Synthesis of a 1α,4′-Di-O-allylated, 2,3,2′,3′- tetra-O-tetradecylated lipid a mimic and its 4-O-(4-methoxybenzyl) precursor

Article abstract of DOI:10.1135/cccc20061532

Compound 18 mimicking lipid A, containing D-glucose instead of D-glucosamine moieties in its gentiobiose skeleton, O-tetradecyl groups at the C-2, C-3, C-2′ and C-3′ instead of the ester- and amide-linked fatty acids, and O-allyl groups at the C-1α and C-

Full text of DOI:10.1135/cccc20061532

1532
Baráth, Petrušová, Hirsch, Petruš:
SYNTHESIS OF A 1α,4′-DI-O-ALLYLATED,
2,3,2′,3′-TETRA-O-TETRADECYLATED LIPID A MIMIC AND
ITS 4-O-(4-METHOXYBENZYL) PRECURSOR
1
2
3
4,
Marek BARÁTH , Mária PETRUŠOVÁ , Ján HIRSCH and Ladislav PETRUŠ *
Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava, Slovakia;
1
2
3
4
e-mail: chemmbar@savba.sk, petrusova@savba.sk, chemhirs@savba.sk, chemlpet@savba.sk
Received July 21, 2006
Accepted Septem ber 22, 2006
Com poun d 18 m im ickin g lipid A, con tain in g D-glucose in stead of D-glucosam in e m oieties in
its gen tiobiose skeleton , O-tetradecyl groups at th e C-2, C-3, C-2′ an d C-3′ in stead of th e es-
ter- an d am ide-lin ked fatty acids, an d O-allyl groups at th e C-1α an d C-4′ replacin g th e
ph osph ate groups, was syn th esized by th e Sch m idt trich loroacetam idate m eth od in a com -
bin ed 8% yield of 13 steps. Allyl 4,6-O-(4-m eth oxyben zyliden e)-α-D-glucopyran oside (1) an d
m eth yl 4,6-O-ben zyliden e-α-D-glucopyran oside (4) were startin g m aterials for preparation of
th e respective O-alkylated an d O-allylated glycosyl don or an d sugar n ucleoph ile. Wh ile
boron trifluoride eth erate in dich lorom eth an e catalysed a h igh ly preferen tial form ation of
th e required β-(1→6)-glycosidic bon d, α-lin ked lipidodisacch aride was a m ajor product wh en
trim eth ylsilyl trifluorom eth an esulfon ate was used as a catalyst, in both cases in depen den tly
of th e an om eric con figuration of th e startin g im idate. Prolon ged treatm en t with acid cata-
lysts in th e couplin g step was exploited also for a on e-pot rem oval of th e in term ediate 4-O-
(4-m eth oxyben zyl) protection of th e target m im ic 18 of lipid A.
Keyw ord s: Carboh ydrates; Lipid A m im ics; Trich loroacetim idate syn th esis; Disacch arides;
Glycosidation s; Liposacch arides; Bacterial toxin s.
Lipid A (Fig. 1a), th e en dotoxin respon sible for toxicity of lipopolysacch a-
ride (LPS) presen t in th e outer surface m em bran e of Gram -n egative bacte-
ria1. Th e lysis of Gram -n egative bacteria causes th em to release LPS th at can
cause various h ealth disorders. On e of th em is sepsis – a severe in flam atory
illn ess wh ich can lead to a serious organ dam age an d dysfun ction an d fi-
n ally to death of th e in ten sive care un it patien ts2. As th ere does n ot exist
an y effective treatm en t of th e leth al sepsis to date, a search for an effective
drug again st th is disease is in evitably n eeded.
Th e basic structure of alm ost all n atural lipid A-related com poun ds
con sists of β-(1→6)-glycosidically lin ked D-glucosam in e disacch aride 1,4′-
diph osph ate with th e α-con figuration of th e an om eric ph osph ate. Recog-
n ized to be essen tial for th e en dotoxic activity of lipid A species are also
Collect. Czech. Chem. Commun. 2006, Vol. 71, Nos. 11–12, pp. 1532–1548
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20061532

2,3,2′,3′-Tetra-O-tetradecylated Lipid A Mimic
1533
several, usually six fatty acid ch ain s per th e disacch aride3. Th e asym m etric,
(4+2) distribution of th ese fatty ch ain s (Fig. 1a), togeth er with th e oth er
structural features, results in an overall con ical sh ape of th e m olecule th at
h as been related to th e stron gly pro-in flam atory, LPS-agon istic activity of
lipid A 4. Less or n o pro-in flam atory properties are ch aracteristic for lipid A
species with respective (3+2) or (2+2) distribution of th e fatty ch ain s. Th e
in teraction of pyrogen ic lipid A species with th eir TLR4 bin din g site m edi-
ated by LBP, CD14 an d MD-2 is believed to in duce production of in -
flam atory m ediators, in cludin g cytokin es, e.g., TNFα 5–8. High levels of th e
released cytokin es cause severe sepsis.
Sin ce Sh iba an d Kusum oto’s total syn th esis of th e m ost toxic Escherichia
coli lipid A 9,10, m an y syn th etic lipid A m im ics h ave been prepared with
both E. coli LPS-agon istic an d an tagon istic properties. In som e of th em
D-glucose replaces D-glucosam in e at th e reducin g en d an d/or at th e n on -
reducin g en d11–13. Especially in spiratory for th e syn th eses of such bis-
1α,4′-O-ph osph orylated LPS-an tagon ists was fin din g of a n atural lipid
A-related com poun d (RsDPLA, Fig. 1b) isolated from Rhodobacter sphaeroides
an d beh avin g as an E. coli LPS-an tagon ist14,15. Based on th at, a com poun d
related to RsDPLA was developed as a poten t an ti-septicem ia drug can di-
date, E5564 (eritoran , Fig. 1c)16, wh ich passed ph ase II trial successfully.
Even tually, th e belief th at th e E. coli LPS-an tagon istic lipid A m im ics h ave
to con tain ph osph ates or oth er an ion ic groups was aban don ed wh en
Rhizobium sin-1 lipid A (Fig. 1d) was isolated17. Wh ile th is un usual m olecule
with (3+2) distribution of th e fatty ch ain s is devoided of 4′-O-ph osph oryl
group an d con tain s 2-am in o-2-deoxy-D-glucopyran olacton e in stead of a
usual D-glucosam in e-1α-ph osph ate un it, it sign ifican tly in h ibits E. coli LPS-
depen den t syn th esis of TNFα 18. A sligh tly weaker in h ibition was observed
with a syn th etic an alogue of Rhizobium sin-1 lipid A with (2+2) distribution
of th e fatty ch ain s18.
In our approach towards poten tial E. coli LPS-an tagon istic lipid A m im ics
we syn th esize sim plified structures of E. coli lipid A, in wh ich its en zym ati-
cally h ydrolyzable lin kages are substituted by th e en zym atically n on -
h ydrolyzable bon ds, usin g gen tiobiose as th e sugar m oiety of th e m im ics.
Th e adm ittan ce of th e substitution of th e D-glucosam in e un its (or 2,3-
diam in o-2,3-dideoxy-D-glucose un its, wh ich occur in som e lipid A struc-
tures as well19) of th e lipid A disacch aride with D-glucose un its is supported,
e.g., by th e observation th at th e substitution of th e Vi-capsular poly-
sacch aride in vaccin es with per-O-acetylated pectin h as n ot caused a ch an ge
in th e im m un ogen icity of th ese vaccin es20. (Vi-capsular polysacch aride dif-
fers from pectin by N-acetylation on C-2 an d O-acetylation on C-3.) Th us,
Collect. Czech. Chem. Commun. 2006, Vol. 71, Nos. 11–12, pp. 1532–1548

1534
Baráth, Petrušová, Hirsch, Petruš:
we h ave prepared m eth yl 6-O-(4,6-di-O-acetyl-2,3-di-O-tetradecyl-β-D-gluco-
pyran osyl)-4-O-(4-m eth oxyben zyl)-2,3-di-O-tetradecyl-α-D-glucopyran oside
– th e first tetra-O-alkylated m im ic of n ative lipid A m olecule syn th esized21
,
wh ich , after its O-deacetylation (Fig. 1e), exh ibits a rem arkable E. coli LPS-
an tagon istic activity22, th ough it does n ot con tain an y an ion ic group.
Sim ilar E. coli LPS-an tagon istic activities h ave been reported for its de-
O-4-m eth oxyben zylated derivative (Fig. 1f) an d a glycosylam in o-lin ked
an alogue (Fig. 1g)23. Th e an tagon istic activity sh own by th e last two disac-
ch arides specifically in volved th e TLR4 receptor.
