Modular Synthesis of Heparan Sulfate Oligosaccharides Having N-Acetyl and N-Sulfate Moieties

DOI: 10.1021/acs.joc.0c01881

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Journal of Organic Chemistry p. 16082 - 16098 (2020)

Update date:2022-08-02

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Authors:

Sun, Lifeng

Chopra, Pradeep

Boons, Geert-Jan

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Article abstract of DOI:10.1021/acs.joc.0c01881

Heparan sulfates are structurally diverse sulfated polysaccharides that reside at the surface of all animal cells where they can interact with a multitude of proteins, thereby modulating a wide range of physiological and disease processes. We describe here a modular synthetic methodology that can provide libraries of heparan sulfate oligosaccharides that have glucosamine residues modified by different patterns of N-acetyl and N-sulfate moieties. It is based on the use of glycosyl donors that are modified at C2 by an azido- or trifluoromethylphenyl-methanimine moiety, which allowed the selective installation of α-glycosides. The amino protecting groups can be selectively unmasked by a reduction or acid treatment, allowing the installation of N-acetyl and N-sulfate moieties, respectively. In combination with the orthogonal hydroxyl protecting groups levulinic (Lev) ester, thexyldimethylsilyl (TDS) ether, allyloxycarbonate (Alloc), and 9-fluorenylmethyl carbonate (Fmoc), different patterns of O-sulfation can be installed. The methodology was applied to prepare four hexasaccharides that differ in the pattern of N- and O-sulfation. These compounds, together with a number of previously prepared HS oligosaccharides, were printed as a glycan microarray to examine the binding selectivities of several HS-binding proteins.

Full text of DOI:10.1021/acs.joc.0c01881

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Article

Modular Synthesis of Heparan Sulfate Oligosaccharides Having

N‑Acetyl and N‑Sulfate Moieties

Lifeng Sun, Pradeep Chopra, and Geert-Jan Boons*

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ABSTRACT: Heparan sulfates are structurally diverse sulfated polysaccharides

that reside at the surface of all animal cells where they can interact with a

multitude of proteins, thereby modulating a wide range of physiological and

disease processes. We describe here a modular synthetic methodology that can

provide libraries of heparan sulfate oligosaccharides that have glucosamine

residues modiﬁed by diﬀerent patterns of N-acetyl and N-sulfate moieties. It is

based on the use of glycosyl donors that are modiﬁed at C2 by an azido- or

triﬂuoromethylphenyl-methanimine moiety, which allowed the selective installa-

tion of α-glycosides. The amino protecting groups can be selectively unmasked by

a reduction or acid treatment, allowing the installation of N-acetyl and N-sulfate

moieties, respectively. In combination with the orthogonal hydroxyl protecting

groups levulinic (Lev) ester, thexyldimethylsilyl (TDS) ether, allyloxycarbonate

(Alloc), and 9-ﬂuorenylmethyl carbonate (Fmoc), diﬀerent patterns of O-sulfation can be installed. The methodology was applied to

prepare four hexasaccharides that diﬀer in the pattern of N- and O-sulfation. These compounds, together with a number of

previously prepared HS oligosaccharides, were printed as a glycan microarray to examine the binding selectivities of several HS-

binding proteins.

relationship studies have employed small numbers of HS

oligosaccharides that cannot properly probe the ligand

selectivities of HS-binding proteins.^{11,20,37−44}

INTRODUCTION

■

Heparan sulfates (HSs) are highly sulfated polysaccharides that

reside on the surface and in the extracellular matrix of virtually

all cells of multicelluar organisms.¹A large number of

endogenous proteins recognize HS, resulting in conformational

changes, the stabilization of receptor−ligand complexes,

protein oligomerization, sequestration, and protection against

degradation. It results in the regulation of many physiological

processes, including embryogenesis, angiogenesis, blood

coagulation, and inﬂammation. The binding of proteins to

HS has also been implicated in many disease processes,

including cancer, viral and bacterial infections, neurological

disorders, and several genetic diseases.¹⁻⁶

A lack of large collections of well-deﬁned HS oligosacchar-

ides is a major hurdle in regard to advancing the understanding

of the biology of HS-binding proteins.⁷⁻⁹Such compounds are

needed to establish the ligand requirements of HS-binding

proteins, determine the substrate speciﬁcities of HS bio-

synthetic enzymes, and develop methods for HS structure

determination. Due to the structural diversity of HS, well-

deﬁned compounds cannot be obtained from natural sources.

Several laboratories have successfully prepared HS oligosac-

charides¹⁰⁻²⁵and, although elegant, these approaches are

mainly focused on the preparation of compounds composed of

the same repeating unit. The enzyme-mediated synthesis of HS

oligosaccharides requires signiﬁcantly fewer steps but cannot

provide a wide variety of structures due to the promiscuity of

the biosynthetic enzymes.²⁶⁻³⁶As a result, structure−activity

Previously, we described a modular synthetic approach that

can provide a wide range of HS oligosaccharides.^12,45−49It

employs common disaccharide building blocks (1 and 2,

Figure 1) that can easily be converted to glycosyl donors and

acceptors for the parallel combinatorial synthesis of HS

oligosaccharides. The anomeric center of the building blocks

is protected as thexyldimethylsilyl (TDS) ether, which can be

removed by treatment with HF, and the resulting lactol can

then be converted to a trichloroacetimidate to give glycosyl

donors. The C4′ hydroxyl of the modular disaccharide building

blocks is protected as 9-ﬂuorenylmethyl carbonate (Fmoc),

which can selectively be cleaved by a hindered base, such as

triethylamine, to give a glycosyl acceptor. Speciﬁc hydroxyls of

the building blocks are protected as Lev esters, which can be

removed after the oligosaccharide assembly to reveal alcohols

for sulfation. The C2 of the glucosamine (GlcN) donors is

masked as an azido moiety, which facilitates the installation of

Special Issue: A New Era of Discovery in Carbohy-

drate Chemistry

Received: August 4, 2020

https://dx.doi.org/10.1021/acs.joc.0c01881

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Figure 1. Modular building blocks.

Scheme 1. Synthesis of Disaccharide Building Blocks

(a) NIS, TMSOTf, DCM, 4 Å MS, 0 °C, 30 min: 8, 85%; 9, 89%. (b) (i) DCM/TFA/H₂O, 30 min; (ii) TEMPO/BAIB, DCM/H₂O, 6 h; and

(iii) TMSCHN₂, MeOH/toluene, 15 min: 10, 61% over three steps; 11, 56% over three steps. (c) AllocCl, TMEDA, DCM, 10 h: 12, 88%; 13,

91%. (d) FmocCl, Py: 18, 89%; 19, 92%. (e) HF·Py, THF: 14, 92%; 16, 88%. (f) PMe₃, THF, H₂O; 2-triﬂuoromethyl benzaldehyde, DCM/Py, 50

°C: 17, 64% over two steps. (g) 2,2,2-Triﬂuoro-N-phenylacetimidoyl chloride, K₂CO₃, DCM: 3, 46% over three steps; 4, 90%; 20, 81% over two

steps; 21, 84% over two steps.

1,2-cis-glycosides because it does not perform neighboring-

group participation during glycosylations. The end stage of the

synthesis involves the reduction of the azide to an amine,

which can then be acetylated or sulfated. Thus, a limitation of

our current approach is that it does not allow the preparation

of HS oligosaccharides that have a combination of GlcNS and

GlcNAc residues. This shortcoming urgently needs to be

addressed because HS has a domain structure in which regions

that are extensively modiﬁed and highly sulfated are ﬂanked by

domains of low or no sulfation and contain GlcNAc residues. It

has been suggested that the transition regions of HS are

important sites for selective protein binding. As a step toward

the synthesis of compounds having a domain structure, it is

critical to be able to diﬀerentiate amines of GlcN residues of

HS precursor oligosaccharides.

We describe here a new set of modular disaccharide building

blocks (3 and 4, Figure 1) that are modiﬁed at C2 by a

triﬂuoromethylphenyl-methanimine moiety to make it possible

to prepare HS oligosaccharides with GlcNS and GlcNAc

moieties. Previous studies have shown that the nickel triﬂate-

promoted activation of glycosyl donors having triﬂuorome-

thylphenyl-methanimine at C2 results in the selective

formation of α-glycosides.⁵⁰It has been proposed that these

glycosylations proceed through a transition state in which the

nickel catalyst chelates the N-phenyl triﬂuoracetimidate at the

anomeric center and the methanimine protecting group at C2,

thereby controlling the anomeric outcome of the glycosylation.

The methanimine-protecting group can be cleaved under mild

acidic conditions to reveal an amine that can be acetylated or

modiﬁed by another derivative, such as a sulfate. We

anticipated that triﬂuoromethylphenyl-methanimine and

azido functions can selectively be converted to an amine,

allowing the installation of GlcNS and GlcNAc moieties.

RESULTS AND DISCUSSION

■

To establish the methodology, we prepared disaccharide

donors 3 and 4, which, in combination with previously

developed modular disaccharides,^12,51,52were employed for the

synthesis of a series of HS tetra- and hexasaccharides. To

minimize the synthetic eﬀorts, we set out to prepare the

modular disaccharides from existing building blocks by

reducing the azido moiety to give an amine, which can then

be protected as a triﬂuoromethylphenyl-methanimine.

First, we focused on the preparation of the conventional

disaccharide building blocks 10 and 11 (Scheme 1). Thus, an

NIS/TMSOTf-mediated glycosylation⁵³of thioglycosyl donor

5 with acceptors 6 and 7 give disaccharides 8 and 9,

respectively. These compounds were treated with a mixture

of DCM/TFA/H₂O to hydrolyze the benzylidene acetal to

give a 4,6-diol that was selectively oxidized using 2,2,6,6-

tetramethyl-1-piperidinyloxy (TEMPO)/(diacetoxyiodo)-

benzene (BAIB)^54,55to give carboxylic acids that were

protected as methyl esters using trimethylsilyldiazomethane

(TMSCHN₂), providing target disaccharides 10 and 11,

respectively. The treatment of 10 and 11 with FmocCl

resulted in the protection of the C4′ hydroxyl to provide 18

and 19, respectively, which were converted to donors 20 and

21 ﬁrst by the removal of the anomeric TDS ether using HF·

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pyridine followed by a reaction with 2,2,2-triﬂuoro-N-phenyl-

acetimidoyl chloride in the presence of K₂CO₃.⁵⁶

used. The failure of the glycosylation reactions is probably due

to the low reactivity of the disaccharide donor and acceptor.

Coupling of 4 with 22 using 0.5 equiv of triﬂic acid as the

promoter in the presence of 4 Å molecular sieves at −40 °C for

1 h resulted in the formation of desired tetrasaccharide 23 but

in a low yield of 19% (entry 3). Employing a stochiometric

amount of triﬂic acid did not improve the yield of the

glycosylation (entry 4). Increasing the concentration from 50

mM to 150 mM gave a slightly higher yield of 24% (entry 5);

however, considerable amounts of glycosyl donor 4 and

glycosyl acceptor 22 were recovered (entries 3−5). Tetra-

saccharide 23 was isolated in an acceptable yield of 58% as

only the α-anomer when the glycosylation was performed at a

higher temperature of −20 °C for 12 h (entry 6). A higher

temperature and prolonged reaction time were required to

consume most of the donor. For each glycosylation, only the

α-anomer was observed and isolated.

Next, attention was focused on the conversion of fully

protected 23 to HS tetrasaccharide 28, having GlcNAc and

GlcNS moieties (Scheme 2). The Alloc protecting group of 23

was selectively removed using Pd(PPh₃)₄, which did not aﬀect

the 2-CF₃−Ph−CHN moiety, providing intermediate 24 in

good yield. The 2-N-[(2-triﬂuoromethyl)benzylidene of 24

was removed by a treatment with hydrochloric acid in THF for

15 min, which resulted in the selective formation of a free

amine without aﬀecting other functionalities. The resulting

intermediate was immediately N- and O-acetylated using acetic

anhydride in pyridine to aﬀord 25. The latter compound was

treated with hydrazine acetate to cleave the Lev esters, which

was followed by the O-sulfation of the resulting hydroxyls

using a sulfur trioxide pyridine complex. Saponiﬁcation and de-

O-acetylation of the O-sulfated product using a mixture of

lithium hydroxide and hydrogen peroxide generated 26 in a

yield of 60%. The azido moiety of 26 was reduced by PMe₃in

the presence of NaOH, resulting in the formation of a free

amine that was immediately sulfated by adding portions of

SO₃·Py every 2 h for 8 h to give 27 in 66% yield over two steps.

Finally, the hydrogenation of the benzyl ethers over Pd(OH)₂/

C gave the target compound 28, which was puriﬁed by size

exclusion chromatography over Biogel P2, followed by an

exchange to a sodium salt by passing it over a Dowex Na⁺

resin.

Next, attempts were made to reduce the azide of 12 and 13

to an amine for protection as triﬂuoromethylphenyl-meth-

animine. Although several conditions were examined, in each

case the cleavage of the Fmoc protecting group was observed.

Therefore, we opted to protect the C4' hydroxyl of 10 and 11

as an allyloxycarbonate (Alloc) because it was expected that

this protecting group would be more stable and compatible

with azide reduction and could be cleaved at a late stage of

synthesis using a Pd catalyst without aﬀecting other sensitive

functional groups, such as sulfate esters. Thus, 10 and 11 were

treated with AllocCl in the presence of N,N,N′,N′-

tetramethylethylenediamine (TMEDA) to give 12 and 13,

respectively, in high yields. The anomeric TDS esters of 12 and

13 were cleaved with HF in pyridine, which was followed by

the reduction of the azido group using a Staudinger reaction

with trimethylphosphine (PMe₃) to give an amine that was

reacted with 2-triﬂuoromethyl benzaldehyde in a mixture of

dichloromethane and pyridine, installing a 2-CF₃−Ph−CH

N moiety to provide 15 and 17, respectively.⁵⁷The required

triﬂuoro-N-phenylacetimidate donors 3 and 4 were obtained

by the treatment of lactols 15 and 17 with 2,2,2-triﬂuoro-N-

phenylacetimidoyl chloride in the presence of K₂CO₃.⁵⁶We

also attempted another sequence of reactions to prepare 15

and 17 by ﬁrst reducing the azide to a free amine, followed by

the installation of the 2-CF₃−Ph−CHN moiety and then

cleavage of the anomeric TDS protecting group. The latter step

was performed by a treatment with HF in pyridine or a TBAF/

AcOH complex, but in each case only decomposition was

observed.

We examined whether glycosyl donor 4, having a 2-CF₃−

Ph−CHN moiety at C2, can be employed for the selective

installation of 1,2-cis-glycosides. For these studies, we used

model acceptor 22 that could readily be prepared from our

standard building blocks (Table 1 and Scheme S1). Nickel

Table 1. Optimization of Glycosylation Conditions for

Donor 4 and Acceptor 22

The new modular donors 3 and 4 and conventional

acceptors 20, 21, 29, and 30 were employed to prepare HS

hexasaccharides 33−36 that resemble sequences spanning NS

and NA domains, having various levels of sulfation at the

nonreducing GlcA-GlcN moiety. Thus, glycosyl donor 29 was

coupled with spacer-modiﬁed acceptor 30 using a 0.5 eq of

TfOH as the promotor to give tetrasaccharide 31 in a yield of

52% as only the α-anomer (Scheme 3). The Fmoc protecting

group of 31 was removed using Et₃N in DCM to give acceptor

32, which was coupled with 3 and 4, having the GlcN C2

amine protected as an imine, and 20 and 21, having the GlcN

C2 amine masked as azide, to provide hexasaccharides 33−36,

respectively, in moderate yields. The Alloc and 2-CF₃−Ph−

CHN protecting groups of 33 and 34 were removed by a

one-pot two-step procedure employing tetrakis-

(triphenylphosphine)palladium(0) in a mixture of THF/

water, followed by the addition of hydrochloric acid. The

resulting free amine of the hexasaccharides was acetylated with

acetic anhydride in pyridine and then treated with hydrazine

acetate to cleave the Lev esters, which was followed by the O-

sulfation of the resulting hydroxyls and the saponiﬁcation of

conc.

4 Å

yield

entry

promotor

equiv

(mM)

(°C) (h)

(%)

Ni(OTf)₂

TfOH

0.15

0.5

1.0

0.5

150

yes

−40

TfOH

0.5

−20

Based on donor 4. Isolated yield. Commercial. Generated in situ

with NiCl₂and AgOTf.

triﬂate [Ni(OTf)₂] was employed as the promoter because

previous studies have demonstrated it has an α-directing eﬀect

by forming a complex with both the anomeric imidate and the

C2 benzylidiene.⁵⁰Unfortunately, tetrasaccharide 23 was not

formed when commercial (Table 1, entry 1) or in situ-

synthesized Ni(OTf)₂(from NiCl₂and AgOTf, entry 2) was

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Scheme 2. Deprotection of Tetrasaccharide 23

(a) Pd(PPh₃)₄(0), THF/H₂O, 2 h. (b) HCl/THF/H₂O, 15 min; Ac₂O, Py, 3 h: 25,: 90% over two steps. (c) NH₂NH₂·AcOH, toluene/EtOH, 2

h. (d) SO₃·Py, DMF, 2 h. (e) 1 M LiOH/H₂O₂, THF/H₂O: 26, 60% over ﬁve steps. (f) PMe₃/NaOH, THF/H₂O, 2 h. (g) SO₃·Py, MeOH/Et₃N:

27, 66% over two steps. (h) Pd(OH)₂/C, H₂, t-BuOH/H₂O, 24 h: 28, 90%.

the esters to provide hexasaccharides 37 and 38. In the case of

hexasaccharides 35 and 36, the reaction sequence was simpler

and entailed the cleavage of the Lev ester followed by O-

sulfation and saponiﬁcation to give compounds 39 and 40,

respectively. The azido moieties of 37−40 were reduced using

PMe₃/NaOH followed by selective N-sulfation using standard

conditions to give 41−44, respectively. The latter compounds

were subjected to hydrogenation over Pd(OH)₂/C in a

mixture of tert-butanol/H₂O (1:1 v/v) to give the target

hexasaccharides 45−48. To avoid the methylation of the free

amine of the linker, it was important to avoid methanol as the

reaction solvent. ¹H NMR spectra of the oligosaccharides were

fully assigned by 1D and 2D NMR spectroscopy, such as

COSY, HSQC, HMBC, TOCSY, and NOESY. The anomeric

CONCLUSION

■

We have developed modular disaccharides that make it

possible to assemble HS oligosaccharides with NHAc and

NS moieties. It was found that the amino protecting groups

triﬂuoromethylphenyl-methanimine and azide and the hydrox-

yl protection by Lev ester, TDS ether, Alloc and Fmoc oﬀer the

opportunity to selectively manipulate functionalities to prepare

high-complex HS oligosaccharides. The methodology will

make it possible to prepare panels of compounds for

structure−activity relationship studies to better understand

the biology of heparan sulfate. A recent paper demonstrated

that triﬂuoromethylphenyl-methanimine can also be employed

to make HS oligosaccharides to modulate heparanase

activity,⁶¹which is an enzyme that can remodel HS and has

been implicated in cancer. The microarray data highlight key

requirements of chemokines to engage with HS. Future studies

will focus on synthesizing HS structures bearing high-aﬃnity

NS domains that connect to NA domain for binding studies.

conﬁguration was conﬁrmed by J_1,2coupling constants (J_1,2

∼

175 Hz for the α-linkage) and ¹³C chemical shifts of C1 (<100

ppm for the α-linkage).

To probe the eﬀect of the NS/NA substitutions, the uronic

acid composition, and the sulfation pattern on HS binding,

compounds 45−48 along with related HS hexasaccharides⁵⁸

(Figure 2, 49−55) were printed on a N-hydroxysuccinamide

(NHS)-activated glass slide. The HS microarray was exposed

to His-tagged IL-8 (1 μg/mL) and His-tagged RANTES (1

μg/mL, Sino Biological), and binding was visualized using the

AlexaFluor647 anti-His antibody (5 μg/mL). These chemo-

tactic cytokines play pivotal roles in neutrophil recruitment,

activation, and migration to the site of injury and

inﬂammation.⁵⁹This process is orchestrated by cell-surface

HS proteoglycans (HSPG’s) by engaging with chemokines to

generate a concentration gradient.⁸Although HSPG is

critically involved in chemokine binding, the structural features

of HS important for binding are still ambiguous.

Our results conﬁrm that IL-8 preferentially binds to HS

having IdoA2S-GlcNS6S sequence (Figure 2B).⁶⁰Compound

55, bearing three of these units, bound potently, and a gradual

decrease of binding was observed when IdoA2S was

sequentially replaced by GlcA moieties (55 vs 54 vs 53). A

complete loss of binding was observed when only GlcA was

present in the backbone (55 vs 49). Also, IL-8 did not bind to

a hexasaccharide in which N-sulfate was replaced by N-acetyl

(e.g., 48 vs 46). RANTES appears to be more promiscuous but

also prefers compounds having IdoA2S in the backbone (e.g.,

55, Figure 2C). A large reduction in binding was observed

when either 6-OS was removed (48 vs 47) or NHAc was

present in the backbone (e.g., 48 vs 46 and 45). Interestingly,

the location of IdoA2S appears to be important and preferably

should be present at the nonreducing end (51 vs 53).

EXPERIMENTAL SECTION

■

General Experimental Procedures. All chemicals were

purchased from commercial sources and used without further

puriﬁcation unless otherwise noted. Molecular sieves (4 Å) were

ﬂame-dried prior to use. All moisture-sensitive reactions were carried

out under an argon atmosphere. All reactions at elevated temperatures

were performed in a silicon oil bath unless otherwise speciﬁed.

