Peptide Modifications

Peptide synthesis can be made with an extensive range of peptide modifications.

The below listed modifications are only examples, and represent the most commonly requested modifications. Several other modifications are available.

Please do not hesitate to request if you need a modification not listed below or if you have any questions about the peptide modifications. You will receive an answer within 24 hours.  

 Headlines:

  • Acetylation/amidation of peptides: Acetylation of N-terminus and/or amidation of C-terminus.
  • Azido-conjugated peptides: Peptide synthesis can be made with an azido group conjugated to the primary epsilon amino group on an inserted lysine or as 5-azidopentanoic acid on the N-terminus.
  • Biotinylation: Peptides can be biotinylated directly at the N-terminus, or at the C-terminus via a C-terminal lysine.
  • Carrier proteins and MAP peptides: KLH or BSA conjugated peptides. MAP peptides.
  • Cell penetrating peptides: Different sequences for increased cell penetration of peptides.
  • Click chemistry activated peptides: Peptides can be activated for click chemistry by conjugation to 5-azidopentanoic acid or the azidogroup can be conjugated to lysine or propargylglycine can be conjugated to the peptide for reactivity with azido groups.
  • Counterions: Peptides are as standard delivered as TFA salts. Peptides can alternatively be counterbalanced by chloride or acetate counterions.
  • Cholesterol conjugated peptides: Cholesterol can be conjugated to a peptide via a N- or
    C-terminal inserted cysteine after peptide synthesis.
  • Cyclization of peptides: Peptide synthesis with disulfide bonds or amide bonds.
  • DOTA, DOPA and DTPA conjugated peptides: Conjugation of DOTA, DOPA and DTPA to the termini.
  • Fatty acid conjugated peptides: Caprylic acid (C8), Capric acid (C10), Lauric acid
    (C12), Myristic acid (C14), Palmitic acid (C16) or Stearic acid (C18) etc.
  • Fluorochrome conjugated peptides: FITC, 5,6 FAM, Rhodamine B etc.
  • Fluorescence/quencher pairs for FRET analysis: Abz/Dnp and EDANS/Dabcyl.
  • Formylation: Formylated N-terminus or lysine.
  • Methylated peptides: Peptide synthesis with mono or di-methylated lysines of monomethylated arginine (symmetric or asymmetric).
  • Phosphorylated peptides: Phosphorylation of tyrosine, serine or threonine.
  • Resin conjugated peptides: Peptides can be delivered fully protected and conjugated to resin for further processing.
  • Side chain protected peptides: Peptide synthesis with various removable side chain protection groups.
  • Stabilization of reactive peptides: DTT can be added if peptides contain several cysteines or other amino acids that are easily oxidized.
  • Sulfated peptides: Sulfation of tyrosine, Tyr(SO3H2).
  • Unnatural amino acids: D-amino acids, Aib, Abu, Ahx, Orn, pGlu, Nle, DAB, Cit, Hyp, Tyr(3-NO2), Met sulfoxide or sulfone.   

 

Detailed descriptions:

Acetylation/amidation of peptides:

Peptide synthesis can be made where peptides are synthesized with acetylated N-termini and/or amidated C-termini. Acetylation and amidation reduce the loadings of the termini, and this can in some cases be an advantage. An acetylated and amidated peptide mimics an internal peptide sequence better than peptides with free termini. Acetylation and amidation
increases the resistance to exonucleases which can be an advantage in cell studies or in vivo experiments.

Lysine(s) can during peptide synthesis be acetylated at the primary epsilon amino group (Lys(Ac)). Acetylation of lysine is relevant in for example epigenetics, where acetylated lysine is involved in binding of peptides/proteins to DNA. Contrary to methylated lysine, acetylated lysine is not positive charged.

Azido-conjugated peptides:

Peptides can be delivered with an azido group conjugated to the primary epsilon amino group on an inserted lysine or azido group can be conjugated as 5-azidopentanoic acid on the N-terminus.

Biotinylation of peptides:

Biotinylation of peptides can during the peptide synthesis be made at the N-terminus or C-terminus. Biotin is conjugated directly to the primary amino group on the N-terminus. Peptide synthesis can also be made where peptides are biotinylated at the C-terminus via the primary epsilon amino group on a C-terminal inserted lysine.

Biotin has a strong affinity for streptavidin and biotinylation of peptides is therefore an efficient method to specifically bind peptides to streptavidin coated surfaces.

