverse chemical structure. They are also useful for the
synthesis of a wide variety of non-oligomeric molecules, especially
those requiring hazardous or expensive materials. In accordance
with the present invention, it has now been found to be highly
desirable to effect chemical reactions upon a reaction support
through the sequential jetting of chemical reagent species upon
predefined sets of reaction sites on such surfaces. The problems
associated with mounting and dismounting of reaction surfaces from
the chemical jetting apparatus is avoided or minimized through
employment of embodiments of the present invention.
[0029] FIG. 1 is a depiction of a chemical jetting apparatus which
may be used with embodiments of the present invention. It will be
appreciated that chemical jetting apparatus suitable for use in the
present invention may be viewed as being essentially similar to
apparatus used in "ink jet" printing. Ink jet printers are known
per se and have achieved a separate status in the patent and other
literature. For example, class 346 of the patent classification of
the United States Patent and Trademark Office contains a large
number of patents directed to ink jet technology, to methodologies
for employment of ink jets, and to apparatus for use therein. All
may be useful in the practice of this invention. While certain
modifications of basic ink jets are preferred for use in accordance
with the present invention--chiefly to render the same inert with
respect to the chemical reactants employed--the basic mechanical
and materials considerations which attend the provision of ink jet
apparatus apply to the manufacture of chemical jetting apparatus as
well.
[0030] Referring now to FIG. 1, chemical jetting devices are
conventionally actuated through piezoelectric devices. A source of
chemical reagent, 10 is provided, conventionally through a pumping
means, 12 to a chamber, 14 in mechanical communication with a
piezoelectric material, 16. The chamber 14, is provided with one or
more orifices, 18 through which droplets of reagent, 28 may be
expressed through the controlled pumping action of the
piezoelectric material. The piezoelectric device is controlled by a
driver, 26 which, in turn, is controlled by controller, 32. In some
apparatuses, droplets, 28 are provided with an electric charge in a
chamber, 24 upon their emergence from the orifice and are
accelerated in one or more planes in an acceleration chamber, 26
under the influence of an applied voltage controlled by the
controller, 32. It will be appreciated that the overall effect of
the foregoing arrangement is to provide a series of droplets at
spaced intervals traveling in predetermined vectors, as showing
established by the controller in response to operator programming.
It is well known to direct individual droplets of liquid, 28 to
various selected locations on a reaction surface, 40 which, in
embodiments of the present invention, is a reaction surface
whereupon chemical reactions take place. Droplets, which are not to
be directed to particular locations on the surface are, in
accordance with this embodiment, directed to a "gutter", 42 for
environmentally approved disposal or recycling.
[0031] The foregoing method of providing droplets and directing the
same to particular locations on a reaction surface, with excess
droplets being diverted to a gutter is known as a "continuous jet"
type of device. It will be appreciated that the elements of reagent
supply, pump, piezoelectric device chambers, orifices, electrodes,
and the like--in short, those elements which are required to
produce droplets of chemical reagent and to direct them in
preselected directions--are conventionally and are conveniently
denominated a "jetting head" or, preferably, a "droplet generator."
These two terms are used interchangeably in the present
application. While droplet generators may not conventionally be
considered to include chemical reservoirs, plumbing, controllers,
connectors and the like, it will be understood that all such
apparatus as may be required to effectuate the delivery of reagent
droplets in accordance with the present invention are included as
needed and will not necessarily be separately recited
hereinafter.
[0032] Another form of chemical jetting is conventionally
denominated a "droplet on demand" device. Such a device is depicted
in FIG. 2. It will be appreciated that rather than have a uniform
stream of chemical reagent droplets provided by the apparatus,
unneeded droplets being directed to a gutter, in droplet on demand
systems, droplets are provided only when actually required for
distribution to the reaction surface, 40. While it is convenient to
use electrostatic directing means under control of a controller to
direct these drops, it is also possible, and in many cases
preferred, to physically move the source of the droplets--the
droplet generator--with respect to the reaction surface or, vice
versa, the reaction surface with respect to the source of the
droplets, thus to deliver droplets to particular locations on the
reaction surface in an imagewise, preselected fashion. Indeed,
combinations of both directing techniques may also be employed. In
some embodiments, it is possible to rely upon the impetus provided
by the piezoelectric material, 16 upon the reagent, forcing the
same through the orifice with sufficient kinetic energy to impact
the reaction surface without additional acceleration. In any event,
the piezoelectric driver, 22 and whatever motion of the droplet
generator or reaction surface 40, may be required is under control
of a controller, 32.
[0033] Irrespective of which form of chemical reagent droplet
generator is selected, it will be appreciated that droplets of
chemical reagent will be provided at a reaction surface in a
fashion which is preselected as to location. Thus, the reagents may
be applied in an imagewise fashion to specific sites on the
reaction surface. In accordance with certain preferred embodiments
of this invention, reagents are applied at sets of such sites such
as in an array defined upon the reaction surface. Thus, a matrix of
sites is conveniently defined upon the reaction surface and one or
more reagents jetted to sets or subsets of such sites in amounts
and in orders of deposition consistent with the chemical synthesis
desired at each site. Any of the chemical jetting apparatus
described above may be used for jetting the reagents to these sites
and, indeed, any of the apparatus, methods, and materials which
have been known heretofore for use in conjunction with ink jet
printing, which are capable of jetting liquids to predefined
locations on a surface, may also be used or easily modified for use
in accordance with the present invention to deposit reagents at
preselected sites upon a reaction surface. It will thus be
appreciated that the present invention is not limited to any
particular droplet generator or control means so long as the
droplet generator and associated control means are effective for
delivering the reagent to preselected locations on the reaction
surface.
[0034] It is preferred to provide the chemical jetting head in
materials that are inert with respect to the chemical reagents
being delivered. Thus, it is preferred that the apparatus be
constructed of materials such as glass, ceramic, porcelain, inert
plastic, inert or passivated metal, and other material which is
consisted with the reagent to be jetted by the particular jetting
head and associated equipment. Persons of ordinary skill in the art
will have no difficulty in determining appropriate materials for
the construction of apparatus useful for the practice of the
present invention upon consideration of the chemical and/or
corrosive nature of the chemical reagents to be dispensed by the
apparatus. It is convenient to employ polytetrafluoroethylene
(PTFE) and other relatively inert polymers for the storage,
transmission, and jetting of chemical reagents in accordance with
this invention. Stainless steel is another preferred material,
especially for the jetting head itself, while glass finds great
utility for the storage of chemical reagents.
[0035] It will be appreciated that many piezoelectric materials are
relatively chemically inert. Accordingly, selection of an
appropriate piezoelectric material for inclusion in the chemical
jetting apparatuses of this invention will be a matter of routine
as well. Polyvinylidine fluoride (PVDF) is one piezoelectric
material which is relatively inert to most chemical species and may
be used in the practice of this invention. Certain other
piezoelectric materials are metallic and are inert with respect to
many reagent and may, accordingly, also be used. All such materials
are contemplated hereby.
[0036] Prior attempts to employ droplet delivery for synthesis of
chemical species at a reaction surface have employed single
chemical delivery heads delivering a single reagent adapted for the
deprotection of chemical moieties found on the reaction surface. It
will be appreciated that this is a highly inefficient technique and
one in which the attainment of high density of chemical synthesis
on the reaction surface cannot easily be achieved. This is so,
inter alia, because the reaction surface must generally be
submitted to other chemical treatments with reagents other than
those provided by the droplet delivery device. It is generally
necessary to dismount the reaction surface from the droplet
delivery apparatus for these further chemical treatments.
Realignment is difficult and time consuming, thus efficiency is
lost.
[0037] FIG. 3 is directed to apparatus which provides great
efficacy in the synthesis of chemical species on reaction surfaces.
A droplet generating head, 50, which may be of the continuous
droplet, droplet on demand, or other form, is provided in fluid
communication with a plurality of chemical reagent reservoirs, 52.
Valving means, 54 is also provided and this means is preferably
electromechanical actuatable under control of a controller, 56. In
accordance with preferred embodiments, the chemical droplet
generating head, 50 is traversable in one or more directions
through electromechanical means as is known for use in connection
with ink jet printing heads. Such traversing means, 58, which is
optional but preferred, permits the deposition of droplets of
reagent at reactions sites, 42 on reaction surface, 40. Traversing
through x or xy planes may be performed as convenient.
[0038] As will be appreciated, use of the present invention will
permit the automation of chemical syntheses since the delivery of
reagents in a sequential fashion to any particular reaction site on
a reaction surface can be controlled through controllers such as
general purpose digital computers or special purpose digital
computers or processors. While it is not essential that the steps
of the reaction sequence be controlled electromechanically by
control means, this is generally preferred. Thus, while the valving
means traversing means, etc., 54 can be actuated manually and still
fall within the spirit of the present invention, it is greatly
preferred that control means e.g. a computer, actuate the valves
electromechanically and traverse the droplet generator in
accordance with programmed demands for particular reagents. A wide
variety of reagent storage, valving, plumbing and other ancillary
apparatus may be employed within the spirit of this invention so
long as the same are generally inert with respect to the reagents
in contact with them. Similarly, the apparatus may be encased in a
special atmosphere, may be operated with exclusion of light or
moisture, and may be oriented in any convenient direction as may be
preferred for any particular synthesis. All such modifications are
contemplated hereby.
[0039] It will be appreciated that it may be necessary to rinse or
otherwise purge the chemical jet reaction system when different
chemical reagents are selected for use. Thus, it is preferred to
provide appropriate solvent means effective to remove one reagent
from the system prior to the provision of a second reagent thereto.
Persons of ordinary skill in the art will have no difficulty
selecting appropriate solvent means and washing steps to effect
this goal. Unwanted materials may be jetted to a dump or gutter for
recycling or disposal.
