Synthesis, separation, and characterization of amphiphilic sulfated oligosaccharides enabled by reversed-phase ion pairing LC and LC-MS methods

Article abstract of DOI:10.1016/j.carres.2011.09.020

Synthesis of amphiphilic oligosaccharides is problematic because traditional methods for separating and purifying oligosaccharides, including sulfated oligosaccharides, are generally not applicable to working with amphiphilic sugars. We report here RPIP-L

Full text of DOI:10.1016/j.carres.2011.09.020

Carbohydrate Research 346 (2011) 2792–2800
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis, separation, and characterization of amphiphilic sulfated
oligosaccharides enabled by reversed-phase ion pairing LC and LC–MS methods
⇑
Amanda M. Fenner, Robert J. Kerns
Division of Medicinal and Natural Products Chemistry, University of Iowa, Iowa City, IA 52242, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2011
Received in revised form 9 September 2011
Accepted 19 September 2011
Available online 24 September 2011
Synthesis of amphiphilic oligosaccharides is problematic because traditional methods for separating and
purifying oligosaccharides, including sulfated oligosaccharides, are generally not applicable to working
with amphiphilic sugars. We report here RPIP-LC and LC–MS methods that enable the synthesis, separa-
tion, and characterization of amphiphilic N-arylacyl O-sulfonated aminoglycosides, which are being pur-
sued as small-molecule glycosaminoglycan mimics. The methods described in this work for separating
and characterizing these amphiphilic saccharides are further applied to a number of uses: monitoring
the progression of sulfonation reactions with analytical RP-HPLC, characterizing sulfate content for indi-
vidual molecules with ESI-MS, determining the degree of sulfation for products having mixed degrees of
sulfation with HPLC and LC–MS, and purifying products with benchtop C18 column chromatography. We
believe that the methods described here will be broadly applicable to enabling the synthesis, separation,
and characterization of amphiphilic, sulfated, and phosphorylated oligosaccharides and other types of
molecules substituted to varying degrees with both anionic and hydrophobic groups.
Keywords:
Biomimetic synthesis
Aliphatic oligosaccharide
Mass spectrometry
Reversed-phase ion-pairing
Sulfation
Electrospray ionization
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
anionic structures has been limited by poor pharmacodynamic
and pharmacokinetic properties and by their nonspecific binding
1.1. Therapeutic potential and limitations of GAG mimics
to many proteins.13–15 Working to overcome these limitations we
recently demonstrated that heparin derivatives substituted with
aromatic residues, in the place of anionic groups to reduce anio-
nic content, bind with high affinity and selectivity to select
HS-binding proteins.12,16,17 The next challenge is to exploit this
discovery and synthesize structurally similar but smaller, oligo-
saccharide-size heparin mimics that would be more drug-like
and would be more selective in binding individual proteins.18
Heparan sulfate (HS) and other glycosaminoglycans (GAGs) are
sulfated, polyanionic polysaccharides located in the extracellular
matrix or on the surface of mammalian cells.1–3 As extracellular
carbohydrates, HS, and other GAGs are bound by many endoge-
nous and exogenous proteins, often termed heparin-binding or
HS-binding proteins.4–10 These carbohydrate–protein interactions
can be inhibited by GAG-mimicking bind-and-block antagonists
for potential therapeutic applications such as: slowing angiogene-
sis, preventing metastasis, inhibiting inflammation, and blocking
numerous pathogens from binding to and invading host cells.5,6,11
To date, the development of molecules that bind HS-binding
proteins and block interaction with HS or other GAGs has primar-
ily focused on preparing persulfated oligosaccharides and polyan-
ionic polysaccharides.4,5,12 Therapeutic application of these highly
1.2. Preparing small amphiphilic oligosaccharides as GAG
mimics
Aminoglycoside antibiotics are small, naturally occurring glyco-
conjugates with multiple hydroxyl and amine groups. The orthogo-
nal nature of the amine and hydroxyl groups provide synthetically
useful handles for chemical modification and the introduction of
structural diversity while the different aminoglycoside core struc-
tures orient these substituent groups in different 3-dimensional
space. Thus we envisioned the aminoglycosides to be readily avail-
able scaffolds for making libraries of relatively small oligosaccha-
ride-size heparin mimics, where the amine groups would be
substituted with structurally diverse N-arylacyl moieties and hy-
droxyl groups would be sulfonated.
Abbreviations: ACN, acetonitrile; cbz, benzyloxycarbonyl; CE, capillary electro-
phoresis; ClSO3H, chlorosulfonic acid; DS, degree of sulfation; ESI, electrospray
ionization; GAG, glycosaminoglycan; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HS,
heparan sulfate; LC–MS, liquid chromatography mass spectrometry; MeOH, meth-
anol; NHS, N-hydroxysuccinamide; PyrꢀSO3, pyridine sulfur trioxide complex; RPIP,
reversed-phase ion pairing; TBAI, tetrabutylammonium iodide; TEA, triethylamine;
TEAA, triethylammonium acetate; TIC, total ion chromatogram; TMAI, tetrameth-
ylammonium iodide.
Toward this end, we set out to use aminoglycosides as scaffolds
for preparing structurally defined oligosaccharides substituted with
both aromatic rings and sulfate groups (see Scheme 1). Although
⇑
Corresponding author. Tel.: +1 319 335 8800; fax: +1 319 335 8766.
E-mail address: robert-kerns@uiowa.edu (R.J. Kerns).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.09.020

A. M. Fenner, R. J. Kerns / Carbohydrate Research 346 (2011) 2792–2800
2793
methods were insufficient for monitoring sulfonation reactions to
establish complete, persulfation. Second, approaches previously
used to separate and purify sulfated oligosaccharides from reaction
byproducts after sulfonation failed with these amphiphilic struc-
tures. Third, incomplete sulfonation reactions produce product mix-
tures that vary by the degree and position of sulfate groups; HPLC
and HPLC-MS methods that were previously used to separate sugars
based on the number of sulfate groups and to characterize the
degree of sulfation, or to demonstrate persulfation, did not work
due to the amphiphilic nature of these molecules.
