Synthesis of 4-nitrophenyl 2-acetamido-2-deoxy-β-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranoside

Article abstract of DOI:10.1135/cccc20030801

The title compounds were synthesized by the selective reduction of the azido group in 4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-mannopyranoside (8) and 4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-mannopyranoside (11), and by subsequent acetylation. Compound 8 was prepared by opening of the epoxide ring in methyl 2,3-anhydro-4,6-O-benzylidene-α-D-glucopyranoside (1) with sodium azide, followed by inversion of the configuration at C-3 in the resulting altropyranoside and glycosidation with 4-nitrophenol.

Full text of DOI:10.1135/cccc20030801

4-Nitrophenyl 2-Acetamido-2-deoxy-D-mannopyranosides
801
SYNTHESIS OF 4-NITROPHENYL 2-ACETAMIDO-2-DEOXY-
β-D-MANNOPYRANOSIDE AND 4-NITROPHENYL
2-ACETAMIDO-2-DEOXY-α-D-MANNOPYRANOSIDE
b
c
a2
Pavel KRISTa1, Marek KUZMA , István F. PELYVÁS , Pavla SIMERSKÁ and
a3,
Vladimír KŘEN
*
a
Laboratory of Biotransformation, Institute of Microbiology,
Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic;
e-mail: krist@biomed.cas.cz, simerska@biomed.cas.cz, 3kren@biomed.cas.cz
Laboratory of Molecular Structure Characterization, Institute of Microbiology,
Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Prague 4,
Czech Republic; e-mail: kuzma@biomed.cas.cz
Research Group of Antibiotics, Hungarian Academy of Sciences, University of Debrecen,
P.O. Box 70, H-4010 Debrecen, Hungary; e-mail: pelyvas@tigris.klte.hu
1
2
b
c
Received Novem ber 12, 2002
Accepted Jan uary 13, 2003
Th e title com poun ds were syn th esized by th e selective reduction of th e azido group in
4-n itroph en yl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-m an n opyran oside (8) an d 4-n itroph en yl
3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-m an n opyran oside (11), an d by subsequen t acetylation .
Com poun d
8 was prepared by open in g of th e epoxide rin g in m eth yl 2,3-an h ydro-
4,6-O-ben zyliden e-α-D-glucopyran oside (1) with sodium azide, followed by in version of th e
con figuration at C-3 in th e resultin g altropyran oside an d glycosidation with 4-n itroph en ol.
Keyw ord s: N-Acetylm an n osam in e; β-Man n opyran osides; Am in osugars; Glycosylation ;
Glycosides; Glycosyl don ors; Im m un ology; Im m un ostim ulan ts.
2-Acetam ido-2-deoxy-D-m an n opyran ose (Man NAc) is a frequen tly occur-
rin g glycosyl residue in a n um ber of bacterial capsular polysacch arides an d
lipopolysacch arides (e.g., Haemophillus influenzae an d Streptococcus pneumo-
niae)1–5. In th e Gram -positive bacteria, such as Staphylococcus aureus H, an d
Bacillus subtilis, th e β-Man NAc residue is a com pon en t of th e “lin kage un it”
attach in g teich oic acids to th e peptidoglycan 4. Recen tly, Man NAc was iden -
tified as th e stron gest m on osacch aridic ligan d for th e n atural killer cell acti-
vatin g protein NKR-P16, an d som e Man NAc-con tain in g sacch arides were
foun d to be stron g im m un ostim ulan ts7. Th e β-Man NAc un its h ave poten t
im plication in th e virulen ce an d path ogen ity of som e bacteria (e.g., S.
pneumoniae 19F an d 19A)4,5. It was sh own th at th e presen ce of m an n os-
Collect. Czech. Chem. Commun. (Vol. 68) (2003)
doi:10.1135/cccc20030801

802
Krist et al.:
am in e derivatives can cause m odification of th e cell wall syn th esis an d
som e ch an ges in m ucin production 8–10
.
Despite its ph ysiological im portan ce, reports on th e preparation of th e
structures con tain in g β-Man NAc residues appeared quite recen tly – as re-
viewed by Gridley an d Osborn 1 in 2000. It is un iversally accepted th at prep-
aration of th e β-m an n opyran osyl (β-Man ), an d particularly, of th e
β-Man NAc glycosidic lin kages still rem ain s on e of th e greatest ch allen ges to
carboh ydrate ch em ists.
Our prim ary goal was to syn th esize 4-n itroph en yl 2-acetam ido-2-deoxy-
β-D-m an n opyran oside (15) as a ch rom ogen ic substrate for a screen in g of
th e h ith erto un kn own β-N-acetylm an n osam in idase activity. Furth erm ore,
our previous studies6,7 sh owed th at th e β-Man NAc m oiety, especially wh en
attach ed to an arom atic system , sh ould h ave stron g affin ity to activation
receptor (NKR-P1) of th e n atural killer cells6 an d th us activate th em again st
m align an t tum ours (e.g., colon carcin om a, m elan om a). Th is requirem en t
sh ould be perfectly m et by com poun d 15. In deed, in vitro tests h ave sh own
th at 4-n itroph en yl 2-acetam ido-2-deoxy-β-D-m an n opyran oside (15) is 10
tim es m ore poten t th an 4-n itroph en yl 2-acetam ido-2-deoxy-β-D-galacto-
pyran oside, so far th e stron gest NK-cell m on osacch aridic activator. More-
over, th e n itro group is suitable for th e con struction of m ultivalen t glyco-
m im etics11. To our kn owledge, com poun d 15 h as n ot been prepared yet.
