Tethered Indoxyl-Glucuronides for Enzymatically Triggered Cross-Linking

Indoxyl-glucuronides, upon treatment with β-glucuronidase under physiological conditions, are well known to afford the corresponding indigoid dye via oxidative dimerization. Here, seven indoxyl-glucuronide target compounds have been prepared along with 22 intermediates. Of the target compounds, four contain a conjugatable handle (azido-PEG, hydroxy-PEG, or BCN) attached to the indoxyl moiety, while three are isomers that include a PEG-ethynyl group at the 5-, 6-, or 7-position. All seven target compounds have been examined in indigoid-forming reactions upon treatment with β-glucuronidase from two different sources and rat liver tritosomes. Taken together, the results suggest the utility of tethered indoxyl-glucuronides for use in bioconjugation chemistry with a chromogenic readout under physiological conditions.


Introduction
The ability to cross-link molecules under physiological conditions may underpin a range of applications in bioorganic chemistry [1][2][3][4][5][6][7][8][9]. One class of molecules that has served well in this regard contains the indoxyl motif, the spontaneous oxidative dimerization of which affords the indigoid dye. The formation of the indoxyl species can be triggered by enzymatic action from the corresponding indoxyl-X compound, where X = phosphate, sulfate, glucoside, or glucuronide (Scheme 1). A comprehensive review of such compounds has been reported by Kiernan [10]. We have been interested in extending the core indoxyl chemistry beyond its chief present use in histology (to identify the location of enzymes in cells and tissues) and enabling applications for bioconjugation chemistry. Such applications require the presence of a tether attached to the indoxyl moiety.

Introduction
The ability to cross-link molecules under physiological conditions may underpin a range of applications in bioorganic chemistry [1][2][3][4][5][6][7][8][9]. One class of molecules that has served well in this regard contains the indoxyl motif, the spontaneous oxidative dimerization of which affords the indigoid dye. The formation of the indoxyl species can be triggered by enzymatic action from the corresponding indoxyl-X compound, where X = phosphate, sulfate, glucoside, or glucuronide (Scheme 1). A comprehensive review of such compounds has been reported by Kiernan [10]. We have been interested in extending the core indoxyl chemistry beyond its chief present use in histology (to identify the location of enzymes in cells and tissues) and enabling applications for bioconjugation chemistry. Such applications require the presence of a tether attached to the indoxyl moiety. The yields of indigoid formation can range considerably depending on the nature of substituents in the indoxyl moiety (Chart 1). The unsubstituted indoxyl-glucoside (I) affords indigo in 17% yield upon treatment with a β-glucosidase (pH 5), whereas the 4chloro-5-bromo derivative (II) affords the corresponding indigoid in 74% yield [5]. We prepared indoxyl-glucosides that bear an alkoxy tether at the indoxyl 5-position [5,6]. For the propargyloxy substituent, the yield was <5% (III), but increased upon inclusion of flanking bromine atoms, affording 56% with one bromine (IV) or an essentially quantitative yield with two bromines (V). Nearly identical results were obtained with the methoxycarbonyl substituent [5], and similar results have been observed with various derivatized PEG groups [9].

Chart 1. Yields of indigoid dye from various indoxyl-glucosides.
In the work leading to the present paper, we sought to pursue two lines of inquiry. In one, we switched from an indoxyl-glucoside to an indoxyl-glucuronide with the objective of increasing the water solubility afforded by the carboxylic acid/carboxylate moiety of the latter. The requisite β-glucuronidase enzymes are known to be prevalent in various tissues, including cancerous tumors [11][12][13][14][15][16][17]. Two, we sought to investigate the utility of tethers positioned at the 5-, 6-, or 7-positions. The thinking behind the latter study was double-pronged: (i) perhaps a substituent positioned more distal to the putative site of enzymatic action (for cleavage of the glycoside) might enable higher yields, and (ii) given that a chief rationale for use of the 5-alkoxy substituent was the convenience of 5-benzyloxyindole-3-carbaldehyde as a commercially available starting material, perhaps starting afresh with the 5-, 6-, or 7-bromoindoxyl-3-carbaldehyde for substitution with an ethyne might enable simplification of the synthesis without requiring flanking halogen substituents.
