Amino acids
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The term ‘Amino Acid’ would denote an organic molecule bearing an amine functionality and an acid functionality. The basic amine moiety could be 1°,2°or 3°, while the acid function could be a sulfonic acid or a carboxylic acid moiety. Molecules bearing a sulfonic acid moiety and an aromatic amine moiety are called sulfanilic acids. The amide derivative of sulfanillic acid, called sulfonamide, is an important class of pharmacophore. These drugs are called sulfa drugs. Hence, the name ‘Amino Acid’ describes only molecules bearing an amine moiety and a carboxylic acid moiety (Fig 1.1).
Fig 1.1: Two classes of compounds bearing an amine moiety and an acid moiety
In the aromatic series, the prototype for amino acids would be the aminobenzoic acids (Fig 1.2). Para – aminobenzoic acid (PABA) is the most important member of this family. PABA is an intermediate in the bacterial synthesis of Folate. PABA is also called Vitamin Bx.
Fig 1.2: α-, and β-amino acids and some important molecules bearing these moieties
When the term ‘amino acid’ is used without any modifier, it generally refers to α-Amino acids. In these molecules, the amine moiety is placed α to the carboxylic acid on an aliphatic chain. The chain could be normal or branched and may carry other functional groups, as shown above for proteinogenic amino acids (Fig 1.2). We would discuss this class of molecules in greater details in this chapter.
The β-Amino Acid functionality is commonly seen in β-lactams – an important class of antibiotics. Penicillins and cephalosporins are some of the most important members of this family (Fig 1.2). The parent molecule is 3-Aminopropionic acid , commonly known as β-Alanine. This molecule forms a fragment (part structure) of Pentatonic Acid (Vitamin B5) and also forms part structure of Coenzyme A (Fig 1.2).
Fig 1.3: Some more amino acids and related molecules of interest
γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter in the central nervous system (CNS). In human, it is directly responsible for the regulation of muscle tone (Fig 1.3).
The δ- amino acid (5-aminopentanoic acid) is derivable from its lactam called caprolactam (Fig 1.3). It finds application in the synthesis of the polymer Nylon-6. Thus, the amino acid family constitutes an important class of organic molecule, which is of great biological and commercial importance.
Note
Nylon 66 is a polyamide obtained by the condensation of adipic acid and hexamethylenediamine
α-Amino Acids: However, the term ‘Amino Acid’ is generally reserved for α-amino acids. They form the backbone for peptides and proteins found in plants and animals. Proteins are an important class of bio-molecules essential for the biochemical processes of life. They are long polymeric chains made up of different α-amino acids. The chain is made by the α-amino function of one amino acid condensing with the acid function of the second amino acid and so on until the desired length is attained. In protein chemistry, these amide bonds are called ‘Peptide Bonds’ (Fig 1.4). Proteins form about half the dryweight of Escherichia Coli cells. The small proteins having a length of about 50 amino acids are called peptides. Peptides upto 20 amino acid units long are called small peptides. The longer analogues are called large peptides. This classification based on length is not strictly defined, but used loosely by the scientists.
Fig 1.4: A peptide bond formed between two amino acids.
Occurrence and History
Amino acids are found in the free state in plants and animals. But the bulk of the amino acids needed for commerce used to come from proteins, derived from animal tissues and hair. When the eukaryatic proteins are hydrolyzed, they yield some of the 20 amino acids called proteinogenic amino acids. The French chemist Nicolas Vauquelin discovered the first amino acid Asparagine in 1806. Wollaston W.H. isolated Cystine in 1810. Glycine and Leucine were discovered by Broconnot H.M. in 1820. The discovery of other amino acids followed soon.