We also in vestigated som e altern atives to th e sin gle described m eth od12
of preparation of th e correspon din g α-an om er 14 from rath er expen sive
startin g m aterial 2-acetam ido-2-deoxy-D-m an n opyran ose.
Th is paper describes n ew m eth ods for th e syn th esis of both α an d β
4-n itroph en yl 2-acetam ido-2-deoxy-D-m an n opyran osides (14 an d 15).
RESULTS AND DISCUSSION
Synthesis of 4-Nitrophenyl 2-Azido-2-deoxy-α-D-mannopyranoside (8)
For th e syn th esis of th e target α-m an n oside (14), com m ercial m eth yl
α-D-glucopyran oside was used as th e precursor. Th is com poun d was tran s-
form ed in to m eth yl 2,3-an h ydro-4,6-O-ben zyliden e-α-D-glucopyran oside13
(1). Open in g of th e epoxide rin g in com poun d 1 with NaN3 in aqueous
m eth oxyeth an -1-ol14 gave m eth yl 2-azido-4,6-O-ben zyliden e-2-deoxy-
α-D-altropyran oside (2), wh ich was con verted in to th e 3-O-(trifluoro-
m eth an esulfon yl) derivative (3) (94%). Triflate displacem en t with tetra-
butylam m on ium acetate in CH2Cl2 yielded 43% of m eth yl 3-O-acetyl-
2-azido-4,6-O-ben zyliden e-2-deoxy-α-D-m an n opyran oside (4) via in version
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

4-Nitrophenyl 2-Acetamido-2-deoxy-D-mannopyranosides
803
of th e con figuration at C-3, an d 39% of th e 2-azidoh ex-3-en opyran oside 5
as a result of com petitive elim in ation facilitated by th e trans arran gem en t
of th e 3-O-triflate leavin g group an d th e adjacen t axial h ydrogen at C-4.
Th e elim in ation process could be suppressed n eith er by ch an gin g th e sol-
ven t (DMF, DMF–CH2Cl2, m eth yl eth yl keton e) n or by variation of th e re-
action tem perature. Th e ch oice of 3-O-triflate as th e leavin g group
com pared to previously described 3-O-(N-im idazolylsulfon yl) group15 gave
com parable yield; h owever, it provided clean er reaction m ixture th at en -
abled isolation of th e h ith erto un kn own side product 5, wh ich was fully
ch aracterized. Acetolysis of 4 allowed sim ultan eous rem oval of th e
ben zyliden e acetal an d th e glycosidic aglycon e to give 1,3,4,6-tetra-
O-acetyl-2-azido-2-deoxy-α-D-m an n opyran ose (6) in h igh yield (92%). Van
N3
O
N3
O
Ph
O
Ph
O
Ph
O
O
a)
b)
O
O
O
O
c)
OH
OMe
Ph
OTf
OMe
OMe
2
3
1
O
N3
O
Ph
O
O
O
N3
+
AcO
O
OMe
OMe
5
4
d)
39%
43%
N3
O
N3
O
N3
O
AcO
AcO
AcO
AcO
AcO
g)
e)
AcO
AcO
AcO
AcO
OAc
Br
O
NO2
6
7
8
f)
a) NaN3, NH4Cl, 2-methoxyethanol–water, reflux15; b) Tf2O, CH2Cl2–pyridine, 0 oC;
c) tetrabutylammonium acetate, CH2Cl2, 0 oC; d) 5% H2SO4 in Ac2O, 0 oC;
e) TiBr4, CH2Cl2–EtOAc, r.t.; f) BF3·Et2O, 4-nitrophenol, CH2Cl2, 0 oC; g) this reaction
was done by three different methods: g-1) Ag-triflate, 4-nitrophenol, CH2Cl2–toluene,
–20 oC; g-2) silver 4-nitrophenolate20, acetonitrile, r.t. or g-3) Ag-silicate, 4-nitrophenol,
acetonitrile, –20 oC
SCHEME 1
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804
Krist et al.:
Boom et al.16 described altern ative syn th esis of in term ediate 6 in two steps
from 2-deoxy-D-m an n osam in e h ydroch loride in overall yield of 88%. How-
ever, our procedure starts from com m ercial (or easily preparable) m eth yl
α-D-glucopyran oside th at is m ore th an 400 tim es ch eaper (Sigm a, Aldrich ).
Th e overall yield of our procedure is on ly 24%, but is com pen sated by a
very low price of th e startin g m aterial. Treatm en t of 6 with TiBr4 in a m ix-
ture of CH2Cl2 an d eth yl acetate15 furn ish ed th e correspon din g 1-α-brom o-
sacch aride17 7 in 93% yield.
To obtain th e desired glycoside 8, th e attach m en t of th e 4-n itroph en yl
glycosidic m oiety to 7 was attem pted usin g eith er silver 4-n itroph en olate
(yield 40%); 4-n itroph en ol in th e presen ce of silver trifluorom eth an e-
sulfon ate (yield 38%), or silver silicate (yield 61%)18,19. Wh en 4-n itroph en ol
was reacted with 6 in th e presen ce of boron trifluoride eth erate, th e yield of
8 was 30% 18,19 (Sch em e 1). Th e yields were rath er low an d, in con trast to
th e literature data17 reportin g th e form ation of β-an om ers or an om eric m ix-
tures by th e above processes, we were able to iden tify solely α-glycoside 8.
