Synthesis of microcin SF608

Article abstract of DOI:10.1021/jo025709y

The first total synthesis of aquatic peptide microcin SF608 is described. Coupling of L-Hpla with the dipeptide L-Phe-L-Choi followed by coupling with agmatine and a deprotection step gave microcin SF608. In addition, the levorotatory character of L-Hpla (5) was thoroughly established, and the conformational analysis of L-Choi containing peptides 1 and 8-10 was performed using NMR spectroscopy to examine the cis-trans isomer equilibrium of the L-Phe-L-Choi amide bond.

Full text of DOI:10.1021/jo025709y

Syn th esis of Micr ocin SF 608
Nativitat Valls,* Merce` Vallribera, Meritxell Lo´pez-Canet, and J osep Bonjoch*
Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028-Barcelona, Spain
bonjoch@farmacia.far.ub.es
Received March 12, 2002
The first total synthesis of aquatic peptide microcin SF608 is described. Coupling of L-Hpla with
the dipeptide L-Phe-L-Choi followed by coupling with agmatine and a deprotection step gave microcin
SF608. In addition, the levorotatory character of L-Hpla (5) was thoroughly established, and the
conformational analysis of L-Choi containing peptides 1 and 8-10 was performed using NMR
spectroscopy to examine the cis-trans isomer equilibrium of the L-Phe-L-Choi amide bond.
In tr od u ction
Many peptides of aquatic origin involving unusual
amino acids have interesting biological activities as well
as unique structures.1 Microcin SF608 was isolated in
Israel from a nontoxic strain of the cyanobacterium
Microcystis aeruginosa and inhibited trypsin with IC50
of 0.5 µg/mL, but not chymotrypsin and neprolysine at
20.0 µg/mL.2 The peptide microcin SF608 is made up of
the two amino acids L-phenylalanine (L-Phe) and the
unusual 2-carboxy-6-hydroxyoctahydroindole (Choi), the
R-hydroxy acid p-hydroxyphenyllactic acid (L-Hpla), and
agmatine (Agma), the decarboxylated arginine (Figure
1). This tetraunit structure resembles that of aerugi-
nosins, a group of aquatic peptides isolated in J apan3 for
which we have recently established a synthetic entry,
having achieved the first total synthesis and reassigned
the structure of aeruginosins 298.4,5 Interestingly, mi-
crocin SF608 (1) has the L configuration in the R-amino
acid attached to the nitrogen of the Choi unit, while
aeruginosins incorporate a D-amino acid at this point.4b,6
Moreover, the Hpla fragment in 1 is also of the L
configuration, in contrast to the D-Hpla derivatives found
in aeruginosins.
F IGURE 1. Subunits of the aquatic peptide microcin SF608.
Resu lts a n d Discu ssion
A P r elim in a r y Qu estion : Th e Hyd r oxyp h en yl-
la ctic Acid (Hp la ). As a prelude to the requisite
coupling reactions for the synthesis of microcin SF608,
we needed the corresponding four subunits (Figure 1).
The preparation of L-Hpla merits some comments, since
there is some confusion in the literature about the
correlation of the nature (d or l) of the rotatory power
with the absolute configuration of L-Hpla. The diprotected
form of the L-isomer of p-hydroxyphenyllactic acid [AcO-
L-Hpla(Bn)-OH] (2), required for the synthetic plan, was
synthesized from O-benzyl-L-tyrosine (Scheme 1) through
a diazotation process, using isoamyl nitrite in AcOH
medium,7 which allows the formation of the protected
form of L-Hpla in only one step. The ee of L-Hpla, which
was 70% after the acetoxylation process, was increased
up to >98% after a crystallization process with (-)-1-
phenylethylamine. The retention of the S configuration
in 2, due to the participation of the neighboring carboxyl
group in the substitution process, was additionally
confirmed by converting 2 into the methoxyphenylacetic
(MPA) ester derivative 38 and deducing the absolute
In this paper we describe the first total synthesis of
microcin SF608 that serves to confirm the structural
assignment of 1 and establishes its absolute configura-
tion.
(1) For an overview: Shioiri, T.; Hamada, Y. Synlett 2001, 184-
201.
(2) (a) Banker, R.; Carmeli, S.Tetrahedron 1999, 55, 10835-10844.
(b) For a new isolation, see: Reshef, V.; Carmeli, S. Tetrahedron 2001,
57, 2885-2894.
(3) Ishida, K.; Okita, H.; Matsuda, H.; Okino, T.; Murakami, M.
Tetrahedron. 1999, 55, 10971-10988.
(4) (a) Valls, N.; Lo´pez-Canet, M.; Vallribera, M.; Bonjoch, J .
J . Am. Chem. Soc. 2000, 122, 11248-11249. (b) Valls, N.; Lo´pez-
Canet, M.; Vallribera, M.; Bonjoch, J . Chem. Eur. J . 2001, 7, 3446-
3460.
(5) For another synthesis of aeruginosin 298-A, see: Wipf, P.;
Methot, J .-L.Org. Lett. 2000, 2, 4213-4216.
(6) For the influence of phenylalanine configuration on Phe-Ψ-Pro
thrombin inhibitors, see Wagner, J .; Kallen, J .; Ehrhardt, C.; Evenou,
J .-P.; Wagner, D. J . Med. Chem. 1998, 41, 3664-3674.
1
configuration of the secondary alcohol from its H NMR
spectra recorded at different temperatures according to
(7) For the use of isoamyl nitrite in acetic medium for the formation
of R-acetoxy acids from R-amino acids, see: Schmidt, U.; Kroner, M.;
Griesser, H. Synthesis 1989, 832-835.
