Design, synthesis, and biological activity of novel triazole amino acids used to probe binding interactions between ligand and neutral amino acid transport protein SN1

Article abstract of DOI:10.1016/j.bmcl.2007.05.061

Novel triazole amino acids were synthesized as probes to investigate ligand-protein binding interactions of the neutral amino acid transporter SN1. The bonding hypothesis to be tested was that the side chains of endogenous substrates are acting as H-bond acceptors. Although limited inhibition of ³H-l-glutamine uptake by SN1 expressing oocytes was observed, the synthetic compounds show a trend that suggests a hydrogen bond interaction just outside the endogenous ligand binding pocket.

Full text of DOI:10.1016/j.bmcl.2007.05.061

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4163–4166
Design, synthesis, and biological activity of novel triazole
amino acids used to probe binding interactions between ligand
and neutral amino acid transport protein SN1
Mariusz Gajewski, Ben Seaver and C. Sean Esslinger*
NIH-COBRE Center for Structural and Functional Neuroscience, Department of Pharmaceutical and Biomedical Sciences,
The University of Montana, Missoula, MT 59812, USA
Received 10 March 2007; revised 15 May 2007; accepted 17 May 2007
Available online 23 May 2007
Abstract—Novel triazole amino acids were synthesized as probes to investigate ligand–protein binding interactions of the neutral
amino acid transporter SN1. The bonding hypothesis to be tested was that the side chains of endogenous substrates are acting
3
as H-bond acceptors. Although limited inhibition of H-L-glutamine uptake by SN1 expressing oocytes was observed, the synthetic
compounds show a trend that suggests a hydrogen bond interaction just outside the endogenous ligand binding pocket.
ꢀ
2007 Elsevier Ltd. All rights reserved.
L-Glutamine is the most abundant free amino acid in
1
throughout the body. It is this glutamine movement in
which we are interested, and particularly transporter
protein facilitated movement.
mammalian blood plasma and cerebral spinal fluid.
As such an abundant bio-molecule, L-glutamine is
2
involved in a variety of metabolic processes. L-Gluta-
3
,4
mine is a major component of muscle, and in times
of post-surgical conditions has been classified as a ‘con-
ditionally essential’ amino acid because of the increased
Neutral amino acid transporter proteins are responsible
for facilitating movement of these highly polar solutes
across the lipophilic cellular membrane. A number of
neutral amino acid transport systems have been identi-
fied, the main transport systems being the sodium
dependent systems A, ASC, N, and the sodium-indepen-
5
metabolic requirements. As a precursor to TCA cycle
intermediates, glutamine is an important source of en-
6
,7
ergy, as well as being an upstream precursor to the
natural reducing agent glutathione, and is thus closely
1
6–18
dent system L.
As these transport systems are
8
related to the redox status of the cell. Glutamine acts
9
responsible for the translocation of the neutral amino
acids (16 of the 20 common mammalian amino acids),
one may suspect there is considerable overlap of sub-
strate specificity. One common thread of these transport
systems is that they all transport L-glutamine.
as a vehicle for both carbon and nitrogen shuttling.
This systemic glutamine shuttling, referred to as the glu-
3
tamine cycle, serves as an excretion route for toxic
0
1
ammonia through complimentary mechanisms bal-
1
1
anced for proper pH buffering of the blood. Glutamine
also acts in a signaling role for a number of biological
Our interest involves the system N transport protein
SN1. This neutral amino acid transporter is found in
1
2
pathways, such as low glutamine concentrations asso-
ciated with a more facile initiation of the apoptotic cas-
1
9
hepatocytes, muscle, and brain. In the brain, the
SN1 transporter is located on astrocytes and is believed
to be responsible for glutamine efflux from astrocytes as
part of the glutamine/glutamate cycle for recycling the
1
3
cade. Glutamine is also accepted as being a major
metabolic precursor for the excitatory neurotransmitter
1
4
L-glutamate
5
and the inhibitory neurotransmitter
GABA. In order for glutamine to participate in all
of these roles, this small polar molecule must move
1
20
neurotransmitter glutamate. Although much has been
discovered about the SN1 transport protein, such as
2
1
22
ionic requirements,
electrogenic behavior,
and
2
3
reversibility, little is known about ligand–protein bind-
ing interactions. It is our goal to elucidate these ligand–
protein binding interactions and develop an accurate
Keywords: L-Glutamine; Amide mimic; H-bond acceptor.