4-Nitrophenyl 2-Azido-2-deoxy-β-D-mannopyranoside (11)
Th e in troduction of th e azido fun ction an d in version of th e con figuration
at C-2 in a suitably protected β-glucopyran oside is a feasible an d in -
expensive m ethod for obtaining 4-nitrophenyl 2-azido-2-deoxy-β-D-m anno-
pyran oside derivatives. Th us, usin g th e Garegg’s procedure20, activation of
th e C-2 h ydroxy group of 4-n itroph en yl 3-O-ben zoyl-4,6-O-ben zyliden e-
β-D-glucopyran oside with triflic an h ydride, subsequen t SN2 displacem en t of
th e resultin g 2-O-triflate ester with NaN3 an d deprotection of th e C-3
h ydroxyl group afforded 4-n itroph en yl 2-azido-4,6-O-ben zyliden e-2-deoxy-
β-D-m an n opyran oside (9). Mild acid h ydrolysis of th e acetal protectin g
N3
O
N3
O
Ph
O
HO
HO
O
a)
O
NO2
O
NO2
HO
HO
9
10
N3
O
AcO
AcO
AcO
b)
O
NO2
11
a) 70% AcOH, 70 oC; b) Ac2O, py, r.t.
SCHEME 1
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4-Nitrophenyl 2-Acetamido-2-deoxy-D-mannopyranosides
805
group in 9 gave 10 (82%), wh ich was subsequen tly peracetylated yieldin g
4-n itroph en yl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-m an n opyran oside (11)
with a 79% overall yield (Sch em e 2).
4-Nitrophenyl 2-Acetamido-2-deoxy-α- and β-D-mannopyranoside
(14 and 15)
Th e C-2 azido group in th e α- an d β-glycosides 8 an d 11 was ch em o-
selectively reduced in th e presen ce of th e n itroph en yl group usin g
triph en ylph ospin e alth ough altern atives (such as reduction with H2S)21
exist. Subsequen t peracetylation (in 85% overall yield) followed by
O-deacetylation (96%) (Sch em e 3) afforded th e title com poun ds 14 an d 15.
Th e couplin gs JH-1,H-2 in α- an d β-m an n ose derivatives are often very
close22 an d un reliable; th erefore, direct couplin g JC-1,H-1 was used to deter-
m in e th e an om eric con figuration s23. Th e respective JC,H extracted from cou-
pled HMQC experim en ts m ade with 14 (178 Hz) an d 15 (166 Hz) allowed
un am biguous an om eric con figuration assign m en ts.
N3
O
NHAc
O
AcO
AcO
AcO
AcO
AcO
AcO
a)
O
NO2
O
NO2
8 (α)
11 (β)
12 (α)
13 (β)
NHAc
O
HO
HO
b)
HO
O
NO2
14 (α)
15 (β)
a) triphenylphosphine, CH2Cl2, 40 oC, H2O, r.t. overnight; AcCl, CH2Cl2–pyridine, 0 oC;
b) NaOMe, MeOH, r.t.
SCHEME 3
CONCLUSIONS
Differen t glycosylation reaction s of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
D-m an n opyran ose or 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-m an n o-
pyran osyl brom ide yielded on ly 4-n itroph en yl 3,4,6-tri-O-acetyl-2-azido-
2-deoxy-α-D-m an n opyran oside. Th is an om er was obtain ed even wh en
usin g silver silicate as a prom oter th at is kn own to catalyse form ation of
β-Man con figuration 17–19. Respective β-an om er can be obtain ed by in direct
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Krist et al.:
route usin g 4-n itroph en yl β-glucopyran oside as th e startin g m aterial via in -
version of th e con figuration at C-2. Th e overall yields were n ot outstan din g
but th e low price of th e startin g m aterial substan tiates th e syn th esis of th e,
h ith erto un kn own com poun d (15). Altern ative, m ore effective syn th eses
were developed for th e respective α-an om er (14). Both n itroph en yl
glycosides can be used for th e screen in g for h ypoth etical α- an d β-N-acetyl-
m an n osam in idase an d also for furth er con struction of m ultivalen t glyco-
m im etic structures for im m un ological studies.
EXPERIMENTAL
Gen eral
NMR spectra were recorded on a Un ity-In ova 400 MHz spectrom eter (399.90 MHz for 1H,
100.55 MHz for 13C) in C6D6, CDCl3, CD3OD or D2O at 30 °C. Ch em ical sh ifts are given in
ppm (δ-scale). Th e assign m en ts were based on COSY, HMQC, HMBC an d differen tial NOE
experim en ts perform ed usin g th e m an ufacturer′s software. Carbon ch em ical sh ifts were de-
rived from 13C NMR or HMQC an d HMBC experim en ts. Aceton e an d residual solven t sign als
(HDO δH 4.508, aceton e δC 30.5, CDCl3 δH 7.265, δC 77.00, C6D6 δH 7.150, δC 128.00,
CD3OD δH 3.330, δC 49.30 ppm ) were used as in tern al stan dards. Couplin g con stan ts (J) are
given in Hz. Digital resolution was 0.0007 an d 0.01 ppm for 1H an d 13C, respectively.
MALDI-TOF m ass spectrom etry: a saturated solution of α-cyan o-4-h ydroxycin n am ic acid
(Sigm a) in aqueous 50% aceton itrile/0.2% TFA was used as th e ion ization m atrix. Sam ple
(2 µl) an d m atrix solution (2 µl) were prem ixed in a tube, 0.5 µl of th e m ixture was placed
on th e sam ple target an d allowed to dry at am bien t tem perature. Positive ion MALDI m ass
spectra were m easured on a Bruker BIFLEX reflectron tim e-of-fligh t m ass spectrom eter
(Bruker–Fran zen , Brem en , Germ an y) equipped with a SCOUT 26 sam ple in let, a gridless de-
layed extraction ion source an d a n itrogen laser (337 n m ; Laser Scien ce, Cam bridge (MA),
U.S.A.). Ion acceleration voltage was 19 kV an d th e reflectron voltage was set to 20 kV. Spec-
tra were calibrated extern ally usin g th e m on oisotopic [M + H]+ ion of α-cyan o-4-h ydroxy-
cin n am ic acid an d a peptide stan dard (an gioten sin II, Aldrich ). Elem en tal an alyses were
perform ed usin g th e Perkin Elm er 2400 II in strum en t. Colum n ch rom atograph y was per-
form ed on Silica Gel 60 (40–63 µm , 230–400 m esh , Merck). TLC was run on precoated Silica
Gel 60 F254 alu m in iu m sh eets, d etection with UV ligh t (254 n m ) an d ch arrin g with
5% H2SO4 in EtOH.
