Solid-phase total synthesis of kahalalide a and related analogues

Article abstract of DOI:10.1021/jm049841x

The marine natural product kahalalide A, a cyclic depsipeptide, was prepared by total synthesis on solid-phase. A backbone cyclization strategy was followed, using the Kenner sulfonamide safety-catch linker. By NMR comparison of synthetic and naturally isolated material, the stereochemistry of the methylbutyrate side chain was established as (S). Several analogues were synthesized and tested for antimycobacterial activity. The results indicate that the alcohol functional group in the serine and threonine residues is important, while the methylbutyrate side chain can be replaced by an achiral hexanoate with an increase in activity.

Full text of DOI:10.1021/jm049841x

1330
J. Med. Chem. 2005, 48, 1330-1335
Solid-Phase Total Synthesis of Kahalalide A and Related Analogues
Line Bourel-Bonnet,†,§ Karumanchi V. Rao,‡ Mark T. Hamann,‡ and A. Ganesan*,†
School of Chemistry, University of Southampton, Southampton, SO17 1BJ, United Kingdom, and Department of
Pharmacognosy and National Center for Natural Products Research, School of Pharmacy, University of Mississippi,
University, Mississippi 38677
Received February 23, 2004
The marine natural product kahalalide A, a cyclic depsipeptide, was prepared by total synthesis
on solid-phase. A backbone cyclization strategy was followed, using the Kenner sulfonamide
safety-catch linker. By NMR comparison of synthetic and naturally isolated material, the
stereochemistry of the methylbutyrate side chain was established as (S). Several analogues
were synthesized and tested for antimycobacterial activity. The results indicate that the alcohol
functional group in the serine and threonine residues is important, while the methylbutyrate
side chain can be replaced by an achiral hexanoate with an increase in activity.
Introduction
kahalalide A by a solid-phase route adaptable for
analogue preparation. Besides the motivation of discov-
ering biologically active compounds, there was a purely
chemical impetus for the total synthesis. The stereo-
chemistry of the methylbutyrate side chain was not
established during the original isolation, and we be-
lieved this uncertainty could be resolved by synthesis.
Tuberculosis (TB), caused by Mycobacterium tuber-
culosis, is among the most important bacterial diseases.1
It is estimated that eight million cases of active TB occur
every year, predominantly in developing countries,
while its prevalence is rising in the Western world
particularly among immunocompromised patients (e.g.
HIV active). Globally, TB is responsible for two to three
million deaths annually. While a number of antimyco-
bacterial medicines are available,2 cost and patient
compliance are major hurdles for successful treatment.
Furthermore, resistance is an increasing problem. Some
strains, resistant to as many as nine drugs, result in
>50% fatality with current therapies. For these reasons,
new antibiotics are desirable, particularly those acting
by a novel mechanism of action.
