Synthesis and biological evaluation of enantiomerically pure glyceric acid derivatives as LpxC inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.01.029

Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent a promising class of novel antibiotics, selectively combating Gram-negative bacteria. In order to elucidate the impact of the hydroxymethyl groups of diol (S,S)-4 on the inhibitory activity against LpxC, glyceric acid ethers (R)-7a, (S)-7a, (R)-7b, and (S)-7b, lacking the hydroxymethyl group in benzylic position, were synthesized. The compounds were obtained in enantiomerically pure form by a chiral pool synthesis and a lipase-catalyzed enantioselective desymmetrization, respectively. The enantiomeric hydroxamic acids (R)-7b (K_i = 230 nM) and (S)-7b (K_i = 390 nM) show promising enzyme inhibition. However, their inhibitory activities do not substantially differ from each other leading to a low eudismic ratio. Generally, the synthesized glyceric acid derivatives 7 show antibacterial activities against two Escherichia coli strains exceeding the ones of their respective regioisomes 6.

Full text of DOI:10.1016/j.bmc.2016.01.029

Accepted Manuscript
Synthesis and biological evaluation of enantiomerically pure glyceric acid de-
rivatives as LpxC inhibitors
Giovanni Tangherlini, Tullio Torregrossa, Oriana Agoglitta, Jens Köhler, Jelena
Melesina, Wolfgang Sippl, Ralph Holl
PII:
S0968-0896(16)30029-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.01.029
BMC 12772
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 November 2015
13 January 2016
15 January 2016
Please cite this article as: Tangherlini, G., Torregrossa, T., Agoglitta, O., Köhler, J., Melesina, J., Sippl, W., Holl,
R., Synthesis and biological evaluation of enantiomerically pure glyceric acid derivatives as LpxC inhibitors,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.01.029
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Synthesis and biological evaluation of enantiomerically pure glyceric acid
derivatives as LpxC inhibitors
Giovanni Tangherlinia,b, Tullio Torregrossaa, Oriana Agoglittaa,c, Jens Köhlera, Jelena
Melesinad, Wolfgang Sippld, Ralph Holla,b,*
aInstitut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-
Universität Münster, Corrensstr. 48, D-48149 Münster, Germany
bCells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Münster,
Germany
cNRW Graduate School of Chemistry, University of Münster, Germany
dInstitut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-
Langenbeck Str. 4, 06120 Halle (Saale), Germany
*To whom correspondence should be addressed. Tel.: +49-251-8333372; Fax: +49-
251-8332144; E-mail: hollr@uni-muenster.de.
1

Graphical Abstract
Key words
LpxC inhibitors, structure–activity relationships, glyceric acid derivatives, chiral pool
synthesis, enantioselective desymmetrization, molecular docking studies
2

Abstract
Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase
(LpxC) represent a promising class of novel antibiotics, selectively combating Gram-
negative bacteria. In order to elucidate the impact of the hydroxymethyl groups of diol
(S,S)-4 on the inhibitory activity against LpxC, glyceric acid ethers (R)-7a, (S)-7a, (R)-
7b, and (S)-7b, lacking the hydroxymethyl group in benzylic position, were
synthesized. The compounds were obtained in enantiomerically pure form by a chiral
pool synthesis and a lipase-catalyzed enantioselective desymmetrization,
respectively. The enantiomeric hydroxamic acids (R)-7b (Ki = 230 nM) and (S)-7b (Ki
= 390 nM) show promising enzyme inhibition. However, their inhibitory activities do
not substantially differ from each other leading to a low eudismic ratio. Generally, the
synthesized glyceric acid derivatives 7 show antibacterial activities against two E. coli
strains exceeding the ones of their respective regioisomes 6.
3

1. Introduction
Antimicrobial resistance poses a severe problem for modern medicine.1,2 Besides
causing high economic cost, antimicrobial-resistant infections claim numerous human
lives, with at least 48,000 people dying from these infections every year in Europe
and the US alone.3 Furthermore, the drastic decrease in the number of novel
antibiotics released onto the market over the last decades has exacerbated the
situation.4,5 Therefore, in order to prevent an accentuation of this scenario, it is
urgently necessary to find new antibacterial targets and to consequently develop
antibiotics possessing novel mechanisms of action, which are able to circumvent
established mechanisms of resistance.6
Especially among Gram-negative bacteria, resistance is a matter of great concern as
the majority of antimicrobial-resistant infections are caused by these bacteria.7-9
Gram-negative bacterial infections are difficult to treat as these bacteria possess an
additional outer membrane, which acts as a permeability barrier shielding the
bacteria from external agents like antibiotics.10,11 The outer monolayer of the outer
membrane of Gram-negative bacteria mainly consists of lipid A, the hydrophobic
membrane anchor of lipopolysaccharides (LPS).12 On the one hand, lipid A is
required for growth and viability of most Gram-negative bacteria.13 On the other, it is
primarily responsible for the biological effects of LPS in the human host, triggering an
innate immune response, which can lead to a life-threatening septic shock.14,15 For
these reasons, the inhibition of the biosynthesis of lipid A represents a promising
strategy for antibacterial drug development.
One of the best characterized but so far clinically unexploited bacterial targets within
the biosynthetic pathway of lipid A is the Zn2+-dependent deacetylase LpxC.16 This
enzyme is present in virtually all Gram-negative bacteria, exhibiting a high sequence
similarity among the orthologues but showing no sequence homology to any
mammalian protein.17 LpxC catalyses the irreversible deacetylation of UDP-3-O-[(R)-
3-hydroxymyristoyl]-N-acetylglucosamine (1), representing the first committed step of
lipid A biosynthesis (Figure 1).18 Structural studies revealed that LpxC displays a “β-
α-α-β sandwich fold”, being made up by two domains with similar topologies.19 At the
interface of these two domains, the conical active site cleft is located.20 The catalytic
Zn2+-ion resides at the bottom of the active site cleft, being coordinated by one
aspartate and two histidine residues.21 Another characteristic structural element of
LpxC is a ~15 Å long, hydrophobic tunnel, which leads out of the active site cleft,
4

binding the 3-O-[(R)-3-hydroxymyristoyl] substituent of the natural substrate 1 during
catalysis.22
Figure 1: LpxC catalyzed deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-
acetylglucosamine (1).
The deacetylase has been validated as an antibacterial target and several classes of
small-molecule LpxC inhibitors have been described so far.23-31 Most of them
possess a Zn2+-binding hydroxamate moiety as well as a structural element
addressing the hydrophobic tunnel of the enzyme. E.g. the N-aroyl-L-threonine
hydroxamic acid derivatives CHIR-090 (3a, Figure 2) and LPC-009 (3b) are able to
potently inhibit diverse LpxC orthologues and exhibit antibacterial activity against
various Gram-negative bacteria.30,21 The compounds could be crystallized with
Yersinia enterocolitica LpxC and Escherichia coli LpxC, respectively, displaying
similar binding modes.32,21 Whereas the lipophilic side chain of the inhibitors
penetrates through the hydrophobic tunnel of LpxC, their hydroxamate moiety
chelates the catalytic Zn2+-ion in the active site and was found to be involved in polar
interactions with the catalytically important residues E78, T191 and H264.22,32,21
CHIR-090 (3a, Figure 2) served as lead compound for the development of
benzyloxyacetohydroxamic acid derivatives, in which the amide group of CHIR-090 is
replaced by an ether moiety.33 In our previous studies, dealing with the synthesis and
biological evaluation of these benzyloxyacetohydroxamic acid derivatives, diol (S,S)-
4, which exhibits a Ki-value of 358 nM against E. coli LpxC, was identified as
promising lead structure for the development of potent LpxC inhibitors.34 Therefore,
its structure was further varied by removing its hydroxymethyl groups. Whereas the
removal of both hydroxymethyl groups of (S,S)-4 was detrimental for the inhibitory
activity of benzyloxyacetohydroxamic acid derivatives 5a and 5b (Table 1), the
truncation of the hydroxymethyl group in α position of the hydroxamate moiety led to
5

