Anal. Chem. 1997, 69, 3851-3853
Technical Notes
Tools for Combinatorial Chemistry: Real-Time Single-Bead Infrared Analysis of a Resin-Bound Photocleavage Reaction Don E. Pivonka* and Thomas R. Simpson
Zeneca Pharmaceuticals, A Business Unit of Zeneca Inc., 1800 Concord Pike, Wilmington, Delaware 19850
The feasibility of using infrared spectroscopy for the realtime, in situ analysis of resin-bound photocleavable linkers from a single bead is illustrated using infrared microscopy. Results indicate this technique to be an extremely powerful tool for the analysis of photocleavage reactions directly on the solid-phase support. This report further details methodology providing a very high degree of automation to eliminate many of the sampling problems associated with batch analysis. Inherent to the recent resurgence of solid-phase synthesis for the production of combinatorial libraries has been a focused attempt to introduce and strengthen the analytical methodologies in support of the synthetic effort. Several previous reports have shown infrared spectroscopy to be a very powerful analytical technique for the analysis of combinatorial reactions through direct on-bead analysis.1-6 The aim of the present work is to expand the basic tools available to synthetic chemists for the analysis of chemical reactions on the solid-phase support, to include real-time infrared analysis of photocleavage reactions from a single bead. Throughout this report, the Scheme 1 photocleavage reaction will be used to demonstrate the application and benchmarks of the infrared methodology presented herein. HARDWARE CONSIDERATIONS The flow-through cell, infrared spectrometer, and infrared microscope used throughout these studies have been previously detailed in a paper describing real-time single-bead analysis of synthetic reactions on the solid-phase support.6 However, adaptation of the referenced cell hardware for photochemical analysis required that a provision for illumination of the sample bead with the appropriate source intensity and frequency be incorporated into the experimental design. Severe physical space limitations imposed by the microscope objective and the flow-through cell prompted an effort to provide a photoillumination source coaxial with the infrared beam via the (1) Yan, B.; Kumaravel, G.; Anjaria, H.; Wu, A.; Petter, R. C.; Jewell, C. F.; Wareing, J. R. J. Org. Chem. 1995, 60, 5736-5738. (2) Yan, B.; Kumaravel, G. Tetrahedron 1996, 52, 843-848. (3) Gremlich, H.-U.; Berets, S. L. Appl. Spectrosc. 1996, 50, 532-536. (4) Direct Monitoring of Combinatorial Chemistry Reactions By Infrared Microspectroscopy; Application Note; Spectra Tech. Inc.: Shelton, CT, 1995. (5) Russell, K.; Cole, D. C.; McLaren, F. M.; Pivonka, D. E. J. Am. Chem. Soc. 1996, 118, 7941-7945. (6) Pivonka, D. E.; Russell, K.; Gero, T. Appl. Spectrosc. 1996, 50, 1471-1478. S0003-2700(97)00084-X CCC: $14.00
© 1997 American Chemical Society
Scheme 1. Analytical Reaction
microscope optics. The two 50 W (top and bottom) microscopeilluminator bulbs were found to provide sufficient UV radiation to drive the Scheme 1 photochemical reaction at rates exceeding those of the Rayonet reactor used for bulk-scale chemistry in synthetic laboratories. Throughout the experiments of this text, however, only the top illuminator was used for two reasons. First, the illuminator intensity imparted onto the bead is sensitive to focus, due to the low f-number of the microscope Cassegrainian objectives. The top objective was relatively simple to reproducibly focus using the visual trinocular image. With the lower objective, it was found to be considerably harder to obtain a reproducible focus, due to distortion of the focal image by the bead, through which the focus had to be viewed. Second, confining irradiation to the top illuminator facilitated measurement and standardization of the input intensity. In this configuration, illuminator intensity was measured by mounting a photodiode directly at the microscope focus for measurement prior to each experiment.7 Day-to-day intensity variations could be compensated for by using the microscope illuminator intensity controls or field stops. Apertures, field stops, and convenient mounts for band-pass filters were already present in the system. A final beneficial feature of the experiment is that, since the illuminator irradiates only the single bead to which the infrared microscope was apertured, duplicate runs are easily obtainable from other beads in the cell by simply moving the microscope focus to an adjacent bead and reinitializing the experiment. SOFTWARE CONSIDERATIONS The NicPlan infrared microscope used in these experiments functions under two modes: view and collect. In the view mode, the user is able to optically view the sample for visual assessment and alignment of the sample. In the collect mode, FT-IR source radiation is directed to the sample for infrared analysis. (7) A Motorola MRD510 PIN photodiode was fitted to a Extech Instruments Model 401025 photometer. An arbitrary unit intensity of 8900 was used in all experiments.
Analytical Chemistry, Vol. 69, No. 18, September 15, 1997 3851
Figure 1. Program schematic.
Due to the design of the infrared microscope, it is not possible to irradiate the sample with the microscope illuminators whenthe microscope is in the collect mode. Hence, the photo experiment was performed as a cyclic experiment in which the sample was irradiated for some preset period of time. The microscope was then switched to collect mode for infrared data acquisition. Subsequent to data acquisition, the microscope was reset to the illuminate mode, and the cycle was repeated throughout the duration of the experiment. Due to the required cycling of the infrared microscope optics between view and collect modes for the photo experiments, the manufacturer’s time-resolved software package cannot be used. In an effort to facilitate, simplify, and automate the photocleavage analysis experiment, a Visual Basic (VB) front end was written to control data acquisition using calls to Nicolet’s Omnic instrument software via the software macro facilities. Under control of the VB front end, the user is prompted for a filename in which to store the time-resolved spectral series of the experiment, a duration time limit for the illumination cycle, a duration time limit for the entire experiment, for peak and baseline frequencies of any analytical peak in question, and to begin the experiment. Subsequent to the user initiation of the experiment, the program automatically sets the infrared microscope to the data collection mode, collects and saves a spectrum, resets the infrared microscope to the timed illumination mode, etc. Hence, the collection-exposure cycle is automated throughout the duration of the experiment. Illumination and collection status are displayed via bar graphs throughout the collection process. During the illumination cycle, the acquired spectrum and its first derivative are calculated and displayed. Peak height for the input peak parameters is calculated and displayed, and the residual fraction (in the case of photocleavage) vs time ) 0 is plotted in the VB program window. Also included in the window is a natural log linearization plot and listing of the resultant kinetic parameters. A provision for calculation of kinetic parameters for cases in which the reaction does not go to completion at infinite time (t∞) is accommodated by allowing user input of a Y∞ parameter. A general schematic of the photo-IR program is illustrated in Figure 1. 3852 Analytical Chemistry, Vol. 69, No. 18, September 15, 1997
Figure 2. (A) Time-resolved data of the CN region. (B) Firstderivative data from (A).
RESULTS AND DISCUSSIONsSINGLE BEAD ANALYSIS Figure 2A illustrates the nitrile band absorbance region for a time-resolved spectral series collected from the Scheme 1 photocleavage reaction. Figure 2B illustrates the first-derivative data calculated “on-the-fly” from the Figure 2A spectra by the VB control program. First-derivative data effectively eliminate baseline drift without the need for baseline correction. Several benefits of single-bead in situ infrared analysis of the photocleavage experiment previously demonstrated in the literature6 are apparent by the quality of data in the Figure 2 spectral series. Aperturing of the infrared beam to the flat parallel surfaces of the bead mated to the cell windows eliminates “false energy” and scattering spectral artifacts inherent to bulk sampling methods, for the 70 µm solid-phase support beads, to produce linear, very high-quality spectra. Parameters associated with interbead homogeneity, path length, focus, source intensity, etc., while of great concern for experimental designs requiring discrete samplings throughout the cleavage reaction, are eliminated in the in situ experiment. Figure 3 illustrates the residual decay profiles for a set of three analyses using three discrete beads in a chloroform solvent stream. In Figure 3, each time-resolved profile was obtained by determination of CN peak height. Kinetic data of this figure were collected by the coaddition of 20 scans/spectrum, with a spectral
Figure 3. Replicate determinations with discrete beads on consecutive days.
resolution of 4 cm-1. A 100 s illumination delay was implemented between each collection scan to drive the photoreaction. The quantity of data obtained and the very low point-to-point scatter of the reaction profile credit in situ analysis from a single sample. Although reaction half-life (t1/2) determinations can be graphically determined from Figure 3, more accurate determinations are obtained from the natural log linearized plot of absorbance vs time, which includes compensation for concentration values other than 0 at t∞. Although the photo-IR program provides onthe-fly kinetic analysis, a spreadsheet template was also developed to provide greater flexibility for postrun data analysis by allowing manual selection of the time region used in the regression calculations and inter-run comparisons. Figure 4 illustrates spreadsheet recalculation of the ln linearized data for the first three half-lives of the photocleavage experiment using time ) ∞ infinity compensation to maximize the R2 correlation coefficient for the 0-40 min data regression. Regression and t1/2 data are presented in Table 1. From Table 1, the excellent correlation between beads and analysis runs is apparent. CONCLUSION This work builds upon the previous report documenting realtime, in situ infrared analysis of reactions on the solid-phase
Figure 4. Ln linearization plots. Table 1. Regression Data sample
slope
R2
t)∞ residual
t1/2 (min)
A B C
-0.0626 -0.0600 -0.0612
0.9996 0.9987 0.9990
34 35.5 35
11.08 11.56 11.32
support,6 to include the single-bead analysis of photolinker cleavage kinetics. The basic methodology presented by analysis of the Scheme 1 reaction provides a pathway for the investigation and optimization of cleavage parameters (solvent, temperature, wavelength and intensity). This method also allows for the rapid measurement of relative t1/2 values for various types of groups to be cleaved in preparation for combinatorial library synthesis.
Received for review January 23, 1997. Accepted July 5, 1997.X AC9700843 X
Abstract published in Advance ACS Abstracts, August 15, 1997.
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