Plague detection by anti-carbohydrate antibodies

Article abstract of DOI:10.1002/anie.201301633

The sugar coat of Black Death betrays it. The plague can be detected by monoclonal anti-carbohydrate antibodies. In a new technique, a plague-specific oligosaccharide antigen (see structure) is synthesized. With the help of lipopolysaccharide (LPS)-specific monoclonal antibodies (mAbs), the presence of the plague pathogen Yersinia pestis can then be detected in serum from patients by using a glycan microarray. Copyright

Full text of DOI:10.1002/anie.201301633

Angewandte
Chemie
Communications
Pathogen Detection
The sugar coat of “Black Death” betrays
it. The plague can be detected by mono-
clonal anti-carbohydrate antibodies. In
a new technique, a plague specific oligo-
saccharide antigen (see structure) is
synthesized. With the help of lipopoly-
saccharide (LPS)-specific monoclonal
antibodies (mAbs) the presence of the
plague pathogen Yersinia pestis can then
be detected in serum from patients by
using a glycan microarray.
C. Anish, X. Guo, A. Wahlbrink,
P. H. Seeberger*
&&&& — &&&&
Plague Detection by Anti-carbohydrate
Antibodies
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DOI: 10.1002/anie.201301633
Pathogen Detection
Plague Detection by Anti-carbohydrate Antibodies**
Chakkumkal Anish, Xiaoqiang Guo, Annette Wahlbrink, and Peter H. Seeberger*
Dedicated to the Bayer company on the occasion of its 150th anniversary
“Black Death” decimated the world population during the
middle ages. In total, three plague pandemics killed more
than 200 million people over the past 1500 years. The Bubonic
plague is caused by the non-sporulating bacterium Yersinia
pestis.[1] Recent plague cases have been reported in Africa and
Asia.[2] Y. pestis is a category-A biothreat agent owing to its
ready person-to-person dissemination and high lethality.[3]
The intentional dissemination of plague by aerosolization
would cause fulminant pneumonia in exposed individuals.[4]
Since pneumonic plague is typically fatal when untreated,
early detection is essential to implement effective preventive
measures.[5] Currently, Y. pestis samples are tested by poly-
merase chain reaction (PCR)-based assays or traditional
phenotyping[6] that are accurate means of detection, but are
complex, expensive, and slow.[7] Antibody binding to surface
protein antigens is a promising and less complicated option
for plague detection.[8] But higher false-positive and negative
readouts as well as differentiation between related bacteria
have made it difficult to create selective, reliable, antibody-
based detection systems.[9] The inner core of Yersinia bacterial
surface lipopolysaccharide (LPS) has a unique structure that,
unlike most Gram-negative LPS, does not contain O-side
chains.[10] The LPS from Y. pestis is immunodominant and
LPS specific antibodies in patient sera are of diagnostic
value.[10a]
Scheme 1. Retrosynthetic analysis of the Y. pestis LPS inner core
heptose trisaccharide.
Herein, we describe the synthesis of a plague-specific
oligosaccharide antigen that facilitates fabricating glycan
arrays to screen patient sera and other biological samples.
LPS-specific monoclonal antibodies (mAbs) serve to detect
Y. pestis bacteria. Incorporation of anti-Y. pestis cell surface
LPS mAbs into point-of-care diagnostic systems would
provide the basis for specific detection.
Since the isolation of LPS from Y. pestis is difficult
because of variations in LPS expression,[11] these antigens
are produced synthetically. The triheptose motif from the
inner core structure (-l-a-d-Hepp-(1!7)-l-a-d-Hepp-(1!
3)-l-a-d-Hepp) served as the antigen target for antibody
generation (Scheme 1). Trisaccharide hapten 1 was designed
to carry a primary amine at the reducing terminus through
a linker for conjugation to a protein carrier. Retrosynthetic
analysis revealed heptoside disaccharide trichloroacetimidate
3 and heptoside 2[12] as key intermediates (Scheme 1).
Disaccharide 3 in turn can be derived from heptose building
blocks 4 and 5.
The synthesis of heptose building blocks 2 and 4
commenced with reduction of lactone 6[12] with lithium tri-
tert-butoxyaluminum hydride to give hemiacetals 7 and 8
(Scheme 2). Migration of the tert-butyldimethylsilyl (TBS)
ether from C-2 to C-3 occurred under the basic conditions.
Desilylation of both 7 and 8 using TBAF and subsequent
acetylation provided intermediate 9. Trichloroacetimidate 4
was obtained in good to excellent yields from a selective
deacetylation of the anomeric position of 9 followed by
reaction with trichloroacetonitrile and 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU). The installation of a linker on
the mono-heptose 4, and deacetylation, provided intermedi-
ate 11. The primary hydroxy group was then masked as
[*] Dr. C. Anish, Dr. X. Guo, A. Wahlbrink, Prof. Dr. P. H. Seeberger
Department of Biomolecular Systems
Max-Planck Institute of Colloids and Interfaces
Am Mꢀhlenberg 1, 14424 Potsdam (Germany)
Prof. Dr. P. H. Seeberger
Institute of Chemistry and Biochemistry
Freie Universitꢁt Berlin
Arnimallee 22, 14195 Berlin (Germany)
E-mail: peter.seeberger@mpikg.mpg.de
Homepage: http://www.mpikg.mpg.de
[**] We thank the Max-Planck Society, German Federal Ministry of
Education and Research, and the Kçrber foundation for generous
financial support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201301633.
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Union of building blocks 4 and 5 yielded disaccharide 16.
Subsequent anomeric deacetylation and conversion into the
glycosyl trichloroacetimidate provided disaccharide glycosy-
lating agent 3. Coupling of 2 and 3 followed by deacetylation,
desilylation, and hydrogenolysis yielded trisaccharide 1.
Carbohydrate antigens elicit
a T cell-independent
immune response, but do not induce an immunoglobulin
class switch, thus, carbohydrate antigens are conjugated to
immunogenic carrier proteins that induce a T cell dependent
immune response.[13] Synthetic hapten 1 was conjugated to
diphtheria toxoid CRM197 as carrier protein.[14] CRM197 is
a constituent of licensed vaccines.[15] The amine group of the
linker moiety in triheptose 1 was treated with one of the N-
hydroxy succinimide (NHS) activated esters of disuccinimido
adipate (DSAP) in DMSO with catalytic amounts of triethyl-
amine to form the corresponding spacer-activated oligosac-
charide (Scheme 4).[16]
Residual DSAP was subsequently removed by extraction
with chloroform. The spacer-activated glycan was coupled
with the CRM197 lysine amino groups in 50 mm phosphate
buffer at pH 7.4 to afford neoglycoconjugate 20. Conjugation
was confirmed by SDS-PAGE and the oligosaccharide/
CRM197 ratio was determined by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS). The mass spectrum of the neoglycocon-
jugate revealed mass peaks between 59.9 and 68.8 kDa
indicating that, on average, seven oligosaccharides were
loaded onto CRM197.
Scheme 2. Synthesis of building blocks 2 and 4. Reagents and con-
ditions: a) LiAlH(OtBu)3, THF, ꢀ20 to ꢀ158C, 7 30%, 8 38%;
b) 1. TBAF, THF; 2. Ac2O, pyridine, quant.; c) NH3, MeOH, quant.;
d) Cl3CCN, DBU, CH2Cl2, 70% to quant; e) 1. N-benzyl-N-benzyloxycar-
bonyl-5-aminopentan-1-ol, TMSOTf, CH2Cl2, ꢀ308C; 2. NaOMe,
MeOH, 35%; f) TBDPSCl, imidazole, DMAP, CH2Cl2, 76%;
g) 1. triethyl orthoacetate, PTSA, toluene; 2. 80% aq. AcOH, THF, 70
to 78%. DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene; DMAP: 4-Dimethyl-
aminopyridine; PTSA: p-Toluenesulfonic acid; TBAF: Tetra-n-butylam-
moniumfluoride; TMSOTf: Trimethylsilyl trifluoromethanesulfonate;
TBDPSCl: tert-Butyl(chloro)diphenylsilane, PBB: p-Bromobenzyl.
a TBDPS ether to give 12. A cyclic orthoacetate was then
formed on the 2,3-diol by treatment of triethyl orthoacetate
and subsequently opened with aqueous acetic acid to
exclusively provide building block 2 in good yield. The
reduction of lactone 13[12] to hemiacetal 14 was carried out
under the same conditions as the reduction of 6. The reaction,
however, was much slower and was not completed even after
two days (Scheme 3, Condition a). Subsequent acetylation of
14 and TBDPS cleavage provided building block 5.
