One-pot access to L-5,6-dihalotryptophans and L-alknyltryptophans using tryptophan synthase

Article abstract of DOI:10.1016/j.tet.2016.02.016

We report, for the first time, the use of tryptophan synthase in the generation of L-dihalotryptophans and L-alkynyltryptophans. These previously unpublished compounds will be useful tools in the generation of probes for chemical biology, in biosynthetic diversification and as convenient building blocks for synthesis.

Full text of DOI:10.1016/j.tet.2016.02.016

Accepted Manuscript
One-Pot Access to L-5,6-Dihalotryptophans and L-Alknyltryptophans Using
Tryptophan Synthase
Michael J. Corr, Duncan R.M. Smith, Rebecca J.M. Goss
PII:
S0040-4020(16)30079-5
10.1016/j.tet.2016.02.016
TET 27486
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 17 November 2015
Revised Date: 11 January 2016
Accepted Date: 4 February 2016
Please cite this article as: Corr MJ, Smith DRM, Goss RJM, One-Pot Access to L-5,6-Dihalotryptophans
and L-Alknyltryptophans Using Tryptophan Synthase, Tetrahedron (2016), doi: 10.1016/
j.tet.2016.02.016.
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One-Pot Access to
L-5, 6-Dihalotryptophans
Leave this area blank for abstract info.
and -Alkynyltryptophans Using
L
Tryptophan Synthase
Michael J. Corr, Duncan R. M. Smith and Rebecca J. M. Goss*

1
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Tetrahedron Letters
journal homepage: www.elsevier.com
One-Pot Access to
L
-5,6-Dihalotryptophans and -Alknyltryptophans Using
L
Tryptophan Synthase
Michael J. Corra,b, Duncan R. M. Smitha,b and Rebecca J. M. Gossa
a
School of Chemistry, University of St Andrews, St Andrews, KY16 9ST, UK
bBoth authors contributed equally to the manuscript.
ARTICLE INFO
ABSTRACT
Article history:
Received
We report, for the first time, the use of tryptophan synthase in the generation of
dihalotryptophans and L-alkynyltryptophans. These previously unpublished compounds will be
L
-
Received in revised form
Accepted
useful tools in the generation of probes for chemical biology, in biosynthetic diversification and
as convenient building blocks for synthesis.
Available online
2009 Elsevier Ltd. All rights reserved
.
Keywords:
Biotransformation
Dihalotryptophan
Alkynyltryptophan
Tryptophan synthase
1. Introduction
enabling the biotransformation of serine 2 and a substituted
indole to give the corresponding substituted tryptophan (Scheme
1). We have previously shown the utility of tryptophan synthase
to convert haloindoles and nitroindoles to the corresponding
tryptophans,9-11 and have even used this approach to access
sterically demanding 7-iodotryptophan.11 Here, we report for the
first time, the generation of 5,6-dihalotryptophans and
Tryptophan is an essential amino acid and a synthetic and
biosynthetic precursor for many natural products of alkaloid and
non-ribosomal peptide origin. These include the anticancer agent
staurosporine1 and diazonamide A,2 indole alkaloids such as
vindoline3 and vinblastine,3 and antibiotics such as daptamycin4
and the calcium-dependent antibiotic (CDA).5 Furthermore,
tryptophans are essential to the structure and function of proteins
and, though present in proteins at only 1.2% frequency,
tryptophan is largely attributed to conferring UV absorbance and
fluorescence in these biomolecules.6 The ability to functionalise
tryptophan is attractive as it provides a handle for selectively
tuning the properties of the free amino acid or the amino acid as a
component of a natural product or a peptide. We have previously
demonstrated a new paradigm in natural product analogue
generation, Genochemetics,7 in which gene installation enables
the biosynthetic introduction of a chemically orthogonal handle
into
a
natural product, enabling selective synthetic
diversification. Specifically, we demonstrated that we could
engineer the production of the antibiotic pacidamycin bearing a
7-chlorotryptophan, the halogen handle enabled synthetic
diversification through Suzuki-Miyaura cross coupling chemistry
to produce a new array of natural product analogues.7
The requirement for halotryptophans has stimulated many
synthetic studies. Since the first chemical synthesis of 7-
chlorotryptophan reported by Rydon and Tweddle,8 despite many
advances, the best methods currently available are multi-step,
lack generality or require specialised procedures. The use of the
Scheme 1: Mechanism of tryptophan synthase showing how it may be
harnessed to enable access to analogues such as L-7-alkynyltryptophan 7
enzyme tryptophan synthase represents a convenient alternative,
———
Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: author@university.eduL

2
Tetrahedron
alkynyltryptophans using tryptophan synthase.
knowledge, the synthesis of alkynyl tryptophans 7 and 13 have
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not previously been reported.
