Chemical Protein Engineering: Synthetic and

SemisyntheticPeptides and Proteins

Ali Hatami

Synthesis of Peptides and Proteins• Synthesis in Solution• Synthesis on Solid Supports• Fragment Condensation Strategies• Chemoselective Ligation• Native Chemical Ligation• Protein Splicing and Expressed Protein Ligation• Amide Bonds Generated by Decarboxylative

Condensation• Staudinger LigationChemical Modification of Proteins• Chemical versus Ribosomal Synthesis• Side Chain ModificationsEnzyme-Mediated Peptide Bond Formation

Peptide synthesis

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

History

Emil Fischer, was the pioneer and founder of peptide chemistry

Fischer anticipated as long ago as 1906 that advances in both peptide and protein research would require the combined efforts of organic chemistry and biology

The methodological advances have provided many refined strategies for essentially routine synthesis in solution and on solid supports of small to medium-sized polypeptides

For peptides containing numerous sensitive amino acids or particular sequences and side chain modifications it is still neither a routine nor a trivial matter

Synthesis in SolutionSynthesis of peptides in solution was pioneered by du Vigneaud with the synthesis of oxytocin in 1953.

The major difficulty encountered in the synthesis of larger peptides was the poor solubility of the growing fully protected polypeptide chains in most of the organic solvents.

Assembly of smaller fully protected fragments: finally enabled the synthesis of ribonuclease A in solution.

Minimum protection: led to the total synthesis of ribonuclease S by Hirschmann

Further progress of this strategy finally led to the most recent procedures based on chemoselective ligation of fully unprotected fragments into target polypeptide chains

Synthesis on Solid Supports

The most innovative discovery in peptide chemistry is the ingenious development of solid-phase peptide synthesis (SPPS) by Merrifield who won the Nobel Prize in Chemistry in 1984 for the invention of solid phase peptide synthesis.

The general principle of SPPS is one of repeated cycles of coupling-wash-deprotection-wash. The free N-terminal amine of a solid-phase attached peptide is coupled 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.

Step 1 Attaching an amino acid to the polymer

Protection

• Step 2- Washing• Step 3- Deprotection• Step 4- Washing• Step 5- Polymer removal

Steps 1 to 4 are repeated as each new amino acid is added onto the chain until the desired peptide has been formed

SPPS 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.

Limitations related to the final yield: incomplete couplings loss of side chain protections partial cleavage of the peptide from

the resin sequence-dependent poor coupling

yields

green resin

yellow linker

red peptide main chain

orange peptide side chain

light blueside chain protecting groups

blue Boc group

magenta activating OBt group

Despite these continuous new improvements, synthesis of peptides with lengths over 50 residues can by no means be classified as routine work. The successful accomplishment of such syntheses may become even more difficult with multiple-cysteine-containing peptides, where the production of correct disulfide connectivities by regioselective disulfide pairing procedures or by oxidative refolding often represents an additional serious challenge.

Fragment Condensation StrategiesAnother method in peptide synthesis is fragment condensation, in which peptide fragments are coupled.

The crown of these efforts is certainly the total chemical synthesis of the 238-membered green fluorescent protein (GFP) by Sakakibara’s group.

The main drawback of these synthetic efforts was the low yield of the correctly folded protein.

Low homogeneity of fragments for coupling

Chemoselective Ligation

Chemical ligation is a set of techniques used for creating long peptide or protein chains.

original chemical ligation: formation of a non-native bond at the ligation site

native chemical ligation: an unprotected peptide-thioester reacts with a Cys-peptide to give a ligation product with a native amide ('peptide') bond at the ligation site.

The chemoselectivity is achieved with pairs of functional groups of complementary reactivity such as hydrazides or aminooxy groups which readily react with aldehydes to form stable hydrazones or oxime bonds, while a thiol and a bromoacetyl group lead to thioester bonds.

Hydrazone and Oxime and Pseudoproline Chemistries

Thioester Bond FormationBy this procedure a fully active HIV-protease analog was synthesized with a pseudo-Gly-Gly sequence at the ligation site

Native Chemical Ligation Native Chemical Ligation of Unprotected Peptides

This strategy, termed “native chemical ligation” (NCL), exploits the reversible intermolecular transesterification between a peptide thioester and the nucleophilic thiol group of an N-cysteinyl peptide to produce the S-(peptidyl)-cysteinyl peptide as an intermediate.

Potentials and Limitations of Native Chemical Ligation

High yield NCL has no rival in the experimental design of

proteins Obligatory requirement of a cysteine as the N-

terminal residue in one ligating fragment. Prevention oxidation of the cysteine thiol group

to the disulfide dimer (which is inactive in ligation).

Protein Splicing and Expressed Protein Ligation

In this process an internal intein domain excises itself from a host protein, the extein. Thereby, the intein is split N- and C-terminally and splicing only occurs on reconstitution of the two extein fragments.

Expressed Protein Ligation: With suitable mutations of the intein segment, the autolytic process can be stopped at the thioester intermediate, which can then be used to cleave the intein variant with excess of suitable mercaptanes to produce the polypeptide thioester as required for chemical ligation with synthetic N-cysteinyl peptides.

