Enzymatically triggered chromogenic cross-linking agents under physiological conditions

Article abstract of DOI:10.1039/c9nj04126e

The ability to cross-link molecules upon enzymatic action under physiological conditions holds considerable promise for use in diverse life sciences applications. Here, an enzymatically triggered "click reaction" has been developed by exploiting the longstanding indigo-forming reaction from indoxyl β-glucoside. The covalent cross-linking proceeds in aqueous solution, requires the presence only of an oxidant (e.g., O₂), and is readily detectable owing to the blue color of the resulting indigoid dye. To achieve facile indigoid formation in the presence of a bioconjugatable tether, diverse indoxyl β-glucosides were synthesized and studied in enzyme assays with four glucosidases including from tritosomes (derived from hepatic lysosomes) and rat liver homogenates. Altogether 36 new compounds (including 15 target indoxyl-glucosides for enzymatic studies) were prepared and fully characterized in pursuit of four essential requirements: enzyme triggering, facile subsequent indigoid dye formation, bioconjugatability, and synthetic accessibility. The 4,6-dibromo motif in a 5-alkoxy-substituted indoxyl-glucoside was a key design feature for fast and high-yielding indigoid dye formation. Two attractive molecular designs include (1) an indoxyl-glucoside linked to a bicyclo[6.1.0]nonyl (BCN) group for Cu-free click chemistry, and (2) a bis(indoxyl-glucoside). In both cases the linker between the reactive moieties is composed of two short PEG groups and a central triazine derivatized with a sulfobetaine moiety for water solubilization. Glucosidase treatment of the bis(indoxyl-glucoside) in aqueous solution gave oligomers that were characterized by absorption, dynamic light-scattering, and ¹H NMR spectroscopy; optical microscopy; mass spectrometry; and HPLC. Key attractions of in situ indigoid dye formation, beyond enzymatic triggering under physiological conditions without exogenous catalysts or reagents, are the chromogenic readout and compatibility with attachment to diverse molecules.

Full text of DOI:10.1039/c9nj04126e

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Enzymatically triggered chromogenic cross-linking
agents under physiological conditions†
Cite this:DOI: 10.1039/c9nj04126e
Hikaru Fujita,
Jinghuai Dou,
Nobuyuki Matsumoto,
Zhiyuan Wu
and
Jonathan S. Lindsey
*
The ability to cross-link molecules upon enzymatic action under physiological conditions holds
considerable promise for use in diverse life sciences applications. Here, an enzymatically triggered
‘‘click reaction’’ has been developed by exploiting the longstanding indigo-forming reaction from indoxyl
b-glucoside. The covalent cross-linking proceeds in aqueous solution, requires the presence only of an
oxidant (e.g., O2), and is readily detectable owing to the blue color of the resulting indigoid dye. To
achieve facile indigoid formation in the presence of
a bioconjugatable tether, diverse indoxyl
b-glucosides were synthesized and studied in enzyme assays with four glucosidases including from
tritosomes (derived from hepatic lysosomes) and rat liver homogenates. Altogether 36 new compounds
(including 15 target indoxyl-glucosides for enzymatic studies) were prepared and fully characterized in
pursuit of four essential requirements: enzyme triggering, facile subsequent indigoid dye formation,
bioconjugatability, and synthetic accessibility. The 4,6-dibromo motif in a 5-alkoxy-substituted indoxyl-
glucoside was a key design feature for fast and high-yielding indigoid dye formation. Two attractive
molecular designs include (1) an indoxyl-glucoside linked to a bicyclo[6.1.0]nonyl (BCN) group for
Cu-free click chemistry, and (2) a bis(indoxyl-glucoside). In both cases the linker between the reactive
moieties is composed of two short PEG groups and a central triazine derivatized with a sulfobetaine
moiety for water solubilization. Glucosidase treatment of the bis(indoxyl-glucoside) in aqueous solution
gave oligomers that were characterized by absorption, dynamic light-scattering, and 1H NMR
spectroscopy; optical microscopy; mass spectrometry; and HPLC. Key attractions of in situ indigoid dye
formation, beyond enzymatic triggering under physiological conditions without exogenous catalysts or
reagents, are the chromogenic readout and compatibility with attachment to diverse molecules.
Received 8th August 2019,
Accepted 28th November 2019
DOI: 10.1039/c9nj04126e
rsc.li/njc
Introduction
Enzyme-triggered processes, where a spontaneous chemical
event follows enzymatic action, are of great interest in the life
sciences, particularly for diagnostic and therapeutic applications.1–4
As a general strategy, a molecule to be released (A) upon action by
the target enzyme is protected with a covalently attached enzyme-
cleavable protecting group (A-PG) (Fig. 1). The molecules of A so-
liberated can undergo myriad processes ranging from formation
of dyes to assembly of nanostructures. A prevalent but less
germane example in the context of the theme reported herein
is enzyme–prodrug therapy, where A-PG constitutes a prodrug
and the released molecule A binds to a receptor or inhibits a
different enzyme.
Fig. 1 Enzyme-triggered reactions of molecule A bearing a protecting
group PG (A-PG). (i) Non-covalent self-assembly of A. (ii) Covalent hetero-
coupling of A with acceptor B. (iii) Covalent homo-coupling of A in the
presence of O2 (this work).
Department of Chemistry, North Carolina State University, Raleigh,
NC 27695-8204, USA. E-mail: jlindsey@ncsu.edu; Tel: +1-919-515-6406
† Electronic supplementary information (ESI) available: 1H and 13C NMR spectra for
all new compounds; single-crystal X-ray data; and further studies and data con-
cerning indigogenic reactions. CCDC 1902488 (18) and 1902489 (17). For ESI and
The formation of dyes entails covalent – and typically
crystallographic data in CIF or other electronic format see DOI: 10.1039/c9nj04126e intramolecular – reaction upon enzymatic removal of one or
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more protecting groups.5,6 The dye-forming reactions are members irreversible owing to the oxidation process [Fig. 1(iii)]. The
of a broad class of covalent self-assembly processes.7 The assembly conversion of indoxyl-glucoside to indigo is accompanied by
of nanostructures entails intermolecular processes that can rely on profound changes in the absorption spectrum, which conveniently
covalent or non-covalent interactions.4 For example, in a process facilitates quantitation.
sometimes referred to as enzyme-instructed self-assembly (EISA),8
The attractive features of indigo formation have led to
a peptide cleaved by an enzyme assembles upon hydrophobic and applications far beyond textile chemistry, namely in histochemistry
hydrogen-bonding interactions to form a hydrogel or nanostruc- and enzyme assays for nearly three quarters of a century.27,28 Indigo
ture [Fig. 1(i)].9,10 In reverse, enzymatic cleavage of large structures is very water-insoluble and precipitates at or near the site of
can cause disassembly, which has been exploited to release formation, enabling histological localization of the glucosidase
constituent drugs as part of novel delivery processes.10,11 Enzymes enzymes. Cotson and Holt studied the kinetics of indigoid-
are widely used to create covalent cross-links yielding hydrogels forming reactions from diverse indoxyl compounds including
and other soft matter; the reactions are largely done in vitro those bearing single bromine atoms at the 4–7 positions.29 The
whereas the resulting materials are used in vivo,12–15 but some bromoindoxyl compounds formed the corresponding indigoid
processes of enzymatic cleavage followed by non-covalent self- dye 2–4 times faster than the unsubstituted indoxyl species (e.g.,
assembly have been performed entirely in vivo.10 An example of 5 - 6, Fig. 3). The 6,60-dibromoindigo dates to antiquity as the
intermolecular covalent bond formation in vivo is provided by the regal natural pigment Tyrian purple (7).30 The attractive features
cyanobenzothiazole–cysteine (CBT–Cys) click reaction,16–18 where of indigoid dye formation thus include enzymatic triggering,
an enzymatically deprotected cysteine (A) reacts with CBT (B) chromogenicity, insoluble deposition from aqueous solution at
[Fig. 1(ii)]. Structures that have been prepared using compounds the site of reaction, and reaction under physiological conditions.
bearing cysteine and CBT moieties include nanorings formed by Histological and bacteriological use has been extended to include
covalent oligomerization followed by non-covalent self-assembly,19 indoxyl derivatives bearing enzymatically cleavable substituents
and nanocrystals immobilized by cross-linking.20 Such enzyme- other than glucosides, including glucuronides, carboxylic esters,
triggered intermolecular covalent-bond forming reactions phosphoesters, phosphodiesters, and sulfoesters.27 Development
remain rare (versus more known intramolecular reactions; e.g., and use of indoxyl-glucosides in the life sciences continue
ref. 5, 6, 21 and 22); the dearth is even more surprising given unabated.31–35 Enzymatically triggered release of substituted
extensive development of click chemistry as a potent means for indoxyl compounds has been described36–42 for molecular cross-
bioconjugation.23
linking and immobilization as part of a novel cancer therapy,43
A far older example of enzyme-triggered covalent bond yet other than an initial set of data39–42 obtained more than a
formation is the natural formation of indigo. Indoxyl-glucoside decade ago, this promising indigoid cross-linking chemistry has
(1, also known as indican) upon action of a glycosidase yields remained fallow. Thus, the venerable history of indigoid chemistry
indoxyl (2); subsequent enol–keto tautomerism affords indoline and incisive use in histochemistry notwithstanding, indoxyl species
(3), which in the presence of air undergoes homo-coupling to give have been little explored as cross-linking agents for biomolecules
indigo (4) (Fig. 2).24 Indigo is quite insoluble in water and typically in vitro or in vivo.
precipitates upon formation in aqueous solution. This chemistry
In this paper, we describe results from lengthy studies aimed
has been exploited to dye textiles dating from antiquity to the at developing indoxyl-based chromogenic covalent cross-linking
present day ‘‘blue jeans’’.25,26 The homo-coupling of two molecules agents of the general design illustrated in Fig. 4. We anticipated
of A to afford indigo (A–A) occurs without an acceptor (B), and is reasonably straightforward molecular designs but were surprised to
find that the linkers we employed for attaching a bioconjugatable
tether thwarted indigoid dye formation upon enzymatic cleavage of
the indoxyl-glucoside. Hence, a first set of studies entailed syntheses
of diverse indoxyl b-glucosides to identify the structural features
compatible with facile indigoid dye formation while bearing a
Fig. 2 Formation of indigo (4) from indoxyl b-glucoside (1) via indoxyl (2)
and tautomer (3).
Fig. 3 Fast indigoid dye formation of bromoindoxyl compounds (top) and
the famous pigment Tyrian purple (7).
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Fig. 4 General structure of indoxyl b-glucosides linked to a chemical/
bioconjugatable handle.
bioconjugatable tether. Next, each structure was examined for
indigoid dye formation upon treatment to several enzymatic
conditions including b-glucosidases, tritosomes, and rat liver
homogenates; from these studies, the 5-alkoxy-4,6-dibromoindoxyl
nucleus was found to give superior results. Finally, oligomerization
via the indigoid dye-forming reaction under physiological con-
ditions was explored to understand the fundamental properties
of this cross-linking moiety. This work establishes part of the
scientific foundation for the longer-term objective wherein the
enzymatically triggerable cross-linking agents yield macromolecular
scaffolds for immobilization of molecular constituents in vivo.
Results and discussion
1. Synthesis of monomeric indoxyl species
The commercially available 5-benzyloxy-3-formylindole (8) provided
the sole indole starting material for all new synthetic indoxyl-
glucosides described herein. Compound 8 was converted in 3 steps
Scheme 1 Synthesis of 5-hydroxyindoxy-glucosides.
to the fully protected 5-hydroxyindoxyl b-glucoside 9 (Scheme 1) in with N-bromosuccinimide (NBS, 1.05 equiv.) afforded 4-bromo-
accord with published protocols.39 Deprotection of the acetyl and indoxyl-glucoside 17 in 75% yield, whereas a larger quantity of
benzyl groups of 9 provided 5-hydroxyindoxyl b-glucoside 10 in NBS (2.3 equiv.) gave 4,6-dibromoindoxyl-glucoside 18 in 83%
89% yield, while debenzylation of 9 afforded acetyl-protected yield. The reactions were carried out in the presence of 2,6-di-tert-
5-hydroxyindoxyl b-glucoside 11 in 99% yield.
butylpyridine (2,6-DTBP), a non-nucleophilic base for proton
1,3,5-Triazine44,45 and carbamate linkers were selected to scavenging.48 Single-crystal X-ray structures of 17 and 18 confirmed
derivatize the phenolic hydroxy group in 11 (Scheme 2). Thus, the sugar stereochemistry and the positions of the bromine
treatment of 11 with 2,4-dichloro-6-methoxy-1,3,5-triazine (12)46 atoms (Fig. 5).
replaced one of the two chlorine atoms to form chlorotriazine 13
Sets of 4,6-unsubstituted, 4-bromo, and 4,6-dibromo indoxyl-
in 87% yield. The remaining chloride was substituted upon pilot glucosides wherein a linker is present at the 5-position were
reaction with morpholine to give morph-13, which retains all of prepared from 10, 11, 17, and 18 (Scheme 4). As indoxyl-
the acetyl protecting groups, in 99% yield. Reaction of 13 with glucosides (and the corresponding indigoid dyes) bearing 5-oxy
heterotelechelic linker 14, which bears an amine on one terminus (e.g., hydroxy, alkoxy, aryloxy) substituents and bromine atoms
and a bicyclo[6.1.0]nonyl (BCN) group47 for Cu-free click chemistry23 have not been reported (although individually substituted examples
on the other, was followed by acetyl removal to give the BCN- are known), we compared the indigogenic reactions among these
tethered indoxyl-glucoside 15 in 93% yield (Scheme 2). Treatment indoxyl-glucosides to investigate the effects of the bromine
of 11 with p-nitrophenyl chloroformate afforded a carbonate substituents. The triazine linker was introduced into indoxyl-
intermediate, which upon reaction with benzylamine gave the glucosides 10, 17, and 18 via the successive substitution of the
carbamate. Reaction with NaOMe caused removal of the acetyl chlorine atoms in dichlorotriazine 12. The reaction of 10 with
groups to give carbamate 16 in 53% yield.
triazine 12 was carried out in DMF rather than the preferred
In initial studies with b-glucosidase from almonds, neither dichloromethane (used for 17 and 18) owing to solubility
15 nor 16 afforded the corresponding indigoid species in good limitations. Thus, 4,6-unsubstituted indoxyl-glucoside 10 was
yield. It appeared that the alkoxy group, necessary for later treated with 12 followed by morpholine and K2CO3/methanol to
bioconjugation, inhibited the indigogenic process. Thus, bromine afford triazine derivative 19 in 59% yield. In a stepwise process,
atoms were introduced onto the indole ring to overcome the acetyl-protected 4-bromoindoxyl-glucoside 17 was reacted with
inhibitory effect of the alkoxy group (Scheme 3). Treatment of 11 12 followed by morpholine to afford the peracetylated triazine
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Scheme 2 Synthesis of 4,6-unsubstituted indoxyl compounds bearing the triazine or carbamate linker.
Scheme 3 Synthesis of 4-bromo and 4,6-dibromo substituted indoxyl-glucosides.
derivative pre-20 (not shown) in 87% yield; subsequent treatment 18 with ethyl bromoacetate in the presence of NaH and sub-
with K2CO3/methanol removed all five acetyl groups yielding the sequent treatment with NaOMe in MeOH. The reaction of 10
triazine 20 in 89% yield (77% overall). The reaction of acetyl- with propargyl bromide in the presence of K2CO3 afforded 4,6-
protected 18 and 12 gave 4,6-dibromoindoxyl-glucoside 21 in unsubstituted indoxyl-glucoside 25 in 30% yield, which bears a
67% yield.
propargyl group for ensuing click chemistry. Propargylation of
Indoxyl-glucosides 22–24 possess a methoxycarbonyl group, acetyl-protected 4-bromo and 4,6-dibromo indoxyl-glucosides 17
which can function as an amine-reactive linker. This linker was and 18 followed by deacetylation with MeONa in MeOH provided
introduced by alkylation of the 5-hydroxy group in 11, 17, and the corresponding 26 and 27 in 84 and 89% yield, respectively.
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Fig. 5 ORTEP drawing of the single-crystal X-ray structures of (A) 17 and (B) 18. All ellipsoids are contoured at the 50% probability level.
Scheme 4 Synthesis of 4,6-unsubstituted, 4-bromo, and 4,6-dibromo indoxyl-glucosides, each bearing a linker at the 5-position.
