Apparatus and method for combinatorial chemistry synthesis

In a first embodiment, this invention includes an integrated robot apparatus for performing combinatorial chemistry synthesis protocols and having interchangeable work-stations, robot arm tools, and reaction vessels and reaction vessel arrays. The work-stations and tools are specialized to perform tasks necessary for the synthesis in a plurality of the reaction vessels grouped in a plurality of the reaction vessel arrays. Preferably, these elements function interchangeably because they have standardized sizes and conformation. The work-stations and tools include those for fluid dispensing or aspirating from individual reaction vessels or from all the reaction vessels in an array simultaneously. The reaction vessels can include, alternatively, stackable, ball-sealed reaction vessels, microtitre-like reaction vessel arrays, arrays of independent reaction vessels, valve-sealed reaction vessels, septum-sealed reaction vessels, and syringe reaction vessels. In alternative embodiments, this invention includes these work-stations, tools, reaction vessels and reaction vessel arrays in various combinations or sub-combinations either for use in partially integrated robots or for manual or standalone use.

1. FIELD OF THE INVENTION 
The field of this invention relates to automated apparatus for chemical 
synthesis; more particularly it relates both to a flexible, 
high-throughput, synthetic robot apparatus for combinatorial chemistry 
synthesis and also, generally, to apparatus for performing various 
manipulations during such synthesis, whether as part of an automated or of 
a manual procedure. 
2. BACKGROUND 
Protocols for combinatorial chemistry synthesis are recently developed 
chemical processes for individually synthesizing a potentially 
combinatorially-large number of chemical compounds. These methods proceed 
by a sequence of steps, each step adding a particular, selected one of a 
plurality of building blocks, i.e. small organic molecules, to a growing, 
intermediate compound. Thereby, the number of potential final compounds is 
a product of terms, one term for each synthesis step representing the 
number of possible building blocks that can be added at that step. For 
example, for peptides, since each step can typically select from the same 
number of amino acid building blocks, the number of potential final 
peptides is the number possible amino acid building blocks raised to a 
power equal to the number of addition steps. 
These addition step reactions typically proceed by combining the 
partially-synthesized, intermediate compound with the building block 
having an attached activating residue. (Hereinafter, building blocks are 
assumed to have necessary activating residues attached.) Also added to an 
addition step reaction are activating reagents and other reagents and 
solvents. The building blocks are added in a molar excess to the partially 
synthesized compound present so that the thermodynamically favorable 
building block addition proceeds substantially to completion. After 
addition of one building block, the intermediate compound is separated 
from the spent reaction solution and prepared for the addition of a 
further building block. Often, the intermediate compound is attached to a 
solid-phase support by, e.g., a cleaveable linking residue, in order to 
simplify separation of intermediate compound from spent addition reaction 
solutions. In such solid-phase protocols a final step of cleaving the 
linking residue frees the final compound. 
Building blocks (activated as necessary), activating and other reagents, 
and reaction conditions have been recently perfected for a wide variety of 
classes of final compounds. Exemplary of such reactions and protocols are 
the following for addition of natural and artificial amino acids to form 
peptides: Lam et al., 1991, A new type of synthetic peptide library for 
identifying ligand-binding activity, Nature 354: 82-84.; U.S. Pat. 
5,510,240 to Lam et al. for Method of screening a peptide library; Lam et 
al., 1994, Selectide technology: Bead-binding screening. Methods: A 
Companion to Methods in Enzymoloqy 6: 372-380. For protocols for the 
synthesis of benzodiazepine moieties, see, e.g.: Bunin et al., 1992, A 
general and expedient method for the solid phase synthesis of 
1,4-benzodiazepine derivatives, J. Amer. Chem. Soc., 114: 10997-10998.; 
U.S. Pat. 288,514 to Ellman for Solid phase and combinatorial synthesis of 
benzodiazepine compounds on a solid support. Also, for protocols for the 
addition of N-substituted glycines to form peptoids, see, e.g., Simon, et 
al., 1992, Peptoids: A modular approach to drug discovery. Proc. Natl. 
Acad. Sci. USA, 89: 9367-9371; Zuckermann et al., 1992, Efficient method 
for the preparation of peptoids [oligo(N-substituted glycines)] by 
submonomer solid-phase synthesis. J. Amer. Chem. Soc., 114: 10646-10647; 
WO PCT94/06,451 to Moos et al. for Synthesis of N-substituted polyamide 
monomers, useful as solvents, additives for food, enzyme inhibitors etc. 
Approaches for synthesis of small molecular libraries were recently 
reviewed by, e.g., Krchnak et al., 1996, Synthetic library techniques: 
Subjective (biased and generic) thoughts and views, Molecular Diversity, 
1: 193-216; Ellman, 1996, Design, synthesis, and evaluation of 
small-molecule libraries, Account. Chem. Res., 29: 132-143; Armstrong et 
al., 1996, Multiple-component condensation strategies for combinatorial 
library synthesis, Account. Chem. Res., 29: 123-131.; Fruchtel et al., 
1996, Organic chemistry on solid supports, Angew. Chem. Int. Ed., 35: 
17-42; Thompson et al., 1996, Synthesis and application of small molecule 
libraries, Chem. Rev., 96 :555-600; Rinnova et al., 1996, Molecular 
diversity and libraries of structures: Synthesis and screening, Collect. 
Czech. Chem. Commun., 61: 171-231; Hermkens et al., 1996, Solid-phase 
organic reactions: A review of the recent literature, Tetrahedron, 52: 
4527-4554. 
Predictable, algorithmic synthesis of a large number of individual 
compounds, which are subsequently collected into a library of compounds, 
is of interest and utility in several fields, in particular in the field 
of pharmaceutical lead-compound selection. A pharmaceutical lead-compound 
for a drug is a compound which exhibits a particular biologic activity of 
pharmaceutical interest and which can serve as a starting point for the 
selection and synthesis of a drug compound, which in addition to the 
particular biological activity has pharmacologic and toxicologic 
properties suitable for administration to humans or animals. It is 
apparent that synthesis of large numbers of compounds and screening for 
their biological activities in a controlled biological system can be of 
assistance in lead compound selection. Instead of turning to botanical or 
other natural sources, the pharmaceutical chemist can use combinatorial 
protocols to generate in the laboratory compounds to screen for desired 
activities. See, e.g., Borman, 1996, Combinatorial Chemists Focus on Small 
Molecules, Molecular Recognition, and Automation, Chemical & Engineering 
News, Feb. 12, 1996, 29-54. 
To achieve the benefits of these recently developed combinatorial 
protocols, automated synthesis apparatus is advantageous. Dealing manually 
with hundreds, thousands, or perhaps tens of thousands of separate 
compounds is expensive, time consuming, and prone to error. Therefore, 
synthesis robots for automating one or more steps of combinatorial 
protocols have been recently developed. Examples of such recently 
developed robots are described in: Cargill et al., 1996, Automated 
Combinatorial Chemistry on Solid-Phase, Laboratory Robotics and 
Automation, 8: 139-148; U.S. Pat. 5,503,805 to Sugarman et al.; U.S. Pat. 
5,252,296 to Zuckermann et al. for Method and apparatus for biopolymer 
synthesis; WO PCT 93/12,427 to Zuckermann et al., for Automated apparatus 
for use in peptide synthesis; Krchnak et al., 1996, MARS--Multiple 
Automatic Robot Synthesizer. Continuous Flow of Peptide, Peptide Res. 9: 
45-49. 
However, these and other existing combinatorial synthesis robots have 
significant limitations. First, they perform such syntheses sequentially 
and batchwise. In other words, at any one time, such robots can process 
only one limited group of synthesis reactions. Typically only 10-96 
reactions can be processed at one time, after which the robotic apparatus 
must be cleaned and reconditioned in order to synthesize the next batch of 
compounds. Second, such robots are often constructed of specialized, 
single-function elements, not otherwise commercially available. For 
example, there can be one set of specially designed and constructed 
automatic manipulators for each single manipulation required by a 
synthetic protocol, thereby requiring a sequential passage of a batch of 
reactions through the robot. Also, typically, the reaction vessels, in 
which the synthetic addition reactions are performed, are specially 
designed, constructed, and arrayed in large, cumbersome, and expensive 
reaction vessel arrays or assemblies. Alternatively, such robots utilize 
complex tubing, valving, and pumping arrangements for fluid distribution, 
which are expensive to manufacture and maintain. A third limitation is 
that these robots typically provide only limited reaction conditions, such 
as limited temperature ranges and an inability to prevent atmospheric 
exposure. 
In summary, current synthesis robots for combinatorial chemistry protocols 
are slow and expensive, limiting the promise of combinatorial chemistry, 
especially as applied to drug selection and design. Existing robots are 
slow because of sequential, batchwise synthesis, because of 
single-function robotic devices, and because of a limited capability to 
process large numbers of synthesis reactions according to multiple 
protocols. They are expensive because their robot actuators, reaction 
vessels, and reaction vessel arrays are specially designed for this one 
application, limiting use of existing, inexpensive, commercially available 
components. 
3. SUMMARY OF THE INVENTION 
It is an object of this invention to provide a combinatorial chemistry 
synthesis robot which overcomes the previously described problems. The 
robot of this invention is capable of the simultaneous, high throughput 
synthesis of compounds according to a plurality of synthesis protocols, 
each such protocol utilizing a plurality of building blocks and reagents. 
The robot is not limited to the synthesis of compounds according to one 
synthesis protocol at one time. The multi-protocol and high throughput 
capabilities of the robot are made possible by a flexible and programmable 
architecture, in which synthetic steps in reaction vessels are performed 
at appropriate modules or work stations and in an appropriate sequence 
according to the particular protocol being performed. In embodiments 
directed toward solid-phase synthesis, the robot is additionally capable 
of the production of combinations of single or multiple compounds per 
solid-phase bead or single or multiple compounds per each reaction vessel. 
The robot provides extended reaction conditions, including temperatures 
from -50 to +150.degree. C., and inert atmospheres, including nitrogen or 
argon. Preferably, the robot is further capable of synthesizing between 
3,000 and 5,000 compounds per day, and less preferably, is capable of 
synthesizing at least 1,000 compounds per day. 
It is a further object of this invention to provide individual elements 
adaptable to such a robot. Such elements can also have utility in the 
manual performance of combinatorial or other chemical synthesis protocols. 
In particular, such elements include reaction vessels and arrays of 
reaction vessels together with appropriate closing and sealing means that 
are preferably built from commercially available and inexpensive 
components. These reaction vessels and arrays are disposable, where 
possible, to avoid any cleaning steps. These elements further include 
tools and work stations directed to the efficient manipulation of such 
reaction vessels and arrays of reaction vessels, such manipulation 
including, e.g., sealing and unsealing, fluid dispensing and removal, 
temperature controlled incubation, and so forth. 
Accordingly, in one embodiment, this invention generally comprises an 
integrated and modular combinatorial chemistry synthesis robot, which is 
based on reaction vessels disposed in standard sized arrays or modules 
together with their novel closing and sealing means. These reaction vessel 
arrays are manipulated by programmable, multipurpose robot arms capable of 
accepting interchangeable tooling, and are processed at specialized work 
stations. These components are contained in an enclosure providing a work 
surface and storage volumes. In various particular embodiments, this 
invention comprises combinations of one or more of the following elements: 
reaction vessels and reaction vessel arrays; sealing means for reaction 
vessels and arrays; one or more robot arms; interchangeable synthesis 
tools adaptable to the robot arms; specialized synthesis work stations for 
processing the reaction vessels and reaction vessel arrays; and an 
enclosure. 
In more detail, the reaction vessels of this invention are preferably 
grouped into arrays or modules of reaction vessels having standard sized 
form factors and arrangements. Standard sized form factors and 
arrangements permit standardized design and interchangeability of work 
stations and tools for processing the reaction vessel arrays. Preferable 
reaction vessels are inexpensive commercially available vessels, 
microtitre plates, and so forth, capable of resisting the solvents and 
reaction conditions used in synthesis protocols. Reaction vessel arrays 
are sealed with various sealing means. One sealing means comprises caps 
with valves, which are arranged in arrays of caps of rectangular or other 
convenient arrangement. The valves are actuated by sliding or rotating rod 
seals or by having a compressible neck and are capable of being opened and 
closed simultaneously. Another sealing means comprises caps with apertures 
which are occluded and sealed by solvent resistent balls made from, e.g., 
Teflon.TM.. Teflon.TM. is used generally herein to specify ethylene 
tetrafluoro-polymers and their compositions or any plastics of equivalent 
chemical and physical properties. In one preferred embodiment, the balls 
are attached to a compliant assembly, each sealing ball being capable of 
tolerating misalignment of a reaction vessel in an array of reaction 
vessels. In another alternative preferred embodiment, individual reaction 
vessels sealed with individual sealing balls can be stacked and retained 
in a cylindrical array for incubation at higher temperatures and internal 
pressures. Alternatively, arrays of reaction vessels can be sealed either 
by an inflatable bag or by conventional rubberized septa. Optionally, 
enhanced septum assemblies are provided with a collapsible, rubber collar 
with a central orifice, which is collapsed and sealed by a screw cap. 
An enclosure according to this invention includes a work surface for 
supporting specialized work stations disposed above, on, or below the work 
surface. Below the work surface, preferably, are work stations for 
temperature controlled incubation of sealed reaction vessel arrays 
together with facilities for shaking or agitating these arrays during 
incubation. Also below the work surface, preferably, are storage volumes 
for storing containers of building block solutions, reagents, and solvents 
necessary for the various synthesis protocols. Below surface work stations 
include elevator means for raising reaction vessel arrays, work stations, 
and storage volumes above the work surface for access by the robot arms. 
Disposed on or above the work surface are specialized work stations for 
filling, emptying, sealing, and shaking reaction vessel arrays. These 
above surface work stations are advantageously directly accessible by the 
robot arms. Reaction vessel arrays rest on the work surface during and 
between their processing steps. The enclosure also optionally includes 
internal sub-enclosures, which are individually capable of maintaining 
reaction vessel arrays in an inert atmosphere during filling, emptying, 
and sealing steps. 
Robot arms adaptable to this invention perform the manipulations needed to 
achieve high throughput combinatorial synthesis. Preferably, they are 
capable of accurate three-dimensional positioning in the enclosure above 
the work surface. Instead of being dedicated to particular tasks, they are 
capable of attaching and controlling specialized and interchangeable 
tooling. One important type of manipulation performed by a robot arm 
gripper tool is the gripping and the moving individual reaction vessels or 
reaction vessel arrays from work station to work station. 
The specialized work stations of this invention perform tasks needed by the 
protocols being implemented by a particular robot. In alternative 
embodiments, certain tasks are performed by specialized tools attached to 
the robot arms. Work stations needing frequent attention by the robot arms 
are preferably disposed above the work surface, whereas work stations not 
requiring frequent attention by the robot arms are preferably disposed 
below the work surface. Accordingly, time and temperature controlled 
incubation, needed during the building block addition steps of certain 
protocols, is performed below the work surface. Reaction vessel arrays are 
placed on elevators by the robot arms, which then descend below the 
surface for temperature controlled incubation and later rise back above 
the surface for access by the robot arms in order to perform subsequent 
processing. Similarly, storage is preferably below the work surface. Items 
are stored in holders or containers with standard sized footprints to 
enable manipulation by standardized tools or stations. It is preferable 
that below work surface storage be provided for at least approximately 600 
storage vessels, e.g., containers such as syringes, or other storage 
vessels, with individual building blocks solutions. In the case of 
protocol reagents, it is preferable that below surface storage be 
available for at least approximately 10 bottles of such reagents. Access 
to these storage bottles can be provided by tubing which connects with 
above work surface workstation or other tools. Optionally, fresh reaction 
vessels and reaction vessel arrays are stored below the surface. 
Above work surface work stations include those for reaction vessel sealing, 
for fluid aspiration, for wash solvent distribution and optionally, for 
fluid dispensing. Work stations for reaction vessel sealing are adapted to 
the various novel sealing means of this invention. Stations for ball-based 
sealing include: those for individually placing sealing balls on the 
apertures of individual reaction vessels and stacking the sealed reaction 
vessels into cylindrical arrays, hereinafter called "hot rods;" those for 
distributing individual balls on an array of reaction vessels; and those 
for placing compliant ball assemblies on arrays of reaction vessels. 
Optionally, hot rods have clips for temporarily retaining reaction vessels 
and sealing balls during assembly and further clips for permanently 
retaining these components during processing. Stations for arrays of 
valved reaction vessels include those for manipulating the valve rod seals 
for simultaneous opening or closing all the valves of the reaction vessels 
in the array. 
Stations for fluid aspiration are based on aspiration through needles, 
either surface aspiration through flat-ended needles or volume aspiration 
through fritted needles of this invention. Such stations have multiple 
needles advantageously arranged in various arrangements, for example 
linear or rectangular arrays, in order to aspirate simultaneously from 
part or all of the reaction vessels in one reaction vessel array. Stations 
for wash solvent distribution, where volumetric accuracy is less 
important, are adapted to more rapid and repetitive fluid distribution. 
Such stations preferably also have a two-dimensional array of dispensing 
tips, which conform to the arrangement of reaction vessels, so that wash 
solvents can be simultaneously dispensed into all the reaction vessels of 
an array. Solvent distribution can be powered by inert gas or fluid 
pressure, and fluid aspiration by vacuum suction. 
Alternatively, this invention includes reaction vessel arrays which are 
arrays of syringes, each syringe including a microporous frit for 
retaining a solid-phase synthesis support while permitting free passage of 
fluids. Such syringe arrays can be constructed either from a block of 
solvent resistant plastic having an array of cylindrical cavities forming 
the syringe bodies or from independent, commercially-available syringes 
held in an array by a support means. However constructed, fluid 
manipulation and distribution can be provided by a network of passageways, 
each such passageway connecting to one syringe body and externally 
terminated either by a needle or by a septum. Such fluid passageways can 
be advantageously placed in a fluid distribution block. In the case of 
needle termination, the array of needles can be inserted in an array of 
fluid reservoirs for dispensing fluids to or removing fluids from the 
syringe bodies. In the case of septum termination, for fluid dispensing to 
the individual syringe bodies, the septums can be penetrated by needles 
containing required fluids. Additionally, dispensing to or removing from 
all the syringe bodies simultaneously can be by means of a common internal 
passageway interconnecting the syringe bodies to a single external fluid 
port, which in turn can be coupled through a further tubing network to 
various fluid reservoirs. Further, plunger means are advantageously 
provided for simultaneously and accurately manipulating, inwardly or 
outwardly, all the syringe plungers in such an array to assist in fluid 
distribution. 
Interchangeable tools according to this invention are adapted to be 
manipulated by the robot arms. Importantly, a robot arm is capable of 
dynamically attaching and controlling different tools, and is, therefore, 
not restricted to a particular task during synthesis. These tools include 
a common attachment base, which permits the robot arm to attach the tool 
and have access to the controls of the tool, such as by providing 
communicating between pneumatic ports or electrical contacts in the tool 
and the robot arm, respectively. These tools are typically used for 
accurate fluid dispensing, such as dispensing building block solutions or 
reagents, and for gripping, such as for gripping reaction vessel arrays or 
compliant ball assemblies. Fluid dispensing tools are preferably 
constructed with one or more dispensing tips through which accurate 
aliquots of a fluid are ejected. In the case of building block solutions, 
where each reaction vessel typically receives a unique building block 
solution in a particular addition step, fluid dispensing is preferably 
done with a single tipped tool. Such a tool can simply be a syringe 
gripper for holding a syringe containing a building block solution and for 
accurately manipulating of the syringe plunger. In the case of reagents, 
which are typically common to those reaction vessels in which synthesis is 
being performed according to a single protocol, it is preferable to use a 
multiple tipped tool for dispensing reagents simultaneously into all such 
reaction vessels. These latter tools include a fluid storage vessel, a 
fluid pump, and piping interconnecting the storage vessel, the pump, and 
the multiple tips. These tools can also be used to dispense solid-phase 
beads, in which case the reagent is replaced with a slurry of the beads. 
Gripper tools are adapted to move reaction vessels, reaction vessel arrays, 
and sealing means for reaction vessel arrays. An important gripper is an 
off-set "U"-shaped gripper, which is adapted both to grip and move 
reaction vessel arrays and to place or remove certain sealing means of 
this invention. Another important gripper is a the previously mentioned 
gripper for distributing building blocks solution. Other grippers can be 
adapted to grip and move other specialized elements of the reaction vessel 
arrays and sealing means. 
A further type of tool dispenses slurries, and can be used for initially 
distributing a solid-phase substrate to reaction vessels as well as for 
mixing and partitioning contents of reaction vessels in order to perform 
split-synthesis according to various protocols. Such a tool includes a 
vertical container with a dependent needle and a source of suction. The 
source of suction draws air through a slurry in the vertical container in 
order to maintain the slurry in suspension, and can also draw a slurry 
from a reaction vessel into the container. A controlled source of air 
volumes can be used to accurately dispense aliquots of the slurry in the 
vertical container, when the suction is interrupted by a valve. 
Optionally, in the case of slurries which must be maintained in an inert 
atmosphere, the dependent needle can be enclosed in a further coaxial tube 
which provides an inert gas to be drawn into the vertical container 
through the dependent needle by the source of suction, and the controlled 
source of air can be replaced by a controlled source of inert gas volumes. 
In further embodiments, this invention comprises other combination and 
sub-combinations of the previously described elements, functioning either 
in conjunction with other robot apparatus or independently of any robot 
apparatus. For example, the reaction vessel arrays with their associated 
sealing means, the fluid aspiration work stations, the fluid dispensing 
work stations, and certain tools have utility in facilitating 
semi-automated or manual performance of combinatorial chemistry synthetic 
protocols. Therefore, each of these elements is independently part of this 
invention.

5. DETAILED DESCRIPTION 
For clarity of disclosure, and not by way of limitation, the detailed 
description of this invention is presented herein with respect to figures 
that illustrate preferred embodiments of elements of this invention. 
However, this invention includes those alternative embodiments of these 
elements performing similar functions in similar manners that will be 
apparent to one skilled in the art from the disclosure provided. 
Additionally, this invention is disclosed with respect to its preferred 
application to solid-phase, combinatorial chemistry synthesis. The 
invention is not so limited, and includes application of the various 
elements disclosed to other chemical protocols having similar functional 
steps, as also will be apparent to one skilled in the art. For example, 
components of this invention can be applied to appropriate liquid-phase, 
combinatorial chemistry synthesis protocols, to other solid- or 
liquid-phase chemical protocols, or to any combination thereof. 
By way of introduction combinatorial chemistry synthesis protocols 
prescribe the stepwise, sequential addition of building blocks to 
intermediate partially-synthesized intermediate compounds in order to 
synthesize a final compound. These protocols are, generally, divided into 
liquid-phase protocols and solid-phase protocols. In liquid-phase 
protocols, final compounds are synthesized in solution. Partially 
synthesized, intermediate compounds are separated from spent reagents 
between building block addition steps by known means, such as 
precipitation, fractionation, and so forth. In solid-phase synthesis, 
final compounds are synthesized attached to solid-phase supports that 
permit the use of simple mechanical means to separate intermediate, 
partially-synthesized intermediate compounds between synthetic steps. 
Typical solid-phase supports include microbeads, of from 30 microns to 
perhaps 300 microns in diameter, which are functionalized in order to 
covalently attach intermediate compounds, and made of, e.g., various 
glasses, plastics, or resins. 
Solid-phase combinatorial synthesis typically proceeds according to the 
following steps. In a first step, reaction vessels are charged with a 
solid-phase support, typically a slurry of functionalized microbeads 
suspended in a solvent. These microbeads are then preconditioned by 
incubating them in an appropriate solvent, and the first of the plurality 
of building blocks or a linker moiety is covalently linked to the 
functionalized beads. Subsequently, a plurality of building block addition 
steps are performed, all of which involve repetitive execution of the 
following substeps, and in a sequence chosen to synthesize the desired 
compound. First, a sufficient quantity of a solution containing the 
building block moiety selected for addition is accurately added to the 
reaction vessels so that the building block moiety is present in a molar 
excess to the intermediate compound. The reaction is triggered and 
promoted by activating reagents and other reagents and solvents, which are 
also added to the reaction vessel. The reaction vessel is then incubated 
at a controlled temperature for a time, typically between 5 minutes and 24 
hours, sufficient for the building block addition reaction to go to 
substantial completion. Optionally, during this incubation, the reaction 
vessel can be intermittently agitated or stirred. Finally, in a last 
substep of building block addition, the reaction vessel containing the 
solid-phase support with attached intermediate compound is prepared for 
addition of the next building block by removing the spent reaction fluid 
and thorough washing and reconditioning the solid-phase support. Washing 
typically involves three to seven cycles of adding and removing a wash 
solvent. Optionally, during the addition steps, multiple building blocks 
can be added to one reaction vessel in order to synthesize multiple 
compounds attached to one solid-phase support, or alternatively, the 
contents of separate reaction vessels can be combined and partitioned in 
order that multiple compounds can be synthesized in one reaction vessel 
with each microbead having only one attached final compound. After the 
desired number of building block addition steps, the final compound is 
present in the reaction vessel attached to the solid-phase support. The 
final compounds can be utilized either directly attached to their 
synthetic supports, or alternatively, can be cleaved from their supports. 
In the latter case, the linker moiety attaching the compound to the 
solid-phase support is cleaved, and the library compound is extracted into 
a liquid phase. 
An exemplary solid-phase combinatorial protocol is that for the synthesis 
of peptides attached to MBHA resin, which proceeds according to Lam et 
al., 1991, A new type of synthetic peptide library for identifying 
ligand-binding activity, Nature 354: 82-84. Another exemplary protocol is 
that for the synthesis of benzodiazepine moieties, which proceeds 
according to Bunin et al., 1992, A general and expedient method for the 
solid phase synthesis of 1,4-benzodiazepine derivatives, J. Amer. Chem. 
Soc., 114: 10997-10998. Exemplary building blocks and reagents are amino 
acids, other organic acids, aldehydes, alcohols, and so forth, as well as 
bifunctional compounds, such as those given in Krchnak et al., 1996, 
Synthetic library techniques: Subjective (biased and generic) thoughts and 
views, Molecular Diversity, 1: 193-216. 
In view of the large potential numbers of final compounds in combinatorial 
libraries, it is advantageous that at least some manipulations needed by 
the synthetic protocols be assisted or performed automatically. In view of 
the exemplary protocol described, a flexible, automated, robot for 
combinatorial chemistry synthesis advantageously includes facilities for 
handling fluids, for manipulating reaction vessels, and for storage of 
reagents and building blocks. Advantageous facilities for fluid handling 
include: facilities to accurately add solutions and slurries, containing, 
for example, building blocks, solid-phase substrates, reagents or 
solvents; facilities to rapidly and repetitively add wash solvents; and 
facilities to rapidly and accurately remove fluid phases from a reaction 
vessel leaving behind the solid-phase support with attached intermediate 
compounds. Facilities for manipulating reaction vessels and reaction 
vessel arrays include: facilities to move reaction vessels and reaction 
vessel arrays between other processing facilities; facilities for time and 
temperature controlled incubation of reaction vessels and reaction vessel 
arrays; and optionally facilities for agitation of reaction vessels during 
incubation. Each such protocol typically uses many building blocks, 
perhaps hundreds, a few activating and other reagents, perhaps 2 to 4, and 
one or two work solvents. Accordingly, there are storage facilities for: a 
large number of building blocks solutions, typically 300 or more building 
blocks solutions or more preferably as many as 600 or more building blocks 
solutions stored, for example, in arrays of syringes; preferably 6 or more 
preferably 12 or more reagents in larger quantities than for building 
block solutions; and preferably 3 or more preferably 6 or more of even 
larger quantities of wash solvents. 
Design of the processing resources, reaction vessels and arrays, and other 
facilities of this invention, advantageously permits simultaneous, 
parallel processing to occur at all apparatus facilities in order to 
achieve high synthesis throughput. This is achieved by designs having a 
few standardized physical sizes and layouts and having a modular nature. 
Thereby, processing resources can be simultaneously applied to multiple 
protocols in many reaction vessel arrays and can be sized to achieve 
required throughput. 
Preferred materials for all elements of this invention in contact with the 
synthetic addition reactions, in particular reaction vessels and their 
sealing means, must resist the reagents, solvents, and reaction conditions 
likely to be encountered in the various protocols. In the following 
detailed description, when solvent resistance is specified and particular 
materials are not specified, the following exemplary general purpose 
solvent resistant materials can be used: Teflon.TM., polypropylene, or 
glass. 
5.1. Integrated Robot Apparatus 
As generally illustrated in FIG. 1, embodiments of the integrated robot 
apparatus of this invention preferably have processing resources and 
facilities with a modular design and standardized spatial form factors and 
spatial layouts or structures. Such modularization and standardization 
enables high throughput, multi-protocol combinatorial synthesis by 
permitting interchangeable parallel use of a small number of standardized 
tools and work stations to process in parallel many inexpensive reaction 
vessel arrays according to a plurality of protocols. For example, 
standardizing physical dimensions of reaction vessel arrays permits one, 
or at most a few, gripper tools to move reaction vessel arrays between 
work stations. Standardizing the structure or layout of reaction vessel 
arrays permits a corresponding standardization of the arrays of fluid 
handling tips in fluid handling work stations. Thereby, one, or at most a 
few, fluid dispensing and aspirating stations per reaction vessel layout 
can provide for multiple simultaneous synthetic protocols. 
Preferred reaction vessel array form factors and array layouts are 
determined by what is inexpensive and commercially available. In the 
following, the terms "form factor" or "footprint" refer to the size and 
shape of a reaction vessel array, and the term "array layout" refers to 
the spatial arrangement of reaction vessels in the reaction vessel array. 
For example, a standard microtiter plate has a rectangular form factor 
with a typical size of 85.times.130 mm and a 8.times.12 rectangular array 
layout of reaction vessel wells. It is advantageous to adapt embodiments 
of the robot of this invention to such a common standard rectangular 
85.times.130 mm form factor of microtiter plates. This form factor can 
accommodate a rectangular 8.times.12 array layout of 96 reaction wells or 
reaction vessels, the microtitre plate, as well as a rectangular 4.times.6 
array layout of independent reaction vessels. A rectangular array of 24 
reaction vessels in a microtitre form factor is particularly adapted to 
commercially available 4 ml reaction vessels. It will be apparent to one 
of skill in the art, that this invention can be adapted to other physical 
array dimensions and array structures by either scaling or rearranging the 
elements disclosed. Thus the currently preferred microtitre, rectangular 
standard can be replaced by other standards that may be developed based on 
squares and rectangles of other sizes with the reaction vessels disposed, 
perhaps, in other array structures, such as hexagonal arrays. Alternative 
standardizations, merely requires changes in fluid handling tip arrays, 
work station sizes, and gripper tools. The principles of the high 
throughput, integrated robot of the invention remain applicable. 
The reaction vessels of this invention are preferably sealed during certain 
synthetic steps, in particular during temperature controlled incubation 
and during agitation. This invention contemplates various sealing means. 
Certain sealing means seal a rectangular array of reaction vessels; other 
sealing means, adapted to higher incubation temperatures and pressures, 
require packing reaction vessels into cylindrical arrays. 
FIG. 1 generally illustrates one embodiment of an integrated, synthetic 
robot according to this invention that incorporates the previously recited 
facilities advantageous for high throughput, multi-protocol combinatorial 
synthesis. This embodiment is adapted to a reaction vessel embodiment in 
which reaction vessels during processing are alternately packed into 
cylindrical modules, such as module 10, or disposed in rectangular arrays, 
such as array 12. The illustrated embodiment generally includes enclosure 
1, robot arms 2, 3 and 4, work surface 5, and various work stations and 
robot arm tools. Enclosure 1 provides mechanical support for work surface 
5 and robot arms 2, 3, and 4. The enclosure illustrated in FIG. 1 has the 
preferred dimensions of 150.times.100.times.50 cm. In general, it can be 
chosen to be of a width and depth sufficient support a sufficient number 
of work stations, tools, and reaction vessel arrays to achieve the desired 
level of synthetic throughput. 
