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. (a) Zimme man, S. S.; Po le, M. S.; Neme hy, G.; Sche aga, H. A.
Mac omolecules 1977, 10, 1. (b) Fische , S.; Dunb ack, R. L., J .; Ka plus,
M. J. Am. Chem. Soc. 1994, 116, 11931. (c) Kang, Y. K. J. Phys. Chem.
1996, 100, 11589. (d) Imp o a, R.; Benzi, C.; Ba one, V. J. Am. Chem.
Soc. 2001, 123, 12568. (e) Benzi, C.; Imp o a, R.; Scalmani, G.; Ba one,
V. J. Compu . Chem. 2002, 23, 341. ( ) Kang, Y. K. J. Phys. Chem. B
2002, 106, 2074. (g) Hudáky, I.; Baldoni, H. A.; Pe czel, A. J. Mol. S uc .
(THEOCHEM) 2002, 582, 233. (h) Hudáky, I.; Pe czel, A. J. Mol. S uc .
(THEOCHEM) 2003, 630, 135. (i) Allen, W.D.; Czinki, E.; Császá , A. G.
Chem. Eu . J. 2004, 10, 4512. (j) Kang, Y. K.; Pa k, H. S. J. Mol. S uc .
(Theochem) 2005, 718, 17. (k) Czinki, E.; Csasza , A. G. Chem. Eu . J.
2003, 9, 1008.
10. Sahai, M.A.; Kehoe, A. K.; Koo, J. C. P.; Se iadi, D. H.; Chass, G. A.;
Viskolcz, B.; Penke, B.; Pai, E. F.; Csizmadia, I. G. J. Phys. Chem. A
2005, 109, 2660.
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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
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Nussino , R. P o eins 2007, 68, 1.
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