Journal of the American Chemical Society
Communication
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the necessary gateway for entering this emerging area of signal
transduction research.
ASSOCIATED CONTENT
* Supporting Information
■
S
Supporting schemes, tables, and figures as well as experimental
procedures and spectroscopic data for all new compounds. This
material is available free of charge via the Internet at http://
(9) (a) Amblard, M.; Fehrentz, J. A.; Martinez, J.; Subra, G. Mol.
Biotechnol. 2006, 33, 239. (b) Sabatino, G.; Papini, A. M. Curr. Opin.
Drug Discovery Dev. 2008, 11, 762.
(10) Exposure of the benzyl-protected precursor of 1 to traditional
piperidine Fmoc-deprotection conditions also resulted in complete
decomposition of the pyrophosphate moiety. See Supp. Scheme S2.
(11) A previous method (ref 6) utilized a convergent peptide
synthesis approach in which the pyrophosphoserine monomer was
coupled to N- and C-terminal peptide fragments. Given the results
obtained in our study, we presume this strategy can yield the desired
products, although with low yield due to a significant amount of cross-
reactivity and decomposition.
AUTHOR INFORMATION
Corresponding Author
■
Author Contributions
†These authors contributed equally.
Notes
The authors declare no competing financial interest.
(12) Attard, T. J.; O’Brien-Simpson, N.; Reynolds, E. C. Int. J. Pept.
Res. Ther. 2007, 13, 447.
ACKNOWLEDGMENTS
■
(13) (a) McMurray, J. S.; Coleman, D. R. T.; Wang, W.; Campbell,
M. L. Biopolymers 2001, 60, 3. (b) Beaucage, S. L.; Caruthers, M. H.
Tetrahedron Lett. 1981, 22, 1859. (c) Cremosnik, G. S.; Hofer, A.;
Jessen, H. J. Angew. Chem. Int. Ed. 2013, DOI: 10.1002/
anie.201306265.
(14) (a) Lohrmann, R.; Orgel, L. E. Nature 1973, 244, 418.
(b) Strenkowska, M.; Wanat, P.; Ziemniak, M.; Jemielity, J.; Kowalska,
J. Org. Lett. 2012, 14, 4782.
(15) For complete method optimization, see Supp. Tables S2−S4.
(16) To assess the generality of Method C, we also subjected
phosphothreonine and phosphotyrosine-containing peptides (21 and
22) to the reaction conditions. In both cases, high conversion to
pyrophosphopeptides 23 and 24, respectively, was observed. (See
Supporting Information.)
(17) Peptides 10a, 11a, and 12a were subjected to standard Pd black
hydrogenolysis conditions. While hydrogenolysis of 10a and 12a
proceeded as expected to peptides 14 and 15, repeated attempts to
hydrogenate the cysteine-containing peptide 11a were unsuccessful
presumably due to catalyst poisioning. Future work will focus on
developing alternative hydrogenolysis procedures or changing the
benzyl protecting group on the phosphorimidazolide reagent.
(18) To further validate the compatibility of Methods A−C with
alcohols, competition experiments with serine-containing peptide 25
and N-/C-protected phosphopeptide 5 were performed. We observed
80−100% conversion of 5 to pyrophosphopeptide 13 for all methods,
while serine-containing peptide 25 showed no reactivity. See
Supporting Information for experimental setup and HPLC assay.
(19) For examples of phosphate as a noninnocent, nucleophilic
buffer, see: (a) Westwood, N. J.; Schofield, C. J.; Claridge, T. D. W. J.
Chem. Soc., Perkin Trans. 1 1997, 2725. (b) Gill, M. S.; Neverov, A. A.;
Brown, R. S. J. Org. Chem. 1997, 62, 7351. For an example of
phosphate as a nucleophilic catalyst, see: (c) Prakash, G. K. S.;
Vaghoo, H.; Panja, C.; Surampudi, V.; Kultyshev, R.; Mathew, T.;
Olah, G. A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 3026.
(20) Sawai, H.; Wakai, H.; Shimazu, M. Tetrahedron Lett. 1991, 32,
6905.
(21) To further validate compatibility with an imidazole functional
group, histidine-containing peptide 26 was synthesized. Subjecting 26
to Method C yielded the desired pyrophosphopeptide 27 with 100%
conversion. (See Supporting Information.)
(22) (a) Park, H. S.; Hohn, M. J.; Umehara, T.; Guo, L. T.; Osborne,
E. M.; Benner, J.; Noren, C. J.; Rinehart, J.; Soll, D. Science 2011, 333,
1151. (b) Heinemann, I. U.; Rovner, A. J.; Aerni, H. R.; Rogulina, S.;
Cheng, L.; Olds, W.; Fischer, J. T.; Soll, D.; Isaacs, F. J.; Rinehart, J.
FEBS Lett. 2012, 586, 3716.
We thank Dr. Zachary Brown for help with peptide synthesis,
Dr. Michael Kagan from Lotus Separations for assistance with
compound purification, and Dr. John Eng for guidance with
mass spectrometry. Furthermore, we thank the Muir and
MacMillan groups for providing access to instruments and
reagents. Financial support from the NIH (R00 GM087306)
and Princeton University is gratefully acknowledged. D.F. is a
Kimmel Scholar and a Rita Allen Scholar.
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