an increase in the biological activity.35 In fact, the phenylethylene glycol derivatives
(S)-6a and (S)-6b showed inhibition of LpxC with Ki-values of 66 nM and 95 nM,
respectively. This latter finding was somewhat surprising, as in case of threonine
derivative LPC-009 (3b) and its analogs, the hydroxy group in Cβ position of the
hydroxamate moiety was shown to undergo favorable hydrogen bonding interactions
with a highly conserved lysine residue (K239) in the active site of the enzyme,
indicating that this functionality is important for inhibitor binding.21
Figure 2: Chemical structures of reported and envisaged LpxC inhibitors.
To learn more about the effect of the hydroxymethyl group in α-position of the
hydroxamate moiety, glyceric acid ethers 7a and 7b, bearing no hydroxymethyl group
in benzylic position, should be synthesized. Moreover, to determine the eutomer,
both stereoisomers of these glyceric acid derivatives should be prepared in
enantiomerically pure form.
6

2. Results and Discussion
2.1. Chemistry
Scheme 1: Reagents and conditions: (a) benzaldehyde, H2SO4, DMF, rt, 3 d, 26 %;
(b) 4-iodobenzyl bromide, LiHMDS, TBAI, THF, Δ, 16 h, 69 %; (c) p-TsOH, propane-
1,3-diol, MeOH, Δ, 4 h, 88 %; (d) 1. NaIO4, MeOH, rt, 2 h, 2. Br2, NaHCO3, MeOH,
H2O, 61 %.
The envisaged enantiomerically pure glyceric acid ethers should be accessed by
performing chiral pool syntheses. For the preparation of the (R)-configured
hydroxamic acids (R)-5a and (R)-5b D-mannitol (8) was used as chiral starting
material (Scheme 1). In the first reaction step, an acid-catalyzed acetalisation of D-
mannitol with benzaldehyde was performed, yielding 1,3:4,6-di-O-benzylidene-D-
mannitol (9).36 Subsequently, the unprotected hydroxy groups of 1,3-dioxane
derivative 9 were benzylated with 4-iodobenzyl bromide in the presence of catalytic
amounts of tetrabutylammonium iodide using lithium bis(trimethylsilyl)amide as base.
The two benzylidene acetals of the resulting bis(4-iodobenzyl) ether 10 were then
cleaved by heating the compound in methanol in the presence of 1,3-propanediol
and p-toluenesulfonic acid to access tetrol 11. In the next reaction step, the C-C bond
between C-3 and C-4 of glycol derivative 11 should be cleaved. Therefore, a glycol
cleavage with sodium periodate was performed yielding two identical (R)-configured
aldehydes, which were directly subjected to a subsequent oxidation. Using bromine
in the presence of sodium bicarbonate dissolved in a 9:1 mixture of methanol and
water, the oxidation directly yielded glyceric acid ester (R)-12.37 As this ester
7

represents a central intermediate of the envisaged synthesis, its optical purity was
determined with a chiral HPLC method. The analysis revealed an enatiomeric excess
(ee) of 98.6 % proofing the effectiveness of the performed chiral pool synthesis for
the preparation of the enantiomerically pure glyceric acid ether (R)-12.
Scheme 2: Reagents and conditions: (a) 1. NaNO2, H2SO4, H2O, 0 °C, 48 h, 2.
trimethyl orthoformate, p-TsOH, MeOH, Δ, 16 h, 23 %; (b) chloromethyl methyl ether,
DIPEA, H3CCN, rt, 16 h, 35 %; (c) 4-iodobenzyl bromide, NaH, TBAI, H3CCN, 80 °C,
16 h, 22 %; (d) HCl (g), MeOH, rt, 16 h, 31 %.
In contrast, the enantiomeric glyceric acid ester (S)-12 should be synthesized in a
chiral pool synthesis using L-serine (13) as starting material (Scheme 2). (S)-
Configured methyl glycerate (14) was prepared according to a literature procedure,
which reported its synthesis in high optical purity.38 Therefore, L-serine was
diazotized in an aqueous medium leading to (S)-configured glyceric acid. The net
retention of configuration in this reaction is due to two subsequent inversions.39,40
Presumably, at first an intermediate α-lactone is formed with an inversion of
configuration. Then, accompanied by a second inversion, the α-lactone reacts with
water, yielding (S)-glyceric acid. Without intermediate purification, this compound was
directly transformed into its methyl ester 14.
In the next reaction step, the primary alcohol of ester 14 was MOM-protected by
reacting glycol 14 with chloromethyl methyl ether. Then, the resulting secondary
alcohol 15 was benzylated with 4-iodobenzyl bromide in acetonitrile at 80 °C using
sodium hydride as base. The reaction yielded benzyl ether 16, whose MOM
8

protective group was subsequently cleaved under acidic conditions to give glyceric
acid ether (S)-12. However, the determination of the optical purity of glyceric acid
derivative (S)-12 revealed that this synthetic route had given the desired compound
with an ee of only 69.8 %. This finding indicated that most probably a partial
racemization had occurred during the diazotisation step.
Scheme 3: Reagents and conditions: (a) benzaldehyde, H2SO4, toluene, Δ, 50 %; (b)
4-iodobenzyl bromide, NaH, DMF, rt, 24 h, 42 %; (c) p-TsOH, THF, MeOH, rt, 16 h,
86 %; (d) IPA, Amano Lipase AK (Pseudomonas fluorescens), TBME, -10 °C, 24 h,
(S)-21 62 %; (e) chloromethyl methyl ether, DIPEA, H3CCN, rt, 21 h, 90 %; (f) K2CO3,
MeOH, rt, 2 h, 87 %; (g) 1. CrO3, H5IO6, H2O, H3CCN, 0 °C, 24 h, 2. HCl (g), MeOH,
Δ, 16 h, 49 %.
Therefore, in order to obtain enantiomer (S)-12 in high enantiomeric excess, a
pathway involving a lipase catalyzed enantioselective desymmetrization was
envisaged. At first, 1,3-dioxane derivative 18 was synthesized in an acid-catalyzed
acetalisation of glycerol (17) and benzaldehyde.41 The remaining hydroxy group of
benzylidene acetal 18 was then benzylated with 4-iodobenzyl bromide to give ether
19. The acetal 19 was subsequently cleaved under acidic conditions yielding diol 20.
In the key step for the introduction of chirality, an enantioselective acetylation of
prochiral diol 20 should be performed.42 In a lipase screening Amano Lipase AK from
Pseudomonas fluorescens was found to be the best catalyst for the enantioselective
desymmetrization of diol 20. Using isopropenyl acetate (IPA) as acylating agent in
tert-butyl methyl ether, the enzyme gave the desired (S)-configured monoacetate (S)-
9

