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Chem 215 F11 Notes – Dr. Masato Koreeda - Page 1 of 7. Date: December 9, 2011
(6) Protein structure: Amino acid sequencing (Chapter 23.6) The structural analysis of proteins, i.e., amino acid sequencing, is primarily achieved by the use of modern mass spectroscopic methods such as MALDI (matrix-assisted laser desorption/ ionization) mass spectroscopy, even proteins with larger than 300,000 molecular weight. However, extremely large protein molecules are often partially digested into mid-sized peptides by using protease enzymes such as trypsin and chymotrypsin and those smaller peptides are analyzed by mass spec. These enzymes are selective, giving cleavage at predictable points in the protein chain. Trypsin: Cleaves the chain at the carboxyl group of the basic amino acids lysine and arginine. Highly specific. Does not cleave when the next AA is proline (to the C-terminus direction). Chymotrypsin: Cleaves the chain at the carboxyl group of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Does not cleave when the next AA is proline (to the C-terminus direction). Show where trypsin and chymotrypsin would cleave the following peptide. Tyr-Ile-Arg-Leu-Gly-Phe-Lys-Asn-Trp-Phe-Gly-Ala-Lys-Gly-Gln-Gln-NH2
N-Terminus determination with Sanger’s reagent, 2,4-dinitrofluorobenzene
Electron deficient aromatic compounds with a leaving group, usually a halogen, undergo a nucleophilic aromatic substitution reaction. For example, 2,4-dinitrofluorobenzene reacts with a basic amine to produce amine-attached aromatic product.
FN
N OO
O
O
2,4-dinitrofluorobenzene(Sanger's reagent)
H2N-R NH-RN
N OO
O
O
The reaction mechanism involves the formation of a carbanion intermediate, by the addition of a nucleophile at the carbon bering a halogen atom, that is highly stabilized by resonance with C=C & two N=O groups.
Chem 215 F11 Notes – Dr. Masato Koreeda - Page 2 of 7. Date: December 9, 2011
FN
N OO
O
O
H2N-R FN
N OO
O
O
N RH H
NN
N OO
O
O
H HR
highly acidicB
NH-RN
N OO
O
OThe negative charge onthe ring highly stabilizedby resoance involving a C=C and two nitro N=Os.
Mechanism:
Application to the N-terminus AA residue determination:
+ +
H2NNH
HN
OH
CH2OHH
O
O
O
H
Ser-Gly-Val
FN
N OO
O
OHN
NH
HN
OH
CH2OHH
O
O
O
H
N
N OO
O
O
H3NOH
O
HN
OH H3NOH
CH2OHH
O O
H
N
N OO
O
O
Cl
ClN-2,4-dinitro-phenylserine
yellow colored
valineglycine
HCl, H2O, Δ
Chem 215 F11 Notes – Dr. Masato Koreeda - Page 4 of 7. Date: December 9, 2011
• The use of the HO group as a nucleophile (as its anion) in this reaction is remarkable; the pKa of HOCH2R is about 16 and the pKa of the conjugate acid of histidine base is around 6-8. This process is made thermodynamically favorable participation of Asp-102 in stabilizing the imidazolium cation of His-57. • The activity of enzymes is a powerful demonstration of the effect that lowering the energy of the transition state can have on the overall rate of the reaction.
Chem 215 F11 Notes – Dr. Masato Koreeda - Page 5 of 7. Date: December 9, 2011 Polynucelotides: DNA and RNA (Chapter 23.9)
(1) Nucleosides: the bicyclic purine (adenine or guanine) or monocyclic pyrimidine base (thymine, uracil, or cytosine) joined from a ring nitrogen to carbon 1 of a pentose sugar; in the ribonucleic acid (RNA) system, the pentose is D-ribose which is locked into a five-membered furanose ring and in the deoxyribonucleic acid (DNA) system the pentose is 2-deoxy-D-ribose.
Chem 215 F11 Notes – Dr. Masato Koreeda - Page 6 of 7. Date: December 9, 2011
(2) The H-bonding between nucleic acid bases and the double helix structure of DNA
NN
N
NN
NN
N
NO
N
NN
N
O
NN
O
OHH
H
HH
C1,C1,
H
HH
T AC GC1,
C1,
Watson-Crick base-pairing for C-G (left) and T-A (right).
(3) Synthesis of ribonucleosides
With heavy metal salts of bases:
N
NN
NHN
O
AcO
AcO
N
NN
NHN Ph
O
HgClO Br
OAcAcO
AcO
OAc
Ph
O
xylene120 °C
N
NN
NNH2
O
HO
HO
OH
NH3MeOH
Ac =H3C
O(acetyl)
Chem 215 F11 Notes – Dr. Masato Koreeda - Page 7 of 7. Date: December 9, 2011
(4) Synthesis of 2-deoxyribonucleosides
N
NN
NCl
NaO
TolO
TolO
ClSN2!
N
NN
NCl
O
TolO
TolO
71% Tol =
OCH3
(5) Formation of oligonucleotide bonds phosphodiester method
N
NN
NHN
O
HO
O
Ph
O
PhH3CO
N
NN
NHN
O
AcO
O
Ph
O
PO
OO
+
SO
OCl
pyridine
2. aqueous workup
1.
N
NN
NHN
O
AcO
O
Ph
O
PO
N
NN
NHN
O
O
O
Ph
O
PhH3CO
O
H3CO
H3CO
N
NN
NNH2
O
HO
O
PO
N
NN
NNH2
O
O
HO
O
1. Cl3CC(=O)OH (trichloroacetic acid)
deprotection of 4,4'-dimethoxytriphenylmethyl
group
2. NH3-CH3OH