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Σχεδιασμός Χημικών Προϊόντων Δημήτρης Χατζηαβραμίδης Σχολή Χημικών Μηχανικών Εθνικό Μετσόβιο Πολυτεχνείο 5/11/2014ΔΧ1.

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Παρουσίαση με θέμα: "Σχεδιασμός Χημικών Προϊόντων Δημήτρης Χατζηαβραμίδης Σχολή Χημικών Μηχανικών Εθνικό Μετσόβιο Πολυτεχνείο 5/11/2014ΔΧ1."— Μεταγράφημα παρουσίασης:

1 Σχεδιασμός Χημικών Προϊόντων Δημήτρης Χατζηαβραμίδης Σχολή Χημικών Μηχανικών Εθνικό Μετσόβιο Πολυτεχνείο 5/11/2014ΔΧ1

2 5/11/2014ΔΧ Μοριακά Προϊόντα Φάρμακα: Φαρμακευτικά δραστικές ουσίες (Αctive Pharmaceutical Ingredients) Νανοϋλικά, νανοσωματίδια Βιολογικά μόρια 2

3 ΠΡΟΒΛΗΜΑΤΑ ΤΟΥ ΣΥΓΧΡΟΝΟΥ ΚΟΣΜΟΥ Υγεία Στην αντιμετώπιση αυτών των προβλημάτων κεντρικό ρόλο παίζει ο σχεδιασμός υλικών με προδιαγεγραμμένες ιδιότητες, καθώς και διεργασιών και προϊόντων βασισμένων σ’ αυτά τα υλικά. Ενέργεια Περιβάλλον Διατροφή Σχεδιασμός υλικών γίνεται σήμερα σε μοριακό επίπεδο «Πριν από περίπου χρόνια, οι άνθρωποι άρχισαν να καλλιεργούν φυτά και να εξημερώνουν ζώα. Τώρα είναι η ώρα να εξημερώσουμε μόρια.» — Susan Lindquist, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology 5/11/2014ΔΧ3

4 Τεχνικές μαθηματικής προτυποποίησης και υπολογιστικής προσομοίωσης υλικών και πρόρρησης των σχέσεων δομής-ιδιοτήτων-επεξεργασίας-επιδόσεων ΕΡΓΑΛΕΙΑ ΜΟΡΙΑΚΟΥ ΣΧΕΔΙΑΣΜΟΥ ΥΛΙΚΩΝ Σύγχρονες πειραματικές τεχνικές διερεύνησης δομής και κίνησης σε μοριακό και υπερμοριακό επίπεδο και μέτρησης ιδιοτήτων (π.χ. σκέδαση ακτίνων Χ και νετρονίων, φασματοσκοπίες ΝΜR, ηλεκτρονική μικροσκοπία, μικροσκοπία σάρωσης φαινομένου σήραγγος, μικροσκοπία ατομικών δυνάμεων, συνεστιακή μικροσκοπία φθορισμού, μικροθερμιδομετρία, τεχνικές high-throughput experimentation) 5/11/2014ΔΧ4

5 Josiah Willard Gibbs ( ) Ludwig Boltzmann ( ) ΠΡΩΤΕΡΓΑΤΕΣ ΤΗΣ ΣΤΑΤΙΣΤΙΚΗΣ ΜΗΧΑΝΙΚΗΣ 5/11/2014ΔΧ5

6 ΓΕΝΙΚΕΣ ΜΕΘΟΔΟΙ ΜΟΡΙΑΚΗΣ ΠΡΟΣΟΜΟΙΩΣΗΣ ΜΟΡΙΑΚΗ ΔΥΝΑΜΙΚΗ (MD) Alder and Wainwright, 1957 Παρακολουθεί χρονική εξέλιξη προτύπου συστήματος επιλύοντας τις εξισώσεις κίνησης για όλα τα σωματίδια. Με συνήθως διαθέσιμα υπολογιστικά μέσα, μπορεί να προσομοιώσει ατομιστικά μοντέλα διαστάσεων της τάξεως 10 nm επί χρόνους μέχρι μερικά μs. Προσομοιώσεις μέχρι ms δυνατές σε ειδικά σχεδιασμένα υπολογιστικά συστήματα. ΜΟNTE CARLO (MC) Metropolis, Rosenbluth, Rosenbluth, Teller, and Teller, 1953 Στοχαστική δειγματοληψία μοριακών απεικονίσεων σύμφωνα με τη συνάρτηση πυκνότητας πιθανότητας ενός στατιστικού συνόλου ισορροπίας. Κάθε απεικόνιση σχηματίζεται από την αμέσως προηγούμενη επιχειρώντας μια στοιχειώδη κίνηση, που γίνεται δεκτή ή απορ- ρίπτεται βάσει κατάλληλα σχεδιασμένων κανόνων επιλογής. 5/11/20146ΔΧ

7 ΚΛΙΜΑΚΕΣ ΜΗΚΟΥΣ ΚΑΙ ΧΡΟΝΟΥ ΣΤΑ ΠΟΛΥΜΕΡΗ Μήκη δεσμών, ατομικές ακτίνες ~ 0.1 nm Μήκος στατιστικού τμήματος (Kuhn) b ~ 1 nm Γυροσκοπική ακτίνα αλυσίδας ~ 10 nm Μέγεθος περιοχών σε φασικά διαχω- ρισμένο υλικό ~ 1  m Δονητικές κινήσεις  s Μεταπτώσεις διαμόρφωσης  s Μέγιστος χρόνος χα- λάρωσης  s Διαχωρισμός σε φάσεις/ μικροφάσεις  1 s Φυσική γήρανση (Τ < Τ g -20 ο C)  1 yr ≈ 10 6 s Τήγμα Υαλώδης κατάσταση 5/11/2014ΔΧ7

8 IΕΡΑΡΧΙΚΗ ΣΤΡΑΤΗΓΙΚΗ ΓΙΑ ΤΗΝ ΥΠΟΛΟΓΙΣΤΙΚΗ ΕΠΙΣΤΗΜΗ ΚΑΙ ΤΕΧΝΙΚΗ ΤΩΝ ΥΛΙΚΩΝ Οι υπολογισμοί κατευθύνουν και συμπληρώνουν πειραματικές προσπάθειες για την ανάπτυξη νέων υλικών, διεργασιών και προϊόντων. Κβαντο- μηχανι- κοί Υπολο- γισμοί Μοριακή γεωμετρία, Ηλεκτρο- νικές ιδιότητες Πεδία δυνάμεων μοριακών αλληλεπι- δράσεων χημική σύσταση Ιδιότητες Υλικού Καταστ. Εξισώσεις, Υλικές Σχέσεις Μοριακή οργάνωση και κίνηση Μικροσκοπικοί μηχανισμοί υπεύθυνοι για μακροσκοπική συμπεριφορά Μοριακές Προσομοι- ώσεις, Εφαρμ. Στατιστική Μηχανική π.χ. Monte Carlo, Μοριακή δυναμική, Μοριακή μηχανική, Θεωρία μεταβατικών καταστάσεων Επεξεργασία Μορφολογία Μικροδομή Αδροποι- ημένες παράμετροι αλληλεπί- δρασης π.χ. Σταθερές ρυθμού, συντελε- στές τριβής Μεσοσκο- πικές Προσομοι- ώσεις π.χ. Κινητική Monte Carlo, Θεωρίες αυτo- συνεπούς πεδίου, Dynamic density functional theory, Dissipative particle dynamics Μακροσκο- πικοί Υπολογισμοί, Σχεδιασμός π.χ. Εφαρμοσμένη θερμοδυναμική, Φαινόμενα μεταφοράς, Χημική κινητική, Μηχανική του συνεχούς, Ηλεκτρομαγνη- τική θεωρία Επιδόσεις υλικού υπό συγκε- κριμένες συνθήκες εφαρμογής 5/11/2014ΔΧ8

9 5/11/2014 ΝΑΝΟΣΩΜΑΤΙΔΙΑ Νανοκλίμακα: 1 nm = m =  m = 10 Å Ατομική κλίμακα (~ Å ) 1  m ) Γιατί είναι σπουδαία; Διότι: 1.Οι κβαντομηχανικές (κυματικές) ιδιότητες των ηλεκτρονίων μέσα στην ύλη επηρεάζονται από μεταβολές της τάξης νανοκλίμακας ⇒ είναι πιθανό να μεταβληθούν οι ιδιότητες των υλικών (π.χ., ειδική θερμότητα, θερμοκρασία τήξης, μαγνητική επαγωγή, κλπ.) χωρίς να μεταβληθεί η χημική τους σύσταση 2.Κύριο χαρακτηριστικό των βιολογικών συστημάτων είναι η αυτό-οργανωμένη δομή της ύλης σε μεγέθη νανοκλίμακας ⇒ δημιουργία νέων προϊόντων με χρήση αυτό-οργανωτικής ικανότητας (self-assembly) της ύλης 3.Τα νανοσυστήματα έχουν πολύ μεγάλο επιφάνεια-προς-όγκο λόγο ⇒ ιδανικά για χρήση σε σύνθετα υλικά (composites), αντιδρώντα συστήματα, χορήγηση φαρμάκων (drug delivery), αποθήκευση ενέργειας (υδρογόνο, φυσικό αέριο) 4.Συστήματα με νανοδομές μπορεί να έχουν υψηλότερες πυκνότητες και καλλίτερες αγωγιμότητες από αυτά με μικροδομές Μακροσκοπικές ιδιότητες (μηχανικές, θερμικές, οπτικές, μαγνητικές, ηλεκτρικές, etc.) για συστήματα σε νανοκλίμακα είναι τελείως διαφορετικές από αυτές για μακροσκοπικά συστήματα 9ΔΧ

10 NANOΣΩΜΑΤΙΔΙΑ Nανοτεχνολογία: Η τεχνολογία επεξεργασίας μακρο- και μικρο-υλικών και δομών με ακρίβεια (precision) σε ατομική κλίμακα Σύμφωνα με τον Feynman (1959), οι νόμοι της φύσης δεν περιορίζουν την ικανότητα μας να εργαζόμαστε σε μοριακό επίπεδο, με κάθε άτομο χωριστά. οι περιορισμοί τίθενται από την έλλειψη των κατάλληλων οργάνων και τεχνικών για να κάνουμε κάτι τέτοιο ⇒ πρόκληση (challenge) για σμίκρυνση (miniaturization) Επιστήμονες στην IBM (1999) είχαν την ιδέα να χρησιμοποιήσουν Atomic Force Microscope στη λιθογραφία (Dip Pen Nanolithography). Η κορυφή (tip) του AFM είναι επικαλυμμένο με μοριακό μελάνι και έρχεται σε επαφή με επιφάνεια που θα διαμορφωθεί (be patterned) Η Θερμοδυναμική και η Στατιστική Μηχανική των μικρών συστημάτων, που αρχικά προτάθηκε από τον T. L. Hill το 1963 και ασχολείται με τα κολλοειδή, τα μακρομόρια και τα μίγματα, μετά τη διατύπωση βασικών αρχών για μη εκτατικά (nonextensive) συστήματα, δηλαδή, για συστήματα μακριά από το θερμοδυναμικό όριο (N → ∞, V → ∞, N / V =  N πεπερασμένο), (C. Tsallis, 1988), έγινε Νανοθερμοδυναμική Διαμοριακά Δυναμικά για πρόβλεψη μακροσκοπικών ιδιοτήτων Άτομα ή -quantum mechanical ab initio απλά μόριαcalculations Macrosystems - classical potentials, e.g.,Coulomb, LJ, for ( particles)covalent and noncovalent interactions Nanosystems-experimental and theoretical development ( finite number of particles) 5/11/2014ΔΧ10

