Synthesis and application of a photocaged-l-lactate for studying the biological roles of l-lactate | Communications Chemistry

Communications Chemistry volume 8, Article number: 104 (2025) Cite this article

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l-Lactate, once considered a metabolic waste product of glycolysis, is now recognized as a vitally important metabolite and signaling molecule in multiple biological pathways. However, exploring l-lactate’s emerging intra- and extra-cellular roles is hindered by a lack of tools to perturb l-lactate concentration intracellularly and extracellularly. Photocaged compounds are a powerful way to introduce bioactive molecules with spatiotemporal precision using illumination. Here, we report the development of a photocaged derivative of l-lactate, 4-methoxy-7-nitroindolinyl-l-lactate (MNI-l-lac), that releases l-lactate upon illumination. We validated MNI-l-lac in cell culture by demonstrating that the photorelease of l-lactate elicits a response from genetically encoded extra- and intracellular l-lactate biosensors (eLACCO1, eLACCO2.1, R-iLACCO1.2). To demonstrate the utility of MNI-l-lac, we employed the photorelease of l-lactate to activate G protein-coupled receptor 81 (GPR81), as revealed by the inhibition of adenylyl cyclase activity and concomitant decrease of cAMP. These results indicate that MNI-l-lac may be useful for perturbing the concentration of endogenous l-lactate in order to investigate l-lactate’s roles in metabolic and signaling pathways.

l-Lactate is an abundant metabolite that is produced from pyruvate, the end-product of glycolysis, through the action of l-lactate dehydrogenase. While l-lactate has traditionally been viewed as a waste byproduct of glucose metabolism1, recent studies suggest it has diverse roles as both an energy source and a signaling molecule in the nervous system2, tumor microenvironment3,4, and immune system5,6. For example, l-lactate activates the G protein-coupled receptor 81 (GPR81), triggering downstream signaling that decreases intracellular cAMP levels through Gi-mediated inhibition of adenylyl cyclase7,8,9. This GPR81-mediated signaling pathway has been demonstrated to be involved in mitigating excitatory damage10,11,12, lipid metabolism7,13, and inflammatory regulation14, highlighting l-lactate’s importance as a regulatory molecule in brain function. The emerging recognition of the roles of l-lactate has implications for physiological and pathological processes that span intracellular15, intercellular2,16, and interorgan17,18 scale environments.

The increased recognition of the various biological roles of l-lactate has highlighted the need for improved tools to study its roles through both its measurement and perturbation in tissues. We and others have recently reported a variety of genetically encoded biosensors for the measurement and imaging of l-lactate in tissues19,20,21,22,23,24,25,26,27,28,29,30,31,32,33. Applications of these measurement tools would be complemented by a tool for perturbing l-lactate concentrations. A molecule that enables the photorelease of l-lactate (that is, a ‘caged’ l-lactate) could be a particularly powerful tool for the spatiotemporal manipulation of l-lactate concentration in tissues. Photocaged compounds have been used as perturbation tools in a wide range of applications34,35,36,37. Since the original reports of o-nitrobenzyl-caged ATP and cAMP in the late 1970s38,39, the demand for alternative or improved caging groups has led to many new types of photocages, including ones that are coumarin-based40, cyanine-based41, and o-nitro-2-phenethyl-based42,43.

One of the most common applications of caged compounds is the study of neuronal processes by one- or two-photon uncaging of 4-methoxy-7-nitroindonyl-L-glutamate (MNI-Glu), which was independently reported by Matsuzaki et al.44 and Canepari et al.45 in 2001. MNI-Glu is the most widely used type of caged glutamate due to its high stability at physiological pH, resistance to hydrolysis, and solubility in physiological buffers46. In addition, MNI-Glu’s uncaging wavelength in the near UV is spectrally compatible with other commonly used chromophores, such as green fluorescent protein (GFP), enabling the combined use of photocaged glutamate and GFP or GFP-based biosensors44. Other MNI-caged compounds have also been developed, including MNI-D-aspartate47 and MNI-auxins48.

In an effort to facilitate studies of l-lactate’s role as a metabolite and a signaling molecule and potentially help resolve ongoing controversies such as the astrocyte to neuron l-lactate shuttle (ANLS) hypothesis2, we undertook the development of an MNI-caged l-lactate (MNI-l-lac). In this work, we report the synthesis and characterization of MNI-l-lac, and validation of l-lactate photorelease in cell culture.

To design a photocaged l-lactate, we considered two key factors: the choice of the photocaging group and the position of attachment to l-lactate. Based on the success and widespread application of MNI-Glu44,45, we chose to pursue an analogous design in which the MNI photocaging group would be linked to the carbonyl carbon of l-lactate.