In th is paper we describe an expeditious syn th esis of an oth er tetra-
O-fatty-alkylated gen tiobiose 18 an d its 4-O-(4-m eth oxyben zyl) precursor
17 as two oth er poten tial E. coli LPS-an tagon ists (Figs 1h , 1i), wh ich are,
OH
OH
O
O
H2O3PO
O
H2O3PO
O
O
O
O
O
O
HO
O
O
HO
HN
HN
O
O
O
O
O
O
HO
HN
HN
OPO3H2
O
OPO3H2
O
O
O
n-C7H15
n-C11H23
O
n-C11H23
n-C13H27
O
OH
n-C11H23
O
OH
O
OH
(CH )
n-C11H23
2 5 n-C7H15
n-C11H23
n-C11H23
n-C11H23
a
b
n-C6H13
OMe
O
OH
O
H2O3PO
O
HO
HO
O
O
O
O
O
O
HO
O
HN
O
HN
O
O
O
O
O
MeO
HN
OPO3H2
O
HO
HN
O
(CH2)9
n-C10H21
n-C7H15
(CH2)m
OH
(CH2)m
(CH2)m
O
O
OH
(CH2)m
n-C6H13
n-C11H23
(CH2)23
OH
c
m = 10, 12 and 14
d
OH
O
e: X = O, R1 = H, R2 = 4-MeO-C6H4, R3 = CH3
f: X = O, R1 = R2 = H, R3 = CH3
g: X = N(OMe), R1 = R2 = H, R3 = CH3
h: X = O, R1 = R3 = CH2-CH=CH2, R2 = 4-MeO-C6H4
i: X = O, R1 = R3 = CH2-CH=CH2, R2 = H
R1O
X
O
R2O
O
O
O
n-C14H29
O
n-C14H29
OR3
n-C14H29
n-C14H29
FIG. 1
Structures of som e n atural lipid A m olecules an d E. coli LPS-an tagon ists. a Escherichia coli lipid A;
b Rhodobacter sphaeroides lipid A (RsDPLA); c E5564 (eritoran , syn th etic E. coli LPS-an tagon ist);
d Rhizobium sin-1 lipid A; e–g gen tiobiose-based tetra-O-tetradecylated E. coli LPS-an tagon ists;
h, i com poun ds 17 an d 18
Collect. Czech. Chem. Commun. 2006, Vol. 71, Nos. 11–12, pp. 1532–1548

2,3,2′,3′-Tetra-O-tetradecylated Lipid A Mimic
1535
m oreover, substituted at th eir C-1α an d C-4′ h ydroxy groups with allyl
groups, m im ickin g ph osph ates an d offerin g possibilities of th eir furth er
tran sform ation s in to oth er groups.
A syn th etic approach based on couplin g n ucleoph ile 3 an d glycosyl do-
n or 12 was used for preparation of th e target disacch arides 17 an d 18. For
syn th esis of 3 (Sch em e 1), startin g allyl 4,6-O-(4-m eth oxyben zyliden e)-
α-D-glucopyran oside24 (1), wh ich already con tain s th e required allyl group,
was treated with NaH an d tetradecyl brom ide in DMF to give dieth er 2.
Its two fatty eth er ch ain s on C-2 an d C-3 were detected in th e 13C NMR
spectrum due to two n ew sign als of th e O-CH2 groups at two of th ree
ch em ical sh ifts (δ in ppm ) 73.5, 71.0 an d 70.8 (on e of th e sign als belon gs to
C-5) as well as due to th e sign als of th e oth er fatty alkyl ch ain carbon s at
1
δ 14.1–31.9. Th e O-alkylation appeared in th e H NMR spectrum of 2 as a
m ultiplet at δ 3.59–3.85 (togeth er with th e skeletal sign als of H-6a an d
H-6b), a broad doublet at δ 1.56, a broad sin glet at δ 1.26 an d a triplet at
δ 0.88. Reductive open in g of th e 4,6-O-(4-m eth oxyben zyliden e) protectin g
group of 2 with LiAlH4/AlCl3 afforded crystallin e n ucleoph ile 3 in an over-
all 76% yield based on 1. Diagn ostic for th e con version was th e appearan ce
of n ew sign als of th e (4-m eth oxyben zyl)m eth ylen e group at δ 77.1 an d at
δ 4.58 an d 4.82 in its respective 13C an d 1H NMR spectra, un der a sim ulta-
n eous disappearan ce of th e 4-m eth oxyben zyliden e acetal atom sign als of
th e reaction substrate at δ 101.9 an d δ 5.50. A broad sin glet at δ 2.02 in th e
1H NMR spectrum con firm ed also th e presen ce of a free OH group at C-6 of
n ucleoph ile 3.
O
O
O
O
1. NaH
DMF
p-MeO-C6H4
O
p-MeO-C6H4
O
OCH2CH=CH2
OCH2CH=CH2
n-OC14H29
2 (88%)
2. n-C14H29Br
HO
OH
n-H29C14O
1
HO
O
1. LiAlH4, THF
2. AlCl3, Et2O
p-MeO-C6H4-CH2O
n-H29C14
OCH2CH=CH2
n-OC14H29
3 (86%)
O
SCHEME 1
Collect. Czech. Chem. Commun. 2006, Vol. 71, Nos. 11–12, pp. 1532–1548

1536
Baráth, Petrušová, Hirsch, Petruš:
Th e startin g com poun d for th e syn th esis of glycosyl don or 12 (Sch em e 2)
was m eth yl 4,6-O-ben zyliden e-α-D-glucopyran oside25 (4), wh ich was alkyl-
ated by th e sam e procedure as com poun d 1; th e kn own fatty dieth er 5 was
obtain ed. Th e 4,6-O-ben zyliden e group of 5 was deprotected by h ydro-
gen olysis on Pd/C in a m ixture of CH2Cl2 an d MeOH to yield diol 6. Both
13C an d 1H NMR spectra of 6 docum en ted disappearan ce of th e sign als of
th e ben zyliden e arom atic an d acetal atom s un der sh iftin g th e skeletal C-4
an d C-6 sign als to th e values δ 69.4 an d 62.4, respectively. A subsequen t
on e-m ol tritylation of 6 with triph en ylm eth yl ch loride in dry pyridin e re-
sulted in protection of its C-6 h ydroxy group. Th is was eviden t again in
1
both 13C an d H NMR spectra of 6 as th e respective sets of th e ph en yl group
sign als at δ 127.0–143.9 an d δ 7.21–7.50. Treatm en t of com poun d 7 with
NaH an d allyl brom ide in DMF fin ally afforded th e design ed 4-O-allylation
of th e glycosyl don or precursor 8. In th e 13C NMR spectrum of 8, a set of
th ree ch aracteristic sign als at δ 134.8, 117.0 an d 78.0 appeared for th e
1
4-O-allyl group, wh ile th e m ost ch aracteristic in th e H NMR spectrum was
a fourth -order m ultiplet of th e allyl CH proton at δ 5.53. Stan dard
O
HO
O
O
1. NaH
DMF
Ph
O
O
Ph
O
H2, Pd/C
O
OMe
HO
OMe
OMe
EtOAc-MeOH
n-OC14H29
2. n-C14H29Br
n-H29C14
O
n-H29C14
O
n-OC14H29
HO
OH
5 (71%)
6 (94%)
4
Ph3CO
Ph3CO
O
O
Ph3CCl
C5H5N
1. NaH, DME
HO
OMe
CH2=CHCH2O
OMe
n-OC14H29
8 (86%)
2. CH2=CHCH2Br
n-H29C14
O
n-OC14H29
n-H29C14O
7 (80%)
HO
AcO
CH2=CHCH2O
n-H29C14
O
O
1. HOAc-H2O
Ac2O, HOAc
CH2=CHCH2O
OMe
n-OC14H29
OAc
2. NaOMe
MeOH
H2SO4
n-H29C14
O
O
n-OC14H29
9 (80%)
10 (77%)
AcO
AcO
O
O
N2H4·HOAc
DMF
DBU, CH2Cl2
CCl3CN
CH2=CHCH2O
OH
n-OC14H29
11 (80%)
CH2=CHCH2O
OC(=NH)CCl3
n-OC14H29
12 (90%)
n-H29C14
O
n-H29C14O
SCHEME 2
Collect. Czech. Chem. Commun. 2006, Vol. 71, Nos. 11–12, pp. 1532–1548

2,3,2′,3′-Tetra-O-tetradecylated Lipid A Mimic
1537
detritylation of 8 followed by acetolysis of in term ediate 9 gave crystallin e
1,6-diacetate 10. In addition to th e obvious skeletal carbon sign als, com -
poun d 10 con tain ed in its 13C NMR spectrum on ly sets of substitution sig-
n als of two tetradecyl eth er ch ain s at δ 74.0, 73.8 an d 14.1–32.0, two
acetoxy groups at δ 170.7, 169.3, 21.0 an d 20.8, an d allyl eth er group at
δ 134.5, 117.5 an d 77.2. Th e required structure of com poun d 10 was con -
1
firm ed also by its H NMR spectrum , wh ich con tain ed two ch aracteristic,
th ree-proton acetate sin glets at δ 2.11 an d 2.06, in addition to th e clear sig-
n al pattern s of th e O-allyl group at δ 5.88, 5.12–2.28 an d 4.02–4.35 as well
as th ose of O-tetradecyl groups, wh ich were sim ilar to th e sign al pattern s of
all th e previous di-O-tetradecylated precursors. Th e value of J1,2 = 3.4 Hz
ch aracteristic of α-glucopyran osyl an om er com pleted th e NMR structure as-
sign em en t of com poun d 10. All th e six syn th etic steps gave good to h igh
yields of th e correspon din g products so th at th e overall yield of 10 from 4
was 30%.
