O-glycosyl amino acids by 2-nitrogalactal concatenation - Synthesis of a Mucin-type O-glycan

Article abstract of DOI:10.1002/ejoc.200390142

Base-promoted Michael-type addition of N-Boc- and N-Fmoc-protected serine and threonine esters to 2-nitrogalactal derivatives 2 and 26 led highly selectively to α-glycosides 4a-d and 27a,c, respectively. Ensuing transformation of threonine derivative 4d a

Full text of DOI:10.1002/ejoc.200390142

FULL PAPER
O-Glycosyl Amino Acids by 2-Nitrogalactal Concatenation ؊ Synthesis of a
Mucin-Type O-Glycan
Gottfried A. Winterfeld,[a] Ahmed I. Khodair,[a][‡] and Richard R. Schmidt*[a]
Keywords: Carbohydrates / Michael additions / Glycosylations / Glycosides / Glycopeptides / Peptidomimetics
Base-promoted Michael-type addition of N-Boc- and N-
Fmoc-protected serine and threonine esters to 2-nitrogalactal
derivatives 2 and 26 led highly selectively to α-glycosides
4a−d and 27a,c, respectively. Ensuing transformation of
threonine tert-butyl esters 31a,c carrying the O-protected
STN-antigen at the hydroxy group. The threonine derivative
31c was then transformed into the N-Fmoc-protected amino
acid building block 33, which was employed for the synthesis
threonine derivative 4d and serine derivatives 4a,b resulted of mucin repeating unit partial structure Ac-GS(STN)-TAP-
in compounds useful as lysine and dipeptide mimetics. 6-O-
Desilylation of 27a,c, then 6-O-sialylation, and transforma-
PAHG-NH2 (1).
tion of the nitro group of the galactose moiety into a 2-aceta- ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
mido functionality, afforded N-Boc-protected serine and
Germany, 2003)
Introduction
plest mucin structure, the TN-antigen, Michael addition to
2-nitrogalactal serves as an efficient alternative
The mucin class of glycopeptides has attracted much at- approach[22Ϫ25] to practically all mucin core structures.
tention, because it consists of numerous structures of fun-
Mucins, a family of highly glycosylated glycoproteins that
damental importance in biological processes like cellϪcell are found in mucus and on cell surfaces of epithelial cells,
adhesion, cell growth, fertilization, parasitic infection, and all have a characteristic unit consisting of a variable number
inflammation.[1Ϫ3] The interest of biologists and chemists of tandem repeat units. The repeating units are rich in ser-
has been sparked especially by the occurrence of aberrant ine and threonine residues that allow for heavy O-glycosyl-
glycoforms of the mucin family in tumours of epithelial tis-
sues.[1,2]
Chemical synthesis of the characteristic α-glycosidic link-
age between 2-acetamido-2-deoxy--galactopyranose and
the side chain hydroxy groups of -serine and -threonine,
however, has proven to be difficult. Despite the endeavours
and achievements of synthetic chemists, most syntheses of
α-O-linked glycopeptides have relied essentially on the
methodology introduced by Paulsen in 1978.[4Ϫ6] The non-
participating azido group as a latent amino function at po-
sition 2 is combined with a good leaving group at the anom-
eric position in the glycosylation reaction. Proceeding from
this concept, many syntheses of mucin structures have been
reported that use different anomeric leaving groups for the
glycosidation step.[7Ϫ18] N-Acetyl-protected galactosamine
donors also have been transformed directly into O-linked α-
glycosyl amino acid derivatives,[19] and enzymatic syntheses
have been investigated successfully in this endeavour.[20,21]
Recently, we have shown that for the synthesis of the sim-
[a]
Fachbereich Chemie, Universität Konstanz, Fach M 725
78457 Konstanz, Germany
Fax: (internat.) ϩ 49-7531/883135
E-mail: Richard.Schmidt@uni-konstanz.de
[‡]
On leave from: Chemistry Department, Faculty of Education,
Figure 1. O-Glycosylated MUC 1 nonapeptide 1 as part of the
Tanta University, Kafr El-Sheikh Branch, Kafr El-Sheikh, MUC 1 tandem repeat unit; the arrow indicates the glycosylation
Egypt site
Eur. J. Org. Chem. 2003, 1009Ϫ1021  2003 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1434Ϫ193X/03/0306Ϫ1009 $ 20.00ϩ.50/0
1009

G. A. Winterfeld, A. I. Khodair, R. R. Schmidt
1
FULL PAPER
ation.[3,40,41] Malignantly transformed cells show increased structural assignment could be derived readily from the H
expression of mucins and, because of incomplete glycosyl- NMR spectra (4aϪd; J1,2 ഠ 4.2, J2,3 ഠ 10.7 Hz).
ation, are covered with shorter carbohydrate chains.[26,27]
Transformation of, for instance, 4d into N-deprotected
One of the prominent, incomplete glycan structures is the threonine derivative 5 (Scheme 2) was accomplished by
STN-antigen. It is an end product in the biosynthetic path- treatment with trifluoroacetic acid (TFA) under standard
way and cannot be converted further into complex struc- conditions. Reaction of 5 with phenyl isocyanate or phenyl
tures.[28] Antibody titres against STN have been reported to isothiocyanate as electrophiles afforded N-carbamoyl deriv-
correlate with improved prognosis in breast cancer patients. atives 6 and 7, respectively, in high yields. Investigation of
The preparation of immunogenic structures eliciting the the base-promoted ring closure to the corresponding hyd-
formation of antibodies, or more importantly cytotoxic T- antoin derivatives led — as shown, for example, upon treat-
lymphocytes, directed against the STN-antigen, therefore, ment with ammonia — to elimination of 2-deoxy-2-nitro-
seems an attractive synthetic and biological challenge.[29,30] α--galactose 8 and formation of 5-(ethylidene)hydantoin 9
Syntheses of an STN glycopeptide fragment of HIV gp 120 and formation of 5-(ethylidene)thiohydantoin 10, respect-
and of STN peptides of the MUC 1 repeating unit, with ively. The (Z) configuration of 9 and 10 was determined
the help of differently prepared STN building blocks, have by comparison with previous studies based on 13C NMR
been reported.[31Ϫ38]
spectroscopic data.[44,45]
In this paper, we extend our studies to the synthesis of
useful GalNAcα(1-O)Ser and GalNAcα(1-O)Thr conjug-
ates — i.e., TN antigen building blocks — and to the syn-
thesis of their 6-O-sialylated derivative, the STN antigen.[39]
This building block is employed for the construction of the
MUC 1 mucin type glycopeptide 1 that is derived from the
tandem repeat eicosapeptide as shown in Figure 1.[40,41]
Results and Discussion
3,4,6-Tri-O-benzyl-2-nitro--galactal (2), which is readily
available from the corresponding galactal derivative,[42] fur-
nished upon reaction with N-Boc-protected serine derivat-
ive 3a the α-galactoside derivative 4a (80%) together with
some β-anomer (13%)[22,43] (Scheme 1). Even direct potas-
sium tert-butoxide-promoted addition of N-Fmoc-protected
serine derivative 3b to 2 was possible, affording α-galacto-
side derivative 4b in 83% yield practically without loss of
the base-sensitive Fmoc group; in addition, 14% of the cor-
responding β-anomer was obtained, which could be separ-
ated readily from 4b.[22] It was confirmed also that no ra-
cemisation had occurred under the reaction conditions. Ad-
dition of threonine derivatives 3c and 3d led to stereoselec-
tive formation of 4c[22] and 4d, respectively; β-anomer
formation was not observed, and thus two new stereogenic
centres were generated stereoselectively in this reaction. The
Scheme 2. Reaction of adduct 4d with heterocumulenes; a) TFA;
b) PhNCX, EtOH, reflux; c) NH3, MeOH
Reduction of the 2-nitro group in 4d with hydrogen and
platinized Raney-Ni T4[46] as catalyst afforded amino deriv-
ative 11, which also can be regarded as a dipeptide mimetic
(Scheme 3). Acid-catalysed removal of the N-Boc protecting
group and subsequent treatment with diphosgene in dichlo-
romethane/pyridine, and then with methanol, furnished
Nα,Nε-bis(methoxycarbonyl) derivative 12. An alternative
reaction sequence led from 11 to Nε-(methoxycarbonyl) de-
rivative 13, which also serves as a starting material for the
synthesis of 12. Hydrogenolytic O-debenzylation of 12 with
palladium on carbon in methanol/acetic acid and then O-
acetylation afforded O-acetyl derivative 14. Similarly, Nα-
(phenylcarbamoyl) derivative 15 was obtained from 13 after
carbamoylation of the unprotected amino group with
phenyl isocyanate, O-debenzylation, and then O-
acetylation.
Scheme 1. Addition of serine and threonine derivatives to 2-nitro-
galactal 2
1010
Eur. J. Org. Chem. 2003, 1009Ϫ1021

O-Glycosyl Amino Acids via 2-Nitrogalactal Concatenation
FULL PAPER
the presence of triethylamine as base furnished Nα-Fmoc-
Nε-Boc-protected dipeptide mimetic 19. Hydrogenolysis of
19 removed both the O-benzyl and N-Fmoc groups; sub-
sequent treatment with Fmoc chloride, in the presence of
triethylamine, furnished the useful O-unprotected derivative
20, which on O-acetylation led to 21. Hydrogenolytic O-
debenzylation of 17 gave O-unprotected tert-butyl ester 22,
which on reaction with Fmoc chloride in the presence of
triethylamine led to Nα-Boc-Nε-Fmoc-protected dipeptide
mimetic 23; O-acetylation furnished per-O-acetyl derivat-
ive 24.
As previously shown, 6-O-tert-butyldiphenylsilyl-
(TBDPS-)protected 2-nitrogalactal 26, which is readily avail-
able from the corresponding galactal 25, afforded exclusively
the α-galactopyranosides 27c and 27a, respectively, not only
with threonine derivative 3c but also with serine derivative
3a (Scheme 5).[22] Obviously, these TN antigen intermediates
are versatile building blocks for the construction, for in-
stance, of mucin-type O-glycopeptides. In order to demon-
strate this versatility in the synthesis of target molecule 1,
the TBDPS groups of 27a,c were removed upon treatment
Scheme 3. Liberation of amino groups in 4d and reaction with elec-
trophiles; a) Raney-Ni T4/Pt, H2, EtOH; b) 1. HCl, Et2O; 2. di-
phosgene, CH2Cl2, Pyr; 3. MeOH; c) 1. diphosgene, CH2Cl2, Pyr;
2. MeOH; 3. HCl, Et2O; d) diphosgene, Pyr; MeOH; e) 1. Pd/C,
H2, MeOH/AcOH; 2. Ac2O, Pyr; f) 1. PhNCO; 2. Pd/C, H2; 3.
Ac2O, Pyr
In order to generate useful peptidomimetics, the nitro
groups in compounds 4a,b were transformed into amino
groups, as described above, thus furnishing 16 and 17 after
Nα-Fmoc and Nα-Boc protection, respectively (Scheme 4).
Acid-catalysed cleavage of the tert-butyl group in the ester
16 furnished the acid 18, which on treatment with 2-(tert-
butoxycarbonyloximino)-2-phenylacetonitrile (BocON) in
Scheme 4. Transformation of 4a,b into lysine and peptido mimetics;
Scheme 5. Synthesis of STN structures by Michael addition and
a) Raney-Ni T4/Pt, H2, EtOH, b) TFA; c) BocON, NEt3, d) Pd/C, sialylation; a) KOtBu, Tol; b) TBAF, THF; c) TMSOTf (0.1 equiv.),
H2; FmocCl, NEt3; e) Ac2O, Pyr; f) Pd/C, H2, AcOH, MeOH; g)
FmocCl, NEt3; h) Ac2O, Pyr
EtCN, Ϫ60 to Ϫ40 °C; d) 1. Raney-Ni T4/Pt, H2, EtOH; 2.
Ac2O, Pyr
Eur. J. Org. Chem. 2003, 1009Ϫ1021
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G. A. Winterfeld, A. I. Khodair, R. R. Schmidt
FULL PAPER
with tetrabutylammonium fluoride (TBAF) in THF/acetic in propionitrile as solvent and with trimethylsilyl trifluoro-
acid affording 6-O-unprotected derivatives 28a,c. Sialylation
methanesulfonate (TMSOTf) as catalyst — furnished STN
antigen derivatives 30a and 30c. Reduction of the nitro
groups under the conditions described above led, despite the
powerful reducing properties of the catalyst, to clean forma-
tion of the amines that, on N-acetylation, furnished the de-
sired building blocks 31a and 31c, respectively. The α-link-
age of the sialyl groups in 30a,c and 31a,c was confirmed
with the known[47,48] sialyl donor 29 — at low temperatures
1
by comparison of the H NMR chemical shifts of 3b-He
and 4b-H, the coupling constants of 7b-H and 8b-H, and
the differences of the chemical shift of 9b-H and 9bЈ-H, with
averages of values reported in the literature.[49]
For the synthesis of target molecule 1, hydrogenolytic O-
debenzylation of 31c in the presence of Pd/C as catalyst was
performed; subsequent treatment with acetic anhydride in
pyridine afforded O-acetylated O-glycosyl threonine deriv-
ative 32 (Scheme 6). Acid-catalysed removal of the N-Boc
and the tert-butyl ester groups, and then treatment with N-
(fluorenylmethoxycarbonyloxy)succinimide (FmocONSu)
Scheme 6. Preparation of an STN building block for glycopeptide
synthesis; a) 1. Pd/C, H2, AcOH, MeOH; 2. Ac2O, Pyr; b) 1. TFA,
CH2Cl2; 2. FmocONSu, NaHCO3
Scheme 7. Solid-phase glycopeptide synthesis of 34 and deprotection to glycopeptide 1: Ac-GS(STN)-TAPPAHG-NH2
1012 Eur. J. Org. Chem. 2003, 1009Ϫ1021

