Peptide Bond Formation: How, Why, Where, Exhaustive Facts around it.

A peptide bond formation takes place when two amino acids combine together and form a bond, so basically, it is the linking of amino acids.

Based on the number of amino acids coming together or combining peptide bond can be classified as dipeptide bond (two amino acids combine to form a peptide bond), tripeptide (three amino acids come together to form a peptide bond). When many amino acids come together and form bond they are called as polypeptides.

Oligopeptides are the short peptides which consist of less than 10 amino acids. Important point to be noted that the dipeptide should not be confused with number of bonds (‘di’ over here does not mean number of bonds, it simply refers to the number of amino acids combining). As we know that amino acids are the monomers to form a protein and they are considered to be the building blocks of proteins (very essential for protein formation).

There are 20 very important amino acids which form bonds together in various different kinds of combinations to form many different proteins. Namely- alanine (ala), arginine (arg), asparagine (asn), aspartic (asp), cystein (cys), glutamine (gln), glutamic acid (glu), glycine (gly), histidine (his), isoleucine (ile), leucine (leu), lysine (lys), methionine (met), phenylalanine (phe), proline (pro), serine (ser), thereonine (thr), tryptophan (trp), tyrosine (tyr), and valine (val).

The scientists who proposed that amino acids combine together and form peptide bond were Hofmeister and Emile Fischer. The bond formation between two amino acids (peptide bond), or two amino acids join to form peptide bond by dehydration, meaning there is loss of water molecule. Hence it is a condensation reaction.

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Peptide Bond formation steps:

The steps involved in peptide bond formation are discussed in the section below in detail .

First dehydration (loss of H2O molecule) will take place and then the amino acids will combine. Let us see how this happens. Consider the example of combination of glycine and alanine (amino acids). The N atoms of ammonia (alanine) has partial positive charge. We can also say alpha carbonyl group of glycine and alpha amino group of alanine.

So nitrogen will act as nucleophile and carbon will act as electrophile. Nitrogen will donate its low pair which will result a bond formation between nitrogen and carbon.

The OH and H attached to carbon and nitrogen respectively will be eliminated out as water (H2O) molecule. The linkage between carbon and nitrogen is known as Amide linkage/bond (wherein amino acids combine together). This special type/kind of bond is called peptide bond.

One end free amino group is known as amino terminus or N terminus and other terminal end free carboxylic group is known as carboxy terminus or C terminus. Each amino acid of the polypeptide/dipeptide formed is referred to as residue.

Characteristics of peptide bond

Double bond character of peptide.

It was deduced by linus Pauling and the other co-workers. Consider the figure, you can notice that in a) the first structure. The carbon nitrogen linkage is a double bond where nitrogen has an unshared pair of electrons and carbon-oxygen has a double bond. Now, coming in to b) the second structure, carbon-oxygen is single bond and carbon-nitrogen is a double bond. So, which one will be correct among the structures?

Image credit : Dr Shobha N

Is carbon nitrogen a single bond or a double bond? The bond length observed for carbon nitrogen single bond is 1.47A and bond length for carbon nitrogen double bond is 1.27 A. But the actual carbon nitrogen bond length in a peptide chain is supposed to be 1.32 A.

So the peptide bond is neither a single bond nor a double bond, the reason for this is because of resonance characteristics or the resonance property of carbon-nitrogen bond that makes it a special bond with 1.32 A and this is restrict rotation because it has a partial double bond character. Hence, it’s neither a single bond nor a double bond, because of this partial double bond character this restrict rotation of the carbon nitrogen bond and thus plays a role in the three dimensional structure of a protein molecule.

A peptide bond due to its partial double bond character is rigid and hence this prevents a free rotation around the carbon nitrogen bond and a peptide bond is planar in nature because all the six atoms that are involved in the peptide bond (starting from C 1 to C 2 they all lie in same plane). Hence a peptide bond is planar. (As all the six atoms lie in the same plane).

Image credit : Dr Shobha N

A peptide bond has Trans Configuration:

The reason it has trans configuration and not a cis configuration because if it is in cis configuration there will be a steric hindrance or steric interference due to the presence of side chains at the r groups. If all the r groups are present on the same side then there will be a steric hindrance, that is why a peptide bond has a trans configuration and it is uncharged but it is polar, though it is uncharged it has a polarity and this polarity is due to resonance or the delocalization of the electrons.

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Peptide bond formation mechanism:

The peptide bond formation mechanism is quite simple one. There are many methods but the one which is convenient is used commonly .

  • We can see that nitrogen is having a lone pair so it act as a nucleophile in the reaction and carbon of other amino acids acts as an electrophile.
  • Nitrogen will donate its lone pair (meaning there will be an attack of nucleophile on the electrophile). This leads to a bond formation between atom of one amino acid and N atom of other amino acid.
  • In this process OH which is attached to carbon and H that is attached to the nitrogen atom are eliminated out as H2O molecule. This is a dehydration/elimination step.
  • The peptide bond formed is stable due to resonance.
  • The resultant peptide bond is rigid and planar.

