guest lecturer: prof. jonathan l. sessler. carbohydrates carbohydrate:carbohydrate: a...
TRANSCRIPT
Guest Lecturer: Prof. Jonathan L. Sessler
Carbohydrates
• Carbohydrate:Carbohydrate: A polyhydroxyaldehyde, a polyhydroxyketone, or a compound that gives either of these compounds after hydrolysis.
• Monosaccharide:Monosaccharide: A carbohydrate that cannot be hydrolyzed to a simpler carbohydrate.
– They have the general formula CCnnHH2n2nOOnn, where nn varies from 3 to 8.
– Aldose:Aldose: a monosaccharide containing an aldehyde group.
– Ketose:Ketose: a monosaccharide containing a ketone group.
Monosaccharides
• Monosaccharides are classified by their number of carbon atoms:
Names and Structures• Monosaccharide: aldehyde or ketone containing at least two additional
hydroxy groups
– Aldehyde - aldose
– Ketone - ketose
– Also named by number of carbons
Monosaccharides• There are only two trioses:
• Often the designations aldo- and keto- are omitted and these compounds are referred to simply as trioses, tetroses, and so forth.
– Although these designations do not tell the nature of the carbonyl group, they at least tell the number of carbons.
Dihydroxyacetone (a ketotriose)
Glyceraldehyde (an aldotriose)
CHO
CHOH
CH2OH
CH2OH
C=O
CH2OH
Monosaccharides
• Glyceraldehyde contains a stereocenter and exists as a pair of enantiomers.
CHO
CH OH
CH2OH
CHO
C
CH2OH
HHO
(S)-Glyceraldehyde(R)-Glyceraldehyde
Fischer Projections• Fischer projection:Fischer projection: A two dimensional representation for
showing the configuration of carbohydrates.– Horizontal lines represent bonds projecting forward. – Vertical lines represent bonds projecting to the rear.– The only atom in the plane of the paper is the
stereocenter.– The more highly oxidized carbon is shown at the top.
C
CHO
CH2OH
OHH
CHO
CH2OH
OHH
(R)-Glyceraldehyde(three-dimensional
representation)
convert to aFischer projection
.
(R)-Glyceraldehyde(Fischer projection)
D,L Monosaccharides
• In 1891, Emil Fischer made the arbitrary assignments of D- and L- to the enantiomers of glyceraldehyde.
• This is an older stereochemical designation that is still used for amino acids and sugars that antedates the Cahn-Ingold-Prelog R/S system.
CHO
H OH
CH2OH
CHO
CH2OH
HHO
D D
L-Glyceraldehyde(S)-Glyceraldehyde
D-Glyceraldehyde(R)-Glyceraldehyde
[α]25 = +13.5 [α]25 = -13.5
D,L Monosaccharides
• According to the conventions proposed by Fischer:– D-monosaccharide:D-monosaccharide: A monosaccharide that has the
same configuration at its penultimate carbon as D-glyceraldehyde; that is, its -OH is on the right when written as a Fischer projection.
– L-monosaccharide:L-monosaccharide: A monosaccharide that has the same configuration at its penultimate carbon as L-glyceraldehyde; that is, its -OH is on the left when written as a Fischer projection.
Note that this designation refers to only one carbon in molecules that often have many stereocenters.
Names and Structures• Sugars are optically active (D vs. L)
– Almost all naturally occurring sugars are D
Fischer projections make it easy to see the “last” carbon.
It is one reason we use them!
• Here are the two most abundant D-aldotetroses and the two most abundant D-aldopentoses in the biological world:
D,L Monosaccharides
D-Erythrose D-Threose D-Ribose 2-Deoxy-D-ribose
CH2OH
CHO
OH
OHH
H
CH2OH
CHO
OH
HHO
H
CH2OH
CHO
OH
OHH
H
CH2OH
CHO
OH
HH
H
OHH OHH
You must know these compounds and their chemistry (and their normal Fischer and Haworth projections)
D,L Monosaccharides
• And the three most abundant hexoses:
CHO
HOHH
HOOHH
CH2OHOHH
HHO
CH2OHOHH
CHO
HOHH
HO
CH2OH
HHOC
OHH
CH2OHOHH
O
D-FructoseD-Glucose D-Galactose
You must know these compounds and their chemistry (and their normal Fischer and Haworth projections)
Amino Sugars
• Amino sugar:Amino sugar: A sugar that contains an -NH2 group in place of an -OH group.– Only three amino sugars are common in nature– N-Acetyl-D-glucosamine is a derivative of D-
glucosamine.
