Análise estrutural e topológica de peptídeos bioativos em meios biomiméticos de membranas

! "

# $

%

&

'

(

(

)

*

/

/

0 1123

%4 5163

% 7

8

7#

9 :

% 7

8

7#

9 :

% 7

8

7#

9 :

% 7

8

7#

9 :

# $

%

&

'

(

(

)

*

%

;

*

<=

>

?

( "

(( ""

( "

Agradecimentos

@ A B ' & # C $

! # ? <= +

D B ; ! 4

E ! FB ' B ( F! ( ! B ! *

E B ! E G F * B + B E E

@ * E ! @ @ 9 7 G 4

# < * B ' ( ; H B

H * ! 4

# < B $* 8 #

7 9 * !

H 4

# < 9'; #' @ * + I 4

# < B J * != 7 +

7 9 $ + H !

4

# < I 7 # = D %

& H 4

@ * B 7 E " +

K ! ! & + I 7K + '

B D 4

# < * + *) + % ; " !=

) + & +

* ; B " * + ; * B B +

B B + B I 4

# < B ( !+ *

* ' B J ) @ " ! + D

! B <= + " 7 ?

) 9 + ) ! & !

( 4

E B B ! ! # * E E @ F B ! !

E E @ * + G ! G! ! G L * * B B B !

+ B J 7 E + B E ! ! F G0! G B 0

9 7 B ! ! !0; E 4

E ! FB ! L G F @ * A # + B ! !

9 7+ '! ( B ! 9 7 +

! F ! * # F + ( * ' + M + 7 BB + !

! F ! E B E G + B ! B ! B !

! G F E ! N

# < * & 4 # @ D

B <H ( + 9 D B + ;

D * * < 4

# < A + + % +

7 * + 8 + < * 4

# < ! + + 8 " O @ P+

( O' F P+ # % + ( G + J + 7 *) I $ + @ = + 9 " !

+ + + H + B + O ! + J $P+ ;

+ +

; = * $ N

# < ; " ! A

7 Q' + ( + 8) R+ " I K + <

Q# + '! ! + + ' ! + + + * + + # 7 9 = R+

+ + B + I * + <

)0 + I % + + $ # 8 " +

<= + Q $

Resumo

* ! <= )

* * + !

Q . ,R <= B

+ <H ) '!E

+ # ? * S ', Q @',R B +

T80'! ,_{U0 Q80'! R T 0'!} ,_{U0 Q 0}

'! R+ ? ) E * +

# "C 4 % $

* ) 0 0 + + !$

; * ? * & 4

+ @! 0 " F 0 Q " F +

.222S @! + .222S + ,---S V + ,---S ' + ,--WR B B

* * & 4 % +

* )

* 4

; = B " 0 )

Q R 7 ? ) 9 Q7 9R <= 4 7

; $ + α0!)

$ B $ 4 + * !

<= $ 0 )

+ = <= * B 4

7 + + B <= α0! + !

; ) + ; <=

B B $ ; * 4 ' @',+

7 9 ,+,+,0 B Q% R

K α0!) * 7 9

B 4 7 9 ' B *)

7 9 B & B " @', B +

* B B .0 0,0

B B Q' ' R .W₉ , _{4 #}

<= & ; .W₉

* ; , _{+ B <H}

* 7 9 <= B

; <= 0 * 4 ' @',+ $ D

7 9 <= B & ; B * +

B ; B ! B&* $ B$

B B ; I B * 4 80'!

0'! + * ! $ & + <H *

B + ; * B 4

* ! " ? <=

0 @', ) % <= ) Q % R4

; <= ; = ) B <=

; + $ $

+ ; <= * + )

<H ! B&*

B$ * B B 4 # $ D *

B ! <=

Abstract

%! G F B ! G ! * 0

B ! * + ! B G !

Q ! . ! ,R E * ! ! B * B ! F B

'!E + B ! ( " # S E ', Q @',R

! ! E T80'! ,_{U0'! E Q80'! R T 0'!} ,_{U0'! E Q 0'! R+}

! B E * + B ! # " B 4 # !

! G * E * ! 0 0 * +

G!E ! G * ! ! !G E !

B G ! * 4 %! G E B !

! ! @! 0 " F 0 Q " F + .222S @! + .222S + ,---S V +

,---S ' + ,--WR+ G! ! F B B ! L !

* 4 ! E !

4

%! B ! ! *E B

! Q R 9 7 Q9 7R4 %! ! G

! G 0 E + B ! ; ! α0!

E G ! ! ! ! ! 4 L + *

! G * G! F ! ! E+

E G 0 B B ! ! ! 4 %!

+ ! G + F α0! B * ! ! ! G B !

! + ! ! ! ! * G ! ! ! !

* * ; * G ! ! G 4 ! @',+ ! B !

! 9 7 ,+,+,0 !

Q% R ! G ! ! Bα0! ! B ! 9 7 ! G

! ! ! ! 4 ! ! E + ! 9 7 '

α0! 0 ! + * ! 0'! ! G ! E ! ! ! 80

'! 4

%! 0 9 7 G B B @', ! G ! E +

* E ! E0 * G ! .W₉ , _{4 ! B}

! .W_{9 ! ! B +} , _{; + G *}

! * B ! B ! 9 7 4

@',+ ! E B * ! 0 9 7 ! G ! !

E G ! ! * E + G E ! ! !E ! *

G ! ! ! ! L ! ! ; 0

! * E L B 4 %! 80'! 0'! + * ! G E E

+ ! B E BB + G! ! ! !

G ! ! * E G! ! ! 4

# ! B ! G F ! E B ! @', 0 E

L ! E ! ! % E Q % R4 %!

! G ! ! ; E ! B !

! ! + B ! ! !

G! ! ! B ! ! * + E *E

!E ! * * G ! 0 ! ! * L !E ! *

4 %! E B ! ! G ! ! 9 7 + G *

B ! * G ! ! *

Sumário

8 #* # C 44444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444. % * # $ 4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444445 # <= 4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444X .4 <= 44444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444W .4.4 44444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444W .4,4 ' # * 444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444.-

.4,4.4 7 <= 0 * 4 444444444444444444444444444444444444444444.X

.454 @ B & ) 4 444444444444444444444444444444444444444444444444444.W

.4X4 44444444444444444444444444444444444444444.1

.4W4 <= 444444444444444444444444444444444444444444444444444444444,. .4W4.4 44444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444,. .4W4,4 7 ? ) 9 <= 44444444444444444444444444444444444444444444444444444444444444444,, .4W454 7 ? ) 9 & 444444444444444444444444444444444444444444444444444444444X-

.4W4X " <= ? <= / *

<= ) Q % R 444444444444444444444444444444444444444444444444444444444444444444444444444444X5 ,4 ) 44444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444XW ,4.4 4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444XW ,4, ) 444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444X6 ,4,4.4 ' 4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444X6 ,4,4,4 @ B ', 44444444444444444444444444444444444444444444444444444444444444444W. ,4,454 7 <= <= 4444444444444444444444444444444444444444444444444444444444444444444444444444444W,

,4,4W4 4444444444444444444444444444444444444444444444444444444444444444444444444444W5

,4,4Y4 ' # $ 7 9 @ <= 444444444444444444444444444444444WX

,4,464 ' <= 7 9 & 444444444444444444444444444444444444444444444444444WW ,4,414 7 9 & 44444444444444444444444444444444444444444444444444444444444444444444444WY

,4,424 @ <= ? 7 9 B & <= <=

* 44444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444W6

,4,4.- ' <= 8 )

% <= Q % R 444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444W1 ,4,4..4 % <= ) 44444444444444444444444444444444444444444444444444444444444444444444444444Y- 54 7 = 4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444Y5

54.4 @ @& ' + ', Q @',R 80

'! 0'! 444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444Y5 54.4.4 @ 4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444Y5 54.4,4 @ ' B <= ', Q @',R 4444444444444444444444444444444444444444444444444444444444444Y2 54.454 @ T80'! ,_{U0 Q80'! R4444444444444444444444444444444444444444444444444444444444444444444444465}

54,4 Q R ' +

', Q @',R 80'! 0'! 44444444444444444444444444444444444444444444444444444444444446W 54,4.4 444444444444444444444444444444444444444444444444444444444444444444444444444444444444446W 54,4,4 ', Q @',R 4444444444444444444444444444444444444444444444444444444444444444444462

5454 <H 7 ? ) 9 Q7 9R @ <=

' + ', Q @',R 80'! 0'! 44444444444444444444444441-

5454.4 <= 7 9 @ <= 4444444444444444444444444444444444441.

5454,4 <= ', Q @',R444444444444444444444444444444444444444444444444444444.-.

545454 <= T80'! ,_{U0 Q80'! R T 0'!} ,_U0

5454X4 $ <= 7 9 @ <= 4444444444444444444..Y

5454W4 % & <= ' 0( B 7 ?

) 9 Q7 9R @& 444444444444444444444444444444444444444444444444444444444444444444444444444.,X

54X4 " <= % ? ' <= ' / *

% <= ) 444444444444444444444444444444444444444444444444444444444444444444444444444444444444444.5X

54X4. <= <= 0 * 44444444444444444444444444444444444444.5X

Lista de Abreviaturas e Acrônomos

# 9 #

#' ( * + # * ' * . , ( 5 % + !

8# B ; B

@V <= +

@@@@ EEEE

9+9L0 *

9+90 B

0'! T 0,_{'! U0}

' B B +

E ! ! !

