Sonogashira diversification of unprotected halotryptophans, halotryptophan containing tripeptides; and generation of a new to nature bromo-natural product and its diversification in water

Article abstract of DOI:10.1039/c6sc04423a

The blending together of synthetic chemistry with natural product biosynthesis represents a potentially powerful approach to synthesis; to enable this, further synthetic tools and methodologies are needed. To this end, we have explored the first Sonogashira cross-coupling to halotryptophans in water. Broad reaction scope is demonstrated and we have explored the limits of the scope of the reaction. We have demonstrated this methodology to work excellently in the modification of model tripeptides. Furthermore, through precursor directed biosynthesis, we have generated for the first time a new to nature brominated natural product bromo-cystargamide, and demonstrated the applicability of our reaction conditions to modify this novel metabolite.

Full text of DOI:10.1039/c6sc04423a

View Article Online
View Journal
Chemical
Science
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. J. Corr, S. V.
Sharma, C. Pubill-Ulldemolins, R. T. Bown, P. Poirot, D. R. M. Smith, C. Cartmell, A. Abou-Fayad and R. J.
M. Goss, Chem. Sci., 2016, DOI: 10.1039/C6SC04423A.
This is an Accepted Manuscript, which has been through the
Volume
7 Number 1 January 2016 Pages 1–812
Royal Society of Chemistry peer review process and has been
accepted for publication.
Chemical
Science
Accepted Manuscripts are published online shortly after
www.rsc.org/chemicalscience
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 2041-6539
EDGE ARTICLE
Francesco Ricci et al.
Electronic control of DNA-based nanoswitches and nanodevices
rsc.li/chemical-science

Page 1 of 8
Chemical Science
View Article Online
DOI: 10.1039/C6SC04423A
Journal Name
ARTICLE
Sonogashira diversification of unprotected halotryptophans,
halotryptophan containing tripeptides; and generation of a
new to nature bromo-natural product and its diversification
in water
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
M. J. Corr,a S. V. Sharma,a C. Pubill-Ulldemolins,a R. T. Bown,a P. Poirot,b D. R. M. Smith,a C.
Cartmell,a A. Abou Fayad,c R. J. M. Gossa*
www.rsc.org/
The blending together of synthetic chemistry with natural product biosynthesis represents a potentially powerful
approach to synthesis; to enable this, further synthetic tools and methodologies are needed. To this end, we have
explored the first Sonogashira cross-coupling to halotryptophans in water. Broad reaction scope is demonstrated
and we have explored the limits of the scope of the reaction. We have demonstrated this methodology to work
excellently in the modification of model tripeptides. Furthermore, through precursor directed biosynthesis, we
have generated for the first time a new to nature brominated natural product bromo-cystargamide, and
demonstrated the applicability of our reaction conditions to modify this novel metabolite.
reactions, we set out to explore the potential applicability of
the Sonogashira cross-coupling reaction.4 In this work, we
wanted to examine, for the first time, whether the
Introduction
Natural products represent a valuable resource for medicinally
relevant compounds, with many antimicrobial and antitumour
reagents entering clinical trials being based on natural
products.1 New approaches to modify natural products and
generate analogues are important, enabling for example
structure activity relationships to be probed, and the natural
product’s bioactivity and bioavailability to be modulated or
enabling natural product tagging for fluorescence detection or
immobilisation.2a-d Palladium cross-coupling reactions are a
powerful tool in synthesis and potentially for late-stage
analogue generation of natural products.3 We previously
reported the first utilisation of Suzuki-Miyaura cross-coupling
for the diversification of free, unprotected halotryptophans.2e
Developing a new GenoChemetic approach to natural product
analogue generation, we showed how, through biosynthetic
pathway manipulation, a new to nature chlorinated antibiotic
could be accessed and reported on the utility of palladium
cross-coupling reactions for synthetic diversification of this
chloro-uridyl peptide antibiotic.2f In order to expand this
GenoChemetic approach and our toolkit of compatible
Sonogashira cross-coupling could be utilised to selectively
diversify unprotected halotryptophans. Through biosynthetic
pathway manipulation, we generated a new-to-nature bromo-
cystargamide, and set out to explore whether the Sonogashira
methodology that we had developed could be utilised in
selectively modifying bromo-tryptophan residues as
a
component of a tripeptide or within our novel bromo natural
product (Scheme 1).