O-Glycosyl Amino Acids via 2-Nitrogalactal Concatenation
FULL PAPER
in acetonitrile/water in the presence of sodium bicarbonate, into dipeptide and lysine mimetics, as well as being em-
afforded STN building block 33, which is suitable for Fmoc- ployed in O-glycopeptide synthesis.
based glycopeptide synthesis.
We chose PyBOP/HOBT activation[50Ϫ52] for glycopep-
tide coupling in the synthesis of target molecule 1 Experimental Section
(Scheme 7), using TentaGel S RAM Gly Fmoc (Rapp Po-
General Remarks: Solvents were evaporated under reduced pressure
while maintaining the water bath temperature below 40 °C. Chro-
matography was performed on silica gel for flash chromatography
40 µm (J. T. Baker) at 3 bar pressure. For thin-layer chromato-
graphy (TLC) plastic sheets of silica gel 60 F254 were used and the
compounds visualized by illumination under UV light at 253 nm
and by treatment with a solution of 5% (NH4)2MoO4 and 0.1%
Ce(SO4)2 in 10% H2SO4 followed by heating to 160 °C. Optical
rotations were measured at 25 °C with a PerkinϪElmer polarimeter
241/MS using the sodium D line. NMR spectra: Bruker AC 250
Cryospec, Bruker DR 600; TMS or the solvent residual peak were
used as internal standard. Coupling constants (3JC,H) were ob-
served in gradient-selected heteronuclear multi-bond correlations
(HMBC). MALDI-MS: Kratos Kompact Maldi 1; 2.5-dihydroxyb-
enzoic acid was used as matrix. FAB-MS: Finnigan MAT 312/
AMD 5000, 790 eV, 70 °C. Where its recovery is stated, yields are
calculated based on reagents consumed.
lymere) as the prefunctionalised resin. Previously, we have
used this system — a Rink amide linker together with the
Tentagel S resin — for the synthesis of a C-linked glyco-
peptide,[53] but with the amino acids used directly as their
Dhbt or Pfp activated esters. In the present study, the free
carboxylic acid is converted into the active ester in situ,
thus shortening the synthesis of the glycopeptide building
block.
Protected glycopeptide 34 was assembled via interme-
diates A and B, using standard procedures. N,N-Dimethyl-
formamide was used as solvent in all coupling steps in the
peptide synthesis, and for washing. Fmoc cleavage was
performed with morpholine and monitored at λ ϭ 360 nm.
Functionalised Fmoc amino acids were added successively
to the peptide chain in the specified sequence. Glycosyl-
ated threonine building block 33 was used in twofold ex-
cess and activated in the same manner as the simple am-
ino acids.
After the peptide had been assembled completely, the
resin-bound peptide was cleaved from the solid phase and
partially deprotected using trifluoroacetic acid/triisopropyl-
silane/water. The crude glycopeptide, still fully protected on
the glycan moiety, was then purified on a column of Se-
phadex LH-20 to give the desired product 34 as a colourless
lyophilisate in 61% yield. This material was characterised by
NMR spectroscopic and mass spectrometric analysis. The
acetyl groups protecting the carbohydrate hydroxy func-
tions were still intact, as was the neuraminic acid methyl
ester. Basic hydrolysis of the acyl groups is a delicate pro-
cedure because the O-glycan 34 can undergo β-elimination
of the carbohydrate moiety. It is important, therefore, to
find hydrolytic conditions that are strong enough to cleave
a tertiary methyl ester, but that do not affect the threonine
glycoside.
As a basis for the deprotection of 34, we examined the
protocol that Kunz et al. had used for the deprotection of
a similar structure.[34] In the first step, the O-acetyl groups
were cleaved within 3 h by treatment with aqueous sodium
hydroxide in methanol at a controlled pH of 10Ϫ11. Methyl
ester hydrolysis was performed in THF/water with aqueous
sodium hydroxide (pH ϭ 11Ϫ11.5). After 4 h, this proced-
N-(tert-Butoxycarbonyl)-O-(3,4,6-tri-O-benzyl-2-deoxy-2-nitro-α-
DL-threonine Methyl Ester (4d): Compounds 3d[54] (1.16 g,
5.00 mmol) and 2 (2.10 g, 5.00 mmol) were dried under high va-
cuum and dissolved in dry toluene (60 mL) under argon. Freshly
˚
activated molecular sieves (3 A, 3 g) were introduced and the mix-
ture stirred for 1 h. Then a solution of 1  potassium tert-butoxide
in THF (0.50 mL, 0.50 mmol) was added and stirring continued
for 2 h. Acetic acid (0.50 mL) was used to acidify the reaction mix-
ture, the molecular sieves were filtered off, and all the solvents were
evaporated. The residue was purified by column chromatography
(petroleum ether/ethyl acetate, 9:1) to furnish 4d (3.34 g, 96%). No
corresponding β-glycoside was detected. TLC (petroleum ether/
1
ethyl acetate, 4:1): Rf ϭ 0.58. [α]D ϭ ϩ75.5 (c ϭ 1.5; CHCl3). H
3
NMR (250 MHz, CDCl3): δ ϭ 1.22 (d, Jγ,β ϭ 6.3 Hz, 3 H, γ-
CH3), 1.49 (s, 9 H, C4H9), 3.26 (m, 2 H, 6-H, 6Ј-H), 3.68 (s, 3 H,
OCH3), 3.95 (m, 2 H, 4-H, 5-H), 4.20Ϫ4.48 (m, 6 H, 3-H,
OCH2Ph, β-CH, α-CH), 4.64 (s, 2 H, OCH2Ph), 4.75 (d, 2J ϭ
11.1 Hz, 1 H, OCH2Ph), 4.86 (dd, 3J2,1 ϭ 4.2, 3J2,3 ϭ 10.8 Hz, 1 H,
2-H), 5.00 (d, 3JNH,α ϭ 8.2 Hz, 1 H, NH), 5.26 (d, 3J1,2 ϭ 4.3 Hz, 1
H, 1-H), 7.11Ϫ7.32 (m, 15 H, Ar-H) ppm. 13C NMR (150.8 MHz,
CDCl3): δ ϭ 18.8 (γ-CH3), 29.01 (CMe3), 53.5 (OCH3), 58.8 (α-C),
69.0 (C-6), 70.7 (C-5, β-C), 73.7 (C-4, 2 ϫ OCH2Ph), 75.8
(OCH2Ph, C-3), 80.9 (CMe3), 85.0 (C-2), 98.3 (C-1), 128.4, 128.5,
128.8, 129.0, 129.2 138.0, 138.4, 138.6 (C-Ar), 156.2 (CO2Me),
171.1 (Boc-CO) ppm. MS (MALDI): m/z ϭ 717 [M ϩ Na]ϩ.
C37H46N2O11 (694.8): calcd. C 63.96, H 6.67, N 4.03; found C
63.87, H 6.60, N 4.17.
ure led to complete methyl ester cleavage with no consider- O-(3,4,6-Tri-O-benzyl-2-deoxy-2-nitro-α-D-galactopyranosyl)-L-
threonine Methyl Ester (5): 3d (1.00 g, 1.44 mmol) was dissolved in
a mixture of trifluoroacetic acid and CH2Cl2 (20 mL, 1:1), the solu-
tion was stirred at room temp. for 12 h, and then the solvents were
evaporated. The residue was dissolved in CH2Cl2, then saturated
aqueous NaHCO3 was added with vigorous stirring. The layers
were separated and the aqueous layer was extracted with CH2Cl2.
The combined organic layers were dried (MgSO4), filtered, concen-
trated under reduced pressure, and purified by column chromato-
graphy (CH2Cl2/MeOH, 98:2) to furnish 5 (0.76 g, 89%). TLC
able loss of material resulting from β-elimination. After
purification on a column of Sephadex LH-20, the depro-
tected glycopeptide 1 was isolated as a lyophilisate in 81%
yield for these last two steps. Deprotected glycopeptide 1
was characterised fully by NMR spectroscopic and mass
spectrometric analyses.
In conclusion, coupling of N-protected serine and
threonine esters to O-protected 2-nitrogalactal gives ready
access to N-acetylgalactosamine-derived α-glycosyl amino
(CH2Cl2/MeOH, 98:2): Rf ϭ 0.20. [α]D ϭ ϩ62.0 (c ϭ 1.0; CHCl3).
3
acids. These versatile building blocks can be transformed 1H NMR (250 MHz, CDCl3): δ ϭ 1.35 (d, Jγ,β ϭ 6.4 Hz, 3 H, γ-
Eur. J. Org. Chem. 2003, 1009Ϫ1021
1013

G. A. Winterfeld, A. I. Khodair, R. R. Schmidt
FULL PAPER
CH3), 3.27 (d, Jα,β ϭ 3.0 Hz, 1 H, α-CH), 3.52 (m, 2 H, 6-H, 6Ј-
H), 3.78 (s, 3 H, OCH3), 3.81 (d, J5,6 ϭ 2.7 Hz, 1 H, 5-H), 4.04
(m, 2 H, 3-H, 4-H), 4.20 (dq, Jβ,α ϭ 3.3, Jβ,γ ϭ 6.5 Hz, 1 H, β-
3
Compound 8: TLC (petroleum ether/ethyl acetate, 2:1): Rf ϭ 0.56.
[α]D ϭ Ϫ70.0 (c ϭ 0.1; CHCl3). 1H NMR (250 MHz, CDCl3): δ ϭ
3
3
3
3
2.50 (br. s, 1 H, OH), 3.42 (dd, J6,5 ϭ 5.7, 2J6,6Ј ϭ 9.4 Hz, 1 H, 6-
3
2
CH), 4.39Ϫ4.52 (m, 5 H, OCH2Ph, NH2), 4.71 (s, 2 H, OCH2Ph), H), 3.54 (dd, J6Ј,5 ϭ 5.7, J6Ј,6 ϭ 9.5 Hz, 1 H, 6Ј-H), 3.82 (m, 2
4.84 (d, 2J ϭ 11.1 Hz, 1 H, OCH2Ph), 4.95 (dd, 3J2,1 ϭ 4.2, 3J2,3 ϭ H, 5-H, 4-H), 4.35Ϫ4.78 (m, 9 H, 3-H, 2-H, 1-H, OCH2Ph),
10.7 Hz, 1 H, 2-H), 5.38 (d, J1,2 ϭ 4.2 Hz, 1 H, 1-H), 7.19Ϫ7.35 7.20Ϫ7.40 (m, 15 H, Ar-H) ppm. 13C NMR (62.8 MHz, CDCl3):
3
(m, 15 H, Ar-H) ppm. MS (MALDI): m/z ϭ 595 [M ϩ H]ϩ.
C32H38N2O9 (594.7): calcd. C 64.63, H 6.44, N 4.71; found C 64.23,
H 6.41, N 4.30.
δ ϭ 65.3 (C-6), 69.8 (C-5, OCH2Ph), 70.8 (C-4), 73.3 (OCH2Ph),
73.5 (OCH2Ph), 74.6 (C-3), 77.9 (C-1), 126.9, 127.6, 127.9, 128.0,
128.2, 128.4, 128.5 137.0, 137.2, 137.5 (C-Ar), 148.8 (C-2) ppm.
EI-MS (positive mode): m/z ϭ 449 [Mϩ Ϫ NO]. C27H29NO7
(479.5): calcd. C 67.63, H 6.10, N 2.92; found C 67.92, H 6.51,
N 3.23.
N-(Phenylaminocarbonyl)-O-(3,4,6-tri-O-benzyl-2-deoxy-2-nitro-α-
D-galactopyranosyl)-L-threonine Methyl Ester (6): A mixture of 5
(450 mg, 0.71 mmol) and phenyl isocyanate (169 mg, 1.42 mmol) in
anhydrous ethanol (3 mL) was heated under reflux for 12 h. The
solvent was evaporated under reduced pressure and the residue was
purified by column chromatography (petroleum ether/ethyl acetate,
4:1) to furnish 6 (0.40 g, 79%). TLC (petroleum ether/ethyl acetate,
4:1): Rf ϭ 0.3. [α]D ϭ ϩ84.8 (c ϭ 0.75; CHCl3). 1H NMR
Compound 9: M.p. 228Ϫ230 °C. TLC (petroleum ether/ethyl acet-
ate, 2:1): Rf ϭ 0.30. 1H NMR (600 MHz, [D6]DMSO): δ ϭ 1.83
(d, J ϭ 7.6 Hz, 3 H, CH3), 5.78 (q, J ϭ 7.6 Hz, 1 H, ϭCH),
7.37Ϫ7.48 (m, 5 H, Ar-H), 10.72 (s, 1 H, N1-H) ppm. 13C NMR
(150.8 MHz, [D6]DMSO): δ ϭ 12.7 (CH3), 108.9 (ϭCH), 126.9,
127.8, 128.7, 129.6, 131.8 (C-Ar), 153.2 (C-2), 161.8 (C-4) ppm.
MS (MALDI): m/z ϭ 203 [M ϩ H]ϩ. C11H10N2O2 (202.2): calcd.
C 65.34, H 4.98, N 13.85; found C 64.93, H 5.14, N 13.69.
3
(250 MHz, CDCl3): δ ϭ 1.32 (d, Jγ,β ϭ 6.4 Hz, 3 H, γ-CH3), 3.52
(m, 2 H, 6-H, 6Ј-H), 3.74 (s, 3 H, OCH3), 4.00 (m, 2 H, 4-H, 5-H),
3
3
3
4.26 (dd, J3,4 ϭ 2.8, J3,2 ϭ 10.7 Hz, 1 H, 3-H), 4.37 (dq, Jβ,α
ϭ
3
2.6, Jβ,γ ϭ 5.8 Hz, 1 H, β-CH), 4.42Ϫ4.47 (m, 4 H, OCH2Ph, α-
CH), 4.64 (d, J ϭ 12.6 Hz, 2 H, OCH2Ph), 4.75 (d, J ϭ 11.2 Hz,
1 H, OCH2Ph), 4.93 (dd, J2,1 ϭ 4.2, J2,3 ϭ 10.7 Hz, 1 H, 2-H),
5.35 (d, J1,2 ϭ 4.3 Hz, 1 H, 1-H), 5.50 (br. s, 1 H, NH), 7.06 (br.
O-3,4,6-Tri-O-benzyl-2-deoxy-2-nitro-α-D-galactopyranose (8) and
(5Z)-5-Ethylidene-3-phenyl-2-thiohydantoin (10): A saturated solu-
tion of NH3 in anhydrous CH3OH (5 mL) was added to a stirred
2
2
3
3
3
suspension of
7 (102 mg, 0.14 mmol) in anhydrous CH3OH
s, 1 H, NHPh), 7.18Ϫ7.40 (m, 20 H, Ar-H) ppm. MS (MALDI):
m/z ϭ 736 [M ϩ Na]ϩ. C39H43N3O10 (713.8): calcd. C 65.63, H
6.07, N 5.89; found C 65.52, H 6.17, N 5.86.
(10 mL) at room temp. and stirring was continued 2 h. The solvent
was evaporated under reduced pressure and the residue was chro-
matographed on silica gel using petroleum ether/ethyl acetate (4:1)
as eluent to give 8 (58 mg, 59%) as a colourless oil and of 10
(26.5 mg, 27%) as a white solid.
N-(Phenylaminothiocarbonyl)-O-(3,4,6-tri-O-benzyl-2-deoxy-2-
nitro-α-D-galactopyranosyl)-L-threonine Methyl Ester (7): A mixture
Compound 10: M.p. 232Ϫ234 °C (ref.[4] 238Ϫ240 °C). TLC (petro-
leum ether/ethyl acetate, 2:1): Rf ϭ 0.40. 1H NMR (250 MHz,
[D6]DMSO): δ ϭ 1.95 (d, J ϭ 7.7 Hz, 3 H, CH3), 5.93 (q, J ϭ
7.7 Hz, 1 H, ϭCH), 7.31Ϫ7.53 (m, 5 H, Ar-H), 12.42 (s, 1 H, N1-
H) ppm. 13C NMR (62.8 MHz, [D6]DMSO): δ ϭ 11.66 (CH3),
112.29 (ϭCH), 127.73, 129.22, 132.21 (C-Ar), 161.20 (C-4), 176.56
(C-2) ppm.
of 5 (300 mg, 0.50 mmol) and phenyl isothiocyanate (135 mg,
1.00 mmol) in anhydrous ethanol (3 mL) was heated under reflux
for 12 h. The solvent was evaporated under reduced pressure and
the residue was purified by column chromatography (petroleum
ether/ethyl acetate, 9:1) to furnish 7 (0.30 g, 82%). TLC (petroleum
ether/ethyl acetate, 2:1): Rf ϭ 0.4. [α]D ϭ ϩ64.1 (c ϭ 0.56; CHCl3).
3
1H NMR (250 MHz, CDCl3): δ ϭ 1.26 (d, Jγ,β ϭ 6.4 Hz, 3 H, γ-
CH3), 3.48 (m, 2 H, 6-H, 6Ј-H), 3.77 (s, 3 H, OCH3), 3.82 (dd,
O-(2-Amino-3,4,6-tri-O-benzyl-2-deoxy-α-
D-galactopyranosyl)-N-
3
3
3J5,6 ϭ 6.6, J5,6Ј ϭ 6.6 Hz, 1 H, 5-H), 3.89 (d, J4,3 ϭ 1.8 Hz, 1
(tert-butoxycarbonyl)- -threonine Methyl Ester (11): 4d (1.00 g,
L
3
3
H, 4-H), 4.00 (dd, J3,4 ϭ 2.3, J3,2 ϭ 10.7 Hz, 1 H, 3-H),
1.44 mmol) was dissolved in ethanol (15 mL) and transferred to a
hydrogenolysis vessel. Platinized Raney-Ni T4 catalyst was freshly
prepared as described previously[46] and the material obtained from
Raney nickel/aluminium alloy (2 g) was suspended in ethanol
(15 mL). A homogeneous suspension of this catalyst (15 mL) was
added to the reaction vessel and the suspension shaken under H2
for 48 h at ambient temp. and pressure. The catalyst was filtered
off and the solvent evaporated. The residue was purified by column
chromatography (CH2Cl2/MeOH, 98:2) to furnish 11 (0.82 g, 86%)
2
4.37Ϫ4.47 (m, β-CH, 4 H, OCH2Ph), 4.53 (d, J ϭ 10.6 Hz, 1 H,
OCH2Ph), 4.58 (d, 2J ϭ 10.6 Hz, 1 H, OCH2Ph), 4.78 (d, 2J ϭ
11.2 Hz, 1 H, OCH2Ph), 4.86 (dd, 3J2,1 ϭ 4.1, 3J2,3 ϭ 10.7 Hz, 1 H,
2-H), 5.31 (d, 3J1,2 ϭ 4.1 Hz, 1 H, 1-H), 5.42 (d, 3Jα,NH ϭ 9.5 Hz, 1
3
H, α-CH), 6.62 (d, JNH,α ϭ 9.4 Hz, 1 H, NH), 7.17Ϫ7.50 (m, 20
H, Ar-H), 8.05 (s, 1 H, NHPh) ppm. 13C NMR (150.8 MHz,
CDCl3): δ ϭ 18.6 (γ-CH3), 53.0 (OCH3), 62.3 (α-C), 68.1 (C-6),
70.0 (C-5), 72.9 (β-C, C-4), 73.7 (OCH2Ph), 73.6 (OCH2Ph), 75.0
(C-3), 75.2 (OCH2Ph), 84.1 (C-2), 97.3 (C-1), 125.0, 127.2, 127.8,
127.9, 127.9, 128.0, 128.1, 128.2, 128.3, 128.5, 128.6, 130.3 135.9,
137.1, 137.5, 137.7 (C-Ar), 170.0 (CO2Me) 181.6 (CϭS) ppm. MS
(MALDI): m/z ϭ 730 [M ϩ H]ϩ. C39H43N3O9S (729.9): calcd. C
64.18, H 5.94, N 5.76; found C 64.11, H 5.80, N 5.55.
as a colourless oil. TLC (CH2Cl2/MeOH, 9:1): Rf ϭ 0.30. [α]D
ϭ
ϩ30.0 (c ϭ 2.0; CHCl3); 1H NMR (250 MHz, CDCl3): δ ϭ 1.28
(d, 3Jγ,β ϭ 6.2 Hz, 3 H, γ-CH3), 1.45 (s, 9 H, C4H9), 3.22 (d, 2J6,6Ј
ϭ
10.5 Hz, 1 H, 6-H), 3.40 (d, 2J6Ј,6 ϭ 10.5 Hz, 1 H, 6Ј-H), 3.50Ϫ3.64
(m, 2 H, 5-H, 4-H), 3.72 (s, 3 H, OCH3), 3.95 (m, 3 H, 3-H, NH2),
4.22 (dq, 3Jβ,α ϭ 3.0, 3Jβ,γ ϭ 5.6 Hz, 1 H, β-CH), 4.30 (d, 3Jα,NH ϭ
O-3,4,6-Tri-O-benzyl-2-deoxy-2-nitro-α-D-galactopyranose (8) and
2
10.3 Hz, 1 H, α-CH), 4.42Ϫ4.58 (m, 5 H, OCH2Ph), 4.72 (d, J ϭ
(5Z)-5-Ethylidene-3-phenylhydantoin (9): A saturated solution of
NH3 in anhydrous CH3OH (5 mL) was added to a stirred suspen-
sion of 6 (100 mg, 0.14 mmol) in anhydrous CH3OH (10 mL) at
room temp. and stirring was continued 2 h. The solvent was evapor-
ated under reduced pressure and the residue was chromatographed
on silica gel using petroleum ether/ethyl acetate (4:1) as eluent to
give 8 (60 mg, 63%) as a colourless oil and 9 (25 mg, 26%) as a
white solid.
11.3 Hz, 1 H, OCH2Ph), 4.83 (m, 2 H, 2-H, 1-H), 5.20 (d, 3JNH,α ϭ
10.0 Hz, 1 H, NH), 7.16Ϫ7.33 (m, 15 H, Ar-H) ppm. MS
(MALDI): 687 [M ϩ Na]ϩ. C37H48N2O9 (664.8): calcd. C 66.85,
H 7.28, N 4.21; found C 67.00, H 7.50, N 3.95.
N-(Methoxycarbonylamino)-O-(3,4,6-tri-O-benzyl-2-deoxy-2-meth-
oxycarbonylamino-2-nitro-α-D-galactopyranosyl)-L-threonine
Methyl Ester (12). ؊ From 11: Compound 11 (664 mg, 1 mmol)
Eur. J. Org. Chem. 2003, 1009Ϫ1021
1014

O-Glycosyl Amino Acids via 2-Nitrogalactal Concatenation
FULL PAPER
was dissolved in dry Et2O (20 mL) and HCl/Et2O (10 mL) was ad-
ded and the mixture stirred at room temp. for 15 min (TLC:
CH2Cl2/MeOH, 10%). Concentration under reduced pressure and
then re-evaporation with toluene (3 ϫ 10 mL) gave the hydro- OCH2Ph), 4.98 (d, JNH,2 ϭ 11.5 Hz, 1 H, NH), 7.25Ϫ7.34 (m, 15
chloride salt of the diamino derivative, which was subjected imme-
diately for further reaction. A solution of diamino derivative CH3), 51.6, 52.4 (2 OCH3), 59.3 (α-C), 69.3 (C-6), 70.5 (C-5), 72.1
(2s, 6 H, 2 OCH3), 3.98 (m, 2 H, 3-H, α-CH), 4.06 (dq, 3Jβ,α ϭ 3.2,
3Jβ,γ ϭ 6.4 Hz, 1 H, β-CH), 4.30Ϫ4.60 (m, 6 H, OCH2Ph, 2-H),
3
4.88 (d, J1,2 ϭ 3.6 Hz, 1 H, 1-H), 4.70 (d, 2J ϭ 12.1 Hz, 1 H,
3
H, Ar-H) ppm. 13C NMR (150.8 MHz, [D6]DMSO): δ ϭ 18.4 (γ-
(664 mg, 1 mmol) and DMAP (cat.) in dry CH2Cl2/pyridine
(25 mL, 4:1) was cooled to 0 °C, then diphosgene (69 µL) was ad-
ded by syringe and the mixture warmed up to room temp. After
stirring for 30 min, methanol (25 mL) was added and the mixture
was stirred further for 5 min. The solvents were then removed in
vacuo, the residue was dissolved in Et2O, then water was added
with vigorous stirring. The layers were separated and the aqueous
phase was extracted with Et2O. The combined organic phases were
dried (MgSO4), filtered, concentrated in vacuo, and purified by
flash chromatography (CH2Cl2/MeOH, 98:2) to furnish 12 (0.51 g,
75%) as a yellow foam. TLC (CH2Cl2/MeOH, 9:1): Rf ϭ 0.28.
[α]D ϭ ϩ49.2 (c ϭ 0.65; CHCl3). 1H NMR (250 MHz, CDCl3):
δ ϭ 1.27 (d, 3Jγ,β ϭ 6.4 Hz, 3 H, γ-CH3), 3.50Ϫ3.60 (m, 2 H, 6-H,
(β-C, C-4), 73.2 (2 OCH2Ph), 74.7 (C-3), 77.8 (OCH2Ph, C-2),
100.4 (C-1), 127.7, 127.8, 127.9, 128.0, 128.4, 128.5, 128.7 138.3,
138.4, 138.9 (C-Ar), 157.1 (CO2Me), 175.0 (HNCOOMe) ppm. MS
(MALDI): m/z ϭ 645 [M ϩ Na]ϩ. C34H42N2O9 (622.7): calcd. C
65.58, H 6.80, N 4.50; found C 65.74, H 6.72, N 4.44.
N-(Methoxycarbonylamino)-O-(3,4,6-tri-O-acetyl-2-deoxy-2-meth-
oxycarbonylamino-α-D-galactopyranosyl)-L-threonine Methyl Ester
(14): Compound 12 (200 mg, 0.29 mmol) was dissolved in meth-
anol/acetic acid (1:1, 8 mL) and Pd/C (0.1 g, 10% Pd) was sus-
pended in the solution. This mixture was stirred for 12 h under H2
at room temp. After complete disappearance of the starting mat-
erial (TLC: CH2Cl2/MeOH, 9:1), the catalyst was filtered off and
all the solvents were evaporated. The residue of O-unprotected
material was treated with pyridine/acetic anhydride (3:2, 10 mL)
and stirred at room temp. for 12 h. All volatiles were evaporated
and the product purified by flash chromatography (petroleum
ether/ethyl acetate, 1:1) to give 14 (132 mg, 85%) as a colourless
oil. TLC (CH2Cl2/MeOH, 95:5): Rf ϭ 0.3. [α]D ϭ ϩ55.9 (c ϭ 0.22;
CHCl3); 1H NMR (250 MHz, CDCl3): δ ϭ 1.28 (d, 3Jγ,β ϭ 6.1 Hz,
3 H, γ-CH3), 1.93, 1.98, 2.09 (3s, 9 H, 3 Ac), 3.62, 3.66, 3.69 (3s, 9
H, 3 OCH3), 4.03 (m, 2 H, 6-H, 6Ј-H), 4.11Ϫ4.21 (m, 3 H, 5-H, 4-
H, β-CH), 4.34 (dd, 3Jα,β ϭ 1.7, 3Jα,NH ϭ 9.7 Hz, 1 H, α-CH), 4.88
3
6Ј-H), 3.68 and 3.72 (2s, 9 H, 3 OCH3), 3.90 (dd, J5,6 ϭ 6.5,
3J5,6Ј ϭ 6.7 Hz, 1 H, 5-H), 3.98 (m, 1 H, 4-H), 4.18 (m, 1 H, β-
CH), 4.33Ϫ4.56 (m, 8 H, α-CH, OCH2Ph, 3-H, 2-H), 4.72 (d, 2J ϭ
3
12.1 Hz, 1 H, OCH2Ph), 4.86 (d, J1,2 ϭ 3.7 Hz, 1 H, 1-H), 4.94
3
3
(d, JNH,α ϭ 11.4 Hz, 1 H, NH), 5.30 (d, JNH,2 ϭ 10.5 Hz, 1 H,
NH), 7.26Ϫ7.40 (m, 15 H, Ar-H) ppm. MS (MALDI): m/z ϭ 703
[M ϩ Na]ϩ. C36H44N2O11 (680.7): calcd. C 63.52, H 6.51, N 4.12;
found C 63.87, H 6.66, N 3.95.
From 13: A solution of 13 (311 mg, 0.50 mmol) in dry CH2Cl2/
pyridine (12.5 mL, 4:1) was cooled to 0 °C, diphosgene (34.50 µL)
was added via syringe, and the mixture was warmed to room temp.
After stirring for 30 min, methanol (12.50 mL) was added and the
mixture was stirred further for 5 min. The solvents were evaporated
in vacuo, the residue was dissolved in diethyl ether, and then water
was added with stirring. The layers were separated and the aqueous
layer was extracted with diethyl ether. The combined organic layers
were dried (MgSO4), filtered, concentrated in vacuo, and then re-
evaporated with toluene. The residue was purified by flash chroma-
tography (CH2Cl2/MeOH, 95:5) to furnish 12 (0.30 g, 88%) as a
yellow foam.
3
3
3
(dd, J3,4 ϭ 3.0, J3,2 ϭ 11.5 Hz, 1 H, 3-H), 5.01 (dd, J2,1 ϭ 2.8,
3J2,3 ϭ 11.5, J2,N,H ϭ 9.4 Hz, 1 H, 2-H), 5.06 (d, 1 H, JNH,α
ϭ
3
3
3
10.1 Hz, NH), 5.32 (d, J1,2 ϭ 2.7 Hz, 1 H, 1-H), 5.69 (d, 1 H,
3JNH,2 ϭ 9.4 Hz, NH) ppm. MS (MALDI): m/z ϭ 559 [M ϩ Na]ϩ.
C21H32N2O14 (536.5): calcd. C 47.01, H 6.01, N 5.22; found C
47.03, H 6.17, N 4.91.
N-(Phenylaminocarbonyl)-O-(3,4,6-tri-O-acetyl-2-deoxy-2-methoxy-
carbonylamino-α-D-galactopyranosyl)-L-threonine Methyl Ester
(15): A mixture of 13 (500 mg, 0.80 mmol) and phenyl isocyanate
(119 mg, 1.00 mmol) in dioxane (15 mL) was heated under reflux
for 2 h. The solvent was evaporated under reduced pressure and
the residue was dissolved in methanol/acetic acid (1:1, 10 mL) and
Pd/C (0.10 g, 10% Pd) was suspended in the solution. This mixture
was stirred for 12 h under H2 at room temp. After complete disap-
pearance of the starting material (TLC: CH2Cl2/MeOH, 95:5), the
catalyst was filtered off and all the solvents were evaporated. The
residue was treated with pyridine/acetic anhydride (3:2, 10 mL) and
stirred at room temp. for 12 h. All volatiles were evaporated and the
product purified by flash chromatography (petroleum ether/ethyl
acetate, 1:1) to give 15 (320 mg, 67%) as a yellow foam. TLC
(CH2Cl2/MeOH, 95:5): Rf ϭ 0.3. [α]D ϭ ϩ30.8 (c ϭ 0.26; CHCl3).
O-(3,4,6-Tri-O-benzyl-2-deoxy-2-methoxycarbonylamino-α-
D-ga-
lactopyranosyl)- -threonine Methyl Ester (13): A solution of 11
L
(664 mg, 1.00 mmol) in dry CH2Cl2/pyridine (25 mL, 4:1) was co-
oled to 0 °C, diphosgene (69 µL) was added via syringe, and the
mixture warmed to room temp. After stirring for 30 min, methanol
(25 mL) was added and the mixture stirred for 5 min. The solvents
were removed in vacuo, the residue was dissolved in diethyl ether,
and then water was added with stirring. The layers were separated
and the aqueous phase was extracted with diethyl ether. The com-
bined organic phases were dried (MgSO4), filtered, concentrated in
vacuo. The residue was dissolved in dry diethyl ether (20 mL), HCl/
Et2O (10 mL) was added, and the mixture stirred at room temp.
for 2 h. The solvents were evaporated under reduced pressure, fol-
lowed by re-evaporation with toluene. The residue was dissolved in
CH2Cl2 followed by addition of saturated aqueous NaHCO3 with
vigorous stirring. The layers were separated and the aqueous phase
was extracted with CH2Cl2. The combined organic phases were
dried (MgSO4), filtered, concentrated under reduced pressure, and
purified by flash chromatography (CH2Cl2/MeOH, 95:5) to furnish
13 (0.51 g, 82%) as a yellow oil. TLC (CH2Cl2/MeOH, 9:1): Rf ϭ
0.28. [α]D ϭ ϩ62.0 (c ϭ 0.3, CHCl3). 1H NMR (250 MHz, CDCl3):
3
1H NMR (250 MHz, CDCl3): δ ϭ 1.33 (d, Jγ,β ϭ 6.3 Hz, 3 H, γ-
CH3), 1.95, 1.98, 2.10 (3s, 9 H, 3 Ac), 3.60, 3.66 (2s, 6 H, 2 OCH3),
4.00 (m, 2 H, 6-H, 6Ј-H), 4.12Ϫ4.29 (m, 3 H, 5-H, 4-H, β-CH),
3
3
3
4.68 (d, Jα,NH ϭ 8.7 Hz, 1 H, α-CH), 4.90 (d, J3,1 ϭ 3.4, J3,2
ϭ
3
3
3
11.3 Hz, 1 H, 3-H), 4.95 (ddd, J2,1 ϭ 2.9, J2,3 ϭ 11.4, J2,NH
ϭ
9.3 Hz, 1 H, 2-H), 5.09 (d, 3JNH,α ϭ 9.6 Hz, 1 H, NH), 5.29 (br. d,
3J1,2 ϭ 3.7 Hz, 1 H, 1-H), 6.18 (d, 3JNH,2 ϭ 9.3 Hz, 1 H, NH), 7.00
(t, J ϭ 7.3 Hz, 1 H, Ar-H), 7.25 (t, J ϭ 7.7 Hz, 2 H, Ar-H), 7.38
(d, J ϭ 7.9 Hz, 2 H, Ar-H), 7.65 (s, 1 H, NHPh) ppm. MS
(MALDI): m/z ϭ 620 [M ϩ Na]ϩ. C26H35N3O13 (597.6): calcd. C
52.26, H 5.90, N 7.03; found C 52.21, H 6.23, N 6.87.
3
δ ϭ 1.32 (d, Jγ,β ϭ 6.4 Hz, 3 H, γ-CH3), 1.93 (br. s, 2 H, NH2),
O-(2-Amino-3,4,6-tri-O-benzyl-2-deoxy-α-D-galactopyranosyl)-N-
3.38 (m, 1 H, 6-H), 3.55 (m, 3 H, 6Ј-H, 4-H, 5-H), 3.69 and 3.70
(fluorenyl-9-methoxycarbonyl)-L-serine tert-Butyl Ester (16): 4b
Eur. J. Org. Chem. 2003, 1009Ϫ1021
1015