In this report we outline the limitations of current approaches
for reversed phase ion pairing liquid chromatography (RPIP-LC)
and LC–mass spectrometry (MS) of various anionic saccharides.
We then describe the adaptation of these approaches to afford LC
and LC–MS methods that facilitated the synthesis and enabled
the characterization of N-arylacyl O-sulfonated aminoglycoside
derivatives. Utility of these methods are exemplified in this work
using aminoglycosides where the amine groups were functional-
ized to N-benzyloxycarbonyl (N-cbz) groups and the hydroxyl
groups were sulfonated to produce N-cbz O-sulfonated apramycin,
kanamycin, and neomycin (Fig. 1). These methods provide a means
to monitor sulfonation of aryl-substituted carbohydrates, to sepa-
rate complex mixtures of the sugars based on degree of aromatic
and/or sulfate groups, and to characterize the structure of these
novel oligosaccharides using LC–MS. The procedures described in
this work are expected to be useful for separating and characteriz-
ing many additional types of amphiphilic oligosaccharides and
other molecules substituted to varying degrees with anionic and
hydrophobic groups.
O
R
NH
O
NH2
HO
HO
O
HO
HO
O
R
HO
HO
O
HO
HN
O
NH2
NH2
O
H
N
a)
HO
R
O
O
OH
OH
OH
OH
O
O
O
HO
O
C
HO
NH
O
NH2
N
O
R
R
O
b) or c)
O
R
NH
O
X
O
O
O
R =
R
O
X
X O
HN
O
H
O
N
O
R
X
O
O
O
X
O
O
CH3
NH
O
X
X
R
X = SO3 or H
Scheme 1. Representative synthesis showing concept for making N-aryl O-sulfo-
nated aminoglycosides; shown is kanamycin. Reagents and conditions: (a) R-NHS,
NaHCO3, H2O, 23 °C, 12–16 h, 30–85%; (b) (i) PyrꢀSO3, DMF, anhydrous pyr, 66 °C, 5–
7 h; (ii) H2O, 10 mM NaOH, 4 °C, 45–65%; (c) (i) ClSO3H, pyr, 57 °C, 4–6 h; (ii) H2O,
NaHCO3, 35–95%.
1.3. RPIP-LC–MS analysis of sulfated oligosaccharides
modification of the aminoglycosides in this manner was expected to
be straightforward, initial efforts in our laboratory to prepare these
amphiphilic, N-arylacyl O-sulfonated oligosaccharides was fraught
with problems. First, standard thin-layer chromatography and HPLC
The separation of sulfated carbohydrates and nucleic acids has
been described by various methods that employ RPIP-LC.19–21
RPIP-chromatography relies on alkyl ammonium salts to separate
H
N
cbz
HN
X-O
zbc
HN
X-O
X-O
X-O
O
cbz
O-X
O
X-O
X-O
cbz
H
O
N
O
X-O
N
cbz
O
O
O
cbz
HN
O-X
O-X
NH
H
O
cbz
O
X-O
X-O
N
cbz
O-X
NH
cbz
N-cbz O-sulfonated Kanamycin
N-cbz O-sulfonated Apramycin
X = SO3 or H
H
N
cbz
HN
X-O
X-O
O
cbz
H
N
cbz HN
cbz
O
N-cbz O-SO3
Core
DS
(X = SO3)
O
O
O-X
aminoglycoside
aminoglycoside
O-X
X-O
H
N
cbz
kz7
kz6
kz5
nz7
az6
az5
az4
kanamycin
kanamycin
kanamycin
neomycin
apramycin
apramycin
apramycin
7
6
5
7
6
5
4
O
O-X
O-X
O
HN
cbz
N-cbz O-sulfonated Neomycin
Figure 1. Sulfonation of N-cbz aminoglycosides (kanamycin, apramycin, and neomycin) with PyrꢀSO3 or ClSO3H resulted in products with varied degrees of sulfation (DS).
RPIP-HPLC methods developed here were used to separate reaction products based on degree and position of sulfate groups. Sulfonation of N-cbz neomycin was complete,
giving persulfated product, nz7. Sulfonation of N-cbz kanamycin and N-cbz apramycin produced per sulfated derivatives kz7 and az6, respectively, as well as under-sulfated
kanamycin derivatives (kz5, kz6) and under-sulfated apramycin derivatives (az4, az5), where the number indicates the number of sulfate groups attached.

2794
A. M. Fenner, R. J. Kerns / Carbohydrate Research 346 (2011) 2792–2800
charged species on reversed phase resin and is particularly useful
for the separation and analysis of sulfated oligosaccharides
because this method is readily compatible with LC–MS.22–25 For
sulfated oligosaccharides, a common problem in MS ionization is
sulfate fragmentation; this complicates structure identification
and renders it difficult to determine the number of sulfate groups
on a saccharide.23,26,27 It has been shown, however, that RPIP with
quaternary ammonium salts decreases sulfate fragmentation dur-
ing electrospray ionization.27 For example, tetraethyl and tetrabu-
tylammonium salts have been used to separate a wide range
of sulfated saccharides and phosphorylated nucleotide sug-
ars.23,27–33 Nevertheless, a limiting factor with these methods is
that the quaternary ammonium ions are non-volatile salts and
are thus difficult to rapidly clear from the mass spectrometer.22
Sulfated oligosaccharides have also been separated using RPIP
methods with volatile salts, including mono-, di-, and trialkylam-
monium salts. The tertiary amines N-hexylaminedibutylamine,
tributylamine, and tripropylamine were shown to be useful for
separating sulfated oligosaccharides based on degree of sulfation,
stereoisomers of sulfate groups, or both.22–24,29,34,35 Unfortu-
nately, application of these methods using tributylammonium
salts did not provide chromatographic resolution of our N-aryl-
acyl O-sulfonated oligosaccharides, which have a considerable
hydrophobic component and thus likely prone to aggregation.
