Standard Custom Peptide Synthesis Services

Peptide Synthesis Modifications:

Modification Examples:

Chemically synthesized peptides carry free amino and carboxy termini. The need for N-terminal acetylation or C-terminal amidation has to be stated explicitly when ordering. It is impossible to perform these modifications after synthesis.

1. N-terminal acetylation / C-terminal amidation (Free of Charge)

Chemically synthesized peptides are delivered with free amino and carboxy termini.

Simple N-terminal acetylation and/or C-terminal amidation increase the stability of the peptide because the terminal acetylation/amidation will generate a closer mimic of the native protein. Thus these modifications may increase the biological activity of a peptide.

Advantages:

• Peptide ends are uncharged so the modifications generate a closer mimic of the native protein thus increasing the permeability of the peptides to cells. This is a good choice of modification for intracellular, in-vivo assay or in-vitro functional studies;
• The modifications increase the metabolic stability of the peptide toward enzymatic degradation by aminopetidases, exopeptidases or synthetase. So the modified peptides can be used as substrates in enzyme assays;
• Amidation of peptides enhances activity of peptide hormones, not only by prolonging their shelf live;
• Reduce influences of charged C or N terminus when ELISA binding assay is conducted.

2. Fluorophores

• Full range of dye labeling on N, C terminus or Lys side chain: Biotin, FITC, 5-FAM, Dansyl, TMR, Luciferin, Dansyl, Dnp, PyBA;
• We recommend N terminus modification (Click here for the reasons): Usually, the dyes such as Biotin, FITC can be introduced either N-terminally or C-terminally. We recommend N terminus modification for higher success rate, shorter turnaround time and easier operation.
• Click to see the comprehensive fluorophore list including absorption and emission peak values.

3. Disulfide Bridges

Disulfide bridges reduce the flexibility of a peptide, making it more rigid and decreasing the number of conformations. Such conformation constraint is important for biological activity and structural stabilization. Effective formation of disulfide bridges includes proper management of cysteine, the protection of this residue, and the methods for protecting group removal and cysteine pairing.

• Successful strategies of peptide synthesis for intramolecular disulfide bridges and intermolecular disulfide bridges;
• Full range of cyclic peptide synthesis experience: triple disulfide bridge, same or different intermolecular disulfide bridges, amide cyclic peptide at ends or side chain, thioester cyclic peptides.

4. Phosphorylation

• Phosphorylation on pSer, pTyr, pThr or D-pSer, D-pTyr, D-pThr;
• Phosphorylation on two sites, tri-sites, 4-sites and 5-sites: For example: NDEpSpTDYEpSERQpTD

5. Other modifications: Methyl amino acids, MAPs and carrier protein modifications such as BSA, OVA or KLH, D-amino acid peptide and other unusual peptide. Please click here for the full modification introduction.

Peptide Synthesis Frequently Asked Questions

Please click here to see more FAQs

1. Why is the peptide delivered at room temperature?

Peptides are shipped at room temperature and highly stable at lyophilized form in sealed vials. Peptides should not be stored in solution for long time storage. More details about handling and storage of synthetic peptides.

2. How should I dissolve peptides?

The solubility of a peptide is determined mainly by its polarity. Acidic peptides can be reconstituted in basic buffers and basic peptides in acids. Hydrophobic peptides or neutral peptides containing large number of polar uncharged amino acids or hydrophobic amino acids should be dissolved in a small amount of organic solvents. More details about how to dissolve synthetic peptides.

3. What is peptide purity?

Peptide purity is the amount of the target peptide as determined by HPLC at 214 nm, where the peptide bond absorbs. Water and residual salts are not detected UV-spectrophotometrically. Other impurities can also be found in the content includes deletion sequences (shorter peptides lacking one or more amino acids of the target sequence), truncated sequences (generated by capping steps to avoid the formation of deletion peptides), and incompletely deprotected sequences (generated during the synthesis or the final cleavage process).

Peptide purity does not include water and salts in the sample. TFA is resulted from the HPLC purification. The free N terminus and other side chain such as Arg, Lys and His will form trifluoroacetates and thus small amounts of TFA may contaminate the peptides. Peptides are usually delivered as trifluoroacetates (TFA) containing residual water. Even in lyophilized peptides, varying amounts of noncovalently-bound water still exist.

4. Is a spacer required for fluorescent modification?

Usually, the dyes such as Biotin, FITC can be introduced either N-terminally or C-terminally. We recommend N terminus modification for higher success, shorter turnaround time and easier operation from LifeTein. The reason is that the peptides are synthesized from C terminus to N terminus. So the N terminus modification is the last step in the SPPS protocol and no more specific coupling steps are required. On the contrary, the C terminus modification needs additional steps and is usually more complex.

Most dyes are large aromatic molecules so the incorporation of such bulky molecules may help to avoid interactions between the label and the peptide. This will help to maintain the peptide conformation and its biological activity. It is recommended to include a flexible spacer such as Ahx, which is a 6 carbon linker, to make the fluorescent label more stable. Otherwise FITC could be easily linked to a cysteine thiol moiety or the amino group of lysine at any position.

Please click here to see more FAQs.

Peptide Synthesis Case Studies

Incomplete deprotection steps and amino acid-coupling reactions can cause big problems for the downstream process. Longer reaction time and/or increased reagent strength could solve the sluggish deprotection reaction problems. However, in some extreme cases, these methods are not sufficient and the protecting group cannot be removed efficiently. Some sequences are prone to undergo self-association by hydrogen bonding because of the side chains. This leads to aggregation and to the formation of beta-sheet-like secondary structures that can make peptide synthesis impossible to continue.

LifeTein's optimized protocol and PeptideSyn technology provide a method for circumventing the difficult sequences problem. The technology can change the peptide structure by protecting some of peptide amide bonds, giving rise to peptides containing tertiary amides at periodic intervals. This leads to better salvation of the peptide chain and to more efficient deprotection and coupling reactions. Using Fmoc/tBu approach on our proprietary resin, the routine synthesis process of difficult sequences is improved.

Case 1: Solid-phase Synthesis of a peptide with formation of both disulfide bridges on the solid support.

A 14 amino acid peptide (molecular weight: 1981.53) with 4 phosphorylation sites at 95% purity was delivered in 3 weeks: xxxpSpTxxxpSxxxpTx

HPLC Results:

MS Results:

Case 2: Client requested a very hydrophobic 68 amino acid peptide (85% purity) with FITC modification at the N terminus. The peptide was successfully synthesized in 4 weeks.

HPLC Results:

MS Results: