Distinct metabolites for photoreactive l-phenylalanine derivatives in Klebsiella sp. CK6 isolated from rhizosphere of a wild dipterocarp sapling

Article abstract of DOI:10.3390/molecules18078393

Photoaffinity labeling is a reliable analytical method for biological functional analysis. Three major photophores-aryl azide, benzophenone and trifluoromethyldiazirine- are utilized in analysis. Photophore-bearing L-phenylalanine derivatives, which are used for biological functional analysis, were inoculated into a Klebsiella sp. isolated from the rhizosphere of a wild dipterocarp sapling in Central Kalimantan, Indonesia, under nitrogen-limiting conditions. The proportions of metabolites were quite distinct for each photophore. These results indicated that photophores affected substrate recognition in rhizobacterial metabolic pathways, and differential photoaffinity labeling could be achieved using different photophore-containing L-phenylalanine derivatives.

Full text of DOI:10.3390/molecules18078393

Molecules 2013, 18, 8393-8401; doi:10.3390/molecules18078393
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Distinct Metabolites for Photoreactive L-Phenylalanine
Derivatives in Klebsiella sp. CK6 Isolated from Rhizosphere of
a Wild Dipterocarp Sapling
Lei Wang 1, Wataru Hisano 1, Yuta Murai 1, Munenori Sakurai 1, Yasuyuki Muto 1,
Haruka Ikemoto 1, Masashi Okamoto 1, Takashi Murotani 1, Reika Isoda 1, Dongyeop Kim 1,
Yasuko Sakihama 1, Irnayuli R. Sitepu 2, Yasuyuki Hashidoko 1, Yasumaru Hatanaka 3 and
Makoto Hashimoto 1,*
1
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University; Kita 9,
Nishi 9, Kita-ku, Sapporo 060-8589, Japan
2
Forest Microbiology Laboratory, Forest and Nature Conservation Research and Development Center,
Forest Research and Development Agency, Jalan Gunung Batu No.5, P.O. Box 165,
Bogor 16610, Indonesia
3
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani,
Toyama 930-0194, Japan
* Author to whom correspondence should be addressed; E-Mail: hasimoto@abs.agr.hokudai.ac.jp;
Tel./Fax: +81-11-706-3849.
Received: 14 June 2013; in revised form: 2 July 2013 / Accepted: 11 July 2013 /
Published: 16 July 2013
Abstract: Photoaffinity labeling is a reliable analytical method for biological functional
analysis. Three major photophores—aryl azide, benzophenone and trifluoromethyldiazirine—
are utilized in analysis. Photophore-bearing L-phenylalanine derivatives, which are used for
biological functional analysis, were inoculated into a Klebsiella sp. isolated from the
rhizosphere of a wild dipterocarp sapling in Central Kalimantan, Indonesia, under
nitrogen-limiting conditions. The proportions of metabolites were quite distinct for each
photophore. These results indicated that photophores affected substrate recognition in
rhizobacterial metabolic pathways, and differential photoaffinity labeling could be
achieved using different photophore-containing L-phenylalanine derivatives.
Keywords: photoaffinity label; phenylalanine; tryptophan; phenyl azide; benzophenone;
trifluoromethyldiazirine; metabolites

