Bai Giang Amino Acid 97-03

8/8/2019 Bai Giang Amino Acid 97-03

1/90

AMINO ACID

Nh ng vin g cht o nn s s ng


2/90

Trnh t amino acidc a insulin b(Cng trnh 10 n m c a Sanger-

nh sinh ho ng i Anh t 2 gi iNobel)

Chu i A c a insulin ng i, l n,th v tinh trng c voi ugi ng nhau

Insulin ki msot l ngng trong

mu v c

dng trong yh c i u trb nh ti u

ng

Chu i B c a insulin b, ch, d v

ng a gi ng nhau


3/90

B nh b u c l do c th thi uL-Thiroxin c trong tuy n gip.


4/90

L-Thiroxin l d n ch t c a m t amino acid c tn l Tyroxin.c ng t Tyroxin ng i ta i u ch cL-Dopa dng

i u tr b nh Parkinson

H

L-Tyroxin


5/90

H 2 N H

CO 2 H

R

NH NGH A

L nh ng h p ch t h u cch a ng th ihai nhm ch c:- Nhm Carboxyl : -COOH- Nhm Amino :-NH

2

H u h t cc amino acid u t n t i d ng mu i ion l ng c c (dipolar or

zwitter ion)


6/90

Trong t nhin c kho ng 300 amino acid (AA) nh ng ch c

20 AA c tm th y trong protein:AA u tin c tm th y l Asparagin(1806) trong h t cc loicy h u.

AA cu i cng trong s 20 aa trn c tm th y l Threonin(1938).


7/90

CC CC

OO

OO

CHCH33SSCHCH22CHCH22

HH

HH33NN++

M ethionineM ethionine(Met or M)(Met or M)

CC CC

OO

OO

CHCH22OHOH

HH

HH33NN++

SerineSerine(Ser or S)(Ser or S)

Ngoi ra ch a cc nhm ch c khc : -SH, -OH,

V d v amino acid


8/90

HistidineHistidine

CC CC

OO

OO

HH

HH33NN++

CHCH22

NHNHNN

(His or H)(His or H)

ProlineProline

CC CC

OO

OO

CHCH22

HH

HH22NN++

HH22CC

CCHH22

(Pro or P)(Pro or P)

V d v amino acid


9/90

20 Amino acid c trong trong protein


10/90

20 amino acid ctrong protein


11/90


12/90

C I M C U T O

Ngo i tr Glycin, t t c cc - AA c phn tch tprotein u ch at nh t m t nguyn t carbon b t ix ng. Do v y, chng quang ho t v chng ch thu c vdy L.

D-amino acid c tm th y r t t, ch y u mng t bo c am t s vi khu n ho c m t s khng sinh.


13/90

1.Valine (Val,V).

2. Leucine ( Leu,L).3. Isoleucine (Ile ho c I).4.Threonine (Thr ho c T).

Amino acid quan tr ng

5.M ethionine (M et,M ).

6.Lysine (Lys, K).7.Phenylalanine (Phe, F).8.Tryptophan (Trp, W).

Nh ng AA thi ty u m cn ng i khng t t ng h p c

Cc aa nhm ny c i u ch trong cng nghi p v cb xung vo trong th c ph m.


14/90

Ngu n b sung AA cho con ng i


15/90

I U CH AMINO ACID


16/90

I. Sinh t ng h p:y

Ch c th c v t v cc vi sinh v t c kh n ng t t ng h p cc AA c n thi t cho c th .y a s cc ng v t khng t t ng h p c cc AA

m ph i c a vo b ng con ng n u ng qu

th c n.y Cc vi sinh v t i t 2- oxoacid (chu trnh citric) t ngh p AA.


17/90

II. CC NGU N CUNG C P AAQUI M CNG NGHI P

Cc aa c s n xu t nhi u nh t theo th t :L-Glutamic (ch t d n truy n th n kinh):250.000 t n/n mDL-Methionin (b xung vo th c n gia sc)L-Lysin(t ng gi tr dinh d ng):25000 t n/n m.

L-Lysin v L-Glutamic thu cch y u t cc qu tr nh ln men.V d :T 1000 kg D-Glucosec th t o ra500 kg L-Glutamic.


18/90

Khc v i cc h p ch t khc, t ng h p amino acid bao gi c ng thu c Racemat (D/L) nn c n ph i phn tch thu c ng phn L.

V d : dng enzym amino acylase th khi thu phn (D,L)-N-acyl amino acid th ch ng phn L b thu phn thu

c L-amino acid t ng ng.

III. T NG H P HA H C


19/90

y Ph ng php Gabriel:y Nguyn li u u l cc acid -halogeno carboxylic

I U CH AM INO ACID


20/90

y i t Kali phtalimid:

Ph ng php ny cho hi u su t cao

I U CH AM INO ACID


21/90

y P H NGP HP Strecker:


22/90

B i t p : Vi t s t ng h p Valineb ng ph ng phpStrecker ?


23/90

TR L I: i t 2-Methylpropanal


24/90

PH NG PHP Erlenmeyer y i t Glycine:


25/90

1. Sinh t ng h p2. Thu phn protein3. T ng h p ho h c

Ph ng php Gabrieli t Kali phtalimid

Ph ng php Strecker Ph ng php Erlenmeyer

TM

T T I U CH AM

INO ACID


26/90

TNH CH T HA H C


27/90

TNH ACID BASE C A AMINO ACID

Khi tan trong n c, m t aa k t tinhs t n t i trong dung d ch d id ng ion l ng c c (dipolar ion or zwitterion ).

N c th ng vai tr nh m t acid:

Ho c c th ng vai tr nh m t base:


28/90

Nh v y, aa l m t ch t l ng tnh (amphoteric)

M i m t aa c m t ng cong chu n c tr ng.D a vo ng cong ny, ng i ta c th bi t c m i linquan gi a l ng i n tch c a aa v i pH c a dung d ch.V d , i v i Glycin:


29/90

ng cong chu n c a dung d ch Glycin 0,1 M25 0C.

i m ng i n pI(Isoelectric point) :L gi tr pH m t i d ng ionl ng c c l l n nh t, t c l t ng i n tch m v i n tchd ng c a aa b ng nhau.V amino acid khng di chuy ntrong i n tr ng.

Gi tr pI c tr ng cho t ngaa.

pI = 1/2* (pK1 + pK2)=1/2* (2,34 + 9,6 )

= 5, 97


30/90

ng cong chu n c a Glutamat

Gi tr pK Ac a nhm R ck hi u l pKR

Gi tr pI l gi tr trung bnhgi a d ng mang i n tch -1

(pK

R) n m cn b ng v i d ngmang i n tch + 1 (pK1).T c l:pI = 1/2*( pK1 + pKR )

= * (2,19 + 4,25)

= 3,22


31/90

ng cong chu n c a Histidin

Bi t p:

D a vo d li u hnh bn,

tnh gi tr pI c aHistidin ?


32/90


33/90

P h n ng c a nhm Amino ( NH2 ):1. Ph n ng v i Ninhydrin:

T NH CH T HA H C

S n ph m cu i cng c mu xanh tm (tr prolin cho s n ph mmu vng). Dng cho phn tch nh tnh v nh l ng aminoacid.


34/90


35/90

2 . Ph n ng v i acid nitr (HNO2) :

y l ph ng php c i n dng nh l ngnhm NH2

T NH CH T HA H C


36/90

3 . Ph n ng acyl ha :

Khi cho m t AA tc d ng v i m tanhydrid acid ho c m thalogenid acid v i s c m t xc tc base s thu cd n ch t N-acyl ha . y ph n ng c s t ng h p

peptid.

T NH CH T HA H C


37/90

4 . Ph n ng ng vng :Cc -amino acid khng b proton ha nhm NH2 khi

un nng t o cc d n ch t2,5 -dioxopiperazin .

Cc -amino acid khi un nng t o ra cc acid khng no

ethylenic

T NH CH T HA H C


38/90

Cc , ho c -amino acid khi ung nng d btch n c ng vng t o amid n i phn tg i l lactam.

T NH CH T HA H C


39/90

5 . Ph n ng v i 1-Fluoro-2,4 -dinitrobenzen :

Ph n ng trn l c s xc nh nhm amino t do cu im ch trong chu i polypeptid ho c protein.

T NH CH T HA H C


40/90

P h n ng c a nhm COOHQuan tr ng l ph n ng ester ha v i alcol trong mi tr ngacid (tr nh c ph n ng ng vng t odicetopiperazin )

C ng c th tr nh ph n ng ng vng b o v b ng cchacyl ha nhm amino t o nhm b o v

T NH CH T HA H C


41/90

T ester c a amino acid ng i ta i u ch c cc d n ch tch a nhm azid -N3, l nh ng nhm r t ho t ng, ng vai trquan tr ng trong t ng h p peptid.

T NH CH T HA H C


42/90

A.P h n ng c a nhm Amino ( NH2 ):1. Ph n ng v i Ninhydrin2. Ph n ng v i acid nitr (HNO2)3. Ph n ng acyl ha4. Ph n ng ng vng5. Ph n ng v i 1-Fluoro-2,4-dinitrobenzenB . P h n ng c a nhm COOHQuan tr ng l ph n ng ester ha v i alcol trong

mi tr ng acid

TMT T V T NH CH T HO H C


43/90

TNH CH T C A M T S NHM CH C KHC Nhm ch a S :

Nhm mercapto d b oxi ha t o lin k t disulfid S-S-, y l lo i lin k t c tm th y nhi u trong peptid v protein nhinsulin,


44/90

RS H RS SROxidation

Reduction

REVERSIBLE DENATURING

Dr. Frederick Sanger,Nobel Prize for Chemistry1958 and 1980Peptide sequencing

Prof. R. B. MerrifieldNobel Prize for Chemistry 1984

Automated Peptide Synthesis

Prof. Linus Pauling


45/90

(CH 2)4NH2

H2N C

HN

O

S

CNH

O

C

Ph

OH

O

S

CNH

O

OHC

O

HN

HOO

H2N

Ph

Lys. Cys. Phe

Phe. Ser. Cys

1. RSH

2. 6 M HCl hydrolysis

Lys + 2 Cys+ 2 Phe + Ser


46/90

PEPTID

Oxytocin l m t trong nh ngs hormon quan tr ng nh t m

c cc thy sau c a tuy n

yn ti t ra.Oxytocin kch thch s co t

cung thc .


47/90

V b n ch t,Oxytocin l m t nonapeptid (chu i g m 9 n vamino acid ), c tch tinh khi t vo n m 1952 .


48/90

Insulin ch a51 n v L-amino acid g mhai m ch Polypeptid.

M ch A ch a21 n v AA.M ch B ch a 30 n v AA.

Insulin ng i, l n, c u, b, ng a,th ng khc nhau v tr 8,9,10 v 30.

ng i:

v tr 8,9,10: Thr -Ser -Ile (l n t ng t ) v tr 30 : Thr. (l n l Ala)T insulin c a l n, i u ch bn t ng h pthay Ala b ngThr thu c insulin c a ng i


49/90

LINK T PEPTID


50/90

S t o thnh lin k t peptid


51/90

Pentapeptid: Ser -Gly-Tyr - Ala-Leu.


52/90

Tetrapeptid: Ala-Glu-Gly-Lys c tnl Alanylglutamylglycyllysine.


53/90

Cc peptid c t o thnh b i s lin k t amid l p i l pl i gi a nhm amino NH2 c a amino acid ny v i nhmcarboxyl COOH c a m t amino acid khc.


