Template-Assisted Enzymatic Synthesis of Oligopeptides from a Polylactide Chain

Peptides are often attached to polymer materials, as bioactive components, for the control of interactions between the material and its surrounding proteins and cells. However, synthesizing peptides and attaching them to polymers can be challenging and laborious. Herein, we describe the grafting of oligopeptides to an aliphatic polyester, using a one-step chemo-enzymatic synthesis with papain as the biocatalySt. To enable enzyme-mediated functionalization of the polyester, ethyl hept-6-enoylalaninate (grafter) was synthesized and attached to polylactide chains using thiol-ene click reactions. The oligopeptides were grafted onto the polylactide chains using two different synthetic routes: the grafting from strategy, in which the grafter was attached to the polyester prior to oligopeptide synthesis, or the grafting to strategy, in which oligopeptides were synthesized on the grafter first, then attached to the polymer chain. The final products were analyzed and their structures were confirmed using nuclear magnetic resonance (NMR). The peptide attachment was evaluated using size exclusion chromatography (SEC), contact angle measurement and energy-dispersive X-ray spectroscopy scanning electron microscopy (EDS-SEM). Furthermore, the mechanistic aspects of the synthesis of the oligopeptides on the grafter were studied using molecular dynamics (MD) simulations. The simulation revealed that hydrogen bonding (between the P1 amide nitrogen of the grafter backbone and the carbonyl oxygen of D158 in the papain) maintain the grafter in a productive conformation to stabilize the transition state of nitrogen inversion, a key step of the biocatalytic mechanism. Apart from being biologically relevant, both experimental and computational results suggest that the designed grafter is a good template for initiating chemo-enzymatic synthesis. The results also showed that the grafting to strategy was more successful compared to the grafting from strategy. Overall, a successful synthesis of predefined peptide functionalized polylactide was prepared, where the oligopeptides were grafted in an easy, time efficient, and environmentally friendly way. QC 2017121

