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Custom Signal Molecule Conjugation

Biomolecule labelings have a variety of biological functions, such as cell signaling molecules, tools in molecular biology, drugs in medicine, and countless other roles. These small, low molecular weight organic compounds such as labels, tags and probes, can be attached to macromolecules via appropriate linkers, macromolecules including antibodies, antibody fragments, enzymes such as alkaline phosphatase and horseradish peroxidase, proteins, peptides and peptidomimetics, oligonucleotides and analogues, ligands, polysaccharide, avidin and streptavidin, polysaccharides, simple and complex lipids for use with immunochemistry, fluorescence in situ hybridization (FISH), cell tracing, receptor labeling, cytochemistry applications, probing biological structure, function and interactions, and also conjugation of biotin and other haptens to biomolecules.

Bio-Synthesis has expertise in synthesizing new small molecules, modification of existing molecules, and advising clients with appropriate methods for activation of small molecules, labeling and conjugation of the small molecules to biomolecules. Our conjugations routinely employ heterobifunctional coupling chemistry, which allows the greatest flexibility in achieving product requirements. As one of the pioneers of Synthetic Biology Service Company, Bio-Synthesis aggressively seeks to develop new technologies to assist clients in developing new bioconjugate reagents for their sensitive detection applications.

To obtain further information regarding custom bioconjugation, contact our National Customer Services Center at 800.227.0627 or contact us online with each inquiry to assist in meeting customer specifications.

Advantages:

  • Superior Technical Support: Assist client from design to delivery
  • High success rate: More than 95%, far higher than competitors
  • Full spectrum of labeling service: Fluorescent dyes, non-fluorescent dyes, redox, nanoparticles, enzymes, custom-made and user supplied small drug compounds
  • Flexible Technologies: Employ solid phase, liquid phase, and microwave technologies
  • Optimized Chemistries: Expertise in hundreds of cross linking modifications, activation and/or conjugation with reactive cross linkers with optimized labeling ratio
  • Guaranteed Quality: All samples are carefully monitored for stability and characterized to ensure batch to batch consistency

Standard Biomolecule Labeling Service: BIOCON20000

Price:

Prices vary based on the project specifications. The list price is for reference only. Our services include materials and labor for conjugation only! Price does not include the cost of biopolymer synthesis and, if deemed necessary, biopolymer modification to introduce additional functional groups, extra linkers, and/or spacers. Please contact us for a quote.

Chemistry:

Coupling of preactivated small molecules and biomolecules with chemical reactive groups such as amine, thiol, carboxylate, hydroxyl, aldehyde, ketone, active hydrogen, photo-chemical and cycloaddition reactions, zero-length cross-linking, homobifunctional, heterobiofunctional, or multifunctional cross-linking chemistries, denrimer and dendrons, and cleavable regent systems.

Some commonly used cross-linking reagents include:

  • Glutaraldehyde - Links carrier molecules to the N-terminus of the peptide
  • Carbodiimide (EDC), - Attaches carrier to the C-terminus of the peptide
  • Succinimide esters (e.g. MBS, SMCC) - Binds free amino group and Cys residues
  • Benzidine (BDB), - Links to Tyr residues
  • Periodate, - Attaches to carbohydrate groups
  • Bioconjugate chemistry may occur through the C- and N-terminals of each polypeptide chain, the carboxylate groups of aspartic and glutamic acids, the ϵ-amine of lysine, the guanidino group of arginine, the sulfhydryl group of cysteine, the phenolate ring of tyrosine, the indol ring of tryptophan, the thioether of methionine, and the imidazole ring of histidine.

Service Specification:

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

Material:

  • Fluorescent, nonfluorescent, redox labels, qdot, enzyme labelng, metal chelators, and other small molecule labeling.
  • Biopolymers
    • Monomers: Amino acids, nucleotides and sugars
    • Protein: Enzymes, antibodies, antigens and cell adhesion molecules
    • Peptides: Synthetic polypeptides
    • Saccharides: Sugars, oligosaccharides and polysaccharides
    • Lipids: Fatty acids, phospholipids, glycolipids and any fat-like substances
    • Ligands: Hormone receptors, cell surface receptors, avidin and biotin, and small molecules
    • Labels: Fluorescent dyes, infrared-absorbing and UV-Vis absorption chromophores, and nonradioactive labels
    • Nucleic Acids and Nucleotides: DNA, RNA, PNA, nucleic acid analogs and genomic DNA
    • Synthetic Polymers: PEG, Nanoparticles, gold particles, dendrimers, dendron and PAMAM
    • Other: Conjugates or mixtures of any the above
    • Solid Supports: Agarose, glass plates, membrane and beads

