An efficient synthesis of alterobactin A; a super siderophore of marine origin

Article abstract of DOI:10.1055/s-1998-5937

Alterobactin A (1), a cyclic depsipeptide and a super siderophore isolated from an open-ocean bacterium, was efficiently synthesized for the first time by a convergent manner with the maximum protection of various functional groups.

Full text of DOI:10.1055/s-1998-5937

SYNTHESIS
April 1998
627
An Efficient Synthesis of Alterobactin A; A Super Siderophore of Marine Origin
Jingen Deng,a Yasumasa Hamada,b Takayuki Shioiri*c
a Union Laboratory of Asymmetric Synthesis, Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China
b Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
c Department of Synthetic Organic Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya
467-8603, Japan
Fax +81(52)8344172; E-mail: shioiri@phar.nagoya-cu.ac.jp
Received 9 September 1997
Abstract: Alterobactin A (1), a cyclic depsipeptide and a super
siderophore isolated from an open-ocean bacterium, was effi-
ciently synthesized for the first time by a convergent manner with
the maximum protection of various functional groups.
extremely low-iron environment may well have evolved
unique structures with exceptional ferric ion affinities.
Alterobactin A (1) is a macrocyclic depsipeptide, which
contains two types of unusual amino acids (Figure 2), two
L-threo-β-hydroxyaspartic acid (3, β-OH-Asp) and one
(3R,4S)-4,8-diamino-3-hydroxyoctanoic acid (4, lysine-
statine, LysSta) attached to a catechol carboxylate at the
Nω-site, through which ferric ion is coordinated to form a
six-coordinate complex.1b Our interest in the exploitation
of new methodology for the synthesis of aquatic natural
products containing non-ribosomal amino acids3 and
siderophores4 led us to investigate the synthesis of altero-
Key words: alterobactin A, depsipeptidic siderophores, macrolac-
tamization, selective OH protection and deprotection
Alterobactin A (1) was isolated together with alterobactin
B (2) by Butler and co-workers from an open-ocean bac-
terium Alteromonas luteoviolacea collected off Chub Cay,
Bahamas and identified as depsipeptidic siderophores1
(Figure 1). Alterobactin A (1) has extraordinary affinity
for ferric ion (KFe = 1049–53), while alterobactin B (2) has
a comparatively lower affinity for iron and may not be a
true siderophore because it is formed by the base hydroly-
sis of alterobactin A (1) in the absence of bound iron(III).
With the possible exception of enterobactin,2 the ferric ion
affinity of alterobactin A (1) is not exceeded by any other
known siderophore and it is likely that the siderophores
produced by open-ocean marine bacteria that live in an
bactin A (1), a super siderophore of marine bacteria ori-
gin. Herein, we report the details of our synthetic work on
alterobactin A (1).5
Figure 2. Structure of the Unusual Amino Acids 3 and 4
Strategy
A [4+3] convergent strategy was adopted in our synthetic
approach to alterobactin A (1). Fragment coupling of the
two segments 28 and 31 was accomplished between Gly
and Arg while macrolactamization was carried out be-
tween Gly and β-OH-Asp,6 because the construction of
the amide bonds at glycine as C-terminus would proceed
without both racemization and steric constraint. Further-
more, a β-turn structure in alterobactin A (1) along Gly-
Arg-β-OH-Asp-Gly sequence1b probably facilitates the
macrolactamization of the linear precursor.7 Alterobactin
A (1) contains various reactive functional groups: one
amino group, one guanidino group, two carboxyl groups,
two phenol groups, and three hydroxyl groups. Thus, the
choice of protecting groups, especially amino and carbox-
yl groups, was viewed as very critical to our ultimate suc-
cess. It is necessary that the γ-amine of LysSta and the β-
carboxylates of two β-OH-Asp should be kept inert in the
amide-coupling process, and the phenol groups and hy-
Figure 1. Structures of Alterobactin A (1) and B (2) and the droxyl groups should also be kept inert in the ester-form-
Disconnection Crucial for the Convergent Synthesis of Alterobactin
A (1)
ing step. The carbobenzoxy (Cbz) group was chosen to
protect the γ-amino group of LysSta because it would per-

SYNTHESIS
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628
mit the selective deprotection of a tert-butyloxycarbonyl
(Boc) group at the N-terminus of the peptide segments un-
der acidic conditions. The benzyl (Bzl) group was chosen
to block the carboxylates of two β-OH-Asp, whereas the
2,2,2-trichloroethyl (Tce) group was chosen to block the
C-terminal carboxylates of the peptide segments, because
the Bzl group would be kept intact under the conditions
which cleave the Tce group with zinc. The tert-
butyldimethylsilyl (TBS) or tert-butyldiphenylsilyl (TB-
DPS) group and the Bzl group were chosen to protect the
other three hydroxyl groups and two phenol groups, respec-
tively. The protection of the two phenol groups and the two
carboxylates by Bzl, protection of the γ-amine by Cbz, and
the three hydroxyl groups by TBS, set the stage for remov-
ing these protecting groups simultaneously through hydro-
genation in aqueous HOAc/THF at the penultimate step of
the synthesis. Although most sulfonyl groups are suitable to
protect the guanidino function of arginine in our approach,
the 2,3,6-trimethyl-4-methoxybenzenesulfonyl (Mtr) group
is considered to be favorable, because removal of the Mtr
group could be readily performed under relatively mild
conditions,8 and also there is no problem in selective depro-
tection of the Boc group.9 The organophosphorus reagents,
diethyl phosphorocyanidate [DEPC, (EtO)2- P(O)CN]10
and pentafluorophenyl diphenylphosphinate [FDPP,
Ph2P(O)OC6F5],11 could be applied here in the fragment-
coupling and macrolactamization processes, respectively.
Scheme 1
(3R,4S)-Lysinestatine:
Another building block (3R,4S)-LysSta derivative 21 was
synthesized by the stereoselective reduction of the β-keto es-
ter 14 and subsequent coupling with the catecholate 17. The
β-keto ester 14 was readily prepared from commercially
available Nα-Cbz-Nω-Boc-lysine (13) according to Rich.16a
Reduction of 14 with sodium borohydride in EtOH/THF at
low temperature afforded a mixture of diastereomers 15,16
whose selectivity was not influenced by the ratio of the sol-
vents: ethanol/tetrahydrofuran (1:3, 64% de; 1:5, 66% de;
1:6, 69% de and 1:10, 66% de). Initially, the hydroxyl group
of 15 was protected as the TBDPS group, and interestingly,
the product 16 enriched in the (3R,4S)-diastereomer (88%
de) was obtained. However, the mixture of diastereomers 16
was inseparable,17 even if 16 was converted to its catecholate
derivative 18 with 84% de (Scheme 2) by coupling with 2,3-
Synthesis
L-Threo-â-hydroxyaspartic Acid:
First, we synthesized the unusual amino acid β-OH-Asp
derivative 12 from commercially available methyl cin-
namate (5) through the stereocontrolled Sharpless
dihydroxylation12 and the transformation of the phenyl
group, a carboxyl synthon,13 to the carboxylate group
(Scheme 1). The diol derivative 6 was obtained by the
Sharpless asymmetric dihydroxylation of 5 by use of AD-
mix-β in 68% yield with >98% ee. The enatiomeric excess
1
(ee) was determined by H NMR analysis of the bis-
MTPA esters.14 The bromo ester 7 was prepared from 6 by
a stereo- and regioselective transformation with 31% HBr/
HOAc15 and then stereoselectively converted into the azido
ester 8 with excess sodium azide. Hydrogenation and si-
multaneous protection of the resulting amino group in a
one-pot process afforded the Boc-amino ester 9 in good
yield and no N-acetyl product was produced.11b Rutheni-
um-catalyzed oxidation13 of 9 and subsequent protection
by using O-tert-butyl-N,N′-diisopropylisourea (BDIU)
smoothly gave the fully protected β-OH-Asp derivative 10
in 71% yield, which was treated with aqueous lithium hy-
droxide in THF, followed by treatment with benzyl bro-
mide and potassium carbonate to give the β-OH-Asp β-
benzyl ester 11. The desired building block, β-OH-Asp de-
rivative 12 was produced in 84% yield by the simultaneous
deprotection of the Boc group and tert-butyl ester, followed
by the selective protection of the amino group as Boc and
the hydroxyl group as the TBS group, respectively.
Scheme 2

SYNTHESIS
April 1998
629
bis(benzyloxy)benzoic acid (17), which was readily pre- The Western Hemisphere (Tetradepsipeptide
pared from 2,3-catecholic acid (19) via protection of both Segment):
the hydroxyl and carboxyl groups and then selective Boc-Gly-OTce (24) was prepared from Boc-Gly-OH (23)
deprotection of the carboxylate group (Scheme 3).
by condensation of 2,2,2-trichloroethanol (TceOH) with
N,N’-dicyclohexylcarbodiimide (DCC) in the presence of
4-(N,N-dimethylamino)pyridine (DMAP). Deprotection
of 24 at the N-terminus with hydrogen chloride and sub-
sequent coupling with Boc-D-Ser-OH (25) with DEPC in
the presence of triethylamine (TEA) gave the dipeptide 26
in a moderate yield of 60%. Because of the non-protection
of the hydroxyl group of 25, phosphonate formation
seems unavoidable even if the ratio of substrates and re-
agent (24/25/DEPC) was changed from 1:1.1:1.3 to
1:1.2:1.2. Although the isolated yield was improved to
70% by using FDPP, phosphinate formation was still not
repressed. The dipeptide 26 was deprotected with hydro-
gen chloride and then coupled with the LysSta derivative
21 with DEPC to give the tripeptide 27. The western
hemisphere (the tetradepsipeptide segment) 28 was
smoothly obtained by condensing 27 with the β-OH-Asp
derivative 12 by the use of 1-ethyl-3-[3-(dimethylami-
no)propyl]carbodiimide (EDCI) and DMAP (Scheme 6).
Scheme 3
Fortunately, the separation of the diastereomers 15 was
effectively accomplished by flash chromatography on sil-
ica gel to provide the unusual amino acid subunit,
(3R,4S)-LysSta derivative 15a as the predominant isomer
with >95% de (Scheme 4). The catecholate derivative 20
was produced by the deprotection of the Boc group of 15a
and subsequent coupling with 17. Conversion of 20 to the
required LysSta building block 21 was effected by hydrol-
ysis of the ethyl ester and subsequent blocking of the hy-
droxyl group as the TBS moiety (Scheme 4). To
determine the stereochemistry, 15a and its diastereomer
15b were converted into oxazolidinones 22a and 22b, re-
spectively, and the configurations at C5-methine of 22a
and 22b were assigned by coupling-constant J4,5 measure-
ments (Scheme 5).16b
Scheme 4
Scheme 6
The Eastern Hemisphere (Tripeptide Segment):
Construction of the eastern hemisphere 31 was also initi-
ated from Boc-Gly-OTce (24), which underwent the Boc-
deprotection followed by coupling with the β-OH-Asp de-
rivative 12 using DEPC in the presence of TEA. However,
Scheme 5

