Preparation of potential inhibitors of the mur-pathway enzymes on solid support using an acetal linker

Article abstract of DOI:10.1016/S0960-894X(03)00043-X

Here, we report a novel strategy for the combinatorial or parallel solid-phase synthesis of potential inhibitors of the mur-pathway enzymes. The strategy involves the efficient use of p-alkoxybenzylidene acetal linker for reversible immobilization of sugar scaffolds to solid phase. This methodology was used to synthesize several glycopeptides on solid phase in good yields.

Full text of DOI:10.1016/S0960-894X(03)00043-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1125–1128
Preparation of Potential Inhibitors of the Mur-Pathway
Enzymes on Solid Support Using an Acetal Linker
Milana Maletic,* Jelena Antonic, Aaron Leeman, Gina Santorelli and Sherman Waddell
Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 30 July 2002; accepted 26 December 2002
Abstract—Here, we report a novel strategy for the combinatorial or parallel solid-phase synthesis of potential inhibitors of the mur-
pathway enzymes. The strategy involves the efficient use of p-alkoxybenzylidene acetal linker for reversible immobilization of sugar
scaffolds to solid phase. This methodology was used to synthesize several glycopeptides on solid phase in good yields.
# 2003 Elsevier Science Ltd. All rights reserved.
The bacterial cell wall was first identified as a target for
antibacterial agents, namely b-lactams, almost half a
century ago.1 However, some of the enzymes involved
in the early, cytoplasmic steps (Fig. 1) of the cell wall
assembly have not been exploited for antibacterial
research until very recently,2 primarily due to the diffi-
culties in obtaining their substrates. Bacterial cell wall is
a polymeric peptidoglycan consisting of alternating
N-acetylmuramic acid (MurNAc) and N-acetyl-
glucosamine (GlcNAc) units that are cross-linked
through the pentapeptide chains. The monomeric pre-
cursor for peptidoglycan biosynthesis (3) is assembled in
the cytoplasm starting with the condensation of UDP-
N-acetylglucosamine (1) and phosphoenolpyruvate
(PEP) catalyzed by MurA. Carboxylate (2), generated
by reduction with NADPH and MurB, serves as the
point of attachment for the pentapeptide chain, which is
in turn synthesized by a series of ATP-dependent amino
acid ligases (MurC-F).3 Recently, a high throughput
coupled enzyme assay has been developed to simulta-
neously screen for an inhibitor of any one of the
enzymes in the pathway.4 With such a potent tool for
identifying inhibitors in hand, the challenge remains to
develop an efficient synthesis of inhibitors for these
enzymes, since they are all valid antibacterial targets.
In the past decade solid-phase combinatorial chemistry
has emerged as a very powerful tool for discovery of
biologically active compounds and for rapid optimiza-
tion of lead structures.5 The split–pool6 technique
allows for rapid synthesis of large libraries of com-
pounds, but assaying mixtures may give false results and
requires labor-intensive deconvolution. Parallel synth-
esis of single compounds avoids these screening pro-
blems, but is limited to the synthesis of smaller
libraries.7 Since each bead in the split-pool combinator-
ial synthesis undergoes a set of unique chemical reac-
tions, at the end of the sequence it carries only a single
Figure 1. Cytoplasmic steps of bacterial cell wall biosynthesis: (a) MurA, PEP; (b) MurB, NADPH; (c) MurC, ATP, l-Ala; (d) MurD, ATP, d-Glu;
(e) MurE, ATP, mDAP; (f) MurF, ATP, d-Ala-d-Ala.
*Corresponding author. Tel.: +1-732-594-5001; fax: +1-732-594-9473; e-mail: milana_maletic@merck.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00043-X

