Controlled Delivery and Light-Induced Release of Magic Spot Nucleotides in Escherichia coli

The “m agic spot nucleotides ” (MSNs) ppGpp and pppGpp (also: (p)ppGpp) are bacterial alarmones central to the conserved stringent response, a stress adaptation mechanism that helps bacteria adapt to stress conditions and hostile environments. Current strategies to manipulate MSN levels rely mainly on genetic or environmental approaches, which are slow and lack temporal control. Chemical tools such as photocaged MSN analogues could provide such temporal control over MSN levels. However, the high negative charge of MSNs prevents spontaneous passage through the complex bacterial cell envelope. Here, we report the synthesis of photocaged, clickable, and isotope -labeled MSN analogues and their delivery into Escherichia coli comparing different approaches . A cyclodextrin -based synthetic nucleotide transporter provides particular advantages. Upon 400 nm irradiation, these probes were photo -released inside living cells, where we tracked their conversion from pppGpp to ppGpp by capillary electrophoresis mass spectrometr y and studied their ability to alter growth .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 2 in a (p)ppGpp⁰ mutant. This work provides the first demonstration that highly charged, photocaged MSNs can traverse the bacterial envelope, be photo -released intracellularly, and be metabolically tracked in real time. These probes lay the foundation for spatially and temporally controlled studies of MSN function and of other highly negatively charged metabolites in bacteria.

When bacteria are exposed to stress conditions like temperature change, pH change, starvation, or antibiotics, alarmones like the magic spot nucleotides (MSN s) guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) accumulate and induce the stringent response (Figure 1A).[1–5] MSNs help bacteria survive by inducing a dormant -like state until conditions improve. The extent of stringency is a function of MSN concentration. Beyond stress adaptation, MSNs also act as second messengers during normal growth, where basal levels are essential for regulating transcription, translation, replication, and metabolism through a wide network of protein interactions .[4,6] To study the concentration-dependent role of MSNs in cellular processes it is useful to be able to manipulate their abundance. Currently this can be achieved by genetic modifications, treatment with small molecule drugs or exposure to stress conditions. [7–9] Additionally, it would be highly desirable to study effects of local, constrained pools of these molecules. Genetically m odifying organisms is time consuming and often causes secondary effects. In addition, MSN-producing enzymes can synthesize both pppGpp and ppGpp , making it difficult to distinguish between different MSN s. To influence concentration levels of single MSNs further genetic modifications are needed. [10] An alternative chemical biology approach for modulation of MSN levels inside bacteria would provide new avenues for complementary experiments and thus enable further investigations into the effects of MSN s on bacterial physiology. The aim of this study is to deliver photocaged MSNs into bacteria l cells thus enabling their controlled release by light (Figure 1B). Since in vitro data of Escherichia coli and Bacillus subtilis MSN synthesizing enzymes RelA and SAS1 show that MSN s can stimulate their own synthesis , the introduction of small amounts of MSN s into the bacteria might be sufficient to induce high cellular concentrations. [8,11–15] .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 3 Figure 1. (A) Chemical structure of ppGpp and pppGpp. (B) Overview of our strategy to modulate MSN concentrations inside living cells. Due to charge repulsion from the negatively charged lipopolysaccharides and lipids, MSNs cannot readily pass through the complex bacterial cell envelope. We tested various strategies to deliver a photocaged MSN analogue into the bacterial cell and release the MSNs by light irradiation. Created in BioRender. Popp, C. (2025) https://BioRender.com/wnqejr2 The main obstacle for this approach is the bacterial cell envelope. In contrast to the lipid bilayer of mammalian cells, bacteria possess a much more challenging barrier . In Gram- negative bacteria the cell wall is composed of several peptidoglycan layers surrounded by an outer membrane containing phospholipids and lipopolysaccharides. MSNs can carry up to seven negative charges that impair translocation through the negatively charged cell envelope.