In itially, th e Koen igs–Kn orr or Helferich m eth od26, based on usin g a cor-
respon din g glycosyl brom ide th at was supposed to be prepared from
diacetate 10, was in ten ded to be used for couplin g with n ucleoph ile 3 as
th e key step of th e syn th esis of th e target lipodisacch aride 18. However,
a stan dard treatm en t of diacetate 10 with h ydrogen brom ide–acetic acid so-
lution 27 did n ot lead to its satisfactory, un am biguous con version to th e ex-
pected glycosyl brom ide, an d a m ixture of several products was form ed.
A probable reason for th e beh aviour was th e 4-O-allyl substitution of th e
brom ide precursor sin ce allyl eth ers are kn own to be un stable in th e pres-
en ce of HBr 28. Th erefore, th e in itial, glycosyl brom ide strategy was aban -
don ed an d a trich loroacetim idate altern ative, kn own as th e Sch m idt
m eth od29, was used for th e couplin g.
Hem iacetal 11, used for th e altern ative approach , was obtain ed in 80%
yield from com poun d 10 by regioselective O-deacetylation with h ydrazin e
acetate in DMF 30. Th e presen ce of a free h em iacetal OH group in its struc-
ture was eviden t from 1H NMR spectrum . In addition to th e ch aracteristic
C-6 acetate th ree-proton sin glet at δ 2.09, it con tain ed two well resolved
doublets of α- an d β-an om eric proton s at δ 5.28 an d 4.60 with couplin g
con stan ts J1,2 = 3.1 an d 7.6 Hz, respectively, an d with th eir total in tegral
in ten sity correspon din g to on e proton . Followin g activation of 11 with
trich loroaceton itrile in dich lorom eth an e usin g 1,8-diazabicyclo[5.4.0]-
un dec-7-en e (DBU) as a base afforded th e isolated 90% yield of α-glycosyl
im idate 12, ch aracterized by its couplin g con stan t J1,2 = 3.4 Hz (δ 6.43),
wh ich was im m ediately coupled with n ucleoph ile 3.
Collect. Czech. Chem. Commun. 2006, Vol. 71, Nos. 11–12, pp. 1532–1548

1538
Baráth, Petrušová, Hirsch, Petruš:
Two distin ct catalysts for couplin g of glycosyl acceptor 3 with im idate 12
were used. Th us, a com m on ly used stron gly acid catalyst, trim eth ylsilyl
trifluorom eth an esulfon ate (TMSOTf) in dich lorom eth an e led to preferen tial
form ation of α-(1→6)-glycosidic bon d between th e couplin g partn ers, disac-
ch aride 13 bein g a m ajor product of th is glycosylation (Sch em e 3). On th e
oth er h an d, a relatively weak Lewis acid catalyst, boron trifluoride eth erate
(BF3·OEt2) used in th e sam e solven t, supported th e h igh ly preferen tial for-
m ation of β-(1→6)-glycosidic bon d. As a result, th e required disacch aride 14
was isolated from th e reaction m ixture in a good (73%) yield. Moreover, it
h as to be also stressed th at, in con trast to th e couplin g catalysts used, th e
an om eric con figuration of glycosyl trich loroacetim idate in th e activation
step did n ot in fluen ce th e fin al an om eric con figuration of th e glycosidic
bon d in th e course of th ese glycosylation s. Th us, practically th e sam e ratios
an d yields of disacch arides 13 an d 14, depen din g on th e aforem en tion ed
catalysts on ly, were obtain ed wh en n ucleoph ile 3 was coupled eith er with
α-im idate 12 or with 6-O-acetyl-4-O-allyl-2,3-di-O-tetradecyl-β-D-gluco-
pyran osyl trich loroacetim idate, wh ich was gen erated in dich lorom eth an e
from trich loroaceton itrile an d K2CO3 as a base, i.e., un der th e con dition s
providin g β-D-glucopyran osyl trich loroacetim idates29.
BF3·OEt2
TMSOTf
15
+
16
3
+
12
13
+
14
13
+
14
(15)
+
16
40 h
24 h
CH2Cl2, 1 h
CH2Cl2, 2 h
68% 9%
<5% 58%
53% 7%
6%
73%
NeOMe
MeOH-Me2CO
NeOMe
18
17
16
14
MeOH-Me2CO
83%
86%
OCH2CH=CH2
O-n-C14H29
O-n-C14H29
R1O
CH2=CHCH2O
O
O
13
15 R1 = Ac, R2 = H
R
1 = Ac, R2 = p-MeO-C6H4
O
R2O
n-H29C14
O
O-n-C14H29
OCH2CH=CH2
O-n-C14H29
O-n-C14H29
R1O
O
14
15 R1 = Ac, R2 = H
16
1 = H, R2 = p-MeO-C6H4
17 R1 = R2 = H
R
1 = Ac, R2 = p-MeO-C6H4
O
CH2=CHCH2O
O
R
R2O
O-n-C14H29
n-H29C14
O
SCHEME 3
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2,3,2′,3′-Tetra-O-tetradecylated Lipid A Mimic
1539
Because of th e acid-labile 4-m eth oxyben zyl group presen t on th e C-4 car-
bon of n ucleoph ile 3 an d th e application of th e acid couplin g catalysts, also
th e reaction tim e of th e glycosylation step played an im portan t role. In th e
1-h glycosylation reaction of com poun ds 3 an d 12 in dich lorom eth an e
usin g BF3·OEt2 as catalyst, th e on ly products were disacch arides 13 an d 14.
However, wh en th e reaction tim e was prolon ged to 2 h , also disacch aride
15 was detected by TLC in th e reaction m ixture. After 24 h , th e rem oval of
th e 4-m eth oxyben zyl group from disacch aride 14 was com plete an d disac-
ch aride 15, obtain ed in a 58% isolated yield, was th e on ly sign ifican t prod-
uct presen t in th e fin al reaction m ixture. A sim ilar ph en om en on of de-O-4-
m eth oxyben zylation in a prolon ged reaction tim e was observed also wh en
TMSOTf was used as a catalyst for couplin g 3 an d 12 in dich lorom eth an e.
Th e last step of th e syn th esis was O-deacetylation of disacch arides 14 an d
16. Th is was accom plish ed by stan dard Zem plen deacetylation an d th e re-
spective targets, β-(1→6)-lin ked disacch arides 17 an d 18, were obtain ed in
th e 86 an d 83% yields, respectively.
Th e an om eric con figuration s of th e lipidodisacch arides 13–18 were as-
1
cribed by 13C an d H NMR spectroscopy. Th us, e.g., in th e 13C NMR spectra,
th e an om eric sign als of 13 were observed at δ 98.2 an d 95.2 an d th ose of 14
at δ 104.0 an d 95.2. Th e ch em ical sh ifts of th e form er sign als of both th e
an om eric carbon atom pairs are th e ch aracteristic values for th e respective
α an d β an om eric carbon atom s C-1′ of th e glycosidic bon d of 1→6 lin ked
glucopyran obioses31, wh ile both th e latter sign als belon g to th e an om eric
carbon s C-1 bearin g th e α-glycosidically lin ked allyl group in troduced in to
th e structures of lipidodisacch arides 13 an d 14 by th e couplin g sugar
n ucleoph ile 3. Even m ore stereoch em ically diagn ostic for th e determ in a-
tion of th e an om eric con figuration s were 1H NMR spectra with th eir well
resolved H-1 an d H-1′ sign als. Th us, th e value of couplin g con stan t J1′,2′
=
3.8 Hz (δH-1′ 4.75) in th e case of disacch aride 13 in dicates its isom altose
α-glycosidic bon d, an d correspon din gly, th e value of couplin g con stan t
J1′,2′ = 7.6 Hz (δH-1′ 4.32) in th e case of disacch aride 14 is typical of a gen tio-
biose β-glycosidic bon d. Th e allyl α-glycosidic con figuration of both disac-
ch arides 13 an d 14 is, in both cases, ch aracterized by th e vicin al couplin g
con stan ts J1,2 = 3.5 Hz (δH-1 4.95). An om eric NMR spectral ch aracteristics of
disacch arides 16–18 were very sim ilar to th ose observed for disacch aride 14.
Practically th e sam e were also th e an om eric NMR spectral ch aracteristics of
disacch arides 13 an d 15.
In th e NMR spectra of disacch arides 13–18, th e ch aracteristic pattern s of
all th eir oth er structural features, skeletal an d substitution al, were observed
as well. Again , th ey were very sim ilar to th ose described above in th e perti-
Collect. Czech. Chem. Commun. 2006, Vol. 71, Nos. 11–12, pp. 1532–1548

1540
Baráth, Petrušová, Hirsch, Petruš:
n en t section s of NMR structure proofs of th e syn th esis of th e m on o-
sacch aride precursors of th e disacch arides. In addition to th e NMR an alyses,
th e structures of all th e products of all th e con version s described were
ch ecked by evaluatin g th eir m olecular weigh ts by MALDI-TOF m ass spec-
trom etry as well as by elem en tal an alysis.
EXPERIMENTAL
All reaction s usin g air or m oisture-sen sitive reagen ts were con ducted in n itrogen or argon
atm osph ere. Com m ercial solven ts were dried an d distilled prior to use. Meltin g poin ts were
determ in ed on a Kofler h ot stage. Optical rotation s were m easured at 20 °C in CHCl3, at
c 1.0 g m l–1, on a Perkin –Elm er 141 polarim eter; [α]D values are given in 10–1 deg cm 2 g–1
.
Elem en tal an alyses were perform ed with a Fison s EA 1108 an alyser. All reaction s were
m on itored by TLC on glass plates precoated with silica gel (Kiesegel G, Merck) by sprayin g
th e ch rom atogram s with a 10% sulfuric acid in eth an ol an d ch arrin g th em on a h ot plate.
Preparative ch rom atograph y was perform ed on dry-packed silica gel (Kieselgel 60,
0.063–0.200 m m , Merck) equilibrated, prior to packin g, with 40% of th e m obile ph ase.
NMR spectra (δ, ppm ; J, Hz) were recorded at 20 °C on a Bruker Avan ce DPX 300 spectrom e-
ter (300.13 MHz, in tern al stan dard sodium 3-(trim eth ylsilyl)propan oate, δ 0.00 for 1H an d
75.47 MHz, in tern al stan dard m eth an ol, δ 50.15 for 13C). Hom o an d h etero n uclear correla-
tion spectroscopy experim en ts were perform ed as well. Mass spectra were obtain ed with a
Sh im adzu-Kratos An alytical MALDI TOF IV in strum en t (m atrix 2,5-dih ydroxyben zoic acid).
Allyl 4,6-O-(4-Meth oxyben zyliden e)-2,3-di-O-tetradecyl-α-D-glucopyran oside (2)
Allyl 4,6-O-(4-m eth oxyben zyliden e)-α-D-glucopyran oside24 (1) (1 g, 2.96 m m ol) was dis-
solved in DMF (40 m l), th e solution was cooled to 0 °C, an d NaH (0.6 g, 25 m m ol) was
slowly added to th e solution . Tetradecyl brom ide (6 m l, 20 m m ol) was th en added dropwise
over a period of 20 m in . Th e solution was warm ed slowly to 60 °C an d stirred overn igh t. Af-
ter coolin g to room tem perature, m eth an ol (5 m l) was slowly added to decom pose an excess
of sodium h ydride. Solven ts were th en evaporated un der reduced pressure an d th e residue
was diluted with eth yl acetate (50 m l), wash ed with saturated aqueous solution of citric acid
(3 × 35 m l), with water (3 × 35 m l), dried (an h ydrous Na2SO4) an d evaporated. Per-O-substi-
tuted sugar 2 (1.9 g, 88%) was isolated by crystallization from eth yl acetate–m eth an ol (1:3).
M.p. 63–65 °C; [α]D +36. For C45H78O7 (731.1) calculated: 73.93% C, 10.75% H; foun d:
73.77% C, 10.63% H. 1H NMR (CDCl3): 7.41 (d, 2 H, J = 8.4, Harom ); 6.88 (d, 2 H, Harom );
5.93 (m , 1 H, CH2=CHCH2-O); 5.50 (s, 1 H, CHAr); 5.19–5.38 (m , 2 H, CH2=CHCH2-O); 4.95
(d, 1 H, J1,2 = 3.7, H-1); 4.22 (dt, 1 H, J4,5 = J5,6a = 9.9, J5,6b = 4.7, H-5); 4.18 (dd, 1 H, J1
=
5.1, CH2=CHCHH-O); 4.08 (dd, 1 H, J1 = 6.6, J2 = 13.0, CH2=CHCHH-O); 3.55–3.89 (m , 7 H,
H-3, H-6a, H-6b, 2 CH2CH2-O); 3.80 (s, 3 H, CH3O); 3.48 (t, 1 H, J3,4 = 9.3, H-4); 3.33 (dd,
1 H, J2,3 = 9.3, H-2); 1.56 (bd, 4 H, 2 CH2CH2-O); 1.26 (bs, 44 H, 2 (CH2)11(CH2)2-O); 0.88 (t,
6 H, J = 7.2, 2 CH3CH2). 13C NMR (CDCl3): 159.6 (Carom ); 133.7 (CH2=CHCH2-O); 132.0
(2 Carom ); 129.8 (Carom ); 118.3 (CH2=CHCH2-O); 114.3 (2 Carom ); 101.9 (CHAr); 95.5 (C-1);
80.9 (C-4); 80.6 (C-2); 73.5 (C-3); 71.0, 70.8, 70.7 (C-5, 2 CH2CH2-O); 68.2 (CH2=CHCH2-O);
62.7 (C-6); 55.6 (CH3O); 31.9, 30.4, 30.0, 29.7, 29.4, 26.1, 22.1 (2 (CH2)12CH2-O); 14.1
(2 CH3CH2). MS (MALDI-TOF), m/z: 756.6 [M + Na+], 772.1 [M + K+].
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2,3,2′,3′-Tetra-O-tetradecylated Lipid A Mimic
1541
Allyl 4-O-(4-Meth oxyben zyl)-2,3-di-O-tetradecyl-α-D-glucopyran oside (3)
Solution of 1 M LiAlH4 in tetrah ydrofuran (11.5 m l) was added dropwise at room tem pera-
ture to a stirred solution of 2 (1.5 g, 2.05 m m ol) in a m ixture of dich lorom eth an e–dieth yl
eth er (1:2, 90 m l). Th en , a solution of AlCl3 (1.6 g, 12 m m ol) in dieth yl eth er (70 m l) was
added dropwise an d th e reaction m ixture was refluxed for an oth er 4 h . Th e m ixture was
cooled to room tem perature, diluted with eth yl acetate (300 m l), wash ed with water (3 ×
400 m l), dried with an h ydrous Na2SO4 an d con cen trated. Th e residue was purified on a
silica gel colum n with h exan e–eth yl acetate (3:1) as eluen t givin g pure com poun d 3 (1.3 g,
86.5%). M.p. 62–64 °C (eth yl acetate–m eth an ol, 1:3); [α]D +51. For C45H80O7 (733.1) calcu-
lated: 73.72% C, 11.00% H; foun d: 73.80% C, 10.87% H. 1H NMR (CDCl3): 7.27 (d, 2 H, J =
7.9, Harom ); 6.87 (d, 2 H, Harom ); 5.90 (m , 1 H, CH2=CHCH2-O); 5.15–5.35 (m , 2 H,
CH2=CHCH2-O); 4.92 (d, 1 H, J1,2 = 2.5, H-1); 4.82 (d, 1 H, J = 10.6, CHHAr); 4.58 (d, 1 H,
CHHAr); 4.14 (dd, 1 H, CH2=CHCHH-O); 4.04 (dd, 1 H, CH2=CHCHH-O); 3.52–3.93 (m ,
8 H, H-3, H-5, H-6a, H-6b, 2 CH2CH2-O); 3.79 (s, 3 H, CH3O); 3.42 (t, 1 H, J3,4 = J4,5 = 9.2,
H-4); 3.28 (dd, 1 H, J2,3 = 9.1, H-2); 2.02 (bs, 1 H, HOCH2); 1.60 (bd, 4 H, 2 CH2CH2-O);
1.26 (bs, 44 H, 2 (CH2)11(CH2)2-O); 0.88 (t, 6 H, J = 7.2, 2 CH3CH2). 13C NMR (CDCl3):
159.3 (Carom ); 133.7 (CH2=CHCH2-O); 130.4 (Carom ); 129.7 (2 Carom ); 118.0 (CH2=CHCH2-O);
113.8 (2 Carom ); 95.4 (C-1); 81.6 (C-3); 80.7 (C-2); 77.1(C-4); 74.5 (CH2Ar); 73.6, 70.7
(2 CH2CH2-O); 71.4 (C-5); 68.0 (CH2=CHCH2-O); 61.9 (C-6); 55.2 (CH3O); 31.9, 30.6, 30.0,
29.7, 29.4, 29.3, 26.2, 22.0, 22.6 (2 (CH2)12CH2-O); 14.0 (2 CH3CH2). MS (MALDI-TOF), m/z:
758.0 [M + Na+], 774.9 [M + K+].
Meth yl 2,3-Di-O-tetradecyl-4,6-O-ben zyliden e-α-D-glucopyran oside (5)
Com poun d 5 was syn th esized, startin g from m eth yl 4,6-O-ben zyliden e-α-D-glucopyran o-
side25 (4) (5 g, 17.7 m m ol), in th e sam e way as com poun d 2. Purification of th e obtain ed
crude product on silica gel colum n with h exan e–eth yl acetate (12:1) as eluen t gave pure
com poun d 5 (9 g, 71.5%). M.p. 79–80.5 °C; [α]D +26 (lit.21 m .p. 79.5–80.5 °C; [α]D +26).
