The study of the reaction of terminated oligomerization in the synthesis of oligo-(β1-6)-glucosamines

Article abstract of DOI:10.1134/S1068162006040108

The applicability of terminated oligomerization to the synthesis of oligo-(β1-6)-glucosamines, fragments of the intercellular polysaccharide adhesin of staphylococci, was studied. The reactions of terminated oligomerization were carried out with mono-, di-, and trisaccharide monomers and N-protected aminopropanol; and spacered mono-and disaccharides as terminating molecules were also attempted. The primary formation of cyclic products of monomer intramolecular glycosylation was observed in almost all the reactions. Only the experiments with the monomer based on the disaccharide bromide under the conditions of the Helferich reaction led to reduced yields (30%) of the cyclic products. However, even in this case, the desired terminated oligosaccharides were generated in approximately 10% yield and mainly were the products of single glycosylation of the terminator by the monomer. These experiments allow the conclusion that, under the examined conditions, the reaction of terminated oligomerization could not result in the synthesis of oligoglucosamines with a high molecular mass. Pleiades Publishing, Inc., 2006.

Full text of DOI:10.1134/S1068162006040108

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 4, pp. 389–399. © Pleiades Publishing, Inc., 2006.
Original Russian Text © M.L. Gening, Yu.E. Tsvetkov, G.B. Pier, N.E. Nifantiev, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 4, pp. 432–443.
The Study of the Reaction of Terminated Oligomerization
in the Synthesis of Oligo-(b1-6)-Glucosamines
M. L. Geninga, Yu. E. Tsvetkova, G. B. Pierb, and N. E. Nifantieva, 1
a
Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 119991 Russia
b
Harvard Medical School, Harvard University, and Channing Laboratory, Brigham and Women’s Hospital,
181 Longwood Ave., Boston, MA, 02115 USA
Received December 28, 2005; in final form, January 12, 2006
Abstract—The applicability of terminated oligomerization to the synthesis of oligo-(β1-6)-glucosamines,
fragments of the intercellular polysaccharide adhesin of staphylococci, was studied. The reactions of terminated
oligomerization were carried out with mono-, di-, and trisaccharide monomers and N-protected aminopropanol;
and spacered mono- and disaccharides as terminating molecules were also attempted. The primary formation
of cyclic products of monomer intramolecular glycosylation was observed in almost all the reactions. Only the
experiments with the monomer based on the disaccharide bromide under the conditions of the Helferich reac-
tion led to reduced yields (30%) of the cyclic products. However, even in this case, the desired terminated oli-
gosaccharides were generated in approximately 10% yield and mainly were the products of single glycosylation
of the terminator by the monomer. These experiments allow the conclusion that, under the examined conditions,
the reaction of terminated oligomerization could not result in the synthesis of oligoglucosamines with a high
molecular mass.
DOI: 10.1134/S1068162006040108
Key words: cyclic oligosaccharides, oligo-(β1-6)-glucosamines, terminated oligomerization
INTRODUCTION
approaches resulted in oligosaccharides (up to nonasac-
charides) in appropriate yields, but the oligosaccharides
were mostly obtained in protected form rather than as
deprotected derivatives necessary for biomedical stud-
ies. None of the published studies demonstrated the
methods for the synthesis of spacered or labeled com-
pounds for biological tests in vivo and nobody of the
authors reported any immunological tests using these
compounds.
Staphylococcus aureus is one of the most frequently
revealed pathogenic bacteria. It is known to cause a
large number of diseases with a high lethality.2 For
example, it is the cause of diseases of the patients with
medical implants, when the implant surfaces are colo-
nized by S. aureus that covers them with adhesive bio-
films, mainly consisting of poly-(β1–6)-N-acetylglu-
cosamine [1]. Immunoassays suggest that the partially
N-deacetylated analogues of this polysaccharide can
induce the production of protective antibodies and,
hence, offer promise as a basis for the design of antista-
phylococcal vaccines. Synthetic oligo-(β1–6)-glu-
cosamines with N-acetyl groups in the predetermined
glucosamine residues are necessary for the elucidation
of structure of the epitopes responsible for the produc-
tion of protective antibodies.
RESULTS AND DISCUSSION
Investigation of a possibility of obtaining the series
of oligomer homologues of the general formula
[D-GlÒN(β1–6)]n-O(CH2)3NH2 containing up to ten
monosaccharide residues and bearing either only free,
or N-acetylated amino groups using the reaction of ter-
minated oligomerization was the first stage of our
study. This reaction consists in the oligomerization of a
bifunctional monomer in the presence of a monofunc-
tional terminator, which is fixed at the reducing end of
the growing oligosaccharide chain. A possibility of
synthesis of homo- and block oligosaccharides through
the reaction of terminated oligomerization of tritylated
1,2-O-cyanoethylidene derivatives of mono- and oli-
gosaccharides had earlier been demonstrated [8, 9]. The
results proved that the monomer–terminator ratio in a
range from 10 : 1 to 5 : 1 gives a possibility to obtain
Only several articles concerning the synthesis of
oligo-(β1–6)-glucosamines have been published up to
now. Various approaches, such as solid phase synthesis
[2], multicomponent coupling using polymeric sup-
ports [3], polymerization of 1,6-anhydro derivatives
[4], and block synthesis [5–7] were used. Some of the
1
Corresponding author: phone/fax: +7 (495) 135-8784,
e-mail: nen@ioc.ac.ru
2
Abbreviations: DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; Fmoc,
fluorenylmethoxycarbonyl; NIS, N-iodosuccinimide; and TfOH,
trifluoromethanesulfonic acid.
389

390
GENING et al.
OAc
O
OH
OTr
OH
O
O
O
ii, iii
i
iv
AcO
AcO
HO
HO
BzO
BzO
BzO
SEt
SEt
SEt
SEt
BzO
Pht<N
Pht<N
Pht<N
Pht<N
(I)
(II)
(III)
(IV)
Reagents: (i) AcCl/MeOH, CH2Cl2; (ii) TrCl/pyridine; (iii) BzCl/pyridine;
(iv) 90% aqueous CF3COOH/CH2Cl2.
Scheme 1.
products with 10–11 monomeric units in approximately which is known not to preclude glycosylation, but can
40% yield [8]. The use of terminating residue allows
the obtaining of oligosaccharides with a particular moi-
ety at the reducing end, which, in our case, would lead
to a series of spacered oligosaccharides necessary for
the further synthesis of conjugates.
create a steric barrier for intramolecular cyclization. In
this case, the reaction resulted in the preferable forma-
tion of glycal (IX) along with cyclic products (VII) and
(VIII).
We hypothesized that the disaccharide dimer should
be less prone to cyclization, which would enable
obtaining the desired oligomeric products. The synthe-
sis of monomeric blocks used in the preparation of di-
saccharides (XVII) and (XVIII) (see Scheme 4) is
given in Scheme 3. The glucosamine derivative (X)
containing N-(2-carboxy)benzoyl group was succes-
sively tritylated and benzoylated to the anomeric mix-
ture (XI). The closure of the phthalimide cycle by treat-
ment with acetic anhydride in pyridine under heating
[13] and subsequent removal of trityl group resulted in
a mixture of anomeric benzoates (XII) and (XIII).
Individual α-isomer (XIII) and β-isomer (XII) were
isolated by column chromatography. Acetylation of
hydroxy derivative (XIII) led to 6-acetate (XIV). Ben-
zoate (XIV) was converted into bromide (XV), which
was put into the reaction of orthogonal glycosylation
with acceptor (IV) to give disaccharide (XVI)
(Scheme 4). The selective removal of acetyl group from
(XVI) by acidic methanolysis [11] resulted in 6'-OH
derivative (XVII). The treatment of thioglycoside
(XVII) with bromine in dichloromethane resulted in
bromide (XVIII).
The first step was the synthesis of thioglycoside
(IV) as a monomeric block (Scheme 1). Thioglycoside
(I) [10] was deacetylated to triol (II) [11], which was
then tritylated and benzoylated to get (III). Detrityla-
tion of (III) with trifluoroacetic acid in dichlo-
romethane resulted in the target block (IV). The protec-
tive groups were chosen taking into account the effec-
tiveness of their introduction and removal, their
stability under the glycosylation conditions, and their
ability to provide the necessary stereoselectivity of for-
mation of trans-(β1
6)-glycosidic bonds. Benzoyl
groups were chosen as the permanent protective groups
for OH functions at C3 and C4, as they are not suscep-
tible to migration unlike acetyl groups [12]. 3-Amino-
propanol derivative (Va) was chosen as a terminating
agent, since its N-protective group can be selectively
removed, which is necessary for the isolation of the ter-
minated products and would allow the further acylation
of the spacer amino group. The monomer–terminator
ratio was 5 : 1 in all experiments.
