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 contact us if you need a modification not listed below or if you have any questions about the modifications. You will receive an answer within 24 hours.
- 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 protein conjugated peptides: Peptides can be conjugated to KLH or BSA carrier protein via a cysteine. There can only be one cysteine in the sequence.
- 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. This is however, only possible for hydrophilic peptides.
- Cyclization of peptides: Peptide synthesis with disulfide bonds or amide bond.
- DOTA, DOPA and DTPA conjugated peptides: Conjugation of DOTA, DOPA and DTPA
- D-Peptides: Peptides with all or some of the amino acids in D-enantiomeric conformation.
- 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, TAMRA etc.
- Fluorescence/quencher pairs for FRET analysis: EDANS/Dabcyl.
- Formylation: Formylated N-terminus or lysine.
- Isotope labelled amino acids. Peptides can be synthesized with a range of isotope labelled amino acids.
- KLH, BSA or MAP peptides: KLH or BSA conjugated peptides. MAP peptides.
- Linkers: A range of different linkers can be inserted between for example biotin or a fluorochrome and the peptide sequence. The most commonly used linkers are Ahx or miniPEG.
- Methylated peptides: Peptide synthesis with mono or di-methylated lysines or 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). The modification is only possible for short peptides.
- Unnatural amino acids: For example: D-amino acids, Aib, Abu, Ahx, Dab, Cit, Orn, pGlu (only at the N-term), Nal, Nle, Pip, Pyr, Hyp, Tyr(3-NO2) and Tle. Many others are possible, please request.
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 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.
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 be made at the N-terminus or C-terminus. Biotin is conjugated directly to the primary amino group on the N-terminus. Peptides can also be biotinylated at the C-terminus via the primary 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 different types of linkers to be inserted between biotin and the peptide sequence. For example aminohexanoic acid (Ahx) or 8-amino-3,6-dioxaoctanoic acid linker (miniPEG).
Carrier protein KLH or BSA conjugated peptides and MAP:
After peptide synthesis peptides can be conjugated to the two carrier proteins KLH or BSA. Carrier proteins are conjugated via the side chain on an inserted 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. There can only be one cysteine in the sequence if carrier protein shall be conjugated to the peptide. Please contact CASLO if you need assistance with finding the right sequence for immunization. If carrier proteins cannot be used for immunizations, peptide synthesis can be designed where peptides are made as branched peptides (MAP) 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. One example is the HIV-TAT sequence (GRKKRRQRRRPQ) placed at the N-terminal part of a peptide, this is the most commonly used sequence of the several options for making peptides more permeable to cells. There are a range of other cell penetrating sequences like for example: RRRRRRRR or LIKLWSHLIHIWFQNRRLKWKKK. Another way is to conjugate peptides to a fatty acid like for example myristic acid at the N-terminus. The fatty 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, the fatty 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 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 inserted in a peptide sequence during the peptide synthesis. Propargylglycine acts as an alkyne and the peptide can thereby be conjugated to azido-conjugated molecules.
Peptides are in general delivered as trifluoroacetate (TFA) salts. Peptide TFA salts can be used for some cell cultures and a some typeof in vivo experiments. There are however, 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 cysteine. Cholesterol can however, only be conjugated to strongly hydrophilic peptides.
Cyclization of peptides:
Peptide synthesis can be made where peptides are cyclized by disulfide bond(s) between cysteines or by amide bond between the N- and C-terminus. Amide bonds can be created on peptides from 5 and up to 20 amino acids, disulfide bonds also on longer peptides
Peptides can be made with some or all of the amino acids in D-enantiomeric conformation. Amino acids in D-enantiomeric conformation are the mirror images of the natural L-enantiomers. D-enantiomeric 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 significantly more stable than peptide containing only L-amino acids. In some cases peptides containing D-amino acids furthermore have higher biological activity than the natural L-form, in other cases they have however, lower activity.
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 or the side chain of lysine. 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.
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 (excitation) 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 (excitation) and emission spectrum peak wavelengths of 492/517 nm.
3) Carboxyfluorescein also defined as just 5-FAM. Absorption (excitation) and emission spectrum peak wavelengths of 492/517 nm.
4) Rhodamine B. Absorption (excitation) and emission spectrum peak wavelengths of 540/625 nm, respectively.
5) TAMRA. Absorbtion (excitation) peak at 552 nm and an emission peak at 578 nm. TAMRA can be excited for example by using a 561 nm laser paired with a 582/15 nm bandpass filter
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 broken, 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 most commonly used fluorescence/quencher pair is EDANS/Dabcyl. Excitation and emission spectrum peak wavelengths of the EDANS fluorochrome are 336/490 nm respectively. Emission max for Dabcyl is 472 nm.
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.
Isotope labelled amino acids:
Peptides can be made with a range of isotope labelled amino acids. Isotope labelled amino acids in protected form, and in high quality, that can be used for peptide synthesis, are expensive, and isotope labelled peptides are therefore more expensive than standard peptides. The most economically isotope labelled amino acids are glycine and alanine. The most expensive are the amino acids with more complex side chains. CASLO do not recommend isotope labelling of more than one or two different amino acids in the sequence. Some of the amino acids can be labelled with one or more 13C and/or 15N isotopes which give different shifts in the molecular weight. Isotope labelling of peptides are typically used to track a peptide for example by mass spectrometry, but they can also be used for spiking or other applications.
A number of different types of linkers can be inserted between active groups and the peptide sequence. For example a linker can be used to create a distance to biotin or a fluorochrome. The most commonly used linkers are aminohexanoic acid (Ahx) and 8-amino-3,6-dioxaoctanoic acid linker (also called OEG or miniPEG).
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.
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)
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.
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. It is only possible to make sulfation on short hydrophilic peptides.
Unnatural amino acids:
It is possible to synthesize peptides with several unnatural amino acids, for example: D-amino acids, Abu, Ahx, Aib, Dab, Cit, Orn, pGlu (but only at the N-terminus), Hyp, Nal, Nle, Pip, Pyr, Tyr(3-NO2) and Tle. Several other unnatural amino acids are possible, please request.