Carbohydrate-based Vaccines Organic Chemistry 12 B Instructor Dr. Adamczeski Presented by Hanna Tong 5/3/06 8#

Carbohydrate-basedCarbohydrate-basedVaccinesVaccines

Organic Chemistry 12 BOrganic Chemistry 12 B

Instructor Dr. AdamczeskiInstructor Dr. Adamczeski

Presented by Hanna TongPresented by Hanna Tong

5/3/065/3/06

8#8#

Bacterial and viral infection is one of the major health problems Bacterial and viral infection is one of the major health problems worldwide. Therefore, the development of new vaccines to combat worldwide. Therefore, the development of new vaccines to combat these types of infections is very important. these types of infections is very important. Vaccines have generally Vaccines have generally been made from weakened or killed pathogens, or from been made from weakened or killed pathogens, or from several several variable carbohydrate molecules,variable carbohydrate molecules, which are extracted from t which are extracted from the cell-he cell-surface of bacteria and viruses. surface of bacteria and viruses. The carbohydrate-based agents from The carbohydrate-based agents from natural sources can be difficult to isolate, and the natural isolates can natural sources can be difficult to isolate, and the natural isolates can have heterogeneity and contamination problems. An alternative would have heterogeneity and contamination problems. An alternative would be to identify antigenic carbohydrates and then synthesize them in the be to identify antigenic carbohydrates and then synthesize them in the laboratory. laboratory.

This seminar will introduce the general outlook of synthetic This seminar will introduce the general outlook of synthetic carbohydrate-based vaccines, the history of development of them, and carbohydrate-based vaccines, the history of development of them, and their application. Some applications include the first approved human their application. Some applications include the first approved human vaccine based on a synthetic carbohydrate Quimi-Hib, cancer-specific vaccine based on a synthetic carbohydrate Quimi-Hib, cancer-specific carbohydrate vaccines: the Globo-H, and multiantigenic vaccine. I will carbohydrate vaccines: the Globo-H, and multiantigenic vaccine. I will also discuss some common pharmaceutical techniques used to also discuss some common pharmaceutical techniques used to synthesize them, including: OPopS, and Automated Solid Phase synthesize them, including: OPopS, and Automated Solid Phase Synthetic Technology, theirs developers, and principles. Synthetic Technology, theirs developers, and principles.

AbstractAbstract

I. IntroductionI. Introduction1.1. What is Vaccines?What is Vaccines?2. What is Antibody?2. What is Antibody?2.2. What is Carbohydrate?What is Carbohydrate?3.3. Classic Carbohydrate Vaccines Classic Carbohydrate Vaccines 4.4. Synthetic Carbohydrate VaccinesSynthetic Carbohydrate Vaccines

II. Why Carbohydrate? II. Why Carbohydrate? III. History of Development of Carbohydrate VaccinesIII. History of Development of Carbohydrate VaccinesIV. Synthetic carbohydrate: Advantages (comparing with carbohydrates IV. Synthetic carbohydrate: Advantages (comparing with carbohydrates

from natural sources) from natural sources)V. SynthesisV. Synthesis: recently developed carbohydrate synthesis techniques : recently developed carbohydrate synthesis techniques

1. Basic Synthesis Technique1. Basic Synthesis Technique2. 2. OPopS (Optimer One-Pot Synthesis)OPopS (Optimer One-Pot Synthesis)33.. Automated Solid-phase Synthetic TechnologyAutomated Solid-phase Synthetic Technology

VIVI. Applications: some carbohydrate-based vaccines:. Applications: some carbohydrate-based vaccines:1.1. The first approved human vaccine based on a The first approved human vaccine based on a

synthetic synthetic carbohydrate: Quimi-Hib carbohydrate: Quimi-Hib 2.2. Cancer: Globo H, multiantigenic agentCancer: Globo H, multiantigenic agent

VII. ConclusionVII. Conclusion

OutlineOutline

What is … What is … Vaccine, Antibody, Vaccine, Antibody, Carbohydrate, & Carbohydrate Carbohydrate, & Carbohydrate Vaccine ?Vaccine ?