One or more general purpose robot arms are present in an embodiment of this 
invention in order to manipulate reaction vessel arrays and tools. They 
are, preferably, capable of full, independent, and unimpeded access to all 
reaction vessels arrays, tools, and work stations disposed above work 
surface 5. The number of robot arms is chosen to provide sufficient 
parallel handling capabilities to maintain the available workstations busy 
in order to achieve the desired throughput. Preferably, the robot arms are 
capable of attaching and actuating interchangeable tools, including tools 
for gripping reaction vessels, reaction vessel arrays, or sealing means 
and tools for accurately dispensing fluids into reaction vessels. Such 
tools can be actuated by, e.g., electric or pneumatic connections between 
the arm and the tool. In FIG. 1, robot arms 2, 3, 4 are positioned by 
means of a two-dimensional linear stepper motors formed by pole arrays 
disposed in upper surface 22 of enclosure 1 and the base of the robot 
arms, such as base 23. This invention is adaptable to other arm 
positioning technologies known in the art, such as jointed arms actuated 
by pneumatic or hydraulic actuators. See, e.g., Rehg, 1997, Introduction 
to Robotics in CIM Systems, Prentice-Hall, Inc., Upper Saddle River, N.J. 
In particular, this invention is adaptable to laboratory robots from, e.g., 
Yaskawa, Inc. (Cypress, Calif.), CRS, Inc. (Burlington, Ontario, Canada), 
Zymark, Inc. (Hopkington, Mass.), or Sagian, Inc. (Indianapolis, Ind.). 
This embodiment includes work stations, reaction vessels, and reaction 
vessel arrays both above and below work surface 5. Facilities needing more 
frequent attention by the robot arms, such as fluid manipulation 
facilities, are preferably disposed above work surface 5, while facilities 
needing less frequent attention, such as storage facilities and incubation 
means, are preferably disposed below work surface 5. This invention is 
adaptable to other distribution of processing resources above and below 
the work surface. An exemplary below-surface storage means, illustrated in 
FIG. 1, is elevator 8, lifting storage shelves 6, supporting holders 7, 
which have a standardized footprint and contain arrays of containers, 
e.g., syringes, with building block solutions, vessels of reagents, 
bottles of solvents, fresh reaction vessels, and so forth. Holder 18, 
having arrays of containers with building block solutions, has been placed 
on work surface 5 by a robot arm using a gripper tool for subsequent 
access by robot arm 3, which is in the process of distributing various 
building block solutions to the reaction vessels in array 12. Elevator 8, 
illustrated in a raised position for robot arms access, can be lowered so 
that its top surface is flush with work surface 5, forming an additional 
area of work surface and not impeding robot arm motion. Raising and 
lowering means adaptable to such elevators are known in the art and, 
optionally, include mechanical, hydraulic, pneumatic or electric means. 
For example, in FIG. 1, the elevator is actuated by a servomotor and a 
rack and pinion drive. Further below work surface stations are temperature 
controlled incubators, in which reaction vessels arrays are placed for 
reactions at a controlled temperature. A temperature range, preferably 
from -50 to +150.degree. C., is created by circulating fluids, preferably 
air, into direct contact with the reaction vessel arrays. 
For rectangular reaction vessel arrays, such as arrays 12 or 13, such 
incubators can be accessible by an elevator similar to elevator 8. For 
cylindrical reaction vessel arrays, such as arrays 10 or 11, such 
incubators can be accessed through surface ports, such as ports 9. 
Cylindrical arrays can be placed in such ports by specialized robot 
gripper tools. Further, these incubation means optionally and preferably 
include means to agitate the reaction vessels during incubation. 
FIG. 1 also illustrates certain above work surface elements, such as 
reaction vessels, reaction vessel arrays, specialized work stations, and 
arm tools. Preferably, reaction vessel arrays have standardized spatial 
footprints or spatial forms (herein also called "form factors"), such as 
the rectangular footprints of arrays 12 or 13 or the cylindrical footprint 
of arrays 10 or 11. Individual work stations performing specialized 
functions are then adapted to reaction vessel arrays with those 
standardized footprints, and thereby can perform their specialized 
functions on all arrays of that footprint, whatever the details of their 
manufacture and independent of the particular synthesis protocol being 
performed. Work stations adapted to cylindrical modules include 
assembly/disassembly station 14, which either assembles cylindrical arrays 
for incubation from individual reaction vessels stored in rectangular 
arrays or disassembles reaction vessels from cylindrical arrays into 
rectangular arrays for fluid manipulation. Below surface incubators having 
access ports 9 are also adapted to cylindrical arrays. Stations adapted to 
rectangular form factors include wash-solvent dispensing work station 15, 
fluid aspiration work station 16, and incubators accessible by elevators 
similar to elevator 8. For example, rectangular reaction vessel array 13 
can be washed by having robot arm 2 position array 13 in fluid dispensing 
station 15 for dispensing of a wash-solvent and then having robot arm 2 
position array 13 in fluid aspiration station 16 for aspiration of the 
wash solvent. A plurality of these manipulations implements a plurality of 
washing steps. Alternatively, one fluid handling work station can be 
adapted to both dispense and aspirate work solvents. 
Not illustrated in FIG. 1 are sub-enclosures capable of retaining an inert 
atmosphere. These sub-enclosures are preferably of rectangular shape, 
having glass or plastic surfaces, with an optional lid, and contain those 
work stations that must manipulate unsealed reaction vessels, such as 
fluid manipulation work stations. These sub-enclosures are charged with a 
heavier than air inert gas, such as argon, and are thereby capable of 
maintaining unsealed reaction vessels in an inert atmosphere, while 
permitting access by robot arms. 
General purpose robot arms 2, 3, and 4 attach interchangeable tools in 
order to perform specialized functions, including gripping and fluid 
manipulation. For example, gripper 19 attached to arm 2 is specialized, 
for among other tasks, gripping and moving reaction vessel arrays having a 
standardized rectangular form factor. Using gripper 19, robot arm 2 moves 
rectangular reaction vessel arrays between the fluid dispensing work 
station, the fluid aspiration work station, and the cylindrical array 
assembly/disassembly work station (see infra.). On the other hand, gripper 
21 is adapted to securely grip long cylindrical reaction vessel arrays, 
such as array 11, and to raise and lower them into below work surface 
incubators through ports 9. Syringe tool 20 is a fluid manipulation tool 
which grips individual syringes and accurately manipulates the syringe 
plunger to dispense controlled aliquots of a contained fluid. Here syringe 
tool 20 attached to arm 3 has removed a syringe containing a building 
block solution from storage array 18, and is dispensing controlled 
aliquots of the building block solution into the reaction vessels in 
reaction vessel array 12. 
Robot arm means refer to any robot arms having specification suitable to 
perform the above-mentioned manipulations. In particular, the embodiment 
of FIG. 1 can be constructed from commercially available robot arm means 
and enclosures. Exemplary robot arm means are manufactured by Yaskawa, 
Inc., as models platform RW 161 using XY motor RM 6210. The work surface 
itself and below work surface work stations are manufactured by SAIC, Inc. 
Further, the embodiment of FIG. 1 advantageously utilizes commercially 
available control hardware and software. (See infra.) Such hardware and 
software is also supplied by SAIC, Inc. 
5.2. Reaction Vessel Embodiments 
The following subsections disclose particular embodiments of reaction 
vessels, reaction vessel arrays, corresponding sealing means, and suitable 
specialized tools and work stations for processing these arrays. These 
embodiments include in the sequence described: arrays of reaction vessels 
sealed by balls and stacked in cylindrical arrays; microtitre plates of 
96, 384, or more wells and other similar modules; rectangular arrays of 24 
independent vessels in a microtitre form factor and sealed by ball 
assemblies or by valves; reaction vessels sealed by various types of 
punctureable septums. The last subsection describes the novel use of 
arrays of standard, commercially available syringes as reaction vessels. 
In the following, container means refers generally to any of these reaction 
vessel embodiments, which are capable of containing reaction mixtures for 
combinatorial chemistry. Further, sealing means generally refers to those 
methods for sealing appropriate to each of these embodiments of container 
means, or reaction vessels. 
5.2.1. Ball-Sealed, Stackable Reaction Vessels 
FIGS. 2, 3, and 19 illustrate generally a preferred structure for 
stackable, ball-sealed reaction vessels and a method of processing such 
reaction vessels. This embodiment is preferred for protocols requiring 
higher incubation temperatures, which generate higher internal pressures 
in the reaction vessels. Cylindrical module 55 of FIG. 3, also known as a 
"hot rod," is particularly adapted to sealing reaction vessels against 
greater internal pressures, because it is able to generate greater forces 
on the sealing balls. 
Turning first to a method of processing such reaction vessels as 
illustrated in FIG. 3, in a first processing step at location 51 in the 
robot apparatus, rectangular reaction vessel array 54 holds a 4.times.6 
array of 24 reaction vessels 60 in an unsealed condition. At this step, 
accurate aliquots of solutions containing building blocks, activating 
reagents, and other reagents are dispensed into the reaction vessels. 
typically, each reaction vessel receives a different building block, while 
all the reaction vessels in an array receive the same activating and other 
reagents. Alternatively, different reaction vessels in one array can 
contain compounds being synthesized according to different protocols, in 
which case different activating and other reagents would also be 
individually dispensed into the reaction vessels according to the 
particular protocol. After all the solutions for a particular synthetic 
addition step are dispensed into the reaction vessels, reaction vessel 
array 54 is transported by a robot arm to a hot rod assembly/disassembly 
station. As represented by step 56, this assembly/disassembly station 
individually places sealing balls over the apertures of each reaction 
vessel, and then stacks, e.g., six reaction vessels into one hot rod 55. 
At the next processing step at location 52, the hot rods are incubated for 
a controlled time at a controlled temperature, during which the building 
block addition reactions proceed substantially to completion. After 
incubation, step 57 represents the transport of each hot rod to the 
assembly/disassembly station, which unstacks the reaction vessels from a 
hot rod into a rectangular reaction vessel array and then removes the 
sealing balls from the reaction vessel apertures. 
At a final processing step at location 53, reaction vessel array 59 is 
repetitively washed and its solid state resin reconditioned by, e.g., 
repetitively positioning the array 59 at a solvent dispensing station for 
dispensing wash solvent into the reaction vessels followed by positioning 
at a solvent aspiration station for selective removal of only the wash 
solvent. Finally, processing step 58 represents transport of a reaction 
vessel array by a robot arm from washing location 53 to fluid dispensing 
location 51 in order to begin another step of building block addition. 
FIGS. 2A-B illustrate in more detail the structure of the reaction vessels 
and the hot rod of this embodiment. Reaction vessel 63 is, preferably, a 
commercially available, solvent resistant, 4 ml vessel, having a size of 
approximately 15 mm outside diameter by 45 mm length. Vessels of such size 
can be placed in a rectangular 4.times.6 reaction vessel array having 
microtitre form factor while leaving separations between the vessels of no 
less than approximately 5 mm. These vessels can be charged with 
approximately 50 mg of solid-phase resin particles to yield approximately 
10 mg of a synthesized compound with molecular weight of approximately 
1000 Da. These vessels include septum 64, having an aperture for access to 
the interior of vial 63, and retainer 65. The aperture of septum 64 is 
capable of being sealed by sealing ball 62 having a diameter preferably at 
least approximately 1.5 to 5 times that of the size of the aperture in 
septum 64. Septum 64 is tightly retained to the lip of vial 63 by retainer 
65. In a preferred embodiment, retainer 65 is of a metal, such as 
aluminum; septum 64 is of a solvent resistant elastomer, such as 
Kalrez.TM., and ball 62 is a solid ball of a rigid solvent resistant 
plastic, such as Teflon.TM.. The aperture in septum 64 is approximately 
from 1 to 4 mm in diameter. In an alternative embodiment, in place of 
sealing ball 62, the aperture in septum 64 can be sealed by a disk which 
protrudes above retainer 65 and is capable of sealing the aperture in 
septum 64. Such a disk can be made of Teflon.TM.. Alternatively, vessels 
in a hot rod can be sealed by use of compliant balls of a solvent 
resistant and flexible elastomer, such as Kalrez.TM. (DuPont, Wilmington, 
Del.). In this alternative, septum 64 and crimp 65 can be omitted. 
Kalrez.TM. is used generally herein to specify any perfluoro-elastomer 
possessing exceptional resistance to degradation by aggressive fluids or 
gases, or any other elastomers of equivalent chemical and physical 
properties. 
FIG. 2B illustrates in more detail a hot rod of this embodiment. Additional 
details are illustrated in FIGS. 19A-C. Tube 66 has an inside diameter 
just sufficient to accommodate the reaction vessels and a length 
sufficient to accommodate, for example, six reaction vessels along with 
spring 65 in a sufficiently compressed condition. Tube 66 is preferably 
made of a metal, such as aluminum. Spring 65 is chosen to compress the 
stacked reaction vessels with a force sufficient to maintain the sealing 
balls sealed against the apertures in the reaction vessel septums despite 
internal vapor pressures generated in the reaction vessels at desired 
incubation temperatures. The required force is determined by the reagents 
and the incubation temperature. During reaction processing, metal clip 67 
retains the reaction vessels in tube 66 against the force of spring 65 by 
occluding a sufficient porreaction ve opening of tube 66 to prevent 
reaction vessels being driven from the tube. During hot rod assembly and 
disassembly, additional temporary retaining means illustrated in FIG. 19A 
temporarily retain the reaction vessels and sealing balls before the metal 
clip 67 is placed in tube 66. As illustrated in FIG. 2B, clip 67 is a 
metal tongue fitting longitudinally through slots in the sides of tube 66 
and being shaped to permit an assembly/disassembly plunger to fit around 
clip 67. Alternatively, clip 67 can be a circular clip retained by 
recesses in the outer surface of tube 66 and having a central aperture, or 
it can be a screw cap having central aperture. 
Reaction vessel array 54 of FIG. 3, also known as a transport block, holds 
the reaction vessels of this embodiment for manipulation steps, including 
fluid dispensing and aspiration steps and hot rod assembly and 
disassembly. This transport block is preferably of the same size and 
structure as the other embodiments of reaction vessel arrays of this 
invention. In an exemplary embodiment, transport block 54 can have a 
microtitre form factor of 85.times.130 mm and hold 24 reaction vessels in 
a 4.times.6 rectangular array and can be made of a plastic material. 
Accompanying this embodiment of reaction vessels and vessel arrays are 
certain specialized work stations and tools, including a hot rod 
assembly/disassembly work station, a specialized incubation work station, 
and a gripper/agitator tool. The hot rod assembly/disassembly station is 
generally illustrated as work station 14 of FIG. 1 and in more detail in 
FIGS. 19B-C. This station takes advantage of further hot rod elements 
illustrated in FIG. 19A. 
FIG. 19A illustrates in detail the retaining means preferably present in 
the base of hot rod 701. First, metal clip 702, here shown inserted 
through slots 703 of hot rod 701, is adapted to securely retain reaction 
vessels with sealing balls during processing of addition reactions. 
Second, clippers 704 are adapted to temporarily retain reaction vessels 
during hot rod assembly and disassembly. The clippers are generally 
rectangular, of metal, and are pivotally mounted on pivot rods 706, which 
are in turn mounted to the base of hot rod 701. Spring assemblies 707 are 
adapted to urge clippers 706 so that their upper segments project into the 
lumen of the hot rod to a limited inward inclination, while permitting 
pivoting about mounting rod 706 outwardly through slots 705 in the side of 
hot rod 701. Thereby, when a reaction vessel or a sealing ball is inserted 
into hot rod 701 through base 708, clippers 704 pivot to freely admit the 
reaction vessel or sealing ball and then incline inwardly after the 
passage to retain the contents of hot rod 701. Further, pressure on the 
outwardly inclined lower segments of clippers 704 pivots them to permit 
the contents of hot rod 701 to drop through base 708. 
