To exclude the possibility of any scrambling of carbon
bridges by ring-opening metathesis and subsequent ring
closure, LC-MS/MS analysis was done on the compound
obtained by cleavage and deprotection of tricyclic derivative
6. This confirmed the absence of crossover products. Thus,
this method is likely to be generally useful for construction
of multiple carbocyclic ring peptides on solid support.
Attempted reduction of olefins on resin-bound 6 using
diimide and Wilkinson’s catalyst gave only the Fmoc-
deprotected 6 and none of the saturated tricycle.
Scheme 2. Solution Synthesis of the Unusual N-Terminus
Sequence
Continuation of SPPS introduced residues (10f15). As
the polymer support turned dark due to repeated exposure
to the RCM catalyst, colorimetric Kaiser tests7 to monitor
the peptide coupling could no longer be used. Consequently,
each Fmoc-deprotection step was monitored using UV at 301
nm8 and the completion of acylation steps was determined
by mass spectrometry. To avoid aggregation caused by nine
hydrophobic side chains in the section of residues 6-15,
N-methylpyrrolidinone (NMP)9 was used as the solvent to
successfully yield 7.
Residues 1-5 of lacticin A2 (1) contain two dehydrobu-
tyrine (Dhb) residues and an N-terminal R-ketoamide as a
result of enzymatic post-translational modification.2,6 Al-
though methods to prepare dehydrobutyrines on solid support
have been reported,10 we chose solution-phase synthesis of
the pentapeptide containing these unusual residues (Scheme
2). This allows facile control of scale as well as use of this
fragment in other syntheses of 1 and its analogues. The
dehydroamino acid moieties in both 8 and 9 were introduced
at the dipeptide stage by treating the corresponding threonine
derivatives with MsCl/DBU11 to obtain the dehydrobutyrine
dipeptides. In both instances, this elimination reaction gave
only the Z stereoisomer, as confirmed by NOE experiments.
DIPCDI/HOBt proved to be the best reagents for the difficult
coupling of the relatively unreactive carboxyl group of
dehydrodipeptide 9 with the secondary amino group of
proline obtained after deprotection of 8. The coupling
proceeded to give 10 in 76% isolated yield. The N-terminal
amino group of 10 was liberated with TFA and then treated
with 4-pyridinecarboxaldehyde acetate salt and DBU.12
Aqueous acidic workup led to transamination with hydrolysis
akin to that catalyzed by pyridoxal phosphate13 and produced
peptide 11 having the desired R-ketoamide with the dehy-
drobutyrines intact. The O-allyl protecting group could be
removed with Pd(PPh3)4 to yield 12 as a single stereoisomer.
To complete the synthesis of 2, the pentapeptide fragment
12 was coupled to the N-terminus of peptide 7 on solid
support (proline, residue 6). This difficult coupling was again
done in the presence of DIPCDI/HOBt (Scheme 3). Cleavage
Scheme 3. Completion of the Synthesis of the Larger Ring
Analogue 2
(5) (a) Ghalit, N.; Rijkers, D. T. S.; Liskamp, R. M. J. J. Mol. Catal.
A-Chem. 2006, 254, 68-77. (b) Stymiest, J. L.; Mitchell, B. F.; Wong,
S.; Vederas, J. C. J. Org. Chem. 2005, 70, 7799-7809. (c) Whelan, A. N.;
Elaridi, J.; Mulder, R. J.; Robinson, A. J.; Jackson, W. R. Can. J. Chem.
2005, 83, 875-881. (d) Elaridi, J.; Patel, J.; Jackson, W. R.; Robinson, A.
J. J. Org. Chem. 2006, 71, 7538-7545. (e) Derksen, D. J.; Stymiest, J. L.;
Vederas, J. C. J. Am. Chem. Soc. 2006, 128, 14252-14253.
(6) For a discussion of oxidative instability of lantibiotics, see: Martin,
N. I.; Sprules, T.; Carpenter, M. R.; Cotter, P. D.; Hill, C.; Ross, R. P.;
Vederas, J. C. Biochemistry 2004, 43, 3049-3056.
of 2 from the resin was achieved using (97.5:2.5) TFA/TIPS.
Water was omitted from the cleavage cocktail, as the dehydro
residues are susceptible to Michael addition by water under
strongly acidic conditions.14 Purification of 2 by reverse-
phase HPLC, followed by analysis using MALDI-TOF MS,
revealed a major peak at 2801.6 Da, corresponding to the
(7) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595-598.
(8) Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2002, 9, 203-206.
(9) Synthesis Notes 3.5. NoVaBiochem Catalog; EMD Biosciences: San
Diego, CA, 2006-2007.
(10) Bonauer, C.; Walenzyk, T.; Konig, B. Synthesis-Stuttgart 2006,
1-20.
(11) Shin, C.-G.; Yonezawa, Y.; Takahashi, M.; Yoshimura, J. Bull.
Chem. Soc. Jpn. 1981, 54, 1132-1136.
(12) Ohta, S.; Okamoto, M. Synthesis-Stuttgart 1982, 756-758.
(13) Eliot, A. C.; Kirsch, J. F. Annu. ReV. Biochem. 2004, 73, 383-415.
(14) Humphrey, J. M.; Chamberlin, A. R. Chem. ReV. 1997, 97, 2243-
2266.
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