Meth yl 2,3-Di-O-tetradecyl-α-D-glucopyran oside (6)
To a suspen sion of 5 (5 g, 7.4 m m ol) in eth yl acetate (140 m l) an d m eth an ol (210 m l) was
added 10% Pd/C (0.15 g) in m eth an ol (25 m l) an d th e m ixture was stirred at room tem pera-
ture un der h ydrogen for 2 h . After filtration an d evaporation of th e solven ts, th e crude
m aterial was purified on silica gel colum n with h exan e–eth yl acetate (3:1) as an eluen t to
obtain pure 6 (4.08 g, 94%). M.p. 75–76 °C (m eth an ol); [α]D +45. For C35H70O6 (586.9) cal-
culated: 71.62% C, 12.02% H; foun d: 71.55% C, 11.93% H. 1H NMR (CDCl3): 4.80 (d, 1 H,
J1,2 = 3.3, H-1); 3.76–3.96 (m , 3 H, H-5, H-6a, H-6b); 3.48–3.65 (m , 6 H, H-3, H-4,
2 CH2CH2-O); 3.42 (s, 3 H, CH3O); 3.28 (dd, 1 H, J2,3 = 9.0, H-2); 2.53 (bs, 1 H, OH); 2.03
(bs, 1 H, OH); 1.58 (bd, 4 H, 2 CH2CH2-O); 1.26 (bs, 44 H, 2 (CH2)11(CH2)2-O); 0.88 (t, 6 H,
2 CH3CH2). 13C NMR (CDCl3): 98.1 (C-1); 80.4 (C-3); 80.2 (C-2); 73.1 (C-4); 71.7, 70.5, 67.6
(C-5, 2 CH2CH2-O); 63.3 (C-6); 55.2 (OCH3); 31.7, 31.4, 30.2, 30.1, 30.0, 29.9, 29.7, 29.2,
26.1, 26.0, 22.7 (2 (CH2)12CH2-O); 13.8 (2 CH3CH2). MS (MALDI-TOF), m/z: 611.3 [M + Na+],
627.9 [M + K+].
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1542
Baráth, Petrušová, Hirsch, Petruš:
Meth yl 2,3-Di-O-tetradecyl-6-O-(triph en ylm eth yl)-α-D-glucopyran oside (7)
Com poun d 6 (6 g, 10.2 m m ol) was dissolved in dry pyridin e (18 m l), trityl ch loride (4.3 g,
15.4 m m ol) was added an d th e reaction m ixture was stirred at 90 °C for 3 h . Th e m ixture
was poured to an ice-cold solution of NaHCO3, th e yellow precipitate was collected by filtra-
tion , dissolved in ch loroform (200 m l) an d successively wash ed with an ice-cold 1 M H2SO4,
saturated aqueous NaHCO3 an d water (3 × 50 m l each ). Th e organ ic layer was dried with an -
h ydrous Na2SO4, filtered an d con cen trated in vacuo. Th e crude m aterial was purified on sil-
ica gel colum n first eluted with toluen e followed by a toluen e–aceton e (10:1) m ixture to
afford com poun d 7 (6.8 g, 80%) as a colorless syrup. [α]D +25. For C54H84O6 (829.2) calcu-
lated: 78.21% C, 10.21% H; foun d: 78.08% C, 9.98% H. 1H NMR (CDCl3): 7.47 (d, 6 H,
H
arom ); 7.18–7.32 (m , 9 H, Harom ); 4.82 (d, 1 H, J1,2 = 3.4, H-1); 3.54–3.92 (m , 8 H, H-4, H-5,
H-6a, H-6b, 2 CH2CH2-O); 3.44 (s, 3 H, CH3O); 3.26–3.42 (m , 2 H, H-2, H-3); 1.57 (bd, 4 H,
2 CH2CH2-O); 1.26 (bs, 44 H, 2 (CH2)11(CH2)2-O); 0.82 (t, 6 H, 2 CH3CH2). 13C NMR
(CDCl3): 143.9, 128.7, 127.8, 127.0 (Carom ); 98.0 (C-1); 86.8 (CPh 3); 81.2, 80.7 (C-2, C-3);
77.2 (C-4); 73.7, 71.3, 70.0 (C-5, 2 CH2CH2-O); 63.9 (C-6); 55.0 (CH3O); 31.9, 30.4, 30.1,
29.7, 29.4, 26.1, 26.0, 22.7 (2 (CH2)12CH2-O); 14.1 (2 CH3CH2). MS (MALDI-TOF), m/z: 853.8
[M + Na+], 877.1 [M + K+].
Meth yl 4-O-Allyl-2,3-di-O-tetradecyl-6-O-(triph en ylm eth yl)-α-D-glucopyran oside (8)
To a solution of 7 (3 g, 3.6 m m ol) in dry 1,2-dim eth oxyeth an e (21 m l) was added NaH
(0.45 g, 18.7 m m ol) at 0 °C. Th en allyl brom ide (0.85 m l, 9.8 m m ol) was added dropwise
an d th e m ixture was stirred at room tem perature for 2 h . Meth an ol (10 m l) was added an d,
after coolin g, th e reaction m ixture was n eutralized with dilute acetic acid (1:1) an d evapo-
rated. Th e residue was diluted with ch loroform an d water, an d th e water layer was wash ed
with CHCl3 (3 × 100 m l). Th e collected organ ic layers were dried with an h ydrous Na2SO4,
evaporated an d th e residue was purified on a silica gel colum n with toluen e as an eluen t
givin g pure syrup 8 (2.7 g, 86%). [α]D +41. For C57H88O6 (869.3) calculated: 78.75% C,
10.20% H; foun d: 78.86% C, 10.07% H. 1H NMR (CDCl3): 7.49 (d, 6 H, Harom ); 7.17–7.32
(m , 9 H, Harom ); 5.53 (m , 1 H, CH2=CHCH2-O); 4.90–5.01 (m , 2 H, CH2=CHCH2-O); 4.86 (d,
1
H, J1,2 = 3.43, H-1); 4.09 (dd, 1 H, CH2=CHCHH-O); 3.73–3.85 (m , 2 H, H-4,
CH2=CHCHH-O); 3.54–3.73 (m , 5 H, H-5, 2 CH2CH2-O); 3.44 (s, 3 H, OCH3); 3.33–3.42 (m ,
3 H, H-2, H-3, H-6a); 3.12 (dd, 1 H, J5,6b = 4.4, J6a,6b = 10.2, H-6b); 1.58 (bd, 4 H,
2 CH2CH2-O); 1.25 (bs, 44 H, 2 (CH2)11(CH2)2-O); 0.88 (t, 6 H, 2 CH3CH2).13C NMR
(CDCl3): 144.0 (Carom ); 134.8 (CH2=CHCH2-O); 128.8, 127.7, 126.9 (Carom ); 116.7
(CH2=CHCH2-O); 97.9 (C-1); 86.2 (CPh 3); 81.8 (C-3); 80.8 (C-2); 78.0 (C-4); 73.8, 71.7
(2 CH2CH2-O); 73.7 (CH2=CHCH2-O); 70.2 (C-5); 62.5 (C-6); 54.8 (OCH3); 31.9, 30.5, 30.1,
29.7, 29.3, 26.2, 26.0, 22.7 (2 (CH2)12CH2-O); 14.1 (2 CH3CH2). MS (MALDI-TOF), m/z: 894.2
[M + Na+], 911.1 [M + K+].
Meth yl 4-O-Allyl-2,3-di-O-tetradecyl-α-D-glucopyran oside (9)
Com poun d 8 (5 g, 5.75 m m ol) was dissolved in acetic acid (25 m l), water (7 m l) was added
dropwise at 90 °C an d th e m ixture was stirred at th is tem perature for 5 h . Th e reaction m ix-
ture was evaporated with a water–toluen e m ixture (3 × 30 m l) an d th e residue, free of acetic
acid, was diluted with m eth an ol (50 m l). Several drops of 1 M solution of MeONa were
added an d th e m ixture was stirred at room tem perature for 30 m in , n eutralized with Dowex
Collect. Czech. Chem. Commun. 2006, Vol. 71, Nos. 11–12, pp. 1532–1548

2,3,2′,3′-Tetra-O-tetradecylated Lipid A Mimic
1543
50W (H+), filtered an d con cen trated. Th e crude m aterial obtain ed was recrystallized from
aceton e–diisopropyl eth er to provide pure com poun d 9 (2.9 g, 80%). M.p. 50–51 °C; [α]D
+61. For C38H74O6 (627.0) calculated: 72.88% C, 11.91% H; foun d: 72.80% C, 11.80% H.
1H NMR (CDCl3): 5.91 (m , 1 H, CH2=CHCH2-O); 5.14–5.32 (m , 2 H, CH2=CHCH2-O); 4.75
(d, 1 H, J1,2 = 3.5, H-1); 4.33 (dd, 1 H, CH2=CHCHH-O); 4.13 (dd, 1 H, CH2=CHCHH-O);
3.56–3.84 (m , 8 H, H-3, H-5, H-6a, H-6b, 2 CH2CH2-O); 3.39 (s, 3 H, CH3O); 3.31 (t, 1 H,
J3,4 = J4,5 = 9.4, H-4); 3.23 (dd, 1 H, J2,3 = 9.6, H-2); 1.75 (bs, 1 H, OH); 1.58 (bd, 4 H,
2 CH2CH2-O); 1.25 (bs, 44 H, 2 (CH2)11(CH2)2-O); 0.88 (t, 6 H, 2 CH3CH2). 13C NMR
(CDCl3): 134.8 (CH2=CHCH2-O); 117.0 (CH2=CHCH2-O); 98.1 (C-1); 81.5 (C-3); 80.7 (C-2);
77.4 (C-4); 73.8 (CH2=CHCH2-O); 73.7, 71.8 (2 CH2CH2-O); 70.6 (C-5); 62.0 (C-6); 55.1
(CH3O); 31.9, 30.5, 30.1, 29.7, 29.5, 29.3, 26.3, 26.2 (2 (CH2)12CH2-O); 14.1 (2 CH3CH2). MS
(MALDI-TOF), m/z: 651.8 [M + Na+], 666.9 [M + K+].