FIG. 19B illustrates details of a workstation adapted to the assembly of 
clipper-equipped hot rods and its method of operation. This workstation 
includes reservoir 720 for holding sealing balls and lifting mechanisms 
722 and 724 actuated by, e.g., electric or pneumatic means. Assembly of 
reaction vessels into vertical rod 726 includes the following two steps: a 
first step in which a sealing ball is placed into hot rod 726, and a 
second step in which a reaction vessel is placed in the hot rod. The first 
step begins when a gripper attached to a robot arm grips a hot rod and 
places it above upper opening 727 in extension 721 of sealing ball 
reservoir 720. Lifting mechanism 722 moves handle 723 upwards through 
lower opening 728 in the reservoir extension. Sealing ball 729 is pushed 
upward till it passes clippers 730 in hot rod 726. The clipper spring 
assembly retains the ball by urging clippers 730 into a closed position, 
i.e., a position that holds reaction vessels and sealing balls inside rod 
726 and prevents them from dropping below the clippers. After sealing ball 
729 is inserted past clippers 730, handle 723 actuated by lifting 
mechanism 722 returns through lower opening 728. For the second step, the 
robot arm moves hot rod 726 above second lifting mechanism 724 actuating 
handle 725 for reaction vessels. A second robot arm grips and positions 
array 731 of reaction vessels so that reaction vessel 732 is placed below 
the hot rod opening. Lifting mechanism 724 moves handle 725 upwards 
through an opening in array 731 and pushes reaction vessel 732 into rod 
726 past clippers 730. The insertion of sealing balls and reaction vessels 
into hot rod 726 repeats as many times as necessary to fill hot rod 726. 
Finally, after the hot rod has been fully loaded with reaction vessels and 
sealing balls, the assembly station places a retaining clip at the base of 
the hot rod to securely retain the reaction vessels in the hot rod during 
reaction processing. 
FIG. 19C illustrates details of a workstation adapted to the disassembly of 
clipper-equipped hot rods and its method of operation. In order to 
disassemble a loaded hot rod and place the contained reaction vessels into 
a rectangular array of reaction vessels, which was previously placed at 
this workstation, a robot arm, first, grips a loaded hot rod and removes 
the retaining clip. Next, the robot arm places hot rod 740 with closed 
clippers 741 over array or holding block 744, and a mechanical actuator 
opens the clippers by pressing their protruding lower ends towards the rod 
axis. Reaction vessel 745 drops from hot rod 740 through open clippers 742 
into array 744. The mechanical actuator then releases the clippers, and 
they again assume closed configuration 743 retaining sealing ball 746 of 
the just released hot rod. Next, the robot arm moves hot rod 740 over a 
sealing ball collection station. Sealing ball 746 is released from rod 740 
by similarly pressing and releasing the clippers. The sealing ball 
collection station can optionally, e.g., wash the balls for reuse. This 
process repeats until the hot rod is empty. 
Further special processing resources of this embodiment include an 
incubation station and a gripper/agitator tool. The incubation station, 
illustrated generally by ports 9 of FIG. 1, comprises circular ports in 
the work surface adopted to receive and support hot rods placed for 
incubation by a robot arm. Below the work surface, the hot rods are 
exposed to a flow of a temperature controlled fluid, such as heated or 
chilled air. During incubation, reaction vessels in the hot rods can be 
agitated by a gripper/agitation tool attached to a robot arm. This tool 
comprises a gripper adapted to grip the top of the hot rods in the ports 
of the incubation station and spin them in an off axis manner. Typically, 
the spinning axis is displaced off-center by a distance of approximately 
1/8 to 3/8 of the radius of the hot rod. 
5.2.2. Microtitre-Style Rraction Vessels 
Another embodiment of reaction vessel arrays of this invention comprises 
various commercially available microtitre-like plates having arrays of 
wells or vessels. Exemplary of such commercially available plates are 
standard microtitre plates with an 85.times.130 mm footprint and having a 
rectangular array of 96, 384, or more wells. Normal or deep well 
microtitre plates made of a solvent resistant material can be used in this 
embodiment. This embodiment is equally adaptable to other similar 
commercially available plates having arrays of wells or vessels. Reaction 
vessel arrays of this embodiment are sealed by various sealing means, 
including free sealing balls, an inflatable bag, a compressible plate, and 
a compliant ball assembly to be described in a subsequent subsection. 
FIGS. 4A-C illustrate exemplary embodiments of a microtitre plate sealed by 
sealing balls unattached to any support. In FIGS. 4A-B, microtitre plate 
100 has reaction vessels, a fraction of which are sealed by unattached 
sealing balls 102. In more detail, FIG. 4A illustrates microtitre plate 
100 having integral wells 101 serving as reaction vessels. A fraction of 
wells 101 have apertures sealed by unattached sealing balls 102. Upon 
complete sealing of a plate, all wells in the plate will be sealed by 
sealing balls. Sealing balls 102 are of a solvent resistant material, such 
as Teflon.TM., and are of a size sufficient to seal apertures of wells 101 
yet not so large that they mutually contact or interfere when all the 
wells of microtitre plate 100 are so sealed. FIG. 4B illustrates an 
alternative embodiment in which plate 100, perhaps a microtitre plate, 
supports reaction vessels 109, which can be vials or test tubes of a 
solvent resistant material such as polypropylene. In this embodiment, 
sealing balls 102 are similarly adapted to seal the aperture of vessels 
109. 
When unattached sealing balls are used, it is preferable to retain these 
balls tightly in place during movement of the reaction vessel array by the 
robot arms and during incubation, perhaps at an elevated temperature. FIG. 
4C illustrates an exemplary means for exerting a sealing and retaining 
force on unattached sealing balls 102, which are sealing the wells of 
microtitre plate 100. The illustrated retaining means comprises rigid 
plate 104, which is for example made of a metal such as aluminum or a 
rigid plastic, and whose face adjacent to the sealing balls is optionally 
covered with a compressible rubber-like layer made of silicone rubber. 
Plate 104 is retained against the sealing balls by spring clips 103 and 
105, which have arms engaging recesses 106 in microtitre plate 100. Clips 
103 and 105 have spring sections 107 for generating sufficient sealing and 
retaining force. This force should be at least approximately sufficient to 
resist internal vapor pressure generated during incubation. These clips 
have sections 108 which can be engaged by a standard gripper on the robot 
arm to permit placement and removal of the sealing means. These clips are 
similar to clips 113 of FIG. 5A-C. 
An alternative embodiment of sealing means for such reaction vessel arrays 
is illustrated in FIGS. 5A-C. The reaction vessel array is here 
illustrated as microtitre block 110 with deep wells 111. It will be 
immediately apparent how to adapt by scaling this sealing means to 
microtitre plates with standard wells. This sealing means comprises 
backing plate 108, inflatable bag 112, and retaining clips 114. Backing 
plate 108 is the base support structure for this sealing means and is 
preferably made of a metal, such as aluminum, or a rigid plastic. Bag 112 
is sized to cover all the well openings of microtitre block 110 and is of 
a flexible, inflatable solvent resistant material capable of tightly 
occluding all the wells of plate 110 when sufficiently inflated. Bag 112 
is preferably made of polypropylene or Teflon.TM. capable of being 
inflated to a pressure sufficient to retain the sealing balls against 
internal vapor pressures in wells. Pneumatic attachment 109 is for 
intermittently connecting to a pneumatic line for inflating or deflating 
bag 112. 
Clips 113 are designed for retaining the sealing means to block 110 and to 
permit the standard robot arm gripper tool to place and to remove the 
sealing means for the reaction vessel array. Accordingly, clips 113 
include horizontal tongues 115 for clipping securely into recesses 118 of 
microtitre plate 110. These clips further include lateral recesses 116 and 
upper horizontal shelf 117 designed to engage with gripper tool 123. 
Spring section 114 of these clips generates a vertical force for retaining 
these sealing means to plate 110 when bag 112 is uninflated and for 
exerting a vertical force against the inflation of bag 112. FIG. 5B 
illustrates bag 112 in an inflated condition, in which it tightly occludes 
all orifices of deep wells 111. FIG. 5C illustrates bag 112 in an 
uninflated condition, in which the sealing is retained against block 110 
by the spring force generated by clips 113. 
FIG. 5A also illustrates a multi-function gripper tool 123, which is 
adapted both for gripping and transporting reaction vessel arrays, such as 
microtitre block 110, and for placing and removing sealing means, such as 
those retained by clips, such as clip 113 or clips 103 of FIG. 4C. Gripper 
tool 123 is one of a plurality of interchangeable, specialized robot arm 
tools which attach to robot arm 120 through a standard attachment base 
contained in tool body 121. This attachment base is adopted to the 
particular robot arm used in an embodiment of this invention, and provides 
both for physical connection between a tool and the arm and for coupling 
of control and feedback lines, such as electrical or pneumatic control 
feeds between a tool and the arm. Tool body 121 also includes activating 
means, e.g., an electrical solenoid or motor or a pneumatic cylinder, for 
moving gripper fingers 122 horizontally. Gripper fingers 122 are 
vertically and horizontally offset from base 121 in order to minimize 
interference between robot arm 120 and any elements gripped by tool 123. 
Commercial suppliers of such a gripping tool include SAIC, Inc. (San 
Diego, Calif.) and Zymark, Inc. (Hopkington, Mass.). 
It is advantageous to minimize the number of specialized tools in an 
embodiment of this invention, both to minimize cost and to improve 
throughput, since tool interchange requires non-productive robot arm time. 
Accordingly, clips 113 are configured so that the standard gripper can 
place and remove sealing means retained by such clips. To place a sealing 
means retained by clips 113, gripper tool 123 engages lateral recesses 116 
of the clips by expanding gripper fingers 122 laterally. The robot arm 
then positions the sealing means over a reaction vessel array so that clip 
tongues 115 can engage array recesses 118. During this positioning, 
gripper fingers 122 can engage horizontal clip shelves 117 in order to 
lift the sealing means against gravity, and can engage fingers 115 to pull 
the sealing means down into position on a reaction vessel array. Then it 
moves fingers 122 inward horizontally, allowing clip tongues 115 to engage 
recesses 118, and then withdraws from engagement with clips 113. To remove 
such a sealing means, gripper tool 123 reverses these steps. Fingers 122 
first engage with lateral recesses 116 of clips 113; fingers 122 then move 
horizontally outward to disengage clip tongues 115 from array recesses 
118; gripper tool 123 finally lifts and removes the sealing means from 
reaction vessel array 110 by engaging against horizontal shelves 117. 
5.2.3. Arrays of Independent Reaction Vessels 
Another embodiment of reaction vessel arrays of this invention uses a 
compliant ball assembly to seal arrays of independent reaction vessels. 
However, such a ball assembly is not so limited and can be used to seal, 
not only the independent reaction vessel arrays of this embodiment, but 
also other arrays of reaction vessels having apertures, such as, e.g., the 
wells of microtitre plate embodiments. Alternately, the arrays of 
independent reaction vessels of this embodiment can be sealed by other 
sealing means, such as, e.g., those adapted also to seal microtitre plate 
embodiments 
FIG. 6A generally illustrates this embodiment. Individual reaction vessels 
150 are supported in a reaction vessel array, or holding block 151, which 
has both a standard form factor and a standard array structure or layout 
for the independent reaction vessels, according to a particular embodiment 
of this invention. In one exemplary embodiment, holding block 151 has a 
standard microtitre physical form factor of 85.times.130 mm and supports 
24 reaction vessels of 4 ml capacity in a standard 4.times.6 rectangular 
array layout. This embodiment is adaptable to other standard reaction 
vessel array layouts, array sizes, and reaction vessels. The reaction 
vessels of a reaction vessel array according to this embodiment can be 
sealed in various manners, including compliant ball assembly 152, and an 
assembly of valved caps, punctureable septum, and so forth, to be 
described in subsequent subsections. 
Generally, a compliant ball assembly comprises a plate to which are 
compliantly fastened an array of sealing balls having an arrangement 
designed to mate with the apertures of the reaction vessels in a reaction 
vessel array. The compliant attachment permits slight lateral motions of 
the sealing balls, in order to accommodate slight misalignments of the 
reaction vessels in the reaction vessel array, and also provides for 
applying a vertical sealing force to the sealing balls, in order to seal 
the apertures of the reaction vessels. The compliant attachment preferably 
permits lateral motions of no more than approximately 1/8 to 1/4 of the 
diameter of a sealing ball. By way of example, FIG. 6B illustrates 
reaction vessel 153, which has a slight lateral misalignment, that is 
nevertheless sealed by sealing ball 154, which has undergone a slight 
lateral motion to accommodate for the slight misalignment of reaction 
vessel 153. As in other sealing ball embodiments, previously, the sealing 
balls are of a solvent resistant material, such as Teflon.TM., and one 
sized to be at least approximately of a diameter equal to 11/8 to 11/4 
times the diameter of the apertures of the reaction vessels to be sealed. 
This embodiment includes alternative compliant attachment means of the 
sealing balls to the support plate that are known in the art. In 
particular, FIGS. 6B-C illustrate two preferred compliant attachment 
means. In the compliant attachment means illustrated in FIG. 6B, each 
sealing ball 154 is attached to a pin comprising pin head 156 and pin 
shaft 157. Aperture 158, pin shaft 157, and plate 160 are of such relative 
sizes that, although pin head 156 can not pass through aperture 158, pin 
shaft 157 has the desired range of lateral motion in aperture 158. Pin 
head 156 can be in recess 165 of plate 160, or alternatively, recess 165 
can be absent, in which case pin head 156 can be retained directly on the 
top surface of plate 160. Compressible layer 162 on the face of plate 160 
provides both a force resisting lateral sealing ball motion, thus 
establishing the compliance of the assembly, and a force for sealing balls 
154 into reaction vessels 153. Preferably plate 160 can be of a metal, 
such as aluminum or a rigid plastic, and compressible layer 162 is of a 
rubber, such as a rubber of foam silicone type. 
FIG. 6C illustrates an alternative compliant attachment means using springs 
163 in place of compressible layer 162. The sealing balls are similarly 
retained by plate 161 with pins having pin shafts 164 and pin heads 159, 
as in the previous compliant attachment means. However, in this means, 
both lateral and vertical force are provided by springs 163. The vertical 
force arises by direct vertical compression of springs 163. The lateral 
force arises due to compression of the springs when the wide pin heads 159 
rotate against the plate surface 161. 
The compliant ball assemblies of this embodiment can be retained to a 
holding block in various ways. In one alternative, the ball assemblies are 
retained to holding block 151 with spring clips in a manner similar to 
that illustrated in FIG. 5A, where spring clips 113 engage recesses 118 in 
block 110. Using such spring clips, the compliant ball assemblies can be 
placed and removed by a standard robot gripper tool in a manner similar to 
that disclosed in the previous embodiment. 
In another alternative, the ball assemblies are retained and sealed to 
reaction vessels by attachment assemblies illustrated in FIG. 6D. 
Generally, holding block 165 is retained by lower assembly 170, while 
compliant ball assembly 178 is retained by upper assembly 171. Both 
assemblies are held together by clips 177, and thereby retain the 
compliant ball assembly and reaction vessels in the holding block in 
sealing engagement. In more detail for the embodiment illustrated, holding 
block 165 holds an 8.times.12 array of independent reaction vessels, one 
of which is reaction vessel 166. Lower assembly 170 retains holding block 
165 in a central position, and has extensions 175 at two ends which in 
turn have recesses for accepting and engaging with clips 177. Clips 177 
automatically engage when the two assemblies are pressed together. 
Further, they have portions protruding from extensions 175 in order that 
they can be released by a gripper tool attached to a robot arm. Compliant 
ball assembly is of the embodiment illustrated in FIG. 6A, and includes 
sealing balls 167 restrained by compressible layer 168 and attached to 
backing plate 169. Illustrated reaction vessel 166 is sealed by one of the 
sealing balls. Upper assembly 171 retains the compliant ball assembly, and 
also has extensions 176 at two ends which in turn have support clips 177 
which engage with recesses in extensions 175 of lower assembly 170. The 
compliant ball assembly is retained to the upper assembly by means of 
spring-loaded tubes 172, which engage the ball assembly. Upper assembly 
171 includes regularly spaced apertures capable of accommodating tubes 
172. These tubes are retained to the upper plate either by a pressure fit 
into these apertures, by u-rings fitted on tubes 172 preventing motion 
through these apertures, or by other means known in the relevant arts. 
