The first-total synthesis of bacterial cell wall precursor UDP-N-acetylmuramyl-pentapeptide: (park nucleotide)

Full text of DOI:10.1021/ja973172d

1916
J. Am. Chem. Soc. 1998, 120, 1916-1917
Scheme 1
The First Total Synthesis of Bacterial Cell Wall
Precursor UDP-N-Acetylmuramyl-Pentapeptide
(Park Nucleotide)
Stephen A. Hitchcock,* Clark N. Eid, James A. Aikins,
Mohammad Zia-Ebrahimi, and Larry C. Blaszczak
Lilly Research Laboratories, A DiVision of Eli Lilly and
Company, Lilly Corporate Center
Indianapolis Indiana 46285
ReceiVed September 10, 1997
During the past 50 years antimicrobial chemotherapy has
revolutionized the treatment of infectious diseases. Several of
the most widely used antibacterial agents, including the â-lactams
and glycopeptides, possess a mode of action which involves
inhibition of bacterial cell wall biosynthesis.1 Recently however,
an alarming increase in antimicrobial resistance has threatened
the clinical efficacy of these and other classes of antibiotic agents.2
To aid in understanding emerging antimicrobial resistance mech-
anisms at the molecular level and to identify new agents to counter
this threat by rational, structure-based design and by devising new
mechanism based screening techniques, access to bacterial cell
wall precursors is invaluable. The importance of these compounds
has been further heightened by recent advances in the determi-
nation of bacterial genome sequences, a development likely to
unveil new targets in the cell wall pathway. Unfortunately the
isolation of cell wall precursors from the bacteria themselves is
a laborious process and is impractical for gram scale quantities.3
For this reason we have embarked on a program to develop
practical total syntheses of these biosynthetic intermediates for
use in the discovery of new antibiotic agents.
Scheme 2
The bacterial cell wall is composed of a framework of
alternating N-acetyl glucosamine (GlcNAc) and N-acetylmuramic
acid (MurNAc) units cross-linked via peptide chains appended
to the muramyl moiety. The first committed steps in bacterial
peptidoglycan biosynthesis begin within the cytoplasm with the
synthesis of UDP-MurNAc-L-Ala-γ-D-Glu-X-D-Ala-D-Ala, the so-
called Park nucleotide,4 6 from UDP-GlcNAc 1 where X is usually
meso-diaminopimelate in Gram-negative bacteria and L-Lys in
Gram-positive bacteria (Scheme 1).5
The non-DNA-encoded peptide chain is established via se-
quential addition of L-Ala, D-Glu, L-Lys (or meso-DAP), and then
D-Ala-D-Ala to UDP-MurNAc 2 by ATP-dependent amino acid
ligases. An undecaprenyl carrier is then attached to 6, and
GlcNAc is added, followed by additional amino acid residues in
some bacteria, before transport through the cell membrane and
incorporation into the cell wall. Descibed herein is the first total
synthesis of UDP-muramyl pentapeptide 6. The convergent
strategy employed should be adaptable to afford both rationally
designed inhibitors of the steps outlined in Scheme 16 as well as
the biosynthetic precursors to 6 (2-5).
glycosyl transferases.7 As a result, both enzymatic8 and chemical9
routes have been explored. Synthetically, the most widely
employed method for construction of these compounds has been
late-stage formation of the diphosphate moiety via coupling of
glycosyl phosphates with nucleoside 5′-morpholidophosphates
(Khorana-Moffatt procedure).10 Retrosynthetically, this first
disconnection reveals monophosphate 7 (Scheme 2).
Traditionally, the Khorana-Moffatt coupling process is per-
formed after the removal of all protecting groups as the final step
in glycosyl nucleotide diphosphate syntheses. The reaction is
(7) For reviews, see: (a) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.;
Kajimoto, T. Angew. Chem., Int. Ed. Engl. 1995, 34, 412. (b) Wong, C.-H.;
Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed. Engl.
1995, 34, 521.
(8) (a) Korf, U.; Thimm, J.; Thiem, J. Synlett 1991, 313. (b) Heidlas, J. E.;
Lees, W. J.; Pale, P.; Whitesides, G. M. J. Org. Chem. 1992, 57, 146. (c)
Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry.
In Tetrahedron Organic Chemistry Series; Baldwin, J. E., Magnus, P. D.,
Eds.; Elsevier Science: Oxford, U.K., 1994; Vol. 12, p 252. (d) Zervoson,
A.; Elling, L. J. Am. Chem. Soc. 1996, 118, 1836.