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M. Maletic et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1125–1128
chemical entity.5e Creating the solid phase library using
the split–pool technique, but separating the beads prior
to cleavage and assay, retains the synthetic advantages
of split–pool technique while also having the analytical
advantages of parallel synthesis.8 In this paper, we
report on our efforts to develop new strategies for com-
binatorial synthesis of glycoside-based potential inhibi-
tors of MurA-F enzymes on solid phase. In this process,
we developed an efficient linker for the reversible
immobilization of monosaccharides to solid phase and,
as a proof of principle, synthesized two small proto-
typical glycoside-based libraries on solid phase.
sugars to solid phase as 4,6-benzylidene acetals
appeared in the literature over 25 years ago,9 the utility
of this linker in solid phase synthesis has not been
widely explored.10
The goal of our first prototype split–pool library was to
evaluate the stability and cleavage conditions of the
benzylidene acetal anchor group and to establish that
good loading and recovery of compound from indivi-
dual beads could be achieved.11 Initially we synthesized
resin bound MurNAc (8) as depicted in Scheme 1, then
functionalized it using the split–pool method (Scheme 2)
to make a nine-compound library. A commercially
available MurNAc derivative (4) was protected as a
2-(trimethylsilyl)ethyl (TMSE) ester using standard
conditions. Treatment of the diol (5) with dimethyl ace-
tal (6) and a catalytic amount of p-toluenesulfonic acid
(TsOH) gave the 9-fluorenylmethyl (Fm)-protected
benzylidene acetal (7a). Following several protecting-
group manipulation steps, acid (7c) was coupled to the
resin using EDC/HOBt conditions. The loading was
assessed qualitatively using the Kaiser test for free
amine residues12 and gravimetrically by releasing the
product off the resin with 90% aqueous acetic acid
It has been shown that the MurNAc moiety is an
important contributor to the potency of some MurD
inhibitors,2a since it displays the UDP, N-acyl, and
pentapeptide moieties around a rigid scaffold in a pre-
dictable orientation. Therefore, in designing our proto-
type libraries we used the unaltered sugar scaffold, but
allowed for modifications at three other sites: the pep-
tide position, the N-acyl position, and the polar
anomeric UDP position. That left the 4,6-diol unit
unfunctionalized, and an ideal point for anchoring to
the resin. Although, an initial report on suspending
Scheme 1. Synthesis of orthogonally protected MurNAc and attachement to the aminomethyl polystyrene resin through the p-alkoxybenzylidene
.
acetal anchor group 3: (a) TMSethanol, EDC, DMAP, cat., 80%; (b) Pd(OH)2, H2, EtOAc, MeOH, 90%; (c) TsOH H2O, DMF, 67%; (d) allyl
chloroformate, Et3N, CH2Cl2, 80%; (e) 7% diethylamine, CH2Cl2, CH3CN; (f) PS-CH2NH2, EDC, HOBt, CH2Cl2, D MF.
Scheme 2. Synthesis of a nine compound library: (a) TBAF, THF; (b) split into three vessels; (c) R1-NH2, EDC, HOBt, DMF; (d) mix; (e)
Pd(PPh3)4, PhSiH3, CH2Cl2; (f) split into three vessels; (g) R2-SO2NCO; (h) mix; (i) TBAF, THF; (j) 15% TFA, CH2Cl2, 1% H2O (or glacial acetic
acid, 40 ꢀC).

