The total synthesis of lipid I

Article abstract of DOI:10.1021/ja016082o

A total synthesis of lipid I (4), a membrane-associated intermediate in the bacterial cell wall (peptidoglycan) biosynthesis pathway, is reported. This highly convergent synthesis will enable further studies on bacterial resistance mechanisms and may prov

Full text of DOI:10.1021/ja016082o

J. Am. Chem. Soc. 2001, 123, 6983-6988
6983
The Total Synthesis of Lipid I
Michael S. VanNieuwenhze,* Scott C. Mauldin, Mohammad Zia-Ebrahimi,
James A. Aikins,‡ and Larry C. Blaszczak
Contribution from DiscoVery Chemistry Research and Chemical Process Research and DeVelopment,
Lilly Research Laboratories, A DiVision of Eli Lilly and Company, Lilly Corporate Center,
Indianapolis, Indiana 46285
ReceiVed April 25, 2001. ReVised Manuscript ReceiVed May 31, 2001
Abstract: A total synthesis of lipid I (4), a membrane-associated intermediate in the bacterial cell wall
(peptidoglycan) biosynthesis pathway, is reported. This highly convergent synthesis will enable further studies
on bacterial resistance mechanisms and may provide insight toward the development of new chemotherapeutic
agents with novel modes of action.
Background and Introduction
total synthesis of UDP-N-acetylmuramyl pentapeptide (Park
Nucleotide7), the immediate biosynthetic precursor to lipid I
(Scheme 1). As part of our continuing studies in bacterial cell
wall biosynthesis, we report herein a total synthesis of lipid I,
the penultimate intermediate utilized by Gram-positive bacteria
in peptidoglycan biosynthesis.8
Bacterial peptidoglycan consists of an intricate network of
â-1,4-linked carbohydrate polymers, which are cross-linked by
pendant peptide chains.1 This rigid macromolecular structure
precisely defines the shape and size of the bacterial cell in both
Gram-positive and Gram-negative bacteria and enables the cell
to resist lysis resulting from high internal osmotic pressure.2
Many of the more widely used antibiotics, â-lactams and
glycopeptides (e.g., vancomycin), function via mechanisms
involving inhibition of key steps in the cell wall biosynthesis
cascade.3 The rapid emergence of antimicrobial resistance has
now begun to erode the once dependable clinical efficacy of
these chemotherapeutic agents.4 In response to this rapid increase
in resistance, a renewed emphasis has been placed on the study
of the bacterial enzymes involved in peptidoglycan biosyn-
thesis. Mechanistic and structural information gained from such
studies could facilitate rational design of new antibacterial agents
with novel mechanisms of action. While efficient access to many
of the biosynthetic enzymes has been gained, the isolation of
their respective precursors from natural sources has proven to
be difficult.5 For study of resistance mechanisms at the
molecular level, access to isolable quantities of the biosyn-
thetic precursors is required. To address this difficulty, we have
established a program to develop practical and efficient syn-
thetic routes to the biosynthetic precursors for use in the
discovery and design of novel antibacterial agents. Previous
efforts6 by workers at Eli Lilly and Company culminated in a
Peptidoglycan biosynthesis is believed to take place in three
distinct stages. The first of these (Scheme 1) occurs in the
cytoplasm of the bacterial cell and begins with the elaboration
of UDP-N-acetylglucosamine (UDP-GlcNAc, 1) into UDP-
MurNAc-pentapeptide (Park Nucleotide, 3).1,2,7 The initial step
in this sequence is mediated by the MurA enzyme and involves
the transfer of an enolpyruvyl group onto the 3-hydroxyl group
of UDP-GlcNAc. Conversion of the enol group into the lactyl
moiety is accomplished via an NADPH-dependent reduction
mediated by the reductase MurB providing UDP-N-acetylmu-
ramic acid (UDP-MurNAc, 2). An ATP-dependent ligase
(MurC) introduces the first amide bond (L-Ala) onto the C(3)-
lactyl moiety. Additional ATP-dependent ligases (MurD-F)
introduce, in succession, D-Glu, L-Lys,8 and D-Ala-D-Ala to
provide the fully elaborated UDP-MurNAc-pentapeptide 3 (Park
Nucleotide).1,2,7
The second stage of peptidoglycan biosynthesis takes place
on the cytoplasmic surface of the bacterial membrane. First,
MraY catalyzes a pyrophosphate exchange reaction in which
UDP-MurNAc-pentapeptide 3 is coupled to a membrane-
anchored lipid (C55) carrier, with concomitant ejection of UMP,
to provide undecaprenylpyrophosphoryl-MurNAc-pentapeptide
4 (lipid I). Second, MurG catalyzes the transfer of GlcNAc from
UDP-GlcNAc to the C(4)-hydroxyl group of the lipid-linked
MurNAc-pentapeptide. The product of this biochemical trans-
formation, lipid II 5, is the final monomeric intermediate in the
cell wall biosynthesis cascade.1,2,8
‡ Chemical Process Research and Development.
