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The custom peptide synthesis service and custom antibody product service company.

Elevating Discoveries: The World of Macrocyclic Disulfide-Rich Peptides

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Bioactive peptides have potential as drug leads. However, one of the peptides’ limitations is their poor stability.

One solution is to graft peptides onto suitable molecular scaffolds. When the peptide scaffolds are rich in disulfide bonds and have no free ends for proteases to attack, the peptide drug would be a stable candidate.

Cyclic disulfide-rich peptides: Stability Matters
Many natural peptides have stable and conserved cyclization. The cyclic chlorotoxin can preferentially bind to tumor cells. The defensin, an antimicrobial peptide, is an 18 amino acid peptide with three disulfide bonds in a laddered arrangement. The cyclotide has about 30 amino acids with three disulfide bonds in a knotted configuration. The cyclic conotoxin cVc1.1 has potent analgesic activity.

disulfide rich cyclic natural product

disulfide-rich cyclic natural product

Molecular grafting
The purpose of molecular grafting is to make a linear peptide sequence into a disulfide-rich peptide with desired stability or oral bioavailability. It was found that the stability of linear peptide epitopes in human serum can be enhanced by grafting them onto a stable scaffold. For example, a crafted peptide comprising an epitope from myelin oligodendrocyte glycoprotein (MOG) is resistant to degradation in human serum (>24 h) and remains intact in strong acid after 24 hours. In the MOG study, the epitope was grafted into the β-turn of Kalata B1 to mimic its native conformation and it turned out to have increased bioactivity and improved affinity and receptor selectivity. A grafted peptide based on the cell-penetrating peptide cyclotide MCoTI-I demonstrated the delivery of the bioactive peptide across the cell membrane and into the intracellular space.

Cyclotide Peptide Synthesis

Cyclotide Peptide Synthesis

How to make molecular grafting
1. Selection of a suitable epitope and scaffold pair. The termini of the epitope should not be essential for its activity. The size of the epitope is an important consideration. Epitopes ranging from 10 amino acids to 20 amino acids are frequently used. Here are a few examples of the epitope size from the disulfide-rich peptide scaffolds by chemical design: cyclic conotoxin (6 amino acids), defensin (6, 7, or 12 amino acids), Kalata B1 (3, 6, or 9 amino acids), McoTI (3, 7, 9, 16, 18, or 21 amino acids), Cyclic chlorotoxin (10 amino acids).

2. Structural and functional characterization.
The peptide epitope should be from the fragments of an interacting protein or functional and bioactive known domains from the screening assays such as a phage display library.

Then where should the epitope be grafted onto the scaffold? There are a few possible suggestions: insertion between two existing residues; substitution of one or more residues in a single loop; replacement of residues that span across connected loops; or replacement of most of the native residues of the scaffold.

3. How to make the grafted peptides?
The grafted peptides have been made using solid-phase chemical peptide synthesis. First, the reduced linear precursors are assembled. They are the sequences from the scaffold and epitope. Then oxidization and cyclization are performed to form the final product. However, synthesis of more complex peptides with multiple disulfide bonds can be challenging. A peptide comprising just three disulfide bonds would have 15 different connectivities. In addition, grafting of an epitope onto a scaffold may not be folded correctly, or it does not have the desired activity.

Future perspectives
It is encouraging that many grafted peptides have exhibited oral activity. The cyclic disulfide-rich scaffolds have enhanced stability. However, not all grafted peptides fold into the desired conformation. So a more detailed understanding of how grafting affects the folding of disulfide-rich peptides would be beneficial.

https://doi.org/10.1038/s41589-018-0039-y

disulfide formation

disulfide formation

Simple method to prepare antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates

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We describe a simple method for preparing antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates and discuss its applications in drug delivery and new drug design. Conjugation is based on alkyne-azide cycloaddition. This Cu-free click reaction starts from the dibenzocyclooctyne (DBCO) moiety-activated antibodies and subsequently linked covalently with an azide-modified peptide, oligonucleotide or compounds. The reaction is performed under physiological conditions and has no adverse effects on antibodies or proteins. This can also be used as the click chemistry fluorescence labeling and the click chemistry in peptide-based drug design.