The Sonogashira cross-coupling reaction of aryl amino acid
derivatives has been shown to be important, enabling the
modification of proteins and peptides through an alkynyl
linker. 5d,5f,5g,5i,5l,8,9 The reaction has also shown applicability in
tagging the proteins with fluorescence labels (Scheme 1A),5a in
tagging for Fluorescence Resonance Energy Transfer (FRET)5c,10
and Positron Emission Tomography (PET) studies.5k Such cross-
coupling investigations within peptides, however, almost
exclusively focused on either the diversification of highly
reactive alkynyl derivatives embedded within peptides or iodo-
phenyl derivatives such as iodophenylalanine5 and iodo-
tyrosine.5a,6 Strategies to exploit the Sonogashira reaction to
modify iodinated or alkynylated nucleic acids have also been
successfully employed (Scheme 1B).5a,7 The reaction frequently
requires the use of protected substrates however Sonogashira
cross-coupling of unprotected amino acids, including iodo and
bromo phenylalanine and tyrosine were also reported.5l,6e
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1

Chemical Science
Page 2 of 8
View Article Online
DOI: 10.1039/C6SC04423A
ARTICLE
Journal Name
The intrinsic fluorescence of tryptophan often determines, or halotryptophans useful are their poor solubility and their
modulates, the spectrophotometric properties of a given propensity to chelate to and deactivate the palladium
peptide or protein; and is a crucial residue for stabilising catalyst.18
secondary and tertiary structure through intra and
intermolecular interactions.11 It is also an important residue in
Results and discussion
many natural products. It is found in its halogenated form in a
wide variety of metabolites including anticancer agents
Rebeccamycin12 and Diazonamide A.13 The possibility of
selective chemical modification of such residues within natural
products and peptides is attractive.14 However, though much
research has been carried out on the Sonogashira modification
We previously reported the Suzuki-Myaura cross-coupling of
bromotryptophan-containing natural product analogues2e
using the Buchwald’s water-soluble ligand sSPhos 11 19
.
Anderson and Buchwald reported the use of water-soluble
ligand sXPhos 12 for the Sonogashira coupling of aryl halides
19
of halophenylalanines an
d halotyrosines, to the best of our
including chloropyridine.
We decided to explore whether
knowledge, there are no prior reports of the utilisation of this
strategy for the Sonogashira diversification of more
challenging halotryptophans, either as a discrete amino acid or
as a component of a peptide or natural product.
this ligand could be utilised to facilitate the Sonogashira
coupling of halotryptophans in water. To start with a slightly
less challenging substrate, we first investigated whether
Sonogashira modification of unprotected 5-bromoindole 15
could be achieved.
Me
SO3Na
SO3Na
Me
PCy2
OMe
PCy2
iPr
Me
MeO
iPr
NaO3
S
P
SO3Na
NaO3S
Me
P
SO3Na
Me
Me
SO3Na
SO3Na
12
11
13
14
Figure 1: Water soluble ligands for Pd mediated cross-coupling. sSPhos 11
sXPhos 12, TPPTS 13 and TXPTS 14
,
Scheme 2: Sonogashira cross-coupling reaction of 5-bromoindole 15 with
phenylacetylene 16. (a) Reagents and conditions are described in Table 1.
Encouragingly we were able to demonstrate the conversion of
5-bromoindole 15 with phenylacetylene 16, in a 1:1 mixture of
water and acetonitrile to its alkynylated derivative 17 in 76%
yield (Scheme 2, and Table 1, entry 1). This provided a good
starting point, however we observed that increasing the
catalyst and ligand loading (from 2.5 to 5 mol% and from 7.5 to
15 mol%, respectively) improved the conversion to 93% (Table
1, entry 2). The reaction could be further improved by
increasing the number of equivalents of alkyne coupling
partner from 1.5 eq to 3.0 eq, leading to complete conversion
of 5-bromoindole 15 to the cross-coupling product 17 (Table 1,
entry 3). With conditions in hand that enabled the cross-
coupling of 5-bromoindole 15 to go to completion, we sought
to investigate if the reaction time could be decreased.
Switching from conventional heating to microwave heating at
Scheme 1:
Sonogashira cross-coupling modification of natural product
analogues. (A) Labelling of iodophenyl derivatives with fluorescent tags.9 (B)
Cross-coupling of iodinated pyrimidine nucleoside.7a (C) Sonogashira cross-
coupling of unprotected bromotryptophans. (D) Sonogashira cross-coupling of 7-
bromotryptophan in a tripeptide system. (E) New to nature bromo-cystargamide.
100 °C proved to be successful, with full conversion being
observed after 2 h heating in the microwave (Table 1, entry 4),
Halotryptophans may be readily accessed through a simple
one-step biotransformation using tryptophan synthase,15,16 or
through a 4-5 step chemical synthesis.17 Challenges that need
to be addressed to render Sonogashira cross-coupling of free
rather than the 18 h previously required.
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx

Page 3 of 8
Chemical Science
View Article Online
DOI: 10.1039/C6SC04423A
Journal Name
ARTICLE
Entry
Substrate
Cat / mol%
Ligand / mol%
Alkyne Loading
/ eq
Heating
Method
Reaction Time /
NMR Conv. / %
h
1
2
3
4
5
15
15
15
15
18
2.5
5.0
5.0
5.0
5.0
7.5
1.5
1.5
3.0
3.0
3.0
Conventional
Conventional
Conventional
Microwave
18
18
18
2
76b
93b
15.0
15.0
15.0
15.0
>99b
>99b
>99c
Microwave
2
respectively). Our results showed that an inert atmosphere
was
preferential for the reaction. However, the reaction still
proceeded to give high conversion without the exclusion of
oxygen from the reaction, showing that the conditions should
work well for reactions where maintaining an inert
atmosphere may prove difficult.
Scheme 3: Investigating the reaction of halo-tryptophans and phenylacetylene
16. (a) Reagents and conditions are described in Table 1 and Table 2.