10.1021/jo025709y CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/07/2002
J . Org. Chem. 2002, 67, 4945-4950
4945

Valls et al.
phenyl)lactic acid and sodium (2S)-(p-hydroxyphenyl)-
lactate, respectively. Moreover the correlation of the
absolute configuration of L-Hpla with its levorotatory
character affords a complementary procedure to that of
the l-menthol method for the structural elucidation of
Hpla-containing natural products.12-14
Syn th esis of Micr ocin SF 608. To assemble the
structure of microcin SF608, having prepared Hpla
derivative 2, we needed the other subunits with the
adequate protecting groups for a peptide synthesis. The
protected agmatine 6 was prepared by guanidinylation
of 1,4-diaminobutane with N,N′-di-tert-butoxycarbonyl
thiourea following the protocol described in the litera-
ture.15 The synthesis of N-Boc-L-Choi(OMe) (7) was
carried out in six steps starting from O-methyl-L-tyrosine
according to our recently described methodology.4b After
obtaining the four units L-Hpla, L-Phe, L-Choi, and Agma
in an appropriate protected form, the synthesis of mi-
crocin SF608 was carried out as outlined in Scheme 2.
F IGURE 2. sp and ap conformations corresponding to the
(R)-MPA ester 3.
SCHEME 1. Syn th esis of L-Hp la
Removal of the Boc group from L-Choi 7 followed by
coupling to Boc-Phe-OH, using PyBOP in the presence
of NMM, gave dipeptide 8 as a separable mixture of
alcohol 8a and its trifluoroacetate 8b in 90% combined
yield. Boc group removal from 8 followed by coupling with
L-Hpla in the same reaction conditions gave peptide 9.
Although peptides 8 and 9 were isolated as a mixture of
compounds with both free and trifluoroacetylated Choi
hydroxyl groups, it did not constitute a synthetic problem,
and the analytical process was only slightly longer since
both compound-types can be easily separated.
Riguera’s method.9 The simplified Riguera protocol relies
on the thermodynamic preference for the sp conformer
rather than the ap conformation associated with MPA
esters (Figure 2). At low temperatures the population of
the sp conformer is increased, resulting in either a
downfield shift [0.07 ppm for the methyl protons of the
methoxycarbonyl group in 3 not shielded by the aryl ring
of (R)-MPA] or an upfield shift (0.04 ppm for the shielded
aromatic protons of Hpla in 3) relative to the observed
shift of the same protons at room temperature.
Having ensured the absolute configuration of 2, we
converted it into L-Hpla, (S)-2-hydroxy-3-(4-hydroxyphe-
nyl)propionic acid (5) (two steps: saponification and
hydrogenolytic cleavage), enabling us to conclude that
L-Hpla is the levorotatory enantiomer. This result allows
us to clarify some mistakes that have appeared in the
literature;10,11 it now being clear that the structure of
natural products isolated from Pterocarpus Marsupium10b
and Aristolochia kaemferi10c should be revised and their
absolute configuration reassigned as (2S)-3-(p-hydroxy-
Saponification of the methyl ester in 9, which induces
concomitant deprotection of the secondary alcohol hy-
droxyl groups, followed by coupling with di-Boc-agmatine
(6) furnished 10. Cleavage of the orthogonal protecting
groups in two high-yielding steps afforded the target
compound 1. The synthetic microcin SF608 coeluted with
authentic material (supplied by Prof. Carmeli) by re-
versed phase analytical HPLC (60:40 H2O/CH3CN; 0.05%
1
TFA), and the H and 13C NMR spectra of the samples
in DMSO were identical,2a although a slight reassign-
ment should be carried out.16
(11) (a) The dextrorotatory isomer of p-hydroxyphenyllactic acid is
of course the R isomer, which agrees with the results reported by
Satake11b and ourselves.4 Unfortunately, the prestigious Beilstein
Institut has wrongly introduced Satake’s data, by including it in the
section corresponding to the S isomer. (b) Satake, T.; Kamiya, K.; Saiki,
Y.; Hama, T.; Fujimoto, Y.; Kitanaka, S.; Kimura, Y.; Uzawa, J .;
Endang, H.; Umar M. Chem. Pharm. Bull. 1999, 47, 1444-1447.
(12) (a) The l-menthol method12b (retention time of menthyl esters
of Hpla in HPLC) has been used to determine the absolute configu-
ration of the Hpla moiety in several structural elucidation of natural
products. (b) Tsukamoto, S.; Painuly, P.; Young, K. A.; Yang, X.;
Shimizu, Y.; Cornell, L. J . Am. Chem. Soc. 1993, 115, 11046-11047.
(13) Murakami has described the synthesis of D- and L-Hpla from
the corresponding p-aminophenylanalines with nearly 30% yield in
each case.3,14 Their absolute configuration was established by the
Shimizu method,12 and there are no comments about the rotatory
power of these enantiomers.
(8) L-Hpla (2) was esterified at room temperature (MeOH/HCl) in
quantitative yield. The resulting hydroxy ester was coupled with (R)-
(-)-2-phenyl-2-methoxyacetic acid in THF using HOBt, pyridine and
DCC, according to the experimental procedure reported in ref 9. The
purified compound 3 (Florisil, hexane/EtOAc 2:1) showed the following
1H NMR data (300 MHz, CDCl3): 2.96 (dd, J ) 13.4, 8.4 Hz, 1H), 3.50
(dd, J ) 14, 4.4 Hz, 1H), 3.379 (s, 3H), 3.71 (s, 3H), 4.83 (s, 1H), 5.01
(s, 2H), 5.21 (dd, J ) 8.5, 4 Hz, 1H), 6.73 (d, J ) 8.7 Hz, 2H), 6.85 (d,
J ) 8.7 Hz, 2H), 7.30-7.42 (m, 10H); (300 MHz, CDCl3) (-50 °C): 3.01
(m, 2H), 3.386 (s, 3H), 3.77 (s, 3H), 4.90 (s, 1H), 5.00 (s, 2H), 5.20 (dd,
J ) 7.9, 4.6 Hz, 1H), 6.72 (d, J ) 8.8 Hz, 2H), 6.81 (d, J ) 8.7 Hz, 2H),
7.35-7.47 (m, 10H).
(14) Matsuda, H.; Okino, T.; Murakami, M.; Yamaguchi, K. Tetra-
hedron 1996, 52, 14501-14506.
(9) Latypov, S. K.; Seco, J .-M.; Quinoa´, E.; Riguera, R. J . Am. Chem.
Soc. 1998, 120, 877-882.