Reactions were monitored by thin-layer chromatography (TLC) on

silica gel-coated aluminum or glass plates (EMD Chemicals, Inc.).

Spots were visualized by UV light (254 nm) when applicable and

charring with 10% sulfuric acid in ethanol or a solution of

(NH₄)₆Mo₇O₂₄·4H₂O (24.0 g, 19.4 mmol) and Ce(NH₄)₂(NO₃)₆

(0.50 g, 0.9 mmol) in sulfuric acid (5%, 500 mL). Column

chromatography was performed on silica gel G60 (Silicycle 60−200

μm, 60 Å). Fractions or reaction mixtures containing sulfated

compounds were concentrated under reduced pressure, with the

water bath temperature less than 25 °C. NMR spectra (¹H, ¹³C,

COSY, HSQC, TOCSY, HMBC, and NOESY) were recorded on

either an Agilent 400-MR DD2 or a Bruker AVANCE-600 MHz

spectrometer at 25 °C. Chemical shifts are reported in parts per

million (ppm) relative to tetramethylsilane (TMS δ 0 ppm),

deuterium oxide (D₂O δ 0 ppm), or methanol-d₄(CD₃OD δ 3.31

ppm) as the internal standard. NMR data are presented as follows:

chemical shift, multiplicity (s = singlet, bs = broad singlet, d =

doublet, bd = broad doublet, t = triplet, dd = doublet of doublet, and

m = multiplet or multiple resonances), integration, and coupling

constant in Hertz (Hz). All the AB quartets were reported as two

doublets due to a large ν_AB/J_ABratio (>4). NMR signals were assigned

on the basis of H NMR, ¹³C{¹H} NMR, COSY, HSQC, HMBC,

TOCSY, or NOESY experiments. Due to the small sample size,

carbon chemical shifts for compounds 26, 27, 28, 37, 38, 39, 40, 41,

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Scheme 3. Synthesis of Hexasaccharide

Scheme 3. continued

39, 35% over ﬁve steps; 40, 38% over ﬁve steps. (h) PMe₃/NaOH,

THF/H₂O, 2 h. (i) SO₃·Py, MeOH/Et₃N; 41, 60% over two steps;

42, 40% over two steps; 43, 46% over two steps; 44, 49% over two

steps. (j)Pd(OH)₂/C, H₂, t-BuOH/H₂O, 24−48 h: 45, 92%, 46, 77%;

47, 88%; 48, 90%.

42, 43, 44, 45, 46, 47, and 48 were collected from the F1 dimension

in the HSQC spectra. Mass spectra were obtained on a Bruker

micrOTOF-QII (ESI LC-MS) or Kratos Analytical Maxima-CFR

MALDI-TOF system (using a 2,5-dihydroxybenzoic acid matrix).

Reported HRMS data were obtained on an Agilent technologies 6560

ion mobility Q-TOF. Optical rotation was recorded on a Jasco P-1010

polarimeter.

General Procedure for the Cleavage of Lev Esters. Hydrazine

acetate (5 equiv per Lev group) was added to a solution of the starting

material in a mixture of DCM and MeOH (1:1 v/v, 0.02 M). The

reaction mixture was stirred at room temperature for 2−4 h until TLC

analysis (petroleum ether/EtOAc, 1:1 to 1:2 v/v) indicated the

completion of the reaction. The reaction mixture was diluted with

DCM (30 mL); washed with water (25 mL), a saturated bicarbonate

solution (2 × 25 mL), and brine (25 mL); dried (Na₂SO₄); and

ﬁltered. The ﬁltrate was concentrated under reduced pressure, and the

residue was puriﬁed by silica gel column chromatography using

petroleum ether and EtOAc as the eluent (1:1 to 1:2 v/v) to give pure

product.

General Procedure for O-Sulfation. To a solution of the

starting material in DMF (0.15 M) was added SO₃·Py (10 equiv per

OH). The reaction mixture was stirred at room temperature for 2−4 h

until TLC (DCM/MeOH, 90:10 v/v) indicated the completion of the

reaction. A mixture of triethylamine and MeOH (1:1 v/v, 1 mL) was

added to the reaction mixture, which continued stirring for 30 min.

The reaction mixture was concentrated under reduced pressure, and

the residue was applied to a column of Iatrobeads (5 g), which was

eluted with a gradient of DCM and MeOH (from 95:5 to 85:15 v/v).

Fractions containing the product were concentrated under reduced

pressure, and the residue was passed through a column of Dowex 50

× 8 Na⁺resin (0.6 cm × 5 cm) using MeOH as the eluent to give

pure product.

General Procedure for the Saponiﬁcation of Methyl Esters

and De-O-acetylation. A premixed solution of a 30% solution of

H₂O₂in H₂O (100 equiv per CO₂Me) and 1 M LiOH (50 equiv per

CO₂Me) was added to a solution of the starting material in THF

(0.02 M). The reaction mixture was stirred at room temperature for

12 h. Then, a 4 M solution of NaOH was added until pH 14. Stirring

continued until LC-MS indicated the completion of the reaction.

After adjusting the pH to 8−9 by the careful addition of AcOH, the

solvents were removed under reduced pressure. The residue was

dissolved in water and applied to a reverse phase C18 column (1.0 cm

× 5 cm), which was eluted with a gradient of H₂O and MeOH (from

90:10 to 40:60 v/v). Fractions containing the product were

concentrated under reduced pressure, and the residue was passed

through a column of Dowex 50 × 8 Na⁺resin (0.6 cm × 5 cm) using

H₂O as the eluent to give pure product.

General Procedure for the Reduction of the Azido Group.

To a solution of the starting material in THF (1.0 mL for 0.013

mmol) was added 0.1 M NaOH (10 equiv per azido group). Then, a 1

M solution of PMe₃in THF (8 equiv per azido group) was added to

the solution. The reaction mixture was stirred at room temperature

for 1 h until LC-MS indicated the completion of the reaction. After

adjusting the pH to 8−9 by the careful addition of AcOH, the

solvents were removed under reduced pressure. The residue was

dissolved in water and applied to a reverse phase C18 column (1.0 cm

× 5 cm), which was eluted with a gradient of H₂O and MeOH (from

90:10 to 40:60 v/v). Fractions containing the product were

concentrated under reduced pressure, and the residue was passed

through a column of Dowex 50 × 8 Na⁺resin (0.6 cm × 5 cm) using

H₂O as the eluent to give the desired product.

(a) TfOH, DCM, 4 Å MS, −40 °C, 1 h: 31, 52%; 35, 55%; 36, 69%.

(b) DCM/Et₃N, 2 h: 32, 90%. (c) TfOH, DCM, 4 Å MS, −20 °C, 12

h: 33, 52%; 34, 48%. (d) (i) Pd(PPh₃)₄(0), THF/H₂O, 2 h; (ii)

HCl/THF/H₂O, 15 min; and (iii) Ac₂O, Py, 3 h. (e) NH₂NH₂·

AcOH, toluene/EtOH, 2 h. (f) SO₃·Py, DMF, 2 h. (g) 1 M LiOH,

30% H₂O₂, THF/H₂O: 37, 43% over six steps; 38, 40% over six steps;

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Figure 2. Microarray binding data of synthetic hexasaccharides to chemokines. (A) Interleukin-8/IL-8/CXCL8 (1 μg/mL), (B) chemokine ligand

5/CCL5/RANTES (1 μg/mL), and (C) structures of synthetic hexasaccharides. Each hexasaccharide was printed at 100 μM as replicates of six.

The hexasaccharides are arranged according to an increasing number of sulfates. C indicates the blank control. Abbreviations are as follows: GlcN,

glucosamine; GlcNAc, N-acetyl glucosamine; IdoA, iduronic acid; and GlcA, glucuronic acid. Data are presented as mean SD (n = 4).

General Procedure for the Selective N-Sulfation Reaction.

SO₃·Py (5 equiv per NH₂) was added to a solution of the starting

material in a mixture of MeOH (1 mL for 0.006 mmol) and Et₃N (0.3

mL). Then, 1 M NaOH was added to adjust the pH to 11. Three

additional portions of SO₃·Py (5 equiv per NH₂) were added to the

solution after 30 min, 1 h, and 2 h, followed by adjusting the pH to 11

by the careful addition of 1 M NaOH for each. The progress of the

reaction was monitored by TLC (silica gel TLC, EtOAc/pyridine/

H₂O/AcOH, 8:5:3:1 v/v/v/v). After stirring for an additional 12 h,

the reaction mixture was co-evaporated with water, and the residue

was passed through a short column of Dowex 50 × 8Na⁺resin (1.0

cm × 5 cm) with H₂O as the eluent. Fractions containing the product

were lyophilized, and the residue was dissolved in water and applied

to a reverse phase C18 silica gel column (1.0 cm × 5 cm), which was

eluted with a gradient of H₂O and acetonitrile (from 98:2 to 85:15 v/

v). Appropriate fractions were lyophilized to give the desired product.

General Procedure for Global Debenzylation. Palladium

hydroxide on carbon (Degussa type, 20%, 1.5× the weight of the

starting material) was added to a solution of the starting material in

tert-butanol and H₂O (1:1 v/v, 1 mL for 1 mg). The mixture was

placed under a hydrogen atmosphere until the completion of the

reaction as indicated by ESI-LC-MS. The mixture was ﬁltered through

a PTFE syringe ﬁlter (Acrodisc, 0.2 μm), and the residue was washed

with a tert-butanol and H₂O mixture (1:1 v/v, 2 mL). The ﬁltrate was

lyophilized to give the ﬁnal product. Biogel P2 (1.5 cm × 50 cm) was

used for puriﬁcation with 0.1 M ammonium bicarbonate as the eluent,

and the fractions containing the compound were lyophilized and

passed through a short column of Dowex 50 × 8Na⁺resin (1.0 cm ×

5 cm) with H₂O as the eluent. Appropriate fractions were lyophilized

to give the desired product.

relative humidity environment); the remaining activated esters were

then quenched with ethanolamine (5 mM) in a Tris buﬀer (pH 9.0,

50 mM) at 50 °C for 1 h. Blocked slides were rinsed with DI water,

spun dry, and kept in a desiccator at room temperature for future use.

Subarrays were incubated with IL-8 and RANTES (1 μg/mL, His-

tagged, Sino Biological). After 1 h, the slide was sequentially washed

by dipping in a TSM wash buﬀer (2 min), a TSM buﬀer (2 min), and

water (2 × 2 min) and spun dry. The subarrays were further

incubated with the Alexa Fluor 647 anti-His tag antibody (5 μg/mL)

for 1 h in the dark, and the same washing sequence was repeated. The

slides were scanned using a GenePix 4000B microarray scanner

(Molecular Devices) at the appropriate excitation wavelength with a

resolution of 5 μM. Various gains and PMT values were employed in

the scanning to ensure that all the signals were within the linear range

of the scanner’s detector and there was no saturation of the signals.

The images were analyzed using GenePix Pro 7 software (ver.

7.2.29.2, Molecular Devices). The data was analyzed with our home-

written Excel macro. The highest and the lowest values of the total

ﬂuorescence intensity of the six replicates were removed, and the

remaining four values were used to calculate the mean value and

standard deviation. The data were ﬁtted using Prism software (Prism

8, GraphPad Software), and bars represent the mean SD for each

compound.

Experimental Procedures and Analytical Data of Products.

Compounds S1, 5, 6, 7, 8, 9, 10, 11, 18, 19, and 30;¹²compounds 20,

21, and 29;⁵²and compound S2⁶²were reported previously. For

oligosaccharide nomenclature for NMR spectroscopy, the mono-

saccharide residues of the HS oligosaccharides were labeled from the

reducing end to the nonreducing end as A, B, C, D, E, and F; the 2-

CF₃−Ph−CHN moiety was labeled as shown in Scheme 1.

Dimethylthexylsilyl O-(Methyl 2-O-Acetyl-3-O-benzyl-4-O-ally-

loxycarbonyl-β-^D-glucopyranosyluronate)-(1 → 4)-O-2-azido-3-O-

benzyl-6-O-acetyl-2-deoxy-β-^D-glucopyranoside (12). To a solution

of compound 10 (1.26 g, 1.57 mmol) in anhydrous DCM (6 mL) was

added N,N,N′,N′-tetramethylethylenediamine (TMEDA) (144 μL,

0.94 mmol). The solution was cooled to 0 °C, followed by the

addition of allyl chloroformate (252 μL, 2.36 mmol). The reaction

mixture was warmed to room temperature and stirred for 10 h. The

reaction mixture was diluted with DCM (25 mL) and washed with a

Glycan Array Screening and Analysis. HS hexasaccharides 45−

55 were printed on NHS-ester-activated glass slides (Nexterion Slide

H, Schott, Inc.) using a Scienion sciFLEXARRAYER S3 instrument.

All samples were printed at a concentration of 100 μM in a sodium

phosphate buﬀer (0.225 M, pH 8.5) in replicates of six with a spot

volume ∼400 pL at 20 °C and 50% relative humidity (each slide has

24 subarrays in a 3 × 8 layout). After printing, the slides were

incubated overnight in a saturated NaCl chamber (aﬀording a 75%

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saturated bicarbonate solution (25 mL) and brine (25 mL). The

organic phase was dried (MgSO₄) and ﬁltered, and the ﬁltrate was

concentrated in vacuo. The residue was puriﬁed by silica gel column

chromatography using petroleum ether and EtOAc as the eluent (4:1

v/v) to give disaccharide 12 as a white amorphous solid (1.22 g,

Hz, 1H, CHHBn), 4.64−4.52 (m, 5H, H1^B, CHHBn, CHHBn,

CH₂CHCH₂), 4.48−4.42 (m, 1H, H6a^A), 4.23−4.14 (m, 1H,

H6b^A), 4.01−3.86 (m, 3H, H3^A, H4^A, H5^B), 3.78−3.63 (m, 2H, H3^B,

H5^A), 3.63−3.55 (m, 1H, H2^A), 3.54 (s, 3H, CO₂CH₃), 2.10 (s, 3H,

CH₃Lev), 1.99 (s, 3H, CH₃Ac). ¹³C{¹H} NMR (151 MHz, CDCl₃):

δ 170.7 (CO Ac), 169.2 (CO Ac), 167.2 (CO₂CH₃), 161.9 (N

CHPhCF₃), 153.8 (CO Alloc), 143.5, 138.4, 137.6, 133.5, 132.0,

131.2 (CH₂CHCH₂), 130.8, 128.8, 128.6, 128.5, 128.2, 128.0,

127.9, 127.6, 127.3, 125.8, 125.7, 124.4, 119.5 (CH₂CHCH₂),

119.4, 101.2 (C1^B), 81.2 (C3^A), 79.6 (C3^B), 78.1 (C4^A), 75.5 (C2^A),

75.0 (C4^B), 74.9 (OCH₂Bn), 74.6 (OCH₂Bn), 73.7 (C5^A), 72.8

(C5^B), 72.5 (C2^B), 69.2 (CH₂CHCH₂), 62.3 (C6^A), 52.8

(CO₂CH₃), 21.0 (CH₃Ac), 20.9 (CH₃Ac). HRMS (ESI): m/z

calcd for C₅₁H₅₁F₆N₂O₁₅[M + H]⁺1045.3188, found 1045.3181;

calcd for C₄₃H₄₈F₃N₂O₁₅[M − [CHPhCF₃] + 3H]⁺899.3001, found

899.3006.

Dimethylthexylsilyl O-(Methyl 2-O-Acetyl-3-O-benzyl-4-O-ally-

loxycarbonyl-β-^D-glucopyranosyluronate)-(1 → 4)-O-2-azido-3-O-

benzyl-6-O-levulinoyl-2-deoxy-β-^D-glucopyranoside (13). To a

solution of compound 11 (0.77 g, 0.90 mmol) in anhydrous DCM

(3.6 mL) was added TMEDA (81 μL, 0.54 mmol). The solution was

cooled to 0 °C, followed by the addition of allyl chloroformate (144

μL, 1.35 mmol). The reaction mixture was warmed to room

temperature and stirred for 10 h. The reaction was monitored by

TLC until completion. The reaction mixture was diluted with DCM

(25 mL) and washed with a saturated bicarbonate solution (25 mL)

and brine (25 mL). The organic phase was dried (MgSO₄) and

ﬁltered, and the ﬁltrate was concentrated in vacuo. The residue was

puriﬁed by silica gel column chromatography using petroleum ether

and EtOAc (4:1 v/v) as the eluent to give disaccharide 13 as a white

amorphous solid (0.77 g, 91%). R_f= 0.55 (petroleum ether/EtOAc,

2:1 v/v). [α]²_D⁰− 10.9 (c = 0.5, CHCl₃). ¹H NMR (400 MHz,

CDCl₃): δ 7.43−7.19 (m, 10H, CH aromatic), 5.95−5.79 (m, 1H,

CH₂CHCH₂), 5.37−5.23 (m, 2H, CH₂CHCH₂), 5.08−5.00 (m,

3H, CHHBn, H2^B, H4^B), 4.78−4.66 (m, 3H, CHHBn, H1^B,

CHHBn), 4.61−4.55 (m, 3H, CHHBn, CH₂CHCH₂), 4.46 (d, J

= 7.5 Hz, 1H, H1^A), 4.29 (dd, J = 11.8, 1.9 Hz, 1H, H6a^A), 4.17 (dd, J

= 11.8, 5.0 Hz, 1H, H6b^A), 4.03 (d, J = 9.8 Hz, 1H, H5^B), 3.86 (t, J =

9.3 Hz, 1H, H3^B), 3.76 (dd, J = 9.6, 8.6 Hz, 1H, H4^A), 3.51 (s, 3H,

CO₂CH₃), 3.44−3.33 (m, 2H, H5^A, H3^A), 3.28 (dd, J = 9.9, 7.7 Hz,

1H, H2^A), 2.90−2.47 (m, 4H, 2 × CH₂Lev), 2.20 (s, 3H, CH₃Lev),

1.98 (s, 3H, CH₃Ac), 1.70−1.59 (m, 1H, CH(CH₃)₂), 0.90−0.83

(4s, 12H, C(CH₃)₂, CH(CH₃)₂), 0.18−0.13 (2s, 6H, Si(CH₃)₂).

¹³C{¹H} NMR (101 MHz, CDCl₃): δ 206.6 (CH₃CO Lev), 172.6

(CH₂CO Lev), 169.4 (CO Ac), 167.4 (CO₂CH₃), 153.9 (CO Alloc),

138.8, 137.8, 131.3 (CH₂CHCH₂), 128.5, 128.4, 127.9, 127.8,

127.6, 127.5, 119.4 (CH₂CHCH₂), 101.1 (C1^B), 97.0 (C1^A), 81.0

(C3^A), 79.7 (C3^B), 78.5 (C4^A), 75.2 (C4^B, OCH₂Bn), 74.7

(OCH₂Bn), 72.7 (C5^A), 72.6 (C2^B), 72.6 (C5^B), 69.2 (CH₂

CHCH₂), 68.7 (C2^A), 62.6 (C6^A), 52.8 (CO₂CH₃), 38.1 (CH₂Lev),

34.1 (CH(CH₃)₂), 30.0 (CH₃Lev), 28.0 (CH₂Lev), 25.0 (C(CH₃)₂),

20.9 (CH₃Ac), 20.1 (CH(CH₃)₂), 20.0 (CH(CH₃)₂), 18.6

(C(CH₃)₂), 18.5 (C(CH₃)₂), −2.0 (Si(CH₃)₂), −3.1 (Si(CH₃)₂).

HRMS (ESI): m/z calcd for C₄₆H₆₃N₃NaO₁₆Si [M + Na]⁺964.3870,

found 964.3874.