21 and diacetate 22 occurred as a side product. In order to optimize the reaction
conditions, the course of the reaction was investigated by performing analytical scale
conversions (5 mg of diol 20). As shown in Figure 3, at a temperature of +25 °C the
monoacetate 21 was produced very fast at the beginning of the reaction (Figure 3,
sample A: 76.1 % (S)-21, 90.5 % ee). However, the enantiomeric excess of (S)-21
increased significantly by formation of diacetate 22, since the undesired monoacetate
(R)-21 was converted preferentially (Figure 3, sample B: 56.6 % (S)-21, 97.5 % ee).
Lowering the temperature to -10 °C led to an additionally increased enantioselectivity
of the lipase (Figure 3, sample C: 82.6 % (S)-21, 94.8 % ee, sample D: 69.4 % (S)-
21, 97.8 % ee). Therefore, large scale conversions of diol 20 (1.0 g, 3.3 mmol) were
performed at -10 °C. Upon isolation the desired (S)-configured monoacetate (S)-21
was obtained in 62 % yield with an ee of 97.6 %.
20
21
22
(S)-21
(S)-21
100,0
80,0
60,0
100,0
95,0
A
B
B
n [%]
[% ee]
90,0
40,0
20,0
0,0
A
85,0
100,0
95,0
0
2
4
6
8
0
2
4
6
8
time [h]
time [h]
(S)-21
20
21
22
100,0
80,0
60,0
C
D
D
C
n [%]
[% ee]
90,0
40,0
20,0
0,0
85,0
0
5
10
15
20
0
5
10
15
20
time [h]
time [h]
Figure 3: Lipase catalyzed conversion of diol 20 using Amano lipase AK from
Pseudomonas fluorescens in a mixture of tert-butyl methyl ether : isopropenyl acetate
(50 : 1, 1.5 mL), left: amount of compounds 20, 21 and 22 (n [%]), right: enantiomeric
10

excess of (S)-21 (% ee), top: carried out at +25 °C (sample A: 76.1 % (S)-21,
90.5 % ee, sample B: 56.6 % (S)-21, 97.5 % ee), bottom: carried out at -10 °C
(sample C: 82.6 % (S)-21, 94.8 % ee, sample D: 69.4 % (S)-21, 97.8 % ee).
The absolute configuration of monoacetate (S)-21 was unequivocally proven after its
transformation into ester (S)-12. At first, MOM-protection of the hydroxy group of
monoacetate (S)-21 gave ester 23, which was then saponified to yield alcohol 24. In
order to obtain ester (S)-12, alcohol 24 was transformed into the corresponding
carboxylic acid using an oxidant solution which contained periodic acid and catalytic
amounts of CrO3 in wet acetonitrile. Heating the intermediately formed carboxylic
acid in methanol under acidic conditions did not only lead to the esterification of the
carboxylate moiety but also caused the cleavage of the MOM protective group
yielding ester (S)-12. In contrast to its dextrorotatory enantiomer (R)-12, which was
obtained from D-mannitol, ester (S)-12 is levorotatory, confirming the (S)-
configuration of the compound and consequently of monoacetate (S)-21. At this
stage of the synthesis the ee was determined again. The ee of (S)-12 was found to
be 96.2 %, indicating that the optical purity did not markedly decrease during the
synthetic transformations of monoacetate (S)-21.
With the two optically pure enantiomes (R)-12 and (S)-12 in hand, the synthesis of
the envisaged hydroxamic acids was pursued. Sonogashira couplings of 4-
iodobenzyl ethers (R)-12 and (S)-12 with phenylacetylene and morpholinomethyl-
substituted phenylacetylene33 gave diphenylacetylene derivatives (R)-25a, (S)-25a,
(R)-25b, and (S)-25b, respectively. The desired hydroxamic acids (R)-7a, (S)-7a, (R)-
7b, and (S)-7b were obtained in a final aminolysis of methyl esters (R)-25a, (S)-25a,
(R)-25b, and (S)-25b with hydroxylamine. Although TLC control of the reactions
indicated nearly complete conversion of the esters, the yields of the obtained
hydroxamic acids were rather poor, as the purification of the compounds was difficult
due to their high polarity.
11

Scheme 4: Reagents and conditions: (a) Pd(PPh3)4, CuI, NEt3, H3CCN, rt, (R)-25a
83 %, (S)-25a 81 %, (R)-25b 88 %, (S)-25b 69 %; (b) H2NOH·HCl, NaOMe, MeOH,
rt, (R)-7a 24 %, (S)-7a 43 %, (R)-7b 20 %, (S)-7b 33 %.
12

2.2. Biological evaluation
zone of inhibition [mm]
MIC [µM]
enzyme assay
IC50 [µM]
compound
E. coli BL21 E. coli D22 E. coli BL21 E. coli D22
Ki [µM]
0.26 ± 0.12
0.27 ± 0.05
0.23 ± 0.04
0.39 ± 0.07
4.4 ± 0.8
(R)-7a
(S)-7a
(R)-7b
(S)-7b
(R)-6a
(S)-6a
(R)-6b
(S)-6b
(S,S)-4
5a
10.3 ± 2.5
11.7 ± 0.6
12.3 ± 0.6
15.7 ± 0.6
9.1 ± 0.4
9.5 ± 0.4
8.7 ± 0.7
13.4 ± 0.5
9.0 ± 0.5
10.6 ± 0.4
16.5 ± 0.4
24.6 ± 1.9
17.0 ± 1.0
20.7 ± 1.7
22.0 ± 1.3
25.8 ± 1.9
13.0 ± 1.7
20.5 ± 0.2
12.3 ± 1.6
21.2 ± 0.6
20.8 ± 0.6
13.2 ± 1.6
20.3 ± 1.0
30.3 ± 2.5
256
128
128
64
8
4
1.87 ± 0.85
1.96 ± 0.36
1.66 ± 0.31
2.82 ± 0.5
31.6 ± 6.0
0.48 ± 0.23
198 ± 12
4
2
256
256
>256
64
32
4
0.066 ± 0.032
27.3 ± 1.7
0.095 ± 0.042
0.36 ± 0.04
-
4
0.5
4
0.69 ± 0.30
2.6 ± 0.3
>256
128
64
4
> 200
5b
1
10.5 ± 2.5
0.058 ± 0.002
1.45 ± 0.35
0.008 ± 0.0003
CHIR-090
1
0.032
Table 1: MIC values as well as the results of the disc diffusion assays and the E. coli LpxC enzyme assay for the synthesized glyceric
acid derivatives 7 and already known benzyloxyacetohydroxamic acid derivatives 4 – 6.35,34
13