Preferably, upper plate 171 has ten regularly spaced apertures for so 
retaining ten tubes 172. Spring loaded tube 173 is illustrated in 
cross-section to reveal internal spring 174, which links the top of tube 
173 with ball assembly 178. The springs and containing tubes are sized 
such that, when the upper and lower assemblies are clipped together, the 
backing plate 169, and thus sealing balls 167, are urged against reaction 
vessels in holding block 165. Thereby, the upper and lower assembly 
achieve a sealing engagement between a compliant ball assembly and 
reaction vessels in a holding block. 
5.2.4. Valved Reaction Vessels 
An alternative sealing means for arrays of independent reaction vessels 
comprises valved caps. Preferred embodiments of such valved caps are 
simply constructed from inexpensive materials and are adapted so that all 
the valved caps in an array of reaction vessels can be simultaneously 
opened and closed by an inexpensive, easily constructed work station. The 
independent reaction vessels and the arrays of such reaction vessels are 
similar to those of the preceding subsection. This subsection describes in 
turn the structure of this sealing means, the opening/closing work 
station, and the method of their use in an integrated robot apparatus. 
FIG. 7 illustrates an exemplary preferred embodiment of a valved cap 
sealing means. Reaction vessels 200 have threaded tops and are constructed 
from glass or solvent resistant plastic. An exemplary reaction vessel, 
adaptable to a 4.times.6 rectangular reaction vessel array occupying a 
microtitre form factor, is preferably of approximately 4 ml capacity and 
of a size of approximately 45 mm long by 15 mm in diameter. The valved top 
includes screw cap 201, valve body 207, and valve rod 206. Alternatively, 
the screw cap and the valve body can be combined into a unitary structure. 
Cap 201, of solvent resistant plastic or metal, is adapted to the threads 
of reaction vessel 200 for retaining and sealing valve body 207 to 
reaction vessel 200. Valve body 207 includes circular base 202, for 
sealing the valve body to the reaction vessel under pressure generated by 
valve cap 201, and cylindrical valve head 203, having central cylindrical 
orifice 204 for permitting communication between the exterior and the 
interior of reaction vessels 200. Orifice 204 is preferably approximately 
one-half the diameter of valve rod 206, or approximately 1 mm in diameter. 
The valve body is preferably constructed of Teflon.TM. or other solvent 
resistant plastic. Valve rod 206 fits sufficiently snugly in orifice 208 
of the valve head to prevent fluid leakage around the rod through this 
orifice, and yet is capable of sliding laterally in this orifice in order 
to open or close the valves of this embodiment. 
In the configuration illustrated in FIG. 7, valve rod 206 occludes orifice 
204, fluid leakage around rod 206 is prevented, and the valve is closed. 
To open the valves, valve rod 206 slides laterally so that orifice 205 in 
the rod 206 is aligned with orifice 204 in valve head 203, thereby 
permitting communication between the exterior and the interior of reaction 
vessel 200. Importantly, all orifices 205 in valve rod 206 are spaced and 
positioned so that all of the valve bodies actuated by rod 206 are 
simultaneously opened at the same lateral position of rod 206. Rod 206 is 
preferably made of poly-vinyl difluoride ("PVDF") or stainless steel and 
is approximately 4 mm in diameter. This sealing means also includes such 
variants as, e.g., those in which valve body 207 is permanently attached 
to reaction vessel 200 by a metal or plastic retaining collar, those in 
which the dimensions recited are scaled to accommodate reaction vessels of 
differing capacities and sizes, and those in which valve rod 206 is 
rotated in order to seal the reaction vessels. 
The valved reaction vessels of this embodiment are disposed in arrays of 
reaction vessels. Preferably, such arrays permit the simultaneous opening 
and closing of the valves of all the reaction vessels in the array, and 
have a size and structure so that the standardized fluid handling work 
stations, incubation work stations, and other elements of this invention, 
which can be used for other reaction vessel array embodiments, can also be 
used for the valved reaction vessel arrays of this embodiment. 
Accordingly, FIG. 8A illustrates one such exemplary array of valved 
reaction vessels. Plate 211 holds by, e.g., a pressure fit twenty-four 
valve bodies 216 and attached valve caps 227 in a 4.times.6 rectangular 
array. Alternatively, valve bodies 216 can be screwed into plate 211 or 
attached by other known means. Using standard 4 ml reaction vessels 
disposed with a minimum of approximately a 5 mm spacing between the 
reaction vessels, this reaction vessel array is supported by holding block 
210 with a microtitre form factor of 85.times.130 mm. This invention is 
equally adaptable to other reaction vessel sizes, reaction vessel array 
sizes, and array structures in a manner apparent to one of skill in the 
art. Plate 211 has longitudinal lips 225 and 226 which are adapted so that 
plate 211 with reaction vessels 213 screwed into the attached valve caps 
can be supported in a specialized opening/closing work station described 
below. Plate 211 is preferably of a metal such as aluminum or a tough and 
rigid plastic such as Delrin.TM. (DuPont, Wilmington, Del.). Delrin.TM. is 
used generally herein to specify any of the Delrin type plastics or any 
plastics of equivalent chemical and physical properties. 
FIG. 8B illustrates a detail of plate 211 with the valve in an open 
configuration. Here, reaction vessel 219 is screwed into valve cap 220, 
which seals valve body 221 to the reaction vessel 219. Valve body 221 is 
retained in plate 211 by a pressure fit. Valve rod 208 slides freely and 
laterally in orifice 222 in plate 211 for opening and closing the valves. 
Valve rod 208 is illustrated in an open position, in which orifices 223 in 
the rod and the valve body are aligned permitting communication between 
the exterior and the interior of reaction vessel 219. To close this valve, 
rod 208 is moved laterally so that these orifices are no longer in 
alignment. 
All valve rods 228 in FIG. 8A actuating all of the valves of the array of 
reaction vessels have control orifices so spaced and are so affixed to 
linkage segment 212 such that all the valves in the array can be 
simultaneously opened or closed by lateral motion of linkage segment 212. 
Linkage segment 212 is preferably of a metal such as aluminum in which 
valve rods 214 are affixed by set screws 215, such as set screws with a 
pointed tip. When linkage segment 212 is so positioned laterally so that 
all the valves are opened, fluid can be aspirated or dispensed into any of 
the reaction vessels 213. For example, syringe 217 is dispensing a 
building block solution into one of the reaction vessels of the array. 
Finally, holding block 210 supports this reaction vessel array when it is 
not in a specialized work station. Holding block 210 has recesses 225 for 
closely accommodating reaction vessels 213 when attached to valve caps 227 
fixed to plate 211. Block 210 preferably affords heat conduction to 
reaction vessels 213, by being constructed of a heat conducting material, 
such as aluminum. 
FIG. 9 illustrates a specialized work station for opening and closing all 
the valves in the array of reaction vessels according to this embodiment. 
This is done by moving laterally linkage segment 241 of the array 231 
between its opened position and its closed position. A controllable 
actuator provides for such relative lateral motion between linkage 241 and 
array 231. In detail, this work station includes support blocks 232 and 
233, which support rods 238 and 239 and actuator 240. Holder 237 retains 
plate 231 supporting the array of reaction vessels in a relatively 
stationary position by engaging lips 243 and 244 of plate 231. Holder 237 
is supported by holder support 242, which slides on support rods 238 and 
239 under a force developed by actuator 240. End plates 235 and 236 limit 
the motion of holder 237, and end plate 236 specially also engages and 
fixes linkage segment 241. Accordingly, as actuator 240 generates a force 
moving holder 237 along support rods 238 and 239, the linkage segment, 
fixed by end plate 236, moves with respect to array 231 and all the valves 
in reaction vessel array 231 simultaneously opened or closed. Actuator 240 
is preferably a pneumatic actuator with pneumatic pressure feed 234, 
although this work station is adaptable to other actuators, such as 
electric actuators. The materials of this work station are preferably 
metal, such as aluminum, or a sufficiently tough and rigid plastic, such 
as Delrin.TM., except for the sliding members, which are preferably of a 
harder wear-resistant material, such as steel. 
An exemplary implementation of this work station is constructed from 
components manufactured by Bimba Manufacturing Company (Monee, Ill.). 
Blocks 232 and 233, rods 238 and 239, and actuator 240 are components of 
part number #UGS-12-02.000-A. Supports 235 and 236 are part number 
#UGS-4-0703. 
The following exemplary methods illustrate the use of the elements of this 
embodiment in an integrated robot during one step of building block 
addition. First the valved reaction vessel array, such as array 231 of 
FIG. 9, is placed by a robot arm in the specialized opening/closing work 
station of FIG. 9. This work station is then actuated to open all the 
valves in the reaction vessel array. The robot arm then places the opened 
reaction vessel array in a holding block, and moves the holding block, to 
wash solvent dispensing and aspirating stations for repetitive solvent 
washing. Next, building block and reagent additions are performed by fluid 
dispensing elements. The robot then moves the holding block with the 
reaction vessel array back to the specialized opening/closing work 
station, and places the reaction vessel array in this work station, which 
is then actuated to close all the valves in the array. The robot arm then 
places the closed reaction vessel array back in the holding block, and 
places the holding block with the reaction vessel array in a temperature 
controlled incubation work station, where it resides for a time and at a 
temperature sufficient for the building block addition reactions to go to 
substantial completion. The incubation work station is preferable below 
the work surface, and its access elevator is raised for robot arm access 
and lowered for incubation. Finally, in preparation for the next building 
block addition step, the robot arm removes the holding block from the 
incubation station and the reaction vessel array from the holding block, 
and places the array back in the opening/closing work station. 
This embodiment is adaptable to other valve means for sealing reaction 
vessels. FIGS. 10A-B depict one such alternative valve sealing means 
having compressible valve body compressed with a rigid valve rod. In FIG. 
10A, base plate 251 has threaded ports for receiving reaction vessels, 
which are preferably disposed in an array layout of a standardized 
structure. Plate 252 has cylindrical recesses 265 for receiving valve 
bodies 258 and for communicating with orifices 255. The orifices are 
aligned with the threaded ports in plate 251. Plates 251 and 252 can be 
made alternatively, of a metal, such as aluminum, or a rigid solvent 
resistant plastic. Compressible valve body 258 includes lips 257 for 
sealing reaction vessel 250 so that valve head 253 with orifice 266 
communicates with orifice 255 in plate 252. Valve body 258 is made of a 
flexible solvent resistant elastomer, such as Kalrez.TM. manufactured by 
DuPont (Wilmington, Del.). Reaction vessel 250 is screwed into the 
threaded port in plate 251 against lips 257 of valve body 258 and is 
thereby sealed. 
Valve rod 254, which moves in longitudinal slot 256 of plate 252, is at 
least sufficiently rigid to compress valve head 253 and thus to occlude 
orifices 255 in valve bodies 258. Preferably, this rod is further 
sufficiently rigid to be able to compress a plurality of valve heads 253 
linearly arranged in the reaction vessel array. FIG. 10A illustrates this 
valve in an open condition, in which valve rod 254 does not compress valve 
head 253, so that orifice 266 is open and communicates with orifice 255, 
permitting communication between the exterior and the interior of reaction 
vessel 250. FIG. 10B illustrates the valve of this embodiment in a closed 
configuration, in which valve rod 260 has moved laterally from open 
position 264, compressed valve head 261, occluded orifice 263, and thereby 
prevented communication between the interior and exterior of reaction 
vessel 267. Similarly to the previous embodiment, these alternative valve 
means and sealed reaction vessels can be supported in a reaction vessel 
array, which is sequentially placed in a specialized opening/closing work 
station or in a holding block during processing. 
5.2.5. Septum-Sealed Reaction Vessels 
A further alternative sealing means for independent reaction vessels 
comprises punctureable septums, either simple septums or septum assemblies 
adapted to resist greater internal reaction vessel pressures. Such a 
sealing means can comprise, alternatively, a single punctureable septum or 
assemblies including one or more punctureable septums together with other 
sealing elements. 
FIG. 11A illustrates a reaction vessel sealed with a single septum. Here 
reaction vessel 300 is preferably of approximately 4 ml capacity, and is 
made of glass or a solvent resistant plastic. Septum 301 is of a solvent 
resistant rubber material capable of being punctured by, e.g., a 14 gauge 
needle and then resealing itself. Septum 301 is preferably made of a 
Teflon.TM. coated rubber or of an elastomer of Kalrez.TM. type. Collar 302 
seals septum 301 to reaction vessel 300, and is of, for example, aluminum 
or plastic. This invention is adaptable to commercially available, 
inexpensive septum-sealed reaction vessels, such as the reaction vessels 
obtained from such suppliers as Phase Separations (Franklin, Mass.) or 
ColePalmer (Niles, Ill.). Septum-sealed reaction vessels are retained for 
processing in arrays of standardized structure by holding hold blocks of 
standardized sizes, as in other reaction vessel embodiments of this 
invention. One exemplary such holding block is holding block 151 of FIG. 
6A. 
These reaction vessels are processed by needle-based fluid manipulation 
tools and workstations. Fluids are dispensed into such reaction vessels 
with a needle-tipped dispensing tool, similar to those to be subsequently 
described. Fluids are preferably separated and removed from a solid-phase 
substrate contained in these reaction vessels by a fritted aspiration 
needle of this invention, illustrated in FIGS. 11A-B. In FIG. 11A, fritted 
needle 303 has been inserted into reaction vessel 300 and is aspirating 
fluid contained therein through a lateral orifice occluded by a fritted 
material. Although fluid can penetrate this fritted material relatively 
freely, solid-phase substrate 304 can not penetrate it, and is thereby 
retained in reaction vessel 300. In FIG. 11B, substantially all contained 
fluid has been aspirated through needle 310, leaving solid-phase substrate 
312 in reaction vessel 313. Any solid-phase substrate adherent to fritted 
needle 310, for example, substrate beads 311, are wiped off needle 310 and 
retained in the reaction vessel as the needle is withdrawn through 
resealable, punctureable septum 314. Alternatively, the fritted material 
may be replaced with other sieving means, such as an array of 
micro-drilled holes. Since adherent solid-phase material is not as fully 
removed from needles with end orifices containing a fritted material, such 
needles are less preferably used with these sealing means. 
FIGS. 12A-B illustrate an alterative septum assembly that achieves more 
thorough sealing and can be adapted to resist greater internal pressures 
in a reaction vessel. Turning to FIG. 12A, this exemplary embodiment of 
the alternative septum assembly, as adapted to reaction vessels 320 having 
threaded necks, includes screw cap 325, punctureable septums 322 and 324, 
compressible collar 323, and rigid seal 321. Screw cap 325 retains and 
seals the septum assembly to reaction vessel 320, and includes aperture 
327 for permitting access by needle-based fluid manipulating elements. 
Rigid seal 321 seals the septum assembly against the top of reaction 
vessel 320 with a rigidity sufficient to withstand pressures developed 
when screw cap 325 is screwed down onto the reaction vessel. It is, 
preferably, of a solvent resistant rigid plastic such as Teflon.TM. and 
has a central aperture for permitting needle access. Punctureable septums 
322 and 324 are standard septums similar to those of the previous 
embodiment, and are also preferably made, from an elastomer of Kalrez.TM. 
type. In a relaxed state, compressible collar 323 has central orifice 328 
for permitting passage of needle-based fluid manipulating elements. 
However, when vertically compressed, collar 323 is of a material such that 
orifice 328 becomes occluded. An exemplary material is the elastomer 
Kalrez.TM. manufactured by DuPont. 
This alternative septum sealing means is used according to the following 
method. When screw cap 325 loosely seals the components of the septum 
assembly against reaction vessel 320, as in FIG. 12A, central orifice 328 
of compressible collar 323 is open, and needle-based fluid handling 
elements can gain access to the interior of reaction vessel 320 through 
punctureable septums 322 and 324. In particular, as in the previous 
embodiment, this embodiment is adapted to the use of fluid removal 
elements based on a fritted aspirating needle, such as needle 326, or, 
when surface aspiration is used for fluid removal, on a flat-ended needle. 
When fluid handling is complete, screw cap 325 is screwed down on the 
threads of reaction vessel 320, vertically compressing the components of 
the septum assembly. Sufficient vertical pressure exerted between screw 
cap 325 and rigid seal 321 acts to compress compressible collar 323 and 
occlude orifice 328. FIG. 12B illustrates the septum assembly in a closed 
state, with collar 330 vertically compressed and orifice 332 occluded. 
