Bioconjugate Chem. 2008, 19, 585–587
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Small-Molecule Reagents for Cellular Pull-Down Experiments Xiang Wang, Brandon S. Imber, and Stuart L. Schreiber* Howard Hughes Medical Institute, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142, and Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138. Received August 7, 2007; Revised Manuscript Received December 1, 2007
Affinity purification of interacting proteins from cellular extracts is a powerful technique for identifying the cellular targets of small molecules. Affinity matrix-based small-molecule reagents are usually prepared by conjugating small molecules of interest to a solid support such as agarose. This protocol describes an efficient and robust method to immobilize small molecules containing a primary alcohol, a common functional group in small molecules, especially in small molecules prepared using diversity-oriented synthesis. This method comprises one element of a systematic approach to the target identification problem in chemical biology.
Small molecules that modulate protein function are valuable tools for studying cell circuitry, among others (1, 2). These small-molecule probes are increasingly being identified in cell-based, phenotypic screens (3). This discovery route requires effective methods for identifying cellular targets, and the technique of affinity chromatography (4), which requires small-molecule-based affinity reagents, has proven to be the most effective approach to accomplish this goal (5, 6). Many methods have been developed to attach small molecules covalently to beaded agarose containing such functional groups as acids, amines, and so forth (7). A complementary pair of functional groups on the beads and small molecules is required
small molecules are extremely common in modern collections of compounds used in high-throughput screening. Whereas amino-containing small molecules are routinely conjugated to beaded agarose for use in affinity chromatography, hydroxylcontaining small molecules have proven to be more challenging. Although methods exist to immobilize hydroxyl-containing ligands using oxiranes (10) or vinyl sulfones (11), these suffer from either the poor reactivity of oxiranes or the poor stability of the sulfones. Diversity-oriented synthesis (DOS, Figure 1) yields small molecules well-suited for the discovery of probes, the optimization of them, and the eventual efficient manufacturing of the optimized variants (12, 13). In our own studies,
Figure 1. General strategy for identifying the targets of biologically active small molecules.
Figure 2. Scheme for immobilizing primary alcohol-containing small molecules on beaded agarose.
for selective immobilization. Recently, Finn and co-workers have shown that small molecules bearing a terminal alkyne or azide can be covalently attached to agarose using a Cu(I)catalyzed 1,3-dipolar cycloaddition reaction (8) with high efficiency and specificity (9). Amino- or hydroxyl-containing * To whom correspondence should be addressed. Phone: (617) 3249603. Fax: (617) 324-9601. E-mail:
[email protected].
we have incorporated a hydroxymethyl group into eVery product, resulting from our diversity syntheses. This functional group is poised for further modifications, such as immobilization on the glass surface for small-molecule microarray screens (14), covalent modification with biasing elements for targeting classes of proteins (15), and, as described here, conjugation to beaded agarose for the identification of cellular targets.
10.1021/bc700297j CCC: $40.75 2008 American Chemical Society Published on Web 01/15/2008
586 Bioconjugate Chem., Vol. 19, No. 3, 2008
Communications
Figure 3. Representative immobilized alcohol-containing small molecules.
Figure 4. Determination of the loading efficiency using HPLC-MS with reserpine as an internal standard. (A) LC trace of the mixed imidazolide and reserpine in DMF. (B) LC trace of the immobilization reaction mixture after incubation at room temperature for 2 days. (C) LC trace of the immobilization reaction mixture after incubation at 50 °C for 16 h. (a) HPLC method: 5–95% acetonitrile in water with 0.1% formic acid over 2.5 min. Diode array wavelength: 210–500 nm.
RESULTS AND DISCUSSION Here, we report an efficient and robust method to immobilize hydroxyl-containing small molecules to beaded agarose. 1,1′Carbonyldiimidazole (CDI 16-18) reagent has been used to immobilize amino-containing small molecules (19). We recognized that the mixed imidazolide intermediate resulting from reaction of this reagent with hydroxyl-containing compounds is inert to most functional groups and stable to a wide range of solvents and temperatures. We reasoned that the resulting carbamate bond would be noncharged and highly stable
under conditions used in cellular pull-downs, and thus wellsuited for many target identification applications. Hence, we chose to use CDI to activate the hydroxyl-containing compounds. The resulting mixed imidazolide was immobilized on amino-containing beaded agarose after the excess CDI was quenched with water (Figure 2). The excess amine on the agarose surface was blocked by acetylation using N-acetoxysuccinamide. This protocol has been used to synthesize dozens of primary alcohol-containing small molecules (see Figure 3 for representa-
Communications
Bioconjugate Chem., Vol. 19, No. 3, 2008 587
Note Added after ASAP Publication: This paper was published ASAP on January 15, 2008 with an incomplete Acknowledgment. The correct version reposted on February 8, 2008.
LITERATURE CITED
Figure 5. Immobilization of a compound known to bind immunophilin proteins and the affinity purification of FKBP12 from A549 whole cell lysate using the immobilized bait. In the Western blot, lane 1, affinitypurified protein sample 1 using immobilized bait compound; lane 2, affinity purified protein sample 2 using immobilized bait compound; lane 3, affinity purified protein sample 3 using blocked blank beads; lane 4, A549 whole cell lysate; lane 5, residue cell lysate 1 after affinity purification using immobilized bait compound; lane 6, residue cell lysate sample 2 after affinity purification using immobilized bait compound; lane 7, residue cell lysate sample 3 after affinity purification using blocked blank beads.
tive molecules A1-A11) conjugated to beaded agarose with over 85% loading efficiency. Secondary alcohol-containing small molecules such as A12 (Figure 3) may also be conjugated to beaded agarose, albeit with significantly lower efficiency (