CHO
OHOHHNH2
HH
HOH
CH2OH
CHO
OHOHHH
HH
HOH2N
CH2OH
CHO
OHOHHNHCCH3
HH
HOH
CH2OH
OCHO
OHHHNH2
HHOHO
H
CH2OH
D-Glucosamine D-Galactosamine N-Acetyl-D-glucosamine
4
2
D-Mannosamine
Physical Properties• Monosaccharides are colorless crystalline solids, very
soluble in water, but only slightly soluble in ethanol.– sweetness relative to sucrose:
Carbohydrate
Fructose
Glucose
Galactose
Sucrose (table sugar)
Lactose (milk sugar)
Honey
SweetnessRelative to Sucrose
1.74
1.000.970.74
0.320.16
Invert sugar 1.25
Artificial Sweetener
SweetnessRelative to Sucrose
Maltose 0.33
Saccharin 450Acesulfame-K 200Aspartame 160
Cyclic Structure
• Monosaccharides have hydroxyl and carbonyl groups in the same molecule and those with five or more carbons exist almost entirely as five- and six-membered cyclic hemiacetals.– Anomeric carbon:Anomeric carbon: The new stereocenter
created as a result of cyclic hemiacetal formation.
– Anomers:Anomers: Carbohydrates that differ in configuration at their anomeric carbons.
Haworth Projections
• Haworth projections– Five- and six-membered hemiacetals are represented
as planar pentagons or hexagons, as the case may be, viewed through the edge.
– They are most commonly written with the anomeric carbon on the right and the hemiacetal oxygen to the back right.
– The designation - means that the -OH on the anomeric carbon is cis to the terminal -CH2OH; α- means that it is trans to the terminal -CH2OH.
Haworth Projections
CHO
OH
H
OH
H
HO
H
H OH
CH2OH
HH OH
HHO
HOH()
OH
H
CH2OHO
C
H OH
HHO
HOH
H
CH2OHOH
O
H
OH(α)H OH
HHO
HH
OH
H
CH2OHO
D-Glucose
β-D-Glucopyranose (β-D-Glucose)
α-D-Glucopyranose (α-D-Glucose)
anomeric carbon
+
anomericcarbon
5
5
1
1
redraw to show the -OH on carbon-5 close to thealdehyde on carbon-1
Hint: Drawn this way, the “non-D” OH’s to the right in a standard Fischer Projection go “down” in the Haworth Projection
Haworth Projections
– Six-membered hemiacetal rings are shown by the infix -pyranpyran-.
– Five-membered hemiacetal rings are shown by the infix -furanfuran-.
OO
PyranFuran
Conformational Formulas
– Five-membered rings are so close to being planar that Haworth projections are adequate to represent furanoses.
OH (α)
H
OH
HOHOCH2
HOH
H
HOCH2
H
OH ( )
HOH H
H HO
α-D-Ribofuranose(α-D-Ribose)
β-2-Deoxy-D-ribofuranose(β-2-Deoxy-D-ribose)
Conformational Formulas
– Other monosaccharides also form five-membered cyclic hemiacetals.
– Here are the five-membered cyclic hemiacetals of D-fructose.
HO
HOCH2 OH
HHO
CH2OH
OHH H
C=O
CH2OH
HOH
CH2OH
OHH
HO HOH
HOHOCH2
HO HCH2OH
OH
D-Fructose
1
2
3
4
5
6
55
1
22
()
-D-Fructofuranose(-D-Fructose)
α-D-Fructofuranose(α-D-Fructose)
(α)
1
Ascorbic Acid (Vitamin C)
• L-Ascorbic acid (vitamin C) is synthesized both biochemically and industrially from D-glucose.
L-Ascorbic acid (Vitamin C)
CH2OH
OHH
HHO
OO
OH
D-Glucose
biochemialand industrial
syntheses
CHO
HOHH
HOOHH
CH2OHOHH
Ascorbic Acid (Vitamin C)
– L-Ascorbic acid is very easily oxidized to L-dehydroascorbic acid.
– Both are physiologically active and are found in most body fluids.
L-Ascorbic acid (Vitamin C)
CH2OH
OHH
HHO
O
OH
L-Dehydroascorbic acid
CH2OH
OHH
HO
O
O
oxidation
reductionOO
Conformational Formulas– For pyranoses, the six-membered ring is more
accurately represented as a chair conformation.