To test the immunogenicity of the heptose trisaccharide
hapten, C57BL/6 mice were immunized with glycoconjugate
20. One prime and two boosting doses of the carbohydrate–
protein conjugates that were formulated with Complete
Freundꢀs adjuvant were delivered. The anti-hapten 1 antibody
titers were monitored by glycan microarray analysis and
showed that all immunized mice formed IgG antibodies that
specifically bound hapten 1 (Figure 1).
Alternatively, when the diol on lactone 13 was initially
masked as the orthoacetate (Scheme 3, Condition c), the
reduction was completed much faster (under 30 min). The
resulting lactol was then treated with aqueous acetic acid and
acetylated to furnish the intermediate 15 in 70% yield over
four steps.
Immunized mice showed robust IgG responses against the
trisaccharide antigen with significantly higher antibody titers
in comparison to pre-immunized serum levels. Serum anti-
bodies showed higher binding to triheptose 1 even in the
presence of 150 mm thiocyanate indicating higher affinities
for the antigen. Sera from immunized mice also contained
antibodies against both the carrier proteins as well the spacer
construct employed in the glycoconjugates (Figure 1). The
response against 1 was comparatively higher and the carrier
protein did not suppress the anti-trisaccharide response.
Glycan array analysis showed the presence of higher amounts
of IgG1 and IgG2 antibodies than IgG3 indicating isotype
switching has occurred. Immunized mice showed a robust
secondary response after boosting as a two-fold increase in
endpoint titers was observed (Figure 1). Both class switch and
robust boosting response can be attributed to transformation
of a T-independent oligosaccharide hapten to a T-dependent
glycoconjugate antigen.[17] T-cell help for triheptose specific
B-cells was recruited and promoted isotype switching and
memory B-cell differentiation.
Scheme 3. Synthesis of building block 5. Reagents and conditions:
a) LiAlH(OtBu)3, THF, room temperature, 40%; b) Ac2O, pyridine,
quant.; c) 1. triethyl orthoacetate, PTSA, toluene; 2. LiAlH(OtBu)3,
THF, ꢀ20 to ꢀ158C; 3. 80% aq. AcOH, THF; 4. Ac2O, pyridine, 70%
(4 steps); d) HF·pyridine, THF, quant.
Mouse splenocytes were isolated for hybridoma develop-
ment. After subcloning and iterative selection, five hybrid-
omas were established. Glycan array analyses at each
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Figure 1. Anti-triheptose IgG responses from sera of immunized mice
determined by glycan array analysis. Endpoint titers are plotted on the
y-axis. Inset: A representative well from glycan array depicting fluores-
cent dots indicating antigen–antibody responses. A) represent anti-
body binding from serum and B) represent binding of purified mAbs.
Triheptose hapten 1 is printed on quadrant 1, CRM 197 in quadrant 2,
an unrelated glycan as a negative control in quadrant 3 and control
spacer construct in quadrant 4. Bars represent mean (ꢁ) standard
deviation of six microarray spots and *** represents Pꢂ0.001 (2-sided
Student’s t-test).
Scheme 4. Synthesis of trisaccharide hapten 1 and conjugation to
carrier protein CRM197 to yield neoglycoconjugate 20. Reagents and
conditions: a) TMSOTf, CH2Cl2, ꢀ35 to ꢀ108C, 65 to 86%; b) hydra-
zine acetate, DMF, 74% or NH3, MeOH, 87%; c) Cl3CCN, DBU,
CH2Cl2, 80%; d) 2, TMSOTf, CH2Cl2, ꢀ40 to ꢀ308C, 68%;
e) 1. NaOMe, MeOH; 2. TBAF, THF, quant.; f) 10% Pd/C, H2, MeOH/
H2O/AcOH 10:10:1, 60 to 95%. g) Et3N, disuccinimido adipate,
DMSO, 378C; h) CRM197 carrier protein, PBS buffer solution
(pH 7.4), 378C. CBz: carboxybenzyl, PBS: Phosphate-buffered saline.
