Synthesis and assignment of stereochemistry of the antibacterial cyclic peptide xenematide

Article abstract of DOI:10.1039/c0ob00315h

The synthesis of the antimicrobial cyclic peptide xenematide was accomplished by Fmoc solid phase peptide synthesis and the key esterification reaction was achieved using a modified Yamaguchi esterification. Comparison of the optical rotation and NMR data

Full text of DOI:10.1039/c0ob00315h

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 236
www.rsc.org/obc
PAPER
Synthesis and assignment of stereochemistry of the antibacterial cyclic
peptide xenematide††
Kuo-yuan Hung, Paul W. R. Harris, Amanda M. Heapy and Margaret A. Brimble*
Received 25th June 2010, Accepted 8th September 2010
DOI: 10.1039/c0ob00315h
The synthesis of the antimicrobial cyclic peptide xenematide was accomplished by Fmoc solid phase
peptide synthesis and the key esterification reaction was achieved using a modified Yamaguchi
esterification. Comparison of the optical rotation and NMR data of the synthesized diastereomers to
that of the natural product confirmed the structure of xenematide to be PA-L-[Thr-L-Trp-D-Trp-b-Ala].
(PA = phenylacetyl).
correct relative and absolute stereochemistry can be unequivocally
assigned.
Xenematide (1) can be synthesized by late stage macrolac-
tam formation after cleavage from the resin, via intramolecular
cyclization, of the N-terminal b-alanine onto the C-terminal
tryptophan residue of resin-bound PA-L-Thr-(L,D)-Trp-(D,L)-Trp-
b-Ala 2. Peptide 2 is obtained via esterification of the secondary
alcohol on threonine of tripeptide 3 with the carboxylic acid
1. Introduction
Xenematide is a cyclodepsipeptide isolated from the bacteria
Xenorhabdus nematophilus in 2008.1 Xenematide exhibits potent
antibacterial activity against several bacterial strains including
Erwinia amylovora, the pathogen which causes fire blight, a
contagious disease that causes the death of apple and pear trees.1,2
Fire blight is an ongoing horticultural problem both interna-
tionally and locally. The presence of fire blight in New Zealand
restricts fruit exportation to foreign markets; thus effective control
over the disease is required. Fire blight is currently controlled
of Boc-b-Ala-OH. Tripeptide 3 in turn is prepared using Fmoc
solid phase peptide synthesis (SPPS) starting from either L-or D-
Trp-2-ClTrt-resin 4 (Scheme 1).6–8 2-Chlorotrityl-resin (2-ClTrt-
with copper solutions, the aminoglycoside antibiotic streptomycin
resin) was chosen in the current synthesis to minimize C-terminal
or the less effective antibiotic oxytetracycline.3 Streptomycin
racemization and the formation of alkylated by-products resulting
is used at concentrations of 50–200 mM and is by far the
most efficient treatment although streptomycin-resistant strains
of E. amylovora have been observed.4,5 Development of novel
and effective antibacterial agents such as xenematide is therefore
crucial in order to prevent outbreaks of this disease in local
horticultural industries. Xenematide has a molecular weight of
662.3 g mol-1 and was shown to consist of one b-alanine (b-
Ala) residue, two tryptophan (Trp) residues, one threonine (Thr)
residue and one phenylacetyl (PA) group. The threonine residue
was determined to be of the L-configuration, whereas both L-and
D-tryptophan residues were present in the structure. The order of
the L-and D-tryptophan residues within the cyclic peptide could
not be assigned at the time of isolation thus two diastereomers
are possible. Chemical synthesis of the two diastereomers, namely
PA-L-[Thr-D-Trp-L-Trp-b-Ala] and PA-L-[Thr-L-Trp-D-Trp-b-Ala]
would allow comparison of their optical rotation and NMR
spectra to that of the isolated natural product such that the
from cations that can be generated from benzyl-based linkers
during TFA-mediated cleavage.9
2. Results and discussion
The synthesis of diastereomer PA-L-[Thr-D-Trp-L-Trp-b-Ala] 11
began with amide coupling of Fmoc-D-Trp(Boc)-OH to com-
mercially available H2N-L-Trp-2-ClTrt-resin 5 using HBTU and
DIPEA in DMF. After Fmoc deprotection with 20% piperidine
solution, dipeptide 6 was coupled to PA-L-Thr-OH prepared
according to literature procedure10 affording tripeptide 7. Esteri-
fication between the carboxylic acid group on Boc-b-Ala-OH and
the hydroxyl group on threonine of tripeptide 7 was then attempted
using DIC/DMAP11 but the reaction did not proceed even after
extended reaction times under microwave irradiation (Scheme 2).
After extensive investigation of the formation of the ester bond
between threonine and Boc-b-Ala-OH, it was found that the use
of modified Yamaguchi esterification conditions12 (BzCl, Et3N
and catalytic DMAP in THF) afforded the desired dipeptide 9
in satisfactory yield in the solution phase reaction (Scheme 3).
Unfortunately, subsequent removal of the benzyl group by hy-
drogenolysis followed by coupling to dipeptide 6 using HBTU
and DIPEA in DMF resulted in the formation of a product
with m/z value of 462.2. This undesired product 10, identified
Department of Chemistry, The University of Auckland, 23 Symonds Street,
Auckland, New Zealand. E-mail: m.brimble@auckland.ac.nz
† Electronic supplementary information (ESI) available: The NMR (1H
and 13C) spectra of PA-L-Thr-OBn, compound 9, isolated xenematide and
the four diastereomers of xenematide, and the HPLC chromatograms of
peptides 8, 10 (after cleavage from resin) and the four diastereomers of
xenematide are provided. See DOI: 10.1039/c0ob00315h
236 | Org. Biomol. Chem., 2011, 9, 236–242
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Scheme 1 Proposed retrosynthesis of xenematide (1).
