An improved synthesis of a fluorescent gabapentin-choline conjugate for single molecule detection

Article abstract of DOI:10.1016/j.tetlet.2009.02.125

Voltage-gated calcium ion channels comprise pore-forming α₁ and auxiliary α₂δ, β, and γ subunits. They are important molecular devices involved in a variety of cell functions. Fluorescently labeled acylcholine analogues are important

Full text of DOI:10.1016/j.tetlet.2009.02.125

Tetrahedron Letters 50 (2009) 2100–2102
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
An improved synthesis of a fluorescent gabapentin–choline conjugate
for single molecule detection
*
Haitao Wu, Gurpreet Kaur, Gary L. Griffiths
Imaging Probe Development Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Voltage-gated calcium ion channels comprise pore-forming a1 and auxiliary a2d, b, and c subunits. They
Received 2 February 2009
Revised 14 February 2009
Accepted 16 February 2009
Available online 21 February 2009
are important molecular devices involved in a variety of cell functions. Fluorescently labeled acylcholine
analogues are important in studies such as ion channel regulation. Cy3-3-acetylcholine has recently been
synthesized for single molecule detection studies; albeit in an extremely low overall yield (0.06%). In this
work, an alternative route to that used in the previous Cy3-3-acetylcholine synthesis was developed with
a 90% yield at a significantly lower material cost.
Keywords:
Calcium ion channel
Gabapentin
Published by Elsevier Ltd.
Single molecule detection
Fluorescence imaging
Voltage-gated Ca2+ channels are important molecular devices
allowing changes in membrane potential to control various Ca2+
dependent cell functions such as muscle contraction, hormone
and neurotransmitter release, neuronal plasticity, and sensory cell
signaling. There are three subfamilies (Cav1, Cav2, and Cav3), 10 dif-
ferent pore-forming a1 subunit isoforms that control the Ca2+ in-
flux to the needs of individual cells, and also accessory subunits
tionalized Cy3 dye moiety (ꢀUS$800/5 mg) was first attached to
4-aminobutyric acid (GABA), and the conjugated acid was esteri-
fied with N,N-dimethylethanolamine via reaction with oxalyl chlo-
ride. Quaternization of the amine with methyl iodide yielded the
desired fluorescent conjugate. The three reactions proceed in yields
-
(a2d, b, and c) involving alternative splicing and regulatory pro-
-O3S
SO3
-O3S
SO3
-
-
teins.1 The nicotinic acetylcholine receptor (nAChR), for example,
which is a well-characterized ligand-gated ion channel,2 plays
important roles in neurotransmission and in Alzheimer’s disease,3
as well as in anti-inflammatory effects.4
i
N
N+
N
N+
H
N
O
O
CO2H
N
Because of the lack of tools for the simultaneous measurement
of ligand binding and channel gating, their relationship has been
inferred only indirectly, and an apparatus was developed recently
to investigate signal transduction processes of channel molecules
at the single molecule level.5 Most recently, a fluorescent GABA–
choline conjugate ligand for single molecule observation of
ligand–channel interactions was developed and binding of this
ligand to nAChR was visualized by total internal reflection fluores-
cence (TIRF) microscopy.6 Although shown to be a powerful tool
for investigation of single channel–ligand interactions, the synthe-
sis of this promising ligand was only performed in an extremely
low yield (0.06%) (Scheme 1).
O
1
O
O
2
ii
-O3S
SO3
O
-
N
N+
H
N
-O3S
SO3
O
-
N
O
iii
O
3
N
N+
H
N
+N
O
The synthesis of the Cy3-3-acetylcholine fluorescent analogue
of acetylcholine, as described by Fujimoto et al., was performed
in three steps (Scheme 1). The commercially available monofunc-
O
4
Scheme 1. Reagents and conditions: (i) 4-aminobutanoic acid (GABA), 0.1 M
sodium borate buffer, pH 8.5, room temperature, 2 h, 41%; (ii) oxalyl chloride, N,N-
dimethylethanolamine, DMF, room temperature, 4 h, 0.1%; (iii) CH3I, DMF, room
temperature, 2 days, 14%.
* Corresponding author.
E-mail address: griffithsgl@mail.nih.gov (G.L. Griffiths).
0040-4039/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2009.02.125