To accommodate the unique differences in polarity between other
sulfated oligosaccharides and our amphiphilic N-arylacyl modified
sulfated oligosaccharides, we looked to methods designed for sep-
arating oligonucleotides. Oligonucleotides have phosphate groups
and hydrophobic bases, and were thus anticipated to have phys-
ical and chromatographic properties more similar to our novel
O-sulfonated N-aryl aminoglycosides.
2. Results and discussion
2.1. Preparing N-cbz O-sulfonated aminoglycosides having
varied degrees of sulfate
The synthesis of O-sulfonated N-arylacyl aminoglycoside deriv-
atives, as exemplified with kanamycin in Scheme 1, is accom-
plished in two steps: (1) selective acylation of the amine group
on the aminoglycoside and (2) the N-arylacyl aminoglycoside is
sulfonated to convert hydroxyl groups into sulfate groups. Using
this methodology, the aminoglycosides apramycin, kanamycin,
and neomycin were converted into their N-benzyloxycarbonyl
(N-cbz) derivatives by coupling with the N-(benzyloxycarbonyl-
oxy)succinimide under alkaline conditions. Subsequently, the N-
cbz aminoglycosides were sulfated with either pyridine sulfur
trioxide complex (PyrꢀSO3) or chlorosulfonic acid (ClSO3H) (Fig. 1).
By using the two different sulfonation reagents under different
reaction conditions we obtained varied results in attempts to gen-
erate per-sulfated products. For example, when PyrꢀSO3 was used
to O-sulfonate N-cbz kanamycin, the resulting product was a mix-
ture of the per-sulfated derivative (with seven hydroxyls sulfo-
nated, kz7) and under-sulfated derivatives: six hydroxyls
sulfonated (kz6) and five hydroxyls sulfonated (kz5). When ClSO3H
was used to O-sulfonate N-cbz neomycin, the reaction produced
N-cbz per-O-sulfonated neomycin (nz7, Fig. 1). In two separate
reactions, both PyrꢀSO3 and ClSO3H were used to O-sulfonate
N-cbz apramycin. Sulfonation with PyrꢀSO3 produced a mixture of
products containing N-cbz per-O-sulfonated apramycin (az6) as
well as under-sulfated products with either five sulfates (az5) or
four sulfates (az4) per molecule. Sulfonation of N-cbz apramycin
with ClSO3H produced per N-cbz O-sulfonated apramycin (az6,
Fig. 1). Thus by using the LC and LC–MS methods developed in this
work to rigorously follow these sulfonation reactions over time and
to characterize product mixtures (methods discussed in Section
2.4), it is shown here that ClSO3H is the superior reagent to achieve
complete, per-sulfation of these glycoconjugates.
1.4. RPIP-LC separation of oligonucleotides
Like oligosaccharides, oligonucleotides have been separated and
characterized with RPIP-HPLC and LC–MS methods. Separation of
oligonucleotides with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) is
very successful when used on conjunction with triethylamine
(TEA).36–39 The HFIP method for separating oligonucleotides was
recently adapted to the separation of sulfated oligosaccharides.25,34
Regardless, HFIP-based methods require column heating, an ultra-
performance LC system, and in some instances high concentrations
of ion pairing reagents. For our analytical and preparative needs,
we desired a single RPIP-separation method that could be used
with HPLC, benchtop C18 chromatography and LC–MS. Single-use
application hampers many methods for RPIP-HPLC-MS of oligonu-
cleotides and sulfated oligosaccharides, such as those that require
a second eluent spray or pre or post-column derivitization prior to
LC-ESI-MS.23,24,27,29,30,40–42 Moreover, other methods require spe-
cial columns such as amino columns, columns designed for hydro-
philic compounds, pre-loaded microcolumns, or capillary
HPLC.22,43–46 These multiple derivitization steps or specialty col-
umns are cost, scale, and time-prohibitive.
In this report, we adapt an oligodeoxyribonucleotide RPIP
method to the HPLC and benchtop chromatographic separation,
and the LCMS characterization, of amphiphilic N-arylacyl O-sulfo-
nated aminoglycosides.47 Specifically reported here are data and
results obtained when developing these methods with N-cbz mod-
ified aminoglycosides. This method is well suited for LC–MS, be-
cause it relies on low salt concentration with common reagents
(10 mM ammonium acetate and <0.1 mM TEA).47 Furthermore,
since separation is achieved with C18 resin and without a column
oven or an ultraperformance LC system, the analytical methods are
shown to be scalable for preparative-scale desalting and benchtop
product purification.
2.2. Optimizing separation of N-cbz O-sulfonated
aminoglycosides with ion pair-RP-HPLC
Since the O-sulfonation of N-cbz aminoglycosides often resulted
in products with varied degrees of sulfation, product characteriza-
tion required a method that would: (1) separate components in
the product mixture based on the degree of sulfation, (2) show
robust ability for separating and characterizing different N-cbz
O-sulfonated aminoglycosides, and (3) minimize sulfate fragmenta-
tion on LC–MS without losing signal intensity so that LC-based sep-
aration could be coupled with MS to characterize the sulfated
products.
