Synthesis of Oligomers Derived from Amide-Linked Neuraminic Acid Analogues

Article abstract of DOI:10.1021/jo035312+

N-Fluoren-9-ylmethoxycarbonyl-protected sugar amino acids derived from α-O-methoxy- and 2,3-dehydroneuraminic acids have been prepared. Incorporation of these monomer units into solid-phase synthesis led to the efficient synthesis of two series of oligomers varying from one to eight units in length. The (1-5)-linked amides of 2,3-dehydroneuraminic acid were further subjected to hydrogenation giving a third series of oligomers with a β-hydrido substituent at the anomeric carbon.

Full text of DOI:10.1021/jo035312+

VOLUME 69, NUMBER 4
FEBRUARY 20, 2004
© Copyright 2004 by the American Chemical Society
Syn th esis of Oligom er s Der ived fr om Am id e-Lin k ed Neu r a m in ic
Acid An a logu es#
Travis Q. Gregar†,‡ and J acquelyn Gervay-Hague*,§
Department of Chemistry, The University of Arizona, Tucson, Arizona 85721, and Department of
Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616
gervay@chem.ucdavis.edu
Received September 5, 2003
N-Fluoren-9-ylmethoxycarbonyl-protected sugar amino acids derived from R-O-methoxy- and 2,3-
dehydroneuraminic acids have been prepared. Incorporation of these monomer units into solid-
phase synthesis led to the efficient synthesis of two series of oligomers varying from one to eight
units in length. The (1f5)-linked amides of 2,3-dehydroneuraminic acid were further subjected to
hydrogenation giving a third series of oligomers with a â-hydrido substituent at the anomeric carbon.
In tr od u ction
In 1998, we first reported that amide-linked homooli-
gomers, derived from neuraminic acid (NeuAc) analogues,
adopt stable secondary structures in solution depending
upon the oligomer length.1 The oligomer series (Figure
1) was composed of (1f5) amide-linked â-O-methoxy
neuraminic acids ranging from one to eight residues in
length. These materials were efficiently prepared using
solid-phase peptide synthesis employing N-fluoren-9-
ylmethoxycarbonyl (Fmoc) protecting groups. A C-termi-
nus ꢀ-amino caproic acid linker was also introduced in
order to prevent fraying of the terminal residue, which
might disturb important hydrogen-bonding interactions
required for stable secondary structure.2
We chose neuraminic acid because it is a readily
available naturally occurring δ amino acid with a trihy-
droxy C-6 side chain, which we anticipated would in-
crease water solubility in higher order oligomers. More-
over, O-glycoside oligomers of NeuAc have stable sec-
ondary structure,3 and it seemed plausible that amide-
linked analogues might exhibit similar properties. In-
deed, these hypotheses proved correct, and our report was
F IGURE 1. Amide-linked â-O-methoxy neuraminic acid oli-
gomer series A.
the first to demonstrate that neutral amide-linked car-
bohydrate-based materials possess stable secondary struc-
tures in water. Shortly thereafter, Fleet and co-workers
reported that furanose-derived amide-linked oligomers
also adopt stable secondary structures in solution,4 and
more recent investigations have demonstrated the gen-
eral utility of sugar amino acids5 in foldamer research.6
In continuing investigations, we have targeted the
synthesis of three other homooligomer series in order to
probe the consequences of introducing variable function-
ality at C-2. In the first series (B) (Scheme 2), conditions
for the synthesis of (1f5) amide-linked R-O-methoxy
neuraminic acids are established in order to compare
reversal in anomeric stereochemistry. The second series
(C) (Scheme 3) introduces a 2,3 dehydro analogue of
NeuAc, which changes the hybridization of the anomeric
carbon. And finally, the third series (D) (Scheme 4)
incorporates a â-hydrogen atom at the anomeric center
#
Taken in part from the dissertation of Travis Q. Gregar, The
University of Arizona, 2001.
* Author to whom correspondence should be addressed. Phone: 530-
754-9577.
† The University of Arizona.
‡ Current address: 3M, 3M Center, Building 201-BS-05, St. Paul,
MN 55144.
§ University of California, Davis.
(3) (a) Brisson, J .-R.; Baumann, H.; Imberty, A.; Pe´rez, S.; J ennings,
H. J . Biochemistry 1992, 31, 4996. (b) Yamasaki, R.; Bacon, B.
Biochemistry 1991, 30, 851.
(4) (a) Smith, M. D.; Claridge, T. D. W.; Tranter, G. E.; Sansom, M.
S. P.; Fleet, G. W. J . J . Chem. Commun. 1998, 2041. (b) Long, D. D.;
Smith, M. D.; Marquess, D. G.; Claridge, T. D. W.; Fleet, G. W. J .
Tetrahedron Lett. 1998, 39, 9293.
(1) Szabo, L.; Smith, B. L.; McReynolds, K. D.; Parrill, A. L.; Morris,
E. R.; Gervay, J . J . Org. Chem. 1998, 63, 1074.
(2) Fraying refers to disruption of secondary structure due to
solvation effects. A hydrophobic linker such as caproamide diminishes
solvation relative to a simple primary amide. Solvation can disrupt
helicity in small oligomers.
10.1021/jo035312+ CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/08/2003
J . Org. Chem. 2004, 69, 1001-1009
1001

Gregar and Gervay-Hague
SCHEME 1
in place of the original methoxy group. Studies leading
to the production of these systems are reported herein.
quantitatively converted to a chloride (3) by the action
of HCl.9 The next critical step in the synthesis employed
a procedure first reported by Magnusson10 in which
methanol displaces the axial chloride with inversion of
stereochemistry providing the R-OCH3 glycoside 4.
Deacetylation of 4 was performed in a two-step process,
which began with Zemple´n11 hydrolysis of the esters to
give 5, followed by the more extreme conditions required
to remove the N-acetyl group.12 The crude sugar amino
acid (6) was neutralized with HCl, filtered, and lyophi-
lized to give a salt. Two different methods were used to
protect the amine. Initially, we employed the procedure
we reported in our first paper,1 which incorporated a 1:1
mixture of 10% NaHCO3 in water with dioxane, but we
encountered reproducibility problems. Further investiga-
tions revealed that using 4 equiv of Hu¨nigs base (N,N-
diisopropylethylamine) in place of aqueous sodium bi-
carbonate greatly increased the yield of 7, possibly due
to the increased solubility of Hu¨nigs base in dioxane
relative to sodium bicarbonate. Subsequent reactions on
the solid-phase support progressed more cleanly and
efficiently using this method of Fmoc protection.
Resu lts a n d Discu ssion
Synthesis of the monomer required for series B began
with acetylation of N-acetyl neuraminic acid (1)7 followed
by methyl ester formation using diazomethane to give 2
(Scheme 1).8 Subsequently, the anomeric acetate was
(5) (a) Van Well, R. M.; Marinelli, L.; Altona, C.; Erkelens, K.;
Siegal, G.; Van Raaij, M.; Llamas-Saiz, A. L.; Kessler, H.; Novellino,
E.; Lavecchia, A.; Van Boom, J . H.; Overhand, M. J . Am. Chem. Soc.
2003, 125, 10822. (b) Chakraborty, T. K.; Srinivasu, P.; Bikshapathy,
E.; Nagaraj, R.; Vairamani, M.; Kumar, S. K.; Kunwar, A. C. J . Org.
Chem. 2003, 68, 6257. (c) van Well, R. M.; Meijer, M. E. A.; Overkleeft,
H. S.; van Boom, J . H.; van der Marel, G. A.; Overhand, M. Tetrahedron
2003, 59, 2423-2434. (d) Xie, J . Carbohydr. Res. 2003, 338, 399. (e)
Gervay-Hague, J .; Weathers, T. M., J r. J . Carbohydr. Chem. 2002, 21,
867 and references therein. (f) Gruner, S. A. W.; Truffault, V.; Voll,
G.; Locardi, E.; Stockle, M.; Kessler, H. Chem.sEur. J . 2002, 8, 4365.
(g) Chakraborty, T. K.; J ayaprakash, S.; Ghosh, S. Comb. Chem. High
Throughput Screening 2002, 5, 373. (h) Stoeckle, M.; Voll, G.; Guen-
ther, R.; Lohof, E.; Locardi, E.; Gruner, S.; Kessler, H. Org. Lett. 2002,
4, 2501. (i) Chakraborty, T. K.; Ghosh, S.; J ayaprakash, S. Curr. Med.
Chem. 2002, 9, 421. Chakraborty, T. K.; J ayaprakash, S.; Srinivasu,
P.; Madhavendra, S. S.; Ravi Sankar, A.; Kunwar, A. C. Tetrahedron
2002, 58, 2853. (j) Gruner, S. A. W.; Locardi, E.; Lohof, E.; Kessler, H.