In order to determine the antibacterial activities of the synthesized hydroxamic acids,
disc diffusion tests were performed and the MIC (minimal inhibitory concentration)
values were determined. Additionally, a LpxC enzyme assay was performed (Table
1). In this fluorescence-based enzyme assay, the extent of the LpxC-catalyzed
deacetylation of the enzyme’s natural substrate 1 in the presence of varying
concentrations of the putative inhibitors is determined by transforming the resulting
primary amine 2 into a fluorescent isoindole.24
In contrast to the ethylene glycol derivatives 6a and 6b, for which a pronounced
difference between the inhibitory activities of the enantiomers had been observed, in
case of the glyceric acid derivatives (R)-7a, (S)-7a, (R)-7b, and (S)-7b, low eudismic
ratios can be observed, indicating that stereochemistry apparently does not play a
key role for the inhibitory activity of these compounds. The finding that the glyceric
acid derivatives show low eudismic ratios was astonishing as in case of the N-aroyl-L-
threonine hydroxamic acid derivatives (S)-configuration in α-position of the
hydroxamate moiety was found to be essential for high antibacterial activity.43 In fact,
with Ki-values of 0.26 µM and 0.23 µM the (R)-configured glyceric acid derivatives
(R)-7a and (R)-7b even exhibit slightly lower Ki-values than their (S)-configured
enantiomes.
Whereas the (S)-configured glyceric acid derivatives (S)-7a and (S)-7b show an
about 4-fold reduced activity compared to the respective ethylene glycol derivatives
(S)-6a and (S)-6b, the inhibitory activities of (R)-7a and (R)-7b are considerably
increased relative to the ones of their (R)-configured regioisomers (R)-6a and (R)-6b.
When comparing the inhibitory activity of diol (S,S)-4 with the ones of hydroxamic
acids (S)-7b and (S)-6b, lacking one of the two hydroxymethyl groups of (S,S)-4, it
can be observed, that the removal of the hydroxymethyl group in α-position of the
hydroxamate moiety yielding ethylene glycol derivative (S)-6b caused a pronounced
increase in inhibitory activity. In contrast, the removal of the hydroxymethyl group in
benzylic position of diol (S,S)-4, leading to glyceric acid derivative (S)-7b, did not
cause a considerable change of the inhibitory activity towards LpxC. These results
indicate, that the presence of both of the two hydroxymethyl groups of diol (S,S)-4 is
not required for LpxC inhibition, with the removal of the group in α-position of the
hydroxamate moiety being rather beneficial as it leads to an increased inhibitory
activity.
14

In the disc diffusion assays, the synthesized glyceric acid derivatives were tested
against E. coli BL21 (DE3) and the E. coli D22 strain, which is more sensitive
towards LpxC inhibition. Against both E. coli strains, all of the newly synthesized
glyceric acid derivatives 7 showed antibacterial properties, which were superior to the
ones of their respective ethylene glycol derivatives 6. Especially the (S)-configured
glyceric acid derivatives (S)-7a and (S)-7b were more active than their (R)-configured
enantiomers (R)-7a and (R)-7b as well as of their respective ethylene glycol
derivatives (S)-6a and (S)-6b, which is in contrast to the results of the LpxC enzyme
assay.
Generally, the same trends, which were observed in the disc diffusion assays, were
also found when determining the MIC values. Only the (S)-configured glyceric acid
derivative (S)-7b, which caused the largest halo of inhibition in the disc diffusion
assay against E. coli D22, showed a 4-fold increased MIC value against this E. coli
strain compared to its regioisomer (S)-6b.
As it had already been observed in the series of the (S)-configured ethylene glycol
derivatives, also in case of the glyceric acid derivatives the presence of a
morpholinomethyl substituent generally resulted in a higher antibacterial activity
against both E. coli strains in comparison to the compounds bearing no substituent at
their distal phenyl ring. Although the presence of the morpholinomethyl substituent
causes increased antibacterial activities, the opposite effect can be observed for the
inhibitory activity, with the unsubsituted compound (S)-7a inhibiting LpxC with a 1.4-
fold lower Ki value than (S)-7b. The same trend had also been observed for the
ethylene glycol derivatives (S)-6a and (S)-6b.
15

2.3. Molecular docking studies
To rationalize the structure-activity relationships of the synthesized LpxC inhibitors,
molecular docking studies were carried out. All compounds show a similar binding
pose in the substrate pocket of E. coli LpxC (Figure 4). The hydroxamic acid chelates
the Zn2+-ion and is involved in hydrogen-bonding to E78 and T191, while the
lipophilic distal part is placed in the hydrophobic tunnel formed by I198, M195, F212
and V217, respectively. The morpholine group (if present) is sticking out of the tunnel
into the solvent. The variability of interactions of distinct compounds with the protein
is observed in the linker region. Only the hydrogen bond between the ether group of
the linker and the hydroxy group of T191 is observed in all cases. Therefore,
according to this binding hypothesis, the relatively high activity of inhibitors CHIR-090
(3a), (S,S)-4, (S)-6a, (S)-6b and 7a,b in comparison to other reported compounds is
due to favorable interactions of their linker groups with surrounding amino acid
residues, especially hydrogen bonding interactions with polar residues of LpxC
(Figure 4).
Figure 4: Putative binding mode of reported LpxC inhibitors CHIR-090 and 4-7b
(cyan carbons) docked to the E. coli LpxC X-ray structure (white carbons, PDB ID
16

3P3G).21 In this and following figures heteroatoms are colored as following: nitrogen
atoms – blue, oxygen atoms – red, sulfur atoms – yellow. The protein backbone is
depicted as white ribbon, the Zn2+-ion as golden ball, conserved water molecules as
red balls and interactions of the ligands with the protein atoms are shown as salmon
lines.
The dramatic difference between the Ki-values of the enantiomers of ethylene glycol
derivatives 6a and 6b (around 70- and 300-fold, respectively) can be explained
partially by their different interactions of the linker region, as suggested previously.35
Another reason might be the displacement of a conserved water molecule by the S-
stereoisomer. As shown by the docking results, the hydroxy group of the R-
enantiomers (R)-6a and (R)-6b makes water-mediated hydrogen bonds with M61
and C63 (Figure 5a). However, the hydroxy group of the S-enantiomers (S)-6a and
(S)-6b instead of interacting with a conserved water molecule, displaces it and
reproduces its hydrogen-bond interactions with M61 and C63 backbone atoms
(Figure 5b). The release of the water molecule may cause a large entropic gain,
which would explain the observed activity cliff. Surprisingly, in case of the glyceric
acid derivatives (R)-7a, (S)-7a, (R)-7b, and (S)-7b no significant difference in the Ki-
values was observed between the enantiomers. Analysis of the docking poses
suggests that this is because, in contrast to compounds 6a and 6b, there is no water
displacement effect. The R-enantiomers (R)-7a and (R)-7b make water-mediated
hydrogen bond interactions with M61 and C63 (Figure 5c) similar to (R)-6a and (R)-
6b (Figure 5a). The difference in the biological activity probably comes from more
favorable linker geometry of (R)-7a and (R)-7b compared to (R)-6a and (R)-6b. The
hydroxy group of the S-enantiomers (S)-7a and (S)-7b is involved in two hydrogen
bonds with the backbone NH and CO of F192 located on the opposite side of the
pocket (Figure 5d). Since R- and S-stereoisomers of 7a and 7b are able to adopt an
equally favorable binding mode, their biological activity is similar. The influence of the
morpholine group is difficult to explain, because it is placed at the entrance of the
pocket and is solvent-exposed.
17

Figure 5: Docking poses of compounds: a) (R)-6a (magenta) and (R)-6b (cyan), b)
(S)-6a (magenta) and (S)-6b (cyan), c) (R)-7a (magenta) and (R)-7b (cyan), d) (S)-7a
(magenta) and (S)-7b (cyan) in the X-ray structure of E. coli LpxC (white carbons,
PDB ID 3P3G).21 Only the region nearby the Zn2+-ion is shown for clarity.
18

Figure 6: Binding mode of (a) LPC-009 (green carbons) in its X-ray structure with E.
coli LpxC (white carbons, PDB ID 3P3G)21 and (b) CHIR-090 (green carbons) in its
X-ray structure with Yersinia enterocolitica LpxC (light gray carbons, PDB ID
3NZK).32
19

Interestingly, the suggested docking poses of compounds 7a and 7b are similar to
the binding modes of known potent LpxC inhibitors. The position of the hydroxy
group of (R)-7a and (R)-7b is the same as the carbonyl group of LPC-009 (3b) in its
crystal structure with E. coli LpxC (Figure 6a).21 In case of (S)-7a and (S)-7b the
hydroxy group is located at the same position as observed for the hydroxy group of
CHIR-090 (3a) in its crystal structure with Yersinia enterocolitica LpxC (Figure 6b).32
This observation suggests that the presented docking poses are realistic.
20

3. Conclusions
In a chiral pool synthesis starting from D-mannitol, glyceric acid ester (R)-12 was
accessed in enantiomerically pure form. Its (S)-configured enantiomer (S)-12 was
obtained in high enantiomeric excess via an enantioselective desymmetrization of
diol 20 using Amano Lipase AK from Pseudomonas fluorescens. From these
intermediates, enantiomerically pure glyceric acid derivatives (R)-7a, (S)-7a, (R)-7b,
and (S)-7b were synthesized and tested for their inhibitory activities against LpxC as
well as their antibacterial activities against two E. coli strains.
The biological evaluation of the synthesized glyceric acid derivatives showed that the
configuration in α-position of the hydroxamate moiety has only a minor effect on the
compounds’ inhibitory activity against LpxC. With Ki-values of 0.27 µM and 0.39 µM,
the (S)-configured hydroxamic acids (S)-7a and (S)-7b show approximately the same
inhibitory activities as their enantiomers (R)-7a and (R)-7b, exhibiting Ki-values of
0.26 µM and 0.23 µM, respectively. These observations are in agreement with
favorable docking poses derived for both classes of isomers.
The comparison of the inhibitory activities of glyceric acid derivatives (S)-7a and (S)-
7b with the ones of their regioisomers (S)-6a and (S)-6b showed, that the shift of the
hydroxymethyl group from the benzylic position (compounds (S)-6a and (S)-6b) to
the α-position of the hydroxamate moiety (compounds (S)-7a and (S)-7b) led to
slightly reduced inhibitory activities of the benzyloxyacetohydroxamic acid
derivatives.
However, the antibacterial properties of the glyceric acid derivatives 7 were generally
higher or at least equal to the ones of the respective ethylene glycol derivatives 6,
thus making these newly synthesized compounds leads for further optimization
steps.
Acknowledgement
This work was supported by the Deutsche Forschungsgemeinschaft (HO 5220/2-1),
which is gratefully acknowledged.
21

4. Experimental Section
4.1. Chemistry, general
Unless otherwise mentioned, THF was dried with sodium/benzophenone and was
freshly distilled before use. Thin layer chromatography (tlc): Silica gel 60 F254 plates
(Merck). Flash chromatography (fc): Silica gel 60, 40 – 64 µm (Macherey-Nagel);
parentheses include: diameter of the column, fraction size, eluent, Rf value. Melting
point (m.p.): Melting point apparatus SMP 3 (Stuart Scientific), uncorrected. Optical
rotation α [deg] was determined with a Polarimeter 341 (Perkin Elmer); path length 1
.
dm, wavelength 589 nm (sodium D line); the unit of the specific rotation
[deg
.
.
mL dm-1 . g-1] is omitted; the concentration of the sample c [mg mL-1] and the
solvent used are given in brackets. 1H NMR (400 MHz), 13C NMR (100 MHz): Agilent
DD2 400 MHz spectrometer; δ in ppm related to tetramethylsilane; coupling
constants are given with 0.5 Hz resolution. IR: IR Prestige-21(Shimadzu). HRMS:
MicrOTOF-QII (Bruker). HPLC methods for the determination of product purity:
Method 1: Merck Hitachi Equipment; UV detector: L-7400; autosampler: L-7200;
pump: L-7100; degasser: L-7614; column: LiChrospher® 60 RP-select B (5 μm);
LiCroCART® 250-4 mm cartridge; flow rate: 1.00 mL/min; injection volume: 5.0 µL;
detection at λ = 210 nm for 30 min; solvents: A: water with 0.05 % (V/V)
trifluoroacetic acid; B: acetonitrile with 0.05 % (V/V) trifluoroacetic acid: gradient
elution: (A %): 0 – 4 min: 90 % , 4 – 29 min: gradient from 90 % to 0 %, 29 – 31 min:
0 %, 31 – 31.5 min: gradient from 0 % to 90 %, 31.5 – 40 min: 90 %. Method 2:
Merck Hitachi Equipment; UV detector: L-7400; pump: L-6200A; column:
phenomenex Gemini® 5 µm C6-Phenyl 110 Å; LC Column 250 x 4.6 mm; flow rate:
1.00 mL/min; injection volume: 5.0 µL; detection at λ = 254 nm for 20 min; solvents:
A: acetonitrile : 10 mM ammonium formate = 10 : 90 with 0.1 % formic acid; B:
acetonitrile : 10 mM ammonium formate = 90 : 10 with 0.1 % formic acid; gradient
elution: (A %): 0 – 5 min: 100 % , 5 – 15 min: gradient from 100 % to 0 %, 15 – 20
min: 0 %, 20 – 22 min: gradient from 0 % to 100 %, 22 – 30 min: 100 %. Method 3:
Merck Hitachi Equipment; diode array detector: L-7455; pump: L-6200A; column:
DiacelChiralpak IA; flow rate: 1.00 mL/min; injection: manual, Rheodyne 7725i;
injection volume: 5.0 µL; detection at λ = 235 nm for 40 min; solvent: isohexane :
ethanol = 9 : 1. Method 4: Merck Hitachi Equipment; diode array detector: L-7455;
22

pump: L-6200A; column: DiacelChiralpak IA; flow rate: 1.00 mL/min; injection:
manual, Rheodyne 7725i; injection volume: 5.0 µL; detection at λ = 235 nm for 60
min; solvent: isohexane : isopropanol = 95 : 5.
23

