Analysis of functionalization of methoxy-PEG as maleimide-PEG

K. Ananda, Parimala Nacharaju, Paul K. Smith, Seetharama A. Acharya, Belur N. Manjula

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The design of the extension arm-facilitated PEGylation (EAFP) of proteins takes advantage of the high selective and quantitative aspects of the thiol-maleimide reaction. However, the efficiency of EAFP with hemoglobin varied with the batches of maleimide-PEG. The low level of functionalization of monomethoxy-PEG (mPEG) as maleimide-PEG has been now investigated as the potential source of this variation. New chemical approaches for the estimation of the functionalization of mPEG using the reaction of the thiol groups of glutathione, dithiothreitol, and hemoglobin with maleimide-PEG have been developed. The single-step modular approach to the synthesis of maleimidophenyl-PEG (MPPEG) that involved the condensation of p-maleimidophenyl isocyanate with mPEG has been optimized to generate a product with an overall purity of 80%. The NMR approach correlates well with the estimates made by the new chemical approaches. Commercial maleimide-PEG reagents synthesized using multiple steps exhibited a lower level of functionalization as reflected by these chemical estimations. The better functionalization of MPPEG increases the efficiency of EAFP as reflected by the generation of hexaPEGylated Hb and the masking of the D antigen of RBCs. This new EAFP protocol is expected to improve the cost effectiveness of the generation of hexaPEGylated Hb, PEGylated albumin, and PEGylated RBCs as new PEGylated therapeutics.

Original languageEnglish (US)
Pages (from-to)231-242
Number of pages12
JournalAnalytical Biochemistry
Volume374
Issue number2
DOIs
StatePublished - Mar 15 2008

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Polyethylene glycols
Sulfhydryl Compounds
Hemoglobins
Isocyanates
Dithiothreitol
Cost-Benefit Analysis
Glutathione
Albumins
maleimide
Antigens
Cost effectiveness
Condensation
Nuclear magnetic resonance
Proteins
Therapeutics

Keywords

  • Chromatography
  • EAF PEGylation
  • Functionalization
  • Maleimidophenyl-PEG
  • Plasma volume expander

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Analysis of functionalization of methoxy-PEG as maleimide-PEG. / Ananda, K.; Nacharaju, Parimala; Smith, Paul K.; Acharya, Seetharama A.; Manjula, Belur N.

In: Analytical Biochemistry, Vol. 374, No. 2, 15.03.2008, p. 231-242.

Research output: Contribution to journalArticle

Ananda, K. ; Nacharaju, Parimala ; Smith, Paul K. ; Acharya, Seetharama A. ; Manjula, Belur N. / Analysis of functionalization of methoxy-PEG as maleimide-PEG. In: Analytical Biochemistry. 2008 ; Vol. 374, No. 2. pp. 231-242.
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