An improved total synthesis of UDP-N-acetyl-muramic acid

Article abstract of DOI:10.1016/j.tetlet.2007.04.098

Biochemical testing for novel inhibitors of Mur ligases requires several commercially unavailable and structurally complex substrates. We describe a modified synthetic strategy for the total chemical synthesis of the MurC ligase substrate UDP-N-acetyl-muramic acid which includes several improvements over published methods, especially with regard to purification procedures. The synthetic strategy is applicable for the synthesis of further Mur ligase substrates.

Full text of DOI:10.1016/j.tetlet.2007.04.098

Tetrahedron Letters 48 (2007) 4403–4405
An improved total synthesis of UDP-N-acetyl-muramic acid
Andrej Babicˇa,* and Slavko Pecˇara,b
aUniversity of Ljubljana, Faculty of Pharmacy, Asˇkercˇeva 7, 1000 Ljubljana, Slovenia
bInstitute Jozef Stefan, Jamova 39, 1000 Ljubljana, Slovenia
Received 25 January 2007; revised 6 April 2007; accepted 17 April 2007
Available online 22 April 2007
Abstract—Biochemical testing for novel inhibitors of Mur ligases requires several commercially unavailable and structurally com-
plex substrates. We describe a modified synthetic strategy for the total chemical synthesis of the MurC ligase substrate UDP-N-
acetyl-muramic acid which includes several improvements over published methods, especially with regard to purification procedures.
The synthetic strategy is applicable for the synthesis of further Mur ligase substrates.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Antibiotic resistance is becoming an overwhelming pub-
lic health problem and there is a need for novel classes of
antimicrobial agents.1 Targeting the enzymes involved
in the biosynthesis of the bacterial cell wall appears to
be an attractive strategy since it should ensure selective
toxicity in humans. Mur ligases are enzymes which cata-
lyse early essential steps of cell wall biosynthesis. They
have been well characterised and constitute a promising
group of targets for antimicrobial research.2–5 However,
biochemical testing of potential Mur ligases inhibitors
requires several commercially unavailable and structur-
ally complex substrates.6,7 UDP-N-acetyl-muramic acid
(UDP-Mur-N-Ac) is the substrate for MurC ligase and
is thus needed for testing inhibition of MurC. It is also
commonly used for further enzymatic synthesis of
MurD, E and F substrates where L-Ala, D-Glu and m-
dap are attached consecutively to the carboxy end of
the peptidoglycan precursor. Here we describe the total
synthesis of UDP-Mur-N-Ac, using a modified synthetic
pathway which could be applied to the total synthesis of
further Mur ligase substrates, as well as exhibiting sev-
eral improvements over the published synthetic and
purification procedures.8–11
and 40-, 60-benzylidene protecting groups were intro-
duced according to known procedures, leaving the 30-
hydroxy group of 4 unprotected.13 The muramic acid
moiety was introduced through Williamson synthesis
using racemic 2-chloropropionic acid. Under appropri-
ate conditions the reaction gave predominantly the
desired R-muramic acid derivative (diastereoisomeric
ratio 4:1). Treatment of the sodium salt of which with
methyl iodide in DMF at room temperature gave methyl
ester 5 in quantitative yield as shown in Scheme 1.
To our knowledge, the only reports on purifying a dia-
stereoisomeric mixture of muramic and isomuramic acid
derivatives make use of complex gradient column chro-
matography with toxic carbon tetrachloride and diox-
ane as eluents.13 Column chromatographic purification
was substantially simplified by using a tri-component
mobile phase with isocratic elution, which eliminated
the need for a stereo-controlled Williamson reaction14
and gave higher overall yield.
For further synthesis of UDP-Mur-N-Ac, compound 6
was synthesised from 5 in three simple and straight-
forward steps, according to reported procedures.9,15
Removal of the benzyl aglycon from lactone 6 under
hydrogenation conditions in methanol, as depicted in
Scheme 2, presented an overwhelming problem. The
reaction gave a dead-end methyl ester derivative 7 with
a free 40-hydroxy group, instead of 8, as the main reac-
tion product. It has been well established that removal
of the benzyl aglycon from muramic acid can sometimes
be problematic15 and our attempts to obtain 8 in good
and reproducible yields failed.
We started the synthesis from the inexpensive a-D-glucos-
amine hydrochloride 1 which was N-acetylated to give
a-D-N-acetyl-glucosamine 2, which is also available
commercially.12 In the two following steps, 1-O-benzyl
Keywords: UDP-Mur-N-Ac; Total synthesis; MurC; Diphosphates.
*
Corresponding author. Tel.: +386 1 476 9500; fax: +386 1 425
8031; e-mail: andrej.babic@ffa.uni-lj.si
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.04.098