4,6-Dibromoindoxyl-glucoside 30, which possesses the BCN group for reaction with the rather hindered phenol, wherein mild
group instead of the propargyl group in 27, was prepared from conditions could be employed to avoid cleavage of the base-labile
18 (Scheme 5). The Mitsunobu reaction of 18 and commercially N-acetyl protecting group and ensuing N-alkylation. Deprotection
available BCN–methanol 28 in the presence of diisopropyl gave the 4,6-dibromoindoxyl-glucoside 33 in 94% yield. To further
diazodicarboxylate (DIAD) and Ph3P gave 29 in 63% yield. extend the triethylene glycol linker, N,N-bis(2-methoxyethyl)aniline
Deacetylation of 29 with K2CO3/MeOH afforded 30 in 99% yield. (34)49 was reacted with cyanuric chloride at elevated temperature
To install a long spacer, the reaction of 2-nitrobenzene- in the presence of 2,6-DTBP, affording the p-anilino-substituted
sulfonyl chloride with a 20-fold excess of triethylene glycol in dichlorotriazine building block 35 in 23% yield. Here, the aniline
the presence of triethylamine afforded triethylene glycol mono- and cyanuric chloride partners undergo respective SEAr and
nosylate (31). Treatment of 31 with 4,6-dibromoindoxyl- SNAr processes with each other. The reaction of 35 and indoxyl-
glucoside 18 gave 32 bearing the triethylene glycol linker in glucoside–PEG-OH 32 gave the mono-chloro triazinyl product 36
89% yield. The nosylate was chosen to provide a superb leaving in 15% yield. Anilinotriazines bearing amino/chloro or diamino
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Scheme 5 Synthesis of 4,6-dibromoindoxyl-glucosides bearing the bicyclononyne or triethylene glycol substituent.
substituents are known to be fluorescent,50,51 which provides a group. Sulfobetaines are stable zwitterions over a wide range of
convenient marker for detection. The reaction of 36 with BCN–PEG- pH. Synthesis of an indoxyl-glucoside bearing a sulfobetaine unit
NH2 (14) gave the 4,6-dibromoindoxyl-glucoside 37, which contains is illustrated in Scheme 6. N-Boc-piperazine (38) was treated with
the BCN group along with the fluorescent anilinotriazine moiety. 1,3-propanesultone to afford 39 in 63% yield. Quaternization
Compound 37 exhibited limited solubility in aqueous buffers of the tertiary nitrogen atom in 39 with 3-bromopropanol
(o10 mM at room temperature) despite the presence of the gluco- gave Boc-protected sulfobetaine 40 in 69% yield. The Boc group
side and PEG units, and was deemed unsuitable for bioconjugation. was cleaved with trifluoroacetic acid (TFA) to afford piperazine-
To improve the water solubility of the indoxyl-glucoside species, TFA salt 41 in 97% yield. Examination of 41 by 13C NMR
a sulfobetaine unit52,53 was incorporated as a water-solubilizing spectroscopy revealed a strong peak at d = 163.1 ppm consistent
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Scheme 6 Synthesis of a BCN-dibromoindoxyl-glucoside equipped with a water-solubilizing group.
with the carbonyl of the TFA anion; however, the expected group of 32 was selectively deprotected with NaHCO3/MeOH in
companion quartet due to the trifluoromethyl carbon was not 84% yield to afford 42.
evident. Further examination by 19F NMR spectroscopy gave a
The traditional method for selective successive substitution
strong peak at d = ꢀ76.9 ppm, consistent with the presence of a of cyanuric chloride relies on temperature control – for example,
TFA species. A series of small peaks in the 1H NMR spectrum of reaction with the first nucleophile at o0 1C, the second at room
41 likely stem from a tert-butyl-related impurity present at the temperature, and the third at Z60 1C.44,45 Here, the piperazine-TFA
few percent level; still, the sample was estimated to be 95% salt 41, triethylene glycol-substituted indoxyl-glucoside 42, and
pure and was used as is in subsequent reactions. The N-acetyl BCN-amine 14 were assembled at a triazine ring via one-flask,
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successive substitution of cyanuric chloride entirely at room the value in DMF/water (2: 1) for all studies here unless noted
temperature to afford 43 in 51% yield. Rather than rely on otherwise.
temperature variation to control substitution, the base 1,10-
Indoxyl-glucosides 19–21 bearing the triazine linker did not
phenanthroline54 was employed to facilitate mono-substitution form the indigoid dye regardless of the presence or absence of a
of cyanuric chloride with the alcohol 42, whereas the stronger bromine atom (entries 5–7, respectively). In the case of 5-[(meth-
base N,N-diisopropylethylamine was used for the second and oxycarbonyl)methoxy]indoxyl-glucosides 22–24, the yield of
third substitutions with secondary amine 41 and primary amine indigoid dye was markedly improved as the number of bromine
14, respectively. Cleavage of the acetyl groups of sulfobetaine 43 atoms increased (entry 8, 22, o1%; entry 9, 23, 68%; entry 10, 24,
provided 44 in 98% yield. Owing to the sulfobetaine unit, 44 122% yield). The same trend was observed for 5-(propargyloxy)-
showed superior solubility (4400 mM at room temperature) indoxyl-glucosides 25–27 (entry 11, 25, o5%; entry 12, 26, 56%;
versus 37 in a 100 mM sodium phosphate (NaPi) buffer entry 13, 27, 105% yield). The calculated yields in excess of
(pH 7.4) containing 100 mM NaCl. Purification of 43 and 44 100% stem from the value employed for the molar absorption
was achieved in part by hydrophilic interaction chromatography coefficient. These results indicated a significant promoting
on diol-functionalized silica, which affords intermediate polar- effect of the bromine atoms on indigoid dye formation. No
ity between bare silica and reverse phase derivatized silica.55 In indigoid product was detected with BCN-indoxyl-glucoside 30
each of the triazine compounds that contain the BCN unit (entry 14) whereas PEG3-indoxyl-glucoside 33 afforded indigoid
(15, 37, 43, 44), examination by NMR spectroscopy revealed a dye in 52% yield (entry 15). In summary, the nature of the
number of duplicate peaks. The duplicate peaks are attributed to 5-substituent significantly affected the indigoid-forming reaction:
rotamers that arise from constrained rotation about the triazinyl 20, 21, and 30 did not afford any indigoid product regardless of
carbon–nitrogen bond to which the BCN–PEG unit is attached.
the presence of bromine atoms. This may be because these
With diverse indoxyl-glucoside compounds in hand, we substrates have low affinity for the enzyme due to the presence
carried out a set of studies to examine indigoid-dye formation of the bulky triazine or BCN moiety.
upon enzymatic cleavage of the glucosyl unit under physiological
The results from the glucosidase survey prompted several
conditions. Altogether, 14 new (15, 16, 19–27, 30, 33, 44) and 2 further experiments. First, a parallel set of studies was carried
known (1, 45) synthetic indoxyl-glucosides (lacking acetyl protect- out with inclusion of several oxidants commonly employed in
ing groups) were examined in an effort to identify suitable histochemical studies, given that the indigogenic process
combinations of substituents to support both bioconjugation requires the presence of an oxidant. No substantial increase
and indigoid dye formation. Ultimately, enzymes from several in yield was observed for the substrates shown in entries 1–15
sources – b-glucosidase from almonds or from Agrobacterium sp.; of Table 1; the results are shown in Fig. S2 and Table S1 (ESI†).
tritosomes; and rat liver homogenate – were employed in corres- Also, the same set of substrates was examined with tritosomes
ponding buffers or media (see the Experimental section).
(lysosomes isolated by loading with a non-ionic detergent) but
the results were uniformly poor, although a low yield of
indigoid dye was obtained from 16 and 19 (Table S1, ESI†).
2. Indigogenic studies of monomeric indoxyl species
In initial studies, b-glucosidase from almonds was employed to The activity of the b-glucosidase (from almonds) was affected
trigger indigoid dye formation (Table 1). Thus, a mixture of this only slightly in the presence of a non-ionic detergent (Fig. S3,
enzyme (1 unit per mL) and an indoxyl b-glucoside (0.1 mmol, ESI†). To verify that the results observed in Table 1 were
1 mM) in sodium acetate buffer (pH 5.0) containing 5% DMF reliable, a 5 mg scale reaction of 33 was carried out to isolate
was incubated at 37 1C for 16–19 h. All indigoid dye was indigoid derivative 46, which was obtained in 66% yield
dissolved (vide infra) in each case for quantitative evaluation. (Scheme 7). The 66% isolated yield corresponded well with
The parent indoxyl-glucoside (1) afforded indigo only in 17% the yield of 52% determined by absorption spectroscopy using
yield under the reaction conditions (entry 1). By contrast, 5-bromo- the molar absorption coefficient for 46 (Table 1, entry 15).
4-chloroindoxyl b-glucoside (45, also known as X-Glu in a chromo-
Next, the b-glucosidase from Agrobacterium sp. was investigated
genic assay for b-glucosides)27 provided the corresponding indigoid as the trigger enzyme for indigoid dye formation. In contrast to
dye in 74% yield (entry 2, yield calculated based on e = 2.00 ꢁ b-glucosidase from almonds, which works chiefly under acidic
104 Mꢀ1 cmꢀ1 reported for 5,50-dibromo-4,40-dichloroindigo).56 conditions57 (optimum pH 5.6),58 b-glucosidase from Agrobacterium
These results are consistent with Holt’s report that such halo has a neutral pH optimum and maintains partial activity under
substituents on the indoxyl nucleus facilitated indigoid dye acidic (pH 4–5) and basic (pH 8–9) conditions as determined by the
formation. The time course of the spectral changes for substrates measured rate of hydrolysis of 4-nitrophenyl b-D-glucopyranoside
1 and 45 are shown in Fig. S1 (ESI†).
(Fig. S4, ESI†).59 Given that the indigoid dye-forming reaction is
No indigoid dye was detected with 15 (entry 3), whereas 16 reported to be faster at a basic rather than an acidic pH,29,60 the pH
formed the corresponding indigoid dye, albeit in low yield effect on indigogenesis was studied with the b-glucosidase from
(24%, entry 4). We measured the molar absorption coefficient Agrobacterium. The reaction was carried out using the enzyme
of the parent indigo 4 in DMF/water (2 : 1) and found the value at (200 nM) and indoxyl-glucoside 33 (100 mM) in NaPi buffers
l
max near 600 nm to be e = 1.27 ꢁ 104 Mꢀ1 cmꢀ1, to be compared (pH 4–9) containing 2% DMF at 37 1C. The progress of indigoid
with e = 1.66 ꢁ 104 Mꢀ1 cmꢀ1 in 1,1,2,2-tetrachloroethane dye formation as a function of pH is illustrated in Fig. 6A. High
reported by Holt and Sadler.56 For consistency, we have used to quantitative yields were attained in 3 h at pH 6–9; within
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Table 1 Indigogenic reactions of diverse indoxyl-glucosides
Yielda (%)
b-Glucosidase
b-Glucosidase
Rat liver
Entry
1
Indoxyl-glucoside
Structure
from almondsb
from Agrobacteriumc
homogenated
1
17
97 ꢂ 5
10
65
2
3
45
15
74e
116 ꢂ 8e
f
f
o1
—
—
f
4
5
16
19
24
—
9
o1
46 ꢂ 5
o5
6
7
20
21
o1
o1
150 ꢂ 3
o5
o5
63 ꢂ 4 (31 ꢂ 2)g
8
22
23
24
o1
30 ꢂ 6
o5
o5
o5
9
68
84 ꢂ 4
10
122 (59)g
209 ꢂ 6 (102 ꢂ 3)g
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Table 1 (continued)
Yielda (%)
b-Glucosidase
b-Glucosidase
Rat liver
Entry
11
Indoxyl-glucoside
Structure
from almondsb
from Agrobacteriumc
homogenated
25
26
27
o5
37 ꢂ 2
11
17
50
12
13
56
79 ꢂ 1
105 (51)g
184 ꢂ 3 (89 ꢂ 2)g
f
14
15
30
33
o1
21 ꢂ 1 (10 ꢂ 0.4)g
—
o5g
81 ꢂ 5g,h
52g
99 ꢂ 5g
f
f
16
44
—
106 ꢂ 4g
—
a
The yield was estimated by absorption spectroscopy with e = 1.27 ꢁ 104 Mꢀ1 cmꢀ1 (DMF/H2O = 2 : 1) measured for 4 (see the ESI) unless otherwise
noted. A yield of o1% implies no color was observed by visual inspection and no absorption was observed spectroscopically. A yield of o5%
b
implies a faint blue color was observed by visual inspection but the absorption was too weak for reliable spectroscopic determination. A mixture
of the indoxyl-glucoside (1 mM) and b-glucosidase from almonds (1 unit per mL) in acetate buffer (pH 5.0, sodium acetate, 50 mM, containing 5%
c
DMF) was incubated at 37 1C for 16–19 h. A mixture of the indoxyl-glucoside (100 mM) and b-glucosidase from Agrobacterium (200 nM) was
d
incubated in 50 mM NaPi buffer (pH 7.0) containing 2% DMF at 37 1C for 2 h. The reaction was repeated three times. The indoxyl-glucoside
e
(1 mM) in rat liver homogenate containing 5% DMF was incubated at 37 1C for 24 h. The yield was estimated from absorption spectroscopy with
g
e = 2.00 ꢁ 104 Mꢀ1 cmꢀ1 reported for 5,50-dibromo-4,40-dichloroindigo.56 f Not conducted. The yield was estimated from absorption spectroscopy
h
with e = 2.6 ꢁ 104 Mꢀ1 cmꢀ1 (DMF/H2O = 2 : 1) measured for 46. The reaction was carried out with 33 (100 mM) and b-glucosidase from
Agrobacterium (200 nM) plus rat liver homogenate containing 2% DMF at 37 1C for 4 h.
this range, pH 7 provided the best result. The reactions at pH 4 the enzymatic activity under the conditions. The effect of concen-
and 5 were also nearly complete in 3 h, although the yields were tration of 33 on the indigoid-forming reaction at pH 7.0 is illustrated
lower: 6% and 38%, respectively.
in Fig. 6C. High yields were maintained when the concentration was
The yields of indigoid dye formation from 33 with different 410 mM (89, 86, 85, and 90% at 56, 32, 18, and 10 mM, respectively),
enzyme concentrations (200 versus 10 nM) at 2 h are shown in while lower yields were obtained at lower concentrations (62, 51, 42,
Fig. 6B (see the Experimental section and ESI† for information and 41% at 5.6, 3.2, 1.8, and 1.0 mM, respectively). Lengthening the
about buffers). Good yields (50–71%) for oxidative dimerization reaction time from 2 h to 14 h improved the yields (88, 79, and 57%
obtained at pH 6–8 with 10 nM enzyme suggested that enzymatic at 3.2, 1.8, and 1.0 mM, respectively). The observation of substantial
cleavage of the sugar of 33 was not the rate-limiting step in indigoid dye formation even at 1 mM augurs well for in vivo
indigoid dye formation. A relatively large decrease in the yield at applications. Note that with 200 nM enzyme and 100 mM
pH 9 with 10 nM enzyme may be attributed to the importance of substrate, complete reaction requires 500 turnovers of each enzyme.
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Scheme 7 Isolation of indigo 46 from a 5 mg scale reaction with 33.
Given that reaction was still observed at 10 nM enzyme, such a
modest turnover appears reasonable. In other words, the cases
where incomplete reaction was observed likely were not due to
limiting enzyme concentration.
With the results in hand for indoxyl-glucoside 33, the 15 other
indoxyl-glucosides shown in Table 1 (100 mM) were similarly
treated with b-glucosidase from Agrobacterium (200 nM) in
50 mM NaPi buffer (pH 7.0) at 37 1C for 2 h. Unsubstituted
indoxyl-glucoside (1) and the 4-chloro-5-bromo derivative 45
provided good yields (97 and 116%, entries 1 and 2, respectively).
In contrast to b-glucosidase from almonds, the enzyme from
Agrobacterium cleaved the glucoside in indoxyl-glucosides con-
taining the triazine linker to give indigoid dye (entry 5 or 6, 46 or
150% yield). In the reactions of 22–27, the order on the basis
of yield was unsubstituted o 4-bromo o 4,6-dibromo indoxyl-
glucosides as observed with b-glucosidase from almonds
(entries 8–13). Indoxyl-glucoside 30 again resulted in low yield
(10%, entry 14), suggesting severe steric hindrance imposed by
the BCN group in the molecule. On the other hand, the indigoid
dye was quantitatively formed from indoxyl-glucosides 33 and 44
(entries 15 and 16, 99 and 106% yield, respectively).
Finally, indigoid-dye formation was carried out in the
presence of rat liver homogenate (Table 1, rightmost column).
Good yields were obtained in the case of 45 (65%, entry 2) and
27 (50%, entry 13). Indoxyl-glucoside 33 did not form an indigoid
product in rat liver homogenate (o5%, entry 15). However, when
b-glucosidase from Agrobacterium (200 nM) was included in the
presence of rat liver homogenate, the indigoid dye was obtained in
81% yield (entry 15).
Fig. 6 Study of reaction conditions for the indigoid-forming reaction
from 33 with b-glucosidase from Agrobacterium. (A) Effect of pH on the
reaction progress [33 (100 mM), enzyme (200 nM), 50 mM NaPi buffers,
n = 3]. (B) Effect of the enzyme concentration [33 (100 mM), 50 mM NaPi
buffer (pH 7.0), 2 h, n = 3]. (C) Effect of the concentration of 33 [enzyme
(200 nM), NaPi–NaCl (pH 7.0), 2 or 14 h, n = 3]. Yields were determined by
absorption spectroscopy of solubilized indigoid dye.