G. A. Winterfeld, A. I. Khodair, R. R. Schmidt
FULL PAPER
(1.66 g, 2.00 mmol) was dissolved in ethanol (15 mL) and trans-
2 H, NH2) 4.18Ϫ4.80 (m, 11 H, Fmoc-CH, Fmoc-CH2, OCH2Ph,
ferred to a hydrogenolysis vessel. Platinized Raney-Ni T4 catalyst α-CH, 2-H), 5.08 (d, 3J1,2 ϭ 2.9 Hz, 1 H, 1-H), 6.98 (br. d, 3JNH,α ϭ
was freshly prepared as described previously[7] and the material ob-
tained from Raney nickel/aluminium alloy (2 g) was suspended in
ethanol (15 mL). A homogeneous suspension of this catalyst
(15 mL) was added to the reaction vessel and the suspension
shaken under H2 for 48 h at ambient temp. and pressure. The cata-
lyst was filtered off and the solvent evaporated. The residue was
purified by column chromatography (CH2Cl2/MeOH, 95:5) to fur-
nish 16 (1.50 g, 94%) as a colourless oil, which was immediately
used in the next step. TLC (petroleum ether/ethyl acetate, 1:1): Rf ϭ
0.30. [α]D ϭ ϩ12.0 (c ϭ 1.0; CHCl3). 1H NMR (250 MHz, CDCl3):
10.7 Hz, 1 H, NH), 7.18Ϫ7.90 (m, 24 H, Ar-H, COOH) ppm. MS
(MALDI): m/z ϭ 781 [M ϩ Na]ϩ. C45H46N2O9 (758.8): calcd. C
71.22, H 6.11, N 3.69; found C 71.42, H 5.86, N 3.47.
N-(Fluorenyl-9-methoxycarbonyl)-O-(3,4,6-tri-O-benzyl-2-tert-but-
oxycarbonylamino-2-deoxy-α-D-galactopyranosyl)-L-serine
(19):
BocON (0.30 g, 1.17 mmol) was added to a mixture of 18 (1.04 g,
1.40 mmol) and triethylamine (0.80 mL, 5.70 mmol) in water
(10 mL) and dioxane (10 mL). The mixture was stirred at room
temp. for 3 h, diluted with water (100 mL), and then extracted with
diethyl ether (2 ϫ 20 mL) and ethyl acetate (2 ϫ 20 mL). The aque-
ous phase was acidified with 1  HCl and extracted with dichloro-
methane (3 ϫ 20 mL). All the organic extracts were combined,
dried (MgSO4), and concentrated. The residue was purified by col-
umn chromatography (CH2Cl2/MeOH, 95:5) to furnish 19 (0.84 g,
84%) as a white foam. TLC (CH2Cl2/MeOH, 9:1): Rf ϭ 0.5. [α]D ϭ
ϩ68.2 (c ϭ 2.0; CHCl3). 1H NMR (250 MHz, [D6]DMSO): δ ϭ
1.38 (s, 9 H, C4H9), 3.54Ϫ3.71 (m, 4 H, 6-H, 6Ј-H, 5-H, 4-H), 3.92
(m, 4 H, β-CH2, 3-H, Fmoc-CH), 4.14 (m, 3 H, Fmoc-CH2, α-
CH), 4.37Ϫ4.61 (m, 7 H, OCH2Ph, 2-H), 4.89 (m, 2 H, 1-H, Boc-
NH), 6.00 (br. s, 1 H, Fmoc-NH), 6.80 (br. s, 1 H, COOH),
7.26Ϫ7.72 (m, 23 H, Ar-H) ppm. 13C NMR (62.8 MHz, CDCl3):
δ ϭ 28.3, 28.4 (CMe3), 47.3 (Fmoc-CH2), 52.0 (α-C), 54.5 (β-C),
67.1 (C-6), 68.6 (Fmoc-CH), 70.1 (C-5, OCH2Ph), 72.5 (C-4), 73.3
(C-3) 73.5 (OCH2Ph), 74.7 (OCH2Ph), 78.7 (OCH2Ph), 79.4
(CMe3), 81.7 (C-2), 98.9 (C-1), 120.0, 125.9, 127.1, 127.5, 127.62,
127.7, 127.8, 128.1, 128.2, 128.3, 128.4, 137.9, 138.7, 141.2, 144.0
(C-Ar), 156.0 (CO2H), 158.5 (Fmoc-CO), 170.3 (Boc-CO) ppm.
MS (MALDI): m/z ϭ 881 [M ϩ Na]ϩ. C50H54N2O11 (858.97):
calcd. C 69.91, H 6.34, N 3.26; found C 69.68, H 6.45, N 3.12.
3
2
δ ϭ 1.58 (s, 9 H, C4H9), 3.44 (dd, J6,5 ϭ 3.5, J6,6Ј ϭ 10.5 Hz, 1
3
2
H, 6-H), 3.52 (dd, J6Ј,5 ϭ 2.3, J6Ј,6 ϭ 10.5 Hz, 1 H, 6Ј-H), 3.70
(m, 2 H, 4-H, 5-H), 4.06Ϫ4.33 (m, 5 H, β-CH2, 3-H, NH2),
4.44Ϫ5.00 (m, 11 H, Fmoc-CH, Fmoc-CH2, OCH2Ph, α-CH, 2-H,
3
1-H), 6.12 (d, JNH,α ϭ 8.5 Hz, 1 H, NH), 7.28Ϫ7.88 (m, 23 H,
Ar-H) ppm. MS (MALDI): m/z ϭ 837 [M ϩ Na]ϩ.
O-(2-Amino-3,4,6-tri-O-benzyl-2-deoxy-α-
D-galactopyranosyl)-N-
(tert-butoxycarbonyl)- -serine tert-Butyl Ester (17): 4a (1.45 g,
L
2.00 mmol) was dissolved in ethanol (15 mL) and transferred to a
hydrogenolysis vessel. Platinized Raney-Ni T4 catalyst was freshly
prepared as described previously[46] and the material obtained from
Raney nickel/aluminium alloy (2 g) was suspended in ethanol
(15 mL). A homogeneous suspension of this catalyst (15 mL) was
added to the reaction vessel and the suspension shaken under H2
for 48 h at ambient temp. and pressure. The catalyst was filtered
off and the solvent evaporated. The residue was purified by column
chromatography (CH2Cl2/MeOH, 98:2) to furnish 17 (1.16 g, 84%)
as a colourless oil, which was immediately used in the next step.
TLC (CH2Cl2/MeOH, 9:1): Rf ϭ 0.50. [α]D ϭ ϩ60.7 (c ϭ 0.3;
CHCl3). 1H NMR (250 MHz, CDCl3): δ ϭ 1.43 (s, 18 H, 2 C4H9),
N-(Fluorenyl-9-methoxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-tert-but-
oxycarbonylamino-2-deoxy-α-D-galactopyranosyl)-L-serine (21): 19
3.26 (dd, 3J6,5 ϭ 3.5, 2J6,6Ј ϭ 10.5 Hz, 1 H, 6-H), 3.42 (dd, 3J6Ј,5
ϭ
2
2.3, J6Ј,6 ϭ 10.5 Hz, 1 H, 6Ј-H), 3.56Ϫ3.62 (m, 2 H, 4-H, 5-H),
3.85Ϫ4.00 (m, 5 H, β-CH2, 3-H, NH2), 4.34Ϫ4.55 (m, 6 H,
(0.10 g, 0.115 mmol) was dissolved in methanol/acetic acid (1:1,
5 mL) and Pd/C (0.04 g, 10% Pd) was suspended in the solution.
This mixture was stirred for 3 h under H2. After complete disap-
pearance of the starting material (TLC: CH2Cl2/MeOH, 8:2), the
catalyst was filtered off and the solvents were evaporated. The res-
idue was suspended in dry CH2Cl2 (25 mL) and cooled to 0 °C.
Et3N (0.06 mL) and FmocCl (0.035 g, 0.115 mmol) were added
dropwise simultaneously. The stirring was continued for 14 h at
room temp., then the reaction mixture was diluted with CH2Cl2
(25 mL), washed with 0.5  HCl (3 ϫ 10 mL) and H2O, dried
(MgSO4), filtered, and the solvents evaporated to dryness. The res-
idue consisting of crude 20 was treated with pyridine/acetic anhyd-
ride (2:1, 6 mL) and stirred at room temp. for 12 h. The solvents
were evaporated and the product purified by column chromato-
graphy (CH2Cl2/MeOH, 19:1) to furnish 21 (0.043 g, 48%) as a
white foam. TLC (CH2Cl2/MeOH, 19:1): Rf ϭ 0.5. [α]D ϭ ϩ6.5
(c ϭ 0.2; CHCl3). 1H NMR (250 MHz, CDCl3): δ ϭ 1.39, 1.41 (2s,
9 H, C4H9), 1.90, 2.01, 2.10 (3s, 9 H, 3 Ac), 3.62 (m, 1 H, 6-H),
3.90 (m, 2 H, 6Ј-H, 5-H), 4.00Ϫ4.50 (m, 7 H, β-CH2, 4-H, α-CH,
Fmoc-CH, Fmoc-CH2), 4.75 (m, 1 H, 3-H), 4.88 (m, 1 H, 2-H),
2
OCH2Ph, α-CH, 2-H), 4.70 (d, J ϭ 11.5 Hz, 1 H, OCH2Ph), 4.81
2
3
(d, J ϭ 11.1 Hz, 1 H, OCH2Ph), 4.85 (d, J1,2 ϭ 2.8 Hz, 1 H, 1-
3
H), 5.48 (d, JNH,α ϭ 8.5 Hz, 1 H, NH), 7.22Ϫ7.40 (m, 15 H, Ar-
H) ppm. 13C NMR (150.8 MHz, CDCl3): δ ϭ 28.0 (CMe3), 28.3
(CMe3), 51.2 (α-C), 54.6 (β-C), 68.7 (C-6), 69.8 (C-5), 70.1 (1C,
OCH2Ph), 71.8 (OCH2Ph), 72.4 (C-4), 73.5 (C-OCH2Ph) 74.5 (C-
3), 79.8 (C-2), 80.9 (CMe3), 82.2 (CMe3), 101.0 (C-1), 127.5, 127.7,
127.8, 128.0, 128.2, 128.4, 128.5 138.0, 138.6 (C-Ar), 155.5 (ester-
CO), 169.4 (Boc-CO) ppm. MS (MALDI): m/z ϭ 715 [M ϩ Na]ϩ.
C39H52N2O9 (692.84): calcd. C 67.61, H 7.56, N 4.04; found C
67.67, H 7.23, N 4.01.
O-(2-Amino-3,4,6-tri-O-benzyl-2-deoxy-α-
D-galactopyranosyl)-N-
(fluorenyl-9-methoxycarbonyl)- -serine (18): 16 (1.78 g, 2.22 mmol)
L
was dissolved in a mixture of trifluoroacetic acid and dichlorome-
thane (50 mL, 1:1), the solution was stirred at room temp. for 6 h,
then the solvents were evaporated and re-evaporated with toluene
several times. The residue was dissolved in CH2Cl2 followed by ad-
dition of saturated aqueous NaHCO3 with vigorous stirring. The
layers were separated and the aqueous phase was extracted with
CH2Cl2. The combined organic phases were dried (MgSO4), fil-
tered, concentrated under reduced pressure, and purified by column
chromatography (CH2Cl2/MeOH, 95:5) to furnish 18 (1.62 g, 96%)
as a white foam, which was used immediately in the next step. TLC
3
5.10 (br. d, JNH,α ϭ 8.0 Hz, 1 H, NH), 5.36 (m, 2 H, 1-H, NH),
6.50 (br. s, 1 H, COOH), 7.23Ϫ7.72 (m, 8 H, Ar-H) ppm. MS
(MALDI): m/z ϭ 737 [M ϩ Na]ϩ.
O-(2-Amino-2-deoxy-α-
D-galactopyranosyl)-N-(tert-butoxycarb-
onyl)- -serine tert-Butyl Ester (22): 17 (0.69 g, 1.00 mmol) was dis-
L
(CH2Cl2/MeOH, 9:1): Rf ϭ 0.4. [α]D ϭ ϩ76.2 (c ϭ 0.32; CHCl3). solved in methanol/acetic acid (1:1, 20 mL) and Pd/C (0.36 g, 10%
3
1H NMR (250 MHz, [D6]DMSO): δ ϭ 3.38 (dd, J6,5 ϭ 3.5,
Pd) was suspended in the solution. This mixture was stirred for 3 h
2J6,6Ј ϭ 10.5 Hz, 1 H, 6-H), 3.54 (m, 2 H, 6Ј-H, 5-H), 3.70 (m, 1 under H2. After complete disappearance of the starting material
H, 4-H), 3.74Ϫ4.15 (m, 3 H, β-CH2, 3-H), 4.00 (br. t, J ϭ 7.1 Hz, (TLC: CH2Cl2/MeOH, 4:1), the catalyst was filtered off, and the
1016
Eur. J. Org. Chem. 2003, 1009Ϫ1021

