Alaninyl variants of the marine natural product halocyamine A and their antibacterial properties

Article abstract of DOI:10.1016/j.tet.2018.10.021

In an effort to explore the antibacterial potential of the marine natural product halocyamine A, a series of analogues including desbromo and alanine-substituted variants were synthesised and evaluated for biological activity against a panel of Gram-positive and –negative bacteria. The analogues were synthesised by a combination of solid-phase peptide synthesis and ruthenium complex/ytterbium triflate catalysed hydroamidation chemistry. Single alanine substitutions ([Ala¹]-halocyamine A and [Ala²]-halocyamine A) gave only modest increases in activity towards Gram-positive bacteria, while di-alaninyl variants exhibited more potent activity with MIC values of 12.5–50 μM towards the Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis. A lipophilic trityl-protected intermediate of [Ala²]-halocyamine was the most active against the Gram-negative bacterium Escherichia coli.

Full text of DOI:10.1016/j.tet.2018.10.021

Tetrahedron xxx (2018) 1e10
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Alaninyl variants of the marine natural product halocyamine A and
their antibacterial properties
Hugo K.H. Fong a, Melissa M. Cadelis a, Jean Michel Brunel b,
,
*
Marie-Lise Bourguet-Kondracki c, David Barker a, Brent R. Copp a
a School of Chemical Sciences, The University of Auckland, Private Bay 92019, Auckland, 1142, New Zealand
b
ꢀ
ꢀ
ꢀ
UMR_MD1 “Membranes et Cibles Therapeutiques”, U1261 INSERM, Aix-Marseille Universite, Faculte de pharmacie, 27 Bd Jean Moulin, 13385, Marseille,
cedex 5, France
c
ꢀ
ꢀ
Laboratoire Molecules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Museum National d'Histoire Naturelle, 57 rue Cuvier
(C.P. 54), 75005, Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to explore the antibacterial potential of the marine natural product halocyamine A, a series of
analogues including desbromo and alanine-substituted variants were synthesised and evaluated for
biological activity against a panel of Gram-positive and enegative bacteria. The analogues were syn-
thesised by a combination of solid-phase peptide synthesis and ruthenium complex/ytterbium triflate
catalysed hydroamidation chemistry. Single alanine substitutions ([Ala1]-halocyamine A and [Ala2]-
halocyamine A) gave only modest increases in activity towards Gram-positive bacteria, while di-alaninyl
Received 14 August 2018
Received in revised form
8 October 2018
Accepted 11 October 2018
Available online xxx
variants exhibited more potent activity with MIC values of 12.5e50 mM towards the Gram-positive
Keywords:
Antibacterial
Halocyamine
Alaninyl analogue
Enamide
bacteria Staphylococcus aureus and Enterococcus faecalis. A lipophilic trityl-protected intermediate of
[Ala2]-halocyamine was the most active against the Gram-negative bacterium Escherichia coli.
© 2018 Elsevier Ltd. All rights reserved.
Marine natural product
1. Introduction
halocyamines A (1) (Fig. 1) and B from hemocytes (blood cells) of
the ascidian Halocynthia roretzi [6,7] and found them to exhibit
The tunichromes are small, modified peptides isolated from the
blood cells of marine organisms belonging to the Class Ascidiacea
[1e3]. Structurally, these natural products are typified by the
antibacterial, antiviral and cytotoxic properties [8]. As part of our
ongoing investigation of the chemistry and biology of tunichromes,
we recently reported the synthesis and structural confirmation of
halocyamine A (1) [9]. In our hands, synthetic halocyamine A only
exhibited modest antibacterial activities. We were interested
however in the potential of this class of natural product to act as a
bioactive scaffold so we sought to use our robust synthetic meth-
odology to prepare novel halocyamine A analogues that may
indeed exhibit more potent biological activities. Our target com-
pounds were a desbromo analogue (2) (Fig. 1) and a sequence of
alanine-scan-like analogues, whereby His and/or DOPA amino acids
in 1 were replaced with alanine. Herein we report the synthesis and
antibacterial activities of these halocyamine A analogues.
presence of a C-terminal decarboxy-D
2,3-enamide fragment. While
the C-terminus amino acid is usually tyrosine, 3,4-
dihydroxyphenylalanine (DOPA) or 3,4,5-trihydroxyphenylalanine
(TOPA)- derived, the halocyamines (e.g. A, 1) are unusual in that
the C-terminus moiety is derived from 6-bromotryptophan and
takes the form of a Z-configuration indolic enamide. A number of
ecological roles have been proposed for the tunichromes, varying
from tunic formation (cross-linking) [4], acting as a primitive
wound repair system, to metal ion sequestration [5] or that they act
as antimicrobial agents [4]. Indeed there is a growing body of
biochemical evidence that many of these roles are at least feasible
[4,5].
2. Results and discussion
In the specific case of the halocyamines, Azumi et al. isolated
In our previously reported synthesis of halocyamine A (1), the
natural product was disconnected at the DOPA-Gly amide bond to
give two fragments, Fmoc-His(Trt)-DOPA(TBDMS)2-OH (3) and Z-
* Corresponding author.
E-mail address: b.copp@auckland.ac.nz (B.R. Copp).
https://doi.org/10.1016/j.tet.2018.10.021
0040-4020/© 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
properties, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.10.021

2
H.K.H. Fong et al. / Tetrahedron xxx (2018) 1e10
Fig. 1. Structures of halocyamine A (1) and desbromohalocyamine A (2).
Scheme 2. Reagents and conditions: (i) EDC$HCl (1.5 eq.), HOBt (2 eq.), DIPEA (6 eq.),
CH2Cl2, 9 h, 39%; (ii) piperidine, DMF, r.t., 20 min, 68%; (iii) Et3N$3HF, THF, 0 ꢀC, 55 min,
69%; (iv) 0.01 N HCl/HFIP, H2O, TIS, r.t., 1 h, 73%.
the protected dipeptide Fmoc-Ala-DOPA(TBDMS)2-OH (95% purity)
(14) which was prepared using standard Fmoc-SPPS methodology
in 81% yield (see experimental). Bromo-indole enamide 4 [9] was
then coupled (EDC, HOBt, DIPEA, 7.5 h) with 14 to give 15 (95Z:5E
mixture) in 42% yield (Scheme 3). Removal of the Fmoc protecting
group (piperidine, DMF, 20 min) gave the chromatographically-
separable (Z)-16 (36%) and (E)-16 (31%). Desilylation of (Z)-16
(triethylamine trihydrofluoride, THF, 55 min) gave [Ala1]-halocy-
amine A 12 (as an inseparable 9:1 Z:E mixture) in 67% yield.
A similar reaction sequence was used to prepare the second Ala-
Fig. 2. Fragments 3e5.
enamide 4 (Fig. 2) [9]. The two fragments were prepared separately
using Fmoc solid phase peptide synthesis (SPPS) and ruthenium-
catalysed hydroamidation of an indole-acetylene, respectively.
In the current project, we sought to explore the influence, if any,
variant 13 with -Ala replacing the DOPA residue. Dipeptide 17,
L
of bromine substitution and each of the amino acids L-His and L-
prepared by Fmoc-SPPS in 88% yield, was coupled with 4 to give 18
in 44% yield (Scheme 4). Deprotection at the N-terminus (piperi-
dine, DMF, 20 min) gave poorly soluble 19 (86%) (as a 4:1 mixture of
Z:E isomers) which followed by removal of the trityl group (HCl/
HFIP, H2O, TIS, 1 h) afforded [Ala2]-halocyamine A (13) (83%) as an
inseparable mixture of Z/E (1:0.9) isomers.
DOPA on the observed antibacterial activity of halocyamine A. To
this end, our first target was desbromo halocyamine A 2, which
required synthesis of indole-enamide 5. The known indolic alkyne
6 [10] was subjected to hydroamidation with Fmoc-glycinamide
using 5 mol% bis(2-methylallyl)(1,5-cyctooctadiene)ruthenium(II)
heated at 70 ꢀC for 32 h to give glycyl-Z-enamide 7 (69%) and by-
product (E)-enyne 8 [9] (18%) (Scheme 1). Enamide 7 was then
deprotected (TFA/CH2Cl2) to give 5 in 89% yield.
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-medi-
ated coupling of enamide 5 and protected dipeptide Fmoc-His(Trt)-
DOPA(TBDMS)2-OH 3 [9] afforded the protected halocyamine
analogue 9 in 39% yield (Scheme 2). Step-wise Fmoc deprotection
(piperidine, DMF, 20 min) to give 10 (68% yield), followed by desi-
lylation (triethylamine trihydrofluoride, THF, 55 min) to give 11
(69% yield) and lastly, removal of the trityl group (1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP), H2O, HCl, triisopropylsilane (TIS),
1 h) afforded desbromo halocyamine A 2 as the dihydrochloride salt
in 73% yield.
A final two analogues were prepared that explored substituting
both His and DOPA residues for either
L-Ala-L-Ala (20) or D-Ala-D-
Ala (21). The requisite Fmoc-dipeptides 22 and 23 were prepared by
SPSS and then coupled with 4 (EDC, HOBt, DIPEA, 8 h) to afford,
respectively 24 (33%) or 25 (34%) (Scheme 5). Subsequent depro-
tection (piperidine, DMF, 20 min) gave [Ala1,Ala2]-halocyamine A
20 (60%) or [D D
-Ala1, -Ala2]-halocyamine A 21 (69%), respectively.
Both 20 and 21 were obtained as an inseparable mixture of Z/E
(1:0.9) isomers.
Desbromo halocyamine A 2, alanine analogues 12 and 13, di-
alaninyl analogues 20 and 21 and a number of the reaction in-
termediates (15, 16, 18, 19, 24, 25) were evaluated against a panel of
Gram-negative (Pseudomonas aeruginosa ATCC 27853, Escherichia
coli ATCC 25922) and Gram-positive (Staphylococcus aureus ATCC
25923, Enterococcus faecalis ATCC 29212) bacteria and for
We next prepared two
L-alanine-substituted variants 12 and 13.
The first of these analogues (12), where Ala replaced His, required
Scheme 1. Reagents and conditions: (i) Fmoc-glycinamide (0.5 eq.), bis(2-methylallyl)(1,5-cyclooctadiene)ruthenium(II) (0.025 eq.), 1,4-bis(dicyclohexylphosphino)butane (0.03 eq.),
yttrium triflate (0.02 eq.), DMF, H2O, 70 ꢀC, 32 h, 69% (7) and 18% (8); (ii) TFA (60 eq.), CH2Cl2, 0 ꢀC, 8 h, 89%.
Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
properties, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.10.021

H.K.H. Fong et al. / Tetrahedron xxx (2018) 1e10
3
Scheme 3. Reagents and conditions: (i) 4 (1 eq.), EDC$HCl (1.5 eq.), HOBt (2 eq.), DIPEA (6 eq.), CH2Cl2, r.t., 7.5 h, 42%; (ii) piperidine, DMF, r.t., 20 min, 36% (Z), 31% (E); (iii) Et3N$3HF,
THF, 0 ꢀC, 55 min, 67%.
Scheme 4. Reagents and conditions: (i) 4 (1 eq.), EDC$HCl (1.1 eq.), HOBt (1.5 eq.), DIPEA (5 eq.), CH2Cl2, DMF, r.t., 8 h, 44%; (ii) piperidine, DMF, r.t., 20 min, 86%; (iii) 0.01 N HCl/HFIP,
H2O, TIS, r.t., 1 h, 83% (Z:E, 1:0.9).
cytotoxicity towards the mammalian L6 rat skeletal myoblast cell
line (Table 1). As we have previously noted, halocyamine A exhibits
poor levels of antibacterial activity, with a modest MIC of 100 mM
towards P. aeruginosa and E. faecalis [9]. In the present study, the
desbromo analogue 2 failed to exhibit any antibacterial activities,
while both of the [Ala1] (12) and [Ala2] (13) analogues (both tested
as an E/Z mixture of isomers) showed a modest increase in activity
these compounds and not for the corresponding de-tritylated
analogue (13) suggests that the presence of a sterically bulky
lipophilic residue at the His-residue position in halocyamine A may
be a useful starting point for the discovery of novel Gram-negative
antibacterials.
