Background of the Invention Traditionally, most chemical reactions for the synthesis of organic compounds have been carried out in liquid phase. However, recent trends in the search for novel chemical and pharmacological agents have concentrated on the preparation of so- called"chemical libraries"as potential sources of new drug candidates. Such chemical libraries are intentionally created collections of differing molecules that can be screened for biological activity. When such libraries are prepared by synthetic chemical reactions, each member of the library is created through a sequential series of chemical reactions, with a different combination of reactions being carried out to create each different member of the library.

To facilitate synthesis and screening of chemical libraries, particularly by automated techniques, different chemical entities within the libraries are often attached to a discrete solid support such as a particle or bead. As a result, a particular compound within that library which is identified as a candidate by the screening technique can be isolated by isolating the discrete solid support to which it is bound.

Chemical synthesis and screening robots for these purposes are known, examples being shown in U. S. Patent No. 5,463,564 to Agrafiotis et al.

Because of the need to construct solid phase combinatorial libraries, reactions that have previously been carried out in liquid phase are now being carried out in

solid phase so that the reaction products are immobilized on a solid support. Solid phase synthesis reactions are typically run for up to 60 hours in order to ensure the reaction goes to completion. The reactions are run for such a long time because making measurements during the reaction required taking a sample and either cleaving the product off of the support or drying the sample. The former approach tends to destroy the product and is time consuming, and the latter approach is time consuming and may degrade the sample. When a large or complex library is desired (as is often the case) the time required for each reaction step significantly slows the construction of the combinatorial library.

In view of the foregoing, there is a need for new techniques for monitoring solid phase synthesis reactions.

Summary of the Invention The present invention provides a method for monitoring a solid phase chemical reaction. The method comprises the steps of (a) providing a reaction mixture comprising a solid support and a liquid reaction medium, (b) contacting an attenuated total reflection element to said reaction mixture; and then (c) monitoring the chemical reaction on said solid support through said attenuated total reflection element. The monitoring step is carried out by attenuated total reflection spectroscopy. Advantageously, the chemical reaction on the solid support may be directly monitored, rather than indirectly monitoring that chemical reaction by monitoring reaction constituents in the liquid reaction medium.

The contacting step is carried out under conditions that cause the progression of the desired reaction, with the appropriate reagents included in the reaction medium.

The monitoring step is preferably carried out by measuring light absorbance by the reaction mixture, with changing light absorbance being positively associated with the progression of the reaction (e. g., increasing light absorbance being positively associated with the progression of a synthesis reaction, or decreasing light absorbance being positively associated with the progression of a degradation reaction).

The foregoing method can be used to directly monitor the progression of chemical reactions as they proceed on solid phase supports. By measuring the light absorbance of the beads (e. g., infra-red, near infra-red, visible, or ultra-violet light absorbance) attached to the probe, the progression of reaction steps on the bead can be

monitored without having to remove a sample or cleave the molecule off of the bead.

While applicable to any type of small or large scale solid phase synthesis, this method is particularly useful for application to solid phase synthesis carried out to generate a solid phase combinatorial library, where the time required for each step to generate the library can be advantageously reduced.

Thus, a further aspect of the present invention is a method of making a combinatorial library preferably comprising at least 100 different compounds, with each of said compounds immobilized on a discrete solid support, by at least two sequential reaction cycles, and with each of said reaction cycles comprising a solid phase chemical reaction, wherein all of the sequential reaction cycles are completed in a total time that averages from not more than 8 or 10 hours for each of the sequential reaction cycles.

The present invention is explained in greater detail in the drawings herein and the specification set forth below.

Brief Description of the Drawings Figure la is a schematic illustration of an apparatus that may be used to carry out the present invention.

Figure lb is a detailed view of a segment of the apparatus of Figure la, showing the adhesion of the solid phase synthesis support beads to the attenuated total reflectance element.

Figure 1 c is a detailed view of the a segment of the apparatus of Figure I b, showing the optical relationship of the solid support beads to the attenuated total reflectance element.

Figure 2 illustrates a solid state Friedel-Crafts alkylation reaction that may be used in carrying out the present invention.

Figure 3 illustrates a Suzuki reaction.

