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Methods, setup and safe handling for anhydrous hydrogen fluoride cleavage in Boc solid-phase peptide synthesis

Nature Protocols volume 10, pages 1067–1083 (2015)Cite this article

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Abstract

Solid-phase peptide synthesis (SPPS) using tert-butyloxycarbonyl (Boc)/benzyl (Bzl) chemistry is an indispensable technique in many laboratories around the globe, and it provides peptides to the pharmaceutical industry and to thousands of scientists working in basic research. The Boc/Bzl strategy has several advantages, including reliability in the synthesis of long and difficult polypeptides, alternative orthogonality regarding protecting groups and ease of producing C-terminal thioesters for native chemical ligation applications. In this process, anhydrous hydrogen fluoride (HF) is used to remove the side chain protecting groups of the assembled peptide and to release the peptide from the resin, a process typically described as 'HF cleavage'. This protocol describes the general methodology, apparatus setup and safe handling of HF, with the aim of providing comprehensive information on the safe use of this valuable, well-studied and validated cleavage technique. We explain the cleavage mechanism, the physicochemical properties and risks of HF, first aid measures and the correct use of the apparatus. In addition, we provide advice on scavenger selection, as well as a troubleshooting section and video material illustrating key steps of the procedure. The protocol comprises precleavage sample preparation (30 min–2.5 h), complete HF cleavage procedure (2 h) and reaction workup (30 min).

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Figure 1: Boc-SPPS procedure overview: a general concept of the in situ neutralization Boc-SPPS procedure.
Figure 2: Examples of type-I HF cleavage apparatus.
Figure 3: Schematic representation of the key steps during the HF cleavage procedure.
Figure 4: Example HF cleavage of α-conotoxin RgIA.

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Acknowledgements

We thank P.A. Huete (Special Reaction Service at Barcelona Science Park, SRE-PCB) and J.B. Blanco-Canosa for help with the videos. We thank D. Thompson, A. Brust, T. Durek, the Peptide Synthesis Service of the Biomedical Networking Center (CIBER-BBN) at Barcelona Science Park, Calgonate, Peptide Institute, CS Bio (D. Slavazza, H.W. Chang), Y. Nishiuchi, P. Alewood and T. Hackeng for their help with photos and proofreading. The work carried out in the Albericio laboratory was partially funded by La Comisión Interministerial de Ciencia y Tecnología (CICYT) (CTQ2012-30930), the Generalitat de Catalunya (2014 SGR 137) and the Institute for Research in Biomedicine Barcelona. M.M. received funding from the Australian Research Council (ARC) Discovery Early Career Award (DECRA) (DE150100784), the European Union Seventh Framework Programme (FP7/2007-2013) under Marie Curie Actions grant agreement no. 254897 and 2013-BP-B-00109, and from the Secretary of Universities and Research of the Department of Economy and Knowledge of the Government of Catalonia. We acknowledge US National Institutes of Health (NIH) grant no. R01 GM098871 (P.E.D.).

Author information

Authors and Affiliations

  1. Institute for Research in Biomedicine Barcelona, Barcelona, Spain

    Markus Muttenthaler & Fernando Albericio

  2. Department of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

    Markus Muttenthaler

  3. The Biomedical Research Networking center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN),

    Fernando Albericio

  4. , Barcelona Science Park, Barcelona, Spain

    Fernando Albericio

  5. Department of Organic Chemistry, University of Barcelona, Barcelona, Spain

    Fernando Albericio

  6. School of Chemistry, Yachay Tech, Yachay City of Knowledge, Urcuqui, Ecuador

    Fernando Albericio

  7. Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA

    Philip E Dawson

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  1. Markus Muttenthaler
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Contributions

M.M. wrote the manuscript and conducted the experiments described. P.E.D. and F.A. edited and improved the manuscript.

Corresponding authors

Correspondence to Markus Muttenthaler or Philip E Dawson.

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Integrated supplementary information

Supplementary Figure 1 Schematic representation of different types of HF cleavage apparatus.

a Classic and most used Type-I HF cleavage apparatus developed by Peptide Institute Inc., Japan, including a large HF collection vessel (CV), two small reaction vessels (RVs) and another large CV/RV. Originally equipped with a mercury manometer, the apparatus are nowadays equipped with standard vacuum gauges. b Type-II HF cleavage apparatus also developed and distributed by Peptide Institute Inc., Japan, without the two large CVs and a vacuum gauge. c Schematic connection setup of apparatus with CaO trap, water trap and vacuum pump.11Adapted from the original HF instruction and operation protocol, edited by Professor Shumpei Sakakibara, with permission of Peptide Institute Inc., Japan.

Supplementary Figure 2 Illustrations of standard safety equipment for HF use.

Standard HF safety equipment includes lab coat, apron, safety shower, emergency eyewash, safety glasses, and elbow-long HF-resistant gloves. HF first aid and spill kit can be obtained from Calgonate Corp.22Adapted from and used by permission of Calgonate Corp. Calgonate Corp. reserves all rights to all images, logos, trademarks and other content provided for use herein.

Supplementary Figure 3 Sample workup/filtration setup.

Supplementary Figure 4 Different HF cylinder setups.

a Dual valve setup with the gas outlet connected to a gooseneck eductor tube sourcing HF from the gas phase and the liquid outlet connected to a full-length eductor tube (FLET) sourcing HF from the liquid phase. The FLET outlet will deliver HF significantly faster than the gooseneck outlet. b Single gooseneck eductor tube - the cylinder can come with a N2 layer, which need to be pulled off through the HF apparatus and CaO trap before HF can be utilized. We recommend a gooseneck eductor tube setup without N2 layer. Do NOT use a FLET HF cylinder with a N2 layer since this delivers HF under pressure and is not compatible with the HF apparatus.

Supplementary Figure 5 Dismantled CaO trap.

a Stainless steel cylinder that can hold 4 kg of CaO with top open. b Inside of a just emptied CaO trap showing the inlet tube going all the way to the bottom. The tube has an outlet for HF at the bottom and small holes in the bottom last third of the tube for a better distribution of HF. c & d The top of the CaO trap displaying the outlet to the vacuum pump as well as the O-ring required for a good seal.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 596 kb)

Supplementary Video 1

Packing of CaO trap. (MP4 26009 kb)

Supplementary Video 2

HF cylinder exchange. (MP4 27717 kb)

Supplementary Video 3

Sample preparation. (MP4 26462 kb)

Supplementary Video 4

HF cleavage procedure. (MP4 25610 kb)

Supplementary Video 5

Sample workup. (MP4 27566 kb)

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Muttenthaler, M., Albericio, F. & Dawson, P. Methods, setup and safe handling for anhydrous hydrogen fluoride cleavage in Boc solid-phase peptide synthesis. Nat Protoc 10, 1067–1083 (2015). https://doi.org/10.1038/nprot.2015.061

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