However, the copper-catalyzed alkyne-azide cycloaddition (CuAAC) is not suitable for applications involving functional biomolecules because copper ions can cause protein denaturation.

Measuring the protein levels directly is challenging. However, the signals can be amplified by immuno-PCR using oligonucleotide-attached antibodies to detect protein indirectly.

Antibody-Conjugate

Antibody-Conjugate

 

Preparing Antibody-Peptide, Antibody Oligonucleotide or Antibody-Compound Conjugates

1. Conjugation of DBCO to the Antibody. The DBCO-PEG5-NHS was used to react with the NH2 groups on the antibody. The inclusion of a PEG5 linker improves the water solubility of the hydrophobic DBCO, introduces a spacer and flexibility between the antibody molecule and the peptide/oligonucleotide or compounds. This will alleviate the steric effect of the antibody on the enzymatic reactions.

2. Prepare the azido-Peptide or azido-oligonucleotide. LifeTein provides click chemistry modified peptide synthesis: N-terminal azide-peptide/oligo or C-terminal peptide/oligo-azide.

3. Covalent attachment of the peptide/oligonucleotide to the antibody. The reaction between DBCO and azide is slow compared to CuAAC reaction. The reaction time of 16–18 h in PBS at 4 °C is ideal to increase the final product yield. The DBCO-antibody in the intermediate reaction is stable.

https://pubs.acs.org/doi/full/10.1021/acs.bioconjchem.5b00613

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Copper-Free Click Chemistry Antibody-DNA Conjugation

Personalized treatment using synthetic peptides

Long peptide synthesis by click chemistry

Post-translational modifications: Methylated peptides

Simple method to prepare antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates

LifeTein Leads the Way in Revolutionary Peptide Conjugation Methods

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In the realm of peptide synthesis and bioconjugation, LifeTein stands at the forefront, offering innovative solutions for linking peptides to other biomolecules. Typically, peptides have three biological functional groups available for conjugation: amino (–NH2), carboxyl (–COOH), and thiol (–SH). Among these, the thiol group, particularly from cysteine residues, is often the most effective for bioconjugation. The reaction between maleimides and thiols is a widely recognized method for the bioconjugation and labeling of biomolecules, and LifeTein has mastered this technique to offer superior results.

Click Chemistry: A Revolution in Peptide Conjugation by LifeTein

LifeTein has embraced Click Chemistry, an efficient method for conjugating peptides with various biomolecules. This technique involves modifying the peptide with azide groups (–N3). A standout feature in LifeTein’s arsenal is the novel Copper-free Click Chemistry, which is based on the reaction of a diaryl cyclooctene moiety (DBCO) with an azide-modified peptide. This reaction is not only rapid at room temperature but also avoids the use of cytotoxic Cu(I) catalysts, leading to almost quantitative yields of stable triazoles.

The DBCO component allows copper-free click chemistry to be safely employed with live cells, whole organisms, and non-living samples, which is a significant advantage in various biological applications. Importantly, within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are abundantly present in many biomolecules. The reaction of the DBCO group with the azide group is notably faster than with the sulfhydryl group (–SH, thiol), making it a preferred choice for many of LifeTein’s clients.

Practical Applications: Peptide Drug Conjugations

A prime example of the application of these techniques is in the creation of antibody-biomolecule conjugates. LifeTein’s protocol for Click chemistry of antibody-DNA conjugation is straightforward and efficient:

  1. Pre-conjugation Preparations: Remove all additives from antibody solutions using methods like dialysis or desalting. It’s crucial to eliminate BSA and gelatin from these solutions and concentrate the antibody post-purification.

  2. Activation with DBCO-NHS Ester: The antibody is mixed with a 20-30 fold molar excess of DBCO-NHS ester dissolved in DMSO and incubated at room temperature or on ice.

  3. Quenching the Activation Reaction: This step involves adding Tris-HCl (50-100mM, pH 8) to the reaction mixture, followed by incubation at room temperature or on ice to stabilize the reaction.

  1. Equilibration and Removal of Non-reactive DBCO-NHS Ester: This is achieved using a Zeba column, following the manufacturer’s instructions to ensure precision and effectiveness.