Meth yl 2-Azido-4,6-O-ben zyliden e-2-deoxy-3-O-(trifluorom eth an esulfon yl)-
α-D-altropyran oside (3)
Triflic an h ydride (165 µl, 0.97 m m ol) in dry CH2Cl2 (2 m l) was added at 0 °C to a stirred so-
lution of 2 (250 m g, 0.81 m m ol)14 in a dry CH2Cl2–pyridin e m ixture (2:1, 6 m l). Th e result-
in g m ixture was kept at 0 °C for 30 m in an d th en left to reach room tem perature. After
an oth er 30 m in , th e reaction was quen ch ed with water (5 m l). Th e organ ic ph ase was sepa-
rated an d th e aqueous ph ase was extracted twice with CH2Cl2. Th e com bin ed extracts were
dried over an h ydrous Na2SO4, evaporated to dryn ess, coevaporated with toluen e, an d puri-
fied (for spectral iden tification ) by flash colum n ch rom atograph y (h exan e–EtOAc, 9:1) to
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

4-Nitrophenyl 2-Acetamido-2-deoxy-D-mannopyranosides
807
yield 3 (335 m g, 94%) as wh ite solid. 1H NMR (C6D6): 2.784 (3 H, s, CH3O); 3.326 (1 H, dd,
J = 10.3, 10.4, H-6a); 3.560 (1 H, dd, J = 3.0, 9.8, H-4); 3.563 (1 H, dd, J = 0.9, 2.9, H-2);
3.951 (1 H, dd, J = 5.2, 10.4, H-6b); 4.079 (1 H, ddd, J = 5.2, 9.8, 10.3, H-5); 4.165 (1 H, m ,
H-1); 4.906 (1 H, dd, J = 2.9, 3.0, H-3); 5.169 (1 H, s, CH(O)O); 7.095 (1 H, m , H-para); 7.169
(2 H, m , H-meta); 7.544 (2 H, m , H-ortho). 13C NMR (C6D6): 55.09 (q, CH3O), 58.62 (d, C-5),
60.95 (d, C-2), 68.73 (t, C-6), 72.71 (d, C-4), 80.16 (d, C-3), 98.32 (d, C-1), 102.50 (d,
CH(O)O), 119.11 (q, JC,F = 319.4, CF3SO2), 126.53 (d, 2 × C-ortho), 128.38 (d, 2 × C-meta),
129.36 (d, C-para). MALDI MS, m/z: 462.2 [M + Na]+
Meth yl 3-O-Acetyl-2-azido-4,6-O-ben zyliden e-2-deoxy-α-D-m an n opyran oside (4) an d
Meth yl 2-Azido-4,6-O-ben zyliden e-2,3-dideoxy-β-D-arabino-h ex-3-en opyran oside (5)
Com poun d
3 (with out purification ) was dissolved in CH2Cl2 (5 m l) an d tetrabutyl-
am m on ium acetate (370 m g, 1.2 m m ol) was added at 0 °C un der stirrin g. After 30 m in th e
reaction m ixture was diluted with CH2Cl2 (15 m l) an d wash ed with aqueous 10% NaCl (3 ×
5 m l), th e organ ic ph ase was separated, dried over an h ydrous Na2SO4 an d con cen trated. Th e
residue was ch rom atograph ed (h exan e–EtOAc, 85:15) to yield 4 (120 m g, 43%) as a colorless
sirup an d 5 (90 m g, 39%) as an am ber sirup.
Compound 4. 1H NMR (C6D6): 1.656 (3 H, s, CH3CO-3); 2.820 (3 H, s, CH3O-1); 3.518
(1 H, dd, J = 10.3, 10.3, H-6a); 3.810 (1 H, dddd, J = 0.6, 4.8, 9.4, 10.3, H-5); 3.854 (1 H, dd,
J = 1.5, 3.9, H-2); 4.024 (1 H, dd, J = 4.8, 10.3, H-6b); 4.092 (1 H, dd, J = 9.4, 10.3, H-4);
4.246 (1 H, dd, J = 0.6, 1.5, H-1); 5.300 (1 H, s, CH(O)O); 5.666 (1 H, dd, J = 3.9, 10.3, H-3);
7.070 (1 H, m , H-para); 7.131 (2 H, m , H-meta); 7.542 (2 H, m , H-ortho). 13C NMR (C6D6):
19.9 (q, CH3CO-3), 54.5 (q, CH3O-1), 62.5 (d, C-2), 64.2 (d, C-5), 68.7 (t, C-6), 70.7 (d, C-3),
76.4 (d, C-4), 99.9 (d, C-1), 102.3 (d, CH(O)O), 126.8 (d, 2 × C-ortho), 128.3 (d, 2 × C-meta),
129.1 (d, C-para), 137.9 (s, C-ipso), 169.5 (s, CH3CO-3). MALDI MS, m/z: 372.4 [M + Na]+.