Figure 4 illustrates a solid state Suzuki reaction that may be used in carrying out the present invention.

Figure 5 illustrates a Mitsunobu reaction.

Figure 6 illustrates a solid state Mitsunobu reaction that may be used in carrying out the present invention.

Figure 7 illustrates the results obtained with a Friedel-Crafts reaction of the present invention. The upper box of this figure is a waterfall plot of absorbance versus time; the lower box of this figure is a contour plot over time.

Figure 8 illustrates the wash steps carried out with the reaction of Figure 7, with a total of 15 washing steps being employed. The upper box of this figure is a waterfall plot of absorbance versus wash number. As can be seen there is a large initial absorbance with the trend towards zero absorbance as the washes progress.

The bottom portion of the figure is a contour plot with the content of the wash indicated at the top of the plot and the wash progression proceeding from right to left.

It can be seen from this plot that the first 5 washes remove a substantial amount of the excess reagent.

Figure 9 illustrates the results obtained with a Suzuki coupling reaction with the present invention.

Figure 10 illustrates the results for the first step of a Mitsunobu reaction in carrying out the present invention.

Figure 11 illustrates the monitoring of chemistry on beads, or the solid phase, and not the reaction solution, in a Suzuki coupling reaction in the present invention.

Figure 12 schematically illustrates a combinatorial library synthesis apparatus employing an attenuated total reflection monitor of the present invention.

Figure 13a illustrates a probe and support structure arrangement for monitoring reactions in a plurality of reaction wells.

Figure 13b illustrates a probe and support structure arrangement for monitoring reactions in a plurality of reaction wells.

Figure 13c illustrates a probe and support structure arrangement for monitoring reactions in a plurality of reaction wells.

Detailed Description of the Preferred Embodiments The present invention is useful for all types of solid phase reactions, including batch reactions, single reactions, or reactions incorporated into a combinatorial synthesis process for making a combinatorial library. Combinatorial libraries are sets or collections of different compounds, which compounds are immobilized on a solid support, with different compounds immobilized on a different discrete solid support

as described below. The compounds may be oligomers or nonoligomers, as also discussed below.

In general, the chemical syntheses and reactions described in the Examples below can be monitored with the apparatus illustrated in Figure 1 a. The apparatus comprises a fiber optic coupled attenuated total reflection (ATR) probe 20 connected to a ultraviolet/visible light spectrograph 25. The ATR probe (obtained from Equitech International, Aiken, South Carolina, USA) was employed a three-bounce design attenuated total reflection element 22 and incorporated 400 um fiber optic fibers as the cable interconnecting the spectrograph to the probe. Two different spectrographs were used for this work depending on the number of channels monitored: A single channel spectrograph using a deuterium light source (Zeiss MCS 501, Custom Sensors and Technology, St. Louis, Missouri, USA) and a four channel spectrograph using a xenon light source (Equitech International, Aiken, SC). The spectrograph was coupled to a personal computer 24 with a standard interface board for data analysis and display. We used the ASPECT data acquisition and display software, available from Zeiss, Germany, and also used LABVIEWTM data acquisition, display and analysis software, available from National Instruments, USA.

Attenuated total reflection elements, monitors and spectroscopy techniques are known and can be carried out in accordance with known techniques, such as described in U. S. Patents Nos. 5,814,565; 5,465,151; 5,070,243; and 3,752,584. In general, the total internal reflection can be carried out with ultraviolet light, near-ultraviolet light, visible light or infrared light. The attenuated total reflection element may be formed from any suitable material that is substantially optically transparent at the wavelength employed, but is generally formed from an inert insoluble inorganic crystal material such as sapphire, glass, quartz, germanium, zinc selenide, diamond (including diamond like carbon), and combinations thereof. The attenuated total reflection element may be formed from composites or layers in accordance with known optical fabrication techniques, and may uncoated or coated with a material that will facilitate adhesion of the solid supports to the attenuated total reflection element In use, the probe 20 is inserted into a solid phase reaction medium 30 comprising a liquid phase 31 and discrete solid supports, preferably polystyrene beads, as the solid support 32. As shown in Figure 1 b, the solid particles 32 adhere or bind to the ATR element 22, with light entering and leaving the element along

paths 35,36. As shown in Figure lc, light 37 from the ATR element 22 is believed to enter and penetrate the solid supports 32 due to the adhesion or binding of the solid supports 32 to the ATR element 22. Note that when the solid support is a separate discrete solid support such as a particle or bead, it is preferred that a major portion, or substantially all of, the exposed surface of the attenuated total reflection element is coated with a continuous layer of said separate discrete solid support that has a thickness of at least one of said separate discrete solid supports, as illustrated in Figure lb.

