Complexion of Boric Acid With 2-Deoxy-D-glucose (DG) As A Novel Boron Carrier For BNCT
Complexion of Boric Acid With 2-Deoxy-D-glucose (DG) As A Novel Boron Carrier For BNCT
Complexion of Boric Acid With 2-Deoxy-D-glucose (DG) As A Novel Boron Carrier For BNCT
MSD
Original Article
Medical Science and Discovery
October 2014, Vol.1, No.3, p:65-71
Doi: 10.17546/msd.74442
Abstract
Objective: Boron neutron capture therapy (BNCT) is an intensive research area for cancer researchers.
Especially the side effects and inabilities of conventional therapies in some cases, directs researchers to find
out a new cancer therapy methods such as BNCT. One of three important problem of BNCT is targeting of
boron to tumor tissue. Borono Phenyl Alanine (BPA) and Borono Sodium Borocaptate (BSH) are already
using in clinical studies as boron carriers. New boron carriers are searching for high yield boron accumulation
in the tumor tissue.
Methods: In this study, a novel 10B carrier was synthesized, ((2R)-4,5,6-trihydroxy-2-
(hydroxymethyl)tetrahydro-2H-pyran-3-yl)boronic acid (10B-DG), for BNCT studies. 10Boric Acid and 2-
Deoxy-d-Glucose was complexed (10B-DG) through a low-high pH reaction and yield of complexion was
tested with FTIR ATR and Liquid Chromatography Mass Spectrometry (LC/MS).
Results: Confirmation studies have been carried out by HPLC and chromatograms have confirmed that
Borono-2-Deoxy-d-Glucose synthesized with % 80 yield.
Conclusions: This compound appears to be an alternative boron carrier for BNCT applications
Introduction
Received: 12 Sept. 2014, Revised 22 Sept. 2014, Accepted 24 Sept. 2014, Available Online 10 Oct. 2014
1Celal Bayar University School of Medicine, Department of Biophysics, Manisa-Turkey
2Celal Bayar University, Faculty of Art and Science, Department of Chemistry, Manisa-Turkey
3Yüzüncü Yıl University School of Medicine, Department of Pharmacology, Van-Turkey
4Marmara University, Faculty of Technology, Department of Textile Engineering, Istanbul-Turkey
5Marmara University, Faculty of Science, Department of Organic Chemistry, Istanbul-Turkey
*Corresponding Author: Zafer Akan E-mail: [email protected]
Akan et al, Doi: 10.17546/msd.74442
glucose transport and glycolytic ATP production DG: Sigma-Aldrich, D8375) solutions were
[3] prepared with the same volumes of deionized water
Positron emitter radioactive 18F complexed and incubated for 1 hour at pH:3 and 50°C.
Deoxy-D-glucose (18F-deoxy-D-glucose: 18FDG) is
routinely used for the detection and staging of Both solutions were then mixed in the same tube
tumors with positron emission tomography (PET). and incubated for 1 hour at pH:3. The pH was
Radioactive positron emitter 18F successfully gradually increased from pH:3 to pH:7 and
targeted to tumor tissue by the Deoxy-D-glucose. stabilized at physiologic pH:7.4. FTIR-ATR
Boric acid B(OH)3 and its anion borate analysis were done for only B(OH)3, only Tiron,
B(OH)4- have solution chemistry that is quite only DG and complexed B-Tiron and B-DG.
different from most other oxyanions. Borate forms Complexation between boric acid and the 2-DG
by the addition of a hydroxyl group to the trigonal may be expressed as eqn.
planar boric acid molecule, forming a tetrahedral
anion. The pK of this reaction is 9.2 [4] B(OH)3 + H2O ↔ B(OH)4⁻ + H⁺ Ka= [B(OH)4⁻][H⁺] / [B(OH)3]
B(OH)4⁻+ DG ↔ B-DG + H⁺ Ka= [B(OH)4⁻][DG] / [B-DG]
B(OH)3 + OH- ↔ B(OH)4- pKa 9.2
HPLC studies
Boric acid and borate both typically exist
as monomers in solution at low concentrations The following quality control studies were done to
(below 25 mM) but at higher concentrations many confirm Boric acid, 2-Deoxy-D-glucose and
poly-borate polymers are known to form [5, 6]. Borono-2-Deoxy-D-glucose. Table 1 shows
Due to simple complexation properties of chromatographic conditions used analytical
borate anions and easy intracellular uptake experiments in HPLC. A low- pressure gradient
properties of 2-DG, the synthesis and complexation HPLC system (LC-10ATvp quaternary pump and
yield of Boric acid with Deoxy-D-Glucose (10B- SPD-10A/V UV detector and a syringe injector
DG) were examined. equipped with a 1 ml loop and 7-µm RP-C-18
column 250 x 4.6 mm I.D. (inner diameter),
Material and Methods Macherey–Nagel), was used for analytical
experiments.