Gram-positive antibacterial activity was observed for both di-
alaninyl analogues with the di-
tivity towards S. aureus (MIC 12.5
analogue 21 exhibiting activity towards both S. aureus and
E. faecalis (both MIC 50 M). Modest cytotoxicity was observed for
these analogues (IC50 115.8 and 142 M, respectively), equating to a
selectivity index (¼ cytotoxicity IC50/antibacterial MIC) in the case
of di- -alaninyl 20 of close to ten. The antibacterial activity for both
di-alaninyl analogues was dependent upon a free amine at the N-
L-alaninyl variant 20 exhibiting ac-
towards Gram-positive bacteria (S. aureus MIC 100
50e100
M). An interesting finding related to the [Ala2] analogue
was the enhanced activity towards the Gram-negative bacterium
E. coli (MIC 25e50 M) for the Fmoc/trityl (18) and trityl-protected
m
M; E. faecalis
m
M) and the di- -alaninyl
D
m
m
m
m
(19) intermediates. Although these same two compounds also
exhibited cytotoxicity towards the L6 cell line (IC50 65.6 and
25.6 mM, respectively) the observation of antibacterial activity for
L
Scheme 5. Reagents and conditions: (i) 4 (1 eq.), EDC$HCl (1.1 eq.), HOBt (1.5 eq.), DIPEA (5 eq.), CH2Cl2, DMF, r.t., 8 h, 33%; (ii) piperidine, DMF, r.t., 20 min, 60%; (iii) 4 (1 eq.),
EDC$HCl (1.1 eq.), HOBt (1.5 eq.), DIPEA (5 eq.), CH2Cl2, DMF, r.t., 8 h, 34%; (iv) piperidine, DMF, r.t., 20 min, 69%.
Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
properties, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.10.021

4
H.K.H. Fong et al. / Tetrahedron xxx (2018) 1e10
Table 1
DOPA(TBDMS)2-OH [11], Fmoc-His(Trt)-DOPA(TBDMS)2-OH 3 [9],
bromo-indole-enamide 4 [9] and indolic alkyne 6 [10], were pre-
pared by published routes and data obtained were in agreement
with those previously reported.
Summary of biological activities observed for compounds 1, 2, 12, 13, 15, 16, 18e21,
24 and 25.
Compound
P. aeruginosaa E. colib S. aureusc E. faecalisd L6e
1
2
100
>200
>200
>200
>200
>200
>200
50
25
200
>200
100
>200
2
>200
>200
100
100
>200
25
>200
25
12.5
50
100
NT
50
100
NT
NT
>200
200
200
50
>200
100
NTf
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
1
>150
82.8
136.8
NT
4.1.1. tert-Butyl (Z)-3-(2-(2-aminoacetamido)vinyl)-1H-indole-1-
carboxylate (7) and di-tert-butyl 3,3'-(but-1-en-3-yne-1,4-diyl)(E)-
bis(1H-indole-1-carboxylate) (8)
Fmoc-Gly-NH2 (0.296 g, 1.00 mmol), tert-butyl 3-ethynyl-1H-
indole-carboxylate (0.482 g, 2.00 mmol), bis(2-methylallyl)(1,5-
12
13g
15
16
18
19
20
21
24
25
4.8
65.6
25.6
115.8
142
48.5
NT
cyclooctadiene)ruthenium(II)
(0.016 g,
0.050 mmol),
1,4-
bis(dicyclohexylphosphino)butane (0.027 g, 0.060 mmol) and
ytterbium triflate (0.025 g, 0.040 mmol) were placed under vacuum
and then flushed with N2 (four times). A degassed DMF (3.00 mL)/
200
100
Colistin
Chloramphenicol
Podophyllotoxin
water (108 mL, 6.00 mmol) mixture was added and the solution
stirred under N2 at 70 ꢀC for 32 h. The reaction was then quenched
with sat. aq. NaHCO3 (30 mL) and the resulting mixture extracted
with EtOAc (5 ꢁ 20 mL). The combined organic layers were washed
with water (30 mL) and brine (30 mL), dried (MgSO4), filtered, and
the solvent removed in vacuo. Purification using silica gel column
chromatography (eluting with n-hexane to n-hexane/EtOAc 9:1)
afforded 8 (0.087 g, 18%) as a brown oil while elution with a more
polar solvent mixture (n-hexane/EtOAc 1:1 to EtOAc) gave 7
(0.220 g, 69%) as a yellow oil.
1.5e3
1.5e3
0.013
a
P. aeruginosa ATCC27853. MIC (
E. coli ATCC25922. MIC (
m
M) values presented as the mean (n ¼ 3).
M) values presented as the mean (n ¼ 3).
S. aureus ATCC25923. MIC (
M) values presented as the mean (n ¼ 3).
M) values presented as the mean (n ¼ 3).
L6 rat skeletal myoblast cell line. IC50 M) values presented as the average
b
c
m
m
m
d
e
E. faecalis ATCC29212. MIC (
(m
(n ¼ 2).
f
Not tested.
Tested as a mixture of E/Z isomers.
g
4.1.1.1. tert-Butyl (Z)-3-(2-(2-aminoacetamido)vinyl)-1H-indole-1-
terminus, as both Fmoc-protected precursors (24 and 25) were
inactive.
carboxylate (7). RG 0.15 (n-hexane/EtOAc 8:2); IR (ATR) nmax 3329,
2973, 2931, 1700, 1451, 1368, 1253, 1149 cmꢂ1 1H NMR (CDCl3,
;
400 MHz)
d
9.71 (1H, d, J ¼ 11.6 Hz, H-4), 8.13 (1H, d, J ¼ 7.9 Hz, H-
3. Conclusions
10), 7.76 (1H, s, H-8), 7.58 (1H, d, J ¼ 7.9 Hz, H-13), 7.36 (1H, td,
J ¼ 7.9, 1.2 Hz, H-11), 7.28 (1H, td, J ¼ 7.9, 1.2 Hz, H-12), 7.06 (1H, dd,
J ¼ 11.6, 9.2 Hz, H-5), 5.86 (1H, d, J ¼ 9.2 Hz, H-6), 3.47 (2H, s, H2-2),
In conclusion, we have established that alaninyl analogues of the
marine natural product halocyamine A exhibit more pronounced
antibacterial activities than the natural product. While single
alanine substitutions gave modest increases in activity (to MIC
1.68 (9H, s, CO2C(CH3)3); 13C NMR (CDCl3, 100 MHz)
d 170.5 (C-3),
149.7 (CO2C(CH3)3), 135.1 (C-9a), 129.9 (C-13a), 125.2 (C-11), 123.0
(C-12),122.6 (C-8),121.9 (C-5),119.4 (C-13),115.9 (C-7),115.3 (C-10),
100.4 (C-6), 84.0 (CO2C(CH3)3), 44.6 (C-2), 28.3 (CO2C(CH3)3);
(þ)-HRESIMS [MþH]þ 316.1650 (calcd. for C17H22N3O3, 316.1656).
50e100
mM), di-alaninyl analogues exhibited MIC values of
12.5e50
mM towards the Gram-positive bacteria S. aureus and
E. faecalis. The finding of activity of a His(Trt)-Ala analogue of hal-
ocyamine A towards E. coli is particularly encouraging, suggesting
that exploration of increasingly lipophilic analogues of [Ala2]-hal-
ocyamine could be worthwhile in the search for new molecules
active against Gram-negative bacteria.
4.1.1.2. Di-tert-butyl
indole-1-carboxylate) (8). RG 0.79 (n-hexane/EtOAc 4:1); IR (ATR)
nmax 2977, 2933, 2865, 1733, 1451, 1368, 1231, 1147, 1093 cmꢂ1 1H
NMR (CDCl3, 500 MHz) 8.18e8.14 (2H, m, H-7 and H-15), 7.78 (1H,
3,3'-(but-1-en-3-yne-1,4-diyl)(E)-bis(1H-
;
d
s, H-13), 7.78e7.73 (2H, m, H-4 and H-18), 7.70 (1H, s, H-2),
7.37e7.32 (2H, m, H-6 and H-16), 7.31e7.26 (2H, m, H-5 and H-17),
7.17 (1H, d, J ¼ 15.8 Hz, H-8), 6.52 (1H, d, J ¼ 15.8 Hz, H-9), 1.66e1.65
4. Experimental
4.1. General
(18H, m, 3H3-21 and 3H3-24); 13C NMR (CDCl3,125 MHz)
d 149.4 (C-
19 or C-22), 149.1 (C-19 or C-22), 136.1 (C-7a), 134.8 (C-14a), 132.6
(C-8), 130.6 (C-18a), 128.5 (C-13), 128.2 (C-3a), 125.2 (C-6 or C-16),
125.0 (C-6 or C-16), 124.8 (C-2), 123.29 (C-5 or C-17), 123.27 (C-5 or
C-17), 120.2 (C-4 or C-18), 120.0 (C-4 or C-18), 118.5 (C-3), 115.6 (C-7
or C-15), 115.3 (C-7 or C-15), 108.0 (C-9), 103.9 (C-12), 92.9 (C-10),
84.24 (C-20 or C-23), 84.17 (C-20 or C-23), 83.4 (C-11), 28.19 (3C-21
or 3C-24), 28.17 (3C-21 or 3C-24); (þ)-HRESIMS [MþNa]þ 505.2091
(calcd. for C30H30N2O4Na, 505.2098).
Infrared spectra were run as dry films on an FTIR infrared
spectrometer fitted with a universal ATR sampling accessory. Op-
tical rotations were recorded using a 0.1 dm cell in methanol. NMR
spectra were recorded at either 500, 400 or 300 MHz for 1H nuclei
and 125, 100 or 75 MHz for 13C nuclei. Proto-deutero solvent signals
were used as internal references (CD3OD: dH 3.31, dC 49.0; CDCl3: dH
TMS 0, dC 77.16; DMSO‑d6: dH 2.50, dC 39.52). 1H NMR data is re-
ported as position (
d
), relative integral, multiplicity (s ¼ singlet,
d ¼ doublet, t ¼ triplet, m ¼ multiplet, br ¼ broad, obs ¼ obscured),
4.1.2. (Z)-N-(2-(1H-Indol-3-yl)vinyl)-2-aminoacetamide (5)
coupling constant (J, Hz), and the assignment of the atom. 13C NMR
A solution of 7 (0.170 g, 0.436 mmol) in CH2Cl2 (2 mL) was stir-
red at 0 ꢀC under N2 for 5 min before TFA (2.00 mL, 26.1 mmol) was
added dropwise. The solution was then stirred for a further 8 h at
0 ꢀC. The reaction was quenched with sat. aq. NaHCO3 (20 mL) and
extracted with EtOAc (4 ꢁ 20 mL). The organic layers were com-
bined and dried in vacuo. The crude black oil was purified by silica
gel column chromatography (eluting with EtOAc to CH2Cl2/MeOH
9:1) to afford 5 (0.13 g, 89%) as a black oil. RG 0.50 (CH2Cl2/MeOH
data are reported as position (d) and assignment of the atom. As-
signments were based on 2D NMR data acquired using standard
COSY, multiplicity edited HSQC and HMBC pulse sequences. MS
data were acquired on a micrOTOF Q II mass spectrometer. Flash
column chromatography was carried out on C2 or C8 (reversed-
phase) or on silica gel (normal phase). All solvents used were
distilled analytical grade or better. Chemical reagents used were
purchased from standard chemical suppliers. Compounds Fmoc-
9:1); IR (ATR) nmax 3403, 3295, 1655, 1535, 1499, 1231 cmꢂ1 1H
;
Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
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H.K.H. Fong et al. / Tetrahedron xxx (2018) 1e10
5
NMR (CDCl3, 400 MHz)
d
9.53 (1H, br s, NH-4), 8.30 (1H, br s, NH-9),
TrtAr), 7.02e7.00 (6H, m, 6H-TrtAr), 6.89 (1H, d, J ¼ 7.8 Hz, NH-10),
6.74e6.70 (2H, m, H-17 and H-24), 6.68e6.67 (1H, m, H-14),
6.58e6.55 (2H, m, H-8 and H-18), 5.87 (1H, d, J ¼ 9.1 Hz, H-25), 4.69
(1H, ddd, J ¼ 7.8, 7.2, 6.4 Hz, H-11), 4.12 (1H, dd, J ¼ 16.8, 6.0 Hz, H2-
21a), 3.83 (1H, dd, J ¼ 16.8, 6.0 Hz, H2-21b), 3.50 (1H, dd, J ¼ 5.2,
5.2 Hz, H-2), 3.05 (1H, dd, J ¼ 14.1, 7.2 Hz, H2-12a), 2.99 (1H, dd,
J ¼ 14.1, 7.2 Hz, H2-12b), 2.85 (1H, dd, J ¼ 14.7, 5.2 Hz, H2-3a), 2.71
(1H, dd, J ¼ 14.7, 5.2 Hz, H2-3b), 0.97e0.96 (18H, m, 2SiC(CH3)3),
7.65 (1H, d, J ¼ 7.5 Hz, H-13), 7.40 (1H, d, J ¼ 8.6 Hz, H-10), 7.35 (1H,
d, J ¼ 2.4 Hz, H-8), 7.27e7.24 (1H, m, H-11), 7.18 (1H, t, J ¼ 7.5 Hz, H-
12), 6.98 (1H, dd, J ¼ 11.5, 9.2 Hz, H-5), 6.00 (1H, d, J ¼ 9.2 Hz, H-6),
3.45 (2H, s, H2-2); 13C NMR (CDCl3, 100 MHz)
d 170.3 (C-3), 135.9 (C-
9a), 126.8 (C-13a), 123.0 (C-11), 121.8 (C-8), 120.3 (C-12), 119.7 (C-
13), 119.5 (C-5), 112.0 (C-7), 111.3 (C-10), 102.6 (C-6), 44.7 (C-2);
(þ)-HRESIMS [MþH]þ 216.1130 (calcd. for C12H14N3O, 216.1131).
0.17e0.16 (12H, m, 2Si(CH3)2); 13C NMR (CDCl3,100 MHz)
d 174.9 (C-
4.1.3. (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-((2-(((Z)-2-(1H-
indol-3-yl)vinyl)amino)-2-oxoethyl)amino)-3-(3,4-bis((tert-
butyldimethylsilyl)oxy)phenyl)-1-oxopropan-2-yl)amino)-1-oxo-3-
(1-trityl-1H-imidazole-4-yl)propan-2-yl)carbamate (9)
To a solution of dipeptide 3 (0.43 g, 0.42 mmol), enamide 5
(0.090 g, 0.42 mmol), EDC$HCl (0.12 g, 0.63 mmol) and HOBt (0.11 g,
0.84 mmol) in CH2Cl2 (3 mL) was added DIPEA (0.44 mL, 2.5 mmol)
under N2 and stirred for 9 h. The reaction mixture was diluted with
CH2Cl2 (15 mL), washed with water (10 mL) and the organic solvent
removed in vacuo. Subsequent purification by silica gel column
9), 172.3 (C-19), 166.7 (C-22), 147.1 (C-15), 146.1 (C-16), 142.2 (3C-
TrtAr), 128.1 (3C-TrtAr), 139.0 (C-6), 136.0 (C-4), 135.8 (C-28a), 129.8
(C-13), 129.7 (6C-TrtAr), 128.3 (6C-TrtAr), 127.1 (C-32a), 123.1 (C-
27),122.6 (C-30),122.3 (C-18),122.1 (C-14),121.3 (C-17),120.6 (C-8),
119.9 (C-31), 118.9 (C-32), 118.6 (C-24), 111.4 (C-29), 110.8 (C-26),
103.6 (C-25), 75.6 (CAr3), 54.40 (C-2 or C-11), 54.37 (C-2 or C-11),
43.8 (C-21), 36.6 (C-12), 33.1 (C-3), 26.1 (2SiC(CH3)3), 18.6
(SiC(CH3)3), 18.5 (SiC(CH3)3), ꢂ3.9 (2Si(CH3)2); (þ)-HRESIMS
[MþH]þ 1002.5152 (calcd. for C58H72N7O5Si2, 1002.5128).
chromatography (eluting with n-hexane/EtOAc 9:1 to n-hexane/
4.1.5. (S)-N-(2-(((Z)-2-(1H-Indol-3-yl)vinyl)amino)-2-oxoethyl)-2-
((S)-2-amino-3-(1-trityl-1H-imidazole-4-yl)propanamido)-3-(3,4-
dihydroxy phenyl)propanamide (11)
27
EtOAc 1:1) afforded 9 (0.20 g, 39%) as a yellow oil. [
a
]
þ16.6 (c
D
1.57, CH2Cl2); RG 0.58 (CH2Cl2/MeOH 9:1); IR (ATR) nmax 2930, 2861,
1714, 1670, 1509, 1447, 1297, 1253, 1155, 1130 cmꢂ1; 1H NMR (CDCl3,
Triethylamine trihydrofluoride (4.59
mL, 28.5 mmol) was added
500 MHz)
d
9.02 (2H, br s, NH-20 and NH-28), 8.22 (1H, d,
to a solution of 10 (9.5 mg, 9.5
m
mol) in THF (0.50 mL) at 0 ꢀC and
J ¼ 10.9 Hz, NH-23), 7.75 (2H, d, J ¼ 6.8 Hz, H-FmocAr), 7.73 (1H, d,
J ¼ 7.3 Hz, H-FmocAr), 7.56 (1H, d, J ¼ 7.7 Hz, H-32), 7.52e7.49 (1H,
m, 2H-FmocAr), 7.39e7.36 (4H, m, H-6, H-29 and 2H-FmocAr),
7.30e7.25 (7H, m, H-FmocAr and 6H-TrtAr), 7.24e7.20 (3H, m, H-27,
H-30 and H-FmocAr), 7.07e7.05 (3H, m, H-31 and 3H-TrtAr),
7.00e6.99 (6H, m, 6H-TrtAr), 6.74e6.73 (2H, m, H-17 and H-24),
6.68 (2H, br s, NH-10 and H-14), 6.56e6.54 (2H, m, H-8 and H-18),
6.01 (1H, d, J ¼ 5.9 Hz, NH-1), 5.88 (1H, d, J ¼ 9.1 Hz, H-25), 4.76 (1H,
br s, H-11), 4.31e4.29 (3H, m, CO2CH2CH and H-2), 4.15e4.12 (2H,
m, CO2CH2CH and H2-21a), 3.76 (1H, dd, J ¼ 16.7, 4.8 Hz, H2-21b),
3.07 (1H, dd, J ¼ 14.2, 7.1 Hz, H2-12a), 3.00e2.94 (2H, m, H2-3a and
H2-12b), 2.65 (1H, br s, H2-3b), 0.97e0.95 (18H, m, 2SiC(CH3)3),
stirred under N2 for 55 min. The reaction mixture was dried under a
stream of N2 and water (15 mL) was added. The aqueous layer was
extracted with CH2Cl2 (4 ꢁ 20 mL) and the organic layers were
combined and solvent was removed in vacuo to give a yellow oil.
Purification by silica gel column chromatography (eluting with
EtOAc to MeOH/CH2Cl2, 1:9), afforded 11 (5.1 mg, 69%) as a yellow
oil. [
a]
22 ꢂ5.1 (c 0.28, CH2Cl2); RG 0.33 (CH2Cl2/MeOH 9:1); IR (ATR)
D
nmax 3312,1657,1493,1445,1281,1155, 1037 cmꢂ1; 1H NMR (CD3OD,
500 MHz)
d
7.52, (1H, d, J ¼ 7.9 Hz, H-32), 7.41 (1H, br s, H-27), 7.36
(1H, d, J ¼ 1.4 Hz, H-6), 7.34e7.31 (10H, m, H-29 and 9H-TrtAr),
7.12e7.09 (1H, m, H-30), 7.09e7.07 (6H, m, 6H-TrtAr), 7.04e7.01
(1H, m, H-31), 6.68 (1H, br s, H-8), 6.64e6.61 (3H, m, H-14, H-17
and H-24), 6.49 (1H, dd, J ¼ 8.1, 2.1 Hz, H-18), 5.99 (1H, d, J ¼ 9.0 Hz,
H-25), 4.54 (1H, dd, J ¼ 9.1, 5.3 Hz, H-11), 3.88 (2H, br s, H2-21), 3.53
(1H, dd, J ¼ 5.8, 5.6 Hz, H-2), 3.01 (1H, dd, J ¼ 14.1, 5.3 Hz, H2-12a),
2.80 (1H, dd, J ¼ 14.7, 5.6 Hz, H2-3a), 2.74e2.67 (2H, m, H2-3b and
0.17e0.16 (12H, m, 2Si(CH3)2); 13C NMR (CDCl3,125 MHz)
d 172.1 (C-
19), 170.6 (C-9), 166.6 (C-22), 156.2 (CO2CH2CH), 147.3 (C-15), 146.3
(C-16), 143.9 (C-FmocAr), 143.7 (C-FmocAr), 141.4 (2C-FmocAr and
3C-TrtAr), 135.9 (C-28a), 129.8 (3C-TrtAr), 129.7 (6C-TrtAr), 129.2
(C-13), 128.4 (6C-TrtAr), 127.9 (2C-FmocAr), 127.2 (2C-FmocAr),
127.1 (C-32a), 125.2 (2C-FmocAr), 122.9 (C-27), 122.7 (C-30), 122.3
(C-18), 122.0 (C-14), 121.3 (C-17), 120.9 (C-8), 120.2 (2C-FmocAr),
120.0 (C-31), 119.0 (C-32), 118.9 (C-24), 111.5 (C-29), 111.1 (C-26),
103.4 (C-25), 67.4 (CO2CH2CH), 54.7 (C-2 and C-11), 47.2
(CO2CH2CH), 43.9 (C-21), 36.6 (C-12), 29.5 (C-3), 26.1 (2SiC(CH3)3),
18.6 (2SiC(CH3)3), ꢂ3.9 (2Si(CH3)2); (þ)-HRESIMS [MþH]þ
1224.5848 (calcd. for C73H82N7O7Si2, 1224.5809).
H2-12b); 13C NMR (CD3OD, 125 MHz)
d 175.7 (C-9), 174.6 (C-19),
169.2 (C-22), 146.3 (C-15), 145.3 (C-16), 143.6 (3C-TrtAr), 139.8 (C-
6), 137.51 (C-4 or C-28a), 137.48 (C-4 or C-28a), 130.8 (6C-TrtAr),
129.7 (C-13), 129.2 (9C-TrtAr), 128.2 (C-32a), 124.7 (C-27), 123.1 (C-
30), 121.7 (C-8 or C-18), 121.6 (C-8 or C-18), 120.5 (C-31), 119.4 (C-
32),118.8 (C-24), 117.3 (C-14),116.3 (C-17),112.4 (C-29),111.0 (C-26),
106.2 (C-25), 76.8 (CAr3), 56.3 (C-11), 55.4 (C-2), 44.1 (C-21), 37.9 (C-
12), 33.8 (C-3); (þ)-HRESIMS [MþNa]þ 774.3411 (calcd. for
C46H47N4O6Na, 774.3388).
