Orthogonal Active-Site Labels for Mixed-Linkage endo-β-Glucanases

pubs.acs.org/acschemicalbiology

Articles

Namrata Jain, Kazune Tamura, Guillaume D é jean, Filip Van Petegem, and Harry Brumer*

Cite This: https://doi.org/10.1021/acschembio.1c00063

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: Small molecule irreversible inhibitors are valuable tools for

determining catalytically important active-site residues and revealing key

details of the speciﬁcity, structure, and function of glycoside hydrolases

(GHs). β-glucans that contain backbone β(1,3) linkages are widespread

in nature, e.g., mixed-linkage β(1,3)/β(1,4)-glucans in the cell walls of

higher plants and β(1,3)glucans in yeasts and algae. Commensurate with

this ubiquity, a large diversity of mixed-linkage endoglucanases

(MLGases, EC 3.2.1.73) and endo-β(1,3)-glucanases (laminarinases,

EC 3.2.1.39 and EC 3.2.1.6) have evolved to speciﬁcally hydrolyze these

polysaccharides, respectively, in environmental niches including the

human gut. To facilitate biochemical and structural analysis of these GHs,

with a focus on MLGases, we present here the facile chemo-enzymatic

synthesis of a library of active-site-directed enzyme inhibitors based on

mixed-linkage oligosaccharide scaﬀolds and N-bromoacetylglycosylamine or 2-ﬂuoro-2-deoxyglycoside warheads. The eﬀectiveness

and irreversibility of these inhibitors were tested with exemplar MLGases and an endo-β(1,3)-glucanase. Notably, determination of

inhibitor-bound crystal structures of a human-gut microbial MLGase from Glycoside Hydrolase Family 16 revealed the orthogonal

labeling of the nucleophile and catalytic acid/base residues with homologous 2-ﬂuoro-2-deoxyglycoside and N-bromoacetylglycosyl-

amine inhibitors, respectively. We anticipate that the selectivity of these inhibitors will continue to enable the structural and

mechanistic analyses of β-glucanases from diverse sources and protein families.

roles in plant cell wall structure and as a store of readily

mobilized glucose for developing seedlings. The agricultural

importance of MLGs is underscored by their direct

contribution to human nutrition and health: MLG is an

important component of the human diet and has been linked

to reducing cholesterol and postprandial blood glucose

levels.³²⁻³⁴In this context, the metabolism of MLGs is

mediated by the gut microbiota,³⁵⁻³⁸which makes a signiﬁcant

contribution to the daily caloric intake in humans.³⁹MLGs

have also been of interest as a component of monocot

feedstocks in large-scale bioethanol production.^28,30

MLGs are composed predominantly of β(1,4)-D-glucopyr-

anosyl linkages that are irregularly interspersed with β(1,3)

linkages (Figure 1), resulting in a kinked polysaccharide chain

that resists crystallization.²⁸In this sense, MLGs are

distinguished from both cellulose (β(1,4)-glucan) and the

all-β-(1,3)glucans that constitute major components of yeast

and algal cell walls (e.g., laminarin), as well as the related plant

callose and bacterial extracellular polysaccharides (e.g.,

INTRODUCTION

■

Glycoside hydrolases (GHs) form the largest class of

carbohydrate-active enzymes (CAZymes), currently consisting

of over 165 sequence-based families.^1,2Both reversible and

irreversible inhibitors have aided in the biochemical and

structural characterization of GHs, by enabling the elucidation

of molecular details of active site-speciﬁcity and the

identiﬁcation of key catalytic residues.³⁻⁶Irreversible inhib-

itors, in particular, have been used in many studies to deﬁne

the mechanisms of GH action and to reveal speciﬁc subsite

recognition elements (see reviews⁷⁻¹²and recent primary

literature¹³⁻²²). Such knowledge is critical to inform muta-

tional and evolutionary analysis, as well as guide enzyme

engineering eﬀorts to produce GHs with altered biochemical

and biotechnological properties. When suitably modiﬁed by

chromophores or aﬃnity tags, irreversible inhibitors also ﬁnd

use in the labeling and identiﬁcation of GHs in complex

biological environments (reviewed in refs 12 and 23; for recent

examples, see refs 24−26).

β(1,3)/β(1,4)-Glucans (mixed-linkage β-glucans, MLGs,

Figure 1) are amorphous matrix polysaccharides found in the

cell walls of cereals and other grasses, early diverging vascular

and nonvascular plants, algae (seaweed), lichens, and some

fungi.²⁷⁻³¹MLGs are particularly abundant in oats and barley,

in which they comprise ca. 70% of the soluble, nonstarch

glycan content of the endosperm.^27,32Thus, MLGs play major

Special Issue: Chemical Glycobiology

Received: January 29, 2021

Accepted: May 4, 2021

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

A

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

Figure 1. Representative structure of mixed-linkage β-glucan (MLG) from cereal crops and other grasses. G and G3 indicate β(1,4)- and β(1,3)-

linked glucosyl residues, respectively. The positions of polysaccharide chain hydrolysis by canonical bacterial mixed-linkage endoglucanases to yield

oligosaccharides GG3G and GGG3G is indicated by dashed lines and magenta coloring.

Scheme 1. Mechanism of Anomeric-Conﬁguration-Retaining Glycoside Hydrolases and Mechanisms of Inhibition: (a)

Canonical Double-Displacement Glycoside Hydrolase Mechanism. (b) Mechanism of Inhibition by 2′,4′-Dinitrophenyl-2-

deoxy-2-ﬂuoroglycoside Inhibitors. (c) Mechanism of Inhibition by N-Bromoacetyl Glycosides

Curdlan).^40,41The ratio of β(1,4)-Glc to β(1,3)-Glc residues

in MLGs from diﬀerent sources generally ranges from 2:1 to

3:1.²⁷Although a small percentage of longer β(1,4)-linked

stretches are present, the β(1,3) linkages typically occur after

every two or three β-(1,4)-linked residues. Thus, hydrolysis

with speciﬁc MLGases, which cleave β(1,4) linkages of 3-

substituted Glc residues, results in a limit-digest comprised of

GG3G and GGG3G (where “G” represents a glucosyl residue

and “3” represents the position of the β(1,3) linkage in the

gluco-oligosaccharide, in the widely used shorthand nomen-

are most commonly associated with Glycoside Hydrolase

Family 16 (GH16; bacterial, fungal, and plant representa-

tives)^43,45and GH17 (plant representatives).⁴²MLGase

activity has also been found in members of the GH5,^44,46

GH6, GH7,⁴⁷GH8, GH9,⁴⁸⁻⁵⁰GH11, GH12,⁵¹GH26,⁵²and

GH51 families (for an updated census by EC number, see

http://www.cazy.org/Glycoside-Hydrolases.html; see ref 1),

comprising both anomeric-conﬁguration-retaining and conﬁg-

uration-inverting enzymes (Scheme 1).⁵³In addition to their

biological importance in carbohydrate metabolism,^{28,35,42,43,54}

MLGases have numerous biotechnological applications in

biomass transformation to high-value products, in animal feed

treatment to improve digestibility, in brewing to reduce mash

viscosity, and for the preparation of deﬁned prebiot-

ics.^{36−38,43,47,55,56}

a

27,35,42−44

,

clature of MLG oligosaccharides).

Because of the ubiquity of MLGs in nature, a broad diversity

of GHs from archaea, bacteria, fungi, and plants has evolved to

hydrolyze this family of polysaccharides. In addition to speciﬁc

MLGases (EC 3.2.1.73; formally, β(1,3)/β(1,4)-^D-glucan 4-

glucanohydrolases, also known as licheninases), MLG can also

be depolymerized by general endo-β(1,4)-glucanases (e.g.,

classic cellulases, EC 3.2.1.4), and endo-β(1,3)-glucanases

(laminarinases, EC 3.2.1.39/EC 3.2.1.6). Speciﬁc MLGases

Despite a long history of enzymological characterization,⁴³

including using bespoke aryl glycoside substrates,⁵⁷⁻⁵⁹there

are few speciﬁc inhibitors for MLGases. These are essentially

limited to nonhydrolyzable thio-oligosaccharide analogues as

B

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

Scheme 2. Synthesis of N-Bromoacetylglycoside Inhibitors

competitive (i.e., noncovalent, reversible) active site inhib-

itors.^60,61On the other hand, epoxyalkyl-glucosides, epox-

yalkyl-cellobiosides, epoxyalkyl-laminaribiosides, and epoxyalk-

yl-mixed-linkage oligosaccharides have been synthesized and

tested as covalent inhibitors of MLGases, in some cases

enabling the solution of enzyme-ligand complexes by X-ray

crystallography.^20,62−64Hence, there is signiﬁcant scope for the

development of novel covalent inhibitors of MLGases based on

MLG oligosaccharide scaﬀolds. In the present study, we have

focused our attention on the synthesis and application of

MLGase inhibitors consisting of two distinct chemical

warheads: 2-deoxy-2-ﬂuoro glycosides⁶⁵and N-bromoacetyl-

glycosylamines (see Scheme 1).⁶⁶

On one hand, 2-deoxy-2-ﬂuoro glycosides (Scheme 1b) are

well-established as inactivators of retaining glycoside hydro-

lases.^65,67,68The replacement of the 2-OH in the natural

glycone with ﬂuorine eliminates key hydrogen-bonding

interactions and inductively destabilizes the transition states

leading to the formation and hydrolysis of the covalent

glycosyl-enzyme intermediate. At the same time, a good

leaving group at the anomeric position such as ﬂuorine (HF,

pK_a3.2) or 2′,4′-dinitrophenyl (dinitrophenol, pK_a4.1)

enhances the rate of enzyme glycosylation, which results in

kinetic trapping of the glycosyl-enzyme intermediate (Scheme

1b). Because they are still able to turnover by hydrolysis or

transglycosylation, 2-deoxy-2-ﬂuoro glycosides are essentially

“slow substrates”, with variable lifetimes of the corresponding

glycosyl-enzyme intermediate, depending on the GH stud-

ied.^21,68−75Nonetheless, these inhibitors have enjoyed wide-

spread success for the speciﬁc labeling and identiﬁcation of the

catalytic nucleophile in retaining GHs from diverse families,

because of their mechanistic mimicry.⁸

mounting evidence suggests that N-bromoacetylglycosylamines

exhibit speciﬁcity for the catalytic acid/base residues when

productively bound by retaining glycosidases (Scheme

1c).^{44,76,80,82,83,88}

Our group has recently reported the straightforward

synthesis of N-bromoacetylglycosylamines and 2-deoxy-2-

ﬂuoroglycosides based on highly branched xyloglucan

oligosaccharides as speciﬁc inhibitors of endoxylogluca-

nases.^13,21A key feature of these syntheses was facile access

to complex oligosaccharide building blocks through speciﬁc

enzymatic hydrolysis of the parent polysaccharide, which

obviated the need for tedious de novo synthesis. In the present

study, we have extended this concept to produce a library of

three N-bromoacetylglycosylamine and two 2-deoxy-2-ﬂuo-

roglycoside inhibitors based on β(1,3)/β(1,4)-gluco-oligosac-

charide scaﬀolds, which are readily obtained in multigram

quantities as enzyme limit-digest products.^35,54,87We also

demonstrated the versatility of these inhibitors with exemplar

GH16 MLGases and a GH158 laminarinase from the human

gut microbiota using inhibition kinetics and intact protein mass

spectrometry. Most notably, crystallography of covalent

complexes with the Bacteroides ovatus MLGase BoGH16

highlighted the ability of GG3G N-bromoacetylglycosylamine

and 2-deoxy-2-ﬂuoroglycoside congeners to orthogonally label

the catalytic acid/base and nucleophile residues, respectively,

in this retaining GH.

RESULTS AND DISCUSSION

■

Chemoenzymatic Syntheses of Candidate Inhibitors.

The stereospeciﬁc and regiospeciﬁc synthesis of oligosacchar-

ides is often laborious and fraught with challenges. Therefore,

to rapidly access oligosaccharide building blocks of the target

inhibitors, the commercially available polysaccharides laminar-

in and MLG were enzymatically hydrolyzed to yield the

congeners G3G, GG3G, GGG3G, and G3GGG (see Scheme

On the other hand, N-bromoacetylglycosylamines (Scheme

1c) consist of an electrophilic warhead appended to a

carbohydrate as an enzyme speciﬁcity determinant. Displace-

ment of bromine by nucleophilic residues such as active site

carboxylates makes N-bromoacetylglycosylamines powerful

probes to label and identify catalytic residues.^{13,66,76−85}

Despite their potential for oﬀ-target protein labeling,^44,86,87

a

2) as starting materials for subsequent functionalization with

reactive warheads. G3G was obtained by hydrolysis of algal

laminarin with the broad-speciﬁcity β-glucanase, BuGH16,

from the human gut symbiont Bacteroides uniformis.⁵⁴This

C

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

Scheme 3. Synthesis of 2-Deoxy-2-Fluoroglycoside Inhibitors

yielded a mixture of glucose, G3G, and a trisaccharide of

undetermined structure,⁵⁴which were readily separable via

column chromatography after per-O-acetylation. Per-O-acety-

lated GG3G and GGG3G were likewise obtained in pure form

from oat MLG, facilitated by the proﬁcient MLGase, BoGH16,

from the human gut symbiont Bacteroides ovatus.³⁵The

tetrasaccharide (per-O-Ac)G3GGG, which diﬀers from the

aforementioned series by the position of the β(1,3) linkage,

was produced from oat MLG by employing the unique

regiospeciﬁcity of the Vitis vinifera GH16 endo-glucanase.⁸⁷

Whereas the per-O-acetyl compounds allow direct entry into

the synthetic routes for 2-deoxy-2-ﬂuoro-glycoside inhibitors,

straightforward Zemplen deprotection liberated the free

oligosaccharides for N-bromoacetyl-glycosylamine syntheses.

To obtain the N-bromoacetyl glycosylamines, we applied

and further optimized a two-step synthetic protocol^13,66,77,81to

install the electrophilic warhead (Scheme 2). Thus, GG3G,

GGG3G, and G3GGG were aminated via dissolution in

ammonium hydroxide and addition of ammonium bicarbonate

at 42 °C (Scheme 2, compounds 1−3). Here, an overnight (20

h) reaction time was suﬃcient, versus two-day reactions

reported previously.^13,89Conveniently, the use of unprotected

oligosaccharides avoids additional, and potentially tedious,

protection and deprotection steps. Following removal of the

solvent and reagents under reduced pressure, amide formation

using bromoacetic anhydride in dimethylformamide (DMF)

was facile in all cases. The resulting N-bromoacetyl glycos-

amines were amenable to ﬂash chromatography on silica gel to

produce pure GG3G-β-NHCOCH₂Br (4, yield = 54%),

GGG3G-β-NHCOCH₂Br (5, yield = 25%), and G3GGG-β-

NHCOCH₂Br (6, yield = 39%) in moderate but suﬃcient

yields.

Thus, per-O-acetylated laminaribiose (G3G) and the MLG

trisaccharide GG3G were converted to their respective α-

glycosyl bromides 7 and 8, using the standard treatment with

HBr/HOAc^58,90in excellent yields. Conversion to the

respective glycals, 9 and 10, with zinc powder²¹was also

straightforward.

