Oxyma as an additive in green solid-phase peptide synthesis

Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate), also known as Oxyma Pure, is a greener alternative to traditional additives used in green solid-phase peptide synthesis and other amide bond-forming reactions. Oxyma Pure is a racemization suppressor and coupling additive used in peptide and organic synthesis. Oxyma Pure forms an Oxyma ester intermediate with carboxyl groups of amino acids, resulting in a more reactive intermediate, less prone to racemization than other intermediates.

Oxyma supports green chemistry principles since it is a safer alternative to (1-hydroxy-benzotriazole/N-[(1H-benzotriazol-1-yl)(dimethyl-amino)-methylene]N-methylmethanaminium hexafluorophosphate N-oxide (HOBt/HBTU) based methods. Coupling additives like HOBt are explosive and hazardous. Oxyma is non-explosive, more stable, less toxic, and safer to handle and store. Also, Oxyma enhances coupling efficiency, reduces the need for excess reagent, minimizing waste, and often improves yields and suppresses racemization in peptide bond formation. Compared to benzotriazole-based additives, Oxyma has a lower environmental and health impact. Oxyma works well in greener solvent systems like ethyl acetate, 2-methyltetrahydrofuran (2-MeTHF), and dimethyl carbonate, supporting a shift away from more harmful solvents like DMF or DCM. Because it reduces side reactions and degradation, it may require fewer solvents as well. However, Oxyma still requires careful disposal, especially in large-scale synthesis.

TFFH is another mild coupling reagent. TFFH (tetramethyl-fluoro-formamidinium hexa-fluoro-phosphate) is a non-hygroscopic salt and stable to handle under ordinary conditions. TFFH is an in-situ reagent to prepare amino acid fluorides during peptide synthesis. TFFH is also an ideal coupling reagent for peptide synthesis in solid and solution phases and organic synthesis.

Additives in peptide synthesis to increase coupling efficiency are very common. Many coupling mixtures contain the additives HOBt and HOAt to minimize configuration loss.

The reactivity of activated esters formed is thought to be related to the stability of the leaving group anions (OBt^- and OAt^-) and their pK_a values. pK_a values for HOBt and HOAt are 4.6 and 3.47, respectively.

Since Wehrstedt et al. in 2005 reported that HOBt has explosive properties, a few years later, Subriros-Funosas et al. in 2009 suggested using Oxyma as a non-explosive coupling reagent in peptide synthesis. Subriros-Funosas et al. found that Oxyma inhibits racemization and increases coupling efficiency in automated and manual synthesis.

El-Faham et al. in 2009 described a new family of uranium-type coupling reagents containing a morpholino group in conjunction with an oxime derivative. The coupling reagent ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) improved the reagents' solubility, stability, and reactivity. The uronium salt derived from Oxyma (COMU) performed well with a less hazardous safety profile than the benzo-triazole-based HMMA and HDMB strategies. The Oxyma moiety contained in COMU suggests a lower risk of explosion than in benzotriazole derivatives.

In the presence of Oxyma, during carboxyl group activation, the amino acid's COOH group reacts with diisopropylcarbodiimide (DIC), forming a reactive O-acylisourea intermediate:

R–COOH + DIC → R–CO–N=C=N–iPr₂ (O-acylisourea intermediate).

Next, the O-acylisourea intermediate reacts with Oxyma, which acts as a nucleophile via its hydroxyimino group:

O-acylisourea + Oxyma → Oxyma ester (R–CO–O–Oxyma) + Urea byproduct.

The Oxyma ester is highly reactive toward amines but less prone to racemization than the O-acylisourea. In the next step, the free amino group of the incoming amino acid or peptide attacks the Oxyma ester, forming the amide bond and releasing the Oxyma leaving group:

R–CO–O–Oxyma + H₂N–R' → R–CO–NH–R' + Oxyma.

Reaction scheme: Mechanism of activation and the role of a coupling additive (HOX = Oxyma).

Incorporating Oxyma in peptide synthesis suppresses racemization, especially at sensitive α-carbon centers, and speeds up coupling reactions. Oxyma is also less toxic and explosive than HOBt or HOAt, now considered a greener reagent.

Sabatino et al. (2020) reported a scalable cGMP multigram synthesis of Eptifibatide, a disulfide-bridge containing cyclopeptide. Eptifibatide is an active pharmaceutical ingredient (API) used as a antithrombotic agent for the treatment of patients with acute coronary syndrome such as unstable angina or myocardial infarct.

References

El-Faham A, Subirós Funosas R, Prohens R, Albericio F. COMU: a safer and more effective replacement for benzotriazole-based uronium coupling reagents. Chemistry. 2009 Sep 21;15(37):9404-16. [Pubmed]

Khattab, S. N.; Hexafluorophosphate (TFFH) to Replace 1-Hydroxybenzotriazole (HOBt) and 1-Hydroxy-7-azabenzotriazole (HOAt) during Peptide Synthesis. Bull. Chem. Soc. Jpn. Vol. 83, No. 11 (2010) 1374-1379. [Bulletin CSJ]

Manne SR, Sharma A, Sazonovas A, El-Faham A, de la Torre BG, Albericio F. Understanding OxymaPure as a Peptide Coupling Additive: A Guide to New Oxyma Derivatives. ACS Omega. 2022 Feb 9;7(7):6007-6023. [PMC]

Subirós-Funosas R, Prohens R, Barbas R, El-Faham A, Albericio F. Oxyma: an efficient additive for peptide synthesis to replace the benzotriazole-based HOBt and HOAt with a lower risk of explosion. Chemistry. 2009 Sep 21;15(37):9394-403. [Chemistry-europe]

Wehrstedt KD, Wandrey PA, Heitkamp D. Explosive properties of 1-hydroxybenzotriazoles. J Hazard Mater. 2005 Nov 11;126(1-3):1-7. [Sciencedirect]

Abbreviations used in organic chemistry and peptide synthesis.

Amino acids, peptides and reagents are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature (J. Biol. Chem. 1972, 247, 977). IUPAC-IUB Commission on Biochemical Nomenclature. Symbols for amino-acid derivatives and peptides. Recommendations (1971). J Biol Chem. 1972 Feb 25;247(4):977-83. PMID: 5010073. (pdf)

4-HDMA: 3-((dimethylamino)- (morpholino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridinium hexafluorophosphate 1-oxide;

6-HDMCB: 6-chloro-1-((dimethylamino)(morpholino)- methylene)-1H-benzotriazolium hexafluorophosphate 3-oxide;

6-HDMFB: 6-trifluoromethyl-1-((dimethylamino)(morpholino)methylene)-1H-benzotriazolium hexafluorophosphate 3-oxide;

Aib: R-aminoisobutyric acid;

Boc: tert-butyloxycarbonyl;

DCM: dicloromethane;

DIEA: N,N-diisopropylethylamine;

DMCH: N-(Chloro(morpholino)methylene)-Nmethylmethanaminium hexafluorophosphate;

DMF: N,N-dimethylformamide;

DMFH: N-(fluoro(morpholino)methylene)-N-methylmethanaminium hexafluorophosphate;

HAM2PipU: O-(1H-1,2,3-triazolo[4,5-b]pyridin-1-yl)-1,1-dimethyl-3,3-pentamethyleneuronium hexafluorophosphate;

HATU: N-[(dimethylamino)- 1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene)-N-methylmethanaminium hexafluorophosphate N-oxide;

HBM2PipU: O-(1H-benzotriazol1-yl)-1,1-dimethyl-3,3-pentamethyleneuronium hexafluorophosphate;

HBTU: N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]N-methylmethanaminium hexafluorophosphate N-oxide;

HDMA: 1-[(Dimethylamino)(morpholino)-methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium 3-oxide hexafluorophosphate;

HDMB: 1-((dimethylamino)(morpholino)-methylene)-1H-benzotriazolium-hexafluoro-phosphate3-oxide);

HDMPfp: 1-((dimethyamino)- (morpholino))oxypentafluorophenyl metheniminium hexafluorophosphate;

HDMS: 1-((dimethyamino)(morpholino))oxypyrrolidine-2,5-dione methanaminium hexafluorophosphate;

HDTMA: 1-((dimethylamino)(thiomorpholino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide;

HDTMB: 1-((dimethylamino)(thiomorpholino)methylene)- 1H-benzotriazolium hexafluorophosphate 3-oxide;

HOAt: 7-aza-1-hydroxybenzotriazole;

HOBt: 1-hydroxybenzotriazole;

HOPfp: pentafluorophenol;

TFA: trifluoroacetic acid;

TFFH, tetramethylfluoroformamidinium hexafluorophosphate;

TMP: 2,4,6-trimethylpyridine (collidine);

Z, benzyloxycarbonyl.

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