Vaccine: a preparation of a weakened or killed pathogen Vaccine: a preparation of a weakened or killed pathogen such as a such as a bacterium or virus, or of a portion of the pathogen's structure that bacterium or virus, or of a portion of the pathogen's structure that upon administration stimulates antibody immunity against the upon administration stimulates antibody immunity against the pathogen but is incapable of causing severe infection.pathogen but is incapable of causing severe infection.

Antibody: protein made by certain white blood cells in response to a Antibody: protein made by certain white blood cells in response to a foreign substance (antigen). foreign substance (antigen).

CarbohydrateCarbohydrate: are sugars or several sugars linked together, : are sugars or several sugars linked together, contains only contains only C, H, and O, usually in the ratio 1:2:1. C, H, and O, usually in the ratio 1:2:1. Carbohydrate Formula: (C)n+(H2O)n = carbon + water = Carbohydrate Formula: (C)n+(H2O)n = carbon + water = carbohydrates.carbohydrates.

Classic carbohydrate-based vaccine: carbohydrate used in the Classic carbohydrate-based vaccine: carbohydrate used in the

vaccine is isolated from natural sources, hence can have vaccine is isolated from natural sources, hence can have heterogeneity and contamination problems. heterogeneity and contamination problems.

Alternative: identify carbohydrates and then synthesize them in the Alternative: identify carbohydrates and then synthesize them in the laboratory.laboratory.

Why Carbohydrate ? Why Carbohydrate ?

Cells of our body have Cells of our body have sensors made out of sensors made out of carbohydrates (on outer carbohydrates (on outer surface of plasma surface of plasma membrane)membrane)

These sensors can detect These sensors can detect many kinds of stimuli, and many kinds of stimuli, and can signal the immune can signal the immune system to respond.system to respond.

Specific carbohydrates Specific carbohydrates that carry appropriate that carry appropriate recognition properties are recognition properties are synthesized and used in synthesized and used in carbohydrate vaccines. carbohydrate vaccines.

http://wong.scripps.edu/

History of Development History of Development In the ‘20s and ‘30s, K. Landsteiner, O. Avery, and W. Goebel In the ‘20s and ‘30s, K. Landsteiner, O. Avery, and W. Goebel

(Rockefeller (Rockefeller University) showed that University) showed that nonimmunogenicnonimmunogenic carbohydrates from bacteria could be carbohydrates from bacteria could be converted into converted into immunogenicimmunogenic (produced protective immune responses)(produced protective immune responses) by attaching by attaching them to them to proteins and inducing antibodies. proteins and inducing antibodies.

Around 1980, two research teams of the University of Rochester Around 1980, two research teams of the University of Rochester and of and of National Institute of Child Health & Human Development (NICHD), National Institute of Child Health & Human Development (NICHD),

Bethesda, Md, working independently, developed a Bethesda, Md, working independently, developed a semisynthetic semisynthetic (natural source carbohydrate combined with (natural source carbohydrate combined with carrier protein) carrier protein) carbohydrate Hib vaccinescarbohydrate Hib vaccines. .

Also around 1980, researcher H. Jennings (National Also around 1980, researcher H. Jennings (National Research Research Council of Canada's Institute of Biological Council of Canada's Institute of Biological Sciences, Ottawa), Sciences, Ottawa), and coworkers, developed a and coworkers, developed a semisynthetic carbohydrate semisynthetic carbohydrate vaccine that protects vaccine that protects infants against bacterial meningitis, the infants against bacterial meningitis, the most common form of most common form of meningitis in young children.meningitis in young children.

The first commercial vaccine made from a The first commercial vaccine made from a synthetic synthetic carbohydrate was approved Nov. 2004 carbohydrate was approved Nov. 2004 in Cuba.in Cuba.

Borman, Stu. Borman, Stu. Carbohydrate VaccinesCarbohydrate Vaccines. . Chemical & Engineering News, Chemical & Engineering News, August 9, 2004, August 9, 2004, Volume 82, Number 32, pp. 31-35Volume 82, Number 32, pp. 31-35

Synthetic Carbohydrate - Synthetic Carbohydrate - AdvantagesAdvantages

Natural carbohydrates and glycopeptides are too scarce and too Natural carbohydrates and glycopeptides are too scarce and too difficult to isolate.difficult to isolate.

Synthetic carbohydrates can be produced as homogeneous single Synthetic carbohydrates can be produced as homogeneous single compounds, while naturally derived carbohydrates are compounds, while naturally derived carbohydrates are heterogeneous mixtures and may include impurities and heterogeneous mixtures and may include impurities and contaminants.contaminants.

Medicinal chemistry techniques can be used to derivatize and Medicinal chemistry techniques can be used to derivatize and modify synthetic carbohydrates to make vaccines that are more modify synthetic carbohydrates to make vaccines that are more immunogenic than those based on natural carbohydrates.immunogenic than those based on natural carbohydrates.

Synthetic carbohydrates can have very precise construct, and the Synthetic carbohydrates can have very precise construct, and the variables of chain length and chain density can be controlled variables of chain length and chain density can be controlled because product is pure.because product is pure.

The advent of synthetic carbohydrate The advent of synthetic carbohydrate vaccines is a triumph of tremendous vaccines is a triumph of tremendous advances in advances in oligosaccharide synthesisoligosaccharide synthesis, , since …since …

"Without the years people have put into "Without the years people have put into synthetic organic chemistry and synthetic organic chemistry and carbohydrate chemistry, you wouldn't be carbohydrate chemistry, you wouldn't be able to make the kinds of investigational able to make the kinds of investigational vaccines you canvaccines you can today.“today.“ Chem. Prof. Samuel J. Danishefsky (Memorial Sloan-Kettering Cancer Center (MSKCC) & Columbia University, N.Y City)Chem. Prof. Samuel J. Danishefsky (Memorial Sloan-Kettering Cancer Center (MSKCC) & Columbia University, N.Y City)

BasicBasic Synthesis TechniqueSynthesis Technique

Condensation reaction.Condensation reaction. --OHOH from the first and from the first and HH- from - from

the second sugar are removed.the second sugar are removed. Glycosidic bondGlycosidic bond (-O- bond (-O- bond

connect. the two sugars) is connect. the two sugars) is formed.formed.

sugarsugar11- - OH OH + + HHOO - sugar - sugar22

sugarsugar11- + - - + - OO - sugar - sugar2 2 + -+ -OH OH + + H-H-

sugarsugar11- - OO - sugar - sugar2 2 + + HHOH OH

http://www.bergen.org/ACADEMY/Bio/molbio/LACTOSE_SYNTH/LactoseSynth.htmlhttp://www.bergen.org/ACADEMY/Bio/molbio/LACTOSE_SYNTH/LactoseSynth.html

Synthesis two beta-glucoses

http://www.bergen.org/ACADEMY/Bio/molbio/LACTOSE_SYNTH/LactoseSynth.htmlhttp://www.bergen.org/ACADEMY/Bio/molbio/LACTOSE_SYNTH/LactoseSynth.html

Carbohydrate Synthesis Technique: Carbohydrate Synthesis Technique: 1. OPopS1. OPopS

Prof. Chi-Huey WongProf. Chi-Huey Wonghttp://wong.scripps.edu/boss.htm

Optimer One-Pot Synthesis. Optimer One-Pot Synthesis. Developed by Dr. Chi-Huey Wong, Developed by Dr. Chi-Huey Wong, Prof. of Chemistry, The Skaggs Prof. of Chemistry, The Skaggs Institute for Chemical Biology & Institute for Chemical Biology & workers.workers. Principle:Principle:

- Put all the chemicals in the pot. - Put all the chemicals in the pot.

- Let them put themselves together, - Let them put themselves together, the most reactive chemical assembles the most reactive chemical assembles first .first . Main Aspects: Main Aspects:

1. Synthesize a number of mono- & 1. Synthesize a number of mono- & disaccharides w/different protecting disaccharides w/different protecting group patterns & measure their group patterns & measure their reactivity. reactivity.

2. A computer program calculates the 2. A computer program calculates the reactivity of involved reactants & reactivity of involved reactants & selects presynthesized building selects presynthesized building blocks. blocks.

3. Computer program analyzes target 3. Computer program analyzes target oligosaccharide and suggests a set of oligosaccharide and suggests a set of presynthesized carbohydrate blocks, presynthesized carbohydrate blocks, combines them from most to least combines them from most to least reactive get desired product in reactive get desired product in one reaction.one reaction.

http://www.scripps.edu/chem/wong/optimer.html

Carbohydrate Synthesis Technique: Carbohydrate Synthesis Technique: 2. Automated Solid-phase 2. Automated Solid-phase SynthesisSynthesis

Prof. Peter. H. Seeberger http://www.seeberger.ethz.ch/people/seeberger

Developed by Dr. Seeberger, Prof. for Organic Chemistry Developed by Dr. Seeberger, Prof. for Organic Chemistry (ETH Zurich, Switzerland), & coworkers. (ETH Zurich, Switzerland), & coworkers.

Inspired by Merrifield's solid-phase peptide synthesis.Inspired by Merrifield's solid-phase peptide synthesis. This method is applied to produce vaccine candidates This method is applied to produce vaccine candidates

for malaria, HIV, tuberculosis, & bacterial infection). for malaria, HIV, tuberculosis, & bacterial infection).

Principle: Principle:

- Attach one side of the sugar to a secure support, the - Attach one side of the sugar to a secure support, the chain grows at the other side. chain grows at the other side.

- Add monosaccharide one by one. - Add monosaccharide one by one.

- After everything is done, cleavage from the solid support- After everything is done, cleavage from the solid support

Main Aspects:Main Aspects:

1. Select ‘solid phase’, a polymer inert to all reaction conditions. Most solid-phase 1. Select ‘solid phase’, a polymer inert to all reaction conditions. Most solid-phase is polystyrene, cross-linked with 1% divinylbenzene.is polystyrene, cross-linked with 1% divinylbenzene.

2. Select linker to attach 1st sugar to the solid support, which inert to all reaction 2. Select linker to attach 1st sugar to the solid support, which inert to all reaction conditions. conditions.

3. Select glycosylating agents: such as thioglycosides, anomeric fluorides, 3. Select glycosylating agents: such as thioglycosides, anomeric fluorides, trichloro-acetimidates, and sulfoxides.trichloro-acetimidates, and sulfoxides.

4. Select protecting group: permanent protection for unoperated hydroxyl, 4. Select protecting group: permanent protection for unoperated hydroxyl, temporary one for involved hydroxyl.temporary one for involved hydroxyl.

5. Repeat the coupling cycles to get the desired sequence.5. Repeat the coupling cycles to get the desired sequence.

6. Remove u6. Remove unreacted reagents at anynreacted reagents at any synthetic step by a wash proceduresynthetic step by a wash procedure..http://pubs.acs.org/cgi-bin/jtextd?chreay/100/12/html/cr9903104.html#tcrx56

Automated Solid-phase Synthesis: StrategiesAutomated Solid-phase Synthesis: Strategies

Donor-bound: Dimethyldioxirane (DMDO) Donor-bound: Dimethyldioxirane (DMDO) converts the double bond into epoxide. OH of converts the double bond into epoxide. OH of acceptor 52 reacts w/ 51 to give the desired -acceptor 52 reacts w/ 51 to give the desired -glycoside 53. Repeat this procedure to get (1 glycoside 53. Repeat this procedure to get (1 6)-linked tetrasaccharide 55. 6)-linked tetrasaccharide 55.

Acceptor-bound: Excess of donor is needed Acceptor-bound: Excess of donor is needed to maximize yield. to maximize yield.

Bidirectional: oligosaccharide grows in both Bidirectional: oligosaccharide grows in both directions. Used to prepare branched structures. directions. Used to prepare branched structures.

..

http://pubs.acs.org/journals/chreay/100/i12/figures/cr9903104h00012.htmlhttp://pubs.acs.org/journals/chreay/100/i12/figures/cr9903104h00012.html



Applications of synthetic Applications of synthetic carbohydrate vaccinecarbohydrate vaccine

"synthetic vaccines against any unique disease-associated carbohydrate structures should be "synthetic vaccines against any unique disease-associated carbohydrate structures should be possible" possible"

(Prof. Seeberger)(Prof. Seeberger)

Cuba-approved vaccine Quimi Hib against Cuba-approved vaccine Quimi Hib against Haemophilus influenzaeHaemophilus influenzae type type b.b.

Globo H: breast cancer vaccine candidate.Globo H: breast cancer vaccine candidate. Unimolecular multivalent vaccine candidate: vaccine with 5 different Unimolecular multivalent vaccine candidate: vaccine with 5 different

cancer antigens.cancer antigens.

Many kinds of vaccine for fungal, bacterial, and protozoan infections etc. Many kinds of vaccine for fungal, bacterial, and protozoan infections etc. have been developed, is developing, or are currently tested in animals.have been developed, is developing, or are currently tested in animals.

Borman, Stu. Borman, Stu. Carbohydrate VaccinesCarbohydrate Vaccines. . Chemical & Engineering News, Chemical & Engineering News, August 9, 2004, August 9, 2004, Volume 82, Number 32, pp. 31-35Volume 82, Number 32, pp. 31-35

Under study: a Under study: a vaccine candidate for vaccine candidate for candidiasis, a type of candidiasis, a type of fungal infection that fungal infection that can affect the skin, can affect the skin, mucous membranes, mucous membranes, or bloodstream.or bloodstream.

http://pubs.acs.org/cen/coverstory/8232/8232vaccines.html

Quimi Hib - Quimi Hib - The first approved The first approved human vaccine based on a synthetic human vaccine based on a synthetic carbohydrate carbohydrate

Prevents Prevents Haemophilus Haemophilus influenzaeinfluenzae type b (Hib), that type b (Hib), that causes pneumonia & causes pneumonia & meningitis in infants and meningitis in infants and young children.young children.

Developed by Cuban and Developed by Cuban and Canadian researchers Canadian researchers

Passed 7 clinical trials. The Passed 7 clinical trials. The efficacy in long-term efficacy in long-term protection in newborns was protection in newborns was almost 100%. Total immunized almost 100%. Total immunized population > 2 000 children. population > 2 000 children.

The carbohydrate component The carbohydrate component is sPRP (poly-ribosylribitol is sPRP (poly-ribosylribitol phosphate oligosaccharide), phosphate oligosaccharide), contains 7 units of contains 7 units of Haemophilus influenzaeHaemophilus influenzae type b type b polysaccharide.polysaccharide.

Applied but simplified existing Applied but simplified existing synthesis process for 5 units to synthesis process for 5 units to get 7 units rPPR.get 7 units rPPR.


sPPR conjugated to a tetanus toxoid sPPR conjugated to a tetanus toxoid (TT) protein.(TT) protein. sPRP-TT mixed with aluminium sPRP-TT mixed with aluminium phosphate (phosphate (immune activator, immune activator, stimulates PRP-specific antibodies) stimulates PRP-specific antibodies) using a process devised by reseachers using a process devised by reseachers of University of Dundee, Scotland.of University of Dundee, Scotland.

Globo H Cancer VaccineGlobo H Cancer Vaccine(OPopS application)(OPopS application)

Cancer carbohydrate vaccines Cancer carbohydrate vaccines evoke antibodies against the evoke antibodies against the carbohydrate antigens of cancer carbohydrate antigens of cancer cells , that aren't produced by cells , that aren't produced by normal cells.normal cells.

Globo H is a carbohydrate which Globo H is a carbohydrate which acts as antigen on breast cancer, acts as antigen on breast cancer, is synthesized by Optimer Pharm.is synthesized by Optimer Pharm.

In Globo H, 4 monosaccharides In Globo H, 4 monosaccharides are linked with 1 disaccharide to are linked with 1 disaccharide to make Globo H hexasaccharide. make Globo H hexasaccharide. This is then linked to a protein This is then linked to a protein carrier to make a candidate carrier to make a candidate breast cancer vaccine. breast cancer vaccine. (Ac = (Ac = acetyl, Bn = benzyl, Bz = benzoyl, acetyl, Bn = benzyl, Bz = benzoyl, Lev = levulinoyl, STol = thiotoluyl, Lev = levulinoyl, STol = thiotoluyl, Troc = 2,2,2-trichloroethoxy-Troc = 2,2,2-trichloroethoxy-carbonyl).carbonyl).