1,6-Di-O-acetyl-4-O-allyl-2,3-di-O-tetradecyl-α-D-glucopyran ose (10)
A stirred suspen sion of 9 (3.5 g, 5.58 m m ol) in acetic acid (50 m l) an d acetic an h ydride
(75 m l) was cooled to –20 °C an d con cen trated H2SO4 (0.5 m l) was added dropwise over a
period of 20 m in . Th e m ixture was warm ed slowly to room tem perature, stirred for 4 h an d
th en poured in to ice water (600 m l). Th e suspen sion was stirred for 3 h an d com poun d 10
was extracted from th e m ixture with ch loroform (3 × 100 m l). Th e com bin ed extract was
wash ed with a saturated aqueous NaHCO3 solution (3×), water (2×), dried over an h ydrous
Na2SO4 an d con cen trated in vacuo. Th e residue was purified on silica gel with a h exan e–
eth yl acetate (10:1) eluen t yieldin g pure com poun d 10 (3 g, 77%), wh ich crystallized from
an diisopropyl eth er–h exan e (1:1) m ixture. M.p. 44–45 °C; [α]D +54. For C41H76O8 (697.0)
calculated: 70.64% C, 10.99% H; foun d: 70.85% C, 10.95% H. 1H NMR (CDCl3): 6.25 (d,
1 H, J1,2 = 3.4, H-1); 5.88 (m , 1 H, CH2=CHCH2-O); 5.12–5.28 (m , 2 H, CH2=CHCH2-O);
4.18–4.37 (m , 3 H, CH2=CHCHH-O, H-6a, H-6b); 4.07 (dd, 1 H, CH2=CHCHH-O); 3.27–3.86
(m , 8 H, H-2, H-3, H-4, H-5, 2 CH2CH2-O); 2.11 (s, 3 H, CH3CO); 2.06 (s, 3 H, CH3CO);
1.40–1.64 (m , 4 H, 2 CH2CH2-O); 1.24 (bs, 44 H, 2 (CH2)11(CH2)2-O); 0.87 (s, 6 H,
2 CH3CH2). 13C NMR (CDCl3): 170.7, 169.3 (2 CH3CO); 134.5 (CH2=CHCH2-O); 117.5
(CH2=CHCH2-O); 89.7 (C-1); 81.3 (C-3); 79.5 (C-2); 77.2 (C-4); 74.0, 71.6 (2 CH2CH2-O);
73.8 (CH2=CHCH2-O); 71.0 (C-5); 62.9 (C-6); 32.0, 30.6, 30.0, 29.7, 29.5, 29.4, 26.2, 26.0
(2 (CH2)12CH2-O); 21.0, 20.8 (2 CH3CO); 14.1 (2 CH3CH2). MS (MALDI-TOF), m/z: 721.9
[M + Na+], 738.0 [M + K+].
6-O-Acetyl-4-O-allyl-2,3-di-O-tetradecyl-α,β-D-glucopyran ose (11)
Com poun d 10 (1 g, 1.43 m m ol) was dissolved in dry DMF (50 m l) an d h ydrazin e acetate
(0.21 g, 2.83 m m ol) was added. Th e reaction m ixture was stirred at room tem perature in
in ert atm osph ere an d, after 90 m in , it was diluted with eth yl acetate (50 m l). Th e organ ic
layer was successively wash ed with a saturated aqueous NaHCO3 solution , 0.1 M HCl, water
(2×), th en dried over an h ydrous Na2SO4 an d con cen trated in vacuo. Th e residue crystallized
from eth an ol givin g pure 11 (0.75 g, 80%). M.p. 67–68 °C; [α]D +29. For C39H74O7 (655.0)
calculated: 71.51% C, 11.39% H; foun d: 71.43% C, 11.22% H. Ch aracteristic NMR data for
both an om ers: 1H NMR (CDCl3): 5.81–5.97 (m , 1 H, (CH2=CHCH2-O)α,β); 5.28 (d, 0.63 H,
J1,2 = 3.1, H-1α); 5.13–5.25 (m , 2 H, CH2=CHCH2-O); 4.60 (d, 0.37 H, J1,2 = 7.6, H-1β);
3.00–4.38 (m , 12 H CH2=CHCH2-O, H-2, H-3, H-4, H-5, H-6a, H-6b, 2 CH2CH2-O); 2.09 (s,
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Baráth, Petrušová, Hirsch, Petruš:
3 H, CH3CO); 1.52–1.66 (m , 4 H, 2 CH2CH2-O); 1.24 (bs, 44 H, 2 (CH2)11(CH2)2-O); 0.87 (t,
H,
CH3CH2). 13C NMR (CDCl3): 170.9 (CH3CO); 134.6 (CH2=CHCH2-O); 117.2
6
2
(CH2=CHCH2-O); 97.3 (C-1β); 91.1 (C-1α); 81.3 (C-3α); 80.6 (C-2α); 77.1 (C-4α); 73.8, 73.7,
73.0, 71.6 (C-5α, CH2=CHCH2-O, 2 CH2CH2-O); 63.2 (C-6α); 31.9, 30.5, 30.0, 29.7, 29.5,
29.4, 26.2, 26.0 (2 (CH2)12CH2-O); 20.8 (CH3CO); 14.1 (2 CH3CH2). MS (MALDI-TOF), m/z:
678.9 [M + Na+], 696.0 [M + K+].
6-O-Acetyl-4-O-allyl-2,3-di-O-tetradecyl-1-O-trich loroacetim idoyl-α-D-glucopyran ose (12)
A m ixture of com poun d 11 (0.5 g, 0.76 m m ol), trich loroaceton itrile (0.6 m l, 5.98 m m ol)
an d 1,8-diazabicyclo[5.4.0]un dec-7-en e (DBU; 0.09 m l, 0.6 m m ol) in dry CH2Cl2 (40 m l) was
stirred un der argon at room tem perature for 90 m in . Th e solution was evaporated an d im -
m ediately ch rom atograph ed on a silica gel colum n with a h exan e–eth yl acetate (5:1) eluen t,
con tain in g also 0.3% of trieth ylam in e, providin g im idate 12 (0.55g, 90%). Ch aracteristic sig-
n als for α an om er: 1H NMR (CDCl3): 8.42 (s, 1 H, NH); 6.43 (d, 1 H, J1,2 = 3.4, H-1); 5.79
(m , 1 H, CH2=CHCH2-O); 5.08–5.50 (m , 2 H, CH2=CHCH2-O); 5.04 (t, 1 H, J3,4 = J4,5 = 9.7,
H-4); 3.48–4.29 (m , 11 H, H-2, H-3, H-5, H-6a, H-6b, CH2=CHCH2-O, 2 CH2CH2-O); 2.05 (s,
3 H, CH3CO); 1.41–1.50 (m , 4 H, 2 CH2CH2-O); 1.11–1.45 (m , 44 H, 2 (CH2)11(CH2)2-O);
0.79–0.96 (m , 6 H, 2 CH3CH2). MS (MALDI-TOF), m/z: 824.5 [M + Na+], 841.8 [M + K+].
Allyl 6-O-(6-O-Acetyl-4-O-allyl-2,3-di-O-tetradecyl-β-D-glucopyran osyl)-
4-O-(4-m eth oxyben zyl)-2,3-di-O-tetradecyl-α-D-glucopyran oside (14)
Procedure A. Im idate 12 (0.5 g, 0.63 m m ol) an d n ucleoph ile 3 (0.4 g, 0.54 m m ol) were dis-
solved in dry CH2Cl2 (15 m l) an d th e m ixture was stirred at room tem perature un der argon
in th e presen ce of 4A m olecular sieves for 15 m in . Th en th e solution was cooled to –20 °C
an d TMSOTf (55 µl, 0.3 m m ol) in CH2Cl2 (1 m l) was added dropwise. Th e m ixture was left
stan din g to warm up to room tem perature, stirred for an oth er 2 h , filtered, wash ed with sat-
urated solution of NaHCO3 an d water, dried over an h ydrous Na2SO4 an d con cen trated in
vacuo. Th e residue was ch rom atograph ed on a silica gel colum n . Elution with h eptan e–eth yl
acetate (10:1) gave first allyl 6-O-(6-O-acetyl-4-O-allyl-2,3-di-O-tetradecyl-α-D-gluco-
pyran osyl)-4-O-(4-m eth oxyben zyl)-2,3-di-O-tetradecyl-α-D-glucopyran oside (13; 0.5 g, 68%).
