Enantiomeric synthesis of natural alkylglycerols and their antibacterial and antibiofilm activities

Article abstract of DOI:10.1080/14786419.2019.1686370

Alkylglycerols (AKGs) are bioactive natural compounds that vary by alkyl chain length and degree of unsaturation, and their absolute configuration is 2S. Three AKGs (5l–5n) were synthesised in enantiomerically pure form, and were characterised for the first time together with 12 other known and naturally occurring AKGs (5a–5k, 5o). Their structures were established using ¹H and ¹³C APT NMR with 2D-NMR, ESI-MS or HRESI-MS and optical rotation data, and they were tested for their antibacterial and antibiofilm activities. AKGs 5a–5m and 5o showed activity against five clinical isolates and P. aeruginosa ATCC 15442, with MIC values in the range of 15–125 μg/mL. In addition, at half of the MIC, most of the AKGs reduced S. aureus biofilm formation in the range of 23%–99% and P. aeruginosa ATCC 15442 biofilm formation in the range of 14%–64%. The antibiofilm activity of the AKGs assessed in this work had not previously been studied.

Full text of DOI:10.1080/14786419.2019.1686370

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Enantiomeric synthesis of natural alkylglycerols
and their antibacterial and antibiofilm activities
Deicy J. Fernández Montoya, Luis A. Contreras Jordan, Bárbara Moreno-
Murillo, Edelberto Silva-Gómez & Humberto Mayorga-Wandurraga
To cite this article: Deicy J. Fernández Montoya, Luis A. Contreras Jordan, Bárbara Moreno-
Murillo, Edelberto Silva-Gómez & Humberto Mayorga-Wandurraga (2019): Enantiomeric synthesis
of natural alkylglycerols and their antibacterial and antibiofilm activities, Natural Product Research,
DOI: 10.1080/14786419.2019.1686370
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1686370
Enantiomeric synthesis of natural alkylglycerols and their
antibacterial and antibiofilm activities
a,b
c
ꢀ
ꢀ
Deicy J. Fernandez Montoya , Luis A. Contreras Jordan , Barbara Moreno-
c
b
c
ꢀ
Murillo , Edelberto Silva-Gomez and Humberto Mayorga-Wandurraga
a
ꢀ
Posgrado Interfacultades de Microbiologıa, Facultad de Ciencias, Universidad Nacional de Colombia,
b
ꢀ
Bogota, Colombia; Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de
Colombia, Bogota, Colombia; Departamento de Quımica, Facultad de Ciencias, Universidad Nacional
de Colombia, Bogota, Colombia
c
ꢀ
ꢀ
ꢀ
ABSTRACT
ARTICLE HISTORY
Received 17 June 2019
Accepted 25 October 2019
Alkylglycerols (AKGs) are bioactive natural compounds that vary
by alkyl chain length and degree of unsaturation, and their abso-
lute configuration is 2S. Three AKGs (5l–5n) were synthesised in
enantiomerically pure form, and were characterised for the first
time together with 12 other known and naturally occurring AKGs
(5a–5k, 5o). Their structures were established using 1H and 13C
APT NMR with 2D-NMR, ESI-MS or HRESI-MS and optical rotation
data, and they were tested for their antibacterial and antibiofilm
activities. AKGs 5a–5m and 5o showed activity against five clinical
isolates and P. aeruginosa ATCC 15442, with MIC values in the
range of 15–125mg/mL. In addition, at half of the MIC, most of
the AKGs reduced S. aureus biofilm formation in the range of
23%–99% and P. aeruginosa ATCC 15442 biofilm formation in the
range of 14%–64%. The antibiofilm activity of the AKGs assessed
in this work had not previously been studied.
KEYWORDS
Natural alkylglycerols; 1-O-
alkyl-sn-glycerols; synthesis;
antibacterial; antibiofilm
1. Introduction
Alkylglycerols (AKGs) are minor constituents of microorganisms, higher plants, and ani-
mals, including humans. Marine sources, such as shark liver oil, contain higher levels
of AKGs as a mixture of alkyl chains of varying lengths and unsaturation levels (Deniau
CONTACT Humberto Mayorga-Wandurraga
hmayorgaw@unal.edu.co
Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2019.1686370.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

