Drug Design, Development and Therapy

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Synthesis and characterization of a novel chemically designed (Globo)3–DTPA–KLH antigen This article was published in the following Dove Press journal: Drug Design, Development and Therapy 22 December 2014 Number of times this article has been viewed

Mehdi Hajmohammadi 1 Seyed Davar Siadat 2 Masoud Ghorbani 3,4,* Mehdi Shafiee Ardestani 5,* Shahram Teimourian 6 Vahid Asgari 3 Reza Ahangari Cohan 3 Mostafa Hajmohammadi 5 Akram Hajmohammadi 7 Ramezan Behzadi 8 Saied Rajab Nezhad 9 Nabiollah Namvar Asl 10 Department of Research and Biotechnology, 2Department of Microbiology, 3Department of Virology, Pasteur Institute of Iran, Tehran Iran; 4 Department of Virology, University of Ottawa, Ottawa, ON, Canada; 5 Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 6 Department of Medical Genetics, Iran University of Medical Sciences, Tehran, 7 Faculty of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, 8 Laboratory Animal Management of North Research Center, Pasteur Institute of Iran, 9Department of Research and Development, Alhavi Pharmaceutical, Tehran, 10Pasteur Institute of Iran, Department of Animal Sciences, Karaj, Iran 1

*These authors contributed equally to this work

Correspondence: Masoud Ghorbani Department of Virology, Pasteur Institute of Iran, Tehran–Karaj Highway, Tehran 31599, Iran Tel +98 26 1610 2999 Fax +98 26 1610 2900 Email [email protected]

Abstract: In recent years, many experiments have been conducted for the production and evaluation of anticancer glycoconjugated vaccines in developed countries and many achievements have been accomplished with Globo H derivatives. In the current experiment, a new chemically designed triplicate version of (Globo H)3–diethylenetriamine pentaacetic acid (DTPA)–KLH antigen was synthesized and characterized. Immunization with (Globo H) 3-DTPA-KLH, a hexasaccharide that is a member of a family of antigenic carbohydrates that are highly expressed in various types of cancers conjugated with DTPA and KLH protein, induced a high level of antibody titer along with an elevated level of IL-4 in mice. Treatment of tumors with the collected sera from immunized mice decreased the tumor size in nude mice as well. None of the immunized mice illustrated any sign of tumor growth after injection of MCF-7 cells compared to the control animals. These findings, based on the newly presented structure of the Globo H antigen, lend exciting and promising evidence for clinical advancement in the development of a therapeutic vaccine in the future. Keywords: (Globo H)3-DTPA-KLH, glycoconjugate vaccines, breast cancer

Introduction Cancer is the second leading cause of death after heart disease worldwide, and is considered a dire threat to human life, with a probability of death in more than 50% of cases. Since most cancers show no clinical symptoms at their early stages of development, diagnosis is very difficult or impossible until they become metastatic, when cure becomes virtually impossible. Therefore, developing a vaccine against cancers to produce strong antibodies against small micrometastatic cells could decrease the chance of development of the cancer.1,2 The design of therapeutic vaccines for the treatment of cancers is underway in many laboratories. These vaccines are intended to shrink tumor cells and prevent their regeneration, as well as delay or stop cancer cell growth.3 Normally, the immune system sees the cancer cells as normal cells within the body, and does not consider them as dangerous or foreign. Therefore, it does not conduct a strong attack against them.4 There are many factors that help cancer cells to escape from the immune system. Cancer cells carry the normal selfantigens that help them to hide, as well as releasing some chemical messages that may suppress the activated anticancer immune responses, which allows them to escape even after being recognized by the immune system.5 Recent studies have indicated that carbohydrates have an obvious and undeniable role in causing tumor malignancy. Carbohydrates on the surface of tumor cells are usually present as glycoprotein, mussing, and glycosphingolipid.1 Researchers have recently discovered the presence of detectable antibodies in the serum

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© 2015 Hajmohammadi et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php

http://dx.doi.org/10.2147/DDDT.S72530

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Hajmohammadi et al

of cancer patients. Their findings indicated a probable immunity and immune response against the cancerous factors and eventually cancer.6 In recent years, many experiments have been conducted for the production and evaluation of anticancer glycoconjugate vaccines, and many achievements have been accomplished.7 It appears that the production of an effective treatment vaccine is even more difficult than developing a cancer-preventive vaccine.8 It has been suggested that the ideal time for the immune system to respond against cancer cells could be the time immediately after the surgery and chemotherapy period, when the anticancer vaccine can train the immune system in order to identify and kill the remains of cancerous cells.6 If sufficient antibody titer is produced against antigens to omit tumor cells from the blood and lymphatic system and to kill micrometastatic cells, a noticeable change will appear in the curing methods of cancer patients. Several preclinical studies have been performed to investigate the production of antibodies against carbohydrate antigens on the surface of tumor cells in order to reduce tumor severity.10,11 A number of clinical experiments have shown that prescribing monoclonal antibodies is clinically efficient. 12,13 Naturally released antibodies against antigens on cell surfaces, especially carbohydrate antigens, have had a close relationship with the development of new cancer therapies at different clinical stages.14–16 So far, various studies have demonstrated that immunization against polysaccharide glycoconjugates is effective, while immunizing against polysaccharides alone is not.17–19 These findings indicate that polysaccharides could induce immunity, but need to be conjugated in order to be recognized by the immune system and provide immunity. Immunization with polyribosylribitol phosphate (PRP) induces immunity in children above 2 years old, whereas in children under 2 years old who are the target population for Haemophilus influenzae type B, PRP needs to be conjugated with another protein, such as tetanus toxin, for the production of PRP-T vaccine.20 Therefore, an effective vaccination against cancers probably requires tumor-specific glycoconjugates to provide strong immunity against tumor cells and their establishment in the body.9 One of the suggested proteins for conjugating polysaccharides to produce anticancer vaccines is the keyhole limpet hemocyanin (KLH) protein.21 KLH is a large, multisubunit, oxygen-carrying metalloprotein, and is found in

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the hemolymph of the giant keyhole limpet – Megathura crenulata. One of the most recently suggested anticancer vaccines is a vaccine against breast cancer, which is the most common cancer in women. “Globo” is one of the known polysaccharides on the surface of cancerous breast cells (MCF-7). Globo can be conjugated with KLH protein to provide a complex that could have an anticancer feature in the humoral immune system against breast cancer.22 Zhu et al23 have been able to stimulate the immune system to elevate IgG and IgM levels against Globo-KLH along with lysis of MCF-7 cells. So far, several derivatives of Globo-KLH vaccine have been derived and evaluated. In all cases, only a single polysaccharide chain of Globo has been used, while the appearance of Globo on the cancer cell surface is a triple chain. The aim of this project was to produce a newer form of Globo-KLH containing three sugar residues, similar to what appears in nature as a triple chain (Figure 1).

Materials and methods All animal protocols were reviewed and approved by the Pasteur Institute of Iran Animal Care Protocol Review Committee. All chemicals were purchased from Sigma-Aldrich, Canada, unless otherwise stated. Forty female CB6F1 mice were purchased from the Pasteur Institute of Iran Animal Care facility. CB6F1 hybrid mice are the F1 progeny result of a cross between female BALB/c (H-2d) and C57BL/6 (B6; H-2b) mice. Mice were divided into five groups of eight, and injected with different vaccine regimens.

Synthesis of (Globo)3–diethylenetriamine pentaacetic acid–KLH All substances were used without purification unless noted. Solvents were distilled under positive-pressure nitrogen stream: tetrahydrofuran (THF) with sodium benzophenone ketyl; ether with LiAlH4, CH2Cl2, and toluene; and benzene with CaH2. All reactions were under N2 pressure. Column chromatography was performed using either silica gel 60 (40–63 μm) (Merck, Ottawa, Canada) or H-type silica gel (10–40 μm) for the normal phase. For the inverse phase, we used LiChroprep RP-18 (12–25 µm) (catalog number 113901; EMD Millipore, USA).

Synthesis of 3-6-di-O-benzyl-d-glucal Both d-glucal (8 g, 54.7 mmol) and (Bu3Sn)2O (30.7 mL, 10 M equivalent) were refluxed for 20 hours in dry C6H6 (150 mL)

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(Globo H)3-DTPA-KLH antigen O CH3

Bn O Bn O

O

O

O

O O Bn

O O O

O TIPS

O

N

O OH PhOSHN

N

O O TIPS

EDC O

O Bn Bn O O

Bn O

Bn O

O

COOH

N

N

O

Bn O

S

O

O

O Bn

O O Bn O

O

O Bn

O Bn O

O Bn Bn O O O

O Bn Bn O

Bn O

O

O Bn

O

O

O Bn

Bn O

O

NHSOPh O O

TIPS O

O Bn O Bn O

CH3

O O

TIPS O O

O O

O

COOH HN O CH3

Bn O Bn O

O O

N

O O Bn

O O

O TPIS NH O O TIPS O O

O O PhOSHN

O O TIPS O O Bn

Bn O O

Bn O Bn O

O O O

O H3C

NH S O Bn O Bn O O BnO O TIPS O O NHSOPh

CH3

Bn O

O Bn O O Bn O O Bn O O Bn

O O

O Bn

O Bn O Bn

O O Bn

O O Bn

O Bn O

O

O Bn

Figure 1 Suggested chemical structure of a stronger derivation than Globo H KLH.

with a Dean–Stark trap apparatus. The mixture was cooled to below boiling point and treated with benzyl bromide (BnBr) (21 mL) and tetra-n butylammonium bromide (TBABr) (35.3 g). The mixture was refluxed for 17 hours and cooled down and dried by evaporation. The residue was then diluted by ethyl acetate (EtOAc; 2×20 mL), and

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washed using distilled water (3×300 mL). It was then filtered, condensed, and dried by Na 2So4. The column chromatography of raw materials was performed using 15%–20% EtOAc in hexanes, which resulted in the desired product (11.66 g, with 65% purity) in the form of a colorless oily residue.

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Synthesis of 1,5-anhydro-6-O-(TIPS)2-deoxy-d-lyxo-hex-1-enopyranose Under a stream of nitrogen (N2), 6.1 g triisopropylsilyl chloride (TIPSCl; 31.8 mmol), 4.2 g d-glucal (28.8 mmol), and 4.3 g imidazole (63.2 mmol) was added to 20 mL di­methylformamide (DMF) at 0°C and mixed appropriately. The mixture was then kept at room temperature and stirred for 24 hours, followed by the addition of 250 mL EtOAc and 250 mL distilled water. After the separation of layers, the blue (aqueous) layer was extracted by the addition of more EtOAc (2×250 mL). The organic layer was dried later using Na2SO4, filtered, and concentrated under vacuum evaporation. The residue was then purified using column chromatography (eluent, 15%–30% EtOAc:hexane) to obtain the desired compound in the form of a light-yellow viscous solution.

Synthesis of 1,5-anhydro-6-O-(TIPS)3,4-O-carbonyl-2-deoxy-d-lyxo-hex-1enopyranose The aforementioned solution (2.4 g, 14.8 mmol) was then mixed with 2.4 g carbonyldiimidazole (14.8 mmol) in 40 mL dry THF under N2 pressure. Three imidazole crystals were then added and the mixture stirred for 30 minutes, followed by the addition of 1.2 g carbonyldiimidazole (7.4 mmol). The mixture was stirred for 90 more minutes. After evaporation of the solvent under vacuum, the residue was mixed with 200 mL chloride and 100 mL distilled water. After separation of the organic layer, it was washed with 100 mL water, filtered, dried, and condensed using Na 2SO 4 under vacuum. The residue was then purified by column chromatography using a 15% EtOAc:hexane mixture to obtain the desired compound in the form of a clear syrup.

Synthesis of lactal carbonate 1 The syrup (22 mg) was mixed with 5 mL methylene chloride, followed by the addition of 30 mL dimethyldioxirane (prepared on the same day, as explained by Robert et al)24 slowly in drops at 0°C. The mixture was then stirred for 1 hour at 0°C and analyzed by thin-layer chromatography to verify the complete expansion of the compound. Then, the mixture was condensed under vacuum and azeotroped using 5 mL benzene. Then, 11.6 g of 3-6-di-O-benzyl-dglucal solution was added and the mixture azeotroped again with 2×5 mL benzene, concentrated under vacuum, and dried using 1.5 mL THF under N2 pressure. A zinc chloride solution (820 μL of a 1 M solution in THF, 0.82 mmol) was

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added slowly in drops and the mixture returned to room temperature and kept for 24 hours. The mixture was then added to 10 mL NaHCO3 (1 M) solution and extracted by EtOAc (3×20 mL). The extracted organic compound was then filtered, condensed, dried by Na 2SO4, and purified by column chromatography (gradient elution, 10%–25% EtOAc:hexanes) to obtain the desired compound.

Synthesis of lactal carbonate One milliliter of a 60% solution of NaOH in water was added to 30 mL DMF containing 2.7 g of lactal carbonate 1 and 574 µL BnBr at 0°C, followed by stirring for 5 minutes. The mixture was then kept at room temperature and stirred for 2 hours. The total mixture content was later transferred to 70 mL cold distilled water, diluted by EtOAc, washed twice with 70 mL distilled water and once with 70 mL salt water, filtered and dried by Na2SO4, followed by column chromatography of raw materials using 10%–12% EtOAc in hexanes to obtain the desired compound (2.479 g) with purity of 81% in the form of a colorless oil (Figure 2).

Synthesis of lactal A lactal carbonate solution of TIPS (5.62 mmol, 4.28 g) was treated with 6.75 mL tetra-n-butylammonium fluoride (TBAF; 1.0 M equivalent, 1.0 M) solution mixed with 25 mL THF and 5 mL MeOH. After 6 hours of incubation at room temperature, 4 mL more TBAF was added and the mixture stirred for an additional 3 hours. The mixture was then condensed and subjected to direct chromatography using 4:1 EtOAc-hexane solution to obtain 2.20 g triol. The remains of the solution contained cyclic carbonate and a mixed carbonate mixture that was hydrolyzed in methanol using MeONa (1.0 mL, 25 wt% in MeOH) and purified by chromatography. The total productivity was 3.02 g (93%). The final product was then used directly in the dibenzylation stage.

Synthesis of disaccharide A mixture containing 2.95 g thioglycol (5.1 mmol), 1.33 g Bu2SnO (1.05 equivalent), and 1.69 mL (Bu3Sn)2O (0.65  equivalent) was refluxed for 5 hours in dry C 6H6 (50 mL) under N2 pressure until all the water was evaporated. The mixture was then cooled to under boiling point, treated with 2.43 mL BnBr (4.0 molar equivalent) and 3.29 g TBABr (2.0 equivalent), and refluxed with 10 mL C6H6 for 16 hours. The mixture was loaded directly on a silica bar and eluted by 15%–20% EtOAc-hexanes to obtain the desired compound (3.48 g) with 91% purity in the form of clear oil.

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(Globo H)3-DTPA-KLH antigen

OH

OH

O TIPS

OH O TIPS Imidazol DMF

HO

O HO

O TIPS O

O 1,2-Carbonyldiimidazole cat. DMAP, CH2CI2, quanti

O

O

3,3′–dimenthyldioxiran CH2Cl2 OH

OH

O

OH

O

(n-Bu3Sn)2O

O

TBIBr BnBr PhH

HO

O TIPS

O Bn

O

Bn O

O

ZnCl2 THF O Bn

O TIPS O

O

O O

O O Bn O

OH

BnBr NaH DMF

O Bn

O TIPS O

O

O O

OH

O Bn O

O Bn

O Bn

O Bn

O Bn

O O Bn O

O

O

OH (n-Bu3Sn)2O PhH BuSnO then TBABr BnBr

O O Bn O TBAF THF then MeOH MeONa

O HO

O Bn

OH

O Bn

O O Bn O

Figure 2 Synthesis of lactal carbonate.

Synthesis of 3-O-(4-methoxybenzyl)-dglucal A suspension of 3.70 g d-glucal (25.3 mmol) and 6.30 g Bu 2SnO (1.0 equivalent) was refluxed for 5 hours in

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dry C6H6 (50 mL) under N2 pressure until the water was evaporated completely. The mixture was cooled down to room temperature and treated with 9.1 g p-methoxybenzyl chloride (1.1 equivalent) and 9.1 g TBABr (1.1 equivalent,

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9.10 g) and refluxed for 4 hours. The mixture was then filtered through a silica bar and eluted with EtOAc:hexanes (4:1). Fractions containing product were condensed and the residues crystallized in hexane to obtain a white solid crystal as product (4.5 g, approximately 70%).

with dimethyldioxirane (0.09 M, 80 mL) under N2 pressure at 0°C. The mixture was stirred until the whole glucal was expanded using thin-layer chromatography containing 30% EtOAc in hexanes. The solution was deazeotroped under N2 pressure at 0°C. The residue was then dissolved in dry THF (30 mL) under N2 pressure at 0°C, followed by treatment with 36 mL TBAF (stored over molecular sieves) and stirred for 20 hours at room temperature. The dark-brown solution was filtered through a silica pad (~4 cm depth) and washed with 200 mL EtOAc, followed by washing twice with 200 mL distilled water, and dried with MgSO4. The residue was then dissolved in 30% EtOAc in hexanes (50 mL) and filtered through a silica bar (10 cm diameter, 4 cm length), and washed again with 1 L of the same solvent. The filtrate was condensed to obtain fluorohydrine in high purity. The residue was then dissolved in dry DMF (30 mL) under N2 pressure at 0°C, treated with BnBr (1.5 equivalent, 958 µL), filtered through basic alumina, and finally was stirred for 30 minutes at 0°C followed by 30 minutes’ stirring at room temperature with NaOH (1.5 equivalent, 60% dispersion, 322 mg). The reaction was stopped after the mixture was poured on 100 g of ice, and the mixture was extracted by EtOAc-hexanes 1:1 (2×150 mL). Column chromatography was performed using 10% EtOAc in hexanes, which resulted in obtaining 2 g (49%) of desired compound in the form of a light-yellow liquid (Figure 3).

4,6-Di-O-benzyl-3-O-(4-methoxybenzyl)d-glucal synthesis A mixture of 3-O-(4-methoxybenzyl)-d-glucal (8.56 mmol, 2.28 g) and BnBr (3.68 molar equivalent, 3.75 mL) solution was prepared and passed through basic alumina treated by NaH 60% (4.0 molar equivalent, 1.37 g) in dry DMF (30 mL) under N2 pressure at 0°C. The mixture was stirred for 30 minutes at 0°C and for 1 hour at room temperature. The mixture was then poured on ice pieces, diluted by 100 mL of distilled water, and extracted by EtOAc-hexanes (1:1, 3×100 mL). Organic extracts was washed by water (2×100 mL) and dried on Na2SO4. Column chromatography of raw materials was performed using 15% EtOAc-hexanes, and resulted in obtaining the desired product in the form of a clear liquid.

Synthesis of the compound containing fluoride A mixture of 4,6-di-O-benzyl-3-O-(4-methoxybenzyl)-d-glucal (7.7 mmol, 3.20 g) solution in dry CH2Cl2 (10 mL) was treated OH

OH

n-BuSnO

O

TBABr PMBCI PhH

HO

OH

OH

BnBr

O

NaH DMF

PMB O

O Bn

O Bn

O PMB O

3,3′-dimethyldioxirane CH2Cl2

O Bn

O Bn

O Bn

BnBr

O PMB O

F O Bn

O Bn

NaH DMF

TBAF

O PMB O

F OH

O Bn

O Bn

O

THF PMB O O

Figure 3 Synthesis of fluoride.

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(Globo H)3-DTPA-KLH antigen

Synthesis of the first trisaccharide containing para-methoxybenzyl

Synthesis of the first trisaccharide without para-methoxybenzyl

The synthesized lactal (1.0 equivalent, 0.791 mmol, 600 mg) and fluorosugar (1.5 equivalent, 679 mg) were synthesized in ether, condensed, dried in vacuum for 2 hours, treated by di-tert-butylpyridine (1.0 equivalent, 177 mL), and dissolved in dry ether (8.5 mL) under N2 pressure. In a separate 25 mL flask, 2 g of 4 Å molecular sieves was added and dried by heat under vacuum and cooled to room temperature. Anhydrous silver perchlorate (1.0 equivalent, 163 mg) and SnCl2 (1.0 equivalent, 150 mg) was added to molecular sieve. The salt mixture was placed in a water bath, and the sugar solution was added to the salt using a double-tipped needle and stirred for 48 hours at room temperature. The mixture was diluted by ether and filtered through a pad of silica, followed by washing with ether. The filtrate (70 mL) was diluted twice with 50 mL sodium bicarbonate solution and dried under Na2SO4. Column chromatography was performed twice using 20% EtOAc in hexanes to obtain 561 mg trisaccharides with a purity of 54%.

A para-methoxybenzyl trisaccharide solution containing 0.428 mg trisaccharide (561 mmol) was treated with 830 μL of distilled water and 126 mg 2,3-dichloro-5,6-dicyano1,4-benzoquinone (1.3 equivalent) at 0°C and stirred for 1 hour at 0°C. The mixture was transferred to 50 mL saturated sodium bicarbonate solution, extracted by EtOAc (2×50 mL), washed with water and saturated sodium bicarbonate solution (2×50 mL), dried with Na2SO4, condensed under vacuum, and purified using column chromatography as previously described to obtain 437 mg of the desired trisaccharide in the form of a colorless oil with 86% purity (Figure 4).

O Bn

O Bn

Glycal disaccharide synthesis TIPS galactal carbonate (3.14 mmol, 4.32 g) was dissolved in 20 mL of CH2Cl2 and cooled to 0°C. The mixture was then mixed with 219 mL dimethyldioxirane (~3.14 mmol) and incubated at 0°C for 20 minutes, followed by condensation using a stream of nitrogen. The residue was then treated with O Bn

OH

O Bn

O PMB O

O F

Bn O

O Bn

Bn O

AgClO4, SnCl2, di-tert-butylpyridine, 4Å, molecular sieves, Et2O

O Bn

O Bn

O Bn

O O

O PMB O O Bn

O Bn O

O Bn O

O Bn O

O Bn

O Bn

O Bn O

DDQ CH2Cl2 H2O

O Bn O

HO O Bn O

O Bn O Bn O

Bn O

O Bn

O O Bn O

Figure 4 Synthesis of trisaccharide.

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20 mL C6H6 and dried by high vacuum power for 30 minutes at 0°C. It was then dissolved in 60 mL THF and cooled to -78°C, followed by the addition of 3.32 g (10.95 mmol) dry TIPS galactal carbonate and 26.3 mL ZnCl2 (1.0 M in ether). The mixture was kept at room temperature and stirred for 24 hours. It was then treated with 40 mL saturated NaHCO3 and extracted with 500 mL ether. The combined organic phase was washed with 300 mL salt water, dried by MgSO4, and the raw product was purified by column chromatography using 20% EtOAc in hexanes. The resulting product was 6.20 g white foam.

Ethyl-thio-sulfonamide synthesis

Glycal trisaccharide synthesis

Hexasaccharide synthesis

A mixture of disaccharide (4.08 mmol, 2.64 g) and fucosyl fluoride (3.77 mmol, 1.64 g) was deazeotroped three times by 10 mL C6H6 and dried by high vacuum power for 1 hour. The residue was then dissolved in 20 mL THF and treated with 2,6-di-tert-butylpyridine (11.31 mmol, 2.16 g). The aforementioned mixture was later added to a flask containing AgClO4 (7.54 mmol, 1.56 g), SnCl2 (7.54 mmol, 1.42 g), and 4 Å molecular sieves (4.0 g), and stirred for 30 minutes at 40°C for 34 hours. After the mixture was treated with 40  mL saturated solution of NaHCO3 at 4°C, the mixture was extracted twice with 300 mL EtOAc. The contents of the organic layer were then washed with 200 mL salt water and dried by MgSO4. The residue was then purified using column chromatography as stated earlier to obtain 1.93 g of 47% purified glycal trisaccharide (Figure 5).

An acceptor mixture containing trisaccharides (1 equivalent, 0.077 mmol, 92 mg), thioglycoside (2 equivalent, 198 mg), and activated molecular sieve (560 mg) suspended in Et2OCH2Cl2 (2:1) under N2 flow was stirred at room temperature for 10 minutes. The mixture was then cooled to 0°C and treated with 52.4 μL methyl triflate (6.0 equivalent). The mixture was then stirred for 4 hours at 0°C and for 1.5 hours at about 15°C (mild temperature). The reaction was later stopped using 10 mL triethylamine. The mixture was then washed and filtered through a silica pad using diethyl ether. The filtrate (70 mL) was washed again with 2×50 mL saturated NaHCO3 solution, dried with Na2SO4, and purified by high-performance liquid chromatography (HPLC) using a MICROSORB Semi-prep Si 80-120-C5 column (Bio-Rad, California, USA), 17% EtOAc in hexanes, at a flow rate of 15 mL/minute and optical density of 260 nm to obtain 158 mg (85% purity) of the desired product.

Synthesis of iodosulfonamide A mixture of glycal trisaccharide (0.11 mmol, 118 mg) and benzenesulfonamide (0.55 mmol, 87 mg) was deazeotroped once by C6H6, dried by high vacuum for 1 hour, and dissolved in 5 mL THF, and then 870 mg freshly activated 4 Å molecular sieves (provided from 4 hours heating in oven at 250°C) was added. I2 (0.22 mmol, 57.5 mg) was sterilized by 100 mg Ag(sym-coll)2 ClO4 (0.22 mmol) in 1 mL THF at room temperature until the brown color of iodine had disappeared and I(sym-coll)2 ClO4 was obtained. It was then added to the flask containing glycal trisaccharide and benzenesulfonamide at 5°C. The mixture was stirred for 1 hour at 5°C and cooled using 10 mL Na2S2O3 solution. After filtration and extraction by EtOAc (80 mL), the organic layer was washed with 20 mL deuterium-depleted water, saturated with 20 mL Cu2SO4, dried by MgSO4, and purified by column chromatography using silica gel (20% EtOAc in hexanes) to obtain 74 mg iodosulfonamide as the result.

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Lithium bis(trimethylsylil)amide (1.0 M in THF, 0.81 mL, 0.81 mmol) was added to 10 mL DMF containing etiolate Ethan solution (1.7 mmol, 102 mg) at -42°C. After 5 minutes’ stirring, it was added to a flask containing iodosulfonamide (0.33 mmol, 448 mg) in 10 mL DMF at -42°C. The mixture was then transferred to room temperature and stirred for another 3 hours. It was then diluted with 200 mL diethyl ether, washed with 10 mL salt water and saturated with 1 N NaHCO3 2×10 mL and dried by MgSO4. The desired product was obtained in the form of a white solid (Figure 6).

Synthesis of azide A mixture of hexasaccharide glycal and freshly activated 4 Å molecular sieve were treated with 1mL dimethyldioxirane (0.07 M) in 1 mL CH2Cl2 at 0°C under nitrogen stream, and the residue was dried for 20 minutes under high vacuum power. Then, an azidohydrin solution was added to epoxide through a container in THF (0.8 mL) and cooled to -40°C. Then, ZnCl2 in Et2O (1 M, 25 μL) was added, the mixture allowed to cool to room temperature, and then stirred for the next 12 hours. Then, the mixture was diluted by EtOAc (50 mL), was washed with NaHCO3 saturated solution (2×20 mL) and salt water (10 mL), dried by Na2SO4, and the raw material purified by column chromatography (10%–22% EtOAc in hexanes) to provide 31.3 mg of coupled product in the form of a clear oil. To the solution of this product (0.013 mmol, 31.3 mg), 4-dimethylaminopyridine (0.013  mmol, 1.6  mg) and triethylamine (0.0219 mmol,

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(Globo H)3-DTPA-KLH antigen O TIPS O

O TIPS

OH

O

O

O

O

HO O

ZnCl2 THF

O TIPS O

O

O O OH

F

O TIPS

OH

H3C

O

O O Bn

O

O Bn

AgClO4 SnCl2 di-tert-butylpyridine

THF

O Bn

O TIPS CH

O

O

O

O TIPS O

O O

O H3C

O O Bn O Bn

O Bn

I(coll)2ClO4 PhSO2NH2 4Å molecular sieves THF O TIPS

OH

O

O

O

O TIPS O

O O

O

NHSO2Ph

H 3C

O O Bn O Bn

O Bn

Figure 5 Synthesis of iodosulfonamide.

3.0 μL) was added in CH2Cl2 (1 mL) and Ac2O (0.013 mmol, 1.2 μL) at 0°C. The mixture was allowed to cool to room temperature and stirred for 1 hour. After the solvent had been evaporated, the residue was diluted by EtOAc (30 mL) and was washed with NaHCO3 diluted solution (285 mL) and salt water and dried by Na2SO4. Column chromatography (20% EtOAc in hexanes) of raw materials provided 30 mg (95%) of the desired product (Figure 7).

stirred for 24 hours at room temperature under H2. The reaction mixture was filtered through a silica pad, washed with 20 mL EtOAc, and concentrated under vacuum. The residue was then purified by HPLC using 20% EtOAc in hexanes at a flow rate of 15 mL/minute and detected at 260 nm wavelength using an ultraviolet detector. The final product was 64 mg colorless oil with 90% purity (Figure 8).

Synthesis of amide

Synthesis of the final product of (Globo)3DTPA-KLH

A mixture containing 66 mg azide (0.023 mmol), 66 mg Lindlar catalyst, and 23 mg palmitic anhydrate (0.46 mmol) was

Approximately 1 mmol of KLH (3 g) was mixed with 2 mmol of S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine

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Hajmohammadi et al O TIPS OH

O

O TIPS

O

O

O

O O

O

NHSO2Ph H3C

O O Bn O Bn

O TIPS

OH

O Bn

O

O

N SO2Ph SEt

or

EtSH, LHMDS, DMF OH

O TIPS O

O HN Ph

O

S O

O TIPS OH

O O

O

O TIPS O

O SEt

O

O

NHSO2Ph H 3C

O O Bn O Bn

O Bn

Figure 6 Synthesis of ethyl-thio-sulfonamide.

pentaacetic acid (p-SCN-Bn-DTPA) in phosphate-buffered saline (PBS) and stirred for 24 hours. The mixture was then transferred to a dialysis bag for separation of SCN-DTPAKLH. The remainder was then added to a mixture containing 10 mL of dissolved DMF, acetic acid anhydride (C4H6O3), and 10 mg pyridine while stirring for 3 hours at 40°C, until the formation of anhydrate. Ten milligrams of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide plus 2 mL triethylamine were then added to the mixture, the contents placed on ice for 2 hours, and kept at room temperature afterward. The synthesized Globo was later dissolved in 10 mL DMF and the pH adjusted to 10.5 using 1 normal NaOH. After the addition of 125 μL of cyanogen bromide solution (0.2 g in 1 mL H2O) to acetonitrile, the pH was adjusted to 10.5. After 2.5 minutes’ incubation at room temperature, 5 mL of 0.5 molar adipic dihydrazide as well as 5 mL bicarbonate (0.5 molar) were added to the mixture and the pH adjusted to 8.5 using 1 N HCl and kept at 4°C for 24 hours. The activated Globo was then added to the anhydrate and stirred for 2 hours. The 226

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conformation of the final product was later determined using 1 H nuclear magnetic resonance (NMR), Fourier-transform infrared, and HPLC techniques.

Animal and biological assays

Immunization of mice with vaccine regimens Mice were divided into five groups of eight, and received different vaccine regimens in 200 µL via intraperitoneal injections. Each mouse received the vaccine four times at 2-week intervals. Group 1 received (Globo)3-DTPA-KLH, group 2 (Globo)3-DTPA-KLH + adjuvant, group 3 was injected with DTPA-KLH as negative control, group 4 received DTPAKLH + adjuvant, and group 5 was injected with PBS alone. All animals were monitored for 4 months. All mice were bled prior to the beginning of the experiment and after each immunization. The blood was collected from the saphenous vein in an Eppendorf tube and centrifuged at 1,500 rpm for 15 minutes. The sera were collected and kept at -20°C until use. Drug Design, Development and Therapy 2015:9

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(Globo H)3-DTPA-KLH antigen O Bn O Bn

O TIPS O O

OH

O

SEt NHSOPh

O

O O

HO

O

O

H3C

O

O TIPS

Bn O

O Bn

O Bn O

Bn O

O Bn

O Bn O Bn

O

O

O Bn O

O Bn

MeOTf, 4Å molecular sieves, Et2OCH2Cl2(2:1)

O TIPS

O O

OH

O

O O

H3C

O O Bn O Bn

O TIPS Bn O O

O

O

O Bn Bn O

O O Bn Bn O

O

(b) Ac2O, DMAP, Et3N, CH2Cl2

N3 (CH2)12CH3

O Bn

H3C

O Bn

O Bn O

HO

O

O O Bn O

NHSOPh

(a) (i) 3,3′-dimethyldioxirane, 4Å molecular sieves, CH2Cl2; (ii)

O Bn

O TIPS

O

O

O

O O

O Bn O Bn

O

ZnCl2, THF

OH O TIPS Bn O O O NHSOPh

O Bn O O Bn O

O Bn Bn O

N3

O Bn

O Bn O O O Bn Bn O

O O Ac

(CH2)12C H3

O

O Bn

Figure 7 Synthesis of azide.

Antibody titration

In vivo tumor-preventive effects

The level of antibodies in the mice sera was detected using enzyme-linked immunosorbent assay (ELISA). The wells of a 96-well plate were coated with either 0.25 µg/well of bovine serum albumin or Globo-DTPA-albumin conjugate separately and incubated overnight. All wells were blocked with 1% skim milk for 1 hour at 37°C. A serial dilution of polled sera (1:50, 1:100, 1:300, 1:900, 1:2,700, and 1:8,100) from each group of mice was prepared, added to the wells, and incubated at 37°C for 1 hour. The plate was then washed three times with PBS containing 0.1% Tween 20. The antibodies were detected using a rabbit antimouse antibody conjugated with horseradish peroxidase at a concentration of 1:1,000 and incubated for 1 hour at 37°C. The plate was then washed three times and 100 μL TMB added to each well and incubated for 15 minutes in the dark. The reaction was stopped by the addition of 100 μL of 0.5 M sulfuric acid to each well, and light intensity was read at 450 nm wavelength using a Bio-Rad ELISA reader.

