Conformational properties of constrained proline analogues and their application in nanobiology

UNIVERSITAT POLITÈCNICA DE CATALUNYA DEPARTAMENT D’ENGINYERIA QUÍMICA “CONFORMATIONAL PROPERTIES OF CONSTRAINED PROLINE ANALOGUES AND THEIR APPLICATION IN NANOBIOLOGY” Alejand a Flo es O ega Supe iso s: D . Ca los Alemán Llansó and D . Jo di Casano as Salas. Ba celona, 27 h Janua y 2009 iii “Chance is a wo d oid o sense; no hing can exis wi hou a cause”. F ançois-Ma ie A oue , Vol ai e “Imagina ion will o en ca y us o wo lds ha ne e we e. Bu wi hou i , we go nowhe e”. Ca l Sagan ACKNOWLEDGEMENTS I would like o acknowledge o D . Ca los Aleman and D . Jo di Cassano as Salas o an in e es ing esea ch heme, and scien i ic suppo . I g a e ully acknowledge o D . Da id Zanuy o in e es ing sugges ions and s ong discussions, wi hou hei suppo his would be an un ul illed ask. Also I, would like o add ess my hanks o all my colleagues in my g oup and depa men , specially Elaine A melin o assi ing me in many di e en ways. I hank no only my iends, bu also colleagues o helping me o o e come he s ess ul ime, wi hou whom i would ha e been di icul o cope up. I wish o exp ess my g a e ulness o my pa en s, specially o my mo he , Ma ía Es he , o all his ca e, and suppo . Also I will like o hanks o my iends and specially Jesus, Me ches, Lau a y A u o. My PhD hesis ha e been inished o all his suppo . I am g ea ly indep ed o D . Ru h Nussino a NCI, D . Ca los Ca i iela a he Uni e si y o Za agoza and Ana I. Jiménez a he “Ins i u o de Ciencias de Ma e iales de A agon” o a collabo a i e e o . I wish o hank all my colleague in he “Chimie e Biochimie Théo iques, Facul é des Sciences e Techniques” in Nancy F ance, I will be g a e ul o ha e wo ked wi h : P . Xa ie .Ass eld and PhD Adele Lau en . I g a e ully acknowledge he inancial suppo p o ided by he In amu al Resea ch P og am o he NIH, Na ional Cance Ins i u e, Cen e o Cance Resea ch. i ii i OBJECTIVES (1) Examine he con o ma ional p e e ences o p oline analogs ha ing one o mo e double bonds in he py olidine ing. Analyze he in luence o he insa u a ions on: (i) he s abili y o he cis a angemen o he pep ide bond in ol ing he py olidine ni ogen; and (ii) he con o ma ional lexibili y o he backbone. (2) Analyze he in insic con o ma ional p e e ences o wo ep esen a i e α- e asubs i u ed p oline analogs (α-me hylp oline and α-phenylp oline) and compa e hem wi h hose o con en ional p oline. Unde s and he e ec s o he subs i uen inco po a ed a he α posi ion on he p e e ed backbone con o ma ion, he pucke ing o he py olidine ing and he cis/ ans disposi ion o he amide bonds. (3) Compa e he con o ma ional p ope ies o di e en amina ed and dime hylamina ed de i a i es o p oline. Examine how he o ma ion o side chain···backbone hyd ogen bonds a ec s no only he con o ma ional lexibili y bu also he ans/cis disposi ion o he pep ide bond in ol ing he py olidine ni ogen. (4) De e mine he con o ma ional p e e ences o he aminop oline analogs p o ona ed a he amino side g oup. Analyze he in luence o he pH on he ela i e s abili y o he di e en possible isome s, he backbone lexibili y and he disposi ion o he pep ide bond. (5) Cha ac e ize he con o ma ional p o ile o he CREKA sequence, which de ines a e y e icien umo -homing pen apen ide, and iden i y he co esponding bioac i e con o ma ion. A sa is ac o y achie emen o his objec i e is essen ial o designing o syn he ic analogs able o p o ide p o ec ion om p o eases, which is an impo an s ep be o e he de elopmen o po en ial applica ions o umo -homing pep ides. (6) Imp o e he biological pe o mance and pha macological p o ile o CREKA by enginee ing an analogue ha inco po a es a non-p o einogenic amino acid. This esidue should be concei ed o e ain he mos ele an cha ac e is ics o he con o ma ional p o ile o he na u al pep ide and simul aneously impa s abili y agains p o eoly ic clea age. i ix x GLOSSARY. A Pucke ing Ampli ude αMeP o Me hylp oline Amp Aminop oline Dipep ides α PhP o Phenylp oline Aze L-aze idine-2-ca boxylic acid azP o azap oline B3 Becke´s h ee-pa ame e hyb id unc ional C Se en Membe edin amolecula Hyd ogen Bond 7 ∆Egp Rela i e Ene gy DFT Densi y Func ional Theo y ∆ Ggp Gibbs ee ene gies in he gas phase Dmp Dime hylaminop oline E Ene gy o he Sys em HF Ha ee-Fock Hyp 4R-Hyd oxyp oline ϕ Fi LYP Lee, Yang and Pa MP Molle -Plesse NHMe N-Me hyl Amide G oup Oxa (S)-oxazolidine-4-ca boxylicacid P S a e o Pucke ing Pip (S)-pipe idine-2-ca boxylic acid P o P oline SA Simula ed Annealing - Molecula Dynamics SCF Sel Consis Field SPIO Dex an-Coa ed I on Oxide Thz ((R)- hiazolidine-4-ca boxylicacid UHF Un es ic ed Ha ee-Fock ZPVE Ze o-Poin Vib a ional Ene gies ρ elec onic densi y Ψ Wa e Func ion ψ Psi 1 INTRODUCTION 3 1.2 Su ey o modi ied P oline esidues. Con o ma ional ea u es Among o he s a egies, he chemical modi ica ion o na u al p o eogenic amino acids has p o en o be an e icien app oach o es ic ing he con o ma ional space o such molecula species.9 This ea u e is o g ea in e es o u he nano echnological applica ions, since a majo pa o he wo k de o ed o edesign na u al biomac omolecules implies exe cising con o ma ional con ol o e such sys ems (ei he as a whole o small pa s o hem).10 The mos cha ac e ized chemical subs i u ion in P oline is he p esence o a unc ional g oup a he posi ion 4 o he ing (C The pa icula con o ma ional ea u es o P oline has made o i a majo a ge o po en ial molecula enginee ing modi ica ions, since i s inne cons i u ional es ic ions aids limi ing i s low ene gy accessible con o ma ions, helping biasing he con o ma ional eedom o he pep ide in which his esidue is included. γ a cha II). This modi ica ion has special ele ance in biological sys ems since he subs i u ion o he P ochi al hyd ogen R in such ca bon by a hyd oxyl g oup (4R-hyd oxy-L-p oline, Hyp) is de ec ed in abou he 50% o he P olines p esen in na u al collagen (Cha III).1 The p esence elec on-wi hd awing g oups signi ican ly a ec s he con o ma ional dynamics o he py olidine ing pucke ing ansi ion. Thus, he p esence o ei he a hyd oxyl g oup o a Fluo ide a om (4R- luo o-L-p oline, Flp) a ec he ansi ion ba ie s be ween he up and down all pucke ing.11 Mo eo e , hese changes also ha e a e lec ion o e he main chain con o ma ional p e e ences, since an inc emen in he popula ion on he up-pucke ed con o ma ion aids he s abiliza ion o he εL con o ma ion (also deno ed polyp oline II-like o PPII), which is e y un a o ed in he unsubs i u ed P o. C 4 C 3 C 2 N1 C 5 OH O H α βγ δ X 12 X= -OH, Hyp X= -F, Flp Figu e 1.3: Cha III 1 INTRODUCTION 4 O he ways o al e ing he con o ma ional ea u es o P oline is playing wi h i s chemical cons i u ion. The ing size can be changed, ei he educing i s lexibili y by diminishing he ing size (elimina ing a me hylene g oup, he L- aze idine-2-ca boxylic acid, Aze) o by inducing he opposi e e ec by enla ging he cyclized segmen (inse ing a new me hylene g oup, he (S)-pipe idine-2- ca boxylic acid, Pip).13 In bo h sys ems, when compa ing wi h he na i e p oline, he e is a educ ion o he ene gy di e ence be ween C7 a angemen (also deno ed γL) and εL, becoming he o me he mos a o ed con o ma ion as he sol en pola i y inc eases. I is wo h no ing ha despi e he no able di e ences be ween he new cons i u ions and he P o he e isn’ g ea di e ences espec o bo h he main chain con o ma ional p e e ences and he cis- ans equilib ium in he pep ide bond. Ano he possibili y is changing he chemical na u e o he py olidine ing: i is possible o hink abou eplacing he me hylene uni o he gamma posi ion by ano he unc ional g oup. Fo ins ance Kang and Pa k 13 14 explo ed he possibili y o con e ing he P o ing in o and he e ocyclic species (see Cha IV) by in oducing in such posi ion ei he an oxygen a om ((S)-oxazolidine-4- ca boxylicacid, named Oxa) o a hiol g oup ((R)- hiazolidine-4-ca boxylicacid, named Thz). In hese pseudo p oline esidues he main chain con o ma ion p e e ences a e e y in luenced by he pola i y o he sol en , as also obse ed o he unmodi ied p oline.15 Thus, in bo h Oxa and Thz he popula ion o γL con o ma ion dec eases as he medium pola i y inc eases, a o ing εL a angemen . The pucke ing p e e ences hough in e hei endencies in he case o he new ings espec o he na u al esidue, since he pola i y p omo es he p esence o up a angemen s o he la e bu un a o s hem o he o me esidues. 1 INTRODUCTION 5 X4C3 C2 N 1 C5 OH O H α βγ δ C4C3 N2 N 1 C5 OH O H α βγ δ X= O, Oxa azP o X= S-H, Thz Figu e 1.4: Cha IV Finally, he p ope backbone o he amino acid can also be modi ied: i he Cα is eplaced by a ni ogen a om, he azap oline (azP o) is ob ained (Cha IV).16 In his case he new elec onic s uc u e esul ing om he inse ion o he azo moie y d as ically changes he new esidue con o ma ional p e e ences, highly s abilizing β-shee like a angemen s (δL con o ma ion). Fu he mo e, he e ec o he non-bonded elec on pai s o he new ni ogen a om aids o e s abilizing he cis a angemen o he second pep ide bond, when azP o is inse ed be ween a dipep ide moie y. As he pola i y o he medium inc eases he p e e ences o his esidue end o mee hose o he na u al p oline by a o ing he εL a angemen o e he δL 1.3 Pep ide design: imp o ing na u e o bionano echnological applica ions one. The ela ionship be ween olding and unc ion among p o eins has long been a sou ce o ascina ion o he molecula ly inclined scien is . The in e play be ween R-amino acid esidue sequence and he h ee-dimensional a angemen o hese subuni s ha esul s om adop ion o a speci ic con o ma ion enables p o eins o mani es an ex ao dina y ange o unc ions. Among such sequences, sho pep ides ha e ound emendous a en ion in di e se aspec s o science anging om a ional d ug design17 o nanoma e ials.18 These di e se applica ions a e due 1 INTRODUCTION 6 o hei dis inc i e p ope ies, such as ease o syn hesis and cha ac e iza ion, in oduc ion o chemical di e si y by simple amino acid subs i u ion, and modula ion o 3D s uc u e by chemical modi ica ion. The applica ion o pep ides as d ugs s ems om hei key ole in many signal ansduc ion pa hways, which makes hem an a ac i e a enue o a ge diseases. Despi e he high ac i i y and ecep o selec i i y o na u ally occu ing bioac i e pep ides (o ac i e p o ein agmen s), hey ha e dis inc disad an ages o p ac ical applica ion in medicine, such as sho hal -li e in i o and lack o o al a ailabili y. The ini ial s ep in d ug esea ch o pep ides is usually simpli ica ion (e.g., educ ion in size), ollowed by pep idomime ic app oaches o ensu e me abolic s abili y, wi h he inal goal o an o ally a ailable, highly ac i e, and selec i e d ug. Whe eas he p elimina y s eps can be done in a a ional way wi h ela i ely high p obabili y o success, he inal, c ucial s ep o con e sion om pep ide in o a d ug is o en mo e p oblema ic. On he o he hand, no all o he amino acids in a pep ide sequence a e essen ial o achie e he biological e ec . The ini ial iden i ica ion o he “bioac i e sequence”, he minimal sequence19 equi ed o achie e he biological ac i i y, is o en done by alanine scanning. This is he sys ema ic subs i u ion o each amino acid by alanine o iden i y he key esidues, ha is, hose whose subs i u ion esul s in educed ac i i y. The nex impo an ac o is he con o ma ion o he pep ide. In he majo i y o such pep ides, a majo obs acle in he s udy o he “bioac i e sequence” is in insic lexibili y. Thus, he ac i e sequence mus be igidi ied in a de ined con o ma ion in o de o achie e he desi ed ac i i y and selec i i y. Reduc ion o con o ma ional space can be achie ed by cycliza ion, esul ing in highly ac i e and selec i e de i a i es when he bioac i e con o ma ion is ma ched.20 This sea ch o ma ching is done by “spa ial sc eening”.21 An al e na i e s a egy can be he sys ema ic explo a ion o he hype su ace o po en ial ene gy o he s udied segmen . Hence, he i s s ep would consis o in silico explo ing all he accessible con o ma ions ha he pep ide can adop unde physiological (o unde hose condi ions ha expe imen al in o ma ion has been eco ded).22 Once he accessible con o ma ions ha e been iden i ied, speci ic chemical modi ica ions can be a ge ed o enhance hose con o ma ions ha con ibu e o he collec i e o bioac i e con o ma ions. 1 INTRODUCTION 7 Ano he majo p oblem in de eloping pep idic d ugs is hei enzyma ic deg ada ion in i o, which e en ually esul s in he lowe ing o he pha macokine ic p o ile (hal -li e, bioa ailabili y, e c.). Medicinal chemis s ha e de eloped an a ay o s a egies o e he yea s o con on his p oblem, such as inco po a ing pep ide bond isos e s,23 pep oids,24 e o-in e so pep ides,25 and pep idomime ics.26 Al hough hese s a egies ha e elegan p ope ies o hei own, hey demand ca e ul design wi h challenging syn heses. Despi e his complexi y, a ge ed modi ica ions o he na u al pep ides p esen high po en ial, since hey can allow o bo h enhance he pep ide inne p ope ies (speci ici y and ac i i y) and o p o ide new physicochemical ea u es o he modi ied segmen , such as esis ance o endogen p o eases. 1 INTRODUCTION 8 1.4 Re e ences 1. Richa dson, J. S.; Richa dson, D. C. P inciples and pa e ns o p o ein con o ma ion. In P edic ion o P o ein S uc u e and he P inciples o P o ein Con o ma ion; Fasman, G. D., Ed.; Plenum P ess: New Yo k, 1989, 98. 2. Vi agliano, L.; Be isio, R.; Mas angelo, A.; Mazza ella, L.; Zaga i, A. P o ein Sci. 2001, 10, 2632. 3. S ewa , D. E.; Sa ka , A.; Wample , J. E. J. Mol. Biol. 1990, 214, 253. 4. Jabs, A.; Weiss, M. S.; Hilgen eld, R. J. Mol. Biol. 1999, 286, 291. 5. Pal, D.; Chak aba i, P. J. Mol. Biol. 1999, 294, 271. 6. Wedemeye , W. J.; Welke , E.; Sche aga, H. A. Biochemis y 2002, 41, 14637. 7. Duga e, C.; Demange, L. Chem. ReV. 2003, 103, 2475. 8. Gibbs, A. C.; Bjo ndahl, T. C.; Hodges, R. S.; Wisha , D. S. J. Am. Chem. Soc. 2002, 124, 1203. 9. C isma, M.; Fo maggio, F.; Mo e o, A.; Toniolo, C. Biopolyme s, 2006, 84, 12. 10. Alemán C.; Zanuy, D.; Jiménez A.I.; Ca i iela, C.; Haspel, N.; Zheng, J.; Casano as, J.; Wol som, H.; Nussino , N. Phys. Biol. 2006, 3, S62. 11. Song, I.K.; Kang, Y.K. J.Phys.Chem.B 2005, 109, 16987. 12. Song, I.K.; Kang, Y.K. J.Phys.Chem.B 2006, 110, 1927. 13. Jhon, J.S.; Kang Y.K. J. Phys. Chem. B 2007, 111, 3507. 14. Kang, Y.K.; Pa k H.S. J.Phys. Chem. B 2007, 111, 12562. 15. Imp o a, R.; Benzi, C.; Ba one, V. J. Am. Chem. Soc. 2001, 123, 12577. 16. Kang K.Y.; Byun B.J. J. Phys. Chem. B 2007, 111, 5385. 17. Ma x, V. Chem. Eng. News 2005, 83, 21. 18. Teixido, M.; Gi al , E. J.Pep . Sci. 2008, 14, 173. 19. Gu a h, M. Cu . Med. Chem. 2001, 8, 1648. 20. Kessle , H. Angew. Chem., In . Ed. 1982, 21, 523. 21. Kessle , H.; G a ias, R.; Hessle , G.; Gu a h, M.; Mulle Pu e Appl. Chem. 1996, 68, 1205. 1 INTRODUCTION 9 22. Ag a io is, D. M.; Gibbs, A. C.; Zhu, F.; Iz aile , S.; Ma in, E. J. Chem. In . Model. 2007, 47, 1086. 23. Houben-Weyl Me hods o O ganic Chemis y; Goodman, M.; Felix, A.; Mo ode , L.; Tonolio, C. Eds.; Geo g Thieme Ve lag: 2002, E22c, 633. 24. Kessle , H. Angew. Chem., In . Ed. 1993, 32, 544. 25. Fle che , M. D.; Campbell, M. M. Chem. Re . 1998, 98, 795. 26. Giannis, A. Angew. Chem., In . Ed. 1993, 32, 1267. 11 2 Me hods 2.1 In oduc ion In his chap e he me hods used h oughou his Thesis, which can be o ganized in quan um mechanical me hods and molecula dynamics simula ions, will be discussed. In he sec ion 2.2, he basic elemen s o quan um mechanical me hods a e p esen ed. Speci ically, he mo e essen ial ends o ab ini io and DFT me hods a e desc ibed. In sec ion 2.3 classical me hods based on Molecula Dynamics simula ions a e b ie ly discussed. Finally, sec ion 2.4. p esen s he basic concep s o he con o ma ional sea ch p ocedu es used in his Thesis. 2.2 Quan um Mechanical Me hods These me hods p o ide a eliable desc ip ion o he ene gies, geome ies and elec onic p ope ies o he sys ems unde s udy. In his app oach, nuclei a e a anged in he space while he co esponding elec ons a e sp ead all o e he sys em in con inuous elec onic densi y and compu ed using he Sch ödinge equa ion. When he Sch ödinge equa ion 2.1 is sol ed, quan um mechanical me hods pos ula e he exis ence o a wa e unc ion, Ψ, ha con ains all he in o ma ion o he sys em: Ψ=Ψ ∧ EH (2.1) whe e ∧ H is he Hamil onian ope a o ha includes he kine ic and po en ial ene gy o he nuclei and elec ons, and E is he ene gy o he sys em. Two basic 2 METHODS 12 quan um mechanical me hodologies a e cu en ly used o s udy chemical p oblems: ab ini io and Densi y Func ional Theo y (DFT), which di e in he p ocedu e o ob ain Ψ . 