Literature Seminar

Peptide and Protein Ligation

M1 Siqi Xi
2018.7.21

Contents➢Introduction➢Extension of Native Chemical Ligation(NCL)➢Enzyme-Mediated Ligation➢SummaryNative Chemical Ligation( NCL)

➢ NCL is an efficient ligation method
for native backbone protein ligation.
➢ NCL is of great use in the cysteine
ligation site.
2. C-terminal

1. N-terminal mercaptan➢ Not only limited to thioester➢ Modification of this part canalso promote the ligation site ofN-terminal.

➢ Only suitable for cysteine.
➢ Modification for expansion ofscope of ligation site.

Brik. A et al. Nature Chem. 2016, 8, 407-418
Bingham. J. P et al. Molecules 2014, 19, 14461-14483

21 Amino Acid ( Ligation Site)➢ Cysteine as the ligation site isachieved.

Contents➢Introduction➢Extension of Native Chemical Ligation(NCL)✓✓✓✓

Auxiliary Mediated Ligation
NCL-Desulfurization
Serine/Threonine Ligation( STL)
α-Ketoacid and a Hydroxylamine( KAHA Ligation)✓ Histidine Ligation➢Enzyme-Mediated Ligation➢SummaryAuxiliary Mediated Ligation➢ C-terminal usually uses thioester.
➢ N-terminal has the auxiliary electron-rich group.
➢ Auxiliary group should be removed, such as TFA, HF.
➢ Requirement is the presence of glycine at one of the ligation sites, due to thebulkiness of the auxiliary group.

Dawson. P. E et al. J. Am. Chem. Soc. 2002, 124, 4642-4646
Cardona. V. M. F et al, J. Peptide Res. 2003, 61, 152-157
Bingham. J. P et al. Molecules 2014, 19, 14461-14483

NCL-Desulfurization

Usual Desulfurization Methods➢ H2/metal reagents, such as Pd/Al2O3, Pd/C, Raney
nickel etc.
➢ Metal-free reductant, VA-044.
Dawson. P. E et al. Isr. J. Chem. 2011, 51, 862 – 867

NCL-Desulfurization
H2/metal reagents➢ H2-mental reagent includes Pd/Al2O3, Pd/BaSO4, Pd/C and Raney nickel,
which are efficient for desulfurization for cysteine.
➢ Cysteine on side chain should be protected by acetamidomethyl( Acm)group.
➢ Undesired side reaction such as hydrogenation of tryptophan anddemethylthiolation of methionine might occur.
➢ Low yield of product due to adsorption on large metal surface.
Dawson. P. E et al. J. Am. Chem. Soc. 2001, 123, 526-533
Danishefsky. S. J et al. Angew. Chem. Int. Ed. 2007, 46, 9248-9252
Bingham. J. P et al. Molecules 2014, 19, 14461-14483

NCL-Desulfurization

Advantages compared to H2/metal➢ Tryptophan and Methionine in side chain is stable.
➢ VA-044 can afford high yield, but sometimes is
not effective enough in some desulfurization
reaction.
Limitation:➢ Cysteine on side chain should also be protectedby acetamidomethyl( Acm) group.
➢ Not efficient for some desulfurization.
Seitz. O C et al. Angew. Chem. Int. Ed. 2008, 47, 6807 –6810
Brik. A et al. Nature Chem. 2016, 8, 407-418
Malins. L. R et al. Chem. Sci. 2014, 5, 260–266

NCL-Desulfurization➢ Development of desulfurization expands the scope of ligation site.
➢ C-terminal is thioester.
➢ N-terminal is not only limited to Cystine, compared to NCL.
➢ Cysteine on side chain should be protected by acetamidomethyl( Acm)group.
Brik. A et al. Nature Chem. 2016, 8, 407-418

NCL-Desulfurization
β-thiolamino acidγ-thiolamino acid

Payne. R. J et al. Curr. Opin. Chem. Biol. 2014, 22, 70-78
Payne. R. J et al. Org. Lett. 2015, 17, 4902−4905
Chuanfa. L et al. Org. Lett. 2016, 18, 2696−2699➢ Chemical ligation, followed bydesulfurization expand the scopeof ligation site to these aminoacids, shown above.
➢ During desulfurization, Cys
needs to be protected.

NCL-Desulfurization( Deselenization)➢ Weak C-Se bond makes
the deselenizaion occur
without radical initiator.
➢ Selective deselenization
can be achieved with theretention of unprotected
cysteine.
➢ Proline selenoesters havesuperior leaving groupability compared to thecorresponding thioester
analogs.

Dawson. P. E et al. Angew. Chem. Int. Ed. 2010, 49, 7049 –7053
Payne. R. J et al. Curr. Opin. Chem. Biol. 2014, 22, 70-78

NCL-Desulfurization
Methionine Ligation using Homocysteine➢ With C-terminal thioester andhomocysteine( Hcy), followedby methylation, we can getmethionine on the ligation site.
➢ Homocysteine can be obtained
from methylation.
➢ Methyl p-nitrobenzenesulfonate
as the methyl donor in the Smethylation step.

