Application and Suggestions of Morpholine Ring as a Lysosome Targeting Group

Abstract

Lysosomes are widely present in eukaryotic cells and play an extremely important role in cell growth and development, and their dysfunction is closely related to a variety of diseases. The development of a precise lysosomal targeting strategies is of great significance for the detection of lysosomal physiological functions and the diagnosis and treatment of related diseases. Morpholino ring modification has become a commonly used lysosomal targeting strategy, but its effects have not been systematically evaluated. This review summarizes the effects of morpholine rings in fluorescent probes in recent years. The results show that morpholine rings as lysosomal targeting groups have excellent structural adaptability, but their localization effect is influenced by the log p value and charge of the overall molecule, and this effect has structural differences. In addition, since the morpholino ring is essentially an acidic microenvironmental targeting moiety, it carries the risk of off-targeting to other acidic sites.

1. Introduction

Lysosomes are acidic organelles with a monolayer membrane structure (pH between 4.5 and 5.5) widely found in eukaryotic cells, which play an extremely important role in cell growth and development [1]. In addition to material degradation and nutrient sensing functions, lysosomes also play important roles in signal transduction, gene expression regulation, cell death, plasma membrane repair, antigen processing and presentation, and organelle quality control [2]. Their dysfunction is closely related to a variety of diseases such as lysosomal storage diseases [3], neurodegenerative diseases [4], cancer [5], and autoimmune diseases [6]. With the study of the relevant mechanisms of lysosomes in the development of various diseases, lysosomes have become a new target for the treatment of various diseases [7].

Lysosomal targeting strategies are mainly categorized as endogenous and exogenous [8]. Lysosomal endogenous targeting is mainly based on the mannose-6-phosphate pathway. Most hydrolytic enzymes in lysosomes are synthesized in the cytoplasm and then modified by endoplasmic reticulum and golgi processing to introduce mannose-6-phosphate residues, which are subsequently recognized by mannose receptors on the Golgi trans-network and packaged into vesicles with clathrin-coated vesicles (CCVs) or transport carriers (TCs) to form lysosomal secretory vesicles [9]. Steven M. Banik et al. modified glycoproteins with mannose 6-phosphate and conjugated them with target protein antibodies, successfully transporting the target protein to lysosomes for degradation [10]. Unfortunately, this approach does not apply to small molecules. Lysosomal exogenous targeting strategies are mainly based on lipophilic tertiary amines as targeting carriers, e.g., morpholine, 4-dimethylaminopyridine, and histamine [11]. The lysosome membrane has highly permeable to neutral weak base, and the major driver of this process is through pH-partitioning [12]. Neutral weak bases are protonated and positively charged in acidic lysosomes, and the latter cannot penetrate the lysosomal membrane, thus enabling lysosomal targeting [13]. The morpholino ring, as the most common lysosome targeting motif, is widely used in the design of fluorescent probes. However, due to the lack of systematic evaluation, morpholines are less used in the design of lysosome-targeted drugs. This review systematically evaluates the application of morpholine molecules in lysosome-targeted fluorescent probes, including structural adaptation, targeting, and toxicity, to provide a reference for the design of lysosome-targeted molecules based on morpholine rings.

2. Morpholine-Modified Molecular Fluorescent Probes

2.1. Lysosome Imaging

The morphology and spatial distribution of lysosomes are closely related to their working status. Long-term and real-time tracking of lysosomal movements and morphological changes will not only help to understand their working status but also provides new therapeutic strategies for the diagnosis, prevention, and treatment of lysosomal dysfunction-related diseases. Modification of fluorescent molecules with morpholine rings is a common strategy for achieving lysosome visualization.

Probe 1 was a morpholino ring-modified coumarin derivative, which could be used for real-time imaging of lysosomes in living cells, with a Mander’s overlap coefficient of up to 0.75 with LysoTracker Red (Figure 1) [14]. Ly Tet (2) was a morpholine ring-modified tetrazole derivative that could generate pyrazoline fluorophore in situ by the intramolecular tetrazole-alkene cycloaddition reaction (“photo-click chemistry”) under radiation. The probe exhibited excellent lysosomal targeting and Pearson correlation coefficient with LysoTracker™ Red DND-99 is 0.85 (Figure 1) [15]. LysoSQ (3) was designed for the visualization of lysosome location and structural integrity based on croconaine and morpholino rings, with a Pearson’s correlation coefficient of up to 0.93 with LysoTracker Yellow (Figure 1) [16]. Probe 4, a morpholine ring-modified rhodaminestilbene derivative, exhibited a significant fluorescence turn-on response in the pH range of 4.0–3.0, which corresponds to the acidic microenvironment of cancer cell lysosomes and could be used to selectively differentiate between cancer cells and normal cells (Figure 1) [17]. Lyso-BS (5) was assembled from morpholine and salicylidenehydrazone ligand by dehydration and dative N-B bond, which could selectively stain HeLa cell lysosomes. Due to the property of free intramolecular rotational bonding in water leading to fluorescence quenching, the probe was a wash-free fluorescent material (Figure 1) [18].

The probes Lyso-NA (6) [19], MPL-NAP (7) [20], and NIMCn (8) [21] prepared by introducing a morpholine ring onto 1,8-naphthalimide could be used for long-term real-time monitoring of lysosomal morphology changes during physiological processes, and they colocalized well with commercially available lysosomal dyes (Figure 1). Among them, NIMCn (8) exhibited different subcellular localization effects by changing the length of the linker. When n = 2–6, 8, 10, all compoundscould be localized in lysosomes, and the Pearson colocalization coefficients were generally higher than 0.89. But when n = 8 (NIMC₈) and 10 (NIMC₁₀), the compounds had lysosomal and endoplasmic reticulum dual localization due to their increased hydrophobicity [21].

Probes 9–14 were designed by introducing a morpholine ring on boron-dipyrromethene (BODIPY) for imaging lysosomes in living cells (Figure 1). Probes 9 and 11 could be used for lysosomal imaging of hypoxic cells (e.g., tumors) due to the presence of nitro. This is because under hypoxic conditions, the overexpression of nitro reductase can cause nitro reduction to produce red fluorescence. Compared to probes 9, 10, and Lyso Red (13) [22], the probes 11, 12, and Lyso NIR (14) contain more morpholine rings. Surprisingly, they did not show a more prominent lysosomal targeting effect. Such as, the colocalization coefficients of probes 9 and 11 with LysoTracker Green were 94% and 96%, respectively [23], and the Pearson colocalization coefficients of Lyso Red (13) and Lyso NIR (14) with LysoTracker Green DND-26 were 0.94 and 0.81, respectively [22].