Having established conditions to enable quantitative
conversion of 5-bromoindole 15 within 2h, we next explored
the reaction with what we had perceived to be the more
challenging unprotected 5-bromotryptophan 18. Pleasingly,
the reaction proceeded remarkably well with the tryptophan
substrate and complete conversion to the product was
observed after microwave heating for 2 h under the same
conditions that we had developed for 5-bromoindole 15 (Table
1, entry 5).
Table 2: Investigating the reaction of halo-tryptophans and phenylacetylene 16
.
Conversions achieved under various conditions: exploring the impact of the
ligand (entries 2-4), reaction in the presence of air (entry 5), concentration
(entries 6 and 7), impact of temperature (entry 8), 5-chlorotryptophan 20 as
substrate in place of 5-bromotryptophan 18 (entry 9)
Entry
Substrate
Substrate
amount in mmol
(concentration)
Ligand
T / °C
NMR
Conv.c
/ %
1
2
18
18
0.1 (50 mM)a
0.1 (50 mM)a
sXPhos
sSPhos
100
100
>99
88
With the successful quantitative conversion of unprotected 5-
bromotryptophan 18 accomplished, we next investigated the
limits of the reaction and specifically the impact of a series of
water-soluble ligands. In order to determine the broader
applicability we also explored the impact of air, the effect of
substrate concentration on conversion, and the influence of
temperature on the reaction (Table 2). To this end, we first
investigated series of commercially available water-soluble
ligands, sSPhos 11 (88%), TPPTS 13 (50%) and TXPTS 14 (83%)
were all shown to mediate reasonable conversions of 5-
bromotryptophan 18 to the expected product 19 (Table 2,
entries 2-4), however, none of the ligands tested matched
sXPhos 12 for performance (Table 2, entry 1). The reactions to
this point had been carried out under inert conditions with de-
gassed solvents, not always simple to achieve for biomaterials.
In order to investigate the impact of air on the cross-coupling,
the reaction was repeated in air using non-degassed solvents
3
4
5
6
7
8
9
18
18
18
18
18
18
20
0.1 (50 mM)a
0.1 (50 mM)a
0.1 (50 mM)b
0.05 (25 mM)a
0.025 (12.5 mM)a
0.1 (50 mM)a
0.1 (50 mM)a
TPPTS
TXPTS
sXPhos
sXPhos
sXPhos
sXPhos
sXPhos
100
100
100
100
100
80
50
83
86
67
34
35
<1
100
Conditions: (a) Substrate (1 eq), PdCl2(CH3CN)2 (5 mol%), Ligand (15 mol%),
Cs2CO3 (2.5 eq), phenylacetylene (3.0 eq), CH3CN/H2O (1:1, 2 mL), microwave
(Table 2, entry 5). The reaction proceeded well in the presence heating, sealed tube, solids and solvents purged with nitrogen. (b) As with (a),
except solids and solvents were not nitrogen purged. (c) Based on ratio of
of oxygen with only a modest decrease in the observed
conversion (86% with respect to >99%, Table 2, entries 5 and 1
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3

Chemical Science
Page 4 of 8
View Article Online
DOI: 10.1039/C6SC04423A
ARTICLE
Journal Name
starting material aromatic peak at δH 7.89 ppm compared to product aromatic
peak at H 7.96 ppm in CD3OD.
terminal alkynes (Scheme 3, 16
,
24-33). In each reaction, the
δ
conversion of the starting halo-tryptophan to the cross-
coupled product was determined by 1H NMR. To obtain
indication as to representative isolable yields for each alkynyl
coupling partner, the product from reaction with the 5-
bromotryptophan 18 was purified to determine the isolated
yield (purified by reverse-phase HPLC and carried out in
triplicate).
We next focused on studying the effect of concentration on
the reaction. The reaction was carried out at 50 mM, 25 mM
and 12.5 mM (Table 2, entries 1, 6 and 7, respectively). As
expected, the conversion is not as efficient at lower
concentrations, however, modest conversion, using the same
catalyst loading, could be observed at a 4-fold dilution. There
may be scope for improving these conversions, by increasing
catalyst loading and coupling partner, when the halogenated
substrate is at high dilution. Finally, the effects of lowering the
temperature of the reaction were investigated. We observed
We first examined the coupling of aromatic-substituted
acetylenes. The three phenylacetylene-based alkynes
(phenylacetylene 16, 3-fluorophenylacetylene 24 and 4-
cyanophenylacetylene 25) all showed very good to excellent
conversions for each tryptophan and high yields for the
isolated products (Scheme 4 and Table 3). The high yield
obtained for the 3-fluorophenylacetylene 24 was particularly
pleasing as this enables a F label to be introduced into a
molecule that can potentially be used to follow the fate of a
molecule. Though current reaction times (2h) are still rather
long, there may be potential to develop this chemistry further
to enable labelling for PET. Similarly, heterocyclic alkyne
ethynylthiophene 26 showed excellent conversions for all the
tryptophans and high yield for the isolated product (Table 3).
that a slight reduction in reaction temperature from 100 °C to
80 C (Table 2, entry 8) significantly decreased the observed
°
conversion from >99% to 35%, indicating that 100
to be close to optimal for this reaction.