The model glycosylation reaction of terminator
(Va) with thioglycoside (I) results in (N-Fmoc)amino-
propyl glycoside (VI) in 85% yield. However, 1,6-
anhydro derivative (VII) obtained in 50% yield turned
out to be the main product of the terminated oligomer-
ization of thioglycoside (IV) in the presence of termi-
nator (Va) (Scheme 2). Cyclic disaccharide (VIII)
(yield 20%) was another reaction product. Note that the
pyranose ring in (VII) has a close to boat conformation,
The resulting monomers (XVII) and (XVIII) and
terminator (Va) were used in a series of reactions of ter-
minated oligomerization, in which we varied monomer
concentration, temperature, solvent, leaving group, and
promoting system (Scheme 5). All the reactions with
thioglycoside (XVII) and bromide (XVIII) in the pres-
ence of silver triflate resulted in cyclic product (VIII) in
almost quantitative yield. Only in the case of oligomer-
ization of bromide (XVIII) under the conditions of the
Helferich reaction, this compound was not the main
reaction product. It should be noted that previously the
same cyclic glucose derivatives had been reported to be
also generated upon oligomerization of 6-é-(2,3,4-tri-
é-acetyl-β-D-glucopyranosyl)-2,3,4-tri-é-acetyl-α-
D-glucopyranosyl bromide in the presence of mercury
salts; however, the yield of cyclic product was only
1
which follows from the coupling constants in its H
NMR spectrum (J1, 2 = 0, J2, 3 = 9.2, J3, 4 = 8.1, J4, 5 = 0,
and JC1, H3 = JC5, H3 = 1.5). This conformation of (VII)
seems to be stable because of equatorial positions of the
bulky substituents at C2, C3, and C4 of the pyranose
ring.
We presumed that the formation of 1,6-anhydro
derivative (VII) can result from the spatial drawing
together of C1 atom and hydroxy group at C6 in mono-
mer (IV). Therefore, the next step was the oligomeriza-
tion of (III) containing the trityl group (Scheme 2), 10% [14].
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006

THE STUDY OF THE REACTION OF TERMINATED OLIGOMERIZATION
391
OAc
O
i
AcO
(I) +
O
NHFmoc
HO
NHR
AcO
Pht<N
(VI), 85%
(Va) R = Fmoc
(Vb) R = Z
Pht<N
O
OBz
O
O
BzO
BzO
Pht<N
OBz
i
O
(IV) + (Va), 0.2 equiv
+
BzO
BzO
O
O
Pht<N
(VIII), 20%
(VII), 50%
OTr
O
BzO
BzO
ii
(III) + (Va), 0.2 equiv
+ (VII), 15% + (VIII), 10%
Pht<N
(IX), 55%
Reagents: (i) NIS, TfOH/CH2Cl2, molecular sieves 4 Å; (ii) MeOTf/CH2Cl2, molecular sieves 4 Å.
Scheme 2.
OH
OTr
OR
OR
O
O
HO
HO
BzO
BzO
O
O
BzO
iii, iv
BzO
i, ii
+
OBz
OH
OBz
BzO
BzO
C(O)NH
C(O)NH
Pht<N
Pht<N
OBz
CO2H
CO2H
(X)
(XI)
(XII) R = H
(XIII) R = H
(XIV) R = Ac
iii
Reagents: (i) TrCl/pyridine; (ii) BzCl/pyridine; (iii) Ac2O/pyridine;
(iv) 90% aqueous CF3COOH/CH2Cl2.
Scheme 3.
The oligomers obtained by (XVIII) + (Va) oligo- generated [16]. We met with difficulties in controlling
merization in the presence of mercury salts were ana-
lyzed as follows. First, cyclic product (VIII) was sepa-
rated by chromatography on silica gel. The remaining
mixture containing terminated products (XXV) was
treated to selectively remove the N-protective group
from the aminopropyl spacer moiety. In this case, we
did not use the classic procedure for the removal of
Fmoc protective group, as it implies the use of an
excess of such basic amines as piperidine and morpho-
line [15] and the resulting mixture of amines should
complicate the subsequent ion exchange chromatogra-
phy. The Fmoc protective group was removed in the
presence of a catalytic amount of DBU and the excess
the proceeding of this reaction in the case of the mix-
ture of oligomers. Therefore, we have proceeded later
to the use of Z-protected terminators. This has not
caused any changes in their reactivity and the yields of
the oligomerization products (see the table). Termi-
nated oligosaccharides with the unprotected amino
function in the aglycone (XXVI) were separated from
the nonterminated products by ion exchange chroma-
tography on Amberlyst 15 cation exchange resin. The
mixture was then subjected to exhausting hydrazinoly-
sis with the formation of totally deprotected spacered
oligosaccharides (XXVII), which were then separated
of ethyl mercaptan necessary for the fixation of fulvene by gel chromatography.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006

392
GENING et al.
OH
O
BzO
BzO
O
NHR
Pht<N
(XIV)
(Va)
or
(Vb)
(XIXa) R = Fmoc
(XIXb) R = Z
+ ii, iii
i
OAc
O
BzO
BzO
Br
Pht<N
(XV)
(XIII) + ii
(IV) + ii
OR2
OR2
O
O
BzO
BzO
BzO
O
O
BzO
Pht<N
Pht<N
O
O
BzO
BzO
BzO
R1
BzO
R1
Pht<N
Pht<N
(XX) R1 = αOBz, R2 = Ac
(XVI) R1 = SEt, R2 = Ac
(XVII) R1 = SEt, R2 = H
(XVIII) R1 = Br, R2 = H
iii
i
(XXI) R1 = Br, R2 = Ac
iv
(IV) + ii, iii
(Vb) + ii, iii
OH
O
OH
BzO
BzO
O
O
BzO
BzO
O
Pht<N
O
Pht<N
BzO
BzO
Pht<N
O
O
BzO
BzO
O
NHZ
O
BzO
BzO
Pht<N
R
Pht<N
(XXII)
(XXIII) R = SEt
(XXIV) R = Br
iv
Reagents: (i) HBr/AcOH, CH2Cl2; (ii) HgBr2, Hg(CN)2/CH3CN; (iii) AcCl/MeOH, CH2Cl2;
(iv) Br2/CH2Cl2.
Scheme 4.
OH
O
BzO
H
O
O
BzO
O
BzO
BzO
(XXVII)
O
NHR
O
Pht<N
+
BzO
R
BzO
Pht<N
0.2 equiv
n
Pht<N
n
(XVIII) n = 1, R = Br
(XXIII) n = 2, R = SEt
(XXIV) n = 2, R = Br
(Va) n = 0, R = Fmoc
(XIXa) n = 1, R = Fmoc
(XIXb) n = 1, R = Z
(XXII) n = 2, R = Z
Scheme 5. Terminated oligomerization involving monomers and terminators of various lengths. The results are given
in table.
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THE STUDY OF THE REACTION OF TERMINATED OLIGOMERIZATION
393
form. Monomer (XVIII) was added in 1-equiv portions
to the terminator at regular intervals sufficient for com-
plete expenditure of monomeric bromide (XVIII) in
order to increase the terminator conversion (table,
experiment no. 3). In this case, conversion of the termi-
nator increased to 75% and the yield of dominating ter-
minated oligomer (XXII, n = 2) was 15% (table, exper-
iment no. 3). Therefore, we followed just the same
order of the addition of the reagents in the further
experiments.
OH
R3O
R3O
O
O
R2N
O
R3O
O
NHR1
R3O
R2N
n
(XXV) R1 = Fmoc, R2 = Pht<, R3 = Bz, n = 1–4
(XXVI) R1 = H, R2 = Pht<, R3 = Bz, n = 1–4
(XXVII) R1 = H, R2 = H2, R3 = H, n = 1–4
The further elongation of the terminating residue up
to disaccharide (XXII) did not result in any change in
the yield of terminated oligosaccharides (table, experi-
ment no. 5). The majority of experiments resulted in the
formation of products of single glycosylation of the ter-
minator with the monomer in low yields. Only in some
cases, negligible amounts of the product of double gly-
cosylation were detected among the isolated terminated
oligomers by mass spectrometry.