Probes 15–21 were a class of lysosomal imaging agents developed based on aggregation-induced emission (AIE) fluorescent materials (Figure 1). These molecules have nonfluorescent or are only weakly fluorescent in good solvents, while in the aggregated state, the intramolecular motion is limited, nonradiative leaps are suppressed, and the energy is released mainly by radiative transitions. [24,25] The main core skeletons of AIE fluorescent materials are triphenylamine (TPA), tetraphenylene (TPE), stilbene, and cyanoethylene. [26] Probes MPAA (15) and MPAN (16) were prepared by introducing a morpholine ring into TPA as the core group of AIE. They have similar lysosomal targeting and higher fluorescence stability compared to LysoTracker Green DND-26 [27]. TPE-MPL (17) [28] and MP-TPEDCH (18) [29] were morpholine ring modified TPE derivatives, and their Pearson correlation coefficients with LysoTracker Red were 0.96 and 0.97, respectively. The probes 2M-DAPS (19) [30] and AIE-LysoY (20) [31] were prepared based on stilbene by introducing a morpholine ring with a six carbon atoms chain length (to ensure probe flexibility), with Pearson correlation coefficients of 0.96 and 0.90 with LysoTracker Red, respectively. Among them, 2M-DAPS (19) could be used to continuously monitor the dynamic changes of lysosomes, including autophagy and mitophagy, as well as to track the process of endocytosis of macromolecules in lysosomes. Lyso-BAM (21) was a morpholine-ring-modified cyanoethylene-based AIEgen, which highly overlaps with the lysosomal-associated membrane protein 1 (LAMP1) mApple fluorescence signal in the A549-Lysosome20 mApple cell line [32,33].

2.2. Lysosome-Targeted Photodynamic Therapy (PDT) Agents

Lysosomes play a crucial role in cell survival and apoptosis. Lysosomal membrane destabilization leads to leakage of protons and hydrolases, which in turn leads to apoptosis. Lysosome-based targeted PDT agents have emerged as a new idea in cancer therapy. Fluo-Mor-NPs (22) were new two-photon fluorene-functional morpholino-based organic nanoparticles that exhibit intense fluorescence and profound PDT activity only in acidic media (Figure 2). In HT-29 cells, the fluorescence of Fluo-Mor NPs was highly overlapped with LysoTracker Reds DND 99 [34]. Aza-BODIPYs (23) was a morpholinomodified hydrophilic aza-boron dipyrromethene that can be used for lysosomal-specific imaging and lysosomal PDT in a range of cancer cell lines (Figure 2) [35]. LysoCR (24) was a morpholine-modified croconaine derivative, which could serve as a laser induced nonfluorescent lysosome targeted photothermal heating probe. In the absence of interference from photodynamic effects, LysoCR (24) could be used to evaluate the sensitivity of lysosomes to photothermal heating (Figure 2) [16].

Probes 25–29 werea class of lysosomal-targeted photodynamic therapy (PDT) agents developed based on morpholine-modified BODIPY (Figure 2). SBOP-Lyso (25) [36] and BOP-Lyso (26) [37] were a class of NIR-lysosomal targeting thiophene-BODIPY photosensitizer, which could produce singlet oxygen with 660 nm LED irradiation to kill cancer cells. MBDP (27), a lysosome-targeted near-infrared (NIR) photosensitizer, showed correlation coefficients of 0.88 and 0.05 with LysoTracker Green DND-26 and Hoechst 33,342, respectively [38]. DP (28) was a dinuclear binuclear platinum (II) BODIPY complex formed by chelating BODIPY containing two catecholate moieties with 1,2-diaminocyclohexane and platinum and could be as a NIR red light PDT agent specifically targeting lysosomes to kill cancer cells [39]. BDPI-lyso (29) was prepared by introducing a morpholine ring through 2-aminothiophenol at the 5-site of 1,3-dimethyl-2,6-diiodo-BODIPY, with a Pearson’s colocalization coefficient of 0.91 with LysoTracker Green in live cells [40].

Probes 30–32 were a class of lysosomal-targeted PDT agents developed based on AIE fluorescent materials (Figure 2). MPAT (30) was prepared by introducing morpholine ring and 1,3-dioxoindene group with TPA. In addition to allowing dynamic long-term tracking of the lysosomes in living cells and zebrafish, MPAT (30) produced large amounts of reactive oxygen species (ROS) under green light irradiation and could also be used as a photosensitizer for cancer tumors in photodynamic therapy (PDT) [27]. MP-TPEDCH (31) [29] was a morpholine ring modified TPE derivative, which could generate large amounts of ROS under NIR irradiation for cancer treatment [29]. MTMM (32) was a cyanoethylene-based AIEgen, which was prepared mainly by integrating morpholinyl and piperidyl moieties with a p-bis (2,2-dicyanovinyl) benzene-based luminogen. It could specifically stain living cellular lysosomes and further ablate cancer cells after white light irradiation [41].

2.3. Lysosome pH Detection

Acidic pH is necessary for lysosomes to promote hydrolase activation and protein function [42]. Abnormal lysosomal pH values can lead to cellular dysfunction and consequently lead to many diseases such as lysosomal storage disorders, neurodegenerative diseases, and cancer [43]. Therefore, it is crucial to dynamically and accurately monitor pH changes in the lysosomes of living cells [44].

Probes 33–37 were a class of morpholine-ring-modified naphthalimide fluorescent probes (Figure 3). BN-lys (33) could be used for measuring the pH of lysosomes in tumor cells, and it was prepared using biotin as a tumor-targeting moiety and morpholine as the lysosome-specific moiety and pH site [45]. NT1 (34) could be used to detect pH changes in lysosomes under heatstroke, with the Pearson correlation coefficient of 0.90 with LysoTracker tracker [46]. MMN (35) was designed based on the intramolecular charge transfer (ICT) strategy, which was highly sensitive to lysosomal pH changes and isoxaflutole [47]. The probe 36 was a ratiometric lysosome-targeted fluorescence resonance energy transfer (FRET) pH probe with a coumarin moiety linked to a naphthalimide moiety via a piperazine group [48]. CN-pH (37) used the hydroxyl and morpholine rings as pH sensing sites and had a Pearson correlation coefficient of 0.82 with LysoTracker Red [49].