°C was likely
Previous investigations of the Sonogashira cross-coupling have
focused predominantly on aryl iodides; and so it is exciting to
see that conditions can be developed to enable quantitative
Sonogashira
conversion
of
the
less
reactive
bromotryptophans. The chloro analogues are even less
reactive, but again in order to determine the limitations of the
reaction conditions, the reaction of 5-chlorotryptophan 20
with phenylacetylene 16 was attempted (Table 2, entry 9).
Unfortunately, this highly unreactive substrate showed no
reaction after heating for 2 h in the microwave, with only
starting material being observed.
Table 3: Reaction of halo-tryptophans with commercially available
a
phenylacetylenes 16
,
24
,
25 and ethynylthiophene 26
.
Entry
Tryptophan
Alkyne
Product
Conversionb
(Yieldc) / %
1
2
5-Br 18
6-Br 21
7-Br 22
7-I 23
16
16
16
16
24
24
24
24
25
25
25
25
26
26
26
26
19
34
35
35
36
37
38
38
39
40
41
41
42
43
44
44
>99 (80)
>99
3
81
4
79
5
5-Br 18
6-Br 21
7-Br 22
7-I 23
>99 (97)
>99
6
7
>99
8
>99
9
5-Br 18
6-Br 21
7-Br 22
7-I 23
>99 (79)
>99
10
11
12
13
14
15
16
>99
>99
5-Br 18
6-Br 21
7-Br 22
7-I 23
>99 (75)
>99
Scheme 4: Exploring the substrate scope of the reaction.
With the optimal conditions for the Sonogashira coupling of
halotryptophans determined, we then sought to examine the
scope of the reaction by varying the halogen and its position,
as well as the alkyne coupling partner (Scheme 4, Tables 3 and
4). The reactions were carried out with 5-bromotryptophan
18, 6-bromotryptophan 21, 7-bromotryptophan 22 and 7-
iodotryptophan 23 with a range of commercially available
>99
>99
Reaction Conditions: (a) Substrate (0.1 mmol, 1.0 eq), alkyne (0.3 mmol, 3.0 eq),
PdCl2(CH3CN)2 (5 mol%), sXPhos (15 mol%), Cs2CO3 (2.5 eq), water/acetonitrile
(1:1, 2 mL, degassed), microwave heating, 100 °C, 2 h. (b) Based on NMR ratios
of starting material to product in CD3OD. (c) Average of three isolated yields.
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx

Page 5 of 8
Chemical Science
View Article Online
DOI: 10.1039/C6SC04423A
Journal Name
ARTICLE
2
3
4
6-Br 21
7-Br 22
7-I 23
27
27
27
46
47
47
>99
81
85
5
6
5-Br 18
6-Br 21
7-Br 22
7-I 23
28
28
28
28
29
29
29
29
30
30
31
32
33
48
49
50
50
51
52
53
53
54
54
55
56
57
63 (26)
>99
74
7
8
39
9
5-Br 18
6-Br 21
7-Br 22
7-I 23
54 (51)
>99
>99
56
10
11
12
13
14
15
16
17
5-Br 18
5-Br 18
5-Br 18
5-Br 18
5-Br 18
16
50 (~25d)
<1
<1
<1
Reaction Conditions: (a) Substrate (0.1 mmol, 1.0 eq), alkyne (0.3 mmol, 3.0 eq),
PdCl2(CH3CN)2 (5 mol%), sXPhos (15 mol%), Cs2CO3 (2.5 eq), water/acetonitrile
Next,
a range of aliphatic alkynes, containing useful
functionality, and designed to introduce different levels of
challenge, were investigated. Though the reaction, would not
proceed with propiolic acid 31 (Table 4, entry 15),
propargylamine 32 (Table 4, entry 16) or TMS acetylene (Table
4, entry 17), the cross-coupling reaction with 4-butyn-1-ol 27
proceeded with high conversion and high isolated yield for
product 45 (Figure 2) and provides an alcohol substituent for
potential further modification. The reaction also proceeded
with 4-phenyl-1-butyne 28 to give moderate to excellent
conversions (Table 4, entries 5 to 8), but low isolated yields of
product 48. 1,7-Octadiyne 29 was chosen as a coupling partner
in order to gain access to a terminal alkyne in the coupled
product, in order to install an alkyne tag for subsequent
alkyne-azide cycloaddition reactions.20 Pleasingly, the reaction
proceeded to give diyne product 51 in 51% yield (average of
three isolated yields), (Table 4, entry 9). 3-Cyclohexyl-1-
propyne 30 was also investigated as a potential coupling
(1:1, 2 mL, degassed), microwave heating, 100
of starting material to product in CD3OD. (c) Average of three isolated yields. (d)
Product co-eluted with sXPhos and could not be isolated pure, the
°C, 2 h. (b) Based on NMR ratios
4
approximate yield is based on a single isolation.