SYNTHESIS
Papers
630
the products were an inseparable mixture of the dipeptide Alterobactin A:
29 and aspartimide derivative 32 in a ratio of 7:1. Fortu- Deprotection of the Tce group of the western hemisphere
nately, the formation of the undesired aspartimide 32 28 was achieved by using zinc and 1M aqueous ammoni-
could be entirely suppressed when TEA was replaced with um acetate, while treatment of the eastern hemisphere 31
N-methylmorpholine (NMM) and the desired dipeptide with p-toluenesulfonic acid monohydrate resulted in the
29 was formed with almost complete selectivity (>120:1 removal of the both Boc and TBS groups.9c Subsequently,
ratio). After selective deprotection of the Boc group of 29 coupling of 33 with 34 provided the expected depsipep-
with trifluoroacetic acid, coupling with Nα-Boc- tide 35 accompanied by the unexpected aspartimide deriv-
Arg(Mtr)-OH (30) afforded 31 in a high yield of 91% ative 36. Furthermore, it was found that 35 slowly
(Scheme 7).
transformed into 36 on a silica gel column and in CDCl3,
an NMR solvent (Scheme 8).
It is well-known that aspartimide formation occurs slowly
or not at all in trifluoroacetic acid,18,19 and thus the selec-
tive deprotection of the Boc group of 31 was accom-
plished by using TFA in dichloromethane without
liberation of the Mtr and TBS groups.8 Condensation of
this deprotected western hemisphere 31 with the eastern
hemisphere 33 proceeded smoothly with FDPP to give the
linear depsipeptide 37 in 86% yield (Scheme 9) and this
implied that the steric protection of the hydroxyl group on
the β-OH-Asp subunit could prevent aspartimide forma-
tion.20 In this particular coupling process, DEPC was less
effective and the yield was ~44–68%. Deprotection of 37
with zinc/1M aqueous ammonium acetate under the same
conditions used for the removal of the Tce group of 28 af-
forded a complicated mixture. As a model experiment,
treatment of the tripeptide 31 under the same condition
gave only the debenzylation products, which were in-
ferred to be the aspartimide derivative and its hydrolyzate
1
based on H NMR spectroscopy and TLC analysis. Sur-
prisingly, however, treatment of 31 with zinc in HOAc
Scheme 7
Scheme 8

SYNTHESIS
April 1998
631
Scheme 9
produced the Tce-cleaved compound as a single product18
(39, Scheme 10). Therefore, after removal of the Tce
group at the C-terminus of 37 with zinc in HOAc and then
removal of the Boc group at the N-terminus with TFA,
which simultaneously cleaved the TBS group on the side
chain,21 the macrolactamization was efficiently achieved
by use of FDPP to give the macrocyclic depsipeptide 38
in a good yield of 53%.9c,11 Cleavage of the protective
groups, except the Mtr group, was effected by a one-pot
catalytic hydrogenation over Pd-carbon in HOAc/THF/
H2O, and then deprotection of the Mtr group was readily
performed with excess trifluoroacetic acid in the presence
of thioanisole.22 HPLC purification and lyophilization
gave alterobactin A (1, Scheme 9), which was identical to
Thus, we have succeeded in synthesizing alterobactin A
(1), a super siderophore, which means that alterobactin B
(2) has been also formally synthesized because alterobac-
tin B is formed by base hydrolysis of 1.1
Discussion
Two independent reports24 have described the stereose-
lective preparation of the protected L-threo-β-hydroxy-
aspartic acids from D- and L-serine, respectively.
Noteworthy features of our synthesis are as follows: (a)
prochiral and commercially available methyl cinnamate
(5) was used as starting material, (b) two stereogenic cen-
ters of L-threo-β-OH-Asp were built at the same time
through Sharpless asymmetric dihydroxylation, (c) the α-
carboxylate of threo-β-OH-Asp was efficiently generated
by ruthenium oxidation of the phenyl group in good yield,
and (d) all reactions proceeded under relatively mild con-
ditions.
the natural product by 500 MHz 1H NMR, 125 MHz 13
C
NMR, high-resolution FAB mass spectra, HPLC and
TLC.23 Interestingly, we found that the pH value of the so-
lution strikingly affects the chemical shifts of the protons
on the two β-OH-Asp moieties and the NMR experiments
of alterobactin A (1) were performed in D2O with pH 1.6.
Alterobactin A (1) contains a β-OH-Asp-Gly sequence,
which is similar to the Asp-Gly sequence. The tendency of
the β-carboxylate in the aspartyl residue to acylate the
amido group of the next amino acid residue in a sequence,
and thereby the production of the aspartimide peptides,
has been often reported.18,19 Aspartimide formation is en-
hanced when the next residue is glycine.18,19a Indeed, as-
partimide formation was frequently encountered in our
approach. The coupling of 12 with the deprotected 24 with
TEA gave a mixture of the dipeptide 29 and the aspartim-
ide 32. However, the subsequent removal of the Boc group
Scheme 10

SYNTHESIS
Papers
632
of 29 with TFA and coupling with Nα-Boc-Arg(Mtr)-OH
(30) in the presence of TEA smoothly afforded the tripep-
tide 31 without any detectable formation of aspartimide.
When the hydroxyl group of the aspartyl residue remains
free, the aspartimide 36 was readily produced in the amide
coupling-process of the deprotected tripeptide 33 and 34.
THF was distilled from sodium/benzophenone ketyl. Et2O was dis-
tilled from LiAlH4. CH2Cl2 was distilled from CaH2. N-Methylmor-
pholine (NMM) was distilled and stored over KOH pellets. Et3N was
dried over sodium wire. DMF was dried over 4Å molecular sieves.
All other commercially available reagents were used as received.
Methyl (2S,3R)-2,3-Dihydroxy-3-phenylpropionate (6):
Furthermore, aspartimide formation predominates in the A 500-mL three-necked flask, equipped with a mechanical stirrer,
was charged with t-BuOH (100 mL), H2O (100 mL), and AD-mix-β
deprotection process of the tripeptide 31 at the C-terminus
[28.0 g, 60.0 mmol of K3Fe(CN)6, 60.0 mmol of K CO , 0.2 mmol of
2
3
with zinc in 1M NH4OAc-THF. Our results have shown
that these aspartimide formations can be entirely sup-
pressed under carefully selected conditions, which use a
less basic amine, NMM, and protection of the hydroxyl