The incorporation of the 2-ﬂuoro group in analogous 2-

deoxy-2-ﬂuoroglycosides has previously been achieved by

electrophilic ﬂuorination of per-O-acetylated glycals with acetyl

or triﬂuoromethyl hypoﬂuorite,^75,91,92xenon ﬂuoride,⁹³or

elemental ﬂuorine.⁹⁴More recently, these highly reactive and

potentially hazardous reagents have been superseded by

S e l e c t ﬂ u o r ( 1 - c h l o r o m e t h y l - 4 - ﬂ u o r o - 1 , 4 -

diazoniabicyclo[2.2.2]octane bis(tetraﬂuoroborate))⁹⁵for this

purpose, because of its inexpensive commercial availability,

ease of handling, high yields, and simultaneous introduction of

a desired anomeric functional group.⁹⁶However, the stereo-

selectivity of ﬂuorine and hydroxyl group addition upon

reaction with Selectﬂuor is dependent on various factors,

including the speciﬁc conﬁguration of the glycal, the nature of

the hydroxyl protecting groups, and the choice of solvent and

temperature.⁹⁷In numerous previous examples, all four

possible stereoisomers were obtained, yielding diﬃcult-to-

separate mixtures,⁹⁸⁻¹⁰²and additional anomeric acetylation or

selective crystallization are often required.^75,103

Such issues are potentially compounded in the synthesis of

ﬂuorinated oligosaccharides, in which the separation of such

diastereomers becomes even more challenging. To avoid this

problem, 2-deoxy-2-ﬂuoro monosaccharides have previously

been extended using trichloroacetimidate chemistry to obtain

2-deoxy-2-ﬂuoro-disaccharide glycosides, such as 2,4-dinitro-

phenyl 4′-azido-2,4′-dideoxy-2-ﬂuoro-β-xylobioside¹⁰⁴and 2,4-

dinitrophenyl 2-acetamido-2-deoxy-β-^D-glucopyranosyl-(1,4)-

2-deoxy-2-ﬂuoro-β-^D-glucopyranoside.¹⁰²Similarly, speciﬁcally

engineered glycosynthase enzymes have been used to extend

the nonreducing end of monosaccharide inhibitors to yield the

corresponding disaccharide and trisaccharide inhibitors.¹⁰⁵On

On the other hand, the syntheses of the 2′,4′-dinitrophenyl-

2-deoxy-2-ﬂuoro-β-glycosides of G3G and GG3G from the

corresponding per-O-acetates required a more extended

synthesis, consisting of α-bromination, glycal formation,

ﬂuorination, glycosylation, and deprotection (Scheme 3).

D

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

Table 1. Apparent Inhibition Kinetics Parameters

inhibitor

enzyme

k_{i (app)}(min⁻¹

)

K_{i (app)}(mM)

k_i/K_{i (app)}(M⁻¹min⁻¹

)

GG3G-β-NHCOCH₂Br (4)

GGG3G-β-NHCOCH₂Br (5)

G3G(2F)-β-DNP (15)

BoGH16

BuGH158

1.45 0.27

−

0.087 0.006

0.374 0.018

1.68 0.47

−

3.88 0.624

860

1920

22.4

119

GG3G(2F)-β-DNP (16)

3.12 0.373

the other hand, we have previously been successful in

stereoselectively 2-ﬂuorinating a complex xyloglucan heptasac-

charide (Xyl₃Glc₄, “XXXG”) glycal with Selectﬂuor in the

synthesis of an endo-xyloglucanase inhibitor,²¹which avoided

tedious stepwise oligosaccharide construction.^106,107Yet, prior

to the present study, the use of Selectﬂuor on glycals

containing β(1,3) glycosidic linkages had not been explored.

Therefore, in an attempt to obtain exclusively the gluco

epimer, small amounts (20 mg each) of 9 and 10 were

subjected to electrophilic ﬂuorination with Selectﬂuor in

several precedented solvents (DMF, acetonitrile, nitrome-

thane/H₂O, and acetone/H₂O mixtures). As exempliﬁed by

the laminaribiose (G3G) glycal (9), reactions using solvents

with MeNO₂or acetone yielded the gluco (α and β anomer)

and manno (α anomer) epimers approximately in the same

ratios (ca. 1:2; see Figure S1 in the Supporting Information).

Reactions performed in DMF or acetonitrile yielded additional

products, which were not further investigated, but were likely

the result of solvent participation, as has been observed

previously.^97,108The absence of the manno β-anomer in all

cases is consistent with previous reports^97,100,103and can be

rationalized by additive anomeric eﬀects of the ring oxygen and

axial 2-ﬂuorine. Notably, in our previous work, the reaction of

a highly branched xyloglucan heptasaccharide glycal aﬀorded

exclusively the gluco epimer with an anomeric α/β mixture (as

conﬁrmed by ¹⁹F nuclear magnetic resonance (NMR)

spectroscopy).²¹

Since we were unfortunately unable to bias the diaster-

eoselectivity of ﬂuorination here, the reactions of glycals 9 and

10 with Selectﬂuor were scaled-up using acetone/water (5:1)

as a convenient solvent system to achieve moderate yields of

diastereomeric mixtures. ¹⁹F NMR spectra indicated that the

diastereomeric ratios were similar to those observed in the

small-scale reactions (11, Figure S27 in the Supporting

Information; 12, Figure S28 in the Supporting Information).

Because of the diﬃculty in separating the three diastereomers

obtained in each case as the free anomers, these mixtures were

directly subjected to reaction with 2,4-dinitroﬂuorobenzene in

the presence of DABCO. Upon workup, TLC of the crude

product mixture showed three individual spots in each case.

Puriﬁcation using ﬂash chromatography (toluene/acetone,

6.5:1) enabled the isolation of fractions corresponding to

spots A2 (R_f= 0.47) and B2 (R_f= 0.38) as the desired β-gluco

diastereomers, 13 and 14, respectively, as ascertained by the

¹⁹F NMR chemical shift and F−H spin coupling constants (see

Figures S33 and S36, respectively, in the Supporting

Information). Spots A1 (R_f= 0.40) and B1 (R_f= 0.34)

correspond to α-manno diastereomers, while spots A3 (R_f=

0.55) and B3 (R_f= 0.47) correspond to the α-gluco

diastereomers, respectively, as also concluded by ¹⁹F NMR

analysis (for ¹⁹F NMR spectra and assignments of A1 and A3,

see Figures S29 and S30, respectively, in the Supporting

Information). Subsequent deprotection of 13 and 14 using

Zemplen conditions and puriﬁcation using ﬂash chromatog-

raphy yielded the target compounds G3G(2F)-β-DNP (15,

yield = 30%) and GG3G(2F)-β-DNP (16, yield = 40%). The

synthesis of the tetrasaccharide congener GGG3G(2F)-β-DNP

was attempted but subsequently abandoned, because of an

inability to separate a similar mixture of per-O-acetylated

diastereomers of this longer oligosaccharide via ﬂash

chromatography.

Evaluation of Inhibitor Potency. N-Bromoacetylglyco-

sylamines. To investigate their potency as inhibitors, the N-

bromoacetylglycosylamines 4, 5, and 6 were incubated with an

exemplar MLGase from glycoside hydrolase family 16,⁴⁵

BoGH16.³⁵A time- and concentration-dependent inactivation

of the enzyme (8.9 μM) was observed upon incubation with

GG3G-β-NHCOCH₂Br (compound 4; see Figure S2 in the

Supporting Information) and GGG3G-β-NHCOCH₂Br (com-

pound 5; see Figure S3 in the Supporting Information) at

concentrations of 0.019−1.25 mM. At high concentrations of

these compounds, the decline in enzyme activity was

particularly rapid. Indeed, complete inhibition of the enzyme

is apparent within the ﬁrst 20 min of incubation of BoGH16

with ≥0.156 mM of GGG3G-β-NHCOCH₂Br.

The covalent nature of the inhibition was demonstrated by

intact-protein mass spectrometry after incubation of BoGH16

with 2.5 mM of compounds 4 and 5. In both cases, a 1:1

labeling stoichiometry is observed with no signs of over-

labeling, as has been observed with N-bromoacetylglycosyl-

amines in some cases.^13,66,77,80Under identical concentrations

of enzyme and G3GGG-β-NHCOCH₂Br (compound 6), there

was no indication of labeling, even with extended incubation

time (Figure S4 in the Supporting Information). These results

are consistent with the known speciﬁcity of BoGH16, which

requires a β(1,3) linkage spanning the −2 and −1 enzyme

subsites³⁵(subsite nomenclature according to ref 109).

Fitting of the Kitz−Wilson model¹¹⁰(see eqs 1−3 in the

section titled “Materials and Methods”) to the time- and

concentration-dependent inhibition data obtained for GG3G-

β-NHCOCH₂Br (Figure S2) enabled the calculation of the

apparent speciﬁc inhibition constants (eq 1, Table 1) and an

apparent k_i/K_ivalue of 860 M⁻¹min⁻¹). These values compare

favorably to those of other N-bromoacetylglycosylamine

inhibitors, which similarly exhibit K_ivalues in the millimolar

range.^{13,44,77−79,81,83−85}However, the inactivation constants, k_i,

for other N-bromoacetylglycosylamine inhibitors are 2−3

orders of magnitude lower.^{13,44,77−79,81,83,85}The large error

on the apparent K_ivalue, in this case, reﬂects an apparently

poor ability to saturate the enzyme and a correspondingly ﬂat

k_appvs [I] curve (see Figure S2). Similarly, saturation

inhibition kinetics were not achieved with the tetrasaccharide

inhibitor GGG3G-β-NHCOCH₂Br (compound 5; see Figure

S3 in the Supporting Information). Thus, a linear ﬁt to the data

was used to obtain an apparent k_i/K_ivalue of 1920 M⁻¹min⁻¹

(Table 1), which is comparable to that of the trisaccharide

inhibitor GG3G-β-NHCOCH₂Br. As discussed in the protein

structure section below, these results are concordant with the

observation of three negative subsites in BoGH16.³⁵

E

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

Figure 2. Stoichiometric labeling of β-glucanases with 2-deoxy-2-ﬂuoroglycosides. Intact protein MS of wild-type enzymes before (upper panel)

and after incubation with 2.5 mM 2-deoxy-2-ﬂuoroglycoside for 2 h: (a) Bacteroides uniformis GH16 (BuGH16) with G3G(2F)-β-DNP (15) (ΔM

expected 327 Da), (b) Bacteroides uniformis GH16 (BuGH16) with GG3G(2F)-β-DNP (16) (ΔM expected 489 Da), and (c) Bacteroides ovatus

GH16 (BoGH16) plus GG3G(2F)-β-DNP (16) (ΔM expected 489 Da). Phosphogluconoylation of each protein resulting from recombinant

production in E. coli is observed at +178 Da.¹³⁷

2′,4′-Dinitrophenyl-2-deoxy-2-ﬂuoro-β-glycosides. Re-

cently, our group elucidated the mechanism of laminarin

digestion by a prominent member of the human gut

microbiota, Bacteroides uniformis, with a particular focus on

the structural and biochemical characterization of the GHs

BuGH158 and BuGH16 encoded within a complex Poly-

saccharide Utilization Locus.⁵⁴BuGH158 was conﬁrmed to be

a highly speciﬁc anomeric conﬁguration-retaining endo-β(1,3)

glucanase with high activity on laminarin from Laminaria

digitata (brown algae) and limited activity on MLG from

barley (ca. 140-fold lower). BuGH16 was found to be a broad-

speciﬁcity endo-β(1,3)/β(1,4)-glucanase with activity on L.

digitata laminarin ∼4 times higher than barley MLG.⁵⁴

As a representative retaining laminarinase/MLGase,

BuGH158 was then used to study the potency of 15 and 16

as potential inhibitors, using the chromogenic substrate 2′-

chloro-4′-nitrophenyl-β-laminaribioside (G3G-β-CNP) to

measure residual activity (K_m= 12.6 1.15 mM, k_cat= 1918

154 min⁻¹; see Figure S5 in the Supporting Information). At

concentrations ranging from 0.31 to 10 mM, both 15 and 16

exhibited a time- and concentration-dependent inactivation of

BuGH158 over 3 h, with rapid and complete inactivation by 16

being particularly apparent (see Figures S6 and S7 in the

Supporting Information). Kitz−Wilson kinetic analysis (eqs

1−3) allowed the calculation of the apparent inhibition

constants for 15 and 16 (Table 1). The apparent k_ivalues

reported here are either in a similar range^75,111−115or 1−2

orders of magnitude lower^{65,68,70,72,116}than similar 2-deoxy-2-

ﬂuoro-dinitrophenyl or 2-deoxy-2-ﬂuoro-glycosyl ﬂuoride

inhibitors. The apparent K_ivalues observed here are in a

comparable millimolar range.

weight of 325.8 and 488.0 Da, respectively, were observed for

15 and 16 upon incubation with the enzyme. These results are

consistent with the addition of a single 2-deoxy-2-ﬂuoro G3G

moiety and a single 2-deoxy-2-ﬂuoro GG3G moiety,

respectively. No trace of the free enzyme was observed for

either inhibitor, indicative of relatively stable glycosyl-enzyme

intermediates with limited turnover over the course of the

incubation. In comparison, the retaining MLGase BuGH16

was labeled by 15 and 16 (Figure 2). However, labeling with

15 was incomplete, suggesting either slow inhibition or slow

turnover.⁷³

To exemplify the versatility of these inhibitors to label a

range of β-glucanases, the mixed-function laminarinase/

MLGase BuGH16 and the MLGase BoGH16 were both

subjected to qualitative intact protein MS analysis following

treatment with 15 and 16 for 2 h. Notably, BuGH16 appeared

to be incompletely labeled by G3G(2F)-β-DNP (15) during

this period, but was completely labeled by GG3G(2F)-β-DNP

(16). Similarly, complete labeling of the MLGase BoGH16 was

observed using 16 (Figure 2) as a prelude to subsequent

crystallography.

Structural Analysis of Covalent Enzyme Complexes.

To provide structural insight into the modes of inhibition, the

complexes formed by the incubation of GGG3G-β-

NHCOCH₂Br and GG3G(2F)-β-DNP with BoGH16 were

solved to resolutions of 2.15 and 1.56 Å, respectively, via X-ray

crystallography (Table S1 in the Supporting Information). The

overall tertiary structure, comprising a canonical β-jelly roll

fold typical of GH16,⁴⁵was virtually identical to previously

determined unliganded and GGG3G product−complexed

structures of BoGH16 (PDB IDs 5NBO and 5NBP,

respectively³⁵) (see Figure 3).

Intact-protein mass spectrometry of BuGH158 upon

incubation with 2.5 mM of 15 or 16 revealed the covalent

nature of inhibition and stoichiometric labeling of the enzyme

after 2 h (Figures S6 and S7). An increase in protein molecular

The GGG3G-β-NHCOCH₂complex structure was success-

fully obtained by soaking crystals in a solution containing 2.5

mM of compound 5 (the same concentration used for intact

F

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

Figure 3. Crystal structure of BoGH16-inhibitor complexes. (A) Overall structure with BoGH16 shown in cartoon color ramped from blue (N-

terminus) to red (C-terminus) with transparent surface in white. Both inhibitors bound in the active site are shown as sticks (GG3G-β-NHCOCH₂

in cyan and GG3G(2F) in rose throughout the ﬁgure), as are catalytic and other key active-site residues. (B) The F_o-F_comit density map

(generated by Privateer¹³⁶) of GG3G-β-NHCOCH₂contoured at 3.0 σ. (C) The F_o-F_comit density map (generated by Privateer¹³⁶) of

GG3G(2F) contoured at 3.0 σ. (D) BoGH16 active site interactions with GG3G-β-NHCOCH₂, with hydrogen bonds (3.0 Å or shorter) shown as

cyan-colored dashed lines. (E) BoGH16 E148A active-site interactions with GG3G(2F), with hydrogen bonds (3.0 Å or shorter) shown as rose-

colored dashed lines. (F) Superposition of the two inhibitors and a GGG3G tetrasaccharide product (yellow sticks; PDB ID: 5NBP) in the

BoGH16 active site, with hydrogen bonds (3.0 Å or shorter) to GGG3G shown as yellow dashed lines.

