Peptide Modification HEADING_TITLE

Some peptides require additional modification to better mimic the native peptide or protein fragment they were modeled on or to introduce elements that enhance their later application.  Most modifications can either be incorporated post-synthetically or during the peptide synthesis by utilizing appropriately derivatized amino acids.  Some of the common modifications are listed:

  • Cyclization
  • Stapling
  • N-Methylation of the peptide backbone
  • Phosphorylation
  • Myristilation
  • Farnesylation
  • PEGylation
  • Biotinylation
  • Fluorescent Labeling
  • Caged Peptides
  • MAP Peptides
  • Thioesters 

Cyclization 

Many natural peptides with interesting biological activity are cyclic.  Cyclization is also used in synthetic peptides to impose a desired conformation in the peptide, especially when the peptide is based on a portion of a much larger peptide or protein.  Cyclic peptides are formed in several ways: sidechain-to-sidechain, terminus-to-sidechain and terminus-to-terminus (Figure 1).  In each case, cyclization typically is performed after the linear peptide has been synthesized. 

Figure 1 - Types of Cyclic Peptides

The most common type of sidechain-to-sidechain cyclization is disulfide bridging of cysteine residues.  This cyclization is introduced by deprotecting a pair of cysteine residues and oxidizing to form the disulfide bond.  Multiple cycles can be selectively formed if selectively removable protecting sulfhydryl protecting groups are utilized.  Cyclization can be performed either in solution following cleavage from the resin or on-resin before cleavage.  On-resin cyclization may be less effective than cyclization in solution, since the resin-bound peptide may not as easily achieve a favorable conformation for cyclization.  Analogs of disulfide bridges have been prepared utilizing two allylglycine residues cyclized by ring closing metathesis.[1]

Another type of sidechain-to-sidechain cyclization is amide formation between and asparic acid or glutamic acid residue and one of the basic amino acids, i.e. lysine (Lys), ornithine (Orn), 2,4-diaminobutyric acid (Dab) and 2,3-diaminopropionic acid (Dap).  These cyclizations require side chain protecting groups that can be selectively removed while the peptide is still attached to the resin or after cleavage.

A third form of sidechain-to-sidechain cyclization is through biaryl ethers of tyrosine or hydroxyphenylglycine.  Natural products with this type of cyclization are exclusively microbial products and often have interesting and desirable pharmaceutical properties.  Preparing these compounds requires unique reactions and conditions and currently not a matter of routine peptide synthesis.

Sidechain-to-terminal cyclization usually involves the C-terminus and the amine function of a lysine or ornithine sidechain or the N-terminal and an aspartic acid or glutamic acid sidechain.  Some cyclic peptides are formed through an ester bond between the C-terminal and a serine or threonine sidechain.  Although forming the cyclic structure by forming the ester bond may seem to be the obvious way to prepare these compounds, higher cyclization yields are usually obtained when the cyclization occurs by forming a peptide amide bond elsewhere in the cyclic peptide.

The third form of cyclization is a terminal-to-terminal, or head-to-tail cyclization.  The linear peptide can be cyclized in solution[2] or while attached to resin through a sidechain.[3]  Cyclization in solution should be performed at low concentrations of the linear peptide to prevent oligomerization of the peptide. 

The yield of head-to-tail cyclized peptide from linear peptide can depend the sequence of the linear peptide.  In a recently published optimization of the protocol for preparing cyclic gramicidin S, the sequence of the linear peptide had a signifcant influence on the success of the cyclization reaction.[4]  Before preparing head-to-tail cyclized peptides on a large scale, a library of possible linear precursor peptides should be prepared and cyclized to find the sequence that provides optimum results.

Stapling

Many times when a synthetic peptide is based upon a segment of a larger peptide or protein, the peptide does not maintain the active conformation of the corresponding segment of the parent protein.  Introducing hydrocarbon bridges, referred to as staples, between residues can help the peptide adopt an alpha-helical structure.  Stapled peptides can have significantly improved activity compared to their non-stapled analogs.  Additional, stapled peptides may be better at penetrating cells and resisting enzymatic hydrolysis.[5]

Staples introduced between one residue and another residue four amino acids away in the sequence (i and i+4) span one loop of the alpha helix, staples between a residue and another residue seven amino acids away (i and i+7) span two loops of the helix.  For an i, i+4 staple, the staple should be six carbons long.  For the longer i, i+7 staple, the staple should be 11 carbon atoms. 

