Synthesis and characterization of a novel chemically designed (Globo)3–DTPA–KLH antigen

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Hajmohammadi, Mehdi, Siadat, Seyed Davar, Ghorbani, Masoud, Shafiee Ardestani, Mehdi, Teimourian, Shahram, Asgari, Vahid, Ahangari Cohan, Reza, Hajmohammadi, Mostafa, Hajmohammadi, Akram, Behzadi, Ramezan, Rajabnezhad, Saeid and Namvar Asl, Nabiollah (2014) Synthesis and characterization of a novel chemically designed (Globo)3–DTPA–KLH antigen. Drug Design, Development and Therapy, 9. pp. 217-239. ISSN 1177-8881

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Open access Full Text article

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

synthesis and characterization of a novel chemically designed (globo)

3–DTPa–Klh antigen

Mehdi hajmohammadi1

seyed Davar siadat2

Masoud ghorbani3,4,*

Mehdi shafiee ardestani5,*

shahram Teimourian6

Vahid asgari3

reza ahangari cohan3

Mostafa hajmohammadi5

akram hajmohammadi7

ramezan Behzadi8

saied rajab nezhad9

nabiollah namvar asl10

1Department of research and Biotechnology, 2Department of Microbiology, 3Department of Virology, Pasteur institute of iran, Tehran iran; 4Department of Virology, University of Ottawa, Ottawa, On, canada; 5Department of radiopharmacy, Faculty of Pharmacy, Tehran University of Medical sciences, Tehran, iran; 6Department of Medical genetics, iran University of Medical sciences, Tehran, 7Faculty of Pharmacy, ahvaz Jundishapur University of Medical sciences, ahvaz, 8laboratory 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

*These authors contributed equally to this work

Abstract: In recent years, many experiments have been conducted for the production and evalu-

ation 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 col-

lected 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

IntroductionCancer 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 devel-

opment, 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 self-

antigens 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

correspondence: Masoud ghorbaniDepartment of Virology, Pasteur institute of iran, Tehran–Karaj highway, Tehran 31599, iranTel +98 26 1610 2999Fax +98 26 1610 2900email mghorbani@irimc.org

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218

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 chemo-

therapy 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 carbo-

hydrate antigens on the surface of tumor cells in order to

reduce tumor severity.10,11 A number of clinical experi-

ments have shown that prescribing monoclonal antibodies

is clinically efficient.12,13 Naturally released antibodies

against antigens on cell surfaces, especially carbohy-

drate 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 polysac-

charides to produce anticancer vaccines is the keyhole

limpet hemocyanin (KLH) protein.21 KLH is a large, mul-

tisubunit, oxygen-carrying metalloprotein, and is found in

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 polysaccha-

rides 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 methodsAll animal protocols were reviewed and approved by the

Pasteur Institute of Iran Animal Care Protocol Review Com-

mittee. 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–KlhAll substances were used without purification unless noted.

Solvents were distilled under positive-pressure nitrogen

stream: tetrahydrofuran (THF) with sodium benzophenone

ketyl; ether with LiAlH4, CH

2Cl

2, and toluene; and benzene

with CaH2. All reactions were under N

2 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-glucalBoth D-glucal (8 g, 54.7 mmol) and (Bu

3Sn)

2O (30.7 mL, 10 M

equivalent) were refluxed for 20 hours in dry C6H

6 (150 mL)

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219

(globo h)3-DTPa-Klh antigen

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

washed using distilled water (3×300 mL). It was then

filtered, condensed, and dried by Na2So

4. 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 color-

less oily residue.

Bn O

Bn O

Bn O

Bn OBn O

Bn O

Bn OBn O

Bn OBn O

Bn O

Bn O

Bn O

Bn O

Bn O

Bn O

Bn OO

O

O O

O

O

O

PhOSHNO TIPSO

O

OO

O

O

O

OO

O

O

O

O

O

O

O

O

OOCH

3

O

O

O

OO

O

OO

OO

O

O Bn

O Bn

O Bn

O Bn

O Bn

O Bn

O Bn O TPIS

PhOSHN

NHSOPh

NH

N

O BnO Bn

CH3

O BnTIPS O

NHSOPh

TIPS O

HN

NHCH

3

COOH

O BnO

S

O

O OOOOO

O O

O

O

O

OO

O

OO

O

O

O

O

O

O

O TIPS

O TIPS

O TIPS

Bn O

Bn OO Bn

O Bn

O Bn

O BnO Bn

O Bn

O Bn

O Bn

O Bn

H3C

O Bn Bn O

O Bn

O Bn

OH

O

O

O

O O

O N

N

NS

COOH

N

EDC

O TIPSO Bn

CH3

Figure 1 suggested chemical structure of a stronger derivation than globo h Klh.