OH
HO
H
HO
H
HOHH
OH
OH
OHH
HO
H
HO
H
HOHH
O
OH
OH
HO
H
HO
H
OHOHH
H
OH
OH
H
HO
H
HO
H
OOHH
H
OH
-D-Glucopyranose(β-D-Glucose)
α-D-Glucopyranose(α-D-Glucose)
rotate aboutC-1 to C-2 bond
Conformational Formulas
– If you compare the orientations of groups on carbons 1-5 in the Haworth and chair projections of -D-glucopyranose, you will see that in each case they are up-down-up-down-up respectively.
OCH2OH
HOHO
OHOH()H
H OH
HHO
HOH()
OH
H
CH2OH
O
-D-Glucopyranose(chair conformation)
β-D-Glucopyranose(Haworth projection)
123
4
5
6
1
23
4
5
6
Cyclic Forms of Monosaccharides - details• Sugars often have a choice in forming intramolecular
hemiacetals
• Sugars form intramolecular hemiacetals– New stereocenter formed at anomeric carbon
– For D sugars, S centers are termed α and R centers are – These diastereomers are termed anomers
Cyclic Forms of Monosaccharides - cont.
Cyclic Forms of Monosaccharides - cont.
Which conformation do you think is the most stable? Why?
Mutarotation• Mutarotation:Mutarotation: The change in specific rotation that occurs
when an α or form of a carbohydrate is converted to an equilibrium mixture of the two.
+80.2
+80.2+52.8
+150.7-D-galactoseα-D-galactose
[α] afterMutarotation[α]Monosaccharide
% Present atEquilibrium
2872
6436α-D-glucose
-D-glucose+112.0+18.7
+52.7+52.7
OHOH
HOHO
CH2OHO O
CH2OH
HO
HOOH
HO
[α]D2 5 +18.7
α-D-Glucopyranoseβ-D-Glucopyranose
(β)
(α)
[α]D25 +112
Mutarotation of Monosaccharides
•Conversion of anomers is termed mutarotation and goes through an open chain form
Glycosides
• Glycoside:Glycoside: A carbohydrate in which the -OH of the anomeric carbon is replaced by -OR.– methyl -D-glucopyranoside (methyl -D-glucoside)
HH OH
HHO
HOH
OH
H
CH2OHO
CH3OH H+
-H2O
OCH2OH
H
OH
OCH3H
HOH
OHH
H
OCH2OH
H
OH
HH
HOH
OHHOCH3
(-D-Glucose)β-D-Glucopyranose Methyl β-D-gluco-
pyranoside(Methyl β-D-glucoside)
++
Methyl α-D-gluco-pyranoside
(Methyl α-D-glucoside)
glycosidicbond
Glycosides
• Glycosidic bond:Glycosidic bond: The bond from the anomeric carbon of the glycoside to an -OR group.
• Glycosides are named by listing the name of the alkyl or aryl group bonded to oxygen followed by the name of the carbohydrate with the ending -e-e replaced by -ide-ide.– methyl -D-glucopyranoside– methyl α-D-ribofuranoside
N-Glycosides• The anomeric carbon of a cyclic hemiacetal also
undergoes reaction with the N-H group of an amine to form an N-glycoside.– N-glycosides of the following purine and pyrimidine
bases are structural units of nucleic acids.
HN
NH
O
O
Uracil
N
NH
O
NH2
Cytosine
HN
NH
O
O
Thymine
CH3N
N N
N
H
NH2
HN
N N
N
H
O
H2N
Adenine Guanine
N-Glycosides
– The -N-glycoside formed between D-ribofuranose and cytosine.
H
H
H
H
OHOCH2
HO OH
NH2
O
N
N
anomericcarbon
a -N-glycosidicbond
Reduction to Alditols
• The carbonyl group of a monosaccharide can be reduced to an hydroxyl group by a variety of reducing agents, including NaBH4 and H2/M.
OHOH
HOHO
CH2OHO
CHOOHHHHOOHH
CH2OHOHH
NaBH4
CH2OHOHHHHOOHH
CH2OHOHH
D-Glucitol(D-Sorbitol)
D-Glucose-D-Glucopyranose
Reduction to Alditols
– Other alditols common in the biological world are:
CH2OH
CH2OH
OHHOHH
CH2OH
CH2OH
OHHHHOOHH
CH2OHHHOHHOOHH
CH2OHOHH
D-Mannitol XylitolErythritol
Oxidation to Aldonic Acids
• The -CHO group can be oxidized to -COOH (reducing sugars). Oxidizing agents for this transformation include bromine in aqueous CaCO3 (Br2, CaCO3, H2O), copper(II) in base (Fehling’s solution), and Tollens’ solution (Ag(NH3)2
+). -- Copper bricks and silver mirrors!
