Anal. Chem. 1999, 71, 4564-4571
Determination of the Absolute Amount of Resin-Bound Hydroxyl or Carboxyl Groups for the Optimization of Solid-Phase Combinatorial and Parallel Organic Synthesis Bing Yan,* Lina Liu, Catherine A. Astor, and Qing Tang
Novartis Pharmaceuticals Corporation, 556 Morris Avenue, Summit, New Jersey 07901
We have developed quantitative methods for the rapid determination of the absolute amount of hydroxyl or carboxyl groups directly on resin support. These methods are based on specific reactions between reagent 9-anthroylnitrile or 1-pyrenyldiazomethane (PDAM) and resinbound hydroxyl or carboxyl groups. After the reaction, the remaining reagent molecules in the supernatant are quantitatively determined by UV-visible spectroscopy. The quantitation can be accomplished by analyzing 2-10 mg of resin sample in 30-60 min. The lowest amount of hydroxyl groups quantitatively analyzed in this work was 0.05 mmol/g of resin. For an array of reference samples, our results agree with the results obtained by the tedious derivatization method. Our methods have also been applied to the analysis of a series of solid-phase organic synthesis samples with various loading levels. The PDAM method is highly specific for carboxylic acid groups and the 9-anthroylnitrile method is also free from interference from most organic functional groups. The later method can also be used for nonhindered primary amine groups directly on resin. Both methods can also be used as a quick qualitative “color” test to identify whether hydroxyl or carboxyl groups are present on the resin. Combinatorial synthesis1 of small organic molecules can greatly enhance our capability to discover new chemical entities with a property of interest (such as new drugs). Solid-phase organic synthesis (SPOS)2 has been a major method for combi* Corresponding author: (tel) 908-277-6023; (fax) 908-277-2785; (e-mail)
[email protected]. (1) (a) Gordon, E. M.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gallop, M. A. J. Med. Chem. 1994, 37, 1385. (b) Fruchtel, J. S.; Jung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17. (c) Thompson, L. A.; Ellman, J. A. Chem. Rev. 1996, 96, 555. (d) DeWitt, D. H.; Czarnik, A. W. Acc. Chem. Res. 1996, 29, 114. (e) Still, W. C. Acc. Chem. Res. 1996, 29, 155. (f) Ellman, J. A. Acc. Chem. Res. 1996, 29, 132. (g) Armstrong, R. W.; Combs, A. P.; Tempest, P. A.; Brown, S. D.; Keating, T. A. Acc. Chem. Res. 1996, 29, 123. (h) Balkenhohl, F.; Bussche-Hunnefeld, C.; Lansky, A.; Zechel, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 2288. (i) Lam, K. S.; Lebl, M.; Krchnak, V. Chem. Rev. 1997, 97, 411. (2) (a) Leznoff, C. C. Acc. Chem. Res. 1978, 11, 327. (b) Akelah, A.; Sherrington, D. C. Chem. Rev. 1981, 81, 557. (c) Frechet, J. M. J. Tetrahedron 1981, 37, 663. (d) Hodge, P. In Synthesis and separations using functional polymers; Sherrington, D. D., Hodge, P., Eds.; Wiley: Chichester, 1988; Chapter 2.
4564 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
natorial organic synthesis. The creative split and pool synthesis3 is a very powerful way of making a large number of compounds with a high molecular diversity. Because the final library is in a one-bead-one-compound format, it is practically impossible to quantitatively characterize all compounds in the final compound library. Therefore, prior reaction optimization in a nonlibrary or trial library format is essential. Even for the discrete library synthesis, the optimization studies will greatly improve the yield and purity of the final library. While qualitative tests are an essential part of the daily routine in monitoring SPOS, quantitative methods are particularly important for the determination of the on-resin loading of the starting resin and the yield of synthetic intermediate and product. Currently, only a few amine quantitation methods inherited from solid-phase peptide synthesis (SPPS) are available. There is a great demand for quantitative methods to analyze the amount of compound on the basis of organic functional groups directly on solid support. Due to the lack of on-support quantitative methods, the synthetic intermediates and products are usually not quantitatively characterized on support, assuming that the excess of reagent and the prolonged reaction time will eventually drive the reaction to completion. Actually, when reaction conditions are not optimal, most reactions cannot reach completion on solid support. The solid-phase synthetic intermediate, unlike the solution-phase counterpart, cannot be purified. The unreacted portion will undergo unwanted side reactions and accumulate until the end of synthesis, increasing impurities in the final cleaved