A concise preparation of the non-proteinogenic amino acid l-kynurenine

Article abstract of DOI:10.1016/j.tetlet.2012.09.055

A concise and practical preparation of the non-proteinogenic amino acid l-kynurenine is reported. The synthetic approach is scalable and provides ready access to this valuable amino acid in either l- or d-stereochemistry starting from l- or d-tryptophan, respectively. In the optimized procedure, two discreet oxidation steps are applied sequentially to convert the tryptophan indole ring into the keto-aniline moiety contained within the kynurenine side chain.

Full text of DOI:10.1016/j.tetlet.2012.09.055

Tetrahedron Letters 53 (2012) 6430–6432
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Tetrahedron Letters
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A concise preparation of the non-proteinogenic amino acid L-kynurenine
⇑
Laurens H. J. Kleijn, Frederike M. Müskens, Sabine F. Oppedijk, Gerjan de Bruin, Nathaniel I. Martin
Department of Medicinal Chemistry & Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CG Utrecht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
A concise and practical preparation of the non-proteinogenic amino acid
L-kynurenine is reported. The
Received 14 August 2012
Revised 1 September 2012
Accepted 13 September 2012
Available online 20 September 2012
synthetic approach is scalable and provides ready access to this valuable amino acid in either
L- or D-ste-
reochemistry starting from - or -tryptophan, respectively. In the optimized procedure, two discreet oxi-
L
D
dation steps are applied sequentially to convert the tryptophan indole ring into the keto-aniline moiety
contained within the kynurenine side chain.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Kynurenine
CNS disease
Tryptophan oxidation
Oxindolylalanine
Kynurenine (Fig. 1) is a non-proteinogenic amino acid with
roles in a number of biochemical signaling pathways and disease
states.1,2 In mammals, kynurenine is produced as the major metab-
olite of tryptophan via the self-named kynurenine pathway (ꢀ99%
of dietary tryptophan is metabolized by this pathway).1 Histori-
cally, study of the kynurenine pathway focused on its role in the
endogenous production of nicotinamide (NAD, vitamin B3). Addi-
tional roles for kynurenine were subsequently brought to light
when it was shown that the kynurenine pathway can also lead to
formation of compounds with either neuroprotective or neurotoxic
properties. These neuroactive metabolites are increasingly recog-
nized as contributors to the pathogenesis of Alzheimer’s3–5 and
Huntington’s6–8 diseases, depression,9,10 schizophrenia,11,12 and
other neuroinflammatory processes. As a signaling molecule, kyn-
urenine has recently been shown to elicit smooth muscle relaxa-
tion via activation of guanylate cyclase, the target of nitric
oxide.13 In addition, kynurenine was also demonstrated to activate
the aryl hydrocarbon (AH) receptor leading to the generation of
regulatory T cells.14,15 Apart from its various roles as a discrete spe-
cies, kynurenine is also found as a building block in certain non-
ribosomal peptides, most notably the clinically used lipopeptide
antibiotic daptomycin.16
Figure 1. Structure of L-kynurenine.
Traditionally, two main approaches have been employed for the
synthesis of kynurenine and its analogues. Among the earliest syn-
theses reported was the condensation of the sodium salt of diethyl
acetamidomalonate with o-nitrophenacyl bromide followed by
hydrolysis, decarboxylation, and reduction to yield kynurenine as
the racemate (Scheme 1A).19 Acetylation of the racemic kynuren-
ine thus obtained, followed by treatment with acylase has been de-
scribed as
a
means of resolving the enantiomers.20,21 This
- or -kynurenine is, however, limited
multistep approach toward
L
D
due to the low yields obtained for the overall process as well as
variability in the activity of the enzyme used in the resolving step.
As an alternative, ozonolysis of N-acetyl-tryptophan has been re-
ported to give stereochemically pure material (Scheme 1B).22,23
While providing a more direct access to kynurenine, the ozonolysis
approach is also limited due to variable yields as well as the
generation of a number of impurities making subsequent purifica-
tions challenging.24,25 In addition to these approaches, the photo-
catalytic decomposition of tryptophan has also been reported to
produce kynurenine, albeit on a scale not suitable for the prepara-
tion of multi-gram quantities of material.26
In pursuing an alternative route for the preparation of kynuren-
ine, we initially drew upon the results of Evano and coworkers who
described an unexpected indole ring cleavage upon treatment of a
protected tryptophan-containing dipeptide with m-chloroperben-
zoic acid (Scheme 2A).27 The resulting N-formyl substituent on
the kynurenine aniline unit can be subsequently removed under
While the majority of investigations into the biological effects
of kynurenine have focused on the
shown that the kynurenine pathway (in mice, rats, and humans)
can also process
-kynurenine.17,18 Given the wide range of interest
in kynurenine, we sought to develop a practical and economical
route for its synthetic preparation (commercial access to -kynu-
renine is expensive while the -enantiomer is available only in mil-
L-enantiomer, it has also been
D
L
D
ligram quantities at even greater expense).
⇑
Corresponding author.
E-mail address: n.i.martin@uu.nl (N.I. Martin).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.09.055

L. H. J. Kleijn et al. / Tetrahedron Letters 53 (2012) 6430–6432
6431
O
O
O
A
B
O
O
O
i
ii
L-tryptophan
Br
EtO
OEt
NHAc
OH
NHCbz
EtO
Na
OEt
OH
HN
NHAc
O
1
NO2
O
O
NO2
H
O
iii
OH
NHCbz
OH
NHCbz
NH2
O
NH2
HN
NH2
O
O
rac-kynurenine
2
3
O
O
O
iv
O3
OH
NHAc
OH
NHAc
OH
NH2 H2SO4
H
NH
O
HN
NH2
O
4
O
O
Scheme 3. Reagents and conditions: (i) CbzCl, 1 M NaOH, H2O, 99%; (ii) DMSO,
concentrated HCl, AcOH, 62%; (iii) air, 1 M NaOH, H2O, 52%; (iv) (a) H2, 10% Pd/C,
1,4-dioxane/aq. HCl; (b) H2SO4, recrystallization from EtOH to H2O, 65%.
OH
NH2
L-kynurenine
O
NH2
HCl in AcOH to yield the expected diastereomeric mixture of
Cbz-protected b-3-oxindolylalanines 2 (Scheme 3). A basic aque-
ous solution of 2 was then aerated, leading to formation of the pro-
tected kynurenine species 3 (conversion can be directly monitored
by TLC). While the isolated yield of 3 was somewhat moderate
(52%), its purification by conventional silica gel chromatography
was straightforward and allowed for the reliable preparation of
this material on large scale. Removal of the Cbz group by routine
hydrogenation, followed by addition of an equimolar quantity of
sulfuric acid and recrystallization from EtOH–H2O yielded pure
kynurenine sulfate 4. In this manner, multi-gram quantities of both
Scheme 1. Previously reported approaches for the synthesis of kynurenine; (A)
preparation of rac-kynurenine via condensation of diethyl acetamidomalonate with
o-nitrophenacyl bromide and (B) preparation of
acetyl- -tryptophan.
L-kynurenine by ozonolysis of N-
L
A
L- and
D-kynurenine were successfully prepared.
A mechanistic rationale for the conversion of Cbz-protected
tryptophan into kynurenine via this two-step oxidation process
can be provided based upon literature precedents (Scheme 4).
The first oxidation step, wherein the indole moiety is converted
into oxindole species 2 via the action of a DMSO/HCl mixture,
has been previously described by Savige and Fontana.29–31 The sec-
ond oxidation process in which the oxindole is converted into the
keto-aniline moiety by simple treatment with air under basic con-
ditions is a less commonly encountered manipulation. A mechanis-
tic explanation for this step can be offered based upon the
analogous reaction of indoles with superoxide anion.32,33 As de-
picted in Scheme 4, hydrogen abstraction by molecular oxygen
leads to formation of the oxindole radical which upon recombina-
tion leads to formation of the hydroperoxide species. Under the ba-
sic conditions employed, an intramolecular nucleophilic addition
then leads to formation of the dioxetane which in turn opens to
yield the corresponding ketone and carbamic acid functionalities.
Decarboxylation then leads to formation of the keto-aniline unit
comprising the kynurenine side chain.
B
Scheme 2. (A) mCPBA oxidation of a tryptophan-containing dipeptide as reported
by Evano and co-workers yielding an N-formyl kynurenine unit and (B) attempted
oxidation of protected tryptophan under similar conditions.
mild conditions using dilute acid.20 As such, we chose to pursue an
analogous approach wherein a suitably protected tryptophan was
exposed to similar oxidizing conditions (Scheme 2B). While the ex-
pected N-formyl kynurenine species could be prepared in this
manner, a large number of undesired side products (attributed to
either under- or over-oxidation) were also formed. Unfortunately,
attempts to perform this reaction under different conditions (vary-
ing time, temperature, or solvent) had little impact on the distribu-
tion of product and side-products formed.
As an alternative approach, a recent report by the group of Hoff-
man demonstrated that b-3-oxindolylalanine, itself an oxidation
product of tryptophan, can be readily oxidized to kynurenine.28
While providing for a concise route to kynurenine, we found the
yields achieved via this procedure to be somewhat variable. Fur-
thermore, the reported use of reverse phase chromatography for
the purification of both the polar intermediate and final product
is not readily amenable to scale-up. To address these issues we
examined the use of Cbz-carbamate protected tryptophan 1 as a
starting material to allow for a more convenient method of moni-
toring the subsequent conversions as well as product isolation. To
this end, Cbz-tryptophan was prepared via standard approaches
after which it was treated with an oxidizing mixture of DMSO/
In summary, a concise and practical synthesis of the non-pro-
teinogenic amino acid kynurenine is reported. This approach al-
lows for the preparation of both
corresponding readily available and inexpensive
L
- and
D
-kynurenine from the
- or -trypto-
L
D
phan. The process developed is operationally straightforward and
can be scaled up to provide multi-gram quantities of material. Gi-
ven the increasing level of interest in kynurenine due to its many
biochemical roles, this methodology serves to greatly improve ac-
cess to this unique amino acid.
Acknowledgements
This work was supported by The Netherlands Organization for
Scientific Research (NWO-VIDI fellowship to N.I.M.). The Utrecht
Institute for Pharmaceutical Sciences and The University of Utrecht
are also gratefully acknowledged for their support.

6432
L. H. J. Kleijn et al. / Tetrahedron Letters 53 (2012) 6430–6432
DMSO + HCl
S
Cl
O
O
O
Cl
Cl
H
OH
NHCbz
OH
OH
NHCbz
NHCbz
N
H
N
H
N
H
OH
1
O
H
H
OH
H
O
O
O
O
O
O
O
O
O
O
H
OH
NHCbz
OH
NHCbz
OH
OH
NHCbz
NHCbz
N
H
N
N
H
N
H
O
OH
O
2
H
O
OH
NHCbz
O
OH
O
-CO2
NHCbz
OH
NHCbz
O
O
O
NH2
O
N
H
NH
3
OH
H
O
O
Scheme 4. Proposed mechanism for the two-step oxidative transformation of Cbz-protected tryptophan 1 into Cbz-protected kynurenine 3 via oxindole 2.
13. Wang, Y.; Liu, H.; McKenzie, G.; Witting, P. K.; Stasch, J. P.; Hahn, M.;
Supplementary data
Changsirivathanathamrong, D.; Wu, B. J.; Ball, H. J.; Thomas, S. R.; Kapoor, V.;
Celermajer, D. S.; Mellor, A. L.; Keaney, J. F., Jr.; Hunt, N. H.; Stocker, R. Nat. Med.
2010, 16, 279–285.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
09.055.
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