ꢀ
D. J. FERNANDEZ ET AL.
2
et al. 2010). Natural AKGs (1-O-alkyl-sn-glycerols) are pure enantiomers with an S con-
figuration at the asymmetric carbon (Pemha et al. 2012; Magnusson et al. 2015). AKGs
display a broad range of beneficial effects on human health, including stimulation of
haematopoiesis, immunological defences and vaccination efficiency; reduction of
tumour growth, metastasis and effects induced by radiotherapy; and increased perme-
ability of therapeutic molecules through the haematoencephalic barrier (Deniau et al.
2010; Pinault et al. 2018). AKGs are precursors of ether phospholipids that are involved
in the structure and function of cell membranes (Dean and Lodhi 2018). There have
been many synthesis studies and industrial applications of AKGs (Sutter et al. 2015),
and some of their antimicrobial properties, although studies on their role in biofilm
formation and bacterial communication remain scarce. Moreover, enantiomerically
pure AKGs are not commercially available (Pinault et al. 2018). Our earlier studies
ꢀ
revealed that AKG 5h showed antibiofilm activity (Martınez et al. 2015), while 5d and
ꢀ
5e exhibited quorum sensing inhibition (QSI) (Barragan et al. 2018). We now report
the synthesis of 15 natural enantiomers of AKGs and the in vitro evaluation of their
antibacterial and antibiofilm activities against clinical bacterial isolates as well as their
activity against Pseudomonas aeruginosa ATCC 15442 as a control reference.
2. Results and discussion
The low abundance of AKGs in natural sources has stimulated interest in their synthe-
sis (Deniau et al. 2010; Sutter et al. 2015; Pinault et al. 2018). To complement this
approach, we synthesised the natural enantiomers of 15 AKGs, 5a–5o, by a four-step
procedure based on the literature with some modifications (Scheme 1), starting from
the commercially available suitable methyl esters 1a–1o and the (R)-solketal, an enan-
tiopure chiral precursor that is known to produce AKGs as a single stereoisomer
(Pemha et al. 2012; Magnusson et al. 2015).
The first step was the nearly quantitative reduction to the respective alcohols
2a–2o with an excess of LiAlH4 that was slowly added to the ester (Parkkari et al.
2006). The second step was the mesylation of the alcohol (Baumann and Mangold
1964; Hanus et al. 2001). For the third stage, after the addition of KOH to the (R)-solke-
tal, the water formed was conveniently removed with Na followed by coupling to
mesylates 3a–3o, leading to the corresponding acetonides 4a–4o, which were finally
deprotected to obtain each AKG (Appendino et al. 2003). We used a method that
enabled us to successfully obtain the unsaturated AKGs and that after purification pro-
vided in good yield (71.3%–89.2%) all desired products 5a–5o. For the spectroscopic
data of the AKGs, see the Supplementary material.
The known compounds were identified through comparison of their spectroscopic
data with those previously published: (2S)-3-O-hexyl-1,2-propanediol 5a and (2S)-3-O-
octyl-1,2-propanediol 5b (Lampe et al. 1998); (2S)-3-O-decyl-1,2-propanediol 5c (Ordaz
et al. 2013); (2S)-3-O-dodecyl-1,2-propanediol 5d, (2S)-3-O-tetradecyl-1,2-propanediol
5e, (2S)-3-O-hexadecyl-1,2-propanediol 5f and (2S)-3-O-octadecyl-1,2-propanediol 5h
0
ꢀ
(Barragan et al. 2018); and (2S)-3-O-(cis-9 -hexadecenyl)-1,2-propanediol 5g and (2S)-3-
O-(cis-90-octadecenyl)-1,2-propanediol 5i (Magnusson et al. 2015). Additionally, the pre-
viously synthesised compounds (2S)-3-O-(cis,cis-90,120-octadecadienyl)-1,2-propanediol

NATURAL PRODUCT RESEARCH
3
5j, (2S)-3-O-(cis,cis,cis-90,120,150-octadecatrienyl)-1,2-propanediol 5k (Sperling and Heinz
1993) and (2S)-3-O-(cis-130-docosenyl)-1,2-propanediol 5o (Ni et al. 2014) were submit-
ted for full unambiguous spectral assignment.
The newly synthesised enantiopure AKG 5m was obtained as a waxy solid. The
HRESI-MS displayed a pseudo-molecular ion peak at m/z 371.3520 [M þ H]þ, which is
consistent with a molecular formula of C23H46O3 (Calcd for C23H47O3, 371.3525) that
1
was supported by its NMR spectra. The H, 13C APT and HMQC NMR spectra showed
signals from a chiral glycerol residue via a proton (dH 3.82–3.89) of hydroxymethine at
dC 70.82 (C-2), four diastereotopic protons (dH 3.71, 3.64, 3.53 and 3.49) belonging to
an oxymethylene at dC 72.29 (C-3) and a hydroxymethylene at dC 64.14 (C-1).
Furthermore, an oxymethylene (dH 3.43–3.48) at dC 71.86 (C-10), one double bond at dC
129.93 and 129.88 (C-110, C-120), a methyl group at dC 14.14 (C-200) and other methy-
lene groups from an alkyl chain were observed. The COSY spectrum revealed two dif-
ferent spin systems and their connectivity was established by the HMBC correlations
between H-10 with C-3 and H-3 with C-10, indicating the attachment of the alkyl chain
to C-3 of the glycerol (Supplementary Figure S1). Other correlations confirmed the
structural assignment. Finally, allylic carbons resonated at dC 27.26 (C-100, C-130), pro-
viding evidence that they are situated in a cis relationship and the positive optical
rotation of [a]20 þ1.8 (c 4.4, CHCl3) that is typical of the S configuration in natural
D
AKGs confirmed its optical purity. Thus, 5m was identified as (2S)-3-O-(cis-110-eico-
senyl)-1,2-propanediol.
In the same way, compound 5l was synthesised as a bright amorphous solid. Its for-
mula, C23H48O3, was determined by HRESI-MS (m/z 395.3507 [M þ Na]þ, calcd for
C23H48NaO3, 395.3501) and in agreement with its positive optical rotation of [a]20
D
þ2.3 (c 4.6, CHCl3) and the NMR spectra; the compound was named (2S)-3-O-(eicosyl)-
1,2-propanediol. Compound 5n was produced as an amorphous solid, and it had the
formula C25H52O3 by HRESI-MS (m/z 423.3805 [M þ Na]þ, calcd for C25H52NaO3,
423.3814). According to its optical rotation of [a]20 þ2.0 (c 4.7, CHCl3) and NMR data,
D
it was called (2S)-3-O-(docosyl)-1,2-propanediol. AKG 5m was previously detected by
coupled chemical and analytical techniques in the liver oil of marine cartilaginous fish,
and natural 5l and 5n were likewise detected in microorganisms, propolis and animal
ꢀ
species, including humans (Karas and Russa 2014; Magnusson et al. 2015). Compound
5n was isolated from propolis; however, its absolute stereochemistry was not deter-
mined (Talla et al. 2016). Furthermore, the preliminary syntheses of 5l and 5n were
carried out as racemic mixtures (Baumann and Mangold 1964; Pinault et al. 2018). To
date, the synthesis and spectroscopic characterisation of the optically pure natural
AKGs 5l, 5m, and 5n has not been reported.
The AKGs showed antibacterial activity in vitro and broad-spectrum against the
strains tested. The MICs were between 15.63 and 250 lg/mL, which are higher than
that of ciprofloxacin (MIC 0.48 lg/mL). Compound 5n was practically inactive (MIC >
500 lg/mL) (Table 1). The range of MIC values suggests that almost all compounds
possess a moderate antibacterial activity against the six clinical isolates.
P. aeruginosa ATCC 15442 was more sensitive to the AKGs than the clinical bacterial
isolates, and AKG 5d showed the best activity against S. aureus and S. epidermidis,
with an MIC of 15.63 mg/mL. It was observed that the most well-known AKGs (5a–5i)

ꢀ
D. J. FERNANDEZ ET AL.
4
Table 1. MIC (mg/mL) of synthesised alkylglycerols against clinical bacterial isolates and
P. aeruginosa ATCC 15442.
Gram-positive bacteria
Gram-negative bacteria
P. aeruginosa
AKG
S. aureus
E. faecalis
S. epidermidis
A. baumannii
K. pneumoniae
62.5
P. aeruginosa
62.5
ATCC 15442
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
CPX
125
125
62.5
15.63
15.63
125
125
125
125
62.5
62.5
125
125
>500
125
62.5
62.5
62.5
62.5
62.5
62.5
62.5
125
62.5
62.5
62.5
125
250
250
62.5
15.63
62.5
125
31.25
125
62.5
62.5
62.5
250
125
125
31.25
62.5
62.5
31.25
31.25
62.5
31.25
62.5
62.5
62.5
62.5
62.5
125
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
125
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
125
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
125
125
>500
125
125
>500
250
>500
125
0.49
>500
125
0.49
>500
125
0.49
>500
125
0.49
0.49
0.49
0.49
AKG: alkylglycerol; CPX: ciprofloxacin.
CPX was the antibacterial agent used as the standard. Values are the average of triplicate determinations.
Table 2. Effects of AKGs on biofilm inhibition of S. aureus clinical isolate and reference strain
P. aeruginosa ATCC 15442.
Percentage inhibition of biofilm formation at 0.5 MIC
AKG
S. aureus
P. aeruginosa ATCC 15442
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
CPX
38.55 0.28
34.96 0.20
23.52 0.13
99.21 0.79
64.66 0.32
28.26 0.14
99.85 0.15
40.38 0.34
85.42 0.66
–
50.35 0.43
54.55 0.39
53.06 0.42
46.99 0.21
52.38 0.58
64.04 0.37
56.96 0.41
61.94 0.34
50.36 0.20
14.18 0.23
52.20 0.44
44.71 0.46
50.36 0.24
–
–
29.70 0.13
65.69 0.56
82.04 0.75
65.00 0.38
–
–
97.39 0.93
CPX: ciprofloxacin.
Values are expressed as the means SE of triplicate determination. The measure of the positive control was 100%
biofilm formation. – No activity detected.
ꢀ
displayed antibacterial properties, confirming earlier reports (Lampe et al. 1998; Karas
ꢀ
and Russa 2014; Sutter et al. 2015; Barragan et al. 2018).
To observe the effects of the optically pure AKGs on S. aureus clinical isolate and P.
aeruginosa ATCC 15442 biofilm formation, the AKGs were tested at 50% of their MIC
values. The AKGs reduced biofilm formation in the range of 23.52%–99.85% for S. aur-
eus and in 14.18%–64.04% for P. aeruginosa ATCC 15442, and each AKG displayed dif-
ferent capacities against each strain. AKGs 5g and 5d possessed the highest potential,
reducing the S. aureus biofilm formation to nearly 100% (Table 2). In comparison, the

NATURAL PRODUCT RESEARCH
5
R = C6H13 (5a); C8H17 (5b); C10H21 (5c); C12H25 (5d); C14H29 (5e); C16H33 (5f); C18H37 (5h); C20H41 (5l);
C22H45 (5n).
Scheme 1. Synthesis of alkylglycerols 5a–5o.
antibiotic ciprofloxacin had no activity against S. aureus but showed high inhibition of
P. aeruginosa ATCC 15442 biofilm formation (97.39%). To the best of our knowledge,
this is the first report on the antibiofilm activity of this AKG series.
3. Conclusion
Fifteen natural AKG enantiomers were synthesised, and their structures elucidated by
spectral analysis, three of which were obtained for the first time. The AKGs demon-
strated antibacterial effects against six clinical isolates and P. aeruginosa ATCC 15442.
Additionally, they depicted promising ability against two biofilm-forming bacteria and
could be considered as new class of biofilm inhibitors. The results increase the known
biological activities of AKGs, and they can be further researched for the development
of novel antimicrobial therapeutic strategies.
Supplementary material
Experimental details relating to this paper are available online alongside
Figures S1–S37.
Acknowledgements
The authors are grateful to the Laboratory of Advanced Analytical Techniques in Natural
Products, at the Universidad de los Andes, for ESI-MS and HRESI-MS analysis.

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D. J. FERNANDEZ ET AL.
6
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
ꢀ
ꢀ
This work was supported by Direccion de Investigacion, Universidad Nacional de Colombia [proj-
ects 20406 and 37110].
ORCID
Humberto Mayorga-Wandurraga
https://orcid.org/0000-0002-2273-3112
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