Commercial importance
The amino acid business is a multi-billion dollar enterprise. All twenty amino acids are sold, although the demand for each amino acid is greatly different. Amino acids lysine, methionine, threonine are used in animal feed as additives. The animal feed segment was worth $472.4 million in 2008. Amino acids are commonly used in food technology. Monosodium glutamate is a common flavor enhancer that gives foods the taste called umami. Amino acids used as flavorings generated an estimated $510.4 million in 2008. There is a huge market for proteins as food / nutrient suppliments. The global market for protein drugs was $1.1 billion in 2008. According to Adolf Togel of Commserv, a Frankfurt-based subsidiary of Hoechst, the world market for amino acids is worth about US $3.5 to $4 billion annually. The second largest consumer of amino acids is the animal feed industry. Lysine, methionine, threonine, tryptophan and others improve the nutritional quality of animal feeds by supplying essential amino acids that may be in low abundance in grain. Using 0.5% lysine in animal feed improves the quality of the feed as much as adding 20% soy meal.
Consumption of the animal feed additives methionine and lysine amounted to 370,000 tonnes and 350,000 tons, respectively, in 1998. Amino acids also find application in the synthesis of biodegradable plastics, drugs and chiral catalysts. The demand for amino acids , small peptides and proteins in the cosmetics industry is growing fast. The commercial demand for amino acids is likely to increase in the coming years. The discovery of a few peptide drugs based on new synthetic amino acid has given impetus to discovery of new amino acids and peptides derives from them. Incorporation of new amino acids through Combinatorial chemistry is playing a vital role in such new drug discoveries.
Classification of Amino Acids
The 20 proteinogenic amino acids have an alkyl group attached to the α-amino acid unit. The only exception to this is Glycine , which has no side chain. Hence glycine constitutes the parent skeleton for amino acids. Based on the polar nature of the side chains, the amino acids could be grouped into three groups: 1) Neutral Amino Acids 2) Basic Amino Acids and 3) Acidic Amino Acids. The neutral amino acids could be 1a) Neutral Nonpolar Amino Acids and 1b) Neutral Polar Amino Acids . The neutral-polar, acidic and basic amino acids are grouped together as polar amino acids. (Table 1.5).
| Amino Acid | 3-Letter | 1-Letter | Side chain polarity | Side chain charge (pH 7.4) |
|---|---|---|---|---|
| Alanine | Ala | A | nonpolar | neutral |
| Arginine | Arg | R | polar | positive |
| Asparagine | Asn | N | polar | neutral |
| Aspartic acid | Asp | D | polar | negative |
| Cysteine | Cys | C | nonpolar | neutral |
| Glutamic acid | Glu | E | polar | negative |
| Glutamine | Gln | Q | polar | neutral |
| Glycine | Gly | G | nonpolar | neutral |
| Histidine | His | H | polar | positive(10%),neutral(90%) |
| Isoleucine | Ile | I | nonpolar | neutral |
| Leucine | Leu | L | nonpolar | neutral |
| Lysine | Lys | K | polar | positive |
| Methionine | Met | M | nonpolar | neutral |
| Phenylalanine | Phe | F | nonpolar | neutral |
| Proline | Pro | P | nonpolar | neutral |
| Serine | Ser | S | Polar | neutral |
| Threonine | Thr | T | polar | neutral |
| Tryptophan | Trp | W | nonpolar | neutral |
| Tyrosine | Tyr | Y | polar | neutral |
| Valine | Val | V | nonpolar | neutral |
The twenty amino acids found in biological systems and their structures are shown below (Fig 1.6).
Fig 1.6: The proteinous amino acids, their structures and 3-letter abbreviations. Source: From Wikipedia: Proteinogenic amino acids.
The peptide backbone folds and coils in different well defined patterns, with the side chains exposed on the surface of the secondary and tertiary structures . These aspects of peptide backbone would be discussed later in this chapter
The nature of the side chain is crucial for several of the bio-chemical reactions of the peptides. When two different proteins interact, the contact is based on neutral-to-neutral and polar-to-polar interaction. Several biochemical reactions are also governed by the placement of appropriate side chains in the required position and chiralities at these centres. For example, peptide bond cleavage occurs in the cell due to enzymes called proteases. Three important members of this group are Trypsin, Chymotrypsin and Elastase (Fig 1.7). All these proteases have
Fig 1.7: Orientation of side chains in some proteases. Thick lines are used for the end residues to focus your attention on these segments.