Stapled peptides

Figure 2 - i, i+4 Staple vs i, i+7 Staple

The staples are typically introduced by incorporating two amino acids with alkenyl sidechains into the appropriate positions of the peptide sequence, then closing the staple by a ring closing metathesis reaction.  Staples may also be formed by Cu(I) catalyzed cycloaddition of azide and alkyne containing residues.[6]

N-Methylation of the Peptide Backbone 

N-Methylation occurs in natural peptides and is introduced in to synthetic peptides to disrupt normal hydrogen-bonding and to make the peptide more resistant to biodegradation and elimination.  The N-methylated backbone is synthesized into the peptide by incorporating the appropriate N-methylated amino acid derivatives.  An alternative method involving a Mitsunobu reaction with an N-(2-nitrobenzenesulfonyl) peptide-resin intermediate and methanol has recently been employed in the preparation of a library of cyclic peptides containing N-methylated amino acids.[7]

Phosphorylation

Phosphorylated peptides play a regulatory role with most protein kinases.  Phosphorylation occurs on tyrosine, threonine and serine residues.  Phosphotyrosine, phosphoserine and phosphothreonine derivatives can be incorporated into a peptide during synthesis or can be formed after the peptide is synthesized.   Phosphoarmidite reagents are preferred for introducing phosphate groups post-synthetically.[8]  Selective phosphorylation can be achieved by using selectively removable protecting groups on the serine, threonine or tyrosine residues to be phosphorylated.

Myristylation/Palmitylation

Acylating the N-terminal with a fatty acid causes peptides and proteins to associate with plasma membranes.  Protein kinases in the src family and retroviral Gag proteins are targeted to plasma membranes by a myristylated sequence at the N-terminal.  Myristylation is also necessary in protein kinase C inhibitors based on substrate analogs.[9]  Peptides utilized in cosmetic formulations may be acetylated with a fatty acid to promote skin absorption of the peptide.

The acids can be coupled using to the resin-peptide N-terminal by standard coupling protocols.[10]  The resulting lipopeptide can be cleaved under standard conditions and purified by RP-HPLC on a C4 column.[11] 

Glycosylation

Glycopeptides like vancomycin and teicoplanin are important antibiotics used to treat bacterial infections that are resistant to other antibiotics.  Other glycopeptides are utilized because they can stimulate the immune system.  Studies with these peptides may lead to improved treatment of infections for many microbial antigens are glycosylated.  Glycopeptides may also play an important role in the study of cancer and the immune defense against tumors, because cancer cells display an abnormal glycosylation of the proteins in the cellular membrane.[12]

Glycopeptides are prepared by Fmoc/tBu protocols.  The glycosylated residues, typically serine or threonine, are often incorporated into the peptide by means of active pentafluorophenol esters of the Fmoc-protected glycosylated amino acid.[13]

Isoprenylation (Farnesylation/Geranylgeranylation)

Isoprenylation of proteins promotes membrane association and contributes to protein-protein interactions. Farnesylated proteins include tyrosine phosphatases, small GTPases, cochaperones, nuclear lamina, and centromere-associated proteins.[14]  Isoprenylation occurs on the sidechain of cysteine residues in the proximity of the C-terminal.

Farnsylated peptides can be prepared by on-resin farnesylation[15] or by incorporating farnyslyated cysteine derivatives.[16]  A novel method involving the SN2 displacement of bromine from a bromoalanine residue by farnesyl mercaptan has been reported.  Characteristic partial structures of human Ras peptides were reported to have been prepared in good yield by this methodology.[17] 

PEGylation

Attaching polyethyleneglycol (PEG) chains to peptides can improve their pharmacological profiles.  The bulky PEG inhibits degradation of the peptide by proteolytic enzymes.  The hydrodynamic radius of a PEGylated peptide is greater than the normal cross section glomerular capillaries, which greatly impedes renal clearance.[18]  These factors combined increase the effective half-life of the peptide in the body.  Therefore a lower, less frequent doses are required to maintain therapeutic levels of the peptide in the body.

PEGylation can have negative effects, too.  The PEG bulk that prevents enzymes from degrading the peptide can also reduce the binding of the peptide to the targeted receptor.  The lower affinity of the PEGylated peptide is usually offset by its longer pharmokinetic half-life.  By remaining in the body longer, there is a greater probability that the PEGylated peptide will be taken up by its target tissue.  The size of the PEG polymer should be optimized for the best results.[19]

Due to their reduced renal clearance, PEGylated peptides can accumulate in the liver, leading to macromolecular syndrome.  Hence, the PEGylation has to be carefully designed if the peptide is to be utilized in drug testing.