[16,17] Several methods have been developed to transport highly negatively charged small molecules like nucleotides into mammalian cells. For example, prodrugs with masked negative charges and/or lipophilic groups, [18–21] positively charged polymers, [22,23] covalently linked cationic groups, [24,25] permeabilization of the plasma membrane with surfactants [26] or by physical and mechanical means [27–30]. In bacteria , however, delivery studies of charged metabolites are very limited and only few examples are known. It is possible to transport non- natural nucleotides into bacterial cells by heterologously expressing importers from other species.[31,32] However, this approach is limited to several non -natural nucleotides and needs genetic modification of the organism. Carlson and co-workers reported the development of a cationic polymer, promoting internalization of an adenosine triphosphate derived chemical .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 4 probe into E. coli and B. subtilis.[33] They proposed that the cationic polymers act as general permeabilization reagents that promote the entry of various molecules into the cytosol. Kraus and coworkers used a cyclodextrin based synthetic nucleotide transporter (SNT).[34] It consists of a per -6-amino-β-cyclodextrin coupled to a peptide consisting of eight aminocaproic acid/arginine pairs. The cyclodextrin binds the nucleotide while the peptide disrupts the membrane to facilitate cellular delivery. They postulate that the bound nucleotide is released in the cytosol d ue to replacement by endogenous nucleotides. Successful delivery of a fluorescently modified deoxyuridine triphosphate into live Mycobacterium smegmatis and E. coli was shown with this approach , yet the study was mainly focused on delivery into mammalian cells.[34] Other highly negatively charged molecule that are routinely transported into bacterial cells are nucleic acids . Transformation is usually done by electroporation or by heat shock of chemically competent cells. [35–38] However, it is also possible to use other methods such as cationic polymers and peptides .[39,40] For mammalian cells several advanced methods have been developed, allowing targeted delivery of nucleic acids in vivo .[41] It has to be noted though that for many applications of nucleic acid delivery, it is sufficient to deliver very small copy numbers, as these will be amplified or are catalytically active. However, i n case of nucleotide transport, high concentrations are desirable. For less polar or cationic substances several further methods have been developed to facilitate delivery.[42,43] One example that showed promising results for cargo delivery into various gram -negative bacteria are siderophores. [44,45] Coupling these Fe(III) -chelating molecules to a cargo allows hijacking of the siderophore -mediated iron uptake pathway. This approach was extensively studied for antibiotic conjugates but was also shown to deliver larger molecules like nucleic acid therapeutics into bacteria. [45–50] Carbohydrate conjugates were used to deliver imaging probes into bacteria through the bacteria -specific maltodextrin transport pathway. [51,52] Other approaches involve the use of nanoparticles [41,53], cell - penetrating peptides [54–56], boron clusters[57], or disulfides.[58,59] An established method to control the release of biologically active molecules inside cells or tissue is the introduction of photolabile protecting groups, so called photocages. [60–64] These structures are covalently bound to the bioactive compound rendering them biologically inert and can be cleaved by light irradiation. The use of light allows temporal and spatial control over the release of the biomolecule. One commonly used caging g roup is based on the coumarin scaffold. The coumarin photocage stands out through high biocompatibility, easy .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 5 synthesis, flexibility of structural modifications and a well -studied mechanism of photocleavage.[65,66] Here, we demonstrate that coumarin caged MSN can be delivered into living E. coli cells using a modified cyclodextrin as additive and that they can be released on demand by irradiation with light.

