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PEPTIDE SYNTHESIS AND PURIFICATION

Frequently Asked Questions on Peptide Synthesis and Purification

How do you synthesize peptides?

With very few exceptions, our peptides are synthesized by chemical methods.

The source (synthetic/recombinant) is explicitly mentioned in the product entry and included in the designation of recombinant peptides (rec).
Bachem’s peptides are obtained either by “classical” solution-phase synthesis or by solid-phase synthesis (SPPS). The peptide is elongated starting from the C-terminus to the N-terminus of the sequence.

What does SPPS mean?

SPPS (solid-phase peptide synthesis) is a standard method for obtaining peptides.

The peptide, linked by its C-terminal amino acid to a carrier resin, an inert, insoluble but swellable polymer, is assembled in the C→ N direction. It is elongated stepwise by coupling suitably protected derivatives of the amino acids constituting its sequence until coupling the N-terminus. After each coupling step, the "temporary" Nα-blocking group has to be removed without affecting the lateral protective groups and the link to the carrier.

The method can be fully automated. Thus, peptides can be obtained by SPPS far more rapidly than in solution, but the method requires large excesses of expensive amino acid derivatives for driving the coupling steps to completion.

For more information, please order our brochure “The Bachem Practice of SPPS“ for free.

What do Boc- and Fmoc-strategy mean?

Two SPPS strategies are now considered standard procedures, the Boc/Bzl and the Fmoc/tBu protection scheme. Fmoc/tBu- and Boc/Bzl-SPPS differ in the choice of the temporary Nα (Boc or Fmoc) and permanent (side-chain: tBu-related or Bzl-related) protecting groups. The type of Nα-blocking group determines the options for side-chain protection and the conditions of final cleavage (removal of the permanent protective groups and splitting from the carrier). Fmoc-SPPS is considered the milder method.

How do you obtain small peptides?

Di- and tripeptides as well as chromogenic and fluorogenic enzyme substrates (e.g. peptide-AMC’s) are usually synthesized in solution.

Do you produce peptides by recombinant methods?

Bachem offers a number of recombinantly produced peptides. They are designated by “rec”

e.g. rec Human Growth Hormone (expressed in E.coli)

How do you purify the peptides?

Peptides obtained by SPPS are usually purified by preparative RP-HPLC. Short peptides obtained by solution synthesis are subjected to counter-current distribution or RP-HPLC.

How pure are your catalog peptides?

The HPLC-purity of most of our peptides is 95% or above. Lower purities have to be expected for labile peptides, e.g. peptides containing free Cys or an N-terminal Gln, or sulfated peptides, and “difficult” peptides. In the latter case, the purity could be steadily improved by optimization of synthetic and purification methods.

Why are peptides sold as salts?

The guanidino group of Arg, a strong base, the ε-amino group of Lys, the free N-terminus, and the weakly basic imidazole moiety of His can form salts with acids. Most peptides contain at least one of these basic functionalities.

Except when mentioned otherwise, our peptides are provided as trifluoroacetate salts. Peptides obtained by SPPS come into contact with trifluoroacetic acid (a strong acid) during cleavage from the carrier resin and purification. Generation of other salt forms requires an additional ion-exchange step.

Acidic peptides, i.e. peptides containing a large proportion of Asp and/or Glu, or phospho- or sulfotyrosine, can form salts with bases. Thus, a few acidic peptides are sold as ammonium salts.

Peptides obtained in solution are offered as acetate salts or inner salts, unless mentioned otherwise.

Long peptides usually do not form stoichiometric salts with acids or bases, as low molecular weight bases or acids do.

Are peptides containing free Cys supplied as monomers?

Yes.
The purity stated on the ADS for peptides containing free Cys residues signifies the monomer content at the time the analytical HPLC was recorded. As air oxidation cannot be completely prevented in the meantime, we suggest reducing the peptide before use by treatment with dithiothreitol (DTT).
For a short description of the cystine reduction using DTT please see our Technical Note “Reversal of Inadvertent Oxidation of Cys-containing Peptides”, which can be downloaded from the “Service & Support” section of our homepage under “Technical Library”.

Do I have to expect batch-to-batch variability?

The impurity profile and especially the peptide content can vary somewhat from batch to batch, even if the protocols of synthesis and purification have not been changed.

What is the difference between “research-grade” and “GMP-API”?

“Research-grade” peptides are intended for labaratory and research purposes only!
They may not be used as drugs. GMP-APIs are products monitored for chemical and biological contaminants applicable to humans.

Can I convert a free peptide into the peptide amide?

Unfortunately, with few exceptions, free peptides cannot be modified, e.g. the C-terminal carboxylic group cannot be converted to a carboxamide. The peptide amide has to be synthesized de novo, i.e. we can offer it as a custom peptide. Just click on the tab “Custom Synthesis” to ask for a quote.

What is the difference between Aβ 1-42 H-1368 and H-6466?

The amyloid peptides differ in the salt form: H-1368 corresponds to the inner salt of Amyloid β-Protein (1-42), whereas H-6466 corresponds to Aβ 1-42 hydrochloride. H-6466 fibrillates more readily than H-1368.

What is a control peptide?

Control peptides are inactive analogs of bioactive peptides for obtaining negatives. Usually, scrambled, reverted or analogous peptides are used as controls, e.g.

 

Amyloid β-Protein (1-42) ↔ Amyloid β-Protein (42-1)

RGDS ↔ RGES

Prion Protein (106-126) (human) ↔Prion Protein (106-126) (human) (scrambled)

What information can I get on your peptides before buying them?

You can download the Analytical Datasheet (ADS) of the currently available lot from the product entry.