Synthesis and immunomodulating activity of lipophilic analogs of N-acetylnormuramoyl-L-2-aminobutanoyl-D-isoglutamine

Article abstract of DOI:10.1135/cccc19980590

N-Acetylnormuramoyl-L-2-aminobutanoyl-D-isoglutamine (7) and its lipophilic 6-O-octadecanoyl (8) and 6-O-(2-tetradecylhexadecanoyl) (9) derivatives were prepared and their immunoadjuvant activity and pyrogenicity were tested. Compounds 8 and 9 are less pyrogenic than muramoyl-dipeptide (MDP) and norMDP analog 7. Both lipophilic derivatives 8 and 9 are better adjuvants than MDP in cell mediated immunity.

Full text of DOI:10.1135/cccc19980590

590
Ledvina, Jezek, Saman, Hribalova:
SYNTHESIS AND IMMUNOMODULATING ACTIVITY OF LIPOPHILIC
ANALOGS OF N-ACETYLNORMURAMOYL-L-2-AMINOBUTANOYL-
D-ISOGLUTAMINE*
a1
a2
b
Miroslav LEDVINA , Jan JEZEK , David SAMANa3 and Vera HRIBALOVA
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
166 10, Prague 6, Czech Republic; e-mail: 1 ledvina@uochb.cas.cz, 2 jezek@uochb.cas.cz,
3 saman@uochb.cas.cz
b National Institute of Public Health, 100 42 Prague 10, Czech Republic; e-mail: bohukriz@mbox.cesnet.cz
Received January 15, 1998
Accepted March 14, 1998
N-Acetylnormuramoyl-L-2-aminobutanoyl-D-isoglutamine (7) and its lipophilic 6-O-octadecanoyl (8)
and 6-O-(2-tetradecylhexadecanoyl) (9) derivatives were prepared and their immunoadjuvant activity
and pyrogenicity were tested. Compounds 8 and 9 are less pyrogenic than muramoyl-dipeptide
(MDP) and norMDP analog 7. Both lipophilic derivatives 8 and 9 are better adjuvants than MDP in
cell mediated immunity.
Key words: Carbohydrates; Glycosides; Aminosugars; Muramyl glycopeptides; Immunoadjuvants.
A recess from natural vaccines and changeover to semisynthetic, recombinant and syn-
thetic vaccines craved a development of nontoxic, structurally defined adjuvants. In
this connection, attention has been focused to the molecule of N-acetylmuramoyl-L-ala-
nyl-D-isoglutamine (muramoyl-dipeptide, MDP), a minimal unit of peptidoglycan of
bacterial cell wall, which still possess immunoadjuvant activity. A large number of
MDP analogs was synthesized with the aim to suppress undesirable side effects, namely
pyrogenicity, and to potentiate immunoadjuvant activity (for review, see refs1,2) Intro-
duction of bulky lipophilic groups onto 6-OH group of the sugar unit led to a decrease
in pyrogenicity and an increase in immunoadjuvant activity, especially for cellular im-
munity2–5. These results and the fact that N-acetylnormuramoyl-L-2-aminobutanoyl-D-
isoglutamine, an analog of MDP modified both in the sugar and peptide parts, is less
pyrogenic and has a higher immunoadjuvant activity6,7 motivated us to prepare its lipo-
philic analogs bearing bulky acyl groups on the primary OH group. The aim was to
* Normuramic acid is the trivial name for 2-amino-3-O-carboxymethyl-2-deoxy-D-glucopyranose. The
symbols and abbreviations obey the published recommendations (Biochemical Nomenclature and
Related Documents. International Union of Biochemistry, London 1978).
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Synthesis of Analogs of Muramyl Glycopeptides
591
obtain new adjuvants with better immunopharmacological parameters compared with
the mentioned lipophilic derivatives of MDP, and to improve their incorporation into
liposomes.