2. Tryptophan Synthase
Naturally, the α-subunit of tryptophan synthase converts
indole-3-glycerolphosphate to indole, with the loss of
glyeraldehyde-3-phosphate, the β-subunit then utilizes this
indole along with L-serine 2 and converts it to L-tryptophan, via
the replacement reaction outlined in scheme 1, using pyridoxal
phosphate (PLP) as a co-factor. When substituted indoles are
provided exogenously, the α-subunit is bypassed and the β-
subunit can be used to convert the indoles to the corresponding
substituted-tryptophans.
To further explore extension of substrate scope, we turned our
attention to di-halotryptophans (Scheme 2). We subjected
commercially-available 5,6-difluoroindole 8 to the tryptophan
synthase reaction and were pleased to see the reaction proceed to
give the corresponding 5,6-difluoro-L-tryptophan 9 in 52% yield
(an average of 3 isolated yields) on a 2 mmol scale. The
unreacted starting indole from the reaction could be recovered
and subjected to additional rounds of reaction with tryptophan
synthase. After 3 rounds of biotransformation with tryptophan
synthase, 5,6-difluoro-L-tryptophan 9 could be isolated in a
synthetically useful 71% yield on a 2 mmol scale.
3. Conclusions
We have expanded the scope of the tryptophan synthase reaction
previously reported9-11 to include the synthesis of di-
halotryptophans and shown the first examples of the enzyme
carrying out the biotransformation of alkyne-containing indoles.
Although the conversion of 5-ethynylindole 13 was poor, 7-
ethynylindole 7 underwent surprisingly high conversion to the
corresponding tryptophan. Perhaps reengineering of the enzyme
might enable useful one step access to other terminal alkyne-
containing tryptophans. These new compounds show that
tryptophan synthase can be used for the production of
synthetically useful building blocks for subsequent chemical
diversification or as useful platforms for precursor directed
biosynthesis.
4. Experimental
4.1. General
All reagents were purchased from commercial suppliers and were
used without further purification unless otherwise stated.
With commercially-available and sterically more demanding 5,6-
dichloroindole 10, the reaction proved more difficult than for the
difluoro-derivative. 5,6-Dichloro-
L-tryptophan 11 was isolated in
Proton NMR (1H), fluorine NMR (19F{1H}) and carbon NMR
(13C) were recorded on a Bruker Advance 500 (500 MHz) or a
Bruker Avance II (400 MHz). Using a deptq sequence or an
HSQC experiment with multiplicity editing, the 13C NMR signals
were assigned to CH3, CH2, CH and C. The NMR experiments
were carried out in deuterochloroform (CDCl3), deuterated
methanol (CD3OD), deuterated water (D2O) or deuterated DMSO
(d6-DMSO). The chemical shifts (δ) are quoted in parts per
million (ppm). Multiplicities are abbreviated as s, singlet; d,
7% yield (average of 4 isolated yields) after 2 mmol scale
reaction with tryptophan synthase. Again, recycling of the
unreacted starting material for 3 rounds with tryptophan synthase
gave 25% isolated yield of 11. With the dihalotryptophans in
hand, we decided to explore the possibility of using this reaction
to process an alkynyl indole (Scheme 3). Incorporation of an
alkyne “tag” into a small molecule, protein or natural product can
enable selective diversification and tagging,12 through an alkyne-
azide cycloaddition reaction, commonly known as “click
chemistry”.13
1
doublet; t, triplet; q, quartet; m, multiplet; b, broad for the H
NMR, 19F{1H} NMR and 13C NMR spectra. Coupling constants
are reported in Hertz (Hz).