Chem. Rev. 2002, 102, 491 and references therein. (k) Suhara, Y.;
Masayuki, I.; Ichikawa, M.; Penno, M.; Ichikawa, Y. Tetrahedron Lett.
1997, 41, 7167. (l) Suhara, Y.; Hildreth, J .; Ichikawa, Y. Chem.
Commun. 1998, 617.
(6) (a) Baldauf, C.; Guenther, R.; Hofmann, H.-J . Helv. Chim. Acta
2003, 86, 2573. (b) Raguse, T. L.; Lai, J . R.; Gellman, S. H. J . Am.
Chem. Soc. 2003, 125, 5592. (c) Garric, J .; Leger, J .-M.; Grelard, A.;
Ohkita, M.; Huc, I. Tetrahedron Lett. 2003, 44, 1421. (d) Tomasini,
C.; Trigari, V.; Lucarini, S.; Bernardi, F.; Garavelli, M.; Peggion, C.;
Formaggio, F.; Toniolo, C. Eur. J . Org. Chem. 2003, 2, 259. (e) de J ong,
R.; Rijkers, D. T. S.; Liskamp, R. M. J . Helv. Chim. Acta 2002, 85,
4230. (f) Cubberley, M. S.; Iverson, B. L. Curr. Opin. Chem. Biol. 2001,
5, 650. (g) Hill, D. J .; Mio, M. J .; Prince, R. B.; Hughes, T. S.; Moore,
J . S. Chem. Rev. 2001, 101, 3893 and references therein.
Coupling of 7 to the resin continued using traditional
solid-phase peptide techniques (Scheme 2). Rink resin13
was employed, because mild acidic conditions sufficiently
cleave the oligomers from the resin without sugar de-
composition. Series B oligomer synthesis began with
condensation of Fmoc-protected ꢀ-amino caproic acid to
the resin, which was accomplished using 5 equiv of the
(9) Meindl, P.; Tuppy, H. Monatsh. Chem. 1965, 96, 802.
(10) Kononov, L. O.; Magnusson, G. Acta Chem. Scand. 1998, 52,
141.
(11) Szurmai, Z.; Kerekgyarto, J .; Harangi, J .; Liptak, A. Carbohydr.
Res. 1987, 164, 313.
(7) Marra, A.; Sinay, P. Carbohydr. Res. 1989, 190, 317.
(8) Kuhn, R.; Lutz, P.; MacDonald, D. L. Chem. Ber. 1966, 99, 611.
(12) Roy, R.; Pon, R. A. Glycoconjugate J . 1990, 7, 3.
(13) Rink, H. Tetrahedron Lett. 1987, 28, 3787.
1002 J . Org. Chem., Vol. 69, No. 4, 2004

Oligomers Derived from Neuraminic Acid Analogues
SCHEME 2
SCHEME 3
SCHEME 4
protected amino acid with 5 equiv of benzotriazol-1-
yloxytris(dimethylamino) phosphonium hexafluorophos-
phate14 (BOP or Castro’s reagent) and 5 equiv of Hu¨nigs
base in NMP for 2 h. After completion of the reaction, as
indicated by the Kaiser test,15 the Fmoc was removed
using 20% piperidine in NMP, and the resin was subse-
quently washed with NMP (5 × 2 mL × 2 min), methanol
(5 × 2 mL × 2 min), and finally with dichloromethane
(14) Nguyen, D. L.; Seyer, R.; Castro, B. J . Chem. Soc., Perkin Trans.
1 1985, 1025.
(15) Kaiser, E.; Colescat, R. L.; Bossinge, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.
J . Org. Chem, Vol. 69, No. 4, 2004 1003

Gregar and Gervay-Hague
7-ene (DBU) to provide the unsaturated sugar 8. A
modification of the procedure to remove the N-acetyl
group was required for this analogue, as 2 N NaOH at
90 °C caused decomposition. In this event, compound 8
was treated with tert-butyloxycarbonyl anhydride
((BOC)2O) and catalytic N,N-(dimethylamino)pyridine
(DMAP) in THF to form the N-acetyl-N-Boc product 9.16
Zemple´n conditions (NaOCH3/CH3OH) removed the N-
acetyl as well as the O-acetates to give 10, a Boc-
protected δ amino acid methyl ester. Treatment of the
protected amino acid with sodium hydroxide in water
converted the methyl ester to a free carboxylate, which
upon neutralization with acid resin and treatment with
1:1 TFA/water provided the amine 11. Fmoc protection
of 11 proceeded nicely to give 12, which we then protected
as the 8,9 acetonide. Protection was necessary because
previous studies in our laboratory indicated that the C-9
hydroxyl undergoes cyclocondensation forming a lactone
under the coupling conditions.17
With the required monomer unit in hand, we focused
on preparing the series C oligomers, which was ac-
complished using the same method as with series B
oligomers. Overall, the coupling times were less for 13,
suggesting enhanced steric accessibility. As anticipated,
the acetonide protecting groups were removed upon
cleavage of the series C oligomers from the solid support,
and purification was performed as previously described
(vide supra).
NMR characterization of the series C oligomers was
also complicated with significant overlap in the proton
and carbon spectra (Figure 3). It was possible to make
complete assignments for the monomer (C-1), dimer (C-
2), and trimer (C-3) proton and carbon resonances.
Beyond the trimer, the HSQC experiment showed that
the carbon peaks were divided into two unique regions:
those assigned to the terminal sugars and all other
internal residues in the oligomer. The remaining carbons
in the sugar chain overlapped with each other. This is
similar to the pattern found in the R-OMe series. How-
ever, the proton spectra show a unique pattern for the
H-3 protons, and it is possible to distinguish three regions
for the H-3 protons including those assigned to the
internal residues (5.78 ppm), the amino terminus residue
(5.83 ppm), and the carboxy terminus sugar attached to
the caproamide linker (5.71 ppm). The utility of H-3 as
a reporter group in conformational studies is currently
under investigation in our laboratories as are circular
dichroism studies.
The R-â unsaturated functionality in the C series
oligomers proved useful in the synthesis of the â-hydrido
series D compounds (Scheme 4). Hydrogenation of the
double bond was effected with a catalytic amount of 10%
Pd-C with the substrate dissolved in a mixture of
methanol and water. Shorter oligomers were soluble in
methanol and did not require the addition of water. As
the length of the oligomer increased, more and more
water was needed to complete the reaction in a 12-h
period. After the reaction was complete, the solution was
filtered through a tight packing of glass wool and the
solvent removed in vacuo. Water was added and subse-
quently removed via lyophilization to give a white solid.
F IGURE 2. Stack plot of 1D proton NMR spectra for B1-8
at 600 MHz in D2O. Note acetone impurity at ∼2.0 ppm.
(5 × 2 mL × 2 min). Compound 7 (2 equiv), BOP (2
equiv), and Hu¨nig’s base (4 equiv) were added to the
caproamide-linked resin in 1.5 mL of NMP, and the
reaction proceeded for 2 h. Routinely, a second coupling
reaction was performed using 1 equiv of the sugar amino
acid (7), 1 equiv of BOP, and 4 equiv of Hu¨nigs base in
1.5 mL of NMP for 1 h to ensure complete loading of the
resin. Reaction completion was confirmed by the Kaiser
test, and the resin was washed with dichloromethane
prior to deprotection of the Fmoc functionality. This
procedure was repeated as required to build the oligomer
library.
Final cleavage of the sugar from the solid support was
accomplished using 30% trifluoroacetic acid (TFA) in
dichloromethane (DCM) for 30 min, which was mild
enough to remove the sugar without causing any decom-
position or epimerization of the OCH3 at the anomeric
center as evidenced by NMR. Purification was accom-
plished by one of two methods. The procedure developed
by Szabo et al.1 incorporating size exclusion chromatog-
raphy on P-10 gel worked well but took about 24 h for
each oligomer. Instead, we found that reverse phase high-
performance liquid chromatography (HPLC) on a C-18
column provided more efficient separation.
Figure 2 shows a stack plot of one-dimensional (1D)
NMR spectra for all eight oligomers (B1-8), which
revealed insightful, yet limited, information. Beginning
with the monomer (B-1), all proton and carbon reso-
nances were assigned by correlation spectroscopy (COSY)
and heteronuclear single quantum coherence (HSQC)
experiments, respectively. The dimer (B-2) and trimer
(B-3) tested the limits of resolution of the NMR 1H
spectra even at 600 MHz, making it impossible to assign
protons in the remaining oligomers.
Having successfully completed the synthesis and pu-
rification of series B, we next turned our attention to the
synthesis of Fmoc-protected 2,3-dehydroneuraminic acid
analogue 13 (Scheme 3). Chloride 3 was dissolved in
benzene and reacted with 1,8-diazabicyclo[5.4.0]undec-
(16) Gervay, J .; Flaherty, T. M.; Nguyen, C. P. Tetrahedron Lett.