O-Glycosyl Amino Acids via 2-Nitrogalactal Concatenation
FULL PAPER
solvents were evaporated. The residue was purified by column chro-
matography (CH2Cl2/MeOH, 9:1) to furnish 22 (0.32 g, 76%) as a
white foam, which was used immediately in the next step. TLC
constant stirring. The external temperature of Ϫ10 °C was used to
maintain the internal temperature in the range of 10Ϫ20 °C during
the addition. Once the addition was complete, the solution was
(CH2Cl2/MeOH, 4:1): Rf ϭ 0.25. [α]D ϭ ϩ91.7 (c ϭ 0.3; CHCl3). cooled to Ϫ50 °C at which point a precipitate was formed. A solu-
1H NMR (250 MHz, CD3OD): δ ϭ 1.41, 1.43 (2s, 18 H, 2 C4H9), tion of galactal[23] 25 (30 g, 0.053 mol) in acetic anhydride (120 mL)
2.72 (br. s, 3 H, NH2, 6-OH), 3.06 (m, 3 H, 6-H, 6Ј-H, 5-H), was added over a period of 10Ϫ15 min, and the mixture was stirred
3.58Ϫ3.88 (m, 6 H, β-CH2, 3-H, 4-H, 3-OH, 4-OH), 4.06 (br. s, 1
at this temperature for 0.5 h. The temperature was then raised to
Ϫ22 °C, at which point the reaction medium became clear. The
3
H, α-CH), 4.32 (br. m, 1 H, 2-H), 4.84 (d, J1,2 ϭ 3.0 Hz, 1 H, 1-
H), 5.60 (d, 1 H, 3JNH,α ϭ 7.9 Hz, NH) ppm. 13C NMR reaction mixture was poured into iced water (500 mL), brine
(62.8 MHz, CDCl3): δ ϭ 28.6, 28.9 (2 CMe3), 52.1 (α-C), 56.0 (β-
C), 62.50 (C-6), 69.9 (C-5), 71.2 (C-3, C-4), 80.6 (CMe3), 83.0 (C- ethyl ether (3 ϫ 300 mL). The combined organic extracts were
2, CMe3), 101.1 (C-1), 156.2 (CO2C4H9), 170.4 (Boc-CO) ppm. MS dried with sodium sulfate and the solvents removed by co-evapora-
(MALDI): m/z ϭ 445 [M ϩ Na]ϩ. C18H34N2O9 (422.5): calcd. C tion with toluene. The crude intermediate 2-nitrogalactopyranose
(250 mL) was added, and the aqueous layer was extracted with di-
51.17, H 8.11, N 6.63; found C 51.30, H 8.32, N 6.59.
was dissolved in dichloromethane (50 mL) and was added slowly
to an ice-cold, stirred solution of triethylamine (22 mL, 0.159 mol)
in dichloromethane (50 mL). The cooling bath was removed and
stirring continued for 20 min. The organic phase was washed with
2  HCl solution and dried with sodium sulfate. Evaporation of the
solvents and column chromatographic purification (toluene/ethyl
acetate, 98:2) of the residue furnished 26 as a light-yellow oil (27 g,
N-(tert-Butoxycarbonyl)-O-[2-deoxy-2-(fluorenyl-9-methoxy-
carbonylamino)-α-D-galactopyranosyl]-L-serine tert-Butyl Ester (23):
22 (0.25 g, 0.5 mmol) was suspended in dry CH2Cl2 (25 mL) and
cooled to 0 °C. Et3N (0.25 mL) and FmocCl (0.15 g, 0.5 mmol)
were added dropwise simultaneously. Stirring was continued for
14 h at room temp., then the reaction mixture was diluted with
CH2Cl2 (25 mL), washed with 0.5  HCl (3 ϫ 10 mL) and H2O,
dried (MgSO4), and filtered. The residue was purified by column
chromatography (CH2Cl2/MeOH, 9:1) to furnish 23 (0.25 g, 77%)
as a white foam, which was used immediately in the next step. TLC
(CH2Cl2/MeOH, 4:1): Rf ϭ 0.35. [α]D ϭ ϩ68.7 (c ϭ 0.3; CHCl3).
1H NMR (250 MHz, CDCl3): δ ϭ 1.40, 1.42 (2s, 18 H, 2 C4H9),
2.52 (br. s, 1 H, 6-OH), 3.42 (m, 1 H, 6-H), 3.87 (m, 6 H, 6Ј-H, 3-
H, 5-H, β-CH2, 4-H), 4.03Ϫ4.18 (m, 5 H, 2-H, α-CH, Fmoc-CH,
Fmoc-CH2), 4.36 (m, 2 H, 3-OH, 4-OH), 4.85 (d, 3J1,2 ϭ 2.9 Hz,
84%). TLC (toluene): Rf ϭ 0.46. [α]D ϭ Ϫ7.5 (c ϭ 1.2; CHCl3); 1H
3
NMR (600 MHz, CDCl3): δ ϭ 1.05 (s, 9 H, C4H9), 3.81 (t, J4,3
ϭ
4.6, 3J4,5 ϭ 4.6 Hz, 1 H, 4-H), 4.14Ϫ4.22 (m, 2 H, 6-H, 6Ј-H), 4.51
(d, 2J ϭ 12.0 Hz, 1 H, OCH2Ph), 4.57Ϫ4.59 (m, 2 H, 5-H,
OCH2Ph), 4.68 (d, 2J ϭ 10.9 Hz, 1 H, OCH2Ph), 4.75 (d, 2J ϭ
3
10.9 Hz, 1 H, OCH2Ph), 4.78 (d, J3,4 ϭ 4.5 Hz, 1 H, 3-H),
7.14Ϫ7.34 (m, 15 H, Ar-H), 7.40Ϫ7.41 (m, 2 H, Ar-H), 7.60Ϫ7.61
(m, 3 H, Ar-H), 7.77 (s, 1 H, 1-H) ppm. 13C NMR (150.8 MHz,
CDCl3): δ ϭ 26.9 (CMe3), 61.5 (C-6), 67.4, 71.9 (C-3), 73.1, 74.6
(C-4), 80.1 (C-5), 95.7 (C-2), 127.4Ϫ128.6 129.7, 129.8, 131.3,
133.3, 133.5, 135.6 136.9, 138.1 (C-Ar), 154.6 (C-1) ppm. FAB-MS:
(positive mode, NBOH/NaI matrix): m/z ϭ 610 [M ϩ H]ϩ, 632 [M
ϩ Na]ϩ.
3
1 H, 1-H), 5.22 (br. d, JNH,α ϭ 8.2 Hz, 1 H, NH), 5.45 (br. d,
3JNH,2 ϭ 8.0 Hz, 1 H, NH), 7.24Ϫ7.75 (m, 8 H, Ar-H) ppm. 13C
NMR (62.8 MHz, CDCl3): δ ϭ 27.9, 28.2 (2 CMe3), 47.0 (Fmoc-
CH2), 51.7 (α-C), 54.4 (β-C), 62.6 (C-6), 67.3 (C-5), 68.9 (Fmoc-
CH), 69.8 (C-3, C-4), 80.1 (CMe3), 82.6 (C-2, CMe3), 98.7 (C-1),
119.8, 125.1, 127.0, 127.6, 141.2 (C-Ar), 155.4 (CO2C4H9), 157.5
(Fmoc-CO), 169.6 (Boc-CO) ppm. MS (MALDI): m/z ϭ 667 [M
ϩ Na]ϩ. C33H44N2O11 (644.7): calcd. C 61.48, H 6.88, N 4.35;
found C 61.35, H 6.47, N 4.06.
N-(tert-Butoxycarbonyl)-O-(3,4-di-O-benzyl-6-O-tert-butyldi-
phenylsilyl-2-deoxy-2-nitro-α-D-galactopyranosyl)-L-serine tert-Bu-
tyl Ester (27a): Nitrogalactal 26 (0.120 g, 0.20 mmol) and serine
derivative 3a (0.077 g, 0.30 mmol) were dried under high vacuum
and dissolved in dry toluene (2 mL) under argon. Freshly activated
˚
molecular sieves (3 A, 0.2 g) were introduced and the mixture
O-[3,4,6-Tri-O-acetyl-2-deoxy-2-(fluorenyl-9-methoxy-carbonyl
stirred for 1 h, then potassium tert-butoxide solution (1  in THF,
20 µL) was added, and stirring was continued for 10 min. Acetic
acid (20 µL) was used to acidify the reaction mixture, the molecular
sieves was filtered off and all solvents were evaporated. The residue
was purified by column chromatography (toluene/ethyl acetate,
20:1) to furnish 27a as colourless oil (0.166 g, 97%). TLC (toluene/
amino)-α-
D-galactopyranosyl]-N-(tert-butoxycarbonyl)-
L-serine tert-Butyl Ester (24): 23 (0.10 g, 0.16 mmol) was treated
with pyridine/acetic anhydride (2:1, 6 mL) and stirred at room
temp. for 12 h. All volatiles were evaporated and the product puri-
fied by column chromatography (CH2Cl2/MeOH, 19:1) to furnish
24 (0.12 g, 97%) as a white foam. TLC (CH2Cl2/MeOH, 19:1): Rf ϭ
0.45. [α]D ϭ ϩ62.7 (c ϭ 0.3; CHCl3). 1H NMR (250 MHz, CDCl3):
δ ϭ 1.43, 1.45 (2s, 18 H, 2 C4H9), 1.92, 2.04, 2.13 (3 s, 9 H, 3 Ac),
3.86 (m, 2 H, 6-H, 6Ј-H), 4.00Ϫ4.50 (m, 9 H, β-CH2, 5-H, 2-H, 4-
1
ethyl acetate, 10:1): Rf ϭ 0.50. [α]D ϭ ϩ55.2 (c ϭ 5.0; CHCl3). H
NMR (600 MHz, CDCl3): δ ϭ 1.05 (s, 9 H, C4H9), 1.43, 1.46 (2s,
18 H, 2 C4H9), 3.73Ϫ3.84 (m, 5 H, 5-H, 6-H, 6Ј-H, β-CH, βЈ-CH),
3.99 (d, 3J4,3 ϭ 2.7 Hz, 1 H, 4-H), 4.29Ϫ4.30 (m, 1 H, α-CH), 4.41
3
H, α-CH, Fmoc-CH, Fmoc-CH2), 4.86 (d, J1,2 ϭ 3.6 Hz, 1 H, 1-
3
3
2
(dd, J3,2 ϭ 10.7, J3,4 ϭ 2.9 Hz, 1 H, 3-H), 4.46 (d, J ϭ 11.0 Hz,
3
3
H), 4.96 (d, JNH,α ϭ 8.2 Hz, 1 H, NH), 5.10 (dd, J2,1 ϭ 3.3,
3J2,3 ϭ 11.3 Hz, 1 H, 2-H), 5.38 (br. d, 3JNH,2 ϭ 8.0 Hz, 1 H, NH),
7.24Ϫ7.76 (m, 8 H, Ar-H) ppm. 13C NMR (62.8 MHz, CDCl3):
δ ϭ 20.6 (3 Ac), 27.9, 28.3 (2 CMe3), 47.1 (Fmoc-CH), 49.6 (α-C),
54.4 (β-C), 61.7 (C-6), 67.2 (C-5), 68.6 (Fmoc-CH2), 69.6 (C-3, C-
4), 80.2 (CMe3), 82.8 (C-2, CMe3), 98.9 (C-1), 120.0, 125.0, 125.1,
127.1, 127.7, 141.3, 143.7, 143.9 (C-Ar), 155.2 (CO2C4H9), 156.0
(Fmoc-CO), 169.1, 170.2, 170.3, 170.6 (Boc-CO, 3 Ac) ppm. MS
(MALDI): m/z ϭ 793 [M ϩ Na]ϩ. C39H50N2O14 (770.8): calcd. C
60.77, H 6.54, N 3.63; found C 60.35, H 6.79, N 3.64.
3
1 H, OCH2Ph), 4.73Ϫ4.83 (m, 3 H, OCH2Ph), 4.94 (dd, J2,1
ϭ
4.2, 3J2,3 ϭ 10.7 Hz, 1 H, 2-H), 5.21Ϫ5.23 (m, J2,1 ϭ 4.2 Hz, 2 H,
1-H, NH), 7.15Ϫ7.43 (m, 16 H, Ar-H), 7.60Ϫ7.63 (m, 4 H, Ar-H)
ppm. 13C NMR (150.8 MHz, CDCl3): δ ϭ 26.9 (CMe3), 27.9
(CMe3), 28.3 (CMe3), 54.1 (α-C), 61.8 (C-6), 69.3 (β-C), 71.5 (C-
5), 73.1, 73.2 (C-4), 75.0, 75.1, 79.9, 82.7 (C-3), 84.1 (C-2), 96.8 (C-
1), 127.7Ϫ137.9 (C-Ar), 155.20 (CO2C4H9), 168.8 (Boc-CO) ppm.
FAB-MS: (positive mode, NBOH/NaI matrix): m/z ϭ 893 [M ϩ
Na]ϩ. C48H62N2O11Si·(H2O)0.5 (880.1): calcd. C 65.51, H 7.21, N
3.18; found C 65.51, H 7.11, N 3.06.
3
1,5-Anhydro-3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-2-deoxy-2-
nitro-D-lyxo-hex-1-enitol (26): Concentrated nitric acid (24 mL,
N-(tert-Butoxycarbonyl)-O-(3,4-di-O-benzyl-6-O-tert-butyldi-
0.38 mol) was added dropwise to acetic anhydride (240 mL) with
phenylsilyl-2-deoxy-2-nitro-α-
D-galactopyranosyl)-L-threonine tert-
Eur. J. Org. Chem. 2003, 1009Ϫ1021
1017