M.p. 58–60 °C (eth yl acetate); [α]D +52. For C84H152O13 (1370.1) calculated: 73.63% C,
11.18% H; foun d: 73.54% C, 11.26% H. 1H NMR (CDCl3): 7.24 (d, 2 H, J = 7.9, Harom ); 6.87
(d, 2 H, Harom ); 5.82–5.98 (m , 2 H, 2 CH2=CHCH2-O); 5.10–5.36 (m , 4 H, 2 CH2=CHCH2-O);
4.95 (d, 1 H, J1,2 = 3.4, H-1); 4.86 (d, 1 H, J1,2 = 3.8, H-1′); 4.82 (d, 1 H, J = 10.7, CHHAr);
4.56 (d, 1 H, CHHAr); 3.97–4.37 (m , 9 H, H-4′, H-5, H-5′, H-6′a, H-6′b, 2 CH2=CHCH2-O);
3.78 (s, 3 H, OCH3); 3.38–3.92 (m , 12 H, H-3′, H-4, H-6a, H-6b, 4 CH2CH2-O); 3.18–3.30 (m ,
3 H, H-2, H-2′, H-3); 2.07 (s, 3 H, CH3CO); 1.54 (bs, 8 H, 4 CH2CH2-O); 1.29 (bs, 88 H,
4 (CH2)11(CH2)2-O); 0.88 (t, 12 H, 4 CH3CH2). 13C NMR (CDCl3): 170.8 (CH3CO); 159.3
(Carom ); 134.6, 133.8 (2 CH2=CHCH2-O); 129.4 (2 Carom ); 128.8 (Carom ); 118.2, 117.3
(2 CH2=CHCH2-O); 113.8 (2 Carom ); 98.2 (C-1′); 95.2 (C-1); 84.7, 82.0, 80.9, 80.5 (C-2, C-3,
C-2′, C-3′); 77.2 (CH2Ar); 74.8, 73.9, 73.8, 73.5, 73.2, 73.2, 72.8, 71.0 (C-5, C-5′,
2 CH2=CHCH2-O, 4 CH2CH2-O); 70.3, 69.0 (C-4, C-4′); 67.9 (C-6); 63.2 (C-6′); 31.9, 30.5,
30.0, 29.7, 29.5, 29.4, 26.2, 26.0 (4 (CH2)12CH2-O); 20.8 (CH3CO); 14.1 (2 CH3CH2); 13.9
(2 CH3CH2). MS (MALDI-TOF), m/z: 1394.5 [M + Na+], 1411.7 [M + K+].
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2,3,2′,3′-Tetra-O-tetradecylated Lipid A Mimic
1545
Secon d fraction con tain ed disacch aride 14 (70 m g, 9.5%). M.p. 63–65 °C; [α]D +28. For
C
84H152O13 (1370.1) calculated: 73.63% C, 11.18% H; foun d: 73.42% C, 10.98% H. 1H NMR
(CDCl3): 7.24 (d,
2 H, J = 7.9, Harom ); 6.87 (d, 2 H, Harom ); 5.82–5.97 (m , 2 H,
2 CH2=CHCH2-O); 5.13–5.35 (m , 4 H, 2 CH2=CHCH2-O); 4.95 (d, 1 H, J1,2 = 3.5, H-1); 4.82
(d, 1 H, J = 10.6, CHHAr); 4.58 (d, 1 H, CHHAr); 4.32 (d, 1 H, J1′,2′ = 7.6, H-1′); 4.00–4.24
(m ,
2 CH2=CHCH2-O, 4 CH2CH2-O); 3.75 (s, 3 H, OCH3); 3.02–3.31 (m , 4 H, H-2, H-2′, H-3,
H-3′); 2.07 (s, H, CH3CO); 1.41–1.50 (m , H, CH2CH2-O); 1.30 (bs, 88 H,
8 H, H-4, H-4′, H-5, H-5′, H-6a, H-6b, H-6′a, H-6′b); 3.41–3.95 (m , 12 H,
3
8
4
4 (CH2)11(CH2)2-O); 0.90 (t, 12 H, 4 CH3CH2). 13C NMR (CDCl3): 170.8 (CH3CO); 159.3
(Carom ); 134.6, 133.8 (2 CH2=CHCH2-O); 130.6 (Carom ); 129.4 (2 Carom ); 118.2, 117.3
(2 CH2=CHCH2-O); 113.8 (2 Carom ); 104.0 (C-1′); 95.2 (C-1); 84.7, 82.0, 80.9, 80.5 (C-2, C-3,
C-2′, C-3′); 77.2 (CH2Ar); 74.8, 73.9, 73.8, 73.5, 73.2, 73.1, 72.8, 71.0 (C-5, C-5′,
2 CH2=CHCH2-O, 4 CH2CH2-O); 70.3, 69.0 (C-4, C-4′); 67.9 (C-6); 63.2 (C-6′); 31.9, 30.5,
30.0, 29.7, 29.5, 29.4, 26.2, 26.0 (4 (CH2)12CH2-O); 20.8 (CH3CO); 14.1 (2 CH3CH2); 13.9
(2 CH3CH2). MS (MALDI-TOF), m/z: 1394.5 [M + Na+], 1411.8 [M + K+].
Procedure B. Im idate 12 (0.5 g, 0.63 m m ol) an d n ucleoph ile 3 (0.4 g, 0.54 m m ol) were dis-
solved in dry dich lorom eth an e (15 m l) an d th e m ixture was stirred at room tem perature un -
der argon in th e presen ce of a 4A m olecular sieve (0.4 g) for 15 m in . Th en th e solution was
cooled to 0 °C an d BF3·OEt2 (0.13 m l) in a m ixture of dieth yl eth er (0.5 m l) an d CH2Cl2
(0.5 m l) was added dropwise. Th e m ixture was left stan din g to warm up to room tem pera-
ture an d stirred for 1 h . Th en NaHCO3 (0.4 g) was added an d, after stirrin g for 10 m in , th e
m ixture was filtered, wash ed with CH2Cl2 an d evaporated. Th e residue, con tain in g
disacch arides 13 an d 14 in approxim ate ratio 1:10 (TLC), was ch rom atograph ed on a silica
gel colum n with h eptan e–eth yl acetate (10:1) as eluen t givin g pure 13 (45 m g, 6%) an d 14
(0.55 g, 73.5%).
Allyl 6-O-(6-O-Acetyl-4-O-allyl-2,3-di-O-tetradecyl-β-D-glucopyran osyl)-
2,3-di-O-tetradecyl-α-D-glucopyran oside (16)
Procedure C. Except for th e 2-h addition al reaction tim e after warm in g th e reaction m ix-
ture to room tem perature, wh ich was in th is case 40 h , procedure A for preparation of
disacch arides 13 an d 14 was followed with th e sam e quan tities of th e reagen ts. Th e syrupy
residue th us obtain ed was ch rom atograph ed on a silica gel colum n with a h eptan e–eth yl
acetate (5:1) elution . First fraction con tain ed allyl 6-O-(6-O-acetyl-4-O-allyl-2,3-di-O-tetra-
decyl-α-D-glucopyran osyl)-2,3-di-O-tetradecyl-α-D-glucopyran oside (15; 0.4 g, 53%). M.p.
59–62 °C (aceton e); [α]D +43. For C76H144O12 (1249.9) calculated: 73.03% C, 11.61% H;
foun d: 73.33% C, 11.90% H. 1H NMR (CDCl3): 5.80–6.00 (m , 2 H, 2 CH2=CHCH2-O);
5.11–5.40 (m , 4 H, 2 CH2=CHCH2-O); 4.91 (d, 2 H, J1,2 = J1′,2′ = 3.5, H-1, H-1′); 3.22–4.36
(m , 24 H, H-2–H-5, H-6a, H-6b, H-2′–H-5′, H-6′a, H-6′b, 2 CH2=CHCH2-O, 4 CH2CH2-O);
2.08 (s, 3 H, CH3CO); 1.58 (bs, 8 H, 4 CH2CH2-O); 1.26 (bs, 88 H, 4 (CH2)11(CH2)2-O);
0.82–0.96 (m , 12 H,
4
CH3CH2). 13C NMR (CDCl3): 170.8 (CH3CO); 134.7, 133.8
(2 CH2=CHCH2-O); 118.4, 117.0 (2 CH2=CHCH2-O); 97.3, 95.0 (C-1, C-1′); 81.6, 81.0, 80.4,
80.3, 77.2, 73.7 (3 C); 72.1, 71.5, 71.1, 69.6, 68.7, 68.3, 68.0 (C-2–C-6, C-2′–C-5′,
2 CH2=CHCH2-O, 4 CH2CH2-O); 63.2 (C-6′); 31.9, 30.6, 30.5, 30.0, 29.7, 29.5, 29.4, 26.2,
26.1 (4 (CH2)12CH2-O); 20.9 (CH3CO); 14.1 (4 CH3CH2). MS (MALDI-TOF), m/z: 1273.7 [M +
Na+], 1290.1 [M + K+].
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1546
Baráth, Petrušová, Hirsch, Petruš:
Secon d fraction con tain ed disacch aride 16 (50 m g, 7%). M.p. 63–64 °C (aceton e); [α]D
+19. For C76H144O12 (1249.9) calculated: 73.03% C, 11.61% H; foun d: 72.88% C, 11.74% H.
1H NMR (CDCl3): 5.82–5.98 (m ,
2
H,
2
CH2=CHCH2-O); 5.13–5.35 (m ,
4
H,
2 CH2=CHCH2-O); 4.96 (d, 1 H, J1,2 = 3.5, H-1); 4.32 (d, 1 H, J1′,2′ = 7.6, H-1′); 4.25–4.36 (m ,
2 H, CH2=CHCHH-O, H-6a); 4.14–4.25 (m , 2 H, H-6b, H-6′a); 4.02–4.14 (m , 3 H, H-4, H-6′b,
CH2=CHCHH-O); 3.45–3.95 (13 H, H-4′, H-5, H-5′, CH2=CHCH2-O, 4 CH2CH2-O); 3.07–3.45
(m , 4 H, H-2, H-2′, H-3, H-3′); 2.07 (s, 3 H, CH3CO); 1.57 (bs, 8 H, 4 CH2CH2-O); 1.39–1.50
(m , 88 H, 4 (CH2)11(CH2)2-O); 0.88 (t, 12 H, 4 CH3CH2). 13C NMR (CDCl3): 170.8 (CH3CO);
134.6, 133.8 (2 CH2=CHCH2-O); 118.2, 117.3 (2 CH2=CHCH2-O); 104.0 (C-1′); 95.2 (C-1);
84.7, 82.0, 81.0, 80.5 (C-2, C-3, C-2′, C-3′); 77.4, 73.9, 73.8, 73.5, 73.2, 72.8, 71.1, 71.0 (C-5,
C-5′, 2 CH2=CHCH2-O, 4 CH2CH2-O); 70.2, 69.0 (C-4, C-4′); 67.9 (C-6); 63.2 (C-6′); 31.9,
31.4, 30.5, 30.4, 30.2, 30.0, 29.7, 29.4, 26.2, 26.1, 22.7 (4 (CH2)12CH2-O); 20.8 (CH3CO);
14.1 (4 CH3CH2). MS (MALDI-TOF), m/z: 1274.1 [M + Na+], 1290.2 [M + K+].
Procedure D. A solution of im idate 12 (0.3 g, 0.38 m m ol) an d n ucleoph ile 3 (0.25 g,
0.34 m m ol) in CH2Cl2 was stirred un der argon in th e presen ce of a 4A m olecular sieves
(0.3 g). After coolin g to 0 °C, a solution of BF3·OEt2 (80 µl) in dieth yl eth er (0.4 m l) an d
CH2Cl2 (0.4 m l) was added dropwise. Th e reaction m ixture was th en left stan din g to warm
to room tem perature an d stirred for 24 h . NaHCO3 (0.3 g) was added an d th e m ixture was
stirred for 15 m in , filtered, wash ed with CH2Cl2 an d con cen trated. Th e residue was purified
on a silica gel colum n with h eptan e–eth yl acetate (5:1) an d pure disacch aride 16 (0.25 g,
58.7%) was obtain ed.
Allyl 6-O-(4-O-Allyl-2,3-di-O-tetradecyl-β-D-glucopyran osyl)-4-O-(4-m eth oxyben zyl)-
2,3-di-O-tetradecyl-α-D-glucopyran oside (17)
Meth an olic 1 M sodium m eth oxide (0.2 m l) was added to a solution of disacch aride 14 (0.3 g,
0.22 m m ol) in dry aceton e (2 m l) an d m eth an ol (10 m l) an d th e solution was stirred at
room tem perature for 4 h . After n eutralization with Dowex 50W (H+) resin , filtration an d
con cen tration , th e residue was tran sferred on to a silica gel colum n an d eluted with h eptan e–
eth yl acetate (7:1). Com poun d 16 (0.25 g, 86%) was obtain ed as a colorless syrup. [α]D +28.
For C82H150O12 (1328.0) calculated: 74.16% C, 11.39% H; foun d: 74.00% C, 11.45% H.
1H NMR (CDCl3): 7.24 (d, 2 H, J = 8.3, Harom ); 6.89 (d, 2 H, Harom ); 5.82–5.97 (m , 2 H,
2 CH2=CHCH2-O); 5.13–5.35 (m , 4 H, 2 CH2=CHCH2-O); 4.95 (d, 1 H, J1,2 = 3.3, H-1); 4.82
(d, 1 H, J = 10.6, CHHAr); 4.57 (d, 1 H, CHHAr); 4.28 (dd, 1 H, CH2=CHCHH-O); 4.22 (d,
1 H, J1′,2′ = 7.6, H-1′); 3.96–4.21 (m , 4 H, H-6′a, CH2=CHCHH-O, CH2=CHCH2-O); 3.42–3.94
(m , 14 H, H-3, H-5, H-5′, H-6a, H-6b, H-6′b, 4 CH2CH2-O); 3.80 (s, 3 H, OCH3); 3.02–3.31
(m , 5 H, H-2, H-2′, H-3′, H-4, H-4′); 2.12 (bs, 1 H, OH); 1.41–1.50 (m , 8 H, 4 CH2CH2-O);
1.27 (bs, 88 H, 4 (CH2)11(CH2)2-O); 0.82–0.95 (m , 12 H, 4 CH3CH2). 13C NMR (CDCl3):
159.2 (Carom ); 134.7, 133.8 (2 CH2=CHCH2-O); 130.6 (Carom ); 129.4 (2 Carom ); 118.1, 117.1
(2 CH2=CHCH2-O); 113.8 (2 Carom ); 103.8 (C-1′); 95.3 (C-1); 84.7, 82.0, 81.6, 80.6 (C-2, C-3,
C-2′, C-3′); 77.4, 77.0, 75.0, 74.6, 73.8, 73.7, 73.6, 73.3, 71.4 (C-5, C-5′, CH2Ar,
2 CH2=CHCH2-O, 4 CH2CH2-O); 70.0, 68.6 (C-4, C-4′); 67.9 (C-6); 63.2 (C-6′); 55.2 (CH3O);
31.9, 30.6, 30.5, 30.4, 30.0, 29.7, 29.3, 26.2, 26.0, 22.6 (4 (CH2)12CH2-O); 14.1 (4 CH3CH2).
MS (MALDI-TOF), m/z: 1352.2 [M + Na+], 1368.4 [M + K+].
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2,3,2′,3′-Tetra-O-tetradecylated Lipid A Mimic
1547
Allyl 6-O-(4-O-Allyl-2,3-di-O-tetradecyl-β-D-glucopyran osyl)-
2,3-di-O-tetradecyl-α-D-glucopyran oside (18)
Com poun d 16 (0.15 g, 0.12 m m ol) was deacetylated in a sim ilar way as disacch aride 14 an d
com poun d 18 (0.12 g, 83%) was isolated from reaction m ixture after work-up by a silica gel
colum n ch rom atograph y with h eptan e–eth yl acetate (3:1) elution . M.p. 59–60 °C (eth yl ace-
tate); [α]D +24. For C74H142O11 (1207.9) calculated: 73.58% C, 11.85% H; foun d: 73.43% C,
11.91% H. 1H NMR (CDCl3): 5.88–5.99 (m , 2 H, 2 CH2=CHCH2-O); 5.12–5.37 (m , 4 H,
2 CH2=CHCH2-O); 4.96 (d, 1 H, J1,2 = 3.4, H-1); 4.28 (dd, 1 H, CH2=CHCHH-O); 4.36 (d,
1 H, J1′,2′ = 7.7, H-1′); 4.28 (1 H, CH2=CHCHH-O); 4.02–4.25 (m , 4 H, H-6′a, CH2=CHCH2-O,
CH2=CHCHH-O); 3.44–3.95 (m , 14 H, H-3, H-5, H-5′, H-6a, H-6b, H-6′b, 4 CH2CH2-O);
3.06–3.33 (m , 5 H, H-2, H-2′, H-3′, H-4, H-4′); 2.67 (bs, 1 H, OH); 2.33 (bs, 1 H, OH); 1.56
(bs, 8 H, 4 CH2CH2-O); 1.26 (bs, 88 H, 4 (CH2)11(CH2)2-O); 0.82–0.96 (m , 12 H, 4 CH3CH2).
13C NMR (CDCl3): 134.7, 133.7 (2 CH2=CHCH2-O); 118.1, 117.1 (2 CH2=CHCH2-O); 104.0
(C-1′); 95.3 (C-1); 84.6, 82.1, 81.0, 80.5 (C-2, C-3, C-2′, C-3′); 77.4, 77.0, 75.0, 73.8, 73.7,
73.5, 73.3, 71.0 (C-5, C-5′, 2 CH2=CHCH2-O, 4 CH2CH2-O); 70.4, 70.1 (C-4, C-4′); 67.9 (C-6);
62.0 (C-6′); 31.9, 30.4, 30.3, 30.0, 29.7, 29.4, 29.3, 26.2, 26.1, 22.6 (4 (CH2)12CH2-O); 14.1
(4 CH3CH2). MS (MALDI-TOF), m/z: 1231.6 [M + Na+], 1247.9 [M + K+].
The present work was supported by the VEGA grant 2/6129/26 and APVT grant 51-039802.
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