Remarkably, our simple scoping reactions revealed that the
developed Sonogashira cross-coupling reaction conditions
could be applied to the modification of halotryptophans and
could be carried out under aqueous conditions without the
need for copper salts and without the need to protect the
amino acid. We carried out analysis of the isolated products, as
a representative set, using Marfey’s reagent (see supporting
information).21 Our analysis revealed no erosion of the
enantiopurity of the tryptophans under the reaction conditions
that we had employed (see supporting information). With
robust conditions for tryptophan diversification via
Sonogashira cross-coupling in hand, we next tested the
reaction with more complex halo-tryptophan-containing
substrates. We chose to design and synthesise two tripeptides
containing an N-terminal 7-bromotryptophan and an internal
7-bromotryptophan. Tripeptides 58 and 59 were synthesised
(see Supporting Information) and were subjected to the
aqueous Sonogashira reaction conditions with 3-
fluorophenylacetylene 24 (Scheme 5), chosen for its high
efficiency as a coupling partner to free halotryptophans and
also due to the benefit of being able to follow the reaction by
19F NMR. The reaction with Ala-(7-Br-Trp)-Phe 58 proceeded to
give product 60 in 47% isolated yield. The reaction of (7-Br-
Trp)-Ala-Phe 59 also gave expected product 61 pleasingly in
quantitative isolated yield. The lower isolated yield for product
partner. After the standard reaction conditions (100
h), 1H NMR showed approximately 16%
°C in
microwave,
2
conversion to the product 54 (Table 4, entry 13). Increasing the
reaction time to 8 h (Table 4, entry 14) improved the
conversion to 50%. The product was purified by reverse-phase
HPLC, but co-eluted with the sXPhos 12 ligand, and it was
challenging to obtain a definitive isolated yield in this case.
Table 4: Reaction of halo-tryptophans with commercially-available alkynes 27
33 a
-
.
Entry
Tryptophan
Alkyne
27
Product
45
Conversionb
(Yieldc) / %
1
5-Br 18
>99 (81)
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5

Chemical Science
Page 6 of 8
View Article Online
DOI: 10.1039/C6SC04423A
ARTICLE
Journal Name
60 could potentially be due to the steric hindrance and/or analysed by LC-MSMS. Results demonstrated that all of the
difficulties associated with purification due to potentially halotryptophans were incorporated into cystargamide,
reduced aqueous solubility. The quantitative conversion of the generating at low level, a series of new to nature halogenated
7-bromotryptophan residue at the N-terminus of this analogues (See SI for details). As judged from the LC-
tripeptide is striking and opens up the possibility of using this absorbance peak area, there was no significant difference in
methodology for the labelling, modification and modulation of the quantities of halo-cystargamide produced across the two
peptides.
tryptophan concentrations that we had carried out the
precursor directed biosynthesis at, therefore, to reduce
wastage of halotrytophan precursor we opted for a 0.25mM
concentration for future experiments. As we simply wanted to
explore and hopefully demonstrate proof of principle by
applying our Sonogashira methodology to the diversification of
a novel “new to nature” natural product we decided to select
just one halotryptophan for scaling up the fermentation with
and feeding.
Scheme 5: Investigating the Sonogashira modification of 7-bromo tryptophan, as
a component of a tripeptide with 3-fluorophenylacetylene 24. (a) Reagents and
conditions: PdCl2(CH3CN)2 (5 mol%), sXPhos 12 (15 mol%), Cs2CO3 (2.5 eq),
H2O:CH3CN (1:1), microwave heating, 100 °C, 2 h
Having demonstrated the ability to apply this methodology to
small peptides we next explored the reaction in the context of
natural products as a potential tool for use in GenoChemetic
analogue generation. The harnessing of biosynthetic pathways
to force the incorporation of a halogen into a natural product
provides a chemically orthogonal handle with the possibility
for selective modification. We have previously demonstrated
that the facile Suzuki-Miyaura cross coupling may be readily
applied to such systems.2f Cystargamide 62 (Scheme 6) is a 5-
hydroxytryptophan containing cyclic lipopeptide isolated from
Kitasatospora cystarginea.22 Though this compound has shown
no notable biological activity to date, a very close structural
analogue Q-6402 has been reported to inhibit phospholipase
A2.23 Cystargamide provides an interesting and challenging
test bed for GenoChemetic analogue generation: firstly
because the naturally incorporated tryptophan shows
hydroxylation and therefore the incorporation of other
analogues of this amino acid may prove challenging, secondly
because the molecule contains a number of functionalities that
could potentially impede the reaction, and thirdly because it is
produced in fairly low titre. 22
Scheme 6: Precursor-directed biosynthesis with K. cystarginea to generate new-
to-nature halo-analogues of cystargamide, a challenging test bed natural product
system. Scale-up culture and Sonogashira reaction of semi-pure 6-bromo-
We set out to explore whether halotryptophans could be
incorporated, through precursor directed biosynthesis, to
generate a halogenated analogue of this natural product
(Scheme 6). To explore this possibility we carried out small
scale feeding experiments with 7-, 6- and 5-chlorotryptophan
and 7-, 6- and 5-bromotryptophan at 0.25 mM and 1.0 mM to
both 4 mL cultures in 24 deep well plates and 50 mL cultures in
250 mL Erlenmeyer flasks. The cultures were extracted and
cystargamide
conditions that we had developed. (a) Reagents and conditions: PdCl2(CH3CN)2
sXPhos 12, Cs2CO3, H2O:CH3CN (1:1), microwave heating, 100 C, 2 h
9 with 3-fluorophenylacetylene 24 using the cross-coupling
°
Our challenge would be to generate sufficient halogenated
natural product on which to explore the cross-coupling. As we
had shown the chlorotryptophans to be too unreactive for
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx

Page 7 of 8
Chemical Science
View Article Online
DOI: 10.1039/C6SC04423A
Journal Name
ARTICLE
Sonogashira cross-coupling, and wanting to avoid placing a be very high yielding with phenylacetylene 16
halogen on C-5 of the indole ring, which is naturally fluorophenylacetylene 24, 4-cyanophenylacetylene 25 and
hydroxylated, and could provide
a future chemically ethynylthiophene 26. The reaction products can be isolated by
orthogonal handle for further functionalization, we set out to reverse-phase HPLC and show no loss of stereochemical
scale up production of 6-bromocystargamide. To this end we integrity under the reaction conditions. The reaction can even
fed 8 L of culture (incubated in batches of 500 mL in 2 L flasks) be carried out in the presence of oxygen with only a small
with 0.26 mM 6-bromotryptophan. Extraction and purification decrease in the observed conversion.