3. Synthesis of dimeric indoxyl species
We sought to carry out an enzyme-triggered oligomerization using
a bis(glucosyl-indoxyl) species bearing a water-solubilization
moiety. A long-term goal beyond the scope of work here is to
use the resulting oligomers as scaffolds for immobilization and Here, the sterically hindered strong base pempidine, which is
localization of molecular constituents in vivo. The synthesis known to facilitate nucleophilic reaction of alcohols with acid
of the monomer for oligomerization is shown in Scheme 8. chlorides,61 was used as the base to facilitate selective disubstitution
Treatment of acetyl-protected dibromoindoxyl-glucoside 32 (two of cyanuric chloride with the alcohol 32. 1,2-Dichloroethane was
molar equiv.) with cyanuric chloride resulted in substitution of two employed as solvent instead of dichloromethane to carry out the
of the three chlorine atoms in the latter to give chlorotriazine 47. reaction at 60 1C. Some loss of the N-acetyl group during
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Table 2 Study of the oligomerization of bis(indoxyl-glucoside) species
dialkoxylation of cyanuric chloride appeared responsible for purifi-
cation challenges that accounted for the isolation of 47 in 53% yield.
Removal of the N-acetyl groups of 47 was achieved in
CH2Cl2/methanol containing i-Pr2EtN, a combination of solvent
and hindered base employed for N-acetyl cleavage (in lieu of the
traditional methanol containing NaHCO3) owing to the poor
solubility of 47 in pure methanol. Subsequent reaction with
water-soluble amine 41 was performed in the presence of the
hindered base 2,6-lutidine (to neutralize HCl and TFA) to afford
48 in 55% yield. Deprotection of the glucosyl O-acetyl groups
provided the target bis(glucosyl-indoxyl) species 49 in 77% yield.
49a
Yield of indigoid dyeb (%)
Entry
[49], mM
Time, h
Supernatant
Precipitate
1c
2c
3c
4c
5e
6e
300
100
50
10
300
50
2
3
2
4
3
3
6
11
10
14
13 ꢂ 0.4d
16 ꢂ 0.6d
22
13
19
6
13
8
a
The reaction was carried out at 37 1C in air with 200 nM Agrobacterium
b
b-glucosidase (see Scheme 8). The yield was calculated from absorption
spectroscopy with e = 2.6 ꢁ 104 Mꢀ1 cmꢀ1
.
The reaction was carried out in
c
4. Oligomerization of dimeric indoxyl species
d
e
NaPi–NaCl. The reaction was repeated three times. The reaction was
carried out in 10 mM NaPi containing DMF (0.1–0.6%).
Oligomerization of 49 was carried out by treatment with
b-glucosidase from Agrobacterium (200 nM) in NaPi buffer (pH 7.0)
at 37 1C for 2–4 h (Scheme 8). Precipitation occurred during the from 300 to 10 mM, although the total yields were in the range
reaction. After centrifugation, the precipitate was separated from the 17–29%. While the reactions in entries 1–4 were carried out
supernatant, washed with H2O, and dried to afford a blue solid.
in NaPi–NaCl (NaPi containing 50 mM NaCl), those of entries 5
The efficacy of the indigogenic oligomerization of 49 was and 6 (with 300 and 50 mM of 49, respectively) were conducted
examined under a variety of conditions. As shown in entries 1–4 in ‘‘low-salt’’ conditions: 10 mM NaPi containing a trace of
in Table 2, the yield of indigoid dye in the supernatant versus DMF. The diminished quantity of NaCl facilitated analysis of
precipitate reversed as the concentration of 49 was decreased the indigoid dye in the supernatant.
Scheme 8 Synthesis and oligomerization of indigogenic bis(indoxyl) derivative 49.
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The time course of the oligomerization was examined under referred to as the supernatant extract. The precipitate and
the reaction conditions listed in entry 5 of Table 2, with 300 mM supernatant extracts were examined by absorption spectroscopy
of 49. The visible course of the reaction is shown in Fig. 7A. and compared with that of 46 (dissolved in DMF) (Fig. 7D). All
Noticeable changes include blue clouding and color deepening three samples showed a characteristic indigoid peak in the
at 20 min. Centrifugation enabled isolation of the precipitate range 550–700 nm. The greater absorbance at ca. 300 nm in the
and the supernatant. Optical microscopic analysis of the pre- precipitate and supernatant extracts versus that of 46 suggested
cipitate suspended in H2O showed small particles of up to contamination by impurities composed of indole derivatives.
several micron dimensions (Fig. 7B). Dynamic light scattering Size-exclusion chromatography (SEC) [DMF/DMSO (9 : 1) as
(DLS) analysis indicated that the particle size was B680 nm eluent] using a three-column sequence62 was applied to the
(number mean, Fig. 7C). The precipitate was dissolved in DMF/ precipitate extract and to the supernatant extract prepared from
DMSO (9 : 1) and is referred to as the precipitate extract. The 300 mM of 49 (Fig. 7E). In each case, the first peak appeared at
supernatant was concentrated to dryness; the resulting residue 18–20 min, and then the second peak at 38.5 min. The ratio of
was suspended in DMF and then filtered to remove salts. peak areas in the supernatant extract was 11 : 89, whereas that
The resulting clear filtrate containing indigoid materials is in the precipitate extract was 93 : 7. On the basis of a calibration
Fig. 7 (A) Time course of oligomerization with 49 under reaction conditions listed in entry 5 of Table 2. (B) Optical microscopic image (ꢁ40) of the
precipitate suspended in H2O. (C) DLS analysis of the precipitate suspended in H2O. (D) Absorption spectra (normalized at 637 nm) of the precipitate
extract in DMF/DMSO (9 : 1) (blue), the supernatant extract in DMF (red), and 46 in DMF (magenta). (E) Analytical SEC traces for the supernatant and
precipitate extracts from 300 mM of 49.
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curve prepared with poly(2-vinylpyridine) standards (Fig. S5, One interpretation is that the indigoid dye exists in distinct
ESI†), the molecular size for the first and second peaks was environments and/or unknown indole derivatives other than
expected to be 4265 and o1 kDa, respectively. The large the indigoid dye and indoxyl b-glucoside are present.
molecular size indicated for the first peak likely implies aggre-
Finally, the oligomerization of 49 was carried out on a larger
gation or assembly of the oligomers. Another possible inter- scale (7.87 mg) under the same ‘‘low-salt’’ reaction conditions as
pretation is that the chromatographic size standards employed those of entry 5 in Table 2. As a result, 3.01 mg of the precipitate
are inappropriate for these indigoid oligomers. The chromato- was obtained, which corresponds to 49% yield based on the
grams for the samples prepared with 50 mM of 49 (Fig. S6, ESI†) monomer formula weight. We attempted to use mass spectrometry
gave similar results but with higher purity compared with those at to gain information about the composition of the oligomeric
higher concentration. Further studies will be required to definitively indigoid products formed upon enzymatic treatment of 49.
gauge the size of the indigoid oligomers prepared herein.
Analysis of the supernatant by electrospray ionization mass
Samples of 33, 49, and the precipitate derived by oligomer- spectrometry (ESI-MS) revealed negative ion peaks at m/z 1214.0
ization of 49 were dissolved in DMSO-d6 and examined by and 2428.9, consistent with monomer cyclization (n = 1) and
1H NMR spectroscopy. The spectra are shown in Fig. 8A. The cyclodimerization (n = 2), respectively (Fig. S7 and S8, ESI†).
lack of signals from the glucosyl group (hydroxyl protons Analysis of the supernatant by matrix-assisted ionization mass
B5.0 ppm; the anomeric proton at 4.65 ppm) in the spectrum spectrometry (MALDI-MS) revealed a progression of broad peaks
of the precipitate is consistent with smooth enzymatic cleavage extending to m/z 4 10 000 with increment of m/z B 1210–1250
of the sugar moiety. On the other hand, the signals in the (Fig. S9, ESI†). Although the progression implies a mixture of
aromatic region of the precipitate were complicated (Fig. 8B). oligomers, the observed m/z values did not match the calculated
Fig. 8 Comparison of 1H NMR spectra (in DMSO-d6) of 33, 46, 49, and the precipitate derived by low-salt oligomerization of 49. (A) Spectral region
(4.5–5.5 ppm) showing sugar and hydroxyl peaks. (B) Spectral region (6.9–7.7 ppm) showing aromatic protons.
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values (Table S2, ESI†). Incomplete purification, decomposition by spectra are reported in parts per million (d) relative to tetramethyl-
laser irradiation (especially the bromoheteroarene units), or com- silane or a solvent signal (CD3OD, d = 3.31 ppm). Chemical shifts
plicated isotopic distribution caused by multiple bromine atoms for 13C NMR spectra are reported in parts per million (d), and
may contribute to the broad peaks. Attempts to use MALDI-MS to spectra were calibrated by using solvent signals [CDCl3, d =
analyze the precipitate, which was very insoluble, were unfruitful.
77.16 ppm; (CD3)2SO, d = 39.52 ppm; CD3OD, d = 49.00 ppm].66
Silica (40 mm), diol-functionalized silica (40–63 mm), and reverse
phase silica (C18, 40–63 mm) were used for column chromato-
graphy. Preparative TLC separations were carried out on Merck
analytical plates precoated with silica 60 F254. Microscopic
analysis was performed on a Zeiss Axio Imager M.2. DLS
analysis was performed on a Zetasizer Nano ZS. Centrifugation
was carried out at 20 000g at 4 1C. Absorption spectroscopy was
performed in the ultraviolet-visible region (200–700 nm).
Outlook
The ability to carry out enzymatically triggered spontaneous
cross-linking reactions under physiological conditions with no
exogenous reagents or catalysts opens the door to a number of
opportunities in the life sciences. Key features of the present
work include the chromogenic readout and the formation of an
aqueous-insoluble product. The four-fold challenge of achieving
facile indigoid formation via enzyme triggering in the presence of a
bioconjugatable tether in a synthetically accessible construct pre-
sented unexpected difficulties but after considerable development
was met with the 5-alkoxy-4,6-dibromoindoxyl-glucoside structures.
Altogether 36 new compounds were synthesized and characterized,
including 15 target indoxyl-glucosides. Two novel molecular designs
that emerged include an indoxyl-glucoside bearing a BCN group for
copper-free click chemistry (44), and a bis(indoxyl-glucoside) (49);
in both cases the intervening linker is composed of two short PEG
groups and a central triazine derivatized with a sulfobetaine for
water solubilization. Within the general designs identified, further
alteration to the 4,6-dibromo-5-alkoxy aglycone moiety may include
(1) replacement of the glucoside with galactosides, glucuronides
or more complex polysaccharides as well as other enzyme-
cleavable moieties; (2) increasing the length of the PEG spacers;
(3) replacement of the BCN group (which upon click reaction
yields isomers) by a functionalized cycloalkyne63 that facilitates
isomer-free generation of ‘‘clicked compounds’’ or other reactive
handles for bioconjugation; and (4) construction of multimeric
analogues of the dimeric 49.
Chemicals
All solvents were reagent grade and were used as received unless
noted otherwise. NBS was recrystallized from water. Na2SO4 was
anhydrous. The CH2Cl2 employed in reactions was commercial
anhydrous grade. Other solvents and reagents were used as
received. The known compounds 9,39 1246 and 3449 were pre-
pared generally following procedures described in the literature.
The ion exchange resin (DOWEX 50WX8-200) was in the proto-
nated form and is designated DOWEX H+.
Enzymes
b-Glucosidase from almonds (lyophilized powder, Z2 units per mg
solid) and peroxidase from horseradish were purchased from Sigma-
Aldrich. b-Glucosidase from Agrobacterium sp. (recombinant,
suspension in 3.2 M (NH4)2SO4) was purchased from Megazyme;
the concentration in solution was determined by absorption
spectroscopy with E0.1% = 2.20 cmꢀ1 at 280 nm.59 Tritosomes
were purchased from XenoTech. Rat liver homogenate was
purchased from MP Biomedicals.
Buffers
A nascent area of chemical biology concerns synthetic
chemistry in vivo, for which the present indigogenic molecules
would seem to be well suited. One recent thrust has been to identify
oxidase enzymes that catalyze the formation of indigoids from
indoles (not indoxyls) bearing diverse functional groups (amino-
methyl, hydroxymethyl, carboxaldehyde, carboxylic acid).64 A second
recent thrust has been to genetically engineer the production of
halogenated indoxyl-glucosides.65 An open issue over the long term
is whether the types of synthetic indoxyl species prepared herein
could also be produced via genetic expression, thereby providing a
counterpart that melds some of the attractive features of fluorescent
proteins and click-chemistry processes with reactions that proceed
under physiological conditions. A more immediate issue is the
examination of the utility of the present structures for the covalent
assembly of macromolecular scaffolds in vivo.
For enzymatic studies, several buffers were prepared. All buffers
contain the sodium cation and were adjusted to a certain pH value
with 1 decimal point before use. The chief buffers were as follows:
(1) acetate buffer – prepared from sodium acetate (pH 5.0, 50 mM);
(2) 50 mM NaPi buffer (pH 7.0); (3) 10 mM NaPi buffer (pH 7.0);
and (4) NaPi–NaCl buffer (pH 7.0, 10 mM NaPi and 50 mM NaCl),
where NaPi refers to sodium/hydrogen phosphate in aqueous
solution at the indicated pH value. Other buffers were prepared
for pH-variation studies. Other constituents often were present in
indigogenic reactions owing to the presence of salts from the
enzyme stock solutions. For example, ammonium sulfate (3.2 M) is
present in the stock solution of b-glucosidase from Agrobacterium.
Also, the first dilution of the enzyme stock solution was carried out
with NaPi–NaCl buffer; thus, regardless of subsequent dilution
into an NaPi buffer, residual NaCl was present. A full description
of the concentrations of all reactants and buffer constituents for
the various indigogenic reactions is provided in the ESI.†
Experimental section
General methods
Bases
1H NMR and 13C NMR spectra were collected at room temperature A number of organic bases were used herein as part of the
in CDCl3 unless noted otherwise. Chemical shifts for 1H NMR triazine and other substitution reactions. The pKa value of the
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conjugate acid of a given base in water is provided in parentheses 131.4, 141.0, 147.6, 168.1, 169.2, 169.4, 170.2, 170.5, 172.5,
as follows: 2,6-di-tert-butylpyridine (3.58),48 1,10-phenanthroline 172.8, 173.2; ESI-MS obsd 665.1499, calcd 665.1492 [(M + H)+,
(4.96),67 2,6-lutidine (5.77),48 triethylamine (10.85),68 pempidine M = C28H29ClN4O13].
(1,2,2,6,6-pentamethylpiperidine, 11.25),69 and N,N-diisopropyl-
1-Acetyl-5-[(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)oxy]-1H-
indol-3-yl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside (morph-13).
ethylamine (11.44).70
5-Hydroxy-1H-indol-3-yl b-D-glucopyranoside (10). A suspension A solution of 13 (10.0 mg, 0.015 mmol) and i-Pr2EtN (3.4 mL,
of 9 (917.4 mg, 1.50 mmol), having 499% stereochemical purity at 0.020 mmol) in CH2Cl2 (150 mL) at room temperature was treated
the anomeric carbon, in MeOH (7.50 mL) at room temperature was with morpholine (1.4 mL, 0.017 mmol). After 2 h, the reaction
treated with sodium methoxide (25 wt% solution in MeOH, mixture was quenched by the addition of AcOH (0.9 mL) and
648 mL, 3.0 mmol). After 2 h, acetic acid (229 mL, 6.00 mmol) directly purified by chromatography (silica, ethyl acetate) to
1
and Pd/C (10 wt%, 79.8 mg, 0.075 mmol) were added. The reaction afford a white solid (10.6 mg, 99%): H NMR (700 MHz, CDCl3)
mixture was stirred for 2 h under a H2 atmosphere (balloon) at d 2.05 (s, 3H), 2.06 (s, 3H), 2.08 (s, 3H), 2.10 (s, 3H), 2.61 (s, 3H),
room temperature and then filtered through a Celite pad. The 3.64–3.75 (m, 6H), 3.81–3.90 (m, 3H), 3.90 (s, 3H), 4.24
filtrate was concentrated and purified by chromatography [silica, (dd, J = 4.5, 12.1 Hz, 1H), 4.29 (dd, J = 2.3, 12.1 Hz, 1H), 5.00–
CH2Cl2/MeOH (7 : 3)] to afford a pale yellow solid (417.6 mg, 89%): 5.06 (m, 1H), 5.14–5.22 (m, 1H), 5.27–5.34 (m, 2H), 7.17 (d, J =
mp darkened 455 1C; 1H NMR [400 MHz, (CD3)2SO] d 3.09–3.29 2.4 Hz, 1H), 7.19 (d, J = 2.4 Hz, 1H), 7.27 (s, 1H), 8.40 (br s, 1H);
(m, 4H), 3.42–3.53 (m, 1H), 3.65–3.76 (m, 1H), 4.47–4.55 (m, 1H), 13C NMR (175 MHz, CDCl3) d 20.7, 20.8, 20.9, 24.0, 44.1, 44.2,
4.59 (br s, 1H), 5.07 (br s, 1H), 5.15 (br s, 1H), 5.38 (br s, 1H), 6.58 54.8, 62.0, 66.7, 66.7, 68.3, 71.1, 72.57, 72.59, 100.8, 110.5, 110.7,
(dd, J = 2.6, 8.6 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.98 (d, J = 2.6 Hz, 117.4, 120.8, 124.7, 131.2, 141.3, 148.6, 166.9, 168.2, 169.4, 169.5,
1H), 7.06 (d, J = 8.6 Hz, 1H), 8.68 (br s, 1H), 10.21 (s, 1H); 13C NMR 170.3, 170.7, 172.5, 172.7; ESI-MS obsd 716.2410, calcd 716.2410
(100 MHz, CD3OD) d 62.6, 71.5, 75.0, 78.02, 78.04, 102.5, 105.8, [(M + H)+, M = C32H37N5O14].
112.9, 113.1, 113.5, 121.9, 130.4, 138.4, 160.0; ESI-MS obsd
334.0894, calcd 334.0897 [(M + H)+, M = C14H17NO7].
5-{[4-({1-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]-3-oxo-2,7,10-
trioxa-4-azadodecan-12-yl}amino)-6-methoxy-1,3,5-triazin-2-yl]oxy}-
1-Acetyl-5-hydroxy-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-b-D-gluco- 1H-indol-3-yl b-D-glucopyranoside (15). A sample of 13 (20.3 mg,
pyranoside (11). A suspension of 9 (6.911 g, 11.3 mmol) and 0.0305 mmol) was added to a solution of (1R,8S,9s)-bicyclo-
Pd/C (10 wt%, 360.8 mg, 0.339 mmol) in ethyl acetate/EtOH [6.1.0]non-4-yn-9-ylmethanol (10.4 mg, 0.0321 mmol) and i-Pr2EtN
(4 : 1, 113 mL) was stirred for 3 h at room temperature under a (6.7 mL, 0.038 mmol) in CH2Cl2 (150 mL) at room temperature. After
H2 atmosphere (balloon). The reaction mixture was filtered 3 h, MeOH (750 mL) and K2CO3 (13.3 mg, 0.096 mmol) were added.
through a Celite pad. The filtrate was concentrated and purified After 1 h, the reaction mixture was quenched by the addition of
by chromatography [silica, CH2Cl2/ethyl acetate (10 : 1)] to afford acetic acid (9.2 mL) and then filtered. The filtrate was concentrated
a pale yellow solid (5.85 g, 99%): mp 88–90 1C; 1H NMR and purified by chromatography [silica, CH2Cl2/MeOH (3 : 1)] to
(400 MHz, CDCl3) d 2.05 (s, 3H), 2.06 (s, 3H), 2.09 (s, 3H), 2.12 afford a pale yellow solid (21.0 mg, 93%): 1H NMR (700 MHz,
(s, 3H), 2.56 (s, 3H), 3.77–3.88 (m, 1H), 4.23 (dd, J = 5.0, 12.4 Hz, CD3OD, B1 : 1 mixture of rotamers) d 0.85–0.98 (m, 2H), 1.25–1.40
1H), 4.93–5.03 (m, 1H), 5.11–5.23 (m, 1H), 5.23–5.34 (m, 2H), (m, 1H), 1.49–1.65 (m, 2H), 2.09–2.30 (m, 6H), 3.10–3.18 (m, 1H),
5.82–6.02 (m, 1H), 6.85–6.96 (m, 2H), 7.10 (br s, 1H), 8.22 3.23–3.28 (m, 1H), 3.29–3.63 (m, 14H), 3.716 (dd, J = 5.6, 11.9 Hz,
(br s, 1H); 13C NMR (100 MHz, CDCl3) d 20.63, 20.66, 20.73, 0.5H), 3.719 (dd, J = 5.7, 11.9 Hz, 0.5H), 3.87–3.93 (m, 2.5H), 3.94
20.8, 23.7, 62.1, 68.3, 71.1, 72.4, 72.6, 101.0, 103.2, 110.9, 115.1, (s, 1.5H), 4.10 (d, J = 8.1 Hz, 0.5H), 4.12 (d, J = 8.2 Hz, 0.5H), 4.68
117.7, 125.4, 128.3, 141.3, 153.0, 168.2, 169.58, 169.63, (d, J = 8.1 Hz, 0.5H), 4.70 (d, J = 8.1 Hz, 0.5H), 6.67–6.73
170.4, 171.0; ESI-MS obsd 544.1430, calcd 544.1426 [(M + Na)+, (m, 0.5H), 6.78–6.84 (m, 0.5H), 6.87 (dd, J = 2.3, 8.7 Hz, 1H),
M = C24H27NO12].
6.90 (dd, J = 2.3, 8.7 Hz, 1H), 7.17 (s, 1H), 7.28 (d, J = 8.7 Hz,
1-Acetyl-5-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)oxy]-1H-indol- 0.5H), 7.29 (d, J = 8.7 Hz, 0.5H), 7.47 (d, J = 2.3 Hz, 0.5H), 7.48
3-yl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside (13). A sample of (d, J = 2.3 Hz, 0.5H); 13C NMR (175 MHz, CD3OD, mixture of
i-Pr2EtN (65.3 mL, 0.375 mmol) was added dropwise over 5 min rotamers) d 18.9, 21.4, 21.9, 30.1, 41.2, 41.5, 41.6, 41.7, 41.9,
to a suspension of 11 (130.4 mg, 0.250 mmol) and 12 (58.5 mg, 55.2, 55.3, 62.6, 63.70, 63.74, 70.0, 70.3, 70.8, 70.88, 70.91, 71.16,
0.325 mmol) in CH2Cl2 (1.25 mL) at 0 1C. The reaction mixture 71.22, 71.5, 75.0, 78.0, 78.16, 78.19, 99.5, 105.9, 106.0, 110.8,
was allowed to warm to room temperature and stirred for 2 h. 111.0, 112.7, 112.9, 114.0, 114.2, 117.3, 117.5, 121.4, 121.5,
The reaction mixture was washed with aqueous citric acid 132.9, 133.0, 139.18, 139.24, 146.4, 146.6, 159.2, 159.2, 169.3,
(10%, 1 mL) followed by brine (1 mL), then dried (Na2SO4) 169.5, 173.4, 173.7, 173.9, 174.1; ESI-MS obsd 743.3249, calcd
and filtered. The filtrate was concentrated and purified by 743.3247 [(M + H)+, M = C35H46N6O12].
chromatography [silica, hexanes/ethyl acetate (2 : 3)] to afford
5-[((Benzylcarbamoyl)oxy)]-1H-indol-3-yl b-D-glucopyranoside
a white solid (188.3 mg, 87%): 1H NMR (300 MHz, CDCl3) d 2.05 (16). Samples of p-nitrophenyl chloroformate (6.0 mg, 0.029 mmol)
(s, 3H), 2.06 (s, 3H), 2.09 (s, 3H), 2.10 (s, 3H), 2.62 (s, 3H), 3.80– and i-Pr2EtN (6 mL, 0.03 mmol) were added to a solution of 11
3.95 (m, 1H), 4.02 (s, 3H), 4.14–4.38 (m, 2H), 4.97–5.09 (m, 1H), (13.0 mg, 0.025 mmol) in CH2Cl2 (1 mL) at room temperature.
5.09–5.40 (m, 3H), 7.10–7.36 (m, 3H), 8.44 (br s, 1H); 13C NMR After 1 h, the reaction mixture was quenched by the addition
(175 MHz, CDCl3) d 20.6, 20.68, 20.71, 23.8, 56.3, 61.9, 68.2, of saturated aqueous NH4Cl (2 mL) and stirred for 30 min at
70.9, 72.37, 72.43, 100.7, 110.2, 111.1, 117.7, 119.7, 124.8, room temperature. Water (2 mL) was added, and then the
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mixture was extracted with Et2O (3 ꢁ 2 mL). The combined [silica, hexanes/ethyl acetate (1 : 1)] followed by recrystallization
organic extract was washed with H2O (2 mL), brine (2 mL), dried from CH2Cl2/MeOH afforded a white solid (6.32 g, 83%): mp
(Na2SO4), and filtered. The filtrate was concentrated under 102–103 1C; 1H NMR (700 MHz, CDCl3) d 2.05 (s, 3H), 2.07
reduced pressure. The residue was dissolved in CH2Cl2 (1 mL). (s, 3H), 2.10 (s, 3H), 2.10 (s, 3H), 2.59 (s, 3H), 3.91 (ddd, J = 2.4,
Benzylamine (3 mL, 0.03 mmol) was added to the solution at 5.2, 9.9 Hz, 1H), 4.22 (dd, J = 5.2, 12.5 Hz, 1H), 4.37 (dd, J = 2.4,
room temperature. After 20 h, the reaction mixture was con- 12.5 Hz, 1H), 5.05 (d, J = 7.6 Hz, 1H), 5.21 (dd, J = 9.5, 9.9 Hz,
centrated under reduced pressure. The residue was dissolved in 1H), 5.31 (dd, J = 9.3, 9.5 Hz, 1H), 5.38 (dd, J = 7.6, 9.3 Hz, 1H),
MeOH (1 mL). NaOMe (25% in MeOH, 5 mL, 0.02 mmol) was 6.02 (s, 1H), 7.22 (br s, 1H), 8.63 (br s, 1H); 13C NMR (100 MHz,
added to the solution at room temperature. After 45 min, the CDCl3) d 20.7, 20.9, 21.0, 23.7, 62.1, 68.4, 71.0, 72.7, 72.8, 98.4,
reaction mixture was quenched by the addition of a sample of 100.3, 108.6, 112.0, 120.0, 122.6, 128.7, 140.2, 146.3, 167.8, 169.4,
ion exchange resin (DOWEX H+) until the pH reached near 7. 169.5, 170.3, 170.6; ESI-MS obsd 699.9645, calcd 699.9636
The mixture was then stirred for 20 min at room temperature [(M + Na)+, M = C24H25Br2NO12]. Suitable crystals for X-ray analysis
followed by filtration. The filtrate was concentrated under were obtained by recrystallization from cyclohexane/acetone.
reduced pressure. Column chromatography [silica, CH2Cl2/MeOH
5-[(4-Methoxy-6-morpholino-1,3,5-triazin-2-yl)oxy]-1H-indol-3-yl
1
(5 : 1)] afforded a colorless oil (5.9 mg, 53%): H NMR (700 MHz, b-D-glucopyranoside (19). A sample of i-Pr2EtN (13.1 mL, 0.075 mmol)
CD3OD) d 3.32–3.36 (m, 1H), 3.40 (t, J = 9.0 Hz, 1H), 3.43 (t, J = was added to a solution of 10 (15.6 mg, 0.050 mmol) and 12 (9.9 mg,
9.0 Hz, 1H), 3.48 (dd, J = 9.0, 8.0 Hz, 1H), 3.71 (dd, J = 12.0, 6.0 Hz, 0.055 mmol) in DMF (125 mL) at room temperature. After 3 h,
1H), 3.90 (dd, J = 12.0, 2.0 Hz, 1H), 4.37 (s, 2H), 4.67 (d, J = 8.0 Hz, morpholine (8.6 mL, 0.10 mmol) was added. After 2 h, the reaction
1H), 6.85 (dd, J = 8.0, 2.0 Hz, 1H), 7.14 (s, 1H), 7.24–7.28 (m, 2H), mixture was passed through silica. The resulting solution was
7.31–7.40 (m, 4H), 7.43 (d, J = 2.0 Hz, 1H); 13C NMR (175 MHz, concentrated under reduced pressure. Column chromatography
CD3OD) d 45.7, 62.6, 71.5, 75.0, 78.0, 78.2, 106.0, 111.0, 112.7, [silica, CHCl3/MeOH (4 : 1)] afforded a white solid (15.0 mg, 59%):
114.2, 117.5, 121.5, 128.2, 128.4, 129.6, 132.9, 139.0, 140.4, 1H NMR (300 MHz, CD3OD) d 3.25–3.93 (m, 14H), 3.92 (s, 3H), 4.67
145.4, 158.6; ESI-MS obsd 467.1419, calcd 467.1425 [(M + Na)+, (d, J = 7.2 Hz, 1H), 6.87 (dd, J = 8.7, 1.8 Hz, 1H), 7.16 (s, 1H), 7.27
M = C22H23N2NaO8].
(d, J = 8.7 Hz, 1H), 7.46 (d, J = 1.8 Hz, 1H); 13C NMR (75 MHz,
1-Acetyl-4-bromo-5-hydroxy-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl- CD3OD) d 45.2, 55.3, 62.6, 67.5, 71.5, 75.0, 78.0, 78.2, 106.0,
b-D-glucopyranoside (17). A solution of NBS in CH2Cl2 (100 mM, 110.7, 112.7, 114.1, 117.3, 121.4, 132.9, 139.2, 146.5, 167.9,
4.20 mL) was added dropwise over 5 min to a solution of 11 173.8, 174.0; ESI-MS obsd 506.1883, calcd 506.1880 [(M + H)+,
(208.6 mg, 0.400 mmol) and 2,6-di-tert-butylpyridine (88 mL, M = C22H27N5O9].
0.40 mmol) in CH2Cl2 (5.80 mL) at ꢀ78 1C. After 1.5 h, the
1-Acetyl-4-bromo-5-[(4-methoxy-6-morpholino-1,3,5-triazin-
reaction mixture was allowed to warm to room temperature 2-yl)oxy]-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside
with stirring for 30 min. The reaction mixture was washed with (pre-20). A sample of i-Pr2EtN (10.5 mL, 0.060 mmol) was added
saturated aqueous Na2S2O3 (3 mL) and brine (5 mL), then dried to a solution of 17 (24.0 mg, 0.040 mmol) and 12 (7.9 mg,
(Na2SO4) and concentrated under reduced pressure. Column 0.044 mmol) in CH2Cl2 (200 mL) at room temperature. After
chromatography [silica, hexanes/CH2Cl2/MeCN (2: 1: 1)] afforded 30 min, morpholine (6.9 mL, 0.080 mmol) was added. After 3 h,
a white solid (180.6 mg, 75%): mp 130 1C (dec.); 1H NMR the reaction mixture was quenched by the addition of acetic acid
(400 MHz, CDCl3) d 2.05 (s, 3H), 2.08 (s, 3H), 2.10 (s, 3H), 2.10 (2.2 mL) and then passed through silica (ethyl acetate as eluent).
(s, 3H), 3.86–3.96 (m, 1H), 4.22 (dd, J = 5.4, 12.3 Hz, 1H), 4.36 The eluent was concentrated under reduced pressure. Column
(dd, J = 2.0, 12.3 Hz, 1H), 5.06 (d, J = 7.8 Hz, 1H), 5.21 (dd, J = 9.2, chromatography [silica, hexanes/ethyl acetate (1 : 2)] afforded
9.2 Hz, 1H), 5.31 (dd, J = 9.2, 9.2 Hz, 1H), 5.40 (dd, J = 7.8, 9.2 Hz, a white solid (27.6 mg, 87%): 1H NMR (400 MHz, CDCl3) d
1H), 5.74 (s, 1H), 7.05 (d, J = 8.8 Hz, 1H), 7.22 (br s, 1H), 8.29 (br s, 3.60–3.76 (m, 6H), 3.78–3.94 (m, 6H), 4.20 (dd, J = 5.2, 12.5 Hz,
1H); 13C NMR (75 MHz, CDCl3) d 20.8, 20.9, 21.0, 23.9, 62.1, 68.3, 1H), 4.37 (dd, J = 2.2, 12.5 Hz, 1H), 5.06 (d, J = 7.6 Hz, 1H),
71.0, 72.6, 72.8, 98.6, 100.3, 111.2, 114.7, 117.1, 122.6, 129.0, 5.19 (dd, J = 9.3, 9.3 Hz, 1H), 5.29 (dd, J = 9.3, 9.3 Hz, 1H), 5.37
140.5, 149.4, 167.9, 169.4, 169.5, 170.4, 170.6; ESI-MS obsd (dd, J = 7.6, 9.3 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 7.30 (br s, 1H),
600.0712, calcd 600.0711 [(M + H)+, M = C24H26BrNO12]. Suitable 8.30–8.50 (m, 1H); 13C NMR (175 MHz, CDCl3) d 20.7, 20.9, 21.0,
crystals for X-ray analysis were obtained by recrystallization from 24.0, 44.06, 44.11, 54.8, 62.0, 66.6, 66.7, 68.3, 70.8, 72.6, 72.7,
cyclohexane/CHCl3.
100.3, 106.4, 112.1, 116.3, 121.5, 123.5, 132.1, 140.8, 146.2,
1-Acetyl-4,6-dibromo-5-hydroxy-1H-indol-3-yl 2,3,4,6-tetra-O- 166.9, 168.1, 169.3, 169.5, 170.3, 170.6, 171.9, 172.6; ESI-MS
acetyl-b-D-glucopyranoside (18). A solution of NBS (3.987 g, obsd 816.1321, calcd 816.1334 [(M + Na)+, M = C32H36BrN5O14].
22.4 mmol) in CH2Cl2 (240 mL) was added dropwise over 1 h to
4-Bromo-5-[(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)oxy]-1H-
a solution of 11 (5.841 g, 11.2 mmol) and 2,6-di-tert-butylpyridine indol-3-yl b-D-glucopyranoside (20). A suspension of pre-20
(2.47 mL, 11.2 mmol) in CH2Cl2 (160 mL) at ꢀ78 1C. The reaction (15.9 mg, 0.020 mmol) and K2CO3 (2.8 mg, 0.020 mmol) in MeOH
mixture was allowed to warm to room temperature with stirring for was stirred at room temperature for 20 min. The reaction mixture
3.5 h. NBS (598 mg, 3.36 mmol) was added. After 1 h, the reaction was quenched by the addition of acetic acid (2.9 mL) and
mixture was washed with aqueous Na2S2O3 (10%, 50 mL) and concentrated under reduced pressure. Column chromatography
brine (50 mL), then dried (Na2SO4) and filtered. The filtrate was [silica, CH2Cl2/MeOH (1 : 2)] afforded a white solid (10.4 mg, 89%):
concentrated under reduced pressure. Column chromatography 1H NMR (300 MHz, CD3OD) d 3.34–4.00 (m, 17H), 4.76
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(d, J = 7.8 Hz, 1H), 7.21–7.34 (m, 2H); 13C NMR (175 MHz, H2O (2 mL) and brine (2 mL), then dried (Na2SO4) and filtered.
