Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-methylguanine nucleotides targeting cap-dependent translation

Article abstract of DOI:10.1016/j.bmc.2020.115523

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5′ cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn⁷GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N⁷-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5′-phosphorothioate (m⁷GMPS), which is a translation inhibitor with in vitro potency higher than bn⁷GMP.

Full text of DOI:10.1016/j.bmc.2020.115523

Journal Pre-proofs
Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-
methylguanine nucleotides targeting cap-dependent translation
Sebastian Golojuch, Michal Kopcial, Dominika Strzelecka, Renata Kasprzyk,
Natalia Baran, Pawel J. Sikorski, Joanna Kowalska, Jacek Jemielity
PII:
S0968-0896(20)30349-7
https://doi.org/10.1016/j.bmc.2020.115523
BMC 115523
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
23 December 2019
23 March 2020
17 April 2020
Please cite this article as: S. Golojuch, M. Kopcial, D. Strzelecka, R. Kasprzyk, N. Baran, P.J. Sikorski, J.
Kowalska, J. Jemielity, Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-
methylguanine nucleotides targeting cap-dependent translation, Bioorganic & Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.bmc.2020.115523
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Exploring tryptamine conjugates as
pronucleotides of phosphate-modified 7-
methylguanine nucleotides targeting cap-
dependent translation
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a,b
a,c,d
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a,c,d
a,e
Sebastian Golojuch , Michal Kopcial , Dominika Strzelecka , Renata Kasprzyk , Natalia Baran , Pawel J.
Sikorski Joanna Kowalska and Jacek Jemielitya,*
a
c,*
,
a
Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097 Warsaw, Poland
Faculty of Chemistry, University of Warsaw, L. Pasteura 1, 02-093 Warsaw, Poland
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, L.
b
c
Pasteura 5, 02-093 Warsaw, Poland
d
College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw,
Banacha 2c, 02-097 Warsaw, Poland
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Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-
methylguanine nucleotides targeting cap-dependent translation
Sebastian Golojuch , Michal Kopcial , Dominika Strzelecka , Renata Kasprzyka,c,d, Natalia Barana,e,
a,b
a,c,d
c
a
c,
a,
Pawel J. Sikorski , Joanna Kowalska and Jacek Jemielity
a
Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097 Warsaw, Poland
Faculty of Chemistry, University of Warsaw, L. Pasteura 1, 02-093 Warsaw, Poland
b
c
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, L. Pasteura 5, 02-093 Warsaw, Poland
d
College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland
Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland
e
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating
translational control of the cell cycle. mRNA 5’ cap analogs targeting eIF4E are small molecules
with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical
utility of typical cap analogs is limited because of their reduced cell membrane permeability.
Transforming the active analogs into their pronucleotide derivatives is a promising approach to
Available online
7
overcome this obstacle. 7-Benzylguanosine monophosphate (bn GMP) is a cap analog that has
been successfully transformed into a cell-penetrating pronucleotide by conjugation of the
phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is
applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides.
7
Keywords:
We report the synthesis of six new tryptamine conjugates containing N -methylguanosine mono-
pronucleotide
and diphosphate and their analogs modified with thiophosphate moiety. These new potential
pronucleotides and the expected products of their activation were characterized by biophysical
and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit
translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract.
The results suggest that compounds containing the thiophosphate moiety may act as
pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5’-
7
-methylguanosine
tryptamine
eIF4E
DcpS
translation inhibition
mRNA cap
7
phosphorothioate (m GMPS), which is a translation inhibitor with in vitro potency higher than
7
bn GMP.
2
019 Elsevier Ltd. All rights reserved.
—
——

Corresponding author. Tel.: +48-22-55-43774; e-mail: jkowalska@fuw.edu.pl

Corresponding author. Tel.: +48-22-55-43774; e-mail: j.jemielity@cent.uw.edu.pl

1
. Introduction
negatively charged triphosphate chain, which significantly impairs
the cell membrane permeability. Unfortunately, except few
examples, typical nucleotide delivery strategies (pronucleotides)
enable delivery of only monophosphorylated nucleoside
Targeting translation initiation is one of viable strategies to
1
fight cancer. One of various approaches explored towards this
goal is aiming at the eukaryotic translation initiation factor 4E
8–10,18,19
derivatives.
2
(eIF4E). eIF4E is a part of the translation initiation complex that
directly interacts with 7-methylguanosine 5’ cap during translation
initiation, which eventually leads to the recruitment of ribosomes
onto mRNA. It has been suggested that translational inhibitors
targeting eIF4E may counteract elevated levels of eIF4E in cancer
cells by selectively downregulating translation of cell-cycle
However, it has been reported that replacing the methyl group
7
in 7-methylguanosine 5’-monophosphate (m GMP) with a bulkier
3
benzyl substituent increases binding affinity for eIF4E from 5.7-
2
0,21
7
to 9.4-fold.
Based on this result Li et al. developed bn GMP
conjugate with tryptamine – 4Ei-1, as a pronucleotide that releases
7
8
controlling mRNAs that contain structured 5’ UTRs (so-called
bn GMP after being taken up by the cells. The release of the active
nucleotide from the prodrug is possible via P-N bond cleavage by
weak mRNAs).4 Many experimental strategies have been
7
employed to develop compounds that inhibit eIF4E-dependent
translation in cancer cells, including antisense oligonucleotides
inhibiting eIF4E expression, inhibitors disrupting its interaction
histidine triad nucleotide binding protein-1 (HINT1). Ghosh et al.
reported that 4Ei-1 prevents the epithelial-to-mesenchymal
2
1
transition without eliciting toxicity in zebrafish embryos. Next,
it has been discovered that the mechanism of 4Ei-1 action is based
on blocking ribosome recruitment to the Snail1 transcript
either with mRNA 5’ cap or scaffold protein eIF4G, and eIF4E-
targeting PROTACs.5–14 7-Methylguanosine nucleotides have
2
2
been often the starting point for the design of compounds targeting
the cap-binding site of eIF4E.14–17 Unfortunately, obtaining drug-
like cap-derived inhibitors is difficult because recognition of the
cap by eIF4E is highly dependent on the interaction with
preventing accumulation of the Snail1 protein in the nucleus. It
has been also shown that treatment of breast and lung cancer cell
lines with 4Ei-1 results in chemosensitization to gemcitabine and
8
induced eIF4E proteasomal degradation.
7
7
7
m GMP derivatives
m GDP derivatives
bn GMP derivatives
NH
NH
O-
N
O-
N
H3C
N+
H3C
NH
O-
N+
N
N
N+
N
O
O
P
O-
O
P
N
N
NH2
NH2
O
*
HN
O
*
X-
O
N
HN
P
O
N
NH2
O
O
X-
HN
P
O-
O
O
OH OH
OH OH
4: X = O
5a, 5b: X = S (*two diastereomers)
OH OH
1
: X = O
4
Ei-1
2
a, 2b: X = S (*two diastereomers)
O-
O-
3
H C
O-
H3C
N+
N+
N+
N
N
N
O
O
P
O-
O
P
N
N
N
NH2
O
N
NH2
N
HO
O
*
X-
O
N
NH2
HO
P
X-
O
O
O
HO
P
X-
O
O
OH OH
OH OH
OH OH
7
7
m GMP: X = O
m GDP: X = O
7
7
: X = O (bn GMP)
7
3
: X = S (m GMPS)
6a, 6b: X = S (*two diastereomers)
7
8
: X = S (bn GMPS)
Figure 1. Compounds studied in this work.
In this work, we aimed to preliminarily explore whether the
tryptamine conjugation strategy is applicable to other m GMP-
the identity of the released metabolites to identify compounds
suitable for further biological evaluation.
7
derived inhibitors of eIF4E (Figure 1). In particular, we were
7
2. Results and discussion
interested in 7-methyguanosine 5’-phosphorothioate (m GMPS),
7
which is an m GMP analog, in which the phosphate group has been
2
.1. Synthesis of 7-methylguanosine analogs and tryptamine-
replaced with 5’-thiophosphate. We expected that such
modification may alter the susceptibility to enzymatic degradation
of the pronucleotide and the corresponding active compound as
well as increase binding affinity for eIF4E protein, as previously
described for 7-methylguanosine 5’-triphosphate analogs.16 This
in turn may affect both the release profile as well as the potency of
the active metabolite. Moreover, we also investigated whether the
tryptamine-conjugation could provide access to pronucleotides of
nucleoside 5’-diphosphates. To these ends, we synthesized a series
of novel tryptamine conjugates with m GMP, m GDP and their
phosphate-modified analogs (Figure 1). We studied the affinity of
those conjugates for eIF4E along with reference compounds that
were the expected to be released as the active metabolites. We also
studied the susceptibility of those compounds to degradation by
enzymes present in blood serum and intracellular histidine triad
hydrolases, Decapping Scavenger (DcpS) and histidine triad
nucleotide binding protein-1 (HINT1). Finally, we investigated the
degradation rates of the compounds in HEK293 cell extracts and
modified nucleotides
The synthesis of tryptamine conjugates 1, 2a and 2b, was first
attempted by a one-pot procedure consisting of phosphorylation or
thiophosphorylation of 7-methylguanosine with the use of
phosphoryl or thiophosphoryl chloride (POCl3 or PSCl3),
respectively, followed by addition of tryptamine and hydrolysis
(Figure 2A). This approach afforded a diastereomeric mixture of
compounds 2a and 2b in 12% isolated yield, while compound 1
was produced only in trace amounts. The diastereomers 2a and 2b
were separated using RP-HPLC and named D1 and D2 or a and b
according to their elution order. The absolute configurations (RP
7
7
and S ) for these compounds remain to be assigned. Compound 1
P
was obtained more efficiently by reacting 7-methylguanosine
P-imidazolide with excess tryptamine (Figure 2B). To that end,
various conditions (DMF, DMSO, and aqueous buffers of various
pH as solvents, various tryptamine excess, the presence of MgCl2
or ZnCl as a reaction mediator) were tested to find that the optimal
2
conversion is achieved over 48 h in 1 M HEPES buffer pH 8.0 with