Compound 5. 1H NMR (C6D6): 2.934 (3 H, s, CH3O-1); 3.334 (1 H, ddd, J = 0.9, 1.0, 5.5,
H-2); 3.672 (1 H, dd, J = 10.1, 10.6, H-6a); 4.174 (1 H, ddd, J = 0.5, 6.3, 10.1, H-6b); 4.255
(1 H, dddd, J = 1.0, 1.1, 6.3, 10.6, H-5); 4.543 (1 H, dd, J = 0.9, 1.1, H-1); 5.140 (1 H, dddd,
J = 0.5, 1.1, 1.1, 5.5, H-3); 5.453 (1 H, s, CHO(O)); 7.084–7.169 (3 H, m , H-meta, H-para);
7.488 (2 H, m , H-ortho). 13C NMR (C6D6): 55.44 (q, CH3O-1), 56.97 (d, C-2), 61.04 (d, C-5),
70.29 (t, C-6), 97.87 (d, C-3), 100.30 (d, C-1), 104.37 (d, CHO(O)), 126.76 (d, 2 × C-ortho),
128.38 (d, 2 × C-meta), 129.49 (d, C-para), 137.12 (s, C-ipso), 156.87 (s, C-4). MALDI MS,
m/z: 311.1 [M + Na]+. For C14H15N3O4 (289.3) calculated: 58.13% C, 5.23% H, 14.53% N;
foun d: 58.39% C, 5.44% H, 14.41% N.
1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-α-D-m an n opyran ose (6)
To a solution of 4 (600 m g, 1.7 m m ol) in acetic an h ydride (30 m l), 4% sulfuric acid in acetic
an h ydride (1 m l) was added. Th e reaction m ixture was stirred at 0 °C for 1 h an d th en it
was diluted with EtOAc (30 m l). Th e organ ic ph ase was wash ed with water (3 × 30 m l), satu-
rated aqueous NaHCO3 (3 × 30 m l) an d saturated aqueous NaCl (20 m l), dried over an h y-
drous Na2SO4, con cen trated an d purified by colum n ch rom atograph y (h exan e–EtOAc, 7:3)
to give 6 (590 m g, 92%) in form of yellowish solid. 1H NMR (CDCl3): 2.048 (3 H, s,
CH3CO-4); 2.087 (3 H, s, CH3CO-6); 2.108 (3 H, s, CH3CO-3); 2.156 (3 H, s, CH3CO-1);
4.000 (1 H, m , H-5); 4.026 (1 H, dd, J = 2.0, 3.4, H-2); 4.090 (1 H, dd, J = 2.4, 12.5, H-6a);
4.238 (1 H, dd, J = 4.5, 12.5, H-6b); 5.366–5.414 (2 H, m , H-3, H-4); 6.108 (1 H, d, J = 2.0,
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H-1). 13C NMR (CDCl3) 20.4 (q, CH3CO-3), 20.5 (q, CH3CO-4), 20.6 (q, CH3CO-6), 20.7 (q,
CH3CO-1), 60.5 (d, C-2), 61.8 (t, C-6), 65.3 (d, C-4), 70.5 (d, C-3), 70.7 (d, C-5), 91.4 (d,
C-1), 168.2 (s, CH3CO-1), 169.4 (s, CH3CO-4), 170.0 (s, CH3CO-3), 170.7 (s, CH3CO-6).
MALDI MS, m/z: 396.2 [M + Na]+.
3,4,6-Tri-O-acetyl-2-azido-2-deoxy-α-D-m an n opyran osyl Brom ide (7)
A solution of 6 (240 m g, 0.64 m m ol) an d TiBr4 (285 m g, 0.77 m m ol) in a m ixture of CH2Cl2
(10 m l) an d EtOAc (2 m l) was stirred at room tem perature for 48 h . Th e reaction m ixture
was diluted with aceton itrile (5 m l). MeONa (200 m g) was added an d stirrin g was con tin ued
un til th e red colour disappeared. Th e m ixture was filtered, evaporated an d th e residue was
ch rom atograph ed with h exan e–EtOAc (7:3) to afford 7 (235 m g, 93%) in a form of yellowish
solid. 1H NMR (CDCl3): 2.079 (3 H, s, CH3CO-4); 2.106 (3 H, s, CH3CO-6); 2.120 (3 H, s,
CH3CO-3); 4.136 (1 H, dd, J = 2.2, 12.5, H-6a); 4.178 (1 H, dddd, J = 0.8, 2.2, 4.4, 10.2, H-5);
4.297 (1 H, dd, J = 4.4, 12.5, H-6b); 4.325 (1 H, dd, J = 1.5, 3.9, H-2); 5.405 (1 H, dd, J = 9.7,
10.2, H-4); 5.708 (1 H, dd, J = 3.9, 9.7, H-3); 6.369 (1 H, dd, J = 0.8, 1.5, H-1). 13C NMR
(CDCl3): 20.45 (q, CH3CO-3), 20.56 (q, CH3CO-4), 20.62 (q, CH3CO-6), 61.19 (t, C-6), 64.97
(d, C-2), 65.05 (d, C-4), 69.99 (d, C-3), 73.05 (d, C-5), 83.80 (d, C-1), 169.33 (s, CH3CO-4),
169.82 (s, CH3CO-3), 170.52 (s, CH3CO-6). MALDI MS, m/z: 416.1 [M + Na]+.