OCH2OH
HOHO
OHOH
COHHHHOOHH
CH2OHOHH
O HC
OHHHHOOHH
CH2OHOHH
O O-
oxidizingagent
D-GluconateD-Glucose-D-Glucopyranose(β-D-Glucose)
basicsolution
Oxidation to Aldonic Acids
• 2-Ketoses (also reducing sugars) are also oxidized to aldonic acids. 3-Ketoses, 4-ketoses, etc. are not. Nor are compounds where the carbonyl is tied up in a glycosidic bond.– Under the conditions of the oxidation, 2-ketoses
equilibrate with isomeric aldoses (Step 1 & 2) by keto-enol tautomerization. The aldose is then oxidized to the aldonic acid (Step 3).
An aldoseAn enediolA 2-ketose
CH2OHC=O
CH2OH
C-OH
CH2OH
CHOH
CHOH
CH2OH
CHO
(CHOH)n (CHOH)n (CHOH)n
An aldonic acid
CHOH
CH2OH
COOH
(CHOH)n
(1) (2) (3)
Oxidation to Uronic Acids
• Enzyme-catalyzed oxidation of the terminal -OH group gives a -COOH group.
CHO
CH2OH
OHHHHOOHHOHH
CHO
COOH
OHHHHOOHHOHH
HOHO
OHOH
COOHO
D-Glucose
enzyme-catalyzedoxidation
D-Glucuronic acid(a uronic acid)
Oxidation to Uronic Acids– In humans, D-gluconic acid is an important
component of the acidic polysaccharides of connective tissue.
– It is also used by the body to detoxify foreign hydroxyl-containing compounds, such as phenols and alcohols; one example is the intravenous anesthetic propofol.
O
OHOHO
OH
COO-
HO
Propofol A urine-soluble glucuronide
Carbohydrates
End of Chapter 25End of Chapter 25
Triglycerides
Beeswax contains a component which is an ester of a fatty acid
Vegetable oils contain mostly unsaturated
fatty acids
Tristearin, a saturated triglyceride
A polyunsaturated triglyceride
A Triglyceride
Soaps and Detergents
Steroids
Tetracycylic ring system characteristic of steroids
Cholesterol
Human gallstones are almost pure cholesterol
(continued)
Biosynthesis of Cholesterol
Amino Acids
• Amino acid:Amino acid: A compound that contains both an amino group and a carboxyl group.
α α-Amino acid:-Amino acid: An amino acid in which the amino group is on the carbon adjacent to the carboxyl group.
– Although neutral α-amino acids are commonly written in the unionized form, they are more properly written in the zwitterionzwitterion (internal salt) form. Needless to say, adding acid or base can lead to conversion to other forms.
RCHCOH
NH2
RCHCO-
NH3+
α-Amino Acid Zwitterion form
OO
Chirality of Amino Acids
• With the exception of glycine, all protein-derived amino acids have at least one stereocenter (the α-carbon) and are chiral.– the vast majority have the L-configuration at their α-
carbon.