product. A less desirable way is to cleave the compound at every step of synthesis. However, chemical changes and the reaction yield on solid support are the most relevant information. To “cleave and analyze” is time-consuming, laborious, and destructive. It is particularly limited when synthetic intermediates are not stable to the cleavage conditions. Quantitative methods for the monitoring of peptide bond formation are well documented.4 Because only the amide bond formation is a concern in SPPS, these methods are primarily based on the quantitation of amines by spectroscopic methods. In comparison with SPPS, the quantitative analysis in SPOS is much (3) (a) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. Int. J. Pept. Protein Res. 1991, 37, 487. (b) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84. (c) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82. 10.1021/ac990685p CCC: $18.00
© 1999 American Chemical Society Published on Web 09/09/1999
more difficult since a variety of diverse functional groups are involved in the synthesis for which an equivalent protocol for their quantitative characterization does not exist. Additionally, because of the small scale of solid-phase synthesis even at the reaction optimization stage, the “cleave-and-weigh” method does not work since there is not enough material to weigh. Furthermore, since the most relevant information is the on-support yield, quantitative analysis must be performed on support in multistep synthesis, increasing the degree of difficulty. We have previously introduced a general principle for direct quantitation of the absolute amount of organic functional groups on polymer support by a spectroscopic method.5 The method employs a specific, efficient, and fast solid-phase reaction of a chromophoric reagent with an organic functional group on solid support and the quantitation of the reagent concentration in the supernatant before and after the reaction. We previously established a routine quantitative method for polymer-bound aldehyde and ketone groups using a spectrofluorometric method.5 Both resin-bound hydroxyl and carboxylic acid groups are also very important reaction intermediates. To continue our efforts in developing analytical methods for quantitative analysis of a wide range of compounds containing diverse organic functional groups, we here report the development of novel methods for both quantitative and qualitative analyses of resin-bound hydroxyl and carboxyl groups directly on solid support. EXPERIMENTAL SECTION Materials. 9-Anthroylnitrile, 1-pyrenyldiazomethane (PDAM), and quinuclidine were purchased from Molecular Probes (Eugene, OR). Wang, carboxy-polystyrene, and PS-PEG carboxyl resins were purchased from NovaBiochem (San Diego, CA). All other solvents or reagents were from Aldrich if not specified. Safety Considerations. 9-Anthroylnitrile, quinuclidine, and PDAM are regarded as hazardous. Skin and eye contact and inhalation should be avoided. Personal protection and mechanical ventilation should be used. Resin beads are considered to be nonhazardous materials, but skin contact or inhalation of the powder should be avoided. Solid-Phase Reactions for Quantitation. Resin beads (210 mg, depending on the loading) were suspended in a 1-mL Supelco filtration tube in 0.5 mL of the reaction solvent, and the tube was mixed on an orbital shaker for 20 min. The resin suspension was then drained on a Visiprep vacuum manifold. The reagent (∼2-fold excess relative to the loading of the resin) was added to the resin and the mixture was rotated on a Glas-Col laboratory rotator (32 rpm) at room temperature until the reaction is complete as detected by single-bead FT-IR.7 In the reaction of (4) (a) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem. 1970, 34, 595. (b) Fontenot, J. D.; Ball, J. M.; Miller, M. A.; David, C. M.; Montelaro, R. C. Pept. Res. 1991, 4, 19. (c) Garden, J., II; Tometsko, A. M. Anal. Biochem. 1972, 46, 216. (d) Krchnak, V.; Vagner, J.; Lebl, M. Int. J. Pept. Protein Res. 1988, 32, 415. (e) Ngo, T. T. Appl. Biochem. Biotechnol. 1986, 13, 213. (f) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981, 117, 147. (g) Tyllianakis, P. E.; Kakabakos, S. E.; Evangelatos, G. P.; Ithakissios, D. S. Anal. Biochem. 1994, 219, 335. (h) Chu, S. S.; Reich, S. H. Bioorg. Med. Chem. Lett. 1995, 5, 1053. (5) Yan, B.; Li, W. J. Org. Chem. 1997, 62, 9354. (6) (a) Kamato, K.; Takahashi M.; Terasima, K.; Nishijima M. J. Chromatogr., A 1994, 667, 113. (b) Ramesha, C. S.; Pickett, W. C.; Murthy, D. V. K. J. Chromatogr. 1989, 491, 37. (c) Goto, J.; Goto, N.; Shama, F.; Saito, M.; Komatsu, S.; Suzaki, K.; Nambara, T. Anal. Chim. Acta 1983, 147, 397.