1997, 38, 1493.
(17) Nguyen, C. P. M.S. Thesis, The University of Arizona, 1998.
1004 J . Org. Chem., Vol. 69, No. 4, 2004

Oligomers Derived from Neuraminic Acid Analogues
F IGURE 3. Stack plot of 1D proton NMR spectra for C1-8 at 600 MHz in D2O. Note acetate and acetone impurity at 1.8 and
2 ppm, respectively.
Hydrogenation of unsaturated sialic acids is reported
to occur from the top face to give the â (axial)-hydride,
and product distribution can be determined by NMR.18
The most discriminating spectroscopic feature is the H2,
H3 coupling constant, which is on the order of 10 Hz for
the â-hydrido anomer (J H2ax,H3ax) and 3.5 Hz for the
equatorial R-hydrido anomer (J H2eq,H3ax). J H2,H3 is g10 Hz
for all of the series D oligomers, indicating that oligomer
conformations do not interfere with stereoselective â-face
hydrogenation.
The NMR spectra for the â-hydrido compounds dis-
played a complicated series of peaks with significant
overlap (Figure 4). The general pattern for regions in the
proton dimension correlated with the unsaturated series.
In a manner similar to the H3 proton of the C series, the
H2 anomeric proton doublets display unique regions for
the amino terminus residue (3.99 ppm), the internal
sugars (4.06 ppm), and the carboxy terminus residue
attached to the caproamide linker (4.14 ppm). As before,
the pattern of increasing the middle anomeric doublet
serves as a reporter group of the conformation of the
oligomer chain. The gradual increase of the middle peak
indicates the gradual increase of defined chemical equiva-
lency within the sugar chain. All sugar residues displayed
similar carbon chemical shifts with the exception of the
terminal residues in a manner consistent with the R-â
unsaturated oligomers.
that might disrupt interactions required for a stable
secondary structure. For the most part, oligomer syn-
thesis proceeded smoothly, although reactions incorpo-
rating 2,3-dehydroneuraminic acid (series C) were most
efficient. Two different series (B and C) of compounds
were prepared with oligomer lengths varying from one
to eight units. A third series (D) was obtained upon
hydrogenation of the 2,3-dehydro oligomers. These reac-
tions yielded only oligomers resulting from axial delivery
of hydrogen. Each component of the three series was
1
characterized by H and 13C NMR. In most cases, resolu-
tion of the peaks for monomer-trimer was possible, as
the amino and carboxy terminal residues have distinct
chemical shifts. The resonances corresponding to the
internal residues were not resolved in longer oligomers
(g4). Importantly, all of the oligomers were soluble in
water and all except C4-8 were soluble in methanol.
Conformational analyses of the series B-D oligomers
using circular dichroism and NH/ND exchange NMR
experiments are currently under investigation in our
laboratories.
Exp er im en ta l Section
N-Acetyl-r-2-O-m eth yl Neu r a m in ic Acid Meth yl Ester
(5). According to the method of Magnusson et al.,10 2-R-O-
methyl-4,7,8,9-tetra-O-acetyl-neuraminic acid methyl ester (4)
(7.41 g, 14.1 mmol) was dissolved in 150 mL of dry methanol.
Sodium methoxide (200 mg, 3.7 mmol) was added. The reaction
vessel was sealed and stirred overnight at room temperature.
After 14 h, the reaction was complete as indicated by TLC (7:3
benzene/acetone) and the solution was neutralized with Dowex
H+ resin and filtered. The solvent was removed in vacuo to
give 4.66 g of 5. Yield: 97%.
r-O-Meth yl-n eu r a m in ic Acid (6). N-Acetyl-R-O-methyl
neuraminic acid methyl ester (5) (500 mg, 1.47 mmol) was
dissolved in 3.5 mL of 2 N NaOH (4.4 mmol) and heated at 90
°C for 48 h. The solution was cooled and checked by TLC (4:1
propanol/H2O). The solution was neutralized to pH ) 7 and
Con clu sion
Two novel Fmoc-protected neuraminic acid analogues
have been prepared and incorporated into solid-phase
synthesis (SPS). A caproamide linker was introduced at
the carboxy terminus in an effort to minimize hydration
(18) (a) Bandgar, B. P.; Zbiral, E. Carbohydr. Res. 1995, 270, 201.
(b) Gervay, J .; Gregar, T. Q. Tetrahedron Lett. 1997, 38, 5921.
J . Org. Chem, Vol. 69, No. 4, 2004 1005

Gregar and Gervay-Hague
F IGURE 4. Stack plot of 1D proton NMR spectra for D1-8 at 600 MHz in D2O. Note acetate, acetone, acetonitrile, and methanol
impurity at 1.8, 2.0, 2.8, and 3.3 ppm, respectively.
freeze-dried. A determination of total yield and mass spectro-
metric analysis were not possible because of the large amount
of sodium chloride salt present. The remaining brown solid
was used, as is, for the next reaction. 1H NMR (D2O, 600
MHz): δ 3.57-3.57 (m, 2H, H8 and H9), 3.60-3.49 (m, 3H,
H4, H7, H9′), 3.37 (d, J ) 9.0 Hz, 1H, H6), 3.24 (at., J ) 8.5
Hz, 1H, H5), 3.22 (s, 3H, OCH3), 2.59 (dd, J ) 4.0 and 10.5
Hz, 1H, H3eq), 1.49 (at., J ) 10.0 Hz, 1H, H3ax). 13C NMR (D2O
referenced to TSP-d6, 125 MHz): δ 176.7, 103.7, 77.3, 74.8,
72.8, 71.6, 65.8, 65.6, 63.4, 57.4, 54.5, 43.5.
solid-phase synthesis. The extent of the reactions was con-
firmed using the Kaiser test. A typical procedure for the
building of oligomers was the following: Fmoc rink resin (100
mg, 0.7 mmol/g) was treated with 2 mL of 20% piperidine in
N-methyl pyrrolidinone (NMP) and agitated to remove Fmoc
(2 × 20 min). The resin was washed with NMP (6 × 2 mL ×
2 min) and finally with CH2Cl2 (1 × 1 mL × 2 min). The Fmoc-
protected ꢀ-amino caproic acid (5 equiv), BOP (5 equiv), and
i-Pr2NEt (5 equiv) was added to the resin in 2 mL of NMP
and agitated for 2 h. Completion was confirmed by the Kaiser
test. Subsequently, the Fmoc was removed per previous
conditions, and the washing step was performed as before.
Addition of the first neuraminic acid unit was accomplished
by the addition, in order, of the following: Fmoc amino acid
(2 equiv), BOP (2 equiv), i-Pr2NEt (4 equiv) in NMP (2 mL).
The reaction mixture was agitated for 2 h at room tempera-
ture, and the procedure was repeated to ensure complete
coupling. Deprotection was the same as before, and the
procedure was repeated as desired. Removal of oligomer from
the resin was accomplished by first washing the resin with
MeOH (2 × 2 mL × 2 min) followed by CH2Cl2 (5 × 2 mL × 2
min). Cleavage was performed with 30% CF3COOH in CH2-
Cl2 (3:7, 40 mL), and the solvent was allowed to pass through
the funnel into a flask below. Seepage through the funnel took
approximately 30 min. The solvent was removed in vacuo, 5
mL of H2O was added, and the filtrate was lyophilized.
Purification of these compounds initially used a size exclu-
sion chromatography gel. A buffer system of 0.03 N NH4HCO3
was used to elute the sugar off the column with a flow rate of
12%, which reflects the percentage of the maximum flow rate
for the fraction collector pump, approximately 0.5 mL/min. The
column had a diameter of 1 in. and a length of 60 in. With
this length and flow rate, each fraction took about 4 min to
fill. The sugar was detected using a UV/vis detector set at 222
nm to detect the amide absorbance. Material eluted at ap-
proximately fraction 30 and ended at approximately fraction
80 depending on the amount of material placed on the column.
N -(9-F lu o r e n y lm e t h o x y c a r b o n y l)-2-r-O -m e t h y l-
n eu r a m in ic Acid (7). R-O-Methyl-neuraminic acid (6) (1.477
g, 5.2 mmol) was dissolved in 35 mL of NaHCO3, 50 mL of
H2O, and 85 mL of dioxane. The solution was stirred briskly
and cooled to 0 °C. 9-Fluorenylmethyl chloroformate was
dissolved in dioxane and added dropwise over 30 min. The
solution was warmed to room temperature and stirred for 16
h. The solution was diluted with water (50 mL) and extracted
with diethyl ether (3 × 20 mL). The aqueous phase was
adjusted to pH ) 3 with the addition of a 10% HCl solution.
The acidic solution was then extracted with EtOAc (3 × 20
mL). The organic layers were combined and dried with MgSO4.