To investigate the antitumor effect of the antibodies in the sera of immunized mice, tumors were developed in two groups of four nude mice (purchased from the Pasteur Institute of Iran), and 20×106 MCF-7 cells were injected into the right limb of each mouse. After the tumor size reached approximately 1 cm, 200 μL of the pooled sera from either the vaccinated mice or the control group was injected around the tumor site in each nude mouse subcutaneously. The tumor size and growth were then monitored during the next 4 weeks.

Cytokine assay Both IL-4 and IFNγ were detected using ab100710–IL-4 (IL-4) and ab46081-IFNγ mouse ELISA kits (Abcam, Cambridge, MA, USA), respectively, for the detection of cytokines in collected sera from immunized mice. Drug Design, Development and Therapy 2015:9

Statistical analysis

Results are presented as means ± standard error of mean. We used InStat software for analyses of variance, followed by the Student–Newman–Keuls post hoc test. Significant differences are based on P0.05.

Results Confirmation of the synthesis of Globo H and Globo H-protected precursor H-NMR (solvent, CDCl3)

1

δ7.67 (2H, d, J =7.2 Hz), 7.35–7.13 (63H, m), 5.65 (1H, d, J =9.0 Hz), 5.63–5.57 (1H, m), 5.43 (1H, br s), 5.33–5.27 (1H, m), 5.11 (2H, t-l1ke, J =4.0 Hz), 4.94–4.90 (2H, m), submit your manuscript | www.dovepress.com

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Hajmohammadi et al

O

O TIPS

O

OH O TIPS O Bn O Bn O O O O Bn NHSO2Ph O

O

O

O

O

H 3C

O

O Bn O Bn

O Bn

Bn O

O Bn O O O BnBn O

N3

O Bn O

(CH2)12 CH3

O O Ac

O Bn

Lindlar’s catalyst, H2, palmitic anhydride, EtOAc

O

O

O TIPS

O

O

O

OH O TIPS O Bn O Bn O O O NHSO Ph O BnO

O

2

O

Bn O

H 3C

O O Bn O Bn O Bn

H O Bn O O O BnBn O

O Bn

N

(CH2)14CH3

O

(CH2)12CH3

O O Ac

O Bn

(a) (i) TBAF, THF; (ii) NaOMe, MeOH, (b) (i) Na, NH3, THF; (ii) Ac2O, Et3N, DMAP, CH2Cl2 (c) NaOMe, MeOH, quantitative

OH HO

OH O

OH O

O H3C

O OH OH

O

OH O

NHAc

HO O

OH O

HO

O

HO

H OH O O OH HO

OH

(CH2)14CH3

N

O

(CH2)12CH3

O OH

OH

OH Figure 8 Synthesis of antigen.

4.88–4.79 (4H, m), 4.76–4.73 (2H, m), 4.71–4.64 (5H, m), 4.62–4.58 (3H, m), 4.56–4.49 (5H, m), 4.44 (1H, dd, J  =8.6 and 2.2 Hz), 4.40 (1H, d, J =7.0 Hz), 4.36–4.32 (2H, m), 4.30–4.11 (9H, m), 4.02–3.88 (7H, m), 3.81–3.65 (8H, m), 3.60–3.50 (4H, m), 3.45–3.36 (4H, m), 3.34–3.23 (5H, m), 3.09 (1H, br s), 2.10–1.98 (4H, m), 2.04 (3H, s), 1.83 (3H, s), 1.51 (2H, m), 1.30–1.23 (49H, m), 1.10–0.97 (42H, m), 0.88 (6H, t, J =6.9 Hz). 13

C-NMR (solvent, CDCl3)

δ172.4, 169.6, 168.5, 153.4, 140.5, 139.3, 139.2, 138.7, 138.6, 138.5, 138.4, 138.3, 138.20, 138.1, 138.0, 136.8, 132.3, 128.9, 128.5, 128.3, 128.3, 128.2, 128.1, 127.9, 127.8, 228

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127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1,127.0, 103.2, 101.1, 99.0, 98.4, 98.1, 79.4, 79.3, 78.8, 77.4, 76.5, 76.3, 76.0, 75.8, 75.2, 74.8, 74.8, 74.3, 74.2, 74.1, 73.5, 73.1, 73.0, 72.9, 72.7, 72.4, 72.3, 72.2, 70.3, 70.2, 69.5, 68.7, 68.3, 68.1, 68.0, 67.9, 67.7, 61.2, 60.9, 53.6, 53.3, 51.5, 36.8, 32.2, 31.9, 29.6, 29.5, 29.4, 29.3, 29.2, 25.6, 22.6, 20.9, 20.7, 18.0, 17.9, 17.9, 17.9, 16.6, 14.0, 11.8 (Figure 9).

Globo H (ceramide)

Fourier transform infrared spectroscopy (FTIR) data (KBr) 3,356.16, 2,920.73, 2,851.92, 1,734.56, 1,652.90, 1,547.93, 1,466.19, 1,372.94, 1,260.95, 1,075.45, 1,042.58 cm-1. Drug Design, Development and Therapy 2015:9

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(Globo H)3-DTPA-KLH antigen

H NMR (solvent, DMSO-4/D2O, 10:1)

N% calculation = (14.0087× nitrogen numbers/MW) ×100

1

δ5.54–5.48 (dt, J =6.7, 15.3 Hz, 1H), 5.37–5.31 (dd, J =7.0, 15.2 Hz, 1H), 4.95 (bs, 1H), 4.81 (d, J =3.7 Hz, 1H), 4.48 (d, J =8.4 Hz, 1H), 4.44 (d, J =8.1 Hz, 1H), 4.25 (d, J =7.6 Hz, 1H), 4.16 (d, J =7.8 Hz, 1H), 4.10–4.04 (m, 2H), 3.98–3.85 (m, 5H), 3.81–3.72 (m, 6H), 3.68–3.35 (m, 22H), 3.32–3.25 (2H, m), 3.04–3.02 (t, J =7.9 Hz, 1H), 2.02 (m, 2H), 1.93 (m, 2H), 1.82 (s, 3H), 1.44 (m, 2H), 1.23 (m, 46H), 1.08 (d, J =6.4, 3H), 0.84–0.83 (t, J =6.6, 6 H).

Calculation of C, H and N numbers from KLH-CHN analysis obtained the data: C:26.73, H:4.12, N:4.35 (reported from CHN analyzer instrument).

C-NMR (solvent, CD3OD)

13

δ176.0, 174.5, 135.2, 131.4, 105.5, 105.4, 104.4, 103.9, 102.8, 101.0, 81.1, 80.4, 80.0, 79.1, 77.9, 76.8, 76.5, 76.4, 76.2, 75.5, 74.9, 74.7, 73.5, 73.0, 72.6, 72.4, 71.5, 70.6, 70.6, 70.3, 69.9, 69.7, 69.7, 68.1, 62.6, 62.6, 61.7, 61.6, 54.8, 54.8, 53.1, 37.4, 33.5, 33.1, 30.9, 30.9, 30.8, 30.8, 30.8, 30.7, 30.7, 30.6, 30.5, 30.5, 30.4, 27.2, 23.8, 23.7, 16.7, 14.4.



KLH MW = ~3,000 KDa



C-numbers =26.73×3,000,000/1,201.07=~66,765



H-numbers =4.12×3,000,000/100.79=~122,631



N-numbers =4.33×3,000,000/1,400.87=~9,268

Calculation of C, H, and N numbers from (Globo)3-KLHCHN analysis obtained the data: C:26.79, H:4.13, N:4.33 (reported from CHN analyzer instrument). We knew that in a conjugate, there is an increase in C, H, and N numbers as follows, respectively. In other words, we expected an increase in CHN numbers as: C =258, H =484, and N =22 (new MW = KLH MW +~5.7 kDa).

Mass-spectrum data [M+]=1,532.6, [M+Na]=1,555.8, [M+2H]2+=778.95, [M+3H]3+=519.6 (Figure 10; original data in Supplementary materials).

(Globo H)n=1,3-p-Bn-SCN-DTPA-KLH synthesis confirmation HPLC procedure



C-numbers =26.79×3,005,700/1,201.07=~67,042



H-numbers =4.13×3,005,700/100.79=~123,162



N-numbers =4.33×3,005,700/1,400.87=~9,292

To confirm the attachment of Globo3 to the complex of DTPA-KLH, we used HPLC (Table 1).

%C-numbers = (67,042–66,765/258) ×100–100=7.3%

CHN analysis



Carbon, hydrogen, and nitrogen analysis showed the total mass of each element residue in the complex, as indicated in Table 2.

The data statistically confirmed the synthesis of our conjugates.