protein MS experiments, which is 2-fold greater than the

highest concentration used for kinetics experiments; see Figure

S3). Notably, the observed electron density corresponding to

the covalently bound inhibitor was superior in chain A. The

lower occupancy observed in chain B was attributed to the

proximity of the active site to a protein chain from a

neighboring asymmetric unit. Nevertheless, the near-complete

glycone was modeled into the active site of chain A, spanning

subsites −1 to −3, including the N-acetylated glucosylamine

residue in subsite −1 (see Figure 3b, as well as Table S2 in the

Supporting Information). The observed electron density

unambiguously revealed that the catalytic acid/base residue,

Glu148, was labeled by nucleophilic substitution of the

bromide (Figure 3d). Notably, the Glu148 side chain

underwent a minor rotation and shift in orientation to

accommodate the covalent linkage between Oε and N-acetyl

group of the inhibitor (Figure 3). The density of the

nonreducing-terminal glucosyl residue corresponding to

subsite −4 was too poor to model (Figure 3b), reminiscent

of the relatively high disorder of this residue in the

BoGH16:GGG3G product complex.³⁵

The inhibitor complex structure clearly revealed that the β

orientation of the N-bromoacetyl group distanced the

electrophilic warhead away from the catalytic nucleophile

Glu143, which is positioned directly below the anomeric

carbon. Instead, the N-acetyl group ﬁlled the space that

typically accommodates a water molecule in the glycosyl-

enzyme intermediate of the normal catalytic cycle, which

ideally positioned the electrophile for nucleophilic attack by

Glu148. The natural role of this residue is to deprotonate a

water molecule for turnover of the glycosyl-enzyme inter-

mediate in the second step of the canonical double

displacement GH mechanism (Figure 1)^53,117The fact that

the catalytic acid/base residue is labeled, rather than the

catalytic nucleophile, is similar to previous observations with

N-bromoacetylglycosylamine inhibitors.^{44,76,80,82,83,88}

Despite extensive eﬀorts, including screening various

concentrations and soaking times, we were unable to obtain

a GG3G(2F)-bound complex structure of wild-type BoGH16,

G

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

because of the apparent turnover of the 2-deoxy-2-

ﬂuoroglycosyl-enzyme, which is a well-known phenomenon

with some glycosidases.⁷³Hence, we replaced the catalytic

acid/base, Glu148, with an alanine to further retard the

turnover of the inhibitor-enzyme complex.⁷³Through this

approach, we successfully captured the inhibitor-bound

complex by soaking crystals of the E148A mutant in a solution

containing 5 mM inhibitor for 20 min. Clear electron density

corresponding to the inhibitor was observed in both molecules

in the asymmetric unit, which allowed modeling of the

complete trisaccharide spanning subsites −1 to −3 in both

chains (see Figure 3b, as well as Table S3 in the Supporting

Information). A covalent bond was clearly observed between

C-1 and the catalytic nucleophile, Glu143, in the glycosyl-

enzyme intermediate (Figure 3b, cf. Figure 1). Relative to the

wild-type, Glu143 is rotated toward the “electrostatic helper”,

Asp145, which is widely conserved in GH16 members.⁴⁵

Asp145, in turn, is swung toward the space normally occupied

by the side chain of Glu148. In wild-type BoGH16, Glu143

likely assumes the normal, unrotated conformation in the

covalent glycosyl-enzyme.

synthesis of N-bromoacetylglycosylamines and 2-deoxy-2-

ﬂuoro glycosides containing β(1,3)-glycosidic linkages. Where-

as the potency of these inhibitors was demonstrated through

enzyme kinetic analysis and intact protein mass spectrometry,

protein crystallography revealed that these two classes of

inhibitors exhibit orthogonal speciﬁcity for the catalytic acid/

base and catalytic nucleophile, respectively, of an exemplar

mixed-linkage endoglucanase. Hence, deploying tandem pairs

of N-bromoacetylglycosylamines and 2-deoxy-2-ﬂuoroglyco-

sides has signiﬁcant scope in the mechanistic analysis of GHs.

This work contributes to the active ﬁeld of small-molecule

covalent probes for GH characterization. We envision that

these inhibitors could be elaborated into probes for activity-

based protein proﬁling^8,9,23by additional chemistry, including

the use of glycosynthases to extend the glycone speciﬁcity

element with reactive handles.¹¹⁸Furthermore, in light of

recent renewed interest in covalent inhibitors as drugs,¹¹⁹such

molecules could potentially be used to control microbial

populations. Such applications will of course be dependent on

the biological environment in which they are deployed, noting

that 2-deoxy-2-ﬂuoroglycosides generally have long hydrolytic

half-lives (tens of days at 37 °C),¹²⁰while N-bromoacetylgly-

cosylamines may be prone to side reactions with nitrogen and

sulfur nucleophiles.^44,66

Other than the notable diﬀerence of labeling diﬀerent

catalytic residues, both inhibitors display essentially identical

interactions with the BoGH16 active-site cleft, including

carbohydrate-aromatic stacking with Trp129 and Trp138 at

the −1 and −2 subsites, respectively, as well as several

hydrogen bonds (Figure 3). Except for the −1 subsite (vide

infra), the individual Glc residues of both inhibitors super-

imposed closely with the GGG3G oligosaccharide in our

previously solved product complex (PDB ID: 5NBP³⁵) (Figure

3f). Interestingly, the same “swung in” rotamer of Tyr181

observed in the GGG3G product complex³⁵is seen with the

GGG3G-NHCOCH₂inhibitor (Figure 3d, cf. Figure 3f).

However, instead of making a hydrogen bond with the

anomeric (C1) hydroxyl of the glucosyl residue in subsite −1

(Figure 3d), the hydroxyl group of Tyr181 hydrogen bonds to

the carbonyl oxygen of the N-acetyl group of the inhibitor

(Figure 3f). This mobile tyrosine did not assume a similar

“swung in” position in the GG3G(2F) covalent complex,

because of a lack of hydrogen bonding partner (Figure 3e).

A further notable diﬀerence between the inhibitors is that, in

the GGG3G-β-NHCOCH₂−complex, the N-acetylated glu-

cose in subsite −1 assumed a ⁴E envelope conformation (Table

S2), whereas the low-energy ⁴C₁chair conformation was

observed in the GG3G(2F) complex (as well as the GGG3G

product complex) (Figure 3). It appears that, due to geometric

constraints of the Glu148 side chain position and the

conservation of active site interactions, the anomeric carbon

is pulled upward by the N-acetyl linkage to Glu148 (Figure

MATERIALS AND METHODS

■

Enzyme Sources. The Vitis vinifera (grape) endo-(xylo)glucanase

variant VvEG16 (ΔV152) was produced as previously described.⁸⁷

The Bacteroides uniformis laminarinase BuGH158 and MLGase

BuGH16 were produced as previously described.⁵⁴The Bacteroides

ovatus MLGase BoGH16 construct in pET28 vector (BoGH16:-

pET28) from a previous study was utilized for wild-type recombinant

protein production.³⁵The catalytic acid/base mutant BoGH16 E148A

was generated by site-directed mutagenesis, using the QuickChange

(Agilent Technologies) protocol with PfuUltra high-ﬁdelity polymer-

ase (Agilent Technologies) and DpnI (New England Biolabs). PCR

ampliﬁcation was conducted using the forward primer 5′-GGG AGA

TTG ATA TTA TGG CTA TGG GAG AAC AGA GCG-3′, reverse

primer 5′-CGC TCT GTT CTC CCA TAG CCA TAA TAT CAA

TCT CCC-3′, and the BoGH16:pET28 plasmid as the template.

Successful generation of clones and site-directed mutants was veriﬁed

by Sanger sequencing (Genewiz).

Recombinant BoGH16 and the BoGH16 E148A mutant were

produced in E. coli BL21 (DE3) and puriﬁed by nickel aﬃnity

chromatography as described previously,³⁵with the exception that

HEPES buﬀers were used instead of sodium phosphate (binding

buﬀer: 20 mM HEPES pH 7.4, 500 mM sodium chloride, 20 mM

imidazole; elution buﬀer: 20 mM HEPES pH 7.4, 500 mM sodium

chloride, 500 mM imidazole). The protein used in crystallography was

further puriﬁed by size exclusion chromatography, using Superdex 75

(GE Healthcare) packed in XK 16/100 column (GE Healthcare) run

with 10 mM HEPES pH 7.0. The fractions were inspected for purity

by SDS-PAGE and pure fractions were pooled and concentrated in

Vivaspin centrifugal ﬁlters (GE Healthcare). Final protein concen-

tration was determined by spectrophotometry at 280 nm in an Epoch

Microplate Spectrophotometer (BioTek) using the extinction

coeﬃcient 54 890 M⁻¹cm⁻¹, which was calculated from the primary

sequence using ProtParam tool from the ExPASy Bioinformatics

Resource Portal.¹²¹

4

3d). Considering these observations together, the E envelope

conformation can be rationalized by a lower energy require-

ment to bring the sugar out of the relaxed ⁴C₁chair

conformation than that needed to compromise the numerous

interactions between the tetrasaccharide and the side chains

that compose the enzyme active site.

CONCLUSION

Inhibitor Synthesis. General. All reagents were analytical or

HPLC grade and were purchased from Sigma−Aldrich, Alfa-Aesar, or

ACROS Organics. Sodium methoxide solution (25 wt % in methanol)

was purchased from Sigma−Aldrich. For anhydrous reactions,

glassware was dried overnight in a 100−150 °C oven and purged

with argon before use. Solvents were dried by stirring with activated 4

Å molecular sieves overnight under argon. Thin-layer chromatography

(TLC) was performed using aluminum sheet TLC plates (0.25 mm)

■

In this work, the synthesis of active-site directed β-glucanase

inactivators, which consist of mixed β(1,3)/(1,4) linkages as

speciﬁcity determinants, was signiﬁcantly enabled by ready

access to the corresponding oligosaccharide building blocks via

enzymatic hydrolysis of bulk polysaccharides. Indeed, to the

best of our knowledge, this is the ﬁrst report of the controlled

H

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

precoated with Merck silica gel 60 F254, using ethyl acetate:/hexanes,

toluene/acetone, water/methanol/ethyl acetate, or water/isopropa-

nol/ethyl acetate as solvent systems (particular solvent ratios speciﬁed

below), and visualized by a UV lamp and/or 10% sulfuric acid in

water with charring using a heat gun. Flash chromatography was

performed using Merck silica gel 60 with the same eluent systems as

used for the corresponding TLC. The Amberlite IR120H⁺resin was

washed with a copious amount of methanol before use.

reduced pressure and subsequent lyophilization, a pale powder was

obtained. This powder was conﬁrmed by HPAEC-PAD to contain a

mixture of glucose, cellobiose, and Glcβ(1,3)-Glcβ(1,4)-Glcβ(1,4)-

Glc (G3GGG) (Figure S9 in the Supporting Information). Isolation

of the pure per-O-acetylated oligosaccharide (per-OAc)G3GGG was

achieved using the acetylation and ﬂash chromatography conditions as

described in the previous section (yield = 700 mg). Zemplen

deprotection (MeOH/DCM (9:1) containing catalytic NaOMe,

followed by stirring at 4 °C overnight), yielded the pure deprotected

oligosaccharide in near quantitative yield.

All ¹⁹F-, ¹³C- and ¹H NMR data were collected on a Bruker Avance

400 MHz spectrometer at RT (100.6 and 376.5 MHz for ¹³C and ¹⁹F,

respectively). ¹H NMR spectra were referenced to CHCl₃at 7.27 ppm

or HOD at 4.79 ppm, ¹³C NMR spectra were referenced to CDCl₃at

77.0 ppm, and ¹⁹F NMR spectra were referenced to CFCl₃at 0.00

ppm.¹²²MALDI-TOF MS data were collected on a Bruker Autoﬂex

instrument in reﬂectron mode over m/z 700−3500, using 6-Aza-2-

thiothymine (ATT) as the matrix. ESCI MS was performed on a

Waters LC-MS system including Waters 2695 HPLC and Waters ZQ

mass spectrometer. HRMS data were obtained using either a Waters

Xevo G2-S Q-TOF or Waters/Micromass LCT TOF mass

spectrometer in positive-ion mode, via direct infusion through an

electrospray ion source. HPAEC-PAD was performed on a Dionex

ICS-5000 system equipped with an AS-AP autosampler and

temperature-controlled sample tray, run in a sequential injection

conﬁguration using CHROMELEON 7 software, as described

previously.^35,54,87

Preparation of Laminaribiose (G3G) per-O-Acetate. Laminar-

ibiose (G3G) was prepared by enzymatic hydrolysis of 3 g of the

commercially available polysaccharide laminarin from Laminaria

digitate, using BuGH16, which yielded a mixture of glucose, G3G,

and a small amount of a mixed-linkage trisaccharide, as described

previously.⁵⁴This mixture was acetylated by dissolving in pyridine (60

mL) and acetic anhydride (40 mL), followed by heating to 60 °C for

3 h. Following workup, the crude mixture was puriﬁed using column

chromatography with toluene/acetone (6.5:1) as the eluent to yield

3.5 g of pure (per-OAc)G3G (R_f= 0.33). The characterization data

were in accordance with literature values.⁹⁰

Preparation of GG3G and GGG3G Mixed-Linkage Oligosacchar-

ides and per-O-Acetates. Oat β-glucan powder (30 g, 70% purity;

Garuda International, CA, USA) was suspended in 25 mM sodium

citrate buﬀer, pH 6.5 (1 L), heated to 50 °C, and stirred for 20 min.

100 μL of 0.45 mM BoGH16 was added to this suspension and the

reaction was stirred overnight at 37 °C, following which the mixture

was concentrated under reduced pressure. The resulting syrup was

then freeze-dried under vacuum to yield a pale powder, conﬁrmed by

HPAEC-PAD chromatography to contain a mixture of trisaccharide

Glcβ-(1,4)-Glcβ(1,3)-Glc (GG3G) and tetrasaccharide Glcβ(1,4)-

Glcβ(1,4)-Glcβ(1,3)-Glc (GGG3G) as the only oligosaccharides

(Figure S8 in the Supporting Information).³⁵

20 g of this mixture was per-O-acetylated at 60 °C using pyridine

(150 mL) and acetic anhydride (100 mL) with N,N-dimethylamino-

pyridine (0.5 g) as a catalyst. After 3 h, the reaction mixture was

concentrated under reduced pressure to near dryness, and

subsequently dissolved in 7 mL of dichloromethane (DCM) and

loaded onto a silica ﬂash column. The peracetylated trisaccharide and

tetrasaccharide were separated using ﬂash chromatography with

isocratic toluene/acetone (6.5:1) as the solvent system ((per-

OAc)GG3G: R_f0.3, yield = 3 g, (per-OAc)GGG3G: R_f0.25, yield

= 1 g). The per-O-acetylated oligosaccharides were stored at RT and

were subsequently deprotected under Zemplen conditions using

catalytic NaOMe in MeOH/DCM (9:1) as a solvent, followed by

stirring at 4 °C overnight), as needed. The characterization of the

resulting deprotected oligosaccharides, as well as their corresponding

peracetylated congeners, was in accordance with previous liter-

ature.^58,123,124

1

(per-OAc)G3GGG. R_f= 0.25 (toluene/acetone: 6.5:1). H NMR

(CDCl₃, 400 MHz; see Figure S10 in the Supporting Information): δ

6.21 (H1α, d, J_H1−H2= 3.69 Hz, 0.45 H), 5.62 (H1β, d, J_H1−H2= 8.39

Hz, 0.54 H), 5.38 (t, J = 9.78, 0.5H), 5.19 (dd, J = 9.27, 8.95, 0.5H),

5.08- 4.78 (m, 7H), 4.52−3.55 (m, 19H), 2.14−1.94 (s, CH₃). ¹³C

NMR: (CDCl₃, 100.6 MHz, Figure S11 in the Supporting

Information): δ 168.56−170.69 (CO), 100.89, 100.73, 100.37

(C1^II, C1^III, C1^IV), 91.64 (C1β), 89.02 (C1α), 78.65, 76.33, 76.04,

75.8, 75.69, 73.65, 73.02, 72.92, 72.86, 72.83, 72.66, 72.6, 72.4, 72.26,

71.84, 71.76, 71.11, 70.85, 70.63, 69.53, 69.47, 68.01, 67.89, 62.37,

61.86, 61.70, 61.32, 20.95−20.44 (COCH₃); monoisotopic m/z

calculated for C₅₂H₇₀O₃₅Na⁺: 1277.35954; LC-MS found: 1277.6;

ESI-HRMS found: 1277.3602

G3GGG. ¹H NMR (CDCl₃, 400 MHz, Figure S12 in the

Supporting Information): δ 5.24 (H1α, d, J_H1−H2= 3.72 Hz

0.40H), 4.76 (H1^II, d, J_H1−H2= 7.95 Hz, 1H), 4.67 (H1β, d, J_H1−H2

= 7.95 Hz, 0.63H), 4.55 (H1^III, H1^IV, d, d, J = 8.14 Hz, 7.95 Hz, 2H),

4.01−3.27 (m, 23 H); ¹³C NMR (D₂O, 100.6 MHz, Figure S13 in the

Supporting Information): δ 102.98, 102.76, 102.52 (C1^II, C1^III, C1^IV),

95.96 (C1β), 92.02 (C1α), 84.14, 78.83, 78.68, 78.59, 78.57, 76.19,

75.79, 75.74, 75.68, 75.01, 74.45, 74.26, 74.1, 73.63, 73.19, 73.15,

71.5, 71.43, 70.32, 69.78, 69.65, 68.19, 60.89, 60.76, 60.20, 60.08;

Monoisotopic m/z calculated for C₂₄H₄₂O₂₁Na⁺: 689.21163; LC-MS

found: 689.2; ESI-HRMS found: 689.2115.