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220

hajmohammadi et al

synthesis of 1,5-anhydro-6-O-(TiPs)- 2-deoxy-d-lyxo-hex-1-enopyranoseUnder a stream of nitrogen (N

2), 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

di methylformamide (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 Na2SO

4, 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-1-enopyranoseThe 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

Na2SO

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 1The syrup (22 mg) was mixed with 5 mL methylene chlo-

ride, 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-D-

glucal 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

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 Na2SO

4, and purified

by column chromatography (gradient elution, 10%–25%

EtOAc:hexanes) to obtain the desired compound.

synthesis of lactal carbonateOne 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 Na2SO

4, followed by column chroma-

tography 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 lactalA 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 dibenzyla-

tion stage.

synthesis of disaccharideA mixture containing 2.95 g thioglycol (5.1 mmol),

1.33 g Bu2SnO (1.05 equivalent), and 1.69 mL (Bu

3Sn)

2O

(0.65 equivalent) was refluxed for 5 hours in dry C6H

6

(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 C6H

6 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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221

(globo h)3-DTPa-Klh antigen

synthesis of 3-O-(4-methoxybenzyl)-d-glucalA suspension of 3.70 g D-glucal (25.3 mmol) and 6.30 g

Bu2SnO (1.0 equivalent) was refluxed for 5 hours in

dry C6H

6 (50 mL) under N

2 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,

OHOH

OO

O

O

O TIPS

O

O

O TIPS

TIPSImidazolDMF

TBIBrBnBrPhH

ZnCl2

THF

BnBrNaHDMF

(n-Bu3Sn)

2O

1,2-Carbonyldiimidazolecat. DMAP, CH

2CI

2,

quanti

3,3′–dimenthyldioxiranCH

2Cl

2

OH

HO

OH

O

OH

HO

HO

OH

OO

O

O

O

O

O

O

O

O Bn

O

O

O

OH

O TIPS

O TIPS

O Bn

Bn O

Bn O

O Bn

OHOH

HO

O Bn

Bn O

O Bn

O Bn

O Bn

Bn OBn O

O Bn

TBAFTHFthen MeOHMeONa

BuSnOthen TBABrBnBr

(n-Bu3Sn)

2O

PhH

O

O

O

O

OH

O

O

OO

O

O

O

O Bn

O

O TIPS

Bn O

Figure 2 synthesis of lactal carbonate.

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222

hajmohammadi et al

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%).

4,6-Di-O-benzyl-3-O-(4-methoxybenzyl)-d-glucal synthesisA mixture of 3-O-(4-methoxybenzyl)-D-glucal (8.56 mmol,

2.28 g) and BnBr (3.68 molar equivalent, 3.75 mL) solu-

tion 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 Na2SO

4. 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 luorideA mixture of 4,6-di-O-benzyl-3-O-(4-methoxybenzyl)-D-glucal

(7.7 mmol, 3.20 g) solution in dry CH2Cl

2 (10 mL) was treated

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 treat-

ment 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).

OHOH

O

PMB O

O BnO Bn

O Bn

O BnO Bn

O BnO Bn

O

F

O

O BnO Bn

PMB O

O

PMB O

PMB O

O

F

OH

O

PMB O

OHOH

O

n-BuSnO

TBABrPMBCIPhH

BnBr

NaHDMF

3,3′-dimethyldioxiraneCH

2Cl

2

TBAFBnBr

NaHDMF

THF

HO

Figure 3 Synthesis of luoride.

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(globo h)3-DTPa-Klh antigen

Synthesis of the irst trisaccharide containing para-methoxybenzylThe 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. Anhy-

drous 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 solu-

tion 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

Na2SO

4. Column chromatography was performed twice using

20% EtOAc in hexanes to obtain 561 mg trisaccharides with

a purity of 54%.

Synthesis of the irst trisaccharide without para-methoxybenzylA 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-dicyano-

1,4-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 Na2SO

4, 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).

glycal disaccharide synthesisTIPS galactal carbonate (3.14 mmol, 4.32 g) was dissolved

in 20 mL of CH2Cl

2 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

PMB O

PMB O

O Bn

O

O

OO

O

O

F

O Bn

O Bn

O Bn

O Bn

Bn OBn OO Bn

O Bn

O

OO

O

O

O Bn

HO

O Bn

Bn O Bn O

O Bn

O Bn

O Bn

Bn O

AgClO4, SnCl

2, di-tert-butylpyridine,

4Å, molecular sieves, Et2O

DDQCH

2Cl

2

H2O

Bn OO Bn

O Bn

O BnO Bn

O BnOH

OO

O

Figure 4 synthesis of trisaccharide.