G. A. Winterfeld, A. I. Khodair, R. R. Schmidt
FULL PAPER
3
Butyl Ester (27c): Nitrogalactal 26 (13 g, 21.3 mmol) and threonine 1H NMR (600 MHz, CDCl3): δ ϭ 1.22Ϫ1.27 (m, Jγ,β ϭ 6.5 Hz,
derivative 3c (7.1 g, 25.6 mmol) were dried under high vacuum and
dissolved in dry toluene (250 mL) under argon. Potassium tert-bu-
toxide solution (1  in THF, 2.1 mL) was added and stirring con-
tinued for 2 h. Acetic acid (2 mL) was used to acidify the reaction
mixture and then all solvents were evaporated. The residue was
3 H, γ-CH3), 1.46, 1.47 (2s, 18 H, 2 C4H9), 3.53 (br. s, 1 H, 6Ј-H),
3
2
3
3.75 (dd, J6,5 ϭ 6.7, J6,6Ј ϭ 11.3 Hz, 1 H, 6-H), 3.88 (t, J5,6
ϭ
3
3
6.0, J5,6Ј ϭ 6.0 Hz, 1 H, 5-H), 3.95 (d, J3,4 ϭ 3.1 Hz, 1 H, 4-H),
4.14 (br. d, 3Jα,NH ϭ 9.6 Hz, 1 H, α-CH), 4.31 (br. d, 3Jβ,γ ϭ 6.5 Hz,
3
3
1 H, β-CH), 4.43 (dd, J3,2 ϭ 10.8, J3,4 ϭ 2.9 Hz, 1 H, 3-H), 4.51
purified by column chromatography (toluene/ethyl acetate, 20:1) to (d, 2J ϭ 11.5 Hz, 1 H, OCH2Ph), 4.73Ϫ4.78 (m, 2 H, OCH2Ph),
furnish 27c as colourless oil (18.3 g, 97%). TLC (toluene/ethyl acet-
4.86 (d, 2J ϭ 11.4 Hz, 1 H, OCH2Ph), 5.00 (dd, 3J2,1 ϭ 4.1, 3J2,3 ϭ
ate, 10:1): Rf ϭ 0.57. [α]D ϭ ϩ53.3 (c ϭ 5.0; CHCl3). 1H NMR 10.8 Hz, 1 H, 2-H), 5.10 (d, JNH,α ϭ 9.6 Hz, 1 H, NH), 5.48 (d,
3
(600 MHz, CDCl3): δ ϭ 1.04 (s, 9 H, C4H9), 1.15 (s, 3 H, γ-CH3), 3J1,2 ϭ 4.1 Hz, 1 H, 1-H), 7.25Ϫ7.36 (m, 10 H, Ar-H) ppm. 13C
1.45, 1.49 (2s, 18 H, 2 C4H9), 3.68 (dd, 3J6Ј,5 ϭ 5.9, 2J6Ј,6 ϭ 10.0 Hz,
NMR (150.8 MHz, CDCl3): δ ϭ 18.2 (γ-CH3), 28.0 (CMe3), 28.3
1 H, 6Ј-H), 3.74 (dd, J6,5 ϭ 7.6, J6,6Ј ϭ 10.3 Hz, 1 H, 6-H), 3.88 (CMe3), 58.0 (α-C), 61.8 (C-6), 71.5 (C-5), 72.7 (OCH2Ph), 73.3
3
2
3
3
(br. t, J5,6 ϭ 6.8, J5,6Ј ϭ 6.8 Hz, 1 H, 5-H), 4.05Ϫ4.06 (m, 2 H,
(OCH2Ph), 74.8 (C-4), 75.2 (β-C), 75.3 (CMe3), 80.0 (CMe3), 82.8
(C-3), 84.3 (C-2), 95.6 (C-1), 128.1Ϫ137.5 (C-Ar), 156.1
3
α-CH, 4-H), 4.23Ϫ4.24 (br. d, 1 H, β-CH), 4.43 (dd, J3,2 ϭ 10.6,
3J3,4 ϭ 2.9 Hz, 1 H, 3-H), 4.50 (d, J ϭ 11.1 Hz, 1 H, OCH2Ph), (CO2C4H9), 170.5 (Boc-CO) ppm. FAB-MS: (positive mode,
2
4.73 (d, J ϭ 11.0 Hz, 1 H, OCH2Ph), 4.77 (d, J ϭ 11.0 Hz, 1 H, NBOH/NaI matrix): m/z ϭ 669 [M ϩ Na]ϩ, 819 [M ϩ 2Na ϩ I]ϩ.
2
2
2
3
OCH2Ph), 4.83 (d, J ϭ 11.0 Hz, 2 H, OCH2Ph), 4.93 (dd, J2,1
ϭ
C33H46N2O11·(H2O)0.5 (664.7): calcd. C 60.44, H 7.22, N 4.27;
found C 60.40, H 7.04, N 4.45.
4.1, 3J2,3 ϭ 10.6 Hz, 1 H, 2-H), 4.96 (d, 3JNH,α ϭ 9.7 Hz, 1 H, NH),
3
5.32 (d, J1,2 ϭ 4.4 Hz, 1 H, 1-H), 7.18Ϫ7.39 (m, 16 H, Ar-H),
7.60Ϫ7.61 (m, 4 H, Ar-H) ppm. 13C NMR (150.8 MHz, CDCl3):
δ ϭ 18.9 (γ-CH3), 26.8 (CMe3), 27.9 (CMe3), 28.4 (CMe3), 58.6 (α-
C), 61.9 (C-6), 71.4 (C-5), 73.1, 73.3 (4-C), 75.2, 75.2 (C-3), 75.5,
79.9, 82.7 (β-C), 84.4 (C-2), 96.7 (C-1), 127.6Ϫ138.0 (Ar-C), 156.2
(CO2C4H9), 169.2 (Boc-CO) ppm. FAB-MS: (positive mode,
N-(tert-Butoxycarbonyl)-O-{O-[methyl (5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-
syl)onate]-(2Ǟ6)-(3,4-di-O-benzyl-2-deoxy-2-nitro-α-
nosyl)-
-serine tert-Butyl Ester (30a): Sialyl donor[47,48] 29 (0.773 g,
D
-glycero-α-
D
-galacto-non-2-ulopyrano-
D-galactopyra-
L
1.26 mmol) and O-glycosylserine 28a (0.400 g, 0.63 mmol) were
carefully dried under high vacuum and dissolved in dry propionitr-
ile (15 mL). The solution was cooled to Ϫ60 °C under nitrogen and
the reaction activated by addition of TMSOTf (23 µL, 0.13 mmol).
The reaction was warmed to Ϫ40 °C over a period of 1 h and then
quenched by addition of solid sodium bicarbonate. The reaction
mixture was filtered, concentrated, and the residue purified by flash
chromatography on silica gel (100 g; toluene/ethanol eluent, 10:1)
to give 30a as a colourless foam (0.332 g, 47%). TLC (toluene/ethyl
NBOH/NaI
matrix):
m/z
ϭ
907
[M
ϩ
Na]ϩ.
C49H64N2O11Si·(H2O)2 (921.17): calcd. C 63.90, H 7.44, N 3.04;
found C 63.74, H 7.35, N 3.26.
N-(tert-Butoxycarbonyl)-O-(3,4-di-O-benzyl-2-deoxy-2-nitro-α-
D-
galactopyranosyl)- -serine tert-Butyl Ester (28a): 27a (0.500 g,
L
0.570 mmol) was dissolved in dry THF (5 mL) and treated with
tetrabutylammonium fluoride (1  in THF, 1.10 mL) and acetic
acid (0.031 mL, 0.550 mmol). The reaction was stirred for 2 h at
1
acetate, 5:1): Rf ϭ 0.39. [α]D ϭ ϩ33.0 (c ϭ 1.0; CHCl3). H NMR
room temp. and then diluted with ethyl acetate and water. The or- (600 MHz, CDCl3): δ ϭ 1.47, 1.49 (2s, 18 H, 2 C4H9), 1.88Ϫ2.14
2
3
ganic layer was separated, dried with sodium sulfate, and concen-
trated. The residue was purified on silica gel (30 g; toluene/ethyl
(m, 16 H, 4 Ac, NHAc, 3bЈ-H), 2.60 (dd, J3,3Ј ϭ 13.0, J3,4
ϭ
4.8 Hz, 1 H, 3b-H), 3.49 (dd, 3J6Ј,5 ϭ 6.2, 2J6Ј,6 ϭ 9.5 Hz, 1 H, 6aЈ-
acetate eluent, 3:1) to give 28a as colourless foam (0.351 g, 97%). H), 3.67 (s, 3 H, OCH3), 3.79Ϫ3.83 (m, 2 H, 6a-H, βЈ-CH),
TLC (toluene/ethyl acetate, 2:1): Rf ϭ 0.38. [α]D ϭ ϩ77.5 (c ϭ 1.0; 3.93Ϫ3.97 (m, 3 H, 4a-H, 5a-H, β-CH), 4.05Ϫ4.15 (m, 3 H, 5b-H,
1
3
CHCl3). H NMR (600 MHz, CDCl3): δ ϭ 1.45, 1.47 (2s, 18 H, 2 6b-H, 9bЈ-H), 4.28Ϫ4.30 (m, 2 H, α-H, 9b-H), 4.42 (dd, J3,2
ϭ
3
2
3
C4H9), 3.54 (d, J6Ј,5 ϭ 5.3, J6,6Ј ϭ 11.4 Hz, 1 H, 6Ј-H), 3.75 (d,
11.0, J3,4 ϭ 2.6 Hz, 1 H, 3a-H), 4.59 (d, 2J ϭ 11.0 Hz, 1 H,
3J6,5 ϭ 6.8, J6,6Ј ϭ 11.4 Hz, 1 H, 6-H), 3.87Ϫ3.89 (m, 2 H, β-H,
OCH2Ph), 4.73 (s, 2 H, OCH2Ph), 4.83 (d, 2J ϭ 11.0 Hz, 1 H,
2
5-H), 3.93Ϫ3.95 (m, 2 H, β-H, 4-H), 4.29Ϫ4.30 (m, 1 H, α-CH), OCH2Ph), 4.88Ϫ4.90 (m, 1 H, 4b-H), 4.96 (dd, 3J2,1 ϭ 4.2, 3J2,3
ϭ
4.37 (dd, J3,2 ϭ 10.6, J3,4 ϭ 2.9 Hz, 1 H, 3-H), 4.53 (d, 2J ϭ
10.7 Hz, 1 H, 2a-H), 5.13Ϫ5.14 (m, JNH,α ϭ 9.8 Hz, 1 H, Ser-
3
3
3
3
11.4 Hz, 1 H, OCH2Ph), 4.73Ϫ4.77 (m, 2 H, OCH2Ph), 4.88 (d, NH), 5.30Ϫ5.33 (m, J1,2 ϭ 4.2 Hz, 3 H, 1a-H, 7b-H, Neu5Ac-
2J ϭ 11.4 Hz, 1 H, OCH2Ph), 4.99 (dd, 3J2,1 ϭ 4.4, 3J2,3 ϭ 10.9 Hz, NH), 5.39Ϫ5.40 (m, 1 H, 8b-H), 7.25Ϫ7.34 (m, 10 H, Ar-H) ppm.
3
3
1 H, 2-H), 5.31 (d, J1,2 ϭ 4.4 Hz, 1 H, 1-H), 5.56 (d, JNH,α
ϭ
13C NMR (150.8 MHz, CDCl3): δ ϭ 20.7Ϫ23.2 (5 Ac), 27.9
(CMe3), 28.4 (CMe3), 37.7 (C-3b), 49.4 (C-5b), 58.2 (OCH3), 54.2
(α-C), 62.3 (C-9b), 63.1 (C-6a), 67.3 (C-7b), 68.5 (C-8b), 68.6 (C-
7.9 Hz, 1 H, NH), 7.25Ϫ7.38 (m, 10 H, Ar-H) ppm. 13C NMR
(150.8 MHz, CDCl3): δ ϭ 27.9 (CMe3), 28.3 (CMe3), 54.4 (α-C),
61.9 (C-6), 70.4 (β-C), 71.6 (C-5), 72.8 (OCH2Ph), 73.3 (OCH2Ph), 4b), 68.7, 69.0 (β-C), 69.8, 72.5 (C-5a), 72.7, 72.9 (C-4a), 74.7
74.8 (C-4), 75.1 (CMe3), 79.9 (CMe3), 82.7 (C-3), 84.0 (C-2), 97.2 (OCH2Ph), 74.9 (OCH2Ph), 82.6 (C-3a), 83.7 (2 CMe3), 83.9 (C-
(C-1), 128.2Ϫ128.6, 137.1, 137.5 (C-Ar), 154.20 (CO2C4H9), 168.8
2a), 96.6 (C-1a), 98.6 (C-2b), 127.7Ϫ128.5, 137.3, 138.1, (C-Ar),
(Boc-CO) ppm. FAB-MS: (positive mode, NBOH/NaI matrix): m/ 155.4, 167.8, 168.8, 170.2, 170.6, 170.9 (8 CO) ppm. FAB-MS:
z ϭ 655 [M ϩ Na]ϩ, 805 [M ϩ 2 Na ϩ I]ϩ.
(positive mode, NBOH/NaI matrix): m/z ϭ 1128 [M ϩ Na]ϩ.
C52H71N3O23·(H2O)2 (1142.2): calcd. C 54.68, H 6.61, N 3.68;
found C 54.80, H 6.65, N 3.53.
N-(tert-Butoxycarbonyl)-O-(3,4-di-O-benzyl-2-deoxy-2-nitro-α-D-
galactopyranosyl)-L-threonine tert-Butyl Ester (28c): 27c (15.0 g,
16.9 mmol) was dissolved in dry THF (100 mL) and treated with
tetrabutylammonium fluoride (1  in THF, 51.0 mL) and acetic
acid (1.4 mL, 25.4 mmol). The mixture was stirred for 2 h at room
temp. and then diluted with ethyl acetate and water. The organic
layer was separated, dried with sodium sulfate, and concentrated.
The residue was purified on silica gel (600 g; toluene/ethyl acetate
eluent, 3:1) to give 28c as a colourless oil (8.9 g, 97%). TLC (tolu-
N-(tert-Butoxycarbonyl)-O-{O-[methyl (5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-
syl)onate]-(2Ǟ6)-(3,4-di-O-benzyl-2-deoxy-2-nitro-α-
nosyl)-
-threonine tert-Butyl Ester (30c): Sialyl donor[47,48] 29
d
-glycero-α-
D
-galacto-non-2-ulopyrano-
D-galactopyra-
L
(7.565 g, 12.37 mmol) and O-glycosylserine 28c (4.000 g,
6.19 mmol) were dried carefully under high vacuum and dissolved
˚
in dry propionitrile (60 mL) together with freshly activated 3-A mo-
ene/ethyl acetate, 10:1): Rf ϭ 0.60. [α]D ϭ ϩ56.3 (c ϭ 3.0; CHCl3). lecular sieves (7 g). The solution was cooled to Ϫ60 °C under nitro-
1018 Eur. J. Org. Chem. 2003, 1009Ϫ1021

O-Glycosyl Amino Acids via 2-Nitrogalactal Concatenation
FULL PAPER
gen and the reaction activated by the addition of TMSOTf
(0.224 mL, 1.24 mmol). The reaction was stirred for 1.5 h at this
temperature and then quenched by the addition of triethylamine.
The reaction mixture was filtered, concentrated, and the residue
passed through a short column of silica gel (toluene/ethanol, 5:1).
This procedure gave a crude product that was separated on Bi-
oBeads SX-3 (toluene) to give a disaccharide-containing fraction,
which was purified finally on silica gel (100 g; toluene/ethanol elu-
ent, 10:1) to give 30c as a colourless foam (4.5 g, 65%). TLC (tolu-
ene/ethyl acetate, 5:1): Rf ϭ 0.50. [α]D ϭ ϩ34.8 (c ϭ 1.0; CHCl3).
1H NMR (600 MHz, CDCl3): δ ϭ 1.32Ϫ1.33 (m, 3 H, γ-CH3),
1.47, 1.48 (2s, 18 H, 2 C4H9), 1.89Ϫ2.14 (m, 16 H, 4 Ac, NHAc,
21.1, 23.2, 23.4 (6 Ac), 28.0 (CMe3), 28.3 (CMe3) 37.8 (C-3b), 48.7
(C-2a), 49.4 (C-5b), 52.8 (OCH3), 54.4 (α-C), 62.3 (C-9b), 63.1 (C-
6a), 67.4 (C-7b), 68.5 (β-C), 68.8 (C-8b), 69.1 (C-4b), 69.8 (C-5a),
71.5, 72.4 (C-4a), 72.7 (C-6b), 74.1 (OCH2Ph), 76.8 (OCH2Ph),
77.1 (3a-C), 80.3 (CMe3), 82.3 (CMe3), 98.7 (C-2b), 99.0 (C-1a),
127.3Ϫ128.4, 138.1, 138.7 (C-Ar), 155.3, 167.8, 169.8, 169.9, 170.1,
170.2, 170.6, 170.9 (9 CO) ppm. FAB-MS: (positive mode, NBOH/
NaI matrix): m/z ϭ 1140 [M ϩ Na]ϩ.
N-(tert-Butoxycarbonyl)-O-{O-[methyl (5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-
syl)onate]-(2Ǟ6)-(2-acetamido-3,4-di-O-benzyl-2-deoxy-α-
pyranosyl)- -threonine tert-Butyl Ester (31c): Nitroglycoside 30c
D
-glycero-α-
D
-galacto-non-2-ulopyrano-
D
-galacto
2
3
L
3bЈ-H), 2.56 (dd, J3,3Ј ϭ 12.9, J3,4 ϭ 4.6 Hz, 1 H, 3b-H), 3.44
(dd, 3J6Ј,5 ϭ 5.3, 2J6Ј,6 ϭ 9.1 Hz, 1 H, 6aЈ-H), 3.68 (s, 3 H, OCH3),
3.94Ϫ4.00 (m, 3 H, 4a-H, 5a-H, 6a-H), 4.03Ϫ4.10 (m, 4 H, 5b-H,
(2.000 g, 1.79 mmol) was dissolved in ethanol (40 mL) and then
transferred to a hydrogenation vessel. Platinized Raney-Ni T4 cata-
lyst was freshly prepared as described previously[46] and the mat-
erial obtained from Raney nickel/aluminium alloy (6 g) was sus-
pended in ethanol (45 mL). A homogeneous suspension of this
catalyst (40 mL) was added to the reaction vessel and the suspen-
sion shaken under hydrogen for 12 h at ambient temp. and pressure.
The catalyst was filtered off and the solvent evaporated. The res-
idue was dissolved in pyridine/acetic anhydride (2:1, 45 mL) and
stirred for 3 h. Removal of the volatiles and column chromato-
graphic purification (toluene/ethanol, 10:1) gave 31c as a colourless
foam (1.710 g, 85%). TLC (chloroform/methanol, 9:1): Rf ϭ 0.59.
[α]D ϭ ϩ38.6 (c ϭ 2.0; CHCl3). 1H NMR (600 MHz, CDCl3): δ ϭ
1.30Ϫ1.32 (m, 3 H, γ-CH3), 1.47, 1.49 (2s, 18 H, 2 C4H9),
1.88Ϫ2.14 (m, 19 H, 4 Ac, 2 NHAc, 3bЈ-H), 2.56 (dd, 2J3,3Ј ϭ 12.9,
3J3,4 ϭ 4.8 Hz, 1 H, 3b-H), 3.45Ϫ4.42 (m, 1 H, 6aЈ-H), 3.57 (dd,
3
2
6b-H, 9bЈ-H, α-CH), 4.26 (dd, J9,8 ϭ 2.6, J9,9Ј ϭ 12.4 Hz, 1 H,
3
3
9b-H), 4.34 (br. d, Jβ,γ ϭ 6.7 Hz, 1 H, β-CH), 4.43 (dd, J3,2
ϭ
10.8, J3,4 ϭ 2.8 Hz, 1 H, 3a-H), 4.58 (d, 2J ϭ 11.1 Hz, 1 H,
3
OCH2Ph), 4.72 (s, 2 H, OCH2Ph), 4.83 (d, 2J ϭ 11.1 Hz, 1 H,
OCH2Ph), 4.87Ϫ4.91 (m, 1 H, 4b-H), 4.95 (dd, 3J2,1 ϭ 4.2, 3J2,3
ϭ
3
10.8 Hz, 1 H, 2a-H), 4.99 (d, JNH,α ϭ 9.8 Hz, Thr-NH), 5.13 (br.
d, JNH,5 ϭ 9.5 Hz, 1 H, Neu5Ac-NH), 5.31 (br. d, 3J7,8 ϭ 8.3 Hz,
3
3
1 H, 7b-H), 5.36Ϫ5.39 (m, 1 H, 8b-H), 5.39 (d, J1,2 ϭ 4.3 Hz, 1
H, 1a-H), 7.26Ϫ7.35 (m, 10 H, Ar-H) ppm. 13C NMR (150.8 MHz,
CDCl3): δ ϭ 18.8 (γ-CH3), 20.7, 20.8, 21.1, 23.2 (5 Ac), 28.0
(CMe3), 28.4 (CMe3) 37.6 (C-3b), 49.4 (C-5b), 52.8 (OCH3), 58.7
(α-C), 62.3 (C-9b), 63.1 (C-6a), 67.3 (C-7b), 68.5 (C8b), 68.9 (C-
4b), 69.9 (C-5a), 72.6 (C-6b), 72.9 (C-4a), 73.1 (2 OCH2Ph), 74.8
(2 CMe3), 75.1 (C-3a), 76.1 (β-C), 82.6, 84.1 (C-2a) 84.2, 96.8 (C-
1a), 98.6 (C-2b), 127.7Ϫ128.5, 137.3, 138.1 (C-Ar), 155.6, 167.9,
169.8, 170.1, 170.2, 170.9 (8 CO) ppm. MS (FAB): m/z ϭ 1142 [M
ϩ Na]ϩ. C53H73N3O23·(H2O) (1138.2) calcd.: C 55.93, H 6.64, N
3.69; found C 55.76, H 6.32, N 3.45.
3
3J3,2 ϭ 10.9, J3,4 ϭ 2.4 Hz, 1 H, 3a-H), 3.65 (m, 3 H, OCH3),
3.88Ϫ3.94 (m, 3 H, 4a-H, 5a-H, 6a-H), 4.05Ϫ4.13 (m, 5 H, 5b-H,
3
2
6b-H, 9bЈ-H, α-CH, β-CH), 4.27 (dd, J9,8 ϭ 2.3, J9,9Ј ϭ 12.4 Hz,
1 H, 9b-H), 4.50 (d, 2J ϭ12.1 Hz, 1 H, OCH2Ph), 4.63 (d, 2J ϭ
11.3 Hz, 1 H, OCH2Ph), 4.71Ϫ4.78 (m, 3 H, 1a-H, 2a-H,
2
N-(tert-Butoxycarbonyl)-O-{O-[methyl (5-acetamido-4,7,8,9-tetra-
OCH2Ph), 4.85Ϫ4.87 (m, 1 H, 4b-H), 4.96 (d, J ϭ 11.3 Hz, 1 H,
3
O-acetyl-3,5-dideoxy-
syl)onate]-(2Ǟ6)-(2-acetamido-3,4-di-O-benzyl-2-deoxy-α-
pyranosyl)- -serine tert-Butyl Ester (31a): Nitroglycoside 30a
D
-glycero-α-
D
-galacto-non-2-ulopyrano-
OCH2Ph), 5.03 (d, JNH,α ϭ 9.0 Hz, 1 H, Thr-NH), 5.18 (d,
3JNH,5 ϭ 9.3 Hz, 1 H, Neu5Ac-NH), 5.31Ϫ5.36 (m, 2 H, 7b-H, 8b-
D
-galacto
3
L
H), 5.64 (d, JNH,2 ϭ 9.8 Hz, 1 H, GalNHAc-NH), 7.24Ϫ7.38 (m,
10 H, Ar-H) ppm. 13C NMR (150.8 MHz, CDCl3): δ ϭ 18.6 (γ-
CH3), 20.7, 20.8, 20.8, 21.0, 21.4, 23.2, 23.5 (6 Ac), 28.1 (CMe3),
28.3 (CMe3) 37.6 (C-3b), 48.6 (C-2a), 49.4 (C-5b), 52.7 (OCH3),
58.7 (α-C), 62.2 (C-9b), 63.4 (C-6a), 67.4 (C-7b), 68.6 (C-8b), 69.0
(C-4b), 70.1 (C-5a), 71.5 (OCH2Ph), 72.6 (C-6b), 72.7 (C-4a), 74.1
(OCH2Ph), 76.7 (β-C), 77.4 (C-3a), 80.1 (CMe3), 82.6 (CMe3), 98.6
(C-2b), 100.6 (C-1a), 125.3Ϫ129.0 138.2, 138.7 (C-Ar), 155.7,
167.9, 169.9, 170.0, 170.2, 170.5, 170.7, 170.9 (9 CO) ppm. FAB-
MS: (positive mode, NBOH/NaI matrix): m/z ϭ 1154 [M ϩ Na]ϩ.
(0.250 g, 0.23 mmol) was dissolved in ethanol (5 mL) and then
transferred to a hydrogenation vessel. Platinized Raney-Ni T4 cata-
lyst was freshly prepared as described previously[46] and the mat-
erial obtained from Raney nickel/aluminium alloy (2 g) was sus-
pended in ethanol (15 mL). A homogeneous suspension of this
catalyst (10 mL) was added to the reaction vessel and the suspen-
sion shaken under hydrogen for 48 h at ambient temp. and pressure.
The catalyst was filtered off and the solvent evaporated. The res-
idue was dissolved in pyridine/acetic anhydride (2:1, 10 mL) and
stirred for 3 h. Removal of the volatiles and column chromato-
graphic purification (toluene/ethanol, 10:1) gave 31a as a colourless
foam (0.189 g, 75%). TLC (chloroform/methanol, 9:1): Rf ϭ 0.78.
N-(tert-Butoxycarbonyl)-O-{O-[methyl (5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-
D
-glycero-α-
D
-galacto-non-2-ulopyrano-
-galac-
-threonine tert-Butyl Ester (32): 31c (1.60 g,
1.41 mmol) was dissolved in methanol/acetic acid (21 mL, 20:1),
(dd, J6Ј,5 ϭ 7.3, J6Ј,6 ϭ 7.3 Hz, 1 H, 6aЈ-H), 3.58Ϫ3.77 (m, 5 H, 10% Pd/C (0.4 g) was added, and the mixture stirred under hydro-
[α]D ϭ ϩ34.8 (c ϭ 1.0; CHCl3). 1H NMR (600 MHz, CDCl3): δ ϭ syl)onate]-(2Ǟ6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-α-
D
1.44, 1.47 (2s, 18 H, 2 C4H9), 1.88Ϫ2.14 (m, 19 H, 4 Ac, NHAc, topyranosyl)-
L
2
3
3bЈ-H), 2.61 (dd, J3,3Ј ϭ 12.9, J3,4 ϭ 4.6 Hz, 1 H, 3b-H), 3.48
3
2
3
3
OCH3, 3a-H, β-CH), 3.80 (t, J5,6 ϭ 6.6, J5,6Ј ϭ 6.6 Hz, 1 H, 5a-
gen for 12 h. The catalyst was filtered off and the volatiles removed.
H), 3.88Ϫ3.98 (m, 2 H, β-CH, 6a-H), 3.96 (br. s, 1 H, 4a-H), The residue was dissolved in pyridine/acetic anhydride (15 mL, 2:1)
4.07Ϫ4.12 (m, 3 H, 5b-H, 6b-H, 9bЈ-H), 4.30Ϫ4.31 (m, 2 H, α-H,
and stirred for 12 h at room temp. The reaction mixture was con-
9b-H), 4.47 (d, 2J ϭ12.1 Hz, 1 H, OCH2Ph), 4.61 (d, 2J ϭ 11.3 Hz, centrated and the crude residue purified by flash chromatography
1 H, OCH2Ph), 4.70Ϫ4.72 (m, 2 H, 2a-H, OCH2Ph), 4.79 (d, (toluene/ethanol, 8:1) to afford 32 as colourless foam (1.46 g,
3J1,2 ϭ 3.2 Hz, 1 H, 1a-H), 4.85Ϫ4.87 (m, 1 H, 4b-H), 4.95 (d, 2J ϭ
quantitative). TLC (chloroform/methanol, 9:1): Rf ϭ 0.58. [α]D ϭ
3
11.3 Hz, 1 H, OCH2Ph), 5.24 (d, JNH,5 ϭ 8.9 Hz, 1 H, Neu5Ac-
ϩ33.8 (c ϭ 2.0; CHCl3). 1H NMR (600 MHz, CDCl3): δ ϭ 1.36
3
3
NH), 5.29 (br. d, 1 H, Ser-NH), 5.33 (br. d, J7,6 ϭ 7.8 Hz, 1 H, (d, Jγ,β ϭ 6.1 Hz, 3 H, γ-CH3), 1.46, 1.49 (2s, 18 H, 2 C4H9),
7b-H), 5.39Ϫ5.43 (m, 2 H, GalNHAc-NH, 8b-H), 7.27Ϫ7.37 (m, 1.87Ϫ2.16 (m, 25 H, 2 NHAc, 6 Ac, 3bЈ-H), 2.51 (dd, 2J3,3Ј ϭ 12.9,
10 H, Ar-H) ppm. 13C NMR (150.8 MHz, CDCl3): δ ϭ 20.7, 20.8,
Eur. J. Org. Chem. 2003, 1009Ϫ1021
3J3,4 ϭ 4.7 Hz, 1 H, 3b-H), 3.26 (dd, J6Ј,5 ϭ 5.3, J6Ј,6 ϭ 10.2 Hz,
2
3
1019

