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J Am Chem Soc. Author manuscript; available in PMC 2008 September 29.
Published in final edited form as: J Am Chem Soc. 2007 April 4; 129(13): 3918–3929. doi:10.1021/ja067710a.

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An enzymatically activated fluorescence probe for targeted tumor imaging
Mako Kamiya†,‡, Hisataka Kobayashi§,*, Yukihiro Hama§, Yoshinori Koyama§, Marcelino Bernardo§, Tetsuo Nagano†,‡, Peter L. Choyke§, and Yasuteru Urano†,¶,* †Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ‡CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan §Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg. 10, Rm. 1B40, 10 Center Dr., Bethesda, MD 20892-1088 ¶PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan

Abstract
β-Galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TGβGal (J. Am. Chem. Soc., 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-βGal, provides a dramatic fluorescence enhancement upon reaction with β-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-βGal, after confirming that tumors in the model could be labeled with avidin-β-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-βGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 μm. This strategy has potential clinical application, for example in video-assisted laparoscopic tumor resection.

Introduction
Marker enzymes are widely used to identify cell types or specific haptens, to examine transcription regulation, or to evaluate the efficiency of transfection experiments. Escherichia coli β-galactosidase is well characterized and extensively used as a marker enzyme in enzymelinked immunosorbent assays (ELISA),1-3 in situ hybridizations,4-6 classification of mycobacteria,7 and gene expression studies.8-11 This enzyme is especially useful for studying gene expression because of its stability, high turnover rate, and ease of conjugation and detection, as well as the absence of endogenous β-galactosidase activity in eukaryotic cells. A range of substrates has been developed including colorimetric,12,13 fluororescence,14-19 chemluminescence,20,21 and paramagnetic probes,22 though they have mostly been used in in vitro systems, with only a few successful applications in vivo.23-25 In our previous work, we focused on simple, rapid, and sensitive detection of β-galactosidase activity, and developed a highly sensitive fluorescence probe for β-galactosidase, TG-βGal (Scheme 1a).26 This probe was rationally designed to show a dramatic fluorescence activation

* To whom correspondence should be addressed. E-mail: kobayash@mail.nih.gov or urano@mol.f.u-tokyo.ac.jp.

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(up to 440-fold) upon reaction with β-galactosidase, and was successfully used to visualize βgalactosidase activity in living cells. We considered that such probes might be suitable to visualize tumors in vivo. For example, in the clinical field, video-assisted laparoscopic surgery is becoming one of the standard methods for resecting metastases,27 and it already relies on optical cameras that could easily be adapted to employ fluorescence imaging.28 Probes such as TG-βGal have appropriate characteristics (high molar extinction coefficient and high fluorescence quantum yield in aqueous media after reaction with the target enzymes) for visualization of surface tumors such as intraperitoneal tumors, for which brightness of the fluorophores is more important than long-wavelength excitability, as the light does not need to penetrate the skin and tissues. Our aim in the present work was to design a TG-βGal-based probe that would be suitable for in vivo tumor imaging and to test its suitability for this purpose in an animal tumor model. TG-βGal itself was unsuitable, as its hydrophobic fluorescent hydrolysis product is easily washed out from cells (Figure 1a). We therefore designed and synthesized a new probe, AM-TG-βGal. Like TG-βGal, AM-TG-βGal shows a dramatic fluorescence enhancement upon hydrolysis with β-galactosidase. Further, the ester moiety of its hydrolysis product, AM-TG, is cleaved by ubiquitous intracellular esterases, generating a hydrophilic product that is well retained within the cells without loss of fluorescence. To examine whether AM-TG-βGal would indeed be suitable for in vivo tumor imaging, we used the following two-step strategy in a mouse tumor model. First, avidin-β-galactosidase conjugate was administered to the mice to target the enzyme to tumors, then AM-TG-βGal was administered. Subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 μm. Our findings demonstrate the feasibility of this approach to tumor imaging, which we believe has potential clinical applicability, for example in video-assisted laparoscopic tumor resection.

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Results and Discussion
Design and synthesis of new probes with improved cellular retention We aimed to modify TG-βGal to obtain sufficient intracellular retention in the living cells by adopting the strategy of introducing an acetoxymethyl (AM) ester group.29,30 We anticipated that the AM-derivatized β-galactosidase probe would be hydrolyzed both by β-galactosidase to show large fluorescence activation, and by ubiquitous intracellular esterases to yield the hydrophilic fluorophore with a free carboxylate group, which would not readily leak from the cells. From the synthetic point of view, we first thought it would be simple to introduce a methoxycarbonyl methoxy group instead of the methoxy group of TG-βGal. According to Scheme 2, we synthesized 1Me-βGal. Although 1Me (the hydrolysis product of 1Me-βGal) is highly fluorescent (Φfl = 0.85), 1Me-βGal had a relatively high background fluorescence (Φfl = 0.069), so the fluorescence enhancement after activation was relatively modest (46-fold) (Table 1, Scheme 1b). In order to understand this result, we have to consider the mechanism underlying the fluorescence off/on switching of TG-βGal, i.e., intramolecular photoinduced electron transfer (PeT), which is a non-radiative pathway from the excited state of the fluorophore. As we demonstrated in our previous paper, fluorescein derivatives, including TokyoGreens, can be considered to consist of two functional components from the viewpoint of fluorescence, namely a benzene moiety which is the electron donor and a xanthene moiety which is the fluorophore (Scheme 1a).31,32 Moreover, the oxidation potential of the benzene moiety can greatly influence the efficiency of PeT, and m-methoxytoluene, the benzene moiety of TG-βGal, has the most suitable oxidation potential as an electron donor (1.66 V vs SCE, Table 2) in order to convert the change in reduction potential of the xanthene moiety induced by β-galactosidaseassisted hydrolysis to the maximal change in fluorescence intensity.26 Therefore, we measured
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the oxidation potential of m-methoxycarbonylmethoxytoluene, the benzene moiety of 1MeβGal (Bn(1Me)), and found that the oxidation potential had been altered to a higher value (1.77 V vs SCE, Table 2) by the introduction of the electron-withdrawing methoxycarbonyl group with a short methylene linker, and this accounted for the increase of the background fluorescence of 1Me-βGal. The calculated energy level of the highest occupied molecular orbital (HOMO), which is known to be linearly correlated with measured oxidation potential, supported this view (-0.02141 hartree for m-methoxytoluene and -0.02207 hartree for Bn (1Me)) (Table 2). Thus, we employed a strategy to lower the oxidation potential of Bn(1Me) to a similar value to that of m-methoxytoluene (1.66 V vs SCE) by introducing an electron-donating group. The oxidation potential of m-methoxycarbonylmethoxyanisole (Bn(2Me)), in which the methyl group of Bn(1Me) is replaced with a methoxy group, proved to be sufficiently small to suppress the background fluorescence (1.57 V vs SCE, Table 2) via the PeT pathway. Thus, we designed and synthesized 2Me-βGal according to Scheme 3. As we expected, 2Me-βGal showed low background fluorescence (Φfl = 0.004), whereas 2Me (the hydrolysis product of 2Me-βGal) showed strong fluorescence (Φfl = 0.67) (Table 1). Thus, the fluorescence enhancement is 410fold. Unfortunately, however, the fluorescence intensity of 2Me was altered by esterase activity, because hydrolysis of the ester group lowered the oxidation potential too much, which led to quenching of the fluorescence output of 2 (Φfl = 0.32), the final fluorescent species generated from 2Me-βGal (Scheme 1b, Table 1, Figure 2). For the reliable fluorescence detection of βgalactosidase activity, the fluorescence should be unaffected by cleavage of the ester. The solution we adopted was to reduce the electron-withdrawing effect of the methoxycarbonyl group of Bn(1Me) by lengthening the methylene chain between the methoxycarbonyl group and the benzene moiety. We calculated the HOMO energy levels of the benzene moiety with various lengths of methylene chain at the B3LYP/6-31G level, and predicted that a HOMO level similar to that of m-methoxytoluene could be achieved by inserting three or four methylene units between the methoxycarbonyl group and the benzene moiety (Table 2). Oxidation potentials of m-methoxycarbonylbutyloxytoluene (Bn(3Me)) measured by cyclic voltammetry were consistent with this expectation (1.62 V vs SCE, Table 2). According to Scheme 4, we synthesized 3Me-βGal with four methylene units. The background fluorescence of 3Me-βGal was successfully suppressed (Φfl = 0.009), while the hydrolysis product of 3MeβGal (3Me) emits sufficiently strong fluorescence (Φfl = 0.85) (Scheme 1b, Table 1). The fluorescence enhancement ratio that can be obtained with 3Me-βGal is approximately 360-fold. Moreover, its fluorescence was independent of the esterase activity (Φfl of 3 was 0.85, Figure 2). Therefore, we derivatized 3Me-βGal to the AM ester, which was designated AM-TGβGal (Scheme 5). As expected, AM-TG-βGal shows almost no fluorescence (Φfl = 0.005), but is converted by β-galactosidase to highly fluorescent AM-TG (Φfl = 0.86), affording a 470fold fluorescent enhancement (Scheme 1c, Table 1, Figure 3). Thus, we finally succeeded in developing a highly sensitive fluorescence probe for β-galactosidase with an esterase-sensitive moiety that could be removed without loss of the strong fluorescence, by finely tuning the oxidation potential to retain the highly activatable character of TG-βGal. Relationship between oxidation potential and fluorescence quantum yield We plotted the fluorescence quantum yields of the anionic and neutral forms of 1Me, 2Me, 3Me (the scaffold fluorophores of 1Me-βGal, 2Me-βGal, 3Me-βGal) on a graph showing the relationship between the oxidation potential and fluorescence quantum yield (Φfl) of previously reported TokyoGreen derivatives (Figure 4, Table 3).26 All the values including that of 1Me, 2Me, 3Me can be fitted with the Marcus equation. It can be seen that 3Me lies between the fluorescence off/on thresholds of the anionic and neutral forms, indicating that it is the most suitable scaffold fluorophore for developing a highly sensitive fluorescence probe with

