The comple
te primary and
three-dimensional solu
tion s
truc
tures of sub
tilosin A (
1), a bac
teriocinfrom
Bacillus subtilis, were de
termined by mul
tidimensional NMR s
tudies on pep
tide produced usingiso
topically labeled [
13C,
15N]medium derived from
Anabaena sp. grown on sodium [
13C]bicarbona
te and[
15N]ni
tra
te. Addi
tional samples of
1 were also genera
ted by separa
te incorpora
tions of [U-
13C,
15N]-
L-phenylalanine and [U-
13C,
15N]-
L-
threonine using o
therwise unlabeled media. The resul
ts demons
tra
te
tha
tin addi
tion
to having a cyclized pep
tide backbone (amide be
tween N and C
termini),
three cross-links areformed be
tween
the sulfurs of Cys13, Cys7, and Cys4 and
the
![](/images/gifchars/alpha.gif)
-posi
tions of Phe22, Thr28, and Phe31,respec
tively. The s
tereochemis
try of all residues in
1 excep
t for
the
three modified ones was confirmed
to be
L by comple
te desulfuriza
tion wi
th nickel boride, acid hydrolysis
to
the cons
ti
tuen
t amino acids,and conversion of
these
to
the corresponding pen
tafluoropropanamide isopropyl es
ters for chiral GC MSanalysis. The s
tereochemis
try a
t the modified residues was de
termined by subjec
ting each of
the eigh
tpossible s
tereoisomers of
1 to eigh
t rounds of ARIA s
truc
ture calcula
tions, s
tar
ting wi
th
the same NMRpeak files and assignmen
ts. The s
tereoisomer wi
th
the
L s
tereochemis
try a
t Phe22 (
![](/images/gifchars/alpha.gif)
-R) and
Ds
tereochemis
try a
t Thr28 (
![](/images/gifchars/alpha.gif)
-S) and Phe31 (
![](/images/gifchars/alpha.gif)
-S) (LDD isomer) fi
t the NMR da
ta, giving
the lowes
t energyfamily of s
truc
tures wi
th
the bes
t rmsd. Thus, biochemical forma
tion of
the unusual
thio links proceedswi
th ne
t re
ten
tion of configura
tion a
t Phe22, and inversion a
t Thr28 and Phe31. Model amino acidderiva
tives bearing a sulfide moie
ty a
t the
![](/images/gifchars/alpha.gif)
-carbon were syn
thesized by reac
tion of
the corresponding
![](/images/gifchars/alpha.gif)
-alkoxy compounds wi
th benzyl
thiol and SnCl
4. Separa
tion of
their pure s
tereoisomers and desulfuriza
tionwi
th nickel boride demons
tra
ted
tha
t the reduc
tion of such compounds proceeds wi
th epimeriza
tion, incon
tras
t to
the previously repor
ted re
ten
tion of s
tereochemis
try for analogous reac
tion of s
teroidal sulfides.However, desulfuriza
tion of sub
tilosin A
to cyclic pep
tide
14, which is inac
tive as an an
timicrobial agen
t,occurs wi
th inversion of s
tereochemis
try a
t the
![](/images/gifchars/alpha.gif)
-carbons of Phe22 and Thr28 and wi
th 4:1 re
ten
tion a
tPhe31. This indica
tes
tha
t the desulfuriza
tion reac
tion proceeds via an N-acyl imine and
tha
t the s
truc
tureof
the surrounding pep
tide con
trols
the geome
try of reduc
tion. Pos
ttransla
tional linkage of a
thiol
to
the
![](/images/gifchars/alpha.gif)
-carbon of an amino acid residue is unpreceden
ted in ribosomally syn
thesized pep
tides or pro
teins, andvery rare in secondary me
taboli
tes. Sub
tilosin A (
1) represen
ts a new class of bac
teriocins.