Membrane receptor probes: Solid-phase synthesis of biotin-Asp-PEG-arvanil derivatives

Article abstract of DOI:10.1021/ol0502578

(Chemical Equation Presented) A modular, flexible solid-phase synthetic route for the preparation of biotinylated cross-linking probes of membrane receptors is described. The route utilizes an orthogonal protection strategy employing a Pd[0] cleavable all

Full text of DOI:10.1021/ol0502578

ORGANIC
LETTERS
2005
Vol. 7, No. 9
1699-1702
Membrane Receptor Probes:
Solid-Phase Synthesis of
Biotin-Asp-PEG-arvanil Derivatives
Cristina Visintin,† Abil E. Aliev,‡ Dieter Riddall,† David Baker,§
|
Masahiro Okuyama,†, Pui Man Hoi, Robin Hiley, and David L. Selwood*,†
The Wolfson Institute for Biomedical Research, Institute of Neurology, and Department
of Chemistry, UniVersity College London, Gower Street, London WC1E 6BT, UK, and
Department of Pharmacology, UniVersity of Cambridge, Cambridge, UK
d.selwood@ucl.ac.uk
Received February 7, 2005
ABSTRACT
A modular, flexible solid-phase synthetic route for the preparation of biotinylated cross-linking probes of membrane receptors is described.
The route utilizes an orthogonal protection strategy employing a Pd[0] cleavable allyl linker attached to the probe via an aspartate residue.
The versatility of the method is illustrated through the synthesis of a number of arvanil-derived cannabinoid receptor ligands displaying either
a photoaffinity or a chemical cross-linking group.
The biotinylation of small molecules exploits the uniquely
tight biotin-avidin interaction for diverse applications such
as detection, visualization, and purification/isolation of
proteins. Innovative technological developments continue to
appear, including the Scavidin gene therapy system for
targeting of biotinylated therapeutics,1,2 extracorporeal af-
finity adsorption3 (for removal of radiolabeled antibodies),
and biotinylated activity-based probes for chemical proteom-
ics.4 In this letter, we describe a flexible solid-phase method
for the synthesis of biotinylated, cross-linking, small-
molecule probes. The synthetic strategy is modular, based
on Fmoc peptide synthesis to allow simple variation of
subunits. The chemistry is conducted manually, using readily
available plastics, and can be carried out in most chemistry
laboratories. The methodology should be applicable to a wide
variety of biotinylation problems. Previously, biotinylated
adenosine 5-triphosphate analogues have been utilized for
the enrichment of membranes carrying the P2Y(1) receptor,5
while bifunctional small-molecule probes (containing biotin
and a cross-linking group) have been used to identify binding
sites in proteins.6 We sought to identify a small-molecule
probe capable of cross-linking partially characterized can-
† The Wolfson Institute for Biomedical Research, University College
London.
‡ Department of Chemistry, University College London.
§ Institute of Neurology, University College London.
| Current address: Mitsubishi Pharma Corporation 1000, Kamoshidacho,
Aoba-ku, Yokohama 227-0033, Japan
University of Cambridge.
(1) Lehtolainen, P.; Taskinen, A.; Laukkanen, J.; Airenne, K. J.; Heino,
S.; Lappalainen, M.; Ojala, K.; Marjomaki, V.; Martin, J. F.; Kulomaa, M.
S.; Yla-Herttuala, S. J. Biol. Chem. 2002, 277, 8545-8550.
(2) Allart, B.; Lehtolainen, P.; Yla-Herttuala, S.; Martin, J. F.; Selwood,
D. L. Bioconjugate Chem. 2003, 14, 187-194.
(3) Wilbur, D. S.; Chyan, M. K.; Hamlin, D. K.; Kegley, B. B.; Nilsson,
R.; Sandberg, B. E.; Brechbiel, M. Bioconjugate Chem. 2002, 13, 1079-
1092.
(4) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Mol. Cell Proteomics
2002, 1, 828-835.
(5) Zundorf, G.; Schafer, R.; Vohringer, C.; Halbfinger, E.; Fischer, B.;
Reiser, G. Biochem. Pharmacol. 2001, 61, 1259-1269.
(6) Hosoya, T.; Hiramatsu, T.; Ikemoto, T.; Nakanishi, M.; Aoyama,
H.; Hosoya, A.; Iwata, T.; Maruyama, K.; Endo, M.; Suzuki, M. Org.
Biomol. Chem. 2004, 2, 637-641.
10.1021/ol0502578 CCC: $30.25
© 2005 American Chemical Society
Published on Web 04/06/2005