Synthesis of N-Bromoacetylglycosylamines. The new N-bromoa-

cetylglycosylamines were obtained by adapting previously established

methods (Scheme 2).^13,77150 mg of GG3G, GGG3G, or G3GGG

was dissolved in 15 mL of 30% ammonium hydroxide, and

ammonium bicarbonate (2 equiv) was added to the mixture. The

mixture was then stirred overnight (20 h) at 42 °C, and thereafter

concentrated under reduced pressure to a powder before lyophiliza-

tion to yield the corresponding β-aminoglycoside as a pale amorphous

solid. Because of their inherent acid lability, these crude compounds

were used directly without isolation.

1

GG3G-β-NH₂(1). H NMR (D₂O, 400 MHz, Figure S14 in the

Supporting Information): δ 4.73 (H1^II, d, J = 9.28 Hz, 1H), 4.51

(H1^III, d, J = 8.27 Hz, 1H), 4.13−3.29 (m, 18 H).

1

GGG3G-β-NH₂(2). H NMR (D₂O, 400 MHz, Figure S15 in the

Supporting Information): δ 4.70 (H1^II, d, J = 9.15 Hz, 1H), 4.50

(H1^III, d, J = 8.60 Hz, 1H), 4.48 (H1^IV, d, J = 7.96 Hz, 1H), 4.10−

3.15 (m, 24H).

1

G3GGG-β-NH₂(3). H NMR (D₂O, 400 MHz, Figure S16 in the

Supporting Information): δ 4.74 (H1^II, d, 1H), 4.55 (H1^III, d, J = 7.85

Hz, 1H), 4.53 (H1^IV, d, J = 8.07 Hz, 1H), 4.13−3.18 (m, 24H).

Each β-aminoglycoside (150 mg) was subsequently dissolved in 7

mL of dry N,N-dimethylformamide (DMF) under an inert

atmosphere and stirred for 10 min to form a pale-yellow suspension.

Bromoacetic anhydride (1.5 equiv) was added to the reaction mixture

as a solution in 0.5 mL dry DMF solution through a syringe.

Thereafter, the solution turned into a homogeneous dark yellow color.

The reaction was monitored via TLC. Generally, the reaction time

was 1.5−2 h. The reaction mixture was concentrated in the presence

of silica (7 g) and dry-loaded onto a ﬂash column. The product was

eluted using a water/isopropanol/ethyl acetate (1:3:6) solvent

system. Fractions containing the product were pooled together,

concentrated, and dried to a pale powder. The yields after puriﬁcation

were 100 mg (yield: 54%, purity estimated by ¹H NMR: >95%) for 1,

Preparation of G3GGG Oligosaccharide and per-O-Acetate.

G3GGG was prepared by scaling-up our previously described enzyme

hydrolysis method,⁸⁷with modiﬁcations. Thus, 5 g of oat β-glucan

powder (70%) was suspended in 25 mM sodium citrate buﬀer, pH 6

(1 L). 100 μL of 600 μM VvEG16 (ΔV152) was added and the

reaction was stirred for 48 h at 37 °C. Upon concentration under

1

44 mg (yield: 25%, purity estimated by H NMR: >95%) for 2, and

1

68 mg (yield: 39%, purity estimated by H NMR: >95%) for 3.

I

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

GG3G-β-NHCOCH₂Br (4). R_f= 0.25 (water/isopropanol/ethyl

acetate 1:3:6); ¹H NMR (D₂O, 400 MHz, Figure S17 in the

Supporting Information): δ 5.05 (H1β, d, J = 9.30 Hz, 1H), 4.84

(H1^II, d, J = 8.06 Hz, 1H), 4.57 (H1^III, d, J = 7.82 Hz, 1H), 4.03 (s,

COCH₂Br, 2H), 3.98−3.37 (m, 18H); ¹³C NMR: (D₂O, 100.6 MHz,

Figure S18 in the Supporting Information): δ 171.31 (CO),

102.77, 102.74, 84.92, 79.76, 78.79, 77.58, 76.73, 76.18, 75.69, 75.05,

74.34, 73.38, 71.65, 69.69, 67.97, 60.83, 60.26, 60.07, 28.16 (CH₂Br);

monoisotopic m/z calculated for C₂₀H₃₄BrNO₁₆Na⁺: 646.09587; LC-

MS found: 646.1; ESI-HRMS found: 646.0948.

GGG3G-β-NHCOCH₂Br (5). R_f= 0.25 (water/isopropanol/ethyl

acetate 1:3:6); ¹H NMR (D₂O, 400 MHz, Figure S19 in the

Supporting Information): δ 5.03 (H1β, d, J = 9.31 Hz, 1H), 4.82

(H1^II, d, 1H), 4.54 (H1^III, H1^IV, d, d, J = 8.36 Hz, J = 8.42 Hz, 2H),

4.00 (s, COCH₂Br, 2H), 4.02−3.33 (m, 24H); ¹³C NMR: (D₂O,

100.6 MHz, Figure S20 in the Supporting Information): δ 171.34

(CO), 102.77, 102.5, 79.74, 78.61, 78.05, 76.72, 76.19, 75.69, 75.1,

75.05, 74.24, 73.35, 73.14, 71.75, 69.66, 60.78, 60.12, 59.95, 27.98

(CH₂Br); monoisotopic m/z calculated for C₂₆H₄₄BrNO₂₁Na⁺:

808.14870; LC-MS found: 808.5; ESI-HRMS found: 808.1477.

G3GGG-β-NHCOCH₂Br (6). R_f= 0.25 (water/isopropanol/ethyl

acetate 1:3:6); ¹H NMR (D₂O, 400 MHz, Figure S21 in the

Supporting Information): δ 5.02 (H1β, d, J = 9.19 Hz, 1H), 4.76

(H1^II, d, 1H) 4.56 (H1^III, d, J = 8.19 Hz, 1H), 4.53 (H1^IV, d, J = 8.09

Hz, 1H), 4.00 (s, COCH₂Br, 2H), 3.95−3.31 (m, 24H); ¹³C NMR:

(D₂O, 100.6 MHz, Figure S22 in the Supporting Information): δ

171.32 (CO), 102.99, 102.77, 102.50, 84.18, 79.75, 78.62, 78.08,

76.72, 76.19, 75.69, 75.04, 74.24, 73.24, 73.64, 73.36, 73.14, 71.75,

69.80, 69.67, 68.21, 60.97, 60.79, 60.13, 59.97, 28.04 (CH₂Br);

monoisotopic m/z calculated for C₂₆H₄₄BrNO₂₁Na⁺: 808.14870; LC-

MS found: 808.1; ESI-HRMS found: 808.1483.

Synthesis of 2′,4′-Dinitrophenyl-2-deoxy-2-ﬂuoro-β-glycosides.

The synthesis of the new 2-deoxy-2-ﬂuoro-2′,4′-dinitrophenyl β-

glycosides proceeded through a ﬁve-step protocol starting from the

corresponding per-O-acetylated oligosaccharides (Scheme 3). Thus,

(per-OAc)G3G (1.2 g) and (per-OAc)GG3G (1 g) were individually

dissolved in 30 mL of dichloromethane and converted to their

respective α-glycosyl bromides 7 and 8, as per previously established

protocols,^58,90using 33% hydrogen bromide in glacial acetic acid. The

characterization data of compounds 7 (81% yield) and 8 (89% yield)

were in accordance with literature values.^58,90

The per-O-acetylated G3G glycal (9) and per-O-acetylated GG3G

glycal (10) were synthesized by adapting previously established

methods.^21,125Thus, the corresponding glycosyl α-bromide (1000 mg

7 or 900 mg 8) was dissolved in 30 mL of acetic acid and slowly

added to a suspension of Zn (31 equiv), NaOAc (18.5 equiv), and

CuSO₄·5H₂O (0.2 equiv) in 20 mL of water, which had been stirring

for 5 min. The solution was stirred vigorously for 3 h at RT and

subsequently ﬁltered through Celite. The mixture was concentrated

under reduced pressure to remove the solvent, redissolved in DCM

(100 mL), and washed with NaHCO₃(3×) and brine (1×). The

DCM layer was concentrated, and the resulting syrup was puriﬁed

using ﬂash chromatography with isocratic ethyl acetate/hexanes (1:1)

as the mobile phase (for 9: R_f= 0.3, for 10: R_f= 0.25). The

evaporation of solvents under reduced pressure yielded 9 (150 mg,

19% yield) and 10 (160 mg, 21% yield) as white solids.

6.26 Hz, 1 H, H1), 5.25−5.23 (m, 1H), 5.19−4.82 (m, 6H), 4.66 (d, J

= 8.05 Hz, 1H), 4.58 (dd, J = 12.01 Hz, J = 1.83 Hz, 1H), 4.52 (d, J =

7.97 Hz, 1H), 4.39−4.33 (m, 3H), 4.13−4.03 (m, 4H), 3.78 (t, J =

9.47 Hz, 1H), 3.68−3.60 (m, 2H), 2.13−1.98 (9s, 27H, COCH₃).

¹³C NMR (Figure S26 in the Supporting Information, 100.6 MHz,

CDCl₃): δ 170.64−169.17 (9 × CO), 145.26 (C1), 100.92 (C1^III),

99.06(C1^II), 97.44 (C2), 76.43, 74.03, 73.07, 73.00, 72.68, 72.13,

71.73, 71.66, 70.05, 68.00, 67.90, 61.79 (C6/C6^II/C6^III), 61.74 (C6/

C6^II/C6^III), 61.67 (C6/C6^II/C6^III), 20.99−20.67 (9 × CH₃);

monoisotopic m/z calculated for C₃₆H₄₈O₂₃Na⁺: 871.24841;

MALDI-TOF MS found: 871.3; ESI-HRMS found: 871.2483.

Utilizing established synthetic protocols^21,97,98with modiﬁcations

(see the Results and Discussion section), the glycals 9 (150 mg) and

10 (160 mg) were converted into a diastereomeric mixture of the

corresponding (per-O-acetylated)-1-hydroxy 2-deoxy-2-ﬂuoro-glyco-

sides by dissolution in a minimum amount of acetone and addition to

a stirring suspension of Selectﬂuor (1.2 equiv) in 18 mL of acetone/

water (5:1). The solution was stirred ﬁrst at RT for 16 h, and then at

60 °C for 3 h. It was subsequently ﬁltered over a Celite plug and

concentrated under reduced pressure. The resulting syrup was

redissolved in minimum DCM and loaded to a silica column for

partial puriﬁcation using ﬂash chromatography, using isocratic ethyl

acetate/hexanes (1.2:1) as the eluent (R_f= 0.25−3.00 for 11 and 12).

This reaction yielded a mixture of diastereomers of 11 (60 mg, 38%

yield) and 12 (90 mg, 54% yield) respectively, because of the addition

of 2-ﬂuoro and 1-hydroxyl functional groups either equatorially or

axially. These mixtures were characterized using ¹⁹F NMR and high-

resolution mass spectrometry and were used in the next step without

further puriﬁcation.

(per-OAc)-1-Hydroxy-2-deoxy-2-ﬂuoro-G3G (11). ¹⁹F NMR (Fig-

ure S27, 376.5 MHz, CDCl₃): δ −199.17 (ddd, J_(F2−H2)= 50.96 Hz,

J_(F2−H3)= 14.47 Hz, J_(F2−H1)= 1.55 Hz, 0.13F, gluco β-anomer),

−200.60 (dd, J_(F2−H2)= 49.39 Hz, J_(F2−H3)= 12.66 Hz, 0.59F, gluco α-

anomer), −205.68 (ddd, J_(F2−H2)= 49.60 Hz, J_(F2−H3)= 27.70 Hz,

J_(F2−H1)= 6.18 Hz, 1F, manno α-anomer); Monoisotopic m/z

calculated for C₂₄H₃₃O₁₆FNa⁺: 619.16504; MALDI-TOF MS found:

619.1; ESI-HRMS found: 619.1650.

(per-OAc)-1-Hydroxy-2-deoxy-2-ﬂuoro-GG3G (12). ¹⁹F NMR

(Figure S28 in the Supporting Information, 376.5 MHz, CDCl₃): δ

−198.84 (ddd, J_(F2−H2)= 51.11 Hz, J_(F2−H3)= 15.11 Hz, J_(F2−H1)

1.91 Hz, 0.06 F, gluco β-anomer), −200.40 (dd, J_(F2−H2)= 49.51 Hz,

_(F2−H3)= 12.63 Hz, 0.39 F, gluco α-anomer), −205.82 (ddd, J_(F2−H2)

=

J

=

49.33 Hz, J_(F2−H3)= 27.53 Hz, J_(F2−H1)= 6.70 Hz, 1 F, manno α-

anomer); monoisotopic m/z calculated for C₃₆H₄₉O₂₄FNa⁺:

907.24956; MALDI-TOF MS found: 907.1; ESI-HRMS found:

907.2496.

The synthesis of the per-O-acetylated 2′,4′-dinitrophenyl-2-deoxy-

2-ﬂuoro-β-glycosides 13 and 14 followed previously reported

glycosylation methods,^21,75with modiﬁcations. The diastereomeric

mixtures comprising 11 (60 mg) or 12 (90 mg) were individually

dissolved in 5 mL of dry DMF containing 4 Å activated molecular

sieves and anhydrous DABCO (5 equiv). A solution of 2 equiv of 2,4-

DNFB in 1 mL of dry DMF was added to the reaction mixture

through an oven-dried steel needle. The reaction was stirred for 3.5 h,

concentrated using a rotary evaporator, redissolved in DCM (100

mL), and washed with NaHCO₃(3×) and brine (1×). TLC analysis

of the product mixtures using toluene/acetone (2.5:1) indicated 3

spots each when visualized under UV lamp (indicating the presence of

aromatic group), as well as on charring after dipping in 10% sulfuric

acid solution [R_f= 0.40 (A1), 0.47 (A2), 0.55 (A3) for the

diastereomeric mixture from 11, and R_f= 0.34 (B1), 0.38 (B2), 0.47

(B3) for the diastereomeric mixture from 12]. The fractions

corresponding to the spots at R_f= 0.47 (A2) and 0.38 (B2) were

separated by column chromatography using isocratic elution with

toluene/acetone (6.5:1) and identiﬁed to be the desired gluco β

diastereomers 13 and 14, respectively. The spots A1/B1 and A3/B3

were identiﬁed to be the manno α and gluco α-diastereomers,

respectively (for ¹⁹F NMR spectra and assignments of A1 and A3, see

Figures S29 and S30, respectively, in the Supporting Information).

The column fractions containing the desired product were pooled and

(per-OAc)G3G Glycal (9). ¹H NMR (Figure S23 in the Supporting

Information, 400 MHz, CDCl₃): δ 6.39 (d, J_(H1−H2)= 6.26 Hz, 1H,

H1), 5.18−5.11 (m, 2H, H1^II, H1^III), 5.00 (t, J = 9.62 Hz, 1H), 4.88

(dd, J = 8.37 Hz, J = 9.20 Hz, 1H), 4.79 (t, J = 5.10 Hz, 1 H), 4.65 (d,

J = 7.97 Hz, 1H), 4.33−4.00 (m, 6 H), 3.69−3.65 (m, 1H), 1.92−

2.01 (6s, 18H, COCH₃). ¹³C NMR (Figure S24 in the Supporting

Information, 100.6 MHz, CDCl₃): δ 171.15−169.31 (6× CO),

145.30 (C1), 98.85 (C1^II), 97.38 (C2), 73.93, 72.93, 72.00, 71.37,

69.96, 68.37, 67.92, 61.90 (C6/C6^II), 61.56 (C6/C6^II), 21.06−20.62

(6× CH₃); monoisotopic m/z calculated for C₂₄H₃₂O₁₅Na⁺:

583.16390; MALDI-TOF MS found: 583.1; ESI-HRMS found:

583.1636.

(per-OAc)GG3G Glycal (10). ¹H NMR (Figure S25 in the

Supporting Information, 400 MHz, CDCl₃): δ 6.45 (d, J_(H1−H2)

=

J

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

concentrated using rotary evaporation to yield pure 13 (24 mg, 33%)

and 14 (26 mg, 24%).

Monoisotopic m/z calculated for C₁₈H₂₃O₁₄N₂FNa⁺: 533.10311;

MALDI-TOF MS found: 533.1; ESI-HRMS found: 533.1018.