(1) Rogers, H. J.; Perkins, H. R.; Ward, J. B. Biosynthesis of Pepti-
doglycan; Chapman and Hall, Ltd.: London, 1980.
(2) (a) Ho¨lt, J.-V. Microbiol. Mol. Biol. ReV. 1998, 62, 181. (b) Bugg,
T. D. H.; Walsh, C. T. Nat. Prod. Rep. 1992, 199.
(3) Gale, E. F.; Cundliffe, E.; Reynolds, P. E.; Richmond, M. H.; Waring,
M. J. The Molecular Basis of Antibiotic Action, 2nd ed.; Wiley-Inter-
science: New York, 1981.
(4) For reviews, see: (a) Chu, D. T. W.; Plattner, J. J.; Katz, L. J. Med.
Chem. 1996, 39, 3853. (b) Morris, A.; Kellner, J. D.; Low, D. E. Curr
Opin. Microbiol. 1998, 1, 524.
(5) See, for example: (a) Anderson, J. S.; Matsuhashi, M.; Haskin, M.
A.; Strominger, J. L. J. Biol. Chem. 1967, 242, 3180. (b) Kohlrausch, U.;
Hoeltje, J.-V. FEMS Microbiol. Lett. 1991, 78, 253. (c) Van Heijenoort,
Y.; Gomez, M.; Derrien, M.; Ayala, J.; van Heijenoort, J. J. Bacteriol. 1992,
174, 3549.
(6) For the first total synthesis of the Park nucleotide, the biosynthetic
precursor to lipid I, see: Hitchcock, S. A.; Eid, C. N.; Aikins, J. A.; Zia-
Ebrahimi, M.; Blaszczak, L. C. J. Am. Chem. Soc. 1998, 120, 1916.
In some Gram-positive bacteria 1-5 amino acid residues may
be successively attached to the ꢀ-amino group of the lysine
residue. Typically, these additional residues are glycine, although
organisms incorporating L-serine, L-threonine, and other amino
acids are known.2,9
(7) Park, J. T. J. Biol. Chem. 1952, 194, 877.
(8) The biochemical pathway illustrated here is that utilized by Gram-
positive bacteria. In the biosynthetic pathway utilized by Gram-negative
bacteria meso-diaminopimelic (meso-DAP) is incorporated into the pen-
tapeptide side chain in place of L-lysine.
(9) Schleifer, K. H.; Kandler, O. Bacteriol. ReV. 1972, 36, 407.
10.1021/ja016082o CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/29/2001

6984 J. Am. Chem. Soc., Vol. 123, No. 29, 2001
VanNieuwenhze et al.
Scheme 1. Biochemical Pathway Utilized in Bacterial Cell Wall Biosynthesis
In the final stage of peptidoglycan biosynthesis, lipid II is
translocated to the extracellular face of the bacterial cell
membrane where it is subsequently polymerized into glycan
strands through the action of transglycosylases. After polym-
erization of the carbohydrate backbone, the undecaprenyl lipid
carrier is translocated back across the cytoplasmic membrane
to be recycled.1,2 The glycan polymers are subsequently cross-
linked via amide bond formation between the terminal amino
group of the lysine residue, or the R-amino terminus of an
attached peptide chain (vide supra), and the penultimate D-Ala
residue of a neighboring chain. The amide bond is established
with concomitant loss of the terminal D-Ala residue. Mature
peptidoglycan is a single macromolecule of muliple layers of
cross-linked glycan strands that precisely defines the size and
shape of the bacterial cell.1,2
To gain a greater understanding of the mechanisms by which
bacteria acquire resistance to cell wall active agents, increasing
effort has been directed toward the isolation and study of the
individual biosynthetic enzymes and their natural substrates.