4-Nitroph en yl 3,4,6-Tri-O-acetyl-2-azido-2-deoxy-α-D-m an n opyran oside (8)
Method A. Com poun d 7 (28 m g, 0.07 m m ol) was slowly added un der stirrin g to a m ixture
of 4-n itroph en ol (10 m g, 0.07 m m ol) an d silver trifluorom eth an esulfon ate (19 m g,
0.07 m m ol) in CH2Cl2–toluen e (1:1, 4 m l) at –30 °C. After 10 m in th e reaction m ixture was
allowed to reach th e room tem perature, an d after an oth er 20 m in it was evaporated in
vacuo, dissolved in CH2Cl2 (10 m l), wash ed with water (3 × 10 m l), dried over an h ydrous
Na2SO4 an d con cen trated. Th e residue was ch rom atograph ed (CH2Cl2–EtOAc, 9:1) to yield 8
(12 m g, 38%) in form of yellowish solid.
Method B. Fresh ly prepared well dried silver 4-n itroph en olate19 (75 m g, 0.30 m m ol) an d 7
(40 m g, 0.10 m m ol) were stirred with aceton itrile (5 m l). Th e reaction m ixture was refluxed
for 10 h . Th en it was filtered, evaporated, dissolved in CH2Cl2, wash ed with water (3 × 10 m l),
dried over an h ydrous Na2SO4 an d evaporated. Th e residue was purified by colum n ch rom a-
tograph y (CH2Cl2–EtOAc, 9:1) to yield 8 (18 m g, 40%).
Method C. A m ixture of 4-n itroph en ol (7.8 m g, 0.05 m m ol), fresh ly prepared well dried
Ag-silicate18 (60 m g), an d 4 Å m olecular sieves (100 m g) in dry MeCN (2 m l) was stirred at
room tem perature for 1 h . Th en 7 (20 m g, 0.05 m m ol) in MeCN (2 m l) was slowly added at
–30 °C. Th e reaction m ixture was allowed to warm up to room tem perature, diluted with
CH2Cl2 (10 m l), filtered, wash ed twice with water (10 m l), dried over an h ydrous Na2SO4 an d
con cen trated. Th e residue was ch rom atograph ed with CH2Cl2–EtOAc (9:1) to yield 8 (14 m g,
61%).
Method D. A solution of 4-n itroph en ol (13 m g, 0.09 m m ol) an d 6 (34 m g, 0.09 m m ol) in
CH2Cl2 (10 m l) was stirred with 4 Å m olecular sieves (100 m g) for 1 h at 0 °C. Th en
BF3·Et2O (100 µl, 0.09 m m ol) was added dropwise. Th e reaction m ixture was stirred over-
n igh t at 0 °C, filtered, wash ed with water (10 m l), saturated NaHCO3 (2 × 10 m l), dried over
an h ydrous Na2SO4, evaporated, an d th e residue was purified by colum n ch rom atograph y
(CH2Cl2–EtOAc, 9:1) to yield 8 (12 m g, 30%). 1H NMR (CDCl3): 2.046 (3 H, s, CH3CO-6);
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4-Nitrophenyl 2-Acetamido-2-deoxy-D-mannopyranosides
809
2.069 (3 H, s, CH3CO-4); 2.160 (3 H, s, CH3CO-6); 3.954 (1 H, ddd, J = 2.3, 5.1, 10.0, H-5);
4.073 (1 H, dd, J = 2.3, 12.4, H-6a); 4.234 (1 H, dd, J = 5.1, 12.4, H-6b); 4.300 (1 H, dd, J =
1.9, 3.8, H-2); 5.434 (1 H, dd, J = 9.9, 10.0, H-4); 5.584 (1 H, dd, J = 3.8, 9.9, H-3); 5.640
(1 H, d, J = 1.9, H-1); 7.210 (2 H, m , H-ortho); 8.238 (2 H, m , H-meta). 13C NMR (CDCl3):
20.4 (q, CH3CO-3), 20.5 (q, CH3CO-4, CH3CO-6), 61.1 (d, C-2), 61.8 (t, C-6), 65.5 (d, C-4),
69.8 (d, C-5), 70.6 (d, C-3), 96.3 (d, C-1), 116.5 (d, 2 × C-ortho), 126.0 (d, 2 × C-meta), 143.1
(s, C-para), 159.9 (s, C-ipso), 169.6 (s, CH3CO-4), 170.1 (s, CH3CO-3), 170.7 (s, CH3CO-6).
JC-1,H-1 = 178. MALDI MS, m/z: 475.0 [M + Na]+.
4-Nitroph en yl 2-Azido-2-deoxy-β-D-m an n opyran oside (10)
Com poun d 9 (250 m g, 0.6 m m ol)20 was stirred with 70% aqueous acetic acid (25 m l) an d
th e resultin g m ixture was warm ed to 70 °C. Th e reaction was com pleted after com plete dis-
solution of th e startin g m aterial. Th e reaction m ixture was diluted with EtOAc (10 m l), or-
gan ic ph ase was wash ed with saturated NaHCO3 (3 × 10 m l) an d water (10 m l), an d dried
over an h ydrous Na2SO4, con cen trated an d th e residue was purified by colum n ch rom atogra-
ph y usin g CH2Cl2–MeOH (9:1) to yield 10 (160 m g, 82%) as a wh ite solid. 1H NMR
(CD3OD): 3.468 (1 H, ddd, J = 2.3, 6.0, 9.7, H-5); 3.585 (1 H, dd, J = 9.2, 9.7, H-4); 3.718
(1 H, dd, J = 6.0, 12.2, H-6a); 3.807 (1 H, dd, J = 3.7, 9.2, H-3); 3.927 (1 H, dd, J = 2.3, 12.2,
H-6b); 4.180 (1 H, dd, J = 1.4, 3.7, H-2); 5.554 (d, J = 1.4, H-1); 7.232 (2 H, m , H-ortho);
8.236 (2 H, m , H-meta). 13C NMR (CD3OD): 62.82 (t, C-6), 66.58 (d, C-2), 68.62 (d, C-4),
74.38 (d, C-3), 79.35 (d, C-5), 98.69 (d, C-1), 117.88 (d, 2 × C-ortho), 126.95 (d, 2 × C-meta),
144.41 (s, C-para), 163.17 (s, C-ipso). MALDI MS, m/z: 349.2 [M + Na]+.
