A simple and efficient route to the FKBP-binding domain from rapamycin

Article abstract of DOI:10.1016/j.tetlet.2011.07.094

A simple and highly efficient route to the FKBP-binding domain (FKBD) from the natural product rapamycin has been developed, which entails a sequence of ozonolysis/Baeyer-Villiger/Wittig reactions. The newly synthesized FKBD may serve as a core to assembl

Full text of DOI:10.1016/j.tetlet.2011.07.094

Tetrahedron Letters 52 (2011) 5070–5072
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A simple and efficient route to the FKBP-binding domain from rapamycin
Wei Li a, Shridhar Bhat a, Jun O. Liu a,b,
⇑
a Department of Pharmacology, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA
b Department of Oncology, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and highly efficient route to the FKBP-binding domain (FKBD) from the natural product rapamy-
cin has been developed, which entails a sequence of ozonolysis/Baeyer–Villiger/Wittig reactions. The
newly synthesized FKBD may serve as a core to assemble hybrid macrocyclic libraries for the discovery
of novel probes of protein function and to synthesize new ligands for the FKBP family of proteins.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 30 June 2011
Revised 20 July 2011
Accepted 21 July 2011
Available online 27 July 2011
Keywords:
FKBP
Rapamycin
FKBP-binding domain
The natural products FK506 and rapamycin possess potent
immunosuppressive and antitumor (rapamycin) activities and they
share a unique and extraordinary mode of action—through inducing
the formation of ternary complexes between an abundant cellular
enzyme known as FKBP and their respective protein targets.1–5
Upon entering cells, both FK506 and rapamycin form binary com-
plexes with FKBP. While the FKBP–FK506 complex binds to and
inhibits the enzymatic activity of the protein phosphatase calcineu-
rin, blocking calcium-dependent intracellular signaling emanating
from the T cell receptor,1 the FKBP-rapamycin complex binds to a
distinct target—the protein kinase mTOR that serves as a nodal
point in signaling leading to the regulation of a multitude of cellular
processes including cell proliferation.2–5 Extensive biochemical and
structural studies have revealed that both FK506 and rapamycin
can be divided into two structural and functional domains, an
FKBP-binding domain (FKBD) and a so-called effector domain. A
comparison of the structures of FK506 and rapamycin revealed that
they share a nearly identical FKBD but each possesses a distinct
effector domain (Fig. 1). In FK506 and rapamycin, Nature has taught
us that by swapping the effector domain of FK506 with that of rap-
amycin, it is possible to change the target from calcineurin to TOR,
which bear no sequence, functional, or structural similarities to
each other.6–9 We were thus inspired to ask the question: Is it pos-
sible to target other proteins by grafting new structures onto FKBD
of rapamycin and FK506?
we needed access to large quantities of FKBD. Although synthetic
versions of FKBD can be employed,10 we felt it necessary to com-
pare the relative affinities of the ‘natural’ FKBD with the synthetic
versions before embarking on the synthesis of libraries with an
optimal version of FKBD. In one of our library designs, we envi-
sioned the use of ring-closing metathesis to achieve the macrocyc-
lization, calling for the use of FKBD that is equipped with a
terminal olefin.
Previous syntheses of FKBD from rapamycin had involved the
initial cleavage of the inherently labile ester bond at C-34 of rapa-
mycin, which necessitated the reassembly of the two resulting
pieces of FKBD.11 Should the same route be followed, it would
not only increase the number of steps, be less elegant and efficient,
but also significantly increase the demand for the not-so-cheap
rapamycin as the starting material. After attempting a number of
potential routes, we developed a simple and efficient method to
prepare the desired FKBD from rapamycin while keeping the entire
FKBD intact throughout the process
Our preparation of FKBD commenced with the treatment of
rapamycin (1) with TBSOTf and Et3N to afford the fully protected
epimers (2) in quantitative yield (Scheme 1). According to an
earlier report by Goulet and Boger,12 protection of the hydroxyl
at the hemiketal center (C-10) of rapamycin is not necessary for
the following ozonolysis step. However, the corresponding C-10
unprotected FKBD fragment was cleaved between C-9 and C-10
when subjected to Baeyer-Villiger oxidation.12 Thus, we deemed
the protection of the C-10 hydroxyl to be an absolute requirement
to succeed in our strategy. The rapamycin derivative 2 was then
subjected to ozonolysis in CH2Cl2 at ꢀ68 °C and subsequently trea-
ted with 35% H2O2 to give an intermediate containing a carboxylic
acid at one end and a peroxyhemiacetal or aldehyde at the other
To answer this question, we will need to synthesize sufficiently
large combinatorial hybrid libraries that display random peptides
or other building blocks on FKBD. As a first step of this exploration,
⇑
Corresponding author. Tel.: +1 410 955 4619; fax: +1 410 955 4620.