Scheme
2 Synthesis of tripeptide 7 and attempted esterification of Boc-b-Ala-OH with L-threonine. Reagents, conditions and yields: i)
Fmoc-D-Trp(Boc)-OH (3 equiv.), HBTU, DIPEA, DMF, rt, 45 min; ii) 20% piperidine/DMF, rt; iii) PA-L-Thr-OH (3 equiv.), HBTU, DIPEA, DMF, rt,
45 min; iv) Boc-b-Ala-OH (3 equiv.), DIC, DMAP, DMF, D, microwave.
as b-Ala-D-Trp-L-Trp, was postulated to form via b-Ala transfer
from threonine to tryptophan during the peptide coupling process
(Scheme 3).
intramolecular cyclization was carried out using BOPCl and
DMAP13 to give the diastereomer PA-L-[Thr-D-Trp-L-Trp-b-Ala]
11 (Scheme 4).
Esterification using modified Yamaguchi conditions was next
performed on resin-bound peptide 7, forming the desired peptide
8 in excellent yield with none of the undesired product 10 being
detected (Scheme 4). CH2Cl2 was found to be a superior solvent
to DMF and use of excess reagents resulted in a shortened
reaction time with quantitative conversion. Following cleavage
from the resin using a mixture of TFA/H2O/TIPS (95 : 2.5 : 2.5),
Alternatively, the other possible diastereomer PA-L-[Thr-L-Trp-
D-Trp-b-Ala] 12 was synthesized in a similar manner starting from
D-Trp(Boc)-2-ClTrt-resin 13 (Scheme 5). Upon comparison of the
optical rotation and NMR data (1H and 13C) of the synthesized
peptides to that of the natural product (Tables 1–3), it was
established that xenematide contains the peptide sequence PA-
L-[Thr-L-Trp-D-Trp-b-Ala] 12.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 236–242 | 237
©

View Article Online
esterification. Comparison of the optical rotation and NMR data
of the synthesized diastereomers to that of the natural product
confirmed the structure of xenematide to be PA-L-[Thr-L-Trp-
D-Trp-b-Ala]. (PA = phenylacetyl). The antibacterial activity of
the synthetic peptides and the design of further peptidomimetic
analogues of xenematide are currently being investigated.
4. Experimental
4.1 Synthesis of (2S,3R)-benzyl 3-[3–(tert-butoxycarbonyl
amino)propanoyloxy]-2-(2-phenylacetamido)butanoate 910,12
4.1.1 Synthesis of (2S,3R)-benzyl 2-hydroxy-2-(2-phenyl
acetamido)butanoate (PA-L-Thr-OBn). To a solution of L-
threonine (5.42 g, 45.49 mmol) in 1 M aqueous NaOH solution
(150 mL) at 0 ◦C was added phenylacetyl chloride (7.85 mL,
59.12 mmol) dropwise, and the reaction was stirred at 0 ◦C for
1 h. Phenylacetyl chloride (7.85 mL, 59.12 mmol) was added at
0 ◦C and the reaction mixture was warmed to room temperature
and stirred for 18 h. After extraction with ethyl acetate (90 mL), the
aqueous layer was acidified with 3 M HCl solution to pH 2 and was
extracted with ethyl acetate (3 ¥ 120 mL). The combined organic
extracts were dried over anhydrous Na2SO4, filtered, concentrated
in vacuo to give a colourless solid which was washed with cold
diethyl ether and used without further purification. To a solution
of the above solid (6.03 g, 25.44 mmol) in DMF (28 mL) at room
temperature was added triethylamine (4.25 mL, 30.49 mmol),
benzyl bromide (6.63 mL, 30.52 mmol), and the reaction mixture
was stirred for 16 h. Water (24 mL) and dichloromethane (60 mL)
were added and the suspension was stirred for 15 min. The organic
layer was separated and the aqueous layer was extracted with
dichloromethane (3 ¥ 60 mL). The combined organic extracts were
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
Washing with cold diethyl ether afforded the desired product
(6.07 g, 73%) as a colourless solid; Rf 0.41 (1 : 1 EtOAc–hexane);
Scheme 3 Esterification using modified Yamaguchi conditions and
formation of undesired product 10. Reagents, conditions and yields: i) Et3N,
BnBr, DMF, rt, 16 h, 73%; ii) Boc-b-Ala-OH, BzCl, Et3N, DMAP, THF,
rt, 88 h, 71%; iii) H2, 10% Pd/C, MeOH, rt, overnight, 93%; iv) compound
6, HBTU, DIPEA, DMF, rt, 45 min; v) TFA : H2O : TIPS (95 : 2.5 : 2.5),
1 h.
In addition to the two possible diastereomers of the natural
cyclic peptide xenematide, two more unnatural diastereomers with
the peptide sequences, PA-L-[Thr-L-Trp-L-Trp-b-Ala] and PA-L-
[Thr-D-Trp-D-Trp-b-Ala], were also synthesized using the same
synthetic strategy. It was envisaged that these additional cyclic
peptides could be used to probe the bioactivity of xenematide.