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35-37 The tumor model in mice was prepared as reported. including SHIN3 cells. As regards the specificity of the staining. owing to the leakage of the fluorescent product derived from TG-βGal (Figure 6e). This difference can be explained in terms of superior retention of the ultimate hydrolysis product of AM-TG-βGal in the living cells. available in PMC 2008 September 29. Although the incubation time required for imaging is likely to vary depending on the size of the tumor. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Cellular retention We next examined whether or not the newly developed AM-TG-βGal is better retained in living cells as compared with TG-βGal. Fluorescence detection of tumors in vivo We next tried to visualize tumors pre-labelled with avidin-β-galactosidase by employing AMTG-βGal. employing a tumor-targeting avidin-βgalactosidase conjugate (avidin-β-galactosidase) to localize β-galactosidase to cancer cells in vivo. 33. The fluorescence enhancement at microfoci over the surrounding tissues on the mesenterium is up to 33-fold (Supporting Figure S1). the organs were harvested and stained with X-Gal. In mice intraperitoneally implanted with SHIN3 and pre-treated with avidin-βgalactosidase (ip). Avidin was chosen based on its high affinity for lectins on the surface of several human ovarian cancer cell lines.38 As a first step. indicating that the fluorescence signal was co-localized with the β-galactosidase activity (Figure 8). Staining of tissue with X-Gal showed a similar distribution to that of the fluorescence signal produced by AM-TG-βGal. suggesting that our strategy of using the hydrolyzable AM moiety did indeed improve cellular retention of the fluorescent product (Figure 7). Small tumors on the mesenterium were clearly seen (Figure 6c). the fluorescence signal produced by AM-TGβGal at the tumors was not decreased by washing. Author manuscript. Targeting of β-galactosidase to tumor cells We next tried to apply AM-TG-βGal to visualize intraperitoneal tumors in mice. Twenty hours later.35 These results suggest that AM-TG-βGal had been hydrolyzed both by the SHIN3-targeted β-galactosidase. For this purpose. and fluorescence microscopy revealed spots as small as 200 μm (Figure 6d). we adopted a two-step procedure. a blue precipitate was observed only in the disseminated tumor. tumor-free mouse treated with avidin-β-galactosidase and AM-TG-βGal (Supporting Figure S2). Indeed. As expected. leading to fluorescence activation.34 followed by the administration of AM-TG-βGal. After further incubation for 2 hours. one-hour incubation produced clearer fluorescence signals than 30-minute incubation in our experiment. in contrast to the case with TG-βGal. HEK293 cells expressing β-galactosidase were incubated with 10 μM AM-TG-βGal. Page 4 enhanced cellular retention. and by intraplasmic esterase. in contrast with the case of TG-βGal. In contrast. resulting in good retention within cells. it has previously been shown that at least 99 % of the small spots in the peritoneal cavity in this tumor model are tumors (Supporting Figure S3). intraperitoneal administration of AM-TG-βGal followed by incubation for 1 hour revealed strongly fluorescent spots in the peritoneal cavity (Figure 6a and 6b). we examined whether avidin-β-galactosidase could be targeted to SHIN3 cells in vivo by injecting it intraperitoneally into mice bearing peritoneal SHIN3 tumors.Kamiya et al. J Am Chem Soc. This result also confirms that the previously reported design strategy is valid for developing new probes with desirable features. but this was unlikely to have been confined to tumors. and fluorescence images were captured before and after washing of the cells (Figure 1b). almost no fluorescence activation could be observed in normal. Mice given TG-βGal also showed a marked fluorescence increase in the peritoneal cavity. washing caused little change of the fluorescence. . confirming that avidin-β-galactosidase can be efficiently targeted to SHIN3 cells in vivo without loss of the enzymatic activity (Figure 5).

Materials and instruments Chemicals used for organic synthesis were of the best grade available. compounds were dissolved in dimethyl sulfoxide (DMSO. to the desired concentration. but with limited success so far. NaOH (Φfl = 0. and is well retained intracellularly after hydrolysis by ubiquitous intracellular esterases without loss of fluorescence. and were used without further purification. unless otherwise specified. All fluorescence spectra were obtained with a LS-55 fluorescence spectrometer (Perkin-Elmer) or a F4500 fluorescence spectrometer (Hitachi). Page 5 Conclusion There is increasing interest in in vivo optical imaging of β-galactosidase in the biological and clinical fields. AM-TG exhibits both highly activated fluorescence and enhanced permeability. Secondly. Studies to examine the feasibility of using this approach in videoassisted laparoscopic surgery are in progress.85) was used as a fluorescence standard.39 β-Galactosidase and porcine liver esterase were purchased from Sigma-Aldrich Japan K. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Experimental Section All procedures were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996). and approved by the National Cancer Institute Animal Care and Use Committee. First. AMTG-βGal is administered.Kamiya et al. and is readily transferred into nearby tumor cells. Dojindo) to obtain 10 mM stock solutions. fluorescein in 100 mM aq. All experiments were carried out at 25 °C. The present results indicate that chemistry-based fine-tuning of chemical properties (both fluorescence and cell permeability) could be the key to the development of small-molecular “magic bullets” in the fields of biology and clinical medicine. and we believe that the range of potential applications of our new probe is enormous. and on a JNM-AL400 (JEOL) instrument at 400 MHz. fluorometric grade. It represents a development of our previously reported fluorescence probe for β-galactosidase. Mass spectra (MS) were measured with a SX-102A (JEOL) for EI. All absorption spectra were obtained with a 8453 UV/Vis spectrometer (Agilent) or with a UV-1600 UV/Vis spectrometer (Shimadzu). Moreover. is administered and binds via avidin to the tumor cell surface. UV/VIS and fluorescence analysis For the absorption or fluorescence measurement. and is hydrolyzed to AM-TG by β-galactosidase bound to the tumor cells. supplied by Tokyo Chemical Industries. . available in PMC 2008 September 29. These stock solutions were diluted with buffer as specified in the figure legends. a MS700 (JEOL) for FAB and a JMS-T100LC (JEOL) for ESI-TOF. Wako Pure Chemical or Aldrich Chemical Company.23-25 We have developed AM-TG-βGal as a highly sensitive fluorescence probe for β-galactosidase that is suitable for targeted tumor imaging in vivo. This procedure provides a tumor-specific. a conjugate of a tumor-targeting molecule and the enzyme. long-lived. 1H-NMR spectra and 13C-NMR spectra were recorded on a JNMLA300 (JEOL) instrument at 300 MHz and 75 MHz. avidin-βgalacotosidase. AM-TGβGal exhibits a drastic fluorescence enhancement upon reaction with β-galactosidase. National Research Council. J Am Chem Soc. where it is hydrolyzed to the free carboxylate by cytoplasmic esterases. For determination of the quac(Φfl). and highly activated fluorescence signal.26 modified by introducing AM ester into TG-βGal through a precisely optimized linker while retaining the advantageous optical properties of TG-βGal. we devised a two-step strategy for highly activatable optical imaging of intraperitoneal tumor cells without the need for prior transfection of reporter enzymes.K. Author manuscript.

Ag/Ag+) were converted to those vs.. especially around the stomach. Melville. Page 6 Cyclic voltammetry Cyclic voltammetry was performed on a 600A electrochemical analyzer (ALS). when numerous small. The main internal organs (tumor. the subphrenic region. pH 7. containing 150 mM NaCl. USA). 10. and the abdominal walls were removed. MA. Obtained potentials (vs. at the hepatic and splenic hila. NY) equipped with an UPLAPO10x objective lens (Olympus USA. and imidazole (3. the mouse was sacrificed under anesthesia.248 V. The images were obtained with an Olympus fluoview system using an IX81 inverted microscope. lung. MD. spleen. 1 mM MgCl2. and 0. Synthesis of 2-Bromo-5-(tert-butyldimethylsilyloxy)toluene (1) A mixture of 4-bromo-3-methylphenol (2 g. and argon was bubbled for 10 min before each measurement. 50 mmol) in dry DMF (10 ml) was stirred NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. confocal fluorescence images and DIC images were captured before and after washing with PSS. 2 mM CaCl2. heart) were removed for routine X-Gal staining.1% glucose (PSS). A threeelectrode arrangement in a single cell was used for the measurements: a Pt wire as the auxiliary electrode. SCE by adding 0. an Ar ion laser.7 mmol). a glassy carbon electrode as the working electrode. Fluorescence images of tumor nodules on the mesentery were captured with a BX51 microscope (Olympus USA. 4 mM KCl. and the emission wavelength was 510-550 nm. 30 mmol). stomach.0 mM sample and 100 mM tetrabutylammonium perchlorate (TBAP) as a supporting electrolyte in acetonitrile. and on the mesentery. Fluorescence imaging of tumors in a mouse tumor model Avidin-β-galactosidase (100 μg) in PBS(-) was injected intraperitoneally into a tumor-bearing mouse and. Author manuscript. 2 × 106 SHIN3 cancer cells suspended in PBS(-) were injected intraperitoneally into female nude mice (National Cancer Institute Animal Production Facility. Experiments with these tumor-harboring mice were performed at 10-13 days after dissemination. kidney. intraperitoneally disseminated tumors had formed.4.6 g. Biodistribution of avidin-β-galactosidase in vivo Avidin-β-galactosidase (100 μg) in PBS(-) was injected intraperitoneally into tumor-bearing mice. Intracellular retention assay HEK293 cells expressing β-galactosidase were incubated with a 10 μM solution of TG-βGal or AM-TG-βGal in physiological salt solution. Melville. Twenty hours later. tert-butyldimethylsilyl chloride (TBDMS-Cl) (4. Mouse preparation The intraperitoneal tumor model was prepared as previously reported. Woburn. which was left for 16-23 hours. USA). mesentery. and a PlanApo 40x/1. The excitation wavelength was 488 nm. intestine. and 0. Tokyo. liver. the mice were sacrificed by exposure to CO2 gas. a 3. Spectral unmixing was performed to obtain the fluorescence unmixed images. Japan). The excitation wavelength was 445-490 nm. The sample solutions contained 1. Then.3 μM solution of AM-TG-βGal was injected. and an Ag/Ag+ electrode as the reference electrode. Fluorescence images in the peritoneal cavity were captured with a Maestro™ In-Vivo Imaging System (CRI Inc. After 1 hour. .38 Briefly. Photochemical calculation HOMO energy levels were calculated at the B3LYP/6-31G level with Gaussian 98W. NY).8 g.1 % DMSO as a cosolvent for 2 hours. available in PMC 2008 September 29. 5 mM HEPES.Kamiya et al. Frederick. Then.40 objective lens (Olympus.

