pharmaceuticals
Article
1H
and 15N NMR Analyses on Heparin,
Heparan Sulfates and Related Monosaccharides
Concerning the Chemical Exchange Regime of
the N-Sulfo-Glucosamine Sulfamate Proton
Vitor H. Pomin 1,2
1
2
Program of Glycobiology, Institute of Medical Biochemistry Leopoldo de Meis,
Federal University of Rio de Janeiro, Rio de Janeiro 21941-590, Brazil; [email protected] or
[email protected]; Tel.: +55-21-3938-2939; Fax: +55-21-3938-2090
University Hospital Clementino Fraga Filho, Federal University of Rio de Janeiro,
Rio de Janeiro 21941-913, Brazil
Academic Editors: Madalena M. M. Pinto, Maria Emília de Sousa and Marta Correia da Silva
Received: 7 July 2016; Accepted: 3 September 2016; Published: 7 September 2016
Abstract: Heparin and heparan sulfate are structurally related glycosaminoglycans (GAGs).
Both GAGs present, although in different concentrations, N-sulfo-glucosamine (GlcNS) as one of
their various composing units. The conditional fast exchange property of the GlcNS sulfamate proton
in these GAGs has been pointed as the main barrier to its signal detection via NMR experiments,
especially 1 H-15 N HSQC. Here, a series of NMR spectra is collected on heparin, heparan sulfate
and related monosaccharides. The N-acetyl glucosamine-linked uronic acid types of these GAGs
were properly assigned in the 1 H-15 N HSQC spectra. Dynamic nuclear polarization (DNP) was
employed in order to facilitate 1D spectral acquisition of the sulfamate 15 N signal of free GlcNS.
Analyses on the multiplet pattern of scalar couplings of GlcNS 15 N has helped to understand the
chemical properties of the sulfamate proton in solution. The singlet peak observed for GlcNS happens
due to fast chemical exchange of the GlcNS sulfamate proton in solution. Analyses on kinetics
of alpha-beta anomeric mutarotation via 1 H NMR spectra have been performed in GlcNS as well
as other glucose-based monosaccharides. 1D 1 H and 2D 1 H-15 N HSQC spectra recorded at low
temperature for free GlcNS dissolved in a proton-rich solution showed signals from all exchangeable
protons, including those belonging to the sulfamate group. This work suits well to the current grand
celebration of one-century-anniversary of the discovery of heparin.
Keywords: heparan sulfate; heparin; NMR; N-sulfo-glucosamine; sulfamate proton
1. Introduction
Heparin and heparan sulfate are structurally correlated glycosaminoglycans (GAGs) endowed
with multiple biomedical functions. They play key roles in coagulation, thrombosis, angiogenesis,
cell proliferation, inflammation, microbial infections, tumor growth, metastasis, and many other
pathophysiological systems [1–3]. Heparin and heparan sulfate are both composed of alternating
4-linked α-L-glucosamine (GlcN) and 4-linked uronic acid units within repeating disaccharide building
blocks [2]. The uronic acid can be either β-D-glucuronic acid (GlcA), which is more abundant in
heparan sulfate, or the C5 epimer α-L-iduronic acid (IdoA) which is dominant in heparin. In heparin,
the IdoA unit is mostly 2-sulfated (IdoA2S). The GlcN units in both GAG types present, very
often, additional O- and/or N-substitutions. The O-substitutions are the rare 3-O-sulfation and
the common 6-O-sulfation. These O-sulfations occur more often in chains of heparin than of heparan
sulfate. The N-substitutions are the N-acetylation (NHCOCH3 ) and the N-sulfation (NHSO3 − )
Pharmaceuticals 2016, 9, 58; doi:10.3390/ph9030058
www.mdpi.com/journal/pharmaceuticals
Pharmaceuticals 2016, 9, 58
Pharmaceuticals 2016, 9, 58
2 of 11
2 of 11
N-sulfation
3−) also
known
as sulfamate.
groups give rise named
to monosaccharides
also
known as(NHSO
sulfamate.
These
groups
give riseThese
to monosaccharides
respectively named
N-acetyl
respectively(GlcNAc)
N-acetyl glucosamine
(GlcNAc) and (GlcNS).
N-sulfo-glucosamine
(GlcNS).
While the former
is
glucosamine
and N-sulfo-glucosamine
While the former
is dominant
in heparan
dominant
in
heparan
sulfate,
the
latter
is
more
abundant
in
heparin
[2].
The
major
repeating
sulfate, the latter is more abundant in heparin [2]. The major repeating disaccharide units of
disaccharide
of heparan
sulfate and
heparin
are respectively
[ → 4)-β-→
D-GlcA-(1
heparan
sulfate units
and heparin
are respectively
[→4)-βD -GlcA-(1
→4)-α-D-GlcNAc-(1
] (Figure→1A)
4)-α-GlcNAc-(1→]
(Figure
1A)
and [→4)-α-→
L-IdoA2S-(1→4)-αand
[→D4)-αL -IdoA2S-(1
→4)-αD -GlcNS6S-(1
] (Figure 1B). D-GlcNS6S-(1→] (Figure 1B).
CH2SO3-
A
O
H
H
OH
H
O
H
H
H
OSO3
-
B
NH
H-OH H-OH
H-OH H-OH
H-OH H-OH
H-OH H-OH
Slow exchange!
O
H
H
OH
H
H
COCH3
H
O
O
H
O
COOOH H
CH2SO3-
O
H
H
H
H
H
O
H
O
COOOH H
OSO3-
O
NH
SO3-
H-OH H-OH
H-OH H-OH
H-OH H-OH
H-OH H-OH
Fast exchange!
Figure
1. 1.Structural
buildingblocks
blocksofofheparan
heparansulfate
sulfate
Figure
Structuralrepresentation
representation of
of the
the main
main disaccharide
disaccharide building
[→[→4)-β4)-β-D-GlcA-(1
→4)-α-D-GlcNAc-(1
→] (A)(A)
andand
heparin
[→4)-αL-IdoA2S-(1
→4)-α-D-GlcNS6S-(1
→] (B).
D-GlcA-(1→4)-αD-GlcNAc-(1→]
heparin
[→4)-αL-IdoA2S-(1→4)-αD-GlcNS6S-(1
Although
unit can
be unit
seencan
in both
GAGintypes,
first
disaccharide
dominant in
sulfate
→] (B). each
Although
each
be seen
both the
GAG
types,
the first is
disaccharide
is heparan
dominant
in
heparan
sulfateiswhile
second in
is heparin.
more abundant
in heparin.
Structures
highlight
the different
while
the second
morethe
abundant
Structures
highlight
the different
chemical
exchange
chemicalofexchange
properties
of theof15the
N-linked
protons
of the N-acetyl
andwith
N-sulfated
groups
withthe
properties
the 15 N-linked
protons
N-acetyl
and N-sulfated
groups
the protons
from
15 N in
thesolvent.
protons Analysis
from the bulk
solvent.
the scalar
coupling
multiplet
pattern
related toofdirect
bulk
on the
scalar Analysis
couplingon
multiplet
pattern
related
to direct
observation
15
in the
GlcNS
is diagnostic
of the
chemical
exchange
regime of the amide proton.
observation
of N of
GlcNS
is diagnostic
chemical
exchange
regime
of the
amide proton.