Modern biochemical and genetics techniques have facilitated
isolation and purification of the biosynthetic enzymes. With
respect to the stage I enzymes, all are soluble and crystal
structures of at least four of these enzymes have been pub-
lished.10-13 Although reports of the chemical synthesis of their
respective substrates have yet to appear, efforts toward the
rational design of inhibitors of the stage I enzymes have been
reported.14 The stage II and stage III enzymes have proven much
more difficult to study. These are membrane-associated proteins
that can make purification difficult and complicate mechanistic
and structural analysis. In addition, the structural complexity
(10) MurA: (a) Schonbrunn, E.; Sack, S.; Eschenburg, S.; Perrakis, A.;
Krekel, F.; Amrhein, N.; Mandelkow, E. Structure 1996, 4, 1065. (b)
Skarzynski, T.; Mistry, A.; Wonacott, A.; Hutchinson, S. E.; Kelly, V. A.;
Duncan, K. Structure 1996, 4, 1465. (c) Skarzynski, T.; Kim, D. H.; Lees,
W. J.; Walsh, C. T.; Duncan, K. Biochemistry 1998, 37, 2572.
(11) MurB: (a) Benson, T. E.; Filman, D. J.; Walsh, C. T.; Hogle, J. M.
Nat. Struct. Biol. 1995, 2, 644. (b) Benson, T. E.; Walsh, C. T.; Hogle, J.
M. Structure 1996, 4, 47.
(12) Mur C: Emanuele, J. J., Jr.; Jin, H.; Jacobson, B. L.; Chang, C. Y.;
Einspahr, H. M.; Villafranca, J. J. Protein Sci. 1996, 5, 2566.
(13) MurD: Bertrand, J. A.; Auger, G.; Fanchon, E.; Martin, L.; Blanot,
D.; van Heijenoort, J.; Dideburg, O. EMBO J. 1997, 16, 3416.
(14) (a) Gegnas, L. D.; Waddell, S. T.; Chabin, R. M.; Reddy, S.; Wong,
K. K. Bioorg. Med. Chem. Lett. 1998, 8, 1643. (b) Tanner, M. E.; Vaganay,
S.; van Heijenoort, J.; Blanot, D. J. Org. Chem. 1996, 61, 1756. (c) Zeng,
B.; Wong K. K.; Pompliano, D. L.; Reddy, S.; Tanner, M. E. J. Org. Chem.
1998, 63, 10081.

The Total Synthesis of Lipid I
J. Am. Chem. Soc., Vol. 123, No. 29, 2001 6985
Scheme 2. Retrosynthetic Analysis for Lipid I
of the natural substrates for these enzymes is significantly greater
than that of the stage I substrates. Moreover, the substrates
themselves are difficult to obtain due to low natural abundances
and difficult isolation procedures.5 For example, experiments
in Escherichia coli have determined lipid I to be present at cell
copy numbers no higher than 700.5c,15
As part of our ongoing effort in the study of peptidoglycan
biosynthesis, we required a supply of lipid I. The inherent
structural complexity of lipid I, coupled with its low natural
abundance, have hampered attempts at its isolation from cell
culture. Recently, in efforts directed toward the purification and
characterization of the MurG enzyme, Walker has reported a
synthesis of lipid I,16 as well as related substrate analogues.17
These results have prompted us to report our total synthesis of
lipid I 4, the penultimate monomeric intermediate utilized by
Gram-positive bacteria in peptidoglycan biosynthesis.
anticipated this linkage to be extremely acid-sensitive thus not
only placing restrictions on the means for introducing the lipid
side chain itself, but also precluding the use of protective groups
requiring acidic conditions for any late stage global deprotection.
Our retrosynthetic analysis is presented in Scheme 2. Our
“protected” version of lipid I (6), employed acetate protective
groups for the peripheral hydroxyls, methyl esters for each
carboxyl group in the pentapeptide side chain, and a trifluoro-
acetate group for the terminal amino group of the lysine residue.
