Chelator Bioconjugation Services

Custom Lipoconjugation Services

As molecular imaging has become an indispensable tool in modern diagnostics in biomedical research and therapeutic fields. Thus the need for highly sensitive and specific molecular imaging probes is still unmet. In order to design diagnostic imaging and radiotherapeutic agents, a high target/background ratio, target uptake and rapid removal of untargeted drug is desirable. Proteins, peptide, antibodies and their fragments are perfect candidates due to high specificity, affinity and selectivity to the target organs.

There are several methods commonly used to label protein, peptide, and antibodies:

• Direct labeling by introducing a radioactive atom to functional groups on the antibody wit out the use of an chemical spacer: example such as: radioiodination through modification of tyrosine side chains or
• Indirect method of radiolabeling by introducing chelating agents into biomolecule which preformed complex with certain radioactive metals.

Types of Bifunctional chelating agents (BCAs) used to create radioimmunoconjugates:

DTPA (Diethylenetriaminepentaacetic Anhydride)

This BCA contains two amine-reactive anhydride groups. This compound reacts with N-terminal and ϵ-amine groups of protein to form amide linkage. The anhydride rings open to create multivalent, metal-chelating arms able to bind tightly metals in a coordination complex. It has 8 coordination sites with metal ions (from 3 nitrogens and 5 oxygens).

DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid)

This BCA containing 4 acetic acid carboxylate group off the 4 nitrogens of its 12 atom cyclic structure and is capable of modifying proteins and binding radioactive metal ions in strong coordination complexes of up to eight dative bonds.

NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid)

NOTA has a smaller ring structure in comparison to DOTA and only has 3 chelating carboxylate groups and 3 nitrogens, it has 6 coordination sites with metal ions.

TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid)

TETA has 4 chelating carboxylate group s and 4 nitrogens. C- and N-functionalized derivatives can be prepare wit TETA. A p-bromoacetamidobenzyle-TETA derivative could be used to label antibodies through sulfhydryl groups.

DTTA N-(p-isothiocyanatobenzyl)-diethylenetriamine-N,N',N'',N'''-tetraacetic acid

This BCA contains four carboxylate groups and 3 nitrogens that can hold metals tightly in a coordination complex of seven dative bonds. This compound is good at chelating lanthanide element series, such as europium, samarium, terbium and dysprosium. This BCA is primarily used for complexing metals to form fluorescent probes for time-resolved fluoroimmunoassays.

Custom Radioimmunoconjugate Service Descriptions

Sample Submission Requirement:

Biomolecules supplied by customers should be sufficiently pure. Please provide 5 mgs of starting material with the necessary data for purity assessment. Commercially available biopolymers can be supplied by customers or synthesized or ordered through Bio-Synthesis.

Price

Price varies based on the proejct specifications. Price does not includes cost of small molecule or biopolymer which required to be order through Bio-Synthesis from a commercial vendor. Some of the small molecules or biomolecules are commerically available in an activated form. For non-active molecules, Bio-Synthesis can assist with the design and, if deemed necessary, biopolymer modification to introduce additional functional groups and extra linkers, spacers. Please contact us for a quote

Discount: 15 % discount price applies to additional conjugates ordered at the same time.

Macrocyclic Ligands

Series of metal chelating ligands can be covalently attached to biopolymer such as oligonucleotide, antibodies, peptide and proteins. This indirect method of biolmolecule labeling with radiolabels utilize organic compounds able to chelate metal ions in a coordination complex. They are widely used in nuclear medicine, MRI, and optical imaging applications.

Macrocyclic Ligands
Cyclen (1,4,7,10-Tetraazacyclododecane)
Cyclam (1,4,8,11-Tetraazacyclotetradecane)
DO2A
DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid)
DOTP (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic) acid)
DOTMA, (1R, 4R, 7R, 10R)-α'α''α'''-Tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) tetrasodium salt
TETA, (1,4,8,11-Tetraazacyclotetradecane-1,4,8, 11-tetraacetic acid)
DOTAM (1,4,7,10-Tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane)
CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4, 11-dicetic acid)
NOTA ((1,4,7-triazacyclononane-N,N',N''-triacetic acid)

Bifunctional Chelating Agent Modification

These bifunctional chelating ligands can be covalently attached to biopolymers such as oligonucleotides, antibodies, peptides and proteins. They are widely used in nuclear medicine, MRI, and optical imaging applications.

Bifunctional chelating agents		Bifunctional chelating agents
p-NO2-Bn-Cyclen		p-NH2-Bn-DTPA-penta (t-Bu ester)
p-NO2-Bn-DOTA		p-SCN-Bn-DTPA
p-NH2-Bn-DOTA		p-NH2-CHX-A"-DTPA
p-NH2-Bn-DOTA-tetra(t-Bu-ester)		CHX-A''-DTPA
p-SCN-Bn-DOTA		DTPA-tetra (t-Bu ester)
DOTA-tris (t-Bu ester)		Maleimido-mono-amide-DTPA
DOTA-mono-NHS-tris(t-Bu ester)		p-NH2-Bn-PCTA
Maleimido-mono-amide-DOTA-tris (t-Bu ester)		p-SCN-Bn-PCTA
Maleimido-mono-amide-DOTA		p-NH2-Bn-oxo-DO3A
Fmoc-L-Lys-mono-amide-DOTA-tris(t-Bu ester)		p-SCN-Bn-oxo-DO3A
2-Aminoethyl-mono-amide-DOTA-tris(t-Bu ester)		p-NH2-Bn-NOTA
Azido-mono-amide-DOTA-tris(t-butyl ester)		p-SCN-Bn-NOTA
DOTA-NHS-ester		Desferrioxamine-p-SCN
Azido-mono-amide-DOTA		DOTA-Biotin-Sarcosine
p-NH2-Bn-DTPA

MRI Contrast Agents

Magnetic Resonance Imaging (MRI) contrast agents are a group of contrast media used to improve the visibility of internal body structures using MRI. We offer agents that are being developed for the ability to measure physiological properties such as pH and temperature.