Recently, 48 structurally diverse marine natural
product and semisynthetic compounds were screened3
for in vitro activity against M. tuberculosis. Within this
set, kahalalide A (1) emerged as a promising lead,
inhibiting 83% of the growth of M. tuberculosis at 12.5
µg/mL. Kahalalide A is one of a family of peptide natural
products isolated4 from the marine mollusk Elysia
rufescens and its algal diet Bryopsis sp. Among these,
the tridecapeptide kahalalide F has attracted the most
attention5 and is currently in phase I clinical trials as
an anticancer and antipsoriatic agent, while kahalalide
B has been the subject6 of a total synthesis. Meanwhile,
the structurally simpler kahalalide A is one of the few
marine-derived cyclic peptides with antimycobacterial
activity,7 in addition to massetolides,8 pitipeptolides,9
cyclomarin and sulfactin.10 Kahalalide A does not have
significant homology to these other antimycobacterial
cyclic peptides. Furthermore, it is devoid of obviously
reactive functional groups, and it is not cytotoxic to
various tumor cell lines, suggesting a selective antibac-
terial target. Here, we report the total synthesis of
There are two main approaches for the ‘head-to-tail’
solid-phase synthesis11 of cyclic peptides and depsipep-
tides. In the first, an amino acid is immobilized by its
side chain with orthogonal protection at the amine and
carboxylic acid. After construction of the linear peptide,
‘head-to-tail’ intramolecular cyclization between the two
ends followed by resin cleavage releases the cyclic
peptide. In the second strategy, immobilization is
through the carboxylic acid, but via a linker that is
stable to the conditions of peptide synthesis. After
assembly of the linear peptide, the site of immobilization
is activated, permitting intramolecular cyclization. The
first approach is fundamentally limited to amino acids
that have suitable side-chain functionality for im-
mobilization, while the second needs a linker that is
robust during linear peptide synthesis and yet selec-
tively activated upon demand. We chose the latter
approach, taking advantage of recent developments with
Kenner’s sulfonamide ‘safety-catch’ linker. Originally
designed12 for linear peptide synthesis, Kenner’s linker
was later popularized13 by Ellman for combinatorial
chemistry applications. Subsequently, it was demon-
strated14 by Yang and Morriello at Merck that the linker
is suitable for the ‘head-to-tail’ synthesis of cyclic
* Corresponding author. Tel: +44 2380593897; Fax: +44 2380596805;
E-mail: ganesan@soton.ac.uk.
† University of Southampton.
‡ University of Mississippi.
§ Present address: Faculte´ de Pharmacie de Lille, UMR 8525 CNRS-
Universite´ de Lille 2, 3 rue du Pr Laguesse, BP 83, 59006 Lille CEDEX,
France.
10.1021/jm049841x CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/12/2005

Solid-Phase Kahalalide Synthesis
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1331
Scheme 1a
a Reagents: (a) (i) 20% piperidine/DMF; (ii) Fmoc-D-Leu-OH, DIC, HOBt (4 equiv each); (iii) 20% piperidine/DMF; (iv) Fmoc-L-Thr(t-
Bu)-OH, DIC, HOBt (4 equiv each); (v) 20% piperidine/DMF; (vi) Fmoc-D-Phe-OH, DIC, HOBt (4 equiv each); (vii) 20% piperidine/DMF;
(b) (S)-2-methylbutyric acid or (()-2-methylbutyric acid, DIC, HOBt (4 equiv each); (c) (i) TFA/i-Pr3SiH/H2O: 95:2.5:2.5; (ii) Fmoc-L-
Ser(t-Bu)-OH, DIC, (4 equiv each), 0.4 equiv of DMAP; (iii) 20% piperidine/DMF; (iv) Fmoc-L-Thr(t-Bu)-OH, DIC, HOBt (4 equiv each); (v)
20% piperidine/DMF; (vi) Fmoc-D-Leu-OH, DIC, HOBt (4 equiv each); (d) (i) 20% piperidine/DMF; (ii) 4 equiv of Trt-Cl, 8 equiv i-Pr2NEt;
(iii) 10 equiv of ICH2CN, 12 equiv of i-Pr2NEt; (iv) 5% TFA/CH2Cl2; 3 equiv of i-Pr2NEt; (e) TFA/i-Pr3SiH/H2O.
peptides, and other groups15 have recently employed
sulfonamide linkers in this manner.
Results and Discussion
The total synthesis of kahalalide A began with the
attachment of Fmoc-D-Phe to the commercially available
sulfonamide resin, giving 2 (Scheme 1). This step was
repeated to ensure high loading, after which peptide
couplings (monitored throughout for completion using
either 2,4,6-trinitrobenzenesulfonic acid (TNBS)16 or
ninhydrin17 colorimetric reagents, and also by quantita-
tive Fmoc removal analysis18) with Fmoc-D-Leu, Fmoc-
L-Thr(t-Bu), Fmoc-D-Phe, and amine deprotection pro-
Figure 1. 1H NMR spectrum in CD3CN, showing the methyl
vided tetrapeptide 3. The stage was now set for addition
region of (a) naturally isolated kahalalide A, (b) (S-MeBu)-
of the methylbutyrate (MeBu) side chain. Unfortunately,
kahalalide A and (c) ((-MeBu)-kahalalide A.
