Synthesis of octa- and dodecamers of d-ribitol-1-phosphate and their protein conjugates

Article abstract of DOI:10.1016/j.carres.2005.10.023

The bacterial cell-wall-associated teichoic acids contain predominantly d-ribitol residues interconnected by phosphodiester linkages. Because of their location, these antigens may be vaccine candidates as part of conjugate vaccines. Here, we describe the

Full text of DOI:10.1016/j.carres.2005.10.023

Carbohydrate
RESEARCH
Carbohydrate Research 341 (2006) 2037–2048
Synthesis of octa- and dodecamers of D-ribitol-1-phosphate and
their protein conjugates
a,b
Aniko Fekete, Peter Hoogerhout,a,c Gijsbert Zomer,c Joanna Kubler-Kielb,a
´
Rachel Schneerson,a John B. Robbinsa and Vince Pozsgaya,*
aNational Institute of Child Health and Human Development, National Institutes of Health, 31 Center Dr. MSC 2423 Bethesda,
MD 20892-2423, USA
bResearch Group for Carbohydrates of the Hungarian Academy of Sciences, H-4010, Debrecen, PO Box 55, Hungary
cUnit Research and Development, Netherlands Vaccine Institute, Bilthoven, The Netherlands
Received 2 September 2005; accepted 20 October 2005
Available online 3 February 2006
´
´
This paper is dedicated to Professor Andras Liptak on the occasion of his 70th birthday
Abstract—The bacterial cell-wall-associated teichoic acids contain predominantly D-ribitol residues interconnected by phosphodies-
ter linkages. Because of their location, these antigens may be vaccine candidates as part of conjugate vaccines. Here, we describe the
synthesis of extended oligomers of D-ribitol-1-phosphate linked to a spacer having an amino group at its terminus. The synthesis
utilized a fully protected D-ribitol-phosphoramidite that was oligomerized in a stepwise fashion followed by deprotection. The free
oligomers were connected to bovine serum albumin using oxime chemistry. Thus, the ribitol phosphate oligomers were converted
into keto derivatives, and the albumin counterpart was decorated with aminooxy groups. Reaction of the functionalized saccharide
and protein moieties afforded conjugates having up to 20 ribitol phosphate chains.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Glycoconjugate; Phosphodiester; Ribitol phosphate; Synthesis; Teichoic acid; Vaccine
1. Introduction
sugars including amino sugars or a D-alanine ester.1 The
size and degree of substitution varies and depends on the
age of the cells and the composition of the growth med-
ia.5 The size of teichoic acids isolated from Bacillus
subtilis and Staphylococcus aureus was reported to be
around 25 kDa accounting for an average range of
45–60 repeating units.6,7 Common are 1,5-poly(D-ribitol
phosphates) found in staphylococci, bacilli and 3,5-
poly(ribitol phosphates) in Nocardiopsis species.8 Patho-
genic Gram-negative bacteria like Haemophilus influen-
zae types a and b also contain ribitol phosphate
moieties in their capsular polysaccharides (CPSÕs). Anti-
bodies elicited by H. influenzae type b CPS, composed of
D-ribosyl-D-ribitol phosphate repeating subunit, were
shown to precipitate poly(D-ribitol phosphates) of S.
aureus, Bacillus pumilus and Lactobacillus plantarum.9
We have shown recently that antibodies induced by a
protein conjugate of an unseparable mixture of
Polymers of D-ribitol phosphate (teichoic acids) are
major cell-wall components of many Gram-positive bacte-
ria. These anionic structures form wall-associated
carbohydrate-containing linear chains, where the
repeating units are connected through phosphodiester
linkages, covalently linked to cell-wall muramic acid
residues in the peptidoglycan. The physiological roles
of teichoic acids include ion exchange and control of
the activity of autolytic enzymes that are important for
growth and division of bacterial cells.1 There are several
types of teichoic acids, and their biosynthesis, function
and compositions have been described.2–4 Typically,
poly(D-ribitol phosphates) are substituted with different
*
Corresponding author. Tel.: +1 301 295 2266; fax: +1 301 295
1435; e-mail: pozsgayv@mail.nih.gov
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.10.023

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A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
poly(D-ribitol phosphate) and poly(glycerol phosphate)
of B. pumilus Sh 18 recognized CPSÕs of H. influenzae
types a and b, as well as those of S. aureus and S. epider-
midis teichoic acids.10 So far we have not been able to
isolate a pure chain of poly(D-ribitol phosphate) from
any Gram-positive bacteria. We note that related struc-
tures, including oligomers of ribosyl-ribitol phosphate,
have been prepared by chemical synthesis either in a
stepwise approach11 or by controlled polymerization.12
However, the synthesis of polymers of ribitol phosphate
has not yet been described. According to our experience,
the size of an oligosaccharide to induce polysaccharide-
specific antibody response when covalently linked to an
immunogenic protein should preferably be in the deca-
mer range.13,14 Therefore, we decided to synthesize an
octamer and a dodecamer of ribitol phosphate to inves-
tigate the role of the chain length in the immunological
properties of ribitol phosphate oligomers.
Here, we report chemical synthesis of compounds 34
and 35, that is, octa- and dodecamers of D-ribitol phos-
phate carrying a reactive amino group that enables their
conjugation to proteins. We also describe the conjuga-
tion of the target compounds to bovine serum albumin
using a method recently reported by Kubler-Kielb and
Pozsgay,15 using thioether16 and oxime chemistries.17–19
Such conjugates will be used for the development of a
single-component experimental vaccine likely capable
of acting against several pathogens carrying D-ribitol
phosphate moieties in their surface antigens.
of a broad range of phosphodiester-linked oligomers of
carbohydrates, was chosen.22
In applying van BoomÕs phosphoramidite protocol, we
have chosen a stepwise sequential approach using D-ribi-
tol derivative 7 as the key building block. In compound
7, the phosphityl moiety serves as the precursor for the
phosphodiester part of the final products, the 4,40-
dimethoxytrityl (DMT) group is a temporary protecting
group at the site of chain extension, whereas the three
benzyl groups provide permanent protection until the last
stages of the synthesis. Preparation of phosphoramidite 7
started with D-ribitol derivative 1 that was synthesized
from D-ribonolactone as described by van Boom and
co-workers.23,24 Compound 1 was treated with trimethyl-
acetyl chloride in pyridine to give the pivaloyl derivative
2 (Scheme 1). The pivaloyl group was chosen over the
conventional O-acetyl protection because of the higher
stability of pivaloyl esters during the tritylation step (see
below). Next, the allyl group at O-5 was removed in a
two-step procedure involving isomerization into the pro-
penyl derivative using (1,5-cyclooctadiene)-bis(methyldi-
phenylphosphine)iridium hexafluorophosphate25,26 (!3)
followed by mercury(II) chloride-assisted hydrolysis to
afford 4 in a 92% combined yield over three steps. The
ribitol alcohol 4 so obtained was converted to the trityl
derivative 5 using 4,40-dimethoxytrityl chloride in the
presence of HunigÕs base to afford compound 5 (85%).
¨
Next, the trimethylacetyl protecting group was removed
under alkaline conditions to give the hydroxy derivative
6 in 90% yield. That this compound was the optically
pure D-isomer was shown by its conversion to either
(ꢀ)-(R)- or (+)-(S)-a-methoxy-a-trifluoromethylphenyl-
acetyl derivative using the corresponding acid chlorides,
to afford the so-called Mosher esters.27 The observation
of only one resonance in the 19F NMR spectrum provides
a proof of 6 being a single stereoisomer; any isomerization
during the preceding steps would have led to an equilib-
2. Results and discussion
For the preparation of the target compounds 34 and 35,
the phosphoramidite method20 which has been devel-
oped for solid-phase oligonucleotide synthesis21 and
adopted by van Boom and co-workers for the synthesis
N
P
HO
PivO
BnO
BnO
RO
O
N
O
BnO
BnO
BnO
BnO
BnO
BnO
a
d
f
OBn
OBn
OBn
OBn
AllO
1
RO
DMTO
DMTO
7
2 R = All
3 R = Prop
4 R = H
5 R = Piv
6 R = H
b
e
c
Scheme 1. Reagents and conditions: (a) 1.05 equiv of pivaloyl chloride, C5H5N, 0 ꢁC, 1.5 h; (b) (1,5-cyclooctadiene)-bis(methyldiphenylphos-
phine)iridium hexafluorophosphate (cat.), THF, 23 ꢁC, 4 h; (c) 1.06 equiv of HgO and 1.05 equiv of HgCl2, acetone–H2O, 23 ꢁC, 0.5 h; (d) 1.25 equiv
of EtN(i-Pr)2, 1 equiv of DMTCl, CH3CN, 23 ꢁC, 1.5 h; (e) 2 equiv of NaOMe, dioxane and MeOH, 23 ꢁC, 72 h; (f) 5 equiv of EtN(i-Pr)2, 2.45 equiv
of chloro-2-cyanoethyl-N,N-diisopropylphosphoramidite, 1,2-dichloroethane, 0–23 ꢁC, 2 h.

A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
2039
rium mixture exhibiting more than one line in the 19F
spectrum. On the other hand, when the same experiment
was performed on the Mosher esters of the corresponding
D,L ribitol derivative (not described in the experimental),
the 19F NMR spectrum showed two signals of equal
intensity, indicating two stereoisomers. Subsequently,
ribitol 6 was phosphitylated using 2-cyanoethyl N,N-di-
N
P
O
P
H
N
OBn
O
N
O
O
O
O
N
O
BnO
BnO
BnO
BnO
OBn
a
OBn
€
isopropylchlorophosphoramidite in a mixture of HunigÕs
base and dichloromethane to afford phosphoramidite 7 in
61% yield.
+
DMTO
HO
7
RO
H
N
In our original strategy, we planned to oligomerize
compound 7 in an automatic solid-phase oligonucleo-
tide synthesizer,24 using standard phosphoramidite
chemistry20 consisting of the following steps: (1) 1H-tetra-
zole-assisted attachment to the glass-attached spacer,
(2) oxidation of the phosphite triester into phosphate tri-
ester using iodine, (3) capping of unreacted ÔacceptorÕ
and (4) cleavage of the DMT group using trichloroacetic
acid. After 6 cycles, the linkage between the solid sup-
port and the spacer was cleaved by the action of ammo-
nia (not described in the experimental). Surprisingly, an
intractable product mixture was obtained that did not
appear to contain the targeted hexamer. This failure
prompted us to reinvestigate the oligomerization using
solution-phase chemistry.
Thus, phosphoramidite 7 was combined with the
spacer 8 by treatment with 1H-tetrazole in MeCN, fol-
lowed by oxidation with iodine to afford phosphotriester
9 in 77% yield (Scheme 2). Attempted removal of the
DMT group from compound 9 by trichloroacetic acid
simultaneously also cleaved the phosphotriester moiety.
This observation explained our failure to use standard
phosphoramidite chemistry in our attempted solid-
phase synthesis as described above. Next, we investi-
gated less aggressive conditions, and eventually we found
that an 85:10:5 mixture of acetic acid, CH2Cl2 and water
selectively cleaved the DMT group in compound 9 and
OBn
9 R = DMT
10 R = H
b
O
8
Scheme 2. Reagents and conditions: (a) 10 equiv of a 0.45 M solution
of tetrazole in CH3CN, CH3CN, 23 ꢁC, 1 h, then 0.5 M solution of
iodine in 2:1 THF–water (excess); (b) AcOH–H2O, 23 ꢁC, 2 h.
afforded 10 in 88% yield without compromising the
integrity of the phosphotriester moiety. However, the
reaction time increased up to 4 h, compared with just
a few minutes for such a cleavage in the standard oligo-
nucleotide synthesis. We hypothesized that the use of
aqueous acetic acid instead of trichloroacetic acid for
the removal of the DMT group would require even long-
er reaction times in solid-phase approach, thus abolish-
ing one of its main attractions, that is, increased speed
over solution-phase chemistry. Therefore, we decided
to use solution-phase chemistry throughout this project.
Next, alcohol 10 was coupled with phosphoramidite 7
as described for the preparation of 9 to afford the dimer
11 from which the 4,40-dimethoxytrityl group was
removed by acetic acid to afford the ÔacceptorÕ 12. Sub-
sequent iterative chain extensions, presented in detail in
Section 4, using the reaction sequence as described
above, afforded the higher membered oligomers of ribi-
tol phosphate up to the dodecamer 32.
O
P
H
N
O
O
Bn
O
O
N
O
BnO
BnO
11 n = 1 R = DMT
12 n = 1 R = H
13 n = 2 R = DMT
14 n = 2 R = H
15 n = 3 R = DMT
16 n = 3 R = H
17 n = 4 R = DMT
18 n = 4 R = H
19 n = 5 R = DMT
20 n = 5 R = H
21 n = 6 R = DMT
22 n = 6 R = H
OBn
23 n = 7 R = DMT
24 n = 7 R = H
25 n = 8 R = DMT
26 n = 8 R = H
27 n = 9 R = DMT
28 n = 9 R = H
29 n = 10 R = DMT
30 n = 10 R = H
31 n = 11 R = DMT
32 n = 11 R = H
O
P
O
O
O
N
BnO
BnO
OBn
O
R
n

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A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
Having assembled extended oligomers of D-ribitol
O
P
NH2
phosphate, we started the removal of the protecting
groups. Hydrolysis of the cyanoethyl group from the
monomeric phosphotriester 10 was uneventful as
expected to provide a phosphodiester that was subjected
to catalytic hydrogenation/hydrogenolysis. Surprisingly,
when the hydrogenolysis was carried out in ethanol, the
only product that was isolated was the N-ethyl deriva-
tive of the targeted ribitol phosphate. While the forma-
tion of the N-ethyl derivative was unexpected,
N-alkylation during hydrogenolysis in primary or sec-
ondary alcohols is not unprecedented and is explained
by the formation of an aldehyde from the solvent by
its catalytic oxidation, presumably by oxygen adsorbed
on the catalyst, followed by Schiff-base formation and
subsequent reduction.28–30 Subsequently, we found that
the formation of the N-ethyl derivative could be com-
pletely suppressed when the hydrogenolysis was carried
out in t-BuOH, affording the free ribitol phosphate 33 as
the only product (Scheme 3). Removal of the protecting
groups from the higher membered oligomers 24 and 32
was performed similarly and the unprotected octa- (34)
and dodecamer (35) were isolated in 66% and 80%
yields, respectively. The structures of compounds 34
and 35 were verified by NMR and mass-spectrometric
methods (see Section 4). Particular proof was provided
by the 31P NMR spectra of 34 and 35, which exhibited
two sets of characteristic resonances in the ratio of 7:1
and 11:1, respectively, indicating a different environment
of just one of the phosphodiester linkages, as expected.
