Biotinylated Anisomycin: A Comparison of Classical and âClick

Bioconjugate Chem. 2007, 18, 1593−1603

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Biotinylated Anisomycin: A Comparison of Classical and “Click” Chemistry Approaches Iain A. Inverarity,† Romain F. H. Viguier,† Philip Cohen,‡ and Alison N. Hulme*,† The School of Chemistry, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh, EH9 3JJ, United Kingdom, and MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, United Kingdom. Received March 13, 2007; Revised Manuscript Received June 14, 2007

Two approaches to the synthesis of biotinylated derivatives of the stress-activated protein kinase (SAPK) pathway activator anisomycin have been investigated. Attachment of the biotin moiety to the central core was achieved either through the use of a classical displacement reaction on R-halo carbonyl derivatives of biotin or through a copper(I)-catalyzed 1,3-dipolar Huisgen cycloaddition (“click”) coupling of biotinylated azides to propargylmarked analogues of anisomycin. In each case, the resultant N-linked molecular probes were found to be active in SAPK pathway immunoblot assays, while their O-linked counterparts were inactive. However, in sharp contrast to the classical coupling approach which results in low coupling yields, the aqueous “click” coupling process was found to deliver high yields of biotinylated probes, making it the conjugation method of choice. A survey of the available methods for the addition of a propargyl marker onto a range of chemical functionalities strongly suggests that this copper(I)-catalyzed 1,3-dipolar Huisgen cycloaddition approach to biotinylation may be generally applied.

INTRODUCTION Despite rapid advances in the generation and testing of small molecule libraries in recent years (1-3), current strategies for the identification of the biological targets of library members in forward chemical genetics screens still require the synthesis of an appropriate molecular probe. The structure of this molecular probe is typically based on an active library member, which has been modified by introduction of a tag (biotin, photoactive, or fluorescent label) (4-7). Selective biotinylation has been widely used over the last 30 years as a method for purification and identification of a range of targets (8). The popularity of this strategy stems from the peculiarly tight binding of biotin to (strept)avidin (Kd of 10-15 M) (9), which means that affinity chromatography methods based around this partnership are particularly useful for the identification of the binding partners of high-affinity small molecules where there is an abundant protein receptor. A notable recent example of this strategy includes the identification of the biological target of L-685,485 (a potent γ-secretase inhibitor) as the PS1 protein (10), while further examples of this approach encompass the biotinylation of a range of biologically active molecules including carbohydrates (11), steroids (12), and a number of drugs such as ezetimibe (a cholesterol inhibitor) (13), and paclitaxel (an anti-cancer compound) (14). The pyrrolidine antibiotic anisomycin 1a was first isolated from Streptomyces roseochromogenes and S. griseolus in 1954 (15). Over subsequent years, a series of studies have confirmed the structure and absolute stereochemistry as 2R,3S,4S for the three stereocentres around the pyrrolidine ring (16-19). Since its discovery, anisomycin has been found to generate many interesting biological responses including anti-fungal activity (20), protein synthesis inhibition (21), and anti-tumor activity in the nanomolar region (22). In addition, over the past fifteen years anisomycin has been used as a chemical stimulant of the * Corresponding author. E-mail: [email protected]. Phone: +44 131 6504711. Fax: +44 131 6506453. † University of Edinburgh. ‡ University of Dundee.

stress-activated protein kinase (SAPK) pathways at subinhibitory levels (Figure 1) (23-28). The SAPK pathway is a distinct signaling branch within the mitogen activated protein kinase (MAPK) superfamily. The core unit of the MAPK pathways is a three-membered protein kinase cascade whose components are highly conserved in structure and organization. In this cascade, the MAPKs are phosphorylated and activated by a MAPK kinase (MAPKK/MKK). The MAPKK are “dualspecific” kinases that catalyze the phosphorylation of MAPKs at Thr and Tyr sites, specifically targeting a Thr-X-Tyr motif on MAPK (where X is proline and glycine for the JNK/SAPK1 and p38/SAPK2 modules, respectively) (29, 30). Regulation of these pathways results in controlled activation of downstream kinases and transcription factors including c-Jun, ATF-2, and MAPKAP-K2. Regulation of the SAPK pathways is of interest across a broad range of fields, as it is thought to play a role in a range of conditions such as ischemic brain injury resulting from stroke episodes (31), cancer (32), and Alzheimer’s disease (33). Although the precise target of anisomycin is unknown, the downstream effects on the SAPK pathways have been welldocumented (29-30). The treatment of mammalian, yeast, and insect cells with anisomycin is known to strongly activate both the JNK/SAPK1 and p38/SAPK2 pathways, resulting in phosphorylation of their substrates, such as JNK and MAPKAPK2, respectively. Previous structure-activity relationship (SAR) studies for activation of the JNK/p38 pathways by anisomycin gave us an insight into the core functionality required to stimulate this phenotypic response, and hence allowed us to hypothesize potential sites for biotinylation. Principally, these studies showed that the presence of an ester functionality at the C(3) position on the pyrrolidine ring was essential for activity, with both the C(3) acetate (anisomycin, 1a) and C(3) propionate ester 1b shown to be active compounds (Figure 2) (34). In addition, both the C(4)-H 2a and C(4)-Me 2b analogues were found to activate the SAPK pathways, with the C(4)-H analogue exhibiting similar levels of activation to that of anisomycin (34). More recent work has shown that, while the N(1)-Bn analogue 1c generates a strong activation of the SAPK pathways (35), simultaneous

10.1021/bc070085u CCC: $37.00 © 2007 American Chemical Society Published on Web 08/17/2007

1594 Bioconjugate Chem., Vol. 18, No. 5, 2007

Figure 1. Stress-activated protein kinase (SAPK) pathways.

Figure 2. Anisomycin and analogues used to determine the structureactivity relationships (SAR) for activation of the SAPK pathways.

dibenzylation of N(1) and C(4)-OH, analogue 1d, is not tolerated. These results suggest that the C(3) and C(4) sites are unsuitable for conjugation as biotinylated probes. However, the N(1) position is tolerant of relatively bulky functionalization, particularly as an amino- rather than an amido/carbamate-type linkage (36). Furthermore, a survey of the literature relating to the bioactivity of anisomycin in other cellular assays led us to postulate that conjugation of the phenolic oxygen might also be possible (22). We therefore set out to synthesize a series of biotinylated molecular probes coupling to the anisomycin scaffold through either the pyrrolidine nitrogen or the phenolic oxygen.

EXPERIMENTAL SECTION General Methods. All reactions involving air- or watersensitive reagents were carried out under an atmosphere of nitrogen using flame- or oven-dried glassware. Unless otherwise noted, starting materials and reagents were obtained from commercial suppliers and were used without further purification. CH2Cl2 and Et3N were distilled from calcium hydride. Anhydrous methanol, DMF, and acetonitrile were used as supplied. Unless otherwise indicated, organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure using a rotary evaporator. Purification by flash column chromatography was carried out using Merck Kieselgel 60 silica gel as the stationary phase. IR spectra were measured on a Perkin-Elmer Paragon 1000 FT-IR spectrometer as thin films unless otherwise stated. 1H and 13C NMR spectra were measured on Bruker DPX360 and Bruker AVA600 instruments; J-values are in hertz. Fast atom bombardment (FAB) mass spectra were obtained using a Kratos MS50TC mass spectrometer at The University of Edinburgh. Complete Protease Inhibitor tablet was obtained from Roche (Lewes, Sussex, UK), cell culture media from Gibco (Paisley,

Inverarity et al.

