Rapid preparation of mycobacterium N-glycolyl lipid i and lipid II derivatives: A biocatalytic approach

Article abstract of DOI:10.1002/chem.201203251

Breaking down barriers: A rapid, inexpensive preparation of the structurally complex mycobacterial N-glycolyl LipidI, LipidII, and their analogues from a range of different synthetic N-glycolyl and N-glycinyl Park's nucleotides is described (see scheme). The biotransformations were catalyzed by a readily available biocatalyst obtained from a bacterial cell-free membrane fraction. The unnatural N-glycinyl LipidII was found to be a substrate of Mycobacterium tuberculosis (Mtb) transglycosylase, PonA, and N-glycolyl LipidI was a weak inhibitor against PonA. Copyright

Full text of DOI:10.1002/chem.201203251

DOI: 10.1002/chem.201203251
Rapid Preparation of Mycobacterium N-Glycolyl Lipid I and Lipid II
Derivatives: A Biocatalytic Approach
Kuo-Ting Chen,[a, b] Ye-Ching Kuan,[a] Wei-Chen Fu,[a] Pi-Hui Liang,[b]
Ting-Jen R. Cheng,[a] Chi-Huey Wong,[a] and Wei-Chieh Cheng*[a]
Tuberculosis (TB), an infectious disease induced by Myco-
bacterium tuberculosis (Mtb), is a leading cause of death
worldwide.[1] The increasing prevalence of drug resistant TB
strains, including multidrug-resistant TB (MDR-TB), and
extensively drug-resistant TB (XDR-TB), is of growing con-
cern. New, more effective antibiotics are urgently required.[2]
The mycobacterial cell wall is essential for viability, and
therefore the enzymes responsible for its biosynthesis are
possible antibiotic targets. For example, mycobacterial peni-
cillin-binding protein (PBP), PonA, located on the external
surface of mycobacterial membranes, possesses two catalytic
domains for transpeptidase and transglycosylase activities
and plays an important role in the last two steps (transglyco-
sylation and transpetidation) of mycobacterial cell wall con-
structions.[3] The transpeptidase responsible for the cross-
linking of peptidoglycans is a known target but the serious
drug resistance has been reported.[4] In contrast, no antibiot-
ics have been developed to target the transglycosylase
(TGase).[5]
PonA is relatively easy to access, essential for mycobacte-
rial viability, without a eukaryotic counterpart, and hence an
attractive target for antibiotic discovery and development.[6]
During transglycosylation, the sugar moiety from the acti-
vated polymeric peptidoglycan (a glycosyl donor) is linked
to the specific hydroxyl group (4-OH) of N-glycolyl Lipid II
(a glycosyl acceptor), and a decaprenyl pyrophosphate is re-
leased (Figure 1). Structurally, N-glycolyl Lipid II consists of
the disaccharide of N-acetylglucosamine (GlcNAc) and N-
glycolyl muramic acid (MurNGlyc), pyrophosphate, decap-
renol lipid tail, and a pentapeptide moiety (l-alanyl-d-glu-
key component, Mtb N-glycolyl Lipid I, consisting of N-gly-
colyl muramic acid, decaprenol lipid tail, and the pentapep-
tide moiety, is a biosynthetic precursor of N-glycolyl Lipi-
d II. MurNGlyc is only observed in mycobacterial cell walls,
and is therefore considered a potential biomarker.[7] The N-
glycolyl groups in peptidoglycan chains may play an critical
role for the resistance to lysozyme and for the innate
immune response during a mycobacterial infection.[8]
During the late stages of peptidoglycan biosynthesis, the
enzyme Mtb MraY catalyzes the transfer of the sugar
moiety from Mtb N-glycolyl Parkꢀs nucleotide (UDP-N-gly-
colyl-muramyl-l-alanyl-d-glutamyl-meso- diaminopimelyl-d-
alanyl-d-alanine) to the decaprenyl phosphate (C50P), to
give N-glycolyl Lipid I. Subsequently, conjugation of Mtb N-
glycolyl Lipid I with UDP-GlcNAc is catalyzed by Mtb
MurG to produce Mtb N-glycolyl Lipid II (Figure 1). It is
noteworthy that both Mtb MraY and Mtb MurG are mem-
brane-associated proteins in mycobacterium.
