Synthesis and antiproliferative activity of glutamic acid-based dipeptides

Article abstract of DOI:10.1007/s00726-015-1987-0

A small and focused library of 22 dipeptides derived from N,N-dibenzylglutamic acid α- and γ-benzyl esters was prepared in a straightforward manner. The evaluation of the antiproliferative activity in the human solid tumor cell lines HBL-100 (breast), HeL

Full text of DOI:10.1007/s00726-015-1987-0

Amino Acids
DOI 10.1007/s00726-015-1987-0
ORIGINAL ARTICLE
Synthesis and antiproliferative activity of glutamic acid‑based
dipeptides
Gastón Silveira‑Dorta1 · Víctor S. Martín1 · José M. Padrón1
Received: 3 March 2015 / Accepted: 11 April 2015
© Springer-Verlag Wien 2015
Abstract A small and focused library of 22 dipeptides
derived from N,N-dibenzylglutamic acid α- and γ-benzyl
esters was prepared in a straightforward manner. The evalu-
ation of the antiproliferative activity in the human solid
tumor cell lines HBL-100 (breast), HeLa (cervix), SW1573
(non-small cell lung), T-47D (breast), and WiDr (colon)
provided γ-glutamyl methionine (GI50 = 6.0–41 μM)
and α-glutamyl proline (GI50 = 7.5–18 μM) as lead com-
pounds. In particular, glutamyl serine and glutamyl proline
dipeptides were more active in the resistant cancer cell line
WiDr than the conventional anticancer drugs cisplatin and
etoposide. Glutamyl tryptophan dipeptides did not affect
cell growth of HBL-100, while in T-47D cells, proliferation
was inhibited. This result might be attributed to the inhibi-
tion of the ATB0,+ transporter.
Introduction
Naturally occurring amino acids represent a powerful
source of relatively inexpensive, widely available and enan-
tiomerically pure building blocks, even on a bulk scale.
They have been largely used in organic chemistry as inter-
mediates in peptide synthesis or in the synthesis of more
complex structures with diverse applications. Of particular
interest to us is the use of amino acids as source for antitu-
mor compounds. In this context, l-glutamic acid is present
in the antitumor drug aminopterin (1a) (Farber et al. 1948)
and its well-known N-methylated derivative methotrex-
ate (1b) (Skeel 2008). Other anticancer drug’s derivatives
of l-glutamic acid are thalidomide (2a) and its analogs
pomalidomide (CC-4047, 2b) and lenalidomide (CC-5013,
2c) (Bartlett et al. 2004). In these compounds, l-glutamic
acid binds through its amino group to the rest of the mol-
ecule forming amide or imide bonds (Fig. 1a). Another
use of l-glutamic acid is as poly-glutamic acid conjugates,
which act as drug carrier because they increase the efficacy
of anticancer drug and decrease its toxicity toward normal
cells (Melancon and Li 2011). In this strategy, the drug
is mostly linked to the γ-carboxyl group through ester or
amide bonds.
Keywords Antitumor agents · Dipeptide · Glutamic acid ·
Structure–activity relationships
Handling Editor: J. D. Wade.
The natural amino acid l-glutamine (3a) is the γ-amide
of l-glutamic acid and it is essential for cell growth and
proliferation. In healthy cells, l-glutamine is synthesized
from l-glutamic acid by l-glutamine synthetase. However,
in neoplastic cells, l-glutamine cannot be produced due
to the lower reactivity of l-glutamine synthetase. In addi-
tion to glucose, cancer cells utilize l-glutamine as a carbon
source for ATP production and biosynthesis. l-Glutamine
can be internalized through cell surface transporters such
as ASCT2 (Pochini et al. 2014). These considerations have
increased the interest of targeting glutamine internalization
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-015-1987-0) contains supplementary
material, which is available to authorized users.
* José M. Padrón
jmpadron@ull.es
1
BioLab, Instituto Universitario de Bio-Orgánica “Antonio
González” (IUBO-AG), Centro de Investigaciones
Biomédicas de Canarias (CIBICAN), Universidad de La
Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La
Laguna, Spain
1 3

G. Silveira-Dorta et al.
Fig. 1 a l-Glutamic acid deriv-
atives with anticancer activity.
