Cancer cell targeting driven by selective polyamine reactivity with glycine propargyl esters

Article abstract of DOI:10.1039/c7cc01934c

Rapidly growing cancer cells have increased levels of intracellular polyamines compared to normal, healthy tissues. Based on the selective reactivity of glycine propargyl esters, probes were synthesized that show evidence for selective polyamine reactivit

Full text of DOI:10.1039/c7cc01934c

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Cancer cell targeting driven by selective polyamine reactivity with
glycine propargyl esters
Received 00th January 20xx,
Accepted 00th January 20xx
Kenward K. H. Vong,a Kazuki Tsubokura,a,b Yoichi Nakao,b Tomonori Tanei,c Shinzaburo Noguchi,c
Shinobu Kitazume,d Naoyuki Taniguchi,d and Katsunori Tanakaa,e,f,
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
O
Rapidly growing cancer cells have increased levels of intracellular
polyamines compared to normal, healthy tissues. Based on the
selective reactivity of glycine propargyl esters, probes were
synthesized that show evidence for selective polyamine reactivity,
which was then applied for selective cancer cell imaging studies.
hydrogenbondacceptor-
containingamine
RHN
O
ex/
OH
GlycinePropargyl Ester
H2N
O
linearprimary alkylamine
ex/
bulky amine
secondaryamine
H2N
ex/
ex/
Polyamines are polycationic alkylamines that exist in virtually
all forms of life, where their major forms (putrescine,
spermidine, and spermine) are typically found within cells at
submillimolar or millimolar concentrations. Despite their
seemingly simplistic nature, polyamines play a profound role in
numerous biological processes;1 perhaps the most significant
being its role in cell growth and proliferation. This is best
exemplified by studies that showed aged cells have decreased
intracellular polyamine levels and biosynthesis compared to
equivalent cells that are younger and still growing.2
Due to the rapid and uncontrolled nature of cancer cell
growth, several studies have confirmed its correlation with
elevated intracellular levels of polyamines.3 In addition,
overexpression of enzymes/proteins related to the polyamine
biosynthetic and transport pathways, such as ornithine
decarboxylase (ODC)4 and polyamine transport system (PTS),5
have been observed.
HN
H2N
O
RHN
N
no to little
reactivity
H
• room temp, H2O compatible
• no base needed, 50-80%
Fig. 1 Summary of glycine propargyl ester reactivity with linear alkylamines.
an extensive list of studies showed cancerous cell growth can
be hindered or reduced.7 In addition, studies have made use of
overactive PTS for selective cancer targeting by employing
drug-polyamine conjugates as antitumor agents,8 and
fluorophore-polyamine conjugates as imaging probes9
Previously,10 our lab discovered that glycine propargyl
ester-based derivatives could selectively react with linear
primary alkylamines (non-secondary amines with long, non-
bulky linear substituents), as shown in Fig. 1. Selectivity can be
mainly attributed to this reaction relying on hydrogen
bonding/intramolecular interactions to stabilize the transition
state, causing the facile amidation of an otherwise moderately
stable leaving group (propargyl alcohol pKa ~ 13.6).
To exploit the relationship between polyamines and cancer,
numerous strategies have been developed. By far the most well-
known example is the ODC-inhibitor difluoromethylornithine
(DMFO).6 When coupled with polyamine transport inhibitors,
Within living systems, there are three major groups of
Monoamine
Neurotransmitters
Amino Acids/Proteins
O
Polyamines
H
a.Biofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi,
Saitama, 351-0198, Japan
H2N
OH
b.School of Advanced Science and Engineering, Department of Chemistry and
Biochemistry, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo, 169-8555,
Japan
H2N
N
H2N
R = various
side chains
OH
H2N
N
dopamine
H
OH
R
spermine
c. Department of Breast and Endocrine Surgery, Graduate School of Medicine,
Osaka University,2-2-E10 Yamadaoka, Suita-shi, Osaka, 565-0871, Japan
d.Disease Glycomics Team, Global Research Cluster, RIKEN-Max Planck Joint
Research Center for Systems Chemical Biology, RIKEN, 2-1 Hirosawa, Wako-shi,
Saitama, 351-0198, Japan
OH
H
N
H
N
OH
OH
N
H2N
H2N
epinephrine
spermidine
N
HN
H
e. Biofunctional Chemistry Laboratory, A. Butlerov Institute of Chemistry, Kazan
Federal University, 18 Kremlyovskaya Street, Kazan, 420008, Russia
f. JST, PRESTO, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan
† Electronic Supplementary Information (ESI) available: Detailed experimental
procedures and characterization data. See DOI: 10.1039/x0xx00000x
histidine
NH
NH2
lysine
H2N
H2N
H2N
H2N
putrescine
NH
N
arginine
Fig. 2 Major classes of biological amines.
histamine
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Table 1 Exploring biological amine selectivity and reactivity
biological amines: polyamines, amino acids/proteins and
monoamine neurotransmitters (Fig. 2). Amino acids act as
important building blocks for not only proteins, but also as
intermediates for a wide range of metabolites. Monoamine
neurotransmitters act as neuromodulators in the central nervous
system and as hormones in the blood circulation. Other minor
groups of biological amines also exist, such as lipids and
sugars, but their free forms are often present in relatively low
abundance.