COO-
CH3
HH3N
L-Alanine
COO-
CH3
H NH3+
D-Alanine
Nonpolar side chains
NH3+
COO-
NH3+
COO-
NH3+
COO-
NH3+
COO-
NH3+
COO-S
NH3+
COO-
NH H
COO-
NH3+
COO-
NH
COO-
NH3+
Alanine (Ala, A)
Glycine (Gly, G)
Isoleucine (Ile, I)
Leucine (Leu, L)
Methionine (Met, M)
Phenylalanine (Phe, F)
Proline (Pro, P)
Tryptophan (Trp, W)
Valine (Val, V)
Polar side chains
NH3+
COO-H2N
O
NH3+
COO-H2N
O
NH3+
COO-HO
NH3+
COO-OH
Asparagine (Asn, N)
Glutamine (Gln, Q)
Serine (Ser, S)
Threonine (Thr, T)
Acidic & Basic Side Chains
NH3+
COO--O
O
NH3+
COO--O
O
NH3+
COO-HS
NH3+
COO-
HO
NH3+
COO-NH
H2N
NH2+
NH3+
COO-N
NH
NH3+
COO-H3N
Cysteine (Cys, C)
Tyrosine (Tyr, Y)
Glutamic acid (Glu, E)
Aspartic acid (Asp, D)
Histidine (His, H)
Lysine (Lys, K)
Arginine (Arg, R)
+
Some Other Amino Acids
NH3+
COO-
NH
H2N
O
NH3+
COO-
H3N
HO O CH2CHCOO-
NH3+
I I
I I
NH3+
-O
O
CitrullineOrnithine
Thyroxine, T4 4-Aminobutanoic acid
(γ- , )Aminobutyric acid GABA
+
Acid-Base propertiespKa ofpKa of
valine 2.29 9.72tryptophan 2.38 9.39
9.102.09threonineserine 2.21 9.15
10.602.00prolinephenylalanine 2.58 9.24
9.212.28methionine9.742.33leucine
isoleucine 2.32 9.76glycine 2.35 9.78
9.132.17glutamine8.802.02asparagine9.872.35alanine
Nonpolar &polar side chains α−NH3
+α−COOH
Acid-Base Properties
pKa ofpKa ofpKa of
10.079.112.20tyrosine
lysine 2.18 8.95 10.536.109.181.77histidine
glutamic acid 2.10 9.47 4.078.0010.252.05cysteine
aspartic acid 2.10 9.82 3.86
arginine 2.01 9.04 12.48
Side Chain
AcidicSide Chains α−NH3
+α−COOH
pKa ofpKa ofpKa ofSide Chain
BasicSide Chains α−NH3
+α−COOH
carboxylcarboxylsufhydrylphenolic
guanidinoimidazole1° amino
SideChainGroup
SideChainGroup
Acidity: α-COOH Groups
• The average pKa of an α-carboxyl group is 2.19, which makes them considerably stronger acids than acetic acid (pKa 4.76).
– The greater acidity is accounted for by the electron-withdrawing inductive effect of the adjacent -NH3
+ group.
NH3+
RCHCOOH H2O
NH3+
RCHCOO- H3O++ pKa = 2.19+
Acidity: side chain -COOH
• Due to the electron-withdrawing inductive effect of the α-NH3
+ group, side chain -COOH groups are also stronger than acetic acid.
– The effect decreases with distance from the α-NH3+
group. Compare:
α-COOH group of alanine (ppKKaa 2.35 2.35)
-COOH group of aspartic acid (ppKKaa 3.86 3.86)
γ-COOH group of glutamic acid (ppKKaa 4.07 4.07)
Acidity: α-NH3+ groups
• The average value of pKa for an α-NH3+ group is 9.47,
compared with a value of 10.76 for a 1° alkylammonium ion.
NH3+
RCHCOO-
H2O
NH3+
CH3CHCH3 H2O
NH2
RCHCOO-
NH2
CH3CHCH3
H3O+
H3O+ pKa = 10.60++
+ pKa = 9.47+
The Guanidine Group of Arg
– This is a special side chain. – The basicity of the guanidine group is attributed to the
large resonance stabilization of the protonated form relative to the neutral form.
CRNH
NH2+
NH2
C
NH2+
NH2
RNH
CRN
NH2
NH2
NH2
CRNH
NH2
H3O+
H2O
+
:
: :
:
:
:
:
:
+
pKa = 12.48
Basicity - Imidazole Group
– The imidazole group is a heterocyclic aromatic amine.
N
NH
H
COO-
NH3+ N
N
H
H
COO-
NH3+
H2O
H3O+
H
COO-
NH3+N
NH3O
+
Not a part of the aromatic sextet;the proton acceptor pKa 6.10+
+
•• +
••
••
••
Titration of Amino Acids• Titration of glycine with NaOH.
Isoelectric Point
• Isoelectric point (pI):Isoelectric point (pI): The pH at which an amino acid, polypeptide, or protein has a total charge of zero.