(Methyl 2-O-Acetyl-3-O-benzyl-4-O-allyloxycarbonyl-β-^D-gluco-

pyranosyluronate)-(1 → 4)-O-2-azido-3-O-benzyl-6-O-levulinoyl-

2-deoxy-α/β-^D-glucopyranoside (16). Hydrogen ﬂuoride pyridine

(70%, 2.1 mL) was added to a solution of compound 13 (0.77 g, 0.82

mmol) in THF (16 mL). The reaction mixture was stirred at room

temperature for 18 h. The progress of the reaction was monitored by

TLC until it showed the completion of the reaction. The mixture was

diluted with dichloromethane (30 mL) and washed with a saturated

bicarbonate solution (2 × 30 mL) and brine (30 mL). The organic

phase was dried (MgSO₄) and ﬁltered, the ﬁltrate was concentrated in

vacuo, and the residue was puriﬁed by silica gel column

chromatography using petroleum ether and EtOAc (3:1 to 1:1 v/v)

as the eluent to give intermediate 16 as a white amorphous solid (0.58

g, 88%). R_f= 0.18 (petroleum ether/EtOAc, 1:1 v/v). ¹H NMR (600

MHz, CDCl₃): δ 7.44−7.10 (m, 10H, CH aromatic), 5.92−5.84 (m,

88%). R_f= 0.42 (petroleum ether/EtOAc, 3:1 v/v). H NMR (600

MHz, CDCl₃): δ 7.39−7.20 (m, 10H, CH aromatic), 5.92−5.84 (m,

1H, CH₂CHCH₂), 5.36−5.24 (m, 2H, CH₂CHCH₂), 5.04 (dd, J

= 9.2, 8.0 Hz, 1H, H2^B), 4.99 (t, J = 9.2 Hz, 1H, H4^B), 4.96 (d, J =

11.5 Hz, 1H, CHHBn), 4.83 (d, J = 11.5 Hz, 1H, CHHBn), 4.68 (d, J

= 11.5 Hz, 1H, CHHBn), 4.62 (d, J = 8.0 Hz, 1H, H1^B), 4.60−4.55

(m, 3H, CHHBn, CH₂CHCH₂), 4.47 (d, J = 7.7 Hz, 1H, H1^A),

4.35 (dd, J = 11.5, 2.0 Hz, 1H, H6a^A), 4.07 (dd, J = 11.5, 6.6 Hz, 1H,

H6b^A), 3.80 (d, J = 9.8 Hz, 1H, H5^B), 3.68 (t, J = 9.2 Hz, 1H, H3^B),

3.64 (dd, J = 9.5, 8.5 Hz, 1H, H4^A), 3.52 (s, 3H, CO₂CH₃), 3.48−

3.44 (m, 1H, H5^A), 3.39 (dd, J = 9.5, 8.5 Hz, 1H, H3^A), 3.29 (dd, J =

9.5, 7.7 Hz, 1H, H2^A), 2.06 (s, 3H, CH₃Ac), 1.98 (s, 3H, CH₃Ac),

1.67−1.61 (m, 1H, CH(CH₃)₂), 0.89−0.85 (4s, 12H, C(CH₃)₂,

CH(CH₃)₂), 0.18−0.15 (2s, 6H, Si(CH₃)₂). ¹³C{¹H} NMR (151

MHz, CDCl₃): δ 170.6 (CO Ac), 169.2 (CO Ac), 167.1 (CO₂CH₃),

153.8 (CO Alloc), 138.7, 137.5, 131.2 (CH₂CHCH₂), 128.5, 128.4,

128.0, 127.8, 127.4, 127.2, 119.5 (CH₂CHCH₂), 101.3 (C1^B), 96.9

(C1^A), 81.0 (C3^A), 79.5 (C3^B), 79.0 (C4^A), 74.9 (C4^B), 74.8

(OCH₂Bn), 74.6 (OCH₂Bn), 72.6 (C5^B, C5^A), 72.5 (C2^B), 69.2

(CH₂CHCH₂), 68.8 (C2^A), 62.7 (C6^A), 52.8 (CO₂CH₃), 34.0

(CH(CH₃)₂), 24.9 (C(CH₃)₂), 20.9 (CH₃Ac), 20.8 (CH₃Ac), 20.0

(CH(CH₃)₂), 19.9 (CH(CH₃)₂), 18.6 (C(CH₃)₂), 18.4 (C(CH₃)₂),

−2.2 (Si(CH₃)₂), −3.2 (Si(CH₃)₂). HRMS (ESI): m/z calcd for

C₄₃H₆₃N₄O₁₅Si [M + NH₄]⁺903.4054, found 903.4072.

(N-Phenyl)-2,2,2-triﬂuoroacetimidate (Methyl 2-O-Acetyl-3-O-

benzyl-4-O-allyloxycarbonyl-β-^D-glucopyranosyluronate)-(1 → 4)-

O-2-N-[(2-triﬂuoromethyl)benzylidene]-3-O-benzyl-6-O-acetyl-2-

deoxy-α/β-^D-glucopyranoside (3). To a solution of compound 12

(1.14 g, 1.29 mmol) in THF (26 mL) was added 70% hydrogen

ﬂuoride pyridine (3.3 mL) . The reaction mixture was stirred at room

temperature for 18 h. The reaction was monitored until TLC analysis

showed the completion of the reaction. The mixture was diluted with

dichloromethane (50 mL) and washed with a saturated bicarbonate

solution (2 × 50 mL) and brine (50 mL). The organic phase was

dried (MgSO₄) and ﬁltered, and the ﬁltrate was concentrated in vacuo.

The residue was puriﬁed by silica gel column chromatography using

petroleum ether and EtOAc (3:1 to 1:1 v/v) as the eluent to give

intermediate 14 as a white amorphous solid (0.88 g, 92%). The

resulting intermediate 14 (300 mg, 0.40 mmol) was dissolved in THF

(8.0 mL), followed by addition of H₂O (73 μL) and 1 M PMe₃in

THF (0.6 mL). The reaction mixture was stirred at room temperature

for 2 h. The presence of amino groups was conﬁrmed using ninhydrin

staining. The mixture was concentrated under reduced pressure, and

the residue was co-evaporated with toluene (3 × 5 mL). The resulting

intermediate was used in the next step without further puriﬁcation.

The amino-containing intermediate was dissolved in anhydrous DCM

(4.0 mL) and anhydrous pyridine (0.4 mL). 2-Triﬂuoromethyl

benzaldehyde (64 μL, 0.48 mmol) was added, and the reaction

mixture was stirred at 50 °C for 12 h. The reaction mixture was

concentrated under reduced pressure, and the residue was dissolved in

DCM and cooled to 0 °C, followed by the addition of K₂CO₃(111

mg, 0.81 mmol) and N-phenyl-triﬂuoroacetimidoyl chloride (129 μL,

0.81 mmol). The reaction mixture was stirred at room temperature for

5 h. The reaction was monitored until TLC analysis showed the

completion of the reaction. The mixture was concentrated under

reduced pressure, and the residue was puriﬁed by silica gel column

chromatography using petroleum ether and EtOAc (3:1 v/v) as the

eluent to give disaccharide 3 as a white foam (194 mg, 46% over three

steps). R_f= 0.26 (petroleum ether/EtOAc, 3:1 v/v). H NMR (600

MHz, CDCl₃): δ 8.64 (s, 1H, NCHPhCF₃), 8.05 (d, J = 7.4 Hz,

1H, H⁴), 7.68 (d, J = 7.2 Hz, 1H, H¹), 7.60−7.52 (m, 2H, H², H³),

7.35−6.71 (m, 15H, CH aromatic), 6.10−5.70 (m, 2H, CH₂

CHCH₂, H1^A), 5.36−5.32 (m, 1H, CHHCHCH₂), 5.28−5.25 (m,

1H, CHHCHCH₂), 5.09 (t, J = 8.5 Hz, 1H, H2^B), 5.02 (t, J = 9.5

Hz, 1H, H4^B), 4.95 (d, J = 11.5 Hz, 1H, CHHBn), 4.69 (d, J = 11.6

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1H, CH₂CHCH₂), 5.36−5.30 (m, 1H, CHHCHCH₂), 5.36−

5.30 (m, 1H, CHHCHCH₂), 5.29−5.22 (m, 1.55H, CHH

CHCH₂, H1^Aα), 5.21−5.00 (m, 3H, CHHBn, H2^B, H4^B), 4.77−4.66

(m, 3H, CHHBn, H1^B, CHHBn), 4.63−4.55 (m, 3H, CHHBn,

CH₂CHCH₂), 4.54 (d, J = 8.2 Hz, 0.45H, H1^Aβ), 4.31−4.24 (m,

2H, H6a^A, H6b^A), 4.17 (d, J = 10.0 Hz, 0.55H, H5^Bα), 4.12 (d, J =

10.0 Hz, 0.45H, H5^Bβ), 4.06 (dt, J = 10.0, 2.9 Hz, 0.55H, H5^Aα),

3.94−3.88 (m, 1.55H, H3^Aα, H3^B), 3.86−3.81 (m, 1H, H4^A), 3.52

and 3.51 (2s, 3H, CO₂CH₃), 3.47−3.44 (m, 0.45H, H5^Aβ), 3.44−3.41

(m, 0.45H, H3^Aβ), 3.37 (dd, J = 10.0, 3.5 Hz, 0.55H, H2^Aα), 3.37

(dd, J = 9.9, 8.2 Hz, 0.45H, H2^Aβ), 3.40−3.33 (m, 1H, H2^A), 2.93−

2.44 (m, 4H, 2 × CH₂Lev), 2.20 (s, 3H, CH₃Lev), 2.00 and 1.98 (2s,

3H, CH₃Ac). ¹³C{¹H} NMR (151 MHz, CDCl₃): δ 207.40 (CH₃CO

Lev), 207.38 (CH₃C′O Lev), 172.76 (CH₂CO Lev), 172.73

(CH₂C′O Lev), 169.4 (CO Ac), 167.50 (CO₂CH₃), 167.46

(C′O₂CH₃), 153.9 (CO Alloc), 138.5, 138.4, 137.80, 137.76, 131.29

(CH₂CHCH₂), 131.27 (CH₂C′HCH₂), 128.63, 128.55, 128.5,

128.4, 128.3, 128.0, 127.92, 127.88, 127.86, 127.82, 127.77, 127.74,

127.72, 127.67, 127.6, 127.5, 119.4 (CH₂CHCH₂), 119.3 (C′H₂

CHCH₂), 100.89 (C1^Bα), 100.87 (C1^Bβ), 96.2 (C1^Aβ), 91.9 (C1^Aα),

81.0 (C3^Aα), 79.60 (C3^Bα), 79.58 (C3^Bβ), 78.6 (C4^Aβ), 77.94

(C4^Aα), 77.92 (C3^Aα), 75.5 (OCH₂Bn α), 75.4 (OCH₂Bn β), 75.24

(C4^Bα), 75.22 (C4^Bβ), 74.7 (OCH₂Bn β), 74.6 (OCH₂Bn α), 72.8

(C5^Aβ), 72.61, 72.60, 72.56, 72.4, 69.1 (CH₂CHCH₂), 68.5

(C5^Aα), 67.3 (C2^Aβ), 63.7 (C2^Aα), 62.4 (C6^Aα), 62.2 (C6^Aβ),

52.7 (CO₂CH₃), 38.2 (CH₂Lev α), 38.1 (CH₂Lev β), 29.95 (CH₃

Lev β), 29.94 (CH₃Lev α), 28.07 (CH₂Lev α), 28.04 (CH₂Lev β),

20.9 (CH₃Ac). HRMS (ESI): m/z calcd for C₃₈H₄₅N₃NaO₁₆[M +

Na]⁺822.2692, found 822.2710.

Alloc), 138.64, 138.59, 138.0, 137.9, 133.8, 133.2, 132.0, 131.4

(CH₂CHCH₂), 130.8, 130.5, 128.5, 128.2, 128.0, 127.8, 127.7,

127.1, 125.7, 125.6, 119.3 (CH₂CHCH₂), 101.02 (C1^Bα), 100.94

(C1^Bβ), 96.0 (C1^Aβ), 93.5 (C1^Aα), 81.0 (C3^Aβ), 79.7 (C3^B), 78.4,

78.3, 78.0, 77.6 (C2^Aβ), 75.6, 75.4, 75.4, 75.2, 74.6 (OCH₂Bn), 74.1

(C2^Aα), 73.1 (C5^Aβ), 72.7 (C2^B), 72.5 (C5^B), 69.1 (CH₂CHCH₂,

C5^Aα), 62.6 (C6^Aα), 62.5 (C6^Aβ), 52.7 (CO₂CH₃), 38.1 (CH₂Lev),

30.0 (CH₃Lev), 28.1 (CH₂Lev), 20.9 (CH₃Ac). HRMS (ESI): m/z

calcd for C₄₆H₅₁F₃NO₁₆[M + H]⁺930.3154, found 930.3176.

(N-Phenyl)-2,2,2-triﬂuoroacetimidate (Methyl 2-O-Acetyl-3-O-

benzyl-4-O-allyloxycarbonyl-β-^D-glucopyranosyluronate)-(1 → 4)-

O-2-N-[(2-triﬂuoromethyl)benzylidene]-3-O-benzyl-6-O-levulinoyl-

2-deoxy-α/β-^D-glucopyranoside (4). Compound 17 (428 mg, 0.46

mmol) was dissolved in DCM (3.0 mL) and cooled to 0 °C, followed

by the addition of K₂CO₃(127 mg, 0.92 mmol) and N-phenyl-

triﬂuoroacetimidoyl chloride (147 μL, 0.92 mmol). The reaction

mixture was stirred at room temperature for 5 h. The reaction was

monitored until TLC analysis showed the completion of the reaction.

The mixture was concentrated under reduced pressure, and the

residue was puriﬁed by silica gel column chromatography using

petroleum ether and EtOAc (3:1 v/v) as the eluent to give

disaccharide 4 as a white foam (456 mg, 90%). R_f= 0.60 (petroleum

ether/EtOAc, 1:1 v/v). ¹H NMR (600 MHz, CDCl₃): δ 8.63 (s, 1H,

NCHPhCF₃), 8.03−8.00 (m, 1H, ArH⁴), 7.70−7.65 (m, 1H, H¹),

7.58−7.52 (m, 2H, H², H³), 7.33−6.66 (m, 15H, CH aromatic),

6.09−5.66 (m, 2H, CH₂CHCH₂, H1^A), 5.36−5.30 (m, 1H,

CHHCHCH₂), 5.27−5.23 (m, 1H, CHHCHCH₂), 5.11 (t, J =

8.7 Hz, 1H, H2^B), 5.08−5.01 (m, 2H, CHHBn, H4^B), 4.81 (d, J = 8.0

Hz, 1H, H1^B), 4.69 (d, J = 11.6 Hz, 1H, CHHBn), 4.62−4.51 (m, 4H,

CHHBn, CHHBn, CH₂CHCH₂), 4.41−4.26 (m, 2H, H6a^A,

H6b^A), 4.24 (d, J = 9.8 Hz, 1H, H5^B), 4.08−3.85 (m, 3H, H4^A,

H3^B, H3^A), 3.82−3.54 (m, 2H, H5^A, H2^A), 3.52 (s, 3H, CO₂CH₃),

2.97−2.47 (m, 4H, 2 × CH₂Lev), 2.21 (s, 3H, CH₃Lev), 2.00 (s, 3H,

CH₃Ac). ¹³C{¹H} NMR (151 MHz, CDCl₃): δ 207.0 (CH₃CO Lev),

172.7 (CH₂CO Lev), 169.3 (CO Ac), 167.6 (CO₂CH₃), 161.9 (N

CHPhCF₃), 154.0 (CO Alloc), 143.5, 138.5, 137.9, 133.6, 132.0,

131.4 (CH₂CHCH₂), 130.7, 129.7, 129.5, 128.8, 128.6, 128.5,

128.11, 128.05, 127.9, 127.7, 127.3, 125.73, 125.70, 125.0, 124.4,

119.4 (CH₂CHCH₂), 100.9 (C1^B), 81.0 (C3^A), 80.0 (C3^B), 77.7

(C4^A), 75.4 (OCH₂Bn), 75.34 (C4^B), 75.28 (C2^A), 74.7 (OCH₂Bn),

73.7 (C5^A), 72.6 (C2^B), 72.5 (C5^B), 69.1 (CH₂CHCH₂), 62.1

(C6^A), 52.7 (CO₂CH₃), 38.1 (CH₂Lev), 30.0 (CH₃Lev), 28.1 (CH₂

Lev), 20.9 (CH₃Ac). HRMS (ESI): m/z calcd for C₅₄H₅₄F₆N₂NaO₁₆

[M + Na]⁺1123.3270, found 1123.3259.

(Methyl 2-O-Acetyl-3-O-benzyl-4-O-allyloxycarbonyl-β-^D-gluco-

pyranosyluronate)-(1

→ 4)-O-2-N-[(2-triﬂuoromethyl)-

benzylidene]-3-O-benzyl-6-O-levulinoyl-2-deoxy-α/β-^D-glucopyra-

noside (17). Compound 16 (575 mg, 0.72 mmol) was dissolved in

THF (14.0 mL), followed by the addition of H₂O (130 μL) and 1 M

PMe₃in THF (1.1 mL). The reaction mixture was stirred at room

temperature for 2 h. The presence of amino groups was conﬁrmed

using ninhydrin staining. The mixture was concentrated under

reduced pressure, and the residue was co-evaporated with toluene

(3 × 5 mL). The resulting intermediate was used in the next step

without further puriﬁcation. The amino-containing intermediate was

dissolved in anhydrous DCM (7.0 mL) and anhydrous pyridine (0.7

mL). 2-Triﬂuoromethyl benzaldehyde (114 μL, 0.86 mmol) was

added, and the reaction mixture was stirred at 50 °C for 12 h. The

reaction mixture was concentrated under reduced pressure, and the

residue was dissolved in DCM (3.0 mL) and cooled to 0 °C, followed

by the addition of K₂CO₃(127 mg, 0.92 mmol) and N-phenyl-

triﬂuoroacetimidoyl chloride (147 μL, 0.92 mmol). The reaction

mixture was stirred at room temperature for 5 h. The reaction was

monitored until TLC analysis showed the completion of the reaction.

The mixture was concentrated under reduced pressure, and the

residue was puriﬁed by silica gel column chromatography using

petroleum ether and EtOAc (1:1 v/v) as the eluent to give

disaccharide 17 as a white foam (428 mg, 64% over two steps). R_f

= 0.20 (petroleum ether/EtOAc, 1:1 v/v). ¹H NMR (600 MHz,

CDCl₃): δ 8.65−8.50 (m, 1H, NCHPhCF₃), 8.09−7.97 (m, 1H,

H⁴), 7.69−7.61 (m, 1H, H¹), 7.56−7.46 (m, 2H, H², H³), 7.34−6.96

(m, 10H, CH aromatic), 5.93−5.82 (m, 1H, CH₂CHCH₂), 5.35−

5.30 (m, 1H, CHHCHCH₂), 5.27−5.23 (m, 1H, CHHCHCH₂),

5.14−4.98 (m, 3.33H, H1^Aα, H2^B, CHHBn, H4^B), 4.94 (d, J = 7.7 Hz,

0.67H, H1^Aβ), 4.81 (d, J = 8.0 Hz, 1H, H1^B), 4.69 (d, J = 11.6 Hz,

1H, CHHBn), 4.62−4.55 (m, 3H, CHHBn, CH₂CHCH₂), 4.50−

4.29 (m, 3H, CHHBn, H6a^A, H6b^A), 4.29−4.17 (m, 1.33H, H5^B,

H5^Aα), 4.00−3.91 (m, 2.33H, H4^A, H3^Aα, H3^B), 4.17 (d, J = 10.0 Hz,

0.55H, H5^Bα), 3.86 (t, J = 8.7 Hz, 0.67H, H3^Aβ), 3.66−3.62 (m,

0.67H, H5^Aβ), 3.53 and 3.51 (2s, 3H, CO₂CH₃), 3.51−3.49 (m,

0.33H, H2^Aα), 3.30 (t, J = 8.7 Hz, 0.67H, H2^Aβ), 2.98−2.46 (m, 4H,

2 × CH₂Lev), 2.21 (2s, 3H, CH₃Lev), 2.02 and 2.01 (2s, 3H, CH₃

Ac). ¹³C{¹H} NMR (151 MHz, CDCl₃): δ 207.14 (CH₃CO Lev),

207.05 (CH₃C′O Lev), 172.8 (CH₂CO Lev), 169.4 (CO Ac), 167.7

(CO₂CH₃), 167.6 (C′O₂CH₃), 161.0 (NCHPhCF₃), 154.0 (CO

Methyl O-(2-O-Levulinoyl-3-O-benzyl-4,6-O-benzylidene-α-^L-

idopyranosyl)-(1 → 4)-O-2-azido-3-O-benzyl-6-O-acetyl-2-deoxy-

α-^D-glucopyranoside (S3). Freshly activated 4 Å molecular sieves

were added to a solution of glycosyl donor S1 (0.85 g, 1.71 mmol)

and glycosyl acceptor S2 (0.50 g, 1.42 mmol) in anhydrous DCM (86

mL, 0.02 M based on the glycosyl donor) (see Scheme S1). After

stirring for 30 min at room temperature, the solution was cooled to 0

°C, followed by the addition of NIS (0.38 g, 1.71 mmol) and

TMSOTf (25 μL, 0.14 mmol). The reaction mixture was stirred for

30 min, followed by quenching with Et₃N. The mixture was ﬁltered,

and the ﬁltrate was concentrated in vacuo. The residue was puriﬁed by

silica gel column chromatography using a stepwise gradient of

petroleum ether and EtOAc (3:1 to 2:1 v/v, 0.1% Et₃N) as the eluent

to give disaccharide S3 as a white amorphous solid (0.77 g, 69%). R_f=

0.39 (petroleum ether/EtOAc, 3:1 v/v). [α]²_D⁰+ 22.8 (c = 0.5,

CHCl₃). H NMR (600 MHz, CDCl₃): δ 7.46−7.24 (m, 15H, CH

aromatic), 5.29 (s, 1H, CH benzylidene), 5.00 (t, J = 2.9 Hz, 1H,

H2^B), 4.95 (bs, 1H, H1^B), 4.84−4.80 (m, 2H, CHHBn, H1^A), 4.77 (d,

J = 11.5 Hz, 1H, CHHBn), 4.66−4.62 (m, 2H, CHHBn, CHHBn),

4.49 (d, J = 12.3 Hz, 1H, H6a^A), 4.18 (dd, J = 12.3, 2.3 Hz, 1H,

H6b^A), 3.89−3.79 (m, 5H, H5^A, H5^B, H4^A, H4^B, H3^A), 3.77 (d, J =

12.8 Hz, 1H, H6a^B), 3.69 (t, J = 3.2 Hz, 1H, H3^B), 3.48 (dd, J = 10.0,

3.6 Hz, 1H, H2^A), 3.45 (s, 3H, OCH₃), 3.08 (dd, J = 12.8, 1.5 Hz, 1H,

H6b^B), 2.66−2.51 (m, 4H, 2 × CH₂Lev), 2.10 (s, 3H, CH₃Ac), 2.03

(s, 3H, CH₃Lev). ¹³C{¹H} NMR (151 MHz, CDCl₃): δ 206.5

(CH₃CO Lev), 172.1 (CH₂CO Lev), 170.8 (CO Ac), 138.2, 138.1,

https://dx.doi.org/10.1021/acs.joc.0c01881

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

137.8, 129.1, 128.6, 128.2, 127.8, 127.5, 126.3, 100.6 (CH

benzylidene), 98.6 (C1^A), 98.1 (C1^B), 79.1 (C3^A), 75.1 (C3^B), 75.0

(OCH₂Bn), 74.1 (C4^A), 73.9 (C4^B), 72.3 (OCH₂Bn), 69.5 (C5^A),

69.2 (C6^B), 67.2 (C2^B), 64.1 (C2^A), 62.4 (C6^A), 60.4 (C5^B), 55.5

(OCH₃), 37.9 (CH₂Lev), 29.8 (CH₃Lev), 28.3 (CH₂Lev), 21.1

(CH₃Ac). HRMS (ESI): m/z calcd for C₄₁H₄₇N₃NaO₁₃[M + Na]⁺

812.3001, found 812.3026.