54/90

Lin k t amid CONH- do c s lin h p c a c p e t do trnnguyn t N v i nhm carbonyl CO- nn lin k tC-N c m t

ph n t

nh ch t c a lin k t i => h n ch s quay t do. di lin k t: lin k t iC=N: 1,25 A0.lin k t nC-N : 1,47 A0. lin k t amidC-N: 1,325 A0.

Cc peptid u tin c u d ng trans.


55/90

PHNTCH AMINO ACID C A CC PEPTID

Phn tch a mino acid : - M t peptid hay protein c phn l p v tinh ch sau

c th y phn thnh cc h p ph n amino acid m c thc phn tch c v nh tnh l n nh l ng.

- B c u tin trong vi c nghin c u c u trc m ch peptid l phn tch nh m cu i, ngh a l nh n bi t nhm n m cu im ch:1) Amino acid ch a nhm amino t do-NH2 cu i m ch.2) Amino acid ch a nhm carboxyl t do COOH cu im ch.


56/90

A. Phn tch a mino acid ch a nh m amino t do NH2cu i m ch (N-ter minal analy s is )

1. Ph ng php FDNB (1-Fluoro-2,4-dinitrobenzen)2. Ph ng php Dansyl chlorid (5-(dimethylamino)naphthalen-1-sulfonyl

chlorid)3. Ph ng php Phenylisothiocyanate (PITC)/ Edman reagent4. Aminopeptidase

B.Phn tch a mino acid ch a nh m carboxyl t do COOH cu i m ch (C-ter minal analy sis )

- Carboxypeptidase

PH NG PHP PHNTCH AMINO ACID


57/90

Nhm amino cu i m ch ph i c th theo cch no m d n ch t nh n c ph i kh b thy phn.M t tc nhn thch h p cho m c ch ny l 1-fluoro-2,4-dinitrobenzen.Khi peptid hay protein b thy phn th nhm 2,4 dinitrophenyl(DNP) v n cn g n vo amino acid cu i m ch.

Ph ng php F DNB (1-Fluoro-2,4 -dinitrobenzen)


58/90

O 2 N


59/90

Ph ng php Dansyl chlorid

S d ng Dansylchlorid

u i m: c nh y cao


60/90

ph ng php ny, Dansylchlorid c cho ph n ng v i

amino acid cu i m ch.

Sau khi th y phn chu ipeptid thu c s n ph m l Dansylamino acid.

Ch t ny pht hu nh quang khib chi u x t ngo i.

S pht hi n b ng ph ngphp ny nh y g p 100 l n sov i ph ng php FDNB.


61/90

Ph ng php Phenylisothiocyanate (PITC) (thoi bi n Edman)

y l qu tr nh thoibi n quan tr ng nh t,theo 3 b c :

B c 1 : t o ra d n ch tPhenylthiocarbo myl-Peptid (PTC-peptid).


62/90

B c 2: C t lin k t peptid t o ra d n ch t c aThiazolinon(d n ch t c a2-Phenylthiazol-5-on).


63/90

B c 3 :Th y phn ch t cu i ny d n n vi c m vng v ngvng l i cho m tPhenylthiohydantoin ( PTH ) b n

c a amino acid cu i m ch.Ch t ny nh n bi t b ngHPLC.


64/90

Qa tr nh ny c th l p l i v i m ch peptid cn l i, t okh n ng ti p t c nh n bi t th t cc AA trongm ch (40-60 AA).

Ph ng php Phenylisothiocyanate (PITC) (thoi bi n Edman)


65/90

T NG H P PEPTIDBi ton :T h n h p g m Glycine(Gly, G) v Alanin(Ala, A)c th thu c bao nhiu dipeptid ?Tr l i: Thu c 4 dipeptid: G- A, A-G, G-G, A- A.S n ph m khng mong mu n:- Peptid cao h n- T o d n ch t dicetopiperazin,

Cu h i:Lm th no t ng h p c G- A-G-G, A-G- A-G- A,t Gl y v Ala ?


66/90

QUY TRNH T NG H P PEPTID1. a nhm b o v c n thi t t ng ng

- B o v nhm Amino (-NH2).- B o v nhmCarboxyl (-COOH).- B o v nhm ch c khc: -SH, -OH,..

2. Ho t ha nhm carboxyl v ti n hnh ph n ng ghp n i.3. M kha, phn c t cc nhm b o v th c hi n b cm i.


67/90

B o v nhm amino (-NH2):

T o nhm Benzyloxycarbonyl (Z hay Cbz: carbobenzoxy)T o nhm t-Butoxycarbonyl (Boc).T o nhm (Fluoren-9-yl)methoxycarbonyl (Fmoc).

B o v nhm carboxyl (-COOH): C ng nhm amino ph n ng c t do, khng c st o mu i.B o v nhm COOH nh ng khng c qu ho t ha.

B o v b ng cch t o ester v i cc alcol: Methylic(Met),

Benzylic(Bn), t-Butylic,* i u quan tr ng i v i nhm b o v c s d ng l c n

ph i lo i b d dng m t khi lin k t peptid c hnh thnhm ng th i khng lm phn li lin k t ny.

M


68/90

CH 3

H 3 N

O

O

Protecting NH 2

O

O ClPh

Benzyl Chloroformate

CH 3

N

O

O

H

O

OPh

CH 3

N

O

O

H

Cbz

Cbz-Cl

H 2 , PtO 2

De-Protect

CH 3

H 3 N

O

O Fmoc-Cl

Base

CH 3

NH

O

OO

O

O

O

Cl Fmoc-Cl=

B O V NH M NH 2

B M


69/90

CH3

N

H

Boct

O

OH=

CH 3

N

H

C

O

OHO

OCH3C

CH 3

CH 3

(Boc) 2O

O

O O

O

O

Di-tert-butyl dicarbonate (Boc-anhydride)

CH 3

H3N

O

O

PEPTIDE SYNTHESIS

PROTECT

De-PROTECT

m ild acid and neutralize

CH 3

H3N

O

N

H

COO

Leu

B O V NHM NH2

B O NHMNH


70/90

CH2OCOCl + H2 NR OH-

25CH2OCONHR + Cl-

C H3 + CO2 +H2 NR C H2Br + CO2 +H2 NR

HBr ace ic acid

(cold) H2 / Pd

(CH3)3COCOC(CH3)3

O+ H2 NR base25 (CH3)3COCNHR

O

+ (CH3)3CHO

tert-Butyloxycarbonylor boc group

(CH3)2C=CH2 + CO2 +H2 NR

Benzyl chloroformateBenzyloxycarbonyl

or Z group

HCl or CF3CO2Hace ic acid, 25

B O V NHMNH2

B O V NHMCOOH


71/90

NH2

CH3CH2O H , H+

NH2

C

O

HO

O

E tO

acid or ba se hydroly s is

Much Milder Conditions are required to Break an ester as compared to an amide bond.

O R

NH2

O

HO

H

NH2

O

O

isobutene in sulfuric acid

H+, H2O

HE

AT

SN1 mechanism

B O V NHMCOOH


72/90

H 2 / Pd25C6H 5CH 2OCR

O

C6H 5CH 3 HOCR

O

+

M KHA NHMB O V -COOH


73/90

Ho t ha nh m carboxyl v ti n hnh ph n ng ghp n i

*Ph n ng c m u b ng s t n cng nucleophin t

nhm amino (-NH2) c a m t AA ny vo nguyn t C c anhm carboxyl (-COOH) c a m t AA khc.* Do nhm OH gy hi u ng p- y e vo lm gi m m t

i n tch d ng trn nguyn t C => p km =>C n anhm ht e vo t ng hi u qu p (Nhm carboxyl ph i

c ho t ha)


74/90

PH NG PHP DDC CH3

N

H

Bot

H2N

O

OR+

N C N

Di yc loh ylca rbodiimid (DCC)

CH3

N

H

Boct

O

N

H O

OR

N C N

H H

ODicyc loh ylur a (DCU)

O

OH


75/90

Z _ NHCHC _ OH

R

O(1) (C2H5)3 N(2)ClCO2C2H5

Z _ NHCH _ C _ O _ C _ OC2H5

OO

R Mix ed a hydr ide(

Ho c t t nh t l dng ph ng php chuy n ha nh m carboxyl AAdng l m b o v thnh h n h p anhydrid

b ng cch s d ng Ethyl chlorofor mat.

Z _ NHCH _ C _ O _ C _ OC2H5

OO

R

H3 N+CHR'CO2- Z _ NHCH _ C _ NHCHCO2H

O

R R'

+ CO2 +C2H5OH


76/90

V d :


77/90

Ph ng php t ng h p peptid t ng(ph ng php Merrifield )


78/90

Ph ng php t ng h p peptid t ng(ph ng php Merrifield )


79/90

PROTEIN


80/90

1 -[4-et y-3-( , -di ydr -1 -met yl-7-oxo-3-pr opyl-1 H -pyr olo[4,3-d ]pyrimidi -5-yl)phenyl lf onyl]-4-methylpiper ine itr te


81/90


82/90


83/90

V iagra c nghin c u nh thu c ch avim h ng.Trong qu tr nh th nghi mng i ta pht hi n ra m thi ung ph c a thu c ny l lm t ng c ng c ng c ad ng v t

ng i dng

NH NGH A:


84/90

NH NGH A:

1 .L h p ch t cao phn t c c u trc ph ct p, c trong m i b ph n c a c th s ng.

2 . Thnh ph n :- C = 54 % ; H = 7 %; O = 22 % ; N = 16 %- Ngoi ra cn c ch a m t s vi l ng

nguyn t khc : S, Fe, Ca, Zn,


85/90

L o iprotein Ch n ng d

Proteinc u trc C u trc, nng

Collagen v lastin t o nn c u trc s i r tb nc a m lin k t, dy ch ng, gn. Keratin t o nnc u trc ch c c a da, lng, mng. Protein t nh n,t t m t o nn b n v ng c a t nh n, v kn

Proteinn yme

Xc tc sinh h c:t ng nhanh, ch n l cccph n ng sinh ha

Cc n yme th yphn trong d dyphn gi ith c n, n yme mylase trong n cb t phngi i tinh b t chn, n yme Pepsin phn gi iProtein, n ymeL ipase phn gi i L ipid

ProteinHormon

i u ha ccho t ngsinh l

Hormone nsulin v lucagon do tbo o t ythu c tuy n t y ti t ra c tc d ng iu ha hml ng ng lucose trong mu ng v t cx ng s ng


86/90

C U TRC:


87/90

Ho t tnh sinh h c c a protein khng nh ng ty thu c vo c utrc ha h c m c

Bi t cn th thu c vo c u trc khng gian tinh vi.PHN LO I THEO C U TRC:1. C u trc b c m t: hay tr nh t cc AA.2. C u trc b c hai :Lin quan n cc cn i v c u d ng c a

cc m ch polypeptid3. C u trc b c ba: (c a cc protein hnh c u) Cc c u trc ny th ng v t xa kh i c utrc b c hai di n s u n g p khng gian th ng c gi

v ng b i cc c u isulfidV cc t ng tc n i phn t khc.4. C u trc b c b n:Khi protein c nhi u chu i polypeptide ph i

h p v i nhau th t o nn c u trc b c b n c a protein.Ccchu i polypeptide lin k t v i nhau nh cc lin k ty u nhlin k t hy ro.


88/90


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90/90

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Bai Giang Amino Acid 97-03