– The pH for glycine, for example, falls between the pKa values for the carboxyl and amino groups.
pI = 12 (pKa α−COOH + pKa α−NH3
+)
= 21 (2.35 + 9.78) = 6.06
Isoelectric Point
6.115.415.656.066.046.045.745.916.305.685.605.886.00
pKa ofpKa ofpKa of
pI
----------------
----------------------------
--------
valine 2.29 9.72tryptophan 2.38 9.39
9.102.09threonineserine 2.21 9.15
10.602.00prolinephenylalanine 2.58 9.24
9.212.28methionine9.742.33leucine
isoleucine 2.32 9.76glycine 2.35 9.78
9.132.17glutamine8.802.02asparagine9.872.35alanine
Side Chain
Nonpolar &polar side chains α−NH3
+α−COOH
Isoelectric Point
10.76
2.98
5.023.08
7.649.74
5.63
pKa ofpKa ofpKa ofpI
10.079.112.20tyrosine
lysine 2.18 8.95 10.536.109.181.77histidine
glutamic acid 2.10 9.47 4.078.0010.252.05cysteine
aspartic acid 2.10 9.82 3.86
arginine 2.01 9.04 12.48
Side Chain
AcidicSide Chains α−NH3
+α−COOH
pKa ofpKa ofpKa of
pISide Chain
BasicSide Chains α−NH3
+α−COOH
Electrophoresis• Electrophoresis:Electrophoresis: The process of separating compounds
on the basis of their electric charge.– electrophoresis of amino acids can be carried out
using paper, starch, polyacrylamide and agarose gels, and cellulose acetate as solid supports.
Electrophoresis– A sample of amino acids is applied as a spot on the paper strip.– An electric potential is applied to the electrode vessels and
amino acids migrate toward the electrode with charge opposite their own.
– Molecules with a high charge density move faster than those with low charge density.
– Molecules at their isoelectric point remain at the origin.– After separation is complete, the strip is dried and developed to
make the separated amino acids visible.– After derivitization with ninhydrin, 19 of the 20 amino acids give
the same purple-colored anion; proline gives an orange-colored compound.
Electrophoresis– The reagent commonly used to detect amino acid is
ninhydrin.
RCHCO-
O OHO
OOH
NH3+
O
O-
N
O
O
O
RCH CO2 H3O++
An α-amino acid
Purple-colored anion
+ +
2+
Ninhydrin
Polypeptides & Proteins
• In 1902, Emil Fischer proposed that proteins are long chains of amino acids joined by amide bonds to which he gave the name peptide bonds.
• Peptide bond:Peptide bond: The special name given to the amide bond between the α-carboxyl group of one amino acid and the α-amino group of another.
Peptide bonds Peptide bonds
Serinylalanine (Ser-Ala)
H2N HO
O
HHOCH2
Serine(Ser, S)
H2NO
OH
HCH3
Alanine(Ala, A)
+
H2NN
OH
HOCH2
H
H
CH3O
H O
Serinylalanine(Ser-Ala, (S-A)
peptide bond
A dipeptide
Peptides
– Peptide:Peptide: The name given to a short polymer of amino acids joined by peptide bonds; they are classified by the number of amino acids in the chain.
– Dipeptide:Dipeptide: A molecule containing two amino acids joined by a peptide bond.
– TripeptideTripeptide: A molecule containing three amino acids joined by peptide bonds.
– PolypeptidePolypeptide: A macromolecule containing many amino acids joined by peptide bonds.
– ProteinProtein: A biological macromolecule of molecular weight 5000 g/mol or greater, consisting of one or more polypeptide chains.
Writing Peptides
– By convention, peptides are written from the left, beginning with the free -NH3
+ group and ending with the free -COO- group on the right.
H3N
OH
NH O
HN
COO-
O-
OC6H5O
+
C-terminalamino acid
N-terminalamino acid
Ser-Phe-Asn
Writing Peptides
– The tetrapeptide Cys-Arg-Met-As– At pH 6.0, its net charge is +1.
HN
NH O
HN
O-
OO
O
NH2
SCH3
NH
NH2+H2N
OH3N
SH C-terminalamino acid
N-terminalamino acid
pKa 12.48
pKa 8.00
+
Primary Structure
• Primary structure:Primary structure: The sequence of amino acids in a polypeptide chain; read from the N-terminal amino acid to the C-terminal amino acid:
• Amino acid analysis:– Hydrolysis of the polypeptide, most commonly carried
out using 6M HCl at elevated temperature.– Quantitative analysis of the hydrolysate by ion-
exchange chromatography.
Ion Exchange Chromatography
• Analysis of a mixture of amino acids by ion exchange chromatography
• Edman degradation:Edman degradation: Cleaves the N-terminal amino acid of a polypeptide chain.
S=C=N-Ph
H2N COO-HN
NO
R
SPh
H3NNH
R
O
COO- +
+
N-terminalamino acid
A phenylthiohydantoin
Phenyl isothiocyanate
+
Edman Degradation
Edman Degradation Mechanism – It will be on the final!
O
O
FMOC
Cytochrome C
And, we are done!!