Peptide synthesis: Wikis

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In organic chemistry, peptide synthesis is the production of peptides, which are organic compounds in which multiple amino acids are linked via peptide bonds which are also known as amide bonds. The biological process of producing long peptides (proteins) is known as protein biosynthesis.

Table of Amino Acids.
Solid-Phase Peptide Syntesis on a Rink amide resin using Fmoc-α-amine-protected amino acid

Contents

Chemistry

Peptides are synthesized by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. The possibility of unintended reactions is obvious; therefore protecting groups are usually necessary. Chemical peptide synthesis starts at the C-terminal end of the peptide and ends at the N-terminus. This is the opposite of protein biosynthesis, which starts at the N-terminal end.

Liquid-phase synthesis

Liquid-phase peptide synthesis is a classical approach to peptide synthesis. It has been replaced in most labs by solid-phase synthesis (see below). However, it retains usefulness in large-scale production of peptides for industrial purposes.

Solid-phase synthesis

Coupling step in solid-phase peptide synthesis

Solid-phase peptide synthesis (SPPS), pioneered by Robert Bruce Merrifield,[1] resulted in a paradigm shift within the peptide synthesis community. It is now the accepted method for creating peptides and proteins in the lab in a synthetic manner. SPPS allows the synthesis of natural peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.

Small solid beads, insoluble yet porous, are treated with functional units ('linkers') on which peptide chains can be built. The peptide will remain covalently attached to the bead until cleaved from it by a reagent such as trifluoroacetic acid. The peptide is thus 'immobilized' on the solid-phase and can be retained during a filtration process, whereas liquid-phase reagents and by-products of synthesis are flushed away.

The general principle of SPPS is one of repeated cycles of coupling-deprotection. The free N-terminal amine of a solid-phase attached peptide is coupled (see below) to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.

The overwhelmingly important consideration is to generate extremely high yield in each step. For example, if each coupling step were to have 99% yield, a 26-amino acid peptide would be synthesized in 77% final yield (assuming 100% yield in each deprotection); if each step were 95%, it would be synthesized in 25% yield. Thus each amino acid is added in major excess (2~10x) and coupling amino acids together is highly optimized by a series of well-characterized agents.

There are two majorly used forms of SPPS -- Fmoc and Boc. Unlike ribosome protein synthesis, solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by these two groups and added onto a deprotected amino acid chain.

Automated synthesizers are available for both techniques, though many research groups continue to perform SPPS manually.

SPPS is limited by yields, and typically peptides and proteins in the range of 70~100 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulty also is sequence dependent; typically amyloid peptides and proteins are difficult to make. Longer lengths can be accessed by using native chemical ligation to couple two peptides together with quantitative yields.

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Fmoc solid-phase peptide synthesis

This method was introduced by Carpino in 1972 and further applied by Atherton in 1978. Fmoc stands for 9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl which describes the Fmoc protecting group, first described as a protecting group by Carpino in 1970. To remove an Fmoc from a growing peptide chain, basic conditions (usually 20% piperidine in DMF) are used. Removal of side-chain protecting groups and peptide from the resin is achieved by incubating in trifluoroacetic acid (TFA), deionized water, and triisopropylsilane. Fmoc deprotection is usually slow because the anionic nitrogen produced at the end is not a particularly favorable product, although the whole process is thermodynamically driven by the evolution of carbon dioxide. The main advantage of Fmoc chemistry is that hydrofluoric acid is not needed and the procedure is easily automated. It is therefore used for most routine synthesis.

t-Boc solid-phase peptide synthesis

When Merrifield invented solid-phase peptide synthesis (SPPS) in 1963, it was according to the t-Boc method. t-Boc (or Boc) stands for (t)ert-(B)ut(o)xy(c)arbonyl. To remove Boc from a growing peptide chain, acidic conditions are used (usually neat TFA). Removal of side-chain protecting groups and the peptide from the resin at the end of the synthesis is achieved by incubating in anhydrous hydrogen fluoride (which can be dangerous or even deadly), although generally safe, and using only small quantities, HF cleavage needs to be done using specialized equipment, so it is generally disfavored. However for complex syntheses t-Boc synthesis is favourable. In addition, when synthesizing nonnatural peptide analogs which are base-sensitive (such as depsipeptides), the t-Boc protecting group is necessary.