The PEG polymers are available in sizes from 1kDa up to 40kDa.  The PEG polymers may be monodisperse or polydisperse.  Monodisperse PEG polymers have a uniform length and molecular weight.  Polydisperse PEG polymers however have a distribution of lengths and molecular weights. 

A number of PEG derivatives have been developed for PEGylating peptides and proteins.  PEG derivatives with acid or activated carbonate functions can be coupled to N-terminal amines or lysine side chains.  Amine functionalized PEG can be coupled to aspartic acid or glutamic acid side chains.  PEG functionalized with malimide will couple to the free thiol moiety of fully deprotected cysteine side chains.[20]

Biotinylation

Biotin binds to avidin or streptavidin with strength that almost approaches a covalent bond.  Biotin-labeled peptides can be used for affinity labeling and affinity chromatography.  Labeled anti-biotin antibodies can also be used to bind biotinylated peptides.

Biotin labels are usually attached to lysine side chains or N-termini.  Often a spacer such as 6-aminohexanoic acid is applied between the peptide and the biotin.  The spacer is flexible and allows better binding to substrates in sterically hindered cases.

Fluorescent Labeling

Fluorescent tags are useful for tracing peptides within living cells.  Fluorescent tags are also very useful in studying enzymes and mechanisms.  Tryptophan is fluorescenct and seldom occurs more than once in common peptides.  Therefore it may be utilized as an intrinsic label.  The emission wavelength of tryptophan is dependent on the surrounding enviroment; the emission wavelength decreases as the solvent polarity decreases[21].  This property is useful for probing peptide structure and binding.[22]  Tryptophan fluorescence can be quenched by protonated Asp and Glu residues, which may limit its utility.

The Dansyl group, when attached to an amine, is highly fluorescent and is commonly used as a fluorescent label on amino acids and proteins.[23]

Excitation and Emission Wavelengths of Common Fluorescent Labels

 

Excitation Wavelength

Emission Wavelength

Trp

280 nm

300-350 nm

2-Abz

315 nm

400 nm

AMC

365-380 nm

430-460 nm

Dansyl

335-339 nm

490-500 nm

EDANS

335 nm

493 nm

Carboxyfluorescein

494 nm

518 nm

Cy3 550 nm 570 nm

Carboxytetramethylrhodamine

555 nm

580 nm

Cy5 650 nm 670 nm

 

Florescence resonance energy transfer (FRET) is quite useful for studying enzymes.  For FRET applications, the substrate peptide contains a fluorescent label and a fluorescence quenching moiety.  The fluorescence of the tag is quenched by the quencher through a non-photonic energy transfer.  When the peptide is cleaved by the enzyme being studied, the tag fluoresces.  Recently an inverted FRET strategy was used to study crenellation of a GTPase.[24]

FRET Donor-Acceptor Pairs

Donor

Acceptor

IAEDANS (1)

DDPM (2)

Dansyl

FITC

Dansyl

Octadecylrhodamine

CF (3)

Texas Red

Fluorescein

Tetramethylrhodamine

FITC

Eosin Thiosemicarbazide

EDANS (4)

Dabsyl

Mca (5)

Dnp

Tryptophan

Dansyl[25]

2-Abz

Tyr(3-NO2)

2-Abz

Dnp

 

 

(1) 5-(2-iodoacetylaminoethyl)aminonaphthalene-1-sulfonic acid

(2) N-(4-dimethylamino-3,5-dinitrophenyl)maleimide

(3) carboxyfluorescein succinimidyl ester

(4) 5-(2-laminoethyl)aminonaphthalene-1-sulfonic acid

(5) 7-methoxycoumarin

Caged Peptides 

Caged peptides have photoremovable protecting groups that block their affinity for a receptor.  The peptide can be activated by UV irradiation.  Because the photoactivation can be controlled in terms of time, amplitude and localization, caged peptides are used to investigate reactions occurring in intracellular pathways.[26] 

The protecting groups most frequently used for caged peptides are based on 2-nitrobenzyl moieties.  These are incorporated by utilizing appropriately protected amino acid derivatives during the peptide synthesis.  Lysine, cysteine, serine, and tyrosine derivatives have been developed.  A recent attempt to utilize 4,5-dimethoxy-2-nitrobenzyl esters on the side chains of aspartic acid and glutamic acid was unsuccessful.  These derivatives proved to be highly prone to cyclization during peptide synthesis and cleavage.[27] 

MAP Peptides

Short peptides usually are not immunogenic and must be conjugated to a carrier protein to prepare antibodies.  Multiple Antigenic Peptides (MAP) are an alternative to preparing peptide-carrier protein conjugates.  MAPs consist of multiple copies of a peptide attached to a lyseine core and typically produce highly efficient immunogens.[28] 

MAP peptides can be prepared by step-wise solid phase peptide synthesis on a MAP resin.  However, incomplete coupling can produce deletion or truncation chains on some of the branches, resulting in a crude MAP peptide that may be difficult to characterize.  Alternatively, the peptide can be prepared and purified separately, then coupled to the MAP core.[29]  The peptide sequence attached to the MAP core is unambiguous and the peptide can be readily characterized by mass spectroscopy.