4.1.4. (S)-N-(2-(((Z)-2-(1H-Indol-3-yl)vinyl)amino)-2-oxoethyl)-2-
((S)-2-amino-3-(1-trityl-1H-imidazole-4-yl)propanamido)-3-(3,4-
bis((tert-butyldimethylsilyl) oxy)phenyl)propanamide (10)
4.1.6. Desbromo halocyamine A dihydrochloride (2)
cocktail solution of 0.01 N aq. HCl/HFIP-TIS/H2O (2 mL,
A
Piperidine (0.18 mL, 1:4 v/v in DMF) was added to 9 (43.9 mg,
35.9 mmol) and stirred at r.t. under N2 for 20 min. Water (10 mL)
95:2.5:2.5) was added to 11 (48.6 mg, 0.0628 mmol) and stirred for
1 h. The reaction was dried under a stream of N2. Purification by C8
column chromatography (eluting with H2O to H2O/MeOH 6:4)
was added to the solution and the aqueous layer was extracted with
EtOAc (4 ꢁ 20 mL). The organic layers were combined and dried in
vacuo. The crude product was purified by silica gel column chro-
matography (eluting with EtOAc to CH2Cl2/MeOH 9:1) to afford 10
afforded 2 (24.4 mg, 73%) as a white powder. m.p. 260 ꢀC (decom-
23
posed); RG 0.87 (butan-1-ol/acetic acid/H2O 2:1:1); [
a]
þ7.8 (c
D
0.43, MeOH); IR (ATR) nmax 3262, 3024, 2928, 1652, 1494, 1445,
(24.5 mg, 68%) as a yellow oil. [
a
]
27 ꢂ12.2 (c 2.66, CH2Cl2); RG 0.62
1258, 1184, 1079, 1032 cmꢂ1; 1H NMR (DMSO‑d6, 500 MHz)
d
11.39
D
(CH2Cl2/MeOH 9:1); IR (ATR) nmax 2931, 2857, 1659, 1509, 1445,
(1H, s, NH-28), 9.06 (1H, d, J ¼ 10.1 Hz, NH-23), 8.71 (1H, d,
J ¼ 18.7 Hz, NH-20), 8.13 (1H, d, J ¼ 6.0 Hz, NH-10), 7.71 (1H, d,
J ¼ 2.0 Hz, H-27), 7.60 (1H, d, J ¼ 8.0 Hz, H-32), 7.55 (1H, s, H-6), 7.39
(1H, d, J ¼ 8.0 Hz, H-29), 7.12 (1H, t, J ¼ 8.0 Hz, H-30), 7.04 (1H, t,
J ¼ 8.0 Hz, H-31), 6.82 (1H, br s, H-8), 6.65 (1H, dd, J ¼ 10.1, 9.6 Hz,
H-24), 6.61e6.58 (2H, m, H-14 and H-17), 6.42 (1H, dd, J ¼ 8.0,
1364, 1251, 1230, 1158, 1132, 1092 cmꢂ1; 1H NMR (CDCl3, 400 MHz)
d
9.40 (1H, br s, NH-28), 8.72 (1H, dd, J ¼ 6.0, 6.0 Hz, NH-20), 8.24
(1H, d, J ¼ 10.9 Hz, NH-23), 7.57 (1H, d, J ¼ 7.8 Hz, H-32), 7.36e7.34
(2H, m, H-6 and H-29), 7.31e7.26 (6H, m, 6H-TrtAr), 7.25e7.23 (1H,
m, H-27), 7.21e7.17 (1H, m, H-30), 7.13e7.06 (4H, m, H-31, and 3H-
Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
properties, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.10.021

6
H.K.H. Fong et al. / Tetrahedron xxx (2018) 1e10
1.8 Hz, H-18), 5.95 (1H, d, J ¼ 9.6 Hz, H-25), 4.44 (1H, br s, H-11),
3.99 (1H, dd, J ¼ 18.7, 5.9 Hz, H2-21a), 3.93 (1H, dd, J ¼ 18.7, 5.9 Hz,
H2-21b), 3.38 (1H, obs, H-2), 2.92 (1H, dd, J ¼ 13.9, 4.2 Hz, H2-12a),
2.80 (1H, dd, J ¼ 14.5, 4.4 Hz, H2-3a), 2.68 (1H, dd, J ¼ 13.9, 9.0 Hz,
H2-12b), 2.53 (1H, d, J ¼ 8.7 Hz, H2-3b); 13C NMR (DMSO‑d6,
0.49 mmol), EDC$HCl (0.14 g, 0.73 mmol) and HOBt (0.13 g,
0.98 mmol) in CH2Cl2 (5 mL) under N2, was added DIPEA (0.51 mL,
2.9 mmol) and the solution was stirred at r.t. for 7.5 h. Water
(10 mL) was added and the crude reaction product was extracted
with EtOAc (4 ꢁ 15 mL). The organic layers were combined, dried
(MgSO4), filtered and solvent removed in vacuo to give a crude oil.
Purification by silica gel column chromatography (eluting with n-
hexane/EtOAc 9:1 to n-hexane/EtOAc 1:1) afforded 15 (95Z:5E
125 MHz)
d 174.0 (C-9), 172.0 (C-19), 167.5 (C-22), 144.8 (C-15),
143.7 (C-16), 135.5 (C-4, C-28a), 134.9 (C-6), 128.4 (C-13), 126.6 (C-
32a), 123.9 (C-27), 121.6 (C-30), 120.0 (C-18), 119.0 (C-8, C-31), 118.2
(C-32), 117.8 (C-24), 116.6 (C-14), 115.2 (C-17), 111.5 (C-29), 109.4 (C-
26),102.8 (C-25), 54.9 (C-2), 53.9 (C-11), 42.6 (C-21), 39.8 (C-3), 36.9
(C-12); (þ)-HRESIMS [MþH]þ 532.2296 (calcd. for C27H30N7O5,
532.2303).
mixture) (112 mg, 42%) as a yellow oil. RG 0.45 (n-hexane/EtOAc
25
1:1); [
a]
þ3.2 (c 0.52, CH2Cl2); IR (ATR) nmax 3280, 2931, 2858,
D
1629, 1504, 1447, 1293, 1252, 1160 cmꢂ1; 1H NMR (CDCl3, 500 MHz)
9.15 (1H, br s, NH-23), 8.36 (1H, d, J ¼ 10.2 Hz, NH-18), 7.74 (2H, d,
d
J ¼ 7.2 Hz, 2H-FmocAr), 7.52 (2H, t, J ¼ 7.2 Hz, 2H-FmocAr), 7.44 (1H,
d, J ¼ 1.2 Hz, H-24), 7.39e7.36 (3H, m, H-27 and 2H-FmocAr), 7.27
(2H, t, J ¼ 7.2 Hz, 2H-FmocAr), 7.21 (1H, d, J ¼ 1.9 Hz, H-22), 7.18 (1H,
dd, J ¼ 8.5, 1.2 Hz, H-26), 7.14 (1H, br s, NH-15), 6.94 (1H, d,
J ¼ 7.5 Hz, NH-5), 6.80 (1H, dd, J ¼ 10.2, 9.6 Hz, H-19), 6.68 (1H, d,
J ¼ 8.2 Hz, H-12), 6.63 (1H, d, J ¼ 2.0 Hz, H-9), 6.51 (1H, dd, J ¼ 8.2,
2.0 Hz, H-13), 5.87 (1H, d, J ¼ 9.6 Hz, H-20), 5.36 (1H, d, J ¼ 4.2 Hz,
NH-1), 4.62 (1H, ddd, J ¼ 7.5, 7.5, 7.5 Hz, H-6), 4.37 (2H, d, J ¼ 6.1 Hz,
CO2CH2CH), 4.15e4.10 (1H, m, CO2CH2CH), 4.09e4.03 (2H, m, H-2
and H2-16a), 3.82e3.78 (1H, m, H2-16b), 2.96 (1H, dd, J ¼ 13.9,
7.5 Hz, H2-7a), 2.86 (1H, dd, J ¼ 13.9, 7.5 Hz, H2-7b), 1.17 (3H, d,
J ¼ 7.0 Hz, H3-3), 0.95e0.93 (18H, m, 2SiC(CH3)3), 0.14e0.13 (12H,
4.1.7. Fmoc-Ala-DOPA(TBDMS)2-OH (14)
A solution of Fmoc-DOPA(TBDMS)2-OH [9] (0.65 g, 1.0 mmol) in
CH2Cl2 (15 mL) was added to 2-chlorotrityl chloride resin (2 g,
loading at 0.5 mmol/g), followed by DIPEA (0.17 mL, 1.0 mmol).
After the resin mixture was agitated for 10 min, DIPEA (0.26 mL,
1.5 mmol) was added and the mixture was further shaken for 1 h.
The solution was drained off and the resin was washed with DMF
(10 mL). A solution of CH2Cl2/MeOH/DIPEA (12.5 mL, 80:15:5) was
added to the mixture and shaken for 23 min. The solution was
drained and the procedure was repeated. The resin was then
washed with DMF (10 mL). Piperidine in DMF (12.5 mL, 1:4) was
added to the resin mixture and shaken for 10 min. The liquid was
drained off and piperidine washing was repeated for another
20 min. The amino acid-loaded resin was thoroughly washed with
DMF (15 mL), isopropanol (15 mL) and n-hexane (15 mL). The resin
was then dried under vacuum for 30 min and placed in a desiccator
overnight. CH2Cl2 (30 mL) was added to the resin and left for 1 h.
m, 2Si(CH3)2); 13C NMR (CDCl3, 125 MHz)
d 172.6 (C-4), 172.3 (C-14),
166.6 (C-17), 156.7 (CO2CH2CH), 147.1 (C-10), 146.3 (C-11), 143.7 (C-
FmocAr), 143.6 (C-FmocAr), 141.42 (C-FmocAr), 141.41 (C-FmocAr),
136.7 (C-23a), 129.2 (C-8), 128.0 (2C-FmocAr), 127.3 (2C-FmocAr),
125.9 (C-27a), 125.0 (2C-FmocAr), 123.5 (C-22), 123.2 (C-26), 122.1
(C-9 or C-13), 122.0 (C-9 or C-13), 121.2 (C-12), 120.2 (C-27 and 2C-
FmocAr), 119.5 (C-19), 116.2 (C-25), 114.4 (C-24), 110.9 (C-21), 103.4
(C-20), 67.3 (CO2CH2CH), 54.6 (C-6), 51.3 (C-2), 47.2 (CO2CH2CH),
44.0 (C-16), 37.4 (C-7), 26.0 (2SiC(CH3)3), 18.6 (SiC(CH3)3), 18.5
(SiC(CH3)3), 18.2 (C-3), ꢂ4.0 (2Si(CH3)2); (þ)-HRESIMS [MþNa]þ
1016.3451 (calcd. for C51H64Na79BrN5O7Si2, 1016.3420).
The solution was drained and
3.75 mmol), HOBt (0.570 g, 3.75 mmol), Fmoc-
a
solution of HBTU (1.42 g,
-Ala-OH (0.62 g,
L
2.0 mmol) and DIPEA (0.87 mL, 5.0 mmol) in DMF (3.75 mL) was
added. The amino acid resin mixture was agitated for 5 h. The so-
lution was then drained and washed with DMF (20 mL), iso-
propanol (20 mL) and n-hexane (20 mL). 2,2,2-Trifluroethanol in
CH2Cl2 (12.5 mL, 1:4) was added to the amino acid-loaded resin and
agitated for 2 h. The solution was drained and the organic solvent
4.1.9. (S)-2-((S)-2-Aminopropanamido)-3-(3,4-bis((tert-
butyldimethylsilyl)oxy)phenyl)-N-(2-((2-(6-bromo-1H-indol-3-yl)
vinyl)amino)-2-oxoethyl)propanamide (16)
was removed in vacuo and dried extensively to afford 14 (95% pu-
25
rity) (0.61 g, 81%) as a yellow foam. m.p. 79e80 ꢀC; [
a
]
þ10.2 (c
Piperidine (0.23 mL, 20% in DMF) was added to 15 (47.0 mg,
0.0473 mmol) and the solution was stirred under N2 at r.t. for
20 min. EtOAc (15 mL) was added and the mixture was washed
with water (5 mL). The aqueous layer was further washed with
EtOAc (3 ꢁ 15 mL) and then the organic layers were combined and
dried in vacuo. The crude product was purified by silica gel column
chromatography (eluting with EtOAc to CH2Cl2/MeOH 9:1), to
afford Z-16 (13.0 mg, 36%) and E-16 (11.5 mg, 31%) as yellow oils.