Bond thickness emphasizes the
l-glutamic acid fragment. b
l-Glutamine and Nγ-alkylated
derivatives. c Statistical analysis
of amino acid residues of the
ACPs listed in the CancerPPD
database
(Wang et al. 2014) and metabolism (Lukey et al. 2013) in
cancer therapy. l-Glutamine derivatives (Fig. 1b) investi-
gated along these lines include the natural product theanine
(3b) (Liu et al. 2009), the glutamine-utilizing enzyme inhib-
itors 3c–3e, or the ASCT2 inhibitor 4 (Schulte et al. 2015).
In addition to enzyme inhibitors or drug carriers, amino
acids constitute peptides that are being used for the ther-
apy of a significant number of diseases (Sato et al. 2006;
Mustata and Dinh 2006). In particular, anticancer peptides
(ACPs) have shown relevant since they exhibit cancer-
selective toxicity while avoiding the shortcomings of the
conventional chemotherapy (Thundimadathil 2012). ACPs
are small peptides with less than 50 amino acids. The Can-
ACPs. The statistical analysis in terms of amino acid resi-
dues shows that small peptides (<5 amino acids) have not
being explored exhaustively as potential anticancer agents
(Fig. 1c). When it comes to the study of anticancer dipep-
tides, the literature is scarce in examples. Although tyros-
ine- and cystine-based dipeptides have been reported to
exhibit antiproliferative activity in human solid tumor cell
lines (Horvat et al. 2006; Banerji et al. 2013), to the best of
our knowledge, there are no reports on the antiproliferative
activity of glutamic acid-based dipeptides.
l-Glutamic and l-aspartic acid are potentially valuable
molecules providing that the two carboxylic groups could
be synthetically differentiated. Recently, we have explored
the regioselective esterification of l-glutamic acid (Sil-
veira-Dorta et al. 2014). Our methodology allows to obtain
cerPPD
(http://www.crdd.osdd.net/raghava/cancerppd/)
database contains over 3400 experimentally verified
1 3

Synthesis and antiproliferative activity…
Fig. 2 Synthesis of glutamic acid-based dipeptides 9–10. Reagents
and conditions: a BnBr, K2CO3-KOH, MeOH-H2O (75:25), Δ, 8 h,
73 %; b BnBr, K2CO3, MeOH-H2O (1:1), Δ, 20 h, 90 %; c NaOH,
MeOH (1:1), Δ, 20 h, 98 %; d CH3SO3H, BnOH, toluene, Δ, 5 h,
70 %; e TBTU, DIPEA, DMF, rt, overnight, 50–70 %
unambiguously both N,N-dibenzylglutamic acid α- (5) and
γ-benzyl esters (6) from commercially available l-glutamic
acid (Fig. 2). As a part of our screening program directed
at the discovery of new biologically active molecules
with antiproliferative activity, we found that both 5 and 6
were active against human solid tumor cells. These results
together with the lack of antiproliferative activity studies
on glutamic acid-based dipeptides encouraged us to explore
further this type of molecules as scaffolds for new antican-
cer drugs. Herein, we report on the synthesis of glutamic
acid-based dipeptides and their antiproliferative activity
against human solid tumor cells.
TBTU (176.6 mg, 1.5 mmol) were dissolved in DMF
(5 mL). The reaction mixture was stirred at room tem-
perature for 30 min. Amino acid methyl ester (71.6 mg,
0.4 mmol) was added and stirring was continued overnight.
The reaction mixture was diluted with water (25 mL) and
extracted with Et2O (3 × 25 mL). The combined organic
phases were dried over magnesium sulfate, filtered, and the
solvent was evaporated. The crude product was purified by
column chromatography (silica gel, Hexane:AcOEt 8:2 or
7:3). The title compound 9 or 10 was obtained in 70–80 %
yield.
Chemosensitivity testing
Experimental section
Cells were inoculated onto 96-well microtiter plates in a
volume of 100 µL per well at densities of 10,000 (HBL-
100, HeLa and SW1573), 15,000 (T-47D), and 20,000
(WiDr) cells per well, based on their doubling times. Com-
pounds 9–10 were initially dissolved in DMSO at 400 times
the desired final maximum test concentration. Control cells
were exposed to an equivalent concentration of DMSO
(0.25 % v/v, negative control). Each agent was tested in
triplicate at different dilutions in the range of 1–100 μM.