and Discussion Synthesis of Caged MSNs The synthesis of caged MSNs followed our previously reported synthetic methods employing chemoselective phosphorylation with phosphoramidites and regioselective RNase T2 hydrolysis (Scheme 1).[67–71] The synthesis started with guanosine or 15N–labeled guanosine, which was treated with pyrophosphoryl chloride. Controlled hydrolysis leads to the 2 ′-3′ cyclophosphate which was regioselectively opened with RNase T2 to form pGp ( 3) or 15N– pGp ( 4).[69,70] Unlabeled and 15N–labeled pGp was phosphorylated with bisfluorenmethyl phosphoramidite (5), oxidized and Fm -deprotected to form ppGpp ( 6) and 15N–ppGpp ( 7). 15N–ppGpp was purified, while unlabeled ppGpp was used without purification. T reatment with RNase T2 and purification by strong anion-exchange chromatography yielded ppGp (8) and 15N–ppGp (9).[69,70] For photocaging, we adopted the 7-(diethylamino)-4-(but-3-yn-1-yl)coumarin (DEACBY) chromophore introduced by Seyfried et al.[72]. We chose to use this photocage because of its easy access and further because the alkyne group would enable late -stage functionalization of the caged MSNs by copper -catalyzed azide-alkyne cycloadditions. [73,74] Treatment of ppGp ( 8) and 15N–ppGp ( 9) with mixed P–amidite 10 containing the photocage DEACBY followed by oxidation with mCPBA led to the 3′-diphosphate 5′- triphosphate nucleo tides with triesters at the terminal phosphates . The triester at the 3 ′-end acts as a leaving group when the molecules are dissolved in MeOH allowing fast and efficient cyclization to the corresponding 2 ′-3′ cyclophosphates. The fluorenmethyl (Fm) group was removed with piperidine and the cyclophosphate was regioselectively opened with RNase T2 leading to 5 ′-DEACBY protected pppGp (11) and 15N – pppGp (12) in 58 % (97 % considering recovered starting material) and 52 % yield, respectively. Bisfluorenmethyl phosphoramidite reacted chemoselective ly with the monoester phosphate. Oxidation and Fm .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 6 deprotection with 1,8-diazabicyclo[5.4.0]undec -7-en led to the caged MSNs DpppGpp (13) and 15N-DpppGpp (14) in 65 % and 41 % yield respectively. The efficiency of this synthesis enabled the production of 13 mg 15N labeled caged pppGpp ( 14) starting from 70 mg 15N- labeled ppGpp (7). Scheme 1. Synthesis of caged and isotope labeled MSN derivatives.[69,70] a) P2Cl4O3, (11 eq), 0 °C, 3 h. b) NaHCO3 (1 M), 0 °C. c) RNase T2, pH 7.5, 37 °C, 12 h. d) 5 (3.0 eq), ETT (5.0 eq), DMF, rt, 15 min. e) mCPBA (3.0 eq), – 20 °C, 10 min. f) DBU, 0 °C→rt, 30 min. g) RNase T2, aq. HCl, pH 5.5, 37 °C. h) 9 (2.3 – 2.4 eq), ETT (5.0 eq), DMF, rt, 1 h. i) MeOH, rt, 5 min j) Piperidine, DMF, rt, 5 min. k) 5 (1.5 – 1.7 eq), ETT (3.0 – 3.5 eq), DMF, rt, 15 min. l) mCPBA (1.5 – 2.1 eq), -20 °C, 10 min. m) Azide (1.5 eq), Na-ascorbate (5.0 eq), CuSO4 (0.1 eq),THPTA (0.5 eq), H2O, rt, 3 h. Abbreviations: brsm: based on recovered starting material, DBU: 1,8- diazabicyclo(5.4.0)undec-7-ene, ETT: 5-(ethylthio)-1H-tetrazole, Fm: fluorenmethyl, Gua: guanine, mCPBA: meta-chloroperbenzoic acid, rt: room temperature. THPTA: tris(3-hydroxypropyltriazolylmethyl)amine. To synthesize the caged magic spot nucleotide DppGpp, pGp was treated with mixed amidite 9, oxidized, cyclized in methanol and deprotected. Regioselective ring -opening with RNase T2 led to DppGp (14) in 77 % yield on a 700 mg scale. Another cycle of P-amidite coupling with bisfluorenmethyl phosphoramidite, that selectively only reacts with terminal phosphates,[75] oxidation and Fm deprotection with DBU led to the caged guanosine tetraphosphate DppGpp (15) in 69 % yield. The alkyne handle on the photocage now offered the possibility to easily introduce structures that could facilitate uptake into bacterial cells , while photocleavage would .