Solid supports used to carry out the present invention are typically discrete solid supports. Discrete solid supports may be separate from one another as described above, or may be discrete regions on a surface portion of a unitary substrate. Such "chip-type"or"pin-type"solid supports are known. See, e. g., U. S. Patent No.

5,288,514 to Ellman (pin-based support) ; U. S. Patent No. 5,510,270 to Fodor et al.

(chip-based support) (the disclosures of all United States patent references cited herein are to be incorporated by reference herein in their entirety). Separate discrete supports such as particles or beads (these terms being used interchangeably herein), disks, fibers, needles or the like, are currently preferred.

The discrete solid supports are formed from a polymer such as polystyrene. In general, the solid substrates are beads, which may be completely solid throughout, porous, deformable or hard. The beads will generally be at least 10,20 or 50 to 250, 500, or 2000 u. m in diameter, and are most typically 50 to 250 um in diameter. Any convenient composition can be used for the particles or beads, including cellulose, pore-glass, silica gel, polystyrene beads such as polystyrene beads cross-linked with divinylbenzene, grafted copolymer beads such as polyethyleneglycol/polystyrene, polyacrylamide beads, latex beads, dimethylacrylamide beads, composites such as glass particles coated with a hydrophobic polymer such as cross-linked polystyrene or a fluorinated ethylene polymer to which is grafted linear polystyrene, and the like.

Where separate discrete solid supports such as particles or beads are employed, they generally comprise from about 1 to 99 percent by weight of the total reaction mixture.

The reaction medium apart from the solid support is, in general, a liquid. In general, the reaction medium will comprise from about 1 to 99 percent by weight of the total reaction medium (where the solid support is separate from the container in which the reaction mixture is held, as in the case of particles or beads). Any suitable

liquid may be employed, including aqueous liquids (water), nonaqueous liquids (e. g., organic solvents), and combinations thereof or mixtures thereof. The liquid may be a single phase or multi-phase solution. Organic solvents used to carry out the present invention may be polar or nonpolar, protic or aprotic, etc. The particular solvent or mixture of solvents depends upon the particular reaction being carried out. Examples of suitable solvents for use in the reaction medium include, but are not limited to, methylene chloride, dimethyl formamide, 1-methyl-2-pyrrolidinone, methanol, ethanol, water, toluene, tetrahydrofuran, dioxane, ethyl acetate, chloroform, acetone, acetic anhydride, pentane, hexane, benzene, carbon tetrachloride, diethyl ether, acetonitrile, etc.

The reaction medium will include the reagents necessary to carry out the reaction desired, such as one or more reactants, a catalyst and/or initiator if necessary for the particular reaction, etc. All can be routinely determined by skilled artisans based on the particular reaction desired. During monitoring, the reaction mixture is held or placed in conditions that cause or permit the reaction to progress. The particular reaction conditions are not critical, depend upon the particular reaction desired, and can be routinely determined by skilled artisans. The reaction may be carried out under atmospheric pressure, elevated pressure, or reduced pressure; the reaction may be carried out at room temperature, elevated temperature, or reduced temperature; the reaction may be carried out at a neutral, acidic, or basic pH; the reaction may be carried out with or without an inert blanketing gas such as nitrogen or argon; etc.

Any suitable reaction can be employed to carry out the solid-phase synthesis that is monitored or observed by the present invention. See, e. g., U. S. Patent No.