Table 1. Quantitation of Resin-Bound Aldehydes and Ketones
sample
weight (mg)
no. of exp
this paper (mmol/g)
fluorescencea
loading (mmol/g)
formyl-PSb TG S-CHOc PS-ketoned TG-ketonee brominated Wangf
9-10 8-10 5-7 8-10 5
6 6 4 3 3
0.54 ( 0.02 0.23 ( 0.02 0.56 ( 0.06 0.30 ( 0.02 0.0 ( 0.09
0.53 ( 0.03 0.24 ( 0.02 0.54 ( 0.02 0.26 ( 0.02 0.0 ( 0.02
0.44 0.27 0.54 0.27 1.05
a See ref 1. b NovaBiochem. c Rapp Polymer. d PS-OCO(CH ) 2 3 COCH3. e Same as c on TG matrix. f PS-PEG-CO-CH(CH3)Br.
carboxyl resin with PDAM, a gas outlet made from a needle inserted into the cap was used throughout of the reaction for the release of nitrogen produced during the reaction. A 5-µL aliquot of supernatant was diluted in 2 mL of DMF (for hydroxyl) or methanol (for carboxyl) in a cuvette for UV measurement. After the reaction, resin beads were rinsed with THF (1 mL × 10) and DCM (1 mL × 10) and then vacuum-dried for 5 min. The dry beads were analyzed by single-bead FT-IR. Synthesis of Compound 10. Wang resin (1 g, 1.17 mmol) was swollen in anhydrous THF (10 mL) for 5 min. To the resin was added succinic anhydride (585 mg, 5.85 mmol) and 4-(dimethylamino)pyridine (DMAP, 143 mg, 1.17 mmol). The reaction mixture was shaken on a Burrell wrist action shaker at room temperature. About 100 mg of resin was removed at 5, 10, 20, 40, and 120 min for single-bead FT-IR and quantitative analyses of the absolute amounts of hydroxyl and carboxyl functional groups. The resin was washed extensively with anhydrous THF (5 mL x 10) and CH2Cl2 (5 mL × 10) and dried in vacuo overnight for quantitative analysis. UV-Visible Spectroscopy. UV spectra were measured on a Shimadzu UV-2101PC UV-visible scanning spectrophotometer. The path length was 1 cm. The concentration of the unreacted reagent was quantitated on the basis of the maximum absorbance at 368 nm for 9-anthroylnitrile and 383 nm for PDAM. Single-Bead FT-IR. All spectra were collected on a Nicolet Magna 550 FT-IR spectrophotometer coupled with a Nic-Plan IR microscope, using OMNIC software. The microscope is equipped with a 15× Cassegrain objective and liquid nitrogen-cooled mercury-cadmium-telluride (MCT) detector. The view mode aided in locating a single bead. The transmission mode was used for the whole-bead measurement. A few resin beads were put on the diamond window (SpectraTech, Shelton, CT) for measurements. The diamond window was used to collect a background spectrum. Data from the bead were collected with 4-wavenumber resolution, and sixty-four scans were averaged. RESULTS AND DISCUSSION To evaluate UV-visible spectrophotometric method as a reliable method for analysis, we first determined the absolute amount of aldehyde and nonhindered ketone groups on resin support using the identical reaction conditions as previously used in a spectrofluometric study (Table 1).5 The comparison demon(7) Goto, J.; Saito, M.; Chikai, T.; Goto, N.; Nambara, T. J Chromatogr. 1983, 276, 289.
Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
4565
Figure 1. UV-visible absorption spectra of the reagent and singlebead FT-IR spectra. (A) UV-visible spectra of the supernatant that contains 9-anthroylnitrile-quinuclidine adduct mixed with polystyrene resin or Wang resin for 20 min. A 5-µL aliquot of supernatant was diluted into 2 mL of DMF for measurement. DMF was used as reference, and the path length was 1 cm. (B) Single-bead FT-IR spectra taken from the starting material Wang resin and the same resin after 20-min reaction with 9-anthroylnitrile. The IR results show the formation of resin-bound ester carbonyl group and the disappearance of hydroxyl group. All IR spectra were taken using the transmission mode at room temperature as described in the Experimental Section.