(15) (a) Expo´sito, A.; Ferna´ndez-Sua´rez, M.; Iglesias, T.; Mun˜oz, L.;
Riguera, R. J . Org. Chem. 2001, 66, 4206-4213. (b) Iwanovicz, E. J .;
Poss, M. A.; Lin, J . Synth. Commun. 1993, 23, 1443-1445.
(16) The NMR data reported for the methylene carbons at C-4 and
C-5 of the Choi nucleus in the isolation of microcin SF6082a should be
reassigned: H-4 (δ 1.40 and 2.03 for the trans rotamer and δ 1.32 and
1.92 for the cis rotamer, all of them as multiplets) and H-5 (δ 1.45 for
both protons in each rotamer as a multiplet signal); C-4 (δ 19.1) and
C-5 (δ 26.1) in both rotamers.
(10) (a) Maurya10b and Wu10c wrongly reported that the configuration
of the levorotatory isomer of 3-p-hydroxyphenyllactic was 2R, and
Hattori10d erroneously claimed that the dextrorotatory isomer was 2S.
(b) Maurya, R.; Ray, A. B.; Duah, F. K.; Slatkin, D.-J .; Schiff, P. J ., J r.
J . Nat. Prod. 1984, 47, 179-181. (c) Wu, T.-S.; Leu, Y.-L.: Chan, Y.-
Y. Chem. Pharm. Bull. 1998, 46, 6, 1624-1626. (d) Hattori, K.;
Takahashi, K. Supramol. Chem. 1993, 2, 209-213.
4946 J . Org. Chem., Vol. 67, No. 14, 2002

Synthesis of Microcin SF608
SCHEME 2. Syn th esis of Micr ocin SF 608 (1)
TABLE 1. P r oton Ch em ica l Sh ifts Va lu es of L-Ch oi
Der iva tives (tr a n s-cis Rota m er s). Com p a r ison betw een
Micr ocin SF 608 Ser ies a n d Aer u gin osin s 298
cates the presence of two predominant conformations
both in CDCl3 and DMSO-d6 in a variable ratio (see
Experimental Section). The assigned isomer geometry
was based on NOESY experiments, and the chemical
shift values for the signals of H-2 and H-7a of L-Choi. In
H-2
trans
H-7a
trans
compound
cis
cis
peptide type
1
the H NMR spectra of 1 and its precursors (8-10) the
1
8a
9a
4.23
4.42
4.26
4.17
4.12
3.90
3.34
3.68
4.63
4.64
4.41
4.42
4.42
4.04
4.02
4.09
4.30
4.14
4.28
4.27
L-Phe-L-Choi
L-Phe-L-Choi
L-Phe-L-Choi
D-Leu-L-Choi
D-Leu-L-Choi
H-2 and H-7a protons appear more deshielded in the
trans isomers than in the cis isomers (e.g., δ 4.23 and
4.41 for trans rotamer and δ 3.90 and 4.09 for cis rotamer
in 1) (Table 1).
298-A4b
298-B4b
The configuration of Xaa-L-Choi has a significant
influence on the chemical shifts of H-2 and H-7a of Choi.
When the configuration is L, as in microcin SF608, a
steric compression of the side chain of Xaa upon the Choi
ring (specially at H-7a) is present in the trans rotamer
(Figure 3) while when the configuration is D, as in the
aeruginosins, the compression (specially at H-2) appears
in the cis rotamers. Consequently, in the microcin SF608
series18 H-7a is downfield in the trans rotamers while in
the aeruginosin series H-2 is downfield in the cis rota-
mers, which agrees with the D configuration of the amino
acid attached to the L-Choi.19 Thus, taking into account
all data reported so far about L-Choi containing peptides,
the NMR signals of H-2 and H-7a of L-Choi can be used
to determine the configuration of the core dipeptide of
natural compounds that embody the L-Choi unit as well
as to assign the cis-trans rotamers.20
F IGURE 3. Amide isomer equilibrium of L-Choi derivatives
(cf. Table 1).
Both synthetic and natural microcin SF608 show two
peaks in their analytical HPLC profiles, in an ap-
proximate ratio of 3:1, and show two set of signals in their
NMR spectra. These data are consistent with the exist-
ence of slow cis-trans rotational isomerism around the
peptide bond preceding Choi (L-Phe-L-Choi). Such rota-
tional isomerism is not commonly observed in chroma-
tography, although the phenomenon is not without prece-
dent, particularly for tertiary amide-containing pep-
tides.17 Rotational isomerism is, however, common enough
on the time scale of NMR experiments and is often ob-
served in spectroscopic studies on peptides and proteins.
NMR Stu d ies a bou t th e Am id e Isom er Equ ilibr i-
u m of th e L-P h e-L-Ch oi Bon d . Several interesting
observations emerge from the 1H NMR data recorded
from L-Choi derivatives 8-10 and microcin SF608 (1).
High-field NMR spectroscopy of these compounds indi-
In summary, the first total synthesis of microcin SF608
has been reported, and its NMR data have been com-
pared with those of aeruginosins to give the NMR
patterns for aquatic peptides containing the L-Choi motif.
(18) The anistrotopic effects of the aromatic ring of the phenylala-
nine residue undoubtedly also contribute to the low chemical shift for
H-2 Choi in the cis rotamers in microcin and its precursors. This
phenomenon has been precedented in the proline series: Poznanski,
J .; Ejchart, A.; Wierzchowski, K. L.; Ciurak, M. Biopolymers 1993, 33,
781-795.
(19) For steric effects on the amide isomer equilibrium of prolyl
peptides, see: (a) Beausoleil, E.; Lubell, W. D. J . Am. Chem. Soc. 1996,
118, 12902-12908. (b) Keller, M.; Sager, C.; Dumy, P.; Schutkowski,
M.; Fischer, G. S.; Mutter, M. J . Am. Chem. Soc. 1998, 120, 2714-
2720. (c) Halab, L.; Lubell, W. D. J . Org. Chem. 1999, 64, 3312-3321.
(d) Halab, L.; Lubell, W. D. J . Am. Chem. Soc. 2002, 124, 2474-2484.