2′,4′-Dinitrophenyl-2-deoxy-2-ﬂuoro-β-GG3G [GG3G(2F)-β-DNP,

16]. ¹H NMR (Figure S40 in the Supporting Information, 400 MHz,

(per-OAc)-2′,4′-Dinitrophenyl-2-deoxy-2-ﬂuoro-β-G3G (13). ¹H

NMR (Figure S31 in the Supporting Information, 400 MHz, CDCl₃):

δ 8.78 (d, J_{(H’3‑H’5)}= 2.70 Hz, 1H, H′3), 8.44 (dd, J_{(H’5‑H’6)}= 9.16 Hz,

D₂O): δ 8.90 (d, J_{(H’3‑H’5)}= 2.50 Hz, 1H, H′3), 8.55 (dd, J_{(H’5‑H’6)}

=

9.33 Hz, J_{(H’5‑H’3)}= 2.50 Hz, 1H, H′5), 7.62 (d, J_{(H’6‑H’5)}= 9.33 Hz,

1H, H′6), 5.76 (dd, J_(H1−H2)= 7.36 Hz, J = 2.21 Hz, 1H, H1), 4.71 (d,

J_{(H1II−H2II)}= 7.40 Hz, 1H, H1^II), 4.51 (d, J_{(H1III−H2III)}= 7.33 Hz, 1H,

H1^III), 4.29−3.29 (m, 18H). ¹³C NMR (Figure S41 in the Supporting

Information, 100.6 MHz, D₂O): δ 153.86 (C′3), 141.72 (C′4),

138.94 (C′5), 129.86 (C′1), 122.19 (C′2), 117.89 (C′6), 102.54,

(C1^III), 102.21 (C1^II), 97.65 (J_(C1−F2)= 24.14 Hz, C1), 91.18 (J_(C2−F2)

= 188.12 Hz, C2), 81.11 (J_(C3−F2)= 16.09 Hz, C3), 78.53, 76.24,

75.94, 75.44, 74.77, 74.05, 73.12, 72.94, 67.50 (C4^II/^III), 67.14

(J_(C4−F2)= 8.05 Hz, C4), 60.54 (C6^II, C6^III), 60.03 (J_(C6−F2)= 11.06

Hz, C6). ¹⁹F NMR (Figure S42 in the Supporting Information, 376.5

MHz, D₂O): δ −199.24 (ddd, J_(F2−H2)= 50.88 Hz, J_(F2−H3)= 15.02

Hz, J_(F2−H1)= 2.42 Hz, gluco β-anomer); Monoisotopic m/z calculated

for C₂₄H₃₃O₁₉N₂FNa⁺: 695.15593; MALDI-TOF MS found: 695.1;

ESI-HRMS found: 695.1552.

J_{(H’5‑H’3)}= 2.70 Hz, 1H, H′5), 7.39 (d, J_{(H’6‑H’5)}= 9.16 Hz, 1H, H′6),

5.29 (dd, J_(H1−H2)= 7.47 Hz, J_(H1−H3)= 3.43 Hz, 1H, H1), 5.25−4.96

(m, 4H), 4.74 (d, J_{(H1II−H2II)}= 7.86 Hz, 1H, H1^II), 4.69−4.64 (m,

1H), 4.34 (dd, J = 12.51 Hz, J = 4.23 Hz, 1H), 4.22 (d, J = 3.76 Hz,

2H), 4.14−4.07 (m, 2H), 3.94−3.89 (m, 1H), 3.72−3.68 (m, 1H),

2.10−2.01 (6s, 18H, COCH₃). ¹³C NMR (Figure S32 in the

Supporting Information, 100.6 MHz, CDCl₃): δ 170.46−169.05 (6 ×

CO), 153.39 (C′3), 142.43 (C′4), 140.38 (C′5), 128.56 (C′1),

121.72 (C′2), 118.39(C′6), 101.39 (C1^II), 98.59 (J_(C1−F2)= 24.93 Hz,

C1), 91.07 (J_(C2−F2)= 190.19 Hz, C2), 79.47, (J_(C3−F2)= 18.13 Hz,

C3), 72.85, 72.56, 71.88, 71.33, 67.97 (C4^II), 67.37 (J_(C4−F2)= 8.31

Hz, C4), 61.70 (C6, C6^II), 20.69−20.33 (6 × CH₃). ¹⁹F NMR

(Figure S33 in the Supporting Information, 376.5 MHz, CDCl₃): δ

−198.81 (ddd, J_(F2−H2)= 50.51 Hz, J_(F2−H3)= 15.27 Hz, J_(F2−H1)

=

3.17 Hz, gluco β-anomer); Monoisotopic m/z calculated for

C₃₀H₃₅O₂₀N₂FNa⁺: 785.16650; MALDI-TOF MS found: 785.2;

ESI-HRMS found: 785.1667.

Enzyme Substrates. The chromogenic substrate GG3G 2′-chloro-

4′-nitrophenyl β-glycoside (GG3G-β-CNP) was purchased from

Megazyme International (Ireland, product code O-CNPBG3). G3G

2′-chloro-4′-nitrophenyl β-glycoside (GG3G-β-CNP) was synthesized

from α-laminaribiosyl bromide (7) by adapting a previously

established protocol,¹²⁶as follows.

(per-OAc)-2′,4′-Dinitrophenyl-2-deoxy-2-ﬂuoro-β-GG3G (14). ¹H

NMR (Figure S34 in the Supporting Information, 400 MHz, CDCl₃):

δ 8.76 (d, J_{(H’3‑H’5)}= 2.38 Hz, 1H, H′3), 8.43 (d, J_{(H’5‑H’6)}= 9.16 Hz,

J_{(H’5‑H’3)}= 2.38 Hz, 1H, H′5), 7.38 (d, J_{(H’6‑H’5)}= 9.16 Hz, 1H, H′6),

To circumvent partial de-O-acetylation often observed with

traditional phase-transfer conditions,¹²⁷the sodium salt of 2-chloro-

4-nitrophenol (NaCNP) was prepared ﬁrst by the addition of NaOH

(400 mg) to an aqueous solution (50 mL) of 2-chloro-4-nitrophenol

(CNP, 1.74 g). The solution was stirred for 10 min and subsequently

acetone (200 mL) was added to precipitate the salt. The precipitated

salt was ﬁltered, washed with acetone several times, and stored as a

dry powder (1.92 g, 98%) at 4 °C until further needed. A solution of

the sodium phenolate salt (1.5 g, 4.5 equiv) in water (3 mL) was

added to a solution of α-laminaribiosyl bromide⁵⁸(7,1.2 g) and

benzyl tri-n-butylammonium chloride (536 mg, 1 equiv) in DCM

(120 mL). The reaction mixture was stirred at RT for 48 h and

monitored via TLC analysis. Subsequently, it was diluted with more

DCM (120 mL), washed with water (2×), dried over MgSO₄, and

concentrated in vacuo. The pure compound (1.1 g, 81%) was isolated

via ﬂash chromatography using ethyl acetate/hexanes (3:4) as eluent

(R_f= 0.25). Zemplen deprotection was performed by dissolving 1.1 g

of the per-O-acetate in MeOH/DCM (9:1) (30 mL) containing

catalytic NaOMe, followed by stirring at 4 °C overnight. The reaction

mixture was then neutralized with Amberlite IR120 hydrogen foam

and puriﬁed by ﬂash chromatography (water/methanol/ethyl acetate

(1:2:9) to aﬀord the pure product (394 mg, 57%).

5.28 (dd, J_(H1−H2)= 7.36 Hz, J = 3.97 Hz, 1H, H1), 5.23−5.00 (m,

5H), 4.91 (t, J = 8.07 Hz, 2H), 4.69 (d, J_{(H1II−H2II)}= 7.68 Hz, 1H,

H1^II), 4.53−3.59 (m, 12H), 2.13−1.98 (9s, 27 H, COCH₃). ¹³C

NMR (Figure S35 in the Supporting Information, 100.6 MHz,

CDCl₃): δ 170.54−169.00 (9 × CO), 153.41 (C′3), 142.36 (C′4),

140.28 (C′5), 128.60 (C′1), 121.78 (C′2), 118.22 (C′6), 101.39

(C1^III), 100.59 (C1^II), 98.47 (J_(C1−F2)= 25.15 Hz, C1), 91.01 (J_(C2−F2)

= 191.14 Hz, C2), 79.58 (J_(C3−F2)= 18.11 Hz, C3), 75.96, 72.89,

72.83, 72.76, 72.16, 72.04, 71.63, 71.50, 67.72, 67.29 (J_(C4−F2)= 7.04

Hz, C4), 61.80 (J_(C6−F2)= 23.14 Hz, C6), 61.50 (C6^II, C6^III), 20.82−

20.43 (9 × CH₃). ¹⁹F NMR (Figure S36 in the Supporting

Information, 376.5 MHz, CDCl₃): δ −198.32 (ddd, J_(F2−H2)= 50.23

Hz, J_(F2−H3)= 15.28 Hz, J_(F2−H1)= 3.54 Hz, gluco β-anomer);

Monoisotopic m/z calculated for C₄₂H₅₁O₂₈N₂FNa⁺: 1073.25102;

MALDI-TOF MS found: 1073.6; ESI-HRMS found: 1073.2513.

The deprotected 2′,4′-dinitrophenyl 2-deoxy-2-ﬂuoro-β-glycosides

15 and 16 were obtained by Zemplen deprotection of 13 (24 mg) or

14 (26 mg), respectively, in 10 mL methanol/DCM (9:1) containing

catalytic NaOMe. The solution was stirred at 4 °C for 16 h and

monitored by TLC. Upon completion, the reaction was neutralized by

adding 3 g of silica and concentrated to dryness under reduced

pressure to yield a pale silica powder to which the product was

adsorbed. This powder was dry loaded on a ﬂash column and pure

product was eluted using water/methanol/ethyl acetate (1:2:18) as

the mobile phase. The fractions containing the product were pooled

together and concentrated to give a syrup, which was redissolved in

water and freeze-dried to give a white, ﬂuﬀy powder 15 (5.1 mg, yield:

30%, purity estimated by ¹H NMR: 95%) and 16 (6.6 mg, yield: 40%,

1

(per-OAc)-2′-Chloro-4′-nitrophenyl β-laminaribioside. H NMR

(Figure S43 in the Supporting Information, 400 MHz, CDCl₃): δ 8.30

(d, J_{(H’3‑H’5)}= 2.63 Hz, 1H, H′3), 8.12 (dd, J_{(H’‑H’6)}= 9.05 Hz, J_{(H’5‑H’3)}

= 2.63 Hz, 1H, H′5), 7.22 (d, J_{(H’6‑H’5)}= 9.05 Hz, 1H, H′6), 5.40 (d,

J

_(H1−H2)= 8.18 Hz, 1H, H1), 5.19−4.90 (m, 5H), 4.65 (d, J_(H2−H1)=

8.18 Hz, 1H, H2), 4.39 (dd, J = 12.31 Hz, J = 4.32 Hz, 1H), 4.25−

4.00 (m, 4H), 3.93−3.88 (m, 1H), 3.72−3.68 (m, 1H), 2.18−2.00

(7s, 21H, COCH₃). ¹³C NMR (Figure S44 in the Supporting

Information, 100.6 MHz, CDCl₃): δ 170.60−168.79 (7 × CO),

157.45 (C′3), 143.38 (C′4), 126.31 (C′5), 125.15 (C′1), 123.66

(C′2), 116.77 (C′6), 101.08 (C1^II), 99.52 (C1), 78.34 (C3), 72.97,

72.73, 71.92, 71.18, 68.12 (C2), 62.07 (C6 or C6^II), 61.82 (C6 or

C6^II), 21.01−20.49 (7 × CH₃); monoisotopic m/z calculated for

C₃₂H₃₈O₂₀NClNa⁺: 814.15735; MALDI-TOF MS found: 814.3; ESI-

HRMS found: 814.1571.

1

purity estimated by H NMR: 95%).

2′,4′-Dinitrophenyl-2-deoxy-2-ﬂuoro-β-G3G [G3G(2F)-β-DNP,

15]. ¹H NMR (Figure S37 in the Supporting Information, 400

MHz, D₂O): δ 8.93 (d, J_{(H’3‑H’5)}= 2.65 Hz, 1H, H′3), 8.57 (dd,

J_{(H’5‑H’6)}= 9.31 Hz, J_{(H’5‑H’3)}= 2.65 Hz, 1H, H′5), 7.64 (d, J_{(H’6‑H’5)}=

9.31 Hz, 1H, H′6), 5.78 (dd, J_(H1−H2)= 7.62 Hz, J = 2.90 Hz, 1H,

H1), 4.74 (d, J_{(H1II−H2II)}= 7.71 Hz, 1H, H1^II), 4.30−4.22 (m, 1H),

4.01−3.33 (m, 11H). ¹³C NMR (Figure S38 in the Supporting

Information, 100.6 MHz, D₂O): δ 154.08 (C′3), 141.97 (C′4),

139.22 (C′5), 130.04 (C′1), 122.40 (C′2), 118.10 (C′6), 102.66

(C1^II), 97.91 (J_(C1−F2)= 23.14 Hz, C1), 91.39 (J_(C2−F2)= 188.12 Hz,

C2), 81.43 (J_(C3−F2)= 17.10 Hz, C3), 76.49, 76.18, 75.72, 73.38 (C5),

69.79 (C4^II), 67.40 (J_(C4−F2)= 7.92 Hz, C4), 60.91 (C6^II), 60.21

(J_(C6−F2)= 20.12 Hz, C6). ¹⁹F NMR (Figure S39 in the Supporting

Information, 376.5 MHz, D₂O): δ −199.39 (ddd, J_(F2−H2)= 50.61 Hz,

J_(F2−H3)= 15.05 Hz, J_(F2−H1)= 2.39 Hz, gluco β-anomer);

2′-Chloro-4′-nitrophenyl β-laminaribioside (G3G-β-CNP). ¹H

NMR (Figure S45 in the Supporting Information, 400 MHz, D₂O):

δ 8.33 (d, J_{(H’3‑H’5)}= 2.75 Hz, 1H, H′3), 8.21 (dd, J_{(H’5‑H’6)}= 9.19 Hz,

J

_{(H’5‑H’3)}= 2.75 Hz, 1H, H′5), 7.46 (d, J_{(H’6‑H’5)}= 9.19 Hz, 1H, H′6),

5.27 (d, J_(H1−H2)= 7.55 Hz, 1H, H1), 4.65 (d, J_{(H1II−H2II)}= 7.93 Hz,

1H, H1^II), 3.95−3.32 (m, 12H). ¹³C NMR (Figure S46 in the

Supporting Information, 100.6 MHz, D₂O): δ 157.46 (C′3),142.67

K

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

(C′4), 126.39 (C′5), 124.49 (C′1), 123.56 (C′2), 115.82 (C′6),

102.98 (C1^II), 99.94 (C1), 83.77 (C3), 76.25, 76.21, 75.74, 73.65,

72.61, 69.80, 67.92, 60.91 (C6 or C6^II), 60.56 (C6 or C6^II);

monoisotopic m/z calculated for C₁₈H₂₄O₁₃NClNa⁺: 520.08339;

MALDI-TOF MS found: 520.0; ESI-HRMS found: 520.0831.

Inhibition Kinetics. The determination of time-dependent enzyme

inhibition kinetics was performed essentially as previously de-

scribed.^13,21Ultrapure water (18.2 MΩ cm⁻¹) was used for all kinetic

experiments. 100 μL solutions of BoGH16 (8.9 μM) were incubated

with a range of concentrations of inhibitor (0.019−1.25 mM) at 37

°C in 50 mM sodium citrate buﬀer pH 6.5 (with 0.1 mg mL⁻¹bovine

serum albumin to prevent nonspeciﬁc loss of activity). A control

experiment was run in parallel, in which no inhibitor was added to the

buﬀered enzyme/BSA solution. Periodically, 10 μL of this solution

was withdrawn and diluted 1:100 in 50 mM sodium citrate pH 6.5

buﬀer, and 100 μL of the diluted incubate was added to 100 μL of the

preincubated solution of chromogenic substrate GG3G-β-CNP in

ultrapure water at 37 °C (0.2 mM ﬁnal substrate concentration in the

assay).