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224

hajmohammadi et al

20 mL C6H

6 and dried by high vacuum power for 30 min-

utes 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 chro-

matography using 20% EtOAc in hexanes. The resulting

product was 6.20 g white foam.

glycal trisaccharide synthesisA 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 C6H

6 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 contain-

ing AgClO4 (7.54 mmol, 1.56 g), SnCl

2 (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).

synthesis of iodosulfonamideA mixture of glycal trisaccharide (0.11 mmol, 118 mg) and

benzenesulfonamide (0.55 mmol, 87 mg) was deazeotroped

once by C6H

6, dried by high vacuum for 1 hour, and dis-

solved 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 ClO

4 (0.22 mmol) in 1 mL THF at room

temperature until the brown color of iodine had disappeared

and I(sym-coll)2 ClO

4 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 Na2S

2O

3 solution. After filtration and extraction

by EtOAc (80 mL), the organic layer was washed with 20 mL

deuterium-depleted water, saturated with 20 mL Cu2SO

4,

dried by MgSO4, and purified by column chromatography

using silica gel (20% EtOAc in hexanes) to obtain 74 mg

iodosulfonamide as the result.

ethyl-thio-sulfonamide synthesisLithium 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).

hexasaccharide synthesisAn acceptor mixture containing trisaccharides (1 equivalent,

0.077 mmol, 92 mg), thioglycoside (2 equivalent, 198 mg),

and activated molecular sieve (560 mg) suspended in Et2O-

CH2Cl

2 (2:1) under N

2 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 satu-

rated NaHCO3 solution, dried with Na

2SO

4, 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 azideA mixture of hexasaccharide glycal and freshly activated 4 Å

molecular sieve were treated with 1mL dimethyldioxirane

(0.07 M) in 1 mL CH2Cl

2 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 Et

2O (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 solu-

tion (2×20 mL) and salt water (10 mL), dried by Na2SO

4,

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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225

(globo h)3-DTPa-Klh antigen

3.0 μL) was added in CH2Cl

2 (1 mL) and Ac

2O (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 Na2SO

4. Column chromatography (20%

EtOAc in hexanes) of raw materials provided 30 mg (95%)

of the desired product (Figure 7).

synthesis of amideA mixture containing 66 mg azide (0.023 mmol), 66 mg Lind-

lar catalyst, and 23 mg palmitic anhydrate (0.46 mmol) was

stirred for 24 hours at room temperature under H2. The reac-

tion 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 the inal product of (Globo)3-

DTPa-KlhApproximately 1 mmol of KLH (3 g) was mixed with

2 mmol of S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine

O

O

O

O

OO

O

O

O

O

O

O

O

O

O

O

O

O

OH

O Bn

O BnO Bn

O Bn

O BnO Bn

O

OO

CH

O

O

O

OH

OH

O

O

HO

ZnCl2

THF

OH

O

O TIPS O TIPS

O TIPSO TIPS

O TIPS

O TIPS

O TIPS

NHSO2Ph

O TIPS

O

H3C

H3C

H3C

O

F

O Bn

O BnO Bn

AgClO4

SnCl2

di-tert-butylpyridine

I(coll)2ClO

4

PhSO2NH

2

4Å molecular sievesTHF

THF

Figure 5 synthesis of iodosulfonamide.

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226

hajmohammadi et al

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-DTPA-

KLH. The remainder was then added to a mixture containing

10 mL of dissolved DMF, acetic acid anhydride (C4H

6O

3),

and 10 mg pyridine while stirring for 3 hours at 40°C, until

the formation of anhydrate. Ten milligrams of 1-ethyl-3-(3-

dimethylaminopropyl) 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 addi-

tion 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

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

infrared, and HPLC techniques.

animal and biological assaysimmunization of mice with vaccine regimens

Mice were divided into five groups of eight, and received

different vaccine regimens in 200 μL via intraperitoneal injec-

tions. 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 DTPA-

KLH + 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.

O

O

O

O

OH

OH O TIPS

O TIPS

EtSH, LHMDS, DMF

OO

HN

PhS

O

O

O

O

O

O

O

O Bn

O BnO Bn

O

O

OHO TIPS

O

SEt

O

N

SO2Ph

SEtor

O

O TIPS

O

O

OHO TIPS

O

O

O Bn

O BnO Bn

O TIPS

H3C

H3C

NHSO2Ph

NHSO2Ph

Figure 6 synthesis of ethyl-thio-sulfonamide.