G. A. Winterfeld, A. I. Khodair, R. R. Schmidt
FULL PAPER
1 H, 6Јa-H), 3.78 (s, 3 H, OCH3), 3.85 (dd, J6,5 ϭ 3.3, J6,6Ј
3
3
ϭ
FAB-MS: (positive mode, NBOH/NaI matrix): m/z ϭ 1124 [M ϩ
10.2 Hz, 1 H, 6a-H), 4.01Ϫ4.08 (m, 3 H, 5b-H, 6b-H, 9bЈ-H), Na]ϩ, 1146 [M ϩ 2 Na Ϫ H]ϩ. C51H63N3O24·(H2O)2.5 (1147.1)
4.14Ϫ4.19 (m, 3 H, α-H, β-H, 5a-H), 4.26 (dd, 3J9,8 ϭ 2.3, 2J9,9Ј
12.4 Hz, 1 H, 9b-H), 4.59 (ddd, J2,1 ϭ 3.7, J2,3 ϭ 10.6, J2,NH
ϭ
calcd.: C 53.40, H 5.98, N 3.66; found C 53.45, H 5.68, N 3.71.
3
3
3
ϭ
N-Acetyl-
O-acetyl-3,5-dideoxy-
onate]-(2Ǟ6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-α-
pyranosyl)- -threonyl- -alanyl- -prolyl- -alanyl- -histidyl-L
L
-glycyl-
L
-seryl-O-{O-[methyl (5-acetamido-4,7,8,9-tetra-
-glycero-α- -galacto-non-2-ulopyranosyl)-
-galacto-
-glycin-
10.3 Hz, 1 H, 2a-H), 4.83Ϫ4.87 (m, 2 H, 1a-H, 4b-H), 5.04 (dd,
3
3
3J3,2 ϭ 11.3, J3,4 ϭ 3.0 Hz, 1 H, 3a-H), 5.11 (d, JNH,5 ϭ 9.6 Hz,
D
D
3
D
1 H, Neu5Ac-NH), 5.22 (d, JNH,α ϭ 9.4 Hz, 1 H, Thr-NH),
3
L
L
L
L
L
5.31Ϫ5.36 (m, 3 H, 4a-H, 7b-H, 8b-H), 5.93 (d, JNH,2 ϭ 9.6 Hz,
1 H, GalNHAc-NH) ppm. 13C NMR (150.8 MHz, CDCl3): δ ϭ
18.5 (γ-CH3), 20.3, 20.8, 21.0, 23.2, 23.3 (8 Ac), 28.1 (CMe3), 28.3
(CMe3), 37.7 (C-3b), 47.2 (C-2a), 49.4 (C-5b), 52.9 (OCH3), 58.6
(α-C), 62.3 (C-9b), 63.5 (C-6a), 67.2 (C-7b), 67.7 (C-4a), 68.1 (C-
8b), 68.4 (C-5a), 68.9 (C-4b), 69.2 (C-3a), 72.6 (C-6b), 77.3 (CMe3),
80.2 (CMe3), 82.8 (β-C), 98.5 (C-2b), 100.1 (C-1a), 155.2,
167.9Ϫ170.9 (11 CO) ppm. FAB-MS: (positive mode, NBOH/NaI
matrix): m/z ϭ 1058 [M ϩ Na]ϩ. C45H69N3O24·H2O (1054.1):
calcd. C 51.28, H 6.79, N 3.99; found C 51.33, H 6.60, N 3.81.
amide (34): Protected glycopeptide 34 (25 mg, 1.57 mmol) was as-
sembled on a semiautomatic peptide synthesiser (NovaSyn Gem,
Novabiochem) using a prefunctional resin (Tentagel S Ram Gly
Fmoc, 310 mg, loading: 0.22 mmol/g, Rapp Polymere) as the solid
phase. N,N-Dimethylformamide for peptide synthesis (Riedel-de
Hae¨n) was used as the solvent in all coupling steps, as well as for
washing. Fmoc cleavage was performed with a 1:1 mixture of
DMF/morpholine (pump speed 3.0 mL/min) and monitored at λ ϭ
360 nm. Functionalised Fmoc amino acids (Novabiochem) were
used in fivefold excess and activated with PyBOP (5 equiv.), HOBt
(5 equiv.), and NMM (10 equiv.). Couplings were monitored using
Coomassie Violett at λ ϭ 600 nm. The standard building blocks
were coupled in automatic mode. Glycosylated threonine building
block 33 was used in twofold excess and activated as described
above. Couplings were performed for 24 h. After the peptide had
been assembled, the resin was washed with DMF, acetic acid/
dichloromethane (1:1), and dichloromethane, and then dried under
high vacuum for 15 h. The peptide was cleaved from the solid phase
and partially deprotected using trifluoracetic acid/triisopropylsil-
ane/water (38:1:1, 8 mL, 3 h). The solution of the peptide was col-
lected and the resin washed with more TFA and dichloromethane.
The combined solutions were concentrated and the residue dried
by co-evaporation with toluene. The crude residue was purified on
a column of Sephadex LH-20 with dichloromethane/methanol (1:1)
as eluent, and the collected material lyophilized from 1,4-dioxane.
This procedure gave 34 (66 mg, 61%) as a colourless lyophilisate.
TLC (butanol/pyridine/acetic acid/water, 4:1:1:2): Rf ϭ 0.33. [α]D ϭ
Ϫ28.8 (c ϭ 1.0; CH3OH). 1H NMR (600 MHz, CD3OD): δ ϭ
1.28Ϫ1.32 (m, 9 H, 3 Thrγ-H, 6 Alaβ-H), 1.76 (t, 2J3Ј,3 ϭ 12.4 Hz,
1 H, 3bЈ-H), 1.80Ϫ2.15 (m, 33 H, 4 Proγ-H, 2 Proβ-H, 3 NHAc,
6 Ac), 2.21Ϫ2.30 (m, 2 H, 2 Proβ-H), 2.57 (dd, 2J3,3Ј ϭ 12.9, 3J3,4 ϭ
4.3 Hz, 1 H, 3b-H), 3.03Ϫ3.09 (m, 1 H, Hisβ-H), 3.18 (br. s, 1 H,
Hisβ-H), 3.25Ϫ3.30 (m, 1 H, 6Јa-H), 3.52Ϫ3.83 (m, 8 H, Serβ-H,
OCH3, 3 Proδ-H, Glyα-H), 3.85Ϫ3.96 (m, 7 H, 5b-H, Proβ-H, 3
N-(Fluoren-9-ylmethoxycarbonyl)-O-{O-[methyl (5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
pyranosyl)onate]-(2Ǟ6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-α-
galactopyranosyl)- -threonine (33): 32 (0.400 g, 0.386 mmol) was
D-glycero-α-D-galacto-non-2-ulo-
D-
L
dissolved in a mixture of trifluoroacetic acid and dichloromethane
(10 mL, 1:1), and the solution stirred at room temp. for 12 h. The
solvents were evaporated, the residue was dissolved together with
FmocONSu (0.260 g, 0.772 mmol) and sodium bicarbonate
(0.649 g. 7.720 mmol) in acetonitrile/water (20 mL, 1:1), and the
mixture stirred for 12 h. The solution was acidified with 2  HCl
solution (150 mL) and the aqueous layer extracted with dichloro-
methane (3 ϫ 100 mL). The combined organic extracts were dried
with sodium sulfate and the volatiles evaporated. Column chroma-
tographic purification (toluene/ethanol/acetic acid, 14:1:1) of the
residue afforded 33 as colourless foam (0.415 g, 98%). TLC (chloro-
form/methanol, 4:1): Rf ϭ 0.44. [α]D ϭ ϩ35.5 (c ϭ 2.5; CHCl3).
3
1H NMR (600 MHz, CDCl3): δ ϭ 1.31 (d, Jγ,β ϭ 5.3 Hz, 3 H, γ-
CH3), 1.87Ϫ2.16 (m, 25 H, 2 NHAc, 6 Ac, 3bЈ-H), 2.53 (dd,
3
2J3,3Ј ϭ 12.6, J3,4 ϭ 4.3 Hz, 1 H, 3b-H), 3.30Ϫ3.32 (m, 1 H, 6Јa-
H), 3.77 (s, 3 H, OCH3), 3.83 (br. t, 1 H, 6a-H), 4.00Ϫ4.10 (m, 4
H, 5a-H, 5b-H, 6b-H, 9bЈ-H), 4.13Ϫ4.53 (m, 7 H, 2a-H, β-H, 9b-
H, α-H, Fmoc-CH2, Fmoc-CH), 4.86 (br. s, 1 H, 4b-H), 5.02 (br.
3
s, 1 H, 1a-H), 5.09 (d, J3,2 ϭ 11.4 Hz, 1 H, 3a-H), 5.17Ϫ5.24 (m,
1 H, NH), 5.30Ϫ5.32 (m, 1 H, 7b-H), 5.38 (br. s, 2 H, 4a-H, 8b-
H), 5.88 (br. s, 1 H, NH), 6.16 (br. s, 1 H, NH), 7.29Ϫ7.40 (m, 4
H, Ar-H), 7.61 (d, J ϭ 7.1 Hz, 2 H, Ar-H), 7.75 (d, J ϭ 7.1 Hz, 2
H, Ar-H) ppm. 13C NMR (150.8 MHz, CDCl3): δ ϭ 18.1 (γ-CH3),
20.7, 20.8, 21.0, 23.2 (8 Ac), 37.7 (C-3b), 47.3 (C-2a), 49.4 (C-5b),
52.9 (OCH3), 59.0 (α-C), 62.4 (C-9b), 64.0 (C-6a), 67.1 (C-7b), 67.6
(C-4a), 68.2 (C-8b), 68.6 (C-5a), 68.8 (C-3a), 69.2 (C-4b), 72.6 (C-
6b), 77.6 (β-C), 98.7 (C-1a), 99.8 (C-2b), 120.0Ϫ127.8, 141.3 (C-
3
2
Glyα-H, Serβ-H, 6a-H), 4.05 (dd, J9Ј,8 ϭ 5.1, J9Ј,9 ϭ 12.3 Hz, 1
3
H, 9bЈ-H), 4.10 (d, J6,5 ϭ 10.5 Hz, 1 H, 6b-H), 4.18Ϫ4.20 (m, 2
H, 2a-H, 5a-H), 4.27 (br. d, 2J9,9Ј ϭ 12.4 Hz, 1 H, 9b-H), 4.35Ϫ4.37
(m, 3 H, Thrβ-H, Serα-H, Alaα-H), 4.50Ϫ4.55 (m, 4 H, Thrα-H,
Proα-H, Proα-H, Alaα-H), 4.67 (br. t, 3Jα,NH ϭ 7.5 Hz, 1 H, Hisα-
H), 4.80Ϫ4.81 (m, 1 H, 4b-H), 5.10Ϫ5.12 (m, 2 H, 1a-H, 4b-H),
5.31Ϫ5.35 (m, 3 H, 3a-H, 7b-H, 8b-H), 7.16 (br. s, 1 H, Hisδ-H),
7.57 (s, 1 H, Hisε-H) ppm. FAB-MS: (positive mode, NBOH/NaI
matrix): m/z ϭ 1596 [M ϩ H]ϩ.
1
Ar), 167.8Ϫ170.9 (11 CO). H NMR (600 MHz, [D6]DMSO): δ ϭ
3
1.16 (d, Jγ,β ϭ 6.4 Hz, 3 H, γ-CH3), 1.84Ϫ2.09 (m, 25 H, 6 Ac, 2
2
3
NHAc, 3bЈ-H), 2.47 (dd, J3,3Ј ϭ 12.4, J3,4 ϭ 4.6 Hz, 1 H, 3b-H),
3.19 (dd, 3J6,5 ϭ 5.3, 2J6,6Ј ϭ 9.9 Hz, 1 H, 6Јa-H), 3.72Ϫ3.76 (m, 5
N-Acetyl-
L
-glycyl-
-galacto-non-2-ulopyranosyl)onic acid]-(2Ǟ6)-(2-acet-
amido-2-deoxy-α- -galactopyranosyl)- -threonyl- -alanyl- -prolyl-
-prolyl- -alanyl- -histidyl- -glycinamide (1): Protected glycopep-
L-seryl-O-{O-[(5-acetamido-3,5-dideoxy-D-
H, α-H, OCH3, 6a-H), 3.92 (dd, 3J5,4 ϭ 10.3, 3J5,6 ϭ 10.0 Hz, 1 H, glycero-α-
D
5b-H), 3.96Ϫ4.00 (m, 2 H, 9bЈ-H, 6b-H), 4.08Ϫ4.09 (m, 1 H, 5a-
H), 4.16Ϫ4.18 (m, 2 H, 9b-H, 2a-H), 4.26Ϫ4.28 (m, 2 H, Fmoc-
D
L
L
L
L
L
L
L
CH, β-CH), 4.40 (dd, 2J ϭ 10.7, 3J ϭ 6.8 Hz, 1 H, Fmoc-CH2), tide 34 was dissolved in methanol (10 mL) and treated with aque-
4.46 (dd, 2J ϭ 10.8, 3J ϭ 6.8 Hz, 1 H, Fmoc-CH2), 4.68 (ddd,
ous sodium hydroxide solution (0.100 mL, 1 ). This solution was
3J4,3 ϭ 4.7, J4,3Ј ϭ 11.0, J4,5 ϭ 10.9 Hz, aH, 4b-H), 4.79 (d, stirred at room temperature for 3 h and then brought to pH 6 by
3
3
3J1,2 ϭ 3.8 Hz, 1 H, 1a-H), 4.98 (dd, J3,2 ϭ 11.6, J3,4 ϭ 3.3 Hz, the addition of acetic acid. All solvents were evaporated, the res-
3
3
3
3
1 H, 3a-H), 5.15 (dd, J7,6 ϭ 8.7, J7,8 ϭ 1.7 Hz, 1 H, 7b-H), 5.24
(br. s, 2 H, 8b-H, 4a-H), 7.32 (t, J ϭ 7.4 Hz, 2 H, Ar-H), 7.41 (t,
idue was redissolved in a mixture of water/THF (4 mL, 1:1), aque-
ous sodium hydroxide (4 mL, 5 m) was added, and the reaction
J ϭ 7.4 Hz, 2 H, Ar-H), 7.72 (d, J ϭ 6.6 Hz, 2 H, Ar-H), 7.89 (d, mixture stirred at room temperature for 4 h. The solution was acidi-
J ϭ 7.4 Hz, 2 H, Ar-H), 11.80Ϫ13.00 (br. m, 1 H, COOH) ppm. fied to pH ϭ 6 by the addition of acetic acid and the solvents were
1020
Eur. J. Org. Chem. 2003, 1009Ϫ1021