The drug treatment started on day 1 after plating. Drug
incubation times were 48 h, after which cells were precipi-
tated with 25 μL ice-cold TCA (50 % w/v) and fixed for
60 min at 4 °C. Then, the SRB assay was performed. The
optical density (OD) of each well was measured at 492 nm,
using BioTek’s PowerWave XS Absorbance Microplate
Reader. Values were corrected for background OD from
All reagents were used as purchased from commercial sup-
pliers without further purification. Solvents were dried and
purified by conventional methods prior to use. The human
solid tumor cell lines HBL-100 (breast), HeLa (cervix),
SW1573 (lung), T-47D (breast), and WiDr (colon) were
used in this study. These cell lines were a kind gift from
Prof. G. J. Peters (VU Medical Center, Amsterdam, The
Netherlands).
General procedure for the preparation of glutamic
acid‑based dipeptides
Monobenzyl ester 5 or 6 (150 mg, 0.4 mmol), N-N-diiso-
propyl ethyl amine (94.2 mg, 0.12 mL, 0.8 mmol), and
1 3

G. Silveira-Dorta et al.
wells only containing medium. The antiproliferative activ-
ity for each compound, expressed as GI50 values, was cal-
culated according to NCI formulas (Monks et al. 1991).
obtained directly from l-glutamic acid. Instead, a three-
step sequence is needed. Briefly, conventional perbenzyla-
tion of l-glutamic acid afforded compound 7 in high yields
as reported earlier (Rodriquez and Taddei 2005). Then,
hydrolysis of the benzyl ester groups of 7 under stand-
ard basic conditions led to diacid 8 in almost quantitative
yield. Finally, esterification under acid conditions (Albert
et al. 1987) allowed us to obtain exclusively the γ-benzyl
ester 6 in 70 % yield. Intermediates 5 and 6 were coupled
successfully with the commercially available methyl ester
hydrochlorides of the amino acids alanine (a), valine (b),
leucine (c), isoleucine (d), phenylalanine (e), tyrosine (f),
tryptophan (g), threonine (h), serine (i), proline (j), and
methionine (k) to provide dipeptides 9a–k and 10a–k,
respectively. In this process, 2-(1H-benzotriazole-1yl)-
1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)
was selected as coupling agent. The reactions were car-
ried out in DMF and N,N-diisopropylethyl amine was used
as based. Yields were in the range of 50–70 %. With this
methodology, we obtained a small library of dipeptides
Results and discussion
During the course of our investigation on the regioselective
benzylation of l-glutamic acid, we analyzed the antiprolif-
erative activity of compounds 5–8. As a model to study the
antiproliferative activity, we selected the panel of represent-
ative human solid tumor cell lines HBL-100 (breast), HeLa
(cervix), SW1573 (non-small cell lung), T-47D (breast),
and WiDr (colon). The in vitro antiproliferative activity was
evaluated using the National Cancer Institute (NCI) pro-
tocol (Monks et al. 1991). The standard anticancer drugs
cisplatin (Dasari and Tchounwou 2014) and etoposide
(Najar and Johri 2014) were used as reference agents. The
results expressed as GI50 are shown in Table 1. We found
that monobenzyl esters 5–6 were able to induce cell growth
inhibition in all cell lines with GI50 values in the range of
22–46 μM, whereas the perbenzylated analog 7 and the
diacid 8 were inactive. In addition, compound 5 showed
slightly more active than compound 6. This result encour-
aged us to synthesize a small library of α- and γ-glutamyl
dipeptides and test their antiproliferative activity.