9H). 6. The inorganic precipitate was filtered off and the NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. dried over Na2SO4 and evaporated.10. 4. 2H. 18.88 (s. a 1 M solution of tetrabutylammonium fluoride in THF (6.64.40. -4.2 Hz). and the red precipitate was collected by filtration. 6. 4. 2.78. Synthesis of 9-[1-(4-Methoxycarbonylmethoxy-2-methylphenyl)]-6-hydroxy-3H-xanthen-3one mono(2’.04 g. found. 431.27 mmol) in distilled THF (10 ml) was cooled down to −78 °C (dry ice-acetone). 160.0 Hz). 122. 1. 1. MS (FAB): m/z 301. methyl bromoacetate (17 μl.16.97 (s. and the residue was chromatographed on silica gel with CH2Cl2-MeOH (100:5 – 100:7) as the eluent to give 3 as an orange powder (1. 1H. The inorganic precipitate was filtered off and the filtrate was concentrated under reduced pressure. 6.65.18 mmol) and Cs2CO3 (400 mg. 2H. 2. 3H). The residue was diluted with water and extracted with CH2Cl2 three times. J = 8. 6H). 1H. 2.6 mmol) and 2. 1. 303 (M+1)+. 2H.18 (s. 6.07 g. . found. 6.7 mg. 168. 0. 2. then 2 N aq.2.01 (s.4 Hz). 1H NMR (300 MHz. 391.7 Hz. J = 9.6-Bis-(t-butyldimethylsilanyloxy)xanthen-9-one (1.13 g. J = 2. CD3OD): δ 7. 138. 2H. 20. J = 9. J = 9. 138. CD3OD): δ 7. 118. 319. 2. The residue was chromatographed on silica gel with CH2Cl2-MeOH (100:5) as the eluent to give 4 as a red powder (41.18 mmol) was added. 58 %).9Hz). 114.37 mmol) dissolved in distilled THF (20 ml) was added dropwise to the mixture using a syringe.60. 0.6’-tetra-O-acetyl-β-D-galactopyranoside) (5) A mixture of 4 (39 mg.2 mmol) in dry dimethylformamide (5 ml). 22. J = 8. 6. Author manuscript.0 Hz. 118.3Hz). 1H. J = 2.32 (s.31 (s.Kamiya et al. 0.4 Hz. 9H). J = 2.3. The combined organic solution was washed with water and saturated aq. The mixture was stirred at room temperature under argon for 2 hours. 7. 0. CDCl3): δ 7.4 Hz). J = 2. 6. J = 8. 1H.62 g. The precipitate was chromatographed on silica gel with CH2Cl2-MeOH (100:3-100:5) as the eluent to give 2 as an orange powder (774. J = 8.03 (s. J = 9. 25. 319.2 ml. 7.29. 6.86 (m. 3H).25. The reaction mixture was stirred at room temperature under argon overnight. 3H).NaCl. 103.84.09703. 2H. CDCl3): δ 7.1 mmol). J = 2.5 ml.76.08 (d. 0. 95 %).15 (d. 431. 1H.5 Hz).63 (dd.73 (s. 6.HCl was added. 6. Synthesis of 9-[1-(4-hydroxy-2-methylphenyl)]-6-hydroxy-3H-xanthen-3-one (3) To a solution of 2 (2.2 mmol) was added.94 (d. J = 8.NaCl. 7. Synthesis of 9-[1-(4-Methoxycarbonylmethoxy-2-methylphenyl)]-6-hydroxy-3H-xanthen-3one (4) To a mixture of 3 (60 mg.48.59. 1H. 132. 121. 1H. 3H).4’.89 (dd.63 (d. 0. available in PMC 2008 September 29.2 Hz.6 Hz.7 Hz).03. HRMS(ESI-): calcd for [M-H]-.80 (dd. 1H. 134. 161. t-Butyllithium in n-pentane solution (4.06 (d.66.72 (d.6 mg. Cs2CO3 (200 mg. 119. J = 8. 6. 2.4. 3.78.98.6 Hz).9 Hz). 2H.06 (s. J = 8. 1H. 391. The solvent was concentrated under reduced pressure. dried over Na2SO4 and evaporated to give 1 as a colorless clear oil (3. then evaporated.2.57 mmol) was added dropwise to the reaction mixture using a syringe.74 (dd. Page 7 at room temperature under argon for 3 hours. 13C NMR (75 MHz.2 mmol) in distilled THF (200 ml). 3H).53 (dd.27 (d. 6.28 g.0 Hz).88.79.11816. 131. The residue was diluted with water and extracted with CH2Cl2 three times.82. 0. 1H NMR (300 MHz.2 Hz.16786. CDCl3): δ 172. Synthesis of 9-{1-[4-(tert-Butyldimethylsilyloxy)-2-methylphenyl]}-6-hydroxy-3H-xanthen-3one (2) A solution of 1 (1.01-6. HRMS (ESI+): calcd for [M+H]+.25 mmol) in dry dimethylformamide (2 ml) was stirred at room temperature under argon overnight.84 (d. J = 9. 2H.4 Hz).6-tetra-O-acetyl-αD-galactopyranosyl bromide (100 mg.11455.10 (d.30.).16636.18. The combined organic solution was washed with water and saturated aq.0 Hz). 1H.75. 57 %).5 Hz). 122. CDCl3): δ 154. 2. found. 3. 82 %). 6H).95 (d. 13C NMR (75 MHz. 6. 1H NMR (300 MHz. 2H).2 Hz). 6H). HRMS (ESI+): calcd for [M+H]+.86 (s. The mixture was stirred at −78 °C under argon for 30 min.3’. 0. 1H NMR (300 MHz.2 Hz).09652. 116.30.78 (d.33 (d. 6.