We wanted to see if alkyne-substituted indoles were amenable to
the tryptophan synthase reaction. 5-Ethynylindole 12 was
synthesised from 5-iodoindole following literature procedure.14
When substrate 12 was subjected to the tryptophan synthase
reaction, alkyne-tryptophan 13 was isolated in low 2% yield
(average of 3 isolated yields) on a 2 mmol scale.
High and low resolution mass spectra were recorded at the
EPSRC National Mass Spectrometry Service, Swansea or at the
University of St Andrews on a Waters Micromass LCT time of
flight mass spectrometer coupled to a Waters 2975 HPLC system
or on an Orbitrap ELOS pro.
Flash chromatography was performed using Davisil silica gel
LC60A (40-63 micron). Thin layer chromatography (TLC) was
performed using aluminium sheets of silica gel 60 F254 and was
visualised under a Mineralight model UVGL-58 lamp (254 nm).
The plates were developed with acidic methanolic vanillin
solutions, ethanolic phosphomolybdic acid solutions or basic
potassium permanganate solutions.
In an analogous manner to 12, 7-ethynylindole 4 was synthesized
from 7-iodoindole. When subjected to the tryptophan synthase
reaction on a small scale (0.9 mmol), the reaction worked with a
much greater efficiency than for the 5-substituted indole and
expected product 7 was isolated in 90% yield. We were unable to
carry out the biotransformations on a larger scale and in triplicate
due to limited availability of the starting 7-iodoindole. To our

3
Purification of the ethynyltryptophans was carried out on a
Biotage Isolera Four using reverse-phase SNAP C18 12 g
column cartridges. The purification was carried out using
water/methanol on the following gradient: 12 mL/min elution,
2% methanol/water for 1.15 min, 2ꢀ15% methanol/water for
1.15 min, 15% methanol/water for 5.15 min, 15ꢀ95%
methanol/water for 15 min, 95% methanol/water for 3.45 min.
The collection wavelength was set a 254 nm. Collection of the
peaks occurring from approx. 4.3 min to 12.0 min gave the
resulting ethynyltryptophans.
J(H,H)= 15.5, 7.1 Hz, CHAHB), 3.28 (1H, dd, J(H,H)= 15.3, 5.0
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Hz, CHAHB), 4.13 (1H, dd, J(H,H)= 7.2, 5.3 Hz, CH), 7.18 (1H,
s, ArH), 7.44 (1H, s, ArH), 7.57 (1H, s, ArH); 13C NMR (125
MHz, D2O) δ = 25.7 (CH2), 53.5 (CH), 106.3 (C), 113.0 (CH),
118.9 (CH), 122.4 (C), 124.7 (C), 126.3 (C), 127.1 (CH), 135.0
+
(C), 172.3 (CO); HRMS (ESI): m/z calcd for C11H11Cl2N2O2
[M+H]+: 273.0192; found: 273.0190.
The reaction was repeated three more times to give
L-5,6-
dichlorotryptophan 11 (68 mg, 12%), (30 mg, 5%) and (26 mg,
5%). Over four runs, this gave an average yield of 7%.
Freeze drying was carried out on a Scanvac CoolSafeTM freeze
dryer.
4.3. tert-Butyl 5-iodo-1H-indole-1-carboxylate
Dialysis tubing cellulose membrane (average flat width 33 mm)
was purchased from Sigma Aldrich. An approximately 20 cm
length of tubing was immersed in water prior to use. One end
was tied in a knot. The thawed cell lysate was inserted into the
tied tubing using a pipette. Excess air was removed from inside
the tubing and the other end was tied to give a cylinder of
membrane sealed at both ends. The off-cuts of the knots were
removed to give a tube length of approximately 5 cm for use in
the reaction.
Di-tert-butyl dicarbonate (3.4 mL, 14.8 mmol, 1.2 eq) was added
to a solution of 5-iodoindole (3 g, 12.3 mmol, 1.0 eq) and 4-
DMAP (0.3 g, 2.5 mmol, 0.2 eq) in DCM (100 mL) at r.t. under
nitrogen and stirred for 2.5 h. The solvent was removed in vacuo.
Purification by column chromatography using silica gel (1:19
ethyl acetate:hexane) gave tert-butyl 5-iodo-1H-indole-1-
1
carboxylate (4.2 g, 100%) as a white solid; H NMR (500 MHz,
CDCl3) δ = 1.68 (9H, s, CH3), 6.50 (1H, dd, J(H,H)= 3.7, 0.6 Hz,
ArH), 7.56 (1H, d, J(H,H)= 3.7Hz, ArH), 7.58 (1H, dd, J(H,H)=
8.7, 1.8 Hz, ArH), 7.90 (1H, d, J(H,H)= 1.8 Hz, ArH), 7.93 (1H,
bd, J(H,H)= 8.7 Hz, ArH); 13C NMR (125 MHz, CDCl3) δ = 28.3
(CH3), 84.3 (C), 86.8 (C), 106.4 (CH), 117.2 (CH), 126.8 (CH),
129.9 (CH), 132.8 (CH), 133.0 (C), 134.7 (C), 149.6 (CO).
4.1.
L-5,6-Difluorotryptophan 9
L-Serine 2 (0.262 g, 2.5 mmol, 1.25 eq) was dissolved in KH2PO4
reaction buffer (0.1 M, pH 7.8, 95 mL). A solution of 5,6-
difluoro-1H-indole 8 (0.306 g, 2.0 mmol, 1.0 eq) in methanol (5
mL) was added to the flask containing the reaction buffer with
vigorous stirring. Tryptophan synthase cell lysate was thawed in
a 37 °C water bath, then stored at 0 °C. The cell lysate (3 mL)
was stored in a cellulose dialysis bag and added to the reaction
flask. The biotransformation was carried out at 37 °C, shaking at
180 rpm for 2 days. The aqueous layer was collected and washed
with ethyl acetate (2 x 50 mL). The aqueous layer was
concentrated to half its volume, then purified on C18 silica. The
aqueous layer was loaded onto 30 g C18 silica, which was then
washed with 200 mL water before elution of the product in
methanol. The methanolic fractions were collected and the
solvent was removed in vacuo. The residue was dissolved in
4.4. 5-Ethynyl-1H-indole 12
Ethynyltrimethylsilane (2.6 mL, 18.5 mmol, 1.5 eq) was added to
a solution of bis(triphenylphosphine)palladium (II) dichloride
(216 mg, 0.3 mmol, 2.5 mol%), copper (I) iodide (117 mg, 0.6
mmol, 5 mol%) and tert-butyl 5-iodo-1H-indole-1-carboxylate
(4.22 g, 12.3 mmol, 1.0 eq) in triethylamine (100 mL) at r.t under
nitrogen and stirred for 18 h. The reaction mixture was filtered
through celite and the solvent was removed in vacuo. The residue
was dissolved in methanol (100 mL) and potassium carbonate
(3.4 g, 24.6 mmol, 2.0 eq) was added. The reaction was stirred at
r.t. for 4 h. The reaction mixture was diluted with water (200 mL)
and extracted with diethyl ether (2 x 200 mL). The combined
organic layers were dried over anhydrous sodium sulfate and the
solvent removed in vacuo. Purification by column
chromatography using silica gel (1:19 ethyl acetate:hexane) gave
5-ethynyl-1H-indole 12 (1.04 g, 60% over 2 steps) as a pale
water (50 mL) and lyophilised to give L-5,6-difluorotryptophan 8
(245 mg, 51%) as a white solid; [α]D -12.4° (c 0.51, MeOH); 1H
NMR (500 MHz, D2O) δ = 3.23 (1H, dd, J(H,H)= 15.5, 7.1 Hz,
CHAHB), 3.28 (1H, dd, J(H,H)= 15.5, 5.4 Hz, CHAHB), 4.18 (1H,
dd, J(H,H)= 6.9, 5.5 Hz, CH), 7.19 (2H, overlapping m, ArH),
7.28 (1H, dd, J(H,F)= 11.2, 7.7 Hz, ArH); 13C NMR (125 MHz,
D2O) δ = 25.6 (CH2), 53.3 (CH), 99.5 (d, J(C,F)= 21.7 Hz, CH),
104.4 (d, J(C,F)= 19.5 Hz, CH), 106.6 (d, J(C,F)= 5.6 Hz, C),
121.7 (d, J(C,F)= 8.1 Hz, C) 126.4 (d, J(C,F)= 3.5 Hz, CH),
131.2 (d, J(C,F)= 10.9 Hz, C), 145.8, (dd, J(C,F)= 198.6, 15.0
Hz, CF), 147.6 (dd, J(C,F)= 200.0, 15.3 Hz, C), 172.0 (CO);
19F{1H} NMR (470 MHz, D2O) δ = -148.4 (d, J(F,F) = 21.1 Hz),
-144.9 (d, J(F,F) - 21.1 Hz); HRMS (ESI): m/z calcd for
C11H11F2N2O2+ [M+H]+: 241.0783; found 241.0782.
1
brown oil; H NMR (500 MHz, CDCl3) δ = 3.01 (1H, s, CH),
6.56 (1H, dd, J(H,H)= 3.2, 2.0 Hz, ArH), 7.25 (1H, dd, J(H,H)=
3.2, 2.4 Hz, ArH), 7.34-7.35 (2H, m, ArH), 7.84-7.85 (1H, m,
ArH), 8.24 (1H, bs, NH); 13C NMR (125 MHz, CDCl3) δ = 74.8
(CH), 85.5 (C), 103.1 (CH), 111.3 (CH), 113.4 (C), 125.4 (CH),
125.5 (CH), 126.2 (CH), 127.8 (C), 135.8 (C); HRMS (EI): m/z
calcd for C10H7N1+• [M]+•: 141.0578; found: 141.0583.
4.5.
L-5-Ethynyltryptophan 13
The reaction was repeated two more times to give
difluorotryptophan 9 (307 mg, 55%) and (233 mg, 49%). Over
three runs, this gave an average yield of 52%.
L-5,6-
L
-Serine 2 (0.262 g, 2.5 mmol, 1.25 eq) was dissolved in KH2PO4
reaction buffer (0.1 M, pH 7.8, 95 mL). A solution of 5-ethynyl-
1H-indole 12 (0.282 g, 2.0 mmol, 1.0 eq) in acetonitrile (5 mL)
was added to the flask containing the reaction buffer with
vigorous stirring. Tryptophan synthase cell lysate was thawed in
a 37 °C water bath, then stored at 0 °C. The cell lysate (3 mL)
was stored in a cellulose dialysis bag and added to the reaction
flask. The biotransformation was carried out at 37 °C, shaking at
180 rpm for 2 days. The aqueous layer was collected and washed
with ethyl acetate (2 x 50 mL). The aqueous layer was
4.2. L-5,6-Dichlorotryptophan 11
Using the biotransformation procedure reported above for 9, 5,6-
dichloro-1H-indole 10 (0.372 g, 2.0 mmol, 1.0 eq) gave 5,6-
dichlorotryptophan 11 (33 mg, 6%) as a white solid; [α]D -14.7°
(c 0.53, MeOH); 1H NMR (500 MHz, D2O) δ = 3.21 (1H, dd,

4
Tetrahedron
concentrated to half its volume, then purified using a Biotage
138.9 (C), 177.5 (CO); MS (ESI) 229 (100) [M+H]+; HRMS: m/z
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column. The aqueous layer was loaded onto a SNAP C18 12 g
cartridge, then 200 mL water was eluted from the cartridge
before loading onto the Biotage Isolera 4 for the purification
gradient. The fractions were collected and the solvent was
removed in vacuo. The residue was dissolved in water (50 mL)
calcd for C13H13N2O2 [M+H]+: 229.0972; found: 229.0969.