However, mechanistic and inhibitor studies for late-stage
peptidoglycan biosynthesis have been hampered by the diffi-
culty in acquiring pure samples of membrane-associated ma-
terials such as C50P, N-glycolyl Lipid I, and N-glycolyl Lipi-
d II. Direct isolation of these materials from mycobacterium
is difficult due to their low natural abundance and structural
complexity.[9] Recently, a multi-step chemical synthesis of
structurally modified N-glycolyl Parkꢀs nucleotide[10] and N-
glycolyl Lipid II,[11] were reported. A similar synthesis of N-
glycolyl Lipid I, however, remains unknown. In previous
work, we demonstrated that the decaprenyl phosphate and
meso-DAP of the natural Mtb N-glycolyl Lipid II could be
substituted by two accessible materials, undecaprenyl phos-
phate (C55P) and l-lysine, respectively, and that this syn-
thetic N-glycolyl Lipid II is recognized as a PonA sub-
strate.[11]
tamyl-meso-diaminopimelyl-d-alanyl-d-alanine).
Another
[a] K.-T. Chen, Y.-C. Kuan, W.-C. Fu, Dr. T.-J. R. Cheng,
Prof. C.-H. Wong, Prof. W.-C. Cheng
Genomics Research Center
As part of our ongoing interest in the development of
new methods for the preparation of mycobacterial cell wall
components, we realized that elaboration of N-glycolyl
Parkꢀs nucleotide to more complex molecules through bioca-
talytic synthesis might be an attractive alternative to con-
ventional chemical synthesis, the utility of which is limited
by tedious chemical transformations such as glycosylation,
pyrophosphate formation, and protection/deprotection
steps.[12] The use of purified or crude enzymes as biocatalysts
for chemical transformations is a promising approach due to
their high chemo-, regio- and enantioselectivity, and mild re-
Academia Sinica
128 Academia Road, Section 2
Nankang,Taipei, 115 (Taiwan)
[b] K.-T. Chen, Prof. P.-H. Liang
Department of Pharmacy
National Taiwan University
1, Jen-Ai Road, Section 1
Taipei 10051 (Taiwan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203251.
834
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Chem. Eur. J. 2013, 19, 834 – 838

COMMUNICATION
Figure 1. Biosynthesis of mycobacterial peptidoglycan.
action conditions.[13] Several biocatalysts have been exten-
sively studied and commercialized for applications including
transesterification, aminolysis, and acidolysis.[14] To the best
of our knowledge, preparation of mycobacterial N-glycolyl
Lipid I, Lipid II, and their analogues through biocatalytic
synthesis has not been extensively explored.[15] Herein, we
describe the preparation of structurally complex mycobacte-
rial N-glycolyl Lipid I and Lipid II-based molecules through
biocatalytic synthesis from N-glycolyl Parkꢀs nucleotide de-
rivatives (Figure 2). The bioactivities of these synthetic mol-
ecules towards PonA are also evaluated.
Our synthesis of N-glycolyl Parkꢀs nucleotide derivatives
started from the protected muramic acid ester
1
(Scheme 1).[11] N-Glycolylation of 1 using N-succinimidyl
acetoxyacetate under basic conditions followed by acetyla-
tion of diols at C4 and C6, and O-debenzylation through
catalytic hydrogenation gave 2 in 63% overall yield over
three steps. Several conditions to accomplish the O-deben-
zylation were investigated; the use of catalytic Pd(OH)2 in
the presence of 0.1% conc. HCl in THF resulted in the best
yield (75%). In contrast, when the reactants were dissolved
in methanol, or when a small amount of glacial acetic acid
was added, no satisfactory results (<40% yield) were ob-
tained (see the Supporting Information, Table S1).
The phosphorylation of 2 was carried out in a phosphity-
lation/oxidation sequence to deliver phosphoryldiester 3 as a
single diastereomer in an excellent yield (90%). The a-con-
1
figuration of 3 was confirmed by H NMR spectroscopy. Se-
lective deprotection of the O-trimethylsilylethyl (TMSE)
group in 3 by treatment with TBAF in THF, followed by
coupling with the tetrapeptide d-GluACTHNUTRGNENG(U OMe)-l-LysAHCUTNGTREN(NGUN TFA)-d-
Ala-d-Ala-COOMe, gave 4. Debenzylation of 4 followed by
conjugation with UMP-morpholine-N,N’-dicyclohexylcar-
boxamidine salt and global deprotection under basic condi-
tions gave N-glycolyl Parkꢀs nucleotide 5 in 39% yield. A
fluorescent probe 6 was prepared from 5 by attaching a ni-
tronbenzoxadiazole (NBD) fluorophore at the terminal-NH2
site of lysine on the peptide side chain.
