Water-Soluble Glucosyl Pyrene Photosensitizers: An Intramolecularly

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Water-Soluble Glucosyl Pyrene Photosensitizers: An Intramolecularly Synthesized 2-C-Glucoside and an O-Glucoside Takashi Kanamori, Akira Matsuyama, Hidenori Naito, Yuki Tsuga, Yoshiki Ozako, Shun-ichiro Ogura, Shigetoshi Okazaki, and Hideya Yuasa J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02066 • Publication Date (Web): 12 Oct 2018 Downloaded from http://pubs.acs.org on October 13, 2018

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The Journal of Organic Chemistry

Water-Soluble Glucosyl Pyrene Photosensitizers: An Intramolecularly Synthesized 2C-Glucoside and an O-Glucoside Takashi Kanamori,† Akira Matsuyama,† Hidenori Naito,† Yuki Tsuga,† Yoshiki Ozako,† Shun-ichiro Ogura,† Shigetoshi Okazaki,‡ and Hideya Yuasa*,† †

School of Life Science and Technology, Tokyo Institute of Technology, J2-10 4259 Nagatsuta,

Midoriku, Yokohama 226-8501, Japan ‡Department

of Medical Spectroscopy, Preeminent Medical Photonics Education & Research Center,

Hamamatsu University School of Medicine, Handayama 1-20-1, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan.

3O 2

hν 1O 2

3O 2

hν 1O 2

ABSTRACT: Prevalent photosensitizing agents for photodynamic therapy (PDT) suffer from their relatively large molecular weights causing photodermatosis. In this regard, low molecular weight pyrene could be an efficient photosensitizer except for its extreme hydrophobicity. To tackle the insolubility of pyrene, we synthesized 1-carboxypyren-2-yl C-glucoside 4 by a tethered C-glucosylation and 1pyrenylmethyl O-glucoside 5 by a simple O-glucosylation. Compounds 4 and 5 showed modest water solubilities of 72 and 47 µg/mL, respectively. Whereas compound 4 partially underwent a cyclization reaction at pH 3 to give the corresponding δ-valerolactone 15b in 31% after 24 h, it is stable at pH 5~9 for at least a week. The 1O2-producing photosensitizabilities of 4 and 5 were sufficient to apply to PDT. Although compound 5 was uptaken by HeLa cells and showed a good PDT activity, compound 4 showed neither a sufficient cell-uptake nor PDT effect. The binding modes of compounds 4 and 5 to concanavalin A were specific and unspecific, respectively. These results demonstrate that compounds 4 and 5 are within a pharmacologically acceptable range as oral drugs, and could be a fluorescence imaging probe for α-glucose/mannose receptors and a photosensitizing agent for PDT, respectively. ■ INTRODUCTION Pyrene has established the status as a chromophore applicable to chemical sensors,1 probes for micelle

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and cell membrane properties,2 organic light-emitting diodes,3 ligands for luminescent coordination complexes,4 surface-coating materials for nanoparticles,5 and photosensitizers (PSs) producing singlet oxygen (1O2).6 Usefulness of pyrene in such various areas comes partly from its peculiar photophysical properties such as capabilities of excimer formation,7 a long fluorescence lifetime,8 and clear vibrational progressions in the fluorescence spectra.9 The photosensitizability of pyrene is ascribed to the relatively long lifetime (about 400 ns) of the singlet excited state (S1), which enables an electron exchanging reaction with oxygen molecule to produce singlet oxygen in a quantum yield of 0.71.6 The fine photosensitizability together with the low molecular weight (202.25) is fascinating as a photosensitizing agent for photodynamic therapy (PDT), since all the available PDT agents suffer from their relatively high molecular weights of more than 500,10 which prohibit oral administration11 and cause long retention in blood12 and then photodermatosis. However, application of pyrene as a PDT agent might have been hindered by its hydrophobic, bioincompatible character. Although pyrene may be misrecognized as much toxic as benzo[a]pyrene, a popular carcinogen, it is actually only 0.1% as toxic13 and a modification of pyrene with hydrophilic groups may lead to an even safer PS.14 Water solubilization of the extremely hydrophobic pyrene, which dissolves in water at not more than 0.16 µg/mL,15 would be a first step to its application in medicine. The most promising method for water solubilization of aromatic compounds is sulfation. The resulting arenesulfonic acid salts are expected to be very soluble in water. However, the use of this reaction to solubilize pharmaceutical drugs is controversial against the risk of contamination of alkyl sulfonates, genotoxic impurities, during their syntheses.16 Furthermore, a tendency of sulfated drug compounds to be quickly excreted from cells and tissues before exerting bioactivity can be another problem.17 We therefore focused on the use of glucose, a milder hydrophilic group than sulfate, for water-solubilization of pyrene. Glucose may become a ligand targeting cancer cells, because cancer cells tend to overexpress glucose transporters (GLUT).18 We therefore designed C- and O-glucosides of pyrene derivatives, the former aiming at the tolerance against endogenous glucosidases that would cleave the biocompatible sugar moiety to revive the innate toxicity of pyrene. C-Glycosylation of pyrene has been reported only with furanosyl donors for the synthesis of pyrenyl C-ribofuranosides as stackable surrogates of nucleotides.19 The absence of pyrenyl Cglycopyranosides in the literature may be not only due to indifference to the structure but also because pyranosyl donors are less reactive than the corresponding furanosyl donors. We thus planned a tethered 2-C-glucosylation of the glycosyl donor 1 as shown in Scheme 1, in order to promote the Friedel-Crafts type glycosidation reaction by increasing the apparent concentrations of the donor and acceptor. The oxidation at the 1-methylene part of the resulting 2-C-glucoside 2 and ring-opening of the lactone 3


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would produce the 1-carboxypyren-2-yl C-glucoside 4. Although the methylene-tethered Cglycosylations of single ring arenes have been studied extensively, none of those of fused polycyclic arenes have been reported except for the naphthyl C-glycosides obtained as side-products.20 Since nontethered, intermolecular C-glycosylations of pyrene via Friedel-Crafts type reactions generally give 1-Cglycosyl pyrene,21 the 2-C-glucosyl pyrene 2 represents unusual regioselectivity.22 The 1-pyrenylmethyl O-glucoside 5 would be obtained by a simple chemical O-glucosylation of 1-pyrenemethanol. In this study, we would assess the potential of the synthesized C-glucosyl and O-glucosyl pyrene derivatives as a photosensitizer for PDT and a fluorescence imaging probe.

+ 1

2

3

4

5

Scheme 1. Strategies for the synthesis of 1-carboxypyren-2-yl C-glucoside 4 and 1-pyrenylmethyl O-glucoside 5.

■ RESULTS AND DISCUSSION Synthesis and Conformational Analyses of Glucosyl Pyrene Derivatives. Tethered 2-Cglucosylation of pyrene was performed with a 2-O-(1-pyrenemethyl)-glucopyranosyl donor 12. Synthesis of 12 was started from the peracetyl-β-glucopyranose 6, which was transformed to the known 1,2-orthoacetate 7 (Scheme 2).23 Without purification, the acetyl protecting groups in 7 were converted into allyl groups to give the compound 8 in 64% yield from 6. Thioglycosidation of the compound 8 gave the 2-O-acetyl-thioglycoside 9 in 57%. The 2-O-acetyl group of 9 was removed to give the compound 10 in 97%. 1-Pyrenylmethyl group was attached at the 2-OH group of the compound 10 to give the compound 11 in 96%, which was then oxidized with m-chloroperoxybenzoic acid (mCPBA) at the sulfur atom to give the sulfoxide 12 in 97%. Intramolecular C-glycosidation at C-2 of the pyrenyl group of 12 was carried out with trifluoromethanesulfonic anhydride (Tf2O) as a promoter to give the Cglycoside 13 in 25%. We thus converted the allyl groups of 13 into acetyl groups, obtaining the acetate 14 in 46%. The ring methylene group attached to the pyrene group of 14 was oxidized with KMnO4 to give the lactone 15a in 51% yield.



b.

a.

c.