L-7-Ethynyltryptophan 7 exhibited some unusual NMR behavior;
When the 1H NMR spectrum was recorded in CD3OD, the signal
for the alkyne proton at δH 3.74 ppm gave a decreased integral to
what was expected (0.2 rather than 1.0). Increasing the relaxation
time of the NMR spectrum (from 1 s to 4 s) did not increase the
intensity of the signal as may have been expected, but removed
the signal for the alkyne proton entirely. When the 13C NMR was
recorded, the alkyne signals were visible at δC 81.21 and 82.23
ppm, along with a new, broader peak at δC 80.76 ppm. When the
spectrum was recorded in d6-DMSO, the alkyne proton signal
was apparent at δH 4.41 ppm and had no decrease in the NMR
integration, relative to the aromatic protons. However, when the
13C NMR was recorded in d6-DMSO, two alkyne signals were
observed (at δC 80.82 and 84.09 ppm), but this time, the carbon
signals for the tryptophan side chain has disappeared. We are
and lyophilised to give L-5-ethynyltryptophan 13 (8.3 mg, 1.8%)
as a white solid; [α]D +64.7° (c 0.37, MeOH); 1H NMR (500
MHz, CD3OD) δ = 3.13 (1H, dd, J(H,H)= 15.1, 9.0 Hz, CHAHB),
3.26 (1H, s, CH), 3.44-3.48 (1H, m, CHAHB), 3.81 (1H, dd,
J(H,H)= 9.0, 4.2 Hz, CH), 7.22 (1H, dd, J(H,H)= 8.5, 1.5 Hz,
ArH), 7.24 (1H, s, ArH), 7.32 (1H, dd, J(H,H)= 8.4, 0.5 Hz,
ArH), 7.91 (1H, bs, ArH); 13C NMR (125 MHz, CD3OD) δ =
28.6 (CH2), 56.8 (CH), 75.6 (CH), 86.4 (C), 110.2 (C), 112.5
(CH), 114.1 (C), 124.0 (CH), 126.4 (CH), 126.5 (CH), 128.4 (C),
138.2 (C), 151.0 (CO); MS (ESI) 229 (100) [M+H]+, 212 (90);
HRMS: m/z calcd for C13H13N2O2 [M+H]+: 229.0972; found:
229.0970.
unsure what the cause of this unusual NMR behaviour. L-5-
ethynyltryptophan 13 exhibited no such behaviour in the NMR
spectra.
The reaction was repeated two more times to give
ethynyltryptophan 13 (5.9 mg, 1.3%) and (7.2 mg, 1.6%). Over
three runs, this gave an average yield of 1.6%.
L-5-
4.6. tert-Butyl 7-iodo-1H-indole-1-carboxylate
Enantiopurity analysis
The enantiopurity of the generated tryptophan derivatives was
analysed using Marfey’s Reagent (1-fluoro-2,4-dinitrophenyl-5-
Following the procedure reported above, 7-iodoindole was
converted to tert-butyl 7-iodo-1H-indole-1-carboxylate; 1H NMR
(500 MHz, CDCl3) δ = 1.68 (9H, s, CH3), 6.54 (1H, d, J(H,H)=
3.7 Hz, ArH), 6.95 (1H, dd, J(H,H)= 7.7, 7.7 Hz, ArH), 7.51 (1H,
d, J(H,H)= 3.7 Hz, ArH), 7.56 (1H, dd, J(H,H)= 7.7, 0.9 Hz,
ArH), 7.85 (1H, dd, J(H,H)= 7.7, 0.8 Hz, ArH); 13C NMR (125
MHz, CDCl3) δ = 28.3 (CH3), 78.6 (C), 84.4 (C), 107.1 (CH),
121.1 (CH), 124.6 (CH), 129.3 (CH), 133.8 (C), 137.2 (CH),
L-alanine amide, FDAA).
The tryptophan was dissolved in HCl solution (1M, 10 mg/mL).
A vial containing the tryptophan (50 µL), FDAA (1% w/v in
actone, 100 µL) and NaHCO3 solution (1M, 70 µL) was mixed
thoroughly and incubated at 37 °C for 1 h. The reaction mixture
was diluted (10 µL in 490 µL water) and centrifuged (13,000
rpm, 5 min) before UPLC analysis.
+
137.7 (C), 148.3 (CO); HRMS (ESI): m/z calcd for C13H18I1N2O2
[M+NH4]+: 361.0407; found: 361.0408.
The UPLC analysis was carried out in acetonitrile/0.1% TFA in
water at a gradient of 20% acetonitrile to 90% acetonitrile over
4.7 minutes. A 3:1 mixture of L-tryptophan to D-tryptophan was
4.7. 7-Ethynyl-1H-indole 4
used as a standard. Two blank runs using acetone were conducted
between each tryptophan sample. In all of the tryptophans
Following the procedure reported above, tert-butyl 7-iodo-1H-
indole-1-carboxylate was converted to 7-ethynyl-1H-indole 4
(0.15 g, 70%) as a brown oil; H NMR (500 MHz, CDCl3) δ =
3.42 (1H, s, CH), 6.62 (1H, dd, J(H,H)= 3.1, 2.2 Hz, ArH), 7.12
(1H, dd, J(H,H)= 7.9, 7.3 Hz, ArH), 7.27-7.28 (1H, m, ArH),
7.41 (1H, d, J(H,H)= 7.3 Hz, ArH), 7.70 (1H, d, J(H,H)= 7.9 Hz,
ArH), 8.51 (1H, bs, NH); 13C NMR (125 MHz, CDCl3) δ = 80.5
(C), 81.4 (CH), 103.5 (CH), 105.0 (C), 119.9 (CH), 122.3 (CH),
124.7 (CH), 126.1 (CH), 127.6 (C), 137.1 (C); HRMS (EI): m/z
calcd for C10H7N1+• [M]+•: 141.0578; found: 141.0582.