MRI Contrast Agents
Tm-DOTA (Thulium (III) (1,4,7,10-tetraazacyclododecane)-1,4,7,10-tetraacetic acid)
Gd-DOTA Gadolinium (III) (1,4,7,10-tetraazacyclododecane)-1,4,7,10-tetraacetate
Tm[DOTP]5- (Thullium (III) (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonate)))
Tm-DOTMA, (Thulium (III) (1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
Eu-DOTA-4AmC, Europium (III) [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamidoacetic acid)]

Chemistry: Coupling of preactivated small molecule and biomolecules with chemical reactive groups such as amine, thiol, carboxylate, hydroxyl, aldehyde and ketone, active hydrogen through use of various cross linkers.

CAUTION: Conjugation with antibody may cause some immunogloublins to lose antigen-binding activity after modification. Pilot experiment to optimize the amount of protein/antibody present and the quantity of metal chelator added to the reaction needs to be done first in order to avoid this type of corsslinking and polymerization.

Service Specification: After standard desalting, or purification, a small percent of heterogeneous products containing single or multi-site conjugate per molecule may exist.

Procedure: After labeling, final conjugates must first be isolated from excess or unreacted reagent by gel filtration or dialysis. Additional purification technique such as stirred cell filtration, tangential flow filtration (TFF), gel filtration chromatography may also be used to either remove excess reagent or isolate and characterized the cross-linked product. For reagents (mostly protein and other biological molecules) that are similar in size or larger than the antibody, one must resort to other purification techniques such as affinity chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography.

Cross-linked target molecule may then be further characterized by biochemical or biophysical techniques. Once the product has been purified, it may be subject to many different types of studies including spectroscopic (MALDI-TOF, ESI, LC-MS Fluorescence), electrophoresis, immunochemical biochemical, enzymatical analysis. QC (quality control) and QA (quality assurance) procedures are also followed independently to offer you double guarantee for the highest quality possible of every delivered conjugates. Moreover, our dedicated technical account managers will guide your project through every step of the process and constantly keep you informed of the latest project progress.

Links and Resources

Reference/Citing:

1. Upadhyay, A. et al.: Immunological Response to Peptide Nucleic Acid and its Peptide Conjugate Targeted to Transactivation Response (TAR) Region of HIV-1 RNA Genome; Oligonucleotides. 18(4) 2008.
2. Olejnik, J. et al.: Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS; Nucleic Acid Researech. 27 (23) 1999.
3. Pai, S. et al.: Proximity ligation assays with peptide conjugate 'burrs' for the sensitive detection of spores; Nucleic Acid Research. 33(18) 2005.

Visit our literature vaults for more references and citings.

Ordering and submitting a request for Bioconjugation Services



For us to better understand your customized project, please complete our Bioconjugation Service Questionnaire. The more our chemists understand your project needs, the more accurate feedback we will be able to provide you.  Providing us with your project details will enable us to recommend the best reagents to use for your own project.  The most useful and readily available tools for bioconjugation projects are cross-linking reagents. A large number of cross-linkers, also known as bifunctional reagents, have been developed.  There are several ways to classify the cross-linkers, such as the type of reactive group, hydrophobicity or hydrophilicity, and the length of the spacer between reactive groups.  Other factors to consider are whether the two reactive groups are the same or different (for example, heterobifunctional or homobifunctional reagents), whether the spacer is cleavable, and whether the reagents are membrane permeable or impermeable.  The most accessible and abundant reactive groups in proteins are the ϵ-amino groups of lysine.  Therefore, a large number of the most common cross-linkers are amino selective reagents, such as imidoesters, sulfo-N-hydroxysuccinimide esters, and N-hydroxysuccinimide esters.  Due to the high reactivity of the thiol group with N-ethylmaleimide, iodoacetate and a-halocarbonyl compounds, new cross-linkers have been developed that contain maleimide and a-carbonyl moieties.  Usually, N-alkylmaleimides are more stable than their N-aryl counterparts.

 

In addition to the reactive groups on the cross-linkers, a wide variety of connectors and spacer arms have also been developed.  The nature of the spacer arm, and also the length, play an important role in the functionality.  Longer spacer arms are generally more effective when coupling large proteins or those with sterically protected reactive side-chains.  Other important considerations are the hydrophobicity, hydrophilicity, and the conformational flexibility.  Long aliphatic chains generally fold on themselves when in an aqueous environment, which makes the actual distance spanned by such linker arms less than expected.  Instead, spacers that contain more rigid structures (for example, aromatic groups or cycloalkanes) should be used.  These structures, however, tend to be very hydrophobic which could significantly decrease the solubility of the modified molecules or even modify some of their properties.  In such cases, it is recommended to choose a spacer that contains an alkylether (PEO) chain.  Bio-Synthesis offers several cross-linkers with PEO chains, such as thiol-binding homobifunctional reagents, heterobifunctional based, and their derivatives.

Once the project scope has collected, we will provide an appropriate quotation within 3-5 days. Orders can be placed with either a PO (Purchase Order) or credit card. We accept POs and major credit cards ( ). Your credit card will be billed under 'Bio-Synthesis, Inc.' Click here to download our credit reference form. For international orders, we must apply the full charge at the time of the order is placed. In the unlikely event that any given order cannot be filled, our guarantee will take the form of a full refund.