Description

MAL-dPEG®24-amido-dPEG®24-TFP ester, product number QBD-11303, is a monodisperse, crosslinking PEGylation reagent that joins sulfhydryl groups to amines through a long (158 atoms, 183.5 Å), single molecular weight, discrete PEG (dPEG®) chain. One end of the crosslinker is functionalized with a maleimidopropyl (maleimide) group for reaction with free thiols, and the other end has the 2,3,5,6-tetrafluorophenyl (TFP) ester of propionic acid for reaction with free amines. The dPEG® linker consists of two dPEG®24 chains joined by an amide bond, making it functionally a dPEG®48 crosslinker with a molecular weight that is slightly more than 2000 Daltons. At pH 6.5 – 7.5, the maleimide group reacts specifically with sulfhydryl (thiol) groups to form a thioether bond. The TFP ester reacts with free amines to form amide bonds at an optimum reaction pH range of 7.5 – 8.0.

Crosslinking free thiols to free amines is one of the most popular and most useful reactions in bioconjugate chemistry. These reactions require heterobifunctional reagents that bridge the two groups. Typical, traditional crosslinkers are short and hydrophobic and frequently trigger aggregation and precipitation of conjugates made from them. In addition, traditional PEG crosslinkers use non-uniform (dispersed) polymer PEGs that consist of an intractable mixture of PEG chain lengths and molecular weights. The complexity arising from non-uniform PEG crosslinkers makes analyzing conjugates a challenging exercise.

In contrast, Vector Laboratories’ dPEG® crosslinking products are water-soluble, amphiphilic, single molecular weight PEG compounds with discrete chain lengths. Our dPEG® products are uniform in chain length and molecular weight (Ð = 1). This uniformity of our dPEG® products eliminates one of the most vexing analytical difficulties faced by users of traditional polymer PEG products.

Specifications

Documents

References

Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, Elsevier, Waltham, MA 02451, 2013, ISBN 978-0-12-382239-0; See Chapter 18, Discrete PEG Reagents, pp. 787-821, for a full overview of the dPEG® products.

Description

Bromoacetamido-dPEG®24-TFP ester, product number QBD-11203, is a long (78 atoms, 91.2 Å), single molecular weight, discrete PEG (dPEG®) crosslinker functionalized with a thiol-reactive bromoacetate group on one end and an amine-reactive 2,3,5,6-tetrafluorophenyl (TFP) ester on the other.

The long, flexible, non-immunogenic dPEG® crosslinker increases the hydrodynamic volumes of conjugates in aqueous media, thereby increasing water solubility. The increased hydrodynamic volume can reduce or eliminate conjugate molecules’ renal clearance and shield conjugates from opsonization.

The bromoacetate moiety is an alternative to the maleimide group, reacting chemoselectively with free thiols at pH ≥8.0. It is useful for situations where the conjugate molecule needs to be kept at a high pH, above the range where maleimides can be used effectively, or in constructs where maleimide groups are unstable.

TFP esters are an alternate reactive group to the N-hydroxysuccinimidyl (NHS) ester and reacts with amines at an optimal pH range of 7.5 – 8.0, higher than the optimal range for NHS esters (pH 7.0 – 7.5). TFP esters are more hydrolytically stable than NHS esters, and they are more reactive toward amines in their optimal pH range than NHS esters are within the pH range 7.0 – 7.5.

Specifications

Documents

References

Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, Elsevier, Waltham, MA 02451, 2013, ISBN 978-0-12-382239-0; See chapter 18, Discrete PEG Reagents, pp.787-821, for a full overview of the dPEG® products.

Description

Bromoacetamido-dPEG®12-TFP ester, product number QBD-11202, is a medium-length (42 atoms, 48.7 Å), single molecular weight, discrete PEG (dPEG®) crosslinker functionalized with a thiol-reactive bromoacetate group on one end and an amine-reactive 2,3,5,6-tetrafluorophenyl (TFP) ester on the other.

The medium-length, flexible, non-immunogenic dPEG® crosslinker increases the hydrodynamic volumes of conjugates in aqueous media, thereby increasing water solubility. The increased hydrodynamic volume can reduce or eliminate conjugate molecules’ renal clearance and shield conjugates from opsonization.

