Synthesis of 13C-labelled sulfated N-acetyl-d-lactosamines to aid in the diagnosis of mucopolysaccharidosis diseases

Article abstract of DOI:10.1002/jlcr.3697

Morquio A syndrome is an autosomal mucopolysaccharide storage disorder that leads to accumulation of keratan sulfate. Diagnosis of this disease can be aided by measuring the levels of keratan sulfate in the urine. This requires the liquid chromatography tandem mass spectrometry (LCMS/MS) measurement of sulfated N-acetyl-d-lactosamines in the urine after cleavage of the keratan sulfate with keratanase II. Quantification requires isotopically-labelled internal standards. The synthesis of these ¹³C₆-labelled standards from ¹³C₆-galactose and N-acetylglucosamine is described. The required protected disaccharide is prepared utilising a regioselective, high yielding β-galactosylation of a partially protected glucosamine acceptor and an inverse addition protocol. Subsequent synthesis of the ¹³C₆-labelled mono and disulfated N-acetyllactosamines was achieved in five and eight steps, respectively, from this intermediate to provide internal standards for the LCMS/MS quantification of keratan sulfate in urine.

Full text of DOI:10.1002/jlcr.3697

Received: 26 September 2018
DOI: 10.1002/jlcr.3697
Revised: 12 November 2018
Accepted: 13 November 2018
R E S E A R C H A R T I C L E
13
Synthesis of C‐labelled sulfated N‐acetyl‐D‐lactosamines to
aid in the diagnosis of mucopolysaccharidosis diseases
Scott A. Cameron1
Phillip M. Rendle1
| Olga V. Zubkova1
| Steven Toms2 | Richard H. Furneaux1
|
1 Ferrier Research Institute, Victoria
University of Wellington, Lower Hutt,
New Zealand
2 GlycoSyn, Callaghan Innovation,
Gracefield, New Zealand
Morquio A syndrome is an autosomal mucopolysaccharide storage disorder
that leads to accumulation of keratan sulfate. Diagnosis of this disease can be
aided by measuring the levels of keratan sulfate in the urine. This requires
the liquid chromatography tandem mass spectrometry (LCMS/MS) measurement
of sulfated N‐acetyl‐D‐lactosamines in the urine after cleavage of the keratan
sulfate with keratanase II. Quantification requires isotopically‐labelled internal
standards. The synthesis of these 13C6‐labelled standards from 13C6‐galactose
and N‐acetylglucosamine is described. The required protected disaccharide is
prepared utilising a regioselective, high yielding β‐galactosylation of a partially
protected glucosamine acceptor and an inverse addition protocol. Subsequent
synthesis of the 13C6‐labelled mono and disulfated N‐acetyllactosamines was
achieved in five and eight steps, respectively, from this intermediate to provide
internal standards for the LCMS/MS quantification of keratan sulfate in urine.
Correspondence
Phillip M. Rendle, Ferrier Research
Institute, Victoria University of
Wellington, Lower Hutt, New Zealand.
Email: phillip.rendle@vuw.ac.nz
KEYWORDS
13C‐labelled, keratan sulfate, lactosamine, Morquio A, MPS IVA, sulfated
1 | INTRODUCTION
Additionally, enzyme activity testing of GALNS is essen-
tial in diagnosing MPS IVA with fibroblasts and leuko-
Morquio A syndrome (mucopolysaccharidosis IVA, MPS
IVA) is a rare autosomal recessive lysosomal storage dis-
ease disorder caused by deficient N‐acetylgalactosamine‐
6‐sulfatase (GALNS; EC 3.1.6.4) activity.1,2 GALNS
catabolises keratan sulfate (KS), a glycosaminoglycan that
consists of sulfated β(1 → 3) repeats of a galactosyl
β(1 → 4)‐N‐acetylglucosamine disaccharide (Figure 1).3,4
Deficiencies in GALNS lead to accumulation of KS
resulting in progressive tissue and organ dysfunction.
Early and accurate diagnosis of this condition is critical
for improved patient outcomes, particularly as enzyme
replacement therapy has recently become available.5 KS
level in urine is a biomarker for MPS IVA and so a liquid
chromatography tandem mass spectrometry (LCMS/MS)
method has been developed that provides a sensitive
and specific means for quantitation of urinary KS.6,7
cytes being the recommended sample types.8 However,
there is still a need in the screening and enzyme replace-
ment therapy dosage monitoring settings for an accurate
and rapid measurement, meaning that LCMS/MS analy-
sis of urine is still a key diagnostic tool.
In this method, keratanase II9 is used to selectively
hydrolyse urinary KS into its key disaccharides, mono
and disulfated N‐acetyllactosamine, and this mixture is
analysed by LCMS/MS. Addition of isotopically‐labelled
versions of these disaccharides is required for quantifica-
tion of the results. Synthetic non‐labelled material is also
valuable for use in calibrating the method. Because of the
appreciable natural abundance of 34S (4.2%), and the fact
that the disulfated N‐acetyllactosamines contains two sul-
fur atoms, there needs to be sufficient isotopic labelling to
increase the mass by five or greater Daltons to fully
J Label Compd Radiopharm. 2018;1–10.
wileyonlinelibrary.com/journal/jlcr
© 2018 John Wiley & Sons, Ltd.
1

2
CAMERON ET AL.
FIGURE 1 Structure of keratan sulfate
separate the internal standard mass peaks from the ana-
lyte natural abundance isotope peaks to facilitate quanti-
fication. Lactosamine derivatives labelled to this extent
are not readily available, and therefore, herein, we
describe syntheses of mono and disulfated N‐
acetyllactosamines (8 and 16, Schemes 3 and 4) from their
constituent monosaccharides, allowing the introduction
of six heavy atom carbons via the use of the readily avail-
able 13C6‐galactose as a key raw material.
SCHEME 1 Synthesis of the key disaccharide (3) from 13C6‐D‐
galactose and N‐acetyl‐D‐glucosamine. Conditions: (i) a. Ac2O/Py,
50°C, 5 h, 20°C overnight; b. ethanolamine, 20°C, overnight; c.
trichloroacetonitrile, DBU, 20°C, 2 h; (ii) TMSOTf, DCM, 4 Å
molecular sieves, ‐50°C, 1 h → 20°C, 1 h. * denotes 13C‐labelling
2 | RESULTS AND DISCUSSION
butyldiphenylsilylation.22 Glycosylation of the TBDPS‐
protected diol acceptor (2) with donor (1) in anhydrous
DCM in the presence of TMSOTf at ‐50°C using a conven-
tional procedure in which the catalyst was added to a
mixture of donor and acceptor yielded very little desired
product. However, when the reaction was carried out
using an inverse addition procedure, in which the acceptor
(2) and TMSOTf were premixed prior to donor (1) addi-
tion,23 it proved to be highly regio‐ and stereo‐selective in
accordance with literature findings.19 The glycosylation
reaction using the inverse addition procedure, under
conditions of excess donor (two equivalents), furnished
the product as a single β‐linked anomer (3) in quantitative
yield after chromatography. As the 13C6‐labelled donor was
deemed valuable, this reaction was modified to use just
one equivalent of donor (1) and then the labelled
disaccharide product was isolated in a reduced but
acceptable yield of 76%. Regio‐ and stereo‐chemistry were
confirmed by nuclear magnetic resonance (NMR).19
Assignment of anomeric protons by a heteronuclear single
quantum correlation (HSQC) experiment revealed a
There have been several syntheses of unlabelled keratan
sulfate oligosaccharide fragments10-14 as well as more spe-
cifically, unlabelled sulfated lactosamines.15-18 Apart
from Ogawa's work,12 these reports generate the final
products as glycosides, or in one case, the oxazoline
rather than the N‐acetyllactosamine required here where
the glucosamine O‐1 is not substituted. As part of our
synthetic plan, for efficiency in monosaccharide building
block preparation, we decided to take advantage of a
selective glycosylation of
a partially protected N‐
acetylglucosamine 3,4‐diol previously employed by
Roy.19 Route development was undertaken using D‐galac-
tose, allowing the generation of the unlabelled variants
for use as calibration controls in urine analysis. The
labelled variants were then produced in an analogous
manner using 13C6‐D‐galactose.
2.1 | Preparation of key intermediate (3)
The galactosyl trichloroacetimidate donor (1) (Scheme 1)
was synthesised in three steps from D‐galactose in 64%
yield. Following established methods for unlabelled
galactose,20 peracetylation of 13C6‐labelled galactose in
anhydrous pyridine followed by anomeric deprotection
with ethanolamine in ethyl acetate, followed by treatment
with trichloroacetonitrile and DBU afforded the donor (1)
as an anomeric mixture. The α‐anomer was isolated
by flash chromatography on silica gel, generating a
highly‐crystalline product as the major product, and this
was subsequently used in the glycosylation reaction.
Following literature procedures, the glycosylation acceptor
(2) was prepared by anomeric O‐benzyl protection of
N‐acetylglucosamine,21 followed by selective 6‐O‐tert‐
1
J1’,2’ = 8.1 Hz in the H NMR, which is indicative of the
desired β‐anomer stereochemistry for the newly formed
glycosidic bond. A large down field shift from 3.80 to
1
5.17 of H‐3 (as assigned by HSQC and H―1H homonu-
clear correlation spectroscopy (COSY)) upon acetylation
of O‐3 in the preparation of compound (4) confirms the
regiochemistry of the glycosylation to be at O‐4.
2.2 | Preparation of 6‐O‐sulfated LacNAc
The target 13C6‐labelled monosulfated LacNAc (8) was
prepared from the key intermediate (3) in five steps

CAMERON ET AL.
3
(Scheme 2). Acetylation of key intermediate (3) gave the
fully‐protected disaccharide (4) in 86% yield. Attempts to
remove the TBDPS protecting group with tetrabutyl-
ammonium fluoride led to deacetylation. Treatment of
disaccharide (4) with HF‐pyridine in THF overnight at
room temperature yielded the desired monohydroxy disac-
charide (5) in good yield. Sulfation of the monohydroxy
disaccharide (5) with sulfur trioxide trimethylamine com-
plex in anhydrous N,N‐dimethylformamide at 50°C gave
the monosulfated disaccharide (6) as the ammonium salt
following chromatographic purification. Zemplén
deacetylation with sodium methoxide in methanol gave
the desired deacetylated product (7) in excellent yield
following chromatography on silica gel. Hydrogenolysis
in the presence of Pd(OH)2/C in ethanol and aqueous
ammonia afforded the target monosulfated LacNAc as
the ammonium salt. Ion exchange chromatography
(Dowex 50WX8‐200, sodium form) of the ammonium
salt provided the final product (8) as the desired
sodium salt.
SCHEME 3 Synthesis of disulfated LacNAc (16). Conditions: (i)
HF·Py, THF, Py, 0°C → 20°C, 2 days; (ii) NaOMe, MeOH, 20°C,
1 day; (iii) TBDMSCl, DMF, imidazole, 0°C, 3 h; (iv) Ac2O, Py,
0°C → 20°C, overnight; (v) HF·Py, THF, Py, 0°C → 20°C, 2 h; (vi)
SO3·NMe3, DMF, 50°C, 20 h; 7 M NH3/MeOH, 20°C 15 min; (vii)
NaOMe, MeOH, 20°C, 1 h; (viii) Pd (OH)2/C, H2, EtOH, aq.
NH4OH, 20°C, overnight, Dowex 50WX8‐200 (Na+ form). * denotes
13C‐labelling
2.3 | Preparation of 6,6‐O‐disulfated
LacNAc
The target 13C6‐disulfated LacNAc (16) was prepared
from the key disaccharide (3) in eight steps
(Scheme 3). Initially, we attempted the preparation
of the unlabelled target material (equivalent of 16)
using similar chemistry to that shown in Scheme 3,
but without first removing the TBDPS group from
disaccharide (3). This led to the unlabelled version
of protected disaccharide (12) where R = TBDPS.
During the silyl deprotection of this material (12,
observed rapid removal of the TBDMS group, but
extended reaction times were required to remove the
remaining TBDPS group. The prolonged reaction time
resulted in significant amounts (approximately 15%)
of a side‐product resulting from an acetyl migration
from O‐4 to O‐6 on the galactosyl unit. Hence, the
synthesis was modified to install two labile TBDMS
groups at the 6‐positions of both sugars (unlabelled
version of 11). In the future, we would trial the prep-
aration and use of the TBDMS‐protected version of the
N‐acetylglucosamine building block (2), to improve
yields and shorten the synthesis of disulfate (16) by
one step.
R
= TBDPS, unlabelled) using HF‐pyridine, we
Therefore, deprotection of disaccharide (3) was car-
ried out using HF‐pyridine and yielded disaccharide
(9) in quantitative yield. Complete de‐O‐acetylation of
disaccharide (9) was achieved under Zemplén condi-
tions (sodium methoxide in methanol). Selective 6‐O
disilyl protection followed by peracetylation afforded
the fully protected LacNAc (12). Desilylation using
HF‐pyridine provided diol (13), which was subsequently
sulfated using sulfur trioxide trimethylamine complex
in anhydrous N,N‐dimethylformamide at 50°C to give
the disulfated product (14), which was isolated as the
ammonium salt. Saponification with sodium methoxide
SCHEME 2 Synthesis of monosulfated LacNAc. Conditions: (i)
Ac2O, Py, 0°C, 3 h → 20°C, overnight; (ii) HF·Py, THF, Py,
0°C → 20°C, overnight; (iii) SO3·NMe3, DMF, 50°C, 16 h; (iv)
NaOMe, MeOH, 20°C, 1 h; (v) Pd (OH)2/C, H2, EtOH, aq. NH4OH,
20°C, 5 h, Dowex 50WX8‐200 (Na+ form). * denotes 13C‐labelling

4
CAMERON ET AL.
in anhydrous methanol at ambient temperature led to
the desired deacetylated product (15) in good yield.
Hydrogenolysis of benzyl glycoside (15) in the presence
of Pd(OH)2/C in ethanol and aqueous ammonia
afforded the target disulfated 13C6‐labelled LacNAc
(16), which was passed as an aqueous solution through
a strong anion‐exchange resin in the sodium form
(Dowex 50WX8‐200) to ensure the product was the
disodium salt.
4.2 | Synthesis of benzyl (2,3,4,6‐tetra‐O‐
acetyl‐β‐13C6‐D‐galactopyranosyl)‐(1 → 4)‐2‐
acetamido‐6‐O‐tert‐butyldiphenylsilyl‐2‐
deoxy‐α‐D‐glucopyranoside (3)
4.2.1 | 2,3,4,6‐Tetra‐O‐acetyl‐β‐13C6‐D‐
galactopyranosyl 2,2,2‐trichloroacetimidate
(1)
Acetic anhydride (12 mL, 7.7 eq) was added to a slurry of
13C6‐D‐galactose (3.00 g, 1 eq) in anhydrous pyridine
(24 mL) at 20°C. The mixture was stirred at 50°C for
5 h then overnight at 20°C. The solvent was removed
under reduced pressure and the resulting residue was
redissolved in ethyl acetate (150 mL) and washed with
0.5 M HCl (100 mL). The aqueous phase was re‐extracted
with ethyl acetate (150 mL), and the combined organic
phases were washed with saturated aqueous NaHCO3
(200 mL) followed by water (200 mL). The organic phase
was dried over MgSO4, filtered and concentrated under
reduced pressure to yield 13C6‐1,2,3,4,6‐penta‐O‐acetyl‐D‐
galactose as a colourless oil (6.40 g, quantitative), which
crystallised over time. HRMS (ESI) calcd for
C1013C6H22O11Na [M + Na]+ m/z 419.1261, found:
419.1252.
3 | CONCLUSIONS
A highly regio‐ and stereo‐selective glycosylation of a
partially protected N‐acetylglucosamine acceptor was
used to prepare a key 13C6‐labelled disaccharide (3).
This key disaccharide (3) was then used as a common
intermediate for the reasonably high‐yielding synthesis
of 13C6‐labelled mono and disulfated N‐acetyllactosa-
mine derivatives for use as internal standards to aid in
the diagnosis of MPS IVA. The synthesis of monosul-
fated N‐acetyllactosamine (8) was achieved in eight
steps with 38% overall yield from 13C6‐labelled galac-
tose, whereas the synthesis disulfated N‐acetyllacto-
samine (16) was achieved in 11 steps with 23% overall
yield from the labelled galactose.
