Synthesis of linear and branched regioisomeric chitooligosaccharides as potential mimetics of natural oligosaccharide ligands of natural killer cells NKR-P1 and CD69 lectin receptors

Article abstract of DOI:10.1135/cccc20041781

Regioisomer of chitobiose 13 with β(1→3) glycosidic bond and branched analog of chitotriose 25 having β(1→4) and β(1→3) glycosidic bonds, were prepared and tested as potential mimetics of natural oligosaccharide ligands for activating lectin receptors NKR

Full text of DOI:10.1135/cccc20041781

Linear and Branched Regioisomeric Chitooligosaccharides
1781
SYNTHESIS OF LINEAR AND BRANCHED REGIOISOMERIC
CHITOOLIGOSACCHARIDES AS POTENTIAL MIMETICS OF
NATURAL OLIGOSACCHARIDE LIGANDS OF NATURAL
KILLER CELLS NKR-P1 AND CD69 LECTIN RECEPTORS
a3
Anna ROHLENOVÁa1, Miroslav LEDVINAa2,*, David ŠAMAN and
b
Karel BEZOUŠKA
a
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Flemingovo nám. 2, CZ-166 10, Prague 6, Czech Republic; e-mail: rohlenova@uochb.cas.cz,
ledvina@uochb.cas.cz, saman@uochb.cas.cz
Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8,
CZ-128 40 Prague 2, Czech Republic; e-mail: bezouska@natur.cuni.cz
1
2
3
b
Received July 2, 2004
Accepted August 20, 2004
Dedicated with due respect to Professor Miloslav Černý on the occasion of his 75th birthday in
recognition of his outstanding contributions to saccharide chemistry.
Regioisom er of ch itobiose 13 with β(1→3) glycosidic bon d an d bran ch ed an alog of ch ito-
triose 25 h avin g β(1→4) an d β(1→3) glycosidic bon ds, were prepared an d tested as poten tial
m im etics of n atural oligosacch aride ligan ds for activatin g lectin receptors NKR-P1A an d
CD69 of n atural killer (NK) cells. Th e structural requirem en ts of NKR-P1 lectin receptor on
effective m im etics of its n atural ligan ds h as been discussed. A sign ifican t bin din g activity of
th e bran ch ed trisacch aride 25 to th e receptor CD69 was observed.
Keyw ord s: Carboh ydrates; Oligosacch arides; Am in osugars; D-Glucosam in e; Glycosylation ;
Glycosides; NK cells; NKR-P1 an d CD69 lectin receptors; Lectin s; Im m un oth erapeutics.
In con n ection with th e recen t in creasin g occurren ce of path ological states
ch aracterized by im m un odeficien cy, con siderable atten tion h as been paid
to th e in vestigation of effective m odulation of in dividual com pon en ts of
th e im m un e system . Natural killer (NK) cells h ave been on e of th e targets.
Th eir activity is m odulated by sign als tran sm itted by in teraction of th eir
surface receptors with appropriate n atural ligan ds. A system atic study of
th e activatin g lectin receptor NKR-P1 sh owed th at com plex oligosac-
ch aride structures on th e surface of tum our cells exert n atural bin din g
affin ity to th is receptor1 an d th ereby in crease th e cytotoxic activity of NK
cells. Th ese n atural oligosacch aride ligan ds are too com plex for practical
Collect. Czech. Chem. Commun. (Vol. 69) (2004)
doi:10.1135/cccc20041781

1782
Rohlenová, Ledvina, Šaman, Bezouška:
use as im m un oth erapeutics. Th is led us to th e search for th eir sim plified
an d h en ce better available m im etics. We h ave reported earlier th at 2-acet-
am ido-2-deoxy-D-h exoses also h ave a sign ifican t bin din g activity2 an d th at
in th e series of ch itooligom ers con sistin g of β(1→4)-lin ked 2-acetam ido-
2-deoxy-D-glucopyran ose un its, th is activity in creases with elon gation of
th e oligosacch aride ch ain to th ree sugar un its3. Biological activity of oligo-
sacch arides h avin g oth er types of lin kages h as n ot been exten sively tested,
except for th e disacch arides with β(1→6)-lin ked N-acetyl-D-h exosam in e
un its, wh ich were sh own to be sligh tly less effective in h ibitors3. With th e
aim to determ in e th e steric requirem en ts of NKR-P1 an d CD69 receptors
with respect to th e ch aracter of glycosidic bon ds an d bran ch in g of oligo-
sacch aride ch ain , we syn th esized an d tested for th e bin din g activity 2-acet-
am ido-2-deoxy-β-D-glucopyran osyl-(1→3)-2-acetam ido-2-deoxy-D-gluco-
pyran ose (13) an d 2-acetam ido-2-deoxy-β-D-glucopyran osyl-(1→3)- [2-acet-
am ido-2-deoxy-β-D-glucopyran osyl-(1→4)]-2-acetam ido-2-deoxy-D-gluco-
pyran ose (25), wh erein 13 represen ts th e ch itobiose an alog with β(1→3)
glycosidic bon d (Sch em e 1) an d 25 represen ts th e bran ch ed ch itotriose an a-
log con tain in g β(1→4) an d β(1→3) glycosidic bon ds (Sch em e 3).
In com parison with oligosacch arides con sistin g of 2-am in o-2-deoxy-
D-glucopyran ose un its lin ked with β(1→4β) or (1→6) glycosidic bon ds, th e
syn th esis of oligosacch arides h avin g β(1→3) glycosidic bon d h as received
less atten tion 4. In th eir syn th esis, com m on m eth ods for th e 1,2-trans-
glycosidic lin kin g of 2-am in o-2-deoxyh exopyran ose residue, i.e., th e
Koen igs–Kn orr5, oxazolin e6–8 an d ph th alim ido9,10 m eth ods as well as th e
m eth od con trolled by 2,2,2-trich loroeth yl carbam ate group at C(2) of th e
glycosyl don or10 were used. Th ese oligosacch arides were also obtain ed by
usin g glycosyl don ors h avin g at C(2) th e n on participatin g azido or 2,5-di-
m eth ylpyrrole group an d (trich loroacetim idoyl)oxy group at C(1) as leavin g
group11,12. Th e syn th esis of bran ch ed oligosacch arides con tain in g β(1→4)
an d β(1→3) glycosidic bon ds h as n ot been in vestigated so far.
RESULTS AND DISCUSSION
In th e syn th esis of th e target bran ch ed trisacch aride β-D-GLcpNAc-(1→3)-
[β-D-GLcpNAc-(1→4)]-D-GLcpNAc (25), we were con fron ted with a sterically
h in dered system . Th erefore, th e ph th alim ide m eth od was applied to th e
glycosylation , because it is con sidered to be on e of th e m ost efficien t
1,2-trans-stereoselective glycosylation processes4 for low-reactive secon dary
OH groups. Th is glycosylation m eth od was also used in th e altern ative syn -
th esis of th e disacch aride β-D-GLcpNAc-(1→3)-D-GLcpNAc (13; lit.8).
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1783
Ben zyl 2-acetam ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (2) was
used as a glycosyl acceptor in th e preparation of target disacch aride 13
(Sch em e 1). Com poun d 2 was obtain ed from ben zyl 2-acetam ido-3-O-allyl-
4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside13 (1) by splittin g off th e allyl
group via its catalytic isom erization to prop-1-en yl group with Wilkin son
catalyst [Rh Cl(Ph 3P)3], followed by acid h ydrolysis. Glycosylation of 2 with
eth yl 3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-1-th io-β-D-glucopyran o-
side14 (3), prom oted by a com bin ation of m eth yl triflate an d silver triflate,
afforded ben zyl 3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran o-
syl-(1→3)-2-acetam ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (6) in
59% yield. Th e un wan ted O-m eth ylation of th e free OH group of glycosyl
BnO
BnO
(i)
O
O
BnO
AllO
BnO
HO
AcHN
AcHN
OBn
OBn
1
2
RO
BnO
BnO
RO
O
O
O
RO
RO
(ii) for 3
2
+
RO
RO
X
O
PhtN
AcHN
PhtN
(iii) for 4 or 5
Y
OBn
6, R = Bn
7, R = Ac
3, R = Bn; X = SEt and Y = H
4, R = Bn; X = Br and Y = H, resp.
5, R = Ac; X = Br and Y = H
(iv) for 6
(v) for 7
BnO
BnO
BnO
RO
RO
(vi)
O
O
O
O
BnO
RO
RO
RO
O
O
RO
AcHN
AcHN
AcHN
NH2
OBn
OBn
10, R = Bn
11, R = Ac
8, R = Bn
9, R = H
(vii) for 10
(viii) for 11
BnO
OH
OH
OH
O
O
(vii)
O
O
BnO
HO
O
HO
HO
HO
HO
O
OH
AcHN
AcHN
AcHN
AcHN
OBn
13
12
(i) RhCl(Ph3P)3in toluene, EtOH and H2O, reflux and then HCOOH, reflux; (ii) MeOTf
and AgOTf in CH2Cl2, r.t.; (iii) AgOTf in CH2Cl2,–45 oC; (iv) Na BH4 in propan-2-ol and
water, r.t. and then AcOH in toluene, 85 oC; (v) BuNH2 in MeOH, 85 oC; (vi) Ac2O in
pyridine, r.t.; (vii) H2 and Pd/C in AcOH, r.t.; (viii) MeONa in MeOH, r.t.
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1784
Rohlenová, Ledvina, Šaman, Bezouška:
acceptor with m eth yl triflate13 was suppressed by silver triflate. Durin g th e
reaction of glycosyl brom ide14 4, obtain ed from 3 by its reaction with bro-
m in e, with glycosyl acceptor 2 in th e presen ce of silver triflate as glyco-
sylation prom otor, n o sign ifican t in crease in th e yield was observed. Th is
reaction was carried with out base at –45 °C (lit.15), because th e base is
kn own to in h ibit glycosylation of less reactive h ydroxy groups16,17. Th e re-
action of glycosyl brom ide 5, protected by less bulky an d electron egative
O-acetyl groups, with glycosyl acceptor 2 un der th e last m en tion ed con di-
tion s gave disacch aride 13 in a yield of 72%. Th e disacch aride 6 was
reductively deph th aloylated (NaBH4), with respect to th e presen ce of
alkali-stable ben zyl group on th e vicin al OH-3 group14. Th e efficien t split-
tin g of th e ph th alim ido group with com m on ly used butylam in e in boilin g
m eth an ol requires participation of free n eigh borin g OH group in th e
deph th aloylation step; th e alkali-labile groups were preferen tially split off
by th e action of butylam in e. Th e free OH group can h en ce participate in
th e deph th aloylation process14. Th e obtain ed crude ben zyl 2-am in o-3,4,6-
tri-O-ben zyl-2-deoxy-β-D-glucopyran osyl-(1→3)-2-acetam ido-4,6-di-O-ben zyl-
2-deoxy-α-D-glucopyran oside (8) was subjected, with out purification , to
N-acetylation with acetic an h ydride in pyridin e to obtain ben zyl 2-acet-
am ido-3,4,6-tri-O-ben zyl-2-deoxy-β-D-glucopyran osyl-(1→3)-2-acetam ido-
4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (10). Subsequen t h ydrogen -
olysis of its ben zyl groups on Pd/C catalyst afforded th e target disacch aride
13. In th e case of partially O-acetylated disacch aride 7, th e alkali-labile
groups were rem oved with butylam in e an d th e form ed ben zyl 2-am in o-
2-deoxy-β-D-glucopyran osyl-(1→3)-2-acetam ido-4,6-di-O-ben zyl-2-deoxy-
α-D-glucopyran oside (9) was con verted, with out purification , to ben zyl
2-acetam ido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyran osyl-(1→3)-2-acet-
am ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (11) by treatm en t with
acetic an h ydride in pyridin e. Th e Zem plén O-deacetylation of 11 followed
by h ydrogen olysis of th e obtain ed ben zyl 2-acetam ido-2-deoxy-β-D-gluco-
pyran osyl-(1→3)-2-acetam ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside
(12) gave com poun d 13. Th e presen ted syn th eses of 13, based on th e use of
fully O-ben zyl-protected glycosyl don ors 3 or 4 an d glycosyl acceptor 2,
m in im ize th e n um ber of reaction steps wh ich are n ecessary to obtain th e
target com poun d in com parison with th e approach es m en tion ed above.
