Optimized Fmoc-Removal Strategy to Suppress the Traceless and Conventional Diketopiperazine Formation in Solid-Phase Peptide Synthesis

Yang, Yi Tien; Hansen, Lena

doi:10.1021/acsomega.2c00214

Cited by 8 publications

(5 citation statements)

References 15 publications

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“…Changing the deprotection cocktail to 2 % DBU and 5 % piperazine in NMP for 2×5 min suppressed the side reaction and was followed by immediate washing and acylation of the free amine. [26] Following allyl deprotection to give the resin-bound acid, a mono-trityl protected PEG2 diamine was installed. This amide coupling reaction was successful using EDC/hydroxybenzotriazole coupling, but not when HATU was employed.…”

Section: Resultsmentioning

confidence: 99%

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Solid‐Phase Synthesis of PROTACs and SNIPERs on Backbone Amide Linked (BAL) Resin

Hales,

Thompson

2023

Chemistry A European J

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Developing straightforward but flexible approaches to PROTAC synthesis that can incorporate the structural elements of emerging designs can improve the quality and efficiency of PROTAC development. Solid‐phase approaches could offer many advantages over conventional PROTAC synthesis if diverse chemistries and topographies can be incorporated. We have exploited the backbone‐amide‐linked (BAL) resin to employ an array of solid‐phase organic reactions, providing access to VHL‐ and IAP‐targeting degraders using the BRD4‐targeting JQ1 conjugates as examples.

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Section: Resultsmentioning

confidence: 99%

“…A deletion product was obtained which lacked both the hydroxyproline and the tert‐ leucine residues. Changing the deprotection cocktail to 2 % DBU and 5 % piperazine in NMP for 2×5 min suppressed the side reaction and was followed by immediate washing and acylation of the free amine [26] …”

Section: Resultsmentioning

confidence: 99%

Solid‐Phase Synthesis of PROTACs and SNIPERs on Backbone Amide Linked (BAL) Resin

Hales,

Thompson

2023

Chemistry A European J

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“…For example, the use of PPZ supplemented with DBU in N-methylpyrrolidone (NMP) has been described to enhance the suppression of DKP formation in some sequences. 23 However, several factors, including limited solubility in DMF, reduced base strength and thus necessitating the addition of another stronger base, and the requirement of additives like hydroxybenzotriazole (HOBt) 24 or formic acid 25 to suppress side reactions, circumscribe the general use of PPZ in Fmoc-SPPS. Likewise, the performance of pyrrolidine was not promising due to its higher base strength, similar to that of PIP, thus influencing side reactions and sometimes causing the degradation of the desired peptide.…”

Section: Introductionmentioning

confidence: 99%

“…In this context, other secondary amines, such as pyrrolidine and piperazine (PPZ), have also been exploited on several occasions. For example, the use of PPZ supplemented with DBU in N‐ methylpyrrolidone (NMP) has been described to enhance the suppression of DKP formation in some sequences 23 . However, several factors, including limited solubility in DMF, reduced base strength and thus necessitating the addition of another stronger base, and the requirement of additives like hydroxybenzotriazole (HOBt) 24 or formic acid 25 to suppress side reactions, circumscribe the general use of PPZ in Fmoc‐SPPS.…”

Section: Introductionmentioning

confidence: 99%

Morpholine, a strong contender for Fmoc removal in solid‐phase peptide synthesis

Mthembu,

Chakraborty,

Schönleber

et al. 2023

Journal of Peptide Science

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Morpholine, which scores 7.5 in terms of greenness and is not a regulated substance, could be considered a strong contender for Fmoc removal in solid‐phase peptide synthesis (SPPS). Morpholine in dimethylformamide (DMF) (50%–60%) efficiently removes Fmoc in SPPS, minimizes the formation of diketopiperazine, and almost avoids the aspartimide formation. As a proof of concept, somatostatin has been synthesized using 50% morpholine in DMF with the same purity as when using 20% piperidine–DMF.

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“…Methods have been developed to inhibit DKP side reaction propagation. ,,− DKP formation can be avoided or minimized by utilizing mild deprotection conditions or incorporating a dipeptide to skip DKP-sensitive intermediates . Goolcharran and Borchardt studied the impact of pH, temperature, and buffer concentration/species on the Phe-Pro- p -nitroaniline (Phe-Pro-pNA) to Phe-Pro-diketopiperazine (Phe-Pro-DKP) reaction .…”

Section: Introductionmentioning

confidence: 99%

Mechanistic Study of Diketopiperazine Formation during Solid-Phase Peptide Synthesis of Tirzepatide

et al. 2022

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This study focused on investigating diketopiperazine (DKP) and the formation of associated double-amino-acid deletion impurities during linear solid-phase peptide synthesis (SPPS) of tirzepatide (TZP). We identified that the DKP formation primarily occurred during the Fmoc-deprotection reaction and post-coupling aging of the unstable Fmoc-Pro-Pro-Ser-resin active pharmaceutical ingredient (API) intermediate. Similar phenomena have also been observed for other TZP active pharmaceutical ingredient (API) intermediates that contain a penultimate proline amino acid, such as Fmoc-Ala-Pro-Pro-Pro-Ser-resin, Fmoc-Pro-Pro-Pro-Ser-resin, and Fmoc-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-resin, which are intermediates for both hybrid and linear synthesis approaches. During post-coupling aging, it is found that Fmoc deprotection can proceed in dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and acetonitrile (ACN) solvents without any piperidine addition. Density functional theory (DFT) calculations showed that a peptide that has a penultimate proline stabilizes the transition state through the C−H•••π interaction during Fmoc decomposition, which causes those peptides to be more prone to cascade-deprotection reactions. Pseudo-reaction pathways are then proposed, and a corresponding macrokinetics model is developed to allow accurate prediction of the TZP peptide intermediate self-deprotection and DKP formation rate. Based on those studies, control strategies for minimizing DKP formation were further investigated and an alternative to Fmoc protection was identified (Bsmoc-protected amino acids), which eliminated the formation of the DKP byproducts. In addition, the use of oxyma additives and lower storage temperature was demonstrated to markedly improve the peptide intermediate stability to DKP degradation pathways.

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Optimized Fmoc-Removal Strategy to Suppress the Traceless and Conventional Diketopiperazine Formation in Solid-Phase Peptide Synthesis

Cited by 8 publications

References 15 publications

Solid‐Phase Synthesis of PROTACs and SNIPERs on Backbone Amide Linked (BAL) Resin

Solid‐Phase Synthesis of PROTACs and SNIPERs on Backbone Amide Linked (BAL) Resin

Morpholine, a strong contender for Fmoc removal in solid‐phase peptide synthesis

Mechanistic Study of Diketopiperazine Formation during Solid-Phase Peptide Synthesis of Tirzepatide

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