There are many methods but here we shall discuss about the solid phase peptide synthesis method and its mechanism. In short it is known as SPPS and was discovered by Robert Bruce Merrifield (in the year 1962). He prepared/synthesized peptide by taking/using polystyrene solid beads as a support.

So SPPS is one of the repeated cycles of N-Terminal deprotection and coupling reaction. In which deprotection, protection and coupling reaction is carried out. In a simplified way we can say amino acid and resin support is taken, so the amino acid will attach to resin support. Protection of NH2 group has to be carried out, where in protection group is attached to N atom. Then you have to couple with another amino acid. Like this many more amino acids are added which will form a chain of amino acids (cycle is repeated again and again).

After this the attached protected group is removed. After synthesis crude peptide is cleaned from solid support. Solid support is a special type of polystyrene in which there are some aromatic rings that have chloromethyl groups. This polymer is known as Merrifield Resin that is formed by copolymerization of styrene with p-Chloro methyl styrene.

STEP 1: Protection of amino acid using protecting groups.

STEP 2: Treatment with solid support Polymer.

STEP 3: BOC is removed by using CF3COOH

STEP 4: II BOC protected amino acid is added to polymer bound amino acid.

STEP 5: BOC protecting group is removed as in step 3

NOTE: Step 4 and 5 are repeated to add many amino acids and long peptide chain is formed.

STEP 6: Completed polypeptide is removed from polymer with HF anhydrous addition.

What catalyzes peptide bond formation?

The enzyme that catalyzes peptide bond formation (during the process of synthesis of protein is peptidyl transferase. Peptidyl transferase (aminoacyltransferase) which is responsible in formation of peptide bonds (between the adjacent amino acids) by making use of tRNA. (During the time of process of translation of biosynthesis of proteins).

We know that peptide bond requires a catalyzing enzyme for the reaction to progress .

Peptide bond formation is a hydrolysis reaction.

A hydrous reaction is referred to a chemical reaction where a water molecule breaks chemical bonds (one or more). Most of the times water will act as nucleophile.

A peptide bond formation is not a hydrolysis reaction but a dehydration reaction where during the process of 2 amino acids combining a water molecule is lost/eliminated. Hence dehydration process takes place. It is a condensation reaction of amino acids (one amino acid is of alpha-amino group of the next amino acid.

So generally in a dehydration reaction what happens is one hydroxyl group and one hydrogen atom of other group combine together and form a water molecule and is eliminated. So in the peptide bond formation similar reaction happens. Hence it is dehydration reaction and not a hydrolysis reaction.

Peptide bond formation in amino acids.

Amino acids may be classified based on functional groups (core structural) like alpha, beta, gamma, and delta. Based on their polarities, levels of pH. The types of side chain groups (aliphatic, hydrophobic, aromatic, presence of sores etc.)

The forward reaction is seen to be dehydrolysis reaction as it is thermodynamically unfavorable. This means that to form a peptide bond, there is a need to input energy. Amino acids come together to form peptide bond. Nucleophile attacks on electrophile (N on C) and a bond is formed. Hydroxy group of carbon and hydrogen of N combines and is eliminated as water.

Image credit : You Tube

Is peptide bond formation spontaneous?

It can be said that peptide bond formation is not a spontaneous process, why not let’s check out.

The formation of peptide bond at a temperature of 25 degrees Celsius in not favorable because of higher enthalpy change (around 1.5 kcal/mol). The reason for this value is as the ionization of free acid and the amine groups takes place at neutral pH. We know that transfer of proton from an acid to the base involves large amount of negative enthalpy change, reversing it to neutralize both will have positive enthalpy change.

As the products are neutral, getting back the energy is not possible. The bond energies (neutral carboxylic acid, amine) are observed to be not that different from water and an amide, hence we can clearly see there is domination of neutralization.

So amide can be formed at a temperature of 60 degrees Celsius as required change of enthalpy can overcome enthalpy change (at that particular temperature).

Where does peptide bond formation occur?

Peptide bond formation is seen to occur in organelle of cell called as ribosome and rRna. It is a chemical bond which occurs between 2 molecules (amino acids).

So, any of the amino acids can come together to form peptide bond and then they form proteins which are very important to us.

We know that almost all our cells in the body are made up of proteins and hence amino acids/peptide bond play a very important role.

Sania Jakati

This is Sania Jakati from Goa. I am an aspiring chemist pursuing my post graduation in organic chemistry. I believe education is the key element that moulds you into a great human being both mentally and physically. I'm glad to be a member of scintillating branch of chemistry and will try my best to contribute whatever I can from my side and Lambdageeks is the best platform where I can share as well as gain knowledge at the same time. LinkedIn-

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