0 @V <= B

; + * 0

@V

@@ X+X0 0X0 0.0 B

&

4 @V @V

<= + E

20B *

( . 0 ! * " "

@', ',

@ ! 0

; + @@@@ 0

!

% <= )

! % E

B <=

80'! T80,_{'! U0}

8 +

8

#8 <= / " <= "

+ #

8 / "

8

8

! + 9999

! BB @@@@ EEEE

' ( * +

' ( F

'A8 7 "= A

7 9 7 ? ) 9

' ' .0 0,0 0B B

' ' .0 0,0 0B B

<= I <=

0

I <= 0

; I <=

0

9 <= ; <H

7 9+ B 9

99 9 7 7

7 +

7

7 @ " ; )

; + 7 @;

+

@# B

@ @ B & + @

E @ !

@ ; +

@

% # $ B )

% ,+,+,0 B

% @ 0

% @V <= +

% %%

% @@@@ EEEE

% C + B B !

<=

Tabela de Aminoácidos

# $

# $

# $

# $ @@@@ **** @@@@ **** &&&&

Z $ # ..W+-,Y

Z ? .,2+-X,

# # # 6.+-56

# 7 # .WY+.-.

# 9 # ..X+-X,

E .-5+--2

'! .X6+-Y1

E W6+-,.

.,1+-W1 .56+-W1 ..5+-1X

8 8 8 ..5+-1X

8 [ 8E .,1+-2X

.5.+-X-' ' ' 26+-W,

@ @ @ 16+-5,

% V %E .Y5+-Y5

% % %! .-.+-X6

% B \ % .1Y+-62

Apresentação

*D <= )

* ; * A + !

Q . ,R <=

B S ', Q @',R B +

T80'! ,_{U0 Q80'! R T 0'!} ,_{U0 Q 0'! R4 %}

$ * ) 0 0 +

+ !$ ; * & 4

+ @! 0

" F 0 Q " F + .222S @! + .222S + ,---S V + ,---S ' + ,--WR

B B * * & 4 %

+ * )

* 4

* ! <= + ; * <=

B & ) " + .S4 '

+ ; ) ,S + * 7

1.Introdução

1.1. Estrutura peptídica

= * ) ; " $

Q .4.+ 4 YR + ) <H

* $ <=

<= 4 <= + <H

B A Q B 0 R+ 90

* Q B 0 * R+ *)

0 4 ' <= + 90 )

0 + B 4 )

D B <= ; $ ; H

<= Q + + 9+ R = + ; ) +

$ + $ + ; I

+ .4. Q9 ] + ,--,R4

.4.4 .4.4 .4.4

.4.4 7 <= ; $ ; +

4

N H₂

NH

NH

OH

O O

R₁

R₂

R₃ O

ligação

peptídica Terminação

amínica

Terminação

.4.4 .4.4 .4.4

.4.4 $

4

= B ; A

$ + $ + $ ; $ 4 0 $

; $ + 90

0 Q( 4+ ,--YR4

# $ B <= <= 4+

B H + ; = α0!)

B ! 0β4 # <= B B I <= ?

Q .4,+ 4 6R ; + "+ ) * B ;

; $ 4

O NH2 C H3 OH Alanina (Ala) A N H O NH NH₂ NH₂ OH Arginina (Arg) R O O N H₂ NH₂ OH Asparagina (Asn) N OH O H NH₂ O O Ácido Aspártico (Asp) D O NH₂ S H OH O O OH O H NH₂ Ácido Glutâmico (Glu) E O O NH₂ NH₂ OH Glutamina (Gln) Q O N H₂ OH Glicina (Gly) G O NH₂ N H N OH Histidina (His) H O NH₂ CH₃ C H₃ OH Isoleucina (Ile) I O NH₂ C H₃ CH₃ OH Leucina (Leu) L O NH₂ N H₂ OH Lisina (Lys) K O NH₂ S C

H₃ OH

Metionina (Met) M O NH₂ OH Fenilalanina (Phe) F O N H OH Prolina (Pro) P O NH₂ O H OH Serina (Ser) S O NH₂ O H CH₃ OH Treonina (Thr) T O NH₂ NH OH Triptofano (Trp) W O NH₂ CH₃ C

H₃ OH

.4,4 .4,4 .4,4

.4,4 7 <= ? <= $ 4

# α0!) Q .45R ) B <H ! + ) <=

* ! <H

4 # <= ! !) $ +

!) + ? W+X ^+ + + 5+Y

$ Q( 4+ ,--YR4

.454 .454 .454

.454 α0!) + B 4 Q RQ RQ RQ R

B <= α0!) S Q*RQ*RQ*RQ*R * + <H

! S Q RQ RQ RQ R α0!) <H + ! 0 *

) S Q RQ RQ RQ R = α0!) < ! Q9

] + ,--,+ 4 .,6R4

# B ! +

! * " <= * " <= α0!) 4 7

& * " B <H 4 7 B

! 4 9 + $ B " +

<= <= − + ; + B

! 4 # ) + ! =

* " <= B ! <H ! 4 9 +

B * B B B ! + B ;

B * + + B B

; ; ! Q9 ] + ,--,R4

# B ! B *)

* 4 # + ) <H

<H $ +

B ! Q9 ] + ,--,R4

9 $ B ! 0β+

<= " 0" 4 0 0 + B

; ! B ! 4 9 + <H ! = B

D 4 # <H !

+ *) B 4 !

<H H B

<= B 0βA B B + * .4X Q 4

2R4 9 B + B 0β 0 $ + ; ; +

+ 0 B ! ;

Q4 49 R4 $ + + =

$ +

4

.4X .4X .4X

.4X4 β + Q RQ RQ RQ R B <= β0

+ ; $ * + ; =

! Q R Q*RQ*RQ*RQ*R B <= β

4 Q9 ] + ,--,+ 4 .,2R4

= $ ) * β Q .4WR+

= B B <H β0 4

# * .1-_+ ;

$ 4 * B <=

! ! " ; ? 4

; = B <= $

Q .4W R4 * B *) <= B <=

+ B .4W* Q9 + ,--,R4

.4W4 .4W4 .4W4

.4W4 Q RQ RQ RQ R * β+ ; $

Q " R ; 4 Q*RQ*RQ*RQ*R B ;

Q R4 Q9 + ,--,+ 4 .5-R4

0 $ D $

4 # $ D

B $ + * D H

4

' D

* 4 " <= )

; $ Q9 ] + ,--,R4

1.2. Peptídeos Antimicrobianos

' * = B

4 @ * <= ) +

; ; * ? <=

4 # !$

+ * ? B ) 0 B 4

* 4 *

* Q#' > # * ' * +

! A// 4 4 /#'/ 4 ! R .W.1 + ; 21

+ XX, BK + ..Y1 * 21

? 4 * B

<= 4 % B <=

) * ; 4 * ? )

B $ ) ) + +

B * + ! Q( ] : BB+ .22-R4

B * Q ] ! +

.26-S BB 4+ .215R4 9 0 *

+ * <

& Q( + .22WS : BB+ ,--,R4 .222+ B

* )

'!E Q( 4+ .222R+ Q( 4+ ,--.R+

! ) + * 0 + D

ENREVPPGFTALIKTLRKCKII NLVSGLIEARKYLEQLHRKLKNCKV

.4Y4 .4Y4 .4Y4

.4Y4 @ ; + <= <= B

4

* & + B 0 ;

* * ) + 0 0

Q( 4+ ,--.R4 # ) + $ B B +

Q R = B ! Q R+ ; <

$ B ! 0β+ )

$ α0!) Q( 4+ ,--.R4 ? ) Q7 9R

; B + ?

α0!) + ) <= ) +

+ < B ) Q7 4+ ,--WR+

B .464

.464 .464 .464

.464 * 7 9 ! $ 4 Q RQ RQ RQ R )

D * + " 0

+ B <= ? ) 4 #

. $ " 0 + ,+ 0 +

* ; 4 Q*RQ*RQ*RQ*R )

) + . , ! Q7

4+ ,--WR4 R

7 + <= * B B B

7 9 & + C 0 B

B <= * 7 9 <= Q7 4+ ,--2R4

; B B <= ; *

B B + B ) Q B B )

* ; R+ <= I +

B ; 90 <H 0 <H B

.414

.414 .414 .414

.414 7 <= Q

" R * Q " R Q7 4+ ,--2R4

* ) B 4

B ) E * +

# "C 4 H ) 0 * A T80,_{'! U0 Q80}

'! R T 0,_{'! U0 Q 0'! R4 + *)}

0 D $ = % * .4.+

+ B 0 B <= B Q ,+ ,_{'! +}

B " R4 9 80'! + 0

C 8 B 0'! + C 4 # ; B

) K B +

90 + D ; ) '! 0'! 0'! 4

# * = <= ) 4

% * .4.4 % *% * .4.4.4.4

% * .4.4 @ ; $ 80'! 0'! + <H

$ 90 I 0

+ <= K >9 ,I <=

$ Q != 4+ ,-.,R B

+ *) + * ! + + *

* * ? 4 <= I +

* * ;

! * )

! B&* + B Q ! * ! + .211S [ + ,--YS ! ! + ,--2R4

# ) B $ ! B&* $ + B * ;

" <= *) ) *

Q8 D+ .21-S + .21WR4

* ? B + " ; *

* )

4 + * & = I B

+ 4

B + * B *

* ; B ; =

= 0 % W+ ; <=

B Q7 + .221S ' "+ ,--,R *

; 4 # B < ;

B * * Q'! 0'! 0'! R+ = B * B <

* ? Q != 4+ ,-.,R4

' + * $

B < 4 0'! " * )

@4 Q <= * & Q R 5,+W 0'! YW+W 80

'! R " `4 ; 80'! Q != 4+ ,-.,R4

; B < B <= * B ,

$ B $ * B + " ; * B

B <= * <=

* * & + B $

.4,4. Q 4 .XR4

% *) ) E * + ',

* * ) 0 0 Q( ! J 4+ ,-..R4 % 0

) + ; !