11 ΝΑΝΟΣΩΜΑΤΙΔΙΑ (ΝΑΝΟPARTICLES) Μοριακές Δομικές Μονάδες (Molecular Building Blocks) Διαμαντοειδή (Diamondoids) Φουλερένια (Buckyballs) Νανοσωλήνες Άνθρακα (Carbon nanotubes) Κυκλοδεξτρίνες (Cyclodextrins) Λιποσώματα (Liposomes) Μονοκλονικά Αντισώματα (Monoclonal Antibodies) Τα διαμαντοειδή, που είναι επίσης γνωστά σαν έγκλειστοι υδρογονάνθρακες (cage hydrocarbons), είναι κεκορεσμένοι, πολυκυκλικοί υδρογονάνθρακες με συν- τηγμένες (fused) δομές παρόμοιες με του αδάμαντα (οι νανοδομές μπορεί να υπερτίθενται -superimposed- στο πλέγμα -lattice- του αδάμαντα) και έχουν ασυνήθεις φυσικές και χημικές ιδιότητες. Ο κοινός τύπος για την ομάδα αυτή είναι C 4n+6 H 4n+12, n=1 για adamantane, n=2 για diamantane, n=3 για trimentane, n=4 για tetramentane, etc.Τα πρώτα 3 μέλη της ομάδας δεν έχουν ισομερή. Όταν n>4, ο αριθμός των ισομερών αυξάνει αισθητά. Χειρικότητα (Chirality), ζεύγος ισο- μερών με συμμετρία όμοια με αυτή των δύο χεριών, εμφανίζεται πρώτα στο tetramantane. Divided into (a) lower diamondoids, diameter 1-2 nm, and (b) higher diamondoids, diameter > 2 nm 5/11/2014 iso anti skew 11ΔΧ

12 5/11/2014ΔΧ NANOPARTICLES Τα διαμαντοειδή, που είναι φυσικά συστατικά στο αργό πετρέλαιο (crude oil), ανακαλύφθηκαν το 1933 στην Τσεχοσλοβακία. Adamantine μπορεί να παραχθεί με σύνθεση με καταλύτες ζεο- λίθους (zeolites). Στην στερεά κατάσταση, τα διαμαντοειδή τήκονται σε πολύ υψηλές θερμοκρασίες από άλλους υδρογονάνθρακες με τον ίδιο αριθμό ατόμων άνθρακα (adamantane, T m = o C; diamantane T m = o C). ΄Εχουν υψηλή πυκνότητα και χαμηλή ενέργεια παραμόρφωσης (low- strain energy), και είναι περισσότερο σταθερά and δύσκαμπτα (stiff). Εφαρμογές διαμαντοειδών Τρία παράγωγα του adamantane, amantadine (1-adamantaneamine hydrochloride), rimantadine (a-methyl-1-adamantane methylamine hydrochloride) και memantine (1-amino- 3,5-dimethyladamantane) χρησιμοποιούνται σαν αντι-ιικά (antiviral) φάρμακα, π.χ., για πρόληψη και θεραπεία των ιογενών λοιμώξεων (infections) της γρίπης (influenza) A. Χρησιμοποιούνται επίσης για θεραπεία της ασθένειας του Parkinson και αναχαίτιση (inhibition) του ιού (virus) της ηπατίτιδας (hepatitis) C (HCV). Έχει παρατηρηθεί ότι είναι αποτελεσματική στην επιβράδυνση της εξέλιξης της ασθένειας του Alzheimer. Ο χρόνος ημιζωής (half-life) των παραγώγων είναι μακράς διαρκείας (adamandine h; rimandine h) Μονοκατιονικά και δικατιονικά παράγωγα του adamantane φράσσουν (block) υποδοχείς (receptors) του τύπου AMPA (A-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid: υποδοχέας για το γλουταμινικό οξύ που βοηθάει στη γρήγορη μεταφορά μηνυμάτων στη σύναψη), NMDA (N-methyl D-aspartate που μιμείται τη δράση του γλουταμινικού στον υποδοχέα) και 5-HT3 (5-Hydroxytryptamine3 που είναι αναστολέας της σεροτονίνης - serotonin inhibitor-). Η πρόσδεση πεπτιδίων με βραχεία αλυσίδα στο adamantane χρησιμοποιείται στην παραγωγή ανταγωνιστών, π.χ., Bradykinin και vasopressin που είναι ανταγωνιστές (antagonists) υποδοχέων

13 5/11/2014ΔΧ NANOΣΩΜΑΤΙΔΙΑ Εφαρμογές διαμαντοειδών Τα παράγωγα του αdamantane μπορούν να χρησιμοποιηθούν σαν μεταφορείς (carriers) για χορήγηση φαρμάκων (drug delivery) και ειδικά στοχευόμενη (targeted) χορήγηση. Λόγω της υψηλής λιποφιλικότητας (lipophilicity) τους, προσθήκη διαμαντοειδών σε φάρμακα με χαμηλή υδροφοβικότητα (hydrophobicity) αναμένεται να οδηγήσει σε αύξηση της διαλυτότητας του φαρμάκου στις μεμβράνες λιπιδίων (lipid) και έτσι αυξάνει την πρόσληψη του φαρμάκου (drug uptake) από τους ιστούς Πεπτίδια με βραχείες αλυσίδες, λιπίδια και πολυσακχαρίτες προσαρτώνται στο adamantine και δημιουργούν μια θέση δέσμευσης (binding site) για σύνδεση μεγαλομοριακών φαρμάκων και μικρών μορίων. Παράδειγμα Φάρμακα για ασθένειες του εγκεφάλου που μπορούν να διαπεράσουν το φράγμα εγκεφάλου- αίματος (Brain Blood Barrier) όπως το κομμάτι (moiety) του 1-Adamantyl που προσδένεται σε AZT (azidothymidine) φάρμακα (για AIDS) μέσω της εστερικής ομάδας Τα παράγωγα του αdamantane που προσδένονται στις αλυσίδες των νουκλεϊκών οξέων (nucleic acid) μέσω συνδετήρα αμιδίου (amide linker) χρησιμοποιούνται για παράδοση γονιδίων (gene delivery) η οποία έχει προβλήματα με την χαμηλή πρόσληψη (uptake) των νουκλεϊκών οξέων από κύτταρα, και αστάθειες στη ροή του αίματος. Το DNA και RNA επιδεικνύουν δεσμευτική επιλεκτικότητα (binding selectivity) σε παράγωγα του polyamine adamantane (DNA και RNA μπορούν να σταθεροποιηθούν με πρόσδεση) 13

14 5/11/2014ΔΧ14 NANOΣΩΜΑΤΙΔΙΑ Εφαρμογές διαμαντοειδών DNA fragments, because of DNA’s unique feature for site-selective immobilization, are used as linkers in DNA-adamantane-protein nanaostructures. Knowledge of protein folding and conformations in biological systems can help us design nanostructures with desired and predictable conformation in a biomimetic way. Thus, adamantane can be used to construct peptide scaffolding and in synthesis of artificial proteins

15 5/11/2014ΔΧ NANOΣΩΜΑΤΙΔΙΑ Φουλερένια (Buckyballs) Το φουλερένιο (Buckyball or Buckminster fullerene molecule), που είναι μια αλλοτροπική (allotropic) μορφή άνθρακα (carbon), είναι η πιο δημοφιλής ανάκαλυψη της Nανοτεχνολογίας For this discovery, Kroto and Smalley were awarded the 1996 Nobel Prize in Chemistry Buckyballs are C n clusters with n>20 ( most common C 60, C 70 ; later fullerenes C 76, C 80, C 240, etc). Originally made by laser evaporation of graphite. More efficient and less expensive methods for making them found later A molecule should have at least 2 linking groups to be considered as M(olecular) B(uilding) B(lock). The presence of 3 linking groups would lead to 2-D or a tubular structure formation. Presence of 4 or more linking groups lead to a 3-D structure. Molecules with 5 linking groups can form a 3-D solid structure; those with 6 linking groups can be attached in a cubic structure. Functionalization of buckyballs with 6 functional groups (for positional or robotic assembly) is presently possible 15

16 5/11/2014ΔΧ NANOΣΩΜΑΤΙΔΙΑ Νανοσωλήνες Άνθρακα (Carbon Nanotubes) Discovered by Iijima in He used an electron microscope while studying cathodic material deposition through vaporizing carbon graphite in an electric arc-evaporation reactor under an inert atmosphere during synthesis of fullerenes They appeared to be made of a perfect network of hexagonal graphite rolled up to form a hollow tube The nanotube diameter range, from one to several nm, is much smaller than its length range, from one to several  m Laser ablation chemical vapor deposition joined with metal-catalyzed disproportionation of suitable carbonaceous feedstock are often used to produce carbon nanotubes Carbon nanotubes exhibit unusual photochemical, electronic, thermal and mechanical properties Single-Walled Carbon NanoTubes could behave as metallic, semi-metallic, or semiconductive 1-D objects. They have high tensile strength, ~ 100 times that of steel 16

17 5/11/2014ΔΧ NANOPARTICLES Cyclodextrins are cyclic oligosaccharides in the shape of a truncated cone with a relatively hydrophobic interior. They have the ability to form inclusion complexes with a wide range of substrates in aqueous solutions ⇒ encapsulation of drugs for drug delivery Liposomes Spherical synthetic lipid bilayer vesicles, created by dispersion of a phospholipid in aqueous salt solutions. Quite similar to micelles with an internal aqueous compartment Used as carriers for a variety of drugs, small molecules, proteins, nucleotides (has nitrogenous base, sugar and phosphate group), even plasmids (extrachromosomal DNA molecule having ability to replicate independently of chromosomal DNA), to tissues and into cells 17

18 5/11/2014ΔΧ NANOPARTICLES Monoclonal Antibodies A monoclonal antibody protein molecule consists of four protein chains, two heavy and two light, which are folded to form a Y-shaped structure. The small size, 10 nm in diameter, ensures that intravenously administered monoclonal antibodies can penetrate small capillaries and reach cells in tissues where they are needed. Nanostructures smaller than 20 nm can transit out of blood vessels 18

19 5/11/2014ΔΧ NANOPARTICLES Nano Thermodynamics and Statistical Mechanics The principles of thermodynamics and statistical mechanics are well defined for macroscopic systems and relations between macroscopic properties and molecular characteristics can be derived Basic concepts in Thermodynamics for macroscopic systems are System & Surroundings State of the System & Equilibrium Process & Reversibility / Irreversibility Energy, Heat and Work Properties (extensive and intensive) & Relations among them Axiomatic Basis, i.e., Laws of Thermodynamics Degrees of Freedom of a System Phase Transitions Basic concepts in Statistical Mechanics for macroscopic systems are Ensembles and Averaging Ergodicity PhaseSpace (coordinates) Ground State Correlated and Interacting Systems 19

20 5/11/2014ΔΧ Nano Thermodynamics and Statistical Mechanics Differences between Macroscopic and Nanoscale systems Macroscopic systems can be in any of the three states of matter, gas, liquid, and solid. Nanoscale systems, i.e., isolated nanostructures and their assemblies, small drops, bubbles, clusters, aggregates, nanocrystals, nanowires, etc., are made of condensed (liquid or solid) matter Macroscopic system size: > 1  m; particles in 1 cm 3 (thermodynamic limit: N → ∞, V → ∞, N / V =  N finite) Nanoscale system size: > 1 nm; N = finite Thermodynamic properties, e.g., temperature, for macroscopic systems are well-defined and their fluctuations in time and space are negligible. This is not the case for nanoscale systems the size of which is of the same order as the size of fluctuations. Pressure in nanosystems is not isotropic and must be treated as a tensor Because of the large size fluctuations in properties, static equilibrium cannot be defined in nanosystems as in macroscopic systems. The states of a nanoscale system can only be in dynamic equilibrium Because of the large size fluctuations in properties, over short periods of time, processes in nanosystems cannot be reversible as in macroscopic systems However, over long periods of time, processes in nanosystems are expected to be closer to reversibility than those in macroscopic systems 20

21 5/11/2014ΔΧ Nano Thermodynamics and Statistical Mechanics Differences between Macroscopic and Nanoscale systems The definitions of extensive, i.e., system-size-dependent, and intensive, i.e., system-size-independent, properties for macroscopic systems don’t seem to be satisfied in nanosystems Thermodynamic property relations in macroscopic systems are independent of their surroundings (environment); they are environment-dependent for nanosystems, e.g., depend on the geometry, size and walls of the confining structure Energy and mass are mutually interchangeable and the laws of their conservation are combined into the First Law of Thermodynamics which is universal. Energy measures the ability of the system to induce a change which is visible at the scale of the system. Work and heat, on the other hand, are means of energy exchange between the system and its surroundings. Transfer of energy through work or heat is a visible phenomenon in macroscopic systems but not in nanosystems ⇒ conversion of thermal to mechanical energy in nanosystems ??? Entropy definition for macroscopic systems cannot be extended to nanosystems If a macroscopic system is divided into parts, the sum of the entropies of its parts is equal to the entropy of the original system. This is not the case for nanosystems. Macroscopic systems are extensive; nanosystems are nonextensive 21