similar structure at the reaction site. They have a cavity in which three amino acids viz., Aspartic acid (a carboxylic acid chain), Histidine ( an imidazole ring) and serine (a hydroxy group) are at the bottom of the cavity with their side chains projected into the cavity. These groups orchestrate and bring about the hydrolysis of only that peptide bond which is permitted to enter the cavity. In the long peptide chains, the peptide bond that is programed for a hydrolytic cleavage, is regulated at the mouth of the cavity. In Trypsin, two Glycine groups line the mouth of the cavity at units 216 and 226. The Aspartic acid group is at 189 and serine is at 190. This arrangement provides a deep pocket enabling large polar amino acid amide units in peptides to enter and stay long enough for hydrolysis to occur. In Chemotrypsin, Glycine units line the mouth at 216 and 226. The pocket is lined with nonpolar side chains, with Aspartic acid at 102, Histidine at 57 and Serine at 195. This arrangement makes the pocket shallow allowing nonpolar side chains to enter and be hydrolyzed. In the case of Elastase, the mouth is lined with Valine at 216 and Threonine at 226. This arrangement allows only peptide units with small side chains as in Glycine and Alanine to enter. Thus, the crucial biochemical processes are selectively carried out at desired points on the peptide chain. This is just one of the several biochemical reactions to show that the side chains play a crucial roll in the control and execution of biochemical reactions.
Another important classification of proteinogenic amino acids is based on whether we humans depend on external supply of any amino acids. For example we cannot biosynthesis the amino acid phenylalanine. This has to be supplied from dietary sources. Hence this amino acid is classified as essential amino acid. However, once this amino acid is supplied, our biosystem could convert it to tyrosine. Hence tyrosine is classified.as nonessential amino acid. The following table (Table 1.8) provides a list of essential and nonessential amino acids.
| Essential | Nonessential |
|---|---|
| Isoleucine | Alanine |
| Leucine | Arginine |
| Lysine | Aspartate |
| Methionine | Cysteine |
| Phenylalanine | Glutamate |
| Threonine | Glutamine |
| Tryptophan | Glycine |
| Valine | Proline |
| Histidine | Serine |
| Tyrosine | Asparagine |
Non-standard amino acids
Aside from the 20 standard amino acids and two special amino acids, selenocysteine and pyrrolysine, which are coded by DNA , there are a large number of nonstandard or non-proteinogenic amino acids. Non-proteinogenic amino acids are either not found in proteins (such as the amino acids carnitine, GABA, or L-DOPA), or they are not coded for in the standard genetic code (like hydroxyproline and selenomethionine) but they may result from the modification of standard amino acids after the protein has been formed in the translation stage of protein synthesis.
Some of these non-standard amino acids have been detected in meteorites, For example, over 79 amino acids were found in the primitive Murchison meteorite, a type of carbonaceous chondrite. Microorganisms and plants can also produce uncommon amino acids that can be found in peptidic antibiotics such as nisin, which is used as a food preservative.
Enantiomerism in Amino Acids
Except glycine, which has no side chain, all 19 proteinogenic amino acids have one asymmetric center at α position. In proteins found in higher animals, the amino acids are L-amino acids (Fig 1.9). D-amino acids found in some proteins are produced by post-translational modification by an enzyme, after translation and translocation to the endoplasmic reticulum. They are found in exotic sea-dwelling organisms such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria. D-Serine may act as a neurotransmitter in the brain. On the basis of R/S nomenclature we note that all L-amino acids are (S)-amino acids, except L-cysteine which is (R)-cysteine.
Note
The amino acids Valine, Leucine, isoleucoine and Threonine have one more asymmetric center on the side chain. Hence these amino acids could exist in 2n enantiomeric forms (where ‘n’ is the number of asymmetric carbons).
Fig 1.9: chirality in amino acids. Both Stereo-projection and Fischer Projection formulae are shown
Exercise
While L-serine is an S-amino acid, L-cysteine is an R-amino acid. The spacial orientations being identify. How does the nomenclature of the amino acids differ?