3
equiv. of tryptamine. Using this approach, compound 1 was
The first method is based on the quenching of intrinsic
isolated in 24% yield. Similar conditions were then employed to
obtain tryptamine conjugates 4, 5a, 5b and 4Ei-1 from
corresponding phosphroimidazolides (Figure 2B); all compounds
were obtained with good conversions and satisfactory isolated
yields (24–32%; for details see Experimental section).
Noteworthy, to enable the synthesis of compounds 5a and 5b we
first had to develop a protocol for the synthesis of imidazolide
derivative of 7-methylguanosine 5’-(1-thiodiphosphate) (9a and
fluorescence of tryptophan residues (λem 337 nm) in eIF4E cap-
2
8
binding pocket upon ligand binding. A typical binding curve for
7-methylguanosine nucleotide is shown in Fig. 3A; the
fluorescence intensity decreases upon titrating eIF4E with
increasing ligand concentration, and after reaching a plateau, the
7
fluorescence increases due to the emission of the free ligand (m G
emission with λmax ~370 nm). The method delivers highly accurate
and reproducible results and there is a lot of affinity data available
in the literature that can be used as a reference. Here, tsFQT
method was used to evaluate the affinity of compounds 3, 6a, 6b,
9
b), which is reported here for the first time (Figure S1A). Despite
our efforts, we were not able to obtain the tryptamine conjugate of
7
7
bn GMPS, which was unstable in our hands and easily
7, 8 and m GTP for eIF4E (Figure 3A and 3B, Table S1).
decomposed upon storage and in aqueous solution.
Unfortunately, in the case of tryptamine-containing compounds 1,
2
a, 2b, 4, 5a and 5b the fluorescence of tryptamine fluorophore
A
O-
O-
O-
H3C
N+
H C
3
H3C
N+
interfered strongly with the tryptophan emission, disabling proper
determination of the binding affinity constants. Hence, to evaluate
the affinity of these compounds, PyFLINT-B assay was applied.
N+
N
N
N
i
ii
S
O
P
S-
N
N
NH2
N
N
NH2
N
N
NH2
HO
Cl
P
O
HN
O
29
O
O
O
Cl
OH OH
7
OH OH
OH OH
2a, 2b
The PyFLINT-B method is based on measuring changes of pyrene
fluorescence in a pyrene-labeled probe upon its competitive
displacement from eIF4E by the studied ligand. The method is less
precise and less accurate than tsFQT, but is a reliable tool for
screening and comparing relative affinities of large sets of ligands.
Therefore, compounds 1-8 and corresponding reference
compounds were subjected to evaluation by PyFLINT-B. The
resulting competitive binding curves were subsequently used to
m G
N
H
O-
O-
N
B
R
O
R
O
N+
N
N+
N
N
N
O
O
iii
O
O
N
N
NH2
NH2
N
P
O-
O
P
X-
O
HN
P
O-
O
P
X-
O
n
n
OH OH
OH OH
7
m GMP-Im: n = 0, R = H, X = O
1: n = 0, R = H, X = O
7
N
m GDP-Im: n = 1, R = H, X = O
H
4: n = 1, R = H, X = O
9
a, 9b: n = 1, R = H, X = S
0: n = 0, R = Ph, X = O
5a, 5b: n = 1, R = H, X = S
4Ei-1: n = 0, R = Ph, X = O
D
calculate the K values (Figure 3C and 3D, Figure S3A, Table S1)
as described previously. For compounds that were evaluated
using both tsFQT and PyFLINT-B methods we observed that the
1
29
Figure 2. Synthesis of tryptamine-modified nucleotides. A) Synthesis of 2a
and 2b, conditions: (i) PSCl lutidine, trimethyl phosphate, 0°C,
ii) 1. tryptamine, 2. (TEAH) B) Coupling P-imidazolides with
3
,
D
K values from PyFLINT-B were consistently higher than those
(
2
CO3 aq.;
from tsFQT. The discrepancies may be explained by lower
accuracy of the PyFLINT-B assay and differences in the
experimental set-ups and models applied to analyze the data (the
model for tsFQT assay includes the active protein concentration as
one of the parameters, whereas PyFLINT-B assay does not).
Nonetheless, the relative compound affinities determined by both
assays were in good agreement. For all studied compounds the
binding affinity increased together with increasing of negative net
charge of the molecule (Figure 3B and 3D), which is in agreement
with previous observations reported for phosphate-unmodified cap
analogs and is associated with an important role of the negatively
charged triphosphate chain in the interaction of mRNA cap with
tryptamine, conditions: (iii) tryptamine, 1 M HEPES buffer, pH 8.0, rt.
Intermediate nucleotide analogs 10 and 11 were synthesized
using previously reported methods with minor modifications.16,23
7
5
-Methylguanosine 5’-thiophosphate (3), and 7-benzylguanosine
’-thiophosphate (8) were obtained from corresponding
7
N -alkylguanosine using PSCl
3
in trimethyl phosphate at 0°C
7
followed by hydrolysis, as previously reported for m GMPS with
minor modifications (Figure S1B).16 Compounds 6a and 6b were
obtained as a mixture of diastereomers from compound 3 via
2
4
coupling reaction with cyanoethyl protected P-imidazolide.
7
7
7
7
Additionally, bn GMP (7), m GMP, m GDP, m GTP and
3
0
7
eIF4E.
m GpppG were synthesized as reference compounds using
reported methods.2
0,25–27
Among nucleoside monophosphates (NMP), which are the
expected products of activation of monophosphorylated prodrugs
2
.2. Affinity to eIF4E
7
4
Ei-1 and 1, compound 7 (bn GMP) had 2.6-fold higher affinity
Binding affinities for murine eIF4E were measured using two
7
D
for eIF4E than m GMP (K 0.460 ± 0.092 and 1.24 ± 0.10 µM by
independent assays: time-synchronized fluorescence quenching
titration (tsFQT) and fluorescence intensity binding assay based
on a pyrene-labeled probe (PyFLINT-B; Figure 3A–D, Figure S3).
tsFQT, respectively; Figure 3B). This finding is in qualitative
agreement with the previous observations that replacing the
methyl in m GMP with benzyl stabilizes the interaction with
7

