Stable cyclopropene-containing analogs of the amino acid neurotransmitter glutamate

Article abstract of DOI:10.1016/j.tetlet.2019.04.046

As a neurotransmitter, the amino acid glutamate has been the subject of efforts to generate structural analogs with unique properties. Here we report a practical, half-gram synthesis of two cyclopropene-containing glutamate analogs. These analogs are stable in solution, in the presence of the biological nucleophile glutathione, upon concentration, and during long-term storage, while maintaining their amenability to photo- or enzyme-caging and reactivity with bioorthogonal reaction partners like s-tetrazine or light-activated tetrazoles.

Full text of DOI:10.1016/j.tetlet.2019.04.046

Accepted Manuscript
Stable cyclopropene-containing analogs of the amino acid neurotransmitter glu-
tamate
Pratik Kumar, Wei Huang, David Shukhman, Frank M. Camarda, Scott T.
Laughlin
PII:
S0040-4039(19)30406-X
https://doi.org/10.1016/j.tetlet.2019.04.046
TETL 50761
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 March 2019
25 April 2019
26 April 2019
Please cite this article as: Kumar, P., Huang, W., Shukhman, D., Camarda, F.M., Laughlin, S.T., Stable
cyclopropene-containing analogs of the amino acid neurotransmitter glutamate, Tetrahedron Letters (2019), doi:
https://doi.org/10.1016/j.tetlet.2019.04.046
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Pratik Kumar, Wei Huang, David Shukhman, Frank M. Camarda, and Scott T. Laughlin

1
Tetrahedron Letters
Stable cyclopropene-containing analogs of the amino acid neurotransmitter glutamate
Pratik Kumar, Wei Huang, David Shukhman1, Frank M. Camarda, and Scott T. Laughlin
Department of Chemistry, Stony Brook University, Stony Brook, NY, 11790, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
As a neurotransmitter, the amino acid glutamate has been the subject of efforts to generate
structural analogs with unique properties. Here we report a practical, half-gram synthesis of two
cyclopropene-containing glutamate analogs. These analogs are stable in solution, in the presence
of the biological nucleophile glutathione, upon concentration, and during long-term storage, while
maintaining their amenability to photo- or enzyme-caging and reactivity with bioorthogonal
reaction partners like s-tetrazine or light-activated tetrazoles.
Available online
Keywords:
Cyclopropene
2019 Elsevier Ltd. All rights reserved.
Glutamate
Bioorthogonal Chemistry
Neurotransmitter
Cyclopropenes have become
a popular choice as a
bioorthogonal reagent due to their small size, inertness to cellular
nucleophiles, and ability to be genetically encoded.1–3 They are
employed as bioorthogonal reaction partners for 1,3-dipoles,4
tetrazines,5,6 1,2,4-triazines,7 and o-quinones.8 Their small size
allows minimum perturbation of the biological system under
study; therefore, they have been appended to a number of
biomolecules (e.g., glycans,9 proteins,10 nucleic acids,11 and
lipids5,12). Recently, photocaged cyclopropenes that allow
spatiotemporal control over their ligations with tetrazines have
been reported.13–15 Additionally, cyclopropenes are important
synthetic intermediates16,17 and find use for polymerization18 and
unnatural amino acid synthesis.19 Surprisingly, neurotransmitters,
an important class of biomolecules, have been largely excluded
from the advances made by biorthogonal reagents. In this report,
we sought to generate cyclopropene-bearing glutamate
neurotransmitters that are stable and straightforward to synthesize.
Fig. 1. Pharmacologically active synthetic analogs of the amino
acid neurotransmitter glutamate.
aminocyclopentane-trans-1,3-dicarboxylate (ACPD) were used
for studying the pharmacology of the metabotropic glutamate
receptors (Fig. 1).20–22 More recently, conformationally restricted
analogs based on cyclopropanes such as L-CCG or DCG have
been identified, which, interestingly, possess biological activity as
each diastereomer.23,24 The high biological activity of a structurally
diverse array of cyclopropane-based glutamate analogs intrigued
us, and, in part, inspired us to develop cyclopropene-containing
glutamate analogs. Such cyclopropene-containing glutamate
analogs will be conformationally restricted, like their
cyclopropane counterparts, but they will have the additional
advantage of acting as reactants for bioorthogonal chemistry
We focused on the primarily excitatory amino acid
neurotransmitter glutamate, which is released from the pre-
synaptic nerve terminals and binds to the metabotropic or
ionotropic glutamate receptors at the post-synaptic sites in the
brain. Synthetic glutamate analogs have helped improve
understanding of the pharmacology of glutamate-receptor binding,
glutamate receptor function and diversity, and the mechanism of
glutamate recycling. For example, N-Methyl-D-aspartate
(NMDA), kainic acid (KA), and 2-amino-(3-hydroxyl-5-methyl-
4-isoxazol-4-yl) propionic acid (AMPA) were critical for
identifying and distinguishing the ionotropic glutamate receptors,
whereas L-2-amino-4-phosphonobutyrate (L-AP4) and 1-
———
 Corresponding author.
E-mail address: scott.laughlin@stonybrook.edu (S. T. Laughlin).
1 Current address: NYIT College of Osteopathic Medicine, Glen Head, NY, 11545, United States.

2
Tetrahedron
Scheme 1. Synthesis of the cyclopropene-containing glutamate
analog with a methylene spacer.
Fig. 2. Improving the stability of cyclopropene-glutamates by
manipulating the acidity of α-proton. (A) First-generation
cyclopropene-glutamate cpGlu is stable in solution but
decomposes upon concentration. Photocaged cyclopropene-
glutamate Nvoc-cpGlu is stable upon concentration and allows
spatitemporal generation of cpGlu upon light-illumination. (B)
Decreasing the acidity of α-proton or substituting it with a methyl
group significantly increases the stability, yield, and synthetic-
4
through Boc2O to obtain the alkyne 1 ([α]20D observed = + 49.7 °
(c 7.88, DCM) similar to previously reported value36) in 93% yield
over two steps (Scheme 1). Rhodium-catalyzed cyclopropenation
of alkyne 1 using ethyl diazoacetate via a syringe pump afforded
mixed-ester N,O-protected cyclopropene 2 as an inseparable
mixture of diastereomers (diastereomeric ratio (dr) = 87:13, Fig.
S1 in the ESI) in a yield of 44% (69% based on starting material
recovery (bsrm)). We found the cyclopropene 2 to be stable to both
silica-gel chromatography and long-term storage. Base-mediated
hydrolysis of mixed-ester 2 in methanol afforded free-acid 3 in
88% yield, and acid mediated Boc-deprotection of 3 afforded 4 in
81% yield. Unlike the first generation, the addition of a methylene
spacer makes the cyclopropene-analog 4 stable to concentration
under reduced pressure. In our hands, we have obtained up to ~0.6
g of 4 (as mixture of inseparable diastereomers) from one reaction
sequence. We determined through a HPLC assay that 4 (5 mM) is
stable as a solution in PBS (pH 7.4) at rt for at least 24 h (Fig. S2
in the ESI). Similarly, using an NMR based assay, we determined
that 4 (10 mM) is also stable at rt for at least 24 h in the presence
of the biological nucleophile L-glutathione up to 10 mM in PBS,
the highest physiologically-relevant concentration37 (Fig. S3 in the
ESI).
scale of second-generation of cyclopropene-glutamates
4 and 11.
through deployment of pro-fluorescent tetrazines25 or tetrazoles.26
Additionally, cyclopropene-glutamates can act as substrates for
unnatural peptide synthesis.
We recently reported the first cyclopropene-analog of the
amino acid neurotransmitter glutamate (Fig. 2A).27 This first-
generation cyclopropene-glutamate expanded the only other
documented report of
a
cyclopropene-neurotransmitter
(cyclopropene-GABA analog by Reissig and coworkers).28 We
obtained this cyclopropene-glutamate, in ten steps, via the
rhodium catalyzed cyclopropenation of a Garner’s aldehyde29
derived alkyne. The decomposition of alkynyl glycine on silica-
gel prohibited the synthesis of cpGlu via a relatively shorter
synthetic route. Unfortunately, the synthesis of cpGlu was tedious,
very low yielding, and the free-amine free-acid analog cpGlu is
only stable in solution as it decomposes to unknown products upon
concentration. Photocaging the amino group to obtain Nvoc-
cpGlu allowed us to circumvent the instability issues of cpGlu
with the added benefit of spatiotemporal control over the
generation of cpGlu, in solution, via non-intrusive light-
illumination. Similar photoactivation strategies are also available
for neurotransmitters such as GABA,30 glutamate,31 and
dopamine.32
Buoyed by the stability of the methylene-spacer cyclopropene-
glutamate analog, we hypothesized that another way bypass α-
proton-induced instability would be to replace it with a methyl
substituent (Scheme 2). Hence, we sought to synthesize the
corresponding alkyne 8 to obtain the cyclopropene 9. The
synthesis of 11 started with commercially available racemic 2-
methyl-DL-serine, which was treated under esterification
conditions with thionyl chloride and methanol followed by Boc-
protection of the amine to obtain 5 in 81% yield. The primary
alcohol 5 was then partially oxidized to the corresponding
aldehyde 6, using Dess-Martin reagent, in 87% yield. Our initial
attempt to utilize the Corey-Fuchs reaction to convert the aldehyde
6 to the terminal alkyne 8 via the dibromo intermediate 7 was
One hypothesis for the decomposition of cpGlu is that it
polymerizes or rearranges to an allene or an alkyne, as observed
for other cyclopropenes,33–35 due to the acidic α-proton (α
corresponding to NH2). Here, we report that decreasing the acidity
of the α-proton by inserting an additional methylene spacer
between the cyclopropene-C1 and α-carbon, or by substituting the
α-proton to an α-methyl significantly improves the stability of both
the starting material alkynes and corresponding cyclopropene-
glutamates to afford a second-generation of cyclopropene-analogs
of glutamate (Fig. 2B). We also show that such tweaking also
stabilizes the corresponding starting material alkynes to silica-gel;
thereby allowing cyclopropene-glutamate synthesis in four (for
methylene-spacer, 4) or six (for methyl-substituent, 11) steps
compared to ten steps for the first-generation. Lastly, we show that
second-generation cyclopropene-glutamates 4 and 11 can be
synthesized in at least ~0.5 g scale quantities which is significantly
higher (>100-folds) than the first-generation cyclopropene-
glutamate cpGlu.
We started our synthesis with the low-cost, commercially
available L-propargylglycine ([α]20 observed = + 24.9 ° (c 7.88,
D
DCM) matches with commercially reported values). We converted
the acid functionality to the corresponding methyl ester using
thionyl chloride and methanol followed by amine-protection
Scheme 2. Synthesis of the cyclopropene-containing glutamate
analog
9 with methyl substitution.

3
unsuccessful (<5% yield). We found the generation of dibromo
species 7, through the ylide from PPh3/CBr4/Et3N, to be <5% at
both 0.2 g and 1.4 g scale reaction. However, stirring the aldehyde
6 with K2CO3 in the presence of Bestmann–Ohira reagent afforded
saturation at around 120 mins. We also observed the TFA
(cyclopropene 11 was used as a TFA salt) and tetrazole ligation
product (Rt = 21 mins). Such carboxylic acid initialized tetrazole
ligation are known and has been utilized for protein ligation.39–41
the silica-gel stable terminal alkyne
8 in 78% yield.
To conclude, we have synthesized the second generation of
cyclopropene-analogs of the amino acid neurotransmitter
glutamate. They are stable in solution, on concentration, and in the
presence of high concentrations of the biological nucleophile L-
glutathione. Compared to the first generation of cyclopropene-
glutamate, inserting a methylene spacer or substituting the α-
proton with a methyl group significantly improves the stability of
cyclopropene-glutamates. This synthetic adjustment also makes
the corresponding starting material alkynes stable to silica-gel;
thereby, reducing the ten steps required for the first-generation
cyclopropene-glutamate synthesis to either four or six steps for the
second-generation. Overall, these improvements combined,
allowed us to obtain the cyclopropene-glutamate 4 and 11 in ~ 0.5
g scale quantities which is >100-fold higher than the first-
generation. Lastly, it opens the possibility of exploring additional
substituents at the α-position, e.g., fluorine, trifluoromethyl, to
study their biological properties. Future efforts in the lab are
dedicated to obtaining isolated diastereomers of the cyclopropene-
Cyclopropenation of the alkyne 8 by rhodium catalyzed addition
of ethyl diazoacetate via a syringe pump afforded the silica-gel
stable mixed-ester N,O-protected cyclopropene 9 as an inseparable
mixture of diastereomers (two major and two minor, Fig. S4 in the
ESI) in moderate yield of 29% (59% bsmr). Hydrolysis of mixed-
ester 9 under basic conditions in methanol afforded the stable free-
acid Boc-cyclopropene 10 in 84% yield. Finally, acid mediated
Boc-deprotection of 10 provided cyclopropene 11 in 80% yield as
a mixture of diastereomers. In our hands, we have obtained up to
~0.45 g of 4 from one reaction sequence. Like the cyclopropene-
analog 4, substitution of original α-proton to a methyl group also
makes this cyclopropene-glutamate analog 11 stable to
concentration, unlike the previous generation of cyclopropene-
glutamate analog.27 Like the cyclopropene-glutamate analog 4, we
determined, using an NMR-based assay, that 11 (10 mM) is also
stable to biological nucleophile reduced L-glutathione (10 mM) in
PBS at rt for at least 24 h (Fig. S5 in the ESI).
With these cyclopropene-glutamate derivatives in hand, we
tested their ability to ligate with popular bioorthogonal reagents
(Fig 3). First, we tested their ability to ligate with a tetrazine (3,6-
Di-2-pyridyl-1,2,4,5-tetrazine) via an inverse electron demand
Diels-Alder reaction by monitoring the characteristic tetrazine
absorbance peak at 520 nm. The ligation rate (second order rate
constant, k2) between Boc- and ester-protected 2 with is 0.0001 M-
1s-1 in MeCN (Fig. S6 in the ESI) which improves by 20-fold (k2 =
0.002 M-1s-1 1:1 PBS/MeOH) for cyclopropene-amino acid 4 (Fig.
S7 in the ESI) possibly due to both a relief in steric strain and
diminished electron-withdrawing ability of carboxylate relative to
the ethyl ester. Such an effect of cyclopropene-C3 substituent’s
electron-withdrawing nature by inductive effect is known.1,38
Next, we subjected the sterically strained (at C1) cyclopropene 9
to tetrazine ligation; however, we could not detect any measurable
ligation between cyclopropenes 9 (or 11) (Fig. S8–S9 in the ESI).
This suggests that inserting methylene units between the
cyclopropene-C1 and substituents at C1 improves the tetrazine
ligation.
glutamates by generating enantiomerically pure alkyne 842,43
,
resolving diastereomers at cyclopropne-C3 using a chiral reagent44
or testing the efficacy of chiral rhodium catalyst45 for mono-
substituted (at C3) cyclopropenes.
Acknowledgments
We thank Dr. Bela Ruzsicska, Director of the analytical
instrumentation laboratory, Stony Brook University, and the Stony
Brook University Institute for Chemical Biology and Drug
Discovery for maintaining and acquiring high-resolution mass
spectrometry data. We thank Stony Brook University NMR
coordinators Francis Picart, Dr. James Marecek and Dr. Fang Liu
for maintaining the NMR facility. We thank Prof. Nicole S.
Sampson for access to the plate-reader for conducting kinetic
experiments, and graduate student Wang Yao in the lab of Prof.
Ming-Yu Ngai for carrying out chiral HPLC measurements.
Lastly, we thank John A. Mannone for assistance with this work.
This work was supported by a grant to S.T.L. from the National
Science Foundation 1451366.
On the other hand, photoclick chemistry presents an alternative
to utilize strained cyclopropenes such as 11 for bioorthogonal
applications.26 For this, we synthesized the 2-(4-methoxyphenyl)-
5-phenyl-2H-tetrazole and assayed its light-dependent ligation
with cyclopropene 11 using an HPLC/MS assay (Fig. S10–12 in
the ESI). We observed the expected ligation product (Rt ~18 mins),
as a mixture of diastereomers, between the cyclopropene-
glutamate 11 and the tetrazole in the first 15 mins, which reached
Supplementary Material
Supplementary data associated with this article can be found, in
the online version, at
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5
Highlights
. Applying bioorthogonal chemistry to
neuroscience with cyclopropene bearing
neurotransmitter analogs
. Shorter and half-gram scale synthesis of
cyclopropene-analogs of glutamate
. Generation of novel forms of structurally
constrained glutamate analogs