Advantages of Discrete PEG (dPEG™)
over conventional alkyl (aliphatic
methylene, X-(CH2)n-Y) spacers and
linkers
PEG (discrete polyethylene glycol ,dPEG™)
crosslinkers, modification reagents
and related products are now available
from Timtec Inc. dPEG compounds and
their derivatives are suitable for
wide range of therapeutic, diagnostic,
and molecular engineering
applications.
Broad range of
crosslinkers and related products that
now contain the discrete polyethylene
glycol (dPEG) based spacers are
preferred substitutes to aliphatic
methylene chain spacers, X-(CH2)n-Y.
Alkyl spacers have serious limitations
and drawbacks in comparison to dPEG
products. Conventional chain spacers
have been widely used for many years
for the lack of batter alternative.
With dPEG
crosslinking products, you now have a
set of tools that not only overcomes
the drawbacks of the alkyl liners and
spacers but also provides many new
creative options for your research
work with chain spacers.
dPEG linkers are
extremely water soluble and
hydrophilic, while the alkyl linkers
are neither. The water
solubility and hydrophilicity of dPEGs
gives them a very large range of
applications.
In contrast the
opposite properties in the alkyl
spacers have severely limited their
actual and potential uses in
biological systems. There are
the aliphatic linker containing
crosslinkers at the sulfo-NHS esters,
which are soluble in water,
commercially available. However, when
the linkers are attached or
crosslinked, the inherent
hydrophobicity of the alkyl spacers
returns, and all the disadvantages
become apparent. The hydrophobic
characteristics of the alkyl linkers
and spacers are most often manifested
through increased aggregation and
precipitation in the modified or
crosslinked products.
dPEG-containing
compounds solve the problem by
significantly decreasing and in some
cases eliminating aggregation and
precipitation. In addition to being
water soluble, the dPEG linkers are
organic soluble and can be used in
organic media when this is desirable.
DPEG likes the normal organics used
for conventional linkers such as DMF
and DMAC, but also especially likes
methylene chloride.
Examples of
application:
Biotinylation
reagents along with LC linker (amino
caproic acid) are a good illustration
of dPEG excellent performance in
comparison to its analog.
Sulfo-NHS-LC-biotin
is probably the most popular
biotinylation currently available on
the market. The hydrophilic LC
linker is introduced to enhance the
binding depth to streptavidin. Once
labeled, the LC-biotin will seek
hydrophobic regions in the protein and
hide in them, making it less available
to the streptavidin. At the same
time, the hydrophobic LC-biotin labels
causes serious agglomeration and
precipitation problems.
There is
agglomeration data available that
compares the sulfo-NHS-LC-biotin with
PN 10200, which has the dPEG4 spacer
(the length of 2x LC). The data
shows that human IgG biotinylated with
the sulfo-NHS-LC biotin precipitates
within a couple of weeks, while human
IgG biotinylated with NHS-dPEG4 biotin
(PN 10200) shows no agglomeration on
the third week.
The comparison is
dramatic. Customers who use MAL-dPEGx-NHS
esters (PN10214, 10274, 10284, dPEG =
4,8,12, resp.) in place of the well
known and widely used SMCC and related
heterobifunctional crosslinkers, have
made similar types of observations.
In addition the use of MAL-dPEGx-NHS
esters often decrease or eliminate
immunogenicity.
Application Note:
Because some of our crosslinkers are
viscous, we often recommend our
customers initially dissolve the
compound in an organic solvent rather
than water. With peptide
synthesis dPEG reagents, as well as
with many of the modification
reagents, the application is already
going into an organic medium, so this
property becomes very valuable to the
application.
Immunogenicity of d-PEG-linked
compounds
PEG is well known
to be non-immunogenic, which makes
dPEG-linked compounds non-immunogenic
as well. This is a huge advantage of
dPEG products. In contrast, the alkyl
linkers containing more than two or
three methylene groups are highly
immunogenic. Oftentimes the alkyl
spacer length needs to be shortened to
hide its undesirable property/
Even if the goal
is a specific immune response, e.g.,
using a carrier protein conjugated to
a peptide with a conventional linker
to generate specific antibodies,
conventional linkers contribute to a
linker dependent response. It is
anticipated that such response is
eliminated with the use of dPEG
linkers.
Control over distance/spacing
For most
applications that involve alkyl
spacers it would be desirable to have
longer spacers to control the distance
of the conjugation species. However,
with all inherent issues related to
poor solubility, a tendency to
exaserbate aggregtion and high
immunogenicity, the control over
conventional spacer distance remained
limited for the last 10-15 years.
dPEG spacer
properties impose absolutely no
restrictions to increasing the length
of the linkers. As already discussed
above, the dPEG spacers are extremely
water soluble, hydrophilic and non-immunogenic.
The use of the
PEG spacer is not new. Yet available
reagents are in high MW ranges (2,000
or 3,400, average n about = 45 and 75,
respectively) have very long spacers
(>250-300 Angstroms), and are
complex polymer mixtures.
Now with the
advent of the smaller dPEGs, which are
single compounds that have spacers in
a length range more useful for most
applications, the opportunities to
optimize length variable for most
applications are readily available.
There is a variety of different
linkers and spacers ranging from dPEG4
to dPEG24, with the option to
customize the length to any specific
ethylene oxide unit number. You
can now select spacer chain lengths
from about 20 to 90 Ĺ. This option
becomes increasingly important as more
and more researchers are using
modeling to predict the physical
requirements of molecules. Also
in a variety of other applications it
is vital to be confident of working
with a single spacer length and not a
range of lengths.
Wide dPEG product
selection and customization option
allows you to find immediate potential
solution for your chemical
modification needs.
Modification reagents
Timtec offers
modification reagents, dPEGx products,
which have no counterparts within
alkyl chains. These products are
specifically designed to be chemically
bonded to a drug, protein or other
biological compounds. Modification
reagents increase water solubility,
decrease immunogenicity and
aggregation, as well as potentially
decrease toxicity. The dPEGx products
are terminated on one end with a
methyl group and with amine (NHS
ester), sulfhydryl (maleimide) or
carbonyl (amine) reactive on the
other. These discrete molecular weight
products are available in the range
from dPEG4 to dPEG24 or from about 200
to over 1000. Product numbers 10211,
10260 and 10262(link to these prducts)
Recently two more
products were added PN10305 and PN
10307), which offer the potential to
be released (e.g., as a pro-drug) or
bonded to functionalities other than
just the amine. For example, there are
products that can bond to acids,
aldehydes, and sulfhydryls. The latter
are of growing interest as molecular
engineers can introduce the sulfhydryl
almost at will using site-directed
mutagenesis.
Note: The
different physical properties of dPEG-containing
crosslinkers and modification reagents
are initially perceived to be
complicated in use. Many of the
lower MW materials are viscous liquids
that can be problematic to handle.
Technical support
is always available to assist our
customers in handling products and
using solvents and solvent systems
efficiently to take full advantage of
reagent properties. Contact
TimTec with product related
questions and for ordering.
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Biotinylation Reagents & Labels