A photocaged, cyclopropene-containing analog of the amino acid neurotransmitter glutamate

Article abstract of DOI:10.1016/j.tetlet.2016.10.106

Substituted cyclopropenes serve as compact biorthogonal appendages that enable analysis of biomolecules in complex systems. Neurotransmitters, a chemically diverse group of biomolecules that control neuron excitation and inhibition, are not among the systems that have been studied using biorthogonal chemistry. Here we describe the synthesis of cyclopropene-containing analogs of the excitatory amino acid neurotransmitter glutamate starting from a Garner's aldehyde-derived alkyne. The deprotected cyclopropene glutamate was stable in solution but decomposed upon concentration. Appending a light-cleavable group improved the stability of the cyclopropene while simultaneously caging the neurotransmitter. This strategy has the potential to permit deployment of cyclopropene-modified glutamate as a bioorthogonal probe of the neurotransmitter glutamate in vivo with spatiotemporal precision.

Full text of DOI:10.1016/j.tetlet.2016.10.106

Tetrahedron Letters 57 (2016) 5750–5752
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A photocaged, cyclopropene-containing analog of the amino acid
neurotransmitter glutamate
⇑
Pratik Kumar, David Shukhman, Scott T. Laughlin
Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Substituted cyclopropenes serve as compact biorthogonal appendages that enable analysis of biomole-
cules in complex systems. Neurotransmitters, a chemically diverse group of biomolecules that control
neuron excitation and inhibition, are not among the systems that have been studied using biorthogonal
chemistry. Here we describe the synthesis of cyclopropene-containing analogs of the excitatory amino
acid neurotransmitter glutamate starting from a Garner’s aldehyde-derived alkyne. The deprotected
cyclopropene glutamate was stable in solution but decomposed upon concentration. Appending a
light-cleavable group improved the stability of the cyclopropene while simultaneously caging the neuro-
transmitter. This strategy has the potential to permit deployment of cyclopropene-modified glutamate as
a bioorthogonal probe of the neurotransmitter glutamate in vivo with spatiotemporal precision.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 5 October 2016
Revised 24 October 2016
Accepted 27 October 2016
Available online 11 November 2016
Keywords:
Cyclopropene
Bioorthogonal
Glutamate
Neurotransmitter
Tetrazine ligation
Cyclopropenes have become popular biorthogonal functional
groups due to their small size, inertness to biological functionality,
and reactivity with tetrazines1 and 1,3-dipoles.2 For example,
cyclopropene installation on monosaccharides3–6 and lipids7 has
promoted these biomolecules’ characterization in challenging
in vivo environs, and their use as chemical handles in proteomics
applications has facilitated analysis of proteomes with improved
spatiotemporal precision.8–10 Additionally, in efforts to employ
cyclopropene tags in studies of protein function, several groups
have synthesized amino acid analogs bearing a cyclopropene moi-
ety at the alpha carbon11 or terminal positions on amino acid side
chains, resulting in novel non-proteinogenic amino acid struc-
tures.2 However, there has been little exploration of cyclopropenes
and bio-orthogonal chemistry to analyze neurotransmitters, an
important and diverse class of biomolecules that modulate neuron
excitation and inhibition to control brain function.
analogs synthesized.13 Several of these structures were confirmed
as substrates for metabotropic glutamate receptors,14,15 which
detect glutamate release from synaptic vesicles at the neural
synapse to propagate neural signals, as well as glutamate trans-
porters that package glutamate into synaptic vesicles or remove
it from the synaptic cleft.13 These findings suggest that cyclo-
propene modified analogs of glutamate might serve as chemically
traceable neurotransmitter mimics. Here we describe the synthesis
of a light-activatable cyclopropene-containing glutamate analog
(Nvoc-cpGlu, Compound 1, Fig. 1), which holds promise to extend
the reach of biorthogonal chemistry into the realm of neurotrans-
mitter biology.
Initially, our strategy for the synthesis of cyclopropene gluta-
mate analogs employed Rh-catalyzed cyclopropenation of ethyl
diazoacetate to protected alkynyl glycine analogs (3a/b, Scheme 1).
Interestingly, our attempts at the preparation of an N-benzoyl alky-
The only documented synthesis of a cyclopropene-containing
neurotransmitter was performed by Reissig and coworkers, who
synthesized cyclopropene-containing analogs of the neurotrans-
mitter gamma-aminobutyric acid (GABA).12 However, there are
numerous neurotransmitters that have structures conducive to
modification with a reactive cyclopropene handle. For example,
glutamate, the subject of this study and an amino acid neurotrans-
mitter that leads to neuron excitation in most situations, has had a
complete panel of saturated, unreactive cyclopropane-containing
nyl glycine by addition of a tributyl tin acetylene to a-chloro-
glycine 2b according to Aldous and coworkers16,17 did not result
in the desired alkyne 3b. Instead, we observed a rapid cycloisomer-
ization-elimination affording oxazole 4b, similar to that reported
for other N-propargyl amides by Ciufolini and coworkers.18 Swap-
ping the amine protecting group from benzoyl to methyl carba-
mate, compound 2a, allowed the formation of the required
alkynyl glycine 3a. Subsequent Rh-catalyzed cyclopropenation of
ethyl diazoacetate produced a reaction mixture whose analysis
by mass spectrometry suggested the formation of cyclopropene
4a. However, our extensive efforts to purify the cyclopropene were
ultimately unsuccessful. The purification challenge resulted from
⇑
Corresponding author.
E-mail address: scott.laughlin@stonybrook.edu (S.T. Laughlin).
http://dx.doi.org/10.1016/j.tetlet.2016.10.106
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

P. Kumar et al. / Tetrahedron Letters 57 (2016) 5750–5752
5751
Fig. 1. Target photo-activatable cyclopropene containing analog of the excitatory
neurotransmitter glutamate.
Scheme 2. Accessing the cyclopropene via a Garner’s aldehyde derived synthon.
by reinstallation of the Boc protecting group proceeded in quanti-
tative yield.
The subsequent oxidation of 7a is a key step in the synthesis of
these cyclopropene-containing glutamate analogs, considering
that oxidations on cyclopropene scaffolds are not well precedented
in the literature. Thus, we sought an effective oxidation strategy for
this scaffold (See conditions tested in Table 1). Our literature
search found successful oxidations of cyclopropene alcohols using
Dess-Martin periodinane (DMP).20 Consistent with these reports,
our attempts to oxidize alcohol 7a with DMP did produce an alde-
hyde. However, DMP oxidation also resulted in the opening of the
cyclopropene ring and formation of an allene at the alpha carbon
(S4 in SI), and our efforts to oxidize this aldehyde to the carboxylic
acid using Pinnick oxidation conditions were unsuccessful.
Another report employed a TEMPO-based oxidation to transform
alcohol to carboxylic acid in a cyclopropenone-containing fatty
acid analog.21 However, multiple efforts to employ TEMPO or
4-acetamido-TEMPO oxidations on cyclopropene 7a were unsuc-
cessful. Chromium based oxidation gave the desired product in
very low yields and produced complex reaction mixtures that pre-
cluded satisfactory purification. Thankfully, periodic acid-based
oxidations with sub-stoichiometric amounts of Chromium, first
described by Reider and coworkers,22 produced the desired pro-
duct with few apparent side products, although in poor yield.
Unfortunately, attempts at ester removal at this stage were unsuc-
cessful, presumably due to deprotonation of the now acidic alpha
carbon proton and subsequent decomposition.
Scheme 1. Efforts to synthesize cpGlu via a alkynyl glycine were unsuccessful.
unreacted alkynyl glycine starting material in the reaction mixture.
This alkynyl glycine is notoriously unstable by virtue of its propen-
sity to form allenes.19 During our attempts at separation via SiO2
Flash chromatography, reversed phase HPLC, and distillation, the
remaining alkynyl glycine in the reaction mixture decomposed
into multiple species that coeluted with the product and precluded
its purification.
We reasoned that avoiding the alkynyl glycine in our synthetic
strategy would be possible if we performed the ethyl diazoacetate
cyclopropenation to alcohol substrates instead of carboxylic acids,
thereby lowering the acidity of the alpha carbon proton and
inhibiting allene formation (Table 1). Alkyne 5, derived from Gar-
ner’s aldehyde, provided an ideal chiral building block for this
strategy. The addition of ethyl diazoacetate to alkyne 5 proceeded
in good yield (55–60%, 85% when calculated based on recovered
starting material) and opening the dimethyloxazolidine followed
Conversely, removing the ester prior to opening the dimethy-
loxazolidine proceeded in excellent yield (9, 90–94%, Scheme 2),
and ester deprotection after ring opening proceeded in slightly
Table 1
Oxidation conditions for accessing cpGlu via a Garner’s aldehyde derived alkyne.
Entry
Oxidant
Solvent
Temp. (°C)
Time
Yield (%)a
Comment
1
2
3
4
5
6
7
H5IO6, CrO3 (cat.)
Dess-Martin periodinane
Jones (CrO3, H2SO4)
Pyridinium dichromate
TEMPO, NaClO2, NaOCl
Acetamido TEMPO, NaOCl
Ruthenium Cl, NaIO4
MeCN, H2O
DCM
Acetone
0
rt
0 ? rt
0
35
rt
rt
20 min
15 min
20 min
3 d
120 min
240 min
4 d
16
5
<5
0
0
0
Decomposed during later NaClO2 oxidation
Unable to purify to >95%
No reaction
No reaction
Decomposition
DMF
MeCN, PBS
MeCN, H2O
CCl4, MeCN, H2O
0
No reaction
a
Isolated yields.

5752
P. Kumar et al. / Tetrahedron Letters 57 (2016) 5750–5752
group, which promotes stability for storage and characterization
as well as precise spatiotemporal control of the molecule’s deploy-
ment in vivo. When coupled with pro-fluorescent tetrazine7 or
tetrazole32,33 reaction partners, these glutamate mimics have the
potential to reveal the anatomy of glutamatergic vesicles in cul-
tured neurons or small transparent models such as drosophila or
zebrafish to promote a more detailed understanding of brain func-
tion. Additionally, the glutamate analogs we described add to the
list of cyclopropene-containing amino acids that could serve as
monomers in the in vitro synthesis of unnatural peptides.
Acknowledgments
We thank Alyssa Preston and Sining Li for helpful discussions
and critical reading of the manuscript, Dr. Bela Ruzsicska and the
Stony Brook University Institute for Chemical Biology and Drug
Discovery for Mass Spectrometry resources and analyses, Dr. James
Marecek and Francis Picart for NMR analysis. This material is based
upon work supported by the National Science Foundation under
CHE – 1451366.
Scheme 3. Generation of a photoactivatable cyclopropene-containing analog of
glutamate.
lower yield but enabled separation of diastereomers via Flash chro-
matography (7b/c, Scheme 3). Oxidation of alcohol 10 using the
previously identified periodic acid/Jones reagent mixture pro-
ceeded in poor yield to produce the penultimate compound S5.
Removal of the Boc group to produce 11 proceeded as expected
based on mass spectrometry analysis of the reaction mixture,
and purification via HPLC was uneventful. However, this molecule
decomposed when concentrated and could not be satisfactorily
characterized. This result is consistent with our and others’ obser-
vations of cyclopropene decomposition upon concentration, likely
through polymerization.7,23,24
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.10.
106.
Interestingly, we noted that deprotected 11 remained present in
crude reaction mixtures after workup and HPLC fractions after
purification, suggesting that it is stable for days in aqueous solu-
tions. Additionally, we observed that Boc-protected compound S5
was stable in solution and after concentration. These observations
suggested a mechanism for deploying cpGlu using a light-activated
prodrug strategy. Essentially, the cpGlu analog could be endowed
with a light cleavable moiety that would facilitate characterization
and storage, while also permitting precise spatiotemporal control
of its release in solution. Similar strategies have been used for con-
trolling the release of natural neurotransmitters, including peptide
neurotransmitters,25 glutamate,26–28 GABA,28,29 dopamine,30 and
serotonin.31
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In conclusion, neurotransmitters are a class of biomolecules
whose analysis in vivo would benefit from bio-orthogonal
approaches, but they have been largely neglected. We have synthe-
sized a cyclopropene-containing analog of the excitatory neuro-
transmitter glutamate with a pendant photocleavable protecting