Tam. J. P et al. Biopolymers, 1998, 46, 319–327

21 Amino Acid ( Ligation Site Achieved)
Short Summary➢ By the method of NCL, NCLdesulfurization, and auxiliary
mediated ligation, most of theligation site are achieved.
➢ Up to now, C-terminal isthioester, and all themodifications are based on Nterminal.
➢ Ligation site of serine, tyrosine,
histidine haven’t been achieved.
Next Section➢ Some special method to expand
the ligation site.

Serine Ligation Site
Serine/Threonine Ligation( STL)
➢ STL originates from achemoselective reaction between
an unprotected peptide with a Cterminal salicylaldehyde (SAL)
ester and another unprotected
peptide with an N-terminal serineor threonine residue.

➢ After acidolysis, the product has
the nature peptide ligation site.
➢ Mild reaction condition and short
reaction time.

Xuechen. L et al. Org. Lett., 2010, 12, 1724-1727
Acc. Chem. Res. 2018, DOI: 10.1021/acs.accounts.8b00151

Serine Ligation Site
➢ The hindered β-branched amino do not retard theformation of product significantly.
➢ Side chain is tolerant to some nucleophilic aminoacid such as lysine, which might react with thealdehyde group, but the reaction is reversible andunable to generate a stable product.
➢ The C-terminal Asn sometime poses a problem inNCL, while under the STL condition, the ligation
proceeded smoothly without significant sideproducts.
➢ The peptide SAL esters with C-terminal
Lys/Asp/Glu could not be prepared as stablecompounds, because the first amino acid contains
nucleophilic side-chain functionalities.
Xuechen. L et al. Front. Chem. 2014, 2, 28.
Acc. Chem. Res. 2018, DOI: 10.1021/acs.accounts.8b00151

Aspartic Acid Ligation Site
Derivates of Serine/Threonine Ligation( STL)

➢ The amino acid bearing vicinal diol functionality was incorporated into theN-terminus of the peptide.
➢ The obtained peptide did undergo imine capture with the peptide SAL ester
followed by 6-endo-trig tautomerization, [1,5] acyl transfer, and acidolysis
to give the peptidic linkage.
➢ Further selective oxidation of the vicinal diol under mild conditions(NaIO4/NaClO2/H2O2)
Xuechen. L et al. Chinese. Chem. Lett. 2018, 29, 1119–1122
Acc. Chem. Res. 2018, DOI: 10.1021/acs.accounts.8b00151

Serine Ligation Site
KAHA Ligation➢ Fmoc-protected Ozt( oxazetidine) is stable, and after removing the Fmoc group,the ozt has high reactivity.
➢ Compared to Opr( oxaproline), reaction condition of Ozt is of lower
concentration and mild temperature.
Bode. J. W et al. Nature. Chem. 2015, 7, 668-672

Serine Ligation Site
➢ The hindered β-branched amino do not retard theformation of product significantly.
➢ Side chain is tolerant to some nucleophilic aminoacid such as lysine, which might react with thealdehyde group, but the reaction is reversible andunable to generate a stable product.
➢ The C-terminal Asn sometime poses a problem inNCL while under the STL condition, the ligation
proceeded smoothly without significant sideproducts.
➢ The peptide SAL esters with C-terminal
Lys/Asp/Glu could not be prepared as stablecompounds, because the first amino acid contains
nucleophilic side-chain functionalities.
Xuechen. L et al. Chinese. Chem. Lett. 2018, 29, 1119–1122
Acc. Chem. Res. 2018, DOI: 10.1021/acs.accounts.8b00151

Serine Ligation Site
KAHA Ligation➢ The ability to synthesis lysine-type α-ketoacid might allow the ligation
between lysine and serine.
Bode. J. W et al. Acc. Chem. Res. 2017, 50, 2104−2115

Histidine Ligation SiteMotivation

Difficulty of Histidine Ligation➢ Imidazole sidechain of histidine, if unprotected, can react with activated carboxylic
acid during coupling thus reducing the amount of activated acid for reaction.
➢ The imidazole moiety of histidine promotes epimerization during coupling andresults in mixtures of enantiomeric products.
➢ Common N-terminal protecting groups like Boc or Fmoc, however, cannot be usedsince protecting group orthogonality with the N-terminal amine need to be achievedfor effective coupling.
Birman. V. B et al. Org. Lett., 2009, 11, 1499-1502
Yuya. A. L et al. Chem. Asian J. 2018, 13, 400 – 403

Histidine Ligation Site
➢ Under the optimized conditions, thisligation enabled the synthesis of arange of histidine-containing
dipeptide, tripeptides andtetrapeptides in moderate to goodyields without the need for protectionof histidine sidechain.
➢ Amino acids such as serine, cysteine,lysine and glutamic acid, whichcontain either nucleophilic, basic or
acidic sidechains, were not tested inhistidine promoted ligation as theunprotected sidechains are highly
likely to interfere with the action of
imidazolate leading to poor ligation
results.