These base-cleavable protective groups offered a potential
solution to our concern regarding the acid-lability of the
anomeric diphosphate, and could be removed in a single
operation as the final step in the synthesis.
Our first disconnection revealed glycosyl monophosphate 7
and undecaprenyl monophosphate 8. A mild method for
introduction of the chemically sensitive lipid diphosphate moiety
would be required.18 We also preferred a late-stage coupling of
these partners to minimize the number of subsequent synthetic
transformations that the diphosphate linkage must withstand.
Another consideration in the design of coupling partner 7
was the choice of protective group for the anomeric phosphate.
This group must be orthogonal to the protective groups on the
periphery of the carbohydrate, as well as those utilized in the
peptide backbone to ensure selective unmasking of the anomeric
phosphate prior to activation for coupling with lipid monophos-
phate 8. For this purpose, we chose benzyl protection thus
revealing monosaccharyl pentapeptide 9 as our next target in
the retrosynthetic direction.
Retrosynthetic Analysis
Managing the interplay of structural complexity, chemical
stability, and protective group strategy required a very carefully
constructed synthetic plan that addressed several key issues. A
major consideration during the development of our synthetic
strategy involved the timing for introduction of the undecaprenyl
side chain and the requisite diphosphate linkage. We were
mindful of the technical obstacles (e.g., solubility, micelle
formation, etc.) that could interfere with chemical transforma-
tions carried out after installation of the lipid side chain. In
addition, introduction of the lipid side chain establishes a
diphosphate moiety that is both glycosidic and allylic. We
The pentapeptide side chain 11 would be prepared using
standard peptide chemistry methods and would be introduced
via coupling with an activated ester intermediate deriving from
a muramic acid derivative 10. Phenylsulfonylethyl protection
of the lactyl ester in 10 would ensure complete protective group
(15) Although the data cited are from a Gram-negative bacterium (E.
coli), it serves to illustrate the difficulty inherent in obtaining lipid I from
cell culture.
(16) Ye, X.-Y.; Lo, M.-C.; Brunner, L.; Walker, D.; Kahne, D.; Walker,
S. J. Am. Chem. Soc. 2001, 123, 3155.
(17) Substrate analogues of Lipid I have been prepared in efforts to
characterize MurG. (a) Ha, S.; Chang, E.; Lo, M.-C.; Men, H.; Park, P.;
Ge, M.; Walker, S. J. Am. Chem. Soc. 1999, 121, 8415. (b) Men, H.; Park,
P. Ge, M.; Walker, S. J. Am. Chem. Soc. 1998, 120, 2484.
(18) Undecaprenyl monophosphate bis-ammonium salt may be purchased
from the Institute of Biochemistry and Biophysics, Polish Academy of
Sciences, ul. Pawinskiego 5A, 02-106 Warszawa, Poland.

6986 J. Am. Chem. Soc., Vol. 123, No. 29, 2001
VanNieuwenhze et al.
Chart 1. Comparison of Phosphitylation/Oxidation
Scheme 3. Synthesis of Phosphomuramyl Pentapeptide 9a
Protocols
to carbohydrate-derived oxazolines has been reported to provide
R-phosphates in some cases,21 although decomposition has been reported
in others.22 Of the available methods utilizing a nucleophilic carbohy-
drate component, we chose a phosphitylation/oxidation sequence for
introduction of the anomeric phosphate.17b,20a
Literature precedent from the Park nucleotide synthesis6 established
the phosphitylation/oxidation of lactol 14, existing predominantly as
the R-anomer, displayed a modest preference (2.5:1) for the desired
R-phosphate 10 (Chart 1). Walker subsequently reported a similar
reaction sequence on a related substrate 16 (Chart 1) that provided an
R-phosphate 17 as the exclusive product in good chemical yield.17b
Although different protection schemes were used in each of these
cases, we felt the enhanced preference for the R-anomer observed in
the latter example may be due to the choice of acid catalyst (tetrazole
vs triazole). Since tetrazole (pKa ) 4.9) is a much better proton donor
than triazole (pKa )10.0), the enhanced R-selectivity in the reaction
employing tetrazole may be the result of a greater equilibrium
concentration of the activated phosphoramidite reagent such that cap-
ture by the lactol R-anomer now occurs at a much faster rate than
anomerization and capture by the more nucleophilic â-anomer.23
Thus, as depicted in Scheme 3, exposure of lactol 14 to dibenzyl-
N,N-diethylphosphoramidite and 1H-tetrazole in dichloromethane fol-
lowed by oxidation of the phosphite intermediate with 30% hydrogen
a Reagents and conditions: (a) EDCI, 2-phenylsulfonylethanol,
DMAP, CH2Cl2, >95% yield; (b) (i) HOAc, H2O, reflux and (ii) Ac2O,
pyridine, 81% yield; (c) H2 (15 psi), Pd/C, HOAc, 53% yield; (d) (i)
dibenzyl-N,N-diethylphosphoramidite, 1H-tetrazole, CH2Cl2 and (ii)
30% H2O2, THF, -78 °C to room temperature, 86% yield; (e) DBU,
i
CH2Cl2, >95%yield; (f) EDCI, NHS, DMF, then 11, Pr2NEt, 75%
yield.
