Membrane-permeable octanoyloxybenzyl-masked cNMPs as novel tools for non-invasive cell assays

Article abstract of DOI:10.3390/molecules23112960

Adenine nucleotide (AN) 2nd messengers, such as 3',5'-cyclic adenosine monophosphate (cAMP), are central elements of intracellular signaling, but many details of their underlying processes remain elusive. Like all nucleotides, cyclic nucleotide monophosphates (cNMPs) are net-negatively charged at physiologic pH which limits their applicability in cell-based settings. Thus, many cellular assays rely on sophisticated techniques like microinjection or electroporation. This setup is not feasible for medium- to high-throughput formats, and the mechanic stress that cells are exposed to raises the probability of interfering artefacts or false-positives. Here, we present a short and flexible chemical route yielding membrane-permeable, bio-reversibly masked cNMPs for which we employed the octanoyloxybenzyl (OB) group. We further show hydrolysis studies on chemical stability and enzymatic activation, and present results of real-time assays, where we used cAMP and Ca²⁺ live cell imaging to demonstrate high permeability and prompt intracellular conversion of some selected masked cNMPs. Based on these results, our novel OB-masked cNMPs constitute valuable precursor-tools for non-invasive studies on intracellular signaling.

Full text of DOI:10.3390/molecules23112960

molecules
Article
Membrane-Permeable Octanoyloxybenzyl-Masked
cNMPs As Novel Tools for Non-Invasive Cell Assays
Alexandra Ruthenbeck 1 , Elisa Marangoni 1,2, Björn-Ph. Diercks 3 , Aileen Krüger 3,
Alexander Froese 3, Nadja I. Bork 3, Viacheslav O. Nikolaev 3 , Andreas H. Guse 3 and
Chris Meier 1,
*
1
Organic Chemistry, Department of Chemistry, Faculty of Sciences, University of Hamburg,
Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany; ruthenbe@chemie.uni-hamburg.de (A.R.);
elisa.marangoni@studenti.unicam.it (E.M.)
School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, via S. Agostino 1,
62032 Camerino, Italy
University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany;
b.diercks@uke.de (B.-P.D.); Krueger-Aileen@web.de (A.K.); a.froese@uke.de (A.F.); n.bork@uke.de (N.I.B.);
v.nikolaev@uke.de (V.O.N.); guse@uke.de (A.H.G.)
2
3
*
Correspondence: chris.meier@chemie.uni-hamburg.de; Tel.: +49-40-42838-4324
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Christian Ducho
Received: 12 October 2018; Accepted: 7 November 2018; Published: 13 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Adenine nucleotide (AN) 2nd messengers, such as 30,50-cyclic adenosine monophosphate
(cAMP), are central elements of intracellular signaling, but many details of their underlying processes
remain elusive. Like all nucleotides, cyclic nucleotide monophosphates (cNMPs) are net-negatively
charged at physiologic pH which limits their applicability in cell-based settings. Thus, many cellular
assays rely on sophisticated techniques like microinjection or electroporation. This setup is not
feasible for medium- to high-throughput formats, and the mechanic stress that cells are exposed
to raises the probability of interfering artefacts or false-positives. Here, we present a short and
flexible chemical route yielding membrane-permeable, bio-reversibly masked cNMPs for which
we employed the octanoyloxybenzyl (OB) group. We further show hydrolysis studies on chemical
stability and enzymatic activation, and present results of real-time assays, where we used cAMP and
Ca2+ live cell imaging to demonstrate high permeability and prompt intracellular conversion of some
selected masked cNMPs. Based on these results, our novel OB-masked cNMPs constitute valuable
precursor-tools for non-invasive studies on intracellular signaling.
Keywords: cyclic nucleotide monophosphate; bio-reversible protection; acyloxybenzyl phosphate ester
1. Introduction
0
0
Among cNMPs, the ubiquitous second messengers 3 ,5 -cyclic adenosine monophosphate (cAMP)
0
0
and 3 ,5 -cyclic guanosine monophosphate (cGMP) constitute the most prominent examples, but further
0
0
cNMPs, such as 3 ,5 -cyclic uridine monophosphate (cUMP), have been reported as related to signaling
processes [ ]. Signaling by cNMPs proceeds via two general pathways. The first relies on direct binding
and regulation of distinctive cyclic nucleotide-gated (CNG) ion channels. The second mechanism is
based on the activation of protein kinases A (PKA) or G to further transduce the signal [ ]. Activated
PKA/G promotes phosphorylation of a variety of proteins, which can be involved in the regulation
of metabolic processes, muscle contraction, and gene transcription [ ]. In contrast, signaling through
1
1–4
4
CNG channels allows a faster processing and implementation of increased cNMP levels. The ion
channels are generally non-selective for cations. However, the entry of sodium ions (Na+) depolarizes
the membrane which promotes the combined influx of Ca2+. Additionally, voltage-gated Ca2+ channels
Molecules 2018, 23, 2960; doi:10.3390/molecules23112960
www.mdpi.com/journal/molecules

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open in response to membrane depolarization and thus enhance the Ca2+ signal further [
2
,3]. Signals
of cAMP and cGMP are ceased through their degradation by phosphodiesterase to AMP and GMP,
respectively [2,3].
Based on the ubiquitous involvement in cellular processes, the role of cNMP signaling in the
context of inflammation and regulation of immune response constitutes a research field of rising
interest. It was, for example, found that regulatory T cells (Treg) exert their suppressive effect on
effector T cells (Teff) through increasing concentrations of cAMP. The rise of cAMP levels in Teff could
either be induced via a paracrine mechanism, or by a direct transfer of cAMP via gap junctions between
Treg and Teff [5,6]. However, the transfer of cAMP between a pair of T cells cannot yet be visualized
directly, and the underlying mechanisms allowing Treg cells to produce such significantly higher cAMP
levels than Teff cells have not been elucidated to date. A loss of the immuno-suppressive mechanism
could contribute to the generation of autoimmune reactions. Understanding the role of cNMPs in
inflammation and T cell regulation and identifying the associated molecular pathways thus could
enable the identification of novel targets in the treatment of autoimmune disease.
However, cell-based studies on 2nd messengers are in our experience difficult to perform as
application of the highly polar compounds is carried out via effortful, single-cell preparative methods
like electroporation, microinjection, or patch clamp. These methods require highly trained staff,
careful preparation and significant amounts of time in advance of each experiment. At the same
time, the invasive application raises the potential for interfering artefacts or false-positives [7]. Thus,
membrane permeable, bio-reversibly modified chemical derivatives of second messengers are highly
desirable compounds to circumvent these drawbacks.
First reports on phosphate-protected cNMPs by Gillen and Nagyvary on alkyl phosphotriesters
of cAMP were followed by Engels et al. who prepared cAMP and cGMP derivatives carrying different
benzyl groups including photo-cleavable moieties at the phosphate (Figure 1) [8–11]. Hughes et al.
reported the synthesis and biologic evaluation of N6,O2-dibutyryl cAMP-acetoxymethyl (AM) ester
(Figure 1) [12]. The synthesis started from N6,O20-dibutyryl cAMP, which was converted with AM
bromide (AM-Br) under DIEA-basic conditions in CH3CN over 4 days at room temperature (rt) to
give the desired product in low yield after demanding chromatography [12]. The obtained masked
nucleotide was used in whole-cell incubation studies where an activation of PKA proceeded, but
only 15 min after the addition of di(Bu)cAMP-AM (10 µM) to the cell medium [12]. In a further
study, Schultz et al. synthesized cAMP-AM [13]. Despite a PKA activating effect of cAMP-AM,
the researchers found that cAMP-AM was less potent than di(Bu)cAMP-AM and was metabolized so
rapidly that it gave only transient signals [13]. The synthesis of AM-esters of cCMP and cUMP has
also been reported [14]. However, cNMP-AMs are in our experience not applicable for all cell types,
seemingly due to their insufficient chemical stability. Further, the variability of the described synthesis
approaches, which started from the respective cNMP, is limited by the availability of these.
Figure 1. Masked cAMP derivatives. Left: Benzyl triesters of cAMP reported by Engels et al. [
8,9
].
cAMP-AM derivatives introduced by Schultz et al. (middle [12,13], Right: structure of the OB-masked
cNMPs disclosed here.