Due to its linear and non-bulky nature, it was hypothesized
that polyamines may have preferential reactivity with glycine
propargyl esters. Furthermore, the imbalance of intracellular
polyamine concentrations between cancer and normal cells is
expected to allow glycine propargyl ester-based imaging probes
to selectively target rapidly growing cancer cells, which was
investigated in this study.
single
linkage
yield1
59%3,
56%4,
double hydrolysis
recovered
entry
1
amine
linkage
yield1
23%3,
18%4,
product
41
17%3,
29%4,
14%5
12
1%3,
N/A4, N/A5
47%5 (2a) 14%5 (3a)
67%3
11%3
2
3
21%3
1%3
7%3
(2b)
(3b)
66%3
N/A3
N/A3
N/A3
22%3
(2c)
Various glycine propargyl ester probes (Fig. 3) were
synthesized and detailed in the supplementary information.
4
5
N/A3
7%3
6%3
92%3
85%3
Cbz-containing glycine propargyl ester
identification studies, while kinetic and cell-based qualitative
assays used the fluorophore probes (linked to 5-((2-
1 was used for product
2%3
9
(2d)
aminoethyl)amino)naphthalene-1-sulfonic acid (EDANS)), and
13 (linked to 5-carboxytetramethylrhodamine (TAMRA)),
1isolated yields via HPLC purification, 2isolation of remaining ester starting
material, 31:1 dioxane/PBS Buffer 7.4, 41:1 dioxane/DMEM media, 51:1
respectively. In the case of probe
9, a quencher 4-((4-
(dimethylamino)phenyl)azo)benzoic acid (DABCYL) is also dioxane/rat serum; N/A
=
speculated product not found. Reactions were
standardized to 0.04 mmol of amine and ester in 0.2 ml of solvent (200 mM).
utilized to make use of Förster resonance energy transfer
(FRET) interactions.
with probe
yields, probe
1
(Fig. 4). As shown by the HPLC trace and isolated
reactivity continues to favour polyamines over
Given that polyamines can be structurally defined as linear
primary alkylamines, it was speculated that glycine propargyl
ester reactivity could be selective for polyamines over other
biological amines, such as amino acids and monoamine
neurotransmitters. To prove this, Table 1 (HPLC traces in Fig.
S1-S3) compares the reactivity of polyamines to representative
molecules of the other biological amine classes (i.e. lysine and
norepinephrine). Results show that when incubated with the
1
norepinephrine and lysine.
To develop an assay that can be used to compare glycine
propargyl ester reactivity for a wide range of substrates, the
FRET-based probe
detailed in Fig. S13, there is a ~13× increase in quantum yield
between probe and its spermine-linked amide product 10. In a
further study, shown in the box insert of Fig. 5, investigation of
the kinetic activity between spermine and probe gave a
9 was synthesized. As shown in Fig. 5 and
9
glycine propargyl ester-based probe 1, the highest yields were
9
obtained with spermine, spermidine and putrescine (59-67%
single amidation product, 11-23% double amidation product,
entries 1-3) compared to norepinephrine (0%, entry 4) and
lysine (2%, entry 5). Observations also show the near complete
calculated apparent second-order rate constant of 19.9 ± 0.1
µM-1min-1 (calculations detailed in Fig. S14-16).
Using this FRET-based probe, various biological amines of
significance from each representative class were tested (Fig. 6
recovery of probe
1 with norepinephrine and lysine, which is in
stark contrast to when polyamines are present. In an additional
experimental scenario, representative molecules of each class
(i.e. spermine, lysine, and norepinephrine) were mixed together
SO3H
EDANS
(fluorophore)
O
CbzHN
O
O
O
1
H
N
HN
N
N
H
O
O
N
O
N
N
N
DABCYL
H
(quencher)
N
HOOC
O
O
O
9
H
N
N
O
H
O
TAMRA
(fluorophore)
13
Fig. 4 HPLC analysis of reaction mixture to observe for polyamine selectivity.
Spermine, L-lysine, and norepinephrine (0.04 mmol) were mixed in 1:1
dioxane/PBS buffer pH 7.4 (0.2 ml) for 1 hr. Isolated yields via HPLC purification.
Fig. 3 Glycine propargyl ester-based probes used in this study.
2 | J. Name., 2012, 00, 1-3
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SO3H
rapidly increases from putrescine to spermidine to spermine.
This is likely due to the increasing presence of secondary
amines, which may play a role in accelerating reactivity
through intramolecular basicity. Future studies will likely look
to confirm these claims.
O
O
H
N
N
HN
N
H
O
O
N
N
H
N
9
To investigate the potential for in vivo polyamine targeting
within a living biological system, cell-based studies with water
soluble probe 13 linked to a cell-penetrable TAMRA moiety
was carried out. Initial studies focused on incubation with the
MCF-7 breast cancer cell line (protocol detailed in
supplementary information). By fluorometric HPLC analysis
(Fig. S6 and S7), a key metabolite found in the cellular extract
may be the 1,3-diaminopropane-TAMRA linked product, which
is formed from spermine oxidase metabolization of spermine-
TAMRA. This result led to speculation that fluorescent probe
13 could be preferentially retained within cancer cells following
polyamine reactivity due to possible interactions with
polyamine biosynthetic and catabolic pathway enzymes.
Another valid reason for cell retention would be that the
fluorescent probe 13 gains an increase in positive charge
following polyamine reaction, thus preventing cell membrane
penetration.
(non-fluorescent)
O
spermine,
1:1 DMSO/H2O,
rt
O
N
O
H
H
H
N
NH2
N
N
HO3S
N
H
N
H
H
O
10
(fluorescent)
O
N
N N
+
NH
HO
ε [M-1 cm-1 1
]
λ
abs [nm]1
420
λ
em [nm]1
ΦF
1,2
9
1325 ± 151
3693 ± 13
420
490
0.0449 ± 0.0002
0.5770 ± 0.0293
10
340
Fig. 5 Photophysical and kinetic parameters of FRET-based probes. 1measured in
1:1 DMSO/H2O, 2relative quantum yields of fluorescence (ΦF) measured using
quinine sulfate as the standard. Box insert shows the kinetic activity of the
reaction done in DMSO.
Studies have shown that there are disproportionately higher
polyamine levels within breast cancer cells compared to their
normal cell counterparts.11 As such, the potential usage of
probe 13 to preferentially image cancer cells over normal cell
lines was next explored. Imaging results show fluorescence
(Fig. 7A and S8-S10) was detected in three different breast
cancer cells lines (MCF7, MDA-MB-231, SK-BR-3) when
incubated with probe 13. In contrast, incubation with normal
cell lines (MCF10A and lymphocytes) showed no fluorescence
(Fig. 7B and S11-S12). This difference is clearly illustrated by
comparing the relative fluorescence intensities normalized
against the background (Fig. 7C).
In conclusion, we have shown glycine propargyl ester-based
compounds have preferential reactivity with polyamines over
other biological amines (amino acids/proteins and monoamine
neurotransmitters). To prove the viability of employing this
reactivity towards biomedical applications, a novel framework
for cancer cell targeting/imaging was developed. Through the
imbalance of intraceullular polyamines typically found within
rapidly growing cells, evidence shows that glycine propargyl
ester-based probes can be applied for selective cancer cell
and S4); amino acids/proteins (albumin, L-arginine, L-histidine,
L-lysine), polyamines (putrescine, spermidine, spermine),
monoamine
neurotransmitters
(epinephrine,
histamine,
dopamine, phenethylamine), as well as sphingosine. As
expected, the highest levels of fluorescence were observed
when polyamines such as spermine and spermidine were tested.
Aware that two primary amines are present on polyamines
compared to one primary amine of most amino acids and
monoamine neurotransmitters, various substrate concentrations
were also tested. For example, comparing the fluorescence
levels of 2 mM lysine with 1 or 0.5 mM of spermine clearly
shows much lower levels of activity. To confirm product
formation (Fig. S5), the reaction mixtures containing L-
arginine, L-lysine, and spermine were further analyzed by
fluorometric HPLC. Observations show that the low levels of
fluorescence for mixtures containing L-arginine and L-lysine
are mainly attributed to ester hydrolysis, whereas the mixture
containing spermine clearly shows desired product, as well as
some ester hydrolysis.
Another important observation is that polyamine reactivity
450
400
350
300
250
200
∗
0 hr
6 hr
†
∗
150
100
∗
†
●
∗
∗
†
●
†
∗
†
●
∗
† ●
†
●
●
50
0
●
∗
●
∗∗ ∗
††●● ∗ †† ● ∗ † ● ∗ ††●●
●●
∗ †
●
† ● ∗ † ●
∗
†
∗ † ● ∗ ††●● ∗ †
∗
∗ † ●
∗
∗ † ●
Albumin
Arginine
Histidine
Lysine
Putrescine
Spermidine
Spermine
Epinephrine
Histamine
Dopamine
Phenethylamine Sphingosine
Amine
Fig. 6 Comparison of FRET-based fluorescence generated from reactivity between probe 9 (100 μM) and various biological amines (indicated as ∗2mM, †1mM, ●0.5mM). Reactions
were done in 1:1 DMSO/PBS buffer pH 7.4 under various time points.
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Fig. 7 A) Cell imaging of breast cancer cell lines MCF7, MDA-MB-231, and SK-BR-3
that were incubated with TAMRA-linked propargyl ester 13 (30 μM). B) Cell
imaging of normal cell lines MCF10A and lymphocytes that were incubated with
TAMRA-linked propargyl ester 13 (30 μM). C) Comparison of the relative
fluorescence intensities normalized against the background.
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This work was supported by JSPS KAKENHI Grant
Numbers JP16H03287, JP16K13104, and JP15H05843 in
Middle Molecular Strategy. K.K.H.V. was supported by the
JSPS Postdoctoral Fellowship for Research Abroad. A part of
this study was done with
a subsidy from the Russian
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