C O M M U N I C A T I O N S
butenyl and pentenyl sulfides (e.g., 7-8 and 11-12) may be
attributed to the formation of unproductive five- or six-membered
chelates, respectively (Scheme 1b).8d,12 Attempts to isolate such
species, however, were unfruitful.13
without Mg2+. Importantly, 18 was an active peptidase and not
denatured over the modification sequence.15 Biologically and
therapeutically relevant glycosylation16 and PEGylation17 were also
achieved by CM (Entries 4-7).
The results in Table 1 led us immediately to pursue Sac
incorporation into proteins. Conveniently, an efficient chemical route
to thioether modified proteins was recently developed in our
laboratory.14 The reaction of O-mesitylenesulfonylhydroxylamine
(MSH) with cysteine rapidly generates dehydroalanine which can
then be reacted with a thiol nucleophile. Application of this
methodology to a single cysteine mutant of the serine protease
subtilisin Bacillus lentus (SBL) allowed efficient incorporation of
Sac into the protein (eq 1).
Finally, efforts in genetic incorporation of allyl sulfide containing
amino acids are also underway to explore their scope as tags for
CM on proteins. Genetic installation ensures stereochemical
homogeneity of the protein backbone and allows strategic flexibility.
This approach was tested using the B834 E. coli strain, a methionine
(Met) auxotroph.4,15 Low level Sac incorporation was verified by
MS-MS analysis in a single Met mutant of Sulfolobus solfataricus
ꢀ-glycosidase expressed in Met-depleted media with Sac as Met
surrogate.15
In conclusion, we have shown that allyl sulfides are effective
substrates in aqueous CM through the use of catalyst 1. Taking
advantage of the enhanced reactivity of allyl sulfides in CM, we
were able to post-translationally modify proteins via carbon-carbon
bond formation. This work is an addition to a growing interest in
metal-mediated protein modifications18 and a new standard in
substrate sensitivity and complexity in olefin metathesis.
Acknowledgment. We gratefully acknowledge the Rhodes Trust
(J.M.C.), the International AIDS Vaccine Initiative (N.F.) and FCT,
Portugal (G.J.L.B.), for financial support.
Ready access to Sac on protein surfaces enabled us to take
advantage of the unique reactivity of allyl sulfides in CM. Initial
attempts were carried out simply by adding excess 1 and 2 to a
solution of SBL-156Sac 17 in 50 mM sodium phosphate (pH 8.0)
(Table 2). LC-MS analysis revealed largely unreacted 17, even after
prolonged reaction time. Nevertheless, we were intrigued by a
minor, yet significant, MS signal that appeared upon the addition
of 1 to 17.15 We speculated this species might be a metalloprotein
derived from metathesis with 1, inactive in CM due to nonproduc-
tive chelation of side chains to ruthenium. MgCl2 was added to the
reaction buffer with the intention of disrupting any such nonproduc-
tive chelation to ruthenium. Fu¨rstner used Ti(OiPr)4 in a similar
fashion to disrupt nonproductive chelation in RCM.12c Gratifyingly,
when MgCl2 was included in the buffer, CM with allyl alcohol
proceeded to >90% conversion at room temperature (Table 2, Entry
2).15 To verify that the effect was due to Mg2+ and not chloride,
NaCl was used as additive (Table 2, Entry 3): no CM was observed
Supporting Information Available: Full experimental details and
compound characterization. This material is available free of charge
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a Determined by LC-MS. b First hour at rt. c First 2 h at RT.
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