As a starting compound for the preparation of the sugar unit 2, we utilized the pre-
viously described benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-methoxycar-
bonylmethyl-α-D-glucopyranoside (1; ref.8) (Scheme 1). Methyl ester 1 was hydrolyzed
with 0.2 M solution of sodium hydroxide in a mixture methanol–water (3 : 2) to give
benzyl 2-acetamido-3-O-carboxymethyl-2-deoxy-4,6-O-isopropylidene-α-D-glucopy-
ranoside (2). Coupling of acid 2 with trifluoroacetate of L-2-aminobutanoyl-D-isoglu-
tamine benzyl ester9 by means of N,N′-dicyclohexylcarbodiimide (DCC) and
1-hydroxybenzotriazole (HOBT) afforded N-[2-O-(benzyl 2-acetamido-2,3-dideoxy-
4,6-O-isopropylidene-α-D-glucopyranosid-3-yl)-glycoloyl]-L-2-aminobutanoyl-D-iso-
glutamine benzyl ester (3) in 66% yield. The isopropylidene protecting group was
removed from the glycopeptide 3 by heating with 50% acetic acid at 60 °C, yielding the
key intermediate N-[2-O-(benzyl 2-acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl)-gly-
coloyl]-L-2-aminobutanoyl-D-isoglutamine benzyl ester (4). Partial O-acylation10 of
compound 4 with stearic acid in the presence of N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (WSC) and 4-dimethylaminopyridine (DMAP) in N,N-
dimethylformamide (molar ratios 1 : 1.3 : 1.9 : 2; WSC and DMAP were added in two
portions) at room temperature gave N-[2-O-(benzyl 2-acetamido-2,3-dideoxy-6-O-octa-
decanoyl-α-D-glucopyranosid-3-yl)glycoloyl]-L-2-aminobutanoyl-D-isoglutamine
O
O
O
O
(CH ) C
3 2
(CH ) C
3 2
O
O
OBzl
NHAc
CH COOR
OBzl
NHAc
O
O
CH CO-L-Abu-D-isoGln(OBzl)
2
2
1, R = CH
3
3
2, R = H
RO
RO
O
O
O
O
OH
OBzl
NHAc
CH CO-L-Abu-D-isoGln(OBzl)
HO
HO
NHAc
CH CO-L-Abu-D-isoGln
2
2
7, R = H
4, R = H
5, R = CH (CH ) CO
8, R = CH (CH ) CO
3
2 16
3
2 16
9, R = [CH (CH ) ] CHCO
6, R = [CH (CH ) ] CHCO
3
2 13 2
3
2 13 2
Bzl = benzyl
SCHEME 1
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592
Ledvina, Jezek, Saman, Hribalova:
benzyl ester (5) in 48% yield. Introduction of fatty acid branched on C-2 required a
higher reaction temperature. In the same way promoted reaction of compound 4 with
2-tetradecylhexadecanoic acid11 at 50 °C afforded the required N-{2-O-[benzyl 2-acet-
amido-2,3-dideoxy-6-O-(2-tetradecylhexa-decanoyl)-α-D-glucopyranosid-3-yl]glycoloyl}-
L-2-aminobutanoyl-D-isoglutamine benzyl ester (6) in 54% yield. Hydrogenolysis of
compound 4 on Pd/C catalyst in acetic acid afforded N-acetylnormuramoyl-L-2-amino-
butanoyl-D-isoglutamine (7). This approach is an alternative to described procedure6. Hy-
drogenolysis of benzyl protecting groups in compounds 5 and 6 as described above
afforded the target 6-O-octadecanoyl-N-acetylnormuramoyl-L-2-aminobutanoyl-D-iso-
glutamine (8) and 6-O-(2-tetradecylhexadecanoyl)-N-acetylnormuramoyl-L-2-amino-
butanoyl-D-isoglutamine (9).
Pyrogenicity was tested on Chinchilla rabbits in doses of 40, 200 and 1 000 nmol per
rabbit. In the tested series of MDP and its derivatives, pyrogenicity decreases in the
order MDP > norMDP analog 7 > stearoyl derivative 8 > 2-tetradecylhexadecanoyl
derivative 9. Compound 9 is apyrogenic even at the highest dose tested.
The adjuvant activity was tested by induction of experimental allergic encephalo-
myelitis, which is, besides the delayed type skin reaction, the most frequently used test
for the adjuvant activity of muramoyl peptides in cell-mediated immunity. In this test,
the adjuvant activity of stearoyl derivative 8 was lower than that of norMDP analog 7 and
higher than that of MDP, whereas the 2-tetradecylhexadecanoyl derivative 9 was the
most active compound from all compounds tested. Details of the above mentioned data
and other immunopharmacological parameters will be published elsewhere.
EXPERIMENTAL
Melting points were determined on a Kofler block and are uncorrected. Optical rotations were
measured on a Perkin–Elmer 141 polarimeter at 22 °C. NMR spectra were recorded with a Varian
UNITY-500 spectrometer in the FT mode at 499.8 MHz (1H) and at 125.6 MHz (13C) in CD3SOCD3,
using the central line of solvent for standardization (δ 2.50 for 1H and 39.5 for 13C). Chemical shifts
are given in ppm (δ-scale) and coupling constants (J) in Hz. Positive-ion FAB mass spectra were
measured on a BEqG geometry mass spectrometer ZAB-EQ (VG Analytical, Manchester, U.K.),
using an M-Scan FAB gun (Xe, energy 8 keV) at an accelerating voltage of 8 kV. Samples were
dissolved in chloroform or methanol, and the mixture glycerol–thioglycerol was used as matrix. Thin-
layer chromatography (TLC) was performed on Silufol UV254 sheets, and column chromatography on
silica gel Silpearl (both Kavalier, Votice, Czech Republic). Analytical RP HPLC was performed with
a Spectra-Physics 8700 apparatus (Darmstadt, Germany) equipped with a column (250 × 4 mm) of
Separon SGX-RPS (C18), particle size 10 nm (Tessek, Prague, Czech Republic). Preparative RP
HPLC was performed with a Knauer apparatus (Bad Homburg, Germany) equipped with a column
(250 × 10 mm) filled with Separon SGX-RPS (C18), particle size 10 nm (Tessek, Prague, Czech
Republic). Solutions were evaporated on rotatory vacuum evaporator. Samples for amino acid analysis
were hydrolyzed in 4 M HCl at 110 °C for 8 h and analyzed on D-500 Durrum Amino Acid Analyzer
(Durrum Corporation, Palo Alto, CA). Analytical samples were dried at 6.5 Pa and 25 °C for 8 h.
Dichloromethane was distilled from phosphorus pentoxide and stored over molecular sieves 4A.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

Synthesis of Analogs of Muramyl Glycopeptides
593
Benzyl 2-Acetamido-3-O-carboxymethyl-2-deoxy-4,6-O-isopropylidene-α-D-glucopyranoside (2)
To a stirred solution of methyl ester8 1 (4.235 g, 10 mmol) in MeOH (60 ml) 0.5 M NaOH (40 ml, 20 mmol)
was added. After 4 h the mixture was neutralized by addition of Dowex 50 (pyridinium cycle). The
ion-exchanger was filtered off, washed with 50% MeOH (3 × 50 ml) and the filtrate was evaporated
in vacuo. The residue was coevaporated with benzene (3 × 100 ml) and lyophilized from benzene.
Yield 4.05 g (99%) of compound 2, TLC-homogeneous in solvent systems ethyl acetate–toluene (2 : 1)
and in butanol–acetic acid–water (4 : 1 : 1), [α]D +90° (c 0.6, chloroform). For C20H27NO8 calcu-
lated: relative molecular mass 409.4, monoisotopic mass 409.1. FAB MS, m/z: 410.1 [M + H]+, 432.1
[M + Na]+. For C20H27NO8 (409.4) calculated: 58.67% C, 6.64% H, 3.42% N; found: 58.75% C,
6.50% H, 3.57% N.
N-[2-O-(Benzyl 2-Acetamido-2,3-dideoxy-4,6-O-isopropylidene-α-D-glucopyranosid-3-yl)-
glycoloyl]-L-2-aminobutanoyl-D-isoglutamine Benzyl Ester (3)
A
solution of tert-butoxycarbonyl-L-2-aminobutanoyl-D-isoglutamine benzyl ester9 (4.22 g,
10.0 mmol) in a mixture of dichloromethane–trifluoroacetic acid (1 : 1, 40 ml) was kept at room
temperature for 30 min. The mixture was evaporated, the syrupy residue was triturated with diethyl-
ether (3 × 50 ml) and the insoluble portion was coevaporated with CH2Cl2 (2 × 50 ml) and dried at
room temperature and 1.32 Pa for 2 h. The syrup obtained was dissolved in DMF (20 ml) and the
resulting solution of the trifluoroacetate of L-2-aminobutanoyl-D-isoglutamine benzyl ester was used
immediately for coupling with the acid 2.
To a stirred solution of 2 (4.094 g, 10 mmol) and 1-hydroxybenzotriazole monohydrate (1.531 g,
10 mmol) in a mixture of DMF (10 ml) and dichloromethane (40 ml) at 0 °C and 1 M solution of
N,N′-dicyclohexylcarbodiimide in dichloromethane (10 ml, 10 mmol), was added. After 1 h at 0 °C,
the precipitated N,N′-dicyclohexylurea was filtered off (2 g, 89%), and the filtrate was concentrated
to approximately 20 ml. The above mentioned solution of the trifluoroacetate of L-2-aminobutanoyl-
D-isoglutamine benzyl ester and N,N-diisopropylethylamine (1.88 ml, 11 mmol) were added at 0 °C
and the mixture was stirred for 1 h at 0 °C and kept overnight at room temperature (pH must be
7–8). Ethyl acetate (500 ml) was added and the overnight precipitated N,N′-dicyclohexylurea was fil-
tered off. The filtrate was cooled in an ice bath and extracted with a saturated solution of sodium
hydrogen carbonate (3 × 100 ml) and 5% solution of sodium chloride (3 × 100 ml), dried over
anhydrous magnesium sulfate and concentrated to approximately 30 ml. Petroleum ether (80 ml) was
added, the mixture was left overnight at +3 °C and the separated crystalline product (6.46 g) was
recrystallized from a mixture of ethyl acetate and petroleum ether. Yield 4.71 g (66%) of compound 3,
TLC homogeneous in solvent systems chloroform–methanol (10 : 1) and butanol–acetic acid–water
(4 : 1 : 1); m.p. 136–137 °C, [α]D +63° (c 0.6, chloroform). For 1H and 13C NMR data, see Tables I and II.
For C36H48N4O11 calculated: relative molecular mass 712.8, monoisotopic mass 712.3. FAB MS, m/z:
713.6 [M + H]+. Amino acid analysis: glutamic acid 1.03, 2-aminobutanoic acid 1.04, normuramic
acid 0.96. For C36H48N4O11 (712.8) calculated: 60.66% C, 6.78% H, 7.86% N; found: 60.49% C,
6.88% H, 7.65% N.
N-[2-O-(Benzyl 2-Acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl)glycoloyl]-L-2-aminobutanoyl-
D-isoglutamine Benzyl Ester (4)
The fully protected compound 3 (6.36 g, 8.92 mmol) was heated under stirring in 50% aqueous
acetic acid (140 ml) at 60 °C for 2 h. The solvents were evaporated and the residue was coevaporated
with toluene (3 × 100 ml). Diethyl ether and petroleum ether were added and the mixture was left in
a refrigerator overnight. The solid product was chromatographed on a silica gel C18 column in a
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Collect. Czech. Chem. Commun. (Vol. 63) (1998)

Synthesis of Analogs of Muramyl Glycopeptides
595
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596
Ledvina, Jezek, Saman, Hribalova:
solvent system water–methanol (linear gradient: 50→80%/60 min), yield 4.8 g (80%) of solid pro-
duct 4, which was crystallized from a mixture methanol–ether–petroleum ether. Yield 4.25 g (71 %)
of compound 4; m.p. 208 °C, [α]D +63° (c 0.2, methanol). For 1H and 13C NMR data, see Tables I
and II. For C33H44N4O11 calculated: relative molecular mass 672.7, monoisotopic mass 672.3. FAB
MS, m/z: 673.4 [M + H]+, 695.3 [M + Na]+. For C33H44N4O11 (672.7) calculated: 58.91% C,
6.59% H, 8.32% N; found: 58.66% C, 6.48% H, 8.40% N.
TABLE II
13C NMR data of compounds 3–9 (CD3SOCD3, chemical shifts in ppm)
Compound
Carbon
3a
4b
5c
6d
7
8e
9f
C-1
97.0
52.8
77.4
73.9
63.6
61.5
70.5
173.0
53.3
25.6
9.7
96.1
52.8
80.8
69.7
73.1
60.5
70.6
173.0
53.9
25.0
9.9
96.2
52.7
80.5
69.9
70.1
63.0
70.7
172.8
53.9
25.1
9.9
96.2
52.7
80.5
70.1
70.2
63.0
70.6
173.0
53.8
25.1
9.9
90.8
53.4
80.7
69.9
72.2
60.7
70.5
174.1
54.0
25.0
10.0
173.3
52.9
26.8
30.5
171.4
169.4
–
90.8
53.2
80.5
69.3
70.0
63.3
70.6
173.8
53.8
25.1
9.9
90.8
53.2
80.5
69.4
69.9
62.7
70.5
173.8
53.9
25.0
9.9
C-2
C-3
C-4
C-5
C-6
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
C-7′
C-8′
C-9′
C-10′
C-11′
CH2Ph
172.1
51.5
27.0
30.1
171.0
169.3
65.5
68.7
169.6
22.5
172.2
51.5
26.7
30.1
171.3
169.5
65.5
68.0
170.5
22.5
172.1
51.6
26.7
30.1
171.3
169.5
65.4
68.3
170.4
22.5
172.1
51.5
26.7
30.1
171.3
169.4
65.4
68.4
170.4
22.4
173.2
51.6
26.8
30.2
171.3
169.4
–
173.1
51.6
26.8
30.2
171.3
169.3
–
–
–
–
NHCOCH3
170.7
22.6
170.5
22.6
170.6
22.6
a
C-1′′ 99.3, C-2′′ 19.1, C-3′′ 29.0, aromatic carbons: 137.6, 136.2, 128.4 (2 C), 128.3 (2 C), 128.0,
b
127.9 (2 C), 127.8 (2 C), 127.7; aromatic carbons: 137.7, 136.2, 128.4 (2 C), 128.3 (2 C), 128.0,
127.9 (2 C), 127.7 (2 C), 127.6; OCOC17H35: 173.1, 33.5, 31.5, 28.9 (9 C), 28.8 (3 C), 28.6, 28.4, 13.9
aromatic carbons: 137.4, 136.2, 128.3 (2 C), 128.2 (2 C), 127.9, 127.8 (4 C), 127.6; OCOC29H59
c
d
:
175.2, 44.7, 31.6, 31.2, 29.0 (10 C), 28.8 (5 C), 28.7 (5 C), 26.6 (3 C), 22.0, 13.8 (2 C), aromatic
e
carbons: 137.3, 136.1, 128.4 (2 C), 128.2 (2 C), 127.9, 127.8 (2 C), 127.7 (2 C), 127.6; OCOC17H35
:
f
172.9, 33.4, 31.2, 29.0 (9 C), 28.7 (3 C), 28.4, 24.4, 13.9; OCOC29H59: 175.4, 44.7, 31.6, 31.2, 29.0
(10 C), 28.9 (5 C), 28.8 (5 C), 26.6 (3 C), 22.0, 13.7 (2 C).
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Synthesis of Analogs of Muramyl Glycopeptides
597
N-[2-O-(Benzyl 2-Acetamido-2,3-dideoxy-6-O-octadecanoyl-α-D-glucopyranosid-3-yl)-glycoloyl]-
L-2-aminobutanoyl-D-isoglutamine Benzyl Ester (5)
To a stirred solution of compound 4 (1.345 g, 2 mmol) in dry N,N-dimethylformamide (80 ml)
stearic acid (740 mg, 2.6 mmol), WSC (728 mg, 3.8 mmol) and DMAP (489 mg, 4 mmol) were
added. After 6 h stirring at room temperature, another portion of WSC (728 mg, 3.8 mmol) and
DMAP (489 mg, 4 mmol) were added and the stirring continued for 14 h. The reaction course was
checked by TLC in chloroform–methanol (10 : 1). Methanol (5 ml) was added to the reaction mix-
ture, the mixture was stirred for 30 min at room temperature and the solvents were distilled off. The
residue was dissolved in CHCl3 (200 ml) and the solution was washed twice with a solution of po-
tassium dihydrogen phosphate, the pH of which was adjusted to 3 with sulfuric acid (KH2PO4 (6 g)
and concentrated H2SO4 (2 ml) in water (40 ml)) and twice with 5% solution of sodium chloride
(60 ml), dried over anhydrous magnesium sulfate and evaporated in vacuo. Chromatography of the
residue on silica gel C18 column in a solvent system water–methanol (linear gradient: 90→100%/60 min)
afforded 900 mg (48%) of solid compound 5, [α]D +33° (c 0.4, chloroform). For 1H and 13C NMR
data, see Tables I and II. For C51H78N4O12 calculated: relative molecular mass 939.2, monoisotopic
mass 938.6. FAB MS, m/z: 939.3 [M + H]+, 961.3 [M + Na]+. For C51H78N4O12 (939.2) calculated:
65.22% C, 8.37% H, 5.96% N; found: 65.41% C, 8.23% H, 6.13% N.
N-{2-O-[Benzyl 2-Acetamido-2,3-dideoxy-6-O-(2-tetradecylhexadecanoyl)-α-D-glucopyranosid-3-yl]-
glycoloyl}-L-2-aminobutanoyl-D-isoglutamine Benzyl Ester (6)
To a stirred solution of compound 4 (1.345 g, 2 mmol) in dry N,N-dimethylformamide (80 ml) 2-tetra-
decylhexadecanoic acid11 (1.177 g, 2.6 mmol), WSC (728 mg, 3.8 mmol) and DMAP (489 mg,
4 mmol) were added and the mixture was stirred at 50 °C for 6 h. Another portion of WSC (728 mg,
3.8 mmol) and DMAP (489 mg, 4 mmol) were added and the stirring continued at the same tempera-
ture for another 6 h. The reaction course was checked by TLC in chloroform–methanol (10 : 1).
After cooling to room temperature, methanol (5 ml) was added, the mixture was stirred at rooom
temperature for 30 min and the solvents were distilled off. The residue was dissolved in CHCl3
(200 ml) and the solution was washed as described above, dried over anhydrous magnesium sulfate
and evaporated in vacuo. Chromatography of the residue on silica gel C18 column in a solvent sys-
tem water–methanol (linear gradient: 95→100%/60 min) afforded 1.2 g (54%) of solid compound 6,
1
[α]D +30° (c 0.6, chloroform). For H and 13C NMR data, see Tables I and II. For C63H102N4O12 calcu-
lated: relative molecular mass 1 107.5, monoisotopic mass 1 106.7. FAB MS, m/z: 1 107.7 [M + H]+,
1 130.3 [M + Na]+. For C63H102N4O12 (1 107.5) calculated: 68.32% C, 9.28% H, 5.05% N; found:
68.52% C, 9.13% H, 5.15% N.
N-Acetylnormuramoyl-L-2-aminobutanoyl-D-isoglutamine (7)
Compound 4 (673 mg, 1 mmol) in acetic acid (150 ml) was hydrogenolyzed in the presence of 10%
Pd/C catalyst (200 mg) at room temperature for 24 h. The catalyst was filtered off, washed with
acetic acid (3 × 30 ml), and the filtrate was lyophilized. The product was chromatographed on a silica
gel C18 column in methanol–water (1 : 19). The homogeneous fractions corresponding to α- and β-
anomer in the ratio 2 : 1 were concentrated in vacuo and the residue was lyophilized from water, to
give 480 mg (91%) of dihydrate 7, [α]D +34° (c 0.5, methanol) and [α]D +11° (c 0.5, water, after 24 h).
Literature6: m.p. 133–144 °C (crystallization with 2.5 molecules of water), [α]D +33° (c 0.3, methanol).
1
For H and 13C NMR data, see Tables I and II. For C19H32N4O11 calculated: relative molecular mass
492.5, monoisotopic mass 492.2. FAB MS, m/z: 493.1 [M + H]+, 515.1 [M + Na]+. Amino acid ana-
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

598
Ledvina, Jezek, Saman, Hribalova:
lysis: glutamic acid 1.05, 2-aminobutanoic acid 1.03, normuramic acid 0.94. For C19H32N4O11 2 H2O
(528.5) calculated: 43.17% C, 6.86% H, 10.60% N; found: 43.21% C, 6.75%H, 10.74% N.
6-O-Octadecanoyl-N-acetylnormuramoyl-L-2-aminobutanoyl-D-isoglutamine (8)
Compound 5 (380 mg, 0.4 mmol) in acetic acid (50 ml) was hydrogenolyzed in the presence of 10%
Pd/C catalyst (400 mg) at room temperature for 16 h and worked up analogously to compound 7.
The lyophilized product was chromatographed on a silica gel C18 column in methanol–water (9 : 1).
The homogeneous fractions were concentrated in vacuo and the residue was lyophilized from acetic
1
acid to give 200 mg (66%) of compound 8, [α]D +17° (c 0.1, acetic acid). For H and 13C NMR data
see Tables I and II. For C37H66N4O12 calculated: relative molecular mass 759.0, monoisotopic mass
758.5. FAB MS, m/z: 759.4 [M + H]+, 781.4 [M + Na]+. For C37H66N4O12 (759.0) calculated:
58.55% C, 8.76% H, 7.38% N; found: 58.67% C, 8.61% H, 7.20% N.
[6-O-(2-Tetradecylhexadecanoyl)-N-acetylnormuramoyl]-L-2-aminobutanoyl-D-isoglutamine (9)
Compound 6 (1.2 g, 1.084 mmol) in acetic acid (50 ml) was hydrogenolyzed in the presence of 10%
Pd/C catalyst (400 mg) at room temperature for 16 h and worked up analogously to compound 7.
The lyophilized product was chromatographed on a silica gel C18 column in methanol–water (9 : 1).
Homogeneous fractions were concentrated in vacuo and the residue was lyophilized from acetic acid
1
to yield 698 mg (69%) of compound 9, [α]D +13° (c 1.0, acetic acid). For H and 13C NMR data, see
Tables I and II. For C49H90N4O12 calculated: relative molecular mass 927.3, monoisotopic mass
926.7. FABMS, m/z: 927.9 [M + H]+, 949.9 [M + Na]+. For C49H90N4O12 (927.3) calculated:
63.46% C, 9.78% H, 6.04% N; found: 63.68% C, 9.96% H, 5.81% N.
The authors would like to express their thanks to Dr V. Pechanec for elemental analyses, Dr K. Ubik
for FAB MS spectra and to Mr J. Zbrozek for amino acid analyses. Part of this work was supported by
grant No. 203/96/0309 of the Grant Agency of the Czech Republic.
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