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227

(globo h)3-DTPa-Klh antigen

antibody titration

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 per-

oxidase 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.

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.

in vivo tumor-preventive effects

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.

statistical analysisResults 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 P0.05.

ResultsConirmation of the synthesis of Globo h and globo h-protected precursor1h-nMr (solvent, cDcl

3)

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),

O

O

O

O

O

O

OO O

O

O

OO

O

O OO

O Ac

O

O Bn

N3

(CH2)

12C

H3

O

O Bn

Bn O O Bn

O BnO Bn

O Bn

O BnBn OBn OO BnO Bn

H3C O

HO

OH

O Bn

N3

(CH2)

12CH

3

ZnCl2, THF

OO

O O

O

OO

O

OO

OO

O

O

O

O

OO

ONHSOPh

NHSOPh

NHSOPh

OH

OH

O Bn

O BnO Bn

O Bn

Bn O O Bn

O Bn

Bn O Bn OO Bn

O Bn

O Bn

O Bn O Bn

Bn OO Bn

O Bn

O Bn

Bn O

MeOTf, 4Å molecular sieves, Et2O-

CH2Cl

2(2:1)

(a) (i) 3,3′-dimethyldioxirane, 4Å molecularsieves, CH

2Cl

2;

(b) Ac2O, DMAP, Et

3N, CH

2Cl

2

(ii)

Bn O

O BnO Bn

O

O TIPS

O TIPS

O TIPS

O TIPSO TIPS

O TIPS

O

H3C

H3C

SEt

O

HO

Figure 7 synthesis of azide.

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228

hajmohammadi et al

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).

13c-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,

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.

O

OO

O

O

O

O

O

OO

OO

O

O

O OO

O

NH

O

(CH2)

14CH

3

(CH2)

12CH

3

O BnO Bn

O Bn

OO O O

O OO

O

OO

OH

O

OO

O

O

OO

OO

OO

O

O

O

O TIPS

O TIPSO TIPS

O TIPSOH

O BnO Bn

O Bn

O Bn

(CH2)

12

CH3

(CH2)

12CH

3

(CH2)1

4CH

3

O Ac

O Ac

O BnBn O Bn O

O Bn

O BnO Bn

O BnO Bn

O Bn

O Bn

O Bn

Bn O Bn O

O BnO Bn

Lindlar’s catalyst, H2, palmitic anhydride,

EtOAc

O Bn

O Bn

H3C

H3C

H3C

NHSO2Ph

NHSO2Ph

N3

(a) (i) TBAF, THF; (ii) NaOMe, MeOH,(b) (i) Na, NH

3, THF; (ii) Ac

2O, Et

3N, DMAP,

CH2Cl

2

(c) NaOMe, MeOH, quantitative

O

NH

H O

H O H O

O H

O H

O HO H

O H

NHAc

O H O H

H O

H O

O H

O HO H

O H

O HO H

Figure 8 synthesis of antigen.

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229

(globo h)3-DTPa-Klh antigen

1h nMr (solvent, DMsO-4/D2O, 10:1)

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).

13c-nMr (solvent, cD3OD)

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.

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 conirmationhPlc procedure

To confirm the attachment of Globo3 to the complex of

DTPA-KLH, we used HPLC (Table 1).

chn analysis

Carbon, hydrogen, and nitrogen analysis showed the total

mass of each element residue in the complex, as indicated

in Table 2.

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

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

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

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).

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-KLH-

CHN 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).

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

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

%H-numbers =(123,162–122,631/484) ×100–100=10.0%

%N-numbers =(9,292–9268/24) ×100=9.0%

The data statistically confirmed the synthesis of our

conjugates.

1h nMr study

As the original results depict in the Supplementary

materials, there was a significant sharp peak regarding

the anomeric hydrogens of Globo H polysaccharide

OO

OO

O

OH3C

OO

OO

O

OO O

O Bn

O Bn O Bn

O BnBn O

Bn O

Bn O

O BnO Bn

O Ac

O CeramideO Bn

Bn O

O TIPS O TIPSA cO

PhSO2HN

Figure 9 Protected antigen.

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hajmohammadi et al

(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)3-

DTPA-KLH as well.

immunization results

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 (P0.05) between (Globo H)3-DTPA-

KLH 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).

Tumor growth

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).

DiscussionFor 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 differ-

ences 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 Sugar-

residue 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 pres-

ent on many cancer cells, including prostate, ovary, lung,

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 0.1% formic acid in water

Mobile phase B 0.1% formic acid in acetonitrile

Gradient

Time (minutes) % B

0 5

5 5

45 50

46 95

50 95

51 5

60 5

Flow rate 0.2 ml/min

Backpressure at starting

conditions

170 bar

Run time 90 minutes

Injection wash solvent Water +0.1% formic acid

Notes: One peak in the KLH digest pattern was deinitely 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.