eIF4E, albeit our result indicate that the stabilization is weaker
than previously reported.20,21 Notably, compound 3 (m GMPS),
carrying the 5’-thiophosphate moiety, had even higher binding
diphosphates
α-thiophosphate modification 6a (K
(K 0.0244 ± 0.0011 µM) had respectively 2.8- and 2.0-fold higher
affinity than m GDP (K
observation has been previously reported for m GTPαS D1,
(NDP),
two
diastereomers
0.0177 ± 0.0007 µM) and 6b
containing
7
D
D
7
7
affinity, which was 4.1-fold higher than that of m GMP and 1.6-
D
0.0490 ± 0.0036 µM). A similar
7
fold higher than that of 7 (K
D D
0.302 ± 0.029 µM). Compound 8 (K
7
7
7
0
.344 ± 0.047 µM), combining the 5’-thiophosphate and N -benzyl
m GTPαS D2, and m GTP, which we also confirmed in our assay
had affinity that was comparable to 7, which indicates that the
complex stabilizing effects of N -benzyl and thiophosphate
(Table S1, Figure 3B, Figure S2) as well as corresponding
m GpppG analogs.
7
7
16,31
modifications are not additive. In the group of nucleoside
Figure 3. Determination of dissociation constants with eIF4E, translation inhibition and DcpS inhibition of the studied compounds; A) tsFQT curves for eIF4E;
B) K
D
values determined for eIF4E at 20C using tsFQT method; C) representative binding curves from PyFLINT-B assay for eIF4E; D) K
D
values determined at
2
0C using PyFLINT-B assay; E) curves from inhibition of cap-dependent translation by the studied 7-methylguanine nucleotides in RRL; F) IC50 values
a
7
7
7
7
16,30
determined from inhibition of cap-dependent translation; KD, tsFQT values for m GMP, m GDP, m GTPS D1 and m GTPS D2 were taken from literature.
indicates K values > 10 µM.
“*”
D
7
Overall, the thiophosphate moiety increased the binding
affinity compared to the unmodified counterpart for all compounds
except 8.
m GDP, making the difference even higher than the differences in
values for eIF4E obtained by the tsFQT assay.
K
D
As expected, the tryptamine-conjugated nucleotides had a
lower binding affinity for eIF4E compared to the expected
products of their enzymatic activation (Figure 3D). Among
monophosphates (NMP), K value for all tryptamine-modified
D
compounds determined by PyFLINT-B was higher than 10 μM. In
2
.4. Susceptibility to degradation and activation by cleavage
We next investigated the enzymatic degradation of tryptamine
conjugates by general and specific phosphohydrolases. We first
determined the general stability of tryptamine conjugates in fetal
bovine serum (FBS), which contains various nucleolytic enzymes,
including pyrophosphatases that are responsible for the
degradation of nucleotide derivatives present in the blood. FBS has
been previously used to study the general stability of modified
the group of diphosphates (NDP), tryptamine-conjugated
compounds 4 (K
b (K 0.49 ± 0.14 µM) had, respectively, 8.1-, 3.3- and 7.0- fold
lower affinity to eIF4E than m GDP (K
D D
0.57 ± 0.01 µM), 5a (K 0.23 ± 0.08 µM) and
5
D
7
D
0.07 ± 0.02 µM), but were
all tighter binders than 7. Hence, it is likely that these compounds
could show biological activity even in the form of tryptamine-
conjugates. The decreased affinity for tryptamine-modified
nucleotides is likely caused by the charge-masking effect of
tryptamine attachment.
32
nucleotides under physiological conditions. To evaluate the
general stability conjugates 1, 2a, 2b, 4, 5a, 5b and 4Ei-1 were
incubated in 10% fetal bovine serum (FBS) at 37 ºC and their
enzymatic turnover was analyzed using RP-HPLC over a period
of 4.5 h (Figure 4A). The content of each conjugate remaining at
different time-points was plotted as a function of time to determine
the half-life values (Table 1).
2
.3. Inhibition of translation
7
To verify the ability of the m GMP analogs of interest to target
All evaluated compounds were more stable in FBS than
eIF4E in a more complex biological system we tested their potency
to inhibit cap-dependent translation in cell lysate (Figure 3E, 3F,
Table S2). The method is based on measurements of luminescence
of luciferase enzyme translated from ARCA-capped mRNA in a
micrococcal nuclease-treated rabbit reticulocyte lysate (RRL). The
inhibition of cap-dependent translation is expected to reduce the
amount of biosynthesized luciferase, which in turn leads to
decrease of luminescence intensity.
7
m GpppG dinucleotide used as a reference in this experiment (t1/2
6
were less stable than corresponding monophosphates, with
phosphate-unmodified compound 4 being the least stable (t1/2 111
7 ± 2 min). Among the tryptamine conjugates, the diphosphates
±
3 min). Analogously phosphate-unmodified compound 1 was the
least stable among monophosphates (t1/2 290 ± 15 min). The
presence of benzyl substituent in compound 4Ei-1 (t1/2 380 ± 23
min) slightly increased the stability compared to compound 1.
However, the highest stability was observed for compounds 2a and
2b containing the thiophosphate modification, which were
virtually unhydrolyzable under the assay conditions (t1/2 >> 80 h
for both). The diphosphates containing α-thiophosphate
modification 5a (t1/2 2773 ± 476 min) and 5b (t1/2 736 ± 44 min)
were less stable than monophosphates but showed higher stability
than all phosphate-unmodified compounds.
The IC50 values obtained in this assay correlated qualitatively
with the K
in potency of inhibition between nucleoside monophosphates
NMP) and nucleoside diphosphates (NDP) were less pronounced
D
values from eIF4E binding assay, albeit the differences
(
than in the case of affinity studies (Figure 3B and 3D). This may
suggest that other factors besides affinity for eIF4E influence the
potency to inhibit translation, such as susceptibility to enzymes
present in RRL. Compound 7 (IC50 14 ± 4 µM) was 1.4-fold better
Phosphoramidate prodrugs are typically converted inside cells
7
inhibitor than m GMP (IC50 20 ± 7 µM), making the difference
3
3
into active metabolites by HINT1. HINT1 is histidine triad (HIT)
superfamily enzyme considered as the primary enzyme
responsible for the hydrolysis of P-N bonds in nucleotides and has
been shown to convert 4Ei-1 to bn GMP in MDA-MB-231, H460,
H838 and H2009 cells. The susceptibility of tryptamine
conjugates 1, 2a, 2b, 4, 5a, 5b to HINT was assesed using an
HPLC-based assay (4Ei-1 was also included in the assay as a
reference). The compounds (50 μM) were incubated with HINT1
D
smaller than expected from comparison of K values for eIF4E and
may suggest problems with selectivity of compound 7 in
interaction with eIF4E and ability of 7 to either bind to other
proteins or undergo degradation. Compound 3 (IC50 5.2 ± 2.0 µM)
7
8
7
was 3.8-fold better inhibitor compared to m GMP, which is in
good agreement with the results obtained from tsFQT assay.
Surprisingly, compound 8 (IC50 1.4 ± 0.4 µM) turned out to be the
strongest inhibitor in the group of nucleoside monophosphates,
(20 nM for 4Ei-1 and 1, 200 nM for 2a, 2b, 4, 5a, 5b) and reaction
7
being 14.3-fold more potent than m GMP and 2.1-fold more potent
progress over time was monitored using RP-HPLC with UV
detection (Figure 4B, S4, Table 1). Among the analyzed
compounds, monophosphates, 4Ei-1 and 1, were hydrolyzed most
rapidly. In the case of monothiophosphates (2a and 2b), much
7
than m GDP (IC50 2.9 ± 1.1 µM). In the group of nucleoside
diphosphates, two diastereomers containing α-thiophosphate
modification 6a (IC50 0.28 ± 0.14 µM) and 6b (IC50 0.52 ± 0.08
µM) were respectively 10.4- and 5.6-fold better inhibitors than

slower hydrolysis was observed, even though the HINT1
concentration was increased by 10-fold. All diphosphates (4, 5a,
potentially be activated via HINT1-catalyzed P-N bond cleavage.
However, compounds 2a and 2b are hydrolyzed at a much slower
rate, which suggests that a completely different release profile can
be expected for those compounds compared to 4Ei-1 and 1.
5
b) were virtually unhydrolyzable under studied conditions. The
results suggest that only pronucleotides 4Ei-1, 1, 2a and 2b can
7
Figure 4. A) Stability of pronucleotides and m GpppG (reference) in 10% fetal bovine serum (FBS); B) Susceptibility of pronucleotides 1 and 2b to hydrolysis by
HINT1. C) Susceptibility of pronucleotides 1 and 2b to hydrolysis by DcpS.
Noteworthy, 7-methylguanine nucleotides are targeted in the
cell by unique degradation pathways, which involve the action of
HPLC with UV detection (Figure 4C, S5, Table 1). Under
conditions in which m GpppG was fully hydrolyzed within 30
7
7
specialized decapping enzymes. Among them, DcpS is an m G-
minutes, only compound 1 was slowly degraded. All other
evaluated compounds were virtually unhydrolyzable. This
indicated that DcpS likely cannot activate these compounds inside
the cells. Thus, the most plausible potential activation pathway for
compounds 1, 2a and 2b is via HINT1 enzyme, whereas for
compounds 4, 5a, 5b is either via chemical hydrolysis or some
other yet to be identified enzymatic activities.
7
specific histidine triad pyrophosphatase hydrolyzing m G-derived
mono- and dinucleotides.34 Hence, we envisaged that the cleavage
7
by DcpS may be an alternative pathway for the conversion of m G-
containing pronucleotides into their active forms. To test this
hypothesis, we studied the susceptibility of compounds 1, 2a, 2b,
4
, 5a, 5b and 4Ei-1 to hydrolysis by recombinant human DcpS.
7
35
m GpppG, a known DcpS substrate, was used as a reference. The
compounds (30 μM) were incubated with DcpS (30 nM) and
sample composition as a function of time was monitored using RP-
Table 1. Susceptibility of obtained tryptamine conjugates and reference compound to enzymatic degradation.
a
b
c
Compound
Half-life in 10% FBS (min)
Susceptibility to hydrolysis by DcpS
Slowly hydrolyzed
Resistant
Susceptibility to hydrolysis by HINT1
1
290 ± 15
Hydrolyzedd
2
2
4
5
5
4
a
>5000
Slowly hydrolyzede
Slowly hydrolyzede
Resistante
b
>5000
Resistant
111 ± 3
Resistant
a
2773 ± 467
736 ± 44
Resistant
Resistante
b
Resistant
Resistante
Ei-1
380 ± 23
Resistant
Hydrolyzedd
7
m GpppG
67 ± 2
Hydrolyzed
Not determined
aDegradation was monitored using RP-HPLC (method A3). Concentrations and conditions: 10% (v/v) FBS, 20% (v/v) PBS (pH 7.4, 140 mM NaCl, 2.7 mM KCl,
0 mM Na HPO , 2 mM KH PO ), 50 M analyzed compound. Further details can be found in experimental section.
1
2
4
2
4
bDegradation was monitored using RP-HPLC (method A3). Concentrations and conditions: 30 μM compound, 30 nM hDcpS, 50 mM Tris-HCl, pH = 7.6, 200
mM KCl, 0.5 mM EDTA buffer at 30C. Further details can be found in experimental section.
c
d
e
Degradation was monitored using RP-HPLC (method A3). Concentrations and conditions: 50 μM compound, 20 nM or 200 nM HINT1, 20 mM HEPES, 1
mM MgCl , pH 7.2 buffer at 25C. Further details can be found in experimental section.
2
2
.5. Inhibition of DcpS
Knowing that the tryptamine conjugates are resistant to
hydrolysis by DcpS, we evaluated them as inhibitors of this