BzO
O
N>Pht
BzO
Pht<N
O
O
OBz
OBz
O
O
BzO
BzO
O
The experiments with trisaccharide monomers
(XXIII) and (XXIV) and terminator (XXII) (table,
experiment nos. 6 and 7) were also carried out. The
yield of cyclic trisaccharide (XXVIII) was 95% upon
oligomerization of thioethyl glycoside (XXIII) and
50% in the case of bromide (XXIV). Previously, the
only one example of the synthesis of similar (β1–6)-
linked cyclic triglucoside obtained by cyclization of the
corresponding peracetylated bromide was reported;
however, the yield of this reaction after optimization
did not exceed 20% [17]. It is possible that a higher ten-
dency of trisaccharide (XXIV) for cyclization as com-
pared with disaccharide (XVIII) can be explained by a
much less tension in the cyclic trisaccharide, which
makes it possible for monosaccharide fragments to
occur in the undistorted minimal energy chair confor-
Pht<N
(XXVIII)
The experiment with bromide (XVIII) and Fmoc-
substituted aminopropanol (Va) was analyzed accord-
ing to the above scheme; the yield of terminated oli-
gosaccharides was 10% (the table, experiment no. 1).
Gel chromatography helped obtain the only spacered
disaccharide (XXVII, n = 1). Such a low involving of
the terminator in the reaction is likely the result of dif-
ferent reactivity of the hydroxy groups in the terminator
and monomer. To adjust the reactivities of the acceptor
hydroxy group of the terminating molecule and that of
the monomer hydroxy group closer to one another, we
passed to the use of terminators based on monosaccha-
rides (XIXa) and (XIXb). The model reaction between
disaccharide (XXI) and terminator (XIXa) led to the
formation of the desired trisaccharide in 85% yield (not
described). During the oligomerization (table, experi-
ment no. 2), the yield of the terminated products was
mation as follows from the coupling constants (J1, 2
=
8.2; J3, 2 = J3, 4 = 10.2; and J4, 5 = 9.5). In the case of the
cyclic disaccharide, the values of coupling constants
(J1, 2 = 4.2; J3, 2 = J3, 4 = 10.5; and J4, 5 = 6.5) correspond
to the distorted conformation of the ring close to the
twist conformation. These data are in agreement with
12%, with 50% of terminator (XIXa) taken in the reac- the results obtained earlier for the similar derivatives of
tion being isolated from the reaction mixture in intact glucose [17].
The results of terminated oligomerization reactions involving monomers and terminators of various lengths
Experiment no.
Monomer
(XVIII)*
(XVIII)*
(XVIII)**
(XVIII)**
(XVIII)**
(XXIII)**
(XXIV)**
Terminator
(Va)
Reaction products (yield, %)
(VIII) (30), (XXVII, n = 1) (10)
(VIII) (30), (XXVII, n = 2) (12)
(VIII) (25), (XXVII, n = 2) (15), (XXVII, n = 4) (<1%)
(VIII) (25), (XXVII, n = 2) (12), (XXVII, n = 4)
(VIII) (25), (XXVII, n = 3) (14)
(XXVIII) (95)
1
2
3
4
5
6
7
(XIXa)
(XIXa)
(XIXb)
(XXII)
(XXII)
(XXII)
(XXVIII) (50)
* The single addition of the monomer.
** The monomer was added in five portions.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006

394
GENING et al.
Thus, under the conditions, the reaction of termi- toluene−ethyl acetate) to yield 1.53 g (95%) of (IV);
1
nated oligomerization does not allow obtaining oligo- [α]D +29°; H NMR: 7.79–7.12 (14 H, m, Ar), 6.38
glucosamines with a rather high molecular mass. (1 H, t, J3, 2 = J3, 4 = 9.8, H3), 5.68 (1 H, d, J1, 2 10.5,
Therefore, the gradient elongation of the oligoglu- H1), 5.55 (1 H, t, J4, 5 9.7, H4), 4.65 (1 H, dd, H2), 3.85
cosamine chain, which we plan to realize in future, (1 H, m, H5), 3.90 (1 H, d, J6a, 6b 11.6, H6a), 3.75 (1 H,
seems to be more promising.
dd, J6b, 5 3.5, H6b), 2.75 (2 H, m, SCH2CH3), 2.60 (1 H,
br. s, OH), 1.40 (3 H, t, J 7.8, SCH2CH3); 13C NMR:
168.14, 167.43, 165.95, and 165.67 (four C=O),
134.31–123.64 (Ar), 81.24 (C1), 78.77 (C5), 71.85
(C3), 70.03 (C4), 61.67 (C6), 54.00 (C2), 24.20
(SCH2CH3), and 14.94 (SCH2CH3).
EXPERIMENTAL
We used reagents from Fluka and Acros. Mass spec-
tra were registered on a Finnigan LCQ mass spectro-
meter at electrospray ionization. The procedures for the
purification of solvents and the conditions for the regis-
tration of NMR spectra (for solutions in CDCl3) and
measurement of coupling constants were similar to
those reported in [18]. The values of optical rotation of
the compounds obtained were measured on a JASCO
DIP-360 (Japan) digital polarimeter in chloroform at
26–30°ë. Silica gel 60 F254 precoated plates (Merck)
were used for TLC. Spots were visualized by spraying
with 10% orthophosphoric acid in ethanol and subse-
quent heating at ~150°ë. Column chromatography was
carried out on silica gel 60–200 µm (Fluka).
3-(9-Fluorenylmethoxycarbonylamino)propanol
(Va). N-Hydroxysuccinimide (0.68 g, 0.6 mmol) and
triethylamine (0.8 ml) were added to a solution of
FmocCl (1.20 g, 4.6 mmol) in dioxane (18 ml). The
resulting precipitate was filtered off, the filtrate was
evaporated, and the residue was dissolved in dioxane.
3-Aminopropanol (0.3 ml, 4 mmol) and triethylamine
(0.1 ml) were added; the reaction mixture was stirred
for 1 h and evaporated. The residue was subjected to
flash chromatography on silica gel (1 : 1 toluene−ethyl
acetate) to give 1.10 g (93%) of (Va); 1H NMR: 7.78–
7.28 (8 H, m, Ar), 5.05 (1 H, br. s, NH), 4.46 [2 H, m,
ëH2 (Fmoc)], 4.22 [1 H, m, CH (Fmoc)], 3.65 (2 H, m,
OCH2CH2CH2NH), 3.31 (2 H, m, OCH2CH2CH2NH),
1.68 (2 H, m, OCH2CH2CH2NH); 13C NMR: 156.24
(OC(O)NH), 143.90, 141.33, 127.66, 127.02, and
124.94 (Ar), 66.67 (CH2 (Fmoc)); 59.63
(OCH2CH2CH2NH); 47.37 [CH (Fmoc)], 37.79
(OCH2CH2CH2NH), 32.60 (OCH2CH2CH2NH).
Ethyl 3,4-di-O-benzoyl-2-deoxy-2-phthalimido-
1-thio-6-O-trityl-b-D-glucopyranoside (III). Trityl
chloride (3.47 g, 12.6 mmol) was added to a solution of
triol (II) [11] (2.22 g, 6.3 mmol) in pyridine (15 ml) at
stirring. The reaction was monitored by TLC, watching
the formation of product with Rf 0.4 (10 : 1 chloroform–
methanol).After the tritylation was over (16 h), benzoyl
chloride (1.74 ml, 15.1 mmol) was added, and the reac-
tion mixture was stirred for 24 h. Then methanol
(10 ml) was added at 0°C; the reaction mixture was
evaporated; and the residue was dissolved in chloroform
(100 ml), washed with 5% aqueous H2SO4 (3 × 25 ml),
water, and saturated solution of NaHCO3 (3 × 50 ml).
The organic layer was dried with Na2SO4 and evapo-
rated. The product was isolated by column chromatog-
raphy on silica gel (40 : 1 toluene—ethyl acetate) to
yield 2.30 g (58%) of (III); [α]D +36° (c 0.33; CHCl3).