The bromoacetate moiety is an alternative to the maleimide group, reacting chemoselectively with free thiols at pH ≥8.0. It is useful for situations where the conjugate molecule needs to be kept at a high pH (≥8.0), above the range where maleimides can be used effectively, or in constructs where maleimide groups are unstable.

TFP esters are an alternate reactive group to the N-hydroxysuccinimidyl (NHS) ester and reacts with amines at an optimal pH range of 7.5 – 8.0, higher than the optimal range for NHS esters (pH 7.0 – 7.5). TFP esters are more hydrolytically stable than NHS esters, and they are more reactive toward amines in their optimal pH range than NHS esters are within the pH range 7.0 – 7.5.

Specifications

Documents

References

Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, Elsevier, Waltham, MA 02451, 2013, ISBN 978-0-12-382239-0; See chapter 18, Discrete PEG Reagents, pp.787-821, for a full overview of the dPEG® products.

Description

Bromoacetamido-dPEG®4-TFP ester, product number QBD-11200, is a short (18 atoms, 20.4 Å), single molecular weight, discrete PEG (dPEG®) crosslinker functionalized with a thiol-reactive bromoacetate group on one end and an amine-reactive 2,3,5,6-tetrafluorophenyl (TFP) ester on the other.

The short, flexible, non-immunogenic dPEG® crosslinker increases the hydrodynamic volumes of conjugates in aqueous media, thereby increasing water solubility. The increased hydrodynamic volume can reduce or eliminate conjugate molecules’ renal clearance and shield conjugates from opsonization.

The bromoacetate moiety is an alternative to the maleimide group, reacting chemoselectively with free thiols at pH ≥8.0. It is useful for situations where the conjugate molecule needs to be kept at a high pH (≥8.0), above the range where maleimides can be used effectively, or in constructs where maleimide groups are unstable.

TFP esters are an alternate reactive group to N-hydroxysuccinimidyl (NHS) esters and react with amines at an optimal pH range of 7.5 – 8.0, higher than the optimal range for NHS esters (pH 7.0 – 7.5). TFP esters are more hydrolytically stable than NHS esters, and they are more reactive toward amines in their optimal pH range than NHS esters are within the pH range 7.0 – 7.5.

Specifications

Documents

References

Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, Elsevier, Waltham, MA 02451, 2013, ISBN 978-0-12-382239-0; See chapter 18, Discrete PEG Reagents, pp.787-821, for a full overview of the dPEG® products.

Description

MAL-dPEG®36-NHS ester, product number QBD-10904, is a crosslinking reagent that joins a sulfhydryl to a free amine through a hydrophilic bridge. The sulfhydryl groups react with a maleimide group via a Michael addition reaction. The amines form amide bonds with the crosslinker by nucleophilic substitution of the N-hydroxysuccinimidyl (NHS) ester of a carboxylic acid group. The maleimide and NHS functional groups on the crosslinking compound sit at either end of a long (118 atoms, 134.8 Å), discrete-length polyethylene glycol (dPEG®) chain.

Reactions that join free amines with free thiols are among the most popular, most useful crosslinking reactions in bioconjugate chemistry. These reactions require heterobifunctional reagents that bridge the two groups. Traditional crosslinkers are hydrophobic molecules. Vector Laboratories’ dPEG® crosslinking products are water-soluble, amphiphilic, single molecular weight PEG compounds with discrete chain lengths.

The conjugation of conventional hydrophobic crosslinking reagents to biomolecules almost inevitably triggers problems such as aggregation and precipitation of the conjugates. These problems do not occur with our water-soluble, non-immunogenic dPEG® crosslinkers.

Because NHS esters hydrolyze readily in water or aqueous buffer, the NHS ester end of the molecule must conjugate to a target molecule before conjugating the maleimide end of the molecule. At pH 7.0 – 7.5, NHS esters react optimally with free amines. However, NHS esters can react with free amines with pH as low as 6.5. As the pH increases, the hydrolysis rate of the NHS ester increases. Indeed, at pH 8 and 25°C, the half-life of an NHS ester in aqueous media is one hour, while at pH 8.6 and 4°C, the half-life falls to ten (10) minutes.[3] Thus, we strongly discourage storing MAL-dPEG®36-NHS ester, product number QBD-10904, in water or aqueous buffer. Instead, we recommend that customers make new solutions of the product as needed, use them immediately, and discard unused solutions after use.