22 5/11/2014ΔΧ SOPT FOPT S Nano Thermodynamics and Statistical Mechanics Differences between Macroscopic and Nanoscale systems Phase transitions in macroscopic systems are different than in nanosystems In first order phase transitions (FOPT) in macroscopic systems, we observe abrupt changes (discontinuities) in entropy and energy associated with the phases, and physically, there is a distinct separating boundary (meniscus in the case of liquid and vapor transition) apparent between the phases. Second order transitions (SOPT), on the other hand, do not exhibit discontinuities in entropy and energy but in their derivatives, e.g., heat capacity = derivative of energy w.r.t. temperature L L 22

23 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics Differences between Macroscopic and Nanoscale systems First Order Phase Transitions in macroscopic systems are different than in nanosystems Small system Large system 23

24 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics Differences between Macroscopic and Nanoscale systems Phase transitions in nanosystems also include 1.Fragmentation, a real phase transition of the first order in nuclei, e.g., boiling, liquid fragmentation when the ratio of viscous-to-capillary forces exceeds a critical value, and 2.Self-assembly, a process in which a set of components or constituents spontaneously forms an ordered aggregate through their global energy minimization 24

25 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics The Laws of Thermodynamics Zeroth Law: establishment of an absolute temperature scale Temperature in a macroscopic system is a well defined property, its fluctuations are negligible and it is a measure of thermal equilibrium or lack of it In nanosystems, space and time fluctuations cannot be neglected In view of temporal scale ~ N (number of particles) spatial scale ~ N logN, accuracy scale ~ N 7 to N! Fluctuations in nanosystems are not yet correlated to their properties Customary in Statistical Mechanics to express fluctuations in properties in terms of distribution functions or as derivatives of other properties First Law: combination of conservation of mass and energy, both of which are interchangeable In macroscopic systems, reversibility is equivalent to thermal equilibrium Nanosystems may be reversible but not in thermal equilibrium Similar to macro systems, nanosystems can be open, closed, adiabatic, isothermal, isochoric (constant volume) or isobaric (constant pressure) 25

26 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics The Laws of Thermodynamics First Law: combination of conservation of mass and energy, both of which are interchangeable dE =  Q +  W(1) Macroscopic systems:  Q = cdT  W = p dV(2a) Nanosystems:  W = t ij de ij  ij V(2b) (t ij external stress tensor; e ij deformation tensor;  ij Kronecker delta,  ij = 1 iff i = j) Second Law: For a closed system entropy production is always nonnegative dS –  Q / T ex > 0(3) Applies both to macroscopic and nano systems, if the concept of entropy is redefined to include nonextensive systems Boltzmann defined the entropy of the systems as S = k B lnW(4) where W is the number of possible configurations of a system of particles consistent with the properties of the system. The Boltzmann entropy has two important features: 1.Non-decrease, i.e. if no heat enters or leaves the system its entropy cannot decrease, 2.Additivity, i.e, the entropy of systems taken together is the sum of their individual entropies 26

27 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics The Laws of Thermodynamics Entropy The Boltzmann entropy, otherwise called entropy of a coarse-grain distribution is for a macroscopic state over a statistical ensemble with equiprobability. For non- equiprobability, Gibbs consider a system of a large number of particles (e.g., molecules), N, distributed in W classes (e.g., energy states) with non equal probability. If p i = W i / W (  W i = W), is the probability of the distribution of i particles in the system, the entropy of the system is given by the Boltzmann-Gibbs formula With equiprobability, p i = 1 / W, Eq.(5) reduces to Eq.(4), i.e., the Gibbs-defined entropy becomes the Boltzmann-defined entropy The problem with these definitions of entropy is that they apply to homogeneous systems with a large number of particles. i.e., systems at the thermodynamic limit. For those systems, the notions of extensivity (additivity) and intensivitiy (averaging) of thermodynamic properties also apply Nanosystems, consist of a finite number of particles and their spatial scale is of the same order of magnitude as the correlation length for their thermodynamic properties 27

28 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics The Laws of Thermodynamics Entropy A new formulation of entropy to include extensive (e.g., macroscopic) as well nonextensive (e.g., nanosystems) was introduced by Tsallis (1988) We need to start with the definition of homogeneous function of degree f(tx 1, tx 2, tx 3, …, tx r ) = t  f(x 1, x 2, x 3, …, x r )(6) and Euler’s theorem x 1 (∂ f / ∂ x 1 ) + x 2 (∂ f / ∂ x 2 ) + … + x r (∂ f / ∂ x r ) = f(7) A thermodynamic variable is intensive when = 0, and extensive if = 1 Tsallis (1988) expressed the entropy as 28

29 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics The Laws of Thermodynamics Entropy q is called entropic index; q >1 represents frequent events; q < 1 represents rare events (p i q < p i ) q is also called extensivity index; q >1 represents superextensivity (superadditivity), q = 1 represents extensivity (additivity of entropy), and q < 1 represents subextensivity (subadditivity) Depending on the entropic index q, Eq.(8) ⇒ 1.For q > 0, S q > 0, i.e., entropy is always positive 2.For q→ 1,, i.e., the Gibbs-Boltzmann formula 3.For q = 1, S 1 = k lnW, i.e., the Boltzmann formula Also 1.For equiprobability, i.e., p i = 1 / W, Eq.(8) ⇒ 2.In the case of certainty, i.e., all but one probabilities vanish, p 1 =1, p i = 0 for i>1, Eq.(8) ⇒ entropy is zero, S q = 0 29

30 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics The Laws of Thermodynamics Entropy 3.When two (statistically) independent systems A and B join, Eq.(8) ⇒ 4.If the set of possibilities W is arbitrarily separated into two subsets W L and W M (W L +W M = W), Eq.(8) ⇒ 30

31 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics The Laws of Thermodynamics Entropy The Tsallis entropy has two more additional properties: 1.Can be tested under translation as well as under dilation The Boltzmann-Gibbs formula satisfies Eq.(8) ⇒ 2.Is always convex, when q 0 This is not the case for other definitions of entropy, e.g., Renyi definition of entrolpy for fractal geometries does not have this property for all values of q 31

32 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics Microcanonical Ensemble for Nonextensive Systems Consider a nanosystem at fixed N, V and T, isolated (no energy exchange) from its surroundings. The entropy of this system, which is given by the Tsallis formula, becomes maximum when the probabilities p i are all equal. If  =  (  ) is the number of states with energies centered around , then p i = 1 /  For this system,  is called degeneracy and is related to entropy through When the system is at equilibrium, dU = T dS – p dV(12) From (11) and (12) ⇒ i.e., k T  q ∂  is related to the energy change dU. Eq.(13) is a fundamental equation of Statistical Mechanics for nonextensive systems 32

33 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics Canonical Ensemble for Nonextensive Systems Consider now a nanosystem at fixed N, V and T, exchanging energy with its surroundings. We’d like to maximize the entropy of this system, under the constraint that the average energy of the system is constant For a nonextensive system, the internal energy constraint is The other constraint on the system is We employ the method of Lagrange multipliers to minimize the function where  has units of inverse temperature and  is dimensionless. Minimization ⇒ 33

34 5/11/2014ΔΧ Nanothermodynamics and Statistical Mechanics Canonical Ensemble for Nonextensive Systems Consider now a nanosystem at fixed N, V and T, exchanging energy with its surroundings. We’d like to maximize the entropy of this system, under the constraint that the average energy of the system is constant Eq.(16) ⇒ where Z q is the canonical ensemble partition function for nonextensive systems Intermolecular Potentials The database of intermolecular potentials of simple fluids and solids to be used in predicting properties of macroscopic systems is rather complete. These potentials cannot be used for predictions of properties of nanosystems The nature and role of intermolecular interactions, needed for formulation of intermolecular potentials, in nanostructures is challenging and not well understood Direct measurements of interparticle force vs. distance data and quantum mechanical ab initio calculations are needed to generate intermolecular poltentials for nanosystems consisting of a few hundred to a few thousand particles 34

35 5/11/2014ΔΧ Simulation Methods for Nanosystems Monte Carlo (MC) simulations generally follow the evolution of a system in which change proceeds not in a predefined but rather a random manner Considering the fact that there are several thousands of atoms or molecules in a 10 nm cube, there are significant challenges in using MC techniques to predict the properties of nanosystems Molecular Dynamics simulations consist of the numerical solution of Newton’s equation of motion for a system of particles (atoms, molecules, aggregates, etc.) to obtain information about their time-dependent properties. MD are an ideal to relate the collective dynamics of a finite number of particles in nanosystems to single-particle Dynamics Optimization methods help us achieve several goals, (a) to develop a controlled simulation scheme to obtain different nanostructures, (b) to study the most stable conditions for nanosystems 35

36 Nanomechanics A branch of science studying fundamental mechanical (elastic, thermal and kinetic) properties of physical systems at the nanoscale Also an applied science area with a focus on the mechanical properties of engineered nanostructures and nanosystems (systems with nanoscale components of importance). Examples include nanoparticles, nanopowders, nanowires, nanorods, nanoribbons, nanotubes, including carbon nanotubes (CNT) and boron nitride nanotubes (BNNTs); nanoshells, nanomebranes, nanocoatings, nanocomposite/nanostructured materials, fluids with dispersed nanoparticles, nanomotors, etc. Some of the well-established fields of nanomechanics are: nanomaterials, nanotribology (friction, wear and contact mechanics at the nanoscale), nanoelectromechanical systems (NEMS), and nanofluidics. Based on: 1) general mechanics principles and 2) specific principles arising from the smallness of physical sizes of the object of study or research General mechanics principles include: Energy and momentum conservation principles Variational Hamilton’s principle Symmetry principles Due to smallness of the studied object, accounts for: Discreteness of the object, whose size is comparable with interatomic distances Plurality, but finiteness, of degrees of freedom in the object Importance of thermal fluctuations Importance of entropic effects (configuration entropy) Importance of quantum effects (quantum machine)

37 Nanomechanics In statistical mechanics, thermal fluctuations are random deviations of a system from its average state, that occur in a system at equilibrium. All thermal fluctuations become larger and more frequent as the temperature increases, and likewise they decrease as temperature approaches absolute zero The volume of phase space Ʋ, occupied by a system of 2m degrees of freedom is the product of the configuration volume V and the momentum space volume. Since the energy is a quadratic form of the momenta for a nonrelativistic system, the radius of momentum space will be E 1/2 so that the volume of a hypersphere will vary as (E 1/2 ) 2m giving a phase volume of Ʋ = (C E) m / Γ(m+1) where C is a constant depending upon the specific properties of the system and Γ is the Gamma function. In the case that this hypersphere has a very high dimensionality, 2m, which is the usual case in thermodymamics, essentially all the volume will lie near to the surface Ω(E) = ∂ Ʋ / ∂E = C m E m-1 / Γ(m) where we used the recursion formula Γ(m+1) = m Γ(m) The surface area Ω(E) has its legs in two worlds: (i) the macroscopic one in which it is considered a function of the energy, and the other extensive variables, like the volume, that have been held constant in the differentiation of the phase volume, and (ii) the microscopic world where it represents the number of complexions that is compatible with a given macroscopic state. It is this quantity that Planck referred to as a 'thermodynamic' probability. ΔΧ37

38 5/11/2014ΔΧ38 Nanomechanics It differs from a classical probability inasmuch as it cannot be normalized; that is, its integral over all energies diverges, but it diverges as a power of the energy and not faster. Since its integral over all energies is infinite, we might try and consider its Laplace transform which can be given a physical interpretation. The exponential decreasing factor, where β is a positive parameter, will overpower the rapidly increasing surface area so that an enormously sharp peak will develop at a certain energy E *. Most of the contribution to the integral will come from an immediate neighborhood about this value of the energy. This enables the definition of a proper probability density according to whose integral is unity. The moments of energy are calculated from Z(β) which is called the partition function In terms of the moments, after expanding, the probability density becomes which is Gaussian. The quantity Ω(Ε) is called the structure function

39 5/11/2014ΔΧ39 Nanomechanics The quantity Ω(Ε) is called the structure function. At E =, If the phase volume increases as E m, its Laplace transform, the partition function, will vary as β -m and With these expressions, the structure function becomes The denominator is exactly Stirling’s approximation for m! = Γ(m+1), and if the structure function retains the same functional dependency for all values of the energy, the canonical probability density belongs to the family of exponential distributions known as gamma densities. Consequently, the canonical probability density falls under the jurisdiction of the local law of large numbers which asserts that a sequence of independent and identically distributed random variables tends to the normal law as the sequence increases without limit.