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The acyloxybenzyl (AB) group constitutes another successfully applied bio-reversible masking
group apart from AM esters. It was introduced originally for nucleoside monophosphate (NMP)
prodrugs and transferred on nucleoside diphosphates (NDPs) and nucleoside triphosphates
(NTPs) [15–19]. The respective prodrugs were shown to efficiently diffuse across cell membranes
and release the corresponding nucleotide intracellularly upon enzymatic activation [16]. Further,
the concept was expanded on compound classes like sugar nucleotides [20]. The broad synthetic
applicability of the AB masking group is complimented by meeting the essential features of a prodrug
concept: (i) high chemical stability; (ii) efficient enzymatic activation; and (iii) sufficient lipophilicity to
enable the passage through cell membranes. Moreover, the two-part composition of the AB-masks
allows variation of e.g., lipophilicity or enzymatic cleavability which adds further versatility to the
concept [15–17,21–23]. Consequently, the AB-concept was investigated for expansion on cNMPs to
provide tools for studies on cellular effects, e.g., calcium signaling in bulk settings on a variety of
cells and without the need for example of microinjection. As a first example, an octanoyloxybenzyl
(OB)-mask was used here since previous studies showed a good chemical stability and sufficiently
high lipophilicity of respectively masked di- and triphosphates (Figure 1) [13–15].
2. Results and Discussion
Inspiration for the synthesis approach towards OB-masked cNMPs was drawn from own studies
on the synthesis of non-symmetric phosphoramidites (PAs) that included an unprotected nucleoside
moiety. In these cases, side reactions were repeatedly observed amongst which the formation of
phosphites was prominent. These phosphites were concluded to result either from an intermolecular
or an intramolecular substitution of the remaining N-diisopropyl group by a second nucleosidic
hydroxy-group. In the latter case, a cyclic nucleoside phosphite would constitute the reaction product,
and further oxidation would result in a cyclic nucleoside monophosphate derivative.
Based on this hypothesis, we set up a synthesis involving different nucleosides and an OB-masked
phosphordiamidite (OBPA2).
2.1. Preparation of AB-Masked cNMPs
2.1.1. Synthesis of Starting Materials
The synthesis of bis(N,N-diisopropylamino)-4-octanoyloxybenzyl phosphordiamidite (OBPA2)
was adapted from Weinschenk et al. who used the building block for the synthesis of non-symmetric
phosphoramidites [24]. The reaction was performed as described in the literature starting from
bis(N,N-diisopropylamino)chlorophosphine
OBPA2 3 in 71% (Scheme 1).
1 and 4-(hydroxymethyl)phenyloctanoate 2, and yielded
Scheme 1. Synthesis of OBPA2 3: a. 1 equiv. 1, 1 equiv. 2, 1.3 equiv. TEA, THF, rt, 18 h, 3: 71%.
0
The nucleosides adenosine (A,
4
), 2 -deoxyadenosine (dA,
5
), and guanosine (G,
6
) were mono-N-
was
via transient silylation of all hydroxy-groups to selectively
introduce the butyryl moiety at the N6-position [25]. The desired protected nucleoside
was obtained
in 61% yield after automated RP flash column chromatography following the procedure described in
butyrylated to add further lipophilicity to the envisaged OB-cNMPs. N6-Butanoyl-adenosine
7
synthesized starting from adenosine
4
7

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the literature [25]. The yield was limited by the formation of N,N-diacylated adenosine and partial
cleavage of the glycosidic bond during concentration of the crude reaction mixture. Analogously,
N6-butanoyl-dA
8
and N2-butanoyl-G
9
were synthesized starting from the respective nucleosides and
obtained in yields of 32% and 48%, respectively (Scheme 2).
◦
Scheme 2. Synthesis of N-butyryl nucleosides 7–9: a. first 2.1–9.0 equiv. TMSCl, 0 C to rt, 18 h, second
1.1 equiv. butanoyl chloride, pyridine/THF or pyridine/CH2Cl2, rt, 5–18 h, third 1 M aq. HCl or
CH3OH, rt, 5 min or 18 h, 7: 61%, 8: 32%, 9: 48%.
2.1.2. Syntheses of OB-cNMPs
First, a model system was set up to study course and outcome of the phosphitylation in more
detail. For this served the reaction towards octanoyloxybenzyl-masked cUMP (OB-cUMP, 10) since
syntheses involving uridine commonly go along with little or no side reactions and complications.
The starting conditions for the synthesis of OB-cUMP 10 were adapted from the approaches
towards non-symmetric phosphoramidites [24]. 4,5-Dicyanoimidazole (DCI, 0.25 M in acetonitrile
(CH3CN)) was used as activator for the PA-coupling, and tBuOOH (5.5 M in n-decane) served as
oxidizing agent. The reaction was carried out in a mixture of CH3CN and DMF due to the limited
solubility of uridine 11 in CH3CN (Scheme 3).
In the first attempt, a solution of nucleoside 11 and OBPA2
3 was treated with 2.2 equivalents of
DCI that were added dropwise at 0 ◦C. The reaction mixture was allowed to warm to rt and stirred
30 min more before tBuOOH was added for oxidation. After further 30 min, all volatile components
were removed under high vacuum and the crude reaction mixture was purified by automated RP
flash column chromatography. The masked cyclic nucleotide 10 was obtained as a mixture of two
diastereomers in a yield of only 13% (Scheme 3).
Prompted by the surprisingly low yield, the reaction course was studied 31P-NMR
spectroscopically (Figure 2). Interestingly, upon just mixing uridine 11 and OBPA2 3, the diamidite
was converted almost quantitatively and a signal at 13.2 ppm formed (Figure 2). Comparison with
literature-data indicated that this signal likely corresponded to an activated amidite [26]. Furthermore,
signals in the range of phosphoramidites (148.4 & 148.3 ppm) and phosphites (141.3 & 141.1 ppm)
were formed already as well (Figure 2, top). The addition of DCI then promoted the formation of the
intermediate phosphoramidite (middle and bottom), but formation of the anticipated cyclic phosphite
seemed to occur only at low proportion. Steric hindrance in the attack of the 30-hydroxy group on

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the phosphorous atom or an insufficient nucleophilicity may be the reasons that led to an insufficient
formation of the cyclic phosphite.
O
O
NH
NH
O
C7H15
O
N
O
HO
N
O
a. - d.
O
O
O
O
(iPr)2N
O
O
+
P
P
N(iPr)2
O
OH
OH
OH
3
11
10
O
O
C7H15
Scheme 3. a. 1.1 equiv.
CH3CN/DMF 5:4, 0 ◦C to rt, 60 min, 10: 13%. b. 1.1 equiv.
in CH3CN), second portion of 1.3 equiv. DCI (0.25 M in CH3CN), 1.5 equiv. tBuOOH (5.5 M in
n-decane), CH3CN/DMF 5:1, rt, 60 min, 10: 15%. c. 1.1 equiv. , first portion of 1.3 equiv. DCI
(0.25 M in CH3CN), second portion of 1.3 equiv. BTT (0.3 M in CH3CN), 1.5 equiv. tBuOOH (5.5 M
in n-decane), CH3CN/DMF 5:1, rt, 60 min, 10: 13–19%. d. 1–1.2 equiv. , first portion of 1–1.2 equiv.
3, 2.2 equiv. DCI (0.25 M in CH3CN), 1.5 equiv. tBuOOH (5.5 M in n-decane),
3, first portion of 1.3 equiv. DCI (0.25 M
3
3
saccharin and 1–1.2 equiv. 1-methylimidazole, second portion of 1–1.2 equiv. saccharin and 1–1.2
equiv. 1-methylimidazole, 1.5 equiv. tBuOOH (5.5 M in n-decane), CH3CN/DMF 5:1, rt, 60 min to 72 h,
10: 16–19%.
Figure 2. 31P-NMR spectra (CH3CN-d3, 162 MHz, 25 ◦C, shifts
between uridine 11 and OBPA2 3.
δ in ppm) of the reaction monitoring
The synthesis of OB-cUMP 10 consequently was repeated with several changes to the protocol in
order to evaluate the influence of the reaction conditions and in particular the impact of the activator
(Scheme 3).