D
1.59, CH2Cl2); RG 0.38 (EtOAc); IR (ATR) nmax 2932, 2858, 1719, 1665,
1509, 1450, 1423, 1297, 1251, 1127 cmꢂ1; 1H NMR (CDCl3, 500 MHz)
d
7.74 (2H, d, J ¼ 7.3 Hz, 2H-FmocAr), 7.57 (2H, d, J ¼ 7.3 Hz, 2H-
FmocAr), 7.38 (2H, t, J ¼ 7.3 Hz, 2H-FmocAr), 7.28 (2H, t, J ¼ 7.3 Hz,
2H-FmocAr), 6.70 (1H, d, J ¼ 8.2 Hz, H-12), 6.65 (1H, d, J ¼ 1.9 Hz, H-
9), 6.59e6.57 (1H, m, H-13), 6.53 (1H, br s, NH-5), 5.56 (1H, br s,
NH-1), 4.75 (1H, ddd, J ¼ 6.2, 6.2, 6.2 Hz, H-6), 4.38e4.36 (2H, m,
CO2CH2CH), 4.23 (1H, br s, H-2), 4.19 (1H, t, J ¼ 7.0 Hz, CO2CH2CH),
3.07 (1H, dd, J ¼ 14.0, 6.2 Hz, H2-7a), 2.98 (1H, dd, J ¼ 14.0, 6.2 Hz,
H2-7b), 1.32 (3H, d, J ¼ 5.7 Hz, H3-3), 0.95e0.94 (18H, m,
2SiC(CH3)3), 0.16e0.14 (12H, m, 2Si(CH3)2); 13C NMR (CDCl3,
4.1.9.1. (S)-2-((S)-2-Aminopropanamido)-3-(3,4-bis((tert-butyldime-
thylsilyl)oxy)phenyl)-N-(2-(((Z)-2-(6-bromo-1H-indol-3-yl)vinyl)
amino)-2-oxoethyl)propanamide (Z-16). RG 0.71 (CH2Cl2/MeOH
22
125 MHz)
d
174.6 (C-14),172.5 (C-4),156.1 (CO2CH2CH),147.0 (C-10),
9:1); [
a
]
D
þ22.1 (c 1.14, CH2Cl2); IR (ATR) nmax 3280, 2931, 1632,
146.3 (C-11), 143.8 (2C-FmocAr), 141.4 (2C-FmocAr), 128.7 (C-8),
127.9 (2C-FmocAr), 127.2 (2C-FmocAr), 125.2 (2C-FmocAr), 122.4
(C-13), 122.1 (C-9), 121.2 (C-12), 120.1 (2C-FmocAr), 67.3
(CO2CH2CH), 53.5 (C-6), 50.6 (C-2), 47.2 (CO2CH2CH), 36.8 (C-7),
26.0 (2SiC(CH3)3), 18.9 (C-3), 18.5 (2SiC(CH3)3), ꢂ3.9 (2Si(CH3)2);
(þ)-HRESIMS [MþNa]þ 741.3381 (calcd. for C39H54NaN2O7Si2,
741.3362).
1508, 1472, 1422, 1295, 1252, 1161, 1127 cmꢂ1
;
1H NMR (CDCl3,
500 MHz)
d
9.20 (1H, br s, NH-23), 8.29 (1H, d, J ¼ 10.4 Hz, NH-18),
7.78 (1H, d, J ¼ 7.8 Hz, NH-5), 7.53 (1H, br s, H-24), 7.43 (1H, d,
J ¼ 8.1 Hz, H-27), 7.23 (2H, br s, H-22 and H-26), 7.01 (1H, br s, NH-
15), 6.83 (1H, dd, J ¼ 10.4, 9.6 Hz, H-19), 6.73 (1H, d, J ¼ 7.9 Hz, H-
12), 6.66 (1H, s, H-9), 6.56 (1H, d, J ¼ 7.9 Hz, H-13), 5.91 (1H, d,
J ¼ 9.6 Hz, H-20), 4.60e4.57 (1H, m, H-6), 4.04 (1H, dd, J ¼ 16.7,
4.8 Hz, H2-16a), 3.81 (1H, d, J ¼ 16.7 Hz, H2-16b), 3.38e3.37 (1H, m,
H-2), 2.98 (1H, dd, J ¼ 13.6, 5.9 Hz, H2-7a), 2.88e2.84 (1H, m, H2-
7b), 1.16 (3H, d, J ¼ 6.4 Hz, H3-3), 0.97 (18H, br s, 2SiC(CH3)3), 0.16
4.1.8. (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-3-(3,4-bis((tert-
butyldimethylsilyl)oxy)phenyl)-1-((2-(((Z)-2-(6-bromo-1H-indol-3-
yl)vinyl)amino)-2-oxoethyl) amino)-1-oxopropan-2-yl)amino)-1-
oxopropan-2-yl) carbamate (15)
(12H, br s, 2Si(CH3)2); 13C NMR (CDCl3, 125 MHz)
d 176.5 (C-4), 172.6
(C-14), 166.5 (C-17), 147.1 (C-10), 146.3 (C-11), 136.7 (C-23a), 129.2
(C-8), 125.9 (C-27a), 123.4 (C-22 or C-26), 123.3 (C-22 or C-26),
To a solution of enamide 4 (0.14 g, 0.49 mmol), 14 (0.36 g,
Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
properties, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.10.021

H.K.H. Fong et al. / Tetrahedron xxx (2018) 1e10
7
122.22 (C-9 or C-13), 122.17 (C-9 or C-13), 121.3 (C-12), 120.3 (C-27),
119.7 (C-19), 116.4 (C-25), 114.4 (C-24), 111.1 (C-21), 103.3 (C-20),
54.3 (C-6), 50.7 (C-2), 43.9 (C-16), 37.3 (C-7), 26.1 (2SiC(CH3)3), 21.4
(6 g, loading at 0.5 mmol/g), followed by DIPEA (0.52 mL,
3.0 mmol). After the resin mixture was agitated for 10 min, DIPEA
(0.78 mL, 4.5 mmol) was added and the mixture was further shaken
for 2 h. The solution was drained off and the resin was washed with
DMF (30 mL). A solution of CH2Cl2/MeOH/DIPEA (20 mL, 80:15:5)
was added to the mixture and shaken for 30 min. The solution was
drained and the procedure was repeated. The resin was then
washed with DMF (20 mL). Piperidine in DMF (15 mL, 1:4) was
added to the resin mixture and shaken for 5 min. The liquid was
drained off and piperidine washing was repeated for another
20 min. The amino acid-loaded resin was thoroughly washed with
DMF (30 mL), isopropanol (30 mL) and n-hexane (30 mL). The resin
was then dried under vacuum overnight. CH2Cl2 (40 mL) was added
to the resin and left for 30 min. The solution was drained and a
solution of HBTU (4.27 g, 11.25 mmol), HOBt (1.72 g, 11.25 mmol),
Fmoc-His(Trt)-OH (4.65 g, 7.5 mmol) and DIPEA (2.61 mL,
15.0 mmol) in DMF (11.3 mL) was added to the resin. The amino
acid resin mixture was agitated for 5 h. The solution was then
drained and washed with DMF (30 mL), isopropanol (30 mL) and n-
hexane (30 mL). 2,2,2-Trifluroethanol in CH2Cl2 (25 mL, 1:4) was
added to the amino acid-loaded resin and agitated for 1.5 h. The
solution was drained and the organic solvent removed in vacuo to
afford 17 (1.82 g, 88%) as a yellow foam. m.p.144.1e145.0 ꢀC; RG 0.55
(C-3), 18.62 (SiC(CH3)3), 18.55 (SiC(CH3)3), ꢂ3.9 (2Si(CH3)2);
79
(þ)-HRESIMS [MþNa]þ 794.2745 (calcd. for C36
H BrNaN5O5Si2,
54
794.2739).
4.1.9.2. (S)-2-((S)-2-Aminopropanamido)-3-(3,4-bis((tert-butyldi-
methylsilyl)oxy)phenyl)-N-(2-(((E)-2-(6-bromo-1H-indol-3-yl)vinyl)
amino)-2-oxoethyl)propanamide (E-16). RG 0.61 (CH2Cl2/MeOH
23
9:1); [
a
]
þ16.8 (c 0.83, CH2Cl2); IR (ATR) nmax 3021, 2881, 1597,
D
1487, 1480, 1429, 1357, 1268, 1179, 1090 cmꢂ1
;
1H NMR (CDCl3,
500 MHz)
d
8.90 (1H, br d, J ¼ 10.2 Hz, NH-18), 8.46 (1H, br s, NH-
23), 7.93 (1H, br s, NH-5), 7.55 (1H, d, J ¼ 8.5 Hz, H-27), 7.48 (1H,
s, H-24), 7.32 (1H, dd, J ¼ 14.7, 10.2 Hz, H-19), 7.18 (1H, dd, J ¼ 8.5,
1.0 Hz, H-26), 7.10 (1H, s, H-22), 6.84 (1H, br s, NH-15), 6.77 (1H, d,
J ¼ 8.1 Hz, H-12), 6.70 (1H, d, J ¼ 1.9 Hz, H-9), 6.65 (1H, dd, J ¼ 8.1,
1.9 Hz, H-13), 6.48 (1H, d, J ¼ 14.7 Hz, H-20), 4.30 (1H, ddd, J ¼ 7.1,
7.1, 7.1 Hz, H-6), 4.13 (1H, dd, J ¼ 16.9, 5.9 Hz, H2-16a), 3.79 (1H, dd,
J ¼ 16.9, 5.9 Hz, H2-16b), 3.51e3.48 (1H, m, H-2), 3.11 (1H, dd,
J ¼ 13.9, 7.1 Hz, H2-7a), 2.93 (1H, dd, J ¼ 13.9, 7.1 Hz, H2-7b), 1.27
(3H, d, J ¼ 7.0 Hz, H3-3), 0.98e0.96 (18H, m, 2SiC(CH3)3), 0.18e0.17
(12H, m, 2Si(CH3)2); 13C NMR (CDCl3, 125 MHz)
d 177.3 (C-4), 172.0
(C-14), 166.3 (C-17), 147.2 (C-10), 146.3 (C-11), 137.5 (C-23a), 129.2
(C-8), 124.6 (C-27a), 123.4 (C-26), 122.6 (C-22), 122.1 (C-9 or C-13)*,
122.0 (C-9 or C-13)*, 121.4 (C-12), 121.0 (C-27), 120.4 (C-19), 116.0
(C-25), 114.4 (C-24), 113.4 (C-21), 107.0 (C-20), 56.3 (C-6), 50.7 (C-2),
43.3 (C-16), 36.5 (C-7), 26.1 (2SiC(CH3)3), 21.2 (C-3), 18.63
(CH2Cl2/MeOH 9:1); [
a]
20 þ10.7 (c 1.01, CH2Cl2); IR (ATR) nmax 3318,
D
3063, 1720, 1652, 1526, 1493, 1447, 1237, 1044 cmꢂ1
;
1H NMR
(CDCl3, 500 MHz)
d
8.53 (1H, br s, NH-10), 7.76 (2H, d, J ¼ 7.4 Hz, 2H-
FmocAr), 7.56 (1H, s, H-8), 7.53 (1H, d, J ¼ 7.8 Hz, H-FmocAr), 7.51
(1H, d, J ¼ 7.8 Hz, H-FmocAr), 7.39 (2H, t, J ¼ 7.4 Hz, 2H-FmocAr),
7.29e7.25 (12H, m, H-6, 2H-FmocAr, 9H-TrtAr), 7.04e7.02 (6H, m,
6H-TrtAr), 6.67 (1H, s, H-8), 5.88 (1H, br s, NH-1), 4.88 (1H, t,
J ¼ 10.7 Hz, H-2), 4.47 (1H, dq, J ¼ 6.6, 6.6 Hz, H-11), 4.38 (1H, dd,
J ¼ 9.4, 7.3 Hz, CO2CH2CH), 4.07 (1H, t, J ¼ 7.3 Hz, CO2CH2CH), 3.98
(1H, dd, J ¼ 9.4 Hz, CO2CH2CH), 3.39 (1H, d, J ¼ 13.2 Hz, H-3a), 2.58
(1H, dd, J ¼ 13.2, 10.7 Hz, H-3b), 1.54 (1H, d, J ¼ 6.6 Hz, H-12); 13C
(SiC(CH3)3), 18.57 (SiC(CH3)3), ꢂ3.9 (2Si(CH3)2); (þ)-HRESIMS
79
[MþNa]þ 794.2731 (calcd. for C36
H BrNaN5O5Si2, 794.2739).