The probes 38–46 were rhodamine spiroamidolactone derivatives containing a morpholino ring, which could be used to simultaneously utilize the pH sensitivity of rhodamine spiroamidolactone and the lysosomal targeting of the morpholino ring for intralysosomal pH monitoring (Figure 3). MSO (38) had a Pearson colocalization coefficient of up to 0.89 with LysoTracker Green DND-26 in HeLa cells [50]. RML (39) exhibited good colocalization with LysoSensor™ Green DND-189 in HeLa cells [51]. FR-Lys (40) was a ratiometric fluorescent probe prepared by introducing an o-hydroxy benzoxazole group at the 2-site of xanthane and had good fluorescence overlap with DND-99 in A549 live cells [52]. Ly-HN₂AM (41) significantly improved the sensitivity to pH by directly bonding to the spironolactone ring (forming a five-membered ring structure with H⁺), with a Pearson coefficient of up to 0.92 with LysoTracker Green DND-26 [53]. Probes 42–46 were a series of novel NIR lysosomal pH probes developed by extending the π-conjugation system of Xanthene. Lyso-hNR (42) was prepared using 7aH-Benzo[a]xanthene instead of Xanthene [54]. NRLH (43) [55], NRH-lyso (44) [56], probe 45 [57], and probe 46 [57] were prepared by modifying the turnip structure at position 5 of Xanthene. Probe 46 introduced mannose residues into probe 45, which improved the biocompatibility and water solubility of the probe [57].

Probes 47–49 were a class of lysosomal pH fluorescent probes developed on the basis of dual fluorescent molecules and morpholine rings (Figure 3). NpRhLys-pH (47) was constructed by coupling a naphthalimide derivative containing a D-Π-A-structured with a rhodamine B fluorophore, which could be used to image lysosomal pH changes in living cells and zebrafish [58]. Probe 48 was an efficient multifunctionalized acidic pH sensitive probe prepared by coupling rhodamine B as a FRET acceptor with naphthalimide fluorophore as a FRET donor using 1,4-diaminobutane as a nonconjugated linker [59]. Probe 49 consists of a 1,3,5,7-tetramethyl-BODIPY donor and a near-infrared rhodamine acceptor bearing a lysosome-targeting morpholine residue [60].

Probes 50–52 were a class of morpholine-ring-modified BODIPY lysosomal pH fluorescent probes (Figure 3). Probe 50 had a Pearson’s coefficient of up to 0.85 with LysoSensor Green DND-189 and could be used to detect pH changes in mammalian cells [61]. The probes 51 and 52 were designed based on the ICT strategy and pH-sensitive p-dimethylaminostyrene unit [62].

Probes 53–56 were a class of ratiometric fluorescent probes for the determination of lysosomal pH changes based on three fluorescent molecules and a morpholine ring (Figure 3). Probe 53 was prepared based on coumarin, NIR hemicyanine, and rhodamine dye. The probe showed only visible fluorescence of the coumarin moiety under physiological and basic conditions, but when the pH decreased to acidic conditions, the spirolactam ring opened and the π-conjugation within the probes was enhanced, resulting in the generation of new hemicyanine near-infrared fluorescence peaks [63]. Probe 54 was designed according to a through-bond energy transfer (TBET) strategy with rhodamine as the core, TPE as the donor, and NIR hemicyanine as the acceptor. And the probe showed an aggregation-induced emission (AIE) property at neutral or basic pH. Probes 55 and 56 were designed based on a π-conjugated modulation strategy and composed of rhodamine, TPE, and hemicyanine moieties [64].

Lyso-APBI-1 (57) and Lyso-APBI-2 (58) were acid-activated fluorescent probes prepared by using perylene bisimide as the fluorescent moiety, introducing morpholine groups on the bay and two pairs of three-unit PEG groups on the imides (Figure 3). The bimorpholine moiety gave the Lyso-APBI-2 (58) probe a higher acid activation rate and better cellular lysosomal specificity compared to Lyso-APBI-1 (57) [65]. The probes 6a (59) and 6b (60) were prepared on the basis of pyrene by introducing quinoline and morpholine rings (Figure 3). Among them, the N atom on quinoline was highly sensitive to pH and could modulate the probe fluorescence intensity in the form of protonation and deprotonation. Due to the structural similarity, both 6a (59) and 6b (60) had similar log p values and good cell permeability for live cell imaging. Surprisingly, 6a (59) demonstrated diffuse intracellular staining, whereas 6b (60) showed very distinct lysosomal localization with a Pearson correlation coefficient of 0.86 with LysoTracker Red DND-99 [66]. Lyso-MPCB (61) was a morpholino-ring-modified benzimidazole fluorescent probe, which enables real-time imaging of lysosomal pH in a dual-channel signaling fashion to observe autophagy (Figure 3) [67]. MIBTAA (62) was prepared based on morpholino ring and imidazole-fused benzothiadiazole and could be used to monitor pH variation in living cells induced by proton-pump inhibitor Baf-A1 and chloroquine (Figure 3) [44]. CQ-Lyso (63) was a lysosomal pH detection ratiometric fluorescent probe based on the chromenoquinoline and the morpholine ring (Figure 3). And the protonation of the quinoline ring induces an enhanced ICT process that forms the basis of the ratiometric fluorescent pH sensor [68].

2.4. Lysosome Reactive Oxygen Species (ROS) Detection

Reactive oxygen species (ROS) play an important role in the pathogenesis of serious diseases like cancer, cardiovascular diseases, and neurodegenerative disorders. The ROS level in lysosomes is tightly related to the redox balance in lysosomes, which is significant to maintain the normal lysosomal function. NH-HOBr (64) and NA lyso (65) were morpholine-modified naphthalimide derivatives and could be used for monitoring HOBr in lysosomes (Figure 4). The morpholine ring in NH-HOBr (64) was both the lysosomal targeting and HOBr-reacting moiety [69]. The 2-methylthiobenzene and amino groups of probe NA lyso (65) underwent cyclization, and the fluorescence intensity decreased dramatically in the presence of HOBr [70]. Lyso-MHC (66) introduced the O²⁻-recognizing trifluoromethanesulfonate moiety and morpholine ring on naphthalimide, which could be used for the specific detection of O²⁻ content in lysosomes (Figure 4) [71]. ML-NAP-DPPEA (67) introduced morpholine ring and the ONOO⁻-recognizing (diphenylphosphino) ethylamine (DPPE) on naphthalimide for highly selective imaging of ONOO⁻ produced by endogenous stimulation of lysosomes in living cells (Figure 4) [72].

Probes 68–70 were fluorescent probes for detecting H₂O₂ in lysosomes (Figure 4). LyNC (68) and 69 were morpholine-modified naphthalimide derivatives. LyNC (68) used catechol as H₂O₂ a recognition group, which could be oxidized by H₂O₂ to o-quinone with electron-deficient properties, thereby inhibiting the intramolecular photoinduced electron transfer (PET) pathway and producing green fluorescence [73]. Probe 69 used p-dihydroxyborylbenzyloxycarbonyl H₂O₂ as a recognition group and could be used to monitor the level of endogenous and exogenous H₂O₂ [74]. Lyso-B-L1 (70) was prepared by a H₂O₂⁻ responsive boronate unit, a lysosome-locating morpholine group, and a pH-activatable benzorhodol fluorophore [75].