eluent to give tetrasaccharide 23 as a white foam (52 mg, 58%). R_f=

0.36 (petroleum ether/EtOAc, 1:1 v/v). ¹H NMR (600 MHz,

CDCl₃): δ 8.51 (d, J = 1.8 Hz, 1H, NCHPhCF₃), 8.16 (d, J = 7.8

Hz, 1H, H⁴), 7.59 (dd, J = 7.2, 1.5 Hz, 1H, H¹), 7.50−7.43 (m, 2H,

H², H³), 7.39−6.80 (m, 20H, 4 × Bn CH aromatic), 5.91−5.84 (m,

1H, CH₂CHCH₂), 5.40 (d, J = 5.9 Hz, 1H, H1^B), 5.35−5.30 (m,

1H, CHHCHCH₂), 5.26−5.23 (m, 1H, CHHCHCH₂), 5.10

(dd, J = 9.6, 8.0 Hz, 1H, H2^D), 5.02 (t, J = 9.5 Hz, 1H, H4^D), 5.00 (d,

J = 12.0 Hz, 1H, CHHBn), 4.98 (d, J = 3.0 Hz, 1H, H1^C), 4.96−4.91

(m, 1H, H2^B), 4.85 (d, J = 12.0 Hz, 1H, CHHBn), 4.79 (d, J = 8.0 Hz,

1H, H1^D), 4.76 (d, J = 3.0 Hz, 1H, H1^A), 4.70 (d, J = 10.5 Hz, 1H,

CHHBn), 4.68 (d, J = 12.0 Hz, 1H, CHHBn), 4.61 (d, J = 12.0 Hz,

1H, CHHBn), 4.58 (t, J = 6.2 Hz, 2H, CH₂CHCH₂), 4.53 (d, J =

12.0 Hz, 1H, CHHBn), 4.48−4.31 (m, 6H, H5^B, CHHBn, H6a^C,

H6a^A, H6b^A, CHHBn), 4.26 (bd, J = 10.0 Hz, 1H, H5^D), 4.24 (dd, J =

12.0, 1.7 Hz, 1H, H6b^C), 4.17−4.13 (m, 1H, H5^C), 4.04−3.99 (m,

2H, H3^B, H4^B), 3.98−3.93 (m, 2H, H4^C, H3^D), 3.92−3.81 (m, 4H,

H5^A, H4^A, H3^C, H3^A), 3.64 (s, 3H, CO₂CH₃), 3.52−3.41 (m, 8H,

H2^C, H2^A, including 2 × CO₂CH₃at 3.48 and 3.41), 2.98−2.90 (m,

1H, CHH Lev), 2.71−2.59 (m, 3H, CHH Lev, CH₂Lev), 2.55−2.44

(m, 3H, CHH Lev, CH₂Lev), 2.25−2.16 (m, 1H, CHH Lev,), 2.21

(s, 3H, CH₃Lev), 2.17 (s, 3H, CH₃Ac), 2.09 (s, 3H, CH₃Lev), 1.99

(s, 3H, CH₃Ac). ¹³C{¹H} NMR (151 MHz, CDCl₃): δ 206.9

(CH₃CO Lev), 206.1 (CH₃CO Lev), 172.8 (CH₂CO Lev), 171.9

(CH₂CO Lev), 171.1 (CO Ac), 170.3 (CO₂CH₃), 169.3 (CO Ac),

167.7 (CO₂CH₃), 160.7 (NCHPhCF₃), 154.1 (CO Alloc), 138.6,

138.2, 138.0, 137.9, 133.49, 132.0, 131.4 (CH₂CHCH₂), 130.6,

129.5, 129.3, 129.2, 129.1, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,

127.9, 127.74, 127.68, 127.3, 127.1, 126.8, 125.6, 119.3 (CH₂

CHCH₂), 100.9 (C1^C, C1^D), 98.6 (C1^A), 97.9 (C1^B), 79.7 (C3^D),

78.5 (C3^A), 78.3 (C4^C), 77.9 (C3^C), 77.3 (C3^B), 76.4 (C4^A), 75.5

(C4^D), 75.4 (OCH₂Bn), 75.2 (OCH₂Bn, C4^B), 74.7 (OCH₂Bn), 74.6

(C2^C), 74.2 (OCH₂Bn), 72.6 (C2^D), 72.5 (C5^D), 71.8 (C5^B), 71.4

(C2^B), 69.8 (C5^C), 69.1 (CH₂CHCH₂), 68.9 (C5^A), 63.4 (C2^A),

62.3 (C6^A), 62.3 (C6^C), 55.4 (OCH₃), 52.6 (CO₂CH₃), 52.2

(CO₂CH₃), 38.2 (CH₂Lev), 37.7 (CH₂Lev), 30.0 (CH₃Lev), 29.8

(CH₃Lev), 28.1 (CH₂Lev), 27.8 (CH₂Lev), 21.1 (CH₃Ac), 20.9

(CH₃Ac). ¹⁹F NMR (565 MHz, CDCl₃): δ −57.00. HRMS (ESI):

m/z calcd for C₈₁H₉₂F₃N₄O₂₉[M + H]⁺1641.5794, found 1641.5823.

Methyl O-(3-O-Benzyl-β-^D-glucopyranosyluronate)-(1 → 4)-O-

(2-acetylamino-3-O-benzyl-6-O-sulfate-2-deoxy-α-^D-glucopyrano-

syl)-(1 → 4)-O-(2-O-sulfate-3-O-benzyl-α-^L-idopyranosyluronate)-

(1 → 4)-O-2-azido-3-O-benzy-2-deoxy-α-^D-glucopyranoside (26).

Tetrakis(triphenylphosphine)palladium(0) (6.6 mg, 5.8 μmol) was

added to a solution of protected tetrasaccharide 23 (9.5 mg, 5.8

μmol) in THF and H₂O (1.1 mL, 10:1 v/v, 5 mM). The reaction

mixture was stirred at room temperature for 1 h. After TLC analysis

indicated the completion of the reaction, a 2 M hydrochloric acid

solution (58 μL, 116 μmol) was added to the reaction mixture, and

stirring was continued for 15 min. The progress of the reaction was

monitored by TLC until it showed the completion of the reaction.

The reaction mixture was co-evaporated with toluene (2 × 3 mL),

and the residue was dissolved in pyridine (0.5 mL), followed by the

addition of Ac₂O (0.2 mL). After stirring for another 3 h at room

temperature, the reaction mixture was concentrated under reduced

pressure, and the residue was puriﬁed by silica gel column

chromatography using petroleum ether and EtOAc (1:1 v/v) as the

eluent to give compound 25 as a colorless amorphous solid (7.7 mg,

90%). Compound 25 (7.7 mg, 5.2 μmol) was subjected to Lev ester

removal, O-sulfation, saponiﬁcation, and de-O-acetylation reactions

according to the general procedures to provide compound 26 as a

Methyl O-(Methyl 2-O-Levulinoyl-3-O-benzyl-α-^L-idopyranosy-

luronate)-(1 → 4)-O-2-azido-3-O-benzyl-6-O-acetyl-2-deoxy-α-^D-

glucopyranoside (22). A solution of a disaccharide S3 (0.74 g, 0.94

mmol) in a mixture of DCM/TFA/H₂O (0.06 M, 16.0 mL, 10:1:0.1

v/v/v) was stirred at room temperature for 30 min. The reaction

mixture was diluted with DCM (25 mL); washed with water (30 mL),

saturated bicarbonate (30 mL), and brine (30 mL); dried (MgSO₄);

and ﬁltered. The ﬁltrate was concentrated under reduced pressure.

The residue was puriﬁed by silica gel column chromatography using

petroleum ether and EtOAc (1:1 v/v) as the eluent to give the pure

product, which was dissolved in a mixture of DCM/H₂O (3.75 mL,

2:1 v/v), followed by the addition of TEMPO (33.2 mg, 0.21 mmol)

and (diacetoxyiodo)benzene (0.82 g, 2.55 mmol). The reaction

mixture was stirred at room temperature for 6 h. The progress of the

reaction was monitored by TLC until completion. The reaction

mixture was quenched by the addition of aqueous Na₂S₂O₃(10%, 10

mL) and extracted with EtOAc (2 × 10 mL). The combined organic

layers were dried (MgSO₄) and ﬁltered, and the ﬁltrate was

concentrated in vacuo. The residue was dissolved in MeOH and

toluene (10 mL, 1:4 v/v) and cooled to 0 °C. An excess of

trimethylsilyldiazomethane (2 M in diethyl ether, 1.0 mL) was added

to the solution slowly until the reaction mixture stayed yellow. Stirring

was continued for 15 min, and the excess trimethylsilyldiazomethane

was quenched by the addition of AcOH until the reaction mixture

became colorless. The mixture was concentrated under reduced

pressure and co-evaporated with toluene (3 × 5 mL), and the residue

was puriﬁed by silica gel column chromatography using petroleum

ether and EtOAc (1:1 v/v) as the eluent to aﬀord compound 22 as a

light yellow amorphous solid (0.40 g, 58% over three steps). R_f= 0.25

(petroleum ether/EtOAc, 1:2 v/v). [α]²_D⁰+ 21.3 (c = 3.3, CHCl₃). ¹H

NMR (600 MHz, CDCl₃): δ 7.39−7.22 (m, 10H, CH aromatic), 5.06

(bs, 1H, H1^B), 4.93 (bs, 1H, H2^B), 4.87 (bs, 1H, H5^B), 4.79 (d, J =

3.6 Hz, 1H, H1^A), 4.77−4.74 (m, 2H, CHHBn, CHHBn), 4.66 (d, J =

10.8 Hz, 1H, CHHBn), 4.63 (d, J = 11.5 Hz, 1H, CHHBn), 4.41 (dd,

J = 12.3, 1.3 Hz, 1H, H6a^A), 4.23 (dd, J = 12.7, 3.6 Hz, 1H, H6b^A),

3.98−3.95 (m, 1H, H4^B), 3.93−3.89 (m, 1H, H4^A), 3.86−3.83 (m,

1H, H5^A), 3.80 (t, J = 9.5 Hz, 1H, H3^A), 3.74 (t, J = 2.6 Hz, 1H,

H3^B), 3.47 (s, 3H, CO₂CH₃), 3.45−3.42 (m, 4H, OCH₃at 3.44,

H2^A), 2.76−2.73 (m, 2H, CH₂Lev), 2.62 (d, J = 10.5 Hz, 1H, OH),

2.58−2.54 (m, 2H, CH₂Lev), 2.18 (s, 3H, CH₃Lev), 2.11 (s, 3H,

CH₃Ac). ¹³C{¹H} NMR (151 MHz, CDCl₃): δ 206.3 (CH₃CO Lev),

171.4 (CH₂CO Lev), 170.7 (CO Ac), 169.6 (CO₂CH₃), 137.8, 137.4,

128.5, 128.2, 128.1, 128.1, 127.5, 127.4, 98.5 (C1^A), 97.9 (C1^B), 78.6

(C3^A), 75.0 (C4^A), 74.8 (OCH₂Bn), 74.7 (C3^B), 72.5 (OCH₂Bn),

69.0 (C5^A), 68.8 (C5^B), 67.7 (C4^B), 67.6 (C2^B), 63.7 (C2^A), 62.2

(C6^A), 55.4 (OCH₃), 52.0 (CO₂CH₃), 37.8 (CH₂Lev), 29.7 (CH₃

Lev), 27.9 (CH₂Lev), 20.9 (CH₃Ac). HRMS (ESI): m/z calcd for

C₃₅H₄₃N₃NaO₁₄[M + Na]⁺752.2637, found 752.2662.

Methyl O-(Methyl-2-O-acetyl-3-O-benzyl-4-O-allyloxycarbonyl-

β-^D-glucopyranosyluronate)-(1 → 4)-O-(2-N-[(2-triﬂuoromethyl)-

benzylidene]-3-O-benzyl-6-O-levulinoyl-2-deoxy-α-^D-glucopyrano-

syl)-(1 → 4)-O-(methyl-2-O-levulinoyl-3-O-benzyl-α-^L-idopyranosy-

luronate)-(1 → 4)-O-2-azido-3-O-benzy-6-O-acetyl-2-deoxy-α-^D-

glucopyranoside (23). Freshly activated 4 Å molecular sieves were

added to a solution of glycosyl donor 4 (73 mg, 66 μmol) and

glycosyl acceptor 22 (40 mg, 55 μmol) in anhydrous DCM (0.44 mL,

0.15 M based on the glycosyl donor). After stirring for 15 min at

room temperature, the solution was cooled to −20 °C, followed by

the addition of TfOH (2.5 μL, 27 μmol). The reaction mixture was

stirred for 12 h and quenched by the addition of Et₃N. The reaction

mixture was ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The residue was puriﬁed by silica gel column chromatog-

raphy using petroleum ether and EtOAc (1:1 v/v, 0.1% Et₃N) as the

white-powder sodium salt (4.3 mg, 60% over three steps). H NMR

(600 MHz, D₂O): δ 7.84 (d, J = 10.2 Hz, 1H, NHCOCH₃), 7.56−

7.33 (m, 20H, CH aromatic), 5.28 (s, 1H, H1^B), 4.98−4.71 (m, 9H, 2

× CH₂Bn, 2 × CHHBn, H1^Aat 4.92, H1^Cat 4.86, H5^Bat 4.79), 4.68

(d, J = 7.9 Hz, 1H, H1^D), 4.61 (d, J = 11.4 Hz, 1H, CHHBn), 4.59 (d,

J = 11.4 Hz, 1H, CHHBn), 4.52 (dd, J = 11.2, 2.4 Hz, 1H, H6a^C),

4.45 (s, 1H, H2^B), 4.31−4.26 (m, 1H, H6b^C), 4.14−4.02 (m, 4H,

H4^B, H5^C, H3^B, H2^C), 3.99−3.85 (m, 4H, H4^C, H4^A, H6a^A, H6b^A),

3.85−3.70 (m, 5H, H3^A, H3^C, H5^A, H5^D, H4^D), 3.68−3.58 (m, 2H,

https://dx.doi.org/10.1021/acs.joc.0c01881

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

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Article

H2^A, H3^D), 3.50 (bt, J = 8.6 Hz, 1H, H2^D), 3 0.42 (s, 3H, OCH₃),

1.84 (s, 3H, NHCOCH₃). ¹³C{¹H} NMR (151 MHz, D₂O): δ 129.0,

128.8, 128.67, 128.66, 102.6 (C1^D), 98.3 (C1^B), 97.8 (C1^A), 93.8

(C1^C), 83.7 (C3^D), 78.6 (C3^C, C3^A), 76.7 (C5^D), 76.0 (C4^C), 75.3

(OCH₂Bn), 74.9 (OCH₂Bn), 74.8 (C4^A), 74.6 (OCH₂Bn), 73.3

(C2^D), 72.3 (OCH₂Bn), 71.7 (C4^D), 71.4 (C2^B), 71.3 (C5^A), 69.4

(C5^C, C3^B), 68.8 (C4^B), 67.9 (C5^B), 66.0 (C6^C), 63.5 (C2^A), 59.9

(C6^A), 54.9 (OCH₃), 51.9 (C2^C), 22.4 (NHCOCH₃). HRMS (ESI):

m/z calcd for C₅₅H₆₅N₄O₂₈S₂[M − 4Na + 3H]⁻1293.3232, found

1293.3221.

The reaction mixture was stirred for 1 h and quenched with pyridine.

The reaction mixture was ﬁltered, and the ﬁltrate was concentrated in

vacuo. The residue was puriﬁed by silica gel column chromatography

using a stepwise gradient of petroleum ether and EtOAc (3:2 to 1:1 v/

v) as the eluent to give disaccharide 31 (264 mg, 52%). R_f= 0.24

(petroleum ether/EtOAc, 1:1 v/v). [α]²_D⁰+16.6 (c = 2.3, CHCl₃). ¹H

NMR (400 MHz, CDCl₃): δ 7.82−7.05 (m, 38H, CH aromatic), 5.25

(d, J = 3.9 Hz, 1H, H1^B), 5.17 (bd, J = 9.5 Hz, 2H, CH₂Cbz), 5.09−

5.04 (m, 2H, H1^C, H1^D), 4.98−4.93 (m, 2H, H4^D, H2^B), 4.89−4.68

(m, 10H, H5^D, H2^D, H1^A, H5^B, 2 × CH₂Bn, 2 × CHHBn), 4.66 (d, J

= 10.7 Hz, 1H, CHHBn), 4.60 (d, J = 10.7 Hz, 1H, CHHBn), 4.53−

4.35 (m, 6H, NCH₂Bn, H6a^C, H6a^A, CH₂Fmoc), 4.29 (d, J = 12.0

Hz, 1H, H6b^A), 4.23−4.14 (m, 2H, CH Fmoc, H6b^C), 4.03 (t, J = 4.6

Hz, 1H, H4^B), 3.98−3.79 (m, 7H, H3^B, H4^C, H5^A, H4^A, H3^D, H3^A,

H5^C), 3.68−3.59 (m, 2H, H3^C, OCHH linker), 3.47 (s, 3H,

CO₂CH₃), 3.44 (s, 3H, CO₂CH₃), 3.42−3.16 (m, 5H, OCHH linker,

H2^A, H2^C, CH₂N linker), 2.86−2.46 (m, 12H, 6 O) × CH₂Lev), 2.17

(s, 3H, CH₃Lev), 2.11 (s, 6H, CH₃Ac, CH₃Lev), 2.04 (s, 3H, CH₃

Lev), 1.70−1.20 (m, 6H, 3 × CH₂linker). ¹³C{¹H} NMR (151 MHz,

CDCl₃): δ 207.0 (CH₃CO Lev), 206.4 (CH₃CO Lev), 206.2

(CH₃CO Lev), 172.3 (CH₂CO Lev), 172.2 (CH₂CO Lev), 171.8

(CH₂CO Lev), 170.9 (CO Ac), 169.5 (CO₂CH₃), 168.6 (CO₂CH₃),

154.4, 143.3, 143.2, 141.43, 141.41, 138.2, 138.1, 137.7, 137.6, 137.3,

128.7, 128.62, 128.59, 128.35, 128.33, 128.27, 128.1, 128.02, 127.98,

127.9, 127.64, 127.61, 127.29, 127.27, 125.18, 125.13, 120.2, 98.2

(C1^B), 97.7 (C1^A), 97.4 (C1^D), 96.9 (C1^C), 78.4 (C3^A), 78.2 (C3^C),

76.0 (C4^A), 74.9 (OCH₂Bn), 74.8 (OCH₂Bn), 74.3 (C3^B, C4^C), 73.6

(OCH₂Bn), 73.51 (OCH₂Bn), 73.45 (C3^D), 72.0 (C4^B), 71.5 (C4^D),

70.3 (CH₂Fmoc), 69.7 (C5^C, C5^B, C2^B), 69.1 (C5^A), 68.3 (OCH₂

linker), 68.2 (C2^D), 67.3 (CH₂Cbz), 67.2 (C5^D), 63.4 (C2^A), 63.3

(C2^C), 62.5 (C6^A), 62.1 (C6^C), 52.3 (CO₂CH₃), 51.9 (CO₂CH₃),

50.7 (NCH₂Bn), 50.4 (NCH₂Bn), 47.2 (CH₂N linker), 46.8 (CH

Fmoc), 46.3 (CH₂N linker), 38.1 (CH₂Lev), 37.8 (CH₂Lev), 37.7

(CH₂Lev), 29.9 (CH₃Lev), 29.8 (CH₃Lev), 29.6 (CH₃Lev), 29.1

(CH₂linker), 28.3 (CH₂Lev), 28.0 (CH₂Lev), 27.8 (CH₂Lev), 27.6

(CH₂linker), 23.4 (CH₂linker), 21.0 (CH₃Ac). HRMS (ESI): m/z

calcd for C₁₀₆H₁₁₇N₇NaO₃₂[M + Na]⁺2022.7635, found 2022.7706.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(Methyl-2-O-

levulinoyl-3-O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-

azido-3-O-benzyl-6-O-levulinoyl-2-deoxy-α-^D-glucopyranosyl)-(1

→ 4)-O-(methyl-2-O-levulinoyl-3-O-benzyl-α-^L-idopyranosyluro-

nate)-(1 → 4)-O-2-azido-3-O-benzy-6-O-acetyl-2-deoxy-α-^D-gluco-

pyranoside (32). A solution of a tetrasaccharide 31 (264 mg, 0.13

mmol) in a mixture of DCM and Et₃N (2.5 mL, 4:1 v/v, 50 mM) was

stirred at ambient temperature for 2 h until TLC analysis indicated

completion of the reaction. The mixture was concentrated under

reduced pressure, and the residue was puriﬁed by silica gel column

chromatography with petroleum ether and EtOAc (1:1 v/v) as the

eluent to give the pure product 32 as a white amorphous solid (211

mg, 90%). R_f= 0.36 (petroleum ether/EtOAc, 1:3 v/v). [α]²_D⁰+21.4 (c

= 2.6, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 7.77−6.56 (m, 30H,

CH aromatic), 5.24 (d, J = 3.5 Hz, 1H, H1^B), 5.17 (bd, J = 10.5 Hz,

2H, CH₂Cbz), 5.05 (d, J = 3.5 Hz, 1H, H1^C), 4.99 (bs, 1H, H1^D),

4.95 (t, J = 3.5 Hz, 1H, H2^B), 4.90−4.55 (m, 12H, H2^D, H1^A, H5^D,

H5^B, 4 × CH₂Bn), 4.49 (bs, 2H, NCH₂Bn), 4.39 (d, J = 12.5 Hz, 2H,

H6a^A, H6a^C), 4.28 (d, J = 12.2 Hz, 1H, H6b^A), 4.19 (d, J = 12.5 Hz,

1H, H6b^C), 4.73 (t, J = 3.3 Hz, 1H, H3^D), 4.04−3.77 (m, 8H, H4^B,

H4^D, H3^B, H5^A, H4^C, H4^A, H3^A, H5^C), 3.68−3.60 (m, 2H, H3^C,

OCHH linker), 3.48 (s, 3H, CO₂CH₃), 3.44 (s, 3H, CO₂CH₃), 3.41−

3.17 (m, 5H, H2^A, H2^C, OCHH linker, CH₂N linker), 2.78−2.46 (m,

12H, 6 × CH₂Lev), 2.17 (s, 3H, CH₃Lev), 2.15 (s, 3H, CH₃Lev),

2.10 (s, 6H, CH₃Lev, CH₃Ac), 1.65−1.26 (m, 6H, 3 × CH₂linker).