3-Benzyloxycarbonylamino)propanol (Vb). Tri-
ethylamine (2 ml) and N-(benzyloxycarbonyloxy)suc-
cinimide (3.40 g, 13.6 mmol) were added to a solution
of 3-aminopropanol (1 g, 13.3 mmol) in a mixture of
ethyl acetate (12 ml) and methanol (20 ml). The mix-
ture was stirred for 30 min and evaporated. The residue
was dissolved in CHCl3 (60 ml) and washed with 1 M
aqueous HCl (3 × 10 ml), water (3 × 10 ml), and
NaHCO3 (2 × 10 ml). The extract was filtered though a
layer of cotton, evaporated, and crystallized from 1 : 1
ethyl acetate–petroleum ether to yield 2.70 g (95%) of
(Vb); 1H NMR: 7.40–7.30 (5 H, m, Ar), 5.12 (3 H, br.
m, NH and CH2Ph), 3.67 (2H, m, OCH2CH2CH2N);
3.33 (2H, m, OCH2CH2CH2N); 2.69 (1 H, br. s, OH),
1.70 (2H, m, OCH2CH2CH2N). 13CNMR: 157.30
[OC(O)NH], 136.46, 128.51, 128.12, and 128.07 (Ar),
66.81 (CH2Ph); 59.59 (OCH2CH2CH2N); 37.78
(OCH2CH2CH2N); 32.54 (OCH2CH2CH2N).
1H NMR: 7.79–7.12 (29 H, m, Ar), 6.25 (1 H, t, J3, 2
=
J3, 4 = 9.8, H3), 5.75 (1 H, d, J1, 2 10.5, H1), 5.70 (1 H,
m, H4), 4.75 (1 H, dd, H2), 4.05 (1 H, m, H5), 3.42 (1
H, dd, J6a, 5 1.5, J6a, 6b 10.5, H6a), 3.30 (1 H, dd, J6b, 5
4.9, H6b), 2.85 (1 H, m, SCH2CH3), 1.40 (3 H, t, J 7.8,
SCH2CH3); 13C NMR: 168.10, 167.33, 165.85, and
164.88 (four C=O), 143.70 (Tr), 134.33–123.74 (Ar),
86.75 (C(Ph)3); 81.13 (C1), 78.10 (C5), 72.44 (C3),
69.96 (C4), 62.77 (C6), 54.25 (C2), 24.38 (SCH2CH3),
and 15.50 (SCH2CH3).
3-(9-Fluorenylmethoxycarbonylamino)propyl-
Ethyl 3,4-di-é-benzoyl-2-deoxy-2-phthalimido- 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-D-glu-
1-thio-b-D-glucopyranoside (IV). Trifluoroacetic copyranoside (VI). A mixture of thioglycoside (I)
acid (10 ml of 90% aqueous solution) was added to a (45 mg, 0.1 mmol), 3-Fmoc-aminopropanol (Va)
solution of 6-é-trityl derivative (III) (2.30 g, 2.9 mmol) (28 mg, 0.1 mmol), freshly distilled dichloromethane
in CH2Cl2 (80 ml). The reaction mixture was stirred for (1 ml), and molecular sieve 4Å (100 mg) was stirred for
2 h at room temperature, diluted with ëHCl3 (70 ml), 1 h under argon at room temperature. The resulting
and washed with saturated NaHCO3 and water. The mixture was cooled to –20°ë, NIS (42 mg, 0.18 mmol)
organic layer was dried with Na2SO4 and evaporated. and TfOH (3 µl, 0.04 mmol)were added, the reaction
The residue was chromatographed on silica gel (6 : 1 mixture was stirred for 1 h at –15°ë, and then pyridine
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006

THE STUDY OF THE REACTION OF TERMINATED OLIGOMERIZATION
395
(10 µl) was added. The resulting suspension was 123.83 (Ar), 90.50 (C1), 75.23 (C5), 70.56 (C3), 69.70
diluted with dichloromethane (20 ml) and filtered (C4), 60.97 (C6), and 53.86 (C2).
though a layer of Celite, which was then washed with
Compound (XIII): [α]D +126°; 1H NMR: 8.38–7.18
dichloromethane (10 ml). The filtrate was washed with
(19 H, m, Ar), 7.30 (1 H, m, H3), 6.63 (1 H, d, J1, 2 3.7,
1 M aqueous Na2S2O3, dried with Na2SO4, and evapo-
H1), 5.57 (1 H, t, J4, 3 = J4, 5 = 9.8, H4), 5.13 (1 H dd,
rated. The residue was chromatographed on silica gel
J2, 3 11.4, H2), 4.30 (1 H, m, H5), 3.83 (1 H, d, J6a, 6b
(10 : 3 toluene−ethyl acetate) to give 57 mg (85%) of
(VI); 1H NMR: 7.89–7.15 (12 H, m, Ar); 5.78 (1 H, t,
J3, 2 = J3, 4 = 9.8, H3); 5.87 (1 H, d, J1, 2 8.8, H1); 5.16 (1
H, t, J4, 5 9.6, H4); 4.38–4.20 [3 H, m, H6a, CH2
(Fmoc)], 4.12 [2 H, m, H6b, CH (Fmoc)], 3.80 (2 H, m,
13.2, H6a), 3.72 (1 H, dd, J6b, 5 3.5, H6b); 13C NMR:
166.38, 164.98, and 164.79 (three C=O), 134.29–
123.71 (Ar), 91.38 (C1), 75.67 (C5), 70.77 (C4), 67.23
(C3), 60.84 (C6), and 52.99 (C2).
H5,
OCH2CH2CH2NH),
3.53
(1
H,
m,
6-O-Acetyl-1,3,4-tri-O-benzoyl-2-deoxy-2-phthal-
OCH2CH2CH2NH), 3.11 (2 H, m, OCH2CH2CH2NH), imido-a-D-glucopyranose (XIV). Pyridine (1.5 ml)
2.08, 2.04, and 1.87 (9 H, 3 s, CH3ë(O)); 1.68 (2 H, m, and acetic anhydride (1 ml) were added to a solution of
OCH2CH2CH2NH); 13C NMR: 170.66, 170.10, and 6-hydroxy derivative (XIII) (2.0 g, 3.2 mmol) in
169.40 (three C=O), 156.24 [OC(O)NH], 143.86, dichloromethane (10 ml). The reaction mixture was
141.20 [arom. H (Fmoc)], 134.29–119.85 (Ar), 98.07 stirred for 16 h at room temperature and then evapo-
(C1), 71.87 (C5), 70.68 (C3), 68.79 (C4), 67.25 (CH2 rated and coevaporated with toluene. The residue was
(Fmoc)); 66.24 (OCH2CH2CH2NH); 61.74 (C6), 54.47 dried at 1 Torr to constant mass to give 2.13 g (100%)
1
(C2), 47.21 [CH (Fmoc)], 37.58 (OCH2CH2CH2NH), of acetate (XIV) (100%). [α]D +89°. H NMR: 8.29–
29.62 (OCH2CH2CH2NH), 20.68, 20.58, and 20.39 7.18 (19 H, m, Ar), 7.25 (1 H, m, H3), 6.62 (1 H, d, J1, 2
(CH3ë(O)).
3.7, H1), 5.70 (1 H, t, J4, 3 = J4, 5 = 10.0, H4), 5.16 (1 H
dd, J2, 3 11.5, H2), 4.55 (1 H, m, H5), 4.37 (1 H, d, J6a, 5
4.1, J6a, 6b 12.4, H6a), 4.21 (1 H, dd, J6b, 5 2.7, H6b),
2.06 (3 H, c, CH3ë(O)). 13C NMR: 170.65, 165.37,
164.99, and 164.84 (four C=O), 134.40–123.81 (Ar),
91.27 (C1), 70.32 (C5, C4), 67.53 (C3), 61.96 (C6),
52.91 (C2), 20.76 (CH3ë(O)).
1,3,4-Tri-O-benzoyl-2-deoxy-2-phthalimido-a- and
b-D-glucopyranoses (XII) and (XIII). Trityl chloride
(11 g, 39.5 mmol) was added to a solution of 2-carboxy-
benzoyl derivative (X) (10 g, 30.5 mmol) in pyridine
(100 ml). The reaction mixture was stirred for 24 h at
room temperature and diluted with anhydrous dichlo-
romethane (100 ml), and a solution of benzoyl chloride
(20 ml, 170 mmol) in dichloromethane (100 ml) was
added dropwise. The resulting suspension was stirred
for 24 h at room temperature, the excess of benzoyl
chloride was quenched with methanol (50 ml), and the
reaction mixture was evaporated. The residue was dis-
solved in chloroform (0.7 l) and washed with 1 M aque-
ous HCl until the complete removal of pyridine (to
acidic pH of the aqueous layer) and saturated aqueous
NaHCO3 solution. The organic layer was dried with
Na2SO4 and evaporated. The residue was dissolved in a
mixture of pyridine (60 ml) and acetic anhydride
(25 ml). The resulting solution was refluxed for 2 h,
poured out onto crushed ice (200 g), and extracted with
chloroform (300 ml). The organic layer was washed
with 1 M aqueous HCl, water, and saturated aqueous
NaHCO3; dried with Na2SO4; and evaporated. The
resulting residue was dissolved in dichloromethane
(100 ml), and 90% aqueous trifluoroacetic acid
(1.87 ml) was added. After 1 h, the TFA excess was
quenched with saturated aqueous NaHCO3; the organic
layer was separated, washed with water, dried with
Na2SO4, and evaporated. The residue was chromato-
graphed on silica gel (50 : 1−5 : 1) to yield 2.1 g of (XII)
and 4.7 g of (XIII) (total yield 33%).
Ethyl 6-O-(6-O-acetyl-3,4-di-O-benzoyl-2-deoxy-
2-phthalimido-b-D-glucopyranosyl)-3,4-di-O-ben-
zoyl-2-deoxy-2-phthalimido-1-thio-b-D-glucopyran-
oside (XVI). Synthesis of bromide (XV). A 33% solu-
tion of HBr in AcOH (2 ml) was added to a solution of
benzoate (XIV) (620 mg, 0.9 mmol) in anhydrous
dichloromethane (5 ml). The mixture was stirred for
16 h. The disappearance of starting benzoate (XIV) (Rf
0.30) and the formation of bromide (XV) as the mixture
of α- and β-anomers (Rf 0.35 and 0.40, respectively)
were monitored by TLC (20 : 3 toluene−ethyl acetate).
The reaction mixture was diluted with dichloromethane
(30 ml) and washed with ice-cold water (3 × 5 ml) and
saturated aqueous NaHCO3 (3 × 5 ml). The organic
layer was filtered through a layer of cotton, concen-
trated, and dried in a vacuum to give 530 mg (91%) of
bromide (XV). Glycosylation. Mercury (II) cyanide
(230 mg, 0.9 mmol) and HgBr2 (100 mg, 0.27 mmol)
were added to a solution of acceptor (IV) (350 mg,
0.62 mmol) in anhydrous acetonitrile (2 ml) under
argon. The mixture was stirred for 10 min at room tem-
perature. Bromide (XV) was dissolved in anhydrous
acetonitrile (5 ml) and added to the solution of the
acceptor. The reaction mixture was stirred for 1 h,
diluted with chloroform (30 ml), and washed with 1 M
1
Compound (XII): [α]D +29°; H NMR: 8.38–7.18 aqueous KBr (10 ml) and saturated NaHCO3 (20 ml).
(19 H, m, Ar), 6.89 (1 H, d, J1, 2 8.9, H1), 6.56 (1 H, t, The organic extract was filtered through a layer of cot-
J3, 2 = J3, 4 = 9.9, H3), 5.65 (1 H, t, J4, 5 9.7, H4), 4.93 ton, concentrated, and dried in a vacuum. The residue
(1 H dd, H2), 4.14 (1 H, m, H5), 3.90 (1 H, d, J6a, 6b was chromatographed on silica gel (15
: 1
13.1, H6a), 3.75 (1 H, dd, J6b, 5 3.4, H6b); 13C NMR: toluene−ethyl acetate) to give 580 mg (85%) of disac-
166.30, 165.59, and 164.36 (three C=O), 134.39– charide (XVI); 1H NMR: 7.89–7.18 (28 H, m, Ar), 6.22
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006

396
GENING et al.
(2 H, t, J3, 2 = J3, 4 = 9.7, H3, H3'), 5.63 (1 H, d, J1, 2 8.4,
H1'), 5.56–5.53 (2 H, m, H4, H1), 5.38 (1H, t, J4', 5 9.6,
H4'), 4.58 (1 H, dd, H2'), 4.53 (1 H, t, H2), 4.30 (1 H,
d, J6a', 5' 5.4, J6a', 6b' 5.23, H6a'), 4.21 (1 H, dd, J6b', 5' 2.6,
H6b'), 4.11–3.95 (3 H, m, H5, H5', H6a), 3.82 (1 H, dd,
J6b, 5 6.8, J6a, 6b 10.9, H6b), 2.55 (2 H, m, SCH2CH3),
1.98 (3 H, s, CH3C(O)); 1.13 (3 H, t, J 7.8, SCH2CH3);
13C NMR: 170.55, 167.83, 167.07, 165.58, and 165.23
(five C=O), 134.21–123.57 (Ar), 98.17 (C1'), 80.84
(C1), 77.50 (C5), 71.95 (C3', C5'), 71.11 (C3), 70.24
(C4'), 69.93 (C4), 68.57 (C6), 62.51 (C6'), 54.73 (C2'),
53.93 (C2), 24.02 (SCH2CH3), 14.92 (SCH2CH3), 20.76
(CH3ë(O)).
3-(9-Fluorenylmethoxycarbonylamino)propyl-
3,3-di-O-benzoyl-2-deoxy-2-phthalimido-b-D-glu-
copyranoside (XIXa) was obtained by glycosylation
as described for disaccharide (XVI) from bromide
(XV) (0.68 mg, 1.1 mmol) and 3-Fmoc-aminopropanol
(Va) (0.3 g, 1 mmol). Deacetylation analogous to that
described for (XVII) yielded 740 mg (88%) of spac-
ered ((XIXa). [α]D –2°. 1H NMR: 7.95–7.18 (22 H, m,
Ar), 6.23 (1 H, t, J3, 2 = J3, 4 = 9.6, H3), 5.63–5.56 (2 H,
m, H1, H4), 5.15 (1 H, br. m, NH), 4.56 (1 H, t, H2),
4.39 [2 H, m, CH2 (Fmoc)], 4.21 [1 H, m, CH (Fmoc)],
3.91–3.60 (5H, m, H5, H6a, H6b, OCH2CH2CH2NH2);
3.15 (2H, m, OCH2CH2CH2NH2); 1.74 (2H, m,
OCH2CH2CH2NH2). 13ë NMR: 165.98 and 165.65
(two C=O), 156.42 [OC(O)NH], 143.98 and 141.30
(Fmoc), 134.24–119.94 (Ar), 98.34 (C1), 74.57, 70.98,
and 70.11 (C3, C4, C5), 67.29 (OCH2CH2CH2NH2);
66.34 [CH (Fmoc)], 61.33 (C6), 54.83 (C2), 47.37
(CH2 (Fmoc)], 37.64 (OCH2CH2CH2NH2); 29.51
(OCH2CH2CH2NH2).
Ethyl 6-O-(3,4-di-O-benzoyl-2-deoxy-2-phthalim-
ido-b-D-glucopyranosyl)-3,4-di-O-benzoyl-2-deoxy-
2-phthalimido-1-thio-b-D-glucopyranoside (XVII).
Absolute methanol (10 ml) and acetyl chloride (0.1 ml)
were added to a solution of 1-acetate (XVI) (520 mg,
0.47 mmol) in dichloromethane (3 ml). The reaction
mixture was stirred for 16 h at room temperature, evap-
orated, and filtered though silica gel (2 : 1 toluene–ethyl
acetate) to give 500 mg (100%) of disaccharide
(XVII); [α]D +15°; 1H NMR: 7.95–7.18 (28 H, m, Ar),
6.24–6.20 (2 H, m, H3, H3'), 5.60 (1 H, d, J1', 2' 8.5,
3-(Benzyloxycarbonylamino)propyl-3,4-di-O-
benzoyl-2-deoxy-2-phthalimido-b-D-glucopyrano-
side (XIXb) was obtained analogously to terminator
(XIXa) from bromide (XV) (530 mg, 0.85 mmol) and
3-Z-aminopropanol (Vb) (160, mg, 0.77 mmol).