Table 2 atomic mass for carbon, hydrogen, and nitrogen

elements

Element Atomic massa

c 12.0107

h 1.0079

n 14.0087

Note: aFrom periodic table.

OO NHAc

O CeramideO

O OO

O

O

O O O

O H O H

O HO H

O HO HO H

O HH O

H O

H O H O H O

H O

H O H O

O H

Figure 10 The molecular structure of globo-h antigen.

Notes: c:26.79, h:4.13, n:4.33 (reported from chn analyzer instrument).

3.5

OD

45

0 (

nm

)

Dilutions

PBS

(Globo)1-KLH

(Globo)3-DTPA-KLH

DTPA-KLH

3

2.5

2

1.5

1

0.5

01/50 1/100 1/300 1/900 1/2,700 1/8,100

Figure 11 antibody titration.

Abbreviations: PBs, phosphate-buffered saline; DTPa, diethylenetriamine pentaacetic

acid; OD, optical density.

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231

(globo h)3-DTPa-Klh antigen

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 desig-

nated 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 ozonoly-

sis 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

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 mol-

ecule. 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 com-

pared 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 spectrom-

etry results and further information related to the structures

16

14

12

10

8

6

4

2

0 0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

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

3-DTPA-KLH

IFNγIL-4

Co

ncen

trati

on

(p

g/m

L)

Co

ncen

trati

on

(n

g/m

L)

Figure 12 concentration of il-4 and iFnγ in immunized mice.

Abbreviations: iFn, interferon; il, interleukin; PBs, phosphate-buffered saline; DTPa, diethylenetriamine pentaacetic acid.

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

1.62145 1.49553

Blood sampling after

fourth injection

12.8276 18.11630526

Blood sampling from

control mice

Untraceable Untraceable

Abbreviations: iFn, interferon; il, interleukin.

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hajmohammadi et al

of the Globo-H compound, please refer to Supplementary

Figures S1-S10 at the end of the article.

DisclosureThe authors report no conflicts of interest in this work.

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Nude mouse injected with pulled sera from control animalNo alteration in tumor size

Nude mouse injected with pulled sera from immunized micewith (Globo)

3-DTPA-KLH

Tumor size reduced significantly to minimum

Figure 13 nude mice injected with pooled sera.

Abbreviation: DTPa, diethylenetriamine pentaacetic acid.

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hajmohammadi et al

Figure S1 globo h mass spectroscopy.

Positive100

90

80

70

60

50

40

30

418.80768.95

[M+2H]2+

512.60[M+3H]3+

1,557.80

1,534.60

[M+Na]

[M+]

20

10

400 500

Sample information:Probe:Nebulizer gas flow:DL:DL temp:

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

–3.5 kV1.0 kV0.2 mL/min50%H20/50%ACNHajmohammadiBlock temp:

ESI1.5 L/min–20.0°C250°C200°C

Date and time:Sample:Inj. volume

2012_6_189: 3,643Far(1Saa)TV-151

600 700 800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900m/z

ppm 7.5

2.0

0

7.6

68

7.3

50

7.3

37

7.3

20

7.3

12

7.3

04

7.2

85

7.2

58

7.2

43

7.2

02

7.1

90

7.1

80

7.1

72

7.1

45

7.1

31

7.1

26

6.9

94

5.6

65

5.6

42

5.6

30

5.5

92

5.4

32

5.3

115.2

98

5.1

24

5.1

13

4.9

13

4.8

16

4.7

86

4.7

32

4.7

00

4.6

81

4.6

73

4.6

58

4.6

19

4.6

10

4.5

91

4.5

82

4.5

43

4.5

36

4.5

17

4.4

89

4.3

93

4.3

19

4.2

68

4.2

43

4.2

17

4.1

60

4.1

30

3.9

92

3.9

81

3.7

93

3.7

80

3.7

43

3.7

28

3.5

55

3.5

32

3.4

38

3.3

20

3.2

96

3.2

27

3.0

85

3.0

00

2.3

48

2.3

31

2.1

02

2.0

41

2.0

12

1.9

94

1.9

76

1.8

28

1.5

58

1.5

111.2

98

1.2

50

1.2

34

1.1

18

1.0

98

1.0

82

1.0

69

1.0

52

1.0

39

1.0

24

1.0

13

1.0

01

0.9

69

0.8

94

0.8

78

0.8

60

0.0

69

0.0

00

0.0

08

Current data parametersNameEXPNDPROCND

F2 – Acquisition parametersDate_Time 10.24INSTRUMPROBHD 5 mm QNP 1H/13PULPROG zg30TD 32,768SOLVENT COC13NS 16DS 0SWH 10,000.000 HzFIDRES 0.305176 HzAQ 1.6385000 secRG 181DW 50.000 usecDE 6.50 usecTE 298.0 KD1 3.00000000 secMCREST 0.00000000 secMCWRK 0.01500000 sec