40 Nanofluidics In 1965, Rice and Whitehead published the seminal contribution to the theory of the transport of electrolyte solutions in long (ideally infinite) nanometer-diameter capillaries. Briefly, the potential, ϕ, at a radial distance, r, is given by the Poisson- Boltzmann equation where κ is the inverse Debye length determined by the ion number density, n, the dielectric constant, ε, the Boltzmann constant, k, and the temperature,T. Knowing the potential, φ(r), the charge density can then be recovered from the Poisson equation, whose solution may be expressed as a modified Bessel function of the first kind, I 0, and scaled to the capillary radius, a. An equation of motion under combined pressure and electrically-driven flow can then be written where F z is the body force driven by the action of the applied electric field, Ez, on the net charge density in the double layer. When there is no applied pressure, the radial distribution of the velocity is given by 5/11/201440

41 5/11/2014ΔΧ Experimental Tools in Nanotechnology S(canning) T(unneling) M(icroscope) discovered by Binning and Rohrer at IBM Zurich (Binning and Rohrer received the Nobel prize in 1986). It allows imaging of solid surfaces with atomic scale precision. Its operation is based on tunneling current which is initiated when a tip mounted on a piezoelectric scanner approaches a conducting surface at a distance of 1 nm It was followed by the S(canning) P(robe) M(icroscope) and the A(tomi) F(orce) M(icroscope) The AFM enables one to study non-conducting surfaces, as it scans van der Waals forces with its “atomic” tips 41

42 5/11/2014ΔΧ Experimental Tools in Nanotechnology Both the STM and AFT are used for positional or robotic assembly, the ultimate goal of which is to build with molecules nanostructures in the same way we build macroscopic structures with macroscopic building blocks, e.g., bricks If we achieve sufficient control over the positioning of the right molecules in the right places, we may be able to alter materials to those with desired properties Assemblers or positional devices are made to position and hold Molecular Building Blocks in positional assemblies. The most basic form of an assembler is the Stewart Platform, a rigid and flexible polyhedron with all its faces being triangular. Two of the faces, designated as base and platform, and are connected by six struts of varying length. Changing the lengths of the struts changes the orientation and position of the platform with respect to the base To restore MBBs to their desired positions, assemblers utilize a spring force, F = s x where x is the distance between the original and the desired position of the MBB and s is the stiffness of the spring The positional uncertainty (mean error in position), e, is given by e 2 = k B T / s The STM has s ~ 10 nm, hence, e ~ 0.02 nm 42

43 5/11/2014ΔΧ Self-Assembly This is a remarkable property of systems at the nanoscale. It is believed to be the basic process that led up to the evolution of the biological world from inanimate matter. There two kinds of self-assembly, (1) occurring on a fluid / solid interface, and (2)occurring in the bulk of a fluid phase An example of self assembly occurring in the bulk of a fluid phase is the micellization of asphaltene macromolecules, followed by self assembly of micelles into micelle- coacervates Asphaltene Micelle Micelle Coacervate Self-assembly on a fluid / solid interface involves immobilization molecules in the fluid as an assembly on a solid surface. It can be achieved via covalent or noncovalent interactions between molecules in the fluid and the molecules of the solid surface 43

44 5/11/2014ΔΧ Self-Assembly Covalent bonds, e.g., between a sulfide and a noble metal, produce irreversible, thus stable, immobilization at all stages. Immobilization through noncovalent bonds is reversible, thus unstable, at the onset of the self assembly process but it achieves stability upon appreciable growth of the assembly. Some common noncovalent bonds involve (1) affinity coupling via antibodies (glycoproteins produced by the immune system in response to invasion of foreign substances called antigens), (2) affinity coupling by biotin-streptavidin (avidin, a glycoprotein, combines with Biotin, a vitamin B; STreptaVidin is a tetrameric protein which has four binding sites for biotin), and (3) Immobilized Metal Ion Complexation (non-covalent binding of biomolecule by formation of a complex with metal ions) Divalent metal ion 44

45 ΝΑΝΟΣΥΝΘΕΤΑ ΠΟΛΥΜΕΡΙΚΗΣ ΜΗΤΡΑΣ Mήτρα: Άμορφο πολυμερές Πληρωτικό: Σφαιρικά νανοσωματίδια, ακτίνας R n  1 nm Κλάσμα όγκου: Συγκέντρωση σωματιδίων: Διεπιφάνεια ανά μονάδα όγκου: Απόσταση επιφανειών γειτον. σωματιδίων: Γυροσκοπική ακτίνα αλυσίδων: 5/11/2014ΔΧ45

46 Ατομιστικό (~ m) Προσομοιώσεις MD Λεπτομέρειες δομής, τοπική δυναμική Δυναμικά Hamaker για αλληλεπιδράσεις νανοσωματιδίου-νανοσωματιδίου και νανοσωματιδίου–πολυμ. τμήματος Αδροποιημένο (~10 -9 m) Monte Carlo μεταβλητής συνδετικότητας Καλά εξισορροπημένες διαμορφώσεις σε συστήματα μακριών αλυσίδων Εμπνευσμένο από τη θεωρία πεδίου (FTiMC) (~10 -7 m) Προσομοιώσεις Monte Carlo με απλοποιημένη Χαμιλτονιανή Μεγάλα νανοσωματίδια, πολλά νανοσωματίδια NAΝΟΣΥΝΘΕΤΑ: EΠΙΠΕΔΑ ΜΟΝΤΕΛΟΠΟΙΗΣΗΣ 5/11/2014ΔΧ46

47 ΕΞΙΣΟΡΡΟΠΗΣΗ ΠΥΚΝΩΝ ΠΟΛΥΜΕΡΙΚΩΝ ΦΑΣΕΩΝ :MONTE CARLO Κινήσεις μεμονωμένων τμημάτων: –Περιστροφή εσωτερικού ατόμου (FLIP) –Ερπυσμός (REPT) Τοπικές ανακατατάξεις διαμόρφωσης: –Μεροληψία απεικόνισης (CB) –Συντονισμένη περιστροφή (CONROT) [1] Μεταβολές συνδετικότητας: –Διπλή γεφύρωση (DB) [2] –Ενδομοριακή διπλή αναγεφύρωση (IDR) [2] Διακύμανση όγκου FLIP REPT 1.L.R. Dodd, T.D. Boone, DNT, Mol. Phys., 78, 961 (1993) 2.N. Karayiannis, V.G. Mavrantzas, DNT, Phys. Rev. Lett. 88, (2002) CB CONROT IDR new chain jch ’ is formed new chain ich ’ is formed DB 5/11/2014ΔΧ47

48 ΕΞΙΣΟΡΡΟΠΗΣΗ ΠΟΛΥΣΤΥΡΕΝΙΟΥ (PS) αυξάνει μονότονα προς μία ασυμπτωτική τιμή. Εξαιρετική συμφωνία με σκέδαση νετρονίων σε μικρές γωνίες (SANS). T. Spyriouni, C. Tzoumanekas, DNT, F. Müller- Plathe, G. Milano, Macromolecules 40, 3876 (2007) G. G. Vogiatzis and DNT, Macromolecules 47, (2014), in press. DOI: /ma402214r R RgRg 5/11/2014ΔΧ48

49 ΝΑΝΟΣΥΝΘΕΤΑ ΠΟΛΥΣΤΥΡΕΝΙΟΥ - C 60 Πεδίο δυνάμεων ενοποιημένων ατόμων για πολυστυρένιο: A.V. Lyulin, M.A.J. Michels, Macromolecules 35, 1463 (2002). Πεδίο δυνάμεων C 60 : S.L. Mayo, B.D. Olafson, W.A. Goddard, J. Phys. Chem. 94, 8897 (1990); L.A. Girifalco, J. Phys. Chem. 96, 858 (1992). αδροποιημένο μοντέλοατομιστικό από αντίστρ. απεικόνιση 5/11/2014ΔΧ49

50  Χρόνος συσχέτισης για τμηματική κίνηση δίνεται από το ολοκλήρωμα:  Προσαρμογή σε τροποποιημένη έκφραση Kohlrausch – Williams – Watts (mKWW): G. G. Vogiatzis and DNT, Macromolecules 47, (2014), in press. DOI: /ma402214r ΔΥΝΑΜΙΚΗ ΤΜΗΜΑΤΩΝ: ΤΗΓΜΑ PS συγκρινόμενο με ΝΑΝΟΣΥΝΘΕΤΟ PS-C 60 5/11/2014ΔΧ50

51 Προσαρμογή εμπειρικής εξίσωσης Williams–Landel–Ferry (WLF) [1] : 1.J.D. Ferry, Viscoelastic Properties of Polymers, 3rd ed., Wiley, New York, S.K. Kumar, R.H. Colby, S.H. Anastasiadis, G. Fytas, J. Chem. Phys. 105, 3777 (1996). 3.J. Hintermeyer, A. Herrmann, R. Kahlau, C. Goiceanu, E.A. Rössler, Macromolecules 41, 9335 (2008). 4.J.M. Kropka, V.G. Sakai, P.F. Green, Nano Lett. 8, 1061 (2008). Συντελεστές WLF σε πολύ καλή συμφωνία με το πείραμα [2]. Πειραματικό σημείο υαλώδους μετάπτωσης ατακτ. πολυστυρενίου: – K [3]. Σύστημα πολυστυρενίου – C 60 επιδεικνύει λίγο υψηλότερο T g από ό,τι το καθαρό πολυστυρένιο. Ανύψωση του T g κατά 1 Κ έχει αναφερθεί από τους Green και συνεργάτες [4]. ΔΥΝΑΜΙΚΗ ΤΜΗΜΑΤΩΝ: ΤΗΓΜΑ PS συγκρινόμενο με ΝΑΝΟΣΥΝΘΕΤΟ PS-C 60 5/11/2014ΔΧ51

52 G. G. Vogiatzis and DNT, Macromolecules 47, (2014), in press. DOI: /ma402214r  Προσαρμογή σε τροποποιημένη έκφραση Kohlrausch – Williams – Watts (mKWW):  Χαρακτηριστικός χρόνος χαλάρωσης δίνεται από το ολοκλήρωμα: 1.Y. He, T.R. Lutz, M.D. Ediger, C. Ayyagari, D. Bedrov, G.D. Smith, Macromolecules 37, 5032 (2004). 2.H.W. Spiess, H. Sillescu, J. Magn. Reson. 42, 381 (1981).  Καλή συμφωνία με μετρήσεις Πυρηνικού Μαγνητικού Συντονισμού (NMR) και παλαιότερες προσομοιώσεις MD [1,2] ΔΥΝΑΜΙΚΗ ΤΜΗΜΑΤΩΝ: ΤΗΓΜΑ PS συγκρινόμενο με ΝΑΝΟΣΥΝΘΕΤΟ PS-C 60 5/11/2014ΔΧ52