A cell-free membrane fraction from Mycobacterium smeg-
matis was first tested as a biocatalyst. A mixture of the fluo-
Figure 2. General strategy for the preparation of mycobacterial N-glycol-
yl Lipid I and Lipid II-based molecules.
Chem. Eur. J. 2013, 19, 834 – 838
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835

W.-C. Cheng et al.
Table 1. Biocatalytic synthesis of N-glycolyl Lipid I and Lipid II.
Entry Substrate Conditions
Product Yield
[%][c]
[mmol]
(membrane fraction source/t [h])
Lipid II Synthesis[a]
M. smegmatis[d]/1
M. smegmatis/24
S. aureus[d]/1
1
2
3
4
5
6
7
6, 2
6, 2
6, 1
6, 1
6, 1
6, 1
5, 1
9
9
9
9
9
9
10
30[e]
50
28
M. smegmatis/1
61
72
M. flavus[d]/1
E. coli/1
M. flavus/1
trace[f]
85
Lipid I Synthesis[b]
M. flavus/3
M. flavus/3
8
9
6, 1
5, 1
7
8
75
80
[a] Reaction mixture contains UDP-GlcNAc, C55P, and cell-free mem-
brane fraction. [b] Reaction mixture contains C55P, and cell-free mem-
brane fraction. [c] Isolated product yield after HPLC purification.
[d] M. smegmatis=Mycobacterium smegmatis; M. flavus=Micrococcus
flavus; S. aureus=Staphylococcus aureus [e] Excess NBD-N-glycolyl
Parkꢀs nucleotide was recovered in 13%. [f] Only a trace of 9 was ob-
served by using TLC.
Figure S1). This result suggested that the fluorescent probe
6 is indeed a biocatalytic substrate, although the conditions
used were not optimized. Extension of the reaction time to
24 h resulted in 9 being isolated in only 50% yield; some
unknown impurities were also observed (Table 1, entry 2).
After the initial amount of 6 was adjusted to the final con-
centration of 1 mmol, the reaction yield of 9 was dramatical-
ly improved to 61% (Table 1, entry 3; see Supporting Infor-
mation Figure S2). These positive results prompted us to fur-
ther systematically investigate the reaction conditions
(Table 1). Then, other cell-free membrane fractions, includ-
ing that from Micrococcus flavus (Gram-(+)), S. aureus
(Gram-(+)), and E. coli (Gram-(À)) were subsequently
evaluated.[16] Several interesting observations should be
noted, for example, S. aureus, M. smegmatis and M. flavus
membrane fractions catalyzed the formation of NBD-N-gly-
colyl Lipid II in a yield of 28, 61 and 72%, respectively, but
no significant product was observed in the reaction mixture
containing E. coli membrane fraction (Table 1, entries 4–6).
This finding suggests that the catalytic efficiency depends on
enzyme capacity. As these membrane fractions are likely to
contain membrane-associated TGases, the TGase inhibitor
moenomycin was added to the reaction mixture to block
transglycosylation and to maximize the yield of Lipid II.
Analysis of reaction progress curves did not show a signifi-
Scheme 1. Synthesis of N-glycolyl Parkꢀs nucleotide and analogues. a) N-
succinimidyl acetoxyacetate, NaHCO3, 1,4-dioxane, RT, 2 h; b) Ac2O,
pyridine, 2 h, 08C to RT; c) Pd(OH)2/H2, THF/0.1% conc. HCl, over-
night, RT, 65% (3 steps); d) 1) iPr2NP
2) tBuOOH, À408C, 84%; e) TBAF, THF, RT, 2 h; f) tetrapeptide (d-
Glu(OMe)-l-Lys(TFA)-d-Ala-d-Ala(OMe)), PyBOP, DIEA, THF/
ACHTUGNTREN(NUNG OBn)2, 1H-tetrazole, CH2Cl2, RT;
A
R
ACHTUNGTRENNUNG
CH2Cl2, RT, 73% (2 steps); g) Pd(OH)2/H2, MeOH, RT, 1 h; h) UMP-
morpholine-N,N’-dicyclohexylcarboxamidine salt, 1H-tetrazole, pyridine,
4 ꢂ molecular sieves, 08C to RT, 48 h; i) LiOH, MeOH, RT, 4 h, 39%
(3 steps); j) NBD-X-OSu, NaHCO3, DMF/H2O, RT, overnight, 90%.