Here we report the synthesis of seven indoxyl-glucuronide target compounds. Four are equipped with 5-alkoxy substituents and flanking bromine atoms (4,6-dibromo substitution). Three are positional isomers with a PEG-ethyne linker at the 5-, 6-, or 7-positions. Each indoxyl-glucuronide has been examined upon treatment with β-glucuronidase to form the corresponding indigoid dye. Characterization of the yields by absorption spectroscopy has enabled evaluation of the effects of the various substituents.

Synthesis of Indoxyl-Glucuronide Scaffolds
We previously prepared 25 indoxyl-glucosides [5,6,9], and here we considered whether indoxyl β-glucosides could be converted directly to the corresponding indoxyl β-glucuronides. The primary hydroxy group (6-position) of a methyl α-glucoside or phenyl β-glucoside was reported to undergo selective oxidation to give the corresponding glucuronide upon treatment with 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and a co-oxidant such as PhI(OAc)2 or t-BuOCl [18,19]. On the other hand, oxidation of indoxyl β-glucoside V with TEMPO/PhI(OAc)2 was unsuccessful (Scheme 2), which we attributed to the presence of the free N-H moiety of the electron-rich indole. In the work leading to the present paper, we sought to pursue two lines of inquiry. In one, we switched from an indoxyl-glucoside to an indoxyl-glucuronide with the objective of increasing the water solubility afforded by the carboxylic acid/carboxylate moiety of the latter. The requisite β-glucuronidase enzymes are known to be prevalent in various tissues, including cancerous tumors [11][12][13][14][15][16][17]. Two, we sought to investigate the utility of tethers positioned at the 5-, 6-, or 7-positions. The thinking behind the latter study was doublepronged: (i) perhaps a substituent positioned more distal to the putative site of enzymatic action (for cleavage of the glycoside) might enable higher yields, and (ii) given that a chief rationale for use of the 5-alkoxy substituent was the convenience of 5-benzyloxyindole-3carbaldehyde as a commercially available starting material, perhaps starting afresh with the 5-, 6-, or 7-bromoindoxyl-3-carbaldehyde for substitution with an ethyne might enable simplification of the synthesis without requiring flanking halogen substituents.
Here we report the synthesis of seven indoxyl-glucuronide target compounds. Four are equipped with 5-alkoxy substituents and flanking bromine atoms (4,6-dibromo substitution). Three are positional isomers with a PEG-ethyne linker at the 5-, 6-, or 7-positions. Each indoxyl-glucuronide has been examined upon treatment with β-glucuronidase to form the corresponding indigoid dye. Characterization of the yields by absorption spectroscopy has enabled evaluation of the effects of the various substituents.

Synthesis of Indoxyl-Glucuronide Scaffolds
We previously prepared 25 indoxyl-glucosides [5,6,9], and here we considered whether indoxyl β-glucosides could be converted directly to the corresponding indoxyl β-glucuronides. The primary hydroxy group (6-position) of a methyl α-glucoside or phenyl β-glucoside was reported to undergo selective oxidation to give the corresponding glucuronide upon treatment with 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and a co-oxidant such as PhI(OAc) 2 or t-BuOCl [18,19]. On the other hand, oxidation of indoxyl β-glucoside V with TEMPO/PhI(OAc) 2 was unsuccessful (Scheme 2), which we attributed to the presence of the free N-H moiety of the electron-rich indole. We decided to apply the same reaction conditions to a fully protected substrate. Thus, indoxyl-glucoside 1 [6] was subjected to selective deacetylation of the 6-O-acetyl group by treatment with [t-BuSnOH(Cl)]2 in methanol [20,21], which afforded 2 in 61% yield (Scheme 3). Attempted oxidation of 2 by treatment with TEMPO/PhI(OAc)2 was unsuccessful as well, which could stem from the presence of the free 5-OH group of the indoxyl moiety. The 5-OH was then protected with a tert-butyldimethylsilyl (TBS) group in the presence of triethylamine to give 3 in 65% yield. Treatment of 3-wherein both indole functional groups are protected-with TEMPO/PhI(OAc)2 resulted in oxidation of the primary hydroxyl group of the glucoside moiety; methylation of the resulting carboxylate with dimethyl sulfate and cleavage of the TBS group with tetra-n-butylammonium fluoride (TBAF) gave the indoxyl-glucuronide methyl ester 4 in 33% yield. We decided to apply the same reaction conditions to a fully protected substrate. Thus, indoxyl-glucoside 1 [6] was subjected to selective deacetylation of the 6-O-acetyl group by treatment with [t-BuSnOH(Cl)] 2 in methanol [20,21], which afforded 2 in 61% yield (Scheme 3). Attempted oxidation of 2 by treatment with TEMPO/PhI(OAc) 2 was unsuccessful as well, which could stem from the presence of the free 5-OH group of the indoxyl moiety. The 5-OH was then protected with a tert-butyldimethylsilyl (TBS) group in the presence of triethylamine to give 3 in 65% yield. Treatment of 3-wherein both indole functional groups are protected-with TEMPO/PhI(OAc) 2 resulted in oxidation of the primary hydroxyl group of the glucoside moiety; methylation of the resulting carboxylate with dimethyl sulfate and cleavage of the TBS group with tetra-n-butylammonium fluoride (TBAF) gave the indoxyl-glucuronide methyl ester 4 in 33% yield. We decided to apply the same reaction conditions to a fully protected substrate. Thus, indoxyl-glucoside 1 [6] was subjected to selective deacetylation of the 6-O-acetyl group by treatment with [t-BuSnOH(Cl)]2 in methanol [20,21], which afforded 2 in 61% yield (Scheme 3). Attempted oxidation of 2 by treatment with TEMPO/PhI(OAc)2 was unsuccessful as well, which could stem from the presence of the free 5-OH group of the indoxyl moiety. The 5-OH was then protected with a tert-butyldimethylsilyl (TBS) group in the presence of triethylamine to give 3 in 65% yield. Treatment of 3-wherein both indole functional groups are protected-with TEMPO/PhI(OAc)2 resulted in oxidation of the primary hydroxyl group of the glucoside moiety; methylation of the resulting carboxylate with dimethyl sulfate and cleavage of the TBS group with tetra-n-butylammonium fluoride (TBAF) gave the indoxyl-glucuronide methyl ester 4 in 33% yield.  In an alternate route (developed to prepare indoxyl-glucosides) [7], a solution of 5-benzyloxyindolinone 5 [6] in toluene/nitromethane was treated with acetobromo-α-Dglucuronic acid methyl ester (6) in the presence of mercury(II) oxide, mercury(II) bromide, and molecular sieves 4 Å at 30 • C to give indoxyl-glucuronide methyl ester 7 in 49% yield (Scheme 3). Debenzylation in CH 2 Cl 2 /ethanol/tetrahydrofuran (THF) containing Pd/C with H 2 (1 atm) gave the 5-hydroxy product 8. While 1 H NMR spectroscopy showed tiny peaks in the 5.55-5.60 ppm region consistent with the α-anomer [6] of 8, such peaks corresponded at most to the 2-3% level and were not observed following recrystallization in hexanes/CH 2 Cl 2 . In this manner, indoxyl-glucuronide 8 was obtained in 75% yield. Treatment of 8 with 2.4 equiv of N-bromosuccinimide (NBS) in CH 2 Cl 2 containing the acid scavenger 2,6-di-tert-butylpyridine (2,6-DTBP) [22] at −78 • C gave 4 in 87% yield.