(9) (a) Ichikawa, Y.; Sim, M. M.; Wong, C.-H. J. Org. Chem. 1992, 57,
2943. (b) Khan, S.; Hindsgaul, O. In Molecular Glycobiology: Frontiers in
Molecular Biology Series; Fukuda, M., Hindsgaul, O., Eds.; Oxford University
Press: Oxford, U.K., 1994; p 206. (c) Klaffke, W. Carbohydr. Res. 1995,
266, 285. (d) Mu¨ller, T.; Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1995,
34, 1328. (e) Arlt, M.; Hindsgaul, O. J. Org. Chem. 1995, 60, 14.
(10) Roseman, S.; Distler, J. J.; Moffatt, J. G.; Khorana, H. G. J. Am. Chem.
Soc. 1961, 83, 659.
Recent advances in enzyme mediated oligosaccharide synthesis
have served to fuel synthetic interest in glycosyl nucleoside
diphosphates since they are used as activated substrates for
(1) Gale, E. F.; Cundliffe, E.; Reynolds, P. E.; Richmond, M. H.; Waring,
M. J. The Molecular Basis of Antibiotic Action, 2nd ed.; Wiley-Interscience:
New York, 1981.
(2) For a review, see: Chu, D. T. W.; Plattner, J. J.; Katz, L. J. Med. Chem.
1996, 39, 9, 3853.
(3) Kohlrausch, U.; Ho¨ltje, J.-V. FEMS Microbiol. Lett. 1991, 78, 253 and
references cited therein.
(4) Park, J. T. J. Biol. Chem. 1952, 194, 877.
(5) For a review, see: Bugg, T. D. H.; Walsh, C. T. Nat. Prod. Rep. 1992,
9, 199.
(6) Tanner, M. E.; Vaganay, S.; van Heijenoort, J.; Blanot, D. J. Org. Chem.
1996, 61, 1756.
S0002-7863(97)03172-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/04/1998

Communications to the Editor
J. Am. Chem. Soc., Vol. 120, No. 8, 1998 1917
Scheme 3a
Thus, the synthesis commenced with the conversion of benzyl
N-acetyl-4,6-benzylidenemuramic acid 11,15 available in three
steps from N-acetyl glucosamine, to the corresponding phenyl-
sulfonylethyl ester 12. Selective acid mediated removal of the
benzylidene group and acetylation next yielded diacetate 13.
The anomeric hydroxyl group was then cleanly unmasked under
hydrogenolytic conditions to deliver lactol 14 in readiness for
introduction of the anomeric phosphate. Treatment of 14 with
dibenzyl N,N-diethylphosphoramidite12b in the presence of 1,2,4-
triazole afforded the corresponding labile anomeric phosphites
15 as an R/â mixture (2.5:1). Although chromatographically
separable at this juncture, oxidation of the anomeric phosphite
mixture followed by rapid chromatography of the resultant
phosphate 9 proved more convenient due to the lability of the
anomeric phosphites. Under these conditions 9 was obtained
solely as the desired R-anomer in 42% overall yield from 14. As
has been noted for other 2-acetamido-2-deoxy glycosyl phos-
phates, the 1,2 trans isomers are typically not isolable due to the
destabilizing effect of the neighboring participating group.16 The
carboxyl group was next unmasked via treatment of 9 with DBU
in preparation for appendage of the pentapeptide fragment 10.
Pentapeptide 10 was, in turn, assembled from commercially
available Cbz-D-Ala-D-Ala using standard Boc protection and
EDC coupling procedures. Coupling of peptide 10 with the
muramyl carboxyl fragment resulted in the corresponding amide
16 in 70% yield. Hydrogenolytic debenzylation of 16 in the
presence of cyclohexylamine then yielded the corresponding
cyclohexylammonium phosphate salt which, to our satisfaction,
proved to be soluble in most organic solvents as anticipated.