3. Conclusions
The synthesis of the antimicrobial cyclic peptide xenematide was
accomplished by Fmoc solid phase peptide synthesis with the key
esterification reaction being achieved using a modified Yamaguchi
Scheme 4 Synthesis of PA-L-[Thr-D-Trp-L-Trp-b-Ala] 11. Reagents, conditions and yields: i) Boc-b-Ala-OH (20 equiv.), BzCl (20 equiv.), Et3N (40 equiv.),
CH2Cl2, rt, 18 h; ii) TFA : H2O :TIPS (95 : 2.5 : 2.5), 1 h; iii) BOPCl, DMAP, CH2Cl2–MeOH, 0 ◦C to rt, 19 h, 8% from compound 5.
238 | Org. Biomol. Chem., 2011, 9, 236–242
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Scheme 5 Synthesis of PA-L-[Thr-L-Trp-D-Trp-b-Ala] 12. Reagents, conditions and yields: i) Fmoc-L-Trp(Boc)-OH (3 equiv.), HBTU, DIPEA, DMF, rt,
45 min; ii) 20% piperidine/DMF, rt; iii) PA-L-Thr-OH (3 equiv.), HBTU, DIPEA, DMF, rt, 45 min; iv) Boc-b-Ala-OH (20 equiv.), BzCl (20 equiv.), Et3N
(40 equiv.), CH2Cl2, rt, 18 h; v) TFA : H2O : TIPS (95 : 2.5 : 2.5), 1 h; vi) BOPCl, DMAP, CH2Cl2–MeOH, 0 ◦C to rt, 19 h, 4% from compound 13.
Table 1 dH and dC(600 MHz; DMSO-d6) of isolated xenematide (1)1
Position
dC, multiplicity
dC (J in Hz)
Position
dC, multiplicity
dC (J in Hz)
b-Ala
169.2, qC
33.9, CH2
Trp–4
–5
–6
–7
–8
109.3, qC
127.0, qC
118.2, CH
118.2, CH
121.0, CH
111.3, CH
136.0, qC
2.52, m
2.40, m
3.39, m
3.32, m
7.55, d (7.8)
7.00, td (7.8, 1.0)
7.08, m
34.6, CH2
b-Ala-NH
Trp–1
–2
7.39, t (6.1)
–9
7.36, bd (8.0)
171.0, qC
54.5, CH
25.7, CH2
–10
–11
–12
Trp-NH
Thr
4.19, ddd (10.0, 7.9, 3.7)
3.18, m
2.87, m
10.7, s
7.09, m
8.81, d (6.7)
–3
123.4, CH
–4
–5
–6
–7
–8
–9
–10
–11
–12
Trp-NH
Trp–1
–2
110.6, qC
126.9, qC
117.9, CH
118.2, CH
120.8, CH
111.3, CH
135.9, qC
170.2, qC
54.0, CH
72.0, CH
16.2, CH3
4.65, dd (9.2, 2.2)
5.11, qd (6.3, 2.3)
1.05, d (6.2)
7.49, d (7.8)
6.96, m
7.05, td (7.8, 1.0)
7.33, bd (8.0)
Thr-NH
PAa
8.09, d (9.5)
170.6, qC
41.7, CH2
3.65, d (14.1)
3.55, d (14.1)
10.6, s
6.96, m
8.72, d (6.7)
123.1, CH
136.3, qC
129.0, CH
128.1, CH
126.1, CH
7.26, m
7.29, m
7.19, m
172.0, qC
54.0, CH
25.7, CH2
4.53, q (7.3)
2.82–2.92, m
–3
a PA = phenylacetyl. [a]2D0 +45.0 (c 0.20 in MeOH).
mp 150.5–152.8 ◦C; vmax(film)/cm-1 3469, 3349, 1705, 1648, 1529,
1279, 1001, 725 and 693; [a]2D0 -3.6 (c 1.11 in CDCl3); dH(300 MHz;
CDCl3) 1.04 (3H, d, J 6.0, Thrb–CH3), 3.55 (2H, s, PhCH2CON),
4.25 (1H, dq, J 3.0 and 6.0, Thrb–CH), 4.48–4.52 (1H, m, Thra–
CH), 5.07 (2H, s, PhCH2CO2), 6.88 (1H, d, CONHCH), 7.17–
7.27 (10H, m, Ph); dC(75 MHz; CDCl3) 19.6 (CH3, Thrb–CH3),
42.95 (CH2, PhCH2CON), 43.0 (CH2, PhCH2CON), 57.6 (CH,
Thra–CH), 57.65 (CH, Thra–CH), 67.1 (CH2, PhCH2CO2), 67.2
(CH, Thrb–CH), 126.9 (CH, Ph), 127.9 (CH, Ph), 128.1 (CH, Ph),
128.2 (CH, Ph), 128.4 (CH, Ph), 128.6 (CH, Ph), 129.0 (CH, Ph),
129.6 (CH, Ph), 132.75 (quat., Ph), 134.4 (quat., Ph), 135.1 (quat.,
Ph), 170.6 (quat., CHCO2CH2), 172.1 (quat., CH2CON), 172.2
(quat., CH2CON); m/z (EI) 328.1531 (MH+, C19H22NO4 requires
328.1543), 328 (MH+, 6%), 350 (MNa+, 100%), 351 (20) and
352 (2).