Covalent attachment of 33, 34 and 35 to bovine serum
albumin (BSA) was performed according to the proce-
dure of Kubler-Kielb and Pozsgay.15 The method is
based on the well-documented oxime formation between
an aldehyde or a ketone and an aminooxy compound
O
O
O-Na+
HO
HO
a,b
OH
10
HO
33
Scheme 3. Reagents and conditions: (a) MeOH, concd NH4OH,
50 ꢁC, 8 h, Dowex 50 · 8–100(Na+); (b) H2, 10% Pd/C, 2:1 t-BuOH–
water, 23 ꢁC, 2 days.
NH2
O
P
O-Na+
O
O
HO
34 n = 4
OH
HO
35 n = 6
O
P
O
O-Na+
O
HO
HO
OH
O
H
n
H
N
CH3
O
P
O
O
H
N
CH3
O
P
O-Na+
O
O
O
O
O-Na+
HO
O
O
OH
HO
O
HO
HO
OH
P
O
O-Na+
O
HO
HO
HO
OH
38
36
O
H
6

A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
2041
H
N
CH3
O
O
O
P
O-Na+
O
O
O
O
O
HO
OH
CH3
CH3
O
HO
O
O
P
O
34
O
O-Na+
a-d
HO
HO
OH
O
H
4
37
Scheme 4. Reagents and conditions: (a) 5-ketohexanoic anhydride (excess), MeOH–H2O, triethylamine, 23 ꢁC, 20 min; (b) Bio-Gel P2
chromatography in 0.02 M C5H5N–AcOH; (c) freeze drying; (d) Dowex 50 · 8–100(Na+), H2O.
Table 1. Conjugates of ribitol phosphates 36, 37 and 38 with bovine serum albumin
Ribitol
phosphate
Amount of the
aminooxy groups
on BSA (lmol)
Amount of
hapten (lmol)
Mass by
MALDI [Da]
Hapten loading
on BSA (mol/mol)
Protein/sugar
ratio (mol/mol)
36
37
38
4
4
1.2
7.3
5.8
1.9
81,249
106,077
106,110
18
15
10
1:0.1
1:0.5
1:0.5
and proceeds under mild conditions. We have chosen to
introduce aminooxy groups in the protein and convert
the carbohydrate counterpart into a keto derivative.
For example, octamer 34 was reacted with 5-ketohexa-
noic anhydride in aqueous MeOH in the presence of tri-
ethylamine (Scheme 4). Purification of the product by
chromatography on a Bio-Gel P2 column, followed by
conversion to the sodium form using Dowex 50 · 8–
100 (Na+), afforded compound 37 in a 85% yield. The
monomer 33 and the dodecamer 35 were similarly acyl-
ated to afford the corresponding ketohexanoyl deriva-
tives 36 and 38. The keto derivatives 36–38 were
allowed to react with aminooxylated BSA in pH 7.4
phosphate buffer as recently reported.15 Table 1 shows
the results of some typical conjugation experiments,
indicating average incorporation levels of 10–18 saccha-
ride units per mole of BSA as determined by MALDI-
TOF mass spectrometry.
meric ribitol phosphoramidite by stepwise polymeriza-
tion. We have also demonstrated that the amino
spacer can serve as a handle to conjugate the oligomers
to proteins under mild conditions using oxime chemis-
try. The immunogenicity and antigenicity of the protein
conjugates described here and conjugates of the
octa- and dodecamers with medically useful proteins
are currently being evaluated.
4. Experimental
4.1. General methods
All chemicals were of commercial grade and used with-
out purification. Solvents for chromatography were dis-
tilled prior to use. Anhydrous solvents were obtained
from Aldrich Chemical Company. Bovine serum albu-
min was purchased from Sigma Chemical Company.
Column chromatography was performed on Silica Gel
60 (0.040–0.063 mm). The 1H and 13C NMR spectra
were recorded at either 500 or 300 MHz and 125 and
75.5 MHz, respectively. Internal references: TMS
(0.000 ppm for 1H for solutions in CDCl3), acetone
3. Conclusions
We constructed spacer-linked, higher membered ribitol
phosphate oligomers from a suitably protected, mono-

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A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
(2.225 ppm for H and 31.00 ppm for 13C for solutions
in D2O), MeOH (3.358 ppm for 1H and 49.68 ppm
for 13C for solutions in D2O) and CDCl3 (77.00 ppm
for 13C for solutions in CDCl3). Coupling constants
are given in hertz. The electrospray-ionization mass
spectra (ESIMS) were recorded at the Laboratory of
Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD.
For the MALDI-TOF mass spectra, the protein conju-
gates were dissolved in 0.1% TFA in 30% aq MeCN
and applied to the target in sinapinic acid matrix.
Elemental analyses were performed by Atlantic Micro-
lab, Inc., Norcross, GA.
phy for analytical purposes. H NMR (CDCl3): d 1.23
1
1
(s, 9H), 1.54 (m, 3H), 3.80–3.95 (m, 5H), 4.15–4.25
(dd, 1H), 4.50–4.80 (m, 8H), 6.15–6.25 (dd, 1H), 7.2–
7.4 (m, 15H). 13C NMR (CDCl3): d 12.6, 27.3, 38.9,
63.7, 68.5, 72.2, 72.5, 73.9, 77.4, 77.5, 78.3, 98.8,
127.0–129.0, 138.2–138.4, 146.6, 178.4. HRESIMS:
Calcd for C34H42LiO6, m/z 553.3145; found, m/z
553.3148. Anal. Calcd for C34H42O6: C, 74.70; H, 7.74.
Found: C, 74.61; H, 7.70.
4.4. 2,3,4-Tri-O-benzyl-1-O-pivaloyl-D-ribitol (4)
To a solution of compound 3 (5.46 g, 10.0 mmol) in
acetone (115 mL) and water (8 mL), HgO (2.29 g,
10.6 mmol) and HgCl2 (2.86 g, 10.5 mmol) were
added.24 The resulting suspension was stirred at room
temperature for 30 min. The solids were removed by
filtration. The filtrate was successively washed with
50% satd aq KI, 1% aq NaHSO3 and 1 M aq NaHCO3,
followed by separation of the phases. The organic
phase was dried (Na2SO4). Removal of the volatiles
under reduced pressure afforded product 4 (4.65 g,
92% for three steps) as an amorphous material that
was used in the next step without further purification.
A small amount was purified by column chromatogra-
4.2. 5-O-Allyl-2,3,4-tri-O-benzyl-1-O-pivaloyl-D-ribitol
(2)
To a stirred solution of 5-O-allyl-2,3,4-tri-O-benzyl-D-
ribitol23,24,31,32 (1) (4.63 g, 10.0 mmol) in dry pyridine
(25 mL) was added dropwise pivaloyl chloride (1.30
mL, 10.5 mmol) at 0 ꢁC within 5 min. Stirring at
0 ꢁC was continued for 25 min, then at 23 ꢁC for
90 min. Water (1 mL) was added, and the reaction mix-
ture was concentrated. A solution of the residue in 1:1
Et2O–hexane was washed successively with water and
with 1 M aq NaHCO3. The organic layer was dried
(MgSO4), filtered and concentrated. The residue was
dissolved in toluene, followed by concentration under
reduced pressure. Compound 2 thus obtained was used
in the next step without further purification. A small
amount was purified by column chromatography for
1
phy for analytical purposes. H NMR (CDCl3): d 1.20
(s, 9H), 2.15 (t, 1H), 3.70–3.85 (m, 4H), 3.85–3.95 (m,
2H), 4.2–4.3 (dd, 1H), 4.5–4.8 (m, 6H), 7.2–7.4
(m, 15H). 13C NMR (CDCl3): d 27.3, 38.9, 61.2,
63.7, 72.1, 72.3, 74.1, 77.4, 78.7, 78.8, 127.0–129.0,
138.0, 138.1, 178.4. HRESIMS: Calcd for C31H38LiO6,
m/z 513.2796; found, m/z 513.2828. Anal. Calcd
for C31H38O6: C, 73.49; H, 7.56. Found: C, 73.19; H,
7.70.