Svällningsorsak i EPDM gummipackningar

The aim of this master thesis was to develop an analysis method which identifies the swelling cause of EPDM rubber gaskets. The method shall facilitate failure investigation of rubber gaskets used in plate heat exchangers which require both determinations of volatile and involatile compounds as well as polar and non-polar compounds. To achieve this goal a combination analysis method of Headspace Gas chromatography Mass spectrometry (GC-MS) and liquid-liquid extraction Fourier Transform Infrared spectroscopy (FT-IR) was chosen. Three extraction fluids were tested to extract the compounds from the EPDM rubber: tetrahydrofuran, 2-propanol and supercritical carbon dioxide. The analysis method was first tested on EPDM rubber samples swelled in four known solvents with different volatility and polarity (hexane, acetic acid, 1-octanol and pentadecane). Then it was tested on EPDM rubber samples swelled in three different mixtures of these four solvents and finally on two EPDM gaskets, swelled by unknown media, that were being assessed in an actual failure investigations. The analysis method was successful in identifying compounds which caused EPDM rubber gaskets to swell but not in a way as it was supposed to. The FT-IR analysis could not be used to identify compounds in a mixture because the spectra of mixtures are very difficult to analyze. However the FT-IR results complimented the results from the GC-MS analysis which due to a poor search database was not good enough to determine the exact composition of the swelling agents by itself. The liquid extraction also gave useful information about how much swelling agents that were absorbed by the EPDM gasket. Tetrahydrofuran and 2-propanol were the most effective for extraction of the solvents. However, supercritical carbon dioxide was very good for selective extraction of non-polar compounds. The method detected both polar and non-polar compounds but non-polar compounds with low boiling point were not detected. It did not interact strongly enough with the GC-column used in this trials, because the polarity of the column was not sufficient. The method detects both volatile and involatile compounds. But to be sure that all volatile swelling agents were detected a special sample handling technique must be developed. To improve the analysis method it is suggested that the method is developed so that only GC-MS analysis is needed. This is accomplished by further GC-MS analysis with other GC-columns and better search databases. It is also suggested that the method is developed so that it is not only a qualitative analysis method but also a quantitative analysis method.  In that way it can be used as a much more effective tool during failure investigations.Syftet med det här examensarbetet var att ta fram en analysmetod för att identifiera svällningsorsaker i EPDM gummipackningar. Metoden utvecklades för att underlätta haveriutredningar av havererade gummipackningar använda i plattvärmeväxlare. Kraven man kan ställa på en sådan metod är att den har ett brett detektionsområde där både polära och opolära föreningar samt lättflyktiga och svårflyktiga föreningar kan analyseras. För att uppnå dessa krav utvecklades en analysmetoden som bestod av en kombination av headspace Gas Kromatografi-Masspektrometri (GC-MS) och vätske-vätske extraktion Fourier Transform-Infraröd spektroskopi (FT-IR). Tre olika extraktionsvätskor testades för att extrahera ut de ämnena som svällt EPDM gummipackningen (2-propanol, tetrahydrofuran och superkritisk koldioxid). Analysmetoden testades först på EPDM packningar som svällt i fyra olika kända lösningsmedel med varierande kokpunkt och polaritet (hexan, ättiksyra, 1-oktanol och pentadecane). Därefter testades metoden på EPDM packningar som svällt i blandningar av dessa kända lösningsmedel och slutligen testades metoden på två EPDM packningar som havererat i plattvärmeväxlare.   Resultaten visade att analysmetoden fungerade för att identifiera ämnen som orsakat svällning i EPDM packningar men inte på det sätt som metoden från början var tänkt att fungera. FT-IR kan inte användas för att analysera de ämnen som inte detekteras med GC-MS. Detta beror framför allt på att det är väldigt svårt att identifiera ämnen i blandningar med FT-IR. Det visade sig dock att resultaten från FT-IR kunde användas för att komplettera resultaten från GC-MS (som var för dåliga på grund av en dålig sökdatabas). Resultaten från vätske-vätske extraktionen gav även användbar information om hur mycket ämnen som absorberats av packningen. Av de tre extraktions vätskor som testades var 2-propanol och tetrahydrofuran mest effektiva för att få ut de ämnen som svällt EPDM packningen. Den superkritiska koldioxiden var inte alls lika effektiv men var och andra sidan bättre om en mer selektiv extraktion av opolära ämnen önskades.   Analysmetoden detekterade både polära och opolära ämnen men opolära ämnen med låg kokpunkt detekterades inte. För att dessa ämnen ska detekteras måste en annan sorts kolonn med mer opolär stationärfas användas i GC-MS. Metoden detekterar även lättflyktiga och svårflyktiga ämnen. Men för att vara säker på att samtliga lättflyktiga ämnen som har orsakat svällningen i EPDM packningen detekteras måste en särskild provhanteringsmetod utvecklas. Detta för att säkerställa att inga ämnen lämnat packningen innan analys av EPDM packningen.   För att förbättra analysmetoden ytterligare föreslås att metoden utvecklas så att den enbart kan genomföras med GC-MS. Detta bör göras genom fler GC-MS analyser med andra sorters kolonner samt med andra bättre sökdatabaser. Det föreslås även att metoden utvecklas så att den förutom kvalitativ bestämning av ämnen som orsakat svällning även kvantitativ bestämma sammansättningen av ämnen i EPDM packningen. På så sätt fås ett ännu bättre analysverktyg vid framtida haveriutredningar

Synthesis of degradable aliphatic polyesters: strategies to tailor the polymer microstructure

Key factors for successful tissue engineering are the synthesis and design of the scaffold materials. Aliphatic polyesters have been studied and often used as scaffold materials for tissue engineering. However, their lack of biological cues and degradation under high-temperature processing (e.g., 3D printing) are a limitation. In this thesis, different synthesis strategies are presented which has the potential to improve the performance of aliphatic polyesters as scaffolds for tissue regeneration. To stimulate interactions between exogenous materials and the surrounding tissue, two different strategies were applied. Either, by designing a two component system in which the different degradation profiles of the polymers allow for sequential release of growth factors. Or, by peptide functionalization of an aliphatic polyester chain using template-assisted chemo-enzymatic synthesis. The results from the studies were successful. A hierarchical system was obtained in which the poly(L-lactide-co-glycolide)-graft-poly(ethylene glycol) methyl ether (PLGA-g-MPEG), hydroxyapatite solution formed a gel around and within the pores of the poly(L-lactide-co-ε-caprolactone) scaffold at 37 ºC, within 1 min, that was stable for 3 weeks. The peptide functionalization was also successful where an aliphatic polyester of L-lactide was functionalized with different oligopeptides using a grafter (ethyl hept-6-enoylalaninate) and chemo-enzymatic synthesis. The thermal properties of poly(L-lactide-co-hydroxybutyrate) were tailored (by modification of the microstructure) to potentially improve the processability of the aliphatic polyester.  The results showed that the yttrium salan catalyst was the most successful, yielding high molecular weight copolymers in shorter time. They also showed that the Tg could be tailored by varying the amount of rac-β-butyrolactone in the copolymer to better suit thermal processing techniques, such as 3D printing.QC 20171207</p