F2 – Processing parameters

NUC1 1HP1 10.50 usecPL1 –3.00 dBSF01 500.1345088 MHZ

SI 32,768SF 500.1300244 MHZWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

======== Channel f1 =======

Spect

20120707

Hajmohammadi721

71.4

3

1.6

8

1.0

1

1.1

6

1.9

3

2.2

44.2

41.8

15.0

83.6

45.8

41.2

11.4

12.3

410.6

2

7.4

6

8.5

5

4.4

5

4.0

7

5.0

4

0.9

9

7.1

8

2.9

0

51.2

4

26.1

819.7

4–6.7

6

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

Figure S2 Protected globo h 1h nMr.

Abbreviation: nMr, nuclear magnetic resonance.

Supplementary materials

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Do

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235

(glo

bo h

)3 -D

TPa

-Klh

antigen

Pro

tecte

d G

lobo, H

ajm

oham

madi

C13N

MR

, CD

Cl3

ppm

ppm

170

160

150

140

130

120

110

100

90

80

70

60

50

40

30

20

10

0

172.41

169.63168.58

153.41140.58138.60138.56138.50138.39138.26138.02128.53128.32128.30128.25128.23128.21128.13128.13127.97127.76127.68127.54127.42127.31127.15

103.27102.23101.1199.0398.4798.17

80.5378.8877.3277.2077.0076.6876.0075.2274.8874.8274.2074.1573.1173.1373.0772.9372.4772.2872.3870.2467.9267.7161.2560.9353.8253.3851.52

36.8132.2731.9029.6929.5229.4429.3429.2725.6722.6620.9120.7918.0317.9817.9517.9016.6614.0911.85

Fig

ure

S3 P

rotected

glo

bo h

13c

nM

r.

Ab

bre

via

tion

: nM

r, n

uclear m

agnetic reso

nan

ce.

ppm Integral

5.51.015

5.54965.53285.5296

5.49125.48805.37645.35895.35165.3136

4.9543

4.8200

4.8107

4.5001

4.47914.45714.43684.26554.24654.17924.15974.10004.04013.98013.85013.81013.7201

3.3612

3.35263.3426

3.3312

3.32263.25263.04523.02543.02262.8726

2.7226

2.52262.4926

2.03332.01331.94301.92001.8201

1.46051.4201

1.24001.22011.08971.0737

0.8488

0.8323

0.8312

3.3700

1.008

1.088

1.011

1.307

1.3321.4602.331

6.253

6.700

26.213

22.535

1.250

5.049

2.521

3.388

3.294

29.029

1.385

9.059

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

Curre

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ata

para

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XP

NO

PR

OC

NO

F2 –

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uisitio

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_T

ime

10.0

0IN

ST

RU

MP

RO

BH

D5 m

m P

AB

BO

BB

–P

ULP

RO

Gzg

TD

32768

SO

LVE

NT

DM

SO

NS

6D

S0

SW

H9014.4

23 H

zF

IDR

ES

0.2

75098 H

zA

Q1.8

175818 se

cR

G90.5

DW

55.4

67 u

sec

DE

6.5

0 u

sec

TE

673.2

KD

11.5

0000000 se

cT

D0

1

F2 –

Pro

cessin

g p

ara

mete

rs

NU

C1

1H

P1

4.0

0 u

sec

PL1

–1.0

0 d

BS

FO

1400.1

005009

MH

Z

SI

16384

SF

400.1

000069

MH

ZW

DW

EM

SS

B0

LB

0.0

0 H

zG

B0

PC

33.0

0

==

==

==

==

Channel f1

==

==

==

=

Spect

20120723

Hajm

oham

ma

di

63

1

Fig

ure

S4 g

lobo h

1h n

Mr

.

Ab

bre

via

tion

: nM

r, n

uclear m

agnetic reso

nan

ce.

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g Design

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ent an

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herapy 2

015:9

sub

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Do

ve

pre

ss

Do

ve

pre

ss

236

hajm

oham

mad

i et al

170

160

150

140

130

120

110

100

90

80

70

60

50

40

30

20

10

0

Hajm

oham

madi-G

lobo-H

16/2

/1391

13C

NM

R-C

DC

l3

176.023174.523

135.212

131.404

105.552105.446104.442103.955102.856101.061

81.17879.12176.84076.57176.44376.26675.57174.96573.59173.05670.69570.66169.72362.66762.61654.68149.67749.56249.50649.39249.33549.22149.16649.05148.88148.71048.54037.42933.54633.16930.95930.91730.88530.86930.84930.79430.72130.58030.53130.48927.25423.83123.75523.52716.75614.54414.458

0.017

Fig

ure

S5 g

lobo h

13c

nM

r.

Ab

bre

via

tion

: nM

r, n

uclear m

agnetic reso

nan

ce.

100

98

96

92

3,356.16

2,920.732,851.92

1,734.56

1,547.93

1,372.94

1,260.95

1,075.451,042.58

860.88

758.46

1,652.90

1,466.19

94

92

90

88

% transmittance

Wave n

um

bers

(cm

–1)

86

84

82

80

78

76

74

72

70

684,0

00

Date

: Wed Ja

n 0

100:3

2:0

2M

r. Hajm

oham

madi

Sca

ns: 4

Reso

lutio

n: 4

.000

3,0

00

2,0

00

1,0

00

Fig

ure

S6 g

lobo h

FTir

.

Ab

bre

via

tion

: FTir

, Fourier-tran

sform

infrared

.

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237

(globo h)3-DTPa-Klh antigen

300 Translocation due to Globo H conjugation

KLH-Globo

KLH

250

200

150

Ab

so

rban

ce

Time (minutes)

100

50

0

–6920.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0

Figure S7 (globo h)3-DTPa-Klh hPlc.

Abbreviations: DTPa, diethylenetriamine pentaacetic acid; hPlc, high-performance liquid chromatography.

Figure S8 Klh 1h nMr.

Abbreviations: DMsO, dimethyl sulfoxide; nMr, nuclear magnetic resonance.

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

ppm

7.5

4183

6.0

920

4.3

809

3.5

545

2.6

008

2.0

000

2.8

258

3.8

858

4.9

028

5.0

536

4.2

621

4.6

443

3.9

098

8.0

920

2.3

809

2.5

545

3.3

521

5.6

560

2.4

103

2.0

035

7.4

8875

7.3

1817

7.2

6783

7.1

7485

7.0

9860

6.8

6981

6.8

5782

4.4

4759

4.3

6623

4.3

3281

3.8

4015

3.5

9591

3.5

1812

3.4

6542

3.4

5420

3.4

3425

3.3

6985

3.3

5792

3.2

7666

3.1

6038

3.1

4715

3.0

8854

3.0

7880

2.5

9508

2.5

3398

2.4

6033

2.3

2170

2.0

4436

2.0

2416

2.0

0752

1.8

2824

1.6

0201

1.5

1670

1.2

8740

Inte

gra

l

ppm 10 8 6 4 2 0

Current data parametersNameEXPNDPROCND

F2 – Acquisition parametersDate_Time 17.17INSTRUMPROBHD 5 mm QNP 1H/13PULPROG zg30TD 32,768Solvent DMSONS 32DS 0SWH 10,000.000 HzFIDRES 0.305176 HzAQ 1.6385000 secRG 812.7DW 50.000 usecDE 6.50 usecTE 298.0 K

D1 5.00000000 sec0.00000000 sec0.01500000 sec

MCRESTMCWRK

F2 – Processing parameters

NUC1 1HP1 10.50 usecPL1 –3.00 dBSF01 500.1345088 MHZ

SI 32,768SF 500.1300244 MHZWDW EMSSB 0

LB 0.30 HzGB 0PC 1.00

1D NMR parametersCX 20.00 cmCY 129.81 cmF1P 11.083 ppmF1 5543.02 HZF2P –0.648 ppmF2 –324.09 HZPPMCM –324.09 ppm/cmHZCM 293.35583 HZ/cm

plot

======== Channel f1 =======

Spect

20120619

Hajmohammadi41

D

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Des

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rapy

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from

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hajmohammadi et al

Current data parametersNameEXPNDPROCND

F2 – Acquisition parametersDate_Time 17.17INSTRUMPROBHD 5 mm QNP 1H/13PULPROG zg30TD 32,768Solvent DMSONS 32DS 0SWH 10,000.000 HzFIDRES 0.305176 HzAQ 1.6385000 secRG 812.7DW 50.000 usecDE 6.50 usecTE 298.0 KD1 5.00000000 sec

0.00000000 sec0.01500000 sec

MCRESTMCWRK

F2 – Processing parameters

NUC1 1HP1 10.50 usecPL1 –3.00 dBSF01 500.1345088 MHZ

SI 32,768SF 500.1300244 MHZWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

