Modular synthesis of diphospholipid oligosaccharide fragments of the bacterial cell wall and their use to study the mechanism of moenomycin and other antibiotics

Article abstract of DOI:10.1016/j.tet.2011.09.114

We present a flexible, modular route to GlcNAc-MurNAc-oligosaccharides that can be readily converted into peptidoglycan (PG) fragments to serve as reagents for the study of bacterial enzymes that are targets for antibiotics. Demonstrating the utility of these synthetic PG substrates, we show that the tetrasaccharide substrate lipid IV (3), but not the disaccharide substrate lipid II (2), significantly increases the concentration of moenomycin A required to inhibit a prototypical PG-glycosyltransferase (PGT). These results imply that lipid IV and moenomycin A bind to the same site on the enzyme. We also show the moenomycin A inhibits the formation of elongated polysaccharide product but does not affect length distribution. We conclude that moenomycin A blocks PG-strand initiation rather than elongation or chain termination. Synthetic access to diphospholipid oligosaccharides will enable further studies of bacterial cell wall synthesis with the long-term goal of identifying novel antibiotics.

Full text of DOI:10.1016/j.tet.2011.09.114

Tetrahedron 67 (2011) 9771e9778
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Modular synthesis of diphospholipid oligosaccharide fragments of the bacterial
cell wall and their use to study the mechanism of moenomycin and other
antibiotics
,
Christian M. Gampe a, Hirokazu Tsukamoto a, Tsung-Shing Andrew Wang a, Suzanne Walker b
,
*
,
Daniel Kahne a
*
a Harvard University, Department of Chemistry and Chemical Biology, 12 Oxford Street, Cambridge, MA 02138, USA
b Harvard Medical School, Department of Microbiology and Molecular Genetics, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 August 2011
Received in revised form 21 September 2011
Accepted 23 September 2011
Available online 29 September 2011
We present a flexible, modular route to GlcNAceMurNAc-oligosaccharides that can be readily converted
into peptidoglycan (PG) fragments to serve as reagents for the study of bacterial enzymes that are targets
for antibiotics. Demonstrating the utility of these synthetic PG substrates, we show that the tetra-
saccharide substrate lipid IV (3), but not the disaccharide substrate lipid II (2), significantly increases the
concentration of moenomycin A required to inhibit a prototypical PG-glycosyltransferase (PGT). These
results imply that lipid IV and moenomycin A bind to the same site on the enzyme. We also show the
moenomycin A inhibits the formation of elongated polysaccharide product but does not affect length
distribution. We conclude that moenomycin A blocks PG-strand initiation rather than elongation or chain
termination. Synthetic access to diphospholipid oligosaccharides will enable further studies of bacterial
cell wall synthesis with the long-term goal of identifying novel antibiotics.
Keywords:
Lipid IV
Moenomycin
Peptidoglycan
Transglycosylation
Carbohydrate synthesis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
antibiotics that inhibit strand polymerization and cross-linking
have been identified (e.g., Scheme 1). These antibiotics function
Bacteria have evolved a cell wall that protects the cell mem-
brane from hostile environments and allows the cells to withstand
varying osmotic pressures.1 The supportive structure of the cell
wall is peptidoglycan (PG), consisting of polysaccharide threads of
alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic
acid (MurNAc) units.2 These polysaccharides are cross-linked
through peptide side chains appended to the MurNAc units to
form a stable polymeric mesh. Many highly effective antibacterial
drugs disrupt the formation of the cell wall, which ultimately re-
sults in cell death. It is important to better understand cell wall
synthesis in order to develop new therapies for bacterial
infections.3
Work over the past several decades has shed light on the
biosynthesis of PG. In the final stages of biosynthesis lipid II (2,
Fig. 1) is polymerized by peptidoglycan glycosyl transferase (PGT)
and the resulting polysaccharide strands are then linked by
transpeptidases (TPs).2 Many different classes of natural product
by three major type of mechanisms: (1) They bind to PGT sub-
strates, such as 2, sequestering them so they are unavailable for
PG-strand synthesis and cross-linking (e.g., vancomycin4 and
ramoplanin5); (2) They acylate TP-domains, inactivating them so
that they are incapable of strand cross-linking (e.g., the beta-
lactams);6 or (3) they bind to PGT domains, preventing PG strand
formation (e.g., moenomycin A (1), Fig. 1).7 Antibiotics having the
first or second mechanism of action have been highly effective in
the clinic but there are no approved drugs with the third
mechanism.8
The natural product moenomycin A (1), which binds to PGTs, has
potent antibacterial activity against Gram-positive pathogens and
has been used as a growth promoter in animals for decades without
engendering resistance.9 Its use in humans has been prevented by
unfavorable pharmacokinetic properties (e.g., a long half-life and
low oral bioavailability)8d but it can serve as a blueprint for the
development of other PGT inhibitors. Hence, a detailed un-
derstanding of how 1 inhibits PGTs is important for exploiting these
crucial enzymes as antibiotic targets.
A major impediment to study antibiotics that inhibit PGTs has
been the lack of access to substrates used by these enzymes.
PGTs catalyze the transformation of lipid II (2) into a polymer. In
* Corresponding authors. Tel.: þ1 617 496 0208; fax: þ1 617 496 0215 (D.K.); tel.:
þ1 617 432 5488; fax: þ1 617 496 0215 (S.W.); e-mail addresses: suzanne_walker@
hms.harvard.edu (S. Walker), kahne@chemistry.harvard.edu (D. Kahne).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.09.114

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C.M. Gampe et al. / Tetrahedron 67 (2011) 9771e9778
polysaccharides of different chain lengths, comprising
nꢀGlcNAceMurNAc-units (cf. 10). We envisioned the use of a di-
saccharide building block 4 that could be converted into both a di-
saccharide donor (5) and acceptor (6), which would then be
coupled to obtain tetrasaccharide 7. A similar sequence of differ-
entiation and coupling would give access to octasaccharide 10 and
higher polysaccharides at will. Additionally, we required a large
amount of Z,Z,Z,Z,E,E-heptaprenyl phosphate 12 for use as an ac-
ceptor substrate. We thus sought a scalable, stereoselective route to
the heptaprenyl alcohol starting from the readily available E,E-far-
nesol. Since current methods for the preparation of pyrophosphates
(13) are inefficient, an effective protocol for the formation of py-
rophosphates by condensation of two monophosphates needed to
be established.
2.1. Synthesis of Z,Z,Z,Z,E,E-heptaprenyl alcohol
For the synthesis of heptaprenyl alcohol 21 we devised a syn-
thetic route that relied on the finding of Casey and co-workers that
b
-keto esters can be stereoselectively converted into their dieth-
ylphosphoroxy enol ethers.11 These, in turn, react stereospecifically
Fig. 1. Molecular structures of moenomycin A (1), lipid II (2), and lipid IV (3).
Scheme 1. The final stages of peptidoglycan biosynthesis involve polymerization of lipid II to make oligosaccharide chains followed by cross-linking via transpeptidation. This
sequence is often performed by bifunctional enzymes containing distinct glycosly transferase (GT) and transpepdidase (TP) domains (depicted in gray). Peptidoglycan synthesis can
be disrupted at different steps by various antibiotics.
the first round of synthesis, two units of lipid II are coupled. In
subsequent rounds of synthesis, lipid II adds in a processive
manner to the elongating glycan strand. In order to dissect the
reaction, it is necessary to have both disaccharide and oligosac-
charide substrates available to discriminate between the glycosyl
acceptor and the glycosyl donor sites of the enzyme. For over
a decade we, and others, have been developing methods to ob-
tain PGT substrates as well as assays using these substrates to
probe the mechanisms and inhibition of PG biosynthetic en-
zymes.10 Here we report a modular synthesis of polysaccharides
to obtain differentiated diphospholipid oligosacharide substrates
that serve as specific mimics of the glycosyl donor strand. Fur-
thermore, we used these substrates to learn more about the
mode of action of moenomycin A.
in a substitution reaction with methylcuprates.12 Thus, E,E-farnesol
(14) was converted into -keto ester 15 in 84% yield (over two
steps). Execution of Casey’s and Weiler’s protocols followed by re-
duction of the ester with DIBAL-H provided E,E,Z-tetraprenyl alco-
hol 17 as the only stereoisomer observed and yields ranged from 30
to 50% over five steps (Scheme 3).11,12
b
Upon iteration of this procedure with 17 we noted, however,
that the Z/E ratio eroded in the third cycle. The desired hexaprenyl
alcohol 19 was obtained in a 7/3 mixture along with its double bond
isomer. Purification by chromatography proved unsuccessful and
we were forced to optimize the reaction conditions with regard to
stereospecificity of the substitution of the phosphate by a methyl
group. To this end we switched from a diethyl phosphate to
a diphenyl phosphate leaving group and were able to derive enol
ether 18 in 60% yield over three steps. Gratifyingly, we found that 18
participated in a highly efficient and stereospecific cross coupling
reaction with MeMgCl using Fe(acac)3 as catalyst.13 After DIBAL-H
reduction we obtained 19 in 75% (over two steps). Moreover, we
found that the iron-catalyzed cross coupling retained its high ste-
reospecificity upon iteration of the sequence to provide the desired
heptaprenyl alcohol 21 in an average yield of 7% from farnesol. With
this procedure in hand we obtained more than 2 g of 21, and we
next turned our attention to the development of an efficient cou-
pling procedure for the formation of pyrophosphates.
2. Results and discussion
An efficient synthesis of lipid IV (3) and its higher homologs, i.e.,
lipid VI, VIII etc., must address a number of challenges associated
with these intriguing, structurally complex, primary metabolites.
First and foremost we had to fundamentally change our previously
described synthetic strategy (Scheme 2).10l,m Whereas the previous
route was specifically tailored to obtain 3, we now required
a
strategy that would easily allow access to a variety of

C.M. Gampe et al. / Tetrahedron 67 (2011) 9771e9778
9773
Scheme 2. Disaccharide 4, equipped with a suitable C4eOH group (red dot) and an anomeric protecting group (black dot), gets converted into both a disaccharide donor and
acceptor (5 and 6). Coupling of 5 and 6 yields tetrasaccharide 7 that can be converted into octasaccharide 10 in a similar sequence via donor 8 and acceptor 9. Repetition of this
sequence followed by coupling to 12 allows access to diphospholipid oligosaccharides 13 with different length of the oligosaccharide.
removal of the protecting groups, pyrophosphate 24 was obtained
in acceptable yields (54%) and was subsequently transformed into
lipid I, showcasing the utility of this protocol for the synthesis of
lipid pyrophosphate saccharides.
Scheme 4. Reagents and conditions: (a) CDI, DMF then MeOH; (b) N-Methyl-
imidazole$HCl (4 equiv), DMF/THF 1/1, rt, 12 h; (c) 1 M LiOH$H2O, THF/MeOH/H2O
(3:1:1), 54%.
Scheme 3. Reagents and conditions: (a) PBr3 (0.37 equiv), Et2O, ꢁ30 ꢂC to rt, 96%; (b)
ethyl acetoacetate (2 equiv), NaH (2.2 equiv), BuLi (2.1 equiv), THF,
0
ꢂC, 87%; (c)
2.3. Modular synthesis of polysaccharides
ClPO(OEt)2 (1.1 equiv), NEt3 (1.1 equiv), DMAP (0.1 equiv), DMPU, 0 ꢂC, 69%; (d) CuI
(3 equiv), MeLi (3 equiv), MeMgCl (5 equiv) THF; (e) DIBAL-H (2.2 equiv), PhMe, 66%
over two steps; (f) PBr3 (0.37 equiv), Et2O, ꢁ30 ꢂC to rt; (g) ethyl acetoacetate (2 equiv),
NaH (2.2 equiv), BuLi (2.1 equiv), THF, 0 ꢂC, 70% (two steps); (h) ClPO(OPh)2 (2 equiv),
NEt3 (2 equiv), DMAP (0.2 equiv), DMPU, 0 ꢂC, 86%; (i) MeMgCl (3 equiv), Fe(acac)3
(0.05 equiv), THF/NMP¼20/1; (j) DIBAL-H (2.2 equiv), PhMe, 75% (two steps); (k) see
fej, 45e60% yield over five steps.
The modular synthesis of the polysaccharide part of the lipid
pyrophosphate sugars started with readily available glucosamine
derivate 25 and took advantage of the bifunctionality of its thio-
phenol group, the phthalimide group, as well as a benzylic acetal at
C4 and C6 (Scheme 5).15 Thiophenol is a powerful protecting group
of the anomeric position but can easily be converted into its sulf-
oxide,16 which in turn is activated with Tf2O under mild conditions
(ꢁ78 ꢂC) to provide a superb glycosyl donor.17 Phthalimides at C2
are known to sterically shield the C3 position, obviating an addi-
tional protecting group for the C3 hydroxy group.18 Phthalimide
groups also provide anchimeric assistance to allow glycosylation to
occur with high ß-selectivity. Finally, a number of procedures have
been put forth for the reduction of a C4/C6 acetal, which allows
selective liberation of the C4 group.19
2.2. N-Methylimidazolium chloride as an effective catalyst for
pyrophosphate formation
We recently reported the identification of N-methyl imidazo-
lium chloride as a catalyst for pyrophosphate formation by con-
densation of two phosphates (Scheme 4).14 This catalyst is superior
to the commonly employed tetrazole with respect to activity, cost,
and safety. Using this catalyst we established a synthesis of lipid I.
Sugar phosphate 22 was first converted to phophorimidazolide 23,
which was coupled with heptaprenyl phosphate 21 in 12 h. After
Glucosamine 25 was thus converted into both glycosyl donor 26,
by protection of C3 and oxidation of the thioether, and glycosyl

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C.M. Gampe et al. / Tetrahedron 67 (2011) 9771e9778
Scheme 5. Reagents and conditions: (a) BnBr (1.1 equiv), NaH (1.2 equiv), Bu4NI (0.1 equiv), DMF, rt, 70%; (b) mCPBA (1 equiv), CH2Cl2, ꢁ78 ꢂC to rt, 88%; (c) HSiEt3 (5 equiv), TFA
(5 equiv), CH2Cl2, 0 ꢂC, 93%; (d) 27 (1.5 equiv), ADMB (5 equiv), DTBMP (5 equiv), Tf2O (1 equiv), CH2Cl2, MS 4 A, 58%; (e) mCPBA (1.1 equiv), CH2Cl2, 77%; (f) HSiEt3 (12 equiv),
ꢀ
ꢀ
BF3$OEt2 (2 equiv), CH2Cl2, 70%; (g) 29 (1.25 equiv), ADMB (6.25 equiv), DTBMP (3.75 equiv), Tf2O (1.25 equiv), CH2Cl2, MS 4 A, 55%. Abbreviations: ADMB¼4-allyl-1,2-
dimethoxybenzene, DTBMP¼2,6-di-tert-butyl-4-methylpyridine.
acceptor 27 by reduction of the acetal using triethylsilane and TFA.
These two building blocks were then coupled under conditions
2.4. Use of synthetic PG substrates to study the mechanism of
moenomycin A inhibition
previously developed in our group to give b-1,4-disaccharide 28 in
57% yield.17 To suppress formation of a trisaccharide (at C3), it
proved beneficial to employ an excess of the glycosyl acceptor,
which could be reisolated after the reaction. Disaccharide 28 was
then subjected to a similar sequence to obtain glycosyl acceptor 30
(HSiEt3, BF3$OEt2, 70%) and glycosyl donor 29 (77%). These di-
saccharides were again subjected to Tf2O-induced glycosylation
and tetrasaccharide 31 was obtained in 55% yield. In this case ex-
cess disaccharide donor proved beneficial and residual acceptor 30
could be reisolated (20%). The regioselectivity of the reaction to give
the C4-glycosylated product was verified by acylation of the C3eOH
group, which leads to a significant 1H NMR downfield shift of the
proton at C3 (ca. 5.7 ppm).20 Furthermore, we found that the car-
bon atom that bears the glycosyl substituent experiences a 13C NMR
downfield shift, which allows facile structure determination (C3
from ca. 70 ppm to 80 ppm; C4 from ca. 75 ppm to ca. 85 ppm, as
determined by HSQC). We obtained precursors to lipid II (28) and
lipid IV (31) as required for the studies reported below but this
synthetic sequence could be further extended to obtain similarly
substituted hexa-, octa-, deca-, etc. saccharides.
To show the utility of this approach we converted 31 into
heptaprenyl-lipid IV (3b) (Scheme 6). To this end we installed the
N-acetates by aminolysis of the phthalimide with ethylenediamine,
followed by acylation of the free amine. The C3-lactic acid sub-
stituent was subsequently installed by SN2-reaction with S-(ꢁ)-2-
bromopropionic acid followed by methylation of the carboxylate
with TMS-diazomethane to obtain 33 in 53% yield from 31.
Substrate 33 intercepted a late stage in our previously published
synthesis and can be converted into lipid IV.10m
We have previously reported that inhibition of the prototypical
PGT, Escherichia coli PBP1a, by moenomycin A cannot be overcome
by increasing the concentration of lipid II.10h The complexity of the
enzymatic reaction, which involves substrate differentiation and
processive elongation, makes it challenging to interpret this result.
Therefore, we have revisited the question of how moenomycin A
inhibits PGT using a different approach that employs both lipid II
and a lipid IV substrate containing a blocked non-reducing end
(Gal-lipid IV).21 The concentration of moenomycin A required to
inhibit by 50% (IC50) the polymerization of lipid II by E. coli PBP1a
(20 nM) was found to be 9.2 nM (Fig. 2); or approximately 1/2 the
enzyme concentration. This IC50 reflects the high affinity of moe-
nomycin A for the enzyme. The addition of 1.4 mM Gal-lipid IV,
which functions only as a glycosyl donor, shifts the IC50 of moe-
nomycin A fivefold.22 The shift implies that Gal-lipid IV and moe-
nomycin A compete for binding to the same site on the enzyme. It
has previously been suggested that moenomycin A is a mimetic of
the elongating glycan strand but the results reported here provide
the first kinetic evidence that the inhibitor and the elongating
substrate occupy the same site.23
Fig. 2. Dose-response curves of the moenomycin A inhibition of peptidoglycan for-
mation. Solutions of E. coli PBP1a (20 nM) were incubated with moenomycin A in
different concentrations and then treated with 14C-lipid II (4
mM). Distribution of lipid
II/polysaccharide was determined by scintilation count after paper chromatography of
the product solution. Blue curve: no Gal-lipid IV added; red curve: PBP1a was pre-
treated with Gal-lipid IV (1.36 mM).
We next examined whether moenomycin A has any effect on the
length distribution of products formed by the PGT catalyzed poly-
merization of lipid II. E. coli PBP1a was incubated with lipid II in the
presence of increasing concentrations of moenomycin A and the
products were analyzed by SDS-PAGE (Fig. 3).10h,24 PG-polymers
could not be detected at moenomycin concentrations equal to or
exceeding the enzyme concentration even at extended reaction
Scheme 6. Reagents and conditions: (a) ethylenediamine, EtOH/MeCN/THF, 60 ꢂC,
48 h; then: Ac2O, MeOH/H2O, 70%; (b) NaH, DMF, S-2-bromopropionic acid, then:
TMSCHN2, MeOH, PhH; 75%; (c) see Ref. 10m.

C.M. Gampe et al. / Tetrahedron 67 (2011) 9771e9778
9775
times. At moenomycin concentrations below the enzyme concen-
tration, products were detected but the length distribution
appeared similar regardless of the inhibitor concentration. Since
the resolution of the page assay is limited for very long products,
we repeated the experiments with a different PGT that was pre-
viously shown to produce much shorter products.10l,m As before,
products were not detected at equimolar enzyme:moenomycin
concentrations. Products formed at lower moenomycin concen-
trations, but the length distribution did not vary significantly as
a function of concentration. These results imply a mechanism for
inhibition in which moenomycin A blocks chain initiation rather
than elongation or termination since effects on the latter stages of
the enzymatic reaction would alter product length (Scheme 7).
oligosaccharide cell wall fragments. We have used these PG frag-
ments, containing diphosphate activated reducing termini, to
evaluate the mechanism of action of moenomycin A. We have
provided the first kinetic evidence that moenomycin A occupies the
same binding site as the elongating PG strand. Since we have pre-
viously shown that PG strands elongate by addition of disaccharide
units to their reducing ends,10l the kinetic data is consistent with
the proposal by Welzel and co-workers that moenomycin A mimics
the growing glycan strand.7 Since PGTs can exist in both open and
closed conformations,23 however, moenomycin A and the growing
chain may not bind to the same conformation. Efficient access to PG
fragments that bind in the donor site may make it possible to obtain
structures of PGTs bound to substrates.10p,r
In addition to providing substrates to study PGTs, the reported
synthetic route enables access to oligosaccharide fragments of PG
to study the transpeptidation reaction. (i.e., peptide cross-linking).
The transpeptidases are the lethal targets of the b-lactams but an
inability to obtain PG oligosaccharides has precluded detailed
mechanistic studies of these enzymes. The methods reported here
will provide the tools necessary to understand the TPs and other
penicillin-binding proteins in detail.
4. Experimental section
4.1. General methods
All reactions were carried out under an argon atmosphere with
solvents dried by passage over activated alumina. Reagents were
used as obtained with the following exceptions: BF3$OEt2 was
ꢀ
distilled prior to use. Ethylenediamine was stirred over MS 4 A for
24 h, then stored over solid KOH and distilled prior to use. TLC was
carried out using SiO2 coated glass plates (Merck,
200ꢀ200ꢀ0.25 mm3) and spots were visualized by fluorescence
quench at 254 nm or by staining with cerium ammonium sulfate or
anisaldehyde solution. Flash column chromatography was per-
Fig. 3. SDS-PAGE of enzymatic 14C-lipid II-polymerization reactions (8
mM) with
varying amounts of moenomycin A (MmA) present. Red lines mark equimolar ratio of
enzyme to moenomycin A. In reactions with c(MmA)ꢃc(PBP) no polymerization of
lipid II is observed. If c(MmA)<c(PBP), polysaccharide is produced. Note that the length
distribution of polysaccharides formed varies with the enzyme employed but is not
influenced by moenomycin A.
ꢀ
formed on silica gel obtained from Sorbent Technologies (60 A,
32e63
(3 MM CHR). Analytical HPLC/MS was carried out on an Agilent
Series 1100 instrument, using a Phenomenex Luna C18 3 m col-
mm). Chromatography paper was obtained from Whatman
m
umn and ESI ionization. NMR spectra were recorded on a Varian
I500, Varian M400, or Varian M300 spectrometer. Chemical shifts
are reported in parts per million as d and referenced to the residual
internal solvent signal (CDCl3: 7.26 (1H) and 77.16 (13C); DMSO-D6:
2.50 (1H) and 39.52 (13C)). High resolution mass spectrometry was
performed on a Bruker microTOF instrument using ESI. Enzymes
were obtained and handled as previously reported.10m 14C-Hep-
taprenyl-lipid II was obtained as previously described.10c Moeno-
mycin
A
was isolated from flavomycin as reported.25
Autoradiography was carried out using storage phosphor screens
by GE Healthcare and a GE Healthcare Typhoon 9400 scanner.
4.2. General procedure for the Z-prenylation of allylic
alcohols 14, 17, 19, and 20
The allylic alcohol was dissolved in Et2O (1 M) and cooled to
ꢁ30 ꢂC before PBr3 (0.37 equiv) was added. MeOH and H2O were
added as TLC analysis indicated completion of the reaction. The
mixture was extracted with hexane and the organic phases were
washed with water, NaHCO3 (satd) and brine, and dried over
MgSO4. Evaporation of the solvent in vacuo provided the allylic
bromide, which was used as such in the next step.
Scheme 7. Model for lipid II-polymerization by peptidoglycan glycosyltransferases
(PGTs) and their inhibition by moenomycin A (MmA). Moenomycin A blocks initiation
of the polymerization of lipid II by binding to the donor site (D) of the PGT.
3. Conclusion
NaH (2.2 equiv) was suspended in THF (1.1 M) cooled to 0 ꢂC
before ethyl acetoacetate (2.0 equiv) were added. After 15 min n-
BuLi (2.5 M, 2.1 equiv) was added and the mixture was stirred for
15 min before a solution of the allylic bromide (1 equiv, 2.0 M in
THF) was added. After 1.5 h, Et2O (1ꢀreaction volume) and 3 M HCl
We report a modular synthesis of diphospholipid oligosaccha-
rides using a bifunctional disaccharide building block. Along with
our improved synthesis of pyrophosphates and of heptaprenyl al-
cohol, this synthetic sequence provides access to higher-order

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C.M. Gampe et al. / Tetrahedron 67 (2011) 9771e9778
(1/2ꢀreaction volume) were added. The phases were separated and
the organic layer was washed with H2O and brine and dried over
MgSO4. Evaporation of the solvent in vacuo and column chroma-
tography provided the desired ketoester.
The ketoester previously obtained (1 equiv), NEt3 (2.0 equiv),
and DMAP (0.2 equiv) were dissolved in DMPU (0.3 M in ketoester)
and cooled to ꢁ30 ꢂC. Diphenyl chlorophosphate (2.0 equiv) was
added and the solution was brought to rt over 3 h NH4Cl (satd) and
Et2O was added, the phases were parted and the organic layer was
washed with H2O and brine and dried over MgSO4. Removal of the
solvent in vacuum and column chromatography provided the de-
sired enol ether.
3H); HRMS (ESI): MþNaþ, found 602.1617; C34H29NO6SNa requires
602.1608.
A solution of this sulfide (3.01 g, 5.19 mmol) in CH2Cl2 (173 mL)
was cooled to ꢁ78 ꢂC and a solution of mCPBA (77% pure, as com-
mercially available, 896 mg) in CH2Cl2 (104 mL) was added over 1 h.
The cloudy reaction mixture was then brought to rt over 45 min
and then quenched with Na2SO3 (10% aqueous solution). The
phases were separated and the organic phase was washed with
NaHCO3 (satd) and brine and dried over Na2SO4. Removal of the
solvent in vacuo and column chromatographic purification of the
residue (SiO2, hexane/EtOAc 6/4) gave the title compound as col-
orless solid and as
a 1/1 mixture of diastereomers (2.72 g,
The above mentioned enol phosphate was dissolved in THF
(0.06 M), NMP (9.0 equiv), and Fe(acac)3 (0.05 equiv) were added,
and the mixture was cooled to ꢁ40 ꢂC. MeMgCl in THF (3.0 equiv)
was added and the solution was stirred for 30 min before the NH4Cl
(satd) was added. The aqueous layer was extracted with Et2O and
the combined organic phases were washed with 1 M HCl, NaHCO3
(satd) and brine and dried over MgSO4. The solvent was evaporated
in vacuo and the residue was dissolved in PhMe (0.5 M), cooled to
ꢁ78 ꢂC and treated with DIBAL-H (1.0 M in PhMe, 2.7 equiv). After
1 h MeOH was added and the mixture was brought to rt. NH4Cl
(satd),1 M HCl, and Et2O were added, the suspension was stirred for
1.5 h and then extracted with Et2O. The organic phases were
washed with water and brine and dried over MgSO4. Evaporation of
the solvent and column chromatography provided the desired
prenylated allylic alcohol. The analytical data obtained matched the
published data.26
4.57 mmol, 88%). Rf (hexane/EtOAc 6/4) 0.25; due to formation of
two sulfoxide diastereomers NMR data is not given; HRMS (ESI):
MꢁHOSPhþNaþ, found 492.1422; C27H24NO7S requires 492.1418.
4.5. Phenyl 4,6-O-benzylidene-3-O-benzyl-2-deoxy-2-
phthalimido-b-
2-phthalimido-1-thio-
D
-glucopyranosyl-(1/4)-6-O-benzyl-2-deoxy-
-glucopyranoside (28)
b-D
Glycosyl acceptor 27 (1.29 g, 2.62 mmol), 4-allyl-1,2-
dimethoxybenzene (1.50 mL, 8.75 mmol), and 2,6-di-tert-butyl-4-
methylpyridine (1.08 g, 5.25 mmol) were dissolved in benzene
and concentrated in vacuo to remove traces of water. The residue
ꢀ
was dissolved in CH2Cl2 (65.5 mL), MS 4 A (400 mg) was added and
the suspension was stirred at rt for 45 min before it was cooled to
ꢁ78 ꢂC. After addition of Tf2O (0.29 mL, 1.8 mmol) a solution of
glycosyl donor 26 (1.04 g, 1.75 mmol) in CH2Cl2 (44 mL) was added
over 2 h by syringe pump. After these 2 h, the solution was stirred
at ꢁ60 ꢂC for 1 h, followed by 1 h at ꢁ40 ꢂC. The reaction was
washed with NaHCO3 (satd) and the organic phase was dried over
Na2SO4. Removal of the solvent in vacuo, followed by column
chromatography on SiO2 (hexane/EtOAc 7/3) yielded disaccharide
28 as colorless solid (970 mg, 1.01 mmol, 58%). Rf (hexane/EtOAc 7/
3) 0.44; dH (500 MHz CDC13) 7.89e7.82 (m, 2H), 7.74e7.68 (m, 4H),
7.51e7.48 (m, 1H), 7.49e7.39 (m, 3H), 7.32e7.26 (m, 4H), 7.19e7.15
(m, 1H), 7.12e7.08 (m, 1H), 7.06 (d, J 6.0 Hz, 1H), 6.96 (d, J 6.5 Hz,
1H), 6.00e6.84 (m, 2H), 5.58 (s, 1H), 5.47 (d, J 10.0 Hz, 1H), 5.31 (d, J
8.0 Hz,1H), 4.75 (d, J 12.5 Hz,1H), 4.48e4.44 (m, 2H), 4.40e4.34 (m,
2H), 4.23e4.16 (m, 2H), 4.08e4.00 (m, 2H), 3.79e3.64 (m, 4H),
3.50e3.47 (m, 1H), 3.26e3.20 (m, 2H); dC (125 MHz, CDCl3) 168.4,
167.8, 138.3, 137.9, 137.2, 134.3, 134.2, 133.1, 132.0, 131.7, 129.4, 129.0,
128.6, 128.4, 128.3, 128.3, 128.1,127.7,127.6,127.6,126.3,124.1,123.7,
123.5, 101.7, 100.0, 83.27, 82.7, 81.6, 78.2, 74.4, 73.1, 71.2, 68.5, 68.2,
66.3, 56.0, 55.2, 30.4; HRMS (ESI): MþNaþ, found 983.2772;
C55H48N2O12SNaþ requires 983.2820.
4.3. Phenyl 6-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-D-
glucopyranoside (27)
Acetal 25 (2.17 g, 4.44 mmol) was dissolved in CH2Cl2 (11.1 mL)
and cooled to 0 ꢂC. Triethylsilane (3.54 mL, 22.2 mmol) and TFA
(1.70 mL, 22.2 mmol) were added and the mixture was stirred for
4 h at 0 ꢂC. Volatile components were removed in vacuo and the
residue was purified by column chromatography on SiO2, eluting
with hexane/EtOAc 1/1 to obtain the title compound 27 as colorless
solid (2.04 g, 4.15 mmol, 93%).15 Rf (hexane/EtOAc 1/1) 0.33; dH
(300 MHz CDC13) 7.83 (br, 2H), 7.73e7.70 (m, 2H), 7.39e7.26 (m,
6H), 7.22e7.19 (m, 3H), 5.60 (d, J 10.2 Hz, 1H), 4.60 (d, J 11.7 Hz, 1H),
4.56 (d, J 11.7 Hz, 1H), 4.36e4.30 (m, 1H), 4.23e4.16 (m, 1H),
3.85e3.75 (m, 2H), 3.70e3.55 (m, 2H), 3.35 (br, 1H), 3.00 (br, 1H);
HRMS (ESI): MþNaþ, found 514.1293; C27H25NO6SNa requires
514.1295.
4.4. Phenyl 4,6-O-benzylidene-3-O-benzyl-2-deoxy-2-
4.6. Phenyl 6-O-benzyl-3-O-benzyl-2-deoxy-2-phthalimido-
phthalimido-1-thio-
b-D
-glucopyranoside S-oxide(26)
b-D-glucopyranosyl-(1/4)-6-O-benzyl-2-deoxy-2-
phthalimido-1-thio-glucopyranoside (30)
Acetal 25 (3.60 g, 7.40 mmol) was dissolved in DMF (37 mL) and
cooled 0 ꢂC before NaH (356 mg, 60% in mineral oil) was added.
After gas evolution had ceased n-Bu4NI (260 mg, 704 mmol) and
BnBr (0.96 mL, 8.1 mmol) were added. After stirring the reaction at
rt for 20 h, MeOH (5 mL) was added dropwise and the resulting
mixture was partitioned between H2O and CH2Cl2. The phases were
separated and the aqueous phase was extracted three times with
CH2Cl2. The combined organic phases were washed with 10% LiCl in
water and dried over Na2SO4. After removal of the volatiles in vacuo
the residue was purified by column chromatography on SiO2
(hexane/EtOAc 9/1/8/2/7/3) to obtain 3-benzyloxy-25 as color-
less solid (3.01 g, 5.18 mmol, 70%).27 Rf (hexane/EtOAc 1/1) 0.81; dH
(300 MHz CDC13) 7.90e7.85 (m, 1H), 7.73e7.64 (m, 4H), 7.53e7.50
(m, 2H), 7.42e7.38 (m, 6H), 7.26e7.24 (m, 2H), 6.99e6.86 (m, 5H),
5.62 (s, 1H), 5.61 (d, J 10.5 Hz, 1H), 4.77 (d, J 12.3 Hz), 4.49 (d, J
12.3 Hz, 1H), 4.47e4.40 (m, 2H), 4.33e4.25 (m, 1H), 3.88e3.70 (m,
Disaccharide 28 (1.35 g, 1.40 mmol) was dissolved in CH2Cl2
(28.1 mL) and cooled to 0 ꢂC. Triethylsilane (2.68 mL, 16.8 mmol)
and BF3$OEt2 (0.36 mL, 2.8 mmol) were added and the mixture was
stirred for 2 h at 0 ꢂC NaHCO3 (satd) was added to the reaction
mixture and the phases were parted. The organic phase was con-
centrated in vacuo and the residue was purified by column chro-
matography on SiO2 (hexane/EtOAc 6/4/1/1) to obtain the title
compound 30 as colorless solid (950 mg, 0.986 mmol, 70%). Rf
(hexane/EtOAc 1/1) 0.53; dH (400 MHz CDC13) 7.92e7.80 (m, 2H),
7.74e7.70 (m, 3H), 7.58e7.55 (m, 1H), 7.35e7.34 (m, 2H), 7.31e7.28
(m, 2H), 7.23e7.22 (m, 3H), 7.17e7.15 (m, 1H), 7.11e7.05 (m, 2H),
7.00e6.97 (m, 2H), 5.49 (d, J 10.5 Hz, 1H), 5.30 (d, J 9.0 Hz, 1H), 4.69
(d, J 12.5 Hz,1H), 4.45e4.42 (m, 5H), 4.33e4.20 (m, 3H), 3.97 (s, 2H),
3.75e3.62 (m, 6H), 3.55e3.50 (m, 1H), 3.25 (m, 2H); 2.62 (m, 1H);
dC (125 MHz, CDCl3) 168.4, 167.7, 138.3, 138.2, 138.5, 134.2, 134.2,

C.M. Gampe et al. / Tetrahedron 67 (2011) 9771e9778
9777
132.9, 132.3, 132.1, 132.0, 131.8, 128.9, 128.8, 128.6, 128.5, 128.5,
128.4,128.1, 128.1,128.0, 127.9,127.8,127.6,127.5,123.9, 123.7,123.5,
99.3, 83.4, 81.9, 79.0, 78.1, 74.8, 73.9, 73.8, 73.5, 73.0, 71.5, 70.1, 68.6,
55.6, 55.2; HRMS (ESI): MþNaþ, found 985.2900; C55H5N2O12SNaþ
requires 985.2977.
tile compound as amorphous solid (422 mg, 289
m
mol, 70%). dH
(500 MHz CDC13) 8.05e8.03 (m, 1H) 8.00e7.97 (m, 1H), 7.93e7.89
(m, 2H), 7.41e7.37 (m, 6H), 7.30e7.17 (m, 26H), 5.69 (s,1H), 4.87 (d, J
12.0 Hz, 1H), 4.80 (d, J 10.5 Hz), 4.70e4.66 (m, 3H), 4.58e4.44 (m,
10H), 4.28 (d, J 12.0 Hz, 1H), 4.24e4.22 (m, 1H), 3.79e3.65 (m, 10H),
3.59e3.31 (m, 11H), 3.21e3.18 (m, 1H), 3.05e3.03 (m, 1H), 1.82 (s,
3H),1.79 (s, 3H), 1.78 (s, 3H), 1.77 (s, 3H); dC (125 MHz, CDCl3) 169.9,
169.9, 169.8, 169.7, 139.9, 139.4, 139.4, 139.2, 139.0, 138.3, 135.7,
129.9, 129.5, 128.9, 128.9, 128.8, 128.8, 128.7, 128.6, 128.1, 128.1,
128.0, 128.0, 127.9, 127.8, 127.7, 127.7, 127.1, 126.7, 101.9, 100.8, 100.6,
86.2, 81.3, 80.9, 79.6, 79.2, 78.2, 75.8, 75.0, 74.7, 74.2, 73.9, 73.8, 72.9,
72.9, 72.8, 72.6, 69.8, 69.4, 69.2, 68.3, 66.4, 56.7, 55.5, 54.4, 39.1,
23.8, 23.7, 23.6, 23.6; HRMS (ESI): MþHþ, found 1461.5986;
C80H93N4O20Sþ requires 1461.6098.
4.7. Phenyl 4,6-O-benzylidene-3-O-benzyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl-(1/4)-6-O-benzyl-2-deoxy-
2-phthalimido-1-thio-glucopyranoside S-oxide (29)
A solution of sulfide 28 (1.69 g, 1.76 mmol) in CH2Cl2 (59 mL)
was cooled to ꢁ78 ꢂC and a solution of mCPBA (77% pure, as com-
mercially available, 434 mg) in CH2Cl2 (39 mL) was added over 1 h.
The cloudy reaction mixture was then brought to rt over 45 min
and quenched with Na2SO3 (10% aqueous solution). The phases
were separated and the organic phase was washed with NaHCO3
(satd) and brine and dried over Na2SO4. Removal of the solvent in
vacuo and column chromatographic purification of the residue
(SiO2, hexane/EtOAc 1/1) gave the title compound as colorless solid
and as a 1/1 mixture of diastereomers (1.33 g, 1.36 mmol, 77%). Rf
(hexane/EtOAc 6/4) 0.30; due to formation of sulfoxide di-
astereomers NMR spectroscopic data is not given.
4.10. GlcNAceMurNAceGlcNAceMurNAc derivate 33
Compound 32 (150 mg, 103 mmol) was dissolved in DMF
(5.15 mL) and cooled to 0 ꢂC. NaH (60% in mineral oil, 102 mg, ca.
25 equiv) was added and the suspension was vigorously stirred for
10 min until gas evolution had ceased. S-2-bromopropionic acid
(93 mL, 1.03 mmol) was added dropwise and the suspension was
allowed to reach rt over 3 h before acetic acid (0.58 mL, 10 mmol)
was carefully added. Volatile components were removed in vacuum
over night and the residue was dissolved in benzene (1.5 mL) and
MeOH (0.5 mL). This solution was cooled to 0 ꢂC and TMS-
diazomethane (2 M in hexane, 1.5 mL) was slowly added. LC/MS
analysis at this point ensured that all free acid had been methylated
and the reaction was quenched by addition of AcOH (0.2 mL). The
mixture was concentrated in vacuum and partitioned between
EtOAc and NaHCO3. The organic phase was dried over Na2SO4,
concentrated in vacuum, and the residue was purified by column
chromatography (SiO2, CH2Cl2/MeOH 96/4) to obtain the title
4.8. Tetrasaccharide 31
Glycosyl acceptor 30 (790 mg, 0.82 mmol), 4-allyl-1,2-
dimethoxybenzene (0.88 mL, 5.1 mmol), and 2,6-di-tert-butyl-4-
methylpyridine (0.63 g, 3.1 mmol) were dissolved in benzene and
concentrated in vacuo to remove traces of water. The residue was
ꢀ
dissolved in CH2Cl2 (20.5 mL), MS 4 A (150 mg) was added and the
suspension was stirred at rt for 45 min before it was cooled to
ꢁ78 ꢂC. After addition of Tf2O (0.17 mL, 1.0 mmol) a solution of
glycosyl donor 29 (1.0 g, 1.0 mmol) in CH2Cl2 (25.5 mL) was added
over 2 h by syringe pump. After these 2 h, the solution was stirred
at ꢁ60 ꢂC for 1 h, followed by 1 h at ꢁ40 ꢂC. The mixture was
washed with NaHCO3 (satd) and the organic phase was dried over
Na2SO4. Removal of the solvent in vacuo, followed by column
chromatography on SiO2 (hexane/EtOAc 6/4) yielded disaccharide
31 as colorless solid (818 mg, 0.451 mmol, 55%). Rf (hexane/EtOAc
6/4) 0.20; dH (500 MHz DMSO-D6) 7.98e6.80 (m, 48H), 6.75e6.60
(m, 3H), 5.59 (s, 1H), 5.43 (d, J 11.0 Hz, 1H), 5.30 (d, J 9.0 Hz,1H), 5.12
(d, J 8.5 Hz, 1H), 5.06 (d J 7.5 Hz, 1H), 4.78 (d, J 12.5 Hz, 1H),
4.49e4.26 (m, 10H), 4.15e3.98 (m, 9H), 3.84e3.42 (m, 7H),
3.45e3.33 (m, 4H), 3.14e3.12 (m, 2H), 3.11e3.07 (m, 1H), 2.75e2.70
(m, 1H); dC (125 MHz, DMSO-D6) 168.8, 168.3, 168.0, 167.7, 138.5,
138.3, 138.1, 138.0, 137.7, 137.2, 134.5, 134.3, 134.2, 133.9, 132.2,
133.9, 132.7, 132.4, 132.1, 132.0, 131.8, 129.4, 128.9, 128.6, 128.5,
128.4,128.3,128.3,128.0, 127.9, 127.8, 127.7,127.6,127.6,127.5,127.4,
127.4, 127.1, 126.3, 124.3, 123.9, 123.7, 123.5, 101.6, 99.9, 99.2, 96.9,
83.4, 82.8, 81.9, 81.3, 78.0, 76.5, 75.7, 74.7, 74.5, 74.4, 74.3, 74.2, 74.2,
73.1, 72.8, 72.8, 71.3, 69.6, 68.5, 68.2, 66.9, 66.2, 56.8, 56.0, 55.9,
55.2; HRMS (ESI): MþHþ, found 1814.5944; C104H93N4O24Sþ re-
quires 1814.5929.
compound as colorless solid (126 mg, 77 mmol, 75%). Rf (CH2Cl2/
MeOH 96/4) 0.33; dH (500 MHz CDC13) 7.60e7.59 (d, 1H), 7.52e7.14
(m, 34H), 5.58 (s, 1H), 4.88 (d, J 12.5 Hz, 1H), 4.79 (d, J 11.5 Hz, 2H),
4.73e4.57 (m, 4H), 4.57e4.34 (m, 10H), 4.26e4.23 (m, 2H), 4.18 (br,
1H), 4.05e3.99 (m, 2H), 3.89 (m, 1H), 3.78e3.41 (m, 18H), 3.77 (s,
3H), 3.70 (s, 3H), 3.35e3.30 (m, 1H), 3.30e3.25 (m, 1H), 3.25e3.20
(m, 1H), 2.04 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H), 1.73 (s, 3H), 1.32 (d J
7.5 Hz, 3H), 1.18 (d J 7.0 Hz, 3H); dC (125 MHz, CDCl3) 176.6, 176.4,
172.7, 172.0, 170.7, 169.9, 162.8, 139.0, 138.6, 138.6, 138.5, 138.0,
137.4, 135.9, 130.7, 129.4, 129.4, 129.0, 129.0, 128.8, 128.7, 128.6,
128.3, 128.2,128.1, 128.0, 127.8,127.8,127.6,126.9, 126.2, 101.7,101.5,
100.7, 100.3, 88.8, 82.8, 82.8, 80.4, 79.6, 79.5, 78.1, 77.0, 76.7, 75.0,
74.6, 74.2, 73.9, 73.7, 73.4, 72.0, 71.9, 70.1, 69.3, 69.0, 67.3, 66.0, 55.8,
55.2, 53.8, 52.5, 52.4, 52.2, 36.7, 31.7, 23.9, 23.6, 23.6, 23.2, 19.0,18.8;
HRMS (ESI): MþHþ, found 1633.6852; C88H105N4O24Sþ requires
1633.6834.
4.11. Determination of the IC50 of moenomycin A
Solutions of PBP1a (E. coli, 20 nM) in 50 mM HEPES pH 7.5,
10 mM CaCl2, 1000 units/mL penicillin G, and 20% DMSO (v/v) were
incubated for 20 min with solutions of moenomycin A of different
concentrations. In the pre-incubation experiments the PBP1a so-
4.9. Tetraacetate 32
Tetraphthalate 31 (750 mg, 0.413 mmol) was dissolved in
a mixture of THF (8.4 mL), MeCN (10.7 mL), EtOH (8.4 mL), and
ethylenediamine (1.05 mL) and was stirred under an atmosphere of
argon for 48 h at 60 ꢂC. Volatile components were evaporated in
vacuo and the residue was dissolved in MeOH (16.8 mL) and H2O
(2.7 mL), cooled to 0 ꢂC, and 10 mL of Ac2O were added in portions
over 6 h. After evaporation of the volatiles in vacuo the residue was
suspended in MeOH (50 mL). The suspension was centrifuged and
the pellet dried over night in vacuo. The supernatant was concen-
trated in vacuo and treated as above (20 mL MeOH) to obtain the
lutions were first incubated with Gal-lipid IV (1.36
m
m
M) for 20 min.
Reactions were initiated by addition of 14C-lipid II (4
M, 3H/1H 1/3)
and were quenched after 20 min (MmA only) and 45 min (þLPIV)
with an equal volume of 10% Triton X-100 solution on ice. The
mixtures obtained were separated by paper strip chromatography
(isobutyric acid/1 M NH4OH 5/3). The paper strips were cut to ob-
tain the top 4/5 (containing monomer) and the lower 1/5 (con-
taining PG). These parts were immersed in EcoLite(þ) scintillation
cocktail (MP Biomedical) and analyzed by an LS 6500 scintillation
counter (Beckman Coulter). The normalized activity (A) was

9778
C.M. Gampe et al. / Tetrahedron 67 (2011) 9771e9778
Markwalder, J. A.; Wang, Y. J. Am. Chem. Soc. 2001, 123, 11638; (e) Lazar, K.;
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Aikins, J. A.; Blaszczak, L. C. J. Am. Chem. Soc. 2002, 124, 3656; (g) Breukink, E.;
van Heusden, H. E.; Vollmerhaus, P. J.; Swiezewska, E.; Brunner, L.; Walker, S.;
Heck, A. J.; de Kruijff, B. J. Biol. Chem. 2003, 278, 19898; (h) Chen, L.; Walker, D.;
Sun, B.; Hu, Y.; Walker, S.; Kahne, D. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5658;
(i) Hu, Y.; Helm, J. S.; Chen, L.; Ye, X. Y.; Walker, S. J. Am. Chem. Soc. 2003, 125,
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calculated relative to a control reaction (no MmA, no LPIV) and
plotted as a function of log(c(MmA)). The IC50 value was extracted
with OriginPro 7.0.
4.12. SDS-page assay
Solutions of the enzymes (0.24 mM or 0.48 mM) in 50 mM HEPES
pH 7.5, 10 mM CaCl2, 1000 units/mL penicillin G were treated with
solutions of MmA in different concentrations for 20 min. The re-
action was initiated by addition of 14C-lipid II (8 M, 14C/13C 3/1)
m
and quenched after 20 min by heat (90 ꢂC, 5 min). The volatiles
were removed by vacuum centrifugation and the residue was taken
24
€
up in buffer (15% SDS, Lammli 6X) and analyzed by SDS-PAGE.
Acknowledgements
Dr. Tania Lupoli and Dr. Matthew Lebar are acknowledged for
their help with the biochemical experiments. This research was
supported by the National Institutes of Health (GM076710 and
GM066174) and by the German Academic Exchange Service (DAAD;
postdoctoral fellowship to C.M.G.).
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NMR spectra of new compounds. Supplementary data associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.tet.2011.09.114.
References and notes
20. See Supplementary data for details.
21. Galactose-lipid IV (Gal-lipid IV) was used since lipid IV itself was found to be
polymerized by GTs. See Ref. 10o,p,r.
22. Polymerization reactions were run for 45 and 20 min with and without lipid IV,
respectively, which leads to maximum conversion in both cases in the absence
of moenomycin see Ref. 10r.
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