selection step helped to identify the clones that secreted
antibodies with high specificity and affinity for 1. Selected
hybridomas were expanded in bioreactors and secreted
antibodies from the supernatant were purified to homoge-
neity using protein G affinity chromatography. To investigate
whether monoclonal anti-1 antibodies recognize the LPS,
fixed Y. pestis bacteria were analyzed by immunofluorescence
and imaged using confocal laser microscopy (CLSM). Specific
bacterial labeling was observed by localized green fluores-
cence of a secondary antibody (anti-mouse IgG FITC). The
green fluorescence co-localized with the blue fluorescence of
DAPI used to stain bacterial DNA. Moreover, the fluores-
cence was associated only with bacteria as evident from the
DIC (Differential interference contrast) images. Purified
mAbs as well as Abs from sera bound specifically to the
Figure 2. Indirect immunofluorescent staining of Y. pestis by anti-
hapten 1 mAbs. CLSM images of immunostained Y. pestis; A) counter
staining of bacterial DNA with DAPI, B) DIC images showing
unstained bacteria, C) FITC specific fluorescence indicating binding of
secondary antibody, and D) overlay of all three layers.
bacterial surface, thus providing the basis for the detection of
Y. pestis in biological samples (Figure 2).
Using synthetic oligosaccharide glycan arrays containing
structures from related bacteria, we determined the binding
specificity and the capability of mAbs to distinguish between
different cell-surface glycans. Monoclonal antibody binding
against LPS oligosaccharides found on Gram-negative bac-
teria was tested using a glycan array. Monoclonal antibodies
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secondary antibody. Increased mean fluorescence intensity of
bacteria-associated fluorescence indicated mAb binding to
the bacteria. Monoclonal antibodies to Y. pestis bound
specifically to this pathogen but not to N. meningitidis and
E. coli bacteria. The mAbs are highly specific, can selectively
detect Y. pestis and differentiate related Gram negative
bacteria.
In conclusion, we demonstrate that synthetic oligosac-
charides based on unique cell surface glycans can be exploited
to create immunological agents for bacterial detection.
Heptose trisaccharide-specific mAbs are the basis for highly
sensitive and specific detection systems for Y. pestis. Efforts to
incorporate these antibodies to point-of-care diagnostic plat-
forms are currently underway.
Figure 3. Anti-hapten 1 mAbs binding to Y. pestis, E. coli, and N. menin-
gitidis. Inset: Representative glycan array image of mAbs binding to
LPS based oligosaccharides from various bacteria A) image and
B) printing pattern. Spot 1: is heptose monosaccharide, 2: Kdo-mono-
saccharide, 3: Heptose-Kdo disaccharide from all Gram-negative
bacteria, 4: Kdo-Kdo-Kdo trisaccharide from C. trachomatis, 5: trihep-
tose 1 from Y. pestis, 6: buffercontrol, 7: inner core tetrasaccharide
from N. meningitidis LPS and 8: conserved trisaccharide from LPS
structures of all Gram negative bacteria). MFI=Mean fluorescence
intensity; bars represent mean (ꢁ) standard deviation of three labeling
experiments.
Experimental Section
1: Disaccharide trichloroacetimidate 3 (64 mg, 0.050 mmol) and
alcohol 2 (38 mg, 0.036 mmol) were dried and dissolved in anhydrous
CH2Cl2 (0.5 mL) and 4 ꢁ MS (100 mg) was added. The mixture was
cooled to ꢀ408C and TMSOTf (4 mL, 0.022 mmol) was added. The
reaction mixture was stirred at ꢀ40 to ꢀ308C for 1 h, and then
quenched with saturated NaHCO3 aqueous solution. The organic
phase was dried over MgSO4, solids removed by filtration, and
concentrated. The crude product was purified by flash column
chromatography (hexane/EtOAc 2:1) to give 18 (52 mg, 67%) as
a colorless oil. NaOMe was added (0.2 mL, 0.5m in MeOH) to
a solution of 18 (50 mg, 0.023 mmol) in MeOH (1.5 mL) and the
reaction mixture was stirred at room temperature for 15 h. After
neutralization by Amberlite IR-120, the mixture was filtered and
concentrated. The residue was then dissolved in THF (1 mL) and
TBAF (0.2 mL, 1m in THF) was added. The reaction mixture was
stirred at room temperature for 48 h and then concentrated in vacuo.