54
4.1.10. (S)-1-(((S)-1-((2-(((Z)-2-(6-Bromo-1H-indol-3-yl)vinyl)
amino)-2-oxoethyl)amino)-3-(3,4-dihydroxy phenyl)-1-oxopropan-
2-yl)amino)-1-oxopropan-2-aminium 2,2,2-trifluoroacetate (12)
A solution of Z-16 (18.2 mg, 0.0236 mmol) in THF (2 mL) was
stirred at 0 ꢀC under N2 for 10 min before triethylamine trihydro-
NMR (CDCl3, 125 MHz)
d 176.8 (C-13), 171.0 (C-9), 156.0
(CO2CH2CH), 144.2 (C-FmocAr), 143.9 (C-FmocAr), 141.6 (3C-TrtAr),
141.4 (2C-FmocAr), 137.8 (C-6), 135.0 (C-4), 129.8 (6C-TrtAr), 128.6
(3C-TrtAr), 128.4 (6C-TrtAr), 127.8 (2C-FmocAr), 127.3 (2C-FmocAr),
125.6 (C-FmocAr), 125.3 (C-FmocAr), 120.4 (C-8), 120.1 (2C-Fmo-
cAr), 76.3 (CAr3), 67.1 (CO2CH2CH), 54.9 (C-2), 50.0 (C-11), 47.3
(CO2CH2CH), 32.8 (C-3), 19.2 (C-12); (þ)-HRESIMS [MþH]þ
691.2900 (calcd. for C43H39N4O5, 691.2915).
fluoride (14.0 mL, 0.0856 mmol) was added dropwise. The solution
was stirred for 55 min and then dried under a stream of N2. Puri-
fication using C2 column chromatography [eluting with H2O/
MeOH/TFA (99.99:0:0.01 to 59.99:39.99:0.02)] afforded 12
(10.4 mg, 67%), a yellow oil, as an inseparable 9:1 Z:E mixture. RG
0.87 (butan-1-ol/acetic acid/H2O 2:1:1); [
a
]
22 þ5.9 (c 0.66, MeOH);
D
IR (ATR) nmax 3253, 2923, 1668, 1532, 1456, 1200, 1129, 1023 cmꢂ1
;
1H NMR (DMSO‑d6, 500 MHz)
d
11.46 (1H, s, NH-23), 9.22 (1H, d,
4.1.12. Fmoc-Trt-[Ala2]-halocyamine A (18)
J ¼ 10.2 Hz, NH-18), 8.53 (1H, d, J ¼ 8.5 Hz, NH-5), 8.39 (1H, dd,
J ¼ 5.7, 5.7 Hz, NH-15), 8.00e7.99 (3H, m, NH3-1), 7.74 (1H, d,
J ¼ 2.4 Hz, H-22), 7.58 (1H, d, J ¼ 1.7 Hz, H-24), 7.56 (1H, d,
J ¼ 8.4 Hz, H-27), 7.17 (1H, dd, J ¼ 8.4, 1.7 Hz, H-26), 6.69e6.67 (2H,
m, H-9 and H-19), 6.61 (1H, d, J ¼ 8.1 Hz, H-12), 6.52 (1H, dd, J ¼ 8.1,
2.0 Hz, H-13), 5.91 (1H, d, J ¼ 9.5 Hz, H-20), 4.51 (1H, ddd, J ¼ 4.4,
8.5, 9.6 Hz, H-6), 3.99 (1H, dd, J ¼ 16.9, 5.7 Hz, H2-16a), 3.93 (1H, dd,
J ¼ 16.9, 5.7 Hz, H2-16b), 3.78e3.76 (1H, m, H-2), 2.89 (1H, dd,
J ¼ 13.9, 4.4 Hz, H2-7a), 2.62 (1H, dd, J ¼ 13.9, 9.6 Hz, H2-7b), 1.32
Fmoc-L-His(Trt)-L-Ala-OH 17 (0.243 g, 0.352 mmol), EDC$HCl
(0.074 g, 0.39 mmol) and HOBt (0.071 g, 0.53 mmol) were dissolved
in CH2Cl2 (1.5 mL) and stirred for 1 h at r.t. under N2. A solution of
enamide 4 (0.103 g, 0.352 mmol) in DMF (0.5 mL) was added to the
mixture followed by DIPEA (0.310 mL, 1.76 mmol). The solution was
stirred at r.t. under N2 for 7 h. The solution was diluted with EtOAc
(10 mL) and washed with water (5 mL). The organic layer was dried
(MgSO4), filtered and the solvent removed in vacuo. The crude
product was purified by silica gel flash column chromatography
(eluting with n-hexane/EtOAc 8:2 to n-hexane/EtOAc/MeOH
(3H, d, J ¼ 7.0 Hz, H3-3); 13C NMR (DMSO‑d6, 125 MHz)
d 171.4 (C-
14), 169.4 (C-4), 167.5 (C-17), 144.9 (C-10), 143.8 (C-11), 136.4 (C-
23a), 128.4 (C-8), 125.7 (C-27a), 124.9 (C-22), 121.9 (C-26), 120.2 (C-
27), 120.0 (C-13), 118.7 (C-19), 116.6 (C-9), 115.3 (C-12), 114.3 (C-25),
114.0 (C-24), 109.8 (C-21), 102.1 (C-20), 54.7 (C-6), 48.0 (C-2), 42.2
5:4.5:0.5) to afford 18 (0.151 g, 44%) as a yellow oil. RG 0.36 (EtOAc);
20
[a
]
þ15.3 (c 1.26, CH2Cl2); IR (ATR) nmax 3303, 2160, 1653, 1494,
D
1448, 1230, 1041, 1002 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d 9.34 (1H,
s, NH-22), 9.13 (1H, dd, J ¼ 6.1, 5.6 Hz, NH-15), 8.23 (1H, d,
J ¼ 10.8 Hz, NH-17), 7.73e7.71 (2H, m, 2H-FmocAr), 7.52 (1H, d,
J ¼ 8.2 Hz, H-FmocAr), 7.49 (1H, d, J ¼ 8.2 Hz, H-FmocAr), 7.47 (1H, s,
H-23), 7.40e7.36 (3H, m, 2H-FmocAr and H-26), 7.34 (1H, s, H-6),
7.29e7.26 (9H, m, 9H-TrtAr), 7.25e7.24 (3H, m, 2H-FmocAr and H-
21), 7.21e7.17 (1 H, m, H-25), 7.03e7.02 (6H, m, 6H-TrtAr),
6.76e6.69 (2H, m, NH-10 and H-18), 6.61 (1H, s, H-8), 6.08 (1H, d,
J ¼ 6.3 Hz, NH-1), 5.80 (1H, d, J ¼ 9.2 Hz, H-19), 4.55e4.51 (1H, m, H-
(C-16), 37.0 (C-7), 17.2 (C-3); (þ)-HRESIMS [MþH]þ 544.1173 (calcd.
79
for C
H BrN5O5, 544.1190). Only signals for the dominant Z-iso-
24 27
mer are reported.
4.1.11. Fmoc-His(Trt)-Ala-OH (17)
A solution of Fmoc-
L-Ala-OH (0.93 g, 3.0 mmol) in CH2Cl2
(25 mL) and DMF (2 mL) was added to 2-chlorotrityl chloride resin
Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
properties, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.10.021

8
H.K.H. Fong et al. / Tetrahedron xxx (2018) 1e10
11), 4.36e4.33 (3H, m, CO2CH2CH and H-2), 4.15 (1H, t, J ¼ 6.3 Hz,
CO2CH2CH), 4.04 (1H, dd, J ¼ 16.5, 6.1 Hz, H2-15a), 3.87 (1 H, dd,
J ¼ 16.5, 5.6 Hz, H2-15b), 3.00e2.84 (2 H, m, H2-3), 1.31 (3 H, d,
135.0 (C-6), 125.7 (C-26a), 124.8 (C-21), 122.1 (C-25), 120.6 (C-8, C-
26), 118.6 (C-18), 114.3 (C-24), 114.0 (C-23), 109.7 (C-20), 102.3 (C-
19), 54.7 (C-2), 48.1 (C-11), 42.5 (C-15), 32.7 (C-3), 18.2 (C-12);
J ¼ 7.3 Hz, H3-12); 13C NMR (CDCl3, 75 MHz)
d 173.5 (C-13), 171.1 (C-
9), 167.0 (C-16), 156.4 (CO2CH2CH), 143.8 (2 ꢁ C-FmocAr), 142.1 (3 ꢁ
C-TrtAr), 141.4 (2 ꢁ C-FmocAr), 138.9 (C-6), 136.6 (C-22a), 135.5 (C-
4), 129.7 (6 ꢁ C-TrtAr), 128.4 (3 ꢁ C-TrtAr), 128.3 (6 ꢁ TrtAr), 128.0
(2 ꢁ C-FmocAr), 127.2 (2 ꢁ C-FmocAr), 125.9 (C-26a), 125.1 (2 ꢁ C-
FmocAr), 123.6 (C-21), 123.2 (C-25), 120.6 (C-8), 120.3 (C-26), 120.2
(2 ꢁ C-FmocAr), 119.4 (C-18), 116.1 (C-24), 114.4 (C-23), 111.0 (C-20),
103.0 (C-19), 75.7 (CAr3), 67.3 (CO2CH2CH), 55.0 (C-2), 49.3 (C-11),
4.1.14.2. [Ala2]-(E)-halocyamine A (E-13). 1H NMR (CDCl3, 300 MHz)
d
11.26 (1H, s, NH-22), 9.71 (1H, d, J ¼ 10.0 Hz, NH-17), 9.07e9.04
(1H, m, NH-14), 8.39 (1H, br, s, NH-10), 7.59e7.54 (3H, m, H-6, H-23
and H-26), 7.47 (1H, d, J ¼ 2.0 Hz, H-21), 7.30 (1H, dd, J ¼ 14.9,
10.0 Hz, H-18), 7.20e7.14 (1H, m, H-25), 6.86e6.85 (1H, m, H-8),
6.49 (1H, d, J ¼ 14.9 Hz, H-19), 4.22e4.15 (1H, m, H-11), 3.86e3.84
(2H, m, H2-15), 3.48e3.40 (1H, m, H-2), 2.92e2.83 (1H, m, H2-3a),
2.77e2.62 (1H, m, H2-3b), 1.28 (3H, d, J ¼ 7.0 Hz, H3-12); 13C NMR
47.2 (CO2CH2CH), 43.9 (C-15), 31.0 (C-3), 17.8 (C-12); (þ)-HRESIMS
79
[MþH]þ 966.2957 (calcd. for C55
H
BrN7O5, 966.2973).
(CDCl3, 75 MHz) d 175.3 (C-9), 172.8 (C-13), 166.5 (C-16), 137.6 (C-4,
49
C-22a), 134.9 (C-6), 124.3 (C-26a), 123.9 (C-21), 121.9 (C-25), 120.1
(C-8, C-26), 120.0 (C-18), 114.3 (C-24), 114.1 (C-23), 111.9 (C-20),
105.7 (C-19), 54.7 (C-2), 49.1 (C-11), 42.2 (C-15), 32.5 (C-3), 17.6 (C-
12).
4.1.13. Trt-[Ala2]-halocyamine A (19)
A flask containing 18 (95.0 mg, 98.4
m
mol) was flushed with N2
before piperidine (20% in DMF, 1 mL) was added. The solution was
stirred at r.t. for 20 min before EtOAc (20 mL) was added and the
mixture was washed with water (10 mL). The aqueous layer was
further washed with EtOAc (3 ꢁ 10 mL) and the organic layers were
combined and dried in vacuo. Purification by silica gel column
chromatography (eluting with EtOAc to CH2Cl2/MeOH 9:1), affor-
4.1.15. Fmoc-
L
-Ala-
L
-Ala-OH (22)
-Ala-OH (0.93 g, 3.0 mmol) in CH2Cl2
A solution of Fmoc-
L
(25 mL) was added to 2-chlorotrityl chloride resin (6 g, loading at
0.5 mmol/g), followed by DIPEA (0.52 mL, 3.0 mmol). After the resin
mixture was agitated for 14 min, DIPEA (0.78 mL, 4.5 mmol) was
added and the mixture was further shaken for 2.5 h. The solution
was drained off and the resin was washed with DMF (20 mL). A
solution of CH2Cl2/MeOH/DIPEA (40 mL, 80:15:5) was added to the
mixture and shaken for 30 min. The solution was drained and the
procedure was repeated. The resin was then washed with DMF
(20 mL). Piperidine in DMF (15.0 mL, 1:4) was added to the resin
mixture and shaken for 10 min. The liquid was drained off and
piperidine washing was repeated for another 40 min. The amino
acid-loaded resin was thoroughly washed with DMF (35 mL), iso-
propanol (35 mL) and n-hexane (35 mL). The resin was then dried
under vacuum for 30 min and placed in a desiccator overnight.