53  Νανοσωματίδια: ◦ Πυριτία με αλυσίδες PS επιφανειακά εμφυτευμένες κατά το ένα άκρο  Μονοδιάσπαρτο ατακτικό PS: ◦ Ελεύθερες αλυσίδες: kg mol -1 ◦ Εμφυτευμένες αλυσίδες: kg mol -1  Προσομοιώσεις NVT υπό T = 500 K  Ακτίνα νανοσωματιδίων: 8 ή 13 nm  Κυβικά κουτιά προσομοίωσης: ◦ Μήκος ακμής nm  Επιφανειακή πυκνότητα εμφύτευσης: 0.2 – 0.7 αλυσίδες nm nm ΔΟΜΗ ΝΑΝΟΣΥΝΘΕΤΩΝ ΠΥΡΙΤΙΑΣ- ΠΟΛΥΣΤΥΡΕΝΙΟΥ G.G. Vogiatzis and DNT Macromolecules 46, 4670 (2013) 5/11/2014ΔΧ53

54 Αλυσίδες: τυχαίοι περίπατοι από στατιστικά τμήματα Kuhn. Κάθε τμήμα Kuhn αντιστοιχεί σε 7 μονομερή PS. Δεσμικές αλληλεπιδράσεις λαμβάνονται υπόψη μέσω του σταθερού μήκους του τμήματος Kuhn ( b =18.3 Å). Μή δεσμικές αλληλεπιδράσεις: – Πολυμερούς-πολυμερούς (όπως στη θεωρία πεδίου) – Πολυμερούς-νανοσωματιδίου (ολοκλήρωση Hamaker των ατομιστικών δυναμικών) – Νανοσωματιδίου-νανοσωματιδίου (Hamaker) Τοπική πυκνότητα πολυμερούς παρακολουθείται χρησιμοποιώντας τρισδιάστατο πλέγμα. Απεικόνιση αλυσίδων και νανοσωμα- τιδίων μεταβάλλεται με κινήσεις MC. Χρησιμοποιούνται και μετακινήσεις του πλέγματος. 60 nm MONTE CARLO ΕΜΠΝΕΥΣΜΕΝΟ ΑΠΟ ΘΕΩΡΙΑ ΠΕΔΙΟΥ 5/11/2014ΔΧ54

55 G.G. Vogiatzis and DNT Macromolecules 46, 4670 (2013) 5/11/2014ΔΧ55

56 FTiMC: ΤΟΠΙΚΗ ΔΟΜΗ  Ακτινική κατανομή πυκνότητας,  Μεταβολές στη θέση και το πάχος της περιοχής αλληλεπικάλυψης εμφυτευμένων και ελεύθερων αλυσίδων συναρτήσει:  Μοριακής μάζας εμφυτευμένων αλυσίδων  Επιφανειακής πυκνότητας εμφύτευσης - Silica nanoparticles, R n =8 nm - Μήτρα ατακτικού πολυστυρενίου - Αραιή διασπορά M g = 20kg/mol M f =100kg/mol σ = 0.5 nm -2 M f = 100kg/mol G.G. Vogiatzis and DNT Macromolecules 46, 4670 (2013) 5/11/2014ΔΧ56

57 FTiMC: Πάχος «ψήκτρας» εμφυτευμένων αλυσίδων G.G. Vogiatzis and DNT, Macromolecules 46, 4670 (2013)  Καλή συμφωνία με πειράματα σκέδασης νετρονίων σε μικρές γωνίες (SANS). [1]  Καλή συμφωνία με θεωρία Daoud-Cotton: [2] 1.Mathias Meyer, Ph. D. thesis, Westfälische Wilhelms- Universität Münster, M. Daoud, J. Cotton, J. Phys. France 43, 531 (1982). SiO 2 σε PS, R n =8 nm M f =100 kg/mol 5/11/2014ΔΧ57

58 FTiMC: Πρόρρηση SANS από εμφυτευμένη στεφάνη Πειράματα : C. Chevigny, J. Jestin, D. Gigmes, R. Schweins, E. Di-Cola, F. Dalmas, D. Bertin, F. Boué, Macromolecules, 43, (2010). SiO 2 in PS, R n =13 nm M g = 25 kg/mol, σ = 0.5 nm -2 SiO 2 in PS, R n =8 nm M g = 20 kg/mol, M f = 100 kg/mol 5/11/2014ΔΧ58

59 ΔΙΑΧΥΤΟΤΗΤΑ ΑΡΩΜΑΤΙΚΩΝ ΜΟΡΙΩΝ ΣΤΟ ΖΕΟΛΙΘΟ ΣΙΛΙΚΑΛΙΤΗ-1 Ζεόλιθοι MFI (Mobil Five): ευρεία χρήση στην πετροχημική βιομηχανία ZSM-5: Καταλύτης για μετατροπές αλκυλαρωματικών μορίων ZSM-5: Καταλύτης για μετατροπή μεθανόλης σε βενζίνη. Τερεφθαλικό οξύ Οξείδωση PET Σιλικαλίτης-1: Μοριακό κόσκινο για διαχωρισμό π-ξυλολίου από άλλα μόρια στη νάφθα, όπως βενζόλιο, τολουόλιο, ο- και m-ξυλόλιο. Ο Σιλικαλίτης-1 είναι η καθαρά πυριτική μορφή του ZSM-5. 5/11/2014ΔΧ59

60 ΣΙΛΙΚΑΛΙΤΗΣ -1 Μοναδιαία κυψελίδα Si 96 O 192 Pnma a = Å b = Å c = Å (δείχνονται 3  3  3 κυψελίδες) Αποτελείται από τετράεδρα SiO 4 που μοιράζονται κορυφές. Ευθύγραμμα (S) και ημιτονοειδή (Z) κανάλια διαμέτρου ≈ 5.5 Å Περιοχές διασταύρωσης καναλιών (I) διαμέτρου ≈ 9 Å x y z a b c 5/11/2014ΔΧ60

61 Η διάχυση αρωματικών μορίων στο σιλικαλίτη-1 είναι βραδεία… Πειραματικός συντελεστής αυτοδιάχυσης, βενζόλιο/Σιλικαλίτης-1 (300 K) Αρωματικό μόριο βρίσκεται υπό ισχυρό περιορισμό στα κανάλια. Προτιμά να εντοπίζεται σε ενεργειακά ευνοϊκές «θέσεις ρόφησης» και σπάνια εκτελεί άλματα μεταξύ αυτών των θέσεων. H. Jobic. M. Bée, S. Pouget J. Phys. Chem.B 104, 7130 (2000). Τόσο χαμηλές διαχυτότητες δεν μπορούν να υπολογιστούν με MD. x z y 5/11/2014ΔΧ61

62 i j state Σταθερά ρυθμού k i  j : Πιθανότητα ανά μονάδα χρόνου να επιτελεστεί μετάβαση προς την κατάσταση j,προϋποτιθεμένου ότι το σύστημα βρίσκεται αρχικά στην κατάσταση i. ΘΕΩΡΙΑ ΜΕΤΑΒΑΤΙΚΩΝ ΚΑΤΑΣΤΑΣΕΩΝ (TST) Ελεύθερη ενέργεια ως συνάρτηση των βαθμών ελευθερίας r που συμμετέχουν στη μετάβαση. Μπορεί να υπολογιστεί ένας δυναμικός παράγοντας διόρθωσης για το k i  j, που ενσωματώνει την επίπτωση φαινομένων αναδιασταύρωσης της διαχωριστικής επιφάνειας, μέσω σύντομων προσομοιώσεων MD που ξεκινούν από τη διαχωριστική επιφάνεια. διαχωριστική επιφάνεια μεταξύ καταστάσεων i και j TST: J. Kärger, D. Ruthven, DNT, Diffusion in Nanoporous Materials, Volume 1, Wiley-VCH, 2012, Chap.9 5/11/2014ΔΧ62

63 Προσδιορισμός A(r): Το ροφημένο μόριο εξαναγκάζεται να δειγματοληπτήσει μικρές αλληλεπικαλυπτόμενες περιοχές μέσα στους πόρους. Χρήση MD παρουσία περιοριστικών «τοίχων». P.D. Kolokathis, E. Pantatosaki, C.-A. Gatsiou, H. Jobic, G.K. Papadopoulos, DNT Molecular Simulation 40, (2014). 5/11/2014ΔΧ63

64 Σταθερές μετάβασης από ΤST: Bενζόλιο στο Σιλικαλίτη-1 Θερμοκρασία Σταθερές ρυθμού TST (s -1 ) 300K465K555K I → S 5.24    10 8 S → I    10 9 I → Z b    10 7 Z b → I    10 9 I → Z a 1.76    10 8 Z a → I3.04    10 9 Δυναμικοί παράγοντες διόρθωσης: 0.81 έως /11/2014ΔΧ64

65 Bενζόλιο και π-ξυλόλιο στο Σιλικαλίτη-1, T =300 K Μονοδιάστατα προφίλ ελεύθερης ενέργειας στο ευθύγραμμο κανάλι: Κάθετα προς δακτύλιο Μίσχος μεθυλίου (κύριος άξονας) 10.5 X(Å) X(Å) ξ (Å) 2 Κατανομή ελεύθερης ενέργειας προσανατολισμού σε διάφορες θέσεις: Χρωματικός κώδικας I S S 5/11/2014ΔΧ65

66 Επίλυση της εξίσωσης Master μέσω αναδρομικής ελάττωσης της διαστατικότητας (MESoRReD) σε σύστημα 2 ν μοναδιαίων κυψελίδων με περιοδικές οριακές συνθήκες στα άκρα: Υπολογισμός του συντελεστή αυτοδιάχυσης D Εξίσωση Master για τη χρονική εξέλιξη των πιθανοτήτων κατάληψης των καταστάσεων: Άνυσμα πιθανοτήτων κατάληψης καταστάσεων LdLd Cell 1 Cell 2 C ell 2 ν -1Ce ll 2 ν Cell 2 ν-1 + 1Cell 2 ν-1 P.D. Kolokathis and DNT J. Chem. Phys 137, (2012) Πίνακας σταθερών ρυθμού 5/11/2014ΔΧ66

67 Τεχνική Neutron Spin-Echo Προσομοιώσεις ΣΥΝΤΕΛΕΣΤΕΣ ΑΥΤΟΔΙΑΧΥΣΗΣ ΣΤΟ ΣΙΛΙΚΑΛΙΤΗ -1 5/11/2014ΔΧ67

68 ΠΕΡΙΛΗΨΗ  Η πολλαπλότητα των κλιμάκων μήκους και χρόνου επιβάλλει την ανάπτυξη στρατηγικών προσομοίωσης σε πολλές κλίμακες.  Προσομοιώσεις Μonte Carlo μεταβλητής συνδετικότητας, μαζί με αδροποίηση και αντίστροφη απεικόνιση, εξισορροπούν πλήρως πολυμερικά τήγματα μεγάλου μοριακού βάρους.  Η επίπτωση νανοσωματιδίων στην τμηματική δυναμική νανοσυνθέτων C 60 -πολυστυρενίου ποσοτικοποιείται με προσομοιώσεις MD.  Προσομοιώσεις Monte Carlo εμπνευσμένες από τη θεωρία πεδίου προβλέπουν τη διαμόρφωση αλυσίδων εμφυτευμένων σε νανοσωματίδια ακτίνων 8 και 13 nm μέσα σε τήγματα πολυστυρενίου.  Η μοριακή προτυποποίηση και προσομοίωση μπορούν να βοηθήσουν σημαντικά στην κατανόηση των ιδιοτήτων νανοδομημένων υλικών.  Υπολογισμός των προφίλ ελεύθερης ενέργειας κατά μήκος των πόρων, σε συνδυασμό με θεωρία μεταβατικών καταστάσεων και MESoRReD, δίνει εκτιμήσεις διαχυτοτήτων αρωματικών μορίων στο Σιλικαλίτη-1.  Το π-ξυλόλιο διαχέεται ταχύτερα από ό,τι το βενζόλιο μέσα στο Σιλικαλίτη-1. Η δυνατότητα αναπροσανατολισμού στις διασταυρώσεις των καναλιών δημιουργεί υψηλότερο φράγμα ελεύθερης ενέργειας εντροπικής προέλευσης για το βενζόλιο. 5/11/2014ΔΧ68

69 5/11/2014ΔΧ Biopharmaceuticals Also known as Biotechnology-based Pharmaceuticals Since 1980 more than 90 recombinant medicines have been approved and 369 are in the pipeline. The majority of these are recombinant versions of proteins found in vivo They are used (1) in replacement therapy, e.g., insulin and growth hormone, (2) as supplement to increase the effect of endogenous proteins, (3) to activate receptors located in target tissues, (4) as therapeutic antibodies, (5) as site-specific carriers of toxic drugs, and (6) as imaging agents Use of therapeutic proteins to replace or supplement endogenous protein molecules has been a long established therapeutic modality for diseases such as diabetes, growth hormone deficiency, and hemophilia. Treatment was often limited by (1) immunlogical responses to heterologous protein molecules, (2) contamination of proteins derived from complex natural sources, and (3) difficulty and expense of obtaining useful quantities of materials of human and animal origin Today recombinant DNA and hybridoma techniques are capable of providing molecules of well defined chemical composition and producing them in cell culture media that can be carefully controlled. Because of these techniques, supply and purity no longer impede development and use of therapeutic biomolecules 69

70 5/11/2014ΔΧ Biopharmaceuticals DNA, because of its role in the replication of new structures and characteristics of living organisms, has widespread use in recapitulating, via viral or non-viral vectors, both desirable and undesirable characteristics of a species to achieve characteristic change or to counteract effects caused by genetic or imposed disorders that affect cellular or organismal processes. Recombinant DNA is a form of artificial DNA that is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences that would not normally occur together. In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organismal genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose, such as immunity. Genetic modification differs from genetic recombination, in that it does not occur through processes within the cell or ribosome, but is exclusively engineered. The recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert Boyer in They published their findings in a 1974 which described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, creating a transgenic bacterium. Recombinant DNA technology was made possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathan, and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine 70

71 5/11/2014ΔΧ Biopharmaceuticals Antibodies are highly specific proteins that bind to antigens, and produced by stimulated B-lymphocytes, each of which secretes antibodies of only one specificity. Polyclonal antibodies are mixtures of many different antibodies of many different specificities, produced in vivo. Monoclonal antibodies are produced from a mixture of antibodies, by isolating B-lymphocytes in tissue culture, growing the isolated B-lymphocytes, and producing clones of identical B-cells. Problems encountered with this procedure are that isolated B-lymphocytes will often not remain viable in culture, don't divide very rapidly, and produce very little antibody. If we combine the properties of a B- lymphocyte, which produces antibodies, and a tumor cell, which exhibits tissue viability and rapid cell division, i.e., we “fuse” a B-lymphocyte and a tumor cell into one cell, this “hybrid" cell continues to grow indefinitely in culture, and undergoes rapid cell division (produces one "clone"). In the same culture, antibody production by one [mono] clone is initiated and large amounts of antibodies can be produced. The hybridoma technique can manipulate genetics of isolated lymphocytes, can select antibodies with certain biological properties, and is much less expensive than maintaining animals that produce antibodies 71

72 5/11/2014ΔΧ Biopharmaceuticals Recombinant DNA technology or Genetic Engineering involves the isolation of cellular DNA fragments that code for proteins of therapeutic interest DNA fragments are inserted into cellular hosts that, by normal replication, make multiple copies of the original sequence. This amplification of the original sequence enables the production of useful quantities of protein in the cell culture medium. By using established biochemical purification techniques, the protein may be isolated in highly purified form Monoclonal antibodies were one of the first proteins, derived from recombinant DNA technology, administered to humans. In the early days, monoclonal antibodies were derived from murine strains using hybridoma techniques. Administration of the murine-derived antibodies elicited a human immune response with the production of endogenous antibodies to neutralize the administered antibody and a short serum half life for the latter. With Genetic Engineering tools developed later, monoclonal antibodies expressing greater sequence homology to endogenous antibodies have been produced. Two main technologies have emerged for production of fully “humanized” proteins, (1) from transgenic animals, and (2) from phage display 72

73 5/11/2014ΔΧ Biopharmaceuticals The most efficient and popular expression systems for recombinantly derived biopharmaceuticals are mammalian cell lines such as Chinese Hamster Ovary and mouse myeloma cells (NSO) Prokaryotic cell lines derived from Escherichia coli and yeast strains are also used to express proteins of therapeutic interest Mammalian expression systems, as opposed to those of nonmammalian origin, have the biochemical machinery to glycosylate proteins during posttranslational biosynthetic events. Recombinantly derived monoclonal antibodies require glycosylation to exhibit a complete spectrum of biological activity. Glycosylation of recombinant antibodies can be controlled during fermentation by adjusting carbohydrate levels in the cell culture where cells are growing. Fermentation conditions for these cell types can also be adapted to express high titers of the therapeutic antibody, at > 1 g/L, which is important for developing efficient and economic processes One can enhance overall product yield of therapeutic proteins by using transgenic animals, e.g., sheep, goats, or pigs Specialized serum-free cell nutritional media, free of bovine-derived products, to remove any possibility of contamination by trace levels of Bovine Spongiform Encephalopathy (BSE), have been developed 73

74 5/11/2014ΔΧ Some Biology What is Life? Defined only in terms of its properties: 1.Cellular organization, assemblages of molecules enclosed within membranes, 2.Sensitivity, ability to respond to stimuli, 3.Growth, assimilate energy and use it to grow (metabolism), 4.Development, systematic gene-directed changes as organisms grow and mature, 5.Reproduction, passing traits to next generation, 6.Regulation, organisms have mechanisms that regulate internal processes, 7.Homeostasis, organisms maintain relatively constant internal conditions, different from their environment Building blocks of life Molecules → Cells → Tissues → Organs → Organisms Cell - smallest living entity, basic unit of organization of all organisms; contains DNA, the hereditary molecule; enclosed and separated from its surroundings by plasma membrane 74

75 5/11/2014 Cells are of two types, (1) procaryotic, i.e., cells that have no nuclear membrane, no organelles, a single chromosome (DNA molecule), and (2) eucaryotic, i.e., cells that have complex internal structure, with a nuclear membrane, a variety of organelles, including mitochondria, endoplasmic reticulum, golgi apparatus and others, and more than one chromosomes in the nucleus Cellular organisms are classified into eucaryotes (have eucaryotic cells) and bacteria (have procaryotic cells). Eucaryotes can be multicellular (extensive differentiation of cells and tissues), e.g., plants, or unicellular (little or no differentiation of cells and tissues), e.g., animals and protists (algae,fungi, protozoa) Bacteria can be divided into eubacteria (most of the bacteria; their cell chemistry is similar to eucaryotes) and archaebacteria (ex. methanogens, halophiles,thermo- acidophiles; have distinctive cell chemistry) Viruses cannot be classified under any category; are very small ( nm) and obligate parasites (not free-living cells) of other cells; contain either DNA or RNA as genetic material and use either RNA or DNA to decode genetic information (in free-living cells all genetic information is contained in DNA). RNA viruses are called retroviruses, e.g., HIV virus ΔΧ75

76 5/11/2014 Some Biology Cell In prokaryotes (bacteria), most of genetic material lies in a single molecule of DNA which resides in area close to cell center, nucleotid; this area in not segregated from the rest of cell’s interior by membranes In eukaryotes, DNA is contained in nucleus which is surrounded by two membranes, (a) A semi-fluid matrix, cytoplasm, fills interior of cell, exclusive of nucleus (eukaryotes) or nucleotid (prokaryotes). Contains sugars, amino acids and proteins for cell’s activities. In eukaryotic cells, cytoplasm contains specialized membrane- bounded compartments, called organelles, and (b) A phospholipid bilayer, plasma membrane, which separates the cell from its sur- roundings ΔΧ76

77 5/11/2014ΔΧ Some Biology Eukaryotic cell structures 1.Cell wall – only in plant cells for protection and support, 2.Cytoskeleton – array of fibrous proteins for structural support and cell movement, 3.Plasma membrane – phospholipid bilayer (hydrophobic end interior, hydrophilic end exterior) regulates what passes in and out of cell, 4.Endoplasmic reticulum - network of membranes in which glycoproteins and lipids are synthesized 5.Nucleus – houses most of cellular DNA; directs protein synthesis and cell reproduction 6.Golgi - packages proteins for export from cell; forms secretory vesicles, 8.Lysosomes - contain enzymes, digest worn-out organelles and cell debris 9.Microbodies - contain enzymes, isolate particular activities from rest of cell, 10.Mitohondria - bacteria-like elements; sites of oxidative metabolism, 11.Chloroplasts - sites of photosynthesis in plant cells, 12.Chromosomes - DNA and protein complex assemblies, contain hereditary information (total DNA in chromosomes of organism is its genome), 13. Nucleolus - site of genes for rRNA synthesis, assembles ribosomes, 14.Ribosomes - complex assemblies of protein and RNA, often found in endoplasmic reticulum; sites of protein synthesis 77

78 5/11/2014ΔΧ Some Biology Molecules of Life Water, inorganic ions, and relatively small organic molecules (e.g., sugars, vitamins, fatty acids) account for 75 – 80% of living matter by weight. Water is most abundant. Water, ions and many small organic molecules are imported into cells. Cells also make and alter many small organic molecules Remaining 20 – 25% of living matter are macromolecules (polymers),including proteins, polysaccharides, and nucleic acids, e.g., DNA (deoxyribonucleic acid), RNA (ribonucleic acid). Cells can acquire macromolecules only by making them MonomerPolymer Amino acid Protein NucleotideNucleic Acid SugarPolysaccharides 78

79 5/11/2014 Amino acids Contain at least one carboxylic group, -COOH, one  -amino group, -NH2, and a side group, R. H | H 2 N - C - COOH | R Building blocks of proteins are 20 common amino acids which, on the basis of side group, R, are classified as: 1.Nonpolar - R groups contain –CH 2 or –CH 3 : alanine, valine, leucine, isoleucine, 2.Polar uncharged - R groups contain O or only H: glycine, serine, threonine, asparagine, glutamine, 3.Ionizable - R groups contain acids or bases: glutamic acid, aspartic acid, histildine, lysine, arginine, 4.Aromatic - R groups contain C rings with alternating single and double bonds: phenylalanine, tryptophan, and 5.Special-function: methionine, proline (causes kinks in chains), cysteine (links chains together) ΔΧ79

80 5/11/2014ΔΧ Amino acids Are optically active and occur in two isomeric forms, L and D H H | H 2 N - C - COOH HOOC - C - NH 2 | | R L-amino acid D-amino acid In gas state are neutral; in liquid state exist as dipolar molecules of the form H | H 3 N + - C - COO - | R zwitterion The pH value at which an amino acid has no net charge is called isoelectric point 80

81 5/11/2014ΔΧ Proteins are the most abundant organic molecules in living cells, 40 – 70% of their dry weight. They are condensation polymers of  -amino acids, joined by peptide bonds. A peptide bond forms when –NH 2 end of one amino acid joins to the -COOH of another The peptide bond is planar. Peptides contain two or more amino acids linked by peptide bonds. Polypeptides are chains containing less than 50 amino acids and larger amino acid chains are proteins Only L amino acids are found in proteins. D amino acids are rare in nature; they are found in the cell walls of some microorganisms and in some antibiotics Conjugated proteins contain organic and/or inorganic components, other than amino acids, which are called prosthetic groups, e.g., hemoglobin is a conjugated protein with four heme groups, i.e., iron-containing organometallic complexes R | H 2 N - C - C - | || H O H R΄ | | N - C - COOH | H 81

82 5/11/2014 Proteins There are two major types of protein conformation, (1) fibrous, and (2) globular Based on their diverse biological functions, proteins can be classified in 5 major classes: 1.Structural, e.g., glycoproteins, collagen, keratin, 2.Catalytic, e.g., enzymes, 3.Transport, e.g., hemoglobin, serum albumin, 4.Regulatory, e.g., hormones (insulin, growth hormone), and 5.Protective, e.g., antibodies, thrombin Enzymes, over 2000 different kinds known, represent the largest class. Most enzymes are globular proteins, with extraordinary catalytic power and high specificity in their function. Every molecule of an enzyme has an active site to which its specific substrate is bound during catalysis Antibodies (Ab) or immunoglobulins (IgG, IgA, IgD, IgE, and IgM), MW = 150 – 900 kD, are found in the blood serum and certain cells of the vertebrae and bind to Antigens (Ag), foreign molecules, to form an Ab-Ag complex. The formation of the complex is called immune response. They are highly specific to foreign proteins that induce their formation. They have 4 polypeptide chains, 2 heavy (430 amino acids) and 2 light (214 amino acids), linked together by disulfide bonds into a Y- shape, flexible structure. Each chain has constant and variable amino-acid-sequence regions. The variable sequence regions of the light and heavy chains compose the binding sites ΔΧ82

83 5/11/2014 Proteins The 3-D structure of proteins, which is critical to their biological activity, can be described at 4 different levels: 1.Primary structure - a unique linear sequence of amino acids with defined composition, 2.Secondary structure - arises from hydrogen bonding of neighboring amino acids, i.e., hydrogen bonds on the same chain form  helix, hydrogen bonds across chains form  -pleated sheet (sheet structure is more stable than the helix structure), 3.Tertiary structure – folding or bending of amino acid chains as a result of covalent, disulfide, or hydrogen bond, or hydrophilic and hydrophobic interactions between R groups (disulfide bonds critical to proper folding), and 4.Quaternary structure – arises from interactions (disulfide bonds or weak interactions) between peptide chains ΔΧ83

84 5/11/2014 Proteins Hemagluttinin: consists of 3 identical subunits, each composed of 2 chains, HA 1 & HA 2 (a)Primary structure - sequence of amino acid residues 68 – 195 of HA 1 (used by influenza virus to bind to animal cells); one- letter amino acid code. Secondary structure - regions of polypeptide chain folded into  -helices (cylinders),  -sheets or strands (arrows), and random coils (bold lines) (b)Tertiary structure - folding of helices and sheets in each subunit; domains with globular (membrane-distal domain), and fibrous conformation (membrane- proximal domain) (c)Quaternary structure – consists of 3 subunits of HA and is stabilized by lateral interactions between subunits ΔΧ84

85 5/11/2014ΔΧ Polysaccharides Carbohydrate - contain C:H:O in molar ratio 1:2:1 C-H bonds release energy when broken Sugars - carbohydrates consisting of six-carbon rings (Fructose, glucose and galactose, isomers with empirical formula C 6 H 12 O 6 ) Polysaccharides – polymers of sugar Organisms convert most of the glucose into fat to store energy over long periods of time Lipids Molecules insoluble in water Fat - composite molecules which consists of glycerol (a 3-C alcohol with each C bearing a OH- group) and 3 fatty acids (long hydrocarbon chains ending in -COOH group); most contain more than 40 C atoms but ratio of energy-storing C-H bonds to C atoms more than twice that of carbohydrates Saturated fats contain maximum possible number of H atoms; unsaturated fats contain double bonds between one or more pairs of successive C atoms; polyunsaturated fats contain more than one double bonds, have low melting point (fatty acid chains bend at double bond preventing molecules from aligning closely with one another) and are usually liquid at room temperature 85

86 5/11/2014 Nucleic acids Nucleic acids - polymers of nucleotides; information storage devices of cells; serve as template to produce precise copies of them-selves, so that information that specifies what an organism is can be copied and passed down to its descendants Nucleotide - consists of (1) a 5-C sugar (ribose in RNA and deoxyribose in DNA), (2) a phosphate group, -PO 4, and (3) an organic N-containing base. When a nucleic acid polymer forms, phosphate group of one nucleotide binds to the hydroxyl group of another, releasing water and forming a phosphodiester bond. Nucleotides contain two types of organic bases: (1) purines, large double- ring molecules found in both DNA and RNA, e.g., adenine (A) and guanine (G), and (2) pyrimidines, smaller single-ring molecules, e.g., cytosine (C, in both DNA and RNA), thymine (T, in DNA only), and uracil (U, in RNA only) Adenine key component of Adenosine TriPhosphate, energy currency of cell; also in Nicotinamide Adenine Dinucleotide (NAD + ) and Flavin Adenine Dinucleotide (FAD + ), carry electrons whose energy is used to make ATP Code of DNA consists of different combinations of 4 nucleotides, A, C, G, T ΔΧ86

87 5/11/2014 Nucleic Acids Nucleotides Double-stranded DNA replicating ΔΧ87

88 5/11/2014ΔΧ Nucleic acids DNA - deoxyribonucleic acid; contains deoxyribose sugar in which –OH is replaced by –H; stores hereditary information as a specific sequence of nucleotide bases; information is used to assemble proteins in a way similar to which letters on a page encode information RNA - ribonucleic acid; contains ribose sugar in which number 2 C is bonded to –OH; similar in structure to DNA; made as a transcript copy of portions of DNA and used by cells to read the DNA-encoded information and direct synthesis of proteins; utilizes uracil instead of thymine in DNA; uracil has same structure as thymine, except one of its C atoms lacks a – CH 3 DNA molecules exist not as single chains (exceptions viruses) but as double chains wound around each other like the outside and inside rails of a circular staircase (double helix) Each step of DNA’s helical staircase is a base-pair, consisting of a base in one chain attracted by hydrogen bonds to a base opposite to it on the other chain RNA molecules are always single-stranded 88

89 5/11/2014 Properties of biological molecules (proteins, peptides, nucleic acids) The diffusion coefficients of particles, of a regular or close to regular shape and size from molecular (in Å ) upwards, in a liquid, with the particles-liquid system being either a suspension or a solution, are given by the Stokes-Einstein equation where D pl is the diffusion coefficient of the particles in the liquid, k B is the Boltzman constant equal to 1.38x erg molecule -1 o K -1 In the dilute regime, where the volumetric fraction of the particles in the particles- liquid sytem is small (< 0.10), such as those found in most biological systems, the motion of particles is caused by thermal energy transferred from the liquid molecules during random collisions between them and particles. This transfer of energy leads to fluid motion which is retarded by the drag of the liquid on the particles. For particles with a characteristic dimension about 10 times greater than that of the solvent molecules, the drag force can be approximated using low-Reynolds-number hydrodynamics, as F D = K. v p (2) where F D is the drag force (vector) acting on a single particle, K is the translation tensor (symmetric, i.e., K ij = K ji ), and v p the velocity of the particle The symmetric tensor K, by appropriate choice of the coordinate axes, can be made so that it has f ij = 0 if i ≠ j and f ij ≠ 0 if i = j. The nonzero main diagonal components of K are called principal friction coefficients and, for the case of isotropic particle, are f 1 = f 2 = f 3 = f ΔΧ89

90 5/11/2014ΔΧ Properties of biological molecules (proteins, peptides, nucleic acids) Proteins under physiological conditions assume their distinctive tertiary structure, native conformation, of minimum free energy, which is a prerequisite for biological function. The native biologically active form of a protein molecule is globular (in synthetic polymers, the random chains may also be coiled into globular structures called spherulites), but protein molecules cannot be treated adequately as spheres. This is clear from the values of the ratios f ̅ / f 0 and the structures and sizes of X-ray Crystallography In solution, a protein is hydrated due to electrostatic and electrodynamic interactions. If hydration were uniform over the protein molecule and occurred in discrete layers, it (hydration) would result in larger molecular size. However, hydration is not uniform over the molecule and is greater close to charged groups. It increases the molecular volume of protein as where v ̅ mp is the molar volume in cm 3 gmol -1, v ̅ sp is specific volume in cm 3 g -1, M is the protein molecular weight, ρ w the density of water, and δ h the extent of hydration in g water (g protein) -1 The Table that follows lists measured and estimated, for different shapes and sizes, diffusion coefficients for the protein lysozyme 90

91 5/11/2014ΔΧ Properties of biological molecules (proteins, peptides, nucleic acids) The previous Table shows that accounting for the shape more accurately, improves the estimate of diffusion coefficient Data from sedimentation in an ultracentrifuge and diffusion can be utilized to estimate the molecular weight of a protein from For a number of proteins, diffusion and sedimentation coefficients at 20 o C, the molecular weight, X-ray crystallographic data for shapes, and measured values of f ̅ / f 0 are known and listed in the Table to follow The method most commonly used to measure the molecular weight of a protein is gel filtration, nowadays. It involves passing the protein solution through a column of packed porous beads. Molecules smaller than the pore size of the beads enter the beads, and travel longer through the column than other molecules do. The smaller the molecule, the more time it takes to go through the column. By measuring the time required to pass through the column a set of standards of known molecular weight, and establishing a molecular weight vs. residence time calibration curve, the molecular weight of the protein is better determined. This method is easier to apply than the ultracentrifuge and does not need pure samples for its operation 91

92 5/11/2014ΔΧ Properties of biological molecules (proteins, peptides, nucleic acids) 92

93 5/11/2014ΔΧ Properties of biological molecules (proteins, peptides, nucleic acids) From analysis of a 3-D random walk, the diffusion coefficient is given by D ij = δ 2 / 6τ(1) where δ is the displacement and τ is the time between collisions A diffusion velocity, v d, is defined as v d = δ /τ(2) The diffusion velocity can be determined by equating the kinetic energy of the particle or molecule to its thermal energy, according to 1/2 m p v d 2 = 3/2 k B T(3) Eqs(1) and (2)  δ = 6D ij /v d (4) Eq.(3)  v d = (3k B T/m p ) 1/2 (5) The Stokes-Einstein equation is D ij = k B T/ f ̅ (6) Substituting from Eqs. (5) and (6) into Eq. (4), results in δ = (12 m p k B T) 1/2 / f ̅ = (12Mk B T/N A ) 1/2 / f ̅ (7) 93

94 5/11/2014 Properties of biological molecules (proteins, peptides, nucleic acids) Polypeptides and polynucleic acids (RNA and DNA) differ from globular proteins and are often treated as random coils For a free-draining (solvent moves freely in area occupied by coil) random coil with freely joined N segments (N is the degree of polymerization) of length l each, the root-mean-square end-to-end distance, 1/2, is 1/2 = N 1/2 l If the coil is fully extended, 1/2 = L c = Nl where L c is the contour length This applies to natural and synthetic random coil macromolecules in solvents with which the mole- cule does not interact (theta solvents) Real macromolecules, like those of polypeptides and polynucleic acids are quite different than the free-draining random coil. These differences are (a)The coil has a finite size, hence, an excluded-volume effect needs to be taken into account, (b)The bond angle, θ, and the bond-rotation angle, φ, of the macromolecule are neither fixed nor changing without constraints, and (c) There is molecule-solvent interaction 1/2 RnRn R n-1 ΔΧ94

95 5/11/2014ΔΧ Properties of biological molecules (proteins, peptides, nucleic acids) To account for these differences, for polypeptides and polynucleic acids (RNA and DNA), the root-mean-square end-to-end distance, 1/2, is 1/2 = (C n N) 1/2 l where C n is the characteristic ratio, equal to the ratio l K / l, and l K being the Kuhn length which, together with number of Kuhn segments, N K < N, parametrize the stiffness of the coil 1/2 = N K 1/2 l K L c = N K l K When N  ∞, C n  C ∞ (finite). Polypeptides without the amino acids glycine or proline have C ∞ = 130 and their root-mean-square end-to-end distance is in nm. The large value of C ∞ indicates significant interaction of the side chains with water and stiffening of the segments For DNA, l K equals 150 nm and l equals 0.34 nm, which indicates that DNA is a stiff chain. For bacterial DNA with 1.5x10 6 base pairs, the root-mean-square end-to-end distance is 8.75 μm, much larger than the size of the bacterium For polyamino acids (polypeptides and proteins) and polynucleotides (RNA and DNA), the friction coefficient can be estimated under the assumption that the random coil behaves as a sphere with radius equal to the radius of gyration, R g 95

96 5/11/2014ΔΧ Properties of biological molecules (proteins, peptides, nucleic acids) The radius of gyration, R g, is defined in two ways, which can be shown to be equivalent and where r k = R k – R k-1 and R k is the vector from the origin of the coordinate system to the k-th joint of the chain For a free-draining, open-ended, random coil For a circular random coil, which can represent well the configuration of a DNA plasmid found in bacteria 96