rescent NBD-labeled Parkꢀs nucleotide 6 (2 mmol), UDP-
GlcNAc (10 mmol), and C55P (1.5 mmol) in the presence of
membrane fraction of M. smegmatis was shaken at room
temperature. After 30 min, the NBD-labeled N-glycolyl Lip-
id II 9 could be detected by TLC and HPLC analysis. After
1 h, the reaction was quenched by the addition of pyridine
acetate buffer (pH 5),[12] though a detectable amount of 6
still remained. Compound 9 was purified by HPLC in 31%
yield based on the initial amount of 6 and 13% of 6 was re-
covered (Table 1, entry 1; see the Supporting Information
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Mycobacterium N-Glycolyl Lipid I and Lipid II Derivatives
COMMUNICATION
Table 2. Preparation of modified Lipid I and Lipid II through the bioca-
talytic method.
cant change, suggesting that either the reaction conditions
or the quality of TGase present were unsuitable for transgly-
cosylation (see the Supporting Information, Figure S1).
Use of the cell-free membrane fraction from M. flavus as
the biocatalyst source for the preparation of Lipid II 9
(Table 1, entry 5) was found to be the most convenient be-
cause of its easy accessibility and the high reaction yield. By
using the same conditions, N-glycolyl Lipid II 10 was also
prepared in a satisfactory yield (85%; Table 1, entry 7).
With these encouraging results and valuable reaction param-
eters in hand, we sought to prepare N-glycolyl Lipid I. Nota-
bly, without adding UDP-GlcNAc, N-glycolyl Parkꢀs nucleo-
tides 6 and 5 were converted to the corresponding NBD-N-
glycolyl-Lipid I (7) and N-glycolyl-Lipid I (8). The reaction
mixtures were extracted and purified by HPLC and charac-
Entry
1
Parkꢀs
nucleotide
R1
R2
Lipid I/
Yield [%]
Lipid II/
Yield [%]
11
l-Ala-d-Glu-
OH
15/68
18/67
l-Lys
l-Ala
l-Ala-d-Glu-
l-Lys(NBD)-
ACHTUNGTNER(NUNG NBD)
2
3
12
13
OH
NH2
–/trace[a]
16/64
–/trace[a]
19/77
1
terized by H NMR spectroscopy to give pure Lipid I 7 and
AHCTUNGTRENNUNG
8 in good yields (75% and 80%, respectively; Table 1, en-
tries 8 and 9). Our results indicate that the fluorescent
probe attached to the e-position of lysine of Parkꢀs nucleo-
tide did not preclude the biosynthesis of Lipid I and Lipid II
analogues.
d-Ala-d-Ala
l-Ala-d-Glu-
4
14
NH2
17/64
20/64
l-Lys
ACHTUNGTNER(NUNG NBD)
[a] Detected by mass spectrometry, not isolated.
Having studied the biocatalytic conditions in detail, new
Parkꢀs nucleotides bearing a N-glycinyl group or different
lengths of the peptide chain were designed and synthesized,
and their conversion into the new corresponding Lipid I and
Lipid II-based molecules was pursued. Following the similar
protocol shown in Scheme 1, N-succinimidyl-2-trifluoro-
acetamidoacetate was utilized instead of N-succinimidyl ace-
toxyacetate to react with 1. Two new fluorescent N-glycinyl
Parkꢀs nucleotides 13 and 14 bearing the tripeptide and pep-
tapeptide, respectively, were prepared (see the Supporting
Information, Scheme S1). In addition, two new N-glycolyl
Parkꢀs nucleotides (11 and 12) with a different length of the
peptide chain were also prepared (Figure 3).
peptide moiety in the substrate disabled the catalytic activi-
ty. Surprisingly, N-glycinyl Parkꢀs nucleotides 13 and 14
were converted to N-glycinyl Lipid I and Lipid II in high
yields (Table 2, entries 3 and 4), proving that the amino
group in 13 or 14 could be tolerated during the biocatalytic
synthesis.
With these structurally modified peptidoglycan precursors
in hand, their biological activity toward PonA was investi-
gated. N-Glycinyl Lipid II analogues 19 (pentapeptide) and
20 (tripeptide) were both found to be PonA substrates with
similar substrate potency. Both were mostly consumed (>
80%) in 8 h during the transglycosylation reaction catalyzed
by Mtb PonA, suggesting the presence of the last two amino
acids (d-Ala-d-Ala) did not significantly affect the sub-
strate-enzyme recognition[17] (see the Supporting Informa-
tion, Figure S4). Kinetic studies revealed that the N-glycolyl
Lipid II 9 and N-glycinyl Lipid II 19 have a similar binding
affinity towards PonA with KM values of 9.7 and 5.4 mm, re-
spectively. Surprisingly, the kcat/KM values of 9 and 19 were
higher than that of the bacterial N-acetyl Lipid II[16] (1.06ꢃ
104 mÀ1sÀ1), indicating that the N-glycolyl or N-glycinyl
group in Lipid II contributes more interactions with PonA
than the N-acetyl group (Table 3; see the Supporting Infor-
mation, Figure S5). Based on the peptidoglycan biosynthesis
Figure 3. Chemical structures of the peptide truncated N-glycolyl Parkꢀs
nucleotides and N-glycinyl Parkꢀs nucleotides.
Table 3. Kinetic parameters of N-acetyl Lipid II,
9
and 19 towards
PonA.[a]
N-Glycolyl and N-glycinyl Parkꢀs nucleotides (11, 12, 13,
and 14) were submitted to the biocatalytic reactions previ-
ously developed (Table 1) and the results are shown in
Table 2. N-Glycolyl Parkꢀs nucleotide 11 was converted to
the corresponding Lipid I 15 and Lipid II 18 in yields of 68
and 67%, respectively. The use of N-glycolyl Parkꢀs nucleo-
tide 12, however, did not result in the detection of any de-
sired products, suggesting that the major truncation of the
N-acetyl-Lipid II[16]
9
19
KM [mm]
26.4Æ6.8
0.28Æ0.03
1.06ꢃ104
9.7Æ1.5
5.4Æ1.6
kcat [sÀ1
kcat/KM
]
0.25Æ0.01
0.23Æ0.02
4.26ꢃ104
2.57ꢃ104
AHCTUNGTERNNUNG ]
[mÀ1 sÀ1
[a] Experiments were performed in 0.085% decyl-PEG, Tris-HCl
(50 mm), pH 8.0, CaCl2 (10 mm), 10% DMSO, 15% MeOH and PonA
(0.07 mm) at 258C and repeated in triplicate.
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W.-C. Cheng et al.
pathway, naturally occurring N-glycolyl Lipid I is located
only on the cytoplasmic surface of the inner membrane, and
therefore cannot interact with PonA. Unexpectedly, N-gly-
colyl Lipid I was found to be a PonA inhibitor with IC50
value of 295 mm (see the Supporting Information, Figure S6).
For comparison purposes, undecaprenyl phosphate (C55P)
and N-glycolyl Parkꢀs nucleotide (6) showed no inhibitory
activity against PonA. These results suggest that a molecule
containing the N-glycolyl-muramyl-pentapeptide pyrophos-
phate moiety linked with a hydrophobic lipid might play an
important role for inhibitory activities against TGases.
In conclusion, a rapid and convenient method for the
preparation of key precursors of mycobacterial cell walls, in-
cluding N-glycolyl Lipid I and Lipid II, has been successfully
developed. This new synthesis starts from diverse N-glycolyl
Parkꢀs nucleotides, and proceeds through a biocatalytic syn-
thesis. The biocatalyst sources, extracted from the crude
membrane fraction of M. flavus, were used directly without
further purification. Reaction optimization resulted in the
development of protocols that are easy to perform and give
satisfactory yields. Structurally new Parkꢀs nucleotides bear-
ing peptide moieties of different lengths are well tolerated
in this biocatalytic method. The unnatural N-glycinyl Lipi-
d II, which bears a versatile amino functional group of po-
tential utility in other chemical applications, was also found
to be a substrate of PonA. Interestingly, N-glycolyl Lipid I
was found to weakly inhibit TGase and is therefore a possi-
ble starting point for the development of new Mtb TGase
inhibitors. The feasibility of this research approach is cur-
rently being investigated.
Keywords: antibiotics · biocatalysis · inhibitors · synthetic
methods · transglycosylase
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Experimental Section
General procedure for Lipid I and Lipid II preparation: UDP-GlcNAc
(10 mmol, only for Lipid II preparation), N-glycolyl Parkꢀs nucleotide de-
rivatives (1 mmol) and C55P (1.5 mmol) were dissolved in reaction buffer
(containing Tris-HCl (10 mm), pH 8, MgCl2 (0.5 mm) and 1% (w/v)
Triton X-100). The cell-free membrane fraction (128 mg) from Micrococ-
cus flavus was added into the reaction buffer and shaken at RT for 3 h.
When Parkꢀs nucleotide was completely consumed, the crude reaction
was extracted by 1-butanol and pyridine acetate. After centrifugation,
the organic layer was collected and purified by RP-HPLC.
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Acknowledgements
This work was supported by National Science Council and Academic
Sinica.
Received: September 16, 2012
Published online: December 11, 2012
838
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Chem. Eur. J. 2013, 19, 834 – 838