The solution was filtered and the solvent was removed in vacuo
1
giving 1.82 g of 7. Yield: 59% from 5. H NMR (CD3OD, 500
MHz): δ 7.78-7.28 (m, 8H, ArH), 4.36 (dd, J ) 4.4 and 8.5
Hz, 2H, Fmoc CH2), 4.21 (t, J ) 6.5 Hz, 1H, Fmoc CH), 3.86-
3.84 (m, 2H, H8, H9), 3.68-3.53 (m, 5H, H4, H5, H6, H7, H9′),
2.67 (dd, J ) 4.6 and 12.7 Hz, 1H, H3eq), 1.70 (at., J ) 12.6
Hz, 1H, H3ax). 13C NMR (CD3OD, 125 MHz): δ 171.8, 159.8,
145.4, 145.2, 142.6, 128.8, 128.2, 126.3, 120.9, 100.0, 75.0, 73.2,
70.2, 68.8, 68.1, 67.4, 55.1, 51.9, 41.7. FABHRMS: [M + H+]
calcd for C25H30O10N, 504.1870; found, 504.1886.
Gen er al P r ocedu r e for (1f5) Am ide-Lin ked Neu r am in -
ic Acid Oligom er s Con ju ga ted to E-Am in o Ca p r oa m id e.
(1f5) Amide-linked oligomers were assembled using Fmoc
chemistry on solid-phase support. Rink resin (4-(2′,4′-dimethoxy-
phenyl-Fmoc-aminomethyl)-phenoxy resin) was used for the
1006 J . Org. Chem., Vol. 69, No. 4, 2004

Oligomers Derived from Neuraminic Acid Analogues
These numbers varied depending on the oligomer length with
the longer oligomers eluting before the shorter chain lengths.
Size exclusion chromatography works well when it is used to
separate two compounds where the molecular weight of one
compound is at least twice that of the other. The separation
of the monomer and dimer sugar proved to be most effective
using this technique. As the oligomer length increased and
the differences in molecular weight decreased, the efficiency
of purification by size exclusion chromatography decreased.
Fractions collected were lyophilized to give a fluffy white
amorphous solid.
25.6, 24.6. FABHRMS: [M + H+] calcd for C46H83O29N6,
1183.5217; found, 1183.5204.
1
P en ta m er (B-5): obtained in 32% purified yield. H NMR
(D2O, 600 MHz): δ 4.00 (d, J ) 10.1 Hz), 3.81-3.70 (m, 4H,
H5), 3.81-3.70 (m, 14H), 3.66 (dd, J ) 5.3, 12.1 Hz), 3.64-
3.51 (m, 14H), 3.28 (s, 12H, OCH3), 3.24 (s, 3H, OCH3), 3.23-
3.13 (m, 3H, H5, CH2 caproamide), 2.69-2.63 (m, 5H, H3eq),
2.19 (t, J ) 7.3 Hz, 2H, CH2 caproamide), 1.75-1.69 (m, 5H,
H3ax), 1.55-1.46 (m, 4H, 2CH2 caproamide), 1.29-1.24 (m, 2H,
CH2 caproamide). 13C NMR (D2O referenced to external TSP
std, 125 MHz): δ 180.0, 170.3, 170.2, 169.9, 168.7, 99.9, 99.8,
73.1, 72.0, 71.2, 71.1, 70.8, 67.8, 67.7, 67.3, 67.2, 67.1, 65.5,
62.7, 62.4, 52.0, 51.8, 51.7, 51.2, 51.1, 51.0, 39.3, 38.6, 38.2,
37.8, 34.8, 27.9, 25.6, 24.6. FABHRMS: [M + H+] calcd for
Reverse phase HPLC was also used to purify these oligomers
as it took less time and resulted in a more uniform separation
between oligomers. A C-18 column was used with a flow rate
of 2.5 mL/min with a UV/vis detector set to 230 to detect the
amide absorbance. The water phase contained 0.1% TFA. A
gradient of 0-10% acetonitrile over 20 min was used to elute
the oligomer with the retention time of approximately 13 min.
Peaks were very broad and generally trailed. The solvent was
then lyophilized to give fluffy white solids.
C
56H100O36N7, 1466.6228; found, 1446.6205.
Hexa m er (B-6): obtained in 24% purified yield. 1H NMR
(D2O, 600 MHz): δ 4.00-3.93 (m, 6H, H6, H5), 3.82-3.70 (m,
17H), 3.65 (dd, J ) 5.4, 12.2 Hz, 1H, H9), 3.64-3.52 (m, 17H),
3.28 (s, 15 H, OCH3), 3.25 (s, 3H, OCH3), 3.24-3.15 (m, 3H,
H5, CH2 caproamide), 2.68-2.63 (m, 6H, H3eq), 2.19 (t, J )
7.3 Hz, 2H, CH2 caproamide), 1.75-1.68 (m, 6H, H3ax), 1.54-
1.46 (m, 4H, 2CH2 caproamide), 1.28-1.24 (m, 2H, CH2
caproamide). 13C NMR (D2O referenced to external TSP std,
150 MHz): δ 180.0, 170.3, 170.2, 170.1, 168.7, 99.9, 99.8, 73.1,
71.2, 71.1, 67.9, 67.7, 67.4, 67.1, 67.0, 62.7, 52.0, 51.8, 51.7,
51.1, 51.0, 39.3, 38.5, 38.3, 38.2, 38.0, 34.7, 27.9, 25.6, 24.6.
FABHRMS: [M + H+] calcd for C66H117O43N8, 1709.7275;
found, 1709.7215.
Hep ta m er (B-7): obtained in 9% purified yield. 1H NMR
(D2O, 600 MHz): δ 8.25 4.02-4.00 (m, 7H, H5, H6), 3.99-
3.93 (m, 22H), 3.67-3.52 (m, 19H), 3.28 (s, 18H, OCH3), 3.25
(s, 3H, OCH3), 3.22-3.13 (m, 3H, H5, CH2 caproamide), 2.76-
2.65 (m, 7H, H3eq), 2.19 (t, J ) 7.3 Hz, 2H, CH2 caproamide),
1.75-1.67 (m, 7H, H3ax), 1.55-1.46 (m, 4H, 2CH2 caproamide),
1.29-1.24 (m, 2H, CH2 caproamide). 13C NMR (D2O referenced
to external TSP std, 125 MHz): δ 183.0, 173.3, 173.2, 172.8,
171.7, 166.1, 165.8, 102.9, 102.8, 76.1, 75.0, 74.2, 74.1, 74.0,
70.9, 70.7, 70.5, 70.3, 70.2, 70.1, 70.0, 69.9, 68.5, 65.7, 65.4,
55.0, 54.8, 54.7, 54.7, 54.2, 54.3, 54.0, 42.3, 41.2, 40.8, 37.7,
30.9, 28.6, 27.6. FABHRMS: [M + H+] calcd for C76H134O50N9,
1972.8220; found, 1972.8329.
1
Mon om er (B-1): obtained in 31% purified yield. H NMR
(D2O, 600 MHz): δ 3.96 (d, J ) 1.7, 10.3 Hz, 1H), 3.85-3.82
(m, 1H, H8), 3.79 (dd, J ) 2.5, 12.2 Hz, 1H), 3.74 (dd, J ) 1.6,
9.1 Hz, 1H, H7), 3.71-3.65 (m, 2H, H4, H9′), 3.25 (s, 3H,
OCH3), 3.19 (t, J ) 7.0 Hz, 2H, CH2 caproamide), 2.69 (dd, J
) 4.7, 13.1 Hz, 1H, H3eq), 2.18 (t, J ) 7.3 Hz, 2H, CH2
caproamide), 1.769 (apt, J ) 12.4 Hz, 1H, H3ax), 1.56-1.47
(m, 4H, 2CH2 caproamide), 1.30-1.24 (m, 2H, CH2 caproam-
ide). 13C NMR (D2O referenced to external TSP std, 125
MHz): δ 178.0, 168.3, 99.8, 72.0, 70.8, 67.2, 65.5, 62.4, 52.0,
51.1, 39.3, 38.2, 34.7, 27.9, 25.5, 24.6. FABHRMS: [M + H+]
calcd for C16H32O8N3, 394.2189; found, 394.2191.
Dim er (B-2): obtained in 12% purified yield. 1H NMR (D2O,
600 MHz): δ 4.00 (d, J ) 1.7 and 10.3 Hz, 1H, H6), 3.96 (t, J
) 10.3 Hz, 1H, H5), 3.83-3.80 (m, 1H, H8), 3.78-3.75 (m, 5H,
H4, H6, H7, H8, H9), 3.66 (dd, J ) 5.1, 12.2 Hz, 1H), 3.61-
3.57 (m, 2H), 3.57 (d, J ) 9.5 Hz, 1H, H9′), 3.28 (s, 3H, OCH3),
3.25 (s, 3H, OCH3), 3.26-3.16 (m, 3H, H5, CH2 caproamide),
2.68 (dt, J ) 4.7 and 13.0 Hz, 2H, H3eq), 2.21 (t, J ) 7.3 Hz,
2H, CH2 caproamide), 1.74 (apt, J ) 12.8 Hz, 1H, H3ax), 1.68
(apt, J ) 12.5 Hz, 1H, H3ax), 1.55-1.45 (m, 4H, 2CH2
caproamide), 1.28-1.23 (m, 2H, CH2 caproamide). 13C NMR
(D2O referenced to external TSP std, 125 MHz): δ 178.0, 169.9,
168.7, 99.8, 73.0, 72.0, 71.0, 70.8, 67.7, 67.3, 66.9, 65.5, 62.6,
62.4, 52.0, 51.8, 51.2, 51.0, 39.3, 38.5, 37.8, 34.7, 27.9, 25.9,
25.6, 24.6. FABHRMS: [M + H+] calcd for C26H49O15N4,
657.3194; found, 657.3196.
Tr im er (B-3): obtained in 15% purified yield. 1H NMR (D2O,
600 MHz): δ 4.00 (d, J ) 10.4 Hz, 1H, H6), 3.98-3.92 (m, 2H,
H5), 3.83-3.53 (m, 17H), 3.29 (s, 3H, OCH3), 3.28 (s, 3H,
OCH3), 3.25 (s, 3H, OCH3), 3.22-3.13 (m, 3H, H5, CH2
caproamide), 2.71-2.64(m, 3H, H3eq), 2.18 (t, J ) 7.3 Hz, 2H,
CH2 caproamide), 1.74-1.65 (m, 3H, H3ax), 1.61-1.47 (m, 4H,
2CH2 caproamide), 1.31-1.27 (m, 2H, CH2 caproamide). 13C
NMR (D2O referenced to external TSP std, 125 MHz): δ 180.0,
170.3, 169.8, 168.7, 99.9, 99.8, 73.1, 73.0, 72.0, 71.1, 70.9, 67.8,
67.7, 67.3, 67.1, 67.1, 65.5, 62.8, 62.7, 62.6, 62.4, 52.0, 51.9,
51.8, 51.7, 51.2, 51.1, 51.0, 39.3, 38.5, 38.2, 37.8, 34.7, 27.9,
25.6, 24.6. FABHRMS: [M + H+] calcd for C36H66O22N5,
920.4205; found, 920.4199.
Tetr a m er (B-4): 1H NMR (D2O, 600 MHz): δ 4.00 (dd, J
) 1.8 and 10.3 Hz, 1H, H6), 3.98 (m, 3H, H5), 3.83-3.70 (m,
13H), 3.66 (dd, J ) 5.6, 12.4 Hz, 1H, H9), 3.63-3.52 (m, 9H),
3.29, 3.28, 3.28, 3.25 (4s, 12H, OCH3), 3.22-3.13 (m, 3H, H5,
CH2 caproamide), 2.69-2.63 (m, 4H, H3eq), 2.19 (t, J ) 7.4
Hz, 2H, CH2 caproamide), 1.76-1.67 (m, 4H, H3ax), 1.56-1.47
(m, 4H, 2CH2 caproamide), 1.29-1.24 (m, 2H, CH2 caproam-
ide). 13C NMR (D2O referenced to external TSP std, 125
MHz): δ 180.0, 170.3, 170.2, 169.8, 168.7, 163.1, 117.4, 115.1,
99.89, 99.87, 99.8, 73.1, 72.0, 71.1, 71.1, 70.8, 67.8, 67.8, 67.7,
67.3, 67.2, 67.1, 67.1, 65.5, 62.7, 62.7, 62.6, 62.4, 52.0, 51.8,
51.7, 51.2, 51.1, 51.0, 39.3, 38.6, 38.2, 38.1, 37.8, 34.7, 27.9,
Octom er (B-8): obtained in 31% yield. 1H NMR (D2O, 600
MHz): δ 4.01-3.95 (m, 8H, H5, H6), 3.82-3.72 (m, 24H),
3.66-3.53 (m, 23H), 3.29 (s, 21H, OCH3), 3.26-3.15 (m, 3H,
H5, CH2 caproamide), 2.68-2.65 (m, 8H, H3eq), 2.19 (t, J )
7.6 Hz, 2H, CH2 caproamide), 1.75 (m, 8H, H3ax), 1.55-1.46
(m, 4H, 2CH2 caproamide), 1.29-1.24 (m, 2H, CH2 caproam-
ide). 13C NMR (D2O referenced to external TSP std, 150
MHz): δ 180.0, 170.3, 179.2, 169.8, 168.7, 99.9, 99.8, 73.1, 72.0,
71.14, 71.09, 70.8, 67.9, 67.8, 67.7, 67.3, 67.2, 67.1, 65.5, 62.7,
62.6, 62.4, 52.0, 51.8, 51.70, 51.67, 51.2, 51.1, 51.0, 39.3, 38.5,
38.2, 37.8, 34.7, 27.9, 25.6, 24.6. MALDI: [M + Na+] calcd for
C
86H149O57N10Na, 2257.9069; found, 2257.8844.
N -(9 -F l u o r e n y l m e t h o x y c a r b o n y l )-2 ,3 -d e h y d r o -
n eu r a m in ic Acid (12). 2,3-Dehydrido neuraminic acid (11)
(1.78 g, 7.16 mmol) was dissolved in 70 mL of H2O, 20 mL of
saturated Na2CO3, and 100 mL of dioxane. The solution was
cooled to 0 °C, and a solution of Fmoc-Cl (2.23 g, 8.62 mmol)
dissolved in 25 mL of dioxane was added dropwise over 30 min.
The solution was warmed to room temperature and stirred for
14 h. Once the reaction was complete as determined by TLC
(4:1 propanol/water), the solution was extracted with diethyl
ether (3 × 20 mL) and the aqueous layer was concentrated to
half its original volume in vacuo. The aqueous solution was
acidified with 10% HCl to pH ) 3, and the sugar was extracted
into ethyl acetate (3 × 30 mL). The solvent was removed in
vacuo giving 2.53 g of a yellow amorphous solid (75% yield).
1H NMR (CD3OD, 600 MHz): δ 7.77-7.28 (m, 8H, Fmoc-Ar),
5.95 (d, J ) 2.4 Hz, 1H, H3), 4.43 (dd, J ) 2.5 and 8.7 Hz, 1H,
H4), 4.38 (d, J ) 6.9 Hz, 2H, CH2-Fmoc), 4.24 (m, 2H, H6,
CH-Fmoc), 3.91 (m, 1H, H8), 3.84 (dd, J ) 3.1 and 4.4 Hz,
1H, H9), 3.82 (t, J ) 10.1 Hz, 1H, H5), 3.67-3.64 (m, 2H, H7,
J . Org. Chem, Vol. 69, No. 4, 2004 1007

Gregar and Gervay-Hague
H9′). 13C NMR (CD3OD, 150 MHz): δ 165.7, 159.4, 145.3,
145.1, 142.6, 128.8, 128.2, 126.18, 120.9, 113.6, 78.2, 71.3, 70.1,
68.2, 68.1, 65.0, 52.9, 48.3. FABHRMS: [M + Na+] calcd for
d, H6d), 4.39 (d, J ) 10.8 Hz, 2H, H6), 4.35 (d, J ) 10.9 Hz,
1H, H6), 4.17-4.11 (m, 3H, H5a-c), 3.88-3.87 (m, 1H, H8d),
3.82-3.75 (m, 10H, H7d, H8a-c, H9a-c, H9′b-d), 3.64 (dd,
J ) 5.5 and 11.9 Hz, 1H, H9d), 3.59-3.51 (m, 4H, H7a-c,
H9′a), 3.53 (t, J ) 8.8 Hz, 1H, H5d), 3.21-3.18 (m, 2H, NCH2),
2.17 (t, J ) 7.4 Hz, 2H, CH2 caproamide), 1.53-1.46 (m, 4H,
2CH2 caproamide), 1.27-1.23 (m, 2H, CH2 caproamide). 13C
NMR (D2O referenced to external TSP std, 125 MHz): δ 183.1,
167.3, 166.7, 166.5, 148.8, 148.5, 148.3, 111.8, 110.9, 79.2, 79.1,
79.1, 77.9, 72.9, 72.8, 70.7, 70.0, 67.5, 65.94, 65.87, 65.8, 65.4,
53.8, 53.1, 53.0, 42.1, 37.8, 30.9, 28.4, 27.7. FABHRMS: [M +
Na+] calcd for C42H66O25N6Na, 1055.4156; found, 1055.4164.
C
24H25O9Na, 494.1427; found, 494.1431.
N-(9-F lu or en ylm eth oxyca r bon yl)-2,3-d eh yd r o-8,9-iso-
p r op ylid in e Neu r a m in ic Acid (13). N-(9-Fluorenylmethox-
ycarbonyl) 2,3-dehydro neuraminic acid (12) (2.53 g, 5.36
mmol) was dissolved in 150 mL of dry acetone. To this, 400
mg Amberlyst H+resin was added and stirred. 2,2-Dimethoxy
propane (2.63 mL, 25.3 mmol) was added via syringe and
stirred for 2 h. The solution was filtered and concentrated
1
giving 1.88 g of white amorphous solid (68% yield). H NMR
1
(acetone-d6, 600 MHz): δ 7.84-7.30 (m, 8H, Fmoc-Ar), 6.91
(d, J ) 8.2 Hz, 1H, CONH), 5.98 (d, J ) 2.1 Hz, 1H, H3), 4.59
(dd, J ) 2.2 and 8.5 Hz, 1H, H4), 4.50 (dd, J ) 7.3 and 10.5
Hz, 1H, CH-Fmoc), 4.39 (dd, J ) 6.7 and 10.6 Hz, 1H, CH2-
Fmoc), 4.31 (dd, J ) 6.2 and 13.2 Hz, 1H, H8), 4.24 (t, J ) 6.9
Hz, 1H, CH2-Fmoc), 4.12-4.09 (m, 2H, H6, H9), 4.01 (dd, J
) 5.1 and 8.4 Hz, 1H, H9′), 3.84 (dt, J ) 8.6 and 10.3 Hz, 1H,
H5), 3.69 (d, J ) 7.8 Hz, 1H, H7). 13C NMR (acetone-d6, 150
MHz): δ 163.8, 159.3, 145.5,142.6, 129.1, 126.6, 113.9, 79.0,
76.1, 75.1, 71.3, 53.7, 49.8, 48.5, 32.5, 27.9, 26.3. FABHRMS:
[M + H+] calcd for C27H30O9N, 512.1921; found, 512.1938.
P en ta m er (C-5): obtained in 66% purified yield. H NMR
(D2O, 600 MHz): δ 5.85 (d, J ) 2.6 Hz, 1H, H3e), 5.80 (s, 3H,
H3b-d), 5.73 (d, J ) 2.2 Hz, 1H, H3a), 4.54-4.49 (m, 6H,
H4a-e, H6e), 4.39 (d, J ) 10.9 Hz, 3H, H6), 4.36 (d, J ) 10.9
Hz, 1H, H6), 4.18-4.11 (m, 4H, H5a-d), 3.90-3.87 (m, 1H,
H8e), 3.84-3.75 (m, 13H, H7e, H8a-d, H9a-d, H9′b-e), 3.64
(dd, J ) 5.5, 11.9 Hz, 1H, H9e), 3.62-3.54 (m, 5H, H7a-d,
H9′a), 3.53 (t, J ) 8.8 Hz, 1H, H5e), 3.24-3.18 (m, 2H, NCH2),
2.17 (t, J ) 7.4 Hz, 2H, CH2 caproamide), 1.54-1.46 (m, 4H,
2CH2 caproamide), 1.28-1.23 (m, 2H, CH2 caproamide). 13C
NMR (D2O referenced to external TSP std, 125 MHz): δ 183.1,
167.3, 166.7, 166.5, 165.7, 148.8, 148.5, 148.3, 111.8, 110.9,
79.1, 79.1, 79.0, 77.9, 72.8, 70.7, 70.0, 67.5, 66.0, 65.9, 65.8,
65.4, 53.8, 53.03, 52.98, 42.1, 37.8, 30.8, 28.4, 27.7. MALDI:
[M + Na+] calcd for C51H81O31N7Na, 1308.4714; found,
1308.4685.
Ser ies C Syn th esis. Assignments for each oligomer are
based upon the following: the sugar chain attached to the
caproamide linker is labeled as sugar a, with the terminal
sugar with the free amine labeled as sugar x. Absolute
assignment is given where possible. AB-X refers to the R-â
unsaturated oligomers with length x.
1
Hexa m er (C-6): obtained in 50% purified yield. H NMR
1
Mon om er (C-1): obtained in 63% purified yield. H NMR
(D2O, 600 MHz): δ 5.85 (d, J ) 2.6 Hz, 1H, H3f), 5.80 (s, 4H,
H3b-e), 5.73 (d, J ) 2.2 Hz, 1H, H3a), 4.54-4.49 (m, 7H,
H4a-f, H6f), 4.39 (d, J ) 10.9 Hz, 4H, H6), 4.36 (d, J ) 10.9
Hz, 1H, H6), 4.18-4.11 (m, 5H, H5a-e), 3.90-3.87 (m, 1H,
H8f), 3.84-3.75 (m, 16H, H7f, H8a-e, H9a-e, H9′b-f), 3.64
(dd, J ) 5.5, 11.9 Hz, 1H, H9f), 3.62-3.54 (m, 6H, H7a-e,
H9′a), 3.53 (t, J ) 8.8 Hz, 1H, H5f), 3.24-3.18 (m, 2H, CH2),
2.17 (t, J ) 7.4 Hz, 2H, CH2 caproamide), 1.54-1.46 (m, 4H,
2CH2 caproamide), 1.28-1.23 (m, 2H, CH2 caproamide). 13C
NMR (D2O referenced to external TSP std, 125 MHz): δ 183.1,
167.3, 166.7, 166.5, 148.8, 148.5, 148.3, 111.8, 110.9, 79.2, 79.0,
77.9, 72.9, 70.7, 70.0, 67.4, 65.9, 65.3, 53.8, 53.0, 42.1, 37.8,
30.8, 28.4, 27.7, 26.8. MALDI: [M + Na+] calcd for C60H93O37N8,
1539.5457; found, 1539.5521.
Hep ta m er (C-7): obtained in 86% purified yield. 1H NMR
(D2O, 600 MHz): δ 5.85 (d, J ) 2.6 Hz, 1H, H3g), 5.80 (s, 5H,
H3b-f), 5.73 (d, J ) 2.2 Hz, 1H, H3a), 4.54-4.49 (m, 8H, H4a-
g, H6g), 4.39 (d, J ) 10.9 Hz, 5H, H6), 4.36 (d, J ) 10.9 Hz,
1H, H6), 4.18-4.11 (m, 6H, H5a-f), 3.90-3.87 (m, 1H, H8g),
3.84-3.75 (m, 19H, H7g, H8a-f, H9a-f, H9′b-g), 3.64 (dd, J
) 5.5 and 11.9 Hz, 1H, H9g), 3.62-3.54 (m, 7H, H7a-f, H9′a),
3.53 (t, J ) 8.8 Hz, 1H, H5g), 3.24-3.18 (m, 2H, NCH2), 2.17
(t, J ) 7.4 Hz, 2H, CH2 caproamide), 1.54-1.46 (m, 4H, 2CH2
caproamide), 1.28-1.23 (m, 2H, CH2 caproamide). 13C NMR
(D2O referenced to external TSP std, 125 MHz): δ 183.1, 167.3,
166.7, 166.5, 148.8, 148.5, 148.3, 111.8, 110.9, 79.2, 79.0, 77.9,
72.9, 70.7, 70.0, 67.50, 65.9, 65.9, 65.4, 65.2, 53.8, 53.0, 42.1,
37.8, 30.8, 28.40, 27.7. FABHRMS: [M + Na+] calcd for
(D2O, 600 MHz): δ 5.77 (d, J ) 2.6 Hz, 1H,), 4.52 (dd, J ) 6.0,
8.4 Hz, 1H, H4), 4.48 (dd, J ) 1.2, 9.6 Hz, 1H, H6), 3.87 (m,
1H, H8), 3.79 (dd, J ) 4.8, 9.0 Hz, 1H, H9), 3.75 (dd, J ) 0.6,
8.8 Hz, 1H, H7), 3.65 (dd, J ) 5.5, 12.0 Hz, 1H, H9′), 3.51 (dd,
J ) 8.6, 9.7 Hz, 1H, H5), 3.20 (t, J ) 6.6 Hz, 2H, NCH2), 2.17
(t, J ) 7.2 Hz, 2H, CH2), 1.53-1.44 (m, 4H, 2CH2), 1.27-1.23
(m, 2H, CH2). 13C NMR (D2O referenced to external TSP std,
125 MHz): δ 183.1, 166.0, 148.7, 110.1, 77.8, 72.8, 70.7, 67.6,
65.5, 53.8, 50.2, 42.2, 42.0, 38.6, 37.8, 30.9, 28.4, 28.0, 27.7.
FABHRMS: [M + H+] calcd for C15H28O7N3, 362.1927; found,
362.1922.
Dim er (C-2): obtained in 77% purified yield. 1H NMR (D2O,
600 MHz): δ 5.87 (d, J ) 2.7 Hz, 1H, H3b), 5.75 (d, J ) 2.4
Hz, 1H, H3a), 4.56-4.52 (m, 3H, H4a,b, H6b), 4.38 (d, J )
10.8 Hz, 1H, H6a), 4.16 (dd, J ) 8.9, 10.7 Hz, 1H, H5a), 3.90
(m, 1H, H8b), 3.84-3.78 (m, 5H, H7b, H8a, H9a, H9′a, H9b),
3.67 (dd, J ) 5.6, 12.0 Hz, 1H, H9′b), 3.60-3.53 (m, 2H, H5b,
H7a), 3.22 (m, 2H, NCH2), 2.19 (t, J ) 7.4 Hz, 2H, CH2
caproamide), 1.56-1.48 (m, 4H, 2CH2 caproamide), 1.30-1.26
(m, 2H, CH2 caproamide). 13C NMR (D2O referenced to external
TSP std, 125 MHz): δ 183.1, 166.7, 166.5, 148.8, 148.3, 110.9,
110.8, 79.0, 77.9, 72.9, 72.8, 70.7, 70.0, 67.5, 65.9, 65.4, 53.8,
53.1, 42.1, 37.8, 30.9, 28.4, 27.7. FABHRMS: [M + H+] calcd
for C24H41O13N4, 593.2670; found, 593.2717.
Tr im er (C-3): obtained in 35% purified yield. 1H NMR
(D2O, 600 MHz): δ 5.83 (d, J ) 2.7 Hz, 1H, H3c), 5.78 (d, J )
2.3 Hz, 1H, H3), 5.71 (d, J ) 2.3 Hz, 1H, H3), 4.53-4.49 (m,
4H, H4a-c, H6c), 4.35 (d, J ) 10.4 Hz, 1H, H6), 4.31 (d, J )
10.9 Hz, 1H, H6), 4.14-4.09 (m, 2H, H5a,b), 3.88-3.86 (m,
1H, H8c), 3.82-3.73 (m, 7H, H7c, H8a,b, H9a,b, H9′b,c), 3.63
(dd, J ) 5.5 and 12.0 Hz, 1H, H9c), 3.56-3.52 (m, 4H, H7a,b,
H9′a), 3.51 (t, J ) 8.3 Hz, 1H, H5c), 3.20-3.17 (m, 2H, NCH2),
2.15 (t, J ) 7.4 Hz, 2H, CH2 caproamide), 1.51-1.44 (m, 4H,
CH2 caproamide), 1.26-1.22 (m, 2H, CH2 caproamide). 13C
NMR (D2O referenced to external TSP std, 125 MHz): δ 183.1,
167.3, 166.7, 166.5, 148.8, 148.5, 148.4, 111.8, 110.9, 79.1, 79.1,
77.9, 72.8, 70.7, 70.0, 67.5, 66.0, 65.8, 65.4, 53.8, 53.1, 53.0,
42.2, 37.8, 30.9, 28.4, 27.7. FABHRMS: [M + H+] calcd for
C
69H106O43N9Na, 1770.6200; found, 1770.6080.
Octa m er (C-8): obtained in 84% purified yield. 1H NMR
(D2O, 600 MHz) δ 5.85 (d, J ) 2.6 Hz, 1H, H3h), 5.80 (s, 6H,
H3b-g), 5.73 (d, J ) 2.2 Hz, 1H, H3a), 4.54-4.49 (m, 9H,
H4a-h, H6h), 4.39 (d, J ) 10.9 Hz, 5H, H6), 4.36 (d, J ) 10.9
Hz, 1H, H6), 4.18-4.11 (m, 6H, H5a-f), 3.90-3.87 (m, 1H,
H8h), 3.84-3.75 (m, 22H, H7h, H8a-g, H9a-g, H9′b-h), 3.64
(dd, J ) 5.5 and 11.9 Hz, 1H, H9g), 3.62-3.54 (m, 6H, H7a-f,
H9′a), 3.53 (t, J ) 8.8 Hz, 1H, H5h), 3.24-3.18 (m, 2H, NCH2),
2.17 (t, J ) 7.4 Hz, 2H, NCH2 caproamide), 1.54-1.46 (m, 4H,
2CH2 caproamide), 1.28-1.23 (m, 2H, CH2 caproamide). 13C
NMR (D2O referenced to external TSP std, 125 MHz): δ 183.0,
167.2, 166.6, 166.4, 148.7, 148.4, 148.3, 111.8, 110.8, 79.1, 79.0,
77.8, 72.8, 70.6, 69.9, 67.4, 65.8, 65.3, 53.7, 52.9, 42.1, 37.8,
C
33H54O19N5, 824.3413; found, 824.3408.
Tetr a m er (C-4): obtained in 35% purified yield. H NMR
1
(D2O, 600 MHz): δ 5.85 (d, J ) 2.5 Hz, 1H, H3d), 5.79 (s, 2H,
H3b,c), 5.72 (d, J ) 1.9 Hz, 1H, H3a), 4.54-4.51 (m, 5H, H4a-
1008 J . Org. Chem., Vol. 69, No. 4, 2004

Oligomers Derived from Neuraminic Acid Analogues
1
30.8, 28.3, 27.6. MALDI: [M + Na+] calcd for C78H118O49N10
-
P en ta m er (D-5): obtained in 82% isolated yield. H NMR
Na, 2001.6943; found, 2001.6901.
(D2O, 600 MHz): δ 4.14 (d, J ) 10.5 Hz, 1H, H2), 4.06 (d, J )
11.7 Hz, 3H, H2), 3.99 (d, J ) 12.0 Hz, 1H, H2), 3.95-3.67
(m, 22H), 3.65-3.54 (m, 4H, H9′), 3.44-3.37 (m, 4H, H7),
3.23-3.08 (m, 3H, H5, NCH2), 2.20-2.16 (m, 5H, H3eq), 2.17
Gen er a l P r oced u r e for t h e Syn t h esis of Ser ies D
Oligom er s. Series C oligomers were dissolved in 5 mL of
methanol. Water was added for the longer oligomers to aid
solubility. To the reaction mixture, 10 mg of 10% Pd-on-carbon
was added to the reaction mixture, and hydrogen gas was
bubbled through the methanol solution for 5 min. The flask
was placed under a balloon of hydrogen for 14 h. The solution
was then filtered through tight glass wool to remove the Pd-
on-carbon. The glass wool was washed several times with
water and methanol. The filtrate was removed in vacuo, and
the remaining sugar was dissolved in water and lyophilized
to give a white amorphous solid.
(t, J ) 7.3 Hz, 2H, CH2 caproamide), 1.55-1.36 (m, 9H, H3ax
,
2CH2 caproamide), 1.26-1.23 (m, 2H, CH2 caproamide). 13C
NMR (D2O referenced to external TSP std, 125 MHz): δ 183.0,
176.9, 176.3, 175.9, 84.6, 78.4, 77.8, 77.7, 77.4, 73.1, 72.9, 72.5,
71.0, 70.7, 65.9, 65.7, 55.4, 54.50, 54.46, 41.6, 39.8, 39.7, 39.6,
37.8, 30.7, 28.2, 27.6. MALDI: [M + Na+] calcd for C51H89O31N7-
Na 1318.5496; found, 1318.5383.
1
Hexa m er (D-6): obtained in 63% isolated yield. H NMR
(D2O, 600 MHz): δ 4.14 (d, J ) 10.7 Hz, 1H, H2), 4.06 (d, J )
11.9 Hz, 4H, H2), 3.99 (d, J ) 12.0 Hz, 1H, H2), 3.92 (m, 1H,
H4), 3.87-3.61 (m, 27H), 3.57-3.54 (m, 5H, H9′), 3.41-3.37
(m, 5H, H7), 3.19-3.13 (m, 3H, H5, CH2 caproamide), 2.33-
2.28 (m, 6H, H3eq), 2.17 (t, J ) 10.8 Hz, 2H, CH2 caproamide),
1.53-1.41 (m, 10H, H3ax, 2CH2), 1.26-1.21 (m, 2H, CH2
caproamide). 13C NMR (D2O referenced to external TSP std,
125 MHz): δ 183.1, 177.0, 176.3, 176.0, 166.1, 84.7, 78.4, 77.9,
77.8, 77.7, 77.3, 73.3, 73.19, 73.15, 72.9, 72.7, 72.6, 71.1, 70.90,
70.85, 70.8, 66.04, 65.95, 65.7, 54.6, 54.5, 54.4, 41.7, 39.9, 39.7,
39.7, 37.8, 30.8, 28.3, 27.7, 20.9. MALDI: [M + Na+] calcd for
1
Mon om er (D-1): obtained in 79% isolated yield. H NMR
(D2O, 600 MHz): δ 4.08 (dd, J ) 2.1, 12.0 Hz, 1H, H2), 3.89
(dt, J ) 4.8, 10.7 Hz, 1H, H4), 3.82-3.76 (m, 2H, H6, H9),
3.64 (m, 2H, H7, H8), 3.16 (m, 2H, H9′, NCH2), 3.13 (t, J )
10.1 Hz, 1H, H5), 2.34 (ddd, J ) 2.2, 4.8, 12.9 Hz, 1H, H3eq),
2.19 (t, J ) 7.4 Hz, 2H, CH2 caproamide), 1.55-1.45 (m, 5H,
H3ax, CH2 caproamide), 1.25 (m, 2H, 2CH2 caproamide). 13C
NMR (D2O referenced to external TSP std, 125 MHz): δ 183.1,
175.5, 77.8, 77.8, 73.0, 71.7, 70.9, 65.8, 55.5, 41.7, 39.6, 37.8,
30.8, 28.3, 27.7. FABHRMS: [M + H+] calcd for C15H30O7N3,
364.2084; found, 364.2068.