97 5/11/2014ΔΧ Information Flow & Control A cell is more than a bag filled with water, lipids, amino acids, sugars, enzymes, and nucleic acids. It must control how these components are made and how they interact with each other. To do so the cell possesses the ability of metabolic regulation, i.e., the ability to coordinate a wide variety of chemical reactions taking place within it Key to metabolic regulation is the flow and control of information All living systems obey the same Central Dogma of Biology when it comes to storage, expression and utilization of information. Information is stored on the DNA molecule. This information can be replicated directly to form a second identical molecule. Further segments of the information can be transcribed to yield RNA molecules. Using a variety of RNAs, the information is translated into proteins which perform a structural or enzymatic role in mediating all the metabolic functions in the cell. The information content of the DNA molecule is static; changes occur slowly through infrequent mutations or rearrangements. The type and the amount of RNA species that are present varies with time and culture conditions. Likewise, the proteins that are present change with time on a scale different than the time scale of the RNA change. Some of the proteins bind to DNA to regulate the transcriptional process to form RNAs 97

98 Information Flow & Control One important, although relatively minor, deviation from the Central Dogma, is the existence of RNA retroviruses that contain an enzyme called reverse transcriptase which enables reverse transcription from RNA to DNA (example: Human Immunodeficiency Virus causing Acquired ImmunoDeficiency Syndrome; one approach to treatment is to inhibit reverse transcriptase) For information storage and exchange to take place, there must be a language. All life is using a 4-letter alphabet made up of nucleotides A, T, C, and G in DNA. In this language, all words, called codons, are 3- letter long. With 4 letters and only 3-letter words, a language can have a maximum of 64 words. When these words are put into a sequence, one can make a sentence, called gene, which, when properly transcribed and translated, is a protein Each step in the information storage and transfer requires a macromolecular template. The success or failure of the processes of genetically engineering therapeutic proteins depends on the choice of the host organism and the expression system. The most important consideration must be whether posttranslational modifications, e.g., glycosylation, i.e., addition of sugars, or phosphorylation, of the product are necessary 5/11/2014ΔΧ98

99 5/11/2014 Protein Stability Proteins under physiological conditions assume their distinctive tertiary structure, native conformation, of minimum free energy, which is a prerequisite for biological function. The native biologically active form of a protein molecule is held together by a delicate balance of noncovalent forces, hydrophobic, van der Waals, and hydrogen bonds In proteins that contain two or more cystine residues, disulfide bonds, which are covalent, contribute substantially to maintaining the native protein conformation By X-ray structure analysis, it has been confirmed that most water-soluble proteins may be grossly described as a hydrophobic core of nonpolar amino acids, surrounded by a hydrophilic shell of solvated polar amino acids. With exposure to certain denaturants and adverse environmental conditions, the noncovalent forces are weakened and broken apart, leading to unfolding (chaperone proteins are important proteins that assist in proper folding of polypeptide chains) and consequent inactivation of the protein Typically, the native structure exhibits only marginal stability that is easily upset by even subtle environmental changes in pressure, temperature, pH, ionic strength, or a combination thereof. The free energy of denaturation of globular proteins rarely exceeds 15 kcal/mol. The complete or partial unfolding of the protein is usually fully reversible, after removal from the antagonistic agent. This reversible unfolding is the precursor to irreversible protein denaturation by covalent and noncovalent reactions. ΔΧ99

100 5/11/2014ΔΧ Protein Stability Covalent Destabilization As with conventional-drug small organic molecules, the chemical reactions involved in protein destabilization are classified as those involving (1) hydrolysis, (2) oxidation, and (3) racemization Disulfide bond cleavage and exchange are also reactions that affect protein stability A striking feature of protein destabilization is that several different reaction mechanisms may proceed simultaneously. Because of the multiple degradation pathways that may take place at elevated temperature, protein stability monitoring data may not conform to the Arrhenius relationship. The chemical reaction mechanisms involved in protein degradation depend on (1) nature of protein, (2) temperature, (3) pH, (4) ionic strength, (5) oxygen vapor pressure, and (6) type and concentration of other solutes 100

101 5/11/2014ΔΧ Protein Stability Covalent Destabilization - Hydrolysis Primary hydrolytic reactions in protein degradation are peptide bond hydrolysis and deamidation Peptide bond hydrolysis occurs readily under strongly acidic conditions or by combination of milder pH and elevated temperature. Complete acid hydrolysis of protein into its amino acids takes place under extreme conditions, 6M HCl, 24 h, 110 o C. Shorter exposures under less acidic conditions, show preferred peptide hydrolysis on aspartic acid residues. Aspartyl-prolyl linkages are especially vulnerable. Loss of oligosaccharide moieties through hydrolysis of glycosidic bonds may also influence protein stability Deamidation is the hydrolysis of the side-chain amide on glutamine and asparagine residues (asparagine residues are more susceptible to deamidation than glutamine residues). Deamidation under physiological conditions proceeds essentially through an imide intermediate. The cyclic imide (succinimide) is rapidly hydrolyzed by water into a mixture of aspartic acid and isoaspartic acid, resulting in the introduction of new negative charge to the protein. The rate of deamidation is affected by the nature of the amino acid residue adjacent to asparagine. The most labile asparagine residues in smaller peptides are followed by glycine residue. Asparagine-serine sequences are next most labile sites of deamidation. In globular proteins, the location of susceptible residue in the folded conformation may be more important in controlling deamidation rate 101

102 5/11/2014 Protein Stability Covalent Destabilization - Oxidation Can occur with a variety of oxidants for amino acids with aromatic side chains, as well as methionine, cysteine, and cystine residues. Molecular oxygen, hydrogen peroxide, and oxygen radicals are all known oxidants of protein Oxidation of methionine residues to their corresponding sulfoxides is associated with loss of biological activity for many peptide hormones and proteins. The thiol group of cysteine can be oxidized in steps successively to a sulfenic acid, RSOH, a disulfide, RSSH, a sulfinic acid, RSO 2 H, and finally a sulfonic acid, RSO 3 H, depending on reaction conditions. Oxidation of thiols occurs readily at basic pH in the presence of transition metal ions such as Cu 2+. When oxidized thiol groups are exposed on the protein surface, because of steric effects, intermolecular disulfide bonds may form, leading to protein aggregation Factors that influence the rate of oxidation include temperature, pH, buffer medium, type of catalyst, and oxygen vapor pressure ΔΧ102

103 5/11/2014ΔΧ Protein Stability Covalent Destabilization - Racemization of the native L-amino acid to the D-enantiomer generally results from base-catalyzed removal of the  -proton to produce a negatively charged planar carbanion. Return of the proton to the carbanion intermediate through reaction with water produces an enantiomeric mixture Rates of racemization depend on the particular amino acid and are influenced by temperature, pH, ionic strength, and metal ion chelation. Aspartic acid and serine residues are the most prone to racemization. An electron-withdrawing group in the side of the amino acid, as in serine, stabilizes the carbanion intermediate, which in turn accelerates the rate of racemization Intermediate succinimide formation plays a major role in racemization at aspartyl and asparaginyl residues. Racemization of amino acids in proteins can generate nonmetabolizable forms of amino acids (D-enantiomers) or create peptide bonds inaccessible to proteolytic enzymes 103

104 5/11/2014 Protein Stability Covalent Destabilization – Disulfide exchange Disulfide bonds provide covalent structural stabilization to proteins. Cleavage and subsequent rearrangement of these bonds can alter the tertiary structure, thereby affecting biological activity. Disulfide exchange is catalyzed by thiols, which can arise by initial hydrolytic cleavage of disulfides, or by  -elimination in neutral or alkaline media At pH 6 and 8, thermal inactivation of ribonuclease at 90 o C is caused primarily by disulfide exchange. This process was inhibited by thiol scavengers, such as N-ethylmaleimide, p-(chloromercuri)benzoate, and copper ion, and accelerated in the presence of thiols, such as with the addition of cysteine. Rates were generally accelerated under alkaline conditions. Widespread formation of Covalent Insulin Dimers (CIDs) in insulin formulation, as a result of disulfide exchange, has been reported ΔΧ104

105 5/11/2014 Protein Stability Noncovalent Destabilization Three major categories of irreversible protein inactivation occur as a result of perturbation of the noncovalent forces that maintain the 3-D native state of proteins, (1) aggregation, (2) macroscopic precipitation, and (3) surface adsorption Noncovalent interactions, electrostatic, hydrogen bond, hydrophobic, and protein hydration, may be altered as a result of thermal or pH effects. The irreversible inactivation proceeds following initial reversible unfolding of the native state With an increase in temperature, a protein molecule in solution will undergo a characteristic transition from native to unfolded state at the melting temperature, T m, defined as the temperature at which 50% of the molecules are unfolded The pH of the solution influences the net charge of the protein, depending on its pI. pH may affect electrostatic interactions, also referred to as salt bridges. At extreme pH values, the net charge of the protein increases with greater charge repulsion, leading to protein unfolding Protein conformation is markedly affected by type and concentration of ionic species in solution. Individual salt effects can be either stabilizing or denaturing. These effects correspond to the Hofmeister lyotropic series: SO 4 - > HPO 4 2- > Oac - > F - > citrate > Cl - > NO 3 - > I - > CNS -, ClO 4 - (CH 3 ) 4 N + > NH 4 + > K +, Na + > Mg 2+ > Ca 2+ > Ba 2+ ΔΧ105

106 5/11/2014ΔΧ Protein Stability Noncovalent Destabilization Anions and cations to the left of the series are the most stabilizing and reduce the solubility of the hydrophobic groups, salting out) on the protein molecule, by increasing the ionic strength of solution. Anions and cations to the right of the series are destabilizing and are known to denature proteins, causing an increase in solubility or salting in Mechanical forces, such as shearing, shaking, and pressure, may also denature proteins. Shaking may lead to deactivation owing to the increase of the gas/liquid interface. Surface denaturation may occur following adsorption of proteins to container walls and filter materials Noncovalent Destabilization -Aggregation and Precipitation Aggregation is a microscopic process of protein molecule association. Aggregates may be dimers or larger oligomers that remain in solution and may affect biological activity. Irreversible aggregation may follow unfolding as a result of incorrect refolding of the protein. Protein unfolding exposes its hydrophobic interior to the solvent, usually water. Interactions between exposed hydrophobic regions of the protein interior drive aggregation. Although a two-state equilibrium model describes, as a general rule, protein unfolding, several examples of intermediate conformational states have been uncovered and may play a role in aggregate formation and subsequent precipitation 106

107 5/11/2014ΔΧ Protein Stability Noncovalent Destabilization -Aggregation and Precipitation The conformational intermediaries have considerable secondary structure but lack tertiary structural interactions. Aggregation results from association of exposed hydrophobic regions on the monomeric intermediaries. Protein concentration may influence the rate of formation of intermediaries. Aggregation depends on both, thermal unfolding and pH Precipitation refers to formation of visible particles which, in addition to altering the appearance of a formulation, reduce the potency and performance in infusion devices Shaking a protein solution may lead to aggregation and precipitation as a result of several mechanisms, such as air oxidation, surface denaturation, adsorption to the container walls, or mechanical stress. Proposed mechanism of inactivation by mechanical stress is through orientation of asymmetrical proteins in the shear flow field, which promotes association of aligned molecules Aggregation and precipitation were observed for human interferon-  and human fibroblast interferon. Insulin aggregation and precipitation was an impediment to the development of implantable devices for insulin delivery. Potential causes for insulin aggregation and precipitation are thermal effects, mechanical stress, nature of materials in contact with insulin, and purity of insulin preparation 107

108 5/11/2014 Protein Stability Noncovalent Destabilization – Surface Adsorption Protein adsorption onto the surfaces of container walls, filter, materials of infusion systems, etc., can be critical when the initial protein concentration in solution is low Surface adsorption results from hydrophobic and electrostatic interactions and depends on the conformational state of protein, pH, ionic strength of solution, as well as the nature of the surface. The interaction of the protein molecule with the surface increases with increasing hydrophobicity of both, the surface and the protein molecule. The tendency of protein to undergo conformational change is time- dependent, and protein- and surface-specific Membrane filtration is the only currently acceptable method of sterilizing protein pharmaceuticals and the adsorption of the protein on the membrane is of particular concern. Nitrocellulose and nylon membranes had extremely high protein adsorption, followed by polysulfone, cellulose diacetate, and hydrophilic polyvinylidene fluoride membranes ΔΧ108


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