The installation of an azido-bearing tether was carried out by treatment of 4 with bromo-PEG 5 -azide (P1) in the presence of K 2 CO 3 in N,N-dimethylformamide (DMF), which enabled alkylation of the phenolic hydroxy group without cleavage of the N-acetyl or O-acetyl protecting groups (Scheme 4). In this manner, the protected azido-PEG-indoxylglucuronide methyl ester 11 was obtained in 44% yield. Global deprotection was carried out under relatively strong basic conditions (aqueous NaOH in THF/methanol) to give target compound 12 in 74% yield. Treatment of 4 with nosyl-PEG 3 -OH (P2) [5] in the presence of N,N-diisopropylethylamine (DIPEA) gave 13 in 79% yield.
To install a longer spacer between the azido group and indoxyl moiety, the alcohol of the short PEG moiety of 13 was activated by treatment with 4-nitrophenyl chloroformate in the presence of pyridine to give carbonate 14 in 97% yield (Scheme 5). The reaction of 14 with the commercially available azido-PEG 4 -amine (P3) gave 15 and 16 in 32% and 26% yield, respectively, upon separation by preparative thin layer chromatography (TLC), where the latter product stems from the loss of the N-acetyl group under these conditions. Treatment of 15 with relatively strong basic conditions (aqueous NaOH in CH 2 Cl 2 /methanol) caused global deprotection and gave target compound 17 in 80% yield.
Molecules 2023, 28, x FOR PEER REVIEW 5 of 22 The installation of an azido-bearing tether was carried out by treatment of 4 with bromo-PEG5-azide (P1) in the presence of K2CO3 in N,N-dimethylformamide (DMF), which enabled alkylation of the phenolic hydroxy group without cleavage of the N-acetyl or O-acetyl protecting groups (Scheme 4). In this manner, the protected azido-PEG-indoxyl-glucuronide methyl ester 11 was obtained in 44% yield. Global deprotection was carried out under relatively strong basic conditions (aqueous NaOH in THF/methanol) to give target compound 12 in 74% yield. Treatment of 4 with nosyl-PEG3-OH (P2) [5] in the presence of N,N-diisopropylethylamine (DIPEA) gave 13 in 79% yield.
To install a longer spacer between the azido group and indoxyl moiety, the alcohol of the short PEG moiety of 13 was activated by treatment with 4-nitrophenyl chloroformate in the presence of pyridine to give carbonate 14 in 97% yield (Scheme 5). The reaction of 14 with the commercially available azido-PEG4-amine (P3) gave 15 and 16 in 32% and 26% yield, respectively, upon separation by preparative thin layer chromatography (TLC), where the latter product stems from the loss of the N-acetyl group under these conditions. Treatment of 15 with relatively strong basic conditions (aqueous NaOH in CH2Cl2/methanol) caused global deprotection and gave target compound 17 in 80% yield.
The indoxyl-glucuronide 13 is equipped with three types of protecting groups: one N-acetyl, three O-acetyl, and one ester O-methyl unit. Selective manipulation was achieved as follows (Scheme 5): • Mild basic conditions (NaHCO 3 suspended in methanol at room temperature for 1.5 h) gave selective cleavage of the N-acetyl group while leaving the O-acetyl groups intact, converting 13 to 18; • Somewhat stronger, but still mild, basic conditions (K 2 CO 3 in CH 2 Cl 2 /methanol at room temperature for 40 min) gave exhaustive cleavage of the N-acetyl and O-acetyl groups, converting 13 to 19; • Stronger basic conditions (0.1 M aqueous NaHCO 3 dissolved in methanol at 60 • C for 18 h) caused saponification of the methyl ester, converting 19 to the target compound 20. In short, the aqueous methanol solution containing dissolved NaHCO 3 affords a stronger basic condition than a suspension of NaHCO 3 in methanol.