2.2.1 Ab Ini io Me hods Fo a sys em o N nuclei and M elec ons, he ∧ H is exp essed as (in a omic uni s): ∑ ∑ ∑∑∑∑∑∑ = = 〉=〉=== ∧++−∇−∇= N i M A M AB AB BA M A N jij N i M AiA A N i A A iR ZZ Z m H 1 1 11111 22 1 2 1 2 1 (2.2) whe e A m is he ela ion o he nucleus mass wi h espec o he elec on mass, A Z is he a omic numbe o he nucleus A, 22 Ai and∇∇ a e ope a o s ha e e o he di e en ia ion be ween he coo dina es o elec on i and he a om A, espec i ely. In equa ion 2.2 each e m is an ope a o de ining he ene gy componen s o he sys em: he i s e m ep esen s he kine ic ene gy o he elec ons, he second e m is he kine ic ene gy o he nucleus, he hi d one is he elec os a ic a ac ion be ween nucleus and elec ons, he ou h one is he elec os a ic epulsion be ween he elec ons and, inally, he i h one co esponds o he elec os a ic epulsion be ween nuclei. Taking in o accoun he high a io be ween nuclea and elec onic masses, he Bo n-Oppenheime app oach allows disca d he second and he i h e ms, he gene al Hamil onian being ans o med in o an elec onic Hamil onian ( el H ∧ ): ∑ ∑ ∑∑∑ = = = 〉= ∧+−∇= N i N i N i N jij M AiA A iel Z H 1 1 1 11 21 2 1 (2.3) In p inciple, i is possible o desc ibe all chemical sys ems by sol ing he Sch ödinge equa ion. Howe e , in p ac ice, only he simples ones may be s udied exac ly using his le el o heo y, and he in oduc ion o some 2 METHODS 19 The pa ame e s Kb, bo Kθ, θo, Kε, εo, Kε, εo, Kφ , η , δ, C6, C12 Many o ce ields a e a ailable. A p ope choice should be made bea ing in mind he p ope ies ha we e used o pa ame iza ion. I one in ends o calcula e he excess ee ene gy o liquid wa e , i.e. he ee ene gy o u ning one mole o wa e in o an ideal gas, a o ce ield designed o p ope ly desc ibe he hea o apo iza ion will be a good candida e. and q a e he o ce ield pa ame e s. Quan um chemical calcula ions can be used o ob ain some o hese, i.e. he molecula geome ies (angles, bond leng hs), o sional po en ials and a omic cha ges, whe eas o he s a e usually ob ained empi ically. In pa icula , he non-bonded e ms a e o en pa ame ized o p ope ly desc ibe he liquid s a e p ope ies, e.g. densi y, second i ial coe icien , hea o apo iza ion, adial dis ibu ion unc ion and/o ime dependen p ope ies such as he di usion coe icien o o a ional co ela ion ime. 2.3.2. Classical Dynamics The mo ion o a oms in a molecula sys em can be simula ed using he classical equa ion o mo ion p o ided ha he o ce ield Φ(R) is a ailable. The classical equa ion ead ))(()( 1 RFmd d iii − = (2.10) )()( d d ii = (2.11) whe e )( i is he eloci y o a om i a ime , mi i F is i s mass and is he o ce on i, i i R F∂ Φ∂ =)( (2.12) Equa ion (2.10) and (2.11) canno be sol ed analy ically and one he e o e is o ced o use ini e di e ence me hods. A simple ini e Taylo expansion o i ( ) a ime poin = n yields14,15 )(!2)2/()2/()()2( 32 2 2 O d d d d n n i i nini ∆+∆+∆±=∆± . (2.13) Sub ac ion o hese wo exp essions and using Eq. (2.10) gi es 2 METHODS 20 )())(()2/()2( 3 1 O RFm ninini ∆+∆+∆−=∆+ − (2.14) Using he same p ocedu e o Taylo expansions o i.( ) a ime poin = n )()2()()( 3 O niini ∆+∆∆++=∆+ + Δ /2 gi es in combina ion wi h Eq. (2.11) (2.15) In his wo k bond leng hs ha e been cons ained using he me hod p oposed by Ryckae e al., which is known as he SHAKE algo i hm16 2.3.3. Pe iodic bounda y condi ions . The MD simula ion o a liquid is pe o med using a simula ion box ha is ypically illed wi h a ew housands o a oms. Dealing wi h mac omolecula sys ems his means ha we usually simula e chains each con aining se e al hund eds o a oms. To emo e he o he wise signi ican wall e ec s, a p ac ical ick, known as he pe iodic bounda y condi ion, is applied. This consis s on su ound he simula ion box by iden ical copies o o m a bulk sys em. This condi ion ensu es ha a oms mo ing ou o he box a one side a e able o e-en e he box a he opposi e side since he eplicas o his pa icle in he neighbou ing boxes mo e in exac ly he same way. This s a egy elimina es he walls a he bounda y o he cen al box and he su ace molecules. 2.3.4. Tempe a u e and p essu e The algo i hm o molecula dynamics desc ibed abo e gene a es he ime e olu ion o he sys em wi h ixed numbe o pa icles N, olume V and ene gy U, whe e he la e is he sum o he kine ic ene gy and he po en ial ene gy. Expe imen ally one o en ob ains in o ma ion a cons an olume and empe a u e (NVT) o a cons an p essu e and empe a u e (NPT). One he e o e would like o ha e an algo i hm ha simula es he dynamics o he sys em a ixed alues o P and o T. Tempe a u e is con olled by adjus ing he eloci ies o he a oms du ing he simula ion. This is done because he a e age kine ic ene gy o he molecules in he sys em de ines empe a u e. The ex en o which he eloci ies need o be adjus ed is made o depend on he ins an aneous alue o he kine ic ene gy a a 2 METHODS 21 gi en ime oge he wi h he desi ed kine ic ene gy ( empe a u e) o he sys em (a la ge di e ence causes a la ge adjus men ). A ixed p essu e means ha olume mus be able o luc ua e; e.g. a o sional ansi ion in a molecule in solu ion a ixed p essu e causes a small sudden olume luc ua ion, o , dissol ing a molecule in a memb ane a ixed p essu e causes he memb ane o swell. In cons an p essu e simula ions one he e o e needs o adjus he olume o he simula ion box by mul iplying he ca esian coo dina es o he a oms wi h an app op ia e alue (which is e y close o 1) a e each ime s ep in he nume ical in eg a ion scheme. The ex en o which his is done again depends on he ins an aneous and he desi ed alues; a la ge di e ence be ween he desi ed p essu e and he ac ual one equi es a la ge olume adjus men . The p essu e is calcula ed using he i ial exp ession. 2.4 Con o ma ional Sea ch Me hods Con o ma ional analysis consis s on he cha ac e iza ion o he s uc u es ha a molecule is able o adop and how hese in luence i s p ope ies. A key componen o he con o ma ional analysis is he con o ma ional sea ch, he objec o which is o iden i y he p e e ed con o ma ions o a molecule, i.e. hose con o ma ions ha de e mine i s beha io . This usually equi es he cha ac e iza ion o con o ma ions ha a e minima on he po en ial ene gy su ace. Fo a pep ide, due o i s high con o ma ional lexibili y in solu ion, he e is a so la ge numbe o minima on he po en ial ene gy su ace ha is imp ac ical o cha ac e ize all hem. Speci ically, mos o he pep ides exis in physiological condi ions as a mix u e o in e changeable con o ma ions wi h simila ene gies popula ed acco ding o he Bol zmann dis ibu ion. I is impo an o emembe ha he s a is ical weigh s o he di e en con o ma ions in ol e also en opic con ibu ions. Sol a ion e ec s may also be impo an , and a ious schemes a e now a ailable o calcula ing he sol a ion ee ene gy o a con o ma ion, ha may be added as an addi ional e m o he in amolecula ene gy. Unde such ci cums ances, i is o en assumed ha he na i e (i.e. na u ally occu ing) con o ma ion is he one wi h he e y lowes alue o ene gy. This con o ma ion is usually e e ed o as he global minimum. Al hough he global minimum 2 METHODS 22 exhibi s he lowes ene gy alue, i may no be highly popula ed because o he con ibu ion o he ib a ional en opy o he s a is ical weigh o each s uc u e. Mo eo e , he global minimum may no be he ac i e (i.e. he unc ional) s uc u e. In his case, i may be e en necessa y o a molecule o adop mo e han one con o ma ion. Fo example, a subs a e migh bind in one con o ma ion o an enzyme and hen adop a di e en con o ma ion p io o eac ion is p oduced. Indeed, in some cases i is possible ha he ac i e con o ma ion does no co espond o any minimum on he ene gy su ace o he isola ed molecule. Compu a ional me hods o he explo a ion o he con o ma ional space o a pep ide s a ed abou hi y yea s ago17. F om hen di e en s a egies ha e been desc ibed and e iewed 18-20 A con o ma ional sea ch me hod ha has shown o be pa icula ly e ec i e o he explo a ion o he con o ma ional space o pep ides is he i e a i e simula ed annealing , and, al hough many e o s ha e been de o ed, his ield o esea ch s ill emains open. Con o ma ional sea ch me hods can be di ided in o he ollowing ca ego ies: sys ema ic sea ch algo i hms, model- building me hods, andom app oaches, dis ance geome y and MD. Independen ly om he s a egy selec ed, ou key elemen s a e needed o ca y ou he explo a ion o a pep ide con o ma ional space. The i s consis s o employing a pep ide model desc ip ion based on classic mechanics, i.e. a o ce ield ha pe mi s o calcula e he ene gy o a de e mined con o ma ion. The second is o ind a me hod capable o gene a ing di e en con o ma ions, in o de o explo e all he low ene gy egions o he con o ma ional space. The hi d key elemen consis s o minimizing he di e en con o ma ions, whe eas he ou h and las elemen is o ind a con e gence c i e ion o assess i he con o ma ional space has been su icien ly explo ed. 21. The me hod has been used in he p esen hesis wo k in chap e 4. The simula ed annealing me hod was i s desc ibed in 198322. This me hod is based on he simila i y ha exis s be ween loca ing he global minimum o he po en ial ene gy unc ion o a molecule and he slow cooling equi ed o ob ain a pe ec c ys al (Figu e 2.1). In ac , c ys al g owing will p obably be pe ec i he sys em is cooled e y slowly by eaching he he modynamic equilib ium when passing h ough es ained egions o he phase space. Applica ion o his concep o he explo a ion o he con o ma ional space can be ansla ed in e ms o s a ing he simula ion a a su icien ly high empe a u e and subsequen ly 2 METHODS 23 dec easing i g adually un il he sys em is ozen in he global minimum. All he s udies ca ied ou using he simula ed annealing me hod ha e demons a ed ha al hough he cooling scheme is no su icien ly slow o ind he global minimum, i is capable o ind local ene gy minima o he egions explo ed. This means ha simula ed annealing combined wi h a sea ching s a egy, which pe mi s o c oss di e en po en ial ene gy ba ie s and o each he low ene gy egions, is a e y e icien me hod o explo e he con o ma ional space. Unde such ci cums ances, a p o ocol based on he simula ed annealing me hod combined wi h MD (SA-MD) ha e been used in his wo k o he explo a ion o he con o ma ional space o pep ides.. This s a egy is schema ically shown in Figu e 2.1. The me hod, which is pa icula ly e icien in he case o la ge pep ide sequences, consis s o pe o ming independen SA-MD cycles and selec ion o a la ge numbe o s uc u es (500) om each SA-MD cycle o ene gy minimiza ion. This p ocedu e is obus enough o loca e he lowe -ene gy minimum s uc u es o he sys em unde s udy, i.e. s uc u es ha a e quasi- degene a e wi h he global minimum bu si ua ed in di e en alleys o he pep ide landscape. The de elopmen o his sampling echnique was inspi ed no only in he wo k o Filizola e al.21 bu also in o he ecen s udies, which demons a ed ha e y low ene gies a e ob ained by minimizing he ene gy o s uc u es gene a ed a he ini ial and in e media e s a es o con en ional SA- MD23,24 . 2.1 Schema ic diag am o he simula ed annealing p o ocol 2 METHODS 24 2.5 Re e ences 1. Binkley, J.S.; Pople, J.A.; Heh e, W.J. J.Am. Chem. Soc. 1980, 102,939. 2. Ha iha an, P.C.; Pople, J.A.; Theo . Chim. Ac a. 1973, 28,213. 3. McLean, A. D.; Chandle , G. S. J. Chem. Phys. 1980, 72, 5639. 4. F isch, M.J.; Pople, J.A.; Binkle, J.S. J. Chem. Phys. 1984, 18, 3265. 5. Mølle , C.; Pless , M.S.; Phys, Re . 1934, 46, 618. 6. Dewa , M.J.S.; Zoebisch, E.G.; Healy, E.F.;S ewa , J.J.P. J.Am. Chem. Soc. 1985, 107, 3902. 7. S ewa , J.J.P. J. Compu . Chem. 1989a, 10, 209. 8. S ewa , J.J.P. J. Compu . Chem. 1989b, 10, 209. 9. Dewa , M.J.S.; Thiel, W. J.Am. Chem. Soc. 1977, 99, 4899. 10. Hohenbe g, P.; Kohn, W. Phys. Re . B. 1964,136, B864. 11.Lee, C.; Yang, W.; Pa , R. G. Phys. Re . B 1993, 37, 785. 12.Becke, A. D. J. Chem. Phys. 1993, 98, 1372. 13.(a) Tomasi, J.; Mennucci, B.; Cammi, R. Chem. Re . 2005, 105, 2999. (b) Tomasi, J.; Pe sico, M. Chem. Re . 1994, 94, 2027. (c) Mie us, S.; Tomasi, J. Chem. Phys. 1982, 65, 239. (d) Mie us, M.; Sc occo, E.; Tomasi, J. Chem. Phys. 1981, 55, 117. 14. Ve le , L. Phys, Re . 1967, 159, 98 15. Ve le , L. Phys, Re . 1968, 165, 201 16. Ryckae , J.P.; Cicco i, G. ; Be endsen. H.J. C. J. Compu , Phys., 1977, 23,237 17. (a) End es, G.F.; Sche aga, H.A. Biochemis y 1968, 7, 4219. (b) Epand, RF.; Sche aga, H.A. Biochemis y 1968, 6, 1383. (c) Epand, R.F.; Sche aga, H.A. Biochemis y, 1968, 6, 1551 (d) Ingwall, R . ; Sche aga, H.A.; Lo an, N. Biochemis y, 1968, 6, 1968. 18. (a) Howa d, A.E; Kollman, P.A. 1988, 110, 7195. (b) Howa d, A.E; Kollman, P.A. J.Med. Chem. 1988, 31, 1669. 19. (a) Van Guns e en, W.F.; Be endsen, H.J.C. Angew. Chem. In . Ed., 1990, 29, 992. 2 METHODS 25 20. (a) Sche aga, H.A. In e na ional Jou nal o Quan um Chemis y, 1992, 21. (a) Filizola, M.; Pe ez, J.J; Palome , A, e al. J. o Mol. G aph. Model., 1997, 15, 290. (b) Filizola, M; Ca eniFa ina, M; Pe ez, J.J. Jou nal o Pep . Res., 1997, 50 42, 1529. (b) Sche aga, H.A. P o . Sci., 1992, 1, 691. (c) Sche aga, H.A. Abs ac s o pape s o he Ame ican Chemical Socie y, 1992, 203, 231. , 22. Ki kpa ick, S.; Gela C.D.; Vecchi, M.P. Science 1983, 220, 671. 55. (c) Filizola, M; Cen eno, N.B; Pe ez, J.J. J. o Pep . Sci., 1997, 3, 85. 23. Baysal, C.; Mei o i ch, H. J. Compu . Chem. 1999, 20, 1659. 24. Simme ling, C.; Elbe , R. J. Am. Chem. Soc. 1994, 116, 2534. 3. In insic Con o ma ional P ope ies o syn he ic P oline Analogues 2 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 35 akes place in he se en-membe ed hyd ogen bonded ing. The pa ame e s o his in e ac ion a e [d(H···O)= 1.971 Å, ∠N-H···O= 146.4º] and [d(H···O)= 1.984 Å, ∠N-H···O= 143.6º], espec i ely. Thus, he γL con o ma ion is equi alen o he ypical γ- u n a angemen . Finally, he hi d minimum was ound o be he - αL[u] (Figu e 3.1.3c). This con o ma ion, which does no in ol e any in amolecula hyd ogen bond, is un a o ed wi h espec o he -γL[d] by 4.0 kcal/mol. The esul s p esen ed in Table 3.1.1 a e in excellen ag eemen wi h hose epo ed by Csizmadia and co-wo ke s32 and Kang34b o he same compound. Thus, hese au ho s ound he same h ee minima using he HF/6- 31G(d), HF/6-31+G(d), B3LYP/6-31G(d) and B3LYP/6-311++G(d,p) me hods, he ela i e ee ene gies (∆G) o he -γL[u] and -αL The i e endocyclic bond angles associa ed wi h he py olidine ing and selec ed bond dis ances o he h ee minimum ene gy con o ma ions o Ac-L-P o- NHMe a e lis ed in Tables 2 and 3, espec i ely. These pa ame e s, which will be compa ed wi h hose ob ained o he analogs s udied in his wo k (see below), do no show any signi ican a ia ion wi h he con o ma ion. [u] a he la e le el o heo y being 1.2 and 4.0 kcal/mol, espec i ely. (a) (b) (c) Figu e 3.1.3: Minimum ene gy con o ma ions o Ac- L -P o-NHMe a he B3LYP/6-31+G(d,p) le el: (a) - γ L[d]; (b) - γ L[u]; and (c) - α L [u]. 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 36 Table 3.1.1 Backbone dihed al angles (in deg ees), pseudo o a ional pa ame e s (A and P, in deg ees), ela i e ene gy ( ∆ E; in kcal/mol) and ela i e ee ene gy ( ∆ G; in kcal/mol) o he minimum ene gy con o ma ions o Ac-L-P o- NHMe wi h he wo pep ide bonds in ans calcula ed a he B3LYP/6-31+G(d,p) le el. # Con . ω ϕ 0 ψ ω (A, P) ∆E ∆G -γL -172.6 [d] -83.4 70.3 -177.7 (37.4, -111.9) 0.0 a 0.0 b c -γL -173.9 [u] -81.6 77.3 -175.9 (37.5, 75.8) 1.0 d 1.3 -αL -171.0 [u] -77.5 -11.5 175.9 (37.8, 89.2) 4.9 e 4.0 a χ0= -13.9º, χ1= 31.4º, χ2= -37.6º, χ3= 28.7º and χ4= -9.3º. b E= -573.315217 a.u. c G= -573.132049 a.u. d χ0= -10.3º, χ1= -13.4º, χ2= 31.0º, χ3= -36.6º and χ4= 29.8º. e χ0= 0.5º, χ1= -22.9º, χ2= 36.1º, χ3= -35.4º and χ4 3.1.3.2 Ac-∆ = 22.0º. α,β Con o ma ional pa ame e s o he wo minimum ene gy con o ma ions ound o Ac-∆ P o-NHMe α,βP o-NHMe wi h he pep ide bond in ans a e lis ed in Table 3.1.4. As can be seen, hese wo minima, which a e sepa a ed by 2.7 kcal/mol, a e signi ican ly di e en om hose p e iously desc ibed o Ac-L-P o-NHMe. The global minimum co esponds o he -γL[u] (Figu e 3.1.4a), which is s abilized by a se en membe ed in amolecula hyd ogen bonded ing wi h pa ame e s [d(H···O)= 1.722 Å, ∠N-H···O= 156.2º]. The dihed al angles ϕ,ψ o his minimum a e signi ican ly close o ze o han hose ound o he -γL[d] and - γL[u] con o ma ions o Ac-L-P o-NHMe. On he o he hand, in e es ingly he se en a oms in ol ed in he in amolecula hyd ogen bonded ing o he -γL[u] con o ma ion o Ac-∆α,βP o-NHMe a e almos in he same plane, i.e. he hyd ogen bonded ing is plana . These s iking con o ma ional ea u es ha e no been de ec ed in o he dehyd oamino acids.54,55 Fo ins ance, he dihed al angles ϕ,ψ o he γL minimum de ec ed o he N-ace yl-N’-me hyl-dehyd oalanineamide a e –66º,27º, he geome y o he se en-membe ed hyd ogen bonded ing being simila o ha de ec ed o Ac-L-P o-NHMe.54,55 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 37 Table 3.1.4 Backbone dihed al angles (in deg ees), pseudo o a ional pa ame e s (A and P, in deg ees), ela i e ene gy ( ∆ E; in kcal/mol) and ela i e ee ene gy ( ∆ G; in kcal/mol) o he minimum ene gy con o ma ions o he N- ace yl-N’-me hylamide de i a i es o he p oline analogs ha ing double bonds in he ing calcula ed a he B3LYP/6-31+G(d,p). In all cases he wo pep ide bonds a e in ans. # Con . ω ϕ 0 ψ ω (A, P) ∆E ∆G Ac-∆ α,β P o-NHMe -γL 180.0 [u] -22.1 7.4 179.3 (21.3, 123.7) 0.0 a 0.0 b -ε c L 179.1 [u] -34.5 126.7 -176.4 (19.3, 125.6) 3.7 d 2.7 Ac-L-∆ β,γ P o-NHMe -γ -172.4 L -80.2 67.0 -179.4 (6.7, 164.3) 0.0 e 0.0 -α g -169.6 L -81.9 -6.6 175.6 (2.9, -180.0) 1.9 h 1.6 Ac-L-∆ γ,δ P o-NHMe -γL -172.0 [d] -81.3 67.2 -178.0 (10.0,-162.2) 0.0 i 0.0 j Ac-Py-NHMe k -γ 180.0 0.0 0.1 179.9 - 0.0 l 0.0 m -ε n 166.5 L -24.3 137.8 179.3 (0.4, 80.6) 3.1 o 2.1 a χ0= -11.8º, χ1= -1.6º, χ2= 13.6º, χ3= -19.6º and χ4= 19.4º. b E= -572.078301 a.u. c G= -571.918781 a.u. d χ0= -11.1º, χ1= -0.9º, χ2= 11.8º, χ3= -17.3º and χ4= 17.8º. e χ0= -6.4º, χ1= 4.1º, χ2= -0.3º, χ3= -3.6º and χ4= 6.3º. E= -572.078932 a.u.. g G= -571.920148 a.u. h χ0= -2.9º, χ1= 2.4º, χ2= -1.1º, χ3= -0.8º and χ4= 2.4º. i χ0= - 9.5º, χ1= 9.1º, χ2= -6.1º, χ3= 0.2º and χ4= 6.1º. j E= -572.084141 a.u.. k G= - 571.924761 a.u. l χ0= 0.0º, χ1= 0.0º, χ2= 0.0º, χ3= 0.0º and χ4= 0.0º. In his case no pseudo o a ional pa ame e has been p o ided because he ing is ideally plana . m E= -570.873298 a.u.. n G= -570.736367 a.u. o χ0= 0.1º, χ1= -0.3º, χ2= 0.4º, χ3= -0.4º and χ4= 0.2º. 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 38 The mos ele an cha ac e is ic o he second minimum, -εL[u] (Figu e 3.1.4b), is ha he wo amide g oups a e a anged pe pendicula ly wi h espec o each o he . On he o he hand, he ex ac ion o wo hyd ogen a oms al e s no only he con o ma ional p e e ences o he backbone bu also he pucke ing o he cyclic side chain. Thus, he pseudo o a ional pa hway ob ained o he minima o Ac-∆α,βP o-NHMe is di e en om hose calcula ed o he minima o Ac-L-P o- NHMe. Fu he mo e, he pucke ing ampli ude is signi ican ly smalle o he minima o he o me dipep ide han o hose o he la e one. On he o he hand, i should be no ed ha due o he achi al na u e o Ac-∆α,βP o-NHMe he -γD[d] and -εD Inspec ion o he geome ic pa ame e s lis ed in Table 3.1.2 indica es ha , as expec ed, he bond angles ∠N-C [d] a e degene a ed minima o hose men ioned abo e. α-Cβ and ∠Cα-Cβ-Cγ a e abou 5º-8º la ge o Ac-∆α,βP o-NHMe han o Ac-L-P o-NHMe due o he double bond be ween Cα and Cβ. Fu he mo e, ∠Cδ-N-Cα is a ew deg ees smalle in he o me dipep ide han in he la e one. This sugges s a change in he conjuga ion pa e n o he modi ied esidue. Thus, inspec ion o he bond leng hs displayed in Table 3.1.3 o Ac-∆α,βP o-NHMe indica es ha he pep ide bond ex ends he conjuga ion o he double bond o he cycle. d(N-Cα) and d(Cα-CXX) a e signi ican ly smalle han in Ac-L-P o-NMe, while he alue o d(Cα-Cβ) is sligh ly la ge han he alue ypically expec ed o a Csp2=Csp2 bond, i.e. 1.332 Å o C2H4 (a) (b) a he B3LYP/6-31+G(d,p) le el. Figu e 3.1.4: Minimum ene gy con o ma ions o Ac- ∆α,β P o-NHMe a he B3LYP/6-31+G(d,p) le el: (a) - γ L[u]; and (b) - ε L [u]. 