*
Corresponding author. Tel.: +1 406 243 4713; fax: +1 406 243
228; e-mail: Christopher.esslinger@umontana.edu
5
0
960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.061

4164
M. Gajewski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4163–4166
binding-model pharmacophore (a model mapping out in
three dimensions the locations of attractive and repul-
sive ligand–protein interactions). Once established, an
accurate binding-model pharmacophore can be used to
develop pharmacological tools for the SN1 transporter
protein. Currently there are no selective inhibitors for
the SN1 transporter.
The SN1 transport protein has three natural substrates:
1
L-glutamine, L-asparagine, and L-histidine (Fig. 1).
9
One possible alignment for these three substrates,
explaining how they interact with the transport protein,
incorporates the side chain acting as a hydrogen-bond
acceptor within the ligand binding site. This proposed
pharmacophore is illustrated in Figure 2, showing the
amide carbonyl oxygen of both L-glutamine and
L-asparagine as well as the non-protonated nitrogen of
L-histidine occupying a similar three-dimensional space
while acting as the hydrogen-bond acceptor.
Figure 2. Overlay of energy-minimized structures of L-glutamine
pink), L-asparagine (green), and L-histidine (yellow) with amide
(
carbonyl (red) or imidazole nitrogen (blue) in similar 3-D space able to
act as a hydrogen-bond acceptor.
In order to test this hydrogen-bond acceptor hypothesis,
the triazole functionality was proposed. The 1,2,3-tria-
zole functionality offers a hydrolysis-stable amide
replacement containing a hydrogen-bond acceptor fit-
ting the alignment above. By incorporating the click
methodology in the synthesis of the 1,2,3-triazoles, the
OH
5
tion with a variety of substituents that may offer addi-
tional insight into the binding pocket properties. The
panel of target compounds designed is shown below
-membered ring can easily be substituted at the 4-posi-
N
N
N
N
N
2
4
N
N
N
N
H N
CO2
H N
CO2
(
Fig. 3.)
3
3
H N
3
CO2
O
1
2
3
Starting with L-serine, the amino and acid functional-
ities are protected as the N-Boc benzylester, respectively.
The benzylating agent 7 (IDC-benzyl alcohol adduct)
O
O
OH
OCH3
NH2
2
5
was used for the ester formation rather than more
standard methods such as acidic alcohol or acid chloride
intermediate in order to minimize serine lactone forma-
tion. Conversion of the hydroxyl to the azide under
N
N
N
N
N
N
N
N
N
H N
CO2
H N
CO2
H N
CO2
3
3
3
2
6
4
5
6
Mitsunobu conditions affords the common azide inter-
mediate for the click triazole formation. Reacting the
chiral azide with the desired acetylene in the presence
of Cu(II) and sodium ascorbate in water/tert-butanol
solvent yielded the corresponding 1,2,3-triazoles in high
Figure 3. Panel of target triazole amino acids for testing as inhibitors
3
of H-L-glutamine uptake by the neutral amino acid transporter SN1.
2
7
yields. Subsequent deprotection via refluxing 6 N HCl
or hydrogenation followed by TFA treatment with sub-
sequent HCl/TFA exchange gave the target compounds
expressing the transporter. Functional expression was
achieved by injecting mRNA encoding for the rSN1
transporter (GenBank Accession No. NM_145776,
Rattus norvegicus SLC38A3) into oocytes, followed by
a four-day period to allow expression. Synthetic com-
2
7
as the HCl salt (Scheme 1).
The compounds were tested as inhibitors of L-glutamine
uptake by the rat SN1 transporter by competition assays
with H-L-glutamine using Xenopus oocytes functionally
3
pounds were tested at 1 mM ( H-L-glutamine at
3
100 lM) at a buffer pH of 8.0 and compared to control
uptake containing no compound. Background levels
were subtracted (identical conditions using un-injected
oocytes) for each data point. For confirmation that
the assay was functioning properly, both the negative
control, L-alanine, and the positive control, L-histidine,
were included in all assays.
