A convenient enzymatic synthesis of L-halotryptophans

Article abstract of DOI:10.1039/b611929h

A scalable and general biotransformation for the generation of a series of l-halotryptophans using the lysate of a commercially available microorganism containing tryptophan synthase. The Royal Society of Chemistry.

Full text of DOI:10.1039/b611929h

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A convenient enzymatic synthesis of L-halotryptophans{
Rebecca J. M. Goss* and Philip L. A. Newill
Received (in Cambridge, UK) 18th August 2006, Accepted 15th September 2006
First published as an Advance Article on the web 5th October 2006
DOI: 10.1039/b611929h
A scalable and general biotransformation for the generation of
a series of L-halotryptophans using the lysate of a commercially
available microorganism containing tryptophan synthase.
The essential amino acid tryptophan is a biosynthetic and synthetic
precursorfor many naturally occurring nonribosomal peptides and
alkaloids.1 Several medicinally important compounds including
Scheme 1 Synthesis of substituted tryptophans.
the anticancer agents Rebeccamycin and Diazonamide A incorpo-
rate chlorinated tryptophans,2,3 and the biosynthesis of the
antifungal, pyrrolnitrin, proceeds via the cleavage of 7-chloro-
tryptophan.4 The seemingly trivial incorporation of a halogen into
a natural product to make new analogues can often result in
dramatic changes in the compounds bioactivity and bioavailabil-
ity.5 A general, rapid and convenient synthesis of halotryptophans
is important to provide starting material for synthetic and biosyn-
thetic incorporation into such natural products. Furthermore, the
generation of such halogenated analogues could enable their
selective functionalisation throughsuch reactionsastheSuzukiand
the Sonogashira coupling. Additionally, fluorinated amino acids
areavaluablelabelingtoolinthestructuralelucidationofproteins.6
The requirement for halotryptophans has stimulated many
synthetic studies but despite many advances since the first synthesis
of 7-chlorotryptophan reported by Rydon and Tweddle,7 the best
methodscurrentlyavailablearemulti-step, lackgeneralityorrequire
specialised procedures. Syntheses of enantiopure halotryptophans,
reported to date, generally involve de-racemisation in the final step
using either an N-acylase8 or through the formation and separation
of diastereoisomers.9 Two notable exceptions are the elegant, but by
no means atom efficient, palladium-mediated heteroannulation of a
Scho¨llkopf chiral auxiliary,10 and the technically specialised electro-
chemical oxidation of proline to 5-hydroxyproline followed by
Fischerindolesynthesis.11Incontrastthedirectenzymaticalkylation
of indole with serine constitutes a direct and potentially efficient
approach and has been demonstrated to be useful in the preparation
of a number of tryptophan analogues.12 This approach has not been
widely adopted because of the need to purify tryptophan synthase.
We report a general, one-step synthesis of fluoro, chloro, bromo
and methyltryptophans using a readily prepared bacterial cell
lysate (Scheme 1). Escherichia coli pretransformed with pSTB7, a
high copy number plasmid expressing tryptophan synthase from
Salmonella enterica, is commercially available (ATCC 37845).
In the first step pyridoxal phosphate (PLP) 1 forms a Shiff base
with L-serine 2 followed by enzyme mediated dehydration to yield
intermediate 3 (Scheme 2). Intermediate 5 results from the
nucleophilic attack of indole 4 on 3. 5 Undergoes subsequent
hydrolysis to release PLP 1 and tryptophan 6.
The cell lysate may be made by sonicating a pellet of E. coli
pSTB7 suspended in buffer. Once obtained, the lysate may be
stored at 5 uC for periods of up to two weeks and treated as a
synthetic reagent. The biotransformation is readily achieved by
addition of an aliquot of cell lysate to a suspension of haloindole
and serine in buffer agitated for 3 d at 37 uC.13 After the reaction,
aggregated proteins remaining in the mixture may be removed by
filtration. Any remaining indole may be extracted into diethyl ether
prior to the aqueous phase being concentrated and the tryptophan
purified by reverse phase silica chromatography. This simple one-
step procedure furnished yields of up to 63%, see Table 1.