4-Nitroph en yl 3,4,6-Tri-O-acetyl-2-azido-2-deoxy-β-D-m an n opyran oside (11)
Com poun d 10 (150 m g, 0.46 m m ol) was dissolved in acetic an h ydride–pyridin e (1:1, 10 m l)
an d stirred at room tem perature overn igh t. Th e reaction m ixture was dissolved in CH2Cl2
(10 m l), wash ed with water (2 × 10 m l), saturated NaHCO3 (3 × 15 m l) evaporated, an d
co-evaporated with toluen e to yield 11 (200 m g, 96%) as a wh ite solid. 1H NMR (CDCl3):
2.080 (3 H, s, CH3CO-6); 2.081 (3 H, s, CH3CO-4); 2.155 (3 H, s, CH3CO-3); 3.844 (1 H, ddd,
J = 2.8, 5.9, 9.5, H-5); 4.199 (1 H, dd, J = 2.8, 12.3, H-6a); 4.282 (1 H, dd, J = 5.9, 12.3,
H-6b); 4.335 (1 H, dd, J = 1.5, 3.7, H-2); 5.123 (1 H, dd, J = 3.7, 9.6, H-3); 5.320 (1 H, dd, J =
9.5, 9.6, H-4); 5.389 (1 H, d, J = 1.5, H-1); 7.119 (2 H, m , H-ortho); 8.226 (2 H, m , H-meta).
13C NMR (CDCl3): 20.55, 20.56, 20.62 (q, CH3CO-3, CH3CO-4, CH3CO-6), 61.21 (d, C-2),
62.13 (t, C-6), 65.41 (d, C-4), 71.42 (d, C-3), 73.02 (d, C-5), 96.84 (d, C-1), 116.49 (d, 2 ×
C-ortho), 125.77 (d, 2 × C-meta), 143.40 (s, C-para), 160.65 (s, C-ipso), 169.24 (s, CH3CO-4),
170.04 (s, CH3CO-3), 170.39 (s, CH3CO-6). MALDI MS, m/z: 475.1 [M + Na]+.
4-Nitroph en yl 2-Acetam ido-3,4,6-tri-O-acetyl-2-deoxy-α-D-m an n opyran oside (12) an d
4-Nitroph en yl 2-Acetam ido-3,4,6-tri-O-acetyl-2-deoxy-β-D-m an n opyran oside (13)
Triph en ylph osph in e (165 m g, 0.63 m m ol) was added at a room tem perature to a stirred so-
lution of com poun d 8 or 11 (190 m g, 0.42 m m ol) in CH2Cl2 (10 m l) an d th e reaction m ix-
ture was kept at 40 °C. After 4 h water (10 m l) was added an d th e stirrin g was con tin ued
overn igh t. Th e organ ic ph ase was separated, con cen trated, an d co-evaporated twice with to-
luen e. Th e residue was dissolved in CH2Cl2–pyridin e (1:1, 10 m l) an d acetyl ch loride (45 µl,
0.63 m m ol) was added at 0 °C. After 1 h th e m ixture was con cen trated an d co-evaporated
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

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Krist et al.:
twice with toluen e. Th e residue obtain ed after evaporation was ch rom atograph ed
(CH2Cl2–EtOAc, 2:3) to afford 12 or 13 (167 m g, 85%), both in form of yellowish solid.
Compound 12. 1H NMR (CDCl3): 2.020 (3 H, s, CH3CO-6); 2.045 (3 H, s, CH3CO-3); 2.062
(3 H, s, CH3CO-4); 2.101 (3 H, s, CH3CONH-2); 4.009 (1 H, ddd, J = 2.8, 5.6, 10.2, H-5);
4.028 (1 H, dd, J = 2.8, 12.4, H-6a); 4.260 (1 H, dd, J = 5.6, 12.4, H-6b); 4.821 (1 H, ddd, J =
1.7, 4.8, 8.4, H-2); 5.201 (1 H, dd, J = 10.2, 10.2, H-4); 5.554 (1 H, dd, J = 4.8, 10.2, H-3);
5.626 (1 H, d, J = 1.7, H-1); 5.942 (1 H, d, J = 8.4, CH3CONH-2); 7.202 (2 H, m , H-ortho);
8.222 (2 H, m , H-meta). 13C NMR (CDCl3): 20.57 (q, CH3CO-4), 20.60 (q, CH3CO-6), 20.66
(q, CH3CO-3), 23.26 (q, CH3CONH-2), 50.42 (d, C-2), 61.97 (t, C-6), 65.63 (d, C-4), 68.38 (d,
C-3), 69.35 (d, C-5), 96.94 (d, C-1), 116.24 (d, 2 × C-ortho), 125.80 (d, 2 × C-meta), 143.21 (s,
C-para), 160.24 (s, C-ipso), 169.66 (s, CH3CO-3), 169.81 (s, CH3CO-4), 170.22 (s, CH3CO-6),
170.33 (s, CH3CONH-2).