%H-numbers = (123,162–122,631/484) ×100–100=10.0% %N-numbers = (9,292–9268/24) ×100=9.0%

H NMR study

1

C% calculation = ( 12.0107× carbon numbers/molecular weight [MW] ×100

As the original results depict in the Supplementary materials, there was a significant sharp peak regarding the anomeric hydrogens of Globo H polysaccharide

H% calculation = (1.0079× hydrogen numbers/MW) ×100 O

O

O TIPS O

O

A cO O

Bn O

O

O PhSO2HN

O H 3C

O TIPS

O Bn

O Bn

O Bn O

O Bn O

Bn O

Bn O

O

O Bn O O Bn

O Bn O Bn O

O

O Ceramide

O Ac

Figure 9 Protected antigen.

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Hajmohammadi et al HO

OH

HO OH

O

O

HO

O O

O

HO

O NHAc

Table 2 Atomic mass for carbon, hydrogen, and nitrogen elements

OH O HO

O OH

OH

O

HO

H OO H

OH

OH O HO

O O Ceramide OH

Element

Atomic massa

C H N

12.0107 1.0079 14.0087

Figure 10 The molecular structure of Globo-H antigen. Notes: C:26.79, H:4.13, N:4.33 (reported from CHN analyzer instrument).

Note: aFrom periodic table.

(4.5–5.5  ppm) in (Globo H) 3-DTPA-KLH and Globo H-KLH with an integral ratio of 3:1, which confirmed the presence of 3 M Globo H in the conjugate of (Globo H)3DTPA-KLH as well.

Tumor growth

Discussion

A significant total of IgM and IgG antibodies was observed only against (Globo H)3-DTPA-KLH and Globo H-KLH compared to other groups (Figure 11). There was also a significant difference (P0.05) between (Globo H)3-DTPAKLH and Globo H-KLH. In general (Globo H)3-DTPA-KLH was found to be more potent than Globo H-KLH.

Cytokine assay IL-4 and IFNγ levels were determined in the collected sera after each injection for all groups. Surprisingly, the cytokines were only detected in the mouse group that received (Globo H)3-DTPA-KLH after the third and fourth injections, as listed in Table 3 (Figure 12). Table 1 Mobile phase and conditions for HPLC analysis Column

Accucore 150-C18, 2.6 µm, 100×2.1 mm 16126-102130

Mobile phase A Mobile phase B Gradient Time (minutes) 0 5 45 46 50 51 60 Flow rate Backpressure at starting conditions Run time Injection wash solvent

0.1% formic acid in water 0.1% formic acid in acetonitrile %B 5 5 50 95 95 5 5 0.2 mL/min 170 bar 90 minutes Water +0.1% formic acid

Notes: One peak in the KLH digest pattern was definitely translocated from ~24 minutes to 29 minutes in the (Globo)3-DTPA-KLH digest pattern. There was a very clear signal of Globo H presence in the KLH structure. Abbreviation: HPLC, high-performance liquid chromatography.

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For successful treatment of a tumor or cancer cells, it is ideal to find molecular targets that are not present on normal cells. Meezan et al25 were among the first to demonstrate the differences of tumor glycans from normal cells. It appears that the glycosylation of sugar residue on cancer cells is structurally atypical to that of healthy cells, due to overexpression of specific structures and omission of some others.26 Sugarresidue association with tumors has also been identified recently by monoclonal antibodies and mass spectrometry.27 Association of glycolipids or glycoproteins has also been reported on the surface of certain types of cancer cells.28 The role of surface carbohydrates and its correlation with tumor malignancy is not known yet. However, it appears that these antigens could activate immune responses. One of the carbohydrates associated with tumors was first isolated from breast cancer MCF-7 cells and called Globo H by Hakomori7 and Bremer et al.29 It is now known that Globo H is present on many cancer cells, including prostate, ovary, lung,

3.5 PBS (Globo)1-KLH DTPA-KLH (Globo)3-DTPA-KLH

3

OD450 (nm)

Immunization results

230

None of the immunized mice showed any sign of cancer after the injection of MCF-7 cells compared to the control animals (see Supplementary materials).

2.5 2 1.5 1 0.5 0

1/50

1/100

1/300

1/900 1/2,700 1/8,100

Dilutions Figure 11 Antibody titration. Abbreviations: PBS, phosphate-buffered saline; DTPA, diethylenetriamine pentaacetic acid; OD, optical density.

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(Globo H)3-DTPA-KLH antigen

Table 3 Amount of IL-4 and IFNγ in the vaccine-administered group after third and fourth injections Vaccine-received group

IL-4 (pg/mL)

IFNγ (pg/mL)

Blood sampling after third injection Blood sampling after fourth injection Blood sampling from control mice

1.62145

1.49553

12.8276

18.11630526

Untraceable

Untraceable

Abbreviations: IFN, interferon; IL, interleukin.

pancreatic, gastric, and colon cancers,24 and high levels of anti-Globo H antibody can be detected in serum samples of breast cancer patients.30,31 These findings indicate that Globo H could be used as a potential target in cancer therapy and cancer-vaccine development. In this study, for the first time, a new chemically designated three-branch structure of Globo H antigen conjugated with KLH was synthesized and characterized. The novelty of this structure was regarding the triplicate structure of Globo H, which is highly similar to the nature of this antigen at its binding sites on cancer cells.17 The next advantage of the proposed structure is indicated by its polyamine linker DTPA, which facilitates its presentation to Globo H-binding sites at the cancerous cell surface as well.18,19 Kudryashov et al among others, previously reported the preparation of Globo H-KLH vaccine.22,24,32 They also investigated the immunogenicity of the Globo H-KLH antigen after ozonolysis of the Globo H aglycone that was followed by reductive amination with the KLH carrier protein to generate about 150 carbohydrate units per protein.24 16

Globo H is a cancer antigen overexpressed in various epithelial cancers. It has been suggested that this antigen can serve as a target in cancer immunotherapy. While vaccines have been developed to elicit antibody responses against Globo H, their anticancer efficacies are unsatisfactory, due to the low antigenicity of Globo H. There is a need for a new vaccine capable of eliciting high levels of immune responses targeting Globo H. In this study, we successfully synthesized a novel three branch Globo H antigen conjugated with a DTPA-KLH molecule. The triplicate Globo H structure allows this antigen to perform with the same antigenic effects as the sugar residue on tumor cells, and therefore to induce a better immune response against cancer cells. Administration of (Globo H) 3-DTPA-KLH in mice subcutaneously, elevated the level of both IgM and IgG antibody titers significantly (Figure 11). In the meantime, IL-4 and IFNγ levels were also elevated significantly in the sera of vaccinated mice with (Globo H)3-DTPA-KLH compared with the control mice (Figure 12). This phenomenon was confirmed after immunotherapy of nude mice with the sera collected from immunized animals, which prevented the growth of tumor cells or reduced the size of tumors that were already developed (Figure 13). Altogether, we concluded that the new synthesized (Globo H)3-DTPA-KLH could be a promising vaccine for treatment of cancers. More studies are required to find the best adjuvant to accompany this vaccine to elicit the best immune response against cancer cells. For mass spectrometry results and further information related to the structures

IL-4

IFNγ 0.4

12

Concentration (ng/mL)

Concentration (pg/mL)

14

10 8 6 4 2 0

PBS

(Globo)3-DTPA-KLH

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

(Globo)3-DTPA-KLH

PBS

Figure 12 Concentration of IL-4 and IFNγ in immunized mice. Abbreviations: IFN, interferon; IL, interleukin; PBS, phosphate-buffered saline; DTPA, diethylenetriamine pentaacetic acid.

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Nude mouse injected with pulled sera from control animal No alteration in tumor size

Nude mouse injected with pulled sera from immunized mice with (Globo)3-DTPA-KLH Tumor size reduced significantly to minimum Figure 13 Nude mice injected with pooled sera. Abbreviation: DTPA, diethylenetriamine pentaacetic acid.

of the Globo-H compound, please refer to Supplementary Figures S1-S10 at the end of the article.

Disclosure The authors report no conflicts of interest in this work.

