Development of fluorescent aryltryptophans by Pd mediated cross-coupling of unprotected halotryptophans in water

Article abstract of DOI:10.1039/b807512c

A convenient and high yielding procedure for the Suzuki-Miyaura cross-coupling of unprotected bromo- and chlorotryptophans in water provides fluorescent aryltryptophans. The Royal Society of Chemistry.

Full text of DOI:10.1039/b807512c

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Development of fluorescent aryltryptophans by Pd mediated
cross-coupling of unprotected halotryptophans in waterw
Abhijeet Deb Roy,a Rebecca J. M. Goss,*a Gerd K. Wagner*b and Michael Winna
Received (in Cambridge, UK) 2nd May 2008, Accepted 27th June 2008
First published as an Advance Article on the web 27th August 2008
DOI: 10.1039/b807512c
A convenient and high yielding procedure for the Suzuki–
Miyaura cross-coupling of unprotected bromo- and chlorotryp-
tophans in water provides fluorescent aryltryptophans.
accept this challenge. While the cross-coupling of boronic
acid-derivatives of phenylalanine has been reported,7 this
functionality is hard to install biosynthetically, and previous
cross-coupling work on tryptophans has been limited to the
use of protected halotryptophans in organic solvents.8 To the
best of our knowledge, there are no examples in the literature
of the cross-coupling of unprotected halotryptophans.
In peptides and proteins, the amino acid tryptophan frequently
stabilises secondary and tertiary structure through selective intra-
and intermolecular interactions.1 In addition, the intrinsic fluo-
rescence of tryptophan often determines, or modulates, the
spectrophotometric properties of a given peptide or protein. This
has been exploited for protein quantification,2a and to study
folding events, ligand binding, and protein–protein interactions.2b
The prospect of a simple chemical method to modify and
intensify the fluorescence of tryptophan is therefore appealing.
Tryptophan is also a biosynthetic precursor for many
naturally occurring non-ribosomal peptides and alkaloids.3
Halotryptophans, for example, are present in numerous im-
portant natural products, including the anticancer agents
rebeccamycin and diazonamide A.4,5 Halotryptophans can
serve as substrates for Suzuki–Miyaura (Scheme 1) and related
Pd-catalysed cross-coupling reactions, which allow the instal-
lation of an aryl, heteroaryl or alkenyl substituent on the
indole ring. Potentially, this synthetic approach can be applied
not only to halotryptophans, but also to halotryptophan-
containing peptides, proteins and natural products.
Recently, we reported the one-pot preparation of a series of
ten halotryptophans using a readily prepared cell free lysate.9 We
have now developed suitable conditions for the Suzuki–Miyaura
cross coupling of unprotected 5- and 7-halotryptophans with a
variety of arylboronic acids in water (Scheme 1). We have also
successfully applied this methodology to the cross-coupling of a
simple dipeptide as a first step towards the use of cross-coupling
reactions for the direct modification of peptides, proteins and
natural products. Importantly, our cross-coupling conditions
allow the cross-coupling of bromo- and chlorotryptophans,
which are less reactive than the corresponding iodo congener,
but more common in natural products.10 The spectrophoto-
metric properties of the cross-coupling products are distinct from
those of the parent tryptophan, and open up the possibility of
using the Suzuki–Miyaura cross-coupling reaction for the site-
selective fluorescent labelling of peptides, proteins and natural
products. In contrast to existing methods for chemical ligation
(e.g. click chemistry), our approach promises to enable selective
functionalisation of a motif that is not uncommon in nature and
without introduction of a heterocyclic scar.
The successful application of this challenging strategy re-
quires the development of cross-coupling conditions which are
mild, compatible with the presence of unprotected carboxy
and amino functionalities, and work in water. Recent progress
in Pd-catalysed reactions in aqueous media6 motivated us to
Halotryptophans 1 and 2 (Scheme 1) were prepared from their
corresponding haloindole and serine, in a simple one-pot enzyme
catalysed reaction as previously reported.9 For the Suzuki–
Miyaura reaction of halotryptophans 1 and 2 in water, we initially
employed a catalytic system composed of the water-soluble Pd
source Na2Cl4Pd and the water-soluble phosphine ligand TPPTS
(tris(3-sulfonatophenyl)phosphine trisodium salt).11 Similar condi-
tions have previously been used for the Suzuki–Miyaura coupling
of other sensitive, water-soluble biomolecules such as nucleo-
tides7b,12 and sugar–nucleotides.13 Cross-coupling reactions were
typically carried out by adding an aqueous solution of the
halotryptophan hydrochloride salt to a degassed mixture of Pd
source, ligand, K2CO3 and boronic acid (Scheme 1 and Table 1).
The reaction mixture was stirred under nitrogen at 80 1C until
TLC indicated completion of the cross-coupling.
Scheme 1 Suzuki–Miyaura cross-coupling of halogenated trypto-
phans. For substituents X and R, synthetic details and yields see
Tables 1 and 2.
a Department of Chemistry, School of Chemical Sciences and
Pharmacy, University of East Anglia, Norwich, UK NR4 7TJ.
E-mail: r.goss@uea.ac.uk; Fax: +44 (0)1603 592003;
Tel: +44 (0)1603 593766
Pleasingly, under these conditions the cross-coupling of un-
protected 5-bromotryptophan 1a with phenylboronic acid gave
the corresponding 5-phenyltryptophan 3a in high yield (Table 1,
entry 1). To test the scope of these conditions, we next used a
number of phenylboronic acid derivatives as cross-coupling part-
ners. Phenylboronic acid derivatives with electron-donating sub-
stituents could be cross-coupled very efficiently (entries 2 and 3),
b Department of Pharmacy, School of Chemical Sciences and
Pharmacy, University of East Anglia, Norwich, UK NR4 7TJ.
E-mail: g.wagner@uea.ac.uk
w Electronic supplementary information (ESI) available: Experimental
procedures, 1H and 13C NMR spectra and HRMS data for all
compounds, fluorescence spectra, and selected UV absorbance spec-
tra. See DOI: 10.1039/b807512c
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while, unsurprisingly, lower yields were obtained from cross-
coupling reactions with electron-poor boronic acids (entries 4
and 5). During the cross-coupling with 4-ethoxycarbonyl phenyl-
boronic acid (entry 4) the ethyl ester was cleaved, and the
corresponding free acid 3d was obtained as the sole product.
Remarkably, the cross-coupling was also successful with less
reactive halotryptophans. Both 5-chlorotryptophan 1b and
7-bromotryptophan 2 were successfully cross-coupled to phenyl-
boronic acid, although the yields from these reactions were
moderate to low (entries 6 and 7). All cross-coupling products
were purified by RP-HPLC and characterised by 1H and 13C
NMR spectroscopy and HRMS.w
Table 2 Suzuki–Miyaura coupling of halotryptophans 1a and 2 with
phenylboronic acid, using TXPTS as the liganda
Reaction
T/1C time/h
Yield
(%)b
Entry Substrate
X
Product
1
2
3
a
1a
1a
2
5-Br
5-Br
7-Br
3a
3a
4
80
40
80
4
22
6
90
70
88
See Scheme 1 for structures; general conditions: phenylboronic acid
(1 equiv.), Na2Cl4Pd (2.5 mol%), TXPTS (10.0 mol%), K2CO3
b
(5 equiv.), H2O. Isolated yields.
With the view towards the potential application of this
methodology to the modification of peptides and proteins,
our next goal was to reduce the reaction temperature for the
cross-coupling of halotryptophans. However, when the reac-
tion of 1a with phenylboronic acid was carried out at lower
temperature (60 1C or below), with TPPTS as the ligand, only
trace amounts of the cross-coupling product 3a were isolated
after 3 h. Even after a prolonged reaction time of 24 h, no
further reaction progress was observed. We therefore set out to
identify a more reactive catalytic system, which we speculated
might also give improved yields for the more challenging
cross-coupling reactions (e.g. entry 7, Table 1).
Scheme 2 Reagents and conditions: (i) N-Boc-L-Ala, EDCꢁHCl (EDC
= N-(dimethylaminopropyl)-N0-ethylcarbodiimide), CH2Cl2, rt, 24 h;
(ii) Na2Cl4Pd (2.5 mol%), TXPTS (10.0 equiv.), K2CO3, phenylboro-
nic acid, H2O, 80 1C, 6 h.
ditions. The resulting halogenated dipeptide 5 was then used as
the substrate for the Suzuki–Miyaura coupling with phenylboro-
nic acid at 80 1C for 6 h, using TXPTS as the ligand. Under these
conditions, the successful cross-coupling reaction was accompa-
nied by cleavage of the methyl ester, but not of the Boc protecting
group. The partially deprotected cross-coupling product 6 was
purified by reverse phase HPLC and isolated in 62% yield.
The installation of a phenyl substituent in position 5 or 7
resulted in a pronounced change in the fluorescence of the
tryptophan derivatives. The alteration of the spectrophotometric
properties of the molecule is particularly interesting as these
novel, non-natural amino acids may be useful for the site-specific,
fluorescent labelling of peptides and proteins. We therefore
systematically investigated the UV absorbance and fluorescence
properties of selected 5- and 7-aryltryptophans 3 and 4.
The water-soluble phosphine ligand TXPTS (tris(4,6-dimethyl-
3-sulfonatophenyl)phosphine trisodium salt) has previously been
used for the Suzuki–Miyaura coupling of nucleosides at room
temperature.11 TXPTS was prepared in two steps as previously
described14 and used as the ligand in the cross-coupling of
5-bromotryptophan 1a with phenylboronic acid (Table 2). At
80 1C, use of TXPTS increased the yield of 5-phenyltryptophan
3a isolated from this reaction to 90% (entry 1).w Gratifyingly, use
of TXPTS also allowed us to reduce the reaction temperature for
this conversion significantly, to temperatures at which proteins
could be expected to remain in their native conformation. At
40 1C, the TXPTS-assisted cross-coupling of 1a gave 3a in 70%
yield after 22 h (entry 2). Finally, in the cross-coupling of
7-bromotryptophan 2 with phenylboronic acid, use of TXPTS
resulted in an increase in the yield of 7-phenyltryptophan 4 from
15% (with TPPTS) to 88% (Table 2, entry 3).
In order to determine suitable fluorescence excitation
wavelengths (lex), UV absorbance spectra were recorded in
methanol for 5- and 7-aryltryptophans as well as 5-bromo-
tryptophan 1a and the parent compound tryptophan. The
spectra of the 5-aryltryptophan derivatives showed only a
single absorbance maximum at 254 nm, while a secondary
maximum at 280 nm was observed, as expected, in the case of
tryptophan and 5-bromotryptophan.w Next, the fluorescence
emission of individual tryptophan derivatives was measured in
different solvents. While 5-bromotryptophan 1a was only
weakly fluorescent, in methanol all aryl tryptophans that were
examined were strong fluorescence emitters (Fig. 1).
In order to assess the applicability of the optimised cross-
coupling conditions to the modification of small peptides, we
prepared a dipeptide containing 5-bromotryptophan (Scheme 2).
The methyl ester of 5-bromotryptophan 1a was coupled with
N-Boc-protected L-alanine under standard peptide coupling con-
Table 1 Suzuki–Miyaura coupling of halotryptophans with various
arylboronic acids R–B(OH)2, using TPPTS as the liganda
Entry Substrate
X
Product
R
Yield (%)b
1
2
3
4
5
6
7
a
1a
1a
1a
1a
1a
1b
2
5-Br 3a
5-Br 3b
5-Br 3c
5-Br 3d
5-Br 3e
5-Cl 3a
5-Phenyl
5-[4-(H3C)C6H4]c
5-[4-(H3CO)C6H4]d 87
5-[4-(CO2H)C6H4
5-[4-(CF3)C6H4]
5-Phenyl
78
90
Importantly, the fluorescence characteristics of the aryltryp-
tophans can be modulated by the nature and position of the
aromatic substituent. 5-Phenyltryptophan 3a showed a stron-
ger emission signal and a considerably larger Stokes shift than
the parent tryptophan (Table 3). Fluorescence intensity
increased further with installation of the electron-rich
4-methoxyphenyl substituent (3c), while the presence of the
electron-withdrawing carboxy group in 3d resulted in the
68
35
40
15
7-Br
4
7-Phenyl
See Scheme 1 for structures; general conditions: arylboronic acid
(1 equiv.), Na2Cl4Pd (2.5 mol%), TPPTS (2.5 mol%), K2CO3
b
c
(5 equiv.), H2O, 80 1C, 6 h. Isolated yields. Reaction time: 4 h.
Boronic acid: R = 5-[4-(CO2C2H5)C6H4].
d
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Fig. 2 Fluorescence emission spectra for tryptophan derivatives 3a
and 4 in water (c = 100 mM unless otherwise indicated).
Fig. 1 Fluorescence emission spectra for tryptophan and derivatives
3a, 3c, 3d and 4 in methanol (3a, 3d, 4: c = 100 mM; 3c: c = 10 mM).
fully to the modification of a dipeptide, potentially opening the
door towards the direct cross-coupling of larger peptides and even
proteins. The cross-coupling products described herein possess
interesting fluorescent properties. Individual analogues showed
strong fluorescence emission in water and at lex 295 nm, and may
therefore be particularly useful for the site-selective, fluores-
cent labelling of peptides, proteins and natural products.
Financial support by the Leverhulme Trust (F/00 204/AF,
to RJMG), BBSRC (BBE0089841) and the EPSRC (First
Grant EP/D059186/1, to GKW) is gratefully acknowledged.
We thank Thomas Pesnot for the synthesis of TXPTS, and the
EPSRC National Mass Spectrometry Service Centre,
Swansea, for the recording of mass spectra.
Table 3 Fluorescence spectroscopic properties of tryptophan analogues
Compound
Solvent
lex/nm
lem/nm
Stokes shifta/cmꢂ1
Trp
3a
3a
3c
3c
3d
3d
4
MeOH
MeOH
Water
MeOH
Water
MeOH
Water
MeOH
Water
280
254
254
254
254
254
254
254
254
348
370
395
353
357
411
443
375
384
6978
12 343
14 054
11 041
11 359
15 039
16 797
12 703
13 328
4
a
Stokes shift = (1/lex ꢂ 1/lem).
largest Stokes shift observed in this series. Interestingly,
7-phenyltryptophan 4 was less strongly fluorescent than its
regioisomer 3a, with a fluorescence intensity comparable to
tryptophan itself but, once again, a larger Stokes shift.
Notes and references
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With a view towards potential biological applications, we
also measured the fluorescence of selected aryl tryptophans
in water and at lex 4 254 nm, in order to minimise, in a
biological context, concomitant excitation of standard organic
fragments (Fig. S1–S3w).w Pleasingly, tryptophan derivatives
3a, 3c and 4 were strongly fluorescent in water at an excitation
wavelength of 254 nm (Fig. S1w). Compared to measurements
in methanol, all analogues showed moderately or significantly
increased Stokes shifts in water (Table 3). Importantly, at lex
295 nm most aryltryptophans were still significantly more
fluorescent than the parent tryptophan (Fig. S2 and S3w).
While the 5-phenyltryptophan derivatives 3a and 3c exhibited
a reduced fluorescence intensity at lex 295 nm, the intensity of
the fluorescence signal of 7-phenyltryptophan 4 did increase
slightly with the longer excitation wavelength (Fig. 2), making
this analogue particularly interesting for further development.
Taken together, these results show that the fluorescence of
aryltryptophans can be modulated by the choice of substituent
and regiochemistry. This flexibility will allow the design of
tryptophan derivatives with spectrophotometric properties
tailored specifically to individual target peptides and proteins.
In conclusion, we have identified suitable conditions for the
Suzuki–Miyaura cross-coupling of unprotected 5- and 7-halo-
tryptophans with a variety of boronic acids. All cross-coupling
reactions were carried out in water, with generally short reaction
times. Importantly, these conditions could also be applied success-
5 N. Lindquist, W. Fenical, C. D. Van Duyne and J. Clardy, J. Am.
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