Access to Galectin-3 Inhibitors from Chemoenzymatic Synthons

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Access to Galectin‑3 Inhibitors from Chemoenzymatic Synthons

́

Christophe Dussouy, Stephane Teletchea, Annie Lambert, Cathy Charlier, Iuliana Botez,

Frederic De Ceuninck, and Cyrille Grandjean *

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ABSTRACT: Chemoenzymatic strategies are useful for providing both regio-

and stereoselective access to bioactive oligosaccharides. We show herein that a

glycosynthase mutant of a Thermus thermophilus α-glycosidase can react with

unnatural glycosides such as 6-azido-6-deoxy-^D-glucose/glucosamine to lead to

β-^D-galactopyranosyl-(1→3)-D-glucopyranoside or β-D-galactopyranosyl-(1→

3)-2-acetamido-2-deoxy-^D-glucopyranoside derivatives bearing a unique azide

function. Taking advantage of the orthogonality between the azide and the

hydroxyl functional groups, the former was next selectively reacted to give rise

to a library of galectin-3 inhibitors. Combining enzyme substrate promiscuity

and bioorthogonality thus appears as a powerful strategy to rapidly access to

sugar-based ligands.

reported the screening of glycoside hydrolase activities from

the GH1 family (CA-zyme) for substrates bearing either an

amino or an azido group at the 3-, 4-, or 6-position of the

glucose.¹⁸The panel of tested enzymes proved rather tolerant

for the replacement of a hydroxyl with an amino group and

when an azido group was present at the 6-position. They were

much less permissive for 3-azido and 4-azido substrates due to

the steric demand of the azido group because of the strong

hydrogen bonds formed between substrates and highly

conserved residues of GH1 enzyme catalytic domains. Finally,

the authors succeeded in synthesizing taggable di- and

trisaccharides from seven glycosynthases derived from these

glycosidases to demonstrate the utility of this approach.

Considering now the acceptor substrate, it is well established

that a wide range of aglycons are tolerated albeit with a

preference for arylated substituents in line with their capability

to form stabilizing hydrophobic interactions with the residues

within the +1,+2 enzyme subsites. Furthermore, the hydroxyl

can be advantageously replaced by a thiol group which can

next be used as the glycosylation site to give rise to

thioglycosides.¹⁹Finally, site-speciﬁc enzymatic α-D-glucosyla-

tion has been performed on a disaccharide whereby an

acetamido group has been replaced by a trichloroacetamide at

a remote position to allow further chemical elongation of the

resulting trisaccharide building block to form an important

INTRODUCTION

■

By essence glycosyltransferases are designed and thus widely

used to achieve glycan synthesis with incomparable selectivity

and eﬃciency.¹⁻⁴The transfer catalyzed by some of these

enzymes can occur eﬃciently not only with their natural donor

substrates but also with synthetic analogues. This considerably

increases the scope of their applications, which is therefore not

limited to the sole preparation of natural glycans. A major

achievement was the advent of metabolic functionalization.⁵

Glycosyltransferase substrate promiscuity has now been

exploited for the synthesis of complex glycans as well as for

inhibitors or ligands. This was achieved starting from

chemoenzymatic synthons, ﬁrst enzymatically used either in

cellulo or in vitro as substrates to form oligosaccharides prior

to their chemical modiﬁcations.⁶⁻⁹Interestingly, a reverse

strategy consisting of using temporary protecting groups on

acceptor substrates to divert and control the reactivity of

glycosyltransferases has been applied to synthesize complex

glycoforms.^10,11trans-Glycosidases, whether natural¹²or

mutant glycoside hydrolases obtained by directed evolution¹³

or rational design,¹⁴as well as glycosynthases¹⁵are powerful

tools for the synthesis of oligosaccharides or glycoconjugates.

For example, the design of a trans-β-N-acetylglucosaminidase

engineered from a GH1 glycoside hydrolase able to hydrolyze

galactose and glucose but not N-acetyl-^D-glucosamine has been

reported.¹⁶Glycosynthases arise from the mutation of the

catalytic nucleophile residue of a retaining glycoside hydrolase

to either Ala, Gly, or Ser. While glycosynthases have lost their

hydrolytic capacity, they are able to catalyze the selective

transfer of ﬂuoro-activated sugar donors to appropriate

acceptors.^17,15However, despite their broad interest, such

enzymes have scarcely been tested for their ability to accept

chemoenzymatic synthons. Withers’ laboratory has recently

Special Issue: A New Era of Discovery in Carbohy-

drate Chemistry

Received: August 9, 2020

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A

The Journal of Organic Chemistry

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Scheme 1. Preparation of the Enzymatic Synthons and Chemoenzymatic Access to β-Galp-(1-3)-6-azido-6-deoxy-β-D-Glcp/

GlcNp Motifs

hapten for vaccination.²⁰As an extension of the use of

chemoenzymatic synthons, we herein report that 6-azidoglyco-

sides are eﬀective glycosynthase acceptor substrates, useful to

give access to glycan-derived inhibitors of relevant therapeutic

targets. In the present work, we focused on the galectin-3

protein as a representative example. Galectins form an

evolutionary conserved family of animal lectins which bind

α-galactoside motifs through one or two carbohydrate binding

domains (CRDs). Human galectin-3 is the only chimera-type

galectin composed of a single CRD at its C-terminus and of a

nonlectin collagen-like domain.²¹Galectin-3 is found in the

nucleus, the cytoplasm, the vicinity of the cell surface, the

extracellular matrices, and even the circulation. Galectin-3 is

expressed in various cell types including epithelial and

endothelial cells, ﬁbroblasts, as well as immune cells.

Galectin-3 has been increasingly recognized as being involved

in highly diverse physiological or pathophysiological processes

such as adhesion, diﬀerentiation, apoptosis, cell division,

proliferation, traﬃcking, growth or regulation of cell signaling,

and gene expression.^22,23Consequently, galectin-3 has

emerged as a potential novel therapeutic target, and sugar-

derived small-molecule inhibitors have been proposed as

galectin-speciﬁc inhibitors and beyond as potential novel drug

candidates.

In their work, Hsieh and co-workers found that galactose

position for both lactosamine types was highly conserved in

galectin-3. This orientation is also identical in more recent

structures of digalactoside galectin inhibitors where the

galactose binding position is strictly conserved.^25,26This

evolution-driven speciﬁcity for galactose recognition involves

two chemical interactions. First, a characteristic direct or

water-mediated hydrogen network is formed by the conserved

amino acids H158, N160, R162, N174, and E184 with the

oxygens O3, O4, O5, and O6 of the galactose moiety. Second,

W181 tightens the binding via a hydrophobic interaction with

galactose atoms C3, C4, C5, and C6. Concerning the glucose

or NAc-glucose recognition for lactosamine type I or II, the

authors observed a rotation of 240° around the glycosidic

angle.²⁴This rotation does not alter one important hydrogen

bond with R162 and E184 since the lactosamine type II C4-

OH and the lactosamine type I C3-OH are superimposable at

this oxygen position. This rotation exchanges C2 and C6

positions between the lactosamines I and II leading to

diﬀerences in their binding aﬃnities, measured K_dbeing,

respectively, 33 and 93 μM, in line with other studies.^24,27

While the carbohydrate core is involved in a dense hydrogen

bond network making the presence of the 4-OH and 3-OH

groups mandatory for the recognition as described above, the

careful choice of the substituents at the other positions

provides a signiﬁcant contribution to both aﬃnity and

speciﬁcity. Tremendous synthetic eﬀorts have been devoted

to the decoration of the lactose/type lI lactosamine core

notably at the C2 position of the glucose/glucosamine

residue.^28,29Modiﬁcations at the C2-position are key since,

RESULTS AND DISCUSSION

■

Rationale. Galectin-3 recognizes both β-Galp-(1→3)-β-D-

GlcpN (lactosamine type I) and β-Galp-(1−4)-β-D-GlcpN

(lactosamine type II) core structures. A close study of

lactosamine I and II binding modes was performed recently.²⁴

B

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Scheme 2. Synthesis of Galectin-3 Inhibitors from Disaccharide 13

for example, aryl substituents have been shown to enhance the

binding aﬃnity toward galectin-3 by 1 order of magnitude

through cation−π interactions with the residue R186 of the

CRD.³⁰By contrast, to our knowledge, only two reports

describe the preparation of type I lactosamine-derived galectin

inhibitors. The ﬁrst one concerns the preparation of N-

acetyllactosamine type 1 oligomers by iterative enzymatic

synthesis.³¹The second one describes the synthesis of a series

of inhibitors bearing modiﬁcations at either the 2′-OH position

of the galactose or the amino position of the glucosamine

residues, obtained according to a classical glycosylation

strategy between a thioglycoside donor and a 4,6-O-

benzylidene-protected glucosamine acceptor.³²Our laboratory

reported some years ago the preparation of the glycosynthase

E338G mutant from Thermus thermophilus glycoside hydrolase

(TTβGly E338G) which was successfully used as a catalyst for

the expeditious synthesis of the β-Galp-(1→3)-β-^D-GlcNp/

Glcp core.³³We reasoned that if this glycosynthase accepts 6-

azidoglycosides as substrate that would oﬀer a unique

opportunity to explore the eﬀect of modiﬁcations at the C6-

position (equivalent to C2 position on type lactosamine/

lactose) on galectin-3 recognition.

Access to β-Galp-(1→3)-6-azido-6-deoxy-β-^D-Glcp/

GlcNp Motifs. To test our working hypothesis, we ﬁrst

synthesized potential donor and acceptor substrates for the

enzyme. α-Galactopyranosyl ﬂuoride 1 was thus prepared in

two steps (90% overall yield) from galactose-pentaacetate

́

upon HF-pyridine treatment followed by Zemplen depro-

tection as described³³except that we omitted the ﬁnal

neutralization step as we noticed that compound 1 could be

stored longer at −20 °C. Synthesis of phenyl 2-acetamido-6-

azido-2,6-dideoxy-1-thio-α-^D-glucopyranoside 2 was preferen-

tially carried out from a 2-phthalimido rather than a 2-

acetamido precursor. Hence, derivative 3³⁴was selectively

mesylated at the C-6 position and next subjected to S_N2

substitution using sodium azide to give compound 4. Acceptor

2 was obtained after ethylene diamine treatment to remove the

phthalimide protecting group from intermediate 4 (46% yield

for the three steps) (Scheme 1).³⁵In parallel, glucose

pentaacetate 5 was glycosylated with thiophenol in the

presence of boron triﬂuoride etherate, treated with sodium

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methoxide in methanol, silylated at the C-6 upon treatment

with the TBDMS-imidazole complex, and submitted to

peracetylation to aﬀord the intermediate 6. Selective

deprotection of the 6-hydroxyl moiety of derivative 6, next

subjected to mesylation followed by sodium azide nucleophile

substitution, conducted to the intermediate 7. Final

deacetylation of 7 gave rise to a second chemoenzymatic

synthon, the phenyl 6-azido-6-deoxy-1-thio-β-^D-glucopyrano-

side 8 in 8 steps and 45% overall yield (Scheme 1).

To our delight, the reaction of α-galactopyranosyl ﬂuoride 1

with acceptor 2 at a 2:1 stoichiometry in phosphate buﬀer in

the presence of TTβGly E338G mutant as described

previously³³gave access to phenyl α-D-galactopyranosyl-(1→

3)-2-amino-2-deoxy-1-thio-α-^D-glucopyranoside 9 as the sole

product in 40% yield (Scheme 2 and Table 1). Addition of 5%

Successful enzymatic access to disaccharides 11 and 12

oﬀered a unique opportunity to further document structure−

activity relationships within a series of sugar-based derivatives

that were further evaluated in a biochemical test to assess their

binding aﬃnity toward galectin-3. The syntheses could be

achieved upon exploiting the unique reactivity of azide

subsituent either directly using click chemistry or after its

reduction to the corresponding amine.

Access to Inhibitors Using Azide Speciﬁc Reactivity in

11. Staudinger reaction or hydrogenation (NaBH₄/catalytic

NiCl₂·6H₂O)³⁷was accompanied by acetate migration when

carried out with intermediate 10. Reduction was thus

attempted on deprotected disaccharide 11 which was treated

with trimethylphosphine. Amine intermediate 13 was not

puriﬁed but further reacted with 2,3-naphthalic anhydride to

give amide 14 (19%) together with the corresponding imide

15 (18%) (Scheme 2).

a

Table 1. Enzymatic Glycosylation Reaction

donor (equiv)

acceptor (equiv)

DMSO (%)

yield (%)

Next, amine 13 was reacted in parallel with a series of acyl

chlorides to give amides 16 (50%), 17 (38%), 18 (65%), 19

(55%), and 20 (60%) (Scheme 2). The methyl ester

compound 20 was further saponiﬁed to give an additional

carboxylic acid derivative 21 in 95% yield. On the one hand, a

carboxylic acid substituent as in 21 was expected to interact

with the Arg186 side-chain residue of galectin-3 CRD. On the

other hand, aromatic substituents at the GlcN-C2 position in

type II lactosamines have been shown to interact through

cation−π interactions with Arg 186, contributing substantially

to the aﬃnity toward galectin-3. This eﬀect was particularly

pronounced with 1-carboxy-2-naphthoyl and 3-methoxyben-

zoyl substituents.^30,27,38,39On the basis of this knowledge, the

3-methoxyaryl motif was next selected to synthesize a new set

of derivatives simply diﬀering by their connectivity to the sugar

moiety. Hence, amine 13 was reacted with 3-methoxysulfonyl

chloride or 3-methoxyisocyanate to aﬀord sulfone 22 and urea

23 with 20% and 47% yield, respectively. Monobenzylated

amine 24 or dibenzylated amine 25 were respectively obtained

upon reductive amination of 13 with either 1.2 equiv of 3-

methoxybenzaldehyde in the presence of sodium cyanobor-

ohydride as the reducing agent or 2.4 equiv of the aldehyde

using sodium borohydride in 56% and 37% yield. Finally,

triazole derivative 26 was obtained with 23% yield according to

a copper-catalyzed azide−alkyne cycloaddition (CuAAC)

reaction from 3-methoxyphenylacetylene and disaccharide 11

(Scheme 3). The low isolated yield was ascribed to the

intrinsic reactivity of compound 11 since, in our hands, these

experimental conditions⁴⁰led to the quantitative trans-

formation of azide 4 used as a model reactant. Alternatively,

using copper(II) sulfate, sodium ascorbate and sodium

carbonate in a H₂O/THF mixture at rt aﬀorded an inseparable

mixture of regioisomers.

1 (2)

1 (1.5)

2 (1)

8 (1)

40

67

78

5

10

b

47

a

TTβGly E338G (3−4.5 mg/mL), phosphate buﬀer (150 mM, pH

7.3), 37 °C, 72 h. Plus inseparable mixture of regioisomers and

excess donor 1

b

or 10% DMSO in the reaction mixture favored the

solubilization of the acceptor 2 without apparent alteration

of TTβGly E338G activity, leading to improved 67% and 78%

yields, respectively (Table 1). Finally, compound 9 was

peracetylated to give intermediate 10 which was then treated

with sodium methoxide in methanol to aﬀord derivative 11.

The optimized enzymatic conditions were next applied to the

enzymatic glycosylation between compounds 1 and 8 to give

phenyl α-^D-galactopyranosyl-(1→3)-1-thio-α-D-glucopyrano-

side 12 in 47% isolated yield (Scheme 1 and Table 1).

Formation of byproducts, presumably corresponding to the

1→2 and/or 1→4 regioisomer disaccharides together with

some unreacted ﬂuoride donor, were detectable on TLC

plates. Similar reactions led to a mixture of both β-(1→3)

disaccharide (96−94%) and β-(1→6) regioisomer (4−6%)

when phenyl 1-thio-β-^D-glucopyranoside was used as the

acceptor substrate. While the C6 position is no longer reactive

due to the replacement of the hydroxyl by the azide, this

observation suggests that the steric demand of the azide

substituent modiﬁes the position of the acceptor within the

(+1 site).

Along this line, our laboratory reported that the nature of

the anomeric substituent could orient the regioselectivity of

the glycosylation catalyzed by TTβGly E338G, in particular,

toward the formation of the β-(1→2) disaccharides.³⁶

Scheme 3. Synthesis of Compound 26 from Disaccharide 11

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Binding Aﬃnity Measurements. We next carried out

biochemical experiments to measure the binding aﬃnities of

these derivatives toward galectin-3 in order to validate the

value of the chemoenzymatic approach for the discovery of

new galectin inhibitors. Dissociation constants were deter-

mined by a ﬂuorescence polarization assay using known 2-

[(ﬂuoresceinyl)thioureido]ethyl 4-O-[3-O-(3-methoxybenzyl)-

β-^D-galactopyranosyl]-β-D-glucopyranoside as ﬂuorescent

probe (Figure S1) and recombinant human galectin-3 (hGal-

3) protein (Table 2).²⁹Two inhibitors previously described in

the literature, the bis{3-deoxy-3-[4-(3-ﬂuorophenyl)-1H-1,2,3-

triazol-1-yl]-β-d-galactopyranosyl}sulfane (usually referred to

as TD139)²⁵and the phenyl β-D-galactopyranosyl-(1→3)-2-

acetamido-2-deoxy-1-thio-β-^D-glucopyranoside 27³³(Figures

S1 and S2), were tested in parallel as positive controls. The

head-to-head comparison toward known inhibitors allows us to

assess the value of the new compounds for targeting galectin-3.

TD139 is among the most potent disaccharide-based galectin-3

inhibitors developed so far. This derivative has been evaluated

in a Phase II clinical trial. Derivative 27 is not modiﬁed at the

C6 position of the glucose residue representing the type I

lactosamine core. This inhibitor is thus useful to assess the

contribution of the substituent at the C6 position of the

glucose to the binding.

K_dfor reference compound 27 was found to be equal to 52

μM by ﬂuorescence polarization, in agreement with the value

measured by isothermal microcalorimetry (32 μM) (see Figure

S2 and Table S1).

The tested compounds could be ranked depending on their

aﬃnity to galectin-3: compounds with similar or less aﬃnity

than the reference 27 (e.g., methoxyphenyl amides 16, 17, and

18), those with slightly improved aﬃnities (amides 14, 19, 20,

and 21, urea 23, and triazole 26 with K_d’s in the range of 28−

42 μM and imide 15, amines 24 and 25 with K_d’s in the range

of 20 μM), and ﬁnally, 3-methoxysulfonamide 22 having a K_d

equal to 10.4 μM.

Table 2. Dissociation Constants of Compounds Tested

a,b

against Galectin-3 at rt

inhibitor

K_d(μM)

inhibitor

K_d(μM)

TD139

14

15

16

17

18

19

20

21

0.22 0.041

28.0 3.6

27

25

26

29

30

31

32

33

34

35

36

51.8 10.3

22.1 3.8

40.2 6.2

35.9 10.9

42.7 6.1

14.9 2.4

9.14 1.9

14.6 2.6

68.5 10.5

19.4 3.4

58.3 8.9

c

64.5 6.8

52.2 9.0

37.6 6.7

c

37.6 5.2

41.5 7.0

c

22

23

24

10.4 2.3

41.0 5.4

c

d

36.8 6.4

225

c

20.8 3.4

Introduction of aromatic substituents at the C6 position of

the glucose residue does not impair the recognition between

the compounds and the galectin-3 CRD, suggesting that the

interactions established by the disaccharide core are main-

tained upon binding. Such observation was expected based on

the results obtained from type II lactosamine derivatives

a

Concentrations were as follows: probe 100 nM, hGal-3 1 μM, and

b

inhibitors tested in serial dilutions from 800 μM to 10 nM. Assays

were carried out in duplicate. An independent assay was repeated in

duplicate for this derivative and conﬁrmed the K_dvalue determined in

the ﬁrst experiment. K_dvalue might be overestimated as compound

c

d

36 precipitated during the assay.

Scheme 4. Synthesis of Galectin-3 Inhibitors from Disaccharide 12

E

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Figure 1. Molecular docking of selected compounds with the galectin-3. Overview of one representative binding pose after docking for 22 (A), 23

(B), 24 (C), and 32 (D). Ligands are displayed in sticks and colored by atom types: carbons in gray, hydrogen in white, oxygens in red, nitrogen in

blue, sulfur in yellow. The receptor surface is colored by aromaticity with the aromatic face in orange (W181) and the aromatic edge in blue

bearing aromatic substituents a the C2 position of the glucose

residue.^30,38Analogously, the overall contribution of the

substituents was equally modest. However, the nature of the

linkage between the substituent and the C6 position of the

glucose seems to modulate the aﬃnity. To conﬁrm the trends

within the diﬀerent structural classes we decided to synthesize

analogues of representative compounds (sulfonamide, amine,

urea, amide) in the β-Galp-(1→3)-β-^D-Glcp series.

Access to Inhibitors Using Azide Speciﬁc Reactivity

of 12. Toward this aim, azide 12 was reduced into the amine

28 upon treatment with trimethylphosphine and next reacted

with 3-methoxybenzoyl chloride, 3-methoxyphenylisocyanate,

3-methoxybenzaldehyde, and 3-methoxysulfonyl chloride to

give derivatives 29, 30, 31, and 32 in 57%, 65%, 61%, and 70%

yields, respectively (Scheme 4). In addition, amine 28 was

condensed with 4-phenyloxyphenylsulfonyl chloride to aﬀord

compound 33 in 55% yield to further exemplify the most

promising sulfonamide family of inhibitor. Moreover, it had

been suggested that a phenyloxyphenyl substituent (at the C2

position of the type II lactosamine) might be oriented such as

to establish cation−π interactions with both Arg186 and

Arg168 residues of the galectin-3 CRD.⁴¹

Furthermore, CuAAC reactions were carried out with azide

12 to prepare triazole inhibitors from methoxyphenylacetylene,

acetylene, or 1-ethynyl-4-phenoxybenzene using either a

catalytic or a stoichiometric amount of copper(I) iodide.²⁵

Derivatives 34, 35, and 36 were obtained in 36%, 85%, and

62% yields.

Binding Aﬃnity Measurements. Galectin-3 binding aﬃn-

ities of the new analogues were measured by a ﬂuorescence

polarization assay as previously described (Table 2). The same

ranking as that reported in the ﬁrst series of compounds was

F

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observed; i.e., the K_dof galectin-3 for urea/amide was 35−45

μM, 15−20 μM for amine, and decreased to 8−10 μM for

sulfonamides.

note, derivatives 22 and 32 lacks substituents at the C3

position of the galactose, this position being known to

contribute signiﬁcantly to the selectivity.⁴²

In silico analysis of docked compounds 22 (sulfonamide),

23 (urea), and 24 (amine) conﬁrms that the core conserved

binding motif characteristic of the lactosamine moieties is

recovered via interactions of all compounds with H158, N160/

R162, N174, and W181 (Figure 1). In all binding modes

observed, the thiophenyl anomeric modiﬁcation does not seem

to bring critical contacts explaining the diﬀerences in binding

energies. The residue E184 is found in interactions with 22 and

24, with small additional hydrophobic interactions provided by

E165 and R186 for 24. The urea part of 23 is also involved in

E184 targeting but through a hydrogen bond. Two small

hydrophobic interactions are detected for 23 with R183 and

R186. In total, the number of interactions between compound

22 (7), 23 (9), and 24 (8) does not seem to be enough to

explain the relative diﬀerences in binding eﬃciency on galectin.

The galactose binding position is, however, displaced from its

location in lactosamine type I structure with an RMSD of 1.58

Å (22), 3.23 Å (23), and 1.55 Å (24), indicative of a

counterbalance between galectin aﬃnity provided by the

galactose and by additional interaction energy provided by

substituents. The equatorial substituent at the C2 position of

Glc/GlcN, which points outward on the CRD, does not impact

the binding. The acetamido present at the C2 position of the

GlcN is not involved in direct interactions with galectin, but it

reduces the potential rotation between Gal and GlcN

compared to Glc. When the acetamido is removed (32), the

galactose can rotate more (Figure 1 D). The substituent in

position 6 is therefore free to have extra hydrophobic

interactions with galectin (close to R186). In this situation,

the lactosamine core is less displaced with a galactose RMSD

of only 0.62 Å. This geometric observation could explain why

compound 32 is measured to be (relatively) better than 22.

The bulky sulfonamide 33 has a similar behavior as inhibitors

22 and 32. The best pose for the bulkier compound 33 is also

less displaced than its acetamido counterpart with an RMSD of

0.33 Å (not shown).

CONCLUSION

■

Substrate promiscuity of enzymes, in particular, that of

glycoenzymes, remains largely overlooked. Glycosyltrans-

ferases, glycoside hydrolases, or transglycosidases can all

show exquisite reactivity not limited to their known natural

substrates but extended to sugars lightly protected or modiﬁed

with biorthogonal functionalities. This peculiarity has been

herein exploited and illustrated using a GH1 glycosynthase to

rapidly access to a library of potential galectin inhibitors.

Further glyco-engineering will feed this enzyme toolbox,

expanding the scope of chemoenzymatic pathways to

synthesize complex oligosaccharides.

EXPERIMENTAL SECTION

■

General Methods. All reagents were purchased from commercial

sources and used without further puriﬁcation. Reactions were

monitored by thin-layer chromatography (TLC) on 0.25 mm silica

gel plates with ﬂuorescent indicator (GF254) and visualized under

UV light. Detection was further achieved by charring with vanillin

(1.5 g) in sulfuric acid/ethanol (1.5:95 mL). Flash chromatography

puriﬁcations were carried out on silica gel columns (4−80 g, 240−400

mesh) using an automated chromatography system equipped by both

ELS (evaporative light scattering) and UV/diode array allowing the

simultaneous use of two customizable wavelengths detectors.

All NMR experiments were performed at 400.13 MHz using a 400

MHz spectrometer equipped with a DUAL+ H/¹³C ATMA grad 5

1

mm probe. Structural assignments were made with additional

information from gCOSY and gHSQC experiments using standard

pulse programs from the Bruker library. Chemical shifts are given

relative to external TMS with calibration involving the residual solvent

signals. High-resolution mass spectra were recorded in positive mode

on a Waters SYNAPT G2-Si HDMS with detection with a hybrid

quadripole time-of-ﬂight (Q-TOF) detector. The compounds were

individually dissolved in MeOH at a concentration of 1 mg·mL⁻¹and

then infused into the electrospray ion source at a ﬂow rate of 10 μL·

min⁻¹at 100 °C. The mass spectrometer was operated at 3 kV while

scanning the magnet at a typical range of 4000−100 Da. The mass

spectra were collected as continuum proﬁle data. Accurate mass

measurement was achieved based on every 5 s autocalibration using

leucine-enkephalin ([M + H]⁺= 556.2771 m/z).

Phenyl 6-Azido-2,6-dideoxy-2-phthalimido-1-thio-β-^D-glucopyr-

anoside 4. Phenyl 2-deoxy-2-phthalimido-1-thio-β-^D-glucopyranoside

3 (2.5 g, 6.25 mmol, 1 equiv) was dissolved in anhydrous pyridine

(120 mL), and the solution was cooled to −20 °C. Mesyl chloride

(2.90 mL, 18.75 mmol, 3 equiv) was added dropwise. After 2 h, the

reaction was quenched upon addition of methanol, and the solvent

was removed under reduced pressure. The residue was diluted with

EtOAc (125 mL) and washed with aqueous 1 N HCl (125 mL), satd

aq NaHCO₃(125 mL) and brine (125 mL). The organic layer was

dried over Na₂SO₄and concentrated under reduced pressure. The

residue was puriﬁed by ﬂash chromatography (cyclohexane/EtOAc 5/

5) to give the mesylated intermediate as a yellow oil (1.93 g, 65%). R_f

0.48 (cyclohexane/EtOAc 2/8). This compound was directly

dissolved in anhydrous DMF (25 mL) and treated with NaN₃(1.31

mg, 20.1 mmol, 5 equiv). The resulting mixture was stirred at 80 °C

for 16 h then was diluted with EtOAc (125 mL) and washed with

H₂O (2 × 60 mL) and satd aq NaHCO₃(60 mL). The organic layer

was dried over Na₂SO₄and concentrated under reduced pressure.

The residue was puriﬁed by ﬂash chromatography (cyclohexane/

EtOAc 7/3) to give compound 4 as a yellow oil (1.54 g, 92%): R_f0.45

(cyclohexane/EtOAc 3/7); ¹H NMR (400 MHz, CDCl₃) δ 7.87 (dd,

J = 3.0, 5.4 Hz 2H, 2 CH Phth), 7.76 (dd, J = 3.0, 5.4 Hz, 2 CH

Phth), 7.44−7.41 (m, 2H, CH SPh), 7.32−7.25 (m, 3H, CH SPh),

5.59 (d, J = 10.2 Hz, 1H, H1), 4.28 (t, J = 9.7 Hz, 1H, H3), 4.16 (t, J

Sulfonamides 22 and 32 were eventually selected to

document the selectivity. To this aim, their dissociation

constants were determined for galectin-1 C3S (a stable mutant

of galectin-1) (hGal-1) and galectin-7 (hGal-7)⁴²in addition

to galectin-3 (Table 3)

Lower aﬃnities were observed for both sulfonamides 22 and

32 for hGal-1 and hGal-7 compared with hGal-3. This resulted

in selectivity factors of 4 and 14 in favor of hGal-3 for

compound 22. Selectivities were further increased to 9 (hGal-3

vs hGal-1) and 18 (hGal-3 vs hGal-7) for compound 32. Of

Table 3. Dissociation Constants (K_ds) of Inhibitors for

a

Galectin-1, Galectin-3, and Galectin-7 at 4 °C

inhibitor

hGal-1 (μM)

hGal-3 (μM)

hGal-7 (μM)

b

TD139

22

32

2.0 0.8

11.7 4.1

23.5 9.4

0.01 0.003

2.66 0.5

2.51 μ 0.2 μM

15.5 6.2

39.4 14.4

46.1 18.0

a

Concentrations were as follows: 2-(ﬂuorescein-5-thiourea)ethyl [3-

O-(2-naphthyl)methyl-β-^D-galactopyranosyl]-(1−4)-2-deoxy-2-(3-

methoxybenzamido)-β-^D-glucopyranoside42 used as probe (500 nM);

hGal-1, 5 μM; hGal-3, 1 μM; hGal-7, 5 μM, and inhibitors tested in

b

serial dilutions from 800 μM to 10 nM. Positive control.

G

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= 10.2 Hz, 1H, H2), 3.63−3.58 (m, 2H, H5, H6a), 3.52 (t, J = 8.8 Hz,

1H, H4), 3.48 (dd, J = 6.2, 13.6 Hz, 1H, H6b). ¹³C NMR (101 MHz,

CDCl₃) δ 162.9 (2 C(O), Phth), 134.3 (2 CH, Phth, 1 C_quat, SPh),

133.1 (2 CH, SPh), 131.6 (2 C_quat, Phth), 128.9 (2 CH, SPh), 128.2

(1 CH, SPh), 123.9 and 123.4 (2 CH, Phth), 83.7 (C1), 78.9 (C5),

72.9 (C3), 72.1 (C4), 55.6 (C6), 51.6 (C2); HRMS (ESI-TOF) m/z

[M + Na]⁺calcd for C₂₀H₁₈N₄O₅SNa 449.0896, found 449.0902.