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 7 eventually remove them again. Various carbohydrates (glucose, galactose, mannose, maltose, lactose, trehalose) and a biomimetic enterobactin analogue[76] were readily coupled to DppGpp via copper-catalyzed azide-alkyne cycloadditions [73,74] with yields of 24–79 % (S1– 8). For control experiments, caged adenosine triphosphate (D -ATP, S9) and caged guanosine diphosphate (D -GDP, S10) were synthesized by treating commercial ADP and GMP with mixed amidite 9. Oxidation, and Fm deprotection, yield ed D-ATP and D-GDP. Photolysis Behavior To study the photolysis behavior of the coumarin photocages, the caged MSNs DppGpp (16) and DpppGpp (13) were irradiated with a 400 nm LED in a small glass vial (25 nmol, 50 µM, 14 mW), and the uncaging was monitored by HPLC ( Figure 2). After 2 min irradiation, only trace amounts of caged MSN s remained and the corresponding free MSN s were formed without detectable nucleotide byproducts indicating a clean and efficient photocleavage. Figure 2. HPLC chromatograms (254 nm) of photolysis of DppGpp (A) and DpppGpp (B). Chromatograms were measured before and after irradiation with a 400 nm LED (25 nmol, 50 µM, 14 mW). Uptake of MSN Probes into E. coli Next, uptake into bacteria was assessed by incubating E. coli cell suspensions with solutions of the caged nucleotides. After incubation, the cells were separated by centrifugation, washed, resuspended and fluorescence of the cell suspension was measured. The intrinsic fluorescence of the DEACBY caging group enabled this rapid initial study of potential delivery. MSN derivatives coupled to carbohydrates and siderophore (S1-8) did not produce .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 8 fluorescence above the autofluorescence of E. coli cells (Figure 3A, S2) under the experimental conditions and thus further optimization was required . Therefore, we studied the synthetic nucleotide transporter ( SNT, 17, Figure 3A) developed by Kraus. [34] This commercially available compound was mixed with DppGpp ( 16) and DpppGpp ( 13) and then incubated with an E. coli suspension. After washing , the cell suspension exhibited a substantial increase in fluorescence , a first indication of efficient delivery (Figure 3B). Flow cytometry confirmed these results ( Figure 3C), demonstrating largely increased fluorescence of nearly all bacterial cells. Figure 3. Uptake measurements of MSN in E. coli. (A) Structure of the synthetic nucleotide transporter (SNT). (B) Cellular uptake determined by measuring fluorescence of cell suspensions. Values are normalized to the autofluorescence of cells treated with DppGpp or DpppGpp alon e. Mean ± standard deviation is shown. (C) Histogram plots of flow cytometry measurements indicate MSN cage fluorescence in nearly every cell. Confocal Microscopy Analysis of Cellular Distribution The previously described experiments cannot distinguish whether the MSN s are just bound to the exterior of the E. coli membrane or if they are internalized into the periplasm or .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 9 cytoplasm. Thus, uptake was additionally evaluated with confocal microscopy under optimized conditions using the high throughput methods described above (Figure 4A+B). The cellular membranes were stained with Nile red. The intensity profiles of Nile red fluorescence compared to DEACBY fluorescence show the distribution of the caged MSN s inside the bacterial cells ( Figure 4C+D). While Nile red fluorescence is maximal at the start and at the end of the intensity profiles representing the cellular membrane, DEACBY fluorescence is maximal between the Nile red maxima. This clearly demonstrates that the caged MSNs enter the cytoplasm of E. coli cells. Additionally, confocal fluorescence images reveal ed heterogeneous caged MSN uptake into different bacterial cells. Some cells exhibit high intracellular fluorescence, while others show low or even no DEACBY fluorescence, contrasting with the flow cytometry measurements in Figure 3C. Cell viability was checked next. It was found that using 50 µl/OD600/ml incubation led to no reduced viability, while 200 µl/OD600/ml led to a decrease of viability to 50% (Figure S6). Figure 4. Microscopy images of E. coli treated with a mixture of DppGpp (16, B + D) or DpppGpp (13, A + C) with SNT (17) and the membrane stain Nile Red (red fluorescence). The fluorescence of the photolabile protecting group DEACBY is shown in cyan. C + D) Intensity profiles through a single cell normalized to the highest and lowest grey values. Scale bar = 2 µm in large images and 1 µm in magnified images. Metabolic Tracking of MSNs In order to confirm that uncaging inside E. coli is possible and to track the fate of the released MSNs , we applied an extraction and quantification assay using capillary electrophoresis mass spectrometr y (CE-MS).