4.2. Synthetic procedures
4.2.1. (2R,2'R,4R,4'R,5R,5'R)-2,2'-Diphenyl-[4,4'-bi(1,3-dioxane)]-5,5'-diol (9)
Benzaldehyde (1.2 mL, 1.3 g, 12 mmol) was added to a stirred solution of D-mannitol
(1.0 g, 5.5 mmol) in DMF (30 mL). After cooling to 0 °C, concentrated sulfuric acid
(0.21 mL, 380 mg, 3.9 mmol) was added dropwise. The reaction was stirred at
ambient temperature for 3 d. Then the solution was poured into a mixture of ice water
(120 mL), potassium carbonate (1.2 g) and petroleum ether (10 mL). The white
precipitate being formed was filtered off and washed with fresh petroleum ether. The
solid residue was purified by flash column chromatography (Ø = 3 cm, h = 15 cm, V =
20 mL, cyclohexane/ethyl acetate = 2/1, Rf = 0.11) to give 9 as colourless solid (500
20
1
 D

mg, 1.4 mmol, 26% yield). m.p. = 180 °C;
= -3.3 (2.9, methanol); H NMR
(DMSO-d6): δ [ppm] = 3.55 (t, J = 10.4 Hz, 2H, OCH2CHOHCHO), 3.75 – 3.85 (m,
2H, OCH2CHOHCHO), 3.91 (d, J = 9.2 Hz, 2H, OCH2CHOHCHO), 4.16 (dd, J =
10.4/5.3 Hz, 2H, OCH2CHOHCHO), 5.35 (d, J = 5.9 Hz, 2H, OCH2CHOHCHO), 5.51
13
(s, 2H, PhCH), 7.31 – 7.46 (m, 10H, Harom.); C NMR (DMSO-d6): δ [ppm] = 58.8
(2C, OCH2CHOHCHO), 71.0 (2C, OCH2CHOHCHO), 78.1 (2C, OCH2CHOHCHO),
100.1 (2C, PhCH), 126.1 (4C, Carom.), 128.0 (4C, Carom.), 128.5 (2C, Carom.), 138.3
(2C, Carom.); IR (neat): [cm-1] = 3476, 2978, 2859, 1447, 1412, 1362, 1223, 1099,
1026, 968, 926, 775, 737, 698, 629; HRMS (m/z): [M+H]+ calcd for C20H23O6,
359.1489; found, 359.1499; HPLC (method 1): tR = 17.3 min, purity 99.2 %.
4.2.2.
(2R,2'R,4R,4'R,5R,5'R)-5,5'-Bis[(4-iodobenzyl)oxy]-2,2'-diphenyl-4,4'-
bi(1,3-dioxane) (10)
Under nitrogen atmosphere, a 1 M solution of lithium hexamethyldisilazane in THF
(3.7 mL, 3.7 mmol) was added to a solution of 9 (550 mg, 1.5 mmol) in anhydrous
THF (50 mL). Tetrabutylammonium iodide (56 mg, 0.15 mmol) and 4-iodobenzyl
bromide (1.0 g, 3.4 mmol) were added and the mixture was heated to reflux
overnight. Then water was added and the mixture was extracted with ethyl acetate
(3×). The combined organic layers were dried (Na2SO4), filtered and the solvent was
removed in vacuo. The residue was purified by flash column chromatography (Ø = 3
cm, h = 15 cm, V = 20 mL, cyclohexane/ethyl acetate = 9/1, Rf = 0.19) to afford 10 as
20

D
colorless solid (840 mg, 1.1 mmol, 69 % yield). m.p. = 118 °C;
= -60.6 (1.5,
24

CH2Cl2); 1H NMR (CDCl3): δ [ppm] = 3.65 (t, J = 10.2 Hz, 2H, OCH2CHCHO), 3.92 –
4.03 (m, 4H, OCH2CHCHO), 4.33 (dd, J = 10.6/4.7 Hz, 2H, OCH2CHCHO), 4.48 (d, J
= 12.0 Hz, 2H, OCH2Ar), 4.53 (d, J = 12.0 Hz, 2H, OCH2Ar), 5.37 (s, 2H, PhCH), 6.97
– 7.02 (m, 4H, 2'-H4-iodophenyl, 6'-H4-iodophenyl), 7.34 – 7.45 (m, 10H, Hphenyl), 7.55 – 7.59
(m, 4H, 3'-H4-iodophenyl, 5'-H4-iodophenyl); 13C NMR (CDCl3): δ [ppm] = 66.9 (2C,
OCH2CHCHO), 69.6 (2C, OCH2CHCHO), 72.0 (2C, OCH2Ar), 77.4 (2C,
OCH2CHCHO), 93.8 (2C, C-4'4-iodophenyl), 101.2 (2C, PhCH), 126.3 (4C, Cphenyl), 128.4
(4C, Cphenyl), 129.2 (2C, Cphenyl), 129.9 (4C, C-2'4-iodophenyl, C-6'4-iodophenyl), 137.6 (4C,
C-1'4-iodophenyl (2C), Cphenyl (2C)), 137.7 (4C, C-3'4-iodophenyl, C-5'4-iodophenyl); IR (neat):
[cm-1] = 2866, 1485, 1450, 1400, 1369, 1219, 1092, 1030, 1007, 972, 926, 795, 748,
737, 694; HRMS (m/z): [M+H]+ calcd for C34H33I2O6, 791.0361; found, 791.0320;
HPLC (method 1): tR = 26.1 min, purity 99.0 %.
4.2.3. (2R,3S,4S,5R)-2,5-Bis[(4-iodobenzyl)oxy]hexane-1,3,4,6-tetraol (11)
p-Toluenesulfonic acid (120 mg, 0.64 mmol) and 1,3-propanediol (2.3 mL, 2.4 g, 32
mmol) were added to a solution of 10 (2.5 g, 3.2 mmol) in methanol (200 mL). The
reaction was heated to reflux for 4 h. Then the solvent was evaporated. The residue
was dissolved in a saturated aqueous solution of NaHCO3 and the mixture was
extracted with ethyl acetate (3×). The combined organic layers were dried (Na2SO4),
filtered and the solvent was removed in vacuo. The residue was purified by flash
column chromatography (Ø = 3 cm, h = 15 cm, V = 20 mL, CH2Cl2/methanol =
9.5/0.5, Rf = 0.27) to give 11 as colourless solid (1.7 g, 2.8 mmol, 88% yield). m.p. =
20
1
 D

151 °C;
= +17.5 (1.7, methanol); H NMR (CD3OD): δ [ppm] = 3.56 – 3.60 (m,
2H, HOCH2CHCHOH), 3.75 (dd, J = 11.9/4.6 Hz, 2H, HOCH2CHCHOH), 3.92 – 3.95
(m, 4H, HOCH2CHCHOH (2H), HOCH2CHCHOH), 4.55 (d, J = 11.7 Hz, 2H,
OCH2Ar), 4.70 (d, J = 11.7 Hz, 2H, OCH2Ar), 7.15 – 7.18 (m, 4H, 2'-H4-iodophenyl, 6'-H4-
iodophenyl), 7.64 – 7.67 (m, 4H, 3'-H4-iodophenyl, 5'-H4-iodophenyl); 13C NMR (CD3OD): δ
[ppm] = 62.1 (2C, HOCH2CHCHOH), 70.1 (2C, HOCH2CHCHOH), 72.7 (2C,
OCH2Ar), 81.5 (2C, HOCH2CHCHOH), 93.4 (2C, C-4'4-iodophenyl), 130.9 (4C, C-2'4-
iodophenyl, C-6'4-iodophenyl), 138.5 (4C, C-3'4-iodophenyl, C-5'4-iodophenyl), 140.0 (2C, C-1'4-
iodophenyl); IR (neat): [cm-1] = 3426, 3306, 2886, 1589, 1481, 1404, 1304, 1234, 1084,
25