only the S-enantiomer of chiral 2-methylbutyric acid is
commercially available. The synthesis was thus carried
out twice, once with the S-enantiomer and once with
racemic 2-methylbutyrate. The latter would ultimately
result in a 50-50 mixture of kahalalide A and its MeBu
diastereomer, while the first would produce a single
diastereomer (possibly kahalalide A). Deprotection of
the Thr side chain in 4 was followed by ester bond
formation with Fmoc-L-Ser(t-Bu). A double coupling was
employed to drive this reaction to completion. Further
peptide extension then afforded the key linear hepta-
peptide 5 for safety-catch activation. According to Yang
and Morriello, this is not compatible with the Fmoc
group, and the nitrogen protection was first switched
to trityl according to their procedure. Sulfonamide
alkylation with iodoacetonitrile then activated the
safety-catch linker, and trityl deprotection to the free
amine resulted in macrocyclative cleavage of depsipep-
tide 6 into solution. Acidic cleavage of the tert-butyl
ethers completed the synthesis of (S-MeBu)-kahalalide
A and ((-MeBu)-kahalalide A. Although the overall
yield was modest (15-20%, unoptimized), the crude
material produced by the cyclative cleavage strategy19
was unaccompanied by any significant peptide impuri-
ties. In this and later cyclizations, we did not observe
the formation of dimers by intermolecular reaction. It
is possible that the yields are dependent on which
residue is chosen as the initial site of immobilization,
although this was not investigated.

1332 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Bourel-Bonnet et al.
MeBu)-kahalalide A and ((-MeBu)-kahalalide A. For
((-MeBu)-kahalalide A, the 2-methyl group protons of
2-methylbutyric acid appeared as a multiplet at lower
chemical shift than the corresponding signal for the
S-isomer. On the other hand, the 2-methyl group
protons of 2-methylbutyric acid for (S-MeBu)-kahalalide
A appeared as a doublet and exactly matched with those
for natural kahalalide A. From this study, it could be
determined that the 2-methylbutyric acid of kahalalide
A has the S-configuration. Our choice of S-methylbutyric
acid for the synthesis, dictated by practical grounds,
fortuitously turned out to provide the right dia-
stereomer.
With the total synthesis successfully accomplished,
we prepared two analogues aimed at probing the
importance of the methylbutyrate side chain. In the
first, the arm containing (MeBu)-Phe was deleted and
replaced by a simple acetyl group. In the second, the
methylbutyrate was replaced by the longer achiral
hexanoate. In all these syntheses, depsipeptides with
protected Ser and Thr residues are first produced by
cyclative cleavage from the resin, and they were tested
alongside with the deprotected material.
The full set of compounds (7-14, Figure 3) was tested
for antimycobacterial activity. The results reveal a
number of structure-activity relationships. First, all
compounds containing protected Ser and Thr tert-butyl
ethers were inactive, indicating the importance of the
free alcohol at these positions. The truncated (Ac)-
kahalalide 12, in which a Phe residue and the methyl-
butyrate side chain are removed, was also inactive.
Meanwhile, (S-MeBu)-kahalalide A and ((-MeBu)-
kahalalide A were equally potent, implying that the
stereochemistry of the methylbutyrate is not important
for antimycobacterial activity. Indeed, replacement by
Figure 2. 1H NMR spectrum of methyl region of a mixture
of (a) naturally isolated kahalalide A and ((-MeBu)-kahalalide
A, and (b) (S-MeBu)-kahalalide A and ((-MeBu)-kahalalide
A.
HPLC separation of the two diastereomers of ((-
MeBu)-kahalalide A with a variety of columns proved
1
unsuccessful. Nevertheless, careful examination of H
NMR spectra allowed assignment of the MeBu stereo-
1
chemistry. The H NMR spectra of naturally isolated
kahalalide A and synthetic (S-MeBu)-kahalalide A
(Figure 1a and 1b, respectively) in CD3CN are closely
identical. These spectra differed significantly from the
spectra of ((-MeBu)-kahalalide A (Figure 1c).
