bis-tetramer, even in the presence of other constituents of the
HbN3 formation reaction. Due to the bioorthogonal10 nature
of the reactive groups, this method overcomes problems
associated with the competing hydrolysis reactions observed
in earlier approaches.
This is the first time click chemistry has been successfully
applied to the derivatization of Hb, providing a new scaffold
from which to develop future Hb-based oxygen-carriers and
other materials.11 The use of a bis-alkyne to connect two azide-
functionalized proteins in sequential cooperative click reactions
extends current protein coupling methods. This approach can
produce a wide variety of novel homodimeric protein-coupled
materials for medicinal and biological applications.
We thank NSERC Canada for support through a Strategic
Project.
Fig. 4 (a) G200 HPLC analysis of reaction mixture at 280 nm—bis-
tetramer appears at 32 min, (b) G200 HPLC analysis of authentic
bis-tetramer.
with the amount of symmetrical bb cross-linked HbN3 present
in the original mixture. Since b-lys82/b0-lys82 cross-linking
occurs in the exposed BPG binding site, the azide is readily
accessible. In contrast, a-lys99/a0-lys99 cross-linkers are
submerged in the inner folds of the protein and are therefore
much less available for reaction. The remaining non-specific
bb cross-linked material is also likely to be less accessible.
It is likely that in the case of the HbPEG1, conjugation
occurs first with the BPG–bb-Hb-positioned azide cross-linker,
followed by slower reaction with the various non-specific
bb-Hb–azides. The failure of the reaction to go to completion
is a consequence of the active Cu(I) complex not reaching the
submerged aa-Hb–azide. In the case of the bis-tetramer
synthesis, steric hindrance between the functionalized
proteins allows only for reaction between intermediate
BPG–bb-Hb–alkyne and unreacted BPG–bb-Hb–N3, with
the remaining material either unable to react with the
bis-alkyne (aa-modified) or reacting once (non-specific bb
modified) but becoming inaccessible for further reaction.
Given this specific reactivity, the bis-tetramers thus produced
are likely to be of higher homogeneity compared to that of the
starting heterogeneous HbN3 material.
Notes and references
z Band appears B10 kDa larger, rather than 5 kDa larger—this
apparent higher mass is
a typical observation when analyzing
PEGylated proteins by SDS-PAGE.12
1 For a general overview of the current status in the field of
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Purification of the 3 : 1 reaction mixture (HbN3–alkyne :Hb–Hb)
to remove bis-tetramer and subsequent reintroduction to the
reaction conditions failed to produce any further bis-tetramer,
supporting our hypothesis that the reaction had gone to
completion. HPLC analysis of the unreactive component
confirms that no further BPG–bb-HbN3 remained (ESIw).
In summary, we have produced an azide-containing
cross-linking reagent that reacts with Hb to form a mixture
of cross-linked HbN3 species. The major component of this
reaction has been shown to be b-lys82/b0-lys82 cross-linked
HbN3. This material reacts rapidly and selectively with
bivalent bis-alkyne reagent 4, in sequential and cooperative
coupling reactions, to provide almost quantitative yields of
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ꢀc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 7315–7317 | 7317