resulted in an approximately 1.0 mg of a partially pure fraction
Tryptophan is an essential amino acid, it is a key component in
containing brominated cystargamides along with other
many natural products and its presence in proteins and
cystargamide derivatives. By comparing the LC-MS extracted
peptides is key to their structural integrity, furthermore the
ion peak areas, it could be estimated that the parent
intrinsic fluorescence of tryptophan often determines, or
unhalogenated cystargamide 62
analogue that lacked the 5-hydroxy on the tryptophan 63 and
bromocystargamide were present in an approximate
, a bromo-cystargamide
modulates, the spectrophotometric properties of a given
peptide or protein, it therefore represents a very exciting
target for diversification. Sonogashira cross-coupling of other
amino acid residues including iodophenylalanine has been
shown to be useful to tagging or modulating the properties of
these peptides. We have demonstrated the first Sonogashira
modification of halotryptophan as a component of a peptide,
this represents a starting point from which milder conditions
must be developed in order to extend the applicability of this
reaction to sensitive proteins.
9,
10:0.2:1.4 ratio. This indicated the ability of the producing
organism to process 6-bromo-tryptophan, though perhaps not
quite as efficiently as unhalogenated tryptophan. Analysis of
the extract also revealed quantities of linear unbrominated
cystargamide in which the ester bond appeared to have been
hydrolysed, and analogues of this and the brominated forms,
which contained an alanine rather than glycine residue (see
supporting information for more details).
Using a simple tripeptide containing a 7-bromotryptophan at
the N-terminus we have been able achieve quantitative
Sonogashira cross-coupling with 3-fluorophenylacetylene 24
within 2h. This is exciting as it opens up the potential for
development for 19F labelling of peptides, with the short
reaction times making this a potentially attractive tool for PET
imaging studies.
A fraction containing predominantly the brominated analogue
(>85%)
9 was subjected to the Sonogashira cross-coupling
conditions developed within this study (Scheme 6). LC-MS
analysis of the crude reaction mixture indicated that all
brominated cystargamide had been consumed and revealed
peaks at m/z 1072 [M+H]+ for product 64 and 1090 [M+H]+ for
hydrolysed product 65 (further confirmed by MSMS),
indicating
successful
cross-coupling
with
3-
We have for the first time generated a series of “new to
fluorophenylacetylene 24 into analogue
9
.
As no
nature” halocystargamides including 6-bromocystargamide 9.
corresponding linear brominated cystargamide had been
present within the starting material, for this reaction, it is likely
that this second product arises from hydrolysis of
cystargamide either during the reaction or work up. 19F{1H}
The enzymes that mediate the biosynthesis of these
compounds are strikingly flexible in enabling the incorporation
of a wide range of halogens at different positions around the
indole ring. Full processing including hydroxylation of the
bromotryptophan observed within the natural product,
demonstrates how readily, even the sterically bulky C-Br, is
tolerated by the biosynthetic enzymes. 6-Bromocystargamide
NMR analysis showed two fluorine peaks at
and -114.43 ppm with a ddd coupling consistent with that
observed for compounds 36 60 and 61. Attempts to separate
δF(D2O) -114.05
,
the very small amounts of the two compounds by HPLC proved
1
to be unsuccessful, as was obtaining sufficient material for H
9
represented the demanding natural product test-bed desired
with which to challenge the cross-coupling conditions that we
had developed. Though the compound is functionally
elaborate and can only be accessed in very small quantities, it
was possible to selectively functionalise the molecule with 3-
fluorophenylacetylene 24 using the conditions that we had
developed. Previously we had exemplified the utility of the
more forgiving Suzuki-Miyaura reaction in the selective
modification of natural products; here we show that the
Sonogashira reaction can be used as a tool for GenoChemetics
and for the selective diversification of “new to nature” natural
products. Such an approach hold the potential to be a useful
tool for diversification for Structure Activity Relationship (SAR)
studies, for modulating the electronics of tryptophan and for
NMR analysis. It was notable though that the conditions that
we had developed could be used to enable the
functionalization of a challenging “new to nature” natural
product even within a chemically demanding test-bed system
with the added challenge of being produced only at very low
level.
Conclusions
We have for the first time demonstrated the Sonogashira
diversification of free unprotected 5-, 6- and 7-bromo
tryptophan and 7-iodotryptophan, and shown the reaction to
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7

Chemical Science
Page 8 of 8
View Article Online
DOI: 10.1039/C6SC04423A
ARTICLE
Journal Name
1, 4713-4719. (e) H. Dibowski and F. P. Schmidtchen, Angew.
Chem. Int. Ed., 1998, 37, 476-478.
For example, see: (a) T. Kottysch, C. Ahlborn, F. Brotzel and
C. Richert, Chem. Eur. J., 2004, 10, 4017-4028. (b) P. Ĉapek,
H. Cahová, R. Pohl, M. Hocek, C. Gloeckner and A. Marx,
Chem. Eur. J., 2007, 13, 6196-6203.
tagging natural products and proteins with labels for
fluorescence tracking, immobilisation tagging and PET imaging.
7
Acknowledgements
8
9
D. T. Bong and M. R. Ghadiri, Org. Lett. 2001, 3, 2509-2511.
S. Hauke, M. Best, T. T. Schmidt, M. Baalmann, A. Krause and
The research leading to these results has received funding
from the European Research Council under the European
Union’s Seventh Framework Programme (FP7/2007-2013/ERC
grant agreement no 614779 (to R.J.M.G), and ERAIB (Grant no.
031A338A)) (to R.J.M.G), and H2020-MSCA-IF-2014 (Grant no.
659399 (to C.P.U). We thank EPSRC National Mass
Spectrometry Service Centre (Swansea, UK) for assistance in
acquiring MS analysis on a number of the lower molecular
weight tryptophan derivatives.
R. Wombacher, Bioconjugate Chem., 2014, 25, 1632-1637.
10 S. J. Miyake-Stoner, A. M. Miller, J. T. Hammill, J. C. Peeler, K.
R. Hess, R. A. Mehl and S. H. Brewer, Biochemistry, 2001, 3,
2509-2511.
11 H. Lodish, A. Berk, S. Lawrence Zipursky, P. Matsudaira, D.
Baltimore and J. Darnell, Molecular Cell Biology, 4th Edition,
Chapter 3- Protein Structure and Function, W. H. Freeman,
New York, 2000.
12 E. Yeh, S. Garneau and C. T. Walsh, Proc. Natl. Acad. Sci. U. S.
A., 2005, 102, 3960-3965.
13 N. Lindquist, W. Fenical, G. D. Van Duyne and J. Clardy, J. Am.
Chem. Soc., 1991, 113, 2303-2304.
Notes and references
D. J. Newman and G. M. Cragg, J. Nat. Prod., 2007, 70, 461-
477.
14 (a) S. De Ornellas, T. J. Williams, C. G. Baumann and I. J. S.
Fairlamb, RSC Catalysis Series No 11, C-H and C-X Bond
Functionalization: Transition Metal Mediation, Chapter 12-
Catalytic C-H/C-X Functionalization of Nucleosides,
Nucleotides, Nucleic Acids, Amino Acids, Peptides and
Proteins, Royal Society of Chemistry, 2013. (b) O. Boutureira
and G. J. L. Bernardes, Chem. Rev., 2015, 115, 2174-2195.
15 (a) R. J. M. Goss and P. L. A. Newill, Chem. Commun., 2006,
4924-4925. (b) M. Winn, A. Deb Roy, S. Grüschow, R. S.
Parameswaran and R. J. M. Goss, Bioorg. Med. Chem. Lett.,
2008, 18, 4508-4510. (c) D. R. M. Smith, T. Willemse, D. S.
Gkotsi, W. Schepens, B. U. W. Maes, S. Ballet and R. J. M.
Goss, Org. Lett., 2014, 16, 2622-2625.
16 (a) A. R. Buller, S. Brinkmann-Chen, D. K. Romney, M. Herger,
J. Merciano-Calles and F. H. Arnold, Proc. Natl. Acad. Sci. U.
S. A., 2015, 112, 14599-14604. (b) M. Herger, P. Van Roye, D.
K. Romney, S. Brinkmann-Chen, A. R. Buller and F. H. Arnold,
J. Am. Chem. Soc., 2016, 138, 8388-8391. (c) J. Murciano-
Calles, D. K. Romney, S. Brinkmann-Chen, A. R. Buller and F.
H. Arnold, Angew. Chem. Int. Ed., 2016, 55, 1-6.
1
2
(a)R. J. M. Goss, S. Shankar and A. A. Fayad, Nat. Prod. Rep.,
2012, 29, 870-889. (b) K. P. P. Mahoney, D. R. M. Smith, E. J.
A. Bogosyan and R. J. M. Goss, Synthesis, 2014, 46, 2122–
2132. (c) A. E. Osbourn, R. J. M. Goss, G. T. Carter, Natural
Products: Discourse, Diversity, and Design, Wiley-Blackwell,
2014, ISBN: 978-1-118-29806-0. (d) A. Kirschning, F. Taft, and
T. Knobloch, Org. Biomol. Chem., 2007, 5, 3245-3259 (e) A.
Deb Roy, R. J. M. Goss, G. K. Wagner and M. Winn, Chem.