A solution of 1,2,3,4,6‐penta‐O‐acetyl‐13C6‐D‐galactose
(6.05 g, 1 eq) and ethanolamine (1.9 mL, 2.0 eq) in ethyl
acetate (100 mL) was stirred at 20°C overnight. The solu-
tion was washed with water and saturated aqueous
NaHCO3, dried over MgSO4, filtered, and the solvent
removed under reduced pressure, giving the tetra‐acetate
as a colourless syrup. This was dissolved in anhydrous
dichloromethane (60 mL), then trichloroacetonitrile
(5.30 mL, 4.88 eq), and 1,8‐diazabicyclo[5.4.0]undec‐7‐
ene (0.080 mL, 0.050 eq) were added. After 2 h, the solu-
tion was diluted with dichloromethane, washed with sat-
urated aqueous NaHCO3 and water, dried over MgSO4,
filtered, and the solvent removed under reduced pressure,
yielding a sticky orange solid. This mixture was purified
by column chromatography (0:1 to 1:1 ethyl acetate:
petroleum ether), giving a white solid 1 (α isomer,
3.36 g, 64%) and then a colourless oil, which slowly
crystallised (β isomer, 0.78 g, 15%). HRMS (ESI) calcd
for C1013C6H20NO10Cl3Na [M + Na]+ m/z 520.0259,
found: 520.0246. Alpha anomer: 1H NMR (CDCl3) δ
(ppm): 2.01 (s, 3H), 2.02 (s, 3H), 2.02 (s, 3H), 2.16 (s,
3H), 4.08 (dd, J = 11.4, 6.6 Hz, 1H), 4.16 (dd, J = 11.3,
6.6 Hz, 1H), 4.44 (td, J = 6.7, 1.3 Hz, 1H), 5.36 (dd,
J = 10.8, 3.5 Hz, 1H), 5.43 (dd, J = 10.8, 3.2 Hz, 1H),
5.56 (dd, J = 3.1, 1.3 Hz, 1H), 6.60 (d, J = 3.5 Hz, 1H),
8.66 (s, 1H). 13C NMR (CDCl3) δ (ppm): 20.8, 20.8, 20.9,
20.9, 61.5 (d, J = 47.0 Hz), 67.2 (m), 67.7 (m), 67.8 (m),
69.3 (m), 91.1, 93.8 (d, J = 42.6 Hz), 161.2, 170.2, 170.3,
4 | EXPERIMENTAL
4.1 | General methods
Thin‐layer chromatography (TLC) was performed on
aluminium sheets coated with 60 F254 silica gel and
visualised under UV light and/or with a ceric ammo-
nium molybdate dip. Chromatography (flash column
or an automated system with continuous gradient
facility) was performed on silica gel (40‐63 μm). NMR
spectra were recorded on a Bruker AdvanceIII 500
instrument at 500 MHz (1H) referenced to TMS δ
0.0 ppm and 125 MHz (13C) referenced to residual sol-
vent peak, CDCl3, δ 77.0 ppm; CD3OD δ 49.0 ppm. High
resolution mass spectra (HRMS) were recorded on a
Waters Q‐TOF Premier Mass Spectrometer with
electro‐spray ionisation (ESI). Unlabelled 1,2,3,4,6‐
penta‐O‐acetyl‐β‐D‐galactopyranose
was
purchased
from Aldrich and used as received. 13C6‐Galactose was
purchased from Omicron Biochemicals, Inc. The 1H
NMR are 13C decoupled. Coupling constants
observed in 13C‐NMR spectra are indicated for the 13C
containing products.

CAMERON ET AL.
5
170.3, 170.5. Beta anomer: 1H NMR (CDCl3) δ (ppm): 1.95
(s, 3H), 1.97 (s, 3H), 1.99 (s, 3H), 2.13 (s, 3H), 4.05–4.10
(m, 2H), 4.14–4.17 (m, 1H), 5.09 (dd, J = 10.4, 3.4 Hz,
1H), 5.42 (dd, J = 3.5, 1.2 Hz, 1H), 5.44 (dd, J = 10.4,
8.3 Hz, 1H), 5.80 (d, J = 8.2 Hz, 1H), 8.71 (s, 1H).
13C NMR (CDCl3) δ (ppm): 20.5, 20.6, 20.6, 20.6, 60.9
(dt, J = 47.3, 4.6 Hz), 66.8 (t, J = 38.9 Hz), 67.8 (dd,
J = 48.5, 42.8 Hz), 70.7 (br. t, J = 40.8 Hz), 71.8 (dd,
J = 47.3, 38.9 Hz), 90.4, 96.1 (dt, J = 48.2, 5.9 Hz),
161.0, 169.0, 170.0, 170.2, 170.3.
1′), 127.8, 128.0, 128.1, 128.6, 130.0, 130.0, 132.9, 133.7,
135.7, 136.1, 137.2, 169.3, 169.9, 170.2, 170.2, 170.5.
4.3 | Synthesis of sodium
2‐acetamido‐2‐deoxy‐4‐O‐(β‐13C6‐D‐
galactopyranosyl)‐D‐glucopyranose‐6‐
sulfate (8)
4.3.1 | Benzyl
2‐acetamido‐3‐O‐acetyl‐2‐deoxy‐6‐O‐tert‐
butyldiphenylsilyl‐4‐O‐(2,3,4,6‐tetra‐O‐
acetyl‐β‐13C6‐D‐galactopyranosyl)‐α‐D‐
glucopyranoside (4)
4.2.2 | Benzyl
2‐acetamido‐2‐deoxy‐6‐O‐tert‐
butyldiphenylsilyl‐4‐O‐(2,3,4,6‐tetra‐O‐
acetyl‐β‐13C6‐D‐galactopyranosyl)‐α‐D‐
glucopyranoside (3)
A solution of 3 (1.98 g, 1 eq) in anhydrous pyridine
(25 mL) was cooled to 0°C. Acetic anhydride (5 mL,
22.8 eq) was added dropwise. The reaction was stirred at
0°C for 3 h, then at 20°C overnight. The solution was con-
centrated and then purified by column chromatography
(1:1 ethyl acetate:petroleum ether), giving the product
(4) as a white foam (2.02 g, 97%). (Unlabelled: HRMS
[ESI] calcd for C47H59NO16SiNa [M + Na]+ m/z
A solution of 1 (5.47 g, 1 eq) in anhydrous dichlorometh-
ane (15 mL) was stirred under argon at 20°C with 4 Å
molecular sieves (2 g) for 30 min, then cooled to −50°C.
TMSOTf (1.89 mL, 1.03 eq) was added and the solution
stirred at −50°C for 15 min. A solution of 2 (6.58 g,
1.34 eq)21,22 in anhydrous dichloromethane (15 mL) was
added dropwise over 30 min, with stirring at −45°C con-
tinued for a further 45 min. The resulting solution was
then allowed to warm to 20°C over 1 h, before excess
solid NaHCO3 and water (3 mL) were added, and the
reaction mixture was stirred for 20 min, diluted with
dichloromethane, filtered, and the solvent removed under
reduced pressure, yielding a white foam. This crude prod-
uct was purified by column chromatography (2:1 ethyl
acetate:petroleum ether), giving the desired product (3)
as a crystalline white solid (6.63 g, 76%). HRMS (ESI)
calcd for C3913C6H57NO15SiNa [M + Na]+ m/z 908.3591,
found: 908.3608. (Unlabelled: HRMS [ESI] calcd for
C45H57NO15SiNa [M + Na]+ m/z 902.3395, found:
1
944.3501, found: 944.3495.) H NMR (CDCl3) δ (ppm):
1.09 (s, 9H, tBu), 1.76 (s, 3H), 1.91 (s, 3H), 1.98 (s, 3H),
2.05 (s, 3H), 2.06 (s, 3H), 2.13 (s, 3H) (6Ac), 3.58 (dt,
J = 9.9, 2.0 Hz, 1H, H‐5), 3.70–3.79 (m, 2H, H‐5′, H‐6′a
or CHaBn), 3.87 (dd, J = 11.6, 2.7 Hz, 1H, H‐6′a or
CHaBn), 4.05–4.15 (m, 3H, H‐4, H‐6′b, CHbBn), 4.25
(ddd, J = 10.9, 9.4, 3.8 Hz, 1H, H‐2), 4.47 (d,
J = 12.0 Hz 1H, H‐6a), 4.61 (d, J = 12.0 Hz, 1H, H‐6b),
4.74 (d, J = 8.0 Hz, 1H, H‐1′), 4.88 (dd, J = 10.3, 3.5 Hz,
1H, H‐3′), 4.93 (d, J = 3.8 Hz, 1H, H‐1), 5.04 (dd,
J = 10.3, 8.0 Hz, 1H, H‐2′), 5.17 (dd, J = 10.9, 9.3 Hz,
1H, H‐3), 5.30 (d, J = 3.5 Hz, 1H, H‐4′), 5.73 (d,
J = 9.4 Hz, 1H, NH), 7.23–7.35 (m, 5H), 7.38–7.50 (m,
6H), 7.69–7.77 (m, 4H) (SiPh, Bn). 13C NMR (CDCl3) δ
(ppm): 20.6, 20.7, 20.8, 20.8, 21.1, 23.3, 27.0, 29.8, 52.4,
61.2, 61.2 (d, J = 47.6 Hz), 67.1 (t, J = 38.8 Hz), 69.5
(dd, J = 48.4, 42.6 Hz), 70.1, 70.7 (dd, J = 47.5,
38.9 Hz), 71.3 (br. t, J = 40.7 Hz), 71.4, 71.5, 74.4, 96.7,
100.4 (dt, J = 48.4, 5.4 Hz), 127.8, 128.1, 128.3, 128.4,
128.7, 130.0, 130.1, 132.5, 133.6, 135.6, 136.1, 137.0,
168.9, 170.1, 170.2, 170.3, 170.4, 171.5.