1038, 1003, 829, 795, 621; HRMS (m/z): [M+H]+ calcd for C20H25I2O6, 614.9735;
found, 614.9725; HPLC (method 1): tR = 18.5 min, purity 95.6 %.
4.2.4. Methyl (S)-2,3-dihydroxypropanoate (14)
Concentrated sulphuric acid (4.1 mL, 7.5 g) was added to a solution of L-serine (12 g,
110 mmol) in water (150 mL) at 0 °C. Afterwards, a solution of NaNO2 (6.8 g, 99
mmol) in water (75 mL) was added over a period of 24 h. Then a cooled solution of
concentrated sulphuric acid (3.3 mL, 6.0 g) in water (20 mL) was added and again a
solution of NaNO2 (6.8 g, 99 mmol) in water (75 mL) was added over a period of 24
h. Afterwards, the solution was stirred at ambient temperature for additional 24 h.
After the volume of the solution had been reduced in vacuo to about 1/3, an aqueous
solution of NaOH (3.8 g, 95 mmol) was added. Then methanol (100 mL) and acetone
(30 mL) were added and the mixture was filtered. After evaporation of the filtrate, the
residue was dissolved in methanol (100 mL). Trimethyl orthoformate (75 mL) and p-
toluenesulfonic acid (1.9 g, 10 mmol) were added and the mixture was heated to
reflux for 16 h. Then a saturated aqueous solution of NaHCO3 was added and the
mixture was extracted with ethyl acetate (3×). The combined organic layers were
dried (Na2SO4), filtered and the solvent was removed in vacuo. The residue was
purified by flash column chromatography (Ø = 8 cm, h = 15 cm, V = 65 mL,
cyclohexane/ethyl acetate = 1/2, Rf = 0.17) to give 14 as colourless oil (3.0 g, 25
20
1
 D

mmol, 23%).
= -2.5 (1.1, methanol); H NMR (CDCl3): δ [ppm] = 3.84 (s, 3H,
CO2CH3), 3.85 (dd, J = 11.7/3.9 Hz, 1H, HOCHCH2OH), 3.91 (dd, J = 11.7/3.3 Hz,
1H, HOCHCH2OH), 4.28 (t, J = 3.6 Hz, 1H, HOCHCH2OH); 13C NMR (CDCl3): δ
[ppm] = 53.1 (1C, CO2CH3), 64.1 (1C, HOCHCH2OH), 71.6 (1C, HOCHCH2OH),
173.6 (1C, CO2CH3); IR (neat): [cm-1] = 3402, 2955, 1732, 1439, 1215, 1115, 1061,
1007, 972, 648; HRMS (m/z): [M+H]+ calcd for C4H9O4, 121.0495; found, 121.0517.
4.2.5. Methyl (S)-2-hydroxy-3-(methoxymethoxy)propanoate (15)
Under N2 atmosphere N,N-diisopropylethylamine (2.5 mL, 15 mmol) and
chloromethyl methyl ether (1.1 mL, 15 mmol) were added to a solution of 14 (1.5 g,
12 mmol) in acetonitrile (50 mL) at 0 °C. After stirring the mixture at ambient
26

temperature for 16 h, water was added and the mixture was extracted with ethyl
acetate (3×). The combined organic layers were dried (Na2SO4), filtered and the
solvent was removed in vacuo. The residue was purified by flash column
chromatography (Ø = 4 cm, h = 15 cm, V = 30 mL, cyclohexane/ethyl acetate = 2/1,
20

D
Rf = 0.18) to give 15 as colorless oil (710 mg, 4.3 mmol, 35%).
= -14.0 (0.7,
1
methanol); H NMR (CDCl3): δ [ppm] = 3.36 (s, 3H, OCH2OCH3), 3.80 (dd, J =
10.8/2.8 Hz, 1H, OCHCH2O), 3.82 (s, 3H, CO2CH3), 3.94 (dd, J = 10.8/3.7 Hz, 1H,
OCHCH2O), 4.32 – 4.34 (m, 1H, OCHCH2O), 4.63 (d, J = 6.7 Hz, 1H, OCH2OCH3),
4.66 (d, J = 6.7 Hz, 1H, OCH2OCH3); 13C NMR (CDCl3): δ [ppm] = 52.8 (1C,
CO2CH3), 55.7 (1C, OCH2OCH3), 70.3 (1C, OCHCH2O), 70.9 (1C, OCHCH2O), 97.2
(1C, OCH2OCH3), 173.1 (1C, CO2CH3); IR (neat): [cm-1] = 3441, 2951, 2889, 1740,
1439, 1211, 1111, 1026, 918; HRMS (m/z): [M+H]+ calcd for C6H13O5, 165.0757;
found, 165.0733.
5.2.6. Methyl (S)-2-[(4-iodobenzyl)oxy]-3-(methoxymethoxy)propanoate (16)
Under N2 atmosphere sodium hydride (55% suspension in paraffin oil, 28 mg, 0.64
mmol) was added to a solution of 15 (70 mg, 0.43 mmol) in acetonitrile (20 mL). Then
4-iodobenzyl bromide (380 mg, 1.3 mmol) and tetrabutylammonium iodide (32 mg,
0.09 mmol) were added and the mixture was stirred at 80 °C for 16 h. Then a
saturated aqueous solution of NaHCO3 was added and the mixture was extracted
with ethyl acetate (3×). The combined organic layers were dried (Na2SO4), filtered
and the solvent was removed in vacuo. The residue was purified by flash column
chromatography (Ø = 2 cm, h = 15 cm, V = 10 mL, cyclohexane/ethyl acetate = 8/2,
20

D
Rf = 0.34) to give 16 as colourless oil (35 mg, 0.09 mmol, 22%).
= -34.6 (0.7,
1
methanol); H NMR (CDCl3): δ [ppm] = 3.34 (s, 3H, OCH2OCH3), 3.78 (s, 3H,
CO2CH3), 3.82 – 3.87 (m, 2H, OCHCH2O), 4.15 (dd, J = 5.1/3.9 Hz, 1H, OCHCH2O),
4.48 (d, J = 12.0 Hz, 1H, OCH2Ar), 4.63 (d, J = 6.6 Hz, 1H, OCH2OCH3), 4.65 (d, J =
6.6 Hz, 1H, OCH2OCH3), 4.73 (d, J = 12.0 Hz, 1H, OCH2Ar), 7.11 – 7.14 (m, 2H, 2'-
13
H4-iodophenyl, 6'-H4-iodophenyl), 7.66 – 7.68 (m, 2H, 3'-H4-iodophenyl, 5'-H4-iodophenyl); C NMR
(CDCl3): δ [ppm] = 52.3 (1C, OCH3), 55.5 (1C, OCH2OCH3), 68.0 (1C, OCHCH2O),
72.1 (1C, OCH2Ar), 77.9 (1C, OCHCH2O), 93.6 (1C, C-4'4-iodophenyl), 96.8 (1C,
OCH2OCH3), 129.9 (2C, C-2'4-iodophenyl, C-6'4-iodophenyl), 137.1 (1C, C-1'4-iodophenyl), 137.7
27