BOP SPPS

The use of BOP reagent was first described by Castro et al. in 1975.

Solid supports

The physical properties of the solid support, and the applications to which it can be utilized, vary with the material from which the support is constructed, the amount of crosslinking, as well as the linker and handle being used.

Polystyrene resin

Polystyrene resin is a versatile resin and it is quite useful in multi-well, automated peptide synthesis, due to its minimal swelling in dichloromethane.

Polyamide resin

Polyamide resin is also a useful and versatile resin. It seems to swell much more than polystyrene, in which case it may not be suitable for some automated synthesizers, if the wells are too small.

PEG hybride polystyrene resin

An example of this type of resin is the Tentagel resin. The base resin is polystyrene onto which is attached long chains (Mw ca. 3000 Da) of polyoxyethylene (PEG). Synthesis is caried out on the distal end of the PEG spacer making it suited for long and difficult peptides. In addition it is also attractive for the synthesis of combinatorial peptide libraries and on resin screening experiments. It does not expand much during synthesis making it a preferred resin for robotic peptide synthesis.

PEG based resin

ChemMatrix(R) is a new type of resin which is based on PEG that is crosslinked. ChemMatrix(R) has claimed a high chemical and thermal stability (is compatible with Microwave synthesis) and has shown higher degrees of swellings in acetonitrile, dichloromethane, DMF, N-methylpyrrolidone, TFA and water compared to the polystyrene based resins. ChemMatrix has shown significant improvements to the synthesis of hydrophobic sequences. ChemMatrix is recommended for the synthesis of difficult and long peptides.

Protecting groups

Due to amino acid excesses used to ensure complete coupling during each synthesis step, polymerization of amino acids is common in reactions where each amino acid is not protected. In order to prevent this polymerization, protecting groups are used. This adds additional deprotection phases to the synthesis reaction, creating a repeating design flow as follows:

  • Protecting group is removed from trailing amino acids in a deprotection reaction
  • Deprotection reagents washed away to provide clean coupling environment
  • Protected amino acids dissolved in a solvent such as dimethylformamide (DMF) are combined with coupling reagents are pumped through the synthesis column
  • Coupling reagents washed away to provide clean deprotection environment

Currently, two protecting groups (t-Boc, Fmoc) are commonly used in solid-phase peptide synthesis. Their lability is caused by the carbamate group which readily releases CO2 for an irreversible decoupling step.

t-Boc protecting group

The t-Boc (tert-butyloxycarbonyl or more simply "Boc") group was commonly used for protecting the terminal amine of the peptide, requiring the use of more acid stable groups for side chain protection in orthogonal strategies. It retains usefulness in reducing aggregation of peptides during synthesis. t-Boc groups can be added to amino acids with t-Boc anhydride and a suitable base.

Boc cleavage

Fmoc protecting group

Fmoc cleavage

Fmoc (9H-fluoren-9-ylmethoxycarbonyl) is currently a widely used protective group that is generally removed from the N terminus of a peptide in the iterative synthesis of a peptide from amino acid units. The advantage of Fmoc is that it is cleaved under very mild basic conditions (e.g. piperidine), but stable under acidic conditions, although this has not always held true in certain synthetic sequences. This allows mild acid labile protecting groups that are stable under basic conditions, such as Boc and benzyl groups, to be used on the side-chains of amino acid residues of the target peptide. This orthogonal protecting group strategy is common in the art of organic synthesis.

FMOC is preferred over BOC due to ease of cleavage; however it is less atom-economical, as the fluorenyl group is much larger than the tert-butyl group. Accordingly, prices for FMOC amino acids were high until the large-scale piloting of one of the first synthesized peptide drugs, enfuvirtide, began in the 1990s, when market demand adjusted the relative prices of the two sets of amino acids.

Because the liberated Fluorenyl group is a chromophore, deprotection by FMOC can be monitored by UV absorbance of the runoff, a strategy which is employed in automated synthesizers.

Benzyloxy-carbonyl (Z) group

The first use of (Z) group as protecting groups was done by Max Bergmann who synthesised oligopeptides.[2]

Another carbamate based group is the benzyloxy-carbonyl (Z) group. It is removed in harsher conditions: HBr/acetic acid or catalytic hydrogenation. Today it is almost exclusively used for side chain protection.