nabinoid receptors.7,8 The endocannabinoid anandamide 1,
SR141716A 2, and WIN55,212-2 3 have affinity9 for these
novel receptors (Figure 1), as does arvanil 4, an anandamide-
reaction between 7 and FmocNH-Asp-OtBu in the presence
of K2CO3 in DMF gave, in good yield, the orthogonally
protected aspartate 8. After removal of the phenacyl group
by treatment with zinc in acetic acid, the acid 9 was reacted
with Argogel-NH2 resin to give the desired aspartate-loaded
resin 10. The synthesis of the arvanil precursor is shown in
Scheme 2. We chose the amide NH as the connection point
Scheme 2. Synthesis of Arvanil Precursor
Figure 1. Cannabinoid ligands.
capsaicin hybrid that activates non-CB1, non-CB2, and non-
VR1 receptors.8 Thus, we chose arvanil as the initial ligand
for tagging. We identified the 6-hydroxy-4-hexenoate allyl
ester linker10 of Nakahara as being acid and base stable and
cleavable under nearly neutral Pd[0] catalysis conditions. The
stability of the arachidonate double bonds in arvanil was
tested under the Pd[0] cleavage conditions; no isomerization
was detected in the olefinic region by NMR (see Supporting
Information). In work on the use of biotin-linked reagents
for antibody pretargeting, Wilbur11 established that a proxi-
mal aspartate gave molecules resistance to biotinidases in
vivo. Incorporation of a proximal aspartate is therefore a
desirable feature and also provides a convenient attachment
point to the solid phase. The synthesis of the complete linker
is shown in Scheme 1. Thus, 6-bromo-hex-4-enoic acid tert-
to biotin, as it was straightforward synthetically and there
was evidence that substitution at this point would not abolish
the biological activity.8 In contrast, the hydroxyl moiety on
the aromatic ring seems to be required. Tips protection of
o-vanillin, 11, followed by reductive amination with amino-
hexan-6-ol provided 12, Fmoc protection to 13, and oxidation
with RuCl3/NaIO4 gave the required intermediate 14. To
establish the optimum linker length,12 we needed to be able
to vary the distance between the biotin and ligand easily.
Fmoc-8-amino-3,6-dioxaoctanoic acid 15 is a convenient
PEG spacer molecule. It was easy to synthesize and could
be inserted using standard peptide synthesis protocols. In the
first instance we prepared three molecular probes, which
differed in the number of spacer units 15 used (Scheme 3).
Thus, the resin-linked aspartate 10 was treated with 20%
piperidine in DMF to remove the Fmoc protection. Then,
Scheme 1. Synthesis of the Allyl Aspartate Linker
(7) Jarai, Z.; Wagner, J. A.; Varga, K.; Lake, K. D.; Compton, D. R.;
Martin, B. R.; Zimmer, A. M.; Bonner, T. I.; Buckley, N. E.; Mezey, E.;
Razdan, R. K.; Zimmer, A.; Kunos, G. Proc. Natl. Acad. Sci. U.S.A 1999,
96, 14136-14141. Ford, W. R.; Honan, S. A.; White, R.; Hiley, C. R. Br.
J. Pharmacol. 2002, 135, 1191-1198.
(8) Di Marzo, V.; Bisogno, T.; De Petrocellis, L.; Brandi, I.; Jefferson,
R. G.; Winckler, R. L.; Davis, J. B.; Dasse, O.; Mahadevan, A.; Razdan et,
al. Biochem. Biophys. Res. Commun. 2001, 281, 444-451.
(9) Breivogel, C. S.; Griffin, G.; Di, M. V.; Martin, B. R. Mol.
Pharmacol. 2001, 60, 155-163.
(10) Nakahara, Y.; Ando, S.; Itakura, M.; Kumabe, N.; Hojo, H.; Ito,
Y.; Nakahara, Y. Tetrahedron Lett. 2000, 41, 6489-6493.
(11) Wilbur, D. S.; Chyan, M. K.; Pathare, P. M.; Hamlin, D. K.;
Frownfelter, M. B.; Kegley, B. B. Bioconjugate Chem. 2000, 11, 569-
583.
(12) Hashimoto, M.; Yang, J.; Holman, G. D. Chembiochem 2001, 2,
52-59.
butyl ester 5 was cleaved to the acid with TFA and reacted
with phenacyl bromide to obtain the ester 7. The coupling
1700
Org. Lett., Vol. 7, No. 9, 2005