Deacetylation resulted in 430 mg (80%) of terminator
(XIXb); [α]D +5°; 1H NMR: 8.78–7.14 (19 H, m, Ar),
6.30 (1 H, t, J3, 2 = J3, 4 = 9.9, H3), 5.60 (1 H, d, J1, 2 8.5,
H1), 5.53 (1 H, t, J4, 5 9.6, H4), 5.60 (2 H, br. s, PhCH2),
4.57 (1 H, dd, H2), 3.97–3.63 (5 H, m, H5, H6a,
H1'), 5.53 (1 H, d, J1, 2 10.5, H1), 5.46 (1 H, t, J4, 3
=
J4, 5 = 9.7, H4), 5.33 (1 H, t, J4', 3' = J4', 5' 9.6, H4'), 4.54
(1 H, t, J2, 3 10.4, H2), 4.42 (1 H, dd, J2', 3' 10.5, H2'),
4.03–4.15 (2 H, m, H5, H6a), 3.76–3.87 (3 H, m, H5',
H6b, H6a'), 3.64 (1 H, dd, J6b', 6a' 12.6, H6b'), 2.58 (2 H,
m, SCH2CH3), 1.13 (3 H, t, J 7.8, SCH2CH3); 13C
NMR: 165.54 (C=O), 133.97–123.54 (Ar), 97.73 (C1'),
80.93 (C1), 77.00 (C5), 74.40, 71.98, 71.00, 70.83, and
69.95 (C5, C4, C4', C3', C3), 68.47 (C6), 61.32 (C6'),
54.64 and 53.96 (C2, C2'), 23.96 (SCH2CH3), 14.93
(SCH2CH3).
OCH2CH2CH2NH2,
H6b),
3.20
(2H,
m,
OCH2CH2CH2NH2); 1.73 (2H, m, OCH2CH2CH2NH2).
13C NMR: 165.86 and 165.65 (two C=O), 156.37
[OC(O)NH], 136.65–123.56 (Ar), 98.34 (C1), 74.71,
71.14, and 70.11 (C3, C4, C5), 67.42 and 66.56
(OCH2CH2CH2N, CH2Ph); 61.38 (C6), 54.88 (C2),
37.83 (OCH2CH2CH2N); 29.56 (OCH2CH2CH2N).
6-O-(3,4-Di-O-benzoyl-2-deoxy-2-phthalimido-
b-D-glucopyranosyl)-3,4-di-O-benzoyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl bromide (XVIII).
A solution of Br2 (0.16 ml) in anhydrous CH2ël2 (1 ml)
was added to a solution of thioglycoside (XVII)
(310 mg, 0.29 mmol). The reaction mixture was stirred
for 1 h in the darkness at room temperature. The disap-
pearance of the starting thioglycoside (XVII) (Rf 0.25)
and the formation of bromide (XVIII) (Rf 0.3) was
monitored by TLC (4 : 1 toluene−ethyl acetate). The
reaction mixture was evaporated and dried in a vacuum
to give 315 mg (100%) of bromide (XVIII); 1H NMR:
7.95–7.18 (28 H, m, Ar), 6.38 (1 H, d, J1, 2 9.5, H1),
6-O-(6-O-Acetyl-3,4-di-O-benzoyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl)-1,3,4-tri-O-
benzoyl-2-deoxy-2-phthalimido-a-D-glucopyra-
nose (XX) was obtained by the Helferich reaction sim-
ilarly to disaccharide (XVI) from benzoate (XIV) (0.95 g)
and acceptor (XIII) (0.75 g); yield 1.10 g (79%); [α]D
+72°; 1H NMR: 8.15–7.18 (33 H, m, Ar), 7.09 (1 H, m,
H3), 6.34 (1 H, d, J1, 2 4.8, H1), 6.22 (1 H, m, J3, 2 10.6,
J3, 4 9.4, H3'), 5.61 (1 H, d, J1', 2' 8.4, H1'), 5.60 (1 H, dd,
J4', 5' 9.7, H4'), 5.45 (1 H, dd, J4, 5 9.7, H4), 5.02 (1 H,
dd, J2, 3 11.5, H2), 4.58 (1 H, dd, H2'), 4.48 (1 H, m,
H5), 4.27 (1 H, dd, J6a', 5' 5.1, J6a', 6b' 12.4, H6a'), 4.22 (1
H, dd, J6b', 5' 2.7, H6b'), 4.15 (1 H, dd, J6a, 5 1.8, J6a, 6b
11.3, H6a), 4.07 (1H, m, H5), 3.77 (1 H, dd, J6b, 5 6.1,
H6b); 2.05 (CH3ë(O)). 13C NMR: 170.60, 167.23,
165.56, 165.14, 165.05, and 164.69 (six C=O), 134.17–
123.30 (Ar), 98.63 (C1'), 91.19 (C1), 71.95, 71.47,
71.11, 70.45, 69.81, and 68.27 (C3, C4, C5, C3', C4',
C5'), 67.49 (C6), 62.43 (C6'), 54.60, 53.04 (C2, C2'),
20.65 (CH3ë(O)).
6.30 (1 H, t, J3', 2' = J3', 4' = 10.4, H3'), 6.14 (1 H, t, J3, 2
=
J3, 4 = 10.0, H3), 5.65 (1 H, d, J1', 2' 8.4, H1'), 5.55 (1 H,
t, J4, 5 9.8, H4), 5.38 (1 H, t, J4', 5' 9.5, H4'), 4.75 (1 H, t
H2), 4.48 (1 H, dd, H2'), 4.18–4.04 (2 H, m, H5, H6a),
3.94–3.78 (3 H, m, H5', H6b, H6a'), 3.66 (1 H, dd, J6b', 5'
4.5, J6b', 6a' 13.0, H6b'); 13C NMR: 165.95, 165.62, and
165.47 (three C=O), 134.65–123.80 (Ar), 98.24 (C1'),
78.04 (C1), 76.60, 74.49 (C5, C5'), 71.04, 70.95, and
69.97 (C3, C4, C3', C4'), 68.52 (C6), 61.26 (C6'),
58.44, and 54.64 (C2, C2').
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THE STUDY OF THE REACTION OF TERMINATED OLIGOMERIZATION
397
3-(Benzyloxycarbonylamino)propyl-6-O-(3,4-di- suspension was diluted with dichloromethane (20 ml)
O-benzoyl-2-deoxy-2-phthalimido-b-D-glucopyra- and filtered through a layer of Celite, which was then
nosyl)-3,4-di-O-benzoyl-2-deoxy-2-phthalimido-b- washed with dichloromethane (40 ml). The filtrate was
D-glucopyranoside (XXII) was obtained as described washed with 1 M aqueous Na2S2O3, dried with Na2SO4,
for terminator (XIXa) from benzoate (XX) (100 mg, and evaporated. The residue was chromatographed on
0.09 mmol) and 3-Z-aminopropanol (Vb) (17 mg, silica gel (9 : 1 toluene−ethyl acetate) to give 220 mg
1
0.08 mmol); yield 80 mg (81%); H NMR: 7.95–7.12 (50%) of anhydro derivative (VII) and 85 mg (20%) of
(33 H, m, Ar), 6.28 (1 H, t, J3', 2' = J3', 4' = 9.4, H3'), 6.13 cyclic disaccharide (VIII).
Compound (VII): MS, m/z 521.9 [M + Na]+ (calcu-
(1 H, t, J3, 2 = J3, 4 = 9.9, H3), 5.60 (1 H, d, J1', 2' 8.4, H1'),
1
5.45 (2 H, m, H4, H1), 5.36 (1 H, t, J4', 5' 9.5, H4'),
lated for C28H21NO8 M 499.13); [α]D −5°; H NMR:
5.14 (1 H, br. m, NH), 5.04 (2 H, s, CH2Ph), 4.47
8.18–7.22 (14 H, m, Ar), 6.22 (1 H, t, J3, 2 9.2, H3), 5.80
(2 H, m, H2, H2'), 4.05 (2H, m, H5, H6a), 3.88–3.77
(1 H, s, H1), 5.22 (1 H, d, J4, 3 8.1, H4), 4.85 (1 H, d,
(3 H, m, H6b, H5', H6a'), 3.74–3.63 (2 H, m,
J5, 6b 5.3, H5), 4.55 (1 H, d, H2), 4.30 (1 H, d, J6a, 6b 7.8,
H6a), 4.00 (1 H, dd,H6b); 13C NMR: 167.58, 166.48,
and 165.93 (three C=O), 134.36–123.71 (Ar), 102.35
(C1), 78.02 (C4), 76.92 (C5), 68.86 (C3), 68.26 (C6),
56.07 (C2).