Th e efficien cy of th e latter of th e presen ted altern ative syn th eses of 13,
n am ely, th e on e em ployin g O-acetyl-protected glycosyl don or 5, is com pa-
rable with th e approach described in lit.8
In th e case of syn th esis of th e bran ch ed trisacch aride 25 (Sch em e 3), ben -
zyl 3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→4)-
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1785
2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (15) was used as th e
glycosyl acceptor at first (Sch em e 2). Th e ch oice of th e glycosyl acceptor 15
bearin g ph th alim ido group in position C-2 of th e secon d sacch aride un it
in stead of an alogous disacch aride with acetam ido group15 in th e sam e posi-
tion was aim ed at decreasin g of th e n egative in fluen ce of in tra- an d
in term olecular H-bridges between OH group an d th e NH group of am ide
fun ction in th e glycosylation process18–20. Com poun d 15 was obtain ed
from ben zyl 3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-
(1→4)-2-acetam ido-3-O-allyl-6-O-ben zyl-2-deoxy-α-D-glu copyran oside14
(14) by deallylation usin g th e above m en tion ed procedure. Th e glyco-
sylation of 15 prom oted by m eth yl triflate in th e presen ce of silver triflate
with O-ben zyl-protected eth yl 1-th ioglycoside 3 gave a com plex m ixture of
reaction products (Sch em e 2). 3,4,6-Tri-O-ben zyl-2-deoxy-2-ph th alim ido-
β-D-glucopyran osyl 3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-gluco-
BnO
BnO
BnO
BnO
(i)
O
O
O
O
BnO
BnO
BnO
BnO
O
AllO
14
O
HO
15
PhtN
PhtN
AcHN
AcHN
OBn
OBn
BnO
OBn
O
NPht
O
OBn
BnO
BnO
O
PhtN
OBn
16
BnO
+
(ii) for 3
RO
O
O
BnO
BnO
RO
RO
X
+ 15
PhtN
PhtN
Y
17
(iii) for 5
3, R = Bn; X = SEt and Y = H
5, R = Ac; X = Br and Y = H
BnO
BnO
O
O
PhtN
O
O
BnO
BnO
O
AcHN
AcO
OBn
AcO
AcO
PhtN
18
(i) RhCl(Ph3P)3 in toluene, EtOH and H2O, reflux and then HCOOH, reflux; (ii) MeOTf
and AgOTf in CH2Cl2, r.t.; (iii) AgOTf in CH2Cl2, –45 o
C
SCHEME 2
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1786
Rohlenová, Ledvina, Šaman, Bezouška:
pyran oside (16) an d 1,5-an h ydro-tri-O-ben zyl-2-deoxy-2-ph th alim ido-
D-arabino-h ex-1-en itol (17) were isolated as th e m ain reaction products, i.e.,
th e product of cross-reaction of two m olecules of th e glycosyl don or an d
th e product of its elim in ation . Th e form ation of sym m etrical disacch aride
with th e β,β(1↔1) glycosidic bon d, sim ilar to β,β-treh alose an d glycal from
BnO
BnO
(i)
O
O
HO
HO
HO
AllO
AcHN
AcHN
OBn
OBn
20
19
AcO
BnO
AcO
O
O
(ii)
AcO
AcO
O
O
AcO
AcO
O
20
+
Br
PhtN
O
AcO
AcHN
AcO
PhtN
OBn
5
AcO
PhtN
21
(iii)
AcO
OH
OH
BnO
BnO
O
O
O
AcHN
O
O
O
HO
HO
AcO
AcO
O
O
AcO
(iv)
O
NH2
AcHN
AcHN
AcO
OBn
OBn
O
HO
HO
AcO
NH2
AcHN
22
23
(v)
OH
OH
BnO
O
OH
O
AcHN
O
O
O
O
HO
HO
HO
HO
O
O
O
(vi)
OH
AcHN
O
AcHN
AcHN
OH
OH
OBn
HO
HO
HO
HO
AcHN
AcHN
25
24
(i) RhCl(Ph3P)3 in toluene, EtOH and H2O, reflux and then HCOOH, reflux; (ii)
AgOTf in CH2Cl2, –45 oC; (iii) BuNH2in MeOH, 85 oC; (iv) Ac2O in pyridine, r.t.; (v)
MeONa in MeOH, r.t.; (vi) H2 and Pd/C in AcOH, r.t.
SCHEME 3
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1787
glycosyl don ors derived from 3,4,6-tri-O-acetyl-2-deoxy-2-ph th alim ido-
D-glucopyran ose in th e case of glycosylation of less reactive glycosyl accep-
tors is kn own from th e literature21,22. Furth erm ore, th e com poun d by its
MS spectrum referred to as 3-O-m eth yl derivative of glycosyl acceptor 15,
was isolated in trace am oun ts. Th e use of glycosyl don or 5 protected by th e
less bulky an d electron egative O-acetyl groups gave th e required bran ch ed
ben zyl 3,4,6-tri-O-acetyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→3)-
[3,4,6-tri-O-ben zyl-2-d eoxy-2-p h th alim id o-β-D-glu cop yran osyl-(1→4)]-
2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (18) in low yield
(11%), alon g with th e un reacted glycosyl acceptor an d un iden tified
products. Th e problem of effective syn th esis of target bran ch ed trisac-
ch aride 25 was solved by application of th e approach based on sim ulta-
n eous glycosylation of m on osacch aride glycosyl acceptor with free 3-OH
an d 4-OH groups with a less bulky glycosyl don or (Sch em e 3). Th e suitable
glycosyl acceptor, ben zyl 2-acetam ido-6-O-ben zyl-2-deoxy-α-D-gluco-
pyran oside (20), was prepared from ben zyl 2-acetam ido-3-O-allyl-
6-O-ben zyl-2-deoxy-α-D-glucopyran oside15 (19) by its deallylation usin g th e
procedure m en tion ed above. Silver triflate-prom oted glycosylation of 20
with O-acetyl protected glycosyl brom ide 5 afforded th e desired ben zyl
3,4,6-tri-O-acetyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→3)-[3,4,6-
tri-O-acetyl-2-d eoxy-2-p h th alim id o-β-D-glu cop yran osyl-(1→4)]-2-acet-
am ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (21) in good yield (64%).
Th e alkali-labile protectin g groups of trisacch aride 21 were rem oved by
treatm en t with butylam in e, an d th e obtain ed crude diam in e 22 was
peracetylated to give ben zyl 2-acetam ido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyran osyl-(1→3)-[2-acetam ido-3,4,6-tri-O-acetyl-2-deoxy-β-D-gluco-
pyran osyl-(1→4)]-2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside
(23). It was th en O-deacetylated to give ben zyl 2-acetam ido-2-deoxy-
β-D-glucopyran osyl-(1→3)-[2-acetam ido-2-deoxy-β-D-glucopyran osyl-(1→4)]-
2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (24). Subsequen t de-
ben zylation (H2/Pd-C) fin ally afforded th e un protected bran ch ed tri-
sacch aride 25.
Biological Activity
Oligosacch arides 13 an d 25 were tested as in h ibitors of th e bin din g of
th e soluble dim eric rat NKR-P1A receptors to th e h igh -affin ity ligan d,
n eoglycoprotein GLcpNAc17BSA. NKR-P1A receptor dem on strates stron g
in teraction with m on osacch arides such as GLcpNAc (IC50 = 10–7 M; Fig. 1),
an d th is in teraction is furth er in creased with th e elon gation of lin ear
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1788
Rohlenová, Ledvina, Šaman, Bezouška:
ch itooligom er-type sequen ces such as ch itobiose (IC50 = 3 × 10–8 M; Fig. 1).
However, th e β(1→3)-lin ked disacch aride β-D-GLcpNAc-(1→3)-D-GLcpNAc
(13) is a m uch worse in h ibitor com pared with β(1→4) liked ch itobiose
β-D-GLcpNAc-(1→4)-D-GLcpNAc. Th ese results in dicate th at th e bin din g
groove for lin ear oligosacch arides occurrin g in NKR-P1A receptor23 accom -
m odates well on ly th e oligosacch arides with (1→4) glycosidic lin kages.
Wh ile th e greater flexibility of th e oligosacch aride ch ain of th e above m en -
tion ed (1→6)-lin ked disacch arides still m akes it possible to bin d such struc-
tures3, it is n ot possible to bin d (1→3)-lin ked lin ear structures. Sim ilarly,
oligosacch aride structures th at are bran ch ed an d n ot sufficien tly flexible
are very poor ligan ds for rat NKR-P1A receptor, such as trisacch aride
β-D-GLcpNAc-(1→3)-[β-D-GLcpNAc-(1→4)]-D-GLcpNAc (25) tested h ere
(IC50 = 2 × 10–4 M; Fig. 1).
Both com poun ds were also tested as poten tial m im etics of th e n atural
ligan ds of activatin g receptor of th e h aem opoietic cells CD69 wh ere th e
bran ch ed trisacch aride 25 exerted a sign ifican t bin din g activity. Th us, in
th e stan dard in h ibition assay, th e trisacch aride 25 was very poten t in h ibi-
tor with IC50 = 10–7 M, th us successfully com petin g with n atural oligosac-
ch arides of m uch greater com plexity, such as th e trian ten n ary octa-
sacch aride N3. Th e details of th is study will be publish ed separately.