[0040] As shown in FIG. 4, it is possible to avoid the need for all
or some of the washing/purging steps which are generally required
when pluralities of reagents are jetted from a single head. This
may be accomplished in accordance with a further embodiment of the
present invention through the employment of a plurality of droplet
generators which are each capable of delivering droplets of reagent
to reaction sites, 42 on a reaction surface, 40. Thus, a plurality
of droplet generators, 50 are provided in fluid communication with
reservoirs of chemical reagents, 52 through the mediation of
valving means, 54. It is preferred that the droplet generators, 50
and valving means, 54 be under control of a controller, 56 which is
preferably either a general purpose digital computer, special
purpose digital computer, or specialized controller.
[0041] The droplet generators, 50 may preferably be arranged in
such a fashion that they can traverse through a space while
distributing reagent droplets, however this is not obligatory. It
is convenient to employ four jetting heads although other
pluralities may be selected. Four is preferred since four head ink
jetting systems for color printing purposes are known as are
control means therefore.
[0042] In accordance with certain preferred embodiments of this
invention, composite droplet generators may be employed. A
composite droplet generator is one which incorporates a plurality
of orifices within one physical structure. Thus, the elements
needed to effect a jetting of reagent droplets may be integrated in
such a fashion that a plurality of droplet orifices share one or
more elements such as reagent reservoirs, pumps, piezoelectric
elements and the like. All such modifications are within the spirit
of the present invention.
[0043] FIG. 5 depicts a preferred aspect of the droplet generator
relationship to the reaction support. One or more droplet
generators, 50 may be caused to be traversable with respect to the
surface of the reaction support, 40. Such droplet generators may
traverse along one axis or, preferably along two axes in the nature
of a plotting device. In such a case, it is not generally necessary
to employ electrostatic direction of droplets to reaction sites,
rather the transverse movement of the droplet generator can
precisely place reagent droplets as required at particular sites,
42. It is preferred that the droplet generators be oriented to
direct droplets in a downward direction; the reaction support is
preferably oriented horizontally. This permits gravity to assist in
the penetration of reagent droplets into the pore volume of
preferred reaction surfaces and to aid in transport of liquid
through such supports as will be described more completely infra in
connection with other preferred embodiments.
[0044] In accordance with other embodiments of the present
invention, reaction supports are provided which are especially
suitable to the practice of the methods of this invention. It will
be appreciated that the techniques as described in the present
invention serve to deliver droplets of chemical reagents to
localized reaction sites on the reaction surface. It has now been
found, however, that great advantages may attained by employment of
particular reaction substrates having significant internal surface
area as reflected by pore volume. Accordingly, it is one aspect of
the present invention to employ reaction supports for chemical
jetting systems, which reaction supports have significant pore
volumes. Such reaction supports are best described by what they do.
Their character of porosity leads to their ability to cause
droplets of reagent to enter into the body of the substrate, within
pores or voids, such that relative large amounts of fluid can be so
accommodated. While objects of this invention can be accomplished
with reaction supports having relatively little porosity, the
larger effective surface areas available from relatively porous
substrates is desirable. By employing porous reaction supports in
accordance with the present invention, it may be seen that
increased volumes of reagent may be delivered to reaction sites on
reaction surfaces since the reagent liquid will be absorbed into
the pore volume of the reaction support through capillary action,
gravity, and otherwise.
[0045] It is preferred to employ reaction supports having a
porosity such that liquid is capable of passing from the surface
upon which reagent impinges to the opposite surface, e.g. the
support is at least partly permeable. Examples of such permeable
reaction supports include permeable and semipermeable membranes,
especially isotropic and anisotropic, polymeric membranes, and
control pore glass. Porated membranes and glasses, e.g. having
surface which have been subjected to ion nuclear bombardment to
effect holes therethrough, and various ceramics are also useful.
Such materials are best described by what they do rather than by
what they are. Thus, preferred reaction supports are those having a
significant pore volume and which are not inconsistent with
reaction chemistries to be practiced upon them. Accordingly,
employment of a reaction support which reacts with or destroys any
of the chemical species which are intended for contact with it
would be contraindicated for that particular chemical reaction
sequence. It is believed that persons of ordinary skill in the art
will have no difficulty in determining appropriate reaction
supports in accordance with the foregoing principles.
[0046] Exemplary reaction supports for use in accordance with the
present invention include CPG (controlled pore glass) available
from various distributors including CPG Inc./Millipore Corp.; RAPP
copolymer, a highly crosslinked polystrene, sold as TentaGel or a
like product HLP (high loaded polystrene) sold by ABI Corp.; Primer
Support, a highly crosslinked polystrene, sold by Pharmacia;
POROS-OS polystrene sold by PerSeptive, MPG (a magnetic pore glass)
sold by CPG Inc.; Nucleic Acid Membrane Support sold by Millipore.
Other useful supports include membranes sold by the Amicon division
of W. R. Grace, Inc., and those sold by Gelman Sciences. Polyether
sulfone, polysulfone, PVDF, PTFE, PVC, polypropylene and nylon
supports may be employed for various applications, as may many
other materials.
[0047] Other membrane supports include membranes as described or
referenced in U.S. Pat. No. 4,923,901 assigned to Millipore Corp.;
various supports as described in patent application WO 94/05394 and
references cited therein; and various supports as described in
patent application WO 90/02749 including activated polystrene layer
on a polyethylene membrane. Further Zeolites, cellulose, cottons,
other polystyrenes that can optionally be crosslinked,
polyacrylamide, which may be optionally crosslinked, latexes,
dimethylacrylamide optionally crosslinked with
N,N'-bis-acryloylethylenediamine can also be mentioned.
[0048] For certain preferred embodiments of the invention, it is
preferred that the reaction support possess porosity in such a
fashion that indiscriminate blotting or wicking is avoided.
Indiscriminate blotting or wicking in this context is defined to
mean the wicking away of liquid from the point of impingement of a
chemical reagent upon a reaction surface in a direction in other
than the direction normal to and through the surface. Thus, it is
desired that the reaction supports transmit liquid impinging upon
their surface through the body of the support and to the opposite
side rather than across the surface or laterally into the body of
the support. While, inevitably, some wicking of reagent will occur,
it is preferred that the fluid be transmitted predominantly in the
normal direction. This concept of wicking is necessarily
qualitative, but is believed to be understood by persons of
ordinary skill in the art.
[0049] Supports which exhibit a diminished tendency towards
laterally wicking or blotting and which transmit fluid normal to
the surface and through the support, are greatly desired for a
number of reasons. First, reliable washing of the support upon
which reagent is absorbed can be attained more easily when lateral
wicking is avoided. Moreover, when lateral wicking is minimized,
reaction sites can be defined more closely together on a surface of
a reaction support than if lateral wicking is significant. The
selection of reaction supports for use in conjunction with the
present invention having diminished tendencies towards lateral
wicking while maintaining high internal pore volume permits greater
efficiencies in performance of the methods of the present
invention. Thus, greater synthetic speed and yield may be evidenced
since it is easier to wash the surfaces contacted by the chemical
reagents while maintaining a high concentration of reaction sites
for reaction.
[0050] Capillary glass has been found to be particularly useful for
the practice of the present invention. Capillary glass is a
material which is known per se and comprises substantially parallel
glass filaments oriented in a direction substantially normal to the
surface. Various capillary glasses are commercially available such
as those that are used for capillary gel electrophoresis. These
include both internally coated and uncoated supports available in
various internal diameters including 50, 75 and 100.mu. that
typically have a 365.mu. outside diameter. One such support is
available for Polymicrotechnologies, Inc. Such materials have
effective large internal porosities compared to surface area and
have little tendency toward lateral wicking. "Bundles" of such
capillary glass filaments are be assembled for use as support of
the processes of the invention.
[0051] A wide variety of other materials may also be used in
accordance with the present invention. The 48 well and the 96 well
versions of GibcoBRL's "The Convertible Filtration Manifold System"
sold by Life Technologies, Gaithersburg, Md. can be used as
separators for reaction area on an appropriate planar membrane such
as the above mentioned Nucleic Acid Membrane Support sold by
Millipore, Corp. In essence, the filtration manifold defines
reaction areas that can be filtered under vacuum to facilitate
reagent and solvent removal. The jetting heads of the invention can
be used to create a single polymeric species within each of the
areas defined by the top plates of these filtration devices or they
can be used to create multiple polymeric species within each area.
Thus in one embodiment of the invention, such a manifold, as for
instance the 96 well manifold, will be used in conjunction with the
jetting head of the invention to create 96 individual polymeric
species whereas in a further embodiment, the jetting head will
deposit, as for instance, as 10 by 10 matrix of individual
polymeric species in each well of the manifold to create a total of
9,600 individual polymeric species over the 96 wells of the
manifold.
[0052] Further preferred support structures of the invention
utilize an array of openings in a matrix support material. Such
structures can be formed utilizing Helix HD-864-PS-50 or
HD-864-PC-50 864 Well High Density Microwell plates (having 864
individual wells, each of a 20 .mu.l volume, located within the
foot print size of a normal 96 well microtiter plate available from
Helix, San Diego, Calif.) loaded with an appropriate reaction
support medium. Each well so modified serves as an individual
reaction vessel that can be charged via the jetting device with
appropriate reagents, wash solvents and the like.
[0053] These same Helix high density microwell plates can be
modified by removing the totality of the bottom surface of the
plates by machining. This creates a structure having a plurality of
parallel capillary tubes suitable of loading with an appropriate
reaction support medium. Such modified plates can be used with an
appropriate commercial vacuum apparatus such as the above described
GibcoBRL Filtration Manifold System from Life Technologies,
Gaithersburg, Md. The modified high density microtiter plates are
supported on a gasket having a hole pattern that matches the hold
pattern of the modified plate. In one embodiment of the invention,
strips of the above-noted Nucleic Acid Membrane Support sold by
Millipore are rolled, in a manner like a cigar, and are inserted in
the so formed capillary tubes. Upon completion of synthesis of the
polymeric compounds, the strips can be remove with the polymeric
materials still attached and used as such for biological testing.