1
902.3385.) H NMR (CDCl3) δ (ppm): 1.08 (s, 9H, tBu),
1.68 (s, 3H), 1.98 (s, 3H), 1.99 (s, 3H), 2.06 (s, 3H), 2.14
(s, 3H), (5Ac), 3.65 (dt, J = 9.2, 2.6 Hz, 1H, H‐5), 3.75–
3.87 (m, 4H, CHaPh, H‐6′a, H‐3, H‐4), 3.90 (td, J = 6.6,
1.2 Hz, 1H, H‐5′), 4.08–4.20 (m, 3H, H‐2, H‐6′a, CHbPh),
4.44 (d, J = 12.0 Hz, 1H, H‐6a), 4.63 (d, J = 12.0 Hz, 1H,
H‐6b), 4.66 (d, J = 8.1 Hz, 1H, H‐1′), 4.94 (dd, J = 10.5,
3.5 Hz, 1H, H‐3′), 4.97 (d, J = 3.7 Hz, 1H, H‐1), 5.19
(dd, J = 10.5, 8.1 Hz, 1H, H‐2′), 5.35 (dd, J = 3.5,
1.2 Hz, 1H, H‐4′), 5.67 (d, J = 8.8 Hz, 1H, NH), 7.22–
7.36 (5H), 7.36–7.49 (m, 6H), 7.68–7.76 (4H) (SiPh, Bn).
13C NMR (CDCl3) δ (ppm): 19.5, 20.4, 20.6, 20.6, 20.7,
23.5, 27.0, 53.4, 61.3 (dt, J = 46.5, 4.4 Hz), 61.8, 67.0 (t,
J = 38.9 Hz), 69.0 (dd, J = 49.0, 42.6 Hz), 69.8, 70.4,
70.7, 71.0 (br. t, J = 40.1 Hz), 71.4 (dd, J = 46.5,
38.9 Hz), 80.6, 96.6 (C‐1), 101.2 (dt, J = 49.0, 5.0 Hz, C‐
4.3.2 | Benzyl
2‐acetamido‐3‐O‐acetyl‐2‐deoxy‐4‐O‐(2,3,4,6‐
tetra‐O‐acetyl‐β‐13C6‐D‐galactopyranosyl)‐α‐
D‐glucopyranoside (5)
Hydrogen fluoride‐pyridine (70% solution in pyridine)
(4 mL) was added to a solution of 4 (2.00 g) in THF
(35 mL) and anhydrous pyridine (3 mL) contained in a

6
CAMERON ET AL.
Nalgene vial at 0°C. The mixture was warmed to 20°C,
then stirred overnight. The solution was diluted with
ethyl acetate, washed with saturated aqueous NaHCO3
and water, then dried over MgSO4, filtered, and the sol-
vent removed under reduced pressure, giving a tan solid.
This material was purified by column chromatography
(2:1 to 1:0 ethyl acetate:petroleum ether), yielding the
product (5) as a white solid (1.32 g, 89%). (Unlabelled:
HRMS [ESI] calcd for C31H41NO16Na [M + Na]+ m/z
2.04 (s, 3H), 2.04 (s, 3H), 2.12 (s, 3H), 3.84 (dt, J = 10.4,
2.2 Hz, 1H), 4.00–4.09 (m, 2H), 4.20 (ddd, J = 11.0, 9.4,
3.7 Hz, 1H), 4.26 (dd, J = 10.9, 1.4 Hz, 1H), 4.34 (dd,
J = 11.2, 2.8 Hz, 1H), 4.50 (d, J = 12.0 Hz, 1H), 4.70 (d,
J = 12.0 Hz, 1H), 4.87 (d, J = 8.0 Hz, 1H), 4.98–5.05 (m,
2H), 5.13–5.21 (m, 2H), 5.33 (d, J = 3.5 Hz, 1H), 5.80 (d,
J = 9.2 Hz, 1H), 7.25–7.38 (m, 5H). 13C NMR (CDCl3) δ
(ppm): 20.7, 20.8, 20.8, 20.9, 21.0, 23.2, 52.2, 61.4 (d,
J = 47.5 Hz), 65.5, 67.5 (t, J = 38.6 Hz), 69.5 (dd,
J = 48.2, 42.5 Hz), 70.0, 70.1, 70.4, (dd, J = 47.4,
38.6 Hz), 71.1 (br t, J = 40.4 Hz), 71.6, 75.0, 96.9, 100.6
(d, J = 48.4 Hz), 128.2, 128.3, 128.7, 137.1, 170.1, 170.3,
170.6, 170.7, 171.2, 171.4.
1
706.2323, found: 706.2318.) H NMR (CDCl3) δ (ppm):
1.88 (s, 3H), 1.97 (s, 3H), 2.05 (s, 3H), 2.06 (s, 3H), 2.06
(s, 3H), 2.14 (s, 3H), 3.68 (dt, J = 9.9, 2.5 Hz, 1H), 3.73
(dd, J = 6.8, 2.6 Hz, 1H), 3.89 (td, J = 10.8, 6.9 Hz, 1H),
3.93 (dd, J = 9.9 Hz, 1H), 4.08 (dd, J = 10.8, 6.9 Hz,
1H), 4.11 (dd, J = 10.8, 6.9 Hz, 1H), 4.18 (ddd, J = 10.8,
9.5, 3.7 Hz, 1H), 4.49 (d, J = 12.0 Hz, 1H), 4.64 (d,
J = 7.9 Hz, 1H), 4.67 (d, J = 12.0 Hz, 1H), 4.89 (d,
J = 3.7 Hz, 1H), 4.99 (dd, J = 10.5, 3.5 Hz, 1H), 5.12
(dd, J = 10.5, 7.9 Hz, 1H), 5.25 (dd, J = 10.8, 9.2 Hz,
1H), 5.34 (dd, J = 3.5, 1.1 Hz, 1H), 5.69 (d, J = 9.5 Hz,
1H), 7.24–7.39 (m, 5H). 13C NMR (CDCl3) δ (ppm): 20.6,
20.7, 20.8, 21.0, 23.2, 52.4, 60.4, 61.1 (dt, J = 47.2,
4.4 Hz), 66.9 (t, J = 38.9 Hz), 69.5 (dd, J = 48.6,
42.5 Hz), 70.3, 70.7 (dd, J = 47.5, 38.7 Hz), 71.1, 71.2
(br. t, J = 40.8 Hz), 71.9, 75.2, 96.8, 101.2 (dt, J = 48.3,
5.5 Hz), 128.2, 126.4, 128.7, 136.8, 169.4, 170.2, 170.2,
170.3, 170.5, 171.2.
4.3.4 | Benzyl 2‐acetamido‐2‐deoxy‐4‐O‐β‐
13C6‐D‐galactopyranosyl‐6‐O‐sulfo‐α‐D‐
glucopyranoside, ammonium salt (7)
The ammonium salt of 6 (470 mg) was dissolved in anhy-
drous MeOH (10 mL) and sodium methoxide in methanol
(30 mass%) was added until pH was 11. After 1 h, the
reaction was complete by TLC and so was neutralised
with Amberlite 15 resin (H+ form). The mixture was fil-
tered, concentrated to remove methanol, and then loaded
onto a column of silica gel and eluted with 7:2:0.5 dichlo-
romethane: MeOH: aq NH4OH (26%), yielding the
ammonium salt of the target material (7) as a clear syrup
(0.341 g, 99%). (Unlabelled: HRMS [ESI] calcd for
C21H30NO14S [M − H]− m/z 552.1387, found: 552.1386.)
1H NMR (CD3OD) δ (ppm): 1.94 (s, 3H), 3.49–3.56 (m,
2H), 3.63 (ddd, J = 7.5, 4.6, 1.0 Hz, 1H), 3.66 (dd,
J = 10.0, 8.5 Hz, 1H), 3.70 (dd, J = 11.5, 4.6 Hz, 1H),
3.76 (dd, J = 11.5, 7.5 Hz, 1H), 3.82–3.84 (m, 1H), 3.87
(dd, J = 10.8, 8.4 Hz, 1H), 3.94 (dd, J = 10.8, 3.6 Hz,
1H), 3.99 (ddd, J = 10.0, 4.3, 2.0 Hz, 1H), 4.27 (dd,
J = 10.9, 2.0 Hz, 1H), 4.35 (dd, J = 11.0, 4.3 Hz, 1H),
4.48‐4.54 (m, 2H), 4.73 (d, J = 12.1 Hz, 1H), 4.85 (d,
J = 3.6 Hz, 1H), 7.26–7.30 (m, 1H), 7.31–7.36 (m, 2H),
7.37–7.40 (m, 2H). 13C NMR (CD3OD) δ (ppm): 22.5,
54.9, 62.5 (br d, J = 44.5 Hz), 67.4, 70.4 (t, J = 38.6 Hz),
70.5, 71.0, 72.8 (dd, J = 46.1, 39.6 Hz), 74.8 (br t,
J = 38.9 Hz), 77.0 (dd, J = 44.7, 38.6 Hz), 80.8, 97.3,
104.7 (d, J = 46.2 Hz), 128.9, 129.3, 129.4, 138.8, 173.4.
4.3.3 | Benzyl
2‐acetamido‐3‐O‐acetyl‐2‐deoxy‐6‐O‐sulfo‐4‐
O‐(2,3,4,6‐tetra‐O‐acetyl‐β‐13C6‐D‐
galactopyranosyl)‐α‐D‐glucopyranoside,
ammonium salt (6)
The starting material (5) and trimethylamine sulfur triox-
ide complex were co‐evaporated with anhydrous acetoni-
trile three times, then put under high vac for 8 h.
Anhydrous N,N‐dimethylformamide (20 mL) was added
to the dried trimethylamine sulfur trioxide complex
(1.20 g, 4.53 eq) and 5 (1.30 g, 1 eq). This mixture was
heated at 50°C for 16 h. MeOH (5 mL) was added, and
the mixture was stirred for 15 min. The solvents were
removed under high vac at 40°C. The crude material
was purified by column chromatography. The silica
column was first equilibrated using 1% NH3 in MeOH,
then flushed with ethyl acetate with 1% of the 1% NH3
in MeOH mixture. At this stage, the material was loaded
onto the column and eluted with 99:1 to 4:1 ethyl acetate:
(1% NH3 in MeOH), yielding a white solid (1.36 g, 92%).
(Unlabelled: HRMS [ESI] calcd for C31H40NO19SNa2
4.3.5 | Sodium 2‐acetamido‐2‐deoxy‐4‐O‐(β‐
13C6‐D‐galactopyranosyl)‐D‐glucopyranose‐
6‐sulfate (8)
Palladium hydroxide (20%) on carbon (65 mg) was added
to a solution of the ammonium salt of 7 (197 mg) in EtOH
(16 mL) and aqueous NH4OH (28%, 4 mL), and the solu-
tion was stirred under a hydrogen gas atmosphere at 20°C
1
[M + 2Na]2+ m/z 404.0850, found: 404.0853.) H NMR
(CDCl3) δ (ppm): 1.82 (s, 3H), 1.94 (s, 3H), 2.03 (s, 3H),

CAMERON ET AL.
7
for 5 h. Celite was washed using the same solvent ratio as
above, and then the mixture was filtered through it. The
filtrate was concentrated to dryness, then passed through
a Dowex 50WX8‐200, Na+ form resin exchange column,
then lyophilized giving the sodium salt of the product
(8, mixture of anomers) as a white foam, (174 mg, quant).
HRMS (ESI) calcd for C813C6H24NO14S [M − Na]− m/z
468.1119, found: 468.1125. (Unlabelled: HRMS [ESI]
calcd for C14H24NO14S [M − Na]− m/z 462.0923, found:
J = 8.8 Hz, 1H), 7.27–7.38 (m, 5H). 13C NMR (CDCl3) δ
(ppm): 20.6, 20.6, 20.7, 20.8, 23.4, 53.1, 60.8, 61.7 (dt,
J = 45.6, 4.7 Hz), 67.1 (t, J = 38.9 Hz), 69.1 (dd,
J = 49.0, 42.7 Hz), 70.2, 70.4, 70.5, 71.0 (br t,
J = 40.8 Hz), 71.5 (dd, J = 45.9, 39.2 Hz), 81.8, 97.0,
102.0 (dt, J = 48.9, 4.7 Hz), 128.1, 128.3, 128.7, 137.1,
169.6, 170.0, 170.2, 170.3, 170.6.
4.4.2 | Benzyl 2‐acetamido‐2‐deoxy‐4‐O‐(β‐
13C6‐D‐galactopyranosyl)‐α‐D‐
glucopyranoside (10)
1
462.0918.) H NMR (D2O) δ (ppm): 2.04 (s, 3H), 3.51–
3.57 (m, 1H), 3.64–3.82 (m, 6H), 3.83–3.96 (m, 2H), 4.17
(d, J = 9.8 Hz, 0.5H), 4.28–4.42 (m, 2H), 4.51–4.55 (m,
1H), 4.65 (under H2O peak, HSQC), 4.73–4.77 (m,
obscured by H2O peak), 5.20 (d, J = 2.9 Hz, 0.5H).
13C NMR (D2O) δ (ppm): 22.1, 22.4, 53.7, 56.2, 61.1 (dt,
J = 44.3, 4.3 Hz), 61.3, 66.5, 66.6, 68.6, 68.7 (t,
J = 38.3 Hz), 68.9, 69.3, 71.1 (dd, J = 46.2, 39.7 Hz),
71.1, 72.4, 72.6, 72.6 (t, J = 39.1 Hz), 72.7, 75.4 (dd,
J = 44.4, 38.3 Hz), 75.4, 77.6, 78.0, 90.7, 95.0, 102.7 (d,
J = 46.2 Hz), 102.8, 174.5, 174.8. Isotope enrichment by
mass spectrometry ion intensities: 99.0%.
To a solution of 9 (0.83 g) in anhydrous MeOH (30 mL),
8 drops of NaOMe in MeOH (5.4 M) was added,
resulting in pH = 10 solution. This was stirred at 20°C
for 20 h, then neutralised by stirring with Amberlyst‐
15 resin (H+ form) for 15 min. The solution was then
filtered through Celite and the solvent removed under
reduced pressure, yielding the product (10) as a white
solid (0.60 g, 98%). HRMS (ESI) calcd for
C1513C6H31NO11Na [M + Na]+ m/z 502.1996, found:
502.1994. 1H NMR (CD3OD) δ (ppm): 1.94 (s, 3H),
3.48 (dd, J = 11.4, 7.4 Hz, 1H), 3.54 (dd, J = 9.7,
7.6 Hz, 1H), 3.58 (ddd, J = 7.4, 4.7, 1.1 Hz, 1H), 3.63
(dd, J = 9.9, 8.3 Hz, 1H), 3.69 (dd, J = 11.4, 4.7 Hz,
1H), 3.76 (dd, J = 11.4, 7.4 Hz, 1H), 3.77 (ddd,
J = 9.7, 4.1, 2.7 Hz, 1H), 3.82 (dd, J = 3.3, 1.0 Hz,
1H), 3.82‐3.90 (m, 3H), 3.93 (dd, J = 10.8, 3.5 Hz, 1H),
4.37 (d, J = 7.6 Hz, 1H), 4.51 (d, J = 12.0 Hz, 1H),
4.72 (d, J = 12.0 Hz, 1H), 4.87 (d, J = 3.5 Hz, 1H),
7.23–7.30 (m, 1H), 7.31–7.35 (m, 2H), 7.36–7.39 (m,
2H). 13C NMR (CD3OD) δ (ppm): 22.5, 55.0, 61.9, 70.3
(t, J = 38.7 Hz), 70.9, 72.6 (dd, J = 46.8, 40.0 Hz),
74.9 (br t, J = 39.2 Hz), 77.1 (dd, J = 44.6, 38.8 Hz),
81.4, 97.4, 105.1 (dt, J = 46.7, 4.8 Hz), 128.8, 129.2,
129.4, 139.0, 173.4. Two natural abundance carbon
peaks are not listed because of being obscured by
high‐abundance 13C signals.