enzyme to determine whether they could target it in vivo in
addition to eIF4E. The inhibitors were evaluated using a
previously developed hydrolytic activity assay based on pyrene-
labeled probe (PyFLINT-HA; Figure 5, Table S3).29 The method
is based on measuring the changes in fluorescence of a pyrene-
labelled turn-on probe occurring upon hydrolysis by DcpS
end, compounds 1, 2a, 2b, 4, 5a, 5b, and 4Ei-1 were incubated in
the extract and aliquots taken at different time-points were
analyzed using LC-MS/MS to determine the amount of remaining
substrate and identify metabolites. The compounds and expected
metabolites (m GMP, 3, m GDP, 6a, 6b, 7, among others) were
quantified based on concentration-dependent calibration curves
(Figure S6).
7
7
2
9
enzyme.
As found previously, 4Ei-1 underwent fast hydrolysis occurring
8
via P-N bond cleavage to release 7. Compound 7 was stable in the
cell extract and accumulated overtime proportionally to 4Ei-1
consumption. Compound 1 also underwent fast conversion into
7
m GMP (Figure 6A), which in contrast to 7 was not stable and
after initial accumulation underwent further degradation. These
7
data indicate that m GMP has significantly lower metabolic
7
stability than 7 (bn GMP). Compounds 2a and 2b were degraded
significantly slower than 4Ei-1 or 1 (Figure 6B and 6C,
respectively), wherein 2b was turned over faster than 2a. The
7
initial product of the degradation of 2b was 3 (m GMPS), but it
7
did not accumulate over time; small concentrations of m GMP
Figure 5. A) representative inhibition curves from PyFLINT-HA activity assay
for DcpS inhibition; B) IC50 values determined from the PyFLINT-HA assay
for DcpS. “*” indicates K values > 100 µM.
I
were also detected. These observations may suggest that 3
generated from 2b, undergoes subsequent slow conversion into
m GMP, which is then rapidly metabolized further. Indeed, it has
been reported that HINT1 – the enzyme responsible for cleavage
of phosphoramidate bonds in nucleotides is also responsible for
7
Among the studied compounds, nucleoside diphosphates
NDP) were better inhibitors than nucleoside monophosphates
NMP; Figure 5B). Introducing benzyl modification notably
(
(
the
intracellular
transformation
of
nucleoside
5’-
decreased inhibition potency as observed for compounds 4Ei-1
and 8 (K values > 100 µM). The introduction of tryptamine moiety
into the nucleoside monophosphate disrupted the inhibition as well
36
monothiophosphates into nucleoside 5’-monophosphates.
Therefore, it is likely that HINT1 is responsible for converting 3
into m GMP in addition to cleaving P-N bond in 2a. In contrast to
the monophosphate conjugates (1, 2a and 2b), the diphosphates (4,
I
7
7
(
compound 1 K
Surprisingly, combining tryptamine modification with
thiophosphate modification in the case of compound 2a (K 9.68 ±
.23 µM) caused 1.4-fold increase of inhibitory potency compared
I I
> 100 µM versus m GMP K 13.8 ± 1.4 µM).
5
a and 5b) can potentially be degraded via two pathways, which
I
involve either the P-N bond cleavage or pyrophosphate bond
cleavage. Thus, the potential metabolites of both these pathways
were monitored by LC-MS. Compound 4 was degraded at a rate
1
to (S
7
P
)-m GDPαS (6a). In contrast, compound 2b (K
in regard to (R )-m GDPαS
6b). The strongest inhibitor in the group of monophosphates was
9.3 ± 0.9 µM), which was 1.5-fold
better inhibitor than m GMP. In the group of diphosphates (NDP),
I
24.5 ± 2.6
7
µM) showed 1.8-fold increase of K
(
compound 3 (m GMPS; K
I
P
7
7
comparable to compound 2b, wherein both m GDP and m GMP
were observed as metabolites. However, comparable levels of
m GDP were also observed in control samples (timepoint 0),
which may indicate that this compound comes solely from the
chemical degradation of 4, while the primary enzymatic
degradation pathway occurs via pyrophosphate bond cleavage
producing m GMP. However, m GMP accumulation was not
observed due to rapid further degradation, similarly as in the case
of compound 1. The α-thiophosphate modification only slightly
stabilized conjugates 5a and 5b but influenced the composition of
metabolites. In the case of compound 5a the sole observed
metabolite was 3. In the case of compound 5b, the presence of 6
was also observed, but it was likely a result of chemical
degradation, similarly as in the case of compound 4.
7
I
7
7
introduction of tryptamine modification increased inhibitory
7
potency by 1.7-fold (compound 4, K
I
0.41 ± 0.04 versus m GDP,
K
I
0.68 ± 0.07 µM), making compound 4 the strongest inhibitor
7
7
among all studied compounds. The introduction of
α-thiophosphate modification within the diphosphate moiety had a
negative impact on inhibition of DcpS for both diastereomers.
I I
Compounds 6a (K 0.87 ± 0.09 µM) and 6b (K 3.11 ± 0.31 µM)
7
were, respectively, 1.3-fold and 4.6-fold less potent than m GDP.
The combination of α-thiophosphate modification with tryptamine
modification caused a slight improvement of inhibitory properties
I I
of 5a (K 0.73 ± 0.11 µM) and 5b (K 0.74 ± 0.07 µM), which both
were more potent than 6a and 6b, respectively.
These results indicate that all studied phosphate-modified
tryptamine conjugates, are metabolized slower than 4Ei-1 and that
they release low, but constant levels of m GMPS (3) as the primary
metabolite. This fact, combined with their exceptionally high
stability in blood serum, makes them potentially useful as
pronucleotides showing sustained-release effect. In turn, the
phosphate-unmodified conjugates of 7-methylguanine rapidly
release m GMP, which undergoes further degradation in contrast
to bn GMP released from 4Ei-1. This fact emphasizes the
importance of the metabolic stability of bn GMP in its efficiency
Overall, the data indicate that in contrast to interaction with
eIF4E, the charge within phosphate moiety is not essential for
interaction with DcpS enzyme and introducing charge-masking
tryptamine to phosphate moiety does not always cause a decrease
in inhibition potency. Thus, some of the tryptamine conjugates
7
(2a, 2b, 4, 5a, 5b) may target DcpS in the cell. Nonetheless, the
7
comparison of K values for DcpS and K values for eIF4E of the
I
D
7
7
7
potential metabolites (m GMP, 3, m GDP, 6a, 6b, 7) indicates that
after hydrolytic removal of the tryptamine moiety the resulting
nucleotides should exhibit selectivity towards eIF4E.
7
for targeting eIF4E.
Finally, to compare the cell membrane permeability of
our conjugates (1, 2a, 2b, 4, 5a, 5b) with 4Ei-1 we applied
Artificial Membrane Permeability Assay (PAMPA). The classic
2
.6. Metabolism in HEK-293 extract and artificial cell membrane
permeability
8
PAMPA utilizing 1% lecithin in dodecane coated PVDF
membrane has been previously shown to be useful to benchmark
permeability of pronucleotides. To determine the concentrations
To gain more insight into the potential degradation pathways of
our compounds inside cells, we studied their turnover in human
embryonic kidney 293 cell extract (HEK-293) (Figure 6). To that
3
7