1
analytical purposes. H NMR (CDCl3): d 1.23 (s, 9H,
CH3), 3.58–3.72 (m, 2H), 3.82–3.92 (m, 3H), 3.94–3.98
(m, 2H), 4.16–4.24 (dd, 1H), 4.50–4.76 (m, 7H), 5.14–
5.25 (m, 2H), 5.82–5.94 (m, 1H), 7.2–7.4 (m, 15H). 13C
NMR (CDCl3): d 27.2, 38.8, 63.9, 69.8, 72.18, 72.20,
72.4, 73.8, 77.5, 78.2, 78.5, 116.8, 127–129, 134.8,
138.2, 138.3, 138.4, 178.3. HRESIMS: Calcd for
C34H42N12LiO6, m/z 553.3145; found, m/z 553.3141.
Anal. Calcd for C34H42O6: C, 74.70; H, 7.74. Found:
C, 74.84; H, 7.65.
4.5. 2,3,4-Tri-O-benzyl-5-O-(4,40-dimethoxytrityl)-1-O-
pivaloyl-D-ribitol (5)
Compound 4 (4.65 g, 9.18 mmol) was dissolved in dry
MeCN, followed by concentration. This procedure was
repeated two more times. To a solution of the residue
in dry MeCN (30 mL) were added at 23 ꢁC N,N-diiso-
propylethylamine (2.0 mL, 11.5 mmol) and 4,40-dimeth-
oxytrityl chloride (3.11 g, 9.18 mmol). After 90 min, the
solution was treated with 1 M aq NaHCO3 (15 mL), fol-
lowed by concentration. A solution of the residue in 1:1
Et2O–hexane was washed with 1 M aq NaHCO3. The
organic layer was dried (MgSO4), filtered and concen-
trated. The residue was purified by column chromato-
graphy using 100:0!85:15 hexane–EtOAc containing
0.5% Et3N as the eluant to afford 5 (6.30 g, 85%) as a
syrup. A small amount was purified by column chroma-
tography for analytical purposes. 1H NMR (CDCl3)
(selected data): d 1.18 (s, 9H), 3.3–3.4 (dd, 1H), 3.5–
3.55 (dd, 1H), 3.7–3.95 (m, 10H), 4.1–4.2 (dd, 1H),
4.45–4.8 (m, 6H), 7.05–7.5 (28H). 13C NMR (CDCl3):
4.3. 2,3,4-Tri-O-benzyl-1-O-pivaloyl-5-O-(propen-1-yl)-
D-ribitol (3)
A solution of compound 2 (5.46 g, 10.0 mmol) in THF
(10 mL) was alternatively degassed and placed under
helium. (1,5-Cyclooctadiene)-bis(methyldiphenylphos-
phine)iridium hexafluorophosphate25,26 (ꢁ5 mg) was
added, followed by degassing as above. A stream of
H2 was passed through the solution for 5 min. Next,
the reaction mixture was degassed, then a gentle stream
of helium was passed through it for 4 h. The reaction
mixture was concentrated, and compound 3 thus
obtained was used in the next step without purification.
A small amount was purified by column chromatogra-

A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
2043
d 27.2, 38.7, 55.1, 63.4, 64.2, 72.3, 72.6, 73.6, 77.8, 78.6,
78.8, 86.1, 110.0–160.0, 178.3. HRESIMS: Calcd for
C52H56LiO8, m/z 815.4135; found, m/z 815.4120. Anal.
Calcd for C52H56O8: C, 77.20; H, 6.98. Found: C,
77.33; H, 7.16.
4.8. [(N-Benzyloxycarbonyl)-2-aminoethyl] 2-cyanoethyl
[2,3,4-tri-O-benzyl-5-O-(4,40-dimethoxytrityl)-1-D-ribityl]
phosphate (9)
To a stirred solution of compound 7 (2.4 g, 2.59 mmol)
and benzyl N-(2-hydroxyethyl) carbamate (8) (1.52 g,
7.78 mmol) in dry MeCN (70 mL) was added a 0.45 M
solution of tetrazole in MeCN (57.6 mL, 25.9 mmol) at
23 ꢁC. After 1 h, dry pyridine (2 mL, 24.7 mmol) was
added, followed by a 0.5 M solution of iodine in 2:1
THF–water until the brown color persisted. The reac-
tion was quenched with satd aq Na2S2O3. The organic
solvent was removed under reduced pressure. A solution
of the residue in CH2Cl2 was washed with water, dried
(Na2SO4) and concentrated. The residue was purified
by column chromatography using 9:1 CH2Cl2–acetone
4.6. 2,3,4-Tri-O-benzyl-5-O-(4,40-dimethoxytrityl)-D-ribi-
tol (6)
Compound 5 (6.30 g, 7.79 mmol) was dissolved in dry
dioxane, and the resulting solution was concentrated
under reduced pressure. This procedure was repeated
two more times. To a solution of the residue in dry diox-
ane (10 mL), dry MeOH (15 mL) and NaOMe in MeOH
(3 mL, 30% w/w, 16 mmol) were added. After 72 h the
mixture was concentrated. A solution of the residue in
a 1:1 mixture of Et2O and hexane was washed with
water and 1 M aq NaHCO3. The organic layer was dried
(MgSO4), filtered and concentrated. The residue was
purified by column chromatography using 9:1!7:3 hex-
ane–EtOAc containing 0.5% Et3N as the eluant to give 6
(5.06 g, 90%) as an amorphous material. A small
amount was purified by column chromatography for
analytical purposes. 1H NMR (CDCl3): d 2.17 (br t,
1H), 3.38 (m, 2H), 3.70–3.78 (m, 9H), 3.8–4.0 (m, 2H),
4.44–4.74 (m, 6H), 6.70–7.50 (28H). 13C NMR (CDCl3):
d 55.2, 61.4, 63.3, 71.9, 72.7, 73.8, 78.6, 78.9, 79.4, 86.2,
110.0–160.0. Anal. Calcd for C47H48O7: C, 77.88; H,
6.67. Found: C, 77.92; H, 6.75.
1
as the eluant to afford 9 (2.06 g, 77%) as a syrup. H
NMR (CDCl3): d 7.09–7.5 (m, 29H), 6.7–6.78 (m, 4H),
5.18–5.26 (m, 1H), 5.05 (br s), 3.74 (br s, 6H), 3.26–
3.4 (m, 4H), 2.33–2.5 (m, 2H). HRESIMS: Calcd
for C60H63N2NaO12P, m/z 1057.4016; found, m/z
1057.3981.
4.9. [(N-Benzyloxycarbonyl)-2-aminoethyl] 2-cyanoethyl
(2,3,4-tri-O-benzyl-1-D-ribityl) phosphate (10)
To a stirred mixture of compound 9 (2 g, 1.94 mmol) in
CH2Cl2 (10 mL) and water (5 mL) was added glacial
HOAc (85 mL). The mixture was stirred at 23 ꢁC for
4 h followed by concentration under vacuum. Toluene
was added to and evaporated from the residue. A solu-
tion of the residue in CH2Cl2 was washed with a satd aq
NaHCO3. The organic phase was dried (Na2SO4) and
concentrated. Column chromatography of the residue
using 9:1 CH2Cl2–acetone as the eluant gave 10
4.7. 2-Cyanoethyl [2,3,4-tri-O-benzyl-5-O-(4,40-dimeth-
oxytrityl)-1-D-ribityl] N,N-diisopropylphosphoramidite
(7)
A solution of compound 6 (2.52 g, 3.48 mmol) in dry
pyridine was concentrated under reduced pressure. To
a solution of the residue in dry 1,2-dichloroethane
(15 mL) was added N,N-diisopropylethylamine (3.0
mL, 17.4 mmol). To the solution thus obtained chloro-
2-cyanoethyl-N,N-diisopropylphosphoramidite (1.9 mL,
8.52 mmol) was added dropwise at 0 ꢁC within 5 min
under stirring. Stirring at 0 ꢁC was continued for
25 min, then at 23 ꢁC for 90 min. The reaction mixture
was concentrated. A solution of the residue in 1:1
EtOAc–hexane was washed with 1 M aq NaHCO3.
The organic layer was dried (MgSO4), filtered and con-
centrated. The residue was purified by column chroma-
tography using 9:1!4:1 hexane–EtOAc containing 0.5%
Et3N as the eluant to give 7 (1.96 g, 61%) as a syrupy
material. 1H NMR (CDCl3): d 1.08–1.18 (m), 2.30–
2.50 (m), 3.76 (s). 13C NMR (CDCl3): d 20.10, 20.13,
20.16, 20.20, 24.5, 42.94, 43.01, 43.06, 43.14, 55.08,
55.09, 58.18, 58.25, 58.37, 58.44, 86.0, 117.58, 117.64.
31P NMR (CDCl3): d 148.8, 149.1. Anal. Calcd for
C56H65N2O8P: C, 72.71; H, 7.08. Found: C, 72.89; H,
7.00.
1
(1.25 g, 88%) as a syrup. H NMR (CDCl3): d 7.2–7.4
(m, 20H), 5.27–5.34 (m, 1H), 5.06 (br s, 2H), 3.65–3.78
(m, 2H) 3.28–3.4 (m, 2H), 2.42–2.57 (m, 2H). HR-
ESIMS: Calcd for C39H46N2O10P, m/z 733.2890; found,
m/z 733.2878.
4.10. Dimer (11)
To a solution of compound 7 (3.16 g, 3.4 mmol) and
compound 10 (1.25 g, 1.7 mmol) in dry MeCN
(130 mL) was added a 0.45 M solution of tetrazole in
MeCN (75.7 mL, 34 mmol) at 23 ꢁC. After stirring for
1 h, dry pyridine (4.3 mL, 53.2 mmol) was added, fol-
lowed by a 0.5 M solution of iodine in 2:1 THF–water
until the brown color persisted. The reaction mixture
was processed as described for the preparation of 9.
The residue was purified by column chromatography
using 95:5!9:1 CH2Cl2–acetone as the eluant to afford
1
11 (2.3 g, 86%) as a syrup. H NMR (CDCl3): d 7.02–
7.48 (m, 44H), 6.67–6.76 (m), 5.22–5.3 (m, 1H), 5.05
(br s, 2H), 3.75 (s, 6H), 3.22–3.4 (m, 4H), 2.35–2.56

2044
A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
1
(m, 2H), 2.1–2.35 (m, 2H). HRESIMS: Calcd
for C89H95N3NaO19P2, m/z 1594.593; found, m/z
1594.5876.
afford 15 (2.12 g, 87%) as a colorless syrup. H NMR
(CDCl3): d 6.92–7.59 (m, 74H), 6.67–6.77 (m, 4H),
5.24–5.33 (m, 1H), 5.06 (br s, 2H), 3.73 (s, 6H), 3.24–
3.43 (m, 4H), 2.37–2.54 (m, 2H), 2.1–2.36 (m, 6H).
HRESIMS: Calcd for
C147H159N5NaO33P4, m/z
4.11. De(dimethoxytrityl)ation of dimer (12)
2668.9766; found, m/z 2668.9802.
Compound 11 (2.3 g, 1.46 mmol) was processed as
described for 10. Column chromatography (9:1!4:1
CH2Cl2–acetone) of the residue gave 12 (1.61 g, 87%)
4.15. De(dimethoxytrityl)ation of tetramer (16)
1
as a syrup. H NMR (CDCl3): d 7.09–7.48 (m, 35H),
Compound 15 (2.12 g, 0.80 mmol) was processed as
described for 10. Column chromatography (9:1!7:3
CH2Cl2–acetone) of the residue gave 16 (1.58 g, 84%)
5.23–5.31 (m, 1H), 5.06 (br s, 2H), 3.63–3.75 (m, 2H),
3.24–3.44 (m, 2H), 2.39–2.55 (m, 2H), 2.23–2.38 (m,
2H). HRESIMS: Calcd for C68H77N3NaO17P2, m/z
1292.4626; found, m/z 1292.4689.
1
as a syrup. H NMR (CDCl3): d 6.96–7.58 (m, 65H),
5.22–5.33 (m, 1H), 5.06 (d, 2H), 3.62–3.76 (m, 2H)
3.27–3.41 (m, 2H), 2.37–2.52 (m, 2H), 2.15–2.36 (m,
6H). HRESIMS: Calcd for C126H141N5NaO31P4, m/z
2366.8459; found, m/z 2366.8696.
4.12. Trimer (13)
To a solution of compound 7 (2.3 g, 2.5 mmol) and
compound 12 (1.61 g, 1.27 mmol) in dry MeCN
(80 mL) was added a 0.45 M solution of tetrazole in
MeCN (55.3 mL, 25 mmol) at 23 ꢁC. After stirring for
1 h, dry pyridine (3.2 mL, 40 mmol) was added, fol-
lowed by a 0.5 M solution of iodine in 2:1 THF–water
until the brown color persisted. The reaction mixture
was processed as described for 9. The residue was puri-
fied by column chromatography (9:1!4:1 CH2Cl2–ace-
4.16. Pentamer (17)
To a solution of compound 7 (1.21 g, 1.32 mmol) and
compound 16 (1.58 g, 0.67 mmol) in dry MeCN
(70 mL) was added a 0.45 M solution of tetrazole in
MeCN (29.2 mL, 13.2 mmol) at 23 ꢁC. After stirring
for 1 h, dry pyridine (1.67 mL, 20.8 mmol) was added,
followed by a 0.5 M solution of iodine in 2:1 THF–
water until the brown color persisted. The reaction
mixture was processed as described for 9. The residue
was purified by column chromatography (9:1!7:3
CH2Cl2–acetone) to afford 17 (1.9 g, 89%) as a syrup.
1H NMR (CDCl3): d 6.94–7.57 (m, 89H), 6.66–6.77
(m, 4H), 5.21–5.32 (m, 1H), 5.06 (d, 2H), 3.75 (s, 6H),
3.26–3.41 (m, 4H), 2.37–2.53 (m, 2H), 2.11–2.36 (m,
1
tone) to afford 13 (2.25 g, 84%) as a syrup. H NMR
(CDCl3): d 6.96–7.58 (m, 59H), 6.68–6.86 (m, 4H),
5.23–5.33 (m, 1H), 5.06 (d, 2H), 3.73, 3.78 (2s), 3.23–
3.42 (m, 4H), 2.37–2.53 (m, 2H), 2.11–2.35 (m). HR-
ESIMS: Calcd for C118H127N4NaO26P3, m/z 2131.7850;
found, m/z 2131.7874.
8H). HRESIMS: Calcd for (C176H191N6Na2O40P5)2+
,
4.13. De(dimethoxytrityl)ation of trimer (14)
m/z 1614.579; found, m/z 1614.5890.