The additional element of this embodiment of septum sealing means--the 
screw cap, the multiple punctureable septum, the collapsible collar, and 
the seal--act to more thoroughly seal the reaction vessel and to increase 
the resistance of this septum assembly to internal pressures generated in 
a reaction vessel. Thus a reaction vessel sealed with this assembly can be 
subjected to higher incubation temperatures, where necessary for a 
particular synthesis protocol. It will be apparent to one of skill in the 
art that variations are possible in this alterative embodiment. For 
example, where more or less sealing is required, there can be only one or 
there may be more than two punctureable septums; where more pressure 
resilience is required, there can be more than collapsible collar. 
5.2.6. Syringe Arrays 
This invention also includes arrays of syringe-like reaction vessels. 
Syringe-like reaction vessels are advantageous because their contents are 
always sealed by plungers, and, therefore, are never exposed to the 
atmosphere, thus avoiding the necessity of separate means to maintain 
reactions in an inert atmosphere. Syringe-like reaction vessels can be 
made in an integral form, using a single solvent-resistant block 
containing an array of cylindrical bodies, or can be assembled from 
individual, commercially-available syringes. Commercially-available 
syringes are advantageous because they are inexpensive and readily 
available. Such commercially-available syringes can be standard syringes 
manufactured for medical applications, having a capacity of approximately 
1-10 ml and made of solvent resistant materials such as polypropylene. 
Syringe-like reaction vessels include porous frits in their bases for 
retaining solid-phase substrates while permitting relatively free movement 
of fluids. A preferred frit is made of polypropylene with a 5-30 micron 
pore size, a porosity of 50%, and capable of retaining solid-phase 
microbeads with a diameter &gt;30 microns. Fluid handling for syringe arrays 
made according to either embodiment can, alternatively, be based on 
aspiration through needles from individual fluid storage vessels or on 
aspiration through a fluid distribution block from common fluid storage 
vessels. Exemplary syringe-like reaction vessel array layouts include a 
linear array of 8 syringes, an array of 24 syringes in a 4.times.6 
rectangular arrangement, and an array of 96 syringes in a 8.times.12 
rectangular arrangement. 
FIG. 17 illustrates an exemplary 1-dimensional array of 8 syringe-like 
reaction vessels formed in a single block and using attached needles for 
fluid handling. Block 550 is formed of a solvent resistent material, e.g., 
Teflon.TM. with holes forming bodies 551 of the syringe-like reaction 
vessels. Each syringe-like reaction vessel, in addition to syringe body 
551, has standard plunger 552, and frit 553 The frit in the base of body 
551 retains a solid-phase substrate. At the base of each syringe body 551, 
straight fluid passageways 554 are formed in block 550. Fluid passageways 
are interrupted by valve rod 555, made preferably of PVDF or stainless 
steel, and mounted for lateral or rotary motion. Valve rod 555 functions 
in a manner similar to that of valve rod 206 of FIG. 7. This rod contains 
fluid passageways spaced and oriented so that they can all be brought into 
alignment with fluid passageways 554 in block 550 in order that fluid 
communication is established between the interior of syringe bodies 551 
and the exterior of block 550. Thereby, by moving laterally or by rotating 
valve rod 554, the syringe bodies can be either sealed from or opened to 
the exterior. Needles 556 are attached to fluid passageways 554 to permit 
drawing and expelling fluids. A further element of this embodiment is a 
plunger holder similar to that illustrated in FIG. 18, which permits all 
the plungers in a syringe array to be accurately manipulated. 
A fluid handling work station adaptable to this embodiment of syringe-like 
reaction vessels includes a support means for securely retaining block 
550, and a plunger activation means for accurately positioning a 
plunger-holder attached to plungers 552 of the syringes. By manipulating 
the plunger-holder, fluids can be drawn into or expelled from all syringe 
bodies 551 of block 550. In this embodiment, solutions containing building 
blocks are stored in individual storage vessels. To dispense particular 
building blocks into particular syringes, the storage vessels containing 
the building block solutions are placed by the robot arm in an array 
having a layout conforming to the layout of needles 556 and in an 
arrangement such that all the correct building block solutions can be 
drawn into all the correct needles simultaneously. Needles 556 are then 
submerged in the building block solution in the storage vessels, and the 
syringe plunger-holder is accurately positioned outward to withdraw 
controlled aliquots of the building block solutions into syringe bodies 
551. Similarly, for reagents and wash solvents, which are typically 
distributed to all the syringes in an array simultaneously, storage 
vessels containing these fluids are arranged in a storage array such that 
needles of the syringe array can also be submerged in all the storage 
vessels of the storage array simultaneously. Outward positioning of all 
the plungers of the syringe array at once then withdraws these fluids into 
the syringes. Such storage vessels can advantageously be similar to the 
septum-sealed reaction vessels of FIG. 11A, and retained in a storage 
array in a holding block similar to the holding block of FIG. 6A. 
Alternatively, these storage vessels and storage arrays can be similar to 
other reaction vessel embodiments previously described. Spent fluids can 
be expelled from the syringes into collecting devices for discarding by 
positioning inward all the plungers of a syringe array. 
During a building block addition reaction, syringe arrays can be optionally 
agitated, or optionally placed in a temperature controlled incubation work 
station. Syringes of this embodiment can be arranged in other one and two 
dimensional array layouts and used with storage arrays having a 
corresponding structure. 
FIG. 18A illustrates a second exemplary embodiment of a 1-dimensional array 
of 8 commercially available syringes attached to a fluid distribution 
block. In this embodiment, the syringes do not use needles for fluid 
handling. This embodiment includes an array of separate syringes 600 
attached to fluid distribution block 601, which is shown partially cut 
away to reveal its internal passageways. Each syringe 600 has plunger 602, 
syringe nipple 606, and porous frit 603 at the bottom. The frit retains a 
solid-phase substrate while allowing relatively free flow of fluids. 
Syringes are preferably made of a solvent resistant plastic material, e.g. 
polypropylene, or glass, with plungers made of polypropylene or 
Teflon.TM.. Plungers are fixed to plunger-holder 604. Manipulation of 
plunger-holder 604 moves all plungers 603 equally and simultaneously for 
drawing or expelling equal volumes of fluid in all syringes 600 in the 
array at the same time. Syringes 600 are mounted on syringe-holder 605, 
which presses syringe nipples 606 tightly into holes in block 601 to 
prevent any fluid leakage. Block 601 contains internal fluid passageway 
607 that connects all syringe nipples 606 to common port 608. Each syringe 
600 is also connected to punctureable septum 610 by straight fluid 
passageway 609. Punctureable septa 610 and passageways 609 allow 
needle-based fluid distribution tools to introduce particular reagents and 
building block solutions separately into each syringe. 
As for the first embodiment, a fluid handling work station adaptable to 
this embodiment of syringe-like reaction vessels includes a support means 
for securely retaining block 601, and a plunger activation means for 
accurately positioning plunger-holder 604 attached to plungers 602 of 
syringes 600. Additionally, external fluid handling means, of which FIG. 
18B illustrates an exemplary embodiment, are attached to common port 608 
of FIG. 18A. First, for distribution of particular fluids to particular 
syringes, such as solutions such as solutions containing building blocks, 
common port valve 619 of FIG. 18B is closed, e.g. by electromagnetic or 
pneumatic means, to seal the common port against entry of unwanted fluids. 
Next, an array of needle based fluid distribution tools, having a needle 
layout conforming to the layout of septa 610 of FIG. 18A, is charged with 
building block solutions in an arrangement such that the correct building 
block solutions can be drawn into all the correct syringes. These 
distribution tools are manipulated so that their needles puncture septa 
610 and advance into passageways 609. Finally, the plunger-holder is 
withdrawn and fluids from the distribution tools are drawn into syringes 
600 as plungers 602 move outward by a determined amount. 
Next, for distribution or removal of fluids from all the syringes of an 
array simultaneously, such as solvents or common reagents, the exemplary 
apparatus illustrated in FIG. 18B can be used. Here, a plurality of 
reservoirs 615, storing common fluids are connected through tube 616 to 
switching valve 617, then through exhaust valve 618, and finally to common 
port 622 in block 623 by tube 620. To dispense common fluids to all the 
syringes, controllable switching valve 617 selects the reservoir from 
which to withdraw fluids; exhaust valve 618 is directed to common port 622 
through tube 620. When the plunger-holder withdraws all the syringe 
plungers, common fluids are drawn into the syringes. To expel fluids from 
all of the syringes, common port valve 619 is opened and exhaust valve is 
directed to fluid exhaust tube 621. When the plunger-holder is manipulated 
to depresses plungers into the syringes, fluids are expelled while the 
solid-phase substrate is retained by the frit in each syringe. The tubing 
used is preferably made of Teflon.TM. or other solvent resistant plastic. 
The fluid handling tubing, passageways, and valving illustrated in FIG. 
18B are exemplary, and this embodiment is adapted to other arrangements of 
these elements which achieve similar results as are apparent to those of 
skill in the art. 
In alternative embodiments, fluid handling means according to FIGS. 18A and 
18B can be adapted to syringes constructed integrally in one block. Also, 
arrays of commercially available syringes are adaptable to the needle 
based fluid handling means as illustrated in FIG. 17. Also, although this 
apparatus was described for use in an automated robotic apparatus, it can 
be adapted for use as a standalone device, in which case it can be 
optionally manually actuated. 
5.3. Fluid Handling Means 
Fluid handling means of this invention, which are adapted to the previously 
described reaction vessel arrays, include specialized work stations and 
robot arm tools for dispensing and aspiration of fluids, including fluids 
containing slurries such as microbeads. Preferably, fluid dispensing means 
are specialized either for solvent washing or for dispensing individual 
building block solutions and reagents. On one hand, fluid handling means 
for solvent washing are preferably specialized for rapid and repetitive 
solvent distribution to most, or to substantially all, of the reaction 
vessels of an array. For solvent washing, rapid fluid distribution and 
removal is more advantageous then accurate fluid distribution. On the 
other hand, dispensing building block solutions and reagents is done once 
per synthesis step with, preferably, greater accuracy. Since, typically, a 
separate building block solution is dispensed to each separate reaction 
vessel in a reaction vessel array whereas the same few reagents are 
dispensed to all reaction vessels in an array in which the same protocol 
is being performed, fluid handling means are advantageously specialized in 
various embodiments of this invention into separate means for building 
block dispensing and for reagent dispensing. 
Accordingly, this section sequentially describes fluid dispensing means of 
this invention, a preferred embodiment of a fluid handling workstation 
means, and fluid and slurry dispensing means. 
5.3.1. Fluid Aspiration Means 
Fluid aspiration work stations or tools of this invention preferably 
aspirate fluids from reaction vessels while leaving behind in the reaction 
vessels substantially all of a solid-phase support to which intermediate 
compounds are attached. Since solid-phase supports include easily 
aspirated microbeads, the fluid removal elements are based on specialized 
aspiration needles or specialized aspiration methods which prevent 
microbead aspiration. 
The specialized aspiration needles of this invention include a filtering or 
sieving means in the needle to prevent aspiration of solid-phase 
microbeads while permitting relatively free flow of fluid in both 
directions. Such a filtering means has a pore or hole size which is 
preferably 5-50% of the typical solid-phase bead size. A typical pore size 
is in the range of 5-50 microns. In one embodiment, such a filtering means 
can be an array of micro-holes drilled by a laser micro-drilling 
apparatus, as is known in the art and is commercially available. Another 
embodiment of a filtering means includes a frit, preferably a 
polypropylene frit with a porosity 35 .mu.m, such as is supplied by 
Bel-Art Products, Inc. (Pequannock, N.J.). These or other filtering means 
can be placed either laterally in the side an aspiration needle, or 
terminally in the end of an aspiration needle. Lateral placement is 
preferred because this configuration is self-cleaning when used with 
septum-sealed reaction vessels. For reactions vessels not sealed with 
septums or with septum-sealed vessels and a needle with a terminally 
placed frit, before an aspiration needle is withdrawn, any adherent 
solid-phase support is advantageously removed by back flushing a small 
amount of the aspirated fluid. 
FIG. 13 illustrates an exemplary preferred aspiration needle structure with 
a laterally placed frit. To aspirate the maximum percentage of fluid from 
a reaction vessel, this needle is preferably long enough to reach to the 
bottom of a reaction vessel and is constructed with a minimum distance 
from the bottom of aperture 351 to the end of tip 353. Exemplary, 
dimension for needle 350 are an inside diameter of approximately 0.039 
inches and an outside diameter of approximately 0.050 inches. Aperture 351 
in needle 350 is lateral, oval shaped, and approximately 0.1 inches long. 
Pointed tip 353 has shank 354 pressure fit into the blunt end of needle 
350, and is of approximately 0.19 inches overall length with a shank of 
approximately 0.06 inches. Cylindrical frit block 352 has a length of 
approximately 0.12 inches and is positioned against the end of shank 354 
in order that the edges 355 of the aperture 351 overlap the cylindrical 
frit block by no less than at least approximately 0.01 inches. Cylindrical 
frit block 352 is of a diameter so that it is held in position in needle 
350 against aspiration suction by static friction between the frit block 
and the inside surface of the needle. The dimensions recited herein are 
not limiting and the fritted needle can be scaled in a manner known in the 
art to dimensions appropriate for needles of other gauges, for example, of 
gauges 12-18. The use of this fritted needle with septum-sealed reaction 
vessels has been described in a previous subsection. 
FIG. 14 illustrates an alternative fluid removal method using a surface 
aspiration method of this invention. This method is based on the discovery 
that aspiration by a blunt ended needle at the surface of a fluid 
containing a settled solid-phase does not disturb that solid-phase, 
because such surface aspiration generates only radially in-flowing fluid 
currents confined to the surface of the fluid. It generates no currents in 
the bulk of the fluid that could disturb the settled solid-phase. Thus, in 
FIG. 14, reaction vessel 365 contains liquid phase 361 with settled 
solid-phase 362, such as solid-phase microbeads. Blunt-toucd needle 360 
just touches surface 363 of liquid phase 361, and aspirates fluid 361 
under an applied partial vacuum. During aspiration, needle 360, reaction 
vessel 365, or both, are moved relatively so that needle 360 remains in 
contact only with surface 363 of liquid phase 361. For example, needle 360 
can be lowered or reaction vessel 365 can be raised. Thereby, this 
aspiration is carefully controlled so that only radially in-flowing 
surface currents 366 in fluid 361 are generated, such surface currents not 
disturbing settled solid-phase 362. Needle 360 can be, e.g., a blunt ended 
stainless steel needle of a gauge approximately from 12-18. 
The relative motions of needle 360 and reaction vessel 365 can be 
controlled in various manners, including closed-loop or open-loop methods. 
In a closed-loop method, these relative motions are adjusted according to 
observations made during aspiration, e.g., of the vacuum present in needle 
360, or of the rate of fluid aspiration in conjunction with the area of 
surface 363. In a preferred open-loop method, these motions are adjusted 
based on prior observations or experiments of surface aspiration under 
controlled conditions. For example, observations of aspiration rates of 
fluids of various viscosities through needles of various gauges subject to 
vacuums of varying amounts can be compiled, and later used to select a 
relative motion for a particular needle aspirating a particular fluid 
subjected to a particular vacuum in a particular embodiment. 
5.3.2. Fluid Handling Workstations 
Fluid removal tools or work stations utilize these previously described 
removal means. For improved throughput, it is preferable to implement 
fluid removal as a work station or tool capable of removing fluid from 
substantially all, or all, of the reaction vessels in a particular 
reaction vessel array in one operation. For example, removal of spent 
reagents at the end of a building block addition step can typically be 
done simultaneously for all the reaction vessels in an array. Also, since 
solvent washing can be done simultaneously for all of the reaction vessels 
in an array, removal of the wash solvent is preferably done simultaneously 
from all reaction vessels in an array. 
Further, for throughput and economy, it is advantageous that a fluid 
removal station can also serve as a wash solvent dispensing station. Such 
a multi-purpose fluid aspiration/dispensing work station can improve robot 
throughput, by eliminating transfers of reaction vessel arrays between 
separate aspiration and dispensing stations during a solvent washing 
steps, and can minimize robot cost by requiring fewer separate components. 
In this regard, both preferred aspiration methods are also adapted to 
fluid distribution. Fluid may be freely distributed through the blunt 
ended needles used in the surface aspiration method, or may be relatively 
freely distributed through the fritted needles of this invention. 