A Phase I clinical trial of this Globo A Phase I clinical trial of this Globo H-protein conjugate & an immune H-protein conjugate & an immune activator was recently carried out.activator was recently carried out.


Multiantigenic agent: Multiantigenic agent: Cancer-specific Carbohydrate VaccineCancer-specific Carbohydrate Vaccine

Includes a single glycopeptide that contains five tumor antigens (Globo H, Includes a single glycopeptide that contains five tumor antigens (Globo H, STn, Tn, Lewis, and TF are tumor antigens; Ac = acetyl). A linker is attached STn, Tn, Lewis, and TF are tumor antigens; Ac = acetyl). A linker is attached to the glycopeptide, followedto the glycopeptide, followed by a deprotection and subsequent conjugationby a deprotection and subsequent conjugation

to two different immunogenic carriers, keyhole limpet hemocyaninto two different immunogenic carriers, keyhole limpet hemocyanin and and NN- -- -palmitoyl-palmitoyl-SS-[2,3-bis(palmitoyloxy)-(2-[2,3-bis(palmitoyloxy)-(2RSRS)-propyl]-L-cysteine,)-propyl]-L-cysteine, resulted in the resulted in the vaccine construct.vaccine construct.

Is considered as next generation synthetic carbohydrate vaccine. Is considered as next generation synthetic carbohydrate vaccine.


ReferencesReferences Borman, Stu. Borman, Stu. Cancer Vaccine is Best in ClassCancer Vaccine is Best in Class. . Chemical & Engineering Chemical & Engineering

News, Volume 83, Number 37, p. 10 (9/News, Volume 83, Number 37, p. 10 (9/2005)2005) Borman, Stu. Borman, Stu. Carbohydrate AdvancesCarbohydrate Advances. . Chemical & Engineering News, Chemical & Engineering News,

Volume 83, Number 32, pp. 41-50 (8/Volume 83, Number 32, pp. 41-50 (8/2005) 2005) Borman, Stu. Borman, Stu. Carbohydrate VaccinesCarbohydrate Vaccines. . Chemical & Engineering News, Chemical & Engineering News,

Volume 82, Number 32, pp. 31-35 (8/Volume 82, Number 32, pp. 31-35 (8/2004) 2004) Borman, Stu. Borman, Stu. Combinatorial ChemistryCombinatorial Chemistry. . Chemical & Engineering News, Chemical & Engineering News,

Volume 80, Number 45, pp. 43-57 (Volume 80, Number 45, pp. 43-57 (11/2002)11/2002) France, Simon. France, Simon. Vaccine Sweetness SynthesizeVaccine Sweetness Synthesize. . Drug Drug

[email protected]@nature.com

http://www.nature.com/http://www.nature.com/drugdiscdrugdisc//resres_high/articles/nrd1507.html_high/articles/nrd1507.html OPopS Technology.OPopS Technology. http://www.optimerpharma.com/core_technology.htmhttp://www.optimerpharma.com/core_technology.htm Professor Chi-Huey Wong.Professor Chi-Huey Wong. http://wong.scripps.edu/http://wong.scripps.edu/ Seeberger Research GroupSeeberger Research Group. . http://www.seeberger.ethz.ch/ Seeberger, Peter H. & Haase, W. Christian. Seeberger, Peter H. & Haase, W. Christian. Solid-Phase Oligosaccharide Solid-Phase Oligosaccharide

Synthesis and Combinatorial Carbohydrate Libraries. Synthesis and Combinatorial Carbohydrate Libraries. ACSACS

http://pubs.acs.org/cgi-in/jtextd?chreay/100/12/html/cr9903104.html#tcrhttp://pubs.acs.org/cgi-in/jtextd?chreay/100/12/html/cr9903104.html#tcrx3x3

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Carbohydrate-based Vaccines Organic Chemistry 12 B Instructor Dr. Adamczeski Presented by Hanna Tong 5/3/06 8#