AltaBioscience
Introduction to Synthetic Peptides
How they are made
Alta Bioscience uses solid phase synthesis to make all of its peptides. Here, the C-terminal amino acid
is anchored to polystyrene based resins and the peptide is grown amino acid by amino acid towards
the amino terminal. When the peptide chain is complete, it is cleaved off the resin with acid, a process
that removes the amino acid side chain protection at the same time. After removal of the acid, the
peptide is ready for QC by HPLC and mass spectrometry. After satisfactory QC, the peptides are
purified by preparative reverse phase HPLC, then freeze dried, packaged and dispatched.
Aspects of purity
Peptide purity
up process, even if the crude material exceeds
The purity of all our purified peptides is
the requested purity. The laboratory makes
determined by reverse phase HPLC. A
extensive use of capping during synthesis, so
wavelength of 215nm is used for the analysis
deletion peptides are very rare. However some
as this is the optimum for the detection of the
truncated material and peptide with modified side
peptide bond and hence detects all peptide
chains could be present.
species present.
It should be noted that the purity value obtained Net peptide content
by this method does not include the presence of
All dried peptides will contain a variable
any water and trifl uoroacetate salt which will be
amount of water plus a fixed amount of the
present in the dried material. Unless specified
peptide counter-ion, usually trifl uoroacetic acid.
in the order, all Alta Bioscience peptides are
Quantitative amino acid analysis is the only
supplied with trifl uoroacetate as the counter-ion,
method which enables the net peptide content to
acetate or chloride can be supplied on request.
be determined. Here, the amount of each amino
Reverse phase chromatography will remove all acid is measured after total acid hydrolysis, the
the reagents used in the cleavage process. All sum total of which gives the amount of peptide
Alta Bioscience peptides that are supplied to a in the product. Typical values for net peptide
content range from 70%  90% but in extreme
specified purity will have been through a clean-
cases can be as low as 20%.
An Introduction to Synthetic Peptides Page 57
AltaBioscience
Levels of purity synthesised.
Three levels of purity are offered, >95%, >90%,
3. Amino acid analysis
>80%, in addition to unpurifi ed material. The
This technique is primarily used to measure the
higher the purity, the higher the cost of the
net peptide content of a product. The peptide
fi nished product. In general, the >95% purity
is acid hydrolysed to its amino acids and these
is only needed when the peptide is to be used
are quantifi ed after separation by ion exchange
as an enzyme substrate or in NMR and X-ray
chromatography and detection with ninhydrin.
crystallography analysis. It is not necessary to
specify high purity for peptides that are to be
4. N-terminal sequencing
used to raise antibodies.
Amino terminal Edman sequencing can be used
If a peptide is requested to a set purity, Alta
to confirm that the sequence of the amino acids
Bioscience will put it through a purification
is correct.
process, even though the crude material passes
the HPLC purity specification. All purified
peptides are supplied with HPLC and MS traces.
Design and structure of peptides
Salt form of peptides
By convention, peptides are written left to right
with the N-terminus at the left and the C-terminus
As peptides are usually purifi ed by HPLC with
at the right. Care must be taken when specifying
acetonitrile gradients and trifluoroacetic acid,
modifi cations. An example of a typical modified
(TFA), as moderator, they exist as their TFA
sequence is shown below.
salts. For most purposes this is not a problem
but when adding peptides to cell cultures, the
acetyl- KLPSSRY pS AGHLLD -amide
TFA can sometimes be toxic. This problem
PhosphoSerine is spelled out as pS with spaces
can be avoided by specifying peptides in either
before and after. The words acetyl and amide
acetate or chloride salt forms.
are separated from the peptide by spaces and
The analysis of peptides hyphens. (Don t forget that both amide and
acetyl spell out a real peptide sequence).
Alta Bioscience has the capability to analyse its
product by a wide range of methods.
Amino acid classification
1. HPLC The following table gives a general classification
of the amino acids
High Performance Liquid Chromatography,
HPLC, is the primary method of analysing
Acidic, polar Asp, Glu
peptide purity. Performed typically on a C18
Basic, polar His, Lys, Arg
reverse phase column, 4.6mm x 250mm with
Polar uncharged Asn, Cys, Gly, Gln,
300Å pore size silica, using an acetonitrile water
Pro, Ser, Thr, Tyr
gradient with TFA, as the acidic species.