Amide Bonds Generated by Decarboxylative Condensation

A decarboxylative condensation of α-keto carboxylic acids and N-alkylhydroxylamine derivatives may well represent a promising amide-forming reaction for ligation chemistry.

It proceeds in water without catalysts or other reagents, generating water and carbon dioxide as the only by-products.

The great advantage of this ligation chemistry would be the absence of specifically required amino acids at the C- or N-termini of the peptide/protein fragments. Most importantly, epimerization of the ketoacid does not occur during the reaction

Chemical Modification of Proteins

Various post-translational covalent modifications enable the regulation of interactions with other proteins and small molecules, and in this way influence, modulate, or change their properties and functions.

Despite the vast diversity of post-translational modifications in nature, there is a rather small number of basic chemical principles behind them.

Chemical versus Ribosomal Synthesis

Side Chain Modifications

reactive amino acid side chains are especially useful for regiospecific conjugation of unprotected polypeptide.

Some other applications of reactive side chains restrict their conformational flexibility enhance their metabolic stability affect receptor binding affinities

Isosteric Replacements: From “Chemical” to “Atomic” Mutations

chemical mutationCysteine which attached enzymatically to its cognate transfer RNA (tRNAcys) was transformed into alanine by the reduction with Raney nickel without affecting its covalent attachment to tRNAcys.

serine protease: Ser Cys

Protease activity Hydrolyses active esters

Trypsin Selenotrypsin

Proteolytic Glutathion peroxidase

In spite of a wide range of possible chemical transformations to generate bespoke proteins, this approach is hampered by difficulties arising from the lack of chemo- and regiospecificity of the reactions applied, which can thus lead to either heterogeneous or hardly reproducible protein product mixtures

site-directed amino acid mutagenesis

using genetic methods which provide very precise tools for dissecting and designing protein functions in the frame of the standard repertoire of 20 amino acids as prescribed by the genetic code.

mutagenesis techniques are limited to the exchange of one canonical amino acid for one of the 19 other canonical amino acids.

atomic mutations

genetically encoded replacements at the level of single atoms such as H/F and CH2/ S/Se/Te are known as “atomic mutations”.

Nonisosteric Chemical ModificationsKaiser (1988) and associates have generated new enzymatic activities via attachment of the flavin cofactor to the active site of the protease papain.

some Nonisosteric Chemical Modifications: Glutharylaldehyde-mediated cross-linking PolyEthyleneGlycol (PEG) modifications of

enzyme surfaces

PEGylation of proteins increases the therapeutic potential of proteins by reducing their toxicity and immunogenicity, increasing their half-life in serum

N-terminal acetylation and C-terminal amidation

biotinylation or fluorophore-conjugation in N-, C-

phosphorylation (phosphoserine, phosphothreonine, and phosphotyrosine)

cyclization via cystine disulfides

Backbone Modifications by Surrogate Peptide Bonds: Peptidomimetics

Peptides modified in this way are often protease-resistant reduced immunogenicity improved bioavailability

All these backbone modifications are accessible by synthesis; and by employing the new procedures of chemical ligations, one can even introduce related peptide fragments at selected positions into proteins.

Scheme 2 Peptide mimicry: some selected types of peptide backbone modifications. Although the mimetic structures are often not stable in acidic or basic aqueous solutions, such backbone modifications differ substantially from the peptide amide structure and thus may affect local conformational geometries. The modifications include exchange of single chain units (A, B, C), double exchange at the peptide bond (D), and extension of the peptide chain (E).

Enzyme-Mediated Peptide Bond Formation

The concept of modifying the active site of proteases for reduction of the proteolytic activity in favor of synthetase properties was first realized by Kaiser et al. (1985) with the successful conversion of the protease subtilisin into a ligase.

Chemical exchange of the active-site serine with cysteine led to thiosubtilisin, which reacts rapidly with an activated peptide to form an acylated enzyme and then transfers the peptide onto another peptide, completing the ligation reaction

sortase

Sortases are bacterial enzymes with their highly selective catalytic properties that are responsible for the covalent attachment of specific proteins to the cell wall of Gram-positive bacteria.

Proteins that are substrates of sortase possess a “sorting signal” at the C-terminus consisting of the LPXTG motif (where X can be any amino acid), a hydrophobic region, and a tail of charged residues.

Sortase is capable of catalyzing a two-step transpeptidation reaction, either in vivo or in vitro.

First, the LPXTG motif is cleaved between threonine and glycine.

Then the threonine is covalently attached to the amino group of a pentaglycine segment of the cell wall peptidoglycan

resulting in a cell-wall-attached protein

With use of a similar experimental set-up with sortase from S. aureus, selective protein–peptide and protein–protein ligations were achieved, confirming the efficiency of this enzyme.

Furthermore, it was shown that nonnative peptide fragments including d-peptides and nonpeptide derivatives (e.g., folate) of glycine, diglycine, or triglycine are efficiently conjugated by sortase to the acceptor protein such as the green fluorescent protein containing the LPXTG motif.

E. coli enzyme biotin ligase is another example of this kind of enzymes which links in sequencespecific mode biotin to a 15-membered acceptor peptide.

since only through daring can the limits of the potentialities of our methods be determined (Fischer 1906)

Thank You

With regards to Nafasam