¹³C{¹H} NMR (101 MHz, CDCl₃): δ 206.8 (CH₃CO Lev), 206.5

Methyl O-(3-O-Benzyl-β-^D-glucopyranosyluronate)-(1 → 4)-O-

(2-acetylamino-3-O-benzyl-6-O-sulfate-2-deoxy-α-^D-glucopyrano-

syl)-(1 → 4)-O-(2-O-sulfate-3-O-benzyl-α-^L-idopyranosyluronate)-

(1 → 4)-O-2-sulfamino-3-O-benzy-2-deoxy-α-^D-glucopyranoside

(27). The azido group of compound 26 (4.3 mg, 3.1 μmol) was

reduced to a free amine, followed by N-sulfation, according to the

general procedure to give compound 27 as a white powder sodium

salt (3.0 mg, 66% over two steps). ¹H NMR (600 MHz, D₂O): δ 7.89

(d, J = 10.2 Hz, 1H, NHCOCH₃), 7.55−7.34 (m, 20H, CH

aromatic), 5.28 (s, 1H, H1^B), 5.02 (d, J = 3.6 Hz, 1H, H1^A), 4.95 (d, J

= 11.4 Hz, 1H, CHHBn), 4.90 (s, 2H, CH₂Bn), 4.85−4.70 (m, 6H,

CH₂Bn, 2 × CHHBn, H1^Cat 4.80, H5^Bat 4.77), 4.68 (d, J = 7.8 Hz,

1H, H1^D), 4.58 (d, J = 12.0 Hz, 1H, CHHBn), 4.53 (dd, J = 11.2, 2.4

Hz, 1H, H6a^C), 4.41 (s, 1H, H2^B), 4.30−4.26 (m, 1H, H6b^C), 4.12−

4.09 (m, 2H, H4^B, H5^C), 4.06−4.01 (m, 2H, H3^B, H2^C), 3.95 (t, J =

9.3 Hz, 1H, H4^C), 3.92−3.87 (m, 2H, H6a^A, H4^A), 3.85 (dd, J = 12.5,

3.9 Hz, 1H, H6b^A), 3.81−3.66 (m, 5H, H5^A, H3^C, H5^D, H3^A, H4^D),

3.63 (t, J = 8.8 Hz, 1H, H3^D), 3.53−3.49 (m, 1H, H2^D), 3 0.43 (s, 3H,

OCH₃), 3.39 (dd, J = 10.4, 3.7 Hz, 1H, H2^A), 1.83 (s, 3H,

NHCOCH₃). ¹³C{¹H} NMR (151 MHz, D₂O): δ 128.9, 128.79,

128.77, 128.48, 128.46, 102.4 (C1^D), 98.23 (C1^B), 98.17 (C1^A), 93.4

(C1^C), 83.6 (C3^D), 78.6 (C3^C), 76.7 (C5^D, C3^A), 75.81 (C4^A), 75.76

(C4^C), 74.7 (OCH₂Bn), 74.5 (OCH₂Bn), 74.3 (OCH₂Bn), 73.2

(C2^D), 72.1 (OCH₂Bn), 71.8 (C4^D), 71.5 (C2^B), 70.6 (C5^A), 68.9

(C3^B), 68.7 (C4^B, C5^C), 68.0 (C5^B), 65.9 (C6^C), 60.2 (C6^A), 57.3

(C2^A), 55.2 (OCH₃), 51.7 (C2^C), 22.2 (NHCOCH₃). HRMS (ESI):

m/z calcd for C₅₅H₆₇N₂O₃₁S₃[M −5Na + 4H]⁻1347.2895, found

1347.2885.

Methyl O-(β-^D-Glucopyranosyluronate)-(1 → 4)-O-(2-acetylami-

no-6-O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sul-

fate-α-^L-idopyranosyluronate)-(1 → 4)-O-2-sulfamino-2-deoxy-α-

D-glucopyranoside (28). Compound 27 (3.0 mg, 2.1 μmol) was

subjected to global deprotection according to the general procedure

to give compound 28 as a white-powder sodium salt (2.0 mg, 90%).

¹H NMR (600 MHz, D₂O): δ 5.23 (bs, 2H, H1^B, H5^B), 5.12 (d, J =

3.6 Hz, 1H, H1^C), 5.03 (d, J = 3.4 Hz, 1H, H1^A), 4.64 (d, J = 8.2 Hz,

1H, H1^D), 4.43 (dd, J = 11.2, 3.0 Hz, 1H, H6a^C), 4.31−4.26 (m, 2H,

H2^B, H6b^C), 4.24 (t, J = 2.6 Hz, 1H, H3^B), 4.09 (bs, 1H, H4^B), 4.06

(dd, J = 10.2, 3.5 Hz, 1H, H2^C), 4.04−4.01 (m, 1H, H5^D), 3.95−3.88

(m, 2H, H5^C, H6a^A), 3.85 (dd, J = 12.0, 4.4 Hz, 1H, H6b^A), 3.82−

3.76 (m, 3H, H3^C, H5^A, H4^C), 3.73−3.66 (m, 2H, H4^A, H3^A), 3.61−

3.56 (m, 2H, H4^D, H3^D), 3 0.42 (s, 3H, OCH₃), 3.40−3.36 (m, 1H,

H2^D), 3.26 (dd, J = 9.9, 3.5 Hz, 1H, H2^A), 2.09 (s, 3H, NHCOCH₃).

¹³C{¹H} NMR (151 MHz, D₂O): δ 101.9 (C1^D), 99.2 (C1^B), 98.2

(C1^A), 94.9 (C1^C), 77.6 (C4^C, C4^A), 74.9 (C3^D), 74.5 (C5^D), 73.2

(C2^B), 72.8 (C2^D), 72.0 (C4^B), 71.4 (C4^D), 70.3 (C5^A), 69.7 (C3^A),

69.5 (C3^C), 69.2 (C5^C), 66.4 (C5^B), 65.8 (C6^C), 64.2 (C3^B), 59.8

(C6^A), 58.1 (C2^A), 55.3 (OCH₃), 52.8 (C2^C), 22.2 (NHCOCH₃).

HRMS (ESI): m/z calcd for C₂₇H₄₂N₂O₃₁S₃[M − 5Na + 3H]²⁻

493.0472, found 493.0489.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-[Methyl-2-O-

levulinoyl-3-O-benzyl-4-O-(9-ﬂuorenylmethyloxycarbonyl)-α-^L-ido-

pyranosyluronate]-(1 → 4)-O-(2-azido-3-O-benzyl-6-O-levulinoyl-

2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(methyl-2-O-levulinoyl-3-

O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-2-azido-3-O-benzy-

6-O-acetyl-2-deoxy-α-^D-glucopyranoside (31). Freshly activated 4 Å

molecular sieves were added to a solution of glycosyl donor 29 (353

mg, 0.30 mmol) and glycosyl acceptor 30 (259 mg, 0.25 mmol) in

anhydrous DCM (6 mL, 0.05 M based on the glycosyl acceptor).

After stirring for 30 min at room temperature, the solution was cooled

to −40 °C, followed by the addition of TfOH (13 μL, 0.15 mmol).

(CH₃CO Lev), 206.1 (CH₃CO Lev), 172.3 (CH₂CO Lev), 172.2

(CH₂CO Lev), 171.5 (CH₂CO Lev), 170.9 (CO Ac), 169.6

(CO₂CH₃), 169.4 (CO₂CH₃), 138.1, 138.0, 137.8, 137.5, 137.3,

128.69, 128.65, 128.6, 128.29, 128.27, 128.10, 127.97, 127.95, 127.9,

127.6, 127.5, 127.4, 127.3, 98.2 (C1^B), 97.9 (C1^D), 97.7 (C1^A), 96.9

(C1^C), 78.3 (C3^A), 78.2 (C3^C), 75.9 (C4^A), 75.3 (C3^D), 74.8

(OCH₂Bn), 74.6 (OCH₂Bn), 74.4 (C5^A), 73.8 (C3^B), 73.5

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(OCH₂Bn), 72.8 (OCH₂Bn), 72.1 (C4^B), 69.7 (C5^C), 69.6 (C5^B),

69.5 (C2^B), 69.1 (C4^C), 69.0 (C5^D), 68.3 (OCH₂linker, C2^D), 67.8

(C4^D), 67.3 (CH₂Cbz), 63.4 (C2^A), 63.3 (C2^C), 62.5 (C6^A), 62.2

(C6^C), 52.1 (CO₂CH₃), 51.9 (CO₂CH₃), 50.7 (NCH₂Bn), 50.4

(NCH₂Bn), 47.2 (CH₂N linker), 46.3 (CH₂N linker), 38.0 (CH₂

Lev), 37.9 (CH₂Lev), 37.8 (CH₂Lev), 29.9 (CH₃Lev), 29.8 (2 ×

CH₃Lev), 29.1 (CH₂linker), 28.08 (CH₂Lev), 28.05 (CH₂Lev),

27.8 (CH₂linker), 23.4 (CH₂linker), 21.0 (CH₃Ac). HRMS (ESI):

m/z calcd for C₉₁H₁₀₇N₇NaO₃₀[M + Na]⁺1800.6955, found

1800.7026.

46.3 (CH₂N linker), 38.1 (CH₂Lev), 37.8 (CH₂Lev), 37.6 (CH₂

Lev), 30.0 (CH₃Lev), 29.8 (CH₃Lev), 29.5 (CH₃Lev), 29.1 (CH₂

linker), 28.1 (CH₂Lev, CH₂linker), 27.8 (CH₂Lev), 27.6 (CH₂Lev),

23.4 (CH₂linker), 21.0 (2 × CH₃Ac), 20.8 (CH₃Ac). HRMS (ESI):

m/z calcd for C₁₃₄H₁₅₆F₃N₉O₄₄[M + H + NH₄]²⁺1326.5110, found

1326.5123.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(Methyl-2-O-

acetyl-3-O-benzyl-4-O-allyloxycarbonyl-β-^D-glucopyranosyluro-

nate)-(1 → 4)-O-2-N-[(2-triﬂuoromethyl)benzylidene]-3-O-benzyl-

6-O-levulinoyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(methyl-2-

O-levulinoyl-3-O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-

azido-3-O-benzy-6-O-levulinoyl-2-deoxy-α-^D-glucopyranosyl)-(1

→ 4)-O-(methyl-2-O-levulinoyl-3-O-benzyl-α-^L-idopyranosyluro-

nate)-(1 → 4)-O-2-azido-3-O-benzy-6-O-acetyl-2-deoxy-α-^D-gluco-

pyranoside (34). Freshly activated 4 Å molecular sieves were added

to a solution of glycosyl donor 4 (36.0 mg, 33 μmol) and glycosyl

acceptor 32 (48.2 mg, 27 μmol) in anhydrous DCM (0.25 mL, 0.13

M based on the glycosyl donor). After stirring for 15 min at room

temperature, the solution was cooled to −20 °C, followed by the

addition of TfOH (1.2 μL, 14 μmol). The reaction mixture was stirred

for 12 h, after which the reaction was quenched by the addition of

Et₃N. The mixture was ﬁltered, and the ﬁltrate was concentrated

under reduced pressure. The residue was puriﬁed by silica gel column

chromatography using petroleum ether and EtOAc (1:1 v/v, 0.1%

Et₃N) as the eluent to give hexasaccharide 34 as a white foam (35.0

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(Methyl-2-O-

acetyl-3-O-benzyl-4-O-allyloxycarbonyl-β-^D-glucopyranosyluro-

nate)-(1 → 4)-O-2-N-[(2-triﬂuoromethyl)benzylidene]-3-O-benzyl-

6-O-acetyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(methyl-2-O-

levulinoyl-3-O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-

azido-3-O-benzy-6-O-levulinoyl-2-deoxy-α-^D-glucopyranosyl)-(1

→ 4)-O-(methyl-2-O-levulinoyl-3-O-benzyl-α-^L-idopyranosyluro-

nate)-(1 → 4)-O-2-azido-3-O-benzy-6-O-acetyl-2-deoxy-α-^D-gluco-

pyranoside (33). Freshly activated 4 Å molecular sieves were added

to a solution of glycosyl donor 3 (67.0 mg, 64 μmol) and glycosyl

acceptor 32 (95.0 mg, 53 μmol) in anhydrous DCM (0.25 mL, 0.13

M based on the glycosyl donor). After stirring for 15 min at room

temperature, the solution was cooled to −20 °C, followed by the

addition of TfOH (2.4 μL, 27 μmol). After stirring the reaction

mixture for 12 h, the reaction was quenched by the addition of Et₃N.