The reaction of the maleimide end of MAL-dPEG®36-NHS ester with a sulfhydryl proceeds optimally at pH 6.5 – 7.5. Use the lowest reasonable pH within this range. Above pH 7.5, free amines compete with free thiols at the maleimide reaction site, which can cause confusing results. Moreover, at higher pH values, the maleimide ring may open to form unreactive maleamic acid.

The use of MAL-dPEG®36-NHS ester, product number QBD-10904, has been published in numerous scientific paper and patents. The following list highlights some of the more interesting uses of this product:
development of extracellular drug conjugates (EDCs), a type of ADC for extracellular markers;
targeted delivery of cancer drugs;
as a long, flexible tether for atomic force microscopy (AFM); and
as a surface coating for amine-functionalized nanoparticle surfaces.

Specifications

Documents

References

Greg T. Hermanson, Bioconjugate Techniques, 2nd Edition, Elsevier Inc., Burlington, MA 01803, April, 2008 (ISBN-13: 978-0-12-370501-3; ISBN-10: 0-12-370501-0); See pp. 276-335 for general description and use of heterobifunctional crosslinkers, as well as his specific discussion with protocols of our MAL-dPEG®x-NHS esters on pp. 718-722.

Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, Elsevier, Waltham, MA 02451, 2013, ISBN 978-0-12-382239-0; See Chapter 18, Discrete PEG Reagents, pp. 787-821, for a full overview of the dPEG® products.

Description

Bis-MAL-Lysine-dPEG®4-TFP ester, product number QBD-10631, is a unique crosslinker from Vector Laboratories, Inc. The alpha (α) and epsilon (ε) amines of lysine are functionalized with sulfhydryl-reactive maleimidopropyl groups. The carboxylic acid tail of lysine is conjugated to a single molecular weight, discrete polyethylene glycol (dPEG®) spacer containing an amine-reactive 2,3,5,6-tetrafluorophenyl (TFP) ester. The distance from the carbonyl carbon of the dPEG® spacer to the reactive site of the α-amine-conjugated maleimide group is 25 atoms (18.773 Å) long. The distance from the carbonyl carbon of the dPEG® spacer to the reactive site of the ε-amine-conjugated maleimide group is 29 atoms (26.105 Å) long.

TFP esters react with primary and secondary amines to form stable amide bonds like the more widely used N-hydroxysuccinimidyl (NHS) esters. However, TFP esters demonstrate superior hydrolytic stability and higher reactivity with amines than NHS esters. The optimum pH for this reaction is 7.5 – 8.0. Thus, the product functions as a heterobifunctional crosslinker.

By design, the two maleimide groups of Bis-MAL-Lysine-dPEG®4-TFP ester can be used to bridge disulfides. For example, this capability may be useful in antibodies following reduction of the disulfide groups. Moreover, the two maleimide groups can be used to dimerize small molecules or peptides, each of which possesses a single free thiol group. At pH 6.5 – 7.5, maleimide groups react chemoselectively with sulfhydryls. However, above pH 7.5, chemoselectivity is lost, as maleimide groups also will react with free amines.

Possible uses for Bis-MAL-Lysine-dPEG®4-TFP ester include the construction of antibody-drug conjugates (ADCs); immobilization of antibodies or antibody fragments on solid supports; and dimerization of sulfhydryl-containing peptides followed by crosslinking to an amine-modified surface.

Specifications

Documents

References

Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, Elsevier, Waltham, MA 02451, 2013, ISBN 978-0-12-382239-0; See Chapter 18, Discrete PEG Reagents, pp. 787-821, for a full overview of the dPEG® products.

Mono-PEGylated Dimeric Exendin-4 as High Receptor Binding and Long-Acting Conjugates for Type 2 Anti-Diabetes Therapeutics.Tae Hyung Kim, Hai Hua Jiang, Seulki Lee, Yu Seok Youn, Chan Woong Park,Youngro Byun, Xiaoyuan Chen, and Kang Choon Lee, Bioconjugate Chem., 2011, 22 (4), pp 625–632 March 14, 2011. DOI: 10.1021/bc100404x.