Phenyl 2-Amino-6-azido-2,6-dideoxy-1-thio-β-^D-glucopyrano-

side 2. Compound 4 (1.49 g, 3.5 mmol, 1 equiv) was stirred with a

solution of ethylene diamine 0.8 M in MeOH (7.4 mL, 5.94 mmol,

1.7 equiv). The mixture was heated at 65 °C for 16 h, and then the

solvent was removed under reduced pressure. The residue was

puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH 95/5) to give

compound 2 as a yellow oil (750 mg, 72%): R_f0.81 (CH₂Cl₂/MeOH

9/1); ¹H NMR (400 MHz, CD3OD) δ 7.63−7.59 (m, 2H, CH SPh),

7.37−7.31 (m, 3H, CH SPh), 4.54 (d, J = 9.8 Hz, 1H, H1), 3.57 (dd,

J = 1.7, 12.7 Hz, 1H, H6a), 3.47−3.42 (m, 1H, H5), 3.41 (dd, J = 5.7,

12.7 Hz, 1H, H6b), 3.30−3.20 (m, 2H, H3, H4), 2.64−2.57 (m, 1H,

H2). ¹³C{¹H} NMR (101 MHz, CD₃OD) δ 132.6 (2CH, SPh), 132.1

(1C_quat, SPh), 128.6 (2CH, SPh), 127.8 (1CH, SPh), 88.3 (C1), 79.1

(C5), 77.5 (C3), 70.8 (C4), 55.6 (C2), 51.6 (C6); HRMS (ESI-

TOF) m/z [M + Na]⁺calcd for C₁₂H₁₆N₄O₃SNa 319.0840, found

319.0834.

Phenyl 2,3,4-Tri-O-acetyl-6-O-tert-butyldimethylsilyl-1-thio-β-^D-

glucopyranoside 6.⁴³Thiophenol (6 mL, 61.50 mmol, 1.2 equiv)

and BF₃·OEt₂(4.21 mL, 35.86 mmol, 0.7 equiv) were added to a

stirred solution of β-^D-glucosapyranose pentaacetate 5 (20 g, 51.2

mmol, 1 equiv) in CH₂Cl₂(200 mL) at 0 °C. After 12 h, the reaction

mixture was quenched by addition of satd aq NaHCO₃(150 mL).

The aqueous layer was separated and extracted with CH₂Cl₂(3 × 75

mL). The combined organic layer was washed with brine, dried over

Na₂SO₄, and evaporated under reduced pressure. The residue was

puriﬁed by precipitation in a mixture of warm EtOAc/cyclohexane to

give phenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-^D-glucopyranoside (18.2 g,

80%); R_f= 0.50 (cyclohexane/EtOAc 7/3). To a solution of this

intermediate (18.2 g, 41.2 mmol, 1 equiv) in MeOH (220 mL) was

added a solution of sodium methoxide 5.4 M (8.24 mmol, 1.5 mL, 0.2

equiv) at rt and stirred for 2 h. Then the mixture was neutralized with

an acidic resin (Amberlyte IR120-H⁺) and ﬁltrated, and the solvent

was removed under reduced pressure to give crude phenyl 1-thio-β-^D-

glucopyranoside which was further used without puriﬁcation.

TBDMS); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₂₄H₃₆O₈SiSNa 535.1798, found 535.1790.

Phenyl 2,3,4-Tri-O-acetyl-6-O-azido-1-thio-β-^D-glucopyranoside

7.⁴⁴Compound 6 (2.96 g, 5.77 mmol, 1 equiv) was stirred for 16 h in

a solution of AcOH/H₂O/THF (3/1/1; 100 mL) at rt. Then THF

was removed under reduced pressure, and the mixture was neutralized

with satd aq NaHCO₃(3 × 70 mL). The organic layer was dried over

Na₂SO₄and concentrated under reduced pressure. R_f0.57 (cyclo-

hexane/EtOAc 1/9). The residue was puriﬁed by ﬂash chromatog-

raphy (cyclohexane/EtOAc 75/25) to give phenyl 2,3,4-tri-O-acetyl-

1-thio-β-^D-glucopyranoside as a colorless oil (1.94 g, 85%). R_f0.32

(cyclohexane/EtOAc 7/3). This intermediate was dissolved in

anhydrous pyridine (32 mL), and the solution was cooled to 0 °C.

Mesyl chloride (750 μL, 9.7 mmol, 2 equiv) was added dropwise.

After 2 h, the reaction was quench by adding methanol, and the

solvent was removed under reduced pressure. R_f0.88 (cyclohexane/

EtOAc 5/5). The residue was directly dissolved in anhydrous DMF

(50 mL) and treated with NaN₃(944 mg, 14.5 mmol, 3 equiv). The

resulting mixture was stirred at 60 °C for 16 h, then diluted in EtOAc

(100 mL) and washed with H₂O (2 × 50 mL) and satd aqNaHCO₃

(50 mL). The organic layer was dried over Na₂SO₄and concentrated

under reduced pressure. The residue was puriﬁed by ﬂash

chromatography (cyclohexane/EtOAc 8/2) to give phenyl 2,3,4-tri-

O-acetyl-6-azido-6-deoxy-1-thio-β-^D-glucopyranoside 7 as a white

solid (1.8 g, 88% over two steps): R_f0.43 (cyclohexane/EtOAc 5/

1

5); H NMR (400 MHz, CDCl₃) δ 7.56−7.50 (m, 2H, CH SPh),

7.37−7.32 (m, 3H, CH SPh), 5.23 (t, J = 9.3 Hz, 1H, H3), 4.99−4.92

(m, 2H, H2, H4), 4.73 (d, J = 10.0 Hz, 1H, H1), 3.70−3.64 (m, 1H,

H5), 3.37 (dd, J = 2.5, 11.5 Hz, 1H, H6a), 3.31 (dd, J = 5.0, 11.5 Hz,

1H, H6b), 2.10 (s, 3H, COCH₃), 2.03 (s, 3H, COCH₃), 2.00 (s, 3H,

COCH₃); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 170.1, 169.4, 169.2

(3C_quat, COCH₃), 133.7 (2CH, SPh), 130.9 (C_quat, SPh), 129.0 (2CH,

SPh), 128.7 (1CH, SPh), 85.7 (C1), 77.0 (C5), 73.8 (C3), 69.9 and

69.3 (C2 and C4), 51.3 (C6), 20.7, 20.6, and 20.6 (3CH₃,); HRMS

(ESI-TOF) m/z [M + Na]⁺calcd for C₁₈H₂₁N₃O₇SNa 446.0998,

found 446.1019.

Phenyl 6-O-Azido-1-thio-β-^D-glucopyranoside 8.⁴⁵To a solution

of compound 7 (690 mg, 1.63 mmol, 1 equiv) in MeOH (10 mL) was

added a solution of sodium methoxide 5.4 M (0.49 mmol, 90 μL, 0.1

equiv) at 0 °C and the mixture stirred for 2 h. Then the mixture was

neutralized with an acidic resin (Amberlyte IR120-H⁺) and ﬁltrated

and the solvent removed under reduced pressure. The residue was

puriﬁed by ﬂash chromatography (cyclohexane/AcOEt 45/55) to give

phenyl 6-azido-6-deoxy-1-thio-β-^D-glucopyranoside 8 as a white solid

The triol was solubilized in anhydrous DMF (100 mL), and then a

solution of tert-butyldimethylsilyl chloride (TBDMSCl, 6.8 g, 45.3

mmol, 1.1 equiv) and imidazole (7.0 g, 103.0 mmol, 2.5 equiv) in

anhydrous DMF (40 mL) was added at 0 °C. After being stirred for

12 h, the mixture was diluted with EtOAc (150 mL) and washed with

H₂O (2 × 100 mL) and satd aq NaHCO₃(100 mL). The organic

layer was dried over Na₂SO₄and concentrated under reduced

pressure; R_f0.57 (cyclohexane/EtOAc 1/9). Then the residue was

solubilized in CH₂Cl₂(100 mL) at 0 °C, treated with Ac₂O (17.5

mL), Et₃N (20.1 mL), DMAP (cat.), and stirred for 16 h. The mixture

was diluted with CH₂Cl₂(100 mL) and washed with H₂O (2 × 100

mL) and satd aq NaHCO₃(100 mL). The organic layer was dried

over Na₂SO₄and concentrated under reduced pressure. The residue

was puriﬁed by ﬂash chromatography (cyclohexane/EtOAc 9/1) to

give phenyl 2,3,4-tri-O-acetyl-6-O-tert-butyldimethylsilyl-1-thio-β-^D-

glucopyranoside 6 as a white solid (16.64 g, 65% over four steps):

1

(455 mg, 94%): R_f0.28 (cyclohexane/AcOEt 4/6); H NMR (400

MHz, CD₃OD) δ 7.62−7.57 (m, 2H, CH SPh), 7.35−7.27 (m, 3H,

CH SPh), 4.60 (d, J = 9.7 Hz, 1H, H1), 3.56 (dd, J = 2.0, 12.9 Hz,

1H, H6a), 3.46−3.35 (m, 3H, H5, H6b, H3), 3.27 (t, 1H, J = 9.0 Hz,

H4), 3.22 (dd, J = 8.7, 9.8 Hz, 1H, H2); ¹³C{¹H} NMR (101 MHz,

MeOD) δ 132.9 (C_quat, SPh), 132.4 (2CH, SPh), 128.5 (2CH, SPh),

127.4 (CH, SPh), 87.8 (C1), 78.9 (C5), 78.0 (C3), 72.2 (C2), 70.6

(C4), 51.6 (C6); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₁₂H₁₅N₃O₄SNa 320.0681, found 320.0694.

Phenyl β-^D-Galactopyranosyl-(1→3)-2-amino-6-azido-2,6-di-

deoxy-1-thio-β-^D-glucopyranoside 9. Donor 1 (184 mg, 1.0 mmol,

2 equiv) and acceptor 2 (150 mg, 0.5 mmol, 1 equiv) were dissolved

in a phosphate buﬀer (150 mmol/L, pH 7, 6 mL) and DMSO (1

mL). Then 3 mL of glycosynthase E338G (4.2 mg·mL⁻¹) was added,

and the reaction was allowed to proceed at 37 °C until complete

consumption of the ﬂuoride (about 72 h). The mixture was acidiﬁed

with HCl 1 M to pH 1 and stirred for 4 h before neutralization with

NaOH 1 M. After removal of the solvent under reduced pressure, the

crude residue was puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH

90/10) to aﬀord disaccharide 9 as a white solid (180 mg, 78%): R_f

0.75 (CHCl₃/MeOH/H₂O/CH₃COOH 60/30/5/3); ¹H NMR (400

MHz, D₂O) δ 7.57−7.52 (m, 2H, CH SPh), 7.36−7.28 (m, 3H, CH

SPh), 4.66 (d, J = 8.3 Hz, 1H, H1), 4.51 (d, J = 7.7 Hz, 1H, H1′),

3.89 (d, J = 3.3 Hz, 1H, H4′), 3.76−3.66 (m, 3H, H5′, H6a′, H6b′),

3.65 (dd, J = 3.3, 9.9 Hz, 1H, H3′), 3.62−3.52 (m, 4H, H2′, H6a, H5,

H3), 3.49 (dd, J = 8.1, 18.5 Hz, 1H, H4), 3.44 (dd, J = 5.7, 12.9 Hz,

1

R_f0.71 (cyclohexane/EtOAc 7/3); H NMR (400 MHz, CDCl₃) δ

7.51−7.49 (m, 2H, CH SPh), 7.30−7.28 (m, 3H, CH SPh), 5.22 (t, J

= 9.3 Hz, 1H, H3), 5.03 (t, J = 9.8 Hz, 1H, H4), 4.96 (t, J = 9.3, 10.0

Hz, 1H, H2), 4.72 (d, J = 10.0 Hz, 1H, H1), 3.75 (dd, J = 2.5, 11.6

Hz, 1H, H6a), 3.70 (dd, J = 5.0, 11.6 Hz, 1H, H6b), 3.58 (ddd, J =

2.5, 5.0, 10.0 Hz, 1H, H5), 2.07 (s, 3H, COCH₃), 2.01 (s, 3H,

COCH₃), 1.98 (s, 3H, COCH₃), 0.90 (s, 9H, 3CCH₃), 0.07 (s, 3H,

CH₃, TBDMS), 0.05 (s, 3H, CH₃, TBDMS); ¹³C{¹H} NMR (101

MHz, CDCl₃) δ 170.2, 169.2, 169.2 (3C_quat, COCH₃), 132.6 (2CH,

SPh), 133.2 (1C_quat, SPh), 128.9 (2CH, SPh), 128.0 (1CH, SPh), 85.6

(C1), 78.9 (C5), 74.4 (C3), 70.1 (C2), 68.6 (C4), 62.4 (C6), 25.8

(3CCH₃), 20.7, 20.6 (3COCH₃), 18.4 (1CCH₃), −5.4, −5.4 (2CH₃,

H

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1H, H6b′), 2.83 (dd, J = 8.3, 11.1 Hz, 1H, H2); ¹³C{¹H} NMR (101

MHz, D₂O) δ 132.7 (2CH, SPh), 130.9 (C_quat, SPh), 129.4 (2CH,

SPh), 128.5 (CH, SPh), 104.1 (C1′), 87.8 (C1), 87.6 (C3), 78.2

(C5), 75.5 (C5′), 72.8 (C3′), 71.2 (C2′), 69.0 (C4), 68.6 (C4′), 60.9

(C6′), 54.6 (C2), 51.3 (C6); HRMS (ESI-TOF) m/z [M + Na]⁺

calcd for C₁₈H₂₆N₄O₈SNa 459.1550, found 459.1558.

glucopyranoside. To facilitate the puriﬁcation, the crude reaction

mixture was suspended in CH₂Cl₂(5 mL), treated with Ac₂O (1 mL),

Et₃N (1 mL), and DMAP (cat.), and stirred for 16 h. Then the

reaction mixture was diluted in CH₂Cl₂(25 mL) and washed with

H₂O (2 × 15 mL) and satd aq NaHCO₃(15 mL). The organic layer

was dried over Na₂SO₄and concentrated under reduced pressure.

The crude residue was puriﬁed ﬁrst by ﬂash chromatography

(cyclohexane/EtOAc 65/35) then by reversed-phase C-18 ﬂash

chromatography (H₂O/MeOH 4/6) to give phenyl 2,3,4,6-tetra-O-

acetyl-β-^D-galactopyranosyl-(1→3)-2,4-di-O-acetyl-6-azido-6-deoxy-1-

thio-β-^D-glucopyranoside as a colorless oil (141 mg, 48% over 2

steps): R_f0.50 (cyclohexane/EtOAc 5/5); ¹H NMR (400 MHz,

CD₃OD) δ 7.55−7.49 (m, 2H, CH SPh), 7.36−7.30 (m, 3H, CH

SPh), 5.34 (dd, J = 1.1, 3.4 Hz, 1H, H4′), 5.21 (t, J = 9.0 Hz, 1H,

H3), 5.10 (dd, J = 3.5, 10.4 Hz, 1H, H3′), 4.98 (dd, J = 7.9, 10.4 Hz,

1H, H2′), 4.92 (d, J = 10.4 Hz, 1H, H1), 4.81 (dd, J = 9.1, 10.1 Hz,

1H, H2), 4.69 (d, J = 7.9 Hz, 1H, H1′), 4.16−4.06 (m, 3H, H5′,

H6a′, H6b′), 3.79 (dd, J = 8.9, 9.8 Hz, 1H, H4), 3.73 (ddd, J = 2.1,

5.3, 9.8 Hz, 1H, H5), 3.63 (dd, J = 2.2, 13.5 Hz, 1H, H6a), 3.42 (dd, J

= 5.3, 13.5 Hz, 1H, H6b), 2.12 (s, 3H, COCH₃), 2.06 (s, 3H,

COCH₃), 2.04 (s, 3H, COCH₃), 2.02 (s, 3H, COCH₃), 2.02 (s, 3H,

COCH₃), 1.92 (s, 3H, COCH₃); ¹³C{¹H} NMR (101 MHz,

CD₃OD) δ 172.0 (COCH₃), 171.9 (COCH₃), 171.7 (COCH₃),

171.4 (COCH₃), 171.1 (COCH₃), 171.0 (COCH₃), 134.5 (2CH,

SPh), 132.6 (C_quat, SPh), 130.0 (2CH, SPh), 129.5 (CH, SPh), 101.9

(C1′),86.1 (C1), 79.2 (C5), 77.5 (C4), 75.5 (C3), 72.4 (C3′), 71.8

and 71.7 (C2 and C5′), 70.7 (C2′), 68.6 (C4′), 62.3 (C6′), 51.9

(C6), 21.1 (COCH₃), 20.8 (COCH₃), 20.7 (CH₃), 20.6 (CH₃), 20.5

(CH₃); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₁₈H₂₅N₃O₉SNa

734.1843, found 734.1854.

Phenyl 2,3,4,6-Tetra-O-acetyl-β-^D-galactopyranosyl-(1→3)-4-O-

acetyl-2-acetamido-6-azido-2,6-dideoxy-1-thio-β-^D-glucopyrano-

side 10. A solution of compound 9 (150 mg, 0.33 mmol, 1 equiv) in

CH₂Cl₂(2.5 mL) was treated with Ac₂O (350 μL), Et₃N (350 μL),

and DMAP (0.03 mmol, 4 mg, 0.1 equiv) and stirred for 16 h. The

mixture was diluted with CH₂Cl₂(25 mL) and washed with H₂O (2

× 12 mL) and satd aq NaHCO₃(12 mL). The organic layer was dried

over Na₂SO₄and concentrated under reduced pressure. The residue

was puriﬁed by ﬂash chromatography (cyclohexane/EtOAc 6/4) to

give compound 10 as a yellow oil (233 mg, 98%): R_f0.50

1

(cyclohexane/EtOAc 3/7); H NMR (400 MHz, CDCl₃) δ 7.52−

7.45 (m, 2H, CH SPh), 7.33−7.27 (m, 3H, CH SPh), 6.07 (d, J = 8.1

Hz, 1H, NH), 5.33 (dd, J = 1.1, 3.4 Hz, 1H, H4′), 5.12 (d, J = 10.2

Hz, 1H, H1), 5.03 (dd, J = 7.5, 10.4 Hz, 1H, H2′), 4.97 (dd, J = 3.3,

10.4 Hz, 1H, H3′), 4.81 (t, J = 9.2 Hz, 1H, H4), 4.62 (d, J = 7.6 Hz,

1H, H1), 4.33 (t, J = 9.2 Hz, 1H, H3), 4.14−4.02 (m, 2H, H6a′,

H6b′), 3.88 (td, J = 1.02, 6.6 Hz, 1H, H5′), 3.63 (ddd, J = 3.0, 6.8, 9.7

Hz, 1H, H5), 3.51 (dd, J = 8.1, 9.4 Hz, 1H, H2), 3.33 (dd, J = 6.8,

13.4 Hz, 1H, H6a), 3.27 (dd, J = 3.0, 13.4 Hz, 1H, H6b), 2.13 (s, 3H,

COCH₃), 2.05 (s, 3H, COCH₃), 2.04 (s, 3H, COCH₃), 2.04 (s, 3H,

COCH₃), 2.03 (s, 3H, COCH₃), 1.95 (s, 3H, COCH₃); ¹³C{¹H}

NMR (100 MHz, CDCl₃) δ 170.5 (COCH₃), 170.4 (COCH₃), 170.1

(2COCH₃), 169.4 (COCH₃), 169.2 (COCH₃), 132.8 (2CH, SPh),

132.1 (C_quat, SPh), 129.0 (2CH, SPh), 128.2 (CH, SPh), 100.6 (C1′),

85.4 (C1), 78.0 (C3), 77.2 (C5), 70.0 (C3′), 70.6 (C5′), 70.1 (C4),

69.3 (C2′), 66.9 (C4′), 61.0 (C6′), 55.9 (C2), 51.6 (C6), 23.6

(COCH₃), 20.8 (2 COCH₃), 20.7 (COCH₃), 20.6 (COCH₃), 20.5

(COCH₃); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₃₀H₃₈N₄O₁₄SNa 733.2003, found 733.2011.

To a solution of phenyl 2,3,4,6-tetra-O-acetyl-β-^D-galactopyranosyl-

(1→3)-2,4-O-diacetyl-6-azido-6-deoxy-1-thio-β-^D-glucopyranoside

(141 mg, 0.20 mmol, 1 equiv) in MeOH (2 mL) was added a solution

of sodium methoxide 5.4 M (0.02 mmol, 3 μL, 0.1 equiv) at 0 °C and

the mixture stirred for 2 h. Then the solvent was removed under

reduced pressure, and the residue was puriﬁed by ﬂash chromatog-

raphy (CH₂Cl₂/MeOH 9/1) to give to give disaccharide 12 as a white

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-6-azido-2,6-

dideoxy-1-thio-β-^D-glucopyranoside 11. To a solution of compound

10 (233 mg, 0.32 mmol, 1 equiv) in MeOH (3.5 mL) was added a

solution of sodium methoxide 5.4 M (0.035 mmol, 8 μL, 0.1 equiv) at

0 °C and the mixture stirred for 2 h. Then the mixture was neutralized

with an acidic resin (amberlyte IR120-H⁺) and ﬁltrated, and the

solvent was removed under reduced pressure. The crude residue was

puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH 8/2) to give

compound 11 as a white solid (135 mg, 85%): R_f0.78 (CHCl₃/

MeOH/H₂O/CH₃COOH 60/30/5/3); ¹H NMR (400 MHz,

DMSO-d₆) δ 7.93 (d, J = 7.6 Hz, 1H, NH), 7.46−7.44 (m, 2H,

CH SPh), 7.35−7.31 (m, 2H, CH SPh), 7.28−7.25 (m, 1H, CH SPh),

5.01 (d, J = 7.7 Hz, 1H, H1), 4.89 (d, J = 1.6 Hz, 1H, OH), 4.79 (br s,

1H, OH), 4.64 (t, J = 5.4 Hz, 1H, OH), 4.48 (m, 2H, 2 × OH), 4.17

(d, J = 6.3 Hz, 1H, H1′), 3.68−3.59 (m, 3H, H2, H3, H4′), 3.56−

3.49 (m, 4H, H6a, H5, H6a′, H6b′), 3.49−3.39 (m, 2H, H5′, H6b),

3.37−3.22 (m, 3H, H2′, H3′, H4), 1.84 (s, 3H, COCH₃); ¹³C{¹H}

NMR (101 MHz, DMSO) δ 170.4 (COCH₃), 134.4 (C_quat, SPh),

130.6 (2CH, SPh), 129.4 (2CH, SPh), 127.4 (CH, SPh), 104.3 (C1′),

85.8 (C1), 85.1 (C3), 78.6 (C5), 76.3 (C5′), 73.5 (C3′), 71.0 (C2′),

70.0 (C4), 68.7 (C4′), 61.1 (C6′), 51.9 (C2), 51.4 (C6), 23.5

(COCH₃); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₂₀H₂₈N₄O₉SNa 523.1474, found 523.1506.

Phenyl β-^D-Galactopyranosyl-(1→3)-6-azido-6-dideoxy-1-thio-

β-^D-glucopyranoside 12. Donor 1 (115 mg, 0.63 mmol, 1.5 equiv)

and acceptor 8 (125 mg, 0.42 mmol, 1 equiv) were dissolved in a

phosphate buﬀer (200 mmol/L, pH 6.8, 6.5 mL) and DMSO (1 mL).

Then glycosynthase E338G (2.5 mL, c = 4.0 mg·mL⁻¹) was added,

and the reaction was allowed to proceed at 37 °C for 72 h. The

mixture was acidiﬁed with HCl 1 M to pH 1 and stirred for 4 h before

neutralization with NaOH 1 M. After removal of the solvent under

reduced pressure, the crude residue was puriﬁed by ﬂash

chromatography (CH₂Cl₂/MeOH 90/10) to aﬀord a mixture of

phenyl β-^D-galactopyranosyl-(1→3)-6-azido-6-deoxy-1-thio-β-D-glu-

copyranoside and unreacted phenyl 6-azido-6-deoxy-1-thio-β-^D-

1

solid (90 mg, 100%): R_f0.60 (CH₂Cl₂/MeOH 9/1); H NMR (400

MHz, CD₃OD) δ 7.61−7.57 (m, 2H, CH SPh), 7.36−7.30 (m, 3H,

CH SPh), 4.64 (d, J = 9.8 Hz, 1H, H1), 4.53 (d, J = 7.7 Hz, 1H, H1′),

3.81 (dd, J = 1.0, 3.3 Hz, 1H, H4′), 3.78 (dd, J = 7.6, 11.4 Hz, 1H,

H6a′), 3.69 (dd, J = 4.5, 11.4 Hz, 1H, H6b′), 3.63−3.55 (m, 4H, H2′,

H3, H5′, H6a), 3.54−3.47 (m, 2H, H3′, H5), 3.44−3.33 (m, 3H,

H6b, H2, H4); ¹³C{¹H} NMR (101 MHz, DMSO-d₆) δ 132.7 (2CH,

SPh), 132.4 (C_quat, SPh), 128.5 (2CH, SPh), 127.5 (CH, SPh), 104.2

(C1′), 87.4 (C3), 87.1 (C1), 78.6 (C5), 75.7 (C5′), 73.3 (C3′), 71.6

(C2′), 17.3 (C2), 69.3 and 68.9 (C4 and C4′), 61.1 (C6′), 51.6 (C6);

HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₁₈H₂₅N₃O₉SNa

482.1209, found 482.1181.

General Procedure for the Reduction of Azide Derivative

11. To a solution of disaccharide 11 (50 mg, 0.1 mmol, 1 equiv) in

THF (5 mL), under an atmosphere of argon, was added PMe₃1 M in

THF (200 μL, 0.2 mmol, 2 equiv). The reaction was stirred at rt and

monitored by TLC (CHCl₃/MeOH/H₂O/CH₃COOH 60/30/5/3).

After completion, H₂O (10 μL) was added, and the mixture was

heated to 50 °C in an oil bath for 16 h. Then solvent was removed

under reduced pressure to give crude phenyl β-^D-galactopyranosyl-

(1→3)-2-acetylamino-6-amino-2,6-dideoxy-1-thio-β-^D-glucopyrano-

side 13 used without further puriﬁcation: R_f0.78 (CHCl₃/MeOH/

H₂O/CH₃COOH 60/30/5/3).

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-2,6-dideoxy-

6-(1-carboxy-2-naphthoyl)amino-1-thio-β-^D-glucopyranoside 14

and Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-2,6-di-

deoxy-6-(2,3-naphthal)imido-1-thio-β-^D-glucopyranoside 15.

Amine 13 was dissolved in anhydrous DMF (2 mL) at 0 °C and

treated with 2,3-naphthalic anhydride (34 mg, 0.17 mmol, 1.7 equiv)

and Et₃N (27 μL, 0.2 mmol, 2.0 equiv). The mixture was stirred 12 h

at rt and then heated at 60 °C in an oil bath. After 12 h, formation of

two products was observed. The reaction was stopped and

concentrated under reduced pressure. The residue was puriﬁed by

I

https://dx.doi.org/10.1021/acs.joc.0c01927

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

reversed-phase C-18 ﬂash chromatography using H₂O/MeOH

gradient mixture as eluant to successively give 14 as a white solid

(11.7 mg, 19%) and 15 as a white solid (12.3 mg, 18%). Compound

14: R_f0.67 (CHCl₃/MeOH/H₂O/CH₃COOH 60/30/5/3); ¹H

NMR (400 MHz, DMSO-d₆) δ 8.64 (br s, 1H, NHCONapht), 8.36

(s, 1H, CH Napht), 8.09 (dd, J = 2.3, 6.5 Hz, 1H CH Napht), 7.95−

7.86 (m, 3H, CH Napht), 7.69−7.60 (m, 2H, CH Napht), 7.44−7.38

(m, 2H, CH SPh), 7.11−6.98 (m, 3H, SPh), 5.06−4.88 (m, 2H, H1,

OH), 4.81 (br s, 2H, 2 × OH), 4.51 (d, J = 3.3 Hz, 2H, 2 × OH),

4.28−4.19 (m, 1H, H1′), 3.88 (dd, J = 7.2, 13.2 Hz, 1H, H6a), 3.78−

3.45 (m, 7H, H2, H3, H5, H4′, H6′a, H6′b, H5′), 3.45−3.14 (m, 4H,

H2′, H3′, H4, H6b), 1.83 (s, 3H COCH₃); ¹³C{¹H} NMR (101

Amine 13 was dissolved in anhydrous DMF (2 mL) at 0 °C and

treated with 3,5-dimethoxybenzoyl chloride (80 mg, 0.44 mmol, 4

equiv) and Et₃N (60 μL, 0.44 mmol, 4 equiv). The mixture was

stirred 8 h at rt where 20% of conversion was observed and at 40 °C

for 16 h where all the starting product was converted. Then the

mixture was concentrated under reduced pressure and the residue was

puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH 9/1) to give

derivative 17 as a white solid (22 mg, 38%); R_f0.67 (CHCl₃/MeOH/

1

H₂O/CH₃COOH 60/30/5/3); H NMR (400 MHz, DMSO-d₆) δ

8.56 (t, J = 5.7 Hz, 1H, NH(3MeOBz)), 7.98 (d, J = 8.3 Hz, 1H,

NH(Ac)), 7.32 (d, J = 7.4 Hz, 2H, CH SPh), 7.08 (d, J = 2.3 Hz, 2H,

CH 3MeOBz), 7.05 (t, J = 7.7 Hz, 1H, CH SPh), 6.92 (t, J = 7.7 Hz,

2H, CH SPh), 6.69 (t, J = 2.3 Hz, 1H, CH 3MeOBz), 4.92−4.90 (m,

1H, OH), 4.86 (d, J = 9.3 Hz, 1H, H1), 4.81 (d, J = 4.9 Hz, 1H OH),

4.69 (t, J = 5.2 Hz, 1H, OH), 4.51 (d, J = 4.7 Hz, 1H, OH), 4.48 (d, J

= 3.2 Hz, 1H, OH), 4.21 (d, J = 6.6 Hz, 1H, H1′), 3.90−3.81 (m, 1H,

H6a), 3.78 (s, 6H, 2 × CH₃3MeOBz), 3.75−3.45 (m, 7H, H2, H3,

H4′, H5, H6a′, H6b′, H5′), 3.41−3.30 (m, 2H, H2′, H3′), 3.30−3.19

(m, 2H, H4, H6b), 1.83 (s, 3H, COCH₃); ¹³C{¹H} NMR (101 MHz,

DMSO-d₆) δ 170.4 (1C, 1C_quat, COCH₃), 166.1 (C_quat, COPh), 160.8

(2C_quat, 3MeO-C), 136.8 (C_quat, 3MeOBz), 135.5 (C_quat, SPh), 129.5

(CH, 3MeOBz), 129.1 (2CH, SPh), 126.7 (CH, SPh), 105.8 (2CH,

3MeOBz), 104.4 (C1′), 103.6 (CH, 3MeOBz), 85.5 (2CH, C1 and

C3), 78.0 (C5), 76.3 (C5′), 73.6 (C3′), 71.5 (C4), 71.1 (C2′), 68.7

(C4′), 61.1 (C6′), 55.9 (2CH₃, 3MeOBz), 52.9 (C2), 41.8 (C6), 23.5

(COCH₃); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₂₉H₃₈N₂O₁₂SNa 661.2043, found 661.2037.

MHz, DMSO-d₆) δ, 170.4 (2C_quat, COCH₃, COOH), 169.0 (C_quat

,

CONapht), 135.3, 134.9, 133.3, 132.7 (4C_quat, Napht, SPh), 130.0

(CH, Napht), 129.5 (2CH, SPh), 129.3 (2CH, SPh), 129.1 (CH,

Napht), 128.6 (CH, Napht), 128.4 (CH, Napht), 128.0 (CH, Napht),

127.6 (CH, Napht), 126.6 (CH, SPh), 104.3 (C1′), 85.6 (C1), 85.3

(C3), 78.4 (C5), 76.3 (C5′), 73.6 (C3′), 71.1, 71.0 (C2′,C4), 68.8

(C4′), 61.1 (C6′), 53.5 (C2), 41.7 (C6), 23.5 (COCH₃); HRMS

(ESI-TOF) m/z [M − H]⁻calcd for C₃₂H₃₅N₂O₁₂S 671.1911, found

671.1920.

Compound 15: R_f0.88 (CHCl₃/MeOH/H₂O/CH₃COOH 60/30/

1

5/3); H NMR (400 MHz, DMSO-d₆) δ 8.52 (s, 2H, CH Napht),

8.29 (dd, J = 3.3, 6.2 Hz, 2H, CH Napht), 7.87 (d, J = 8.2 Hz, 1H,

NH), 7.81 (dd, J = 3.3, 6.3 Hz, 1H, CH Napht), 7.07−7.02 (m, 2H,

CH SPh), 6.54−6.45 (m, 3H, CH SPh), 5.03 (d, J = 1.7 Hz, 1H, OH),

4.82 (d, J = 4.9 Hz, 1H, OH), 4.78 (d, J = 8.0 Hz, 1H, H1), 4.70 (t, J

= 5.1 Hz, 1H, OH), 4.56 (d, J = 3.7 Hz, 1H, OH), 4.53 (d, J = 4.7 Hz,

1H, OH), 4.21 (d, J = 6.8 Hz, 1H, H1′), 3.96 (dd, J = 3.4, 14.0 Hz,

1H, H6a), 3.89 (dd, J = 9.6, 14.1 Hz, 1H, H6b), 3.75−3.59 (m,

4H,H2, H3, H5, H4′), 3.57−3.46 (m, 3H, H6′a, H6′b, H5′), 3.40−

3.30 (m, 3H, H2′, H3′, H4), 1.83 (s, 3H COCH₃); ¹³C{¹H} NMR

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-2,6-dideoxy-

6-(3-methoxyphenylacetamido)-1-thio-β-^D-glucopyranoside 18.

Amine 13 was dissolved in anhydrous DMF (2 mL) at 0 °C and

treated with 3-methoxyphenylacetyl chloride (16 μL, 0.10 mmol, 1.1

equiv) and Et₃N (14 μL, 0.10 mmol, 1.1 equiv). The mixture was

stirred 3 h and concentrated under reduced pressure. The residue was

puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH 9/1) to give

derivative 18 as a white solid (37 mg, 66%): R_f0.65 (CHCl₃/MeOH/

(101 MHz, DMSO-d₆) δ 170.3 (C_quat, COCH₃), 167.7 (2C_quat

,

N(CO)₂), 135.5 (2C, 2C_quat, Napht), 134.4 (1C, 1C_quat, SPh), 130.8

(CH, SPh), 130.1 (2CH, Napht), 129.8 (2CH, SPh), 128.8 (2CH,

Napht), 127.7 (2C_quat, Napht), 126.6 (CH, SPh), 125.0 (2CH,

Napht), 104.4 (C1′), 86.0 (C1), 85.3 (C3), 76.3 (C5), 76.2 (C5′),

73.6 (C3′), 71.8 and 71.0 (C2′ and C4), 68.8 (C4′), 61.5 (C6′), 53.3

(C2), 40.6 (C6), 23.5 (COCH₃); HRMS (ESI-TOF) m/z [M + Na]⁺

calcd for C₃₂H₃₄N₂O₁₁SNa 677.1781, found 677.1773.

1

H₂O/CH₃COOH 60/30/5/3); H NMR (400 MHz, DMSO-d₆) δ

8.11 (t, J = 5.8 Hz, 1H, NH(3MeOPh)), 8.01 (d, J = 8.0 Hz, 1H,

NHAc), 7.42 (d, J = 7.1 Hz, 2H, CH SPh), 7.29 (d, J = 8.0 Hz, 2H,

CH SPh), 7.23 (d, J = 7.0 Hz, 1H, CH SPh), 7.17 (t, J = 7.8 Hz, 1H,

CH 3MeOPh), 6.83−6.79 (m, 2H, CH 3MeOPh), 6.78 (dd, J = 3.1,

8.1 Hz, 1H, CH 3MeOPh), 4.91 (d, J = 9.8 Hz, 1H, H1),4.86−4.79

(m, 2H, 2 × OH), 4.68 (t, J = 5.3 Hz, 1H, OH), 4.51 (d, J = 4.7 Hz,

1H, OH), 4.48 (d, J = 3.7 Hz, 1H, OH), 4.19 (d, J = 6.3 Hz, 1H,

H1′), 3.71 (s, 3H, CH₃3MeOPh), 3.69−3.56 (m, 4H, H2, H3, H6a,

H4′), 3.55−3.37 (m, 6H, H6a′, H6b′, H5′, H5, CH₂3MeOPhCH₂),

3.37−3.28 (m, 2H, H2′, H3′), 3.24−3.17 (m, 1H, H4′), 3.11 (dd, J =

5.9, 14.0 Hz, 1H, H6b), 1.84 (s, 3H COCH₃); ¹³C{¹H} NMR (101

MHz, DMSO-d₆) δ 170.1 (C_quat, COCH₂Ph), 170.4 (C_quat, COCH₃),

159.6 (C_quat, 3MeO-C), 138.3 (C_quat, 3MeOPh), 135.3 (C_quat, SPh),

129.8 (2CH, SPh), 129.6 (CH, 3MeOPh), 129.4 (2CH, SPh), 127.0

(CH, SPh), 121.7 (CH, 3MeOPh), 115.2 (CH, 3MeOPh), 112.3

(CH, 3MeOPh), 104.3 (C1′), 86.0 (C1), 85.3 (C3), 78.4 (C5), 76.3

(C5′), 73.6 (C3′), 71.0 (C2′), 70.8 (C4), 68.7 (C4′), 61.0 (C6′),

55.4 (CH₃, 3MeOPh), 53.4 (C2), 45.9 (CH₂, 3MeOPhCH₂), 40.9

(C6), 23.5 (COCH₃); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₂₉H₃₈N₂O₁₁SNa 645.2094, found 645.2088.

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-6-

(piperonyloyl)amido-2,6-dideoxy-1-thio-β-^D-glucopyranoside 19.

Amine 13 was dissolved in anhydrous DMF (2 mL) at 0 °C and

treated with piperonyloyl chloride (20 mg, 0.11 mmol, 1.1 equiv) and

Et₃N (15 μL, 0.11 mmol, 1.1 equiv). The mixture was stirred for 12 h

and concentrated under reduced pressure. The residue was ﬁrst

puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH 75/25) and then

by reversed-phase C-18 ﬂash chromatography using a H₂O/MeOH

gradient mixture as eluent to give derivative 19 as a white solid (33

mg, 55%): R_f0.65 (CHCl₃/MeOH/H₂O/CH₃COOH 60/30/5/3);

¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (t, J = 5.7 Hz, 1H,

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-2,6-dideoxy-

6-(3-methoxybenzamido)-1-thio-β-^D-glucopyranoside 16. Amine

13 was dissolved in anhydrous DMF (2 mL) at 0 °C and treated

with 3-methoxybenzoyl chloride (15 μL, 0.11 mmol, 1.1 equiv) and

Et₃N (15 μL, 0.11 mmol, 1.1 equiv). The mixture was stirred for 2 h

and concentrated under reduced pressure. The residue was puriﬁed by

ﬂash chromatography (CH₂Cl₂/MeOH 9/1) to give derivative 16 as a

white solid (29 mg, 50%): R_f0.72 (CHCl₃/MeOH/H₂O/CH₃COOH

1

60/30/5/3); H NMR (400 MHz, DMSO-d₆) δ 8.55 (t, J = 5.7 Hz,

1H, NH(3MeOBz)), 7.98 (d, J = 8.3 Hz, 1H, NHAc), 7.50−7.44 (m,

2H, CH 3MeOBz), 7.40 (t, J = 7.9 Hz, 1H, CH 3MeOBz), 7.32 (t, J =

4.5 Hz, 2H, CH SPh), 7.13 (dd, J = 3.4, 8.5 Hz, 1H, CH 3MeOBz),

7.04 (t, J = 7.4 Hz, 1H, CH SPh), 6.91 (t, J = 7.6 Hz, 2H, CH SPh),

4.92−4.90 (m, 1H, OH), 4.86 (d, J = 9.5 Hz, 1H, H1), 4.81 (d, J = 4.4

Hz, 1H OH), 4.69 (t, J = 5.2 Hz, 1H, OH), 4.52 (d, J = 4.7 Hz, 1H,

OH), 4.49 (d, J = 3.3 Hz, 1H, OH), 4.22 (d, J = 6.4 Hz, 1H, H1′),

3.90−3.82 (m, 1H, H6a), 3.80 (s, 3H, CH₃3MeOBz), 3.77−3.44 (m,

7H, H2, H3, H4′, H5, H6a′, H6′b, H5′), 3.42−3.22 (m, 4H, H2′,

H3′, H6b, H4), 1.84 (s, 3H, COCH₃); ¹³C{¹H} NMR (101 MHz,

DMSO-d₆) δ 170.3 (C_quat, COCH₃), 166.3 (C_quat, COPh), 159.6

(C_quat, 3MeO-C), 136.2 (C_quat, 3MeOBz), 135.5 (C_quat, SPh), 129.8

(CH, 3MeOBz), 129.6 (2CH, SPh), 129.1 (2CH, SPh), 126.7 (CH,

SPh), 120.0 (CH, 3MeOBz), 117.5 (CH, 3MeOBz), 113.0 (CH,

3MeOBz), 104.3 (C1′), 86.2 (C1), 85.5 (C3), 78.0 (C5), 76.2 (C5′),

73.6 (C3′), 71.5 (C4), 71.1 (C2′), 68.7 (C4′), 61.1 (C6′), 55.8 (CH₃,

3MeOBz), 53.5 (C2), 41.7 (C6), 23.5 (COCH₃); HRMS (ESI-TOF)

m/z [M + Na]⁺calcd for C₂₈H₃₆N₂O₁₁SNa 631.1938, found

631.1941.

NH(C(O)Ph)), 8.02 (d, J = 8.2 Hz, 1H, NH(Ac)), 7.47 (d, J = 8.0

Hz, 1H, Ph), 7.41 (br s, 1H, Ph), 7.33 (d, J = 7.7 Hz, 2H, CH SPh),

7.08 (t, J = 7.4 Hz, 1H, CH SPh), 7.03−6.93 (m, 3H, CH, SPh, Ph),

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-2,6-dideoxy-

6-(3,5-dimethoxybenzamido)-1-thio-β-^D-glucopyranoside 17.

J

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J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

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Article

6.11 (s, 2H, OCH₂O), 4.98−4.79 (m, 3H, 2 × OH, H1), 4.71 (br s,

1H, OH), 4.52−4.50 (m, 2H, 2 OH), 4.21 (d, J = 6.4 Hz, 1H, H1′),

3.82 (dd, J = 6.3, 12.8 Hz, 1H, H6a), 3.76−3.60 (m, 3H, H2, H3,

H4′), 3.60−3.44 (m, 4H, H5, H6′a, H6′b, H5′), 3.44−3.29 (m, 2H,

H2′, H3′), 3.29−3.18 (m, 2H, H4, H6b), 1.84 (s, 3H, COCH₃);

¹³C{¹H} NMR (101 MHz, DMSO-d₆) δ 170.4 (C_quat, COCH₃), 165.7

(C_quat, COPh), 150.1 (C_quat, COCH₂), 147.7 (C_quat, COCH₂), 135.4

CH 3MeOPh), 4.74 (d, J = 10.5 Hz, 1H, H1), 4.27 (d, J = 7.4 Hz, 1H,

H1′), 3.86 (s, 3H, CH₃3MeOPh), 3.82−3.73 (m, 3H, H2, H4′,

H6a′), 3.73−3.61 (m, 2H, H6b′, H3), 3.59−3.49 (m, 2H, H5′, H2′),

3.49−3.40 (m, 2H, H3′, H6a), 3.38−3.33 (m, 1H, H5), 3.22 (dd, J =

8.1, 9.4 Hz, 1H, H4), 3.02 (dd, J = 7.6, 13.8 Hz, 1H, H6b), 1.99 (s,

3H, COCH₃); ¹³C{¹H} NMR (101 MHz, CD₃OD) δ 172.6 (C_quat

,

COCH₃), 160.1 (C_quat, 3MeO-C), 141.8 (C_quat, 3MeOPh), 133.5

(C_quat, SPh), 131.5 (2CH, SPh), 129.9 (CH, 3MeOPh), 128.6 (2CH,

SPh), 127.2 (CH, SPh), 118.7 (CH, 3MeOPh), 118.2 (CH,

3MeOPh), 111.6 (CH, 3MeOPh), 104.2 (C1′), 86.4 (C1), 84.3

(C3), 78.4 (C5), 75.7 (C5′), 73.2 (C3′), 70.9 (C2′), 70.4 (C4), 68.8

(C4′), 61.1 (C6′), 54.8 (CH₃, 3MeOPh), 55.0 (C2), 44.2 (C6), 21.7

(COCH₃); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₂₇H₃₆N₂O₁₂S₂Na 667.1607, found 667.1614.

(C_quat, SPh), 129.6 (2CH, SPh), 129.1 (2CH, SPh), 128.8 (C_quat

,

CCONH), 126.8 (CH, SPh), 122.7 (CH, Ph), 108.2 (CH, Ph), 107.9

(CH, Ph), 104.3 (C1′), 102.1 (CH₂, OCH₂O), 86.1 (C1), 85.5 (C3),

78.0 (C5), 76.3 (C5), 73.6 (C3′), 71.5 (C4), 71.0 (C2′), 68.7 (C4′),

61.0 (C6′), 53.5 (C2), 41.3 (C6), 23.5 (COCH₃); HRMS (ESI-TOF)

m/z [M + Na]⁺calcd for C₂₈H₃₄N₂O₁₂SNa 645.1730, found 645.1738.

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-6-(methox-

yoxoacetamido)-2,6-dideoxy-1-thio-β-^D-glucopyranoside 20.

Amine 13 was dissolved in anhydrous DMF (2 mL) at 0 °C and

treated with methyl chlorooxoacetate (10 μL, 0.11 mmol, 1.1 equiv)

and Et₃N (15 μL, 0.11 mmol, 1.1 equiv). The mixture was stirred 12 h

and concentrated under reduced pressure. The residue was puriﬁed

ﬁrst by ﬂash chromatography (CH₂Cl₂/MeOH 7/3) then by

reversed-phase C-18 ﬂash chromatography using a H₂O/MeOH

gradient as eluent to give derivative 20 as a white solid (33 mg, 60%):

R_f0.75 (CHCl₃/MeOH/H₂O/CH₃COOH 60/30/5/3); ¹H NMR

(400 MHz, DMSO-d₆) δ 8.91 (t, J = 6.1 Hz, 1H, NH(C(O)-

CO₂Me)), 7.90 (d, J = 7.8 Hz, 1H, NHAc), 7.38−7.32 (m, 2H, CH

SPh), 7.25−7.21 (m, 3H, CH SPh), 4.90 (br s, 1H, OH), 4.85−4.80

(m, 2H, H1 and OH), 4.68 (t, J = 5.0 Hz, 1H, OH), 4.17 (d, J = 6.8

Hz, 1H, H1′), 3.79 (s, 3H, CH₃MeO), 3.72−3.44 (m, 8H, H3, H4′,

H6a, H2, H5′, H6′a, H6′b, H5), 3.34−3.22 (m, 3H, H2′, H3′, H6b),

3.22 (t, J = 8.6 Hz, 1H, H4), 1.83 (s, 3H, COCH₃); ¹³C{¹H} NMR

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-2,6-dideoxy-

6-(3-methoxyphenylureido)-1-thio-β-^D-glucopyranoside 23. Amine

13 was dissolved in anhydrous DMF (2 mL) at 0 °C and treated with

3-methoxyphenyl isocyanate (16 μL, 0.12 mmol, 1.2 equiv) and Et₃N

(17 μL, 0.12 mmol, 1.1 equiv). The mixture was stirred for 12 h and

concentrated under reduced pressure. The residue was ﬁrst puriﬁed

by ﬂash chromatography (CH₂Cl₂/MeOH 85/15) then by reversed-

phase C-18 ﬂash chromatography using a H₂O/MeOH gradient

mixture to give derivative 23 as a white solid (29 mg, 47%): R_f0.89

(CHCl₃/MeOH/H₂O/CH₃COOH 60/30/5/3); ¹H NMR (400

MHz, DMSO-d₆) δ 8.77 (s, 1H, NH(3MeOPh)), 7.99 (d, J = 8.0

Hz, 1H, NHAc), 7.44−7.38 (m, 2H, CH SPh), 7.26−7.20 (m, 2H,

CH SPh), 7.20−7.10 (m, 2H, CH SPh, 3MeOPh), 7.13 (dd, J = 5.7,

13.9 Hz,1H, CH 3MeOPh), 6.86 (dd, J = 1.9, 8.1 Hz, 1H, CH

3MeOPh), 6.48 (dd, J = 2.5, 8.2 Hz, 1H, CH 3MeOPh), 6.33 (t, J =

6.0 Hz, 1H, NHCONH), 4.94−4.77 (m, 3H, H1, 2 × OH), 4.76−

4.64 (m, 1H, OH), 4.52 (d, J = 11.8 Hz, 2H, 2 × OH), 4.20 (d, J = 5.2

Hz, 1H, H1′), 3.71 (s, 3H, CH₃3MeOPh), 3.68−3.58 (m, 4H, H2,

H3, H6a, H4′), 3.52 (d, J = 6.1 Hz, 2H, H6′a, H6′b), 3.49−3.28 (m,

4H, H5′, H5, H2′, H3′), 3.22 (t, J = 9.3 Hz, 1H, H4), 3.19−3.10 (m,

1H, H6b), 1.83 (s, 3H COCH₃); ¹³C{¹H} NMR (101 MHz, DMSO-

(101 MHz, DMSO-d₆) δ 170.3 (C_quat, COCH₃), 161.5 (C_quat

,

COOMe), 157.3 (C_quat, NHCOCO₂Me), 135.1 (C_quat, SPh), 129.9

(2CH, SPh), 129.3 (2CH, SPh), 127.0 (CH, SPh), 104.4 (C1′), 85.8

(C1), 85.5 (C3), 77.4, 76.3 (C5′, C5), 73.5 (C3′), 71.2 (C4), 71.0

(C2′), 68.7 (C4′), 61.1 (C6′), 53.3 (CH₃, MeO), 50.9 (C2), 41.4

(C6), 23.5 (COCH₃); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₂₃H₃₂N₂O₁₂SNa 583.1574, found 583.1582.

d₆) δ, 170.4 (C_quat, COCH₃), 160.1 (C_quat, 3MeO-C), 155.6 (C_quat

,

CONHPh), 142.3 (C_quat, 3MeOPh), 135.2 (C_quat, SPh), 130.0 (2CH,

SPh), 129.9 (CH, 3MeOPh), 129.4 (2CH, SPh), 127.0 (CH, SPh),

110.3 (CH, 3MeOPh), 106.9 (CH, 3MeOPh), 104.3 (C1′), 103.7

(CH, 3MeOPh), 86.2 (C1), 85.4 (C3), 78.6 (C5), 76.3 (C5′), 73.5

(C3′), 71.0 (C2′), 70.7 (C4), 68.7 (C4′), 61.0 (C6′), 55.3 (CH₃,

3MeOPh), 53.4 (C2), 41.1 (C6), 23.5 (COCH₃); HRMS (ESI-TOF)

m/z [M + Na]⁺calcd for C₂₈H₃₇N₃O₁₁SNa 646.2046, found 646.2043.

Phenyl β-^D-Galactopyranosyl-(1−3)-2-acetamido-6-N-(3-

methoxybenzyl)amino-2,6-dideoxy-1-thio-β-^D-glucopyranoside

24. Amine 13 was dissolved in anhydrous DMF (1 mL) and treated

with 3-methoxybenzaldehyde (7.3 μL, 0.06 mmol, 1.2 equiv) and

Et₃N (7 μL, 0.05 mmol, 1 equiv). The mixture was stirred for 12 h at

60 °C, and the reaction was monitored by TLC. When all starting

product was consumed, the mixture was cooled to rt and NaBH₄(2.3

mg, 0.06 mmol, 1.2 equiv) was added. After completion of the

reaction (2 h), the mixture was concentrated under reduced pressure.

The residue was ﬁrst puriﬁed by ﬂash chromatography (CH₂Cl₂/

MeOH 8/2) and then by reversed-phase C-18 ﬂash chromatography

using a H₂O/MeOH gradient mixture to give derivative 24 as a white

solid (16.7 mg, 56%): R_f0.76 (CHCl₃/MeOH/H₂O/CH₃COOH 60/

30/5/3); ¹H NMR (400 MHz, DMSO-d₆) δ 7.92 (d, J = 8.1 Hz, 1H,

NHAc), 7.44−7.38 (m, 2H, CH SPh), 7.29−7.17 (m, 4H, CH SPh,

3MeOPh), 6.88 (dd, J = 1.4, 2.8 Hz, 1H, CH 3MeOPh), 6.85 (dt, J =

1.2, 7.6 Hz, 1H, CH 3MeOPh), 6.80 (ddd, J = 1.0, 2.7, 8.3 Hz, 1H,

CH 3MeOPh), 4.95 (d, J = 9.5 Hz, 1H, H1), 4.85−4.72 (m, 2H,

NHCH₂, OH), 4.62 (t, J = 5.1 Hz, 1H, OH), 4.48 (d, J = 4.7 Hz, 1H,

OH), 4.46 (br s, 1H, OH), 4.17 (d, J = 7.1 Hz, 1H, H1′), 3.73 (s, 3H,

CH₃3MeOPh), 3.71−3.56 (m, 5H, NHCH₂, H2, H3, H4′), 3.55−

3.38 (m, 4H, H6′a, H6′b, H5′, H5), 3.38−3.28 (m, 2H, H2′, H3′),

3.22 (t, J = 8.8 Hz, 1H, H4), 2.92 (dd, J = 2.6, 12.7 Hz, 1H, H6a),

2.64 (dd, J = 7.8, 12.7 Hz, 1H, H6b), 1.83 (s, 3H COCH₃); ¹³C{¹H}

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-6-(carboxyox-

oacetamido-2,6-dideoxy-1-thio-β-^D-glucopyranoside 21. A solu-

tion of ester derivative 20 (15 mg, 0.027 mmol, 1 equiv) in a mixture

of aq. NaOH 2 M (1 mL) and MeOH (1 mL) was stirred 2 h at 0 °C.

Then the mixture was acidiﬁed with HCl 1 M to pH 1 and

concentrated under reduced pressure. The residue was puriﬁed by

reversed-phase C-18 ﬂash chromatography using a H₂O/MeOH

gradient mixture to give acid 21 as a colorless solid (14 mg, 95%); R_f

0.25 (CHCl₃/MeOH/H₂O/CH₃COOH 60/30/5/3); ¹H NMR (400

MHz, DMSO-d₆) δ 8.31 (br s, 1H, NH(C(O)CO₂Na)), 7.95 (d, J =

8.2 Hz, 1H, NHAc), 7.40 (d, J = 7.6 Hz, 2H, CH SPh), 7.31 (t, J = 7.5

Hz, 2H, CH SPh), 7.22 (t, J = 7.2 Hz, 1H, CH SPh), 4.92−4.83 (m,

2H, H1 and OH), 4.52 (br s, 1H, OH), 4.17 (d, J = 6.4 Hz, H1′),

3.73−3.55 (m, 4H, H3, H4′, H6a, H2), 3.55−3.41 (m, 4H, H5′,

H6′a, H6′b, H5), 3.40−3.28 (m, 2H, H2′, H3′), 3.26−3.14 (m, 2H,

H4, H6b), 1.83 (s, 3H, COCH₃); ¹³C{¹H} NMR (101 MHz, DMSO-

d₆) δ 170.3 (2C_quat, COCH₃, COONa), 165.9 (C_quat, NHCOCO₂Na),

135.0 (C_quat, SPh), 130.2 (2CH, SPh), 129.6 (2CH, SPh), 127.1 (CH,

SPh), 104.4 (C1′), 86.1 (C1), 85.3 (C3), 78.0 (C5), 76.3 (C5′), 73.4

(C3′), 71.0 (2C, C4 and C2′), 68.6 (C4′), 61.0 (C6′), 53.5 (C2),

41.1 (C6), 23.4 (COCH₃); HRMS (ESI-TOF) m/z [M - Na]⁻calcd

for C₂₂H₂₈N₂O₁₂S 545.1441, found 545.1437.

Phenyl β-^D-galactopyranosyl-(1→3)-2-acetamido-2,6-dideoxy-

6-(3-methoxyphenylsulfonamido)-1-thio-β-^D-glucopyranoside 22.

Amine 13 was dissolved in anhydrous DMF (2 mL) at 0 °C and

treated with 3-methoxyphenylsulfonyl chloride (24 μL, 0.17 mmol,

1.6 equiv) and Et₃N (24 μL, 0.17 mmol, 1.6 equiv). The mixture was

stirred 24 h at rt and concentrated under reduced pressure. The

residue was puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH 9/1)

to give derivative 22 as a white solid (14 mg, 21%): R_f0.70 (CHCl₃/

MeOH/H₂O/CH₃COOH 60/30/5/3); ¹H NMR (400 MHz,

CD₃OD) δ 7.55−7.61 (m, 2H, CH SPh), 7.45−7.39 (m, 3H, CH

3MeOPh), 7.37−7.28 (m, 3H, CH SPh), 7.16 (dt, J = 2.3, 7.4 Hz, 1H,

NMR (101 MHz, DMSO-d₆) δ, 170.4 (C_quat, COCH₃), 159.7 (C_quat

,

3MeO-C), 134.7 (C_quat, 3MeOPh), 130.4 (2CH, SPh), 129.6 (CH,

3MeOPh), 129.4 (2CH, SPh), 127.2 (CH, SPh), 120.5 (CH,

K

https://dx.doi.org/10.1021/acs.joc.0c01927

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

3MeOPh), 113.8 (CH, 3MeOPh), 112.6 (CH, 3MeOPh), 104.3

(C1′), 85.6 (C1), 85.5 (C3), 79.3 (C5), 76.3 (C5′), 73.5 (C3′), 71.0

(C2′), 70.9 (C4), 68.7 (C4′), 61.1 (C6′), 55.4 (CH₃, 3MeOPh), 53.6

(C2), 53.1 (CH₂, NHCH₂), 40.7 (C6), 23.5 (COCH₃); HRMS (ESI-

TOF) m/z [M + Na]⁺calcd for C₂₈H₃₉N₂O₁₀SNa 595.2325, found

595.2352.

(C4′), 61.1 (C6′), 55.6 (CH₃, 3MeOBz), 53.2 (C2), 51.6 (C6), 23.5

(COCH₃); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₂₉H₃₆N₄O₁₀SNa 655.2050, found 655.2053.

General Procedure for the Reduction of Azide Derivative

12. To a solution of disaccharide 12 (30 mg, 0.065 mmol, 1 equiv) in

THF (3 mL), under an atmosphere of argon, was added PMe₃1 M in

THF (130 μL, 0.13 mmol, 2 equiv). The reaction was stirred at rt and

monitored by TLC (CHCl₃/MeOH/H₂O/CH₃COOH 60/30/5/3).

After completion, H₂O (60 μL) was added, and the mixture was

heated to 60 °C in an oil bath for 16 h. Then solvent was removed

under reduced pressure to give crude phenyl β-^D-galactopyranosyl-

(1→3)-6-amino-6-deoxy-1-thio-β-^D-glucopyranoside 28, used without

further puriﬁcation: R_f0.60 (CHCl₃/MeOH/H₂O/CH₃COOH 60/

30/5/3).

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetamido-6-N,N′-di-(3-

methoxybenzyl)amino-2,6-dideoxy-1-thio-β-^D-glucopyranoside

25. Amine 13 was dissolved in anhydrous DMF (2 mL) and treated

with 3-methoxybenzaldehyde (29.0 μL, 0.24 mmol, 2.4 equiv) and

Et₃N (28 μL, 0.2 mmol, 2 equiv). The mixture was stirred for 12 h at

60 °C, and the reaction was monitored by TLC. When all starting

product was consumed, the mixture was cooled to rt, and then

NaBH₃CN (12.6 mg, 0.2 mmol, 2 equiv) and AcOH (11.4 μL, 0.2

mmol, 2 equiv) were added. After 2 h, the reaction mixture was

concentrated under reduced pressure. The residue was ﬁrst puriﬁed

by ﬂash chromatography (CH₂Cl₂/MeOH 8/2) and then by reversed-

phase C-18 ﬂash chromatography using a H₂O/MeOH gradient

mixture to give derivative 25 as a white solid (26.6 mg, 37%): R_f0.85

(CHCl₃/MeOH/H₂O/CH₃COOH 60/30/5/3); ¹H NMR (400

MHz, DMSO-d₆) δ 7.93 (d, J = 7.8 Hz, 1H, NHAc), 7.48−7.43

(m, 2H, CH SPh), 7.29−7.22 (m, 2H, CH SPh), 7.22−7.13 (m, 3H,

CH SPh, 3MeOPh), 6.90−6.84 (m, 4H, CH 3MeOPh), 6.79−6.73

(m, 2H, CH 3MeOPh), 5.06 (d, J = 8.6 Hz, 1H, H1), 4.83 (br s, 1H,

OH), 4.72 (s, 1H, OH), 4.63 (d, J = 7.6 Hz, 1H, OH), 4.54−4.51 (br

s, 2H, 2x OH), 4.18 (d, J = 6.4 Hz, 1H, H1′), 3.69 (s, 6H, 2 × CH₃

3MeOPh), 3.66−3.58 (m, 6H, N(CH₂)₂, H2, H3, H5, H4′), 3.53−

3.41 (m, 5H, N(CH₂)₂, H6′a, H6′b, H5′), 3.39−3.26 (m, 2H, H2′,

H3′), 3.16−3.08 (m, 1H, H4), 2.91 (d, J = 13.8 Hz, 1H, H6a), 2.58

(dd, J = 8.1, 14.1 Hz, H6b, 1H), 1.84 (s, 3H COCH₃); ¹³C{¹H} NMR

Phenyl β-^D-Galactopyranosyl-(1→3)-6-deoxy-6-(3-methoxyben-

zamido)-1-thio-β-^D-glucopyranoside 29. Amine 28 was dissolved in

anhydrous DMF (1.5 mL) at 0 °C and treated with 3-methoxybenzoyl

chloride (11 μL, 0.08 mmol, 1.2 equiv) and Et₃N (11 μL, 0.08 mmol,

1.2 equiv). The mixture was stirred for 12 h and concentrated under

reduced pressure. The residue was puriﬁed by ﬂash chromatography

(CH₂Cl₂/MeOH 9/1) then by reversed-phase C-18 ﬂash chromatog-

raphy using a H₂O/MeOH gradient mixture to give derivative 29 as a

white solid (21.2 mg, 57%): R_f0.42 (CH₂Cl₂/MeOH 9/1); ¹H NMR

(400 MHz, DMSO-d₆) δ 8.51 (t, J = 5.8 Hz, 1H, NH(3MeOBz)),

7.51−7.35 (m, 5H, 3 CH 3MeOBz, 2 CH SPh), 7.16−7.11 (m, 1H,

CH 3MeOBz), 7.08 (m, 1H, CH SPh), 7.00−6.93 (m, 2H, CH SPh),

5.49 (d, J = 4.6 Hz, 1H, OH), 5.01 (d, J = 3.3 Hz, 1H, OH), 4.97 (d, J

= 2.5 Hz, 1H, OH), 4.83 (d, J = 5.5 Hz, 1H, OH), 4.72−4.68 (m, 1H,

OH), 4.68 (d, J = 9.6 Hz, 1H, H1), 4.53 (d, J = 4.5 Hz, 1H, OH), 4.31

(d, J = 7.5 Hz, 1H, H1′), 3.87−3.80 (m, 1H, H6a), 3.80 (s, 3H, CH₃

3MeOBz), 3.64−3.54 (m, 2H, H4′, H5), 3.51 (m, 2H, H6a′, H6b′),

3.50−3.38 (m, 3H, H3, H5′, H2′), 3.38−3.29 (m, 2H, H3′, H2),

3.28−3.15 (m, 2H, H6b, H4); ¹³C{¹H} NMR (101 MHz, DMSO-d₆)

(101 MHz, DMSO-d₆) δ 170.4 (C_quat, COCH₃), 159.6 (2C_quat

,

3MeO-C), 141.5 (2C_quat, 3MeOPh), 134.9 (C_quat, SPh), 130.0 (2CH,

SPh), 129.5 (2CH, 3MeOPh), 129.4 (2CH, SPh), 127.0 (CH, SPh),

121.0 (2CH, 3MeOPh), 114.1 (2CH, 3MeOPh), 112.7 (2CH,

3MeOPh), 104.2 (C1′), 85.7 (C1), 85.4 (C3), 78.8 (C5), 76.2

(C5′), 73.5 (C3′), 71.0 (C2′), 71.0 (C4), 68.7 (C4′), 61.0 (C6′),

58.1 (2CH₂, N(CH₂)₂), 55.3 (2CH₃, 3MeOPh), 55.1 (C2), 40.7

(C6), 23.5 (COCH₃); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₃₆H₄₇N₂O₁₁SNa 715.2901, found 715.2868.

δ 166.3 (C_quat, COPh), 159.6 (C_quat, 3MeO-C), 136.2 (C_quat

,

3MeOBz), 134.9 (C_quat, SPh), 130.3 (CH, 3MeOBz), 129.8 (2CH,

SPh), 129.1 (2CH, SPh), 126.9 (CH, SPh), 120.0 (CH, 3MeOBz),

117.5 (CH, 3MeOBz), 113.0 (CH, 3MeOBz), 104.6 (C1′), 89.2

(C3), 86.6 (C1), 78.0 (C5), 76.1 (C5′), 73.3 (C3′), 71.6 and 71.5

(C2 and C2′), 71.1 (C4), 68.5 (C4′), 60.9 (C6′), 55.8 (CH₃,

3MeOBz), 41.7 (C6); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₂₆H₃₃NO₁₁SNa 590.1672, found 590.1671.

Phenyl β-^D-Galactopyranosyl-(1→3)-2-acetylamino-2,6-di-

deoxy-6-[4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl]-1-thio-β-^D-

glucopyranoside 26. To a solution of azide 11 (20 mg, 0.04 mmol)

in H₂O/^tBuOH (1/1 2 mL) were added 3-methoxyphenylacetylene

(11.4 μL, 0.08 mmol, 2 equiv), o-phenylenediamine (16 μL of a 375

mM solution in H₂O, 6 μmol, 0.15 equiv), sodium ascorbate (16 μL

of a 250 mM solution in H₂O, 4 μmol, 0.1 equiv), and copper sulfate

pentahydrate (16 μL of a 125 mM solution in H₂O, 2.0 μmol, 0.05

equiv). The reaction mixture was stirred at rt under an argon

atmosphere for 12 h, by which time TLC (cyclohexane/EtOAc 9/1)

showed complete conversion. Then activated charcoal was added to

the reaction mixture, which was stirred for 16 h. The reaction mixture

was then ﬁltered through a Celite plug and eluted with water, and the

solvent evaporated. The residue was puriﬁed by ﬂash chromatography

(CH₂Cl₂/MeOH 1/0 to 5/5) to give derivative 26 as a white solid

(6.7 mg, 23%): R_f0.52 (CH₂Cl₂/MeOH 85/15); ¹H NMR (400

MHz, DMSO-d₆) δ 8.51 (s, 1H, C = CH triazole), 7.95 (d, J = 8.0 Hz,

1H, NH(3MeOBz)), 7.44−7.34 (m, 3H, CH 3MeOBz), 7.05−7.00

(m, 2H, CH SPh), 6.99−6.90 (m, 4H, CH 3MeOBz, 3 CH SPh), 5.13

(br s, 1H, OH), 4.89−4.77 (m, 3H, H1, H6a, OH), 4.71 (t, J = 5.0

Hz, 1H, OH), 4.58−4.52 (m, 3H, H6b, 2 OH), 4.21 (d, J = 5.9 Hz,

1H, H1′), 3.89−3.82 (m, 1H, H5), 3.81 (s, 3H, CH₃3MeOBz),

3.74−3.64 (m, 2H, H2, H3), 3.62 (d, J = 3.7 Hz, 1H, H4′), 3.58−3.51

(m, 2H, H6a′, H6b′), 3.51−3.46 (m, 1H, H5′), 3.40−3.29 (m, 3H,

H2′, H3′, H4), 1.82 (s, 3H, COCH₃).; ¹³C{¹H} NMR (101 MHz,

DMSO-d₆) δ 170.4 (C_quat, COCH₃), 160.1 (C_quat, 3MeO-C), 146.6

(C=CH triazol), 134.6 (C_quat, 3MeOBz), 132.6 (C_quat, SPh), 130.5

(CH, 3MeOBz), 130.0 (2CH, SPh), 129.1 (2CH, SPh), 127.0 (CH,

SPh), 123.0 (C = CH triazol), 118.0 (CH, 3MeOBz), 114.0 (CH,

3MeOBz), 110.9 (CH, 3MeOBz), 104.5 (C1′), 86.1 (C1), 85.1 (C3),

78.2 (C5), 76.3 (C5′), 73.5 (C3′), 71.0 and 70.6 (C2′ and C4), 68.7

Phenyl β-^D-Galactopyranosyl-(1→3)-6-deoxy-6-(3-methoxyphe-

nylureido)-1-thio-β-^D-glucopyranoside 30. Amine 28 was dissolved

in anhydrous DMF (1.5 mL) at 0 °C and treated with 3-

methoxyphenyl isocyanate (10 μL, 0.08 mmol, 1.2 equiv) and Et₃N

(10 μL, 0.08 mmol, 1.2 equiv). The mixture was stirred for 12 h and

concentrated under reduced pressure. The residue was ﬁrst puriﬁed

by l-phase ﬂash chromatography (CH₂Cl₂/MeOH 9/1) then by

reversed-phase C-18 ﬂash chromatography using a H₂O/MeOH

gradient mixture to give derivative 30 as a white solid (24.3 mg,65%):

1

R_f0.40 (CH₂Cl₂/MeOH 9/1); H NMR (400 MHz, DMSO) δ 8.69

(s, 1H, NH(3MeOPh)), 7.53−7.44 (m, 2H, CH SPh), 7.32−7.07 (m,

5H, 3 CH SPh, 2 CH 3MeOPh), 6.90−6.82 (m, 1H, CH 3MeOPh),

6.52−6.44 (m, 1H, CH 3MeOPh), 6.22 (t, J = 6.2 Hz, 1H,

NHCONH), 5.44 (d, J = 4.5 Hz, 1H, OH), 4.98 (d, J = 3.3 Hz, 1H),

4.91 (br s, 1H, OH), 4.82 (d, J = 5.3 Hz, 1H, OH), 4.74 (d, J = 9.7

Hz, 1H, H1), 4.69 (t, J = 5.3 Hz, 1H, OH), 4.50 (d, J = 4.6 Hz, 1H,

OH), 4.28 (d, J = 7.4 Hz, 1H, H1′), 3.71 (s, 3H, CH₃3MeOPh),

3.69−3.57 (m, 2H, H4′, H6a), 3.52−3.50 (m, 2H, H6a′, H6b′),

3.49−3.39 (m, 4H, H5′, H3, H5, H2′), 3.39−3.25 (m, 2H, H3′, H2),

3.21−3.06 (m, 2H, H4, H6b); ¹³C{¹H} NMR (101 MHz, DMSO) δ

160.1 (C_quat, 3MeO-C), 155.5 (C_quat, CONHPh), 142.2 (C_quat

,

3MeOPh), 134.4 (C_quat, SPh), 130.9 (2CH, SPh), 129.9 (CH,

3MeOPh), 129.4 (2CH, SPh), 127.2 (CH, SPh), 110.3 (CH,

3MeOPh), 106.9 (CH, 3MeOPh), 105.1 (C1′), 103.7 (CH,

3MeOPh), 89.2 (C3), 86.5 (C1), 78.6 (C5), 76.1 (C5′), 73.3

(C3′), 71.5 and 71.3 (C2 and C2′), 70.3 (C4), 68.6 (C4′), 61.0

(C6′), 55.3 (CH₃, 3MeOPh), 40.9 (C6); HRMS (ESI-TOF) m/z [M

+ Na]⁺calcd for C₂₆H₃₄N₂O₁₁SNa 605.1781, found 605.1785.

L

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J. Org. Chem. XXXX, XXX, XXX−XXX

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Article

Phenyl β-D-Galactopyranosyl-(1→3)-6-deoxy-6-N-(3-methoxy-

benzyl)-1-thio-β-^D-glucopyranoside 31. Amine 28 was dissolved in

anhydrous DMF (1.5 mL) and treated with 3-methoxybenzaldehyde

(9.6 μL, 0.08 mmol, 1.2 equiv) and Et₃N (11 μL, 0.08 mmol, 1.2

equiv). The mixture was stirred for 12 h at 60 °C, and the reaction

was followed by TLC. When all starting product was consumed, the

mixture was cooled to rt and NaBH₄(5.9 mg, 0.16 mmol, 2.4 equiv)

was added. After 2 h, the reaction mixture was concentrated under

reduced pressure. The residue was ﬁrst puriﬁed by ﬂash chromatog-

raphy (CH₂Cl₂/MeOH 9/1) and then by reversed-phase C-18 ﬂash

chromatography using a H₂O/MeOH gradient mixture to give

derivative 31 as a white solid (22.1 mg, 61%): R_f0.75 (CHCl₃/

MeOH/H₂O/CH₃COOH 60/30/5/3); ¹H NMR (400 MHz,

CD₃OD) δ 7.56−7.50 (m, 2H, CH SPh), 7.29−7.22 (m, 4H, CH

SPh, 3MeOPh), 6.92−6.84 (m, 1H, 3H, CH 3MeOPh), 4.67 (d, J =

9.8 Hz, 1H, H1), 4.53 (d, J = 7.6 Hz, 1H, H1′), 3.86−3.73 (m, 7H,

H6a′, H4′ CH₃3MeOPh, NHCH₂), 3.69 (dd, J = 4.5, 11.4 Hz, 1H,

H6b′), 3.65−3.54 (m, 3H, H2′, H5′, H3), 3.5−3.45 (m, 2H, H3′,

H5), 3.43 (dd, J = 8.5, 9.9 Hz, 1H, H2), 3.26 (t, J = 9.2 Hz, 1H, H4),

3.11 (dd, J = 2.0, 12.9 Hz, 1H, H6a), 2.78 (dd, J = 8.6, 12.7 Hz, 1H,

H6b); ¹³C{¹H} NMR (101 MHz, CD₃OD) δ, 160.0 (C_quat, 3MeO-

C), 139.3 (C_quat, 3MeOPh), 132.6 (2CH, SPh), 132.5 (C_quat, SPh),

129.3 (CH, 3MeOPh), 128.6 (2CH, SPh), 127.5 (CH, SPh), 120.5

(CH, 3MeOPh), 113.8 (CH, 3MeOPh), 112.7 (CH, 3MeOPh), 104.2

(C1′), 87.3 (C3), 86.9 (C1), 77.6 (C5), 75.7 (C5′), 73.3 (C3′), 71.6

and 71.6 (C2 and C2′), 70.6 (C4), 68.9 (C4′), 61.2 (C6′), 54.3

(CH₃, 3MeOPh), 53.4 (C2), 52.3 (CH₂, NHCH₂), 49.6 (C6); HRMS

(ESI-TOF) m/z [M + H]⁺calcd for C₂₆H₃₆NO₁₀S 554.2060, found

554.2068.

Phenyl β-^D-Galactopyranosyl-(1→3)-6-deoxy-6-(3-methoxyphe-

nylsulfonamido)-1-thio-β-^D-glucopyranoside 32. Amine 28 was

dissolved in anhydrous DMF (1.5 mL) at 0 °C and treated with 3-

methoxyphenylsulfonyl chloride (12 μL, 0.08 mmol, 1.2 equiv) and

Et₃N (12 μL, 0.08 mmol, 1.2 equiv). The mixture was stirred for 8 h

at rt and concentrated under reduced pressure. The residue was

puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH 88/12) and then

by reversed-phase C-18 ﬂash chromatography using a H₂O/MeOH

gradient mixture to give derivative 32 as a white solid (27.8 mg, 70%):

R_f0.48 (CH₂Cl₂/MeOH 9/1); ¹H NMR (400 MHz, CD₃OD) δ

7.59−7.55 (m, 2H, CH SPh), 7.47−7.39 (m, 3H, CH 3MeOPh),

7.37−7.29 (m, 3H, CH SPh), 7.17 (dt, J = 2.4, 7.2 Hz, 1H, CH

3MeOPh), 4.51 (d, J = 7.5 Hz, 1H, H1′), 4.48 (d, J = 9.89 Hz, 1H,

H1), 3.86 (s, 3H, CH₃3MeOPh), 3.80 (dd, J = 1.0, 3.4 Hz, 1H, H4′),

3.77 (dd, J = 7.2, 10.9 Hz, 1H, H6a′), 3.69 (dd, J = 4.4, 11.5 Hz, 1H,

H6b′), 3.61−3.47 (m, 4H, H2′, H5′, H3, H3′), 3.42 (dd, J = 2.6, 13.8

Hz, 1H, H6a), 3.38−3.29 (m, 2H, H2, H5), 3.21 (dd, J = 8.6, 9.7 Hz,

1H, H4), 2.99 (dd, J = 7.7, 13.8 Hz, 1H, H6b); ¹³C{¹H} NMR (101

MHz, CD₃OD) δ 160.1 (C_quat, 3MeO-C), 141.8 (C_quat, 3MeOPh),

132.9 (C_quat, SPh), 132.1 (2CH, SPh),130.0 (CH, 3MeOPh), 128.6

(2CH, SPh), 127.3 (CH, SPh), 118.7 (CH, 3MeOPh), 118.2 (CH,

3MeOPh), 111.5 (CH, 3MeOPh), 104.2 (C1′), 87.3 (C3), 87.1 (C1),

78.2 (C5), 75.7 (C5′), 73.3 (C3′), 71.6 and 71.3 (C2 and C2′), 70.0

(C4), 68.9 (C4′), 61.2 (C6′), 54.8 (CH₃, 3MeOPh), 44.2 (C6);

HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₅H₃₃NO₁₂S₂

626.1342, found 626.1337.