OCH2CH2CH2NH2,
H6b'),
3.46
(1H,
m,
OCH2CH2CH2NH2); 3.05 (2H, m, OCH2CH2CH2NH2);
1.56 (2H, m, OCH2CH2CH2NH2). 13C NMR: 167.42,
165.84, 165.05, and 165.59 (four C=O), 134.14–125.27
(Ar), 98.15 (C1), 97.71 (C1'), 74.42 (C5'), 73.15 (C5),
71.17 (C3), 70.85 (C3'), 70.55 (C4), 69.93 (C4'), 67.93
(C6), 67.34 (OCH2CH2CH2NH2); 66.37 (CH2Ph);
61.21 (C6'), 54.76, 54.57 (C2, C2'), 37.86
(OCH2CH2CH2NH2); 29.36 (OCH2CH2CH2NH2).
Compound (VIII): MS, m/z 1021.1 [M + Na]+ (cal-
1
culated for C56H42N2O16 M 998.26); [α]D –39°; H
NMR: 7.94–7.18 (14 H, m, Ar), 6.40 (1 H, t, J3, 2
=
J3, 4 = 10.5, H3), 6.05 (1 H, dd, J4, 5 6.5, H4), 5.50 (1 H,
d, J1, 2 4.2, H1), 5.03 (1 H, dd, H2), 4.42 (1 H, d, J5, 4
6.5, H5), 4.05 (1 H, d, J6a, 6b 10.4, H6a), 3.98 (1 H, d,
H6b); 13C NMR: 167.47, 165.71, 165.34 (three C=O),
134.06–123.68 (Ar), 98.47 (C1), 78.37 (C5), 70.54
(C4), 68.77(C3), 66.08 (C6), 55.13 (C2).
Ethyl
6-O-[6-O-(3,4-di-O-benzoyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl)-3,4-di-O-ben-
zoyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl]-
3,4-di-O-benzoyl-2-deoxy-2-phthalimido-1-thio-b-
D-glucopyranoside (XXIII) was synthesized as
described for disaccharide (XVI) from benzoate (XX)
(250 mg, 0.22 mmol) and acceptor (IV) (115 mg,
Oligomerization(III) + (Va). A mixture of mono-
mer (III) (100 mg, 0.12 mmol), terminator (Va) (8 mg,
0.025 mmol), freshly distilled dichloromethane (1 ml),
and molecular sieves 4 Å (200 mg) was stirred for 1 h
under argon at room temperature. Methyl triflate (82 µl,
0.75 mmol) was then added. After 1 h, TLC (5 : 1
toluene−ethyl acetate) showed the absence of the start-
ing monomer (Rf 0.70) and prevalence of the product
(Rf 0.65. The reaction mixture was evaporated and
chromatographed on silica gel (50 : 1 – 10 : 1 toluene–
ethyl acetate) to give 50 mg (55%) of glycal (IX),
13 mg (15%) of (VII), and 8 mg (10%) of (VIII).
1
0.2 mmol); yield 246 mg (75%); H NMR: 7.95–7.05
(42 H, m, Ar), 6.24 (1 H, t, J3'', 2'' = J3, H3''), 6.18 (1 H,
t, J3, 2 = J3, 4 = 9.8, H3), 6.11 (1 H, t, J3', 2' = J3', 4' = 9.5,
H3'), 5.58 (1 H, d, J1'', 2'' 8.4, H1''), 5.54 (1 H, d, J1, 2
10.5, H1), 5.49 (1 H, d, J1', 2' 8.4, H1'), 5.37 (2 H, m, H4,
H4'), 5.29 (1 H, t, J4'', 5'' 9.6, H4''), 4.54 (1 H, t, H2), 4.41
(1 H, dd, H2'), 4.34 (1 H, dd, H2''), 4.05 (1 H, dd, J6a', 5'
4.1, J6a', 6b' 10.7, H6a'); 4.01–3.94 (3 H, m, H6a, H5,
H5'), 3.90 (1 H, m, H5''), 3.85–3.79 (2 H, m, H6a'',
H6b'), 3.71 (1 H, dd, J6b'', 5'' 5.2, J6b'', 6a'' 12.5, H6b''), 2.60
(2 H, m, SCH2CH3), 1.15 J 8, SCH2CH3). 13C NMR:
167.09, 165.56, and 165.13 (three C=O), 134.05–
123.50 (Ar), 97.86 and 97.42 (C1', C1''), 80.83 (C1),
74.24, 72.60, 71.99, 71.06, 70.83, 70.33, and 69.82
(C3–C5, C3'–C5', C3''–C5''), 68.21, and 67.88 (C6,
C6'), 61.27 (C6''), 54.68, 54.60, and 53.87 (C2, C2,
C2''), 23.92 (SCH2CH3), 14.96 (SCH2CH3).
1
Compound (IX): [α]D –70°; H NMR: 8.08–7.15
(29 H, m, Ar), 6.90 (1 H, s, H1), 5.88 (1 H, d, J3, 4 3.3,
H3), 5.77 (1 H, t, J4, 5 4.0, H4), 4.57 (1 H, m, H5), 3.90
(1 H, dd, J6a, 5 7.4, J6a, 6b 10.9, H6a), 3.58 (1 H, dd, J6b, 5
4.0, H6b); 13C NMR: 167.66, 165.67, and 164.83 (three
C=O), 143.62 (Tr), 134.15–123.63 (Ar, C2), 105.00
(C1), 87.31 (C(Ph)3), 76.34 (C5), 67.67 (C4), 66.70
(C3), 61.59 (C6).
Terminated oligomerization (IV) + (Va). A mix-
ture of monomer (IV) (430 mg, 0.77 mmol), 3-Fmoc-
Terminated oligomerization (XVII) or (XVIII) +
aminopropanol (Va) (45 mg, 0.15 mmol), freshly dis- (Va) (a series of experiments). Experiment no. 1. The
tilled dichloromethane (10 ml), and molecular sieves reaction was carried out as described for (IV) + (Va)
4 Å (1 g) was stirred for 1 h under argon at room tem- using monomer (XVII) (30 mg, 30 µmol), terminator
perature. The resulting mixture was cooled to –20°ë, (Va) (2 mg, 6 µmol), freshly distilled dichloromethane
NIS (345 mg, 1.53 mmol) and TfOH (27 µl, 0.3 mmol) (250 µl), NIS (13 mg), and TfOH (2 µl) in the presence
were added, and the reaction mixture was stirred for 1 h of molecular sieves 4 Å at –15°C. The disappearance of
under argon at –15°ë. According to TLC (3 : 2 toluene– the starting monomer (Rf 0.35) and the generation of
ethyl acetate), the starting monomer (Rf 0.55) disap- almost the only product (visualization with H3PO4)
peared, and the reaction mixture contained the products with Rf 0.45, which was identified on the basis of NMR
with Rf 0.7 (the main product) and Rf 0.6. Pyridine spectrum as the cyclic disaccharide (VIII), were moni-
(50 µl) was added to the reaction mixture; the resulting tored by TLC (2 : 1 toluene–ethyl acetate).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006

398
GENING et al.
Experiment no. 2. The reaction was carried out as solved in ethanol (5 ml); hydrazine hydrate (0.5 ml)
described for (IV) + (Va) using monomer (XVII) was added, and the reaction mixture was refluxed for
(50 mg, 47 µmol), terminator (Va) (3 mg, 9 µmol), 1 h. Then the reaction mixture was evaporated and
freshly distilled dichloromethane (125 µl), NIS dried in a vacuum. The residue was chromatographed
(13 mg), and TfOH (2 µl) in the presence of molecular on TSK HW-40(S) (25–40 µm) gel column (80 ×
sieves 4 Å at –40°C.According to TLC, the results were 2.5 cm) in 0.1 M AcOH to give 12 mg (10%) of disac-
identical to those obtained in the Experiment no. 1.
Experiment no. 3. (XVIII) + (Va) terminated oligo-
merization.
charide (XXVII, n = 1); 1H NMR: 4.75 and 4.77 (2 H,
2 d, H1, H1'), 4.31 (1 H, t, J6a, 6b 10.1, H6a), 4.10 (1 H,
m, OCH2CH2CH2NH2), 4.04–3.97 (2 H, m, H6b, H6a'),
3.88 (1 H, m, OCH2CH2CH2NH2), 3.84 (1 H, dd, J6b', 5'
=
Experiment no. 4. The reaction was carried out as
described for (IV) + (Va) using monomer (XVIII)
(30 mg, 28 µmol), terminator (Va) (2 mg, 6 µmol),
freshly distilled dichloromethane (175 µl), and AgOTf
(15 mg) in the presence of molecular sieves 4 Å at
−15°C. According to TLC of the reaction mixture, the
results were identical to those obtained in Experiment
no. 1.