Initially, different mobile phase compositions that varied in al-
kyl ammonium salts, pH, and acetonitrile gradient were evaluated
to identify a method that would elute N-cbz per-O-sulfonated neo-
mycin as a single, separable peak. Representative results for a
number of different methods tested are shown in (Fig. 2). In each
chromatogram shown, the mobile phase contained 10 mM ammo-
nium acetate. In chromatograms A–D, the counter ion was a qua-
ternary ammonium salt, tetramethyl ammonium iodide (TMAI):
10 mM with pH 7.3 (Fig. 2A), 0.8 mM with pH adjusted to 5.0
(Fig. 2B), 0.8 mM with pH 7.4 (Fig. 2C) and 0.8 mM with pH ad-
justed to 8.0 (Fig. 2D). For each of these analyses, A–D, the run gra-
dient was 10–80% ACN in water over 20 min then isocratic elution
at 80% ACN until the end of run time. Another quaternary ammo-
nium salt, tetrabutylammonium iodide (TBAI), was evaluated at
10 mM, pH 7.3, with gradient elution of 10–70% ACN for 20 min
followed by isocratic elution at 70% ACN for the remaining run

A. M. Fenner, R. J. Kerns / Carbohydrate Research 346 (2011) 2792–2800
2795
100
(A)
788.51
400
300
200
100
0
A
75
50
25
0
B
C
D
E
828.12
800
F
-100
700
900
1000
1100
1200
10
15
20
25
30
35
40
Time (min)
100
1047.20
(B)
1030.60
Figure 2. Representative comparison of analytical HPLC chromatograms from
various RPIP conditions eluting persulfated N-cbz neomycin. Each chromatogram
was generated from a 20 min gradient increase in percent ACN, and the ending
percent ACN was then maintained isocratic for an additional 22 min. (A) 10–80%
ACN; 10 mM ammonium acetate, 10 mM TMAI, pH 7.3. (B) 10–80% ACN; 10 mM
ammonium acetate, 0.8 mM TMAI, pH 5.0. (C) 10–80% ACN; 10 mM ammonium
acetate, 0.8 mM TMAI, pH 7.4. (D) 10–80% ACN; 10 mM ammonium acetate, 0.8 mM
TMAI, pH 8.0. (E) 10–70% ACN; 10 mM ammonium acetate, 10 mM TBAI, pH 7.3. (F)
10–30% ACN; 10 mM ammonium acetate, 0.8 mM TEA, pH 8.3.
1063.67
1072.20
1021.80
75
50
25
0
868.00
788.33
907.93
947.93
828.00
1097.13
1106.07
1114.33
time (Fig. 2E). In each of these chromatograms (Fig. 2A–E), N-cbz
O-sulfonated neomycin eluted as a broad or undefined peak. How-
ever, using a trialkyl ammonium salt, triethylamine, as the alkyl
ammonium ion pairing agent afforded elution of N-cbz O-sulfo-
nated neomycin as a single sharp peak (Fig. 2F).
700
800
900
1000
1100
1200
m/z
As shown, optimal separation on HPLC was achieved with
adjusting the mobile phase to pH 8.3 with triethylamine (TEA,
ꢁ0.8 mM) and using a gradient elution of 10–30% ACN over
20 min, followed by isocratic elution at 30% ACN until the end of
the run. Moreover, the triethylammonium salt is better suited for
LC–MS analyses than the quaternary ammonium salts, because ter-
nary salts are more quickly cleared from the instrument thus
allowing for more frequent injections. Other studies have de-
scribed the use of triethylammonium acetate (TEAA) ion pairing
for the separation of oligonucleotides.37,48 In these reported meth-
ods, TEA is used at concentrations two orders of magnitude greater
than the concentration of TEA in the optimized method for amphi-
philic saccharides described here. When ion pairing with TEAA was
used to separate and characterize N-cbz O-sulfonated aminoglyco-
sides, it produced sharp peaks and similar elution profiles on HPLC,
but often resulted in decreased ionization on LC–MS. This finding
agrees with the previous observation that increasing TEA in the
elution buffer decreases signal intensity in MS detection.47
Figure 3. Tuning of the ESI-ion trap mass spectrometer is critical for decreasing
sulfate fragmentation and detecting the many ionic states of highly sulfated
products. Panel A: Before tuning, direct inject ESI of persulfated N-cbz neomycin,
nz7, detects only desulfated ions m/z = 788.51 [Mꢂ5SO3+5H]ꢂ2 and m/z = 828.12
[Mꢂ4SO3+4H]2. Panel B: After tuning, higher molecular weight ions are more
abundant and persulfated nz7 is observed in various adducts with ion pairing
reagents (denoted in Table 1).
the low abundance initially observed for persulfated material. Fur-
thermore, even after tuning the instrument to decrease fragmenta-
tion, loss of sulfate during ionization still occurred, and ions were
observed for the various fragmented states (Table 1 and Fig. 3).
Table 1
N-cbz per O-sulfonated neomycin (nz7) adducts and in source sulfate fragmentation
observed in ESI direct inject spectra (Fig. 3B)
m/z Theoretical
m/z Observed
Interpretation
D
Mr
788.22
828.19
868.68
908.15
948.13
788.33
828.00
868.00
907.93
947.93
[Mꢂ5SO3+5H]ꢂ2
0.11
[Mꢂ4SO3+4H]2
ꢂ0.19
ꢂ0.68
ꢂ0.22
ꢂ0.20
0.42
[Mꢂ3SO3+3H]ꢂ2
[Mꢂ2SO3+2H]ꢂ2
[MꢂSO3+H]ꢂ2
2.3. LC–MS detection of N-cbz O-sulfonated aminoglycosides
988.11
996.62
988.53
996.20
[M+5H]ꢂ2
[M+4H+NH4]ꢂ2
ꢂ0.42
Initial analysis of N-cbz O-sulfonated aminoglycosides on LC–MS
with the optimized TEA-containing ammonium acetate buffer
(Section 2.2) detected primarily di-sulfate, doubly-charged prod-
ucts for all peaks observed in the total ion chromatogram (TIC). As
1005.13
1013.64
1022.15
1030.67
1038.67
1047.18
1055.69
1064.20
1072.71
1089.23
1097.74
1106.25
1114.76
1005.13
1014.13
1021.80
1030.60
1038.27
1047.20
1055.13
1063.67
1072.20
1089.87
1097.13
1106.07
1114.33
[M+3H+2NH4]ꢂ2
[M+2H+3NH4]ꢂ2
[M+H+4NH4]ꢂ2
0.00
0.49
ꢂ0.35
ꢂ0.07
ꢂ0.40
0.02
ꢂ0.56
ꢂ0.53
ꢂ0.51
0.64
ꢂ0.61
ꢂ0.18
ꢂ0.43
[M+5NH4]ꢂ2
shown for nz7 in
Figure 3A, initially, only m/z = 788.51
[M+4H+TEA]ꢂ2
[Mꢂ5SO3+5H]ꢂ2 and m/z = 828.12 [Mꢂ4SO3+4H]2 were observed.
However, decreasing both capillary temperature and auxiliary gas
flow and increasing the tube lens offset captured persulfated species
(M = nz7) in high abundance (Fig. 3B). As shown, the persulfated
material is not concentrated in a single ionic species but rather dis-
[M+3H+TEA+NH4]ꢂ2
[M+2H+TEA+2NH4]ꢂ2
[M+H+TEA+3NH4]ꢂ2
[M+TEA+4NH4]ꢂ2
[M+3H+2TEA]ꢂ2
[M+2H+2TEA+NH4]ꢂ2
[M+H+2TEA+2NH4]ꢂ2
[M+2TEA+3NH4]ꢂ2
tributed across
a
wide range of distinct ions. These
per-sulfated ions of nz7 were detected in association with hydro-
gen, triethylammonium, ammonium, or combinations thereof
(Table 1). The various ion states of the persulfated product and the
high propensity for sulfate fragmentation in ESI likely account for
nz7 forms adducts with cations: H+, TEA = (CH3CH2)3NH+, NHþ4
observed in negative mode and are doubly-charged.
M = C71H75N6O46S77ꢂ, Mr = 1972.83.
.
All ions are

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A. M. Fenner, R. J. Kerns / Carbohydrate Research 346 (2011) 2792–2800
2.4. Characterizing N-cbz O-sulfonated aminoglycoside
mixtures using LC–MS
A
B
Tuning the MS to nz7 as outlined in Section 2.3 allowed for the
first time characterization of these sulfonation reaction products.
The O-sulfonation of N-cbz kanamycin with PyrꢀSO3 resulted in a
variety of under-sulfated products that were only now possible
to separate based on both degree of sulfation and position of sul-
fate groups using the RPIP LC–MS (Fig. 4). All peaks detected by
the ion trap mass analyzer are shown in the total ion chromato-
gram (TIC, Fig. 4A). From the TIC, ion chromatograms were ex-
tracted by selecting m/z ratios calculated for various ions
corresponding to specific degrees of sulfation (Fig. 4B–D); an
example of how m/z ratios were calculated for the various ions is
shown in Table 1. Ions corresponding to persulfated compound,
kz7, (m/z = 839.6 0.5) correlate to the peak eluting at 24.10 min
in the TIC (Fig. 4B). Likewise, ions that have a mass to charge ratio
of kz6 (m/z = 799.6 0.5) are found within peaks eluting at 24.58,
25.34, 26.22, and 27.11 min (Fig. 5C). Ions with a mass to charge
ratio of kz5 (m/z = 759.6 0.5) are found within the peaks eluting
at 27.58, 28.03, 28.72, and 31.18 min (Fig. 4D). Additionally, there
is evidence of in-source sulfate fragmentation: the persulfated
product peak at 24.10 min also appears with the loss of one or
two sulfates in chromatograms C and D, corresponding to kz6
and kz5, respectively. Peaks within each TIC were assigned a de-
gree of sulfation based on extracted ion chromatograms; the ion
representing the maximum degree of sulfation detected within a
particular peak was assigned as the degree of sulfation.
C
D
Figure 5. RPIP-HPLC-MS total ion chromatograms (TICs) of reaction products from
the sulfonation of N-cbz aminoglycosides. Shown are product mixtures from the
sulfonation of kanamycin and apramycin with PyrꢀSO3 (A and B, respectively) and
the sulfonation of neomycin and apramycin with ClSO3H (C and D, respectively). A
and B demonstrate separation of different sulfated states of N-cbz kanamycin
(DS = 5–7) and N-cbz O-sulfonated apramycin (DS = 4–6), respectively. O-
Sulfonation with ClSO3H afforded persulfated products: (C) nz7 and (D) az6.
degree of sulfation for each peak within the product mixtures
(Figs. 4 and 5). Next, the relative concentration of each sulfated
compound was calculated by integrating peak area with UV detec-
tion at each compound’s k max between 254 and 258 nm. Peak areas
for compounds having the same number of sulfate groups were
added together to then calculate the average degree of sulfation
for the entire product mixture (Table 2). The average degree of sul-
fation was notably lower in sulfonation reactions with PyrꢀSO3 than
with ClSO3H (Table 2). This is most evident in the O-sulfonation of N-
cbz apramycin: O-sulfonation with ClSO3H produced persulfated
product (az6) that eluted as a single peak on LC–MS (Fig. 5D) while
O-sulfonation with PyrꢀSO3 provided a mixture of sulfated com-
pounds that varied in both positioning and degree of sulfation, as
ascertained by LC–MS (Fig. 5B).
2.5. Determining degree of sulfation
The methods employed for O-sulfonation with PyrꢀSO3 resulted
in mixtures of products having varied degrees of sulfation for both
N-cbz kanamycin (Fig. 5A) and N-cbz apramycin (Fig. 5B). For each
of these sulfated compounds, MS analysis was used to assign a
kz6
100
TIC
kz5
A
kz7
kz5
kz6
kz6
50
kz6
0
2.6. Monitoring O-sulfonation reactions and product
purification using RP-IP-HPLC
100
-2
kz7
B
C
[
+ TEA]
m/z 839.6
50
Once methods had been established to characterize the reaction
products using LC–MS, subsequent O-sulfonation reactions could
be monitored over time to drive synthesis to the persulfated prod-
uct. The utility of these methods for monitoring sulfonation reac-
tions is exemplified in Figure 6, which shows the RPIP HPLC
analysis of reaction aliquots at various times during the sulfonation
of N-cbz neomycin with ClSO3H. In these chromatograms, N-cbz
per-O-sulfonated neomycin correlates to the peak at 28 min. With-
in 5 min of adding ClSO3H to N-cbz neomycin, a number of
partially sulfated products appear, including a small amount of
hexasulfated product at 29 min (Fig. 6A). Fifteen minutes after
adding ClSO3H, the reaction shows nearly 70% of the product to
be persulfated (Fig. 6B). After 20 min, 77% of the N-cbz neomycin
is persulfated (Fig. 6C) and by 275 min about 90% of the saccharide
is persulfated (Fig. 6E).
0
[kz6 + TEA]-2
m/z 799.6
100
50
0
100
-2
kz5
[
+ TEA]
D
m/z 759.6
50
0
20
22
24
26
28
30
32
34
Time (min)
Figure 4. Separation of N-cbz O-sulfonated kanamycin derivatives that differ by
degree of sulfation and the position of sulfate groups using RPIP-HPLC interfaced
with an ESI-ion trap mass analyzer. Peaks for all sulfonation products are shown in
the total ion chromatogram (TIC, A). The extracted ion chromatograms (B–D) were
extracted based on m/z ratios that fall within the range predicted for a degree of
sulfation (5, 6, or 7 sulfate groups); see Table 1 for an example of predicted m/z
ratios. Ion chromatograms were extracted from the TIC for the indicated degrees of
sulfation (DS): (B) persulfated, DS = 7, kz7; (C) DS = 6, kz6; (D) DS = 5, kz5.
2.7. Purifying and desalting of N-cbz O-sulfonated
aminoglycosides with RPIP benchtop chromatography
After aqueous quenching of the sulfonation reactions, the desired
N-cbz O-sulfonated aminoglycoside products are in an aqueous-

A. M. Fenner, R. J. Kerns / Carbohydrate Research 346 (2011) 2792–2800
2797
Table 2
Calculated degree of sulfation (DS) for reaction product mixtures afforded by O-sulfonation of N-cbz kanamycin (A), N-cbz apramycin (B and D), and N-cbz neomycin (C)
Rxn
Starting material
Sulf method
Avg DS
% DS 7SO4
% DS 6SO4
% DS 5SO4
% DS 4SO4
A
B
C
D
N-cbz kanamycin
N-cbz apramycin
N-cbz neomycin
N-cbz apramycin
PySO3
PySO3
ClSO3H
ClSO3H
6.0
4.9
7
17.8
NA
100
NA
66.0
18.4
—
16.2
55.5
—
—
26.1
—
6
100
—
—
Two different sulfating reagents were employed, PyꢀSO3 and ClSO3H. The percent at each sulfated state is expressed as %DS and the number of sulfates per molecule.
NA, not applicable; —, below limit of detection.
nz7
(E)
nz6
89%
700
(D)
80%
500
(C)
77%
300
(B)
69%
(A)
0%
100
Figure 7. Adaptation of RPIP-HPLC method developed here to separate sulfonated
products from reaction mixtures by gravity flow benchtop C18 resin. N-cbz O-
sulfonated neomycin was eluted in 10 mM ammonium acetate, initially in 100%
22
24
26
28
30
32
34
water, and then with addition of 30% ACN as indicated by the dashed arrow. 1 mL
fractions were collected and analyzed for UV absorbance at 258 nm. UV-absorbing
fractions were pooled, freeze-dried and injected on analytical HPLC, detected at
258 nm.
Time (min)
Figure 6. RPIP-HPLC to follow reaction progression over time for O-sulfonation of
N-cbz neomycin. Following addition of ClSO3H, reaction aliquots were analyzed at:
(A) 5 min, (B) 15 min, (C) 20 min, (D) 50 min, (E) 275 min. Percent of reaction
product that is persulfated product (nz7) is indicated next to the nz7 peak at
28 min.
with RPIP conditions used for sulfated oligosaccharides having no
aryl substituents.
organic mixture with reaction by-products including sodium
sulfate, trace amounts of pyridine, and at times under-sulfated
N-cbz aminoglycosides. To remove pyridine and sodium sulfate, sul-
fated N-aryl aminoglycosides were separated using a benchtop C18
employing the RPIP buffer conditions developed here for LC–MS
separation (Fig. 7). In this example, the column was loaded with
the aqueous reaction mixture from the O-sulfonation of N-cbz
neomycin with ClSO3H. After elution with ꢁ20 column volumes of
the LC buffer (sans organic solvent), 30% of ACN was added to the
mobile phase and elution continued (Fig. 7, dashed arrow indicates
ACN in elution buffer). UV-absorbing product was eluted off the
column in one peak; absorbing fractions were pooled, dried, and
confirmed on LC–MS to be persulfated N-cbz neomycin. The product
was analyzed for the presence of sodium sulfate by CE using indirect
detection; no sodium sulfate was detected in product purified with
the optimized RPIP separation (data not shown).
This separation method is shown to be applicable to LC–MS
characterization of single compounds in complex product mix-
tures, useful in monitoring sulfation reaction progression, adapt-
able to using UV detection combined with LC–MS for calculating
the degree of sulfation, and useful for purifying amphiphilic sac-
charides by benchtop chromatography. We believe the methods
described here will be useful for the separation of other sulfated
or phosphorylated amphiphilic small molecules. We are currently
employing this methodology in the preparation, purification, and
characterization of structurally diverse O-sulfonated N-aryl amino-
glycosides for further evaluation as bind-and-block antagonists of
heparin-binding proteins.
4. Experimental section
4.1. General
3. Conclusions
Neomycin sulfate, apramycin sulfate, and pyridine sulfur triox-
ide complex were from Sigma Aldrich (St. Louis, MO, USA); kana-
mycin sulfate was from Bristol Laboratories Inc. (Syracuse, NY,
USA). N-(Benzyloxycarbonyloxy)succinimide and chlorosulfonic
acid were purchased from Acros Organics (Morris Plains, NJ,
USA). HPLC-grade acetonitrile (ACN) and triethylamine (TEA) were
from Fisher Scientific (Hampton, NH, USA). Filtered, deionized
water used with all buffers was from a Barnstead Nanopure Dia-
mond system, Thermo Fisher Scientific (Hampton, NH, USA).
Ammonium acetate (99.999% metal basis) was obtained from Sig-
ma Aldrich. All buffers were filtered and degassed prior to use. A
Fisher Accumet AB15 pH meter was used for all pH determinations.
Described here is the optimization and exploitation of a highly
useful methodology for separating and characterizing amphiphilic,
sulfated N-aryl substituted oligosaccharides. Compared with other
RPIP-HPLC methods for the separation of sulfated carbohydrates,
the sulfated N-cbz aminoglycosides presented here were best re-
solved by adaptation of a method originally developed for oligonu-
cleotide separation. The aryl substituents on the amphiphilic
sugars likely enhance interaction with the C18 resin; thus lower
concentrations of ion pairing reagents were sufficient, even re-
quired, to achieve separation of various sulfated states compared

2798
A. M. Fenner, R. J. Kerns / Carbohydrate Research 346 (2011) 2792–2800
Analytical HPLC used a Phenomenex Luna C18 100 Å LC col-
to find a way to efficiently follow the sulfonation reactions and to
separate final products. The method to monitor the sulfonation
reactions described below is the method used once we developed
the RPIP-LC–MS system described in this report. Thus analytical
HPLC was used to monitor O-sulfonation of the N-cbz aminoglyco-
umn (4.6 mm ꢃ 250 mm) (Torrance, CA, USA) with a Shimadzu
chromatography system consisting of an LC-10AT VP pump and
a SPD-M10A VP photodiode array detector (Kyoto, Japan). Semi-
preparative HPLC used a Phenomenex Luna 10 lm C18(2) 100 Å
LC Column (250 ꢃ 21.2 mm) with a Shimadzu HPLC system: dual
LC-10AT VP pumps and SPD-M10A VP diode array detector. Cen-
trifugation was done in a Fisher Marathon 21000R, Fisher Scien-
tific. Cation exchange chromatography used Amberlite IR 120
resin, Sigma Aldrich. Benchtop column C18 chromatography used
Silica Gel 60 RP-18 resin, EMD Chemicals (Gibbstown, NJ, USA).
Product collected from benchtop chromatography was identified
by UV absorbance in 96-well plates using a BioTek Synergy 2
Multimode plate reader (Winooski, VT, USA). Capillary electro-
phoresis (CE) was employed to determine presence/absence of
inorganic sulfate using inverse detection with a UV detector at
214 nm on a Beckman Coulter P/ACE MDQ CE system (Brea, CA,
USA). For LC–MS characterization of compounds, LC separation
was performed with either a Thermo Fisher Scientific Surveyor
LC system or a Dionex UltiMate 3000 (Sunnyvale, CA, USA) cou-
pled to a Thermo Fisher Scientific LCQ Deca ESI-quadrupole- ion
trap mass spectrometer.
sides as follows: as reactions progressed, 10
tion mixture were diluted with 20 L aq NaHCO3, 20
60 L H2O and mixed, followed by loading 100 L of this solution
l
L aliquots of a reac-
l
lL ACN and
l
l
onto the analytical column. The mobile phase for elution consisted
of two buffers: buffer A: 10 mM ammonium acetate, 10% ACN, pH
adjusted to 8.3 with addition of TEA; buffer B: 10 mM ammonium
acetate, 30% ACN, pH adjusted to 8.3 with addition of TEA. The mo-
bile phase gradient was: 0–20 min, 0–100% B; 20–38 min, 100% B;
38–40 min, 100–0% B; 40–45 min, 100% A. The flow rate was held
at 0.5 mL/min. Each N-cbz O-sulfated aminoglycoside was detected
at 210 and 254 nm.
4.4. Sulfonation of N-cbz aminoglycosides
All glassware was oven-dried; all reactions were performed un-
der argon gas. Two of the sulfonation methods were compared in
the results here. In one method, aminoglycoside (100 mg) was dis-
solved in anhydrous DMF (1 mL) and stirred at 66 °C. To this solu-
tion was added PyrꢀSO3 (3 equiv per aminoglycoside hydroxyl)
dissolved in DMF (1 mL) with anhydrous pyridine (1 mol equiv).51
The reaction was stirred at 66 °C for 12–16 h and monitored by
analytical HPLC as outlined in Section 4.3. The completed reaction
was cooled to 4 °C with an ice bath. Residual PyrꢀSO3 was quenched
by the addition of water (2 mL), and the resulting aqueous solution
4.2. Synthesis of N-cbz aminoglycosides
Synthesis and characterization of per N-cbz derivatives of neo-
mycin, kanamycin, and apramycin have been reported.49,50 Synthe-
sis of the per-N-cbz derivatives of these aminoglycosides used
N-(benzyloxycarbonyloxy)succinimide (NHS-cbz) as previously
reported for introducing cbz groups onto free amine groups of N-
desulfonated heparin.17 Briefly, aminoglycoside (0.2–0.5 mmol)
was dissolved in aqueous NaHCO3 and stirred at room tempera-
ture. Aliquots of NHS-cbz (1.2 equiv per amine moiety) dissolved
in 4.5 mL DMF were then added to the above solution fraction-wise
(0.9 mL) at 60–90 min intervals. During the reaction, a white solid
precipitate was formed. The reaction was monitored using analyt-
made alkaline with cold 10 M NaOH (added in 10 lL increments).
Alternatively, ClSO3H was used to sulfonate the N-cbz aminoglyco-
sides.52,53 With this method, ClSO3H (8 equiv per hydroxyl) was
added drop-wise to anhydrous pyridine (3 mL) stirring at room
temperature in a round bottom flask and the mixture then heated
to 57 °C. Separately, the N-cbz aminoglycoside (50 mg) was dis-
solved in anhydrous pyridine and evaporated for azeotropic re-
moval of residual water; this procedure was repeated 2–3 times
before dissolving the N-cbz aminoglycoside in anhydrous pyridine
(2 mL) and cannulating the resulting solution into the stirring
ClSO3H/pyridine solution. Heating was maintained at 57 °C and
reaction progress monitored by analytical HPLC as described in
Section 4.3. Upon reaction completion, about 4–6 h, the mixture
was cooled in an ice bath and adjusted to pH 8 with addition of
aqueous NaHCO3 (7.5 mL). The resulting aqueous solution was
transferred to a separatory funnel and extracted with CH2Cl2
(7–10 ꢃ 15 mL) to remove pyridine. The water layer was then con-
densed on a rotary evaporator to remove residual organic solvent.
Desalting and product separation were accomplished by loading
the aqueous product on to a benchtop C18 column as described
in Section 4.5.
ical HPLC: 20
ACN and 40
l
L of the reaction mixture and was diluted in 80
lL
lL water; 100 L of this solution was injected and
l
eluted with a gradient of 10–95% ACN in water (0.1% TFA) over
40 min at 1 mL/min. After complete acylation of amine groups,
ꢁ5–7 h, the reaction was diluted with water (4 mL) to give addi-
tional white precipitate. The precipitate was collected by centrifu-
gation (20 min, 4 °C, 3500 rpm) and decanting followed by
washing the solid with cold water (10 mL), centrifugation and
decanting. This wash procedure was repeated seven times fol-
lowed by lyophilization of a final suspension to give dry white so-
lid. In reactions where the N-acylation was incomplete, as detected
by analytical HPLC, under-reacted product was removed by dis-
solving the product in ACN/water (3:1) and passing the solution
through a column of amberlite cation (H+) exchange resin (4 mL
bed volume).
In some reactions, over-acylated product (having at least one O-
cbz) was detected by ESI-MS; the desired product was then puri-
fied using semi-preparative HPLC. To this end, the product mixture
was dissolved in 3:1 ACN/water and replicate aliquots were in-
jected and separated. The per-N-cbz product was collected after
about 20 min, depending on the parent aminoglycoside, when
eluted with 67% ACN in water with 0.1% TFA, 7 mL/min. Product
purified with cation exchange or semi-prep chromatography was
rotary evaporated to remove organic solvent and the resulting
aqueous suspension lyophilized to give a white solid.
4.5. RPIP-benchtop C18 chromatography
The aqueous mixture of sulfonation reaction product(s) was
loaded onto a benchtop column packed with C18 silica resin
(1 ꢃ 5.8 cm, 6.5 mL bed volume). Non-volatile salts were first eluted
with the ammonium acetate buffer (10 mM ammonium acetate ad-
justed to pH 8.3 with TEA) under gravity flow. Eluent was collected
in fractions (1 mL) and analyzed at 210 and 258 nm to detect elution
of sodium sulfate and sugar, respectively. As determined with
210 nm detection, the sodium sulfate eluted within the first
100 mL; this was verified with CE (see Section 4.6). Then, sulfated
product was eluted by the addition of 10% ACN. As determined with
258 nm detection, fractions containing sulfated N-Cbz aminoglyco-
side were pooled, and ACN was removed en vacuo. The remaining
water and volatile salts were removed by lyophilization to give
4.3. RPIP-HPLC for monitoring sulfonation reaction progression
Many sulfonation procedures as well as TLC and HPLC methods
were employed throughout the early stages of this work in efforts

A. M. Fenner, R. J. Kerns / Carbohydrate Research 346 (2011) 2792–2800
2799
product as a white solid. Products were analyzed by CE to show com-
Acknowledgments
plete removal of sulfate and LC–MS to determine product purity or
composition (methods described in Section 4.6).
We are grateful to Dr. Lynn Teesch, the University of Iowa High
Resolution Mass Spectrometry Facility, for her insightful advice in
developing and troubleshooting ESI-MS and LC–MS methods in
this work. We are also grateful to Professor Lei Geng and Claudiu
Brumaru, the University of Iowa Department of Chemistry, for ad-
vice and assistance with CE. This work was supported in part by an
NIH Predoctoral Training Grant in the Pharmacological Sciences to
A.F. (T32 GM067795), an ACS Division of Medicinal Chemistry Pre-
doctoral Fellowship sponsored by Bristol-Myers Squibb to A.F., and
a Predoctoral Fellowship from the American Foundation for Phar-
maceutical Education to A.F.
4.6. Capillary electrophoresis to demonstrate removal of sulfate
N-Cbz O-sulfonated aminoglycoside was dissolved in DI water
to give a stock solution (1 mg/mL). Similarly, a stock solution of
Na2SO4 was prepared and serially diluted to generate a standard
curve of sulfate concentrations between 0 and 25 mM Na2SO4. An
autosampler was used to inject samples over 10 s at 0.5 psi. Sepa-
ration was performed at 20.0 kV (25 °C, 50 cm capillary, 5 lm
linear diameter, 4 Hz) using a buffer composed of 10 mM 5-sul-
fosalicyic acid pH 3.0.54,55 Sodium sulfate was detected by indirect
absorbance at 214 nm. Sodium sulfate concentration was calcu-
lated from the standard curve, and products desalted until sulfate
was undetectable.
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