Chem 27 - Exam 1 Review

Wednesday Feb. 22, 2006Science Center Hall D

K.C. O’Brien

Carol Fang

Walter Kowtoniuk

Outline of Topics

• 1) Conformational Analysis of amino acids• 2) Protein Folding

• 3) Edman Degradation(-like) chemistry• 4) Cyanogen bromide(-like) chemistry

• 5) Peptide Coupling/Synthesis• 6) Biosynthesis of Proteins

Conformational Analysis

K.C. O’Brien

Amino Acid Structure

-ONH3

+

O

RH

•Amino acids are chiral molecules

•Stereochemistry at -carbon always as shown (R group coming out)

•All natural amino acids have S configuration, except cysteine

•pKa’s: NH3+ is about 9

COO- is about 2.2

•Hydrophobic, polar and charged side chains

Staggered vs. Eclipsed Conformation

HH

H

H

H H

H

HH

H

HH

+3.0 kcal/mol

staggeredeclipsed

• Hyperconjugation C-HC-H• Newman projections help visualize interactions

Gauche interactions

CH3

H H

CH3

HH

+0.9 kcal/mol

anti gauche

H

H CH3

CH3

HH

Cyclohexane Chair Conformations

H

H3C

H

H3C

CH3

H

H

CH3

• Ring flip changes groups from axial to equatorial• Lower energy conformation has large groups equatorial• A values are used to quantify the energy difference

between the axial and equatorial positions

Syn-pentane Interaction

• Syn-pentane > 3.7 kcal /mol

• 1,3-diaxial groups generate a syn pentane interaction H

H

H

H

H

H

H's cause steric strain

A1,3 Strain

H RR"R'

12

3

HHH

H

HHH

H

• H is in the same plane as double bond

• If R=R’=R”=Me, A1,3=3.5 kcal/mol

• Minimize A1,3 in amide bonds

Template Projection of Amino Acids

NH

H

O

NH

H

O

NH R1

H

R2

O

R1 R3

NH H

H

HR1 O

R2

• Amino acid template projection is based on cyclohexane chair structure• Add up gauche and syn-pentane interactions to find the lowest energy conformation• R1>R2>R3 is a good place to start, but consider other conformations• Make sure you don’t invert the stereochemistry of the amino acid or its side chain!!!!

Protein Folding: Hydrogen Bonds

O H N

R

RR

R

:

• 1-4 kcal/mol• Directionality is

important• N-H-----O=C• Stabilize -helices,

-sheets and turns

Protein Folding: -helix

• stabilized by hydrogen bonding• 3.6 amino acids per turn

Protein Folding: -sheet

• NH’s of one strand H-bond to C=O of next strand• R groups alternate on opposite sides of the plane

Protein Folding: -turn

• C=O and N-H are 10 atoms apart

• Changes the direction of the main chain

Protein Folding: Electrostatic Interactions

O

O-R

N

NR

H

NH2H• Between oppositely

charged amino acids• Most important in

the interior of the protein

• Neutralizes charges

Protein Folding:

Hydrophobic Interactions:

• Hydrophobic amino acids pack into the interior of the protein

• Folding increases the disorder of the solvent

• Positive H is overcome by positive S

Disulfide Bonds:

• Dihedral angle 90o

• ns donates into *S-R

• Two Cys oxidized to form a disulfide bond

Edman Degradation

Carol Fang

N

S

NH

Ph

O

H2N

H

R1 N

S

NH

Ph

R1

O

+ NH2

O

R2

Nucleophilic Amine (primary and secondary)

E and Nu are 5 atoms apartRotatable bond

Thiazolinone DerivativeKinetic product New N-terminal

S

C

N

Ph

H2N

HN

R1

O R2

O

N

HN

R1

O R2

O

N

S

Ph

A H

H

H

B

N

HN

R1

O R2

O

NH

S

Ph

H

1

2

34

5NN

H

S

Ph

H

1

23

4

HN

R1

5O

H A

NNH

S

Ph

H

1

23

4

HN

R1

5O

H

N

S

NH

Ph

O

HN

H

H

H A

R1 N

S

NH

Ph

O

H2N

H

R1

B B

Enol Formation

Potential racemization

PTH, to be detected by HPLCThermodynamic Product

Pre-note

N

S

NH

Ph

R1

O H+

N

S

NH

Ph

R1

OH

N

S

NH

Ph

R1

OH

H+

N

S

NH

Ph

R1

O

+

N

S

NH

Ph

R1

O

H2O

N

S

NH

Ph

R1

O

OH

HB

H A

NH

NO

SR1

Ph

Frame of Reaction

H2N

HN

R1

O R2

O

H3N

R2

O

NH

NO

S

R1

Ph

+

When racemization is taken care of

Brain teasers:

a) a peptide is not reactive to Edman Degradation

b) After a round of Edman degradation, only one fragment is obtained c) After a round of Edman degradation, two PTH products are obtained

d) Bicyclic PTH product from Edman Degradation

c) Special case: Lysine

No nucleophilic amine Cyclic peptide D05

Breaking the peptide bond does not break the molecule

Presence of Nu amine; Cyclic D10, D12

2 Nu amines at both ends / 1 PTH end and 1 Nu amine endD10, D12

A ring before Edman degradationD02, D04

A more protonated amine D09

Cyanogen Bromide Cleavage

NH

HN

O

S

Me

Br C N

NH

HN

O

S

MeC

N

Br

NH

O

NH

S

MeCN

- MeSCNO

HNNH

H2O

O

ONH

NH

H A

HHBH

O

ONH2

NH

HBH

O

NH

O

+ H2N

1

34

5

2

12

34

5

-Br

Nucleophilic SNu and E 5 atoms apartRotatable bond

Met (C) N cleaved

Brain teasers:

1) A peptide gives only one fragment after CNBr cleavage a) A cyclic peptide

b) C-terminal Methionine

2) It is known that a peptide has n Met. It gives n pieces of fragments

3) How about (n+1) fragments?

H3N

NH

HN

NH

NH2

O

OS

NN

O

O

O H

H

HN

O

OH

NHO

O

OH

NH2

How this reacts with CNBr? (2004 Exam 1)

Why S / C=O combo can be so different in these two

reactions?

S

O

S

O

Edman Degradation CNBr Cleavage

C=S bond, S is Nucleophilic3 C-S bond, S has an extra Covalent bond; adjacent C is ready for SN2

Peptide Syntheses

Walter Kowtoniuk

Amide Bond Synthesis

- Synthesis of an amide bond using the corresponding carboxylic acid and amine.

- Use DCC to both activate the acid and serve as a dehydrating agent

HN C

R

O

O

H

N

C

N

R2

NH2

HN C

R

NH

O

R2

NH

CNH

O

Amide Bond Synthesis

HN C

R

O

O

H

N

C

N

HA

B-

HN C

R

O

O

N

C

N

H

HN C

R

O

O

NC

N

H

HA

HN C

R

O

O

NC

N

H

H

R2

NH2

HN C

R

O

O

NH

CNH

R2N

HH

B-

HN C

R

O

O

NC

N

H

H

R2

H2N

Amide Bond Synthesis

HN C

R

O

O

NH

CNH

R2N

HH

B-

HN C

R

O

R2NH

Protecting GroupsWhy do we need them?

Protecting Groups

Lecture Notes pg33

Protecting Groups

• t-Boc Synthesis

• t-Boc Deprotection

H3C

H3C

H3C

OH

O

Cl

Cl

H3CH3C

CH3

O

O

ClR

NH3C

H3C

CH3

O

O

H HH2N

R

B-

RNHH3C

H3C

CH3

O

O

RNHH3C

H3C

CH3

O

OTFA

H+

RNHH3C

H3C

CH3

O

OH

RNHCH3

H2C

CH3

O

OH

H+

RH2N

CO2

(gas)

HE1

CH3H2C

CH3

Protecting Groups

• Cbz follows the same mechanism as shown for t-Boc

Cl Cl

O OH

OCl

O

ONH

O

R

or

OO

O

R

ONH

O

R TFA

H2C

NH2R

CO2 H3CEt3Si-H

Protecting Groups

• Ts synthesis

• Ts Deprotection

H2NC

OH

O

HNNH2

NH CH3S

O

Cl

O

H2NC

OH

O

HNN

NH2

CH3

S

O

OH

B-

H2NC

OH

O

HNN

NH2

CH3

S

O

O

H2NC

OH

O

HNN

NH2

CH3

S

O

OH

F

HFF-

H2NC

OH

O

HNNH

NH2CH3S

O

O

F

Protecting Groups

• DNP synthesis

• DNP deprotection

H2N

OHO

HNN

NO2

NO2

HS H2N

OHO

HNN

NO2

NO2S

H2N

OHO

HNN

H

B-

H2N

OHO

HNN Cl

O2N NO2

H2N

OHO

HNN

NO2

NO2Cl

H2N

OHO

HNN

NO2

NO2

C to N Directionalitywhy not N to C?

NH

O

R2

N

O O

R1

HN

NH

Cy

Cy

B-

H

NH

O

R2

N

O

R1

H

B-

NH

O

R2

N

O

R1NH

O

R2

N

O

R1

H

HA

Solid Phase Peptide Synthesis

Solid Phase Peptide Synthesis

Peptide Fragment Coupling

• Thioester

• True coupling

O

NH

O

RO

NH

O

RSH

O

NH

O

RSH2N

SHO

HN

O

R

N

O

NH

O

R

HH

H+

O

NH

O

RHN

HS

O

NH

O

RS

O

HN

O

R

NHH

SH

H

B-

O

NH

O

R

SO

HN

O

RN

H H

H+

B-

O

NH

O

RO

NH

O

RS

Br

B-

H

O

NH

O

RS

O

NH

O

RS

O

NH

O

R

Determining Yield

• Synthesizing a 100mer requires 99rxns, thus n=99

• If we factor in the initial coupling to the solid phase, the 100mer requires 100rxns, thus n=100

• For convergent synthesis we are concerned with the longest linear sequence of steps. In this case the yield of each individual reaction is multiplied

Translation

Biological Carbonyl Activation

Biological Carbonyl Activation

Biological Carbonyl Activation

Ribosome

Role of A2486