Experiment no. 5. The reaction was carried out as
described in Experiment no. 1 using freshly distilled
acetonitrile (250 µl) as a solvent in the presence of
molecular sieves 3Å. According to TLC of the reaction
mixture, the results were identical to those obtained in
Experiment no. 1.
Terminated oligomerization (XVIII) + (Va). Mer-
cury(II) cyanide (100 mg, 0.4 mmol) and HgBr2
(40 mg, 0.11 mmol) were added to a solution of termi-
nator (Va) (18 mg, 0.06 mmol) in anhydrous CH3CN
(0.3 ml) under argon; the mixture was stirred for 10 min
at room temperature; a solution of bromide (XVIII) in
anhydrous CH3CN (0.5 ml) was added to the solution
of the acceptor; and the reaction mixture was stirred for
additional 1 h. According to TLC (2 : 1 toluene−ethyl
acetate), the reaction mixture contained no starting bro-
mide (XVIII) (Rf 0.50) and exhibited a separate spot
with Rf 0.55 and a lane of spots from the start line to Rf
0.40. The product with Rf 0.55 was isolated by chroma-
tography on silica gel to give 87 mg (30%) of cyclic di-
saccharide (VIII).
Analysis of the oligomerization results
(Method 1). Removal of Fmoc group. The mixture of
oligomers (240 mg) obtained after the (XVIII) + (Va)
terminated oligomerization and separation of the cyclic
product was dissolved in anhydrous THF (1 ml),
ethanethiol (0.07 ml, 0.9 mmol) and DBU (3 µl,
0.02 mmol) were added, and the mixture was stirred for
20 min. Thin layer chromatography in 5 : 1 ëHCl3–
MeOH demonstrated the appearance of ninhydrin-pos-
itive spots with Rf 0.1–0.3. Acetic acid (20 µl) was
added, the reaction mixture was evaporated, the residue
was dissolved in 1 : 4 ëH2Cl2–MeOH, and Amberlyst
15 (Fluka) ion-exchange resin (H+) was added up to
complete sorption of ninhydrin-positive compounds
J6b', 6a' = 12.4, H6b'), 3.75 (1 H, m, H5), 3.68 (2 H, m,
H3, H3'), 3.63 (1 H, m, J4, 3 9.3, H4), 3.59–3.52 (2 H, m,
H5', H4'), 3.21 (2 H, m, CH OCH2CH2CH2NH2), 3.09–
3.01 (2 H, m, H2, H2'), 2.08 (2 H, m,
OCH2CH2CH2NH2); 13C NMR: 101.25 (C1, C1'), 77.34
(C5'), 75.78 (C5), 73.98 and 73.89 (C3, C3'), 70.82
(C4'),
70.59
(C4),
69.50
(C6),
68.94
(OCH2CH2CH2NH2), 61.54 (C6'), 56.95 (C2, C2'),
38.41 (OCH2CH2CH2NH2), 27.95 (OCH2CH2CH2NH2).
Oligomerization(XVIII) + (XIXa). The reaction
was carried out similarly to (XVIII) + (Va) oligomer-
ization using bromide (XVIII) (70 mg) and terminator
(XIXa) (11 mg). According to TLC, the result of the
reaction was analogous to that of the (XVIII) + (Va)
oligomerization.
Analysis of the results of oligomerization
(Method 2). The products obtained by oligomerization
(XVIII) + (XIXa) were analyzed as follows: gel chro-
matography on BioBeads SX-3 (BioRad) in toluene of
the reaction mixture helped isolate the monosaccharide
fraction consisting of the unreacted terminator (5 mg),
the disaccharide fraction containing cyclic disaccharide
(VIII) and the product of monomer hydrolysis (XVII),
and an oligomeric fraction, which was then analyzed as
described in Method 1. Trisaccharide (XXVII, n = 2)
was obtained; yield 4 mg (12%); MS, m/z: 559.2 [M +
H]+ (calculated for C21H42N4O13 M 558.3).
Oligomerization (XVIII) + (XIXa) with graduate
addition of monomer. Bromide (XVIII) (100 mg,
0.1 mmol in 300 µl of anhydrous acetonitrile) was
added in 60 µl portions at 30-min intervals to a solution
of terminator (XIXa) (15 mg, 0.02 mmol), Hg(CN)2,
(24 mg, 0.1 mmol), and HgBr2 (10 mg, 0.03 mmol) in
anhydrous acetonitrile (50 µl) at stirring under argon.
The reaction mixture was treated as described for the
(XVIII) + (Va) oligomerization. The reaction products
were analyzed by Method 2. Yield: 4 mg of terminator
(XIXa), 7 mg (15%) of terminated trisaccharide
(XXVII, n = 2), and traces of pentasaccharide (XXVII,
n = 4); MS, m/z: 881.3 [M + H]+ (calculated for
C33H64N6O21 M 880.4).
Terminated oligomerization (XVIII) + (XIXb).
(TLC monitoring). The suspension of cation exchanger The reaction was carried out and analyzed using mono-
was transferred to a chromatographic column and mer (XVIII) (100 mg, 0.1 mmol) and terminator
washed with 1 : 4 ëH2Cl2–MeOH. The terminated (XIXb) (14 mg, 0.02 mmol) as described for the
products were eluted with a mixture of MeOH (XVIII) + (XIXa) oligomerization. Removal of the
(100 ml), ëH2Cl2 (25 ml), and concentrate HCl (5 ml). Fmoc group. The mixture obtained after oligomeriza-
The resulting mixture of terminated oligomers was dis- tion was dissolved in 20 : 30 : 0.8 : 0.5 i-PrOH–THF–
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THE STUDY OF THE REACTION OF TERMINATED OLIGOMERIZATION
399
AcOH–H2é mixture, a catalytic amount of 10% Pd/C
REFERENCES
was added, and the mixture was stirred for 16 h under
hydrogen at room temperature. The reaction mixture
was filtered through a layer of Celite; the filtrate was
evaporated; and then the reaction products were ana-
lyzed by Method 2. Yield: 5.5 mg (12%) of terminated
trisaccharide (XXVII, n = 2) and traces of pentasaccha-
ride (XXVII, n = 4).
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Terminated oligomerization (XVIII) + (XXII).
The reaction was carried out using monomer (XVII)
(50 mg, 0.05 mmol) and terminator (XXII) (9 mg,
0.007 mmol) as described for the (XVII) + (XVIII) oli-
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Terminated oligomerization (XXIII) + (XXII).
The reaction was carried out using trisaccharide mono-
mer (XXIII) (30 mg, 0.02 mmol) and terminator
(XXII) (5 mg, 4 µmol) as described for (XVII) + (Va)
oligomerization; yield 27 mg (95%) of cyclic trisaccha-
ride (XXVIII). [α]D +44°. 1H NMR: 7.90–7.18 (42 H,
m, Ar), 6.15 (1 H, t, J3, 2 = J3, 4 = 10.2, H3), 5.77 (1 H,
d, J1, 2 8.2 H1), 5.56 (1 H, t, J4, 5 9.52, H4), 4.68 (1 H,
dd, H2), 4.19 (1 H, dd, J6a, 5 5.4, J6a, 6b 12.6, H6a), 4.14
(1 H, m, H5), 3.90 (1 H, dd, H6b); 13C NMR: 165.80
and 164.74 (two C=O), 133.94–123.55 (Ar), 97.28
(C1), 73.78 (C5), 71.37 (C3), 69.75 (C4), 68.41 (C6),
55.02 (C2).
Terminated oligomerization (XXIV) + (XXII).
The reaction was carried out using trisaccharide
(XXIV) (75 mg, 46 µmol) and terminator (XXII)
(10 mg, 8 µmol) as described for oligomerization
(XVIII) + (XIXa); yield 36 mg (50%) of cyclic trisac-
charide (XXVIII)
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ACKNOWLEDGMENTS
This work was supported by CRDF grant no. RUB1-
2639-MO-05 and Russian Foundation for Basic
Research, project no. 05-03-08107.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006