$ + [

Q E 4+ ,--2R & B 4 # ) + <= )

D <= Q .42R ; + B α0! +

B $ + + +

* * 4

% * .4,4 % *% * .4,4.4,4

% * .4,4 @ ; $ @',+ <=

$ 90 I 0 +

<= K >9 , I <= K

.424 .424 .424

.424 7 <= @', D <= +

+ +

+ = 0 " 4

1.2.1. Relação estrutura-atividade de peptídeos antioicrobianos.

B B B

; ; = *

<= 4 $ * ? *

* * Q( ! + .222S 4+ .222R4

B B α0! ; " * Q\ ! + .212S

\a ! !+ .212S ] * + .22.R4 # 0 ; * " <= * )

B <= $ Q\ ! BB 4+ .212R4

;

* ) @! 0 " F 0 Q@ R Q " F + .222S @! + .222S V

4+ ,---R ; H <= * + " <=

+ <= * + +

) 4 + = * B ! +

$ <= * 4 ' + $ ! & +

" <= * Q\ ! BB+ J 4+ .212R+ B <=

Q( * ] @ ! + .21WS V + \ 4+ ,---R+ <= <=

! ; Q( * ] @ ! + .21WR+

* Q " F + .222R <= ) " <=

Q[ 4+ ,--.R4

% + B + <=

+ ) $ <= <H

; " B & 4

1.3. Síntese em fase sólida de peptídeos pela estratégia Fmoc.

# B & * 0 <= +

+ * & K 4 ) A

* $

B <= " + <= <= 4

) $ + <H

90 + " 4 ' B

" + = +

+ 4 + +

; B K

" + ; <H $ +

+ B <H + " 0 ;

B$ <= B " <= 4

" B ) % [ Q% ] + .2W5R+ ; B

$ <= <= 4

% B & + ) +

B 4 ' + * ) K

+ $ + + ; = $

0 K 4 % $ "

Q * R =

<H $ 4 $

Q * R $* ; = <=

+ B ; K

$ ) B + 4

$ ; ) I = + ; +

B + 0 4 M B + = +

<= * ; & 4 # B

+ ) B + 0 + + +

<= 4 #

.4.-<= ; $ B & + <= I

+ ; ) " 4

.4.-4 .4.-4.4.-4

.4.-4 7 <= ; $ B & 4

# ) ) ; 20B *

Q + .4..R α0 4 ) B

*$ + ; ; $*

$ 4 + " 0 <= <= + B +

0 $ B ) Q% #R+ ; ; * <=

+ ) * ?

$ + <= 4 J

$ = " + ;

* ; $ 90 4

+ " 0 * + B BC C

! * " " 4 " )

= .4., Q 4 .1R4

.4..4 .4..4 .4..4

.4.,4 .4.,4 .4.,4

.4.,4 7 * " B &

+ ) 4

1.4. Características angulares de estruturas de proteínas e

peptídeos

# * ) *

B + ; B <= * <= )

+ B + H ) + +

<H $ + \ <H ! +

4 + D + I * + <=

<H + + * <H ?

Q + ω_{R = 4}

? ω_{) ? I <= + $} − ’ −

B <= B ? + <=

$ <= 4 B ; <= = ) + ;

<= 4 9 +

? ω _{.1-_+ " B <= 4 + B <H}

Qω _{b -_R *) = + = B <=}

4 7 + + B <= +

90 + B .4W Q 4 2R4 B +

; * <H ? + ; + +

5-c <H

$ ; ; B + ; ; .+Wc <H

; = D B <= Q\ 4+

.221R4

? <= ) ; +

+ * <= ω 4 # $

) B <= 0` ; 0 =

? <= ) ∼W+Y_+ ; ) .,_ Q 4+

.22,S # ! ] %! + .22YS \ 4+ .221R4

? + "+ $

* ) 4 @ * <H "

$ + ) + $ 4 *

<H ) + 7 ! * Q7 ! 4+ .2Y5R

; * <H $ =

B + ; " ! 7 !

.4.54 .4.54 .4.54

.4.54 7 ! B QQQQ RRRR E+ *R*R*R ' + RRRR*R + RRRR

# QQQQ RRRR 7 ! " 4 QQQQ RRRR QQQQ RRRR+ "

H B + + H

+ " H * + = * 4

QQQQ RRRR ! + + 0 * H B +

+ * 4 QQQQ RRRR QQQQ RRRR *

& ' ( .#,X B QQQQ RRRR * '7 [+

& ' ( .#.'4

# ) <H ) + ? * B * <H

; $ +

+ 7 ! ? $

& 4

1.5. Determinação de estruturas de proteínas e peptídeos

*D * ! B <H * &

<= + 0 <=

<H 4 = ) ; = "

B B <H + ) 7 ? ) 9 Q7 9R 0

" B * <=

4 ' ) + 7 9+ = "

Q R *D * B <H

$ ; * B B

* 7 94

1.5.1. Dicroísoo Circular

# Q R * 0 * <= B

" 0 " &B

4 + "

B < B Q( ! ] ( ! + .21-S

] % ! BB+ .21,R4 B < ) + +

+ B * 4

# ) " 4 9

* ? + &B ? ) − = − −

+ I <= 4 # B $

I $ + ) $

* *? <H ; B B <H

$ 4

# B I * <H &B

? Q .4,+ 4 6R4 ; ?

? <H + <=

<H ; B " + * <H =

B <H 4

# ) + 0 B * ?

$ α0!) B ! 0β = Q

C R4 $ Q R

<H C ; * *? +

Qλ_{R4 # <= $}

$ ) ) " <= H

D $ = ! * B 4 <= )

" $ <= Q + .21,S @ + ,---R+ ;

; ) * <= B

" $ 4 H

* <H <H = .4.X4

.4.X4 .4.X4.4.X4

.4.X4 = + ; α B Iα0!) S +

C + B ! 0β4

1.5.2. Ressonância Magnética Nuclear eo solução

# ? ) Q7 9R ) ) ; B

<= * ) + $

) Q ! 4+ ,--YR+

* <= + ) B B <H

? + * <= 4 # 7 9

Negativo a 222 n_→π*

Negativo a 209 π_→π*

Positivo a 192 π_→π*

α-hélice

Negativo a 218 n_→π*

Positivo a 196 π_→π*

Folhaβ

Negativo a 195 π_→π*

Positivo a 212 n→π*

Randômico

λ/nm Transição Estrutura

Negativo a 222 n_→π*

Negativo a 209 π_→π*

Positivo a 192 π_→π*

α-hélice

Negativo a 218 n_→π*

Positivo a 196 π_→π*

Folhaβ

Negativo a 195 π_→π*

Positivo a 212 n→π*

Randômico

B

* + B ; & 4

# & <= B 7 *

.252 Q + .216R+ <= * ? ? B "

& ; Q a + .221R4 .2WX+ J * * "

" 7 9 <= * ) + B

9# QJ * 4+ .2WXR4 9 7 9

. _{+ B 0 ; <H 9# 0 I B <=}

! <= <= I $ 4 .2W6 B *

7 9 . _{+ * Q@ 4+ .2W6R4 # )}

.2YW B * 5- * ! <H 7 9 * ) 4 9

) .26- ! ,-- * <H + .21-+ K

X4---* <H + " B <H 7 9 = Q7 9 . R

B Q a + .221R4

* ! ; " 7 9 * <=

* ) ) .21-4 # <=

Q( 4+ .21.R B =

<= ; Q# 4+ .21XR B " 0 7 9

<H B 4 * ! B " : G

* + ; B B

α0! Q: G 4+ .21XR4 ' + B " <=

0 + I * B <= 0` Q 4+ .21YR

<= * # Q\ 4+ .21WR4

# ) .21-+ B 0 7 9

H Q7 9 , R $ * ) 4 # =

* 7 9 B <H =

+ ) + B *

<H & + B 4 )

<= & <=

7 9 Q[ 4+ .21YR+ * 0` Q'B ! 4+ .21YR4

9 ) + \a ! ! " * <= ; + *

Q\a ! !+ .21YR4 # ) C 0 " * <= B <H

7 9 , 4 = + 7 9

<= * ) 0 + * 0

! <H

* <= 4

B <= ) 7 9 * )

B <= * ) + D B =

$ B B 4 # + =

7 9 <= + *

<H B 7 9+ B <H

7 9 , + $ $ B <H +

* <H B =

7 9+ = ? * B <H +

$ & " <=

$ = B 4

1.5.2.1 Atribuição Sequencial

) * <= ; B [ \a ! ! .21Y Q\a ! !+

.21YR + " * <= * + * <=

B <H * 0 $ ! 4

@V % @V * + ) <H

Q JR+ + B + B <=

; 4 9 9 @V+ * =

) < Q I <= " 0 R+

; 0 Q _," .4.W*+ 4 ,WR4 9 + = B

<H 9 ; Q , 1 + , 1 , 1 R+ ;

B ; $ = 4 # ) + = "