4.1.2 Synthesis of (2S,3R)-benzyl 3-[3-(tert-butoxycarbonyl
amino)propanoyloxy]-2-(2-phenylacetamido)butanoate 9. To
a
solution of PA-L-Thr-OBn (1.70 g, 5.19 mmol), Boc-b-Ala-OH
(0.98 g, 5.19 mmol), and benzoyl chloride (0.61 mL, 5.25 mmol)
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 236–242 | 239
©

View Article Online
Table 2 dH(300 MHz; DMSO-d6) and dC(75 MHz; DMSO-d6) of PA-L-[Thr-D-Trp-L-Trp-b-Ala] 11
Position
dC, multiplicity
dC (J in Hz)
Position
dC, multiplicity
dC (J in Hz)
b-Ala
169.70, qC
34.77, CH2
35.21, CH2
Trp–4
–5
–6
–7
–8
109.98, qC
127.59, qC
118.77, CH
118.77, CH
121.39, CH
111.85, CH
136.47, qC
2.28–2.44, m
3.55–3.59, m
7.22–7.35, m
7.46, d (7.8)
6.91–6.95, m
6.91–6.95, m
7.22–7.35, m
b-Ala-NH
Trp–1
–2
–3
–4
–5
–6
–7
–8
171.52, qC
55.32, CH
26.45, CH2
110.93, qC
127.59, qC
118.54, CH
118.54, CH
121.33, CH
111.85, CH
136.47, qC
4.30–4.34, m
2.74–2.95, m
–9
–10
–11
–12
Trp-NH
Thr
10.69, s
6.91–6.95, m
8.44–8.59, m
123.66, CH
7.41, d (7.8)
6.91–6.95, m
6.91–6.95, m
7.22–7.35, m
171.30, qC
56.59, CH
70.43, CH
16.95, CH3
4.44–4.48, m
5.41–5.43, m
1.03, d (6.0)
7.82, d (7.8)
–9
–10
–11
–12
Trp-NH
Trp–1
–2
10.69, s
6.91–6.95, m
8.44–8.59, m
Thr-NH
PAa
123.66, CH
171.45, qC
42.56, CH2
136.85, qC
129.48, CH
128.75, CH
126.90, CH
b
—
171.76, qC
53.70, CH
27.04, CH2
4.53–4.57, m
3.12–3.17, m
7.22–7.35, m
7.22–7.35, m
7.22–7.35, m
–3
a PA = phenylacetyl. b dH Peaks for PhCH2CON are not included as they are obscured by DMSO-d6. [a]2D0 -36.9 (c 0.52 in MeOH).
Table 3 dH(300 MHz; DMSO-d6) and dC(75 MHz; DMSO-d6) of PA-L-[Thr-L-Trp-D-Trp-b-Ala] 12
Position
dC, multiplicity
dC (J in Hz)
Position
dC, multiplicity
dC (J in Hz)
b-Ala
169.78, qC
34.48, CH2
Trp–4
–5
–6
–7
–8
109.82, qC
127.49, qC
118.81, CH
118.81, CH
121.53, CH
111.90, CH
136.54, qC
2.37–2.49, m
3.39–3.41, m
7.17–7.42, m
7.54, d (7.8)
6.93–7.09, m
6.93–7.09, m
7.17–7.42, m
35.13, CH2
b-Ala-NH
Trp–1
–2
–3
–4
–5
–6
–7
–8
–9
171.61, qC
55.07, CH
26.25, CH2
111.17, qC
127.40, qC
118.49, CH
118.81, CH
121.38, CH
111.84, CH
136.54, qC
–10
–11
–12
Trp-NH
Thr
4.17, ddd (10.2, 6.6, 3.6)
3.16–3.21, m
10.72, s
6.93–7.09, m
8.82, d (6.9)
123.96, CH
170.83, qC
54.53, CH
72.54, CH
16.70, CH3
7.48, d (7.8)
6.93–7.09, m
6.93–7.09, m
7.17–7.42, m
4.62–4.65, m
5.09–5.11, m
1.04, d (6.0)
8.13, d (6.9)
–9
Thr-NH
PAa
–10
–11
–12
Trp-NH
Trp–1
–2
171.20, qC
42.17, CH2
10.63, s
6.93–7.09, m
8.76, d (6.9)
3.65, d (14.1)
3.54, d (14.1)
123.70, CH
136.83, qC
129.55, CH
128.64, CH
126.78, CH
172.58, qC
54.44, CH
26.25, CH2
7.17–7.42, m
7.17–7.42, m
7.17–7.42, m
4.52, q (7.2)
2.82–2.95, m
–3
a PA = phenylacetyl. [a]2D0 +61.0 (c 0.58 in MeOH).
in dry THF (47 mL) at room temperature under N2 was added
dropwise triethylamine (1.45 mL, 10.38 mmol) and DMAP (0.16 g,
1.31 mmol), and the reaction mixture was stirred for 88 h.