Resolution of Amino Acids
When a new amino acid which bears atleast one asymmetric centre is chemically synthesised in the laboratory, the amino acid is always as a recemic mixture. The classical method of resolution is by diastereomeric salts. The amino acid is protected at the amine end and the acid is converted to a salt of optically active morphine or any other suitable alkaloid. These salts would be diastereomeric salts. They could be seperated by crystallisation. In this way, both the enantiomers could be obtained in a purified form.
Note
This would be so unless special techniques are adapted in the synthetic protocol by the scientists for asymmetric synthesis. We would later discuss some of them.
A new approach is based on enzymatic hydrolysis, using the principle of Kinetic resolution. An example of one such resolution is given below:
Fig 1.10: Kinetic resolution by enzymatic hydrolysis
Acid-Base properties of amino acids
When acids and bases react, they form salts. Amino acids have an amine function and an acid function in the same molecule. They are therefore amphoteric. The amine function and the acid function react to form a carboxylate ion and an ammonium ion. Functionalities that carrying a positive and negative charge in the same molecule (separated by one or more carbons) are called ‘zwitterions’ . In these molecules, the structure of the molecule depends on the pH of the solution. When such a molecule is titrated with acid or base, the structure depends on the pH. In acid pH, say close to pH = 0, the carboxylate ion is protanated to a neutral species, while the amine function is protanated to a positive ammonium salt. The molecule bears a net positive charge. At basic pH, say around pH 11, the ammonium salt looses its proton and so does the carboxylic acid moiety. The net result is an amine function and a carboxylate function, leaving a net negative charge on the molecule. At neutral pH, say around pH 7, the amino acid is bipolar (i.e. the acid function is carboylate and the base function is an ammonium ion – a zwitterion). This is summarized in Equation 1.11. As seen in the equation, the amino acids cannot exist as an uncharged structure.
Note
When the charges are next to each other, as in the case of a Wittig Reagent, they are called ylides.
What happens when an amino acid at pH 0 is titrated with sodium hydroxide - a strong alkali? As we proceed from acid pH, the first inflection point comes around 2 and the second appears around 9-10. In the case of alanine, they appear at 2.4 and 9.7 respectively. In acidic and basic amino acids an extra inflection point is notices. The resultant titration curve is therefore characteristic of the class to which the amino acid belongs. The types of titiation curves they provide could help us to recognize the acidic, basic and neutral amino acids. Let us have a close look at such titration curves of some amino acids.
Alanine has methyl group as a side chain and is therefore a neutral amino acid. The titration curve of this amino acid with alkali shows two inflection points. The pH values at the inflection points are 2.4 and 9.7. We proceed from pH 0, where the amino acid molecules are in the cationic form 1.11A (Equation 1.11). As the titration proceed, the most acidic group, viz., the –COOH looses the proton. This causes the first inflection point at 2.4. As we procees further, the molecule is progressively converted to the zwitterions. The pH increases sharply in this region. At 6.1 (the midpoint of this region) the entire molecules in the titration flask would be as zwitterions 1.11B. This point is called the Isoelectric point (pl) of the amino acid. As we proceed with the titration, the next inflection point occurs at 9.87 for alanine. At this point, the ammonium ion looses its proton (1.11C). At pl point, all the acidic and basic functional groups in the molecule would be in the charged form. This is an important property of amino acids. At the isoelectric point, the amino acid molecule is in the most polar form and totally insoluble in common organic solvents. In a mixture of amino acids (say, obtained by the hydrolysis of a protein), the different amino acids could be separated by adjusting the pH value to different pl values of the amino acids and extracting the amino acids one by one in the aqueous layer.
In the case of aspartic acid, the molecule has a carboxylic acid on the side chain. For this molecule, the titration curve exhibits a broad, slowly rising curve in the acid region of pH. On the other hand, the basic amino acid lysine exhibits such behaviour in the basic region of pH. Thus, the pH titration curve gives an indication of the nature of the amino acid. It also helps us to identify the pl. pK1, pK2, pK3 etc.. The titration curves for alanine, aspartic acid, lysine and histidine are shown in Fig 1.12. The pKa values of the proteinogenic amino acids are listed in Table 1.13.
Fig 1.12 Titration curves of alanine, aspartic acid, lysine and histidine
| Amino Acid | pI | pK1(α-COOH) | p+K2(α-NH3) |
|---|---|---|---|
| Alanine | 6.01 | 2.35 | 9.87 |
| Cysteine | 5.05 | 1.92 | 10.07 |
| Aspartic acid | 2.85 | 1.99 | 9.90 |
| Glutamic acid | 3.15 | 2.10 | 9.47 |
| Phenylalanine | 5.49 | 2.20 | 9.31 |
| Glycine | 6.06 | 2.35 | 9.78 |
| Histidine | 7.60 | 1.80 | 9.33 |
| Isoleucine | 6.05 | 2.32 | 9.76 |
| Lysine | 9.60 | 2.16 | 9.06 |
| Leucine | 6.01 | 2.33 | 9.74 |
| Methionine | 5.74 | 2.13 | 9.28 |
| Asparagine | 5.41 | 2.14 | 8.72 |
| Proline | 6.30 | 1.95 | 10.64 |
| Glutamine | 5.65 | 2.17 | 9.13 |
| Arginine | 10.76 | 1.82 | 8.99 |
| Serine | 5.68 | 2.19 | 9.21 |
| Threonine | 5.60 | 2.09 | 9.10 |
| Valine | 6.00 | 2.39 | 9.74 |
| Tryptophan | 5.89 | 2.46 | 9.41 |
| Tyrosine | 5.64 | 2.20 | 9.21 |
Note
In view of the experimental difficulties in exactly reproducing such experiments, the pKa values slightly differ and depend on the source of the information. To avoid such errors, an experimentalist always introduces one or more reference molecules (whose pKa values are reported) in his own work and compares with his own values.
Example
Carbocylic acids have pKa values around 4-5. In the case of amino acids, the typical pKa value is approximately 2. What is the orgin of this increased acidity?
The titration curve of histidine is unique. The various protonated structures and the pH value from the titration curve are given below
- Below pH = 1.7 (pKa1) H3A
- At pH = 1.8 there is a equal amount of H3A and H2A
- At pH = 6.02 there is equal amounts of H2A and HA.
- At pH = 9.08 there is equal amounts of HA and A
- A is the dominant species above pH = 9.1.
Fig 1.14: Different protonated structures of histidine.
Histidine is the only proteinogenic amino acid that has an inflection point at pH=6. It has three inflection points at pH = 1.8, 6.0 and 9.2. No other amino acid has an ionizable side chain with a pKa value near enough to pH 7.0 to be an effective physiological buffer.
For amino acids having polar side chains, Isoelectric points could be calculated from the known values of pKa. For neutral amino acids, the calculation is shown for alanine. For polar amino acids, the calculation is based on the values of the pKa values of identicallly charged groups or neutral to anionic charged groups as shown below.
Purification and Identification of Amino acids
Amino acids, peptides and proteins could be purified and identified by chromatographic techniques. The popular techniques are discussed below.
Electrophoritic separation of amino acids
The bipolar nature of amino acids provides an option of separation by Electrophoresis (Fig 1.15). In this technique, a rectangular filter paper of appropriate length is spotted at the mid point with a mixture of lysine, aspartic acid and alanine. The filter paper is wetted with an aqueous solution of an appropriate buffer, kept in tanks in either sides of the paper. In the experiment shown below, the pH of the buffer is 5. When a DC electric current is passed at the poles at either end of the filter paper, lysine moves to the cathode, while aspartic acid moves to the anode. At this pH, alanine is mostly in the zwitterionic form and hence moves only very minimally. After an appropriate length of time, the DC current is switched off and the paper is developed with ninhydrin spray. A purple colour is developed at the points where the amino acids have migrated.