analysed (with the exception of
single peak for the -enantiomer was observed (see supporting
information). In the case of -5-ethynyltryptophan 13,
L-5-ethynyltryptophan 13), only a
1
L
L
decomposition appeared to occur when subjected to the analysis
conditions. Given the similar optical rotations between 7 and 13,
we are confident that no racemization of the stereocentre had
occurred during the biotransformation reaction.
Use of DL-serine
The biotransformation was carried out on indole using either L-
serine (2.5 eq) or DL-serine (2.5 eq). The number of equivalents
of serine was doubled from a normal biotransformation to ensure
4.8. L-7-Ethynyltryptophan 7
Using the procedure reported above for 13, 7-ethynyl-1H-indole
4 (0.128 g, 0.9 mmol, 1.0 eq) gave -7-ethynyltryptophan 7 (0.19
that enough
L-serine was present for the reaction to go to full
L
conversion (for the case of DL-serine). In both reactions,
g, 90%) as a white solid; [α]D +64.2° (c 0.55, MeOH); 1H NMR
(500 MHz, d6-DMSO) δ = 2.83 (1H, bs, CHAHB), 3.20 (1H, bd,
J(H,H)= 13.2 Hz, CHAHB), 3.32 (1H, bs, CH), 4.41 (1H, s, CH),
6.96 (1H, dd, J(H,H)= 7.6, 7.5 Hz, ArH), 7.19-7.20 (2H, m,
ArH), 7.61 (1H, bd, J(H,H)= 7.8 Hz, ArH), 11.03 (1H, bs, NH);
1H NMR (500 MHz, CD3OD) δ = 3.05-3.10 (1H, m, CHAHB),
3.39-3.43 (1H, m, CHAHB), 3.72-3.74 (1H, m, CH), 3.74 (1H, s,
CH), 7.01 (1H, dd, J(H,H)= 7.8, 7.5 Hz, ArH), 7.24 (1H, s, ArH),
7.25 (1H, d, J(H,H)= 7.3 Hz, ArH), 7.75 (1H, dd, J(H,H)= 8.0,
0.8 Hz, ArH); 13C NMR (125 MHz, CD3OD) δ = 30.0 (CH2),
57.1 (CH), 80.8 (C), 81.2 (C), 82.2 (CH), 106.6 (C), 111.6 (C),
119.8 (CH), 120.9 (CH), 125.7 (CH), 126.6 (CH), 128.7 (C),
comparable quantities of
tryptophan (0.120 g, 29%) was isolated from the reaction with
serine,
reaction with DL-serine. Analysis of the enantiopurity of the
tryptophans using Marfey’s reagent showed that in both cases,
only L-typtophan was produced. The use of DL-serine is possible
in the reaction and shows no detriment to yield or enantiopurity.
L
-tryptophan were isolated;
L
-
-
L
L
-tryptophan (0.153 g, 37%) was isolated from the
Acknowledgments
We thank the European Research Council under the European
Union’s Seventh Framework Programme (FP7/2007-2013/ERC
grant agreement no 614779, and the University of St Andrews for

5
5. Hopwood, D. A.; Wright, H. M. J. Gen. Microbiol. 1983, 129,
a studentship (to D. R. M. Smith) We thank the EPSRC National
Mass Spectrometry Service and Mrs Caroline Horsburgh
(University of St Andrews) for mass spectrometry analysis.
ACCEPTED MANUSCRIPT
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Supplementary Material
Supplementary data (1H, 19F{1H} and 13C NMR, as well as
Marfey’s reagent analysis) associated with this article can be
found, in the online version, at xxxx.
References and notes
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