! . ₊.5 _{0 @}

. ₊.W_{90 @ + 4 D * *}

N C C N H _H CH _O H α ββββ i i N-Terminal _C-Terminal N C C H2 C O N C C H2 C O N C C H2 C O N C C H2

H H H H H H H

C O

N

H H

αααα αααα αααα

dαN

dαN(i,i+2) dαN(i,i+3)

dαN(i,i+4)

dNN

dβN

dαβ(i,i+3)

i i i+1 i+1 i+2 i+2 i+3 i+3 i+4

.4.W4 .4.W4.4.W4

.4.W4 7 <= <H $ ! Q RQ RQ RQ R4

; <= 9 $ α0!) Q*RQ*R4Q*RQ*R

% $ + 0 I

* <= <H 9 $ 4 * ! +

0 $ α0!) + .4.W

9 $ 4

' B + * I $ + 0 I

* <= 9 B $ ; $ 4 B

= H $ + * <=

+ * + ) * * ! +

$ " * ! 4

1.5.2.2 Conversão de dados de RMN para inforoações estruturais

tridioensionais

# $ ) B <= + &

7 9 = ) <= $ $

4 ' + <H ) B =

+ * ; + $ Q\a ! !+ .21YS ! 4+

,--YR RRRR

<H ;

A <H ? + <H ? Q ? <= R

<H 4

1.5.2.2.1 Restrições de distância

# <H ? A <H

9 <H <= ! + B 0

" ; <= 7 9 ; B $ B

4

<= + <H 9

B + B ! + B " <= < +

; K + ; D

$ 4 # ) + $ 9 )

) ? % K

& ; + B ' (₎ + ; B *

+ B ; <= .4.4

$ = 〈%+,_{〉' (}

) Q ;4 .4.RQ ;4 .4.RQ ;4 .4.RQ ;4 .4.R

* D 9 ; <=

.4.+ ) B 9 A B + )

B Q F E 4+ .221R+ + + ?

,+1-+ 5+X- W+-- ^4 * + I +

; = ; <= .4.+ B <= ; 9 B !

" B B = ? + *

<= * <H Q9 * 4+ ,--XR4 M

; $ = " = B +

B +

) $ $ Q ! 4+ .22YS + .221R4

# <H <= ! *) $

? + ) + $ <H ? <H 9 +

D <H ) D + B .4.Y Q 4

,6R4 $ ? = B ; $ ;

! ? & ; <= ! Q .+1

,+- ^R4 α0!) + <= ) + " ; =

* <H ! 0 +

3S , 4 , 5+ $ ! * + B

.4.64

.4.Y4 .4.Y4 .4.Y4

.4.Y4 8 ? <H <= !

<H 4

.4.64 .4.64 .4.64

.4.64 α0!) + <= + *

+ <H <= ! 4

Q9 * + @ F 4+ ,--XR4

$ ; <= !

; B $ ! ; = B

) Q ; ; R & Q\ ]

\ ! !+ .21,S [ 4+ .222R+ ; Q\ ! 4+ .22,R+

1 _′– 41 _{′– Q ] " F+ .222S}

4+ .222R4

H

N

O

1,8 Å < d_OH< 2,0 Å

2,7 Å < d_OH< 3,0 Å H

N

O

1,8 Å < d_OH< 2,0 Å

1.5.2.2.2. Restrições de ângulos diedros

# ? C + ? *)

+ * 7 94 % +

" 0 4 + ;

<= [ Q[ + .2W2R+ ; <= .4,A

=

5 _{6 cos : cos Q ;4 .4,RQ ;4 .4,RQ ;4 .4,RQ ;4 .4,R}

; θ _{? <= ; $}

? 6+ : = + " 0

B <= 0`4

4 _{= * 7 9} .

+ ; !$ <= B + ) 0

@V+ % 0 @V 4 @V Q 4+ .21WS D * 4+ .222R '! @

@V Q 4+ ,--.R = 4 + 7 9 .

= " $ + I * <=

0 @V+ % 0 @V 4 @V = B

B & + ; * ! ) * ? +

; + " + <H " = * B

* H Q 4+ ,--.R4 # ) + =

<H B B + <= =

+ I B ; " = 4 9 '!

@ @V+ ) " = 4

<H + ; ;

<H + ) * " 4

# <H + + *

; $ I 4

; " = = %#8 @ Q 4+ .222R =

%#8 @d Q@! 4+ ,--2R4 # * * 0 B ;

; = " * ; ;

K Q F ] 7 + ,--2R4 # + * <H ?

H * ) 4 B " *

; $ D B

<= + " $ =

<H 4

1.5.2.2.3. Restrições orientacionais

% <H ) = 4 *

" D B <= *

* + = * )

4 9 + B "0 $ * <= B <H )

Q 4+ .222R4 "

7 9 ; * <= B <H " )

K 4 # ? ) ;

<H <= ) ;_<4 + <H

& + <= ;_< ) +

B * " = ; <= "= <=

) * G Q8 " ] %D + ,--XR4

9 B + ) + I B <H

* ) + * ! ) <= ;_<+

= + B

$ 7 94 + " B )

* B $ 4 9 + )

; B ; * + ; B " ;

D ; $ +

Q7 R4 0 I H

) + <=

$ Q%D ] ( + .226R4 7

. ₊.W_{90 @} . ₊.5 _{0 @ = *}

.4.14 .4.14 .4.14

.4.14 . ₊.W_{90 @ A Q RQ RQ RQ R * H +}

* .W₉₀. _{= * S Q*RQ*RQ*RQ*R =} .W_{9+ <=}

& + * .W₉₀. .W₉₀. _{Qe2, "RS Q R}

= .W_{9+ + *} .W₉₀.

7 4

1.5.2.2.4. Construção dos modelos estruturais a partir dos dados de restrição

conformacional: Arrefecimento Simulado

# & * <H B + = " $

4 $ B "

* ! <= 7 9+ ;

= $ ? Q( 4+ .21.S ] \a ! !+

.21XR $ B <H 0 $ Q( ] + .21WR B +

*) ! = Q9 4+ .211S (

4+ .226R4 # " + ) B 4

B ) " <= *

? 4 ) )

$ $ ; $

B 4 $ + <=

) + 0 * B * <=

$ Q( 4+ .22-R4 # .4.2 Q 4 5.R <

B <= B <= @#4 = *

.4.24 .4.24.4.24

.4.24 7 <= < B )

$ " A Q RQ RQ RQ R B <= Q*RQ*RQ*RQ*R Q9 + ,--.R4

# B * B $ *

* + $ ) B B

; ; ) 4 ;a <=

* + ; = " + " ; B

$ * <= * ) 4 ' + ) $

! ? ; " B

+ B $ & <= 4

1.5.2.2.5. Análise, Validação e Qualidade Estrutural.

# = B <H 7 9

+ * * ;

B $ 4 @ B <H = B B *

B B <H + =

D ) ; * !

B & 4 8 & * D + = * I

$ + = BC +

B ; D + B + = ) 4

1.3.2.2.6. Violações de Restrições

<= <= B ; " = B " <H

B <H 4 # $

alta temperatura baixa temperatura

alta temperatura baixa temperatura

Q R Q R Q R Q R

<= 4 # <= C ) B B

$ + H B B <H * 4

B $ ) = B <H +

<H C = $ B 4 +

? B <H B

B ; "

= B B B B <H 4

# <H =

6Wc B <H D B + B ! H

B + * <= 7 94 <=

+ * <= + * * <=

9 + < C ; B

; 4 K <H <= D

B <H ?

& 4 + <= K <H B

) <= B <H * 4

B <= <H " ?

4 # ? ? * = =

<H + ) + ; = <H 4

+ D B + !$ <H <H 4 9

+ " <= * <H

<H + $ 4 # + 0

*) B <= <H <=

* 4

# B <H 7 9

<H + H H B D

+ ) + $ 4 B +

<= * B <H 7 9 0

; $ B ! B & ; 4

" $ ;

; $ 4 = ) +

= B < + B ; <= B G " $

Q@ F 4+ ,--XR4 # <= ?

4 ; <= B + )

<= " ) I ! <= 4 '

"+ = ? &

+ B < ; + I = <= + *

B G + + )

Q + .221R4

1.5.2.7. Conteúdo de Informação como Parâmetro de Qualidade

B <= 7 9

<H ) $ +

; ; B K <H " + ! ) ;

4 9 ) 4 B

' ( Q9 * 4+ ,--5R+ * 0 ; 5-c <H

? = + & I <H I

? Q <= $ ! ;

; R4 + ; D <H I

? B $ 5-c B <H + D

$ -+Xc B <= 4 + D

<H <= ! + ; + $ + 6+Wc <H + B

$ 5-c B <= 4

B < B <= * D B

0 B ; $

D$ " + B <= + ! * " <=

<H & D$ B 4 ' + B <H

? $ B $ 4 # <H ;

B <= + $0 <H 4

# <H ; ! B <=

+ ; ; + " ! + = $ $

) 4 <H = <H

+ K <=

) 4

# <= K B <= D <H ) "

9 Q B 9 7 7 R Q9 * +

@ F 4+ ,--5R4 $ B <= ) B * %

B <= 4 @! Q@! + .2X1R <

? + " < 4

" <=

B <= <H $ B * ! + + B B +

) " <= " % B <= * ;

B <= " $ Q R+ ;

" Q _{=> "? ?"@}R+ ; " C = "

Q ! + 9 * 4+ ,--5R4 ' B + B <=

QA_{B @}+ ;4.45R D <H ) ; <=

.45A

AB @ = => "? ?"@|D− => "? ?"@|E Q ; .45RQ ; .45RQ ; .45RQ ; .45R ; _{=> "? ?"@|D} ) " ! <= => "? ?"@|E ) " <H 4

@ + B <= QA"+ ;4.4XR K <= %

B A

A"= => "? ?"@− => "? ?"@|" Q ; .4XQ ; .4XRQ ; .4XQ ; .4X

; _{=> "? ?"@|"} ) " + <= % _{=> "? ?"@}+ " <= <= %4

' K $ A

B <= QA_{? ,"}+ ;4.4WR B <= ) QA_{F= ,"}+ ;4.4YR4 # + B <=

K + B <= % ) B B <= <= +

0 ! <H Q − %R D A

# ? <= + 0 D <H + )

* 0 K ) B <H D

QA_F= R4 # B <= ) <= D

<H ) K B <= <=

<H A

AF= ," = 〈 => "? ?"@− => "? ?"@|"〉 Q ;4 .4YRQ ;4 .4YRQ ;4 .4YRQ ;4 .4YR

" = " B <H

D <H Q9 * 4+ ,--WR + ;

+ D + " ; B <H

; K B B <=

4 9 + ! + + <H ;

B <= + ; = + $ = $ "

B $0 <H B <= Q 4 4 <H G , 4

G , − 4 + ; D K <H α0!)