NaCl.7 Hz.00 (dd.NaOH 1 ml (2 mmol) was added.7 Hz). J = 9.04. 6.3H).16771. The residue was diluted with water and extracted with CH2Cl2 three times. 3H).08779.8 g.92 (s. J = 7. J = 8.5 Hz). 1H NMR (300 MHz. 2H). 6.19.3 Hz). 2. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. 1H.22-4.4 mg.05(s. 2. 1H. 13C NMR (75 MHz. J = 3.06.6 mmol) in dist MeOH (100 ml). 103. J = 8.40 (d. dried over Na2SO4 and evaporated. 1H NMR (300 MHz. 3.88 (d.9 μmol) in methanol (2 ml). The mixture was stirred at room temperature for 1 hour.0 Hz).40 (d.6 Hz. 5.73 (s.19309. 1H.Kamiya et al. then neutralized with 2 N aq.9 Hz). 2.3 Hz). 58 %). 2. 553. 3H). 1H.33 (d. 113. 1H. J = 8.7. CDCl3): δ 7. available in PMC 2008 September 29. 2H. 3H). 1H. HRMS (ESI-): calcd for [M-H]-. J = 9. The Amberlite IR-120 was filtered off.92 (d. -4. J = 8. 1H. J = 9. 2H). 2N aq. 2H. CDCl3): δ 156.0.45 (d. The organic solution was washed with water and saturated aq. 2. 8. 1. The methanol was evaporated off. 3H).7.17099. Synthesis of 9-[1-(4-Carboxymethoxy-2-methylphenyl)]-6-hydroxy-3H-xanthen-3-one (7) To a solution of 4 (15 mg.08686.2 Hz). The solution was refluxed for 1.25. J = 2. 3.12 (m.3 Hz). The mixture was stirred at room temperature for 4 hours.5 Hz). 7H).57 g. 4.73-3. and the filtrate was evaporated.88 (m. quant. 3.). 5.62 (dd.57 (dd. 3. 1H NMR (300 MHz. 6.36 (d. The residue chromatographed on silica gel with CHCl3-MeOH (100:10) as the eluent to give 6 as an orange powder (2.21 g. 1H NMR (300 MHz. 2H).86 (s.3H).2. J = 2. and the residue was chromatographed on silica gel with CH2Cl2-MeOH (100:5) as the eluent to give 7 as an orange powder (12 mg. and the reaction mixture was refluxed overnight again. 7. J = 2. 62 %). 1H. J = 9.2.03 (s.15 (d. J = 2. 6.39 (s.6 Hz). 375. 1H). 3. 5. 6.1 Hz). 6. 11.1 Hz).7 Hz). 2. 553. 7. 6.93-6. 1H.3H).NaOH (20 ml) was added. 7.12 (m.6 Hz). a 5 M methanol solution of NaOMe (2 μl.6 Hz). 743. J = 8.08 g.64. 25. Author manuscript. and the residue was diluted with H2O and extracted with CH2Cl2. HRMS (ESI+): calcd for [M+H]+. 7.45. then the solvent was evaporated. 204 (M+).41 (d.8 mg. 1H.6 Hz).6. 3.3 ml. J = 8.6 Hz). 6. 133. J = 2. 2.9 Hz).NaCl. 83 %). Synthesis of 2-Bromo-5-(toluene-4-sulfonyloxy)anisole (8) A mixture of 4-bromoresorcinol (2. 6.47 (d. J = 1. 1H.48 (m. 6.32 (dd.3H). 4. 38 μmol) in methanol (4 ml). Page 8 filtrate was concentrated under reduced pressure. The inorganic precipitate was filtered off and the filtrate was concentrated under reduced pressure.0 Hz).4. 7.57-5.13 (s. 1H.17 (d. 7. 10 μmol) was added. and K2CO3 (4. 21 mmol) was added. 1H. CD3OD): δ 7.03 (s.3H). 743.72 (d.08. 3.18 (d. 2H.45 (s. 1H. 4H). 2H). 1H NMR (300 MHz. 89 %).2. 3H). J = 9.80 (m. J = 8. MeI (1.00 (d. found. The obtained residue was neutralized with 2 N aq. 4. 7.34 (d.06 (d. 3. 2.07-7.5 Hz). The combined organic solution was washed with water and saturated aq.3H). 1H. 2H.10 (dd.89 (m. p-toluenesulfonyl chloride (2. 56. then neutralized with Amberlite IR-120 (H+). 1H.4 Hz).NaCl. 1H. The residue was diluted with water and extracted with AcOEt three times. The residue was chromatographed on silica gel with CH2Cl2-MeOH (100:3) as the eluent to give 5 as an orange powder (41. 375.6 Hz. 1H. MS (EI): 356. found.5 mmol).03. 7.84 (s.19. 4. J = 9. CDCl3): δ 7. 2. 156. J = 2.3H). 105.61 (dd.4 g. 2 M aq.0 Hz). .40 (dd.5 hrs.6 mmol).HCl.19 (s.59 (d. CDCl3): δ 7. HRMS (ESI+): calcd for [M+Na]+.1. 2H). 2. Synthesis of 9-[1-(4-Methoxycarbonylmethoxy-2-methylphenyl)]-6-hydroxy-3H-xanthen-3one mono-β-D-galactopyranoside (6) To a solution of 5 (5 mg.0 Hz). The combined organic solution was washed with water and saturated aq.HCl and extracted with CH2Cl2 three times. 6. 32 mmol) in dry acetone (150 ml) was refluxed overnight. dried over Na2SO4 and evaporated to give 9 as clear oil (1. Synthesis of 4-Bromo-3-methoxyphenol (9) To a solution of 8 (3. 1H. 2H). 2H.10-6. found.07 (s.16 (m. 6.6 Hz). dried over Na2SO4 and evaporated to give 8 as light pink oil (3. MS (EI): 202. J = 8. 3H).87 (s.47. J = 8.18-5. J = 9.0 g. 10. 6.44 (dd.82 (s. CD3OD): δ 7. 18.4 Hz).19519.78 (s. The combined organic layer was evaporated. 358 (M+). 1H.

6. found.7 %). 6.56 (d.HCl was added. 1H. 6. CDCl3): δ 7. 68 μmol).61. J = 9. CDCl3): δ 156. 6.88 g.0 Hz.2 Hz.3 Hz). 56. HRMS (ESI+): calcd for [M+H]+.97. 1H NMR (300 MHz. 9H). 129.Kamiya et al. 2.57 mmol) was added dropwise to the reaction mixture.18 (d.0 Hz).19 g.11006.32 (d.0 Hz).17842.98 (s.6-bis-(t-butyldimethylsilanyloxy)xanthen-9one (1.2 Hz. CDCl3): δ 7.87 (s.NaCl. 1H. 149. The combined organic solution was washed with water and saturated aq.20 (s. J = 8. 132.NaCl.03. 3H). 204 μmol) was stirred at room temperature under argon overnight. The residue was chromatographed on silica gel with CH2Cl2-MeOH (100:5) as the eluent to give 13 as an orange powder (17.70 (dd. J = 8.0 Hz).70 (s. J = 9. then 3. 6H). J = 8. J = 9.57 g.75 (dd.6 g. 6. The residue was diluted with water and extracted with CH2Cl2 three times.4 Hz).2 Hz).6 Hz). J = 8.59 (dd. Author manuscript. Page 9 Synthesis of 2-Bromo-5-(tert-butyldimethylsilyloxy)anisole (10) A mixture of 9 (1.70. 3. 2H.04 (s.02 (d. 6. MS (EI): 316. 4.NaCl.7 mg. 318 (M+). Synthesis of 9-[1-(4-Hydroxy-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (12) To a solution of 11 (43. tert-Butyllithium in n-pentane solution (4. 3. 3. 7.18 (d. 2. 1H NMR (300 MHz. 2.6 Hz). 1H. 1H. 2. J = 9. 4. The residue was diluted with water and extracted with CH2Cl2 three times.11308. . 0. 109.0 Hz).92. 407. found. 449.7 mmol). 335. CDCl3): δ 7. 2. CD3OD): δ 7. 1H. 2H.20. HRMS (ESI+): calcd for [M+H]+. 106. 3H). The combined organic solution was washed with water and saturated aq. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. which was diluted with water and extracted with CH2Cl2 three times.08862. J = 8. 2H). 1H.33 (d. 1. 39 mmol) in dry DMF (10 ml) was stirred at room temperature under argon overnight.7 mg. 1H. 63 %).85 (s. 6. 3H). J = 2. found.09195. J = 8. J = 9.76 (d. 2H . 145. 3H). 335. 1H NMR (300 MHz. 133.).69 (s.39 g. dried over Na2SO4 and evaporated. 23 mmol). The obtained residue was chromatographed on silica gel with CH2Cl2-MeOH (100:5) as the eluent to give 12 as a red powder (22.8. 1H. 3H).3 Hz.74 (s. TBDMS-Cl (3.6 mg. 2.4 mg.0 Hz). 3. J = 2.16 (d.80.6 Hz).HCl (2 ml) was added.0 Hz).5 mg. 2H.3 Hz.59. The combined organic solution was washed with water and saturated aq.5 g. 6H).3 Hz). 2H.34.0 Hz). 70 %). 4H). 1H. Synthesis of 9-{1-[4-(tert-Butyldimethylsilyloxy)-2-methoxyphenyl]}-6-hydroxy-3Hxanthen-3-one (11) 10 (1.6 mmol) dissolved in dist THF 20 ml was further added dropwise. 7. 9H).3 Hz.60 (dd.34 (dd.7 %).6 Hz. 128. 0. 115. 2 N aq. J = 2.3 Hz). The residue was chromatographed on silica gel with hexane-AcOEt (4:1) as the eluent to give 10 as a light yellow clear oil (1. 2. 407. 2H.41 (d. 7.10 (d. The solvent was evaporated under reduced pressure to leave a residue. and the mixture was refluxed for 5 hrs. 68 μmol). 6. 3. 3H). 13C NMR (75 MHz.75 (m.28. 6.17790. 76. and Cs2CO3 (66. J = 2. 6. 97 μmol).81 (m. J = 8.46. 21. dried over Na2SO4 and evaporated. 1H. 7. 6. available in PMC 2008 September 29. 1H. HRMS (ESI+): calcd for [M +H]+.6 mg. The solvent was evaporated under reduced pressure. 0. The precipitate was chromatographed on silica gel with CH2Cl2-MeOH (100:5) as the eluent to give 11 as an orange powder (43. The solvent was concentrated under reduced pressure.5 ml. Synthesis of 9-[1-(2-Methoxy-4-methoxycarbonylmethoxyphenyl)]-6-hydroxy-3H-xanthen-3one (13) A mixture of 12 (22. The inorganic precipitate was filtered off and the filtrate was concentrated under reduced pressure. The mixture was stirred at −78 °C under argon for 30 min.24 (dd.80-6.55. 7.27 μl. and imidazole (2.67 (s.4 mmol) was dissolved in distilled THF (10 ml) and the solution was cooled to −78 °C (dry ice-acetone). 7. 449. bromomethyl acetate (6.31 (s. 1H NMR (300 MHz.4 Hz).82 (d. dried over Na2SO4 and evaporated. and 2 N aq. 3. J .