5,565,324. Specific examples of suitable reactions include, but are by no means limited to, Mitsunobu, Freidel-Crafts, Suzuki, Merrifield (for the synthesis of polypeptides) (see, e. g., Merrifield, J. Am. Chem. Soc. 85,2149 (1963); Merrifield, Science 150,178 (1965), Hofmann, Grignard, Cannizarro, Clemmensen, Knovenagel, Perkin, Wittig, Wolff-Kishner, Ruff, Reimer-Tiemann, Kiliani-Fisher, Markonikov, Curtius, Lossen, Schoniger, Williamson, Bogert-Cook, and Hell-Volhard-Zellinsky reactions, etc.

The reaction or reactions employed in carrying out the present invention may thus be selected to produce a variety of products, the progression of the synthesis of

which products can be monitored as described herein. Such products are, in general, non-oligomers, oligomers, or combinations thereof.

Non-oligomer reaction products include a wide variety of organic molecules, such as heterocyclics, aromatics, alicyclics, aliphatics and combinations thereof, comprising steroids, antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids, opioids, terpenes, porphyrins, toxins, catalysts, as well as combinations thereof.

Oligomer reaction products include oligopeptides, oligonucleotides, oligosaccharides, polylipids, polyesters, polyamides, polyurethanes, polyureas, polyethers, and poly (phosphorus derivatives), e. g. phosphates, phosphonates, phosphoramides, phosphonamides, phosphites, phosphinamides, etc., poly (sulfur derivatives) e. g., sulfones, sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for the phosphorous and sulfur derivatives the indicated heteroatom for the most part will be bonded to C, H, N, O or S, and combinations thereof.

The present invention provides a direct monitoring technique rather than an indirect monitoring technique. By directly monitoring the progression of the reaction with the methods described herein, the reaction is advantageously monitored in real time as the reaction occurs. Thus the progression of the reaction need not be inferred from other measurements, and it is not necessary to have an intervening sampling, handling, or other processing step that intervenes between the reaction event or events and analysis. Further, the reaction need not be"opened"or otherwise disrupted as atmosphere, pressure, temperature, concentration, volume and/or stirring is changed by the sampling or processing step. As a result, individual solid state reactions can be efficiently and effectively monitored so that the reaction materials and equipment can be optimized for a particular combinatorial chemistry and synthesis program, allowing the time and effort required to carry out the combinatorial synthesis to be reduced.

It will be appreciated that, while the present invention is primarily concerned with monitoring the progression of synthesis reactions, and while the object of a series of reactions may be to synthesize a compound, that a given reaction may involve the addition, removal (e. g., degradation), or substitution of a chemical unit from the core compound in solid phase, and that the progression of all such reactions may be monitored by the present invention.

The method of the present invention can be employed to monitor single batch reactions, as described in connection with Figure 1 above, or can be used to monitor sequential and/or concurrent reactions, such as employed in the synthesis of a combinatorial chemical library, where the reaction product may be oligomers or nonoligomers as described above. Such procedures are readily automated, and an apparatus for generating a solid phase combinatorial library while utilizing the methods of the present invention is schematically illustrated in Figure 12. The apparatus includes a chemical synthesis robot 101 configured to receive a plurality of reaction wells, as discussed in greater detail below. A reagent repository 102 is operatively associated with the chemical synthesis robot in accordance with standard techniques. An attenuated total reflection element as described above (illustrated in Figures 13a-13c) is operatively associated with the chemical synthesis robot for insertion into at least one, and preferably a multiplicity or even all of the reaction wells. An attenuated total reflection monitor 103 is connected to each attenuated total reflection element, which are configured to monitor a solid phase synthesis reaction in the corresponding reaction well by attenuated total reflection spectroscopy. A synthesis controller 104 is operatively associated with both the reagent repository and the chemical synthesis robot. The synthesis controller is configured through the implementation of hardware and/or software instructions to control the construction of a solid phase combinatorial library in the plurality of reaction wells. Synthesis robots, reagent repositories and controllers therefore are known in the art and can be implemented in accordance with standard techniques. See, e. g., U. S. Patent No.

5,463,564 to Agrafiotis et al (the disclosures of all United States patent references cited herein are to be incorporated herein by reference). The synthesis controller is operatively associated with the attenuated total reflection monitor through an appropriate interface, with the synthesis controller configured through hardware and/or software instructions to end a particular synthetic step or reaction upon detection of completion of a chemical reaction, so that the overall combinatorial synthesis scheme can proceed to the next cycle of reaction steps.