E-mail address: joliu@jhu.edu (J.O. Liu).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.07.094

W. Li et al. / Tetrahedron Letters 52 (2011) 5070–5072
5071
OH
OH
O
MeO
MeO
FKBD
O
FKBD
O
O
O
33
H
N
H
N
30
OH
29
9
O
OMe
O
Calcineurin
O
TOR
O
O
HO
OH
OH
10
OMe
O
H
O
O
H
18
17
Effector Domain
Effector Domain
MeO
OMe
Rapamycin (1)
FK506
OH
MeO
FKBD
O
H
R4
O
O
N
H
O
N
HN
O
R3
O
OH
New Targets?
New Effector Domains
O
O
H
NH
R2
O
O
N
H
NH
R1
Figure 1. Rapamycin, FK506, their FKBP-binding domains (blue), effector domains (pink and red) and generic structures of the proposed new hybrid combinatorial cyclic
libraries.
Deprotection of the TBS protecting groups with trifluoroacetic acid
and H2O at 0 °C gave the final product FKBD (4) in 96% yield.17 It is
OTBS
noteworthy that neither TABF nor HFꢁpyridine were able to accom-
plish a clean deprotection of the two silyl groups. Thus, FKBD was
prepared from rapamycin in just four steps with 52% overall yield.
We were unable to isolate and completely characterize the
intermediates after ozonolysis and the Baeyer–Villiger reaction
due in part to the complexity of the reaction products. The com-
plexity was further compounded by the propensity of the FKBD
to exhibit rotamerism and in this case we were also dealing with
a pair of epimers. The most likely intermediates in the course of
these two tandem steps (ozonolysis/Baeyer–Villiger) can be pre-
dicted (Scheme 2). Ozonolysis of the C29-C30 double bond gave
rise to aldehyde intermediate (5) that could undergo facile enoliza-
tion to give 6. This rendered 6 with an exclusive migratory aptitude
in the Baeyer–Villiger rearrangement to give ester 7. Hydrolysis of
7 led to the formation of the carboxylic group in 3. Concurrently,
ozonolysis of C17-C18 double bond yielded the ketone intermedi-
ate 8. Baeyer–Villiger oxidation of 8 led to the formation, possibly
via ester 9, of peroxyhemiacetal (10) that, upon hydrolysis, gave
the corresponding aldehyde 11, which is ready to undergo Wittig
reaction to form the terminal olefin in 3. An interesting feature
of this procedure is that the same set of reactions occurred con-
comitantly at both ends of FKBD, but led to the formation of dis-
tinct functional groups—a carboxylic acid and an olefinic group.
This simultaneous chemical transformation of two groups signifi-
cantly reduced the number of steps required to prepare FKBD,
hence the high overall yield.
MeO
N
O
O
O
a
1
O
OMe
O
TBSO
TBSO
O
O
2
OR
MeO
N
O
O
b, c
O
OH
O
O
RO
O
R = TBS 3
R = H 4
d
In addition to serving our purpose for the synthesis of FKBD-
containing hybrid combinatorial libraries, high-affinity ligands of
FKBP isoforms may find use as probes for a number of biological
processes, as FKBP themselves have been implicated in the regula-
tion of neuronal cell differentiation, ion channels, and Ras post-
translational modification among others.18–22 The easy access to
FKBD could thus allow for the synthesis of potent and possibly iso-
form-selective FKBP ligands that are devoid of immunosuppressive
and antiproliferative activity of FK506 and rapamycin.
