A R T I C L E S
Peddibhotla et al.
proliferation assay using Jurkat T cells.34 These derivatives were
also found to lead to disruption of the Golgi complex using
confocal fluorescence microscopy indicating a similar mecha-
nism of action as BFA.34
(150 Å, 20 × 20, 1000 µm) and developed with appropriate solvent.
Bands were identified by UV, and silica was scraped off the plate and
placed into 20-mL scintillation vials. Organic residues were extracted
by suspending the silica gel in 5:95 methanol/ethyl acetate. This was
followed by two more elutions with 5:95 methanol/ethyl acetate. The
combined washings were filtered through Gelman Nylon Acrodisc (0.45
µm) to remove fine particles of silica gel. Unless indicated otherwise,
deuterochloroform (CDCl3) served as an internal standard (7.26 ppm)
for all 1H spectra and (77.0 ppm) for all 13C spectra. Mass spectra were
obtained at the Center for Chemical Characterization and Analysis
(Texas A&M University).
Conclusion
A mild, chemosite-selective and site-nonselective O-H
insertion process using alkynyl diazo esters and Rh2(OAc)4 was
developed that enables simple, two-step, simultaneous arming
and SAR studies of bioactive natural products. A subsequent
conjugation step involving the Sharpless-Hu¨isgen [3 + 2]
cycloaddition allows attachment of various probes including
biotin. Importantly, variation in chemosite selectivity was
possible even with achiral catalysts between a secondary and
tertiary alcohol in gibberellic acid methyl ester and two
secondary alcohols in BFA. This method was applied to the
synthesis of HPBA ethers of a structurally diverse set of natural
products and derivatives bearing alcohols with varied steric
environment and electronics, enabling initial guidelines for
applying this process to other natural products and derivatives.
The described method is demonstrated in several cases to be
superior in terms of mildness of reaction conditions and to be
flexible in terms of modifying site selectivity based on reagent
control (i.e., modifying catalysts or diazo reagent). These studies
open possibilities for further modifying chemosite selectivity
through the agency of chiral ligands employing a type of “double
diastereodifferentiation”. An O-H insertion/[3 + 2] cycload-
dition sequence with FK506 provided access to a novel FK506-
biotin conjugate 20, which was successfully employed to pull-
down the ternary complex involved in immunosuppression by
FK506 from RKO cell lysates. The concept of simultaneous
arming and SAR was demonstrated with BFA in this case simply
by modifying achiral ligands on rhodium. These studies initiate
an important area in the biological chemistry of natural products,
involving the development of both chemosite-selective and also
site-nonselective monofunctionalizations toward a rapid, sys-
tematic approach for fully exploiting natural products for
chemical genetics. The described overall strategy (1) enables
rapid arming and SAR studies of novel natural products,
requiring minimal structural information (e.g., presence of
alcohols) and (2) provides a mild, versatile synthesis of natural
product conjugates that should expedite the identification of
natural product cellular receptors, facilitate their continued use
as modulators of cellular function, and contribute to their
continued potential as enduring leads for drug discovery. The
application of this method to novel natural products including
the use of chiral rhodium catalysts to further modulate site-
selectivity constitutes our ongoing studies in this area, and the
results of these studies will be reported in due course.
Representative Procedure for the Rh(II)-Mediated O-H Inser-
tion As Described for the Preparation of Gibberellic Acid Methyl
Ester 3-HBPA Ether, 3g. Gibberellic acid methyl ester (20 mg, 0.056
mmol) and rhodium acetate (1.3 mg, 0.003 mmol, 0.05 equiv) were
weighed into a flame-dried Schlenk flask under a nitrogen atmosphere.
The solids were suspended in dry CH2Cl2 (4 mL), and the mixture was
stirred for 5 min to dissolve the methyl ester (Note: the rhodium catalyst
does not dissolve) at 23 °C. A solution of 5-hexynyl-R-(4-bromophen-
yl)-diazo acetate 2g (36 mg, 0.112 mmol) in dry CH2Cl2 (2 mL) was
prepared and added via syringe. The reaction mixture was stirred for 1
h at 22 °C. The solvent was evaporated, and the residue was purified
by flash chromatography on SiO2 (pentane/ether, 20:80) to afford ether
3g (20 mg, 55% yield) and recovered gibberellic acid methyl ester 1
(8 mg, 38%). Rf 0.39 (hexanes/Et2O, 20:80). IR (thin film) cm-1: 2117,
1772, 1733. LRMS (MALDI): calculated for C34H37BrO8 (M + Na)
) 675.2 and observed (M + Na) ) 675.2 (see Supporting Information
1
for H and 13C NMR line listings and NMR spectra).