CD3OD) d 45.1, 45.3, 55.29, 55.34, 62.65, 62.69, 67.5, 71.6, 75.3, The filtrate was concentrated under reduced pressure. The
78.1, 78.3, 105.2, 106.1, 112.2, 114.7, 118.3, 119.8, 133.6, 139.2, residue was dissolved in MeOH (0.5 mL). NaOMe (25% in
143.8, 167.9, 173.4, 173.8; ESI-MS obsd 584.0986, calcd MeOH, 5 mL, 0.02 mmol) was added to the solution at room
584.0987 [(M + H)+, M = C22H26BrN5O9].
temperature. After 45 min, the reaction mixture was quenched
4,6-Dibromo-5-[(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)oxy]- by the addition of a sample of ion exchange resin (DOWEX H+)
1H-indol-3-yl b-D-glucopyranoside (21). A sample of i-Pr2EtN until the pH reached near 7. The mixture was then stirred for
(7.3 mL, 0.042 mmol) was added to a solution of 18 (19.0 mg, 20 min at room temperature followed by filtration. The filtrate
0.028 mmol) and 12 (5.54 mg, 0.031 mmol) in CH2Cl2 (140 mL) was concentrated and purified by chromatography [silica, CH2Cl2/
1
at 0 1C. The reaction mixture was allowed to warm to room MeOH (5 : 1)] to afford a colorless oil (5.4 mg, 52%): H NMR
temperature with stirring for 1 h. Morpholine (4.8 mL, 0.056 mmol) (300 MHz, CD3OD) d 3.34–3.56 (m, 4H), 3.72 (dd, J = 12.0, 5.0 Hz,
was added. After 3 h, MeOH (560 mL) and K2CO3 (19.3 mg, 1H), 3.80 (s, 3H), 3.92 (d, J = 12.0 Hz, 1H), 4.66 (s, 2H), 4.75
0.14 mmol) were added. The reaction mixture was heated at (d, J = 7.0 Hz, 1H), 6.91 (dd, J = 9.0, 1.0 Hz, 1H), 7.20 (dd, J = 9.0,
35 1C for 1 h and then allowed to cool to room temperature. The 1.0 Hz, 1H), 7.24 (s, 2H); 13C NMR (175 MHz, CD3OD) d 52.5,
reaction mixture was quenched by the addition of acetic acid 69.7, 69.8, 71.6, 75.4, 78.3, 103.3, 105.3, 112.2, 113.9, 115.0,
(16 mL) and filtered. The filtrate was concentrated under reduced 120.3, 132.5, 138.9, 149.7, 171.7; ESI-MS obsd 484.0205, calcd
pressure. Column chromatography [silica, CH2Cl2/MeOH (6: 1)] 484.0214 [(M + Na)+, M = C17H20BrNO9].
afforded a white solid (12.5 mg, 67%): 1H NMR (300 MHz,
4,6-Dibromo-5-(methoxycarbonyl)methoxy-1H-indol-3-yl b-D-
CD3OD) d 3.34–4.02 (m, 17H), 4.77 (d, J = 7.2 Hz, 1H), 7.30 (s, glucopyranoside (24). Ethyl bromoacetate (3.0 mL, 27 mmol) and
1H), 7.56 (s, 1H); 13C NMR (175 MHz, CD3OD) d 45.1, 45.3, 55.4, NaH (1.0 mg, 42 mmol) were added to a solution of 18 (12.7 mg,
55.5, 62.6, 67.4, 71.5, 75.2, 78.1, 78.3, 105.00, 105.02, 107.6, 111.2, 0.019 mmol) in DMF (1 mL) at room temperature. After 1 h, the
115.2, 115.7, 119.5, 133.6, 139.2, 140.6, 167.9, 172.6, 173.9; ESI- reaction mixture was quenched by the addition of saturated
MS obsd 662.0098, calcd 662.0092 [(M + H)+, M = C22H25Br2N5O9]. aqueous NH4Cl (2 mL) and stirred for 20 min at room temperature.
5-(Methoxycarbonyl)methoxy-1H-indol-3-yl b-D-glucopyranoside Water (2 mL) was added, then the mixture was extracted with
(22). Ethyl bromoacetate (5.0 mL, 45 mmol) and NaH (2.0 mg, Et2O (3 ꢁ 2 mL). The combined organic extract was washed with
83 mmol) were added to a solution of 11 (10.0 mg, 0.019 mmol) H2O (2 mL) and brine (2 mL), then dried (Na2SO4) and filtered.
in DMF (1 mL) at room temperature. After 1 h, the reaction The filtrate was concentrated under reduced pressure. The
mixture was quenched by the addition of saturated aqueous residue was dissolved in MeOH (1 mL). NaOMe (25% in MeOH,
NH4Cl (2 mL) and stirred for 10 min at room temperature. 5 mL, 0.02 mmol) was added to the solution at room temperature.
Water (2 mL) was added, then the mixture was extracted with After 45 min, the reaction mixture was quenched by the addition
Et2O (3 ꢁ 2 mL). The combined organic extract was washed with of a sample of ion exchange resin (DOWEX H+) until the pH
H2O (2 mL) and brine (2 mL), then dried (Na2SO4) and filtered. reached near 7. The mixture was then stirred for 20 min at room
The filtrate was concentrated under reduced pressure. The temperature followed by filtration. The filtrate was concentrated
residue was dissolved in MeOH (1 mL). NaOMe (25% in MeOH, and purified by chromatography [silica, CH2Cl2/MeOH (5 :1)] to
5 mL, 0.02 mmol) was added to the solution at room tempera- afford a colorless oil (8.3 mg, 82%): 1H NMR (700 MHz, CD3OD) d
ture. After 45 min, the reaction mixture was quenched by the 3.37–3.41 (m, 2H), 3.45 (t, J = 9.0 Hz, 1H), 3.53 (dd, J = 9.0, 8.0 Hz,
addition of a sample of ion exchange resin (DOWEX H+) until 1H), 3.70 (dd, J = 12.0, 5.0 Hz, 1H), 3.84 (s, 3H), 3.92 (d, J = 12 Hz,
the pH reached near 7. The mixture was then stirred for 20 min 1H), 4.61 (s, 2H), 4.74 (d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.51 (s, 1H);
at room temperature followed by filtration. The filtrate was 13C NMR (175 MHz, CD3OD) d 52.6, 62.7, 70.3, 71.6, 75.3, 78.2,
concentrated and purified by chromatography [silica, CH2Cl2/ 78.3, 105.1, 107.6, 111.6, 115.4, 116.1, 119.9, 133.2, 139.1,
1
MeOH (5 : 1)] to afford a colorless oil (3.9 mg, 53%): H NMR 145.6, 170.6; ESI-MS obsd 561.9310, calcd 561.9319 [(M + Na)+,
(300 MHz, CD3OD) d 3.34–3.56 (m, 4H), 3.72 (dd, J = 12.0, M = C17H19Br2NO9].
5.0 Hz, 1H), 3.80 (s, 3H), 3.91 (dd, J = 12.0, 2.0 Hz, 1H), 4.66
5-Propargyloxy-1H-indol-3-yl b-D-glucopyranoside (25). A
(d, J = 7.5 Hz, 1H), 4.71 (s, 2H), 6.82 (dd, J = 9.0, 2.5 Hz, 1H), 7.09 suspension of 10 (15.6 mg, 0.050 mmol), propargyl bromide
(s, 1H), 7.16–7.22 (m, 2H); 13C NMR (75 MHz, CD3OD) d 52.5, (18.6 mL, 80% in toluene, 0.125 mmol), and K2CO3 (17.2 mg,
62.7, 67.0, 71.5, 75.1, 78.0, 78.2, 101.5, 106.0, 113.3, 113.7, 113.9, 0.124 mmol) in DMF (125 mL) was heated to 80 1C for 2.5 h. The
153.1, 172.1; ESI-MS obsd 406.1109, calcd 406.1109 [(M + Na)+, reaction mixture was allowed to cool to room temperature and
M = C17H21NO9].
then passed through silica (CH2Cl2/MeOH = 1 : 1 as eluent). The
4-Bromo-5-(methoxycarbonyl)methoxy-1H-indol-3-yl b-D-gluco- eluent was concentrated under reduced pressure. Preparative
pyranoside (23). Ethyl bromoacetate (4.0 mL, 36 mmol) and NaH thin layer chromatography [silica, 0.25 mm, 20 ꢁ 20 cm, CHCl3/
(1.0 mg, 42 mmol) were added to a solution of 17 (15 mg, MeOH (4 : 1)] afforded a brown solid (5.3 mg, 30%): 1H NMR
0.025 mmol) in DMF (0.5 mL) at room temperature. After 1 h, (400 MHz, CD3OD) d 2.88 (t, J = 2.4 Hz, 1H), 3.32–3.54 (m, 4H),
the reaction mixture was quenched by the addition of saturated 3.73 (dd, J = 5.0, 11.8 Hz, 1H), 3.92 (dd, J = 2.2, 11.8 Hz, 1H), 4.69
aqueous NH4Cl (2 mL) and stirred for 10 min at room temperature. (d, J = 7.6 Hz, 1H), 4.71 (d, J = 2.4 Hz, 2H), 6.79 (dd, J = 2.4,
Water (2 mL) was added, then the mixture was extracted with 8.8 Hz, 1H), 7.09 (s, 1H), 7.18 (d, J = 8.8 Hz, 1H), 7.28 (d, J =
Et2O (3 ꢁ 2 mL). The combined organic extract was washed with 2.4 1H); 13C NMR (100 MHz, CD3OD) d 57.6, 62.7, 71.5, 75.1,
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76.1, 78.0, 78.2, 80.5, 102.2, 105.9, 113.1, 113.5, 114.1, 121.4, 20.9, 21.1, 21.7, 23.9, 29.5, 62.0, 68.3, 70.9, 72.0, 72.6, 72.7, 99.1,
131.0, 139.0, 152.8; ESI-MS obsd 372.1055, calcd 372.1054 100.3, 107.9, 112.0, 116.6, 120.4, 123.2, 131.0, 140.5, 150.1,
[(M + Na)+, M = C17H19NO7].
168.0, 169.4, 169.6, 170.4, 170.6; ESI-MS obsd 810.0761, calcd
4-Bromo-5-propargyloxy-1H-indol-3-yl b-D-glucopyranoside 810.0755 [(M + H)+, M = C34H37Br2NO12].
(26). Propargyl bromide (8.9 mL, 80% in toluene, 0.080 mmol)
5-{[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]methoxy}-4,6-dibromo-
was added to a suspension of 17 (30.0 mg, 0.050 mmol) and 1H-indol-3-yl b-D-glucopyranoside (30). K2CO3 (2.8 mg, 0.020 mmol)
K2CO3 (8.3 mg, 0.060 mmol) in DMF (0.80 mL) at room was added to a solution of 29 (16.2 mg, 0.020 mmol) in MeOH/THF
temperature. The mixture was stirred for 18 h and then treated (4 : 1, 200 mL) at room temperature. After 1 h, the reaction mixture
with MeOH (0.40 mL) and NaOMe solution (30 mL, 0.5 M in MeOH, was diluted with CH2Cl2 and passed through silica [CH2Cl2/MeOH
0.015 mmol). After 1 h, the reaction mixture was quenched by the (2 : 1) as an eluent] to afford a white solid (11.9 mg, 99%): 1H NMR
addition of acetic acid (20 mL) and concentrated under reduced (700 MHz, CD3OD) d 0.97–1.06 (m, 2H), 1.65–1.77 (m, 3H), 2.14–
pressure. Column chromatography [silica, CH2Cl2/MeOH = 9 : 1 to 2.21 (m, 2H), 2.21–2.34 (m, 4H), 3.37–3.44 (m, 2H), 3.44–3.52
17 : 3] afforded a pale-brown solid (18 mg, 84%): mp 146–148 1C; (m, 1H), 3.55 (dd, J = 8.1, 8.9 Hz, 1H), 3.68–3.75 (m, 1H), 3.92
1H NMR (400 MHz, CD3OD) d 2.90 (t, J = 2.4 Hz, 1H), 3.34–3.52 (d, J = 11.8 Hz, 1H), 4.08 (d, J = 7.8 Hz, 2H), 4.74 (d, J = 7.7 Hz,
(m, 3H), 3.55 (dd, J = 8.2, 8.2 Hz, 1H), 3.65–3.75 (m, 1H), 1H), 7.24 (s, 1H), 7.49 (s, 1H); 13C NMR (175 MHz, CD3OD)
3.92 (dd, J = 1.2, 11.8 Hz, 1H), 4.71 (d, J = 2.4 Hz, 2H), 4.74 d 20.1, 21.7, 22.0, 30.6, 62.7, 71.5, 72.8, 75.3, 78.1, 78.3,
(d, J = 8.0 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 8.8 Hz, 1H), 99.6, 105.2, 107.9, 112.5, 115.2, 116.0, 119.9, 132.8, 139.0,
7.23 (s, 1H); 13C NMR (100 MHz, CD3OD) d 60.2, 62.7, 71.5, 75.3, 146.9; ESI-MS obsd 600.0238, calcd 600.0227 [(M + H)+,
76.7, 78.0, 78.2, 80.2, 103.6, 105.2, 112.0, 114.5, 114.9, 120.2, M = C24H28Br2NO7].
132.5, 138.8, 149.2; ESI-MS obsd 450.0155, calcd 450.0159
[(M + Na)+, M = C17H19BrNO7].
2-[2-(2-Hydroxyethoxy)ethoxy]ethyl 2-nitrobenzenesulfonate
(31). Triethylamine (1.53 mL, 11.0 mmol) was added to a suspension
4,6-Dibromo-5-propargyloxy-1H-indol-3-yl b-D-glucopyranoside of 2-nitrobenzenesulfonyl chloride (2.216 g, 10.0 mmol) in
(27). Propargyl bromide (18 mL, 80% in toluene, 0.16 mmol) was triethylene glycol (26.7 mL, 200 mmol) at 0 1C. The reaction
added to a suspension of 18 (68 mg, 0.10 mmol) and K2CO3 mixture was allowed to warm to room temperature. After
(17 mg, 0.12 mmol) in DMF (0.80 mL) at room temperature. The 30 min, the reaction mixture was diluted with CH2Cl2 (50 mL),
mixture was stirred at room temperature for 2 h and then washed with aqueous citric acid (10%, 100 mL) and brine
treated with MeOH (0.40 mL) and NaOMe solution (60 mL, (50 mL), then dried (Na2SO4) and filtered. The filtrate was
0.5 M in MeOH, 0.030 mmol). After 1 h, the reaction mixture concentrated and purified by chromatography [silica, hexanes/
was quenched by the addition of acetic acid (40 mL) and acetone (2 : 3)] to afford a clear pale yellow oil (2.929 g, 87%):
concentrated under reduced pressure. Column chromatography 1H NMR (700 MHz, CDCl3) d 2.41 (br s, 1H), 3.54–3.59 (m, 2H),
[silica, CH2Cl2/MeOH, 9 : 1 to 17 : 3] afforded a white solid 3.59–3.66 (m, 4H), 3.66–3.75 (m, 2H), 3.76–3.83 (m, 2H), 4.38–
1
(45 mg, 89%): mp 179–181 1C; H NMR (700 MHz, CD3OD) d 4.47 (m, 2H), 7.74–7.79 (m, 1H), 7.79–7.85 (m, 2H), 8.13–8.20 (m,
2.94 (t, J = 2.6 Hz, 1H), 3.37–3.42 (m, 2H), 3.42–3.49 (m, 1H), 3.54 1H); 13C NMR (175 MHz, CDCl3) d 61.9, 68.7, 70.4, 70.9, 71.2,
(dd, J = 7.9, 9.1 Hz, 1H), 3.68–3.74 (m, 1H), 3.92 (dd, J = 1.5, 72.5, 125.0, 129.9, 131.5, 132.5, 134.9, 148.4; ESI-MS obsd
11.9 Hz, 1H), 4.68 (d, J = 2.6 Hz, 2H), 4.75 (d, J = 7.8 Hz, 1H), 7.25 336.0735, calcd 336.0748 [(M + H)+, M = C12H17NO8S].
(s, 1H), 7.50 (s, 1H); 13C NMR (175 MHz, CD3OD) d 61.6, 62.7,
1-Acetyl-4,6-dibromo-5-[1-hydroxy-3,6,9-trioxanon-9-yl]-1H-indol-
71.6, 75.3, 76.8, 78.1, 78.3, 79.5, 105.1, 108.1, 112.3, 115.2, 115.9, 3-yl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside (32). A sample of
119.8, 133.1, 139.1, 145.8, ESI-MS obsd 527.9260, calcd 527.9264 i-Pr2EtN (66 mL, 0.38 mmol) was added to a suspension of 18
[(M + Na)+, M = C17H17Br2NO7].
(172.0 mg, 0.253 mmol) and 31 (110.4 mg, 0.329 mmol) in
1-Acetyl-5-{[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl]methoxy}-4,6- CH2Cl2 (253 mL) at room temperature. The reaction mixture was
dibromo-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside heated to 35 1C for 24 h and then allowed to cool to room
(29). Diisopropyl azodicarboxylate (39.4 mL, 0.20 mmol) was temperature. The reaction mixture was diluted with ethyl acetate
added to a solution of 18 (67.9 mg, 0.10 mmol), 28 (16.5 mg, (2 mL), washed with aqueous HCl (1 M, 2 mL) and brine (2 mL),
0.11 mmol), and PPh3 (52.5 mg, 0.20 mmol) in CH2Cl2 (0.50 mL) dried (Na2SO4), and filtered. The filtrate was concentrated and
at room temperature. After 1.5 h, the reaction mixture was passed purified by chromatography (silica, ethyl acetate as an eluent) to
through silica (ethyl acetate as eluent). The eluent was concen- afford a white solid (182.7 mg, 89%): 1H NMR (700 MHz, CDCl3) d
trated and further purified by chromatography [silica, hexanes/ 2.05 (s, 3H), 2.07 (s, 3H), 2.09 (s, 3H), 2.10 (s, 3H), 2.44 (br s, 1H),
acetone (2 : 1) followed by hexanes/ethyl acetate (1 : 1)] to afford 2.60 (s, 3H), 3.64 (t, 2H), 3.71–3.78 (m, 4H), 3.78–3.84 (m, 2H),
a white solid (51.1 mg, 63%): 1H NMR (400 MHz, CDCl3) d 3.89 (ddd, J = 2.5, 5.2, 9.9 Hz, 1H), 4.17–4.23 (m, 2H), 3.97 (t, 2H),
1.03–1.10 (m, 2H), 1.60–1.82 (m, 3H), 2.05 (s, 3H), 2.07 (s, 3H), 4.20 (dd, J = 5.2, 12.4 Hz, 1H), 4.38 (dd, J = 2.5, 12.4 Hz, 1H), 5.05
2.09 (s, 3H), 2.11 (s, 3H), 2.18–2.40 (m, 6H), 2.60 (s, 3H), 3.89 (d, J = 7.6 Hz, 1H), 5.20 (dd, J = 9.6, 9.9 Hz, 1H), 5.30 (dd, J = 9.4,
(ddd, J = 2.3, 5.1, 9.7 Hz, 1H), 4.10 (d, J = 7.2 Hz, 2H), 4.20 9.6 Hz, 1H), 5.38 (dd, J = 7.6, 9.4 Hz, 1H), 7.25 (s, 1H), 8.69 (br s, 1H);
(dd, J = 5.1, 12.5 Hz, 1H), 4.38 (dd, J = 2.3, 12.5 Hz, 1H), 5.06 13C NMR (175 MHz, CDCl3) d 20.7, 20.9, 21.1, 23.9, 61.9, 62.0, 68.3,
(d, J = 7.6 Hz, 1H), 5.21 (dd, J = 9.2, 9.7 Hz, 1H), 5.31 (dd, J = 9.2, 70.3, 70.6, 70.9, 71.0, 72.6, 72.7, 100.3, 107.7, 112.1, 116.3, 120.4,
9.2 Hz, 1H), 5.39 (dd, J = 7.6, 9.2 Hz, 1H), 7.25 (s, 1H), 123.2, 131.1, 140.5, 149.8, 168.0, 169.4, 169.5, 170.3, 170.6; ESI-MS
8.70 (br s, 1H); 13C NMR (175 MHz, CDCl3) d 19.2, 20.7, 20.8, obsd 832.0399, calcd 832.0422 [(M + Na)+, M = C30H37Br2NO15].
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4,6-Dibromo-5-[1-hydroxy-3,6,9-trioxanon-9-yl]-1H-indol-3-yl
NJC
4-{(1-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]-3-oxo-2,7,10-trioxa-
b-D-glucopyranoside (33). A suspension of 32 (10.2 mg, 0.013 mmol) 4-azadodecan-12-yl)amino}-6-{4-[bis(2-methoxyethyl)amino]phenyl}-
and K2CO3 (0.4 mg, 0.003 mmol) in MeOH (250 mL) was stirred for 2-{10-[1-acetyl-3-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyloxy)-4,6-
30 min at room temperature. The reaction mixture was quenched dibromo-1H-indol-5-yl]-1,4,7,10-tetraoxadec-1-yl}-1,3,5-triazine (37).
by the addition of AcOH (0.4 mL), diluted with CH2Cl2, and then Samples of 14 (2.4 mg, 7 mmol) and i-Pr2EtN (1.2 mL, 7 mmol) were
passed through silica gel [CH2Cl2/MeOH (2 : 1) as eluent]. The added to a solution of 36 in MeCN (47 mL) at room temperature.
eluent was concentrated under reduced pressure. The residue was After 1 h, MeOH (188 mL) and K2CO3 (1.3 mg, 9 mmol) were added.
triturated with MeOH/ethyl acetate/hexanes to afford a white solid After 2.5 h, the reaction mixture was diluted with CH2Cl2 and
1
(7.1 mg, 94%): H NMR (700 MHz, CD3OD) d 7.49 (s, 1H), 7.25 passed through a silica pad [CH2Cl2/MeOH (5 : 1) as eluent]. The
(s, 1H), 4.74 (d, J = 7.7 Hz, 1H), 4.14 (t, J = 4.9 Hz, 2H), 3.95 eluent was concentrated under reduced pressure. Preparative TLC
(t, J = 4.9 Hz, 2H), 3.92 (d, J = 11.8 Hz, 1H), 3.82–3.77 (m, 2H), [silica, 0.25 mm, 20 ꢁ 20 cm, CH2Cl2/MeOH (15 : 1)] afforded a pale
3.74–3.65 (m, 5H), 3.59 (t, J = 4.8 Hz, 2H), 3.54 (dd, J = 7.8, yellow solid (3.9 mg, 69%): 1H NMR (700 MHz, CD3OD, mixture of
9.2 Hz, 1H), 3.50–3.43 (m, 1H), 3.43–3.37 (m, 2H); 13C NMR rotamers) d 0.82–0.91 (m, 2H), 1.26–1.36 (m, 1H), 1.46–1.58 (m, 2H),
(175 MHz, CD3OD) d 146.8, 139.0, 132.9, 119.9, 116.0, 115.2, 2.06–2.25 (m, 6H), 3.25–3.33 (m, 1H), 3.35 (s, 6H), 3.36–3.43
112.2, 107.7, 105.1, 78.3, 78.1, 75.3, 73.7, 73.6, 71.7, 71.54, (m, 1H), 3.43–3.81 (m, 29H), 3.86–3.95 (m, 3H), 4.04–4.17
71.50, 71.3, 62.7, 62.3; HRMS (ESI-TOF) m/z: [M + Na]+ calcd (m, 3H), 4.53–4.64 (m, 3H), 4.72–4.77 (m, 1H), 6.71–6.78
for C20H27Br2NNaO10 621.9894; found 621.9891.
(m, 2H), 7.22–7.26 (m, 1H), 7.46 (s, 1H), 8.11–8.24 (m, 2H);
6-{4-[Bis(2-methoxyethyl)amino]phenyl}-2,4-dichloro-1,3,5- 13C NMR (175 MHz, CD3OD, mixture of rotamers) d 18.9, 21.3,
triazine (35). A mixture of 34 (523.2 mg, 2.50 mmol), cyanuric 21.9, 30.1, 41.6, 41.7, 41.7, 51.9, 59.3, 62.7, 63.7, 67.1, 67.3, 70.5,
chloride (553.2 mg, 3.00 mmol), and 2,6-di-tert-butylpyridine 70.6, 70.7, 71.0, 71.27, 71.31, 71.4, 71.6, 71.8, 73.7, 75.3, 78.1,
(1.10 mL, 5.00 mmol) was heated to 140 1C for 18 h. The reaction 78.3, 99.5, 105.1, 105.2, 107.7, 112.0, 112.3, 115.0, 115.1, 116.0,
mixture was cooled to room temperature and directly purified 119.9, 123.9, 124.2, 131.4, 131.6, 132.9, 139.0, 146.8, 152.5, 152.6,
by column chromatography [silica, hexanes/acetone (18 : 1)] 159.2, 168.4, 168.7, 171.8, 172.1, 173.6, 174.0; ESI-MS obsd
followed by recrystallization (hexanes) to afford a yellow solid 1208.3363, calcd 1208.3397 [(M + H)+, M = C52H71Br2N7O16]; labs
(207.2 mg, 23%): mp 73–75 1C; 1H NMR (700 MHz, CDCl3) d (DMF) 341 nm; lem (DMF) 397 nm.
3.36 (s, 6H), 3.59 (t, J = 6.0 Hz, 4H), 3.69 (t, J = 6.0 Hz, 4H),
3-[4-(tert-Butoxycarbonyl)piperazin-1-yl]propane-1-sulfonic acid
6.74, (d, J = 8.9 Hz, 2H), 8.30, (d, J = 8.9 Hz, 2H); 13C NMR (39). A sample of 1-(tert-butoxycarbonyl)piperazine (38, 2.011 g,
(175 MHz, CDCl3) d 51.2, 59.2, 70.0, 111.5, 119.5, 132.4, 10.8 mmol) was added to a solution of 1,3-propane sultone
153.3, 171.0, 173.8; ESI-MS obsd 357.0888, calcd 357.0880 (1.319 g, 10.8 mmol) in 1,4-dioxane (5.40 mL) at room temperature.
[(M + H)+, M = C15H184Cl2N4O2]; labs (DMF) 369 nm; lem The reaction mixture was heated to 60 1C for 1 h and then allowed
(DMF) 423 nm.
to cool to room temperature. The precipitate was filtered and
(10-[6-{4-[Bis(2-methoxyethyl)amino]phenyl}-4-chloro-1,3,5- washed with ethyl acetate to afford a white solid (2.106 g, 63%): mp
1
triazin-2-yl]-1,4,7,10-tetraoxadec-1-yl)-1-acetyl-4,6-dibromo-1H- 210 1C (dec.); H NMR (700 MHz, D2O) d 1.47 (s, 9H), 2.17–2.28
indol-3-yl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside (36). A mixture (m, 2H), 3.02 (t, J = 7.3 Hz, 2H), 3.20–3.39 (m, 2H), 2.65–3.91
of 35 (12.6 mg, 0.035 mmol), 32 (31.2 mg, 0.038 mmol), and (m, 6H), 4.23 (br s, 2H); 13C NMR (175 MHz, D2O) d 20.3, 28.5, 41.5,
1
powdered molecular sieves 3 Å (16.0 mg) in MeCN (160 mL) was 48.7, 52.5, 56.4, 83.8, 156.5; H NMR (500 MHz, DMSO-d6) d 1.42
treated with i-Pr2EtN (16.7 mL, 0.096 mmol) at room temperature. (s, 9H), 2.00 (tt, J = 6.8, 6.5 Hz, 2H), 2.65 (t, J = 6.5 Hz, 2H),
The reaction mixture was heated to 50 1C for 2.5 h, 80 1C for 8 h, 2.86–3.14 (m, 4H), 3.20–3.26 (m, 2H), 3.49 (d, J = 15.0 Hz, 2H), 4.03
90 1C for 9.5 h, and 110 1C for 3 h. After allowing to cool to room (d, J = 15.0 Hz, 2H), 9.96 (s, 1H); ESI-MS obsd 309.1474, calcd
temperature, the reaction mixture was passed through a silica pad 309.1479 [(M + H)+, M = C12H24N2O5S].
(acetone as eluent). The eluent was concentrated under reduced
3-(4-(tert-Butoxycarbonyl)-1-(3-hydroxypropyl)piperazin-1-ium-
pressure. Column chromatography [silica, hexanes/acetone (1 : 1)] 1-yl)propane-1-sulfonate (40). 3-Bromopropanol (2.17 mL, 24 mmol)
1
afforded a pale yellow solid (5.9 mg, 15%): H NMR (700 MHz, was added to a mixture of 39 (1.234 g, 4.00 mmol), NaHCO3 (2.688 g,
CDCl3) d 2.04 (s, 3H), 2.07 (s, 3H), 2.09 (s, 3H), 2.10 (s, 3H), 2.59 (s, 32.0 mmol), and KI (132.8 mg, 0.80 mmol) in H2O (1.09 mL) at
3H), 3.36 (s, 6H), 3.58 (t, J = 6.0 Hz, 4H), 3.67 (t, J = 6.0 Hz, 4H), room temperature. The reaction mixture was heated to 80 1C for
3.76–3.83 (m, 4H), 3.86–3.99 (m, 6H), 4.14–4.22 (m, 3H), 4.38 15 h, allowed to cool to room temperature, and washed with Et2O
(dd, J = 2.5, 12.4 Hz, 1H), 4.63–4.68 (m, 2H), 5.05 (d, J = 7.6 Hz, (20 mL). The residue was suspended in CH2Cl2/MeOH (4 : 1, 25 mL)
1H), 5.20 (dd, J = 9.2, 9.7 Hz, 1H), 5.30 (dd, J = 8.5, 9.2 Hz, 1H), 5.38 and filtered. The filtrate was concentrated and purified by chroma-
(dd, J = 7.6, 8.5 Hz, 1H), 6.72 (d, J = 8.8 Hz, 2H), 7.25 (s, 1H), tography [silica, CH2Cl2/MeOH (4 : 6)] to afford a white solid (1.011 g,
8.30 (d, J = 8.8 Hz, 2H), 8.68 (br s, 1H); 13C NMR (175 MHz, 69%): 1H NMR (700 MHz, CD3OD) d 1.48 (s, 9H), 1.95–2.03 (m, 2H),
CDCl3) d 20.7, 20.9, 21.1, 23.9, 51.1, 59.2, 62.0, 67.8, 68.3, 69.1, 2.14–2.24 (m, 2H), 2.90 (t, J = 6.6 Hz, 2H), 3.46–3.60 (m, 6H), 3.64–
70.1, 70.3, 70.9, 71.0, 71.1, 72.6, 72.66, 72.74, 100.3, 107.7, 3.72 (m, 4H), 3.81 (br s, 4H); 13C NMR (175 MHz, CD3OD) d 18.7,
111.2, 112.2, 116.4, 120.4, 121.0, 123.2, 131.0, 131.8, 140.5, 25.5, 28.5, 37.8, 39.0, 48.4, 57.4, 57.7, 59.3, 82.4, 155.5, ESI-MS obsd
149.9, 152.4, 167.9, 169.4, 169.5, 170.3, 170.6, 170.9, 171.7, 367.1896, calcd 367.1897 [(M + H)+, M = C15H30N2O6S].
174.3; ESI-MS obsd 1130.1635, calcd 1130.1643 [(M + H)+,
M = C45H54Br2ClN5O17].
3-(1-(3-Hydroxypropyl)piperazin-1-ium-1-yl)propane-1-sulfonate
trifluoroacetic acid salt (41). A sample of 40 (980.2 mg, 2.67 mmol)
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was dissolved in trifluoroacetic acid (1.78 mL) at room temperature. 7.62 (s, 1H), 10.3 (s, 1H); 13C NMR (175 MHz, CDCl3, mixture of
After 2 h, the reaction mixture was concentrated under reduced rotamers) d 17.8, 17.9, 20.1, 20.7, 20.9, 21.1, 21.5, 24.6, 29.1,
pressure. The residue was triturated with EtOH/Et2O to afford a 36.8, 40.5, 40.7, 40.8, 41.5, 47.4, 53.5, 56.4, 57.0, 58.3, 61.9, 62.7,
pale yellow solid (985.4 mg, 97%): 1H NMR (700 MHz, CD3OD) d 66.0, 66.1, 68.4, 69.3, 69.4, 69.7, 69.8, 70.1, 70.20, 70.23, 70.7,
1.95–2.10 (m, 2H), 2.15–2.30 (m, 2H), 2.97 (t, J = 6.5 Hz, 2H), 70.8, 71.1, 71.9, 72.5, 73.0, 98.9, 101.1, 106.2, 111.6, 114.7, 115.7,
3.60–3.95 (m, 14H); 13C NMR (175 MHz, CD3OD) d 18.9, 118.3, 131.5, 136.5, 145.6, 156.9, 165.6, 165.8, 166.7, 167.2,
25.6, 38.8, 48.2, 56.4, 58.3 (br s), 58.8 (br s), 59.1, 163.1 169.5, 169.6, 170.2, 170.4, 170.8; ESI-MS obsd 717.1884, calcd
(q, J = 34.5 Hz); 19F (564 MHz, CD3OD) d ꢀ76.9 ppm (versus 717.1888 [(M + 2H)2+, M = C58H82Br2N8O22S].
the reference CFCl3); ESI-MS obsd 267.1371, calcd 267.1373
[(M – CF3CO2H + H)+, M = C12H23F3N2O6S].
4-{(1-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]-3-oxo-2,7,10-trioxa-
4-azadodecan-12-yl)amino}-6-[4-(3-hydroxypropyl)-4-(3-sulfopropyl)-
4,6-Dibromo-5-[1-hydroxy-3,6,9-trioxanon-9-yl]-1H-indol-3-yl piperazin-1-yl]-2-{10-[[b-D-glucopyranosyloxy]-4,6-dibromo-1H-indol-
2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside (42). A suspension of 5-yl]-1,4,7,10-tetraoxadec-1-yl}-1,3,5-triazine (44). A sample of K2CO3
32 (811.4 mg, 1.00 mmol) and NaHCO3 (8.4 mg, 0.10 mmol) in (0.3 mg, 2 mmol) was added to a solution of 43 in MeOH/CH2Cl2
MeOH (5.00 mL) was stirred for 3.5 h at room temperature. The (25 : 6, 310 mL) at room temperature. After 2 h, the reaction mixture
reaction mixture was concentrated under reduced pressure. was passed through diol-functionalized silica [CH2Cl2/MeOH (2 : 1)
The residue was suspended in ethyl acetate. The suspension as eluent]. The eluent was concentrated under reduced pressure to
1
was passed through silica (ethyl acetate as eluent). The eluent afford a white solid (12.5 mg, 98%): H NMR (700 MHz, CD3OD,
was concentrated under reduced pressure to afford a white mixture of rotamers) d 0.85–0.97 (m, 2H), 1.28–1.41 (m, 1H),
solid (650.1 mg, 84%): 1H NMR (700 MHz, CDCl3) d 2.04 1.50–1.63 (m, 2H), 1.88–1.99 (m, 2H), 2.07–2.29 (m, 8H), 2.79–
(s, 3H), 2.05 (s, 3H), 2.096 (s, 3H), 2.103 (s, 3H), 2.50 (br s, 2.91 (m, 2H), 3.24–3.33 (m, 2H), 3.37–4.25 (m, 44H), 4.42–4.61
1H), 3.62–3.68 (m, 2H), 3.71–3.78 (m, 4H), 3.78–3.84 (m, 3H), (m, 2H), 4.78 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 6.8 Hz, 1H), 7.53
3.94–4.00 (m, 2H), 4.13–4.20 (m, 2H), 4.24 (dd, J = 4.8, 12.3 Hz, (s, 1H); 13C NMR (175 MHz, CD3OD, mixture of rotamers) d
1H), 4.27 (dd, J = 2.8, 12.3 Hz, 1H), 4.97 (d, J = 7.8 Hz, 1H), 5.19 18.7, 19.0, 21.4, 21.9, 22.0, 25.5, 30.2, 37.9, 38.0, 41.5, 41.7, 48.3,
(dd, J = 9.5, 9.6 Hz, 1H), 5.29 (dd, J = 9.3, 9.5 Hz, 1H), 5.37 49.5, 54.8, 55.1, 57.6, 57.7, 59.3, 59.46, 59.52, 62.56, 62.62, 63.7,
(dd, J = 7.8, 9.6 Hz, 1H), 7.07 (d, J = 2.7 Hz, 1H), 7.45 (s, 1H), 67.2, 67.3, 70.6, 70.7, 71.0, 71.1, 71.3, 71.4, 71.50, 71.55, 71.85,
7.94 (br s, 1H); 13C NMR (175 MHz, CDCl3) d 20.6, 20.7, 20.8, 71.88, 73.8, 75.3, 78.2, 78.30, 78.32, 99.6, 105.1, 105.2, 107.7,
21.1, 61.7, 61.9, 68.4, 70.2, 70.4, 70.7, 71.0, 71.9, 72.4, 72.5, 112.4, 116.2, 132.9, 139.0, 146.8, 159.2, 166.9, 167.2, 168.2,
72.9, 101.0, 106.6, 112.0, 114.5, 115.2, 118.7, 131.3, 136.7, 168.6, 171.8, 172.2; ESI-MS obsd 633.1670, calcd 633.1677
145.9, 169.5, 169.6, 170.3, 170.7; ESI-MS obsd 768.0494, calcd [(M + 2H)2+, M = C50H74Br2N8O18S].
768.0497 [(M + H)+, M = C28H35Br2NO14].
4,40,6,60-Tetrabromo-5,50-bis[1-hydroxy-3,6,9-trioxanon-9-yl]-
4-{(1-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]-3-oxo-2,7,10-trioxa- indigo (46). Samples of 33 (4.98 mg, 8.28 mmol) in DMF
4-azadodecan-12-yl)amino}-6-[4-(3-hydroxypropyl)-4-(3-sulfopropyl)- (414 mL), b-glucosidase in water (from almonds, 10 units/mL,
piperazin-1-yl]-2-{10-[1-acetyl-3-(2,3,4,6-tetra-O-acetyl-b-D-gluco- 828 mL), and acetate buffer (pH 5.0, 50 mM + 5% DMF, 7038 mL)
pyranosyloxy)-4,6-dibromo-1H-indol-5-yl]-1,4,7,10-tetraoxadec-1- were mixed at room temperature. The reaction mixture was
yl}-1,3,5-triazine (43). Cyanuric chloride (20.3 mg, 0.11 mmol) incubated at 37 1C under air for 22 h and then allowed to cool to
was added to a mixture of 42 (76.9 mg, 0.10 mmol), 1,10- room temperature. The reaction mixture was diluted with H2O
phenanthroline (36.0 mg, 0.20 mmol), and powdered molecular (20 mL) and extracted with CH2Cl2 (20 mL). The organic layer
sieves 4 Å (50.0 mg) in CH2Cl2 (0.50 mL) at room temperature. was washed with brine (10 mL), dried (Na2SO4), and filtered.
After 16 h, 41 (49.4 mg, 0.13 mmol) in DMF (0.50 mL) and The filtrate was concentrated under reduced pressure. Preparative
i-Pr2EtN (70 mL, 0.40 mmol) were added. After 3 h, a solution of thin layer chromatography [silica, 0.25 mm, CHCl3/MeOH (12 : 1)]
1
14 (35.7 mg, 0.11 mmol) in CH2Cl2 (300 mL) and a sample of afforded an indigo-blue solid (2.4 mg, 66%): H NMR (700 MHz,
i-Pr2EtN (35 mL, 0.20 mmol) were added. After 4 h, i-Pr2EtN CDCl3/CD3OD = 9 : 1) d 3.60–3.66 (m, 4H), 3.69–3.76 (m, 8H),
(35 mL, 0.20 mmol) was added. After 15 h, the reaction mixture 3.79–3.84 (m, 4H), 3.94–4.00 (m, 4H), 4.16–4.21 (m, 4H), 7.31 (s,
was diluted with CH2Cl2 (3 mL) and filtered. The filtrate was 1H); 13C NMR (175 MHz, CDCl3/CD3OD = 9 : 1) d 61.6, 70.2,
washed with aqueous citric acid (10%, 3 mL) and brine (3 mL), then 70.4, 70.9, 72.78, 72.79, 115.3, 116.0, 118.1, 122.3, 127.4, 147.9,
dried (Na2SO4) and filtered. The filtrate was concentrated under 149.6, 185.7; ESI-MS obsd 870.8692, calcd 870.8707 [(M + H)+,
reduced pressure. Column chromatography [diol-functionalized M = C28H30Br4N2O10].
silica (ethyl acetate/MeOH = 19 : 1 to CH2Cl2/MeOH = 5 : 1) followed
2,4-Bis{10-[[1-acetyl-3-(2,3,4,6-tetra-O-acetyl-b-D-glucopyrano-
by silica (CH2Cl2/MeOH = 5 : 1)] enabled removal of multiple syloxy)-4,6-dibromo-1H-indol-5-yl]-1,4,7,10-tetraoxadec-1-yl]}-6-
1
byproducts and afforded a white solid (72.5 mg, 51%): H NMR chloro-1,3,5-triazine (47). Pempidine (38.1 mL, 0.21 mmol) was
(700 MHz, CDCl3, mixture of rotamers) d 0.91 (m, 2H), 1.26–1.38 added to a mixture of cyanuric chloride (11.1 mg, 0.060 mmol),
(m, 1H), 1.54 (br s, 2H), 1.80–2.00 (m, 2H), 2.01 (s, 3H), 2.04 32 (102.6 mg, 0.13 mmol), and powdered molecular sieves 4 Å
(s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.11–2.33 (m, 9H), 2.90 (12.0 mg) in 1,2-dichloroethane (120 mL) at room temperature.
(br s, 2H), 3.15–4.47 (m, 42H), 4.75 (br s, 1H), 4.92 (s, 1H), The reaction mixture was heated to 60 1C for 13 h, allowed to
5.16 (t, J = 9.1 Hz, 1H), 5.21–5.36 (m, 2H), 5.43 (s, 0.5H), cool to room temperature, and passed through a silica pad
5.53 (s, 0.5H), 5.81 (br s, 0.5H), 6.05 (br s, 0.5H), 7.13 (s, 1H), (ethyl acetate as eluent). The eluent was concentrated under
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reduced pressure. The putative N-deacetylated byproduct showed 114.9, 117.9, 131.0, 137.4, 144.8, 166.4, 171.5; ESI-MS obsd
similar chromatographic properties as the title compound. 770.5642, calcd 770.5653 [(M + 2H)2+, M = C53H73Br4N7O24S].
Preparative thin layer chromatography [silica, 1.0 mm, 20 ꢁ
Procedure for e determination
20 cm, hexanes/acetone (6 : 4)] afforded a white solid (55.7 mg,
1
53%): H NMR (700 MHz, CDCl3) d 2.04 (s, 6H), 2.07 (s, 6H), (1) Indigo (13.1 mg, 50 mmol) was dissolved in DMF (200 mL) to
2.091 (s, 6H), 2.093 (s, 6H), 2.60 (s, 6H), 3.70–3.73 (m, 4H), 3.73– prepare a 250 mM solution. An aliquot (320 mL) was withdrawn
3.82 (m, 4H), 3.83–3.94 (m, 6H), 3.94–3.97 (m, 4H), 4.13–4.23 from the solution and diluted with DMF/H2O (1680 : 1000,
(m, 6H), 4.38 (dd, J = 1.5, 12.3 Hz, 2H), 4.55–4.62 (m, 4H), 5.05, 2680 mL) to prepare a 40.0 mM solution. The absorption spectrum
(d, J = 7.6 Hz, 2H), 5.17–5.23 (m, 2H), 5.30 (dd, J = 9.3, 9.3 Hz, was recorded at room temperature. The average of two runs was
2H), 5.34–5.41 (m, 2H), 7.25 (s, 2H), 8.68 (br s, 2H); 13C NMR calculated.
(175 MHz, CDCl3) d 20.7, 20.9, 21.1, 23.8, 62.0, 68.4, 68.5, 68.9,
(2) Indigoid derivative 46 (1.6 mg, 1.8 mmol) was dissolved in
70.3, 70.9, 71.0, 72.6, 72.7, 100.3, 107.7, 112.2, 116.3, 120.3, CHCl3/MeOH (2 : 1, 2.93 mL). An aliquot (64.0 mL) was withdrawn
123.1, 131.0, 140.5, 149.8, 167.9, 169.4, 169.5, 170.3, 170.6, from the solution and concentrated under reduced pressure. The
172.5, 172.7; ESI-MS obsd 1730.0752, calcd 1730.0757 residue was dissolved in DMF/H2O (2 : 1, 1000 mL) to prepare a
[(M + H)+, M = C63H72Br4ClN5O30].
40 mM solution. The absorption spectrum was recorded at room
2,4-Bis{10-[[3-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyloxy)-4,6- temperature. e631nm = 2.6 ꢁ 104 Mꢀ1 cmꢀ1
.
dibromo-1H-indol-5-yl]-1,4,7,10-tetraoxadec-1-yl]}-6-[4-(3-hydroxy-
propyl)-4-(3-sulfopropyl)piperazin-1-yl]-1,3,5-triazine (48).
Procedures for experiments of Fig. 6
A
sample of i-Pr2EtN (19.2 mL, 0.11 mmol) was added to a solution
Effect of pH. A solution of 33 in DMF (2 mL, 5 mM) and a
of 47 (190.8 mg, 0.11 mmol) in CH2Cl2/MeOH (5 : 1, 0.84 mL) at solution of b-glucosidase from Agrobacterium in NaPi–NaCl
room temperature. After 4 h, 41 (46.0 mg, 0.12 mmol) in MeOH buffer (2 mL, 10 mM, pH 7.0) were mixed with various 50 mM
(0.70 mL) and 2,6-lutidine (25.5 mL, 0.22 mmol) were added. sodium phosphate buffers (96 mL, pH 4.0, 5.0, 6.0, 7.0, 8.0, or
After 4 h, the reaction mixture was quenched by the addition 9.0). The reaction mixture was incubated at 37 1C for 10, 20, 40,
of acetic acid (12.6 mL, 0.22 mmol) and concentrated under 60, 90, 120, or 180 min and then diluted with DMF (200 mL) at
reduced pressure. Column chromatography [silica, CH2Cl2/ 0 1C. After centrifugation for 1 min, the supernatant was
MeOH (7 : 1 to 5 : 1)] followed by trituration with H2O afforded analyzed by absorption spectroscopy. All the experiments were
a pale yellow solid (114.7 mg, 55%): 1H NMR (700 MHz, CDCl3) repeated three times.
d 1.78 (br s, 2H), 1.96–2.16 (m, 26H), 2.88 (br s, 2H), 3.15 (br s,
Effect of the enzyme concentration. A solution of 33 in DMF
2H), 3.23 (br s, 2H), 3.36 (br s, 2H), 3.54 (br s, 4H), 3.68–3.99 (m, (2 mL, 5 mM) and a solution of b-glucosidase from Agrobacterium
22H), 4.04 (br s, 4H), 4.16–4.52 (m, 9H), 4.89 (d, J = 7.3 Hz, 2H), in NaPi–NaCl buffer (1 mL, 1 mM, pH 7.0) were mixed with
5.16 (dd, J = 9.4, 9.4 Hz, 2H), 5.22–5.33 (m, 4H), 7.13 (s, 2H), 50 mM NaPi (97 mL, pH 7.0). The reaction mixture was incubated
7.59 (s, 1H), 7.59 (s, 1H), 9.96 (s, 1H), 9.99 (s, 1H); 13C NMR at 37 1C for 2 h and then diluted with DMF (200 mL) at 0 1C.
(175 MHz, CDCl3) d 17.9, 20.7, 20.9, 21.2, 24.6, 37.0, 47.4, 56.4, After centrifugation for 1 min, the supernatant was analyzed
56.8, 58.0, 58.2, 61.9, 67.0, 68.4, 69.2, 70.2, 70.6, 70.7, 71.1, 71.8, by absorption spectroscopy. All the experiments were repeated
72.4, 73.0, 101.2, 0106.2, 111.6, 114.9, 115.7, 118.3, 131.4, 136.5, three times.
145.5, 166.4, 169.6, 169.6, 170.2, 170.8, 171.6; ESI-MS obsd
1876.2076, calcd 1876.2079 [(M + H)+, M = C69H89Br4N7O32S].