CH2Cl2 (50 mL) was added to the resin and left for 1 h. The solution
was drained and a solution of HBTU (4.27 g, 11.25 mmol), HOBt
ded 19 (62.8 mg, 86%) as a yellow oil. RG 0.17 (CH2Cl2/MeOH 9:1);
21
[
a]
ꢂ10.1 (c 0.24, CH2Cl2); IR (ATR) nmax 3276, 1652, 1533, 1492,
D
1445, 1231, 1130, 1039 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d 9.80 (1H,
s, NH-22), 8.76 (1H, dd, J ¼ 5.8, 5.6 Hz, NH-14), 8.27 (1H, d,
J ¼ 10.2 Hz, NH-17), 7.48 (1H, d, J ¼ 1.5 Hz, H-23), 7.38 (2H, d, J ¼ Hz,
H-6 and H-26), 7.32e7.28 (11H, m, NH-10, H-21 and 9H-TrtAr), 7.17
(1H, dd, J ¼ 8.4, 1.5 Hz, H-25), 7.06e7.03 (6H, m, 6H-TrtAr), 6.74 (1H,
dd, J ¼ 10.2, 9.8 Hz, H-18), 6.64 (1H, s, H-8), 5.81 (1H, d, J ¼ 9.8 Hz,
H-19), 4.48e4.43 (1H, m, H-11), 4.05 (1H, dd, J ¼ 16.9, 5.8 Hz, H2-
15a), 3.93 (1H, dd, J ¼ 16.9, 5.6 Hz, H2-15b), 3.66 (1H, s, H-2), 2.94
(1H, dd, J ¼ 14.6, 4.8 Hz, H2-3a), 2.84 (1H, dd, J ¼ 14.6, 5.5 Hz, H2-
3b), 1.32 (3H, d, J ¼ 7.2 Hz, H3-12); 13C NMR (CDCl3, 75 MHz)
d 174.7
(C-9), 173.6 (C-13), 167.1 (C-16), 142.0 (3C-TrtAr), 139.0 (C-6), 136.7
(C-4), 136.2 (C-22a), 129.7 (6C-TrtAr), 128.3 (6C-TrtAr), 128.2 (3C-
TrtAr), 126.0 (C-26a), 123.8 (C-21), 123.1 (C-25), 120.5 (C-8), 120.2
(C-26),119.3 (C-18),116.0 (C-24),114.4 (C-23),110.8 (C-20),103.2 (C-
(1.72 g, 11.25 mmol), Fmoc-L-Ala-OH (2.34 g, 7.5 mmol) and DIPEA
(2.61 mL, 15.0 mmol) in DMF (11.3 mL) was added. The amino acid
resin mixture was agitated for 4 h. The solution was then drained
and washed with DMF (30 mL), isopropanol (30 mL) and n-hexane
(30 mL). 2,2,2-Trifluroethanol in CH2Cl2 (15 mL, 1:4) was added to
the amino acid-loaded resin and agitated for 2 h. The solution was
drained and the organic solvent removed in vacuo to afford 22
19), 75.7 (CAr3), 54.6 (C-2), 49.4 (C-11), 43.8 (C-15), 33.1 (C-3), 17.8
79
(C-12); (þ)-HRESIMS [MþH]þ 744.2307 (calcd. for C40
H BrN7O3,
39
744.2292).
4.1.14. [Ala2]-halocyamine A (13)
A
cocktail solution of 0.01 N aq. HCl/HFIP-TIS/H2O (1 mL,
(0.90 g, 78%) as a yellow foam. m.p. 195e196 ꢀC; RG 0.53 (CH2Cl2/
20
95:2.5:2.5) was added to 19 (62.8 mg, 0.0844 mmol) and stirred for
1 h. The reaction was dried under a stream of N2. Subsequent pu-
rification by C8 column chromatography (eluting with H2O to H2O/
MeOH 25:75) afforded 13 as an inseparable 1Z:0.9E mixture (yellow
oil) (39.9 mg, 83%).
MeOH 9:1); [
a]
ꢂ23.9 (c 0.14, MeOH); IR (ATR) nmax 3297, 2918,
D
1692, 1650, 1533, 1450, 1318, 1229 cmꢂ1; 1H NMR (CDCl3, 500 MHz)
d
8.09 (1H, d, J ¼ 7.2 Hz, NH-5), 7.89 (2H, d, J ¼ 7.4 Hz, 2H-FmocAr),
7.74 (1H, d, J ¼ 7.4 Hz, 1H-FmocAr), 7.72 (1H, d, J ¼ 7.4 Hz, 1H-
FmocAr), 7.50 (1H, d, J ¼ 7.5 Hz, NH-1), 7.41 (2H, t, J ¼ 7.4 Hz, H-
FmocAr), 7.33 (2H, td, J ¼ 7.4, 0.9 Hz, 2H-FmocAr), 4.27e4.15 (4H, m,
CO2CH2CH, CO2CH2CH, H-6), 4.08 (1H, d, J ¼ 7.5, 7.2 Hz, H-2), 1.27
(3H, d, J ¼ 7.3 Hz, H3-7), 1.22 (3H, d, J ¼ 7.2 Hz, H3-3); 13C NMR
The mixture had the following properties: RG 0.59 (butan-1-ol/
21.6
acetic acid/H2O 2:1:1); [
a
]
ꢂ0.6 (c 1.03, MeOH); IR (ATR) nmax
D
3240, 2963, 1646, 1533, 1410, 1259, 1004 cmꢂ1; (þ)-HRESIMS
79
[MþH]þ 502.1195 (calcd. for C21
H
BrN7O3, 502.1197). NMR as-
(CDCl3, 125 MHz) d 174.2 (C-8), 172.2 (C-4), 155.6 (CO2CH2CH), 143.9
25
signments for each of the isomers were discerned from 2D NMR
data.
(C-FmocAr), 143.8 (C-FmocAr), 140.7 (2C-FmocAr), 127.6 (2C-Fmo-
cAr), 127.1 (2C-FmocAr), 125.3 (2C-FmocAr), 120.1 (2C-FmocAr),
65.6 (CO2CH2CH), 49.7 (C-2), 47.5 (C-6), 46.7 (CO2CH2CH), 18.2 (C-
3), 17.3 (C-7); (þ)-HRESIMS [MþNa]þ 405.1424 (calcd. for
4.1.14.1. [Ala2]-(Z)-halocyamine A (Z-13). 1H NMR (CDCl3, 300 MHz)
d
11.54 (1H, s, NH-22), 9.06 (1H, d, J ¼ 9.9 Hz, NH-17), 8.80 (1H, br, s,
C21H22NaN2O5, 405.1421).
NH-14), 8.20 (1H, d, J ¼ 4.4 Hz, NH-10), 7.71 (1H, s, H-21), 7.59e7.54
(3H, m, H-6, H-23 and H-26), 7.20e7.14 (1H, m, H-25), 6.86e6.85
(1H, m, H-8), 6.67 (1H, dd, J ¼ 9.9, 9.7 Hz, H-18), 5.92 (1H, d,
J ¼ 9.7 Hz, H-19), 4.33e4.31 (1H, m, H-11), 3.97e3.94 (2H, m, H2-
15), 3.48e3.40 (1H, m, H-2), 2.92e2.83 (1H, m, H2-3a), 2.77e2.62
(1H, m, H2-3b), 1.24 (3H, d, J ¼ 7.1 Hz, H3-12); 13C NMR (CDCl3,
4.1.16. Fmoc-[Ala1,Ala2]-halocyamine A (24)
Fmoc- -Ala- -Ala-OH 22 (0.176 g, 0.461 mmol), EDC$HCl
L
L
(0.097 g, 0.51 mmol) and HOBt (0.093 g, 0.69 mmol) was dissolved
in DMF (1.5 mL) and the mixture was stirred for 1 h at r.t. under N2.
A solution of 4 (0.135 g, 0.461 mmol) in DMF (1.5 mL) was added to
the mixture followed by DIPEA (0.40 mL, 2.3 mmol). The solution
75 MHz)
d 174.1 (C-9), 173.1 (C-13), 167.6 (C-16), 136.4 (C-4, C-22a),
Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
properties, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.10.021

H.K.H. Fong et al. / Tetrahedron xxx (2018) 1e10
9
was stirred at r.t. under N2 for 7 h. The solution was diluted with
EtOAc (10 mL) and washed with water (5 mL). The organic layer was
dried (MgSO4), filtered and the solvent removed in vacuo. The crude
product was purified by silica gel flash column chromatography
(eluting with n-hexane/EtOAc 8:2 to n-hexane/EtOAc/MeOH
(2C-FmocAr), 127.6 (2C-FmocAr), 127.1 (2C-FmocAr), 125.3 (2C-
FmocAr), 120.1 (2C-FmocAr), 65.6 (CO2CH2CH), 49.7 (C-2), 47.4 (C-
6), 46.6 (CO2CH2CH), 18.2 (C-3), 17.2 (C-7); (þ)-HRESIMS [MþNa]þ
405.1423 (calcd. for C21H22NaN2O5, 405.1421).
5:4.5:0.5) to afford 24 (0.10 g, 33%) as a yellow oil. RG 0.34 (EtOAc);
4.1.19. Fmoc-[
D
-Ala1,
D
-Ala2]-halocyamine A (25)
21
[
a
]
ꢂ6.7 (c 0.36, MeOH); IR (ATR) nmax 3019, 2978, 1595, 1488,
Fmoc- -Ala-D
D
-Ala-OH (23) (0.102 g, 0.266 mmol), EDC$HCl
D
1451, 754 cmꢂ1
;
1H NMR (DMSO‑d6, 300 MHz)
d
11.43 (1H, s, NH-
(0.056 g, 0.29 mmol) and HOBt (0.054 g, 0.40 mmol) were dissolved
in DMF (1.5 mL) and stirred at r.t. for 1 h under nitrogen. A solution
of 4 (0.078 g, 0.27 mmol) in DMF (1.5 mL) was then added followed
by DIPEA (0.230 mL, 1.33 mmol). The solution was further stirred at
r.t. for 7 h. EtOAc (10 mL) was added to the mixture and washed
with water (5 mL). The organic layer was dried (MgSO4), filtered
and the solvent removed in vacuo. The crude product was purified
by silica gel flash column chromatography (eluting with n-hexane/
EtOAc 8:2 to n-hexane/EtOAc/MeOH 5:4.5:0.5) to afford 25
17), 9.13 (1H, d, J ¼ 10.0 Hz, NH-12), 8.14 (1H, t, J ¼ 5.7 Hz, NH-9),
8.03 (1H, d, J ¼ 7.4 Hz, NH-5), 7.88 (2H, d, J ¼ 7.3 Hz, 2H-FmocAr),
7.73e7.40 (3H, m, 2H-FmocAr and H-16), 7.58e7.54 (2H, m, H-18
and H-21), 7.52 (1H, d, J ¼ 7.7 Hz, NH-1), 7.41 (2H, t, J ¼ 7.3 Hz, 2H-
FmocAr), 7.32 (2H, t, J ¼ 7.3 Hz, 2H-FmocAr), 7.16 (1H, dd, J ¼ 8.4,
1.7 Hz, H-20), 6.66 (1H, dd, J ¼ 10.0, 9.7 Hz, H-13), 5.91 (1H, d,
J ¼ 9.7 Hz, H-14), 4.35e4.31 (1H, m, H-6), 4.27e4.18 (3H, m,
CO2CH2CH and CO2CH2CH), 4.10e4.05 (1H, m, H-2), 3.94 (2H, d,
J ¼ 5.7 Hz, H2-10), 1.23 (3H, d, J ¼ 6.7 Hz, H3-7), 1.21 (3H, d,
(60.0 mg, 34%) as a yellow oil. RG 0.21 (n-hexane/EtOAc/MeOH
22
J ¼ 6.9 Hz, H3-3); 13C NMR (DMSO‑d6, 75 MHz)
d
172.7 (C-8), 172.2
5:4.5:0.5); [
a
]
þ5.6 (c 0.32, MeOH); IR (ATR) nmax 3367, 2974,
D
(C-4), 167.5 (C-11), 155.7 (CO2CH2CH), 143.9 (C-FmocAr), 143.8 (C-
FmocAr), 140.7 (2C-FmocAr), 136.4 (C-17a), 127.6 (2C-FmocAr),
127.1 (2C-FmocAr), 125.7 (C-21a), 125.3 (2C-FmocAr), 124.9 (C-16),
121.9 (C-20), 120.1 (2C-FmocAr and C-21), 118.7 (C-13), 114.3 (C-19),
114.0 (C-18), 109.8 (C-15), 102.2 (C-14), 65.6 (CO2CH2CH), 50.0 (C-2),
48.1 (C-6), 46.6 (CO2CH2CH), 42.3 (C-10), 18.2 (C-3 or C-7), 18.1 (C-3
1656, 1494, 1447, 1231, 1130 cmꢂ1
;
1H NMR (DMSO‑d6, 300 MHz)
d
11.43 (1H, s, NH-17), 9.13 (1H, d, J ¼ 9.9 Hz, NH-12), 8.14 (1H, d,
J ¼ 5.8 Hz, NH-9), 8.03 (1H, d, J ¼ 7.7 Hz, NH-5), 7.89 (2H, d,
J ¼ 7.2 Hz, 2H-FmocAr), 7.74e7.70 (3H, m, 2H-FmocAr and H-16),
7.58e7.54 (2H, m, H-18 and H-21), 7.52 (1H, d, J ¼ 7.1 Hz, NH-1), 7.41
(2H, t, J ¼ 7.2 Hz, 2H-FmocAr), 7.33 (2H, t, J ¼ 7.2 Hz, 2H-FmocAr),
7.16 (1H, dd, J ¼ 8.5, 1.8 Hz, H-20), 6.67 (1H, d, J ¼ 9.9, 9.7 Hz, H-13),
5.91 (1H, d, J ¼ 9.7 Hz, H-14), 4.35e4.31 (1H, m, H-6), 4.27e4.21
(3H, m, CO2CH2CH and CO2CH2CH), 4.10e4.05 (1H, m, H-2), 3.94
(2H, d, J ¼ 5.8 Hz, H2-10), 1.23 (3H, d, J ¼ 6.9 Hz, H3-7), 1.21 (3H, d,
or C-7); (þ)-HRESIMS [MþNa]þ 680.1474 (calcd. for
79
C
H BrNaN5O5, 680.1479).