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8394
1. Introduction
Photoaffinity labeling is a useful biochemical method used to reveal structural and functional
relationships between low molecular weight bioactive compounds and biomolecules [1–6], and, for
this purpose various photophores, such as phenyl diazirine, phenyl azide and benzophenone, are
commonly used. Although comparisons of the chemical and physiological properties of these
photophores have been reported [7], there are no reports concerning comparative analysis of
metabolites resulting from inoculation of these photophores contained in precursors. A rhizobacterium,
Klebsiella sp. CK6, which was isolated from a dipterocarp sapling in Central Kalimantan, Indonesia, is
able to grow in a low-nitrogen medium supplemented with L-tryptophan [8]. Some bacteria use
transaminase for the oxidative deamination reaction of L-tryptophan to yield indol-3-acetic acid (IAA)
via intermediary indole-3-pyruvic acid [9]. Supplementation of L-tryptophan into the culture medium
of Klebsiella sp. CK6 afforded IAA, tryptophol (TOL), and indole-3-lactic acid (ILA) under nitrogen-
limiting conditions [Scheme 1(A)]. However, the other aromatic α-amino acids were not tested as
nitrogen sources. Phenylalanine is one of the most fundamental aromatic α-amino acids and plays an
important role in hydrophobic interactions responsible for substrate binding to biomolecules.
Photoreactive phenylalanine derivatives can be applied to the biological functional analysis of α-amino
acids and bioactive peptides instead of phenylalanine. These strategies are premised on the fact that
photoreactive phenylalanine derivatives act in almost the same way as phenylalanine itself. Currently,
there are no reports that analyze comparatively the metabolites of photoreactive precursors inoculated
into microorganisms. In this paper, we report that L-phenylalanine acts as nitrogen source, but the
resulting metabolites are slightly different from those of L-tryptophan, and each photoreactive
L-phenylalanine derivative affords different proportions of metabolites in Klebsiella sp. CK6 under
nitrogen-limiting condition.
2. Results and Discussion
2.1. Inoculation of L-Phenylalanine as a Nitrogen Source for Klebsiella sp. CK6
L-Phenylalanine as nitrogen source was inoculated into Klebsiella sp. CK6 in a standing culture at
28 °C for one week under nitrogen-limiting conditions, and cell growth did not change compared to
when the L-tryptophan was used as nitrogen source. The inoculate mixture was centrifuged to remove
planktonic bacterial cells, and the pH of the supernatant was adjusted to 2.5 with 1N HCl. Then, the
metabolites were extracted with ethyl acetate. After drying over MgSO4 and filtration, the organic
layer was concentrated to afford crude extracts. The crude extracts were subjected to silica column
chromatography. Phenyllactic acid (PLA) and phenylacetic acid (PAA) were the major metabolites
from L-phenylalanine supplemented at pH 6. However, phenylethanol (PE), which is deaminated,
decarboxylated, then reduced, was not detected, even though tryptophol (TOL), which undergoes a
similar sequence after L-tryptophan inoculation, was easily detected [Scheme 1(B)]. These results
indicated substrate specificity, since the reduction from aldehyde to alcohol recognized the indole ring.
D-Phenylalanine was less metabolized to PLA and PAA as compared to the L-isomer. No substantially
different metabolites were observed after one week under pH conditions between 5 and 6, with or

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without rotation of the L-phenylalanine inoculum. These results indicated that the metabolic pathways
of L-tryptophan and L-phenylalanine were slightly different due to their chemical structures.
Scheme 1. Metabolic pathway for L-tryptophan and L-phenylalanine inoculated into
Klebsiella sp. CK6 under nitrogen-limiting conditions.
OH
CHO
(A)
N
N
H
H
tryptophol (TOL)
COOH
NH2
COOH
COOH
O
N
N
H
H
COOH
OH
N
L-Trypyophan (L-Try)
H
indolacetic acid (IAA)
N
H
indolactic acid (ILA)
OH
(B)
CHO
phenylethanol (PE)
COOH
COOH
NH2
O
COOH
COOH
L-Phenylalanine (L-Phe)
phenylacetic acid (PAA)
OH
phenyllactic acid (PLA)
2.2. Inoculation of Photoreactive L-Phenylalanine Derivatives for Klebsiella sp. CK6
2.2.1. Inoculation of 4-Azido-L-phenylalanine as a Nitrogen Source for Klebsiella sp. CK6
4-Azido-L-phenylalanine (1a) was prepared in a similar manner as described by Wieland et al. with
some slight modifications [10]. Briefly, phenylalanine was nitrated with nitric acid (fuming) in sulfuric
acid. The 4-nitro derivative was reduced by hydrogenation with Pd-BaSO4 to afford the aniline
derivative. Diazotization, followed by azidation, afforded the target 4-azido-L-phenylalanine in
moderate yield. Inoculation of the 4-azido-L-phenylalanine into Klebsiella sp. CK6 in the standing
culture afforded an azide-substituted PAA derivative 1b as the main metabolite. IR measurements of
the main product indicated that the azide group (2,140 cm−1) persisted after inoculation. No reduction
of the azide to an amine or other functional group was observed under direct culture analysis (Scheme 2).
2.2.2. Inoculation of 4-benzoyl-L-Phenylalanine as a Nitrogen Source for Klebsiella sp. CK6
4-Benzoyl-L-phenylalanine (2a) was prepared by Friedel-Crafts benzoylation of L-phenylalanine
with benzoyl chloride in trifluoromethanesulfonic acid (TfOH). Direct Friedel-Crafts benzoylation of
L-phenylalanine has not been reported, presumably due to the low solubility of unprotected α-amino
acids in many organic solvents. The solubility of α-amino acids and the properties of the Friedel-Crafts