18-5. 4H). J = 9.7 mg. 6.74 (d.47. 2H.04. J = 2.57 (dd. 2H.8 Hz).13 (m. 0. 2H). 152. 94.75 (m. J = 8.49 (m.90. 1H). J = 9. HRMS (ESI-): calcd for [M-H]-. HRMS (ESI+): calcd for [M+H]+. 13C NMR (75 MHz. 3H). The inorganic precipitate was filtered off and the filtrate was concentrated under reduced pressure.35 (d.25 mmol) in dry DMF (2 ml) was stirred at room temperature under argon for 4.18 mmol) was added. 6.25-4.0 Hz). 3H).3’.4 mg.2 Hz. 10 μmol) in methanol (1 ml).65 (s. 3. The residue was chromatographed on silica gel with CH2Cl2-MeOH (100:3) as the eluent to give 14 as an orange powder (6. 121.4 Hz). 1H NMR (300 MHz.72 (m.70 (s. 737. 3H).0 Hz).NaCl.7 Hz. 1H. J = 8.20 Hz). a 5 M methanol solution of NaOMe (2 μl. 1H NMR (300 MHz.5 hrs. 2H). Page 10 Synthesis of 9-[1-(2-Methoxy-4-methoxycarbonylmethoxyphenyl)]-6-hydroxy-3H-xanthen-3one mono(2’.8 Hz.20816. 1H.1 ml (0.4 Hz).93 (d. 1H NMR (300 MHz. available in PMC 2008 September 29.3H).73 (s.3 Hz). J = 2.2 %).16590. The residue was diluted with water and extracted with CH2Cl2 three times.2 Hz. 3.8.78 (s. and evaporated. CDCl3): δ 7. 3.3 Hz).70-3.07(s.07947. The reaction mixture was stirred at room temperature under argon overnight.39 (d. 2. J = 9. 3. 6. 4H). 62 %). 1H.8 mg.07 (d.43. 1H.42.17 Hz).80.06 (m. 7. 1H.3.7 μmol) in methanol (1 ml). found. 124.03(s.74 mg. 7.11 (dd. 0. 3. Author manuscript. 5.5 Hz).25 Hz).4’.20 (s. 174.88 (d.73 (s.66-6. CDCl3): δ 175.08178. J = 8.2 Hz). The combined organic solution was washed with water and saturated aq. 2H. and 2. 2H).46 (d. J = 2. J = 2. 2.84 (m. 6. and the filtrate was evaporated.20 Hz). The inorganic precipitate was filtered off and the filtrate was concentrated under reduced pressure. 1H. 1H. 3. found.12 (d.21.73.2H). 1H.27 (d. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc.3H). 4H).HCl.2 Hz).4 Hz).30 (d. and the residue was subjected to reverse-phase preparative TLC (RP18W) with acetonitrile/water (1:1) as the eluent to give 16 as an orange powder (3 mg. dried over Na2SO4. 2. then neutralized with 2 N aq. 1. The residue was chromatographed on silica gel with CH2Cl2-MeOH (100:5) as the eluent to give 17 as a red powder (53. J = 7.NaCl.0 Hz). 3.16846. CD3OD): δ7. The residue was diluted with water and extracted with CH2Cl2 three times.3.25 (d. J = 8. J = 9. 2H).02 (s. 2. J = 8.3 Hz). J = 8. J = 9. 6.67.62 (dd. 6. 1H NMR (400 MHz. J = 9. 1H. 391. 7.8 Hz).3.86 (s. 2.70 (s.3 Hz).08.20 mmol) and Cs2CO3 400 mg (1. found.2 mmol) in dry dimethylformamide (2 ml). 1H. Synthesis of 9-{1-[4-(4-Methoxycarbonylbutyloxy)-2-methylphenyl]}-6-hydroxy-3Hxanthen-3-one (17) To a mixture of 3 63. methyl 5-bromovalerate (21 μl. 3H). 4.2 mmol) was added.45 (m. 137. J = 2.2 Hz).88 (m.10 (dd. 130.20860. 2. 2. 6. The mixture was stirred at room temperature for 4 hours.91-6. Cs2CO3 (38.3H).17 (m. 737.1 mg.56-5.4. 1H.14 (s. 4H). 4. 6. J = 1. 2. 3H).69 (m.6’-tetra-O-acetyl-β-D-galactopyranoside)(14) A mixture of 13 (3. 1.4 Hz). 117 μmol). J = 9. 2. 6. 1H. Synthesis of 9-[1-(2-Methoxy-4-methoxycarbonylmethoxyphenyl)]-6-hydroxy-3H-xanthen-3one mono-β-D-galactopyranoside)(15) To a solution of 14 (2 mg.NaOH 0. 3. 2.4 mg. 2.81 (dd. 6.19 (d. 5 μmol) was added.57. 3. 1H. 5. 391.6-tetra-O-acetylα-D-galactopyranosyl bromide (100 mg. 1H. 2H).3 Hz). 130. 7. The residue chromatographed on silica gel with CHCl3-MeOH (100:10) as the eluent to give 15 as an orange powder (0. 9.06 (m. The mixture was stirred at room temperature for 1 hour.3 μmol). 5. CDCl3): δ 7.3H).Kamiya et al. The combined organic solution was washed with water and saturated aq. 1H. J = 8. J = 2. 2.13-7. 6. 1H. 2H). 4.3. The solvent was evaporated off. 76 %).81 (dd. 1H. 157. .25. 159. The Amberlite IR-120 was filtered off. then neutralized with Amberlite IR-120 (H+). 6. 4. 1H.89-3.8 mg (0. 3H). 7.77 (dd. 1H. 1. 3H).52 (s. 569.10 (d. dried over Na2SO4 and evaporated.59 (dd. 7. HRMS (ESI+): calcd for [M+H]+.8 Hz). CD3OD): δ 7. 2. 2. 48 %). 6. J = 9. 4.74 (s. 6. 115. 569. 2 N aq. 3H).77. 1H.4H). Synthesis of 9-[1-(4-Carboxymethoxy-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (16) To a solution of 13 (4. 116.84 (d.74 (dd.

Cs2CO3 (250 mg. HRMS (ESI+): calcd for [M +H]+. J = 8. 7.3 Hz.0 Hz). 50 μmol) was added.77 mmol) and 2. then neutralized with 2 N aq.08 (d. 2. J = 9.62 (dd. 78 %). a 5 M methanol solution of NaOMe (10 μl.48 (m. Synthesis of 9-{1-[4-(4-Carboxybutyloxy)-2-methylphenyl]}-6-hydroxy-3H-xanthen-3-one mono(2’. The residue was diluted with water and extracted with CH2Cl2 three times.4. 595.26019. J = 8.7 Hz. 53.6 Hz). 763. The residue was subjected to reverse-phase preparative TLC (RP18W) with acetonitrile/water (1:1) as the eluent to give 21 as an orange powder (15. 595. . 7. 3H).06 (m.78 (m.70-6. 1.3H). 28. J = 2.4 Hz.6 Hz).2. 2.40 (t.6-tetra-O-acetylα-D-galactopyranosyl bromide (100 mg.13 (s.02 (s. 3.92-1. 2. Synthesis of 9-{1-[4-(4-methoxycarbonylbutyloxy)-2-methylphenyl]}-6-hydroxy-3Hxanthen-3-one Mono-β-D-galactopyranoside (19) To a solution of 18 (28 mg.99 (dd.8 Hz.7 Hz. 4H). found. 3.26 (d. CDCl3): δ 7. 7. 2. 2H. 6.74 (m.3’.8 Hz. found. 1H NMR (300 MHz. 1H). 5. 2.3 Hz.10 (m. then extracted with CH2Cl2. 4.55. 3H). 1H. 3H). 1H.83. CD3OD): δ 7. 3. 78 %).8 Hz).10 (t. 417.57 (dd.049 mmol) in methanol/water (1. dried over Na2SO4 and evaporated. 21. 4. The Amberlite IR-120 was filtered off. 6. J = 8. The inorganic precipitate was filtered off and the filtrate was concentrated under reduced pressure.6 Hz).7 Hz. 1H).7 mg.4’.84. HRMS (ESI+): calcd for [M+H]+.NaOH 1 ml (2 mmol) was added.47 (d.2 Hz).1H).19 (d. and the filtrate was evaporated. then neutralized with Amberlite IR-120 (H+). J = 8. 1H). 53 %).1H). 433. 7. available in PMC 2008 September 29.15 (d.22-4.1 Hz. 1H).11 (m.10 (dd.0 Hz.35.21862. 2H).68. 5. Author manuscript. 3.4H). J = 5.5 Hz). 1H.18-5. Synthesis of 9-{1-[4-(4-Carboxybutyloxy)-2-methylphenyl]}-6-hydroxy-3H-xanthen-3-one (20) To a solution of 17 (9. 9. 6. then neutralized with Amberlite IR-120 (H+). 4.03 (d. 37 μmol) in methanol (5 ml). 7.5 Hz. 2.NaCl.0 Hz.5 ml (1 mmol) was added. J = 1.19 (s. HRMS (ESI-): calcd for [M-H]-.10 (d. 2H). 3.07 (s.25 mmol) in dry dimethylformamide (1 ml) was stirred at room temperature under argon overnight. J = 3.37 (d. 3H).8 Hz. 34 %). 3H). 433. 3H).4 Hz.6’-tetra-O-acetyl-β-D-galactopyranoside) (18) A mixture of 17 (18.88-3. 6. 1H).4’. 3H).4.39 (d. 6. 4H). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Synthesis of 9-{1-[4-(4-methoxycarbonylbutyloxy)-2-methylphenyl]}-6-hydroxy-3Hxanthen-3-one Mono(2’. The combined organic layer was evaporated.16511. J = 9.0 Hz). 0. 1H NMR (300 MHz. 2H).75 (m.HCl. 3H). 5.16853.03 (d. 103. Page 11 111.0 Hz.88 (m. 1H). The residue was chromatographed on silica gel with AcOEt as the eluent to give 18 as an orange powder (25 mg. 417. 4H).4 mg. J = 2. 3.5 μmol) in methanol (2 ml). 6. 19. 1.03 (s. 0. 7.16 (d. 2. 21.5 ml: 0. 2H).5 Hz. CD3OD): δ 7. The mixture was stirred at room temperature for 4 hours.21794.2 mg.1H). 7. J = 2.13084.6’-tetra-O-acetyl-β-D-galactopyranoside) (21) To a solution of 18 (37. CD3OD): δ 7.54. 7. 33.70 (s. 67.1 mg. The mixture was stirred at room temperature for 1 hour. 1H NMR (300 MHz. 1H NMR (300 MHz.92 (d.1H.98 (dd.3. J = 9. 2H). 1. 1H. J = 8. 1H). 4H). 0. 1H.08-6.3 Hz. 1H. 1H). and the filtrate was evaporated. 763.41. found. found. 1. and the residue was chromatographed on silica gel with CH2Cl2-MeOH (100:5) as the eluent to give 20 as an orange powder (7 mg.88 (m. 4. J = 2.NaOH 0. 2H.13381.06 (s.11-7. 2. J = 2. 6.61 (dd. HRMS (ESI+): calcd for [M+H]+.5 ml). and the residue was diluted with H2O.3 mg.45 (m. The mixture was stirred at 0 °C for 30 minutes. 2 M aq.3’. 2. J = 8. 3.16 (m.12 (m. The methanol was evaporated off.11 (d.58.6 J Am Chem Soc. The combined organic solution was washed with water and saturated aq. The Amberlite IR-120 was filtered off.56-5. The residue was subjected to reverse-phase preparative TLC (RP18W) with acetonitrile/water (1:1) as the eluent to give 19 as an orange powder (7. J = 6.80 (m.67 (s. 7. 2 M aq.7 Hz. 7H).Kamiya et al. 3H).25543. 2. J = 9. 1H). 2. 42 μmol).