7
of compounds on both sides of the membrane, HPLC with UV
detection was applied. Unfortunately, for all compounds,
including 4Ei-1, the concentration of compounds that penetrated
the membrane was below the limit of detection for the applied
method (data not shown). This observation may suggest a different
mechanism of permeation for 4Ei-1 than passive diffusion or assay
bias in the case of compounds with such structure leading to false-
negative result as has been previously observed for Acyclovir.38
the 5’-phosphate moiety in m GMP with 5’-thiophosphate moiety
stabilizes the interaction with eIF4E and the effect is even stronger
than that previously reported for the N -benzyl group, which was
used in the first tryptamine conjugated eIF4E-targeting
pronucleotide, 4Ei-1. Second, we show that the pronucleotides
containing the O-to-S substitution in the phosphate moiety have
higher stability in blood serum than 4Ei-1 and are also not
7
7
degraded by the cellular N -methylguanosine specific
pyrophosphatase, DcpS, but are slowly hydrolyzed by HINT1.
Finally, we show that the phosphate-modified tryptamine
Therefore, further studies are needed to evaluate the cellular
uptake and the potential of these novel-tryptamine conjugates to
inhibit eIF4E in vivo.
conjugates are metabolized in HEK-293 cell extract slower than
7
4
Ei-1 and that they release m GMPS as the main metabolite. We
thus envisage that compounds 2a and 2b, as well as 5a and 5b
could be potentially employed as pronucleotides releasing
7
m GMPS in a sustainable manner. In turn, the phosphate
unmodified conjugates of 7-methylguanine rapidly release
7
m GMP, which undergoes further degradation in contrast to
7
bn GMP released from 4Ei-1. This provides new insights into the
reasons underlying the previously reported high biological activity
of 4Ei-1, emphasizing the importance of metabolic stability of
7
bn GMP for efficient eIF4E targeting. Further studies are needed
to evaluate the potential of the novel-tryptamine conjugates as
translational inhibitors targeting eIF4E in vivo.
4
. Experimental
4
.1. Starting materials and chemical reagents
Guanosine and guanosine 5’-monophosphate (sodium salt)
were purchased from Carbosynth company. Iodomethane,
thiophosphoryl chloride, 2,6-dimethylpyridine, tryptamine, benzyl
bromide, imidazole, triphenylphosphine, 2,2’-dithiodipyridine,
triethylamine, and sodium perchlorate were purchased from
Sigma-Aldrich company. HEPES was purchased from Carl Roth
Company. Sodium bicarbonate and ammonia solution were
purchased from Chempur company. Solvents were purchased from
Sigma-Aldrich Company and used without further purification.
7
7
m G, ammonium and/or triethylammonium salts of bn GMP (7),
7
7
7
7
7
m GTP, m GDP, m GMP, m GpppG, m GMPS (compound 3),
7
m GDPS S
P
and R
P
(compounds 6a, 6b, respectively), sodium
7
7
salts of m GMP-Im and m GDP-Im were obtained as described in
2
0,23–25,39
the literature or similarly.
For syntheses under anhydrous
conditions solvents were additionally dried over 4 Å molecular
sieves over 24 h.
4
.2. Conversion of compounds into triethylammonium salts
The sodium salt of GMP purchased from a commercial supplier
was converted into triethylammonium salt passing its aqueous
+
3
solution (ca. 1 g/20 mL) through Dowex 50W X8 (Et NH form).
The collected eluate was evaporated under reduced pressure with
repeated additions of ethanol and acetonitrile to dryness yielding
the triethylammonium salt of GMP as a white solid.
4
.3. Ion-exchange chromatography (IEC)
Figure 6. Metabolism of tryptamine conjugates in HEK 293 cell extract
7
monitored using LC-MS/MS. A) Compound 1 and metabolite – m GMP; B)
Synthesized nucleotides were purified by ion-exchange
7
7
Compound 2a and metabolites: 3 (m GMPS) and m GMP; C) Compound 2b
-
chromatography on DEAE Sephadex A-25 (HCO
3
form)
7
7
and metabolites: 3 and m GMP; D) Compound 4 and metabolites: m GDP and
pressurized column. After loading the column with reaction
mixture and washing it with water, the products were eluted using
different gradients of TEAB in deionized water: 0 - 0.7 M for
7
7
7
m GMP; E) Compound 5a and metabolites: 6a (m GDPS D1), 3 and m GMP.
F) Compound 5b and metabolites: 6b (m GDPS D2), 3 and m GMP. G)
Reference compound 4Ei-1 and metabolite – 7 (bn GMP).
7
7
7
nucleoside monophosphates,
0 - 1.0 M for nucleoside
diphosphates or 0-1.2 M for nucleoside triphosphates. Fractions
containing the desired product were collected together after
spectrophotometric (UV-detection at 260 nm) and RP HPLC
analysis. Evaporation under reduced pressure with repeated
additions of 96% and then 99.8% ethanol resulted in the isolation
of nucleotide analogs as triethylammonium salts.
3
. Conclusions
In this work, we investigated the properties of tryptamine
conjugates with 7-methylguanosine 5’-monophosphate and 5’-
diphosphate analogs as potential tools for delivery of these
nucleotides into living cells. First, we demonstrate that replacing

