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Chemical Protein
Engineering: Synthetic and
Semisynthetic
Peptides 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 Ligation
Chemical Modification of Proteins
• Chemical versus Ribosomal Synthesis
• Side Chain Modifications
Enzyme-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 Solution
Synthesis 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-washdeprotection-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
Chemical protein engineering synthetic and semisynthetic
•
•
•
•

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 Dproteins, 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 blue

side 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 Strategies
Another 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
Chemical protein engineering synthetic and semisynthetic
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 Formation
By 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 Ncysteinyl 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 Nterminal 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 Cterminally 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 mutation
Cysteine 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
Protease activity

Cys
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 Modifications
Kaiser (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.
Chemical protein engineering synthetic and semisynthetic
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

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Chemical protein engineering synthetic and semisynthetic

  • 1. Chemical Protein Engineering: Synthetic and Semisynthetic Peptides and Proteins Ali Hatami
  • 2. 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 Ligation Chemical Modification of Proteins • Chemical versus Ribosomal Synthesis • Side Chain Modifications Enzyme-Mediated Peptide Bond Formation
  • 3. 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.
  • 4. History Emil Fischer, was the pioneer and founder of peptide chemistry
  • 5. 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
  • 6. 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
  • 7. Synthesis in Solution Synthesis 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
  • 8. 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.
  • 9. The general principle of SPPS is one of repeated cycles of coupling-washdeprotection-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.
  • 10. Step 1 Attaching an amino acid to the polymer
  • 13. • • • • 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
  • 14. 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 Dproteins, which consist of D-amino acids.
  • 15. 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
  • 16. green resin yellow linker red peptide main chain orange peptide side chain light blue side chain protecting groups blue Boc group magenta activating OBt group
  • 17. 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.
  • 18. Fragment Condensation Strategies Another 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.
  • 19.  The main drawback of these synthetic efforts was the low yield of the correctly folded protein.  Low homogeneity of fragments for coupling
  • 21. 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.
  • 22. 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.
  • 23. Hydrazone and Oxime and Pseudoproline Chemistries
  • 24. Thioester Bond Formation By this procedure a fully active HIV-protease analog was synthesized with a pseudo-Gly-Gly sequence at the ligation site
  • 25. 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 Ncysteinyl peptide to produce the S-(peptidyl)-cysteinyl peptide as an intermediate.
  • 26.  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 Nterminal residue in one ligating fragment.  Prevention oxidation of the cysteine thiol group to the disulfide dimer (which is inactive in ligation).
  • 27. 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 Cterminally 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.
  • 28. 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.
  • 29. 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
  • 30. 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.
  • 32. 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
  • 33. Isosteric Replacements: From “Chemical” to “Atomic” Mutations chemical mutation Cysteine 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.
  • 34.  serine protease: Ser Protease activity Cys 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
  • 35. 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.
  • 36. 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”.
  • 37. Nonisosteric Chemical Modifications Kaiser (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
  • 38.  N-terminal acetylation and C-terminal amidation  biotinylation or fluorophore-conjugation in N-, C phosphorylation (phosphoserine, phosphothreonine, and phosphotyrosine)  cyclization via cystine disulfides
  • 39. 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.
  • 40. 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).
  • 41. 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
  • 42. 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.
  • 43. 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
  • 44. 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.
  • 46. 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.
  • 47. since only through daring can the limits of the potentialities of our methods be determined (Fischer 1906) Thank You With regards to Nafasam