Synthesis of degradable aliphatic polyesters: strategies to tailor the polymer microstructure

Key factors for successful tissue engineering are the synthesis and design of the scaffold materials. Aliphatic polyesters have been studied and often used as scaffold materials for tissue engineering. However, their lack of biological cues and degradation under high-temperature processing (e.g., 3D printing) are a limitation. In this thesis, different synthesis strategies are presented which has the potential to improve the performance of aliphatic polyesters as scaffolds for tissue regeneration. To stimulate interactions between exogenous materials and the surrounding tissue, two different strategies were applied. Either, by designing a two component system in which the different degradation profiles of the polymers allow for sequential release of growth factors. Or, by peptide functionalization of an aliphatic polyester chain using template-assisted chemo-enzymatic synthesis. The results from the studies were successful. A hierarchical system was obtained in which the poly(L-lactide-co-glycolide)-graft-poly(ethylene glycol) methyl ether (PLGA-g-MPEG), hydroxyapatite solution formed a gel around and within the pores of the poly(L-lactide-co-ε-caprolactone) scaffold at 37 ºC, within 1 min, that was stable for 3 weeks. The peptide functionalization was also successful where an aliphatic polyester of L-lactide was functionalized with different oligopeptides using a grafter (ethyl hept-6-enoylalaninate) and chemo-enzymatic synthesis. The thermal properties of poly(L-lactide-co-hydroxybutyrate) were tailored (by modification of the microstructure) to potentially improve the processability of the aliphatic polyester.  The results showed that the yttrium salan catalyst was the most successful, yielding high molecular weight copolymers in shorter time. They also showed that the Tg could be tailored by varying the amount of rac-β-butyrolactone in the copolymer to better suit thermal processing techniques, such as 3D printing.QC 20171207</p

Synthesis of degradable aliphatic polyesters: strategies to tailor the polymer microstructure

Key factors for successful tissue engineering are the synthesis and design of the scaffold materials. Aliphatic polyesters have been studied and often used as scaffold materials for tissue engineering. However, their lack of biological cues and degradation under high-temperature processing (e.g., 3D printing) are a limitation. In this thesis, different synthesis strategies are presented which has the potential to improve the performance of aliphatic polyesters as scaffolds for tissue regeneration. To stimulate interactions between exogenous materials and the surrounding tissue, two different strategies were applied. Either, by designing a two component system in which the different degradation profiles of the polymers allow for sequential release of growth factors. Or, by peptide functionalization of an aliphatic polyester chain using template-assisted chemo-enzymatic synthesis. The results from the studies were successful. A hierarchical system was obtained in which the poly(L-lactide-co-glycolide)-graft-poly(ethylene glycol) methyl ether (PLGA-g-MPEG), hydroxyapatite solution formed a gel around and within the pores of the poly(L-lactide-co-ε-caprolactone) scaffold at 37 ºC, within 1 min, that was stable for 3 weeks. The peptide functionalization was also successful where an aliphatic polyester of L-lactide was functionalized with different oligopeptides using a grafter (ethyl hept-6-enoylalaninate) and chemo-enzymatic synthesis. The thermal properties of poly(L-lactide-co-hydroxybutyrate) were tailored (by modification of the microstructure) to potentially improve the processability of the aliphatic polyester.  The results showed that the yttrium salan catalyst was the most successful, yielding high molecular weight copolymers in shorter time. They also showed that the Tg could be tailored by varying the amount of rac-β-butyrolactone in the copolymer to better suit thermal processing techniques, such as 3D printing.QC 20171207</p

Short One-Pot Chemo-Enzymatic Synthesis of l‑Lysine and l‑Alanine Diblock Co-Oligopeptides

Amphiphilic diblock co-oligopeptides are interesting and functional macromolecular materials for biomedical applications because of their self-assembling properties. Here, we developed a synthesis method for diblock co-oligopeptides by using chemo-enzymatic polymerization, which was a relatively short (30 min) and efficient reaction (over 40% yield). Block and random oligo­(l-lysine-<i>co</i>-l-alanine) [oligo­(Lys<i>-co-</i>Ala)] were synthesized using activated papain as enzymatic catalyst. The reaction time was optimized according to kinetic studies of oligo­(l-alanine) and oligo­(l-lysine). Using ¹H NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we confirmed that diblock and random co-oligopeptides were synthesized. Optical microscopy further revealed differences in the crystalline morphology between random and block co-oligopeptides. Plate-like, hexagonal, and hollow crystals were formed due to the strong impact of the monomer distribution and pH of the solution. The different crystalline structures open up interesting possibilities to form materials for both tissue engineering and controlled drug/gene delivery systems