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In a second approach, the ratio between CH3CN and DMF was altered to 5:1 and the mode of
addition of reagents was changed. This time, the nucleoside was placed in the reaction flask and
dissolved in CH3CN/DMF 5:1. A separate solution of OBPA2
3 in CH3CN and one equivalent of DCI
were added slowly and dropwise to nucleoside 11 at rt. Once the addition was completed, a second
equivalent of DCI was added, and the reaction mixture stirred at rt for 60 min. After the successive
oxidation and final purification, OB-cUMP 10 was obtained in 15% yield. Consequently, the outcome
of the reaction was almost identical to that of the attempt before.
Next, the activator was changed to 5-(benzylthio)-1H-tetrazole (BTT) which displays a higher
acidity and lower nucleophilicity than DCI, and the reaction protocol was varied as follows: uridine 11
was dissolved in CH3CN/DMF 5:1 and successively treated with a solution of OBPA2
3 in CH3CN
and one equivalent of DCI. Both reagents were added in small portions over a period of 30 min. Upon
completed addition, the reaction mixture was stirred another 30 to 120 min at rt. Then, one equivalent
of BTT was added slowly and dropwise over 10 min, and the reaction was kept stirring for further 15
to 60 min. After the subsequent oxidation and final purification, OB-cUMP 10 was isolated in yields
between 13–19% which again constituted no significant improvement of the reaction outcome. Lastly,
an alternative activator-system composed of saccharine and 1-methylimidazole was tested as it was
reported to efficiently mediate reactions between PAs and poorly nucleophilic alcohols like tertiary
alcohols [27]. For this, saccharine and 1-methylimidazole were dissolved in a 1:1 ratio in CH3CN prior
to the reaction to generate the activating salt. Simultaneously with OBPA2 3, one equivalent of the
activator salt was added to a solution of uridine 11 in CH3CN/DMF 5:1. Upon completion of the first
addition, the reaction mixture was stirred for 30 min, then treated with a second equivalent of the
activator solution and successively stirred for another 60 min to 72 h. After oxidation and automated RP
column chromatography, the desired product 10 was obtained in yields between 16–19%. Monitoring
of the reaction course via 31P-NMR spectroscopy indicated an incomplete activation of OBPA2
3 even
after 72 h. Further, the activated intermediates were again not converted efficiently to the desired
phosphite as concluded from the persistence of the respectively attributed phosphorous signals [27].
This alternative approach thus constituted no improvement in comparison to the previous protocols. In
summary, the isolation of OB-cUMP 10 succeeded repeatedly despite low yields, and thus the reaction
protocols were transferred on the N-butyrylated nucleosides 7–9 (Scheme 4).
Scheme 4. Syntheses of and N-butyrylated OB-cNMPs 12
1.3 equiv. DCI (0.25 M in CH3CN), second portion of 1.3 equiv. BTT (0.3 M in CH3CN), 1.5 equiv.
tBuOOH (5.5 M in n-decane), CH3CN/DMF 3:1, rt, 60 min, 12: 14%. To 13 a. 1.1 equiv. , first portion
of 1.3 equiv. DCI (0.25 M in CH3CN), second portion of 1.3 equiv. DCI (0.25 M in CH3CN), 1.5 equiv.
tBuOOH (5.5 M in n-decane), CH3CN/DMF 1:1, rt, 60 min, 13: 13%. To 14 a. 1.1 equiv. , first portion
–14: To 12: b. 1.1 equiv. 3, first portion of
:
3
:
3
of 1.5 equiv. DCI (0.25 M in CH3CN), second portion of 1.5 equiv. DCI (0.25 M in CH3CN), 1.5 equiv.
tBuOOH (5.5 M in n-decane), CH3CN/DMF 1:1, rt, 60 min, 14: 4%.
The N-butyrylated OB-cNMPs 12–14 were prepared following the synthesis protocols b. and
c. described above. The yields obtained for OB-N(Bu)-cAMP 12 and OB-N(Bu)-c(dAMP) 13

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were in a similar range as found for OB-cUMP 10 with 14% and 13%, respectively (Scheme 4).
The reaction towards OB-N(Bu)-cGMP 14 proceeded to a lower extent and accordingly, the desired
product was isolated in a comparably low yield of 4% (Scheme 4). Interestingly, in the case of 14
,
the formation of only one of the two possible diastereomers seemed favored as crude 31P-NMR spectra
indicated. Here, only one of the possible two phosphate signals was prominent, and consequently,
OB-N(Bu)-cGMP 14 was isolated as a single diastereomer. Additionally, to the N-acylated derivative
12, the NH2-unmodified OB-cAMP 15 was prepared. The synthesis was performed analogously to
OB-cUMP 10 starting from nucleoside
4 and following synthesis variant a. in which a stepwise addition
of the in total applied 2.4 equivalents DCI was performed (Scheme 5).
Scheme 5. Preparation of OB-cAMP 15: a. 1.1 equiv. 3, 2.4 equiv. DCI (0.25 M in CH3CN) added in
two portions, 1.5 equiv. tBuOOH (5.5 M in n-decane), CH3CN/DMF 1:1, rt, 60 min, 15: 12%.
In summary, despite the need of further yield optimization, five different OB-cNMPs were
successfully and reliably synthesized. With regard to the overall similar outcomes of the various
preparations, the scope of the reaction can likely be expanded on further nucleosides and/or differently
masked phosphoramidites. This makes the chosen approach very flexible, particularly in contrast to
the previously reported procedures involving AM esters.
Concluding the syntheses, the functional evaluation of selected OB-cNMPs was performed next.
2.2. Functional Evaluation of Selected OB-cNMPs
Ideally, masked precursors of bio-active compounds are biologically inactive and display a stability
in physiologic media that on the one hand exceeds their specific activation significantly, and on the
other hand guarantees sufficient time for their approximation to their respective target structure. Once
activated, the previously inactive compound regains its biologic activity back and should accordingly
displays respective effects from target interaction. These demands applied for the prepared OB-cNMPs
equally as e.g., for nucleotide prodrugs. Consequently, the chemical stability of the OB-cNMPs
under physiologic conditions as well as the efficiency of the enzymatic activation of the OB-mask by
an exemplary esterase was evaluated. In complementation, the masked nucleotides were applied
in various cell assays to analyze their biologic effects e.g., in the context of Ca2+ mobilization and
cell activation.
2.2.1. Investigation of Chemical Stability and Enzymatic Activation by PLE
Stability determinations for OB-cNMPs 10, 12, and 13 (as 5 mM stock sol. in DMSO) were
performed in PBS (50 mM, pH 7.3) as a physiologic pH mimic. The compounds (2 mM, final conc. in
PBS/DMSO) were incubated for 120 to 200 h at 37 ◦C. Hydrolysis samples were taken at distinctive
times and analyzed via HPLC/MS (Figures 3 and 4). The recorded HPLC chromatograms and ESI
mass spectra showed that the OB-cNMPs released only the respective parent cNMP caused by the
cleavage of the OB-mask without the formation of further cleavage byproducts (Figure 4).

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Figure 3. Course of the chemical hydrolysis of OB-N(Bu)-cAMP 12 to N(Bu)-cAMP 15 given as
normalized values for each time point analyzed. Similar hydrolysis courses were measured for
OB-cNMPs 10 and 13.
1
It was found that after approximately 8 h, 50% of the OB-cNMPs were hydrolyzed (10: t ⁄
=
2
1
1
8.6 h, 12: t ⁄ = 7.4 h, 13: t ⁄ = 7.5 h) (Figure 3). This half-life implied a stability of the OB-cNMPs that
2
2
should facilitate convenient setups of cell-based assays and allow experimental set-ups to run, for
example, even (pre-)incubation experiments with the masked nucleotides. The hydrolysis behavior of
10, 12, and 13 was assessed further, and the relative areas of signals for OB-cNMP and cNMP were
determined (in percent). These values were normalized and then averaged for all three hydrolyses
under determination of their standard deviation. By this procedure, the individual hydrolysis courses
were compared with another since the progress of hydrolyses was expected to be similar as the
dissociation of the OB-mask should constitute the determinant process.
The average deviation of the hydrolysis progress for OB-cNMPs 10, 12, and 13 at the time points
measured was 5% with a single value maximum of 12%. From these calculations, it was deduced
that the hydrolysis course was indeed analog for all OB-cNMPs tested and subject to the pace of the
cleavage of the masking group and was almost independent of the nucleotide.
After successfully probing the chemical stability of the OB-cNMPs, their enzymatic activation
by pig liver esterase (PLE) as a model esterase was evaluated. The incubations of OB-cNMPs 10
and 13 (2 mM final conc. in PBS/DMSO) with PLE were carried out with 0.05 u PLE per hydrolysis
sample (V = 20 L) which enabled good traceability of the enzymatic conversion (Figure 5). Again,
, 12,
µ
no further cleavage products except the corresponding cNMPs were detected. The N-butyryl group
of 12 and 13 was not cleaved by PLE, even at longer incubation times (up to 60 min), as expected.
The chromatograms were processed as described for the chemical hydrolysis to compare the progress
of the incubations with PLE (Figure 6).
Half-lives of the studied OB-cNMPs were found to be around 5 min under the applied conditions,
which, however, depended significantly on the amount of esterase present. More importantly in this
context, the enzymatic activation of OB-cNMPs proceeded even at low PLE concentrations significantly
faster than their decomposition in PBS by a factor of approximately 100. In addition, the enzymatic
hydrolyses showed analog progression as indicated by a mean deviation of normalized signal areas for
OB-cNMPs and cNMPs of 4% with a maximum deviation of 9% for single time point values (Figure 6).
This lead again to the conclusion that the enzymatic reaction was almost independent of the type of
nucleotide and relied on the OB-mask applied.

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Figure 4. HPLC chromatograms and ESI mass spectra of the chemical hydrolysis of OB-N(Bu)-cAMP
12 in PBS (50 mM, pH 7.3) at 0 h, 1 h, 8 h, and 50 h. The two signals of the diastereomers vanished over
time while the signal for N(Bu)-cAMP 15 increased. The compounds were assigned from the mass
spectra recorded at the retention times coinciding with the signals found in the HPLC chromatograms.
In summary, the results of both hydrolysis studies under chemical or enzymatic conditions
went well along with the initial criteria as they showed that the stability of the OB-cNMPs was
significantly higher than the rate of enzymatic activation. Further, the masked nucleotides proved to
be stable enough for application in cell-based assays as their stabilities allow for incubations even over
several hours.
Encouraged by the promising hydrolysis properties, the ability of the OB-cNMPs to cross cellular
membranes as well as their potential to induce cellular processes was studied next.