In a further embodiment, the polymeric materials are released from
the support membranes and used in solution. Alternately such high
density microtiter plates can be modified by the drilling an
opening, as for instance a 0.1 to 0.5 mm opening, in the bottom of
the individual wells. A porous plug is then located in the bottom
of each well and the well loaded with an appropriate support
medium.
[0054] In a further preferred support, glass whiskers can be
repeated sonicated to create porosity therein. Such porous whiskers
are then aligned axially and are imbedded in a spaced array in an
inert matrix material, as for instance polyethylene, to form a
filamentous structure having a plurality of parallel aligned porous
glass rods. These porous glass rods can then be derivitized with
linkers. Usable as a linker is one of the many linkers known for
use with CPG and other glass supports. The linkers, in turn, are
used to attach the first monomer unit of a oligomeric compound in
the same manner as is practice with common CPG support
materials.
[0055] Further preferred support materials are anisotropic
polymeric membranes. These are known per se and are widely
available such as from the Amicon Division of W. R. Grace. Such
anisotropic membranes have a first surface with relatively "tight"
pores which communicate with increasingly larger pores in the
membrane. At the distal surface of the membrane, the pores are
quite large and provide no hydraulic impedance of fluid movement.
Application of chemical reagent to the tight surface of such
membranes, called the skin, with concomitant migration to the large
pore volumes just below the surface is highly advantageous in the
practice of this invention. Such membranes are available in a large
number of chemical forms including polyether sulfones,
polysulfones, polyvinylidene fluorides, nylons, PTFEs, acrylics and
many others. Such materials generally exhibit a desirably small
lateral wicking and are, accordingly, preferred for use in some
embodiments of the invention.
[0056] Other preferred membrane supports utilizes a polyvinylidene
difluoride membrane as described above that is a treated with
diaminopropane in DMF e.g. utilizing the procedures of Example 1 of
U.S. Pat. No. 4,923,901 to form a polymeric membrane suitable for
use to form polynucleotide type oligomers. A further particular
preferred membrane, particularly useful for synthesis of peptide
and peptide like (peptoid, polycarbamate and the like) polymeric
compound libraries, utilizes a polypropylene membrane that is
derivitized with hydroxypropylacrylate by coating the polypropylene
membrane with crosslinked polyhydroxylproplyacrylate. This membrane
was described by Daniels, et. al, in poster # T81 at the Protein
Society meeting, San Diego, Calif., 1989. Particularly useful for
synthesis of oligonucleotides and the like is a membrane support
described by Fitzpatrick, et. al., presented in a paper entitled
"Membrane Supports for DNA Synthesis", at the 1993 "Innovations and
Perspectives in Solid-Phase Synthesis" conference at the University
of Oxford. This support utilizes a PTFE (polytetrafluoroethylene)
membrane that is coated with a terpolymer coating consisting of
methylene-bis-acrylamide, N,N-dimethylacrylamide and
aminopropylmethacrylamide. Nitrophenylsuccinates of appropriate
first monomeric units are reacted with the primary amine groups of
the coating. The density of the growing oligomer is controlled by
the spacing of the aminopropylmethacrylamide monomer of the
coating. Steric hinderance can be prevented by infrequently
incorporating this monomer in the coating.
[0057] It will be appreciated that syntheses similar in many
chemical respects to existing "solid state" synthesis are preferred
for use in connection with some embodiments of this invention. In
such cases attachment of an initial reactant, chemical substrate or
moiety to a solid, here the reaction surface of the reaction
support, is preferred. The materials selected for the reaction
support should preferably be capable of functionalization by such
an initial chemical moiety. A large variety of appropriate
materials are known in this context such as glasses, ceramics, many
polymers and other species. Following synthesis, it is generally
the case that the synthesized chemical species are conventionally
cleaved from the solid and recovered through washing. Selection of
reaction supports stable to such practices is greatly
preferred.
[0058] It is also preferred in some embodiments to effect partial
synthesis of chemical species. Thus, for example, oligomers can be
elaborated through iterative solid phase chain elongation reactions
and then cleaved and recovered. Post processing, such as to remove
protecting groups, may then be performed as desired.
[0059] A matrix of reaction sites is preferably defined on the
surface of the reaction support such sites being intended for the
deposition of droplets of chemical reagent. Such sites are not
generally marked visibly, but rather are defined geometrically and
addressably by the control means for deposition of reagents.
Pluralities of sites may be used for the same reaction series or
different reactions may occur at each in accordance with the
designs of the operator.
[0060] In accordance with other embodiments of the invention,
chemical reaction apparatuses are provided that are particularly
adapted to the present methods. A reaction support having a
reaction surface for the desired chemical reactions is provided. A
collection plate is also provided, such plate being adapted for
lying adjacent to the reaction support at the surface distal from
the reaction surface. The collection plate is also preferably
provided in such a fashion as to have a plurality of collection
wells which are arrayed in a matrix isomorphic with the matrix of
reaction sites extant upon the reaction surface of the reaction
support. The assembly is such that when a fluid is placed upon the
reaction surface of the reaction support at a reaction site
thereof, it will pass through the reaction support, arrive at the
second surface distal from the first surface, and collect in a well
of the collection plate corresponding to the reaction site. It is
apparent that the array of reaction sites and collection wells on
the collection plate may be structured in any geometric pattern as
may be convenient. A rectangular array is convenient and a
conventional ninety-six well plate can be used to good effect.
[0061] It is also possible and, in some cases preferred, to
associate reaction sites with collection wells in a fashion other
than one-to-one. For example, a plurality of reaction sites may be
identified which are associated with a single collection well on
the collection plate. For example, a matrix of reaction sites on
the surface of the reaction support may lead to a common collection
well. This may be done for a number of reasons including the desire
to improve the volume of chemicals processed. A further, and in
some cases preferred utility for such an arrangement is to provide
libraries of chemical species collected within particular
collection wells. Accordingly, a matrix of reaction sites, e.g. a
10.times.10 matrix, can be associated with a single collection well
and the reagents jetted to the 10.times.10 matrix controlled both
as to identity and timing in such a manner as to provide one
hundred different chemical moieties to be collected in a single
collection well associated with the 10.times.10 matrix. Since the
sequence and identity of chemical reagents jetted to the particular
reaction sites is known in all cases, the same having been
determined through the programming of the control means, the
composition of the chemical library resident in the particular
collection well is known with certainty. It is thus possible, in
the example given, to assay one hundred chemical species in a
particular chemical, biological, or other assay, knowing with
certainty the identity of all one hundred chemical species whose
performance is to be monitored in the assay. Chemical libraries are
inherently useful and valuable. The same are in great commercial
demand and should be viewed as a commercially useful article per
se.
[0062] The foregoing library procedure is of obvious benefit in the
screening of new drugs and diagnostics. It is also of great benefit
in the identification of chemicals which have agricultural,
therapeutical, medicinal, industrial, or other practical uses.
Indeed, the present invention provides an unambiguous, rapid, and
powerful method for the generation of such libraries without the
ambiguity that random synthesis provides. As such, it represents a
great advance over prior methods for library creation.
[0063] FIG. 6 shows one example of a preferred arrangement.
Reaction support, 40 surmounts collection plate 44 having
collection wells 46. The reaction surface, 43 of reaction support,
40 is impinged by reagent droplets, 20 at one predefined reaction
site, 42 on the reaction surface, 43 of the reaction support, 40.
Internal porosity, 47 is shown although the exact geometry of such
pores will rarely be known. Such pores or voids may be those from
nuclear bombardment from anisotropic synthesis, or as otherwise
known or as described herein. In any event, such pores preferably
communicate with the second, distal surface, 45 of the reaction
support, 40 preferably without undue lateral wicking, such that
liquid impinging a particular site, 42 on the reaction surface, 43
of the reaction support will be transported to the collection well,
46 of collection plate, 44 which is isomorphic with such reaction
site. Such liquid is indicated, 49.
[0064] FIGS. 7A and 7B are cross section and plan views of
additional preferred embodiments of this invention. In this
embodiment, the reaction support is present in subportions located
within reaction wells, 62 on a shaped body, 60. The shaped body is
any solid inert with respect to the chemical reactions to take
place and capable of appropriate shaping, sterilizing, cleaning and
the like as may be desired. Polymer, e.g. nylon or PTFE, glass,
ceramic or metal are exemplary materials. The reaction wells, 62
are preferably molded or machined into a surface of the shaped
body, 60 in any convenient manner, such as by milling. The reaction
wells are preferably in fluid communication with a second surface
of the shaped body, and are optionally but preferably adapted to
funnel liquid from a larger portion of the well to a smaller or
funnel portion, 64. The funnel portions are preferably located to
cooperate with collection wells, 46 of a collection plate 44
adapted to lie adjacent the shaped body distal from the reaction
wells. Chemical reactions are performed on the reaction support, 40
in each well which can be easily rinsed through the well funnel
portions. Following completion of synthesis, the completed chemical
species can be directed through the funnel portions for collection
in the collection wells. While the apparatus thus described is
useful with chemical jetting techniques as set forth herein, other
synthetic techniques may also be used therewith.
[0065] FIG. 7B shows a plan view of a portion of a shaped body, 60
with one complete well, 62. An array of reaction sites, 42 are
shown. The reaction support may be any material herein described
or, indeed, any other as may be benefitted from the reaction well
and funnel aspects of the invention.