group on the β-OH-Asp for the amide coupling, and use of
a relatively weak acid, TFA and HOAc, for the deprotec-
tion at the N- and C-terminus, respectively.
(DHQD)2-PHAL, and 0.04 mmol of K2OsO2(OH)4]. Stirring at r.t.
produced two clear phases with the lower aqueous phase appearing as
bright yellow. MeSO2NH2 (1.90 g, 20.0 mmol) was added at this
point. The mixture was cooled in an ice-bath whereupon some of the
dissolved salts precipitated. Methyl cinnamate (5; 3.24 g, 20.0 mmol)
was added at once in one portion, and the heterogeneous slurry was
stirred vigorously at this temperature for 12 h and then warmed to r.t.
for 12 h. After the mixture was recooled in an ice-bath, Na2SO3 (30 g,
238 mmol) was added and the mixture was allowed to warm to r.t. and
stirred for 1 h. EtOAc was added to the reaction mixture, and after
separation of the organic layer, the aqueous phase was further extract-
ed three times with EtOAc. The combined organic extracts were
washed with H2O, dried (Na2SO4), and concentrated in vacuo. The
crude residue was purified by chromatography (1:1 EtOAc/hexane) to
afford the 1,2-diol 6 as white crystals (2.67 g, 68%; >98% ee, by 1H
NMR analysis of the (+)-MTPA-diester); mp 80–81°C (EtOAc/hex-
ane); [α]D26 –9.7 (c = 1.07, CHCl3).
Two glycyl residues and two β-OH-Asp residues are in-
cluded in alterobactin A (1), and we have applied the same
building units, 24 and 12, for the construction of the pep-
tide skeleton. In addition, the selected protective groups
for the various reactive functional groups in alterobactin
A (1), kept intact in the deprotection and the coupling of
the peptide segments, were successfully removed in two
steps under relatively mild conditions. Surprisingly, a se-
lective deprotection of the Boc group in the tripeptide 31
which includes an Nω-Mtr blocked arginine was achieved
by using TFA in CH2Cl2 at 0°C. To our knowledge, the
selective deprotection is little known.8
IR (KBr): ν = 3492, 3384, 1717, 1458, 1323, 1308, 1275, 1223, 1111,
1049, 722, 700 cm–1.
1H NMR (270 MHz, CDCl3): δ = 7.31–7.42 (m, 5 H, arom), 5.02 (dd,
J = 2.8, 6.8 Hz, 1 H), 4.38 (dd, J = 3.0, 5.9 Hz, 1 H), 3.82 (s, 3 H,
OCH3), 3.10 (d, J = 5.9 Hz, 1 H), 2.72 (d, J = 6.9 Hz, 1 H).
Anal. C10H12O4 (196.2): calcd C, 61.22; H, 6.16; found: C, 61.14; H,
6.19.
Our synthesis of alterobactin A (1) has not only proved the
proposed structure, but also promises availability of this
super siderophore in quantities which will be useful for
the elucidation of the siderophoric mechanism in marine
bacteria.
Methyl (2R,3S)-2-Acetoxy-3-bromo-3-phenylpropionate (7):
To the diol 6 (2.67 g, 13.6 mmol) was added 31% HBr/HOAc
(21 mL), and the mixture was heated at 45°C for 30 min. After cool-
ing to r.t., the mixture was quenched by slowly pouring into an ice-
saturated aq NaHCO3 solution, and the aqueous layer was extracted
three times with Et2O. The combined organic extracts were washed
once with H2O and twice with brine, dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by chromatography
(1:12.5 EtOAc/hexane) to give 7 as white crystals (3.19 g, 78%); mp
78–79°C (EtOAc/hexane); [α]D26 +100.3 (c = 1.36, CHCl3).
IR (KBr): ν = 1763, 1748, 1493, 1451, 1433, 1377, 1260, 1219, 1198,
1090, 702 cm–1.
Melting points were determined on a YAMATO MP-21 and are un-
corrected. IR spectra were recorded on a SHIMADZU FTIR-8100
1
spectrometer. H NMR spectra were recorded on a JEOL EX-270
(270 MHz), JEOL GSX-400 (400 MHz), or JEOL α-500 (500 MHz)
spectrometer with TMS, or sodium 3-(trimethylsilyl)propanoate-d ,
4
or solvent as an internal standard, unless otherwise indicated. Abbre-
viations for NMR: s = singlet, d = doublet, t = triplet, q = quartet, dd
= doublet of doublets, m = multiplet, and br = broad. 13C NMR spectra
were obtained at 125 MHz on a JEOL α-500 (500 MHz) spectrometer
1H NMR (270 MHz, CDCl3): δ = 7.43–7.45 (m, 2 H), 7.31–7.35 (m,
3 H), 5.64 (d, J = 6.3 Hz, 1 H), 5.34 (d, J = 6.6 Hz, 1 H), 3.71 (s, 3 H,
OCH3), 2.11 (s, 3 H, OCCH3).
with sodium 3-(trimethylsilyl)propanoate-d as an internal standard.
4
Fast atom bombardment mass spectra (FAB-MS) were recorded on a
JEOL JMS-HX110A or JEOL JMS AX505HA high resolution spec-
trometer by using an argon beam in a glycerol or nitrobenzyl alcohol
(NBA) matrix. Accurate mass measurement was obtained by using a
JEOL JMS-HX110A high resolution spectrometer in the FAB mode
(glycerol and NBA matrices). Optical rotations were measured on a
JASCO DIP-140 digital polarimeter with a sodium lamp (λ = 589 nm,
D line). Microanalysis was carried out on a YANACO CHN CORD-
ER NT-5. TLC separations were conducted on 250 µm silica plates
(Merck Art. 5715, Kieselgel 60 F254) with visualization by UV fluo-
rescence, and ninhydrin, or anisaldehyde, or phosphomolybdic acid
staining by heating. Chromatography was carried out on a silica gel
column with Fuji Silysia BW-820 or 200, and flash chromatography
was accomplished on silica gel, Fuji Silysia BW-300. HPLC was car-
Anal. C12H13O4Br (301.2): calcd C, 47.86; H, 4.35; found: C, 47.73;
H, 4.33.
Methyl (2S,2R)-2-Acetoxy-3-azido-3-phenylpropionate (8):
To a solution of 7 (2.93 g, 9.72 mmol) in DMF (35 mL) was added
NaN3 (2.53 g, 38.9 mmol), and the mixture was stirred at r.t. for 3 h
and then heated at 40°C for 2 h. After cooling to r.t., the mixture was
diluted with EtOAc, and the organic layer was washed once with H2O
and brine, dried (Na SO ), filtered, and concentrated in vacuo. The
2
4
crude product was purified by chromatography (1:10 EtOAc/hexane)
to afford the azide 8 as a colorless oil (2.20 g, 86 %); [α]D26 –104.2 (c
= 2.33, CHCl3).
IR (neat): ν = 2109, 1825, 1755 (br), 1455, 1439, 1375, 1221 (br),
702 cm–1.
ried out on YMC-Pack C (preparative column: 250 × 20 mm I. D.,
4
1H NMR (270 MHz, CDCl3): δ = 7.35–7.40 (m, 5 H, arom), 5.23 (d,
J = 5.0 Hz, 1 H, α-H), 5.06 (d, J = 5.0 Hz, 1 H, β-H), 3.69 (s, 3 H,
OCH3), 2.14 (s, 3 H, OCCH3).
S-5 µm, 120Å; analytical column: 150 × 4.6 mm I. D., S-5 µm, 120Å)
connected in series, 280 nm or 320 nm on a SHIMADZU SPD-10A
UV-vis detector.

