Sulfated Polyserines: A Potential Heparin and Dermatan Sulfate Mimetic
(2022) KEML10 20212Department of Chemistry
- Abstract
- All around in our bodies – inside and on top of cells, as well as in the extracellular matrix (ECM) – complex carbohydrate chains known as glycosaminoglycans (GAGs) are to be found. While some of their properties are known, many functions and biosynthetic pathways remain undiscovered. Heparin is one of the GAGs whose function in the body is not completely known, however, it has been used as an anticoagulant since the 1930s after the discovery that it inhibits the blood clotting cascade in multiple steps, primarily by enhancing the effects of antithrombin (AT), a factor Xa inhibitor. Nowadays, heparin makes out the biggest part of a 13.8-billion-dollar industry, with most of it originating from China. Heparin is not synthesized but... (More)
- All around in our bodies – inside and on top of cells, as well as in the extracellular matrix (ECM) – complex carbohydrate chains known as glycosaminoglycans (GAGs) are to be found. While some of their properties are known, many functions and biosynthetic pathways remain undiscovered. Heparin is one of the GAGs whose function in the body is not completely known, however, it has been used as an anticoagulant since the 1930s after the discovery that it inhibits the blood clotting cascade in multiple steps, primarily by enhancing the effects of antithrombin (AT), a factor Xa inhibitor. Nowadays, heparin makes out the biggest part of a 13.8-billion-dollar industry, with most of it originating from China. Heparin is not synthesized but extracted from pig’s intestines, and to satisfy the yearly demand hundreds of millions of animals are needed. Other anticoagulants are present on the market, but nothing has significantly challenged heparin yet. In 2019, a study from Tykesson et al. discovered that recombinant dermatan sulfate GAGs (more specifically 2,4-sulfated) exhibit anticoagulant effects like heparin by a strong interaction with the thrombin inhibitor heparin cofactor II (HCII).
In this project we hypothesize that HCII binds recDS-2,4 because of the specific pattern of charges and that such a pattern can also be achieved by a fully O-sulfated polyserine. Synthesis of persulfated compounds is challenging because of the charge density and polarity of the product, which complicates traditional work-up. In this project, a tri- and pentaserine synthesized by solid-phase peptide synthesis (SPPS) are used as targets for sulfation. The sulfation strategy was using trimethylsilyl chlorosulfonate, with the expectation that this would avoid the problem with anionic crowding, as well as produce a non-polar molecule that could be worked up by flash chromatography. The reagent was first investigated on benzyl β-L-arabinose with good result: all three hydroxyl groups were sulfated despite steric crowding and the product could easily be purified due to its highly apolar nature – however, it did not seem to be stable when deprotected. The sulfation on the polyserine chains did not give any detectable product – a lack of starting material forcing the reactions to be done on a small scale, probably combined with a low yield, seemed likely to be the main reasons for this. Since the sulfation reagent remains promising due to its ability to fully sulfate benzyl β-L-arabinose, a fully sulfated polyserine should be possible to synthesize in the future with some optimizations of the reaction and can then be tested for inhibiting properties on the blood clotting cascade. (Less) - Popular Abstract (Swedish)
- Kolhydrater är livsviktiga för människor och djur av fler anledningar än för att generera energi; överallt i kroppen –på cellmembran, inuti, och utanför celler – hittas långa kolhydratkedjor som kallas glukosaminoglykaner. Dessa kedjor bär många negativa laddningar, till stor del i form av sulfatgrupper, och kan vara självständiga eller kovalent bundna till ett protein – det senare fallet inkluderar proteoglykaner, där ungefär 50% av massan utgörs av linjära glukosaminoglykaner.Trots att dessa kolhydratkedjor är frekvent förekommande i kroppen är området i stora drag outforskat.
En typ av glukosaminoglykan är heparin, vilken tack vare sina antikoagulerande egenskaper används som intravenöst blodförtunnande medel sedan 30-talet och är... (More) - Kolhydrater är livsviktiga för människor och djur av fler anledningar än för att generera energi; överallt i kroppen –på cellmembran, inuti, och utanför celler – hittas långa kolhydratkedjor som kallas glukosaminoglykaner. Dessa kedjor bär många negativa laddningar, till stor del i form av sulfatgrupper, och kan vara självständiga eller kovalent bundna till ett protein – det senare fallet inkluderar proteoglykaner, där ungefär 50% av massan utgörs av linjära glukosaminoglykaner.Trots att dessa kolhydratkedjor är frekvent förekommande i kroppen är området i stora drag outforskat.
