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Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation

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Abstract

The deuterohemin-peptide conjugate, DhHP-6 (Dh-β-AHTVEK-NH2), is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species (ROS). In this study, specific multi-site N-methylated derivatives of DhHP-6 were designed and synthesized to improve metabolic stability and intestinal absorption, which are important factors for oral delivery of therapeutic peptides and proteins. The DhHP-6 derivatives were tested for (1) scavenging potential of hydrogen peroxide (H2O2); (2) permeability across Caco-2 cell monolayers and everted gut sacs; and (3) enzymatic stability in serum and intestinal homogenate. The results indicated that the activities of the DhHP-6 derivatives were not influenced by N-methylation, and that tri-N-methylation of DhHP-6 could significantly increase intestinal flux, resulting in a two- to threefold higher apparent permeability coefficient. In addition, molecules with N-methylation at selected sites (e.g., Glu residue) showed high resistance against proteolytic degradation in both diluted serum and intestinal preparation, with 50- to 140-fold higher half-life values. These findings suggest that the DhHP-6 derivatives with appropriate N-methylation could retain activity levels equivalent to that of the parent peptide, while showing enhanced intestinal permeability and stability against enzymatic degradation. The tri-N-methylated peptide Dh-β-AH(Me)T(Me)V(Me)EK-NH2 derived from this study may be developed as a promising candidate for oral administration.

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Abbreviations

ROS:

Reactive oxygen species

APx:

Ascorbate peroxidase

PEG:

Polyethylene glycol

DMF:

N,N-dimethylformamide

HOBT:

1-Hydroxy-1H-benzotriazole

NMM:

N-methylmorpholine

HATU:

O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

HOAt:

3H-[1,2,3]-triazolo[4,5-b]pyridin-3-ol

DIEA:

N,N-diisopropylethylamine

NMP:

N-methylpyrrolidone

PyBop:

Benzotriazole-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate

TFA:

Trifluoroacetic acid

R-CHCA:

R-cyano-4-hydroxycinnamic acid

DMEM:

Dulbecco’s Modified Eagle’s Medium

TEER:

Transepithelial electrical resistance

HBSS:

Hank’s balanced salt solution

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 30901863, 20872048) and China Postdoctoral Science Foundation (No. 20110491321). We gratefully acknowledge Hui Cai and Xu Yang (Changchun BCHT Co. Ltd.) for their assistance with peptide synthesis.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. National Engineering Laboratory for AIDS Vaccine, College of Life Science, Jilin University, Qianjin Street 2699, Changchun, 130012, Jilin, People’s Republic of China

    Qing-Guang Dong, Yong Zhang, Meng-Shu Wang, Jiao Feng, Hai-Hong Zhang, Yong-Ge Wu, Tie-Jun Gu, Xiang-Hui Yu, Chun-Lai Jiang, Yan Chen, Wei Li & Wei Kong

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  1. Qing-Guang Dong
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  2. Yong Zhang
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  3. Meng-Shu Wang
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  4. Jiao Feng
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  9. Chun-Lai Jiang
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  10. Yan Chen
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  11. Wei Li
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  12. Wei Kong
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Corresponding authors

Correspondence to Yan Chen or Wei Kong.

Additional information

Q.-G. Dong and Y. Zhang contributed equally to this work.

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Dong, QG., Zhang, Y., Wang, MS. et al. Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation. Amino Acids 43, 2431–2441 (2012). https://doi.org/10.1007/s00726-012-1322-y

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