Alternatively, and less preferably fluid removal by either the fritted 
needle or the surface aspiration method can be implemented as a tool or 
work station for removing fluid from a single reaction vessel or from only 
part of a reaction vessels array. 
FIG. 15 illustrates an exemplary embodiment of a multi-purpose fluid 
aspiration/dispensing work station implementing these preferred features. 
This station includes a support base and a fluid aspiration/distribution 
assembly with an array of needles. In the case of aspiration by using the 
surface aspiration method, this station also optionally can include means 
for providing relative motion between the aspiration needle array and the 
reaction vessel array. Therefore, base 407 provides for overall support of 
this work station, including rigid attachment of support rods 409. Fluid 
aspiration/distribution assembly 400 can be variably positioned on support 
rods 409 by, e.g., thumb screws 408. Distribution assembly 400 includes 
top plate 401, bottom plate 402, and plenum 403. Top plate 401 provides 
for the attachment of distribution assembly 400 to support rods 409. Top 
plate 401 also carries an optional number of sensors, which can be used in 
closed-loop methods of controlling surface aspiration, or simply for 
monitoring fluid handling. Illustrated here is vacuum sensor 410 for 
monitoring effective fluid aspiration vacuum. 
Bottom plate 402 carries an array of aspiration/dispensing needles 412 
arranged and spaced to match the arrangement and spacing of apertures of 
reaction vessel arrays. Therefore, one embodiment of this work station has 
bottom plate 402 with 96 needles 412 spaced in a 8.times.12 rectangular 
array to match reaction vessel arrays having 96 rectangularly arranged 
wells, and another embodiment has bottom plate 402 with a 4.times.6 array 
of needles conforming to reaction vessels arrays having 24 rectangularly 
placed reaction vessels. Needles 412 attached to bottom plate 403 can be, 
alternatively, fritted needles, flat ended needles for surface aspiration, 
or needles adapted to other fluid aspiration or distribution methods. 
This workstation can be adapted to various arrangements of reaction vessels 
in arrays by merely replacing bottom plate 402, with its array of needles 
412, with other bottom plates having needle arrays conforming to the 
reaction vessel array layouts to be serviced. For this interchangeability, 
it is advantageous in an embodiment of this invention, that reaction 
vessel arrays preferably have one standard footprint size. 
Plenum 403 communicates between the base of needles 412 and a source of 
fluid for dispensing or a source of vacuum for aspiration. In one 
embodiment, a controlled valve can switch the plenum between a fluid 
source and a vacuum source. Here, plenum 403 is illustrated as permanently 
connected via connecting tube 404 to vacuum source 406 through fluid 
collecting reservoir 405. 
In an alternative embodiment, this multipurpose workstation is instead 
specialized into either a fluid dispensing or a fluid aspiration function. 
In the case of a fluid dispensing workstation, plenum 403 is 
advantageously made smaller, so that less fluid is in transit during the 
dispensing process. In the case of a fluid aspirating workstation, plenum 
403 is advantageously made larger, so that the vacuum or suction is 
maintained more evenly across all of the aspirating needles. 
This multipurpose fluid aspiration/dispensing work station is used with 
reaction vessel arrays according to the following method. Reaction vessel 
array 414 is placed in an aspiration/dispensing work station with a 
conforming array of needles 412 by a robot arm with an attached gripper 
tool. For fluid dispensing and for fluid aspiration with fritted needles, 
the robot arm alone can position a reaction vessel array 414 in the work 
station and raise reaction vessel array 414 so that the needles penetrate 
fully into the reaction vessels. Alternatively, the work station can be 
provided with a means to raise and lower reaction vessel arrays. 
Preferably, a reaction vessel array is positioned so that the needles 412 
will penetrate substantially to the bottom of the reaction vessels. 
Various mechanical or electrical feedback means can assist this 
positioning. Here illustrated is a mechanical feedback means composed of 
rod assembly 411, which provides a mechanical stop limiting motion of the 
reaction vessel array at the point of maximum needle penetration. 
For fluid aspiration according to the surface aspiration method controlled 
according to an open-loop method, the reaction vessel array is raised at a 
fixed and pre-determined rate during aspiration. Again, this can be done 
by the robot arm alone, if it has sufficient position resolution to raise 
the reaction vessel array accurately at the pre-determined rate. 
Alternatively, the work station can have means for automatically and 
accurately raising the reaction vessel array at the pre-determined rate. 
Illustrated here is an embodiment of a raising means advantageous for use 
of this work station in a stand-alone and manual environment. Standard 
laboratory jack 415 is manually adjusted to raise reaction vessel array 
414 at the correct rate for fluid aspiration. 
5.3.3. Fluid and Slurry Dispensing Means 
Fluid dispensing means are advantageously adapted to handling fluids alone 
or to handling fluids with slurries. Concerning dispensing means for 
fluids alone, as previously described, those for building block solutions, 
activating reagents, and other reagents are specialized differently from 
those for solvent dispensing. Since each reaction vessel in a reaction 
vessel array typically receives a separate one of perhaps hundreds of 
building block solutions, it is advantageous that building block solutions 
be dispensed by one, or at most a few common tools or work stations 
capable of dispensing solutions from one of a plurality of storage vessels 
of similar structure. Other embodiments, for example with a separate robot 
arm tool or work station for each building block solution, are less 
desirable in that such alternatives would typically require more storage 
volume or more work surface and cost more than the preferred embodiment. 
Accordingly, and in a preferred embodiment, building block solutions are 
stored in commercially available syringe bodies having a capacity of, 
e.g., from approximately 10 to 50 ml. The robot arm gripper/dispensing 
tool removes such a syringe body from a storage rack, positions it for 
dispensing building block solutions into a correct reaction vessel, and 
then actuates the syringe plunger to accurately dispense an aliquot of the 
solution. When not being used, a syringe body containing a building block 
solution is stored in a below work surface storage station, which is 
raised above the work surface when needed for access by the robot arm. 
Robot arm tools or work stations for distributing activating reagents or 
other reagents are advantageously designed for simultaneously and 
accurately dispensing such fluids to the several reaction vessels in an 
array, or to all the reaction vessels in an array, in which a single 
protocol is being performed. Since each synthesis protocol typically 
requires only a few common reagents, e.g., 2 to at most 6, it is 
advantageous for embodiments of this invention to have specialized reagent 
distribution tools or work stations for each required reagent. Therefore, 
a preferred exemplary reagent distribution tool, has a one or two 
dimensional array of fluid dispensing tips arranged and spaced to dispense 
reagents into the preferred reaction vessel arrays. Thus, there are 
separate embodiments of these tools for the rectangular reaction vessel 
arrays having 24 or 96 reaction vessels. In FIG. 16A, tool 460 has a 
one-dimensional array of dispensing tips, and in FIG. 16B, tool 470 has a 
two-dimensional array of dispensing tips. 
Generally, these tools include an internal storage vessel for a fluid, a 
precisely controllable fluid pump, an array of dispensing tips, and tubing 
interconnecting the storage vessel, the pump and the array of tips. An 
exemplary fluid pump is a piston fluid pump manufactured by Cavro 
Scientific Instruments, Inc. (Sunnyvale, Calif.), with a model number 
XL3000. Finally, although it is preferable to distribute wash solvent at 
work stations such as that illustrated in FIG. 15, optionally, wash 
solvents can be distributed by tools similar to those illustrated in FIGS. 
16A-B. 
An additional class of fluid handling tools dispenses and aspirates 
slurries, for example, slurries of microbeads in a solvent. Such tools can 
be used to distribute fresh solid-phase substrate to reaction vessels 
prior to performing a synthesis protocol. Also, such tools permit the 
solid-phase substrates from a selected reaction vessels to be partially 
transferred to other selected reaction vessels, during, for example, 
various split synthesis protocols, in which multiple compounds are 
synthesized attached to one solid-phase microbead or in which one reaction 
vessel contains a mixture of microbeads, each microbead having attached a 
single synthesized compound. 
Generally, solid-phase substrate in the form of microbeads are dispensed 
similarly to reagents. Such microbeads are maintained in a slurry in an 
appropriate solvent, and aliquots of the solvent containing the slurry are 
dispensed by a fluid distributing tool for slurries. A suitable solvent is 
dimethylformamide, and a suitable means to maintain the beads in a slurry 
is to bubble an inert gas, such as nitrogen, into a storage vessel 
containing the microbeads and the solvent. 
In more detail, FIGS. 20A-B illustrate an exemplary apparatus for 
aspirating and dispensing slurries. This apparatus includes slurry 
container 800, made of glass or an inert plastic material, source of 
suction 801, fluid delivery module 802, valve 803, and connecting tubing 
807. Container 800 has two openings, opening 804 located at the bottom 
with an attached narrow plastic or steel tube 806, and opening 805 at the 
top of the container connected to suction source 801 via valve 803. Valve 
803 can be electromagnetically or pneumatically actuated. Tube 806 can be 
a standard needle, e.g., a 14 gauge needle. Fluid delivery module 802, 
which can be a syringe or a piston pump, is attached to connecting tubing 
807 by a "T" piece. 
Combining slurries from one or more reaction vessels into one or more other 
reaction vessels proceeds by a first step, in which slurries are aspirated 
from reaction vessels, and a second step, in which the aspirated slurries 
are dispensed. For the first step, valve 803 connecting container 800 with 
a suction source 801 is opened, and tube 806, attached to container 800, 
is immersed into reaction vessel 808 containing slurry 809, e.g., of resin 
beads. The suction draws slurry 809 into container 800 from reaction 
vessel 808. Next, container 800 with tube 806 is moved into those further 
reaction vessels, if any, whose slurries are to be combined, and this 
process is repeated. Distribution of a fresh solid-phase substrate into 
reaction vessels prior to a synthesis proceeds similarly, except that this 
first step is replaced by aspiration of the fresh substrate from a storage 
container holding fresh substrate, e.g., microbeads in a solvent. 
For the second step, fluid delivery module 802 is adjusted to dispense a 
volume of air equal to the volume of slurry to be distributed from 
container 800 into a reaction vessel, e.g., reaction vessel 808. Then 
container 800 with tube 806 is moved above this reaction vessel, valve 803 
is closed, and the determined and adjusted volume of air is introduced 
into container 800 by fluid delivery module 802. Thereby, an equal volume 
of slurry 810 from container 800 is dispensed into reaction vessel 808. 
After the first portion of the slurry is delivered, valve 803 is opened 
again, to facilitate mixing of the resin by the suction. 
To prevent non-uniform distribution, the slurry can be preferably 
distributed in 2 or 3 phases. In a first phase, preferably 90-95% of 
slurry 810 is distributed into reaction vessels according to the previous 
process, leaving approximately 5-10% of the slurry remaining in container 
800. Then this 5-10% residual slurry is diluted to the original volume by 
solvent, and a second distribution phase is performed, which leaves 
approximately only 0.25-1% of the slurry remaining. For even more complete 
distribution, this process can be repeated for a third phase. 
The suction produced by suction source 801 serves two purposes. First, it 
serves to draw a slurry into container 800. Second, it serves to keep 
slurry 810 in container 800 mixed and homogeneous during the distribution 
process by drawing air or inert gas bubbles through the slurry. Therefore, 
suction source 801 should generate a moderate suction sufficient to 
accomplish these purposes, yet not strong enough to draw any slurry 810 
back into connecting tubing 807. Further, a solvent used for phased 
distribution should be chosen among those with a high boiling point to 
prevent evaporation during distribution, e.g., dimethylformamide. 
FIG. 20B illustrates an alternative embodiment of this apparatus for 
handling those slurries that must be protected from air. Such slurries 
include, for example, hydrides. In this embodiment single tube 806 is 
replace by coaxial structure 815 including and inner tube 811 and a 
coaxial outer tube 812. Inner tube 811 communicates with the interior of 
container 800 for aspirating and dispensing slurries. Outer, coaxial tube 
812 surrounds inner tube 811 and protrudes beyond the end of inner tube 
811. It includes a port 813 through which an inert gas, for example argon 
or nitrogen, can be introduced under a small pressure. Thereby, inner tube 
811 is maintained in an inert atmosphere, and suction source 801 draws 
only this inert atmosphere through the slurry. Additionally, only this 
inert gas is introduced into delivery module 802. In this manner, a slurry 
can be exposed only to the required inert atmosphere. 
In the exemplary embodiment described, this apparatus is configured as a 
tool for attachment to a robot arm. Thus, array 814 of reaction vessels is 
stationary, and container 800 with attached tube 806 is moved form one 
reaction vessel to another by the robot arm. Alternatively, this apparatus 
can be configured as a stationary workstation. In such an embodiment, 
container 800 is fixed by a support, and array 814 of reaction vessels is 
moved under tube 806 and then lifted for access to individual reaction 
vessels by, e.g., a robot arm with a gripper tool. A work station 
embodiment is preferred if arrays of reaction vessels are moved between 
other work stations by robot arms. 
All the tools and work stations adapted to fluid handling, which have been 
described in embodiments directed to robotic synthesis systems, can be 
easily and usefully adapted to use in bench environments in which manual 
operations replace robotic operations. In such a bench environment, manual 
actuation of work station controls can replace automatic actuation where 
useful. In particular, the slurry distribution tool can be configured for 
standalone use without robot arms. In this case, valve 803 and suction 
source 801 can be either manually or automatically controlled. 
Additional robot arm tools and work stations adapted to the previously 
described reaction vessel arrays include additional types of robot arm 
grippers, temperature controlled incubation stations, agitators for 
reaction vessel arrays, tools for tightening or loosening reaction vessel 
caps, and so forth. 
5.4. Control Elements 
It is advantageous that substantially all processing elements of an 
automatic synthesis robot according to this invention be under automatic 
control having certain facilities. Individual processing resources or 
facilities--for example, robot arms, work station, and tools--should be 
controllable in order that final compounds can be completely and 
automatically synthesized from input building blocks and reagents 
according to various and different synthesis protocols. The automatic 
control should also be sufficiently general that a different final 
compound can be synthesized in each reaction vessel of each array of 
reaction vessels present in the robot, and that a different combinatorial 
synthesis protocol can be performed in the reaction vessels of each 
reaction vessel array present in the robot. Finally, the automatic control 
should be able to manage a plurality of reaction vessels, arrays of 
reaction vessels, work stations, robot arms, and robot arm tools so that 
all of the processing resources or facilities of an embodiment of this 
robot are optimally engaged or facilities in performing tasks for the 
synthesis being carried out by the robot. One exemplary criterion for 
optimal employment of robot processing resources is that all such 
resources be utilized, or busy, for approximately the same fraction of 
processing or synthesis time. In this manner no one resource is a 
bottle-neck causing under-utilization of other resources or processing 
delays for compound synthesis. 
Although fully automatic control is preferable, this invention is adapted 
to controls requiring manual intervention for certain, or even all, 
processing steps. 
These automatic control facilities are supported by certain hardware and 
software elements. General hardware elements preferably include one or 
more general control computers, an optional number of specialized control 
processors, and electrical interfaces to all controlled processing 
resources of the robot. In a manner known in the art, all the directly and 
indirectly controlled resources of the robot can be provided with 
electrical interfaces having certain standardized electrical 
characteristics. Certain of these low-level hardware interfaces are 
directly linked from their standardized interfaces to interfaces of the 
general control computers. Optionally, for complex resources, such as 
complex work stations or the robot arms, an intermediate level of 
specialized control processors is interposed between the general control 
computers and the low-level electrical interfaces of such resources. 
The general control computers can be sufficiently capable personal 
computers provided with such specialized electrical interfaces. An 
exemplary personal computer includes an Intel Pentium.TM. processor 
running at 133 Mhz, a 1 gigabyte or greater hard drive, 16 megabytes or 
more of memory, and commercially available interface boards providing 
interfaces such as D/A or on/off output circuits or links to standard 
instrument control buses. These hardware control elements are provided by 
such commercial suppliers as SAIC, Inc. 