The reaction mixture was ﬁltered, and the ﬁltrate was concentrated

under reduced pressure. The residue was puriﬁed by silica gel column

chromatography using petroleum ether and EtOAc (1:1 v/v, 0.1%

Et₃N) as the eluent to give hexasaccharide 33 as a white foam (73.2

mg, 52%). [α]²_D⁰+42.0 (c = 1.2, CHCl₃).¹H NMR (600 MHz,

CDCl₃): δ 8.50 (d, J = 1.8 Hz, 1H, NCHPhCF₃), 8.18 (d, J = 7.6

Hz, 1H, ArH⁴), 7.58 (d, J = 7.7 Hz, 1H, H¹), 7.51−7.44 (m, 2H, H²,

H³), 7.41−6.77 (m, 40H, 7 × Bn, Cbz CH aromatic), 5.91−5.84 (m,

1H, CH₂CHCH₂), 5.38 (d, J = 5.9 Hz, 1H, H1^D), 5.36−5.30 (m,

2H, CHHCHCH₂, H1^B), 5.28−5.25 (m, 1H, CHHCHCH₂),

5.19−5.15 (m, 2H, CH₂Cbz), 5.08 (dd, J = 9.3, 8.0 Hz, 1H, H2^F),

5.06 (d, J = 3.4 Hz, 1H, H1^C), 5.02−4.99 (m, 1H, H4^F), 4.97 (d, J =

3.4 Hz, 1H, H1^E), 4.94 (t, J = 5.1 Hz, 1H, H2^B), 4.91−4.87 (m, 3H,

H2^D, CHHBn, CHHBn), 4.84−4.78 (m, 2H, H1^A, H5^B), 4.76−4.47

(m, 14H, 3 × CH₂Bn, CHHBn, H1^Fat 4.59, CH₂CHCH₂,

CHHBn, H5^D, NCH₂Bn), 4.45 (d, J = 11.8 Hz, 2H, CHHBn, H6a^C),

4.41 (dd, J = 11.9, 1.6 Hz, 1H, H6a^E), 4.37 (d, J = 11.6 Hz, 2H,

CHHBn, H6a^A), 4.31 (dd, J = 12.0, 2.6 Hz, 1H, H6b^A), 4.26 (dd, J =

12.6, 2.6 Hz, 1H, H6b^C), 4.23 (dd, J = 12.0, 3.4 Hz, 1H, H6b^E), 4.14−

4.10 (m, 1H, H5^E), 4.07 (t, J = 5.1 Hz, 1H, H4^B), 4.02−3.98 (m, 2H,

H4^D, H3^D), 3.95−3.80 (m, 9H, H3^B, H5^C, H5^A, H3^E, H5^F, H4^C, H4^A,

H3^A, H4^E), 3.71 (t, J = 9.3 Hz, 1H, H3^F), 3.70−3.59 (m, 5H, H3^C,

OCHH linker, CO₂CH₃), 3.52 (s, 3H, CO₂CH₃), 3.48 (s, 3H,

CO₂CH₃), 3.44 (dd, J = 9.6, 3.1 Hz, 1H, H2^E), 3.42−3.31 (m, 1H,

OCHH linker), 3.31−3.18 (m, 4H, H2^C, H2^A, CH₂N linker), 2.85−

2.39 (m, 12H, 6 × CH₂Lev), 2.21 (s, 3H, CH₃Lev), 2.11 (s, 6H,

CH₃Ac, CH₃Lev), 2.07 (s, 3H, CH₃Ac), 1.98 (s, 3H, CH₃Ac), 1.97

(s, 3H, CH₃Lev), 1.67−1.26 (m, 6H, 3 × CH₂linker). ¹³C{¹H}

NMR (151 MHz, CDCl₃): δ 206.7 (CH₃CO Lev), 206.2 (CH₃CO

Lev), 206.1 (CH₃CO Lev), 172.6 (CH₂CO Lev), 172.1 (CH₂CO

Lev), 171.8 (CH₂CO Lev), 171.0 (CO Ac), 170.7 (CO Ac), 170.3

(CO₂CH₃), 169.9 (CO₂CH₃), 169.1 (CO Ac), 167.2 (CO₂CH₃),

160.6 (NCHPhCF₃), 153.9 (CO Alloc), 138.4, 138.2, 138.03,

137.98, 137.8, 137.74, 137.66, 132.0, 131.2 (CH₂CHCH₂), 130.7,

129.2, 129.1, 128.7, 128.6, 128.4, 128.3, 128.1, 128.1, 128.03, 127.98,

127.9, 127.8, 127.6, 127.5, 127.3, 127.2, 126.7, 119.5 (CH₂

CHCH₂), 101.2 (C1^F), 100.8 (C1^E), 98.2 (C1^B), 97.9 (C1^D), 97.7

(C1^A), 97.2 (C1^C), 79.7 (C3^F), 78.5 (C3^A), 78.3 (C4^E), 78.0 (C3^E),

77.9 (C3^C), 77.4 (C3^D), 76.3 (C4^A), 76.0 (C4^C), 75.2 (C3^B), 75.2

(C4^F, OCH₂Bn), 75.1 (C4^D, OCH₂Bn), 74.8 (C2^E, OCH₂Bn), 74.7

(OCH₂Bn), 74.2 (OCH₂Bn), 73.9 (OCH₂Bn), 72.8 (C5^F), 72.5

(C2^F), 72.3 (C4^B), 71.8 (C5^D), 71.5 (C2^D), 70.2 (C2^B, C5^B), 69.9

(C5^E), 69.8 (C5^C), 69.2 (CH₂CHCH₂), 69.0 (C5^A), 68.3 (OCH₂

linker), 67.3 (CH₂Cbz), 63.3 (C2^A), 63.0 (C2^C), 62.5 (C6^A), 62.11

(C6^E), 62.08 (C6^C), 52.8 (CO₂CH₃), 52.3 (CO₂CH₃), 52.0

(CO₂CH₃), 50.7 (NCH₂Bn), 50.4 (NCH₂Bn), 47.2 (CH₂N linker),

mg, 48%). R_f= 0.36 (petroleum ether/EtOAc, 1:2 v/v). H NMR

(600 MHz, CDCl₃): δ 8.50 (d, J = 1.8 Hz, 1H, NCHPhCF₃), 8.15

(d, J = 6.7 Hz, 1H, H⁴), 7.58 (d, J = 7.7 Hz, 1H, H¹), 7.49−7.43 (m,

2H, H², H³), 7.38−6.75 (m, 40H, 7 × Bn, Cbz CH aromatic), 5.91−

5.83 (m, 1H, CH₂CHCH₂), 5.38 (d, J = 6.2 Hz, 1H, H1^D), 5.34−

5.31 (m, 2H, CHHCHCH₂, H1^B), 5.26−5.22 (m, 1H, CHH

CHCH₂), 5.21−5.13 (m, 2H, CH₂Cbz), 5.10 (dd, J = 9.4, 8.4 Hz, 1H,

H2^F), 5.05 (d, J = 3.4 Hz, 1H, H1^C), 5.04−4.96 (m, 3H, H4^F,

CHHBn, H1^E), 4.94 (t, J = 4.9 Hz, 1H, H2^B), 4.92−4.84 (m, 3H,

H2^D, CHHBn, CHHBn), 4.84−4.77 (m, 3H, H1^A, H5^B, H1^F), 4.77−

4.51 (m, 9H, 2 × CH₂Bn, CHHBn, CHHBn, CH₂CHCH₂,

CHHBn), 4.51−4.47 (m, 2H, NCH₂Bn), 4.46−4.20 (m, 10H, H6a^C,

H5^D, CHHBn, H6a^E, CHHBn, H6a^A, H6b^A, H6b^C, H5^F, H6b^E),

4.16−4.12 (m, 1H, H5^E), 4.07 (t, J = 5.3 Hz, 1H, H4^B), 4.01−3.82

(m, 11H, H4^D, H3^D, H4^E, H3^F, H3^B, H5^C, H5^A, H4^A, H4^C, H3^E,

H3^A), 3.69−3.58 (m, 5H, H3^C, OCHH linker, CO₂CH₃), 3.52 (s, 3H,

CO₂CH₃), 3.50−3.30 (m, 5H, CO₂CH₃, H2^E, OCHH linker), 3.30−

3.17 (m, 4H, H2^C, H2^A, CH₂N linker), 2.97−2.38 (m, 16H, 8 × CH₂

Lev), 2.21 (s, 3H, CH₃Lev), 2.20 (s, 3H, CH₃Lev), 2.11 (s, 6H, CH₃

Ac, CH₃Lev), 1.98 (s, 3H, CH₃Ac), 1.97 (s, 3H, CH₃Lev), 1.67−

1.26 (m, 6H, 3 × CH₂linker). ¹³C{¹H} NMR (151 MHz, CDCl₃): δ

206.9 (CH₃CO Lev), 206.6 (CH₃CO Lev), 206.2 (CH₃CO Lev),

206.0 (CH₃CO Lev), 172.8 (CH₂CO Lev), 172.6 (CH₂CO Lev),

172.1 (CH₂CO Lev), 171.8 (CH₂CO Lev), 171.0 (CO Ac), 170.3

(CO₂CH₃), 170.0 (CO₂CH₃), 169.2 (CO Ac), 167.7 (CO₂CH₃),

160.7 (NCHPhCF₃), 154.1 (CO Alloc), 138.6, 138.3, 138.03,

137.97, 137.9, 137.8, 132.0, 131.4 (CH₂CHCH₂), 130.6, 130.2,

129.9, 128.7, 128.6, 128.4, 128.1, 128.0, 127.91, 127.87, 127.7, 127.5,

127.4, 127.3, 127.1, 126.7, 125.61, 125.57, 119.3 (CH₂CHCH₂),

101.01 (C1^E), 100.95 (C1^F), 98.2 (C1^B), 97.9 (C1^D), 97.8 (C1^A),

97.2 (C1^C), 79.7 (C3^F), 78.3 (C3^A, C4^E), 77.9 (C3^E, C3^C), 77.6

(C3^D), 76.4 (C4^A), 76.2 (C4^C), 75.5 (C4^F), 75.3 (C4^D, C3^B,

OCH₂Bn), 75.2 (OCH₂Bn), 75.1 (OCH₂Bn), 74.7 (C2^E), 74.5

(OCH₂Bn), 74.3 (OCH₂Bn), 73.9 (OCH₂Bn), 72.6 (C2^F), 72.5

(C5^F), 72.4 (C4^B), 72.0 (C5^D), 71.8 (C2^D), 70.3 (C2^B, C5^B), 69.8

(C5^E, C5^C), 69.1 (CH₂CHCH₂), 69.0 (C5^A), 68.3 (OCH₂linker),

67.3 (CH₂Cbz), 63.3 (C2^A), 63.0 (C2^C), 62.5 (C6^A), 62.3 (C6^E),

62.1 (C6^C), 52.6 (CO₂CH₃), 52.3 (CO₂CH₃), 52.0 (CO₂CH₃), 50.7

(NCH₂Bn), 50.4 (NCH₂Bn), 47.2 (CH₂N linker), 46.3 (CH₂N

linker), 38.2 (CH₂Lev), 38.1 (CH₂Lev), 37.8 (CH₂Lev), 37.6 (CH₂

Lev), 30.0 (CH₃Lev), 29.8 (2 × CH₃Lev), 29.5 (CH₃Lev), 29.1

(CH₂linker), 28.1 (CH₂Lev, CH₂linker), 27.8 (CH₂Lev), 27.6 (CH₂

Lev), 27.4 (CH₂Lev), 23.4 (CH₂linker), 21.0 (CH₃Ac), 20.9 (CH₃

Ac). HRMS (ESI): m/z calcd for C₁₃₇H₁₅₉F₃N₉NaO₄₅[M + Na +

NH₄]²⁺1365.5151, found 1365.5145.

https://dx.doi.org/10.1021/acs.joc.0c01881

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-[Methyl-2-O-

acetyl-3-O-benzyl-4-O-(9-ﬂuorenylmethyloxycarbonyl)-β-^D-gluco-

pyranosyluronate]-(1 → 4)-O-(2-azido-3-O-benzyl-6-O-acetyl-2-

deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(methyl-2-O-levulinoyl-3-O-

benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-azido-3-O-benzyl-6-

O-levulinoyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(methyl-2-O-

levulinoyl-3-O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-2-

azido-3-O-benzy-6-O-acetyl-2-deoxy-α-^D-glucopyranoside (35).

Freshly activated 4 Å molecular sieves were added to a solution of

glycosyl donor 20 (47.5 mg, 36 μmol) and glycosyl acceptor 32 (53.5

mg, 30 μmol) in anhydrous DCM (1 mL). After stirring for 30 min at

room temperature, the solution was cooled to −40 °C, followed by

the addition of TfOH (2.3 μL, 15 μmol). The reaction mixture was

stirred for 1 h, after which the reaction was quenched with pyridine.

The reaction mixture was ﬁltered, and the ﬁltrate was concentrated in

vacuo. The residue was puriﬁed by silica gel column chromatography

using a stepwise gradient of petroleum ether and EtOAc (3:1 to 1:1 v/

v) as the eluent to give hexasaccharide 35 as a white foam (43.8 mg,

55%). R_f= 0.54 (petroleum ether/EtOAc, 1:2 v/v). [α]²_D⁰+33.6 (c =

puriﬁed by silica gel column chromatography using petroleum ether

and EtOAc (1:1 v/v) as the eluent to give hexasaccharide 36 as a

white foam. (90 mg, 69%). [α]²_D⁰+25.1 (c = 1.4, CHCl₃) H NMR

(400 MHz, CDCl₃): δ 7.78−7.11 (m, 48H, CH aromatic), 5.30−5.26

(m, 2H, H1^B, H1^D), 5.22 (d, J = 11.3 Hz, 1H, CHHBn), 5.30−5.26

(m, 2H, CH₂Cbz), 5.13−5.07 (m, 2H, H2^F, H4^F), 5.06−5.04 (m, 2H,

H1^C, H1^E), 4.95 (d, J = 4.6 Hz, 1H, H2^B), 4.91−4.78 (m, 5H, H2^D,

CHHBn, CHHBn, H1^Aat 4.83, H5^B), 4.77−4.62 (m, 9H, H1^Fat 4.76,

2 × CH₂Bn, CHHBn, CHHBn, CHHBn, CHHBn), 4.59 (d, J = 11.8

Hz, 1H, CHHBn), 4.52−4.47 (m, 3H, NCH₂Bn, H5^D), 4.44−4.27

(m, 7H, H6a^C, H6a^A, H6a^E, H6b^A, CH₂Fmoc, H5^F), 4.26−4.19 (m,

2H, H6b^C, CH Fmoc), 4.17 (dd, J = 12.7, 2.0 Hz, 1H, H6b^E), 4.06 (t,

J = 4.9 Hz, 1H, H4^B), 4.02−3.82 (m, 11H, H3^Fat 4.00, H3^D, H4^D,

H3^B, H4^E, H5^A, H5^C, H5^E, H4^A, H4^C, H3^A), 3.69−3.59 (m, 3H, H3^C,

H3^E, OCHH linker), 3.58 (s, 3H, CO₂CH₃), 3.50 (s, 3H, CO₂CH₃),

3.45 (s, 3H, CO₂CH₃), 3.41−3.18 (m, 6H, OCHH linker, CH₂N

linker, H2^C, H2^A, H2^E), 2.99−2.40 (m, 16H, 8 × CH₂Lev), 2.22 (s,

3H, CH₃Lev), 2.17 (s, 3H, CH₃Lev), 2.12 (s, 6H, CH₃Lev, CH₃

Ac), 2.06 (s, 3H, CH₃Lev), 1.97 (s, 6H, CH₃Ac), 1.64−1.26 (m, 6H,

3 × CH₂linker). ¹³C{¹H} NMR (151 MHz, CDCl₃): δ 207.0

(CH₃CO Lev), 206.6 (CH₃CO Lev), 206.2 (CH₃CO Lev), 206.2

(CH₃CO Lev), 172.7 (CH₂CO Lev), 172.5 (CH₂CO Lev), 172.1

(CH₂CO Lev), 172.0 (CH₂CO Lev), 171.0 (CO Ac), 169.9

(CO₂CH₃), 169.8 (CO₂CH₃), 169.2 (CO Ac), 167.6 (CO₂CH₃),

154.2 (CO Fmoc), 143.5, 143.2, 141.41, 141.39, 138.3, 138.2, 138.0,

137.8, 137.7, 137.0, 136.9, 129.2, 128.7, 128.6, 128.5, 128.4, 128.3,

128.2, 128.0, 127.92, 127.87, 127.8, 127.7, 127.6, 127.5, 127.4, 127.29,

125.28, 125.2, 120.2, 100.6 (C1^F), 98.2 (C1^D), 98.1 (C1^B), 97.7 (C1^A,

C1^E), 97.0 (C1^C), 79.4 (C3^F), 78.4 (C3^A), 78.1 (C3^C), 77.7 (C4^E),

77.6 (C3^E), 76.2 (C4^A), 75.8 (C4^C), 75.5, 75.0, 74.6, 74.2, 73.7, 73.1,

72.5 (C2^F), 72.4 (C5^F), 72.2 (C4^B), 70.7 (C5^D, C2^D), 70.5 (CH₂

Fmoc), 70.0 (C5^B), 69.7 (C2^B), 69.2 (C5^E), 69.0 (C5^C), 68.3 (OCH₂

linker), 67.3 (CH₂Cbz), 63.3 (C2^C), 63.1 (C2^A), 62.7 (C2^E), 62.5

(C6^A), 62.1 (C6^C), 61.9 (C6^E), 52.7 (CO₂CH₃), 52.4 (CO₂CH₃),

52.0 (CO₂CH₃), 50.7 (NCH₂Bn), 50.4 (NCH₂Bn), 47.2 (CH₂N

linker), 46.7 (CH Fmoc), 46.3 (CH₂N linker), 38.2 (CH₂Lev), 38.0

(CH₂Lev), 37.8 (CH₂Lev), 37.7 (CH₂Lev), 30.0 (CH₃Lev), 29.9

(CH₃Lev), 29.8 (CH₃Lev), 29.6 (CH₃Lev), 29.1 (CH₂linker), 28.1

(CH₂Lev), 28.0 (CH₂Lev), 27.8 (CH₂Lev, CH₂linker), 27.6 (CH₂

Lev), 23.4 (CH₂linker), 21.0 (CH₃Ac), 20.8 (CH₃Ac). HRMS

(ESI): m/z calcd for C₁₄₀H₁₆₄N₁₂O₄₅[M + 2NH₄]²⁺1367.0468,

found 1367.0444.

2.7, CHCl₃). H NMR (400 MHz, CDCl₃): δ 8.26−6.64 (m, 48H,

CH aromatic), 5.29 (d, J = 4.5 Hz, 1H, H1^B), 5.27 (d, J = 4.9 Hz, 1H,

H1^D), 5.17 (bd, J = 9.5 Hz, 2H, CH₂Cbz), 5.12 (d, J = 11.0 Hz, 1H,

CHHBn), 5.10−5.00 (m, 4H, H2^F, H4^F, H1^E, H1^C), 5.00−4.52 (m,

17H, H2^B, H2^D, CHHBn, H1^Aat 4.82, H5^B, 5 × CH₂Bn, H5^D, H1^Fat

4.53), 4.50 (bs, 2H, NCH₂Bn), 4.47−4.12 (m, 9H, H6a^C, H6a^E,

H6a^A, H6b^A, H6b^E, H6b^C, CH₂Fmoc, CH Fmoc), 4.06 (t, J = 4.9 Hz,

1H, H4^B), 4.03−3.64 (m, 15H, H4^D, H3^B, H3^D, H5^F, H5^E, H5^C, H5^A,

H4^C, H4^E, H3^A, H4^A, H3^F, H3^E, H3^C, OCHH linker), 3.59 (s, 3H,

CO₂CH₃), 3.51 (s, 3H, CO₂CH₃), 3.46 (s, 3H, CO₂CH₃), 3.44−3.18

(m, 6H, OCHH linker, CH₂N linker, H2^C, H2^E, H2^A), 2.82−2.42 (m,

12H, 6 × CH₂Lev), 2.17 (s, 3H, CH₃Lev), 2.11 (s, 6H, CH₃Lev,

CH₃Ac), 2.07 (s, 3H, CH₃Lev), 2.05 (s, 3H, CH₃Ac), 1.95 (s, 6H,

CH₃Ac), 1.64−1.26 (m, 6H, 3 × CH₂linker). ¹³C{¹H} NMR (101

MHz, CDCl₃): δ 206.6 (CH₃CO Lev), 206.2 (CH₃CO Lev), 206.1

(CH₃CO Lev), 172.5 (CH₂CO Lev), 172.1 (CH₂CO Lev), 172.0

(CH₂CO Lev), 170.9 (CO Ac), 170.5 (CO Ac), 169.8 (CO₂CH₃),

169.7 (CO₂CH₃), 169.0 (CO Ac), 167.0 (CO₂CH₃), 154.0, 143.4,

143.1, 141.43, 141.39, 138.2, 138.04, 137.99, 137.7, 137.63, 137.55,

128.7, 128.59, 128.55, 128.53, 128.4, 128.32, 128.30, 128.11, 128.09,

128.02, 128.00, 127.96, 127.91, 127.89, 127.77, 127.74, 127.68,

127.65, 127.6, 127.3, 125.2, 125.1, 120.2, 100.9 (C1^F), 98.2 (C1^D),

98.1 (C1^B), 97.7 (C1^A), 97.5 (C1^E), 97.0 (C1^C), 79.5 (C3^F), 78.4,

78.1, 77.9, 77.6, 76.2, 75.7, 75.3 (OCH₂Bn), 75.2 (C4^F), 75.0 (C3^B,

C3^D, OCH₂Bn), 74.6 (OCH₂Bn), 74.1 (OCH₂Bn), 73.7 (OCH₂Bn),

72.9, 72.8 (C4^D), 72.4 (C2^F), 72.2 (C4^B), 70.6 (C5^D, CH₂Fmoc),

70.5 (C2^D), 70.0 (C2^B, C5^B), 69.7, 69.3, 69.1, 68.3 (OCH₂linker),

67.3 (CH₂Cbz), 63.3 (C2^C), 63.1 (C2^A), 62.8 (C2^E), 62.5 (C6^A),

62.1 (C6^E), 61.7 (C6^C), 52.8 (CO₂CH₃), 52.3 (CO₂CH₃), 52.0

(CO₂CH₃), 50.7 (NCH₂Bn), 50.4 (NCH₂Bn), 47.2 (CH₂N linker),

46.7 (CH Fmoc), 46.3 (CH₂N linker), 38.0 (CH₂Lev), 37.8 (CH₂

Lev), 37.7 (CH₂Lev), 29.9 (CH₃Lev), 29.8 (CH₃Lev), 29.6 (CH₃

Lev), 29.1 (CH₂linker), 28.0 (CH₂Lev), 27.8 (CH₂Lev), 27.6 (CH₂

linker), 23.4 (CH₂linker), 21.0 (CH₃Ac), 21.0 (CH₃Ac), 20.7 (CH₃

Ac). HRMS (ESI): m/z calcd for C₁₃₇H₁₆₀N₁₂O₄₄[M + 2NH₄]²⁺

1339.0337, found 1339.0395.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(3-O-Benzyl-β-

D-glucopyranosyluronate)-(1 → 4)-O-(2-acetamino-3-O-benzyl-2-

deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-benzyl-α-L-

idopyranosyluronate)-(1 → 4)-O-(2-azido-3-O-benzy-6-O-sulfate-

2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-benzyl-α-

L-idopyranosyluronate)-(1 → 4)-O-2-azido-3-O-benzy-2-deoxy-α-

D-glucopyranoside (37). Tetrakis(triphenylphosphine)palladium(0)

(30.0 mg, 27 μmol) was added to a solution of protected

hexasaccharide 33 (70.0 mg, 27 μmol) in THF and H₂O (5.5 mL,

10:1 v/v, 5 mM). The reaction mixture was stirred at room

temperature for 1 h. After TLC analysis indicated the completion

of the reaction, 2 M hydrochloric acid (0.27 mL, 0.53 mmol) was

added to the reaction mixture. Stirring continued for 15 min. The

reaction was monitored by TLC until completion. The reaction

mixture was co-evaporated with toluene (2 × 3 mL), and the residue

was dissolved in pyridine (1.0 mL), followed by the addition of Ac₂O

(0.5 mL). After stirring for another 3 h at room temperature, the

reaction mixture was concentrated under reduced pressure, and the

residue was puriﬁed by silica gel column chromatography using

petroleum ether and EtOAc (1:1 v/v) as the eluent to aﬀord a

hexasaccharide intermediate as a white amorphous solid (57.3 mg,

87%). The resulting compound (42.0 mg, 17 μmol) was subjected to

the Lev ester removal, O-sulfation, saponiﬁcation, and de-O-

acetylation reactions according to the general procedures to provide

compound 37 as a white-powder sodium salt (19.8 mg, 50% over

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-[Methyl-2-O-

acetyl-3-O-benzyl-4-O-(9-ﬂuorenylmethyloxycarbonyl)-β-^D-gluco-

pyranosyluronate]-(1 → 4)-O-(2-azido-3-O-benzyl-6-O-levulinoyl-

2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(methyl-2-O-levulinoyl-3-

O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-azido-3-O-ben-

zyl-6-O-levulinoyl-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(meth-

yl-2-O-levulinoyl-3-O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-

2-azido-3-O-benzy-6-O-acetyl-2-deoxy-α-^D-glucopyranoside (36).

Freshly activated 4 Å molecular sieves were added to a solution of

glycosyl donor 21 (65.0 mg, 59 μmol) and glycosyl acceptor 32 (86.0

mg, 48 μmol) in anhydrous DCM (0.4 mL, 0.15 M based on the

glycosyl donor). After stirring for 30 min at room temperature, the

solution was cooled to −40 °C, followed by the addition of TfOH

(2.1 μL, 24 μmol). The reaction mixture was stirred for 1 h, after

which the reaction was quenched with pyridine. The mixture was

ﬁltered, and the ﬁltrate was concentrated in vacuo. The residue was

three steps). H NMR (600 MHz, D₂O): δ 7.56−7.16 (m, 40H, CH

aromatic), 5.30 (s, 1H, H1^D), 5.27 (s, 1H, H1^B), 5.17−5.06 (m, 3H,

H1^C, CH₂Cbz), 4.95−4.73 (m, 10H, CHHBn, 2 × CH₂Bn, CHHBn,

https://dx.doi.org/10.1021/acs.joc.0c01881

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

H5^B, H5^D, H1^E), 4.73−4.38 (m, 11H, 2 × CH₂Bn, CHHBn, CHHBn,

H1^F, H2^B, H2^D, NCH₂Bn), 4.35 (d, J = 10.6 Hz, 1H, H6a^C), 4.30 (d, J

= 10.6 Hz, 1H, H6b^C), 4.25 (s, 1H, H3^B), 4.20−4.13 (m, 1H, H4^B),

4.10−4.04 (m, 2H, H5^C, H4^D), 4.02−3.34 (m, 22H, H3^D, H4^C, H2^E,

H4^A, H5^E, H3^C, H4^E, H6a^A, H6b^A, H6a^E, H6b^E, H3^A, H5^A, H4^F, H3^E,

H5^F, H3^F, H2^F, OCH₂linker, H2^C, H2^A), 3.30−3.11 (m, 2H, CH₂N

linker), 1.82 (s, 3H, CH₃NHAc), 1.60−1.20 (m, 6H, 3 × CH₂

linker). ¹³C{¹H} NMR (151 MHz, D₂O): δ 128.8, 128.7, 128.6,

128.53, 128.45, 102.8 (C1^F), 98.3 (C1^B), 97.9 (C1^D), 96.8 (C1^A),

94.0 (C1^C), 93.9 (C1^E), 83.5 (C3^F), 79.0 (C3^E), 78.9 (C3^C), 77.8

(C3^A), 76.6 (C5^F), 76.2 (C4^E), 75.4 (OCH₂Bn), 75.2 (OCH₂Bn),

75.2 (OCH₂Bn), 74.6 (OCH₂Bn), 74.5 (C4^A), 73.2 (C2^F), 72.4

(C4^C), 72.1 (OCH₂Bn), 71.9 (OCH₂Bn, C5^A), 71.5 (C2^D, C4^F), 71.1

(C2^B), 70.9 (C5^E), 70.5 (C4^B), 69.7 (C3^D), 69.4 (C3^B, C5^C, C4^D),

67.9 (C5^B, C5^D), 67.6 (CH₂Cbz), 67.6 (OCH₂linker), 66.5 (C6^C),

63.3 (C2^C), 62.9 (C2^A), 59.7 (C6^A, C6^E), 52.2 (C2^E), 50.5

(NCH₂Bn), 46.9 (NCH₂linker), 27.5 (CH₂linker), 27.1 (CH₂

linker), 23.0 (CH₂linker), 22.2 (CH₃NHAc). HRMS (ESI): m/z

calcd for C₁₀₀H₁₁₄N₈O₄₃S₃[M − 6Na + 4H]²⁻1105.8093, found

1105.8076.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(3-O-Benzyl-β-

D-glucopyranosyluronate)-(1 → 4)-O-(2-azido-3-O-benzyl-2-

deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-benzyl-α-

L-idopyranosyluronate)-(1 → 4)-O-(2-azido-3-O-benzyl-6-O-sul-

fate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-ben-

zyl-α-^L-idopyranosyluronate)-(1 → 4)-O-2-azido-3-O-benzy-2-

deoxy-α-^D-glucopyranoside (39). A solution of a hexasaccharide 35

(43.8 mg, 17 μmol) in a mixture of DCM and Et₃N (3 mL, 4:1 v/v, 10

mM) was stirred at ambient temperature for 2 h. The reaction was

monitored until TLC analysis indicated the completion of the

reaction. The reaction mixture was concentrated under reduced

pressure, and the residue was dissolved in pyridine (2.0 mL), followed

by the addition of acetic anhydride (0.5 mL). Stirring continued for 3

h until TLC analysis indicated the completion of the reaction. The

reaction mixture was concentrated under reduced pressure, and the

residue was dissolved in EtOAc (25 mL); washed with water (25

mL), a 1 N HCl solution (25 mL); water (25 mL); saturated

bicarbonate (25 mL); and brine (25 mL), dried (Na₂SO₄), and

ﬁltered, and the ﬁltrate was concentrated under reduced pressure. The

residue was subjected to the Lev ester removal, O-sulfation,

saponiﬁcation, and de-O-acetylation reactions according to the

general procedures to provide compound 39 as a white-powder

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(3-O-Benzyl-β-

D-glucopyranosyluronate)-(1 → 4)-O-(2-acetamino-3-O-benzyl-6-

O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-

O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-azido-3-O-benzy-

6-O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-

3-O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-2-azido-3-O-

benzy-2-deoxy-α-^D-glucopyranoside (38). Tetrakis-

(triphenylphosphine)palladium(0) (13.7 mg, 12 μmol) was added

to a solution of protected hexasaccharide 34 (32.0 mg, 12 μmol) in

THF and H₂O (5.5 mL, 10:1 v/v). The reaction mixture was stirred at

room temperature for 1 h. After TLC analysis indicated the

completion of the reaction, a 2 M hydrochloric acid solution (0.12

mL, 0.24 mmol) was added directly to the reaction mixture. Stirring

continued for 15 min. The progress of the reaction was monitored by

TLC until it showed the completion of the reaction. The mixture was

co-evaporated with toluene (2 × 3 mL), and the residue was dissolved

in pyridine (1.0 mL), followed by the addition of Ac₂O (0.5 mL).