Alloc protecting group

The allyloxycarbonyl (alloc) protecting group is often used to protect a carboxylic acid, hydroxyl, or amino group when an orthogonal deprotection scheme is required. It is sometimes used when conducting on-resin cyclic peptide formation, where the peptide is linked to the resin by a side-chain functional group. The alloc group can be removed using tetrakis(triphenylphosphine)palladium(0) along with a 37:2:1 mixture of methylene chloride, acetic acid, and N-Methylmorpholine (NMM) for 2 hours. The resin must then be carefully washed 0.5% DIPEA in DMF, 3x10 ml of 0.5% sodium diethylthiocarbamate in DMF, and then 5x10 ml of 1:1 DCM:DMF.

Lithographic protecting groups

For special applications like protein microarrays lithographic protecting groups are used. Those groups can be removed through exposure to light.

Activating groups

For coupling the peptides the carboxyl group is usually activated. This is important for speeding up the reaction. There are two main types of activating groups: carbodiimides and triazolols. However the use of pentafluorophenyl esters (FDPP[3] , PFPOH[4]) and BOP-Cl[5] are useful for cyclising peptides.

Carbodiimides

Alanine attaching to DCC

These activating agents were first developed. Most common are dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC). Reaction with a carboxylic acid yields a highly reactive O-acyl-urea. During artificial protein synthesis (such as Fmoc solid-state synthesizers), the C-terminus is often used as the attachment site on which the amino acid monomers are added. To enhance the electrophilicity of carboxylate group, the negatively charged oxygen must first be "activated" into a better leaving group. DCC is used for this purpose. The negatively charged oxygen will act as a nucleophile, attacking the central carbon in DCC. DCC is temporarily attached to the former carboxylate group (which is now an ester group), making nucleophilic attack by an amino group (on the attaching amino acid) to the former C-terminus (carbonyl group) more efficient. The problem with carbodiamides is that they are too reactive and that they can therefore cause racemization of the amino acid.

Triazolols

HOBt
HOAt
Neighbouring group effect of HOAt

To solve the problem of racemization, triazolols were introduced. The most important ones are 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt). Others have been developed. These substances can react with the O-acylurea to form an active ester which is less reactive and less in danger of racemization. HOAt is especially favourable because of a neighbouring group effect.[6] Recently, HOBt has been removed from many chemical vendor catalogues; although almost always found as a hydrate, HOBt may be explosive when allowed to fully dehydrate and shipment by air or sea is heavily restricted. Alternatives to HOBt and HOAt has been introduced. One of the most promising is Oxyma Pure, ethyl 2-cyano-2-(hydroxyimino)acetate which is not explosive and has a reactivity of that in between HOBt and HOAt.

Newer developments omit the carbodiimides totally. The active ester is introduced as a uronium or phosphonium salt of a non-nucleophilic anion (tetrafluoroborate or hexafluorophosphate): HBTU, HATU, HCTU, TBTU, PyBOP. Two uronium types of the coupling additive of Oxyma Pure is also available as Como or Toto reagent.

Synthesizing long peptides

Stepwise elongation, in which the amino acids are connected step-by-step in turn, is ideal for small peptides containing between 2 and 100 amino acid residues. Another method is fragment condensation, in which peptide fragments are coupled. Although the former can elongate the peptide chain without racemization, the yield drops if only it is used in the creation of long or highly polar peptides. Fragment condensation is better than stepwise elongation for synthesizing sophisticated long peptides, but its use must be restricted in order to protect against racemization. Fragment condensation is also undesirable since the coupled fragment must be in gross excess, which may be a limitation depending on the length of the fragment.

A new development for producing longer peptide chains is chemical ligation: Unprotected peptide chains react chemoselectively in aqueous solution. A first kinetically controlled product rearranges to form the amide bond. The most common form of native chemical ligation uses a peptide thioester that reacts with a terminal cystein residue.