UK), precast Bis-Tris gradient SDS-polyacrylamide gels, running buffer, and transfer buffer from Invitrogen (Paisley, UK), and enhanced chemiluminescence (ECL) reagents from Amersham (Bucks, UK). Phospho-specific antibodies that recognize JNK1/2 phosphorylated at Thr183 or unphosphorylated JNK1/2 were purchased from Biosource (Nivelles, Belgium), while horseradish peroxidase-conjugated secondary antibodies were from Pierce (Cheshire, UK). Coupling to C2 Biotinylated Iodoacetamide 4. 2′-NBiotinylamino-eth-1-yl 4′-(2′′-acetoxyeth-1′′-yl)phenyloxyacetamide 5. To biotinylated iodoacetamide 4 (20.0 mg, 40.0 µmol) in DMF (1.5 mL) was added phenol 3 (10.0 mg, 40.0 µmol), followed by K2CO3 (6.00 mg, 40.0 µmol). The solution was stirred for 48 h at 80 °C. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [10% MeOH/CH2Cl2] to give phenol ether 5 as a colorless solid (17.0 mg, 34.4 µmol, 86%). Rf [10% MeOH/CH2Cl2] ) 0.30. 1H NMR δ (360 MHz, DMSO) 8.12 (1H, t, J 5.2, N17H), 7.87 (1H, t, J 5.2, N14H), 7.17 (2H, d, J 8.7, ArH), 6.89 (2H, d, J 8.7, ArH), 6.42 (1H, br s, N6H), 6.36 (1H, br s, N4H), 4.42 (2H, s, C19H2), 4.30-4.27 (1H, m, C3H), 4.15 (2H, t, J 7.0, C26H2), 4.12-4.08 (1H, m, C7H), 3.15 (4H, dt, J 9.9, 5.6, C15H2, C16H2), 3.10-3.04 (1H, m, C8H), 2.81 (2H, t, J 7.0, C25H2), 2.80 (1H, dd, J 12.3, 5.2, C2HAHB), 2.56 (1H, d, J 12.3, C2HAHB), 2.04 (2H, t, J 7.3, C12H2), 1.97 (3H, s, OAc), 1.64-1.23 (6H, m, C9H2, C10H2, C11H2). 13C NMR δ (90.7 MHz, DMSO) 172.3 (1C, C13, Q), 170.2 (1C, OAc, Q), 167.8 (1C, C18, Q), 162.6 (1C, C5, Q), 156.6 (1C, Ar, Q), 130.4 (1C, Ar, Q), 129.5 (2C, ArH, CH), 114.4 (2C, ArH, CH), 66.7 (1C, C19, CH2), 64.2 (1C, C26, CH2), 60.7 (1C, C7, CH), 59.0 (1C, C3, CH), 55.1 (1C, C8, CH), 39.6 (1C, C2, CH2), 38.3 (1C, C16, CH2), 37.8 (1C, C15, CH2), 34.9 (1C, C12, CH2), 33.2 (1C, C25, CH2), 28.0 (1C, C10, CH2), 27.8 (1C, C9, CH2), 24.9 (1C, C11, CH2), 20.5 (1C, OAc, CH3). m/z (FAB, THIOG) 507 ([M + H]+, 29%), 91 (75). HRMS (FAB, THIOG) (found: [M + H]+, 507.2281. C24H35N4O6S requires m/z, 507.2277). C2-Classical O-Linked Boc C(4)-H Anisomycin Biotin Molecular Probe 7a. To biotinylated iodoacetamide 4 (50.0 mg, 111 µmol) in DMF (2.5 mL) was added C(4)-H analogue 6 (37.0 mg, 111 µmol), followed by K2CO3 (18.0 mg, 130 µmol). The solution was stirred for 48 h at 80 °C. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [5% to 15% MeOH/CH2Cl2] to give carbamate 7a as a colorless solid (23.0 mg, 34.0 µmol, 31%). Rf [20% MeOH/CH2Cl2] ) 0.27. 1H NMR δ (600 MHz, CDCl3, 323 K) 7.23 (1H, br t, J 4.6, N17H), 7.14 (2H, d, J 8.0, ArH), 6.85 (2H, d, J 8.0, ArH), 6.80 (1H, br s, N14H), 6.35 (1H, br s, N6H), 5.45 (1H, br s, N4H), 5.11 (1H, br q, J 6.6, C27H), 4.52-4.45 (3H, br s, C3H, C19H2), 4.31-4.29 (1H, m, C7H), 4.24 (1H, br q, J 6.1, C26H), 3.48-3.43 (5H, m, C15H2, C16H2, C29HMHN), 3.35-3.31 (1H, m, C29HMHN) 3.14-3.12 (1H, m, C8H), 2.952.92 (1H, m, C25HSHT), 2.89 (1H, dd, J 12.7, 4.4, C2HAHB), 2.82 (1H, dd, 13.5, 5.5, C25HSHT), 2.71 (1H, d, J 12.7, C2HAHB), 2.21 (2H, br q, J 6.5, C12H2), 2.10-2.05 (1H, m, C28HEHF), 2.00 (3H, s, OAc), 1.82-1.65 (5H, m, C9H2, C11H2, C28HEHF), 1.46 (9H, s, tBuO), 1.30-1.27 (2H, m, C10H2). 13C NMR δ (151.1 MHz, CDCl3, 323 K) 174.3 (1C, COtBu, Q), 174.2 (1C, C13, Q), 170.3 (1C, OAc, Q), 169.9 (1C, C18, Q), 164.1 (1C, C5, Q), 156.2 (1C, Ar, Q), 132.7 (1C, Ar, Q), 130.9 (2C, ArH, CH), 114.9 (2C, ArH, CH), 80.1 (1C, tBu, Q), 73.3 (1C, C27, CH), 67.7 (1C, C19, CH2), 61.9 (1C, C7, CH), 60.4 (1C, C3, CH), 59.5 (1C, C26, CH), 55.7 (1C, C8, CH), 43.3 (1C, C29, CH2), 40.7 (1C, C2, CH2), 39.6 (1C, C16, CH2), 39.6 (1C, C15, CH2), 35.9 (1C, C12, CH2), 34.4 (1C, C25, CH2), 29.8 (1C, C10, CH2), 29.0 (1C, C28, CH2), 28.4 (3C, tBu, CH3), 28.4 (1C, C9, CH2), 25.5 (1C, C11, CH2), 21.0 (1C, OAc, CH3). m/z (FAB, THIOG) 662 ([M + H]+, 3%), 562 (27) 91 (22). HRMS (FAB,

Biotinylated Anisomycin

THIOG) (found: [M + H]+, 662.3229. C32H48N5O8S requires m/z, 662.3224). C2-Classical O-Linked C(4)-H Anisomycin Biotin Molecular Probe 7b. To a solution of carbamate 7a (19.0 mg, 31.0 µmol) in CH2Cl2 (2 mL) was added trifluoroacetic acid (23.0 µL, 310 µmol), and the solution was stirred for 3 h at RT. The reaction was concentrated in Vacuo to give O-linked probe 7b (20.0 mg, 29.0 µmol, 94%) as a colorless foam. Rf [20% MeOH/CH2Cl2] ) 0.36. 1H NMR δ (360 MHz, DMSO) 8.16 (1H, t, J 5.3, N17H), 7.90 (1H, t, J 5.3, N14H), 7.19 (2H, d, J 8.6, ArH), 6.93 (2H, d, J 8.6, ArH), 6.45 (1H, br s, N6H), 6.38 (1H, br s, N4H), 5.15 (1H, t, J 3.7, C27H), 4.43 (2H, s, C19H2), 4.31-4.28 (1H, m, C3H), 4.14-4.10 (1H, m, C7H), 3.88-3.82 (1H, m, C26H), 3.33-3.23 (2H, m, C29H2), 3.20-3.10 (4H, m, C15H2, C16H2), 3.08 (1H, ddd, J 8.7, 6.2, 4.5, C8H), 2.98-2.86 (2H, m, C25H2), 2.80 (1H, dd, J 12.4, 5.0, C2HAHB), 2.57 (1H, d, J 12.4, C2HAHB), 2.29-2.18 (1H, m, C28HEHF), 2.12 (3H, s, OAc), 2.05 (2H, t, J 7.4, C12H2), 2.02-1.97 (1H, m, C28HEHF), 1.65-1.25 (6H, m, C9H2, C10H2, C11H2). 13C NMR δ (90.7 MHz, DMSO) 172.2 (1C, C13, Q), 169.4 (1C, OAc, Q), 167.7 (1C, C18, Q), 162.5 (1C, C5, Q), 156.4 (1C, Ar, Q), 129.7 (2C, ArH, CH), 128.9 (1C, Ar, Q), 114.8 (2C, ArH, CH), 72.6 (1C, C27, CH), 66.8 (1C, C19, CH2), 63.1 (1C, C26, CH), 60.8 (1C, C7, CH), 59.0 (1C, C3, CH), 55.2 (1C, C8, CH), 42.5 (1C, C29, CH2), 39.6 (1C, C2, CH2), 38.3 (1C, C16, CH2), 37.8 (1C, C15, CH2), 35.0 (1C, C12, CH2), 30.8 (1C, C25, CH2), 30.2 (1C, C28, CH2), 28.1 (1C, C10, CH2), 27.9 (1C, C9, CH2), 25.0 (1C, C11, CH2), 20.6 (1C, OAc, CH3). m/z (FAB, THIOG) 562 ([M + H]+, 80%), 91 (100). HRMS (FAB, NOBA) (found: [M + H]+, 562.2705. C27H40N5O6S requires m/z, 562.2699). C2-Classical N-Linked Anisomycin Biotin Molecular Probe 8. To biotinylated iodoacetamide 4 (25.0 mg, 55.0 µmol) in DMF (5 mL) was added anisomycin 1a (15.0 mg, 55.0 µmol), followed by K2CO3 (7.60 mg, 55.0 µmol). The solution was stirred for 48 h at 80 °C. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [5% to 15% MeOH/CH2Cl2] to give N-linked probe 8 as a colorless solid (11.0 mg, 18.7 µmol, 34%). Rf [14% MeOH/CH2Cl2] ) 0.33. 1H NMR δ (360 MHz, DMSO) 7.87 (2H, br s, N14H, N17H), 7.08 (2H, d, J 8.6, ArH), 6.81 (2H, d, J 8.6, ArH), 6.43 (1H, br s, N6H), 6.38 (1H, br s, N4H), 4.60 (1H, dd, J 4.2, 1.5, C23H), 4.31-4.28 (1H, m, C3H), 4.13-4.09 (1H, m, C7H), 3.95-3.90 (1H, m, C22H), 3.70 (3H, s, OMe), 3.49-3.35 (2H, m, C19HXHY, C21HMHN), 3.17-3.05 (5H, m, C8H, C15H2, C16H2,), 3.00-2.85 (3H, m, C19HXHY, C21HMHN), 2.80 (1H, dd, J 12.5, 5.0, C2HAHB), 2.77-2.71 (1H, m, C25HSHT), 2.66-2.60 (1H, m, C25HSHT), 2.57 (1H, d, J 12.5, C2HAHB), 2.08 (3H, s, OAc), 2.06 (2H, t, J 7.6, C12H2), 1.76-1.22 (6H, m, C9H2, C10H2, C11H2). 13C NMR δ (151.1 MHz, DMSO) 172.5 (1C, C13, Q), 171.1 (1C, C18, Q), 169.7 (1C, OAc, Q), 162.8 (1C, C5, Q), 157.7 (1C, Ar, Q), 130.2 (1C, Ar, Q), 129.7 (2C, ArH, CH), 113.5 (2C, ArH, CH), 78.5 (1C, C23, CH), 72.5 (1C, C22, CH), 69.6 (1C, C24, CH), 60.8 (1C, C7, CH), 60.8 (1C, C21, CH2), 59.0 (1C, C3, CH), 58.2 (1C, C19, CH2), 55.2 (1C, C8, CH), 54.7 (1C, OMe, CH3), 39.5 (1C, C2, CH2), 38.2 (1C, C16, CH2), 37.8 (1C, C15, CH2), 35.1 (1C, C12, CH2), 32.4 (1C, C25, CH2), 28.2 (1C, C10, CH2), 27.8 (1C, C9, CH2), 25.0 (1C, C11, CH2), 20.7 (1C, OAc, CH3). m/z (FAB, THIOG) 592 ([M + H]+, 52%), 91 (100). HRMS (FAB, THIOG) (found: [M + H]+, 592.2804. C28H42N5O7S requires m/z, 592.2805). Coupling to C6 Biotinylated Iodoacetamide 9. 6′-NBiotinylamino-hex-1-yl 4′-(2′′-acetoxyeth-1′′-yl)phenyloxyacetamide 10. To biotinylated iodoacetamide 9 (20.0 mg, 47.0 µmol) in DMF (5.0 mL) was added 3 (8.60 mg, 47.0 µmol), followed by K2CO3 (6.60 mg, 47.0 µmol). The solution was stirred for 48 h at 80 °C. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [5% to 15%

