Peptoid Ligation

Reflecting work in the Kirshenbaum Group

Published here April 4, 2026

Native Chemical Ligation of Peptoid Oligomers

Matthew R. Seraydarian, Michael D. Connolly, Ronald N. Zuckermann, and Kent Kirshenbaum

Biochemistry. 2026, ASAP

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Peptide chemists have long used native chemical ligation, NCL, to overcome a fundamental limitation: the longer the chain, the lower the yield. By joining shorter, high-yielding fragments into full-length proteins, NCL enables ambitious syntheses that would otherwise fail. The strategy relies on selective reaction between a C-terminal thioester and an N-terminal cysteine, producing a native amide bond without protecting groups. Peptoid chemists have envied this capability for years, but a critical obstacle has blocked progress. Peptoid synthesis uses primary amines as building blocks under basic conditions, incompatible with the acid-labile thioesters required for NCL. Without fragment condensation methods, peptoid chemists face a stark trade-off between sequence control and chain length.

Researchers in the Kirshenbaum Group at New York University, published in Biochemistry, adapted the hydrazide ligation technique recently developed for peptides to enable native chemical ligation of peptoid oligomers. The team synthesized peptoid fragments bearing C-terminal hydrazides using standard solid-phase submonomer chemistry, initiating synthesis from hydrazine resin with an Fmoc-protected amino acid to circumvent the incompatibility of bromoacetic acid coupling. Partner fragments incorporated an N-terminal cysteamine-derived side chain to provide the thiol nucleophile. Oxidation of the hydrazide with sodium nitrite at pH 3 generated an acyl azide intermediate, which was converted to a thioester in situ using 4-mercaptophenylacetic acid. Subsequent ligation at neutral pH proceeded through the canonical five-membered ring intermediate, with the S-to-N acyl shift mediated by the secondary amine backbone characteristic of peptoids.

Model oligomer ligations demonstrated broad tolerance for side chain diversity. Short peptoid hexamers and heptamers incorporating aromatic, aliphatic, and charged side chains ligated efficiently, with some conversions approaching 90 percent. The protocol accommodated variation in both the C-terminal residue, with N-benzylglycine substituting for sarcosine, and the N-terminal thiol-bearing monomer, with 3-mercaptopropylglycine successfully replacing cysteamine. Internal thiols did not interfere with ligation selectivity. Isolated yields following purification reached 32 percent for model sequences. Radical desulfurization successfully converted the ligation-site thiol to an N-ethylglycine residue, demonstrating that the method can produce native peptoid sequences without residual functional groups. The chemistry also enabled intramolecular cyclization of a linear hexamer bearing both termini, achieving near-quantitative conversion to the macrocycle.

The team applied NCL to synthesize functional peptoid nanomaterials, ligating charged anchor blocks to hydrophilic loop cassettes to create 38-mer sequences capable of self-assembling into two-dimensional nanosheets at the air-water interface. Sequential ligation of three fragments demonstrated modular construction, where stable anchor blocks can be stored and combined with diverse loop sequences to generate libraries of functional nanosheets. Fluorescence microscopy confirmed that ligated products assembled into micron-scale sheets indistinguishable from those synthesized by conventional routes. One unexpected challenge emerged: peptoids bearing N-terminal amine side chains formed N-nitrosamines during hydrazide oxidation, adding 29 daltons to the product mass. Redesigning the charge orientation of nanosheet sequences eliminated this side reaction.

This work provides peptoid chemists with the first convergent ligation method that preserves native backbone connectivity. Unlike previous conjugation strategies relying on triazoles, disulfides, or other non-native linkages, NCL produces only tertiary amide bonds at ligation sites. The modular approach enables incorporation of low-yielding monomers into long sequences by confining them to short, purifiable fragments. Sequential ligation strategies promise accelerated discovery of functional peptoid macromolecules, including structures with defined tertiary architecture and biomimetic properties previously inaccessible due to synthetic limitations. The method opens routes to proteomimetic systems that rival natural biopolymers in complexity and function.

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Additional Authors

Author

Michael Connolly received his BS in Chemistry and Biochemistry and conducted graduate studies at Texas A&M University. In 1999, he joined Chiron Corporation, later Novartis, where he worked in the Bioorganic Chemistry group with Ron Zuckermann developing combinatorial chemistry and high-throughput screening approaches using peptoid biopolymers for drug discovery, vaccines, and diagnostics. Since 2006, he has been at the Molecular Foundry at Lawrence Berkeley National Laboratory, where he leads biopolymer synthesis efforts and develops technologies for the rapid discovery of protein-mimetic nanostructured materials.

Author

Ronald Zuckermann is a Researcher Emeritus at the Molecular Foundry at the Lawrence Berkeley National Laboratory, where he studies the mimicry of biomolecular architectures using bio-inspired polymers. He received his BS in Chemistry in 1984 from Harvey Mudd College, and a Ph.D. in Organic Chemistry in 1989 from UC Berkeley under Prof. Peter Schultz. After receiving the first Schultz group Ph.D. in 1989, he became one of the founding chemists at Protos Corp., a combinatorial drug discovery start-up in Emeryville, CA. There he helped discover a novel class of sequence-defined heteropolymers called peptoids. Chiron Corp. acquired Protos in 1991 where this work continued and he applied peptoids to small molecule drug discovery, diagnostics, biomimetic materials, and nucleic acid delivery. Dr. Zuckermann was promoted to Research Fellow in 2003. In early 2006, he left Chiron to direct the Biological Nanostructures Facility of the Molecular Foundry at Lawrence Berkeley National Laboratory, and worked at the interface of chemistry, biology and nanoscience. He served as Sr. Scientist and Director of the Biological Nanostructures Facility, where he pioneered the field of peptoid nanoscience, and continues to study the folding of sequence-defined peptoid polymer chains into protein-like nanoarchitectures. He is the organizer of the Peptoid Summit conferences, the founder of the International Community for the Advancement of Peptoids, and has published over 200 papers and is co-inventor on 40 patents.

Author

Kent Kirshenbaum is a Professor in the Department of Chemistry at New York University. Kent obtained his Bachelor’s degree in Chemistry at Reed College in Portland, Oregon. His Ph.D. was in Pharmaceutical Chemistry, from the University of California, San Francisco. Kent conducted his post-doctoral studies in protein chemistry at the California Institute of Technology in Pasadena. At NYU, Kent teaches Biochemistry and Medicinal Chemistry. He also leads a research group that is studying sequence-structure-function relationships in biomimetic polymers. Kent is a member of the Cancer Center at NYU's School of Medicine. Kent was the co-founder of the Experimental Cuisine Collective at NYU, along with Professor Amy Bentley, Food Studies, and Chef Will Goldfarb, Pastry. His science outreach efforts include appearances with the Food Network, the Cooking Channel, the Science Channel, Sid the Science Kid, NBC-Learn, and the Discovery Channel.

Peptoid Ligation

Author Spotlight

Author

Matthew Seraydarian is a Ph.D. candidate in the Kirshenbaum group at New York University. He received his BS in Biochemistry and Molecular Biology from the University of South Carolina with honors from the South Carolina Honors College in 2021. His research interests include method development for the synthesis of sequence-defined peptoid macromolecules. At NYU, Matthew demonstrated that peptoid oligomers can be used in native chemical ligation reactions using the hydrazide ligation method. Matthew continued to develop peptoid native chemical ligation at the Lawrence Berkeley National Laboratory's Molecular Foundry and demonstrated the modular synthesis of peptoid nanosheet-forming sequences using ligation chemistry.