; + + B <= + = = B

$ + R4 ? B <=

) + + = <= B$ <H

+ ) " B <H * +

D 4

1.5.2.2.8. Parâmetros estatísticos utilizados na análise da qualidade estrutural

# ) ; ; " "

; B * <= ) + *

<= <= 4 &

? " $ ; B

* ! 4

.4W4,4,414.4 : .4W4,4,414.4 :.4W4,4,414.4 : .4W4,4,414.4 :0000

:0 ) ? " \ #% +

* D ;

? * <= * Q ; <= .46R4 9 ; <= +

〈HI 〉 J HI = ) = 4 ' :0

* 0 ; = Q R+ D + ; = $

4

K =

H − 〈HI

_{J HI}

〉

7 @ :0 $ ; :0 + B

* <= ; )

; <= .414 ? K ) :0 B ; <= .46 * <= &+

; ) K * <H 4

LMK = N∑ P K Q ;4 .41RQ ;4 .41RQ ;4 .41RQ ;4 .41R

.4W4,4,414,4 $ ) Q7 @

.4W4,4,414,4.4W4,4,414,4 $$ )) Q7 @Q7 @

.4W4,4,414,4 $ ) Q7 @ >>>> 7777 @;@;@;@; RRRR

; $ ) Q7 @ R ) " " ; B

B < D & 4 $ 7 @

B B <= 4 # B <= * 0! C

<= ? B 4 9 B <= O <

; P+ = ; B

B Q( + # 4+ .226R+ ; <= .424 ? _Q ) ?

<= F R+ %_Q>? <= S %_Q T )

B <= S4 # ) _" <H ?

9 4 + 0 ) B < <H $

4

LM UV = W 1

" FX X ΔZ[

\

P

]

QP

^

Q > %Q>? ΔZ[= ` Q − %Q>? a

%_Q T< ≤ %_Q>? ∆Q = 0

%Q < %Q T ∆Q = `%Q T− Qa

Q ;4 .42RQ ;4 .42RQ ;4 .42RQ ;4 .42R

7 @ = B B <H * +

< B <H

+ <H + ; B

B $ 4 ; =

4 +

; / B * ) $ I =

+ ; + 7 @4 9 + 7 @

) 5 ^ B Q% + ,--,R4 + )

; * 7 @ = ) C D

; 4 @ D <H " $ <H

+ <H = ; H

D B + B " ; 7 @ * +

4 M ; 7 @ * + +

= ) D$ 4 * ; 7 9 B

<= + + 0 ; ; ; B

B ) <= 4 + 0 ;

) * 4 + *

7 @ B ; = ) +

<= ) 4 B + D$ B ; + ;

*) + 7 @ D " +

+ B Q@ F 4+

,--5R4

.4W4,4,41454 * <= f .4W4,4,41454.4W4,4,41454 * <=* <= ff .4W4,4,41454 * <= f

* <H ? = ;

B <= H B 4 B +

* <H ? = B ) H

7 ! Q .4.5 + 4 ,-R4 # $ ; ) B

* B <H * <H ? ; H

A H B $ + +

* 4 # = B <H

C B $ 4 # = B

B <H ; = ! B * ? <= 4

+ ; B * <= 7 ! +

" 0 :0 ; 4

9 + " 0 :0 * I <= $B

7 ! 4 # .4,- <= * <= ?

7 ! :0 4

.4,-4 .4,-4 .4,-4

.4,-4 7 <= :0 * <=

7 ! 4 Q RQ RQ RQ R 7 ! :0 b .+1 Q*RQ*RQ*RQ*R :0 b 01+54 #

H B $ + + * + =

+ + " 0 + " 0 ) + " 0 * 4

Q@ F+ 9 * 4+ ,--XR4

H * B B <H

$ + ; $ B $ 4 0

$ = H * ; =

4 % + H 7 !

= B 4 + :0 +

* = + <= 4

<= * *) ? ω_{4 B}

+ ? -g QB R .1-g QB R

$ <= * <= + )

* 0 + ) .,_4 9 + < )

D$ D + ; ; ) *

7 94 B $

" + ω _{B 4 # ) + B <}

B <= ) ; + ; B

$ + <H = B 4

.4W4,4,414X4 9 '

.4W4,4,414X4 9.4W4,4,414X4 9 ''

.4W4,4,414X4 9 '

" * ! B 4 % 0

? B <= + $

4 * <= +

<= * <= 4 $ ?

<H ; +

<H _G ; * B 4 #

) K ;

* 4 ' <= + = " +

$ 1- " 4

1.5.2.2.9. Refinamento de estruturas

B $ 7 9 *

* 0 $ ; B < +

<H ) 4 0

+ " ; *

7 9 = $ B < $ 4

) ; + B

<= + $ B <H ; B + + +

<H $ \ + ;

7 9 K * <H B $

H <= ! <= & 4

B B B Q8

4+ ,--5R ) $ + ) ) B

" <= <= <H C + ) B <

Q@ !G 4+ ,--5S @ !G 4+ ,--YR4 # *

<H ) + <H + B

" 4

1.5.3. Ressonância Magnética Nuclear no estado sólido

9 & = " <H 7 9 B &

* B B <=

) 4 * <=

7 9+ = " + B +

* = 4

' * B B <=

) C + 7 9 B & "

" <= * ) 4 ' =

.W₉ , _{+ ; + * Q + .226S}

4+ ,--,S # * E 4+ ,-.-R4

* 7 9 .W₉ , _{+ ) *}

B <H * <= ! <= I * ) +

)+ * <= 0 * 4 * <= + +

<= ? & + *

I * + <= * & 4

1.5.3.1 Fundaoentação Teórica

# 7 9 B & " B <=

I <=

) 4 ) B B <H <= I ? + *

C + I " <H 0 I

Q( ! 4+ .222R4 # 7 9 B & )

B ; I * <H & ; <H

; Q8 G 4+ ,--,R4

7 9 B & !

I <H 0 * *

+ " <= & + 7 9

+ K ) ; = "

<H Q[ ! 4+ .22XR4

; 7 9 & )

+ & .4,. ***4 * )

B ;

QJ + J J₄₄R+ B .4,. + K .W₉

! 4 ;

C 7 9 ) J_ff+ B D <=

J44 ) ;< QQQQ .4,.*R*R*R4*R B 0

? QΦ

+

+ 0 ! I <= J_ff Φ

Θ

; <= .4.-4

.4,.4 .4,.4 .4,.4

.4,.4 7 <= ; .W_{9 Q RQ RQ RQ R}

B Q*RQ*RQ*RQ*R+ ? Φ Θ

; +J_ffQ( ! ] @ " + ,--5R4

Jff= J sen Θcos Φ J sen Θsen Φ J44cos Θ Q ; .4.-RQ ; .4.-RQ ; .4.-RQ ; .4.-R

B J₄₄

!) α0! Q7 R+ ) + + B ? Θ

? B !) ) ;_<4444 '

; * B B $ ! ;_<+ 0

B + = + ; Θ B 0 ? !)

* B B 4 <=

; .W_{9+ " ) <=}

!) <= I * + B B <H

<= 0 * Q( ! ] @ + .22.R4 ' α0!

* * ? .W_{9 ; ;}

,--Q 4 .4,, R+ ; I B *

B B = " ; Y- 1- Q .4,,****R

Q# * E ] ( ! + ,--XR4

.4, .4, .4,

.4,,,,,4444 7 <H α0! B * Q RQ RQ RQ R

I B * B B Q*RQ*RQ*RQ*R D

) ;_<4 ' * <H = + I +

7 9 .W_{9 Q( ! ] @ " + ,--5R4}

; ; .W_{9 B}

B <H <= !) <= I *

B B + $ 7 9 ,

+ $ ) +

+ B <= <= <= α — m <=

!) 4 ) B $

I * + B " ; K , _D

; ; ; D

<= α — m4

) * ! + ! B

* + B ; <= α — m 0

0? <= + ? <= 4

# " 7 9 ,

* B B ) ; ) Q∆n_oR+ ;

? ; ? ; <= *

<= ) ;_<+ B ; <= .4..+

RRRR

Δno = 3₂q r_ℎ 3 cos − 1₂ 3 cos − 1₂ Q ;4 .4..RQ ;4 .4..RQ ;4 .4..RQ ;4 .4..R

; q r ℎ⁄ ) ; ) 4

* 7 9 .W₉ , _{= +}

" <= ! <= I * B B +

) <= $ Θ Q ;4 .4.-+ 4 X.R Q ;4 .4..R4 <=

! <= ) ) ,4,41+ 4 WY4

1.5.4 Caracterização teroodinâoica do processo de interação

peptídeo/oeobrana por calorioetria de titulação isotéroica (ITC)

# <= ) Q % R 0 )

" <H <= + ; ) * B ?

<H + <H B * Q@ + .226S \! 4+ .221R4

# $ B ) ? ? * +

* B 0;

<= + ) 4

" <= <= 0 * ) ! "

<= + $0 $ Qu ∆ R

& Qu ∆MR+ *) * <H Q∆u vR+

B ; ) <= QwR <= Q R4 9

<= ) + ? ? = *

* $ 4

9 <= ) ) + B

<= + "H A Q'A8R 4 9 + +

<= + ) ) <= + ; B

A # "= 'A8 * + = <=

* 4 * 0 B ;

! <= 4 * * D <= )

I <= 0 + 0 * <= Q R

+ * + + B ; <= .4.,

* A

= − (Eq. 1.12)

# <= <= ∆_=x ) "=

<= K Qw ₌ D <= + B ; <=

.4.5A

∆=x = w

=

(Eq. 1.13)

9 <= ) )

B = 4 B 0 <= ; ;

" + ; ;

) & D <= = 4 # ; ;

* D <= <= $

<= $ <= <= * 4 9

B <= B 0 + " + = + ;

* ; !$ 4 # + K

<= = + <H B

2. Materiais e Métodos

2.1. Materiais

% B B * < Q( +

( R+ # .YY+ = -+---.4

+ B " 7 F0 + ( ! *

Q F G "+ # ! R * <= -+Y5 / 4

$ 90

B ; ( ! * + 9 * ! 0 F Q +

# ! R @ 0# ! Q@ 4 8 + R4

# <= B B

B Q R+ Q R ,0 Q '#R 4

" B @V9% + B <=

= " 4

" B @V9% Q@= ' + ( R 7 [ Q +

# ! R 4

'# " B @V9% 4

9 + " 0 .0

! * " " Q (%R+ 9 * ! 0 F+ 9+9L0 *

Q R+ @ 0# !4 " * " <=

B + 4

' <= 90 + " 0

<= ,-c / X0 0 Q FR 4

' <= ; <H <= + B "

[ Q[ 4+ .26-R+ ; <H A [ 9 -+-. + B -+1

.A,A.+ + + + ; 0 .--g

X 4

' <=

+ " 0 Q% @R+ ( ! * "

<= $ B ) Q% #R $ + <=

.2A. Q A R4

# B <= B " B ;

B Q 8# R4 ) B <= * ? " B <=

B ,.-0,.W 4 # B <= B " + ;

; B " * B 4

' B ; + " 0 & B Q@ +

R+ ' @ ,.-+ = ' @

55-$ D <= 07! E Q F * + R

# " B * .--0W .1 ,W-hX+Y

" B E .--0W .1 ,W-h.- + ! +

+ Wµ8 ,W-µ84

' B & 8# B " <H -+.c / % #+ $

88 0 + -+-1c / % #+ + " 4

# $ " 0 B * ! '#[+ 0

F Q( + R4

Q R B * ! J#@ J0

1.- Q + R+

' J ' 0X,W@4

B B " Q % R

+ " 22c+ B ; # ' 8 + 4 Q# * +

R4 ' <= B " .0 0,0 0 0

050B B Q' ' R+ .0 0,0 0 0 050B B Q' ' R

7 9 . .5 _{B * 9 7 ?}

) 9 Q 97 9R+ @ K 7J ;

( F # #9 + B ; 1--+.5 " ! 4

+ B " <H ,+,+,0 B 0 ,Q% R $ 0 +

< = <H ; < B B Q ' R

<= .- 4 ' ; <= + " 0 <= X

" <= B -+W 84 =

" B B ,+,0 0,0 0W0 B & Q @@R

Q# !R . .5 ₄

' + B " % @V+ " 0

; 8 + 9 @V . ₊.5 _{0 @ +}

B 4

; <= , . _h. _{9 @V B}

.,- 4 % @V 1- 4

7 9 B & B * @ * +

8 * & 7 9 ( B * + ; ( F # #9 X-- G 0

* Q 5._' .W_{9R ( F # #9 5--G 0* Q} , _R4

2.2 Métodos

2.2.1. Procedioento Geral

% B " B & + " )

Q ! + ,---R+ 0 $ 0 <= I

90 + B * 0 4 #

B " B " I = &

2.2.1.1. Preparação da Resina

<= 4 B B

0 Q7 F0 + -+Y5 / R ;

<= 4 + 0 * ; + B

; B * 4 # B

.- 4 # B + 0 <= $

B " 4 ) +

+ ; B $ 4

2.2.1.2. Desproteção da Resina

# & + 0 I <= 4 #

$ + ; 0

0 4 # <= B B " 0

<= X0 0 9+90 B Q R+ <= ,-c / 4

<= <= B <= + B

* 4 @ * 0 I <= + " .W +

+ 0 <= X0 0 <= $ 4

<= + ; = ! D <= B

<= 4 + 0 + ,0 Q '#R +

+ " 4 ' B " 0 [ <=

4 9 + + D + = * " 4

0 ; ,4.4

; ,4.4

; ,4.4

; ,4.4

; ,4.4 ' <= + +

2.2.1.3. Síntese dos peptídeos

<= + 0 I + ;

$ I 4 # + 0

* <= $ Q 0 $ R

* ? <= .A. Q A R4 '

+ " 0 .0! * " " Q ( R 9+9L0

* Q R+ ; B $

; ; 4 B =

* I <= <= 4 # B + " 0 ;

+ '# + ; B <=

+ + [ ; + & <= +

+ D + = + 4 @

B + * 0 I <= K

4 $ + 0 <= ) ; D 4

# <= B B " 0 <= ,-c / X0 *

+ B * 0 4 B + +

* I <= 4 + <= ) " .,

+ ; <= <= ) B 4

9 + " 0 [ + + 0 &

$ 4 # 0 ) ; $

B 4 # B + Q R ) * I

<= + * + = +

0 4 ) ; ,4,4

; ;;

; ,4,4,4,4,4,4,4,4 " 4 # 0

2.2.1.4. Clivageo dos peptídeos

% B <= 2W+-c $ B ) Q% #R+

,+Wc $ " Q 0 R ,+Wc Q% @R+ 0

= ) Q ! + ,---R4 ' "

+ " 0 <= .- ,W 8/ 0 4 # 0

B ; 0 B *

<= <= + ; )+ + .+W! 5 !4 # B +

0 * " <= D

<= &B ;

* $ = I 4 @ 0 + = + B ; + B 0

* * 0 <= % #+ B

+ B ; + * $ + $ 4

0 ; ! <= ; <= =

" 4 = * Q .- 8R )

B * ! 4 ) B B &

; = 4 9 + 0 ) & +

"+ B 0 0 ; & + 0

" 4 & * + B $

" B + B + B " + " B 4

$ ; ,454

; ,45

; ,45

; ,45

2.2.2. Síntese das fenilseptinas e da Hilaseptina P2

# B Q80'! 0'! R ', Q @',R+ D ; 0

% * ,4.+ B " " 0 .X5+Y 7 F0 +

* <= -+Y5 / + * .W- * B

4 " 0 + + ; ;

; <= I ; B 4 B

" + + WW+X .0! * " " Q ( R W6µ8

9+9L0 * Q R4 " $

; $ 4 ' 7 9 & +

B " + ; +

80'! 0'! + B 802 Q.W_{9R #0.- Q}, _{5R + @',+ #0}

.Y Q 0, _{5R 80.1 Q}.W_{9R+ B % * ,4.4}

% * ,4.4

% *% * ,4.4,4.4

% * ,4.4 @ ; + ; 80 0 +

+ $ B C 8

B + 09 , B ; <=

0 4 # 0 = B 4

$ .W_{9 * ! +}

9 99

9 @ ;@ ;@ ;@ ; ''''

80'! 0 Q80 R %8[9888# [8 #8%09 ,

0'! 0 Q 0 R %8[98888# [ #8%09 ,

@', 0 8[98#[## [##888 #8 @809 ,

# <H <= B + 0

$ " 4 <=

B 5+W ! + + B .+W !4

# & ) + 0 I <=

4 # B " " 0 <=

2.2.3. Reação de Foroação da Distinctina

! ) B " <= <=

B <= <= B . ,+ "

B & + ) 4 # " <= 0 <= + * 0

+ B + <= W / 8 + <=

+ .- * * C + 1+54 # <= B ,X

! I * + + " $ ) 4

# B <= B ! 8# + D 0 ;

! + 4 ' ! <= +

" 0 % * ,4,+ ; . , +

+ <= ,-+- ,-+6 4

,,+, Q " <= R

+ ! <= I . ,4

# B <= <= B

B + 0.1 Q E i _{,.1%'WXR4 B <= B B "}

#8 0% /% + ( F 4

% * ,4,4 % *% * ,4,4,4,4

% * ,4,4 " ! ) <= " <=

4 B B .+- 8/

Tempo (min) TFA, 0,1% em água (%) TFA, 0,08% em água (%)

0 100 0

15 60 40

55 40 60

60 0 100

2.2.4. Preparação de Vesículas para Experioentos de Dicroísoo

Circular (CD)

# B B .0 0,0 0 0 050B B Q' ' R .0

0,0 0 0 050B B Q' ' R B

0 ; * ? + 0 <H

) ! 4 B B

<= I = " 4 B <= $

. ! B B + + ! $ 4

B * 0 =

; + ; $ ,6 _ 4 #

= B + = + * = + .- " + )

* * .-- 4

2.2.5. Experioentos de Dicroísoo Circular

B ; < ' ' A' ' 5A. Q A R

% . , + J 06.W+ " 0

* ; " + ! & .+- $ " 4 %

B ; B ,Y- .2- + " 0 *

W+-,+- % + <=

-+, + .-- / X 4 ! *

B " * 4

% * ) + * Q ! *

! " R * 4

' + 0 <H ; <H X1- j

. , X5- j + * B B

Q. R4 # <H B + + 5-0X-µ +

,,- R4 $ B ; <= ; + <=

I <= 4

# <= % ; +

B " ' Q@ ] \ E+ ,--XR B

$ 4

2.2.6. Processaoento e Análise de Experioentos de RMN eo Solução

# = 7 9 B B 9 7'

Q 4+ .22WR4 # <= B B

9 7 G W4- QJ ! + .22XR4 % " B

B 8 + B * <= +

H 2+ .- ..4

# <H ? B * 9 4

B <= + " 0

* <= a * Q a 4+ .22.R4 B B

9 7 G W4-+ ;

4

# <H B * %#8 @d Q@! + 4+

,--2R+ $ ; $

α+ α+ β+ 9+ 9 L+ <= * ;

$ <= 4

$ D * $ 7 9 B " " 0

` 09 = ,4.6 Q@ !G 4+ ,--5R B

Q R k G4 +

B < ? <= k 4 Q7 ]

( + .22XS @ 4+ .226R4 " 0 C

k 4 " <H 4 + ;

$ B ?

.--- [+ K Q R .X4---+ K

B Y4---4 B B B W- W- [ ) ;

! .-- [4 $ B " A

<= <H ? -+W ^

W_+ + -+5 ^ 5_

B + <= -+. ^ ._4 0 $

; $ = <H

$ ! 4

' $ D <H ) + " 0 9

Q9 * 4+ ,--5R+ I <= <H <H

) 4

% $ B " ,-- 4

K + B ,- +

8 8 Q[ 4+ .22YR+ B * ! * <=

4 * + B * 7 @ Q7 @;

+ ; $ ) R <= $ 4

# <= $ B B ) $

Q! A// 4 * 4 4 / / 4! lB R+ " 0

'7 [ Q8 F G F 4+ .22YR \ #% Q + .22-R+ * )

B <= 9 Q B B 9 7 @ R4

2.2.7. Preparação das aoostras para RMN no estado sólido

' 7 9 & + B "

* .0 0,0 0 0 050B B Q' ' R+

B <H B B + B

% * ,454 % *% * ,454,454

% * ,454 7 <= B B Q' ' R "

7 9 & +

' ' ' ' ' ' ' ' ' ' ' ' ' '' ' ' ' ' ' ' ' ' '

80'! ., .W. 5c

0'! .. .51 5c

@', ., .,, 5c

+ 0 , 8 ,+,+,0 B Q% R

0 " ) 4 ' + * 0 ; ;

<= & * + & <= + B

$ "

! <= 4 B = D 6 <H $

! & & . /84 + <= + 0 ;

' ' <= B * B * ) ;

4 + * 0

B B * .W ,W B Q1 h ,, m+

B + 8 0[n ! B + # ! R * 0 = "

% 4 # B = B

? ! <= 25c ! 4

' B + B ! B B $ 4

2.2.8. Experioentos de RMN no estado sólido

7 9 5._{' ! *}

B " .Y, " C ( F # `X-- G 0

* + " 0 $ ? 7 9

& 4 B B <= *

) 4 ; ! B 4

? B " A D 6W F "+ ; <= .5+Y + ,4-X1

W YX 4 <= $ B B& 1Wc Q R B

" B Q- R4

7 9 .W_{9 ! B ; X- "}

C ( F # `X-- G 0* + " 0 0B

? 7 9 & 4 B " <=

* ) 4 ? B

A 2-_ 1 µ + ! 6-- µ +

5+W + W., + .14--- ; <H

D 55 F "4 " 0 B .W_{9 X D}

!$ X.+W 4

7 9 , _{B & B * C ( F #}

`X--G 0* Q 80'! R+ C ( F # 5-- G 0* Q

R " 0 $ ? 4 B

" <= * ) 4

" 0 ; ; ? A , _{(. W- F "+}

X-µ + D .-- F "+ X4-2Y +

1-4---<= . 4 B * <= I, _,

-+ ; B B

4 <H " <= !

5-- " B B 4

2.2.9. Sioulação de parâoetros da RMN eo fase sólida e deteroinação

da orientação dos peptídeos eo bicaoadas lipídicas

! B 0

! Q\ B 7 !+ 4R ' ( B

<H + " 0 'E 8 Q%! 'E 8

! @E + 4 -422+ @ ! n + 8 R4 ' <= +

! 0 !) "+ + + " 0

<H B ? <= ? <= Q ,4.R+

,4.4 ,4.4,4.4

,4.4 7 <= α0! + <= "

Q * R y+ B !) 4

? Θ+ ? <= + ) B <= y <= "+ + ?

<= + ) 0? B <= <= − _m

* B B 4

2.2.10 Preparação de Lipossooas para Experioentos de Calorioetria

Isotéroica de Titulação (ITC)

' <= + B " ' ' ' ' 5A.

Q A R B B 4 # * ) Q R B *

B ? .-- 4 # <=

; B 4

2.2.10.1. Obtenção do filoe lipídico

# ; B B " B B <=

5W 4 B B B B& B

B * 9, 4 B * & <= B

* = " + + 5-° + 5!+ 4

2.2.10.2. Hidratação do filoe lipídico

B B ! <= = % 0 + 6+Y ;

<= B

* = + * 0 B Q 8 R4

2.2.10.3. Ciclos de aquecioento/congelaoento (“Freeze/Taw”)

# = B B B B * .W 8

Q 8 R B * 0

QoB " 0 GLRA ! 0 * ; $

= + + * ! $ " I B )

4

2.2.10.4. Extrusão

# & 0 + = B * $

" + 5- ° + " Q ,4,R+ ; *

* Q9 + ' + #+ R ?

4 # = B B * = 9,+ "

* X-- ,-- + * = X 1 9,+ + " "

* .-- + * = ., 9,+ * <=

Q8 R4

,4,4 ,4,4 ,4,4

,4,4 .- 8 Q8 ( * + + ( +

$R+ B <= * !

$ " 4 # = 9,Q R $ )

# = B * B <= X ) " <= +

., !4 # H B

& <= + = ! ) 4

# <= B <= + ) ( + *)

! ) B B * Q9 G+ .22-R4 # * <= !

Q8 R * & = B !

" ? Q E 8 ! @ > 8@R4

2.2.11. Calorioetria de Titulação Isotéroica

# <= ) ) B B '0

% 0 ! Q' G E+ R4 ' B " <=

? * / + B B

% A

+ = <= ) )

) + <= 4 9 + "=

/ ) * + = <= <H B

B * 4 # ; ) D <H

Q * R )

<= Q∆ R $ ) * + K

D <= 4

9 + <= ) ) )

<= * ; <= = D 4 # <=

B <= + * 0 <=

$ <= ;

<= 4 # <= *

<= + <= <= Q∆ R4

# <= + ; <=

Q <= R ) = + <H

* D <H B <= + ) * D +

B <= / <= + *D I <= <=

4 # & * <= <= * 0

<= + ; = ? <= ;

.

2.2.11.1. Titulações efetuadas no caloríoetro VP-ITC

9 '0 % B ,45 Q 4 Y,R B B <H

' ' A' ' Q5A.+ A R+ @',4 9 <=

) + B +

* = = A

# ) B B

! Q ,45+ 4 Y,R4 # D <= B 5 µ8+

; D <H * ; + ; $ !

= D <= + ) + !$ B =

* " <= <= 4 D <= = ) <=

) + * ; D D 4

# ) B ! ) 4 # <=

4

+ 0 ; .- 5- " ; <=

* 4

? = ' G ; B " ; <=

<= ! 4 0 % Q 0 ! +

' G E+ R B + B $

<= * + Q + I !

