Efficient access to the non-reducing end of low molecular weight heparin for fluorescent labeling

Article abstract of DOI:10.1039/c4cc00708e

A novel thiol fluorophore was synthesized to be selectively attached to the non-reducing end of low molecular weight heparin (LMWH) via a Michael addition. Double labeling of LMWH was demonstrated to be a feasible approach for the determination of heparinase II activity by FRET. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00708e

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Efficient access to the non-reducing end of
low molecular weight heparin for fluorescent
labeling†
Cite this: Chem. Commun., 2014,
50, 7004
Received 28th January 2014,
Accepted 2nd April 2014
Zongqiang Wang, Chen Shi, Xuri Wu and Yijun Chen*
DOI: 10.1039/c4cc00708e
www.rsc.org/chemcomm
A
novel thiol fluorophore was synthesized to be selectively Michael acceptor,11 and the lack of a suitable fluorophore makes
attached to the non-reducing end of low molecular weight heparin this selective chemical functionalization of the unsaturated uronic
(LMWH) via a Michael addition. Double labeling of LMWH was acid (DUA) double bond on GAGs extremely difficult.
demonstrated to be a feasible approach for the determination of
heparinase II activity by FRET.
In order to develop a selective method for the functionaliza-
tion of the non-reducing ends of GAGs, we aimed to design
a new fluorophore to selectively attack the D4,5-unsaturated
The glycosaminoglycans (GAGs) are a family of linear, anionic carboxylic acid. After screening various conjugated systems, we
polysaccharides comprised of a repeating disaccharide, which found that a thiol group can be conjugated to the D4,5-unsaturated
is found on the surface of animal cells and within extracellular carboxylic acid of a GAG via Michael addition with high effi-
matrices. There are four groups of GAGs: (1) hyaluronan, ciency.12,13 To obtain a fluorophore with a reactive thiol group, we
(2) keratin sulfate, (3) chondroitin sulfate, and (4) heparan sulfate, then synthesized sulfhydryl acetaldehyde (2) by the condensation of
the most structurally complex GAG.1,2 In particular, low molecular chloroacetaldehyde (1) and sodium bisulfide. Next, reductive amina-
weight heparin (LMWH) has been widely used and intensively tion of 2 by 2-aminobenzamide gave the dimeric compound 3. The
1
investigated among GAGs. LMWH possesses a variety of biological structure of 3 was confirmed by H-NMR (Fig. S1, ESI†), 13C-NMR
activities, including being an anticoagulant, having anti-cancer (Fig. S2, ESI†) and IR (Fig. S3, ESI†). Finally, reduction of 3 by
activity, and affecting regulation of the biological functions dithiothreitol in DMSO resulted in the monomeric fluorophore 2-(2-
of morphogens, growth factors, cytokines, chemokines and mercaptoethylamino)benzamide (4, 2-MEAB) in an isolated yield of
1
enzymes.3,4
75%. The structure of 4 was confirmed by H-NMR (Fig. S4, ESI†),
Fluorescent labeling of proteins or nucleic acids has been a 13C-NMR (Fig. S5, ESI†) and IR (Fig. S6, ESI†). This newly synthesized
well-established approach for studying protein–protein inter- fluorophore contained a strong nucleophilic thiol group, and exhi-
actions,5,6 and DNA and RNA folding.7 To date, it has not been bited a high fluorescence intensity with excitation and emission
possible to utilize this approach for GAGs due to the lack of wavelengths of 340 nm and 440 nm respectively (Scheme 1).
suitable methodology to selectively label the non-reducing ends
LMWH (MW 2000–8000) 5 bearing a D4,5-unsaturated carboxylic
of GAGs with fluorophores.8,9 Thus, developing a method to acid at the non reducing end was used as a model for the
selectively functionalize the non-reducing ends of GAGs, such
as LMWH, is attractive for various applications.
In general, specific enzymes selectively hydrolyze GAGs to
produce oligosaccharides containing a D4,5-unsaturated carboxylic
acid at the non-reducing terminus.10 This unique feature in the
heavily sulfated GAG chain provides an opportunity for selective
functionalization via Michael addition. However, the carboxylic
acids on the non-reducing ends of GAGs are relatively poor
State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology,
China Pharmaceutical University, 24 Tongjia Street, Nanjing, Jiangsu Province,
210009, People’s Republic of China. E-mail: yjchen@cpu.edu.cn;
Fax: +86 25 83271031; Tel: +86 25 83271031
Scheme 1 Synthetic route to 2-(2-mercaptoethyl-amino)benzamide.
Reagents and conditions: (a) NaSH, H2O; (b) 2-aminobenzamide, NaCNBH3,
ZnCl2, MeOH, 3 Å sieves; (c) dithiothreitol, DMSO.
† Electronic supplementary information (ESI) available: Detailed methods and
supplementary figures. See DOI: 10.1039/c4cc00708e
7004 | Chem. Commun., 2014, 50, 7004--7006
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GAG oligosaccharides. To address the poor reactivity of the
carboxylic acid at the non-reducing end of LMWH, a three-step
process, including protection, a 1,4-Michael addition, and
deprotection, to transform the carboxylic acid of LMWH to
a carboxylic benzyl ester was implemented.14 First, LMWH
sodium salt was changed to the form of benzethonium chloride
salt. Then, PhCH2Cl was used to esterify the LMWH benzethonium
chloride salt to give LMWH benzyl ester 6 with a D4,5-unsaturated
benzyl ester at the non-reducing end. Next, LMWH benzyl ester 6
was reacted with 2-MEAB and boric acid in formamide at 50 1C for
24 h, and the resulting mixture was dialyzed (MWCO 500) against
deionized water to remove any unreacted 2-MEAB and formamide,
and then lyophilized to yield the 2-MEAB labeled LMWH benzyl
ester 7 (Scheme 2).
LMWH benzyl ester 6 and 2-MEAB labeled LMWH benzyl
ester 7 were then separated by HPLC, and compounds 6 and 7
were monitored by UV (232 nm) and a fluorescence detector
(340/440 nm) as shown in Fig. 1. Before the thia-Michael
addition, LMWH benzyl ester 6 at 1.7 min showed UV absorbance
from the double bond at the non-reducing end (Fig. 1a).15
However, when LMWH benzyl ester 6 was dissolved in water
and detected by the fluorescence detector, only a small amount of
impurity from the solvent was observed (Fig. 1b). After the thia-
Michael addition, the 2-MEAB labeled LMWH benzyl ester 7
exhibited both UV absorbance (232 nm) and fluorescence absor-
bance (340/440 nm). In addition, the chromatogram at 232 nm
indicated that nearly all of the unreacted 2-MEAB and formamide
had been removed, and that the relatively pure 2-MEAB labeled
LMWH benzyl ester 7 had been obtained (Fig. 1c). Moreover,
Fig. 1d clearly confirmed that the conjugation between 2-MEAB
and the non-reducing end of LMWH benzyl ester 7 had been
accomplished to show strong fluorescence compared to Fig. 1b.
To cleave the benzyl protecting group from 7, 0.1 N NaOH was
used to hydrolyze 7 at 60 1C for 1 h, and product 8 was
Fig. 1 HPLC chromatograms of unlabeled and labeled low molecular
weight heparin. (a) LMWH ester 6 and 2-MEAB were dissolved in formamide
and monitored at UV 232 nm. (b) LMWH ester 6 was dissolved in water and
monitored by a fluorescence detector at 340/440 nm. (c) After dialysis,
2-MEAB-LMWH 7 was monitored at UV 232 nm. (d) After dialysis, 2-MEAB-
LMWH 7 was monitored by a fluorescence detector at 340/440 nm. Peak 1:
LMWH derivatives; peak 2: formamide; peak 3: 2-MEAB.
precipitated in methanol. The fluorescence intensity of the
2-MEAB labeled LMWH 8 was detected to be very strong (Fig. S7,
ESI†). The high fluorescence intensity of the 2-MEAB labeled
LMWH clearly demonstrated that the conjugation between the
novel fluorophore 2-MEAB and the non-reducing end of the
LMWH via thia-Michael addition is a feasible approach for
the fluorescent labeling of LMWH.
To further confirm the utility of this novel fluorophore, LMWH
with double fluorescence labeling was prepared in order to inves-
tigate the interaction between GAGs and proteins by fluorescence
resonance energy transfer (FRET) as illustrated in Fig. 2a. Given
that 2-MEAB possesses a new emission wavelength of 440 nm,
with a 90% overlap with the excitation wavelength of a commer-
cially available fluorophore, 2-aminoacridone (2-AMAC), and
labeling at the reducing end was easier to achieve (Fig. S8,
ESI†),16 double labeling of LMWH on the reducing and non-
reducing ends by these two fluorophores would create a donor
and acceptor pair for FRET in order to determine the heparinase
activity because the length of the LMWH is less than 10 nm.17
Subsequently, to obtain the double fluorophore labeled LMWH
9, 2-MEAB-LMWH 8 was dissolved in deionized water and mixed
with a solution of 2-AMAC dissolved in 85% (v/v) acetic acid–
DMSO. The mixture was incubated at 37 1C for 16 h and dialyzed
Scheme 2 Synthetic route to the double fluorescent labeling of low
Fig. 2 Heparinase II activity assay by FRET. (a) Schematic illustration of the
activity assay of heparinase II. (b) Time courses of the FRET. FRET ratios
(340–538 nm/340–440 nm) of the control (dotted line) and the activity
assay (solid line). Data are an average of 3 independent determinations with
standard deviations.
molecular weight heparin. Reagents and conditions: (a)
C27H42NO2,
PhCH2Cl, CH2Cl2; (b) boric acid, formamide, 50 1C; (c) NaOH, 60 1C;
(d) 2-aminoacridone, NaCNBH3, acetic acid–DMSO (1 : 3, v/v); X = SO3Na
or H; Y = COCH3, SO3Na or H.
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against deionized water to remove any unreacted 2-AMAC, and opening of another sugar ring by Hg2+ 21
Communication
.
In conclusion, we have
then lyophilized to yield 9. Since heparinase II (HepII), from synthesized a novel fluorophore and selectively conjugated it
Pedobacter heparinus, is able to degrade heparin from a long with the non-reducing end of LMWH; this approach could be
chain to short chains in an endolytic manner,18 recombinant used to investigate the conformational changes of GAG oligo-
HepII was then prepared for an activity assay by FRET. The gene saccharides and the interactions between GAGs and proteins.
encoding his-tagged HepII was synthesized and transformed
This work was supported by the ‘‘111 Project’’ from the
into E. coli BL21 (DE3) cells. One liter cultures were grown Ministry of Education of China and the State administration of
at 37 1C in a Luria-Bertani (LB) medium supplemented with Foreign Expert Affairs (No. 111-2-07) and the research grant
50 mg mlÀ1 kanamycin and protein expression was induced by from the State Key Laboratory of Natural Medicines, China
the addition of 500 mM isopropyl 1-thio-b-D-galactopyranoside Pharmaceutical University (SKLNMZZCX No. 201301).
for 8–12 h at 15 1C. Purification of the rHepII was performed as
described in the literature.19 Briefly, recombinant HepII was
purified by two chromatographic steps, including immobilized
Notes and references
metal-chelate chromatography using a 1 ml His-trap FF column
(GE Healthcare) and a cation exchange column using a 1 ml
Hitrap-SP column. The protein purity was assessed by SDS-
PAGE as shown in Fig. S9 (ESI†). For the determination of the
HepII activity by FRET, LMWH was double labeled with two
fluorophores on the reducing- and non-reducing ends. Then,
the degradation of the LMWH double labeled with fluoro-
phores was measured by time-resolved fluorescence after the
addition of the purified HepII. The control consisted of 100 ml
of 9 (1 mg mlÀ1) and 100 ml potassium phosphate buffer
(pH 7.4) in a 96 black well plate. After incubating at 37 1C for
24 h, the FRET ratio (340–538 nm/340–440 nm) did not change
(Fig. 2b, dotted line), indicating that no LMWH had been
degraded. However, when 9 and HepII were mixed and incu-
bated under the same conditions, the FRET ratio decreased in a
time-dependent manner (Fig. 2b, solid line). The results
demonstrated that 2-MEAB had been selectively conjugated to
the non-reducing end of the LMWH, and can be used as a FRET
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