H. Wu et al. / Tetrahedron Letters 50 (2009) 2100–2102
2101
attempts to isolate a pure gabapentin–choline conjugate were
not successful due to the relative instability of the conjugate
during work-up. A Boc-protected gabapentin–choline conjugate
precursor was then tried but also proved useless due to instabil-
ity of intermediates during work-ups as well as having the
inconvenience of the lack of a suitable UV absorption for reac-
tion monitoring and analyses. An Fmoc-protected gabapentin–
choline conjugate and its intermediates would have proper UV
absorption for detection and can be deprotected under mild con-
ditions. Furthermore, we intended to perform the final coupling
reaction in an anhydrous organic phase using an in situ genera-
tion of gabapentin–choline from a stable lipophilic precursor in
order to diminish competing hydrolysis of the activated Cy3
dye and reduce effects due to the observed instability of free
gabapentin–choline. This route proved successful with high
yields for each step. One challenge was purification of the
Fmoc-protected gabapentin–N,N-dimethylaminoethyl ester9 as
purity is vital for quantitative conversion to the Fmoc-protected
gabapentin–choline conjugate, but a quick wash of its ethyl ace-
tate solution with cold saturated aqueous sodium bicarbonate
solution during work-up proved safe for the base sensitive Fmoc
protecting group. Then, methylation with methyl iodide pro-
ceeded quantitatively to yield the Fmoc-protected gabapentin–
N
N
OH
N
Fmoc-O N
i
FmocHN
ii
Fmoc-Cl
FmocHN
O
5
iii
I-
+N
O
O
iv
FmocHN
O
O
7
6
Scheme 2. Reagents and conditions: (i) HOBt, pyridine, DCM, room temperature,
1 h; (ii) gabapentin, DIPEA, DMF, room temperature, 5 h, 94%; (iii) N,N-dimethyl-
aminoethanol, 2-dipyrodinyl carbonate, DMAP, toluene, room temperature, 24 h,
72%; (iv) CH3I, CHCl3, room temperature, 24 h, quantitative.
I-
I-
O
O
N+
H2N
N+
FmocHN
i
O
O
7
7
-O3S
SO3
-
i
choline
conjugate
which
was
used
without
further
N
N+
1
+
purification.10,11
H
O
N+
N
In the final Cy3 coupling step, choice of an appropriate base
which would remove the Fmoc protecting group, while not
interfering with the concomitant coupling reaction, was crucial
since the initially tried piperidine, even at low concentration
(5%) in DMF, seriously competes with in situ-generated gaba-
pentin–choline for the Cy3 monofunctional ester. A 10% dimeth-
ylaminopyridine (DMAP) solution in DMF, which served as both
base and solvent, proved to be safe. The DMAP slowly cleaves
off the Fmoc group and the slowly formed and unstable gaba-
pentin–choline conjugate immediately reacts with the Cy3
monofunctional ester that is present to form the stable final
product. Eventually, the final product was isolated in ꢀ90%
yield by injecting the reaction mixture directly onto a prepara-
tive HPLC and then lyophilizing the product fraction. Finally, at-
tempted coupling reactions with a cyanine dye monoacid to an
Fmoc-protected gabapentin–choline conjugate catalyzed by
HBTU, HATU, or EDC coupling agents in situ with Fmoc depro-
tecting agents such as piperidine, DIPEA, or TBAF were all tried
but proved less successful due to higher by-product levels and
lower yields.
O
O
8
Scheme 3. Reagents and conditions: (i) 10% DMAP in DMF, room temperature, 12 h,
90%.
of 41%, 0.1%, and 14%, respectively, with the 0.1% yield obtained
during the esterification reaction making the reaction sequence
impracticable for scale-up and prohibitively expensive in light of
the cost of the starting Cy3 dye. As such agents are likely to be
important in future single molecule studies, we designed an alter-
native ‘one-step’ synthesis strategy [from the expensive Cy3 mono-
functional ester] for a similar acylcholine agent based on a
gabapentin analogue. The calcium ion channel regulation study
using this agent is in progress.
The FDA-approved anticonvulsant drug gabapentin, an ana-
logue of GABA, down-regulates activity of voltage-activated cal-
cium channels by specifically interacting with the extracellular
auxiliary a2d subunit, and its mechanisms of action have been re-
viewed.7 We were asked to synthesize a fluorescent derivative of
gabapentin for voltage-gated calcium channel studies and devel-
oped the alternative synthetic route shown (Schemes 2 and 3).
The synthesis used the succinimido-activated monofunctional es-
ter of Cy3, 1, and the Fmoc-protected gabapentin–choline conju-
gate, 7, to give the final conjugate product, 8, in one step in an
isolated yield of 90%. This route is particularly attractive since it
not only gives a high yield of the final product but also mitigates
the need for time-consuming chromatography of intermediates
as the Fmoc-protected intermediates are synthesized in organic
solvents and are isolated and purified by simple extractive work-
ups.
In conclusion, we have developed an efficient synthesis of the
Cy3-gabapentin–choline conjugate, using an approach which
should also prove useful for the generation of other similar fluores-
cent dye conjugates of biologically important acylcholine
analogues.
Acknowledgement
This work was supported by the NIH Roadmap for Medical Re-
search Initiative through its establishment of the Imaging Probe
Development Center, administered by the National Heart, Lung,
and Blood Institute.
Attaching the expensive Cy3 dye during the last step of the
synthesis is very attractive in order to mitigate costs, even with-
out the extremely poor esterification yield obtained in the
Fujimoto route.6 In an alternative strategy we decided to first
make a gabapentin–choline conjugate. Direct coupling of GABA
and choline was successful using hydrogen chloride-saturated
anhydrous dioxane,8 and although this method might have
proved applicable to a gabapentin–choline analogue in our hands
Supplementary data
Experimental procedures and complete spectral data and the
copies of 1H and 13C NMR spectra of all intermediates and final
product are available. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.tetlet.2009.02.125.

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H. Wu et al. / Tetrahedron Letters 50 (2009) 2100–2102
5. Ide, T.; Takeuchi, Y.; Aoki, T.; Yanagida, T. Jpn. J. Physiol. 2002, 52, 429.
References and notes
6. Fujimoto, K.; Yoshimura, Y.; Ihara, M.; Matsuda, K.; Takeuchi, Y.; Aoki, T.; Ide, T.
Bioorg. Med. Chem. Lett. 2008, 18, 1106.
7. Taylor, C. P.; Gee, N. S.; Su, T. Z.; Kocsis, J. D.; Welty, D. F.; Brown, J. P.; Dooley, D.
J.; Boden, P.; Singh, L. Epilepsy Res. 1998, 29, 233.
8. Kuriaki, K.; Kohiga, M.; Kadoo, G.; Yatabe, K. JP36017707, 1961.
9. Narula, P.; Patel, B.; Vaid, S. WO 2008/015693 A2, 2008.
10. Jurss, R.; Maelicke, A. J. Biol. Chem. 1983, 258, : 10272.
11. Abood, L. G. US 4,966,916, 1990.
1. Catterall, W. A.; Perez-Reyes, E.; Snutch, T. P.; Striessnig, J. Pharmacol. Rev. 2005,
57, 411.
2. Hille, B. In Ion Channels of Excitable Membranes, 3rd ed.; Sinauer Associates:
Sunderland, MA, 2001; p 169.
3. Wang, H.-Y.; Lee, D. H. S.; D’Andrea, M. R.; Peterson, P. A.; Shank, R. P.; Reitz, A.
B. J. Biol. Chem. 2000, 275, 5626.
4. Gallowitsch-Puerta, M.; Tracey, K. J. Ann. N.Y. Acad. Sci. 2005, 1062, 209.