Compound 13. 1H NMR (CDCl3): 2.043 (3 H, s, CH3CO-6); 2.070 (3 H, s, CH3CO-3); 2.101
(3 H, s, CH3CO-4); 2.116 (3 H, s, CH3CO-2); 3.924 (1 H, ddd, J = 3.4, 6.5, 8.3, H-5); 4.189
(1 H, dd, J = 3.4, 12.2, H-6a); 4.289 (1 H, dd, J = 6.5, 12.2, H-6b); 4.990 (1 H, ddd, J = 2.1,
3.9, 9.0, H-2); 5.107 (1 H, dd, J = 3.9, 9.0, H-3); 5.162 (1 H, dd, J = 8.3, 9.0, H-4); 5.416 (1 H,
d, J = 2.1, H-1); 5.945 (1 H, d, J = 9.0, CH3CONH-2); 7.069 (2 H, m , H-ortho); 8.206 (2 H, m ,
H-meta). 13C NMR (CDCl3): 20.59 (q, CH3CO-6), 20.65 (q, CH3CO-4), 20.70 (q, CH3CO-3),
23.34 (q, CH3CO-2), 49.39 (d, C-2), 62.33 (t, C-6), 65.80 (d, C-4), 70.72 (d, C-3), 73.03 (d,
C-5), 96.22 (d, C-1), 116.49 (d, 2 × C-ortho), 125.75 (d, 2 × C-meta), 143.24 (s, C-para),
160.90 (s, C-ipso), 169.58 (s, CH3CO-4), 170.11 (s, CH3CO-3), 170.23 (s, CH3CO-6), 170.60
(s, CH3CO-2). MALDI MS, m/z: 491.2 [M + Na]+.
4-Nitroph en yl 2-Acetam ido-2-deoxy-α-D-m an n opyran oside (14)
To a solution of 12 (50 m g, 0.11 m m ol) in m eth an ol (5 m l), sodium m eth oxide (2 M in
m eth an ol) was added to reach pH 10, an d th e m ixture was stirred for 2 h un til th e com plete
con version of 12 (TLC; CH2Cl2–EtOAc, 1:4). Th e reaction m ixture was n eutralized with
Dowex 50W-X2 (H form ), filtered an d con cen trated to give 14 (35 m g, 96%) as a wh ite
solid. 1H NMR (D2O): 1.880 (3 H, s, CH3CONH-2); 3.445 (1 H, dd, J = 2.4, 4.6, 10.0, H-5);
3.521 (1 H, dd, J = 9.6, 9.8, H-4); 3.533 (1 H, dd, J = 2.4, 12.4, H-6a); 3.587 (1 H, dd, J = 4.6,
12.4, H-6b); 4.053 (1 H, dd, J = 4.8, 9.6, H-3); 4.386 (1 H, dd, J = 1.6, 4.8, H-2); 5.487 (1 H,
d, J = 1.6, H-1); 5.942 (1 H, d, J = 8.4, CH3CONH-2); 7.034 (2 H, m , H-ortho); 8.013 (2 H, m ,
H-meta). 13C NMR (D2O): 22.07 (q, CH3CONH-2), 52.40 (d, C-2), 60.25 (t, C-6), 66.55 (d,
C-4), 68.93 (d, C-3), 73.52 (d, C-5), 96.92 (d, C-1), 116.76 (d, 2 × C-ortho), 126.16 (d, 2 ×
C-meta), 142.48 (s, C-para), 160.84 (s, C-ipso), 175.06 (s, CH3CONH-2). [α ]2D2 +53 (c 0.69
g/m ol, water). For C14H18N2O8 (342.3) calculated: 49.12% C, 5.30% H, 8.18% N; foun d:
49.33% C, 5.45% H, 8.21% N.
4-Nitroph en yl 2-Acetam ido-2-deoxy-β-D-m an n opyran oside (15)
Com poun d 15 was prepared from 13 (50 m g, 0.11 m m ol), usin g an an alogous procedure for
com poun d 14, in th e yield of 35 m g (96%) as a wh ite solid. 1H NMR (D2O): 1.925 (3 H, s,
CH3CONH-2); 3.44 (1 H, m , H-5); 3.45 (1 H, m , H-4); 3.646 (1 H, dd, J = 4.6, 12.4, H-6a);
3.737 (1 H, dd, J = 1.8, 12.4, H-6b); 3.74 (1 H, m , H-3); 4.51 (1 H, m , H-2); 5.378 (d, J = 1.7,
H-1); 6.939 (2 H, m , H-ortho); 8.006 (2 H, m , H-meta). 13C NMR (D2O): 22.32 (q, CH3CO-2),
53.14 (d, C-2), 60.58 (t, C-6), 66.83 (d, C-4), 71.93 (d, C-3), 77.00 (d, C-5), 96.74 (d, C-1),
116.77 (d, 2 × C-ortho), 126.38 (d, 2 × C-meta), 142.86 (s, C-para), 161.53 (s, C-ipso), 175.96
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4-Nitrophenyl 2-Acetamido-2-deoxy-D-mannopyranosides
811
(s, CH3CO-2). MALDI MS, m/z: 365.1 [M + Na]+. [α ]D22 –139 (c 0.69 g/m ol, water). For
14H18N2O8 (342.3) calculated: 49.12% C, 5.30% H, 8.18% N; foun d: 49.20% C, 5.39% H,
8.11% N.
C
Support from the Volkswagen Foundation and the Czech National Science Foundation
(No. 203/01/1018), by the Ministry of Education, Youth and Sports of the Czech Republic (project ME
371), institutional research concept (AV0Z5020903), the grant from the Hungarian Academy of Sci-
ences (OTKA 029027), and by a bilateral project of the Czech and Hungarian Academies of Sciences is
gratefully acknowledged.
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