[0066] FIG. 8 is a schematic depiction of one system in accordance
with this invention. Chemical droplet generating head, 50, in this
depiction attached to a plurality of reagent sources, 52, is
arranged to deposit chemical species upon reaction surface, 43, of
reaction supports, 40 disposed in reaction wells in first surface,
63 of shaped body, 60. In accordance with this embodiment,
pluralities of matrices of reaction sites, here 10.times.10 sites,
shown in FIG. 8A, are defined on the reaction surface of the
reaction supports, 40 such that each set of one hundred reaction
sites generally communicates with one area on the second, distal
surface, 65 of shaped body 60. A collection plate, 44 having
collection wells, 46 is also provided in an isomorphic fashion such
that, in this example, for each 10.times.10 matrix of reaction
sites leading to a generally common area on the distal surface, 65
of the shaped body, 60, one well is provided into which liquid from
the reaction sites can flow in a common fashion. FIG. 8B depicts
reaction support, 40 in a well of shaped body, 60.
[0067] Throughput of liquid from the reaction sites to the second
surface of the reaction support may optionally be encouraged in a
number of ways. In accordance with preferred embodiments, a partial
vacuum is applied in such a fashion that liquid is extracted from
the reaction support. Subsequent washing with an appropriate
solvent can ensure complete transfer of material from the reaction
support to the collection plate. Alternatively, application of
solvent to the reaction sites on the reaction surface of the
reaction support will have a washing effect causing translocation
of chemical species to the collection wells.
[0068] Once the products of the chemical reaction have been
transferred to the collection wells, they may be used in further
synthesis, may be used per se, may be subjected to one or more
assays, or may otherwise be employed in many ways known to persons
of ordinary skill in the art.
[0069] In accordance with certain preferred embodiments, it is
preferred to adapt the apparatus of the present invention so as to
direct liquid from the reaction support to the collection wells in
as highly efficient a fashion as possible. Thus, the reaction
support may be formed of a composite or may be adapted in various
ways to encourage this goal. It is within the spirit of the present
invention to provide composite supports for elaboration of chemical
species. Thus, a shaped body containing pluralities of wells may be
elaborated, such wells being shaped so as to effectively funnel
liquid from a first surface to a second surface thereof as
described above. This arrangement is adapted either to be
surmounted by a reaction support where chemical reactions are to
take place or to contain in reaction wells, reaction support for
such reactions. In any event, the elaboration of appropriately
shaped structures facilitates the cooperation of reaction support
with collection wells in a collection plate to accomplish preferred
goals of the present invention. It will be appreciated that large
numbers of variations are possible in this context and all are
within the spirit of this invention.
[0070] It will be appreciated that these aspects of the invention
are not limited by the particular type of chemical synthesis that
may be employed and while both single head jetting systems as well
as plural head jetting systems are contemplated hereby other
reagent application techniques are also comprehended by this
invention.
[0071] It will also be understood that a wide variety of
atmospheres may be imposed upon the synthetic methodologies of the
present invention to permit the elaboration of chemically
sensitive, corrosive or reactive moieties. Thus, it is often
preferred to employ an inert atmosphere, such as argon, and to
exclude moisture.
[0072] Determination of preferred conditions of time and
temperature as well as reagent concentration and catalysis is
within the skill of the routineer in the synthetic art. For the
synthesis of oligomers, conditions generally similar to those
employed in automated synthesis equipment known heretofore provides
a useful point of departure from which selection of appropriate
conditions may easily be determined for any particular apparatus or
method in accordance with this invention.
[0073] In accordance with some embodiments it is preferred to
orient the synthetic systems such that the effects of gravity may
be invoked to transmit liquid from the reaction sites through the
reaction support to the second surface of the reaction support for
collection in collection wells. It will also be appreciated that
evaporation of solvent from the collection wells may be
accomplished through the application of gas to permit sequential
washing steps and the like to occur without overflowing the
collection wells.
[0074] Through the use of the collection well embodiments of the
present invention, interface of the methodologies of this invention
with previously known techniques in chemistry, biotechnology, and
other disciplines may be attained. Thus, once predetermined species
are known to be present in collection wells of the collection
plate, various traditional analytical techniques may be applied.
These include testing protocols based upon cell based and
biochemical assays. These can be used in association with other
techniques such as ELISA assays and the like. Testing protocols can
be used to measure various parameters including enzyme/substrate
interaction, protein/protein interaction, substrate/transcription
factor interaction, ligand/receptor interaction.
[0075] A very important use of the present invention is for the
generation of highly accurate, libraries of chemical compounds,
specially oligomeric libraries. Exemplary uses of such libraries
are abundant. Illustrative cell based assays and brief descriptions
of the assays are:
[0076] HIV
[0077] CEM-SS cells are infected with live virus (HIV-1) in
presence of library subsets; assay measures protection of the cells
by the library subsets from virus-induced cytopathic effects.
Tuberculosis Bacteriocidal effects of the library subsets on the
mycobacterium are measured.
[0078] Tumor Necrosis Factor
[0079] Inhibition by library subsets of TNF induction of
inflammatory cascade in NHDF cells is monitored using ICAM-1
induction as endpoint.
[0080] Interleukin 1-Beta
[0081] Inhibition by library of IL1-.beta. induction of
inflammatory cascade in NHDF cells is monitored using ICAM-1 as
endpoint.
[0082] LPS
[0083] Inhibition by library subsets of LPS induction of
inflammatory cascade in NHDF cells is monitored using ICAM-1
induction as endpoint.
[0084] Malaria
[0085] Inhibition of parasite replication in blood cells by library
subsets is measured.
[0086] Interleukin-6
[0087] Inhibition by the library subsets of interaction of IL-6 and
its receptor expressed on live cells is monitored using an antibody
specific for IL-6.
[0088] MRP/MDR
[0089] Enhancement by the library subsets of the toxic effects of
chemotherapeutic drugs on mammalian cells expressing either MRP or
MDRI is monitored using an MTT assay.
[0090] PDGF
[0091] Library subset inhibition of the radioactively-labeled
ligand interaction with membrane-bound receptor is measured. The
membrane is partially purified from guinea pig spleen. Complement
C5.sub.A Library subset inhibition of the radioactively-labeled
ligand interaction with membrane-bound receptor is measured. The
membrane is partially purified from guinea pig spleen.
[0092] LTB.sub.4
[0093] Library subset inhibition of the radioactively-labeled
ligand interaction with membrane-bound receptor is measured. The
membrane is partially purified from guinea pig spleen.
[0094] PLA.sub.2
[0095] Library subset inhibition of enzymatic activity of type II
phospholipase A.sub.2 is measure. The substrate is E. coli with a
radioactively-labeled fatty acid in the membrane.
[0096] TAT/tar
[0097] Biotinylated TAR RNA is bound to streptavidin-coated wells
of a 96-well microtiter plate. Inhibition by the library subsets of
the interaction of the tat protein with the TAR RNA is monitored in
an ELISA-type assay using a tat-specific antibody.
[0098] In addition to the above specific uses, other use for the
compounds that comprise the libraries of this invention are as
general use enzyme inhibitors, additives for foodstuffs, as agent
used in affinity chromatography, and as probes and diagnostic
agents in kits and the like with or without the addition of
suitable labels including fluorescent agent, radioactive agents, or
enzymes labels. Other uses will also be apparent.
[0099] As will be appreciated, the apparatuses and techniques of
the present invention are amenable to a wide variety of chemical
and biochemical synthetic schemes. Thus, it is possible to employ
nearly any type of chemical reaction save, possibly, those which
take place exclusively in the gaseous phase and those which require
biphasic catalysis. In general, it is preferred to attach a first
species to the reaction surface, both at the actual surface of the
reaction support and to a greater or lesser extent at the surface
of the internal pore volume, followed by subsequent chemical
reactions. It is convenient to attach the first chemical reactant
universally over the entire reaction support since the same may be
accomplished through immersion of the support in appropriate
reactants. Subsequent, dropwise application of reagents effect
chemical reactions locally at the reaction sites under the control
of control means. It is also possible and in many applications
preferred, to perform all reactions in a dropwise fashion through
chemical jetting.
[0100] Oligomers are preferred species for synthesis in accordance
with the present invention. Thus, oligonucleotides, polypeptides,
and oligosaccharides may be so synthesized. Such oligomers may be
either synthesized in relative bulk, where many or all of the
reaction sites are caused to experience the same reaction
conditions and to lead to the same reaction products, or may be
individually determined for subsets of reaction sites.
[0101] In accordance with other embodiments, it is preferred to
employ the apparatuses and methods of the present invention to
prepare nonoligomeric chemical moieties. Thus, "classical"
chemistry may be employed to synthesize such chemicals either "in
bulk" or with different chemical species being synthesized at
different subsets of reaction sites. While it is preferred in some
cases to physically attach the developing chemical moiety to the
reaction surfaces during the course of synthesis, it is also
possible in some embodiments to avoid this step. In such case, it
is generally preferred to effect careful reaction and washing
conditions so as to retain the intermediate chemical species on the
surface for further reaction. This may generally be done through
careful selection of the reaction support, chemical reagents, and
washing solvents in view of the molecules to be synthesized.
Persons of ordinary skill in the art will know how to effect such
selections in view of the objects to be attained in particular
synthesis.
[0102] It is believed to be possible to effect asymmetric synthesis
through employment of the present invention. If a chiral synthetic
support is adopted, in some circumstances growing chemical
intermediates will adopt preferred stereochemical configurations
leading to the synthesis of one enantiomer over another without the
use of chiral reagents. The asymmetric synthesis of chemical
species on asymmetric surfaces is known per se and such techniques
may be adopted here. The methods of the invention may be used to
produce oligomeric species having a variety of different monomeric
subunits. Thus, the methods of the invention may advantageously be
employed in the synthesis of polymers of nucleotides
(oligonucleotides or nucleic acids), peptides, proteins, peptide
nucleic acids (PNAs), and other polymeric species synthesizable by
iterative addition of synthons to adducts on the reaction
surface.