Complexation reaction of B(OH)3 and 2-DG and
FT-IR/ATR measurements Table 1. Chromatographic conditions used analytical
experiments in HPLC
Column in analytical exp.: RP-C18(250x4.6mm)
The complexation reaction of boric acid Flow speed in analytical exp.: 0.7 mL/min
with polyhydroxyl compounds, such as tiron, has Wave length: 240 nm
been studied, and the reaction has been well defined Temperature: 30 oC
Pressure: 76 bar
in previous studies [6]. In same reaction conditions Mobile phase in analytical exp.: 18 mM NaOH
were applied for B(OH)3 and 2-DG complexation.
A Perkin Elmer PE100 Infrared
Spectrophotometer with Universal ATR Sampling LC-MS
Accessory was used for spectroscopic studies. All
spectra were measured in the range between 1600 Liquid chromatography mass spectrometry
and 750 cm−1, at resolution of 4 cm−1 [7]. Distilled (LC-MS) chromatograms were taken using a
water was used as background and to clean the HCTultra LC-MS instrument. Chromatographic
diamond probe between each sample. All conditions used in this study were given in Table 2.
measurements were realized at room temperature. The parameters were optimized and set as followed.
Deionized water prepared with a Milli-Q SP system Ion source Type ESI pos and ESI neg, Mass
(Millipore). Range Mode Ultra Scan (26000 m/z/s), Column
0.1 M Boric acid (B(OH)3; Sigma- No column, direct infussion, Capillary pos -4000 V
Aldrich, B6768) and 0.5 M Tiron (4,5-Dihydroxy- and neg +4000 V, Drying gas tempreture 300 oC,
1,3-benzenedisulfonic acid disodium salt, Sigma- Drying gas pressure 5 psi, Nebulizing gas pressure
Aldrich, D7389) solutions were prepared with the 10 psi .
same volume of deionised water and incubated for
1 hour at pH:3 and 50°C.
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Akan et al, Doi: 10.17546/msd.74442
Table 2. Chromatographic conditions for LC-MS Table 3. LC–MS/MS Spectrum (m/z) Values for Borono-2-
experiments Deoxy-D-glucose Compounds and Some Different
Ion source type: ESI pos and ESI neg Fragments and Proposed Structures of Selected Fragments
Mass range mode: Ultra Scan (26000 m/z/s)
Column: No column direct infussion Frag. Structure m/z
Capillary: pos -4000V and neg +4000V H3C O OH
Drying gas tempreture: 300 oC
Drying gas pressure: 5 psi
Nebulizing gas pressure: 10 psi
1 164,1
HO OH
OH
Results
H3C O
Complexation reaction of B(OH)3 and 2-DG
2 187,7
FT-IR/ATR results of 10B-Tiron and 10B-
(HO) 2B B(OH) 2
DG (Fig. 1) have similar peak shifts which indicate
complexation due to literature results (Fig. 2, 3), H3C O
[7]. The IR spectra of these solutions of B(OH)3, 2-
deoxy glucose (2-DG) and 10B-DG showed that 3 143,9
formation bonding between 10B and 2-DG by the
disappearance of asymmetric stretching of B(OH) 3 B(OH) 2
at 1413 cm-1 and decreasing peaks intensity of 2- +
DG solution at 1264 cm-1 (O-H blending of Na
deoxyglucose) and 1067 cm-1, 1029 cm-1 and H3C
O
1015cm-1 (C-O stretching of deoxyglucose).
4 182,9
Figure 1. Chemical synthesis of Borono-Deoxy-D-glucose
B(OH) 2
OH
O
HO
5 132,1
HO
+
Na
H3C O OH
6 171,1
HO
OH
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Akan et al, Doi: 10.17546/msd.74442
Shao and coworkers have shown that Boric investigated by IR Spectra. The IR spectra of
acid - Tiron (1,2-dihydroxybenzene-3,5-disulfonic solutions Tiron, B, B-Tiron were taken. The IR
acid disodium salt monohydrate) complex spectra (Figure 2) showed that the disappearance of
characterization by 11B NMR spectra and proved asymmetric stretching of B(OH)3 at 1407 cm-1 like
forming complex between Boric acid and Tiron [6] in Figure 3. This indicates that boric acid form
In this work Boric acid-Tiron complex were complex with deoxyglucose like Tiron.
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Akan et al, Doi: 10.17546/msd.74442
Results of HPLC and LC-MS studies hydrogen, so that it cannot undergo further
glycolysis. As such, it acts to competitively inhibit
HPLC chromatograms confirmed that the production of glucose-6-PO4 from glucose at the
Borono-2-Deoxy-D-glucose synthesized with 80% phosphoglucoisomerase level [10].
yield. Three peaks were detected for Borono-2- 2-DG is easily uptaken by the glucose
Deoxy-D-glucose HPLC analyses, the retention transporters of the cell. Therefore, cells with higher
times of related compounds were different from glucose uptake, for example tumor cells, have also
each other as is seen in Figure 4 and Figure 5. a higher uptake of 2-DG.
Retention times are 3.68, 4.18 and 4.87 min for Inhibition of glycolysis by the small
Boric acid, 2-Deoxy-D-glucose and Borono-2- glycolysis inhibitors (GI) brought up to the use of
Deoxy-D-glucose, respectively. combine usage of glycolysis inhibitors with
LC–MS spectrum (m/z) values for Borono- chemotherapeutics in the treatment of malignant
2-Deoxy-D-glucose compounds and some different tumors therefore affectivity research of 2-DG -
fragments and proposed structures of selected Chemotherapeutic combine treatments were
fragments (m/z) values are 162,1 : 187 : 143,9 : recently started in clinical trials [11].
182,9 : 132,1 : 169,1. Due to higher glucose uptake of tumor
cells, radiolabelled 2-DG (18F-DG) is also routinely
Discussion using for tumor imaging and staging with positron
emitting tomography since 1990’s (PET) [12].
Most cancer cells exhibit increased Even if alternative reactions can be used for 2-DG
glycolysis and use this metabolic pathway and boric acid complexation which enrolled in the
(anaerobic pathway) for generation of ATP as a study as alternative boron carrier; 2-DG and Boric
main source of their energy supply because of Acid thought to be complexed rapidly and easily
delayed angiogenesis. This phenomenon is known via low-high pH reactions due to poly-hydroxyl
as the Warburg effect and is considered as one of components of two molecules. Low-high pH
the most fundamental metabolic alterations during reactions have been recommended in the literature
malignant transformation. Although delayed for similar boric acid reactions [6].
angiogenesis seen as a chance to delay for If compare with low-high pH
metastases, makes malignancies chemotherapy- complexation reaction results, yield is very low for
resistant. Beside of chemotherapeutic resistant, other reactions and reaction time is not reasonable.
oxygen-free environment due to malignant For example, nucleophilic substitution reaction is
transformation makes cancer cells resistant to more widely used for 18F, 2-DG complexation
radiotherapy too [8] Importantly, the increased reaction and electrophilic fluorination reaction has
dependence of cancer cells on glycolytic pathway an important place in the synthesis of 18F-FDG.
for ATP generation provides a biochemical basis Synthesis of 18F-FDG in radio-fluorination
for the design of therapeutic strategies to reactions, triflates produces a moderate consistent
preferentially kill cancer cells by pharmacological yield at about 50 to 60% [13].
inhibition of glycolysis. Several small molecules Boric acid reacts with polyhydroxyl
have emerged that exhibit promising anticancer compounds as a Lewis acid to form complex in
activity in vitro and in vivo, as single agent or in aqueous solution. 2-Deoxy-D-glucose has the 2-
combination with other therapeutic modalities [9]. hydroxyl group therefore simple pH complexation
2-Deoxy-D-glucose is a glucose molecule reaction was designed as defined in previous
which has the 2-hydroxyl group replaced by studies [6].
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Figure 4. HPLC chromatograms of 2-Deoxy-D-glucose and Borono-2-Deoxy-D-glucose (first pink peak belongs to 2-Deoxy-D-
glucose, Rt (Retention time):4.18 min and the second black peak is Borono-2-Deoxy-D-glucose, Rt (Retention time): 4.87 min)
Figure 5. HPLC chromatograms of Boric acid B(OH)3, Rt (Retention time): 3,68 min
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1998;1(1):15-30.
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Copyright © 2014 The Author(s); This is an open-access article distributed under the terms of the Creative Commons Attribution
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