The solvent was removed in vacuo and the residue was purified
by flash column chromatography (EtOAc–hexane 1 : 2) to give
compound 9 (1.85 g, 71%) as a yellow oil; Rf 0.39 (1 : 2 EtOAc–
hexane); vmax(film)/cm-1 3302, 1739, 1663, 1519, 1164, 731 and
697; [a]2D0 +17.3 (c 2.61 in CDCl3); dH(300 MHz; CDCl3) 1.17
(3H, d, J 6.0, Thrb–CH3), 1.44 [9H, s, C(CH3)3], 2.21–2.30 (2H,
m, CO2CH2CH2NH), 3.16–3.27 (2H, m, CO2CH2CH2NH), 3.67
(2H, s, PhCH2CON), 4.79 (1H, d, J 9.0, Thra–CH), 5.09 (2H,
d, PhCH2CO2), 5.38 (1H, d, J 3.0, Thrb–CH), 7.26–7.37 (10H,
m, Ph); dC(75 MHz; CDCl3) 16.9 (CH3, Thrb–CH3), 28.3 [CH3,
C(CH3)3], 34.6 (CH2, b–Ala–CH2), 35.9 (CH2, b–Ala–CH2), 43.3
(CH2, PhCH2CON), 55.5 (CH, Thra–C), 67.5 (CH2, PhCH2CO2),
70.6 (CH, Thrb–C), 79.4 [quat., C(CH3)3], 127.3 (CH, Ph), 128.3
(CH, Ph), 128.5 (CH, Ph), 128.6 (CH, Ph), 128.8 (CH, Ph), 129.3
(CH, Ph), 129.9 (CH, Ph), 133.1 (CH, Ph), 134.5 (quat., Ph), 134.9
(quat., Ph), 155.8 (quat., NCO2C), 169.5 (quat., CHCO2CH2,
CHCO2CH2), 170.5 (quat., CH2CON); m/z (EI) 521.2256 (MNa+,
C27H34N2NaO7 requires 521.2258), 521 (MNa+, 100%), 522 (48),
523 (10), 537 (20) and 538 (4).
240 | Org. Biomol. Chem., 2011, 9, 236–242
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
4.2 Synthesis of H2N-D-Trp(Boc)-2-ClTrt-aminomethyl
polystyrene resin 136–8
(0.13 g, 0.51 mmol) and DMAP (0.11 g, 0.92 mmol) were added,
and the reaction was stirred for 19 h. 1 M HCl solution (30 mL) was
added and the aqueous layer was extracted with dichloromethane
(3 ¥ 80 mL). The combined organic extracts were dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. Purification
by semi-preparative RP–HPLC (using a linear gradient of 40% B
to 70% B) yielded the title compound (8% from H2N-L-Trp-2-ClTrt-
resin) as an off-white amorphous solid in >99% purity according
to analytical RP-HPLC; Rt 10.77 min (XTerra C18, 4.6 ¥ 150 mm,
30% to 75% B over 15 min, 1 mL min-1); mp 150.2–156.4 ◦C;
To aminomethyl polystyrene resin (0.1 g) was added a mixture
of 2-chloro-4¢-carboxytriphenylmethanol (68.8 mg, 0.2 mmol)
and N,N-diisopropylcarbodiimide (21.37 mL, 0.2 mmol) in DMF
(1 mL), and the reaction was stirred for 1 h. The resin was washed
with DMF (2 ¥ 1 mL), dichloromethane (2 ¥ 1 mL), methanol
(3 ¥ 1 mL), diethyl ether (2 ¥ 1 mL) and then dried under N2. To
the above resin was added dry thionyl chloride/dichloromethane
(1 : 1 v/v, 4 mL) dropwise, and the reaction was gently stirred
at room temperature for 3 h. The resin was washed with DMF
(2 ¥ 1 mL), dichloromethane (3 ¥ 1 mL) and then dried for
10 min. To this resin was added a solution of Fmoc-D-Trp(Boc)-
OH (0.12 g, 0.2 mmol) and DIPEA (92.1 mL, 0.5 mmol) in dry
dichloromethane (2 mL), and the reaction was gently stirred at
room temperature for 30 min. After washing with DMF (2 ¥ 1 mL),
a solution of CH2Cl2/MeOH/DIPEA (80 : 15 : 5 v/v, 10 mL) was
added, the reaction was stirred for 10 min and repeated. After
washing with DMF (3 ¥ 1 mL), a 20% piperidine/DMF solution
(v/v, 10 mL) was added and the reaction was stirred for 3 min and
then repeated for 20 min. The resin was washed with DMF (6 ¥
1 mL), iPrOH (3 ¥ 1 mL), hexane (4 ¥ 1 mL), air dried for 15 min
and then dried under N2. The loading of Fmoc-D-Trp(Boc)-OH
was found to be 0.39 mmol g-1 (theoretical loading = 0.58 mmol g-1,
67%) using the Fmoc assay.14
v
max(film)/cm-1 3315, 1736, 1649, 1514, 1165, 1060, 743 and 697;
[a]2D0 -36.9 (c 0.52 in MeOH); dH(300 MHz; DMSO-d6)* 1.03 (3H,
d, Thrb–CH3), 2.28–2.44 (2H, m, b–Ala–CH2), 2.74–2.95 (3H,
m, Trpb–CH2), 3.12–3.17 (1H, m, Trpb–CH2), 3.55–3.59 (2H,
m, b–Ala–CH2), 4.30–4.34 (1H, m, Trpa–CH), 4.44–4.48 (1H,
m, Thra–CH), 4.53–4.57 (1H, m, Trpa–CH), 5.41–5.43 (1H, m,
Thrb–CH), 6.91–6.95 (6H, m, Trp–H7, Trp–H8, Trp–H12), 7.22–
7.35 (8H, m, b–Ala–NH, Trp–H9, PA–Ph), 7.41 (1H, d, J 7.8,
Trp–H6), 7.46 (1H, d, J 7.8, Trp–H6), 7.82 (1H, d, J 7.8, Thr–
CONH), 8.44–8.59 (2H, m, Trp–CONH), 10.69 (2H, s, Trp–H11);
dC(75 MHz, DMSO-d6) 16.95 (CH3, Thrb–CH3), 26.45 (CH2,
Trpb–CH2), 27.04 (CH2, Trpb–CH2), 34.77 (CH2, b-Ala–CH2),
35.21 (CH2, b-Ala–CH2), 42.56 (CH2, PhCH2CON), 53.70 (CH,
Trpa–CH), 55.32 (CH, Trpa–CH), 56.59 (CH, Thra–CH), 70.43
(CH, Thrb–CH), 109.98 (quat., Trp–C4), 110.93 (quat., Trp–C4),
111.85 (CH, Trp–C9), 118.54 (CH, Trp–C6 and C7), 118.77 (CH,
Trp–C6 and C7), 121.33 (CH, Trp–C8), 121.39 (CH, Trp–C8),
123.66 (CH, Trp–C12), 126.90 (CH, Ph), 127.51 (quat., Trp–C5),
127.59 (quat., Trp–C5), 128.75 (CH, Ph), 129.48 (CH, Ph), 136.47
(quat., Trp–C10), 136.85 (quat., PA–Ph), 169.70 (quat., b–Ala–
CON), 171.30 (quat., Thr–CON), 171.45 (quat., PhCH2CON),
171.52 (quat., Trp–CON), 171.76 (quat., Trp–CON); m/z (EI)
663.2918 (MH+, C37H39N6O6 requires 663.2926), 663 (MH+, 21%),
682 (30), 685 (MNa+, 100%), 686 (40), 687 (9) and 701 (42). (* 1H
peaks for PhCH2CON are not included as they are obscured by
DMSO-d6.)