Commun. 2008, 4831-4833. (f) A. Deb Roy, S. Grüschow, N.
Cairns and R. J. M. Goss, J. Am. Chem. Soc. 2010, 132, 12243-
12245.
3
(a) K. C. Nicolaou, P. G. Bulger and D. Sarlah, Angew. Chem.
Int. Ed., 2005, 44, 4442-4489. (b) X. Chen, K. M. Engle, D.-H.
Wang and J.-Q. Yu, Angew. Chem. Int. Ed. 2009, 48, 5094-
5115. (c) J. Wencel-Delford and F. Glorius, Nature Chem.,
2013, 5, 369-375.
4
5
K. Sonogashira, J. Organometallic Chem., 2002, 65, 46-49.
For selected reports of Sonogashira cross-coupling on
iodophenylalanine derivatives, see: (a) G. T. Crisp and J.
Gore, Tetrahedron, 1997, 53, 1523. (b) B. Kayser, J. Altman
and W. Beck, Tetrahedron, 1997, 53, 2475-2484. (c) S.
17 For example, see: (a) Y. Konda-Yamada, C. Okada, K. Yoshida,
U. Yasuyuki, S. Arima, N. Sato, T. Kai, H. Takayanagi and Y.
Harigaya, Tetrahedron, 2002, 58, 7851-7861. (b) C. Ma, X.
Liu, X. Li, J. Flippen-Anderson, S. Yu and J. Cook, J. Org.
Chem., 2001, 66, 4525-4542.
Nampalli, M. Khot and S. Kumar, Tetrahedron Lett., 2000, 41,
8867-8871. (d) V. Hoffmanns and N. Metzler-Nolte,
Bioconjugate Chem., 2006, 17, 204-213. (e) C. Corona, B. K.
Bryant and J. B. Arterburn, Org. Lett., 2006, 8, 1883. (f) A.
Takasu, S. Kondo, A. Ito, Y. Furukawa, M. Higuchi, T.
Kinoshita and I. Kwon, Biomacromolecules, 2011, 12, 3444-
3452. (g) S. Zhang, K. H. Chan, R. K. Prud’Homme and A. J.
18 M. A. Carvalho, B. C. Souza, R. E. F. Paiva, F. R. G. Bergamini,
A. F. Gomes, F. C. Gozza, W. R. Lustri, A. L. B. Formiga, G.
Rigatto and P. P. Corbi, J. Coordination Chem., 2012, 65
1700-1711.
,
19 K. W. Anderson and S. L. Buchwald, Angew. Chem. Int. Ed.,
2005, 44, 6173-6177.
20 K. Thirumurgan, D. Matosiuk and K. Jozwaik, Chem. Rev.,
2013, 113, 4905-4979.
Link, Mol. Pharmaceutics, 2012, 9, 2228-2236. (h) M. Peña-
López, L. A. Sarandeses and J. P. Sestelo, Eur. J. Org. Chem.,
21 R. Bhushan and H. Brückner, J. Chromatogr. B, 2011, 879
3148-3161.
,
2013, 2545-2554. (i) E. M. Tookmaman, E. E. Fenlon and S. H.
Brewer, RSC Advances, 2015, 5, 1274. (j) N.-D. Doan, M. P. de
22 K. A. Gill, F. Berrue, J. C. Arens and R. G. Kerr, J. Nat. Prod.,
2014, 77, 1372-1376.
Molliens, M. Létourneau, A. Fournier and D. Chatenet, Eur. J.
Med. Chem., 2015, 104, 106-114, (k) P. Krapf, R. Richarz, E. A.
Urusova, B. Neumaier and B. D. Zlatopolskiy, Eur. J. Org.
Chem., 2016, 430-434. (l) K. Kodama, D. Fukuzawa, H.
Nakayama, K. Sakamoto, R. Kigawa, T. Yaburi, N. Matsuda,
M. Shirouza, K. Takio, S. Yokoyana and K. Tachiban,
23 Hiramoto, M.; Niwa, A.; Miyake, A.; Yamamoto, H.;
Takebayashi, Y.; Nishikawa, T.; Shibazaki, M.; Nagai, K.
Symposium on the Chemistry of Natural Products;
Yamanouchi Pharmaceutical Co., Ltd.: Tokyo, Japan, 1993;
pp 678−684.
ChemBioChem, 2007, 8, 232-238.
6
For selected reports of Sonogashira cross-coupling on
iodotyrosine derivatives, see: (a) A. Khatyr and R. Ziessel,
Synthesis, 2001, 1665-1670. (b) A. Khatyr and R. Zeissel, Org.
--------------------------------------------------------
Lett., 2001, 3, 1857-1860. (c) W. H. Walker IV and S. E.
Rokkita, J. Org. Chem., 2003, 68, 1563-1566. (d) X. Liu, J. Jiao,
X. Jiang, J. Lie, Y. Cheng and C. Zhu, J. Mater. Chem. C, 2013,
8
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx