Amino Acids, Peptides and Proteins | Organic Chemistry 3

Amino acids, peptides, and proteins are studied in this chapter: structure and properties of amino acids, synthesis of amino acids (Hell-Volhard-Zelinsky reaction, amidomalonate synthesis, Strecker synthesis, enantiopure synthesis), structure and properties of peptides, peptide sequencing by Edman degradation and enzymatic cleavage, synthesis of peptides, Merrifield solid-phase peptide synthesis, structures and functions of proteins.

Structure and Properties of Amino Acids

Amino acids:

Organic compounds containing both an amino group (-NH2) and a carboxyl group (-COOH), along with a side chain (R group) specific to each amino acid. Amino acids are the building blocks of proteins and are essential for their function. They form proteins by linking together through amide bonds, also called peptide bonds.


α-amino acids: 

Amino acids in which the amino group is attached to the carbon atom that is α to the carboxylic acid moiety. Many different amino acids are observed in nature, but only 20 α-amino acids are abundant in proteins. They differ from each other by the R group attached to the α-carbon.

With the exception of glycine (R = H), all α-amino acids have a chirality center on the α-carbon. Common, naturally occurring amino acids have an S configuration (except for cysteine) and are called L-amino acids because their Fischer projections are similar to those of L-sugars.


Acid-base properties of amino acids:

Amino acids are both basic and acidic due to the presence of amino (pKa ~ 9-10) and carboxylic acid (pKa ~ 2-3) groups. The form of an amino acid is pH dependent:

At physiological pH, amino acids exist as salts: the amino group is protonated while the carboxylic acid group is deprotonated ⇒ this salt is called a zwitterion (a net neutral compound that exhibits charge separation). Amino acids are therefore water soluble and have high melting points.


Isoelectric point (pI): 

pH at which an amino acid exists primarily in its neutral form. At the isoelectric point, the number of positive charges is equal to the number of negative charges in the molecule, resulting in an overall neutral charge. Each amino acid has a unique pI.

  • For neutral amino acids, the pI is calculated from the average of the pKa values of the amino and carboxylic acid groups:

pI = pKa COOH + pKa NH22


  • For amino acids with acidic or basic side chains, the pI is calculated from the average of the 2 pKa values of the 2 acidic groups (acidic side chain) or the 2 basic groups (basic side chain).

Synthesis of Amino Acids

Hell-Volhard-Zelinsky reaction:

Conversion of a carboxylic acid into a racemic mixture of α-amino acids via an α-haloacid.


  1. Halogenation of the α-carbon to form a racemic mixture of α-haloacids.
  2. Nucleophilic attack of NH3 (SN2 reaction) to form a racemic mixture of α-amino acids.


Polyalkylation of the primary amine is not observed because the amino group in the product is less basic and more sterically hindered than other primary amines.


Amidomalonate synthesis:


Conversion of diethyl acetamidomalonate into a racemic mixture of α-amino acids.

Mechanism: similar to the malonic ester synthesis.

  1. Deprotonation of diethylacetamidomalonate to form a doubly stabilized enolate.
  2. Nucleophilic attack of the enolate on the alkyl halide resulting in alkylation of the enolate.
  3. Acid-catalyzed hydrolysis of the esters and the amide to form an amino-1,3-dicarboxylic acid.
  4. Decarboxylation of the amino-1,3-dicarboxylic acid at high temperature to form the desired α-amino acid.



Strecker synthesis:


Conversion of an aldehyde into a racemic mixture of α-amino acids via an α-amino nitrile.


First part: Conversion of the aldehyde into an α-amino nitrile.

  1. Protonation of the aldehyde, making it even more electrophilic.
  2. Nucleophilic attack of NH3 on the carbonyl group to form a tetrahedral intermediate.
  3. Deprotonation of the ammonium group to form a neutral intermediate.

  4. Protonation of the hydroxyl group to form a good leaving group.
  5. Loss of the leaving group, a molecule of water to form an iminium.
  6. Nucleophilic attack of a cyanide ion to form a racemic mixture of α-amino nitriles.


Second part: Hydrolysis of the α-amino nitrile into an α-amino acid.

Synthesis of Enantiopure Amino Acids

Resolution of a racemic mixture:

Optically pure amino acids can be obtained by separating a racemic mixture, a process called resolution. However, this technique is expensive, time-consuming, and half of the reaction product is useless.


Enantioselective synthesis of amino acids:

To promote the formation of one desired enantiomer over the other, another strategy is to use a chiral reagent. This approach has found application in the preparation of amino acids with very high enantioselectivity in the presence of a chiral catalyst, such as Rh and Ru catalysts, carrying a BINAP moiety. The rigid 3D shape of BINAP renders it non-superimposable on its mirror image, thus favoring one configuration over the other.




This approach provides a more efficient and selective method for obtaining optically pure amino acids compared to traditional resolution techniques.

Structures of Peptides


A peptide is a short chain of amino acids linked together by amide bonds, called peptide bonds, resulting in the reaction between COOH and NH2:


  • The amino acid with the free amino group is called the N-terminal amino acid, while the one with the free carboxy group is called the C-terminal amino acid. By convention, peptides are always drawn with the N-terminus on the left.
  • The sequence of amino acid residues in a peptide can be abbreviated with three-letter abbreviations starting with the N-terminus.
  • Polypeptides with more than 40 amino acids are called proteins.


Geometry of peptide bonds:

  • The carbonyl carbon of an amide is sp2 hybridized and has a trigonal planar geometry.
  • Due to electron delocalization, the nitrogen atom of a peptide bond has a partial double bond character and is therefore also an sp2 hybridized:


As a result, there are 2 possible conformations for a peptide bond: s-trans and s-cis. The s-trans conformation is typically more stable because it lacks steric hindrance: the two bulkier R groups are oriented on opposite sides of the C-N bond.

A second consequence of resonance stabilization is that all atoms involved in the peptide bond are in the same plane.


Disulfide Bridges:

Cysteine is the only amino acid found in proteins that contains a thiol group. 2 cysteines can be joined via an oxidation process of their thiol moiety to form a disulfide.

Disulfide bridges between the same strand or different strands greatly affect the 3D structure and properties of peptides and proteins.

Peptide Sequencing

The Edman degradation:

In the Edman degradation, amino acids are cleaved one at a time from the N-terminal amino acid, the identity of the amino acid is determined, and the process is repeated to sequence the entire peptide.

In this process, phenyl isothiocyanate is used to convert the N-terminal amino acid into a phenylthiohydantoin (PTH) derivative resulting in a new peptide with one fewer amino acid.


  1. Nucleophilic attack of the N-terminal amino group on the carbon of phenyl isothiocyanate to form a thiolate ion. 
  2. Intramolecular nucleophilic attack of the thiolate on the carbonyl group of the first peptide bond.
  3. Proton transfers to form a neutral intermediate.
  4. Protonation of the amine of the former first peptide bond followed by the loss of a leaving group, the peptide, to form a 5-membered thiazolinone ring.
  5. Rearrangement of the thiazolinone ring to form a more stable phenylthiohydantoin derivative.


Sequencing of large peptides:

Automated peptide sequencers are capable of sequencing a peptide chain of up to 50 amino acid residues. To sequence larger peptides, they are first cleaved into smaller fragments by:

  • Partial hydrolysis under acidic conditions to randomly form smaller fragments
  • Enzymatic cleavage using peptidases, enzymes that can selectively hydrolyze peptide bonds.

There are a variety of peptidases with different cleavage sites:

  • Carboxypeptidase catalyzes the hydrolysis of the amide bond closest to the C-terminal end ⇒ used to identify the C-terminal amino acid.
  • Chymotrypsin catalyzes the hydrolysis of the amide bond with a carbonyl group from Phe, Tyr, or Trp.
  • Trypsin catalyzes the hydrolysis of the amide bond with a carbonyl group from Arg or Lys.

Peptide Synthesis

Peptide bond formation:

The synthesis of a specific dipeptide consists of coupling an amino-protected amino acid and a carboxyl-protected amino acid. An additional reagent, a dehydrating agent such as dicyclohexylcarbodiimide (DCC), is used to promote amide formation by converting the carboxyl OH group into a better leaving group.



  1. Nucleophilic attack of the OH of the carboxylic acid on the carbodiimide.
  2. Proton transfer to form a neutral activated carboxy group.

  3. Nucleophilic attack of the amine on the carbonyl to form a tetrahedral intermediate.
  4. Loss of the leaving group, the dicyclohexylurea, to re-form the carbonyl group.
  5. Proton transfer to form an amide and a urea.


Protection of other reactive amino and carboxyl groups is necessary to prevent formation of undesired polypetides ⇒ The protected amino acids can be coupled regioselectively followed by removal of the protecting groups.


Protection and deprotection of the amino group:

  • Protection:

An amino group can be protected by converting it into a carbamate. There are 2 commonly used protecting groups: the tert-butoxycarbonyl group (Boc) and the fluorenylmethylcarbonyl group (Fmoc)



  1. Nucleophilic attack of the amino group on the carbonyl group.
  2. Loss of the leaving group to re-form the carbonyl group.
  3. Deprotonation of the ammonium ion to form the desired protected amino acid.


  • Deprotection of the Boc group:


  1. Protonation of carbonyl group with trifluoroacetic acid.
  2. Loss of the leaving group, a carbamic acid. The by-product, a carbocation, undergoes an elimination reaction to form isobutylene.
  3. Deprotonation of carbamic acid with trifluoroacetic acid.
  4. Protonation of the amine followed by loss of CO2 to form the deprotected amino acid.
  5. Under acidic conditions, the amino group of the amino acid is protonated.


This process produces isobutylene and CO2, both of which are gases ⇒ this drives the reaction to completion.


  • Deprotection of the Fmoc group:


  1. Deprotonation of the benzylic proton to form a resonance-stabilized anion.
  2. Protonation of the amino group followed by loss of CO2 and dibenzofulvene.


Protection and deprotection of the carboxyl group:

  • Protection:

A carboxylic acid moiety can be protected by converting it into an ester, commonly methyl esters or benzyl esters.


  • Deprotection:

An ester protecting group can be removed with an aqueous base.

Benzyl esters can also be removed by hydrogenolysis with H2 in the presence of Pt, or with HBr in acetic acid:

Automated Peptide Synthesis

The Merrifield synthesis:

Large peptides can be prepared using the Merrifield synthesis, a solid-phase synthesis. In this method, an amino acid is attached to an insoluble polymer, usually a polystyrene derivative, and Fmoc-protected amino acids are added sequentially one at a time. In the final step, the polypeptide chain is cleaved from the polymer.


  1. A Fmoc-protected amino acid is attached to the polymer.

  2. The Fmoc group is removed and an amide bond is formed with another Fmoc-protected amino acid using DCC.

  3. Step 2 is repeated as many times as necessary to build the desired peptide.
  4. At the end, the protecting group is removed and the peptide is cleaved from the polymer using HF.

The Merrifield synthesis can be fully automated.

Protein Structure and Function

Protein structure:

Polypeptides are characterized by their amino acid sequence. They are organized into 4 levels of structure:

  • Primary structure: the linear sequence of amino acids. The specific arrangement of amino acids determines the unique identity of the protein and sets the foundation for higher order structures. A small change in the primary structure can lead to a large change in function.
  • Secondary structure: the 3D conformations of localized regions of the protein. 2 particularly stable arrangements are the α-helix, a portion of the protein that twists into a clockwise spiral, and the β-pleated sheet, a portion of the protein in which 2 or more protein chains lie side by side.
  • Tertiary structure: the 3D arrangement of the entire polypeptide chain. Interactions such as hydrophobic interactions, hydrogen bonding, disulfide bridges, and van der Waals forces contribute to the stability of the tertiary structure. The unique folding of a protein's tertiary structure determines its specific function and activity. A protein can unfold under conditions of mild heating in a process called denaturation.
  • Quaternary structure: a functional, multi-subunit protein complex resulting from the interaction of multiple polypeptide chains (subunits).

Based on their shape, proteins are often classified as fibrous (linear chains that are bundled together) or globular (chains that are coiled into compact shapes).


Protein function:

Proteins play a variety of roles that are critical to cellular function:

  • Structural proteins: Fiibrous proteins that provide support and maintain the structural integrity of cells and tissues.
  • Enzymes: Proteins that serve as biological catalysts, accelerating all cellular processes without being consumed. Each enzyme is highly specific, recognizing and interacting with a specific substrate.
  • Transport proteins: Proteins that facilitate the movement of molecules across biological membranes. For example, hemoglobin transports oxygen in the blood, while ion channels and carriers regulate the passage of ions and molecules across cell membranes.

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The side chain, or R-group, of amino acids greatly influences their properties by affecting attributes such as polarity, charge, and hydrophobicity. Nonpolar, aliphatic R-groups make the amino acid hydrophobic, suitable for the interior of proteins away from water, while polar or charged R-groups render the amino acid hydrophilic, often found on the protein surface. Moreover, the R-group can determine the amino acid's role in protein structure; for instance, amino acids with bulky aromatic side chains often stabilize the protein through hydrophobic interactions, and those with charged side chains can form ionic bonds or contribute to the active site in enzymes.

The chirality of amino acids is significant because it affects how they can interact with other chiral molecules, which is crucial in biological systems. Proteins, which are made up of L-amino acids, have specific three-dimensional structures and functions that are determined by the spatial arrangement of these chiral units. Enzymes, which are chiral, also recognize substrates based on chirality, which plays a vital role in the metabolic processes of living organisms.

Zwitterions are molecules that contain both positive and negative charges but are overall electrically neutral. In the context of amino acids, at a specific pH known as the isoelectric point (pI), the amino group (-NH3+) is protonated and the carboxyl group (-COO-) is deprotonated, making the amino acid exist as a zwitterion. This dual charge state significantly affects the solubility and overall chemical behavior of amino acids.

Enantiopure amino acids, also known as optically pure or single-enantiomer amino acids, are essential in biological systems because they are the building blocks of proteins, which are vital for countless biological functions. Organisms typically use L-amino acids to synthesize proteins, and these proteins exhibit a high degree of specificity and efficiency due to their three-dimensional structures, which are determined by the configuration of the constituent amino acids. Introducing the wrong enantiomer (D-form) of an amino acid into a protein can disrupt its structure and function, possibly leading to loss of activity or even adverse effects in a biological context.

The Hell-Volhard-Zelinsky reaction converts carboxylic acids into a racemic mixture of α-amino acids via α-haloacids. It involves the halogenation of the α-carbon followed by nucleophilic attack of NH3, resulting in the formation of α-amino acids. Polyalkylation of the primary amine is not observed because the amino group in the product is less basic and more sterically hindered than other primary amines.

The Amidomalonate synthesis converts diethyl acetamidomalonate into α-amino acids. This process involves deprotonation of diethylacetamidomalonate, nucleophilic attack of the enolate on the alkyl halide, acid-catalyzed hydrolysis of esters and amide, and decarboxylation to form the desired α-amino acid.

The Strecker synthesis converts aldehydes into a racemic mixture of α-amino acids via α-amino nitriles. It involves several steps, including protonation of the aldehyde, nucleophilic attack of NH3, deprotonation, protonation of the hydroxyl group, loss of water, and nucleophilic attack of a cyanide ion. The α-amino nitrile is then hydrolyzed into the desired α-amino acid.

Enantioselective synthesis involves the selective formation of one enantiomer of an amino acid over the other. This process is achieved using chiral reagents or catalysts that favor the desired stereochemistry during the synthesis.

Chiral catalysts such as Rhodium (Rh) and Ruthenium (Ru) complexes carrying ligands like BINAP (2,2'-Bis(diphenylphosphino)-1,1'-binaphthyl) are commonly employed. These catalysts exhibit high selectivity, promoting the formation of specific enantiomers.

The primary structure of a peptide refers to the linear sequence of amino acids linked by peptide bonds. This sequence determines the three-dimensional structure and ultimately the function of the protein. The primary structure is usually determined by methods such as Edman degradation or tandem mass spectrometry (MS/MS), where the peptide is sequentially cleaved and the amino acids are identified.

Peptide bond formation in chemical synthesis, also known as condensation reaction, is typically carried out by coupling the carboxyl group of one amino acid to the amino group of another, with the release of a molecule of water. This is commonly achieved using coupling reagents such as dicyclohexylcarbodiimide (DCC) or carbodiimides that activate the carboxyl group, facilitating nucleophilic attack by the amino group. Protecting groups are often used to guard reactive side chains and the terminal amino group which can interfere with the intended condensation reaction.

Protecting groups are essential in amino acid synthesis because they prevent unwanted reactions at reactive sites during chemical transformations. For instance, when synthesizing peptides, amino group protecting groups like the Boc (tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl) are used to ensure that only the desired amino and carboxyl groups react to form peptide bonds. Without protecting groups, side reactions could occur, leading to a complex mixture of products rather than the desired peptide.

Peptide sequencing involves determining the order of amino acids in a peptide chain. The most common technique is Edman degradation, where the peptide is sequentially treated with phenyl isothiocyanate under mildly alkaline conditions to selectively remove the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative, which can then be identified.

Automated peptide sequencers can sequence peptides of up to 50 amino acid residues. They work by sequentially cleaving amino acids from the peptide chain and identifying them. However, for larger peptides, fragmentation into smaller fragments is necessary. This can be achieved through partial hydrolysis under acidic conditions or enzymatic cleavage using peptidases.

The Merrifield synthesis is a solid-phase peptide synthesis method used to prepare large peptides. The process begins with the attachment of an Fmoc-protected amino acid to the polymer. The Fmoc group is then removed, and an amide bond is formed between the attached amino acid and another Fmoc-protected amino acid using DCC (dicyclohexylcarbodiimide) as a coupling agent. This process is repeated iteratively until the desired peptide sequence is achieved. Finally, the peptide is cleaved from the polymer using HF (hydrofluoric acid).

Primary structure determines the sequence of amino acids, secondary structure defines local folding patterns, tertiary structure shapes the overall protein molecule, and quaternary structure involves the arrangement of multiple subunits. Together, these levels of organization dictate the protein's functionality, including its enzymatic activity, ligand binding specificity, and structural role in cellular processes.

Denaturation is the process by which a protein loses its native structure and function due to external stressors such as heat, pH changes, or exposure to certain chemicals. Denaturation disrupts the intricate folding of the protein's tertiary structure, leading to loss of function. However, denatured proteins may still retain their primary structure.