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 39 Table 3.1.2 Selec ed angles (in deg ees) o he minimum ene gy con o ma ions o he N-ace yl-N’-me hyl de i a i es o p oline and i s analogs ha ing double bonds in he ing cha ac e ized a he B3LYP/6-31+G(d,p) le el. ∠N-C α -C ∠C β α -C β -C ∠C γ β -C γ -C ∠C δ γ -C δ ∠C-N δ -N-C α Ac- L -P o-NHMe -γL 103.1 [d] 103.6 103.6 103.7 112.1 -γL 104.4 [u] 105.4 103.1 102.8 110.7 -αL 104.0 [u] 104.2 102.9 103.3 111.5 Ac-∆α,βP o-NHMe -γL 109.7 [u] 112.1 102.1 104.5 107.2 -εL 111.3 [u] 110.4 102.5 103.8 108.5 Ac-L-∆ β,γ P o-NHMe -γ 102.1 L 111.8 111.7 102.4 111.8 -α 102.3 L 111.7 111.6 102.5 111.6 Ac-L-∆ γ,δ P o-NHMe -γL 104.0 [d] 103.7 110.7 111.8 108.8 Ac-Py-NHMe -γ 106.5 109.5 107.2 109.3 109.3 -ε 107.6 L 108.5 107.3 108.5 108.5 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 40 Table 3.1.3 Selec ed dis ancesa (in Å) o he minimum ene gy con o ma ions o he N-ace yl-N’-me hyl de i a i es o p oline and i s analogs ha ing double bonds in he ing cha ac e ized a he B3LYP/6-31+G(d,p) le el. C Ac C =O Ac N-C -N C α α -C C XX α -C C β β -C C γ γ -C C δ δ -N Ac- L -P o-NHMe -γL 1.241 [d] 1.360 1.483 1.553 1.533 1.538 1.536 1.4770 -γL 1.241 [u] 1.361 1.485 1.554 1.543 1.540 1.533 1.471 -αL 1.241 [u] 1.375 1.480 1.534 1.550 1.535 1.534 1.476 Ac-∆ α,β P o-NHMe -γL 1.239 [u] 1.367 1.449 1.520 1.343 1.500 1.539 1.490 -εL 1.228 [u] 1.378 1.415 1.507 1.342 1.514 1.551 1.481 Ac-L-∆ β,γ P o-NHMe -γ 1.240 L 1.360 1.484 1.557 1.506 1.333 1.504 1.476 -α 1.230 L 1.375 1.476 1.545 1.510 1.334 1.505 1.478 Ac-L-∆ γ,δ P o-NHMe -γL 1.239 [d] 1.366 1.493 1.558 1.549 1.509 1.336 1.417 Ac-Py-NHMe -γ 1.223 1.403 1.433 1.508 1.374 1.425 1.363 1.404 -ε 1.212 L 1.422 1.397 1.497 1.376 1.431 1.370 1.394 a CAc and CXX deno e he ca bon a oms o he ca bonyl g oups in he ace yl and he P o esidue (o i s analogue), espec i ely. 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 41 3.1.3.3 Ac-L-∆β,γ Two minimum ene gy con o ma ions ha e been cha ac e ized o Ac-L- ∆ P o-NHMe β,γP o-NHMe when he wo pep ide bonds a e in ans (Table 3.1.4). The lowes ene gy minimum co esponds o a γL backbone con o ma ion (Figu e 3.1.5a) wi h backbone dihed al angles simila o hose o he global minimum o Ac-L-P o- NHMe. The hyd ogen bonding pa ame e s associa ed wi h he s abilizing in amolecula in e ac ion ound in he -γL con o ma ion a e [d(H···O)= 1.939 Å, ∠N-H···O= 147.0º]. The second minimum was ound o be -αL (Figu e 3.1.5b), which was p e iously de ec ed in Ac-L-P o-NHMe bu no in Ac-∆α,β (a) (b) P o-NHMe. This s uc u e is des abilized by 1.6 kcal/mol wi h espec o he global minimum. On he o he hand, a de ailed inspec ion o Figu e 3.1.5 indica es ha he cyclic side chain adop s an almos plana a angemen in he wo minima. This is con i med by he low pucke ing ampli ude pa ame e s calcula ed om he endocyclic dihed al angles (see Table 3.1.4). Figu e 3.1.5: Minimum ene gy con o ma ions o Ac- L- ∆β,γ P o-NHMe a he B3LYP/6-31+G(d,p) le el: (a) - γ L; and (b) - α L . On he o he hand, Table 3.1.2 indica es ha only he angles cen e ed a he Cβ and Cγ a oms di e om hose ob ained o Ac-L-P o-NHMe, wi h no esonance be ween he backbone amide g oup and he side chain double bond being de ec ed. The la e ea u e is ully consis en wi h he bond leng hs lis ed in Table 3.1.3. Speci ically, d(N-Cα) and d(Cα-CXX) a e e y simila o hose ound o Ac-L-P o-NMe, while he d(Cβ-Cγ) is la ge han he d(Cα-Cβ) calcula ed o Ac-∆α,βP o-NHMe. Tables 5 analyzes he ela i e s abili ies be ween Ac-∆α,βP o- NHMe and Ac-L-∆β,γP o-NHMe isome s in e ms o ene gies and ee ene gies. The -γL con o ma ion o he la e isome is 0.9 kcal/mol mo e s able han he - γL[u] o he o me one. 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 42 3.1.3.4 Ac-L-∆γ,δ The i s no iceable esul o his isome is ha only one minimum ene gy con o ma ion was ound when he wo pep ide bonds a e a anged in ans. This co esponds o a γ P o-NHMe L (Figu e 3.1.6) con o ma ion, which is cha ac e ized by a se en-membe ed hyd ogen bonded ing wi h he pa ame e s [d(H···O)= 1.938 Å, ∠N-H···O= 147.3º]. Thus, he posi ion o he double bond es ic s he con o ma ional lexibili y o he Ac-L-∆γ,δP o-NHMe wi h espec o he o he isome s. The s uc u al pa ame e s lis ed in Table 3.1.4 indica e ha he ing p esen s an incipien [d] pucke ing, which is mani es ed by he low alue o A. Figu e 3.1.6: Minimum ene gy con o ma ion ( - γ L[d]) o Ac-L- ∆γ,δ P o- NHMe a he B3LYP/6-31+G(d,p) le el. Inspec ion o he bond angles and dis ances lis ed in Tables 2 and 3 sugges s ha he pa ial sp2 cha ac e o he amide ni ogen is sligh ly smalle in Ac-L- ∆γ,δP o-NHMe han in he dehyd op oline isome s p esen ed abo e. This is pa icula ly e idenced by he d(N-Cα) bond leng h, which is e en la ge han hose ound o he minimum ene gy con o ma ions o Ac-L-P o-NHMe. Inspec ion o Table 3.1.5 e eals ha Ac-L-∆γ,δP o-NHMe is he mos s able N- ace yl-N’-me hylamide de i a i e o P o analogs ha ing one double bond in he ing. Thus, he global minimum o Ac-L-∆β,γP o-NHMe and Ac-∆α,βP o-NHMe is des abilized by 2.9 and 3.8 kcal/mol, espec i ely, wi h espec o he -γL[d] con o ma ion o Ac-L-∆γ,δP o-NHMe. 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 43 Table 3.1.5 Rela i e s abili y a he B3LYP/6-31+G(d,p) among he h ee isome s calcula ed in his wo k. Rela i e ene gy ( ∆ E; in kcal/mol) and ela i e ee ene gy ( ∆ G; in kcal/mol) o he minimum ene gy con o ma ions ob ained o he N-ace yl-N’-me hylamide de i a i es o p oline analogs ha ing one double bond in he ing. Compound ∆E ∆G Ac-∆ α,β -γP o-NHMe L 3.7 [u] 3.8 -εL 7.4 [u] 6.4 Ac-L-∆ β,γ -γP o-NHMe 3.3 L 2.9 -α 5.2 L 4.5 Ac-L-∆ γ,δ -γP o-NHMe L 0.0 [d] 0.0 3.1.3.5 Ac-Py-NHMe As can be seen in Table 3.1.4, he con o ma ional p e e ences o Ac-Py- NHMe a e ela i ely simila o hose ound o Ac-∆α,βP o-NHMe. Thus, wo minimum ene gy con o ma ions we e cha ac e ized when he wo pep ide bonds a e a anged in ans. The ϕ,ψ dihed al angles o he global minimum a e ze o, which ep esen s a small bu non-negligible educ ion o he dihed als ound o he global minimum o Ac-∆α,βP o-NHMe. As e idenced in Figu e 3.1.7a, his minimum p esen s a pe ec ly plana se en-membe ed hyd ogen bonded ing. The co esponding geome ic pa ame e s, [d(H···O)= 1.767 Å, ∠N-H···O= 153.5º], sugges s ha he in amolecula hyd ogen bond is e y s ong in his case. Fu he mo e, he global minimum o Ac-Py-NHMe, which is no ound in p o eogenic amino acids, is be ween he γL and γD a angemen s. Acco dingly, his con o ma ion has been deno ed he ea e -γ, i.e. he L o D cha ac e ypically a ibu ed o he γ con o ma ion has been omi ed. The second minimum is he - εL (Figu e 3.1.7b), which is dis a o ed by 2.1 kcal/mol wi h espec o he -γ. The mos impo an di e ence be ween Ac-Py-NHMe and Ac-∆α,βP o-NHMe was ound o be he a angemen o he cyclic side chain. Thus, as expec ed, he py ole ing adop s a plana con o ma ion in he wo minima. On he o he hand, i 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 44 should be no ed ha -εD (a) (b) is he only degene a ed minimum expec ed o Ac-Py- NHMe because o he pa icula dihed al angles ound o he -γ global minimum. Figu e 3.1.7: Minimum ene gy con o ma ion o Ac- Py-NHMe a he B3LYP/6-31+G(d,p) le el: (a) - γ ; and (b) - ε L . On he o he hand, inspec ion o Table 3.1.2 indica es ha he bond angles a e essen ially hose expec ed o a plana py ole ing, no signi ican con o ma ional dependence being de ec ed. Indeed, hey do no di e oo much om hose calcula ed o Ac-L-P o-NHMe wi h he excep ion o ∠Cγ-Cδ-N, which is abou 6º la ge in Ac-Py-NHMe. Howe e , bond leng hs illus a e an opposi e beha io in Table 3.1.3. Thus, he bond leng h be ween he ca bonyl ca bon a om o he Ac g oup and he ni ogen o he py ole ing, d(CAc-N) is e y la ge, whe eas he d(N-Cα) and d(Cα-CXX) a e e y sho . These ea u es combined wi h he alues o he endocyclic bonds leng hs sugges ha conjuga ion e ec s ex end om he py ole ing o he dipep ide backbone. Fu he mo e, he di e ences ound be ween he bond leng hs o he -γ and -εL 3.1.3.6 Rela i e S abili y o he Cis Con o me s minima indica e ha his elec onic p ocess depends on he con o ma ion. The dihed al angle ω 0 o he h ee minimum ene gy con o ma ions cha ac e ized o Ac-L-P o-NHMe was changed om he alues displayed in Table 3.1.1 o 0º. The esul ing con o ma ions we e used as s a ing poin s o ull geome y op imiza ions a he B3LYP/6-31+G(d,p) le el. The con o ma ional pa ame e s o he new minima a e lis ed in Table 3.1.6. Resul s indica e ha he s a ing con o ma ions c-γL[d] and c-γL[u] e ol e owa ds wo comple ely di e en con o ma ions. These a e he c-αL[d] and c-εL[u], which a e dis a o ed wi h espec o he global minimum o Ac-L-P o-NHMe ( -γL[d] in Table 3.1.1) by 2.3 and 5.0 kcal/mol, espec i ely. On he o he hand, he s a ing c-αL[u] con o ma ion was e ained as ene gy minimum a e comple e geome y 3.1 CONFORMATION OF PROLINE ANALOGS HAVING DOUBLE BONDS IN THE RING 51 Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian, Inc., Pi sbu gh PA, 2003. 49. Becke, A. D. J. Chem. Phys. 1993, 98, 1372. 50. Lee, C.; Yang, W.; Pa , R. G. Phys. Re . B 1993, 37, 785. 51. McLean, A. D.; Chandle , G. S. J. Chem. Phys. 1980, 72, 5639. 52. Baldoni, H. A.; Rod iguez, A. M.; Zama bide, G.; En iz, R. D.; Fa kas, O¨ .; Csasza , P.; To day, L. L.; Sosa, C. P.; Jakli, I.; Pe czel, A.; Hollosi, M.; Csizmadia, I. G. J. Mol. S uc . (THEOCHEM) 1999, 465, 79. 53. Pe czel, A.; Angyan, J. G.; Kaj a , M.; Vi iani, W.; Ri ail, J.-L-; Ma coccia, J.-F.; Csizmadia, I. G. J. Am. Chem. Soc. 1991, 113, 6256. 54. Alemán, C.; Casano as, J. Biopolyme s 1995, 36, 71. 55. Tho mann, M.; Ho mann, H.-J. J. Mol. S uc . (THEOCHEM) 1998, 431, 79. 53 3.2 Con o ma ional P e e ences o α-Subs i u ed P oline Analogues DFT calcula ions a he B3LYP/6-31+G(d,p) le el ha e been used o in es iga e how he eplacemen o he α hyd ogen by a mo e s e ically demanding g oup a ec s he con o ma ional p e e ences o p oline. Speci ically, he N-ace yl-N’-me hylamide de i a i es o L-p oline, L- α - me hylp oline and L- α -phenylp oline ha e been calcula ed, wi h bo h he cis/ ans isome ism o he pep ide bonds and he pucke ing o he py olidine ing being conside ed. The e ec s o sol a ion ha e been e alua ed using a Sel Consis en Reac ion Field model. As expec ed, e asubs i u ion a he α ca bon des abilizes he con o me s wi h one o mo e pep ide bonds a anged in cis. The lowes ene gy minimum has been ound o be iden ical o he h ee compounds in es iga ed, bu impo an di e ences a e obse ed ega ding o he ene ge ically accessible backbone con o ma ions. The esul s ob ained p o ide e idence ha he dis inc s e ic equi emen s o he subs i uen a C α may play a signi ican ole in modula ing he con o ma ional p e e ences o p oline.* 3.2.1 In oduc ion The inco po a ion o con o ma ionally cons ained amino acids in o a pep ide chain is a powe ul ool o educe i s in insic lexibili y. Among he esidues whose s uc u al igidi y can be exploi ed in he design o pep ides wi h well- de ined backbone con o ma ions a e α- e asubs i u ed α-amino acids.1 The simples α- e asubs i u ed analogue o a p o einogenic amino acid ha can be conside ed is ha esul ing om he eplacemen o he α hyd ogen by a me hyl g oup. In he las wo decades, ex ensi e e o s ha e been di ec ed a he de elopmen o e icien me hodologies o he syn hesis o he α-me hyl de i a i es o all gene ically coded amino acids 2 (glycine excluded, since i leads o alanine). The simples one is α-me hylalanine (α-aminoisobu y ic acid, Aib), whose con o ma ional p ope ies ha e been deeply in es iga ed and a e well es ablished.1,3,4 * The wo k desc ibed in his chap e p e iously appea ed in J. O g. Chem, 2008 , 73, 3418-3427 In compa ison, he α-me hyla ed analogues o all o he 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 54 p o einogenic amino acids ha e been much less s udied, mainly due o syn he ic di icul ies: α-me hyla ion o Ala gi es ise o a symme ic achi al esidue, whe eas wo enan iome ic o ms a e possible o all o he esidues. Al hough no as ex ensi ely as o Aib, he s udy o he con o ma ional p ope ies o he α- me hyl de i a i es o o he p o einogenic amino acids (mainly aline, leucine and phenylalanine) has been add essed.1c,e,5 The unique p ope ies o p oline make he s udy o i s α-me hyla ed de i a i e (in gene al, α-subs i u ed analogues) pa icula ly in iguing. The singula i y o p oline lies in i s cyclic s uc u e, which includes he amino unc ion. As a consequence, o a ion abou he N—C In gene al, hese α-me hyla ed esidues beha e as he p o o ype Aib, al hough hey p esen pa icula con o ma ional ea u es de i ed om hei chi al na u e. α bond is p ohibi ed and he ϕ o sion angle is con ined o alues a ound –60º. Acco dingly, p oline is o e whelmingly ound in he α-helical [( ϕ , ψ ) ≈ ( –60º,–30º)] and semi-ex ended [( ϕ , ψ ) ≈ ( –60º,140º)] egions o he con o ma ional map.6 In addi ion, p oline shows a highe p opensi y o p omo e γ- u n con o ma ions [( ϕ , ψ ) ≈ ( –70º,60º)] han o he p o einogenic amino acids.6d,7 Ano he e ec de i ed om i s cyclic s uc u e is ha he pep ide bond p eceding p oline ( ha in ol ing he py olidine ni ogen) has a ela i ely high p obabili y o accommoda ing a cis a angemen 8 as compa ed o o he pep ide bonds, o which he cis o m is almos inexis en . Recen s udies in p oline dipep ides e idenced ha he cis/ ans isome iza ion is a en halpy d i en p ocess ha depends on he pola i y o he en i onmen .9 Due o i s pa icula s uc u al p ope ies, p oline plays a key ole in he s uc u e and biology o pep ides and p o eins, and, hence, α-subs i u ed de i a i es a e o g ea in e es . The con o ma ional p e e ences o he α- me hyla ed analogue (αMeP o) emain li le explo ed. Thus, al hough he elec onic e ec s ha s abilize he cis o m become enhanced in pola en i onmen s, he cis/ ans o a ional ba ie s inc eases wi h he pola i y o he en i omen . 10,11 S udies on he N-ace yl- N’-me hylamide de i a i e indica ed a p e e ence o he γ- u n con o ma ion in solu ion,10c,d whe eas an α-helical s uc u e was ound in he solid s a e.10b Spec oscopic and compu a ional s udies on o he pep ides con aining αMeP o sugges ed a s abiliza ion o he βI- u n in compa ison wi h p oline.11 In con as o 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 55 he sca ce s uc u al s udies, he la ge numbe o pape s11,12 and pa en s13 In his wo k, we ha e in es iga ed he in insic con o ma ional p e e ences o α-me hylp oline (αMeP o) and α-phenylp oline (αPhP o) using Densi y Func ional Theo y (DFT) me hods. Calcula ions we e pe o med on he N-ace yl- N’-me hylamide de i a i es o he L-amino acids, he ea e deno ed as Ac-L- αMeP o-NHMe and Ac-L-αPhP o-NHMe(Figu e 3.2.1), espec i ely. The in luence o he me hyl and phenyl g oups has been de e mined by compa ison wi h he p oline de i a i e Ac-L-P o-NHMe, which has been in es iga ed o compa a i e pu poses using he same quan um mechanical me hod. Speci ically, we ha e examined how he subs i uen inco po a ed a he α posi ion a ec s he p e e ed backbone con o ma ion, he pucke ing o he py olidine ing and he cis/ ans disposi ion o he amide bonds. On he o he hand, as was men ioned abo e, he ole o he en i onmen , in pa icula o he sol en , in he cis/ ans o a ional isome ism o p oline was epo ed o be c ucial. dealing wi h he inco po a ion o αMeP o in o bioac i e pep ides and o he biologically ele an sys ems p o ide e idence o he eno mous po en ial o his amino acid. Howe e , he exploi a ion o αMeP o and o he α- e asubs i u ed p oline analogues in he design o pep ides wi h con olled old in he backbone elies on he p e ious knowledge o hei con o ma ional p opensi ies. 9 NMe MeCO CONHMe Ac-L-α-MeP o-NHMe NPh MeCO CONHMe Ac-L-α-PhP o-NHMe In spi e o his, no in o ma ion abou he sol en e ec s on he isome iza ion o he α-subs i u ed p oline analogs has been p o ided ye . Acco dingly, we decided o e alua e he in luence o he sol en pola i y on he con o ma ional p e e ences o he compounds unde s udy using a Sel Consis en Reac ion Field me hod. Figu e 3.2.1: α -subs i u ed P oline Analogues s udied in his wo k 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 56 3.2.2 Me hods 3.2.2.1 Compu a ional De ails. DFT calcula ions we e ca ied ou using he Gaussian 03 compu e p og am,14 combining he Becke´s h ee-pa ame e hyb id unc ional (B3)15 wi h he Lee, Yang and Pa (LYP)16 exp ession o he nonlocal co ela ion (B3LYP). This me hod p o ides a e y sa is ac o y desc ip ion o he con o ma ional p ope ies o cyclic cons ained amino acids, including P o and pseudop olines.17,18 Acco dingly, all he calcula ions p esen ed in his wo k we e pe o med using he B3LYP me hod combined wi h he 6-31+G(d,p) basis se ,19 e en al hough some addi ional single poin calcula ions on selec ed con o ma ions we e pe o med using he aug-cc-pVTZ20 The backbone ( ω basis se . 0, ϕ , ψ , ω ) and side chain (χi; endocyclic) dihed al angles o he N-ace yl-N’-me hylamide de i a i es o P o, αMeP o and αPhP o a e de ined in Figu e 3.2.2. Since ϕ is ixed by he geome y o he i e-membe ed ing, only h ee minima may be an icipa ed o he po en ial ene gy su aces E=E( ψ ) o he dipep ides o a gi en a angemen o he pep ide bonds. The lexible angle ψ is expec ed o ha e h ee minima, i.e. gauche+ (60º), ans (180º) and gauche– (– 60º), while each amide bond ( ω 0, ω ) can be a anged in cis o ans. I should be no ed ha only he pep ide bond p eceding p oline ( ha in ol ing he py olidine ni ogen, co esponding o he ω 0 o sion angle) is likely o adop a cis con igu a ion. Howe e , we conside ed also he cis and ans s a es o he amide bond o med by he p oline ca bonyl ( he me hylca boxamide g oup, –CONHMe, gi en by ω ) wi h he aim o explo ing how α-me hyla ion a ec s he isome ism o his amide linkage. Fo he αPhP o de i a i e, only he cis/ ans a angemen o ω 0 was conside ed. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 57 R = H R = Me R = Ph χ 1 χ 2 χ 3 χ 4 N C δ C γ C β C α R H 3 CC O ω 0 CN O CH 3 H ψ ω χ 0 φ L -P o L -α-MeP o L -α-PhP o Figu e 3.2.2: Dihed al angles used o iden i y he con o ma ions o he N- ace yl-N’-me hylamide de i a i es o p oline and i s α -subs i u ed analogues s udied in his wo k. The dihed al angles ω 0, ϕ , ψ and ω a e de ined using backbone a oms while he endocyclic dihed al angles χ i a e gi en by he a oms o he i e-membe ed ing. In pa icula , he sequence o a oms used o de ine ϕ and χ 0 a e C(=O)–N–C α –C(=O) and C δ –N–C α –C β , espec i ely. compounds s udied in his wo k. The cyclic side chains o he compounds unde s udy may adop wo main di e en con o ma ional s a es, co esponding o he down and up pucke ing o he i e-membe ed ing. They a e de ined as hose in which he Cγ a om and he ca bonyl g oup o he P o esidue (o analogue) lie on he same and opposi e sides, espec i ely, o he plane de ined by he Cδ, N and Cα Acco dingly, o Ac-L-P o-NHMe and Ac-L-αMeP o-NHMe, 3( ψ backbone) × 2( ω a oms. 0 cis-o - ans) × 2( ω cis-o - ans) × 2(cyclic side chain) = 24 s uc u es we e conside ed as s a ing poin s o comple e geome y op imiza ions a he B3LYP/6-31+G(d,p) le el. Rega ding Ac-L-αPhP o-NHMe, ω was kep in he ans con igu a ion, while o he a angemen o he phenyl subs i uen h ee di e en o ien a ions we e conside ed. The e o e, he numbe o s a ing s uc u es o geome y op imiza ions we e 3( ψ backbone) × 2( ω 0 cis-o - ans) × 2(cyclic side chain) × 3(Ph subs i uen ) = 36. F equency analyses we e ca ied ou o e i y he na u e o he minimum s a e o all he s a iona y poin s ob ained and o calcula e he ze o-poin ib a ional ene gies (ZPVE) wi h bo h he mal and en opic co ec ions, he la e s a is ical e ms being used o compu e he con o ma ional Gibbs ee ene gies in he gas phase (∆Ggp) a he B3LYP/6- 31+G(d,p) le el. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 58 To ob ain an es ima ion o he sol a ion e ec s on he ela i e s abili y o he di e en minima, single poin calcula ions we e also conduc ed on he B3LYP/6- 31+G(d,p) op imized s uc u es using a Sel -Consis en Reac ion Field (SCRF) model. SCRF me hods ea he solu e a he quan um mechanical le el, while he sol en is ep esen ed as a dielec ic con inuum. Speci ically, he Pola izable Con inuum Model (PCM) de eloped by Tomasi and co-wo ke s was used o desc ibe he bulk sol en .21 This me hod in ol es he gene a ion o a sol en ca i y om sphe es cen e ed a each a om in he molecule and he calcula ion o i ual poin cha ges on he ca i y su ace ep esen ing he pola iza ion o he sol en . The magni ude o hese cha ges is p opo ional o he de i a i e o he solu e elec os a ic po en ial a each poin calcula ed om he molecula wa e unc ion. The poin cha ges may, hen, be included in he one-elec on Hamil onian, hus inducing pola iza ion o he solu e. An i e a i e calcula ion is ca ied ou un il he wa e unc ion and he su ace cha ges a e sel -consis en . PCM calcula ions we e pe o med using he s anda d p o ocol and conside ing he dielec ic cons an s o ca bon e achlo ide (ε = 2.228), chlo o o m (ε = 4.9), me hanol (ε= 32.6) and wa e (ε= 78.4). The con o ma ional ee ene gies in solu ion (∆G#sol#, whe e #sol# e e s o he sol en ) we e compu ed using he classical he modynamics scheme, ha is, he ee ene gies o sol a ion p o ided by he PCM model we e added o he ∆Ggp alues. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 59 3.2.2.2 Nomencla u e and Pseudo o a ional Pa ame e s. The minimum ene gy con o ma ions o he h ee dipep ides s udied in his wo k ha e been deno ed using a ou -label code ha speci ies he a angemen o he wo pep ide bonds, he ( ϕ , ψ ) backbone con o ma ion and he pucke ing o he i e-membe ed ing. The i s le e e e s o he ans ( ) o cis (c) a angemen o he pep ide bond p eceding p oline ( ω 0). The second label iden i ies he backbone con o ma ion using he nomencla u e in oduced by Pe czel e al.22 mo e han i een yea s ago. Acco dingly, nine di e en backbone con o ma ions can be dis inguished in he po en ial ene gy su ace E=E( ϕ , ψ ) o amino acids: γD, δD, αD, εD, βL, εL, αL, δL and γL. In he case o p oline, only he γL (γ- u n o C7), αL (α-helical), and εL (polyp oline II-like) con o ma ions a e accessible due o ϕ being ixed in he neighbo hood o –60º. Nex , he up o down pucke ing o he i e-membe ed ing is indica ed using he [u] and [d] labels, espec i ely. In pa icula , he down ing pucke ing was iden i ied when χ1 and χ3 we e posi i e while χ2 and χ4 we e nega i e. Con e sely, he up ing pucke ing is cha ac e ized by nega i e alues o χ1 and χ3 and posi i e alues o χ2 and χ4 The pucke ing o he i e-membe ed ing was desc ibed using he classical pseudo o a ional algo i hm, which uses a e y simple model based on only wo pa ame e s, as p e iously applied o p oline by Pe czel e al. . Finally, he las le e indica es he ans ( ) o cis (c) a angemen o he amide bond in ol ing he p oline ca bonyl g oup ( ω ). 23 ( ) 20 2)(PsinAA χ += The pseudo o a ional pa ame e s A and P, which desc ibe he pucke ing ampli ude and he s a e o he pucke in he pseudo o a ion pa hway, espec i ely, a e de i ed om he endocyclic dihed al angles as ollows: , whe e )º72sinº144(sin2 PsinA4321 +− −+− = χχχχ (3.2.1) and        <− ≥ = 0PsinAi , A a ccos 0PsinAi , A a ccos P0 0 χ χ (3.2.2) 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 60 Acco dingly, pa ame e A is de ined o be posi i e while P alls be ween – 180º and 180º. 3.2.3 Resul s and Discussion 3.2.3.1 Ac-L-P o-NHMe. Table 3.2.1 lis s he mos ele an s uc u al pa ame e s oge he wi h he ela i e ene gy (∆Egp) and ee ene gy (∆Ggp) in he gas phase o he 14 minimum ene gy con o ma ions cha ac e ized o Ac-L-P o-NHMe (Figu e 3.2.3). These minima a e dis ibu ed acco ding o he disposi ion o he pep ide bonds (de ined by he ω 0 and ω angles, Figu e 3.2.2) as ollows: bo h amide moie ies adop a ans a angemen in 3 minima ( ans- ans con o me s), one pep ide bond is cis in 7 minima (4 cis- ans and 3 ans-cis con o me s) and, inally, bo h pep ide bonds exhibi a cis con igu a ion in 4 minima (cis-cis con o me s). I is wo h no ing ha he s uc u al da a and ∆Egp alues displayed in Table 3.2.1 o he 14 minima cha ac e ized o Ac-L-P o-NHMe a e in excellen ag eemen wi h he esul s ecen ly epo ed by Csizmadia24 and Kang8b a he B3LYP/6-31G(d) and B3LYP/6-311++G(d,p) le els, espec i ely. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 67 Figu e 3.2.4: Rep esen a ion o he minimum ene gy con o ma ions cha ac e ized o Ac-L- α MeP o-NHMe a he B3LYP/6-31+G(d,p) le el. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 68 Table 3.2.3: Backbone dihed al angles (in deg ees), pseudo o a ional pa ame e s (A and P; in deg ees) and ela i e ene gy ( ∆ Egp; in kcal/mol) and ee ene gy ( ∆ Ggp # Con . ; in kcal/mol) o he minimum ene gy con o ma ions cha ac e ized o Ac-L- α MeP o-NHMe a he B3LYP/6-31+G(d,p) le el in he gas phase. ωϕ 0 ψ ω (A, P) ∆E∆G gp gp -γ L -172.2 [d]- -77.2 57.1 178.7 (35.0, -108.7) 0.0 a 0.0 b c -γ L -176.2 [u]- -69.8 60.7 178.8 (31.2, 86.9) 1.6 d 1.7 -ε L 175.4 [u]- -55.1 123.4 -174.0 (31.3, 91.7) 3.3 e 2.8 -α L -172.6 [u]- -64.2 -20.1 177.6 (33.6, 72.8) 4.2 3.2 c-αL 10.8 [d]- -77.7 -17.1 -179.1 (37.3, -94.3) 4.5 g 3.3 c-αL 3.0 [u]- -63.5 -28.1 -177.9 (37.6, 74.9) 4.6 h 3.1 c-εL -3.0 [u]- -48.5 141.5 176.4 (37.8, 74.9) 7.6 i 6.9 c-εL 0.0 [d]- -66.3 149.5 178.3 (36.8, -100.0) 7.7 j 6.6 -εL 175.3 [u]-c -55.5 125.9 -32.4 (38.3, 88.5) 10.3 k 11.2 -εL 177.8 [d]-c -61.1 127.7 -32.4 (36.1, -94.3) 10.6 l 11.5 -αL -174.4 [u]-c -54.6 -41.1 15.8 (36.8, 68.3) 10.6 m 11.1 -εL 173.9 [d]-c -67.2 164.4 -2.0 (36.1, -116.5) 11.6 n 11.7 -αL -170.2 [d]-c -73.7 -18.9 18.6 (37.7, -95.5) 11.8 o 11.7 c-αL -0.2 [u]-c -56.5 -40.0 3.5 (36.4, 71.2) 10.6 p 10.8 c-αL 12.9 [d]-c -64.9 -38.6 -8.4 (32.2, -68.8) 12.8 q 12.7 c-εL -2.5 [d]-c -70.0 177.0 -4.1 (37.7, -111.0) 13.9 14.1 c-εL -11.7 [u]-c -57.5 177.3 3.7 (37.9, 94.5) 14.6 s 14.4 a χ0= -11.2º, χ1= 30.9º, χ2= -39.4º, χ3= 31.6º and χ4= -12.8º. b E= -612.629968 a.u. c G= -612.419998 a.u. d χ0= -0.9º, χ1= -22.5º, χ2= 37.1º, χ3= -36.8º and χ4= 23.8º. e χ0= 1.7º, χ1= -24.0º, χ2= 37.0º, χ3= -35.1º and χ4= 21.2º. χ0= 9.9º, χ1= - 29.4º, χ2= 38.1º, χ3= -31.4º and χ4= 13.4º. g χ0= -2.8º, χ1= 24.4º, χ2= -36.5º, χ3= 33.9º and χ4= -19.7º. h χ0= 9.8º, χ1= -29.1º, χ2= 37.9º, χ3= -31.2º and χ4= 13.4º. i χ0= 9.8º, χ1= -29.3º, χ2= 38.0º, χ3= -31.4º and χ4= 13.6º. j χ0= -6.4º, χ1= 26.6º, χ2= -36.7º, χ3= 32.1º and χ4= -16.2º. k χ0= 1.0º, χ1= -23.5º, χ2= 36.9º, χ3= -35.4º and χ4= 21.8º. l χ0= -2.3º, χ1= 22.5º, χ2= -33.7º, χ3= 31.3º and χ4= -18.2º. m χ0= 13.6º, χ1= -30.9º, χ2= 317.1º, χ3= -28.3º and χ4= 9.1º. n χ0= -16.1º, χ1= 31.7º, χ2= -36.0º, χ3= 25.7º and χ4= -5.9º. o χ0= -3.6º, χ1= 25.2º, χ2= -37.1º, χ3= 33.9º and χ4= -19.2º. p χ0= 11.3º, χ1= -29.3º, χ2= 36.7º, χ3= -29.3º and χ4= -11.2º. q χ0= 11.6º, χ1= 8.8º, χ2= -24.9º, χ3= 31.3º and χ4= -27.5º. χ0= -13.5º, χ1= 31.5º, χ2= - 38.0º, χ3= 29.2º and χ4= -9.7º. s χ0= -3.0º, χ1= -20.1º, χ2= 35.1º, χ3= -36.1º and χ4= 25.0º. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 69 The lowes ene gy con o ma ion cha ac e ized o Ac-L-αMeP o-NHMe in he gas phase co esponds o a -γL[d]- con o me , which was also iden i ied as he global minimum o Ac-L-P o-NHMe. The geome ic pa ame e s o he hyd ogen bond associa ed wi h his con o ma ion [d(H···O) = 1.874 Å, ∠N–H···O = 151.4º] indica e ha his in amolecula in e ac ion is s onge in he α-me hyl de i a i e. The o he wo ans- ans con o me s ound o Ac-L-P o-NHMe, - γL[u]- and -αL[u]- (Table 3.2.1), we e also loca ed as ene gy minima o Ac-L- αMeP o-NHMe (Table 3.2.3), wi h simila geome ies and ene gies. Thus, he main di e ence be ween P o and αMeP o when bo h pep ide bonds exhibi a ans a angemen is he cha ac e iza ion o a minimum in he εL egion o he α- me hyla ed compound. No such semi-ex ended backbone con o ma ion was de ec ed as an ene gy minimum o Ac-L-P o-NHMe. This could be indica i e o his backbone con o ma ion being mo e a o able o αMeP o han o he pa en amino acid, which is con a y o he gene al obse a ion ha semi-ex ended and ully ex ended con o ma ions a e mo e s able o p o einogenic amino acids han o hei α-me hyla ed coun e pa s.1c,e,3-5 This singula i y is speci ically e idenced in Figu e 3.2.5, whe e he po en ial ene gy cu es E= E( ψ ) o Ac-L-P o-NHMe and Ac-L-αMeP o-NHMe o ans pep ide bonds and an up-pucke ed ing a e compa ed. As can be seen, he wo p o iles di e almos uniquely in he la egion ha appea s o he la e compound a ψ alues anging om 120º o 150º, ha is, whe e he ε L semi- ex ended con o ma ion is loca ed. Howe e , as al eady men ioned, con o ma ions in he εL egion a e e y o en obse ed expe imen ally6 o P o-con aining pep ides longe han ha conside ed in he p esen wo k. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 70 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 060 120 180 240 300 360 ψ ∆E(kcal/mol) 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 060 120 180 240 300 360 ψ ∆E(kcal/mol) Figu e 3.2.5: Po en ial ene gy cu es E=E( ψ ) c oss sec ions o he con o ma ional po en ial ene gy su aces o Ac-L-P o-NHMe ( illed squa es and solid lines) and Ac-L- α MeP o-NHMe (emp y squa es and dashed lines). In bo h compounds, he py olidine ing is up-pucke ed and he pep ide bonds a e a anged in ans. Also cis- ans αL and εL con o me s simila o hose obse ed o p oline we e cha ac e ized o αMeP o. They a e dis a o ed wi h espec o he global minimum by abou 3 and 7 kcal/mol, espec i ely, he in luence o he py olidine ing pucke ing being negligible (Table 3.2.3). Compa ison be ween he ∆Ggp alues ob ained o he cis- ans con o me s o Ac-L-P o-NHMe and Ac-L- αMeP o-NHMe indica es ha , in gene al, α-me hyla ion p oduces a des abiliza ion o 1–2 kcal/mol. This esul is no unexpec ed since he α-me hyl g oup inc eases he s e ic hind ance a ound Cα, hus dis a o ing he cis disposi ion be ween he ace yl me hyl g oup and he α ca bon ( ω 0 ≈ 0º). The e ec o α-me hyla ion in he des abiliza ion o cis amide bonds becomes mo e e iden o he –CONHMe moie y (co esponding o ω ). In ac , all ans-cis and cis-cis con o me s exhibi ∆Ggp Table 3.2.4 shows he e ec s o sol a ion on he 17 minima o Ac-L-αMeP o- NHMe. As can be seen, he -γ alues abo e 10.8 kcal/mol (Table 3.2.3) and signi ican ly highe han hose ob ained o he equi alen con o me s o Ac-L- P o-NHMe (Table 3.2.1). L[d]- is he mos s able con o ma ion no only in 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 71 he gas phase bu also in ca bon e achlo ide and chlo o o m solu ions. Signi ican di e ences a e obse ed be ween he esul s ob ained o Ac-L-P o- NHMe (Table 3.2.2) and Ac-L-αMeP o-NHMe (Table 3.2.4) in chlo o o m. Speci ically, o he la e pep ide, he ∆GCHCl3 alues o all ou ans- ans con o me s lie below 1.6 kcal/mol, whe eas all he cis- ans con o me s show ∆GCHCl3 Table 3.2.4: Rela i e ee ene gy in he gas-phase ( ∆ G alues abo e his limi , indica ing ha only ans- ans a angemen s a e ene ge ically accessible in chlo o o m. This is in sha p con as wi h he esul s ob ained o Ac-L-P o-NHMe, o which ce ain cis- ans con o me s we e ound o exhibi a high s abili y. gp; in kcal/mol) and in ca bon e achlo ide, chlo o o m. me hanol and aqueous solu ions ( ∆ GCCl4, ∆ GCHCl3, ∆ GCH3OH and ∆ GH2O # Con . , espec i ely; in kcal/mol) o he minimum ene gy con o ma ions o Ac-L- α MeP o-NHMe a he B3LYP/6- 31+G(d,p) le el. ∆G∆G gp ∆G CCl4 ∆G CHCl3 ∆G CH3OH H2O -γL 0.0 [d]- 0.0 0.0 0.9 1.4 -γL 1.7 [u]- 1.5 1.4 2.4 2.8 -εL 2.8 [u]- 2.1 1.6 1.7 2.0 -αL 3.3 [u]- 2.4 1.2 0.2 0.3 c-α L 3.3 [d]- 1.1 1.9 1.1 0.5 c-α L 3.1 [u]- 0.8 1.7 0.4 0.0 c-εL 6.9 [u]- 2.7 4.5 0.0 0.6 c-εL 6.6 [d]- 2.3 4.3 0.5 0.6 -εL 11.2 [u]-c 8.9 8.0 7.7 7.5 -εL 11.5 [d]-c 9.4 8.6 8.5 8.4 -αL 11.1 [u]-c 7.2 4.7 2.6 2.2 -εL 11.7 [d]-c 8.5 6.7 5.1 5.1 -αL 11.7 [d]-c 8.2 6.2 4.8 4.5 c -αL 10.8 [u]-c 7.1 5.4 4.8 4.5 c -αL 12.7 [d]-c 9.0 7.2 6.7 6.2 c-εL 14.1 [d]-c 9.8 7.2 5.2 4.8 c-εL 14.4 [u]-c 10.0 7.4 5.5 5.0 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 72 Al hough he s abili y o he cis- ans con o me s in Table 3.2.4 is p obably o e es ima ed in ca bon e achlo ide, he gene al endencies de i ed om PCM calcula ions in non-pola en i onmen s a e ully consis en wi h da a om NMR expe imen s, which showed no cis con o me s o Ac-L-αMeP o-NHMe in chlo o o m solu ion.10d In good ag eemen , ou calcula ions p edic he pep ide in ol ing he py olidine ni ogen ( ω 0 Finally, analysis o he esul s ob ained o Ac-L-αMeP o-NHMe in me hanol and aqueous solu ion indica es ha he cis- ans con o me s a e he mos a o ed in hese pola en i onmen s. Thus, he c-ε ) o exhibi a conside ably smalle p obabili y o adop ing a cis disposi ion in αMeP o han in P o. L[u]- and c-αL[u]- a e he lowes ene gy minimum in me hanol and wa e , espec i ely, and, in addi ion, he ∆GCH3OH and ∆GH2O o he emaining h ee cis- ans con o me s a e lowe han 1.5 kcal/mol. These esul s clea ly e idence ha he s abili y o he cis con igu a ion o he pep ide bond in ol ing he αMeP o ni ogen ( ω 0 3.2.3.3 Ac-L-αPhP o-NHMe. ≈ 0º) is signi ican ly o e es ima ed by he PCM me hod when pola sol en s a e conside ed. Table 3.2.5 shows he s uc u al pa ame e s oge he wi h he ∆Egp and ∆Ggp alues o he 8 minimum ene gy con o ma ions ound o he αPhP o-con aining pep ide (Figu e 3.2.6). Speci ically, 4 minima wi h wo ans amide bonds we e cha ac e ized, while he o he 4 co espond o cis- ans con o me s. I should be no ed ha he cis a angemen o he –CONHMe pep ide bond (co esponding o he ω angle) was no conside ed o his compound. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 73 Figu e 3.2.6: Rep esen a ion o he minimum ene gy con o ma ions cha ac e ized o Ac-L- α PhP o-NHMe a he B3LYP/6-31+G(d,p) le el. Table 3.2.5: Backbone dihed al angles (in deg ees), pseudo o a ional pa ame e s (A and P; in deg ees) and ela i e ene gy ( ∆ Egp; in kcal/mol) and ee ene gy ( ∆ Ggp # Con . ; in kcal/mol) o he minimum ene gy con o ma ions cha ac e ized o Ac-L- α PhP o-NHMe a he B3LYP/6-31+G(d,p) le el in he gas phase. ωϕ 0 ψ ω (A, P) ∆E∆G gp gp -γL -174.9 [d]- -75.4 59.1 178.9 (39.1, -107.0) 0.0 a 0.0 b c -γL -179.6 [u]- -67.9 66.2 -179.9 (39.2, 89.8) 1.7 d 1.3 -εL 172.0 [u]- -46.5 120.3 -172.0 (39.9, 76.6) 2.6 e 2.6 -εL 176.7 [d]- -61.2 161.7 176.6 (37.8, -98.4) 7.8 7.5 c-γL 10.8 [d]- -85.5 5.5 -176.4 (38.7, -103.8) 4.5 g 4.0 c-αL 3.2 [u]- -59.9 -28.1 -175.9 (38.6, 68.2) 5.1 h 3.8 c-ε L -5.0 [u]- -43.3 129.6 -179.5 (39.9, 66.0) 5.6 i 5.5 c-ε L -4.7 [d]- -73.3 -162.6 -177.4 (39.7, -118.2) 8.2 j 7.6 a χ0= -11.4º, χ1= 31.2º, χ2= -39.5º, χ3= 31.7º and χ4= -12.7º. b E= -804.371070 a.u. c G= -804.113301 a.u. d χ0= 0.1º, χ1= -23.4º, χ2= 37.5º, χ3= -36.6º and χ4= 23.1º. e χ0= 9.3º, χ1= -30.3º, χ2= 40.2º, χ3= -33.7º and χ4= 15.3º. χ0= -5.5º, χ1= - 26.6º, χ2= -37.4º, χ3= 34.0º and χ4= -17.6º. g χ0= -9.2º, χ1= 29.5º, χ2= -38.9º, χ3= 32.6º and χ4= -14.6º. h χ0= 14.3º, χ1= -32.4º, χ2= 38.9º, χ3= -29.7º and χ4= 9.4º. i χ0= 16.2º, χ1= -34.3º, χ2= 40.1º, χ3= -29.7º and χ4= 8.2º. j χ0= -18.8º, χ1= 35.4º, χ2= -39.5º, χ3= 27.6º and χ4= -5.2º. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 74 As obse ed be o e o P o and αMeP o, he γL backbone con o ma ion wi h all ans pep ide bonds is he mos s able a angemen o αPhP o, wi h he down pucke ing o he py olidine ing being p e e ed. Thus, -γL[d]- appea s as he global minimum while -γL[u]- is des abilized by 1.3 kcal/mol. In spi e o his pa allelism, he con o ma ional p o ile o he α-phenyl de i a i e shows impo an di e ences wi h espec o hose desc ibed abo e o P o and αMeP o. The semi- ex ended s uc u e -εL[u]- cha ac e ized as an ene gy minimum o he α- me hyla ed compound, bu no o he pa en amino acid, was also loca ed o αPhP o, 2.6 kcal/mol abo e he global minimum (Table 3.2.5). Mo eo e , an addi ional εL minimum wi h a down pucke ing was ound o he la e compound, al hough his a angemen o he i e-membe ed ing p o ed e y un a o able ene ge ically. The o e all o hese esul s sugges s ha con o ma ions in he εL egion could be mo e a o ed o α-subs i u ed p oline de i a i es han o p oline i sel , con a y o he gene al beha io expec ed o α- e asubs i u ed amino acids in compa ison wi h hei p o einogenic coun e pa s. Ano he dis inc ea u e in he con o ma ional map o Ac-L-αPhP o-NHMe is he disappea ance o ans- ans minima o he α-helical ype. Thus, he -α 1c,e,3–5 L The e ec o α-subs i u ion on he cis/ ans isome ism desc ibed abo e o Ac-L-αMeP o-NHMe is also obse ed o αPhP o. The cis- ans con o me s in Table 3.2.5 exhibi ∆G [u]- , which was cha ac e ized o bo h Ac-L-P o-NHMe and Ac-L-αMeP o-NHMe, was no a minimum in he po en ial ene gy hype su ace o Ac-L-αPhP o-NHMe. Al hough calcula ions on small pep ide sys ems like hese in he p esen s udy a e known o unde es ima e he s abili y o α-helical con o ma ions (in gene al, o hose lacking an in amolecula hyd ogen bond) in a o o γ- u ns, his inding is highly ema kable. gp alues anging om 3.8 o 7.6 kcal/mol, e idencing a des abiliza ion o he cis disposi ion o he ω 0 Table 3.2.6 compa es he sol a ion e ec s es ima ed o he 8 minima cha ac e ized o Ac-L-αPhP o-NHMe. As can be seen, he con o ma ional p ope ies p edic ed in ca bon e achlo ide and chlo o o m solu ions a e e y simila o hose ob ained in he gas phase. The ans- ans con o me s a e sca cely amide bond wi h e e ence o ha obse ed o P o. 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 75 a ec ed by sol a ion, while he ela i e ee ene gy o he cis- ans con o me s dec eases on going om he gas phase o solu ion, and wi h he sol en pola i y. In spi e o such s abiliza ion, minima wi h a cis pep ide bond emain inaccessible a oom empe a u e. In ac , only he -γL[d]- and -γL[u]- con o me s p esen ene gies below 2.0 kcal/mol in bo h sol en s and a e he e o e p edic ed o be popula ed. Howe e , esul s in me hanol and aqueous solu ions e lec again he limi a ions o he PCM model o desc ibe he cis/ ans isome ism o ω 0 Table 3.2.6: Rela i e ee ene gy in he gas-phase ( ∆ G in pola en i onmen s. gp; in kcal/mol) and in ca bon e achlo ide, chlo o o m, me hanol and aqueous solu ions ( ∆ GCCl4, ∆ GCHCl3, ∆ GCH3OH and ∆ GH2O # Con . , espec i ely; in kcal/mol) o he minimum ene gy con o ma ions o Ac-L- α PhP o-NHMe a he B3LYP/6-31+G(d,p) le el. ∆G∆G gp ∆G CCl4 ∆G CHCl3 ∆G CH3OH H2O -γL 0.0 [d]- 0.0 0.0 0.0 0.3 -γL 1.3 [u]- 1.2 1.3 1.4 1.6 -εL 2.6 [u]- 2.4 2.2 1.6 1.3 -εL 7.5 [d]- 6.9 6.1 4.5 4.4 c-γL 4.0 [d]- 2.8 2.5 2.3 2.1 c-αL 3.8 [u]- 2.4 2.0 1.4 0.9 c-εL 5.5 [u]- 3.5 2.5 1.3 2.8 c-εL 7.6 [d]- 4.8 2.8 0.5 0.0 3.2.4 Conclusions Quan um mechanical calcula ions a he B3LYP/6-31+G(d,p) le el ha e been used o explo e he con o ma ional p e e ences o Ac-L-αMePhe-NHMe and Ac- L-αPhP o-NHMe. Compa ison o he esul s wi h hose ob ained o Ac-L-P o- NHMe a he same heo e ical le el allows us o d aw he ollowing conclusions: (i) Replacemen o he α hyd ogen in p oline by a mo e bulky g oup des abilizes he cis con igu a ion o he amide bond in ol ing he py olidine ni ogen. The pe cen age o cis con o me s usually 3.2 CONFORMATIONAL PREFERENCES OF α -SUBSTITUTED PROLINE ANALOGUES 76 obse ed o he pep ide bond p eceding p oline, i any, is hus p edic ed o be much in e io o α- e asubs i u ed p oline de i a i es. (ii) Ano he gene al s uc u al end associa ed wi h Cα- e asubs i u ion seems o be he s abiliza ion o he semi-ex ended polyp oline II con o ma ion (εL (iii) Al hough α- e asubs i u ion esul s in gene al con o ma ional changes like hose ou lined abo e, mo e sub le bu equally impo an di e ences seem o be associa ed wi h he pa icula na u e o he subs i uen inco po a ed a C ), which was iden i ied as an ene gy minimum o bo h αMeP o and αPhP o bu no o he p o einogenic amino acid. α. Thus, e en i he γ- u n (γL) is he lowes ene gy minimum o bo h Ac-L-αMePhe-NHMe and Ac-L- αPhP o-NHMe in all he en i onmen al condi ions examined, he α- helical con o ma ion (αL (i ) PCM calcula ions in solu ion indica e ha he s abili y o he con o me s wi h a cis con igu a ion o he pep ide bond in ol ing P o ni ogen inc eases wi h he pola i y o he en i onmen . Howe e , in his case esul s in solu ion mus be analyzed wi h cau ion since SCRF calcula ions o e es ima e his e ec signi ican ly, especially when pola sol en s (as wa e o me hanol) a e conside ed. ) wi h ans amide bonds was also ound o be accessible o he α-me hyl de i a i e bu was no loca ed as an ene gy minimum o he αPhP o-con aining pep ide. 3.2.5 Re e ences 1. (a) Toniolo, C.; Fo maggio, F.; Kap ein, B.; B ox e man, Q. B. Synle 2006, 1295. (b) Venka aman, J.; Shanka amma, S. C.; Bala am, P. Chem. Re . 2001, 101, 3131. (c) Toniolo, C.; C isma, M.; Fo maggio, F.; Peggion, C. Biopolyme s (Pep . Sci.) 2001, 60, 396. (d) Kaul, R.; Bala am, P. Bioo g. Med. Chem. 1999, 7, 105. (e) Benede i, E. Biopolyme s (Pep . Sci.) 1996, 40, 3. ( ) Toniolo, C.; Benede i, E. Mac omolecules 1991, 24, 4004. 2. (a) Ca i iela, C.; Díaz-de-Villegas, M. D. Te ahed on: Asymme y 2007, 18, 569. (b) Vog , H.; B äse, S. O g. Biomol. Chem. 2007, 5, 406. (c) Pa k, 83 3.3 Con o ma ional P e e ences o β− and γ−Amina ed P oline Analogues Quan um mechanical calcula ions ha e been used o in es iga e how he inco po a ion o an amino g oup o he C β - o C γ -posi ions o he py olidine ing a ec s he in insic con o ma ional p ope ies o he p oline. Speci ically, a con o ma ional s udy o he N-ace yl-N’- me hylamide de i a i es o ou isome s o aminop oline, which di e no only in he β - o γ -posi ion o he subs i uen bu also in i s cis o ans ela i e disposi ion, has been pe o med. In o de o u he unde s and he ole o he in amolecula hyd ogen bonds be ween he backbone ca bonyl g oups and he amino side g oup, a con o ma ional s udy was also pe o med on he co esponding ou analogues o dime hylaminop oline. In addi ion, he e ec s o sol a ion on aminop oline and dime hylaminop oline dipep ides ha e been e alua ed using a Sel Consis en Reac ion Field model, and conside ing ou di e en sol en s (ca bon e achlo ide, chlo o o m, me hanol and wa e ). Resul s indica e ha he inco po a ion o he amino subs i uen in o he py olidine ing a ec s he con o ma ional p ope ies, wi h backbone···side chain in amolecula hyd ogen bonds de ec ed when i is inco po a ed in a cis ela i e disposi ion. In gene al, he inco po a ion o he amino side g oup ends o s abilize hose s uc u es whe e he pep ide bond in ol ing he py olidine ni ogen is a anged in cis. The aminop oline isome wi h he subs i uen a ached o he C γ -posi ion wi h a cis ela i e disposi ion is he mos s able in he gas-phase and in chlo o o m, me hanol and wa e solu ions. Replacemen o he amino side g oup by he dime hylamino subs i uen p oduces signi ican changes in he po en ial ene gy su aces o he ou in es iga ed dime hylaminop oline- con aining dipep ides. Thus, hese changes a ec no only he numbe o minima, which inc eases conside ably, bu also he backbone and pseudo o a ional p e e ences. In spi e o hese e ec s, compa ison o he con o ma ional p e e ences, i.e. he mo e a o ed con o me s, calcula ed o di e en isome s o aminop oline and dime hylaminop oline dipep ides showed a high deg ee o consis ency o he wo amilies o compounds.* 3.3.1 In oduc ion P oline (P o) is unique among na u ally occu ing amino acids in ha i s side chain is bonded o bo h he α-ca bon and i s p eceding amide ni ogen. As a consequence, o a ion abou he N—Cα * The wo k desc ibed in his chap e p e iously appea ed in J. Phys. Chem. B 2008 , 112, 14045–14055 bond is p ohibi ed and he ϕ o sion angle 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 84 is con ined o alues a ound –60º. Acco dingly, P o is o e whelmingly ound in he α-helical ( ϕ , ψ ≈ –60º,–30º) and semi-ex ended ( ϕ , ψ ≈ –60º,140º) egions o he con o ma ional map.1 In addi ion, P o shows a highe p opensi y o p omo e γ- u n con o ma ions ( ϕ , ψ ≈ –70º,60º) han o he p o eogenic amino acids.1d,2 Ano he e ec de i ed om i s cyclic s uc u e is ha he pep ide bond p eceding P o ( ha in ol ing he py olidine ni ogen) has a ela i ely high p obabili y o accommoda ing a cis a angemen 3 as compa ed o o he pep ide bonds, o which he cis o m is almos inexis en . Recen s udies in P o dipep ides obse ed ha he cis/ ans isome iza ion is an en halpy d i en p ocess ha depends on he pola i y o he en i onmen .4 Thus, al hough he elec onic e ec s ha s abilize he cis o m a e enhanced in pola en i onmen s, he cis/ ans o a ional ba ie s inc ease wi h he pola i y o he en i onmen . These s uc u al ea u es play a undamen al ole in di ec ing he seconda y s uc u e o p o eins,5 inducing special mo i s like e e se u ns and bends.6 Fu he mo e, he cis- ans isome iza ion o P o has been specula ed o play a ole no only in impo an biological p ocesses7 bu also in he a e de e mining s eps o olding and e olding o some p o eins. 4R-Hyd oxyp oline (Hyp) is a hyd oxyla ed de i a i e o P o ha sha es he same ea u es as i s pa en amino acid. I is o med by a pos - ansla ional modi ica ion whe e a P o esidue is con e ed o Hyp by an enzyme wi h a e ous ion a i s ac i e si e, called p olyl hyd oxylase. Bo h Hyp and P o, along wi h glycine, a e ound in collagen, he mos abundan p o ein in e eb a es. As a consequence o hei impo ance, he in insic con o ma ional p e e ences o P o 8 3a,9-12 and Hyp13 ha e been examined in de ail on he co esponding dipep ide analogues using ad anced heo e ical me hods. In e es ingly, in spi e o he capabili ies o he hyd oxyl side g oup o o m in amolecula hyd ogen bonds able o induce signi ican s uc u al dis o ions, he minimum ene gy con o ma ions ound o he N-ace yl-N’-me hylamide de i a i es o P o and Hyp (Ac-P o-NHMe and Ac-Hyp-NHMe dipep ides, espec i ely) we e e y simila . Speci ically, a s ong co ela ion was obse ed be ween he op imized dihed al angles o hese dipep ides. Indeed, he la ges e ec p oduced by hyd oxyla ion o P o was de ec ed in he pucke ing o he py olidine ing. Thus, he down pucke ing is p e e ed o Ac-P o-NHMe, while he up pucke ing wi h he 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 85 hyd oxyl g oup occupying an equa o ial posi ion is he mos s able o Ac-Hyp- NHMe. In ecen yea s we ha e been in ol ed in a b oad p ojec de o ed o he design and applica ion o syn he ic amino acids wi h es ic ed con o ma ional mobili y in di e en ields o nanobiology. Non-p o eogenic amino acids ha e been ound o be e y use ul o he e-enginee ing o physical p o ein modules and he gene a ion o nanode ices.14 Mo e speci ically, we obse ed ha inse ion o chemically cons ained esidues wi h sui able backbone con o ma ional endencies enhance he he modynamic s abili y o he nano ubula s uc u es cons uc ed by sel -assembling p o ein agmen s wi h a β-helical con o ma ion.15 We ha e u he selec i ely inco po a ed syn he ic amino acids o impa esis ance agains p o eases no only a he mu a ed posi ion bu also a neighbo ing amino acids.16 In his wo k, we in es iga e he in insic con o ma ional p e e ences o di e en amina ed de i a es o P o. These non- p o eogenic amino acids, which ha e been al eady used o cons uc β-pep ides wi h helical seconda y s uc u es,17 Theo e ical calcula ions based on Densi y Func ional Theo y (DFT) me hods ha e been used o in es iga e he con o ma ional p ope ies o he N-ace yl-N’- me hylamide de i a i es o Amp ha inco po a e an amino g oup o he C a e expec ed o be o po en ial in e es in many nanobiological applica ions. This is because he opological cha ac e is ics o he amino and hyd oxyl g oups a e di e en and, he e o e, in amolecula hyd ogen bonds in aminop oline (Amp) de i a i es a e expec ed o al e signi ican ly he s uc u al p ope ies o P o. β- o Cγ - posi ions o he py olidine ing, bo h he cis and ans isome s being conside ed in each case. Acco dingly, calcula ions we e pe o med on he ou compounds displayed in Figu e 3.3.1: Ac-β Amp-NHMe, Ac-βcAmp-NHMe, Ac-γ Amp- NHMe and Ac-γcAmp-NHMe. 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 86 H 3 CN C N H CH 3 OO H 3 CN C N H CH 3 OO R R H 3 CN C N H CH 3 OO R H 3 CN C N H CH 3 OO R Ac-β Amp-NHMe Ac-βcAmp-NHMe Ac-γ Amp-NHMe Ac-γcAmp-NHMe Ac-β Dmp-NHMe Ac-βcDmp-NHMe Ac-γ Dmp-NHMe Ac-γcDmp-NHMe R= NH 2 R= N(CH 3 ) 2 R= NH 2 R= N(CH 3 ) 2 Figu e 3.3.1: Compounds s udied in his wo k In o de o p o ide a be e unde s anding o he c ucial ole o in amolecula hyd ogen bonds, he s udy has been u he ex ended o he ou dipep ides cons uc ed by eplacing he Amp esidue by he co esponding dime hylaminop oline (Dmp) analogue: Ac-β Dmp-NHMe, Ac-βcDmp-NHMe, Ac-γ Dmp-NHMe and Ac-γcDmp-NHMe in Figu e 3.3.1. In addi ion we ha e examined how he inco po a ion o amino and dime hylamino subs i uen s a he β and γ posi ions o P o a ec s he ans/cis disposi ion o he pep ide bond in ol ing he py olidine ni ogen. Finally, he in luence o he en i onmen , in pa icula o he sol en , on he con o ma ional p e e ences o he di e en Amp- and Dmp-con aining dipep ides has been e alua ed using a Sel Consis en Reac ion Field (SCRF) me hod. Resul s ha e been compa ed wi h hose ecen ly epo ed o Ac-P o-NHMe,12b 3.3.2 Me hods which we e calcula ed using he same heo e ical p ocedu es. All calcula ions we e ca ied ou using he Gaussian 03 compu e p og am.18 DFT calcula ions we e pe o med using he ollowing combina ion: he 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 87 Becke´s h ee-pa ame e hyb id unc ional (B3)19 wi h he local unc ional de eloped by Lee, Yang and Pa (LYP),20 which is g adien co ec ed. Thus, all he calcula ions p esen ed in his wo k we e pe o med using he B3LYP me hod combined wi h he 6-31+G(d,p) basis se .21 This compu a ional p ocedu e p o ided a e y sa is ac o y desc ip ion o he con o ma ional p ope ies o cyclic cons ained amino acids, including P o and i s dehyd o- and α-subs i u ed de i a i es.22,12 The backbone (ω 0,ϕ,ψ,ω) and side chain (χi; endocyclic) dihed al angles o he N-ace yl-N’-me hylamide de i a i es o con en ional P o, Amp and Dmp a e de ined in Figu e 3.3.2. Since ϕ is ixed by he geome y o he i e-membe ed ing, only h ee minima may be an icipa ed o he po en ial ene gy su aces E=E( ψ ) o he dipep ides o a gi en a angemen o he pep ide bonds. Thus, he lexible angle ψ is expec ed o ha e h ee minima, i.e. gauche+ (60º), ans (180º) and gauche– (–60º), while each amide bond ( ω 0, ω ) can be a anged in ans o cis. I should be no ed ha only he pep ide bond in ol ing he py olidine ni ogen, which co esponds o he ω 0 o sion angle, is likely o adop a cis con igu a ion. The e o e, bo h he ans and cis s a es we e conside ed o ω 0, while he amide bond in ol ing he N-me hylamide blocking g oup (gi en by ω ) was a anged in ans only. The cyclic side chains o he compounds unde s udy may adop wo main di e en con o ma ional s a es ha co espond o he down and up pucke ing o he i e-membe ed ing. They a e de ined as hose in which he Cγ a om and he ca bonyl g oup o he P o esidue (o analogue) lie on he same and opposi e sides, espec i ely, o he plane de ined by he Cδ, N and Cα Acco dingly, o each o he eigh dipep ides unde s udy (Figu e 3.3.1), 3( ψ backbone) × 2( ω a oms. 0 ans-o -cis) × 2(cyclic side chain) = 12 s uc u es we e conside ed as s a ing poin s o comple e geome y op imiza ions a he B3LYP/6-31+G(d,p) le el. F equency analyses we e ca ied ou o e i y he na u e o he minimum s a e o all he s a iona y poin s ob ained and o calcula e he ze o-poin ib a ional ene gies (ZPVE) as well as bo h he mal and en opic co ec ions, hese s a is ical e ms being used o compu e he con o ma ional Gibbs ee ene gies in he gas phase (∆Ggp) a 298 K. 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 88 Me CNCNMe OO H ω0ϕψω χ0 χ1 χ2 χ3 χ4 α β γ δ Figu e 3.3.2: Dihed al angles used o iden i y he con o ma ions o he N- ace yl-N’-me hylamide de i a i es o he Amp and Dmp analogues s udied in his wo k. The dihed al angles ω 0, ϕ , ψ and ω a e de ined using backbone a oms while he endocyclic dihed al angles χ i a e gi en by he a oms o he i e-membe ed ing. In pa icula , he sequence o a oms used o de ine ϕ and χ 0 a e C(=O)–N–C α –C(=O) and C δ –N–C α –C β , espec i ely To ob ain an es ima ion o he sol a ion e ec s on he ela i e s abili y o he di e en minima, single poin calcula ions we e conduc ed on he B3LYP/6- 31+G(d,p) op imized s uc u es using a Sel -Consis en Reac ion Field (SCRF) model. SCRF me hods ea he solu e a he quan um mechanical le el, while he sol en is ep esen ed as a dielec ic con inuum. Speci ically, he Pola izable Con inuum Model (PCM) de eloped by Tomasi and co-wo ke s was used o desc ibe he bulk sol en .23 This me hod in ol es he gene a ion o a sol en ca i y om sphe es cen e ed a each a om in he molecule and he calcula ion o i ual poin cha ges on he ca i y su ace ep esen ing he pola iza ion o he sol en . The magni ude o hese cha ges is p opo ional o he de i a i e o he solu e elec os a ic po en ial a each poin calcula ed om he molecula wa e unc ion. The poin cha ges may, hen, be included in he one-elec on Hamil onian, hus inducing pola iza ion o he solu e. An i e a i e calcula ion is ca ied ou un il he wa e unc ion and he su ace cha ges a e sel -consis en . PCM calcula ions we e pe o med using he s anda d p o ocol implemen ed in Gaussian 0318 and conside ing he dielec ic cons an s o ca bon e achlo ide (ε = 2.228), chlo o o m (ε = 4.9), me hanol (ε= 32.6) and wa e (ε= 78.4). The con o ma ional ee ene gies in solu ion (∆G#sol#, whe e #sol# e e s o he 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 89 sol en ) we e compu ed using he classical he modynamics scheme, i.e. he ee ene gies o sol a ion p o ided by he PCM model we e added o he ∆Ggp 3.3.2.1 Nomencla u e and Pseudo o a ional Pa ame e s alues.. The minimum ene gy con o ma ions o he dipep ides s udied in his wo k ha e been deno ed using a h ee-labels code ha speci ies he a angemen o he i s pep ide bond, he backbone con o ma ion and he pucke ing o he i e membe ed ing. The i s le e e e s o he ans ( ) o cis (c) a angemen o ω o. The second label iden i ies he backbone con o ma ion using he nomencla u e in oduced by Pe czel e al.53 mo e han i een yea s ago. Acco dingly, nine di e en backbone con o ma ions can be ound in he po en ial ene gy su ace E=E( ϕ , ψ ) o amino acids: γD, δD, αL, εD, βL, εL, αD, δL and γL. Finally, he up o down pucke ing o he i e-membe ed ing is indica ed using he labels [u] and [d], espec i ely. In pa icula , he [d] ing pucke ing was iden i ied when χ 1 and χ 3 a e posi i e while χ 2 and χ 4 a e nega i e. The e o e, he [u] ing pucke ing is cha ac e ized by nega i e alues o χ 1 and χ 3 and posi i e alues o χ 2 and χ 4 The pucke ing o he i e-membe ed ing was desc ibed using he classical pseudo o a ional algo i hm, which uses a e y simple model based on only wo pa ame e s, as was p e iously applied o P o by Hudaky and Pe czel. . We no e ha he cyclic side chain adop s a plana a angemen in wo o he s udied molecules, and no indica ion o he pucke ing was included in he code used o hese cases. 