Unnatural Amino Acids Conjugation

Unnatural Amino Acids Conjugation

BOC Sciences can synthesize site-specific antibody-drug conjugate (ADC) using unnatural amino acids (uAAs) that have orthogonal chemical reactivity with standard amino acids. We introduce unnatural amino acid residues in the antibody to upregulate drug conjugation specificity. With our extensive experience in antibody engineering and pharmaceuticals, BOC Sciences' professional research team is capable of providing customers with various personalized services.

What are Unnatural Amino Acids?

Unnatural amino acids are a special class of amino acids that are not restricted by genetic codes. They have unique spatial configurations and chemical properties, providing new resources and opportunities in multiple fields. Especially in synthetic proteins, they break the limitations of 20 natural amino acids, giving proteins more stable performance and broader applications. Currently, uAAs can be synthesized through two methods: chemical synthesis and biosynthesis. Compared with chemical synthesis, biosynthesis has higher efficiency, stereoselectivity, lower cost and pollution, and is a potential synthesis method. uAAs have broad prospects in protein probes, enzyme engineering, antibody drug conjugates, antimicrobial peptides, etc.

What are Unnatural Amino Acids in Novel Antibody Conjugates?

In addition to modification of known amino acids in antibodies, uAAs can also be integrated into the protein sequence of antibodies through genetic code expansion (GCE) to specifically modify the binding site of the antibody. Antibody-drug site-specific conjugation can be achieved through uAAs, that is, unnatural amino acids can be introduced into specific sites of any target protein in the organism. The residues on these unnatural amino acids can be combined with linkers to form site-specific ADCs. Compared with traditional ADC drugs, the introduction of unnatural amino acid antibody and drug linkers can achieve targeted and quantitative coupling. The obtained ADC has high efficacy, good stability, long circulation half-life, high safety, and uniform drug-to-antibody ratio (DAR).

Unnatural amino acids conjugationFig. 1. Unnatural amino acids conjugation strategies.

Since the introduction of unnatural amino acids into antibodies may bring immunogenicity risks, the selection of unnatural amino acids is very critical for the construction of ADCs. Generally speaking, unnatural amino acids must be structural analogs of natural amino acids, and they should also have active sites that are easy to bind to linkers, such as carbonyl and azide functional groups to facilitate further connection. The unnatural amino acids currently introduced are usually acetylphenylalanine (pAF), azidomethyl-L-phenylalanine (pAMF) and azidolysine. For example, Kularatne et al. symmetrically integrated the amino acid part of p-acetyl-phenylalanine (p-AcF) into the two heavy chains of the antibody, and the other end was coupled with the linker by constructing a ketone oxime bond to produce a homogeneous ADC with DAR of 2. Several ADCs generated by introducing uAAs have entered clinical trials.

Unnatural Amino Acids Conjugation Services

BOC Sciences' expertise in unnatural amino acid synthesis allows us to design and synthesize custom linker molecules that meet the specific requirements of your ADC project. Our chemists can work with you to select the optimal unnatural amino acids and linker functionality for your application, and we can scale up synthesis to deliver the quantities of linker molecules required for preclinical and clinical studies.

  • Unnatural Amino Acid Synthesis

BOC Sciences supports the in vitro synthesis of unnatural amino acids that meet pharmaceutical needs through medicinal chemistry. We offer a wide range of unnatural amino acid synthesis services, including the design and synthesis of novel amino acids with specific chemical properties. In particular, we support the rapid synthesis of acetylphenylalanine, azidomethyl-L-phenylalanine, and azidolysine. Our experienced team of chemists can work with you to develop a custom synthesis protocol that meets your specific needs, whether you require milligram quantities for research purposes or larger quantities for drug development. We can also provide analytical services to ensure the purity and identity of the final product, including HPLC, mass spectrometry and NMR analysis.

  • Orthogonal Translation of Unnatural Amino Acids

The orthogonal translation technology of unnatural amino acids is also called genetic code expansion technology. Protein synthesis on the ribosome proceeds through the recognition of tRNA anticodons and mRNA codons. We can introduce tRNA that can specifically recognize unnatural amino acids and its corresponding aminoacyl-tRNA synthetase. Under the action of aminoacyl-tRNA synthetase, tRNA combines with the corresponding unnatural amino acid to form aminoacyl-tRNA. Then, by complementing its anticodon with the codon on the mRNA, the unnatural amino acid is integrated into the polypeptide chain, and a recombinant antibody containing the unnatural amino acid is synthesized. In this process, BOC Sciences is able to perform rapid and parallel synthesis of numerous variants utilizing a large number of different sites, enabling analysis early in discovery to define structure-activity relationships and locate optimal sites for uAA incorporation according to:

  • Antibody expression
  • Incorporation efficiency of uAA
  • Conjugate efficiency of linker and payload
  • Stable payload during in vivo circulation
  • Cell killing efficiency
  • Target targeting efficiency
  • Internalized traits
  • Site-specific Conjugation of Linkers

The unnatural amino acids introduced into the recombinant antibody synthesized in the previous step become predictable coupling sites for the payload. The ketone and azide functional groups on unnatural amino acids can react chemically with the drug linker to obtain an ADC with uniform DAR.

  • Ketones Unnatural Amino Acids Modification

Studies have successfully integrated p-acetylphenylalanine (pAcF) as an unnatural amino acid residue in the antibody, and it contains a ketone functional group does not present in natural amino acids. An oxime bond can be formed between the ketone group and the alkoxyamine, which is highly stable under physiological conditions. Auristain glycoside payload is effectively coupled to the antibody through oxime linkage, generating a chemically uniform ADC. BOC Sciences' ADCs service platform can provide a variety of ketone-containing unnatural amino acids modification strategies for our customers to choose from.

  • Azides Unnatural Amino Acids Modification

Azides containing unnatural amino acids is another commonly used technique for research and development. Under physiological conditions, the widely used p-azidopiperidine (pAzF) can quickly perform CuAAC or SPAAC reactions, and this strategy can be used to successfully conjugate glucocorticoid payloads on anti-CD74 antibodies. In addition to pAcF technology, azide-containing lysine analogs were successfully introduced into the antibody to produce site-specific ADCs with Auristain glycoside payload, PBD dimer, or tubulin payloads. The ADCs service platform of BOC Sciences provides advanced and comprehensive azides unnatural amino acids modification services for your research projects.

  • Cyclopropenes Unnatural Amino Acid Modifications

Lysine cyclopropene derivatives and naturally occurring atypical amino acids such as selenocysteine (Sec) have been successfully integrated into the antibody. The resulting ADC showed good stability, selectivity, as well as in vitro and in vivo activities. As a leading service provider in the antibody-drug conjugate field, BOC Sciences has offered a full range of cyclopropenes unnatural amino acids modification services to our clients worldwide for years.

Why Choose BOC Sciences?

  • Advanced site-specific ADC coupling technology support
  • Optimized support for linkers and payloads
  • Comprehensive therapeutic endpoints and safety assessment
  • Dozens of project experience and flexible production capabilities
  • Cost-saving and fast turnaround one-stop service
  • Complete risk assessment and management system
  • Comprehensive confidentiality agreements and measures
1. How are unnatural amino acids made?

Unnatural amino acids (uAAs) are a type of non-protein optical amino acids, which are obtained through chemical modification, gene site-directed mutagenesis and computer-aided protein design of 20 natural amino acids. Unnatural amino acids have diverse functional groups, such as ketone groups, aldehyde groups, alkenyl groups, nitro groups, amide groups, phosphate groups, etc., and can undergo reactions such as photochemistry, click chemistry, glycosylation, and fluorescence color development.

At present, there are several methods to add unnatural amino acids to proteins: chemical synthesis; designing tRNA corresponding to the stop codon and aminoacyl-tRNA synthetase carrying unnatural amino acids; substitute common 20 amino acids in auxotrophic strains with close structural homologs; designed to recognize four-codon tRNA.

Protocol 1 Genetic code expansion and unnatural amino acid insertions

The genetic code is expanded and uAA is inserted into the protein of living cells. This system mainly includes the following steps:

  1. Synthetic uAA: Has better specificity and stability than natural amino acids.
  2. Orthogonal tRNA synthetase: Only catalyzes the loading of uAA onto orthogonal tRNA and does not recognize natural amino acids and tRNA.
  3. Orthogonal tRNA: Only orthogonal tRNA synthetase and uAA are recognized.
  4. Unique codons: The triplet codons encoding natural amino acids are expanded, and the codons required to be inserted into the uAA site are mutated into amber stop codons.
  5. Introduction of uAA at specific sites in the protein: DNA, orthogonal tRNA and orthogonal tRNA synthetase are transferred into the genome of the cell. DNA is transcribed into target site gene mRNA, tRNA and orthogonal tRNA synthetase mRNA, which is further translated into orthogonal tRNA synthetase. uAA introduced into the cell culture medium is recognized and loaded onto orthogonal tRNA by orthogonal tRNA synthetase. Orthogonal tRNA brings uAA to the ribosome for linear assembly with 20 natural amino acids according to the instructions of the gene's mRNA. When the pre-mutated amber stop codon is reached, it is recognized by the orthogonal tRNA, and then the uAA loaded on the orthogonal tRNA is integrated into this position. The protein translation process continues unaffected.


  1. Kotschy, A. et al. The Chemistry Behind ADCs. Pharmaceuticals (Basel). 2021, 14(5): 422.
  2. Wiemer, A.J. et al. Stepping forward in antibody-drug conjugate development. Pharmacology & Therapeutics. 2021, 107917.
* Only for research. Not suitable for any diagnostic or therapeutic use.
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