Hidden Champion – The Tetrahydropyranyl Protective Group

In theory, peptide synthesis sounds very simple by growing the peptide chain in a C- to N-direction on an insoluble support. This is achieved via combination of the deprotected, free alpha amino group of the growing peptide (which is anchored on the resin) with the free carboxy group – typically supplied as an active ester – of the next amino acid. However, side chain functional groups can interfere and lead to undesired side reactions. Thus, protecting groups play a pivotal role during peptide synthesis.

In terms of serine, threonine, and cysteine, the side chain hydroxyl and thiol reactive moieties, respectively, might undergo dehydration and acylation, followed by O/S→N migration after amino deprotection, especially when activating reagents like carbodiimides are present, as used in standard SPPS protocols. To prevent these side reactions, typically, tert. butyl (OtBu) and trityl (Trt) are used in the case of the mentioned amino acids, whereas tetrahydropyranyl (THP) is rarely used by peptide chemists, even though it has some advantages over the tBu and Trt protection of hydroxyls and thiols.

The acetal is easy to introduce and stable towards strong bases, acylating and alkylating agents. It is compatible with Fmoc as well as tBu SPPS. Due to the high acid lability of THP esters, carboxyl functions do not need to be protected, because the corresponding hemiacetal ester is unstable. Furthermore, as THP is non-aromatic and non-bulky, fewer inter- and intra-chain interactions are expected, and purer products can be obtained. Thus, the THP (thio)ethers of serine, threonine and cysteine can be particularly useful for the synthesis of peptides which are rich in these amino acids, as the THP-protected building blocks impart greater solubility to the resulting peptide chain than the tBu- and Trt-protected analogs.

Schematic representation of THP side chain protection of threonine, serine and cysteine in SPPS (model peptide sequence: -ThrValSerAlaCys-).

In the case of cysteine, racemization and C-terminal 3-(1-piperidinyl)-alanine formation were minimized when the side chain was protected with THP instead of Dpm, Acm, Trt, or StBu. Remarkably, THP-protected Cys is stable at 1% TFA in DCM, suggesting the use of the tetrahydropyranyl group for the controlled formation of cyclic peptides with (multiple) disulfide bridges.

THP acetals are racemic, giving rise to mixtures detectable by HPLC. However, these stereocenters are transient (while the THP protection is attached to the peptide), and no isomers are observed after deprotection.

Tetrahydropyranyl (THP) can easily be removed by TFA in the presence of scavengers, e.g., by applying commonly used SPPS cleavage protocols such as TFA/TIS/DCM (10:1.5:88.5) within 5 min, but also higher concentrations of trifluoroacetic acid (TFA/TIS/TCM 95:2.5:2.5) are tolerated. As a TFA-free protocol, deprotection with HCl in dioxane (12:88) for 2 h is also possible. Compared to Trt, the degree of racemization is reported to be significantly lower with less than 1% compared to over 3%.

→ For larger quantities and THP-protected homologues and analogues of Ser and Thr, please get in contact! 

References:

Tetrahydropyranyl: A Non-aromatic, Mild-Acid-Labile Group for Hydroxyl Protection in Solid-Phase Peptide Synthesis; A. Sharma, I. Ramos-Tomillero, A. El-Faham, H. Rodriguez, B G. de la Torre, F. Albericio; ChemistryOpen 2017; 6(2): 206-210. http://dx.doi.org/10.1002/open.201600157

Understanding Tetrahydropyranyl as a Protecting Group in Peptide Chemistry; A, Sharma, I. Ramos-Tomillero, A. El-Faham, E. Nicolás, H. Rodríguez, B. G. de la Torre, F. Albericio; ChemistryOpen 2017; 6(2): 168-177. https://doi.org/10.1002/open.201600156

Tetrahydropyranyl, a Nonaromatic Acid-Labile Cys Protecting Group for Fmoc Peptide Chemistry; I. Ramos-Tomillero, H. Rodriguez, F. Albericio; Organic Letters 2015; 17(7): 1680-1683. https://doi.org/10.1021/acs.orglett.5b00444

Ready to Use Cysteine Thiol Protecting Groups in SPPS, A Practical Overview; A. Chakraborty, S. N. Mthembu, B. G. de la Torre, F. Albericio; Org Process Res. Devel. 2024; 28(1): 26-45. https://doi.org/10.1021/acs.oprd.3c00425

Greene’s Protective Groups in Organic Synthesis; P. G. Wuts, T. W. Greene; John Wiley & Sons, Hoboken, NJ, 2006. ISBN:9780471697541 (Online ISBN:9780470053485). https://doi.org/10.1002/0470053488