For comparison, 1H NMR spectra of a mixture of
natural kahalalide A and ((-MeBu)-kahalalide A (Fig-
ure 2a), and a mixture of synthetic (S-MeBu)-kahalalide
A and ((-MeBu)-kahalalide A (Figure 2b) were also
recorded. The 2-CH, 3-CH2, 4-CH3, and 5-CH3 protons
of the 2-methylbutyric acid moiety showed similar NMR
patterns, but there were significant differences between
the chemical shift sets for these signals in the two
molecules. From these observations, it was possible to
establish unambiguously the differences between (S-
Figure 3. Compounds prepared, with antimycobacterial activity indicated.

Solid-Phase Kahalalide Synthesis
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1333
Scheme 2
mmol) is activated in 5 mL of DMF by DIC (2.24 mmol, 351
µL) and HOBt (2.24 mmol, 351 mg). The solution is filtrated
in case of precipitation, before addition to the deprotected
peptidyl resin. Reaction completion is checked by TNBS or
ninhydrin. The coupling of acetic, 2-methylbutyric, or hexanoic
acids was carried out under identical conditions.
Depsipeptide bond formation: The threonine tert-butyl ether
is deprotected by a TFA/TIS/water (95/2.5/2.5) mixture for 2
min. The procedure was repeated for 30 min and the resin
washed (CH2Cl2, THF). The alcohol resin was incubated with
DMAP (27.3 mg, 0.224 mmol) and reacted in 4 mL of THF
with Fmoc-L-Ser(t-Bu)-OH (859 mg, 2.24 mmol) and DIC (351
mg, 2.24 mmol). This coupling was first done for 2 h and
repeated overnight to ensure complete esterification as quanti-
fied by Fmoc removal analysis. After resin washing (3 × THF,
3 × DMF), elongation of the linear peptide is continued, until
the Fmoc group on the last residue is removed.
Safety-catch activation and cyclative cleavage of cyclic
depsipeptides: The amine resin is treated with trityl chloride
(624 mg, 2.24 mmol) and Hu¨nig’s base (782 µL, 4.48 mmol) in
2.5 mL of CH2Cl2 for 2 h or left overnight. After CH2Cl2
washings and TNBS test, the sulfonamide linker is activated
by iodoacetonitrile (405 µL, 5.6 mmol) and Hu¨nig’s base (1.73
mL, 6.72 mmol) in 2.5 mL of N-methylpyrrolidinone for 12 h.
This activation step was repeated under the same conditions.
After resin washing (N-methylpyrrolidinone, CH2Cl2), the trityl
group is removed by 5% TFA in CH2Cl2 treatment for 2 min.
The deprotection was repeated for 2 h, followed by a TNBS
test. The linear peptidyl resin is then washed (3 × CH2Cl2, 3
× THF, once by 1% Hu¨nig’s base in THF). Immediately
afterward, the resin is swollen in THF and Hu¨nig’s base (293
µL, 1.68 mmol) added. The cyclization proceeds overnight,
releasing the cyclic depsipeptide.
Peptide purification: The supernatant from cyclative cleav-
age is concentrated to the crude cyclic peptide, whose identity
is checked by ES-MS and purity by HPLC and TLC. The
compound is then purified by column chromatography (silica)
to give peptides with protected Ser/Thr residues. Side-chain
deprotection is performed by TFA/i-Pr3SiH/water (95/2.5/2.5)
for 30 min, followed by precipitation of the peptide in Et2O
dried over CaH2. Deprotected peptides are further purified by
preparative RP-HPLC (C-18 Nucleosil 300 mm × 25 mm, 3
mL/min, detection at 230 nm, eluent A: Water/TFA: 99.95/
0.05, eluent B: acetonitrile/water/TFA 80/19.95/0.05, gradient
B from 0 to 100% in 100 min).
the achiral hexanoate (14) resulted in a 2-fold increase
in potency over the natural product.