100
80
60
40
20
0
–9
–8
–7
–6
–5
–4
–3
10
10
10
10
10
10
10
–1
Concentration of inhibitor, mmol l
FIG. 1
Plate in h ibition assay for th e evaluation of biological activity of th e syn th esized oligosac-
ch arides: ᭹ β-D-GlcpNAc-(1→4)-D-GlcpNAc, ᭺ D-GlcpNAc, ᭢ β-D-GlcpNAc-(1→3)-D-GlcpNAc,
᭞ β-D-GlcpNAc-(1→3)-[β-D-GlcpNAc-(1→4)]-D-GlcpNAc, ᭿ D-Man p
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1789
In sum m ary, th is paper provides n ew fin din gs about th e structural re-
quirem en ts of NKR-P1 lectin receptor for effective m im etics of its n atural
ligan ds, an d, in th e case of bran ch ed trisacch aride 25, th e first efficien t m i-
m etic of n atural oligosacch aride ligan ds of CD69 lectin receptor. Th ese re-
sults, th erefore, open th e way to a m ore exact design of n ew effective lig-
an ds of both n am ed lectin receptors an d h en ce of poten tial im m un o-
th erapeutics.
EXPERIMENTAL
Meltin g poin ts were determ in ed on a Kofler block an d are un corrected. Optical rotation s
were m easured on a Perkin –Elm er 141 polarim eter at 25 °C an d are given in 10–1 deg cm 2 g–1
.
Th e IR spectra were recorded on a Bruker IFS 88 (FTIR) spectrom eter, waven um bers are given
in cm –1. NMR spectra were recorded usin g a Bruker Avan ce spectrom eter in th e FT m ode at
500.1 MHz (1H) an d at 125.8 MHz (13C) in deuterioch loroform , usin g tetram eth ylsilan e as
in tern al stan dard for 1H NMR spectra an d deuterioch loroform (δ 77.0) as stan dard for
13C NMR spectra. Ch em ical sh ifts are given in ppm (δ-scale) an d couplin g con stan ts (J) in
Hz. For un am biguous assign m en t of sign als in 13C NMR spectra of com poun ds 6, 7, 10, 11,
15, 21, an d 24, th e h eterocorrelated 2D NMR spectra were m easured by th e HSQC tech n ique
usin g th e stan dard pulse sequen ce delivered by th e m an ufacturer of th e spectrom eter. Th e
followin g set of ch aracteristic param eters was used: spectral width in both f1 an d f2 dim en -
sion s 4500 an d 17 000 Hz, respectively, n um ber of scan s 32, n um ber of in crem en ts in f1
dim en sion 256, recycle delay 1 s, acquisition tim e 0.2 s, data m atrix for processin g 2048 ×
2048 datapoin ts. For processin g sin e squared weigh tin g fun ction was used. NMR data (if n ot
given in th e text) are sum m arized in Tables I–VI. Positive-ion FAB m ass spectra were m ea-
sured on a BEqG geom etry m ass spectrom eter ZAB-EQ (VG An alytical, Man ch ester, U.K.), us-
in g an M-Scan FAB gun (Xe, en ergy 8 keV) at an acceleratin g voltage of 8 kV. Sam ples were
dissolved in ch loroform or m eth an ol an d th e m ixture glycerol–th ioglycerol or 3-n itroben zyl
alcoh ol was used as m atrix. For com plex m ixtures, an A P 4000 pum p with SCM 1000
vacuum degasser (all Fin n igan , In c., U.S.A.) were used for pum pin g th e flow. Th e m obile
ph ase con sisted of 90% MeOH, th e flow rate was 0.7 m l m in –1. An am oun t of 5 µl of an alyte
solution was directly in troduced to an LCQ m ass spectrom etric detector (Fin n igan , In c.,
U.S.A.) equipped with an ion -trap an alyzer. +APCI ion ization was used for recordin g spectra.
Th e spectrom eter worked in a full-scan m ode with th e m/z 50–2000 Dalton ran ge of an a-
lyzed ion s. Th e capillary was h eated at 200 °C, th e ion source was h eated to 475 °C, dis-
ch arge curren t was set to 4.5 µA, 24 V on capillary. Th in -layer ch rom atograph y (TLC) was
perform ed on Silufol UV254 sh eets (Kavalier, Votice, Czech Republic) or DC-Alufolien
Kieselgel 60 F254 (Merck, Darm stadt, Germ an y), an d colum n ch rom atograph y on silica gel
Fluka Silica gel 60 (40–63 µm ; Fluka, Neu-Ulm , Switzerlan d). An alytical RP HPLC was per-
form ed with a Waters Allian ce HPLC System (PDA 996 detector, software Millen n ium 32
Ch rom atograph y Man ager, Waters, Milford, Massach usetts, U.S.A.) equipped with a colum n
(150.0 × 3.9 m m ) filled with Nova-Pak C18 (4 µm ; Waters). Preparative RP HPLC was per-
form ed with a Kn auer apparatus (Bad Hom burg, Germ an y) equipped with a colum n (250 ×
25 m m ) filled with LiCh rosorb RP-18 (5 µm ; Merck, Darm stadt, Germ an y). Solution s were
evaporated on a rotatory evaporator. An alytical sam ples were dried at 6.5 Pa an d at 25 °C
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1790
Rohlenová, Ledvina, Šaman, Bezouška:
for 8 h . Dich lorom eth an e was distilled from ph osph orus pen toxide an d stored over m olecu-
lar sieve 4A. Silver trifluorom eth an esulfon ate was recrystallized from toluen e.
Preparation an d Radioiodin ation of Soluble Dim eric Rat NKR-P1A Receptor
Soluble dim eric rat NKR-P1A receptor (NKR358) was expressed in Escherichia coli, refolded,
cleaved with en terokin ase, an d purified essen tially as described previously3. Th is protein was
radioiodin ated as reported2, with carrier-free Na125I (Am ersh am ) to a specific activity of
107 cpm per µg protein .
Plate In h ibition Assays
Bin din g an d in h ibition assays were perform ed as described previously2 with m in or m odifica-
tion s. Briefly, 96-well poly(vin yl ch loride) m icroplates (Titertek Im m un o Assay-Plate, ICN
Flow, Irvin e, U.K.) were coated overn igh t at 4 °C with 50 µl of GLcpNAc17BSA (10 µg/m l,
Sigm a) in TBS+C buffer (10 m M Tris-HCl pH 8.0 with 150 m M NaCl, 1 m M CaCl2, an d 1 m M
NaN3). Plates were blocked with 1% BSA (Sigm a) in TBS+C at 4 °C for 2 h , an d in cubated
with 125I-NKR358 correspon din g to h alf of th e saturation am oun t an d th e in dicated dilu-
tion s of th e tested com poun ds in a total reaction volum e of 100 µl. Plates were wash ed
th ree tim es with TBS+C, drain ed, dried, an d 100 µl of a scin tillation solution was added to
each well. Radioactivity in wells was determ in ed in a β-coun ter Microbeta (Wallac). All ex-
perim en ts were perform ed in duplicate. Th e degree of in h ibition was calculated relative to
wells with out an y in h ibitor (addition of water).
Microtitre plates were coated with a h igh -affin ity ligan d, an d th e bin din g of th e radio-
labeled NKR-P1A protein to th is ligan d was in h ibited by serial dilution s of th e tested oligo-
sacch arides as described in Experim en tal. D-GLcpNAc an d D-Man p were used as a positive
an d n egative con trol, respectively. Ch itobiose (β-D-GLcpNAc-(1→4)-D-GLcpNAc) was used as
th e h igh -affin ity ligan d. Th e results are average values from duplicate experim en ts with in
th e ran ge in dicated by error bars.
Ben zyl 2-Acetam ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (2)
Ben zyl 2-acetam ido-3-O-allyl-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside13 (1; 1.035 g,
2 m m ol) an d tris(triph en ylph osph in e)rh odium (I) ch loride (233 m g, 0.25 m m ol) were
refluxed in a m ixture of eth an ol–toluen e–water (7:3:2; 60 m l) un der stirrin g for 4 h . Form ic
acid (2 m l) was added an d reflux was con tin ued for an oth er 3 h . Th e m ixture was evapo-
rated in vacuo an d th e residue was ch rom atograph ed on a silica gel colum n (40 m l) in
toluen e–eth yl acetate (2:1) to give 698 m g (71%) of com poun d 2. An an alytical sam ple was
prepared by crystallization from eth yl acetate–petroleum eth er, m .p. 134–135 °C, [α]D +69
(c 0.4, ch loroform ); lit.24 [α]D +65 (c 1.1, ch loroform ). 1H NMR spectrum agrees with th e
data for th e auth en tic sam ple prepared by procedure described in lit.24 For 2 (C29H33NO6)
calculated: relative m olecular m ass 491.6, m onoisotopic m ass 491.2. FAB MS, m/z: 492 [M + H]+.
For C29H33NO6 (491.6) calculated: 70.86% C, 6.77% H, 2.85% N; foun d: 70.77% C, 6.82% H,
2.78% N.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1791
Ben zyl 3,4,6-Tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→3)-
2-acetam ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (6)
Method A: Eth yl 3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-1-th io-β-D-glucopyran oside14 (3;
624 m g, 1 m m ol), com poun d 2 (246 m g, 0.5 m m ol), silver trifluorom eth an esulfon ate
(129 m g, 0.5 m m ol) an d crush ed m olecular sieve 4A (1 g) were dried in an apparatus
equipped with a septum at room tem perature an d 1.32 Pa for 10 h an d th en th e apparatus
was flush ed with argon (2×). Dry dich lorom eth an e (5 m l) was added th rough th e septum
un der stirrin g an d th e m ixture was stirred at room tem perature for 1 h . Th en m eth yl
trifluorom eth an esulfon ate (113 µl, 1 m m ol) was added th rough th e septum an d stirrin g was
con tin ued at room tem perature for 12 h . Th e addition of th e sam e am oun t of m eth yl
trifluorom eth an esulfon ate followed by 12-h stirrin g was repeated twice m ore. After coolin g
to –20 °C, dry pyridin e (1 m l) was added th rough th e septum an d th e m ixture was stirred at
–15 °C for 30 m in an d th en at room tem perature for 1 h . Th e m ixture was diluted with
ch loroform (150 m l) an d filtered th rough Celite. Th e filtrate was wash ed with 10% aqueous
NaHSO4, saturated aqueous NaHCO3, an d 5% aqueous NaCl (30 m l each ). Th e organ ic ph ase
was dried over an h ydrous MgSO4 an d evaporated in vacuo. Ch rom atograph y of th e solid
residue on a silica gel colum n (70 m l) in toluen e–eth yl acetate (2:1) followed by HPLC sepa-
ration on a silica gel colum n C18 in th e solven t system water–m eth an ol (lin ear gradien t
75→90%/70 m in ) afforded 310 m g (59%) of solid com poun d 6.
Method B: Eth yl 3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-1-th io-β-D-glucopyran oside14 (3;
624 m g, 1 m m ol) was dried in an apparatus equipped with septum at room tem perature an d
1.32 Pa for 10 h . Th e apparatus was flush ed with argon (2×) an d dry dich lorom eth an e
(2.3 m l) was added th rough th e septum . After dissolution , th e m ixture was cooled to 0 °C
an d 1 M solution of brom in e in dry dich lorom eth an e (1.1 m l) was added th rough th e sep-
tum un der stirrin g. Th en th e m ixture was stirred at 0 °C for 1 h an d th en at room tem pera-
ture for 1 h . In th e sam e apparatus th e solven ts were evaporated in vacuo (water pum p)
with exclusion of m oisture. Th e residue was co-evaporated with toluen e (3 × 2 m l) at 20 Pa,
added th rough th e septum an d th e residue was dissolved in dry dich lorom eth an e (1.3 m l).