Nonpolar and Ala, Ile, Leu, Met,
2. MALDI-TOF
hydrophobic Phe, Trp, Val
A  matrix assisted laser desorption and
Solubility
ionisation  time of flight mass spectrometer is
Solubility, or primarily the lack of it, is the cause
used to determine the molecular weight of the
of the majority of problems when working with
peptides. Highly accurate, fast and requiring
peptides. In general, peptides with a large
small amounts of sample, it is the ideal method
proportion of nonpolar amino acids will be
to ascertain that the target peptide has been
Page 58 An Introduction to Synthetic Peptides
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difficult to dissolve in aqueous solutions, the
NH2
O
C
more polar residues that are present, the easier
H2
CH2 C
O
it will be to dissolve a peptide. Peptides that are
C CH2 O +
CH2 O NH3
acidic, i.e. contain more acidic amino acids than
H2N CH C HN CH C
basic, will be more soluble at higher pH and visa
versa, peptides that are overall basic will be most
soluble at lower pH. Figure 1. Mechanism of pyroGlu formation
*ð ð
Peptides containing long strings of Valine
Length of peptides
or Isoleucine are virtually impossible to
Although Alta Bioscience has made some very
synthesise and work with.
long peptides of over 80 amino acids, the solid
*ð ð
A peptide with no charged or polar groups
phase method essentially has a realistic upper
may be very insoluble.
limit of about 50 amino acids. Above this length,
*ð ð
Multiple additions of phospho amino acids
the high risk of failure tends to make a synthesis
can cause major synthesis and purification
fi nancially uneconomic. As the length increases,
problems. The peptides can be made but
so does the number of impurities that have to
the costs rise steeply with each additional
be removed from the target sequence, thus the
phospho group.
absolute purity of the product will be lower. A
*ð ð
If possible, it is best to avoid cysteine when
longer peptide will also have a higher chance of
designing peptides for raising antibodies.
containing a sequence region that is difficult to
In proteins, cysteine usually exists as a
make.
disulphide bridge so it would present a very
The ease of synthesis of any peptide is entirely
different shape if presented as the monomer,
dependent on its sequence, a difficult sequence
as shown in figure 6.
can easily prevent even a short peptide of 10
*ð ð
These amino acids decrease solubility:-
amino acids being made. Alta Bioscience will
Trp, Val, Ile, Phe
freely give as much help as possible concerning
the viability of a synthesis. Peptides that are
potentially difficult, could cost more to make than
easy ones.
If in doubt please ask, we are happy to
give advice free of charge
Things to avoid
Some sequences can be particularly difficult
and if they can be avoided in some way, the
Things to include if possible
synthesis will be much easier or even made
*ð ð
Proline breaks up beta sheet formation
possible.
and although non-polar, helps to solubilise
*ð ð
N-terminal Gln should be avoided at
peptides.
all costs. It is very unstable and rapidly
*ð ð
A spacer between a dye or tag and the
forms the cyclic pyroglutamic acid as shown
rest of the peptide sequence is usually
in the illustration. It is best to add either
advantageous. A range of spacers can be
pyroglutamic acid itself, or include an
used. Ahx, amino hexanoic acid is a simple,
acetyl group at the N-terminal Glutamine.
useful spacer. SGSG is a hydrophilic
sequence designed by Alta Bioscience for
use as a biotin spacer. A range of PEG
spacers are available with varying numbers
An Introduction to Synthetic Peptides Page 59
AltaBioscience
of atoms. 13C and 15N can be incorporated into peptides
for use in quantitative mass spectrometry. It
*ð ð
It is always cheaper to put a dye or tag at the
is advised to focus on the amino acids with
N-terminus rather than the C-terminus.
nonreactive side chains, such as Val and Phe.
*ð ð
These amino acids increase solubility:- Lys,
The more complex amino acids tend to be
His, Arg, Asp, Glu, Ser, Thr.
prohibitively expensive, if available at all.
*ð ð
Unnatural amino acids
Modifi cations and unnatural amino
Compounds such as phenylglycine, napthyl
acids alanine, nor leucine and beta alanine are readily
incorporated into peptides.
There is a huge number of modifications
Spacers
possible, listed below are the more common *ð ð
ones. The structures of many of these unusual
These are used to pull dyes and tags away
amino acids are shown in the accompanying
from the active site of a peptide, some common
paper,  Table of the amino acids .
examples are shown here:-
*ð ð
Phosphorylated amino acids
Hydrophobic aminohexanoic acid
Phosphorylated Ser ,Thr and Tyr can be placed
Hydrophilic SGSG a short peptide sequence
at any specified site in a peptide. However,
Hydrophilic PEG, ranging from 9 to 88 atoms
multiple incorporations can cause synthesis and
purification problems.
*ð ðerminus modifications
T
Please let us know if you need
N-terminal acetyl and C-terminal amides remove
a compound that isn t in the above
the charges at the ends of a peptide and make it list of modifications
much more like the parent protein.
*ð ð
Methylation
*ð ð
Biotin.
Mono, di and tri methylated Lys, mono and
O
dimethyl Arg are found in histone proteins,
C
these methylated amino acids can be easily
HN NH
incorporated at specific positions.
O
CH CH
H2 H2
C C C
*ð ð
D amino acids
OH
H2C
CH C C
H2 H2
S
All the D amino acids can be added at any
position. Binds irreversibly to streptavidin and is used
extensively in screening assays and to bind
*ð ð
Analogues
peptide to substrates.
Amino acids with longer or shorter versions of
*ð ð
Desthiobiotin.
the side chain length are available. For example,
O
homoserine and homoarginine are longer
C
variants of serine and arginine while ornithine
HN NH
and diamino butyric acid are shorter analogues
O
CH HC
H2 H2
of lysine. These are very useful in fi ne tuning the C C C
H3C C
OH
H2 C C
shape of peptides. H2 H2
Binds to streptavidin but can be displaced
*ð ð
Isotopes
by biotin. Useful when you need to get your
Amino acids enriched with the stable isotopes
peptide out of a binding experiment.
Page 60 An Introduction to Synthetic Peptides
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Cross linked peptides
Peptides with Dyes
Many bioactive peptides contain several
A very wide range of dyes and tags are available,
disulphide bridges. Alta Bioscience has had
a short list of the more common ones is shown
considerable success in the synthesis of these
here. The accompanying paper,  Introduction to
complex compounds.
dyes, labels and tags describes these more fully.
*ð ð
FAM
Cyclic with a peptide bond
*ð ðamra
T
Either the two ends of a peptide or specific 
*ð ð
The DyLight"! range of dyes
CO2H and  NH2 residues can be reacted
to form a peptide bond, resulting in a cyclic
*ð ð
Dansyl
compound. Care must be taken in the design of
*ð ð
NBD
the peptide for this method to work well.
*ð ð
Edans
*ð ð
Dabcyl
*ð ð
Mca
Cyclic peptides
Alta Bioscience can synthesise both cyclic and
cross linked peptides.
Cyclic disulphide
If a peptide is made with two cysteine residues,
careful oxidation in solution will result in a cyclic
Figure 2. Cyclised with a peptide bond
compound, created as the cysteines bridge to
form their dimer, cystine. This reaction generally
Cyclic thioethers
proceeds smoothly with good yield and minimal
These are useful when designing peptide
polymer formation. The bridge can be broken
libraries where the peptide needs to be
under physiological conditions.
presented as a constrained shape. The
cyclisation process proceeeds smoothly, in
good yield. The thioether bond is stable under
physological conditions.
Figure 1. Diagram of a peptide with a disulphide
bridge.
Figure 3. Diagram of a thioether cyclic peptide
An Introduction to Synthetic Peptides Page 61
AltaBioscience
triggers the antibody system, which recognises
Peptides for raising antibodies
the attached peptides. The most popular carrier
In general, synthetic peptides are too small to
protein is keyhole limpet heamocyanin, KLH,
elicit an antibody response, Alta Bioscience
which elicits a strong antibody response and
uses two methods to convert its peptides into a
contains a very large number of lysine residues
suitable form.
which are used to attach the peptide antigen.
1 MAP peptides
This particular approach can be used to attach
the peptide in any orientation, i.e. at either the N
MAP peptides are octomeric molecules with
or the C terminus. However, it is not suitable for
the peptide chains branching out from a central
any peptide containing cysteine, as that amino
poly-lysine core, as shown in figure 4. The eight
acid is added to the sequence to act as the linker
peptide chains increase the molecular weight
to the protein.
of the compound sufficiently for it to be easily
recognised as an antigen. It provides an easy
and flexible method for antibody production.
K
K
K
K
K
K
K
K
K
K
K
K
K
K
Figure 5. Diagram of a peptide-protein conjugate.
Figure 4. Diagram of an octomeric MAP peptide
Antigen design considerations
It is also possible to make chimeric MAPs with
two different peptides sequences, each forming
In general, peptides for antibodies will be
four of the chains.
hydrophilic and flexible, coming from the exterior
of the parent protein. A hydophilicity plot will
The MAP method however, isn t suitable for
indicate which parts of the protein are likely to
peptides which come from the C-terminus of a
be on the outside of the structure. The Kyte-
protein, as that particular amino acid is the one
Doolittle or the Hopp-Woods algorithms will be
conjugated to the core peptide and thus not
very useful here.
exposed.
Structure predictions can be done with Chou-
Dialysis through a 2-3kDa membrane is the only
Fasman plots. The best source for the data
purification method which is required for these
would be the European Bioinformatics Institute.
molecules.
Cysteine should be avoided where possible.
2 Peptide  protein conjugates
The following illustration in fi gure 6, shows that
Here, a synthetic peptide with a free cysteine
a single cysteine would present a very different
residue, is covalently attached to the lysines in a
shape to the immune system compared with the
protein carrier molecule. The size of the protein
disulphide bridged, cystine.
Page 62 An Introduction to Synthetic Peptides
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Handling peptides
Storage
Alta Bioscience supplies all its peptides as freeze
dried materials and these can be regarded as
stable compounds for shipping purposes. For
Cys SH
Cys S--S Cys
long term storage however, it is recommended
to store them in a deep freeze at -20°C. When
taking them out of the freezer, it is important
to allow the bottles/vials to warm up to room
temperature before opening the container. This
is because peptides are often hygroscopic and it
Figure 6. Differences in shape between the
avoids condensation of atmospheric water on the
disulphide bridge in a protein and a linear
peptide.
peptide.
Peptides in solution can degrade, primarily due
to oxidation of Cys, Met and Trp residues but
they are also susceptible to attack by microbes,
Peptides for micro arrays
so it is advised to store solutions at -20°C when
Virtually any type of sequence can be printed
not in use. It is difficult to predict the storage
onto a micro array. To reduce steric hindrance
life of a peptide as it is highly dependent on its
effects, it is helpful to specify a spacer such as
amino acid content and sequence.
Ahx or a PEG between the peptide sequence
Dissolving peptides
and any biotin which is used to anchor the
peptide onto the array slide. The biotin is
This can be a very difficult operation.
usually added at the N-terminus but there are no
*ð ð
Always try to use volatile materials such as
synthesis difficulties in having either a C-terminal
dilute acetic acid and ammonia solutions
biotin or it anywhere along the peptide chain.
when fi rst dissolving an unknown peptide. If
If a cysteine is being used as the linker amino
everything fails, the buffers can be removed
acid for binding to maleimide surfaces, then the
by lyophilisation and the dissolution attempted
array peptide must not contain any sequence
again.
cysteines. If two Cys residues were present,
*ð ð
If the peptide is acidic, i.e. contains more
there would be no control over which of them
Asp and Glu residues than His, Lys or Arg,
would act as the linker.
then fi rst attempt to dissolve the peptide in
It is advisable to specify the linker group to be
dilute ammonia solution, e.g. 0.5%
at the N-terminal of the peptide. The synthesis
ammonium hydroxide. Do not use this
proceeds C to N with capping, so only the full
method if your peptide has disulphide
length peptide would contain the linker. All failure
bridges, the high pH may cause them to
sequences would be washed away and take no
unfold.
part in the binding.
*ð ð
If the peptide is basic, i.e. contains and
excess of His, Lys and Arg groups, then
try and dissolve the peptide in something like
10% acetic acid.
*ð ð
DMSO is a very good solvent and has the
advantage of being tolerated by cells, it is
An Introduction to Synthetic Peptides Page 63
AltaBioscience
however difficult to remove by drying. Add a
References
small amount of a high purity grade DMSO
The original paper
to the stock peptide solution until it dissolves.
Once dissolved, water or buffer solution can
1. Merrifi eld R. B.  Solid Phase Peptide
be added very slowly to dilute the DMSO
Synthesis . J. Am. Chem. Soc. 85, 2149 (1963)
content. Stop the water addition if the peptide
starts to precipitate out. DMSO isn t suitable Recent reviews
for peptides containing single Cys as it
2. Cheng W., White P. D.  Fmoc Solid Phase
promotes disulphide bridge formation.
Peptide Synthesis: A Practical Approach Oxford
*ð ð
Gentle warming and sonication are
University Press, 2000
useful tactics in getting peptides to dissolve.
3. Albericio F,. Kates S. A.  Solid-Phase
*ð ð
Peptides originating from the transmembrane
Synthesis: A Practical Guide CRC Press, 2000
regions of proteins will certainly be difficult to
dissolve.
© Copyright by AltaBioscience
August 2011
Reproduction forbidden without permission
AltaBioscience is a leading manufacturing laboratory providing analysis and synthesis of DNA, proteins and other biochemical
molecules to clients world-wide. Founded in 1973 at the University of Birmingham, England, we offer a well established and
comprehensive range of synthetic, sequencing and analytical methodologies, which are available to academia and commercial
clients. The following internationally recognised accreditations position AltaBioscience amongst the few laboratories world-
wide working to such high standards. ISO 9001:2008 Quality management system for the laboratory as a whole, and ISO
17025:2005 Technical competence in amino acid analysis and protein sequencing.
This publication is one of a series presenting answers to questions frequently asked by established researchers, as well as
those new to their field. Should you have a question which is not dealt with, or if you find an item lacking clarity, we invite you to
bring it to our attention by sending an email to E: [e-mail]
AltaBioscience, Building Y10, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom
T: [phone] F: [phone] E: [e-mail] W: www.altabioscience.com
Page 64 An Introduction to Synthetic Peptides