Description

Bis-MAL-Lysine-dPEG®4-acid, product number QBD-10630, is a unique crosslinker from Vector Laboratories, Inc. The two amines of lysine are functionalized with maleimidopropyl groups that react with sulfhydryl groups. The carboxylic acid tail of lysine is conjugated to a single molecular weight, discrete polyethylene glycol (dPEG®) spacer containing a carboxylic acid terminus that can be reacted with free amines or used to provide negative surface charge. The distance from the carbonyl carbon of the dPEG® spacer to the reactive site of the α-amine-conjugated maleimide group is 25 atoms (17.5 Å) long. The distance from the carbonyl carbon of the dPEG® spacer to the reactive site of the ε-amine-conjugated maleimide group is 29 atoms (27.9 Å) long.

By design, the two maleimide groups of Bis-MAL-Lysine-dPEG®4-acid can be used to bridge disulfides. For example, this capability may be useful in antibodies following reduction of the disulfide groups. Moreover, the two maleimide groups can be used to dimerize small molecules or peptides, each of which possesses a single free thiol group. At pH 6.5 – 7.5, maleimide groups react chemoselectively with sulfhydryls. However, above pH 7.5, chemoselectivity is lost, as maleimide groups also will react with free amines.

The carboxylic acid end of the molecule couples with free amines using EDC chemistry to form stable amide bonds. However, if the acid terminus of the molecule is left unreacted, the acid can act as a charge modifier for the molecule to which it is conjugated. Construction of antibody-drug conjugates (ADCs); immobilization of antibodies and antibody fragments; and dimerization of sulfhydryl-containing peptides are some of the possible uses for this product.

Specifications

Documents

References

Mono-PEGylated Dimeric Exendin-4 as High Receptor Binding and Long-Acting Conjugates for Type 2 Anti-Diabetes Therapeutics.Tae Hyung Kim, Hai Hua Jiang, Seulki Lee, Yu Seok Youn, Chan Woong Park,Youngro Byun, Xiaoyuan Chen, and Kang Choon Lee, Bioconjugate Chem., 2011, 22 (4), pp 625–632 March 14, 2011. DOI: 10.1021/bc100404x.

Description

MAL-dPEG®6-TFP ester, product number QBD-10556, is a crosslinking reagent that joins free amines to free thiols (sulfhydryl groups) across a medium-length (28 atoms), single molecular weight, discrete PEG (dPEG®) linker. The sulfhydryl groups react with a maleimido group via a Michael addition reaction. The amines form amide bonds with the crosslinker by nucleophilic substitution of the 2,3,5,6-tetrafluorophenyl (TFP) ester of the terminal propionic acid group.

The conjugation of free amines to free thiols is one of the most popular, most useful crosslinking reactions in bioconjugate chemistry is the conjugation of free amines to free thiols. These reactions require heterobifunctional reagents that bridge the two groups. Typical crosslinkers are hydrophobic. Vector Laboratories’ dPEG® crosslinking products are water-soluble, amphiphilic, single molecular weight PEG compounds with discrete chain lengths. These hydrophilic, non-immunogenic, dPEG® crosslinkers prevent the problems with aggregation that arise when using traditional, hydrophobic crosslinkers.

Published studies have shown that TFP esters are more hydrolytically stable in aqueous buffers than N-hydroxysuccinimidyl (NHS) esters and are more reactive toward free amines. TFP esters react under the same conditions as NHS esters. However, the optimal pH range for TFP esters (7.5 – 8.0) is slightly higher than for NHS esters (7.0 – 7.5). Amide bond formation between the TFP-activated propionic acid group of MAL-dPEG®6-TFP ester and a free amine will be slightly slower at a suboptimal pH compared to the reaction rate within the optimum pH range.

The maleimide end of MAL-dPEG®6-TFP ester, product number QBD-10556, reacts optimally with a sulfhydryl at pH 6.5 – 7.5. Conduct the conjugation at the lowest reasonable pH within this range. Above pH 7.5, free amines compete with free thiols at the maleimide reaction site, which may confound the data. Moreover, at higher pH values, the maleimide ring may open to form unreactive maleamic acid.