Scheme 1
strates that the UV-visible spectrophotometric method is a highly reliable method, while the fluorometric method can be used when high sensitivity is needed or when only a very small amount of sample is available for analysis. From the synthetic chemist’s point of view, the UV-visible method is very advantageous because the operation is simpler, the spectral reading is more stable, and the instrument is widely available. Additionally, the amount of sample is usually more than enough for UV-visible spectroscopic measurements in the reaction optimization stage of combinatorial chemistry. Quantitative Determination of Resin-Bound Hydroxyl Groups. Resin-bound hydroxyl compounds are important intermediates involved in SPOS. They can be transformed into esters, ethers, phosphonates, carbamates, aldehydes, ketones, and halides. So far there is no available quantitative method for the determination of the amount of hydroxyl groups on solid support except for the indirect derivatization analysis, which requires two reaction steps and many hours for a single analysis. We first studied reactions between m-dansylaminophenylboronic acid and 9-anthroylnitrile with hydroxyl resins. The former reaction did not finish in an hour, but the latter reaction was complete and 4566 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Figure 2. (A) Single-bead FT-IR spectra at various times during the reaction shown in Scheme 1. Spectra were taken from a single flattened bead at 0, 1, 2, 4, 8, and 20 min after the initiation of the reaction. The hydrogen-bonded and unbonded hydroxyl stretching at 3458 and 3578 cm-1 disappears as the ester carbonyl band at 1722 cm-1 increases with time. (B) Reaction time course obtained by plotting areas of the IR bands at 1722 (open circles) and 3578 + 3458 (closed circles) cm-1 against time.
appeared quantitative as monitored by single-bead FT-IR. We, therefore, selected 9-anthroylnitrile for further study. Reaction of 9-Anthroylnitrile with Polystyrene-Based Hydroxyl Resin. Compounds containing hydroxyl group(s) react with 9-anthroylnitrile to form an ester (Scheme 1). Condensation of a hydroxyl compound with 9-anthroylnitrile in solution is efficient in the presence of an organic base.6,7 Quinuclidine was reported to accelerate the reaction in solution.7 In the solid-phase version of this reaction, we added a ∼2-fold excess of 9-anthroylnitrile to the suspension of a hydroxyl resin (5-10 mg of resin in 0.2 mL of DMF with quinuclidine) or the same amount of polystyrene resin as a control. The UV spectra of the supernatant in the polystyrene resin mixture and the hydroxyl resin mixture were taken after 20 min (Figure 1A). Figure 1A shows the decreased absorbance indicating a decrease in the concentration of 9-anthroylnitrile in the supernatant after a 20-min reaction. The complete formation of the solid-phase reaction product was confirmed by single-bead FT-IR analysis8 (Figure 1B). The hydroxyl bands at 3578 and 3458 cm-1 disappear and a new band at 1722 cm-1 corresponding to the ester carbonyl in the product 3 emerges after a 20-min reaction.
Table 2. Absolute Amount of Hydroxyl Groups on PS Resinsa sample
weight (mg)
no. of exp
this paper (mmol/g)
loadingb
Wang resin 1 2 3 4
10 8.3-8.6 7.8-8.2 5.5-6.1
2 2 2 2
0.54 ( 0.01 0.64 ( 0.01 0.85 ( 0.04 1.17 ( 0.04
0.54 0.63 0.86 1.17
a The reaction time was 20 min for all polystyrene-based resins. Values from NovaBiochem: Wang resin was first converted to a Fmoc derivative and then measured photometrically by the amount of Fmoc chromophore released upon treatment with piperidine/DMF.
b
Figure 3. UV-visible absorption spectra of 9-anthroylnitrile and the 9-anthroylnitrile/quinuclidine adduct. The measurements were performed at room temperature, and the path length was 1 cm. Spectrum 1 is the UV absorption spectrum of 9-anthroylnitrile at a concentration of 1.46 × 10-4 M. Spectrum 2 is the absorption spectrum of the 9-anthroylnitrile/quinuclidine adduct. The concentrations of 9-anthroylnitrile and quinuclidine were 1.46 × 10-4 and 5.25 × 10-4 M.