Coupling Efficiency Vs. Peptide Length

Peptide

Length Coupling Efficiency Coupling Efficiency Coupling Efficiency Coupling Efficiency Coupling Efficiency
0 0.995 0.99 0.98 0.97 0.96
5 0.98 0.95 0.92 0.89 0.85
10 0.96 0.91 0.83 0.76 0.69
15 0.93 0.87 0.75 0.65 0.56
20 0.91 0.83 0.68 0.56 0.46
25 0.89 0.79 0.62 0.48 0.38
30 0.86 0.75 0.56 0.41 0.31
35 0.84 0.71 0.50 0.36 0.25
40 0.82 0.67 0.45 0.30 0.20
45 0.80 0.63 0.41 0.26 0.17
50 0.78 0.60 0.37 0.22 0.14
55 0.76 0.58 0.34 0.19 0.11
60 0.74 0.55 0.30 0.17 0.09
65 0.73 0.53 0.27 0.14 0.07
70 0.71 0.50 0.25 0.12 0.06

Microwave assisted peptide synthesis

Although microwave irradiation has been around since the late 1940s, it was not until 1986 that microwave energy was used in organic chemistry. During the end of the 1980s and 1990s, microwave energy was an obvious source for completing chemical reactions in minutes that would otherwise take several hours to days. Through several technical improvements at the end of the 1990s and beginning of the 2000s, microwave synthesizers have been designed to provide both low and high energy pockets of microwave energy so that the temperature of the reaction mixture could be controlled. The microwave energy used in peptide synthesis is of a single frequency providing maximum penetration depth of the sample which is in contrast to conventional kitchen microwaves.

In peptide synthesis, microwave irradiation has been used to complete long peptide sequences with high degrees of yield and low degrees of racemization[7]. Microwave irradiation during the coupling of amino acids to a growing polypeptide chain is not only catalyzed through the increase in temperature, but also due to the alternating electromagnetic radiation[citation needed] to which the polar backbone of the polypeptide continuously aligns to. Due to the this phenomenon, the microwave energy can prevent aggregation and thus increases yields of the final peptide product. There is however no clear evidence that microwave is better than simple heating and some peptide laboratories regard microwave just as a convenient method for rapid heating of the peptidyl resin. Heating to above 50-55 degrees celcius also prevents aggregation and accelerates the coupling.

Despite the main advantages of microwave irradiation of peptide synthesis, the main disadvantage is the racemization which may occur with the coupling of cysteine and histidine. A typical coupling reaction with these amino acids are performed at lower temperatures than the other 18 natural amino acids. A number of peptides does not survive microwave synthesis of heating in general. One of the more serious side effects is dehydration (loss of water) which for certain peptides can be almost quantitative like pancreatic polypeptide (PP). This side effect is also seen by simple heating without the use of microwave.

As of January 2009, over 200 microwave peptide synthesizers are in use with the rate of acceptance increasing.[citation needed]

Cyclic peptides

On resin cyclization

Solution phase cyclization

References

  1. ^ R. B. Merrifield (1963). "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide". J. Am. Chem. Soc. 85 (14): 2149–2154. doi:10.1021/ja00897a025. 
  2. ^ Max Bergmann, Leonidas Zervas (1932). "Über ein allgemeines Verfahren der Peptid-Synthese". Berichte der deutschen chemischen Gesellschaft 65 (7): 1192–1201. doi:10.1002/cber.19320650722. 
  3. ^ K. C. Nicolaou (1998). "Total Synthesis of Vancomycin Aglycon - Part 1: Synthesis of Amino Acids 4-7 and Construction of the AB-COD Ring Skeleton". Angew. Chem. Int. Ed. 37 (19): 2708–2714. doi:10.1002/(SICI)1521-3773(19981016)37:19<2708::AID-ANIE2708>3.0.CO;2-E. 
  4. ^ Schmidt (1998). "Synthetic studies of 14-membered cyclopeptide alkaloids". Tetrahedron Lett. 39 (40): 7211–7214. doi:10.1016/S0040-4039(98)01589-5. 
  5. ^ R. Baker, J. L. Castro (1989). "The total synthesis of (+)-macbecin I". Chem. Commun. (6): 378–381. doi:10.1039/C39890000378. 
  6. ^ L. A. Carpino (1993). "1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive". J. Am. Chem. Soc. 115 (10): 4397–4398. doi:10.1021/ja00063a082. 
  7. ^ Stacey A. Palasek, Zachary J. Cox, Jonathan M. Collins (2007). Journal of Peptide Science 13 (3): 143–148. doi:10.1002/psc.804. 
  • Atherton, E.; Sheppard, R.C. (1989). Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press. ISBN 0199630674. 
  • Stewart, J.M.; Young, J.D. (1984). Solid phase peptide synthesis (2nd ed.). Rockford: Pierce Chemical Company. p. 91. ISBN 0935940030. 

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