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Figure 5. HPLC chromatograms and ESI mass spectra of the enzymatic hydrolysis of OB-N(Bu)-cAMP
12 in with PLE (0.05 u/hydrol. sol.) in PBS (50 mM, pH 7.3) at 1, 5, and 10 min. The two signals of
the diastereomers vanished over time while the signal for N(Bu)-cAMP 15 increased. The compounds
were assigned from mass spectra recorded at the retention times coinciding with the signals found in
the HPLC chromatograms.
Figure 6. Enzymatic hydrolyses of OB-N(Bu)-cAMP 12 to N(Bu)-cAMP 15 given as normalized values
for each time point analyzed. Similar hydrolysis courses were measured for OB-cNMPs 10 and 13.
2.2.2. Performance of Selected OB-cNMPs in Cell-Based Settings
Primary mouse cardiomyocytes carrying a FRET-sensor with a cAMP binding site were used to
examine the membrane-permeability of OB-N(Bu)-cAMP 12 in particular. The binding of intracellular
cAMP to the FRET sensor is indicated by a decreasing FRET-signal and an increasing fluorescence
ratio between cyan-fluorescent protein (CFP) and yellow-fluorescent protein (YFP) [28]. FRET-sensors
carrying mouse cardiomyocytes were incubated with OB-N(Bu)-cAMP 12 (20 mM DMSO stock sol.,

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20
µ
M final conc. in DMSO/buffer, at t
≈
60 s) (Figure 7A,B). Immediately after addition of 12 to
the extracellular medium, the ratio between CFP and YFP started to increase and reached a steady
maximum state at circa 50 s (Figure 7A) which was comparable with the previously well-established
cell permeable cAMP analogue 8-Br-20-O-Me-cAMP-AM (Figure 7B) (Figure 7A,B) [28]. Likewise,
the concentration–response dependencies were largely comparable for both analogues and reached the
maximum at 20 µM (Figure 7C). These results imply that OB-N(Bu)-cAMP 12 instantaneously crossed
the cell membrane and was also rapidly activated by intracellular esterases. Further, the product
of the activation process was successfully recognized by the cAMP binding site of the FRET sensor.
For the studied substrate, OB-N(Bu)-cAMP 12, this implicated that the N6-butyryl group was either
removed by enzymatic hydrolysis, or that its presence had no detrimental effect on the FRET
biosensor interaction. Indeed, as expected from 6-monobutyryl derivatives of cAMP, incubation
of cardiomyocytes with 12 led to almost instantaneous of phospholamban phosphorylation by the
cAMP dependent protein kinase (Figure 7D). These immunoblots suggest that OB-N(Bu)-cAMP might
activate phospholamban phosphorylation in a reversible manner. It can be expected from the chemical
structure of 6-N(Bu)-cAMP that it should be degradable by intramolecular phosphodiesterases, in a way
that high activity of these enzymes in cardiomyocytes leads to a gradual decline of cAMP dependent
protein kinase substrate phosphorylated over time.
In a second setup, Jurkat T cells were loaded with the Ca2+-sensitive fluorescent dye Fura-2. Upon
intracellular elevation of Ca2+, the absorption ratio between the two excitation wavelengths of Fura-2
at 340 nm and 380 nm increases. This effect correlates directly with the amount of free cytosolic Ca2+
.
Jurkat T cells loaded with Fura-2 were stimulated with OB-cNMPs 10
, 12, and 13 (20 µM in DMSO,
at t 120 s) added to their extracellular medium (Figure 8). The Fura-2 ratio rose rapidly almost
≈
immediately after addition of OB-N(Bu)-cAMP 12, and reached its maximum after approximately
200 s. Then, the Ca2+ signal slowly decreased as indicated by the degression of the signal. A similar
trend was observed for OB-cUMP 10 but the induced Ca2+ signal was significantly reduced compared
to 12 (Figure 8). In the case of OB-N(Bu)-c(dAMP) 13, no initial increase of the intracellular Ca2+
concentration was measured. However, the ratio seemed to increase slightly over time (Figure 8).
The results confirmed again that the OB-masked cNMPs were able to cross the cell membrane
and, importantly, immediately triggered cellular responses. In this context, it was concluded that
de-masked cNMPs promoted the observed effects based on the results of the previous hydrolysis
studies and the substrate specify Ca2+ signaling events display.
The hydrolysis product of 12 acted as would be expected for cAMP, supporting the assumption
that the N6-butyryl group was either removed enzymatically, too, or that its presence did not impede
the FRET sensor activation.
Comparison of the measured effects with those evoked by NH2-unmodified OB-cAMP 15 and,
e.g., further nucleobase derivatives of adenosine or uridine in combination with incubation studies in
cell homogenate could help to finalize the analysis and clarify whether the N6-butyryl group is cleaved
or the interacting receptors and binding sites lack selectivity in the corresponding region. Studies
along these lines are currently being performed in our laboratories.
In summary, the performed cell assays confirmed excellent membrane-permeability of the selected
OB-cNMPs. Further, the cellular effects observed allow the conclusion that the bio-reversible protection
at the phosphate was removed rapidly and efficiently. A fast enzymatic activation of the prepared
OB-cNMPs was shown analogously in hydrolysis studies using pig liver esterase. The esterase cleaved
the OB-mask even at low concentration within very short time, so that similar effects can be expected
to proceed in cells. Finally, the observed FRET-sensor binding site interaction and induced Ca2+
mobilization proved that biologically active compounds were released out of the masked cNMPs.
Moreover, the masked nucleotides triggered processes like those attributed to their parent cNMPs
(if existent in nature/identified yet).

Molecules 2018, 23, 2960
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A
B
C
D
Figure 7. Normalized FRET ratio (between CFP and YFP) over the course of OB-N(Bu)-cAMP
12 addition (20 M) in ( M) in ( ) to mouse
) or of 8-Br-20-O-Me-cAMP-AM application (20
µ
A
µ
B
cardiomyocytes expressing Epac1-camps biosensor for intracellular cAMP. An instantaneous increase
of FRET ratio after addition of 12 to the extracellular medium indicated intracellular release of cAMP
and its binding to the FRET biosensor. Representative experiments (n = 5). (
dependencies to both analogues show similar potencies (mean SEM, n = 5 for each point).
) cardiomyocyte incubation with OB-N(Bu)-cAMP led to rapid phosphorylation of the cAMP
C) concentration–response
±
(
D
dependent protein kinase substrate phospholamban (PLN). Cells were incubated for 30, 90, or 180 s
with OB-N(Bu)-cAMP and lysed for immunoblot analysis. Alternatively, cells were preincubated
for 20 min with 10
µM of the kinase inhibitor H-89 before 180 s treatment with OB-N(Bu)-cAMP.
Representative immunoblots (n = 3).

Molecules 2018, 23, 2960
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**
stimulation
thapsi
***
0.8
0.6
0.4
0.2
0.5
0.4
0.3
0.2
0.1
0.0
*
0
200
400
600
800 1000 1200
Time (s)
Figure 8. Stimulation of the of Jurkat T cells with OB-cNMPs 10
were stimulated after 120 s with the respective OB-cNMPs (20 M) or DMSO (as negative control).
Furthermore, as positive control Thapsigagarin (1.67 M) was added after 900 s. Mean signal ratio
, 12, and 13. Left: Jurkat T cells
µ
µ
between 340 nm and 380 nm from single cells are shown (DMSO n = 26; OB cAMP n = 77; OB cUMP
n = 37; OB c(dAMP) n = 14). The addition of 12 and 10 resulted in a transient increase of the Ca2+
concentration, while no transient increase is visible for 13 or DMSO. Right: Statistical analysis of the
mean delta peak for the OB-cNMPs and DMSO (data represent mean
effect is measured for 12 and statistically significant differences between are marked by asterisks
(* p < 0.05, ** p < 0.01, *** p < 0.001, Kruskal–Wallis Test).
±
SEM). The most pronounced
3. Materials and Methods
All reactions involving water-sensitive reagents were conducted under anhydrous conditions
and a dry atmosphere of nitrogen. Reagents were used as purchased from commercial suppliers.
Anhydrous N,N-dimethylformamide (DMF) was purchased and stored over 4 Å molecular sieves.
All other anhydrous solvents were purified and dried using a solvent purification system (MB
SPS-800 from Braun) and stored over appropriate molecular sieves. Solvents for normal phase
(NP) chromatography were distilled prior to use. Acetonitrile was purchased in HPLC grade for
reversed phase (RP) chromatography and HPLC. Evaporation of solvents was performed under
reduced pressure on a rotary evaporator or using a high vacuum pump. Reactions were monitored via
thin layer chromatography (TLC) carried out on pre-coated Macherey-Nagel TLC plates Alugram®
Xtra SIL G/UV254, and compounds stained with Vanillin (Vanillin (5 g), 1000 mL MeOH/AcOH 9:1,
35 mL H2SO4) under heating. For automated NP or RP chromatography two flash systems (Interchim
Puriflash 430 (Montlucon, France) or Büchi Sepacore® Flash System (Essen, Germany), combined
with Macherey-Nagel Chromabond® Flash RS 80 SiOH (NP) or RS40 C18 ec (RP) columns (Düren,
Germany)) were used. For purifications of phosphordiamidites, a chromatotron (Harrison Research
7924T) with glass plates coated with 2 or 4 mm layers of VWR60 PF254 silica gel containing a fluorescent
indicator (VWR no. 7749) was used. Analytical RP-High Performance Liquid Chromatography-Mass
Spectrometry (RP-HPLC/MS) was performed with an Agilent 1260 Infinity instrument, Waldbronn,
Germany (pump G1311B, autosampler G1329B, column compartment G1316A, diode array detector
G4221B, column Agilent Poroshell 120 EC-C18, 2.7 mm, 4.6
(Advion expressionL CMS, Harlow, U.K.). Ultrapure water was generated by a Sartorius Arium® pro
unit (Sartopore 0.2 m, UV, Göttingen, Germany). As elution buffer served a tetra-n-butylammonium
×
50 mm) coupled with single-quad MS
µ
acetate solution (10 mM, pH 7.2). HPLC/MS runs were performed according to the following method:
◦
0–15 min: water/acetonitrile gradient (2–98% B) with a flow of 0.5 mL/min, 20 C column temperature
and UV detection at 259 nm and 270 nm, MS scans from 150 to 1100 m/z. Nuclear magnetic resonance
(NMR) spectra were recorded at room temperature on Bruker Fourier 300 (300 MHz for 1H acquisitions),