4
.4. Reversed phase (RP) HPLC
1H), 6.96 (t, J = 7.8 Hz, 1H), 6.80 (t, J = 7.8 Hz, 1H), 5.82 (d, J =
4
–
.1 Hz, 1H), 4.40 (t, J = 4.5 Hz, 1H), 4.21 (t, J = 4.5 Hz, 1H), 4.14
Analytical HPLC was performed on Agilent Tech. Series 1200
using Supelcosil LC-18-T HPLC column (4.6 x 250 mm, flow rate
4.09 (m, 1H), 3.78 (s, 3H), 2.96 (dt, J = 17.1, 7.9 Hz, 2H), 2.71
31
(t, J = 7.9 Hz, 2H); P NMR (162 MHz, DMSO-d
6
, 25C, proton
1
1
5
.3 mL/min). MeOH + 0.05 M ammonium acetate buffer (pH 5.9)
:1 (v/v) gradient in 0.05 M ammonium acetate buffer (pH 5.9); 0-
0% in 7.5 min (method A1); 0-100% in 7.5 min (method A2) or
MeOH + 0.1 M phosphate buffer (pH 6.0) 1:1 (v/v) gradient in 0.1
M phosphate buffer (pH 6.0); 0-50% in 15 min (method A3). UV-
detection at 254 nm.
decoupling) δ 3.96 (s).
4
.7.2. Compounds 2a, 2b
Dry 7-methylguanosine (50.1 mg, 1696 mODU, 0.149 mmol)
was suspended in trimethyl phosphate (762 μL) and placed on an
ice-bath. After cooling to 0C, 2,6-dimethylpyridine (86.2 μL,
0
μL, 0.593 mmol) were added. The reaction was stirred at 0C until
the integration of signal from 7-methylguanosine on the HPLC
profile dropped below 10%. Then tryptamine (118.7 mg,
.744 mmol) and freshly distilled thiophosphoryl chloride (60.2
All nucleotides used in biophysical and biological assays were
purified on HPLC. Preparative HPLC was performed on Agilent
Tech. Series 1200 using Discovery RP-Amide C16 HPLC column
(
21.2 x 250 mm, flow rate 5.0 mL/min) or Hichrom Vydac Denali
0
.741 mmol) was added, and the reaction mixture was stirred at
C18 HPLC column (10 x 150 mm, flow rate 5 mL/min). CH CN
3
room temperature for 2 hours. The reaction was quenched by
addition of triethylammonium bicarbonate buffer (10 mL, 1.2 M,
pH 8.5). Then the mixture was washed three times with ethyl
acetate (3 x 20 mL) and filtered using syringe filter. Two
diastereomers of the final product were separated and purified
using preparative RP-HPLC and isolated as ammonium salts (12%
gradient in 0.05 M ammonium acetate buffer (pH 5.9). UV-
detection at 254 nm.
4
.5. Yields and concentrations determination
Yields of nucleotide analogs after ion-exchange or preparative
RP-HPLC purifications and the concentrations of mono- and
dinucleotide analogs solutions used for biophysical and biological
experiments were determined on the basis of absorbance
measurements performed in 0.1 M phosphate buffer pH 6.0 for 7-
methylguanine and 7-benzylguanine nucleotide analogs and in 0.1
M phosphate buffer pH 7.0 for guanine mononucleotide analogs
and dinucleotide analogs. The quantities of obtained ion-exchange
purified products were expressed as optical density milliunits
yield). D1 (compound 2a): 4.6 mg, 0.0084 mmol; R
t
(method A1)
-
=
14.1 min; HRMS ESI (-) calculated m/z for C21
H
25
N
7
O
6
PS [M-
H] 534.13301, found 534.13336; H NMR (500 MHz, DMSO-d
5C) δ 10.73 (s, 1H), 10.08 (s, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.28
-
1
6
,
2
(d, J = 7.8 Hz, 1H), 7.08 (s, 1H), 7.00 (dd, J = 7.8 Hz, 1H), 6.87
(dd, J = 7.6 Hz, 1H), 5.86 (d, J = 3.8 Hz, 1H), 5.63 (s, 1H), 5.33
(d, J = 4.1 Hz, 1H), 4.44 (t, J = 7.9, 3.8 Hz, 1H), 4.25 – 4.20 (m,
1
–
H), 4.16 – 4.12 (m, 1H), 3.99 (s, 3H), 3.96 – 3.84 (m, 2H), 3.07
(mODU = absorbance of the solution volume in mL). For
31
2.98 (m, 2H), 2.77 (t, J = 7.6 Hz, 2H); P NMR (202 MHz,
calculations of yields and concentrations following molar
1
DMSO-d
5
calculated m/z for C21
5
1
7
6
, 25C, H decoupling) δ 55.86 (s). D2 (compound 2b):
-
1
-1
3
extinction coefficients (M cm ) were employed: ε258 = 11.4 * 10
.4 mg, 0.0098 mmol; R
t
(method A1) = 15.1 min; HRMS ESI (-)
7
7
17
3
at pH 6.0 (m G and Bn G mononucleotides),
pH 6.0 (m G and Bn G mononucleotides coupled with tryptamine,
determined via UV absorbtion measurment of standard solutions),
ε
262 = 12.1 * 10 at
-
-
H
25
N
7
O
6
PS [M-H] 534.13301, found
7
7
1
34.13327; H NMR (500 MHz, DMSO-d , 25C) δ 10.73 (s, 1H),
6
0.10 (s, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H),
.10 (s, 1H), 7.00 (t, J = 7.8 Hz, 1H), 6.87 (t, J = 7.4 Hz, 1H), 5.85
3
40
ε
1
258 = 12.08 * 10 at pH 7.0 (G mononucleotides), ε255 = 22.6 *
3
7
41
0 at pH 7.0 (m GpppG).
(d, J = 3.5 Hz, 1H), 5.65 (s, 1H), 5.45 – 5.41 (m, 1H), 4.45 – 4.41
(
1
m, 1H), 4.25 – 4.21 (m, 1H), 4.16 – 4.12 (m, 1H), 4.09 – 4.04 (m,
4
.6. NMR and MS
H), 3.99 (s, 3H), 3.80 (dd, J = 11.5, 5.4 Hz, 1H), 3.11 – 2.98 (m,
3
1
NMR spectra were recorded at 25 °C on a Bruker Avance III
2H), 2.78 (t, J = 7.5 Hz, 2H); P NMR (202 MHz, DMSO-d
25C, H decoupling) δ 56.45 (s).
6
,
1
31
1
HD at 500.24 MHz ( H NMR) and 202.49 MHz ( P NMR) or on
a Varian UNITY-plus spectrometer at 399.90 MHz ( H NMR) and
1
reported to sodium 3-trimethylsilyl-[2,2,3,3-D4]-propionate (TSP)
in D
shifts were reported to 20% phosphorus acid in D
as an internal standard. The raw NMR files were processed using
MestReNova (Mestrelab Research S.L.).
1
4
.7.3. Compound 4
3
1
1
61.89 MHz ( P NMR). The H NMR chemical shifts were
7
To sodium salt of m GDP-Im (34.9 mg, 855 mODU, 0.075
3
1
mmol) solution in HEPES buffer (635 μL, 1 M, pH 8.0),
tryptamine (31.9 mg, 0.199 mmol) was added. Then the reaction
mixture was stirred at room temperature for 48 hours. After
diluting the reaction with water (6.0 mL), the product was purified
using preparative RP-HPLC and isolated as an ammonium salt
2
O (δ = 0 ppm) as an internal standard. The P NMR chemical
O (δ = 0 ppm)
2
High-resolution mass spectra (HRMS) were recorded on
Thermo Scientific LTQ Orbitrap Velos in a negative [MS ESI (-)]
or a positive [MS ESI (+)] ion mode. Analytical (low-resolution)
mass spectra were recorded on AB Sciex API 3200 spectrometer.
(15.1 mg, 0.024 mmol, 32% yield). R (method A1) = 10.3 min;
t
-
-
HRMS ESI (-) calculated m/z for C H N O P
2
1
1
26
7
10
2
[M-H]
1
598.12219, found 598.12238;
H NMR
H NMR (400 MHz,
Deuterium Oxide, 25C) δ 8.83 (s, 1H), 7.43 (ddd, J = 7.4, 0.9 Hz,
1
8
H), 7.25 (ddd, J = 8.1, 0.9 Hz, 1H), 7.13 (s, 1H), 7.04 (ddd, J =
.1, 7.1, 1.0 Hz, 1H), 6.95 (ddd, J = 7.4, 0.9 Hz, 1H), 5.76 (d, J =
4
4
.7. Chemical syntheses
.7.1. Compound 1
2.0 Hz, 1H), 4.41 (ddd, J = 11.8, 3.6, 2.0 Hz, 1H), 4.34 – 4.29 (m,
3H), 4.20 (ddd, J = 11.8, 4.7, 1.8 Hz, 1H), 3.72 (s, 3H), 3.23 – 3.12
7
To sodium salt of m GMP-Im (48.5 mg, 1169 mODU, 0.103
3
1
(m, 2H), 2.89 – 2.82 (m, 2H); P NMR (162 MHz, Deuterium
mmol) solution in HEPES buffer (1.0 mL, 1 M, pH 8.0),
tryptamine (50.4 mg, 0.315 mmol) was added. Then the reaction
mixture was stirred at room temperature for 48 hours. After
diluting the reaction with water (10.0 mL), the product was
purified using preparative RP-HPLC and isolated as an ammonium
salt (13.1 mg, 0.025 mmol, 24% yield). R
HRMS ESI (-) calculated m/z for C21
5
2
Oxide, 25C) δ -2.88 (ddd, J = 21.0, 10.0 Hz), -11.48 (ddd, J =
2
1.0, 11.0, 4.0 Hz).
4
.7.4. Compounds 5a, 5b
To sodium salt of compounds 9a, 9b (mixture of diastereomers,
3.2 mg, 0.081 mmol) solution in HEPES buffer (735.2 μL, 1 M,
t
(method A2) = 9.7 min;
4
-
-
H
25
N
7
O
7
P
[M-H]
pH 8.0), tryptamine (35.7 mg, 0.223 mmol) was added. Then the
reaction mixture was stirred at room temperature for 48 hours.
After diluting the reaction with water (15.0 mL), the product was
1
18.15531, found 518.15600; H NMR (400 MHz, DMSO-d6,
5C) δ 7.32 (d, J = 7.8 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.04 (s,