To synthesize MNI-l-lac, we expected to be able to employ a synthetic strategy analogous to the one commonly reported for MNI-Glu44,45. Specifically, we planned to couple 4-methoxyindoline to l-lactate with an amide bond-forming reaction. We would then perform a nitration reaction to introduce the 7-nitro group at the desired position, para to the 4-methoxy group. Following this plan, we succeeded in coupling 4-methoxyindoline with l-lactate. However, when we attempted the nitration reaction, we found that the nitration occurred preferentially on the hydroxyl group of l-lactate rather than at the desired position on the 4-methoxyindoline (Supplementary Fig. 1). To circumvent this problem, we tried nitrating 4-methoxyindoline first before coupling the nitrated product to free l-lactate. However, we were unable to obtain the desired MNI-l-lactate, possibly due to the electron-withdrawing nature of the nitro group attenuating the indoline’s nucleophilicity.

Ultimately, we obtained the desired product by first protecting the hydroxyl group of ethyl l-lactate with tert-butyldimethylsilyl (TBDMS) to give compound 2 (Fig. 1a). 4-methoxyindoline (compound 3) was prepared by reducing commercially available 4-methoxyindole with sodium cyanoborohydride in acetic acid. Compound 3 was then successfully coupled with compound 2 using HATU/EDAC coupling conditions to give compound 4. Compound 4 was nitrated using silver nitrate and acetyl chloride, which gave a mixture of the ortho- and para- nitro-substituted regioisomers (compounds 5 and 6, respectively). These regioisomers were separated by flash chromatography using silica gel to yield the ortho-isomer as an oil (compound 5) and the para-isomer as a yellow solid (compound 6). The desired regioisomer, compound 6, was deprotected with TBAF/AcOH to afford MNI-l-lac (compound 7) as a yellow solid. The deprotection was performed under acidic conditions because standard basic conditions resulted in cleavage of the amide bond, yielding free 7-nitroindoline as the major product. This lability under basic conditions suggests that MNI-l-lac is susceptible to amide bond cleavage, which may explain the poor yield for the coupling reaction between unprotected l-lactate and indoline in the initial (and unsuccessful) synthetic strategy.

a Synthetic scheme of MNI-l-lac. b Schematic representation of the photo-uncaging of MNI-l-lac to release l-lactate, a proton, and the nitrosoindole by-product. c Absorbance spectra were recorded during the progressive photolysis of MNI-l-lac (40 μM solution in l-lactate (-) buffer (30 mM MOPS, 100 mM KCl, 1 mM CaCl2, pH 7.2) containing 10% DMSO). Dotted red curve: MNI-l-lac, λmax (ε) = 330 nm (5400 M−1cm−1). Black curve: nitrosoindole by-product after photolysis, λmax (ε) = 403 nm (7200 M−1cm−1). The arrows indicate the effect of increasing irradiation time for photolysis.

To investigate whether the expected uncaging reaction49 occurred upon UV illumination of MNI-l-lac, we measured the UV-Vis absorption spectra of the compound exposed to 365 nm UV light for increasing durations (Fig. 1b). The progressive photolysis of MNI-l-lac in aqueous neutral buffer resulted in a bathochromic shift in the absorption maximum, with distinct isosbestic points (Fig. 1c). Based on this data, we estimate that the conversion is 90% complete within 10 min illumination with 365 nm light (810 μW/cm2). We also observed the color of the MNI-l-lac solution turn from clear to yellow upon UV illumination, consistent with the bathochromic shift in the absorption spectra and the conversion of the starting MNI-l-lac (λmax = 330 nm) into the 4-methoxynitrosoindole byproduct (λmax = 403 nm)50,51,52.

To determine if l-lactate was released upon UV illumination of MNI-l-lac, we used a commercial l-lactate assay kit to quantify the amount of l-lactate released in both buffered and unbuffered aqueous solutions at two concentrations (Supplementary Fig. 2 and Supplementary Table 1). The results indicated that l-lactate was released upon illumination to over 86 ± 8% (mean ± SD) of the theoretically expected concentration. The difference between the uncaging efficiency in buffered versus unbuffered solution was insignificant (one-way ANOVA, p = 0.858).

We envisioned that our previously reported genetically encoded l-lactate biosensors, such as eLACCO1 (Table 1), would provide an effective means of characterizing and validating the utility of MNI-l-lac in the context of cultured cells23. Before proceeding with cell-based experiments, we first needed to determine if MNI-l-lac could be uncaged in the presence of the purified eLACCO1 biosensor (Fig. 2a)23.

a Schematic representation of the eLACCO1 biosensor’s fluorescence response. eLACCO1 consists of cpGFP + l-lactate binding domain (LBD). b eLACCO1 fluorescence emission (λex = 460 nm) with MNI-l-lac (40 µM) with different UV illumination times (average of n = 3). The arrow indicates the effect of increasing illumination time. c Overlay of l-lactate (gray) and uncaged MNI-l-lac (black) titrations using the eLACCO1 fluorescence biosensor (n = 3, error bars are SEM). MNI-l-lac solutions in varying concentrations were illuminated for 10 min.