The increased conformational constraints placed by
converting a linear peptide into a cyclic skeleton usually
have a marked effect on its properties and biological
activity. To test this in the present context, a small-
scale methanolysis was carried out with synthetic
kahalalide A to cleave the depsipeptide linkage (Scheme
2). The resulting ‘linear’ kahalalide A proved to be
devoid of antimycobacterial activity, and this avenue
was not pursued further.
To summarize, an efficient solid-phase total synthesis
of kahalalide A was achieved. To the best of our
knowledge, this is the first example where the Kenner
safety-catch linker was used for the preparation of cyclic
depsipeptides. By careful NMR analysis of synthetic
kahalalide A containing either (S-MeBu) or ((-MeBu),
we have established that the natural product con-
tains a (S)-2-methylbutyrate subunit, thus resolving the
only ambiguity in its structure. Our route is readily
amenable to the preparation of libraries for antimyco-
bacterial testing, by amino acid substitutions during the
synthesis. The data from our first analogues highlight
the importance of the free Ser and Thr side chains and
the constrained depsipeptide framework for biological
activity. The methylbutyrate side chain can be replaced
by other hydrophobic groups, as evidenced by increased
activity with the hexanoate. On the basis of these
preliminary results, we plan the synthesis of additional
analogues and mechanistic studies to identify the target
of action.
Experimental Methods
Materials. All Fmoc amino acids, coupling reagents, and
resin were purchased from Novabiochem, and other chemicals
are from Aldrich. Dichloromethane and triethylamine were
distilled over CaH2 and THF distilled over Na in the presence
of benzophenone. The inside surfaces of peptide synthesis
reactors were pretreated by dichlorodimethylsilane, then
rinsed with methanol and dichloromethane.
(R/S)-Methyl-2-butyryl-D-Phe-cyclo[L-Thr-D-Leu-D-Phe-
D-Leu-L-Thr(t-Bu)-L-Ser(t-Bu)] (7). Overall yield: 19% (104
mg). ES-MS: 1028.5 [M + Na]+; HPLC purity: 98% by ELSD,
90% at 220 nm; TLC (CH2Cl2/AcOEt/TEA: 6/4/0.05) Rf: 0.3.
(R/S)-Methyl-2-butyryl-D-Phe-cyclo[L-Thr-D-Leu-D-Phe-
D-Leu-L-Thr-L-Ser] (((-MeBu)-kahalalide A, 8). Deprotec-
tion yield: 95%, overall yield: 18% (17 mg). ES-MS: 916.7
[M + Na]+; HPLC purity: >99% by ELSD, 87% at 220 nm.
(S)-Methyl-2-butyryl-D-Phe-cyclo[L-Thr-D-Leu-D-Phe-D-
Leu-L-Thr(t-Bu)-L-Ser(t-Bu)] (9). Overall yield: 15% (85
mg). ES-MS: 1029.0 [M + Na]+; HPLC purity: 98% by ELSD,
>99% at 220 nm; TLC (CH2Cl2/AcOEt/NH4OH aq: 7/3/0.05)
Rf: 0.3.
General Procedure for Depsipeptide Synthesis. Resin
loading: To the 4-sulfamylbutyryl AM resin (Novabiochem,
500 mg, 0.56 mmol) preswollen in DMF was added Fmoc-D-
Phe-OH (697 mg, 1.8 mmol), PyBOP (936 mg, 1.8 mmol) and
Hu¨nig’s base (942.8 µL, 5.4 mmol) in 5 mL of DMF. The
reaction mixture was left overnight, and the coupling repeated
twice more. The efficiency of loading was determined by Fmoc
removal analysis.
Peptide coupling: The Fmoc-peptidyl resin, swollen in DMF,
was deprotected by 20% piperidine in DMF for 2 min, and the
deprotection repeated for 20 min. The resin is washed (3 ×
DMF) and the presence of the amino group checked with
TNBS. At the same time, the next Fmoc amino acid (2.24

1334 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Bourel-Bonnet et al.