[0103] Synthetic techniques such as solid phase peptide synthesis
and solid phase nucleic acid synthesis utilize the ability to
selectively protect and deprotect specific functional groupings.
Protecting groups are conveniently characterized as either
"temporary" or "permanent." "Temporary" protecting groups are
quantitatively removed at each step of the synthesis to allow
coupling of the next synthon. "Permanent" protecting groups are
stable to the conditions of the iterative elongation cycle, and
therefore protect side chain, nucleobase, or other functional
groups which do not participate in, but may interfere with chain
elongation. Typically, permanent protecting groups are chosen such
that conditions required for their removal are equivalent to those
required for cleavage of the completed chain from the reaction
support, affording concomitant removal.
[0104] The methods of the present invention may be used to
synthesize peptides by standard solid phase peptide synthesis
(SPPS) methodologies (see, e.g., Merrifield, J. Am. Chem. Soc.,
1963, 85, 2149 and Science, 1986, 232, 341). Media suitable for use
as reaction supports in connection with peptide and synthetic
applications of the invention include aminomethyl polystyrene
resins, various polyamide support materials, membranes, cotton and
other carbohydrates, controlled-pore silica glass, and other media
known to those in the art for use in peptide synthesis as solid
supports. See Synthetic Peptides A Users Guide, Gregory A. Grant,
Ed. Oxford University Press 1992.
[0105] The initial functionalization of the reaction support may be
achieved by any of the more than fifty methods which have been
described in connection with traditional solid-phase peptide
synthesis (see, e.g., Barany and Merrifield in "The Peptides" Vol.
2, Academic Press, New York, 1979, pp. 1-284, and Stewart and
Young, "Solid Phase Peptide Synthesis", 2nd Ed., Pierce Chemical
Company, Ill., 1984).
[0106] Typically, SPPS (solid phase peptide synthesis) is performed
in the "C to N" direction. Thus, anchoring linkers are designed
such that cleavage at the end of the synthetic regime produces a
C-terminal acid or amide. In preferred embodiments a linker
containing an activated carboxyl group is keyed to amino groups on
the reaction support. In some preferred embodiments of the
invention protected amino acid derivatives having linkers attached
(so called "preformed handles") are keyed to the reaction support.
See Synthetic Peptides A Users Guide, supra, at pages 105-119.
[0107] Any of the several "temporary" protecting groups routinely
used in the art are suitable for use in the present invention.
Preferred among these are the widely used BOC (t-butoxycarbonyl)
and FMOC (N.sup..alpha.-9-fluorenylmethyloxycarbonyl) groups. Other
suitable amino protecting groups include
2-(4-biphenyl)propyl[2]oxycarbonyl (Bpoc),
2-(3,5,-dimethoxyphenyl)propyl[2]oxycarbonyl (Ddz),
1-(1-adamantyl)-1-methylethoxycarbonyl (Adpoc) and
4-methoxybenzyloxycarbonyl (Moz). Other suitable protecting groups
will be apparent to those skilled in the art, based on their
experience and knowledge.
[0108] After keying of a linker and/or first monomeric synthon to
the reaction support, the iterative process of chain elongation
occurs. This may proceed, in accordance with the invention, by any
of the several methods known in the art for the formation of
peptide bonds. Representative of such methods are the use of in
situ coupling reagents, active esters, preformed symmetrical
anhydrides and acid halides.
[0109] Representative in situ coupling reagents suitable for use in
the present invention include N,N'-dicyclohexylcarbodiimide (DCC),
and N,N'-diisopropylcarbodiimide (DIPCDI), especially in
conjunction with the use of scavenging agents (so called
accelerators or additives) such as 1-hydroxybenzotriazole (HOBt),
benzotriazol-1-yl-oxy-tris (dimethylamino)phosphonium
hexafluorophosphate (BOP), and
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU). A list of suitable in situ coupling
agents may be found in Synthetic Peptides A Users Guide, supra.
[0110] Appropriately selected sites on the first surface of a
reaction support are jetted with a solvent as a washing or rinsing
step, and then the temporary protecting group on the terminal
synthon is cleaved by jetting a deprotection reagent onto the
preselected sites of the reaction support. This is optionally
followed by the jetting of one or more conventional rinsing
reagents. The next protected monomeric synthon is then jetted in a
suitable solvent such as dimethyl formamide. Coupling or activating
agents, including accelerators or additives such as HOBt,
optionally may be jetted with the protected monomeric synthon or
alternatively may be jetted independently in an appropriate
solvent.
[0111] After a suitable reaction time, the iterative cycle is
repeated until the desired amino acid sequence is achieved.
Cleavage of the completed product from the reaction support is
achieved by jetting a cleaving reagent onto the reaction support
surface. The cleaving reagent also typically functions to remove
the "permanent" protecting groups from the amino acid side chains.
Choice of a specific cleavage reagent will necessarily be
determined by the particular synthetic chemistry employed. For
example, Boc SPPS chemistry will typically employ strong acid such
as hydrogen fluoride for cleavage, while in Fmoc SPPS chemistry,
the same result is typically accomplished with trifluoroacetic
acid. See Synthetic Peptides A Users Guide, supra, at pages
130-136.
[0112] The methods on the invention may similarly be employed in
the synthesis of non-peptide polymeric species which consist of
monomers linked by traditional peptide chemistries. Representative
of these species are peptide nucleic acids (PNAs), which are
disclosed in WO 92/20702. In PNAs ligands are linked to a polyamide
backbone through aza nitrogen atoms. U.S. application Ser. No.
08/054,363, filed Apr. 26, 1993 and a corresponding PCT Application
PCT/IB94/00142 filed Apr. 25, 1994 discloses peptide nucleic acids
in which their recognition moieties are linked to the polyamide
backbone additionally through amido and/or ureido tethers.
Additional PNAs and methods for their synthesis are also disclosed
in U.S. application Ser. No. 08/088,658, filed Jul. 2, 1993 and a
corresponding PCT Application PCT/US94/07319 filed Jul. 2,
1994.
[0113] These PNAs are synthesized by adaptation of standard peptide
synthesis procedures. The synthons used are unique monomer amino
acids or their activated derivatives, which are protected by
standard protecting groups. Thus, the synthesis of these PNAs may
be accomplished according to the methods of the present invention
in similar fashion to protocols specified above for synthesis of
peptides.
[0114] For example, the reaction support may be functionalized
according to methodologies specified above to incorporate
Boc-L-Lys(2-chlorobenylox- ycarbonyl). An excess of a desired
monomer to be coupled may be jetted on the reaction support,
followed by jetting of a coupling reagent such as
dicyclohexylcarbodiimide in a suitable solvent such as 50% DMF in
dichloromethane. Boc deprotection may then be accomplished by
jetting of trifluoroacetic acid. After completion of the desired
chain, the PNA may be cleaved from the reaction support by jetting
of a mixture of trifluoromethanesulfonic acid:trifluoroacetic
acid:meta-cresol (1:8:1 v:v:v). The product is precipitated from
the solution by the addition of diethyl ether.
[0115] The methods of the present invention may be advantageously
employed to synthesize deoxyribonucleic acid (DNA) or ribonucleic
acid (RNA) (together "oligonucleotide") species by any of the
several chemistries for solid phase DNA or RNA synthesis, including
phosphite triester, (phosphoramidite) synthesis and H-phosphonate
synthesis. See Nucleic Acids in Chemistry and Biology, M. Blackburn
and M. Gait, Eds., IRL Press 1990, at pages 112-130. In principle,
the methods of the present invention will be useful in the practice
of any iterative nucleic acid synthetic technique. In preferred
embodiments of the invention oligonucleotides are synthesized by
the phosphoramidite method. Representative solid-phase synthetic
methodologies useful in the present invention may be found in
Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707;
4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. Nos.
4,725,677 and Re. 34,069.
[0116] Media suitable for use as reaction supports in connection
with oligonucleotide synthetic applications of the invention
include controlled-pore silica glass (CPG); oxalyl-controlled pore
glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19,
1527); RAPP copolymer highly crosslinked polystyrene, TENTAGEL
Support, (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34,
3373); HLP, a high loaded polystrene available from ABI; POROS, a
polystyrene resin available from Perceptive Biosystems, and other
media known to those in the art for use in oligonucleotide
synthesis as solid supports.
[0117] For phosphoramidite synthesis according to the invention,
the reaction support is preferably functionalized with spacer
groups according to methods known in the art. Typical of such
spacer groups are long chain alkylamines. The spacer groups may be
keyed to preselected portions of the reaction support, or to the
entire reaction support surface.
[0118] Keying of the initial monomeric synthon may be accomplished
by jetting appropriately derivitized nucleoside monomers (having
protecting groups on any exocyclic amine functionalities present)
onto the reaction support. For example, a
5'-O-DMT-3'-O-(4-nitrophenyl) succinate monomer may be jetted onto
the reaction support, keying the initial monomer to the reaction
support through the alkylamine spacer. Typically, monomeric
synthons bear temporary protecting groups at appropriate nucleobase
or 2'-O positions. See Nucleic Acids in Chemistry and Biology,
supra.
[0119] Solid phase nucleic acid synthetic techniques employ
"temporary" and "permanent" protecting groups in analogous fashion
to solid phase peptide synthesis. Base labile protecting groups are
used to protect the exocyclic amino groups of the heterocyclic
nucleobases during the synthesis. This type of protection is
generally achieved by acylation with acylating reagents such as
benzoylchloride and isobutyrylchloride. Acid labile protecting
groups are used to protect the nucleotide 5' hydroxyl during
synthesis. Representative hydroxyl protecting groups commonly used
in the art may be found in Beaucage, et al., Tetrahedron 1992, 48,
2223. These include the dimethoxytrityl, monomethoxy trityl,
trityl, and 9-phenyl-xanthene (pixyl) groups. Dimethoxytrityl
protecting groups are widely used owing to their great acid
lability, which affords efficient removal by dilute acid (e.g. 3%
trichloroacetic acid).