orthogonality and would allow mild conditions for unmasking
of the carboxyl group for peptide coupling. The phenylsulfo-
nylethyl ester 10 would in turn be prepared from a differentially
protected muramic acid derivative 12, the starting material for
our total synthesis of lipid I.19
peroxide provided the desired R-phosphate 10 in 86% yield (3JH1H2
)
3.4 Hz).24 The lactyl carboxyl group, the anchor for introduction of
the pentapeptide side chain, was liberated through treatment of the
phenylsufonylethyl ester with DBU (>95% yield). The stage was now
set for introduction of the pentapeptide side chain. The pentapeptide
fragment was conveniently prepared utilizing standard peptide synthesis
protocols (Scheme 4). Carboxyl group activation of muramic acid
derivative 15 was achieved through its conversion to the corresponding
NHS-ester. Addition of a DMF solution of pentapeptide 11 to a solution
of the NHS-ester deriving from muramic acid derivative 15 and
iPr2NEt in DMF provided the protected muramyl pentapeptide 9 in
excellent yield (75% from 15).
Synthesis of Phosphomuramyl Pentapeptide 9
Our synthesis began (Scheme 3) with conversion of muramic acid
derivative 1219 to a phenylsulfonylethyl ester (> 95% yield). Subsequent
acid-mediated cleavage of the 4,6-O-benzylidene acetal followed by
acetylation of the liberated hydroxyl groups provided diacetate 13 in
81% yield. Hydrogenolytic cleavage of the anomeric benzyl protective
group was achieved through exposure of 13 to H2 (15 psi) and Pd/C in
acetic acid solvent. This provided lactol 14 (predominantly R) in 53%
yield and set the stage for introduction of the anomeric phosphate.
Methods for preparation of glycosyl monophosphates can be divided
into two classes, wherein the carbohydrate component may function
either as the nucleophilic or as the electrophilic component.20 In the
latter case, carbohydrate substrates bearing a functional group capable
of neighboring group participation at C(2) generally favor formation
of 1,2-trans linked glycosyl phosphates. This would not provide a
glycosyl monophosphate with the proper anomeric stereochemistry for
completion of the lipid I total synthesis. Addition of phosphate diesters
Thus we achieved efficient access to a differentially protected phos-
phomuramyl pentapeptide derivative 9 which set the stage for comple-
(21) (a) Khorlin, A. Y.; 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.
(22) Srivastava, G.; Alton, G.; Hindsgaul, O. Carbohydr. Res. 1990, 207,
259.
(23) While we have attributed the enhanced R-selectivity observed in
1H-tetrazole mediated phosphitylation/oxidation of 16 to the enhanced
acidity of tetrazole compared to triazole, we could not discount the additional
possibility that a torsional effect, exerted by the 4,6-O-benzylidene acetal,
could also influence the observed preference for the R-anomer. See, for
example: Fraser-Reid, B.; Wu, Z.; Andrews, C. W.; Skowronski, E.; Bowen,
J. P. J. Am. Chem. Soc. 1991, 113, 1434.
(24) For mechanistic studies on the role of 1H-tetrazole in phosphora-
midite coupling reactions, see: (a) Dahl, B. H.; Nielsen, J.; Dahl, O. Nucleic
Acids Res. 1987, 15, 1729. (b) Berner, S.; Muehlegger, K.; Seliger, H.