The reaction wells may be incorporated into the robot, but are typically provided in a separate structure that is insertable into or carried by the robot, as is known in the art. Microtiter plates, individual containers, columns, gels, Teraski plates, flasks, Merrifield synthesis vessels, etc., can be employed. Preferred are

structures that contain a plurality of reaction wells, such as microtiter plates, which typically contain from 96 up to 2,304 reaction wells, or more. Filter plates that match the support structure can be used to drain the liquid contents of the reaction well while retaining the solid supports therein, in accordance with known techniques.

The attenuated total reflection element can be associated with the reaction wells in a combinatorial synthesis apparatus as described in Figure 12 in any of a variety of ways, some of which are illustrated in Figures 13a-c. In Figure 13a, the well support structure 120a contains a plurality of reaction wells 121a. The bottom of each well is plugged and sealed by a probe 122a that terminates with the attenuated total reflection element 123a. The probes are thus connected to the robot stage with the support structure removably connected thereto, and with each well sealed by the reflection element. The contents of each well can be pipetted therefrom for subsequent reactions.

An alternate approach is illustrated in Figure 13b, which again comprises a support structure 120b containing a plurality of reaction wells 121b. Here, however, the total reflection elements 123b are rigidly and sealably connected into the support structure 120b, while the probe elements 122b are removably contacte to the reflection elements. Thus the entire support structure can be simply secured through an appropriate stage to the probe elements, which again are connected to the synthesis robot.

Still another approach is illustrated in Figure 13c, which again comprises a support structure 120c having a plurality of reaction wells 121 c, with a plurality of probes 122e, each carrying an attenuated total reflection element 123c, inserted into each reaction well. This structure can employ a conventional microtiter plate. The probes are carried by the synthesis robot, which can be structured so that the probes are placed in the reaction well before, during, or after the addition of supports, reaction medium or liquid, and other reagents into each well.

In use, the foregoing apparatus provides a method of making a combinatorial library by solid phase chemical synthesis carried out by the following steps: (a) combining (typically in a reaction well) a solid support, a liquid reaction medium and reaction reagents to produce a reaction mixture; (b) contacting an attenuated total reflection element to the reaction mixture; then

(c) monitoring the chemical reaction on the solid support through the attenuated total reflection element to thereby detect completion of said reaction, then (d) separating the solid support from said liquid reaction medium and said reaction reagents upon detecting completion of said reaction ; and then (e) repeating steps (a) through (d) with the separated solid supports produced in step (d) above.

The repeating step is carried out cyclically until the desired number of compounds is produced as the combinatorial library. Again, the library so produced may be comprised of oligomer or nonoligomer compounds, as described above, by any type of solid phase reaction, including but not limited to those described above.

In the context of combinatorial chemistry and the present invention, completion of the reaction is achieved when the reaction is sufficiently complete to achieve reasonable uniformity of the product population for a given library constituent, or group of constituents, to achieve the desired result of producing a useful combinatorial library. Preferably, completion of the reaction occurs when the reaction reaches equilibrium.

Typically, the repeating of steps (a) through (d) above is carried out with a different reagent or substrate, or any other technique useful for generating a diverse combinatorial library. For example, with the apparatus illustrated in Figures 13a-13c, steps (a) through (d) above are concurrently carried out in a plurality of different reaction wells, and then a new set initiated in step (e), again typically in a plurality of different reaction wells. The steps (a) through (d) may be cyclically repeated, individually or as sets of concurrent reactions, as many times as necessary to produce the desired combinatorial library, typically 2,3 or 4 repetitions up to 10,20,30 or 40 repetitions or more.

Depending upon the particular apparatus employed, each reaction cycle may be concurrently carried out in 10,20 or 30 up to 400,600 or 800 or more wells or reaction vessels. Current formats include 96 well and 384 well microtiter plates, but it is not necessary that every well in the plate be used.