[0120] The first step in the iterative chain elongation cycle
according to the phosphoramidite technique is the removal
5'-O-protecting group (deprotection) of the initial monomer by
jetting an appropriate deprotecting reagent onto the preselected
portions of the reaction support. This is followed by the jetting
of a rinsing reagent. Suitable reagents for deprotection include
Lewis acids such as ZnBr.sub.2, AlCl.sub.3, BF.sub.3 and TiCl.sub.4
in solvents such as nitromethane, tetrahydrofuran, and mixed
solvents such as nitromethane and lower alkyl alcohols, such as
methanol. Protic acids such as acetic acid, dichloroacetic acid,
trifluoroacetic acid, and toluenesulfonic acid may also be used in
an appropriate solvent, typically dichloromethane.
[0121] Chains are lengthened by jetting and reaction of activated
5'-O-protected monomeric synthons. In the phosphoramidite technique
a
5'-DMTr-deoxynucleoside-3'-O-(N,N-diisopropylamino)-.beta.-cyanoethylphos-
phite is jetted onto the reaction support. Phosphoramidites of
numerous nucleosides are commercially available (for example, from
Applied Biosystems Inc. and Millipore Corp.).
[0122] A mild organic acid catalyst, typically tetrazole, is jetted
onto the reaction support either together with the phosphoramidite
or independently thereafter. Commonly used commercially available
activating agents are disclosed in U.S. Pat. No. 4,725,677 and in
Berner, S., Muhlegger, K., and Seliger, H., Nucleic Acids Research
1989, 17:853; Dahl, B. H., Nielsen, J. and Dahl, O., Nucleic Acids
Research 1987, 15:1729; and Nielson, J. Marugg, J. E., Van Boom, J.
H., Honnens, J., Taagaard, M. and Dahl, O., J. Chem. Research 1986,
26, all of which are herein incorporated by reference. The coupling
reaction is followed by the jetting of a rinsing solvent, typically
anhydrous acetonitrile.
[0123] After rinsing, a capping reagent is jetted onto the
preselected portions of the reaction support to cap free hydroxyl
species remaining due to incomplete reaction of phosphite monomers.
The capping reagent, which is typically a solution of an acid
anhydride, also functions to reverse any inadvertent
phosphitylation of guanine O-6 positions.
[0124] Oxidation of the resulting phosphite triester to the
corresponding phosphate triester may be accomplished by jetting
oxidants known in the art to be suitable, such as a solution of
alkaline Iodine.
[0125] The methods of the present invention may be employed in the
synthesis of oligonucleotides having the naturally occurring
nucleobases adenine (A), thymine (T), guanine (G), cytosine (C) and
uracil (U), as well as non-naturally occurring nucleobases.
Non-naturally occurring nucleobases are molecular moieties which
are known in the art to mimic the function of naturally occurring
nucleobases in their biological role as components of nucleic
acids. Examples of non-naturally occurring nucleobases are
disclosed in, for example, Antisense Research and Applications,
Crooke and Lebleu, eds., CRC Press, Boca Raton, 1993.
[0126] Further details of methods useful for preparing
oligonucleotides may be found in Sekine, M., etc. al., J. Org.
Chem., 1979, 44, 2325; Dahl, O., Sulfur Reports, 1991, 11, 167-192;
Kresse, J., et. al., Nucleic Acids Research, 1975, 2, 1-9;
Eckstein, F., Ann. Rev. Biochem., 1985, 54, 367-402; and Yau, E. K.
U.S. Pat. No. 5,210,264 entitled
"S-(2,4-Dichlorobenzyl)-.beta.-Cyanoethyl Phosphorothioate
Diester".
[0127] Oligonucleotide species having a wide variety of
modifications to nucleobases, sugars, or inter-sugar linkages can
be prepared in accordance with the methods of the invention, which
are generally applicable to the synthesis of any oligomer
synthesizable by solid phase techniques. For example, the methods
of the present invention may be employed in the synthesis of
S-phosphorodithioates, phosphorothioates, methyl phosphonates,
phosphoramidates, phosphorotriesters, thiophosphotriesters,
thiophosphoramidates, methylphosphonothioates, and cyclic
phosphorothioates, phosphorothioates, and phosphorodithioates.
These modifications are disclosed as set forth in Antisense
Research and Applications, supra, and in U.S. patent applications
assigned to a common assignee hereof, entitled "Backbone Modified
Oligonucleotide Analogs," Ser. No. 703,619 and "Heteroatomic
Oligonucleotide Linkages," Ser. No. 903,160, the disclosures of
which are incorporated herein by reference.
[0128] The polymers produced according to the methods of the
invention may be composed of more than one type of monomeric
subunit (e.g., amino acids, peptide nucleic acids, nucleotides,
sugars, etc.) and may possess more than one type of inter-subunit
linkage. Illustrative polymers produced according to the methods of
the invention include peptides, peptoids (N-alkylated glycines),
.alpha.-polyesters, polythioamides, N-hydroxy amino acids,
.beta.-esters, polysulfonamides, N-alkylates polysulfonamides,
sulfonamides, polyureas, urethanes, peptide nucleic acids,
nucleotide, polysaccharides, polycarbonates, oligonucleotide,
oligonucleosides and the like and chimeric molecules that contain
one or more of these polymers joined together as a single
macro-molecule.
[0129] Libraries of monomeric analog species can also prepared by
the methods of the invention. These include benzodiazepine
libraries and other such analog libraries including, but not
limited to, antihypertensive agents, e.g. enalapril,
.beta.-blockers, e.g. proproanol; antiulcer drugs (H.sub.2-receptor
antagonists) e.g. cimetidine and ranitidine; antifungal agents
(cholesterol-demethylase inhibitors, e.g. isoconazole; anxiolytics,
e.g. diazepam; analgesics, e.g. aspirin, phenacetamide, and
fentanyl; antibiotics, e.g. vancomycin, penicillin and
cephalosporin; antiinflammatories, e.g. cortisone; contractives,
e.g. progestins; abortifacients, e.g. RU-456; antihistamines, e.g.
chlorphenamine; antitussives, e.g. codeine; sedatives, e.g.
barbitol and well as many others that will be suggested by this
disclosure. Illustrative are the benzodiazepine and the
hetero-Diels-Alder libraries as described in published PCT
application WO 94/08051; the benzodiazepine and prostaglandins
described in U.S. Pat. No. 5,288,514; the dipeptides, hydantoins,
benzodiazepins, quinolones, keto-ureas, benzamido-5-oxopentanoic
acids, diketopeperazines, 2H-pyranones, N-aryl piperazines,
benzoisothiazolones, spirosuccimides, pilocarpine analogs,
benzopyrans, pyrimidinediones and tepoxalin analogs described in
U.S. Pat. No. 5,324,483.
EXAMPLES
Example 1
Synthesis of Library of Compounds Incorporating P.sup.v Linkages
Reagents
[0130]
1 Abbreviations and Definitions: DCM dichloromethane Deblocking
agent 3% trichloroacetic acid in DCM ACN acetonitrile 1st Capping
Sol. N-methylimidazole 2nd Capping Sol. acetic anhydride Oxidizer
Iodine for (P = O) or Beaucage reagent for (P = S) Activator
1-H-tetrazole Amidite One of various reactant species activated as
a phosphoramidite Position set A predetermined set of positions
where a reaction is to take place - may vary for one to all
positions wherein jetting head is targeted to dispense reagents All
positions Every position where jetting head is targeted to dispense
reagents
[0131] Support
[0132] Membrane Carrier
[0133] Membrane strips are positioned on the base plate and gasket
and overlaid with the top plate of a 96 well, 6-mm dot GibcoBRL
"The Convertible Filtration Manifold System" device from Life
Technologies, Gaithersburg, Md. The "well" vacuum line of the
carrier is modified to include an electromechanical in line off-on
value for control of the vacuum below the membranes. The membrane
carrier is located vertically below a droplet generator for jetting
reagent droplets. Actuation of the in line vacuum value is via the
controller 32.
[0134] Cycle
[0135] 1. Deliver deblocking agent to all positions on the
support
[0136] 2. Wait 30 seconds
[0137] 3. Remove by vacuum
[0138] 4. Deliver ACN to all positions and remove by vacuum
[0139] 5. Repeat step 4 five times
[0140] 6. Deliver amidite A to first designated position set
[0141] 7. Deliver activator to same designated position set
[0142] 8. Wait 1-5 minutes as required for current amidite being
used
[0143] 9. Remove amidite and activator by vacuum
[0144] 10. Deliver ACN to same designated position set
[0145] 11. Remove by vacuum
[0146] 12. Repeat steps 6-11 for further amidite B to second
designated position set, amidite C to third designated position
set, etc. for further amidites at designated position sets
[0147] 13. Deliver ACN to all positions and remove by vacuum
[0148] 14. Deliver 1st and 2nd capping solutions to all
positions
[0149] 15. Wait 30 seconds
[0150] 16. Remove by vacuum
[0151] 17. Deliver ACN to all positions and remove by vacuum
[0152] 18. Repeat step 15 five times
[0153] 19. Deliver oxidizer to all positions
[0154] 20. Wait 30 seconds
[0155] 21. Deliver ACN to all positions and remove by vacuum
[0156] 22. Repeat step 19 five times
[0157] The cycle is repeated n times to achieve oligomer n residues
long.