7

6

f.

12

9

h.

13

e.

d.

8

g.

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10

i.

14

11

k.

j.

15a (R = Ac) 15b (R = H)

4

Scheme 2. a. 30% HBr-AcOH/CH2Cl2 2,6-dimethylpyridine (4.0 eq), tetrabutylammonium bromide (0.4 eq)/MeOH7.6 eq), AllCl (4.8 eq)/DMF (64%), c. EtSH (2.1 eq), BF3 CH2Cl2, b. NaOMe (0.3 2 (1.1 eq)/CH2Cl2 (57%), d. NaOMe (0.4 eq)/MeOH (97%), e. NaH (3 eq), PyCH2Cl (1.2 eq)/DMF (96%), f. mCPBA (1 eq)/CH2Cl2, (97%), g. Tf2O (1.5 eq), 2,6-di-tert-butyl-4-methylpyridine (4.0 eq)/CH2Cl2 (25%), h. Rh(PPh3)3Cl (0.6 eq), p-TsOH (1 eq)/CH2Cl2triethylamine/CH2Cl22O-pyridine (46%), i. KMnO4 (6 eq), benzyltriethylammonium chloride (6 eq)/CH2Cl2 (51%), j. NaHCO3/ MeOH-CH2Cl2 (87%), k. NaOH/H2O (100%).

The regio- and stereoselectivity of the tethered C-glycosidation of 12 was confirmed by structural analyses of the lactone derivative 15a by NMR techniques (Figures 1A, S1~S4): (i) There are HMBC cross peaks indicating the proposed connections of pyrene and glucose: C3/H1’, H3/C1’, and C11/H2’ (Figure S3). C11 also shows a weak four-bond HMBC cross peak with H3, which is most likely due to the W-shaped arrangement of C11-C1-C2-C3-H3. (ii) NOESY cross peaks (Figure S4) are observed for H3/H1’ and H3/H5’, indicating the proposed orientation of pyrenyl plane with H3 located between H1’ and H5’. The α-configuration of the C-glycoside is also supported by the absence of NOEs between H1’ and the axial sugar ring protons (H3’ and H5’). Whereas most of the NMR results are supportive of the suggested structure, the J-value between H1’ and H2’ might be somewhat too large (6.6 Hz) for an αglucopyranoside. It should be noted that a pyrenyl-C-2-deoxyribofuranoside has a larger cis H1-H2a coupling constant (J = 7.2 Hz) than that (J = 5.0 Hz) of the corresponding phenyl-C-furanoside while the corresponding trans H1-H2b vicinal protons maintain an identical J-value of 10.8 Hz.19a Thus the relatively large J-values for these cis vicinal protons are ascribable to the increased electronegativity of pyrene as compared to benzene.24 In addition, the sugar ring conformation of 15a may be distorted at the anomeric carbon to some extent by the constrained lactone, causing a decrease of the H1’-C1’-C2’-H2’ dihedral angle and thus a shift of the J-value upwards along the Karplus cosine curve.25 It is also noteworthy that H10 on the pyrenyl ring is abnormally at a high field (9.60 ppm) probably because the lactone carbonyl group is in a close proximity to H10 causing a positive internal magnetic field effect by the carbonyl ring current.


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a.

b.

6

µg/mL,

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16

5

µM)

µg/mL

µg/mL

µM)

µg/mL

µM)

Figure 2. HPLC analysis of the cyclization reaction

δ-vale


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This cyclization reaction was confirmed by a HPLC analysis of the solution of 4 left to stand at pH 3 for 30 h (Figure 2). The yield of 15b was determined at a reaction time of 24 h to be 31%. The cyclization process could be monitored by absorption spectra, because specific absorptions of 15b appeared in the wavelength region between 350 and 450 nm (Figure 3). By plotting the absorbance at 396 nm, we confirmed a continuous increase of 15b at pH 3 for 24 h (Figure S13). It was also found that the cyclization reaction did not occur at pH 5~9 within a week, suggesting that the compound 4 is expected to be stable as a drug in physiological conditions (Figure S14). Photophysical Properties. Data for photophysical properties of pyrenyl derivatives are listed in Tables 1 and S2. The absorption spectrum of 4 was very similar to that of 1-pyrenylcarboxylic acid (PyCOOH) as shown in Figure 3, indicating that the attached glucose and sodium salt have virtually no effects on the structures of excited states. In contrast, the lactone derivative 15b has largely red-shifted bands at 393, 372, and 362 nm, which are unexplainable simply by the ester formation. Fluorescence spectra of the pyrene derivatives in a citrate buffer indicate that only 15b mainly produces excimer complex as demonstrated by the broad band at 440 nm (Figure 4A). In DMSO, the fluorescence spectrum of 15b includes a small excimer band centered at 447 nm within the right shoulder of a large band centered at 424 nm as demonstrated by the multiple Gaussian fitting (Figures 4B and S17). It is thus suggested that 15b partially existed in DMSO as a charge transfer (CT) complex in a dimer, such as the one in Figure 1B, or in aggregates, affording a CT absorption at 393 nm and an excimer fluorescence at 447 nm. The dimer formation of pyrenes in the ground state (GS) has been thoroughly studied both experimentally and theoretically.29 The GS dimer of a pyrene derivative is estimated to be 100-fold less stable than the excited state dimer (excimer complex) in water.29a Hydrophobic and π-π stacking interactions have been suggested to cause the GS dimer formation. The GS dimers of structurally unrestricted pyrenes have a parallel or antiparallel sandwich structure with a thickness of approximately 0.34 nm.29b,c,d Serial π-π stackings in the direction perpendicular to the pyrene plane produces columnar structures, which would cause insoluble aggregation.30 Thus the NMR, absorption, and fluorescence spectra of 15b are all reasonably interpretable by the formation of dimer and/or larger aggregates. Although the NOESY spectrum of the C-glucosyl pyrene 4 in D2O suggested the formation of a dimer or aggregates to a great extent, no significant excimer fluorescence emissions were observed in the citrate buffer (Figure 4A). The different observations are ascribable to the difference in the solution concentrations, i.e., 4.9 mM for NOESY and 10 µM for fluorescence. The colloidal aggregation formation in high concentrations may be advantageous for a drug, since a high dose exceeding the drug’s solubility may form a dispersed solution without formation of precipitates, enabling delivery to the target.



PyCOOH 4 15b

Absorbance

0.4

362 nm 372 nm 393 nm

0.2

0 250

300

350

400

450

Wavelength /nm Figure 3. Absorption spectra of 4, 15b, and pyrenylcarboxylic acid (PyCOOH) in DMSO (10 µM).