Residual activities were determined by measuring the rate of release

of the chromophore 2-chloro-4-nitrophenolate (ε = 16.57 mM⁻¹

cm⁻¹) over 1−2 min in a 1 cm quartz cuvette maintained at 37 °C at

405 nm in an Agilent Cary 60 UV−vis spectrophotometer with a

Peltier temperature-controlled cell holder. The inhibition kinetics

constants K_iand k_iwere determined according to the Kitz−Wilson

kinetics model (eq 1)¹¹⁰by ﬁtting eqs 2 and 3 to the data by

nonlinear regression using Origin Pro software, as previously

described.^13,88

dinitrophenyl β-glycoside inhibitor were 605 equiv (with respect to

BuGH158), 962 equiv (with respect to BoGH16), and 590 equiv

(with respect to BuGH16). Negative controls omitting the inhibitors

from the respective incubates were run in parallel.

Protein Crystallography. To reproduce previous crystallography,³⁵

crystals of unliganded recombinant BoGH16 were obtained by ﬁrst

screening sitting drops in 96-well format at RT (setup using a Phoenix

robot, Art Robbin). A hit observed in the JCSG+ screen (Qiagen)

condition G7 was pursued for optimization by screening around this

condition, varying the PEG concentration in one dimension and the

buﬀer pH in the other (24-well format hanging drops, set up by

hand). Large, orthorhombic crystals were readily reproduced by

mixing 27.2 mg mL⁻¹protein solution one-to-one with reservoir

solution comprised of 0.1 M succinic acid pH 7.2 and 15% (w/v)

PEG3350. Crystals of the BoGH16 E148A mutant were reproduced in

identical concentration and conditions as the wild-type.

To obtain the BoGH16:GGG3G-β-NHCOCH₂− complex struc-

ture, wild-type crystals were soaked for 1 h in mother liquor

containing 2.5 mM of compound (5), after which time they were

cryoprotected in mother liquor supplemented with 20% ethylene

glycol. X-ray data from these crystals were collected at Advanced

Photon Source beamline 23 ID-B. To obtain the BoGH16:GG3G(2F)

complex structure, crystals of BoGH16E148A were soaked for 20 min

in mother liquor containing 5 mM inhibitor, after which time they

were cryoprotected in mother liquor supplemented with 25% ethylene

glycol. X-ray data from these crystals were collected at Stanford

Synchrotron Radiation Lightsource beamline BL12-2.

Datasets were indexed and integrated using XDS,¹²⁹and scaled and

merged using AIMLESS.¹³⁰The structures were determined by

molecular replacement using PHASER from the CCP4i2 software

suite¹³¹with the original unliganded BoGH16 structure (PDB ID:

5NBO)³⁵as the search model. After density modiﬁcation using

PARROT,¹³²iterative rounds of manual model building and

reﬁnement were performed in COOT¹³³and REFMAC5,¹³⁴

respectively. The quality of the model was monitored using

MOLPROBITY¹³⁵and sugar conformations were validated using

PRIVATEER.¹³⁶

K_i

k_i

(1)

(2)

E + I F E·I → E·

I

ν = ν₀e^−k

_appt

k_i[I]

K_i+ [I]

k_app

=

(3)

The inhibition of the laminarinase BuGH158 in the presence of

compounds 15 and 16 was measured analogously, using the

chromogenic substrate G3G-β-CNP to measure residual activity.

The Michaelis−Menten kinetic parameters for G3G-β-CNP were ﬁrst

determined by directly ﬁtting the Michaelis−Menten equation to data

from duplicate assays over the concentration range of 0.078−7.5 mM

in 50 mM sodium citrate buﬀer, pH 6, at 50 °C (optimum

temperature of BuGH158 activity⁵⁴). Inhibition kinetics were

performed in this same buﬀer and at the same temperature. Thus,

100 μL solution of 0.13 mM enzyme in buﬀer was incubated with the

inhibitor (with 0.1 mg mL⁻¹bovine serum albumin added to prevent

nonspeciﬁc loss of activity). The concentration range of each inhibitor

was 10−0.31 mM. At various times up to 180 min, a 10 μL aliquot of

this incubate was withdrawn and diluted 1:100 in the same buﬀer and

100 μL of this diluted enzyme−inhibitor incubate was mixed with 100

μL of a solution of G3G-β-CNP(0.25 mM after dilution). This

solution was added to a 1 cm quartz cuvette that had been warmed to

50 °C and residual activity of the enzyme at that time interval was

determined by measuring the rate of release of 2-chloro-4-nitro-

phenolate at 405 nm in an Agilent Cary 60 UV-vis spectrophotometer

(ε = 15.8 mM⁻¹cm⁻¹). As above, inhibition kinetics parameters K_i

and k_iwere determined according to the Kitz−Wilson kinetics model.

Intact-Protein Mass Spectrometry. Intact-protein masses were

determined on a Waters Xevo LC-ESI-MS Q-TOF with a NanoAcuity

UPLC system using Masslynx 4.0 software, essentially as previously

described.¹²⁸For N-bromoacetylglycosylamine oligosaccharides, a 20

μL solution of BoGH16 (3.27 μM in 50 mM pH 6.5 sodium citrate

buﬀer) containing 2.5 mM inhibitor (765 eq. with respect to

BoGH16) was incubated for 3 h (for compounds 4 and 5) or 7 h (for

compound 6) at 37 °C. For the 2-deoxy-2-ﬂuoro-2′,4′-dinitrophenyl

β-glycosides, a 20 μL solution of BuGH158 (4.13 μM), BoGH16

(2.60 μM), or BuGH16 (4.24 μM) in 10 mM sodium citrate buﬀer,

pH 6, containing 2.5 mM inhibitor was incubated at 50 °C for 2 h.

The ﬁnal equivalent molar values for each 2-deoxy-2-ﬂuoro-2′,4′-

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acschembio.1c00063.

■

sı

Compound characterization checklist (XLS)

Crystallographic data tables for covalent enzyme

complexes, supplementary protein mass spectrometry

and kinetic data, HPLC analysis of oligosaccharides, and

NMR spectra of synthetic intermediates and products

(PDF)

Accession Codes

PDB accession 6VHO: Glycoside hydrolase family 16

endoglucanase from Bacteroides ovatus in complex with

G4G4G3G-β-NHCOCH₂Br. PDB accession 7KR6: Glycoside

hydrolase family 16 endoglucanase from Bacteroides ovatus in

complex with G4G3G(2F)-β-DNP

AUTHOR INFORMATION

Corresponding Author

■

Harry Brumer − Michael Smith Laboratories, University of

British Columbia, Vancouver, British Columbia V6T 1Z4,

Canada; Department of Chemistry, University of British

Columbia, Vancouver, British Columbia V6T 1Z1, Canada;

Department of Biochemistry and Molecular Biology,

University of British Columbia, Vancouver, British Columbia

V6T 1Z3, Canada; orcid.org/0000-0002-0101-862X;

Phone: (+1) 6048273738; Email: brumer@msl.ubc.ca;

Fax: (+1) 6048222114

L

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

Authors

G3GGG β-^D-glucopyranosyl-(1,3)-β-D-glucopyranosyl-(1,4)-

β-^D-glucopyranosyl-(1,4)-D-glucopyranose.

Namrata Jain − Michael Smith Laboratories, University of

British Columbia, Vancouver, British Columbia V6T 1Z4,

Canada; Department of Chemistry, University of British

Columbia, Vancouver, British Columbia V6T 1Z1, Canada

Kazune Tamura − Michael Smith Laboratories, University of

British Columbia, Vancouver, British Columbia V6T 1Z4,

Canada; Department of Biochemistry and Molecular Biology,

University of British Columbia, Vancouver, British Columbia

V6T 1Z3, Canada

Guillaume Déjean − Michael Smith Laboratories, University

of British Columbia, Vancouver, British Columbia V6T 1Z4,

Canada

Filip Van Petegem − Department of Biochemistry and

Molecular Biology, University of British Columbia,

Vancouver, British Columbia V6T 1Z3, Canada

REFERENCES

■

(1) Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M.,

and Henrissat, B. (2014) The carbohydrate-active enzymes database

(CAZy) in 2013. Nucleic Acids Res. 42, D490−D495.

(2) CAZypedia Consortium. (2018) Ten years of CAZypedia: A

living encyclopedia of carbohydrate-active enzymes. Glycobiology 28,

3−8.

(3) Borges de Melo, E., da Silveira Gomes, A., and Carvalho, I.

(2006) α - and β -Glucosidase inhibitors: chemical structure and

biological activity. Tetrahedron 62, 10277−10302.

(4) Gloster, T. M., and Davies, G. J. (2010) Glycosidase inhibition:

assessing mimicry of the transition state. Org. Biomol. Chem. 8, 305−

320.

(5) Legler, G. (1990) Glycoside Hydrolases: Mechanistic

Information from Studies with Reversible and Irreversible Inhibitors.

Adv. Carbohydr. Chem. Biochem. 48, 319−384.

(6) Driguez, H. (2001) Thiooligosaccharides as tools for structural

biology. ChemBioChem 2, 311−318.

(7) Kallemeijn, W. W., Witte, M. D., Wennekes, T., and Aerts, J. M.

F. G. (2014) Mechanism-based inhibitors of glycosidases: Design and

applications, In Advances in Carbohydrate Chemistry and Biochemistry

(Horton, D., and Horton, D., Eds.), pp 297−338, Academic Press,

New York.

(8) Rempel, B. P., and Withers, S. G. (2008) Covalent inhibitors of

glycosidases and their applications in biochemistry and biology.

Glycobiology 18, 570−586.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acschembio.1c00063

Author Contributions

N.J. performed the chemo-enzymatic synthesis of inhibitors,

active site labeling, and inhibition kinetics experiments. K.T.

performed recombinant protein production of BoGH16, X-ray

crystallography, analyzed enzyme structures, and provided

advice on enzyme kinetics. G.D. produced BuGH158 and

BuGH16 and provided advice on biochemistry. F.v.P.

supervised crystallography. H.B. devised the overall study

and supervised research. N.J., K.T., and H.B. cowrote the

article.

(9) Witte, M. D., Van Der Marel, G. A., Aerts, J. M. F. G., and

Overkleeft, H. S. (2011) Irreversible inhibitors and activity-based

probes as research tools in chemical glycobiology. Org. Biomol. Chem.

9, 5908−5926.

Funding

(10) Willems, L. I., Jiang, J., Li, K. Y., Witte, M. D., Kallemeijn, W.

W., Beenakker, T. J. N., Schröder, S. P., Aerts, J. M. F. G., van der

Marel, G. A., Codée, J. D. C., and Overkleeft, H. S. (2014) From

covalent glycosidase inhibitors to activity-based glycosidase probes.

Chem. - Eur. J. 20, 10864−10872.

(11) Xu, Y., Uddin, N., and Wagner, G. K. (2018) Covalent Probes

for Carbohydrate-Active Enzymes: From Glycosidases to Glycosyl-

transferases. In Methods in Enzymology (Pyle, A. M., Christianson, D.

W., Pyle, A. M., and Christianson, D. W., Eds.), pp 237−265,

Cambridge.

(12) Wu, L., Armstrong, Z., Schröder, S. P., de Boer, C., Artola, M.,

Aerts, J. M. F. G., Overkleeft, H. S., and Davies, G. J. (2019) An

overview of activity-based probes for glycosidases. Curr. Opin. Chem.

Biol. 53, 25−36.

(13) Fenger, T. H., and Brumer, H. (2015) Synthesis and Analysis of

Specific Covalent Inhibitors of endo-Xyloglucanases. ChemBioChem

16, 575−583.

(14) Jiang, J., Beenakker, T. J. M., Kallemeijn, W. W., van der Marel,

G. A., van den Elst, H., Codée, J. D. C., Aerts, J. M. F. G., and

Overkleeft, H. S. (2015) Comparing Cyclophellitol N-Alkyl and N-

Acyl Cyclophellitol Aziridines as Activity-Based Glycosidase Probes.

Chem. - Eur. J. 21, 10861−10869.

(15) Chakladar, S., Wang, Y., Clark, T., Cheng, L., Ko, S., Vocadlo,

D. J., and Bennet, A. J. (2014) A mechanism-based inactivator of

glycoside hydrolases involving formation of a transient non-classical

carbocation. Nat. Commun. 5, 5590.

(16) Artola, M., Wu, L., Ferraz, M. J., Kuo, C. L., Raich, L., Breen, I.

Z., Offen, W. A., Codée, J. D. C., Van Der Marel, G. A., Rovira, C.,

Aerts, J. M. F. G., Davies, G. J., and Overkleeft, H. S. (2017) 1,6-

Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase

Inhibitor. ACS Cent. Sci. 3, 784−793.

(17) Adamson, C., Pengelly, R. J., Shamsi Kazem Abadi, S.,

Chakladar, S., Draper, J., Britton, R., Gloster, T. M., and Bennet, A. J.

(2016) Structural Snapshots for Mechanism-Based Inactivation of a

Glycoside Hydrolase by Cyclopropyl Carbasugars. Angew. Chem., Int.

Ed. 55, 14978−14982.

Work in the Brumer Laboratory was generously supported by

the Natural Sciences and Engineering Research Council of

Canada (NSERC, Discovery Grant Nos. RGPIN 435223-13

and RGPIN-2018-03892), the Canadian Institutes of Health

Research (CIHR, Operating Grant Nos. MOP-137134 and

MOP-142472), as well as infrastructure support from the

Canadian Foundation for Innovation (Project No. 30663) and

the British Columbia Knowledge Development Fund. Work in

the Van Petegem Lab was supported by the Canadian

Institutes of Health Research (No. MOP-119404). K.T. was

partially supported by a four-year doctoral fellowship from the

University of British Columbia.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank N. McGregor (Brumer Group, UBC) for producing

VvEG16(ΔV152) and J. Rogalski (UBC Proteomics Core

Facility) for assistance with intact-protein MS analysis. H.B.

gratefully acknowledges Waters Corporation for the provision

of the LC-MS system used in this study.

ADDITIONAL NOTE

Throughout, the widely used shorthand for mixed-linkage

■

a

glucan oligosaccharides, is used, in which “3” indicates the

position of the β(1,3) linkage between two glucosyl residues

“G′′. A β(1,3) linkage is otherwise indicated by default. Thus,

G3G represents β-^D-glucopyranosyl-(1,3)-D-glucopyranose

(trivial name laminaribiose), GG3G represents β-^D-glucopyr-

anosyl-(1,4)-β-^D-glucopyranosyl-(1,3)-D-glucopyranose,

GGG3G represents β-^D-glucopyranosyl-(1,4)-β-D-glucopyra-

nosyl-(1,4)-β-^D-glucopyranosyl-(1,3)-D-glucopyranose, and

M

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

(18) Ren, W., Pengelly, R., Farren-Dai, M., Shamsi Kazem Abadi, S.,

Oehler, V., Akintola, O., Draper, J., Meanwell, M., Chakladar, S.,

Swiderek, K., Moliner, V., Britton, R., Gloster, T. M., and Bennet, A. J.

(33) Wood, P. J. (2007) Cereal β -glucans in diet and health. J. Cereal

Sci. 46, 230−238.

́

(34) El Khoury, D., Cuda, C., Luhovyy, B. L., and Anderson, G. H.

(2012) Beta glucan: Health benefits in obesity and metabolic

syndrome. J. Nutr. Metab. 2012, 1−28.

(2018) Revealing the mechanism for covalent inhibition of glycoside

hydrolases by carbasugars at an atomic level. Nat. Commun. 9, 3243.

(19) Beenakker, T. J. M., Wander, D. P. A., Offen, W. A., Artola, M.,

Raich, L., Ferraz, M. J., Li, K. Y., Houben, J. H. P. M., Van Rijssel, E.

R., Hansen, T., Van Der Marel, G. A., Codée, J. D. C., Aerts, J. M. F.

G., Rovira, C., Davies, G. J., and Overkleeft, H. S. (2017) Carba-

cyclophellitols Are Neutral Retaining-Glucosidase Inhibitors. J. Am.

Chem. Soc. 139, 6534−6537.

(20) Hoj, P. B., Rodriguez, E. B., Iser, J. R., Stick, V. R., and Stone, B.

A. (1991) Active site-directed inhibition by optically pure epoxyalkyl

cellobiosides reveals differences in active site geometry of two 1,3 −

1,4-β -D-glucan 4-glucanohydrolases: The importance of epoxide

stereochemistry for enzyme inactivation. J. Biol. Chem. 266, 11628−

11631.

(21) Jain, N., Attia, M. A., Offen, W. A., Davies, G. J., and Brumer,

H. (2018) Synthesis and application of a highly branched,

mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor of

endo-xyloglucanases. Org. Biomol. Chem. 16, 8732−8741.

(22) Kwan, D. H., Jin, Y., Jiang, J. B., Chen, H. M., Kotzler, M. P.,

Overkleeft, H. S., Davies, G. J., and Withers, S. G. (2016)

Chemoenzymatic synthesis of 6-phospho-cyclophellitol as a novel

probe of 6-phospho-beta-glucosidases. FEBS Lett. 590, 461−468.

(23) Stubbs, K. A. (2014) Activity-based proteomics probes for

carbohydrate-processing enzymes: Current trends and future outlook.

Carbohydr. Res. 390, 9−19.

(24) Armstrong, Z., Kuo, C. L., Lahav, D., Liu, B., Johnson, R.,

Beenakker, T. J. M., de Boer, C., Wong, C. S., van Rijssel, E. R.,

Debets, M. F., Florea, B. I., Hissink, C., Boot, R. G., Geurink, P. P.,

Ovaa, H., van der Stelt, M., van der Marel, G. M., Codee, J. D. C.,

Aerts, J., Wu, L., Overkleeft, H. S., and Davies, G. J. (2020) Manno-

epi-cyclophellitols Enable Activity-Based Protein Profiling of Human

alpha-Mannosidases and Discovery of New Golgi Mannosidase II

Inhibitors. J. Am. Chem. Soc. 142, 13021−13029.

(25) McGregor, N. G. S., Artola, M., Nin-Hill, A., Linzel, D., Haon,

M., Reijngoud, J., Ram, A., Rosso, M. N., van der Marel, G. A., Codee,

J. D. C., van Wezel, G. P., Berrin, J. G., Rovira, C., Overkleeft, H. S.,

and Davies, G. J. (2020) Rational Design of Mechanism-Based

Inhibitors and Activity-Based Probes for the Identification of

Retaining alpha-L-Arabinofuranosidases. J. Am. Chem. Soc. 142,

4648−4662.

(26) Jariwala, P. B., Pellock, S. J., Goldfarb, D., Cloer, E. W., Artola,

M., Simpson, J. B., Bhatt, A. P., Walton, W. G., Roberts, L. R., Major,

M. B., Davies, G. J., Overkleeft, H. S., and Redinbo, M. R. (2020)

Discovering the Microbial Enzymes Driving Drug Toxicity with

Activity-Based Protein Profiling. ACS Chem. Biol. 15, 217−225.

(27) Burton, R. A., and Fincher, G. B. (2009) (1,3;1,4)-β -D-glucans

in cell walls of the poaceae, lower plants, and fungi: A tale of two

linkages. Mol. Plant 2, 873−882.

(35) Tamura, K., Hemsworth, G. R., Dejean, G., Rogers, T. E.,

Pudlo, N. A., Urs, K., Jain, N., Davies, G. J., Martens, E. C., and

Brumer, H. (2017) Molecular Mechanism by which Prominent

Human Gut Bacteroidetes Utilize Mixed-Linkage Beta- Glucans,

Major Health-Promoting Cereal Polysaccharides. Cell Rep. 21, 417−

430.

(36) Dong, J.-l., Yu, X., Dong, L.-e., and Shen, R.-l. (2017) In vitro

fermentation of oat β -glucan and hydrolysates by fecal microbiota and

selected probiotic strains. J. Sci. Food Agric. 97, 4198−4203.

(37) Hughes, S. A., Shewry, P. R., Gibson, G. R., McCleary, B. V.,

and Rastall, R. A. (2008) In vitro fermentation of oat and barley

derived β -glucans by human faecal microbiota. FEMS Microbiol. Ecol.

64, 482−493.

(38) Jaskari, J., Kontula, P., Siitonen, A., Jousimies-Somer, H.,

Mattila-Sandholm, T., and Poutanen, K. (1998) Oat β -glucan and

xylan hydrolysates as selective substrates for Bi f i dobacterium and

Lactobacillus strains. Appl. Microbiol. Biotechnol. 49, 175−181.

(39) McNeil, N. I. (1984) The contribution of the large intestine to

energy supplies in man. Am. J. Clin. Nutr. 39, 338−342.

(40) Stone, B. A. (2009) Chemistry of β-Glucans, In Chemistry,

Biochemistry, and Biology of 1−3 Beta Glucans and Related

Polysaccharides (Bacic, A., Fincher, G. B., and Stone, B. A., Eds.),

pp 5−46, Academic Press, San Diego, CA.

(41) Fincher, G. B., and Stone, B. A. (2004) Chemistry of Nonstarch

Polysaccharides. In Encyclopedia of Grain Science (Wrigley, C., Ed.), pp

206−223, Elsevier, Oxford.

(42) Hrmova, M., and Fincher, G. B. (2001) Structure-function

relationships of β -^D-glucan endo- and exohydrolases from higher

plants. Plant Mol. Biol. 47, 73−91.

(43) Planas, A. (2000) Bacterial 1,3 −1,4-β -glucanases: Structure,

function and protein engineering. Biochim. Biophys. Acta, Protein

Struct. Mol. Enzymol. 1543, 361−382.

(44) McGregor, N., Morar, M., Fenger, T. H., Stogios, P., Lenfant,

N., Yin, V., Xu, X., Evdokimova, E., Cui, H., Henrissat, B., Savchenko,

A., and Brumer, H. (2016) Structure-function analysis of a mixed-

linkage β -glucanase/xyloglucanase from the key ruminal bacteroidetes

prevotella bryantii B14. J. Biol. Chem. 291, 1175−1197.

(45) Viborg, A. H., Terrapon, N., Lombard, V., Michel, G., Czjzek,

M., Henrissat, B., and Brumer, H. (2019) A subfamily roadmap of the

evolutionarily diverse glycoside hydrolase family 16 (GH16). J. Biol.

Chem. 294, 15973−15986.

(46) Aspeborg, H., Coutinho, P. M., Wang, Y., Brumer, H., and

Henrissat, B. (2012) Evolution, substrate specificity and subfamily

classification of glycoside hydrolase family 5 (GH5). BMC Evol. Biol.

12, 186.

(28) Fincher, G. B. (2009) Exploring the evolution of (1,3;1,4)-β -D-

glucans in plant cell walls: comparative genomics can help! Curr. Opin.

Plant Biol. 12, 140−147.

(29) Salmeán, A. A., Duffieux, D., Harholt, J., Qin, F., Michel, G.,

Czjzek, M., Willats, W. G. T., and Hervé, C. (2017) Insoluble (1 →

3), (1 → 4)-β -D-glucan is a component of cell walls in brown algae

(Phaeophyceae) and is masked by alginates in tissues. Sci. Rep. 7,

2880.

(30) Vogel, J. (2008) Unique aspects of the grass cell wall. Curr.

Opin. Plant Biol. 11, 301−307.

(31) Popper, Z. A., Michel, G., Hervé, C., Domozych, D. S., Willats,

W. G. T., Tuohy, M. G., Kloareg, B., and Stengel, D. B. (2011)

Evolution and Diversity of Plant Cell Walls: From Algae to Flowering

Plants. Annu. Rev. Plant Biol. 62 (1), 567−590.

̂

(47) Bernardi, A. V., de Gouvea, P. F., Gerolamo, L. E., Yonamine,

D. K., de Lourdes de Lima Balico, L., Uyemura, S. A., and Dinamarco,

T. M. (2018) Functional characterization of GH7 endo-1,4-β -

glucanase from Aspergillus f u migatus and its potential industrial

application. Protein Expression Purif. 150, 1−11.

(48) Yoshida, K., and Komae, K. (2006) A rice family 9 glycoside

hydrolase isozyme with broad substrate specificity for hemicelluloses

in type II cell walls. Plant Cell Physiol. 47, 1541−1554.

(49) Tamoi, M., Kurotaki, H., and Fukamizo, T. (2007) β -1,4-

Glucanase-like protein from the cyanobacterium Synechocystis

PCC6803 is a β -1,3 −1,4-glucanase and functions in salt stress

tolerance. Biochem. J. 405, 139−146.

(50) Foley, M. H., Déjean, G., Hemsworth, G. R., Davies, G. J.,

Brumer, H., and Koropatkin, N. M. (2019) A Cell-Surface GH9

Endo-Glucanase Coordinates with Surface Glycan-Binding Proteins to

Mediate Xyloglucan Uptake in the Gut Symbiont Bacteroides ovatus.

J. Mol. Biol. 431, 981−995.

(32) Collins, H. M., Burton, R. A., Topping, D. L., Liao, M. L., Bacic,

A., and Fincher, G. B. (2010) Variability in fine structures of

noncellulosic cell wall polysaccharides from cereal grains: Potential

importance in human health and nutrition. Cereal Chem. 87, 272−282.

N

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

(51) Bauer, M. W., Driskill, L. E., Callen, W., Snead, M. A., Mathur,

E. J., and Kelly, R. M. (1999) An Endoglucanase, EglA, from the

Hyperthermophilic Archaeon Pyrococcus . J. Bacteriol. 181, 284.

(52) Taylor, E. J., Goyal, A., Guerreiro, C. I. P. D., Prates, J. A. M.,

Money, V. A., Ferry, N., Morland, C., Planas, A., Macdonald, J. A.,

Stick, V. R., Gilbert, H. J., Fontes, C. M. G. A., and Davies, G. J.

(2005) How family 26 glycoside hydrolases orchestrate catalysis on

different polysaccharides: Structure and activity of a Clostridium

thermocellum lichenase, Ct Lic26A. J. Biol. Chem. 280, 32761−32767.

(53) Sinnott, M. L. (1990) Catalytic Mechanisms of Enzymic

Glycosyl Transfer. Chem. Rev. 90, 1171−1202.

(54) Déjean, G., Tamura, K., Cabrera, A., Jain, N., Pudlo, N. A.,

Pereira, G., Viborg, A. H., Van Petegem, F., Martens, E. C., and

Brumer, H. (2020) Synergy between Cell Surface Glycosidases and

Glycan-Binding Proteins Dictates the Utilization of Specific Beta-

(1,3)-Glucans by Human Gut Bacteroides. mBio 11, 1−21.

(55) Chaari, F., and Chaabouni, S. E. (2019) Fungal β -1,3 −1,4-

glucanases: production, proprieties and biotechnological applications.

J. Sci. Food Agric. 99, 2657−2664.

(56) Von Wettstein, D., Mikhaylenko, G., Froseth, J. A., and

Kannangara, C. G. (2000) Improved barley broiler feed with

transgenic malt containing heat-stable (1,3 −1,4)-β -glucanase. Proc.

Natl. Acad. Sci. U. S. A. 97, 13512−13517.

(57) Faijes, M., Pérez, X., Pérez, O., and Planas, A. (2003)

Glycosynthase Activity of Bacillus licheniformis 1,3 −1,4-β -Glucanase

Mutants: Specificity, Kinetics, and Mechanism. Biochemistry 42,

13304−13318.

(58) Malet, C., Vallés, J., Bou, J., and Planas, A. (1996) A specific

chromophoric substrate for activity assays of 1,3 −1,4-β -D-glucan 4-

glucanohydrolases. J. Biotechnol. 48, 209−219.

(70) Miao, S., Ziser, L., Aebersold, R., and Withers, S. G. (1994)

Identification of Glutamic Acid 78 as the Active Site Nucleophile in

Bacillus subtilis Xylanase Using Electrospray Tandem Mass Spectrom-

etry. Biochemistry 33, 7027−7032.

(71) Withers, S. G., Warren, A. J., Street, I. P., Rupitz, K., Kempton,

J. B., and Aebersold, R. (1990) Unequivocal Demonstration of the

Involvement of a Glutamate Residue as a Nucleophile in the

Mechanism of a “Retaining ” Glycosidase. J. Am. Chem. Soc. 112,

5887−5889.

(72) Gebler, J. C., Aebersold, R., and Withers, S. G. (1992) Glu-537,

not Glu-461, is the nucleophile in the active site of (lac Z) β -

galactosidase from Escherichia coli . J. Biol. Chem. 267, 11126−11130.

(73) Vocadlo, D. J., Davies, G. J., Laine, R., and Withers, S. G.

(2001) Catalysis by hen egg-white lysozyme proceeds via a covalent

intermediate. Nature 412, 835−838.

(74) Hart, D. O., He, S. M., Chany, C. J., Withers, S. G., Sims, P. F.

G., Sinnott, M. L., and Brumer, H. (2000) Identification of Asp-130 as

the catalytic nucleophile in the main alpha-galactosidase from

Phanerochaete chrysosporium , a family 27 glycosyl hydrolase.

Biochemistry 39, 9826−9836.

(75) McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D.,

and Withers, S. G. (1993) Syntheses of 2-deoxy-2-fluoro mono- and

oligo-saccharide glycosides from glycals and evaluation as glycosidase

inhibitors. Carbohydr. Res. 249, 77−90.

(76) Tull, D., Burgoyne, D. L., Chow, D. T., Withers, S. G., and

Aebersold, R. (1996) A Mass Spectrometry-Based Approach for

Probing Enzyme Active Sites: Identification of Glu 127 in

Cellulomonas f i mi Exoglycanase as the Residue Modified by N-

Bromoacetyl Cellobiosylamine. Anal. Biochem. 234, 119−125.

(77) Black, T. S., Kiss, L., Tull, D., and Withers, S. G. (1993) N-

Bromoacetyl-glycopyranosylamines as affinity labels for a β -

glucosidase and a cellulase. Carbohydr. Res. 250, 195−202.

(78) Fekete, C. A., and Kiss, L. (2013) A new approach in the active

site investigation of an inverting β -^D-xylosidase from Thermobi f i da

f u sca TM51. Protein J. 32, 97−105.

(59) Planas, A., Abel, M., Millet, O., Palasi, J., Pallares, C., and

Viladot, J.-L. (1998) Synthesis of aryl 3-O-β -cellobiosyl-β -d-

glucopyranosides for reactivity studies of 1,3- 1,4- β -glucanases.

Carbohydr. Res. 310, 53−64.

(60) Moreau, V., Viladot, J. L., Samain, E., Planas, A., and Driguez,

H. (1996) Design and chemoenzymatic synthesis of thiooligosac-

charide inhibitors of 1,3:1,4-β -D-glucanases. Bioorg. Med. Chem. 4,

1849−1855.

(79) Jäger, S., and Kiss, L. (2005) Investigation of the active site of

the extracellular β -^D-glucosidase from Aspergillus carbonarius . World J.

Microbiol. Biotechnol. 21, 337−343.

(61) Money, V. A., Cartmell, A., Guerreiro, C. I. P. D., Ducros, V.

M.-A., Fontes, C. M. G. A., Gilbert, H. J., and Davies, G. J. (2008)

Probing the β -1,3:1,4 glucanase, CtLic26A, with a thio-oligosacchar-

ide and enzyme variants. Org. Biomol. Chem. 6, 851−853.

(62) Hoj, P. B., Condron, R., Traeger, J. C., McAuliffe, J. C., and

Stone, B. A. (1992) Identification of glutamic acid 105 at the active

site of Bacillus amyloliquefaciens 1,3 −1,4-β -D-glucan 4-glucanohydro-

lase using epoxide-based inhibitors. J. Biol. Chem. 267, 25059−25066.

(63) Hoj, P. B., Rodriguez, E. B., Stick, V. R., and Stone, B. A.

(1989) Differences in active site structure in a family of β -glucan

endohydrolases deduced from the kinetics of inactivation by

epoxyalkyl β -oligoglucosides. J. Biol. Chem. 264, 4939−4947.

(64) Keitel, T., Simon, O., Borriss, R., and Heinemann, U. (1993)

Molecular and active-site structure of a Bacillus 1,3 −1,4-β -glucanase.

Proc. Natl. Acad. Sci. U. S. A. 90, 5287−5291.

(65) Withers, S. G., Street, I. P., Bird, P., and Dolphin, D. H. (1987)

2-Deoxy-2-fluoroglucosides: A Novel Class of Mechanism-Based

Glucosidase Inhibitors. J. Am. Chem. Soc. 109, 7530−7531.

(66) Yariv, J., Wilson, K. J., Hildesheim, J., and Blumberg, S. (1971)

Labelling of the Active Site of β -galactosidase by N-bromoacetyl β -^D-

galactopyranosylamine. FEBS Lett. 15, 24−26.