SYNTHESIS
April 1998
633
Anal. C12H13N3O4 (263.2): calcd C, 54.75; H, 4.97; N, 15.96; found:
C, 54.89; H, 5.14; N, 16.04.
cold 0.2 N aq HCl and EtOAc. The organic layer was separated and
washed once with H2O and brine, and dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by chromatography
(gradient from 1:9 to 1:7 EtOAc/hexane) to afford the alcohol 11 as a
white solid (500 mg, 63%); mp 100–101°C (EtOAc/hexane); [α]D22
+19.6 (c = 1.08, CHCl3).
Methyl (2S,3R)-2-Acetoxy-3-(tert-butoxycarbonylamino)-3-phe-
nylpropionate (9):
A suspension of 5% Pd/C (532 mg) in EtOAc (7 mL) was stirred at
r.t. under an H2 atmosphere for 15 min, whereupon a solution of di-
tert-butyl dicarbonate (4.36 g, 20 mmol) and the azide 8 (1.32 g,
5.0 mmol) in EtOAc (3 mL) was added.. The mixture was stirred un-
der an H2 atmosphere for 6 h, and then filtered through a pad of Celite.
The organic phase was washed with ice-cold 1M aq KHSO4, sat.
NaHCO3, and brine, dried (Na2SO4) and concentrated in vacuo. The
residue was purified by chromatography (gradient from 1:5 to 1:4
EtOAc/hexane) to give 9 as a colorless oil (1.14 g, 68%); [α]D26 +27.6
(c = 1.25, CHCl3).
IR (KBr): ν = 3407, 2984, 1744, 1698, 1509, 1370, 1350, 1298, 1258,
1206, 1152, 1096, 1064, 747, 698 cm–1.
1H NMR (400 MHz, CDCl3): δ = 7.27–7.43 (m, 5 H, arom), 5.24
(overlapping, 1 H, NH), 5.19 (ABq, J = 12.0 Hz, 2 H, CH2Ph), 4.69
(overlapping, 1 H, α-H), 4.67 (dd, J = 2.2, 6.6 Hz, 1 H, β-H), 3.12 (d,
J = 6.2 Hz, 1 H, β-OH), 1.49 (s, 9 H), 1.43 (s, 9 H).
Anal. C20H29NO7 (395.5): calcd C, 60.75; H, 7.39; N, 3.54; found: C,
60.76; H, 7.43; N, 3.45.
IR (neat): ν = 3375, 1755, 1751, 1717, 1514, 1499, 1370, 1229 (br),
(2S,3S)-N-Boc-â-(tert-Butyldimethylsilyloxy)aspartic Acid â-
Benzyl Ester (12):
1169, 1086, 758, 702 cm–1.
1H NMR (270 MHz, CDCl3): δ = 7.25–7.37 (m, 5 H, arom), 5.40 (br
s, 2 H, NH, α-H), 5.30 (s, 1 H, β-H), 3.77 (s, 3 H, OCH3), 2.08 (s, 3
H, OCCH3), 1.43 [s, 9 H, OC(CH3)3].
Anal. C17H23NO6 (337.4): calcd C, 60.53; H, 6.87; N, 4.15; found: C,
60.54; H, 7.16; N, 4.10.
To a stirred solution of the alcohol 11 (466 mg, 1.18 mmol) in CH2Cl2
(3.5 mL) cooled in an ice-bath was added dropwise trifluoroacetic
acid (TFA, 3.5 mL). The resulting mixture was stirred at this temper-
ature for 1 h and allowed to warm to r.t. over 4 h. The mixture was
concentrated in vacuo, and the residual TFA was removed twice by
diluting with CH2Cl2 (1 mL) and benzene (3 mL) and concentrating
in vacuo. The resulting amine salt was dissolved in H2O (6 mL) and
dioxane (6 mL) and cooled in an ice-bath, whereupon Et3N (0.41 mL,
3.0 mmol) was added dropwise with stirring, followed by Boc2O in
one portion. The mixture was stirred at this temperature for 20 min
and allowed to warm to r.t. for 4 h, and then concentrated in vacuo.
α-tert-Butyl â-Methyl (2S,3S)-N-Boc-â-Acetoxyaspartic Diester
(10):
To a stirred solution of 9 (1.14 g, 3.38 mmol) in EtOAc (10 mL) and
MeCN (10 mL) was added a suspension of NaIO4 (18.07 g, 84.5 mmol)
in H2O (70 mL) followed by RuCl3·H2O (46 mg, 0.20 mmol), and the
heterogeneous slurry was vigorously stirred at r.t. for 11 h. The mixture
was diluted with EtOAc, and after separation of the layers, the aqueous
layer was further extracted twice with EtOAc. The combined extracts
were dried (Na2SO4), filtered, and concentrated in vacuo. The residue
was dissolved in Et2O, and the solution was filtered through a pad of
Celite. The solvent was evaporated in vacuo to give a brown foam (981
mg). The crude acid was dissolved in CH2Cl2 (3.5 mL) and t-BuOH (14
mL) was added followed by BDIU (3.2 mL, 13.5 mmol). The mixture
was heated at 50°C for 11 h under an argon atmosphere, and after cool-
ing to r.t., it was filtered through a pad of Celite and concentrated in
vacuo. The residue was dissolved in EtOAc, and the solution was fil-
tered through a pad of Celite and then washed with ice-cold 1 M aq
KHSO4, sat. NaHCO3 and brine, dried (Na2SO4), and concentrated in
vacuo. The crude product was purified by chromatography (gradient
from 1:7 to 1:6 EtOAc/hexane) to afford the acetoxy diester 10 as a col-
orless wax (854 mg, 70%); mp 51–52°C (EtOAc/hexane); [α]D24 +47.2
(c = 0.57, CHCl3).
Ice–saturated NaHCO solution (v/v, 1:1, 20 mL) was added to the
3
residue, and then the aqueous layer was washed twice with Et2O and
acidified with 6 N aq HCl to pH 4 in an ice-bath and subsequently ex-
tracted twice with CH2Cl2. The combined extracts were dried
(Na SO ), filtered, and concentrated in vacuo to give a white foam
2
4
(468 mg). The resulting acid and imidazole (803 mg, 11.8 mmol)
were dissolved in DMF (4 mL) and cooled in an ice-bath, whereupon
TBSCl (889 mg, 5.9 mmol) was added in one portion. The mixture
was stirred at this temperature for 30 min and allowed to warm to r.t.
over 40 h under an argon atmosphere. Subsequently, the mixture was
poured into ice/1 M aq KHSO4 (v/v, 1:1) and the aqueous layer was
extracted three times with EtOAc. The combined organic extracts
were washed once with brine, dried (Na2SO4), filtered, and concen-
trated in vacuo. The crude product was purified by chromatography
(gradient from 1:8 to 1:2 EtOAc/hexane) to give 12 as an oily white
solid (448 mg, 84%); [α]D24 –6.8 (c = 1.88, CHCl3).
IR (neat): ν = 1761, 1723, 1667, 1501, 1256, 1165, 1132, 839,
IR (KBr): ν = 3337, 2980, 1761, 1750, 1742, 1700, 1528, 1512, 1370,
781 cm–1.
1
1283, 1233, 1225, 1154, 1092, 1065 cm–1.
H NMR (500 MHz, CDCl3): δ = 7.31–7.38 (m, 5 H, arom), 5.26 (d,
1H NMR (500 MHz, CDCl3): δ = 5.53 (d, J = 2.4 Hz, 1 H, β-H), 5.22
(d, J = 9.8 Hz, 1 H, NH), 4.86 (d, J = 9.8 Hz, 1 H, α-H), 3.76 (s, 3 H,
OCH3), 2.13 (s, 3 H, OCCH3), 1.45 (s, 9 H), 1.43 (s, 9 H).
Anal. C16H27NO8 (361.4): calcd C, 53.18; H, 7.53; N, 3.88; found: C,
53.02; H, 7.61; N, 3.85.
J = 8.6 Hz, 1 H, NH), 5.15 (ABq, J = 11.9 Hz, 2 H, CH2Ph), 4.84 (br
s, 2 H, α-H, β-H), 1.43 [s, 9 H, OC(CH3)3], 0.86 [s, 9 H, SiC(CH3)3],
0.08 (s, 3 H, SiCH3), 0.01 (s, 3 H, SiCH3).
Anal. C22H35NO7Si (453.6): calcd C, 58.26; H, 7.77; N, 3.09; found:
C, 57.73; H, 7.76; N, 3.06.
α-tert-Butyl â-Benzyl (2S,3S)-N-Boc-â-Hydroxyaspartic Diester
(11):
Ethyl (4S)-4-(Benzyloxycarbonylamino)-8-(tert-butyloxycarbon-
ylamino)-3-oxooctanoate (14):
To a solution of 10 (722 mg, 2.0 mmol) in THF (45 mL) cooled in an
ice-bath was added a 0.31 N aq LiOH solution (15 mL). The mixture
was stirred at this temperature for 3 h, and then quenched by adding
1.0 N aq HCl (4.6 mL). The aqueous layer was extracted three times
with EtOAc and the combined extracts were dried (Na2SO4) and fil-
tered. To remove the residual HOAc, the combined filtrate was con-
centrated to about 15 mL, followed by azeotropic removal of the
HOAc in vacuo with heptane (15 mL). The resulting acid was dis-
solved in DMF (7 mL) and cooled in an ice-bath, whereupon benzyl
bromide (0.48 mL, 4.0 mmol) was added. Then, ground K2CO3
(41.5 mg, 3.0 mmol) was added to the mixture, and the suspension
was stirred at this temperature for 5 h and partitioned between ice-
To a stirred solution of Nα-Cbz-Nω-Boc-Lys-OH (13; 1.90 g,
5.0 mmol) in THF (15 mL) at 0°C was added carbonyldiimidazole
(CDI; 973 mg, 6.0 mmol). The mixture was stirred at this temperature
for 1 h and at r.t. for 3 h, and then cooled to –18°C before the magne-
sium enolate solution in THF (15 mL) [prepared from ethyl hydrogen
malonate (1.32 g, 10.0 mmol) and 2 M i-PrMgCl (30 mmol) in THF
(12.5 mL) at –18°C for 45 min and at r.t. for 2.5 h] was added drop-
wise. After the mixture was stirred at –18°C for 1 h and allowed to
warm to r.t., stirring was continued for 4.5 h, and then the mixture was
poured into ice/1 M aq KHSO4. The aqueous layer was extracted
three times with EtOAc and the combined organic phases were
washed with sat. NaHCO3 and brine, dried (Na2SO4), and evaporated