The resulting residue was purified by a Sephadex LH-20 column
chromatography (MeOH) to give 19 as colorless oil. Pd/C (10%,
100 mg) was added to a solution of 19 (37.5 mg, 0.022 mmol) in
MeOH/H2O/AcOH (2 mL, 50:50:1). The mixture was stirred under
H2 atmosphere for 48 h, filtered and concentrated in vacuo. The crude
product was purified by Sephadex LH-20 column chromatography
(H2O) to give 1 (9 mg, 60%) as a white powder after lyophilisation.
Conjugation of trisaccharide 1 to CRM197 carrier protein: A
solution of trisaccharide 1 (4.27 mmol) in DMSO was added to
a solution of disuccinimido adipate (0.43 mmol) and triethylamine
(10 mL) in DMSO (100 mL), at room temperature. After 1.5 h, 0.5 mL
of phosphate buffer (0.1m pH 7.4) was added to the reaction mixture
and the residual linker extracted with chloroform. The extraction
procedure was repeated three times and the resultant aqueous layer
was centrifuged (300 g, 5 min) to separate traces of chloroform. The
aqueous layer was separated and added to 1 mL of protein solution
(CRM197, 1 mgmLꢀ1 in 0.1m phosphate buffer pH 7.4). The reaction
was allowed to continue for 5–6 h with gentle stirring at pH 7.4. The
glycoconjugate was purified either by using size exclusion chroma-
tography or by ultrafiltration.
to 1 exclusively bound to antigen 1 (Figure 3, spot 5) and none
of the other synthetic glycans on the array. One of the
antibodies weakly bound to the heptose monosaccharide. The
binding specificity was also tested on native isolated LPS from
related bacteria by a surface plasmon resonance (SPR) based
binding assay. Isolated LPS from E. coli, Salmonella typhi,
and Neisseria meningitidis were used in the study. The mAbs
were captured using anti-IgG surfaces and used for binding
isolated LPS. Capturing mAbs and binding LPS in solution
allowed the variations arising from differential immobiliza-
tion of structurally different LPSs to be limited. Compared to
the synthetic Y. pestis LPS derivative, binding to the mAbs by
E. coli O55 and N. meningitidis LPS was negligible. Even
a delipidated derivative of E. coli O55:B5 LPS did not bind to
the mAb. Since E. coli show strain specific LPS expression,
LPS isolated from another strain, E. coli O127:B8, was used in
the binding study. Both LPS from E. coli O127:B8 and S. typhi
showed significant cross reactivity to the anti-Y. pestis-mAbs
but the binding was weaker than to the Y. pestis LPS
derivative (Supporting Information). The specificity of bind-
ing of mAbs to Y. pestis LPS can be explained by unique
structural features of Y. pestis LPS. The inner core region of
E. coli and N. meningitidis LPS is often decorated with
nonstoichiometric additions of other glycans and with phos-
phate, pyrophosphorylethanolamine, or phosphorylcholine
residues.[18] Preliminary saturation-transfer difference (STD)
NMR spectroscopy studies of mAb binding to Y. pestis LPS
derivative 1 indicated a role for the side-chain hydroxy groups
in antibody recognition (data not shown). These groups are
often derivatized in N. meningitidis and E. coli LPS which
serves to explain how mAbs discriminate the different LPSs.
To further determine the binding specificity, the inter-
action of the mAbs to E. coli as well as N. meningitidis, was
analyzed by flow cytometry using an anti-mouse IgG-FITC
Received: February 25, 2013
Revised: May 14, 2013
Published online: && &&, &&&&
Keywords: lipopolysaccharides · antibodies · oligosaccharides ·
.
pathogen detection · yersinia pestis
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[10] a) A. A. Byvalov, Y. S. Ovodov, Russ. J. Bioorg. Chem. 2011, 37,
400 – 410; b) Y. A. Knirel, A. P. Anisimov, Acta naturae 2012, 4,
46.
[11] Y. A. Knirel, B. Lindner, E. V. Vinogradov, N. A. Kocharova,
S. N. Senchenkova, R. Z. Shaikhutdinova, S. V. Dentovskaya,
N. K. Fursova, I. V. Bakhteeva, G. M. Titareva, S. V. Balakho-
nov, O. Holst, T. A. Gremyakova, G. B. Pier, A. P. Anisimov,
Biochemistry 2005, 44, 1731.
[1] S. Haensch, R. Bianucci, M. Signoli, M. Rajerison, M. Schultz, S.
Kacki, M. Vermunt, D. A. Weston, D. Hurst, M. Achtman, E.
Carniel, B. Bramanti, Plos Pathog. 2010, 6, e1001134.
[2] R. D. Perry, J. D. Fetherston, Clin. Microbiol. Rev. 1997, 10, 35.
[3] a) T. V. Inglesby, D. T. Dennis, D. A. Henderson, J. G. Bartlett,
M. S. Ascher, E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer,
J. F. Koerner, M. Layton, J. McDade, M. T. Osterholm, T.
OꢀToole, G. Parker, T. M. Perl, P. K. Russell, M. Schoch-Spana,
K. Tonat, W. G. C. Biodefense, JAMA J. Am. Med. Assoc. 2000,
283, 2281; b) T. V. Inglesby, D. A. Henderson, T. OꢀToole, D. T.
Dennis, JAMA J. Am. Med. Assoc. 2000, 284, 1649.
[4] M. B. Prentice, L. Rahalison, Lancet 2007, 369, 1196.
[5] K. V. Sergueev, Y. X. He, R. H. Borschel, M. P. Nikolich, A. A.
Filippov, Plos One 2010, 5, e11337.
[6] a) D. M. Engelthaler, K. L. Gage, J. A. Montenieri, M. Chu,
L. G. Carter, J. Clin. Microbiol. 1999, 37, 1980 – 1984; b) J.
Guarner, W. J. Shieh, P. W. Greer, J. M. Gabastou, M. Chu, E.
Hayes, K. B. Nolte, S. R. Zaki, Am. J. Clin. Pathol. 2002, 117, 205.
[7] H. Neubauer, H. Meyer, J. Prior, S. Aleksic, A. Hensel, W.
Splettstosser, J. Vet. Med. Ser. B 2000, 47, 573.
[12] T. Ohara, A. Adibekian, D. Esposito, P. Stallforth, P. H.
Seeberger, Chem. Commun. 2010, 46, 4106.
[13] a) F. Y. Avci, D. L. Kasper, Annu. Rev. Immunol. 2010, 28, 107;
b) M. L. Hecht, P. Stallforth, D. V. Silva, A. Adibekian, P. H.
Seeberger, Curr. Opin. Chem. Biol. 2009, 13, 354.
[14] Pfenex Ltd. (Reagent proteins, http://www.reagentproteins.com)
is gratefully acknowledged for providing CRM197 at discounted
price.
[15] M. Brçker, P. Costantino, L. DeTora, E. D. McIntosh, R.
Rappuoli, Biological 2011, 39, 195.
[16] A. Kabanova, I. Margarit, F. Berti, M. R. Romano, G. Grandi, G.
Bensi, E. Chiarot, D. Proietti, E. Swennen, E. Cappelletti, P.
Fontani, D. Casini, R. Adamo, V. Pinto, D. Skibinski, S. Capo, G.
Buffi, M. Gallotta, W. J. Christ, A. S. Campbell, J. Pena, P. H.
Seeberger, R. Rappuoli, P. Costantino, Vaccine 2010, 29, 104.
[17] P. Costantino, R. Rappuoli, F. Berti, Expert Opin. Drug
Discovery 2011, 6, 1045.
[8] O. Lazcka, F. J. Del Campo, F. X. Munoz, Biosens. Bioelectron.
2007, 22, 1205.
[9] N. Gilmartin, R. OꢀKennedy, Enzyme Microb. Technol. 2012, 50,
87 – 95.
[18] C. R. H. Raetz, C. Whitfield, Annu. Rev. Biochem. 2002, 71,
635 – 700.
6
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