Nucleic Acids Res. 1989, 17, 853
(19) Jeanloz, R. W.; Walker, E.; Sinay, P. Carbohydr. Res. 1968, 6, 184.
(20) For leading references, see: (a) Sim, M. M.; Kondo, H.; Wong,
C.-H. J. Am. Chem. Soc. 1993, 115, 2261. (b) Boons, G.-J.; Burton, A.;
Wyatt, P. Synlett 1996, 310. (c) Schmidt, R. R.; Braun, H.; Jung, K.-H.
Tetrahedron Lett. 1992, 33, 1585. (d) Sabesan, S.; Neira, S. Carbohydr.
Res. 1992, 223, 169. (e) Schmidt, R. R.; Wegmann, B.; Jung, K.-H. Liebigs
Ann. Chem. 1991, 121. (f) P. Pale, P.; Whitesides, G. M. J. Org. Chem.
1991, 56, 4547. (g) Veeneman, G. H.; Broxterman, H. J. G.; Van der Marel,
G. A.; Van Boom, J. H. Tetrahedron Lett. 1991, 32, 6175. (h) Bannwarth,
W.; Trzeciak, A. HelV. Chim. Acta 1987, 70, 175.

The Total Synthesis of Lipid I
J. Am. Chem. Soc., Vol. 123, No. 29, 2001 6987
Scheme 4. Synthesis of Pentapeptide 11a
Scheme 5. Lipid I Total Synthesis: The Final Stagesa
a Reagents and conditions: (a) (i) DCC, NHS, THF, 2 h and (ii)
i
D-Ala-D-Ala-OMe (TFA salt), Pr2NEt, THF, 72% yield; (b) CH2Cl2:
TFA (1:1), then add to a solution of Boc-D-iso-Glu-γ-NHS-OMe 20,
iPr2NEt, 56% yield; (c) CH2Cl2:TFA (1:1), then add to a solution of
Boc-L-Ala-OBT, iPr2NEt, 85% yield; (d) CH2Cl2:TFA (1:1), quantitative
yield.
a Reagents and conditions: (a) H2, 10% Pd/C, MeOH, then pyridine;
(b) (i) 1,1′-carbonyldiimidazole, THF/DMF (4:1), (ii) MeOH, and (iii)
THF/DMF (4:1), undecaprenyl monophosphate bis-ammonium salt 8,
1H-tetrazole, 4 d; (c) 1 N NaOH, dioxane/H2O (1:1); 40% yield overall
from 9.
tion of the lipid I total synthesis. The remaining technical hurdles
involved introduction of the lipid diphosphate linkage followed by
global deprotection and purification. Our solutions to these issues are
discussed in the following section.
as electrophiles for the addition of nucleophilic phosphate salts. The
phosphoric anhydride protocol has been used to establish a carbohydrate-
lipid diphosphate linkage that is also allylic. Of these procedures,
however, only the phosphoroimidazolidate protocol proceeds via initial
activation of the carbohydrate component. This was particularly
attractive to us in that it would allow use of undecaprenyl monophos-
phate directly in a coupling reaction with an activated carbohydrate
component, the end result being installation of the chemically sensitive
diphosphate linkage under relatively mild reaction conditions.
Encouraged by promising results with model systems,31 our attention
turned toward application of the phosphoroimidazolidate method to
complete the lipid I total synthesis. Cleavage of the benzyl groups from
the anomeric phosphotriester was readily achieved via hydrogenolysis
(Scheme 5). The anomeric phosphate was isolated as the monopyridyl
salt 23 and used directly without purification.30a Addition of 1,1′-
carbonyldiimidazole to a solution of 23 in THF/DMF (4:1) provided
the intermediate phosphoroimidazolidate. After quenching of excess
carbonyldiimidazole via careful addition of methanol, a solution of
undecaprenyl monophosphate bis-ammonium salt 8 and tetrazole in
THF/DMF (4:1) was added carefully via syringe. Reaction progress
was monitored by mass spectrometry until complete disappearance of
the phosphoroimidazolidate intermediate was observed. The crude
reaction solution was concentrated in vacuo, dissolved in a dioxane-
water solvent mixture (1:1), and treated with 1 N NaOH. After being
stirred for 2 h at room temperature, the mixture was filtered and purified
by reverse-phase HPLC.32 Lyophilization of the pure fractions provided
pure lipid I in 40% overall yield from muramyl pentapeptide 9.