Probes 71–89 were fluorescent probes for detecting HClO in lysosomes (Figure 4). Probes 71–75 were designed based on naphthalimide and morpholino ring. PT-1 (71) used the phenothiazine as the HClO recognition moiet, which could be oxidized to sulfoxide by HClO to block the PET process [76]. The probe 72 used (2-aminoethyl) thiourea as the HClO recognition group, which underwent an HClO-induced intramolecular cyclization reaction that hindered the ICT process [77]. TPFP (73) used phenylthiourea as the HClO recognition moiety, which could be oxidized to urea and inhibited the intramolecular PET effect [78]. L1 (74) was a reversible fluorescent probe whose methyl sulfide group could be oxidized by HClO to methyl sulfoxide and reduced by the GHS in the cell [79]. The probe 75 used a hydrazone bond as the ClO⁻ recognition moiety, which could be hydrolyzed by ClO⁻ to produce blue fluorescent coumarin aldehydes and nonfluorescent 1,8-naphthoimide derivatives [80]. Probes 76–86 were morpholine-modified rhodamine derivatives. They were designed based on the instability of spironolactone in rhodamine to HClO, such as the hydrazone structure of Lyso-1 (76) [81], the thio-lactone structure of HN4MS (77) [82], the thioamino structure of LR1 (78) [83], the bishydrazide structure of FL-HA (79) [84], and the monothio-bishydrazide structures of IRh-Ly (80) [85], RL1 (81) [86], RIL (82) [87], Lyso-R-HClO (83) [88], Lyso-NIR-HCIO (84) [88], and CR-Ly (85) [89]. Lyso-HA-HS (86) introduced an azide group that could be reduced by H₂S based on CR-Ly (85), thus achieving dual-channel imaging of HClO (blue fluorescence) and H₂S (red fluorescence) in the lysosome [90]. MN-BODIPY (87) was developed by introducing a morpholine ring and an aldoxime unit to BODIPY. When the aldoxime was specifically oxidized to an aldehyde by ClO⁻, its fluorescence was significantly enhanced [91]. CS (88) was developed on the basis of coumarin and morpholine rings, whose N, N-dimethyl-thiocarbamate acted as the HClO-recognizing group that could be separated by HClO oxidation [92]. Probe 89 was a morpholine-modified 2H-benzo[h]chromenopyridine derivative whose CH = CH bond could undergo an addition reaction with ClO⁻ to produce a compound containing Cl. The heavy atom effect of the Cl atoms leads to fluorescence quenching [93].

2.5. Lysosome Metal Ion Detection

Metal ions play important roles in protein function, signal transduction, osmotic pressure regulation, and gene expression regulation. Metal ion homeostasis in lysosomes has important implications for health and disease; for instance, copper ion homeostasis in lysosomes is strongly associated with aging, mutations within the ATP7B sequence, Wilson’s Menkes, and Alzheimer’s diseases [94,95], and Alzheimer’s disease, osteoporosis, rickets, anemia, and memory loss are all possible as a result of aluminium toxicity [96].

Probes 90–94 could be used for detecting Zn²⁺ in lysosomes (Figure 5). DDP 9 (90) [97] and LysoDPP-C4 (91) [98] were designed with a diketopyrrolidine scaffold as the chromophore, di-(2-picolyl)amine as the Zn²⁺ chelator, and a morpholine ring as the lysosomal targeting moiety. Among them, LysoDPP-C4 (91) could be used to identify malignant prostate tissue, as the levels of Zn²⁺ in malignant prostate tissue were significantly reduced compared to normal prostate tissue [98]. DR (92) was a morpholine-modified naphthalimidefluorescent probe that used N, N-bis(2-pyridylmethyl)ethylenediamine (BPEN) as the Zn (II) receptor and could be used to observe the endogenous release of Zn²⁺ from lysosomes of SH-SY5Y cells under H₂O₂ stimulation [99]. CBL2 (93) was a class of self-assembly driven fluorescent nanoprobe prepared by introducing a 2,2′-bipyridine molecule for Zn²⁺ binding on the basis of carbazole. The N atoms at different sites of the molecule undergo deprotonation or protonation reactions due to pH changes that lead to the aggregation to form nanoparticles of different sizes [100]. Lys-NBD-TPEA (94) was prepared by introducing a morpholine ring onto a commercially available Zn²⁺ detection probe NBD-TPEA with an overlap coefficient of up to 0.85 with the lysosomal tracker [101].

The probes 95–98 were a class of morpholine-modified naphthalimide fluorescent probes that could be used for detecting Fe³⁺ in lysosomes (Figure 5). The N-methylpiperazine group of MNP (95) coordinated with Fe³⁺ ions to produce a protonated [MNPH]⁺ that overlaps well with LysoTracker Red [102]. The probes MP-Gal-1 (96), MP-Gal-2 (97), and MP-Gal-3 (98) could be used to monitor Fe³⁺ levels in hepatocyte lysosomes. Among them, the galactose group acted as the hepatocyte-targeting group, and the nitrogen atom at the end of piperazine selectively bound to Fe³⁺ to block the PET effect of the probes [103].

Probes 99–101 were lysosomal Cu²⁺ detection probes designed based on the lysosomal targeting of the morpholine ring (Figure 5). Lyso-CS₂ (99) was a naphthylamide-based lysosomal ratiometric fluorescent Cu²⁺ probe, which had two picolylamine arms for the specific chelation of divalent Cu²⁺ [104]. NC-Cu (100) was prepared by introducing coumarin via a hydrazone bond on 1,8-naphthylimide. The hydrazone bond could be hydrolyzed by Cu²⁺ to produce blue-fluorescent coumarin aldehydes and nonfluorescent 1,8-naphthoimide derivatives [105]. Probe 101 was prepared by the condensation reaction of formyl-1H-pyrrole and rhodamine 6G arylhydrazone. It could be coordinated to Cu²⁺ via deprotonation of pyrrole N atoms, imine N atoms, and carbonyl O atoms, causing a rhodamine ring-opening reaction and FRET between pyrrole and rhodamine [106].

Probes 102 and 103 were a class of fluorescent probes that respond specifically to Hg²⁺ in lysosomes (Figure 5). Lyso-HGP (102) was prepared using 4-methyl-2,6-diformylphenol as the fluorescent backbone, the thioacetal linkage of the 1,3-dithiolane moiety as the Hg²⁺-responsive moiety, and the morpholine ring as the lysosomal targeting moiety [107]. Probe 103 was an NBD-based fluorescent probe, and its morpholine ring acted as both a lysosomal targeting group and a ligand for Hg²⁺ [108]. Interestingly, Bao-Xiang Zhao et al. reported that probe 103 was a lysosomal-targeted pH detection probe [109]. Although the sensitivity of probe 103 to Hg²⁺and H⁺ is still controversial, it could be confirmed that probe 103 containing a morpholine ring had an excellent lysosomal localization effect.

NIC (104) was a morpholine-modified naphthalimide fluorescent probe that used oxygen atoms on hydroxyl group and amide as coordination site for Ni²⁺ and could be used for lysosomal Ni²⁺ detection (Figure 5) [110]. CMN (105) was the first fluorescent probe to selectively image trivalent metal ions in lysosomes of living cells, with excellent responsiveness to Fe³⁺, Al³⁺, and Cr³⁺. The N atoms on secondary amines and morpholine of CMN (105) were used as complexation sites (Figure 5) [111]. Probe 106 was a morpholine-modified salicylaldehyde acylhydrazone derivative that could fluorescently respond to Al³⁺ in lysosomes (Figure 5). As a hydrazine-based probe characterized by O and N donor groups, it selectively forms a coordination complex with Al³⁺, thereby impeding C double bond N isomerization and emitting strong fluorescence [96].

2.6. Lysosome Sulfide Detection

Probes 107–114 were a class of lysosomal H₂S detection probes (Figure 6). Lyso-C (107) was prepared by introducing a morpholine ring on 7-azido-4-methylcoumarin (AzMC, a commercially available fluorescent probe) and exhibited excellent lysosomal targeting [112]. CMDN (108) was prepared using coumarin as the fluorescent backbone and 2,4-dinitrobenzenesulfonyl as the H₂S recognition group [113]. Lyso-NPNM (109) was prepared using N-annulated perylene as a fluorescent molecule and electron-donor group and nitro as an H₂S recognition and electron-absorbing group [114]. Rh Lyso H₂S (110) was a rhodamine-based fluorescent probe that was designed based on the sensitivity of thiophenecarboxylate to H₂S [115]. ANp-Rh-Lys (111) was designed by the TBET strategy and could logically detect H₂S, H⁺, and H₂S/H⁺ in lysosomes with different fluorescence signals. Among them, the spirolactam of rhodamine B acted as a pH-responsive group, the azide group modified on naphthylamine acted as an H₂S recognition group, and the morpholine ring acted as a lysosomal targeting group [116]. LyNP-H₂S (112) [117], probe 113 [118], and SN-N₃ (114) [119] were morpholine-modified naphthalimide derivatives for lysosomal H₂S monitoring, which were prepared by introducing dinitrobenzene ether (DNB), NBD, and azide as the reactive sites for H₂S, respectively.

Na-SO₂₋Lyso (115) [120] and NA-CHO (116) [121] were naphthalimide-based fluorescent probes for detecting of SO₂ derivatives in lysosomal (Figure 6). They were designed on the basis of the nonradiative transition induced by C double bond N isomerization and the recognition of SO₂ derivatives by the aldehyde group. Although the different alkane chains resulted in slightly different log p values for the probes, they exhibited the same colocalization effect with LysoTracker Red.

Probes 117–119 were lysosomal H₂S_n detection probes (Figure 6). The probe 117, as a morpholine-modified naphthalimide derivative, used 2-chloro-5-nitrobenzoate as the H₂S_n recognition group that could undergo substitution reactions with the sulfur atom of H₂S_n [122]. Lyso-NRT-HP (118) [123] and LR-H₂S (119) [124] were prepared by introducing 2-fluoro-5-nitrobenzoic acid phenylester and 4-azidophenyl via carbamate on naphthalimide, respectively. The carbamate bond acted as a H₂S_n recognition group that released fluorescent molecules upon reaction with H₂S_n.

Probes 120–125 were lysosomal cysteine (Cys) detection probes (Figure 6). Lyso-DCHO (120) was prepared by introducing two benzaldehyde groups and a morpholine ring on the carbazole. The aldehyde group as a Cys-recognizing group could react with Cys by cyclization [125]. Probe 121 [126] and LFA (122) [127] were prepared with acrylate as the Cys recognition moiety, morpholine ring as the lysosomal targeting moiety, and naphthalimide and chromone as the fluorescent backbone, respectively. DCICA (123) [128] and Ly-1 (124) [129] were morpholine-modified coumarin derivatives prepared using acrylate and α, β-unsaturated ketone as the Cys-responsive moiety, respectively. MNPO (125) was prepared using α, β-unsaturated ketone as the Cys-responsive moiety and morpholine-modified naphthalimide derivative as chromophores [130].

Probes 126–131 were lysosomal biothiol detection probes (Figure 6). The probe 126 was prepared using morpholine-modified BODIPY as the fluorophore a 2,4-dinitrobenzenesulfonyl group as the thiol reaction recognition group [131]. BISMORX (127), also known as bis(7-(N-(2-morpholinylethyl)sulfamoyl) benzo[c] [1,2,5]-oxadiazol-5-yl)thiophane), could be used for the detection of nonprotein thiols in lysosomes of living cells. The probe itself shows no fluorescence and reacts readily with a nonprotein thiols to form a same fluorescent thiol adduct [132]. The probe 128 was prepared by using morpholine-modified naphthalimide as the fluorescent backbone and 2,4-dinitrobenzene-sulfonyl (DNBS) as the thiol-recognition group [133]. Lyso-O-NBD (129) was prepared by ether bonding NBD and morpholine ring modified coumarin. In the presence of Cys, homocysteine (Hcy), and glutathione (GSH), the ether bond of Lyso-O-NBD (129) underwent cleavage to produce the blue-fluorescent compound Lyso-OH and the weakly fluorescent thiolate derivative NBD-S-Cys/Hcy/GSH [134]. Among them, NBD-S-Cys/Hcy would subsequently undergo a rapid intramolecular rearrangement to form the amino-substituted NBD derivative NBD-N-Cys/Hcy to display green fluorescent, while the NBD-S-GSH remained in the nonfluorescence state [135,136]. DCIMA (130) consists of a dicyanoisophorone core, an acrylate group for biothiol detection, and a morpholine group for lysosome targeting, which could be used for lysosome-targeted imaging of Cys and GSH in living cells [137]. Lyso-RC (131) used a morpholine ring as the lysosome targeting group and connected 7-diethylaminocoumarin and resorufin through ether bonds. When incubated with biothiols, the ether bond of Lyso-RC (131) may be cut off to release red-emitting resorufin and sulfydryl coumarin with different fluorescence characteristics, such as nonfluorescent sulfydryl coumarin Lyso-C-SH (132), green-emitting sulfydryl coumarin Lyso-C-S-GSH (133), and blue-emitting aminocoumarin Lyso C-S Cys/Lyso C-S-Hcy (134) (Figure 6) [138].