References

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13. Jones PC, Sze LL, Liu PY, Morton DL, Irie RF. Prolonged survival for melanoma patients with elevated IgM antibody to oncofetal antigen. J Natl Cancer Inst. 1981;66(2):249–254. 14. Livingston PO, Ritter G, Srivastava P, et al. Characterization of IgG and IgM antibodies induced in melanoma patients by immunization with purified GM2 ganglioside. Cancer Res. 1989;49(24 Pt 1):7045–7050. 15. Livingston PO, Wong GY, Adluri S, et al. Improved survival in stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside. J Clin Oncol. 1994;12(5): 1036–1044. 16. MacLean GD, Reddish MA, Koganty RR, Longenecker BM. Antibodies against mucin-associated sialyl-Tn epitopes correlate with survival of metastatic adenocarcinoma patients undergoing active specific immunotherapy with synthetic STn vaccine. J Immunother Emphasis Tumor Immunol. 1996;19(1):59–68. 17. Springer GF. Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy. J Mol Med. 1997;75(8):594–602. 18. Dalmau J, Gultekin HS, Posner JB. Paraneoplastic neurologic syndromes: pathogenesis and physiopathology. Brain Pathol. 1999;9(2):275–284. 19. Porter CW, Miller J, Bergeron RJ. Aliphatic chain length specificity of the polyamine transport system in ascites L1210 leukemia cells. Cancer Res. 1984;44(1):126–128. 20. Seiler N. Thirty years of polyamine-related approaches to cancer therapy. Retrospect and prospect. Part 2. Structural analogues and derivatives. Curr Drug Targets. 2003;4(7):565–585. 21. Pohjanpelto P. Putrescine transport is greatly increased in human fibroblasts initiated to proliferate. J Cell Biol. 1976;68(3):512–520. 22. Kudryashov V, Ragupathi G, Kim L, et al. Characterization of a mouse monoclonal IgG3 antibody to the tumor-associated Globo H structure produced by immunization with a synthetic glycoconjugate. Glycoconj J. 1998;15(3):243–249. 23. Zhu J, Warren JD, Danishefsky SJ. Synthetic carbohydrate-based anticancer vaccines: the Memorial Sloan-Kettering experience. Expert Rev Vaccines. 2009;8(10):1399–1413. 24. Robert C, Guilpin C, Limoge A. Review of neural network applications in sleep research. J Neurosci Methods. 1998;79(2):187–193. 25. Meezan E, Wu HC, Black PH, Robbins PW. Comparative studies on the carbohydrate-containing membrane components of normal and virus-transformed mouse fibroblasts II. Separation of glycoproteins and glycopeptides by Sephadex chromatography. Biochemistry. 1969;8(6): 2518–2524. 26. Gabius HJ. Biological information transfer beyond the genetic code: the sugar code. Naturwissenschaften. 2000;87(3):108–121. 27. Shriver Z, Raguram S, Sasisekharan R. Glycomics: a pathway to a class of new and improved therapeutics. Nat Rev Drug Discov. 2004;3(10): 863–873.

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Dovepress 28. Dube DH, Bertozzi CR. Glycans in cancer and inflammation – potential for therapeutics and diagnostics. Nat Rev Drug Discov. 2005;4(6): 477–488. 29. Bremer EG, Levery SB, Sonnino S, et al. Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland. J Biol Chem. 1984;259(23):14773–14777. 30. Kim SK, Wu X, Ragupathi G, et al. Impact of minimal tumor burden on antibody response to vaccination. Cancer Immunol Immunother. 2011;60(5):621–627.

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(Globo H)3-DTPA-KLH antigen 31. Wang CC, Huang YL, Ren CT, et al. Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer. Proc Natl Acad Sci U S A. 2008;105(33):11661–11666. 32. Slovin SF, Keding SJ, Ragupathi G. Carbohydrate vaccines as immunotherapy for cancer. Immunol Cell Biol. 2005;83(4):418–428.

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Supplementary materials Positive 100 512.60

90

[M+3H]3+

80 70 60 50 40 30

768.95

418.80

[M+2H]2+

20

1,557.80

[M+Na]

10 [M+]

1,534.60 400

500

600

700

800

900

1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 Probe: Nebulizer gas flow: DL: DL temp: Block temp:

Sample information: 2012_6_189: 3,643 Far(1Saa)TV-15 Date and time: 1 Sample: Inj. volume

Probe bias: Detector: T.flow: B.conc:

ESI 1.5 L/min –20.0°C 250°C 200°C

m/z

–3.5 kV 1.0 kV 0.2 mL/min 50%H20/50%ACN Hajmohammadi

0.069 0.000 0.008

5.665 5.642 5.630 5.592 5.432 5.311 5.298 5.124 5.113 4.913 4.816 4.786 4.732 4.700 4.681 4.673 4.658 4.619 4.610 4.591 4.582 4.543 4.536 4.517 4.489 4.393 4.319 4.268 4.243 4.217 4.160 4.130 3.992 3.981 3.793 3.780 3.743 3.728 3.555 3.532 3.438 3.320 3.296 3.227 3.085 3.000 2.348 2.331 2.102 2.041 2.012 1.994 1.976 1.828 1.558 1.511 1.298 1.250 1.234 1.118 1.098 1.082 1.069 1.052 1.039 1.024 1.013 1.001 0.969 0.894 0.878 0.860

7.668 7.350 7.337 7.320 7.312 7.304 7.285 7.258 7.243 7.202 7.190 7.180 7.172 7.145 7.131 7.126 6.994

Figure S1 Globo H mass spectroscopy.

Current data parameters Hajmohammadi Name 72 EXPND PROCND 1 F2 – Acquisition parameters 20120707 Date_ Time 10.24 Spect INSTRUM PROBHD 5 mm QNP 1H/13 PULPROG zg30 TD 32,768 SOLVENT COC13 NS 16 DS 0 SWH 10,000.000 Hz FIDRES 0.305176 Hz AQ 1.6385000 sec RG 181 DW 50.000 usec DE 6.50 usec TE 298.0 K D1 3.00000000 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== Channel f1 ======= NUC1 1H P1 10.50 usec PL1 –3.00 dB SF01 500.1345088 MHZ F2 – Processing parameters SI 32,768 SF 500.1300244 MHZ WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00

1.0

0.5

0.0

26.18 19.74 –6.76

1.5 51.24

2.0 2.90

2.5

7.18

0.99

3.0 5.04

3.5 4.45 4.07

4.0 8.55

4.5

7.46

5.0

2.24 4.24 1.81 5.08 3.64 5.84 1.21 1.41 2.34 10.62

5.5

1.93

6.0

1.01 1.16

6.5

1.68

7.0 71.43

2.00

ppm 7.5

Figure S2 Protected Globo H 1H NMR. Abbreviation: NMR, nuclear magnetic resonance.

234

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Drug Design, Development and Therapy 2015:9

Drug Design, Development and Therapy 2015:9 9.059

1.5 1.385

1.4605 1.4201

0.8312

0.8323

1.2400 1.2201 1.0897 1.0737 0.8488

60

2.0333 2.0133 1.9430 1.9200 1.8201

Figure S3 Protected Globo H 13C NMR. Abbreviation: NMR, nuclear magnetic resonance. 70

29.029

2.0

3.294

2.5

80

3.388

3.0

90

5.049 2.521

3.5 100

2.5226 2.4926

110

2.7226

ppm

50 40

1.0

36.81 32.27 31.90 29.69 29.52 29.44 29.34 29.27 25.67 22.66 20.91 20.79 18.03 17.98 17.95 17.90 16.66 14.09 11.85

80.53 78.88 77.32 77.20 77.00 76.68 76.00 75.22 74.88 74.82 74.20 74.15 73.11 73.13 73.07 72.93 72.47 72.28 72.38 70.24 67.92 67.71 61.25 60.93 53.82 53.38 51.52

103.27 102.23 101.11 99.03 98.47 98.17

153.41 140.58 138.60 138.56 138.50 138.39 138.26 138.02 128.53 128.32 128.30 128.25 128.23 128.21 128.13 128.13 127.97 127.76 127.68 127.54 127.42 127.31 127.15

169.63 168.58

172.41

Protected Globo, Hajmohammadi C13NMR, CDCl3

1.250

3.3312 3.3226 3.2526 3.0452 3.0254 3.0226 2.8726

3.3526 3.3426

120

22.535

4.0

26.213

130

6.700

4.5

6.253

4.4791 4.4571 4.4368 4.2655 4.2465 4.1792 4.1597 4.1000 4.0401 3.9801 3.8501 3.8101 3.7201 3.3700 3.3612

140

1.332 1.460 2.331

5.0 4.5001

4.8107

4.8200

150

1.307

ppm 5.5 4.9543

160

1.011

170

1.088

5.4912 5.4880 5.3764 5.3589 5.3516 5.3136

5.5496 5.5328 5.5296

ppm

1.008

1.015

Integral

Dovepress (Globo H)3-DTPA-KLH antigen

30 20 10 0

Current data parameters Hajmohammadi Name 63 EXPNO PROCNO 1

F2 – Acquisition parameters 20120723 Date_ Time 10.00 INSTRUM Spect PROBHD 5 mm PABBO BB– PULPROG zg TD 32768 SOLVENT DMSO NS 6 DS 0 SWH 9014.423 Hz FIDRES 0.275098 Hz AQ 1.8175818 sec RG 90.5 DW 55.467 usec DE 6.50 usec TE 673.2 K D1 1.50000000 sec TD0 1 ======== Channel f1 ======= NUC1 1H P1 4.00 usec PL1 –1.00 dB SFO1 400.1005009 MHZ

F2 – Processing parameters SI 16384 SF 400.1000069 MHZ WDW EM SSB 0 LB 0.00 Hz GB 0 PC 33.00

0.5

Figure S4 Globo H 1H NMR. Abbreviation: NMR, nuclear magnetic resonance.