purified using ion-exchange chromatography on Sephadex resin
followed by preparative RP-HPLC and isolated as ammonium
salts (29% yield). D1 (compound 6a): 6.75 mg, 0.0104 mmol;
room temperature for 24 hours. The final product was precipitated
after addition of a solution of anhydrous NaClO (145.1 mg, 2.02
mmol) in dry acetone (25 mL). The obtained suspension was
4
R
t
(method A2) = 8.5 min; HRMS ESI (-) calculated m/z for
cooled to 4C, and the precipitate was filtered off and washed
-
-
1
C
(
21
H
26
N
7
O
9
P
2
S [M-H] 614.09934, found 614.09999; H NMR
500 MHz, Deuterium Oxide, 25C) δ 8.92 (s, 1H), 7.42 (d, J = 7.7
Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.10 (s, 1H), 7.03 (t, J = 7.8 Hz,
H), 6.93 (t, J = 7.7 Hz, 1H), 5.76 (d, J = 2.3 Hz, 1H), 4.45 – 4.37
m, 1H), 4.38 – 4.32 (m, 1H), 4.33 – 4.28 (m, 1H), 4.28 – 4.23 (m,
H), 3.76 (s, 3H), 3.19 – 3.10 (m, 2H), 2.86 – 2.80 (m, 2H); 31
4
triply with cold, dry acetone and dried overnight over P O10 in a
vacuum. The final product was obtained as a white solid (84.4 mg,
0.16 mmol, 64% yield). R (method A2) = 9.8 min; HRMS ESI (-)
t
-
-
1
(
2
calculated m/z for C20
502.12489.
21 7 7
H N O P [M-H] 502.12456, found
P
4
.7.8. Compound 11
To guanosine (6.0g, 21.2 mmol) solution in DMSO (53 mL),
NMR (202 MHz, Deuterium Oxide, 25C) δ 42.24 (d, J = 29.0
Hz), -1.86 (dt, J = 29.0, 10.0 Hz). D2 (compound 6b): 8.35 mg,
benzyl bromide (20.1 mL, 169.2 mmol) was added. The reaction
mixture was stirred at room temperature for 3 hours. Then 25%
ammonia solution in water was being added dropwise until pH
value of the reaction mixture reached value 7. In the next step
acetone (250 mL) was added and continuously additional portions
of 25% ammonia solution in water was being added dropwise until
pH value of the mixture was stabilized at value 7. Meanwhile the
product was being precipitated from the solution. The obtained
suspension was cooled to 4C, and the precipitate was filtered off
and washed triply with cold acetone and dried overnight over
0
.0129 mmol; R
t
(method A2) = 8.7 min; HRMS ESI (-) calculated
-
-
1
m/z for C21H N O P S [M-H] 614.09934, found 614.09934; H
NMR (500 MHz, Deuterium Oxide, 25C) δ 8.88 (s, 1H), 7.38 (d,
J = 7.9 Hz, 1H), 7.21 (d, J = 8.1 Hz, 1H), 7.10 (s, 1H), 6.99 (t, J =
8
1
3
26 7 9 2
.1, 7.4 Hz, 1H), 6.91 (t, J = 7.9, 7.4 Hz, 1H), 5.76 (d, J = 2.0 Hz,
H), 4.48 – 4.43 (m, 1H), 4.37 – 4.29 (m, 2H), 4.28 – 4.24 (m, 2H),
.72 (s, 3H), 3.25 – 3.10 (m, 2H), 2.89 – 2.76 (m, 2H); 31P NMR
(
(
202 MHz, Deuterium Oxide, 25C) δ 41.70 (d, J = 27.0 Hz), -2.06
dt, J = 27.0, 9.3 Hz).
4
.7.5. Compound 8
4
P O10 in a vacuum. If a color of the obtained solid after drying was
Dry compound 11 (90.3 mg, 0.242 mmol) was suspended in
yellow, then it was washed and dried again. Presence of bromine
or bromides in the final product may lead to reduction of yield in
tiophosphorylation reaction. The final product was obtained as a
trimethyl phosphate (1.2 mL) and placed on ice-bath. After cooling
to 0C, 2,6-dimethylpyridine (140.0 μL, 1.209 mmol) and freshly
distilled thiophosphoryl chloride (97.8 μL, 0.964 mmol) was
added. The reaction was stirred at 0C until the integration of
signal from 7-methylguanosine on the HPLC profile dropped
below 10%. The reaction was quenched by addition of water (6.0
mL) and methanol (6.0 mL) to the reaction mixture followed by
adjustment of pH value to 6 by addition of 0.1 M NaOH solution.
The product was purified using preparative RP-HPLC and isolated
as an ammonium salt (41.7 mg, 0.083 mmol, 34% yield).
white solid (5.6 g, 15.0 mmol, 71% yield). R
t
(method A2) = 9.0
+
+
min; HRMS ESI (+) calculated m/z for C17
H
20
N
5
O
5
[M+H]
1
374.14590, found 374.14582; H NMR (400 MHz, DMSO-d
6
) δ
9.43 (s, 1H), 7.49 – 7.45 (m, 2H), 7.42 – 7.33 (m, 3H), 6.98 (s,
2H), 5.83 (d, J = 4.1 Hz, 1H), 5.67 (s, 1H), 5.70 – 5.59 (m, 2H),
5.30 (s, 1H), 5.28 (s, 1H), 4.45 (t, J = 4.6, 4.1 Hz, 1H), 4.13 (t, J =
5.0, 4.6 Hz, 1H), 3.99 (q, J = 5.0, 3.3 Hz, 1H), 3.71 (dd, J = 12.2,
3.3 Hz, 1H), 3.59 (dd, J = 12.2, 3.3 Hz, 1H).
t
R (method A1) = 9.0 min; HRMS ESI (-) calculated m/z for
4
.7.9. 4Ei-1
-
-
1
C
(
5
=
17
H
18
N
5
O
7
PS [M-H] 468.07483, found 468.07463; H NMR
400 MHz, Deuterium Oxide, 25C) δ 9.77 (s, 1H), 7.43 – 7.27 (m,
H), 6.07 (d, J = 3.8 Hz, 1H), 5.68 (q, J = 14.8 Hz, 2H), 4.70 (t, J
4.8, 3.8 Hz, 1H), 4.53 (t, J = 4.8 Hz, 1H), 4.46 – 4.41 (m, 1H),
.28 (ddd, J = 11.9, 5.4, 2.4 Hz, 1H), 4.11 (ddd, J = 11.9, 5.4, 2.2
To sodium salt of compound 10 (46.3 mg, 1130 mODU, 0.099
mmol) solution in HEPES buffer (995 μL, 1 M, pH 8.0),
tryptamine (47.5 mg, 0.296 mmol) was added. Then the reaction
mixture was stirred at room temperature for 72 hours. After
diluting the reaction with water (25.0 mL) and methanol (1.0 mL),
the product was purified using preparative RP-HPLC and isolated
as an ammonium salt (17.2 mg, 0.028 mmol, 28% yield).
4
3
1
1
Hz, 1H); P NMR (162 MHz, Deuterium Oxide, 25C,
decoupling) δ 45.95 (s).
H
4
.7.6. Compounds 9a, 9b
Triethylammonium salt of compounds 6a, 6b (a mixture of
t
R (method A2) = 14.4 min; HRMS ESI (-) calculated m/z for
C
-
-
1
27
H
29
N
7
O
7
P [M-H] 594.18661, found 594.18760; H NMR (400
diastereomers, 113.4 mg, 1626 mODU, 0.143 mmol) was
dissolved in DMF (1.4 mL). Then imidazole (97.2 mg, 1.427
mmol), 2,2’-dithiodipyridine (94.28 mg, 0.428 mmol),
triphenylphosphine (112.98 mg, 0.431 mmol) and triethylamine
MHz, CD OD, 25C) δ 7.33 – 7.24 (m, 7H), 7.06 (s, 1H), 7.01
3
(ddd, J = 8.1, 7.0, 1.0 Hz, 1H), 6.82 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H),
5.94 (d, J = 4.7 Hz, 1H), 5.25 (q, J = 14.6 Hz, 2H), 4.60 (t, J = 4.7
Hz, 1H), 4.40 (t, J = 4.7 Hz, 1H), 4.32 (ddd, J = 4.7, 4.2, 3.7 Hz,
1H), 4.12 (ddd, J = 11.8, 4.2, 2.3 Hz, 1H), 3.98 (ddd, J = 11.8, 3.7,
(
80.0 μL, 0.574 mmol) were added. The reaction mixture was
stirred at room temperature for 5 hours. The final product was
precipitated after addition of a solution of anhydrous NaClO (81.9
3
1
2.3 Hz, 1H), 3.19 – 3.07 (m, 2H), 2.85 – 2.78 (m, 2H); P NMR
1
4
(162 MHz, CD
3
OD, 25C, H decoupling) δ 10.11 (s).
mg, 0.669 mmol) in dry acetone (15.0 mL). The obtained
suspension was cooled to 4C, and the precipitate was filtered off
and washed triply with cold, dry acetone and dried overnight over
4
.8. Determination of dissociation constants with murine eIF4E
protein (time-synchronized fluorescence quenching titration,
tsFQT)
P
4
O
10 in a vacuum. The final product was obtained as a mixture of
sodium salts of diastereomers (68.3 mg, 0.120 mmol, 84% yield).
(method A2) = 5.6 min (D1), 5.8 min (D2); HRMS ESI (-)
Determination of dissociation constants of obtained compounds
without tryptamine modification was carried out according to
R
t
-
-
calculated m/z for C14
H
18
N
7
O
9
P
2
S [M-H] 522.03674, found
previously described time-synchronized fluorescence quenching
5
22.03683 (D1), 522.03683 (D2).
.7.7. Compound 10
Triethylammonium salt of compound 7, 139.8 mg, 0.25 mmol)
titration method.28 The measurements were carried out on a Cary
Eclipse BioMelt (Agilent Technologies) fluorescence
spectrophotometer using thermostated at 20 C couvette
containing stirring bar in 50 mM HEPES/ KOH (pH 7.2), 100 mM
KCl, 0.5 mM EDTA, 1 mM DTT. Aliquots of 1 L increasing
concentration of compound solutions (2 x 2 M, 2 x 5 M, 8 x 10
4
was dissolved in DMF (2.5 mL). Then imidazole (171.0 mg, 2.53
mmol), 2,2’-dithiodipyridine (167.0 mg, 0.76 mmol),
triphenylphosphine (198.8 mg, 0.76 mmol) and triethylamine (142
μL, 1.01 mmol) were added. The reaction mixture was stirred at

