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C O M M U N I C A T I O N S
Table 2. Modification of Lysozyme (3a) By Reductive Alkylationa
Figure 2. Conjugation of PEG to lysozyme using a simple two-step
procedure. (a) Commercially available PEG alcohols can be oxidized to
afford aldehydes before conjugation to proteins via reductive alkylation.
(b) SDS-PAGE analysis of 6 shows the formation of singly and doubly
alkylated conjugates. Control reactions run in the absence of catalyst (lane
2) or aldehyde (lane 3) afforded no conjugates. MW ladder: 25, 20, and
15 kD (from top). Lysozyme MW ) 14.3 kD.
aldehyde (4j) to remaining alcohol. The crude material was then
added directly to aqueous formate/phosphate buffer for protein
modification. In the presence of 20 µM 1b and 1 mM 4j, lysozyme
was modified to afford single and double polymer conjugates with
59% overall conversion, as determined by quantitative SDS-PAGE
analysis (Figure 2b). As was observed previously, no modification
of the protein was observed in the absence of 1b. In addition to
preparing protein conjugates with PEG polymers that are not
available as NHS-esters, we are exploring this technique for the
attachment of alternative polymers bearing primary hydroxyl groups.
In conclusion, a convenient new method has been described for
the reductive alkylation of proteins under mild conditions. The
reaction is capable of functionalizing proteins with similar efficien-
cies to NHS-ester- and sodium cyanoborohydride-based techniques,
but without the use of water-sensitive materials or harsh reagents.
As this reaction represents one of the few transition metal based
methods for bioconjugation, it also provides an important design
lead for the development of future reductive modification strategies.
a Conditions: 100 µM lysozyme, 1 mM 4b-i, 20 µM 1b, 25 mM sodium
formate, 50 mM K2HPO4, pH 7.4, 22 °C, 22 h. Product distributions were
determined using ESI-MS analysis.
indicated that the catalyst can be completely removed from protein
solutions by gel filtration.8
To date, the reaction has shown a wide tolerance for both the
protein and the aldehyde components. With respect to protein scope,
many substrates have been modified successfully, including cyto-
chrome c (3b, 19 lysines), R-chymotrypsinogen A (3c, 14 lysines),
myoglobin (3d, 19 lysines), ribonuclease A (3e, 10 lysines), and
an intact virus (bacteriophage MS2, 3f, 6 lysines per monomer)
(Table 1). Although the overall reactivity changes with the structure
of the substrate, the reaction conditions can be adjusted in each
case to achieve the desired levels of modification. Numerous
aldehydes have been screened, a selection of which are summarized
in Table 2. Although simple alkyl aldehydes (such as 4b) generally
exhibit low levels of reactivity, substrates possessing aromatic
groups have afforded the highest levels of conversion to product.
This is likely due to the enhanced stability of the imine intermediates
arising from hydrophobic interactions between these groups and
the proteins and/or catalyst.9 Electron-withdrawing substituents
generally increase the reactivity of these substrates (e.g., 4d,f,h,i),
although useful modification can still be obtained without these
functional groups (4c,e). No reaction has been observed using
ketone substrates, such as 4g or acetone (not shown). This allows
substrates bearing both a ketone and an aldehyde (such as 4h,i) to
be coupled chemoselectively, leaving the ketone for further
elaboration. After reductive alkylation with these substrates, we
have found that proteins can be further modified with alkoxyamines
to form oximes.8
Compared to lysine acylation with NHS-esters, reductive alky-
lation strategies offer several key advantages. First, the overall
charge state of the protein remains unaltered after the modification
takes place, thus minimizing changes in protein solubility. This
method also avoids competitive hydrolysis pathways that can be
problematic for some activated esters, potentially allowing less
reagent to be used. Similarly, the aldehyde feedstock materials that
are used in this technique are frequently more convenient to prepare
and store than the corresponding NHS-esters.
As an example of the latter case, we have developed a simple
two-step oxidation/reductive alkylation method for the attachment
of unfunctionalized poly(ethylene glycol) (PEG) to proteins.
Conversion of commercially available PEG alcohol 5 to the
corresponding aldehyde 4j can be accomplished conveniently
through exposure to Dess-Martin periodinane (DMP) in CH2Cl2
(Figure 2a). The resulting polymer can be isolated by precipitation
from ethyl ether, affording an unoptimized 1:1.7 mixture of
Acknowledgment. We would like to thank Dr. Arnold Falick
for his assistance with MS/MS experiments. We also acknowledge
the NIH (GM072700-01) and the DOE Nanoscale Science, Engi-
neering, and Technology Program for generous financial support,
as well as the University of California, Berkeley.
Supporting Information Available: Full experimental procedures
and characterization data are available for all compounds. This material
References
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(7) A similar reactivity trend was observed for a related Cp*Ir(phenanthrine)2+
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(8) See Supporting Information for details and spectra.
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