A benzoboroxole-functionalized monolithic column for the selective enrichment and separation of cis-diol containing biomolecules

Article abstract of DOI:10.1039/c2cc30230f

A benzoboroxole-functionalized monolithic column was synthesized, which exhibited the best specificity and affinity towards cis-diol containing biomolecules as compared with the boronate affinity monolithic columns reported as well as significant secondar

Full text of DOI:10.1039/c2cc30230f

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COMMUNICATION
A benzoboroxole-functionalized monolithic column for the selective
enrichment and separation of cis-diol containing biomoleculesw
Hengye Li, Heye Wang, Yunchun Liu and Zhen Liu*
Received 11th January 2012, Accepted 4th March 2012
DOI: 10.1039/c2cc30230f
6
called ‘‘teamed boronate affinity’’ has reduced the binding
c
A benzoboroxole-functionalized monolithic column was synthesized,
which exhibited the best specificity and affinity towards cis-diol
containing biomolecules as compared with the boronate affinity
monolithic columns reported as well as significant secondary
separation capability under acidic conditions.
pH to neutral or medium acidic conditions. However, Wulff-type
6g
6c
boronic acid and ‘‘teamed boronate affinity’’ based monolithic
columns failed to capture glycoproteins. Furthermore, almost all
of the existing boronate affinity monoliths are associated with
relatively low binding capacities. So it is very necessary to further
explore new boronic acid ligands to resolve all these issues.
Benzoboroxoles are a unique class of boronic acids which
have showed excellent water solubility and improved sugar-
Boronate affinity (BA) is a unique means for selective capture
and enrichment of cis-diol containing biomolecules. The principle
is that boronic acids form five or six-membered cyclic esters with
cis-diol molecules under high pH conditions (usually alkaline
conditions) and the cyclic esters dissociate when the environmental
medium is changed to acidic pH. This pH switch property
7
binding capacity, even superior to Wulff-type boronic acid, in
8
a,b
neutral water.
They have been used as binding ligands for
8
c,d
8e
and oligosaccharide and as
the detection of saccharide
9
1
inhibitors for HIV entry. This class of boronic acids may
provide an ideal solution to the obstacles mentioned above.
However, such feasibility has not been investigated yet.
Here we report the synthesis of a benzoboroxole functionalized
hydrophilic polymeric macroporous monolithic column. This
boronate affinity monolithic column showed the best specificity
and affinity towards cis-diol containing molecules among all
makes boronic acids excellent ligands in saccharide sensing
2
and separation science.
Many cis-diol biomolecules are of great biological significance.
For example, glycoproteins play important roles in molecular
recognition, inter- and intracell signaling, immune response
and sperm–egg interaction while modified nucleosides have
3
been considered as potential tumor markers for many diseases.
6
boronate affinity monolithic columns, as well as high binding
Besides, cis-diol compounds in biosamples usually exist in very
low abundance along with abundant interfering components.
Recently, BA-based techniques have drawn great attention in
proteomics and metabolomics for the selective capture and
enrichment of cis-diol containing biomolecules of interest in
capacity under neutral or acidic conditions. These features
overcome all the disadvantages of the previous monolithic
columns. It also exhibited significant secondary separation
capability towards cis-diol compounds under acidic conditions.
4
3
-Carboxy-benzoboroxole was synthesized through a simple
complex biosamples.
8e,10
procedure relative to the reported methods,
as shown in
BA monolithic columns, which inherit the virtues of boronate
5
affinity and those of monolith materials (easy fabrication, low
Scheme 1A. Its structure was confirmed by NMR and MS. Its
pK value was measured to be approximately 6.92 (Fig. S2 in the
back pressure and fast convective mass transfer), have developed
6
quickly in recent years. However, several issues may be severe
a
ESIw). The hydrophilic base monolithic capillary was prepared
by copolymerization of glycidyl methacrylate (GMA) and
methylene bisacrylamide (MBAA), as shown in Scheme 1B.
3-Carboxy-benzoboroxole was then immobilized onto the base
monolith surface using the procedure depicted in Scheme 1C.
Scanning electron microscopy (SEM) analysis revealed that
the functionalized monolith exhibited a well-controlled skeleton
and a well-distributed macroporous open-channel network
obstacles for the wide applications of BA monolithic columns.
First, the specificity towards glycoproteins is poor on some
6
b
columns, and it was found that reversed-phase interaction is a
6b,d
major factor that degrades the specificity towards glycoproteins.
Second, conventional boronic acids require a basic pH for strong
binding, which gives rise to the risk of degradation of labile
molecules. The employment of sulfonyl substituted phenylboronic
6
f
6g
(
(
Fig. S5 in the ESIw). Using the Brunauer–Emmett–Teller
BET) method, the average pore size and specific surface area
acid and Wulff-type boronic acid as well as a new strategy
2
ꢀ1
were measured to be 6.12 nm and 11.40 m g , respectively.
FT-IR and XPS characterization verified the presence of
compound 3 on the monolith (Fig. S6 and S7 in the ESIw).
The boronate affinity properties of the functionalized column
were evaluated in terms of the chromatographic retention of
deoxyadenosine (4) and adenosine (5). Adenosine has a cis-diol
State Key Laboratory of Analytical Chemistry for Life Science,
School of Chemistry and Chemical Engineering, Nanjing University,
Nanjing 210093, China. E-mail: zhenliu@nju.edu.cn;
Fax: +86-25-8368-5639; Tel: +86-25-8368-5639
w Electronic supplementary information (ESI) available: Full experi-
mental details. See DOI: 10.1039/c2cc30230f
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4115–4117 4115

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Fig. 2 Isocratic separation of a mixture of standard nucleosides.
Mobile phase, 100 mM HOAc (pH 2.7); sample: seven nucleosides
Scheme 1 (A) The synthesis of 3-carboxy-benzoboroxole; (B) the
preparation of the base monolithic column; (C) the immobilization
of compound 3 onto the surface of the base monolith.
ꢀ1
and deoxyadenosine (0.03 mg mL each) dissolved in the mobile
0
phase; detection at 260 nm; peak identity: 1, 2 -deoxyadenosine; 2, cytidine;
3, adenosine; 4 + 5, uridine and 5-methyluridine; 6, inosine; 7, guanosine;
8
, xanthosine.
moiety in the structure while deoxyadenosine does not. When
the binding pH was 7.0, deoxyadenosine was not retained
(
RSD) for retention time of adenosine, which was 0.28, 2.35
(
traces a and c in Fig. 1), but adenosine was (traces b and c in
and 4.87%, respectively. This indicates good repeatability.
The separation capability of the functionalized column
under isocratic acidic elution conditions was further investi-
gated with a more complex sample, a mixture containing seven
nucleosides and deoxyadenosine. As shown in Fig. 2, the
mixture was separated into seven peaks on the functionalized
column by isocratic elution with 100 mM HOAc (pH 2.7).
Deoxyadenosine was not retained by the functionalized column
and eluted at dead time while the seven nucleosides were separated
into six peaks (5-methyluridine and uridine were co-eluted).
The mechanism for the secondary separation was apparently
different from hydrophilic interaction chromatography (HILIC).
First, the elution order was completely different from those on
Fig. 1). This indicates the excellent specificity of the functionalized
monolithic column towards cis-diol containing compounds.
Interestingly, similar experimental results were also observed
when the binding pH decreased from 6.5 to 5.0 (traces d–g in
Fig. 1). The complete retention of adenosine at pH 5.0 (trace g in
Fig. 1) reveals that a stable cyclic ester between benzoboroxole
and adenosine can form even at pH 5.0. To our best knowledge,
this is the lowest pH at which boronate affinity chromatography
could function. More interestingly, when the pH of the binding
buffers was further reduced from 5.0 to 3.0, adenosine still
exhibited apparent retention, but the retention decreased upon
decreasing the binding pH (traces h–k in Fig. 1). Even by
isocratic elution with 100 mM HOAc (pH 2.7), adenosine was
still separated from deoxyadenosine by the functionalized
column (trace l in Fig. 1) while the mixture was not separated
on the non-functionalized column (trace m in Fig. 1). To
further confirm the strong interaction between the functionalized
column and other cis-diol compounds under acidic pH condi-
tions, retention behaviour of quinol and catechol was also
investigated and the same trend was observed (Fig. S8 in the
ESIw). Additionally, the run-to-run, column-to-column and
batch-to-batch repeatability of the functionalized monolithic
column was investigated in terms of relative standard deviation
11
typical HILIC columns. Second, a mobile phase of high-organic
content is a precondition for HILIC while an aqueous mobile
phase with zero-organic solvent was used on the current column.
When attempts were made to reduce the pH value required
6c,f,g
for strong binding,
specific binding capability towards
glycoproteins was not an inevitable outcome. The ability of
specific capture of glycoproteins of the functionalized monolithic
column was evaluated. As shown in Fig. 3, five glycoproteins
(
traces a–e in Fig. 3) were captured by the column under neutral
aqueous conditions until the mobile phase was switched to
00 mM HOAc. While three non-glycoproteins (traces f–h in
1
Fig. 3) were not retained by the column. These results demon-
strate the specific binding capability of the functionalized
monolithic column towards glycoproteins. Mixtures of standard
glycoproteins and non-glycoproteins were further used as samples.
The results further confirmed the excellent specificity of the
functionalized monolithic column towards glycoproteins at
neutral pH (Fig. S9 in the ESIw).
Binding capacity is a crucial factor for affinity chromatography.
The binding capacities of the functionalized monolithic column for
small molecules at different pH values were measured using frontal
chromatography with adenosine as the test molecule (Table S1 in
the ESIw). To our best knowledge, the value at each specific pH,
Fig. 1 The chromatographic retention of deoxyadenosine (4) and
adenosine (5) on the functionalized (a–l) and the non-functionalized
ꢀ
1
ranging from 5.30 to 22.33 mmol mL , was the highest as
compared with those of the boronate affinity monolithic
(m) columns. Mobile phase: (a–k) 30 mM sodium phosphate buffer at
pH 7.0, 7.0, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5 and 3.0, respectively,
6b–d,f,g]
columns reported.
With HRP as the test analyte, the binding
ꢀ
switching to 100 mM HOAc (pH 2.7) at 15 min; (l–m) 100 mM HOAc;
ꢀ1
1
.
ꢀ1
capacity for glycoprotein was measured to be 0.033 mmol mL
sample: (a) deoxyadenosine (0.2 mg mL ), (b) adenosine (0.4 mg mL ),
ꢀ
1
c–m) adenosine (0.4 mg mL ) and deoxyadenosine (0.2 mg mL
ꢀ1
(
)
The feasibility of applications of the functionalized mono-
lithic column to real complex samples was examined in terms
dissolved in responding loading buffers; detection at 260 nm.
4
116 Chem. Commun., 2012, 48, 4115–4117
This journal is c The Royal Society of Chemistry 2012

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towards nucleosides and glycoproteins, as well as high binding
capacity. This boronate affinity monolith could function at
pH as low as pH 5.0, which is the lowest among all boronate
affinity chromatographic columns reported in the literature.
The feasibility of application to real complex samples was
demonstrated with the selective enrichment of urinary nucleosides.
Furthermore, the boronate affinity monolith exhibited apparent
secondary separation capability towards nucleosides under acidic
elution conditions, which may provide a new opportunity for the
development of LC techniques for the separation of nucleosides
and other cis-diol containing compounds.
Fig. 3 Specific capture of glycoproteins on the modified column and
comparison with non-glycoproteins. Mobile phase, 20 mM HEPES
We gratefully acknowledge the financial support from
the National Natural Science Foundation of China (Grants
No. 21075063 and 21121091), the Natural Science Foundation
of Jiangsu Province (Grant No. KB2011054), the Ministry
of Science and Technology of China (Grant No. 34-8) and
Dionex China.
2
containing 100 mM MgCl (pH 7.0), switching to 100 mM HOAc (pH 2.7)
ꢀ1
at 15 min; samples (a–h) 1 mg mL responding proteins dissolved in
loading buffer, (i) the loading buffer as blank control; detection at 214 nm.
The proteins used: (a) RNase B, (b) HRP, (c) anti-AFP monoclonal
antibody, (d) anti-CEA polyclonal antibody, (e) anti-PSA monoclonal
antibody; (f) RNase A, (g) cytochrome C, (h) b-lactoglobin.
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0
Urine sample: (b) un-extracted and (b ) extracted by the functionalized
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4, 15, 17 and 21) in original urine were completely removed while
other components (peaks 1, 3, 4, 5, 6, 7, 8, 10, 11, 16, 19 and 20)
were effectively enriched. Especially, some components (peaks 1,
7
8
3
, 16 and 19) could not be observed if the sample was not
extracted by the monolithic column. While, after extraction,
they were clearly observed. The identities of some of the peaks
(1, uridine; 3, cytidine; 4, xanthosine; 5, adenosine or guanosine;
9
1
7, inosine) confirmed the excellent specificity of the functionalized
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1 Y. Kawachi, T. Ikegami, H. Takubo, Y. Ikegami, M. Miyamoto
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2
The benzoboroxole-functionalized monolithic column synthe-
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1
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This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4115–4117 4117