A

B 404 nm

PyCOOH 4 15b

800 600 440 nm 400 200 0 350

400

450

500

550

Wavelength /nm

600

Fluorescence intensity

800

1000

Fluorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Observed (15b) Simulation Peaks 1~3 Peak 4 (Excimer)

424 nm

600 1

400

447 nm

2

200 4 3

0 350

400

450

500

550

Wavelength /nm

Figure 4. (A) Fluorescence spectra of 4, 15b, and pyrenylcarboxylic acid (PyCOOH) (10 µM) in citrate-phosphate buffer (pH 7.0, 1% DMSO). Excitation wavelengths for 4, 15b, and PyCOOH were 342, 363, and 342 nm, respectively. (B) Fluorescence spectrum of 15b in DMSO (orange solid line), the simulated spectrum (black broken line), and Gaussian functions for peaks 1~3 (grey broken lines) and peak 4 (red broken line). The fitting was performed for the wavenumberbased spectrum (Figure S17) and the obtained curves were converted back to the wavelength-based spectra. Excitation wavelengths for 15b was 362 nm.

Fluorescence emission decays of the pyrene derivatives were analyzed by fitting to single exponential equations (Figure S18). The obtained lifetimes (τ) indicate a tendency that pyrenylcarboxylate derivatives (PyCOOH and 4) have shorter lifetimes than pyrenylmethyleneoxy derivatives (PyCH2OH and 5) (Tables 1 and S2). The established theory ascribes the relatively long lifetime of pyrene to the forbidden dipole S0-S1 transition, which is actually the synthesis of two transitions with the opposite dipoles that are canceled out each other.8 It is therefore conjectured that a symmetry break in the π orbitals would result in alleviation of the dipole cancellation (forbiddeness),


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shortening fluorescence lifetime. A carboxylate group is more electron withdrawing than a methyleneoxy group, perturbing symmetry of the molecular orbitals to a greater extent. It is therefore quite reasonable that the symmetrically more perturbed PyCOOH and 4 have shorter lifetimes than those of PyCH2OH and 5. It is interesting to note that very weak absorptions at 382 nm corresponding to the S0-S1 transition can be observed for 4 and PyCOOH, which are scarcely seen for 5 and PyCH2OH (Figure S15A). These observations in the absorption spectra are also ascribable to alleviation of forbiddeness in the S0-S1 transition dipole. Table 1. Selected photophysical properties of pyrene derivatives.a Compound

λmax /nmb

λem /nmc

ΦFd

τ /nse

Φ ∆f

PyCH2OH

341

376

0.28

129

0.25

PyCOOH

342

382

0.35

34

0.14

4

342

381

0.29

70

0.47

5

342

376

0.33

129

0.34

The data except those of the 1O2 quantum yields were measured for the 10 µM solutions in citrate-phophate buffer (pH 7.0, 1% DMSO). bMaximum absorption wavelength. cMaximum fluorescence wavelength with the irradiation at the wavelength of maximum absorption. dFluorescence quantum yield. e Fluorescence lifetime. f 1O2 quantum yield for the solution in H2O (1% DMSO) irradiated at 355 nm. a

Photosensitization of Pyrene Derivaives. The photosensitizability (Φ∆: the relative 1O2 generation rate per standard irradiation power) was determined for 4, 5, PyCH2OH, and PyCOOH in three conditions: 266 nm irradiation (S0-S3) in H2O, 341 nm (S0-S2) in methanol (Table S2), and 355 nm (S0S1) in H2O (Table 1). In H2O, the C-glucosyl pyrene 4 tends to indicate the best Φ∆, 0.44 and 0.47 for S0S3 and S0-S1 excitations, respectively, among the four compounds. This result is probably because the best water solubility of 4 hinders the dimer or aggregation formation that can quench the excited states. This hypothesis is also supported by the higher Φ∆ of the four compounds (0.47~0.67) in methanol, a more soluble solvent. There is also a tendency that carboxylate group increase the 1O2-producing efficiency, e.g., from 0.47 (PyCH2OH) to 0.67 (PyCOOH) and from 0.51 (5) to 0.56 (4) in methanol. This tendency contradicts the supposition described in the introductory section that the relatively long lifetime of S1 allows pyrene to undergo an electron exchanging reaction with oxygen molecule to produce 1O2. Since carboxypyrenes tend to have much shorter S1 lifetime than methyleneoxypyrenes as mentioned above, 1O2-producing efficiencies of the carboxypyrenes, PyCOOH and 4, should be much smaller than those of PyCH2OH and 5 in the S1-driven mechanism alone. It is more likely that the carboxypyrene derivatives react with O2 mainly in a triplet excited state (T1). The short S1 lifetime of carboxypyrene might be due to a substantial S1-to-T1 intersystem crossing rate assisted by a spin-orbit coupling of the carbonyl group. This hypothesis is quite consistent with the facts that the addition of an



Φ∆ values for the four compounds are smaller than the reported Φ∆ of pyrene (0.71)

Figure 5. µM pyrene derivatives (containing 0.5% DMSO) for 2 h. PyCH2OH,


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uptaking of the O-glucosylpyrene 5 (Figure S20). The uptake of 5 by diffusion mechanism is reasonable, since the molecular weight of 5 (394) is less than 500, which is a criterion of oral drugs to penetrate through cell membrane.11 The pyrenylcarboxylate derivatives 4 and PyCOOH were barely uptaken by HeLa cells as opposed to the corresponding neutral analogs 15b and PyCH2OH, respectively, probably owing to the repulsion of the negative charge of the COOH group with that on the cell surface. Photodynamic Therapy Effect of Pyrene Derivatives. The pyrene derivatives 4, 5, PyCH2OH, and PyCOOH were tested for PDT effect against HeLa cells (Figure 6). These compounds indicated no dark toxicity (Figure S21) and 3-min irradiation in the absence of the compounds showed no phototoxicity (Figure 6). Only in the presence of 5 or PyCH2OH, the irradiation resulted in significant cell death with viabilities of about 40%. The result is consistent with the fact that these are the compounds that were well uptaken by HeLa cells. Compound 5 did not decompose in physiological conditions (pH 5~9) for a week. The compound 4 showed no significant PDT effects. 140

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


120 100 80 60 40 20 0

UV− UV– − –

UV+ UV+ − –

UV+ UV+ 4 4

UV+ UV+ UV+ UV+ UV+ UV+ 5 PyMeOH PyCOOH 5 PyCH2OH PyCOOH

Figure 6. Viabilities of HeLa cells showing photodamaging effects in the presence of pyrenemethanol derivatives under an excitation light. HeLa cells were incubated with 150 µM pyrene derivatives (4, 5, PyCH2OH, PyCOOH) for 2 h and after washing with PBS, irradiated for 0 min or 3 min with an ark lamp (>300 nm) with a power at 340 nm of 9.2 mW/cm2. Cell viability was evaluated by MTT assay (n = 27 and 4 for UV- and UV+, respectively).