4.3 Synthesis of PA-L-[Thr-D-Trp-L-Trp-b-Ala] 11
4.3.1 Synthesis of PA-L-Thr-D-Trp-L-Trp-2-ClTrt-resin 7. To
H2N-L-Trp-2-ClTrt-resin (0.56 mmol g-1) (0.18 g, 0.1 mmol) was
added a mixture of Fmoc-D-Trp(Boc)-OH (0.16 g, 0.31 mmol),
HBTU (0.11 g, 0.29 mmol) and DIPEA (0.11 mL, 0.61 mmol)
in DMF (1 mL), and the reaction was agitated for 45 min. The
resin was washed with DMF (6 ¥ 1 mL), isopropanol (3 ¥ 1 mL),
hexane (4 ¥ 1 mL) and then dried under N2. A solution of 20%
piperidine/DMF (v/v, 10 mL) was added and the reaction was
gently agitated for 5 min. The resin was washed with DMF (6 ¥
1 mL), and the same procedure was repeated for 10 min. After
washing with DMF (6 ¥ 1 mL), a mixture of PA-L-Thr-OH (0.08 g,
0.33 mmol), HBTU (0.11 g, 0.29 mmol) and DIPEA (0.11 mL,
0.61 mmol) in DMF (1 mL) was added, and the reaction was
agitated for 45 min. The resin was washed with DMF (6 ¥ 1 mL),
isopropanol (3 ¥ 1 mL), hexane (4 ¥ 1 mL) and then dried under
N2.
4.4 Synthesis of PA-L-[Thr-L-Trp-D-Trp-b-Ala] 12
The title compound [4% from resin 13 (0.39 mmol g-1) (0.25 g,
0.1 mmol)] was obtained as a colourless amorphous solid in >99%
purity according to analytical RP-HPLC; Rt 10.70 min (XTerra
C18, 4.6 ¥ 150 mm, 30% to 75% B over 15 min, 1 mL min-1);
mp 181.9–184.6 ◦C; vmax(film)/cm-1 3276, 1739, 1634, 1547, 1261,
1233, 1187 and 742; [a]2D0 +61.0 (c 0.58 in MeOH); dH(300 MHz;
DMSO-d6) 1.04 (3H, d, J 6.0, Thrb–CH3), 2.37–2.49 (2H, m,
b–Ala–CH2), 2.82–2.95 (3H, m, Trpb–CH2), 3.16–3.21 (1H, m,
Trpb–CH2), 3.39 (2H, m, b–Ala–CH2), 3.54 (1H, d, J 14.1,
PhCH2CON), 3.65 (1H, d, J 14.1, PhCH2CON), 4.17 (1H, ddd, J
10.2, 6.6 and 3.6, Trpa–CH), 4.52 (1H, q, J 7.2, Trpa–CH), 4.62–
4.65 (1H, m, Thra–CH), 5.09–5.11 (1H, m, Thrb–CH), 6.93–7.09
(6H, m, Trp–H7, Trp–H8, Trp–H12), 7.17–7.42 (8H, m, b–Ala–
NH, Trp–H9, PA–Ph), 7.48 (1H, d, J 7.8, Trp–H6), 7.54 (1H, d, J
7.5, Trp–H6), 8.13 (1H, d, J 6.9, Thr–CONH), 8.76 (1H, d, J 6.9,
Trp–CONH), 8.82 (1H, d, J 6.6, Trp–CONH), 10.63 (1H, s, Trp–
H11), 10.72 (1H, s, Trp–H11); dC(75 MHz; DMSO-d6) 16.70 (CH3,
Thrb–CH3), 26.25 (CH2, Trpb–CH2), 34.48 (CH2, b–Ala–CH2),
35.13 (CH2, b–Ala–CH2), 42.17 (CH2, PhCH2CON), 54.44 (CH,
Trpa–CH), 54.53 (CH, Thra–CH), 55.07 (CH, Trpa–CH), 72.54
4.3.2 Synthesis of PA-L-Thr(Boc-b-Ala)-D-Trp-L-Trp-2-ClTrt-
resin 8. To peptide 7 was added a mixture of Boc-b-Ala-OH
(0.39 g, 2.05 mmol), benzoyl chloride (0.24 mL, 2.04 mmol), tri-
ethylamine (0.57 mL, 4.08 mmol) and DMAP (4.7 mg, 0.04 mmol)
in dichloromethane (10 mL) and the reaction was agitated for 18 h.
The resin was washed with DMF (6 ¥ 1 mL), isopropanol (3 ¥
1 mL), hexane (4 ¥ 1 mL) and dried under N2.
4.3.3 Synthesis of PA-L-[Thr-D-Trp-L-Trp-b-Ala] 1113. To
peptide
8
was added
a
mixture of TFA/H2O/TIPS
(95 : 2.5 : 2.5 v/v, 10 mL) and the reaction was agitated for 1 h.
The solution was filtered and concentrated in vacuo. To the
resultant yellow residue (69.5 mg, 0.1 mmol) was dissolved in
dichloromethane–methanol (4 : 1 v/v, 139 mL) at 0 ◦C, BOPCl
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 236–242 | 241
©

View Article Online
1527, 1457, 1166, 1061, 743 and 698; [a]2D0 +51.1 (c 0.99 in MeOH);
dH(300 MHz; DMSO-d6) 0.92 (3H, d, J 6.6, Thrb–CH3), 2.33–2.36
(2H, m, b-Ala–CH2), 2.88–3.03 (2H, m, Trpb–CH2), 3.05–3.19
(2H, m, Trpb–CH2), 3.42–3.54 (2H, m, b–Ala–CH2), 3.78–3.83
(2H, m, PhCH2CON), 4.35–4.44 (3H, m, Trpa–CH and Thra–
CH), 5.48–5.54 (1H, qd, J 6.6 and 4.5, Thrb–CH), 6.40 (2H, s,
Trp–H8), 6.80 (2H, s, Trp–H12), 7.04–7.10 (2H, m, Trp–H9), 7.12–
7.21 (2H, m, Trp–H6), 7.27–7.42 (7H, m, Trp–H7 and PA–Ph);
dC(75 MHz; DMSO-d6) 16.09 (CH3, Thrb–CH3), 25.26 (CH2,
Trpb–CH2), 26.19 (CH2, Trpb–CH2), 34.57 (CH2, b–Ala–CH2),
35.36 (CH2, b–Ala–CH2), 42.84 (CH2, PhCH2CON), 54.13 (CH,
Trpa–CH), 54.94 (CH, Trpa–CH), 56.11 (CH, Thra–CH), 69.50
(CH, Thrb–CH), 108.22 (quat., Trp–C4), 111.17 (CH, Trp–C9),
111.32 (CH, Trp–C9), 117.74 (CH, Trp–C6), 118.43 (CH, Trp–C6),
118.93 (CH, Trp–C7), 119.47 (CH, Trp–C7), 121.61 (CH, Trp–C8),
121.88 (CH, Trp–C8), 122.82 (CH, Trp–C12), 123.44 (CH, Trp–
C12), 126.94 (quat., Trp–C5), 127.21 (CH, Ph), 127.38 (quat., Trp–
C5), 128.72 (CH, Ph), 128.81 (CH, Ph), 134.66 (quat., PA–Ph),
135.98 (quat., Trp–C10), 136.05 (quat., Trp–C10), 169.99 (quat.,
b–Ala–CON), 171.39 (quat., Thr–CON and PhCH2CON), 171.89
(quat., Trp–CON), 172.63 (quat., Trp–CON); m/z (EI) 663.2920
(MH+, C37H39N6O6 requires 663.2926), 663 (MH+, 100%), 664
(40), 665 (10), 666 (2), 685 (MNa+, 60%), 686 (32) and 687 (8).
(CH, Thrb–CH), 109.82 (quat., Trp–C4), 111.17 (quat., Trp–C4),
111.84 (CH, Trp–C9), 111.90 (CH, Trp–C9), 118.49 (CH, Trp–
C6), 118.81 (CH, Trp–C6 and C7), 121.38 (CH, Trp–C8), 121.53
(CH, Trp–C8), 123.70 (CH, Trp–C12), 123.96 (CH, Trp–C12),
126.78 (CH, Ph), 127.40 (quat., Trp–C5), 127.49 (quat., Trp–C5),
128.64 (CH, Ph), 129.55 (CH, Ph), 136.45 (quat., Trp–C10), 136.54
(quat., Trp–C10), 136.83 (quat., PA–Ph), 169.78 (quat., b–Ala–
CON), 170.83 (quat., Thr–CON), 171.20 (quat., PhCH2CON),
171.61 (quat., Trp–CON), 172.58 (quat., Trp–CON); m/z (EI)
663.2913 (MH+, C37H39N6O6 requires 663.2926), 663 (MH+, 20%),
685 (MNa+, 100%), 686 (32), 687 (8) and 701 (35).
4.5 Synthesis of PA-L-[Thr-L-Trp-L-Trp-b-Ala]
The title compound (6% from H2N-L-Trp-2-ClTrt-resin) was ob-
tained as an off-white amorphous solid in >99% purity according
to analytical RP-HPLC; Rt 10.46 min (XTerra C18, 4.6 ¥ 150 mm,
30% to 75% B over 15 min, 1 mL min-1); mp 162.6–167.4 ◦C;
v
max(film)/cm-1 3324, 1727, 1649, 1522, 1457, 1177, 1069, 742 and
703; [a]2D0 -30.7 (c 0.81 in MeOH); dH(300 MHz; DMSO-d6) 1.13
(3H, d, J 6.3, Thrb–CH3), 2.29–2.37 (1H, m, b-Ala–CH2), 2.60–
2.70 (1H, m, b–Ala–CH2), 2.93–3.03 (2H, m, Trpb–CH2), 3.05–
3.10 (1H, m, Trpb–CH2), 3.24–3.35 (1H, m, Trpb–CH2), 3.36–3.41
(2H, m, b–Ala–CH2), 3.61 (2H, m, PhCH2CON), 4.15–4.28 (2H,
m, Trpa–CH), 4.61–4.65 (1H, m, Thra–CH), 5.03–5.06 (1H, qd,
J 6.3 and 1.5, Thrb–CH), 6.53 (2H, s, Trp–H8), 6.83 (2H, s, Trp–
H12), 7.02–7.08 (1H, m, Trp–H9), 7.12–7.17 (1H, m, Trp–H9),
7.19–7.38 (7H, m, Trp–H7 and PA–Ph), 7.43–7.47 (2H, m, Trp–
H6); dC(75 MHz; DMSO-d6) 16.16 (CH3, Thrb–CH3), 24.48 (CH2,
Trpb–CH2), 25.80 (CH2, Trpb–CH2), 34.28 (CH2, b–Ala–CH2),
34.99 (CH2, b–Ala–CH2), 42.85 (CH2, PhCH2CON), 54.61 (CH,
Thra–CH), 55.22 (CH, Trpa–CH), 55.94 (CH, Trpa–CH), 72.25
(CH, Thrb–CH), 108.70 (quat., Trp–C4), 110.68 (quat., Trp–C4),
111.00 (CH, Trp–C9), 111.15 (CH, Trp–C9), 117.94 (CH, Trp–
C6), 118.09 (CH, Trp–C7), 118.72 (CH, Trp–C6), 118.95 (CH,
Trp–C7), 121.32 (CH, Trp–C8), 121.62 (CH, Trp–C8), 122.90
(CH, Trp–C12), 123.20 (CH, Trp–C12), 126.71 (quat., Trp–C5),
127.12 (quat., Trp–C5), 127.21 (CH, Ph), 128.61 (CH, Ph), 129.03
(CH, Ph), 134.24 (quat., PA–Ph), 136.00 (quat., Trp–C10), 136.12
(quat., Trp–C10), 169.96 (quat., b–Ala–CON), 171.06 (quat., Thr–
CON), 171.42 (quat., PhCH2CON), 171.99 (quat., Trp–CON),
172.08 (quat., Trp–CON); m/z (EI) 663.2913 (MH+, C37H39N6O6
requires 663.2926), 663 (MH+, 20%), 685 (MNa+, 100%), 686 (32),
687 (8) and 701 (35).
Acknowledgements
We thank the Maurice Wilkins Centre for Molecular Discovery
for financial support.
Notes and references
1 G. Lang, T. Kalvelage, A. Peters, J. Wiese and J. F. Imhoff, J. Nat. Prod.,
2008, 71, 1074–1077.
2 J. M. Crawford, R. Kontnik and J. Clardy, Curr. Biol., 2010, 20, 69–74.
3 J. L. Norelli, A. L. Jones and H. S. Aldwinckle, Plant Dis., 2003, 87,
756–765.
4 E. Badosa, Peptides, 2007, 28, 2276–2285.
5 J. E. Loper, Plant Dis., 1991, 75, 287–290.
6 M. Quibell, L. C. Packman and T. Johnson, J. Am. Chem. Soc., 1995,
117, 11656–11668.
7 M. Harre, K. Nickisch and U. Tilstam, React. Funct. Polym., 1999, 41,
111–114.
8 K. Barlos and D. Gatos, in Fmoc Solid Phase Peptide Synthesis, ed.
W. C. Chan and P. D. White, Information Press, Oxford, U. K., 2000,
ch. 9, pp. 216.
9 K. Barlos, O. Chatzi and D. Gatos, Int. J. Peptide Protein Res., 1991,
37, 513–520.
10 G. P. Nora, M. J. Miller and U. Mo¨llmann, Bioorg. Med. Chem. Lett.,
2006, 16, 3966–3970.
4.6 Synthesis of PA-L-[Thr-D-Trp-D-Trp-b-Ala]
11 M. Stawikowski and P. Cudic, Tetrahedron Lett., 2006, 47, 8587–
The title compound [8% from resin 13 (0.39 mmol g-1) (0.25 g,
0.1 mmol)] was obtained as an off-white amorphous solid in
>90% purity according to analytical RP-HPLC; Rt 10.53 min
(XTerra C18, 4.6 ¥ 150 mm, 30% to 75% B over 15 min,
1 mL min-1); mp 155.2–159.2 ◦C; vmax(film)/cm-1 3315, 1727, 1657,
8590.
12 I. Dhimitruka and Jr. J. SantaLucia, Org. Lett., 2006, 8, 47–50.
13 A. K. Ghosh and C. Liu, Org. Lett., 2001, 3, 635–638.
14 W. C. Chan and P. D. White, in Fmoc Solid Phase Peptide Synthesis, ed.
W. C. Chan and P. D. White, Information Press, Oxford, U. K., 2000,
ch. 9, pp. 62.
242 | Org. Biomol. Chem., 2011, 9, 236–242
This journal is
The Royal Society of Chemistry 2011
©