C
60H104O37N8Na, 1551.6396; found, 1551.6317.
Dim er (D-2): obtained in 94% isolated yield. 1H NMR (D2O,
600 MHz): δ 4.14 (dd, J ) 1.9 and 11.9 Hz, H2), 3.99 (d, J )
11.9 Hz, 1H, H2), 3.93 (dt, J ) 4.8 and 10.7 Hz, 1H, H4), 3.87-
3.69 (m, 7H, H4, H5, H6, H8, H9), 3.64-3.61 (m, 3H, H7, H8,
H9), 3.54 (dd, J ) 5.9, 11.8 Hz, 1H, H9′), 3.37 (d, J ) 9.2 Hz,
1H, H7), 3.19 (t, J ) 10.2 Hz, 1H, H5), 3.15 (m, 2H, NCH2),
2.37 (ddd, J ) 2.6, 4.8, 10.8 Hz, 1H, H3eq), 2.29 (d, J ) 12.5
Hz, 1H, H3eq), 2.17 (t, J ) 7.3 Hz, 2H, CH2 caproamide), 1.53-
1.42 (m, 5H, H3ax, 2CH2 caproamide), 1.23 (m, 2H, CH2
caproamide). 13C NMR (D2O referenced to external TSP std,
125 MHz): δ 183.1, 176.3, 176.0, 78.4, 77.9, 77.8, 77.3, 73.1,
72.9, 72.7, 71.1, 71.0, 70.8, 66.0, 65.7, 55.4, 54.6, 41.7, 39.8,
37.8, 30.8, 28.3, 27.7. FABHRMS: [M + H+] calcd for
Hep ta m er (D-7): obtained in 52% isolated yield. 1H NMR
(D2O, 600 MHz): δ 4.14 (d, J ) 9.7 Hz, 1H, H2), 4.07 (d, J )
11.8 Hz, 6H, H2), 3.99 (d, J ) 12.0 Hz, 1H, H2), 3.92 (m, 1H,
H4), 3.87-3.60 (m, 32H), 3.57-3.51 (m, 6H, H9), 3.43-3.39
(m, 6H, H7), 3.18-3.13 (m, 3H, H5, CH2 caproamide), 2.33-
2.28 (m, 7H, H3eq), 2.17 (t, J ) 7.3 Hz, 2H, CH2 caproamide),
1.53-1.42 (m, 11H, H3ax, 2CH2 caproamide), 1.26-1.18 (m,
2H, CH2 caproamide). 13C NMR (D2O referenced to external
TSP std, 125 MHz): δ 180.1, 174.0, 173.3, 173.0, 163.1, 162.8,
75.6, 75.4, 74.9, 74.8, 74.7, 74.3, 71.5, 70.3, 69.9, 69.6, 68.1,
67.9, 67.8, 63.0, 62.7, 52.4, 51.5, 38.7, 36.9, 36.7, 36.6, 34.8,
27.8, 25.3, 24.7. MALDI: [M + Na+] calcd for C69H119O43N9-
Na, 1784.7295; found, 1784.7273.
Octa m er (D-8): obtained in 85% isolated yield. 1H NMR
(D2O, 600 MHz): δ 4.13 (d, J ) 12.1 Hz, 1H, H2), 4.06 (d, J )
12.1 Hz, 6H, H2), 3.99 (d, J ) 13.5 Hz, 1H, H2), 3.85 (m, 1H,
H4), 3.81-3.37 (m, 37H), 3.57-3.54 (m, 7H, H9′), 3.41-3.37
(m, 7H, H7), 3.19-3.13 (m, 3H, H5, CH2 caproamide), 2.37-
2.28 (m, 8H, H3eq), 2.17 (t, J ) 7.4 Hz, 2H, CH2 caproamide),
1.53-1.44 (m, 12H, H3ax, 2CH2 caproamide), 1.26-1.22 (m,
2H, CH2 caproamide). 13C NMR (D2O referenced to external
TSP std, 125 MHz): δ 174.0, 75.4, 74.9, 74.7, 70.3, 69.6, 68.1,
63.0, 62.7, 52.4, 51.5, 38.7, 36.9, 34.8, 27.8, 25.3, 24.7.
MALDI: [M + Na+] calcd for C78H134O49N10Na, 2017.8195;
found, 2017.8375.
C
24H45O13N4, 597.2983; found, 597.3000.
Tr im er (D-3): obtained in 100% isolated yield. 1H NMR
(D2O, 600 MHz): δ 4.14 (dd, J ) 2.0, 11.0 Hz, 1H, H2), 4.06
(dd, J ) 1.8, 12.0 Hz, 1H, H2), 3.99 (dd, J ) 1.5, 11.9 Hz, 1H,
H2), 3.93 (dt, J ) 4.8, 10.6 Hz, 1H, H4), 3.87-3.61 (m, 12H),
3.57-3.50 (m, 2H, H9′), 3.40-3.37 (m, 2H, H7), 3.18 (t, J )
10.2 Hz, 1H, H5), 3.15 (m, 2H, NCH2), 2.38-2.28 (m, 3H, H3eq),
2.17 (t, J ) 7.4 Hz, 2H, CH2 caproamide), 1.56-1.42 (m, 7H,
H3ax, 2CH2 caproamide), 1.23 (m, 2H, CH2 caproamide). 13C
NMR (D2O referenced to external TSP std, 125 MHz): δ 183.1,
177.0, 176.3, 176.0, 78.4, 77.9, 77.9, 77.8, 77.3, 73.2, 72.9, 72.7,
72.6, 71.1, 71.1, 70.9, 70.8, 66.0, 65.9, 65.7, 55.4, 54.6, 41.7,
39.9, 39.8, 39.7, 37.8, 33.2, 30.8, 28.3, 27.7. FABHRMS: [M +
H+] calcd for C33H60O19N5, 830.3883; found, 830.3886.
1
Tetr a m er (D-4): obtained in 70% isolated yield. H NMR
Ack n ow led gm en t. This work was supported by
National Science Foundation Grant CHE-0196482. The
NSF CRIF program (Grant CHE-9808183), NSF Grant
OSTI 97-24412, and NIH Grant RR11973 provided
funding for the NMR spectrometers used in this project.
(D2O, 600 MHz): δ 4.13 (d, J ) 11.9 Hz, 1H, H2), 4.06 (d, J )
11.9 Hz, 2H, H2), 3.99 (d, J ) 12.0 Hz, 1H, H2), 3.92 (dt, J )
6.0, 10.7 Hz, 1H, H4), 3.87-3.61 (m, 17H), 3.57-3.53 (m, 3H,
H9′), 3.48-3.37 (m, 3H, H7), 3.15 (m, 3H, H5, NCH2), 2.37-
2.28 (m, 4H, H3eq), 2.17 (t, J ) 7.4 Hz, 2H, CH2 caproamide),
1.55-1.41 (m, 8H, H3ax, 2CH2 caproamide), 1.23 (m, 2H, CH2
caproamide). 13C NMR (D2O referenced to external TSP std,
125 MHz): δ 183.0, 176.9, 176.2, 175.9, 78.3, 77.82, 77.78, 77.7,
77.2, 73.2, 73.1, 72.8, 72.6, 72.5, 71.0, 70.8, 70.7, 65.9, 65.8,
65.6, 55.3, 54.5, 54.5, 41.6, 39.8, 39.7, 39.6, 37.8, 30.7, 28.2,
27.6. MALDI: [M + Na+] calcd for C42H74O25N6Na, 1085.4597;
found, 1085.4540.
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal procedures and carbon NMR spectra for 6, 7, B1-8, 12,
13, C1-8, and D1-8. This material is available free of charge
via the Internet at http://pubs.acs.org.
J O035312+
J . Org. Chem, Vol. 69, No. 4, 2004 1009