Recognition of C-Glucosyl Pyrene by ConA. The emission strength in the fluorescence spectrum of 4 was comparable to the pyrene carboxylic acid in a citrate buffer (Figure 4A), demonstrating that the capability of pyrene as an imaging probe was not impaired by the attachment of C-glucoside. The absence of excimer bands suggests that 4 is dispersed as monomers in water. Although the C-glucoside 4 has α-configuration, we were uncertain if this “α”-like C-glucoside is recognized as a mimic of αglucosides by sugar-recognizing proteins regardless of the bulky pyrene aglycon. We thus carried out a binding assay for a lectin, concanavalin A (ConA), by fluorescent depolarization method. The C-glucside 4 indicated a specific binding to ConA with a Kd value of 24 µM (Figure 7A), which is better than that of



methyl α-glucoside (Kd 588 µM)32 by about 25-fold. This is in sharp contrast to the result for the pyrenylmethyl β-O-glucoside 5, which showed a typical unspecific binding: a linear correlation between ConA concentration and the anisotropy change (Figure 7B). This unspecific binding is most likely due to a very weak specific binding of β-glycons, as typically exemplified by that of methyl β-glucoside (Kd 14 mM).32 These results indicate that ConA strictly recognizes the anomeric configuration even of the Cglycoside with a large aglycon and its hydrophobic surface assists the binding of 4. The recognizability of 4 by α-glucoside-recognizing proteins is advantageous for a fluorescence imaging of cancer tissues, because cancer tissues often include the tumor-associated macrophages with lectins to recognize exogenous glycans.33

A

B

0.04 0.03

∆Anisotropy

∆Anisotropy

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0.02 0.01 0 0 10 20 30 40 50 60 70

0.05 0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 0

[ConA] /µM

20

40

60

80 100

[ConA] /µM

Figure 7. Fluorescent polarization anisotropy assays for the binding of 4 (A) and 5 (B) with Con A. The solutions include 0.2 µM ligand (4 or 5), 900 mM NaCl, 1 mM CaCl2, and 1 mM MnCl2 in 100 mM HEPES (pH 7.2). Polarized fluorescence emissions at 395 nm with polarized excitations at 342 nm were analyzed for anisotropy. The Kd value of 24 µM was deduced from the fitting an isotherm equation to the plots. For the unspecific binding, the linear relationship, “∆Anisotropy” = 4×10-4×[ConA] + 8×10-4 (r2 = 0.980), was obtained.

■ CONCLUSIONS The modified pyrene derivatives 4 and 5 showed modest water solubilities of 72 and 47 µg/mL, respectively, which were more than 290-fold that of unmodified pyrene. The photosensitizabilities (the relative 1O2 generation rate per a standard irradiation power) of 4 and 5 were sufficient to apply to PDT. Whereas 4 showed poor results in cell-uptake and PDT assays on HeLa cells, 5 showed much better results. Overall, we found a water-soluble, pyrene-based photosensitizer 5 that is applicable to PDT, but further scrutinization is necessary for the development of 5 as a cancer-targeting PS, because 5 seemed to be uptaken not through GLUT but by a diffusion mechanism. The C-glucoside 4 was boundable to an α-glucoside binding protein, concanavalin A (ConA), with a Kd value of 24 µM, demonstrating a potential as a fluorescence imaging probe for α-glucoside-binding lectins.


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■ EXPERIMENTAL SECTION General. All reagents and materials were obtained from commercial suppliers and used as received. Chemical reagents were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA), Kanto Chemical Co., Inc. (Tokyo, Japan), Wako Pure Chemical Industries Ltd. (Osaka, Japan), and Tokyo Chemical Industry Co., LTD. (Tokyo, Japan). Thin-layer chromatograms (TLC) were performed on precoated silica gel Merck 60-F254 plates and visualized by charring after immersing in 1% Ce(SO4)2·5% (NH4)6Mo7O24·4H2O in 10% H2SO4 solution. Column chromatography was usually performed on Merck Kieselgel 60 (Art 7734) with the solvent systems specified. Where Wako gel C300 or Kanto Silica gel 60N (spherical, neutral) was used, it was specified as C300 or 60N. 1H NMR spectra were recorded at 500 MHz on a Biospin Avance HD or a Varian Unity INOVA 500 spectrometer. Solvent peaks were used as standards in CHCl3 (δ = 7.26 ppm), CHD2(SO)CD3 (δ = 2.50 ppm), and DOH (δ = 4.79 ppm). Chemical shifts are expressed in ppm with reference to the standards. The multiplicity of the signals is abbreviated as follows: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, ddd = doublet of doublets of doublets, br. = broad signal, m = multiplet. 13C NMR spectra were recorded at 126 MHz on a Biospin Avance HD, 126 MHz on a Varian Unity INOVA 500, or 101 MHz on a Bruker Biospin Avance III sepctrometer. Solvent peaks (δ = 77.16 ppm in CDCl3, and δ = 39.52 ppm in (CD3)2SO) were used as standards. High-resolution mass spectra (HRMS) were recorded on a Bruker ESI-TOF MS spectrometer. Optical rotations were determined at room temperature on a Horiba SEPA-200 high sensitive polarimeter with a 1 dm length cell. Absorption spectra were measured on a SHIMAZU UV2600 spectrophotometer with a 1 cm length cell. Fluorescence spectra were measured on a JASCO FP8500 fluorometer with a 1 cm × 0.3 cm micro cell. The citrate-phosphate buffer solutions (pH 3, 6, 7) were prepared by mixing 0.1 M citric acid and 0.1 M Na2HPO4. The borate buffer solutions (pH 8, 9) were prepared by adding NaOH to a 1.0 M boric acid solution and diluting it by 10-fold. Synthesis of 3,4,6-Tri-O-allyl-α-D-glucopyranose 1,2-(Methyl Orthoacetate) (8). To a solution

of 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose (24.19 g, 61.97 mmol) in dry CH2Cl2 (200 mL) was added 30% HBr-acetic acid (120 mL) and the solution was stirred at room temperature for 1 h. The solution was poured into ice-water and extracted with CH2Cl2. The organic layer was dried over MgSO4, filtered, evaporated, and co-evaporated with toluene. To a solution of the residue in dry CH2Cl2 (500 mL) was added dry methanol (15 mL, 370 mmol), 2,6-dimethylpyridine (28.5 ml, 246 mmol), and tetrabutylammonium bromide (8.0 g, 24.8 mmol). The mixture was stirred for 3 days at room temperature and then 3 h at 40 ºC. The mixture was diluted with CH2Cl2 and washed with water. The organic layer was dried over MgSO4 and evaporated. The residue was chromatographed on a silica gel



Page 14 of 25

column (hexane–ethyl acetate, 5:1) to give a syrupy compound 7 (about 18.6 g). To a solution of 7 (13.9 g, 38.4 mmol) in methanol (300 mL) was added 1 M NaOCH3 in methanol (11.7 mL, 11.7 mmol). The reaction mixture was stirred for 50 min at room temperature and evaporated to give a solid compound. The mixture of the compound and 60% NaH (11.6 g, 290 mmol) in dry DMF (130 mL) was stirred at room temperature for 0.5 h. To this mixture was added allyl chloride (15.0 mL, 184 mmol) and it was further stirred at room temperature for 24 h. The reaction mixture was diluted with CH2Cl2 and methanol (15.8 mL, 390 mmol) was added to decompose NaH. The mixture was neutralized with NaH2PO4 (23.4 g, 195 mmol), filtered, and evaporated. The residue was dissolved into CH2Cl2 and washed with sat. NaHCO3 and brine. The organic layer was dried over MgSO4 and evaporated. The residue was chromatographed on a silica gel column (hexane–ethyl acetate, 4:1) to give compound 8 (10.68 g, 64% in 4 steps) as a colorless syrup: [α]D25 +53.3° (c = 0.98 in CHCl3 Rf 0.39 (hexane–ethyl acetate, H NMR (500 MHz, CDCl3, 298K) δ 5.92–5.80 (m, 3H, -CH=CH2), 5.67 (d, J = 5 Hz, 1H, H-1), 5.31–

1

5.19 (m, 3H, -CH=CHH), 5.19–5.06 (m, 3H, -CH=CHH), 4.27 (dd, J = 5, 3 Hz, 1H, H-2), 4.21–4.12 (m, 2H, -OCH2-), 4.12–3.92 (m, 4H, -OCH2-), 3.72–3.64 (m, 2H, H-3, H-4), 3.64–3.56 (m, 2H, H-6a, H-6b), 3.50 (dd, J = 9.5, 4.8 Hz, 1H, H-5), 3.22 (s, 3H, -OCH3), 1.61 (s, 3H, -CH3

