A convenient one-step synthesis of l-aminotryptophans and improved synthesis of 5-fluorotryptophan

Article abstract of DOI:10.1016/j.bmcl.2008.07.053

A one-pot biotransformation for the generation of a series of l-aminotryptophans using a readily prepared protein extract containing tryptophan synthase is reported. The extract exhibits remarkable stability upon freeze-drying, and may be stored and used for long periods after its preparation without significant loss of activity.

Full text of DOI:10.1016/j.bmcl.2008.07.053

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4508–4510
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A convenient one-step synthesis of L-aminotryptophans and improved
synthesis of 5-fluorotryptophan
Michael Winn a, Abhijeet Deb Roy a, Sabine Grüschow a, Raj S. Parameswaran b, Rebecca J. M. Goss a,
*
a School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK
b Bharathiar University, Coimbatore 641 046, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A one-pot biotransformation for the generation of a series of
L-aminotryptophans using a readily pre-
Received 26 June 2008
Revised 11 July 2008
Accepted 13 July 2008
Available online 17 July 2008
pared protein extract containing tryptophan synthase is reported. The extract exhibits remarkable stabil-
ity upon freeze-drying, and may be stored and used for long periods after its preparation without
significant loss of activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Aminotryptophan
Tryptophan synthase
Halotryptophan
Biotransformation
Preparation
Tryptophan is an essential amino acid that plays a crucial role in
the structure and function of proteins. Due to its low natural abun-
dance of approximately 1%, it is ideally suited for replacement with
analogues to probe function or alter enzyme properties. For exam-
ple, incorporation of fluoro- and amino-substituted tryptophans
making designer aminotryptophan-containing natural products
arises. For these reasons, a convenient preparation of amino and
fluorotryptophans is required.
Whilst a wealth of syntheses of halotryptophans are reported in
the literature, there are very few reports of the generation of amin-
otryptophans. Typically, aminotryptophans are prepared from
their corresponding nitroindoles via nitrogramine (Scheme 1).
Reduction of the nitro group is performed either before or after es-
ter hydrolysis. Racemic mixtures of 4-, 5-, 6- and 7-aminotrypto-
phans have been prepared in this manner.10 Using
enantiomerically pure starting material, Moriya et al. were able
into proteins has been used to probe
p
-cation interactions1 or to
study folding pathways.2 The incorporation of an electron-donat-
ing group such as an amine into an indole ring system has a signif-
icant effect upon its electronic and physical properties, which, in
turn, influence the absorption and emission characteristics of tryp-
tophan.3,4 This has been exploited in the modification of green-
fluorescent protein to generate a variant with a significant red-
shifted emission.5
to obtain 6-amino-D-tryptophan from D-tryptophan via nitration
followed by reduction.11 A procedure using tryptophanase has also
been described for the synthesis of 5-aminotryptophan.12 The syn-
In addition to its proteinogenic role, tryptophan is also the pre-
cursor for numerous metabolites in eukaryotes such as plant alka-
loids and the neurotransmitter serotonin.6 Of particular interest
are microbial tryptophan-containing natural products that exhibit
a range of biological activities.7 In the non-ribosomal peptide
gramicidin, fluorotryptophan derivatives have been used to study
the orientation of the antibiotic in the membrane using 19F NMR
and to investigate the effects on ion channel function.8 Interest-
ingly, no natural products containing an aminotryptophan appear
to have been isolated to date. However, precursor-directed biosyn-
thesis and mutasynthesis provide convenient methods for access-
ing novel natural products derivatives.9 The opportunity of
thesis of L-amino-tryptophans using tryptophan synthase has been
alluded to; however, no details have been published.2,3
The enzyme tryptophan synthase can be utilised to access a
range of tryptophan derivatives using serine and indole derivatives
(Scheme 2).13,14 Tryptophan synthase consists of two subunits,
a
and b, and uses pyridoxal phosphate (PLP) as co-factor. The
a sub-
* Corresponding author. Tel.: +44 01603 593 766; fax: +44 01603 592 003.
E-mail address: r.goss@uea.ac.uk (R.J.M. Goss).