We also studied the reaction kinetics using single-bead IR method (Figure 2). Intensities of the O-H stretch bands at 3578 and 3458 cm-1 decrease gradually and the intensity of the carbonyl band at 1722 cm-1 from the product increases with time. Using a resin band at 1945 cm-1 as an internal reference, changes in peak areas of above-mentioned bands were plotted against time and the time course fitted to a pseudo-first-order reaction rate equation with a rate constant of ∼6.6 × 10-3 s-1. The above experiments have shown that the reaction of the hydroxyl resin with 9-anthroylnitrile is fast and efficient. The remaining reagent can be easily quantified by UV-visible spectrophotometric measurement. Therefore, this procedure is suitable for a quantitative analysis of the amount of hydroxyl groups on resin. Absorption Spectrum of 9-Anthroylnitrile in the Presence of Quinuclidine. The reaction between 9-anthroylnitrile and resinbound hydroxyl groups is facilitated by quinuclidine. A spectroscopically stable adduct of 9-anthroylnitrile with the quinuclidine was formed when quinuclidine was added to the 9-anthroylnitrile solution as observed by UV-visible spectroscopic measurements. The absorption spectrum of this adduct shows fine structures with absorption maximums at 386, 366, 350, and 335 nm (Figure 3), while the 9-anthroylnitrile alone has a broad band at 442 nm. Because quinuclidine itself has no absorption from 190 to 700 nm and the UV spectrum of the supernatant was always measured in the presence of quinuclidine, we always used the excess amount of quinuclidine in our experiments and UV-visible measurements. Correction for the Amount of Noncovalently Bound 9-Anthroylnitrile Molecules. During the reaction, resin is capable of absorbing the reagent molecules without a covalent bond. For quantitative analysis, the amount of reagent molecules noncovalently trapped into the resin matrix was corrected by subtracting the amount of reagent molecules absorbed in the same amount of 1% divinyl(8) (a) Yan, B.; Kumaravel, G.; Anjaria, H.; Wu, A.; Petter, R.; Jewell, C. F., Jr.; Wareing, J. R. J. Org. Chem. 1995, 60, 5736. (b) Yan, B.; Kumaravel, G. Tetrahedron 1996, 52, 843. (c) Yan, B.; Fell, J. B.; Kumaravel, G. J. Org. Chem. 1996, 61, 7467. (d) Yan, B.; Sun, Q.; Wareing, J. R.; Jewell, C. F. J. Org. Chem. 1996, 61, 8765.
Chart 1
benzene cross-linked polystyrene resin under identical conditions. It is appropriate to question here whether it is justified to use such a control because the absorbing capability of hydroxyl resin may not be the same as that of polystyrene resin. To compare the 9-anthroylnitrile-absorbing capability of the hydroxyl resin with that of the polystyrene resin, we studied the binding of a nonreactive 9-anthroylnitrile analogue 7 (Chart 1) to both resins. Compound 7 binds to the hydroxyl resin and the polystyrene resin with a similar affinity as determined by the absorption of reagent from supernatant as observed in the decrease of the UV-visible absorption spectrum of 7 in the presence of resin. On the basis of these measurements, it is justifiable to use polystyrene resin as a control for correcting the absorption capability of the hydroxyl resin. Analysis of Various Hydroxyl Resin Samples. The absolute amount of hydroxyl groups in various resin samples was then determined by the reagent consumption deduced from UV spectroscopic measurements. It was calculated from the decrease in 9-anthroylnitrile concentration in the supernatant. The noncovalently bound 9-anthroylnitrile in the resin was corrected and estimated to be ∼10% under our conditions. The experimental results are shown in Table 2. Results by our method agree well within experimental error with the loading values determined by Fmoc-derivatization method. The advantages of the method reported above over the Fmoc-derivatization method will be discussed in a later section. Reactivity of 9-Anthroylnitrile with Other Organic Functional Groups. We further investigated the reactivity of 9-anthroylnitrile with other organic functional groups, which might pose interference or complications. We specifically tested a hindered and nonhindered primary amine resin, a phenol resin, a secondary amine resin, and two amide resins as shown in Table 3. The aminomethylated PS resin reacted with 9-anthroylnitrile quantitatively. It gave a 100% yield in 20 min and a new IR band at 1662 cm-1 corresponding to the carbonyl group of the amide product was detected. The sterically hindered primary amine MBHA resin Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
4567
Table 3. Tests for Hydroxyl Interferencea sample
weight (mg)
aminomethylated 7 polystyrene MBHA 4.5-10.2 4-iodobenzene 7 sulfonamide rink amide Rink amide 16.1 prolinol 2-chlorotrityl 6.9
no. of exp
this paper (mmol/g)
loading (mmol/g)
4
0.91 ( 0.02
0.83b
3 2
0.047 ( 0.02 0.048 ( 0.025
0.57b 0.81c
2 2
0.025 ( 0.00 -0.031 ( 0.002
0.34d 0.82
a TG S Br resin was used as a control for PS-PEG resins and 1% DVB polystyrene for PS resins. The reaction time was 20 min. b The loading was determined by elemental analysis of the nitrogen content. c The loading was calculated from the amount of product released upon treatment with TFA/DCM. d The loading was determined from the amount of Fmoc chromophore released upon treatment with piperidine/DMF.
Scheme 2
did not react with 9-anthroylnitrile in three independent experiments. After the 20-min reaction, no decrease in reagent concentration in the supernatant and no IR band corresponding to the amide product in the IR spectrum were detected. Since amino and hydroxyl groups rarely coexist in the same SPOS intermediate, this reagent provides a useful quantitative analytical method for either of the hydroxyl or nonhindered primary amine groups. Phenol resin reacted with the reagent only partially (∼20%). This should be considered when phenol and hydroxyl groups coexist in the molecule. Amide and secondary amine resins did not react with 9-anthroylnitrile. Therefore, the hydroxyl quantitation can be done in the presence of most organic functional groups except for the nonhindered primary amine group. Phenol only poses a small interference when present. This method can also be conveniently used to selectively quantify nonhindered primary amine groups. This reagent can provide a satisfactory way to analyze hydroxyl group in most synthesis steps. Hydroxyl Group on PS-PEG Resins. PS-PEG resins are notoriously absorbent for moisture. A quantitative method using 9-anthroylnitrile is moisture sensitive and, therefore, cannot be used for quantitative analysis of PS-PEG resins. The conversion of PS-PEG hydroxyl resins to a Fmoc derivative, then, becomes an appropriate alternative for analysis. However, this reagent provides a qualitative “color” test for analyzing PS-PEG-based resin samples to detect the existence of hydroxyl groups (see discussion later). Quantitative Determination of Resin-Bound Carboxyl Groups. Like aldehyde, ketone, and hydroxyl functional groups, carboxyl is also an important functional group involved in SPOS. Resin-bound carboxylic acid is the precursor of esters, amides, anhydrides, and other compounds. Carboxylic acid can be involved in the Ugi reaction, Curtius rearrangement, and Mitsunobu reaction. Quantitative analysis of the resin-bound carboxyl group is necessary for general organic synthesis on solid support. 4568
Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Current analyses rely on indirect analysis such as derivative analysis. The direct titration analysis suffers from serious errors due to two probable reasons: (1) the incompatibility of the hydrophobic bead with the aqueous titration and (2) the charge repulsion effect. The second factor would not affect titration in solution. However, when negative charges are clustered in a bead, the further entrance of negative ions would be prohibited, and therefore, the complete titration reaction is impossible. PDAM was used as a fluorescence-labeling reagent for chromatographic analysis in the past.10 It has never been used in solidphase reactions to our knowledge. The solid-phase reaction between PDAM and carboxyl resin was, therefore, optimized first. The formation of nitrogen gas was released by inserting a needle on the cap of the reaction tube. Different solvents were compared in their effects on reaction rate as monitored by single-bead FTIR. Ethyl acetate was found to be a better solvent compared with DMF, DCM, and acetonitrile. Because of the lower solubility of PDAM in ethyl acetate, a lower concentration of the reagent and a smaller amount of resin (2 mg) were used for the analysis. Reaction of PDAM with Carboxyl Resins. The 1-pyrenyldiazomethane 5 (PDAM) reacts with a carboxyl compound to form an ester product (Scheme 2).10b A 2-fold excess of PDAM reacted with resin 4 in ethyl acetate. The kinetics of this reaction was studied by the single-bead FT-IR method as shown in Figure 4. The IR spectrum of the starting carboxyl resin (Figure 4A, spectrum at 0 min) exhibits two carbonyl bands at 1688 and 1722 cm-1 corresponding to associated and free carboxyl groups.9 After a 33-min reaction, both bands disappear while an ester carbonyl band in the product appears coincidentally at 1722 cm-1. Besides these spectral changes, the acid OH group stretching signals at 2500-3500 cm-1 also disappear after 33 min. The changing area of the acid carbonyl band at 1688 cm-1 was integrated after a peakfit procedure using PeakFit program (Jandel Scientific). The rate constant was determined to be ∼3.0 × 10-3 s-1. The analysis of reaction kinetics is shown in Figure 4B. From kinetic analysis, the reaction is complete in 30-40 min under our reaction conditions. Figure 5 shows the UV-visible absorption spectra of PDAM in the supernatant after mixing with carboxyl resin (lower spectrum) or polystyrene resin (upper spectrum) for 50 min. On the basis of these studies, quantitative analysis of resinbound carboxyl compounds is feasible by UV-visible spectroscopic measurement alone. Correction for the Amount of Noncovalently Bound PDAM Molecules. To calibrate noncovalently bound PDAM molecules in the resin, polystyrene resin was used as a control as in the case of the hydroxyl resin study. To examine whether polystyrene resin is a valid control for carboxyl resins in the noncovalent absorption of reagent 5, the noncovalent absorption of a nonreactive PDAM analogue 8 (Chart 1) was compared in both resins. The absorption of 8 into both resin 4 and the polystyrene resin is nearly the same as determined by the extent of reagent absorption from supernatant as monitored by the decrease in the UV-visible absorption (9) Letsinger, R. L.; Kornet, M. J.; Mahadevan, V.; Jerina, D. M. J. Am. Chem. Soc. 1964, 86, 5163. (10) (a) Iwamura, M.; Ishikawa, T.; Koyama, Y.; Sakuma, K.; Iwamura, H. Tetrahedron Lett. 1987, 28, 679. (b) Nimura, N.; Kinoshita, T.; Yoshida, T.; Uetake, A.; Nakai, C. Anal. Chem. 1988, 60, 2067. (c) Yoshida, T.; Uetake, A.; Nakai, C.; Nimura, N.; Kinoshita, T. J. Chromatogr. 1988, 456, 421. (d) Iohan, F.; Monder, C.; Cohen, S. J. Chromatogr. 1991, 564, 27. (e) Schneede, J.; Ueland, P. M. Anal. Chem. 1992, 64, 315.