To determine if the MNI-modified l-lactate was indeed “caged” with respect to binding to the eLACCO1 biosensor, we performed a fluorescence assay using purified eLACCO1 protein along with MNI-l-lac exposed to varying durations of UV light. The result revealed that the fluorescence intensity of eLACCO1 with MNI-l-lac (t = 0 min; no light illumination) and the l-lactate (-) control condition (lactate-free state) was very similar (Fig. 2b). This indicated that MNI-l-lac itself did not induce a meaningful fluorescence response for eLACCO1. Additionally, as the illumination time of MNI-l-lac increased, so too did the fluorescence response of eLACCO1. This increase in fluorescence indicates eLACCO1’s response to the release of free l-lactate from MNI-l-lac upon light illumination. The fluorescence response of the eLACCO1 biosensor reached its maximum after 8 minutes of UV illumination. Accordingly, for all later experiments, we used 10 minutes of illumination time to ensure near-complete uncaging of MNI-l-lac.

To further investigate whether lactate released from MNI-l-lac is indeed l-lactate (as opposed to D-lactate), we performed a titration experiment against eLACCO1 to determine the apparent dissociation constant (Kd) value. If MNI-l-lac releases l-lactate, we expect identical Kd values and maximum fluorescence responses (ΔF/F) for completely uncaged MNI-l-lac (i.e., illuminated for 10 min with 365 nm light) and native l-lactate. The fluorescence intensity of l-lactate titration using sodium l-lactate and uncaged MNI-l-lac were plotted against log [l-lactate or uncaged MNI-l-lac] (µM), which was fitted to the Hill Equation to find the Kd values (Fig. 2c). The apparent Kd values were calculated to be 4.0 µM and 3.3 µM for l-lactate (previously reported values were 4.1 µM for l-lactate, 120 μM for D-lactate) and uncaged MNI-l-lac, respectively 23. Similarly, the ∆F/F0 of eLACCO1 at the maximum concentration of 100 µM was calculated to be 7.2 for l-lactate and 5.7 for uncaged MNI-l-lac, which were close to the literature value of 623. There appeared to be a substantial divergence in the fluorescence response with standard l-lactate versus uncaged MNI-l-lac at concentrations above 10 µM, which may be due to fluorescence attenuation by an inner filter effect. This is consistent with the data in Fig. 1c, where the byproduct 4-methoxynitrosoindole has some absorbance at 450 nm that partly overlaps the excitation of eLACCO1.

Genetically encoded fluorescent biosensors enable precise and dynamic imaging of l-lactate in either the extracellular or intracellular environments, allowing for detailed spatial and temporal analysis. We have previously reported the green-fluorescent eLACCO series of biosensors for extracellular l-lactate23,30, and the red-fluorescent R-iLACCO series for intracellular l-lactate (Table 1)30. Our in vitro results were consistent with the conclusion that illumination of MNI-l-lac releases l-lactate, which, in turn, binds to and causes the high-affinity eLACCO1 biosensor to respond.

We next aimed to determine if MNI-l-lac could be used to perturb the l-lactate concentration in the environment of cultured mammalian cells. To determine if uncaging of MNI-l-lac could perturb the extracellular l-lactate concentration, we prepared HeLa cells expressing the l-lactate biosensor eLACCO2.1 or the non-lactate responsive deLACCO1 control on the cell surface. Relative to eLACCO1, eLACCO2.1 has a higher Kd (Table 1)23,30 which is close to the basal extracellular l-lactate concentration in mouse brain (approximately 0.2 mM)53 and close to the resting serum concentration (approximately 1 mM)54. We performed fluorescence imaging to determine the response of deLACCO1 and eLACCO2.1 on the surface of HeLa cells to uncaging of MNI-l-lac (1 mM, 1 s, 405 nm, 2.4 mW/cm2). The intensity and duration of the 405 nm pulse are comparable to values typically used for uncaging of MNI-Glu55. The deLACCO1 variant is not responsive to l-lactate but retains the pH sensitivity of eLACCO2.123,30. Controlling for pH changes in this experiment is essential due to the fact that uncaging of MNI-l-lac releases l-lactate plus a proton (Fig. 1b). In addition, lactate is transported across the mammalian cell membrane by proton-dependent monocarboxylate transporters (MCTs), such that l-lactate import is necessarily accompanied by proton import and a decrease in cytosolic pH56,57.

