Identification of N epsilon-[(R)-1-carboxyethyl]-L-lysine in, and the complete structure of, the repeating unit of the O-specific polysaccharide of Providencia alcalifaciens O23.

Article abstract of DOI:10.1016/S0008-6215(98)00083-4

N epsilon-[(R)-1-Carboxyethyl]-L-lysine was released by acid hydrolysis from the O-specific polysaccharide of Providencia alcalifaciens O23 and identified by 1H and 13C NMR spectroscopy, GLC-MS after conversion to a di-N-acetylated dimethyl ester, and by comparison with the authentic sample. Solvolysis of the polysaccharide with anhydrous HF resulted in an amide of D-glucuronic acid with N epsilon-[(R)-1-carboxyethyl]-L-lysine. These and published data allowed the determination of the full structure of the repeating unit of the O-specific polysaccharide.

Full text of DOI:10.1016/S0008-6215(98)00083-4

Carbohydrate Research 309 (1998) 131±133
Note
Identi®cation of N"-[(R)-1-carboxyethyl]-l-lysine
in, and the complete structure of, the repeating
unit of the O-speci®c polysaccharide of
Providencia alcalifaciens O23
Nina A. Kocharova, Evgeny V. Vinogradov, Svetlana A. Borisova,
Alexander S. Shashkov, Yuriy A. Knirel *
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow 117913, Russian
Federation
Received 9 January 1998; accepted 10 March 1998
Abstract
N"-[(R)-1-Carboxyethyl]-l-lysine was released by acid hydrolysis from the O-speci®c poly-
1
saccharide of Providencia alcalifaciens O23 and identi®ed by H and 13C NMR spectroscopy,
GLC-MS after conversion to a di-N-acetylated dimethyl ester, and by comparison with the
authentic sample. Solvolysis of the polysaccharide with anhydrous HF resulted in an amide of d-
glucuronic acid with N"-[(R)-1-carboxyethyl]-l-lysine. These and published data allowed the
determination of the full structure of the repeating unit of the O-speci®c polysaccharide. # 1998
Elsevier Science Ltd. All rights reserved
Keywords: Providencia alcalifaciens; O-speci®c polysaccharide; O-antigen; N"-[(R)-1-Carboxyethyl]-l-lysine;
d-Glucuronic acid amide
Providencia is a genus within the family Entero-
bacteriaceae. On the basis of somatic antigens
(lipopolysaccharides) two species, P. alcalifaciens
and P. stuarti, were classi®ed into 62 O-serogroups
[1]. Providencia is among the least studied entero-
bacteria with respect to the lipopolysaccharide
structure. Recently, we have found an amide of d-
glucuronic acid with N"-(1-carboxyethyl)lysine in
the O-speci®c polysaccharide chain (O-antigen) of
the P. alcalifaciens O23 lipopolysaccharide [2±4].
The structure of the polysaccharide was established
by 2D NMR spectroscopy and selective degrada-
tions (partial acid hydrolysis and solvolysis with
anhydrous HF) [3,4], but the con®guration of the
unusual amino acid remained unknown. Now, we
report on the identi®cation of this component,
including the determination of the absolute
con®guration.
The O-speci®c polysaccharide was isolated as
described [4] and hydrolyzed with 2 M CF3COOH
ꢀ
(121 C, 2 h) to give d-Glc, d-Gal, d-GalN and
d-GlcA as well as a neutral amino acid 1 which was
isolated by preparative PC using the solvent system
5:5:1:3 ethyl acetate±pyridine±acetic acid±water.
* Corresponding author.
0008-6215/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00083-4

132
N.A. Kocharova et al./Carbohydrate Research 309 (1998) 131±133
1
The H NMR spectrum of 1 revealed spin systems
for lysine and alanine (Table 1). Correspondingly,
the 13C NMR spectrum of 1 (Table 1) contained
signals for both amino acids but those for C-6 of
lysine and C-20 of alanine were shifted signi®cantly
down®eld to ꢀ 46.97 and 59.05, as compared with
their positions at ꢀ 40.6 and 51.6 in the spectra of
the corresponding free amino acids.
present in the spectrum of a mixture of 1 and N"-
[(S)-1-carboxyethyl]-l-lysine, the most marked dif-
ference being observed for the C-4 chemical shifts
(Table 1)1. Therefore, the amino acid released from
the O-speci®c polysaccharide of P. alcalifaciens
O23 is N"-[(R)-1-carboxyethyl]-l-lysine.
Cleavage of the polysaccharide with anhydrous
ꢀ
HF (20 C, 2 h) gave an amide 3 isolated by GPC
These data suggested that 1 is N"-(1-carboxy-
ethyl)lysine, which was con®rmed by GLC±MS
analysis of a di-N-acetylated dimethyl ester 2
derived from 1. CIMS revealed for 2 the expected
molecular mass of 330 a.m.u. The EI mass spec-
trum of 2 showed peaks at m/z 330 (M), 298 (M±
MeOH), 287 (M±Ac), 271 (M±COOCH3), 229 (M±
COOCH3±CH2CO), and 211 (M±COOCH3±HOAc).
A positive optical rotation value for 1, [ꢁ]d+4.9ꢀ
(c 0.5, water), showed that the lysine residue has
the l con®guration {compare published data [5]:
[ꢁ]d+9.7ꢀ and +11.6ꢀ (water) for N"-[(R)-1-car-
boxyethyl]-l-lysine and N"-[(S)-1-carboxyethyl]-l-
lysine, respectively}. In order to determine the
con®guration of the 1-carboxyethyl group, both
stereoisomers of N"-(1-carboxyethyl)-l-lysine were
synthesized by condensation of Na-carbobenzoxy-
l-lysine with (S)- and (R)-2-bromopropionic acid
followed by deprotection essentially as described
[5].
on TSK HW-40 in water. The H and 13C NMR
1
spectra of 3 contained signals for ꢁ-GlcpA, ꢂ-
GlcpA and N"-(1-carboxyethyl)lysine. The signal
for H-2 of the lysine residue was shifted down®eld
to ꢀ 4.4, as compared with its position at ꢀ 3.78 in
the spectrum of 1, thus indicating acylation at N-2.
Accordingly, C-6 of GlcA resonated at ꢀ 170.0 that
is characteristic for hexuronamides (e.g., ref 7).
The structure of 3 was ®nally con®rmed by GLC±
MS analysis of a gulonamide derivative 4 derived
from 3. CIMS proved for 4 the molecular mass of
676 a.m.u., and EIMS revealed the same fragmen-
tation in the amino acid moiety as in 2 with no
signi®cant fragmentation in the gulonic acid resi-
due.
The synthetic diastereomers and the natural
amino acid 1 were converted into ammonium salts
by absorption on Dowex 50Â4 (H+ form) resin
followed by elution with aq 5% ammonia, and
then studied by 13C NMR spectroscopy (for refer-
ence data, see [6]). The spectrum of a mixture of 1
and N"-[(R)-1-carboxyethyl]-l-lysine and the spec-
tra of the individual compounds were indis-
tinguishable, while two series of signals were
Table 1
1
500-MHz H and 125-MHz 13C NMR data (ꢀ, ppm). Spectra
were run for solutions of NH4-salts in D2O at 20 ꢀC, chemical
shifts are referred to acetone (ꢀH 2.225, ꢀC 31.45)
Proton
H-2
H-3 H-4 H-5 H-6 H-20 H-30
Amino acid 1
3.78
1.94 1.52 1.80 3.09 3.70
1.52
C-10
Carbon
C-1
C-2 C-3 C-4 C-5 C-6
C-20 C-30
N"-[(R)-1-Carboxyethyl]-l-lysine and amino acid 1
176.15a 55.70 31.11 22.77 26.64 46.97 175.82a 59.05 16.28
N"-[(S)-1-Carboxyethyl]-l-lysine
1 We found that the assignment of the C-3 and C-5 signals
previously reported for these compounds [6] was erroneously
intercharged.
176.15a 55.70 31.14 22.82 26.67 46.99 175.82a 59.08 16.28
a
Assignment could be interchanged.

N.A. Kocharova et al./Carbohydrate Research 309 (1998) 131±133
133
Therefore, the polysaccharide studied contains
N"-[(R)-1-carboxyethyl]-Na-(d-glucuronoyl)-l-lysine
(d-GlcA6AlaLys). Taking into account the struc-
ture of the carbohydrate backbone of the poly-
saccharide established earlier by 2D NMR
spectroscopy and chemical methods [4], it was
concluded that the repeating unit of the O-antigen
of P. alcalifaciens O23 has the following structure:
scopy and GLC-MS, respectively. This work was
supported by grant 96-04-50460 of the Russian
Foundation for Basic Research.
References
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!6†-ꢂ-d-Galp-ꢀ1!6†-ꢂ-d-Glcp-ꢀ1!3†-
ꢂ-d-GalpNAc-ꢀ1!4†-ꢂ-d-GlcpA6AlaLys-ꢀ1!
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contain N"-[(R)-1-carboxyethyl]-l-lysine. A dia-
stereomeric amino acid, N"-[(S)-1-carboxyethyl]-l-
lysine, has been found to be produced by Strepto-
coccus lactis K1 during growth in an arginine-de®-
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unknown con®guration has been reported as a
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Acknowledgements
The authors thank Mr. G.V. Zatonsky (N.D.
Zelinsky Institute of Organic Chemistry, Moscow,
Russia) and Mr. H. Moll (Forschungszentrum
Borstel, Germany) for help with NMR spectro-
[8] A.S. Shashkov, F.V. Toukach, S.N. Senchenkova,
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