(S)-Methyl-2-butyryl-D-Phe-cyclo[L-Thr-D-Leu-D-Phe-D-
Leu-L-Thr-L-Ser] (kahalalide A, 10). tert-Butyl ether depro-
tection yield: 75%, overall yield: 11% (12 mg). 1H NMR δ 8.21
(Thr2-NH, d, J ) 9.59 Hz), 7.63 (Phe1-NH, d, J ) 9.45 Hz),
7.63 (Thr1-NH, d, J ) 9.45 Hz), 7.45 (Leu2-NH, d, J ) 9.37
Hz), 7.37 (Phe2-NH, d, J ) 4.68 Hz), 7.25 (Phe2-H5,5′,6,6′,7, Phe1-
H5,5′,6,6′,7, m), 7.23 (Leu1-NH, m), 6.87 (Ser-NH, d, J ) 5.70
Hz), 5.44 (Thr2-H3, dq, J ) 6.16, 2.18 Hz), 5.04 (Phe2-H2, dt,
J ) 7.98, 4.90 Hz), 4.77 (Phe1-H2, m), 4.70 (Leu1-H2, m), 4.48
(Thr1-H3, dq, J ) 6.49,1.89 Hz), 4.40 (Thr2-H2, dd, J ) 9.56,
2.16 Hz), 4.29 (Leu2-H2, q, J ) 9.27 Hz), 4.02 (Thr1-H2, m),
4.02 (Ser-H2, m), 3.52 (Ser-H3, dd, J ) 5.16, 1.63 Hz), 3.24
(Phe1-H3, dd, J ) 14.18, 5.13 Hz), 3.00 (Phe2-H3, dd, J ) 14.18,
5.13 Hz), 2.80 (Phe1-H3′, dd, J ) 14.27, 10.4 Hz), 2.4 (MeBu-
H2, m), 1.65 (Leu1-H4, m), 1.55 (MeBu-H3, m), 1.50 (Leu1-H3,
m), 1.29 (Thr1-H4, d, J ) 6.51 Hz), 1.20 (Leu2-H3, t, J ) 7.63
Hz), 1.13 (MeBu-H5, d, J ) 6.90 Hz), 1.02 (leu2-H4, m), 0.91
(Leu1-H5, d, J ) 6.42 Hz), 0.90 (MeBu-H4, t, J ) 7.40 Hz),
0.89 (Leu1-H6, d, J ) 6.42 Hz), 0.74 (Leu2-H5, d, J ) 6.53 Hz),
0.68 (Leu2-H6, d, J ) 6.53 Hz), 0.61 (Thr2-H4, d, J ) 6.58 Hz);
13C NMR δ 180.6 (MeBu-C1), 175.3 (Leu1-C1), 174.3 (Phe2-
C1), 172.1 (Leu2-C1), 171.9 (Phe1-C1), 170.9 (Thr1-C1), 170.0
(Ser-C1), 169.3 (Thr2-C1), 138.0 (Phe1-C4), 137.4 (Phe2-C4),
130.2 (Phe2-C5,5′), 129.7 (Phe1-C5,5′), 129.3 (Phe2-C6,6′), 129.3
(Phe1-C6,6′), 127.8 (Phe2-C7), 127.6 (Phe1-C7), 69.9 (Thr2-C3),
66.4 (Thr1-C3), 61.7 (Ser-C3), 60.8 (Thr1-C2), 57.2 (Ser-C2),
56.86 (Phe2-C2), 56.86 (Thr2-C2), 55.5 (Phe1-C2), 54.2 (Leu2-
C2), 52.4 (Leu1-C2), 43.4 (Leu1-C3), 42.9 (MeBu-C2), 42.3 (Leu2-
C3), 39.8 (Phe1-C3), 37.7 (Phe2-C3), 28.3 (MeBu-C3), 25.5 (Leu2-
C4), 25.2 (Leu1-C4), 23.0 (Leu2-C6), 22.8 (Leu1-C6), 22.6 (Leu1-
C5), 21.9 (Leu2-C5), 20.8 (Thr1-C4), 17.9 (MeBu-C5), 16.0 (Thr2,
C4), 12.3 (MeBu-C4); ES-MS: 916.17 [M + Na]+; HPLC
purity: 97% by ELSD, >99% at 220 nm.
(Thr2-H2, dd, J ) 9.57, 2.17 Hz), 4.31 (Leu2-H2, q, J ) 9.4
Hz), 4.02 (Thr1-H2, m), 3.99 (Ser-H2, dd, J ) 6.93, 1.63 Hz),
3.54 (Ser-H3, d, J ) 4.89 Hz), 3.23 (Phe1-H3, dd, J ) 14.04,
4.91 Hz), 3.02 (Phe2-H3, dd, J ) 7.85, 1.79 Hz), 2.86 (Phe1-
H4, dd, J ) 14.10, 10.25 Hz), 2.3 (Hex-H2, t, J ) 7.5 Hz), 1.66
(Hex-H3, m), 1.59 (Leu1-H4, m), 1.50 (Leu1-H3, m), 1.37 (Hex-
H4,5, m), 1.32 (Thr1-H4, d, J ) 6.48 Hz), 1.22 (Leu2-H3, m),
1.06 (Leu2-H4, m), 0.96 (Leu1-H5, d, J ) 6.37 Hz), 0.92 (Hex-
H6, t, J ) 7.1 Hz), 0.92 (Leu1-H6, d, J ) 6.49 Hz), 0.79 (Leu2-
H5, d, J ) 6.53 Hz), 0.73 (Leu2-H6, d, J ) 6.51 Hz), 0.61 (Thr2-
H4, d, J ) 6.56 Hz); 13C NMR δ 177.0 (Hex-C1), 174.8 (Leu1-
C1), 173.6 (Phe2-C1), 171.6 (Leu2-C1), 171.4 (Phe1-C1), 170.4
(Thr1-C1), 169.6 (Ser-C1), 168.7 (Thr2, C1), 137.6 (Phe1-C4),
136.8 (Phe2-C1), 129.8 (Phe2-C5,5′), 129.4 (Phe1-C5,5′), 128.9
(Phe2-C6,6′), 128.9 (Phe1-C6,6′), 127.4 (Phe2-C7), 127.2 (Phe1-
C7), 69.5 (Thr2-C3), 65.9 (Thr1-C3), 61.3 (Ser-C3), 60.5 (Ser-
C2), 56.7 (Thr1-C2), 56.6 (Phe2-C2), 56.3 (Thr2-C2), 55.2 (Phe1-
C2), 53.8 (Leu2-C2), 51.9 (Leu1-C2), 42.6 (Leu1-C3), 42.0 (Leu2-
C3), 39.2 (Phe1-C3), 37.1 (Phe2-C3), 36.2 (Hex-C2), 31.4 (Hex-
C4), 26.5 (Hex-C3), 25.0 (Leu1-C4), 24.8 (Leu2-C4), 22.8 (Leu1-
C6), 22.52 (Hex-C5), 22.51 (Leu2-C6), 22.3 (Leu1-C5), 21.6
(Leu2-C5), 20.2 (Thr1-C4), 17.6 (Thr2-C4), 15.5 (Hex-C6); ES-
MS: 930.6 [M + Na]+, 946.7 [M + K]+; HPLC purity: 96% by
ELSD, 77% at 220 nm.
Acknowledgment. L.B. and A.G. acknowledge the
Faculty of Pharmacy, University of Lille 2, for financial
support. We are grateful to Mrs. Joan Street and Dr.
John Langley at Southampton for NMR and MS sup-
port, respectively; the late Professor Paul J. Scheuer at
the University of Hawaii for a sample of naturally
isolated kahalalide A; Professor Christian Roussel at the
University of Marseille and Professor Daniel Armstrong
and Dr. Alain Berthod at Iowa State University for
attempted separation of ((-MeBu)-kahalalide A; and Dr.
Fangqiu Zhang and Professor Scott Franzblau at the
College of Pharmacy, University of Illinois at Chicago,
for antimycobacterial testing. K.V.R. and M.T.H. ac-
knowledge the NIH for financial support and thank
Frank Wiggers and Chuck Dunbar from the National
Center for Natural Products Research for assistance in
recording NMR spectra.
Ac-cyclo[L-Thr-D-Leu-D-Phe-D-Leu-L-Thr(t-Bu)-L-Ser(t-
Bu)] (11). Overall yield: 5% (26 mg). ES-MS: 855 [M + K]+;
HPLC purity: >99% by ELSD, 68% at 220 nm; TLC (CH2Cl2/
AcOEt/TEA: 5/5/0.05) Rf: 0.3.
Ac-cyclo[L-Thr-D-Leu-D-Phe-D-Leu-L-Thr-L-Ser] (12).
Deprotection yield: 59%, overall yield: 3% (7 mg). 1H NMR δ
9.08 (Thr1-NH, d, J ) 7.57 Hz), 8.12 (Phe1-NH, m), 8.05 (Leu1-
NH, d, J ) 6.94 Hz), 7.90 (Ser-NH, m), 7.78 (Thr2-NH, d, J )
9.18 Hz), 7.33 (Leu2-NH, m), 7.25 (Phe-H5,5′,6,6′,7, m), 5.28
(Thr2-H3, d, J ) 5.76 Hz), 4.63 (Thr2-H2, d, J ) 8.93 Hz), 4.59
(Leu1-H2, d, J ) 6.95 Hz), 4.29 (Phe-H2, m), 4.27 (Thr1-H3,
dd, J ) 6.62, 2.45 Hz), 4.16 (Leu2-H2, m), 4.02 (Thr1-H2, m),
3.53 (Ser-H2,3, m), 3.17 (Phe-H3′, dd, J ) 13.92, 5.00 Hz), 3.00
(Phe-H3, dd, J ) 13.83, 10.39 Hz), 1.56 (Leu1-H3, Leu2-H3, m),
1.35 (Leu2-H4, m), 1.09 (Thr1-H4, d, J ) 6.44 Hz), 1.04 (Thr2-
H4, d, J ) 6.72 Hz); 0.91 (Leu1-H5, d, J ) 6.42 Hz), 0.90 (Leu1-
H4, m), 0.79 (Leu1-H6′, d, J ) 6.02 Hz), 0.74 (Leu1-H6, d, J )
5.94 Hz), 0.55 (Leu2-H5′, d, J ) 6.54 Hz), 0.42 (Leu2-H5, d, J
) 6.55 Hz); 13C NMR δ 174.0, 172.4, 171.5, 171.2, 170.8, 170.6,
170.1 (all C1), 138.0, 130.2, 129.8, 129.1, 128.9, 127.2 (Phe1-
C4, C5,5′, C6,6′ and C7), 71.3 (Thr2-C3), 66.4 (Thr1-C3), 61.3 (Ser-
C2), 61.2 (Ser-C3), 60.4 (Thr1-C2), 56.5 (Phe1-C2), 56.4 (Leu2-
C2), 55.4 (Thr2-C2), 52.2 (Leu1-C2), 42.9 (MeBu-C2), 40.6 (Leu1-
C3), 40.4 (Leu2-C3), 37.3 (Phe1-C3), 25.1, 24.8, 23.8; 23.5, 23.4,
23.3, 23.1, 22.9, 22.7, 221.4, 21.1 (Leu2-C4, Leu1-C4, Leu2-C6,
Leu1-C6, Leu1-C5, Leu2-C5, Thr1-C4, Thr2-C4, Ac-C2); ES-
MS: 727.7 [M + Na]+; HPLC purity: 96% by ELSD, 77% at
220 nm.
Supporting Information Available: HPLC data and 1H
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
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