Effect of the concentration of 33. A solution containing 33
(1.00, 1.78, 3.16, 5.62, 10.0, 17.8, 31.6, or 56.2 mM) and
2,4-Bis{10-[[3-(b-D-glucopyranosyloxy)-4,6-dibromo-1H-indol- b-glucosidase from Agrobacterium (200 nM) in NaPi–NaCl buffer
5-yl]-1,4,7,10-tetraoxadec-1-yl]}-6-[4-(3-hydroxypropyl)-4-(3-sulfo- (pH 7.0; containing 2% DMF) was incubated at 37 1C for 2 h.
propyl)piperazin-1-yl]-1,3,5-triazine (49). A sample of K2CO3 The total volume was 4000, 2000, 1500, 1200, 300, 300, 300, or
(0.6 mg, 4 mmol) was added to a solution of 48 (39.3 mg, 300 mL for the reaction at 1.00, 1.78, 3.16, 5.62, 10.0, 17.8, 31.6,
0.020 mmol) in MeOH/CH2Cl2 (5 : 1, 600 mL) at room temperature. or 56.2 mM, respectively. After the incubation, the reaction
After 15 min, H2O (50 mL) was added. After 2 h, H2O (150 mL) and mixture was centrifuged for 5 min. Any precipitate was separated
K2CO3 (2.2 mg, 16 mmol) were added. After 1 h, reverse phase silica from the supernatant and dissolved in DMF (200–300 mL) followed
(320 mg) was added. The mixture was dried under reduced by analysis by absorption spectroscopy. Additionally, the reaction
pressure. The residue was purified by column chromatography with 33 at 1.00, 1.78, or 3.16 mM was carried out for a longer
[reverse phase silica, H2O to MeOH/H2O (4 : 1)] to afford a pale reaction time (14 h). All the experiments were repeated three times.
yellow solid (23.9 mg, 77%): 1H NMR [700 MHz, (CD3)2SO] d
Procedures for indigogenic reactions in Table 1
1.78–1.87 (m, 2H), 1.93–2.02 (m, 2H), 2.47–2.56 (m, 2H), 3.15
(t, J = 9.1 Hz, 2H), 3.22–3.36 (m, 6H), 3.38–3.55 (m, 11H), 3.55–
Reactions with b-glucosidase from almonds. A solution of an
3.69 (m, 11H), 3.69–3.78 (m, 6H), 3.78–3.86 (m, 4H), 3.93–4.05 indoxyl-glucoside in DMF (5 mL, 20 mM) and a solution of
(m, 6H), 4.05–4.15 (m, 2H), 4.36–4.45 (m, 4H), 4.56–4.64 (m, 2H), b-glucosidase from almonds in H2O (10 mL, 10 units per mL)
4.65 (d, J = 7.6 Hz, 2H), 4.78 (br s, 1H), 4.95–5.14 (m, 6H), 7.23 were mixed with acetate buffer (85 mL, pH 5.0). The reaction
(s, 2H), 7.55 (s, 2H), 10.92 (s, 2H); 13C NMR [175 MHz, (CD3)2SO] mixture was incubated at 37 1C for 16–19 h and then allowed to
d 17.7, 24.1, 36.8, 47.3, 56.9, 57.6, 60.9, 66.4, 68.4, 69.4, 69.8, cool to room temperature. DMF (300 mL for the reactions of 15,
69.9, 70.0, 72.4, 73.5, 76.8, 77.2, 99.5, 103.3, 106.1, 110.4, 113.3, 16, 1, 19, 22, and 25; or 900 mL for the reactions of 45, 20, 21, 23,
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24, 26, 27, 30, and 31, respectively) was added to dissolve any fractions (fraction A, B, and C) were withdrawn. Fractions A–C
indigoid precipitate. The resulting solution was analyzed by were individually centrifuged for 5 min, separated from the
absorption spectroscopy.
supernatant, and dried under reduced pressure to afford a blue
Reactions with b-glucosidase from Agrobacterium. A solution solid in each case. The solid from Fraction A was dissolved in
of an indoxyl-glucoside in DMF (2 mL, 5 mM) and a solution of DMF/DMSO (9 : 1, 500 mL) to prepare a sample for absorption
b-glucosidase from Agrobacterium in NaPi–NaCl buffer (2 mL, spectroscopy. The solid from Fraction B was dissolved in DMF/
pH 7.0) were mixed with 50 mM NaPi buffer (96 mL, pH 7.0). The DMSO (9 : 1, 500 mL), and the resulting solution was filtered
reaction mixture was incubated at 37 1C for 2 h and then through a membrane filter (polytetrafluoroethylene, 0.45 mm) to
centrifuged for 3 min. Any precipitate was separated from the prepare a sample (‘‘precipitate extract’’) for HPLC analysis. The
supernatant and dissolved in DMF (200 mL). The resulting solid from Fraction C was suspended in H2O (500 mL) to prepare a
solution was analyzed by absorption spectroscopy. The experiment sample for optical microscopy, from which 40 mL was withdrawn
was repeated three times.
and diluted 10-fold with H2O to prepare a sample for DLS
Reactions in rat liver homogenate. A solution of an indoxyl- analysis. The combined supernatant was concentrated under
glucoside in DMF (5 mL, 20 mM) was mixed with rat liver reduced pressure. The residue was suspended in DMF. The
homogenate (95 mL). The reaction mixture was incubated at suspension was filtered through cotton. The total volume of the
37 1C for 24 h. After allowing to cool to room temperature, the filtrate was adjusted to 600 mL by adding DMF to prepare a
reaction mixture was diluted with DMF (900 mL). The mixture solution (‘‘supernatant extract’’) for analysis. An aliquot of the
was heated at 70 1C for 2 min and then centrifuged for 2 min. The supernatant extract (30 mL) was diluted 10-fold with DMF to
supernatant was separated from any precipitate. This extraction prepare a sample for absorption spectroscopy. The supernatant
procedure was repeated two or three times with DMF (100– sample solution (200 mL) was filtered through a membrane
500 mL). The combined supernatant (1500–1800 mL) was analyzed filter (polytetrafluoroethylene, 0.45 mm) to prepare a sample for
by absorption spectroscopy.
HPLC analysis. The supernatant sample solution (100 mL) was
Reaction of 33 with b-glucosidase from Agrobacterium in rat concentrated under reduced pressure to prepare a sample for
liver homogenate. A DMF solution of 33 (2 mL, 5 mM) and a MALDI-MS and ESI-MS analysis.
1
solution of b-glucosidase from Agrobacterium in NaPi–NaCl
To prepare a sample for H NMR analysis, the reaction was
buffer (2 mL, pH 7.0) were mixed with rat liver homogenate repeated on the same reaction scale. Instead of dividing the
(96 mL). The reaction mixture was incubated at 37 1C for 4 h and precipitate suspension in H2O (1000 mL) into fractions A–C, the
then centrifuged for 3 min. Any precipitate was separated from entire suspension was centrifuged for 5 min. The precipitate
the supernatant and then suspended in DMF (200 mL). The was separated from supernatant and dried under reduced pressure
suspension was centrifuged for 3 min. The supernatant was to afford a blue solid, which was dissolved in DMSO-d6 (300 mL).
analyzed by absorption spectroscopy.
To determine a gravimetric yield of the precipitate, the
reaction was carried out on a 5.10 mmol (7.87 mg) scale using
102 mL of 49 in DMF (50.0 mM). After the washing with H2O, a
Oligomerization procedures
Oligomerization of 49 in NaPi–NaCl buffer. A solution of 49 blue precipitate (3.01 mg) was obtained, which corresponds to 49%
(300, 100, 50, or 10 mM) and a solution of b-glucosidase from based on the monomer formula weight (1215.55, C41H49Br4N7O14S).
Agrobacterium (200 nM) in NaPi–NaCl buffer were mixed and
HPLC analysis. Analytical size-exclusion chromatography
incubated at 37 1C for 2–4 h. The total volume was 2000, 250, 300, (SEC) was performed in DMF/DMSO (9 : 1, flow rate = 0.6 mL minꢀ1
)
or 2000 mL for the 300, 100, 50, or 10 mM reaction, respectively. at room temperature with three analytical SEC columns62 in order
After the incubation, the reaction mixture was centrifuged for (PL gel 5 mm 500 Å, 500 Å, and 1000 Å; 7.5 mm i.d. ꢁ 300 mm) using
5 min. The supernatant was separated from any precipitate an HPLC instrument with absorption spectral detection. A
and analyzed by absorption spectroscopy. The precipitate was calibration curve (Fig. S5, ESI†) was prepared with eight standard
suspended in H2O (the same volume as the reaction volume) poly(2-vinylpyridine) samples (PSS polymer, MW = 1110–256 000 Da,
and then centrifuged for 5 min. The precipitate was separated Mw/Mn = 1.04–1.24) in DMF (1 mg mLꢀ1).
from the supernatant and dried under reduced pressure. The
residue was dissolved in DMF/DMSO (4 : 1) to prepare a sample
for absorption spectroscopy.
Conflicts of interest
Low-salt oligomerization of 49. To minimize the NaCl content
Patent applications have been filed encompassing this work for
and thereby facilitate purification and analysis of the product, a
which authors HF, NM, ZW and JSL are coinventors.
solution of 49 in DMF (12 mL, 50.0 mM) and a solution of
b-glucosidase from Agrobacterium in NaPi–NaCl buffer (40 mL,
10 mM) were mixed with 10 mM NaPi buffer (1948 mL, pH 7.0).
The reaction mixture was incubated at 37 1C for 3 h and then
Acknowledgements
centrifuged for 5 min. After the supernatant was removed, any This work was supported by NC State University. Mass spectro-
precipitate was suspended in H2O (1000 mL) and then centrifuged metric analyses were carried out in the Molecular Education,
for 5 min. The supernatant was removed again. The precipitate Technology, and Research Innovation Center (METRIC) at NC
was suspended in H2O (1000 mL), from which 3 ꢁ 250 mL State University.
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¨
33 S. Bottcher and J. Thiem, RSC Adv., 2014, 4, 10856–10861.
Notes and references
¨
34 S. Bottcher and J. Thiem, J. Visualized Exp., 2015, 99, e52442.
1 Y.-H. Yang, H. Aloysius, D. Inoyama, Y. Chen and L.-Q. Hu,
Acta Pharm. Sin. B, 2011, 1, 143–159.
2 R. de la Rica, D. Aili and M. M. Stevens, Adv. Drug Delivery
Rev., 2012, 64, 967–978.
35 H. M. Burke, T. Gunnlaugsson and E. M. Scanlan, Chem.
Commun., 2015, 51, 10576–10588.
36 S. Rose, US5816259, 1998.
37 S. Rose, US6080383, 2000.
3 Q. Hu, P. S. Katti and Z. Gu, Nanoscale, 2014, 6, 12273–12286.
4 J. Mu, J. Lin, P. Huang and X. Chen, Chem. Soc. Rev., 2018,
47, 5554–5573.
38 S. Rose, US6468503B2, 2002.
39 L. R. Schroeder and J. W. Green, J. Chem. Soc. C, 1966,
´
530–531; A. S. Bourlot, E. Desarbre and J. Y. Merour, Synthesis,
5 A. Romieu, Org. Biomol. Chem., 2015, 13, 1294–1306.
6 T. He, H. He, X. Luo, Y. Yang and Y. Yang, Sci. Sin.: Chim.,
2017, 47, 945–954.
7 J. S. Lindsey, New J. Chem., 1991, 15, 153–180.
8 J. Zhou and B. Xu, Bioconjugate Chem., 2015, 26, 987–999.
9 C. S. McKay and M. G. Finn, Chem. Biol., 2014, 21, 1075–1101.
10 Z. Feng, T. Zhang, H. Wang and B. Xu, Chem. Soc. Rev., 2017,
46, 6470–6479.
11 Y. Li, L. Du, C. Wu, B. Yu, H. Zhang and F. An, Curr. Top.
Med. Chem., 2019, 19, 74–97.
12 J. Hu, G. Zhang and S. Liu, Chem. Soc. Rev., 2012, 41,
5933–5949.
13 L. S. M. Teixeira, J. Feijen, C. A. van Blitterswijk, P. J. Dijkstra
and M. Karperien, Biomaterials, 2012, 33, 1281–1290.
14 X. Wang, S. Chen, D. Wu, Q. Wu, Q. Wei, B. He, Q. Lu and
Q. Wang, Adv. Mater., 2018, 30, 1705668.
15 D. Wang, X. Yang, Q. Liu, L. Yu and J. Ding, J. Mater. Chem.
B, 2018, 6, 6067–6079.
1994, 411–416; A. Andreani, A. Cavalli, M. Granaiola,
M. Guardigli, A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi,
M. Recanatini and A. Roda, J. Med. Chem., 2001, 44, 4011–4014;
G. L. Mayers, D. Lee and H.-L. Chin, US7615221B2, 2009.
40 G. L. Mayers, S. Rose and L. Rose, US7807136B2, 2010.
41 G. L. Mayers, S. Rose and D. Rose, US2011/0142756A1, 2011.
42 G. L. Mayers, Drug Dev. Res., 2006, 67, 94–106.
43 S. Rose, J. Theor. Biol., 1998, 195, 111–128.
44 G. Blotny, Tetrahedron, 2006, 62, 9507–9522.
45 K. Bretterbauer and C. Schwarzinger, Curr. Org. Synth., 2012,
9, 342–356.
46 H. Tanaka, A. Wada, M. Shiro, K. Hioki, D. Morisaki and
M. Kunishima, Heterocycles, 2009, 79, 609–616.
47 J. Dommerholt, S. Schmidt, R. Temming, L. J. A. Hendriks,
F. P. J. T. Rutjes, J. C. M. van Hest, D. J. Lefeber, P. Friedl and
F. L. van Delft, Angew. Chem., Int. Ed., 2010, 49, 9422–9425.
48 H. C. Brown and B. Kanner, J. Am. Chem. Soc., 1966, 88,
986–992.
16 G. Liang, H. Ren and J. Rao, Nat. Chem., 2010, 2, 54–60.
17 Y. Yuan and G. Liang, Org. Biomol. Chem., 2014, 12, 865–871.
18 M. Zhang and G. Liang, Sci. China: Chem., 2018, 61, 1088–1098.
19 Y. Yuan, J. Zhang, M. Wang, B. Mei, Y. Guan and G. Liang,
Anal. Chem., 2013, 85, 1280–1284.
20 X. Ai, C. J. H. Ho, J. Aw, A. B. E. Attia, J. Mu, Y. Wang, X. Wang,
Y. Wang, X. Liu, H. Chen, M. Gao, X. Chen, E. K. L. Yeow, G. Liu,
M. Olivo and B. Xing, Nat. Commun., 2016, 7, 10432.
21 S. Debieu and A. Romieu, Org. Biomol. Chem., 2015, 13,
10348–10361.
49 R. Gotor, P. Ashokkumar, M. Hecht, K. Keil and K. Rurack,
Anal. Chem., 2017, 89, 8437–8444.
50 D. J. Cowley, E. O’Kane and R. S. J. Todd, J. Chem. Soc.,
Perkin Trans. 2, 1991, 1495–1500.
51 V. S. Padalkar, V. S. Patil, R. D. Telore and N. Sekar,
Heterocycl. Commun., 2012, 18, 127–134.
52 S.-L. Niu, G. Ulrich, P. Retailleau, J. Harrowfield and
R. Ziessel, Tetrahedron Lett., 2009, 50, 3840–3844.
53 A. Natrajan, D. Sharpe and D. Wen, Org. Biomol. Chem.,
2012, 10, 1883–1895.
22 S. Debieu and A. Romieu, Org. Biomol. Chem., 2017, 15,
2575–2584.
54 H. Fujita, N. Hayakawa and M. Kunishima, J. Org. Chem.,
2015, 80, 11200–11205.
23 P. Thirumurugan, D. Matosiuk and K. Jozwiak, Chem. Rev.,
2013, 113, 4905–4979.
24 G. A. Russell and G. Kaupp, J. Am. Chem. Soc., 1969, 91,
3851–3859.
25 E. S. B. Ferreira, A. N. Hulme, H. McNab and A. Quye, Chem.
Soc. Rev., 2004, 33, 329–336.
26 N. R. Krishnaswamy and C. N. Sundaresan, Resonance, 2012,
17, 1022–1033.
27 J. A. Kiernan, Biotech. Histochem., 2007, 82, 73–103.
28 S. Trifonov, Y. Yamashita, M. Kase, M. Maruyama and
T. Sugimoto, Anat. Sci. Int., 2016, 91, 56–67.
29 S. Cotson and S. J. Holt, Proc. R. Soc. London, Ser. B, 1958,
148, 506–519.
55 P. Jandera, Anal. Chim. Acta, 2011, 692, 1–25.
56 S. J. Holt and P. W. Sadler, Proc. R. Soc. London, Ser. B, 1958,
148, 495–505.
57 E. M. Bowers, L. O. Ragland and L. D. Byers, Biochim.
Biophys. Acta, 2007, 1774, 1500–1507.
58 M. P. Dale, H. E. Ensley, K. Kern, K. A. R. Sastry and
L. D. Byers, Biochemistry, 1985, 24, 3530–3539.
59 J. B. Kempton and S. G. Withers, Biochemistry, 1992, 31,
9961–9969.
60 D. J. Rabiger, M. Y. Chang, S. Matsukawa and K. C. Tsou,
J. Heterocycl. Chem., 1970, 7, 307–311.
61 K. Ishihara, H. Kurihara and H. Yamamoto, J. Org. Chem.,
1993, 58, 3791–3793.
30 C. J. Cooksey, Molecules, 2001, 6, 736–769.
62 M. del Rosario Benites, T. E. Johnson, S. Weghorn, L. Yu,
P. D. Rao, J. R. Diers, S. I. Yang, C. Kirmaier, D. F. Bocian,
D. Holten and J. S. Lindsey, J. Mater. Chem., 2002, 12, 65–80.
¨
´ ´
31 S. Bottcher, M. Hederos, E. Champion, G. Dekany and
J. Thiem, Org. Lett., 2013, 15, 3766–3769.
¨
¨
32 S. Bottcher and J. Thiem, Eur. J. Org. Chem., 2014, 564–574. 63 C. Grost and T. Berg, Org. Biomol. Chem., 2015, 13, 3866–3870.
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019

View Article Online
NJC
Paper
64 M. Sadauskas, R. Statkeviciute, J. Vaitekunas, V. Petkevicius, 67 W. Robb and C. G. Nicholson, S. Afr. J. Chem., 1978, 31,
V. Casaite, R. Gasparaviciute and R. Meskys, Dyes Pigm.,
2020, 173, 107882.
1–5.
68 D. D. Perrin, Dissociation Constants of Organic Bases in
Aqueous Solution: Supplement 1972, Butterworth & Co. Ltd,
London, 1972.
¨
65 S. Frabel, B. Wagner, M. Krischke, V. Schmidts, C. M. Thiele,
A. Staniek and H. Warzecha, Metab. Eng., 2018, 46, 20–27.
66 G. R. Fulmer, A. J. M. Miller, N. H. Sherden, H. E. Gottlieb, 69 H. K. Hall Jr., J. Am. Chem. Soc., 1957, 79, 5444–5447.
A. Nudelman, B. M. Stoltz, J. E. Bercaw and K. I. Goldberg, 70 T. Fujii, H. Nishida, Y. Abiru, M. Yamamoto and M. Kise,
Organometallics, 2010, 29, 2176–2179.
Chem. Pharm. Bull., 1995, 43, 1872–1877.
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019