Scheme 1. Reagents and conditions: Preparation of FKBD from rapamycin. (a)
TBSOTf, Et3N, CH2Cl2, rt, 100%; (b) O3, CH2Cl2, ꢀ68 °C; then 35% H2O2, rt; (c)
CH3PPh3Br, t-BuOK, THF, 0 °C (54% b,c); (d) CF3COOH, H2O, 0 °C (96%).
end of the FKBD fragment.13,14 After a brief flash-column chroma-
tography over silica gel, the crude product was directly subjected
to Wittig olefination to yield product 3 in 54% combined yield.15,16

5072
W. Li et al. / Tetrahedron Letters 52 (2011) 5070–5072
O
O
O
O
Bayer-Villiger
Oxidation
Isomerization
hydrolysis
O
OH
33
3
HO
7
HO
O
H
5
6
2
Ozonolysis
OMe
OMe O
OMe
OOH
10
O
H+
H2O2
Wittig
Bayer-Villiger
Oxidation
O
17
O
9
H
H
11
3
8
Scheme 2. Proposed reaction intermediates.
CDCl3): d 6.39ꢀ5.82 (m, 4 H), 5.52ꢀ4.91 (m, 4 H), 4.45ꢀ3.98 (m, 2 H), 3.83 (d,
J = 4.0 Hz, 1 H), 3.40 (s, 3 H), 3.27 (s, 3 H), 3.10 (s, 3 H), 2.89ꢀ2.83 (m, 1 H),
2.66ꢀ2.40 (m, 3 H), 2.23ꢀ2.19 (m, 3 H), 1.70 (s, 3 H), 0.17ꢀ0 (m, 18 H); 13C
NMR (100 MHz, CDCl3): d 210.3, 206.6, 197.9, 169.5, 167.0, 139.3, 139.0, 136.6,
132.0, 130.8, 127.4, 126.7, 126.4, 101.7, 86.6, 84.3, 83.2, 78.0, 75.7, 74.3, 67.7,
58.1, 57.8, 56.1, 51.4, 45.8, 44.1, 41.4, 40.8, 40.6, 40.2, 38.2, 36.8, 35.7, 34.0,
33.9, 33.0, 32.0, 30.1, 27.1, 26.6, 26.3, 26.0, 25.9, 25.8, 25.7, 24.9, 22.0, 21.0,
19.3, 18.2, 18.1, 15.7, 15.5, 14.0, 13.9, 11.0, ꢀ2.5, ꢀ3.2, ꢀ4.5, ꢀ4.6, ꢀ4.7, ꢀ4.9;
HR-ESIMS calcd for C69H121O13NSi3Na [M + Na]+ 1278.8043, found 1278.8049.
(3)To a solution of silyl derivative 2 (1.20 g, 0.95 mmol) in CH2Cl2 (15 mL) at
ꢀ68 °C was bubbled O3 until the blue color persisted. 35% H2O2 (15 mL) was
then added and the stirring was continued for another 14 h at rt. The solution
was diluted with EtOAc (120 mL), washed with brine, dried and concentrated
to afford an oil, which was purified using silica gel chromatography (hexane-
EtOAc, 4:1, then CH2Cl2-MeOH, 20:1) to give an epimeric mixture (710 mg) as a
white solid. The epimeric mixture from the ozonolysis reaction stated above
(210 mg, 0.26 mmol) was dissolved in anhydrous THF (2 mL), and then added
to a freshly prepared Wittig reagent at 0 °C, which in turn was prepared from
CH3PPh3þBrꢀ (560 mg, 1.57 mmol) and t-BuOK (140 mg, 1.25 mmol) in
anhydrous THF (5 mL). After stirring for 10 min, the reaction mixture was
quenched with 5% HCl. The resulting mixture was diluted with EtOAc (60 mL),
washed with brine, dried and concentrated. The crude residue was purified by
silica gel chromatography (toluene-EtOAc, 4:1, then toluene-EtOAc-AcOH,
4:1:0.025) to provide 3 (124 mg, 54%) as a white solid: 1H NMR (400 MHz,
CDCl3): 5.84ꢀ5.68 (m, 1 H), 5.29ꢀ4.89 (m, 4 H), 4.42ꢀ4.29 (m, 1 H), 4.06ꢀ3.85
(m, 1 H), 3.40 (s, 3 H), 2.93ꢀ2.48 (m, 4 H), 2.34ꢀ2.13 (m, 4 H), 0.87 (s, 9 H),
0.19ꢀ0.02 (m, 12 H); 13C NMR (100 MHz, CDCl3): d 197.7, 197.4, 176.0, 175.4,
169.7, 169.2, 167.2, 166.4, 135.3, 134.3, 117.2, 116.6, 102.0, 101.6, 84.5, 84.4,
75.7, 75.2, 74.6, 70.5, 70.3, 58.1, 58.0, 56.6, 56.5, 51.8, 44.4, 40.3, 38.9, 38.6,
36.5, 36.4, 36.2, 36.0, 35.1, 34.7, 33.9, 33.34, 33.25, 33.1, 31.4, 30.9, 30.6, 29.7,
29.4, 27.6, 27.3, 26.84, 26.76, 26.6, 26.3, 25.9, 24.8, 24.4, 22.8, 21.3, 20.7, 19.4,
18.2, 15.82, 15.78, 15.3, 14.9, ꢀ2.5, ꢀ2.7, ꢀ2.9, ꢀ3.0, ꢀ4.5, ꢀ4.7; HR-ESIMS
calcd for C42H75O10NSi2Na [M + Na]+ 832.4827, found 832.4833.