(20) For the influence of chirality of the preceding acyl moiety on
the cis/ trans ratio of the proline peptide bond, see: Breznik, M.;
Grdadolnik, S. G.; Giester, G.; Leban, I.; Kikelj, D. J . Org. Chem. 2001,
66, 7044-7050. See also ref 19d.
(17) J ou, G.; Gonza´lez, I.; Albericio, F.; Lloyd-Williams, P.; Giralt,
E. J . Org. Chem. 1997, 62, 354-366.
J . Org. Chem, Vol. 67, No. 14, 2002 4947

Valls et al.
14.2, 4 Hz, 1H), 4.36 (dd, J ) 7.0, 4.4 Hz, 1H), 5.04 (s, 2H),
6.91 (d, J ) 8.8 Hz, 2H), 7.18 (d, J ) 8.8 Hz, 2H), 7.41 (m,
5H), 13C NMR (50 MHz, CDCl3+CD3OD): 40.7 (t), 70.8 (t), 72.8
(d), 115.6 (d), 128.5 (d), 128.7 (d), 129.4 (d), 131.0 (s), 131.5
(d), 138.8 (s), 158.9 (s), 177.2 (s). Anal. Calcd for C16H16O4‚1/
2H2O: C 68.30, H 6.05. Found: C 68.22, H 6.24.
Exp er im en ta l Section
Gen er a l. All reactions were carried out under an argon
atmosphere with dry, freshly distilled solvents under anhy-
drous conditions. Unless otherwise noted, analytical thin-layer
chromatography was performed on SiO2 (silica gel 60 F254), and
the spots were located with iodoplatinate reagent or 1%
aqueous KMnO4. Chromatography refers to flash chromatog-
raphy and was carried out on SiO2 (silica gel 60, SDS, 230-
240 mesh ASTM). Drying of organic extracts during workup
of reactions was performed over anhydrous Na2SO4. Evapora-
tion of solvent was accomplished with a rotatory evaporator.
Chemical shifts of 1H and 13C NMR spectra are reported in
ppm downfield (δ) from Me4Si. The 13C NMR spectra, when
unambiguous assignation was not available, are reported as
follows: chemical shift (multiplicity determined from DEPT
spectra). Only noteworthy IR absorptions (cm-1) are listed.
Melting points were determined in a capillary tube. Mi-
croanalyses and HRMS were performed by the Centro de
Investigacio´n y Desarrollo (CSIC), Barcelona. Reversed-phase
HPLC was performed using a Waters Nova-Pak C18 (15 × 0.39
cm, 4 µm, 60 Å) column and detection was by ultraviolet
absorption at 238 nm. Abbreviations: NMM, (N-methylmor-
pholine); PyBOP (benzotriazol-1-yloxy)tripyrrolidinophospho-
nium hexafluorophosphate.
(S)-2-Hydr oxy-3-(4-h ydr oxyph en yl)pr opion ic Acid [(-)-
â-(4-Hyd r oxyp h en yl)la ctic Acid , l-Hp la ] (5). Pd/C (10%, 12
mg) was added to a solution of 4 (63 mg, 0.23 mmol) in EtOAc/
MeOH 1% (1 mL), and the mixture was stirred under atmo-
spheric hydrogen pressure for 20 h. The catalyst was removed
by filtration through a short pad of Celite, which was washed
with MeOH (15 mL). The filtrate and washings were concen-
trated to give L-Hpla 5 (30 mg, 70%) as a white solid, which
was recrystrallized with CHCl3/MeOH (9:1) to give white
needles: Rf 0.42 (EtOAc/MeOH 1:1); mp 158-160 °C; [R]D: -10
(c 0.58, MeOH) IR (KBr): 3251, 1737, 1236; 1H NMR: (200
MHz, CDCl3+CD3OD): 2.90 (dd, J ) 14, 8 Hz, 1H, H-3), 3.10
(dd, J ) 14.4, 4.4 Hz, 1H, H-3), 3.4 (br s, 1H, OH), 4.34 (dd, J
) 7.8, 4.4 Hz, 1H, H-2), 6.75 (d, J ) 8.4 Hz, 2H, H-6 and H-8),
7.10 (d, J ) 8.8 Hz, 2H, H-5 and H-9); 13C NMR (50 MHz,
CDCl3+CD3OD): 40.7 (C-3), 73.0 (C-2), 116.0 (C-6 and C-8),
129.5 (C-4), 131.5 (C-5 and C-9), 157.0 (C-7), 177.5 (C-1). Anal.
Calcd for C9H10O4‚1/2H2O: C 56.53, H 5.76. Found: C 56.56,
H 6.02.
(S)-2-Acetyloxy-3-(4-ben zyloxyp h en yl)p r op ion ic Acid
[AcO-L-Hp la (Bn )-OH] (2). To a solution of O-benzyl-L-ty-
rosine (2 g, 7.4 mmol) and NaOAc (2.2 g, 26.6 mmol) in glacial
AcOH was slowly added isoamyl nitrite (3.7 mL, 27.4 mmol)
at room temperature. The reaction mixture was stirred for 66
h. Hexane was added, and the mixture was concentrated to
remove AcOH. The residue was dissolved in EtOAc (100 mL),
and H2O (70 mL) and concentrated HCl (20 mL) were added.
The organic layer was washed with H2O (5 × 30 mL) and
extracted with saturated solution of NaHCO3. The aqueous
phase was acidified with HCl until pH 1 and extracted with
EtOAc (3 × 30 mL). The dried organic extracts were concen-
trated, and the residue was purified by chromatography (1%
AcOH in CH2Cl2) to give 1.5 g (64%) of acid 2: Rf 0.50 (hexane/
CH2Cl2/AcOH 3:6:1); mp 99-101 °C (H2O); [R]D: -6.6 (c 0.6,
CHCl3); IR (KBr) 2926-3213, 1760, 1706, 1248, 1218; 1H NMR
(200 MHz, CDCl3): 2.10 (s, 3H), 3.06 (dd, J ) 14.6, 8.4 Hz,
1H), 3.20 (dd, J ) 14.4, 4 Hz, 1H), 5.04 (s, 2H), 5.20 (dd, J )
8.6, 4.2 Hz, 1H), 6.92 (d, J ) 8.4 Hz, 2H), 7.16 (d, J ) 8.8 Hz,
2H), 7.34 (m, 5H); 13C NMR (50 MHz, CDCl3): 20.5 (q), 36.2
(t), 70.0 (t), 79.0 (d), 114.8 (d), 127.5 (d), 128.0 (d), 128.0 (s),
128.5 (d), 130.3 (d), 137.0 (s), 158.0 (s), 170.6 (s), 174.8 (s).