Bioconjugate Chem., Vol. 18, No. 5, 2007 1595

MeOH/CH2Cl2] to give phenol ether 10 as a colorless solid (20.0 mg, 33.8 µmol, 72%). Rf [10% MeOH/CH2Cl2] ) 0.16. 1H NMR δ (600 MHz, DMSO) 8.03 (1H, t, J 5.7, N21H), 7.72 (1H, t, J 5.7, N14H), 7.17 (2H, d, J 8.5, ArH), 6.88 (2H, d, J 8.5, ArH), 6.41 (1H, br s, N6H), 6.35 (1H, br s, N4H), 4.42 (2H, s, C23H2), 4.31-4.29 (1H, m, C3H), 4.15 (2H, t, J 6.9, C30H2), 4.13-4.11 (1H, m, C7H), 3.10 (2H, br q, J 6.8, C20H2), 3.093.07 (1H, m, C8H), 3.00 (2H, br q, J 6.7, C15H2), 2.60 (1H, dd, J 12.5, 5.0, C2HAHB), 2.81 (2H, t, J 6.7, C29H2), 2.57 (1H, d, J 12.5, C2HAHB), 2.04 (2H, t, J 7.4, C12H2), 1.97 (3H, s, OAc), 1.64-1.21 (14H, m, C9H2, C10H2, C11H2, C16H2, C17H2, C18H2, C19H2). 13C NMR δ (151.5 MHz, DMSO) 171.6 (1C, C13, Q), 170.1 (1C, OAc, Q), 167.3 (1C, C22, Q), 162.5 (1C, C5, Q), 156.2 (1C, Ar, Q), 130.4 (1C, Ar, Q), 129.7 (2C, ArH, CH), 114.5 (2C, ArH, CH), 66.9 (1C, C23, CH2), 64.4 (1C, C30, CH2), 60.9 (1C, C7, CH), 59.0 (1C, C3, CH), 55.2 (1C, C8, CH), 39.7 (1C, C2, CH2), 38.1 (1C, C15, CH2), 38.0 (1C, C20, CH2), 35.1 (1C, C12, CH2), 33.3 (1C, C29, CH2), 29.0, 28.9, 28.8, 28.1, 27.9, 25.9, 25.2 (7C, C9, C10, C11, C16, C17, C18, C19, CH2), 20.6 (1C, OAc, CH3). m/z (FAB, THIOG) 563 ([M + H]+, 49%), 91 (70). HRMS (FAB, NOBA) (found: [M + H]+, 563.2907. C28H43N4O6S requires m/z, 563.2903). C6-Classical N-Linked Anisomycin Biotin Molecular Probe 11. To biotinylated iodoacetamide 9 (20.0 mg, 47.0 µmol) in DMF (5.0 mL) was added anisomycin 1a (12.6 mg, 47.0 µmol), followed by K2CO3 (6.60 mg, 47.0 µmol). The solution was stirred for 48 h at 80 °C. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [5% to 15% MeOH/CH2Cl2] to give N-linked probe 11 as a colorless solid (11.0 mg, 16.5 µmol, 35%). Rf [10% MeOH/CH2Cl2] ) 0.19. 1H NMR δ (360 MHz, DMSO) 7.74 (1H, t, J 5.6, N14H), 7.68 (1H, t, J 5.6, N21H), 7.06 (2H, d, J 8.6, ArH), 6.80 (2H, d, J 8.6, ArH), 6.43 (1H, br s, N6H), 6.36 (1H, br s, N4H), 5.29 (1H, d, J 4.8, OH), 4.59 (1H, dd, J 4.9, 2.2, C27H), 4.32-4.28 (1H, m, C3H), 4.14-4.10 (1H, m, C7H), 3.89-3.85 (1H, m, C26H), 3.70 (3H, s, OMe), 3.42-3.38 (1H, m, C25HMHN), 3.26 (1H, d, J 15.9, C23HXHY), 3.19-3.13 (1H, m, C28H), 3.112.98 (5H, m, C8H, C15H2, C20H2), 2.97 (1H, d, J 15.9, C23HXHY), 2.81 (1H, dd, J 12.3, 5.1, C2HAHB), 2.75-2.72 (1H, m, C29HSHT), 2.59-2.57 (1H, m, C29HSHT), 2.57 (1H, d, J 12.3, C2HAHB), 2.31 (1H, dd, J 10.4, 3.5, C25HMHN), 2.06 (3H, s, OAc), 2.04 (2H, t, J 7.4, C12H2), 1.49-1.25 (14H, m, C9H2, C10H2, C11H2, C16H2, C17H2, C18H2, C19H2). 13C NMR δ (90.7 MHz, DMSO) 171.7 (1C, C13, Q), 169.9 (1C, C22, Q), 169.6 (1C, OAc, Q), 162.5 (1C, C5, Q), 157.4 (1C, Ar, Q), 130.7 (1C, Ar, Q), 129.7 (2C, ArH, CH), 113.6 (2C, ArH, CH), 78.9 (1C, C27, CH), 72.9 (1C, C26, CH), 65.6 (1C, C28, CH), 60.8 (1C, C7, CH), 60.1 (1C, C25, CH2), 59.0 (1C, C3, CH), 57.1 (1C, C23, CH2), 55.2 (1C, C8, CH), 54.7 (1C, OMe, CH3), 39.7 (1C, C2, CH2), 38.2 (1C, C20, CH2), 37.9 (1C, C15, CH2), 35.0 (1C, C12, CH2), 32.8 (1C, C29, CH2), 29.2, 28.9, 28.0, 27.8, 25.9, 25.5, 25.2 (7C, C9, C10, C11, C16, C17, C18, C19, CH2), 20.9 (1C, OAc, CH3). m/z (FAB, NOBA) 648 ([M + H]+, 5%), 307 (50), 77 (70). HRMS (FAB, THIOG) (found: [M + H]+, 648.3435. C32H50N5O7S requires m/z, 648.3431). Synthesis of Biotinylated Azides 15 and 16. Safety in the handling of sodium azide and other azides: (37) Sodium azide is toxic (LD50 oral (rats) ) 27 mg kg-1) and can be absorbed through the skin. It decomposes explosively upon heating to above 275 °C; hence, its use in airbags in the automotive industry. Sodium azide reacts vigorously with CS2, bromine, nitric acid, dimethyl sulfate, and a series of heavy metals, including copper and lead. In reaction with water or Brønsted acids, the highly toxic and explosive hydrogen azide is released. It has been reported that sodium azide and polymer-bound azide reagents form explosive di- and triazidomethane with CH2Cl2 and CHCl3, respectively.


Heavy-metal azides that are highly explosive under pressure or shock are formed when solutions of NaN3 or HN3 vapors come into contact with heavy metals or their salts. Heavy-metal azides can accumulate under certain circumstances, for example, in metal pipelines and on the metal components of diverse equipment (rotary evaporators, freeze drying equipment, cooling traps, water baths, waste pipes), and thus lead to violent explosions. Some organic and other covalent azides are classified as toxic and highly explosive, and appropriate safety measures must be taken at all times. 2-Azido-1-ethylamine 17. To a solution of 2-chloro-1-ethylamine (500 mg, 4.31 mmol) in water (5 mL) was added sodium azide (840 mg, 12.9 mmol), and the reaction mixture was heated at 80 °C for 15 h. The solution was basified with KOH (solid) and extracted with ether. The organics were dried and concentrated to give amino azide 17 as a volatile colorless oil (371 mg, 4.31 mmol, 100%). νmax(neat)/cm-1 3375, 2104. 1H NMR δ (360 MHz, CDCl3) 3.30 (2H, t, J 5.7, CH2), 2.79-2.74 (2H, m, CH2), 1.43 (2H, s, NH2). 13C NMR δ (90.7 MHz, CDCl3) 54.2 (1C, CH2), 40.9 (1C, CH2). m/z (ESI+) 194 ([2M + H]+). All spectroscopic data were in good agreement with that of the literature (38). 6-Azido-1-hexylamine 18. A solution of 6-amino-hexan-1-ol (1.17 g, 10.0 mmol) and SOCl2 (3.28 mL, 45.0 mmol) in toluene (10 mL) was heated at reflux for 1 h. The solvent was removed in Vacuo, and the intermediate 6-chloro-1-hexylamine was obtained as a hygroscopic solid. m/z (ESI+, MeOH) 136 ([M + H]+, 100), 138 ([M + H]+, 33). To a solution of sodium azide (1.95 g, 30.0 mmol) in water (10 mL) was added 6-chloro1-hexylamine and the reaction was stirred at 90 °C for 2 h. The solution was basified (pH 12-14) with KOH (solid), extracted with CH2Cl2, dried (MgSO4), and concentrated in Vacuo. The amino azide 18 was obtained as a colorless solid (1.28 g, 9.01 mmol, 90% over two steps). 1H NMR δ (360 MHz, DMSO) 3.40-3.20 (4H, m, CH2NH2, CH2N3), 1.75-1.45 (4H, m, 2CH2), 1.41-1.22 (4H, m, 2CH2). 13C NMR δ (90.6 MHz, DMSO) 51.0 (1C, CH2), 39.4 (1C, CH2), 29.6 (1C, CH2), 29.5 (1C, CH2), 27.2 (1C, CH2), 26.8 (1C, CH2). m/z (ESI+, MeOH) 143.0 ([M + H]+, 100%). HRMS (FAB, NOBA) (found: [M + H]+, 143.1297. C6H15N4 requires m/z, 143.1297). N-Biotinyl-2-azido-1-ethylamine 15. To a solution of biotin succinimidyl ester 19 (60.0 mg, 170 µmol) in methanol (2 mL) was added triethylamine (73.0 µL, 530 µmol) and 1-azidoethylamine 17 (90.0 mg, 1.06 mmol). The reaction mixture was stirred at RT for 6 h. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [8% to 12% MeOH/CH2Cl2] to give biotinylated azide 15 as a colorless solid (48.0 mg, 160 µmol, 94%). Rf [8% MeOH/CH2Cl2] ) 0.19. νmax(neat)/cm-1 3286, 2103, 1694, 1651. 1H NMR δ (360 MHz, DMSO) 8.06 (1H, t, J 5.8, N14H), 6.44 (1H, br s, N6H), 6.38 (1H, br s, N4H), 4.31-4.28 (1H, m, C3H), 4.16-4.12 (1H, m, C7H), 3.32 (2H, t, J 5.8, C16H2), 3.23 (2H, t, J 5.8, C15H2), 3.09-3.07 (1H, m, C8H), 2.87 (1H, dd, J 12.5, 5.1, C2HAHB), 2.56 (1H, d, J 12.5, C2HAHB), 2.07 (2H, t, J 7.4, C12H2), 1.521.28 (6H, m, C9H2, C10H2, C11H2). 13C NMR δ (90.7 MHz, DMSO) 172.7 (1C, C13, Q), 163.0 (1C, C5, Q), 61.2 (1C, C7, CH), 59.4 (1C, C3, CH), 55.7 (1C, C8, CH), 50.2 (1C, C16, CH2), 40.1 (1C, C2, CH2), 38.4 (1C, C15, CH2), 35.4 (1C, C12, CH2), 28.4 (1C, C10, CH2), 28.3 (1C, C9, CH2), 25.4 (1C, C11, CH2). m/z (FAB, THIOG) 313 ([M + H]+, 48%), 45 (100). HRMS (FAB, THIOG) (found: [M + H]+, 313.1445. C12H21N6O2S requires m/z, 313.1447). N-Biotinyl-6-azido-1-hexylamine 16. To a solution of 6-azido1-hexylamine 18 (316 mg, 1.46 mmol) and triethylamine (202 µL, 1.46 mmol) in methanol (20 mL) was slowly added a solution of biotin succinimidyl ester 19 (500 mg, 1.46 mmol) in methanol (10 mL). The reaction was stirred overnight at RT.

Inverarity et al.

The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [5% MeOH/CH2Cl2] to give biotinylated azide 16 as a pale yellow solid (479 mg, 1.30 mmol, 89%). Rf [10% MeOH/CH2Cl2] ) 0.11. νmax(neat)/cm-1 3301, 2099, 1704, 1638. 1H NMR δ (360 MHz, DMSO) 7.76 (1H, t, J 5.5, N14H), 6.44 (1H, br s, N6H), 6.38 (1H, br s, N4H), 4.324.29 (1H, m, C3H), 4.15-4.11 (1H, m, C7H), 3.31 (2H, t, J 6.9, C20H2), 3.12-3.06 (1H, m, C8H), 3.01 (2H, dt, J 12.5, 6.7, C15H2), 2.82 (1H, dd, J 12.5, 5.1, C2HAHB), 2.58 (1H, d, J 12.5, C2HAHB), 2.04 (2H, t, J 7.4, C12H2), 1.54-1.25 (14H, m, C9H2, C10H2, C11H2, C16H2, C17H2, C18H2, C19H2,). 13C NMR δ (90.7 MHz, DMSO) 172.1 (1C, C13, Q), 163.0 (1C, C5, Q), 61.3 (1C, C7, CH), 59.4 (1C, C3, CH), 55.7 (1C, C8, CH), 50.8 (1C, C20, CH2), 39.9 (1C, C2, CH2), 38.5 (1C, C15, CH2), 35.5 (1C, C12, CH2), 29.3, 28.5, 28.3, 26.2, 26.1, 25.6, 25.5 (7C, C9, C10, C11, C16, C17, C18, C19, CH2). m/z (FAB, THIOG) 369 ([M + H]+, 58%), 258 (46). HRMS (FAB, THIOG) (found: [M + H]+, 369.2074. C16H29N6O2S requires m/z, 369.2073). Coupling to C2 Biotinylated Azide 15. 1-(N-Biotinyl-2aminoeth-1-yl)-4-[4′-(2′′-acetoxyeth-1′′-yl)phenyloxymethyl]-1H[1,2,3]triazole 20. To biotinylated azide 15 (30.0 mg, 90.0 µmol) in H2O/tBuOH (2 mL, 1:1) was added propargyl ether 12 (24.0 mg, 100 µmol), followed by copper(II) sulfate (2.00 mg, 10 mol %) and sodium ascorbate solution (20.0 µL, 1 M solution, 20 mol %). The solution was stirred for 15 h at RT. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [8% to 15% MeOH/CH2Cl2] to give probe 20 as a colorless solid (41.0 mg, 77.4 µmol, 86%). Rf [20% MeOH/ CH2Cl2] ) 0.30. 1H NMR δ (600 MHz, DMSO) 8.18 (1H, s, C18H), 8.00 (1H, t, J 5.9, N14H), 7.17 (2H, d, J 8.6, ArH), 6.96 (2H, d, J 8.6, ArH), 6.44 (1H, br s, N6H), 6.38 (1H, br s, N4H), 5.09 (2H, s, C20H2), 4.42 (2H, t, J 5.9, C16H2), 4.28-4.27 (1H, m, C3H), 4.16 (2H, t, J 7.0, C27H2), 4.14-4.09 (1H, m, C7H), 3.48 (2H, br q, J 5.9, C15H2), 3.10-3.05 (1H, m, C8H), 2.81 (2H, t, J 7.0, C26H2), 2.79 (1H, dd, J 12.2, 5.2, C2HAHB), 2.54 (1H, d, J 12.2, C2HAHB), 2.03 (2H, t, J 7.4, C12H2), 1.98 (3H, s, OAc), 1.56-0.86 (6H, m, C9H2, C10H2, C11H2). 13C NMR δ (151.1 MHz, DMSO) 173.5 (1C, C13, Q), 171.2 (1C, C29, Q), 163.6 (1C, C5, Q), 157.6 (1C, Ar, Q), 143.4 (1C, C19, Q), 131.0 (1C, Ar, Q), 130.6 (2C, ArH, CH), 125.6 (1H, C18, CH), 115.5 (2C, ArH, CH), 65.6 (1C, C27, CH2), 62.1 (1C, C20, CH2), 61.9 (1C, C7, CH), 60.2 (1C, C3, CH), 56.3 (1C, C8, CH), 49.8 (1C, C16, CH2), 40.8 (1C, C2, CH2), 38.7 (1C, C15, CH2), 36.0 (1C, C12, CH2), 34.4 (1C, C26, CH2), 29.3 (1C, C10, CH2), 28.0 (1C, C9, CH2), 26.0 (1C, C11, CH2), 21.6 (1C, OAc, CH3). m/z (FAB, THIOG) 531 ([M + H]+, 27%), 270 (22) 227 (16), 45 (100). HRMS (FAB, THIOG) (found: [M + H]+, 531.2390. C25H35N6O5S requires m/z, 531.2387). C2-Click O-Linked Boc-Protected C(4)-H Anisomycin Biotin Molecular Probe 21a. To biotinylated azide 15 (50.0 mg, 160 µmol) in H2O/tBuOH (3 mL, 1:1) was added O-propargyl analogue 13 (60.0 mg, 170 µmol), followed by copper(II) sulfate (3.5 mg, 10 mol %) and sodium ascorbate solution (37.0 µL, 1 M solution, 20 mol %). The solution was stirred for 15 h at RT. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [5% to 15% MeOH/CH2Cl2] to give carbamate 21a as a colorless oil (76.0 mg, 111 µmol, 70%). Rf [10% MeOH/CH2Cl2] ) 0.19. 1H NMR δ (360 MHz, CDCl3, 323 K) 7.71 (1H, s, C18H), 7.11 (2H, d, J 8.4, ArH), 6.89 (2H, d, J 8.4, ArH), 5.14 (2H, s, C20H2), 5.10 (1H, br q, J 7.0, C28H), 4.54-4.57 (3H, m, C3H, C16H2), 4.36-4.31 (1H, m, C7H), 4.26-4.21 (1H, m, C27H), 3.70-3.76 (2H, q, J 5.8, C15H2), 3.48-3.41 (1H, m, C30HMHN), 3.35-3.28 (1H, m, C30HMHN), 3.16-3.11 (1H, m, C8H), 3.03-2.96 (1H, m, C26HSHT), 2.89 (1H, dd, J 13.0, 4.9, C2HAHB), 2.80 (1H, dd, J 13.8, 8.6, C26HSHT), 2.70 (1H, d, J 13.0, C2HAHB), 2.18 (2H, t, J 6.5, C12H2), 2.09-2.01 (1H, m, C29HEHF), 1.97 (3H, s, OAc),


1.85-1.76 (1H, m, C29HEHF), 1.72-1.38 (6H, m, C9H2, C10H2, C11H2), 1.45 (9H, s, tBuO). 13C NMR δ (90.7 MHz, CDCl3, 323 K) 174.3 (1C, COtBu, Q), 172.7 (1C, C13, Q), 170.3 (1C, OAc, Q), 156.9 (1C, C5, Q), 155.5 (1C, Ar, Q), 144.3 (1C, C19, Q), 131.7 (1C, Ar, Q), 130.7 (2C, ArH, CH), 124.1 (1C, C18, CH), 115.1 (2C, ArH, CH), 80.0 (1C, tBu, Q), 73.5 (1C, C28, CH), 62.3 (1C, C7, CH), 62.2 (1C, C20, CH2), 60.7 (1C, C3, CH), 59.6 (1C, C27, CH), 55.7 (1C, C8, CH), 49.8 (1C, C16, CH2), 43.7 (1C, C30, CH2), 40.6 (1C, C2, CH2), 39.5 (1C, C15, CH2), 35.8 (1C, C12, CH2), 34.3 (1C, C26, CH2), 29.8 (1C, C29, CH2), 28.7 (3C, tBu, CH3), 27.9 (1C, C10, CH2), 27.8 (1C, C9, CH2), 25.1 (1C, C11, CH2), 20.0 (1C, OAc, CH3). m/z (FAB, THIOG) 686 ([M + H]+, 16%), 586 (60). HRMS (FAB, THIOG) (found: [M + H]+, 686.3336. C33H48N7O7S requires m/z, 686.3336). C2-Click O-Linked C(4)-H Anisomycin Biotin Molecular Probe 21b. To a solution of carbamate 21a (25.0 mg, 36.0 µmol) in CH2Cl2 (2 mL) was added trifluoroacetic acid (27.0 µL, 360 µmol), the solution was stirred for 4 h at RT. The reaction was concentrated in Vacuo to give O-linked probe 21b (23.0 mg, 32.9 µmol, 91%) as a colorless foam. Rf [20% MeOH/CH2Cl2] ) 0.16. 1H NMR δ (360 MHz, DMSO) 8.19 (1H, s, C18H), 8.01 (1H, t, J 5.6, N14H), 7.20 (2H, d, J 8.6, ArH), 7.02 (2H, d, J 8.6, ArH), 6.76 (1H, br s, N6H), 6.44 (1H, br s, N4H), 5.16 (1H, t, J 3.7, C28H), 5.10 (2H, s, C20H2), 4.42 (2H, t, J 6.0, C16H2), 4.31-4.28 (1H, m, C3H), 4.13-4.10 (1H, m, C7H), 3.87-3.84 (1H, m, C27H), 3.49 (2H, q, J 5.8, C15H2), 3.333.23 (2H, m, C30H2), 3.11-3.06 (1H, m, C8H), 2.98-2.85 (2H, m, C26H2), 2.80 (1H, dd, J 12.4, 5.0, C2HAHB), 2.57 (1H, d, J 12.4, C2HAHB), 2.30-2.20 (1H, m, C29HEHF), 2.12 (3H, s, OAc), 2.03 (2H, t, J 7.4, C12H2), 2.01-1.97 (1H, m, C29HEHF), 1.64-1.21 (6H, m, C9H2, C10H2, C11H2). 13C NMR δ (90.7 MHz, DMSO) 172.1 (1C, C13, Q), 169.3 (1C, OAc, Q), 162.1 (1C, C5, Q), 157.1 (1C, Ar, Q), 142.1 (1C, C19, Q), 129.7 (2C, ArH, CH), 128.3 (1C, Ar, Q), 124.5 (1C, C18, CH), 114.5 (2C, ArH, CH), 72.5 (1C, C28, CH), 63.0 (1C, C27, CH), 60.7 (1C, C20, CH2), 60.7 (1C, C7, CH), 58.9 (1C, C3, CH), 55.1 (1C, C8, CH), 48.7 (1C, C16, CH2), 42.5 (1C, C30, CH2), 39.6 (1C, C2, CH2), 38.5 (1C, C15, CH2), 34.9 (1C, C12, CH2), 30.7 (1C, C26, CH2), 30.1 (1C, C29, CH2), 27.9 (1C, C10, CH2), 27.7 (1C, C9, CH2), 24.8 (1C, C11, CH2), 20.5 (1C, OAc, CH3). m/z (FAB, THIOG) 586 ([M + H]+, 29%). HRMS (FAB, THIOG) (found: [M + H]+, 586.2812. C28H40N7O5S requires m/z, 586.2812). C2-Click N-Linked Anisomycin Biotin Molecular Probe 22. To biotinylated azide 15 (27.0 mg, 90.0 µmol) in H2O/tBuOH (2 mL, 1:1) was added N-propargyl anisomycin 14 (17.0 mg, 50 µmol), followed by copper(II) sulfate (2.0 mg, 10 mol %) and sodium ascorbate solution (20.0 µL, 1 M solution, 20 mol %). The solution was stirred for 15 h at RT. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [8% to 15% MeOH/CH2Cl2] to give N-linked probe 22 as a colorless solid (22.0 mg, 35.5 µmol, 71%). Rf [20% MeOH/CH2Cl2] ) 0.35. 1H NMR δ (360 MHz, DMSO) 7.98 (1H, t, J 5.4, N14H), 7.96 (1H, s, C18H), 7.11 (2H, d, J 8.6, ArH), 6.82 (2H, d, J 8.6, ArH), 6.43 (1H, br s, N6H), 6.38 (1H, br s, N4H), 5.10 (1H, d, J 3.9, OH), 4.48 (1H, dd, J 4.5, 1.8, C24H), 4.39 (2H, t, J 6.0, C16H2), 4.32-4.28 (1H, m, C3H), 4.15-4.10 (1H, m, C7H), 3.92 (1H, d, J 14.1, C20HXHY), 3.813.78 (1H, m, C23H), 3.71 (3H, s, OMe), 3.63 (1H, d, J 14.1, C20HXHY), 3.48 (2H, br q, J 5.8, C15H2), 3.18 (1H, dd, J 10.3, 6.2, C22HMHN), 3.09 (1H, ddd, J 8.2, 6.1, 4.5, C8H), 3.012.93 (2H, m, C25H, C26HSHT), 2.81 (1H, dd, J 12.4, 5.0, C2HAHB), 2.60-2.53 (2H, m, C2HAHB, C26HSHT), 2.24 (1H, dd, J 10.3, 4.5, C22HMHN), 2.04 (3H, s, OAc), 2.02 (2H, t, J 7.4, C12H2), 1.48-0.80 (6H, m, C9H2, C10H2, C11H2). 13C NMR δ (90.7 MHz, DMSO) 173.5 (1C, C13, Q), 169.7 (1C, OAc, Q),


162.7 (1C, C5, Q), 157.5 (1C, Ar, Q), 143.0 (1C, C19, Q), 131.0 (1C, Ar, Q), 129.8 (2C, ArH, CH), 123.8 (1C, C18, CH), 113.7 (2C, ArH, CH), 79.5 (1C, C24, CH), 72.9 (1C, C23, CH), 64.8 (1C, C25, CH), 61.0 (1C, C7, CH), 59.2 (1C, C3, CH), 55.4 (1C, C22, CH2), 49.9 (2C, CH, CH3, C8, C32), 48.7 (1C, C16, CH2), 47.0 (1C, C20, CH2), 39.8 (1C, C2, CH2), 38.7 (1C, C15, CH2), 35.0 (1C, C12, CH2), 32.2 (1C, C26, CH2), 28.1 (1C, C10, CH2), 28.0 (1C, C9, CH2), 25.1 (1C, C11, CH2), 20.8 (1C, OAc, CH3). m/z (FAB, THIOG) 615 ([M]+, 19%). HRMS (FAB, THIOG) (found: [M + H]+, 616.2916. C29H42N7O6S requires m/z, 616.2917). Coupling to C6 Biotinylated Azide 16. 1-(N-Biotinyl-6aminohex-1-yl)-4-[4′-(2′′-acetoxyeth-1′′-yl)phenyloxymethyl]1H- [1,2,3] triazole 23. To biotinylated azide 16 (50.0 mg, 140 µmol) in H2O/tBuOH (5 mL, 1:1) was added O-propargyl analogue 12 (30.0 mg, 140 µmol), followed by copper(II) sulfate (4.0 mg, 15 mol %) and sodium ascorbate solution (28.0 µL, 1 M solution, 20 mol %). The solution was stirred for 24 h at RT. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [2% to 5% MeOH/CH2Cl2] to give probe 23 as a colorless solid (62.0 mg, 105 µmol, 75%). Rf [10% MeOH/CH2Cl2] ) 0.08. 1H NMR δ (360 MHz, DMSO) 8.30 (1H, s, C22H), 7.72 (1H, br s, N14H), 7.16 (2H, d, J 8.5, ArH), 6.95 (2H, d, J 8.5, ArH), 6.40 (1H, br s, N6H), 6.35 (1H, br s, N4H), 5.09 (2H, s, C24H2), 4.34 (2H, t, J 7.1, C20H2), 4.32-4.30 (1H, m, C3H), 4.15 (2H, t, J 6.9, C31H2), 4.14-4.10 (1H, m, C7H), 3.12-3.06 (1H, m, C8H), 3.01 (2H, dt, J 12.5, 6.7, C15H2), 2.81 (2H, t, J 6.9, C30H2), 2.80 (1H, dd, J 12.2, 5.1, C2HAHB), 2.58 (1H, d, J 12.2, C2HAHB), 2.04 (2H, t, J 7.3, C12H2), 1.97 (3H, s, OAc), 1.80 (2H, qn, J 6.9, C19H2), 1.66-1.16 (12H, m, C9H2, C10H2, C11H2, C16H2, C17H2, C18H2). 13C NMR δ (90.7 MHz, DMSO) 171.7 (1C, C , Q), 170.1 (1C, 13 OAc, Q), 162.6 (1C, C5, Q), 156.6 (1C, Ar, Q), 142.5 (1C, C23, Q), 130.2 (1C, Ar, Q), 129.6 (2C, ArH, CH), 124.2 (1C, C22, CH), 114.5 (2C, ArH, CH), 64.4 (1C, C31, CH2), 61.0 (1C, C24, CH2), 60.8 (1C, C7, CH), 59.0 (1C, C3, CH), 55.3 (1C, C8, CH), 49.0 (1C, C20, CH2), 39.6 (1C, C2, CH2), 38.1 (1C, C15, CH2), 35.0 (1C, C12, CH2), 33.2 (1C, C30, CH2), 29.4 (1C, C19, CH2), 28.8, 28.0, 25.8, 25.7, 25.2, 25.1 (6C, C9, C10, C11, C16, C17, C18, CH2), 20.5 (1C, OAc, CH3). m/z (FAB, THIOG) 587 ([M + H]+, 18%), 91 (78). HRMS (FAB, THIOG) (found: [M + H]+, 587.3020. C29H43N6O5S requires m/z, 587.3016). C6-Click O-Linked Boc-Protected C(4)-H Anisomycin Biotin Molecular Probe 24a. To biotinylated azide 16 (41.0 mg, 111 µmol) in H2O/tBuOH (2 mL, 1:1) was added O-propargyl analogue 13 (45.0 mg, 120 µmol), followed by copper(II) sulfate (3.0 mg, 10 mol %) and sodium ascorbate solution (25.0 µL, 1 M solution, 20 mol %). The solution was stirred for 18 h at RT. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [5% to 15% MeOH/CH2Cl2] to give carbamate 24a as a colorless oil (62.0 mg, 84.0 µmol, 76%). Rf [10% MeOH/CH2Cl2] ) 0.24. 1H NMR δ (360 MHz, CDCl3, 323 K), 7.60 (1H, s, C22H), 7.10 (2H, d, J 8.5, ArH), 6.88 (2H, d, J 8.5, ArH), 5.16 (2H, s, C24H2), 5.11 (1H, dt, J 6.5, 14.1, C32H), 4.52-4.46 (1H, m, C3H), 4.34 (2H, t, J 7.0, C20H2), 4.33-4.29 (1H, m, C7H), 4.29-4.21 (1H, m, C31H), 3.47-3.27 (2H, m, C34H2), 3.25-3.14 (3H, m, C8H, C15H2), 3.01-3.92 (1H, m, C30HSHT), 2.89 (1H, dd, J 12.6, 4.3, C2HAHB), 2.81 (1H, dd, J 13.8, 8.4 C30HSHT), 2.72 (1H, d, J 12.6, C2HAHB), 2.19 (2H, t, J 6.9, C12H2), 2.09-2.00 (1H, m, C33HEHF), 1.97 (3H, s, OAc), 1.91 (2H, br t, J 6.6, C19H), 1.841.74 (1H, m, C33HEHF), 1.71-1.56 (4H, m, C9H2, C11H2), 1.44 (9H, s, tBuO), 1.49-1.27 (8H, m, C10H2, C16H2, C17H2, C18H2). 13C NMR δ (90.7 MHz, CDCl , 323 K) 173.1 (1C, C , Q), 3 13 170.7 (1C, COtBu, Q), 169.7 (1C, OAc, Q), 162.1 (1C, C5, Q), 156.8 (1C, Ar, Q), 144.0 (1C, C23, Q), 131.4 (1C, Ar, Q), 130.3 (2C, ArH, CH), 122.5 (1C, C22, CH), 114.7 (2C, ArH, CH),


79.7 (1C, tBu, Q), 73.0 (1C, C32, CH), 62.1 (1C, C24, CH2), 61.8 (1C, C7, CH), 60.2 (1C, C3, CH), 59.1 (1C, C31, CH), 55.4 (1C, C8, CH), 50.1 (1C, C20, CH2), 43.0 (1C, C34, CH2), 40.3 (1C, C2, CH2), 39.1 (1C, C15, CH2), 35.8 (1C, C12, CH2), 33.9 (1C, C30, CH2), 29.8 (1C, C19, CH2), 29.4, 29.2, 28.5, 28.3, 28.3, 28.1, 25.8, 25.4 (10C, C9, C10, C11, C16, C17, C18, C33 all CH2’s and tBu CH3’s), 20.7 (1C, OAc, CH3). m/z (FAB, THIOG) 742 ([M + H]+, 20%), 642 (57), 91 (61). HRMS (FAB, THIOG) (found: [M + H]+, 742.3960. C37H56N7O7S requires m/z, 742.3962). C6-Click O-LinkedC(4)-H Anisomycin Biotin Molecular Probe 24b. To a solution of carbamate 24a (41.0 mg, 55.0 µmol) in CH2Cl2 (2 mL) was added trifluoroacetic acid (41.0 µL, 550 µmol), the solution was stirred for 4 h at RT. The reaction was concentrated in Vacuo to give O-linked probe 24b (40.0 mg, 52.9 µmol, 96%) as a colorless foam. Rf [20% MeOH/CH2Cl2] ) 0.38. 1H NMR δ (360 MHz, DMSO) 8.23 (1H, s, C22H), 7.76 (1H, t, J 5.6, N14H), 7.19 (2H, d, J 8.6, ArH), 7.00 (2H, d, J 8.6, ArH), 6.44 (1H, br s, N6H), 6.38 (1H, br s, N4H), 5.16 (1H, t, J 3.8, C32H), 5.10 (2H, s, C24H2), 4.42 (2H, t, J 7.0, C20H2), 4.31-4.28 (1H, m, C3H), 4.13-4.10 (1H, m, C7H), 3.87-3.81 (1H, m, C31H), 3.34-3.22 (2H, m, C34H2), 3.08 (1H, ddd, 8.5, 6.1, 4.8, C8H), 2.99 (2H, br q, J 6.0, C15H2), 3.333.23 (2H, m, C30H2), 2.80 (1H, dd, J 12.5, 5.1, C2HAHB), 2.57 (1H, d, J 12.5, C2HAHB), 2.30-2.18 (1H, m, C33HEHF), 2.12 (3H, s, OAc), 2.03 (2H, t, J 7.3, C12H2), 2.01-1.97 (1H, m, C33HEHF), 1.80 (2H, qn, J 7.1, C19H), 1.65-1.19 (12H, m, C9H2, C10H2, C11H2, C16H2, C17H2, C18H2). 13C NMR δ (90.7 MHz, DMSO) 172.0 (1C, C13, Q), 169.8 (1C, OAc, Q), 162.9 (1C, C5, Q), 157.3 (1C, Ar, Q), 142.9 (1C, C23, Q), 130.3 (2C, ArH, CH), 128.8 (1C, Ar, Q), 124.8 (1C, C22, CH), 115.1 (2C, ArH, CH), 73.0 (1C, C32, CH), 63.5 (1C, C31, CH), 61.2 (1C, C7, CH), 61.2 (1C, C24, CH2), 59.4 (1C, C3, CH), 55.7 (1C, C8, CH), 49.5 (1C, C20, CH2), 42.8 (1C, C34, CH2), 40.0 (1C, C2, CH2), 38.4 (1C, C15, CH2), 35.5 (1C, C12, CH2), 31.1 (1C, C30, CH2), 30.6 (1C, C33, CH2), 29.9 (1C, C19, CH2), 29.1, 28.5, 28.3, 26.0, 25.7, 25.6 (6H, C9, C10, C11, C16, C17, C18, CH2), 21.0 (1C, OAc, CH3). m/z (FAB, THIOG) 642 ([M + H]+, 30%), 91 (69). HRMS (FAB, THIOG) (found: [M + H]+, 642.3437. C32H48N7O5S requires m/z, 642.3438). C6-Click N-Linked Anisomycin Biotin Molecular Probe 25. To biotinylated azide 16 (47.0 mg, 130 µmol) in H2O/tBuOH (3 mL, 1:1) was added N-propargyl anisomycin 14 (42.0 mg, 140 µmol), followed by copper(II) sulfate (3.0 mg, 10 mol %) and sodium ascorbate solution (28.0 µL, 1 M solution, 20 mol %). The solution was stirred for 15 h at RT. The solution was concentrated in Vacuo, and the residue was purified by flash chromatography [8% to 15% MeOH/CH2Cl2] to give N-linked probe 25 as a colorless solid (64.0 mg, 95.0 µmol, 73%). Rf [20% MeOH/CH2Cl2] ) 0.35. 1H NMR δ (360 MHz, 323 K, DMSO) 8.13 (1H, br s, C22H), 7.58 (1H, t, J 5.6, N14H), 7.09 (2H, d, J 8.6, ArH), 6.83 (2H, d, J 8.6, ArH), 6.26 (1H, br s, N6H), 6.23 (1H, br s, N4H), 5.06 (1H, br s, OH), 4.58 (1H, dd, J 4.9, 2.1, C28H), 4.33 (2H, t, J 7.0, C20H2), 4.32-4.28 (1H, m, C3H), 4.15-4.12 (1H, m, C7H), 3.98 (1H, br d, J 13.9, C24HXHY), 3.90-3.86 (1H, m, C27H), 3.75 (1H, br d, J 13.9, C24HXHY), 3.72 (3H, s, OMe), 3.25-3.10 (2H, m, C26HMHN, C29H), 3.08 (1H, ddd, J 8.1, 6.6, 4.8, C8H), 2.98 (2H, br q, J 6.6, C15H), 2.91 (1H, dd, J 13.3, 4.6, C30HSHT), 2.83 (1H, dd, J 12.4, 5.1, C2HAHB), 2.60 (1H, d, J 13.3, C30HSHT), 2.81 (1H, d, J 12.4, C2HAHB), 2.41-2.32 (1H, m, C26HMHN), 2.05 (2H, t, J 7.3, C12H2), 2.04 (3H, s, OAc), 1.82 (2H, qn, J 7.0, C19H2), 1.70-1.20 (12H, m, C9H2, C10H2, C11H2, C16H2, C17H2, C18H2,). 13C NMR δ (90.7 MHz, DMSO) 171.8 (1C, C , Q), 169.5 (1C, 13 OAc, Q), 162.6 (1C, C5, Q), 157.6 (1C, Ar, Q), 142.3 (1C, C23, Q), 130.4 (1C, Ar, Q), 129.6 (2C, ArH, CH), 123.4 (1C, C22, CH), 113.5 (2C, ArH, CH), 78.9 (1C, C28, CH), 72.3 (1C, C27,

Inverarity et al.

CH), 64.7 (1C, C29, CH), 60.9 (1C, C7, CH), 59.0 (1C, C3, CH), 58.8 (1C, C26, CH2), 55.0 (1C, C8, CH), 54.7 (1C, OMe, CH3), 49.0 (1C, C20, CH2), 47.3 (1C, C24, CH2), 39.5 (1C, C2, CH2), 38.0 (1C, C15, CH2), 34.9 (1C, C12, CH2), 31.9 (1C, C30, CH2), 29.4 (1C, C19, CH2), 28.6, 27.9, 27.8, 25.5, 25.2, 25.0 (6H, C9, C10, C11, C16, C17, C18, CH2), 20.4 (1C, OAc, CH3). m/z (FAB, THIOG) 672 ([M + H]+, 38%), 91 (88). HRMS (FAB, THIOG) (found: [M + H]+, 672.3540. C33H50N7O6S requires m/z, 672.3543). Immunoblot Assays. (34, 35, 39) Cell Culture and Stimulation. Human embryonic kidney (HEK) 293 cells were cultured at 37 °C, 95% air/5% CO2, in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. Anisomycin 1a was dissolved at 10 mg/mL (38 mmol L-1) in DMSO, while the other compounds (5, 7b, 8, 10, 11, 20, 21b, 22, 23, 24b, 25) were dissolved in DMSO at 38 mmol L-1. Cells (9 mL of cell culture) were incubated with the library members by addition of 9 µL of anisomycin solution, anisomycin analogue in DMSO, or DMSO as a control. Cell Lysis. After stimulation for 30 min, the media was aspirated and the cells lysed in 50 mM Tris/HCl, pH 7.5, containing 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 0.27 M sucrose, 1% (v/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol, and Complete Protease Inhibitor cocktail (one tablet per 50 mL). Lysates were centrifuged at 13 000 g for 10 min at 4 °C and the supernatants (termed “cell extract”) were removed. Protein concentrations were determined according to the method of Bradford (40). Immunoblotting. Samples were denatured in SDS, run on polyacylamide gels, and transferred to nitrocellulose membranes. The membranes were incubated for 1 h at room temperature in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2% (v/v) Tween, and 5% (w/v) skimmed milk powder. Primary antibodies were added to 10 mL of the previous buffer and incubated at 4 °C overnight. The membranes were then washed four times with buffer (5 min per wash) to remove the excess primary antibody. The membranes were then incubated with the secondary antibody at room temperature for 1 h. After washing six times with buffer to remove the excess secondary antibody (5 min per wash), immunoreactive proteins were visualized Via enhanced chemiluminescence reagent according to the manufacturer’s instructions.

RESULTS AND DISCUSSION Traditional approaches to the design of functional biotin molecular probes are often based on the formation of peptide and ester bonds (8). However, for biotinylation of anisomycin at either the pyrrolidine nitrogen (N-linked) or phenolic oxygen (O-linked), this approach was ruled out first due to the SAR data, which suggests that only amine derivatives are able to activate the SAPK pathways, and second due to the known poor stability of phenolic esters. We therefore turned our attention to the classical nucleophilic displacement reaction of R-halo carbonyl derivatives of biotin by each of these functionalities. In designing appropriate biotin derivatives for coupling, two linker lengths were identified for the carbon chain between the active small molecule (anisomycin) and the functional tag (biotin): a short commercially available C2 linker, and a longer C6 linker. To allow us to establish whether any false positives were caused in the immunoblot assays from activation by these linkers, the synthesis of a series of structurally related, but SAPK-inactive, control probes was also envisaged. The monoacetate of 2-(4-hydroxyphenyl)ethanol 3 was chosen as a simple model of the anisomycin core (41), allowing initial



Table 1. Classical Nucleophilic Displacement Reactions of Anisomycin and Analogues to Give O-Linked and N-Linked Biotinylated Molecular Probes

investigations into the conditions required for the nucleophilic displacement coupling to give O-linked biotinylated probes. Promising initial results were obtained in the reaction of 3 with commercially available iodoacetyl biotin 4, which occurred readily upon heating at 80 °C in DMF in the presence of potassium carbonate, to give the desired product 5 in 86% yield (Table 1). However, rather disappointingly, heating the analogous Boc-protected C(4)-H analogue of anisomycin 6 (35) gave only a low yield of coupled material 7a (31%), while Nalkylation of anisomycin 1a under the same conditions was found to be only marginally more successful, giving 34% of the C2-biotinylated probe 8 (Table 1). The longer C6-linked iodoacetyl biotin derivative 9 was shown to exhibit a similar pattern of reactivity when coupled to the simple model 3 (72%) and anisomycin 1a (35%) under identical conditions (Table 1) to give biotin-conjugated analogues 10 and 11, respectively. With these more-complex substrates, the highly polar nature of the starting materials and resultant biotinylated products, combined with low conversions, gave rise to difficult separation procedures. Thus, while the coupling of simple substrates can be achieved in good yield using this classical nucleophilic displacement methodology, on more complex substrates it was found to be highly unsatisfactory. In a contrasting approach, the synthesis of a number of biotinylated probes was envisaged using an extension of our recent marked library strategy (35). In this strategy, a biocompatible marker is attached onto the small molecule’s scaffold; this marker plays no role in the screening process itself, but facilitates the formation of a range of molecular probes from active marked library members. Having previously utilized the highly popular copper(I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction as a means of formation of fluorescent molecular probes from marked library members (35), we sought to extend

Scheme 1. Preparation of C2 and C6 Biotinylated Azidesa

a Reagents and Conditions: (a) NaN , H O, 80 °C, 15 h (n ) 1, X 3 2 ) Cl; 100%); (b) (i) SOCl2, PhCH3, 110 °C, 1 h; (ii) NaN3, H2O, 90 °C, 2 h (n ) 5, X ) OH; 90% over two steps); (c) biotin NHS (19), MeOH, Et3N, rt, 6-15 h (n ) 1, 94%; n ) 5, 89%).

this strategy to the synthesis of biotinylated probes (42). To this end, propargylation of the monoacetate of 2-(4-hydroxyphenyl)ethanol 3, the Boc-protected C(4)-H analogue of anisomycin 6, and anisomycin 1a was carried out in DMF to give the O-linked (12, 13) and N-linked (14) “click” precursors in excellent yield (77%, 99% (35), and 99% (35), respectively). The C2 15 and C6 16 biotinylated azides required by this approach are readily prepared by coupling alkylamino azides 17 or 18 to biotin N-hydroxy succinimidyl ester 19 in high yield (94% and 89%, respectively; Scheme 1). Using standard coupling conditions [10 mol % CuSO4, 20 mol % NaAsc, tBuOH/H2O (1:1)] for the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction (43-44), the propargyl derivative of the model of the anisomycin core 12 was coupled to the C2 biotinylated azide 15 to give 20 in high yield (Table 2). Applying the same reaction conditions to the O-linked (13) and N-linked (14) precursors resulted in gratifyingly high yields of products (21a and 22, Table 2). When the linker chain length was extended through coupling to the related C6 azide


Inverarity et al.

Table 2. Copper(I) Catalyzed Huisgen 1,3-Dipolar Cycloaddition Coupling Reactions of O-Linked and N-Linked Propargyl Anisomycin Derivatives to Give Biotinylated Molecular Probes

16, comparable reactivity was observed, and the control 23, O-linked 24a, and N-linked 25 probes were also isolated in excellent yields (Table 2). In all cases, the products were isolated as discreet chemical entities which could be fully characterized using a range of spectroscopic techniques (1D and 2D NMR experiments, including COSY, HSQC, and HMBC), and which were shown to be single regioisomers around the triazole core. The high purity and high yields of conjugation that are possible using this “click” methodology, combined with the relative ease of production of a range of linker lengths and functionalities for the biotinylated azide, make this an extremely attractive synthetic approach to biotinylation. The relative levels of activation of the SAPK1 pathway by the classical and “click” biotinylated probes generated in this study were screened using an immunoblot assay for phosphorylation of JNK1/2 in HEK-293 cells (Figure 3). For this study, Boc-protected pyrrolidines 7a, 21a, and 24a were deprotected (TFA, CH2Cl2) in high yield (91-96%) to give the corresponding pyrrolidines 7b, 21b, and 24b (Tables 1 and 2) which were used in the SAPK pathway activation assays. The observed activation levels were scaled against anisomycin and DMSO (strong activator and control respectively). These assays showed that, independent of the coupling strategy used, the O-linked biotinylated probes (C2: 5, 7b, 20, 21b; C6: 10, 23, 24b) were inactive, whereas their N-linked counterparts (C2: 8, 22; C6: 11, 25) were found to be comparable in the levels of activation they induced to anisomycin 1a. Levels of SAPK1 activation also appear to be relatively unaffected by the length of linker (C2 or C6) in both classical and “click” series; although we anticipate that the nature (all-carbon, PEG, etc.) and length (C2, C6, etc.) of linker is likely to be of far greater significance in

future affinity chromatography studies using these biotinylated molecular probes. Overall, these results were in good accord with our previous studies into the synthesis of fluorescent molecular probes (35). A survey of the methods available for the selective derivatization of a range of chemical functionalities (Table 3), suggests that this “click”-based approach to biotinylation might be applicable to a wide range of substrates. Chemical coupling of a propargyl marker to small molecules may be readily achieved using commercially available (or easily prepared) reagents, and can be tailored to fit diverse synthetic strategies. By matching the chemical reactivity of the propargyl reagent to the known SAR profile of a small molecule, both in terms of site of attachment (e.g., O-linked or N-linked probes in this study) and resultant functionality (e.g., amine, amide, or carbamate formation; see Table 3), a series of propargyl derivatives may be readily synthesized. Subsequent coupling of these propargyl derivatives with a biotinylated azide under standard “click” coupling conditions allows the rapid and high-yielding synthesis of biotinylated probes.

CONCLUSIONS The attachment of a propargyl group (or “marker”) to a small molecule core may be achieved using a wide range of reagents to common chemical functionalities. Through examining the known SAR of the SAPK pathway activator anisomycin, two potential sites for the rapid development of molecular probes were identified. In these, phenolic methyl and pyrrolidine N-benzyl groups were replaced by the propargyl marker to give small molecules which retain comparable activity to their parent


Bioconjugate Chem., Vol. 18, No. 5, 2007 1601 Table 3. Survey of Propargylation Methodologies for the Selective Functionalization of Alcohol, Amine, Thiol, Ketone/Aldehyde, and Acid Functionalities

Figure 3. Effect of anisomycin and biotinylated conjugates on the phosphorylation of JNK1/2 isoforms in HEK-293 cells. The cells were exposed to DMSO (lanes 1 and 2), anisomycin 1a (lanes 3 and 4), and the analogues indicated (lanes 5-10), each dissolved in DMSO: (a) classical C2 probes; (b) classical C6 probes; (c) click C2 probes; (d) click C6 probes. The cells were lysed and an aliquot (20 µg of lysate protein) was denatured in SDS, subjected to electrophoresis on a 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with an antibody that recognized JNK1/2 phosphorylated at Thr183 or with an antibody that recognized phosphorylated and unphosphorylated JNK1/2 equally well.

structures. Of these two lead compounds, the N-linked precursor was found to allow the development of active biotinylated probes, while derivatization of the O-linked precursor led to a loss of phenotypic response. Although these assay results were mirrored by molecular probes formed using classical nucleophilic displacement coupling strategies, low reaction yields were shown to be inherent to this alternate methodology. Thus, the versatility of precursor synthesis, mild coupling conditions, high yields, and excellent regioselectivity found using the “click”based strategy renders it the method of choice in conjugative routes to biotinylated molecular probes.

ACKNOWLEDGMENT We thank the BBSRC (Studentship to IAI), and MRC for financial support of this work; and the Scottish Executive/Royal Society of Edinburgh (Research Fellowship to ANH). We thank

Dr. Ian Sadler and Juraj Bella for assistance with NMR studies and Dr. Simon Morton for technical assistance with the immunoassays. Supporting Information Available: Experimental for the preparation of C6 biotinylated iodoacetamide 9; synthesis of compound 12; NMR spectra for N-linked molecular probes 8, 11, 22, and 25; NMR spectra for biotinylated azides 15 and 16. This information is available free of charge via the Internet at http://pubs.acs.org.

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