4404
A. Babicˇ, S. Pecˇar / Tetrahedron Letters 48 (2007) 4403–4405
OH
OH
OH
O
iii
O
i, ii
O
HO
HO
HO
HO
HO
HO
NH
NH
H3N+
OH
O
Ph
Ph
OH
Ac
Ac
Cl
1
2
3
O
O
Ph
Ph
O
O
O
O
v, vi
O
iv
HO
NH
NH
O
Ac
Me
O
Ph
Ac
O
O
4
5
Scheme 1. Reagents and conditions: (i) Na/MeOH (ii) (Ac)2O, rt, 98%; (iii) BnOH, PTSA, benzene, reflux, 12 h, 63%; (iv) Ph-CHO, (EtO)3CH,
PTSA, DMF, dioxane, rt, 12 h, 76%; (v) NaH, (R,S)-CH3CHClCOOH, 50 ꢁC, dioxane, 4 h; (vi) MeI, DMF, rt, 6 h, 63% (for steps v and vi).
Ac
Ac
Ac
O
O
O
O
O
O
O
O
O
i
O
HO
O
O
O
+
NH
OH
NH
NH
OH
Ac
Me
Ac
O
Ph
Ac
O
O
6
7
8
Scheme 2. Reagents and conditions: (i) H2, 10% Pd/C, MeOH, rt.
At this stage, we hoped that a very simple modification
of the muramic acid protecting groups would make the
further synthesis straightforward, bypassing the rela-
tively labile internal lactone 6.
The phenyl groups of 11 were deprotected under hydro-
genation conditions using platinum dioxide as the cata-
lyst19 immediately followed by hydrolysis with lithium
hydroxide which removed the remaining hydroxyl and
carboxy protecting groups. The triethylamine salt of
12 was obtained via elution through a Dowex 50W-X2
ion-exchange column.
The methyl ester moiety was hence left intact and served
as a carboxy protective group in further synthetic steps.
Instead, the benzylidene group was removed in aqueous
acetic acid and the free 40- and 60-hydroxyl groups acet-
ylated with acetic acid anhydride in dry pyridine, giving
9 as depicted in Scheme 3. Compound 9 represents a
synthetic substitute for the internal lactone 6. Indeed,
methyl ester 9 was stable under hydrogenation condi-
tions in THF and 10 was obtained in a very short reac-
tion time and in an excellent yield (96%) compared to
reported procedures for removing the anomeric benzyl
protective group.15,16 Phosphorylation of the aglycon
hydroxyl group catalysed by 4-pyrrolidinopyridine gave
only the a-anomer of 1117 in good yield.18
In a fashion similar to the procedure reported by Dini
et al.,9 coupling of 12 with uridine-50-monophospho-
morpholidate proceeded in sufficiently good yield only
when considerable attention was given to drying the
reaction glassware and both reagents over phosphorus
pentoxide under high vacuum. The reaction was per-
formed under argon using anhydrous DMF. Interest-
ingly, a good reaction yield was achieved despite the
fact that no tetrazole was used as the catalyst.20,21
HPLC analysis of the crude product revealed UDP-
Mur-N-Ac 13 as the major product with small amounts
Ac
O
Ac
O
Ac
Ph
O
O
O
Ac
O
O
O
O
O
iii
O
i, ii
O
O
NH
NH
OH
NH
O
Ph
Ac
Me
Ac
Me
O
Ph
Ac
Me
O
O
O
O
O
5
9
10
Ac
O
HO
HO
Ac
O
O
iv
O
v, vi, vii
O
O
O
P
O
NH
NH
P
Ph
Ac
Me
O
Ac
O
O
O
O
O
O
O
O
O
Ph
12
11
Scheme 3. Reagents and conditions: (i) 60% AcOH, H2O, 100 ꢁC; (ii) (Ac)2O, pyridine, 92%; (iii) H2, Pd/C, THF, 96%; (iv) ClPO(OPh)2, 4-
pyrrolidinopyridine, CH2Cl2, ꢀ30 ꢁC, 55%; (v) H2, PtO2, THF; (vi) 1 M LiOH, THF, rt; (vii) DOWEX 50W-X2-TEA form.

A. Babicˇ, S. Pecˇar / Tetrahedron Letters 48 (2007) 4403–4405
4405
O
NH
O
O
P
O
O
N
N
O
O
N
O
HO
HO
HO
HO
O
NH
O
O
P
O
O
P
O
OH OH
O
O
P
NH
O
NH
Ac
O
O
O
O
O
Ac
O
O
O
O
O
O
O
OH OH
13
12
˚
Scheme 4. Reagents and conditions: DMF, 4 A molecular sieves, 70 ꢁC, 20 h, 51%.
ˇ
5. Humljan, J.; Kotnik, M.; Boniface, A.; Solmajer, T.;
Urleb, U.; Blanot, D.; Gobec, S. Tetrahedron 2006, 62,
10980–10988.
of UMP and UMP-UMP dimer present. A prolonged
reaction time of 20 h compared to reported procedures
and the use of a smaller excess of uridine-5’-monophos-
phomorpholidate (1.3 equiv) improved the outcome of
the reaction. After work-up, crude product 13 was puri-
fied by gel filtration. It was applied consecutively to G-
10 and G-15 Sephadex columns equilibrated with bidis-
tilled water. Very good separation was achieved by care-
ful control of the flow rate and the amount of crude
product applied to the columns. Fractions containing
the product were pooled and lyophilized, thereby omit-
ting the usual preparative HPLC purification (Scheme
4).
6. Silver, L. L. Biochem. Pharmacol. 2006, 71, 996–1005.
7. Deng, G.; Gu, R. F.; Marmor, S.; Fisher, S. L.; Jahic, H.;
Sanyal, G. J. Pharm. Biomed. Anal. 2004, 35, 817–828.
8. Blanot, D.; Auger, G.; Liger, D.; van Heijenoort, J.
Carbohydr. Res. 1994, 252, 107–115.
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Chem. Lett. 2000, 10, 143–145.
10. Benson, T. E.; Marquardt, J. L.; Marquardt, A. C.;
Etzkorn, F. A.; Walsh, C. T. Biochemistry 1993, 32, 2024–
2030.
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Pompliano, D. L. J. Am. Chem. Soc. 1999, 121, 1175–
1178.
In conclusion, we have presented an improved total
synthesis of UDP-Mur-N-Ac. Several key synthetic
intermediates were modified and novel purification
methods for muramic acid derivative 5 and UDP-
Mur-N-Ac were introduced. Perhaps most importantly,
the synthetic strategy is applicable to the synthesis of
other Mur ligase substrates. This is currently under
investigation in our laboratory and will be published
in due course.
12. Yoshiyuki, I.; Konoshin, O.; Shozaburo, K.; Tokuji, K.
Bull. Inst. Chem. Res. Kyoto Univ. 1955, 33, 270–271.
13. Gross, P.; Rimpler, M. Liebigs Ann. Chem. 1986, 1, 37–
45.
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Waddell, S. Bioorg. Med. Chem. Lett. 2003, 13, 1125–
1128.
15. Osawa, T.; Sinay, P.; Halford, M.; Jeanloz, R. W.
Biochemistry 1969, 8, 3369–3375.
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M.; Aikins, J. A.; Blaszczak, L. C. J. Am. Chem. Soc.
2001, 123, 6983–6988.
17. Spectroscopic data for 11: Yield 55%; colourless oil; IR
(KBr) tmax/cmꢀ1: 2956, 1745, 1594, 1490, 1376, 1213,
1092, 1035, 916, 777; 1H NMR 300 MHz [CDCl3], dH 1.36
(d, 3H, CH3, J = 7.0 Hz), 2.02 (s, 3H, CH3CONH), 2.08
(s, 3H, CH3CO), 2.09 (s, 3H, CH3CO), 3.82 (s, 3H, CH3–
OCO), 3.71–4.03 (m, 4H, H-2, H-3, H-5, H-6), 4.10 (dd,
1H, H-60, J = 4.5, J = 12.5 Hz), 4.27 (q, 1H, CHCH3,
J = 7.0 Hz), 5.18 (t, 1H, H-4, J = 9.5 Hz), 6.38 (dd, 1H,
H-1, J = 2.8, 6.0 Hz), 7.17–7.38 (m, 10H, H-Ar), 7.88 (d,
1H, NH, J = 3.9 Hz); 13C NMR 75 MHz [CDCl3], dC
18.72, 20.63, 20.79, 22.77, 53.88, 61.28, 70.37, 71.06, 74.56,
75.35, 96.96, 119.94, 120.00, 120.12, 120.19, 120.33,
125.49, 129.48, 129.80, 129.85, 169.02, 170.71, 171.41,
175.12. 31P NMR 121 MHz [CDCl3], dP ꢀ 13.29. MS
(ESI) 646 (M+Na)+; HRMS Calcd for C28H34NO13NaP
m/z: 646.1665 (M+Na)+, found 646.1677. C28H34NO13-
P1 · 1.4 H2O calcd: C 51.79, H 5.67, N 2.16. Found: C
52.18, H 6.08, N 2.14.
Acknowledgements
This work was supported by the European Union FP6
Integrated Project EUR-INTAFAR (Project No.
LSHM-CT-2004-512138) under the thematic priority
Life Sciences, Genomics and Biotechnology for Health,
and the Ministry of Education, Science and Sport of the
Republic of Slovenia. The authors thank Dr. Roger
Pain for critical reading of the manuscript.
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