1D NMR plot parametersCX 20.00 cmCY 129.81 cmF1P 11.083 ppmF1 5,543.02 HzF2P –0.648 ppmF2 –324.09 HzPPMCM 0.58656 ppm/cmHZCM 293.35583 Hz/cm

======== Channel f1 =======

Spect

20120619

Hajmohammadi21

ppm 10 8 6 4 2 0

Inte

gra

lp

pm

5.9

870

5.5

91

34

7.5

79

84

7.4

61

67

7.3

54

21

7.3

01

52

7.1

87

43

7.1

28

03

6.9

71

47

6.9

14

20

4.4

4357

4.3

602

84.3

3188

3.8

4415

3.5

949

33.5

171

2

3.4

684

23.4

5420

3.4

3425

3.3

698

5

3.3

579

23.2

766

63.1

6038

3.1

4715

3.0

8854

3.0

7880

2.5

950

82.5

3377

2.4

6037

2.3

2170

2.0

4403

2.0

2414

2.0

075

21.8

282

41.6

0215

1.5

1674

1.2

8780

5.4

44

69

4.7

75

57

4.9

95

76

4.9

114

24

.88

02

0

4.7

01

52

4.5

46

02

4.4

414

3.6

692

0.3

010

0.2

873

0.3

602

0.2

527

0.6

369

0.2

855

2.8

985

2.5

155

2.1

592

3.7

052

4.8

508

5.9

025

4.1

668

4.4

273

3.4

555

8.0

524

2.4

271

2.5

840

3.7

296

5.8

701

2.5

826

2.0

72

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

Figure S9 globo h-Klh 1h nMr.

Abbreviations: DMsO, dimethyl sulfoxide; nMr, nuclear magnetic resonance.

Current data parametersNameEXPNDPROCND

F2 – Acquisition parametersDate_Time 17.17INSTRUMPROBHD 5 mm QNP 1H/13PULPROG zg30TD 32,768Solvent DMSONS 32DS 0SWH 10,000.000 HzFIDRES 0.305176 HzAQ 1.6385000 secRG 812.7DW 50.000 usecDE 6.50 usecTE 298.0 KD1 5.00000000 sec

0.00000000 sec0.01500000 sec

MCRESTMCWRK

F2 – Processing parameters

NUC1 1HP1 10.50 usecPL1 –3.00 dBSF01 500.1345088 MHZ

SI 32,768SF 500.1300244 MHZWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

1D NMR plot parametersCX 20.00 cmCY 129.81 cmF1P 11.083 ppmF1 5,543.02 HzF2P –0.648 ppmF2 –324.09 HzPPMCM 0.58656 ppm/cmHZCM 293.35583 Hz/cm

======== Channel f1 =======

Spect

20120619

Hajmohammadi21

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

ppm 10 8 6 4 2 0

Inte

gra

lppm

6.3

222

5.5

91

83

7.5

7984

7.4

6167

7.3

5421

7.3

0152

7.1

8743

7.1

2803

6.9

7147

6.9

1420

4.4

4357

4.3

6028

4.3

3188

3.8

4415

3.5

9493

3.5

1712

3.4

6842

3.4

5420

3.4

3425

3.3

6985

3.3

5792

3.2

7666

3.1

6038

3.1

4715

3.0

8854

3.0

7880

2.5

9508

2.5

3377

2.4

6037

2.3

2170

2.0

4403

2.0

2414

2.0

0752

1.8

2824

1.6

0215

1.2

8780

1.5

1674

5.4

44

41

4.7

75

48

4.9

95

20

4.9

112

34

.88

03

6

4.7

01

55

4.5

46

03

5.0

413

0.9

135

0.8

985

0.8

052

1.1

592

0.9

135

0.9

135

2.8

985

2.5

155

2.1

592

3.7

052

4.8

508

5.9

025

4.1

668

4.4

273

3.4

555

8.0

920

2.4

271

2.5

840

3.7

296

5.8

701

2.5

826

2.0

72

3.9

403

Figure S10 (globo h)3-DTPa-Klh 1h nMr.

Abbreviations: DMsO, dimethyl sulfoxide; DTPa, diethylenetriamine pentaacetic acid; nMr, nuclear magnetic resonance.

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rug

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(globo h)3-DTPa-Klh antigen

D

rug

Des

ign,

Dev

elop

men

t and

The

rapy

dow

nloa

ded

from

http

s://w

ww

.dov

epre

ss.c

om/ b

y 13

9.18

4.66

.129

on

12-S

ep-2

017

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per

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