General software elements executed by the general control computers include 
operating system software, low-level moment-to-moment control and 
monitoring software, robot scheduling and monitoring software, and 
synthesis planning software. At the lowest software level is the operating 
system software of the general control computers, which in an exemplary 
embodiment, can be Unix.TM. or Windows NT.TM. (Microsoft Corporation). The 
low-level moment-to-moment control and monitoring software inputs scripts 
describing in detail robot actions to perform and outputs electrical 
control signals to the controlled processing resources through the 
interfaces attached to the general control computers. These signals cause 
robot and work station actions to be performed. At the next software level 
is robot scheduling software, which inputs a description of the synthetic 
steps to be performed, the locations of stored building blocks and 
reagents, the location and type of available work stations, the location 
and type of available interchangeable tools, and so forth, and outputs the 
detailed command scripts controlling robot functions. These scripts are 
interpreted by the moment-to-moment control and monitoring software. At 
the highest software level is chemical synthesis planning software, which 
inputs a description of the synthetic protocols available in a particular 
embodiment of this robot and the desired compounds to be synthesized, and 
then outputs the synthetic steps necessary to synthesize the desired 
compounds in a form usable by the scheduling software. An exemplary 
embodiment the low-level moment-to-moment control software and the 
scheduling software are supplied by SAIC, Inc. 
Tables 1, 2, and 3 illustrates exemplary robot control scripts, such as 
those output by the scheduling software and input by the moment-to-moment 
control software in order to generate electrical signals actuating 
controlled elements of the robot. These scripts are directed to the 
embodiment of the robot illustrated in FIG. 1 processing ball-sealed, 
stackable reaction vessels according to the method illustrated in FIG. 3. 
These scripts provide for arm control by using such elementary commands 
as: grip, for having a robot arm grip a reaction vessel array with a 
gripper tool; transfer, for causing a robot arm to move a gripped array 
between two locations automatically avoiding intervening obstacles; 
interchange, for having a robot arm attach a new arm tool; and activate, 
for having a robot arm activate and use a particular tool. 
TABLE 1 
______________________________________ 
ARM NO. 2 (in FIG. 1) 
Step Action 
______________________________________ 
1 Grip the holding block with unsealed reaction 
vessels from the assembly/disassembly station 14 
2 Transfer the holding block with reaction 
vessels to the solvent dispensing work station 15 
3 Transfer the holding block with reaction 
vessels to the solvent aspiration work station 16 
4 Repeat washing steps 2 and 3 for N times 
5 Transfer the holding block with reaction vessels 
to the building block distribution location 12 
6 Grip the holding block with fully charged reaction vessels 
7 Transfer the holding block with reaction vessels 
to the assembly/disassembly work station 14 
______________________________________ 
According to the exemplary control script in Table 1, robot arm 2 prepares 
a reaction vessel array for a new building block addition step. First, it 
grips a reaction vessel array that has just been disassembled from a hot 
rod at assembly/disassembly work station 14. Then it transfers this array 
repetitively between solvent aspiration work station 16 and solvent 
dispensing work station 15, for preforming a selected number of wash 
cycles. It then transfer this reaction vessel array to location 12 for 
building block and reagent distribution. Finally, it grips and transfers a 
fully charged reaction vessel array at this location back to 
assembly/disassembly station work 14, for assembling the reaction vessels 
into a hot rod. 
TABLE 2 
______________________________________ 
ARM NO. 3 (in FIG. 1) 
Step Action 
______________________________________ 
1 Activate a building block dispensing tool to 
distribute building blockS, and a reagent 
dispensing tool to dispense reagents to the 
reaction vessels in an array; during this 
process interchange tools and grip building 
block syringes as needed 
2 Interchange tools, attaching a hot rod 
agitation tool 
3 Activate a hot rod agitation tool to couple 
the motor to the hot rods in the incubation 
ports 9. 
4 Activate a hot rod agitation tool to spin the 
hot rod for 5 sec 
5 Activate a hot rod agitation tool to decouple 
the motor from the hot rod 
6 Repeat step 3, 4 and 5 for all hot rods in 
the incubation ports 9 
7 Interchange tools, attaching building block 
and reagent dispensing tools 
______________________________________ 
Concurrently, arm 3 performs two functions, distributing building block 
solutions and reagents and agitating hot rods during their incubation. 
First, arm 3, using building block distribution tool for gripping syringes 
20, grips syringes with building block solutions, and distributes then to 
reaction vessels in array 12. It also distributes activating and other 
reagents to the same reaction vessel array using appropriate reagent 
distribution tools. Then it interchanges tools, attaching an agitation 
tool, moves to the incubation array ports 9, and couples to and agitates 
intermittently all the hot rods being incubated. 
TABLE 3 
______________________________________ 
ARM NO. 4 (in FIG. 1) 
Step Action 
______________________________________ 
1 Grip a hot rod from incubator ports 9 
2 Transfer the hot rod to the assembly/disassembly 
work station 14 
3 Grip the assembled hot rod from the assembly/disassembly 
work station 
4 Transfer the assembled hot rod to incubator ports 
______________________________________ 
9 
Finally, during the activities of arms 2 and 3, arm 4 grips and transfers 
assembled hot rods from the assembly/disassembly work station 14 to the 
incubator ports 9 for incubation, and then returns them to the 
assembly/disassembly station after incubation for disassembly into a 
rectangular reaction vessel array in a holding block. 
These robot arm commands and command sequences are exemplary, and this 
invention is adaptable to other such similar commands and command 
sequences as can be output by the scheduling software in response to 
commands requesting the synthesis of particular compounds. 
5.5. Standalone Embodiments 
Although this invention has been described primarily with respect to use of 
the disclosed reaction vessels, reaction vessel arrays, work stations, and 
tools in an integrated and automatic robot apparatus, this invention is 
not so limited. All the reaction vessel embodiments, workstations, and 
tools have use in other environments, such as only partially integrated 
robots or entirely manual use on a laboratory benchtop. Accordingly, this 
invention further comprises each of these elements and sub-elements 
individually for use in such manual and semi-automated environments. In 
these environments, the manipulations described as being performed by 
robot arms can instead be performed by hand. Further, the various elements 
can optionally have a more limited range of automatic actuators, requiring 
manual attention to, e.g., actuate valves and so forth. 
6. EXAMPLES 
The invention is further described in the following example which is in no 
way intended to limit the scope of the invention. 
6.1. Example 1 
Tetrahydroisoquinolinone Library Synthesis 
A library of 2,280 different tetrahydroisoqinolinone amides was synthesized 
according to the chemistry illustrated below by using the stations and 
tools previously described. 
##STR1## 
In general, a separate tetrahydroisoqinolinone amide was synthesized 
attached to solid-phase support in separate reaction vessels by performing 
three addition steps: a first step in which one of 10 amino acid building 
blocks was added; a second step in which one of 38 aldehyde building 
blocks was added; and a third step in which one of 6 amine building blocks 
were added (10*38*6=2280). The building blocks used are listed in Table 4. 
The synthesis steps described in this example were performed using the 
work stations and tools with a combination of manual and robotic 
assistance. 
TABLE 4 
______________________________________ 
LIBRARY BUILDING BLOCKS 
______________________________________ 
Amino Acid Solutions - 0.3M Fmoc protected amino 
acid in a solution of 0.3M N-hydroxybenzotriazole and 
0.3M diisopropylcarbodiimide 
MW (Fmoc) 
______________________________________ 
1 2-Aminoethanoic Acid 297 
2 3-Aminopropionic Acid 311 
3 5-Aminopentanoic Acid 339 
4 7-Aminoheptanoic Acid 367 
5 (S)2,3-Diaminopropionic Acid (Fmoc on side chain) 
426 
6 (S)-2,6-Diaminohexanoic Acid (Fmoc on side chain) 
469 
7 (S)/(R)-3-Amino-2-methylpropionic Acid 
325 
8 2-(2-Aminoethoxyethoxy)acetic Acid 
385 
9 trans-4-(Aminomethyl)cyclohexanecarboxylic Acid 
380 
10 4-(Aminomethyl)benzoic Acid 
373 
______________________________________ 
Aldehyde Solutions - 0.5M aldehyde solution in 
dimethylformamide MW 
______________________________________ 
1 1,4-Benzodioxan-6-carboxaldehyde 
164 
2 1-Methylindole-3-carboxaldehyde 
159 
3 2,3-Difluorobenzaldehyde 142 
4 2-Bromobenzaldehyde 185 
5 2-Chloro-5-nitrobenzaldehyde 
186 
6 2-Furaldehyde 96 
7 2-Imidazolecarboxaldehyde 96 
8 2-Naphthaldehyde 156 
9 2-Pyridinecarboxaldehyde 107 
10 2-Thiophenecarboxaldehyde 112 
11 3,4-Dichlorobenzaldehyde 175 
12 3,5-bis(trifluoromethyl)benzaldehyde 
242 
13 3,5-Dihydroxybenzaldehyde 138 
14 3,5-Dihydroxybenzaldehyde 166 
15 3,5-Dimethyl-4-hydroxybenzaldehyde 
150 
16 3-(4-Methoxyphenoxy)benzaldehyde 
228 
17 3-Furaldehyde 96 
18 3-Hydroxybenzaldehyde 122 
19 3-Methyl-4-methoxybenzaldehyde 
150 
20 3-Methylbenzaldehyde 120 
21 3-Nitrobenzaldehyde 151 
22 3-Pyridinecarboxaldehyde 107 
23 3-Thiophenecarboxaldehyde 112 
24 4-(3-Dimethylaminopropoxy)benzaldehyde 
207 
25 4-(Dimethylamino)benzaldehyde 
149 
26 4-(Methylthio)benzaldyde 152 
27 4-(Trifluoromethyl)benzaldehyde 
174 
28 4-Biphenylcarboxaldehyde 182 
29 4-Bromo-2-thiophenecarboxaldehyde 
191 
30 4-Cyanobenzaldehyde 131 
31 4-Methoxy-1-Naphthaldehyde 186 
32 4-Nitrobenzaldehyde 151 
33 4-Pyridinecarboxaldehyde 107 
34 5-(Hydroxymethyl)-2-furaldehyde 
126 
35 5-Bromo-4-hydroxy-3-methoxybenzaldehyde 
231 
36 5-Nitor-2-furaldehyde 141 
37 6-Methyl-2-pyridinecarboxaldehyde 
121 
38 Benzaldehyde 106 
______________________________________ 
Amine Solutions - 1.0M amine solution in 
Amine 
dimethylformamide mg/ml 
______________________________________ 
1 Tryptamine 9.6 
2 (+/-)-3-(1-Hydroxyethyl)aniline 
8.22 
3 3,4,5-Trimethoxyaniline 10.98 
4 3,5-Dimethoxyaniline 9.18 
5 (+/-)-endo-2-Aminonorbornane 
8.88 
6 4-(Dimethylamino)benzylamine 
13.38 
______________________________________ 
In a pre-processing step, the reaction vessels were arrayed and charged 
with solid-phase support. Each of 24 microtitre-plate holding blocks was 
filled with 96 polypropylene test-tube reaction vessels of 1.2 ml volume, 
for a total of 2,280 test tube reaction vessels. (The last tube in each 
microtitre-plate was not used.) Then twenty-four grams of 
polyoxyethylene-grafted polystyrene beads, having an average size of 90 
.mu.m, a substitution capacity of 0.24 mmol/g, and attached acid labile 
linker (TentaGel S RAM), were equally distributed to all the test tubes 
using the resin distribution tool described herein. 
The amino acid addition step was performed using the freshly charged 
reaction vessels. Microtitre plates were placed, eight at a time, on the 
surface of the robotic platform at a building block distribution station, 
and one of the ten amino acid solutions were distributed into individual 
test tube reaction vessels from storage containers. The 96 wells of each 
microtitre plate received 100 .mu.l of the 0.3 M amino acid solutions. 
Just prior to the distribution to the individual test tubes, 0.5 ml of 
diisopropylcarbodiimide was added to the stock solutions of the amino 
acids. A computer generated protocol based on the compounds to be 
synthesized and the available building blocks, determined which building 
blocks, here which amino acids, to distribute to which test tubes. 
After distribution of amino acids, the microtitre plates were capped with a 
compliant array of sealing balls and shaken overnight. Microtitre plates 
with settled resin beads were then transferred to the previously described 
solvent-removal station configured with an array of 96 needles conforming 
to the microtitre plate layout, and solutions were aspirated 
simultaneously from all 96 test tubes in one microtitre plate. The 
microtitre plates were then transferred to a 96 needle wash station, as 
previously described, and 25 ml per microtitre plate of dimethylformamide 
("DMF") was dispensed into the test tubes in such a way that the resin was 
thoroughly mixed by the DMF. After the resin settled, the DMF was 
aspirated by the solvent-removal station. This DMF washing cycle of 
dispensing and aspirating of the work stations was repeated three times. 
The microtitre plates were transferred to a 96 needle solvent-distribution 
station and a solution of 50% piperidine in DMF was added simultaneously 
to all the test tubes of a microtitre plate in order to remove the Fmoc 
protecting groups. The microtitre plates were capped with a compliant 
array of sealing balls and shaken for 15 minutes. The piperidine solution 
was then removed with a 96 needle solvent-removal station, and DMF wash 
cycles of dispensing and aspirating were repeated 5 times. 
Next, the aldehyde addition step was performed. Microtitre plates were 
transferred to a building block distribution station, and the 0.5 M 
aldehyde solution was distributed from storage containers to each of the 
96 wells of each microtitre plate, each well receiving 100 .mu.l of 
solution. Next, a solution of trimethylorthoformiate in DMF, 100 .mu.l per 
microtitre plate well, was added to all wells for a final concentration of 
0.25 M aldehyde and 0.5 M trimethylorthoformiate. The microtitre plates 
were capped with a compliant array of sealing balls and placed on a 
shaker. After 3 hours of shaking, the plates were washed, again in the 96 
needle aspirating and dispensing wash station, with 0.2 M 
trimethylorthoformiate in DMF (2.times.). Then homophthalic anhydride 
solution was added to the test tubes, 125 .mu.l per well of 0.4 M 
anhydride solution with 0.03 M diisopropylethylamine in DMF. The base was 
added immediately prior to the anhydride solution distribution. After 
shaking at room temperature overnight, the plates were washed 3 times with 
DMF, two times with water, and 3 times with DMF in the 96 needle 
aspirating and dispensing wash station. Each wash used 25 ml of wash 
solution per microtitre plate. 
Finally, the amine addition step was performed. To each test tube, a 100 
.mu.l solution of 0.3 M HATU in DMF was distributed and then aspirated 
after a 20 minute incubation. After one wash with DMF at the 96 needle 
aspiration and dispensing wash station, 150 .mu.l per microtitre plate 
well of one of the amine solutions was distributed from storage containers 
to each of the test tubes in the microtitre plates. Plates were shaken for 
1 hour at room temperature and washed twice with DMF. Then the HATU 
solution was distributed as previously, and removed after a 20 minute 
incubation. After one wash with DMF, the amine solutions were distributed 
again as previously, and plates were capped with a compliant array of 
sealing balls and shaken overnight. Next, the amine solutions were removed 
from the microtitre plates, and solid phase was washed at the 96 needle 
aspirating and dispensing wash station: three times with DMF, once with 
water, 3 times with DMF, and 3 times with tertbutylmethylether. 
The microtitre plates were dried in vacuo. Trifluoroacetic acid, 200 .mu.l 
per test tube, was distributed to the microtitre plates. After incubation 
for 2 hours at room temperature, the test tube contents were evaporated in 
an evacuated centrifuge, such as the SpeedVac from Savant (Holbrook, 
N.Y.). A methanol extraction was then performed. Methanol, 300 .mu.l per 
test tube, was distributed into the dry resin in microtitre plates and the 
methanolic solution was transferred into new microtitre plates. This 
process was repeated three times. 
In a post-processing step, the resulting library compounds were analyzed. 
Twenty micro-liter samples were taken from each test tube containing the 
combined methanolic extractions for liquid chromatographic and mass 
spectrometric analysis. The remainder of the solution was evaporated to 
dryness in the SpeedVac. 
The present invention is not to be limited in scope by the specific 
embodiments described herein. Indeed, various modifications of the 
invention in addition to those described herein will become apparent to 
those skilled in the art from the foregoing description and accompanying 
figures. Such modifications are intended to fall within the scope of the 
appended claims. 
Various publications are cited herein, the disclosures of which are 
incorporated by reference in their entireties.