Amino Acids

, Volume 43, Issue 6, pp 2431–2441

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

  • 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
Original Article

DOI: 10.1007/s00726-012-1322-y

Cite this article as:
Dong, QG., Zhang, Y., Wang, MS. et al. Amino Acids (2012) 43: 2431. doi:10.1007/s00726-012-1322-y

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.

Keywords

DhHP-6 ROS N-methylated peptide Permeability Enzymatic stability 

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

Supplementary material

726_2012_1322_MOESM1_ESM.doc (1.8 mb)
Supplementary material 1 (DOC 1808 kb)

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Qing-Guang Dong
    • 1
  • Yong Zhang
    • 1
  • Meng-Shu Wang
    • 1
  • Jiao Feng
    • 1
  • Hai-Hong Zhang
    • 1
  • Yong-Ge Wu
    • 1
  • Tie-Jun Gu
    • 1
  • Xiang-Hui Yu
    • 1
  • Chun-Lai Jiang
    • 1
  • Yan Chen
    • 1
  • Wei Li
    • 1
  • Wei Kong
    • 1
  1. 1.National Engineering Laboratory for AIDS Vaccine, College of Life ScienceJilin UniversityChangchunPeople’s Republic of China

Personalised recommendations