Example 2
Synthesis of Library of Compounds Incorporating Amide Linkages--BOC
Chemistry
[0158] Regents, Abbreviations and Definitions
[0159] DCM
[0160] dichloromethane
[0161] DMF
[0162] dimethylforamide
[0163] TFA
[0164] trifluoroacetic acid
[0165] HATU
[0166] O-(7-azabenzotriazol-1-yl)-1,1,2,2-tetramethyl uronium
hexafluorophosphate
[0167] MDCHA
[0168] N-methyldicyclohexylamine
[0169] Deblocking agent
[0170] TFA/m-cresol, 95/5, v/v
[0171] Cap
[0172] acetic anhydride/collidine/DMF, 5/6/89, v/v/v
[0173] Pyridine
[0174] Pyridine/DMF, 5/95, v/v
[0175] Piperdine
[0176] Piperdine/DMF, 5/95, v,v
[0177] Activator A
[0178] 0.18 M HATU in DMF, 855 mg plus 12 mL DMF
[0179] Activator B
[0180] 0.3 M HATU in DMF, 570 mg plus 4.7 mL DMF
[0181] Position set
[0182] A predetermined set of positions where a reaction is to take
place--may vary for one to all positions wherein jetting head is
targeted to dispense reagents
[0183] All positions
[0184] Every position where jetting head is targeted to dispense
reagents
[0185] Monomers
[0186] One of various reactant species capable of being linked
together via amide linkages
[0187] Support
[0188] Membrane Carrier
[0189] Membrane are strips positioned on the base plate and gasket
and overlaid with the top plate of a 96 well, 6-mm dot GibcoBRL
"The Convertible Filtration Manifold System" device from Life
Technologies, Gaithersburg, Md. The "well" vacuum line of the
carrier is modified to include an electromechanical in line off-on
value for control of the vacuum below the membranes. The membrane
carrier is located vertically below with the jetting head.
Actuation of the in line vacuum value is via the controller 32.
[0190] Cycle
[0191] 1. Deliver DMF/DCM to all positions and remove by vacuum
[0192] 2. Wait 10 seconds
[0193] 3. Deliver TFA to all positions
[0194] 4. Wait 10 seconds
[0195] 5. Deliver TFA to all positions
[0196] 6. Wait 180 seconds
[0197] 7. Deliver TFA to all positions
[0198] 8. Wait 180 seconds
[0199] 9. Remove by vacuum
[0200] 10. Deliver DMF/DCM wash and remove by vacuum
[0201] 11. Deliver pyridine to all positions
[0202] 12. Deliver DMF/DCM to all positions
[0203] 13. Deliver monomer A and HATU activator to first designated
position set
[0204] 14. Wait 840 seconds
[0205] 15. Deliver DMF/DCM wash to first designated position
set
[0206] 16. Remove by vacuum
[0207] 17. Repeat, in parallel, steps 13-16 for further monomer B
to second designed position set, etc. for further monomers at
designated position sets
[0208] 18. Deliver DMF/DCM wash to all positions
[0209] 19. Deliver cap to all positions
[0210] 20. Wait 300 seconds
[0211] 21. Deliver piperidine/DMF to all positions
[0212] 22. Wait 60 seconds
[0213] 23. Remove by vacuum
[0214] 24. Deliver DMF/DCM wash to all positions and remove by
vacuum
[0215] The cycle is repeated n times to achieve oligomer n-residues
long.
[0216] Optional cleavage of library compounds from support is
effected using a cleavage cocktail of
m-cresol/thioanisole/TFMSA/TFA, 1/1/2/6, v/v/v/v. The membrane is
treated with the cleavage cocktail for one hour followed by removal
from the membrane by vacuum into individual wells of an appropriate
matrix collection plate.
Example 3
Synthesis of Library of Compounds Incorporating Amide
Linkages--FMOC Chemistry
[0217] Regents, Abbreviations and Definitions
[0218] DCM
[0219] dichloromethane
[0220] DMF
[0221] dimethylforamide
[0222] TFA
[0223] trifluoroacetic acid
[0224] HATU
[0225] O-(7-azabenzotriazol-1-yl)-1,1,2,2-tetramethyluronium
hexafluorophosphate
[0226] MDCHA
[0227] N-methyldicyclohexylamine
[0228] Deblocking agent
[0229] 20% piperdine in DMF
[0230] Cap
[0231] acetic anhydride/collidine/DMF, 5/6/89, v/v/v
[0232] Pyridine
[0233] Pyridine/DMF, 5/95, v/v
[0234] Piperdine
[0235] Piperdine/DMF, 5/95, v,v
[0236] Activator A
[0237] 0.18 M HATU in DMF, 855 mg plus 12 Ml DMF
[0238] Activator B
[0239] 0.3 M HATU in DMF, 570 mg plus 4.7 Ml DMF
[0240] Position set
[0241] A predetermined set of positions where a reaction is to take
place--may vary for one to all positions wherein jetting head is
targeted to dispense reagents
[0242] All positions
[0243] Every position where jetting head is targeted to dispense
reagents
[0244] Monomers
[0245] One of various reactant species capable of being linked
together via amide linkages
[0246] Support
[0247] Membrane Carrier
[0248] Membrane are strips positioned on the base plate and gasket
and overlaid with the top plate of a 96 well, 6-mm dot GibcoBRL
"The Convertible Filtration Manifold System" device from Life
Technologies, Gaithersburg, Md. The "well" vacuum line of the
carrier is modified to include an electromechanical in line off-on
value for control of the vacuum below the membranes. The membrane
carrier is located vertically below with the jetting head.
Actuation of the in line vacuum value is via the controller 32.
[0249] Cycle
[0250] 1. Deliver DMF/DCM to all positions and remove by vacuum
[0251] 2. Wait 10 seconds
[0252] 3. Deliver TFA to all positions
[0253] 4. Wait 10 seconds
[0254] 5. Deliver TFA to all positions
[0255] 6. Wait 180 seconds
[0256] 7. Deliver TFA to all positions
[0257] 8. Wait 180 seconds
[0258] 9. Remove by vacuum
[0259] 10. Deliver DMF/DCM wash and remove by vacuum
[0260] 11. Deliver pyridine to all positions
[0261] 12. Deliver DMF/DCM to all positions
[0262] 13. Deliver monomer A and HATU activator to first designated
position set
[0263] 14. Wait 840 seconds
[0264] 15. Deliver DMF/DCM wash to first designated position
set
[0265] 16. Remove by vacuum
[0266] 17. Repeat, in parallel, steps 13-16 for further monomer B
to second designed position set, etc. for further monomers at
designated position sets
[0267] 18. Deliver DMF/DCM wash to all positions
[0268] 19. Deliver cap to all positions
[0269] 20. Wait 300 seconds
[0270] 21. Deliver piperidine/DMF to all positions
[0271] 22. Wait 60 seconds
[0272] 23. Remove by vacuum
[0273] 24. Deliver DMF/DCM wash to all positions and remove by
vacuum
[0274] 25. Repeat cycle by starting at step 13
[0275] The cycle is repeated n times from step 13 to achieve
oligomer n-residues long.
[0276] Optional cleavage of library compounds from support is
effected using a cleavage cocktail of 95% trifluoroacetic acid
containing 5% scavenger. The membrane is treated with the cleavage
cocktail for one hour followed by removal from the membrane by
vacuum into individual wells of the matrix collection plate.
Example 4
Synthesis of Library of Compounds Incorporating Hydroxylamine
Linkages
[0277] Regents, Abbreviations and Definitions
[0278] DCM
[0279] dichloromethane
[0280] Deblocking agent
[0281] 3% N-methyl hydrazine in DCM:methanol (9:1, v:v)
[0282] GAA
[0283] Glacial Acetic Acid
[0284] Reducing reagent
[0285] NaCNBH.sub.3
[0286] Alkylating reagent
[0287] 20% Formaldehyde
[0288] TBAF
[0289] Tetrabutylammonium fluoride
[0290] THF
[0291] Tetrahydrofuran
[0292] Intermediate Monomer
[0293] One of various
5'-O-phthalimido-3'-C-aldehydo-3'-deoxynucleosides
[0294] First Monomer
[0295] One of various
5'-O-phthalimido-3'-O-(succinyl)nucleosides
[0296] Final Monomer
[0297] One of various 5'-t-butyldiphenylsilyl-3'-aldehyde-3'-deoxy
nucleosides
[0298] Membrane activator
[0299] Pentachlorophenol
[0300] Position set
[0301] A predetermined set of positions where a reaction is to take
place--may vary for one to all positions wherein jetting head is
targeted to dispense reagents
[0302] All positions
[0303] Every position where jetting head is targeted to dispense
reagents
[0304] Support
[0305] Membrane Carrier
[0306] Membrane strips are positioned on the base plate and gasket
and overlaid with the top plate of a 96 well, 6-mm dot GibcoBRL
"The Convertible Filtration Manifold System" device from Life
Technologies, Gaithersburg, Md. The "well" vacuum line of the
carrier is modified to include an electromechanical in line off-on
value for control of the vacuum below the membranes. The membrane
carrier is located vertically below with the jetting head.
Actuation of the in line vacuum value is via the controller 32.
[0307] Cycle
[0308] 1. Deliver DCM solution of First Monomer A and Membrane
activator to a first designated position set
[0309] 2. Wait 15 seconds
[0310] 3. Wash with DCM and remove by vacuum
[0311] 4. Repeat steps 1-3 for further First Monomer B to second
designated position set, etc. for further First Monomers to
designated position sets
[0312] 5. Deliver Deblocking agent to a first designated position
set
[0313] 6. Wait 120 seconds
[0314] 7. Wash with DCM for 240 seconds and remove by vacuum
[0315] 8. Deliver a DCM solution of a selected Intermediate monomer
and GAA to this first designated position set
[0316] 9. Wait 60 seconds
[0317] 10. Wash with DCM and remove by vacuum
[0318] 11. Repeat steps 5 to 10 for further Intermediate monomer B
to second designated position set, etc. for further Intermediate
monomers to designated position sets
[0319] 12. Repeat steps 5 to 11 n-2 times for oligomer n-residues
long
[0320] 13. Deliver a DCM solution of a first Final monomer to a
first designated position set
[0321] 14. Wait 60 seconds
[0322] 15. Wash with DCM and remove by vacuum
[0323] 16. Repeat steps 13 to 15 for further Final monomer B to
second designated position set, etc. for further Intermediate
monomers to designated position sets
[0324] 17. Wash with DCM and remove by vacuum
[0325] 18. Deliver mixture of Reducing agent, Alkylating reagent
and GAA to all positions
[0326] 19. Wait 180 seconds
[0327] 20. Wash with DCM and remove by vacuum
[0328] 21. Deliver TBAF in THF at all positions to de-block all
Final nucleosides
[0329] 22. Wait 30 seconds
[0330] 23. Wash with DCM and remove by vacuum
[0331] The oligomers are removed from membrane by treating with 30%
ammonium hydroxide.
Example 5
Synthesis of Peptide Nucleic Acid Library
[0332] A library of peptide nucleic acids, wherein individual
peptide nucleic acid are 6-mers, is synthesizer utilizing the
protocol of Example 2. Four nucleobase monomers are used in
construction the library. The monomers incorporate normal
nucleobases attached to the peptide nucleic acid backbone wherein A
is an adenine peptide nucleic acid monomer, G is a guanine peptide
nucleic acid monomer, C is a cytosine nucleic acid monomer and T is
a thymine nucleic acid monomer. The monomers are used at a 0.2M
concentration. BOC (terminal amine groups) and Z (nucleobases)
protection is utilized. The monomers and HATU are purchased from
Millipore Corp., Bedford Mass. The membrane is crosslinked PEPS
film that is aminomethylated. The aminomethylated film is further
modified with a BOC-Try(BrZ)Pam linder from Star Chemicals coupled
as a preformed HOBt ester at about 0.15 mmol amino groups per gram
of film with the remaining amino groups capped by acetylation with
acetic anhydride.
[0333] Monomers
[0334] (0.2 M Monomer, 0.2 M MDCHA and 0.3 M Collidine)
2 Monomer Weight (mg) MDCHA (.mu.l) collidine (.mu.L) Solvent (mL)
A 528 214 198 4.3 G 544 214 198 4.3 T 384 214 198 4.3 C 504 214 198
4.3 The monomers are appropriately solubilized in either DMF or
N-methylpyrrolidinone.
Example 6
Synthesis of Peptide Library
[0335] A library of 8 mer peptides is synthesized utilizing the
protocol of Example 3. The complexity of the library is based upon
the 8 amino acids, alanine (A), arginine (R), glycine (G), leucine
(L), lysine (K), serine (S), tyrosine (Y) and histidine (H). The
monomer are used at 0.3M concentration. FMOC protection is
utilized. The monomers and HATU are purchased from Millipore Corp.,
Bedford Mass. The membrane is a polypropylene coated with
polyhydroxypropylacrylate as described in poster #T81 and
accompanying poster #T80 of the 1989 Protein Society meeting, San
Diego, Calif. Standard peptide oligomerization protocol are
followed.
Example 7
Synthesis of Phosphate Oligonucleotide Library
[0336] A library of 10 mer oligonucleotides is synthesized
utilizing the protocol of Example 1 with iodine as the oxidizer.
The membrane used is a "Nucleic Acid Membrane Support", (from
Millipore Corp.--a polyvinylidene difluoride polymeric membrane
derivitized with diamine propane). Monomer are standard
deoxyribonucleotides purchased from Millipore, Corp., Bedford,
Mass. or Glen Research, Sterling, Va.
Example 8
Synthesis of Phosphorothioate Oligonucleotide Library
[0337] A library of 10 mer oligonucleotides is synthesized
utilizing the protocol of Example 1 with Beaucage reagent as the
oxidizer. The membrane used is a "Nucleic Acid Membrane Support",
(from Millipore Corp.--a polyvinylidene difluoride polymeric
membrane derivatized with diamine propane). Monomer are standard
deoxyribonucleotides purchased from Millipore, Corp., Bedford,
Mass. or Glen Research, Sterling, Va.
Example 9
Synthesis of Oligoribonucleotide Library
[0338] A library of 6 mer oligoribonucleotides is synthesized
utilizing the protocol of Example 1 with iodine as the oxidizer.
The membrane used is a "Nucleic Acid Membrane Support", (from
Millipore Corp.--a polyvinylidene difluoride polymeric membrane
derivatized with diamine propane). Monomer are standard
deoxyribonucleotides available from either Millipore, Corp.,
Bedford, Mass. or Glen Research, Sterling, Va.
Example 10
Synthesis of Chimeric Phosphate
Oligoribonucleotide-Phosphorothioate Oligodeoxyribonucleotide
Library
[0339] A library of 8 mer oligonucleotides having an internal
section of 4 consecutive phosphorothioate deoxyribonucleotides
flanked by 2 mer ribonucleotides is synthesized utilizing the
protocol of Example 1 with either iodine or Beaucage reagent used
as the oxidizer as appropriate. The ribonucleotides are selected as
2'-O-methylribonucleotides. The nucleobases are A, C, G and T for
the deoxy portion of the chimera and A, C, G and U for the ribo
portions. The membrane used is a "Nucleic Acid Membrane Support",
(from Millipore Corp.--a polyvinylidene difluoride polymeric
membrane derivatized with diamine propane). Monomers are standard
deoxyribonucleotides or 2'-O-methyl ribonucleotides available for
either Millipore, Corp., Bedford, Mass. or Glen Research, Sterling,
Va.
Example 11
Synthesis of Oligomeric Library Incorporating Propane-1,2-diol
Monomeric Units Connected Via Phosphate Linkers
[0340] A library of 8 mer oligomers based on a propane-1,2-diol
backbone linked via phosphate linkages and that incorporates 4
nucleobase for diversity is synthesized utilizing the protocol of
Example 1 with iodine as the oxidizer. The membrane used is a
"Nucleic Acid Membrane Support", (from Millipore Corp.--a
polyvinylidene difluoride polymeric membrane derivatized with
diamine propane). Monomer are 1{{N-{2-[9-(N2-isobutyroyl-
)guanine]acetyl}amino}}-3-O-dimethyoxytritylmethyl-1-amino-2-O-[(N,N-disio-
propylamino)-2-cyanoethoxyphosphite]propane,
1{{N-{2-[9-(N6-benzoly)adenin-
e]acetyl}amino}}-3-O-dimethyoxytritylmethyl-1-amino-2-O-[(N,N-disiopropyla-
mino)-2-cyanoethoxyphosphite]propane,
1{{N-{2-[9-(N4-benzoly)cytosine]acet-
yl}amino}}-3-O-dimethyoxytritylmethyl-1-amino-2-O-[(N,N-disiopropylamino)--
2-cyanoethoxyphosphite]propane and
1{N-{2-[1-thymidine)acetyl[amino}-3-O-d-
imethyoxytritylmethyl-1-amino-2-O-[(N,N-disiopropylamino)-2-cyanoethoxypho-
sphite]propane.
Example 12
Synthesis of Oligomeric Library Incorporating 3-Hydroxypyrrolidine
Monomeric Units Connected Via Phosphate Linkers
[0341] A library of 6 mer oligomers base on a hydroxypyrrolidine
backbone linked via phosphate linkages and that incorporates 6
diversity moieties having various characteristics is synthesized
utilizing the protocol of Example 1 with iodine as the oxidizer.
The membrane used is a "Nucleic Acid Membrane Support", (from
Millipore Corp.--a polyvinylidene difluoride polymeric membrane
derivatized with diamine propane). Monomer are
N.sup.1-palmitoyl-5-dimethoxytrityloxymethylpyrrolidine-3-O-[(N,N-dii-
sopropylamino)-2-cyanoethoxyphosphite,
N.sup.1-phenylacetyl-5-dimethoxytri-
tyloxymethylpyrrolidine-3-O-[(N,N-diisopropylamino)-2-cyanoethoxyphosphite-
,
N.sup.1-(fluorenylmethylsuccinoyl)-5-dimethoxytrityloxymethylpyrrolidine-
-3-O-[(N,N-diisopropylamino)-2-cyanoethoxyphosphite,
N.sup.1-(N-Fmoc-3-aminopropionoyl)-5-dimethoxytrityloxymethylpyrrolidine--
3-O-[(N,N-diisopropylamino)-2-cyanoethoxyphosphite and
N.sup.1-(N-imidazolyl)-5-dimethoxytrityloxymethylpyrrolidine-3-O-[(N,N-di-
isopropylamino)-2-cyanoethoxyphosphite to give a six fold diversity
incorporated in the oligomers.
Example 13
Synthesis of Oligonucleoside Library Incorporating Nucleoside
Monomeric Units Connected Via Hydroxylamine Linkages
[0342] A library of 6 mer oligonucleosides linked via
methylenehydroxylamino linkages and that incorporates 4 nucleobases
as diversity moieties is synthesized utilizing the protocol of
Example 4. The membrane used is a "Nucleic Acid Membrane Support",
(from Millipore Corp.--a polyvinylidene difluoride polymeric
membrane derivatized with diamine propane). Monomers are
N4-benozyl-3'-deoxy-3'-C-formyl-5'-O-phtha-
limido-5-methylcytosine,
N6-benozyl-3'-deoxy-3'-C-formyl-5'-O-phthalimido-- adenosine,
N2-isobutryl-3'-deoxy-3'-C-formyl-5'-O-phthalimido-guanosine and
3'-deoxy-3'-C-formyl-5'-O-phthalimido-thymidine.
* * * * *