It will be appreciated that diversity in the library can be achieved by any means of solid phase or solid state synthesis, such as by carrying out different series of syntheses in different wells, by pooling the solid supports from multiple different

reaction wells (including some or all) after a synthetic cycle, splitting that pool back into different reaction wells for a further set of reactions, and then repeating that cycle, in accordance with known techniques. See, e. g., U. S. Patents No. 5,656,324 to Still et al.; PCT Application No. W097/37953 to Geysen et al. Reactions, reaction conditions and reaction products include but are not limited to those described above.

By optimizing a plurality of the reaction cycles (or even a majority of or all of the reaction cycles) through the application of the monitoring steps described herein, combinatorial libraries containing at least 100,500,1,000 or 5,000 different compounds up to 10,00,20,000,50,000,100,000, up to 106 different compounds or more, can be produced more rapidly than by prior techniques. Depending upon the intended purpose of the combinatorial library the library may be relatively large or relatively small, but the present invention is useful in the preparation of both relatively small libraries and relatively large libraries on a more expeditious basis.

The process of synthesizing a combinatorial library may be varied from that described above. In general, when the synthesis of a combinatorial library is carried out, a plurality of, more preferably a majority of, and most preferably all of the reaction cycles are terminated upon completion of the chemical reaction being carried out in that cycle. Completion can be determined by attenuated total reflection spectroscopy as described above. In a particular library synthesis procedure, the attenuated total reflection spectroscopy step may be carried out in situ during the chemical reaction cycle of interest, and completion directly detected. In the alternative, the attenuated total reflection spectroscopy step may be carried out a priori on a model reaction system prior to the chemical reaction cycles of interest to, the reaction optimized and a necessary reaction time determined. Then, during the combinatorial library synthesis, the reaction cycle that incorporates a reaction step based upon the reaction model (and for which a reaction time has been determined a priori) may be terminated upon completion of the previously determined reaction time. In a particular combinatorial synthesis, a plurality of, a majority of, or all of the reaction cycles may be carried out to completion, with completion being determined by either in situ measurement or a priori measurement as described above, or combinations thereof. It will also be appreciated that not every reaction in a particular combinatorial synthesis need by optimized by the techniques described herein, so long as the overall library synthesis time is advantageously reduced. Further, where a time

to completion is determined a priori from a model reaction, that model reaction can be the same as the reaction carried out in the synthesis of the combinatorial library, or may differ in one or more parameters, with the reaction time for the combinatorial synthesis reaction being interpolated from the model reaction. When a time to completion is interpolated from a different reaction, it is preferably interpolated from a plurality of different reactions in accordance with known techniques.

In general, by use of the present invention in conjunction with making a combinatorial library, the time to synthesize the library can be substantially reduced.

In general, all of the reaction cycles may be completed in a time that averages not greater than 6,8 or 10 hours per reaction cycle (typically, an average time of at least 1 or 2 hours per reaction cycle is employed). Thus, for example, when a library is synthesized in three consecutive reaction cycles, the cycles having reaction times of 2, 4 and 6 hours respectively, the average reaction time per cycle is 4 hours. Note that in determining the average time any resting time or periods of inactivity between cycles is not counted, but the time for all aspects of each reaction, including mixing and washing steps, is included.

In combinatorial syntheses, the compounds on the solid supports or the solid supports themselves may be tagged for later decoding by any suitable technique in accordance with known procedures, including but not limited to oligonucleotide tags and oligopeptide tags, see, e. g., S. Brenner and R. Lerner, Proc. Natl. Acad. Sci. USA 89,5381 (1992); J. Kerr et al., J. Am. Chem. Soc. 115,2529 (1993), nonsequential tags, such as described in U. S. Patent No. 5,565,324 to Still, or mass-based encoding such as described in PCT Application W097/37953 to Geysen et al. Tagging reactions may be carried out concurrently with the reactions employed to synthesis the members of the combinatorial library in the same reaction medium.

The present invention is explained in greater detail in the following non- limiting examples.