Scheme 3. Synthesis of the Biotin-PEG Ligands without Cross-Linkers
the free amine compound was coupled with biotin using
21. Resins 19-21, were coupled under the usual conditions
with the acid 14 to give, after Fmoc deprotection, the
intermediates 22-24, respectively. Two cycles of the last
coupling reaction, with arachidonic acid, were necessary,
using HATU as a coupling reagent in the presence of DIPEA,
in DMF. Tips deprotection with triethylamine trihydrofluo-
ride in THF followed by cleavage from the solid support
using Pd(Ph3P)4 and morpholine in CHCl3 gave 25-27.
These molecular probes were purified by HPLC and char-
acterized using mass spectrometry techniques. For 26, the
PyBop and N-methyl morpholine in DMF. From the resin
16, the tert-butyl protection was removed by treatment with
50% TFA in CH2Cl2, containing 2.5% of tri-n-propylsilane
as scavenger. The resin was then reacted with the PEG-
diamine 17 in the presence of PyBop and N-methyl mor-
pholine in DMF. The intermediate 18 was then coupled with
the spacer 15 using the above coupling conditions to give
19. Appropriate repetition of this coupling procedure gave,
after Fmoc deprotection, the resin-linked compounds 20 and
Scheme 4. Synthesis of Biotin-PEG Ligands with a Cross-Linking Group
Org. Lett., Vol. 7, No. 9, 2005
1701

1
structure was confirmed by two-dimensional H NMR (see
Supporting Information). To consolidate the binding between
a small-molecule ligand and its receptor, a covalent cross-
link is frequently required.13 We designed a series of com-
pounds containing a lysine unit, allowing attachment of a
thiol-specific group such as malemide (for cross-linking to a
cysteine residue). Alternatively, a benzophenone unit, which
is commonly employed for photoaffinity labeling, was incor-
porated.14,15 The optimal distance of the cross-linking group
from the ligand was explored with a series of four mole-
cules using the spacer 15 as a repeating unit (Scheme 4).
Coupling of the amino acids with 18 gave, following the
route described above, four potential cross-linking ligands:
34-37. Preliminary biological evaluation used a standard
cannabinoid CB1 binding assay employing displacement of
[3H] SR141716A 2 in rat brain membranes. In this case, we
are utilizing CB1 as a model for the novel cannabinoid
receptors. When no cross-linker was present, the binding was
relatively insensitive to the number of units of 15 (see Table
1 compounds 25-27). With either the 4-PhCO-phenylalanine
or lysine cross-linking groups (compounds 34 and 35) one
unit of 15 was optimal.
Table 1. Radioligand CB1 Binding Assay
probe
units of 15
cross-linker
IC50 µM
25
26
27
34
35
36
37
1
2
3
1
1
2
2
none
none
none
4PhCOPh
Lys
0.7
3.3
1.0
4.0
21.4
2.6
NDa
4PhCOPh
Lys
a ND not determined.
sensitive to both capsaicin and capsazepine (showing interac-
tion with vanilloid receptors), and in some preparations it
was SR141716A 2 sensitive, indicating interaction with the
abnormal cannabidiol receptor.
In summary, we have developed a flexible solid-phase
synthesis for biotinylation utilizing a stable allyl-Asp linker.
The synthetic method is compatible with a wide range of
reaction conditions, including Fmoc and Boc peptide chem-
istry, and should have wide applicability.
36 also relaxed precontracted rat small mesenteric artery
(EC50 30 nM), which was in part endothelium-dependent,
Acknowledgment. This work was supported by a re-
search grant from the Brain Research Trust (C.V.).
(13) Haugland, R. P Crosslinking and Photoreactive Reagents. In
Handbook of Fluorescent Probes and Research Chemicals; Spencer, M. T.
Z., Ed.; Molecular Probes, Inc.: Eugene, OR; 1995.
Supporting Information Available: Detailed synthetic
methods and analytical data. This material is available free
of charge via the Internet at http://pubs.acs.org.
(14) Fleming, S. A. Tetrahedron 1995, 51, 12479-12520.
(15) Li, Y. M.; Xu, M.; Lai, M. T.; Huang, Q.; Castro, J. L.; DiMuzio-
Mower, J.; Harrison, T.; Lellis, C.; Nadin, A.; Neduvelil, J. G.; Register,
R. B.; Sardana, M. K.; Shearman, M. S.; Smith, A. L.; Shi, X. P.; Yin, K.
C.; Shafer, J. A.; Gardell, S. J. Nature 2000, 405, 689-694.
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