13C

NMR (101 MHz,

CDCl3, 298K) δ 134.69, 134.66, 134.4, 121.2, 117.5, 117.3, 117.2, 97.8, 79.0, 76.0, 74.8, 72.5, 72.1, 71.1, 70.5, 69.2, 50.5, 21.4. HRMS (ESI): m/z calcd for C18H28O7Na+: 379.1728 [M+Na]+ 379.1721. Synthesis of Ethyl 2-O-Acetyl-3,4,6-tri-O-allyl-1-thio-β-D-glucopyranoside (9). Compound 8

(985 mg, 2.76 mmol) was dissolved in CH2Cl2 (20 mL) and the solution was cooled to 0 °C. To this solution was added ethanethiol (0.430 mL, 5.81 mmol) and BF3 Et2O (0.365 mL, 2.9 mmol). The reaction mixture was warmed to room temperature and stirred for 3 h. It was then diluted with CH2Cl2 and washed with sat. NaHCO3 and brine. The organic layer was dried over MgSO4 and evaporated. The residue was chromatographed on a silica gel column (hexane–toluene–ethyl acetate, 15:15:1) to give compound 9 (606 mg, 57%) as a colorless syrup: [α]D25 +25.2° (c = 0.94 in CHCl3 Rf 0.44 (hexane–ethyl acetate,

1

H

NMR (500 MHz, CDCl3, 298K) δ 5.96–5.81 (m, 3H, -CH=CH2), 5.32–5.20 (m, 3H, -CH=CHH), 5.20– 5.11 (m, 3H, -CH=CHH), 4.94–4.86 (m, 1H, H-2), 4.35–4.19 (m, 2H, -OCH2-, H-1), 4.16–3.98 (m, 4H, OCH2-), 3.74–3.67 (m, 1H, H-6a), 3.63 (dd, J = 11.1, 4.6 Hz, 1H, H-6b), 3.51–3.43 (m, 2H, H-3, H-4), 3.42–3.35 (m, 1H, H-5), 2.74–2.60 (m, 2H, -SCH2CH3), 2.09 (s, 3H, -OCOCH3), 1.24 (t, J = 7.4 Hz, 3H, -SCH2CH3

13C

NMR (126 MHz, CDCl3, 298K) δ 169.7, 134.9, 134.8, 134.7, 117.3, 117.03, 117.00, 84.0,

83.5, 79.6, 77.6, 74.1, 74.0, 72.6, 71.8, 69.0, 23.9, 21.2, 15.0. HRMS (ESI): m/z calcd for C19H30O6SNa+: 409.1657, [M+Na]+ Synthesis of Ethyl 3,4,6-Tri-O-allyl-1-thio-β-D-glucopyranoside (10). To a solution of


Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


compound 9 (7.97 g, 20.6 mmol) in dry methanol (100 mL) was added 1.5 M NaOCH3 in dry methanol (5.6 mL, 8.4 mmol) and the solution was stirred for 16 h at room temperature. The reaction mixture was evaporated and the residue was chromatographed on a silica gel column (hexane–ethyl acetate, 9:1) to give compound 10 (6.9 g, 97%) as a colorless syrup: [α]D25 −20.6° (c = 1.0 in CHCl3 Rf 0.25 (hexane– ethyl acetate, 4:1) 1H NMR (500 MHz, CDCl3, 298K) δ 6.02–5.84 (m, 3H, -CH=CH2), 5.34–5.21 (m, 3H, -CH=CHH), 5.21–5.12 (m, 3H, -CH=CHH), 4.37 (dd, J = 12.6, 5.8 Hz, 1H, -OCHH-), 4.34–4.25 (m, 3H, -OCH2-, H-1), 4.12 (dd, J = 12.3, 5.8 Hz, 1H, -OCHH-), 4.06 (dd, J = 12.9, 5.6 Hz, 1H, -OCHH-), 4.00 (dd, J = 13.0, 5.7 Hz, 1H, -OCHH-), 3.70 (d, J = 10.9 Hz, 1H, H-6a), 3.66–3.59 (m, 1H, H-6b), 3.45–3.34 (m, 4H, H-2, H-3, H-4, H-5), 2.77–2.65 (m, 2H, -SCH2CH3), 2.47–2.41 (m, 1H, -OH), 1.30 (t, J = 7.4 Hz, 3H, -SCH2CH3

13C

NMR (101 MHz, CDCl3, 298K) δ135.3, 134.9, 134.8, 117.1, 117.03, 116.99, 86.2,

85.7, 79.6, 77.4, 74.1, 74.0, 73.1, 72.6, 69.2, 24.5, 15.5. HRMS (ESI): m/z calcd for C17H28O5SNa+: 367.1551, [M+Na]+

367.1543.

Synthesis of Ethyl 2-O-(1-Pyrenylmethyl)-3,4,6-tri-O-allyl-1-thio-β-D-glucopyranoside (11).

The mixture of 10 (2.0 g, 5.81 mmol) and 60% NaH (697 mg, 17.4 mmol) in dry DMF (60 mL) was stirred for 0.5 h. To this mixture was added 1-(chloromethyl)pyrene (1.75 g, 6.98 mmol) and the mixture was further stirred for 3 h at room temperature. The reaction mixture was then slowly poured into ice water and evaporated. The residue was dissolved in CH2Cl2 and filtered. The filtrate was evaporated and the residue was chromatographed on a silica gel column (hexane–ethyl acetate, 19:1) to give compound 11 (3.11 g, 96%) as a yellow amorphous solid: mp 89– (hexane–ethyl acetate,

[α]D25 +51.3° (c = 0.75 in CHCl3 Rf 0.40

H NMR (500 MHz, CDCl3, 298K) δ 8.62 (d, J = 9.2 Hz, 1H, Pyrene), 8.22–

1

7.98 (m, 8H, Pyrene), 6.03–5.88 (m, 3H, -CH=CH2), 5.67 (d, J = 10.0 Hz, 1H, Pyrene-CH2-), 5.36–5.23 (m, 4H, Pyrene-CH2-, -CH=CH2), 5.22–5.13 (m, 3H, -CH=CH2), 4.48 (d, J = 9.6 Hz, 1H, H-1), 4.39 (dd, J = 12.5, 5.4 Hz, 1H, -OCHH-), 4.34–4.28 (m, 2H, -OCH2-), 4.16 (dd, J = 12.2, 5.9 Hz 1H, -OCHH-)

4.13–4.01 (m, 2H, -OCH2-), 3.77–3.70 (m, 1H, H-6a), 3.65 (dd, J = 10.9, 4.9 Hz, 1H, H-6b), 3.56 (t, J = 8.9 Hz, 1H, H-2), 3.53–3.44 (m, 2H, H-3, H-4), 3.41–3.35 (m, 1H, H-5), 2.90–2.76 (m, 2H, -SCH2CH3), 1.39 (t, J = 7.4 Hz, 3H, -SCH2CH3

13C

NMR (101 MHz, CDCl3, 298K) δ 135.3, 134.9, 131.6, 131.5,

131.4, 131.0, 129.8, 127.9, 127.8, 127.6, 127.5, 126.0, 125.3, 125.1, 124.9, 124.7, 124.3, 117.1, 117.0, 116.5, 86.3, 85.3, 81.8, 79.3, 78.0, 74.5, 73.9, 73.8, 72.6, 69.3, 25.3, 15.4. HRMS (ESI): m/z calcd for C34H38O5SNa+: 581.2334, [M+Na]+

581.2321.