O-Glycosyl Amino Acids via 2-Nitrogalactal Concatenation
FULL PAPER
O. Seitz, ChemBioChem 2000, 1, 214Ϫ246.
H. Herzner, T. Reipen, M. Schultz, H. Kunz, Chem. Rev. 2000,
100, 4495Ϫ4537; and references therein.
D. Qiu, R. R. Koganty, Tetrahedron Lett. 1997, 38, 961Ϫ964;
[17]
[18]
lyophilised. The residue was purified on a column of Sephadex LH-
20 with dichloromethane/methanol (1:1) as eluent and the collected
material lyophilized from water/1,4-dioxane. This procedure gave 1
(17 mg, 81%) as a colourless lyophilisate. TLC (butanol/pyridine/
acetic acid/water, 4:1:1:2): Rf ϭ 0.09. [α]D ϭ Ϫ40.8 (c ϭ 1.0; H2O).
1H NMR (600 MHz, D2O): δ ϭ 1.18Ϫ1.27 (m, 9 H, 6 Alaβ-H, 3
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
and references therein.
`
S. Bay, O. Berthie-Vergues, V. Biberovic, D. Cantacuzene, Car-
bohydr. Res. 1997, 303, 25Ϫ31.
2
3
Thrγ-H), 1.59 (t, J3Ј,3 ϭ 12.3, J3Ј,4 ϭ 12.3 Hz, 1 H, 3bЈ-H),
1.71Ϫ1.74 (m, 1 H, Proβ-H), 1.81Ϫ1.83 (m, 1 H, Proβ-H),
1.92Ϫ2.04 (m, 11 H, 4 Proγ-H, 3 NHAc), 2.17Ϫ2.22 (m, 1 H, Proβ-
´
G. Dudziak, N. Bezay, T. Schwientek, H. Clausen, H. Kunz,
A. Liese, Tetrahedron 2000, 56, 5865Ϫ5869.
G. A. Winterfeld, Y. Ito, T. Ogawa, R. R. Schmidt, Eur. J. Org.
Chem. 1999, 1167Ϫ1171.
G. A. Winterfeld, R. R. Schmidt, Angew. Chem. 2001, 113,
2718Ϫ2721; Angew. Chem. Int. Ed. 2001, 40, 2654Ϫ2657.
J. Geiger, Diploma Thesis, University of Konstanz, 2001; J.
Geiger, R. R. Schmidt, to be published.
2
3
H), 2.26Ϫ2.29 (m, 1 H, Proβ-H), 2.63 (dd, J3,3Ј ϭ 12.2, J3,4
ϭ
4.1 Hz, 1 H, 3b-H), 3.03Ϫ3.08 (m, 2 H, 2 Hisβ-H), 3.42Ϫ3.69 (m,
7 H, 7b-H [3.43], 6aЈ-H [3.53], Proδ-H [3.53], Proδ-H [3.55], 9bЈ-H
[3.56], 4b-H [3.58], 6b-H [3.62]), 3.70Ϫ3.86 (m, 10 H, Proδ-H
[3.70], Proδ-H [3.73], 5b-H [3.74], Glyα-H [3.76], 3a-H [3.76], 9b-
H [3.79], 8b-H [3.79], Serβ-H [3.81], 6a-H [3.83], Glyα-H [3.84]),
3.88Ϫ3.98 (m, 3 H, 2 Glyα-H [3.88, 3.88], 4a-H [3.90]), 4.00Ϫ4.01
(m, 2 H, 2a-H [4.00], 5a-H [4.01]), 4.15Ϫ4.17 (m, 1 H, Alaα-H),
H. Paulsen, Angew. Chem. 1990, 102, 851Ϫ867; Angew. Chem.
Int. Ed. Engl. 1990, 29, 823Ϫ839.
T. F. Orntoft, N. Harving, N. C. Langklide, Int. J. Cancer 1990,
45, 666Ϫ672.
R. Kushima, S. Jancic, T. Hattori, Int. J. Cancer 1993, 55,
3
3
4.24Ϫ4.25 (m, 1 H, Thrβ-H), 4.29 (t, Jα,β ϭ 7.0, Jα,βЈ ϭ 7.0 Hz,
1 H, Proα-H), 4.48Ϫ4.60 (m, 5 H, Thrα-H [4.48], Alaα-H [4.48],
Hisα-H [4.52], Proα-H [4.56], Serα-H [4.58]), 4.85 (br. d, 1 H, 1a-
H), 6.98 (s, 1 H, Hisδ-H), 7.86 (br. d, 1 H, Hisε-H) ppm. 13C NMR
(150.8 MHz, D2O) (excerpt): δ ϭ 15.5 (C-Alaβ), 16.5 (C-Alaβ),
18.7 (C-Thrγ), 24.9 (2 C-Proγ), 28.0 (C-Hisβ), 28.2 (C-Proβ), 29.4
(C-Proβ), 40.4 (C-3b), 42.3 (C-Glyα), 42.6 (C-Glyα), 47.7 (C-Alaα),
47.7 (C-Proδ), 47.9 (C-Proδ), 49.7 (C-2a), 50.0 (C- Alaα), 52.0 (C-
5b), 53.6 (C-Hisα), 55.4 (C-Serα), 57.4 (C-Thrα), 58.6 (C-Proα),
60.3 (C-Proα), 61.4 (C-Serβ), 62.8 (C-9b), 64.1 (C-6a), 68.3 (C-3a),
68.5 (C-4b) 68.5 (C-7b), 68.8 (C-4a), 69.8 (C-6b), 70.2 (C-5a), 72.0
(C-8b), 76.5 (C- Thrβ), 99.1 (C-1a) ppm. FAB-MS: (positive mode,
NBOH/NaI matrix): m/z ϭ 1351.9 [M ϩ Na Ϫ H]ϩ, 1373.6 [M ϩ
2 Na Ϫ H]ϩ.
904Ϫ908.
[28]
[29]
I. Brockhausen, Biochem. Soc. Trans. 1997, 25, 871Ϫ874.
G. Ritter, P. O. Livingston, Seminars Cancer Biol. 1991, 2,
401Ϫ409.
[30]
H. F. Oettgen, L. J. Old, in: Biologic Therapy of Cancer (Eds.:
V. DeVita, S. Hellmann, S. A. Rosenberg), J. B. Lippincott
Company, Philadelphia, PA, 1991.
[31]
[32]
[33]
M. Elofson, L. A. Salvador, J. Kihlberg, Tetrahedron 1997,
53, 369Ϫ390.
S. K. George, T. Schwientek, B. Holm, C. A. Reis, H. Clausen,
J. Kihlberg, J. Am. Chem. Soc. 2001, 123, 11117Ϫ11125.
B. Liebe, H. Kunz, Angew. Chem. 1997, 109, 629Ϫ631; Angew.
Chem. Int. Ed. Engl. 1997, 36, 618Ϫ621.
B. Liebe, H. Kunz, Helv. Chim. Acta 1997, 80, 1473Ϫ1482.
S. Keil, C. Claus, W. Dippold, H. Kunz, Angew. Chem. 2001,
113, 379Ϫ382; Angew. Chem. Int. Ed. 2001, 40, 366Ϫ369.
Y. Nakahara, H. Iijima, S. Shibayama, T. Ogawa, Carbohydr.
Res. 1991, 216, 211Ϫ225.
J. B. Schwarz, S. D. Kuduk, X.-T. Chen, D. Sames, P. W. Glunz,
S. J. Danishefsky, J. Am. Chem. Soc. 1999, 21, 2662Ϫ2673.
For a recent review on glycopeptide synthesis, see: H. Herzner,
T. Reipen, M. Schulz, H. Kunz, Chem. Rev. 2000, 100,
4495Ϫ4537.
G. A. Winterfeld, Dissertation, University of Konstanz, 2000.
Part of this work has been reported in preliminary form; see
ref.[23]
P. L. Devine, I. F. C. McKenzie, BioEssays 1992, 14, 619Ϫ625.
J. R. Gum, Jr., Biochem. Soc. Trans. 1995, 23, 795Ϫ799.
J. Das, R. R. Schmidt, Eur. J. Org. Chem. 1998, 1609Ϫ1613.
The amount of β-anomer can be varied with the nature of the
base; see ref.[28]
A. A. El-Barbary, A. I. Khodair, E. B. Pedersen, J. Org. Chem.
1993, 58, 5994Ϫ5999; and references therein.
S.-F. Tan, K.-P. Ang, Y.-F. Fong, J. Chem. Soc., Perkin Trans.
2 1986, 1941Ϫ1944.
S. Nishimura, Bull. Chem. Soc. Jpn. 1959, 32, 61Ϫ64.
T. J. Martin, R. R. Schmidt, Tetrahedron Lett. 1992, 33,
6123Ϫ6126.
T. J. Martin, R. Brescello, A. Toepfer, R. R. Schmidt, Glycocon-
jugate J. 1993, 10, 16Ϫ25.
[34]
[35]
[36]
[37]
[38]
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinsch-
aft, the European Community (Grant No. HRPN-CT-2000-00001/
GLYCOTRAIN), and the Fonds der Chemischen Industrie. A. I.
K. is grateful for an Alexander von Humboldt Fellowship. We are
grateful to Prof. Dr. A. Geyer and Ms. Anke Friemel for their help
in the structural assignments by NMR experiments.
[39]
[40]
[41]
[42]
[43]
[1]
A. Varki, Glycobiology 1993, 3, 97Ϫ130.
[2]
I. Brockhausen, Biochem. Biophys. Acta 1999, 1473, 67Ϫ95.
[3]
C. Brocke, H. Kunz, Bioorg. Med. Chem. 2002, 10, 3085Ϫ3112.
[4]
H. Paulsen, W. Stenzel, Chem. Ber. 1978, 111, 2334Ϫ2347; H.
[44]
[45]
Paulsen, W. Stenzel, Chem. Ber. 1978, 111, 2348Ϫ2357.
[5]
H. Paulsen, C. Kolar, W. Stenzel, Chem. Ber. 1978, 111,
2358Ϫ2369.
[6]
H. Paulsen, C. Kolar, Chem. Ber. 1979, 112, 3190Ϫ3202.
[7]
[46]
[47]
B. Ferrari, A. A. Pavia, Carbohydr. Res. 1980, 79, C1ϪC7.
[8]
H. Paulsen, J.-P. Hölck, Carbohydr. Res. 1982, 109, 89Ϫ107.
[9]
G. Grundler, R. R. Schmidt, Liebigs Ann. Chem. 1984,
[48]
[49]
[50]
1826Ϫ1847.
[10]
W. Kinzy, R. R. Schmidt, Carbohydr. Res. 1987, 164, 265Ϫ276.
H. Paulsen, W. Ranwald, U. Weichert, Liebigs Ann. Chem.
[11]
O. Kanie, M. Kiso, A. Hasegawa, J. Carbohydr. Chem. 1988,
7, 501Ϫ506.
B. Castro, J. R. Dormoy, G. Evin, C. Selve, Tetrahedron Lett.
1975, 16, 1219Ϫ1222.
D. Hudson, J. Org. Chem. 1988, 53, 617Ϫ624.
J. Coste, D. Le-Nguyen, B. Castro, Tetrahedron Lett. 1990,
31, 205Ϫ208.
1988, 75Ϫ86.
[12]
W. Kinzy, R. R. Schmidt, Carbohydr. Res. 1989, 193, 33Ϫ47.
[13]
Y. Nakahara, H. Iijima, S. Sibayama, T. Ogawa, Tetrahedron
[51]
[52]
Lett. 1990, 31, 6897Ϫ6900.
S. Hanessian, D. Qiu, H. Prabhanjan, G. V. Reddy, B. Lau,
[14]
Can. J. Chem. 1996, 74, 1738Ϫ1747.
X.-T. Chen, D. Sames, S. J. Danishefsky, J. Am. Chem. Soc.
[15]
[53]
[54]
T. Fuchss, Dissertation, University of Konstanz, 1999.
3d is commercially available.
1998, 112, 7760Ϫ7769.
[16]
M. Elofson, L. A. Salvador, I. Kehlberg, Tetrahedron 1997,
Received September 2, 2002
53, 369Ϫ390.
[O02493]
Eur. J. Org. Chem. 2003, 1009Ϫ1021
1021