Thioesters

Peptide thioesters are reactive intermediates used to form cyclic peptides and to prepare large peptides and small proteins through native chemical ligation.  Peptide thioesters can be readily prepared on thiol-linked resins utilizing Boc protocols.[30]  When Fmoc chemistry is used, the thioester has to be incorporated last since piperdine will react with thioesters to form the piperdine amide.  A number of strategies have been developed, from coupling a thiol to the C-terminal while the peptide is anchored to the resin through an amino acid side chain[31] to using a safety-catch resin and displacing the peptide from the resin with the C-terminal amino acid thioester.[32] 

Recently N-alkyl cysteine-assisted thioesterification was reported which utilizes N to S migration.[33]  Peptides with a C-terminal N-alkyl cyteine are in an acid catalysed equilibrium between the amide and thioester forms as illustrated.  Addition of 3-mercaptoproionic acid cleanly converts the peptide to the thioester of 3-mercatopropionic acid.

N-S Migration

     Figure 3 – N – S Migration

 


[1] Mollica, A.; Guardidani, G.; Davis, P.; Ma, S.-W., Porreca, F.; Lai, J.; Marrina, L.; Sobolev, A. P.; Hruby, V. J. J. Med. Chem., 2007, 50, 3138-3142.

[2] For a recent example see Cyr, J. E.; Pearson, D. A.; Wilson, D. M.; Nelson, C. A.; Guaraldi, M.; Azure , M. T.;m Lister-James, J.; Dinkelborg, L. M.; Dean, R. T. J. Med. Chem. 2007, 50, 1354-1364.

[3] Dartois, V.; Sanchez-Quesada, J.; Cabezas, E.; Chi, E.; Dubbelde, C.; Dunn, C.; Granja, J.; Gritzen, C.; Weinberger, D.; Ghadiri, M. R.; Parr, T. R., Jr. Antimicrob. Agents Chemother., 2005, 49, 3302-3310; Berthelot, T.; Gonçalves, M.; Laïn, G.; Estieu-Gionnet, K.; Deleris, G.. Tetrahedron, 2006, 62, 1124-1130; Xiao, Q. Pei, D. J. Med. Chem., 2007, 50, 3132-3137.

[4] Wadhwani, P.; Afonin, S.; Ieronimo, M.; Buerck, J.; Ulrich, A. S. J. Org. Chem., 2006, 71, 55-61.

[5] Verdine, G.L.; Hilinski, G.J., Methods Enzymol2012503, 3-33; Schafmeister, C.;, Po, J.; Verdine, G., J. Am. Chem. Soc.2000122, 5891-5892.

[6] Jacobsen, Ø.; Maekawa, H.; Ge, N.-H.; Görbitz, C.H.; Rongved, P.; Otterson, O.P.; Amiry-Moghaddam, M.; Klaveness, J., J. Org. Chem., 2011, 76, 1228-1238; Kawamoto, S.A.; Coleska, A.; Ran, X.; Yi, H.; Yang, C.-Y.; Wang, S., J. Med. Chem., 2012, 55, 1137-1146.

[7] Ueda, S.; Oishi, S.; Wang, Z.; Araki, T.; Tamamura, H.; Cluzeau, J.; Ohno, H.; Kusano, S.; Nakashima, H.; Trent, J. O.; Peiper, S. C.; Fujii, N. J. Med. Chem. 2007, 50, 192-198.

[8] Hoffmann R, Wachs WO, Berger RG, Kalbitzer HR, Waidelich D, Bayer E, Wagner-Redeker W, Zeppezauer M. Int J Pept Protein Res. 1995, 45,26-34.

[9] O'Brian, C. A; Ward, N. E; Liskamp, R. M; de Bont, D. B; van Boom, J. H. Biochem Pharmacol. 1990, 39, 49-57; Ward, N. E; O'Brian, C. A. Biochemistry, 1993, 32,11903-9.