[69,77] After incubation with DpppGpp ( 13) and SNT (17) and successive washing, the E. coli cells were resuspended in medium the first .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 10 sample for MSN extraction was taken and the cell suspension was irradiated by a 400 nm LED (14 mW, 3 min) to release the free nucleotides (Figure 5A). The culture was then incubated at 37 °C for 1 h and samples were collected a t predetermined time points before and after irradiation . The samples were lysed, spiked with heavy isotope -labeled standards and extracted using weak anion exchange. Quantification was subsequently performed by CE- MS. After irradiation with 400 nm light , we observed a direct and sharp increase in pppGpp levels, confirming successful photolysis (Figure 5B). Over time , pppGpp levels decreased while ppGpp increased, consistent with phosphatase -mediated hydrolysis. In E. coli GppA is the phosphatase responsible for the conversion of pppGpp to ppGpp. In fact, ppGpp ist the main alarmone in E. coli due to GppA activity. [78,79] This was confirmed using 15N-DpppGpp (14): we detected a corresponding increase in [ 15N]5-ppGpp as [ 15N]5-pppGpp declined (Figure 5C), indicating that the ppGpp originates mostly from the uncaged 15N-labeled pppGpp and not through indirect effects of the uncaging process . Bacteria treated with caged GDP or bacteria that were not irradiated with 400 nm light did not show this change in MSN levels (Figure 5B). .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 11 Figure 5. MSNs extracted from E. coli after incubation with caged MSNs. (A) Experimental timeline of extraction experiments. (B) pppGpp and ppGpp concentrations extracted from cell suspension of E. coli cells incubated with DpppGpp/SNT or D-GDP/SNT. Cell suspension was irradiated with 400 nm light before t = 0 min. (C) Ratio of 15N labeled pppGpp and ppGpp extracted from E. coli cell suspension incubated with 15N-DpppGpp/SNT and irradiated with 400 nm light. Before irradiation, no 15N-labeled nucleotides were detected. (D) pppGpp and ppGpp concentrations extracted from cell suspension of E. coli ΔgppA mutant incubated with DpppGpp/SNT or D - GDP/SNT. Cell suspension was irradiated with 400 nm light before t = 0 min. (E) Ratio of 15N labeled pppGpp and ppGpp extracted from E. coli cell suspension that was incubated with 15N-DpppGpp/SNT and irradiated with 400 nm light. After uncaging, cells were separated by centrifugation (5 min, 5 kG) and then immediately lysed in 1 M formic acid. To further validate this assumption, we also performed the extraction experiment with a ΔgppA mutant[80] (Figure 5D). In this strain, pppGpp levels remained stable due to the absence of GppA , however, ppGpp still accumulated, consistent with previous reports that pppGpp allosterically activates RelA, thereby enhancing ppGpp synthesis .[12,15] We performed another control: Bacteria were treated with DpppGpp ( 13) and SNT (17) and [15N]5-pppGpp was spiked into the medium directly after uncaging . The conversion of the heavy -isotope labeled [15N]5–pppGpp to [ 15N]5–ppGpp resembled the conversion of pppGpp to ppGpp (Figure S7), even though [ 15N]5–pppGpp should only be present in the medium, while pppGpp should be located inside the bacterial cells. These results could imply that periplasmic or extracellular phosphatases , such as PhoA[81] and/or phosphatases released .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 12 from damaged cells can also process MSN s. Furthermore, damaged cells may release MSNs into the culture medium or take up 15N-labelled MSNs from the medium. To better understand which portion of the quantified nucleotides were truly intracellular, we separated the cell pellets and the culture supernatants after photolysis by centrifugation and analyzed both fractions. We could detect MSNs in both fractions, but s urprisingly, the supernatant contained more pppGpp but less ppGpp than the pellet fraction (Figure S8). This shows that MSNs leak into the medium, potentially through the action of the SNT . However, rapid intracellular metabolism could reduce detectable MSN levels inside the cells, exaggerating the relative abundance measured in the supernatant. When we repeated these experiments with 15N-DpppGpp ( 14), we found that pppGpp -to-ppGpp conversion was indeed markedly faster in the pellet fraction, whereas extracellular conversion was slower (Figure 5E). Next, we investigated whether a drug efflux system could be responsible for MSN leakage into the medium.