Introduction of the Lipid Diphosphate: Lipid I Endgame
Numerous methods, both chemical25 and enzymatic,26 have been
exploited for the preparation of glycosyl diphosphates. Perhaps the most
widely used method for construction of anomeric diphosphates is the
Khorana-Moffatt27 protocol, the coupling of glycosyl monophosphates
with phosphoromorpholidates (usually nucleoside 5′-phosphoromor-
pholidates). Although this reaction has enjoyed great utility in the
construction of glycosyl nucleoside diphosphates, to the best of our
knowledge, it has not been used for the construction of prenyl-linked
glycosyl diphosphates. Moreover, application of this method would
require independent synthesis of a phosphoromorpholidate deriving from
either the carbohydrate or lipid fragment. Thus, in recognition of the
potential instability of such activated glycosidic or allylic phosphate
intermediates, and for synthetic ease and efficiency, we chose to
investigate in situ activation protocols.
Several procedures have been developed for the construction of lipid-
linked glycosyl diphosphates. For example, phosphoric anhydrides,28
phosphoryl dichlorides,29 and phosphoroimidazolidates30 have been used
(25) (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) Muller, T.; Schmidt, R. R. Angew. Chem., Int. Ed.
Engl. 1995, 34, 1328. (e) Arlt, M.; Hindsgaul, O. J. Org. Chem. 1995, 60,
14.
(26) (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) Zervosen, A.; Elling, L. J. Am. Chem. Soc. 1996, 118, 1836.
(27) Roseman, S.; Distler, J. J.; Moffatt, J. G.; Khorana, H. G. J. Am.
Chem. Soc. 1961, 83, 659.
(29) Imperiali, B.; Zimmerman, J. W. Tetrahedron Lett. 1990, 31, 6485.
(30) (a) Fang, X.; Gibbs, B. S.; Coward, J. K. Bioorg. Med. Chem. Lett.
1995, 5, 2701. (b) Danilov, L. L.; Maltsev, S. D.; Shibaev, V. N.; Kochetkov,
N. K. Carbohydr. Res. 1981, 88, 203.
(31) For example, coupling reactions utilizing the phosphoroimidazolidate
deriving from the anomeric phosphate (R-configuration) of N-acetyglu-
cosamine-3,4,6-triacetate and farnesyl monophosphate proceeded cleanly
to the desired product as judged by mass spectrometry.
(32) Please refer to the Supporting Information for details regarding the
HPLC purification of lipid I.
(28) Warren, C. D.; Jeanloz, R. W. Biochemistry 1972, 11, 2565.

6988 J. Am. Chem. Soc., Vol. 123, No. 29, 2001
VanNieuwenhze et al.
Conclusions
events in bacterial cell wall biosynthesis and, ultimately, lead
to the identification of new chemotherapeutic agents with novel
modes of action.
The emergence of bacterial resistance to antimicrobial agents
that inhibit bacterial cell wall biosynthesis has hastened the
search for novel, yet to be exploited, targets for chemothera-
peutic intervention. Detailed study of the bacterial cell wall
biosynthesis enzymes, as well as the resistance mechanisms
themselves, has been slow to develop due to the limited
availability of the natural substrates. Organic synthesis can
provide a powerful solution to this problem as has already been
demonstrated with the synthesis of Park Nucleotide, the im-
mediate biosynthetic precursor to lipid I.6 It is our anticipation
that this total synthesis of lipid I will provide another valuable
biochemical tool that may advance the understanding of key
Acknowledgment. We gratefully acknowledge the important
contributions of Dr. Brian Winger, Dr. David Peake, and Mr.
James Gilliam (mass spectrometry), Mr. Robert Boyer (NMR
spectroscopy), and Mr. William Alborn (biochemistry).
Supporting Information Available: Complete experimental
procedures and spectral data are available for all compounds
(PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
JA016082O