MAL-dPEG®6-TFP ester, product number QBD-10556, can be used the same way and in the same applications as the equivalent NHS esters. Possible uses for this product include the following:
Coating nanoparticle surfaces;
Tethering antibodies to atomic force microscopy (AFM) probes;
Constructing antibody-drug conjugates (ADCs);
Adding flexibility and water solubility to a peptide;
Increasing the water solubility and hydrodynamic volume of hydrophobic biomolecules;
Building supramolecular constructs; and,
Conjugating the TFP ester end of the molecule a liposomal surface and then using the free maleimide end of the molecule to attach a small molecule drug, peptide, or antibody for targeted delivery of a diagnostic or therapeutic package.

Specifications

Documents

References

Greg T. Hermanson, Bioconjugate Techniques, 2nd Edition, Elsevier Inc., Burlington, MA 01803, April, 2008 (ISBN-13: 978-0-12-370501-3; ISBN-10: 0-12-370501-0). Specifically see pp. 726-729 in his Chapter 18 on discrete PEG compounds for pegylation applications.

Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, Elsevier, Waltham, MA 02451, 2013, ISBN 978-0-12-382239-0; See chapter 18, Discrete PEG Reagents, pp.787-821, for a full overview of the dPEG® products.

Description

MAL-dPEG®24-TFP ester, product number QBD-10554, is a crosslinking reagent that joins free amines to free thiols (sulfhydryl groups) across a long (87 atoms), single molecular weight, discrete PEG (dPEG®) linker. The sulfhydryl groups react with a maleimido group via a Michael addition reaction. The amines form amide bonds with the crosslinker by nucleophilic substitution of the 2,3,5,6-tetrafluorophenyl (TFP) ester of the terminal propionic acid group.

The conjugation of free amines to free thiols is one of the most popular, most useful crosslinking reactions in bioconjugate chemistry is the conjugation of free amines to free thiols. These reactions require heterobifunctional reagents that bridge the two groups. Typical crosslinkers are hydrophobic. Vector Laboratories’ dPEG® crosslinking products are water-soluble, amphiphilic, single molecular weight PEG compounds with discrete chain lengths. These hydrophilic, non-immunogenic, dPEG® crosslinkers prevent the problems with aggregation that arise when using traditional, hydrophobic crosslinkers.

Published studies have shown that TFP esters are more hydrolytically stable in aqueous buffers than N-hydroxysuccinimidyl (NHS) esters and are more reactive toward free amines. TFP esters react under the same conditions as NHS esters. However, the optimal pH range for TFP esters (7.5 – 8.0) is slightly higher than for NHS esters (7.0 – 7.5). Amide bond formation between the TFP-activated propionic acid group of MAL-dPEG®24-TFP ester and a free amine will be slightly slower at a suboptimal pH compared to the reaction rate within the optimum pH range.

The maleimide end of MAL-dPEG®24-TFP ester, product number QBD-10554, reacts optimally with a sulfhydryl at pH 6.5 – 7.5. Conduct the conjugation at the lowest reasonable pH within this range. Above pH 7.5, free amines compete with free thiols at the maleimide reaction site, which may confound the data. Moreover, at higher pH values, the maleimide ring may open to form unreactive maleamic acid.

MAL-dPEG®24-TFP ester, product number QBD-10554, can be used the same way and in the same applications as the equivalent NHS esters. Possible uses for this product include the following:
Coating nanoparticle surfaces;
Tethering antibodies to atomic force microscopy (AFM) probes;
Constructing antibody-drug conjugates (ADCs);
Adding flexibility and water solubility to a peptide;
Increasing the water solubility and hydrodynamic volume of hydrophobic biomolecules;
Building supramolecular constructs; and,
Conjugating the TFP ester end of the molecule a liposomal surface and then using the free maleimide end of the molecule to attach a small molecule drug, peptide, or antibody for targeted delivery of a diagnostic or therapeutic package.

Specifications

Documents

References

Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, Elsevier, Waltham, MA 02451, 2013, ISBN 978-0-12-382239-0; See Chapter 18, Discrete PEG Reagents, pp. 787-821, for a full overview of the dPEG® products.