Ring-opening polymerization with synergistic co-monomers: Access to a boronate-functionalized polymeric monolith for the specific capture of c/s-diol-containing biomolecules under neutral conditions

Full text of DOI:10.1002/anie.200902469

Communications
DOI: 10.1002/anie.200902469
Monolithic Materials
Ring-Opening Polymerization with Synergistic Co-monomers: Access
to a Boronate-Functionalized Polymeric Monolith for the Specific
Capture of cis-Diol-Containing Biomolecules under Neutral
Conditions**
Lianbing Ren, Zhen Liu,* Yunchun Liu, Peng Dou, and Hong-Yuan Chen
Polymeric monoliths, which show fast convective mass trans-
fer between the monolith bed and the surrounding solution,
have already become important materials in separation
science and (bio-)catalysis.[1] Although some purely polymeric
monoliths (such as acrylate-based monoliths) are excellent
separation media, functionalization is of utmost importance
in most situations. Conventional functionalization can be
classified into two strategies: 1) copolymerization of func-
tional monomers[2] and 2) postpolymerization functionaliza-
tion.[3] In both cases, the designed functionalities depend on
the structure and properties of functional monomers. To
obtain a certain functionality, a high-purity monomer with
appropriate structure and properties is indispensable.
Boronate affinity chromatography (BAC) has been a
useful means for the specific capture and separation of cis-
diol-containing biomolecules, such as saccharides, nucleo-
sides, and glycoproteins, since the early 1980s.[4] The principle
relies on reversible covalent complex formation/dissociation
between boronic acids and cis diols in an alkaline/acidic
aqueous solution. Recently, boronate-functionalized mono-
liths were synthesized by copolymerization[5] and postpoly-
merization functionalization.[6] However, as for other BAC-
based techniques, an apparent disadvantage is that the
chromatography in aqueous solution has to be performed in
alkaline media and can lead to the degradation of labile
compounds. Thus, boronate-functionalized monoliths that
function at neutral pH would be highly desirable for
physiological samples.
amino group capable of B–N coordination into the ligand
molecules (Wulff-type boronic acids),[8] or 3) the replacement
of intramolecular B–N coordination with intramolecular B–O
coordination (improved Wulff-type boronic acids).[9] Accord-
ing to these strategies, to make a boronate-functionalized
monolith that is functional under neutral conditions, a boronic
acid monomer with a low pKa value, if not commercially
available, must be synthesized and purified first through
tedious procedures. Herein, we present a new approach—
ring-opening polymerization with synergistic co-monomers—
for the preparation of a boronate-functionalized polymeric
monolith that functions under neutral conditions, without the
synthesis and purification of a single functional monomer.
The main synthetic route is based on the ring-opening
polymerization protocol established recently by Tanaka and
co-workers.[10] An epoxy resin, a diamine curing agent, and a
porogenic solvent are required for the preparation of the
monolith. To obviate the inconvenience of the synthesis of a
new diamine monomer, we took advantage of the coordina-
tion of m-aminophenylboronic acid (mPBA, 1) with 1,6-
hexamethylenediamine (HMDA, 2) to form a stable complex
À
3 with a B N bond (Scheme 1). The B–N-coordinated
complex was used as a diamine curing agent in a ring-opening
polymerization reaction with the epoxy resin tris(2,3-epoxy-
propyl)isocyanurate (TEPIC, 4) to form a macroporous
monolith. Since the conformation of the coordinated complex
is “frozen” by the polymerization, the coordinated complex
A conventional solution to this problem is to decrease the
pKa value of the ligands by synthesizing novel boronic acids
with exquisite structures through: 1) the introduction of an
electron-withdrawing group, such as a sulfonyl group, into the
ligand molecules,[7] 2) the introduction of a neighboring
[*] L. Ren, Dr. Z. Liu, Y. Liu, P. Dou, Prof. H. Y. Chen
Key Laboratory of Analytical Chemistry for Life Science
School of Chemistry and Chemical Engineering
Nanjing University
22 Hankou Road, Nanjing 210093 (China)
Fax: (+86)25-8368-5639
E-mail: zhenliu@nju.edu.cn
[**] We gratefully acknowledge the financial support of a General Grant
(No. 20675038) from the National Natural Science Foundation of
China, a Key Grant of 973 Program (No. 2007CB914102), an
International Science & Technology Cooperation Grant
(2008DFA01910) from the Ministry of Science and Technology of
China, and a General Grant (No. KB2008258) to Z.L. from the
Natural Science Foundation of Jiangsu Province.
Scheme 1. I) B–N coordination and II) ring-opening polymerization
with synergistic co-monomers.
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Chemie
on the resulting monolith 5 can function like a Wulff-type
boronic acid ligand and was expected to be capable of
capturing cis-diol-containing molecules under neutral condi-
tions.
The mechanism for reversible capture/release upon
changes in the pH value of the medium is illustrated in
Scheme 2. In an aqueous medium at pH 7, the nitrogen-
coordinated boronic acid reacts covalently with a cis-diol-
containing compound to form a five- or six-membered ring.
When the medium is made more acidic, the amine group is
protonated, and the B–N coordination is broken, which
results in the release of the cis diol from the monolith. When
the medium is neutralized again, the protonated amine group
is deprotonated, with recovery of the B–N coordination. Thus,
the pH-controlled capture/release mechanism can be
employed for affinity chromatographic separation at physio-
logical pH values.
Figure 1. 11B NMR spectra of mPBA dissolved in a) CDCl3, b) D2O,
c) CDCl3 containing HMDA, and d) D2O containing HMDA.
To verify the expected intermolecular B–N coordination,
11B NMR spectroscopic measurements were carried out for
surement indicated that macropores of
approximately 3 mm in diameter contrib-
uted
a total intraparticle porosity of
64.3%, whereas mesopores contributed a
total interparticle porosity of 18.6%. The
average size of mesopores was estimated
to be 9.6 nm by a Brunauer–Emmett–
Teller (BET) measurement corresponding
to a pore volume of 0.047 cm3 gÀ1 and a
specific surface area of 19.9 m2 gÀ1. These
results suggest that the prepared monolith
followed the typical bicontinuous pore-
distribution model.[11] The loading capacity
of the prepared monolithic capillaries at
pH 7.0 was measured to be 1.56 mmolmLÀ1
by frontal chromatography with catechol
Scheme 2. Intermolecular synergistic action for the reversible capture/release of cis-diol-
containing compounds.
mPBA in different media. mPBA was first dissolved in
PEG 200 (the porogenic solvent) with and without the
addition of HMDA in a 1:1 molar ratio. The mixture was
then dissolved in an aprotic (CDCl3) or a protic solvent
(D2O). A signal was observed at d = 9.8 ppm in CDCl3, and
the same signal was observed along with another signal at d =
À42.1 ppm in D2O (D2O-solvated mPBA; Figure 1). Upon
the addition of HMDA, the chemical shifts of mPBA at d =
9.8 and À42.1 ppm disappeared, and a new peak at À14.2 ppm
was observed. This peak can be assigned to the nitrogen-
coordinated aryl boronic acid. These results indicate that the
expected intermolecular B–N coordination did occur under
conditions similar to those used for the ring-opening poly-
merization.
With coordinated mPBA and HMDA as the co-mono-
mers, TEPIC as the crosslinker, and PEG 200 as the
porogenic solvent, boronate-functionalized monolithic capil-
laries were prepared by the proposed approach. The obtained
monoliths exhibited a well-controlled skeletal network and a
well-distributed macroporous open-channel network. Scan-
ning electron microscopy (SEM) images of a representative
monolith are shown in Figure 2. A mercury intrusion mea-
Figure 2. Scanning electron microscopy photographs of the cross-
section of the prepared monolithic capillary at different magnifications:
a) 1200ꢀ (distance between scale lines bottom right: 40.0 mm);
b) 5000ꢀ (distance between scale lines: 10.0 mm); c) 30000ꢀ
(distance between scale lines: 1.00 mm).
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Communications
as the test compound. For comparison, the value for a
boronate-functionalized monolithic column without intermo-
lecular coordination was zero.[5]
The expected functionality of the prepared monolithic
column was first evaluated on the basis of the affinity
chromatographic separation of catechol and quinol. Catechol
is a cis 1,2-diol, whereas quinol is a non-cis diol analogue for
control experiments. Quinol showed no retention on the
monolithic column at all the pH values investigated. In
contrast, catechol was not retained at pH 2.7, but its retention
was enhanced significantly when the pH value was increased
(Figure 3). At pH 7.0 and 7.5, catechol was completely
trapped by the column, and its elution needed an acidic
Figure 4. Chromatographic retention and enrichment of adenosine on
the boronate-functionalized monolithic column. Mobile phase:
a) 100 mm HAc (pH 2.7); b) 10 mm sodium phosphate buffer
(pH 7.4); the mobile phase was changed to 100 mm HAc (pH 2.7) at
15 min. Sample: adenosine (1 mgmLÀ1) in 10 mm sodium phosphate
buffer (pH 7.4).
capture cis-diols under neutral conditions, which should
facilitate the application to physiological samples. Unlike
conventional strategies for monolith preparation and the
lowering of the pH values at which they function, the
proposed approach substitutes two coordinated functional
co-monomers for a single functional monomer and obviates
tedious synthesis and purification procedures. Thus, the
concept of intermolecular synergism provides a new method
for the functionalization of polymer monoliths and Wulff-type
boronic acids. Monoliths prepared by the proposed approach
may be promising for glycocapture in glycoproteomics and
glycomics.
Figure 3. Chromatographic retention of quinol (6) and catechol (7) on
the boronate-functionalized monolithic column. Mobile phase:
a) 100 mm HAc (pH 2.7); b–e) 10 mm sodium phosphate buffer at
pH 6.0, 6.5, 7.0, and 7.5, respectively; the mobile phase was changed
to 100 mm HAc (pH 2.7) at 30 min. Sample: Quinol and catechol were
dissolved in the mobile phase at a concentration of 1 mgmLÀ1 each.
mobile phase (pH 2.7). In contrast, it was reported that
commercial mPBA–agarose affinity materials and a poly(4-
vinylphenylboronic acid-co-ethylene glycol dimethacrylate)
monolith did not capture catechol at pH < 8.0.[5,12] Thus, the
pH value at which capturing was possible was decreased by at
least one pH unit by the monolith with synergistic co-
monomers.
The enrichment of adenosine (a cis 1,2-diol) demonstrates
the usefulness of the boronate-functionalized monolith.
When an acidic mobile phase (pH 2.7) was used, adenosine
showed no retention and exhibited a relatively broad peak
(Figure 4). When the sample was prepared with a PBS buffer
(10 mm sodium phosphate, pH 7.4), loaded onto the column
with the same buffer, and then eluted with the acidic mobile
phase, adenosine was enriched, and the peak became sharper
(the enrichment of catechol needs a mobile phase containing
an organic solvent at a certain concentration to offset the
hydrophobic retention).
Experimental Section
A capillary with an inner diameter of 75 or 150 mm was rinsed
sequentially with 1m NaOH, water, 1m HCl, and water for 30 min
each. The capillary was then dried with a stream of nitrogen in a GC
oven at 608C. A 1:1 (v/v) mixture of tetrahydrofuran and (3-
aminopropyl)triethoxysilane were pumped through the capillary at
808C for 24 h. The modified capillary was then washed with methanol.
TEPIC (0.32 g), mPBA monohydrate (0.055 g), and HMDA (0.041 g)
were dissolved completely in PEG 200 (2.8 g). This solution was
injected into the modified capillary with a syringe. The polymeri-
zation reaction was carried out at 808C for 12 h, and the capillary was
then washed with water and methanol.
A TriSep-2100 pCEC instrument (Unimicro Technologies, Pleas-
anton, CA, USA) with a UV-absorbance detector was used for the
chromatographic separations. A boronate-functionalized monolithic
column with a total length of 50 cm (effective length: 35 cm) was used
in all experiments. A flow rate of 0.15 mLminÀ1 was used with the
splitting ratio set at 99:1; the UV absorbance was monitored at
275 nm. The volume of each sample was 20 nL.
11B NMR spectra were recorded on a Bruker Avance DMX
500 MHz instrument at 160 MHz. A solution of BF3OEt2 was used as
an external reference (d = À18.2 ppm). mPBA monohydrate (0.055 g)
was dissolved in PEG 200 (0.5 g) with or without the addition of
HMDA (0.041 g), and the solution was added to CDCl3 or D2O
(0.4 mL) for measurement.
In summary, a new approach to the preparation of
boronate-functionalized monoliths has been developed:
ring-opening polymerization with synergistic co-monomers.
The prepared boronate-functionalized monoliths were able to
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Chemie
For characterization of the pore-size distribution and specific
surface area, a monolith bed was synthesized in an empty regular
HPLC column. The monolithic column was washed with water and
methanol until the residual reagents had been flushed out. The
obtained monolith bed was dried and removed from the column for
characterization.
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Keywords: boronate affinity chromatography ·
intermolecular synergism · molecular recognition ·
monolithic materials · ring-opening polymerization
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