[10] For a recent example see Cirioni, O.; Giacometti, A.; Ghiselli, R.; Kamysz, W.; Silvestri, C.; Orlando, F.; Mocchgiani, F.; Della Vittoria, A.; Kamysz, E.; Saba, V.; Scalise, G. Peptides, 2007, 28, 1299-1303.

[11] Avrahami, D.; Shai, Y.  J. Biol. Chem., 2004, 279, 12277-12285.

[12] Hakomori, S. Adv. Cancer Res. 1989, 52, 257-331.

[13] For a review of glycopeptide synthetic methodology, see Buskas, T.; Ingale, S.; Boons, G.-J. Glycobiology, 2006, 16, 113R-136R.

[14] Basso, A. D.; Kirschmeier, P.; Bishop, W. R. J Lipid Res., 2006, 47, 15-31.

[15] Ludolph, B.; Eisele, F.; Waldmann, H. J. Am. Chem. Soc., 2002, 124, 5954 -5955.

[16] Durek, T.; Alexandrov, K.; Goody, R. S.; Hildebrand, A. ; Heineman, I.; Waldmann, H. J. Am. Chem. Soc., 2004, 126, 16368-16378.

[17] Pachamuthu, K.; Zhu, X.; Schmidt, R. S. J. Org. Chem., 2005, 70, 3720-3723.

[18] Chapman, A. P. Advanced Drug Delivery Reviews, 2002, 54, 531-545.

[19] León-Tamariz, F.; Verbaeys, I.; Van Boven, M.; De Cuyper, M.; Buyse, J.; Clynen, E.; Cokelaere, M. Peptides, 2007, 28, 1003-1011.

[20] For a recent application of PEG-malimide, see Chen, C.-P.; Park, Y.; Rice, K. G.;  J. Pept. Res., 2004, 64, 237-243.

[21] Ward, L. D. Methods Enzymol. 1985, 117, 400-414.

[22] Doyle, T. C.; Hansen, J. E.; Reisler, E. Biophys. J. 2001, 80, 427-434; Verheyden, S.; Sillen, A.; Gils, A.; Declerck, P. J.; Engelborghs, Y. Biophys. J. 2003, 85, 501-510.

[23] Kinoshita, S.; Iinuma, F.; Tsuji, A. Analytical Biochemistry 1974, 61, 632-637.

[24] Walker, J. W.; Gilbert, S. H.;  Drummong, R. M.; Yamada, M. Sreeumar, R.; Carraway, R. E; et al. Proc. Natl. Acad. Sci. USA, 1998, 95, 1568-1573; Watai, Y.; Sase, I.; Shiono, H.; Nakano, Y. FEBS Lett., 2001, 488, 39-44; Durek, T.; Alexandrov, K.; Goody, R. S.; Hildebrand, A. ; Heineman, I.; Waldmann, H. J. Am. Chem. Soc., 2004, 126, 16368-16378.

[25] Gustiananda, M; Liggins, J. R.; Cummins, P. L.; Gready, J. L. Biophys. J., 2004, 86, 2467-2483.

[26] Rothman, D. M.; Shults, M. D.; Imperiali, B. Trends Cell Biol., 2005, 15, 502-510.

[27] Bourgault, S.; Létourneau, M.; Fournier, A. Peptides, 2007, 28, 1074-1082.

[28] Tam, J. P. Proc Natl. Acad. Sci. U S A. 1988, 85, 5409-5413; Posnett, D. N., Tam, J.P.. Methods Enzymol., 1989;178, 739-46; Tam, J. P. Methods Enzymol. 1989;168, 7-15.

[29] Defoort, J.P.; Nardelli, B.; Huang, W.; Tam, J.P. Int. J. Pept. Protein Res. 1992, 40, 214-221.

[30] For examples see Lu, Y-A; Tam, J. P. Org. Lett., 2005, 7, 5003-5006; Camarero, J. A.; Cotton, G. J.; Adeva, A.; Muir, T. W. J. Pept. Res. 1998, 51, 303-316.

[31] Li, L.; Wang, P. Tetrahedron Lett. 2007, 48, 29-32.

[32] Camarero, J. A.; Hackel, B. J.; de Yoreo, J. J.; Mitchell, A. R. J. Org. Chem., 2004, 69, 4145-4151; Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc., 1999, 121, 11369 –11374; Quaderer, R.; Hilvert, D. Org. Lett., 2001, 3, 3181-3184.

[33] Hojo, H.; Onuma, Y; Nakahara, Y.; Nakahara, Y. Tetrahedron Lett., 2007, 48, 25-28.