Template-Assisted Enzymatic Synthesis of Oligopeptides from a Polylactide Chain

Peptides are often attached to polymer materials, as bioactive components, for the control of interactions between the material and its surrounding proteins and cells. However, synthesizing peptides and attaching them to polymers can be challenging and laborious. Herein, we describe the grafting of oligopeptides to an aliphatic polyester, using a one-step chemo-enzymatic synthesis with papain as the biocatalyst. To enable enzyme-mediated functionalization of the polyester, ethyl hept-6-enoylalaninate (grafter) was synthesized and attached to polylactide chains using thiol–ene click reactions. The oligopeptides were grafted onto the polylactide chains using two different synthetic routes: the grafting from strategy, in which the grafter was attached to the polyester prior to oligopeptide synthesis, or the grafting to strategy, in which oligopeptides were synthesized on the grafter first, then attached to the polymer chain. The final products were analyzed and their structures were confirmed using nuclear magnetic resonance (NMR). The peptide attachment was evaluated using size exclusion chromatography (SEC), contact angle measurement and energy-dispersive X-ray spectroscopy-scanning electron microscopy (EDS-SEM). Furthermore, the mechanistic aspects of the synthesis of the oligopeptides on the grafter were studied using molecular dynamics (MD) simulations. The simulation revealed that hydrogen bonding (between the P1 amide nitrogen of the grafter backbone and the carbonyl oxygen of D158 in the papain) maintain the grafter in a productive conformation to stabilize the transition state of nitrogen inversion, a key step of the biocatalytic mechanism. Apart from being biologically relevant, both experimental and computational results suggest that the designed grafter is a good template for initiating chemo-enzymatic synthesis. The results also showed that the grafting to strategy was more successful compared to the grafting from strategy. Overall, a successful synthesis of predefined peptide functionalized polylactide was prepared, where the oligopeptides were grafted in an easy, time efficient, and environmentally friendly way

Statement on methods in sport injury research from the 1st METHODS MATTER Meeting, Copenhagen, 2019

High quality sports injury research can facilitate sports injury prevention and treatment. There is scope to improve how our field applies best practice methods-methods matter (greatly!). The 1st METHODS MATTER Meeting, held in January 2019 in Copenhagen, Denmark, was the forum for an international group of researchers with expertise in research methods to discuss sports injury methods. We discussed important epidemiological and statistical topics within the field of sports injury research. With this opinion document, we provide the main take-home messages that emerged from the meeting.Funding Agencies|BJSM</p

Statement on methods in sport injury research from the first methods matter meeting, Copenhagen, 2019

SYNOPSIS: High-quality sports injury research can facilitate sports injury prevention and treatment. There is scope to improve how our field applies best-practice methods-methods matter (greatly!). The first METHODS MATTER meeting, held in January 2019 in Copenhagen, Denmark, was the forum for an international group of researchers with expertise in research methods to discuss sports injury methods. We discussed important epidemiological and statistical topics within the field of sports injury research. With this opinion document, we provide the main take-home messages that emerged from the meeting. Meeting participants agreed that the definition of sport injury depends on the research question and context. It was considered essential to be explicit about the goal of the research effort and to use frameworks to illustrate the assumptions that underpin measurement and the analytical strategy. Complex systems were discussed to illustrate how potential risk factors can interact in a nonlinear way. This approach is often a useful alternative to identifying single risk factors. Investigating changes in exposure status over time is important when analyzing sport injury etiology, and analyzing recurrent injury, subsequent injury, or injury exacerbation remains challenging. The choice of statistical model should consider the research question, injury measure (eg, prevalence, incidence), type and granularity of injury data (categorical or continuous), and study design. Multidisciplinary collaboration will be a cornerstone for future high-quality sport injury research. Working outside professional silos in a diverse, multidisciplinary team benefits the research process, from the formulation of research questions and designs to the statistical analyses and dissemination of study results in implementation contexts. This article has been copublished in the British Journal of Sports Medicine and the Journal of Orthopaedic & Sports Physical Therapy