Anal. Calcd for C18H18O5: C, 68.77; H, 5.71. Found: C, 68.38;
H, 5.81.
The optical rotation value corresponds to that of a crystal-
lized sample, which was obtained as follows: (S)-(-)-1-phe-
nylethylamine (0.09 mL, 0.70 mmol) was added to a solution
of 2 (201 mg, 0.64 mmol) in CHCl3 (3 mL), the mixture was
stirred for 20 min, concentrated, and crystallized (Et2O/EtOH)
to give the corresponding salt of 2 (de g 98%): 1H NMR (300
MHz, CDCl3) 1.56 (d, J ) 6.6 Hz), 1.96 (s, 3H), 2.78 (dd, J )
14.5, 9.8 Hz, 1H), 2.93 (dd, J ) 14.3, 3.1 Hz, 1H), 4.25 (q, J )
6.8 Hz, 1H), 4.89 (dd, J ) 9.8, 3.6 Hz, 1H), 5.04 (s, 2H), 6.89
(d, J ) 8.5 Hz, 2H), 7.08 (d, J ) 8.5 Hz, 2H), 7.30-7.44 (m,
10H). A solution of the above crystals in CH2Cl2 (8 mL) was
washed with 1 N HCl. The organic layer was dried and
concentrated to give enantiopure 2.
Boc-L-P h e-L-Ch oi-OMe (8a ) a n d Its Tr iflu or oa ceta te
8b. Trifluoroacetic acid (5 mL) was added to a solution of 74b
(391 mg, 1.31 mmol) in CH2Cl2 (10.6 mL). The reaction mixture
was stirred at 0 °C for 2 h and concentrated. To a solution of
the resulting material and Boc-L-Phe-OH (452 mg, 1.7 mmol)
in CH2Cl2 (26 mL), cooled at 0 °C, PyBOP (885 mg, 1.7 mmol)
and NMM (462.2 mg, 4.57 mmol, 0.55 mL) were added. After
the solution had been stirred for 30 min at 0 °C and 22 h at
room temperature, CH2Cl2 (30 mL) was added, and the
solution was washed with 5% aqueous NaHSO4 (3 × 25 mL),
saturated aqueous NaHCO3 (3 × 25 mL), and brine (1 × 15
mL). The organic layer was dried and concentrated, and the
residue was purified by chromatography (hexane/EtOAc 1:1).
The first eluate gave dipeptide 8b (358 mg, 0.66 mmol) as a
colorless syrup, and then a mixture of 8b and 8a (1:1 ratio, 20
mg) was eluted. A later elution gave dipeptide 8a (225 mg,
0.51 mmol) as a white foam, the combined yield being 92%.
Com p ou n d 8a : Rf ) 0.47 (EtOAc); [R]D: -37.7 (c 0.35,
CHCl3); IR (film): 3432-3304, 1748, 1710, 1634; 1H NMR (500
MHz, CDCl3, COSY, NOESY): trans-cis rotamer 1.6:1 ratio,
trans rotamer: 1.30 (s, 9H, C(CH3)3), 1.47 (m, 4H, H-7, 2H-5
and H-4 Choi), 1.77 (m, 1H, H-7 Choi), 1.88 (ddd, J ) 13, 12.5,
10 Hz, 1H, H-3 Choi), 2.11 (m, 2H, H-3 and H-4 Choi), 2.38
(dddd, J ) 13, 6.5, 6.5, 6.5 Hz, 1H, H-3a Choi), 2.85 (dd, J )
14, 8 Hz, 1H, H-3 Phe), 3.10 (dd, J ) 14, 6 Hz, 1H, H-3 Phe),
3.24 (br, 1H, OH-6 Choi), 3.71 (s, 3H, CO2Me), 3.98 (br s, 1H,
H-6 Choi), 4.42 (dd, J ) 10.5, 8.5 Hz, 1H, H-2 Choi), 4.42 (m,
1H, H-7a Choi), 4.60 (ddd, J ) 8, 8, 6 Hz, 1H, H-2 Phe), 5.33
(d, J ) 9 Hz, 1H, NH); cis rotamer: 1.40 (s, 9H, C(CH3)3), 2.27
(m, 1H, H-3a Choi), 2.84 (dd, J ) 13.5, 8 Hz, 1H, H-3 Phe),
2.97 (dd, J ) 13.5, 5.5 Hz, 1H, H-3 Phe), 3.34 (dd, J ) 8.5, 8.5
Hz, 1H, H-2 Choi), 3.69 (s, 3H, CO2Me), 4.02 (br s, 1H, H-6
Choi), 4.23 (ddd, J ) 8, 8, 6 Hz, 1H, H-2 Phe), 4.30 (ddd, J )
12, 6, 6 Hz, 1H, H-7a Choi), 5.46 (d, J ) 9 Hz, 1H, NH); 13C
NMR (75 MHz, CDCl3, HMQC): trans-cis rotamer 2.5:1 ratio,
trans rotamer: 18.9 (C-4 Choi), 25.6 (C-5 Choi), 28.2 (C(CH3)3),
30.3 (C-3 Choi), 34.0 (C-7 Choi), 36.9 (C-3a Choi), 39.3 (C-3
Phe), 52.1 (CO2Me), 52.6 (C-2 Phe), 54.3 (C-7a Choi), 59.0 (C-2
Choi), 65.3 (C-6 Choi), 79.6 (C(CH3)3), 126.5 (C-7 Phe), 128.1
(C-6,6’ Phe), 129.6 (C-5,5’ Phe), 136.5 (C-4 Phe), 170.5 (C-1
Phe), 172.6 (CO2Me); cis rotamer: 18.9 (C-4 Choi), 25.8 (C-5
Choi), 28.1 (C(CH3)3), 29.5 (C-3 Choi), 34.0 (C-7 Choi), 34.7
(C-3a Choi), 41.2 (C-3 Phe), 52.7 (C-2 Phe), 54.0 (C-7a Choi),
58.5 (C-2 Choi), 65.3 (C-6 Choi), 79.2 (C(CH3)3), 126.7 (C-7
Phe), 128.3 (C-6,6’ Phe), 129.2 (C-5,5’ Phe), 136.2 (C-4 Phe),
(S)-3-(4-Ben zyloxyph en yl)-2-h ydr oxypr opion ic Acid (4).
LiOH (0.1 N, 6.9 mL) was added to a cooled (0 °C) solution of
2 (72 mg, 0.23 mmol) in THF (2 mL), and the mixture was
stirred at room temperature for 20 h. The mixture was
extracted with Et2O (2 × 4 mL), and the aqueous phase was
acidified with 1 N HCl to pH 1. Extraction of the aqueous layer
with CHCl3/iPrOH (4:1, 4 × 10 mL) gave an organic extract
which provided 4 as a white solid (62 mg, 99%); Rf 0.42
(EtOAc/MeOH 1:1); [R]D: -12.2 (c 0.62, MeOH); mp 140-142
°C; IR (KBr): 3448-2928, 1727, 1253; 1H NMR (200 MHz,
CDCl3+CD3OD): 2.87 (dd, J ) 13.8, 7 Hz, 1H), 3.08 (dd, J )
4948 J . Org. Chem., Vol. 67, No. 14, 2002

Synthesis of Microcin SF608
154.4 (NCO2), 170.2 (C-1 Phe), 172.6 (CO2Me). Anal. Calcd for
Anal. Calcd for C37H42N2O8‚H2O: C 67.25, H 6.41, N 4.23.
Found: C 67.68, H 6.89, N 4.22.
C
24H34N2O6: C 64.55, H 7.67, N 6.27. Found: C 64.44, H 7.93,
N 6.03.
Com p ou n d 8b: Rf ) 0.59 (EtOAc); [R]D: -34.3 (c 0.4,
Com p ou n d 9b: mp 48-51 °C; [R]D: -34 (c 0.45, CHCl3);
1H NMR (200 Mz, CDCl3) (1.7:1 mixture of trans and cis
rotamers): trans rotamer: 1.39-2.25 (m, 8H), 2.04 (s, 3H),
2.44 (dddd, J ) 12, 6, 6, 6 Hz, 1H), 2.64-3.12 (m, 4H), 3.74 (s,
3H), 4.34 (m, 1H), 4.42 (dd, J ) 10, 8 Hz, 1H), 4.71 (ddd, J )
8, 8, 6 Hz, 1H), 5.01 (s, 2H), 5.11 (br s, 1H); 5.31 (dd, J ) 6, 4
Hz, 1H), 6,57 (d, J ) 8.4 Hz, 1H), 6.85 (d, J ) 8.8 Hz, 2H),
7.03 (d, J ) 8,8 Hz, 2H), 7.32 (m, 10H); cis rotamer: 2.11 (s,
3H), 2.64-3.12 (m, 4H), 3.73 (s, 3H), 5.23 (br s, 1H), 6.75 (d,
J ) 8.4 Hz, 1H); 13C NMR (50 MHz, CDCl3) (2:1 mixture of
trans and cis rotamers): trans rotamer: 19.2 (t), 20.8 (q), 22.7
(t), 30.0 (t), 30.4 (t), 36.3 (d), 36.7 (t), 39.9 (t), 51.4 (d), 52.2
(q), 53.9 (t), 58.1 (d), 69.7 (t), 73.9 (d), 114.4 (d), 126.9-129.3
(doublets), 130,5 (d), 135.6 (s), 135.7 (s), 136.7 (s), 157.6 (s),
168.3 (s), 169.1 (s), 169.5 (s), 169.6 (s), 172.3 (s); cis rotamer:
20.9 (q), 22.9 (t), 34.2 (d), 40.5 (t), 51.5 (d), 53.0 (q), 53.4 (d),
58.2 (d), 74.2 (d), 114.5 (d), 126.9-129.3 (doublets), 156.1 (s).
HO-L-Hp la (Bn )-L-P h e-L-Ch oi-d iBoc-Agm a (10). A cooled
solution of LiOH (0.1 N, 4.2 mL, 0.42 mmol) was added
dropwise to a cooled (0 °C) solution of peptide 9a (89 mg, 0.14
mmol) in THF (3.5 mL). After having been stirred at room
temperature for 20 h, the solution was washed with Et2O (2
× 7 mL) and acidified to pH 1 with 1 N HCl. The aqueous
solution was extracted with CHCl3/iPrOH (4:1, 6 × 7 mL). The
combined extracts were washed with brine, dried, and con-
centrated to give the corresponding acid (70 mg, 86%) as a
white solid (structure not shown); Rf 0.47 (EtOAc/MeOH 1:1);
1H NMR (200 MHz, CDCl3+drops CD3OD): trans-cis rotamer
10:1 ratio, trans rotamer: 1.27-2.45 (m, 9H), 2.53-3.14 (m,
4H), 3.38-4.88 (m, 5H), 5.02 (s, 2H), 6.89 (d, J ) 8.4 Hz, 2H),
7.10 (d, J ) 8.4 Hz, 2H), 7.21-7.39 (m, 5H); 13C NMR (50 MHz,
CDCl3+drops CD3OD): trans-cis rotamer 10:1 ratio, trans
rotamer: 18.8 (t), 25.5 (t), 30.2 (t), 33.7 (t), 36.7 (d), 38.6 (t),
39.4 (t), 51.0 (d), 54.9 (d), 59.2 (d), 65.1 (d), 69.9 (t), 72,5 (d),
114.5 (d), 126.8-129.5 (doublets), 130.3 (d), 135.5 (s), 135.7
(s); 136.8 (s); 157.3 (s), 170.5 (s), 173.5 (s), 174.0 (s); cis
rotamer: 29,6 (t), 34.7 (t), 51.4 (d), 54.2 (d), 58.5 (d), 130.5
(d).
PyBOP (62.5 mg, 0.12 mmol) and NMM (34 µL, 0.30 mmol)
were added to a cooled (0 °C) solution of the above acid (70
mg, 0.12 mmol) and agmatine 615 (56 mg, 0.16 mmol) in DMF
(2 mL). After the solution had been stirred for 30 min at 0 °C
and 22 h at room temperature, EtOAc (70 mL) was added, and
the solution was washed with saturated aqueous NaHCO3 (1
× 50 mL) and H2O (5 × 60 mL). The organic layer was dried
and concentrated, and the residue was purified by chroma-
tography (EtOAc/MeOH 95:5) to give 10 (83 mg, 77%) as a
white foam: Rf 0.77 (EtOAc/MeOH 1:1); [R]D: -24.2 (c 0.65,
CHCl3); IR (film): 3333, 1636; 1H NMR (200 MHz, CDCl3)
1.46-2.4 (m, 13H), 1.47 and 1.49 (2s, 9H each), 2.6-3.4 (m,
8H), 4.02 (br s, 1H), 4.24 (br, 1H), 4.4 (m, 2H), 4.89 (ddd, J )
8, 8, 6 Hz, 1H), 5.02 (s, 2H), 6.9 (d, J ) 8.6 Hz, 2H), 7.13 (d,
J ) 8.6 Hz, 2H), 7.1-7.4 (m, 12H), 8.3 (m, 1H); 13C NMR (75
MHz, CDCl3): 19.0 (t), 25.8 (t), 26,5 (t), 26.8 (t), 28.0 (q), 28.3
(q), 29.7 (t), 33.4 (t), 36.5 (d), 39.1 (t), 39.4 (t), 39.7 (t), 40.5 (t),
51.5 (d), 55.4 (d), 59.9 (d), 65.8 (d), 69.9 (t), 72.8 (d), 79.2 (s),
83.0 (s), 127-130.5 (doublets), 135.8 (s), 136.8 (s), 153.1 (s),
156.0 (s), 157.6 (s), 163.4 (s), 170.7 (s), 172.8 (s). Anal. Calcd
for C49H66N6O10: C 65.47, H 7.35, N 9.35. Found: C 65.18, H
7.56, N 9.11.
L-Hp la -L-P h e-L-Ch oi-Agm [Micr ocin SF 608] (1). A solu-
tion of 10 (32 mg, 0.035 mmol) in CH3CN (3 mL) and 6 N HCl
(0.6 mL) was stirred at room temperature for 6 h, then
concentrated to give the hydrochloride of the intermediate
L-Hpla(Bn)-L-Phe-L-Choi-Agm, as an amorphous solid. To a
solution of this tetrapeptide (27 mg) in EtOAc/MeOH (1:1, 3
mL) was added Pd/C (10%, 16.5 mg and an additional 27 mg
after 6 h), and the mixture was stirred under hydrogen
atmospheric pressure for 24 h. The catalyst was removed by
filtration with Celite, which was washed with MeOH (30 mL).
CHCl3); 1H NMR (200 MHz, CDCl3) (1.3:1 mixture of trans
and cis rotamers): trans rotamer: 1.37 (s, 9H), 1.40-1.99 (m,
6H), 2.48 (dddd, J ) 13, 6.5, 6.5, 6.5 Hz, 1H), 2.86 (dd, J )
13.6, 6.6 Hz, 1H), 3.09 (dd, J ) 13.6, 7.8 Hz, 1H), 3.74 (s, 3H),
4.36 (m, 3H), 5.14 (br s, 1H, H-6 Choi), 5.24 (d, J ) 8.8 Hz,
1H), 7.12-7.23 (m, 5H); cis rotamer: 1.43 (s, 9H), 2.21 (dddd,
J ) 13, 6.5, 6.5, 6.5 Hz, 1H), 5.24 (br s, 1H), 5.40 (d, J ) 8.8
Hz, 1H);13C NMR (200 MHz, CDCl3): (1.3:1 mixture of trans
and cis rotamers): trans rotamer: 19.1 (t), 22.6 (t), 28.1 (q),
29.9 (t), 36.3 (d), 39.1 (t), 52.0 (q), 53.2 (d), 53.6 (d), 58.9 (d),
74-3 (d), 79.2 (s), 126.5-129.2 (doublets), 136.2 (s), 154.2 (s),
170.3-172.4 (singlets); cis rotamer: 19.0 (t), 22.9 (t), 27.9 (q),
34.1 (d), 41.2 (t), 58.3 (d), 74.2 (d), 79.4 (s), 136.1 (s), 154.7 (s).
AcO-L-Hp la (Bn )-L-Leu -L-Ch oi-OMe (9a ) a n d Its Tr if-
lu or oa ceta te 9b. TFA (2 mL, 53 mmol) was added to a cooled
(0 °C) solution of 8b (350 mg, 0.65 mmol) in CH2Cl2 (9 mL),
and the mixture was stirred for 50 min and concentrated. A
solution of the resulting material in CH2Cl2 (3.6 mL) with
NMM (108.34 µL, 0.97 mmol) was added to a cooled (0 °C)
solution of the L-Hpla derivative 2 (245 mg, 0.78 mmol) in CH2-
Cl2 (4.3 mL), which had been previously stirred at this
temperature with PyBOP (406 mg, 0.78 mmol) and NMM ((153
µL, 1.3 mmol) for 30 min. The mixture was stirred at room
temperature for 22 h. CH2Cl2 (20 mL) was added, and the
organic layer was successively washed with HCl (1 N, 3 × 25
mL), saturated NaHCO3 solution (3 × 25 mL), and brine (1 ×
10 mL). The concentrated dried extract was purified by
chromatography (hexane/EtOAc 1:1) to give the trifluoroac-
etate 9b (236 mg) and then the alcohol 9a (120 mg), both as
white solids, the combined yield being 78%.
Com p ou n d 9a : mp 81-84 °C; [R]D: -39.4 (c 0.5, CHCl3);
IR (film): 3417-3296, 1746, 1627; 1H NMR (500 MHz, DMSO,
COSY, NOESY) trans-cis rotamer 10:1 ratio, trans rotamer:
1.39 (m, 5H, 2H-5, 2H-4, H-7 Choi), 1.51 (br d, J ) 13.5 Hz,
1H, H-7 Choi), 1.82 (ddd, J ) 12.5, 12, 11 Hz, 1H, H-3â Choi),
1.93 (s, 3H, CH3CO), 2.09 (ddd, J ) 12.5, 7.5 7 Hz, 1H, H-3R
Choi), 2.18 (dddd, J ) 12, 6, 6, 6 Hz, 1H, H-3a Choi), 2.76 (dd,
J ) 14.5, 9 Hz, 1H, H-3 Phe), 2.84 (dd, J ) 14.5, 8 Hz, 1H,
H-3 Hpla), 2.89 (dd, J ) 14.5, 4.5 Hz, 1H, H-3′ Hpla), 2.94
(dd, J ) 14, 6 Hz, 1H, H-3 Phe), 3.62 (s, 1H, OMe), 3.80 (br s,
1H, H-6 Choi), 4.26 (dd, J ) 10, 8.5 Hz, 1H, H-2 Choi), 4.42
(ddd, J ) 12.5, 6, 6 Hz, 1H, H-7a Choi), 4.50 (d, J ) 2.5 Hz,
1H, OH-6 Choi), 4.57 (ddd, J ) 8, 8, 6 Hz, 1H, H-2 Phe), 5.03
(dd, J ) 8.5, 4 Hz, 1H, H-2 Hpla), 5.04 (s, 2H, CH2Ar), 6.88
(d, J ) 8.5 Hz, 2H, H-6 and H-8 Hpla), 7.07 (d, J ) 8.5 Hz,
2H, H-5 and H-9 Hpla), 7.29 (m, 10H, H-5, 5′, 6, 6′, 7 Phe and
Ar), 8.53 (d, J ) 7.5 Hz, 1H, NH); cis rotamer: 1.94 (s, 3H,
CH3CO), 3.58 (s, 3H, OMe), 3.68 (dd, J ) 8.5, 8.5 Hz, 1H, H-2
Choi), 3.83 (br s, 1H, H-6 Choi), 4.14 (ddd, J ) 12.5, 6, 6 Hz,
1H, H-7a Choi), 4.31 (m, 1H, H-2 Phe), 5.11 (dd, J ) 8.5, 3.5
Hz, 1H, H-2 Hpla), 6.88 (d, J ) 8 Hz, 2H, H-6, H-8 Hpla), 7.07
(d, J ) 8 Hz, 2H, H-5, H-9 Hpla), 8.29 (d, J ) 9 Hz, 1H,
NH);13C NMR (75 MHz, CDCl3, HMQC): trans-cis rotamer
4:1 ratio, trans rotamer: 18.9 (C-4 Choi), 20.9 (CH3CO), 25.8
(C-5 Choi), 30.3 (C-3 Choi), 34.0 (C-7 Choi), 36.8 (C-3 Phe),
36.9 (C-3a Choi), 39.0 (C-3 Hpla), 50.8 (C-2 Phe), 52.1 (CO2-
Me), 54.6 (C-7a Choi), 59.1 (C-2 Choi), 65.5 (C-6 Choi), 69.8
(OCH2Ar), 74.0 (C-2 Hpla), 114.5 (C-6 and C-8 Hpla), 127.4-
129.8 (C-5, 5′, 6, 6′, 7 Phe and Ar), 128.0 (C-4 Hpla), 130.5
(C-5 and C-9 Hpla), 135.7 (C-7 Phe, ipso-Ar), 136.8 (C-4 Phe),
157.6 (C-7 Hpla), 168.4, 169.2, 169.5, 172.4 (CO); cis rotamer:
18.9 (C-4 Choi), 25.8 (C-5 Choi), 29.6 (C-3 Choi), 31.9 (C-7
Choi), 34.7 (C-3a Choi), 36.8 (C-3 Phe), 40.5 (C-3 Hpla), 51.5
(C-2 Phe), 53.0 (CO2Me), 54.1 (C-7a Choi), 58.3 (C-2 Choi), 65.5
(C-6 Choi), 74.0 (C-2 Hpla), 114.5 (C-6,8 Hpla), 127.4-129.8
(C-5.5′,6,6′, 7-Phe, Ar), 130.5 (C-5, 9 Hpla), 135.8 (ipso-Ar),
136.8 (C-4 Phe), 157.6 (C-7 Hpla), 167.8, 169.3, 169.4 (CO).
J . Org. Chem, Vol. 67, No. 14, 2002 4949

Valls et al.
The organic solution was concentrated to give microcin SF608
(1) (25 mg, 99%); RP-HPLC tR 1.13 min (cis rotamer) and 1.47
min (trans rotamer), isocratic elution with A/B 60:40 for 30
min, where A is H2O and B is CH3CN/0.05% TFA; coelution
of 1 with natural microcin SF608 gave the same two peaks;
[R]D: -27.4 (c 1.25, MeOH). The 1H NMR (500 MHz, DMSO-
d6, COSY, NOESY), trans-cis rotamer 3:1 ratio, and 13C NMR
(100 MHz, DMSO-d6, HSQC) data of 1 matched those reported
in the literature for the natural product.2a,16 FABMS for
C32H44N6O6: 609.3 ([M + H]+, 100), 567.2 (12), 461.2 (10), 383.3
(40).
Ack n ow led gm en t. This work was supported by the
MCYT, Spain (project BQU2001-3551). Thanks are also
due to the DURSI, Catalonia, for Grant 2001SGR-
00083. M.V. is a recipient of a fellowship (MEC, Spain).
We thank Professor Shmuel Carmeli (Tel Aviv Univer-
sity) for a generous supply of natural microcin SF608
for comparison as well as copies of NMR spectra of the
natural product.
J O025709Y
4950 J . Org. Chem., Vol. 67, No. 14, 2002