Table 4. Quantitation of Carboxyl Groups on Resin Supportsa sample
weight (mg)
no. of exp
this paper (mmol/g)
loadingb (mmol/g)
A15709 (PS) A17819 (PS) A17115 (PS-PEG) A18514 (PS-PEG)
2-3 2-3 9-10 9-10
6 4 4 4
0.90 ( 0.02 1.03 ( 0.08 0.26 ( 0.02 0.24 ( 0.01
0.89 0.98 0.26 0.26
a The reaction time was 50 min for both PS and PS-PEG resins. Values from NovaBiochem calculated from the aminoethyl precusor resins.
b
Table 5. Tests for Carboxyl Interferencea sample
product peak at 1680-1776 cm-1
loading (mmol/g)
aminomethylated polystyrene prolinol 2-chlorotrity Wang oxime Rink acid
no no no no no
0.83 0.82 0.86 0.57 0.49
a The possible amide or ester product should have a carbonyl band in 1680-1776-cm-1 region.
Figure 4. (A) Single-bead FT-IR spectra taken at various times during the reaction shown in Scheme 2. (B) Reaction time course obtained by plotting areas of the IR band at 1688 cm-1 from peak-fit analysis against time.
Figure 5. UV-visible absorption spectra of PDAM in the supernatant before and after a 50-min reaction.
spectrum of 8 in the presence of resin. The results suggest that using PS resin as a control to correct PDAM’s absorption property in carboxyl resins is appropriate. Analysis of Various Carboxyl Resin Samples. We then analyzed the absolute amount of carboxyl groups in various PS- and PSPEG-based resin samples, in which the loading values have been obtained by indirect analysis. In our measurements, the amount of carboxyl groups was calculated from the decrease in PDAM
concentration in the supernatant after a 50-min reaction. The noncovalently bound PDAM was corrected based on the PS resin control experiment and estimated to be ∼10% under our reaction conditions. Multiple analyses of two batches of PS-based carboxyl resin and two batches of PS-PEG-based carboxyl resins were carried out. Results are shown in Table 4. Results obtained with our method are in agreement within experimental error with results obtained by derivative analysis. The Reactivity of PDAM with Other Organic Functional Groups. A variety of resins (aminomethyl, prolinol-2-chlorotrityl, oxime-, Rink acid, and Wang resins) were tested for their reactivity with PDAM (Table 5), and no UV-visible spectral change in the supernatant and no formation of ester or amide products in the resin were detected by both spectrophotometric and single bead IR measurements. Results have shown that the PDAM is a specific reagent for quantifying carboxyl groups on resins. Quantitative Monitoring of a Solid-Phase Organic Reaction. As presented above, both quantitation methods were successfully used in the analysis of the reference samples, in which the loading was determined by the tedious derivatization analysis. We further carried out the analysis of a series of SPOS samples using both quantitative methods. We have monitored the changes in the amounts of hydroxyl and carboxyl groups on resin in the reaction depicted in Scheme 3. During the course of the reaction, a very low level of carboxyl groups at the very beginning of the reaction (Scheme 3) and a very low level of hydroxyl groups near the end of the reaction will be encountered. These experiments give us an opportunity to analyze samples containing various levels of functional groups and estimate detection limits of our methods. In this reaction, the starting hydroxyl resin has a loading of 1.17 mmol/g determined using 9-anthroylnitrile method. During the reaction, the amount of hydroxyl groups decreases as the amount of carboxyl groups increases. The product should be a carboxyl resin with a loading of 1.09 mmol/g after correcting for Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
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Figure 6. Single-bead FT-IR spectra at various times during the reaction shown in Scheme 3. Spectra were taken from a single flattened bead at 0, 5, 10, 20, 40, and 120 min.
Figure 7. Changes in the absolute amounts of hydroxyl and carboxyl groups on the resin at various times during the reaction depicted in Scheme 3. Closed circles represent the amounts of hydroxyl groups and open circles the amount of carboxyl groups. Triangles represent the area integrations of the IR band at 1712 and 1735 cm-1.