The Sonogashira coupling reaction [26] was carried out by treating each indoxyl β-D-glucuronide (24-Br 5 , 24-Br 6 , or 24-Br 7 ) with propargyl-PEG3-OMe (P4) in the presence of palladium(0) tetrakis(triphenylphosphine) and copper iodide at 60 °C. Subsequent exposure to a basic condition (a suspension of K2CO3 in methanol at room temperature for 4 h) caused exhaustive deacetylation as well as saponification to afford the 5-, 6-, or 7-PEGylated indoxyl β-D-glucuronide (25, 26, or 27) in 8%, 23%, or 30% yield, respectively (Scheme 7). The hydrolysis of the methyl ester was surprising, albeit desirable, and may stem from adventitious water in the methanol employed. The presence of a byproduct was observed after Sonogashira coupling and exhaustive deacetylation reactions. In the synthesis of 25, normal phase silica chromatography was not effective for separating 25 and the byproduct due to their similar chromatographic mobilities; however, C18-reversed phase silica chromatography (H2O/acetonitrile) enabled better separation. The byproduct in crude form was assigned to the structure 25-elim on the basis of 1 Figure S4). A byproduct of this type is precedented [27] in glucuronide chemistry and is readily understood to form by deprotonation of the acidic proton α-to the carbonyl group followed by β-elimination (shown for acetate elimination in Scheme 8). The 1 H NMR data for the crude reaction product following deprotection was observed for each reaction leading to 25-27. While we did not study this issue in depth, the elimination is believed to The presence of a byproduct was observed after Sonogashira coupling and exhaustive deacetylation reactions. In the synthesis of 25, normal phase silica chromatography was not effective for separating 25 and the byproduct due to their similar chromatographic mobilities; however, C 18 -reversed phase silica chromatography (H 2 O/acetonitrile) enabled better separation. The byproduct in crude form was assigned to the structure 25-elim on the basis of 1 Figure S4). A byproduct of this type is precedented [27] in glucuronide chemistry and is readily understood to form by deprotonation of the acidic proton αto the carbonyl group followed by β-elimination (shown for acetate elimination in Scheme 8). The 1 H NMR data for the crude reaction product following deprotection was observed for each reaction leading to 25-27. While we did not study this issue in depth, the elimination is believed to occur during the Sonogashira reaction. In the reaction of 24-Br 7 , the ratio of the corresponding 27 and 27-elim was 4:1 upon reaction at 60 • C versus 2:3 at 80 • C, as indicated by LC-MS data (see the Supplementary Materials, Figure S7). occur during the Sonogashira reaction. In the reaction of 24-Br 7 , the ratio of the corresponding 27 and 27-elim was 4:1 upon reaction at 60 °C versus 2:3 at 80 °C, as indicated by LC-MS data (see the Supplementary Materials, Figure S7). Scheme 8. Proposed elimination process leading to an unsaturated glucuronide.

Enzymatic Tests
Enzymatic assays were performed to evaluate the efficacy of indigoid formation depending on the nature of the various substituents. Three enzyme treatments were employed: β-glucuronidase from bovine liver (at pH 5.0), β-glucuronidase from Escherichia coli (at pH 7.0), and rat liver tritosomes (hepatic lysosomes that have been loaded with Triton WR 1339; examined at pH 4.9). The reaction time and enzyme concentration were determined by time course and enzyme concentration-dependent studies (see the Supplementary Materials, Figures S1-S3). In general, 40 U/mL of enzyme sufficed, and the halflife for indigoid formation was 5 h (pH 5.0) or <30 min (pH 7.0). A mixture of indoxylglucuronide (100 µM) and β-glucuronidase (40 U/mL) or rat liver tritosomes (0.125 mg protein/mL) in the corresponding buffer with 1% dimethyl sulfoxide (DMSO) was incubated at 37 °C for 24 h. The resulting indigoid dye often precipitates, in which case the product was dissolved in an organic solvent for quantitative evaluation. The yields of indigoid are listed in Table 1.
The key findings are as follows: • The standard control compound (VI, 4-chloro-5-bromo-1H-indol-3-yl β-D-glucopyranosiduronic acid, the glucuronide analogue of II) gave yields of 75%, 133%, and 35% under the three conditions (β-glucuronidase from bovine liver or E. coli and use of rat tritosomes, respectively). The yield >100% must reflect inaccuracies in the molar absorption coefficient value or experimental error.