Enantiomeric purity was confirmed using a binol derived chiral
shift reagent for amino acids.14
Furthermore, it was determined that almost all of the indole
that was not converted into tryptophan could be recovered by
School of Chemical Sciences and Pharmacy, University of East Anglia,
Norwich, UK NR4 7TJ. E-mail: r.goss@uea.ac.uk;
Tel: +44 (0)1603 593 766
{ Electronic supplementary information (ESI) available: full experimental
procedures are provided. See DOI: 10.1039/b611929h
Scheme 2 The tryptophan synthase mediated conversion of serine and
haloindole (where X = F, Cl, Br) to tryptophan with PLP.
4924 | Chem. Commun., 2006, 4924–4925
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Table 1 Yields for the transformation of indole analogue and serine
to tryptophan
Haloindole
Yield after one cycle with enzyme (%)
7-Fluoro
6-Fluoro
5-Fluoro
7-Chloro
6-Chloro
5-Chloro
7-Bromo
6-Bromo
5-Bromo
4-Bromo
7-Methyl
6-Methyl
5-Methyl
4-Methyl
2-Methyl
Indole
41
62, 82a
63b
9
54
Fig. 1 Nile red.
50, 61a
8
40
steric factors with the 5 and 6-substituted indoles bearing smaller
substituents resulting in excellent yield of the analogue. Our
procedure requires no purification of the enzyme and makes the
reaction readily accessible to synthetic chemists. The generality of
the transformation enables the rapid preparation of synthetically
useful quantities of halotryptophans under environmentally
friendly conditions. By recycling the unconsumed indole and
reintroducing this to fresh enzyme almost quantitative yields of
several of the halotryptophans may be obtained.
16, 26a
3
50
38
42
12
41
53
a
Yields reported for cell-free lysate contained within dialysis tubing.
Improved to 100% yield after extracting unreacted indole and
b
exposing it to two further cycles of reaction.c All products were fully
characterised, see electronic supplementary information, ESI.
The Royal Society is gratefully acknowledged for a Royal
Society BP Dorothy Hodgkin Fellowship (R. J. M. G.).
extraction into diethyl ether. By reintroducing this material to the
lysate and serine mixture we were able to obtain almost
quantitative yields over the course of three cycles. The synthesis
of tryptophans by cell lysate contained within dialysis tubing, tied
to form a bag, was also investigated. This method had the effect of
concentrating the enzyme and we observed a further improvement
in yield, attributed to improved stabilisation of the enzyme. The
contained cell lysate could be removed from the reaction, by
removal of the dialysis bag. The contained enzyme proved still to
be active and could be reused as catalyst in further reactions. Using
dialysis tubing, the reactions were scaled up to enable the
generation of gram quantities of tryptophans in one pot.
Notes and references
1 For example see D. R. Dalton, The Alkaloids, Marcel Dekker, Inc.,
New York, 1979, pp. 415–628; C. Kempter, D. Kaiser, S. Haag,
G. Nicholson, V. Gnau, T. Walk, K. H. Gierling, H. Decker, H. Zahner,
G. Jung and J. W. Metzger, Angew. Chem., Int. Ed. Engl., 1997, 36, 498.
2 E. Yeh, S. Garneau and C. T. Walsh, Proc. Natl. Acad. Sci. U. S. A.,
2005, 102, 3960.
3 N. Lindquist, W. Fenical, C. D. Van Duyne and J. Clardy, J. Am.
Chem. Soc., 1991, 113, 2303.
4 (a) S. Kirner, S. Krauss, G. Surry, S. T. Lam, J. M. Ligon and K.-H. van
Pee, Angew. Chem., Int. Ed. Engl., 1997, 36, 2012; (b) C. Dong, S. Flecks,
S. Unversucht, C. Haupt, K.-H. van Pe´e and J. H. Naismith, Science,
2005, 309, 2216.
5 N. L. Neidleman and J. Geigert, Biohalogenation: Principles, basic roles
and applications, Ellis Horwood Ltd., Chichester, 1986.
6 For example see S. L. Grage, J. Wang, T. A. Cross and A. S. Ulrich,
Biophys. J., 2002, 83, 3336.
