ORIGINAL ARTICLES
clear light yellow solution. The solvent was evaporated under reduced pres-
the coupling reaction (Wittmann and Wong 1997). The
solvent was removed and the resulting residue dissolved
in 100 mM ammonium formate, lyophilized and directly
applied to a preparative reverse phase column to afford
UDP-MurNAc (1) in good yield (65%).
sure, and the oily residue was taken up in water (60 mL). Evaporation of
water removed the last traces of acetic acid and benzaldehyde, and gave a
white solid, which was dried by addition of dry toluene and distillation. The
white solid was dissolved in pyridine (75 mL) and Ac2O (60 mL), and the
mixture was stirred at room temperature overnight. The solvents were evapo-
rated, and the last traces of the solvents were removed by addition of toluene,
followed by evaporation. The residue was recrystallized from ethanol to af-
ford 6 as a white solid (10.51 g, 92%). Rf ¼ 0.70 (EtOAc : acetone ¼ 2 : 1);
m.p. 168–170 ꢁC (lit. 170–172 ꢁC (Osawa et al. 1969)); [a]2D3 þ 189 (c
0.135, MeOH); IR (KBr, cmꢀ1) 3314, 2939, 1751, 1653, 1549, 1237, 1038
and 697; 1H NMR (300 MHz, CDCl3) d ¼ 1.50 (d, 3 H, J ¼ 7.0 Hz,
CH3CH), 1.99 (s, 3 H, CH3CON), 2.12 (s, 3 H, CH3COO), 3.83 (dd, 1 H,
J ¼ 10.5 and 9.1 Hz, CH), 3.92–4.02 (m, 1 H, CH), 4.22 (dd, 1 H,
J ¼ 12.3 and 4.3 Hz, CH), 4.30–4.44 (m, 3 H, CH2 and CH), 4.48–4.76
(2ꢂd, 2 H, J ¼ 11.7 Hz, CH2Ph), 4.71 (q, 1 H, J ¼ 7.0 Hz, CHCH3), 4.97
(d, 1 H, J ¼ 3.7 Hz, CH), 5.60 (d, 1 H, J ¼ 9.4 Hz, NH) and 7.28–7.45 (m,
5 H, Ph); m/z (ESþ) 408.1659 ([M þ H]þ C20H26N08 requires 408.1658);
HPLC purity 98% (t ¼ 16.6 min).
2.3. Preparative HPLC purification of UDP-MurNAc
UDP-MurNAc (1) is fairly hydrophilic and thus poorly re-
tained on a conventional reversed-phase column. Atlantis
dC18, a fully endcapped stationary phase, appeared to be
the most appropriate column for an efficient purification
with a relatively short run time (Fig. 2). The best separa-
tion was scaled up to a 30ꢂ150 mm, 5 mm, Atlantis dC
18 OBD preparative column. Ammonium formate mobile
phase at pH ¼ 4 was selected to achieve good separation,
as well as good fraction volatility during lyophilization.
To improve sample load and throughput, we increased the
sample size and actually overloaded the column until
peaks started to overlap and purity was compromised.
3.2.2. 2-Acetamido-6-O-acetyl-3-O-[(R)-1-carboxyethyl]-2-deoxy-a-D-glu-
copyranoside 10,4-lactone (7)
A solution of benzyl lactone 6 (5.10 g, 12.52 mmol) in anhydrous THF
(150 mL) was hydrogenated for 24 h in the presence of 10% Pd/C (1.50 g).
The catalyst was filtered off, the solvent was evaporated under reduced pres-
sure, and the residue was purified by dry-column flash chromatography
through 15 parts of silica (ethyl acetate/acetone ¼ 2/1) to afford lactone 7 as
a white solid (3.77 g, 95%). Rf ¼ 0.45 (EtOAc : acetone ¼ 2 : 1); m.p. 188–
190 ꢁC (lit. 197–199 ꢁC (Osawa et al. 1969)); [a]2D3 þ 118 (c 0.165, MeOH);
IR (KBr, cmꢀ1) 3427, 3292, 2945, 1759, 1660, 1552, 1375, 1233, 1127 and
1048; 1H NMR (300 MHz, CDCl3 : CD3OD ¼ 1 : 1) d = 1.48 (d, 3 H,
J ¼ 7.2 Hz, CH3CH), 1.99 (s, 3 H, CH3CON), 2.06 (s, 3 H, CH3COO), 3.85
(dd, 1 H, J ¼ 10.4 and 9.2 Hz, CH), 4.10ꢀ4.53 (m, 5 H, 3ꢂCH and CH2),
4.68 (q, 1 H, J ¼ 7.2 Hz, CHCH3) and 5.15 (d, 1 H, J ¼ 3.6 Hz, CH); m/z
(ESþ) 318.1196 ([M þ H]þ C13H20NO8 requires 318.1189).
2.4. Conclusions
In this work the synthesis of UDP-MurNAc was opti-
mized, enabling a robust procedure to be performed on a
multi-gram scale. In addition, a coupling study of mura-
myl phosphate and UMP-morpholidate and the optimal
conditions obtained for the reaction provide a good start-
ing point towards more efficient coupling of nucleoside
monophosphates with various glycosylphosphates.
3.2.3. 2-Acetamido-6-O-acetyl-3-O-[(R)-1-carboxyethyl]-2-deoxy-a-D-glu-
copyranoside-1-a-(diphenyl phosphate) 10,4-lactone (8)
A solution of 4-pyrrolidinopyridine (16.30 g, 109.98 mmol) in anhydrous
CH2Cl2 (20 mL) was added to a solution of 7 (3.10 g, 9.77 mmol) in anhy-
drous CH2Cl2 (250 mL) at ꢀ30 ꢁC under argon. The mixture was stirred
for 20 min, followed by dropwise addition of diphenyl chlorophosphate
(15.0 mL, 72.03 mmol) and stirring was continued for 5 h between ꢀ25 to
ꢀ30 ꢁC. The mixture was then diluted with CH2Cl2 (100 mL) and washed
with ice cold H2O (2ꢂ150 mL), saturated NaHCO3 (2ꢂ150 mL), and
brine (100 mL). The organic phase was dried over MgSO4, filtered and
evaporated under reduced pressure at room temperature. The resulting resi-
due was purified by flash column chromatography on silica, eluting with
EtOAc : hexane ¼ 2 : 1 and then EtOAc : acetone ¼ 10 : 3 to afford the a-
anomer of 8 as a colourless solid. The product 8 was dried with anhydrous
ether and stored at ꢀ15 ꢁC (3.49 g, 65%). 1H NMR was in agreement with
reference (Dini et al. 2000). Rf ¼ 0.70 (EtOAc : acetone ¼ 2 : 1); m.p. 50–
57 ꢁC; [a]2D3 þ 105 (c 0.500, MeOH), IR (KBr, cmꢀ1) 3427, 3071, 1747,
1591, 1490, 1228, 962, 776 and 690; 1H NMR (300 MHz, CDCl3)
d ¼ 1.53 (d, 3 H, J ¼ 7.0 Hz, CH3CH), 1.88 (s, 3 H, CH3CON), 2.03 (s,
3 H, CH3COO), 3.87 (dd, 1 H, J ¼ 10.6 and 9.3 Hz, CH), 3.98–4.07 (m,
1 H, CH), 4.13 (dd, 1 H, J ¼ 12.5 and 3.5 Hz, CH2), 4.26 (dd, 1 H,
J ¼ 12.5 and 3.5 Hz, CH2), 4.39–4.53 (m, 2 H, 2ꢂCH), 4.72 (q, 1 H,
J ¼ 7.0 Hz, CHCH3), 5.57 (d, 1 H, J ¼ 9.0 Hz, NH), 6.00 (dd, 1 H, J ¼ 6.1
and 3.3 Hz, CHOPO3, a anomer) and 7.10–7.45 (m, 10 H, 2ꢂPh); m/z
(ESþ) 572.1303 ([M þ Na]þ C25H28NO11NaP requires 572.1298); HPLC
purity 92% (t ¼ 19.3 min).
3. Experimental
3.1. Materials
Anhydrous solvents were purchased from Fluka and used without further
purification. Chemicals from Sigma-Aldrich, Acros Organics or Merck were
of analytical or HPLC grade and were used without further purification.
Deionised water for preparative HPLC was purified by a MilliQ water puri-
fication system (Millipore Corporation, Massachusetts, USA). Analytical
TLC was performed on Merck silica (60F254) plates (0.25 mm); compounds
were visualized with ultraviolet light or by heating the plate at 250 ꢁC. Col-
umn chromatography was carried out on silica 60 (particle size 240–400
mesh). Cation exchange resin Dowex 50WX2 100–200 mesh (Hþ form)
was purchased from Acros and converted to Et3N salt form prior to use.
Melting points were determined on a Reichert hot stage microscope and are
uncorrected. IR spectra were obtained with a Nicolet FT-IR Nexus spectro-
meter and optical rotation was measured on a Perkin Elmer 1241 MC po-
larimeter. 1H NMR and 31P NMR spectra were recorded on a Varian Ino-
va300 spectrometer in CDCl3, DMSO-d6, CD3OD or D2O solution, with
TMS as the internal standard and phosphoric acid as the external standard.
Mass spectra were obtained using a Autospec Q Micromass mass spectro-
meter. HPLC analyses were performed using a Waters 2695 Separation
Module with a Waters 2996 PDA detector. For intermediates 3–6 and 8
analytical column XTerra RP C18 (150ꢂ4.6 mm I.D., 3.5 mm) and a gradi-
ent elution method combining mobile phase A with 25 mM ammonium
acetate (pH ¼ 6)/acetonitrile (95/5 v/v) and mobile phase B with 25 mM
ammonium acetate (pH ¼ 6)/acetonitrile (10/90 v/v) was used. For com-
pounds 1, 11, 12 and fractions from preparative HPLC analytical column
Atlantis dC18 OBD (100ꢂ4.6 mm I.D., 5 mm) and isocratic elution method
with mobile phase 50 mM ammonium formate (pH ¼ 4) was used. The pre-
parative chromatography was performed using a Varian PrepStar SD-1
(Walnut Creek, USA) HPLC solvent delivery system, Atlantis dC18 OBD
(150ꢂ30 mm I.D., 5 mm) preparative column, UV detector (Model ProStar
320) and a Rheodyne preparative injection valve (Model 3725i-038) with a
5 mL sample loop. The elution was carried out using 50 mM ammonium
formate (pH ¼ 4) with the flow rate 40 mL/min. The main fractions were
neutralized with a 25% ammonia solution, as soon as they were eluted,
and then lyophilized. All reported yields are yields of purified products.
3.2.4. Triethylammonium muramyl phosphate (9)
PtO2 hydrate (Pt content 79–84%, 200 mg) was added to a solution of di-
phenyl phosphate 8 (2.50 g, 4.55 mmol) in THF (40 mL) under argon. Hy-
drogen was bubbled into the reaction mixture for 15 min, and then a posi-
tive hydrogen pressure was maintained in the flask overnight. After the
catalyst was filtered, 0.6 M aq. LiOH (40 mL) was added immediately. The
resulting mixture was stirred for 24 h at room temperature and then evapo-
rated to dryness under reduced pressure. The residue was redissolved in
water and lyophilized to give lithium muramyl phosphate. The solution of
lithium muramyl phosphate in water (5 mL) was slowly adsorbed onto a
resin bed of Dowex 50WX2, 100–200 mesh (Et3NHþ form, 3.0ꢂ25 cm)
and slowly washed with water. Fractions containing the desired compound
were pooled and lyophilized. The procedure of lyophilization was repeated
to afford 9 as a highly hygroscopic colourless solid (2.34 g, 82%). The
content of Et3N was determined to be 2.5 equiv (1H NMR). Rf ¼ 0.35
(i-PrOH : H2O : 25% NH4OH ¼ 6 : 1 : 3); IR (KBr, cmꢀ1) 3375, 2977, 2937,
2676, 2491, 1652, 1568, 1398, 1037 and 950; 1H NMR (300 MHz, D2O)
d ¼ 1.23 (t, 9 H, J ¼ 7.3 Hz, Et3N), 1.31 (d, 3 H, J ¼ 7.0 Hz, CH3CH),
2.00 (s, 3 H, CH3CON), 3.15 (q, 6H, J ¼ 7.3 Hz, Et3N), 3.50–3.88 (m,
3.2. Synthetic procedures
3.2.1. Benzyl 2-acetamido-6-O-acetyl-3-O-[(R)-1-carboxyethyl]-2-deoxy-a-
D-glucopyranoside 10,4-lactone (6)
Compound
5 (13.21 g, 28.04 mmol) was suspended in 60% AcOH
(130 mL), and heated under reflux for 2 h, during which time it became a
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