If a distance between biotin and the peptide is important, for example to avoid sterical hindrance, CASLO offers that an inert six carbon linear aminohexanoic (Ahx) linker can be inserted between biotin and the peptide sequence.

Carrier proteins and MAP conjugated peptide synthesis:

After peptide synthesis peptides can be conjugated to the two carrier proteins KLH or BSA, Carrier proteins can be conjugated to N- or C-termini of peptides via inserted N- or C-terminal cysteine. KLH or BSA conjugated peptides are primarily used for immunizations. Peptides are poor stimulators of the cell mediated immune response, but when peptides are conjugated to carrier protein, the cell mediated immune response is increased significantly. If carrier proteins cannot be used for immunizations, peptide synthesis can be designed where peptides are made as branched peptides (MAP peptides) to increase the cell mediated immune response.

Cell penetrating peptides:

There are several cell penetrating amino acid sequences, most of them are positively loaded sequences. Cell penetrating sequences can be used as extensions to peptide sequences thereby making them more permeable to cell membranes, or cell penetrating peptides can be used to make other molecules permeable to cells. One example is the HIV-TAT sequence (GRKKRRQRRRPQ) placed at the N-terminal part of a peptide, this is one of the many options for making peptides more permeable to cells, and the most commonly used method. There are a range of other cell penetrating sequences like: 1) ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cys, 2) ac-GALFLGFLGAA-GSTMGAWSQPKSKRKV-cys, 3) RRRRRRRR or 4) LIKLWSHLIHIWFQNRRLKWKKK. Another way is to conjugate peptides to Myristic acid (also called tetradecanoic acid) at the N-terminus. The myristic acid has a sufficiently high hydrophobicity to become incorporated into the fatty acyl core of the phospholipid bilayer of the plasma membrane of eukaryotic cells. In this way, myristic acid acts as a lipid anchor in biomembranes.

Click chemistry activated peptides:

Peptides can be delivered conjugated to 5-azidopentanoic acid for "click chemistry". Alternatively the azido group can be conjuagted to the epsilon primary amino group on an inserted lysine. The azido group reacts with alkynes in the presence of Cu/CuSO4 yielding triazoles. This is for example used for conjugation of peptides to alkyne conjugated DNA oligonucleotides. Propargylglycine can also be conjugated dring the peptide synthesis. Propargylglycine acts as an alkyne and the peptide can thereby be conjugated to azido-conjugated molecules.

Counterions:

Peptides are in general delivered as trifluoroacetate (TFA) salts. Peptide TFA salts can be used for most cell cultures and a range of in vivo experiments. There are however, some cell cultures that are sensitive to the TFA counterion, and in some in vivo experiments TFA salts cannot be used. For these applications CASLO recommend that peptides are delivered as chloride or acetate salts which are natural counterions.

Cholesterol conjugated peptides:

Cholesterol can be conjugated to a peptide via a N- or C-terminal inserted cysteine.

Cyclization of peptides:

Peptide synthesis cn be made where peptides are cyclized by disulfide bond(s) between cysteines or by amide bond between the N- and C-terminus.

DOTA, DOPA and DTPA conjugated peptides:

DOTA, DOPA and DTPA conjugated peptides are primarily used in renal science. The modifications can be made at the N-terminus or at the C-terminus via a C-terminal inserted
lysine.

Fatty acid conjugated peptides:

Fatty acid conjugated peptides can be used for a number of different applications, for example antibacterial activity or eukaryotic cell toxicity. Peptide synthesis can be arranged where fatty acids are conjugated to the N-terminus of peptides. Peptides can be conjugated to fatty acids like: Caprylic acid (C8), Capric acid (C10), Lauric acid (C12), Myristic acid (C14), Palmitic acid (C16) or Stearic acid (C18) etc. Furthermore cysteines in peptides can be palmitoylated.

Fluorochrome conjugated peptides:

Fluorochrome conjugated peptides can be visualized by fluorescence microscopy or other fluorescence visualisation techniques. Peptides can be conjugated to fluorophores directly at the N-terminus during peptide synthesis, (FITC always via an aminohexanoic acid (Ahx) linker). Conjugation can also be made to the C-terminus via an inserted lysine. Peptides can be delivered conjugated to the following fluorochromes:

1) Fluorescein isothiocyanate (FITC). Absorption and emission spectrum peak wavelengths of approximately 495 nm/521 nm.