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Hajmohammadi et al

170

160

150

140

130

120

110

100

90

80

70

60

50

40

30

20

10

0.017

81.178 79.121 76.840 76.571 76.443 76.266 75.571 74.965 73.591 73.056 70.695 70.661 69.723 62.667 62.616 54.681 49.677 49.562 49.506 49.392 49.335 49.221 49.166 49.051 48.881 48.710 48.540 37.429 33.546 33.169 30.959 30.917 30.885 30.869 30.849 30.794 30.721 30.580 30.531 30.489 27.254 23.831 23.755 23.527 16.756 14.544 14.458

105.552 105.446 104.442 103.955 102.856 101.061

131.404

135.212

16/2/1391

176.023 174.523

Hajmohammadi-Globo-H 13CNMR-CDCl3

0

Figure S5 Globo H 13C NMR. Abbreviation: NMR, nuclear magnetic resonance.

100 98

82 80 78

1,652.90

76 74 72

758.46

1,075.45 1,042.58 1,260.95

84

2,920.73

86

1,547.93

88

1,372.94

1,734.56

2,851.92

90 3,356.16

% transmittance

92

860.88

92

94

1,466.19

96

70 68 4,000

3,000

Date: Wed Jan 0100:32:02 Scans: 4 Resolution: 4.000

2,000

Wave numbers (cm–1)

1,000

Mr. Hajmohammadi

Figure S6 Globo H FTIR. Abbreviation: FTIR, Fourier-transform infrared.

236

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Drug Design, Development and Therapy 2015:9

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(Globo H)3-DTPA-KLH antigen

300

Translocation due to Globo H conjugation

250

Absorbance

200 150 KLH-Globo

100 KLH

50 0

–69 20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

Time (minutes) Figure S7 (Globo H)3-DTPA-KLH HPLC. Abbreviations: DTPA, diethylenetriamine pentaacetic acid; HPLC, high-performance liquid chromatography.

4.44759 4.36623 4.33281 3.84015 3.59591 3.51812 3.46542 3.45420 3.43425 3.36985 3.35792 3.27666 3.16038 3.14715 3.08854 3.07880 2.59508 2.53398 2.46033 2.32170 2.04436 2.02416 2.00752 1.82824 1.60201 1.51670 1.28740

ppm

7.54183 7.48875 7.31817 7.26783 7.17485 7.09860 6.86981 6.85782

1 1H NMR in DMSO at 298 k 91/3/29 Current data parameters Name Hajmohammadi EXPND 4 PROCND 1 F2 – Acquisition parameters Date_ 20120619 Time 17.17 INSTRUM Spect PROBHD 5 mm QNP 1H/13 PULPROG zg30 TD 32,768 Solvent DMSO NS 32 DS 0 SWH 10,000.000 Hz FIDRES 0.305176 Hz AQ 1.6385000 sec RG 812.7 DW 50.000 usec DE 6.50 usec TE 298.0 K D1 MCREST MCWRK

5.00000000 sec 0.00000000 sec 0.01500000 sec

======== Channel f1 ======= NUC1 1H P1 10.50 usec PL1 –3.00 dB SF01 500.1345088 MHZ

ppm

10

8

6

4

2

1D NMR plot CX CY F1P F1 F2P F2 PPMCM HZCM

2.0035

2.4103

8.0920 2.3809 2.5545 3.3521 5.6560

2.6008 2.0000 2.8258 3.8858 4.9028 5.0536 4.2621 4.6443 3.9098

4.3809 3.5545

Integral

6.0920

F2 – Processing parameters SI 32,768 SF 500.1300244 MHZ WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 parameters 20.00 cm 129.81 cm 11.083 ppm 5543.02 HZ –0.648 ppm –324.09 HZ –324.09 ppm/cm 293.35583 HZ/cm

0

Figure S8 KLH H NMR. Abbreviations: DMSO, dimethyl sulfoxide; NMR, nuclear magnetic resonance. 1

Drug Design, Development and Therapy 2015:9

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3.46842 3.45420 3.43425 3.36985 3.35792 3.27666 3.16038 3.14715 3.08854 3.07880 2.59508 2.53377 2.46037 2.32170 2.04403 2.02414 2.00752 1.82824 1.60215 1.51674 1.28780

4.44357 4.36028 4.33188 3.84415 3.59493 3.51712

4.70152 4.54602

5.59134 5.44469 4.77557 4.99576 4.91142 4.88020

ppm

7.57984 7.46167 7.35421 7.30152 7.18743 7.12803 6.97147 6.91420

3 1H NMR in DMSO at 298 k 91/3/29 Current data parameters Hajmohammadi Name 2 EXPND 1 PROCND F2 – Acquisition parameters 20120619 Date_ Time 17.17 Spect INSTRUM PROBHD 5 mm QNP 1H/13 PULPROG zg30 TD 32,768 Solvent DMSO NS 32 DS 0 SWH 10,000.000 Hz FIDRES 0.305176 Hz AQ 1.6385000 sec RG 812.7 DW 50.000 usec DE 6.50 usec TE 298.0 K D1 5.00000000 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== Channel f1 ======= NUC1 1H P1 10.50 usec PL1 –3.00 dB SF01 500.1345088 MHZ

ppm

8

6

4

1D NMR plot parameters CX 20.00 cm CY 129.81 cm F1P 11.083 ppm F1 5,543.02 Hz F2P –0.648 ppm F2 –324.09 Hz PPMCM 0.58656 ppm/cm HZCM 293.35583 Hz/cm

2.072

2.5826

2.5840 3.7296 5.8701

2.4271

8.0524

2.8985 2.5155 2.1592 3.7052 4.8508 5.9025 4.1668 4.4273 3.4555

0.3010 0.2873 0.3602 0.2527 0.6369 0.2855

3.6692

5.9870 10

4.4414

Integral

F2 – Processing parameters SI 32,768 SF 500.1300244 MHZ WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00

2

0

Figure S9 Globo H-KLH 1H NMR. Abbreviations: DMSO, dimethyl sulfoxide; NMR, nuclear magnetic resonance.

4.70155 4.54603

4.44357 4.36028 4.33188 3.84415 3.59493 3.51712 3.46842 3.45420 3.43425 3.36985 3.35792 3.27666 3.16038 3.14715 3.08854 3.07880 2.59508 2.53377 2.46037 2.32170 2.04403 2.02414 2.00752 1.82824 1.60215 1.51674 1.28780

5.59183 5.44441 4.77548 4.99520 4.91123 4.88036

ppm

7.57984 7.46167 7.35421 7.30152 7.18743 7.12803 6.97147 6.91420

2 1H NMR in DMSO at 298 k 91/3/29

Current data parameters Name Hajmohammadi EXPND 2 PROCND 1 F2 – Acquisition parameters Date_ 20120619 Time 17.17 INSTRUM Spect PROBHD 5 mm QNP 1H/13 PULPROG zg30 TD 32,768 Solvent DMSO NS 32 DS 0 SWH 10,000.000 Hz FIDRES 0.305176 Hz AQ 1.6385000 sec RG 812.7 DW 50.000 usec DE 6.50 usec TE 298.0 K D1 5.00000000 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== Channel f1 ======= NUC1 1H P1 10.50 usec PL1 –3.00 dB SF01 500.1345088 MHZ

ppm

10

8

6

4

2

2.072

0.9135 0.8985 0.8052 1.1592 0.9135 0.9135 2.8985 2.5155 2.1592 3.7052 4.8508 5.9025 4.1668 4.4273 3.4555 8.0920 2.4271 2.5840 3.7296 5.8701 2.5826

6.3222 5.0413 3.9403

Integral

F2 – Processing parameters SI 32,768 SF 500.1300244 MHZ WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00

0

1D NMR plot parameters CX 20.00 cm CY 129.81 cm F1P 11.083 ppm F1 5,543.02 Hz F2P –0.648 ppm F2 –324.09 Hz PPMCM 0.58656 ppm/cm HZCM 293.35583 Hz/cm

Figure S10 (Globo H)3-DTPA-KLH 1H NMR. Abbreviations: DMSO, dimethyl sulfoxide; DTPA, diethylenetriamine pentaacetic acid; NMR, nuclear magnetic resonance.

238

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