When MNI-l-lac was uncaged in the presence of HeLa cells, we observed distinctly different fluorescent responses for cells expressing eLACCO2.1 versus deLACCO1 (Fig. 3a, b). For cells expressing the deLACCO1 control biosensor, uncaging produced a rapid 37 ± 1% (mean ± SEM, n = 10) decrease in fluorescence followed by a recovery to baseline within about one minute. For cells expressing the eLACCO2.1 biosensor, uncaging produced a smaller 10 ± 3% (mean ± SEM, n = 9) decrease in fluorescence followed by an increase to a value that was 19 ± 4% (mean ± SEM, n = 9) greater than the baseline (Fig. 3c). We rationalize these responses as follows. The first sudden decrease in ∆F/F immediately after photoactivation is likely due to the release of protons from the uncaging reaction of MNI-l-lac, causing a fluorescence decrease in these pH-sensitive biosensors23. Second, the transient increase in fluorescence observed with eLACCO2.1 but not with deLACCO1 suggests that the local concentration of l-lactate has been substantially increased due to the released l-lactate from MNI-l-lac. The later slight decrease in fluorescence may be due to the diffusion of l-lactate over time.

a Schematic of the experimental design where eLACCO2.1 was used to detect the uncaging of MNI-l-lac (1 mM). b deLACCO1 or eLACCO2.1 expressed on the cell surface of HeLa cells before and after MNI-l-lac photoactivation at 1 minute (405 nm, 1 s, 2.4 mW/cm2). Photoactivation was performed over the entire field of view. Images in the ΔF/F column are pseudocolored as (F - F0)/F0 to illustrate the change in fluorescence upon photo-uncaging (F0 is for t = 0.16 min and F is for t = 1.6 min). Scale bars represent 20 µm. c Fluorescence response of eLACCO2.1 to uncaged MNI-l-lac over time. n = 10 and 9 cells for deLACCO1 (dashed, grey line) and eLACCO2.1 (solid, cyan line), respectively (n = 3 biological replicates, mean ± SEM). d Schematic of the experimental design where R-iLACCO1.2 was used to detect the uncaging of MNI-l-lac. e R-diLACCO1 or R-iLACCO1.2 in HeLa cells under the same conditions as in b). Scale bars represent 20 µm. f Fluorescence response of R-iLACCO1.2 (solid, magenta line) and R-diLACCO1 (dashed, grey line) to uncaged MNI-l-lac over time. n = 12 and 20 cells, respectively (n = 3 biological replicates, mean ± SEM).

As an additional control, the imaging experiment was performed in the absence of MNI-l-lac. The initial decrease of ∆F/F by 2 ± 1% (mean ± SEM, n = 13) for eLACCO2.1 and 14 ± 0.2% (mean ± SEM, n = 12) for deLACCO1 was still evident after photoactivation but was less than in the experiment with MNI-l-lac (Supplementary Fig. 3). A possible explanation for this decrease is transient quenching of GFP fluorescence due to heating or photoswitching. Furthermore, while we did not observe a transient increase in fluorescence for HeLa cells expressing eLACCO2.1, there was a gradual increase up to the value of 18 ± 2% (mean ± SEM, n = 13) over time post-photoactivation (Supplementary Fig. 3). This increase is attributed to the efflux of endogenous l-lactate from cells to extracellular space as cells continually produce l-lactate through glycolysis over the course of measurement. The reasoning is also supported by a small but steady increase of eLACCO2.1 (not observed in deLACCO1) fluorescence even before photoirradiation (Fig. 3c, 0–1 min). In summary, these results support the conclusion that uncaging of MNI-l-lac releases l-lactate at a sufficient concentration to induce the fluorescence response of eLACCO2.1.

Next, we explored whether we could observe an increase in intracellular l-lactate concentration upon uncaging MNI-l-lac with HeLa cells expressing R-iLACCO1.2 and R-diLACCO1 (Fig. 3d, e). Contrary to the eLACCO2.1 cell-based characterization, we imaged HeLa cells expressing R-iLACCO1.2 under glucose-starved conditions to reduce the intracellular l-lactate concentration and to better observe the change upon uncaging of MNI-l-lac (5 mM). In addition, R-iLACCO1.2 imaging was performed with and without AR-C15585858, an inhibitor of the MCT1 and MCT2 lactate transporters. With no inhibitor, following illumination both R-iLACCO1.2 and R-diLACCO1 exhibited an initial decrease in fluorescence of 5 ± 3% (mean ± SEM, n = 12) and 38 ± 1% decrease (mean ± SEM, n = 20) respectively (Fig. 3f). This initial decrease is consistent with the results using eLACCO2.1 and is likely due to transient acidification due to the release of protons from the uncaging of MNI-l-lac. This transient acidification would quench the fluorescence of the pH-sensitive R-iLACCO. Following this initial decrease, the fluorescence of R-iLACCO1.2 increased to 21 ± 5% (mean ± SEM, n = 12) above its initial baseline (Fig. 3f). Based on a previously reported characterization of R-iLACCO1.2 in HeLa cells treated with l-lactate30, a ~ 20% increase in red fluorescence is consistent with an extracellular l-lactate concentration in the range of 10 s of μM. This suggests that only a small fraction of MNI-l-lac is uncaged, and/or only a small fraction of either MNI-l-lac or the released l-lactate is entering the cells, under the illumination conditions used for these experiments (405 nm, 1 s, 2.4 mW/cm2).

R-iLACCO1.2 exhibited no fluorescence increase above baseline when MNI-l-lac was uncaged in the presence of AR-C155858 (Supplementary Fig. 4). The result suggests that MNI-l-lac has poor cell membrane permeability and remains primarily outside the cell until it is uncaged. This poor membrane permeability may limit the utility of MNI-l-lac to applications in which extracellular uncaging is appropriate. While our results demonstrate that l-lactate that is produced in the extracellular environment is taken up by cells relatively rapidly, a caged form of l-lactate that is membrane permeable may have advantages for some applications.

As an additional control experiment, we subjected cells expressing R-diLACCO1 or R-iLACCO1.2 to our standard uncaging conditions, but in the absence of MNI-l-lac (Supplementary Fig. 5). Under these conditions, we did not observe the initial transient decrease in fluorescence that we always observe when MNI-l-lac is present (Fig. 3f and Supplementary Fig. 4). This is consistent with the initial decrease being caused by transient acidification due to the release of protons from the uncaging of MNI-l-lac. However, we did observe a persistent decrease in fluorescence for R-iLACCO1.2 after photoactivation, likely caused by photobleaching. A similar photobleaching effect is observed for R-diLACCO1 with MNI-l-lac (Fig. 3f) and for both R-diLACCO1 and R-iLACCO1.2 with MNI-l-lac and MCT inhibitor (Supplementary Fig. 4). We suggest that this photobleaching effect is occurring in all of our uncaging experiments, but can be masked by the l-lactate-dependent fluorescent increase when it occurs (i.e., for R-iLACCO1.2 in Fig. 3f). A corollary of this is that the observed fluorescence intensity increases due to lactate influx are likely smaller than they would otherwise be. For reasons that remain unclear to us, we did not observe this same photobleaching with R-diLACCO1 when illuminated in the absence of MNI-l-lac (Supplementary Fig. 5). Notably, mApple-based constructs, such as R-diLACCO1, can exhibit complex photoswitching behavior upon illumination with blue light59. Overall, this control experiment provides further support for the conclusion that the observed response of R-iLACCO1.2 to uncaging of MNI-l-lac is due to the MCT-mediated import of released l-lactate from the extracellular environment.

l-Lactate has been proposed to serve as an important signaling molecule in the brain and central nervous system8,60. For example, activation of GPR81 by l-lactate induces Gi-mediated downregulation of adenylyl cyclase, leading to a subsequent decrease in cAMP levels7,8,9. Expecting that the photo-uncaging of MNI-l-lac could serve as a means to artificially activate this pathway, we explored whether we could decrease intracellular cAMP levels in GPR81-expressing cells, as visualized using the genetically encoded cAMP indicator, cAMPinG1 (Fig. 4a)61.

a Schematic showing the activation of GPR81-cAMP signaling pathway from uncaging MNI-l-lac. b Images of the cAMPinG1 expressing HeLa cells before and after photoactivation at 1 min (405 nm, 1 s, 2.4 mW/cm2). Photoactivation and pseudocoloring (ΔF/F column) were done as described in Fig. 3b with F0 at t = 0.8 min and F at t = 5 min. Scale bars represent 20 µm. c Fluorescence response of cAMPinG1 versus time in HeLa cells expressing both cAMPinG1 and GPR81-mCherry in the presence (solid, cyan line) or absence (dashed, grey line) of 5 mM MNI-l-lac. n = 24 for both conditions (n = 3 biological replicates, mean ± SEM).

To determine if uncaging of MNI-l-lac could activate GPR81, we prepared HeLa cells expressing both the intracellular green fluorescent cAMP biosensor cAMPinG1 and membrane-associated GPR81-mCherry2. The mCherry2 fluorescent tag was added at the end of the GPR81 sequence to assess the cotransfection efficiency. We confirmed that treatment with micromolar concentrations of l-lactate caused a decrease in cAMPinG1 fluorescence only in cells expressing GPR81, consistent with a decrease in cAMP concentration (Supplementary Fig. 6). Uncaging of MNI-l-lac (5 mM) resulted in an immediate decrease in the fluorescence of cAMPinG1 followed by a rapid increase to a level that was 36 ± 4% (mean ± SEM, n = 24) less than the initial baseline (Fig. 4b, c). This time course is consistent with the immediate biosensor quenching being caused by the release of protons, as we had seen before with the cell-based imaging. The fact that the fluorescence did not return to the original baseline is consistent with the inhibition of cAMP production, leading to a decrease in cAMPinG1’s fluorescence. In the absence of MNI-l-lac, there was no substantial decrease in cAMPinG1’s fluorescence (Fig. 4c). We did observe a gradual increase in fluorescence over time, possible due to the production of cAMP over the course of experiment.

To obtain further evidence to support the conclusion that the initial transient decrease in cAMPinG1 fluorescence was due to proton release upon uncaging of MNI-l-lac, we performed a control experiment with HeLa cells coexpressing GPR81 and pHuji, a red fluorescent protein-based pH indicator62. Illumination to uncage MNI-l-lac resulted in an immediate and transient decrease in the fluorescence of pHuji, followed by a gradual return to baseline over time. (Supplementary Fig. 7). This observation aligns with our previous observations using both the l-lactate and cAMP biosensors and provides further evidence in support of transient acidification causing the transient decrease in fluorescence. Presumably, there is a delay between when protons are released and when the resting pH of the extracellular buffer, and the cytosol, is re-established (as reported by the pH indicator or biosensor). Overall, these results substantiate MNI-l-lac’s potential in perturbing the concentration of endogenous l-lactate to investigate its roles in metabolism and signaling pathways.

In this study, we have reported the synthesis, characterization, and validation of a photocaged l-lactate molecule, 4-methoxy-7-nitroindolinyl-l-lactate (MNI-l-lac). The progressive photolysis studies, along with the quantification of released l-lactate using a commercial assay, demonstrated the ability of MNI-l-lac to release free l-lactate upon illumination. Genetically encoded l-lactate biosensors were then used to validate the release of l-lactate in vitro. The results revealed a correlation between the biosensor response and the uncaging of MNI-l-lac, demonstrating compatibility between these complementary tools for perturbation (MNI-l-lac) and visualization (lactate biosensors). Furthermore, we demonstrated the combined use of MNI-l-lac to perturb the l-lactate concentration in cultured cells and visualize the change of l-lactate concentration with l-lactate biosensors. The results from utilizing both intracellular and extracellular l-lactate biosensors are consistent with the conclusion that MNI-l-lac is not highly membrane permeable. However, l-lactate that is released in the extracellular environment is efficiently transported into cells. Finally, we used uncaging of MNI-l-lac to activate GPR81 which was expected to lead to inhibition of intracellular cAMP production. Indeed, we observed that uncaging of extracellular MNI-l-lac resulted in the activation of GPR81 leading to a decrease in intracellular cAMP levels in cultured cells expressing GPR81, as visualized using a cAMP biosensor. One limitation of MNI-l-lac is poor membrane permeability which hinders its application for intracellular uncaging. Development of a caged l-lactate that is membrane-permeable could be an important direction for future research. In summary, we have demonstrated that MNI-l-lac is a practically useful new tool that will enable researchers to explore the spatial and temporal dynamics of l-lactate as a signaling molecule and metabolite. Although the biological applications in this initial study have been limited to cultured cells, the precedent of MNI-Glu suggests that MNI-l-lac should be well-suited to applications in more complex tissue preparations such as organotypic or acute brain slices.

All chemicals and solvents were purchased from Tokyo Chemical Industries, Wako Pure Chemical, and Aldrich Chemical Co. HeLa cell line was purchased from ATCC (#CCL-2). Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JNM-ECS400 or JEOL JNM-ECZ500R/M3 instrument. Mass spectra were measured with a Bruker Compact System. Products were purified by flash column chromatography using Isolera-1SW (Biotage) and Biotage Sfär Silica HC D column. Absorbance spectra were measured using the Shimadzu UV1800 spectrometer. Fluorescence emission spectra were recorded on a Spark plate reader (Tecan).

The details of the chemical synthesis and characterization of MNI-l-lac is described in the Supplementary Methods. For the NMR spectra of new compounds, see Supplementary Fig. 8–14.

The gene encoding eLACCO1 cloned into pBAD-HisB with an N-terminus 6×His tagged was expressed in E. coli strain DH10B (Thermo Fisher Scientific). A single colony from freshly transformed E. coli was inoculated into two tubes of 5 mL of Terrific Broth (1 μL mL−1 ampicillin) at 37 °C overnight. The seed culture was then added to 200 mL of fresh Terrific Broth (2% Luria-Bertani Broth supplemented with additional 1.4% tryptone, 0.7% yeast extract, 54 mM K2PO4, 16 mM KH2PO4, 0.8% glycerol) with ampicillin (100 μL/100 mL of Terrific Broth) in a 500 mL flask, and incubated by shaking at 37 °C and 250 rcf for 1 h to reach the exponential growth phase (OD > 0.6). Once the OD600 reached 0.6, L-arabinose (200 µL/100 mL TB) was added to induce expression for another 16 h in a 25 °C shaker at 250 rcf. Bacteria were then harvested and lysed using a sonicator (QSonica) for 2 cycles of 60 s sonication with a 60 s break in between at a 50% duty cycle. After centrifugation at 12,000 rcf for 20 min, the supernatant was purified with Ni-NTA affinity agarose beads (G-Biosciences) on a column (Thermo Fisher ScientificTM PierceTM Centrifuge Columns 10 mL). The eluted sample was further concentrated and desalted with an Amicon Ultra-15 Centrifugal Filter Device (Merck).

For the progressive uncaging experiment, a stock solution of MNI-l-lac (10 mM) was diluted to a final concentration of 100 µM with l-lactate (-) buffer (30 mM MOPS, 100 mM KCl, 1 mM CaCl2, pH 7.2). The solution was aliquoted into 5 tubes, and each tube was illuminated with 365 nm UV for 0 min, 0.5 min, 1 min, 4 min, and 8 min, respectively. To conserve protein structure, the eLACCO1 biosensor itself was not illuminated. The fluorescence response of 5 µM eLACCO1was measured in the presence of 40 µM of MNI-l-lac (25 μL protein solution and 20 µL MNI-l-lac were added to 5 μL lac (-) buffer). All fluorescence measurements using a plate reader were performed using the same conditions (λex = 460 ± 20 nm, λem = 545 ± 20 nm).

For l-lactate titration experiments, sodium l-lactate buffers were prepared by diluting an l-lactate (+) buffer (l-lactate (-) buffer + 100 mM l-lactate, pH 7.2) in an l-lactate (-) buffer to provide l-lactate concentrations ranging from 0 to 100 µM at 25 °C. The fluorescence intensities of 5 μL purified eLACCO1 protein in 45 μL lac (+) buffer were measured in various sodium l-lactate concentrations ranging from 10 nM to 100 µM. For the MNI-l-lac titration, a stock solution of MNI-l-lac (10 mM) was diluted with an l-lactate (-) buffer to provide MNI-l-lac concentrations ranging from 10 nM to 100 µM at 25 °C. Each solution was illuminated with a 365 nm UV lamp for 10 min to fully uncage MNI-l-lac. The fluorescence intensities of 5 µL purified eLACCO1 protein mixed with 45 µL illuminated MNI-l-lac were measured. Fluorescence intensities were plotted against both MNI-l-lac and sodium l-lactate concentrations separately and fitted to a modified Hill Equation (Eq. 1) in GraphPad Prism (equation name: log(agonist) vs. response – Variable slope) to determine the apparent Kd. All fluorescence measurements using a plate reader were performed using the same conditions (emission mode, λex = 460 ± 20 nm, λem = 545 ± 20 nm).

Where min and max are the minimum and maximum plateaus of the curve. [L] is the concentration of l-lactate, and n is the Hill slope. Kd is the dissociation constant and the l-lactate concentration where half the binding sites are occupied.

HeLa cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Nakalai Tesque) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% penicillin-streptomycin (Nakalai Tesque) at 37 °C and 5% CO2. Cells were seeded in 35 mm glass-bottom cell-culture dishes (Iwaki) and transiently transfected with the eLACCO2.1 and deLACCO1 using polyethyleneimine (Polysciences) prior to imaging. Transfected cells were imaged using an IX83 wide-field fluorescence microscopy (Olympus) equipped with a pE-300 LED light source (CoolLED), a 40× objective lens (numerical aperture (NA) = 1.3; oil), an ImagEM X2 EM-CCD camera (Hamamatsu), Cellsens software (Olympus), and an STR stage incubator (Tokai Hit). The filter sets used in live cell imaging had the following specifications. For eLACCO variants, we used excitation 470/20 nm, dichroic mirror 490 nm dclp, and emission 518/45 nm. For R-iLACCO variants, we used excitation 545/20 nm, dichroic mirror 565-nm dclp, and emission 598/55 nm. DMD-assisted 405 nm photoactivation uncaging experiment was performed with a multi-pattern LED illumination system (Opto-line; LEOPARD2) equipped with a DMD (Mightex; Polygon 1000) and LED light source (Prizmatix; UHP-F-405 LED, 100% power). The entire sample was illuminated, and a perfusion system was not used. Fluorescence images were analyzed with ImageJ software (National Institutes of Health).