Procedure:

All custom synthesis of biomolecules, modification, or bioconjugation services are manufactured under strict quality control (QC) processes. Analytical HPLC and MS analyses are performed in every development cycle. Final target conjugates must first be isolated from excess or unreacted reagent. In many cases, simple dialysis may suffice to remove unreacted reagent from the reaction solution. Depending on the project scope, size-exclusion chromatography (SEC) or HPLC may also be used to either remove excess reagent or to isolate and characterize the cross-linked product. The 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 various characterization methods including spectometric (MALDI-TOF, ESI, LC-MS), fluorescence, electrophoresis, and immunochemical, biochemical, and enzymatic analysis. QC (quality control) and QA (quality assurance) procedures are also followed independently to ensure the highest quality possible. Moreover, our dedicated technical account managers will guide you through every step of the process and keep you informed of the latest progress.

Delivery Specifications:

The typical delivery consists of a lyophilized sample in an individual, fully labeled vial. The shipment also contains COA, MS HPLC and/or other analytical data. Additional analytical data is also available upon request.

Small molecule labeling generally refers to any form of cross-linking or modification whose, purpose is to conjugate a chemical group (e.g. fluorescent and chemi-luminescent compound, redox labels, biotin, haptens, nanoparticles or user supplied compound having target functional group) with target molecules such as proteins, nucleic acids, and other biomolecules through single, bifunctional or heterofunctional cross linkers to aid in detection.

Choosing the correct cross-linking chemistry sometimes appears overwhelming as it requires a careful understanding of target molecule structure and reactivity. These cross-linking and modifying agents can be applied to alter the native state and function of peptides and proteins, sugars and polysaccharides, nucleic acids and oligonucleotides, lipids, and almost any other imaginable molecule that can be chemically derivatized. Therefore, Bio-Synthesis carefully designs each conjugation. 

Bio-Synthesis not only offers synthesis of small molecule and biopolymers, but also assists in the functionalization or activation of compounds ready to cross link with pre-activated small molecules. These small molecules are either supplied by customers or they are prepared in-house. These pre-activated small molecules can be created with an amine, acid, hydrazine, aldehyde/ketone, hydroxylamine, maleimide/alkylhalide, and sulfhydryl functional group in a polymer. After labeling of biopolymer with small molecules, a standard desalting, purification, quality check and final concentration and labeling ratio are determined.

Sample Requirements

Non-commercial small molecule supplied by customer

User-supplied, non-commercial small or macromolecule should be sufficiently pure (≥95% pure). Please provide the QC data (typically HPLC, MS, ESI or NMR data etc.) and MSDS (if any) along with your compound. We can assist with the purification and acquisition of analytical data.  These compounds must contain functional groups that can be targeted for crosslinking including carboxylate groups, primary amine groups, aldehyde/ketone residues, hydroxyl, hydrazine, hydrazide, aminoxy,  saccharide /glycan groups or thiol reactive functional group(s). Coupling can also be non-selective using a photoreactive phenyl azide cross-linker. If necessary, we can also assist with the creation and activation of specific functionalities. Any sample supplied by the client requires a quality check by our analytical team prior to conjugation.

Commercially available small molecules

Commercially available small molecules can be supplied by customers or ordered through Bio-Synthesis.  Common small molecules such as fluorescent dyes, biotin-NHS, and other hetero-bifunctional cross-linkers are available at Bio-Synthesis. For in-stock compounds, Bio-Synthesis will charge customers only the amount needed for conjugation. If the small molecules are to be purchased separately, the cost of acquiring this material will be added to the invoice along with a $50 administration charge per order.

Non-commercially available small molecules

We can custom synthesize small molecules in-house . A quotation will be prepared for such syntheses.

Oligonucleotide, oligomimetics, and custom gene or genomic DNA

Oligo or custom genes such as DNA, RNA, and PNA can be synthesized and conjugated through amino, thiol, or 5'-phosphate modification. Oligos supplied by the customer should be HPLC or gel purified (>90% pure). Please provide the QC data needed for purity assessment. Extra charges may apply for analyzing the starting materials at Bio-Synthesis.