Compound 13 (2.25 g, 1.06 mmol) was processed as
described for 10. Column chromatography (9:1!4:1
CH2Cl2–acetone) of the residue gave 14 (1.67 g, 87%)
4.17. De(dimethoxytrityl)ation of pentamer (18)
1
as a syrup. H NMR (CDCl3): d 6.96–7.6 (m, 50H),
Compound 17 (1.9 g, 0.59 mmol) was processed as
described for 10. Column chromatography (9:1!7:3
CH2Cl2–acetone) of the residue gave 18 (1.45 g, 85%)
5.23–5.33 (m, 1H), 5.06 (br s, 2H), 3.63–3.76 (m, 2H)
3.24–3.41 (m, 2H), 2.37–2.58 (m, 2H), 2.23–2.38 (m,
4H); HRESIMS: Calcd for C97H109N4NaO24P3, m/z
1829.6542; found, m/z 1829.6611.
1
as a syrup. H NMR (CDCl3): d 6.93–7.56 (m, 80H),
5.22–5.32 (m, 1H), 5.06 (d, 2H), 3.61–3.75 (m, 2H)
3.26–3.41 (m, 2H), 2.38–2.53 (m, 2H), 2.14–2.36 (m,
8H). HRESIMS: Calcd for (C155H173N6O38P5H2)2+
,
4.14. Tetramer (15)
m/z 1441.532; found, m/z 1441.5338.
To a solution of compound 7 (1.67 g, 1.82 mmol) and
compound 14 (1.67 g, 0.92 mmol) in dry MeCN (80
mL) was added a 0.45 M solution of tetrazole in MeCN
(40.3 mL, 18.2 mmol) at 23 ꢁC. After stirring for 1 h, dry
pyridine (2.3 mL, 28.7 mmol) was added, followed by a
0.5 M solution of iodine in 2:1 THF–water until the
brown color persisted. The reaction mixture was pro-
cessed as described for 9. The residue was purified by
column chromatography (9:1!4:1 CH2Cl2–acetone) to
4.18. Hexamer (19)
To a solution of compound 7 (0.9 g, 0.98 mmol) and
compound 18 (1.45 g, 0.50 mmol) in dry MeCN
(60 mL) was added a 0.45 M solution of tetrazole in
MeCN (21.7 mL, 9.8 mmol) at 23 ꢁC. After stirring for
1 h, dry pyridine (1.24 mL, 15.4 mmol) was added, fol-
lowed by a 0.5 M solution of iodine in 2:1 THF–water

A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
2045
until the brown color persisted. The reaction mixture
was processed as described for 9. The residue was puri-
fied by column chromatography (9:1!7:3 CH2Cl2–
acetone) to afford 19 (1.59 g, 85%) as a syrup. 1H
NMR (CDCl3): d 6.94–7.58 (m, 104H), 6.67–6.77 (m,
4H), 5.24–5.33 (m, 1H), 5.06 (br s, 2H), 3.73 (s, 6H),
3.25–3.42 (m, 4H), 2.38–2.53 (m, 2H), 2.12–2.37 (m,
10H). HRESIMS: Calcd for C205H223Li2N7O47, m/z
1867.201; found, m/z 1867.205.
1 h, dry pyridine (0.78 mL, 9.7 mmol) was added, fol-
lowed by a 0.5 M solution of iodine in 2:1 THF–water
until the brown color persisted. The reaction mixture
was processed as described for 9. The residue was puri-
fied by column chromatography (9:1!7:3 CH2Cl2–ace-
1
tone) to afford 23 (1.36 g, 90%) as a syrup. H NMR
(CDCl3): d 6.94–7.56 (m, 134H), 6.67–6.76 (m, 4H),
5.23–5.32 (m, 1H), 5.06 (d, 2H), 3.73 (s, 6H), 3.24–
3.41 (m, 4H), 2.37–2.53 (m, 2H), 2.11–2.36 (m, 14H).
HRESIMS: Calcd for (13C2C261H287N9O61P8)2+, m/z
2405.3957; found, m/z 2405.4067. Anal. Calcd for
4.19. De(dimethoxytrityl)ation of hexamer (20)
C
263H287N9O61P8: C, 65.84; H, 6.03. Found: C, 65.63;
Compound 19 (1.59 g, 0.43 mmol) was processed as
described for 10. Column chromatography (9:1!7:3
CH2Cl2–acetone) of the residue gave 20 (1.34 g, 91%)
H, 6.01.
4.23. De(dimethoxytrityl)ation of octamer (24)
1
as a syrup. H NMR (CDCl3): d 6.93–7.59 (m, 95H),
5.23–5.32 (m, 1H), 5.07 (br s, 2H), 3.62–3.76 (m, 2H)
3.26–3.43 (m, 2H), 2.38–2.53 (m, 2H), 2.15–2.37 (m,
Compound 23 (1.36 g, 0.28 mmol) was processed as
described for 10. Column chromatography (9:1!7:3
CH2Cl2–acetone) of the residue gave 24 (1.1 g, 87%) as
a syrup. 1H NMR (CDCl3): d 6.94–7.57 (m, 125H),
5.24–5.33 (m, 1H), 5.07 (d, 2H), 3.63–3.74 (m, 2H)
3.27–3.40 (m, 2H), 2.38–2.53 (m, 2H), 14H). HRESIMS:
Calcd for (13C2C240H269Li2N9O59P8)2+, m/z 2254.331;
10H). HRESIMS: Calcd for (C184H205N7O45P6H2)2+
,
m/z 1710.128; found, m/z 1710.141.
4.20. Heptamer (21)
To a solution of compound 7 (0.7 g, 0.76 mmol) and
compound 20 (1.34 g, 0.39 mmol) in dry MeCN
(50 mL) was added a 0.45 M solution of tetrazole in
MeCN (16.8 mL, 7.6 mmol) at 23 ꢁC. After stirring for
1 h, dry pyridine (0.96 mL, 11.9 mmol) was added, fol-
lowed by a 0.5 M solution of iodine in 2:1 THF–water
until the brown color persisted. The reaction mixture
was processed as described for 9. The residue was puri-
fied by column chromatography (9:1!7:3 CH2Cl2–ace-
found, m/z 2254.3398. Anal. Calcd for C242H269-
N9O59P8: C, 64.65; H, 6.03. Found: C, 64.51; H, 6.10.
4.24. Nonamer (25)
To a solution of compound 7 (0.288 g, 0.31 mmol) and
compound 24 (0.94 g, 0.21 mmol) in dry MeCN (25
mL) was added a 0.45 M solution of tetrazole in MeCN
(7 mL, 3.14 mmol) at 23 ꢁC. After stirring for 1 h, dry
pyridine (0.4 mL, 4.94 mmol) was added, followed by a
0.5 M solution of iodine in 2:1 THF–water until the
brown color persisted. The reaction mixture was pro-
cessed as described for 9. The residue was purified by col-
umn chromatography (9:1!7:3 CH2Cl2–acetone) to
1
tone) to afford 21 (1.48 g, 89%) as a syrup. H NMR
(CDCl3): d 6.93–7.57 (m, 119H), 6.67–6.77 (m, 4H),
5.23–5.32 (m, 1H), 5.07 (br s, 2H), 3.74 (s, 6H), 3.26–
3.41 (m, 4H), 2.38–2.52 (m, 2H), 2.12–2.37 (m, 12H).
HRESIMS: Calcd for (C234H255N8O54P7H2)2+, m/z
2152.272; found, m/z 2152.260.
1
afford 25 (0.95 g, 85%) as a syrup. H NMR (CDCl3): d
6.93–7.56 (m, 149H), 6.68–6.76 (m, 4H), 5.22–5.29
(m, 1H), 5.06 (d, 2H), 3.74 (s, 6H), 3.23–3.42 (m, 4H),
2.38–2.52 (m, 2H), 2.10–2.35 (m, 16H). HRESIMS: Calcd
for (C292H319N10O68PLi2)2+, m/z 2674.4929; found, m/z
2674.5158. Anal. Calcd for C292H319N10O68P9: C, 65.73;
H, 6.03. Found: C, 65.46; H, 6.02.
4.21. De(dimethoxytrityl)ation of heptamer (22)
Compound 21 (1.48 g, 0.35 mmol) was processed as
described for 10. Column chromatography (9:1!7:3
CH2Cl2–acetone) of the residue gave 22 (1.25 g, 90%)
1
as a syrup. H NMR (CDCl3): d 6.92–7.58 (m, 110H),
5.24–5.31 (m, 1H), 5.06 (d, 2H), 3.63–3.76 (m, 2H)
3.27–3.41 (m, 2H), 2.37–2.52 (m, 2H), 2.15–2.36 (m,
4.25. De(dimethoxytrityl)ation of nonamer (26)
12H). HRESIMS: Calcd for (C213H237N8O52P7H2)2+
,
Compound 25 (0.95 g, 0.18 mmol) was processed as
described for 10. Column chromatography of the residue
using 9:1!7:3 CH2Cl2–acetone as the eluant gave
26 (0.75 g, 83.7%) as a syrup. 1H NMR (CDCl3): d
6.95–7.56 (m, 140H), 5.22–5.28 (m, 1H), 5.06 (d, 2H),
3.65–3.72 (m, 2H) 3.29–3.36 (m, 2H), 2.38–2.52
(m, 2H), 2.14–2.36 (m, 16H). HRESIMS: Calcd for
(C271H301Li3N10O66P9)3+, m/z 1684.6237; found, m/z
1684.6179.
m/z 1979.225; found, m/z 1979.236.
4.22. Octamer (23)
To a solution of compound 7 (0.57 g, 0.62 mmol) and
compound 22 (1.25 g, 0.31 mmol) in dry MeCN
(50 mL) was added a 0.45 M solution of tetrazole in
MeCN (13.7 mL, 6.2 mmol) at 23 ꢁC. After stirring for

2046
A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
4.26. Decamer (27)
20H). Anal. Calcd for C329H365N12O80P11: C, 64.69;
H, 6.02. Found: C, 64.54; H, 6.17.
To a solution of compound 7 (0.205 g, 0.224 mmol) and
compound 26 (0.75 g, 0.15 mmol) in dry MeCN (18 mL)
was added a 0.45 M solution of tetrazole in MeCN
(5 mL, 2.24 mmol) at 23 ꢁC. After stirring for 1 h, dry
pyridine (0.286 mL, 3.54 mmol) was added, followed
by a 0.5 M solution of iodine in 2:1 THF–water until
the brown color persisted. The reaction mixture was
processed as described for 9. The residue was purified
by column chromatography (9:1!7:3 CH2Cl2–acetone)
to afford 27 (0.77 g, 88%) as a syrup. 1H NMR (CDCl3):
d 6.93–7.45 (m, 164H), 6.65–6.77 (m, 4H), 5.20–5.29 (m,
1H), 5.06 (d, 2H), 3.72 (s, 6H), 3.26–3.40 (m, 4H), 2.38–
2.52 (m, 2H), 2.10–2.35 (m, 18H). Anal. Calcd for
4.30. Dodecamer (31)
To a solution of compound 7 (0.112 g, 0.121 mmol) and
compound 30 (0.495 g, 0.081 mmol) in dry MeCN
(10 mL) was added a 0.45 M solution of tetrazole in
MeCN (2.72 mL, 1.21 mmol) at 23 ꢁC. After stirring
for 1 h, dry pyridine (0.156 mL, 1.93 mmol) was added,
followed by a 0.5 M solution of iodine in 2:1 THF–
water until the brown color persisted. The reaction
mixture was processed as described for 9. The residue
was purified by column chromatography (9:1!7:3
CH2Cl2–acetone) to afford 31 (0.466 g, 83%) as a syrup.
1H NMR (CDCl3): d 6.93–7.54 (m, 194H), 6.69–6.77 (m,
4H), 5.22–5.28 (m, 1H), 5.06 (d, 2H), 3.74 (s, 6H), 3.23–
3.42 (m, 4H), 2.39–2.52 (m, 2H), 2.10–2.38 (m, 22H).
Anal. Calcd for C379H415N13O89P12: C, 65.52; H, 6.02.
Found: C, 65.61; H, 6.24.
C
321H351N11O75P10: C, 65.65; H, 6.02. Found: C,
65.63; H, 6.26.
4.27. De(dimethoxytrityl)ation of decamer (28)
Compound 27 (0.77 g, 0.13 mmol) was processed as
described for 10. Column chromatography (9:1!7:3
CH2Cl2–acetone) of the residue gave 28 (0.63 g, 86%)
4.31. De(dimethoxytrityl)ation of dodecamer (32)
1
Compound 31 (0.466 g, 0.067 mmol) was dissolved in
CH2Cl2 (10 mL) and water (5 mL). Glacial HOAc
(50 mL) was added, and the mixture was stirred at room
temperature overnight. The reaction mixture was pro-
cessed as described for 10. Column chromatography
(9:1!7:3 CH2Cl2–acetone) of the residue gave 32
(0.373 g, 83.8%) as a syrup. 1H NMR (CDCl3): d
6.93–7.55 (m, 185H), 5.22–5.28 (m, 1H), 5.06 (d, 2H),
3.64–3.73 (m) 3.28–3.39 (m, 2H), 2.39–2.52 (m, 2H),
2.12–2.38 (m, 22H). Anal. Calcd for C358H397N13O87P12:
C, 64.70; H, 6.02. Found: C, 64.74; H, 6.16.
as a syrup. H NMR (CDCl3): d6.95–7.56 (m, 155H),
5.22–5.27 (m, 1H), 5.06 (d, 2H), 3.65–3.72 (m, 2H)
3.29–3.38 (m, 2H), 2.39–2.52 (m, 2H), 2.12–2.38 (m,
18H). Anal. Calcd for C300H333N11O73P10: C, 64.68;
H, 6.03. Found: C, 64.53; H, 6.26.
4.28. Undecamer (29)
To a solution of compound 7 (0.156 g, 0.17 mmol) and
compound 28 (0.63 g, 0.11 mmol) in dry MeCN
(13.5 mL) was added a 0.45 M solution of tetrazole in
MeCN (3.8 mL, 1.7 mmol) at 23 ꢁC. After stirring for
1 h, dry pyridine (0.217 mL, 2.68 mmol) was added, fol-
lowed by a 0.5 M solution of iodine in 2:1 THF–water
until the brown color persisted. The reaction mixture
was processed as described for 9. The residue was puri-
fied by column chromatography (9:1!7:3 CH2Cl2–ace-
tone) to afford 29 (0.605 g, 83.5%) as a syrup. 1H
NMR (CDCl3): d 6.94–7.56 (m, 179H), 6.69–6.76 (m,
4H), 5.21–5.29 (m, 1H), 5.07 (d, 2H), 3.72 (s, 6H),
3.24–3.40 (m, 4H), 2.38–2.51 (m, 2H), 2.10–2.36 (m,
20H). Anal. Calcd for C350H383N12O82P11: C, 65.58;
H, 6.02. Found: C, 65.51; H, 6.14.
4.32. 2-(Aminoethyl) (1-D-ribityl) phosphate (33)
To a solution of compound 10 (210 mg, 0.286 mmol) in
MeOH (4 mL) was added concd NH4OH (2 mL). The
mixture was stirred under reflux for 8 h. After concen-
tration, the residue was purified on Sephadex LH-20
using 1:1 MeOH–CH2Cl2 as the eluant. A solution of
the resulting syrup in MeOH was treated with Dowex
50 · 8–100 (Na+), followed by filtration and removal
of the volatiles from the filtrate under reduced pressure.
A solution of residue in 1:1 t-BuOH–H2O (10 mL) was
stirred under hydrogen at 200 psi for 2 days in the pres-
ence of 10% Pd/C (ꢁ200 mg). The catalyst was removed
by filtration through layer of Celite, and the filtrate was
concentrated. A solution of the residue was freeze dried
to afford amorphous 33 (68 mg, 88.0%). 1H NMR
(D2O): d 4.10–4.04 (m, 3H), 3.98 (dd, 1H, J 5.0,
11.5 Hz), 3.93 (m, 1H), 3.84 (m, 1H), 3.80 (dd, 1H, J
3.2, 11.4 Hz), 3.74 (t, 1H, J 5 Hz), 3.65 (dd, 1H, J 7.0,
11.7 Hz), 3.19 (t, 2H, J 4.7 Hz); 13C NMR d 72.7,
72.3, 71.5 (d, J 8.0 Hz), 67.3 (d, J 5.6 Hz), 63.8 (d, J
4.29. De(dimethoxytrityl)ation of undecamer (30)
Compound 29 (0.605 g, 0.094 mmol) was processed as
described for 10. Column chromatography (9:1!7:3
CH2Cl2–acetone) of the residue gave 30 (0.495 g, 86%)
1
as a syrup. H NMR (CDCl3): d 6.93–7.55 (m, 170H),
5.21–5.29 (m, 1H), 5.06 (d, 2H), 3.63–3.72 (m, 2H)
3.29–3.39 (m, 2H), 2.38–2.52 (m, 2H), 2.13–2.36 (m,

A. Fekete et al. / Carbohydrate Research 341 (2006) 2037–2048
2047
5.2 Hz), 63.0, 40.9 (d, J 8.9 Hz); 31P NMR d 1.77. HR-
ESIMS: Calcd for C7H18LiNNaO8P, m/z 305.0828;
found, m/z 305.0824.
1.84 (m, 2H); 31P NMR d 2.39 (7P), 1.97 (1P). Ribitol
phosphates 33 and 35 were converted to their respective
5-ketohexanoyl derivatives (36 and 38) in a similar man-
ner and were obtained in similar yields.
4.33. Deprotection of octamer 24 (34)
4.36. Conjugation of ketohexanoyl derivatives 36, 37 and
38 to aminooxylated bovine serum albumin
To a solution of compound 24 (380 mg, 0.084 mmol) in
MeOH (4 mL) was added concd NH4OH (2 mL). The
mixture was stirred under reflux for 8 h, then was pro-
cessed as described for 33 to afford amorphous 34
(110 mg, 66.2%). 1H NMR (D2O): d 4.16–4.11 (m,
1H), 4.11–4.04 (m), 4.04–3.92 (m) 3.86 (ddd, 1H),
3.84–3.78 (m), 3.76 (t, 1H, J 6.0 Hz), 3.66 (dd, 1H, J
7.1, 12.0 Hz), 3.29 (t, 2H, J 4.8 Hz); 13C NMR d 72.8,
72.5, 71.95, 71.90, 71.7, 71.6, 67.4 (d, J 5.4 Hz), 67.3
(d, J 5.0 Hz), 67.3, 67.2, 63.1, 62.6 (d, J 4.7 Hz), 40.8
(d, J 8.1 Hz); 31P NMR d 2.35 (7P), 1.62 (1P). Anal.
Calcd for C42H87NNa8O57P8: C, 25.87; H, 4.50. Found:
C, 25.96; H, 4.60.
Bovine serum albumin was derivatized with O-(3-thiol-
propyl)hydroxylamine as described.15 Average incorpo-
ration of the linker was 30 mol/1 mol of protein, as
assayed by MALDI-TOFMS using sinapinic acid as
the matrix. To a solution of aminooxy-BSA (10 mg,
0.135 lmol containing 4 lmol of aminooxy-linker) in
0.1 M phosphate buffer containing 1 mM EDTA and
0.1% glycerol at pH 7.4 was added either the monomer
36 (3.0 mg, 7.3 lmol) or the octamer 37 (15 mg,
5.8 lmol), respectively. In another experiment, amino-
oxy-BSA (3 mg, 0.04 lmol containing 1.2 lmol amino-
oxy-linker) was reacted with dodecamer 38 (6 mg,
1.9 lmol). All reactions were carried out at pH 6.5, at
23 ꢁC, for 18 h, in 2.5 mL of total volume. After 18 h
the conjugation mixture was applied to a Sephadex
G50 column made up in 0.2 M NaCl. The fractions
containing the conjugates were pooled and analyzed
by MALDI-TOFMS (Table 1).
4.34. Deprotection of dodecamer 32 (35)
Compound 32 (210 mg, 0.031 mmol) was dissolved in
MeOH (4 mL) and concd NH4OH (2 mL). The mixture
was stirred at reflux for 8 h, then was processed as de-
1
scribed for 33 gave 35 (72 mg, 80%). H NMR (D2O):
d 4.15–4.40 (m), 4.04–3.92 (m) 3.86 (ddd, 1H), 3.84–
3.77 (m), 3.75 (t, 1H, J 6.0 Hz), 3.65 (dd, 1H, J 7.3,
12.2 Hz), 3.28 (t, 2H); 13C NMR d 72.8, 72.5, 71.9
(11C), 71.7 (11C), 71.6 (11C), 67.4 (d, J 4.5 Hz), 67.3
(11C), 67.2 (11C), 63.1, 62.9 (d, J 4.6 Hz), 40.9 (d,
J 7.7 Hz); 31P NMR 2.39, 2.37, 2.35, 2.33, 1.64.
MALDI-TOFMS: Calcd for C62H127NNa13O85P12,
m/z 2917.19; found, m/z 2917.0. Anal. Calcd for
C62H127NNa12O85P12: C, 25.73; H, 4.42. Found: C,
25.8; H, 4.38.
Acknowledgements
We thank Dr. Martin Hamzink, for his help with some
of the NMR experiments. This work was supported by
the Intramural Research Program of the National Insti-
tute of Child Health and Human Development.
Supplementary data
4.35. Preparation of the N-(5-ketohexanoyl) derivatives of
ribitol phosphates 33, 34 and 35
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2005.10.023.
To a solution of ribitol phosphate 34 (19 mg) in H2O
(0.3 mL) was added MeOH (1.5 mL) followed by Et3N
(0.1 mL). This solution was treated with 5-ketohexanoic
anhydride (ꢁ35 mg) at 23 ꢁC. After 20 min, the volatiles
were removed in a stream of air. The residue was puri-
fied on a Bio-Gel P2 column (80 · 2 cm) using 1:1
0.02 M C5H5N–0.02 M HOAc as the eluant. Fractions
containing the product were pooled and freeze dried.
To a solution of the residue in water was added Dowex
50 · 8–100 (Na+) in excess, and the mixture was gently
mixed for 30 min, followed by filtration and freeze dry-
ing of the filtrate to afford ketone 37 as a glass (17 mg,
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