2) 5-(and-6)-Carboxyfluorescein (5-(and-6)-FAM,mixed isomer) also defined as just 5,6-FAM. Absorption and emission spectrum peak wavelengths of 492/517 nm.

3) Rhodamine B, Absorption and emission spectrum peak wavelengths of 540/625 nm, respectively.

CASLO offers peptides conjugated to other fluorochromes, but the above listed four fluorochromes are the fluorochromes that function best with synthetic peptides.

Fluorescence/quencher pairs for FRET analysis:

Peptides synthesis can be arranged where peptides are conjugated with fluorochromes and quenchers for FRET analysis. Fluorescence/quencher pairs must have a perfect spectral overlap between the emission spectrum of the fluorochrome and absorbance spectrum of the quencher. When a fluorochrome and a quencher are conjugated to the same peptide, with a limited distance, the quencher blocks the emission of the fluorochrome. When however, the peptide is divided, for example by enzymatic degradation, the distance is increased and the fluorochrome is activated. The intensity of the fluorescence is therefore proportional with the degradation of the peptide.

The two fluorescence/quencherpairs CASLO offers for synthetic peptides are:

1) Abz/Dnp

2) EDANS/Dabcyl

Abz/Dnp is the pair that works best for peptides and also the Fluorescence/quencher pair that most often is used. Abz/Dnp is the pair that has the higest success rate at peptide synthesis. Abz is the fluorochrome which is conjugated to the N-terminus and has an Excitation and emission spectrum of 320/420 nm, respectively. Dnp quencher is always conjugated either internally in the sequence, or to the C-terminus, and always via the primary amino group on a lysine.

EDANS/Dabcyl is an alternative to Abz/Dnp, but it is significantly more expensive, and more complicated to conjugate during peptide synthesis than Abz/Dnp. Excitation and emission spectrum peak wavelengths of the EDANS fluorochrome are 336/490 nm respectively. Emission max for Dabcyl is 472 nm.

Formylation:

Formylation of proteins or peptides has a wide range of applications in protein science. CASLO can deliver peptides formylated at the N-terminus, or at other locations via an inserted lysine.

Methylated peptides:

CASLO offers peptides with methylated lysines or arginines. Peptide synthesis can be made where lysines can be mono- di- or trimethylated. Arginine can be monomethylated and can also be symmetric or asymmetric dimethylated. Methylated peptides can be used for a number of applications,. Methylated peptides and proteins play an important role in gene expression, as methylation of a number of proteins change the binding affinity to DNA or alter the histone pathway.

Phosphorylated peptides:

Peptides can be phosphorylated by phosphorylation of tyrosine, serine or threonine. Peptides can be made with one or two phosphorylation sites, some peptides can be made with more sites but it depends on the length and sequence.

Peptides conjugated to resin:

Peptides can be delivered fully protected and conjugated to resin solid phase for further peptide synthesis or processing by customer. Detailed descriptions of the resin and detailed instructions for cleavage will be provided.

Side chain protected peptides:

Peptides can be delivered with side chain groups protected with various protection groups which can be removed:

Cys(Acm) or Cys(tBu)

Lys(Dde) (Lysine can also be delivered methylated or acetylated, but these groups cannot be removed)

Met(Se)

Stabilization of reactive peptides:

If the peptide sequence contains several cysteines, or other reactive amino acids, which are easily oxidized, CASLO offers to deliver the peptide with traces of the strong reductant DTT. The peptide is only delivered with DTT if this is specifically permitted by the customer.

Sulfated peptides:

Peptides can be sulfated by sulfation of tyrosine, Tyr(SO3H2). Sulfation of tyrosine increase interactions to other proteins or peptides. Proteins that are dependend on strong bonds to other proteins are therefore often sulfated like adhesion proteins and proteins like some receptors, hormones etc.

Unnatural amino acids:

D-amino acids are the mirror images of the natural L-isomers. D-isomeric amino acids are used for a range of applications. Most often D-amino acids are used to increase the resistance against a range of degradation enzymes. Peptides containing D-amino acids are therefore significantly more stable than peptide containing only L-amino acids. In some cases peptides containing D-amino acids have higher biological activity than the natural L-form. Peptides can be made with all the 20 natural amino acids as D-isomers (except for glycine where the L- and D-isomers are the same).

Other unnatural amino acids that can be built into peptides are:

Aib,  Abu,  Ahx,  Orn,  pGlu,  Nle,  DAB,  Cit,  Hyp,  Tyr(3-NO2),  Met sulfoxide or sulfone.