7 H. N. Rydon and J. C. Tweddle, J. Chem. Soc., 1955, 3499.
8 Y. Konda-Yamada, C. Okada, K. Yoshida, U. Yasuyuki, S. Arima,
N. Sato, T. Kai, H. Takayanagi and Y. Harigaya, Tetrahedron, 2002,
58, 7851.
9 C. W. Perry, A. Brossi, K. H. Deitcher, W. Tautz and S. Teitel,
Synthesis, 1977, 492.
10 C. Ma, X. Liu, X. Li, J. Flippen-Anderson, S. Yu and J. Cook, J. Org.
Chem., 2001, 66, 4525.
Thisone-stepenzymaticsynthesiscomparesveryfavourablywith
chemical syntheses reported in the literature. Syntheses of racemic
5-fluoro and 6-fluoro tryptophan starting in three steps from the
corresponding indoles have overall yields of 79 and 61% respec-
tively.15,16 A two step synthesis 6-bromo D-tryptophan involving an
acylase to effect de-racemisation is reported in overall 36% yield.8
A variety of factors contribute to the trend in yields. In the series
of halotryptophans the major contribution to the efficiency of the
reaction is from steric factors. The shorter the carbon–halogen
bond the greater the ease of conversion. For example 5-fluoro,
5-chloro and 5-bromo indole are converted to tryptophan in yields
of 63, 54 and 16% respectively. In order to reach the active site of
the b subunit of tryptophan synthase, indole must first enter the a
11 K. Irie, A. Ishida, T. Nakamuru and T. Oh-Ishi, Chem. Pharm. Bull.,
1984, 32, 2126.
12 (a) M. Lee and R. S. Phillips, Bioorg. Med. Chem. Lett., 1992, 2, 1563;
(b) R. S. Phillips, Tetrahedron: Asymmetry, 2004, 15, 2787.
13 General procedure for biotransformations: L-serine (0.127 g, 1.21 mmol),
haloindole (1 eq., 1.21 mmol), PLP (0.8 mg), and Buffer B, were added
to a 250 mL Erlenmeyer flask and cooled to 5 uC. Cell lysate (2 mL at
5 uC) was then added to the flask. Reactions were incubated in an
orbital shaker (37 uC, 180 rev min21, 3 d). The reaction mixture was
filtered and extracted with diethyl ether (2 6 50 mL) to remove any un-
reacted indole. The combined organic layers were dried (MgSO4) and
the solvent removed under reduced pressure to give un-reacted starting
material. The aqueous layer was reduced in volume to 20 mL under
reduced pressure prior to the material being purified by reverse-phase
chromatography.
14 J. Chin, D. C. Kim, F. B. Panosyan and K. M. Kim, Org. Lett., 2004, 6,
2591.
15 E. Hoffmann, R. Ikan and A. B. Galun, J. Heterocycl. Chem., 1965, 2,
289.
16 E. D. Bergmann and E. Hoffmann, J. Chem. Soc., 1962, 2827.
17 S. B. Ruvinov, X.-J. Yang, K. D. Parris, B. Utpal, A. S. Ahmed and
E. W. Miles, J. Biol. Chem., 1995, 270, 6357.
˚
subunit then pass through a 25 A long tunnel. A turn in this tunnel
has been noted to prevent the passage of Nile red, a molecule
17
˚
12 6 6 6 3.4 A in dimension (Fig. 1).
Indoles substituted at the 7 and 4 positions will have the greatest
˚
˚
˚
˚
widths (6.0 A for Br, 5.8 A for methyl, 5.8 A for Cl, 5.3 A for F)
compared to the width of indole unsubstituted in the 4 or 7 position
˚
(5.0 A). Comparatively lower yields in each series of halotrypto-
phans for the 7 and 4 analogues could be postulated to be due to
steric difficulties with passage of these molecules through the
enzyme’s tunnel to the active site.
To summarise, the general and scalable synthesis of a range of
tryptophan analogues, using a readily prepared cell-free lysate, is
reported. The efficiency of the reaction is predominantly related to
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Chem. Commun., 2006, 4924–4925 | 4925