Th e solution of glycosyl brom ide14 4 was used for th e con den zation with com poun d 2.
Com poun d
2 (246 m g, 0.5 m m ol) an d silver trifluorom eth an esulfon ate (129 m g,
0.5 m m ol) were dried in an apparatus equipped with septum at room tem perature an d
1.32 Pa for 10 h . Th e apparatus was wash ed with argon (2×) an d dry dich lorom eth an e
(1.3 m l) was added th rough th e septum . After dissolution , th e m ixture was cooled to –45 °C
an d a solution of glycosyl brom ide 4 was added un der stirrin g th rough th e septum durin g
1 h . Th e m ixture was stirred at –45 °C for an oth er 30 m in an d th en at –20 °C for 1 h . Pyri-
din e (0.4 m l) was added th rough th e septum an d th e m ixture was stirred at –20 °C for
20 m in . After warm in g to room tem perature, th e m ixture was diluted with ch loroform
(100 m l), filtered an d th e filtrate was wash ed with saturated aqueous NaHCO3 (25 m l) an d
water (3 × 25 m l). Th e organ ic ph ase was separated, dried over an h ydrous MgSO4, evapo-
rated in vacuo an d th e residue was co-distilled with toluen e. Th e residue was worked up us-
in g th e sam e procedure as given in m eth od A to afford 331 m g (63%) of solid com poun d 6,
m .p. 150 °C, [α]D +41 (c 0.3, ch loroform ). For 6 (C64H64N2O12) calculated: relative m olecular
m ass 1053.2, m on oisotopic m ass 1052.5. FAB MS, m/z: 1053.4 [M + H]+, 1075.3 [M + Na]+.
For C64H64N2O12 (1053.2) calculated: 72.99% C, 6.12% H, 2.66% N; foun d: 72.71% C,
6.29% H, 2.48% N.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1792
Rohlenová, Ledvina, Šaman, Bezouška:
TABLE I
1H NMR param eters of com poun ds 6, 7, 10–12
Param eter
6
7
10
11
12
δ(H-1)
δ(H-2)
δ(H-3)
δ(H-4)
δ(H-5)
δ(H-6a)
δ(H-6b)
δ(H-1′)
δ(H-2′)
δ(H-3′)
δ(H-4′)
δ(H-5′)
δ(H-6a′)
δ(H-6b′)
4.63 d
4.64 d
4.78 d
4.79 d
4.81 d
4.01 ddd
4.15 dd
4.04–4.07 m
4.04–4.07 m
3.51 dd
3.80 ddd
3.58 dd
3.62 dd
5.47 d
4.24 dt
4.14 dd
3.58 dd
3.85 ddd
3.65 dd
3.72 dd
4.68 d
4.25 ddd
4.06 dd
3.59 dd
3.84 ddd
3.63 dd
3.70 dd
4.67 d
4.31 ddd
4.04 dd
3.66 dd
3.84 ddd
3.66 dd
3.78 dd
4.39 d
3.44 dd
3.80 dt
3.60 d
3.60 d
5.29 d
4.16 dd
4.28 dd
5.86 dd
5.12 dd
3.83 ddd
4.05 dd
4.30 dd
3.72 dd
3.61 dd
3.72 dd
3.46 ddd
3.55 dd
3.75 dd
3.95 dt
5.01 dd
5.09 t
3.47 dd
3.37 ddd
3.41 ddd
3.27 ddd
3.46 dd
3.76 dd
4.44–4.46 m
3.62–3.65 m
3.62–3.65 m
3.51–3.54 m
3.78 dd
3.60 ddd
4.04 dd
4.25 dd
J(1,2)
3.8
10.5
8.5
3.8
4.0
9.9
4.1
9.8
4.1
9.9
a
J(2,3)
J(3,4)
8.4
10.1
2.2
8.4
8.4
8.7
J(4,5)
10.1
3.3
10.2
2.1
10.1
2.1
10.1
1.9
J(5,6a)
J(5,6b)
J(6a,6b)
J(1′,2′)
J(2′,3′)
J(3′,4′)
J(4′,5′)
J(5′,6a′)
J(5′,6b′)
J(6a′,6b′)
3.3
a
4.2
4.3
4.3
3.8
10.6
8.3
10.7
8.3
10.7
8.4
10.6
8.1
8.3
10.7
a
10.7
9.0
9.5
10.4
9.3
9.5
8.5
8.4
a
a
10.2
2.6
9.5
9.8
9.2
2.2
2.7
3.4
1.3
4.5
5.5
4.4
6.4
10.5
12.2
10.6
12.2
11.9
a
Value n ot determ in ed. Addition al NMR param eters an d param eters of substituen ts: 6 – arom . H: 6.86–7.33 m ,
NHAc: 5.58 d (1 H, J = 9.8), OCH2C6H5: 5.01 d (1 H, J = 11.1), 4.81 d (1 H, J = 11.0), 4.76 d (1 H, J = 11.9), 4.60 d
(1 H, J = 11.8), 4.60 d (1 H, J = 11.8), 4.50 d (1 H, J = 12.2), 4.49 d (1 H, J = 11.6), 4.48 d (1 H, J = 11.1), 4.42 d
(1 H, J = 11.6), 4.41 d (1 H, J = 12.2), 4.40 d (1 H, J = 11.9), 4.34 d (1 H, J = 11.8), NHAc: 1.85 s (3 H); 7 – arom .
H: 7.24–7.89 m , NHAc: 5.45 d (1 H, J = 9.8), OCH2C6H5: 5.02 d (1 H, J = 11.1), 4.64 d (1 H, J = 11.6), 4.52 d (1 H,
J = 12.1), 4.48 d (1 H, J = 11.1), 4.46 d (1 H, J = 12.1), 4.35 d (1 H, J = 11.6), OAc: 2.00 s (3 H), 1.93 s (3 H), 1.92 s
(3 H), NHAc: 1.84 s (3 H); 10 – arom . H: 7.12–7.35 m , NHAc: 6.07
d (1 H, J = 7.8), 5.58 d (1 H, J = 9.2),
OCH2C6H5: 4.99 d (1 H, J = 10.9), 4.77 d (1 H, J = 10.9), 4.76 d (1 H, J = 11.0), 4.70 d (1 H, J = 11.0), 4.69 d (1 H,
J = 11.7), 4.59 d (1 H, J = 12.1), 4.53 d (1 H, J = 10.9), 4.52 d (1 H, J = 12.1), 4.46 d (1 H, J = 12.1), 4.44 d (1 H, J =
10.9), 4.43 d (1 H, J = 11.7), 4.41 d (1 H, J = 12.1), NHAc: 1.97 s (3 H), 1.90 s (3 H); 11 – arom . H: 7.20–7.39 m ,
NHAc: 6.22 d (1 H, J = 8.2), 5.94 d (1 H, J = 9.3), OCH2C6H5: 4.97 d (1 H, J = 10.7), 4.70 d (1 H, J = 11.6), 4.58 d
(1 H, J = 12.1), 4.51 d (1 H, J = 12.1), 4.43 d (1 H, J = 11.6), 4.41 d (1 H, J = 10.7), OAc: 2.01 s (3 H), 2.00 s (3 H),
1.99 s (3 H), NHAc: 1.98 s (3 H), 1.93 s (3 H); 12 – arom . H: 7.18–7.40 m , 3′-OH: 6.42 d (1 H, J = 2.1), NHAc:
6.11 d (1 H, J = 9.3), OCH2C6H5: 4.84 d (1 H, J = 10.4), 4.72 d (1 H, J = 11.7), 4.64 d (1 H, J = 12.1), 4.53 d (1 H, J =
12.1), 4.47 d (1 H, J = 11.7), 4.43 d (1 H, J = 10.4), 4′-OH: 3.00 d (1 H, J = 2.0), NHAc: 2.17 s (3 H), 1.96 s (3 H).
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1793
Ben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→3)-
2-acetam ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (7)
Com poun d 2 (492 m g, 1 m m ol) an d silver trifluorom eth an esulfon ate (520 m g, 2.02 m m ol)
were dried in an apparatus equipped with septum at room tem perature an d 1.32 Pa for 10 h .
Th e apparatus was flush ed with argon (2×) an d th en dry dich lorom eth an e (2 m l) was added
th rough th e septum . After dissolution , th e m ixture was cooled to –45 °C an d a solution of
3,4,6-tri-O-acetyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl brom ide15 (5; 996 m g, 2 m m ol)
in dry dich lorom eth an e (2 m l) was added un der stirrin g th rough th e septum durin g 1 h .
Th en th e m ixture was stirred at –45 °C for an oth er 30 m in an d at –20 °C for 1 h . Pyridin e
(0.4 m l) was added th rough th e septum an d th e m ixture was stirred at –20 °C for 20 m in .
After warm in g to room tem perature, th e m ixture was diluted with ch loroform (250 m l), fil-
tered an d th e filtrate was wash ed with saturated aqueous NaHCO3 (50 m l) an d water (3 ×
50 m l). Th e organ ic ph ase was separated, dried over an h ydrous MgSO4, evaporated in vacuo
an d th e residue was co-distilled with toluen e. Ch rom atograph y of th e solid residue on a sil-
ica gel colum n (60 m l) in toluen e–eth yl acetate (1:1) afforded 657 m g (72%) of com poun d 7
as a solid foam , [α]D +11 (c 0.2, ch loroform ). For 7 (C49H52N2O15) calculated: relative m olec-
TABLE II
a
13C NMR ch em ical sh ifts of com poun ds 6, 7, 10–12
Carbon
6
7
10
11
12
1
96.73
52.76
76.47
76.17
70.70
68.90
97.11
56.76
79.21
80.00
75.08
69.11
96.89
52.63
77.75
76.17
70.70
68.74
97.64
55.14
70.23
69.27
71.39
62.17
96.78
53.31
77.66
76.09
70.49
68.73
99.12
56.62
78.56
82.49
75.67
69.46
96.78
53.30
79.08
72.02
70.21
68.40
99.55
54.23
73.49
68.61
75.60
62.08
96.63
53.08
79.20
72.72
70.43
68.30
99.60
58.27
77.72
75.52
75.43
62.59
2
3
4
5
6
1′
2′
3′
4′
5′
6′
a
Ch em ical sh ifts for arom atic carbon s from protectin g groups are n ot given . Addition al 13C
NMR ch em ical sh ifts of substituen ts: 6 – OCH2C6H5: 75.11 t, 74.92 t, 74.92 t, 73.47 t,
73.33 t, 69.46 t, NHAc: 169.82 s, 23.32 q; 7 – OCH2C6H5: 75.14 t, 73.39 t, 69.67 t, NHAc:
169.54 s, 23.46 q, OAc: 170.67 s, 170.14 s, 170.00 s, 20.60 q, 20.60 q, 20.40 q; 10 –
OCH2C6H5: 75.11 t, 74.92 t, 74.81 t, 73.52 t, 73.52 t, 69.65 t, NHAc: 171.18 s, 170.49 s,
23.63 q, 23.52 q; 11 – OCH2C6H5: 75.21 t, 73.56 t, 69.79 t, NHAc: 169.27 s, 170.74 s, 23.85 q,
23.15 q, OAc: 171.41 s, 170.74 s, 170.74 s, 20.69 q, 20.61 q, 20.58 q; 12 – OCH2C6H5: 75.30 t,
73.61 t, 69.87 t, NHAc: 173.29 s, 171.43 s, 23.68 q, 22.65 q.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1794
Rohlenová, Ledvina, Šaman, Bezouška:
ular m ass 908.9, m on oisotopic m ass 908.3. FAB MS, m/z: 909 [M + H]+, 932 [M + Na]+. For
49H52N2O15 (908.9) calculated: 64.75% C, 5.77% H, 3.08% N; foun d: 64.59% C, 5.87% H,
2.95% N.
C
Ben zyl 2-Acetam ido-3,4,6-tri-O-ben zyl-2-deoxy-β-D-glucopyran osyl-(1→3)-
2-acetam ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (10)
Sodium boroh ydride (287 m g, 7.6 m m ol) was gradually added durin g 20 m in to a stirred
suspen sion of com poun d 6 (480 m g, 0.46 m m ol) in a m ixture of propan -2-ol–water (6:1;
20 m l) at room tem perature an d th e m ixture was stirred for 6 h . Th en an oth er portion of so-
dium boroh ydride (28.7 m g, 0.76 m m ol) was added an d th e stirrin g was con tin ued for an -
oth er 6 h . Th e solven ts were evaporated in vacuo an d th e residue was taken between ch loro-
form (50 m l) an d 5% aqueous NaCl (5 m l). Th e organ ic layer was separated, wash ed with
5% aqueous NaCl (2 × 5 m l), dried over an h ydrous MgSO4 an d evaporated in vacuo. Th e
residue was co-distilled with toluen e (2 × 5 m l) an d dissolved in a m ixture of toluen e–acetic
acid (6:1; 7 m l) an d th e solution was h eated at 70 °C for 6 h . Th e solven ts were evaporated
in vacuo an d th e residue was co-distilled with toluen e (2 × 5 m l) to give crude ben zyl
2-am in o-3,4,6-tri-O-ben zyl-2-deoxy-β-D-glucopyran osyl-(1→3)-2-acetam ido-4,6-di-O-ben zyl-
2-deoxy-α-D-glucopyran oside (8), wh ich was used with out furth er purification for th e prepa-
ration of com poun d 10.
Th e obtain ed com poun d 8 was dissolved in a m ixture of dich lorom eth an e–pyridin e (10:1;
5 m l). To th e stirred solution at room tem perature, acetic an h ydride (0.2 m l) was slowly
added an d stirrin g was con tin ued for 2 h . Th e solven ts were evaporated in vacuo an d th e
residue was co-distilled with toluen e (2 × 5 m l). Ch rom atograph y of th e residue on a silica
gel colum n (50 g) in toluen e–eth yl acetate (1:1) gave 270 m g (61%) of solid com poun d 10.
An an alytical sam ple was prepared by crystallization from toluen e–petroleum eth er, m .p.
164–166 °C, [α]D +41 (c 0.4, ch loroform ). For 10 (C58H64N2O11) calculated: relative m olecu-
lar m ass 965.1, m on oisotopic 964.5. FAB MS, m/z: 965.0 [M + H]+, 987 [M + Na]+. For
C
58H64N2O11 (965.1) calculated: 72.18% C, 6.68% H, 2.90% N; foun d: 72.27% C, 6.76% H,
2.79% N.
Ben zyl 2-Acetam ido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyran osyl-(1→3)-
2-acetam ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (11)
A solution of com poun d 7 (650 m g, 0.72 m m ol) in a m ixture of dry m eth an ol–butylam in e
(4:1; 15 m l) was h eated in a pressure bottle at 85 °C for 10 h . After coolin g, th e m ixture was
evaporated an d th e solid residue was extracted with eth er (3 × 50 m l). Th e in soluble residue
was dissolved in a m ixture of m eth an ol–water (9:1; 5 m l), th e solution was adjusted to pH 4
with form ic acid an d poured on to a colum n of ion -exch an ge resin Dowex 50 (in H+ form )
(150 m l). Th e colum n was wash ed with a m ixture of m eth an ol–water (9:1; 250 m l) an d th e
product was eluted with a m ixture of m eth an ol–25% aqueous am m on ia (7:1; 500 m l). Evap-
oration of th e eluate in vacuo yielded 241 m g (51.3%) of syrupy crude ben zyl 2-am in o-
2-deoxy-β-D-glucopyran osyl-(1→3)-2-acetam ido-4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside
(9), wh ich was used with out an y furth er purification for th e preparation of com poun d 11.
For 9 (C35H45N2O10) calculated: relative m olecular m ass 652.7, m on oisotopic m ass 652.3.
FAB MS, m/z: 653 [M + H]+.
A solution of com poun d
9 (210 m g, 0.322 m m ol) in a m ixture of pyridin e–acetic
an h ydride (2:1; 5 m l) was kept at room tem perature for 20 h . Meth an ol (5 m l) was added
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1795
un der stirrin g at 0 °C to decom pose excess of acetic an h ydride. After 20 m in stan din g at
room tem perature, th e m ixture was evaporated in vacuo an d th e residue was co-distilled
with toluen e (3 × 10 m l). Ch rom atograph y of th e residue on a silica gel colum n (60 m l) in
ch loroform –eth yl acetate (3:2) followed by crystallization from eth yl acetate an d ch loroform
afforded 180 m g (68%) of com poun d 11, m .p. 165–167 °C, [α]D +52 (c 1, m eth an ol). For 11
(C43H52N2O14) calculated: relative m olecular m ass 820.9, m on oisotopic m ass 820.3. FAB MS,
m/z: 821 [M + H]+, 843 [M + Na]+, 713 [M – OBn + H]+. For C43H52N2O14 (820.9) calculated:
62.92% C, 6.38% H, 3.41% N; foun d: 62.78% C, 6.45% H, 3.30% N.
Ben zyl 2-Acetam ido-2-deoxy-β-D-glucopyran osyl-(1→3)-2-acetam ido-
4,6-di-O-ben zyl-2-deoxy-α-D-glucopyran oside (12)
A suspen sion of com poun d 11 (130 m g, 0.158 m m ol) in 0.01 M CH3ONa in m eth an ol (4 m l)
was stirred at room tem perature for 2 h an d th e obtain ed solution kept at –3 °C overn igh t.
Th e m ixture was n eutralized by addition of Dowex 50 (pyridin ium form ), th e ion exch an ger
was filtered off, wash ed with m eth an ol (20 m l) an d th e filtrate was evaporated in vacuo.
Th e HPLC separation of th e residue on a silica gel colum n C18 in th e solven t system
water–m eth an ol (lin ear gradien t 55→75%/60 m in ) afforded 109 m g (99%) of solid com -
poun d 12, [α]D +73 (c 0.4, m eth an ol). For 12 (C37H46N2O11) calculated: relative m olecular
m ass 694.8, m on oisotopic m ass 694.3. FAB MS, m/z: 695 [M + H]+. For C37H46N2O11 (694.8)
calculated: 63.96% C, 6.67% H, 4.03% N; foun d: 64.08% C, 6.73% H, 3.88% N.
2-Acetam ido-2-deoxy-β-D-glucopyran osyl-(1→3)-2-acetam ido-
2-deoxy-D-glucopyran ose (13)
Com poun d 10 (193 m g, 0.2 m m ol) or com poun d 12 (139 m g, 0.2 m m ol) was h ydrogen o-
lyzed in acetic acid (15 m l) over 10% palladium catalyst on carbon (200 m g) at room tem -
perature for 20 h . After th is tim e th e vessel was flush ed with argon , th e catalyst was filtered
off, wash ed with acetic acid (30 m l) an d th e filtrate was lyoph ilized. Th e solid residue was
separated by HPLC on a silica gel colum n C18 in water followed by perm eatin g ch rom atog-
raph y on Toyopearl HW 40F (450 m l) in water. Th e h om ogen ous fraction was evaporated in
vacuo an d th e residue was lyoph ilized from water, to give an α/β-an om eric m ixture of com -
poun d 13. Yield 65 m g (76%) of α/β-an om eric m ixture (5:3) from 10. Yield 56 m g (66%) of
α/β-an om eric m ixture (5:3) from 12. Th e ratio of an om ers was determ in ed by 1H NMR spec-
tra. [α]D –17 (c 0.2, water); lit.25 [α]D +14.5 → +6.5 (water) an d lit.8 [α]D +40 → +4 (water) .
1H NMR (500 MHz, D2O, 300 K): 5.20 d (1 H, J = 3.5, H-1); 4.74 d (1 H, J = 8.3, H-1); 4.67 d
(1 H, J = 8.3, H-1′); 4.66 d (1 H, J = 8.3, H-1′); 4.06 m (1 H, H-2); 3.81 m (1 H, H-2); 3.78 m
(2 H, H-2′); 3.53–4.02 m (10 H, H-3, H-4, H-5, H-6a, H-6b, H-3′, H-4′, H-5′, H-6′a, H-6′b);
2.31 s, 2.30 s, 2.21 s, 2.18 s (4 × 3 H, NHAc). 13C NMR (125 MHz, D2O, 300 K): 174.50 s,
174.46 s, 173.80 s (2 C); 101.14 d (2 C, C-1′); 95.08 d (C-1); 90.94 d (C-1); 81.53 d, 79.02 d,
75.86 d, 75.79 d, 75.50 d, 73.37 d, 73.35 d, 71.19 d, 69.85 d (2 C); 68.61d, 68.60 d, 60.85 t
(C-6); 60.71 t (2 C, C-6); 60.64 t (C-6); 55.81 d (C-2′); 55.78 d (C-2′); 55.68 d (C-2); 53.03 d
(C-2); 22.39 q, 22.38 q, 22.33 q, 22.15 q. For 13 (C16H28N2O11) calculated: relative m olecu-
lar m ass 424.4, m on oisotopic m ass 424.2. FAB MS, m/z: 425 [M + H]+. For C16H28N2O11
(424.4) calculated: 45.28% C, 6.65% H, 6.60% N; foun d: 45.13% C, 6.76% H, 6.48% N.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1796
Rohlenová, Ledvina, Šaman, Bezouška:
Ben zyl 3,4,6-Tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→4)-
2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (15)
Ben zyl 3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→4)-2-acetam ido-
3-O-allyl-6-O-ben zyl-2-deoxy-α-D-glucopyran oside14 (14; 1 g, 1 m m ol) an d tris(triph en yl-
ph osph in e)rh odium (I) ch loride (0.1 g, 0.11 m m ol) were refluxed in a m ixture of eth an ol–
toluen e–water (7:3:2; 30 m l) un der stirrin g for 5 h . Form ic acid (0.7 m l) was added an d
th e reflux was con tin ued for an oth er 2 h . Th e m ixture was evaporated in vacuo an d th e
residue was ch rom atograph ed on a silica gel colum n (120 m l) in toluen e–eth yl acetate
(10:4) to give 482 m g (50%) of com poun d 15 as a solid foam , [α]D +99 (c 0.3, ch loroform ).