In conclusion, a simple and highly efficient process for the prep-
aration of FKBD from rapamycin has been developed using a one-
pot ozonolysis and Baeyer–Villiger reaction as the key step.
Acknowledgment
This work was supported by the NIH Director’s Pioneer Award
(DP1OD006795 to JOL). We are grateful to Drs. Zufeng Guo, Man-
isha Das and members of the Liu Lab for helpful discussions. We
thank Drs. Carol Greider and Paul Talalay for generous provision
of temporary lab space for this work.
References and notes
1. Liu, J.; Farmer, J. D., Jr.; Lane, W. S.; Friedman, J.; Weissman, I.; Schreiber, S. L.
Cell 1991, 66, 807–815.
2. Heitman, J.; Movva, N. R.; Hall, M. N. Science 1991, 253, 905–909.
3. Brown, E. J.; Albers, M. W.; Shin, T. B.; Ichikawa, K.; Keith, C. T.; Lane, W. S.;
Schreiber, S. L. Nature 1994, 369, 756–758.
4. Sabatini, D. M.; Erdjument-Bromage, H.; Lui, M.; Tempst, P.; Snyder, S. H. Cell
1994, 78, 35–43.
5. Chen, Y.; Chen, H.; Rhoad, A. E.; Warner, L.; Caggiano, T. J.; Failli, A.; Zhang, H.;
Hsiao, C. L.; Nakanishi, K.; Molnar-Kimber, K. L. Biochem. Biophys. Res. Commun.
1994, 203, 1–7.
6. Bierer, B. E.; Somers, P. K.; Wandless, T. J.; Burakoff, S. J.; Schreiber, S. L. Science
1990, 250, 556–559.
7. Griffith, J. P.; Kim, J. L.; Kim, E. E.; Sintchak, M. D.; Thomson, J. A.; Fitzgibbon, M.
J.; Fleming, M. A.; Caron, P. R.; Hsiao, K.; Navia, M. A. Cell 1995, 82, 507–522.
8. Kissinger, C. R.; Parge, H. E.; Knighton, D. R.; Lewis, C. T.; Pelletier, L. A.;
Tempczyk, A.; Kalish, V. J.; Tucker, K. D.; Showalter, R. E.; Moomaw, E. W., et al
Nature 1995, 378, 641–644.
9. Choi, J.; Chen, J.; Schreiber, S. L.; Clardy, J. Science 1996, 273, 239–242.
10. Holt, D. A.; Juengo, J. I.; Yamashita, D. S.; Oh, H.; Konialian, A. L.; Yen, H.;
Rozamus, L. W.; Brandt, M.; Bossard, M. J.; Levy, M. A.; Eggleston, D. S.; Liang, J.;
Schultz, L. W.; Stout, T. J.; Clardy, J. J. Am. Chem. Soc. 1993, 115, 9925–9938.
11. Chakraborty, T. K.; Weber, H. P.; Nicolaou, K. C. Chem. Biol. 1995, 2, 157–161.
12. Goulet, M. T.; Boger, J. Tetrahedron Lett. 1990, 31, 4845–4848.
13. Moulines, J.; Bats, J.-P.; Lamidey, A.-M.; Silva, N. D. Helv. Chim. Acta 2004, 87,
2695–2705.