4.4 | Synthesis of 2‐acetamido‐2‐deoxy‐6‐O‐
sulfo‐4‐O‐(6‐O‐sulfo‐β‐13C6‐D‐
galactopyranosyl)‐α‐D‐glucopyranose,
disodium salt (16)
4.4.1 | Benzyl 2‐acetamido‐2‐deoxy‐4‐O‐
(2,3,4,6‐tetra‐O‐acetyl‐β‐13C6‐D‐
galactopyranosyl)‐α‐D‐glucopyranoside (9)
Hydrogen fluoride‐pyridine (70% solution in pyridine)
(3 mL) was added to a solution of 3 (1.14 g) in THF
(20 mL) and anhydrous pyridine (2 mL) contained in a
Nalgene vial at 0°C. The mixture was warmed to 20°C,
then stirred for 2 days. The solution was diluted with
ethyl acetate, washed with saturated aqueous NaHCO3
and water, then dried over MgSO4, filtered, and the sol-
vent removed under reduced pressure, yielding a tan
solid. This material was purified by column chromatogra-
phy (2:1 to 1:0 ethyl acetate:petroleum ether), giving the
product (9) as colourless oil. On further drying, this gave
a white solid (0.80 g, quantitative). HRMS (ESI) calcd for
C2313C6H39NO15Na [M + Na]+ m/z 670.2413, found:
4.4.3 | Benzyl
2‐acetamido‐2‐deoxy‐6‐O‐tert‐
butyldiphenylsilyl‐4‐O‐(6‐O‐tert‐
butyldimethylsilyl‐β‐13C6‐D‐
galactopyranosyl)‐α‐D‐glucopyranoside (11)
1
670.2413. H NMR (CDCl3) δ (ppm): 1.96 (s, 3H), 1.98
(s, 3H), 2.05 (s, 3H), 2.10 (s, 3H), 2.14 (s, 3H), 3.63 (d,
A solution of 10 (0.52 g, 1 eq) in anhydrous N,N‐
dimethylformamide (15 mL) under argon was cooled to
0°C. Imidazole (0.30 g, 4.1 eq) was added followed by
tert‐butyldimethylsilyl chloride (0.38 g, 2.3 eq). The
resulting solution was stirred at 0°C for 3 h. The solvent
was removed under high vac. The crude material was
purified by column chromatography (1:0 petroleum
J
= 11.7 Hz, 1H), 3.64–3.70 (m, 2H), 3.71 (d,
J = 11.7 Hz, 1H), 3.81–3.86 (m, 1H), 3.97–4.04 (m, 2H),
4.08–4.16 (m, 1H), 4.11 (ddd, J = 10.5, 8.8, 3.7 Hz, 1H),
4.46 (d, J = 11.8 Hz, 1H), 4.65 (d, J = 8.0 Hz, 1H), 4.67
(d, J = 11.8 Hz, 1H), 4.95 (d, J = 3.6 Hz, 1H), 5.00–5.05
(m, 1H), 5.20–5.26 (m, 1H), 5.36–5.40 (m, 1H), 5.63 (d,

8
CAMERON ET AL.
ether:ethyl acetate to 1:0 ethyl acetate:MeOH to 10:1
ethyl acetate:MeOH), giving several fractions containing
the desired product. These fractions were combined
and the solvent removed under reduced pressure, yield-
ing the product (11) as a colourless oil (0.59 g, 77%).
HRMS (ESI) calcd for C2713C6H59NO11Si2Na [M + Na]
J = 3.7 Hz, 1H), 4.95 (dd, J = 10.3, 3.5 Hz, 1H), 5.06
(dd, J = 10.4, 7.9 Hz, 1H), 5.18 (dd, J = 10.8, 9.3 Hz,
1H), 5.45 (dd,
J = 3.4, 1.1 Hz, 1H), 5.78 (d,
J = 9.5 Hz, 1H), 7.28–7.38 (m, 5H). 13C NMR (CDCl3)
δ (ppm): −5.7, −5.6, −5.3, −5.0, 18.0, 18.3, 20.6, 20.7,
20.8, 20.9, 23.2, 25.7, 25.9, 52.1, 60.0 (d, J = 47.8 Hz),
60.9, 66.7 (t, J = 39.2 Hz), 69.8 (dd, J = 48.5,
42.6 Hz), 71.5 (br t, J = 41.0 Hz), 73.2 (dd, J = 47.7,
39.5 Hz), 96.6, 100.5 (dt, J = 48.4, 5.4 Hz), 128.1,
128.1, 128.5, 136.9, 169.0, 169.9, 170.0, 170.1, 171.1. Four
natural abundance carbon peaks are not listed because
of being obscured by high‐abundance 13C signals.
+
1
m/z 730.3726, found: 730.3726. H NMR (CD3OD) δ
(ppm): 0.09 (s, 6H), 0.11 (s, 3H), 0.11 (s, 3H), 0.91 (s,
9H), 0.93 (s, 9H), 1.94 (s, 3H), 3.45 (dd, J = 9.7,
3.3 Hz, 1H), 3.53 (dd, J = 6.5, 1.1 Hz, 1H), 3.55 (dd,
J = 9.7, 7.7 Hz, 1H), 3.64 (dd, J = 9.9, 8.4 Hz, 1H),
3.75 (ddd, J = 9.8, 4.3, 1.8 Hz, 1H), 3.79 (dd, J = 10.2,
6.4 Hz, 1H), 3.81–3.87 (m, 3H), 3.90 (dd, J = 11.5,
1.8 Hz, 1H), 3.92 (dd, J = 10.8, 3.5 Hz, 1H), 3.99 (dd,
J = 11.5, 4.2 Hz, 1H), 4.38 (d, J = 7.7 Hz, 1H), 4.49
(d, J = 12.0 Hz, 1H), 4.70 (d, J = 12.0 Hz, 1H), 4.85
(d, J = 3.5 Hz, 1H), 7.26–7.30 (m, 1H), 7.31–7.37 (m,
4H). 13C NMR (CD3OD) δ (ppm): −5.3, −5.2, −5.0,
−4.9, 22.5, 26.4, 26.5, 31.7, 36.9, 55.1, 62.9, 62.9 (dt,
J = 46.7, 4.2 Hz), 69.6 (t, J = 38.9 Hz), 70.4, 70.8, 72.5
(dd, J = 46.9, 40.0 Hz), 75.0 (t, J = 39.4 Hz), 76.9 (dd,
J = 46.7, 39.1 Hz), 80.8, 97.5, 105.0 (dt, J = 47.0,
4.8 Hz), 128.8, 129.1, 129.4, 139.0, 173.3. Two natural
abundance carbon peaks are not listed because of being
obscured by high‐abundance 13C signals.
4.4.5 | Benzyl
2‐acetamido‐3‐O‐acetyl‐2‐deoxy‐4‐O‐(2,3,4‐
tri‐O‐acetyl‐β‐13C6‐D‐galactopyranosyl)‐α‐D‐
glucopyranoside (13)
Hydrogen fluoride‐pyridine (70% solution in pyridine)
(1.5 mL) was added to a solution of 12 (0.58 g) in
THF (6.7 mL) and anhydrous pyridine (0.70 mL)
contained in a Nalgene vial at 0°C. The mixture was
taken out of the ice bath and stirred for 2 h. TLC (in
ethyl acetate) indicated complete reaction after 2 h.
The solution was diluted with ethyl acetate, cooled to
0°C, washed with saturated aqueous NaHCO3 and
water, then dried over MgSO4, filtered, and the solvent
removed under reduced pressure, giving a yellow syrup.
This crude material was purified by column chromatog-
raphy (solid loaded, 1:0 to 4:1 ethyl acetate: MeOH),
giving the product (13) as a colourless oil, which on
drying gave a white solid (0.38 g, quantitative). HRMS
(ESI) calcd for C2313C6H39NO15Na [M + Na]+ m/z
670.2419, found: 670.2421. (Unlabelled: HRMS (ESI)
calcd for C29H39NO15Na [M + Na]+ m/z 664.2217,
found: 664.2220.) 1H NMR (CDCl3) δ (ppm): 1.89 (s,
3H), 1.97 (s, 3H), 2.05 (s, 3H), 2.07 (s, 3H), 2.13 (s,
3H), 3.49 (dd, J = 11.6, 5.2 Hz, 1H), 3.63–3.81 (m,
5H), 4.01 (t, J = 9.5 Hz, 1H), 4.22 (ddd, J = 10.8, 9.4,
3.7 Hz, 1H), 4.46 (d, J = 12.0 Hz, 1H), 4.66 (d,
J = 11.9 Hz, 1H), 4.70 (d, J = 7.8 Hz, 1H), 4.85 (d,
J = 3.6 Hz, 1H), 5.02 (dd, J = 10.4, 3.4 Hz, 1H), 5.11
(dd, J = 10.4, 7.8 Hz, 1H), 5.24 (dd, J = 10.8, 9.2 Hz,
4.4.4 | Benzyl
2‐acetamido‐3‐O‐acetyl‐2‐deoxy‐6‐O‐tert‐
butyldiphenylsilyl‐4‐O‐(2,3,4‐tri‐O‐acetyl‐6‐
O‐tert‐butyldimethylsilyl‐β‐13C6‐D‐
galactopyranosyl)‐α‐D‐glucopyranoside (12)
To a solution of 11 (0.59 g) in anhydrous pyridine
(10 mL) at 0°C was added acetic anhydride (5 mL)
dropwise. The solution was stirred at 20°C overnight.
TLC (1:1 petroleum ether:ethyl acetate) indicated a
small amount of intermediate was still present and so
the reaction was heated at 40°C for 2 h to drive it to
completion. The solvent was removed under reduced
pressure, and the crude material purified by column
chromatography (2:1 to 1:1 petroleum ether:ethyl ace-
tate), giving the product (12) as a clear syrup, which
on drying gave a white foam (0.58 g, 79%). HRMS
(ESI) calcd for C3513C6H68NO15Si2 [M + H]+ m/z
876.4329, found: 876.4339. 1H NMR (CDCl3) δ (ppm):
0.01 (s, 3H), 0.03 (s, 3H), 0.86 (s, 9H), 0.93 (s, 9H),
1.88 (s, 3H), 1.96 (s, 3H), 2.02 (s, 3H), 2.03 (s, 3H),
2.11 (s, 3H), 3.54 (t, J = 8.9 Hz, 1H), 3.57–3.64 (m,
2H), 3.68–3.74 (m, 2H), 3.79 (dd, J = 11.5, 3.3 Hz,
1H), 3.89 (t, J = 9.6 Hz, 1H), 4.18 (ddd, J = 10.8, 9.5,
3.8 Hz, 1H), 4.48 (d, J = 12.0 Hz, 1H), 4.63 (d,
J = 7.9 Hz, 1H), 4.67 (d, J = 12.0 Hz, 1H), 4.85 (d,
1H), 5.33 (dd,
J = 3.4, 1.0 Hz, 1H), 5.76 (d,
J = 9.4 Hz, 1H), 7.27–7.38 (m, 5H). 13C NMR (CDCl3)
δ (ppm): 20.7, 20.8, 20.9, 21.1, 23.2, 52.3, 60.3, 61.1 (d,
J = 43.4 Hz), 68.0 (t, J = 38.6 Hz), 69.9 (dd, J = 48.1,
42.4 Hz), 70.2, 71.1, 71.3 (br. t, J = 40.4 Hz), 72.4, 74.2
(dd, J = 43.7, 38.5 Hz), 74.6, 96.7, 100.9 (dt, J = 48.1,
5.0 Hz), 128.2, 128.4, 128.8, 136.8, 169.6, 170.1, 170.4,
170.9, 171.6.

CAMERON ET AL.
9
yielding the sodium salt of the product (15) as a white
solid (0.095 g, 97%). HRMS (ESI) calcd for
C1513C6H29NO17S2Na [M − H]− m/z 660.0981, found:
660.0971. (Unlabelled: HRMS [ESI] calcd for
4.4.6 | Benzyl
2‐acetamido‐3‐O‐acetyl‐2‐deoxy‐6‐O‐sulfo‐4‐
O‐(2,3,4‐tri‐O‐acetyl‐6‐O‐sulfo‐β‐13C6‐D‐
galactopyranosyl)‐α‐D‐glucopyranoside,
diammonium salt (14)
C21H30NO17S2 [M
−
H]− m/z 632.0955, found:
632.0950.) 1H NMR (CD3OD) δ (ppm): 1.95 (s, 3H),
3.50–3.57 (m, 2H), 3.64 (dd, J = 10.0, 8.0 Hz, 1H), 3.85‐
3.91 (m, 3H), 3.93 (dd, J = 10.8, 3.4 Hz, 1H), 3.99 (ddd,
J = 10.0, 4.4, 2.0 Hz, 1H), 4.18 (d, J = 6.3 Hz, 2H), 4.28
(dd, J = 11.0, 2.1 Hz, 1H), 4.35 (dd, J = 11.0, 4.4 Hz,
1H), 4.51 (m, 2H), 4.74 (d, J = 12.0 Hz, 1H), 4.86 (d,
J = 3.4 Hz, 1H), 7.26–7.30 (m, 1H), 7.31–7.36 (m, 2H),
7.37–7.40 (m, 2H). 13C NMR (CD3OD) δ (ppm): 22.6,
54.8, 67.5, 67.9 (dt, J = 46.3, 4.3 Hz), 70.1 (t,
J = 38.4 Hz), 70.4, 70.5, 71.1, 72.7 (dd, J = 46.3,
39.6 Hz), 74.5 (br t, J = 38.7 Hz), 74.6 (dd, J = 46.2,
38.5 Hz), 81.5, 97.3, 104.9 (dt, J = 46.4, 4.7 Hz), 128.9,
129.3, 129.4, 138.8, 173.6.
The diol starting material and sulfating reagent were
dried under high vac for 5 h. A mixture of 13 (0.38 g,
1 eq) and sulfur trioxide trimethylamine complex
(0.20 g, 2.4 eq) in anhydrous N,N‐dimethylformamide
(6.5 mL) were heated at 50°C for 20 h. MeOH (5 mL)
and 7 M NH3 in MeOH (1 mL) was added and the mix-
ture was stirred for 15 min. The solvent was removed
under high vac. The resulting crude material was purified
by column chromatography (dry loaded, 65:35 CHCl3:
MeOH), giving the ammonium salt of the product (14)
as a white solid (0.43 g, 87%). HRMS (ESI) calcd for
C2313C6H37NO21S2Na [M − H]− m/z 828.1403, found:
828.1389. (Unlabelled: HRMS (ESI) calcd for
C29H38NO21S2 [M
−
H]− m/z 800.1378, found:
1
800.1379.) H NMR (CD3OD) δ (ppm): 1.89 (s, 3H), 1.91
(s, 3H), 2.07 (s, 3H), 2.09 (s, 3H), 2.12 (s, 3H), 3.83 (ddd,
J = 10.1, 4.1, 2.0 Hz, 1H), 3.88 (dd, J = 10.1, 8.6 Hz,
1H), 4.01 (dd, J = 9.8, 7.7 Hz, 1H), 4.06 (dd, J = 9.8,
5.7 Hz, 1H), 4.12 (ddd, J = 7.2, 5.7, 1.2 Hz, 1H), 4.16
(dd, J = 11.0, 5.8 Hz, 1H), 4.17 (d, J = 11.0 Hz, 1H),
4.22 (dd, J = 10.9, 4.1 Hz, 1H), 4.59 (d, J = 12.1 Hz,
1H), 4.74 (d, J = 12.1 Hz, 1H), 4.81 (d, J = 3.7 Hz, 1H),
4.85 (d, J = 7.9 Hz, 1H), 5.00 (dd, J = 10.4, 7.9 Hz, 1H),
5.10 (dd, J = 10.4, 3.5 Hz, 1H), 5.20 (dd, J = 11.0,
8.6 Hz, 1H), 5.44 (dd, J = 3.5, 1.2 Hz, 1H), 7.27–7.32 (m,
1H), 7.33–7.38 (m, 2H), 7.39–7.44 (m, 2H). 13C NMR
(CD3OD) δ (ppm): 20.5, 20.6, 20.9, 21.4, 22.4, 53.0, 65.7
(dt, J = 47.7, 4.1 Hz), 66.5, 68.8 (t, J = 38.4 Hz), 70.7
(dd, J = 48.5, 42.8 Hz), 70.8, 70.9, 72.2 (dd, J = 47.5,
38.7 Hz), 72.3, 72.8 (br t, J = 40.3 Hz), 77.0, 97.3, 101.7
(dt, J = 48.5, 5.5 Hz), 129.0, 129.5, 129.6, 138.6, 171.4,
171.6, 172.0, 172.6, 173.3.
4.4.8 | 2‐Acetamido‐2‐deoxy‐6‐O‐sulfo‐4‐O‐
(6‐O‐sulfo‐β‐13C6‐D‐galactopyranosyl)‐α‐D‐
glucopyranose, disodium salt (16)
Palladium hydroxide (20%) on carbon (100 mg) was
added to a solution of disodium salt of 15 (0.310 g) in
EtOH (20 mL) and aqueous NH4OH (28%) (5 mL), and
the solution was stirred under a hydrogen gas atmosphere
at ambient temperature overnight. Celite was washed
using the same solvent ratio as above. The mixture was
filtered through this, and the filtrate was concentrated
to dryness. The resulting residue was dissolved in water,
passed through a Dowex exchange column (Na+ form,
50WX8‐200), then lyophilized (approximately 60 mL of
water), giving the disodium salt of the product (16) as a
white solid (0.255 g, 93%). HRMS (ESI) calcd for
C813C6H23NO17S2Na [M − Na]− m/z 570.0506, found:
570.0514. (Unlabelled: HRMS [ESI] calcd for
C14H23NO17S2Na [M − Na]− m/z 564.0311, found:
564.0300.) 1H NMR (D2O) δ (ppm): 2.03 (s, 3H), 3.53
(ddd, J = 9.9, 7.8, 4.4 Hz), 3.69 (dt, J = 9.9, 3.4 Hz),
3.71–3.77 (m), 3.82 (ddd, J = 9.6, 5.1, 2.3 Hz), 3.88–3.92
(m), 3.95–4.00 (m), 4.14‐4.22 (m), 4.28 (dd, J = 11.1,
5.1 Hz), 4.30–4.37 (m), 4.40 (dd, J = 11.1, 2.1 Hz), 4.54
(d, J = 7.9 Hz, 4.73 (s), 5.21 (d, J = 2.5 Hz). 13C NMR
(D2O) δ (ppm): 22.0, 22.3, 53.7, 56.2, 66.7, 67.1 (dt,
J = 46.3, 4.6 Hz), 68.3 (t, J = 38.2 Hz), 68.4, 69.3, 70.9
(dd, J = 46.2, 39.8 Hz), 72.3, 72.4 (br t, J = 39.5 Hz),
72.7, 72.8 (dd, J = 46.6, 38.0 Hz), 78.8, 79.2, 90.6, 95.0,
102.8, 103.0 (ddt, J = 46.3, 21.0, 5.2 Hz), 174.5, 174.8. Iso-
tope enrichment by mass spectrometry ion intensities:
98.7%.
4.4.7 | Benzyl
2‐acetamido‐2‐deoxy‐6‐O‐sulfo‐4‐O‐(6‐O‐
sulfo‐β‐13C6‐D‐galactopyranosyl)‐α‐D‐
glucopyranoside, disodium salt (15)
To a solution of 14 (0.120 g) in anhydrous MeOH (5 mL),
8 drops of NaOMe in MeOH (5.4 M) was added, resulting
in a pH = 10–11 solution. The solution was left at 20°C
for 1 h, at which point TLC indicated complete reaction.
The solution was neutralised by stirring with Amberlyst‐
15 (H+ form) resin for 15 min (added in small portions
until pH neutral). The solution was filtered through
Celite and the solvent removed under reduced pressure,

10
CAMERON ET AL.
11. Bhaduri S, Pohl NLB. Fluorous‐tag assisted syntheses of sulfated
Keratan sulfate oligosaccharide fragments. Org Lett.
2016;18(6):1414‐1417.
ACKNOWLEDGEMENT
The authors wish to thank Drs Herbert Wong and
Yinrong Lu (Callaghan Innovation, New Zealand) for
assistance with NMR spectroscopy and for obtaining the
high resolution mass spectra.
12. Kobayashi M, Yamazaki F, Ito Y, Ogawa T. A regio‐ and stereo‐
controlled synthesis of β‐D‐Glcp NAc6SO3‐(1→3)‐β‐D‐Galp6SO3‐
(1→4)‐β‐D‐GlcpNAc6SO3‐(1→3)‐D‐Galp, a linear acidic glycan
fragment of keratan sulfate I. Carbohydr Res. 1990;201(1):51‐67.
13. Misra AK, Agnihotri G, Madhusudan SK, Tiwari P. Practical
synthesis of sulfated analogs of lactosamine and sialylated
lactosamine derivatives. J Carbohydr Chem. 2004;23(4):191‐199.
ORCID
Scott A. Cameron https://orcid.org/0000-0003-2669-8622
Olga V. Zubkova https://orcid.org/0000-0002-6892-8812
14. Mende M, Bednarek C, Wawryszyn M, et al. Chemical synthesis
of glycosaminoglycans. Chem Rev. 2016;116(14):8193‐8255.
Richard H. Furneaux
https://orcid.org/0000-0003-0973-
15. Ohmae M, Sakaguchi K, Kaneto T, Si F, Kobayashi S.
Keratanase II‐catalyzed synthesis of keratan sulfate oligomers
by using sugar oxazolines as transition‐state analogue substrate
monomers: a novel insight into the enzymatic catalysis mecha-
nism. Chembiochem. 2007;8(14):1710‐1720.
2606
Phillip M. Rendle https://orcid.org/0000-0002-2825-8746
REFERENCES
16. Pazynina GV, Severov VV, Maisel ML, Belyanchikov IM, Bovin
NV. Synthesis of mono‐, di‐ and tri‐O‐sulfated N‐
acetyllactosamines in a form suitable for glycochip printing.
Mendeleev Commun. 2008;18(5):238‐240.
1. Harmatz P, Mengel KE, Giugliani R, et al. The Morquio A clin-
ical assessment program: baseline results illustrating
progressive, multisystemic clinical impairments in Morquio A
subjects. Mol Genet Metab. 2013;109(1):54‐61.
17. Takeda N, Tamura J‐i. Synthesis of biotinylated keratan sulfate
2. Tomatsu
S,
Montano
AM,
Oikawa
H,
et
al.
repeating
2014;78(1):29‐37.
disaccharides.
Biosci
Biotechnol
Biochem.
Mucopolysaccharidosis type IVA (Morquio A disease): clinical
review and current treatment: a special review. Curr Pharm
Biotechnol. 2011;12(6):931‐945.
18. Lange B, Šimonová A, Fischöder T, Pelantová H, Křen V, Elling
L. Towards keratan sulfate—chemoenzymatic cascade synthesis
of sulfated N‐acetyllactosamine (LacNAc) glycan oligomers. Adv
Synth Catal. 2016;358(4):584‐596.
3. Funderburgh JL. Keratan sulfate: structure, biosynthesis. And
Function Glycobiology. 2000;10(10):951‐958.
4. Pomin VH. Keratan sulfate: an up‐to‐date review. Int J Biol
Macromol. 2015;72:282‐289.
19. Gan ZH, Cao SD, Wu QQ, Roy R. Regiospecific syntheses of N‐
acetyllactosamine derivatives and application toward a highly
practical synthesis of Lewis X trisaccharide. J Carbohydr Chem.
1999;18(7):755‐773.
5. Hendriksz CJ, Burton B, Fleming TR, et al. Efficacy and safety
of enzyme replacement therapy with BMN 110 (elosulfase alfa)
for Morquio A syndrome (mucopolysaccharidosis IVA): a phase
3 randomised placebo‐controlled study. J Inherited Metab Dis.
2014;37(6):979‐990.
20. Schmidt RR, Stumpp M. Glycosylimidates.8. Synthesis of 1‐
thioglycosides. Liebigs Ann Chem. 1983;(7):1249‐1256.
21. Yeager AR, Finney NS. Synthesis of fluorescently labeled UDP‐
GlcNAc analogues and their evaluation as chitin synthase sub-
strates. J Org Chem. 2005;70(4):1269‐1275.
6. Martell LA, Cunico RL, Ohh J, Fulkerson W, Furneaux R, Foehr
ED. Validation of an LC‐MS/MS assay for detecting relevant
disaccharides from keratan sulfate as a biomarker for Morquio
A syndrome. Bioanalysis. 2011;3(16):1855‐1866.
22. Munneke S, Painter GF, Gainsford GJ, Stocker BL, Timmer
MSM. Total synthesis of Lewis(X) using a late‐stage crystalline
intermediate. Carbohydr Res. 2015;414:1‐7.
7. Auray‐Blais C, Lavoie P, Maranda B, Boutin M. Evaluation of
urinary keratan sulfate disaccharides in MPS IVA patients using
UPLC–MS/MS. Bioanalysis. 2016;8(3):179‐191.
23. Schmidt RR, Toepfer A. Glycosylimidates.50. Glycosylation with
highly reactive glycosyl donors–efficiency of the inverse proce-
dure. Tetrahedron Lett. 1991;32(28):3353‐3356.
8. Wood TC, Harvey K, Beck M, et al. Diagnosing
mucopolysaccharidosis IVA.
J
Inherited Metab Dis.
2013;36(2):293‐307.
9. Steward M, Berezovskaya Y, Zhou H, et al. Recombinant,
truncated B. circulans keratanase‐II: description and
characterisation of a novel enzyme for use in measuring urinary
keratan sulphate levels via LC‐MS/MS in Morquio A syndrome.
Clin Biochem. 2015;48(12):796‐802.
How to cite this article: Cameron SA, Zubkova
OV, Toms S, Furneaux RH, Rendle PM. Synthesis
of 13C‐labelled sulfated N‐acetyl‐D‐lactosamines to
aid in the diagnosis of mucopolysaccharidosis
diseases. J Label Compd Radiopharm. 2018;1–10.
https://doi.org/10.1002/jlcr.3697
10. Hahm HS, Broecker F, Kawasaki F, et al. Automated glycan
assembly of oligo‐N‐acetyllactosamine and keratan sulfate probes
to study virus‐glycan interactions. Chem. 2017;2(1):114‐124.