Yuya. A. L et al. Chem. Asian J. 2018, 13, 400 – 403

Short Summary➢ By investigating kind ofchemical ligation, we greatlyenlarged the scope ofligation site, both on the Nterminal and C-terminal.
➢ Some amino acid, withspecial substrates, canachieve ligation using more
appropriate methods.

Contents➢Introduction➢Extension of Native Chemical Ligation(NCL)➢ Enzyme-Mediated Ligation✓ Sortases✓ Butelase✓ Subtiligase➢SummaryEnzyme-Mediated Ligation➢ Compared to chemical peptide ligation, enzymatic ligation has theadvantages like mild reaction conditions, high regioselectivity, smalltoxicity, racemization-free.
➢ Enzymes, that mediate peptide and protein hydrolysis, also known asproteases are abundantly available from nature. On the contrary,
peptide ligases, enzymes that catalyze the peptide bond formation arevery rare.

➢ With the long-standing efforts, a few peptide ligases have been
discovered from nature, such as sortases, butelase and etc.

Junfeng. Z et al. Chin. Chem. Lett, 2018, 29, 1009–1016
Chunmao. H et al. Chin. Chem. Lett, 2018, 29, 1017–1021

Sortase A➢ Sortases are a class of transpeptidase enzymes that are responsible for ‘sorting’and covalently anchoring virulence factors to the cell wall of Gram-positivebacteria, while the sortase A is the most widely used one among sortases.
➢ For sortase A, five amino acid recognition sequence LPXTG, X can be any
amino acid, is necessary at the C-terminus.
➢ N-terminus consists of aminoglycine, Gn.

Ploegh. H. L et al. Curr. Opin. Struc. Biol, 2016, 38, 111–118

SrtA ( Sortase A) with Modified Condition➢ The reaction can be rendered effectively irreversible if the scissile peptide
bond (i.e., the amide bond between the threonine and glycine residues) is
replaced by an ester linkage.
➢ Substoichiometric molar equivalents of SrtA required
Turnbull. W. B et al. Nature Protocols. 2014, 9, 253-262

Butelases➢ A tripeptide recognition sequence, Asx–His–Val, must be present at the C-terminus.
➢ For the N-terminus, butelase 1 accepts 20 natural amino acids at the X1 position,
except for Pro, and acidic amino acids (Asp, Glu). And it highly favors bulkyhydrophobic amino acids such as Ile, Leu, Val and, to some extent, Cys at the X2position.
➢ The enzyme is usually added in very small quantities.
Tam. J. P et al. Nature Protocols, 2016, 17, 1977-1987

Subtiligase➢ It does not require a certain recognition motif at the termini of any reaction
partners.
➢ The sequence properties of the substrates do have great influence on thecatalytic performance.
Liminations:➢ The sequence of each target protein/ peptide and modifier pair has to be
optimized to obtain satisfactory yields.
➢ A large excess of one ligation partner and the presence of Ca 2+ is necessary.

Chunmao. H et al. Chin. Chem. Lett, 2018, 29, 1017–1021

Contents➢Introduction➢Extension of Native Chemical Ligation(NCL)➢Enzyme-Mediated Ligation➢SummarySummary➢ By investigating kind ofchemical ligation and someenzyme-mediated ligation,
we greatly enlarged thescope of ligation site.
➢ Some amino acid, withspecial substrates, canachieve ligation using more
appropriate methods.
➢ Introduction of enzymemediated ligation gives anew version for peptide andprotein ligation.

Appendix: Remove Acm group

Removal of the Acm group from the purified ligation product, H-Cys(Acm)(57–131)-OH, was achieved quantitatively by dissolving the Cys-protected
peptide at 0.5 mg/ml in 0.1 M citric acid/0.2 M HCl, to which 5 mM iodine (inMeOH) was added dropwise to a final concentration of 0.05 mM or until astable yellow color was observed.
Zhibin. W et al. PNAS. 2004, 101, 11587-11592

Appendix: Desulfurization with H2-metalDawson. P. E et al. J. Am. Chem. Soc. 2001, 123, 526-533

Appendix: β-thiol Val Ligation

Seitz. O et al. Angew. Chem. Int. Ed. 2008, 47, 6807-6810

Appendix: γ-thiol Val Ligation

Danishefsky. S. J et al. Angew. Chem. Int. Ed. 2008, 47, 8521 –8524

Appendix: β-thiol Arg Ligation

Payne. R. J et al. Chem. Bio. Chem. 2013, 14, 559 – 563

Appendix: β-thiol Asp Ligation

Zhongping. T et al. Org. Lett. 2013, 15, 6128-6131

Appendix: γ-thiol Glu Ligation

Payne. R. J et al. Org. Lett. 2014, 16, 290−293

Appendix: Isoleucine Ligation SiteChuanfa. L et al. Org. Lett. 2016, 18, 2696−2699

Appendix: Serine/Threonine Ligation➢ Product has the nature peptide ligation site, which is generated from Sfive-member intermediate.

Xuechen. L et al. Chinese. Chem. Lett. 2018, 29, 1119–1122
Acc. Chem. Res. 2018, DOI: 10.1021/acs.accounts.8b00151