Chemoenzymatic synthesis of the two enantiomers of 7-azatryptophan

Article abstract of DOI:10.1016/S0957-4166(98)00130-X

7-Azatryptophan is an unnatural α-amino acid with a very potent fluorescent activity. It is used as a vehicle for probing the structure and dynamics of proteins and peptides. Diastereoselective alkylation, diastereoselective protonation and enzymatic resolution have been tested for preparing enantiomerically pure 7-azatryptophan.

Full text of DOI:10.1016/S0957-4166(98)00130-X

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 1753–1758
Chemoenzymatic synthesis of the two enantiomers of
7-azatryptophan
L. Lecointe, V. Rolland-Fulcrand,∗ M. L. Roumestant, Ph. Viallefont and J. Martinez
Laboratoire Aminoacides, Peptides et Protéines, Université Montpellier I & II, Place E. Bataillon, 34095 Montpellier Cedex 5,
France
Received 24 March 1998; accepted 27 March 1998
Abstract
7-Azatryptophan is an unnatural α-amino acid with a very potent fluorescent activity. It is used as a vehicle
for probing the structure and dynamics of proteins and peptides. Diastereoselective alkylation, diastereoselective
protonation and enzymatic resolution have been tested for preparing enantiomerically pure 7-azatryptophan.
© 1998 Elsevier Science Ltd. All rights reserved.
1. Introduction
In 1955, Robison et al. described the synthesis of racemic 7-azatryptophan via 3-(N-
dimethylaminomethyl)-7-azaindole or 7-azagramine.1 The first known biological activity of 7-
azatryptophan was the inhibition of the Tetrahymena pyriformis growth, through inhibition of tryptophan
metabolism,2 while replacement of tryptophan by 7-azatryptophan in enzyme sequences resulted in
inactivation.3
On the other hand, tryptophan and its derivatives are well known for their fluorescent activity4 due to
the indole ring. Negrerie et al. proved the lone (395 nm) and exponential signal of 7-azatryptophan. Rich
et al. used N1-Me-7-azatryptophan as an optical standard.5 7-Azatryptophan is now used as a vehicle for
probing the structure and dynamics of proteins and peptides.
The first enantioselective synthesis of (−)-(R)-7-azatryptophan was published in 19926 by Fallis et al.
via alkylation of the (1R,4R)-camphor imine of tert-butylglycinate. The diastereoisomeric excess was
greater than 98% but the yield of alkylation was only 27%.
One enzymatic synthesis was described in 1989: tryptophan synthase [EC.4.2.1.20], which is a non-
commercial recombinant enzyme, catalysed the direct condensation of 7-azaindole with (L)-serine in 8
days and in 88% yield. This enzymatic condensation allowed the preparation of enantiomerically pure 4,
∗
Corresponding author. E-mail: rolland@crit.univ-montp2.fr
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00130-X

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5 and 6-azatryptophans. However, this method is limited by the price of the recombinant enzyme and the
necessary use of cofactors (pyridoxal phosphate).7
2. Results
Two different diastereoselective methods were tested, diastereoselective alkylation and diastereo-
selective protonation (deracemization) using 2-hydroxypinan-3-one (abbreviated to H.P.) as the chiral
auxiliary (Scheme 1).
Scheme 1.
Diastereoselective alkylation is a well known procedure in our laboratory,8 however the synthesis of
the alkylating reagent (3-bromomethyl-7-azaindole) needed several steps and proceeded with very poor
yields.
Diastereoselective protonation is a recent method9 carried out in two steps. 7-Azatryptophan methyl
ester was quantitatively obtained from the racemic mixture 2; the Schiff base was prepared using the
chiral auxiliary (S,S,S)-2-hydroxypinan-3-one in toluene with a catalytic amount of BF3·Et2O. The
diastereoisomers 3 were not separated at this stage, but when the N1 is protected the separation of both
diastereoisomers 4 was easily achieved.
The Schiff base is deprotonated with a base in THF; several protecting groups (Boc, Z) and bases (tert-
BuOK, LDA, KHMDS) were tested at −80°C (Table 1). The best results were obtained with 2 equivalents
of KHMDS. The protonation was carried out with an NH4Cl saturated solution in quantitative yield. The
diastereoisomeric excess was evaluated by 1H NMR and gave in the best case a d.e. of 66%.
Table 1

L. Lecointe et al. / Tetrahedron: Asymmetry 9 (1998) 1753–1758
1755
We tested as a third method the enzymatic resolution with an efficient acylase, Aspergillus genus
acylase (Scheme 2).
Scheme 2.
From compound 1, we carried out a saponification and a decarboxylation under controlled pH to
obtain the racemic mixture of N-Ac-7-azatryptophan 6. Resolution was achieved with the purified
Aspergillus genus acylase in 36 hours. The separation step between (S)-7-azatryptophan 7 and (R)-N-Ac-
7-azatryptophan 8 was carried out by precipitation of 7 in MeOH at pH 3. (R)-N-Ac-7-Azatryptophan 8
was hydrolysed to yield 9.
The yields were excellent and for easier manipulation the enzyme was immobilized on different
supports:
(i) by copolymerization with long chain amphipatic agents such as PEG;10
(ii) by a covalent bond on alumina preactivated with colamine-O-phosphate and glutaraldehyde;11 and
(iii) by a covalent bond on Eupergit C (Rhom Society).
The first immobilization method produced the same yields as using the soluble enzyme; however the
reaction time was longer than with the soluble acylase (48 h) and the enzyme elimination by gel filtration
was replaced by a simple filtration. Moreover, this system allowed the automation of the process and the
reuse of the immobilised enzyme that was stabilized.
3. Conclusion
In our hands, the best method for preparing the two enantiomerically pure 7-azatryptophan isomers is
the enzymatic resolution with Aspergillus genus acylase.
4. Experimental
Thin-layer chromatography (TLC) was performed on Merck precoated silica gel 60F254 plates and
spots were visualised by ultraviolet light and/or iodine vapour. Column chromatography was performed
using Geduran silica gel. Chiral TLC was performed on Macherey–Nagel plates and spots were revealed

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by 0.2% ninhydrin solution. Chiral HPLC was performed on a Waters HPLC system with a 486 detector
apparatus on a Crownpack CR+ column. Melting points were obtained on a Büchi 510 apparatus and
were not corrected. Optical rotations were recorded with a Perkin–Elmer 141 polarimeter at the sodium
1
D line. Mass spectra were measured on Jeol JMS DX 100 and DX 300 apparatus. H NMR spectra were
recorded on a Brucker spectrometer AC 250.
4.1. Literature preparation1 of racemic 7-azatryptophan from the commercially available 7-azaindole
The intermediate azagramine synthesis (yield=87%), the alkylation of diethyl acetamidomalonate with
azagramine (yield=75%) and the acidic total hydrolysis gave racemic 7-azatryptophan 2 in 50% overall
yield. The racemic mixture crystallized with one molecule of water. Mp1=262–264°C; mp=260–262°C.
4.2. 7-Azatryptophan methyl ester hydrochloride: 7-azaTrpOMe·HCl
7-azaTrpOH 2 (11 mmol) was dissolved in ethanol (100 ml) under argon and ClSiMe3 (88 mmol, 11.2
ml) was added dropwise. The mixture was stirred at 40°C over 36 hours. The solvent was concentrated
under vacuum (yield=95%). Mp=222–225°C.
4.3. Schiff base of 7-azatryptophan methyl ester: 3
7-Azatryptophan methyl ester (5.25 mmol, 1.16 g) was neutralized by NH3(g) bubbling in Et2O. The
solvent was evaporated under vacuum. The ester and (S,S,S)-2-hydroxypinan-3-one (3.5 mmol, 0.59
g) were dissolved in toluene (75 ml) and a catalytic amount of BF3·Et2O was added and the mixture
was refluxed for 2 hours with elimination of water (Dean–Stark apparatus). The solvent was evaporated
and the orange oil was purified by chromatography on silica gel (petroleum ether/Et2O gradient); the
diastereoisomers were not separated (yield=80%). Rf=0.35 (Et2O) for both diastereoisomers.
4.4. Protection of the Schiff base: 4
Schiff base 3 (5.3 mmol, 1.95 g) was dissolved in DCM (100 ml) and Boc2O (7.7 mmol, 1.68 g) was
added. The reaction was stirred at room temperature for 12 hours. The solvent was concentrated under
vacuum and the diastereoisomers were separated on silica gel. After purification, A and B were obtained
in 33 and 37% yield, respectively: Rf A=0.66 (Et2O); Rf B=0.46 (Et2O).
A: 1H NMR C6D6 δ ppm: 0.68 (s, 3H); 1.03 (s, 3H); 1.47 (s, 9H); 1.58 (s, 3H); 1.80–2.40 (8H); 3.15
(dd, 1H, J1=8.04 Hz, J2=10.989 Hz); 3.26 (s, 3H); 3.30 (dd, 1H, J1=4.85 Hz, J2=11 Hz); 4.30 (dd, 1H,
J1=4.81 Hz, J2=8.04 Hz); 6.77 (dd, 1H, J1=4.74 Hz, J2=7.84 Hz); 7.58 (dd, 1H, J1=1.63 Hz, J2=7.84 Hz);
7.75 (s, 1H); 8.42 (dd, 1H, J1=1.62 Hz, J2=4.73 Hz).
B: 1H NMR C6D6 δ ppm: 0.72 (s, 3H); 1.08 (s, 3H); 1.5 (s, 9H); 1.66 (s, 3H); 1.80–2.40 (6H); 3.25
(dd, 1H, J1=4.17 Hz, J2=13.00 Hz); 3.3 (s, 3H); 3.37 (dd, 1H, J1=8.24 Hz, J2=12.6 Hz); 4.35 (dd, 1H,
J1=4.6 Hz, J2=8.08 Hz); 6.8 (dd, 1H, J1=4.70 Hz, J2=7.86 Hz); 7.6 (dd, 1H, J1=1.60 Hz, J2=7.20 Hz);
7.8 (s, 1H); 8.5 (dd, 1H, J1=1.50 Hz, J2=4.70 Hz).
4.5. Diastereoselective protonation (deracemization): 5
2.5 N KHMDS in toluene (4.2 ml, 2.1 equiv.) was diluted in anhydrous THF (20 ml) at −80°C.
Schiff base 4 (5 mmol, 1 equiv.) was diluted in anhydrous THF (20 ml) and added dropwise to the