Scheme 3
the change in the “molecular weight” of resin after the addition of 1.17 mmol of building block onto 1 g of resin. First, the reaction in Scheme 3 was monitored by single-bead FT-IR (Figure 6). As the reaction progresses, the hydroxyl stretching bands at 3578 and 3450 cm-1 are diminishing and two carbonyl-stretching bands at 1735 and 1712 cm-1 as well as the broad feature from 3300 to 2500 cm-1 emerging. These singlebead FT-IR measurements confirmed the interconversion between the hydroxyl and carboxyl groups during the reaction. The peak areas of the two carbonyl bands in the product at various times were integrated and normalized to the corresponding loading value and plotted against time (triangles in Figure 7). The absolute amounts of hydroxyl and carboxyl groups at various times during the reaction were then determined by quantitative methods described above and plotted (closed and open circles in Figure 7). The data points were obtained from duplicate measurements. These results demonstrate that an accurate determination can be achieved at various functional group loading levels. The lowest amount of hydroxyl groups was determined to be 0.05 mmol/g in this study. The flexibility and the dynamic range of these methods are extremely useful for monitoring the synthesis of a product and the disappearance of a starting material in SPOS. Comparison with Other Quantitative Methods. Organic functional groups such as aldehyde, hydroxyl, and carboxyl groups can be measured indirectly by first converting them into a derivative containing a Fmoc protection group and then cleaving Fmoc in a basic solution. The UV absorbance of the released Fmoc groups is proportional to the amount of functional groups to be detected. Two reaction steps are involved, and a large amount of resins is needed. This method is not suitable for routine reaction monitoring in ordinary synthesis laboratories for several reasons. First, it is very slow. For example, the derivatization method 4570 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
requires two reaction steps and can take many hours to a day to analyze a sample. Second, it is prone to errors. Results obtained from the derivatization method highly depend on the completeness of both derivatization and Fmoc cleavage reactions. An example is that the aldehyde quantitation value obtained by our method is consistently higher than the Fmoc derivatization method probably due to the incomplete derivatization/cleavage reactions.4 Third, it consumes much more sample. In this aspect, although the method may be useful for resin manufacturing laboratories where plenty of resin samples is available for analysis, it is not suitable for ordinary synthetic laboratories where only small-scale synthesis is practiced. “Color Tests” for Qualitative Analysis of Aldehyde, Hydroxyl, and Carboxyl Groups on Resin. In the solid-phase reaction optimization process, it is sometimes desirable to confirm quickly whether the starting material is totally converted to a product. A simple “color” test is very useful. A few drops of dansylhydrazine, 9-anthroylnitrile, or PDAM were added to resin-bound aldehyde, hydroxyl, or carboxyl compounds. After 2 min, resin beads were completely washed with solvent to remove excess reagents and viewed under a UV lamp. Under 350-365-nm UV illumination, beads became fluorescent if the targeted organic functional group was present. As a control, the nonfunctionalized polystyrene resin bead was not fluorescent. This qualitative “color” test can be used for both polystyrene- and PS-PEG-based resins using all three reagents. CONCLUSION In this paper, we report the development and application of two novel methods for rapid determination of the absolute amount of hydroxyl or carboxyl groups directly on resin supports. These methods can also be used as a fast qualitative “color” test. The method for the analysis of resin-bound hydroxyl groups is based on the rapid and efficient reaction of 9-anthroylnitrile with resin-bound hydroxyl groups. A spectroscopic study of the supernatant concentration of the reagent determines the loading on the resin after subtracting the noncovalently absorbed amount of reagent molecules in the resin. Approximately 5-10 mg of resin and an analysis time of 30 min are needed. Most organic functional
groups do not interfere with the analysis except for the nonhindered primary amine group. The phenol group may pose a small interference. This method can also be used to determine the absolute amount of nonhindered primary amine groups on solid supports. The method for the analysis of resin-bound carboxyl groups is based on the rapid and efficient reaction of both PS- and PSPEG-based carboxyl resins with PDAM. Only 2 mg of resin is required, and the analysis takes ∼1 h. Amines, amides, and acidic hydroxyl groups do not interfere with the quantitation.
Compared with the Fmoc-derivatization analysis method, methods reported here are much more rapid, consume much less resin, and provide more accurate results. By incorporating the easy operation of the widely available UV-visible spectrometer and simple calculations, these methods are designed to provide synthetic chemists with routine methods to optimize solid-phase organic synthesis in combinatorial chemistry. Received for review June 21, 1999. Accepted August 12, 1999. AC990685P
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