Orgo 2: Amino Acid Reactions

Mechanism to break proteins into amino acids

Hydrolysis

Acid catalyzed and base catalyzed hydrolysis mechanism

Nucleophilic Acyl Substitution

Process of acid catalyzed nucleophilic acyl substitution

The molecule that’s the electrophile (leaving group) is substituted out for water that’s the nucleophile

Process of base catalyzed nucleophilic acyl substitution

The molecule that’s the electrophile (leaving group) is substituted out for OH that’s the nucleophile

Function of amino acid analysis

Determines the composition and amounts of amino acids

Steps of amino acid analysis

1. Acid hydrolysis

2. Separation by ion-exhange HPLC (column chromatography)

3. Amino acids reacted with ninhydrin to form a chromophore and measured by UV-VIS spectroscopy

Method for reducing protein disulfides

By using an excess thiol (mercaptoethanol)

Chromophore formed via ninhydrin reaction

Ruhemann’s Purple

Form of ninhydrin that reacts

Triketone form

Reaction of ninhydrin (triketone) with amino acid mechanism and product prior to Ruhemann’s Purple

Nucleophilic addition to imine

What kind of reaction is Edman Degradation

N-Terminal sequencing

N-terminal sequencing mechanism

Nucleophilic acyl substitution

In Edman degradation, what is the peptide cleaved as?

Phenylthiohydantoin (PTH) derivative that’s specific to each amino acid

Which amino acid can’t form Ruhemann’s purple and why?

Proline because it doesn’t have a primary amine

Mechanism for N-terminal identification?

Nucleophilic aromatic substitution

What reagent is used for N-terminal identification?

Dinitroflurobenzene (DNFB, Sanger’s Reagent)

What does N-terminal identification do?

Tags the N-terminal amino group and any amino group on side chain as dinitrophenyl (DNP), then hydrolyzed and analyzed by HPLC

What enzyme is used for C-terminal sequencing?

Carboxypeptidase

What does C-terminal sequencing do?

Identifies the C-terminal amino acid by using carboxypeptidase to remove the c-terminal amino acids

How are the released amino acids identified and quantified in C-terminal sequencing?

HPLC, top is the c-terminal and going down is closer to the N-terminal

Amino acid that CNBr (cyanogen bromide)

Methionine

Amino acids that trypsin cleaves after

Lysine and arginine

Amino acids that chymotrypsin cleaves after

Phenylalanine, tyrosine and tryptophan

COOH protecting group, what it’s made from and mechanism

Benzyl ester made from benzyl alcohol through fischer esterification

NH2 protecting group, what it’s made from and mechanism

t-butyloxycarbamate (carbamate, BOC) made from t-butyloxycarbonyl anhydride through nucleophili acyl substitution

Deprotection of COOH reactant and mechanism

Hbr/HOAc (acetic acid) through acid hydrolysis

Deprotection of NH2 reactant and mechanism

TFA (trifluroacetic acid) through decarboxylation

Which one is more acid labile, BOC or ester

BOC

What does decarboxylation do?

Removes carboxyl group and releases CO₂

Why must protecting groups be used?

To ensure amino acids are joined in the correct order to make the correct protein.

What is used as a coupling reagent and why?

DCC is the coupling reagent and is sued to make COOH more reactive so an amide bond can form

What does DCC convert COOH to and using what mechanism?

Anhydride by nucleophilic acyl substitution

Solution Phase Synthesis steps

1. Protect amino acids

2. Use coupling reagent DCC to make COOH more reactive using nucleophilic acyl substitution. Amide bond formed.

3. Deprotect NH2 end using TFA to get ammonium, and then reacted with Et₃N to get amide.

4. Then use DCC to add the next amine protected residue

5. Repeat

6. When done, remove both protecting groups using Hbr/HOAc

Why can’t you turn COOH into an acid halide?

Risk enolization of amino acid, destroying the chiral centre

Drawback of solution phase synthesis

Tedious purification

What type of synthesis is Merrifield Synthesis?

Solid phase synthesis

Benefit of solid phase synthesis

Easier to purify because of insoluble chloromethylpolystyrene attached to COOH as protecting group; great for automation

What bond is between the bead and COOH?

Ester bond

Limitation to polypeptide synthesis

Steps and coupling must occur with high efficiency because small changes in yield can impact the final yield