33 32
4.1.17. [Ala1,Ala2]-halocyamine A (20)
To 24 (19.0 mg, 0.0289 mmol) was added piperidine (20% in
DMF, 1 mL) and flushed with nitrogen. The solution was stirred at
r.t. for 20 min before EtOAc (20 mL) was added and the mixture was
washed with water (10 mL). The aqueous layer was further washed
with EtOAc (3 ꢁ 10 mL) and the organic layers were combined and
dried in vacuo. Purification by silica gel column chromatography
(eluting with EtOAc to CH2Cl2/MeOH 8:2), afforded 20 (7.54 mg,
60%), a yellow oil, as an inseparable mixture of Z/E (1:0.9) isomers.
J ¼ 7.2 Hz, H3-3); 13C NMR (DMSO‑d6, 75 MHz)
d 172.7 (C-8), 172.2
(C-4), 167.4 (C-11), 155.7 (CO2CH2CH), 143.9 (C-FmocAr), 143.8 (C-
FmocAr), 140.7 (2C-FmocAr), 136.4 (C-17a), 127.6 (2C-FmocAr),
127.1 (2C-FmocAr), 125.7 (C-21a), 125.3 (2C-FmocAr), 124.8 (C-16),
121.9 (C-20), 120.1 (2C-FmocAr and C-21), 118.7 (C-13), 114.3 (C-19),
114.0 (C-18), 109.7 (C-15), 102.1 (C-14), 65.6 (CO2CH2CH), 49.9 (C-2),
48.0 (C-6), 46.6 (CO2CH2CH), 42.3 (C-10), 18.2 (C-3 or C-C-7), 18.1
(C-3 or C-C-7); (þ)-HRESIMS [MþNa]þ 680.1464 (calcd. for
79
RG 0.20 (CH2Cl2/MeOH 8:2); [
a
]
19 þ1.6 (c 0.26, MeOH); IR (ATR) nmax
C H BrNaN5O5, 680.1479).
33 32
D
3393, 2258, 1655, 1048, 1024 cmꢂ1
;
1H NMR (DMSO‑d6, 300 MHz)
d
11.49 (1H, br s, NH-17), 9.11 (1H, d, J ¼ 10.1 Hz, NH-12), 8.26 (1H, d,
4.1.20. [D-Ala1, -Ala2]-halocyamine A (21)
D
J ¼ 5.7 Hz, NH-9), 8.08 (1H, br s, NH-5), 7.70 (1H, s, H-16), 7.58 (1H,
d, J ¼ 1.8 Hz, H-18), 7.56 (1H, d, J ¼ 8.4 Hz, H-21), 7.16 (1H, dd, J ¼ 8.4,
1.8 Hz, H-20), 6.66 (1H, dd, J ¼ 10.1, 9.7 Hz, H-13), 5.91 (1H, d,
J ¼ 9.7 Hz, H-14), 4.33 (1H, d, J ¼ 7.2 Hz, H-6), 3.93 (2H, d, J ¼ 5.7 Hz,
H2-10), 3.29 (1H, obs, H-2), 1.23 (3H, d, J ¼ 7.2 Hz, H3-7), 1.13 (3H, d,
Piperidine (20% in DMF, 1 mL) was added to 25 (34.2 mg,
0.520 mmol) and stirred at r.t. under N2 for 20 min. EtOAc (20 mL)
was added and the mixture was washed with water (10 mL). The
aqueous layer was further washed with EtOAc (3 ꢁ 10 mL) and the
organic layers were combined and dried in vacuo. Purification by
silica gel column chromatography (eluting with EtOAc to CH2Cl2/
MeOH 8:2), afforded 21 (15.7 mg, 69%), a clear film, as an insepa-
J ¼ 6.9 Hz, H3-3); 13C NMR (DMSO‑d6, 75 MHz)
d 175.1 (C-4), 172.9
(C-8), 167.5 (C-11), 136.4 (C-17a), 125.6 (C-21a), 124.9 (C-16), 121.9
(C-20), 120.1 (C-21), 118.7 (C-13), 114.3 (C-19), 114.0 (C-18), 109.7 (C-
rable mixture of Z/E (1:0.9) isomers. RG 0.22 (CH2Cl2/MeOH 8:2);
21
15), 102.2 (C-14), 50.1 (C-2), 47.7 (C-6), 42.3 (C-10), 21.1 (C-3), 18.5
[a
]
ꢂ0.5 (c 1.59, CH2Cl2); IR (ATR) nmax 3274, 2921, 2854, 1646,
D
79
(C-7); (þ)-HRESIMS [MþH]þ 436.0970 (calcd. for C18
H
BrN5O3,
1532, 1449, 1398, 1235, 1054 cmꢂ1 1H NMR (CD3OD, 300 MHz)
;
23
436.0979).
d
7.55 (1H, d, J ¼ 1.5 Hz, H-18), 7.51 (1H, d, J ¼ 0.8 Hz, H-16), 7.47 (1H,
d, J ¼ 8.5 Hz, H-21), 7.18 (1H, dd, J ¼ 8.5, 1.5 Hz, H-20), 6.76 (1H, d,
J ¼ 9.2 Hz, H-13), 6.00 (1H, dd, J ¼ 9.2, 0.76 Hz, H-14), 4.36 (1H, d,
J ¼ 7.1 Hz, H-6), 3.97 (2H, s, H2-10), 3.48 (1H, d, J ¼ 6.9 Hz, H-2), 1.33
(3H, d, J ¼ 7.1 Hz, H3-7), 1.26 (3H, d, J ¼ 6.9 Hz, H3-3); 13C NMR
4.1.18. Fmoc-D-Ala-D-Ala-OH (23)
The synthesis of 23 used Fmoc-
D
-Ala-OH and the same pro-
cedure as described for the synthesis of Fmoc-L-Ala-L-Ala-OH (22),
to give the product (0.45 g, 79%) as a white solid. m.p. 196e197 ꢀC;
(CD3OD, 75 MHz) d 177.0 (C-3), 175.7 (C-8), 169.4 (C-11), 138.4 (C-
20
RG 0.53 (CH2Cl2/MeOH 9:1); [
a]
þ21.8 (c 0.15, MeOH); IR (ATR)
17a), 127.2 (C-21a), 125.5 (C-16), 123.6 (C-20), 121.0 (C-21), 120.0 (C-
13), 116.4 (C-19), 115.3 (C-18), 111.4 (C-15), 105.5 (C-14), 51.2 (C2),
D
nmax 3293, 2990, 1688, 1653, 1534, 1450, 1319, 1262 cmꢂ1; 1H NMR
(CDCl3, 400 MHz)
d
12.52 (1H, br s, OH), 8.13 (1H, d, J ¼ 7.2 Hz, NH-
50.4 (C-6), 43.9 (C-10), 20.6 (C-3), 17.9 (C-7); (þ)-HRESIMS
79
5), 7.89 (2H, d, J ¼ 7.4 Hz, 2H-FmocAr), 7.73 (1H, d, J ¼ 7.4 Hz, 1H-
FmocAr), 7.72 (1H, d, J ¼ 7.4 Hz, 1H-FmocAr), 7.50 (1H, d, J ¼ 7.5 Hz,
NH-1), 7.41 (2H, t, J ¼ 7.4 Hz, H-FmocAr), 7.32 (2H, td, J ¼ 7.4, 0.9 Hz,
2H-FmocAr), 4.26e4.17 (4H, m, CO2CH2CH, CO2CH2CH, H-6), 4.08
(1H, d, J ¼ 7.5, 7.2 Hz, H-2), 1.27 (3H, d, J ¼ 7.3 Hz, H3-7), 1.22 (3H, d,
[MþNa]þ 458.0800 (calcd. for C18
H BrNaN5O3, 458.0798).
22
4.2. Antibacterial assays [12]
The antibacterial activity of the compounds was studied by
determination of minimum inhibitory concentrations (MICs) using
the standard broth dilution method in accordance with the NCCLS
J ¼ 7.2 Hz, H3-3); 13C NMR (CDCl3, 100 MHz)
d 174.0 (C-8), 172.3 (C-
4), 155.6 (CO2CH2CH), 143.9 (C-FmocAr), 143.8 (C-FmocAr), 140.7
Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
properties, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.10.021

10
H.K.H. Fong et al. / Tetrahedron xxx (2018) 1e10
guidelines M7-A2. Briefly, the MICs were determined with an
inoculum of 105 CFU in 200
L of MH broth containing twofold
Acknowledgments
m
serial dilutions of each drug. The MIC was defined as the lowest
concentration of drug that completely inhibited visible growth af-
ter incubation for 18 h at 37 ꢀC. To determine all MICs, the mea-
surements were independently repeated at least three times.
Minimum inhibitory concentration of positive control: colistin
The authors thank Dr. M. Schmitz and Mr T. Chen (University of
Auckland) for assistance with NMR and MS data acquisition, and Dr
M. Kaiser (Swiss Tropical Public Health Institute, Basel,
Switzerland) for cytotoxicity data. We gratefully acknowledge
funding from the University of Auckland.
[P. aeruginosa (1
m
M), E. coli (2
M), E. coli (21.5 M), S. aureus (21.5
M)] and chloramphenicol [S. aureus (1.5e3
M)].
m
M)], streptomycin [P. aeruginosa
M), and E. faecalis
M), and
(21.5
(21.5
m
m
m
m
Appendix A. Supplementary data
m
E. faecalis (1.5e3
m
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2018.10.021.
4.3. In vitro cytotoxicity assay [13]
Assays were performed in 96-well microtiter plates, each well
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Please cite this article in press as: H.K.H. Fong, et al., Alaninyl variants of the marine natural product halocyamine A and their antibacterial
properties, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.10.021