8
M, 8 x 20 M, 4 x 50 M, 4 x 100 M, 4 x 200 M, 4 x 500 M,
x 1000 M) were added to 1.4 mL of 30 M protein solutions.

Fluorescence intensities (excitation at 280 nm with 2.5-nm
bandwidth and detection at 337 nm with 4-nm bandwidth and 290
nm cutoff filter) were corrected taking into account sample
dilution and the inner filter effect. Equilibrium association
constants (KAS) were determined by fitting the theoretical
dependence of fluorescence intensity on the total concentration of
cap analog to the experimental data points, according to the
equation described previously.28 The concentration of protein was
fitted as a free parameter of the equilibrium equation showing the
amount of “active” protein. KAS were calculated as a weighted
average of 3 independent titrations, with the weights taken as the
reciprocals of the numerical standard deviations squared. The final
30°C), and inhibitor (half log dilutions of Clig. from 100 µM to
0.003 µM). The reagents without protein were pre-incubated for
15 min at 30°C and stirred at 300 rpm. After addition of the
enzyme point fluorescence detection (exc. 345 nm, em. 378 nm)
was applied. To the first points of the fluorescence intensity
measured over time linear curves were fitted, and initial rates V
calculated as curve slopes. To calculate IC50 from the dependences
0
of the initial rate V to initial rate with no inhibitor V on inhibitor
concentration the following equation was fitted:
푉
퐴2 ― 퐴1
= 퐴1 +
푝
,
푉0
퐶
lig.
1 +
(
퐼퐶
)
50
KD, tsFQT values were reported as reciprocals of KAS. Numerical
where: A1 is bottom asymptote, A2 is top asymptote, Clig. is the
nonlinear least-squares regression analysis was performed using
Origin 9.6 (OriginLab Corporation).
inhibitor concentration, p is hill slope and V/V
0
is the ratio of the
initial reaction rate with inhibitor to the initial rate without
I
inhibitor. Then, we calculated inhibition constants K from
determined IC50, following Cheng-Prusoff equation:
4
.9. Determination of dissociation constants with murine eIF4E
protein (pyrene fluorescence intensity binding method, PyFLINT-
B)
퐼퐶50
퐾 =
퐼
,
[퐿]
+ 퐾
1
To determine dissociation constants of the ligands binding to
the eIF4E we used previously described pyrene fluorescence
intensity binding method.29 Briefly, the competitive binding
experiments were performed using plate reader (Synergy
H1MFDG Microplate Reader Biotek) in 96-well, black, non-
M
where: K
concentration. All curves were fitted using Origin 2017
OriginLab Corp., Northampton, MA, USA) software.
M
is Michaelis-Menten constant, [L] – fluorescent probe
®
(
4
.11. Inhibition of translation
binding assay plates with point fluorescence measurements (λexc
45 nm; λem = 378 nm). Each well contained a buffer (50 mM
Hepes/KOH pH 7.2 containing 100 mM KCl and 0.5 mM EDTA),
0 nM of pyrene-labeled 7-methylguanosine pentaphosphate
=
3
The ability of the phosphate-modified 7-methylguanine
nucleotides to inhibit cap-dependent translation was assayed in a
rabbit reticulocyte lysate (RRL) system. In vitro translation
reaction (10 µl total volume) contained a tested nucleotide
1
probe, non-fluorescent ligand (half-log dilutions of Clig. from 100
μM to 0.003 μM), and 75 nM of eIF4E protein. The reagents and
protein were pre-incubated for 15 min at 20°C and stirred at 300
rpm. Measurements were performed at three different
temperatures: 20, 30, and 37°C in three repetitions. To the
registered dependences of fluorescence intensity on ligand
concentration the following equation was fitted:
(inhibitor) and an in vitro transcribed, ARCA-capped mRNA
encoding Gaussia luciferase at 1 µg/ml final concentration. The
nucleotides at a concentrations falling in the range of 0.05 to 100
μM and mRNA were added to the reaction mixture at the same
time. Reactions were incubated at 30 ºC for 60 min and stopped
afterwards by freezing in liquid nitrogen. To detect luminescence
from Gaussia luciferase, 50 μl of 10 ng/ml h-coelenterazine
(NanoLight) in PBS was added to 10 μl of RRL and the
luminescence was measured on Synergy H1 (BioTek) microplate
reader. The IC50 were determined by plotting the luminescence
values as a function of ligand concentration and fitting a following
equation to the data:
퐹max ― 퐹0
퐹 = 퐹 +
푝,
0
퐶
lig.
퐾
D, app. PyFLINT ― B)
1
+
(
where KD, app. PyFLINT-B is the inhibitor concentration required to
replace 50% of the fluorescent probe from the protein binding site,
C
slope. To fit the curve we used Origin 2017 (OriginLab Corp.,
Northampton, MA, USA) software. To calculate the inhibition
lig. is the concentration of non-fluorescent ligand and p is the hill
®
푇표푝 ― 퐵표푡푡표푚
푦 = 퐵표푡푡표푚 +
푥
1
+
(IC50)
(dissociation) constants KD, PyFLINT-B of a competitive ligands from
K
D, app. PyFLINT-B s the following formula was used:
4
.12. Stability in Fetal Bovine Serum
퐾
D, probe
퐾D, app. PyFLINT ― B ―
[퐿]
[
푃t
]
+
[
푃L]50( [퐿]
+ 1)
5
0
For all analyzed compounds, 1 mL of water solution containing
10% (v/v) FBS, 20% (v/v) PBS (pH 7.4, 140 mM NaCl, 2.7 mM
퐾D, PyFLINT ― B
=
,
[푃]
5
0
0
+ 1
퐾
+ 퐾
KCl, 10 mM Na HPO , 2 mM KH PO ) and 50 M analyzed
D, probe
D, probe
2
4
2
4
where KD, probe is the pyrene-labelled probe dissociation constant,
] is the total protein concentration, [P 50 is the protein–probe
complex concentration at 50% inhibition, [L]50 is the free probe
compound was prepared. Additionally, for all analyzed
compounds, 100 L of analogic solution without addition of FBS
was prepared. Afterwards all solutions containing FBS were
incubated at 37 C. After 15, 30, 60, 90, 120, 150, 210, 270
minutes of incubation, 100 L aliquot from each reaction was
thermally deactivated by heating at 95 C for 3 minutes and
centrifuged. Aliquots not containing FBS (0 time point) and
containing FBS (other time points) were analyzed using 1260
Infinity (Agilent Technologies Inc.) HPLC with a diode array
detector and an automated sample injector (method A3).
Percentage content of remained compounds was determined based
on integrals of signals from absorbance at 254 nm ratios
[P
t
L
]
0
concentration at 50% inhibition, and [P] is the free protein
concentration without inhibition.29
4
.10. Determination of inhibition constants with human DcpS
enzyme (PyFLINT-HA)
For inhibition parameters determination of enzymatic hydrolysis
catalyzed by DcpS enzyme we used previously described
2
9
method. The experiments were performed using plate reader
Synergy H1MFDG Microplate Reader Biotek) in 96-well, black,
non-binding assay plates. Each well contained appropriate buffer
50 mM Tris/HCl pH 7.6 containing 200 mM KCl and 0.5 mM
EDTA), substrate (5 µM of probe pyrene-labeled 7-
methylguanosine triphosphate), enzyme (10 nM of DcpS dimer at
(
(corresponding time point sample signal integral to 0 time point
sample signal integral). For each compound 3 independent
repetitions of experiment were performed. To calculate half-life
values, the following equation: Y = (Y - Plateau) * exp(-K * X) +
0
(
–

Plateau, where Y
0
is Y value at 0 time point, Plateau is bottom
PVDF membrane (MultiScreen IP Filter Plate, Millipore). The
membrane was coated with 5 μL/well 1% suspension of lecithin in
dodecane. The donor plate was filled with 125 μL 300 μM PBS
solutions of tryptamine conjugates: 1, 2a, 2b, 4, 5a, 5b, 4Ei-1 and
asymptote and K is decay constant, was fitted to data using
GraphPad Prism 8 (GraphPad Software, Inc.). The final half-life
values were calculated from the equation t1/2 = ln(2) / K as
weighted averages, with the weights taken from reciprocal
standard deviations squared.
7
nucleotides: m GMP, 7 as a control. The acceptor plate was filled
with 200 μL of PBS buffer. The plates were assembled and
incubated (600 rpm, room temperature) for 16 hours. The
concentrations of compounds in donor and acceptor plate were
determined using 1260 Infinity (Agilent Technologies Inc.) HPLC
with a diode array detector and an automated sample injector
4
.13. Susceptibility to hydrolysis by human DcpS enzyme
For all analyzed compounds, 800 L of solution containing 30
µM compound and 30 nM DcpS enzyme were prepared in 50 mM
Tris/HCl, 200 mM KCl, 0.5 mM EDTA, pH 7.6 buffer.
Additionally, for all analyzed compounds, 100 L of analogic
solution without addition of DcpS was prepared. Afterwards all
solutions containing enzyme were incubated at 30 C. After 10,
(method A3).
Acknowledgments
We thank J. Oledzki (IBB, Polish Academy of Sciences) for
HRMS measurements, P. Wanat, M. Chrominski, and M.
Baranowski (University of Warsaw) for recording NMR spectra,
K. Kaczmarska (University of Warsaw) for preparing HEK-293
cell extracts and A. Guranowski (Poznan University of Life
Sciences) for providing HINT1 enzyme.
2
0, 30, 45, 60, 90, 120 minutes of incubation, 100 L aliquot from
each reaction was thermally deactivated by heating at 95 C for 3
minutes and centrifuged. Aliquots not containing enzyme (0 time
point) and containing enzyme (other time points) were analyzed
using 1260 Infinity (Agilent Technologies Inc.) HPLC with a
diode array detector and an automated sample injector (method
A3).
This work was supported by the Foundation for Polish Science
(
(
TEAM/2016-2/13) to J.J. and National Science Centre, Poland
UMO-2019/32/T/ST4/00073) to D.S.
4
.14. Susceptibility to hydrolysis by human HINT1 enzyme
For all analyzed compounds, 800 L of solution containing 50
µM compound and 20 nM (for compounds 1 and 4Ei-1) or 200nM
Appendix A. Supplementary data
Supplementary data to this article can be found online at
References
(for compounds 2a, 2b, 4, 5a, 5b) HINT1 enzyme were prepared
in 20 mM HEPES, 1 mM MgCl , pH 7.2 buffer. Additionally, for
2
all analyzed compounds, 100 L of analogic solution without
addition of HINT1 was prepared. Afterwards all solutions
containing enzyme were incubated at 25 C. After 10, 20, 30, 45,
6
0, 90, 120 minutes of incubation, 100 L aliquot from each
1
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point) and containing enzyme (other time points) were analyzed
using 1260 Infinity (Agilent Technologies Inc.) HPLC with a
diode array detector and an automated sample injector (method
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Declaration of interests
The authors declare that they have no known
☒
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
☐
The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: