Complexion of Boric Acid with 2-Deoxy-D-glucose ( DG ) as a novel boron carrier for BNCT

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: zafer_akan@hotmail.com Complexion of Boric Acid with 2-Deoxy-D-glucose (DG) as a novel boron carrier for BNCT


Introduction
Radiotherapy is a long-standing treatment method which makes use of ionizing radiation for the treatment of patients with malignancies.Ionizing radiations are absorbed by the all encountered tissues during radiotherapy.Side effects of ionizing radiation are unavoidable [1] Even different modalities and approaches are advancing the radiotherapy more radical changes such as BNCT are needed for patient life quality.glucose transport and glycolytic ATP production [3] Positron emitter radioactive 18 F complexed Deoxy-D-glucose ( 18 F-deoxy-D-glucose: 18 FDG) is routinely used for the detection and staging of tumors with positron emission tomography (PET).Radioactive positron emitter 18 F successfully targeted to tumor tissue by the Deoxy-D-glucose.
Boric acid B(OH) 3 and its anion borate B(OH) 4 -have solution chemistry that is quite different from most other oxyanions.Borate forms by the addition of a hydroxyl group to the trigonal planar boric acid molecule, forming a tetrahedral anion.The pK of this reaction is 9.2 [4] B(OH)3 + OH -↔ B(OH)4 -pKa 9.2 Boric acid and borate both typically exist as monomers in solution at low concentrations (below 25 mM) but at higher concentrations many poly-borate polymers are known to form [5,6].
Due to simple complexation properties of borate anions and easy intracellular uptake properties of 2-DG, the synthesis and complexation yield of Boric acid with Deoxy-D-Glucose ( 10 B-DG) were examined.

Complexation reaction of B(OH) 3 and 2-DG and FT-IR/ATR measurements
The complexation reaction of boric acid with polyhydroxyl compounds, such as tiron, has been studied, and the reaction has been well defined in previous studies [6].In same reaction conditions were applied for B(OH) 3 and 2-DG complexation.
A Perkin Elmer PE100 Infrared Spectrophotometer with Universal ATR Sampling Accessory was used for spectroscopic studies.All spectra were measured in the range between 1600 and 750 cm −1 , at resolution of 4 cm −1 [7].Distilled water was used as background and to clean the diamond probe between each sample.All measurements were realized at room temperature.Deionized water prepared with a Milli-Q SP system (Millipore).
Both solutions were then mixed in the same tube and incubated for 1 hour at pH:3.The pH was gradually increased from pH:3 to pH:7 and stabilized at physiologic pH:7.4.
FTIR-ATR analysis were done for only B(OH) 3 , only Tiron, only DG and complexed B-Tiron and B-DG.Complexation between boric acid and the 2-DG may be expressed as eqn.

HPLC studies
The following quality control studies were done to confirm Boric acid, 2-Deoxy-D-glucose and Borono-2-Deoxy-D-glucose.Table 1 shows chromatographic conditions used analytical experiments in HPLC.A low-pressure gradient HPLC system (LC-10ATvp quaternary pump and SPD-10A/V UV detector and a syringe injector equipped with a 1 ml loop and 7-µm RP-C-18 column 250 x 4.6 mm I.D. (inner diameter), Macherey-Nagel), was used for analytical experiments.

LC-MS
Liquid chromatography mass spectrometry (LC-MS) chromatograms were taken using a HCTultra LC-MS instrument.Chromatographic conditions used in this study were given in Table 2.The parameters were optimized and set as followed.Ion source Type ESI pos and ESI neg, Mass Range Mode Ultra Scan (26000 m/z/s), Column No column, direct infussion, Capillary pos -4000 V and neg +4000 V, Drying gas tempreture 300 o C, Drying gas pressure 5 psi, Nebulizing gas pressure 10 psi .

Complexation reaction of B(OH) 3 and 2-DG
FT-IR/ATR results of 10 B-Tiron and 10 B-DG (Fig. 1) have similar peak shifts which indicate complexation due to literature results (Fig. 2, 3), [7].The IR spectra of these solutions of B(OH) 3 , 2deoxy glucose (2-DG) and 10 B-DG showed that formation bonding between 10 B and 2-DG by the disappearance of asymmetric stretching of B(OH) 3 at 1413 cm -1 and decreasing peaks intensity of 2-DG solution at 1264 cm -1 (O-H blending of deoxyglucose) and 1067 cm -1 , 1029 cm -1 and 1015cm -1 (C-O stretching of deoxyglucose).Shao and coworkers have shown that Boric acid -Tiron (1,2-dihydroxybenzene-3,5-disulfonic acid disodium salt monohydrate) complex characterization by 11 B NMR spectra and proved forming complex between Boric acid and Tiron [6] In this work Boric acid-Tiron complex were investigated by IR Spectra.The IR spectra of solutions Tiron, B, B-Tiron were taken.The IR spectra (Figure 2) showed that the disappearance of asymmetric stretching of B(OH) 3 at 1407 cm -1 like in Figure 3.This indicates that boric acid form complex with deoxyglucose like Tiron.

Results of HPLC and LC-MS studies
HPLC chromatograms confirmed that Borono-2-Deoxy-D-glucose synthesized with 80% yield.Three peaks were detected for Borono-2-Deoxy-D-glucose HPLC analyses, the retention times of related compounds were different from each other as is seen in Figure 4

Discussion
Most cancer cells exhibit increased glycolysis and use this metabolic pathway (anaerobic pathway) for generation of ATP as a main source of their energy supply because of delayed angiogenesis.This phenomenon is known as the Warburg effect and is considered as one of the most fundamental metabolic alterations during malignant transformation.Although delayed angiogenesis seen as a chance to delay for metastases, makes malignancies chemotherapyresistant.Beside of chemotherapeutic resistant, oxygen-free environment due to malignant transformation makes cancer cells resistant to radiotherapy too [8] Importantly, the increased dependence of cancer cells on glycolytic pathway for ATP generation provides a biochemical basis for the design of therapeutic strategies to preferentially kill cancer cells by pharmacological inhibition of glycolysis.Several small molecules have emerged that exhibit promising anticancer activity in vitro and in vivo, as single agent or in combination with other therapeutic modalities [9].
2-Deoxy-D-glucose is a glucose molecule which has the 2-hydroxyl group replaced by hydrogen, so that it cannot undergo further glycolysis.As such, it acts to competitively inhibit the production of glucose-6-PO 4 from glucose at the phosphoglucoisomerase level [10].
2-DG is easily uptaken by the glucose transporters of the cell.Therefore, cells with higher glucose uptake, for example tumor cells, have also a higher uptake of 2-DG.
Inhibition of glycolysis by the small glycolysis inhibitors (GI) brought up to the use of combine usage of glycolysis inhibitors with chemotherapeutics in the treatment of malignant tumors therefore affectivity research of 2-DG -Chemotherapeutic combine treatments were recently started in clinical trials [11].
Due to higher glucose uptake of tumor cells, radiolabelled 2-DG ( 18 F-DG) is also routinely using for tumor imaging and staging with positron emitting tomography since 1990's (PET) [12].Even if alternative reactions can be used for 2-DG and boric acid complexation which enrolled in the study as alternative boron carrier; 2-DG and Boric Acid thought to be complexed rapidly and easily via low-high pH reactions due to poly-hydroxyl components of two molecules.Low-high pH reactions have been recommended in the literature for similar boric acid reactions [6].
If compare with low-high pH complexation reaction results, yield is very low for other reactions and reaction time is not reasonable.For example, nucleophilic substitution reaction is more widely used for 18F, 2-DG complexation reaction and electrophilic fluorination reaction has an important place in the synthesis of 18F-FDG.Synthesis of 18F-FDG in radio-fluorination reactions, triflates produces a moderate consistent yield at about 50 to 60% [13].
Boric acid reacts with polyhydroxyl compounds as a Lewis acid to form complex in aqueous solution.2-Deoxy-D-glucose has the 2hydroxyl group therefore simple pH complexation reaction was designed as defined in previous studies [6].
We have accomplished the effective and simple synthesis of ((2R)-4,5,6-trihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)boronic acid by the low-high pH reaction in high yields and short time.This method also could be applied to synthesis of (6-fluoro-2,4,5-trihidroxyoxan-3-yl) boronic acid.Complexation of B(OH) 3 with 2-DG was observed with FT-IR/ATR also proved with LC-MS and yield of complexation was measured by the HPLC.Peak areas of HPLC chromatograms confirmed that Borono-2-Deoxy-D-glucose synthesized with a higher consistent yield at about 80%.
In this work, due to successfully carrier properties, 2-DG thought to be alternative Boron carriers for BNCT application and boron ( 10 B) successfully complexed with 2-DG.

Table 2 .
Chromatographic conditions for LC-MS

Table 3 .
LC-MS/MS Spectrum (m/z) Values for Borono-2-Deoxy-D-glucose Compounds and Some Different Fragments and Proposed Structures of Selected Fragments