•
The 4,6-dibromo-5-alkoxyindoxyl-glucuronide methyl esters (13, 18, and 19) bearing 4, 3, or 0 acetyl groups, respectively, gave little or no indigoid formation, indicating the necessity for the carboxylic acid of the glucuronide moiety. No indigoid was observed even with the inclusion of an esterase in a cocktail experiment (Table S1). On the other hand, the fully deprotected analogue 4,6-dibromo-5-alkoxyindoxyl-glucuronide (20) gave a good yield of indigoid under the three conditions. • Compound 20 contains a short PEG group. Two other 4,6-dibromo-5-alkoxyindoxylglucuronides bearing PEG groups (12 and 17) gave reasonable yields, but the shorter linker design of 12 afforded up to a 2.5-fold higher indigoid yield compared to the longer PEG linker compound (17) under acidic conditions. A more pronounced distinction occurred with the BCN-indoxyl-glucuronide (10), which gave no observable indigoid, an effect attributed to the presence of the bulky BCN group. In general, indigoid formation is more favorable (higher yield and shorter t1/2) under neutral (pH 7.0) or basic versus acidic (pH 5.0) conditions [28,29]. The time course for indigoid formation upon reaction of 12 is shown in Figures S2 and S3.

Enzymatic Tests
Enzymatic assays were performed to evaluate the efficacy of indigoid formation depending on the nature of the various substituents. Three enzyme treatments were employed: β-glucuronidase from bovine liver (at pH 5.0), β-glucuronidase from Escherichia coli (at pH 7.0), and rat liver tritosomes (hepatic lysosomes that have been loaded with Triton WR 1339; examined at pH 4.9). The reaction time and enzyme concentration were determined by time course and enzyme concentration-dependent studies (see the Supplementary Materials, Figures S1-S3). In general, 40 U/mL of enzyme sufficed, and the half-life for indigoid formation was 5 h (pH 5.0) or <30 min (pH 7.0). A mixture of indoxyl-glucuronide (100 µM) and β-glucuronidase (40 U/mL) or rat liver tritosomes (0.125 mg protein/mL) in the corresponding buffer with 1% dimethyl sulfoxide (DMSO) was incubated at 37 • C for 24 h. The resulting indigoid dye often precipitates, in which case the product was dissolved in an organic solvent for quantitative evaluation. The yields of indigoid are listed in Table 1.
The key findings are as follows: • The standard control compound (VI, 4-chloro-5-bromo-1H-indol-3-yl β-D-glucopyranosiduronic acid, the glucuronide analogue of II) gave yields of 75%, 133%, and 35% under the three conditions (β-glucuronidase from bovine liver or E. coli and use of rat tritosomes, respectively). The yield >100% must reflect inaccuracies in the molar absorption coefficient value or experimental error.

•
The 4,6-dibromo-5-alkoxyindoxyl-glucuronide methyl esters (13, 18, and 19) bearing 4, 3, or 0 acetyl groups, respectively, gave little or no indigoid formation, indicating the necessity for the carboxylic acid of the glucuronide moiety. No indigoid was observed even with the inclusion of an esterase in a cocktail experiment (Table S1). On the other hand, the fully deprotected analogue 4,6-dibromo-5-alkoxyindoxyl-glucuronide (20) gave a good yield of indigoid under the three conditions. • Compound 20 contains a short PEG group. Two other 4,6-dibromo-5-alkoxyindoxylglucuronides bearing PEG groups (12 and 17) gave reasonable yields, but the shorter linker design of 12 afforded up to a 2.5-fold higher indigoid yield compared to the longer PEG linker compound (17) under acidic conditions. A more pronounced distinction occurred with the BCN-indoxyl-glucuronide (10), which gave no observable indigoid, an effect attributed to the presence of the bulky BCN group. In general, indigoid formation is more favorable (higher yield and shorter t 1/2 ) under neutral (pH 7.0) or basic versus acidic (pH 5.0) conditions [28,29]. The time course for indigoid formation upon reaction of 12 is shown in Figures S2 and S3.
