Protein engineering

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

11. Protection

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.

31. Chemical versus Ribosomal Synthesis

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