Synthesis of Ethyl 2-O-Pyrenylmethyl-3,4,6-tri-O-allyl-1-sulfinyl-β-D-glucopyranoside (12). To

a solution of compound 11 (3.0 g, 5.37 mmol) in CH2Cl2 (100 mL) was added a solution of

m-

chloroperoxybenzoic acid (1.29 g, 65%~75%, 4.86~5.61 mmol) in CH2Cl2 (10 mL) at −20 °C and the solution was stirred for 2.5 h. The reaction mixture was diluted with CH2Cl2 and it was washed with



Page 16 of 25

aqueous sodium thiosulfate and brine. The organic layer was dried over MgSO4 and evaporated and the residue was chromatographed on a silica gel column (hexane–ethyl acetate, 2:3) to give compound 12 (3.0 g, 97%) as a diastereomeric mixture. One of the diastereomers of 12: mp 112– 0.1 in CHCl3 Rf 0.24 (hexane–ethyl acetate,

[α]D25 −45° (c =

H NMR (500 MHz, CDCl3, 298K) δ 8.44 (d, J = 9.1

1

Hz, 1H, Pyrene), 8.20 (m, 2H, Pyrene), 8.17–8.13 (m, 2H, Pyrene), 8.10–7.99 (m, 4H, Pyrene), 6.08–5.99 (m, 1H, -CH=CH2), 5.98–5.84 (m, 2H, -CH=CH2), 5.62–5.54 (m, 2H, Pyrene-CH2-), 5.39–5.33 (m, 1H, CH=CHH), 5.33–5.24 (m, 2H, -CH=CH2), 5.23–5.16 (m, 3H, -CH=CH2), 4.44 (d, J = 5.5 Hz, 2H, -OCH2-), 4.34–4.23 (m, 2H, -OCHH-, H-1), 4.17 (dd, J = 12.3, 5.8 Hz, 1H, -OCHH-), 4.08–3.97 (m, 2H, -OCH2-), 3.97–3.90 (m, 1H, H-2), 3.76–3.68 (m, 2H, H-6a, H-3), 3.65 (dd, J = 11.1, 4.1 Hz, 1H, H-6b), 3.55–3.45 (m, 2H, H-4, H-5), 2.92–2.82 (m, 1H, -SCHHCH3), 2.20–2.09 (m, 1H, -SCHHCH3), 0.94 (t, J = 7.5 Hz, 3H, -SCH2CH3

13C

NMR (101 MHz, CDCl3, 298K) δ 134.9, 134.73, 134.69, 131.6, 131.4, 131.0, 129.7,

128.0, 127.8, 127.5, 126.1, 125.44, 125.41, 125.0, 124.9, 124.6, 124.0, 117.3, 117.2, 116.9, 92.5, 86.3, 79.8, 76.0, 74.3, 73.8, 72.63, 72.56

HRMS (ESI): m/z calcd for C34H38O6SNa+ : 597.2283,

[M+Na]+ Synthesis of (8R,9S,10S,10aR,6bR)-8-allyloxymethyl-9,10-diallyloxy-6b,8,10,11-tetrahydropyreno[2’,1’:4,5]pyrano[3,2-b]pyran (13). Compound 12 (460 mg, 0.80 mmol) was co-evaporated three

times with dry toluene. To the mixture of compound 12 and 2,6-di-tert-butyl-4-methylpyridine (658 mg, 3.20 mmol) in dry CH2Cl2 (8 mL) was added dropwise a solution of trifluoromethanesulfonic anhydride (202 µL, 1.20 mmol) in CH2Cl2 (1.8 mL) at -78 °C under nitrogen atmosphere. Then the mixture was warmed to room temperature and stirred for 2 h. The reaction mixture was diluted with CH2Cl2 and it was washed with sat. NaHCO3 and brine. The organic layer was dried over MgSO4 and evaporated and the residue was chromatographed on a silica gel column (hexane–ethyl acetate, 10:1) to give compound 13 (99.4 mg, 25%) as a yellow amorphous solid: mp 124– 0.42 (hexane–ethyl acetate,

1

[α]D25 +115.8° (c = 1.0 in CH2Cl2 Rf

H NMR (500 MHz, CDCl3, 298K) δ 8.33 (s, 1H, H-6), 8.20 (m, 2H,

Pyrene), 8.15 (d, J = 9.2 Hz, 1H, Pyrene), 8.06–7.99 (m, 4H, Pyrene), 7.91 (d, J = 9.2 Hz, 1H, Pyrene), 6.10–5.97 (m, 2H, -CH=CH2), 5.88–5.79 (m, 1H, -CH=CH2), 5.63–5.58 (m, 2H, H-6b, H-12a), 5.54 (d, J = 16.1 Hz, 1H, H-12b), 5.37 (d, J = 17.1 Hz, 2H, -CH=CH2), 5.28–5.13 (m, 3H, -CH=CH2), 5.08 (d, J

= 10.4 Hz, 1H, -CH=CHH), 4.47 (dd, J = 12.4, 5.7 Hz, 1H, -OCHH-), 4.41–4.34 (m, 2H, -OCHH-, H8a), 4.29 (dd, J = 12.5, 5.7 Hz, 1H, -OCHH-), 4.17 (dd, J = 12.8, 5.6 Hz, 1H, -OCHH-), 4.13–4.06 (m, 2H, -OCH2-), 3.80–3.73 (m, 2H, H-9, -OCHH- at C-8), 3.69 (dd, J = 10.5, 4.1 Hz, 1H, -OCHH- at C-8), 3.63 (t, J = 9.1 Hz, 1H, H-9), 3.47–3.41 (m, 1H, H-8

13C

NMR (101 MHz, CDCl3, 298K) δ 135.5,

134.92, 134.89, 131.4, 130.8, 130.7, 130.2, 128.4, 128.2, 127.6, 127.5, 126.2, 125.9, 125.7, 125.6, 124.7, 123.9, 122.4, 121.2, 117.3, 117.0, 116.7, 77.94, 77.90


HRMS

Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(ESI): m/z calcd for C32H32O5Na+:519.2143, [M+Na]+

519.2137.

Synthesis of (8R,9S,10S,10aR,6bR)-8-acetyloxymethyl-9,10-diacetyloxy-6b,8,10,11-tetrahydropyreno[2’,1’:4,5]pyrano[3,2-b]pyran (14). To a solution of compound 13 (68 mg, 0.14 mmol) in ethanol

(4 mL) and CH2Cl2 (2 mL) were added triethylamine (96 µL, 0.69 mmol) and a solution of Rh(Ph3P)3Cl (38 mg, 0.041 mmol) in benzene (1 mL). The reaction mixture was stirred at 80 °C for 6 h. After evaporation of the reaction mixture, ethanol (4 mL), CH2Cl2 (2 mL), triethylamine (96 µL, 0.69 mmol), and Rh(Ph3P)3Cl (38 mg, 0.041 mmol) in benzene (1 mL) were added to the residue and the mixture was stirred at 80 °C for 5 h. The reaction mixture was evaporated and the residue was chromatographed on a silica gel column (hexane–ethyl acetate, 10:1) to give a syrupy intermediate (47 mg). To a solution of the intermediate in methanol (1 mL) and CH2Cl2 (1 mL), was added p-toluenesulfonic acid monohydrate (18 mg, 0.095 mmol). The reaction mixture was stirred at room temperature for 21 h. To the mixture was added triethylamine (13 µL, 0.093 mmol) and the mixture was evaporated. To the residue were added pyridine (1 mL) and acetic anhydride (1 mL). The mixture was stirred at room temperature for 2 h and then evaporated. The residue was chromatographed on a silica gel column (hexane–ethyl acetate, 5:1) to give [α]D25 +189.1° (c =

compound 14 (32 mg, 46%, 3 steps) as a white amorphous solid: mp 242– 1

0.49 in CH2Cl2 Rf 0.68 (hexane–ethyl acetate,

H NMR (500 MHz, CDCl3, 298K) δ 8.25 (s, 1H, H-

6), 8.18 (m, 2H, Pyrene), 8.07 (d, J = 9.1 Hz, 1H, Pyrene), 8.05–7.98 (m, 3H, Pyrene), 7.96 (d, J = 8.9 Hz,1H, Pyrene), 7.81 (d, J = 9.1 Hz, 1H, Pyrene), 5.69 (d, J = 6.8 Hz, 1H, H-6b), 5.62 (d, J = 16.2 Hz, 1H, Pyrene-CHH-), 5.48–5.36 (m, 2H, Pyrene-CH2-, H-10), 5.19 (t, J = 9.5 Hz, 1H, H-9), 4.54–4.47 (m, 1H, H-10a), 4.32 (dd, J = 12.1, 5.0 Hz, 1H, -OCHH- at C-8), 4.20 (d, J = 11.2 Hz, 1H, -OCHH- at C-8), 3.72– 3.63 (m, 1H, H-8), 2.22 (s, 3H, -COCH3), 2.18 (s, 3H, -COCH3), 1.91 (s, 3H, -COCH3

13

C NMR (126

MHz, CDCl3, 298K) δ 170.8, 170.7, 169.7, 131.4, 130.9, 130.8, 128.6, 128.4, 128.2, 127.9, 127.3, 126.5, 126.2, 125.89, 125.88, 124.5, 124.1, 121.5, 121.4, 71.8, 70.1, 69.7, 69.2, 69.0, 62.6, 62.0 HRMS (ESI): m/z calcd for C29H26O8Na+: 525.1521, [M+Na]+

.

Synthesis of (8R,9S,10S,10aR,6bR)-8-acetyloxymethyl-9,10-diacetyloxy-6b,8,10,11-tetrahydropyreno[2’,1’:4,5]pyrano[3,2-b]pyran-12-one (15a). To a solution of compound 14 (123 mg, 0.245 mmol)

in CH2Cl2 (12 mL) were added benzyltriethylammonium chloride (336 mg, 1.48 mmol) and KMnO4 (233 mg, 1.47 mmol) at 0 °C. The mixture was stirred at room temperature for 15 h and diluted with CH2Cl2, which was washed with aqueous sodium thiosulfate and brine. The organic layer was dried over MgSO4 and evaporated. The residue was chromatographed on a silica gel column (hexane–ethyl acetate, 3:2) to give compound 15a (64 mg, 51%) as a yellow amorphous solid: mp 100– in CHCl3 Rf 0.30 (hexane–ethyl acetate, 1

1

[α]D25 +279.7° (c=0.8

H NMR (500 MHz, CDCl3, 298K) δ 9.60 (d, J = 9.5 Hz,

1H, H-13), 8.40–8.35 (m, 2H, H-1, H-14), 8.34 (d, J = 7.6 Hz, 1H, H-3), 8.30 (d, J = 8.8 Hz, 1H, H-4),



Page 18 of 25

8.23 (s, 1H, H-6), 8.14 (t, J = 7.6 Hz, 1H, H-2), 8.09 (d, J = 8.9 Hz, 1H, H-5), 5.98 (d, J = 6.6 Hz, 1H, H6b), 5.29 (t, J = 9.5 Hz, 1H, H-10), 5.16 (t, J = 9.5 Hz, 1H, H-9), 4.88 (dd, J = 9.7, 6.6 Hz, 1H, H-10a), 4.40 (dd, J = 12.2, 5.2 Hz, 1H, -OCHH- at C-8), 4.30–4.24 (m, 1H, -OCHH- at C-8), 3.96–3.91 (m, 1H, H-8), 2.24 (s, 3H, -COCH3), 2.14 (s, 3H, -COCH3), 1.90 (s, 3H, -COCH3

13C

NMR (126 MHz, CDCl3,

298K) δ 170.8, 170.4, 169.5, 161.6, 135.9, 134.0, 133.7, 131.7, 131.5, 130.9, 130.6, 127.6, 127.5, 127.2, HRMS (ESI): m/z calcd for

C29H24O9Na+:

539.1313,

[M+Na]+

nd: 539.1304.

Synthesis of (8R,9S,10S,10aR,6bR)-8-hydroxymethyl-9,10-dihydroxy-6b,8,10,11-tetrahydropyreno[2’,1’:4,5]pyrano[3,2-b]pyran-12-one (15b). To a solution of compound 15a (20 mg, 0.039 mmol)

in methanol 2 mL and CH2Cl2 0.5 mL was added NaHCO3 (11 mg, 0.13 mmol). The mixture was vigorously stirred at room temperature for 25 h. After the mixture was evaporated, water (2 mL) was added to the residue and the resulting mixture was sonicated. The mixture was then centrifuged (8000 rpm) at 4 °C for 10 min. The precipitate was washed in the same way by water (2 mL) and ethyl acetate (2 mL) to give compound 15b (13 mg, 87%) as a yellow amorphous solid: mp 281 °C (decomp. [α]D23 250.6° (c = 0.16 in CH2Cl2–methanol

Rf 0.10 (hexane–ethyl acetate, 1:3

1H NMR (500 MHz, DMSO-d , 298K) 6

δ 9.49 (d, J = 9.4 Hz, 1H, H-13), 8.50–8.41 (m, 5H, Pyrene), 8.27 (d, J = 8.9 Hz, 1H, Pyrene), 8.20 (t, J = 7.6 Hz, 1H, H-2), 5.96 (d, J = 6.8 Hz, 1H, H-6b), 5.57–5.50 (m, 1H, OH at C-10), 4.93 (d, J = 6.3 Hz, 1H, OH at C-9), 4.89–4.83 (m, 1H, -CH2OH), 4.61–4.53 (m, 1H, H-10a), 3.84–3.77 (m, 1H, -OCHH- at C-8), 3.62–3.55 (m, 1H, -OCHH- at C-8), 3.26–3.19 (m, 1H, H-9

13C

NMR (126 MHz, DMSO-d6, 298K) δ

162.1, 136.9, 134.8, 131.7, 130.9, 130.6, 130.4, 129.7, 127.4, 127.2, 127.10, 127.07, 124.4, 123.6, 123.0, HRMS (ESI): m/z calcd for C23H18O6Na+: 413.0996, [M+Na]+ Synthesis of Sodium 2-(α-D-1-Deoxy-glucopyranos-1-yl)-1-pyrenecarboxylate (4). To a solution

of compound 15b (8.0 mg, 0.020 mmol) in water (0.6 mL) was added 0.1M NaOH (0.21 mL, 0.021 mmol). The reaction mixture was stirred at 80 °C for 14 h. Then the mixture was diluted with water and washed with CH2Cl2. The water layer was concentrated and dried in vacuo to give compound 4 (9.7 mg, quant.) as a pale yellow amorphous solid (too hygroscopic to measure melting point): [α]D25 +242.5° (c = 0.08 in DMSO Rf 0.16 (CH2Cl2–methanol,

1

H NMR (500 MHz, D2O, 298K) δ 8.58 (s, 1H, Pyrene-H), 8.24

(t, J = 6.9 Hz, 2H, Pyrene-H), 8.20–8.09 (m, 4H, Pyrene-H), 8.05 (t, J = 7.6 Hz, 1H, Pyrene-H), 5.77 (d, J = 4.7 Hz, 1H, H-1’), 4.32 (t, J = 7.4, 7.1* Hz, 1H, H-3’, *obtained by decoupling), 4.18 (dd, J = 7.4, 4.7

Hz, 1H, H-2’), 3.88 (dd, J = 12.4, 6.4 Hz, 1H, H-6’), 3.76 (dd, J = 12.5, 2.7 Hz, 1H, H-6’), 3.73 (t, 7.8*, 6.9* Hz, 1H, H-4’, *obtained by decoupling), 3.67–3.62 (m, 1H, H-5

1H

NMR (500 MHz, DMSO-d6,

298K) δ 8.58 (s, 1H, Pyrene-H), 8.27 (d, J = 9.2 Hz, 1H, Pyrene-H), 8.18 (t, J = 8.0 Hz, 2H, Pyrene-H),


Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8.07–7.96 (m, 4H, Pyrene-H), 6.00 (s, 1H, -OH), 5.64 (d, J = 4.7 Hz, 1H, H-1’), 5.42 (d, J = 8.1 Hz, 1H, -OH), 5.19 (s, 1H, -OH), 5.01 (s, 1H, -OH), 3.99–3.89 (m, 2H, H-2’, H-3’), 3.55–3.48 (m, 1H, H-6’a), 13C

3.46–3.40 (m, 1H, H-6’b), 3.20–3.13 (m, 1H, H-4’)

NMR (126 MHz, DMSO-d6) δ 173.0, 142.2,

132.1, 130.9, 130.6, 127.7, 127.3, 127.1, 125.9, 125.8, 125.7, 125.51, 125.48, 124.3, 124.1, 123.1, 77.2, 73.7, 73.6, 72.9, 71.4, 61.8 HRMS (ESI): m/z calcd for C23H19O7Na2+: 453.0921 [M+Na]+ 453.0922. m/z calcd for C23H19O7 -: 407.1137 [M−Na]-

407.1144.

Synthesis of 1-Pyrenylmethyl 2,3,4,6-Penta-O-acetyl-β-D-glucopyranoside (16). To a solution of

1-pyrenemethanol (630 mg, 2.71 mmol) and Ag2CO3 (813 mg, 2.95 mmol) in CH2Cl2 (10 mL), was added 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (700 mg, 1.70 mmol). The mixture was stirred at room temperature for 5 days and then filtered through celite. The filtrate was evaporated and the residue was chromatographed on a silica gel column (C300, hexane–ethyl acetate, 5:1). The resulting elute was not pure enough probably owing to concomitant production of an orthoester derivative. We thus added tetrahydrofuran (5 mL), acetic acid (0.5 mL), and H2O (0.5 mL) to the residue of evaporation of the elute to hydrolyze the orthoester group and kept it at room temperature overnight. The mixture was evaporated and the residue was chromatographed on a silica gel column (C300, hexane–ethyl acetate, 5:1) to give compound 16 (347 mg, 36%) as a yellow amorphous solid: mp 67–90 CH2Cl2 Rf 0.30 (hexane–ethyl acetate,

[α]D25 −55.9° (c = 0.32 in

¹H NMR (500 MHz, CDCl3, 298K) δ 8.32 (d, J = 9.2 Hz,

1H, Pyrene), 8.22 (d, J = 7.5 Hz, 2H, Pyrene), 8.18–8.01 (m, 5H, Pyrene), 7.96 (d, J = 7.7 Hz, 1H, Pyrene), 5.64 (d, J = 12.3 Hz, 1H, Pyrene-CH2-), 5.31 (d, J = 12.3 Hz, 1H, Pyrene-CH2-), 5.16–5.01 (m, 3H, H-2’, H-3’, H-4’), 4.53 (d, J = 7.7 Hz, 1H, H-1’), 4.33 (dd, J = 12.2, 4.8 Hz, 1H, H-6’a), 4.25 (d, J = 12.1 Hz, 1H, H-6’b), 3.68–3.63 (m, 1H, H-5’), 2.17 (s, 3H, -COCH3), 1.99 (s, 3H, -COCH3), 1.94 (s, 3H, -COCH3), 3,

1.70 (s, 3H, -COCH3

298K) δ 170.9, 170.3, 169.5, 169.4, 132.0, 131.4,

130.8, 130.0, 129.2, 128.1, 128.0, 127.8, 127.5, 126.3, 125.7, 125.6, 125.1, 124.7, 124.6, 123.4, 98.5, 72.9, 72.0, 71.3, 69.2, 68.6, 62.2, 21.0, 20.72, 20.70

31H30O10Na

+:

585.1732

[M+Na]+ Synthesis of Pyrenylmethyl β-D-Glucopyranoside (5). To a solution of compound 16 (347 mg,

0.62 mmol) in methanol (4 mL) and CH2Cl2 (4 mL) was added 1 M NaOMe in methanol (0.5 mL, 0.5 mmol). The mixture was stirred at room temperature for 13 h under N2 atmosphere. The mixture was evaporated and the residue was chromatographed on a silica gel column (60N, CH2Cl2–methanol, 5:1) to give compound 5 (223 mg, 91%) as a pale yellow amorphous solid: mp 204–

[α]D25 −41.4° (c =

0.22 in methanol Rf 0.82 (CH2Cl2–methanol, 4:1 ¹H NMR (500 MHz, DMSO-d6, 298K) δ 8.46 (d, J = 9.2 Hz, 1H, Pyrene), 8.34–8.17 (m, 7H, Pyrene), 8.09 (t, J = 7.6 Hz, 1H, Pyrene), 5.58 (d, J = 12.1 Hz, 1H, Pyrene-CH2-), 5.31 (d, J = 12.1 Hz, 1H, Pyrene-CH2-), 5.12 (d, J = 4.8 Hz, 1H, -OH), 5.00–4.93 (m,



Page 20 of 25

2H, -OH), 4.69–4.63 (m, 1H, -OH), 4.41 (d, J = 7.6 Hz, 1H, H-1’), 3.82–3.75 (m, 1H, H-6’a), 3.59–3.51 (m, 1H, H-6’b), 3.22–3.06 (m, 4H, H-2’, H-3’, H-4’, H-5 ¹³C NMR (126 MHz, DMSO-d6, 298K) δ 131.6, 131.0, 130.9, 130.6, 129.1, 127.9, 127.73, 127.66, 126.7, 125.7, 125.6, 125.0, 124.2, 124.1, 102.2, + 23H22O6Na :

417.1309 [M+Na]+

417.1301. ■ ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 1H

and 13C NMR spectra, experiments for chemical and photophysical properties, and cell assays (PDF)

■ AUTHOR INFORMATION

Corresponding Author *Tel/Fax: +81-45-924-5850. E-mail: [email protected]. ORCID Hideya Yuasa: 0000-0002-2864-3185 Shigetoshi Okazaki: 0000-0001-5041-6301 Shun-ichiro Ogura: 0000-0002-4673-8882 Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS

This work was partly supported by JSPS KAKENHI Grants No 16H04176 and 17K19194 (to H.Y.). The authors thank Masato Koizumi for ESI-TOF-MS analysis and Yoshihisa Sei for NMR measurements, both at Suzukakedai Materials Analysis Division, Technical Department, Tokyo Institute of Technology. ■ REFERENCES

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Water-Soluble Glucosyl Pyrene Photosensitizers: An Intramolecularly

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