(67) Withers, S. G., Rupitz, K., and Street, I. P. (1988) 2-Deoxy-2-

fluoro-^D-glycosyl fluorides. A new class of specific mechanism-based

glycosidase inhibitors. J. Biol. Chem. 263, 7929−7932.

(80) Keresztessy, Z., Kiss, L., and Hughes, M. A. (1994)

Investigation of the active site of the cyanogenic β -^D-glucosidase

(linamarase) from Manihot esculenta Crantz (Cassava). II. Identi-

fication of Glu-198 as an active site carboxylate group with acid

catalytic function. Arch. Biochem. Biophys. 315, 323−330.

(81) Kiss, T., Erdei, A., and Kiss, L. (2002) Investigation of the

active site of the extracellular β -^D-xylosidase from Aspergillus

carbonarius . Arch. Biochem. Biophys. 399, 188−194.

(82) Vocadlo, D. J., Wicki, J., Rupitz, K., and Withers, S. G. (2002) A

case for reverse protonation: Identification of Glu160 as an acid/base

catalyst in Thermoanaerobacterium saccharolyticum β -xylosidase and

detailed kinetic analysis of a site-directed mutant. Biochemistry 41,

9736−9746.

(83) Chir, J., Withers, S., Wan, C.-F., and Li, Y.-K. (2002)

Identification of the two essential groups in the family 3 β -glucosidase

from Flavobacterium meningosepticum by labelling and tandem mass

spectrometric analysis. Biochem. J. 365, 857−863.

(84) Legler, G., Sinnott, M. L., and Withers, S. G. (1980) Catalysis

by β -Glucosidase A3 of Aspergillus wentii . J. Chem. Soc., Perkin Trans.

2, 1376−1383.

(85) Viratelle, M., Yon, J. M., and Yariv, J. (1977) The inactivation

of β -galactosidase by N-bromoacetyl-β -^D-glucosylamine. FEBS Lett.

79, 109−112.

(86) Naider, F., Bohak, Z., and Yariv, J. (1972) Reversible alkylation

of a methionyl residue near the active site of β -galactosidase.

Biochemistry 11, 3202−3208.

(68) Street, I. P., Kempton, J. B., and Withers, S. G. (1992)

Inactivation of a β -glucosidase through the accumulation of a stable 2-

deoxy-2-fluoro-α -^D-glucopyranosyl-enzyme intermediate: A detailed

investigation. Biochemistry 31, 9970−9978.

(69) Blanchard, J. E., and Withers, S. G. (2001) Rapid screening of

the aglycone specificity of glycosidases: Applications to enzymatic

synthesis of oligosaccharides. Chem. Biol. 8, 627−633.

(87) McGregor, N., Yin, V., Tung, C. C., Van Petegem, F., and

Brumer, H. (2017) Crystallographic insight into the evolutionary

origins of xyloglucan endotransglycosylases and endohydrolases. Plant

J. 89, 651−670.

O

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

(88) Attia, M. A., Nelson, C. E., Offen, W. A., Jain, N., Davies, G. J.,

Gardner, J. G., and Brumer, H. (2018) In vitro and in vivo

characterization of three Cellvibrio japonicus glycoside hydrolase

family 5 members reveals potent xyloglucan backbone-cleaving

functions. Biotechnol. Biofuels 11, 45−61.

(107) Dallabernardina, P., Schuhmacher, F., Seeberger, P. H., and

Pfrengle, F. (2016) Automated glycan assembly of xyloglucan

oligosaccharides. Org. Biomol. Chem. 14, 309−313.

(108) Dax, K., Albert, M., Ortner, J., and Paul, B. J. (2000) Synthesis

of deoxyfluoro sugars from carbohydrate precursors. Carbohydr. Res.

327, 47−86.

(89) Xue, J., Guo, M., Gu, G., and Guo, Z. (2012) A Facile Synthesis

of N γ -Glycosyl Asparagine Conjugates and Short N-Linked

Glycopeptides. J. Carbohydr. Chem. 31, 105−113.

(109) Davies, G. J., Wilson, K. S., and Henrissat, B. (1997)

Nomenclature for sugar-binding subsites in glycosyl hydrolases.

Biochem. J. 321, 557−559.

(110) Kitz, R., and Wilson, I. B. (1962) Esters of methanesulfonic

acid as irreversible inhibitors of acetylcholinesterase. J. Biol. Chem.

237, 3245−3249.

(111) Mackenzie, L. F., Sulzenbacher, G., Divne, C., Jones, T. A.,

Woldike, H. F., Schulein, M., Withers, S. G., and Davies, G. J. (1998)

Crystal structure of the family 7 endoglucanase I (Cel7B) from

Humicola insolens at 2.2 Å resolution and identification of the catalytic

nucleophile by trapping of the covalent glycosyl-enzyme intermediate.

Biochem. J. 335, 409−416.

(112) Wang, Q., Tull, D., Meinke, A., Gilkes, N. R., Warren, R. A. J.,

Aebersold, R., and Withers, S. G. (1993) Glu280 is the nucleophile in

the active site of Clostridium thermocellum CelC, a family A endo-β -

1,4-glucanase. J. Biol. Chem. 268, 14096−14102.

(113) Ziser, L., Setyawati, I., and Withers, S. G. (1995) Syntheses

and testing of substrates and mechanism-based inactivators for

xylanases. Carbohydr. Res. 274, 137−153.

(90) Marca, E., Valero-Gonzalez, J., Delso, I., Tejero, T., Hurtado-

Guerrero, R., and Merino, P. (2013) Synthesis of O- and C-glycosides

derived from β -(1,3)-^D-glucans. Carbohydr. Res. 382, 9−18.

(91) Adam, M. J., Pate, B. D., Nesser, J. R., and Hall, L. D. (1983) A

rapid, stereoselective synthesis of fluorinated carbohydrates: Addition

of acetyl hypofluorite to vinyl ether derivatives of sugars. Carbohydr.

Res. 124, 215−224.

(92) Adamson, J., Foster, A.B., Hall, L.D., Johnson, R.N., and Heese,

R.H. (1970) Fluorinated Carbohydrates: 2-deoxy-2-fluoro-^D-glucose

and 2-deoxy-2-fluoro-^D-mannose. Carbohydr. Res. 15, 351−359.

(93) Korytnyk, W., Valentekovic-Horvath, S., and Petrie, C. R.

(1982) A convenient synthesis of 1,2-difluoro-1, 2-dideoxyhexoses

using xenon fluoride. Tetrahedron 38, 2547−2550.

(94) Ido, T., Wan, C. N., Fowler, J. S., and Wolf, A. P. (1977)

Fluorination with F2. A Convenient Synthesis of 2-Deoxy-2-fluoro-^D-

glucose. J. Org. Chem. 42, 2341−2342.

(95) Abdul-Ghani, M., Banks, E. R., Besheesh, M. K., Sharif, I., and

Syvret, R. G. (1995) N-Halogeno compounds. Part 14. “Transfer

fluorination ” of quinuclidine using F-TEDA-BFc (Selectfluor

reagent): laboratory synthesis of N-fluoroquinuclidinium salts not

requiring the use of elemental fluorine. J. Fluorine Chem. 73, 255−257.

(96) Burkart, M. D., Zhang, Z., Hung, S. C., and Wong, C. H.

(1997) A new method for the synthesis of fluoro-carbohydrates and

glycosides using selectfluor. J. Am. Chem. Soc. 119, 11743−11746.

(97) Vincent, S. P., Burkart, M. D., Tsai, C. Y., Zhang, Z., and Wong,

C. H. (1999) Electrophilic fluorination-nucleophilic addition reaction

mediated by Selectfluor: Mechanistic studies and new applications. J.

Org. Chem. 64, 5264−5279.

(114) McCarter, J. D., Adam, M. J., and Withers, S. G. (1992)

Binding Energy and Catalysis. Fluorinated and deoxygenated

glycosides as mechanistic probes of Escherichia coli (lacZ) β -

galactosidase. Biochem. J. 286, 721−727.

(115) Shaikh, F. A., Mullegger, J., He, S., and Withers, S. G. (2007)

̈

Identification of the catalytic nucleophile in Family 42 β -

galactosidases by intermediate trapping and peptide mapping: YesZ

from Bacillus subtilis . FEBS Lett. 581, 2441−2446.

(116) Namchuk, M. N., and Withers, S. G. (1995) Mechanism of

Agrobacterium β -Glucosidase: Kinetic Analysis of the Role of

Noncovalent Enzyme/Substrate Interactions. Biochemistry 34,

16194−16202.

(98) Albert, M., dax, K., and Ortner, J. (1998) A novel direct route

to 2-deoxy-2-fluoro-aldoses and their corresponding derivatives.

Tetrahedron 54, 4839−4848.

(117) Ly, H. D., and Withers, S. G. (1999) Mutagenesis of

glycosidases. Annu. Rev. Biochem. 68, 487−522.

(118) Armstrong, Z., Liu, F., Chen, H. M., Hallam, S. J., and

Withers, S. G. (2019) Systematic Screening of Synthetic Gene-

Encoded Enzymes for Synthesis of Modified Glycosides. ACS Catal. 9,

3219−3227.

(119) Halford, B. (2020) Covalent drugs go from fringe field to

fashionable endeavor. Chem. Eng. News 98, 28−33.

(120) Namchuk, M. N., McCarter, J. D., Becalski, A., Andrews, T.,

and Withers, S. G. (2000) The role of sugar substituents in glycoside

hydrolysis. J. Am. Chem. Soc. 122, 1270−1277.

(121) Artimo, P., Jonnalagedda, M., Arnold, K., Baratin, D., Csardi,

G., de Castro, E., Duvaud, S., Flegel, V., Fortier, A., Gasteiger, E.,

Grosdidier, A., Hernandez, C., Ioannidis, V., Kuznetsov, D., Liechti,

R., Moretti, S., Mostaguir, K., Redaschi, N., Rossier, G., Xenarios, I.,

and Stockinger, H. (2012) ExPASy: SIB bioinformatics resource

portal. Nucleic Acids Res. 40, W597−W603.

(122) Gottlieb, H. E., Kotlyar, V., and Nudelman, A. (1997) NMR

chemical shifts of common laboratory solvents as trace impurities. J.

Org. Chem. 62, 7512−7515.

(123) Abel, M., Segade, A., and Planas, A. (2009) Synthesis of an

aryl 2-deoxy-β -glycosyl tetrasaccharide to probe retaining endo-

glycosidase mechanism. Tetrahedron: Asymmetry 20, 847−850.

(124) Malet, C., Jimenez-Barbero, J., Bernabe, M., Brosa, C., and

Planas, A. (1993) Stereochemical course and structure of the products

of the enzymic action of endo-1,3 −1-4-β -D-glucan 4-glucanohydro-

lase from Bacillus licheniformis . Biochem. J. 296, 753−758.

(125) Xu, X., Tan, Q., and Hayashi, M. (2008) Versatile and mild

synthesis of Di- and trisaccharidic 2-enopyranosyl cyanides by

cyanation of per-O-acetylglycals with trimethylsilyl cyanide catalyzed

by palladium(II) acetate. Synthesis 2008, 770−776.

̀

(99) Benito, D., Matheu, M. I., Morere, A., Díaz, Y., and Castillón, S.

(2008) Towards the preparation of 2 ″-deoxy-2 ″-fluoro-adenophostin

A. Study of the glycosylation reaction. Tetrahedron 64, 10906−10911.

(100) Dohi, H., Périon, R., Durka, M., Bosco, M., Roué, Y., Moreau,

F., Grizot, S., Ducruix, A., Escaich, S., and Vincent, S. P. (2008)

Stereoselective glycal fluorophosphorylation: Synthesis of ADP-2-

fluoroheptose, an inhibitor of the LPS Biosynthesis. Chem. - Eur. J. 14,

9530−9539.

(101) Ortner, J., Albert, M., Weber, H., and Dax, K. (1999) Studies

on the Reaction of ^D-Glucal and its Derivatives with 1-Chloromethyl-

4-Fluoro-1,4-Diazoniabicyclo[2.2.2]Octane Salts. J. Carbohydr. Chem.

18, 297−316.

(102) Vocadlo, D. J., and Withers, S. G. (2005) The chemical

synthesis of 2-deoxy-2-fluorodisaccharide probes of the hen egg white

lysozyme mechanism. Carbohydr. Res. 340, 379−388.

(103) Wolf, S., Berrio, R. M., and Meier, C. (2011) Synthesis of

nonnatural nucleoside diphosphate sugars. Eur. J. Org. Chem. 2011,

6304−6313.

(104) Williams, S. J., Hekmat, O., and Withers, S. G. (2006)

Synthesis and testing of mechanism-based protein-profiling probes for

retaining endo-glycosidases. ChemBioChem 7, 116−124.

(105) Goddard-Borger, E. D., Fiege, B., Kwan, E. M., and Withers, S.

G. (2011) Glycosynthase-Mediated Assembly of Xylanase Substrates

and Inhibitors. ChemBioChem 12, 1703−1711.

(106) Sakai, K., Nakahara, Y., and Ogawa, T. (1990) Total synthesis

of nonasaccharide repeating unit of plant-cell wall xyloglucan an

endogenous hormone which regulates cell-growth. Tetrahedron Lett.

31, 3035−3038.

P

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

pubs.acs.org/acschemicalbiology

Articles

(126) Ibatullin, F. M., Baumann, M. J., Greffe, L., and Brumer, H.

(2008) Kinetic Analyses of Retaining endo-(Xylo)glucanases from

Plant and Microbial Sources Using New Chromogenic Xylogluco-

Oligosaccharide Aryl Glycosides. Biochemistry 47, 7762−7769.

(127) Kleine, H. P., Weinberg, V. D., Kaufman, R. J., and Sidhu, R. S.

(1985) Phase-transfer-catalyzed synthesis of 2,3,4,6-tetra-O-acetyl-β -

d-galactopyranosides. Carbohydr. Res. 142, 333−337.

(128) Sundqvist, G., Stenvall, M., Berglund, H., Ottosson, J., and

Brumer, H. (2007) A general, robust method for the quality control of

intact proteins using LC-ESI-MS. J. Chromatogr. B: Anal. Technol.

Biomed. Life Sci. 852, 188−194.

(129) Kabsch, W. (2010) XDS. Acta Crystallogr., Sect. D: Biol.

Crystallogr. 66, 125−132.

(130) Evans, P. R., and Murshudov, G. N. (2013) How good are my

data and what is the resolution? Acta Crystallogr., Sect. D: Biol.

Crystallogr. 69, 1204−1214.

(131) McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn,

M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic

software. J. Appl. Crystallogr. 40, 658−674.

(132) Cowtan, K. (2010) Recent developments in classical density

modification. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 470−478.

(133) Emsley, P., and Cowtan, K. (2004) Coot: Model-building

tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr.

60, 2126−2132.

(134) Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S.,

Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and Vagin, A. A.

(2011) REFMAC5 for the refinement of macromolecular crystal

structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 67, 355−367.

(135) Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral,

G. J., Wang, X., Murray, L. W., Arendall, W. B., Snoeyink, J.,

Richardson, J. S., and Richardson, D. C. (2007) MolProbity: All-atom

contacts and structure validation for proteins and nucleic acids.

Nucleic Acids Res. 35, W375−W383.

(136) Agirre, J., Iglesias-Fernández, J., Rovira, C., Davies, G. J.,

Wilson, K. S., and Cowtan, K. D. (2015) Privateer: Software for the

conformational validation of carbohydrate structures. Nat. Struct. Mol.

Biol. 22, 833−834.

(137) Geoghegan, K. F., Dixon, H. B. F., Rosner, P. J., Hoth, L. R.,

Lanzetti, A. J., Borzilleri, K. A., Marr, E. S., Pezzullo, L. H., Martin, L.

B., LeMotte, P. K., McColl, A. S., Kamath, A. V., and Stroh, J. G.

(1999) Spontaneous alpha-N-6-phosphogluconoylation of a “His tag ”

in Escherichia coli: The cause of extra mass of 258 or 178 Da in fusion

proteins. Anal. Biochem. 267, 169−184.

Q

https://doi.org/10.1021/acschembio.1c00063

ACS Chem. Biol. XXXX, XXX, XXX−XXX

Article Doi

Orthogonal Active-Site Labels for Mixed-Linkage endo-β-Glucanases

DOI: 10.1021/acschembio.1c00063

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acschembio.1c00063

Full text of DOI:10.1021/acschembio.1c00063

Products guided by the article

R&D Labs maybe for 7231-15-4

Relevant to this article

Hot Product