En typ av glukosaminoglykan är heparin, vilken tack vare sina antikoagulerande egenskaper används som intravenöst blodförtunnande medel sedan 30-talet och är ledande på marknaden. Heparin har dock sina nackdelar, bland annat på grund av att det inte produceras syntetiskt, utan utvinns från grisar, till största del i Kina. Mellan 2007–2008 cirkulerade förorenat heparin på marknaden vilket resulterade i nästan 100 dödsfall där den bakomliggande orsaken tros vara Kinas bristfälliga regleringar kring djurhållningen i livsmedel- och hälsoindustrin.
Forskning kring en annan glukosaminoglykan, dermatansulfat, ledde till upptäckten att även den (med ett visst mönster av sulfatgrupper) uppvisade blodförtunnande egenskaper likt heparin, vilket ledde till hypotesen att en molekyl med sulfatgrupper på samma positioner som dermatansulfat också skulle kunna verka antikoagulerande. Molekylen som teoretiskt sett passade in på denna beskrivning var en kedja av aminosyran serin, med en sulfatgrupp på varje serindel.
Fokus i detta projekt har varit att skapa den sulfaterade serinkedjan för att sedan undersöka dess effekt på blodkoagulationen; då sulfatering i praktiken för med sig en hel del svårigheter kan detta vara lättare sagt än gjort. Sulfatering testades först på en sockermolekyl, vilket gav lovande resultat, men fungerade inte lika bra på serinkedjan; möjligen var det alldeles för lite produkt som skapades för att ge ett tydligt resultat. Serinkedjan var även något förorenad, vilket störde analysen. Då sulfaterings-strategin fungerade på sockermolekylen är det möjligt att det även kan göra det på serinkedjan, men reaktionen bör då ske i en större skala, och eventuella föroreningar bör avlägsnas i förväg. Endast en mycket liten mängd produkt krävs för vidare testning, så länge den är detekterbar och fri från föroreningar, med de förbättringar som nämnts kan teorin om den antikoagulerande molekylen testas inom en snar framtid. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9072357
- author
- Zuleta Sjögren, Isolde LU
- supervisor
- organization
- course
- KEML10 20212
- year
- 2022
- type
- M2 - Bachelor Degree
- subject
- keywords
- Organisk kemi, Kolhydrat, Heparin
- language
- English
- id
- 9072357
- date added to LUP
- 2022-01-19 14:05:26
- date last changed
- 2022-01-19 14:06:41
@misc{9072357,
abstract = {{All around in our bodies – inside and on top of cells, as well as in the extracellular matrix (ECM) – complex carbohydrate chains known as glycosaminoglycans (GAGs) are to be found. While some of their properties are known, many functions and biosynthetic pathways remain undiscovered. Heparin is one of the GAGs whose function in the body is not completely known, however, it has been used as an anticoagulant since the 1930s after the discovery that it inhibits the blood clotting cascade in multiple steps, primarily by enhancing the effects of antithrombin (AT), a factor Xa inhibitor. Nowadays, heparin makes out the biggest part of a 13.8-billion-dollar industry, with most of it originating from China. Heparin is not synthesized but extracted from pig’s intestines, and to satisfy the yearly demand hundreds of millions of animals are needed. Other anticoagulants are present on the market, but nothing has significantly challenged heparin yet. In 2019, a study from Tykesson et al. discovered that recombinant dermatan sulfate GAGs (more specifically 2,4-sulfated) exhibit anticoagulant effects like heparin by a strong interaction with the thrombin inhibitor heparin cofactor II (HCII).
In this project we hypothesize that HCII binds recDS-2,4 because of the specific pattern of charges and that such a pattern can also be achieved by a fully O-sulfated polyserine. Synthesis of persulfated compounds is challenging because of the charge density and polarity of the product, which complicates traditional work-up. In this project, a tri- and pentaserine synthesized by solid-phase peptide synthesis (SPPS) are used as targets for sulfation. The sulfation strategy was using trimethylsilyl chlorosulfonate, with the expectation that this would avoid the problem with anionic crowding, as well as produce a non-polar molecule that could be worked up by flash chromatography. The reagent was first investigated on benzyl β-L-arabinose with good result: all three hydroxyl groups were sulfated despite steric crowding and the product could easily be purified due to its highly apolar nature – however, it did not seem to be stable when deprotected. The sulfation on the polyserine chains did not give any detectable product – a lack of starting material forcing the reactions to be done on a small scale, probably combined with a low yield, seemed likely to be the main reasons for this. Since the sulfation reagent remains promising due to its ability to fully sulfate benzyl β-L-arabinose, a fully sulfated polyserine should be possible to synthesize in the future with some optimizations of the reaction and can then be tested for inhibiting properties on the blood clotting cascade.}},
author = {{Zuleta Sjögren, Isolde}},
language = {{eng}},
note = {{Student Paper}},
title = {{Sulfated Polyserines: A Potential Heparin and Dermatan Sulfate Mimetic}},
year = {{2022}},
}