R+ <H ) + & D <= 4

+ B B D ; + I

,454 Q#R ,454 Q#R ,454 Q#R

,454 Q#R <=

; <= 4 Q(RQ(RQ(RQ(R '0 % & 4 Q R

! * * $ + <= B <=

4444

# <= ) B "

<H 5-05W ) +

<H 5X-µ , 4 K D <H .-

W-D <H W ,6µ84 B ) ) .+X,XX

84 <= B B ,W 5W 4

# <= Q _zR <=

<= / * B * <= Q 4 .5YR4

#

3. Resultados e Discussão

3.1. Síntese em Fase Sólida dos Peptídeos Distinctina,

Hilaseptina P2 (HSP2) e Fenilseptinas L-Phes e D-Phes

3.1.1. Síntese da Distinctina

# B " + 0 B

& Q $ % * 54.+ " 0 )

4 # 0 <= B <= <= B

.2+ .+ XW+ ,4

% * 54.4 % *% * 54.454.4

% * 54.4 @ ; C . ,

Monômero Sequência Peptídica

Cadeia 1 H-ENREVPPGFTALIKTLRKCKII-NH2

Cadeia 2 H-NLVSGLIEARKYLEQLHRKLKNCKV-NH2

# B " + ! " Q,--1R4 #

<= ! 0 <= <= 4 ' +

B , 8 <H = 9 X 5 .- + 1+5+

Q . ,R I <= W / 84 # <H

B = K B * <= +

+ ,X !4 <= $ +

* ) + ; B B 8# 4

! B " D <H + 0

% * 54, Q 4 YXR 'C 0 * + +

<= * <= I . , Q

<= ,-+- ,-+6 + R B <= * <=

% * 54,4 % *% * 54,454,4

% * 54,4 " ! ) <= " <=

4 B B .+- 8/

Tempo (min) TFA, 0,1% em água (%) TFA, 0,08% em acetonitrila (%)

0 100 0

15 60 40

55 40 60

60 0 100

65 0 100

54.4 54.4 54.4

54.4 8# ! <= B <= !

<= +

<= . , Q,-+- ,-+6 + R <=

! Q,,+, R4

B <= C &

* $ $B * <H +

0 ; <= * I

4 # B <= <= ,,+, B

#8 0% /% Q 54,+ 4 YWR4 +

* T Ud_{b WX61 / I +}

* 0 *) + $ +

,,+,+ B <= )+ + 4

54,4 54,454,4

54,4 <= <= B <=

<= ,,+, ) ; X F / Q RQ RQ RQ R

) ; X F / Q*RQ*R4Q*RQ*R = ; ) +

/"W2-Y+ WX61+ ,2X2 ,W,,4 5477.836 5905.959 0 200 400 600 800 In te n s . [a .u .]

5000 6000 7000 8000 9000 10000

m /z 2948.671 2522.457 0 500 1000 1500 2000 In te n s . [a .u .]

1500 2000 2500 3000 3500

# B <= ,,+, B = * $

$B 4 "+ " 0 & B 8 + " 0

.-c 1-c -+-1c % # B -+X 8/ + " 0

+ X- 1- _ + .- _ + B

5454 K * * * 0

4 B ;

* $ 4

+ <H $ <= = ; * + 0 +

+ B <= B ) ;

H B <= 4

5454 5454 5454

5454 8 <= <= B <=

<= ,,+, + B

B <= $ 4 # B <H $

= K . > W4

% B <H B

# 0% /% 4 # 54X Q 4 Y6R B <=

+ * )

54X* Q 4 Y6R+ B )

54X4 54X4 54X4

54X4 B <H . > W $ 8 + <=

+ ) ; X F / Q RQ RQ RQ R ) ; X

F / Q*RQ*R4Q*RQ*R

' $ 54X * $ ) T Ud_b

WX1- / + 4 # )

* T Ud_{b 5-.- / + T d U}d _b

RRRR

*R *R*R *R

.

,

5

X

W

.

,

5

X

5-.. / + T U,d_{b .W-W / T d U},d_{b .W-Y / 4 # T U}d ₎

B WY / , QT Ud_{b ,2WX / R+}

0* Q ( R

$ 4 ' + = B B

,4

B T d Ud _{b W2-2 / + * ! B}

<= ) ,4 # ) + B

I . , B ) T Ud_{b ,W,6 / T U}d_{b ,2WX / +}

4 # ) B <= = % * 5454 *

D * $ ; ) D ; ;

B <= + K B <= D <= B <=

) B B <= W4 % * 5454

% *% * 54545454

% * 5454 7 <= ) B <= $B + $ 1-_

<= 54X Q 4 Y6R

Fração Espécie presente*

1 CH2-t_Bu

2 CH2-t_Bu

3 CH2-t_Bu, Homodim_-CH2

4 Dtn, Homodim_-CH2

5 Dtn, CH1*, CH2*

* ,0 ( b , 0*

$ S 0 , b B <=

) ,S > S . b .S , b ,4

9 B <H . 5+ ) * B , 0* Q ,0

( R4 # + "+ B B B <H X W4 *

* B <= X+ $ + K

B <= ; ) " $ $ ) B <= W4

3.1.2. Síntese e Purificação da Hilaseptina P2 (HSP2)

# @', B B + ) Q ! ] \! +

,---R4 ) % * 54X4 <=

B ! [ + ; B <=

$ 4 # + < ; )

K = ; ) " 4 B *) % * 54X+ B

" .W_{9 8.1+} , ₅

#.Y " <= 7 9 B &

Q 5454X4.+ 4 ..YR4

% * 54X4

% *% * 54X454X4

% * 54X4 @ ; @',+

$ + $ .W₉

$ , _{5 * !}

Peptídeo Sequência peptídica

HSP2 G I G D I L K N L A K A A G K A A L H A V G E S L

sentido do acoplamento

# % * 54W ; " + *

<= ) [ 4

+ B $ B & [ +

B % * 54Y Q 4 6-R4 # & = + B "

+ " 0 <= Q 4 XYR * 4 * ,1Y

* B + 2Wc 4

% * 54W4 % *% * 54W454W4

% * 54W4 " @',

Reagente/Suporte Polimérico Quantidade

7 7 F -+Y5 / 21+X

(

# % * 54Y !

4 ! B "0 $ B <H ;

= [ 4 9 < ) "

<= " 4 B + B

<= + B " [ 4

% * 54Y

% *% * 54Y54Y

% * 54Y4 # ! @',

Ordem do Acoplamento

Derivado de

Aminoácido Excesso

Tempo de reação (h)

Teste de Kaiser

Acoplamento Desproteção

1 Fmoc-Leu-OH 4 3 ---- +++

2 Fmoc-Ser(t_{Bu)-OH 4 1,5 ---- +++}

3 Fmoc-Glu(tBu)-OH 4 1,5 ---- +++

4 Fmoc-Gly-OH 4 1,5 ---- +++

5 Fmoc-Ala-OH 4 1,5 ---- +++

6 Fmoc-Ser(t_{Bu)-OH 4 1,5 ---- +++}

7 Fmoc-His(Trt)-OH 4 1,5 ---- +++

8 Fmoc-Leu-OH 4 1,5 +++ ×

Reacoplamento Fmoc-Leu-OH 4 2,5 ---- +++

9 Fmoc-Ala-OH 4 1,5 ---- +++

10 Fmoc-Ala-OH 4 2,5 ---- +++

11 Fmoc-Lys(Boc)-OH 4 1,5 +++ ×

Reacoplamento Fmoc-Lys(Boc)-OH 4 2 --- +++

12 Fmoc-Gly-OH 4 2 ---- +++

13 Fmoc-Ala-OH 4 2 ---- +++

14 Fmoc-Ala-OH 4 2 ---- +++

15 Fmoc-Lys(Boc)-OH 4 2 ---- +++

16 Fmoc-Ala-OH 4 2 ---- +++

17 Fmoc-Leu-OH 4 2 ---- +++

18 Fmoc-Asn(Trt)-OH 4 2 +++ ×

Reacoplamento Fmoc-Asn(Trt)-OH 4 2,5 +++ ×

19 Fmoc-Lys(Boc)-OH 4 2 ---- +++

20 Fmoc-Leu-OH 4 2 ---- +++

21 Fmoc-Ile-OH 4 2 ++- ×

Reacoplamento Fmoc-Ile-OH 4 2,5 ---- +++

22 Fmoc-Asp(tBu)-OH 4 2 ---- +++

23 Fmoc-Gly-OH 4 2 ---- +++

24 Fmoc-Ile-OH 4 2 +++ ×

Reacoplamento Fmoc-Ile-OH 4 2 ---- +++

25 Fmoc-Gly-OH 4 2 ---- +++

d 7 + < 4 0 7 +

4 h <= = " I 4

# <= B " $ Q% #R

? <= 0 <= 4 9

+ <= = ;

+ " ; B ; B

<H <= <= ; $

<H * $ ; <= ;

* $ 4 9 + <H @', =

% * 5464 % * 5464 % *% * 54645464

% * 5464 <= @',

Peptídeo Tempo de Reação(h) Mistura de Clivagem

HSP2 2

95%TFA, 2,5% água,

2,5% TIS

8 & + B B " +

* 2.c4 + B * I $

8# 4 <= <= . / 8 B D ,-µ8 <=

& B 4 B & " 0 % * 541 Q 4

% * 5414 % *% * 54145414

% * 5414 " QB .+- 8/ R $ *

@',

Tempo (min) Água 0,1% TFA (%) Acetonitrila 0,08% TFA (%)

0 95 5

10 75 25

30 60 40

35 0 100

40 0 100

# 54W @',4 9 B +

0 * <= .6+. +

5,c <= -+-1c % #4 + * 0

; " +

* * + B * 4

54W4 54W4 54W4

54W4 @',+ " 0

% * 541 Q * .1+ , A% # -+.c # A% #

-+-1cR4

# 54Y Q 4 65R * * 4

@', /" ,YX- /

90 4

54Y4 54Y4 54Y4

54Y4 @',4

3.1.3. Síntese da [L-Phe

_{]-Fenilseptina (L-Phes)}

# 80'! B B B $ I @',+ ; ;

+ * <= +

0 % * 5424

% * 5424

% *% * 54245424

% * 5424 @ ; 80'! +

$ + $ .W₉

$ , _{5 * !}

Peptídeo Sequência peptídica

L-Phes F F F D T L K N L A G K V I G A L T