L. Lecointe et al. / Tetrahedron: Asymmetry 9 (1998) 1753–1758
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KHMDS solution. The mixture was stirred for 30 min at −80°C and the protonation was carried out with
a saturated solution of NH4Cl. The temperature was allowed to reach room temperature. The aqueous
phase was washed several times with Et2O. The organic phase was dried on MgSO4 and concentrated
under vacuum: yield=95%, d.e.=66%.
4.6. Racemic N-Ac-7-azatryptophan 6
Compound 1 (4 mmol, 1.796 g) was dissolved in a mixture of dioxane/2N NaOH (9/1, v/v; 50 ml),
and stirred for one day at 40°C. The saponification was followed by TLC in a DCM/20% MeOH solvent
system. The mixture was neutralized until pH 8 with a concentrated HCl solution; the solvent was
evaporated and NaCl removed by precipitation in MeOH (yield=95%). The salt was dissolved in dioxane
(40 ml), concentrated HCl solution was added until pH 4 and the solution was refluxed for 15 hours.
The mixture was neutralized until pH 7 and the solvent was evaporated. NaCl was removed with the
1
same treatment in MeOH as for the saponification. Compound 6 was obtained in 90% yield. H NMR
DMSO-d6 δ ppm: 1.76 (s, 3H); 3.15 (d, 2H, J=6.02 Hz); 4.3 (t, 1H, J=6.04 Hz); 7.3 (dd, 2H, J=6.03 and
7.99 Hz); 7.7 (d, 1H, J=6 Hz); 7.8 (s, 1H); 8.3 (dd, 1H, J=6.04 and 1.04 Hz); 11.9 (s, 1H).
4.7. Enzymatic resolution with Aspergillus genus acylase (TCI)
The commercially available enzyme from TCI contains only 30% of protein and has to be purified by
gel filtration on Sephadex G50 with water and a solution of CoCl2 (5×10−4 M) as eluent.
N-Ac-7-Azatryptophan 6 (4 mmol) was dissolved in phosphate buffer (0.1 M, pH 7.2) with CoCl2
solution (5×10−4 M, 25 ml). Then acylase (12 mg) was added and the pH was maintained by adding 6
N NaOH. The reaction was vigourously stirred at 40°C and can be monitored with a pH stat. After one
day of agitation, enzyme (6 mg) was added and stirred overnight. When the pH was stable, the kinetic
enzymatic hydrolysis of the (S) isomer was quenched with HCl until pH 4. The enzyme was removed by
gel filtration on Sephadex G50 with water as eluent.
The reaction was followed by chiral TLC on Macherey–Nagel plates using as the eluent a mixture of
MeOH (12 ml), CH3CN (10 ml), H2O (5.5 ml), EtOH/NH4OH (4/1, 2.5 ml) and the plates were revealed
with ninhydrin. Chiral HPLC was carried out on a Crownpack CR+ column with aqueous HClO4 as an
eluent at 0°C.12
As described in the literature,13 derivatization with Marfey reagent was tested to analyse diastereo-
isomers. The derivatization did not succeed.
(R)-N-Ac-7-Azatryptophan 8 was less soluble at pH 4 than the (S)-7-azatryptophan 7; it precipitated
at 0°C and was separated from 7 by filtration. This operation was repeated until obtention of one spot by
TLC. (S)-7-Azatryptophan 7 was crystallized in a water/ethanol system in 45% yield.
Rf (R)=0.60; Rf (S)=0.69; 1H NMR (D2O) δ ppm: 3.6 (d, 2H, J=6.14 Hz); 4.3 (t, 1H, J=6.10 Hz); 7.66
(dd, 1H, J1=7.50, J2=6.20 Hz); 7.75 (s, 1H); 8.5 (dd, 1H, J1=6.04, J2=1.05 Hz); 8.7 (dd, 1H, J1=7.99,
J2=1.05 Hz). Mp=261–262°C, [α]D=+11 (c=10 mg/ml in H2O/HCl, pH 3).
4.8. (R)-N-Ac-7-Azatryptophan 8 hydrolysis: (R)-7-azatryptophan 9
(R)-N-Ac-7-Azatryptophan (1.5 mmol) was refluxed in 6 N HCl for 4 hours. The mixture was
neutralized and concentrated under vacuum. NaCl was removed by precipitation in MeOH.
(R)-7-Azatryptophan 9 was crystallized from a water/ethanol system in 40% yield. Mp=262–263°C,
[α]D=−11 (c=10 mg/ml in H2O/HCl, pH 3).

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4.9. Table of chiral analysis results on Crownpack CR+ column
Product
tR (min)
6
12.5
7
3
9
4
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