2.7. Lysosome Viscosity Detection

Lysosomal viscosity monitoring is important for understanding cellular-state-based functions. During autophagy, the autophagosome membrane was fuseed with the lysosome membrane, and some biological substances required to be degraded in the autophagosome were released into the lysosome, thus leading to an increase in lysosomal viscosity accordingly. MC-1 (135), Lyso-MC (136), and JIND-Mor (137) were prepared based on an indolium merocyanine fluorescent scaffold and 1,2-dicyanoethylene molecular rotor (Figure 7). Compared to MC-1 (135) [139] without a morpholine ring, Lyso-MC (136) [140] and JIND-Mor (137) [141] were found to have excellent lysosome targeting properties.

The probe 138 was prepared by introducing a morpholine ring and a rotatable trifluoromethyl group (CF₃) on BODIPY (Figure 7). When the rotation of the CF₃ group was restricted by viscosity, the probe 138 could produce strong NIR fluorescence [142]. NCIC-VIS (139) was prepared by introducing a morpholine ring and a vinylated isophorone on coumarin (Figure 7). Isophorone, as the core viscosity sensing unit, would have its dihedral angle rotation limited by viscosity [143]. Lyso-B (140) was a BODIPY derivative modified with a morpholine ring, in which the morpholine moiety serves as both a lysosome-anchoring group and a viscosity-sensitive group (Figure 7) [144]. Lys-V (141) was prepared by introducing two morpholine rings into the structure of p-hydroxybenzylideneimidazolidinone (Figure 7). When the two C=C twisting double bonds between phenol and imidazolidinone were restricted, the fluorescence emission of Lys-V (141) was significantly enhanced [145]. PIM (142) was a lysosomal pH and endogenous viscosity monitoring probe prepared by introducing a morpholine ring through Schiff base condensation reaction on the basis of pyrene (Figure 7). The fluorescence intensity of PIM (142) was closely related to the rotation of its C single bond C and C double bond N bonds and the stability of the Schiff base [146]. BDP-1 (143) was a morpholine-ring-modified BODIPY derivative that could be used to determine the temperature of living cells (Figure 7). The fluorescence of this probe was quenched by intramolecular rotation at room temperature and was gradually enhanced during heating due to an increase in microviscosity around the fluorophore [147].

2.8. Lysosome Polarity Detection

The local micropolarity at the cellular level controls biochemical reactions and the stability of biomolecular complexes and lipid membranes of different organelles. Any unusual change in lysosomal micropolarity triggers severe disruption of apoptosis and hindrance of various enzymatic reactions; thus, lysosomal polarity plays a key role in its function. Lyso-OSC (144) and LyPol (145) were a class of fluorescent probes designed based on the ICT strategy and morpholine ring lysosome targeting for real-time monitoring of lysosomal polarity changes (Figure 7). Lyso-OSC (144) was a D-A type of two-photon fluorescent probe prepared by introducing 1-vinyl-4-methoxybenzene and morpholine on coumarin [148]. LyPol (145) was prepared by the solvatofluorochromic propellerocein chromophore and the lysosome-targeting morpholine moiety [149].

2.9. Lysosome Oxidate Detection

Probes 146–149 were prepared by introducing a morpholine ring and an NO-reactive group into the fluorescent backbone and could be used for NO monitoring in lysosomes (Figure 7). Lyso-DHP (146) was prepared by introducing a morpholine ring and a Hantzsch ester on BODIPY. The Hantzsch ester could react with NO and restore the fluorescence of BODIPY by blocking the PET process [150]. LysoNO-Naph (147) [151] and MBTD (148) [152] were respectively designed based on nphalimide-fused o-phenylenediamino scaffold and thiadiazole-fused o-phenylenediamino scaffold, where o-phenylenediamine served as the NO recognition group and could react with NO to form a triazole ring. The spironolactam of Lyso-SiRB-NO (149) underwent a ring-opening reaction in the presence of NO derivatives to produce the highly fluorescent product SiR-benzotriazole [153].

Rh-NO (150) was a water-soluble and turn-on fluorescent NO donor prepared by introducing the NO-releasing unit N-nitroso group and the lysosomal localization motif morpholino ring at the 6-position N of rhodamine (Figure 7). The probe was irradiated at 525 nm and produced NO and a ring-opening product with red fluorescence [154].

Na-FA-Lyso (151) used 1,8-naphthalimide as the fluorescent chromophore and hydrazine as the interaction site for observation of endogenous HCHO in lysosomes of living cells (Figure 7) [155]. Lyso-HNO (152), a morpholine ring-modified naphthalimide derivative, used 2-(diphenylphosphine)-benzoate as the HNO recognition group, which could be hydrolyzed by HNO to produce green fluorescence for detecting exogenous HNO in HeLa cell lysosomes (Figure 7) [156].

2.10. Lysosome Enzyme Detection

Mela TYR (153) was a melanosome-targeting tyrosinase fluorescent probe with morpholine as a melanosome-targeting group and 4-aminophenol as a tyrosinase reaction group (Figure 7). The probe exhibited an accurate targeting ability toward the acidic organelles of melanosomes and lysosomes but fluoresced only after oxidative cleavage by tyrosinase [157]. Lyso-NTR (154) [158] and Lyso FP NO₂ (155) [159] were prepared by introducing nitro groups at the 4 and 3 site of the naphthalene ring, respectively (Figure 7). They mainly utilize the lysosomal targeting of the morpholine ring and the characteristic of nitro being easily reduced to detect nitroreductase and CO in lysosomes. MPBOD (156) could be used to monitor the carboxylesterase 1 activity in lysosomes of living cells due to the introduction of the morpholine ring and methyl benzoate on BODIPY (Figure 7) [160]. Probe 157 enabled fluorescent imaging of lysosomal cathepsin B (CTB) in living cells by incorporating a CTB-recognitive peptide substrate Cbz-Lys-Lys-p-aminobenzyl alcohol (Cbz-Lys-Lys-PABA) and a lysosome locating group morpholine into an aminoluciferin scaffold (Figure 7). The probe could be selectively hydrolyzed by CTB in acidic lysosomal environments and produced an intense green fluorescence [161].

2.11. Others

Lyso-Flipper (158) was a mechanosensitive lysosomal fluorescent probe that responded to the change of plasma membrane tension by altering the fluorescence lifetime (Figure 7). The probe consists of two dithienothiophene “flippers” that are twisted out of coplanarity due to the repulsion of the methyl groups with the σ holes on the endocyclic sulfurs. The headgroup of flippers contains an essential triazole and a basic morpholine [162].

Probe 159–161 were lysosomal targeted fluorescent anion transporters and prepared on the basis of coumarin by introducing a morpholino ring with lysosomal targeting and a squaramido group for facilitating the transport of anions (Figure 7) [163]. Compared to probe 159, probes 160 and 161 introduced trifluoromethyl groups with electron-withdrawing effects on the arylsquaramide benzene ring, significantly improving their anion transport efficiency [164].

Lyso-BADY (162) was a lysosome-selective Raman probe made by conjugating bisphenylbutadiyne with morpholine (Figure 7). The Raman peak intensity of this probe was 28 times higher than that of 5-ethynyl-2′-deoxyuridine [165].

Se-1 (163) was a lysosomal H₂Se fluorescent probe prepared with a H₂Se-capturing benzoselenadiazole, a lysosome-targeting morpholine, and fluorophore naphthalimide (Figure 7). The content of H₂Se in lysosomes was positively correlated with the degree of environmental hypoxia [166].

3. Morpholine-Modified Ionic Fluorescent Probes

Mitochondria are the only negatively charged organelles in the cell, and their membrane potential can reach −150 to −180 mv. Driven by the mitochondrial membrane potential, lipophilic cations are easily enriched in mitochondria. Thus, cationic compounds that utilize morpholine ring modifications for lysosomal targeting face greater obstacles than molecular compounds.

3.1. Lysosomal Localization

Probes 164–167 were morpholino-modified cationic fluorescent probes for lysosomal pH determination in living cells (Figure 8). Probe 164 was a morpholine-modified phenoxazinium derivative [167], and probe 165 was a morpholine-modified hemicyanine derivative [168], both of which were designed based on the PET strategy for pH monitoring in lysosomes. The N atom on the morpholine ring changes from electron-donating amino groups (deprotonation) to the electron-withdrawing ammonium groups (protonation) by combining with H⁺, hindering the PET process and thus enabling pH detection. CPH (166) was a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe for the ratiometric detection of lysosomal pH. Despite the absence of the morpholino ring, CPH (166) was also enriched in lysosomes with a Pearson correlation coefficient of up to 0.825 with LysoTracker Deep Red [169]. CPY (167) was prepared by introducing a morpholine ring on the basis of CPH (166) with a Pearson’s correlation coefficient of 0.898 with LysoTracker Deep Red. Moreover, CPH (166) maintained excellent lysosomal targeting in the presence of increased lysosomal membrane permeability and pH due to heat shock stimulation [170].

Lyso-Gal (168) was a lysosome-targeting NIR fluorescent probe for detection and visualization of endogenous β-galactosidase in ovarian cancer cells (Figure 8). The galactopyranoside group acts as a recognition group for β-galactosidase, which can be cleaved by β-galactosidase to produce Lyso-OH with bright NIR fluorescence [171]. Probe 169 was prepared by introducing a morpholine ring and ester groups recognizing carboxylesterases on the basis of merocyanine, which could be used to detect the activity of carboxylesterases in lysosomes of C6 cells (Figure 8) [172].

M-PT-RGD (170) was an active tumor- and lysosome-targeted organic photothermal agent based on cyanine dyes, which could be used for cancer therapy (Figure 8). The morpholino ring acted as the lysosomal targeting moiety, cyanine acted as photosensitizer, and cRGD (a ligand for integrin, ανβ3, and ανβ5 overexpressed in tumor cells) was the tumor-targeting moiety [173]. Probe 171 was a morpholine-modified hemicyanine derivative for 3D imaging and real-time tracking of live cell lysosomes (Figure 8). Under acidic pH conditions, the visible closed oxazolidine form can be switched to the highly conjugated NIR Cy-7 form through ring opening of the oxazolidine moiety [174].

3.2. Dual Localization of Lysosomes and Mitochondrian

Unlike the previous fluorescent probes, the morpholine ring-containing cationic fluorescent probes DML-P (172), Mito-SO2-Lyso (173), and Cy-S-NP (174) had dual lysosomal and mitochondrial targeting (Figure 8). DML-P (172) was a fluorescent probe developed based on semicyanine, naphthalimide, and morpholine motifs for detecting endogenous SO₂ in live cell lysosomes and mitochondria. The semicyanine unit serves as a mitochondrial targeting group and SO₂ recognition site, while the morpholine ring serves as a lysosomal targeting group. The corresponding Pearson’s correlation coefficients of this probe with MitoTracker Deep Red and LysoTracker Blue were 0.90 and 0.89, respectively [175]. Mito-SO2-Lyso (173) was a SO₃²⁻ NIR fluorescent probe prepared based on a FRET modulation strategy, which used naphthalimide as the energy donor, benzopyranium salt unit as the energy acceptor and SO₂ recognition site, O⁺ as the mitochondrial targeting site, and a morpholine ring as the lysosomal targeting site. The Pearson’s coefficients of this probe with MitoTracker Green and LysoTracker Blue were 0.88 and 0.80, respectively [176]. Cy-S-NP (174) was a glutathione specific NIR fluorescent probe composed of a mitochondrial targeted near-infrared cyanine moiety, lysosome-targeted morpholine-containing naphthalimide, and a thioether-type moiety as a thiol-reactive site. The Pearson’s correlation coefficients of Cy-S-Np (174) with MitoTracker Green FM and LysoTracker Green DND-26 were 0.93 and 0.85, respectively [177].

3.3. Mitochondria Localization

Probes 175, 176 and PM-Mor-OH (177) were morpholino-modified cationic fluorescent probes that were only localized on mitochondria (Figure 8). Probes 175 and 176 were two ratiometric near-infrared fluorescent probes used for detecting pH changes based on highly efficient through-bond energy transfer (TBET) from cyanine donors to near-infrared hemicyanine acceptors. Despite containing both a morpholino ring structure with lysosomal localization and a semicarbocyanine cation structure with mitochondrial targeting, probes 175 and 176 could only be used for mitochondrial pH monitoring. The Pearson’s correlation coefficients of probe 175 and probe 176 with LysoSensor Blue were 0.23 and 0.34, respectively, while those with MitoView Blue were 0.92 and 0.93, respectively [178]. PM-Mor-OH (177) was a unique mitophagy based on the lipophilic morpholine ligand-conjugated pyridinium derivative of “IndiFluors”, which could be used to quantify the mitochondrial pH and internal pH alteration. Unlike morpholine conjugated probes typically located in lysosomes, PM-Mor-OH (177) could selectively localize in mitochondria, with Pearson correlation coefficients of 0.91 and 0.77 for MitoTracker Deep Red and Mito⁺LysoTracker Deep Red, respectively [179].