28,29 ( ) 20 2)(PsinAA χ += The pseudo o a ional pa ame e s a e A and P, which desc ibe he pucke ing ampli ude and he s a e o he pucke in he pseudo o a ion pa hway, espec i ely. The pa ame e s a e de i ed om he endocyclic dihed al angles as ollows: , whe e )º72sinº144(sin2 PsinA4321 +− −+− = χχχχ (3.1.1) and 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 90        <− ≥ = 0PsinAi , A a ccos 0PsinAi , A a ccos P0 0 χ χ (3.1.2) Acco dingly, pa ame e A is de ined o be posi i e while P alls be ween – 180º and 180º. 3.3.3 Resul s and Discussion 3.3.3.1 Aminop oline (Amp) dipep ides This sec ion epo s he esul s ob ained a he B3LYP/6-31+G(d,p) le el o he ou Amp-con aining dipep ides displayed in Figu e 3.3.1, which ha e been compa ed o he dipep ide o con en ional P o ha was ecen ly epo ed a he same le el o heo y.12b Table 3.3.1 lis s he mo e ele an s uc u al pa ame e s oge he wi h he ela i e ene gy (∆Egp) o he 4, 6, 3 and 7 minimum ene gy con o ma ions cha ac e ized o Ac-β Amp-NHMe, Ac-βcAmp-NHMe, Ac- γ Amp-NHMe and Ac-γcAmp-NHMe, espec i ely, selec ed minima being displayed in Figu es 3.3.3 and 3.3.4. The ela i e s abili y o he ou dipep ides is indica ed in Table 3.3.1 h ough ∆E#gp#, which co esponds o he ene gy ela i e o he lowes ene gy con o ma ion o he mos s able isome . Table 3.3.2 compa es he ela i e ee ene gies in he gas-phase (∆Ggp), ca bon e achlo ide (∆GCCl4), chlo o o m (∆GCHCl3), me hanol (∆GMeOH) and wa e (∆GH2O) solu ions o he minima o he ou dipep ides men ioned abo e. Calcula ions in solu ion we e pe o med by applying he PCM me hod o he geome ies op imized in he gas phase. Thus, p e ious s udies on simple o ganic and bio-o ganic compounds indica ed ha solu e geome y elaxa ions in solu ion and single poin calcula ions on he op imized geome ies in he gas phase gi e almos iden ical ee ene gies o sal a ion,26 e en al hough nuclea elaxa ion in solu e has been ound o be essen ial in some speci ic cases.27 Finally, Table 3.3.3 compa es he ela i e s abili y o he ou Amp-con aining dipep ides by showing he ee ene gies in he di e en en i onmen s calcula ed in each case wi h espec o he 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 91 con o ma ion o lowes ee ene gy o he mos s able isome : ∆G#gp#, ∆G#CCl4#, ∆G#CHCl3#, ∆G#MeOH# and ∆G#H2O# Table 3.3.1 Backbone dihed al angles (in deg ees), pseudo o a ional pa ame e s (A and P; in deg ees), ela i e ene gy ( ∆ E . gp; in kcal/mol) and ela i e ene gy wi h espec o he lowes ene gy con o ma ion o he mos s able dipep ide ( ∆ E#gp# # Con . ; in kcal/mol) o he minimum ene gy con o ma ions cha ac e ized o Ac- β Amp-NHMe, Ac- β cAmp-NHMe, Ac- γ Amp-NHMe and Ac- γ cAmp-NHMe a he B3LYP/6-31+G(d,p) le el in he gas phase. ωϕ 0 ψ ω (A, P) ∆ E ∆ E gp #gp# Ac-β Amp-NHMe -γL -174.0 [d] -83.1 71.9 -177.6 (38.4, -117.2) 0.0 a 1.4 b c-εL 1.2 [u] -66.7 178.8 175.9 (38.2, 92.2) 2.8 c 4.2 c-αL 6.8 [u] -69.6 -35.1 179.4 (37.8, 82.2) 5.9 d 7.3 c-εL -0.6 [d] -77.5 145.8 176.0 (37.5, 75.8) 5.9 e 7.3 Ac- β cAmp-NHMe -γL -174.1 [d] -83.6 78.6 -175.5 (39.8,118.8) 0.0 0.5 g - αL -170.5 [d] -88.7 -7.5 173.9 (39.2, -112.5) 4.0 h 4.5 c-αL 10.4 [d] -84.3 -17.4 -177.2 (36.2, 104.0) 4.3 i 4.8 c- εL -3.0 [d] -74.5 172.9 177.8 (39.3, -126) 5.0 j 5.5 c-αL 8.2 [u] -96.2 -0.7 178.6 (42.8, -122.2 ) 5.6 k 6.1 - αL -170.8 [u] -70.5 -19.3 175.2 (40.2, 78.1) 8.6 l 9.1 Ac-γ Amp-NHMe -γL -170.8 [u] -83.7 75.2 -176.6 (37.3, 102.6) 0.0 m 1.3 n c-α L 10.5 [d] -91.6 -3.9 -179.9 (37.8, -112.5) 2.8 o 4.1 c-εL -0.8 [u] -62.5 147.7 175.7 (39.3, 93.1) 5.9 p 7.2 Ac-γcAmp-NHMe 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 92 -γL -172.8 [d] -82.9 76.6 -176.0 (32.73,-116.5) 0.0 q 0.0 -γ L -174.5 [u] -81.8 77.4 -176.2 (37.8, 109.3) 1.5 s 1.5 c-αL 8.5 [d] -68.5 -47.6 -176.4 (34.1, -93.2 ) 2.7 2.7 c-α L 8.0 [u] -79.0 -18.9 -177.6 (37.2, 89.4) 5.2 u 5.2 -αL -170.9 [u] -79.3 -9.2 175.5 (37.9, 93) 5.7 5.7 c-εL 3.8 [d] -70.8 148.6 174.6 (35.4, -104.1) 6.0 w 6.0 c-εL 0.9 [u] -62.5 148.2 176.9 (39, 89.3 ) 7.9 x 7.9 a χ0= -17.5º, χ1= 33.5º, χ2= -38.0º, χ3= 27.4º and χ4= -6.0º. b E= -628.667446 a.u. c χ0= -1.4º, χ1= -21.6º, χ2= 35.7º, χ3= -36.2º and χ4= 23.9º. d χ0= 5.1º, χ1= - 26.01º, χ2= 37.3º, χ3= -33.9º and χ4= 18.1º. e χ0= -10.3º, χ1= -13.4º, χ2= 31.0º, χ3= -36.6º and χ4= 29.8º. χ0= -18.7º, χ1= 34.5º, χ2= -38.3º, χ3= 27.0º and χ4= -5.0o. g E= -628.668896 a.u. h χ0= -15.0º, χ1= 32.9º, χ2= -39.2º, χ3= 30.2º and χ4= -9.2º. i χ0= -8.8º, χ1= 27.5º, χ2= -36.3º, χ3= 30.7º and χ4= -13.7º. j χ0= -23.1º, χ1= 36.5º, χ2= -37.4º, χ3= 23.8º and χ4= -0.1º. k χ0= -22.8º, χ1= 39.0º, χ2= -41.6º, χ3= 28.0º and χ4= -3.0º. l χ0= 8.3º, χ1= -29.5º, χ2= 39.9º, χ3= -35.0º and χ4= 16.7º. m χ0= - 8.2º, χ1= -15.5º, χ2= 31.9º, χ3= -36.3º and χ4= 28.4º. n E= -628.667515 a.u. o χ0= - 14.4º, χ1= 31.0º, χ2= -37.7º, χ3= 28.7º and χ4= -9.1º. p χ0= -2.1º, χ1= -21.9º, χ2= 36.6º, χ3= -37.1º and χ4= 25.1º. q χ0=-14.6º, χ1= 28.9º, χ2= -32.4º, χ3= 23.1º and χ4= -5.6º. E= -628.669674 a.u. s χ0= -12.5º, χ1= -11.8º, χ2= 29.9º, χ3= -36.8º and χ4= 31.3º. χ0= -1.9º, χ1= 21.8º, χ2= -33.0º, χ3= 31.1º and χ4= -18.7º. u χ0= 0.4º, χ1= -22.5º, χ2= 35.4º, χ3= -34.6º and χ4= 21.9º. χ0= -2.0º, χ1= -21.2º, χ2= 35.2º, χ3= -35.9º and χ4= 24.1º. w χ0= -8.6º, χ1= 27.4º, χ2= -35.3º, χ3= 29.6º and χ4= - 13.4º. x χ0= 0.5º, χ1= -23.7º, χ2= 37.2º, χ3= -36.3º and χ4= 22.8º. 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 99 (Figu e 3.3.4c) o Ac-β Amp-NHMe, Ac-γ Amp-NHMe and Ac-γcAmp-NHMe, espec i ely. The only excep ion o his beha iou was o Ac-βcAmp-NHMe, in which he lowes ene gy minimum co esponds o he -γL[d] con o ma ion (Figu e 3.3.3c). Howe e , i should be no ed ha in his case he -αL[d] and c- εL[d] s uc u es a e des abilized by only 0.2 and 0.3 kcal/mol, espec i ely. On he o he hand, he ∆GCHCl3 2.084 Å 149.5 º 2.289 Å 118.4 º (a) (b) (c) (d) (e) 2.084 Å 149.5 º 2.289 Å 118.4 º (a) (b) (c) (d) (e) alue o he leas s able con o me is 2.5, 2.9, 0.5 and 2.3 kcal/mol o Ac-β Amp-NHMe, Ac-βcAmp-NHMe, Ac-γ Amp-NHMe and Ac- γcAmp-NHMe, espec i ely, sugges ing ha chlo o o m induces a s ong s abilizing e ec in all he s uc u es. Figu e 3.3.4: Rep esen a ion o selec ed minimum ene gy con o ma ions cha ac e ized o he Amp-con aining dipep ides s udied in his wo k: (a) c- ε L[d] o Ac- β Amp-NHMe; (b) c- ε L[u] o Ac- γ Amp-NHMe; (c) c- α L[d] o Ac- γ cAmp-NHMe; (d) c- ε L[d] o Ac- β cAmp-NHMe; (e) c- ε L [u] o Ac- γ Amp- NHMe. These minima a e especially ele an because hey a e ela i ely s able in chlo o o m, me hanol and/o aqueous solu ion. The c-εL is he mos s able con o ma ion in bo h me hanol and aqueous solu ions o all he Amp-con aining dipep ides, he only di e ence be ween hem being he pucke ing o he ing. Thus, he wo β-amina ed dipep ides p e e a 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 100 down pucke ing, while he ing is a anged up when he subs i uen is in oduced a he γ-ca bon a om. The con o ma ional cha ac e is ics o hese s uc u es a e displayed in Figu e 3.3.4. Howe e , he mos ema kable esul in pola en i onmen s is he des abiliza ion o he emaining s uc u es, especially hose wi h ω 0 a anged in ans. This ea u e is ully consis en wi h heo e ical es ima ions p e iously epo ed o he P o dipep ide.3 Al hough he s abili y o he cis con o me s in solu ion was ound o be o e es ima ed by PCM o P o de i a i es, especially in p o ic sol en s able o o m speci ic hyd ogen bonds wi h he solu e, he gene al endencies p o ided by his heo e ical me hod desc ibe e y sa is ac o ily he expe imen al obse a ions om a quali a i e poin o iew. Thus, i was ound ha he elec onic e ec ha s abilize he cis o m o he pep ide bond become enhanced in pola en i onmen s, e en hough he cis/ ans o a ional ba ie s inc ease wi h he pola i y o he sol en . 12 Thus, in a ecen s udy PCM calcula ions p edic ed ha ω 0 exhibi s a conside ably smalle p obabili y o adop ing a cis disposi ion in α-me hylp oline and α-phenylp oline han in P o,12b which was in good ag eemen wi h expe imen al in o ma ion.29 Compa ison o he esul s p o ided in Table 3.3.2 o Amp-con aining dipep ides wi h hose epo ed o Ac-P o-NHMe a he same heo e ical le el sugges s ha he inco po a ion o he subs i uen o he py olidine ing enhances, in gene al, he s abili y o he con o me s wi h ω 0 a anged in cis. Thus, al hough he c-εL[u] con o ma ion was p edic ed as he mos a o ed o Ac-P o-NHMe in bo h chlo o o m and wa e , he lowes ene gy s uc u e wi h ω 0 in ans was un a o ed by only 0.3 kcal/mol ( -γL[d]) and -αL[u], espec i ely).12b Table 3.3.3 shows he ee ene gies ela i e o he lowes ene gy minimum o he mos s able Amp-con aining isome o each en i onmen . As can be seen he mos a o ed isome in he gas-phase, chlo o o m, me hanol and wa e solu ions is he Ac-γcAmp-NHMe dipep ide, e en hough as e lec ed in Table 3.3.2, he p e e ed con o ma ion depends on he pola i y o he en i onmen . Mo eo e , in he gas-phase he mos s able con o ma ion o each isome shows ∆G Table 3.3.2 illus a es ha his ene gy di e ence is highe o he in es iga ed Amp-con aining dipep ides. Howe e , cau ion is equi ed in he analysis o PCM esul s, especially when p o ic sol en s able o o m speci ic solu e-sol en in e ac ions a e conside ed. #gp#< 1.5 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 101 kcal/mol indica ing ha he ela i e s abili y o he o he h ee dipep ides is s ill signi ican . Howe e , in chlo o o m, me hanol and aqueous solu ions only one isome , he Ac-β Amp-NHMe dipep ide, sa is ies such condi ion. These esul s clea ly indica e ha he s abili y o Ac-βcAmp-NHMe and Ac-γ Amp-NHMe dec eases wi h he pola i y o he en i onmen . Finally, i should be no ed ha he Ac-γ Amp-NHMe is he lowes ene gy isome in ca bon e achlo ide solu ion. Acco dingly, i can be concluded ha Ac-β Amp-NHMe is s abilized by he a o able elec os a ic in e ac ions be ween he solu e and he sol en , while Ac- γ Amp-NHMe is p e e ed in non-pola o ganic sol en whe e solu e-sol en in e ac ions a e domina ed by non-elec os a ic e ms, i.e. an de Waals and ca i a ion. 3.3.3.2 Dime ylaminop oline (Dmp) dipep ides Resul s p o ided by B3LYP/6-31+G(d,p) calcula ions o he ou Dmp- con aining dipep ides (Figu e 3.3.1) a e epo ed in Tables 3.3.4, 3.3.5 and 3.3.6, a omis ic pic u es o he mo e ele an minima being displayed in Figu es 3.3.5 and 3.3.6. The ∆Egp alues displayed in Table 3.3.4 indica e ha he con o ma ional p e e ences o he Dmp-con aining dipep ides a e comple ely di e en om hose desc ibed in he p e ious sec ion o he Amp-con aining ones. Se en minimum ene gy con o ma ions, including hose wi h he pep ide bond ω 0 a anged in cis, we e ob ained o Ac-β Dmp-NHMe, while ou we e ound o Ac-β Amp- NHMe. The only s uc u es de ec ed o he o me dipep ide below a ela i e ene gy h eshold alue o 1.5 kcal/mol we e he -γL[d] and -γL[u] (Figu es 3.3.5a and 3.3.5b), which a e almos isoene ge ic and p esen a se en-membe ed hyd ogen bonded ing. This ep esen s ano he impo an di e ence wi h espec o Ac-β Amp-NHMe, since o his compound he only s uc u e wi h ∆Egp < 1.5 kcal/mol was he -γL[d]. The ela i e s abili y o he c-εL[u] con o ma ion (Figu e 3.3.5c), which is he mos s able s uc u e wi h ω 0 in cis, wi h espec o he global minimum is simila o bo h Ac-β Dmp-NHMe and Ac-β Amp-NHMe. This is a su p ising ea u e since in he o me dipep ide, his con o ma ion p esen s a s abilizing hyd ogen bond be ween he N-H o he NHMe blocking g oup and he ni ogen o he dime hylamino subs i uen ha was no de ec ed in he la e . 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 102 Fu he mo e, ema kable di e ences appea in he ∆Egp o he minima wi h c-αL Table 3.3.4 Backbone dihed al angles (in deg ees), pseudo o a ional pa ame e s (A and P; in deg ees), ela i e ene gy ( ∆ E backbone con o ma ion. Thus, hese a e mo e s able in Ac-β Dmp-NHMe han in he co esponding β Amp-con aining analogue by abou 3 kcal/mol. gp; in kcal/mol) and ela i e ene gy wi h espec o he lowes ene gy con o ma ion o he mos s able dipep ide ( ∆ E#gp# # Con . ; in kcal/mol) o he minimum ene gy con o ma ions cha ac e ized o Ac- β Dmp-NHMe, Ac- β cDmp-NHMe, Ac- γ Dmp-NHMe and Ac- γ cDmp-NHMe a he B3LYP/6-31+G(d,p) le el in he gas phase. ω ϕ 0 ψ ω (A, P) ∆ E ∆ E gp #gp# Ac-β Dmp-NHMe -γ L -170.6 [d] -85.0 72.0 -176.9 (40.5, -110.2) 0.0 a 2.3 b -γL -175.1 [u] -81.5 78.8 -175.8 (37.5, 110.2) 0.2 c 2.5 c-ε L 0.1 [u] -67.4 179.9 176.0 (38.7, 95.0) 2.9 d 5.2 c-αL 12.0 [d] -94.1 -4.9 179.3 (39.4, -111.2) 3.2 e 5.5 c-α L 3.9 [u] -84.1 -17.2 179.8 (35.6, 113.7) 3.4 5.7 c-εL 3.6 [d] -79.6 141.4 177.8 (39.1, -111.9) 5.0 g 7.3 c-ε L -0.2 [u] -74.8 120.7 -178.5 (38.4, 106.5) 6.3 h 8.6 Ac-β c Dmp-NHMe -γL 179.1 [d] -77.8 122.0 -174.1 (39.2, -123.2 ) 0.0 i 4.0 j c-εL 0.9 [d] -87.3 -137.3 -177.6 (43.1, -134.6) 1.6 k 5.6 -αL -171.8 [d] -81.7 -23.9 174.4 (39.6, -106.7) 1.7 l 5.7 -εL 175.7 [d] -92.9 -159.7 180.0 (46.2, -142.2) 1.8 m 5.8 c-αL 7.6 [d] -83.7 -27.2 178.0 (38.2, -110.9) 2.0 n 6.0 -εL 179.5 [u] -65.1 143.8 -176.5 (32.5, 99.3) 4.2 o 8.2 c-εL -1.5 [d] -81.8 138.3 174.1 (40.6, -125.2) 4.4 p 8.4 -αL -172.1 [u] -65.9 -26.9 175.9 (42.6, 75.3) 5.6 q 9.6 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 103 c-αL 9.8 [u] -66.0 -32.0 -175.4 (41.8, 76.2) 5.6 9.6 c-ε L 2.3 [u] -62.0 156.9 171.6 (31.8, 88.3) 6.6 s 10.6 Ac-γ Dmp-NHMe -γL -171.6 [d] -83.9 69.5 -178.1 (37.1, -109.5) 0.0 0.0 u -γL -171.0 [u] -83.1 75.7 -176.5 (40.3, 98.3) 3.1 3.1 c-αL 8.9 [u] -89.4 -4.5 -179.3 (37.2, -111.4) 3.4 w 3.4 c-εL 1.0 [d] -73.1 148.6 -175.9 (36.1, -112.2) 6.5 x 6.5 -αL -168.1 [u] -81.0 -6.9 175.3 (40.8, 87.9) 6.7 y 6.7 c-εL 4.1 [u] -65.0 143.8 177.7 (41.8, 76.2) 8.0 z 8.0 Ac-γcDmp-NHMe -γL -173.6 [u] -81.5 74.5 -176.9 (38.1, 105.2) 0.0 aa 1.3 bb -γL -171.1 [d] -79.2 47.8 175.4 (36.7, -105.2 ) 2.0 cc 3.3 c-αL 8.6 [u] -79.0 -18.8 -176.1 (37.1, 89.1) 2.7 dd 4.0 c-αL 7.8 [d] -67.5 -44.5 -176.3 (38.1, -92.7) 3.2 ee 4.5 -αL -170.8 [u] -77.5 -11.3 -176.0 (37.5, 88.6) 3.5 4.8 c-αL 9.7 [d] -90.8 3.7 -175.5 (38.1, -111.7) 3.6 gg 4.9 c-εL 1.1 [u] -62.1 147.5 177.0 (37.4, 87.6) 5.5 hh 6.8 c-εL -0.1 [d] -76.4 -178.8 178.6 (40.8, -117.9) 8.9 ii 10.2 a χ0= -14.0º, χ1= 32.9º, χ2= -40.5º, χ3= 32.3º and χ4= -11.3º. bE= -707.281408 a.u. c χ0= -13.0º, χ1= -10.8º, χ2= 29.3º, χ3= -36.7º and χ4= 31.6º. d χ0= -3.4º, χ1= - 20.3º, χ2= 35.4º, χ3= -37.0º and χ4= 25.8º. e χ0= -14.3º, χ1= 32.4º, χ2= -39.3º, χ3= 31.1º and χ4= -10.3º. χ0= -14.3º, χ1= -8.1º, χ2= 26.5º, χ3= -34.7º and χ4= 30.9º. g χ0= -14.6º, χ1= 32.3º, χ2= -39.0º, χ3= 30.6º and χ4= -9.7º. h χ0= -10.9º, χ1= -13.3º, χ2= 31.6º, χ3= -37.7º and χ4= 30.7º. i χ0= -21.4º, χ1= 35.9º, χ2= -37.9º, χ3= 25.1º and χ4= -2.1º. j E= -707.278763 a.u. k χ0= -30.3º, χ1= 41.5º, χ2= -38.6º, χ3= 20.8º and χ4= 6.4º. l χ0= -11.4º, χ1= 31.2º, χ2= -39.8º, χ3= 32.7º and χ4= -13.2º. m χ0= - 36.5º, χ1= 44.8º, χ2= -38.0º, χ3= 17.0º and χ4= 12.7º. n χ0= -13.6º, χ1= 31.6º, χ2= - 38.3º, χ3= 30.0º and χ4= -10.0º. o χ0= -5.3º, χ1= -14.8º, χ2= 28.8º, χ3= -31.8º and χ4= 23.4º. p χ0= -23.4º, χ1= 37.6º, χ2= -38.8º, χ3= 25.0º and χ4= -0.7º. q χ0= 10.8º, χ1= -32.3º, χ2= 42.4º, χ3= -36.4º and χ4= 15.6º. χ0= 10.0º, χ1= -31.3º, χ2= 41.7º, 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 104 χ3= -36.2º and χ4= 15.9º. s χ0= 0.9º, χ1= -19.3º, χ2= 30.4º, χ3= -29.9º and χ4= 18.2º. χ0= -12.4º, χ1= 30.6º, χ2= -37.2º, χ3= 29.2º and χ4= -10.7º. u E=- 707.285136 a.u. χ0= -5.8º, χ1= -19.6º, χ2= 35.8º, χ3= -38.6º and χ4= 28.6º. w χ0= -13.6º, χ1= 31.2º, χ2= -37.1º, χ3= 28.6º and χ4= -9.6º. x χ0= -13.7º, χ1= 30.6º, χ2= - 36.0º, χ3= 27.5º and χ4= -8.8º. y χ0= 1.5º, χ1= -25.8º, χ2= 38.9º, χ3= -37.5º and χ4= 23.2º. z χ0= 10.0º, χ1= -31.3º, χ2= 41.7º, χ3= -36.2º and χ4= 15.9º. aa χ0= -10.0º, χ1= -14.3º, χ2= 31.7º, χ3= -37.1º and χ4= 30.0º. bb E= -707.283071 a.u. cc χ0= - 9.6º, χ1= 29.01º, χ2= -36.7º, χ3= 30.1º and χ4= -13.3º. dd χ0= 0.6º, χ1= -22.7º, χ2= 35.3º, χ3= -34.5º and χ4= 21.6º. ee χ0= -1.8º, χ1= 24.4º, χ2= -36.6º, χ3= 34.7º and χ4= -21.4º. χ0= 0.9º, χ1= -23.2º, χ2= 35.8º, χ3= -34.8º and χ4= 21.6º. gg χ0= - 14.1º, χ1= 32.4º, χ2= -38.0º, χ3= 29.0º and χ4= -9.6º. hh χ0= 1.6º, χ1= -23.7º, χ2= 36.0º, χ3= -34.4º and χ4= 21.0º. ii χ0= -19.0º, χ1= 36.4º, χ2= -40.0º, χ3= 28.5º and χ4 = -6.1 º 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 105 2.337 Å 107.3 º 2.297 Å 108.3 º 1.975 Å 146.3 º 2.011 Å 142.6 º 2.929 Å 108.3 º 2.173 Å 127.8 º 1.968 Å 146.5 º 1.963 Å 145.0 º (a) (b) (c) (d) (e) ( ) (g) (h) 2.277 Å 134.0 º 1.949 Å 140.8 º (i) 2.337 Å 107.3 º 2.297 Å 108.3 º 1.975 Å 146.3 º 2.011 Å 142.6 º 2.929 Å 108.3 º 2.173 Å 127.8 º 1.968 Å 146.5 º 1.963 Å 145.0 º (a) (b) (c) (d) (e) ( ) (g) (h) 2.277 Å 134.0 º 1.949 Å 140.8 º (i) Figu e 3.3.5: Rep esen a ion o selec ed minimum ene gy con o ma ions cha ac e ized in he gas-phase o he Dmp-con aining dipep ides s udied in his wo k: (a) - γ L[d], (b) - γ L[u] and (c) c- ε L[u] o Ac- β Dmp-NHMe; (d) - γ L[d] and (e) c- ε L[d] o Ac- β cDmp-NHMe; ( ) - γ L[d] and (g) c- α L[u] o Ac- γ Dmp-NHMe; (h) - γ L[u] and (i) c- α L [u] o Ac- γ cDmp-NH. A o al o 10 minimum ene gy con o ma ions we e cha ac e ized o Ac- βcDmp-NHMe, 5 o each a angemen o ω 0. Su p isingly, he lowes ene gy con o ma ion co esponds o a -γL[d] wi h he dihed al angles ϕ and ψ signi ican ly dis o ed owa ds hose o a con en ional -εL (Figu e 3.3.5d). As indica ed by he co esponding geome ic pa ame e s, i.e. dH···O= 2.929 Å and 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 106 ∠N-H···O= 108.3º, his s uc u e is s abilized by a e y weak in amolecula in e ac ion ha de ines a se en-membe ed hyd ogen bonded ing be ween he N- H o NHMe and he C=O o he Ac. Indeed, a s anda d -γL con o ma ion wi h a s ong in amolecula hyd ogen bond o ming a se en-membe ed ing is no possible o he Ac-βcDmp-NHMe dipep ide. This is because such combina ion o ϕ and ψ dihed al angles leads o a s ong epulsi e in e ac ion be ween he lone pai o he dime hylamine g oup and he ca bonyl oxygen o he Dmp esidue. The ∆Egp o he o he ou con o ma ions wi h ω 0 a anged in ans anges om 1.7 ( - αL[d]) o 5.6 ( -αL[u]) kcal/mol, hese ene gy alues being signi ican ly lowe han hose ound o Ac-βcAmp-NHMe, i.e. in he la e dipep ide he ∆Egp o he i s ( -αL[d]) and he las ( -αL[u]) local minimum we e 4.5 and 9.1 kcal/mol, espec i ely. On he o he hand, he c-εL[d] is he lowes ene gy con o ma ion wi h ω 0 a anged in cis, his s uc u e being des abilized wi h espec o he global minimum by 1.6 kcal/mol. Figu e 3.3.5e e eals ha his con o ma ion is s abilized by an in amolecula hyd ogen bond be ween he N-H o he NHMe blocking g oup and he ni ogen o he dime hylamino subs i uen . Compa ison wi h he esul s lis ed in Table 3.3.1 o Ac-βcAmp-NHMe indica es ha he eplacemen o he amino subs i uen by he dime hylamino g oup also al e s he po en ial ene gy hype su ace o he dipep ide wi h he pep ide bond a anged in cis. Thus, he leas a o ed cis minimum o Ac-βcDmp-NHMe (c-εL[u]) is des abilized by 5.0 kcal/mol wi h espec o ha o c-εL[d], whe eas in Ac- βcAmp-NHMe he mos (c-αL[d]) and he leas (c-αL[u]) s able con o ma ions wi h ω 0 Six minimum ene gy con o ma ions, h ee wi h ω a anged in cis a e sepa a ed by only 1.3 kcal/mol. 0 a anged in ans, we e ound o Ac-γ Dmp-NHMe. The lowes ene gy one co esponds o he -γL[d] (Figu e 3.3.5 ), he -γL[u], which was he global minimum o Ac-γ Amp-NHMe, being un a o ed by 3.1 kcal/mol. In e es ingly, he lowes ene gy con o ma ion and he -αL[u], which is des abilized by 6.7 kcal/mol, we e no ound as ene gy minima in Ac-γ Amp-NHMe. Rega ding he h ee con o ma ions wi h ω 0 in cis, he mos s able, c-αL[u] (Figu e 3.3.5g), is s abilized by a i e-membe ed in amolecula hyd ogen bonded ing in ol ing he backbone ni ogen a om o he γ Dmp esidue and he N-H o he NHMe blocking g oup. This s uc u e is 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 107 un a o ed by 3.4 kcal/mol wi h espec o he global minimum, while he ∆Egp alues o he o he wo cis con o me s a e 6.5 (c-εL[d]) o 8.0 kcal/mol (c-εL Eigh minimum ene gy con o ma ions we e cha ac e ized o Ac-γcDmp- NHMe. The lowes ene gy one co esponds o he -γ [u]). L[u] (Figu e 3.3.5h), while he o he s uc u es wi h ω 0 in ans, -γL[d] and -αL[u], a e un a o ed by 2.0 and 3.0 kcal/mol, espec i ely. These h ee con o ma ions a e s abilized by an in amolecula hyd ogen bond. Thus, a se en-membe ed hyd ogen bonded ing in ol ing he NHMe and Ac blocking g oups is shown by he wo -γL s uc u es, whe eas in he -αL[u] con o ma ion he ni ogen o he γcDmp esidue and he N- H o he NHMe g oup o ms a i e-membe ed in amolecula hyd ogen bonded ing. Compa ison wi h he minima lis ed in Table 3.3.1 o Ac-γcAmp-NHMe indica es ha he inco po a ion o he me hyl g oups in o he amino subs i uen mainly a ec s o he pucke ing o he py olidine ing, i.e. he ela i e s abili y be ween -γL[u] and -γL[d] is exchanged and he -αL[u] minimum ans o m in o he -αL On he o he hand, ω [d]. 0 is a anged in cis in he emaining i e minima o Ac- γcDmp-NHMe, he mos s able cis s uc u e being he c-αL[u] (Figu e 3.3.5i). This con o ma ion is 2.7 kcal/mol less s able han he global minimum, and is s abilized by an in amolecula hyd ogen bond simila o ha desc ibed o he - αL[u] minimum. In e es ingly, he Ac-γcDmp-NHMe dipep ide shows wo minima wi h c-αL[d] con o ma ion, which di e in he ni ogen a om ha ac s as accep o in he s abilizing in amolecula hyd ogen bond. In he mos a o ed con o ma ion ha is 3.2 kcal/mol less s able han he global minimum, he ni ogen o he γcDmp esidue pa icipa es in such in e ac ion, whe eas he in e ac ion in he second con o ma ion which is 0.4 kcal/mol less a o ed han he i s one, in ol es he ni ogen o he dime hylamino side g oup. Finally, he ∆Egp alues o he c-εL[u] and c-εL The ∆G [d] s uc u es a e 5.5 and 8.9 kcal/mol, espec i ely. gp alues lis ed in Table 3.3.5 show ha he e ec s p oduced by he inco po a ion o he ZPVE and he he mal and en opic co ec ions a e less d ama ic o Dmp-con aining dipep ides han o he Amp ones. Thus, he addi ion o hese s a is ical e ms ep esen s ela i e a ia ions ypically smalle han 1 kcal/mol. Speci ically, he la ges change ound in Ac-β Dmp-NHMe, Ac- 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 108 βcDmp-NHMe, Ac-γ Dmp-NHMe and Ac-γcDmp-NHMe, is -0.9 (c-εL[u]), -1.1 (c-αL[d]), -1.3 (c-εL[u]) and -1.4 kcal/mol (c-εL[u]), espec i ely. Inspec ion o he ela i e ee ene gies in ca bon e achlo ide, also displayed in Table 3.3.5, indica es ha in gene al, solu e-sol en in e ac ions end o s abilize he minimum ene gy con o ma ions wi h ω 0 Table 3.3.5 Rela i e ee ene gy in he gas-phase ( ∆ G in cis. In spi e o his, he lowes ene gy con o ma ion in ca bon e achlo ide solu ion and in he gas-phase is he same o he ou Dmp-con aining dipep ides. This is an impo an di e ence wi h espec o he ou Amp-con aining dipep ides since, as we p e iously showed, his o ganic sol en is able o al e he con o ma ional p e e ences o Ac-βcAmp- NHMe and Ac-γcAmp-NHMe (Table 3.3.2). gp; in kcal/mol) and in ca bon e achlo ide, chlo o o m. me hanol and aqueous solu ions ( ∆ GCCl4, ∆ GCHCl3, ∆ GCH3OH and ∆ GH2O # Con . , espec i ely; in kcal/mol) o he minimum ene gy con o ma ions o Ac- β Dmp-NHMe, Ac- β cDmp-NHMe, Ac- γ Dmp-NHMe and Ac- γ cDmp-NHMe a he B3LYP/6-31+G(d,p) le el. ∆ G ∆ G gp ∆ G CCl4 ∆ G CHCl3 ∆ G CH3OH H2O Ac-β Dmp-NHMe -γL 0.0 [d] 0.0 a 0.4 3.0 4.0 -γL 0.4 [u] 0.6 0.8 3.0 4.0 c-εL 2.6 [u] 1.0 0.0 0.7 1.4 c-αL 2.6 [d] 1.8 1.4 0.7 3.0 c-αL 2.7 [u] 1.7 1.6 3.1 3.5 c-εL 5.3 [d] 3.4 4.3 0.5 0.6 c-εL 5.4 [u] 3.6 1.0 0.0 0.0 Ac-β c Dmp-NHMe -γL 0.0 [d] 0.0 b 0.0 0.4 1.6 c-εL 0.8 [d] 0.2 0.2 0.7 1.9 -αL 2.6 [d] 2.4 2.0 1.5 2.2 -εL 2.4 [d] 1.7 1.2 0.8 2.4 c-αL 0.9 [d] 1.1 1.5 2.1 2.9 -εL 4.8 [u] 4.0 3.4 2.7 3.8 c-εL 2.9 [d] 2.0 1. 2 0.0 0.4 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 115 9. 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J. Chem. Phys. 1993, 98, 1372. 20. Lee, C.; Yang, W.; Pa , R. G. Phys. Re . B 1993, 37, 785. 21. McLean, A. D.; Chandle , G. S. J. Chem. Phys. 1980, 72, 5639. 22. (a) Alemán, C.; Jiménez, A. I.; Ca i iela, C.; Pé ez, J. J.; Casano as, J. J. Phys. Chem. B 2002, 106, 11849. (b) Casano as, J.; Zanuy, D.; Nussino , R.; Alemán, C. Chem. Phys. Le . 2006, 429, 528. (c) Casano as, J.; Jiménez, A. I.; Ca i iela, C.; Pé ez, J. J.; Alemán, C. J. Phys. Chem. B 2006, 110, 5762. 3.3 CONFORMATIONAL PREFRENCES OF β-AND-γ ANIMATED PROLINE ANALOGUES 117 23. (a) Tomasi, J.; Mennucci, B.; Cammi, R. Chem. Re . 2005, 105, 2999. (b) Tomasi, J.; Pe sico, M. Chem. Re . 1994, 94, 2027. (c) Mie us, S.; Tomasi, J. Chem. Phys. 1982, 65, 239. (d) Mie us, M.; Sc occo, E.; Tomasi, J. Chem. Phys. 1981, 55, 117. 24. Pe czel, A.; Angyan, J. G.; Kaj a , M.; Vi iani, W.; Ri ail, J.-L.; Ma coccia, J.-F.; Csizmadia, I. G. J. Am. Chem. Soc. 1991, 113, 6256. 25. (a) Hudáky, I.; Pe czel, A. J. Mol. S uc . (THEOCHEM) 2003, 630, 135. (b) Hudáky, I.; Baldoni, H. A.; Pe czel, A. J. Mol. S uc . (THEOCHEM) 2002, 582, 233. 26. (a) I iba en, J. I.; Casano as, J.; Zanuy, D.; Alemán, C. Chem. Phys. 2004, 302, 77. (b) Jang, Y. H.; Godda d III, W. A.; Noyes, K. T.; Sowe s, L. C.; Hwang, S.; Chung, D. S. J. Phys. Chem. B 2003, 107, 344. (c) Hawkins, G. D.; C ame , C. J.; T uhla , D. G. J. Chem. Phys. B 1998, 102, 3257. (d) O ozco, M.; Luque, F. J. J. Am. Chem. Soc. 1995, 117, 1378. (e) Alemán, C.; Na a o, E.; Puiggalí, J. J. O g. Chem. 1995, 60, 6135. 27. (a) Ass eld, X.; Ruiz-Lopez, M. F.; González, J.; Lopez, R.; So do, J. A.; Tomas, T. L. J. Comp. Chem. 1994, 15, 479. (b) Ass eld, X.; Ruiz-Lopez, M. F.; Ga cía, J. I.; Mayo al, J. A.; Sal a ella, L. J. Chem. Soc. Chem. Comm. 1995, 13, 1371. 28. Delaney, N. G.; Madison, V. J. Am. Chem. Soc. 1982, 104, 6635. 119 3.4 P o ona ion o he side g oup in β−and γ− Amina ed P oline Analogues: E ec s on he Con o ma ional P e e ences Densi y Func ional Theo y calcula ions ha e been pe o med on he N- ace yl-N’-me hylamide de i a i es o he ou possible isome s o aminop oline p o ona ed a he amino side g oup. Compa ison o he esul s ob ained o hese isome s, which di e in he β - o γ -posi ion o he subs i uen and i s cis o ans ela i e disposi ion, wi h hose epo ed o he co esponding neu al analogues [J. Phys. Chem. B 2008, 112, 14045] has allowed o each he ollowing conclusions: (i) p o ona ion o he amino g oup p oduces a educ ion o he backbone con o ma ional lexibili y and a des abiliza ion o he cis con igu a ion o he amide bond in ol ing he py olidine ni ogen; (ii) he plana i y o he pep ide bond is b oken in some cases o o m s ong side chain···backbone in e ac ions, which induce a e y signi ican py amidiliza ion a he amide ni ogen a om; (iii) as was also de ec ed o he neu al analogues, he o ma ion o side chain···backbone in a esidue in e ac ions a ou he cis disposi ion o he subs i uen ; and (i ) p o ona ion o he amino side g oup inc eases he ene gy gaps ha sepa a e he di e en in es iga ed isome s, which esul s in an enhancemen o he des abiliza ion o he dipep ides wi h he subs i uen a ached in ans ∗ 3.4.1 In oduc ion . The design and applica ion o syn he ic amino acids wi h es ic ed con o ma ional mobili y in di e en ields o nanobiology, e.g. he e-enginee ing o physical p o ein modules and he gene a ion o nanode ices,1,2 is a opic o g owing in e es . Wi hin his con ex , we ecen ly obse ed ha he inse ion o chemically cons ained esidues wi h sui able backbone con o ma ional endencies enhance he he modynamic s abili y o he nano ubula s uc u es cons uc ed by sel -assembling p o ein agmen s. Among he la ge a ie y o amino acids ha can be designed, hose achie ed by in oducing chemical modi ica ions o P oline (P o) a e pa icula ly a ac i e. This is because he side chain o P o is bonded o bo h he α-ca bon and i s 3 ∗ Submi ed o Publica ion. 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 120 p eceding amide ni ogen p o iding con o ma ional p ope ies ha a e unique among na u ally occu ing amino acids. As a consequence, o a ion abou he N— Cα bond is p ohibi ed and he ϕ o sion angle is con ined o alues o a ound –60º. Acco dingly, P o is o e whelmingly ound in he α-helical ( ϕ , ψ ≈ –60º,–30º) and semi-ex ended ( ϕ , ψ ≈ –60º,140º) egions o he con o ma ional map.4 In addi ion, P o shows a highe p opensi y o p omo e γ- u n con o ma ions ( ϕ , ψ ≈ –70º,60º) han o he p o eogenic amino acids.4d,5 Ano he e ec de i ed om i s cyclic s uc u e is ha he pep ide bond p eceding P o ( ha in ol ing he py olidine ni ogen) has a ela i ely high p obabili y o accommoda ing a cis a angemen 6 as compa ed o o he pep ide bonds, o which he cis o m is almos inexis en . The con o ma ional p ope ies o a ela i e wide numbe o syn he ic P o de i a i es ha e been epo ed. These compounds we e ob ained by inco po a ing a subs i uen a he Cα a om (α-subs i u ed P o analogues)7,8 o in he py olidine ing (e.g. hyd oxyla ed, luo ina ed and amina ed P o analogues),9-11 o al e ing he chemical na u e o he own py olidine ing (e.g. diminishing o enla ging he ing size,12 eplacing a ca bon a om by an he e oa om,13 and inco po a ing a double bond h ough a deshyd ogena ion14). Wi hin his con ex , we ecen ly epo ed he in insic con o ma ional p ope ies o di e en aminop oline (Amp) de i a i es,11 which ha e been al eady used o cons uc β- and γ-pep ides wi h helical seconda y s uc u es.15 Speci ically, we in es iga ed he N-ace yl-N’- me hylamide de i a i es o bo h he cis and ans Amp isome s ha inco po a e an amino g oup o he Cβ- o Cγ In -posi ions o he py olidine ing. Theo e ical calcula ions based on Densi y Func ional Theo y (DFT) me hods on hese ou compounds, which we e deno ed Ac-β Amp-NHMe, Ac-βcAmp-NHMe, Ac- γ Amp-NHMe and Ac-γcAmp-NHMe (Figu e 3.4.1), e idenced ha he inco po a ion o he amino g oup educes he in insically low con o ma ional lexibili y o con en ional P o. Fu he mo e, he s abili y o con o ma ions wi h he pep ide bond in ol ing he py olidine ni ogen a anged in cis was, in gene al, highe o Amp de i a i es han o P o. This was a ibu ed o he o ma ion in such con o ma ions o s able in amolecula hyd ogen bonds wi h he ni ogen o he amino subs i uen ac ing as accep o . Al hough hese esul s sugges ed ha Amp de i a i es ha e po en ial in e es o many nanobiological applica ions, he con o ma ional p ope ies o 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 121 hese amino acids may be easily al e ed by ans o ming he amino g oup, a easonably weak base, in o he posi i ely cha ged ammonium g oup, i.e. amines eac eadily wi h acids. This p o eoli ic equilib ium is especially impo an in aqueous solu ion, in which he pH can be used o con ol he con o ma ion o he Amp de i a i es. Howe e , he con o ma ional p e e ences o he p o ona ed Amp de i a i es, he ea e deno ed Ac-β AmH+p-NHMe, Ac-βcAmH+p-NHMe, Ac-γ AmH+p-NHMe and Ac-γcAmH+ H 3 CN C N H CH 3 OO H 3 CN C N H CH 3 OO R R H 3 CN C N H CH 3 OO R H 3 CN C N H CH 3 OO R Ac-β Amp-NHMe Ac-βcAmp-NHMe Ac-γ Amp-NHMe Ac-γcAmp-NHMe Ac-β AmH + p-NHMe Ac-βcAmH + p-NHMe Ac-γ AmH + p-NHMe Ac-γcAmH + p-NHMe R= NH 2 R= NH 3+ R= NH 2 R= NH 3+ p-NHMe (see Figu e 3.4.1) emain o ally unknown ye . In his wo k we ha e used DFT calcula ions o explo e sys ema ically he po en ial ene gy hype su aces o hese ou dipep ides, he in luence o he sol en being been e alua ed h ough a Sel Consis en Reac ion Field (SCRF) me hod. Fu he mo e, we ha e also examined he in luence o he p o eoli ic equilib ium on bo h he ans/cis disposi ion o he pep ide bond in ol ing he py olidine ni ogen and he ela i e s abili y o he ou isome s gene a ed by he subs i u ion a di e en posi ions. Figu e 3.4.1: Compounds s udied in his wo k 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 122 3.4.2 Me hods All calcula ions we e ca ied ou using he Gaussian 03 compu e p og am.16 DFT calcula ions we e pe o med using he B3LYP me hod17 combined wi h he 6-31+G(d,p) basis se ,18 which was p e iously employed o s udy he N-ace yl-N’-me hylamide de i a i es o con en ional P o7 and Amp.11 To sion angles o he backbone and side chain o he AmH+p de i a i es s udied in his wo k a e de ined in Figu e 3.4.2. Since each lexible backbone dihed al angle is expec ed o ha e h ee minima, i.e. gauche+ (60º), ans (180º) and gauche- (–60º), and ϕ is ixed by he geome y o he i e-membe ed ing, he numbe o minima ha may be an icipa ed o he po en ial ene gy su ace E= E(ψ) o each AmH+p dipep ide is 3. Addi ionally, each amide bond (gi en by he o sional angles ω 0 and ω ) can be a anged in ans o cis, e en hough only he pep ide bond in ol ing he py olidine ni ogen ( ω 0) is likely o adop a cis con igu a ion. The e o e, bo h he ans and cis s a es we e conside ed o ω 0, while he amide bond in ol ing he N-me hylamide blocking g oup ( ω ) was a anged in ans only. Fu he mo e, due o he cyclic na u e o he side chain, wo pucke ing s a es (deno ed down and up) a e expec ed o each backbone minimum ene gy con o ma ion. The down and up a angemen s a e de ined as hose in which one a om o he py olidine ing and he ca bonyl g oup o he AmH+p esidue lie on he same and opposi e sides, espec i ely, o he plane de ined by he emaining ou a oms o he py olidine ing. 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 123 H3CCNCNCH3 OO H ω0ϕψω χ0 χ1 χ2 χ3 χ4 α β γ δ H Figu e 3.4.2: Dihed al angles used o iden i y he con o ma ions o he N- ace yl-N’-me hylamide de i a i es o he AmH+p analogues s udied i n his wo k. The dihed al angles ω0, ϕ, ψ and ω a e de ined using backbone a oms while he endocyclic dihed al angles χ i a e gi en by he a oms o he i e- membe ed ing. In pa icula , he sequence o a oms used o de ine ϕ and χ 0 a e C(=O)–N–C α –C(=O) and C δ –N–C α –C β Acco dingly, o each o he ou dipep ides unde s udy (Figu e 3.4.1), 3( ψ backbone) × 2( ω , espec i ely. 0 ans-o -cis) × 2(cyclic side chain) = 12 s uc u es we e conside ed as s a ing poin s o comple e geome y op imiza ions a he B3LYP/6-31+G(d,p) le el. F equency analyses we e ca ied ou o e i y he na u e o he minimum s a e o all he s a iona y poin s ob ained and o calcula e he ze o-poin ib a ional ene gies (ZPVE) as well as bo h he mal and en opic co ec ions. These s a is ical e ms we e used o e alua e he con o ma ional Gibbs ee ene gies in he gas phase (∆Ggp To examine he sol a ion e ec s on he molecula geome y and con o ma ional s abili y, single poin calcula ions we e conduc ed on he B3LYP/6-31+G(d,p) op imized s uc u es using a sel -consis en eac ion- ield (SCRF) model. SCRF me hods ea he solu e a he quan um mechanical le el, and he sol en is ep esen ed as a dielec ic con inuum. Speci ically, we chose he pola izable con inuum model (PCM) de eloped by Tomasi and co-wo ke s o desc ibe he sol en . ) a 298 K. 19 The PCM ep esen s he pola iza ion o he liquid by a cha ge densi y appea ing on he su ace o he ca i y c ea ed in he sol en , i.e. he solu e/sol en in e ace. This ca i y is buil using a molecula shape algo i hm. 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 124 PCM calcula ions we e pe o med in he amewo k o he B3LYP/6-31+G(d,p) le el using he s anda d p o ocol, and conside ing he dielec ic cons an o wa e (ε= 78.1). The con o ma ional ee ene gies in solu ion (∆GWAT) we e es ima ed by adding he ee ene gies o sol a ion o he ∆Ggp alues. The minimum ene gy con o ma ions o he ou dipep ides s udied in his wo k ha e been deno ed using he same h ee-label code ha was used o he Amp de i a i es,11 which speci ies he a angemen o he ω 0 pep ide bond, he ( ϕ , ψ ) backbone con o ma ion and he pucke ing o he i e-membe ed ing. The i s le e e e s o he ans ( ) o cis (c) a angemen o he pep ide bond in ol ing he py olidine ni ogen. The second label iden i ies he backbone con o ma ion using he nomencla u e in oduced by Pe czel e al.20 mo e han i een yea s ago. In he case o P o and i s de i a i es, only he γL (γ- u n o C7), αL (α-helical), and εL (polyp oline II-like) con o ma ions a e accessible o he backbone since ϕ is ixed in he neighbo hood o –60º. Finally, he up o down pucke ing o he i e-membe ed ing is indica ed using he [u] and [d] labels, espec i ely. The pucke ing o he ing was desc ibed using he classical pseudo o a ional algo i hm, which uses a e y simple model based on he pucke ing ampli ude and he s a e o he pucke in he pseudo o a ion pa hway. This model was p e iously applied by Pe czel e al.21 3.4.3 Resul s and Discussion o desc ibe con en ional P o. Calcula ions a he B3LYP/6-31+G(d,p) le el led o 3, 5, 5 and 3 minimum ene gy con o ma ions cha ac e ized o Ac-β AmH+p-NHMe, Ac-βcAmH+p- NHMe, Ac-γ AmH+p-NHMe and Ac-γcAmH+p-NHMe, espec i ely. Table 3.4.1 lis s he mo e ele an s uc u al pa ame e s oge he wi h he ela i e ene gy (∆Egp) o all hese s uc u es, which a e displayed in Figu es 3.4.3-3.4.6. Table 3.4.1 also shows he ela i e s abili y o he ou dipep ides (∆E#gp#), which co esponds o he ene gy ela i e o he lowes ene gy con o ma ion o he mos s able isome . Table 3.4.2 compa es he ela i e ee ene gies in he gas-phase (∆Ggp) and aqueous solu ion (∆GH2O) o he minima o he ou dipep ides. 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 131 (a) (b) (c) 1.921 Å 147.2º 1.643 Å 145.9º -γ L[d] 1.867 Å 144.7º - γL[u] 1.590 Å 147.9º c-ε L [u] 1.693 Å 146.1º (d) (e) Figu e 3.4.4: Rep esen a ion o he minimum ene gy con o ma ions cha ac e ized in he gas-phase o he Ac- β cAmH+ p-NHMe dipep ide. The c-αL[d] (Figu e 3.4.4c) is he mos s able minimum o Ac-βcAmH+p- NHMe wi h he pep ide bond ω 0 a anged in cis. Al hough his con o ma ion p esen s a s ong side chain···backbone in a esidue in e ac ion, i is un a o ed by 7.2 kcal/mol wi h espec o he lowes ene gy minimum. These esul s a e ully consis en wi h hose showed abo e o Ac-β AmH+p-NHMe, which indica ed ha he s uc u es wi h ω 0 in cis become s ongly des abilized upon p o ona ion o he amino g oup a ached o he Cβ a om. Thus, o Ac-βcAmp-NHMe he ene gy o he c-αL[d] was highe han ha o he global minimum by 4.3 kcal/mol only. On he o he hand, he o he wo minima ound o Ac-βcAmH+p-NHMe co espond o he c-αL[u] (Figu e 3.4.4d) and c-εL[u] (Figu e 3.4.4e) wi h ∆Egp alues o 10.3 and 11.0 kcal/mol, espec i ely. In e es ingly, he ϕ,ψ alues o he wo minima ha show a αL c-εL[u] 1.693 Å 146.1º con o ma ion a e signi ican ly dis o ed wi h espec o hose expec ed o an ideal con o ma ion. These de o ma ions, which we e also c-αL[u] 1.705 Å 140.5º 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 132 de ec ed in he wo c-αL minima o Ac-β AmH+p-NHMe, a e consequence o he in e ac ion be ween he ammonium g oup and he pep ide bond ω . Inspec ion o he ∆Ggp alues indica es ha he s a is ical co ec ions added o he elec onic ene gies p oduce a ela i e des abiliza ion o he -γL[u], c-αL[d] and c-εL[u] s uc u es, which ange om 0.6 o 2.8 kcal/mol. In opposi ion, he c- αL[u] con o ma ion becomes mo e s able by 1.2 kcal/mol, e en al hough i s popula ion in he gas-phase is negligible. Thus, he only s uc u e o Ac- βcAmH+p-NHMe ha p esen s a signi ican popula ion (97.6%) in he gas-phase is he -γL[d]. Simila ly, his minimum is he only popula ed con o ma ion in aqueous solu ion, he ∆GH2O o he o he ou con o me s anging be ween 3.2 and 3.9 kcal/mol. The ac ha he global minimum in he gas-phase is also he mos a o ed con o ma ion in wa e ep esen s a ema kable di e ence wi h espec o he Ac-βcAmp-NHMe dipep ide. Thus, o he la e sys em he -γL[d] was des abilized by 3.3 kcal/mol in aqueous solu ion, he c-εL[d] becoming he mos a o ed in his pola en i onmen . These esul s a e ully consis en wi h he des abiliza ion o he s uc u es wi h ω 0 a anged in cis discussed abo e o Ac- β AmH+ Ac- γ AmH p-NHMe. +p-NHMe. The con o ma ional p e e ences displayed in Table 3.4.1 o Ac-γ AmH+p-NHMe a e unique. As can be seen, wo almos isoene ge ic minima wi h he pep ide bond ω 0 a anged in a gauche+ con o ma ion (labeled as g+) a e he mos a o ed o his dipep ide. The dis o ion om he plana i y o he pep ide bond in hese s uc u es, g+-δL[u] (Figu es 3.4.5a and 3.4.5b), mus be a ibu ed o he s eng h o he side chain···backbone in e ac ion, which shows a a o able geome y because o he N-exo (NE) con o ma ion o he py olidine ing, i.e. his en elope con o ma ion b eaks he plana disposi ion o he pep ide bond. This dis o ion is e idenced by a no able py amidaliza ion o he amide ni ogen, he sum o he alence angles a ound his a om ( θ ) being 338.5 and 340º o he wo g+-δL[u] minima. This la ge de o ma ion e idences ha he py amidaliza ion o ω 0 in he g+-δL[u] s uc u es is simila , o e en highe , han ha obse ed o he bicyclic amide ni ogen o highly cons ained P o analogues.23 In e es ingly, he wo g+-δL[u] minima only di e in he dihed al angle ψ, which de ines he o ien a ion o pep ide bond ω . The o ien a ion o he 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 133 pola -CONH- moie y in such minima does no a ec o hei in insic s abili y in he gas-phase, i.e. he ∆Ggp alues di e by 0.2 kcal/mol only, e en hough hei ela i e s abili ies in aqueous solu ion a e comple ely di e en . Thus, he s eng h o he solu e···sol en a ac i e in e ac ions inc eases wi h he accessibili y o his pep ide g oup o he sol en , i.e. he g+-δL[u] con o ma ion wi h ψ= 104.6º is a o ed by 11.1 kcal/mol, which explains he di e ence ound in hei ∆GH2O (a) alues (Table 3.4.2). (b) (c) 1.721 Å 156.9º g+-δL[d] 1.724 Å 157.7º g+-δL[d] 2.204 Å 133.6º -γL[d] (d) (e) Figu e 3.4.5: Rep esen a ion o he minimum ene gy con o ma ions cha ac e ized in he gas-phase o he Ac- γ AmH+ p-NHMe dipep ide. The nex wo minima, which co espond o he con en ional -γL[d] (Figu e 3.4.5c) and -γL[u] (Figu e 3.4.5d) con o ma ions, a e des abilized by 2.0 and 2.8 kcal/mol, espec i ely. These s uc u es, in which he wo pep ide bonds adop a plana ans a angemen , a e s abilized by he backbone···backbone in amolecula hyd ogen bond only, no side chain···backbone in e ac ion being de ec ed. This ea u e explains he lowe s abili y o he wo -γL c-ε L [d] con o ma ions 1.724 Å 157.7º g+-δL[d] 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 134 wi h espec o he wo g+-δL minima. Thus, he s ong side chain···backbone in e ac ion o he la e , which is mo e a ac i e han he se en-membe ed in amolecula hyd ogen bond, almos compensa es he ene gy penal y associa ed o he geome ic de o ma ion o he pep ide bond ω 0. Finally, he leas s able con o ma ion, c-εL[d] (Figu e 3.4.5e), which is un a o ed by 4.7 kcal/mol wi h espec o he global minimum, co esponds o he only con o ma ion wi h ω 0 a anged in cis. As can be seen, his s uc u e does no show any N-H···O in amolecula in e ac ion. Compa ison o he ∆Egp and ∆Ggp alues e eals ha , in his case, he in luence o he s a is ical co ec ions is e y small. Thus, wi h excep ion o wo minima o highe ene gy, which become s abilized by 0.4 ( -γL[u]) and 1.8 kcal/mol (c-εL[d]) by he addi ion o he he mal and en opic e ms, he ela i e s abili y o he o he h ee s uc u es emained unal e ed. On he o he hand, he ela i e ee ene gy o de unde goes a d as ic change in aqueous solu ion. Speci ically, he mos s able s uc u e in wa e is he c-εL[d], which was he leas a o ed in he gas-phase. This ea u e is consis en wi h he o e es ima ion o he con o me s wi h ω 0 a anged in cis p e iously a ibu ed o he PCM sol a ion model. Fu he mo e, he lowes ene gy minimum in he gas-phase is he leas a o ed in aqueous solu ion, his ea u e being consequence o he poo in e ac ion be ween he C=O g oup o he pep ide bond ω and he sol en (Figu e 3.4.5a). In con as , he second minimum wi h a dis o ed pep ide bond esul s less a o ed in aqueous solu ion han he c-εL[d] by 1.0 kcal/mol only, which is due o he e y a o able in e ac ions o he pep ide bond ω wi h he en i onmen . Finally, he s abili y in wa e o he wo s uc u es wi h he -γL backbone con o ma ion is simila o ha ob ained in he gas-phase. The o e all o hese esul s indica e ha he con o ma ional popula ions p edic ed o Ac-γ AmH+p-NHMe in aqueous solu ion, conside ing Bol zmann dis ibu ion o he iden i ied minima, a e: 81.7 % c-εL[d], 15.0 % g+-δL[d], and 3.3% -γL Ac- γ cAmH [d]. +p-NHMe. Only h ee minima we e de ec ed o Ac-γcAmH+p- NHMe e idencing ha he p o ona ion o he amino side g oup educes d as ically he con o ma ional lexibili y o neu al Ac-γcAmp-NHMe.11 Thus, se en minima wi h ela i e ene gies o up 7.9 kcal/mol we e ound o he la e dipep ide: h ee 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 135 wi h ω 0 a anged in ans and ou in cis. The lowes ene gy minimum ound o Ac-γcAmH+p-NHMe co esponds o he -γL[d] (Figu e 3.4.6a), which p esen s bo h backbone···backbone and side chain···backbone a o able in e ac ions, he la e being acili a ed by he βE a angemen o he py olidine ing. This con o ma ion was also iden i ied as he mos s able con o ma ion o Ac-γcAmp- NHMe, e en al hough he -γL[u] and -αL (a) [d] minima annihila e when he amino g oup o his dipep ide ans o ms in o ammonium. (b) (c) Figu e 3.4.6: Rep esen a ion o he minimum ene gy con o ma ions cha ac e ized in he gas-phase o he Ac- γ cAmH + p-NHMe dipep ide. The second minimum o Ac-γcAmH+p-NHMe shows a c-εL[d] s uc u e (Figu e 3.4.6b) wi h he py olidine ing a anged like in he global minimum, i.e. βE con o ma ion. This s uc u e, which is un a o ed by 4.3 kcal/mol, is simila o he leas s able minimum o he Ac-γ AmH+p-NHMe dipep ide, e en al hough he cis disposi ion o he subs i uen allows he o ma ion o an a ac i e side chain···backbone in e ac ion ha was no possible in he la e compound. Finally, he las minimum, c-αL[d] (Figu e 3.4.6c), is s ongly des abilized, i.e. ∆Egp= 15.9 kcal/mol. This should be a ibu ed o he simul aneous combina ion o a numbe o ac o s: (i) he cis a angemen o ω 0; (ii) he lack o backbone···backbone in amolecula hyd ogen bond; and, especially, (iii) he na u e o side chain···backbone in e ac ion, which is o he N-H···N ype. Thus, he s abilizing e ec p o ided by he la e in e ac ion is lowe han ha achie ed h ough he N- H···O(=C) one.24 I is wo h no ing ha he c-εL[d] and c-αL[d] minima o Ac- γcAmp-NHMe we e un a o ed by 6.0 and 2.7 kcal/mol,11 c-αL[d] 1.990 Å 158.7 º espec i ely, which 1.662 Å 160.3º c-ε L [d] 2.316 Å 124.4º 1.631 Å 162.8º -γ L [d] 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 136 e lec s he la ge change ha he ioniza ion o he side g oup p oduces in he po en ial ene gy su ace o his dipep ide. The mos signi ican change p oduced by he ans o ma ion o ∆Egp in o ∆Ggp is he des abiliza ion o he c-αL[d] con o ma ion, which inc eases 5.0 kcal/mol. Acco dingly, he -γL[d] is p edic ed o be he only popula ed con o ma ion in he gas-phase. On he o he hand, inspec ion o he ∆GH2O alues indica es again ha he PCM model p oduces a conside able s abiliza ion o he c-εL[d] s uc u e. Thus, he la e con o ma ion becomes he mos a o ed in aqueous solu ion, whe eas he -γL[d] is highe in ene gy by 1.3 kcal/mol, i.e. he popula ions o he c-εL[d] and -γL[d] con o ma ions in aqueous solu ion a e 90.1% and 9.9%, espec i ely. These p e e ences a e signi ican ly di e en om hose epo ed o Ac-γcAmp-NHMe, in which he c-εL[d] was p edic ed o be only con o ma ion wi h a signi ican popula ion in aqueous solu ion,11 Rela i e S abili y o he ou isome s. The ∆E i.e. all he o he minima we e des abilized by mo e han 2 kcal/mol. #gp# and ∆G#gp# alues displayed in Tables 3.4.1 and 3.4.2, espec i ely, indica e ha he Ac-γcAmH+p-NHMe is he mos s able isome , he Ac-βcAmH+p-NHMe being un a o ed by only 1.7 kcal/mol (2.8 kcal/mol in e ms o ∆E#gp#). The s abili y o hese isome s, which is signi ican ly highe han ha o he analogues wi h a ans disposi ion o he cha ged side g oup, should be a ibu ed o he o ma ion o side chain···backbone in e ac ions. Thus, in a esidue in e ac ions a e clea ly s onge when he subs i u ion is a ached in cis. This ea u e is e lec ed by he H···O dis ances displayed in Figu es 3.4.4 and 3.4.6, which a e ∼1.65 Å o he minima o he γcAmH+p- and βcAmH+p-con aining dipep ides. In con as , hese dis ances a e 1.770 and 1.890 Å o he wo minima o Ac-β AmH+p-NHMe ha p esen a side chain···backbone in e ac ion (Figu es 3.4.3a and 3.4.3b), his isome being un a o ed by 7.6 kcal/mol (6.2 kcal/mol in e ms o ∆E#gp#) wi h espec o he mos s able. Simila ly, he alues o he ∠N-H···O angles a e consis en wi h a mo e a o able in e ac ion when he subs i uen is a ached in cis. The Ac- γ AmH+p-NHMe is no able o o m side chain···backbone in e ac ions wi hou induce a ema kable dis o ion o he pep ide bond, which p oduces a signi ican 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 137 ene gy penal y. Consequen ly, his isome is un a o ed by 17.4 kcal/mol (13.1 kcal/mol in e ms o ∆E#gp#) wi h espec o he Ac-γcAmH+p-NHMe dipep ide. The ela i e s abili y o de ob ained o he neu al Amp-con aining dipep ides was iden ical, i.e. Ac-γcAmp-NHMe > Ac-βcAmp-NHMe > Ac-β Amp-NHMe > Ac-γ Amp-NHMe.11 Howe e , in his case he ene gy di e ences among he di e en isome s we e signi ican ly lowe han hose ob ained o he AmH+p de i a i es. Thus, he lowes ∆G#gp# alue o he βcAmp-, β Amp- and γ Amp- con aining dipep ides was 0.6, 1.0 and 1.4 kcal/mol, espec i ely. The ema kable ene ge ic di e ence be ween Amp and AmH+p dipep ides should be a ibu ed o he s eng h o he in a esidue in e ac ion, which is signi ican ly highe when he side g oup is ionized. On he o he hand, ∆G#H2O# alues indica e ha he sol en does no al e he p e e ences by he isome s wi h he ammonium g oup a ached in cis wi h espec o hose in ans. Thus, he Ac-γcAmH+p-NHMe is he mos s able in wa e ollowed by he Ac-βcAmH+p-NHMe, which is un a o ed by 3.6 kcal/mol. Acco dingly, he sol en des abilizes he la e isome 1.9 kcal/mol wi h espec o he gas-phase. Rega ding o he Ac-β Amp-NHMe and Ac-γ Amp-NHMe, hei ela i e s abili ies a e exchanged wi h espec o he gas-phase, he la e being a o ed wi h espec o he o me by 3.2 kcal/mol. Thus, hese isome s a e 5.4 and 7.6 kcal/mol, espec i ely, less s able han he Ac-γcAmH+p-NHMe one. Compa ison wi h he esul s epo ed in aqueous solu ion o he neu al Amp- con aining dipep ides e eals conside able di e ences. Speci ically, he ela i e ene gy o de epo ed o hese dipep ides we e:11 Ac-γcAmp-NHMe ≈ Ac- β Amp-NHMe (∆G#H2O#= 0.2 kcal/mol) > Ac-γ Amp-NHMe (∆G#H2O#= 1.7 kcal/mol) > Ac-βcAmp-NHMe (∆G#H2O#= 5.9 kcal/mol). Acco dingly, he p o ona ion o he amino subs i uen in wa e p oduces a p onounced s abiliza ion o he isome s subs i u ed in cis wi h espec o hose wi h he subs i uen in ans. 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 138 3.4.4 Conclusions DFT calcula ions a he B3LYP/6-31+G(d,p) le el ha e been used o examine he con o ma ional p e e ences o Ac-β AmH+p-NHMe, Ac-βcAmH+p-NHMe, Ac-γ AmH+p-NHMe and Ac-γcAmH+ (i) P o ona ion o he amino g oup a ached o he β- o γ-posi ion o he py olidine ing educes he backbone con o ma ional lexibili y o he Amp de i a i es, which was low compa ed o ha con en ional P o. Speci ically, he numbe o minima iden i ied o he ou AmH p-NHMe, bo h in he gas-phase phase and aqueous solu ion, which ha e been compa ed wi h hose epo ed o he neu al analogues Ac-β Amp-NHMe, Ac-βcAmp-NHMe, Ac-γ Amp-NHMe and Ac- γcAmp-NHMe. Resul s allow us o d aw he ollowing conclusions: + (ii) The s abili y o con o ma ions wi h ω p- con aining dipep ides was smalle han ha ound o hei Amp analogues. Fu he mo e, he ela i e ene gies and ee ene gies inc ease upon p o ona ion o he side g oup. 0 in cis is signi ican ly lowe o AmH+p han o Amp. This is a e y ema kable esul because he inco po a ion o he non-p o ona ed amino g oup o he py olidine ing o con en ional P o p oduced a s abiliza ion o such con o ma ions. Acco dingly, he popula ion o cis con o me s in Amp/AmH+ (iii) The in insic con o ma ional p e e ences o he Ac-γ AmH p de i a i es could be easily con olled wi h he pH. + (i ) The Ac-γcAmH p-NHMe dipep ides show ha he s eng h o he side chain···backbone a ac i e in e ac ion allows compensa e he ene gy penal y associa ed o he de o ma ion o he pep ide bond. Thus, in o de o each such in e ac ions, his compound ends o b eak he plana i y o he pep ide bond inducing a la ge py amidaliza ion o he amide ni ogen a om. +p-NHMe dipep ide, ollowed by he Ac-βcAmH+p- NHMe, a e he mo e s able isome s in bo h he gas-phase and aqueous solu ion. The cis disposi ion o he subs i uen is a o ed because o he a ac i e side chain···backbone in e ac ions. Compa ison, be ween 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 139 Amp and AmH+p de i a i es e eals ha he ene gy gap be ween he di e en isome s inc eases upon ioniza ion o he side g oup. This should be a ibu ed o he s eng h o he in a esidue in e ac ions, which is highe when a posi i ely cha ged ammonium g oup is in ol ed. 3.4 PROTONATION OF THE SIDE GROUP IN β-AND γ-AMINATED PROLINE ANALOGUES: EFFECTS ON THE CONFORMATIONAL PREFERENCES 140 3.4.5 Re e ences 1. Alemán, C.; Zanuy, D.; Jiménez, A. 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