H6a′), 3.70 (dd, J = 4.4, 11.5 Hz, 1H, H6b′), 3.61−3.55 (m, 2H, H2′,

H5′), 3.55−3.49 (m, 2H, H3, H3′), 3.45 (dd, J = 2.5, 13.9 Hz, 1H,

H6a), 3.37 (dd, J = 8.6, 9.8 Hz, 1H, H2), 3.32−3.25 (m, 1H, H5),

3.23 (dd, J = 8.3, 9.7 Hz, 1H, H4), 3.02 (dd, J = 7.4, 13.8 Hz, 1H,

H6b); ¹³C{¹H} NMR (101 MHz, CD₃OD) δ 161.3 and 155.4 (2C,

2C_quat, PhenoxyPh), 134.5 (1C, 1C_quat, PhenoxyPh), 132.9 (C_quat

,

SPh), 132.3 (2CH, SPh), 129.2 (2CH, PhenoxyPh), 128.9 (2CH,

PhenoxyPh), 128.6 (2CH, SPh), 127.4 (2CH, SPh), 124.6 (CH,

PhenoxyPh), 119.9 (2CH, PhenoxyPh), 117.3 (2CH, PhenoxyPh),

1074.2 (C1′), 87.4 (C3), 87.2 (C1), 78.0 (C5), 75.7 (C5′), 73.3

(C3′), 71.6 and 71.4 (C2′ and C2), 70.0 (C4), 68.9 (C4′), 61.2

(C6′), 44.2 (C6); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₃₀H₃₅NO₁₂S₂Na 688.1498, found 688.1515.

Phenyl β-^D-Galactopyranosyl-(1→3)-6-deoxy-6-[4-(3-methoxy-

phenyl)-1H-1,2,3-triazol-1-yl]-1-thio-β-^D-glucopyranoside 34. To a

solution of azide 12 (30 mg, 65 μmol) in DMF (5 mL) were added 3-

methoxyphenylacetylene (27.9 μL, 0.20 mmol, 3 equiv), copper

iodide (1.24 mg, 6.5 μmol, 0.1 equiv), and Et₃N (9.1 μL, 65 μmol, 1

equiv). The reaction mixture was stirred at rt under an argon

atmosphere for 12 h. After completion, the mixture was concentrated

under reduced pressure and puriﬁed by ﬂash chromatography

(CH₂Cl₂/MeOH 1/0 to 9/1) to give derivative 34 as a white solid

(13.9 mg, 36%): R_f0.31 (CH₂Cl₂/MeOH 9/1); ¹H NMR (400 MHz,

CD₃OD) δ 8.22 (s, 1H, C = CH triazol), 7.40−7.32 (m, 3H, CH

3MeOBz), 7.28−7.20 (m, 2H, CH SPh), 7.05−6.97 (m, 3H, CH

SPh), 6.97−6.93 (m, 1H, CH 3MeOBz), 4.96 (dd, J = 2.4, 14.4 Hz,

1H, H6a), 4.64 (d, J = 9.9 Hz, 1H, H1), 4.62−4.54 (m, 2H, H6b and

H1′), 3.87 (s, 3H, CH₃3MeOBz), 3.87−3.78 (m, 3H, H6a′, H4′,

H5), 3.74 (dd, J = 4.4, 11.4 Hz, 1H, H6b′), 3.68 (t, J = 8.7 Hz, 1H,

H3), 3.65−3.60 (m, 2H, H2′, H5′), 3.54 (dd, J = 3.4, 9.7 Hz, 1H,

H3′), 3.44 (dd, J = 8.7, 9.9 Hz, 1H, H2), 3.40−3.34 (m, 1H, H4);

¹³C{¹H} NMR (101 MHz, CD₃OD) δ 160.2 (C_quat, 3MeO-C), 147.2

(C=CH triazol), 132.6 (C_quat, 3MeOBz), 131.5 (C_quat, SPh), 131.5

(2CH, SPh), 129.7 (CH, 3MeOBz), 128.5 (2CH, SPh), 127.1 (CH,

SPh), 122.4 (C = CH triazol), 117.8 (CH, 3MeOBz), 113.7 (CH,

3MeOBz), 110.7 (CH, 3MeOBz), 104.3 (C1′), 87.2 (C1 and C3),

77.8 (C5), 75.7 (C5′), 73.3 (C3′), 71.6 and 71.5 (C2′ and C2), 69.9

(C4), 68.9 (C4′), 61.2 (C6′), 54.5 (CH₃, 3MeOBz), 51.4 (C6);

HRMS (ESI-TOF) m/z [M + Na]⁺calcd for C₂₇H₃₃N₃O₁₀SNa

614.1784, found 614.1803.

Phenyl β-^D-Galactopyranosyl-(1→3)-6-deoxy-6-(4-phenyl-1H-

1,2,3-triazol-1-yl)-1-thio-β-^D-glucopyranoside 35. To a solution of

azide 12 (30 mg, 65 μmol) in DMF (1 mL) were added

phenylacetylene (28.0 μL, 0.20 mmol, 3 equiv), copper iodide

(12.45 mg, 65.4 μmol, 1 equiv), and Et₃N (10 μL, 72 μmol, 1.1

equiv). The reaction mixture was stirred at rt under an argon

atmosphere for 12 h. After completion the mixture was concentrated

under reduced pressure and puriﬁed by ﬂash chromatography

(CH₂Cl₂/MeOH 1/0 to 9/1) and then by reversed-phase C-18

ﬂash chromatography using a H₂O/MeOH gradient mixture to give

derivative 35 as a white solid (30 mg, 85%): R_f0.18 (CH₂Cl₂/MeOH

9/1); ¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (s, 1H, C = CH

triazol), 7.82−7.79 (m, 2H, CH Ph), 7.46 (t, J = 7.6 Hz, 2H, CH Ph),

7.35 (t, J = 7.4 Hz, 1H, CH Ph), 7.17−7.12 (m, 2H, CH SPh), 7.00−

6.95 (m, 3H, CH SPh), 5.51 (d, J = 4.7 Hz, 1H, OH), 5.18 (br s, 1H,

OH), 4.98 (br s, 1H, OH), 4.84−4.74 (m, 3H, 2 OH, H6a and H1),

4.69 (t, J = 5.2 Hz, 1H, OH), 4.54−4.47 (m, 2H, OH, H6b), 4.31 (d,

J = 7.4 Hz, 1H, H1′), 3.90−3.84 (m, 1H, H5), 3.64−3.62 (m, 1H,

H4′), 3.59−3.46 (m, 4H, H6a′, H6b′, H3, H5′), 3.46−3.35 (m, 2H,

H2′, H3′), 3.35−3.22 (m, 2H, H2, H4); ¹³C{¹H} NMR (101 MHz,

Phenyl β-^D-Galactopyranosyl-(1→3)-6-deoxy-6-[(4-phenoxy)-

phenylsulfonamido]-1-thio-β-^D-glucopyranoside 33. Amine 28

was dissolved in anhydrous DMF (5 mL) at 0 °C and treated with

4-phenoxyphenylsulfonyl chloride (35 mg, 0.13 mmol, 1.2 equiv) and

Et₃N (18 μL, 0.13 mmol, 1.2 equiv). The mixture was stirred for 8 h

at rt and concentrated under reduced pressure. The residue was

puriﬁed by ﬂash chromatography (CH₂Cl₂/MeOH 93/7) and then by

reversed-phase C-18 ﬂash chromatography using a H₂O/MeOH

gradient mixture to give derivative 33 as a white solid (38.9 mg, 54%):

R_f0.31 (CH₂Cl₂/MeOH 9/1); ¹H NMR (400 MHz, CD₃OD) δ

7.83−7.77 (m, 2H, CH PhenoxyPh), 7.58−7.56 (m, 2H, CH SPh),

7.47−7.42 (m, 2H, CH PhenoxyPh), 7.35−7.27 (m, 3H, CH SPh),

7.27−7.22 (m, 1H, CH PhenoxyPh), 7.12−7.07 (m, 2H, CH

PhenoxyPh), 7.03−6.98 (m, 2H, CH PhenoxyPh), 4.51 (d, J = 7.6

Hz, 1H, H1′), 4.47 (d, J = 9.8 Hz, 1H, H1), 3.81−3.76 (m, 2H, H4′,

DMSO-d₆) δ 146.6 (C=CH triazol), 133.9 (C_quat, SPh), 131.3 (C_quat

,

Ph), 130.8 (2CH, SPh), 129.3 and 129.1 (2 × 2CH, SPh and Ph),

128.3 (CH, Ph), 127.1 (CH, SPh), 125.6 (2CH, Ph), 122.7 (C = CH

triazol), 105.2 (C1′), 88.9 (C3), 86.3 (C1), 77.9 (C5), 76.2 (C5′),

73.3 (C3′), 71.5 and 71.3 (C2′ and C2), 70.2 (C4), 68.6 (C4′), 61.0

(C6′), 51.5 (C6); HRMS (ESI-TOF) m/z [M + Na]⁺calcd for

C₂₆H₃₁N₃O₉SNa 584.1679, found 584.1684.

Phenyl β-^D-Galactopyranosyl-(1→3)-6-deoxy-6-[4-(4-phenoxy-

phenyl)-1H-1,2,3-triazol-1-yl]-1-thio-β-^D-glucopyranoside 36. To a

solution of azide 12 (30 mg, 65 μmol) in DMF (1 mL) were added 4-

M

https://dx.doi.org/10.1021/acs.joc.0c01927

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

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Article

phenoxyphenylacetylene (23.7 μL, 0.13 mmol, 2 equiv), copper

iodide (12.45 mg, 65.4 μmol, 1 equiv), and Et₃N (10 μL, 72 μmol, 1.1

equiv). The reaction mixture was stirred at rt under an argon

atmosphere for 12 h. After completion, the mixture was concentrated

under reduced pressure and puriﬁed by normal phase ﬂash-

chromatography (DCM/MeOH 1/0 to 9/1) and then by reversed-

phase C-18 ﬂash chromatography using a H₂O/MeOH gradient

mixture to give derivative 36 as a white solid (26.7 mg, 63%): R_f0.18

Authors

Christophe Dussouy − Université de Nantes, CNRS, Unité

Fonctionnalité et Ingénierie des Protéines (UFIP), UMR 628,

F-44000 Nantes, France

́

Stephane Teletchea − Université de Nantes, CNRS, Unité

Fonctionnalité et Ingénierie des Protéines (UFIP), UMR 628,

F-44000 Nantes, France

Annie Lambert − Université de Nantes, CNRS, Unité

Fonctionnalité et Ingénierie des Protéines (UFIP), UMR 628,

F-44000 Nantes, France

Cathy Charlier − Université de Nantes, CNRS, Unité

Fonctionnalité et Ingénierie des Protéines (UFIP), UMR 628,

F-44000 Nantes, France; Université de Nantes, CNRS,

Plateforme IMPACT, UMR 6286, F-44000 Nantes, France

Iuliana Botez − Institut de Recherches Servier, Croissy-sur-

Seine, 78290 Croissy, France

1

(CH₂Cl₂/MeOH 9/1); H NMR (400 MHz, DMSO-d₆) δ 8.33 (s,

1H, C = CH triazol), 7.84−7.77 (m, 2H, CH PhenoxyPh), 7.45−7.38

(m, 2H, CH PhenoxyPh), 7.22−7.12 (m, 3H, CH SPh), 7.12−7.03

(m, 4H, CH PhenoxyPh), 7.03−6.96 (m, 3H, CH PhenoxyPh, SPh),

5.60 (br s, 1H, OH), 5.21 (br s, 1H, OH), 4.83−4.71 (m, 1H, H6a),

4.74 (d, J = 9.8 Hz, 1H, H1), 4.50 (dd, J = 8.8, 14.4 Hz, 1H, H6b),

4.32 (d, J = 7.4 Hz, 1H, H1′), 3.86 (ddd, J = 1.9, 6.0, 11.1 Hz, 1H,

H5), 3.64 (dd, J = 1.3, 3.2 Hz, 1H, H4′), 3.58−3.47 (m, 4H, H6a′,

H6b′, H5′, H3), 3.47−3.34 (m, 2H, H2′, H3′), 3.34−3.23 (m, 2H,

H2, H4); ¹³C{¹H} NMR (101 MHz, DMSO-d₆) δ 157.1 and 156.7

(2C_quat, PhenoxyPh), 146.2 (C=CH triazol), 134.0 (C_quat, SPh), 130.7

(2CH, SPh), 130.6 (2CH, PhenoxyPh), 129.1 (2CH, SPh), 127.4

(2CH, PhenoxyPh), 127.1 (CH, PhenoxyPh), 126.7 (C_quat, Phenox-

yPh), 124.0 (CH, SPh), 122.4 (C = CH triazol), 119.5 and 119.1

4CH, PhenoxyPh), 105.2 (C1′), 89.0 (C3), 86.4 (C1), 77.9 (C5),

76.2 (C5′), 73.3 (C3′), 71.5 and 71.3 (C2′ and C2), 70.2 (C4), 68.6

(C4′), 61.0 (C6′), 51.5 (C6); HRMS (ESI-TOF) m/z [M + Na]⁺

calcd for C₃₂H₃₅N₃O₁₀SNa 676.1941, found 676.1918.

́

Frederic De Ceuninck − Institut de Recherches Servier,

Croissy-sur-Seine, 78290 Croissy, France

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.joc.0c01927

Notes

The authors declare no competing ﬁnancial interest.

Molecular Modeling. Lactosamine derivatives were built using

ACKNOWLEDGMENTS

■

̀

Discovery Studio 4.1, Dassault Systemes BIOVIA, San Diego or from

the Glycam Web Server.⁴⁶The docking experiments were performed

using the CDOCKER protocol⁴⁷as previously described.^30,29Brieﬂy,

the lactosamine binding domain of the 3ZSJ structure⁴⁸was deﬁned

as the center of a 18 Å sphere where the ligand could freely explore

the protein hypersurface. The protocol has a two-step procedure: the

ﬁrst phase involves a rapid ligand−protein evaluation with limited

precision for interaction energy evaluation, and the second phase

involves a full molecular dynamics simulation using the CHARMm

force ﬁeld. During the ﬁrst phase, 250 binding modes were evaluated,

25 poses were reﬁned in the second full potential phase, and the 10

best poses were kept. The best pose for each compound was selected

as presenting the best binding interaction energy in the CDOCKER

protocol and the lowest deviation of the galactose moiety from the

location found in the original crystal structure. The root-mean-square

deviation of the galactose location compared to the crystallographic

galactose was assessed using the Find Most Common Sub-Structure

implemented in rdkit.⁴⁹Poses analysis and illustrations were

performed in Discovery Studio and PyMol.⁵⁰Detailed ligand−

receptor interaction analysis were done with LigPlot+.⁵¹

Les Laboratoires Servier are acknowledged for a postdoctoral

fellowship to C.D. and research funding. We also thank the

mass spectrometry core facility of CRNH for exact mass

determinations.

REFERENCES

■

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* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.joc.0c01927.

(6) Bull, C.; Heise, T.; van Hilten, N.; Pijnenborg, J. F. A.;

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Interactions on Living Cells Using Bioorthogonal Chemistry. Angew.

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Experimental procedures for compound 1 precursors,

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■

Corresponding Author

Cyrille Grandjean − Université de Nantes, CNRS, Unité

Fonctionnalité et Ingénierie des Protéines (UFIP), UMR 628,

F-44000 Nantes, France; Email: cyrille.grandjean@univ-

nantes.fr

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