[82–84] Therefore, we compared knockout strains lacking the major drug efflux components AcrA, AcrB and TolC with the wild type. [80] In a simple experiment , we incubated the cell suspensions with DpppGpp/SNT and performed four washing steps where we centrifuged the cell suspension, removed the supernatant and resuspended the pellet. We then analyzed the washing solution for DEACBY fluorescence to measure DpppGpp leakage. Even after four washing steps, significant amounts of DpppGpp could still be detected in the washing solution of all the strains investigated (Figure S9). This finding suggests that SNT - induced membrane perturbation, as opposed to active export of internalized caged MSN , serve as the predominant contributors to the observed phenomenon . Taken together, these data suggest that the extracellular MSN pool arises largely from membrane damage, which releases (p)ppGpp into the medium. At the same time, rapid intracellular turnover depletes the internal MSN pool, leading to lower intracellular levels relative to extracellular ones. Consequently, while CE -MS provides robust tracking of MSN photo-release and metabolism, careful interpretation is required, as a portion of the detected signal may not reflect true intracellular pools. Notwithstanding, confocal microscopy unambiguously showed internalization of the probes into a fraction of cells. Taken together, our current interpretation is that part of the probe is released inside of the cells, while another fraction is leaking from cells due to SNT action. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 13 Bacterial Growth Assay The bacterial growth rate is apparently regulated by MSN. [85–87] As simple read -out we envisioned that photo-release of caged MSN should slow down bacterial growth . Therefore, the growth of E. coli was monitored after incubation with the D(p)ppGpp/SNT mixture with and without following photo -release. For wild type E. coli, we could not detect this projected difference in growth rate. As shown in the confocal microscopy experiments described above, uptake of the caged MSN s was heterogeneous. We therefore attribute the absence of a measurable population -level effect to this cell-to-cell variability: while some cells internalized high amounts of D(p)ppGpp, others likely contained only minimal levels, resulting in no apparent change in overall growth. To overcome this limitation , we switched to a mutant strain incapable of synthesizing MSNs ((p)ppGpp0) due to deletion of the relA and spoT genes . As basal levels of MSNs are important to maintain cellular component homeostasis, t his mutant has multiple disabilities in cell division, transcription and translation and can have difficulties to adapt to environmental changes .[4,6] Due to the absence of MSNs in this mutant, the influence of artificially added MSNs on the growth rate should be pronounced , but in this case potentially increase the growth rate due to compensation of above -mentioned defects. We observed that treatment with SNT alone decreases the bacterial growth rate, likely due to its toxicity by damaging the bacterial envelope. To evaluate the influence of DppGpp and DpppGpp mixed with SNT on the growth rate of the E. coli (p)ppGpp0 strain, we used D-ATP (S9) mixed with SNT as a control . Interestingly, incubation of (p)ppGpp 0 with SNT and caged (p)ppGpp already recovered the loss in growth rate caused by SNT. Caged ATP did not show this effect (Figure 6A). One possible conclusion is that caged MSNs, due to their versatile binding patterns,[88] can already interact with some of the binding partners of MSN and thus restore growth. In line with this interpretation, uncaging of (p)ppGpp did not further affect the recovery of the growth rate ( Figure 6B). This interpretation is in line with previous findings for caged molecules.[63,89,90] Future studies will now address the increase of bulkiness in the cage through click chemistry on the alkyne handle to abrogate unwanted binding prior to uncaging.[91] .