The yield was estimated from absorption spectroscopy following centrifugation and solubilization of any precipitates in DMF/H 2 O (2:1) with ε = 2.6 × 10 4 M −1 cm −1 (measured for 4,4 6,6 -tetrabromo-5,5 -bis(8-hydroxy-3,6-dioxaoctyloxy)indigo) [5] for the dominant red-region absorption band in each case unless noted otherwise. b The precipitate (indigoid) was dissolved in DMF for absorbance measurements. c Calculated with ε = 2.00 × 10 4 M −1 cm −1 reported for 5,5 -dibromo-4,4 -dichloroindigo [30]. d Not conducted. e No blue color was observed by visual inspection, and no absorption peak was observed spectroscopically.  (Figure 1). The isolated yields of 93%, 94%, or 90% were in accordance with those determined by absorption spectroscopy using a generic value of the molar absorption coefficient (26,000 M −1 cm −1 ) in Table 1 Table 1 were then calculated using the obtained molar absorption coefficient. Under the neutral condition (pH 7.0), the yield of indigoids was almost the same for all three substrates. However, 26 gave a better yield for the indigogenic reactions under the acidic condition compared to that of 25 or 27, up to 3-fold.  1 H NMR and 13 C{ 1 H} NMR spectra were collected at room temperature unless noted otherwise. Chemical shifts for 1 H NMR spectra are reported in parts per million (δ) relative to tetramethylsilane or a solvent signal [CD3OD, δ = 3.31 ppm] [31]. Chemical shifts for 13 C{ 1 H} NMR spectra are reported in parts per million (δ) relative to tetramethylsilane or a solvent signal [CD3OD, δ = 49.00 ppm; CDCl3, δ = 77.16 ppm] [31].
All solvents were reagent grade and were used as received unless noted otherwise. m-Chloroperbenzoic acid (mCPBA, 75%) was purchased from Oakwood Chemical and purified [32] prior to use, unless noted otherwise.
β-glucuronidase from bovine liver and β-glucuronidase from E. coli were purchased from Sigma. The enzymatic activity of β-glucuronidase was determined by the standard substrate, phenolphthalein β-D-glucuronide. Rat liver tritosomes and 10X catabolic buffer were purchased from Xenotech.
All solvents were reagent grade and were used as received unless noted otherwise. m-Chloroperbenzoic acid (mCPBA, 75%) was purchased from Oakwood Chemical and purified [32] prior to use, unless noted otherwise.
β-glucuronidase from bovine liver and β-glucuronidase from E. coli were purchased from Sigma. The enzymatic activity of β-glucuronidase was determined by the standard substrate, phenolphthalein β-D-glucuronide. Rat liver tritosomes and 10X catabolic buffer were purchased from Xenotech.
Commercial compounds were used as received, unless noted otherwise. Known compounds 1, 5, and P2 were prepared as described in the literature [5,6].

Outlook
The synthesis of an indoxyl-glucuronide from an indoxyl-glucoside has been demonstrated in the case where the indole nitrogen and three secondary hydroxy groups of the glucoside are protected with acetyl groups. Conditions have been deployed for selective cleavage of the N-acetyl group in the presence of O-acetyl groups and the methyl ester, for selective cleavage of the O-acetyl groups in the presence of the methyl ester, and for saponification of the glucuronide methyl ester in the presence of a variety of functional groups, including hydroxy (PEG termini and glucosyl), azide, alkyne, carbamate, and the unprotected indole. The placement of the PEG-ethynyl group at the 6-position provides yields of indigoid dye comparable to those of the 4,6-dibromo-5-alkoxy substitution pattern upon treatment with β-glucuronidase at neutral conditions. The indigoid yield from the 6-PEGylated indoxyl-glucuronide (26) was 3-fold higher than that of the 5-and 7-positional isomers (25 and 27) under acidic conditions. Conversion of indoxyl-glucuronides to the corresponding indigoid dye has an absolute requirement for full display of unprotected hydroxy and carboxylic acid groups on the glucuronide moiety. The indoxyl-glucuronides bearing functional tethers described herein (10, 12, 17, and 20), with further development, may support applications in the life sciences that require cross-linking under physiological conditions.