O
NH2
O
N
NH
NH2
H N
CO2
H N
CO2
H3N
CO2
3
3
As illustrated in Figure 4 below, the panel of triazole
amino acids exhibited limited inhibition of H-L-gluta-
L-Glutamine
L-Asparagine
L-Histidine
3
mine uptake into SN1 expressing oocytes. Uptake in
the presence of compounds 1–6 was not significantly dif-
Figure 1. Endogenous substrates of the neutral amino acid transporter
SN1.

M. Gajewski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4163–4166
4165
OH
OH
have discovered a hydrogen-bond interaction with pro-
tein or protein-bound water just outside the endogenous
ligand binding site that may offer future possibilities.
Boc O, Et N
2
3
iPrOH/H O
2
H N
3
CO2
BocHN
CO H
2
73% yield
L-Serine
Although the panel of triazole amino acids is essen-
tially inactive at the SN1 transporter protein, there
are a number of other glutamine utilizing proteins
with which these compounds may interact signifi-
cantly. Our laboratories are currently continuing to
explore the binding requirements for the SN1 trans-
porter protein and pursuing active hydrolysis-stable
amide mimics.
H
N
Ph P, DEAD,
O
3
OH
CO Bn
N
7
HN THF
3
o
-
78 C - RT
BocHN
8
8% yield
2
78% yield
R
R, CuSO ,
N3
4
Sodium ascorbate
N
BuOH/H O
N
2
N
BocHN
CO Bn
2
4
6-99% yield
8
6
Acknowledgments
BocHN
CO Bn
2
R
We give special thanks to Professor Robert H. Edwards
for contributing the SN1 clone, Frederick J. Rhoderick
for his sub-cloning work, Professor Michael P. Kava-
naugh for his oocyte plasmid, Professor Richard
Bridges, Director of the Center for Structural and Func-
tional Neuroscience, and the Department of Chemistry,
The University of Montana and NSF for the use of the
NMR instrument. This research was funded by NIH
NINDS NS045704 (C.S.E.) and NIH NCRR P20
RR15583 (M.G.).
N HCl
or
N
1
2
) H Pd/C
N
N
2
HCl
) TFA/HCl
8
2% - quant.
H N
CO H
2
2
yield
1-6
Scheme 1. Synthesis of substituted triazoles starting with L-serine. R-
groups are shown in Figure 3.
Glutamine Uptake Inhibition of SN1 Expressing Oocytes
1
1
1
40
20
00
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.05.061.
8
6
4
2
0
0
0
0
0
References and notes
1
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987, 12, 135.
2. J. Nutr. 2001, 131, 2447S.
1
Control
1
2
3
4
5
6
His
Ala
Compound
3. Rennie, M. J.; Hundal, H. S.; Babij, P.; MacLennan, P.;
Taylor, P. M.; Watt, P. W.; Jepson, M. M.; Millward, D.
J. Lancet 1986, 2, 1008.
3
Figure 4. Inhibition of H-L-glutamine uptake of substituted triazoles
3
using mSN1 expressing oocytes. All [compounds] = 1 mM, [ H-L-
4
. Boelens, P. G.; Nijveldt, R. J.; Houdijk, A. P. J.; Meijer,
S.; Van Leeuwen, P. A. M. J. Nutr. 2001, 131, 2569S.
. Labow, B. I.; Souba, W. W. World J. Surg. 2001, 24, 1503.
. Chaudhry, F. A.; Reimer, R. J.; Krizaj, D.; Barber, D.;
Storm-Mathisen, J.; Copenhagen, D. R.; Edwards, R. H.
Cell 1999, 99, 769.
glutamine] = 100lM in Nd96 buffer at pH 8.0, incubation time = 10 -
min. Background using un-injected oocytes under same conditions was
subtracted. Each data point (n) is the average of five oocytes, n P 3,
reported as % of control ± SEM. Assay details are given in the
Supplementary material.
5
6
7
. Varoqui, H.; Zhu, H.; Yao, D.; Ming, H.; Erickson, J. D.
J. Biol. Chem. 2000, 275, 4049.
ferent from control values using one-way ANOVA. The
figure does show a trend by the phenyl substituted tria-
zole 3, the ester substituted triazole 5, and the amide
substituted triazole 6 that there may be a hydrogen-
bond occurring with these groups. These functional
groups, although too far removed to be glutamine amide
mimics, can all act as hydrogen bond acceptors.
8
. Mates, J. A.; Perez-Gomez, C.; Nunez de Castro, I.;
Asenjo, M.; Marquez, J. Int. J. Biochem. Cell Biol. 2002,
34, 439.