M. Maletic et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1125–1128
1127
(AcOH) containing 1% trifluoroacetic acid (TFA).
Released product showed no detectible cleavage of the
acid labile anomeric allyloxycarbonyl (Alloc) group.
TsOH.15 MurNAc derivative (5) was directly attached
onto the activated resin (13) by reacting only two
equivalents of sugar with respect to the reactive sites on
the resin to accomplish high loading.16
Using a split–pool combinatorial technique we prepared
a 1 Â 3 Â 3 library of compounds following Scheme 2.
The carboxylate of the resin bound MurNAc derivative
(8) was unmasked with tetrabutylammonium fluoride
(TBAF) in THF. The resin was split into three vessels
and coupled with different protected amino acids (b-
alanine, l-serine and d-glutamate) (9), then mixed and
the anomeric position deprotected. Next, the resin was
split into three vessels and treated with three aryl-sulfo-
nyl isocyanates (phenyl, o-toluyl, and p-chlorophenyl)
(10). Finally, the resin was pooled, treated with TBAF
and the compounds (11) released from solid phase using
either TFA in methylene chloride at room temperature,
or glacial acetic acid at 40 ꢀC. These cleavage conditions
were sufficiently mild to leave the acid-labile anomeric
acylsulfonamides intact.13 Average yield was deter-
mined gravimetrically to be 53%.14 By successful pre-
paration of this 9-component library, we demonstrated
the usefulness and versatility of the alkoxybenzylidene
acetal linker, its stability during synthesis, and desirable
susceptibility to weak acid that allows for reversible
immobilization of sugars to solid phase.
To showcase the usefulness of this approach in immo-
bilizing not very readily available mono-saccharides on
solid phase and to synthesize useful building blocks for
a larger library of mur-pathway inhibitors, we prepared
four analogues of 5 (17a–d) by diversifying the acyl
moiety (Scheme 4). Commercial 2-acetamido-2-deoxy-
glucopyranoside (15), was deacetylated using potassium
hydroxide (KOH) in ethanol at elevated temperature.
Using standard conditions, the amino group was selec-
tively acylated, in 75–85% yields, to give four amides
(16a–d) which were then elaborated to triols (17a–d).
Each of the glycosides was attached to the solid phase
using procedures outlined in Scheme 3. These resins
were then subjected to the series of steps analogous to
the ones described in Scheme 2 to elaborate fully to the
potential inhibitors (18 a–e) shown in Figure 2.
Having to functionalize the monosaccharide with the
linker (7c) prior to resin attachment makes this metho-
dology less useful when preparing larger libraries, due
to the laborious synthesis of the appropriately protected
sugars. A more efficient approach would involve con-
densing the resin-bound linker directly with the 4,6-diol
of the monosaccharide. In order to employ that
approach, we developed methodology outlined in
Scheme 3. The resin-bound 2-(4-formylphenoxy) acetate
(12) was activated as a dimethyl acetal using standard
methods, trimethylorthoformate (TMOF) and catalytic
Figure 2. Potential inhibitors of the mur-pathway enzymes prepared
on solid phase using the procedures outlined in Schemes 2 and 3.
Scheme 3. An alternative method for immobilization of MurNAc to the resin using a preattached linker: (a) EDC, HOBt, DMF; (b) TMOF, TsOH,
CH2Cl2; (c) TsOH, DMF; (d) allylchloroformate, CH2Cl2.
Scheme 4. Synthesis of acyl derivatives of 5: (a) KOH, EtOH, 130 ꢀC (87%); (b) RCOCl, CH2Cl2, pyridine (75–85%); (c) NaH, dioxane (30–50%);
(d) EDC, DMAP, TMSethanol (80–95%); (e) Pd(OH)2/C, EtOAc, MeOH, 40 psi.

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M. Maletic et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1125–1128
In conclusion, we developed a new synthetic strategy for
the combinatorial or parallel synthesis of muramic acid
glycopeptides as potential inhibitors of the mur- path-
way enzymes. The strategy involves reversible attach-
ment of the sugars through the 4,6-diol to the resin
using an acid-sensitive linker. This efficient methodol-
ogy for immobilizing sugars to a resin, which reduces
the number of synthesis steps prior to attachment,
makes this strategy very appealing for immobilizing not
very readily available sugar scaffolds. Work is in pro-
gress to miniaturize the mur-pathway enzyme assay and
to develop usable tagging methodology that will facil-
itate identification of compounds on bead. Coupled
with these powerful technological tools, this synthetic
strategy could allow for identification of novel inhibi-
tors for some of the mur-pathway enzymes.
6. (a) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. Int. J.
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Acknowledgements
We thank Dr. Yusheng Xiong for developing the meth-
odology for characterization of the libraries.
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References and Notes
13. The library was characterized by single bead cleavage of
compounds followed by LC/MS analysis. Beads were picked,
suspended in 50 mL of cleavage cocktail for 15 min, then dried
under a nitrogen stream, resuspended in 15 mL of acetonitrile
and analyzed by LC/MS. Individual cleavage of 20 beads
resulted in finding all of the nine desired compounds. The
HPLC trace of each compound confirmed the high purity
(>90%) or each analogue.
14. Forty milligrams of resin were treated with 15%TFA in
methylene chloride for 10 min. The resin was filtered off and
washed with three washes of methylene chloride and acetoni-
trile. The filtrates were combined and solvent was removed
under reduced pressure to yield 9 mg of oily residue (53%
yield).
15. While this conversion could be followed by IR (observing
the disappearance of the carbonyl stretch) on the Wang alde-
hyde resin, it could not be followed by the same method on the
methyl amino resin due to the presence of the amide bond.
Instead, the optimized conditions for the Wang aldehyde resin
were applied to the aminomethyl polystyrene resin.
16. Loading was assessed gravimetrically, by weighing the
cleaved product from the known amount of resin (assum-
ing 1.1 mmol/g loading) and also by comparison of clea-
vage product HPLC trace with the cleavage product of
resin 8.
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