Enzyme-Primed Native Chemical Ligation Produces Autoinducing Cyclopeptides in Clostridia.

Clostridia coordinate many important processes such as toxin production, infection, and survival by density-dependent communication (quorum sensing) using autoinducing peptides (AIPs). Although clostridial AIPs have been proposed to be (thio)lactone-containing peptides, their true structures remain elusive. Here, we report the genome-guided discovery of an AIP that controls endospore formation in Ruminiclostridium cellulolyticum. Through a combination of chemical synthesis and chemical complementation assays with a mutant strain, we reveal that the genuine chemical mediator is a homodetic cyclopeptide (cAIP). Kinetic analyses indicate that the mature cAIP is produced via a cryptic thiolactone intermediate that undergoes a rapid S→N acyl shift, in a manner similar to intramolecular native chemical ligation (NCL). Finally, by implementing a chemical probe in a targeted screen, we show that this novel enzyme-primed, intramolecular NCL is a widespread feature of clostridial AIP biosynthesis.

Surprisingly Different Reaction Behavior of Alkali and Alkaline Earth Metal Bis(trimethylsilyl)amides toward Bulky N-(2-Pyridylethyl)-N'-(2,6-diisopropylphenyl)pivalamidine.

N-(2,6-Diisopropylphenyl)-N'-(2-pyridylethyl)pivalamidine (Dipp-N=C(tBu)-N(H)-C2 H4 -Py) (1), reacts with metalation reagents of lithium, magnesium, calcium, and strontium to give the corresponding pivalamidinates [(tmeda)Li{Dipp-N=C(tBu)-N-C2 H4 -Py}] (6), [Mg{Dipp-N=C(tBu)-N-C2 H4 -Py}2 ] (3), and heteroleptic [{(Me3 Si)2 N}Ae{Dipp-N=C(tBu)-N-C2 H4 -Py}], with Ae being Ca (2 a) and Sr (2 b). In contrast to this straightforward deprotonation of the amidine units, the reaction of 1 with the bis(trimethylsilyl)amides of sodium or potassium unexpectedly leads to a ß-metalation and an immediate deamidation reaction yielding [(thf)2 Na{Dipp-N=C(tBu)-N(H)}] (4 a) or [(thf)2 K{Dipp-N=C(tBu)-N(H)}] (4 b), respectively, as well as 2-vinylpyridine in both cases. The lithium derivative shows a similar reaction behavior to the alkaline earth metal congeners, underlining the diagonal relationship in the periodic table. Protonation of 4 a or the metathesis reaction of 4 b with CaI2 in tetrahydrofuran yields N-(2,6-diisopropylphenyl)pivalamidine (Dipp-N=C(tBu)-NH2 ) (5), or [(thf)4 Ca{Dipp-N=C(tBu)-N(H)}2 ] (7), respectively. The reaction of AN(SiMe3 )2 (A=Na, K) with less bulky formamidine Dipp-N=C(H)-N(H)-C2 H4 -Py (8) leads to deprotonation of the amidine functionality, and [(thf)Na{Dipp-N=C(H)-N-C2 H4 -Py}]2 (9 a) or [(thf)K{Dipp-N=C(H)-N-C2 H4 -Py}]2 (9 b), respectively, are isolated as dinuclear complexes. From these experiments it is obvious, that ß-metalation/deamidation of N-(2-pyridylethyl)amidines requires bases with soft metal ions and also steric pressure. The isomeric forms of all compounds are verified by single-crystal X-ray structure analysis and are maintained in solution.

Solid-Phase-Based Total Synthesis and Stereochemical Assignment of the Cryptic Natural Product Aurantizolicin.

The total synthesis and stereochemical assignment of the polyazole cyclopeptide aurantizolicin was achieved by connecting the solution synthesis of building blocks with solid-phase peptide synthesis. Macrothiolactonization and an aza-Wittig reaction provided the natural product macrocycle in high yield as well as key stereoisomers. NMR comparison as well as isolation of the natural product from the producer organism Streptomyces aurantiacus confirmed the presence and sequence of one l-Ile and one d- allo-Ile residue in aurantizolicin.

Synthesis and catalytic activity of tridentate N-(2-pyridylethyl)-substituted bulky amidinates of calcium and strontium.

Metalation of the formamidine Dipp-N[double bond, length as m-dash]C(H)-N(H)-C2H4-Py (1a) and benzamidine Dipp-N[double bond, length as m-dash]C(Ph)-N(H)-C2H4-Py (1b) with [(thf)2M{N(SiMe3)2}2] (M = Ca, Sr) yields the corresponding homoleptic complexes [M{Dipp-N[double bond, length as m-dash]C(R)-N-C2H4-Py}2] (M/R = Ca/H (2a), Ca/Ph (2b), and Sr/Ph (3b)) regardless of the applied stoichiometry. Only during calciation of Dipp-N[double bond, length as m-dash]C(H)-N(H)-C2H4-Py (1a), the heteroleptic intermediate [{(Me3Si)2N}Ca{Dipp-N[double bond, length as m-dash]C(R)-N-C2H4-Py}]2 (2a') has been observed. The formamidinate complex of strontium crystallizes as a tmeda adduct of the type [(tmeda)Sr{Dipp-N[double bond, length as m-dash]C(H)-N-C2H4-Py}2] (3a). Metalation of the pivalamidine Dipp-N[double bond, length as m-dash]C(tBu)-N(H)-C2H4-Py (1c) leads to the formation of heteroleptic mononuclear [{(Me3Si)2N}M{Dipp-N[double bond, length as m-dash]C(tBu)-N(H)-C2H4-Py}] (M = Ca (2c) and Sr (3c)) with a side-on bonding of the Dipp group to the alkaline earth metals. Calciation of chiral Dipp-N[double bond, length as m-dash]C(tBu)-N(H)-CH2CH(Me)-Py (R)-1d and (S)-1d with [(thf)2Ca{N(SiMe3)2}2] yields the homoleptic complexes [Ca{Dipp-N[double bond, length as m-dash]C(tBu)-N-CH2CH(Me)-Py}2] with the enantiomeric forms (R,R)-2d and (S,S)-2d regardless of the applied stoichiometry. The complexes 2c and 2d catalyze the intramolecular hydroamination of the aminoalkene 2,2-diphenylpent-4-enylamine yielding 2-methyl-4,4-diphenylpyrrolidine but the stereochemistry cannot be influenced by the chiral compounds (R,R)-2d and (S,S)-2d.