J = 8. 5. 2.07 (s. 4H).74 (m. 1H. 5. 3.7 Hz. then diluted with H2O.02 (s.11 (t. found. available in PMC 2008 September 29. 3. 675.53 (dd. CD3OD): δ 7.3 Hz). The combined organic layer was concentrated under reduced pressure.9 mg.5 Hz).97 (dd. pH 6. J = 8.00-7. The inorganic precipitate was filtered off and the filtrate was concentrated under reduced pressure. 1H.82-3. 7. J = 2. 1H. Author manuscript. 3. 13. 1H. 1. J = 8. 6.17493. 3H). a solution of bromomethyl acetate (6. 1. J = 9. CDCl3): δ 7.7 Hz. MS (EI): m/z 180 (M+). 603. 6.03 (d. 2.18423.3H).5) and eluent B (acetonitrile) (A/B = 65/35) to give 22 as an orange powder (10. a solution of bromomethyl acetate (68. 3H). 1H.94 (s.02 (m. 2H).2 Hz).64 (m. 1H. 5. 6. 675.5 μl. final (20 minutes): 30 % A / 70 % B) to give 23 as an orange powder (0.N-diisopropylethylamine (22. 1H. 27 mmol) in DMF (100 ml) was stirred at room temperature under argon for 4 hours. 3. quant. 6. 1H. 2H).72.5 mmol) in DMF (100 ml) was stirred at room temperature under argon for 4 hours.2H). 3H). 4H).8 mg. 2. Synthesis of m-methoxycarbonylmethoxytoluene (Bn(1Me)) A mixture of m-cresol (3 g.79 (d. 1H NMR (300 MHz. pH 6. J = 3.94 (d.1 Hz). 6. 6. found. J = 8.0 Hz).6 Hz).5 Hz.19. 2H). 4H).4H). 1H.65 (m.05 (m. 1H.11 (d. 157.53 (dd.00 (dd.5 ml) was added. 603.5 mmol).65 (s.8 μl. 7. J = 7. 30.3 μl. 1H. HRMS (ESI+): calcd for [M +Na]+.). 491. 491. 2H). J =2.62 (s.72 g. found. 690 μmol) and N. 13C NMR (75 MHz. 1H. 27 %).0 Hz).2 μl.5 ml). 3.57.N-diisopropylethylamine (2. 6.6 Hz). Synthesis of 9-{1-[4-(4-acetoxymethoxycarbonylbutyloxy)-2-methylphenyl]}-6-hydroxy-3Hxanthen-3-one mono-β-D-galactopyranoside (22) To a solution of 21 (10 mg. 1H. 5. CD3OD): δ 7. bromomethyl acetate (2. 21.88 (m. Synthesis of m-methoxycarbonylmethoxyanisole (Bn(2Me)) A mixture of m-methoxyphenol (1. 7. HRMS (ESI+): calcd for [M+H]+.8 Hz).67 g.18242.5 Hz.73-6.49. 2.83 (d. 111. 2H.50 (t. J = 8. 6. 2.6 Hz).79-3. 1H. and extracted with ethyl acetate.88 mg.1 Hz. 27.7 mmol). 3H). dried over Na2SO4.2 μmol) in methanol/acetonitrile (1 ml: 2ml). J = 9. 122.13-6.01 (m. 15. washed with water and saturated aq. J = 7.8 mmol). . and Cs2CO3 (18 g.3. 2. and evaporated. 139. 4. 1H.93 (s.64 (m.2. 3.01 (dd. 6H).NaCl. J = 5. washed with water and NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. 4.83 (d. 4. 7.53 (dd. The mixture was stirred at room temperature under argon for 10 hrs.17 (t. 115.2 Hz). 2. 1.9 Hz).2 Hz).99 (s. 4. 1. J = 9. 2.3 Hz).3 Hz).28. 3H). 6.79-6.2 Hz.0 Hz).7 Hz.33 (s.3 Hz).34 g. 3.42 (m.3 Hz). 13 μmol) in dimethylformamide (0.20536. .38 (dd. 52.78 (m.89 (dd. 2. J = 3.90-1. 3H). 3. 97 %). J = 9.5) and eluent B (acetonitrile) (initial: 70 % A / 30 % B. 1H).22. The residue was diluted with CH2Cl2 three times. J = 4.7 μmol) in dimethylformamide (0. The residue was purified by semipreparative reverse-phase HPLC using eluent A (100 mM triethylammonium acetate buffer.58 Hz). J = 2.14. The inorganic precipitate was filtered off and the filtrate was concentrated under reduced pressure. 65.41. 1H NMR (300 MHz. 6.68 (m. 1H.20359. J = 6. 1H).84 (m.37 (d. 3H. 6.17 (m.72-6. The residue was purified by semi-preparative reverse-phase HPLC with a linear gradient of eluent A (100 mM triethylammonium acetate buffer. 1. 2. 129. 1H). 6.02 (dd. 2. then concentrated under reduced pressure. The mixture was stirred at room temperature under argon overnight. bromomethyl acetate (4.48 (s. 2H). 130 μmol) in acetonitrile (2ml) was added slowly. 1. 4H).0 Hz). 1H.53 (dd. HRMS (ESI+): calcd for [M+Na]+. CDCl3): δ 169. Page 12 Hz). 1H NMR (300 MHz.Kamiya et al. 69 μmol) and N.59-6. 1H.4H). J = 2. 3.17059.3 Hz). 55.47.84 (m.6 Hz.28 (d. Synthesis of 9-{1-[4-(4-acetoxymethoxycarbonylbutyloxy)-2-methylphenyl]}-6-hydroxy-3Hxanthen-3-one (23) To a solution of 20 (2. The residue was chromatographed on silica gel with CH2Cl2 as the eluent to give Bn(1Me) as a colorless clear oil (5 g. 2H.2 mmol).64.76 (m. 17. J = 8. 2H). 2. The residue was diluted with CH2Cl2 three times. and Cs2CO3 (9 g.

9. Synthesis of m-methoxycarbonylbutyloxytoluene (Bn(3Me)) A mixture of m-cresol (1 g. Fiering S. Goodfellow M. CDCl3): δ 7. 33. and evaporated. Rubenstein JL. Zentralbl Bakteriol 1994. Magee JG. Page 13 saturated aq.161:77–83. 10.27. The inorganic precipitate was filtered off and the filtrate was concentrated under reduced pressure. 1H. 4.61. Robson IS.82. from the Ministry of Education. 16651106. 52. Center for Cancer Research.08. Link G. [PubMed: 7851157] 11. Science and Technology of the Japanese Government.50:1–6.98 g. Sturm KS.. N. [PubMed: 7940758] 7. 121.74-6. Anal Biochem 1985.83. [PubMed: 3128790] 9. dried over Na2SO4.78 (m. Nicolas JF. Fiering S. 3H). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Supplementary Material Refer to Web version on PubMed Central for supplementary material. 3.NaCl. CDCl3): δ 7. 3.30:336– 340. Culture.93:10315– 10320. and by grants from Hoansha Foundation to T. Hershko CJ. 106.78 (s. Fields S. 16370071 and 16659003) to T. 2H).43.85:2603–2607. 101. 160.280:476–87. [PubMed: 8816797] 6. The residue was diluted with CH2Cl2 three times. 3.36. Glickstein H. 2H. Acknowledgements This study was supported in part by the Intramural Research Program of the NIH. Herzenberg LA. 2. 2. Parks DR. N. Fattaey A. N. Ullman EF.68-6. [PubMed: 1959571] 4. Proc Natl Acad Sci U S A 1963. Brachmann RK. [PubMed: 3102226] 13. 2H. 99%). Immunol Methods 1982.92.Kamiya et al. 1H NMR (300 MHz. [PubMed: 13975398] J Am Chem Soc. and Cs2CO3 (6 g.5 mmol) in DMF (50 ml) was stirred at room temperature under argon for 4 hours. 2H).9 Hz). Yang S. [PubMed: 8293887] 12.12:291– 301. James AL. [PubMed: 2547163] 5. Baruch D. Proc Natl Acad Sci U S A 1996a. 5-bromovaleric acid methyl ester (1.24.60. 1H. research grants (Grant Nos.33 (s. National Cancer Institute.53 (m.57-6. 4H). 6. Perry JD.30. Dev Biol 1994. Minnikin DE. Harlow E.1 mmol). 13CNMR (75 MHz. Orr KE. The residue was chromatographed on silica gel with CH2Cl2 as the eluent to give Bn(2Me) as a colorless clear oil (2. 65. J = 5. 129.54:297–307.. [PubMed: 8061408] 8. Sports.68 (s.86-1. dried over Na2SO4. research grants (grant Nos. and from Kowa Life Science Foundation to Y. Hopkins N.80 (m. Cabantchik ZI. Trends Genet 1994. U. 18. [PubMed: 1905992] 10. 6.0 Hz).73:440–450. Chun J. Konijn AM. 28.29. 51.08.146:211–219.30. Nicolas JF. U. 1H). and evaporated. Armstrong L. CDCl3): 173. MS (EI): m/z 222 (M+). Cytometry 1991. 3H). Tan S-S. Sanes JR. 2.24. Nature 1989. [PubMed: 10792658] 14. Armenta R. Chilvers K. 158. Gibbons I. 3H). washed with water and saturated aq. 111. 1.34. 3H). and 16689002) to Y. 21. 98%). Nolan GP. Proc Natl Acad Sci USA 1988. 13C NMR (75 MHz.2 mmol). Rotman B. Cytometry 1994. 1H NMR (300 MHz. Zderic JA. CDCl3): δ 169.43. 6.18 (t. Tarnowski T. 55.45 (m.5:3133–3142. U. Berger CN. Lin S. Boeke JD. Hamid ME.66 g. Levy R. [PubMed: 3922243] 3.10:286–292.1 Hz). Lett Appl Microbiol 2000.17:216–223.50-6.. 2H). 1H).96 (t.80 (s. EMBO J 1986. 3. .41 (t. Herzenberg LA. 67.74 (m.45. J = 7. 139. Roederer M. References 1. Nolan GP.19. 21.89.340:245–246. J = 7.16 (t.7 Hz). 158.NaCl. Fields S.65. Exp Parasitol 1991. Micklem DR. 115.24. 13NP0401) to Y. 13557209. and a grant for the Advanced and Innovational Research Program in Life Sciences to T. 129. MS (EI): m/z 196 (M+). available in PMC 2008 September 29. The residue was chromatographed on silica gel with CH2Cl2 as the eluent to give Bn(3Me) as a colorless clear oil (2 g. Vidal M. Sternglanz R. Edelstein M.95. Song OK. Author manuscript. a Grant-in-Aid for Creative Scientific Research (No. 6. J = 8.61 (s. 107.

Tsien RY. Schellingerhout D. Perry JD. Hellström I. Paeker CA. Togashi K. Anal Biochem 1998. Ishimori T. [PubMed: 15796553] 27.91:1118–1130. MacDonald RC. Takeda A. Ahrens ET.279:84–88. Parker JA. Haugland RP. Louie AY. Saulnier MG. Mere L. Nagano T. Kingsley SD. Kanda K.83:317–325. Kamiya M. Miura T. Bawendi MG. Dutko FJ.127:4888–4894. Dor DM. Page 14 15. Ishimori T. Upson RH. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. [PubMed: 11705883] 39. Clinical Cancer Research 2001. Paik RS. Brechbiel MW. available in PMC 2008 September 29. Kim S. Zeng Q. Kobayashi H. Chilvers KF.22:93–97. [PubMed: 14661026] 29.7:3606–3612. Cancer Research 2004. Rakhmanova VA. Saga T.264:8171–8178. J Natl Cancer Inst 1998. Rothbächer U. Saga T. Nagano T. Maeda M. [PubMed: 14996712] 24. Klaubert DH. Brechbiel MW. Author manuscript. Kim DE. . Kikuchi K. J Am Chem Soc 2005. Tung C-H. Ueno T. Nakayama A. [PubMed: 11744170] 35. [PubMed: 16554835] 26. [PubMed: 10700150] 25. [PubMed: 12848574] 33.273:41–48. Choyke PL. Konishi J. [PubMed: 11456921] 32. Tanaka K. Nuclear Medicine and Biology 2003. Ryan KA. Proc Nati Acad Sci USA 1988.8:607–612. Burres N. Soltesz EG. [PubMed: 2414365] 21. [PubMed: 10452797] 19.123:2530–2536. Nakamura H.3:45–48. Fraser SE. Corey PF. Weissleder R.85:587–600. Biddlecom WG. Nat Biotechnol 2004. Konishi J. Koyama Y. Alauddin MM. [PubMed: 1725950] 22. Konishi J. Krutzik PO. Frangioni JV. [PubMed: 9514778] 16. Hellström KE. Schreiber GJ. Clinical Cancer Research 2003. Whitney M. Rees WT. [PubMed: 9428779] 37. Cohn LH. Kao JPY. Anal Biochem 1991. Louie AY.18:321–325. [PubMed: 8297011] 17. Lee J. Manabe S. Senter PD. 30. Shah K. Roemer K.3:295–301. Jacobs RE. Mihaljevic T. Sun WC. Fukuzumi S.9:3756–3762. Bernardo M.30:261–265. Ohkubo K. [PubMed: 9417030] 31. Fujibayashi Y. Kobayashi H. Tsuji A. Umezawa N. Maeda M. Sato N. [PubMed: 14506168] 38. Gee KR. Bhalgat MK. Science 1998. Angew Chem Int Ed Engl 1991. Tsien RY. Sakahara H. James AL. Wehrman TS.199:238–42. [PubMed: 16867223] 36. Feng L. Knapp TE. Latham KA. Kamiya M. Arakawa H. Advanced drug delivery reviews 2001. 34. Mitsui T. Arano Y. Analyst 1960. Nagano T. Yao Z. Zhang M. Krishna MC. Laurence RG. Nolan GP. Mamede M. Urano Y. J Am Chem Soc 2003. Meade TJ. Urano Y. J Appl Microbiol 2001. J Boil Chem 1989. Miura T. Minta A. Degenfeld Gv. [PubMed: 12745017] 23. Sato N.257:234–237. Reed RH. Kobayashi H. Springer CJ. Takayasu S. Hirose K.53:247–264.215:24–30. Higuchi T.30:1646–1648. Blau HM. Brown JP. Hüber MM. Nakamoto Y. Tsuji A.64:1579–1583. Urano Y. Neoplasia 2006. 28. De Grand AM. J Immunol Methods 1985. Tanaka K. Moats R. Gynecol Surg 2006.125:8666– 8671. 20. Hama Y. Trimmer RW. Senter PD. Conti PS. Anal Biochem 1993. Zlokarnik G. Meade TJ. Anal Biochem 1999. Nat Methods 2006.85:4842–4846. Hirschberg DL. Young DC. J Am Chem Soc 2001. [PubMed: 11851821] 18. Urano Y. Nat Biotechnol 2000. Shahinian A.90:25–29.Kamiya et al. Lim YT. Saga T. Higashi T. Negulescu PA.

4. Author manuscript. and 0. pH 7. A clear decrease of fluorescence was observed in the case of cells incubated with TG-βGal. confocal fluorescence images and DIC images were captured before (Upper) and after (Lower) washing with PSS. . available in PMC 2008 September 29. containing 150 mM NaCl. 2 mM CaCl2.Kamiya et al.1 % DMSO as a cosolvent for 2 hours. while little change was observed in the case of cells incubated with AM-TG-βGal.1% glucose (PSS). Fluorescence microscopic imaging of β-galactosidase activity in living cells with TG-βGal and AM-TG-βGal and comparison of their intracellular retention. 1 mM MgCl2. HEK293 cells expressing βgalactosidase were incubated with a 10 μM solution of TG-βGal (a) or AM-TG-βGal (b) in physiological salt solution. Page 15 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. 5 mM HEPES. 4 mM KCl. Then. Figure 1. and 0.

. J Am Chem Soc.1 % DMSO as a cosolvent. Page 16 NIH-PA Author Manuscript Figure 2. containing 0. available in PMC 2008 September 29.Kamiya et al. pH 7. and 3Me in 100 mM sodium phosphate buffer. NIH-PA Author Manuscript NIH-PA Author Manuscript Fluorescence change of 1Me. 2Me.4. Author manuscript. respectively. 2Me. 3Me upon reaction with esterase. Porcine liver esterase (10 units) was added at 0 minute to solutions of 1Me. Excitation and emission wavelengths were 491 nm and 510 nm.

Excitation and emission wavelengths were 492 nm and 509 nm.4. The fluorescence increase of 1 μM AM-TG-βGal in 100 mM sodium phosphate buffer.Kamiya et al. containing 1 mM MgCl2.1 % DMSO. 14. Author manuscript. 0. . In vitro reaction of AM-TG-βGal with β-galactosidase. available in PMC 2008 September 29. pH 7.3 mM 2-mercaptoethanol. Figure 3. and 6 U of β-galactosidase at 37 °C is shown. respectively. Page 17 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc.

Author manuscript. Data for previously reported TokyoGreen derivatives26 and the newly developed 1Me. Relationship between the oxidation potential of the benzene moiety and Φfl of the anion form (red) and neutral form (blue).Kamiya et al. Figure 4. The curve represents the best fit to the Marcus equation. available in PMC 2008 September 29. Page 18 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. 2Me. and 3Me are depicted on the same graph. .

lung (Lu). kidney (K). muscle (Mu). Avidinβ-galactosidase (100 μg) in PBS(-) was injected intraperitoneally into a SHIN3-implanted mouse. available in PMC 2008 September 29. heart (H). In vivo targeting of avidin-β-galactosidase to intraperitoneal SHIN3 tumors in mouse. and after 20 hours the mouse was sacrificed. Page 19 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 5. spleen (Sp).Kamiya et al. J Am Chem Soc. bone (B)) were removed and stained with X-Gal for 2 hours. intestine (I). . The main internal organs (tumor (T). Author manuscript. liver (Li). mesentery (Me). stomach (St).

spleen. K. following pretreatment with avidin-β-galactosidase. Tumors (Arrows) were clearly visualized with a high tumor-tonontumor signal ratio. lung. Sp. following pretreatment with avidin-β-galactosidase. Author manuscript. Page 20 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 6. Strong fluorescence signals reveal tiny peritoneal tumor nodules (Arrows). Strong fluorescence was observed in the J Am Chem Soc.Kamiya et al. (a) Tumors were imaged with AM-TG-βGal. (c) Magnified image of a peritoneal tumor. . Li. kidney. St. tumor. (d) Fluorescent microfoci as small as 200 μm in diameter can be seen by using fluorescence microscopy. available in PMC 2008 September 29. H. mesentery. Lu. intestine. stomach. T. Ex vivo fluorescence imaging of SHIN3-targeted β-galactosidase activity with AM-TGβGal or TG-βGal. liver. heart. (b) Ex vivo fluorescence imaging of the main internal organs of the mouse. Me. I. (e) Tumor imaging with TG-βGal.

available in PMC 2008 September 29. The unmixed fluorescence images (Left) and white light images (Right) of (a). NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. owing to the rapid leakage of its fluorescent product. Page 21 peritoneal cavity. To obtain the unmixed fluorescence images. NY). USA). this was unlikely to have been confined to tumors. MA. spectral division was performed using the autofluorescence of intestine (Yellow) and the fluorescence at the tumor (Green). Melville. The fluorescence image (Left) and DIC image (Right) of (d) were captured with a BX51 microscope (Olympus USA. however..Kamiya et al. (c) and (e) were captured with a Maestro™ In-Vivo Imaging System (CRI Inc. Author manuscript. (b). Woburn. .

Author manuscript. Page 22 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 7. NIH-PA Author Manuscript J Am Chem Soc. before and after washing with PBS.Kamiya et al. . available in PMC 2008 September 29. Fluorescence images of SHIN3 tumors removed from a mouse after incubation with TG-β Gal (a) or AM-TG-βGal (b).

Avidin-β-galactosidase was intraperitoneally injected into SHIN3-implanted mouse. Distribution of β-galactosidase activity in the removed tumor. . then AM-TG-βGal was administered. available in PMC 2008 September 29. Author manuscript. Page 23 NIH-PA Author Manuscript Figure 8.Kamiya et al. The tumor was removed and stained with X-Gal for 1 hour. NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc.

which is well retained in the cells without loss of fluorescence. Author manuscript. Scheme 1. with β-galactosidase. available in PMC 2008 September 29. (b) Newly developed β-galactosidase substrates bearing an esterase-sensitive methylester. and is further hydrolyzed by intracellular esterase to the free carboxylate. Reaction schemes of our fluorescence probes with β-galactosidase and intracellular esterase. (c) Our newly developed fluorescence probe. 2Me-βGal. (a) Reaction of our previously reported β-galactosidase probe. TG-βGal. which shows a large fluorescence increase upon reaction with β-galactosidase. Page 24 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc.Kamiya et al. AM-TGβGal. 1Me-βGal. and 3Me-βGal. .

Reagents.4. (e) 2. Synthetic scheme of 1Me-βGal. imidazole. MeOH.3. (d) Methyl bromoacetate. DMF.6-tetra-O-acetyl-α-D-galactopyranosyl bromide. (a) TBDMS-Cl.6-Bis-(tbutyldimethylsilanyloxy)xanthen-9-one.NaOH. DMF. THF. . Page 25 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. THF. (f) NaOMe. (b) 3. Author manuscript. (c) TBAF. Cs2CO3. DMF. MeOH. Scheme 2. t-BuLi. Cs2CO3. (g) aq. available in PMC 2008 September 29.Kamiya et al.

Cs2CO3. Page 26 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. . MeOH. t-BuLi. 2) MeI. MeOH. K2CO3.6-tetraO-acetyl-α-D-galactopyranosyl bromide. Cs2CO3. (a) 1) TsCl. DMF. THF. MeOH. Author manuscript. (h) NaOMe. Scheme 3.4. (e) TBAF.6-Bis-(t-butyldimethylsilanyloxy)xanthen-9one. acetone. (g) 2. THF. (f) Methyl bromoacetate. imidazole. (c) TBDMS-Cl.Kamiya et al. available in PMC 2008 September 29. (d) 3.NaOH. (i) aq. DMF. DMF.NaOH.3. Synthetic scheme of 2Me-βGal. Reagents. (b) aq.

(c) NaOMe. Reagents.4. NIH-PA Author Manuscript NIH-PA Author Manuscript Synthetic scheme of 3Me-βGal.6-tetra-O-acetyl-α-D-galactopyranosyl bromide. Cs2CO3. (d) aq.Kamiya et al. Cs2CO3. .NaOH. J Am Chem Soc. Author manuscript. MeOH. DMF.3. DMF. Page 27 NIH-PA Author Manuscript Scheme 4. available in PMC 2008 September 29. (a) Methyl bromovalerate. (b) 2. MeOH.

NIH-PA Author Manuscript NIH-PA Author Manuscript J Am Chem Soc. Page 28 NIH-PA Author Manuscript Scheme 5. DIEA. DMF. Synthetic scheme of AM-TG-βGal. available in PMC 2008 September 29. (a) aq. .NaOH. MeOH/MeCN. (c) Bromomethyl acetate. Author manuscript. DIEA. Reagents.Kamiya et al. MeOH/H2O. (b) Bromomethyl acetate.

85 0.85 0. 2. Data for 1Me.0. 2Me. 3Me-βGal.009 0.83 0. and AM-TG-βGal were measured in 100 mM sodium phosphate buffer. Compound 1Me-βGal 2Me-βGal 3Me-βGal AM-TG-βGal 1Me 1 2Me 2 3Me 3 AM-TG a Absorption maximum (nm)a 454 452 452 453 492 491 494 494 491 491 491 Emission maximum (nm)a 515 527 512 518 511 510 516 515 510 510 510 Fluorescence quantum yieldb 0. pH 9. 2Me-βGal. pH 7.Kamiya et al.86 Fluorescence enhancementc 46 410 370 470 - NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Data for 1Me-βGal. 1.1 % DMSO as a cosolvent. available in PMC 2008 September 29. 3.32 0. b For determination of the fluorescence quantum yield. Page 29 Table 1 Absorption and fluorescence properties of the newly developed β-galactosidase substrates and their esterase and/or βgalactosidase-catalyzed hydrolysis products. containing 0.004 0. and AM-TG were measured in 100 mM sodium phosphate buffer. Author manuscript. fluorescein in 100 mM aq. J Am Chem Soc.67 0.069 0. 3Me. .005 0.85) was used as a fluorescence standard.39 c Fluorescence enhancement represents the ratio of fluorescence intensity at 509-511 nm before and after hydrolysis by β-galactosidase.1 % DMSO as a cosolvent.4.85 0. containing 0. NaOH (0.

d.d. and 3Me-βGal. not determined. 2Me βGal..2207 -0. and 3MeβGal. Page 30 Table 2 Oxidation potential and calculated HOMO energy level of the benzene moiety of 1Me-βGal.d.2107 a All data were measured in 100 mM TBAF MeCN.62 -0.57 -0. Benzene moiety Oxidation potential (V vs SCE)a m-methoxytoluene 1. 2Me βGal.2152 Bn(2Me) 1. HOMO (hartrees) b NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript -0.2156 -0.2141 n = 1: Bn(1Me) n = 2: n = 3: n = 4: Bn(3Me) 1. and Bn(3Me) correspond to the benzene moiety of 1Me-βGal. Author manuscript. Bn(2Me). available in PMC 2008 September 29. J Am Chem Soc. with Ag/Ag+ as a reference electrode.2198 -0.Kamiya et al. .77 n. Bn(1Me). respectively.c n.c 1.66 Calcd. b c n. HOMO level was calculated at the B3LYP/6-31G level.

pH 9. Author manuscript.4) b 0. containing 0. Page 31 Table 3 Fluorescence quantum yields of anionic and neutral forms of 1Me.85 0.67 0.1 % DMSO as a cosolvent. available in PMC 2008 September 29.4. Data for neutral form were measured in 100 mM sodium phosphate buffer. For determination of the fluorescence quantum yield. Compound Φfl (pH 9. containing 0.005 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript 1Me 2Me 3Me a b Data for anionic form were measured in 100 mM sodium phosphate buffer. pH 3. .008 0.Kamiya et al.0.85 Φfl (pH 3.13 0.0) a 0.85) was used as a fluorescence standard. fluorescein in 100 mM aq.39 J Am Chem Soc.1 % DMSO as a cosolvent. 2Me. 3Me. NaOH (0.

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