(2C, C-3'4-iodophenyl, C-5'4-iodophenyl), 170.9 (1C, CO2CH3); IR (neat): [cm-1] = 2947,
2886, 1748, 1485, 1439, 1265, 1207, 1115, 1038, 1007, 918, 799; HRMS (m/z):
[M+H]+ calcd for C13H18IO5, 381.0193; found, 381.0187; HPLC (method 1): tR = 19.3
min, purity 97.1 %.
4.2.7. cis-2-Phenyl-1,3-dioxan-5-ol (18)
Concentrated sulfuric acid (3 drops) was added to a mixture of glycerol (44 mL, 55 g,
0.60 mol) and freshly distilled benzaldehyde (48 mL, 50 g, 0.47 mol) in toluene (69
mL). The resulting mixture was heated to reflux in a Dean-Stark water separator.
When the separation of water was complete (8.5 mL), the reaction mixture was
allowed to cool to room temperature and the solvent removed in vacuo. The resulting
white solid was recrystallized from isopropyl ether/petroleum ether. The precipitate
was filtered off and purified by flash column chromatography (Ø = 6 cm, h = 15 cm, V
= 50 mL, cyclohexane/ethyl acetate = 2/1, Rf = 0.16) to give 18 as colourless solid
1
(42.6 g, 0.24 mol, 50% yield). m.p. = 81 °C; H NMR (DMSO-d6): δ [ppm] = 3.48 –
3.52 (m, 1H, OCH2CHOH), 3.91 – 3.97 (m, 2H, OCH2CHOH), 4.01 – 4.07 (m, 2H,
OCH2CHOH), 4.99 (d, J = 4.8 Hz, 1H, OH), 5.53 (s, 1H, CHPh), 7.33 – 7.40 (m, 3H,
3'-Hphenyl, 4'-Hphenyl, 5'-Hphenyl), 7.42 – 7.47 (m, 2H, 2'-Hphenyl, 6'-Hphenyl); 13C NMR
(DMSO-d6): δ [ppm] = 62.4 (1C, OCH2CHOH), 71.4 (2C, OCH2CHOH), 100.2 (1C,
CHPh), 126.2 (2C, C-2'phenyl, C-6'phenyl), 127.9 (2C, C-3'phenyl, C-5'phenyl), 128.5 (1C, C-
4'phenyl), 138.9 (1C, C-1'phenyl); IR (neat): [cm-1] = 3271, 2916, 2851, 1450, 1385,
1339, 1277, 1153, 1084, 995, 976, 806, 741, 694; HRMS (m/z): [M+H]+ calcd for
C10H13O3, 181.0859; found, 181.0878; HPLC (method 1): tR = 11.7 min, purity 95.4
%.
4.2.8. cis-5-[(4-Iodobenzyl)oxy]-2-phenyl-1,3-dioxane (19)
4-Iodobenzyl bromide (5.0 g, 17 mmol) and sodium hydride (55 % suspension in
paraffin oil, 0.81 g, 19 mmol) were added to a solution of 18 (2.6 g, 14 mmol) in DMF
(27 mL). After stirring the mixture at ambient temperature for 24 h, a saturated
aqueous solution of NaHCO3 was added and the mixture was extracted with ethyl
acetate (4×). The combined organic layers were dried (Na2SO4), filtered and the
solvent was removed in vacuo. The residue was purified by flash column
28

chromatography (Ø = 8 cm, h = 15 cm, V = 60 mL, cyclohexane/ethyl acetate = 9/1,
Rf = 0.18) to give 19 as colourless solid (2.4 g, 6.1 mmol, 42% yield). m.p. = 125 °C;
1H NMR (DMSO-d6): δ [ppm] = 3.38 – 3.41 (m, 1H, OCH2CHO), 4.02 – 4.08 (m, 2H,
OCH2CHO), 4.19 – 4.25 (m, 2H, OCH2CHO), 4.56 (s, 2H, OCH2Ar), 5.58 (s, 1H,
CHPh), 7.19 – 7.23 (m, 2H, 2'-H4-iodophenyl, 6'-H4-iodophenyl), 7.33 – 7.43 (m, 5H, Hphenyl),
7.70 – 7.74 (m, 2H, 3'-H4-iodophenyl, 5'-H4-iodophenyl); 13C NMR (DMSO-d6): δ [ppm] = 68.2
(2C, OCH2CHO), 68.7 (1C, OCH2Ar), 69.9 (1C, OCH2CHO), 93.2 (1C, C-4'4-iodophenyl),
100.1 (1C, CHPh), 126.0 (2C, C-2phenyl, C-6phenyl), 127.9 (2C, C-3phenyl, C-5phenyl),
128.6 (1C, C-4phenyl), 129.7 (2C, C-2'4-iodophenyl, C-6'4-iodophenyl), 137.0 (2C, C-3'4-
iodophenyl, C-5'4-iodophenyl), 138.6 (1C, C-1'4-iodophenyl), 138.7 (1C, C-1phenyl); IR (neat):
[cm-1] = 2970, 2859, 1481, 1454, 1377, 1335, 1277, 1238, 1153, 1096, 1007, 980,
799, 745, 698; HRMS (m/z): [M+H]+ calcd for C17H18IO3, 397.0295; found, 397.0309;
HPLC (method 1): tR = 23.1 min, purity 99.7 %.
4.2.9. 2-[(4-Iodobenzyl)oxy]propane-1,3-diol (20)
p-Toluenesulfonic acid monohydrate (230 mg, 1.2 mmol) was added to a solution of
19 (2.4g, 6.1 mmol) in a mixture of methanol (80 mL) and tetrahydrofuran (1 mL).
After stirring the mixture at ambient temperature for 16 h, a saturated aqueous
solution of NaHCO3 and brine were added and the mixture was extracted with ethyl
acetate (4×). The combined organic layers were dried (Na2SO4), filtered and the
solvent was removed in vacuo. The residue was purified by flash column
chromatography (Ø = 5 cm, h = 15 cm, V = 30 mL, cyclohexane/ethyl acetate = 1/2,
Rf = 0.15) to give 20 as colourless solid (1.6 g, 5.3 mmol, 86% yield). m.p. = 95 °C;
1H NMR (CDCl3): δ [ppm] = 3.56 – 3.61 (m, 1H, HOCH2CHO), 3.73 (dd, J = 11.7/4.9
Hz, 2H, HOCH2CHO), 3.80 (dd, J = 11.7/4.4 Hz, 2H, HOCH2CHO), 4.61 (s, 2H,
OCH2Ar), 7.09 – 7.12 (m, 2H, 2'-H4-iodophenyl, 6'-H4-iodophenyl), 7.67 – 7.71 (m, 2H, 3'-H4-
iodophenyl, 5'-H4-iodophenyl); 13C NMR (CDCl3): δ [ppm] = 62.6 (2C, HOCH2CHO), 71.4
(1C, OCH2Ar), 79.5 (1C, HOCH2CHO), 93.6 (1C, C-4'4-iodophenyl), 129.8 (2C, C-2'4-
iodophenyl, C-6'4-iodophenyl), 137.8 (2C, C-3'4-iodophenyl, C-5'4-iodophenyl), 137.9 (1C, C-1'4-
iodophenyl); IR (neat): [cm-1] = 3167, 2913, 1481, 1462, 1339, 1119, 1069, 1038, 1003,
887, 795, 679; HRMS (m/z): [M+H]+ calcd for C10H14IO3, 308.9982; found, 308.9989;
HPLC (method 1): tR = 15.4 min, purity 98.5 %.
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