Molecules 2013, 18
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reaction catalyzed by TfOH enabled us to directly benzoylate L-phenylalanine. The inoculation of
4-benzoyl-L-phenylalanine into Klebsiella sp. CK6 in standing culture afforded benzoyl-substituted
PAA and phenylethanol (PE) derivatives 2b and 2c as the main metabolites. No PLA derivative was
detected in the extract (Scheme 2).
2.2.3. Inoculation of 4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]-L-phenylalanine as a Nitrogen Source
for Klebsiella sp. CK6
4-[(3-Trifluoromethyl)-3H-diazirin-3-yl]-L-phenylalanine (3a) was prepared in an asymmetric
manner from corresponding diazirinyl benzyl bromide, which was consistent with our previous
reports [11,12]. The inoculation of 4-(3-trifluoromethyl)-L-phenylalanine into Klebsiella sp. CK6 in
standing culture afforded trifluormethyldiazirinyl-substituted PLA 3d as the sole product. The UV
spectrum of the isolated metabolite indicated a broad adsorption around 350 nm, suggesting that the
diazirinyl photophore was still intact. No optical rotation was observed for the metabolite. PAA and PE
type metabolites were not detected in the extract (Scheme 2).
Scheme 2. Metabolic pathway for 4-photophore-substituted and 4-nitro-substituted
phenylalanine derivatives inoculated into Klebsiella sp. CK6.
OH
CHO
R
L-Phe derivatives
COOH
(2c)
O
COOH
PE derivative
NH2
O
R
R
R
1a N3
2a
COOH
COOH
OH
(1b)
O
N3
F3C
N N
COOH
(3d)
COOH
OH
3a
F3C
(2b)
PAA derivatives
O
N
N
O2N
(4d)
4a NO2
PLA derivatives
2.2.4. Inoculation of 4-nitro-L-Phenylalanine as a Nitrogen Source for Klebsiella sp. CK6
4-Nitro-L-phenylalanine (4a), which was a synthetic intermediate for the preparation of
4-azide-L-phenylalanine, was fed to Klebsiella sp. CK6 to reveal a substitution effect for the three
photophores. The nitrated PLA derivative 4d was identified as the sole metabolite, similar to that of
4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-L-phenylalanine. No optical rotation was observed for the
metabolite. These results indicated that electron-withdrawing substitution at the 4-position promoted
the formation of PLA-type compounds as major metabolites (Scheme 2).
The electron-donating group at the 4-position of the phenyl ring (azide and benzoyl) promoted
stabilization of the α-keto acid skeletons by their conjugations via the phenyl ring, and decarboxylation
(phenylacetaldehyde) was preferred over reduction of keto group to hydroxyl group (phenyllactic
acid). No phenyllactic acid derivatives were observed from the supplementation of these two

Molecules 2013, 18
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photophore-containing L-phenylalanine derivatives. Phenylacetaldehyde intermediates were oxidized
to phenylacetic acid in the same metabolic pathway as unsubstituted L-phenylalanine. However, the
bicyclic skeleton in 4-benzoyl-L-phenylalanine promoted the reduction of an aldehyde to a hydroxyl
group, which was only observed in the L-tryptophan metabolic pathway. 4-Benzoyl-L-phenylalanine
was metabolized to phenylethanol and phenylacetic acid. On the other hand, 4-[3-(trifluoromethyl)-
3H-diazirin-3-yl] substitution represents an electron-withdrawing group [13]. This property promoted
the reduction of the α-keto group, which was preferred over decarboxylation, and afforded phenyllactic
acid derivatives as the sole product. 4-Nitro-L-phenylalanine, which has an electron-withdrawing
group, was also metabolized to a phenyllactic acid skeleton.
3. Experimental
3.1. General Procedures
NMR spectra were measured on JEOL EX-280 or Bruker AMX500 spectrometers. All solvents
were of reagent grade and purified using the appropriate methods. ESI-TOF-MS and FD-MS data were
obtained with a Waters UPLC ESI-TOF and JEOL JMS-T100GCV mass spectrometer, respectively.
Chiral HPLC for synthetic photoreactive L-phenylalanine derivatives was achieved using a Chirobiotic
T column (Astec, Bellefonte, PA, USA) in 10% CH3OH-H2O.
3.2. Synthesis of 4-azido-L-phenylalanine (1a, Scheme 3)
A mixed solution of concentrated HNO3 (60%) and concentrated H2SO4 (1.4:1.1 v/v) was prepared
and chilled to 10 °C. Then, a portion of this solution (3.5 mL) was added dropwise under stirring to a
solution of L-phenylalanine (4.139 g, 25.1 mmol) in H2SO4 (98%, 12.5 mL). The reaction mixture was
stirred at 10 °C for 2.5 h. The reaction solution was then adjusted to pH 5 with NH4OH. The pale
yellow precipitate formed was collected by filtration, washed with a small volume of water and
CH3CN, and then dried to yield 4-nitro-L-phenylalanine as a yellow powder (4.748 g, 90%).
4-Nitro-L-phenylalanine (2.006 g, 9.54 mmol) was suspended in water (35 mL) and subjected to
hydrogenation at room temperature for 3 h in the presence of 0.2 g of 5% palladium-barium sulfate.
The catalyst was filtered through a Celite pad, and the pale brown filtrate was concentrated. The
residue was washed with CH3CN to afford a pale brown mass (1.460 g, 85%). No further purification
was performed for the following step. The 4-amino-L-phenylalanine derivative (0.060 g, 0.33 mmol)
was dissolved in 6N HCl (4 mL). Sodium nitrate in water (0.030 g/0.5 mL) was added at 0 °C. The
reaction mixture was stirred at the same temperature for 40 min and diluted with 6N HCl (0.25 mL).
Subsequently, sodium azide in water (0.033 g/0.75 mL) was added at 0 °C. The reaction mixture was
stirred at same temperature for 5 min, and then warmed to room temperature for 1 h, then
concentrated. The residue was reprecipitated from CH3CN to afford a pure colorless amorphous mass
(0.0435 g, 64%). Analytical and spectroscopic data were identical to those reported in the literature [10].
3.3. Synthesis of 4-benzoyl-L-phenylalanine (2a, Scheme 3)
Trifluoromethanesulfonic acid (0.5 mL) was added to L-phenylalanine (16.5 mg, 0.1 mmol) in a
tube fitted with a screw cap and PTFE-faced rubber liner. After the solution became homogeneous,

Molecules 2013, 18
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benzoyl chloride (8 equivalents) was added at 0 °C. The reaction mixture was stirred at room
temperature for 12 h, then poured into cold water and ethyl acetate. The organic layer was washed with
saturated NaCl, and dried over MgSO4, then filtrated. The filtrate was concentrated and the residue
was subjected to silica column chromatography (ethyl acetate-MeOH-H2O = 8:1:0.5) to afford
colorless amorphous mass (7.5 mg, 28%, 98% ee). 1H-NMR (CD3OD): δ 7.76 (d, 2H, J = 8.6 Hz, Ar),
7.75 (d, 2H, J = 8.6 Hz, Ar), 7.64 (t, 1H, J = 7.4 Hz, Ar), 7.52 (t, 2H, J = 7.4 Hz, Ar), 7.47 (d, 2H,
J = 8.0 Hz, Ar), 3.83 (m, 1H, Hα), 3.35 (m, 1H, Hβ), 3.09 (m, 1H, Hβ).
Scheme 3. Preparations of p-photophore substituted L-phenylalanine derivatives.
1) HNO3, H2SO4, <10 °C, 2.5h
2) H2 / Pd-BaSO4, H2O, rt, 3h
COOH
3) NaNO2, 6M HCl, 0 °C, 1h
4) NaN3, 6M HCl, rt, 1h
NH2
N3
1a
2a
COOH
NH2
COOH
NH2
BzCl
L-Phenylalanine (L-Phe)
TfOH, rt, 12 h
O
tBu
Br
1)
Br
N
P
N
N
H
F3C
N
N
N
5
O
N N
+
,
COOH
NH2
CH2Cl2, -78 ¡C, 8 h
7
8
F3C
N N
Ph
N
COOH
Ph
6
2) TFA, 0 °C, 2h
3a
3.4. Synthesis of 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-L-phenylalanine (3a, Scheme 3)
The synthesis of title compound was performed according to our previous asymmetric synthesis
protocols with slightly modifications [11,12]. Briefly, the diazirinyl benzyl bromide (5, 30.0 mg,
0.10 mmol) was asymmetrically condensed with diphenyliminoglycine t-butyl ester (6, 40.8 mg,
0,14 mmol) in the presence of catalytic cinchonidium salt (8) and phosphazene base BTPP (7) at −78 °C.
The reaction mixture was directly deprotected with TFA to minimize losses due to purification. The
reaction mixture was subjected to silica column chromatography (ethyl acetate: MeOH: H2O = 8:1:0.5)
1
to afford colorless solid (7.7 mg, 28%, 98% ee). H-NMR (CD3OD): δ 7.47 (d, 2H, J = 7.6 Hz, Ar),
7.26 (d, 2H, J = 7.6 Hz, Ar), 3.84 (m, 1H, Hα), 3.40 (m, 1H, Hβ), 3.10 (m, 1H, Hβ).
3.5. Culture Medium and Growth Conditions
Culture medium and growth conditions were described previously [8]. Phenylalanine derivatives in
DMSO: H2O (1:1; 75 mg/L) were filtered using a filter unit and then supplemented. The inoculated
mixture formed the standing culture under conditions of 28 °C and darkness for 7 days.

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3.6. Extraction of Metabolites
After incubation, cultured media were centrifuged at 3,500 × g for 10 min. The supernatant was
acidified with aqueous HCl to pH 2.5 and it was then extracted with equal volumes of ethyl acetate
three times. The organic layer was dried over MgSO4, filtered, and concentrated to afford crude
extracts. The crude extracts were subjected to silica column chromatography (pre-washed with
methanol and ethyl acetate-methanol-water in a ratio of 4:1:1) to afford pure metabolites.
3.7. 2-(4-Azidophenyl)acetic Acid (1b)
Inoculation of 4-azido-L-phenylalanine (1a, 5.0 mg) afforded a phenylacetic acid type metabolite
1
(2.3 mg). The analytical data was identical with previous reports [14]. H-NMR (CDCl3): δ 7.28
(d, 2H, J = 7.8 Hz, Ar), 7.01 (d, 2H, J = 7.8 Hz, Ar), 3.65 (m, 2H, 2-H), IR (film): 2150 cm−1, ESI-MS
(negative): m/z 176 [M−H]−.
3.8. 2-(4-Benzoylphenyl)acetic acid (2b) and (4-(2-hydroxyethyl)phenyl)(phenyl)methanone (2c)
Inoculation of 4-benzoyl-L-phenylalanine (2a, 10.0 mg) afforded phenylacetic acid (2.3 mg) and
phenylethanol type metabolites (2.2 mg). Analytical and spectroscopic data were identical to those
reported in the literature [15].
2-(4-Benzoylphenyl)acetic acid (2b) 1H-NMR (CDCl3): δ 7.80 (m, 4H, Ar), 7.58 (d, 1H, J = 7.8 Hz, Ar),
7.49 (d, 2H, J = 7.8 Hz, Ar), 7.42 (d, 2H, J = 7.8 Hz, Ar), 3.68 (m, 2H, 2-H), FD-MS: m/z 240 [M]−.
1
(4-(2-Hydroxyethyl)phenyl)(phenyl)methanone (2c) H-NMR (CDCl3): δ 7.77 (m, 4H, Ar), 7.60 (d,
1H, J = 7.8 Hz, Ar), 7.48 (d, 2H, J = 7.8 Hz, Ar), 7.36 (d, 2H, J = 7.8 Hz, Ar), 3.93 (t, 2H, J = 7.8 Hz,
CH2), 2.97 (m, 2H, J = 7.8 Hz, CH2), FD-MS: m/z 226 [M]−.
3.9. 2-Hydroxy-3-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]propanoic Acid (3d)
Inoculation of 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-L-phenylalanine (3a, 10.0 mg) afforded a
1
phenyllactic acid type metabolite (2.3 mg). H-NMR (CD3OD): δ 7.38 (d, 2H, J = 7.8 Hz, Ar), 7.12
(d, 2H, J = 7.8 Hz, Ar), 4.20 (m, 1H, 2-H), 3.46 (m, 1H, 3-Ha), 3.15 (m, 1H, 3-Hb), ESI-MS
(negative): m/z 273 [M−H]−, 246 [M−N2−H]−. λmax (ε) (CH3OH) 355 (380).
3.10. 2-Hydroxy-3-(4-nitrophenyl)propanoic Acid (4d)
Inoculation of 4-nitro-L-phenylalanine (4a, 15.0 mg) afforded a phenyllactic acid type metabolite
(2.3 mg). 1H-NMR (CD3OD): δ 8.15 (d, 2H, J = 7.8 Hz, Ar), 7.51 (d, 2H, J = 7.8 Hz, Ar), 4.37 (m, 1H,
2-H), 3.56 (m, 1H, 3-Ha), 3.03 (m, 1H, 3-Hb), ESI-MS (negative): m/z 210 [M−H]−.
4. Conclusions
Inoculation of L-phenylalanine afforded different metabolites in Klebsiella sp CK6 compared with
L-tryptophan. Both lactic acid (ILA and PLA) and acetic acid (IAA and PAA) skeletons are common

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structural units between L-phenylalanine and L-tryptophan. However, no ethanol skeleton was detected
after the inoculation of L-phenylalanine. These results indicated that a bicyclic (indole) moiety was
important for the enzymatic reduction of acetaldehyde moiety under TOL-type metabolism conditions.
The stereochemistry of the precursor phenylalanine preferred the L-configuration rather than the
D-configuration. Based on the analysis of phenylalanine metabolites, the photophore-substituted
L-phenylalanines participated in the metabolic pathway. The photochemical properties of the
photophores—phenyl azide, benzophenone, and trifluoromethylphenyldiazirine—have been compared
to assess the success of photoaffinity labeling to target biomolecules. There is no doubt that
photophore-introduced phenylalanines afforded the same metabolites, and, in addition, most of the
photoreactive derivatives acted in an identical manner as phenylalanine. This study presents the first
observations of a comprehensive metabolite analysis of different photophores containing L-phenylalanine
derivatives and the different metabolites afforded by each photophore. These results indicated that
there is possibility for different photoaffinity labeling of metabolic enzymes, which can be achieved
using different photophore-containing phenylalanine derivatives.
Acknowledgments
This research was partially supported by the Ministry of Education, Science, Sports and Culture
Grant-in-Aid for Scientific Research (C), (19510210, 21510219 to MH). MH also thanks the Fugaku
Foundation for financial support. Part of this work was performed under the Cooperative Research
Program of “Network Joint Research Center for Materials and Devices”.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1a–4a are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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