After stirring for another 3 h at room temperature, the reaction

mixture was concentrated under reduced pressure and puriﬁed by

silica gel column chromatography using petroleum ether and EtOAc

(1:1 v/v) as the eluent to aﬀord the hexasaccharide intermediate as a

white amorphous solid (25.6 mg, 85%). The resulting compound

(25.6 mg, 10 μmol) was subjected to the Lev ester removal, O-

sulfation, saponiﬁcation, and de-O-acetylation reactions according to

the general procedures to provide compound 38 as a white powder

sodium salt (13.4 mg, 35% over ﬁve steps). H NMR (600 MHz,

D₂O): δ 7.59−7.17 (m, 40H, CH aromatic), 5.30 (s, 2H, H1^B, H1^D),

5.21−5.10 (m, 3H, CH₂Cbz, H1^C), 5.08 (d, J = 10.8 Hz, 1H,

CHHBn), 5.02−4.84 (m, 6H, CH₂Bn, CHHBn, CHHBn, H1^A, H1^E),

4.84−4.66 (m, 6H, H5^B, CHHBn, H5^D, CHHBn, CHHBn, CHHBn),

4.65−4.40 (m, 8H, CHHBn, H1^Fat 4.58, CHHBn, H2^B, H2^D,

CHHBn, NCH₂Bn), 4.39−4.31 (m, 2H, H6a^C, H6b^C), 4.28−4.13 (m,

4H, H4^B, H3^B, H4^D, H3^D), 4.10−4.06 (m, 1H, H5^C), 4.02−3.70 (m,

14H, H3^E, H4^C, H4^A, H4^E, H5^E, H3^C, H6a^A, H6b^A, H6a^E, H6b^E, H3^A,

H4^F, H5^F, H5^A), 3.70−3.24 (m, 9H, OCHH linker, H3^F, H2^C, H2^F,

H2^A, H2^E, OCHH linker, CH₂N linker), 1.63−1.29 (m, 6H, 3 × CH₂

linker). ¹³C{¹H} NMR (151 MHz, D₂O): δ 129.0, 128.7, 129.6,

128.5, 128.4, 102.7 (C1^F), 98.3 (C1^B, C1^D), 96.9 (C1^A), 94.4 (C1^E),

94.0 (C1^C), 83.6 (C3^F), 78.7 (C3^C, C3^E), 77.6 (C3^A), 76.8 (C5^F),

75.7 (C4^E), 75.5 (3 × OCH₂Bn), 74.6 (OCH₂Bn), 74.5 (C4^A), 73.3

(C2^F), 72.8 (C4^C), 72.3 (2 × OCH₂Bn), 71.6 (C5^A, C4^F), 71.4

(C2^D), 71.2 (C5^E), 71.0 (C2^B), 70.6 (C4^D), 70.2 (C3^B, C3^D), 69.7

(C5^C), 69.3 (C4^B), 67.9 (C5^D), 67.8 (OCH₂linker), 67.74 (C5^B),

67.73 (CH₂Cbz), 66.5 (C6^C), 63.2 (C2^C), 62.9 (C2^A, C2^E), 59.6

(C6^A, C6^E), 50.5 (NCH₂Bn), 47.1 (NCH₂linker), 27.3 (CH₂linker),

23.0 (CH₂linker). HRMS (ESI): m/z calcd for C₉₈H₁₁₂N₁₀O₄₂S₃[M

− 6Na⁺+ 4H⁺]²⁻1097.7993, found 1097.7996.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(3-O-Benzyl-β-

D-glucopyranosyluronate)-(1 → 4)-O-(2-azido-3-O-benzyl-6-O-sul-

fate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-ben-

zyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-azido-3-O-benzyl-6-O-

sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-

benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-2-azido-3-O-benzy-2-

deoxy-α-^D-glucopyranoside (40). A solution of a hexasaccharide 36

(80.0 mg, 30 μmol) in a mixture of DCM and Et₃N (3 mL, 4:1 v/v, 10

mM) was stirred at ambient temperature for 2 h. The reaction was

monitored until TLC analysis indicated the completion of the

reaction. The reaction mixture was concentrated under reduced

pressure, and the residue was dissolved in pyridine (2 mL), followed

by the addition of acetic anhydride (0.5 mL). Stirring was continued

for 3 h until TLC analysis indicated the completion of the reaction.

The mixture was concentrated under reduced pressure, and the

residue was dissolved in EtOAc (25 mL); washed with water (25

mL), a 1 N HCl solution (25 mL), water (25 mL), saturated

bicarbonate (25 mL), and brine (25 mL); dried (Na₂SO₄); and

ﬁltered, and the ﬁltrate was concentrated under reduced pressure, the

residue was subjected to Lev ester removal, O-sulfation, saponiﬁca-

tion, and de-O-acetylation reactions according to the general

procedures to provide compound 40 as a white powder sodium salt

(27.3 mg, 38% over ﬁve steps). ¹H NMR (600 MHz, D₂O): δ 7.54−

7.17 (m, 40H, CH aromatic), 5.26 (s, 2H, H1^B, H1^D), 5.18−5.07 (m,

3H, CH₂Cbz, H1^C), 5.05 (d, J = 10.8 Hz, 1H, CHHBn), 4.95−4.79

(m, 8H, CH₂Bn, CHHBn, CHHBn, CHHBn, H1^A, H1^E, H5^B), 4.73

(s, 1H, H5^D), 4.71−4.39 (m, 13H, CHHBn, CHHBn, H1^Fat 4.64,

sodium salt (11.6 mg, 47% over three steps). H NMR (600 MHz,

D₂O): δ 7.56−7.18 (m, 40H, CH aromatic), 5.33 (s, 1H, H1^D), 5.31

(s, 1H, H1^B), 5.21−5.08 (m, 3H, H1^C, CH₂Cbz), 5.04−4.83 (m, 7H,

CHHBn, 2 × CH₂Bn, CHHBn, H1^A), 4.82−4.78 (m, 2H, H5^B, H5^D),

4.75 (d, J = 2.7 Hz, 1H, H1^E), 4.73−4.66 (m, 3H, CH₂Bn, H1^F),

4.65−4.40 (m, 9H, CH₂Bn, CHHBn, CHHBn, H6a^E, H2^B, H2^D,

NCH₂Bn), 4.38 (d, J = 10.6 Hz, 1H, H6a^C), 4.33 (d, J = 10.6 Hz, 1H,

H6b^C), 4.28 (s, 1H, H3^B), 4.25−4.16 (m, 2H, H6b^E, H4^B), 4.14−3.84

(m, 11H, H5^C, H5^E, H4^D, H3^D, H4^C, H2^E, H4^A, H4^E, H3^C, H6a^A,

H6b^A), 3.82−3.56 (m, 8H, H3^E, H3^A, H5^F, H5^A, H4^F, H3^F, OCHH

linker, H2^C), 3.49 (d, J = 8.3 Hz, 1H, H2^F), 3.45−3.24 (m, 4H,

OCHH linker, H2^A, CH₂N linker), 1.85 (s, 3H, CH₃NHAc), 1.60−

1.26 (m, 6H, 3 × CH₂linker). ¹³C{¹H} NMR (151 MHz, D₂O): δ

128.7, 128.60, 128.55, 128.47, 128.45, 128.4, 102.3 (C1^F), 98.3

(C1^B), 97.9 (C1^D), 96.9 (C1^A), 93.9 (C1^C), 93.5 (C1^E), 83.6 (C3^F),

78.8 (C3^A, C3^C, C3^E), 76.6 (C5^F), 75.5 (OCH₂Bn), 75.3 (OCH₂Bn),

75.2 (C4^E), 74.6 (2 × OCH₂Bn, C4^A), 73.1 (C2^F), 72.6 (C4^C), 72.1

(2 × OCH₂Bn), 71.6 (C2^D, C5^A, C4^F), 71.0 (C2^B), 70.5 (C4^B), 69.5

(C3^D, C3^B, C5^C, C5^E), 67.9 (C5^B, C5^D), 67.7 (CH₂Cbz), 67.6 (OCH₂

linker), 66.5 (C6^C), 65.9 (C6^E), 63.3 (C2^C), 62.7 (C2^A), 59.8 (C6^A),

51.8 (C2^E), 50.4 (NCH₂Bn), 47.0 (NCH₂linker), 27.3 (CH₂linker),

23.0 (CH₂linker), 22.9 (CH₂linker), 22.2 (CH₃NHAc). HRMS

(ESI): m/z calcd for C₁₀₀H₁₁₄N₈O₄₆S₄[M − 7Na + 5H]²⁻1145.7877,

found 1145.7866.

https://dx.doi.org/10.1021/acs.joc.0c01881

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

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Article

CH₂Bn, CHHBn, NCH₂Bn, CHHBn, H6a^E, H2^B, H2^D, CHHBn),

4.36−4.29 (m, 2H, H6a^C, H6b^C), 4.26 (s, 1H, H4^B), 4.20−4.12 (m,

3H, H6b^E, H3^B, H4^D), 4.10−4.02 (m, 3H, H3^D, H5^C, H5^E), 4.00−

3.82 (m, 7H, H4^C, H3^E, H4^A, H4^E, H3^C, H6a^A, H6b^A), 3.80−3.21 (m,

13H, H3^A, H4^F, H5^F, H5^A, OCHH linker, H3^F, H2^C, H2^F, H2^A, H2^E,

OCHH linker, CH₂N linker), 1.60−1.20 (m, 6H, 3 × CH₂linker).

¹³C{¹H} NMR (151 MHz, D₂O): δ 128.9, 128.60, 128.55, 128.5,

NHAc), 1.54−1.20 (m, 6H, 3 × CH₂linker). ¹³C{¹H} NMR (151

MHz, D₂O): δ 129.0, 128.47, 128.46, 128.4, 127.7, 102.2 (C1^F), 98.7

(C1^C), 97.8 (C1^D, C1^B), 97.0 (C1^A), 93.3 (C1^E), 83.6 (C3^F), 78.7

(C3^E), 77.2 (C3^C), 77.0 (C3^A), 76.6 (C5^F), 75.2 (OCH₂Bn), 75.0

(OCH₂Bn, C4^A, C4^B), 74.6 (2 × OCH₂Bn, C3^B), 73.1 (C4^C, C2^F),

72.4 (OCH₂Bn), 71.8 (C2^B, OCH₂Bn), 71.6 (C5^A, C4^F), 71.0 (C2^D),

69.5 (C3^D), 68.9 (C4^D), 68.2 (C5^C, C5^E), 68.0 (OCH₂linker), 67.6

(C5^D, C5^B, CH₂Cbz), 66.5 (C6^C), 65.7 (C6^E), 60.2 (C6^A), 58.3

(C2^C), 57.4 (C2^A), 51.7 (C2^E), 50.5 (NCH₂Bn), 47.1 (NCH₂linker),

28.0 (CH₂linker), 27.0 (CH₂linker), 22.8 (CH₂linker), 22.2 (CH₃

NHAc). HRMS (ESI): m/z calcd for C₁₀₀H₁₁₈N₄Na₂O₅₂S₆[M -7Na +

5H]²⁻1221.7360, found 1221.7337.

128.4, 102.1 (C1^F), 98.3 (C1^B, C1^D), 96.8 (C1^A), 93.84 (C1^C), 93.76

(C1^E), 83.6 (C3^F), 78.9 (C3^C), 78.4 (C3^E), 77.6 (C3^A), 76.8 (C5^F),

75.5 (OCH₂Bn), 75.3 (2 × OCH₂Bn), 74.9 (C4^A, C4^E), 74.6

(OCH₂Bn), 73.1 (C2^F), 72.7 (C4^C), 72.3 (OCH₂Bn), 72.0

(OCH₂Bn), 71.7 (C5^A, C4^F), 71.5 (C2^D), 71.2 (C2^B), 70.4 (C3^B),

69.8 (C4^D), 69.7 (C3^D), 69.4 (C5^C, C5^E), 69.3 (C4^B), 68.0 (C5^B),

67.8 (C5^D), 67.6 (CH₂Cbz), 67.5 (OCH₂linker), 66.4 (C6^C), 65.8

(C6^E), 63.2 (C2^C), 62.4 (C2^A, C2^E), 59.8 (C6^A), 50.5 (NCH₂Bn),

47.0 (NCH₂linker), 27.3 (CH₂linker), 23.0 (CH₂linker). HRMS

(ESI): m/z calcd for C₉₈H₁₁₀N₁₀O₄₅S₄[M − 7Na + 5H]²⁻1137.7777,

found 1137.7744.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(3-O-Benzyl-β-

D-glucopyranosyluronate)-(1 → 4)-O-(2-sulfamino-3-O-benzyl-2-

deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-benzyl-α-

L-idopyranosyluronate)-(1 → 4)-O-(2-sulfamino-3-O-benzyl-6-O-

sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-

benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-2-sulfamino-3-O-

benzy-2-deoxy-α-^D-glucopyranoside (43). The azido group of

compound 39 (13.4 mg, 5.8 μmol) was reduced to a free amine,

followed by N-sulfation according to the general procedures, to give

compound 43 as a white-powder sodium salt (6.8 mg, 46% over two

steps). ¹H NMR (600 MHz, D₂O): δ 7.58−7.14 (m, 40H, CH

aromatic), 5.49 (s, 1H, H1^B), 5.39 (s, 1H, H1^D), 5.32−5.27 (m, 2H,

H1^C, H1^E), 5.20−5.09 (m, 2H, CH₂Cbz), 5.00 (bd, 1H, H1^A), 4.91−

4.64 (m, 11H, H5^B, H5^D, 4 × CH₂Bn, CHHBn), 4.60 (s, 2H, H2^B,

H2^D), 4.56−4.42 (m, 7H, H1^Fat 4.54, NCH₂Bn, CH₂Bn, CHHBn,

H6a^C), 4.36−4.22 (m, 4H, H6b^C, H3^B, H4^B, H3^D), 4.18 (s, 1H, H4^D),

4.12−4.08 (m, 1H, H5^C), 3.97−3.49 (m, 16H, H4^C, H5^E, H4^E, H4^A,

H6a^A, H6b^A, H3^E, H6a^E, H6b^E, H5^A, H3^C, H4^F, H5^F, H3^A, H3^F,

OCHH linker), 3.44−3.30 (m, 4H, OCHH linker, H2^C, H2^E, H2^F),

3.30−3.20 (m, 3H, CH₂N linker, H2^A), 1.60−1.18 (m, 6H, 3 × CH₂

linker). ¹³C{¹H} NMR (151 MHz, D₂O): δ 129.3, 129.2, 128.79,

128.75, 128.7, 128.60, 128.3, 102.7 (C1^F), 98.5 (C1^E, C1^C), 97.9

(C1^B), 97.4 (C1^D), 97.1 (C1^A), 83.6 (C3^F), 78.0 (C3^E), 77.7 (C3^C),

76.5 (C5^F, C3^A), 75.5 (C4^A, C4^E), 75.4 (OCH₂Bn), 75.14 (C3^D),

75.06 (OCH₂Bn, C4^D), 75.0 (C3^B, C4^B), 74.5 (OCH₂Bn), 73.3, 72.62

(C4^C), 72.56 (OCH₂Bn), 72.3 (C2^B, C2^D), 71.5 (C5^A, C4^F), 71.2

(C5^E), 69.8 (C5^C), 68.6 (C5^D), 68.3 (C5^B), 68.1 (OCH₂linker), 67.7

(CH₂Cbz), 66.9 (C6^C), 60.2 (C6^Aor C6^E), 59.9 (C6^Aor C6^E), 58.1

(C2^C, C2^E), 57.4 (C2^A), 50.6 (NCH₂Bn), 47.2 (NCH₂linker), 27.3

(CH₂linker), 23.0 (CH₂linker). HRMS (ESI): m/z calcd for

C₉₈H₁₁₆N₄O₅₁S₆[M − 9Na⁺+ 7H⁺]²⁻1178.7488, found 1178.7486.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(3-O-Benzyl-β-