SYNTHESIS
Papers
634
in vacuo. The crude product was purified by chromatography (gradi-
ent from 1:2.5 to 1:2 EtOAc/hexane) to give the β-keto ester 14 as a
white solid (1.86 g, 83%); mp 70–71°C (EtOAc/hexane); [α]D26 –19.9
(c = 1.97, MeOH).
with Et2O and then acidified with 1 N aq HCl (24 mL) to pH ca. 4,
and then extracted three times with CH2Cl2. The combined organic
phases were dried (Na2SO4), filtered, and concentrated in vacuo. The
crude product was recrystallized from EtOAc/hexane to provide the
acid 17 as white crystals (1.84 g, 92%); mp 113–114°C (EtOAc/hex-
ane).
IR (KBr): ν = 3357, 1736, 1717, 1686, 1678, 1526, 1277, 1252, 1169,
1032 cm–1.
1H NMR (270 MHz, CDCl3): δ = 7.36 (br s, 5 H, arom), 5.54 (d, J =
6.3 Hz, 1 H, 4-NH), 5.11 (s, 2 H, CH2Ph), 4.58 (br s, 1 H, 8-NH),
4.40–4.47 (m, 1 H, 4-H), 4.19 (q, J = 6.9 Hz, 2 H, OCH2Me), 3.55
(ABq, J = 15.8 Hz, 2 H, 2-CH2), 3.07–3.11 (m, 2 H, 8-CH2),
1.88–1.95 (m, 1 H, 5-H), 1.32–1.67 (m, 5 H, 5-H, 6- and 7-CH2), 1.42
[s, 9 H, OC(CH3)3], 1.27 (t, J = 6.9 Hz, 3 H, CH3).
IR (KBr): ν = 3032, 1694 (br), 1578, 1474, 1456, 1416, 1377, 1314
(br), 1262, 1086, 752, 698 cm–1.
1H NMR (270 MHz, CDCl3): δ = 11.34 (br s, 1 H, CO2H), 7.73–7.76
(dd, J = 1.8, 9.8 Hz, 1 H, catechol 4-H ), 7.16–7.50 (m, 12 H, arom),
5.27 (s, 2 H, CH2Ph ), 5.20 (s, 2 H, CH2Ph).
Anal. C21H18O4 (334.4): calcd C, 75.44; H, 5.42; found: C, 75.40; H,
5.43.
Anal. C23H34N2O7 (450.5): calcd C, 61.32; H, 7.60; N, 6.22; found:
C, 61.25; H, 7.49; N, 6.24.
Ethyl (3R,4S)-4-(Benzyloxycarbonylamino)-8-[2,3-bis(benzyl-
oxy)-benzoylamino]-3-hydroxyoctanoate (20):
Ethyl (3R,4S)-4-(Benzyloxycarbonylamino)-8-(tert-butyloxycar-
bonylamino)-3-hydroxyoctanoate (15a):
The lysinestatine derivative 15a (1.58 g, 3.5 mmol) was treated with
4 N HCl/dioxane (15 mL) at 0°C for 1 h and then at r.t. for 45 min.
The mixture was concentrated in vacuo, and the residual HCl was re-
moved by diluting with dioxane (3 mL) and toluene (9 mL) and evap-
orating in vacuo. The resulting amine salt (a white solid) and 2,3-
bis(benzyloxy)benzoic acid (17; 1.46 g, 4.4 mmol) were dissolved in
DMF (15 mL) and cooled in an ice-bath, whereupon DEPC (0.67 mL,
4.4 mmol) was added dropwise with stirring. After 15 min, Et3N
(1.12 mL, 8.1 mmol) was added, and the mixture was stirred at 0°C
for 4 h and subsequently allowed to warm to r.t. over 13 h. The mix-
ture was diluted with EtOAc/benzene (v/v, 3:1; 60 mL), and the or-
ganic layer was washed twice with ice-cold 1 M aq KHSO4, once with
sat. NaHCO3, and once with brine, dried (Na2SO4), filtered, and con-
centrated in vacuo. The residue was purified by chromatography (gra-
dient from 1:1 to 1.5:1 EtOAc/hexane) to give 20 as a white solid
(2.02 g, 86%); mp 114–115°C (EtOAc/hexane); [α]D26 –9.3 (c = 1.22,
CHCl3).
To a stirred solution of the oxo ester 14 (2.70 g, 6.0 mmol) in EtOH/
THF (v/v, 6:1; 105 mL) at –78°C was added portionwise NaBH4
(248 mg, 7.5 mmol). The mixture was stirred at this temperature for
1.5 h, and the reaction was quenched by pouring the mixture into ice-
cold 10 % aq citric acid (50 mL). The mixture was concentrated in
vacuo, and then diluted with H2O (50 mL). The aqueous layer was ex-
tracted three times with EtOAc and the combined organic phase was
washed once with brine, dried (Na2SO4), filtered, and concentrated in
vacuo to give a mixture of the starting material 14 and diastereomers
1
of the alcohol 15 [(3R,4S)/(3S,4S) 85:15 by H NMR analysis] as a
white solid. The crude product was purified by flash chromatography
(2:1 Et2O/hexane) to recover 14 (245 mg, 9%) and provide the desired
diastereomer (3R,4S)-15a as a white solid (1.80 g, 66%, 73 % conver-
1
sion; >95% de by H NMR analysis); mp 91–92°C (Et O/hexane).
2
The analytical sample was recrystallized twice from Et2O/hexane
(>98% de); [α]D26 –9.4 (c = 1.10, CHCl3).
IR (KBr): ν = 3355, 3326, 1732, 1690, 1541, 1281, 1244, 1175, 1061,
1019 cm–1.
IR (KBr): ν = 3306, 1728, 1684, 1639, 1576, 1541, 1246, 1057, 747,
696 cm–1.
1
H NMR (500 MHz, CDCl3): δ = 7.29–7.39 (m, 5 H, arom), 5.10 (s,
1H NMR (400 MHz, CDCl3): δ = 7.94 (br s, 1 H, LysSta 8-NH), 7.72
(dd, J = 1.6, 7.8 Hz, 1 H, catechol 6-H), 7.28–7.48 (m, 15 H,
3 × C6H5), 7.12 (d, J = 6.9 Hz, 1 H, catechol 4-H), 7.06 (t, J = 7.9 Hz,
1 H, catechol 5-H), 5.15 (s, 2 H, CH2Ph), 5.09 (br s, 1 H), 5.06 (s, 2
H, CH2Ph), 5.05 (ABq, J = 12.0 Hz, 2 H, CH2Ph), 4.16 (q, J = 7.1 Hz,
2 H, OCH2CH3), 3.98–4.01 (m, 1 H, 3-H), 3.57–3.60 (m, 1 H, 4-H),
3.41 (d, J = 4.0 Hz, 1 H, 3-OH), 3.32–3.39 (m, 1 H, 8-H), 3.11–3.17
(m, 1 H, 8-H), 2.46–2.51 (m, 2 H, 3-CH2), 1.54–1.57 (m, 1 H),
1.24–1.44 (m, 5 H), 1.27 (t, J = 7.1 Hz, 3 H, OCH2CH3).
2 H, CH2Ph), 4.97 (d, J = 7.7 Hz, 1 H, NH), 4.56 (br s, 1 H, NH), 4.16
(q, J = 7.1 Hz, 2 H, OCH2CH3), 4.14–4.19 (m, 1 H, 3-H), 3.63–3.65
(m, 1 H, 4-H), 3.35 (br s, 1 H, 3-OH), 3.09 (br s, 2 H, 8-CH2),
2.45–2.54 (m, 2 H, 2-CH2), 1.59–1.62 (m, 3 H), 1.42 [s, 9 H,
OC(CH3)3], 1.30–1.49 (m, 3 H), 1.26 (t, J = 7.1 Hz, 3 H, OCH2CH3).
Anal. C23H36N2O7 (452.6): calcd C, 61.05; H, 8.01; N, 6.19; found:
C, 60.82; H, 7.84; N, 6.13.
2,3-Bis(benzyloxy)benzoic Acid (17):
Anal. C39H44N2O8 (668.9): calcd C, 70.04; H, 6.63; N, 4.19; found: C,
69.86; H, 6.62; N, 4.01.
To a solution of catecholic acid (19; 1.54 g, 10.0 mmol) and benzyl
bromide (5.4 mL, 45.0 mmol) in DMF (20 mL) was added pulverized
K2CO3 (8.29 g, 60.0 mmol). The mixture was stirred at r.t. for 5 h and
then poured into ice-water. The aqueous layer was extracted three
times with EtOAc and the combined organic phases were washed
with ice-cold 1 M aq KHSO4, H2O, and brine, dried (Na2SO4), and
concentrated in vacuo. The residue was purified by chromatography
(gradient from 1:15 to 1:10 EtOAc/hexane) to give a tribenzylated
product as a white wax (4.15 g, 98%); mp 40°C (EtOAc/hexane).
IR (KBr): ν = 1709 (br), 1578, 1497, 1469, 1456, 1375, 1366, 1319,
1283, 1258, 1132, 1035, 750, 746, 696 cm–1.
(3R,4S)-4-(Benzyloxycarbonylamino)-8-[2,3-bis(benzyloxy)ben-
zoylamino]-3-(tert-butyldimethylsilyloxy)octanoic Acid (21):
To a stirred solution of the ester 20 (1.20 g, 1.79 mmol) in THF
(35 mL) was added dropwise an aq solution of 0.24 N LiOH at 0°C,
and then the mixture was stirred at this temperature for 3 h and diluted
with H2O (35 mL). Aq 1N HCl (0.31 mL) was added to the mixture
to quench the reaction, and the aqueous layer was subsequently satu-
rated with NaCl and extracted three times with EtOAc/benzene (v/v,
1:1). The combined extracts were dried (Na2SO4), filtered, and con-
centrated in vacuo to give a white solid (1.14 g). The resulting acid
and imidazole (1.22 g, 17.9 mmol) were dissolved in DMF (6 mL)
and cooled in an ice-bath, whereupon TBSCl (1.35 g, 8.97 mmol) was
added in one portion. The mixture was stirred at 0°C for 15 min and
allowed to warm to r.t. over 17.5 h under an argon atmosphere. The
reaction was quenched by partitioning between ice-cold 1 M aq
KHSO4 solution and EtOAc. The organic layer was separated and the
aqueous layer was further extracted twice with EtOAc. The combined
organic extracts were washed with ice-cold 1 M aq KHSO4 and brine,
dried (Na2SO4), filtered and concentrated in vacuo. The residue was
dissolved THF/MeOH (v/v, 2:1, 24 mL), and an aq solution of K2CO3
1H NMR (270 MHz, CDCl3): δ = 7.24–7.45 (m, 16 H, arom), 7.14
(dd, J = 1.8, 7.9 Hz, 1 H, catechol 4-H), 7.08 (dd, J = 8.3, 15.9 Hz, 1
H, catechol 5-H), 5.31 (s, 2 H, CH2Ph), 5.13 (s, 2 H, CH2Ph), 5.06 (s,
2 H, CH2Ph).
Anal. C28H24O4 (424.5): calcd C, 79.23; H, 5.70; found: C, 79.12; H,
5.78.
The resulting tribenzylated product (2.53 g, 5.96 mmol) was dis-
solved in THF (75 mL) and cooled in an ice-bath, whereupon a solu-
tion of LiOH•H2O (1.00 g, 23.8 mmol) in H2O (25 mL) was added.
The mixture was allowed to warm to r.t. and stirred for 3 days, and
then diluted with H2O (75 mL). The aqueous layer was washed twice