9. Bode, B. P.; Souba, W. W. J. Parenter. Enteral. Nutr.
999, 23, S33.
1
0. Haussinger, D. Metabolism 1989, 38, 14.
1. Bourke, E.; Haussinger, D. Contrib. Nephrol. 1992, 100,
5
1
1
8.
1
2. Curi, R.; Lagranha, C. J.; Doi, S. Q.; Sellitti, D. F.;
Procopio, J.; Pithon-Curi, T. C.; Corless, M.; Newsholme,
P. J. Cell. Physiol. 2005, 204, 392.
The results of this study suggest the binding of ligand to
the neutral amino acid transport protein SN1 requires
interactions other than the ligand acting only as a
hydrogen-bond acceptor on the amino acid side chain
functionality within the ligand binding site. We may
1
3. Fuchs, B. C.; Bode, B. P. J. Surg. Res. 2006, 131, 26.
14. Deitmer, J. W.; Broer, A.; Broer, S. J. Neurochem. 2003,
87, 127.

4166
M. Gajewski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4163–4166
1
5. Rae, C.; Hare, N.; Bubb, W. A.; McEwan, S. R.; Broer,
A.; McQuillan, J. A.; Balcar, V. J.; Conigrave, A. D.;
Broer, S. J. Neurochem. 2003, 85, 503.
6. Broer, S.; Brookes, N. J. Neurochem. 2001, 77, 705.
7. Chaudhry, F. A.; Schmitz, D.; Reimer, R. J.; Larsson, P.;
Gray, A. T.; Nicoll, R.; Kavanaugh, M.; Edwards, R. H.
J. Neurosci. 2002, 22, 62.
Compound 1: 1H NMR (400 MHz, deuterium oxide) d
ppm 8.10 (1H, s), 7.90 (1H, s), 5.01 (2H, dd, J = 9.5,
5.9 Hz), 4.61 (1H, dd, J = 9.5, 3.7 Hz). 13C NMR
(100 MHz, CD3OD), d ppm 167.27 (C), 134.97 (CH),
1
1
123.35 (CH), 52.19 (CH
for C : 157.0726, found: 157.0715 (6.7 ppm).
½aꢀ ꢁ 10 (0.070, H O).
2
), 50.42 (CH). HR-MS calculated
5
9 4 2
H N O
23
0
D
2
1
8. Bode, B. P. J. Nutr. 2001, 131, 2475S.
6
Compound 2: 1H NMR (400 MHz, DMSO-d ) delta ppm
1
9. Boulland, J. L.; Osen, K. K.; Levy, L. M.; Danbolt, N. C.;
Edwards, R. H.; Storm-Mathisen, J.; Chaudhry, F. A.
Eur. J. Neurosci. 2002, 15, 1615.
0. Chaudhry, F. A.; Reimer, R. J.; Edwards, R. H. J. Cell
Biol. 2002, 157, 349.
1. Br o¨ er, A.; Albers, A.; Setiawan, I.; Edwards, R. H.;
Chaudhry, F. A.; Lang, F.; Wagner, C. A.; Br o¨ er, S.
J. Physiol. 2002, 539, 3.
2. Chaudhry, F. A.; Krizaj, D.; Larsson, P.; Reimer, R. J.;
Werden, C.; Storm-Mathisen, J.; Copenhagen, D. R.;
Kavanaugh, M.; Edwards, R. H. EMBO J. 2001, 20, 7041.
3. Boulland, J. L.; Rafiki, A.; Levy, L. M.; Storm-Mathisen,
J.; Chaudhry, F. A. Glia 2005, 41, 260.
8.81 (1H, br. s), 8.02 (1H, s), 7.52, 7.39, 7.26 (1H, 3 · s),
4.90 (2H, m), 4.51 (1H, m), 4.49 (2H, s). 13C NMR
(100 MHz, DMSO-d
124.77 (CH), 55.63 (CH
11 4 3
MS calculated for C H N O
6
) d ppm 168.89 (C), 148.72 (C),
), 52.48 (CH ), 48.93 (CH). HR-
: 187.0831, found: 187.0840
(4.7 ppm). ½aꢀ ꢁ 6 (0.050, H O).
2
2
2
6
0
23
2
D
2
3
Compound 3: 1H NMR (400 MHz, CD OD) d ppm 8.25
(1H, s), 7.59–7.69 (2H, m), 7.21–7.29 (2H, m), 7.14–7.20
(1H, m), 4.90 (2H, d, J = 5.1 Hz), 4.55 (1H, t, J = 5.1 Hz).
2
13C NMR (100 MHz, DMSO-d ) d ppm 167.48 (C),
6
147.97 (C), 129.95 (CH), 128.89 (2 · CH), 128.54 (CH),
2
2
2
125.64 (2 · CH), 122.60 (C), 52.54 (CH
2
), 49.02 (CH).
HR-MS calculated for C H N O : 233.1039, found:
1
1
13
4
2
2
D
3
0
4. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem.,
Int. Ed. 2001, 40, 2004.
5. Mathias, L. J.; Fuller, W. D.; Nissen, D.; Goodman, M.
Macromolecules 1978, 11, 534.
6. Mitsunobu, O. Synthesis 1981, 1.
233.1049 (4.5 ppm).½aꢀ 20 (c 0.090, TFA).
Compound 4: 1H NMR (400 MHz, deuterium oxide) d
ppm 8.54, 8.43, 8.31 (1H, 3 · s; rotamers), 3.93–4.15 (1H,
m), 3.20–3.47 (2H, m). 13C NMR (100 MHz, DMSO-d ) d
6
2
ppm 168.77 (C), 162.22 (C), 140.34 (C), 131.09 (CH), 52.37
2
7. General procedure for Click chemistry Azide 8 480 mg
(
2 6 9 4 4
(CH ), 50.78 (CH). HR-MS calculated for C H N O :
23
0
1.5 mmol) and 1 equivalent of alkyne were dissolved in a
201.0624, found: 201.0623 (0.4 ppm). ½aꢀ 10 (0.100,
D
mixture of 1 ml t-BuOH and 0.5 ml water. With vigorous
stirring, sodium ascorbate (20 mg, 20 mol%) was added
followed by copper sulfate (8 mg, 10 mol%) solution in
H O).
2
3
Compound 5: 1H NMR (400 MHz, CD OD) d ppm 8.43
(1H, s), 4.89 (2H, d, J = 4.4 Hz), 4.53 (1H, t, J = 4.4 Hz),
3.68 (3H, s). 13C NMR (101 MHz, DMSO-d ) d ppm
0
2
.5 ml water. The mixtures were stirred at rt for 10 min–
4 h. The solvents were then evaporated to dryness and
6
167.33 (C), 161.04 (C), 139.64 (C), 129.98 (CH), 52.45
(CH ), 51.60 (CH), 49.16 (CH3). HR-MS calculated for
the residue taken up in 50 ml AcOEt. The suspension was
washed 2–3 times with 0.5 N HCl until the color was
mostly gone (copper removal), and then with 50 ml water.
2
C H N O : 215.0780, found: 215.0783 (1.3 ppm).
7 11 4 4
23
0
½aꢀ 15 (0.095, MeOH).
D
Compound 6: 1H NMR (400 MHz, DMSO-d
Drying with Na
recrystallization from either Et
2
SO
4
, evaporation of the solvent, and
O/hexane or AcOEt/hex-
6
) d ppm
+
2
8.88 (3H, br s NH3 ), 8.57 (1H, s), 7.90 (1H, s, NH2a),
7.50 (1H, s, NH2b), 4.99 (2H, ddd, J = 22.3, 15.0, 5.1 Hz),
4.54 (1H, dd, J = 5.1 Hz) 13C NMR (101 MHz, DMSO-
ane yielded the pure compounds. The triazole was then
refluxed in 6 N HCl for 1–48 h to yield targets 1–4, or
hydrogenated (Pd/C/H
treatment with TFA (50% in CH
2
(1 atm) in MeOH) followed by
Cl , 15 min.) with
subsequent counterion exchange (0.1 N HCl, 24 h) and
6
d ) d ppm 168.74 (C), 162.07 (C), 143.48 (C), 128.76 (CH),
2
2
52.39 (CH
2
), 49.25 (CH) HR-MS calculated for
C H N O : 200.0784, found: 200.0776 (3.8 ppm).
6 10 5 3
0
23
evaporated to yield targets 5 and 6.
½aꢀ 27 (c 0.055, DMSO).
D