Although slow (14 days), the coupling of the cyclohexylammo-
nium phosphate salt with uridine 5′-monophosphomorpholidate
under anhydrous conditions in DMF cleanly afforded the corre-
sponding protected UDP-N-acetylmuramyl pentapeptide. Rapid
deprotection with aqueous sodium hydroxide11 and chromato-
graphic purification by reverse phase HPLC finally afforded pure
UDP-N-acetylmuramyl pentapeptide 6 in 32% yield from 16 as
a hygroscopic white powder. The structure of 6 was confirmed
by analysis of spectral data (see Supporting Information). Further
structural corroboration was obtained by HPLC and mass spectral
comparison with an authentic sample of 6 obtained from Sta-
phylococcus aureus17 and from the observation that synthetic 6
was successfully converted into polymerized peptidoglycan using
a bacterial enzyme preparation.18
a Reagents: (a) DCC, NHS, 2-(phenylsulfonyl)ethanol, THF 96%;
(b) (i) AcOH, H2O, reflux; (ii) Ac2O, pyridine, 85%; (c) H2, Pd/C,
AcOH, 95% (d) dibenzyl-N,N-diethylphosphoramidite, 1,2,4-triazole,
CH2Cl2; (e) 30% H2O2, THF -78 °C to room temperature, 42% from
14; (f) (i) DBU, CH2Cl2, (ii) DCC, NHS, 10, 70%; (g) (i) H2, Pd/C,
cyclohexylamine, MeOH, (ii) 8, DMF, 45 °C, 14 days; then NaOH,
H2O, 32% from 16.
often limited therefore by the solubility of the unmasked glycosyl
monophosphates in suitable organic solvents. Conscious that the
pentapeptide chain present in our muramyl coupling partner was
likely to exacerbate this problem, we proposed to perform the
coupling reaction on a protected form of peptidomuramyl
phosphate (7). The choice of protection for the peptidosugar was
then dictated by the need to unmask the final molecule, maintain-
ing the labile anomeric diphosphate moiety intact. To address
this goal, acetate was chosen to block the hydroxyl groups, methyl
ester to protect the carboxylic acid moieties, and trifluoroaceta-
mide to mask the amino functionality. Thus, global deprotection
could be accomplished by exposure to hydroxide as the final
step.11
Approaches to glycosyl monophosphates12 fall broadly into two
categories wherein the sugar is either the electrophilic component
(nucleophilic addition of phosphate anion) or the nucleophile
(phosphorylation or phosphitylation/oxidation at the anomeric
hydroxyl group). In the former case, carbohydrates bearing a
neighboring participating group at C-2, favor the formation of
1,2-trans linked glycosyl phosphates (â-anomer in the case of
GlcNAc). Although addition of phosphate diesters to carbohy-
drate derived oxazolines under thermodynamic control has been
reported to deliver R-phosphates in some cases,8b,13 decomposition
has been reported in others.14 The milder conditions offered by
phosphitylation of carbohydrate lactols and subsequent oxidation12b
suggested a more attractive alternative considering the complex
substrate dictated by target 6.
The identification of new antibiotics that disrupt steps in
bacterial cell wall biosynthesis not targeted by existing agents is
an attractive counter-offensive strategy in the evolutionary battle
against emerging antimicrobial drug resistance. The synthesis
of UDP-N-acetylmuramyl pentapeptide outlined here should be
amenable to other intermediates in the bacterial cell wall pathway
which, in addition to 6, are useful biochemical tools for the
discovery of antibiotics with novel modes of action.
Supporting Information Available: Experimental details and spectral
data for all compounds are provided (22 pages). See any current masthead
page for ordering and Web access instructions.
(11) Sjo¨lin, P.; Elofsson, M.; Kihlberg, J. J. Org. Chem. 1996, 61, 560.
(12) (a) Boons, G.-J.; Burton, A.; Wyatt, P. Synlett 1996, 310. (b) Sim, M.
M.; Kondo, H.; Wong C.-H. J. Am. Chem. Soc. 1993, 115, 2261 and references
cited therein.
JA973172D
(13) (a) Khorlin, A. Ya.; Zurabyan, S. E.; Antonenko, T. S. Tetrahedron
Lett. 1970, 4803. (b) Warren, C. D.; Herscovics, A.; Jeanloz, R. W. Carbohydr.
Res. 1978, 61, 181. (c) Inage, M.; Chaki, H.; Kusumoto, S.; Shiba, T.
Tetrahedron Lett. 1981, 22, 2281.
(14) Srivastava, G.; Alton, G.; Hindsgaul, O. Carbohydr. Res. 1990, 207,
259.
(15) Jeanloz, R. W.; Walker, E.; Sinay, P. Carbohydr. Res. 1968, 6, 184.
(16) Sabesan, S.; Neira, S. Carbohydr. Res. 1992, 223, 169.
(17) The authors wish to thank Dr. Norris Allen (Lilly Research Labora-
tories) for supplying an authentic sample of 6.
(18) The authors wish to thank William Alborn (Lilly Research Labora-
tories) for this information.