Scheme 1. Chemical synthesis of racemic aminotryptophans.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.053

M. Winn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4508–4510
4509
biotransformation mixture. By recycling the indole two times, we
were able to obtain very good overall yields even for poorly con-
verting substrates. Interestingly, the yields for 4- and 7-substituted
methyl or halotryptophans were significantly lower than those for
the corresponding 5- and 6-substituted tryptophans.13 This is the
opposite of our observation for the amino derivatives. At present,
it is unclear whether the observed substituent effects are reflective
of changes in reactivity of the indole or if they are the result of
interactions with the enzyme.
We also wanted to investigate how freeze-drying would affect
the activity of the protein extract. For this purpose, the crude extract
waspreparedasbeforebutPMSFandb-mercaptoethanolwereomit-
ted from the lysis buffer.17 Aliquots were lyophilised immediately
after cell lysis and removal of the cell debris. The freeze-dried mate-
rial was redissolved in the original volume of water and then used as
described above. For up to 2 months of storage at 4 °C, the crude ex-
tract retained all of its activity relative to non-lyophilised extract
stored at À80 °C (Table 1, entries 5–7). Notably, the yields for two
out of three independent experiments were above 80%.
Scheme 2. Tryptophan synthase-mediated conversion of serine and aminoindole to
give aminotryptophan. The reaction cycle starts with enzyme-bound PLP. Serine
replaces the active site lysine and undergoes dehydration to give the aminoacrylate
intermediate. The ensuing b-addition by aminoindole yields co-factor bound
aminotryptophan that is liberated by transamination with the active site lysine.
This method provides a convenient and scalable means of
accessing L-aminotryptophans from commercially available start-
ing materials. The crude protein extract containing tryptophan
synthase is readily prepared and may be freeze-dried for storage.
It is then utilised as a reagent by simply resuspending it in water.
unit provides the indole from indole 3-glycerolphosphate and
channels it through a tunnel to the b subunit where the condensa-
tion between indole and serine occurs.15 The reaction cycle of the b
subunit is shown in Scheme 2. We recently reported a convenient
one-step synthesis of a series of methyl- and halotryptophans util-
ising a readily prepared bacterial protein extract, rather than the
purified enzyme improving the ease of utility of this reaction by or-
ganic chemists.13 We also demonstrated that yields were signifi-
cantly higher when the extract was contained within a dialysis bag.
Escherichia coli pre-transformed with pSTB7, a high copy num-
ber plasmid expressing tryptophan synthase from Salmonella enter-
ica, is commercially available (ATCC 37845).16 The cell lysate was
prepared as previously described.13 Typically, 80 ml protein extract
was obtained from 2 L of a E. coli/pSTB7 overnight culture.17 Ali-
quots of the extract were either stored at À80 °C or lyophilised.
Freeze-dried material was stored at 4 °C and reconstituted, to its
original volume, with water before use. For the biotransformation,
the crude extract containing tryptophan synthase was sealed into a
dialysis bag and introduced into buffered aqueous solution of ser-
ine, the corresponding indole, and the co-factor PLP.18 After three
days of incubation at 37 °C with gentle shaking, indoles were re-
moved by extraction with ethylacetate or ether. The aqueous phase
was concentrated in vacuo and loaded onto C18 reverse-phase silica
gel. Serine and PLP were removed by washing with water, and the
tryptophan derivative was then eluted in methanol.19
Acknowledgments
We gratefully acknowledge financial support by the Lever-
hulme Trust (F/00 204/AF, to S.G.) and BBSRC (BBE0,089,841 to
A.D.R.). We thank the EPSRC National Mass Spectrometry Service
Centre, Swansea, for the recording of mass spectra.
Supplementary data
1H NMR and 13C NMR spectra for aminotryptophans. Supple-
mentary data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2008.07.053.
References and notes
1. Zhong, W.; Gallivan, J. P.; Zhang, Y.; Li, L.; Lester, H. A.; Dougherty, D. A. Proc.
Natl. Acad. Sci. U.S.A. 1998, 9, 12088.
2. Rubini, M.; Lepthien, S.; Golbik, R.; Budisa, N. Biochim. Biophys. Acta 2006, 1764,
1147.
3. Budisa, N.; Rubini, M.; Bae, J. H.; Weyher, E.; Wenger, W.; Golbik, R.; Huber, R.;
Moroder, L. Angew. Chem. Int. Ed. 2002, 41, 4066.
4. Sinha, H. K.; Dogra, S. K.; Krishnamurthy, M. Bull. Chem. Soc. Jpn. 1987, 60, 4401.
5. Bae, J. H.; Rubini, M.; Jung, G.; Wiegand, G.; Seifert, M. H. J.; Azim, M. K.; Kim, J.
S.; Zumbusch, A.; Holak, T. A.; Moroder, L.; Huber, R.; Budisa, N. J. Mol. Biol.
2003, 328, 1071.
6. (a) Dalton, D. R. In The Alkaloids; Marcel Dekker, Inc.: New York, 1979; pp 415–
628; (b) Kempter, C.; Kaiser, D.; Haag, S.; Nicholson, G.; Gnau, V.; Walk, T.;
Gierling, K. H.; Decker, H.; Zahner, H.; Jung, G.; Metzger, J. W. Angew. Chem. Int.
Ed. 1997, 36, 498; (c) Gaddum, J. H.; Giarman, N. J. Br. J. Pharmacol. Chemother.
1956, 11, 88.
Both 4- and 7-aminotryptophans were obtained in excellent
yield (Table 1, entries 1 and 4). Indoles with the amino substituent
in the 5- or 6-position were converted less efficiently (Table 1, en-
tries 2 and 3). However, if large quantities are required, the ex-
tracted indole derivative can be reintroduced into
a fresh
7. (a) Baltz, R. H.; Miao, V.; Wrigley, S. K. Nat. Prod. Rep. 2005, 22, 717; (b) Kaneko,
I.; Kamoshida, K.; Takahashi, S. J. Antibiot. 1989, 42, 236; (c) Nettleton, D. E.;
Doyle, T. W.; Krishnan, B.; Matsumoto, G. K.; Clardy, J. Tetrahedron Lett. 1985,
26, 4011.
Table 1
Yields of tryptophan derivatives obtained through biotransformation
8. (a) Grage, S. L.; Wang, J. F.; Cross, T. A.; Ulrich, A. S. Biophys. J. 2002, 83, 3336; (b)
Andersen, O. S.; Greathouse, D. V.; Providence, L. L.; Becker, M. D.; Koeppe, R. E.
J. Am. Chem. Soc. 1998, 120, 5142.
9. (a) Weissman, K. J. Trends Biotechnol. 2007, 25, 139; (b) Weist, S.; Süssmuth, R.
D. Appl. Microbiol. Biotechnol. 2005, 68, 141.
10. (a) Casini, G.; Goodman, L. Can. J. Chem. 1964, 42, 1235; (b) Melhado, L. L.;
Leonard, N. J. J. Org. Chem. 1983, 48, 5130; (c) Goodman, L.; Spencer, R. R.;
Casini, G.; Crews, O. P.; Reist, E. J. J. Med. Chem. 1965, 8, 251.
11. Moriya, T.; Hagio, K.; Yoneda, N. Bull. Chem. Soc. Jpn. 1975, 48, 2217.
12. Yamada, H.; Kumagai, H. Pure Appl. Chem. 1978, 50, 1117.
13. Goss, R. J. M.; Newill, P. L. A. Chem. Commun. 2006, 4924.
14. Phillips, R. S. Tetrahedron: Asymmetry 2004, 15, 2787.
Entry
Tryptophan
Conditions
Yielda (%)
1
2
3
4
5
6
7
7-Amino
6-Amino
5-Amino
4-Amino
5-Fluoro
5-Fluoro
5-Fluoro
Crude extract
Crude extract
Crude extract
Crude extract
Crude extract
Freeze-dried, 1 day storage
Freeze-dried, 2 months storage
65
35
37
70
83
77 12b
72 19b
a
Yields are relative to theoretical maximum based on the amount of indole used
for the biotransformation.
15. (a) Miles, E. W.; Bauerle, R.; Ahmed, S. A. Methods Enzymol. 1987, 142, 398; (b)
Miles, E. W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991, 64, 93.
b
Yields from three independent experiments with standard deviation.

4510
M. Winn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4508–4510
16. (a) Ruvinov, S. B.; Yang, X. J.; Parris, K. D.; Banik, U.; Ahmed, S. A.; Miles, E. W.;
Sackett, D. L. J. Biol. Chem. 1995, 270, 6357; (b) Miles, E. W.; Kawasaki, H.;
Ahmed, S. A.; Morita, H.; Morita, H.; Nagata, S. J. Biol. Chem. 1989, 264, 6280.
17. Pelleted cells were washed with saturated NaCl, then resuspended in lysis
buffer for cell lysis by sonication. Cell debris was removed by centrifugation.
Lysis buffer: Tris–HCl (0.5 M), b-mercaptoethanol (10 mM), EDTA (5 mM),
PMSF (1 mM), pH 7.8.
75 MHz) d 25.2, 53.6, 104.0, 112.8, 114.3, 118.9, 121.1, 121.5, 126.8, 137.5,
172.5; HRMS (ESI, +ve) m/z 220.1084 [M+H]+, Calcd for C11H14N3O2 m/z
220.1081. 5-Amino-L
-tryptophan: 1H NMR (D2O, 400 MHz) d 3.18–3.35 (m,
2H), 4.05 (t, 1H, J = 6.4 Hz), 7.00 (d, 1H, J = 8.4 Hz), 7.25 (s, 1H), 7.40 (d, 1H,
J = 8.8 Hz), 7.50 (s, 1H); 13C NMR (D2O, 75 MHz) d 24.9, 52.9, 106.5, 112.0,
112.7, 115.9, 121.3, 126.3, 127.0, 135.3, 171.5; HRMS (ESI, +ve) m/z
220.1079 [M+H]+, Calcd for C11H14N3O2 m/z 220.1081. 6-Amino-
L-
18. Per 100 ml potassium phosphate buffer (0.1 M, pH 7.8): serine (0.127 g,
1.21 mmol), aminoindole (0.160 g, 1eq), PLP (0.8 mg), 2 ml protein extract in
dialysis tubing. Ref. 13 gives a detailed procedure.
tryptophan: 1H NMR (D2O, 400 MHz)
d 3.21 (dd, 1H, 7.6, 15.6 Hz), 3.27
(dd, 1H, J = 5.6, 15.6 Hz), 3.99 (t, 1H, J = 6.0 Hz), 6.91 (d, 1H, J = 8.0 Hz), 7.20
(s, 1H), 7.32 (s, 1H), 7.58 (d, 1H, J = 8.4 Hz); 13C NMR (D2O, 75 MHz) d 25.0,
53.0, 106.0, 106.5, 113.5, 119.1, 123.7, 126.3, 126.7, 135; HRMS (ESI, +ve) m/
19. Analytical data: 5-Fluoro-L
-tryptophan hydrochloride: 1H NMR (D2O,
400 MHz) d 3.10–3.15 (dd, 1H, J = 7.2, 15.6 Hz), 3.16–3.22 (dd, 1H, J = 5.6,
15.6 Hz), 4.08 (t, 1H, J = 6.2 Hz), 6.76–6.81 (dt, 1H, J = 2.4, 9.2 Hz), 7.07 (d,
1H, J = 2.4 Hz), 7.10 (s, 1H), 7.18–7.22 (dd, 1H, J = 4.6, 9.0 Hz); MS (EI, +ve)
z L-
220.1079 [M+H]+, Calcd for C11H14N3O2 m/z 220.1081. 7-Amino-
tryptophan: 1H NMR (D2O, 400 MHz)
d 3.22–3.34 (m, 2 H), 4.10 (t, 1H,
J = 5.6 Hz), 7.01 (s, 1H), 7.07 (t, 1H, J = 7.6 Hz), 7.23 (s, 1H), 7.58 (d, 1H,
J = 8.0 Hz); 13C NMR (D2O, 75 MHz) d 24.9, 52.8, 100.1, 107.3, 113.8, 116.1,
118.9, 119.2, 126.2, 128.8, 171.7; HRMS (ESI, +ve) m/z 220.1078 [M+H]+,
Calcd for C11H14N3O2m/z 220.1081.
m/z 222.95 (M+). 4-Amino- -tryptophan: 1H NMR (D2O, 400 MHz) d 3.25 (dd,
L
1H, J = 4.8, 16.4 Hz), 3.37 (dd, 1H, J = 5.6, 16.0 Hz), 4.03 (t, 1H, J = 6.2 Hz),
7.03–7.06 (m, 2H), 7.22 (s, 1H), 7.37 (dd, 1H, J = 0.40, 8.0 Hz); 13C NMR (D2O,