SYNTHESIS
April 1998
635
(273 mg, 1.97 mmol) in H2O (8 mL) was added to this solution at
0°C. The mixture was stirred at this temperature for 1.5 h, and then
poured into ice-cold 0.2 N aq HCl to quench the reaction. The aque-
ous layers were extracted three times with EtOAc, and the combined
extracts were washed once with brine, dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by chromatography
(gradient from 1:4 to 2:1 EtOAc/hexane) to give the acid 21 as an oily
white solid (1.17 g, 87%); [α]D24 –10.1 (c = 1.11, CHCl3).
Tripeptide 27:
Boc-D-Ser-Gly-OTce (26; 673 mg, 1.71 mmol) was treated with 4 N
HCl/dioxane (8 mL) at 0°C. The mixture was stirred at this temper-
ature for 30 min and then at r.t. for 1.5 h, and concentrated in vacuo.
The residue was dissolved in dioxane (2 mL) and toluene (6 mL),
and the volatiles were evaporated in vacuo. The resulting amine salt
and the acid component 21 (1.17 g, 1.55 mmol) were dissolved in
DMF (5 mL), and DEPC (0.26 mL, 1.71 mmol) was added to this
solution at 0°C. After the mixture was stirred for 15 min, NMM
(0.36 mL, 3.26 mmol) was added, and the mixture was stirred at 0°C
for 40 h and then diluted with EtOAc/benzene (v/v, 3:1). The organ-
ic phases were washed twice with ice-cold 1 M aq KHSO4 solution,
once with sat. NaHCO3 solution, and brine, dried (Na2SO4), and
concentrated in vacuo. The crude product was purified by chroma-
tography (gradient from 1.5:1 to 3.5:1 EtOAc/hexane) to recover the
acid component 21 as a colorless oil (400 mg, 34% recovery) and to
afford the tripeptide 27 as a white solid (813 mg, 51%, 77% conver-
sion); mp 42–43°C (EtOAc/hexane); [α]D23 +9.2 (c = 0.96, MeOH).
IR (KBr): ν = 3380 (br), 2953, 2930, 1771, 1701, 1649 (br), 1576,
1536 (br), 1454, 1262, 1175, 1082 (br), 837, 777, 733, 698 cm–1.
1H NMR (270 MHz, CDCl3): δ = 7.98 (t, J = 5.1 Hz, 1 H, LysSta 8-
NH), 7.69 (dd, J = 2.0, 7.6 Hz, 1 H, catechol 6-H), 7.66 (t, J = 3.6 Hz,
1 H, Gly NH), 7.28–7.49 (m, 15 H, 3 × C6H5), 7.05–7.15 (m, 2 H, cat-
echol 5-H, 4-H), 5.19 (s, 2 H, CH2Ph), 5.08 (overlapping d, J = 5.3
Hz, 1 H, Ser NH), 5.05 (s, 2 H, CH2Ph), 5.01 (s, 2 H, CH2Ph), 4.70
(ABq, J = 11.9 Hz, 2 H, CH2CCl3), 4.54–4.55 (m, 1 H, Ser α-H), 4.20
(dd, J = 6.0, 17.9 Hz, 1 H, Gly α-H), 4.14 (overlapping dd, J = 4.3 Hz,
1 H, Ser β-H), 4.07 (overlapping m, 1 H, LysSta 3-H), 4.00 (dd, J =
5.1, 18.0 Hz, 1 H, Gly α-H), 3.67 (overlapping dd, J = 4.6 Hz, 1 H,
Ser β-H), 3.59–3.66 (m, 1 H, LysSta 4-H), 3.17–3.34 (m, 2 H, LysSta
IR (CHCl3): ν = 3378, 1717 (br), 1653, 1647, 1576, 1539, 1456, 1262,
1217, 1084, 756, 698 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.97 (br s, 1 H, 8-NH), 7.71 (dd, J
= 1.8, 7.9 Hz, 1 H, catechol 6-H), 7.29–7.47 (m, 15 H, 3 × C6H5), 7.13
(d, J = 6.7 Hz, 1 H, catechol 4-H), 7.08 (t, J = 7.9 Hz, 1 H, catechol
5-H), 5.15 (s, 2 H, CH2Ph), 5.06 (s, 2 H, CH2Ph), 5.05 (ABq, J = 11.9
Hz, 2 H, CH2Ph), 4.93 (d, J = 8.5 Hz, 1 H, 4-NH), 4.15 (br s, 1 H, 3-
H), 3.60 (br s, 1 H, 4-H), 3.28 (br s, 1 H, 8-H), 3.20 (br s, 1 H, 8-H),
2.44–2.51 (m, 2 H, 3-CH2), 1.56 (br s, 1 H), 1.22–1.33 (m, 5 H), 0.87
[s, 9 H, SiC(CH3)3], 0.06 (s, 3 H, SiCH3), 0.05 (s, 3 H, SiCH3).
Anal. C43H54N2O8Si (755.0): calcd C, 68.41; H, 7.21; N, 3.71; found:
C, 68.17; H, 7.30; N, 3.69.
Boc-Gly-OTce (24):
To a stirred solution of Boc-Gly-OH (23; 4.96 g, 28.3 mmol), 2,2,2-
trichloroethanol (3.4 mL, 35.4 mmol) and DMAP (864 mg,
7.1 mmol) in CH2Cl2 (150 mL) at 0°C was added DCC (6.72 g,
32.5 mmol) in one portion. The mixture was stirred at this tempera-
ture for 2 h, allowed to warm to r.t., stirred for 18 h, and then filtered
through a pad of Celite. The filtrate was washed with ice-cold 1 M aq
KHSO4 solution, sat. NaHCO3 solution and brine, dried (Na2SO4),
and concentrated in vacuo. The residue was purified by chromatogra-
phy (gradient from 1:8 to 1:6 EtOAc/hexane) to give the ester 24 as a
white solid (7.98 g, 92% ); mp 70–71°C (EtOAc/hexane).
IR (KBr): ν = 3416, 1782, 1771, 1690 (br), 1518 (br), 1371, 1281,
1262, 1154 (br), 819, 723 cm–1.
8-CH ), 2.53 (br s, 2 H, LysSta 2-CH2), 2.00 (br s, 1 H, Ser OH), 1.89
2
(br s, 1 H), 1.22–1.69 (m, 5 H), 0.89 [s, 9 H, SiC(CH3)3], 0.08 [s, 6 H,
Si(CH3)2].
Anal. C50H63Cl3N4O11Si (1030.5): calcd C, 58.28; H, 6.16; N, 5.44;
found: C, 58.08; H, 6.16; N, 5.48.
1H NMR (270 MHz, CDCl3): δ = 5.01 (br s, 1 H, NH), 4.80 (s, 2 H,
CH2Tce), 4.07 (d, J = 5.6 Hz, 2 H, CH2N), 1.46 [s, 9 H, OC(CH3)3].
Anal. C9H14NO4Cl3 (306.6): calcd C, 35.26; H, 4.60; N, 4.57; found:
C, 35.11; H, 4.54; N, 4.57.
Depsitetrapeptide 28:
The tripeptide 27 (421 mg, 0.41 mmol), the acid component 12
(161 mg, 0.36 mmol), and DMAP (6 mg, 0.05 mmol) were dissolved
in CH2Cl2 (2.5 mL) and cooled in an ice-MeOH bath, whereupon
EDCI (102 mg, 0.53 mmol) was added. The mixture was stirred at this
temperature for 30 min, and then in an ice-bath for 24 h. After dilution
with EtOAc, the organic layer was washed with ice-cold 1 M aq
KHSO4 solution, sat. NaHCO3 solution, and brine, dried (Na2SO4),
and concentrated in vacuo. The residue was purified by chromatogra-
phy (3:5 then 1:1 EtOAc/hexane) to recover the tripeptide 27 (88 mg,
21% recovery) and give the depsipeptide 28 as a white solid (427 mg,
82%; 87% conversion); mp 53–54°C (EtOAc/hexane); [α]D23 +2.8 (c
= 1.06, CHCl3).
IR (KBr): ν = 3393, 2955, 2932, 1763, 1719, 1701, 1686, 1655, 1647,
1638, 1541, 1509, 1499, 1260, 1163, 1132, 839, 781, 735, 698 cm–1.
1H NMR (270 MHz, CDCl3): δ = 7.95 (br s, 1 H, LysSta 8-NH), 7.69
(dd, J = 2.3, 7.3 Hz, 1 H, catechol 6-H), 7.30–7.49 (m, 22 H, 4 × C6H5,
Gly NH, β-OHAsp NH), 7.07–7.13 (m, 2 H, catechol 5-H, 4-H), 5.36
(d, J = 9.2 Hz, 1 H, Ser NH), 5.14 (s, 2 H, CH2Ph), 5.13 (s, 2 H,
CH2Ph), 5.09 (overlapping br s, 1 H, LysSta 4-NH), 5.05 (d, J = 1.3
Hz, 2 H, CH2Ph), 5.02 (s, 2 H, CH2Ph), 4.81–4.84 (m, 3 H, Ser α-H,
β-OHAsp α-H, β-H), 4.66 (s, 2 H, CH2CCl3), 4.51 (br s, 1 H, Ser α-
H), 4.42 (dd, J = 5.3, 11.2 Hz, 1 H, Ser β-H), 4.04–4.15 (m, 3 H, Lys-
Sta 3-H, Gly α-CH2), 3.59 (br s, 1 H, LysSta 4-H), 3.30 (br s, 1 H,
LysSta 8-H), 3.19 (br s, 1 H, LysSta 8-H), 2.46 (br s, 2 H, LysSta 2-
CH2), 1.64 (br s 1 H), 1.41 [s, 9 H, OC(CH3)3], 1.27–1.42 (m, 5 H),
0.86 [s, 9 H, SiC(CH3)3], 0.84 [s, 9 H, SiC(CH3)3], 0.07 [s, 6 H,
Si(CH3)2], 0.05 (s, 3 H, SiCH3), –0.03 (s, 3 H, SiCH3).
Boc-D-Ser-Gly-OTce (26):
Boc-Gly-OTce (24; 2.30 g, 7.5 mmol) was treated with 4 N HCl/di-
oxane (15 mL) at 0°C. The mixture was stirred at this temperature for
1 h, then warmed to r.t. for 1.5 h, and concentrated in vacuo. The res-
idue was dissolved in dioxane (3 mL) and toluene (9 mL), and the vol-
atiles were removed in vacuo. The resulting amine salt and Boc-D-
Ser-OH (25; 1.69 g, 8.25 mmol) were dissolved in DMF (20 mL), and
then DEPC (1.5 mL, 9.75 mmol) at 0°C was added to this solution
with stirring. After 15 min, Et3N (2.3 mL, 16.9 mmol) was added and
the mixture was stirred at 0°C for 5 h and then at r.t. for 2 h, and di-
luted with EtOAc/benzene (v/v, 3:1). The organic layer was washed
twice with ice-cold 1 M aq KHSO4 solution, once with sat. NaHCO3
solution, and brine, dried (Na2SO4), and concentrated in vacuo. The
crude product was purified by chromatography (gradient from 1:1 to
1.5:1 EtOAc/hexane) to afford the dipeptide 26 as a white solid (1.77
g, 60%); mp 92–93°C (EtOAc/hexane); [α]D26 +27.8 (c = 1.04,
CHCl3).
IR (KBr): ν = 3310 (br), 3092, 1767, 1680 (br), 1541, 1368, 1298,
1254, 1057, 927 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.22 (br s, 1 H, Gly NH), 5.56 (br s,
1 H, Ser NH), 4.78 (ABq, J = 11.9 Hz, 2 H, CH2CCl3), 4.26 (dd, J =
6.1, 18.3 Hz, 1 H, Gly α-H), 4.24 (br s, 1 H, Ser α-H), 4.17 (dd, J =
5.5, 18.3 Hz, 1 H, Gly α-H), 4.12 (br s, 1 H, Ser β-H), 3.69 (br s, 1H,
Ser β-H), 2.91 (br s, 1 H, OH), 1.46 [s, 9 H, OC(CH3)3].
Anal. C12H19Cl3N2O6 (393.6): calcd C, 36.62; H, 4.86; N, 7.12;
found: C, 36.51; H, 4.82; N, 7.08.
Anal. C72H96Cl3N5O17Si2 (1466.1): calcd C, 58.99; H, 6.60; N, 4.78;
found: C, 58.79; H, 6.44; N, 4.87.

SYNTHESIS
Papers
636
Dipeptide 29:
IR (KBr): ν = 3450, 3350, 2953, 2932, 1759, 1721, 1655, 1649, 1576,
1541, 1499, 1456, 1260, 1213, 1163, 1130, 839, 781, 698 cm–1.
1H NMR (270 MHz, CDCl3): δ = 8.20 (t, J = 5.4 Hz, 1 H, LysSta 8-
NH), 7.66 (dd, J = 3.1, 6.7 Hz, 1 H, catechol 6-H), 7.24–7.48 (m, 20
H, 4 × C6H5), 7.12–7.20 (m, 2 H, catechol 5-H, 4-H), 7.02 (t, J =
4.6 Hz, 1 H, Gly NH), 5.62 (d, J = 9.9 Hz, 1 H, β-OHAsp NH), 5.29
(d, J = 9.2 Hz, 1 H, NH), 5.15 (s, 2 H, CH2Ph), 5.08–5.12 (m, 4 H,
CH2Ph, CHPh, NH), 5.04 (s, 2 H, CH2Ph), 4.90 (d, J = 12.5 Hz, 1 H,
CHPh), 4.79 (d, J = 9.9 Hz, 1 H, β-OHAsp α-H), 4.74 (d, J = 2.3 Hz,
1 H, β-OHAsp β-H), 4.67–4.71 (m, 1 H, Ser α-H), 4.52–4.57 (m, 1 H,
Ser β-H), 4.35 (dd, J = 6.3, 11.2 Hz, 1 H, Ser β-H), 4.19–4.20 (m, 1
H, LysSta 3-H), 3.99–4.07 (m, 1 H, Gly α-H), 3.72–3.79 (m, 1 H, Gly
α-H), 3.49–3.54 (m, 1 H, LysSta 4-H), 3.20–3.31 (m, 2 H, LysSta 8-
CH2), 2.43–2.59 (m, 2 H, LysSta 2-CH2), 1.62–1.65 (m, 1 H), 1.40 [s,
9 H, OC(CH3)3], 1.17–1.46 (m, 5 H), 0.87 [s, 9 H, SiC(CH3)3], 0.84
[s, 9 H, SiC(CH3)3], 0.10 (s, 3 H, SiCH3), 0.08 (s, 3 H, SiCH3), 0.05
(s, 3 H, SiCH3), 0.02 (s, 3 H, SiCH3).
To a stirred solution of the tripeptide 31 (38 mg, 0.038 mmol) in
CH2Cl2 (1 mL) at 0°C was added dropwise TFA (1 mL). The mixture
was stirred at 0°C for 2 h, and concentrated in vacuo. The residue was
dissolved in CH2Cl2 (1 mL) and benzene (3 mL), and the volatiles
were removed in vacuo. The resulting amine salt and the above acid
(33; 44 mg, 0.033 mmol) were dissolved in DMF (0.2 mL), and NMM
(16 µL, 0.15 mmol) at 0 °C was added dropwise, followed by solid
FDPP (38 mg, 0.098 mmol) in one portion. The mixture was stirred at
0°C for 42 h, diluted with EtOAc. The organic layer was washed with
ice-cold 1 M aq KHSO4 solution, sat. NaHCO3 solution, and brine,
dried (Na2SO4), and concentrated in vacuo. The crude product was
purified by chromatography (gradient from 1:1 to 2:1 EtOAc/hexane)
to give the depsiheptapeptide 37 as a white solid (63 mg, 86%); mp
81–82°C (EtOAc/hexane); [α]D25 +2.5 (c = 0.86, CHCl3).
A mixture of Boc-Gly-OTce (24; 115 mg, 0.38 mmol) and 4 N HCl/
dioxane (2 mL) was stirred at 0°C for 30 min and at r.t. for 1 h, and
then concentrated in vacuo. The residue was dissolved in dioxane
(1 mL) and toluene (3 mL), and the volatiles were removed in vacuo.
The resulting amine salt and the acid component 12 (138 mg,
0.3 mmol) were dissolved in DMF (1 mL), whereupon DEPC (68 µL,
0.45 mmol) at 0°C was added to this solution. After the mixture was
stirred for 15 min, NMM (79 µL, 0.72 mmol) was added, and then the
mixture was further stirred at 0°C for 20 h. After dilution with ben-
zene/EtOAc (v/v, 1:3, 20 mL), the organic layer was washed with ice-
cold 1 M aq KHSO4 solution, sat. NaHCO3 solution, and brine, dried
(Na2SO4), and concentrated in vacuo. The crude product was purified
by chromatography (1:8 EtOAc/hexane) to give the dipeptide 29 as an
1
oily white solid (165 mg, 86%; 29, aspartimide 32 >120:1 from H
NMR).
IR (neat): ν = 3422, 3359, 2955, 2932, 1759, 1723, 1717, 1682, 1498,
1368, 1256, 1165, 1129, 839, 782, 727 cm–1.
1H NMR (270 MHz, CDCl3): δ = 7.31–7.35 (m, 5 H, arom), 7.01 (br
s, 1 H, Gly NH), 5.36 (br s, β-OHAsp NH), 5.13 (ABq, J = 11.9 Hz,
2 H, CH2Ph), 4.94 (d, J = 3.0 Hz, β-OHAsp β-H), 4.79 (ABq, J = 11.9
Hz, CH2CCl3), 4.66 (d, J = 8.3 Hz, β-OHAsp α-H), 4.18 (br s, 2 H,
Gly α-CH2), 1.44 [s, 9 H, OC(CH3)3], 0.86 [s, 9 H, SiC(CH3)3], 0.08
(s, 3 H, SiCH3), 0.02 (s, 3 H, SiCH3).
FAB-MS: m/z = 641, 643, 645 (MH+).
Tripeptide 31:
To a solution of the dipeptide 29 (151 mg, 0.24 mmol) in CH2Cl2
(1.5 mL) at 0°C was added dropwise TFA (1.5 mL). The mixture
was stirred at 0°C for 2 h, and then concentrated in vacuo. The res-
idue was dissolved in CH2Cl2 (1 mL) and benzene (3 mL), and the
volatiles were removed in vacuo. The resulting amine salt and Boc-
Arg(Mtr)-OH (143 mg, 0.29 mmol) were dissolved in DMF (1 mL),
whereupon DEPC (54 µL, 0.36 mmol) at 0°C was added. After
15 min, TEA (79 µL, 0.57 mmol) was added and the mixture was
stirred at 0°C for 16 h, then diluted with EtOAc/benzene (v/v, 3:1,
20 mL). The organic layer was washed with ice-cold 1 M aq KHSO4
solution, sat. NaHCO3 solution, and brine, dried (Na2SO4), and con-
centrated in vacuo. The crude product was purified by chromatogra-
phy (1:1 then 1.5:1 EtOAc/hexane) to give the tripeptide 31 as a
white solid (217 mg, 91%); mp 73–74°C (EtOAc/hexane); [α]D24
–13.3 (c = 1.00, CHCl3).
IR (KBr): ν = 3416, 3343, 2955, 2934, 1759, 1686, 1624, 1586,
1551, 1508, 1474, 1458, 1368, 1308, 1256, 1163, 1121, 839 cm–1.
1H NMR (270 MHz, CDCl3): δ = 7.53 (br s, 1 H, NH), 7.27–7.31 (m,
6 H, C6H5, NH), 6.94 (d, J = 8.9 Hz, 1 H, β-OHAsp NH), 6.53 (s, 1
H, Mtr 5-H), 6.19 (br s, guanidino), 5.11 (ABq, J = 12.0 Hz, 2 H,
CH2Ph), 4.97 (s, 1 H, β-OHAsp β-H), 4.90 (d, J = 8.6 Hz, β-OHAsp
α-H), 4.75 (s, 2 H, CH2CCl3), 4.01–4.14 (m, 3 H, Arg α-H, Gly α-
CH2), 3.81 (s, 3 H, Mtr OCH3), 3.12–3.14 (m, 2 H, Arg δ-CH2), 2.70
(s, 3 H, Mtr 2-CH3), 2.63 (s, 3 H, Mtr 6-CH3), 2.12 (s, 3 H, Mtr 3-
CH3), 1.27–1.67 (m, 4 H, Arg β-CH2, γ-CH2), 1.42 [s, 9 H,
OC(CH3)3], 0.88 [s, 9 H, SiC(CH3)3], 0.07 (s, 3 H, SiCH3), 0.05 (s, 3
H, SiCH3).
IR (KBr): ν = 3351 (br), 2953, 2932, 1759, 1721, 1675 (br), 1541,
1501, 1260, 1163, 1123, 839, 781, 698 cm–1.
1H NMR (270 MHz, CDCl3): δ = 8.10 (br s, 1 H, LysSta 8-NH), 7.78
(br s, 1 H, Gly NH), 7.62 (d, J = 7.3 Hz, 1 H, catechol 6-H), 6.96–7.45
(m, 31 H, arom, NH), 6.50 (s, 1 H, Mtr 5-H), 6.20 (br s, 3H, guanidi-
no), 5.83 (br s, 1 H, NH), 5.34 (d, J = 8.9 Hz, 1 H, NH), 4.95–5.14 (m,
12 H, 5 × CH2Ph, 2 × β-OHAsp α-H), 4.75 (s, 2 H, CH2CCl3),
4.60–4.72 (m, 3 H, 2 × β-OHAsp β-H, Ser α-H), 4.46 (d, J = 7.9 Hz,
1 H, Ser β-H), 3.96–4.24 (m, 5 H, Ser β-H, 3 × Gly α-H, LysSta 3-H),
3.79 (s, 3 H, Mtr OCH3), 3.70 (d, J = 14.4 Hz, 1 H, Gly α-H), 3.43 (br
s, 1 H, LysSta 4-H), 3.29 (br s, 1 H, LysSta 8-H), 3.12 (br s, 3 H,
LysSta 8-H, Arg δ-CH2), 2.70 (s, 3 H, Mtr 2-CH3), 2.63 (s, 3 H, Mtr
6-CH3), 2.43–2.53 (m, 2 H, LysSta 2-CH2), 2.11 (s, 3 H, Mtr 3-CH3),
1.75 (br s, 1 H), 1.41 [s, 9 H, OC(CH3)3], 1.25–1.47 (m, 9 H), 0.88 [s,
9 H, SiC(CH3)3], 0.83 [s, 18 H, 2 × SiC(CH3)3], 0.06 [s, 6 H,
Si(CH3)2], 0.05 (s, 3 H, SiCH3), 0.01 (s, 3 H, SiCH3), –0.03 [s, 6 H,
Si(CH3)2].
Anal. C107H148Cl3N11O26SSi3 (2227.8): calcd C, 57.71; H, 6.69; N,
6.92; found: C, 57.52; H, 6.66; N, 6.98.
Cyclic Depsiheptapeptide 38:
Linear depsiheptapeptide 37 (100 mg, 0.045 mmol) was dissolved in
HOAc followed by zinc powder (294 mg, 4.5 mmol). The suspension
was vigorously stirred at r.t. for 4.5 h, diluted with EtOAc, and fil-
tered through a pad of Celite. The filtrate was washed with ice-cold
1 M aq KHSO4 solution, and brine, dried (Na2SO4), and filtered. The
organic solvent was concentrated in vacuo to 15 mL followed by
azeotropic removal of HOAc in vacuo with heptane (15 mL) to afford
the free acid as a white solid (90 mg, 96%).
Anal. C42H63Cl3N6O12SSi (1010.9): calcd C, 49.92; H, 6.28; N, 8.32;
found: C, 50.05; H, 6.33; N, 8.46.
Linear Depsiheptapeptide 37:
To a stirred solution of the depsitetrapeptide 28 (172 mg, 0.12 mmol)
in THF (1.5 mL) was added zinc powder (460 mg, 7.04 mmol), fol-
lowed by 1 M aq NH4OAc (0.3 mL). The heterogeneous slurry was
vigorously stirred at r.t. for 20 h, then diluted with EtOAc (20 mL),
and filtered through a pad of Celite. The combined filtrate was
washed with ice-cold 1 M aq KHSO4 solution, and brine, dried
(Na2SO4), and concentrated in vacuo. The residue was purified by
chromatography (1:1 EtOAc/hexane then 25:1 CH2Cl2/MeOH) to
give the acid 33 as a white solid (136 mg, 87%).
1H NMR (270 MHz, CDCl3): δ = 8.10 (br s, 1 H, NH), 7.72 (br s, 1
H, NH), 7.62 (d, J = 7.6 Hz, 1 H, catechol 6-H), 7.05–7.47 (m, 31 H,
arom, catechol, and NH), 6.50 (s, 1 H, Mtr 5-H), 6.35 (br s, guanidi-
no), 5.60 (br s, 1 H, NH), 5.33 (d, J = 8.9 Hz, 1 H, NH), 4.92–5.18 (m,
12 H, 5 × CH2Ph, 2 × β-OHAsp α-H), 4.74–4.78 (m, 3 H, 2 × β-
OHAsp α-H, Ser α-H), 4.52–4.57 (m, 1 H), 4.37 (br s, 1 H),
4.01–4.20 (m, 3 H), 3.80 (br s, 1 H), 3.79 (s, 3 H, Mtr OCH3), 3.48 (br

SYNTHESIS
April 1998
637
s, 1 H, LysSta 4-H), 3.10 (br s, 4 H, LysSta 8-CH2), 2.67 (s, 3 H, Mtr
2-CH3), 2.60 (s, 3 H, Mtr 6-CH3), 2.51 (br s, 2 H, LysSta 2-CH2), 2.10
(s, 3 H, Mtr 3-CH3), 1.25–1.66 (m, 10 H), 1.39 [s, 9 H, OC(CH3)3],
0.85 [s, 9 H, SiC(CH3)3], 0.81 [s, 9 H, SiC(CH3)3], 0.80 [s, 9 H,
SiC(CH3)3], 0.06 (s, 3 H, SiCH3), 0.03 (s, 3 H, SiCH3), 0.02 [s, 6 H,
Si(CH3)2], –0.04 (s, 3 H, SiCH3), –0.06 (s, 3 H, SiCH3).
H), 7.08 (d, J = 7.9 Hz, 1 H, catechol 4-H), 6.87 (t, J = 7.9 Hz, 1 H,
catechol 5-H), 5.16 (d, J = 3.1 Hz, 1 H, β-OHAsp α-H), 5.03 (d, J =
3.1 Hz, 1 H, β-OHAsp α-H), 4.96 (d, J = 3.1 Hz, 1 H, β-OHAsp β-
H), 4.86 (d, J = 3.1 Hz, 1 H, β-OHAsp β-H), 4.70 (overlapping m, 1
H, Ser α-H), 4.64 (dd, J = 5.2, 11.3 Hz, 1 H, Ser β-H), 4.40 (dd, J =
1.8, 10.9 Hz, 1 H, Ser β-H), 4.35 (m, 1 H, LysSta 3-H), 4.12 (overlap-
ping m, 1 H, Arg α-H), 4.08 (ABq, J = 17.1 Hz, 2H, Gly α-CH2), 4.03
(ABq, J = 17.1 Hz, 2 H, Gly α-CH2), 3.39 (m, 3 H, LysSta 4-H and
8-H), 3.20 (t, J = 6.7 Hz, 2 H, Arg δ-CH2), 2.58 (d, J = 5.5 Hz, 2 H,
LysSta 2-CH2), 1.77–1.86 (m, 2 H, Arg β-CH2), 1.60–1.76 (m, 6 H,
Arg γ-CH2, LysSta 5-CH2 and 7-CH2), 1.54–1.58 (m, 1 H, LysSta 6-
H), 1.41–1.48 (m, 1 H, LysSta 6-H).
The resulting acid (90 mg, 0.043 mmol) was treated with TFA
(2.5 mL) in CH2Cl2 (2.5 mL) at 0°C. The mixture was vigorously
stirred at this temperature for 2.5 h, and concentrated in vacuo. The
residue was dissolved in CH2Cl2 (1 mL) and benzene (3 mL), and the
volatiles were removed in vacuo. The deprotected product was dis-
solved in DMF (10 mL). NMM (24 µL, 0.22 mmol) at 0°C was added
to this mixture, followed by a solution of FDPP (33 mg, 0.086 mmol)
in DMF (1 mL). The mixture was stirred at 0°C for 30 min and then
allowed to warm to r.t. for 38 h. After removal of DMF by distillation
below 20°C, the residue was dissolved in EtOAc and the organic lay-
er was washed with ice-cold 1 M aq KHSO4 solution, sat. NaHCO3
solution, and brine, dried (Na2SO4), and concentrated in vacuo. The
crude product was purified by chromatography (25:1 CH2Cl2/MeOH)
to give the cyclic depsipeptide 38 as white crystals (42 mg, 53%); mp
108–109°C (CH2Cl2/hexane); [α]D25 –28.8 (c = 0.83, CHCl3).
IR (KBr): ν = 3335 (br), 2951, 2932, 1744, 1620 (br), 1539 (br), 1456,
1379, 1308, 1260 (br), 1121, 889, 783, 698 cm–1.
13C NMR (125 MHz, D2O): δ = 177.5 (Arg C=O), 177.3 (OHAsp β-
C=O), 176.9 (OHAsp β-C=O), 175.4 (LysSta C=O), 175.2 (Gly
C=O), 173.9 (Gly C=O), 173.7 (Ser C=O), 173.3 (OHAsp α-C=O),
173.0 (DHB C=O), 172.3 (OHAsp α-C=O), 159.6 (Arg C=NH),
149.6 (DHB C-2), 147.4 (DHB C-3), 122.5 (DHB C-5), 122.2 (DHB
C-4), 121.7 (DHB C-6), 119.9 (DHB C-1), 73.0 (OHAsp Cβ), 72.8
(OHAsp Cβ), 70.0 (LysSta C-3), 67.7 (Ser Cβ), 58.9 (OHAsp Cα),
58.3 (Arg Cα), 58.2 (OHAsp Cα), 58.0 (LysSta C-4), 54.5 (Ser Cα),
45.3 (Gly Cα), 45.2 (Gly Cα), 43.2 (Arg Cδ), 41.6 (LysSta C-8), 40.7
(LysSta C-2), 30.9 (LysSta C-7), 30.3 (Arg Cβ), 29.2 (LysSta C-5),
26.9 (Arg Cγ), 25.0 (LysSta C-6).
1H NMR (400 MHz, DMSO-d6): δ = 8.81 (br s, 0.5 H, guanidino),
8.56 (d, J = 4.4 Hz, 1 H, Arg NH), 8.27 (d, J = 8.3 Hz, 1 H, β-OHAsp
NH), 8.13 (t, J = 5.4 Hz, 1 H, LysSta 8-NH), 8.04 (br s, 1H, Gly NH),
7.75 (d, J = 7.8 Hz, 1 H, Ser NH), 7.49 (d, J = 7.1 Hz, 1 H, catechol
6-H), 7.10–7.45 (m, 29 H, arom, Gly NH, β-OHAsp NH), 6.97(d, J =
8.8 Hz, 1 H, LysSta 4-NH), 6.67 (s, 1 H, Mtr 5-H), 6.41 (br s, guani-
dino), 5.19 (s, 2 H, CH2Ph), 5.10 (ABq, J = 12.5 Hz, 2 H, CH2Ph),
5.05 (s, 2 H, CH2Ph), 5.01 (s, 2 H, CH2Ph), 4.97 (ABq, J = 10.6 Hz,
2 H, CH2Ph), 4.85 (d, J = 5.0 Hz, 2 H, CH2Ph), 4.85 (d, J = 5.0 Hz, 2
H, β-OHAsp α-H, LysSta 3-OH), 4.83 (d, J = 3.7 Hz, 1 H, β-OHAsp
α-H), 4.76 (d, J = 4.6 Hz, 1 H, β-OHAsp β-H), 4.67 (br s, 2H, β-
OHAsp β-H, Ser α-H), 4.24 (d, J = 10.5 Hz, 1 H, Ser β-H), 4.11–4.13
(m, 1 H, Ser β-H), 4.06 (dd, J = 7.8, 12.2 Hz, 1 H, Gly α-H),
3.82–3.85 (m, 2 H, Arg α-H, Gly α-H), 3.77 (s, 5 H, Mtr OCH3, Lys-
Sta 3-H, Gly α-H), 3.67 (d, J = 13.4 Hz, 1 H, Gly α-H), 3.31–3.32
(overlapping m, 1 H, LysSta 4-H), 3.21–3.24 (overlapping m, 1 H,
LysSta 8-H), 3.11–3.13 (m, 1 H, LysSta 8-H), 3.01 (d, J = 4.4 Hz, 2
H, Arg α-CH2), 2.59 (s, 3 H, Mtr 2-CH3), 2.52 (s, 3 H, Mtr 6-CH3),
2.30–2.33 (d, J = 12.5 Hz, 1 H, LysSta 2-H), 2.21 (dd, J = 9.16, 15.0
Hz, 1 H, LysSta 2-H), 2.04 (s, 3 H, Mtr 3-CH3), 1.15–1.55 (m, 10 H),
0.79 [s, 9 H, SiC(CH3)3], 0.78 [s, 9 H, SiC(CH3)3], –0.01 (s, 3H,
SiCH3), –0.02 (s, 3 H, SiCH3), –0.05 [s, 6 H, Si(CH3)2].
FAB-MS in H2O: m/z = 928 (MH+).
FAB-HRMS (NBA) in H2O: C36H54N11O18 (MH+): m/z calcd
928.3649, found 928.3723.
We thank Professor Alison Butler of University of California, Santa
Barbara, for kindly sending us the sample of natural alterobactins
and their NMR spectra. The high resolution FAB mass of alterobac-
tin A was determined by Dr. Hideaki Murata of Meijo University, to
whom the authors' thanks are due. One of the authors (J. D.) is grate-
ful to the Japan Society for a Promotion of Science and the Miyata
Foundation for a postgraduate fellowship. This work was financially
supported in part by the Ministry of Education, Science, Sports and
Culture, Japan.
(1) a) For isolation and purification of alterobactins, see: Reid, R.
T.; Butler, A. Limnol. Oceanogr. 1991, 36, 1783.
b) For structural determination and iron affinity, see: Reid, R.
T.; Live, D. H.; Faulkner, D. J.; Butler, A. Nature 1993, 366,
455. (see also: Chem. Brit. 1994, 30, 174).
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ya, 1995.
Anal. C94H123N11O23SSi2 (1864.0): calcd C, 60.60; H, 6.65; N, 8.27;
found: C, 60.52; H, 6.79; N, 8.42.
Alterobactin A (1):
To a solution of the cyclic depsipeptide 38 (37 mg, 0.020 mmol) in
HOAc/THF/H2O (v/v/v, 3:1:1; 4.5 mL) was added 5% Pd/C. The sus-
pension was stirred at r.t. for 12.5 h under a H2 atmosphere, filtered
through a pad of Celite, and the pad was washed twice with HOAc/
THF/H2O (3:1:1) and three times with THF/H2O (v/v, 1:1). The com-
bined filtrate was concentrated in vacuo and any residual HOAc and
H2O were removed by adding heptane and toluene, and concentrating
in vacuo. The residue was triturated and washed three times with Et2O
to afford the product retaining the Mtr group as a white solid (24 mg).
The above Mtr-protected product was treated with TFA (6 mL), fol-
lowed by thioanisole (0.24 mL). The mixture was vigorously stirred
at r.t. for 7 h and concentrated in vacuo. The residual thioanisole was
removed under high vacuum. The crude product was triturated and
washed three times with Et2O and then further purified by reverse-
phase HPLC (C4; MeCN/H2O/TFA, 10:90:0.1; 7.5 mL/min) and ly-
ophilized to afford alterobactin A (1) as a grey-white solid (15 mg,
81%); Rf 0.32 (silica gel, BuOH/H2O/AcOH, 3:3:1).
c) Wipf, P. Chem. Rev. 1995, 95, 2115.
d) For recent examples of our work, see Noguchi, H.; Aoyama,
T.; Shioiri, T; Tetrahedron Lett. 1997, 38, 2883.
e) Sasaki, S.; Hamada, Y.; Shioiri, T. Tetrahedron Lett. 1997,
38, 3013.
(4) a) Shioiri, T.; Hamada, Y.; Matsuura, F. Tetrahedron 1995, 51,
3939.
b) see also: Deng, J. Chemistry (The Chinese Chem. Soc., Tai-
pei) 1997, 55, 81.
(5) a) For a preliminary account of the synthesis of alterobactin A,
see: Deng, J.; Hamada, Y.; Shioiri, T. J. Am. Chem. Soc. 1995,
117, 7824.
b) see also: Deng, J.; Hamada, Y.; Shioiri, T. In Peptide Chem-
istry 1995, Nishi, N., Ed.; Protein Research Foundation: Osaka,
1996, p 109.
(6) Symbols and abbreviations are in accordance with the recom-
mendations of the IUPAC-IUB Commission on Biochemical
Nomenclature: Eur. J. Biochem. 1984, 138, 9.
1H NMR (500 MHz, D2O): δ = 7.21 (d, J = 7.9 Hz, 1 H, catechol 6-

SYNTHESIS
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