For 15 (C57H58N2O12) calculated: relative m olecular m ass 963. 1, m on oisotopic m ass 962.4.
FAB MS, m/z: 963.7 [M + H]+. For C57H58N2O12 (963.1) calculated: 71.09% C, 6.07% H,
2.91% N; foun d: 70.90% C, 6.12% H, 3.02% N.
An Attem pt of Preparation of Ben zyl 3,4,6-Tri-O-ben zyl-2-deoxy-2-ph th alim ido-
β-D-glucopyran osyl-(1→3)-[3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-
(1→4)]-2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside
Eth yl 3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-1-th io-β-D-glucopyran oside14 (3; 262 m g,
0.42 m m ol), silver trifluorom eth an esulfon ate (54 m g, 0.21 m m ol) an d crush ed m olecular
sieve 4A (0.3 g) were dried in an apparatus equipped with a septum at room tem perature
an d 1.32 Pa for 10 h an d th en th e apparatus was twice flush ed with argon . A solution of
com poun d 15 (200 m g, 0.21 m m ol) in dry dich lorom eth an e (3 m l) was added th rough th e
septum un der stirrin g an d th e m ixture was stirred at room tem perature for 1 h . Th en m eth -
yl trifluorom eth an esulfon ate (0.05 m l, 0.42 m m ol) was added th rough th e septum an d stir-
rin g was con tin ued for 12 h . Th e addition of th e sam e am oun t of m eth yl trifluoro-
m eth an esulfon ate followed by 12-h stirrin g was repeated th ree m ore tim es. After coolin g to
–15 °C, dry pyridin e (0.63 m l) was added th rough th e septum an d th e m ixture was stirred at
–15 °C for 30 m in an d at room tem perature for 1 h . Th e m ixture was diluted with ch loro-
form (80 m l) an d filtered th rough a Celite pad. Th e filtrate was wash ed with 10% aqueous
NaHSO4 (30 m l), saturated aqueous NaHCO3 (30 m l), 5% aqueous NaCl (30 m l). Th e organ ic
ph ase was dried over an h ydrous MgSO4 an d evaporated in vacuo. Ch rom atograph y of th e
residue on a silica gel colum n (600 m l) in toluen e followed by HPLC separation on a silica
gel colum n C18 in th e solven t system water–m eth an ol (lin ear gradien t 75→90%/70 m in )
afforded 120 m g (59%) of glycosyl acceptor 15, 82 m g (17%) of syrupy com poun d 16 an d
33 m g (14%) of syrupy com poun d 17.
3,4,6-Tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl
3,4,6-Tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran oside (16)
Syrup: [α]D +30 (c 0.4, ch loroform ). For 16 (C70H64N2O13) calculated: relative m olecular
m ass 1141.3, m on oisotopic m ass 1140.4. FAB MS, m/z: 1142 [M + H]+, 1163.7 [M + Na]+. For
C
70H64N2O13 (1141.3) calculated: 73.67% C, 5.65% H, 2.45% N; foun d: 73.55% C, 5.71% H,
2.38% N.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1797
TABLE III
1H NMR param eters of com poun ds 15–17
Param eter
15
16
17
δ(H-1)
δ(H-2)
δ(H-3)
δ(H-4)
δ(H-5)
δ(H-6a)
δ(H-6b)
δ(H-1′)
δ(H-2′)
δ(H-3′)
δ(H-4′)
δ(H-5′)
δ(H-6a′)
δ(H-6b′)
4.91 d
5.34 d
3.67 d
4.03 ddd
3.78 dd
4.03 dd
4.30 dd
3.63 dd
3.47 ddd
3.37 dd
3.65 dd
5.34 d
–
4.55 dt
3.60 dd
4.06 dd
3.64 ddd
3.10 dd
4.38 bddd
3.83 dd
3.18 dd
3.93 dd
5.30 d
–
–
–
–
–
–
–
4.20 dd
4.03 dd
4.30 dd
3.63 dd
3.47 ddd
3.37 dd
3.65 dd
4.39 dd
3.70 dd
3.74–3.76 m
3.64–3.66 m
3.74–3.76 m
J(1,2)
3.7
10.6
7.9
8.7
10.7
8.7
–
J(2,3)
–
J(3,4)
5.3
7.3
3.4
5.5
10.8
–
J(4,5)
9.9
9.9
J(5,6a)
J(5,6b)
J(6a,6b)
J(1′,2′)
J(2′,3′)
J(3′,4′)
J(4′,5′)
J(5′,6a′)
J(5′,6b′)
J(6a′,6b′)
1.6
1.9
4.1
3.4
11.0
8.5
11.2
8.7
10.7
8.5
10.7
8.7
–
–
9.6
a
9.9
–
1.9
–
a
a
3.4
–
11.2
–
a
Not determ in ed. Addition al NMR param eters an d param eters of substituen ts: 15 – arom . H:
6.79–7.38 m , NHAc: 5.53 d (1 H, J = 8.4), OCH2C6H5: 4.81 d (1 H, J = 11.0), 4.77 d (1 H, J =
12.1), 4.59 d (1 H, J = 11.9), 4.59 d (1 H, J = 11.0), 4.57 d (1 H, J = 11.7), 4.50 d (1 H, J =
11.9), 4.40 d (1 H, J = 12.1), 4.33 d (1 H, J = 11.7), 3.94 d (1 H, J = 12.0), 3.88 d (1 H, J =
12.0), NHAc: 1.91 s (3 H); 16 – arom . H: 6.80–7.32 m , OCH2C6H5: 4.68 d (1 H, J = 12.3),
4.68 d (1 H, J = 10.9), 4.53 d (1 H, J = 10.9), 4.36 d (1 H, J = 12.3), 4.20 d (1 H, J = 11.8),
4.05 d (1 H, J = 11.8); 17 – arom . H: J1,3 = 1.2, J3,5 = 0.8, 6.92–7.80 m , OCH2C6H5: 4.78 d
(1 H, J = 11.5), 4.68 d (1 H, J = 11.5), 4.60 s (2 H), 4.57 d (1 H, J = 12.0), 4.31 d (1 H, J =
12.0).
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1798
Rohlenová, Ledvina, Šaman, Bezouška:
1,5-An h ydrotri-O-ben zyl-2-ph th alim ido-2-deoxy-D-arabino-h ex-1-en itol (17)
Syrup: [α]D +52 (c 0.4, ch loroform ). For 17 (C35H31NO6) calculated: relative m olecular m ass
561.6, m on oisotopic m ass 561.2. FAB MS, m/z: 561 [M + H]+, 584.6 [M + Na]+. For
C
35H31NO6 (561.6) calculated: 74.85% C, 5.56% H, 2.49% N; foun d: 75.11% C, 5.63% H,
2.43% N.
Ben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→3)-
[3,4,6-tri-O-ben zyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→4)]-
2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (18)
Com poun d 15 (200 m g, 0.21 m m ol) an d silver trifluorom eth an esulfon ate (126 m g,
0.5 m m ol) were dried in an apparatus equipped with a septum at room tem perature an d
1.32 Pa for 20 h . Apparatus was flush ed with argon (2×) an d dry dich lorom eth an e (1 m l)
was added th rough th e septum . After dissolution , th e m ixture was cooled to –45 °C an d
a solution of 3,4,6-tri-O-acetyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl brom ide15 (5;
210 m g, 0.42 m m ol) in dry dich lorom eth an e (1 m l), was added th rough th e septum un der
stirrin g durin g 2 h . Th en th e m ixture was stirred at –45 °C for an oth er 30 m in an d at –20 °C
for 1 h . Dry pyridin e (0.2 m l) was added th rough th e septum at –20 °C an d after warm in g to
room tem perature th e m ixture was diluted with ch loroform (150 m l) an d filtered. Th e fil-
trate was wash ed with saturated aqueous NaHCO3 (40 m l) an d water (2 × 40 m l), dried over
TABLE IV
13C NMR ch em ical sh ifts of com poun ds 15–17a
Carbon
15
16
17
1
96.46
53.03
70.53
81.37
69.58
68.34
98.86
55.92
79.50
78.93
74.32
68.76
96.88
55.49
78.83
78.93
75.29
68.62
96.88
55.49
78.83
78.93
75.29
68.62
146.50
2
107.96
3
74.13
4
77.65
5
74.26
6
67.93
1′
2′
3′
4′
5′
6′
–
–
–
–
–
–
a
Ch em ical sh ifts for arom atic carbon s from protectin g groups are n ot given . Addition al 13C
NMR ch em ical sh ifts of substituen ts: 15 – OCH2C6H5: 75.00 t, 74.95 t, 73.45 t, 72.67 t,
69.60 t, NHAc: 170.10 s, 23.34 q; 16 – OCH2C6H5: 74.77 t, 74.53 t, 73.54 t; 17 – OCH2C6H5:
73.51 t, 73.27 t, 72.35 t.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1799
an h ydrous MgSO4, evaporated in vacuo an d th e residue was co-distillated with toluen e (2×).
Ch rom atograph y of th e residue on a silica gel colum n (80 m l) in toluen e–eth yl acetate
(10:1) followed by HPLC separation on a silica gel colum n C18 in th e solven t system
water–m eth an ol (85% m eth an ol/20 m in an d th en lin ear gradien t 85→90%/60 m in ) afforded
35 m g (11%) of solid com poun d 18, m .p. 103–105 °C, [α]D +44 (c 0.14, ch loroform ). For 18
(C77H77N3O21) calculated: relative m olecular m ass 1380.4, m on oisotopic m ass 1379.5.
FAB MS, m/z: 1402.5 [M + Na]+. For C77H77N3O21 (1380.4) calculated: 66.99% C, 5.62% H,
3.04% N; foun d: 66.79% C, 5.68% H, 2.96% N.
Ben zyl 2-Acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (20)
Ben zyl 2-acetam ido-3-O-allyl-6-O-ben zyl-2-deoxy-α-D-glucopyran oside15 (19; 5 g, 11.33 m m ol)
an d tris(triph en ylph osph in e)rh odium (I) ch loride (1.2 g, 1.34 m m ol) were refluxed in a m ix-
ture of eth an ol–toluen e–water (7:3:2; 300 m l) un der stirrin g for 4 h . Form ic acid (10 m l)
was added an d th e reflux was con tin ued for an oth er 2 h . Th en th e m ixture was evaporated
in vacuo. Ch rom atograph y of th e residue on a silica gel colum n (500 m l) in ch loroform –
m eth an ol (30:1) followed by crystallization from eth an ol gave 3.45 g (76%) of com poun d
20, m .p. 187–188 °C, [α]D +52 (c 0.3, m eth an ol); lit.26 m .p. 183 °C (m eth an ol), [α]D +45
(c 1.0, m eth an ol). 1H NMR spectrum agrees with th e data for an auth en tic sam ple prepared
by th e procedure described in lit.26 For 20 (C22H27NO6) calculated: relative m olecular m ass
401.5, m on oisotopic m ass 401.2. FAB MS, m/z: 402 [M + H]+. For C22H27NO6 (401.5) calcu-
lated: 65.82% C, 6.78% H, 3.49% N; foun d: 65.70% C, 6.83% H, 3.54% N.
Ben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→3)-
[3,4,6-tri-O-acetyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl-(1→4)]-
2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (21)
Com poun d 20 (100 m g, 0.25m m ol) an d silver trifluorom eth an esulfon ate (257 m g, 0.1 m m ol)
were dried in an apparatus equipped with a septum at room tem perature an d 1.32 Pa for
20 h . Apparatus was flush ed with argon (2×) an d dry dich lorom eth an e (1 m l) was added
th rough th e septum . After dissolution , th e m ixture was cooled to –45 °C an d th e solution of
3,4,6-tri-O-acetyl-2-deoxy-2-ph th alim ido-β-D-glucopyran osyl brom ide15 (5; 996 m g, 2 m m ol)
in dry dich lorom eth an e (1 m l) was added un der stirrin g th rough th e septum durin g 1 h .
Th en th e m ixture was stirred at –45°C for an oth er 30 m in an d at –20 °C for 1 h . Dry pyri-
din e (0.4 m l) was added th rough th e septum at –20 °C an d, after warm in g to room tem pera-
ture, th e m ixture was diluted with ch loroform (150 m l) an d filtered. Th e filtrate was wash ed
with saturated aqueous NaHCO3 (30 m l) an d water (2 × 30 m l), dried over an h ydrous
MgSO4, evaporated in vacuo an d th e residue was co-distilled with toluen e (2 × 20 m l). Ch ro-
m atograph y of th e residue on a silica gel colum n (60 m l) in toluen e–eth yl acetate (3:1) fol-
lowed by lyoph ilization from ben zen e afforded 238 m g (64%) of com poun d 21, [α]D +12
(c 0.25, ch loroform ). For 21 (C62H65N3O24) calculated: relative m olecular m ass 1236.2,
m on oisotopic m ass 1235.4. FAB MS, m/z: 1258.5 [M + Na]+. For C62H65N3O24 (1236.2) calcu-
lated: 60.24% C, 5.30% H, 3.40% N; foun d: 60.19% C, 5.38% H, 3.33% N.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1800
Rohlenová, Ledvina, Šaman, Bezouška:
Ben zyl 2-Acetam ido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyran osyl-(1→3)-
[2-acetam ido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyran osyl-(1→4)]-
2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (23)
A solution of com poun d 21 (600 m g, 0.49 m m ol) in a m ixture of absolute m eth an ol–
butylam in e (4:1; 10 m l) was h eated in a pressure bottle at 85 °C for 7 h . After coolin g, th e
m ixture was evaporated an d th e solid residue was extracted with eth er (3 × 30 m l). Th e in -
soluble residue was dissolved in a m ixture of m eth an ol–water (9:1; 5 m l), th e solution was
adjusted to pH 4 with form ic acid an d poured on to an ion -exch an ge resin Dowex 50 (in H+
form ) colum n (150 m l). Th e colum n was wash ed with a m ixture of m eth an ol–water (9:1;
500 m l) an d th e product was desorbed with a m ixture of m eth an ol–25% aqueous am m on ia
TABLE V
1H NMR param eters of com poun ds 18, 21, 23, 24
Param eter
18
21
23
24
δ(H-1)
4.54 d
4.58 d
4.82 d
4.77 d
δ(H-2)
3.97 ddd
3.80 dd
3.89 dd
3.53 ddd
3.92 dd
4.05 dd
5.14 d
3.97 ddd
3.70 dd
3.86 dd
3.47 ddd
3.38 dd
3.42 dd
5.29 d
4.20 dt
3.92 dd
3.85 dd
3.76 ddd
3.57 dd
3.65 dd
4.82 d
4.19 dd
4.21 dd
4.00 dd
3.91 ddd
3.74 dd
3.79 dd
4.80 d
δ(H-3)
δ(H-4)
δ(H-5)
δ(H-6a)
δ(H-6b)
δ(H-1′)
δ(H-2′)
δ(H-3′)
δ(H-4′)
δ(H-5′)
δ(H-6a′)
δ(H-6b′)
δ(H-1′′)
δ(H-2′′)
δ(H-3′′)
δ(H-4′′)
δ(H-5′′)
δ(H-6a′′)
δ(H-6b′′)
4.40 dd
5.82 dd
5.39 dd
3.81 ddd
4.34 dd
4.72 dd
5.37 d
4.39 dd
5.83 dd
5.33 dd
3.56 ddd
4.22 dd
4.81 dd
5.30 d
3.60 ddd
5.34 dd
5.02 dd
3.63 ddd
3.96 dd
4.53 dd
4.61 d
3.63 dd
3.51 dd
3.27 dd
3.24 ddd
3.65 dd
3.87 dd
4.69 d
4.44 dd
4.25 dd
3.80 dd
3.49 dt
4.40 dd
5.84 dd
5.35 dd
3.83 ddd
4.29 dd
4.87 dd
3.69 ddd
5.15 dd
4.98 dd
3.51 ddd
4.05 dd
4.54 dd
3.63 dd
3.51 dd
3.40 dd
3.22 ddd
3.76 dd
3.82 dd
3.36–3.38 m
3.36–3.38 m
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1801
TABLE V
(Continued)
Param eter
18
21
23
24
J(1,2)
3.9
3.9
3.9
3.8
J(2,3)
9.7
8.6
10.5
8.6
9.7
8.7
9.7
7.3
J(3,4)
J(4,5)
9.8
10.1
3.5
9.5
8.9
J(5,6a)
3.9
2.1
2.3
J(5,6b)
J(6a,6b)
J(1′,2′)
1.8
1.6
3.4
4.5
10.5
8.2
10.9
7.3
10.8
8.1
10.9
8.3
J(2′,3′)
10.9
9.2
11.0
9.2
10.7
9.2
10.5
9.3
J(3′,4′)
J(4′,5′)
10.0
2.1
10.0
1.5
10.0
2.2
9.8
J(5′,6a′)
J(5′,6b′)
J(6a′,6b′)
J(1′′,2′′)
J(2′′,3′′)
J(3′′,4′′)
J(4′′,5′′)
J(5′′,6a′′)
J(5′′,6b′′)
J(6a′′,6b′′)
2.1
3.6
3.1
5.0
6.1
12.3
8.1
12.6
8.2
12.3
8.3
12.1
8.2
10.8
8.2
10.9
9.3
10.7
9.2
10.4
8.7
9.0
10.0
1.6
10.1
2.4
9.7
3.4
2.3
3.4
a
3.1
5.5
4.6
12.6
12.3
11.9
a
Not determ in ed. Addition al NMR param eters an d param eters of substituen ts: 18 – arom . H:
6.82–7.83 m , NHAc: 5.36 d (1 H, J = 9.7), OCH2C6H5: 5.01 d (1 H, J = 12.0), 4.73 d (1 H, J =
11.9), 4.66 d (1 H, J = 11.8), 4.64 d (1 H, J = 12.0), 4.50 d (1 H, J = 11.8), 4.41 d (1 H, J =
11.9), 4.38 d (1 H, J = 12.0), 4.38 d (1 H, J = 11.8), 4.38 d (1 H, J = 11.8), 4.20 d (1 H, J =
12.0), OAc: 2.17 s (3 H), 2.03 s (3 H), 1.66 s (3 H), NHAc: 1.84 s (3 H); 21 – arom . H:
7.13–7.88 m , NHAc: 5.30 d (1 H, J = 9.8), OCH2C6H5: 4.63 d (1 H, J = 11.8), 4.52 d (1 H, J =
11.8), 4.48 d (1 H, J = 12.0), 4.27 d (1 H, J = 12.0), OAc: 2.31 s (3 H), 2.30 s (3 H), 2.02 s (3
H), 2.01 s (3 H), 1.81 s (3 H), 1.79 s (3 H), NHAc: 1.87 s (3 H); 23 – arom . H: 7.23–7.48 m ,
NHAc: 6.40 bd (1 H, J = 8.3), 2-NHAc: 5.93 bd (1 H, J = 9.2), 2′′-NHAc: 5.55 bd (1 H, J = 8.9),
OCH2C6H5: 4.75 d (1 H, J = 12.0), 4.68 d (1 H, J = 11.8), 4.52 d (1 H, J = 12.0), 4.46 d (1 H,
J = 11.8), OAc: 2.14 s (3 H), 2.07 s (3 H), 2.03 s (3 H), 2.00 s (3 × 3 H), NHAc: 1.98 s (3 H),
1.93 s (3 H), 1.84 s (3 H); 24 – arom . H: 7.25–7.41 m , OCH2C6H5: 4.68 d (1 H, J = 12.1),
4.64 d (1 H, J = 11.8), 4.60 d (1 H, J = 11.8), 4.49 d (1 H, J = 12.1), NHAc: 1.98 s (3 H),
1.97 s (2 × 3 H).
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1802
Rohlenová, Ledvina, Šaman, Bezouška:
(7:1; 500 m l). Evaporation of th e eluate in vacuo yielded 271 m g (77%) of syrupy crude ben -
zyl 2-am in o-2-deoxy-β-D-glucopyran osyl-(1→3)-[2-am in o-2-deoxy-β-D-glucopyran osyl-(1→4)]-
2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (22), wh ich was used with out furth er
purification for th e preparation of com poun d 23. For 22 (C34H49N3O14) calculated: relative
m olecular m ass 723.8, m on oisotopic m ass 723.3. FAB MS, m/z: 724 [M + H]+.
A solution of com poun d 22 (260 m g, 0.36 m m ol) in a m ixture of pyridin e–acetic an h y-
dride (2:1; 8 m l) was kept at room tem perature for 20 h . Meth an ol (5 m l) was added un der
TABLE VI
13C NMR param eters of com poun ds 18, 21, 23, 24a
Param eter
18
21
23
24
δ(C-1)
δ(C-2)
δ(C-3)
δ(C-4)
δ(C-5)
δ(C-6)
δ(C-1′)
δ(C-2′)
δ(C-3′)
δ(C-4′)
δ(C-5′)
δ(C-6′)
δ(C-1′′)
δ(C-2′′)
δ(C-3′′)
δ(C-4′′)
δ(C-5′′)
δ(C-6′′)
95.98
52.24
75.66
73.23
70.86
68.31
97.83
54.79
71.09
68.76
72.62
61.95
97.00
56.32
79.63
79.61
71.70
68.96
96.51
52.43
75.75
72.85
70.34
68.00
98.01
54.37
70.64
68.26
71.85
61.28
96.65
54.66
70.94
68.43
71.74
61.52
96.60
52.64
73.64
73.39
70.58
67.74
98.81
54.78
71.58
68.93
72.45
61.83
98.43
55.01
71.90
68.89
72.16
61.91
96.97
54.36
74.75
74.10
73.02
70.03
99.64
57.81
75.68
72.20
78.04
62.67
100.68
57.92
75.68
71.74
77.77
62.04
a
Ch em ical sh ifts for arom atic carbon s from protectin g groups are n ot given . Addition al 13C
NMR ch em ical sh ifts of substituen ts: 18 – OCH2C6H5: 75.11 t, 74.92 t, 74.49 t, 72.76 t,
69.45 t, NHAc: 170.02 s, 23.29 q, OAc: 171.33 s, 169.81 s, 169.38 s, 21.03 q, 20.69 q,
20.29 q; 21 – OCH2C6H5: 72.64 t, 69.66 t, NHAc: 169.88 s, 23.35 q, OAc: 171.54 s, 171.47 s,
169.88 s, 169.81 s, 169.46 s, 164.41 s, 20.95 q, 20.95 q, 20.64 q, 20.63 q, 20.42 q, 20.40 q;
23 – OCH2C6H5: 73.44 t, 69.90 t, NHAc: 170.16 s, 169.54 s, 169.45 s, 23.53 q, 23.31 q,
23.23 q, OAc: 171.22 s, 171.14 s, 170.91 s, 170.55 s, 170.51 s, 170.47 s, 20.84 q, 20.75 q,
20.67 q, 20.64 q, 20.64 q, 20.62 q; 24 – OCH2C6H5: 74.35 t, 70.63 t, NHAc: 173.91 s,
173.78 s, 173.55 s, 23.42 q, 23.27 q, 22.82 q.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

Linear and Branched Regioisomeric Chitooligosaccharides
1803
stirrin g at 0 °C to decom pose excess acetic an h ydride. After 20 m in stan din g at room tem -
perature, th e m ixture was evaporated in vacuo an d th e residue was co-distilled with toluen e
(3 × 20 m l). Ch rom atograph y of th e residue on a silica gel colum n (60 m l) in toluen e–
aceton e (2:1) followed by HPLC separation on a silica gel colum n C18 in solven t system
water–m eth an ol (lin ear gradien t 63→70%/30 m in , th en 70% m eth an ol/30 m in an d fin ally
lin ear gradien t 70→75%/30 m in ) afforded 250 m g (66%) of com poun d 23, [α]D +19 (c 0.7,
m eth an ol). For 23 (C50H65N3O22) calculated: relative m olecular m ass 1060.1, m on oisotopic
m ass 1059.4. FAB MS, m/z: 1082.1 [M + Na]+. For C50H65N3O22 (1060.1) calculated:
56.65% C, 6.18% H, 3.96% N; foun d: 56.78% C, 6.13% H, 3.88% N.
Ben zyl 2-Acetam ido-2-deoxy-β-D-glucopyran osyl-(1→3)-[2-acetam ido-2-deoxy-
β-D-glucopyran osyl-(1→4)]-2-acetam ido-6-O-ben zyl-2-deoxy-α-D-glucopyran oside (24)
A suspen sion of com poun d 23 (200 m g, 0.189 m m ol; dried at a room tem perature an d
1.32 Pa for 10 h ) in 0.01 M CH3ONa in m eth an ol (4 m l) was stirred at room tem perature for
2 h an d th e solution was allowed to stan d at –3 °C overn igh t. Th e m ixture was n eutralized
by addition of Dowex 50 (pyridin ium form ). Th e resin was filtered off, wash ed with m eth a-
n ol (20 m l) an d th e filtrate was con cen trated in vacuo. Th e HPLC separation of th e residue
on a silica gel colum n C18 in th e solven t system water–m eth an ol (lin ear gradien t 50→60%/
60 m in ) afforded 118 m g (77%) of syrupy com poun d 24, [α]D +34 (c 0.5, m eth an ol). For 24
(C38H53N3O16) calculated: relative m olecular m ass 807.8, m on oisotopic m ass 807.3. FAB MS,
m/z: 808 [M + H]+, 830 [M + Na]+. For C38H53N6O13 (807.8) calculated: 56.50% C, 6.61% H,
5.20% N; foun d: 56.48% C, 6.75% H, 5.28% N.
2-Acetam ido-2-deoxy-β-D-glucopyran osyl-(1→3)-[2-acetam ido-2-deoxy-
β-D-glucopyran osyl-(1→4)]-2-acetam ido-2-deoxy-D-glucopyran ose (25)
Com poun d 24 (88 m g, 0.11 m m ol) was h ydrogen olyzed in acetic acid (10 m l) over 10% pal-
ladium catalyst on carbon (100 m g) at room tem perature for 20 h . Th e vessel was th an
flush ed with argon , th e catalyst was filtered off, wash ed with acetic acid (30 m l) an d th e fil-
trate was lyoph ilized. Th e solid residue was separated by HPLC on a silica gel colum n C18 in
water followed by perm eatin g ch rom atograph y on Toyopearl HW 40F (450 m l) in water. Th e
obtain ed h om ogen ous fraction was evaporated in vacuo an d th e residue was lyoph ilized
from water to give 46 m g (67%) of an α/β-an om eric m ixture (3:2) of com poun d 25, [α]D –52
(c 0.7, water). Th e ratio of an om ers was determ in ed by 1H NMR spectra. 1H NMR (500 MHz,
D2O, 300 K): 5.15 d (1 H, J = 3.1, H-1); 4.78 d (1 H, J = 8.4); 4.738 d (1 H, J = 8.3); 4.733 d
(1 H, J = 8.1); 4.717 d (1 H, J = 8.1); 4.704 d (1 H, J = 8.3, H-1′ an d H-1′′); 3.42–4.37 m
(18 H, H-2, H-3, H-4, H-5, H-6, H-2′, H-3′, H-4′, H-5′, H-6′, H-2′′, H-3′′, H-4′′, H-5′′ an d H-6′′);
2.18 s, 2.15 s, 2.140 s, 2.136 s, 2.136 s, 2.126 s (6 × 3 H, NHAc). 13C NMR (125 MHz, D2O,
300 K): 174.92 s, 174.76 s, 174.57 s, 174.54 s, 174.27, 173.84 s, 102.71 d, 101.34 d, 99.91 d,
99.91 d, 98.97 d, 94.76 d, 79.06 d, 76.95 d, 75.98 d, 75.92 d, 75.83 d, 75.83 d, 75.76 d,
75.73 d, 75.58 d, 74.25 d, 74.16 d, 73.88 d, 73.82 d, 73.73 d, 73.68 d, 73.63 d, 73.55 d,
73.40 d, 62.49 t, 60.83 t, 60.79 t, 60.71 t, 60.71 t, 60.59 t, 55.93 d, 55.90 d, 55.81 d, 55.80 d,
51.89 d, 51.79 d, 22.35 q, 22.32 q, 22.31 q, 22.30 q, 22.24 q, 22.21 q. For 25 (C24H41N3O16
)
calculated: relative m olecular m ass 627.6, m on oisotopic m ass 627.3. FAB MS, m/z: 628
[M + H]+. For C24H41N3O16 (627.6) calculated: 45.93% C, 6.58% H, 6.70% N; foun d: 45.99% C,
6.53% H, 6.78% N.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

1804
Rohlenová, Ledvina, Šaman, Bezouška:
We are thankful for the support to the Grant Agency of Charles University in Prague (grant
No. 416/2004/B-CH/PrF), the Grant Agency of Czech Republic (grant No. 203/00/0071), the Ministry
of Agriculture (grant No. QF3115/2003) and the research project No. Z4 055 905.
REFERENCES
1. Bezouška K., Yuen C. T., Obrien J., Childs R. A., Chai W. G., Lawson A. M., Drbal K.,
Fišerová A., Pospíšil M., Feizi T.: Nature 1994, 372, 150.
2. Bezouška K., Vlahas G., Horvath O., Jinochová G., Fišerová A., Giorda R., Chambers
W. H., Feizi T., Pospíšil M.: J. Biol. Chem. 1994, 269, 16945.
3. Krist P., Herkommerová-Rejnochová E., Rauvolfová J., Semeňuk T., Vavrušková P.,
Pavlíček J., Bezouška K., Petrus L., Křen V.: Biochem. Biophys. Res. Commun. 2001, 287,
11.
4. Banoub J., Boullanger P., Lafont D.: Chem. Rev. 1992, 92, 1167.
5. Excoffier G., Gagnaire D., Utille J. P., Vignon M.: Tetrahedron 1975, 31, 549.
6. Blatter G., Beau J. M., Jacquinet J. C.: Carbohydr. Res. 1994, 260, 189.
7. Kiso M., Anderson L.: Carbohydr. Res. 1985, 136, 309.
8. Zurabyan S. E., Antonenko T. S., Khorlin A. Y.: Carbohydr. Res. 1970, 15, 21.
9. Mehta S., Pinto B. M.: J. Org. Chem. 1993, 58, 3269.
10. Hashimoto S., Umeo K., Sano A., Watanabe N., Nakajima M., Ikegami S.: Tetrahedron
Lett. 1995, 36, 2251.
11. Kinzy W., Schmidt R. R.: Liebigs Ann. Chem. 1985, 1537.
12. Bowers S. G., Coe D. M., Boons G. J.: J. Org. Chem. 1998, 63, 4570.
13. Zyka D.: Ph.D. Thesis. Charles University, Prague 2000.
14. Ledvina M., Zyka D., Ježek J., Trnka T., Šaman D.: Collect. Czech. Chem. Commun. 1998,
63, 577.
15. Farkaš J., Ledvina M., Brokeš J., Ježek J., Zajíček J., Zaoral M.: Carbohydr. Res. 1987, 163,
63.
16. Vanboeckel C. A. A., Beetz T., Vos J. N., Dejong A. J. M., Vanaelst S. F., Vandenbosch
R. H., Mertens J. M. R., Vandervlugt F. A.: J. Carbohydr. Chem. 1985, 4, 293.
17. Paulsen H., Himpkamp P., Peters T.: Liebigs Ann. Chem. 1986, 664.
18. Crich D., Dudkin V.: J. Am. Chem. Soc. 2001, 123, 6819.
19. Fowler P., Bernet B., Vasella A.: Helv. Chim. Acta 1996, 79, 269.
20. Vasella A., Witzig C.: Helv. Chim. Acta 1995, 78, 1971.
21. Kantoci D., Keglevic D., Derome A. E.: Carbohydr. Res. 1987, 162, 227.
22. Maloisel J. L., Vasella A.: Helv. Chim. Acta 1992, 75, 1491.
23. Pavlíček J., Bezouška K.: Chem. Pap. 1998, 52, 327.
24. Udodong U. E., Rao C. S., Fraser-Reid B.: Tetrahedron 1992, 48, 4713.
25. Heyns K., Harrison R., Paulsen H.: Chem. Ber. 1967, 100, 271.
26. Becker B., Thiem J.: Tetrahedron: Asymmetry 1994, 5, 2339.
Collect. Czech. Chem. Commun. (Vol. 69) (2004)