Peptides, peptidomimetics, and expressed protein

Peptides and proteins can be synthesized or expressed in-house at Bio-Synthesis. We label or conjugate peptides through N-terminal, C-terminal or internal side chains of an amino acid. Peptides supplied by the customer should be HPLC purified (>90% pure) and should contain a single modification site. We can make any standard peptide with functional groups incorporated through peptide synthesis. Please provide the peptide sequence of the desired peptide on the order form. 

Antibodies

Commercial antibodies can be supplied by customers or ordered through Bio-Synthesis with an additional fee plus the cost of antibody. Non-commercial biopolymers supplied by customers should be affinity purified.  Please provide gel electrophoresis data along with your antibody.  Bio-Synthesis also assists customers with antibody production, purification, modification, and fragmentation prior to any cross linking reaction

Functional target modification services

We offer post-synthetic modification of ligands to be immobilized on various solid supports, which allow the immobilization process to occur selectively in the presence of common functional groups, including amines, thiols, carboxylic acids, and alcohols. Bio-Synthesis offers functional group modification and derivatization.

  • Amino acids, peptides and protein modification
  • Modification of sugars, polysaccharides, and glycoconjugates
  • Modification of nucleic acids and oligonucleotides
  • Creating Specific functionalities such as sulfhydryl, carboxylate, primary amine, aldehyde/ketone, hydrazine or hydrazide,saccharide, and glycan groups
  • Blocking functional groups such as amine, sulfhydryl, aldehyde or a carboxylate group

See more information on Biomolecule Modification

Chemistry of Reactive Group Used

Every chemical modification or conjugation process involves the reaction of one functional group with another, resulting in the formation of a covalent bond. The creation of bioconjugate reagents with spontaneously reactive, or selectively reactive functional groups, forms the basis for simple and reproducible crosslinking or tagging of target molecules. Our well-trained chemists assist clients from project scope collections to design, and to determine appropriate homobiofunctional or heterobifunctional cross-linking chemistries We have delivered thousands of custom conjugated biopolymers and are fully capable of meeting the ever-increasing bioconjugation needs in biological and drug discovery research. Hundreds of reaction systems have been applied in our organic laboratories.

  • Amine Reaction: NHS ester, imidoester, hydroxymethyl phosphine, guanidination of amine, fluorophenyl esters, carbodiimides, anhydrides, arylating agents, carbonates, aldehydes, and glyoxals
  • Thiol Reactions: Maleimide, Haloacetyl, Pyridyldisulfide, Vinyl sulfone, Thiol-disulfide exchange
  • Carboxylate reactions: Carbodiimides
  • Hydroxyl Reactions: Isocyanates, enzymatic oxidation, Carbonyldiimidazole
  • Aldehyde and Ketone Reaction: Hydrazine derivative, Schiff Base formation, reductive amination
  • Active Hydrogen Reaction: Iodination reaction
  • Photo-chemical Reactions: Psoralen compounds, aryl azides and halogenated aryl azides, bensophenones, anthraquinones
  • Cycloaddition Reaction: Chemoselective ligation such as Click chemistry, Diels-Alder reaction

Custom synthesized biopolymers such as peptides and oligonucleotides from Bio-Synthesis are manufactured under strict quality control processes. We use mass spectrometry (MS) for all single custom peptides, oligonucleotides, and purified biopolymers. We also analyze by RP-HPLC and other quality check methods are available upon request.

Bio-Synthesis is committed to Total Quality Management (TQM) to assure complete satisfaction. MS and HPLC analyses are performed following the completion of biopolymer synthesis, purification and QC (quality control). QA (quality assurance) procedures are also followed independently to double guarantee the quality of every delivered peptide. Bio-Synthesis's Total Quality Management System (TQM) has been successfully upgraded to fully comply with ISO 9001:2008 regulations.

Case Study:

Sequence: 5' GCT CGG ATA CAT ATT TAG TGA CAC TAT A-3'
Modification: Marina Blue
Conjugate MW: 9019.12
MS and HPLC results:

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 Requests 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.  Provide us with your project details will enable to us to recommend the best reagents to use for your 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 aremore 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 and length of the spacer arm 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.

Within 3-5 days upon receiving your project scope, we will provide you an appropriate quotation. An order can be placed with PO (Purchase Order) or major credit cards ( ). Your credit card will be billed under Bio-Synthesis, Inc.

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