  2. Copper-Free Click Reaction: The DBCO-NHS ester labeled antibody is then mixed with a 2-4 times molar excess of azide-modified oligos. This mixture is incubated overnight at 4°C or for a few hours at room temperature, facilitating the conjugation process.

  3. Validation and Purification: The final step involves validating the conjugation and purifying the product using HPLC, ensuring the high quality and efficacy of the conjugate.

LifeTein’s expertise in peptide synthesis and conjugation is further exemplified by their application of Click Chemistry and thiol-maleimide bioconjugation techniques. These methods are not only efficient but also versatile, opening up new possibilities in the field of peptide-based therapeutics and research.

Selected References:

  • Simon et al. (2012). Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23(2):279.
  • Arumugam et al. (2011). [18F]Azadibenzocyclooctyne ([18F]ADIBO): A biocompatible radioactive labeling synthon for peptides using catalyst-free [3+2] cycloaddition. Bioorg. Med. Chem. Lett. 21:6987.
  • Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50:11117.

Through these advanced techniques, LifeTein continues to be a leader in the field of peptide synthesis and bioconjugation, contributing significantly to the advancement of biomedical research and therapeutic development.

One example of peptide drug conjugations is the antibody-biomoleule conjugate.

click chemistry: DBCO-azide

click chemistry: DBCO-azide

A simple protocol: Click chemistry of antibody-DNA conjugation

Pre-conjugation considerations

  • Remove all additives from antibody solutions using dialysis or desalting.
  • Remove BSA and gelatin from antibody solutions.
  • Concentrate the antibody after dialysis or purification.

Activation of antibodies with DBCO-NHS ester

  • Mix antibody with 20-30 fold molar excess over antibody of DBCO-NHS ester dissolved in DMSO.
  • Incubates at room temperature for 30 min or 2 hours on ice.

Quenching activation reaction

  • Add Tis-Hcl (50-100mM, pH 8) to the reaction.
  • Incubate at RT for 5 min or 15 minutes on ice.

Equilibration and removal of non-reactive DBCO-NHS ester by Zeba column (Follow the manufacturer’s instruction)

Copper-Free click reaction

  • Mix DBCO-NHS ester labeled antibody with 2-4 times molar excess of azide-modified Oligos.
  • Incubated overnight (around 10-12 hours) at 4°C or 3-4 hours at room temperature.

Validation of conjugation and purification by HPLC

A simple protocol: Maleimide labeling of peptide and other thiolated biomolecules

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The reaction of maleimides with thiols is widely used for bioconjugation and labeling of biomolecules such as proteins and peptides. Maleimides are electrophilic compounds which show high selectivity towards thiols.

The Reaction of Maleimides With Thiols

1. Dissolve the peptide or other biomolecules containing thiol in degassed buffer (PBS, Tris, or HEPES) at pH 7-7.5. 2. Add a 100x molar excess of TCEP (tris-carboxyethyl phosphine) reagent to reduce disulfide bonds. 3. Dissolve maleimide in DMSO or fresh DMF (1-10mg in 100uL). 4. Add dye solution such as cy5 maleimide to thiol solution (20x fold excess of dye), flush with an inert gas, and close tightly. 5. Mix thoroughly and keep at room temperature or 4C overnight. 6. Purify by gel filtration, HPLC, FPLC, or electrophoresis.

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Personalized treatment using synthetic peptides

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personalized medicine using synthetic peptides

personalized medicine using synthetic peptides

Interest in personalized treatment has been fuelled by the concept to tailor therapy with the best response and highest safety margin to ensure better patient care. Personalized medicine holds promise for improving health care while also lowering costs.

Synthetic Peptides for Personalized Treatment

An immunogenic personal neoantigen vaccine for melanoma patients using the synthetic peptides provides an opportunity to develop agents that are targeted to patient groups that do not respond to medications as intended and for whom the traditional health systems have otherwise failed. The T cell epitopes with tumor-specific expression arising from non-silent somatic mutations are not expressed in normal tissues. These neoantigens are mutated peptides with the high-affinity binding of autologous HLA molecules. The vaccination with neoantigens can induce new T cell specificities in cancer patients. Using the synthetic peptides as a personalized vaccine, researchers found that of 6 vaccinated patients, 4 had no recurrence at 25 months post-vaccination. The T cells discriminated mutated from wildtype peptide antigens, and directly recognized autologous tumor. From this study, immunizing peptides were selected based on HLA binding predictions. Each patient received up to 20 long peptides in 4 pools.

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Long peptide synthesis by click chemistry

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Some fusion protein or chimeric proteins could never be produced from the e.coli expression system, especially when several hydrophobic sequences are involved in the functional domains. Obtaining peptides sized 100–200 amino acids using chemical synthesis is much faster and cheaper than cloning and overexpressing in Escherichia coli. In addition, the resulting peptide is always correct. Chemical synthesis can be used to incorporate non-genetically encoded structures, such as D-amino acids, into the protein in a completely regular fashion. Synthetic peptides eliminate problems such as poor or no expression, cloning errors, tags like FLAG or 6-His, or the mistranslation of non-preferred codons in prokaryotic hosts. Artificial amino acids that have isosteric side chains can be used to investigate the functional importance of specific residues. All these chimeric proteins can be achieved by the peptide design and synthesis using the click chemistry.
Long peptide synthesis by click chemistry

Long peptide synthesis by click chemistry

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Exploring the Role of Methylated Peptides in Histone Methylation: A LifeTein Perspective

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Post-translational modifications (PTMs) of histone proteins, such as acetylation, methylation, and phosphorylation, are pivotal in regulating chromatin dynamics. Among these, the role of methylation, particularly at arginine or lysine residues, stands out for its complexity and significance. LifeTein, a leader in peptide synthesis, has contributed significantly to this field by synthesizing mono-, di-, or tri-methylated peptides. These peptides are instrumental in studying protein-protein interactions, especially in the context of histone methylation.

Histone methylation, a process that can signal either transcriptional repression or activation, is increasingly recognized for its interrelation with DNA methylation in mammals. For instance, the targeting of DNA methylation is intricately linked to H3K9 methylation, a key regulatory mechanism in gene expression. The p53 gene, known as the guardian of the genome and frequently mutated in human cancers, is regulated by various PTMs, including methylation.

LifeTein’s contribution to this research is highlighted in a study focusing on the ASHH2 CW domain, which is responsible for recognizing the methylation state at lysine 4 of histone 3 N-terminal tails. This domain is crucial in recruiting the ASHH2 methyltransferase enzyme to histones. The study utilized H3 histone tail mimicking peptides, specifically monomethylated (ARTK(me1)QTAR), dimethylated (ARTK(me2)QTAR), and trimethylated (ARTK(me3)QTAR) peptides, all synthesized by LifeTein with a remarkable 95% purity as confirmed by mass spectrometry.

The research documented the assignment of a shortened ASHH2 CW construct, CW42, which showed similar binding affinity and better expression yields than previous constructs. This advancement is significant in understanding how different methylation states affect protein-peptide interactions. The study also performed 1H–15N HSQC-monitored titrations to determine the saturation point of the protein-peptide complex. The findings revealed that the CW42 domain, when bound to the monomethylated histone tail mimic, showed similar perturbations in shifts as the di- and tri-methylated instances.

In summary, LifeTein’s synthetic methylated peptides have been instrumental in advancing our understanding of histone methylation. Their high-purity peptides have enabled researchers to delve deeper into the complexities of chromatin dynamics and gene regulation, paving the way for future discoveries in epigenetic therapies and cancer treatment.

Read the full article on SpringerLink](https://link.springer.com/article/10.1007/s12104-018-9811-x) for more detailed insights into this groundbreaking research.

Noble metal gold and silver nanoparticle are conjugated with peptides for cellular imaging

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Noble metal gold (Au) and silver (Ag) nanoparticle (NPs) are used to conjugate with M3 peptides. The AuNPs-sGFP andAuNPs-M3 peptide form SERS active hot spot through self-assembly and GFP complementation. The nanoparticles self-assemble into surface-enhanced Raman-scattering (SERS) nanoclusters. The nanocluster can be used as contrast agents for multimodal SERS and photoacoustic microscopy with single-cell sensitivity.

AuNPs coated with M3 peptides-GFP

AuNPs coated with M3 peptides-GFP

Reference: M3 peptide was purchased from LifeTein.

Cellular imaging by targeted assembly of hot-spot SERS and photoacoustic nanoprobes using split-fluorescent protein scaffolds

https://www.nature.com/articles/s41467-018-03046-w

 

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Custom Chemical Synthesis Other Posts: How to generate highly stable D-amino acid analogs of bioactive helical peptides? A six-mer synthetic peptide (AT1002) showed enhanced nasal drug delivery A simple protocol: Maleimide labeling of peptide and other thiolated biomolecules

How to generate highly stable D-amino acid analogs of bioactive helical peptides?

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Using D-amino acids as the building blocks for bioactive peptides can dramatically increase their potency. In this study, the authors generated a database of ∼2.8 million D-peptides using a mirror image of every structure in the Protein Data Bank (PDB). The critical or hotspot residues were studied. Residues critical to target binding and activity can then be ideally done experimentally such as alanine scanning mutagenesis. It can also be carried out computationally such as thermodynamic integration or free energy perturbation.

D-Amino Acids for Bioactive Peptides

Two peptides were tested to prove the concept: GLP-1 and Parathyroid Hormone. Both (L)- and (D)-peptides were synthesized by Lifetein LLC.

  1. GLP-1 is a helical GPCR agonist as a diabetes mellitus and obesity treatment. Hotspot and junction residues are annotated in green and blue, respectively. The authors investigated the ability of (D)-GLP1 peptide to induce activation of GLP1R and compared the response with native (L)-GLP1 peptide. It was found that the D-GLP-1 performed well and resistance to protease degradation. The retro-inversion (RI) reversing the (D)-peptide sequence was used in the experiment.
D amino acid peptides

D amino acid peptides

Glucagon-Like Peptide 1, GLP – 1 (7 – 36), amide, human: 
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR – NH2

2.  Parathyroid Hormone (PTH) is an FDA-approved treatment for osteoporosis. The (D)-PTH activates PTH1R with a potency and efficacy comparable to (L)-PTH. And more than 85% of the (D)-PTH analog is still detectable at six hours.

PTH 1-34: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF

Conclusion: The D-Protein Data Bank (PDB) can be used to search and find therapeutically active topologies. The D-PDB could be a key tool for finding stable lead molecules in early-stage drug discovery.

Hot spot residues for receptor binding

Hot spot residues for receptor binding

Reference: 

Method to generate highly stable D-amino acid analogs of bioactive helical peptides using a mirror image of the entire PDB

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A six-mer synthetic peptide (AT1002) showed enhanced nasal drug delivery

A simple protocol: Maleimide labeling of peptide and other thiolated biomolecules

A six-mer synthetic peptide (AT1002) showed enhanced nasal drug delivery

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Zonula occludens toxin (Zot) and its biologically active fragment, delta G, have been shown to reversibly open tight junctions (TJ) in endothelial and epithelial cells. AT1002, a six-mer synthetic peptide H-FCIGRL-OH of ZO toxin was identified and synthesized that retains the Zot permeating effect on intercellular TJ. It was found that AT1002 disrupts the epithelial barrier while larazotide acetate restores barrier function by rearrangement of actin. In addition, AT1002 enhances the transport of molecular weight markers or agents with low bioavailability with no cytotoxicity. So this synthetic peptide AT1002 is a tight junction modulator with promising permeation-enhancing activity.

A Synthetic Peptide Showed Enhanced Nasal Drug Delivery

The C-terminal amidated AT1002 FCIGRL-NH2 showed enhanced nasal drug delivery and may lead to the development of a practical drug delivery technology for drugs with low bioavailability. The synthetic peptide AT1002 was synthesized by LifeTein.
Peptide amidation

Peptide amidation

https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-018-0481-z https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383222/

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Our Services: COVID-19 Services & Products Custom Antibody Services Rush Peptide Synthesis Peptide Nucleic Acids (PNAs) Custom Peptide Synthesis Services Gene Synthesis Service Custom Chemical Synthesis Other Posts: Noble metal gold and silver nanoparticle are conjugated with peptides for cellular imaging How to generate highly stable D-amino acid analogs of bioactive helical peptides? A simple protocol: Maleimide labeling of peptide and other thiolated biomolecules