Synthesis of FK506-Biotin Conjugate 20 by Sharpless-Hu1isgen
Cycloaddition. (+)-Biotin- (PEO)4-propionyl azide 19 (2.3 mg, 0.004
mmol) was dissolved in 0.2 mL of dry acetonitrile in a vial flushed
with nitrogen and equipped with a magnetic stir bar. FK506 32-HBPA
ether 14b (4.0 mg, 0.004 mmol) was dissolved in 0.2 mL of dry
acetonitrile and then transferred to the reaction vial followed by CuI
(0.1 mL, 0.004 µM, stock solution in dry acetonitrile) and Hu¨nig’s
base (10 µL, 0.6 µM, stock solution in dry acetonitrile), and the mixture
was stirred at 22 °C overnight. The solvent was evaporated, and the
residue was purified by preparative TLC (20 × 10 cm2, 1000-µm
thickness SiO2, MeOH/EtOAc, 30:70) to afford the FK506-biotin
conjugate 20 (3.7 mg, ∼60% yield): Rf 0.35 (EtOAc/MeOH, 50:50).
HRMS (MALDI): calcd for C82H125BrN8O21S (M + H) ) 1691.7942;
1
found (M + H) ) 1691.7959 (see Supporting Information for H and
13C NMR line listings and NMR spectra).
Cell Culture. HeLa, Jurkat, and RKO cells were grown in DMEM
or RPMI1640 medium with 10% fetal bovine serum and cultured at
5% CO2 and 37 °C in a humidified atmosphere.
Interleukin 2 Reporter Gene Assay. The transient transfection was
performed by electroporation using minimal IL-2 promoter driven
luciferase reporter plasmid (Promega). Jurkat T cells (1 × 107) were
harvested, washed once in RPMI-1640 medium, resuspended in 300
µL of RPMI, and mixed with 2 µg of IL-2 plasmid. The electroporation
was carried out by applying an electric pulse (150 V, 960 µF using the
Bio-Rad Gene Pulser II). The cells were allowed to rest for 10 min
before they were transferred back to the culture medium (RPMI with
10% fetal calf serum and 2 µM glutamine) and incubated at 37 °C for
24 h, and then aliquoted into 24 well plates. FK506 (12), 32-HBPA
FK506 13a,b, and biotin-FK506 conjugate 20 dissolved in DMSO
were added to cultured cells and incubated with the cell cultures for 1
h before PMA (final concentration 40 nM) and ionomycin (final
concentration of 1 µM) were added. After another 6 h of incubation,
cells were harvested and prepared for luciferase assay according to
manufacturer’s protocol (Promega).
Experimental Section
Reactions were carried out in flame-dried glassware under a nitrogen
or an argon atmosphere, unless otherwise noted. Commercial solvents
and reagents were used as received. Anhydrous solvents were dried
over neutral alumina (MBraun system). All reactions were magnetically
stirred and monitored by TLC, performed using glass-backed silica gel
60F254 (Merck, 250-µm thickness). Yields refer to chromatographically
and spectroscopically pure compounds unless otherwise stated. Flash
column chromatography was performed using 60 Å silica gel (Baker,
230-400 mesh, or Silacycle, 230-400 mesh) as a stationary phase.
Preparative TLC was performed using Partisil PKL5F silica gel plate
Affinity Pull-Down Experiment. RKO cells were cultured in RPMI-
1640 with 10% FBS medium and treated with or without PMA (final
concentration 40 nM) and ionomycin (final concentration of 1 µM)
before harvesting. Cells were homogenized using a dounce homogenizer
9
12230 J. AM. CHEM. SOC. VOL. 129, NO. 40, 2007