For the cell-based imaging of MNI-l-lac, adherent HeLa cells seeded onto a glass-bottom dish were transfected with pcDNA3.1 R-iLACCO1.2/diLACCO1 or pcDNA3.1 eLACCO2.1/deLACCO1 variants. Forty-eight hours after transfection, the eLACCO2.1/deLACCO1 imaging was performed in Hanks’ Balanced Salt Solution (HBSS (+) Nakalai Tesque) supplemented with 10 mM HEPES, 1 μM AR-C155858 (Tocris) with 1 mM MNI-l-lac (1% DMSO) as the imaging buffer. HeLa cells expressing R-iLACCO1.2 or R-diLACCO variants were incubated with DMEM containing no glucose (Nacalai Tesque) for 3 h prior to imaging. The imaging in the absence of an MCT inhibitor was performed in HBSS (+) no glucose solution supplemented with 10 mM HEPES and 5 mM MNI-l-lac (1% DMSO, 0.04% Pluronic F127). In contrast, imaging in the presence of MCT inhibitor was performed in HBSS (+) no glucose solution supplemented with 10 mM HEPES, and 1 μM AR-C155858 (Tocris) with 5 mM MNI-l-lac (1% DMSO, 0.04% Pluronic F127).

For the cell-based imaging of MNI-l-lac in the GPR81-cAMP signaling pathway, adherent HeLa cells seeded onto a glass-bottom dish were co-transfected with pcDNA3.1 cAMPinG1 and pcDNA3.1 GPR81 mCherry2 variants (mCherry fused to the C-terminus of GPR81). Forty-eight hours after transfection, fluorescence imaging was performed in Hanks’ Balanced Salt Solution (HBSS (+) Nakalai Tesque) supplemented with 10 mM HEPES in the presence or absence of 5 mM MNI-l-lac (1% DMSO, 0.04% Pluronic F127). The filter sets and photoactivation setting used for cAMPinG1 imaging were the same as used for eLACCO variants. To confirm how MNI-l-Lac uncaging influences the pH, the same protocol was used except adherent HeLa cells were transfected with pcDNA3.1 pHuji. The filter sets and photoactivation setting used were the same as for R-iLACCO1.2.

All data are expressed as mean ± SD or mean ± SEM, as specified in figure legends. Sample sizes (n) are listed with each experiment. Statistical analysis was performed using one-way analysis of variance (ANOVA) using Rstudio. Microsoft Excel software was used to plot the figures.

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

The authors declare that the data supporting the findings of this study are available within the paper, the Supplementary Information, and the Supplementary Data files. Reasonable requests for reagents, or raw data files in another format, should be directed to a corresponding author.

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The authors thank M. Sakamoto, Kyoto University for kindly sharing plasmids (pCAG-cAMPinG1). This research was supported by the Japan Society for the Promotion of Science (JSPS) (21K14738 and 23H04151 to Y.N., 21H00273 and 23H02101 to T.T., and 19H05633, 22H04743, 24H00489, and 24H02267 to R.E.C.), and the Japan Science and Technology Agency (JST) PRESTO program (JPMJPR22E9 to Y.N.). K.K.T. is supported by the Human Frontier Science Program Cross Disciplinary Fellowship (LT000333/2021-C). I.M. is supported by JST SPRING (JPMJSP2108) and the Forefront Physics and Mathematics Program to Drive Transformation (FoPM), a World-leading Innovative Graduate Study (WINGS) Program of The University of Tokyo.

Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Ikumi Miyazaki, Kelvin K. Tsao, Yuki Kamijo, Yusuke Nasu, Takuya Terai & Robert E. Campbell

Global Standard Science Education Division, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Kelvin K. Tsao

PRESTO, Japan Science and Technology Agency, Chiyoda-ku, Tokyo, Japan

Yusuke Nasu

Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan

Yusuke Nasu

CERVO, Brain Research Center and Department of Biochemistry, Microbiology, and Bioinformatics, Université Laval, Québec, Québec, Canada

Robert E. Campbell

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I.M. synthesized MNI-l-lac and performed in vitro characterization. I.M. and T.T. decided on the photocaging group and design. I.M. and K.K.T. established the synthetic strategies for MNI-l-lac. I.M., K.K.T., Y.N., and R.E.C. designed experiments for in vitro and cell-based characterization and analyzed data. I.M. performed cell-based imaging under supervision from Y.K. and Y.N.; I.M., K.K.T., Y.N., T.T., and R.E.C. wrote the manuscript. K.K.T., T.T., and R.E.C. supervised research.

Correspondence to Kelvin K. Tsao, Takuya Terai or Robert E. Campbell.

The authors declare no competing interests.

Communications Chemistry thanks James A. Frank and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Miyazaki, I., Tsao, K.K., Kamijo, Y. et al. Synthesis and application of a photocaged-l-lactate for studying the biological roles of l-lactate. Commun Chem 8, 104 (2025). https://doi.org/10.1038/s42004-025-01495-1

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Received: 27 September 2024

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DOI: https://doi.org/10.1038/s42004-025-01495-1

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