4. Structure–Activity Relationships Discussion

The results of our qualitative conformational relationship analysis are as follows: (i) The morpholine ring as a lysosomal targeting moiety has strong structural adaptability. Whether it is naphthalimide, BODIPY, coumarin, rhodamine, or other fluorescent frameworks, lysosomal targeting can be achieved after modification by the morpholine ring. (ii) The lysosomal localization of the morpholine ring is not affected by the relative molecular weight. According to the prediction of Chemdaw software 23.0, the exact masses of Lyso-C (107) [112] and probe 50 [62] were 286.1066 and 1714.9218, respectively. They all showed excellent lysosomal localization properties. (iii) The flexible and rigid conformations of the molecules had no significant effect on the lysosomal targeting efficiency of the morpholinemodified compounds, such as CQ-Lyso (63) [68]/probe 50 [62] and NCIC-VIS (139) [143]/Lyso-B (140) [144]. (iv) The introduction sites of morpholine ring and functional groups have no significant effect on the lysosomal targeting effect of the compounds, such as LyNP-H₂S (112) [117]/probe 113 [118], CMDN (108) [113]/DCICA (123) [128], and IRh-Ly (80) [85]/RL1 (81) [86]. (v) Hydrophobicity significantly affects the lysosomal targeting effect of morpholine ring-modified compounds. For example, the log p value of morpholinering-modified NIMCn progressively increases with the lengthening of the carbon chain of the linker arm. When the carbon chains were 8 and 10, compounds NIMC₈ (log p = 4.55) and NIMC₁₀ (log p = 5.39) had dual lysosomal and endoplasmic reticulum (ER) targeting effects [21]. This may be due to the fact that the ER, a major site for lipid synthesis and metabolism, is a hydrophobic environment. Lipophilic compounds can be induced to enrich in the endoplasmic reticulum under the hydrophobic driving force within the ER [21]. Notably, no apparent linear correlation was observed between the log p values of morpholine-modified small molecules and their lysosomal targeting efficiency. For example, BN-lys (33) (log p = 0.3) [45], LysoNO-NaPh (147) (log p = 0.02) [151], RML (39) (log p = 5.46) [51], and probe 4 (log p = 6.66) [17] all exhibited excellent lysosomal targeting. In contrast, probes NIMC₈ (log p = 4.55) and NIMC₁₀ (log p = 5.39) displayed dual localization in both lysosomes and the endoplasmic reticulum [21], while probe 6a (59) (log p = 6.32) showed diffuse intracellular staining [66]. (vi) When morpholine-ring-modified small molecules are cationic or susceptible to protonation under physiological conditions, compounds can exhibit lysosomal or/and mitochondrial targeting due to their different lipophilicity, such as Lyso-Gal (168) [171], Mito-SO₂-Lyso (173) [176] and PM-Mor-OH (177) [179]. This is because esterophilic cations can be enriched in mitochondria driven by the mitochondrial membrane potential. (iv) No significant correlation between the number of morpholino groups in a molecule and its lysosomal localization effect, such as MPL-NAP (7) [20]/MMN (35) [47] and Lyso NIR (14) [22]/Lyso Red (13) [22]. (vii) Some compounds still exhibit diffuse intracellular staining after being modified with morpholine rings, such as 6a (59) [66].

5. Conclusions

As a lysosomal targeting molecule, the morpholine ring has remarkable structural adaptations. The molecular backbone, size and conformation, number of morpholine rings, modification sites, linker arms, and functional groups have no significant effect on the lysosomal targeting efficiency of the morpholine ring. The hydrophobicity of the compounds was closely related to the lysosomal targeting efficiency. Specifically, highly hydrophobic neutral compounds tend to accumulate in the endoplasmic reticulum, while hydrophobic cationic compounds preferentially localize to mitochondria. It is also worth noting that (i) the morpholine ring is essentially an acidic microenvironment targeting group, which can not only target lysosomes but also target organelles with pH values similar to lysosomes, such as probe Mela TYR (153) that can localize to melanosomes. (ii) The accumulation of weakly alkaline morpholine rings in lysosomes may lead to an increase in their pH value, resulting in cell toxicity. For example, the probe CPY (167) containing the morpholine ring exhibited significant cytotoxicity to HeLa cells at 70 μM, which could be eliminated by reducing the concentration of CPY (167). In contrast, the CPH (166) probe without a morpholine ring did not exhibit significant cytotoxicity. (iii) The morpholine ring is not a perfect lysosome targeting group, and some compounds still exhibit diffuse intracellular staining after being modified with morpholine rings, such as probe 6a (59). Overall, morpholino substituents, as lysosomal targeting carriers, have excellent structural adaptability and lysosomal targeting effects, which are important for the development of lysosome-related drugs.

6. Future Directions

Growing evidence implicates important role of lysosomes in the pathology of a wide range of diseases. Lysosomal dysfunction not only underlies classic lysosomal storage disorders such as sphingolipidoses, mucopolysaccharidoses, glycoproteinoses, cystinosis and GM2 gangliosidosis but also plays a pivotal role in the pathogenesis of various major diseases, including atherosclerosis, neurodegenerative disorders, autoimmune diseases, pancreatitis, and malignancies. Given the central position of lysosomes in cellular metabolic regulation, developing strategies to modulate lysosomal function has emerged as a crucial therapeutic approach. This review systematically discusses the structural adaptation and lysosomal targeting efficiency of morpholino as a lysosomal targeting moiety, which lays a solid foundation for the research and development of lysosomal-targeted drugs. However, some challenges and opportunities remain to be explored: (i) Optimization of the hydrophobicity (log p) and charge properties of morpholine derivatives using computational modeling to improve lysosome-specific targeting and reduce off-organelle accumulation. (ii) The development of dual-functional probes combining morpholine with pH-stabilizing moieties (e.g., carboxylates) to effectively mitigate morpholine-induced lysosomal alkalinization cytotoxicity while maintaining targeting efficacy. (iii) Since morpholine targets acidic organelles, its utility could be extended to disease-specific acidic microenvironments, such as tumor lysosomes or inflamed tissues. (iv) Multiplexed organelle probes could be designed by coupling morpholine with other targeting groups to study organelle crosstalk. (v) For morpholine-modified compounds with poor lysosomal retention, structural modifications such as rigid linkers or additional lysosome-anchoring groups and may enhance specificity. (vi) High-throughput screening of morpholine derivatives could identify subfamilies with universally high lysosomal targeting efficiency. (vii) Morpholine-based lysosome-targeted delivery systems (e.g., ERTs and antibody–morpholine conjugates) require systematic pharmacokinetic evaluation to facilitate clinical translation for cancers and lysosomal storage disorders. (viii) The integration of AI-driven drug design with morpholine-functionalized nanocarriers (e.g., pH-responsive liposomes) can accelerate the development of lysosome-targeted delivery systems with enhanced specificity and reduced toxicity. In summary, the morpholine ring, as a versatile lysosome-targeting moiety, holds immense potential for both basic research (e.g., organelle imaging) and therapeutic applications (e.g., targeted drug delivery). Future work should prioritize selectivity enhancement, toxicity reduction, and multidisciplinary integration (chemistry, biology, and computational tools) to unlock its full clinical potential. By addressing current limitations, morpholine-based strategies could revolutionize the treatment of lysosome-related disorders, cancers, and neurodegenerative diseases.