D-glucopyranosyluronate)-(1 → 4)-O-(2-sulfamino-3-O-benzyl-6-

O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-

O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-sulfamino-3-O-

benzyl-6-O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-

sulfate-3-O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-2-sulfami-

no-3-O-benzy-2-deoxy-α-^D-glucopyranoside (44). The azido group

of compound 40 (22.0 mg, 9.1 μmol) was reduced to a free amine,

followed by N-sulfation according to the general procedures, to give

compound 44 as a white-powder sodium salt (11.9 mg, 49% over two

steps). ¹H NMR (600 MHz, D₂O): δ 7.62−7.14 (m, 40H, CH

aromatic), 5.48 (s, 1H, H1^B), 5.39 (s, 1H, H1^D), 5.29 (d, J = 3.0 Hz,

2H, H1^C, H1^E), 5.17−5.09 (m, 2H, CH₂Cbz), 5.00 (bd, 1H, H1^A),

4.88−4.65 (m, 11H, 4 × CH₂Bn, CHHBn, H5^B, H5^D), 4.64 (d, J =

7.3 Hz, 1H, H1^F), 4.60 (s, 2H, H2^D, H2^B), 4.56−4.40 (m, 7H,

NCH₂Bn, CH₂Bn, CHHBn, H6a^E, H6a^C), 4.34−4.16 (m, 6H, H6b^C,

H3^B, H4^B, H3^D, H4^D, H6b^E), 4.08 (d, J = 9.0 Hz, 1H, H5^C), 4.05 (d, J

= 9.6 Hz, 1H, H5^E), 3.97−3.77 (m, 6H, H4^E, H4^C, H4^A, H3^E, H6a^A,

H6b^A), 3.74−3.51 (m, 7H, H4^A, H4^F, H5^F, H3^C, H3^A, H3^F, OCHH

linker), 3.44 (dd, J = 10.8, 3.3 Hz, 1H, H2^E), 3.42−3.31 (m, 4H,

OCHH linker, H2^C, H2^F), 3.30−3.19 (m, 3H, CH₂N linker, H2^A),

1.60−1.18 (m, 6H, 3 × CH₂linker). ¹³C{¹H} NMR (151 MHz,

D₂O): δ 129.2, 129.0, 128.61, 128.58, 128.4, 128.1, 102.0 (C1^F), 98.1

(C1^E, C1^C), 97.7 (C1^B), 97.3 (C1^D), 96.9 (C1^A), 83.6 (C3^F), 77.6

(C5^F), 77.4 (C3^E), 76.5 (C3^C), 76.4 (C3^A), 75.4 (OCH₂Bn), 75.0

(OCH₂Bn), 74.9 (C4^A), 74.78 (C4^B, C3^D), 74.75 (C4^D), 74.71

(C3^B), 74.65 (C4^E), 74.5 (OCH₂Bn), 73.7 (OCH₂Bn), 73.1 (C2^F),

72.5 (OCH₂Bn), 72.39 (OCH₂Bn), 72.38 (C4^C), 72.1 (C2^B, C2^D),

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(3-O-Benzyl-β-

D-glucopyranosyluronate)-(1 → 4)-O-(2-acetamino-3-O-benzyl-2-

deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-benzyl-α-L-

idopyranosyluronate)-(1 → 4)-O-(2-sulfamino-3-O-benzy-6-O-sul-

fate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-O-ben-

zyl-α-^L-idopyranosyluronate)-(1 → 4)-O-2-sulfamino-3-O-benzy-2-

deoxy-α-^D-glucopyranoside (41). The azido group of compound 37

(14.1 mg, 6.0 μmol) was reduced to a free amine, followed by N-

sulfation according to the general procedures, to give compound 41 as

a white-powder sodium salt (8.9 mg, 60% over two steps). H NMR

(600 MHz, D₂O): δ 7.49−7.16 (m, 40H, CH aromatic), 5.46 (s, 1H,

H1^D), 5.31 (s, 1H, H1^C), 5.29 (s, 1H, H1^B), 5.18−5.08 (m, 2H,

CH₂Cbz), 5.00 (bd, 1H, H1^A), 4.92 (d, J = 11.4 Hz, 1H, CHHBn),

4.87−4.61 (m, 12H, 4 × CH₂Bn, CHHBn, H5^Bat 4.77, H1^Eat 4.75,

H5^Dat 4.71), 4.56−4.41 (m, 6H, H1^Fat 4.53, CH₂Bn, NCH₂Bn, H2^D

at 4.48), 4.39−4.27 (m, 3H, H6a^C, H6b^C, H3^B), 4.25 (s, 1H, H4^B),

4.12−4.07 (m, 1H, H5^C), 4.03 (s, 2H, H3^D, H4^D), 3.96−3.50 (m,

17H, H2^E, H4^C, H5^E, H4^A, H4^E, H6a^A, H6b^A, H6a^E, H6b^E, H5^A, H3^E,

H3^C, H4^F, H5^F, H3^A, H3^F, OCHH linker), 3.48 (t, J = 8.6 Hz, 1H,

H2^F), 3.44−3.31 (m, 2H, OCHH linker, H2^C), 3.30−3.20 (m, 3H,

CH₂N linker, H2^A), 1.81 (s, 3H, CH₃NHAc), 1.60−1.20 (m, 6H, 3 ×

CH₂linker). ¹³C{¹H} NMR (151 MHz, D₂O): δ 129.0, 128.60,

128.59, 128.5, 102.9 (C1^F), 98.6 (C1^C), 98.1 (C1^D), 97.8 (C1^B), 97.1

(C1^A), 93.8 (C1^E), 83.6 (C3^F), 79.2 (C3^E), 77.9 (C3^C), 76.81 (C3^A),

76.75 (C5^F), 76.3 (C4^E), 75.2 (C4^B), 75.1 (OCH₂Bn, C4^A), 75.01

(OCH₂Bn), 74.98 (OCH₂Bn), 74.9 (C3^B), 74.6 (OCH₂Bn), 73.5

(C4^C), 73.2 (C2^F), 72.5 (OCH₂Bn), 72.0 (C2^B), 71.8 (OCH₂Bn),

71.5 (C5^A, C4^F), 71.2 (C2^D, C5^E), 69.7 (C5^C), 69.3 (C3^D, C4^D), 68.3

(C5^B), 68.1 (OCH₂linker), 68.0 (C5^D), 67.6 (CH₂Cbz), 66.7 (C6^C),

59.9 (C6^A, C6^E), 58.3 (C2^C), 57.5 (C2^A), 52.3 (C2^E), 50.5

(NCH₂Bn), 47.2 (NCH₂linker), 27.3 (CH₂linker), 22.9 (CH₂

linker), 22.3 (CH₃NHAc). HRMS (ESI): m/z calcd for

C₁₀₀H₁₁₈N₄O₄₉S₅[M − 8Na + 6H]²⁻1159.7756, found 1159.7742.

N-(Benzyl)-benzyloxycarbonyl-5-aminopentyl O-(3-O-Benzyl-β-

D-glucopyranosyluronate)-(1 → 4)-O-(2-acetamino-3-O-benzyl-6-

O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-3-

O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-sulfamino-3-O-

benzy-6-O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-

sulfate-3-O-benzyl-α-^L-idopyranosyluronate)-(1 → 4)-O-2-sulfami-

no-3-O-benzy-2-deoxy-α-^D-glucopyranoside (42). The azido group

of compound 38 (9.4 mg, 3.8 μmol) was reduced to a free amine,

followed by N-sulfation according to the general procedures, to give

compound 42 as a white-powder sodium salt (4.0 mg, 40% over two

steps). ¹H NMR (600 MHz, D₂O): δ 7.56−7.11 (m, 40H, CH

aromatic), 5.46 (s, 1H, H1^D), 5.31 (s, 1H, H1^C), 5.26 (s, 1H, H1^B),

5.17−5.08 (m, 2H, CH₂Cbz), 5.04−4.98 (m, 1H, H1^A), 4.92−4.61

(m, 15H, 5 × CH₂Bn, CHHBn, H5^B, H5^D, H1^Eat 4.73, H1^Fat 4.64),

4.59 (s, 1H, H2^B), 4.55−4.44 (m, 5H, CHHBn, H6a^E, H2^D,

NCH₂Bn), 4.37−4.27 (m, 3H, H6a^C, H6b^C, H3^B), 4.24 (s, 1H,

H4^B), 4.20 (d, J = 11.0 Hz, 1H, H6b^E), 4.12−4.02 (m, 3H, H5^C, H5^E,

H3^D), 3.99−3.81 (m, 7H, H4^D, H2^E, H4^C, H4^A, H4^E, H6a^A, H6b^A),

3.78−3.22 (m, 14H, H5^A, H3^E, H3^C, H4^F, H5^F, H3^A, H3^F, OCHH

linker, H2^F, H2^C, OCHH linker, H2^A, CH₂N linker), 1.80 (s, 3H, CH₃

https://dx.doi.org/10.1021/acs.joc.0c01881

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

pubs.acs.org/joc

Article

71.5 (C5^A, C4^F), 69.6 (C5^C), 69.4 (C5^E), 68.3 (C5^D), 68.1 (C5^B),

67.9 (OCH₂linker), 67.6 (CH₂Cbz), 66.7 (C6^C), 60.1 (C6^E, C6^A),

58.2 (C2^C), 57.3 (C2^A), 57.2 (C2^E), 50.4 (NCH₂Bn), 47.1 (NCH₂

linker), 27.1 (CH₂linker), 22.9 (CH₂linker). HRMS (ESI): m/z

calcd for C₉₈H₁₁₄N₄Na₂O₅₄S₇[M − 8Na + 6H]²⁻1240.7091, found

1240.7044.

(600 MHz, D₂O): δ 5.45 (d, J = 3.6 Hz, 1H, H1^C), 5.41 (d, J = 3.5

Hz, 1H, H1^E), 5.29 (d, J = 3.6 Hz, 1H, H1^B), 5.22 (d, J = 3.0 Hz, 1H,

H1^D), 5.14 (d, J = 3.5 Hz, 1H, H1^A), 4.82 (1H, H5^D), 4.70 (d, J = 2.9

Hz, 1H, H5^B), 4.52 (d, J = 7.9 Hz, 1H, H1^F), 4.40 (dd, J = 11.5, 2.9

Hz, 1H, H6a^C), 4.35−4.30 (m, 2H, H2^D, H2^B), 4.28 (dd, J = 11.5, 2.2

Hz, 1H, H6b^C), 4.22 (dd, J = 5.8, 3.7 Hz, 1H, H3^D), 4.19 (dd, J = 6.6,

3.9 Hz, 1H, H3^B), 4.12 (t, J = 3.6 Hz, 1H, H4^B), 4.09 (t, J = 3.3 Hz,

1H, H4^D), 4.03−4.00 (m, 1H, H5^C), 3.97−3.69 (m, 13H, H6a^A,

H6b^A, H6a^E, H6b^E, H5^E, H5^A, H3^A, H4^C, H5^F, H3^E, H4^A, H4^E,

OCHH linker), 3.66−3.61 (m, 1H, H3^C), 3.58−3.49 (m, 3H, OCHH

linker, H4^F, H3^F), 3.44−3.38 (m, 1H, H2^F), 3.32−3.22 (m, 3H, H2^C,

H2^E, H2^A), 3.05 (t, J = 7.3 Hz, 2H, CH₂N linker), 1.77−1.47 (m, 6H,

3 × CH₂linker). ¹³C{¹H} NMR (151 MHz, D₂O): δ 102.2 (C1^F),

99.2 (C1^D), 97.8 (C1^B), 96.9 (C1^A), 96.5 (C1^E), 96.1 (C1^C), 77.5

(C4^E, C4^A), 76.7 (C2^B), 75.9 (C2^D), 75.9 (C4^B), 75.8 (C4^D), 75.8

(C4^C, C5^F), 75.1 (C4^F), 72.8 (C2^F), 71.8 (C3^F), 70.6 (C5^E), 70.4

(C5^A), 69.8 (C5^B), 69.6 (C3^C), 69.5 (C3^E), 69.4 (C3^B), 69.3 (C5^C),

69.2 (C5^D), 69.1 (C3^D), 68.5 (C3^A), 67.7 (OCH₂linker), 66.4 (C6^C),

59.6 (C6^A, C6^E), 57.9 (C2^C, C2^E, C2^A), 39.5 (NCH₂linker), 28.0

(CH₂linker), 26.4 (CH₂linker), 22.6 (CH₂linker). HRMS (ESI): m/

z calcd for C₄₁H₆₈N₄O₄₉S₆[M − 9Na + 7H]²⁻796.0644, found

796.0635.

5-Aminopentyl O-(β-^D-Glucopyranosyluronate)-(1 → 4)-O-(2-

acetamino-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-

α-^L-idopyranosyluronate)-(1 → 4)-O-(2-sulfamino-6-O-sulfate-2-

deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-α-L-idopyrano-

syluronate)-(1 → 4)-O-2-sulfamino-2-deoxy-α-^D-glucopyranoside

(45). Compound 41 (5.2 mg, 2.1 μmol) was subjected to global

deprotection according to the general procedure to give the title

compound 45 as a white-powder sodium salt (3.3 mg, 92%). ¹H

NMR (600 MHz, D₂O): δ 5.43 (s, 1H, H1^C), 5.26 (s, 1H, H1^B), 5.16

(s, 1H, H1^D), 5.12 (d, J = 3.0 Hz, 2H, H1^A, H1^E), 4.91 (s, 1H, H5^D),

4.67 (bs, 1H, H5^B), 4.47 (d, J = 7.7 Hz, 1H, H1^F), 4.32−4.23 (m, 5H,

H2^D, H2^B, H6a^C, H6b^C, H3^D), 4.17−4.14 (m, 1H, H3^B), 4.09 (t, J =

3.3 Hz, 1H, H4^B), 4.04−3.96 (m, 3H, H5^C, H4^D, H2^E), 3.92−3.66

(m, 13H, H6a^A, H6b^A, H6a^E, H6b^E, H5^E, H3^A, H3^E, H4^C, H5^A, H5^F,

H4^A, H4^E, OCHH linker), 3.66−3.61 (m, 1H, H3^C), 3.55−3.47 (m,

3H, OCHH linker, H4^F, H3^F), 3.39−3.35 (m, 1H, H2^F), 3.32−3.22

(m, 2H, H2^C, H2^A), 3.01 (t, J = 7.1 Hz, 2H, CH₂N linker), 2.03 (s,

3H, CH₃NHAc), 1.76−1.42 (m, 6H, 3 × CH₂linker). ¹³C{¹H} NMR

(151 MHz, D₂O): δ 102.1 (C1^F), 98.9 (C1^D), 97.8 (C1^B), 96.9

(C1^A), 95.7 (C1^C), 93.2 (C1^E), 77.4 (C4^A, C4^E), 76.9 (C2^B), 75.8

(C5^A, C4^C, C5^F), 75.7 (C4^B), 75.1 (C3^F), 73.7 (C2^D), 72.8 (C2^F),

71.7 (C4^F), 70.8 (C4^D), 70.5 (C5^E), 69.8 (C3^E), 69.7 (C5^B), 69.6

(C3^B), 69.5 (C3^C), 69.2 (C5^C), 68.3 (C3^A), 67.8 (C5^D), 67.6 (OCH₂

linker), 66.3 (C6^C), 64.1 (C3^D), 59.4 (C6^A, C6^E), 57.9 (C2^C, C2^A),

52.9 (C2^E), 39.5 (CH₂N linker), 28.0 (CH₂linker), 26.4 (CH₂

linker), 22.6 (CH₂linker), 22.1 (CH₃NHAc). HRMS (ESI): m/z

calcd for C₄₃H₆₉N₄NaO₄₇S₅[M − 7Na + 5H]²⁻788.0822, found

788.0804.

5-Aminopentyl O-(β-^D-Glucopyranosyluronate)-(1 → 4)-O-(2-

sulfamino-6-O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-

O-sulfate-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-sulfamino-6-O-

sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-α-L-

idopyranosyluronate)-(1 → 4)-O-2-sulfamino-2-deoxy-α-^D-gluco-

pyranoside (48). Compound 44 (6.2 mg, 2.3 μmol) was subjected to

global deprotection according to the general procedure to give the

title compound as a white-powder sodium salt (4.0 mg, 90%). H

NMR (600 MHz, D₂O): δ 5.43−5.38 (m, 2H, H1^C, H1^E), 5.26 (bs,

1H, H1^B), 5.20 (s, 1H, H1^D), 5.11 (d, J = 3.2 Hz, 1H, H1^A), 4.80 (s,

1H, H5^D), 4.68 (s, 1H, H5^B), 4.58 (d, J = 7.9 Hz, 1H, H1^F), 4.46 (d, J

= 11.0 Hz, 1H, H6a^E), 4.38 (d, J = 10.4 Hz, 1H, H6a^C), 4.33−4.27

(m, 2H, H2^D, H2^B), 4.25 (d, J = 10.5 Hz, 1H, H6b^C), 4.21 (d, J = 10.5

Hz, 1H, H6b^E), 4.19−4.15 (m, 2H, H3^D, H3^B), 4.11−4.05 (m, 3H,

H4^B, H4^D, H5^C), 4.01−3.97 (m, 1H, H5^E), 3.89−3.65 (m, 10H,

H6a^A, H6b^A, H4^A, H3^A, H4^C, H4^E, H5^F, H3^C, H5^A, OCHH linker),

3.65−3.58 (m, 1H, H3^E), 3.55−3.46 (m, 3H, OCHH linker, H3^F,

H4^F), 3.33 (bt, J = 8.5 Hz, 1H, H2^F), 3.26 (dd, J = 10.4, 3.0 Hz, 2H,

H2^C, H2^E), 3.22 (dd, J = 10.1, 3.1 Hz, 1H, H2^A), 3.01 (t, J = 7.1 Hz,

2H, CH₂N linker), 1.78−1.42 (m, 6H, 3 × CH₂linker). ¹³C{¹H}

NMR (151 MHz, D₂O): δ 101.8 (C1^F), 99.1 (C1^D), 97.7 (C1^B), 96.9

(C1^A), 96.4 (C1^C), 96.2 (C1^E), 77.1 (C5^A), 76.6 (C2^B), 75.80 (C2^D,

C4^C, C4^E, C5^F), 75.77 (C4^B, C4^D), 75.0 (C3^F), 72.9 (C2^F), 71.9

(C4^F), 70.4 (C4^A), 69.6 (C5^B), 69.5 (C3^E), 69.3 (C3^C), 69.2 (C5^E),

69.1 (C5^D, C3^B, C3^D), 68.7 (C5^C), 68.5 (C3^A), 67.7 (OCH₂linker),

66.4 (C6^C), 65.8 (C6^E), 60.2 (C6^A), 57.7 (C2^C, C2^E, C2^A), 39.5

(CH₂N linker), 28.0 (CH₂linker), 26.3 (CH₂linker), 22.6 (CH₂

linker). HRMS (ESI): m/z calcd for C₄₁H₆₈N₄O₅₂S₇[M − 10Na +

8H]²⁻836.0428, found 836.0469.

5-Aminopentyl O-(β-^D-Glucopyranosyluronate)-(1 → 4)-O-(2-

acetamino-6-O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-

(2-O-sulfate-α-^L-idopyranosyluronate)-(1 → 4)-O-(2-sulfamino-6-

O-sulfate-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-α-L-

idopyranosyluronate)-(1 → 4)-O-2-sulfamino-2-deoxy-α-^D-gluco-

pyranoside (46). Compound 42 (2.4 mg, 0.9 μmol) was subjected to

global deprotection according to the general procedure to give the

title compound 46 as a white-powder sodium salt (1.3 mg, 77%). H

NMR (600 MHz, D₂O): δ 5.42 (bs, 1H, H1^C), 5.26 (s, 1H, H1^B),

5.16 (s, 1H, H1^D), 5.14−5.10 (m, 2H, H1^E, H1^A), 4.92 (s, 1H, H5^D),

4.68 (s, 1H, H5^B), 4.56 (d, J = 7.9 Hz, 1H, H1^F), 4.44 (d, J = 10.7 Hz,

1H, H6a^E), 4.33−4.21 (m, 6H, H2^D, H2^B, H6a^C, H6b^C, H6b^E, H3^D),

4.18−4.15 (m, 1H, H3^B), 4.11−4.07 (m, 2H, H4^B, H5^E), 4.04−3.99

(m, 3H, H4^D, H5^C, H2^E), 3.88−3.66 (m, 10H, H6a^A, H6b^A, H4^A,

H3^A, H3^E, H4^C, H4^E, H5^F, H5^A, OCHH linker), 3.66−3.60 (m, 1H,

H3^C), 3.55−3.46 (m, 3H, OCHH linker, H3^F, H4^F), 3.34 (t, J = 8.4

Hz, 1H, H2^F), 3.26 (dd, J = 10.0, 3.1 Hz, 1H, H2^C), 3.22 (dd, J =

10.1, 3.1 Hz, 1H, H2^A), 3.01 (t, J = 7.1 Hz, 2H, CH₂N linker), 2.04 (s,

3H, CH₃NHAc), 1.77−1.42 (m, 6H, 3 × CH₂linker). ¹³C{¹H} NMR

(151 MHz, D₂O): δ 101.8 (C1^F), 99.0 (C1^D), 97.7 (C1^B), 96.9

(C1^A), 95.9 (C1^C), 93.4 (C1^E), 77.1 (C5^A), 76.8 (C2^B), 76.5 (C4^C,

C4^E), 75.9 (C5^F), 75.7 (C4^B), 74.9 (C3^F), 73.8 (C2^D), 72.9 (C2^F),

71.9 (C4^F), 71.1 (C4^D), 70.4 (C4^A), 69.70 (C5^B), 69.65 (C3^E), 69.6

(C3^C), 69.4 (C3^B), 69.2 (C5^C), 68.8 (C5^E), 68.5 (C3^A), 67.8 (C5^D),

67.6 (OCH₂linker), 66.3 (C6^C), 65.8 (C6^E), 64.2 (C3^D), 60.2 (C6^A),

57.9 (C2^C, C2^A), 52.8 (C2^E), 39.5 (CH₂N linker), 28.0 (CH₂linker),

26.4 (CH₂linker), 22.7 (CH₂linker), 22.2 (CH₃NHAc). HRMS

(ESI): m/z calcd for C₄₃H₆₉N₄NaO₅₀S₆[M − 8Na + 6H]²⁻828.0606,

found 828.0595.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.joc.0c01881.

■

sı

Copies of NMR spectra (PDF)

AUTHOR INFORMATION

Corresponding Author

■

Geert-Jan Boons − Department of Chemical Biology and Drug

Discovery, Utrecht Institute for Pharmaceutical Sciences, and

Bijvoet Center for Biomolecular Research, Utrecht University,

3584 CG Utrecht, The Netherlands; Complex Carbohydrate

Research Center and Department of Chemistry, University of

Georgia, Athens, Georgia 30602, United States; orcid.org/

0000-0003-3111-5954; Email: g.j.p.h.boons@uu.nl,

gjboons@ccrc.uga.edu

5-Aminopentyl O-(β-^D-Glucopyranosyluronate)-(1 → 4)-O-(2-

sulfamino-2-deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-α-

L-idopyranosyluronate)-(1 → 4)-O-(2-sulfamino-6-O-sulfate-2-

deoxy-α-^D-glucopyranosyl)-(1 → 4)-O-(2-O-sulfate-α-L-idopyrano-

syluronate)-(1 → 4)-O-2-sulfamino-2-deoxy-α-^D-glucopyranoside

(47). Compound 43 (6.8 mg, 2.7 μmol) was subjected to global

deprotection according to the general procedure to give the title

compound 47 as a white-powder sodium salt (4.2 mg, 88%).¹H NMR

https://dx.doi.org/10.1021/acs.joc.0c01881

J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Authors

Lifeng Sun − Department of Chemical Biology and Drug

Discovery, Utrecht Institute for Pharmaceutical Sciences, and

Bijvoet Center for Biomolecular Research, Utrecht University,

3584 CG Utrecht, The Netherlands

Pradeep Chopra − Complex Carbohydrate Research Center,

University of Georgia, Athens, Georgia 30602, United States;

orcid.org/0000-0002-6003-4574

pubs.acs.org/joc

Article

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X. Chemical synthesis of syndecan-3 glycopeptides bearing two

heparan sulfate glycan chains. Angew. Chem., Int. Ed. 2014, 53 (34),

9051−9058.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.joc.0c01881

Notes

The authors declare no competing ﬁnancial interest.

(20) Dulaney, S. B.; Xu, Y. M.; Wang, P.; Tiruchinapally, G.; Wang,

Z.; Kathawa, J.; El-Dakdouki, M. H.; Yang, B.; Liu, J.; Huang, X. F.

Divergent synthesis of heparan sulfate oligosaccharides. J. Org. Chem.

2015, 80 (24), 12265−12279.

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J. M. Making the longest sugars: a chemical synthesis of heparin-

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ACKNOWLEDGMENTS

■

The research was supported by the Chinese Scholarship

Council (to L.S.) and Utrecht University (to G.J.B.).

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