EXAMPLE 1 Solid State Friedel-Crafts Reactions In general, a Friedel-Crafts reaction is an electrophilic substitution The general reaction steps are as follows (r. Morrison and R. Boyd, Organic Chemistry (Allyn and

Bacon, Inc., 1973): The electrophile is typically a carbonium ion and is formed in an acid-base equilibrium, as illustrated in Scheme 1 below: Scheme 1 For the example discussed herein the chemistry involved is more particularly illustrated Figure 2. In brief, in an illustrative reaction, Biorad S-X1 resin, 5.0 grams, is suspended in 200 mL of methylene chloride to which N-chloromethyl phthalimide, 6.85 grams, is added and stirred. Solid iron is added, 0.8 grams, and the reaction is stirred at room temperature.

Example 2 Solid State Suzuki Reactions Suzuki couplings involve the palladium-catalyzed couplings of unsaturated halides with boronic acids or esters. Suzuki coupling reactions are known in the art. See, e. g., G. Smith et al., J. Org. Chem. 59,8151 (1994). The general Suzuki coupling is shown in Figure 3. An example of this type of reaction applied to solid phase synthesis is shown in Figure 4. In brief, in an illustrative reaction, precursor resin, 0.4 grams, is suspended in 6 mL of methylene chloride and stirred. To this phenylboronic acid, 0.2.4 grams, 2M sodium carbonate solution, 0.95 mL, and tetrakis (triphenyl phosphine) palladium, 44 milligrams, are added and refluxed.

EXAMPLE 3 Solid State Mitsunobu Reactions Mitsunobu reactions refer to the reaction of hydroxy compounds with acids (pK, < 11) in the presence of triphenylphosphine and diethyl azodicarboxylate. See, e. g., M. Varasi et al., J. Org. Chem. 52,4235 (1987). This scheme is used for the functionalization of alcohols and related compounds. The general Mitsunobu reaction is shown in Figure 5. A specific solid phase Mitsunobu reaction is shown in Figure 6. In brief, in an illustrative reaction, Sasrin resin, 2.16 grams, is suspended in 50 mL of tetrahydorfuran and gently agitated for 30 minutes under a blanket of nitrogen. N- hydroxyphthalimide, 1.60 grams, and triphenylphosphine, 2.52 grams, is added and the mixture is agitated until these reagents are dissolved. Diisopropyl azodicarboxylate, 2.0 mL, is added and the mixture stirred at room temperature.

EXAMPLE 4 Monitoring of a Solid State Friedel-Craft Reaction The spectral results for a Friedel-Craft reaction, step 1, as described above as monitored by an apparatus described in Figures la-c above, are presented in Figure 7.

W-vis data are displayed as waterfall and contour plots. Note the increase in absorbance over time. It was found that the reaction finished quite quickly. In addition, the actual number of washes specified in the literature to complete the reaction procedure was discovered to be in excess of what is truly required to clean the beads. The typical wash cycle called for 15 washes; by monitoring the wash cycle through this number of washes, as shown in Figure 8, it became is clear that fewer washes were sufficient.

EXAMPLE 5 Monitoring of a Solid State Suzuki Reaction Results from the Suzuki coupling reaction shown in Figure 4, as monitored by an apparatus described in Figures la-c, are presented in Figure 9. As in Example 4 above, again note the increase in absorbance over time during the progression of the reaction.

EXAMPLE 6 Monitoring of a Solid State Mitsunobu Reaction The results for the first step of the Mitsunobu reaction described above, as monitored by an apparatus as described in Figures la-c, are shown in Figure 10.

Again, note the increase in absorbance over time during the progression of the reaction.

EXAMPLE 7 Direct Monitoring of Solid Phase Chemistrv Figure 11 illustrates that the present invention directly monitors the synthesis on the beads, and not indirectly through the reaction medium. Again, an apparatus as described in Figures la-c was employed. The"last reaction spectrum" (middle trace) is typical of the waterfall traces collected during the reaction. The probe was removed from the reaction vessel and allowed to air dry. A thin film of residue, determined to be beads, was observed on the sapphire probe tip. The spectrum collected after removal of the probe from solution is slightly shifted due to drying, but closely matches that of the reaction spectrum. (As it was weaker than that of the reaction scan, it is plotted at two times the actual intensity.) Next, the probe was wiped clean with a KIMWIPETM paper. No spectral response was observed in the spectrum of the wiped probe. These data show that the increase in absorbance over time, as described in Examples 4 to 6 above, represents a direct monitoring of the synthesis reaction.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.