Structural analyses on heparin and heparan sulfates are crucial, especially to establish further
Structural analyses on heparin and heparan sulfates are crucial, especially to establish further
correlations with their biological roles. Nuclear magnetic resonance (NMR) spectroscopy is the most
correlations with their biological roles. Nuclear magnetic resonance (NMR) spectroscopy is the most
explored and informative analytical technique available so far for structural analyses of GAGs. Most
explored
and informative analytical technique available so far for structural analyses of GAGs. Most of
of these analyses are based on the 1H and 13C nuclei. However, a great content of works using the less
1
these
analyses
are based
on the
and 13 C nuclei.
However,
a great
content
workscan
using
the less
15N have
employed
isotope
beenHappearing
in the literature
lately
[4–24].
This of
nucleus
be found
15
employed
isotope
N
have
been
appearing
in
the
literature
lately
[4–24].
This
nucleus
can
be
found
at the composing amino sugars of GAGs such as GlcN, GlcNAc, GlcNS and N-acetyl galactosamine.
at the
composing
amino
sugars
of
GAGs
such
as
GlcN,
GlcNAc,
GlcNS
and
N-acetyl
galactosamine.
15
The lower exploration of the N in NMR analyses of GAGs, as compared to the two other magnetic
15N. Recent
Theactive
lowernuclei,
exploration
of the 15
in NMR
as compared
to thesuch
two as
other
relies mostly
onNthe
lower analyses
sensitive of
of GAGs,
developments
the magnetic
advent
active
mostly
on ultrahigh
the lower magnetic
sensitive fields,
of 15 N.cryoprobe
Recent developments
as the
advent
and nuclei,
spreadrelies
of the
use of
technology, such
isotopic
labeling
and
spread
of
the
use
of
ultrahigh
magnetic
fields,
cryoprobe
technology,
isotopic
labeling
techniques,
techniques, novel combinations of 2D pulse sequences and other modern techniques such as
dynamic
nuclear polarization
have, and
however,
facilitating
the assessment
of 15N-related
novel
combinations
of 2D pulse(DNP)
sequences
otherbeen
modern
techniques
such as dynamic
nuclear
15
resonances(DNP)
in NMR
analyses
of GAGs
polarization
have,
however,
been[4].
facilitating the assessment of N-related resonances in NMR
Here,
a series
analyses
of GAGs
[4]. of NMR-based experiments, mostly those involving 15N, has been applied to
heparin,
heparan
sulfates
and related
monosaccharides,
mostlyinvolving
GlcNS. It 15
has
demonstrated
Here, a series of
NMR-based
experiments,
mostly those
N, been
has been
applied to
that the
uronic sulfates
acid type
(GlcA
or monosaccharides,
IdoA) linked to the
observable
of heparin and
heparin,
heparan
and
related
mostly
GlcNS.amino
It hassugars
been demonstrated
that
1H-15N heteronuclear single quantum coherence
heparan
sulfates
can
be
properly
identified
via
the uronic acid type (GlcA or IdoA) linked to the observable amino sugars of heparin and heparan
(HSQC)
Further
NMR analyses,
via 1D 15single
N signal
acquisition
assisted
by DNP
on
sulfates
canspectra.
be properly
identified
via 1 H-15especially
N heteronuclear
quantum
coherence
(HSQC)
spectra.
the standard GlcNS monosaccharide, employed
here
as
a
model
compound
to
the
correspondent
Further NMR analyses, especially via 1D 15 N signal acquisition assisted by DNP on the standard
unit in heparin and heparan sulfate, have greatly supported the phenomenon of fast chemical
GlcNS monosaccharide, employed here as a model compound to the correspondent unit in heparin
exchange of the GlcNS sulfamate proton in solution, although the regime of the chemical exchange is
and heparan sulfate, have greatly supported the phenomenon of fast chemical exchange of the GlcNS
highly conditional to other parameters such as pH, temperature and Ka. Analysis of the scalar
sulfamate proton in solution, although15the regime of the chemical exchange is highly conditional to
coupling multiplet pattern of the 1D N in GlcNS will help to understand the phenomenon of fast
other parameters such as pH, temperature and Ka. Analysis of the scalar coupling multiplet pattern of
exchange of the sulfamate proton in solution. Although other works exist in the field regarding the
thechemical
1D 15 N in
GlcNS will
help
to understand
exchange ofofthe
proton
15N NMR for of
exchange
of the
sulfamate
proton,the
1Dphenomenon
thefast
investigation
thissulfamate
phenomenon
in solution.
exist in
the field regarding
chemical
exchange
of the
sulfamate
has never Although
been used other
befor. works
1D 1H NMR
experiments
were herethe
explored
to (1)
understand
kinetics
of
15 N NMR for the investigation of this phenomenon has never been used befor. 1D 1 H NMR
proton,
1D
anomeric mutarotation of some related monosaccharides, including GlcNS; (2) confirm the fast
experiments
were hereofexplored
to (1)
understand
kinetics
of anomeric
mutarotation
of some
exchange property
the GlcNS
sulfamate
proton
at regular
experimental
conditions;
andrelated
(3)
monosaccharides,
(2)detection
confirm of
thethis
fastsulfamate
exchangeproton
property
of spectrum
the GlcNSofsulfamate
demonstrate the including
possibility GlcNS;
of signal
when
GlcNS
proton
at regular
experimental
conditions;
andat(3)
demonstrate
the
possibility
of signal
dissolved
in a proton-rich
solution
is recorded
low
temperature.
This
NMR-based
studydetection
suits well of
thistosulfamate
whenofspectrum
GlcNS dissolved
in a proton-rich
solution is recorded at low
the currentproton
celebration
the 100thof
anniversary
of the discovery
of heparin.
temperature. This NMR-based study suits well to the current celebration of the 100th anniversary of
the discovery of heparin.
Pharmaceuticals 2016, 9, 58
Pharmaceuticals 2016, 9, 58
3 of 11
3 of 11
Resultsand
andDiscussion
Discussion
2. 2.Results
15N
1 H15 N
HSQC
2.1.
RecognitionofofGlcNAc-Linked
GlcNAc-LinkedUronic
Uronic Acid
Acid Types
Types in
in Heparin and
2.1.
Recognition
and Heparan
HeparanSulfates
Sulfatesvia
via1HSpectra
HSQC
Spectra
15N HSQC spectrum of a heparin-based sample dissolved
The
onlytwo
twocross-peaks
cross-peaksseen
seenin
inaa11HH-15
The
only
N HSQC spectrum of a heparin-based sample dissolved
mMsodium
sodium acetate
acetate buffer
2OO
4.5)4.5)
0.1%
sodium
azide
(final(final
concentration
of ~10of
inin5050mM
buffer 12.5%
12.5%DD
(pH
0.1%
sodium
azide
concentration
2 (pH
mg/mL)
are
highlighted
in
Figure
2A
by
a
continuous-line
circle.
These
cross-peaks
resonating
with
~10 mg/mL) are highlighted in Figure 2A by a continuous-line circle. These cross-peaks resonating
δ
H
/δ
N
at
8.32–8.37/123.4–123.8
and
at
8.24–8.29/123.2–124.1
ppm
belong
to
the
amide
group
of
with δH /δN at 8.32–8.37/123.4–123.8 and at 8.24–8.29/123.2–124.1 ppm belong to the amide group
ofofthese
ofGlcNAc
GlcNAcunits
unitsasasassigned
assignedininprevious
previousworks
works[4–6].
[4–6].The
Thereason
reasonforforthe
theappearance
appearance
thesetwo
two
resonances
with
different
intensities
has
not
been
explained
before.
Relative
abundance
of
these
resonances with different intensities has not been explained before. Relative abundance of these peaks
peaks
hasa shown
a proportion
of 82%:18%
(Figure
2B). The difference
of percentages
forofthe
pair of
has
shown
proportion
of 82%:18%
(Figure 2B).
The difference
of percentages
for the pair
resonances
1H dimension can be explained in terms of the composition of
resonances distinguished solely
on
the
distinguished solely on the 1 H dimension can be explained in terms of the composition of the adjacent
the adjacent uronic acid types linked to the GlcNAc units.
uronic acid types linked to the GlcNAc units.
77.0
A
81.0
85.0
89.0
93.0
97.0
101.0
15N
105.0
– chemical shift (ppm)
110.0
114.0
117.0
121.0
125.0
129.0
133.0
137.0
141.0
145.0
9.5
9.0
8.5
8.0
7.5
7.0
1
B
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
H – chemical shift (ppm)
118.0
C
D
118.5
119.0
119.5
15N
120.0
– chemical shift (ppm)
120.5
121.0
121.5
122.0
122.5
123.0
123.5
124.0
8.5
8.4
8.3
8.2
8.5
8.4
1H –
8.3
8.2
8.5
8.4
8.3
8.2
124.5
8.1
chemical shift (ppm)
1 H-1515 N HSQC spectra of heparin (A,B) and heparan sulfates isolated from
Figure
Figure2.2. 2D
2D NMR 1HN HSQC spectra of heparin (A and B) and heparan sulfates isolated from
Chinese
Hamster
Expansions are
are δδHH/δ/δ
NN
Chinese
HamsterOvarian
Ovariancells
cells(C)
(C)and
andfrom
fromthe
thebivalve
bivalveNodipecten
Nodipecten nodosus (D). Expansions
3.0–9.5/77.0–147.0
8.1–8.5/118.0–124.6ppm
ppm(B–D)
(B–D)for
forsamples
samples(10
(10mg/mL)
mg/mL)
/δNN 8.1–8.5/118.0–124.6
3.0–9.5/77.0–147.0 ppm
ppm (A)
(A) and
and δδ
HH/δ
dissolved
O (pH
(pH 4.5),
4.5), 0.1%
0.1%sodium
sodiumazide.
azide.The
Thesolid
solid
and
and
dissolvedinin5050mM
mMsodium
sodiumacetate
acetate buffer,
buffer, 12.5%
12.5% D
D22O
dashedcircles
circleshighlight
highlightthe
theobserved
observedand
andtheoretical
theoretical regions
regions for the 11Hdashed
H-1515NNcross-peaks
cross-peaksofofN-acetyl
N-acetyl
glucosamine(GlcNAc)
(GlcNAc)and
andN-sulfo-glucosamine
N-sulfo-glucosamine (GlcNS)
glucosamine
(GlcNS)units,
units,respectively.
respectively.Spectra
Spectrawere
wererecorded
recorded
◦
18.8
and2525 C.
°C.
at at
18.8
T Tand
Pharmaceuticals 2016, 9, 58
4 of 11
Heparin is well-known for bearing ≥70% IdoA units and the counter-balance of GlcA units [25].
The two GlcNAc-related 1 H-15 N cross-peaks result, therefore, from the GlcA/IdoA content in
heparin structure. To support this interpretation and the assignments of the 1 H-15 N pairs in
heparin as IdoA-linked and GlcA-linked GlcNAcs (Figure 2A,B); 1 H-15 N HSQC spectra of two
heparan sulfates structurally distinct in terms of IdoA/GlcA ratios were included in the analyses
(Figure 2C,D). The structures of these heparan sulfates were characterized in previous works [5,26].
The heparan sulfates were extracted from Chinese Hamster Ovarian (CHO) cells [5] and from the
bivalve Nodipecten nodosus [26]. The IdoA percentage in mammalian-derived heparan sulfates has
commonly a range of 30%–50% regardless of the origins and pathophysiological conditions [25].
The invertebrate heparan sulfate shows, on the other hand, a unique structure composed of chains
entirely constituted of GlcA units [26]. The assignments of the 1 H-15 N cross-peaks with distinct
percentages in the 15 N-HSQC spectra of the two structurally distinct heparan sulfate samples can
be associated with the differential GlcA/IdoA ratios reported to these two samples in the previous
works [5,26]. As a consequence, the cross-peaks observed in the 1 H-15 N spectra for GlcNAc of heparin
and heparan sulfates (Figure 2B–D) distinguished solely in terms of 1 H-chemical shifts were attributed
and assigned to a GlcA-linked GlcNAc NH resonance with downfield δH , and to an IdoA-linked
GlcNAc resonance with upfield δH .
2.2. Fast Exchange Nature of the Sulfamate Proton in GlcNS from Heparin and Heparan Makes Difficult Its
Signal Observation through 1 H-15 N HSQC Spectra at Regular Experimental Conditions
The cross-peaks observed in the 1 H-15 N HSQC spectra of heparin and heparan sulfates were
assigned and attributed to the GlcNAc units (Figure 2). However, based on the background regarding
the structure of these GAG species, GlcNAc (Figure 1A) is not the only the GlcN type present in their
chains (Figure 1). The N-sulfated GlcN unit (Figure 1B), usually represented as GlcNHSO3 − or GlcNS,
is also present, and the GlcNAc/GlcNS ratio varies significantly among heparin and heparan sulfates
(Figure 1A vs. Figure 1B). Based on the knowledge regarding the mammalian-derived GAGs, it has
been generally accepted that while GlcNS unit occurs in heparin within a concentration of ≥80%,
in heparan sulfates it happens in a range of 40%–60% [25]. Despite the significant concentrations of
GlcNS units in both GAG types, 1 H-15 N cross-peaks derived from this unit have not been detected
through the series of 1 H-15 N HSQC spectra depicted at Figure 2. Many previous works have, however,
observed and identified the 1 H-15 N cross-peak of the GlcNS unit [7,16,18–20,23,24]. The 1 H-15 N pair
of GlcNS resonates to a far upfield region than GlcNAc in both 1 H and 15 N dimensions, more exactly
close to δH /δN at 5.5/93.5 ppm [16]. This region has been highlighted in the 1 H-15 N HSQC spectrum
of Figure 2A by a dashed-line circle. The NH signal of GlcNS can be detected at natural abundance by
choosing the proper pH condition in which the exchange with the bulk solvent can be reduced [7].
2.3. Non-polarized and Hyperpolarized 1D 15 N and 1 H Direct-observe of 15 N-Gln, GlcNS and
Other Monosaccharides
Figure 3 displays 1D 15 N signals for 15 N-labeled side chain Gln (panels A and B) and GlcNS at
isotopic natural abundance (panel C) acquired at non-polarized (panel A) and hyperpolarized (panels B
and C) conditions. The 15 N signals of 15 N-Gln (Figure 3A,B) and GlcNS (Figure 3C) resonate with δN
at 109.93 and at 93.1 ppm, respectively. Theoretical calculations lead to a factor of four-thousand-fold
enhancement from the non-polarized (Figure 3A) to the hyperpolarized (Figure 3B) condition. A time
course of one month-long acquisition in average would be necessary to record a 1D 15 N spectrum with
same signal-to-noise ratio of the hyperpolarized 15 N-Gln (Figure 3B) via a non-polarized experiment
(Figure 3A) on the same magnetic field (500 MHz). This indicated that sensitivity of 15 N direct-observe
through the non-polarized method, even on a 15 N-labeled molecule of low-molecular weight such as
15 N-Gln, can be very difficult. To achieve the optimal sensitivity, the experiment would be unreasonably
time-consuming. On the other hand, resolution can be enhanced in a faster way for both labeled
(Figure 3B) and unlabeled compounds (Figure 3C) via the hyperpolarization technique. 15 N-Gln was
Pharmaceuticals 2016, 9, 58
5 of 11
Pharmaceuticals 2016, 9, 58
5 of 11
used initially here for setting up the experimental parameters and to understand the potentiality of the
DNP
in enhancing
for spectral
acquisition.
ThisDNP
standard
turned out sensitivity
to be usefulfor
as aspectral
tool in
parameters
and tosensitivity
understand
the potentiality
of the
in enhancing
15
comparative
of the multiplicity
the as
N peak
of GlcNS
(Figure 3C)analyses
as compared
to
acquisition. analyses
This standard
turned outpatterns
to be of
useful
a tool
in comparative
of the
15 N-Gln (Figure 3B).
multiplicity patterns of the 15N peak of GlcNS (Figure 3C) as compared to 15N-Gln (Figure 3B).
A
120
B
115
110
105
100
120
115
110
105
100
C
15N
– chemical shift (ppm)
Figure
Figure 3.
3. Comparison
Comparison of
of the
the1D
1DNMR
NMRspectra
spectra1515N
N direct-observe
direct-observe of
ofthe
the1515N-isotopically labeled
labeled side
side
15
15
15
15
chain
N-Gln)atat2323mM
mMwith
with
98%N abundance
N abundance
(A,B)B)versus
98%
(A and
versusN-sulfo-glucosamine
N-sulfo-glucosamine
chain glutamine (( N-Gln)
15N natural abundance (0.37%) (C) at
(GlcNS)
(GlcNS) at
at 20
20 mM
mM with
with15
at non-polarized
non-polarized (A)
(A) and
and hyperpolarized
hyperpolarized
◦ C.
conditions
werewere
recorded
at 11.7
andT37
conditions (B,C).
(B andAll
C).spectra
All spectra
recorded
at T11.7
and
37 °C.
15N-Gln displays a resonance with splitting
The spectrum
spectrum from
from the
the 1515N
N direct-observe
direct-observe of
of 15
The
N-Gln displays a resonance with splitting
characterized by
by aa triplet
triplet pattern
pattern accompanied
accompanied by
by aa small
small triplet
triplet signal
signal on
on left
left due
due to
to isotopic
isotopic shift
shift
characterized
15N. On
15
(Figure
3B).
The
triplet
pattern
occurs
due
to
the
presence
of
two
amide
protons
coupled
to
(Figure 3B). The triplet pattern occurs due to the presence of two amide protons coupled to N. On the
15N-direct observation (Figure
the other
hand,
the GlcNS
produces
a single
singlet
other
hand,
the GlcNS
produces
just ajust
single
singlet
peakpeak
withwith
the 15the
N-direct
observation (Figure 3C).
3C).
This
singlet
pattern
occurs
due
to
fast
exchange
of
the
sulfamate
proton
the solvent
under
This singlet pattern occurs due to fast exchange of the sulfamate proton withwith
the solvent
under
the
the
conditions
of
the
experiment.
The
exchange
property
of
the
amide
protons
in
Gln
as
compared
to
conditions of the experiment. The exchange property of the amide protons in Gln as compared to
the one
one from
from GlcNS
GlcNS is
is much
much slower
slower due
due to
to the
the absence
absence of
of aa nearby
nearby electronegative
electronegative chemical
chemical group
group
the
like
sulfation.
like sulfation.
2.4.
2.4. Kinetic of the α-β
α-β Mutarotation Monitored
Monitored through
through Anomeric
Anomeric 11H Resonances
After questioning
questioning about the chemical properties of the sulfamate
sulfamate proton of
of the
the GlcNS
GlcNS units
units
After
(either as
as composing
composing unit
unit in
in the
the backbones
backbones of
of GAGs
GAGs or
or as
as free
free monosaccharide
monosaccharide in
in solution)
solution) and
and
(either
15N signal pattern of this
analyzing the
the DNP-assisted
DNP-assisted 1D
1D 15
this monosaccharide,
monosaccharide, questions
questions about
about the
the
analyzing
anomericmutarotation
mutarotation
kinetics
this
unit compared
to other
related monosaccharides
and commoner
anomeric
kinetics
of thisofunit
compared
to other related
and commoner
monosaccharides
also raised.
To addresspoint
this particular
point
of the α↔β
anomers
GlcNS
also
raised. To address
this particular
kinetics of
the kinetics
α↔β anomers
of GlcNS
wasoffurther
was further investigated comparatively with other Glc-based standards, via the commoner and
faster 1D 1H NMR method (Figure 4). As opposed to the three first standards (Glc, GlcN and
GlcNAC) that showed faster kinetics in the first hours after sample dissolution, the α:β ratio of
Pharmaceuticals 2016, 9, 58
6 of 11
investigated comparatively with other Glc-based standards, via the commoner and faster 1D 1 H NMR
method
(Figure
Pharmaceuticals
2016,4).
9, 58As opposed to the three first standards (Glc, GlcN and GlcNAC) that showed
6 of 11
faster kinetics in the first hours after sample dissolution, the α:β ratio of GlcNS changes, interestingly,
GlcNS
changes,
just slightly
fromofthe
great
dominance
of to
thethe
α-form
at 10
just
slightly
frominterestingly,
the initial great
dominance
theinitial
α-form
(90%
at 10 min)
point(90%
which
the
min) to the point
which(80:20
the equilibrium
reached
the α:β ratio
days
dissolution).
equilibrium
is reached
for the α:β is
ratio
after 2(80:20
daysfor
dissolution).
Asafter
seen,2 the
GlcNS
presents
seen,anomeric
the GlcNSconformation
presents a major
anomeric
conformation
in solution
which
is themonosaccharide
α-form (Figure
aAs
major
in solution
which
is the α-form
(Figure
4). These
4).
These
monosaccharide
standards
were
used
in
the
investigation
rather
than
heparin-derived
standards were used in the investigation rather than heparin-derived disaccharides because of their
disaccharides
because ofand
their
commercial
availability
and reduced structural variations.
commercial
availability
reduced
structural
variations.
Glc
αH1
GlcN
ring Hs
GlcNAc
ring Hs
αH1
HOD
75%
88%
10%
91%
βH1
βH1
25%
12%
9%
67%
85%
83%
30 min
HOD
αH1
HOD
βH1
βH1
10 min
ring Hs
αH1
HOD
90%
GlcNS
ring Hs
91%
33%
17%
15%
65%
9%
60%
70%
86%
40%
35%
1,5 h
30%
14%
54%
65%
53%
46%
86%
35%
47%
2,5h
14%
52%
67%
60%
2 days
33%
5.0
1H
48%
81%
40%
4.0
– chemical shift (ppm)
5.0
1H
19%
4.0
3.0
– chemical shift (ppm)
5.0
1H
4.0
5.0
3.0
– chemical shift (ppm)
1H
4.0
3.0
– chemical shift (ppm)
Figure4.
4. Kinetics
Kinetics of
of anomeric
anomeric mutarotation
Figure
mutarotation observed
observed for
forglucose
glucose(Glc)
(Glc)and
andGlc-based
Glc-basedstandards
standardssuch
such
as
glucosamine
(GlcN),
N-acetyl
glucosamine
(GlcNAc)
and
N-sulfo-glucosamine
(GlcNS)
as glucosamine (GlcN), N-acetyl glucosamine (GlcNAc) and N-sulfo-glucosamine (GlcNS)measured
measured
through 1D
1D 11H
different
time
courses
after
dissolution
in 100%
D2O D
(10O
through
H NMR
NMRspectra
spectrarecorded
recordedwithin
within
different
time
courses
after
dissolution
in 100%
2
mg/mL).
Anomeric
proton
signals
(αH1
and
βH1),
their
respective
relative
percentages
and
ring
(10 mg/mL). Anomeric proton signals (αH1 and βH1), their respective relative percentages and ring
protonsignals
signalsare
are
indicated
in the
panels.
denotes
residual
water signals.
were
proton
indicated
in the
panels.
HODHOD
denotes
residual
water signals.
Spectra Spectra
were recorded
◦
recorded
at
18.8
T
and
25
°C.
at 18.8 T and 25 C.
2.5. Reducing the Fast Exchange Rates of the GlcNHSO3− to Enable Proper NMR Detection
2.5. Reducing the Fast Exchange Rates of the GlcNHSO3 − to Enable Proper NMR Detection
Since the
the proton
proton of
of the
the sulfamate group
Since
group in
in GlcNS
GlcNS free
free in
in solution
solutionor
oras
ascomposing
composingunits
unitsofof
heparin/heparan sulfate
heparin/heparan
sulfate chains
chainsisis not
not easily
easilyobserved
observedthrough
throughNMR
NMRspectra
spectra(Figures
(Figures3C
3Cand
and2A)
2A)
◦ C)
◦ C),
recorded respectively
respectively at
°C)
and
physiological
temperatures
(37 °C),
development
of an
recorded
atroom
room(25
(25
and
physiological
temperatures
(37the
the development
experimental
condition
for detection
of this of
particular
proton proton
throughthrough
NMR spectroscopy
seems
of
an experimental
condition
for detection
this particular
NMR spectroscopy
valuable.
This condition
reliesrelies
on dissolving
the the
GlcNS
standard
in in
a mixture
seems
valuable.
This condition
on dissolving
GlcNS
standard
a mixtureofof10%:20%:70%
10%:20%:70%
◦ C.The
O/acetone/H2O2 Osolution
DD22O/acetone/H
solutionand
andthen
thentotorecord
recordNMR
NMRspectra
spectraatat3 3°C.
Thelow
lowtemperature
temperaturehelps
helpstoto
slow
down
the
rapid
chemical
exchange
of
the
labile
protons
during
the
NMR
experiment.
The
slow down the rapid chemical exchange of the labile protons during the NMR experiment. The residual
D2O in the solution was used for deuterium-lock in the instrument, acetone was used for
Dresidual
2 O in the solution was used for deuterium-lock in the instrument, acetone was used for avoiding
avoiding
of the
sample
at low temperature
and abundance
H2O was
to force
freezing offreezing
the sample
at low
temperature
and abundance
of H2 O wasofexplored
toexplored
force protonation.
protonation.
The 1D NMR spectrum of the GlcNS sample obtained after following this experimental strategy
is displayed in Figure 5A. This spectrum demonstrates that not only the sulfamate proton resonance,
but in fact, all exchangeable protons of GlcNS have become accessible. Signals of the exchangeable
Pharmaceuticals 2016, 9, 58
7 of 11
The 1D NMR spectrum of the GlcNS sample obtained after following this experimental strategy
Pharmaceuticals 2016, 9, 58
7 of 11
is displayed in Figure 5A. This spectrum demonstrates that not only the sulfamate proton resonance,
but
in fact,
all exchangeable
protons protons)
of GlcNSwere
haverather
become
of the exchangeable
protons
(nitrogenor oxygen-linked
lessaccessible.
sharp thanSignals
the unexchangeable
ones.
protons
(nitrogenor
oxygen-linked
protons)
were
rather
less
sharp
than
the
unexchangeable
ones.
The line-broadening and low intensity of the exchangeable proton resonances arise from a residual
The
line-broadening
and
low
intensity
of
the
exchangeable
proton
resonances
arise
from
a
residual
effect from the continued chemical exchange and dynamics, even at reduced temperature.
effect
from the continued
chemical
exchange and
even
at reduced
temperature.
Nonetheless,
Nonetheless,
peaks were
well-resolved
anddynamics,
intensities
were
sufficient
for chemical
shift
peaks
were
well-resolved
and
intensities
were
sufficient
for
chemical
shift
measurements
and to
measurements and to attempt further 2D spectral acquisition on the GlcNS. Although the detection
attempt
further 2D
spectral
acquisition
on theofGlcNS.
detection
and assignment
of the
and assignment
of the
exchangeable
protons
GlcNSAlthough
have beenthe
reported
in previous
works [8,22],
1 H NMR
1
exchangeable
protons
of
GlcNS
have
been
reported
in
previous
works
[8,22],
the
current
1D
the current 1D H NMR data present here is useful to demonstrate the differential multiplicities of
data
present
here is useful
the differential
of theregime
αH2 resonance
withas
δH
the αH2
resonance
with δHtoatdemonstrate
~3.2 ppm upon
low and fastmultiplicities
proton exchange
(Figure 5B,C)
atdiscussed
~3.2 ppmfurther.
upon low and fast proton exchange regime (Figure 5B,C) as discussed further.
Assignments
spectrum (Figure
(Figure 5)
5) were
were accomplished
accomplishedby
bytracing
tracing
Assignments of
of all
all resonances
resonances in this 1D spectrum
◦ C for the GlcNS
spin-spin
connectivities
(spin
systems)
in
the
2D
TOCSY
spectrum
recorded
also
at
3
spin-spin connectivities (spin systems) in the 2D TOCSY spectrum recorded also at 3 °C for the
sample
(Figure (Figure
S1A). InS1A).
this TOCSY
spectrum,
the labile
from hydroxyl
and amide
groups
GlcNS sample
In this TOCSY
spectrum,
theprotons
labile protons
from hydroxyl
and amide
are
accordingly
labeled as
OH and
NHand
together
with thewith
unexchangeable
protonsprotons
ascribed
as αH1,
groups
are accordingly
labeled
as OH
NH together
the unexchangeable
ascribed
1 H resonances
βH1,
αH2,
βH2,
αH3
andαH3
αH4.and
Chemical
shift values
thesefor
are plotted
in Tablein
S1.
as αH1,
βH1,
αH2,
βH2,
αH4. Chemical
shiftfor
values
these 1H resonances
are plotted
13
◦
13
Acquisition
of a C-HSQC
of this GlcNS
sample
3 C temperature
was alsowas
achieved
Table S1. Acquisition
of a spectrum
C-HSQC spectrum
of this
GlcNSatsample
at 3° C temperature
also
achieved
(Figure
S1B). Assignments
of 1H-13C cross-peaks
based
on the identified
previously1 H-chemical
identified
(Figure
S1B).
Assignments
of 1 H-13 C cross-peaks
were basedwere
on the
previously
13
1H-chemical shifts obtained via TOCSY spectrum. The chemical
13
shifts
forsubsequently
all C-atoms
were
shifts obtained via TOCSY spectrum. The chemical shifts for all C-atoms
were
obtained
subsequently
obtained
after
the 1H-chemical
after
the 1 H-chemical
shift
assignments
(Tableshift
S1).assignments (Table S1).
A
B
C
3.4
3.5
1H –
7.0
6.5
5.5
6.0
1H
5.0
4.5
4.0
3.5
3.3
3.2
3.1
chemical shift (ppm)
3.0
– chemical shift (ppm)
1 NMR spectra of N-sulfo-glucosamine (GlcNS). Expansions are 2.9–7.3 ppm (A) and
Figure
Figure5.5.1D
1D 1H
H NMR spectra N-sulfo-glucosamine (GlcNS). Expansions are 2.9–7.3 ppm (A) and
3.10–3.55
ppm
for C)
GlcNS
(10 mg/mL)
dissolved
in 10%:20%:70%
D2 O/acetone/H
O for
3.10–3.55 ppm(B,C)
(B and
for GlcNS
(10 mg/mL)
dissolved
in 10%:20%:70%
D2O/acetone/H
2 O for 2spectra
◦ C (A,B) or 37 ◦ C (C). Spectrum in panel B is just a close-up
recorded
in
18.8
T
NMR
instrument
at
3
spectra recorded in 18.8 T NMR instrument at 3 °C (A and B) or 37 °C (C). Spectrum in panel B is just
window
of window
the correspondent
region in spectrum
panel A.of panel A.
a close-up
of the correspondent
region inof
spectrum
2.6. Fast Chemical Exchange of GlcNHSO3− and Validation of the Low-Temperature-and-Proton-Rich-Solution
Condition for NMR Detection as Seen by 1H Coupling
Note that in panel B of Figure 5 there is a triplet of doublets on the resonance of αH2 of GlcNS
(δH at 3.16–3.22 ppm). This is likely due to the splitting phenomenon caused by 3JH2-H1, 3JH2-H3 and
3JH2-HN at 3 °C temperature. However, when the temperature is raised to the physiological one
Pharmaceuticals 2016, 9, 58
8 of 11
2.6. Fast Chemical Exchange of GlcNHSO3 − and Validation of the Low-Temperature-and-Proton-Rich-Solution
Condition for NMR Detection as Seen by 1 H Coupling
Note that in panel B of Figure 5 there is a triplet of doublets on the resonance of αH2 of GlcNS
(δH at 3.16–3.22 ppm). This is likely due to the splitting phenomenon caused by 3 JH2-H1 , 3 JH2-H3
and 3 JH2-HN at 3 ◦ C temperature. However, when the temperature is raised to the physiological
one (Figure 5C), the multiplet profile of the αH2 resonance moves to a doublet of doublets (3 JH2-H1
and 3 JH2-H3 of same constant values) demonstrating thus that the 3 JH2-HN was lost due to enhanced
dynamics and faster chemical exchange of the sulfamate proton. Although chemical shifts are highly
sensitive to temperature [14], the comparative analyses on the multiplet patterns in the spectra recorded
in this work at different temperatures (3 ◦ C at Figure 5B and 37 ◦ C at Figure 5C) have not compromised
the interpretation and conclusions of the results.
After designing, testing and probing the experimental protocol for assessing exchangeable protons
in GlcNS (Figure 5 and Figure S1), a 15 N-HSQC spectrum of the GlcNS sample was recorded following
the same strategy (Figure S2). The spectrum of Figure S2 is represented in two different thresholds
because of the proximity of the upfield peak of the NH pair of α-GlcNS (δH /δN at 5.36/93.9 ppm) with
the broad residual water noise peak (δH ranging from 5.35 to 4.87 ppm). The 1 H-15 N cross-peak of the
NH pair of β-GlcNS resonates more downfield in the spectrum, with δH /δN exactly at 5.91/93.6 ppm.
Percentage of these amide resonances relative to the α- and β-configurations of GlcNS was measured
based on integral values of the 1 H-15 N cross-peaks. The values obtained are in total accordance
with the expected α/β-anomeric ratio observed for GlcNS in equilibrium as measured previously by
1D 1 H-NMR (Figure 4). Hence, the special protocol for decreasing rapid chemical exchange of the
sulfamate protons in GlcNS and to enable signal detection via both 1D 1 H (Figure 5A) and 15 N-HSQC
NMR spectra (Figure S2), both recorded at 3 ◦ C after dissolving powder GlcNS in a mixture of 10:20:70:
D2 O:acetone:H2 O solution, was probed to be functional.
3. Materials and Methods
3.1. General Materials
The sodium salt of heparin (unfractionated heparin) was gently provided by Eurofarma,
Itapevi, Brasil. The 3 and 8 mm glass NMR tubes were purchased from Tedia, Rio de Janeiro,
Brasil. 15 N-labeled side chain glutamine (15 N-Gln with 15 N at 98%) and deuterium oxide “100%”
(D99.96%) were purchased from Cambridge Isotope Laboratories, In. (Andover, MA, USA).
The GlcNS (D-Glucosamine-2-N-sulfate sodium salt) was purchased from Carbosynth (Berkshire, UK).
The heparan sulfate from CHO K1 cells and from the bivalve Nodipecten nodosus were the same utilized
in the previous publications [5,26]. Glucose (Glc) (D-(+)-Glucose ≥99.5%), GlcN (D-(+)-Glucosamine
hydrochloride ≥99%, crystalline) and GlcNAc (N-Acetyl-D-glucosamine ≥99%) and reagents for buffer
preparation like sodium acetate, sodium azide, dibasic sodium phosphate, citric acid and acetone were
purchased from Sigma Aldrich Co. (St. Louis, MO, USA).
3.2. NMR Experiments, Instrumentation and Related Reagents
For each hyperpolarization experiment, a solution of 23 mM 15 N-Gln or 20 mM GlcNS was
obtained dissolving gently the powder compounds in 25:25:50 (v/v/v) D2 O:H2 O:glycerol containing
15 mM trityl radical (GE-Healthcare, Buckinghamshire, UK) in heating (70 ◦ C). This mixture was
added to a polyether ether ketone plastic cup and lowered into an Oxford Hypersense 3.35 T DNP
polarizer (Oxfordshire, UK). Samples were cooled to 1.4–1.5 ◦ C then irradiated for 1–2 h at 94.007 GHz
and 100 mW. The hyperpolarized samples were then quickly melted and dissolved in a buffer 25 mM
citric acid, 25 mM dibasic sodium phosphate (pH previously adjusted to ~7.0). The dissolved material
was automatically flushed into a waiting 8 mm NMR tube in a Varian 11.7 T Inova spectrometer
(Santa Clara, CA, USA) equipped with an XH probe operating at a 37 ◦ C sample temperature (∼5 s
transfer time). Each sample was analyzed and experimentally performed separately.
Pharmaceuticals 2016, 9, 58
9 of 11
To assess the kinetics of anomeric mutarotation of Glc-based standards, approximately 1.5 mg of
the standard monosaccharides (dried weight) was dissolved in 160 µL 100% D2 O and transferred into
3 mm NMR tubes for 1D 1 H spectral acquisition. To assess exchangeable proton resonances in GlcNS,
approximately 1.5 mg of powder GlcNS was dissolved in 160 µL 10:20:70% D2 O:acetone:H2 O (the pH
after dissolution was measured as ~6.5) and transferred into a 3 mm NMR tube for 1D 1 H spectral
acquisition. For 1 H-1 H Total Correlation SpectroscopY (TOCSY), 1 H-15 N HSQC and 1 H-13 C HSQC
spectral acquisition of samples (GlcNS, heparin or heparan sulfates) around 1.5 mg of dried weight
material was dissolved in either 160 µL 10%:20%:70% D2 O/acetone/H2 O (the pH after dissolution
was measured as ~6.5) or in 160 µL 50 mM sodium acetate buffer 12.5% D2 O (pH 4.5) 0.1% sodium
azide (as indicated in figure captions) and transferred into a 3 mm NMR tube.
All NMR experiments were recorded on Varian Inova spectrometer (Santa Clara, CA, USA),
with a triple resonance cold probe operating at 800 MHz (18.8 T) or 500 MHz (11.7 T) for the 1 H Lamor
frequency. During NMR spectra collection, temperatures of 3 (protonated sulfamate), 25 or 37 ◦ C
(unprotonated sulfamate) were used as indicated in figure captions. The 1D 1 H spectra were recorded
with 128 scans with a spectral width of 7 kHz, carrier position at the HOD peak (4.8 ppm), acquisition
time set to 2 s, and water presaturation pulse (when used) set to the position of the carrier for a period
equal to the recovery delay (1.5 s). The 1D 15 N-direct-observe spectra of 15 N-Gln were recorded using
the 15 N channel with similar parameters used for 1D 1 H except 90◦ pulse width for 15 N and the lack
of presaturation pulse.15k scans were used for 1D 15 N direct-observe in the non-polarized sample.
The 1 H-1 H TOCSY spectra were run with spectral widths of 6 kHz, and acquisition time of 175 ms
using 96 scans per t1 increment (64 points) to achieve a time domain matrix of 2110 × 128 complex
points, using a spin-lock field of 9 kHz, and a mixing time of 60 ms. 1 H-13 C HMQC spectra were run
with acquisition time of 0.128s using 144 scans per t1 increment (128 points) to achieve a time domain
matrix of 1366 × 256 complex points. 1 H-15 N HSQC spectra were recorded with acquisition time of
0.095s and with192 scans per t1 increment (128 points) to achieve a time domain matrix of 1366 × 256
complex points. All processing was done with NMRPipe software [27]. All spectra were apodized
in both dimensions with the automatic function cosine-bells, together with automatic zero-filling to
double the sizes followed by rounding to the nearest power of 2. Reported chemical shifts for 1 H, 13 C
and 15 N are relative to the trimethylsilylpropionic acid, methanol and liquid ammonia, respectively.
4. Conclusions
In this approach, a series of NMR spectra have been collected for heparin, heparan sulfates
and related monosaccharides. The standard GlcNS monosaccharide was the most analyzed sample,
especially via methods involving the less used and less sensitive 15 N isotope. The standard GlcNS
monosaccharide was used here as a model compound to the composing GlcNS unit in heparin and
heparan sulfate. DNP was employed to facilitate 1D signal acquisition of 15 N, and spectra recorded at
low temperature after dissolution of the samples in a proton-rich solution were studied. Data obtained
from this current NMR-based study have strongly supported the conception that the sulfatame proton
in free standard GlcNS monosaccharide (Figures 3C and 5C) as well as composing GlcNS units of the
heparin and heparan sulfate chains, analyzed at either room (Figure 2) or physiological temperatures
(Figure 5C), occurs in fast chemical exchange in solution under the pH achieved here by just dissolving
the sample in the used solvents. The rapid chemical exchange of the sulfamate proton is pH-dependent
and choosing the correct pH range for work, detection of the GlcNS NH is possible [7]. Besides pH,
temperature is another contributing factor to the slow-fast exchange regimes of the sulfamate proton as
well as for the other exchangeable protons. This work has shown a strategy based on low temperature
to reduce the fast chemical exchange property of GlcNHSO3 − to a point where proper signal detection
can also be achieved by NMR. The current investigation on this key structural element of heparin is
of great relevance in light of the ongoing grand celebration of one-hundred-year-anniversary of the
discovery of heparin. As known, GlcNS is a functional unit in chains of heparin and its sister GAG
heparan sulfate, especially inside biding sequences responsible for interactions with antithrombin and
Pharmaceuticals 2016, 9, 58
10 of 11
growth factors during events of anticoagulation and cell division. Heparin is the most therapeutic
carbohydrate, largely employed as anticoagulant in the clinic. The structural studies on the biologically
active structural elements of this GAG type are therefore valuable.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/9/3/58/s1,
Figure S1: 2D NMR 1 H-1 H TOCSY (A) and 1 H-13 C HSQC (B) spectra of N-sulfo-glucosamine (GlcNS) (10 mg/mL)
dissolved in 10%:20%:70% D2 O/acetone/H2 O, recorded at 18.8 T and 3 ◦ C (A); Figure S2: 2D NMR 1 H-15 N
HSQC spectrum of GlcNS (5 mg/mL) dissolved in 10%:20%:70% D2 O/acetone/H2 O recorded at 18.8 T and 3 ◦ C
displayed at higher (A) and lower (B) counter levels; Table S1: Chemical shifts of carbon-attached unexchangeable
1 H from both α and β-anomeric configurations, oxygen-linked exchangeable 1 H from α-anomeric configuration,
nitrogen-linked exchangeable 1 H from α- and β-anomeric configurations, and 13 C of α-anomeric configuration of
GlcNS as assigned in spectra of Figures S1A, S2A and S2B.
Acknowledgments: This study was supported by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro
(FAPERJ). VHP immensely thanks Prof. James Prestegard, Drs. John Glushka and David Live, all from Complex
Carbohydrate Research Center (CCRC), University of Georgia (UGA) for their assistance during his investigations
on GAGs by 15 N-NMR spectroscopy. VHP also acknowledges Dr. Angélica Maciel Gomes (former PhD candidate
at IBqM, UFRJ) and Prof. Lianchun Wang (also from CCRC, UGA) for providing respectively the bivalve and
CHO heparan sulfates.
Conflicts of Interest: The author declares no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Sasisekharan, R.; Venkataraman, G. Heparin and heparan sulfate: Biosynthesis, structure and function.
Curr. Opin. Chem. Biol. 2000, 4, 626–631. [CrossRef]
Rabestein, D.L. Heparin and heparan sulfate: structure and function. Nat. Prod. Rep. 2002, 19, 312–331.
[CrossRef]
Mulloy, B.; Hogwood, J.; Gray, E.; Lever, R.; Page, C.P. Pharmacology of Heparin and Related Drugs.
Pharmacol. Rev. 2016, 68, 76–141. [CrossRef] [PubMed]
Pomin, V.H. Advances in glycosaminoglycanomics by 15 N-NMR spectroscopy. Anal. Bioanal. Chem. 2013,
405, 3035–3048. [CrossRef] [PubMed]
Pomin, V.H.; Sharp, J.S.; Li, X.; Wang, L.; Prestegard, J.H. Characterization of glycosaminoglycans by 15 N
NMR spectroscopy and in vivo isotopic labeling. Anal. Chem. 2010, 82, 4078–4088. [CrossRef] [PubMed]
Pomin, V.H. NMR chemical shifts in structural biology of glycosaminoglycans. Anal. Chem. 2014, 86, 65–94.
[CrossRef] [PubMed]
Langeslay, D.J.; Young, R.P.; Beni, S.; Beecher, C.N.; Mueller, L.J.; Larive, C.K. Sulfamate proton solvent
exchange in heparin oligosaccharides: Evidence for a persistent hydrogen bond in the antithrombin-binding
pentasaccharide Arixtra. Glycobiology 2012, 22, 1173–1182. [CrossRef] [PubMed]
Beecher, C.N.; Young, R.P.; Langeslay, D.J.; Mueller, L.J.; Larive, C.K. Hydroxyl-proton hydrogen bonding
in the heparin oligosaccharide Arixtra in aqueous solution. J. Phys. Chem. B 2014, 118, 482–491. [CrossRef]
[PubMed]
Mobli, M.; Nilsson, M.; Almond, A. The structural plasticity of heparan sulfate NA-domains and hence
their role in mediating multivalent interactions is confirmed by high-accuracy 15 N-NMR relaxation studies.
Glycoconj. J. 2008, 25, 401–414. [CrossRef] [PubMed]
Almond, A.; DeAngelis, P.L.; Blundell, C.D. Dynamics of hyaluronan oligosaccharides revealed by 15 N
relaxation. J. Am. Chem. Soc. 2005, 127, 1086–1087. [CrossRef] [PubMed]
Blundell, C.D.; Deangelis, P.L.; Almond, A. Hyaluronan: the absence of amide-carboxylate hydrogen bonds
and the chain conformation in aqueous solution are incompatible with stable secondary and tertiary structure
models. Biochem. J. 2006, 396, 487–498. [CrossRef] [PubMed]
Almond, A.; Deangelis, P.L.; Blundell, C.D. Hyaluronan: the local solution conformation determined by
NMR and computer modeling is close to a contracted left-handed 4-fold helix. J. Mol. Biol. 2006, 358,
1256–1269. [CrossRef] [PubMed]
Mobli, M.; Almond, A. N-Acetylated amino sugars: the dependence of NMR 3 J(H N H 2 )-couplings on
conformation, dynamics and solvent. Org. Biomol. Chem. 2007, 5, 2243–2251. [CrossRef] [PubMed]
Pharmaceuticals 2016, 9, 58
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
11 of 11
Blundell, C.D.; Almond, A. Temperature dependencies of amide 1 H- and 15 N-chemical shifts in hyaluronan
oligosaccharides. Magn. Reson. Chem. 2007, 45, 430–433. [CrossRef] [PubMed]
Blundell, C.D.; DeAngelis, P.L.; Day, A.J.; Almond, A. Use of 15 N-NMR to resolve molecular details
in isotopically-enriched carbohydrates: sequence-specific observations in hyaluronan oligomers up to
decasaccharides. Glycobiology 2004, 14, 999–1009. [CrossRef] [PubMed]
Zhang, Z.; McCallum, S.A.; Xie, J.; Nieto, L.; Corzana, F.; Jiménez-Barbero, J.; Chen, M.; Liu, J.; Linhardt, R.J.
Solution structures of chemoenzymatically synthesized heparin and its precursors. J. Am. Chem. Soc. 2008,
130, 12998–13007. [CrossRef] [PubMed]
Sattelle, B.M.; Shakeri, J.; Roberts, I.S.; Almond, A. A 3D-structural model of unsulfated chondroitin from
high-field NMR: 4-Sulfation has little effect on backbone conformation. Carbohydr. Res. 2010, 345, 291–302.
[CrossRef] [PubMed]
Limtiaco, J.F.; Langeslay, D.J.; Beni, S.; Larive, C.K. Getting to know the nitrogen next door: HNMBC
measurements of amino sugars. J. Magn. Reson. 2011, 209, 323–331. [CrossRef] [PubMed]
Langeslay, D.J.; Beni, S.; Larive, C.K. Detection of the 1H and 15 N NMR resonances of sulfamate groups
in aqueous solution: A new tool for heparin and heparan sulfate characterization. Anal. Chem. 2011, 83,
8006–8010. [CrossRef] [PubMed]
Langeslay, D.J.; Beni, S.; Larive, C.K. A closer look at the nitrogen next door: 1 H-15 N NMR methods for
glycosaminoglycan structural characterization. J. Magn. Reson. 2012, 216, 169–174. [CrossRef] [PubMed]
Langeslay, D.J.; Beecher, C.N.; Naggi, A.; Guerrini, M.; Torri, G.; Larive, C.K. Characterizing the
microstructure of heparin and heparan sulfate using N-sulfoglucosamine 1 H and 15 N NMR chemical
shift analysis. Anal. Chem. 2013, 85, 1247–1255. [CrossRef] [PubMed]
Beecher, C.N.; Larive, C.K. Methods for measuring exchangeable protons in glycosaminoglycans.
Methods Mol. Biol. 2015, 1229, 173–187. [PubMed]
Beecher, C.N.; Larive, C.K. 1 H and 15 N NMR characterization of the amine groups of heparan sulfate related
glucosamine monosaccharides in aqueous solution. Anal. Chem. 2015, 87, 6842–6848. [CrossRef] [PubMed]
Beecher, C.N.; Manighalam, M.S.; Nwachuku, A.F.; Larive, C.K. Screening enoxaparin tetrasaccharide SEC
fractions for 3-O-sulfo-N-sulfoglucosamine residues using [1 H,15 N] HSQC NMR. Anal. Bioanal. Chem. 2016,
408, 1545–1555. [CrossRef] [PubMed]
Esko, J.D.; Kimata, K.; Lindahl, U. Proteoglycans and sulfated glycosaminoglycans. In Essentials of
Glycobiology, 2nd ed.; Varki, A., Cummings, R.D., Esko, J.D., Freeze, H.H., Stanley, P., Bertozzi, C.R.,
Hart, G.W., Etzler, M.E., Eds.; Cold Spring Harbor Laboratory Press: Woodbury, NY, USA, 2009; Chapter 16;
pp. 243–244.
Gomes, A.M.; Kozlowski, E.O.; Pomin, V.H.; de Barros, C.M.; Zaganeli, J.L.; Pavão, M.S. Unique extracellular
matrix heparan sulfate from the bivalve Nodipecten nodosus (Linnaeus, 1758) safely inhibits arterial thrombosis
after photochemically induced endothelial lesion. J. Biol. Chem. 2010, 285, 7312–7323. [CrossRef] [PubMed]
Delaglio, F.; Grzesiek, S.; Vuister, G.W.; Zhu, G.; Pfeifer, J.; Bax, A. NMRPipe: A multidimensional spectral
processing system based on UNIX pipes. J. Biomol. NMR. 1995, 6, 277–293. [CrossRef] [PubMed]
© 2016 by the author; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).