Note
When ninhydrin test is applied for such chromatography experiments, no heating is needed. The quantity of amino acid in the test spot is often about a milligram. On spraying the reagent, the colour appears at the amino acid spot(s).This is marked with pencil and the paper stored for records.
Fig 1.15: Electrophoresis of a mixture of Ala, Lys and Asp at pH=6
Ion-exchange chromatography
When a protein is to be analysed, it is first heated with acid to hydrolyse all the peptide bonds. When such a mixture of amino acids is to be purified and estimated quantitatively, ion-exchange chromatography is the technique of choice. Fully automated amino acid analyzers are now available, which are equipped with a solvent pump to deliver the required buffer(s) in a programmed manner. There is a column, filled with Dowex 50 resin (Fig 1.16) . This solid support is made up of polymeric beads. Chemically speaking they are polymers bearing arylsulfonic acid groups. The cation exchange resin helps in the separation of amino acids. In a typical run (Fig 1.17), the eluent is a buffer. The pH value of the buffer could be varied as step elution or as gradient elution. The chromatogram shown in Fig 1.17 is a chromatogram run with gradient elution technique, using ninhydrin as the post column treatment. The detector is a UV detector scanning the wavelengths 570 nm and 440 nm.
Fig 1.16: A Cation Rasin like Dowex 50 is a polymeric bead bearing aryl sulfonic acid groups
Fig 1.17: Some typical chromatograms from an amino acid analyzer
Size Exclusion Chromatography: Special polymer beads are synthesized, which have uniform size of pores on the surface. When such beads are loaded on a columns and the mixture is eluted with a suitable solvent mixture, these molecules would enter the pores. The smaller molecules would enter deep into the pores, while the larger molecules would stay on the top of the pore. During elution, the molecules that have moved deeper into the pores would stay for longer time and therefore get eluted slowly. The largest molecules would be eluted very quickly. The result would be a separation, which is solely based on the size of the molecules. The size exclusion chromatograohy is most suited for polymers, proteins and nucleotides.
Affinity Chromatography: Proteins have high affinities for their substrates or co-factors or prosthetic groups or receptors or antibodies raised against them. This affinity can be exploited in the purification of proteins. A column of beads bearing the high affinity compound can be prepared and a solution of protein passed through the column. Elution is done using a wash buffer run through the column and the elution buffer subsequently applied to the column and collected.
High Performance Liquid Chromatography (HPLC): This is an advanced liquid chromatography technique. Separation can be acheived on an alumina column or a silica column. Most often a reverse phase (RP) column is preferred.
Centrifugation of Proteins
Proteins will sediment through a solution in a centrifugal field dependent upon their mass. Analytical Centrifugation measures the rate of proteins sedimentation. The most common solution used is a linear gradient of sucrose (generally from 5–20%). Proteins are layered atop the gradient in an ultracentrifuge tube and then subjected to centrifugal fields in excess of 100,000 x g. The sizes of unknown proteins can then be determined by comparing their migration distance in the gradient with those of known standard proteins.
Ninhydrin Test for amino acids
Amino acids could be characterized by the Ninhydrin test. Amino acids are colourless compounds, very soluble in water. When a solution of ninhydrin (Fig 1.18) is added to a solution of amino acid and the mixture gently warmed, a purple colour develops. The colour is due to the following reaction (Fig 1.19)
Fig 1.18: Structure of Nindydrin
Note
Stable ninhydrin reagents are now commercially available, which are stable at room temperatures for about a year.
Fig 1.19: The mechanism of ninhydrin colour test
Exercise
In the above reaction, the amine groups appear to react at C2 carbone of the ninhydrin molecule. How do you rationalize this reaction
Exercise
The final product is the dye responsible fro teh colr. However, this structure does not have an extensive conjugation needed for a deep color to develop. Use tautomeric forms to explain the observed color.
Contributors
- Prof. R Balaji Rao (Department of Chemistry, Banaras Hindu University, Varanasi) as part of Information and Communication Technology