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Membrane-Permeable Octanoyloxybenzyl-Masked Cnmps As Novel Tools for Non-Invasive Cell Assays

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Membrane-Permeable Octanoyloxybenzyl-Masked Cnmps As Novel Tools for Non-Invasive Cell Assays 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; [email protected] (A.R.); [email protected] (E.M.) 2 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, via S. Agostino 1, 62032 Camerino, Italy 3 University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany; [email protected] (B.-P.D.); [email protected] (A.K.); [email protected] (A.F.); [email protected] (N.I.B.); [email protected] (V.O.N.); [email protected] (A.H.G.) * Correspondence: [email protected]; Tel.: +49-40-42838-4324 Academic Editor: Christian Ducho Received: 12 October 2018; Accepted: 7 November 2018; Published: 13 November 2018 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 Among cNMPs, the ubiquitous second messengers 30,50-cyclic adenosine monophosphate (cAMP) and 30,50-cyclic guanosine monophosphate (cGMP) constitute the most prominent examples, but further cNMPs, such as 30,50-cyclic uridine monophosphate (cUMP), have been reported as related to signaling processes [1]. 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 [1–4]. 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 [4]. In contrast, signaling through 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 MoleculesMolecules 20182018, ,2323, ,x 2960 FOR PEER REVIEW 22 of of 20 20 (Na+) depolarizes the membrane which promotes the combined influx of Ca2+. Additionally, voltage- 2+ gatedopen inCa response2+ channels to open membrane in response depolarization to membrane andthus depolarization enhance the and Ca thussignal enhance further the [2 Ca,3].2+ Signalssignal furtherof cAMP [2,3]. and Signals cGMP of are cAMP ceased and through cGMP are their ceased degradation throughby their phosphodiesterase degradation by phosphodiesterase to AMP and GMP, torespectively AMP and GMP, [2,3]. respectively [2,3]. BasedBased on on the the ubiquitous ubiquitous involvement involvement in in cellular cellular processes, processes, the the role role of of cNMP cNMP signaling signaling in in the the contextcontext of of inflammation inflammation and and regulation regulation of of immune immune response response constitutes constitutes a a research research field field of of rising rising interest. It was, for example, found that regulatory T cells (Treg) exert their suppressive effect on interest. It was, for example, found that regulatory T cells (Treg) exert their suppressive effect on effector T cells (T ) through increasing concentrations of cAMP. The rise of cAMP levels in T could effector T cells (Teffeff) through increasing concentrations of cAMP. The rise of cAMP levels in Teff could eithereither be inducedinduced viavia a a paracrine paracrine mechanism, mechanism, or byor aby direct a direct transfer transfer of cAMP of cAMP via gap via junctions gap junctions between Treg and T [5,6]. However, the transfer of cAMP between a pair of T cells cannot yet be visualized between Tregeff and Teff [5,6]. However, the transfer of cAMP between a pair of T cells cannot yet be directly, and the underlying mechanisms allowing Treg cells to produce such significantly higher cAMP visualized directly, and the underlying mechanisms allowing Treg cells to produce such significantly levels than T cells have not been elucidated to date. A loss of the immuno-suppressive mechanism higher cAMP efflevels than Teff cells have not been elucidated to date. A loss of the immuno-suppressive mechanismcould contribute could tocontribute the generation to the generation of autoimmune of autoimmune reactions. Understandingreactions. Understanding the role of the cNMPs role of in cNMPsinflammation in inflammation and T cell and regulation T cell regulation and identifying and identifying the associated the associated molecular molecular pathways pathways thus could thus couldenable enable the identification the identification of novel of novel targets targ inets the in treatment the treatment of autoimmune of autoimmune disease. disease. However,However, cell-based cell-based studies studies on on 2nd 2nd messengers messengers ar aree in in our our experience experience difficult difficult to to perform perform as as applicationapplication of of the the highly highly polar polar compounds compounds is is carried carried out out via via effortful, effortful, single-cell single-cell preparative preparative methods methods likelike electroporation, electroporation, microinjection, microinjection, or or patch patch clam clamp.p. These These methods methods require require highly highly trained trained staff, staff, carefulcareful preparation preparation and and significant significant amounts amounts of time of time in advance in advance of each of each experiment. experiment. At the At same the time, same thetime, invasive the invasive application application raises raises the potential the potential for forinterfering interfering artefacts artefacts or orfalse-positives false-positives [7]. [7]. Thus, Thus, membranemembrane permeable, permeable, bio-reversibly bio-reversibly modified modified chemical chemical derivatives derivatives of of second second messengers messengers are are highly highly desirabledesirable compounds compounds to to circumvent circumvent these these drawbacks. drawbacks. FirstFirst reports reports on on phosphate-protec phosphate-protectedted cNMPs cNMPs by by Gillen Gillen and and Nagyva Nagyvaryry on on alkyl alkyl phosphotriesters phosphotriesters ofof cAMP cAMP were were followed followed by by Engels Engels et et al. al. who who prepared prepared cAMP cAMP and and cGMP cGMP derivatives derivatives carrying carrying different different benzylbenzyl groups groups including including photo-cleavable photo-cleavable moieties moieties at at the the phosphate phosphate (Figure (Figure 1)1)[ [8–11].8–11]. Hughes Hughes et et al. al. 6 2 reportedreported the the synthesis synthesis and and biologic biologic evaluation of N6,,OO2-dibutyryl-dibutyryl cAMP-acetoxymethyl cAMP-acetoxymethyl (AM) (AM) ester ester 6 20 (Figure(Figure 11))[ [12].12]. The The synthesis synthesis started started from from NN6,O,O2′-dibutyryl-dibutyryl cAMP, cAMP, which which was was converted converted with with AM AM bromide (AM-Br) under DIEA-basic conditions in CH CN over 4 days at room temperature (rt) to bromide (AM-Br) under DIEA-basic conditions in CH3CN3 over 4 days at room temperature (rt) to givegive the the desired desired product product in in low low yield yield after after dema demandingnding chromatography chromatography [12]. [12]. The The obtained obtained masked masked nucleotidenucleotide was was used used in in whole-cell whole-cell incubation incubation stud studiesies where where an activation an activation of PKA of PKAproceeded, proceeded, but only but 15only min 15 after min the after addition the addition of di(Bu)cAMP-AM of di(Bu)cAMP-AM (10 µM) (10 toµ theM) cell to the medium cell medium [12]. In [ 12a further]. In a furtherstudy, Schultzstudy, Schultzet al. synthesized et al. synthesized cAMP-AM cAMP-AM [13]. Despite [13]. Despite a PKA a activating PKA activating effect effectof cAMP-AM, of cAMP-AM, the researchersthe researchers found found that that cAMP-AM cAMP-AM was was less less potent potent than than
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