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 14 Figure 6. Growth rate of the E. coli (p)ppGpp0 mutant treated with SNT and different nucleotides. (A) Comparison of the different treatment conditions without light irradiation. Analyzed by Brown-Forsythe and Welch ANOVA test with Dunnett′s T3 multiple comparisons test; individual variances computed for each comparison. ns: p =0.125; **: p = 0.0054. (B) Comparison of samples irradiated or not irradiated with 400 nm light. Analyzed with two-way ANOVA with Šídák multiple comparisons test, with a single pooled variance. ns: p > 0.99. Mean and standard error of mean of at least four separate experiments is shown. Summary and Conclusion In this study , we present the first photocaged derivatives of the MSNs pppGpp and ppGpp. DppGpp (16), Dpp pGpp (13), and the heavy isotope labeled 15N-DpppGpp (14) were efficiently synthesized in high yields using a chemoenzymatic synthesis approach . By comparing different delivery strategies, we achieve the delivery of highly negatively charged, caged MSNs through the complex cell envelope of living E. coli. Several analytical approaches combined provide evidence for intracellular delivery into a subpopulation of cells . Upon 400 nm irradiation, these probes release free nucleotides, enabling tracking of pppGpp - to-ppGpp conversion by CE -MS. Moreover, initial growth curve studies in wild type and (p)ppGpp0 strains demonstrate the potential of these probes for in vivo studies after further optimization: While photolysis and metabolic conversion were clearly demonstrated, extraction experiments revealed substantial leakage of MSNs into the extracellular medium, complicating accurate quantification of intracellular levels. Growth studies with a (p)ppGpp⁰ strain confirmed that caged MSNs can modulate bacterial physiology, but no light -dependent growth effect was yet observed, likely due to residual activity of the caged species. Future work should prioritize improved transporter strategies and the development of bulkier, biologically inert cages. Single -cell studies of cells that have received large amounts of caged nucleotide could help attribute heterogeneous uptake to specific cellular states . Despite its current limitations, this study provides first significant strategies how to deliver .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 15 highly charged signaling molecules into living bacteria and control cellular concentrations on a minute time-scale through photo -uncaging.

We thank Dr. Guizhen Liu, Dr. Mengsi Lu and Dr. Anuj Shukla for CE -MS measurements. Furthermore, w e thank the staff of the Life Imaging Center (LIC) of the University of Freiburg, in particular I. Bierschenk, A. Neumann and R. Nitschke for help with their microscopy resources, and the excellent support in image recording and analysis. The following instrument funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) was used: ZEISS LSM 880 Observer / Fast Airyscan with inverted microscope (Instrument Grant Number 317784314). In addition, we thank D. Herchenbach and M. Follo of the Lighthouse Core Facility, funded in part by the Medical Faculty, University of Freiburg (Project Number 2023/B3 -Fol). Hans-Georg Koch acknowledges funding by the SFB1381 (Project-ID 403222702). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 864246, to H.J.J.). This study was supported by the Deutsche Forschungsgemeinschaft (DFG) under Germany's excellence strategy (CIBSS, EXC-2189, Project ID 390939984, to H. J. J ) Author Contributions Christoph Popp: Synthesis, Investigation, Formal analysis, Visualization, Writing; Patrick Moser: Synthesis of 15N-DpppGpp (14); Xinwei Liu: Supervision and discussion of in vivo experiments; Johannes Freitag: Supervision and discussion of in vivo experiments; Isabel Prucker: Discussion of CE-MS results; Robert Zscherp: Synthesis of S11; Xuan Wang: Synthesis of S13; Philipp Klahn: Supervision and discussion ; Hans-Georg Koch: Supervision and discussion; Gert Bange: Supervision and discussion of in vivo experiments; Henning J. Jessen: Conceptualization, Supervision, Funding acquisition, Writing – review & editing .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 22, 2025. ; https://doi.org/10.1101/2025.10.22.683773doi: bioRxiv preprint 16 Conflict of Interest The authors declare no conflict of interest.

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