(4) To a mixture of CF3CO2H (0.8 mL) and H2O (0.2 mL) at 0 °C was added olefin
3 (32 mg, 0.040 mmol). After stirring for 2 h, the mixture was concentrated in
vacuo and purified by silica gel chromatography (CH2Cl2–MeOH–AcOH,
20:1:0.2) to offer 4 (22 mg, 96%) as a white solid: 1H NMR (400 MHz, CDCl3):
5.82ꢀ5.68 (m, 1 H), 5.27ꢀ4.90 (m, 4 H), 4.45ꢀ4.32 (m, 1 H), 4.02ꢀ3.85 (m, 1
H), 3.40 (s, 3 H), 2.99ꢀ2.94 (m, 1 H), 2.65ꢀ2.51 (m, 2 H); 13C NMR (100 MHz,
CDCl3): d 197.8, 194.8, 175.1, 175.0, 171.0, 170.0, 169.8, 169.34, 169.26, 167.6,
166.8, 165.7, 135.1, 134.8, 134.3, 117.4, 117.1, 116.7, 99.3, 98.5, 97.7, 84.4,
75.7, 75.2, 75.0, 73.9, 70.2, 70.1, 56.5, 56.4, 51.5, 44.5, 43.1, 41.5, 41.4, 40.2,
39.1, 38.9, 35.1, 35.0, 34.4, 34.3, 34.1, 33.4, 33.2, 31.3, 30.8, 30.6, 29.7, 27.5,
27.1, 26.6, 26.4, 26.3, 25.0, 24.6, 24.4, 21.1, 20.9, 20.8, 16.7, 16.1, 15.9, 15.7,
15.3, 15.1, 15.0; HR-ESIMS calcd for C30H47O10NNa [M + Na]+ 604.3098, found
604.3093.
14. Note: Shortly after the addition of H2O2 the reaction mixture attains a pH of 2.
Oxidative cleavage of the ozonides at C-17, C-19 and C-21 ought to produce
two equivalents of oxalic acid. With the excess of hydrogen peroxide around
peroxalic acid can be generated in situ and it can mediate the Baeyer–Villiger
18. Lyons, W. E.; George, E. B.; Dawson, T. M.; Steiner, J. P.; Snyder, S. H. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 3191–3195.
rearrangements. We have observed this to be
a convenient and efficient
19. Brillantes, A. B.; Ondrias, K.; Scott, A.; Kobrinsky, E.; Ondriasova, E.; Moschella,
M. C.; Jayaraman, T.; Landers, M.; Ehrlich, B. E.; Marks, A. R. Cell 1994, 77, 513–
523.
20. Shou, W.; Aghdasi, B.; Armstrong, D. L.; Guo, Q.; Bao, S.; Charng, M. J.; Mathews,
L. M.; Schneider, M. D.; Hamilton, S. L.; Matzuk, M. M. Nature 1998, 391, 489–
492.
21. Xin, H. B.; Senbonmatsu, T.; Cheng, D. S.; Wang, Y. X.; Copello, J. A.; Ji, G. J.;
Collier, M. L.; Deng, K. Y.; Jeyakumar, L. H.; Magnuson, M. A.; Inagami, T.;
Kotlikoff, M. I.; Fleischer, S. Nature 2002, 416, 334–338.
22. Ahearn, I. M.; Tsai, F. D.; Court, H.; Zhou, M.; Jennings, B. C.; Ahmed, M.;
Fehrenbacher, N.; Linder, M. E.; Philips, M. R. Mol. Cell 2011, 41, 173–185.
process rather than isolating the ozonolysis product and subjecting to Baeyer–
Villiger reaction using peracetic acid or mCPBA.
15. Getty, S. J.; Berson, J. A. J. Am. Chem. Soc. 1990, 112, 1652–1653.
16. Wong, J. C. Y.; Lacombe, P.; Sturino, C. F. Tetrahedron Lett. 1999, 40, 8751–8754.
17. Synthetic Procedures: (2) To a solution of rapamycin (999 mg, 1.09 mmol) and
anhydrous Et3N (0.6 mL, 4.45 mmol) in anhydrous CH2Cl2 (3 mL) was added
TBSOTf (0.85 mL, 3.79 mmol). The solution was stirred for 1 h at rt, and then
quenched with H2O. The resulting mixture was diluted with EtOAc (120 mL),
washed with saturated NaHCO3 and brine, dried and concentrated in vacuo.
The crude residue was subjected to silica gel column chromatography (hexane-
EtOAc, 8:1) to afford 2 (1.37 g, 100%) as a yellow solid: 1H NMR (400 MHz,