Molecules 2018, 23, 2960
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1
Bruker AMX 400 (Karlsruhe, Germany) (400 MHz for H 101 MHz for 13C and 152 MHz for 31P
1
acquisitions) or Bruker AVIII 600 (Karlsruhe, Germany) (600 MHz for H and 151 MHz for 13C
acquisitions) spectrometers in automation mode. All chemical shifts (δ) are given in parts per million
(ppm) with the solvent resonance as internal standard. Coupling constants J are given in Hertz (Hz).
Two-dimensional NMR experiments (HSQC, HMBC) were used for the assignment of quaternary
carbons. For mass spectrometric (MS) analysis, spectra were acquired on an Agilent 6224 ESI-TOF
spectrometer (Waldbronn, Germany) in positive and negative mode as required. Infrared spectroscopy
(IR) was carried out with a Bruker Alpha P FT-IR (Bremen, Germany) in attenuated total reflection
(ATR) mode at room temperature ranging from 400 cm−1 to 4000 cm−1. For FRET measurements,
primary mouse ventricular cardiomyocytes were isolated from Epac1-camps biosensor expressing
transgenic mice [29] as described [28] and plated onto laminin coated glass cover slides. Measurements
were performed 1–2 h after plating using a Nikon Ti microscope based FRET imaging system containing
pE-100 440 nm light source (CoolLED), DV2 Dual View and ORCA-03G charge-coupled device camera
(Hamamatsu, Japan), and analyzed as described [28]. Cells were kept in a buffer containing 144 mM
NaCl, 5.4 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 10 mM Hepes (pH 7.3) and stimulated with OB-cNMPs
or 8-Br-20-O-Me-cAMP-AM (purchased from BIOLOG Life Science Institute, Bremen, Germany)
dissolved 1:1000 in the same buffer from a freshly made 20 mM DMSO stock solution. For Ca2+
mobilization assays, Jurkat T cells were incubated with the membrane-permeable AM ester of the Ca2+
dye Fura-2 (4
µ
M, Calbiochem). Therefore, about 2
×
106 cells were centrifuged at 500 g for 5 min
and resuspended in 1 mL of freshly supplemented RPMI medium containing Fura-2 AM. Cells were
incubated for 30 min at 37 C. After centrifugation, cells were washed and resuspended in Ca2+ buffer
◦
(140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM Hepes (pH 7.4), 1 mM NaH2PO4, 5 mM
glucose) and kept for 20 min at rt for de-esterification. Cells were added on prepared coverslips and
allowed to adhere before measurement. Slides were mounted onto a Leica IRBE microscope (100-fold
magnification) and after 120 s the respective OB-cNMPs (20
As positive control, Thapsigagarin (1.67 M, Calbiochem) was added after 900 s. A Sutter DG-4 was
used as a light source, and frames were acquired with an electron-multiplying charge-coupled device
camera (C9100-13, Hamamatsu). Images (512 512 pixels) were acquired in 16-bit mode with the
µM) or DMSO (as control) were added.
µ
×
following filter sets (AHF Analysentechnik) (excitation (ex), beam splitter (bs), and emission (em),
all in nanometers): Fura-2 (ex, HC 340/26, HC 387/11; bs, 400DCLP; em, 510/84).
For immunoblot analysis, freshly isolated cardiomyocytes sedimented in stop buffer without
serum were preincubated for 30 min either with vehicle or with 10 µM H-89 (Sigma). Next, 20 µM of
OB-N(Bu)-cAMP were added to various time points (30, 90, and 180 s), after which cells were lysed and
processed for immunoblot analysis. Homogenized proteins were quantified using BCA Protein Assay
(Pierce). Samples (20 µ
g protein per lane) were boiled at 95 ◦C for 5 min to disrupt phospholamban
pentamers, loaded onto 15% SDS–polyacrylamide gel and immunoblotting using the anti-P-Ser16
phospholamban (Badrilla 1:5.000), and total anti-PLN A-1 antibodies (Badrilla).
3.1. General Procedures
GP I: N-butanoylation of nucleosides via transient TMS-protection:
The respective nucleoside (adenosine, guanosine, or 20-deoxyadenosine) was co-evaporated
three times and then dissolved in anhydrous pyridine (2–5 mL/mmol), and diluted either with the
same volume of THF or double the volume of CH2Cl2. At 0 ◦C, TMSCl (2.1–9.0 equiv.) was added.
The reaction mixture was allowed to warm up to rt and stirred for 5–18 h. Successively, butyryl
chloride (1.1 equiv.) was added slowly and the reaction mixture stirred for another 6 h at rt. Cleavage
of TMS ethers was promoted by the addition of either 1 M HCl (0.5 mL/mmol) under vigorous stirring
for 5 min, or methanol (2–5 mL/mmol) and stirring at rt for further 12 h. The reaction was terminated
by removal of all volatile components under high vacuum. The crude residue was co-evaporated with
toluene and CH2Cl2 several times, and then taken up in acetonitrile/demin. water. Purification was

Molecules 2018, 23, 2960
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performed by means of automated RP flash column chromatography on C18 modified silica gel with
an acetonitrile gradient in water (0% to 100%).
GP II: 30,50-Phosphorylation of nucleosides to their OB-masked cNMP analogues:
Under an atmosphere of nitrogen, the respective nucleoside was dissolved in DMF/CH3CN
(25 mL/mmol). In a separate flask, bis(N,N-diisopropylamino)-4-octanoyloxybenzyl phosphoramidite
(1 equiv.) was dissolved in acetonitrile (25 mL/mmol total volume). The phosphor diamidite
solution and DCI (0.25 M in CH3CN, 1.3–1.5 equiv.) were added slowly and dropwise in five to
ten portions to the nucleoside solution. The addition of more DCI (0.25 M in CH3CN, 1.2–1.5 equiv.) or
5-(benzylthio)-1H-tetrazole (BTT, 0.3 M in CH3CN, 1.3 equiv.) followed, and the reaction mixture was
stirred 1 h further. Then, tBuOOH (5.5 M in n-decane, 1.5 equiv.) was added and the solution stirred
for 10 min more. Successively, all volatile components were removed in vacuum, and the obtained
residue was taken up in CH3CN/demin. water and purified by means of an automated RP flash
column chromatography on C18 modified silica gel with an CH3CN gradient in water (0% to 100%).
3.2. Syntheses
3.2.1. Synthesis of bis(N,N-diisopropylamino)-4-octanoyloxybenzyl phosphordiamidite 3
1.00 g (3.75 mmol) bis(N,N-diisopropylamino)chlorophosphine
anhydrous THF. In a separate flask, 0.68 mL (4.87 mmol, 1.3 equiv.) NEt3 and 0.94 g (3.75 mmol,
1 equiv.) 4-(hydroxymethyl)phenyloctanoate were mixed with 7 mL anhydrous THF, and the mixture
1 were dissolved in 15 mL
2
was added dropwise to the chlorophosphine. The reaction mixture was stirred at rt for 20 h, then
filtrated and the filtrate concentrated to dryness in vacuum. The remaining residue was purified by
NP chromatography on silica gel with PE/TEA 98:2 as eluents, and the desired product obtained as
colorless syrup.
Yield: 1.28 g (2.67 mmol, 71%).
2
1H-NMR (600 MHz, chloroform-d):
δ
[ppm] = 7.36 (d, JH,H = 8.2 Hz, 2H), 7.14–6.95 (m, 2H),
4.63 (d, 2JH,H = 7.2 Hz, 2H), 3.66–3.51 (m, 4H), 2.54 (t,
J
= 7.5 Hz, 2H), 1.75 (p,
J
= 7.4 Hz,
H,H
2,3
2,3
H,H
2
2
2,3
2H), 1.51–1.23 (m, 8 H), 1.21 (d, JH,H = 2.7 Hz, 12H), 1.20 (d, JH,H = 2.8 Hz, 12H) 0.88 (t,
J
H,H
= 7.3 Hz, 3H); 13C{1H}-NMR (151 MHz, chloroform-d):
65.8, 44.69, 44.56, 34.6, 31.8, 29.2, 29.1, 25.1, 24.8, 24.7, 24.1, 24.0, 22.75, 14.22; 31P{1H}-NMR (162
MHz, chloroform-d):
[ppm] = 123.5. IR (ATR): v in [cm−1] = 2963.3, 2927.8, 2861.4, 2079.0, 2025.5,
δ [ppm] = 172.6, 149.7, 138.2, 127.9, 121.5,
δ
˜
1761.3, 1607.8, 1507.4, 1457.5, 1416.5, 1390.1, 1361.6, 1300.3, 1194.2, 1184.8, 1162.9, 1140.3, 1116.2, 1045.2,
1016.9, 952.7, 916.5, 866.3, 779.6, 748.7, 706.7, 642.7, 566.1, 527.6. MS (MALDI): m/z [M-H] calcd. for
C27H48N2O3P-: 479.340, found: 479.245.
3.2.2. Synthesis of 6-N-butanoyl-adenosine 7
In accordance with GP I, 1.40 g (5.26 mmol) adenosine 4 were dissolve in 34 mL pyridine/THF
1:1 and converted with 2.11 mL (16.5 mmol, 3.2 equiv.) TMSCl and 0.60 mL (5.78 mmol, 1.1 equiv.)
butyryl chloride. After 6 h stirring at rt, 2.5 mL 1 M HCl (aq.) was added to cleave the TMS ethers, and
after 5 min, all volatile components were removed under vacuum. Upon final purification of the crude
product via automated RP flash column chromatography on C18 modified silica gel with an CH3CN
gradient in water (0% to 100%), the product was obtained as colorless powder.
Yield: 1.09 g (3.22 mmol, 61%).
1H-NMR (500 MHz, DMSO-d6):
δ
[ppm] = 10.63 (s, 1H), 8.69 (s, 1H), 8.65 (s, 1H), 6.01 (d, 3JH,H
= 5.8 Hz, 1H), 5.53 (d, 3JH,H = 5.8 Hz, 1H), 5.23 (d, 3JH,H = 4.8 Hz, 1H), 5.12 (t, 3JH,H = 5.6 Hz, 1H),
4.63 (q, 3JH,H = 5.4 Hz, 1H), 4.19 (q, 3JH,H = 4.3 Hz, 1H), 3.98 (q, 3JH,H = 3.9 Hz, 1H), 3.76–3.64 (m, 1H),
3.58 (ddd, 2JH,H = 11.9 Hz, 3JH,H = 6.1 Hz, 3JH,H = 4.0 Hz, 1H), 2.55 (t,
J
= 7.3 Hz, 2H), 1.63 (h,
H,H
2,3
2,3JH,H = 7.4 Hz, 2H), 0.94 (t, 2,3JH,H = 7.4 Hz, 3H); 13C-NMR (126 MHz, DMSO-d6):
151.7, 151.6, 149.7, 142.7, 123.9, 87.6, 85.7, 73.7, 70.3, 61.3, 38.0, 18.2, 13.5. IR (ATR): v in [cm−1] = 3273.2,
δ [ppm] = 171.5,
˜

Molecules 2018, 23, 2960
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2963.3, 2934.0, 2875.0, 1716.3, 1685.0, 1613.3, 1587.0, 1521.9, 1460.3, 1409.0, 1356.6, 1327.0, 1224.3, 1124.1,
1082.2, 1056.7, 984.6, 902.4, 866.3, 799.6, 745.0, 704.7, 643.7, 547.3. MS (ESI-HR): m/z [M + H]+ calcd. for
C14H20N5O5+: 338.1459, found: 338.1469.
3.2.3. Synthesis of 6-N-butanoyl-20-deoxyadenosine 8
0
Following GP I, 1.14 g (4.24 mmol) 2 -deoxyadenosine
5 were dissolve in 24 mL pyridine/CH2Cl2
1:2. At 0 ◦C, 1.13 mL (8.91 mmol, 2.1 equiv.) TMSCl were added, and the reaction mixture was stirred
for 18 h at rt. Successively, 0.48 mL (4.67 mmol, 1.1 equiv.) butyryl chloride were added. After further
3 h stirring at rt, the TMS ethers were removed by addition of 8 mL CH3OH at 0 ◦C. After further
5 h at rt, the reaction was terminated by removing all volatile components under vacuum. The crude
product was taken up in water containing little amount of CH3CN and purified via automated RP
flash column chromatography on C18 modified silica gel with an CH3CN gradient in water (0% to
100%) to afford the desired product as colorless powder.
Yield: 0.43 g (1.35 mmol, 32%).
1H-NMR (400 MHz, methanol-d4):
δ [ppm] = 8.66 (s, 1H), 8.62 (s, 1H), 6.61–6.52 (m, 1 H), 4.64 (dt,
3JH,H = 6.1 Hz, 3JH,H = 3.1 Hz, 1H), 4.10 (q, 3JH,H = 3.4 Hz, 1H), 3.88 (dd, 2JH,H = 12.2 Hz, 3JH,H = 3.4 Hz,
1H), 3.79 (dd, 2JH,H = 12.2 Hz, 3JH,H = 3.9 Hz, 1H), 2.88 (ddd, 2JH,H = 13.4 Hz, 3JH,H = 7.4 Hz, 3JH,H
=
2,3
6.0 Hz, 1H), 2.67 (t,
J
= 7.4 Hz, 2H), 2.51 (ddd, 2JH,H = 13.5 Hz, 3JH,H = 6.2 Hz, 3JH,H = 3.3 Hz,
H,H
2,3
2,3
1H), 1.82 (h,
J
= 7.4 Hz, 2H), 1.08 (t,
J
= 7.4 Hz, 3H); 13C{1H}-NMR (101 MHz, methanol-d4):
H,H
H,H
δ
[ppm] = 174.4, 152.9, 150.7, 144.3, 123.2, 89.7, 86.7, 72.7, 63.4, 41.5, 39.9, 19.6, 14.0. IR (ATR): v in
˜
[cm−1] = 3336.8, 2964.6, 2933.1, 2875.3, 2592.1, 2330.9, 1682.2, 1612.5.1585.9, 1522.5, 1459.6, 1402.8,
1354.8, 1329.2, 1223.8, 1093.3, 1058.5, 993.8, 941.1, 867.1, 799.6, 749.6, 984.8, 644.4, 585.3, 561.2, 542.4,
527.4, 509.6, 464.7. MS (ESI-HR): m/z [M + H]+ calcd. for C14H20N5O4+: 322.1510, found: 322.1521.
3.2.4. Synthesis of 2-N-butanoyl-guanosine 9
According to GP I, 1.55 g (5.48 mmol) guanosine 6 w◦ ere co-evaporated with pyridine three times
and then dissolved in 81 mL pyridine/CH2Cl2 1:2. At 0 C, 6.28 mL (49.4 mmol, 9 equiv.) TMSCl were
added, and the reaction mixture was stirred for 4 h at rt. Successively, 0.62 mL (6.03 mmol, 1.1 equiv.)
butyryl chloride were added. After 3 h stirring at rt, the cleavage of the TMS ethers was induced by
addition of 27 mL CH3OH, and after further 12 h at rt, the reaction was terminated and all volatile
components were removed under vacuum. The crude residue was taken up in water containing
little amount of CH3CN and finally purified via automated RP flash column chromatography on C18
modified silica gel with an CH3CN gradient in water (0% to 100%) to afford the desired product as
colorless powder.
Yield: 0.92 g (2.61 mmol, 48%).
1H-NMR (400 MHz, DMSO-d6):
δ [ppm] = 12.07 (bs, 1H), 11.71 (bs, 1H), 8.26 (s, 1 H), 5.80 (d,
3JH,H = 5.7 Hz, 1H), 5.47 (d, 3JH,H = 5.7 Hz, 1H), 5.17 (d, 3JH,H = 4.5 Hz, 1H), 5.03 (t, 3JH,H = 5.4 Hz, 1H),
4.43 (d, 3JH,H = 5.2 Hz, 1H), 4.20–4.05 (m, 1H), 3.90 (q, J = 3.9 Hz, 1H), 3.64 (dt, 2JH,H = 11.9 Hz, 3JH,H
=
=
4.8 Hz, 1H), 3.55 (dt, 2JH,H = 11.9 Hz, 3JH,H = 4.7 Hz, 1H), 2.45 (t, 2,3JH,H = 7.3 Hz, 2H), 1.62 (h,
J
H,H
2,3
2,3
7.4 Hz, 2H), 0.92 (t,
J
= 7.5 Hz, 3H); 13C{1H}-NMR (101 MHz, DMSO-d6):
δ
[ppm] = 176.2, 154.8,
H,H
148.8, 148.0, 137.6, 120.1, 86.6, 85.3, 73.9, 61.1, 37.8, 17.9, 13.4. IR (ATR): v in [cm−1] = 3364.4, 3279.5,
˜
2968.3, 2941.1, 1680.2, 1608.8, 1564.3, 1554.1, 1536.0, 1481.4, 1469.2, 1449.9, 1402.5, 1251.7, 1204.2, 1179.4,
1127.9, 1089.0, 1060.2, 993.3, 976.4, 901.1, 863.3, 818.6, 801.0, 762.7, 737.4, 717.0, 680.4, 643.7, 607.0, 589.1,
562.3, 510.2, 484.2. MS (ESI-HR): m/z [M + H]+ calcd. for C14H19N5O6+: 354.1408, found: 354.1400.
3.2.5. Synthesis of Uridine-30,50-(4-octanoyloxybenzyl)cyclophosphate 10
According to GP II, 26 mg (0.11 mmol) uridine 10 were dissolved in 2 mL DMF and reacted with
57 mg (0.12 mmol, 1.1 equiv.) OB-PA2
(0.13 mmol, 1.3 equiv.) DCI (0.25 M in CH3CN) and 0.45 mL (0.13 mmol, 1.3 equiv.) BTT (0.3 M
in CH3CN). The addition of 32 L (0.16 mmol, 1.5 equiv.) tBuOOH (5.5 M in n-decane) followed
3, dissolved in 2.5 mL CH3CN, in the presence of 0.54 mL
µ

Molecules 2018, 23, 2960
17 of 20
successively. After purification by automated RP flash column chromatography on C18 modified silica
gel with a CH3CN gradient in water (0% to 100%) and lyophilization, the product was obtained as
colorless cotton and in two fractions containing each of the stereoisomers.
Yield: 11 mg (0.02 mmol, 19%).
3
1H-NMR (400 MHz, methanol-d4):
δ
[ppm] = 7.66–7.51 (m, 2H), 7.47 (d, JH,H = 8.1 Hz, 1H),
7.22–7.08 (m, 2H), 5.69 (d, 3JH,H = 8.0 Hz, 1H), 5.64 (d, 3JH,H = 0.9 Hz, 1H), 5.24–5.14 (m, 2H), 4.64 (ddd,
3JH,P = 22.5 Hz, 2JH,H = 9.3 Hz, 3JH,H = 4.6 Hz, 1H), 4.39 (ddd, 3JH,H = 10.2, 2JH,H = 9.4, 3JH,H = 0.8 Hz,
2,3
2,3
1H), 4.35–4.25 (m, 2H), 4.25–4.10 (m, 1H), 2.58 (t,
1.53–1.22 (m, 8H), 1.05–0.82 (m, 3H)
and
J
= 7.4 Hz, 2H), 1.73 (p,
J
= 7.4 Hz, 2H),
H,H
H,H
7.63 (d, 3JH,H = 8.1 Hz, 1H), 7.53–7.47 (m, 2H), 7.19–7.10 (m, 2H), 5.73 (d, 3JH,H = 1.2 Hz, 1H), 5.71
(d, 3JH,H = 8.1 Hz, 1H), 5.19 (d, 2JH,H = 8.7 Hz, 2H), 4.81 (ddd, 3JH,P = 9.8 Hz, 3JH,H = 5.1 Hz, 3JH,H
=
1.0 Hz, 1H), 4.67 (ddd, 3JH,P = 14.2 Hz, 2JH,H = 9.2 Hz, 3JH,H = 5.5 Hz, 1H), 4.62–4.47 (m, 2H), 4.42 (td,
2JH,H = 10.1, 3JH,H = 5.5 Hz, 1H), 2.60 (t, 2,3JH,H 7.4 Hz, 2H), 1.74 (p, 2,3JH,H 7.4 Hz, 2H), 1.50–1.26 (m,
8H), 1.02–0.84 (m, 3H); 13C{1H}-NMR (101 MHz, methanol-d4):
δ [ppm] = 173.8, 165.9, 151.5, 150.1,
143.7, 134.3, 131.2, 123.3, 103.0, 97.5, 80.1, 72.4, 71.5, 71.2, 70.3, 35.0, 32.9, 30.2, 30.1, 25.0, 23.7, 14.4
and
172.8, 164.9, 151.7, 150.5, 143.5, 133.0, 130.2, 122.8, 103.1, 97.7, 79.3, 72.4, 71.2, 71.1, 70.6, 35.0,
32.8, 30.2, 30.1, 26.0, 23.7, 14.4; 31P{1H}-NMR (162 MHz, methanol-d4):
δ
[ppm] =
−
3.93,
−
5.01. MS
(ESI-HR): m/z [M + Na]+ calcd. for C24H31N2O10PNa+: 561.1609, found: 561.1445.
3.2.6. Synthesis of 6-N-butanoyl-adenosine-30,50-(4-octanoyloxybenzyl)cyclophosphate 12
According to GP II, 48 mg (0.14 mmol) 6-N-butanoyl-adenosine
and treated with a solution of 76 mg (0.16 mmol, 1.1 equiv.) OB-PA2
7
3
were dissolved in 4 mL DMF
in 4 mL CH3CN and 0.97 mL
(0.24 mmol, 1.7 equiv.) DCI (0.25 M in CH3CN) as well as 0.62 mL (0.19 mmol, 1.3 equiv.) BTT (0.3 M
in CH3CN). Then, 43 L (0.21 mmol, 1.5 equiv.) tBuOOH (5.5 M in n-decane) were added. After
µ
purification by automated RP flash chromatography on C18 modified silica with a CH3CN gradient in
water (0% to 100%), the product was obtained as colorless cotton and mixture of two diastereomers.
Yield: 13 g (0.02 mmol, 14%).
1H-NMR (400 MHz, methanol-d4):
7.55–7.49 (2 m, 4H), 7.17–7.12, 7.13–7.06 (2
3JH,H = 5.1 Hz, 1H) 5.23 (2 d, 2JH,H = 10.4 Hz & 8.1 Hz, 4H), 5.05 (ddd, 3JH,P = 9.6 Hz, 3JH,H = 5.1 Hz,
3JH,H = 1.6 Hz, 1H), 4.89–4.86 (m, 1H), 4.73–4.34 (m, 7H), 2.71–2.55 (m, 6H,), 2.51 (t, 2,3JH,H = 7.4 Hz,
2H), 1.86–1.70 (m, 6H), 1.68–1.57 (m, 2H), 1.53–1.25 (m, 16H), 1.05 (2
t, 2,3JH,H = 7.4 Hz, 6H), 0.98–0.95
(m, 6H); 13C{1H}-NMR (151 MHz, methanol-d4):
[ppm] = 173.8, 173.6, 152.8, 152.4, 150.8, 143.1, 133.3,
133.2, 130.7, 130.6, 124.5, 123.1, 123.0, 88.8, 81.7, 80.4, 69.3, 69.2, 68.5, 59.8, 39.9, 34.5, 31.8, 29.2, 29.1, 25.0,
22.8, 18.5,14.2, 13.9; 31P{1H}-NMR (162 MHz, methanol-d4):
[ppm] = 3.80, 4.90. MS (ESI-HR): m/z
[M + H]+ calcd. for C29H39N5O9P+: 632.2848, found: 632.2848.
δ
[ppm] = 8.68, 8.58 (2
×
s, 2H), 8.49, 8.38 (2
×
s, 2H), 7.62–7.56,
×
×
m, 4H), 6.20, 6.12 (2
×
s, 2H), 5.47 (dd, 3JH,P = 9.1 Hz,
×
×
δ
δ
−
−
3.2.7. Synthesis of 6-N-butanoyl-20-deoxyadenosine-30,50-(4-octanoyloxybenzyl)cyclophosphate 13
According to GP II, 50 mg (0.15 mmol) 6-N-butanoyl-2’-deoxyadenosine were dissolved in
4 mL DMF and reacted with 82 mg (0.17 mmol, 1.1 equiv.) OB-PA2 , dissolved in 4.3 mL CH3CN,
in the presence of 1.60 mL (0.40 mmol, 2.6 equiv.) DCI (0.25 M in CH3CN) in total and 50
8
3
µL
(0.25 mmol, 1.6 equiv.) tBuOOH (5.5 M in n-decane). After purification by automated RP flash
column chromatography on C18 modified silica gel with a CH3CN gradient in water (0% to 100%) and
lyophilization, the product was obtained as colorless cotton and mixture of two diastereomers.
Yield: 12 mg (0.02 mmol, 13%).
1H-NMR (400 MHz, methanol-d4):
7.65–7.57, 7.55–7.48 (2
m, 2H), 7.19–7.10 (m, 4H), 6.59 (dd, 3JH,H = 9.0 Hz, 3JH,H = 2.0 Hz, 1H), 6.55
(dd, 3JH,H = 6.7 Hz, 3JH,H = 4.0 Hz, 1H), 5.64 (q, 3JH,H/P = 9.2 Hz, 1H), 5.33 (q, 3JH,H/P = 9.2 Hz, 1H),
δ [ppm] = 8.69 (s, 1H), 8.61 (s, 1H), 8.47 (s, 1H), 8.37 (s, 1H),
×

Molecules 2018, 23, 2960
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5.23 (2
×
d, 2JH,H = 13.1 Hz & 3JH,H = 12.9 Hz, 4H), 4.70–4.40 (m, 4H), 4.22 (td, 3JH,H = 9.9 Hz, 3JH,H
= 5.6 Hz, 1H), 4.08 (td, 3JH,H = 9.9 Hz, 3JH,H = 4.7 Hz, 1H), 2.98 (ddd, 2JH,H = 13.1 Hz, 3JH,H = 7.7 Hz,
3JH,H = 1.9 Hz, 1H), 2.83–2.70 (m, 3H), 2.69–2.57 (m, 6H), 2.54 (t,
6H), 1.66 (q, 2,3JH,H = 7.3 Hz, 2H), 1.51–1.26 (m, 16H), 1.06 (2 x t, 2,3JH,H = 7.4, 6H), 0.98–0.86 (m, 6H);
13C{1H}-NMR (101 MHz, methanol-d4):
[ppm] = 174.5, 174.4, 173.8, 173.6, 153.4, 153.2, 152.8, 152.7,
J
= 7.4 Hz, 2H), 1.90–1.70 (m,
H,H
2,3
δ
152.4, 152.3, 150.8, 150.7, 144.6, 144.5, 134.4, 134.3, 131.1, 130.7, 123.2, 123.1, 122.6, 85.8, 85.6, 79.3, 79.2,
75.7, 75.6, 71.7, 71.6, 71.4, 71.3, 40.0, 39.9, 35.0, 34.9, 32.8, 30.1, 30.0, 25.9, 25.9, 23.7, 19.7, 19.6, 14.4, 14.1,
14.0; 31P{1H}-NMR (162 MHz, methanol-d4):
calcd. for C29H39N5O8P+: 616.2531, found: 616.2534.
δ
[ppm] =
−
4.14,
−
5.50. MS (ESI-HR): m/z [M + H]+
3.2.8. Synthesis of 2-N-butanoyl-guanosine-30,50-(4-octanoyloxybenzyl)cyclophosphate 14
In accordance with GP II, 52 mg (0.15 mmol) 6-N-butanoyl-guanosine were dissolved in 4 mL
DMF and treated with a solution of 77 mg (0.16 mmol, 1.1 equiv.) OB-PA2 in 4 mL CH3CN, 1.75 mL
(0.44 mmol, 3 equiv.) DCI (0.25 M in CH3CN) in total and finally, 44 L (0.21 mmol, 1.5 equiv.) tBuOOH
9
3
µ
(5.5 M in n-decane). After purification by automated RP flash column chromatography on C18 modified
silica gel with a CH3CN gradient in water (0% to 100%) and lyophilization, the product was obtained
as colorless cotton and a single diastereomer.
Yield: 4 mg (5.6 µmol, 4%).
1H-NMR (400 MHz, methanol-d4):
δ
[ppm] = 7.92 (s, 1H), 7.56 (d, 3JH,H = 8.2 Hz, 2H), 7.07 (d,
3JH,H = 8.3 Hz, 2H), 6.01 (s, 1H), 5.23 (d, 2JH,H = 11.6 Hz, 2H), 4.69 (ddd, 3JH,H = 22.2 Hz, 3JH,P = 9.4 Hz,
3JH,H = 4.8 Hz, 1H), 4.55 (t, 3JH,H = 10.0 Hz, 1H), 4.35 (dd, 3JH,H = 10.0 Hz, 3JH,H = 4.8 Hz, 1H), 4.32 (d,
3JH,H = 4.7 Hz, 1H), 4.18 (dd, 3JH,P = 9.9 Hz, 3JH,H = 4.7 Hz, 1H), 2.51 (t, 2,3JH,H = 7.4 Hz, 2H), 2.46 (t,
2,3
2,3
2,3
J
= 7.1 Hz, 2H), 1.74 (h,
J
= 7.4 Hz, 2H), 1.62 (t,
J
= 7.1 Hz, 2H), 1.49–1.25 (m, 8H,), 1.02
H,H
H,H
2,3
H,H
(t,
J
= 7.4 Hz, 3H), 0.98–0.86 (m, 3H); 13C{1H}-NMR (101 MHz, methanol-d4):
δ
[ppm] = 177.7,
H,H
173.6, 152.2, 150.0, 149.8, 137.5, 134.4, 131.4, 123.3, 120.2, 93.4, 80.3, 73.3, 71.6, 71.5, 70.5, 39.4, 34.8, 32.9,
30.1, 25.8, 23.7, 19.3, 14.4, 13.9; 31P{1H}-NMR (162 MHz, methanol-d4):
m/z [M + H]+ calcd. for C29H39N5O10P+: 648.2429, found: 648.2720.
δ
[ppm] =
−
4.93. MS (ESI-HR):
3.2.9. Synthesis of adenosine-30,50-(4-octanoyloxybenzyl)cyclophosphate 15
Following GP II, 21 mg (78 mol) adenosine were dissolved in 2 mL DMF and treated with
a solution of 41 mg (85 mol, 1.1 equiv.) (OB)PA2 101 in 2 mL CH3CN as well as 0.74 mL (0.19 mmol,
2.4 equiv.) DCI (0.25 M in CH3CN). Lastly, 23 L (0.12 mmol, 1.5 equiv.) tBuOOH (5.5 M in n-decane)
µ
µ
µ
were added for oxidation. After purification by automated RP flash column chromatography on C18
modified silica gel with a CH3CN gradient in water (0% to 100%) and lyophilization, the product was
obtained as a colorless cotton and mixture of two diastereomers.
Yield: 5 mg (10 µmol, 12%).
1H-NMR (400 MHz, acetonitrile-d3):
δ [ppm] = 8.19 (s, 1H), 8.09 (s, 1 H), 7.93 (s, 1H), 7.88 (s, 1H),
7.55–7.46 (m, 2H), 7.45–7.41 (m, 2H), 7.12–7.02 (m, 4H), 5.99 (bs, 4H), 5.98 (s, 1H), 5.93 (s, 1H), 5.47 (dd,
3JH,P = 9.1 Hz, 3JH,H = 5.1 Hz, 1H), 5.27 (ddd, 3JH,P = 9.4 Hz, 3JH,H = 4.9 Hz, 3JH,H = 1.0 Hz, 1H), 5.19
(ddd, 3JH,P = 9.3 Hz, 3JH,H = 5.0 Hz, 3JH,H = 1.6 Hz, 1H), 5.14–5.06 (m, 4H), 4.72 (d, 3JH,H = 4.9 Hz, 1H),
4.60 (d, 3JH,H = 5.1 Hz, 1H), 4.58–4.14 (m, 6H), 2.50 (2 x t, 3JH,H = 7.5 Hz, 4H), 1.64 (h, 3JH,H = 7.6 Hz,
4H), 1.47–1.14 (m, 16H), 0.89–0.78 (m, 6H); 13C{1H}-NMR (151 MHz, acetonitrile-d3):
156.9, 153.9, 151.3, 149.4, 140.9, 133.3, 130.6, 130.7, 123.1, 119.8, 93.6, 81.0, 72.7, 71.5, 69.4, 34.7, 32.4, 29.6,
δ [ppm] = 173.3,
25.5, 23.3, 20.1, 14.3; 31P{1H}-NMR (162 MHz, acetonitrile-d3):
δ
[ppm] =
−
4.37,
−
6.03. MS (ESI-HR):
m/z [M + H]+ calcd. for C25H33N5O8P+: 562.2061, found: 562.2068.
4. Conclusions
In sum, although the yields still need improvement, we have identified a flexible and reliable
synthesis approach towards membrane-permeable, bio-reversibly masked cNMPs and prepared

Molecules 2018, 23, 2960
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OB-cNMPs that fulfilled all requirements in terms of synthetic flexibility of the approach, chemical
stability, and enzymatic activation to be valuable new tools for the setup of novel cellular assays.
Author Contributions: Conceptualization, A.R. and C.M.; methodology, A.R., E.M., B.-P.D., A.K., V.O.N., N.I.B.
and A.F.; formal analysis, A.R.; investigation, A.R., E.M., B.-P.D., A.K., V.O.N., N.I.B. and A.F.; resources, C.M.;
writing—original draft preparation, A.R.; writing—review and editing, A.R., B.D., A.H.G. and C.M.; project
administration, A.R., B.-P.D. and C.M.; funding acquisition, A.H.G. and C.M.
Funding: This research was funded by the Universität Hamburg and the Deutsche Forschungsgemeinschaft
(DFG), grant number SFB1328.
Acknowledgments: E.M. is grateful for a fellowship by Advanced School C. Urbani, University of Camerino.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).