Table 2 Antiproliferative activity (GI50) against human solid tumor
cells of compounds 9–10
Compound Cell line
HBL-100 HeLa
SW1573 T-47D
WiDr
Synthesis of glutamic acid‑based dipeptides
9a
24 ( 2.2) 22 ( 4.4) 36 ( 2.6) 22 ( 2.9) 24 ( 1.2)
22 ( 8.5) 19 ( 2.7) 65 ( 26) 38 ( 10) 40 ( 4.8)
35 ( 2.8) 25 ( 8.2) 30 ( 9.8) 33 ( 2.6) 40 ( 3.1)
22 ( 0.8) 23 ( 5.5) 26 ( 4.5) 21 ( 1.9) 26 ( 2.8)
36 ( 6.2) 27 ( 7.0) 14 ( 4.1) 21 ( 0.1) 36 ( 9.0)
20 ( 7.2) 14 ( 1.6) 18 ( 6.1) 22 ( 7.7) 20 ( 4.9)
The general synthetic pathway for the preparation of the
glutamic acid-based dipeptides is outlined in Fig. 2. The
direct regioselective benzylation of commercially avail-
able l-glutamic acid afforded N,N-dibenzylglutamic acid
α-benzyl ester (5) in 73 % yield. We have reported recently
that the reaction conditions play a critical role in the out-
come of the products (Silveira-Dorta et al. 2014). However,
N,N-dibenzylglutamic acid γ-benzyl ester (6) cannot be
9b
9c
9d
9e
9f
9g
>100
18 ( 4.0) 20 ( 7.1) 33 ( 2.5) 40 ( 11)
9h
37 ( 7.1) 31 ( 2.0) 44 ( 7.2) 39 ( 8.4) 33 ( 8.2)
17 ( 2.2) 15 ( 2.2) 17 ( 4.4) 18 ( 2.5) 18 ( 5.6)
20 ( 4.6) 17 ( 1.3) 24 ( 4.2) 19 ( 1.1) 21 ( 1.1)
6.0 ( 1.1) 7.9 ( 1.2) 41 ( 9.6) 11 ( 2.3) 13 ( 5.3)
17 ( 1.2) 10 ( 6.5) 31 ( 0.1) 27 ( 0.4) 32 ( 0.3)
9.1 ( 1.2) 9.0 ( 6.5) 23 ( 7.8) 23 ( 3.3) 17 ( 2.4)
16 ( 6.0) 12 ( 5.1) 25 ( 4.1) 24 ( 6.5) 33 ( 2.1)
16 ( 0.1) 19 ( 2.8) 41 ( 21) 33 ( 4.9) 36 ( 5.2)
24 ( 9.6) 35 ( 14) 7.4 ( 0.9) 48 ( 3.5) 37 ( 15)
18 ( 2.9) 19 ( 2.4) 26 ( 2.0) 30 ( 1.4) 37 ( 5.6)
9i
9j
9k
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
Table 1 Antiproliferative activity (GI50) against human solid tumor
cells of compounds 5–8
Compound Cell line
HBL-100 HeLa
SW1573 T-47D
WiDr
5
6
7
8
39 ( 2.6) 22 ( 6.6) 38 ( 4.1) 29 ( 5.6) 29 ( 7.8)
46 ( 9.0) 30 ( 6.8) 40 ( 4.4) 34 ( 7.2) 44 ( 9.4)
>100
21 ( 6.3) 31 ( 6.8) 36 ( 7.8) 57 ( 2.8)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
19 ( 4.0) 18 ( 2.1) 30 ( 4.6) 31 ( 5.9) 24 ( 2.7)
16 ( 1.3) 16 ( 0.3) 18 ( 2.1) 19 ( 2.3) 18 ( 1.7)
7.5 ( 0.6) 9.3 ( 2.9) 13 ( 4.9) 18 ( 4.4) 10 ( 4.0)
14 ( 5.9) 17 ( 1.8) 25 ( 0.5) 31 ( 2.7) 39 ( 8.6)
Cisplatin 1.9 ( 0.2) 2.0 ( 0.3) 3.0 ( 0.4) 15 ( 2.3) 26 ( 5.3)
Etoposide 2.3 ( 0.9) 3.0 ( 0.9) 15 ( 1.5) 22 ( 5.5) 23 ( 3.1)
Values are given in μM and are means of two to five experiments;
Values are given in μM and are means of two to three experiments;
standard deviation is given in parentheses
standard deviation is given in parentheses
1 3

Synthesis and antiproliferative activity…
comprising two sets of regioisomers, the γ- (9a–k) and the
α-glutamyl series (10a–k).
cancer cell lines HBL-100 and T-47D might relate to the
ATB0,+ (SLC6A14, solute carrier family 6 member 14)
transporter. ATB0,+ accepts tryptophan as a substrate with
high affinity and is up-regulated markedly in some types
of cancer (Gupta et al. 2005, 2006). Diverse tryptophan
derivatives inhibit ATB0,+ causing antiproliferative effects
in cell lines overexpressing the transporter such as MCF-7,
ZR-75.1, and T-47D (Karunakaran et al. 2008). However,
in HBL-100, HMEC, and MCF10A cells, the expression of
ATB0,+ is undetectable and the tryptophan derivatives do
not affect cell growth. Our findings for 9g and 10g are con-
sistent with these results.
When considering the aliphatic side chain in the
α-glutamyl series 10a–d, valine dipeptide 10b stands out
against all cell lines. However, in the γ-glutamyl set 9a–d,
this trend was not observed. The antiproliferative activity
of the compounds with an aromatic side chain (9e–g and
10e–g) did not show clear tendency. In peptides containing
a hydroxyl group, serine dipeptides (i) resulted more active
than the corresponding threonine derivative (h) in all cell
lines tested. Finally, proline dipeptide (j) in terms of activ-
ity was significant in the γ-glutamyl set, while methionine
dipeptide (k) was relevant in the γ-glutamyl series.
Biological evaluation of glutamic acid‑based dipeptides
The antiproliferative activity of γ-glutamyl (9a–k) and
α-glutamyl dipeptides (10a–k) was evaluated and the
results are shown in Table 2 and Fig. 3. Although the set
of compounds is not large, some structure–activity relation-
ships could be inferred from the antiproliferative data. The
data showed a clear difference in potency between both set
of compounds. Based on average GI50 values, α-glutamyl
dipeptides (10a–k) were more active than the γ-glutamyl
derivatives (9a–k) in HBL-100, HeLa, and SW1573 cells.
In the more resistant cell lines T-47D and WiDr, the overall
effect favored the γ-glutamyl derivatives. The most potent
compound of both series was the α-glutamyl dipeptide 10j
and exhibited GI50 values in the range of 7.5–18 μM. In
the γ-glutamyl set, the lead in terms of GI50 values was 9k
with GI50 values in the range of 6.0–41 μM. However, in
SW1573 cells, none of the designated lead was the most
potent compound. Instead, the phenylalanine dipeptide
(e) was the most active. It is noteworthy that when com-
pared to the standard anticancer drugs etoposide and cispl-
atin, dipeptides 9i, 9j, 10i, and 10j were more active in the
resistant cell line WiDr.
Conclusion
All compounds were able to inhibit cell growth in all
cell lines with GI50 values in the range of 6–65 μM with
the exception of compounds 9g and 10g (tryptophan dipep-
tides) against HBL-100 cells. We speculate that the differ-
ence in activity observed against the mammary epithelial
In summary, we have reported the synthesis of a novel class
of N,N-dibenzylglutamic acid-based dipeptides. Commer-
cially available amino acids were coupled to the appropri-
ate carboxylic acid of glutamic acid to generate the α- and
Fig. 3 Mean graph plots for the antiproliferative activity of glutamic acid-based dipeptides 9a–k (top) and 10a–k (bottom). The middle line rep-
resents the median GI50 (μM) value of each set of compounds against each individual cell line. GI50 values >40 μM are indicated with asterisk
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G. Silveira-Dorta et al.
V (2006) Up-regulation of the amino acid transporter ATB0,+
(SLC6A14) in carcinoma of the cervix. Gynecol Oncol 100:8–
13. doi:10.1016/j.ygyno.2005.08.016
the γ-glutamyl series of dipeptides. The dipeptides were
tested for their antiproliferative activity against five human
solid tumor cell lines. Overall, the compounds show active
against all cancer cell lines tested. Remarkably, some
dipeptides were more active in the resistant cancer cell
line WiDr than conventional anticancer drugs. From the
data on growth inhibition, γ-glutamyl methionine 9k and
α-glutamyl proline 10j were identified as lead compounds.
More experiments are needed to establish the scope and
limitations of N,N-dibenzylglutamic acid derivatives as
novel anticancer agents, as well as to identify their mecha-
nism of antiproliferative activity. Further research involv-
ing novel derivatives of N,N-dibenzylglutamic acid is in
progress and will be reported elsewhere. Finally, the results
obtained for the tryptophan dipeptides 9g and 10g in breast
cancer cells merit further investigation, which remain
beyond the scope of our study. In particular, the ability of
the tryptophan dipeptides to inhibit the ATB0,+ transporter.
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Acknowledgments Co-financed by the EU Research Poten-
tial (FP7-REGPOT-2012-CT2012-31637-IMBRAIN), the Euro-
pean Regional Development Fund (FEDER), the Spanish MINECO
(CTQ2011-28417-C02-01 and Instituto de Salud Carlos III
PI11/00840). G.S.-D. thanks the EU Social Fund (FSE) and the
Canary Islands ACIISI for a predoctoral grant.
Mustata G, Dinh SM (2006) Approaches to oral drug delivery for
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Biosci 39:139–144
Conflict of interest The authors declare that they have no conflict
of interest.
Ethical standard This article does not contain any studies with
human participants or animals performed by any of the authors.
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transporters for the special amino acid glutamine: structure/func-
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