Hexafluoroacetone as a protecting and activating reagent. N- and O-glycosylation of isoserine and isocysteine *

Article abstract of DOI:10.1007/s00706-004-0248-9

Starting from HFA-protected malic and thiomalic acid a series of O- and N-glycoconjugates suitable for peptide and depsipeptide modification has been synthesized. Springer-Verlag 2004.

Full text of DOI:10.1007/s00706-004-0248-9

Monatshefte f€ur Chemie 136, 577–595 (2005)
DOI 10.1007/s00706-004-0248-9
Hexafluoroacetone
as a Protecting and Activating Reagent.
N- and O-Glycosylation of Isoserine
ꢀ
and Isocysteine
1
1;2
1
¨
Christoph Bottcher , Jan Spengler , Lothar Hennig ,
Fernando Albericio2, and Klaus Burger1;#
1
Department of Organic Chemistry, University of Leipzig, D-04103 Leipzig, Germany
2
ꢀ
Institut de Recerca Biomedica de Barcelona, Parc Cientific Barcelona,
08028 Barcelona, Spain
Received June 1, 2004; accepted (revised) August 5, 2004
Published online November 23, 2004 # Springer-Verlag 2004
Summary. Starting from HFA-protected malic and thiomalic acid a series of O- and N-glycoconjugates
suitable for peptide and depsipeptide modification has been synthesized.
Keywords. Malic acid; Thiomalic acid; Hexafluoroacetone; Isocyanates; Glycosylamines;
Glycoconjugates.
Introduction
O- and N-Glycosylation are important co- and post-translational modifications of
proteins [1–3]. Glycoconjugates play a key role in various biological recognition
processes, including cell to cell communication and adhesion of bacteria or of
viruses to cell-surface proteins [4]. Glycosylation improves certain properties
of peptides and proteins, i.a. solubility, thermal and proteolytical stability [5].
Furthermore, glycosylation influences conformation and folding [6]. This suggests
an enormous potential of glycoconjugates in drug design [7].
In glycoconjugates the carbohydrate and peptide part are linked together by
glycosidic bonds, which are potentially acid-sensitive and in some cases labile even
under moderate basic conditions [8]. In general, glycoconjugates isolated from
biological sources or produced by gentechnological methods are microheteroge-
nous. Therefore, the development of new methodologies for the assembly of
Dedicated to Prof. Dr. K. Schulze on the occasion of his 70th birthday
ꢀ
#
Corresponding author. E-mail: burger@organik.chemie.uni-leipzig.de

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C. Bottcher et al.
578
Scheme 1. Non-natural linkages in glycoconjugates
homogeneous and more robust glycopeptide mimetics by chemoselective ligation
is of current interest.
Multivalency [9] has attracted attention not only of chemists interested in drug
design. Multivalent carbohydrate ligands have advantages over their monovalent
counterparts, which generally bind their receptors weakly. The development of low
molecular weight compounds that bind strongly to carbohydrate receptors is a
challenge to medicinal chemists [10]. Cross-linking of multivalent carbohydrates
to proteins can lead to supramolecular assemblies. To study the factors governing
the strength of these cluster effects, new methodologies have to be developed for
the synthesis of oligosaccharide and glycoconjugate mimetics [11].
In this context, glycoconjugates have been synthesized where O- and N-glyco-
sidic linkages are replaced by non-natural linkages, like carbon–carbon [12],
carbon–sulfur [13], and carbon–aminooxy units [14] (Scheme 1). Alternative con-
cepts proposed by H. Kessler et al. are the replacement of the amide group of N-
glycosylated species by a retroamide subunit [15] or an ethylene isoster [16],
and by B. Imperiali et al. are the incorporation of alanine-ꢀ-hydroxylamine and
alanine-ꢀ-hydrazide [17] as asparagine surrogates. Recently, a new approach to
glycopeptide mimetics was disclosed by Y. Ichikawa et al. [18]. They replaced O-
and N-glycosidic linkages by urea-glycosyl bonds. Urea-glycosyl bonds can be
constructed from glycosyl isocyanates and the corresponding amines and vice
versa from glycosylamines and amino acid moieties with an isocyanate group in
the side chain. An alternative approach via glycosyl phosphinimines, obtained from
glycosyl azides and triphenylphosphine was described by I. Pinter et al. [19].
The ꢀ-amino-ꢁ-hydroxycarboxylate unit can be found in many naturally occur-
ring, biologically active compounds, like GABOB [20], carnitin [21], and statin
[22]. Isoserine was found as a constituent of antibiotics like edeine [23] and tatu-
min [24] produced by bacillus brevis Vm4 strains. Biological activity of antibiotics,
e.g. butirosin [25] and gentamycin [26], was improved by incorporation of isoser-
ine into strategical positions of the molecule.

Hexafluoroacetone as a Protecting and Activating Reagent
579
In the case of glycopeptides, even low molecular oligomers have been docu-
mented to have significant activities [27]. In addition, some isosters exhibit
increased stability [28]. Like ꢁ-peptides, ꢀ-peptides fold into helices, sheets, and
turns which are the main structural elements of proteins. In some instances ꢀ-
peptides exhibited higher biological activity than their parent ꢁ-peptides [29].
Furthermore, ꢀ-peptides show improved resistance to peptidases and proteases.
Consequently, conjugates comprised of a carbohydrate and a ꢀ-amino acid unit
are valuable building blocks for drug design [30].
Results and Discussion
N-Glycosylation of Isoserine
Based on Ichikawa’s results, we developed a new route for constructing urea-
glycosyl bonds via reaction of glycosylamines with isocyanate 4, using hexafluoro-
acetone (HFA) as protecting and activating reagent.
Isocyanates of type 4 are readily obtained from HFA-protected malic, citrama-
lic, and thiomalic acid in a two step procedure [31] (Scheme 2). Compounds 3 and
4 belong to a new class of preparatively highly interesting dielectrophiles where the
lactone moiety plays a double role. In the first step of the reaction sequence the
lactone acts as protective group for the ꢁ-hydroxy as well as for the ꢁ-carboxy
group. In the second step it acts as an activated ester. The isocyanate and the acid
chloride are considerably more reactive than the lactone moiety. At 4ꢁC they react
Scheme 2
Scheme 3

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C. Bottcher et al.
580
readily with weak nucleophiles, like glycosylamines 5, in a site-specific way to
give the N-glycosylated isoserine derivatives 6 (Scheme 3). During this transfor-
mation the lactone remains unaffected. Compounds 6 are obtained as anomeric
mixtures, with the ꢀ-anomers being the main products (anomeric ratios see
Scheme 3), while in the case of GlcNAc derivatives 6c and 6f the pure ꢀ-anomers
crystallize from the reaction mixture.
ꢀ-Anomers 6a-b, 6d-b, and 6e-b have been isolated from the crude reaction
mixtures by flash chromatography. However, experiments to separate the anomeric
pair 6b-b=6b-a failed so far.
On heating a solution of 6a-b in chloroform in the presence of pyridine an
intramolecular cylcocondensation reaction takes place to form 3-glycosylated 5-
hydroxy-2,4-dioxo-5,6-dihydropyrimidine 7a (Scheme 4). A similar type of ring
closure was observed when N-glycosylated HFA-protected malic acid derivatives
were treated under the same conditions [32]. While the transformation of the
glucose derivative 6a gives acceptable yields of 7a, the mannose derivative 6b
decomposed on prolonged heating.
When a solution of 6-b in dichloromethane (DCM) was stirred with trifluoro-
acetic acid (TFA, 2 equiv) a slow anomerization process was initiated. Equilib-
1
rium ratios were taken from H and 19F NMR spectra after 36 h (Scheme 5).
Scheme 4
Scheme 5

Hexafluoroacetone as a Protecting and Activating Reagent
581
Scheme 6
Compounds 6 are ꢁ-carboxy-activated species. Therefore, they can be directly
submitted to reactions with nucleophiles (Scheme 6). Cleavage of the lactone ring
is always coupled with a simultaneous deprotection of the ꢁ-hydroxy group, which
can be functionalized afterwards. Compounds 6 represent versatile building blocks
suitable for the construction of libraries of N-glycosylated peptide hybrides and
depsipeptides. Deprotection of the hydroxy groups of the carbohydrate moiety can
be achieved without anomerization applying Zemplꢁen conditions (8a=b ! 9a=b)
[33].
N-Glycosylation of Isocysteine
Analogously, HFA-protected thiomalic acid 11 can be transformed into a doubly
activated isocysteine synthone 13 via Curtius rearrangement (Scheme 7). The iso-
cyanate 13 provides access to a series of new mercapto acid derivatives. At 4ꢁC
Scheme 7

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C. Bottcher et al.
582
glycosylamines 5 add selectively across the isocyanate group. Since the reaction
sequence was started with racemic thiomalic acid, mixtures of diastereomers were
formed.
Reactivity of N-glycosylated 2,2-bis(trifluoromethyl)-1,3-oxathiolan-5-ones
14 is similar to that of N-glycosylated 2,2-bis(trifluoromethyl)-1,3-dioxolan-4-
ones 6 providing ready access to new types of peptide and depsipeptide hybrids
(14 ! 15).
O-Glycosylation of Isoserine
Isoserine represents an ꢁ-functionalized ꢀ-amino acid. Therefore, O-glycosylated
isoserine derivatives represent new interesting building blocks for the construction
of glycosylated ꢀ-peptides [34].
9-Fluorenylmethanol adds regioselectively across the isocyanate function of
compound 4 when heated in chloroform to give fully protected, carboxy group
activated isoserine derivative 16, which on treatment with an excess of allyl alcohol
readily reacts to give the corresponding allyl ester 17 (Scheme 8). Concomitantly,
the hydroxy group is deblocked, and can be glycosylated in a consecutive step, e.g.
by R. R. Schmidt’s imidate strategy [35]. Compounds 18 are O-glycosylated orthog-
onally protected ꢀ-alanine derivatives. Deblocking of the carboxyl moiety can be
achieved by applying the protocol of Kunz and Waldmann [36] to obtain O-glyco-
sylated N-Fmoc-protected isoserine derivatives 19. Via a slightly modified proce-
dure compound 4 can be transformed into N- and C-protected di- and tripeptides
Fmoc-Ise-Xaa-OMe and Fmoc-Xaa-Ise-Yaa-OMe, respectively [31a], which were
transformed into O-glycosides. However, even on application of the imidate
method the yields of the glycosylation step are low (20–30%) [37].
N-, O-Diglycosylated Isoserines
Another new type of building block, namely N-,O-diglycosylated isoserine deriva-
tive 21, is obtained on O-glycosylation of 20 (Scheme 9). Since the aminolytic ring
cleavage can also be performed with ꢀ-amino acid esters diglycosylated ꢀ-dipep-
tides become readily available by this route.
Scheme 8. i) 9-Fluorenylmethanol, CHCl3, reflux; ii) allyl alcohol, CHCl3, reflux; iii) ꢁ-D-Ac4-
˚
Glc{Gal}-O-C(¼NH)CCl3, cat. TMSOTf, MS 4 A, DCM; iv) (Ph3P)4Pd, N-methyl-aniline, THF

Hexafluoroacetone as a Protecting and Activating Reagent
583
˚
Scheme 9. i) Allyl alcohol, CHCl3, reflux; ii) ꢁ-D-Ac4-Glc-O-C(¼NH)CCl3, cat. TMSOTf, MS 4 A,
DCM
Conclusion
Starting from HFA-protected malic and thiomalic acid a series of new glycosylated
and diglycosylated monomers suitable for ꢁ-peptide, ꢀ-peptide, and depsipeptide
modification has been described.
Experimental
1
NMR spectra were recorded on Varian Gemini 300 or Bruker DRX 600 spectrometers. H chemical
shifts are referenced to residual protic solvent (CDCl3, ꢂH ¼ 7.26ppm; DMSO-d6, ꢂH ¼ 2.49ppm). 13
C
chemical shifts were referenced to the solvent signals (CDCl3, ꢂC ¼ 77.16; DMSO-d6, ꢂC ¼ 39.50 ppm)
and 19F chemical shifts to CF3COOH (external, ꢂF ¼ 0.00 ppm). IR spectra were obtained with a FT-IR
spectrometer (Genesis ATI Mattson=Unicam). Mass spectra were recorded on a Finnigan ZAB-HSQ
€
spectrometer (matrix: 3-NBA). Melting points (uncorrected) were determined on a Boetius heating
table. Optical rotation indices were measured with a Schmidt & Haensch Polartronic-D polarimeter.
Solvents were purified and dried prior to use. Reagents were used as purchased. Isocyanates 4 and 13
were prepared according to known procedures [31]. Glycosylamines 5a–5c were obtained by hydro-
genation of the corresponding glycosylazide in the presence of 10% Pd=C in ethyl acetate. Glycosy-
lamines 5d–5f were prepared starting from the peracetylated glycosylazides by Zemplꢁen-deacetylation,
benzylation, and careful hydrogenation in the presence of 10% Pd=C in methanol [38]. TLC was
performed on Silica Gel 60 F254 (Merck) with detection by UV light or phosphomolybdic
acid=ceric sulphate in 5% aqueous sulfuric acid followed by heating. Flash chromatography was per-
formed using silica gel (32–63ꢃm) with solvent systems given in the text. Elemental analyses were
performed with a CHNO-S-Rapid apparatus (Fa. Heraeus); their results agreed favourably with the
calculated values.
Synthesis of N-Glycosylated Isoserine Derivatives
Protocol 1: A solution of isocyanate 4 (1.05 equiv) in dry DCM (5 cm3 per 1 mmol) was cooled to 0ꢁC.
After addition of the corresponding ꢀ-D-glycosylamine 5 (1.00 equiv) the reaction mixture was kept
for 18 h in a fridge at 4ꢁC. Then the solvent was evaporated in vacuo and the remaining foamy solid
was purified by flash chromatography.
Protocol 2: A solution of the HFA-protected isoserine derivative 6 (1.0 equiv) in dry DCM
(20 cm3 per 1 mmol) was stirred for 36 h at room temperature after addition of TFA (2.0 equiv).
Then DCM (40 cm3 per 1 mmol) was added and the organic layer was washed with sat. NaHCO3
solution and sat. NaCl solution (15 cm3 per mmol). After drying the organic layer with MgSO4,
the solvent was removed in vacuo and the anomers were separated by flash chromatography or
crystallization.

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C. Bottcher et al.
584
Protocol 3: To a solution of an amino acid tert-butylester hydrochloride (1.2 equiv) and N-
ethylmorpholine (NEM) (1.2 equiv) in dimethylformamide (DMF) (5cm3 per 1 mmol) compound
6 (1.0 equiv) was added. The mixture was stirred at room temperature until TLC-analysis showed
complete consumption of the starting material (2–3 d). Then the solvent was evaporated in vacuo and
the residue dissolved in ethyl acetate (50 cm3 per 1 mmol). The organic layer was washed with citric
acid (10%), water, and brine (10 cm3 per 1 mmol). After drying with MgSO4, the organic solvent was
removed and the residue purified by column chromatography.
Protocol 4: Under an atmosphere of argon a solution of compound 7 (1.0 equiv) in dry methanol
(5cm3 per 0.3mmol) was stirred with NaOMe (0.1 equiv per O-acetyl group) for 3 h at room tem-
perature. After neutralization with Dowex 50X8 and filtration, the solvent was evaporated and the
residue dried in vacuo.
1-[(5S)-4-Oxo-2,2-bis(trifluoromethyl)-1,3-dioxolan-5-ylmethyl]-3-
(2,3,4,6-tetra-O-acetyl-ꢀ-D-glucopyranosyl)urea (6a-b, C21H24F6N2O13)
Ac4-ꢀ-D-Glc-NH2 (5a, 1.20 g, 3.46mmol) was reacted with isocyanate 4 (1.02 g, 3.64mmol) due to
protocol 1. Purification by flash chromatography (ethyl acetate:petroleum ether¼ 1:1, Rf(ꢀ) ¼ 0.28,
Rf(ꢁ) ¼ 0.22) provides 1.04 g (48%) 6a-b and 351 mg (16%) of a diastereomeric mixture of 6a-b and
6a-a (ratio 3:1) as foamy solids.
1
6a-b ½ꢁꢂD ¼ ꢃ4ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.3, CHCl3); H NMR (CDCl3, 300 MHz, COSY): ꢂ ¼ 2.01
(s, 3H), 2.03 (s, 3H), 2.04 (s, 3H), 2.07 (s, 3H) (OAc), 3.64 (dt, 1H, J ¼ 5.7, 14.8 Hz, CH2Ise), 3.80–
3.90 (m, 2H, CH2Ise, 5-H), 4.07 (dd, 1H, J ¼ 1.8, 12.6Hz, 6-Ha), 4.30 (dd, 1H, J ¼ 4.5, 12.6Hz, 6-Hb),
4.79 (t, 1H, J ¼ 5.7 Hz, CHIse), 4.89 (dd, 1H, J ¼ 9.6, 9.6 Hz, 2-H), 5.05 (dd, 1H, J ¼ 9.6, 9.9 Hz, 4-H),
5.11 (dd, 1H, J ¼ 9.3, 9.6 Hz, 1-H), 5.30 (dd, 1H, J ¼ 9.6, 9.6Hz, 3-H), 5.61 (t, 1H, J ¼ 6.3 Hz, NHIse),
5.88 (d, 1H, J ¼ 9.0 Hz, 1-NH) ppm; 19F NMR (CDCl3, 282MHz): ꢂ ¼ ꢃ3.18 (m, 3F), ꢃ2.84 (m, 3F)
ppm; 13C NMR (CDCl3, 75MHz, HETCOR): ꢂ ¼ 20.86 (2C), 20.93, 20.98 (OAc), 40.48 (CH2Ise),
62.36 (6-CH2), 68.86 (4-CH), 70.97 (2-CH), 73.37 (3-CH), 73.68 (5-CH), 74.81 (CHIse), 80.42 (1-CH),
98.00 (sept, J ¼ 36 Hz), 119.22 (q, J ¼ 287 Hz), 119.97 (q, J ¼ 289 Hz), 157.05, 167.25, 170.46,
170.55, 171.45, 171.55 ppm; IR (KBr): ꢄꢂ¼ 1850, 1751, 1667, 1565cmꢃ1; MS (FAB): m=z ¼ 649.1
[Mþ Na]þ, 627.1 [Mþ H]þ.
1-[(5S)-4-Oxo-2,2-bis(trifluoromethyl)-1,3-dioxolan-5-ylmethyl]-3-
(2,3,4,6-tetra-O-acetyl-D-mannopyranosyl)urea (6b-b, C21H24F6N2O13)
Ac4-ꢀ-D-Man-NH2 (5b, 993 mg, 2.86mmol) was reacted with isocyanate 4 (840 mg, 3.01 mmol)
due to protocol 1. Purification by flash chromatography (ethyl acetate:petroleum ether ¼ 3:2,
Rf(ꢀ) ¼ Rf(ꢁ) ¼ 0.35) provides 1.15 g (64%) of a diastereomeric mixture of 6b-b and 6b-a (ratio
15:1) as foamy solid.
6b-b: ½ꢁꢂD ¼ ꢃ20ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.5, CHCl3); 1H NMR (DMSO-d6, 300 MHz, COSY):
ꢂ ¼ 1.90 (s, 3H), 2.00 (s, 3H), 2.01 (s, 3H), 2.18 (s, 3H) (OAc), 3.49 (dt, 1H, J ¼ 4.8, 15.0Hz,
CH2Ise), 3.83 (m, 1H, 5-H), 3.92–3.98 (m, 2H, CH2Ise, 6-Ha), 4.10 (dd, 1H, J ¼ 5.4, 12.6Hz, 6-Hb),
4.98 (dd, 1H, J ¼ 9.9, 10.2Hz, 4-H), 5.12 (dd, 1H, J ¼ 1.2, 3.3 Hz, 2-H), 5.31–5.36 (m, 2H, CHIse
,
3-H), 5.55 (dd, 1H, J ¼ 1.2, 9.9 Hz, 1-H), 6.48 (t, 1H, J ¼ 5.7 Hz, NHIse), 6.81 (d, 1H, J ¼ 9.9 Hz, 1-
NH) ppm; 19F NMR (DMSO-d6, 282 MHz): ꢂ ¼ ꢃ2.16 (m, 3F), ꢃ1.72 (m, 3F) ppm; 13C NMR
(CDCl3, 75MHz, HETCOR): ꢂ ¼ 20.69, 20.86 (2C), 20.93 (OAc), 40.27 (CH2Ise), 62.56 (6-CH2),
65.61 (4-CH), 70.20 (2-CH), 71.65 (3-CH), 73.81 (5-CH), 74.39 (CHIse), 77.90 (1-CH), 98.31 (sept,
J ¼ 37 Hz), 118.87 (q, J ¼ 287 Hz), 119.62 (q, J ¼ 290 Hz), 156.36, 166.93, 170.16, 170.34, 170.62,
171.15 ppm; IR (KBr): ꢄꢂ¼ 1851, 1751, 1668, 1556 cmꢃ1; MS (FAB): m=z ¼ 649.0 [Mþ Na]þ, 627.1
[Mþ H]þ.

Hexafluoroacetone as a Protecting and Activating Reagent
585
1-[(5S)-4-Oxo-2,2-bis(trifluoromethyl)-1,3-dioxolan-5-ylmethyl]-3-
(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)urea (6c-b, C21H25F6N3O12)
Ac3-ꢀ-D-GlcNAc-NH2 (5c, 955 mg, 2.76mmol) was reacted with isocyanate 4 (818 mg, 2.93 mmol)
due to protocol 1. The analytically pure ꢀ-anomer crystallizes from the reaction mixture. After
filtration and careful trituration with cold DCM 1.11 g (64%) of 6c-b were obtained as color-
less crystals. Mp 224ꢁC (dec); ½ꢁꢂD ¼ þ5ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.3, DMF); 1H NMR (DMSO-d6,
300 MHz, COSY): ꢂ ¼ 1.77 (s, 3H, NHAc), 1.93 (s, 3H), 1.98 (s, 3H), 2.02 (s, 3H) (OAc), 3.42 (dt,
J ¼ 4.2, 15.3Hz, CH2Ise), 3.79 (5-H), 3.85–3.97 (m, 3H, CH2Ise, 2-H, 6-Ha), 4.19 (dd, 1H, J ¼ 4.2,
12.3Hz, 6-Hb), 4.83 (dd, 1H, J ¼ 9.6, 9.9 Hz, 4-H), 5.00 (dd, 1H, J ¼ 9.6, 9.6 Hz, 1-H), 5.09 (dd, 1H,
J ¼ 9.9, 9.9 Hz, 3-H), 5.36 (t, 1H, J ¼ 4.2 Hz, CHIse), 6.69–6.72 (m, 2H, 1-NH, NHIse), 8.05 (d, 1H, J ¼
9.3 Hz, 2-NH) ppm; 19F NMR (DMSO-d6, 282 MHz): ꢂ ¼ ꢃ2.18 (q, 3F, J ¼ 9.0 Hz), ꢃ1.57 (q, 3F,
J ¼ 9.0 Hz) ppm; 13C NMR (DMSO-d6, 75MHz, HETCOR): ꢂ ¼ 20.98, 21.05, 21.15 (OAc), 23.04
(NHAc), 39.40 (CH2Ise), 52.47 (2-CH), 62.65 (6-CH2), 69.36 (4-CH), 72.43 (5-CH), 74.02 (3-CH),
76.11 (CHIse), 80.60 (1-CH), 97.22 (sept, J ¼ 36Hz), 119.23 (q, J ¼ 286 Hz), 120.09 (q, J ¼ 289 Hz),
157.48, 167.44, 170.01, 170.21, 170.75, 171.14 ppm; IR (KBr): ꢄꢂ¼ 1849, 1745, 1659, 1566cmꢃ1; MS
(FAB): m=z ¼ 626.1 [M þ H]þ.
1-[(5S)-4-Oxo-2,2-bis(trifluoromethyl)-1,3-dioxolan-5-ylmethyl]-3-
(2,3,4,6-tetra-O-benzyl-ꢀ-D-glucopyranosyl)urea (6d-b, C41H40F6N2O9)
Bzl4-ꢀ-D-Glc-NH2 (5d, 1.01 g, 1.87 mmol) was reacted with isocyanate 4 (550 mg, 1.97 mmol)
due to protocol 1. Purification by flash chromatography (ethyl acetate:CHCl3 ¼ 1:4, Rf(ꢁ) ¼
0.55, Rf(ꢀ) ¼ 0.30) provides 234 mg (16%) of 6d-a and 714 mg (46%) of 6d-b as crystalline
solids.
6d-b: Mp 164–166ꢁC; ½ꢁꢂD ¼ ꢃ21ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.5, CHCl3); 1H NMR (CDCl3, 300 MHz,
COSY): ꢂ ¼ 3.32 (dd, 1H, J ¼ 9.0, 9.0Hz, 2-H), 3.40 (dt, 1H, J ¼ 5.7, 14.4Hz, CH2Ise), 3.50–3.64 (m,
4H, 4,5-H, CH2Ise, 6-Ha), 3.68 (dd, 1H, J ¼ 1.8, 10.4 Hz, 6-Hb), 3.77 (dd, 1H, J ¼ 8.7, 9.0 Hz, 3-H),
4.44 (d, 1H, J ¼ 12.3Hz, CH2Ph), 4.50 (d, 1H, J ¼ 10.8 Hz, CH2Ph), 4.52 (d, 1H, J ¼ 12.3Hz, CH2Ph),
4.70 (d, 1H, J ¼ 11.7Hz, CH2Ph), 4.75 (t, 1H, J ¼ 5.7 Hz, CHIse), 4.84 (d, 1H, J ¼ 10.8Hz, CH2Ph),
4.85 (d, 1H, J ¼ 11.7Hz, CH2Ph), 4.90–4.94 (m, 3H, CH2Ph, 1-H), 5.05 (d, 1H, J ¼ 8.4Hz, 1-NH),
5.10 (br.s, 1H, NHIse), 7.13–7.41 (m, 20H, Harom) ppm; 19F NMR (CDCl3, 282 MHz): ꢂ ¼ ꢃ3.17
(q, 3F, J ¼ 9.0 Hz), ꢃ2.89 (q, 3F, J ¼ 9.0 Hz) ppm; 13C NMR (CDCl3, 75MHz, HETCOR, APT):
ꢂ ¼ 40.64 (CH2Ise), 68.96 (6-CH2), 73.61 (CH2Ph), 74.20 (CHIse), 74.79 (CH2Ph), 75.08 (CH2Ph),
75.88 (CH2Ph), 76.04 (5-CH), 78.18 (4-CH), 81.00 (2-CH), 81.50 (1-CH), 85.70 (3-CH), 97.71 (sept,
J ¼ 36 Hz), 118.98 (q, J ¼ 286 Hz), 119.70 (q, J ¼ 288Hz), 127.9–128.7, 137.39, 137.97 (2C), 138.41,
157.05, 166.48 ppm; IR (KBr): ꢄꢂ¼ 1847, 1651, 1574 cmꢃ1; MS (FAB): m=z ¼ 841.3 [Mþ Na]þ, 819.3
[Mþ H]þ.
1-[(5S)-4-Oxo-2,2-bis(trifluoromethyl)-1,3-dioxolan-5-ylmethyl]-3-
(2,3,4,6-tetra-O-benzyl-ꢁ-D-glucopyranosyl)urea (6d-a, C41H40F6N2O9)
6d-b (500 mg, 0.61 mmol) was subjected to acid catalyzed anomerization due to protocol 2. 6d-a was
separated from the anomeric mixture by flash chromatography (ethyl acetate:CHCl3 ¼ 1:4). Yield
262 mg (52%) 6d-a, crystalline solid, mp 160–162ꢁC; ½ꢁꢂD ¼ þ55ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.5, CHCl3);
1H NMR (CDCl3, 300 MHz, COSY): ꢂ ¼ 3.30 (dt, 1H, J ¼ 6.3, 14.6Hz, CH2Ise), 3.59 (m, 1H, 4-H),
3.67 (dd, 1H, J ¼ 5.1, 10.4Hz, 6-Ha), 3.71–3.81 (m, 4H, 2,3-H, 6-Hb, CH2Ise), 3.93 (ddd, J ¼ 2.2, 5.1,
9.9 Hz, 5-H), 4.47 (d, 1H, J ¼ 11.5 Hz, CH2Ph), 4.53 (d, 1H, J ¼ 11.0Hz, CH2Ph), 4.60 (d, 1H,
J ¼ 11.5Hz, CH2Ph), 4.66 (d, 1H, J ¼ 11.5Hz, CH2Ph), 4.71 (d, 1H, J ¼ 11.5 Hz, CH2Ph), 4.78
(t, 1H, J ¼ 5.8 Hz, CHIse), 4.84 (d, 1H, J ¼ 11.0Hz, CH2Ph), 4.86 (d, 1H, J ¼ 11.0Hz, CH2Ph),

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4.94 (d, 1H, J ¼ 10.7 Hz, CH2Ph), 5.13 (br.s, 1H, 1-H), 5.42 (d, 1H, J ¼ 1.7 Hz, 1-NH), 6.07 (t, 1H,
J ¼ 6.1 Hz, NHIse), 7.15–7.32 (m, 20H, Harom) ppm; 19F NMR (CDCl3, 282 MHz): ꢂ ¼ ꢃ3.13 (q, 3F,
J ¼ 9.0 Hz), ꢃ2.90 (q, 3F, J ¼ 9.0 Hz) ppm; 13C NMR (CDCl3, 75MHz, HETCOR, APT): ꢂ ¼ 40.46
(CH2Ise), 68.59 (6-CH2), 70.01 (5-CH), 73.22 (CH2Ph), 73.67 (CHIse), 73.70 (CH2Ph), 75.09
(CH2Ph), 75.96 (CH2Ph), 77.34 (4-CH), 78.04 (2-CH), 78.17 (1-CH), 81.95 (3-CH), 97.61 (sept,
J ¼ 36 Hz), 118.89 (q, J ¼ 286 Hz), 119.64 (q, J ¼ 288 Hz), 127.8–128.7, 137.07, 137.7, 138.08,
138.33, 158.44, 166.52ppm; IR (KBr): ꢄꢂ¼ 1849, 1681, 1554 cmꢃ1; MS (FAB): m=z ¼ 841.3
[Mþ Na]þ, 819.3 [Mþ H]þ.
1-[(5S)-4-Oxo-2,2-bis(trifluoromethyl)-1,3-dioxolan-5-ylmethyl]-3-
(2,3,4,6-tetra-O-benzyl-ꢀ-D-mannopyranosyl)urea (6e-b, C41H40F6N2O9)
Bzl4-ꢀ-D-Man-NH2 (5e, 943 mg, 1.65mmol) was reacted with isocyanate 4 (485mg, 1.74 mmol) due
to protocol 1. Purification by flash chromatography (ethyl acetate:petroleum ether¼ 1:2, Rf(ꢁ) ¼ 0.26,
Rf(ꢀ) ¼ 0.21) provides 163 mg (12%) of a mixture of 6e-a and 6e-b (ratio 1:1) and 778 mg (56%) of
6e-b as foamy solids.
6e-b: ½ꢁꢂD ¼ þ5ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.0, CHCl3); 1H NMR (CDCl3, 300 MHz, COSY): ꢂ ¼ 3.21
(dt, 1H, J ¼ 6.0, 14.4 Hz, CH2Ise), 3.40–3.47 (m, 2H, CH2Ise, 6-Ha), 3.54 (m, 1H, 5-H), 3.60 (dd,
1H, J ¼ 1.8, 12.0 Hz, 6-Hb), 3.66 (dd, 1H, J ¼ 2.7, 9.6 Hz, 3-H), 3.76 (dd, 1H, J ¼ 1.5, 2.7 Hz, 2-H),
3.77 (dd, 1H, J ¼ 9.3, 9.6 Hz, 4-H), 4.40 (d, 1H, J ¼ 11.4 Hz, CH2Ph), 4.42 (d, 1H, J ¼ 11.1 Hz,
CH2Ph), 4.48 (d, 1H, J ¼ 11.4 Hz, CH2Ph), 4.56 (d, 1H, J ¼ 12.0 Hz, CH2Ph), 4.67 (t, 1H,
J ¼ 6.0 Hz, CHIse), 4.75 (s, 2H, CH2Ph), 4.83 (d, 1H, J ¼ 11.1 Hz, CH2Ph), 5.03 (d, 1H, J ¼ 9.6 Hz,
1-H), 5.08 (br.t, 1H, NHIse), 5.15 (d, 1H, J ¼ 12.0 Hz, CH2Ph), 5.49 (d, 1H, J ¼ 9.3 Hz, 1-NH),
7.12–7.40 (m, 20H, Harom) ppm; 19F NMR (CDCl3, 282 MHz): ꢂ ¼ ꢃ3.24 (m, 3F), ꢃ3.03 (m, 3F)
ppm; 13C NMR (CDCl3, 75 MHz, HETCOR, APT): ꢂ ¼ 40.28 (CH2Ise), 69.83 (6-CH2), 73.20
(CH2Ph), 73.75 (CH2Ph), 74.15 (CHIse), 74.83 (CH2Ph), 75.31 (4-CH), 75.46 (CH2Ph), 76.24
(5-CH), 77.14 (2-CH), 78.81 (1-CH), 83.93 (3-CH), 97.50 (sept, J ¼ 36 Hz), 118.92 (q,
J ¼ 287 Hz), 119.62 (q, J ¼ 289 Hz), 127.9–128.8, 137.47, 138.22, 138.34, 139.02, 156.46,
167.21 ppm; IR (KBr): ꢄꢂ¼ 1847, 1649, 1568 cmꢃ1; MS (FAB): m=z ¼ 841.3 [M þ Na]þ, 819.3
[M þ H]þ.
1-[(5S)-4-Oxo-2,2-bis(trifluoromethyl)-1,3-dioxolan-5-ylmethyl]-3-
(2,3,4,6-tetra-O-benzyl-ꢁ-D-mannopyranosyl)urea (6e-a, C41H40F6N2O9)
6e-b (500mg, 0.61mmol) was subjected to acid catalyzed anomerization due to protocol 2. 6e-a
was separated from the anomeric mixture by crystallization (DCM=petroleum ether, ꢃ28ꢁC). Yield
131 mg (26%) 6e-a, colorless needles, mp 170–171ꢁC; ½ꢁꢂD ¼ þ20ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.0, CHCl3);
1H NMR (CDCl3, 600 MHz, COSY): ꢂ ¼ 3.08 (dt, 1H, J ¼ 6.0, 14.6 Hz, CH2Ise), 3.67 (dt, 1H,
J ¼ 6.0, 14.6 Hz, CH2Ise), 3.69 (dd, 1H, J ¼ 2.4, 10.5 Hz, 6-Ha), 3.72 (t, 1H, J ¼ 2.9 Hz, 2-H), 3.75
(dd, 1H, J ¼ 4.8, 10.5Hz, 6-Hb), 3.80 (dd, 1H, J ¼ 2.9, 8.1Hz, 3-H), 3.85 (dd, 1H, J ¼ 8.4, 8.4Hz,
4-H), 3.91 (m, 1H, 5-H), 4.45 (d, 1H, J ¼ 11.5Hz, CH2Ph), 4.51 (d, 1H, J ¼ 11.2Hz, CH2Ph), 4.54
(d, 1H, J ¼ 11.5Hz, CH2Ph), 4.58 (d, 1H, J ¼ 2.0 Hz, CH2Ph), 4.59 (m, 1H, CHIse), 4.61 (d, 1H,
J ¼ 12.0Hz, CH2Ph), 4.66 (d, 1H, J ¼ 12.3Hz, CH2Ph), 4.68 (d, 1H, J ¼ 12.3 Hz, CH2Ph), 4.79
(d, 1H, J ¼ 11.2Hz, CH2Ph), 5.14 (t, 1H, J ¼ 3.0 Hz, 1-H), 5.78 (br.s, 1H, 1-NH), 6.12 (br.t, 1H,
NHIse), 7.12–7.30 (m, 20H, Harom) ppm; 19F NMR (CDCl3, 282 MHz): ꢂ ¼ ꢃ3.12 (m, 3F), ꢃ2.94
(m, 3F) ppm; 13C NMR (CDCl3, 150 MHz, HMQC): ꢂ ¼ 40.49 (CH2Ise), 68.91 (6-CH2), 72.06 (5-
CH), 72.62 (CH2Ph), 72.75 (CH2Ph), 73.58 (CH2Ph), 74.06 (CHIse), 74.49 (2-CH), 74.69 (CH2Ph),
74.84 (4-CH), 78.80 (3-CH), 78.96 (1-CH), 97.58 (sept, J ¼ 36Hz), 118.78 (q, J ¼ 286 Hz), 119.56
(q, J ¼ 288 Hz), 127.7–128.6, 137.67, 138.07, 138.15, 138.27, 158.56, 166.30ppm; IR (KBr):
ꢄꢂ¼ 1850, 1675, 1558 cmꢃ1
.

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1-[(5S)-4-Oxo-2,2-bis(trifluoromethyl)-1,3-dioxolan-5-ylmethyl]-3-
(3,4,6-tri-O-benzyl-2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)urea (6f-b, C36H37F6N3O9)
Bzl3-ꢀ-D-GlcNAc-NH2 (5f, 900 mg, 1.83mmol) was reacted with isocyanate 4 (538mg, 1.93 mmol)
due to protocol 1. The product precipitates from the reaction mixture. After filtration and careful
trituration with ethyl acetate 1.06 g (75%) of 6f-b were obtained as colorless crystals. Mp 218–219ꢁC;
1
½ꢁꢂD ¼ þ1ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.2, DMF); H NMR (DMSO-d6, 300 MHz, COSY): ꢂ ¼ 1.82 (s, 3H,
NHAc), 3.36–3.50 (m, 3H, 4,5-H, CH2Ise), 3.60–3.64 (m, 3H, 3-H, 6-H2), 3.78 (m, 1H, 2-H), 3.97
(ddd, 1H, J ¼ 3.9, 7.5, 15.3 Hz, CH2Ise), 4.46–4.56 (m, 3H, CH2Ph), 4.67–4.75 (m, 3H, CH2Ph), 4.81
(dd, 1H, J ¼ 9.9, 9.3 Hz, 1-H), 6.54 (d, 1H, J ¼ 9.6Hz, 1-NH), 6.77 (t, 1H, J ¼ 7.5 Hz, NHIse),
7.18–7.36 (m, 15H, Harom), 8.16 (d, 1H, J ¼ 9.3 Hz, 2-NH) ppm; 19F NMR (DMSO-d6, 282MHz):
ꢂ ¼ ꢃ2.16 (q, 3F, J ¼ 9.0 Hz), ꢃ1.51 (q, 3F, J ¼ 9.0Hz) ppm; 13C NMR (DMSO-d6, 75 MHz,
HETCOR): ꢂ ¼ 22.54 (NHAc), 39.08 (CH2Ise), 53.24 (2-CH), 68.74 (6-CH2), 72.30 (CH2Ph), 73.91
(CH2Ph), 74.08 (CH2Ph), 75.26 (4=5-CH), 75.53 (CHIse), 77.93 (4=5-CH), 80.23 (1-CH), 83.12
(3-CH), 96.44 (sept, J ¼ 35 Hz), 118.58 (q, J ¼ 286 Hz), 119.43 (q, J ¼ 289 Hz), 127.3–128.2,
138.08, 138.15, 138.57, 156.96, 166.75, 169.22ppm; IR (KBr): ꢄꢂ¼ 1844, 1653, 1568cmꢃ1; MS
(FAB): m=z ¼ 792.2 [M þ Na]þ, 770.2 [Mþ H]þ.
(5S)-5-Hydroxy-3-(2,3,4,6-tetra-O-acetyl-ꢀ-D-glucopyranosyl)-5,6-
dihydropyrimidin-2,4-dione (7a, C18H24N2O12)
6a-b (626mg, 1.00 mmol) and pyridine (1.98 g, 25.0 mmol) were heated under reflux for 24h in dry
CHCl3 (10 cm3). After removal of the volatiles, the residue was redissolved in CHCl3. The organic
layer was washed twice with 0.5 M HCl, sat. NaCl solution and dried with MgSO4. After removal of
the solvent the product was purified by flash chromatography (ethyl acetate, Rf ¼ 0.26). Yield 192 mg
(42%) 7a, colorless foam; ½ꢁꢂD ¼ ꢃ15ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.0, CHCl3); 1H NMR (CDCl3, 300 MHz,
COSY): ꢂ ¼ 2.03 (s, 3H), 2.06 (s, 3H), 2.08 (s, 3H), 2.12 (s, 3H) (OAc), 3.26 (m, 1H, CH2Ise), 3.63 (m,
1H, CH2Ise), 3.85 (m, 1H, 5-H), 4.20–4.25 (m, 2H, 6-H2), 4.31 (m, 1H, CHIse), 5.24 (dd, 1H, J ¼ 9.3,
9.6 Hz, 4-H), 5.31 (dd, 1H, J ¼ 9.6, 9.3 Hz, 3-H), 5.70 (d, 1H, J ¼ 9.0 Hz, 1-H), 5.96 (br.s, 1H, NH),
6.05 (dd, 1H, J ¼ 9.0, 9.3 Hz, 2-H) ppm; 13C NMR (CDCl3, 150 MHz): ꢂ ¼ 20.69 (2C), 20.72, 20.95
(OAc), 41.49 (CH2Ise), 61.97 (6-CH2), 65.56 (CHIse), 68.05 (4-CH), 69.12 (2-CH), 73.57 (3-CH), 74.66
(5-CH), 81.02 (1-CH), 153.05, 169.58, 169.85, 170.24, 170.83, 171.43ppm; IR (KBr): ꢄꢂ¼ 3400, 1752,
1700, 1640 cmꢃ1; MS (FAB): m=z ¼ 461.1 [Mþ H]þ.
tert-Butyl N-{(2S)-2-Hydroxy-1-oxo-3-[3-(2,3,4,6-tetra-O-acetyl-ꢀ-D-glucopyranosyl)-
ureido]propyl}phenylalaninate (8a, C31H43N3O14)
6a-b (626 mg, 1.00 mmol) was reacted with HCl ꢀ H-Phe-OtBu (309 mg, 1.20mmol) due to protocol 3.
Purification by flash chromatography (ethyl acetate, Rf ¼ 0.27). Yield 586 mg (86%) 8a, colorless
1
needles from acetone, mp 116–117ꢁC; ½ꢁꢂD ¼ þ1ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.1, THF); H NMR (DMSO-
d6, 300 MHz, COSY): ꢂ ¼ 1.36 (s, 9H, CH3tBu), 1.96 (s, 3H), 1.99 (s, 3H), 2.00 (s, 3H), 2.01 (s, 3H)
(OAc), 2.93 (m, 1H, CH2Ise), 2.99–3.05 (m, 2H, ꢀ-CH2Phe), 3.38 (m, 1H, CH2Ise), 3.87 (m, 1H, CHIse),
3.95 (dd, 1H, J ¼ 2.1, 12.0Hz, 6-Ha), 4.03 (m, 1H, 5H), 4.14 (dd, 1H, J ¼ 4.5, 12.0Hz, 6-Hb), 4.45 (m,
1H, ꢁ-CHPhe), 4.78 (dd, 1H, J ¼ 9.6, 9.6 Hz, 2-H), 4.90 (dd, 1H, J ¼ 9.6, 9.9 Hz, 4-H), 5.24 (dd, 1H,
J ¼ 9.9, 9.6Hz, 1-H), 5.35 (dd, 1H, J ¼ 9.6, 9.6Hz, 3-H), 5.97 (d, 1H, J ¼ 5.7 Hz, OH), 6.28 (t, 1H,
J ¼ 5.7 Hz, NHIse), 6.94 (d, 1H, J ¼ 9.9 Hz, 1-NH), 7.20–7.34 (m, 5H, Harom), 7.82 (d, 1H, J ¼ 8.1 Hz,
NHPhe) ppm; 13C NMR (CDCl3, 75 MHz, HETCOR): ꢂ ¼ 21.01, 21.10, 21.14, 21.22 (OAc), 28.25
(CH3tBu), 37.67 (ꢀ-CH2Phe), 44.11 (CH2Ise), 53.92 (ꢁ-CHPhe), 62.56 (6-CH2), 68.85 (4-CH), 71.15
(2-CH), 71.57 (CHIse), 72.29 (5-CH), 73.62 (3-CH), 79.46 (1-CH), 81.81 (CtBu), 127.26, 128.89,
130.00, 137.62, 157.51, 170.05, 170.19, 170.73, 170.95, 172.60ppm; IR (KBr): ꢄꢂ¼ 3400, 1749,
1660, 1560, 1531 cmꢃ1; MS (FAB): m=z ¼ 682.3 [Mþ H]þ.

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tert-Butyl N-{(2S)-2-Hydroxy-1-oxo-3-[3-(2,3,4,6-tetra-O-acetyl-ꢀ-D-mannopyranosyl)-
ureido]propyl}phenylalaninate (8b, C31H43N3O16)
6b-b (626mg, 1.00mmol) was reacted with HCl ꢀ H-Phe-OtBu (309mg, 1.20mmol) due to protocol 3.
Purification by flash chromatography (ethyl acetate, Rf ¼ 0.29). Yield 565 mg (83%) 8b, colorless
1
foam; [ꢁ]D ¼ ꢃ26ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.3, CHCl3); H NMR (DMSO-d6, 300 MHz, COSY): ꢂ ¼
1.36 (s, 9H, CH3tBu), 1.92 (s, 3H), 2.01 (s, 3H), 2.03 (s, 3H), 2.20 (s, 3H) (OAc), 2.92 (m, 1H,
CH2Ise), 2.99–3.05 (m, 2H, ꢀ-CH2Phe), 3.39 (m, 1H, CH2Ise), 3.86–3.97 (m, 3H, CHIse, 5-H, 6-Ha),
4.11 (dd, 1H, J ¼ 5.4, 12.6Hz, 6-Hb), 4.45 (m, 1H, ꢁ-CHPhe), 5.00 (dd, 1H, J ¼ 9.9, 9.9 Hz, 4-H), 5.16
(d, 1H, J ¼ 3.3 Hz, 2-H), 5.33 (dd, 1H, J ¼ 3.3, 9.9 Hz, 3-H), 5.53 (d, 1H, J ¼ 9.9 Hz, 1-H), 6.04 (d, 1H,
J ¼ 5.7 Hz, OH), 6.29 (br.t, 1H, NHIse), 6.84 (d, 1H, J ¼ 9.9Hz, 1-NH), 7.19–7.32 (m, 5H, Harom), 7.83
(d, 1H, J ¼ 7.8 Hz, NHPhe) ppm; 13C NMR (DMSO-d6, 75 MHz, HETCOR): ꢂ ¼ 21.05, 21.23, 21.27,
21.50 (OAc), 28.24 (CH3tBu), 37.55 (ꢀ-CH2Phe), 43.94 (CH2Ise), 53.90 (ꢁ-CHPhe), 62.94 (6-CH2),
66.13 (4-CH), 70.82 (2-CH), 71.43 (CHIse), 71.65 (3-CH), 72.56 (5-CH), 77.46 (1-CH), 81.80 (CtBu),
127.26, 128.89, 130.01, 137.59, 156.82, 170.14, 170.30, 170.77, 170.84, 170.97, 172.59 ppm; IR
(KBr): ꢄꢂ¼ 3400, 1747, 1655, 1554, 1533cmꢃ1; MS (FAB): m=z ¼ 704.3 [M þ Na]þ, 682.3 [Mþ H]þ.
tert-Butyl N-{(2S)-2-Hydroxy-1-oxo-3-[3-(3,4,6-tri-O-acetyl-2-acetamido-2-
deoxy-ꢀ-D-glucopyranosyl)ureido]propyl}phenylalaninate (8c, C31H44N4O13)
6c-b (624 mg, 1.00 mmol) was reacted with HCl ꢀ H-Phe-OtBu (309mg, 1.20mmol) due to protocol 3.
Purification by flash chromatography (DCM:iPrOH¼ 10:1, Rf ¼ 0.20). Yield 516 mg (76%) 8c, cry-
stalline solid, mp 213–214ꢁC; [ꢁ]D ¼ þ12ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.3, DMF); 1H NMR (DMSO-d6,
300 MHz, COSY): ꢂ ¼ 1.37 (s, 9H, CH3tBu), 1.79 (s, 3H, NHAc), 1.93 (s, 3H), 1.98 (s, 3H), 2.05
(s, 3H) (OAc), 2.89 (m, 1H, CH2Ise), 3.00–3.04 (m, 2H, ꢀ-CH2Phe), 3.38 (m, 1H, CH2Ise), 3.78 (m, 1H,
5-H), 3.84–3.96 (2-H, 6-Ha, CHIse), 4.17 (dd, 1H, J ¼ 3.9, 12.6Hz, 6-Hb), 4.46 (m, 1H, ꢁ-CHPhe), 4.83
(dd, 1H, J ¼ 9.6, 9.6 Hz, 4-H), 5.00 (dd, 1H, J ¼ 9.6, 9.9 Hz, 1-H), 5.08 (dd, 1H, J ¼ 9.9, 9.9 Hz, 3-H),
5.98 (d, 1H, J ¼ 5.4 Hz, OH), 6.37 (t, 1H, J ¼ 5.7 Hz, NHIse), 6.81 (d, 1H, J ¼ 9.6 Hz, 1-NH), 7.20–7.33
(m, 5H, Harom), 7.81 (d, 1H, J ¼ 7.8 Hz, NHPhe), 8.02 (d, 1H, J ¼ 9.6Hz, 2-NH) ppm; 13C NMR
(DMSO-d6, 75 MHz, HETCOR): ꢂ ¼ 20.33, 20.40, 20.50 (OAc), 22.67 (NHAc), 27.53 (CH3tBu), 36.96
(ꢀ-CH2Phe), 43.37 (CH2Ise), 51.89 (2-CH), 53.17 (ꢁ-CHPhe), 61.92 (6-CH2), 68.56 (4-CH), 71.04
(CHIse), 71.67 (5-CH), 73.56 (3-CH), 79.96 (1-CH), 81.07 (CtBu), 126.53, 128.16, 129.28, 136.89,
157.02, 169.30, 169.38, 169.55, 170.03, 170.23, 171.86 ppm; IR (KBr): ꢄꢂ¼ 3400, 1740, 1655, 1564,
1535 cmꢃ1; MS (FAB): m=z ¼ 681.3 [Mþ H]þ.
tert-Butyl N-{(2S)-2-Hydroxy-1-oxo-3-[3-(2,3,4,6-tetra-O-benzyl-ꢀ-D-glucopyranosyl)-
ureido]propyl}phenylalaninate (8d, C51H59N3O10)
6d-b (409mg, 0.50mmol) was reacted with HCl ꢀ H-Phe-OtBu (155mg, 0.60mmol) due to protocol 3.
Purification by flash chromatography (ethyl acetate:CHCl3 ¼ 5:4, Rf ¼ 0.20). Yield 323 mg (74%) 6d-
1
b, crystalline solid, mp 150–151ꢁC; [ꢁ]D ¼ ꢃ25ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.2, CHCl3); H NMR (DMSO-
d6, 300 MHz, COSY): ꢂ ¼ 1.37 (s, 9H, CH3tBu), 2.99–3.04 (m, 2H, ꢀ-CH2Phe), 3.08 (m, 1H, CH2Ise),
3.32 (dd, 1H, J ¼ 9.0, 9.6 Hz, 2-H), 3.40–3.52 (m, 3H, 4,5-H, CH2Ise), 3.63–3.66 (m, 2H, 6-H2), 3.70
(dd, 1H, J ¼ 9.0, 9.0Hz, 3-H), 3.94 (m, 1H, CHIse), 4.45–4.56 (m, 4H, ꢁ-CHPhe, CH2Ph), 4.66–4.85
(m, 5H, CH2Ph), 4.91 (dd, 1H, J ¼ 9.0, 9.6 Hz, 1-H), 6.01 (d, 1H, J ¼ 5.4 Hz, OH), 6.18 (t, 1H,
J ¼ 5.7 Hz, NHIse), 7.16–7.36 (m, 26H, 1-NH, Harom), 7.85 (d, 1H, J ¼ 7.5 Hz, NHPhe) ppm;
13C NMR (DMSO-d6, 75 MHz, HETCOR): ꢂ ¼ 27.53 (CH3tBu), 36.94 (ꢀ-CH2Phe), 43.58 (CH2Ise),
53.21 (ꢁ-CHPhe), 68.70 (6-CH2), 71.09 (CHIse), 72.33 (CH2Ph), 73.83 (CH2Ph), 73.91 (CH2Ph), 74.50
(CH2Ph), 75.02, 77.79 (4,5-CH), 80.50 (1-CH), 81.03 (CtBu), 81.40 (2-CH), 84.74 (3-CH),
126.4–129.3, 136.86, 138.18, 138.20, 138.45, 138.65, 157.09, 170.17, 171.94 ppm; IR (KBr):
ꢄꢂ¼ 3400, 1732, 1651, 1570, 1525 cmꢃ1; MS (FAB): m=z ¼ 896.4 [Mþ Na]þ, 874.4 [Mþ H]þ.

Hexafluoroacetone as a Protecting and Activating Reagent
589
tert-Butyl N-{(2S)-2-Hydroxy-1-oxo-3-[3-(2,3,4,6-tetra-O-benzyl-ꢀ-D-mannopyranosyl)-
ureido]propyl}phenylalaninate (8e, C51H59N3O10)
6e-b (409 mg, 0.50 mmol) was reacted with HCl ꢀ H-Phe-OtBu (155 mg, 0.60 mmol) due to protocol 3.
Purification by flash chromatography (ethyl acetate:petrolether ¼ 4:1, Rf ¼ 0.40). Yield 319 mg (74%)
1
8e, foamy solid; [ꢁ]D ¼ ꢃ9ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 2.0, CHCl3); H NMR (CDCl3, 300 MHz, COSY):
ꢂ ¼ 1.39 (s, 9H, CH3tBu), 2.99–3.13 (m, 2H, ꢀ-CH2Phe), 3.32–3.36 (m, 2H, CH2Ise), 3.42–3.48 (m,
2H, 5-H, 6-Ha), 3.62–3.67 (m, 2H, 3-H, 6-Hb), 3.75 (m, 1H, 2-H), 3.86 (dd, 1H, J ¼ 9.3, 9.3Hz,
4-H), 4.01 (m, 1H, CHIse), 4.40 (d, 1H, J ¼ 12.0 Hz, CH2Ph), 4.46 (d, 1H, J ¼ 11.1Hz, CH2Ph), 4.53
(d, 2H, J ¼ 11.7 Hz, CH2Ph), 4.73 (s, 2H, CH2Ph), 4.74 (m, 1H, ꢁ-CHPhe), 4.84 (d, 1H, J ¼ 10.5Hz,
CH2Ph), 5.05 (d, 1H, J ¼ 9.9 Hz, 1-H), 5.12 (d, 1H, J ¼ 11.7Hz, CH2Ph), 5.62 (br.t, 1H, NHIse), 5.82
(d, 1H, J ¼ 9.9 Hz, 1-NH), 5.85 (br.s, 1H, OH), 7.14–7.37 (m, 25H, Harom), 7.58 (d, 1H, J ¼ 8.7 Hz,
NHPhe) ppm; 13C NMR (CDCl3, 75 MHz, HETCOR): ꢂ ¼ 28.17 (CH3tBu), 38.89 (ꢀ-CH2Phe), 45.01
(CH2Ise), 53.55 (ꢁ-CHPhe), 69.27 (6-CH2), 72.94 (CHIse), 73.07 (CH2Ph), 73.55 (CH2Ph), 74.96
(CH2Ph), 75.11 (4-CH), 75.34 (CH2Ph), 76.45 (5-CH), 77.09 (2-CH), 78.97 (1-CH), 82.39 (CtBu),
84.06 (3-CH), 127.2–129.8, 136.44, 137.82, 138.27, 138.47, 138.82, 159.11, 170.27, 172.32 ppm;
IR (KBr): ꢄꢂ¼ 3400, 1732, 1651, 1562, 1520cmꢃ1; MS (FAB): m=z ¼ 896.3 [M þ Na]þ, 874.4
[Mþ H]þ.
tert-Butyl N-{(2S)-2-Hydroxy-1-oxo-3-[3-(3,4,6-tri-O-benzyl-2-acetamido-2-
deoxy-ꢀ-D-glucopyranosyl)ureido]propyl}phenylalaninate (8f, C46H56N4O10)
6f-b (384mg, 0.5 mmol) was treated with HCl ꢀ H-Phe-OtBu (155mg, 0.60 mmol) due to protocol 3.
Purification by flash chromatography (CHCl3:MeOH¼ 10:1, Rf ¼ 0.26). Yield 355 mg (86%) 8f, color-
less crystals, mp 208–209ꢁC; [ꢁ]D ¼ ꢃ4ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.1, DMF); 1H NMR (DMSO-d6,
300 MHz, COSY): ꢂ ¼ 1.37 (s, 9H, CH3tBu), 1.83 (s, 3H, NHAc), 2.89 (m, 1H, CH2Ise), 3.00–3.05
(m, 2H, ꢀ-CH2Phe), 3.36–3.50 (m, 3H, 4,5-H, CH2Ise), 3.58–3.64 (m, 3H, 3-H, 6-H2), 3.77 (m, 1H,
2-H), 3.87 (m, 1H, CHIse), 4.43–4.54 (m, 4H, ꢁ-CHPhe, CH2Ph), 4.66–4.75 (m, 3H, CH2Ph), 4.82 (dd,
1H, J ¼ 9.6, 9.6Hz, 1-H), 5.99 (d, 1H, J ¼ 5.7Hz, OH), 6.43 (t, 1H, J ¼ 5.1 Hz, NHIse), 6.64 (d, 1H,
J ¼ 9.6 Hz, 1-NH), 7.18–7.36 (m, 20H, Harom), 7.82 (d, 1H, J ¼ 7.8 Hz, NHPhe), 8.10 (d, 1H,
J ¼ 9.3 Hz, 2-NH) ppm; 13C NMR (DMSO-d6, 75MHz, HETCOR): ꢂ ¼ 22.54 (NHAc), 27.51
(CH3tBu), 36.98 (ꢀ-CH2Phe), 43.45 (CH2Ise), 53.16 (ꢁ-CHPhe), 53.43 (2-CH), 68.76 (6-CH2), 71.20
(CHIse), 72.29, 73.90, 74.09 (3ꢄCH2Ph), 75.29, 77.97 (4,5-CH), 80.37 (1-CH), 81.04 (CtBu), 83.43
(3-CH), 126.5–129.3, 136.87, 138.14, 138.21, 138.62, 157.43, 169.30, 170.22, 171.87ppm; IR (KBr):
ꢄꢂ¼ 1731, 1651, 1568, 1531 cmꢃ1; MS (FAB): m=z ¼ 825.4 [Mþ H]þ.
tert-Butyl N-{(2S)-2-Hydroxy-1-oxo-3-[3-(ꢀ-D-glucopyranosyl)ureido]propyl}phenyl-
alaninate (9a, C23H35N3O10)
8a (205 mg, 0.30mmol) was treated with NaOMe (6.5mg, 0.12mmol) due to protocol 4. Yield 150 mg
(>95%) 9a, foam; [ꢁ]D ¼ ꢃ17ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.0, MeOH); 1H NMR (DMSO-d6, 300MHz,
Phe
COSY): ꢂ ¼ 1.38 (s, 9H, CH3tBu), 2.83–2.96 (m, 2H, CH2Ise, 2-H), 3.02–3.10 (m, 4H, ꢀ-CH2
,
4,5-H), 3.18 (m, 1H, 3-H), 3.37–3.44 (m, 2H, CH2Ise, 6-Ha), 3.63 (m, 1H, 6-Hb), 3.88 (m, 1H, CHIse),
4.46 (m, 1H, ꢁ-CHPhe), 4.50 (t, 1H, J ¼ 6.0 Hz, 6-OH), 4.60 (dd, 1H, J ¼ 9.3, 9.3 Hz, 1-H), 4.88 (d, 1H,
J ¼ 4.8 Hz, 4-OH), 4.91 (d, 1H, J ¼ 5.4 Hz, 2-OH), 4.99 (d, 1H, J ¼ 4.5 Hz, 3-OH), 6.05 (d, 1H,
J ¼ 5.4 Hz, OHIse), 6.12 (br.t, 1H, NHIse), 6.77 (d, 1H, J ¼ 9.3 Hz, 1-NH), 7.20–7.34 (m, 5H, Harom),
7.85 (d, 1H, J ¼ 8.1 Hz, NHPhe) ppm; 13C NMR (DMSO-d6, 75MHz, HETCOR): ꢂ ¼ 28.27 (CH3tBu),
37.61 (ꢀ-CH2Phe), 44.18 (CH2Ise), 53.89 (ꢁ-CHPhe), 61.62 (6-CH2), 70.71 (4-CH), 71.96 (CHIse), 73.67
(2-CH), 78.44 (3-CH), 78.68 (5-CH), 81.79 (1-CH, CtBu), 127.03, 128.92, 130.03, 137.63, 158.21,
170.99, 172.74 ppm; IR (KBr): ꢄꢂ¼ 3400, 1728, 1660, 1561, 1527cmꢃ1; MS (FAB): m=z ¼ 552.2
[Mþ K]þ, 536.2 [Mþ Na]þ, 514.2 [M þ H]þ.

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tert-Butyl N-{(2S)-2-Hydroxy-1-oxo-3-[3-(ꢀ-D-mannopyranosyl)-
ureido]propyl}phenylalaninate (9b, C23H35N3O10)
8b (205 mg, 0.30 mmol) was treated with NaOMe (6.5mg, 0.12mmol) due to protocol 4. Yield 150 mg
(>95%) 9b, foam; [ꢁ]D ¼ ꢃ10ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.0, MeOH); 1H NMR (DMSO-d6, 300MHz,
COSY): ꢂ ¼ 1.37 (s, 9H, CH3tBu), 2.82 (m, 1H, CH2Ise), 3.00–3.07 (m, 3H, ꢀ-CH2Phe, 5-H),
3.29–3.48 (m, 4H, CH2Ise, 3,4-H, 6-Ha), 3.54 (m, 1H, 2-H), 3.65 (m, 1H, 6-Hb), 3.87 (m, 1H, CHIse),
4.43–4.48 (m, 2H, 6-OH, ꢁ-CHPhe), 4.71 (d, 1H, J ¼ 4.8 Hz, 4-OH), 4.82 (d, 1H, J ¼ 4.8Hz, 3-OH),
4.86 (d, 1H, J ¼ 4.8 Hz, 2-OH), 4.91 (d, 1H, J ¼ 9.9 Hz, 1-H), 6.03 (d, 1H, J ¼ 6.0 Hz, OHIse), 6.66
(t, 1H, J ¼ 5.7 Hz, NHIse), 6.73 (d, 1H, J ¼ 9.9 Hz, 1-NH), 7.20–7.34 (m, 5H, Harom), 7.80 (d, 1H,
J ¼ 8.1 Hz, NHPhe) ppm; 13C NMR (DMSO-d6, 75MHz, HETCOR): ꢂ ¼ 28.27 (CH3tBu), 37.63
(ꢀ-CH2Phe), 44.20 (CH2Ise), 53.81 (ꢁ-CHPhe), 62.02 (6-CH2), 67.50 (4-CH), 71.88 (2-CH), 72.13
(CHIse), 75.05 (3-CH), 79.05 (5-CH), 79.18 (1-CH), 81.85 (CtBu), 127.30, 128.90, 130.03, 137.59,
157.85, 170.97, 172.66 ppm; IR (KBr): ꢄꢂ¼ 3400, 1727, 1650, 1561, 1529cmꢃ1; MS (FAB):
m=z ¼ 552.2 [M þ K]þ, 536.2 [Mþ Na]þ, 514.2 [Mþ H]þ.
Synthesis of N-Glycosylated Isocysteine Derivatives
1-[5-Oxo-2,2-bis(trifluoromethyl)-1,3-oxathiolan-4-ylmethyl]-3-
(2,3,4,6-tetra-O-benzyl-D-glucopyranosyl)urea (14, C41H40F6N2O8S)
Bzl4-ꢀ-D-Glc-NH2 (5d, 866 mg, 1.61mmol) was reacted with isocyanate 13 (502mg, 1.70 mmol)
due to protocol 1. Purification by flash chromatography (ethyl acetate:CHCl3 ¼ 1:5, Rf(ꢁ) ¼ 0.43,
Rf(ꢀ) ¼ 0.18) provides 201 mg (15%) of 14-a and 809mg (60%) of 14-b as foamy solids.
1
14-a: Diastereomeric mixture (ratio 1:1); H NMR (CDCl3, 300 MHz): ꢂ ¼ 3.29–3.39 (m, 2H,
CH2Icy), 3.50–3.81 (m, 12H, CH2Icy, 2,3,4-CH, 6-CH2), 3.89–3.94 (m, 2H, 5-H), 4.35 (t, 1H,
J ¼ 6.0 Hz, CHIcy), 4.40 (t, 1H, J ¼ 6.0 Hz, CHIcy), 4.42–4.70 (m, 10H, CH2Ph), 4.80 (d, 2H,
J ¼ 11.1Hz, CH2Ph), 4.82 (dd, 2H, J ¼ 10.5 Hz, CH2Ph), 4.90 (d, 2H, J ¼ 11.1Hz, CH2Ph), 5.09
(br.s, 2H, 1-H), 5.39 (d, 1H, J ¼ 2.1 Hz, 1-NH), 5.40 (d, 1H, J ¼ 2.1 Hz, 1-NH), 6.19 (br.t, 1H,
J ¼ 5.7 Hz, NHIcy), 6.24 (br.t, 1H, J ¼ 5.7 Hz, NHIcy), 7.18–7.40 (m, 40H, Harom) ppm; 19F NMR
(CDCl3, 282 MHz): ꢂ ¼ 0.76–0.88 (m, 6F), 1.95–2.13 (m, 6F) ppm; 13C NMR (CDCl3, 75 MHz, APT):
ꢂ ¼ 41.73, 41.89 (CH2Icy), 46.24, 46.32 (CHIcy), 68.61, 68.68 (6-CH2), 69.96, 69.99 (5-CH), 73.14,
73.23, 73.64, 73.71, 75.11, 75.92 (CH2Ph), 77.35 (4-CH), 78.02, 78.08, 78.18 (1,2-CH), 81.87, 81.90
(3-CH), 83.31 (sept, J ¼ 36 Hz), 120.82 (q, J ¼ 288 Hz), 121.41 (q, J ¼ 284 Hz), 128.0–128.9, 137.06,
137.09, 137.63, 137.94, 138.30, 158.47, 158.63, 169.43, 169.67ppm; IR (KBr): ꢄꢂ¼ 1813, 1666,
1554 cmꢃ1; MS (FAB): m=z ¼ 857.2 [Mþ Na]þ, 835.2 [Mþ H]þ.
14-b: Diastereomeric mixture (ratio 1:1); 1H NMR (CDCl3, 300 MHz): ꢂ ¼ 3.33 (dd, 1H, J ¼ 9.0,
9.0 Hz, 2-H), 3.34 (dd, 1H, J ¼ 9.0, 9.0 Hz, 2-H), 3.42–3.79 (m, 14H, CH2Icy, 3,4,5-H, 6-H2), 4.21 (t,
1H, J ¼ 6.3 Hz, CHIcy), 4.37 (t, 1H, J ¼ 6.0 Hz, CHIcy), 4.38–4.53 (m, 6H, CH2Ph), 4.66–4.93 (m, 12H,
1-H, CH2Ph), 5.30 (d, 2H, J ¼ 9.0 Hz, 1-NH), 5.56 (m, 2H, NHIcy), 7.12–7.35 (m, 40H, Harom) ppm; 19
F
NMR (CDCl3, 282 MHz): ꢂ ¼ 0.81–0.93 (m, 6F), 1.91–2.07 (m, 6F) ppm; 13C NMR (CDCl3, 75 MHz,
APT): ꢂ ¼ 41.85, 42.77 (CH2Icy), 46.29 (CHIcy), 68.85, 68.96 (6-CH2), 73.55, 74.66, 75.09, 77.81
(CH2Ph), 75.85 (5-CH), 77.99 (4-CH), 81.00, 81.05, 81.35, 81.42 (1,2-CH), 83.36 (sept, J ¼ 36 Hz),
85.47, 85.54 (3-CH), 120.81 (q, J ¼ 284 Hz), 121.39 (q, J ¼ 285 Hz), 127.9–128.8, 137.16, 137.24,
137.75, 137.78, 137.80, 137.84, 138.26, 138.27, 157.11, 157.18, 169.83, 169.96 ppm; IR (KBr):
ꢄꢂ¼ 1811, 1645, 1572 cmꢃ1; MS (FAB): m=z ¼ 857.2 [Mþ Na]þ, 835.2 [Mþ H]þ.
tert-Butyl N-{2-Mercapto-1-oxo-3-[3-(2,3,4,6-tetra-O-benzyl-ꢀ-D-glucopyranosyl)ureido]-
propyl}phenylalaninate (15, C51H59N3O9S)
14-b (236mg, 0.28 mmol) was reacted with HCl ꢀ H-Phe-OtBu (87 mg, 0.34mmol) due to protocol 3.
The reaction mixture was flushed with argon for 30min. Purification by flash chromatography (ethyl

Hexafluoroacetone as a Protecting and Activating Reagent
591
acetate:petrolether¼ 1:1, Rf ¼ 0.29, Rf ¼ 0.25). Yield 181 mg (71%) 15, foam, diastereomeric mixture
(ratio 1:1); 1H NMR (CDCl3, 300 MHz, COSY): ꢂ ¼ 1.42 (s, 9H, CH3tBu), 1.45 (s, 9H, CH3tBu), 2.03
(br.s, 2H, SH), 3.06–3.14 (m, 4H, ꢀ-CH2Phe), 3.31–3.82 (m, 18H, 2,3,4,5-H, 6-H2, CHIcy, CH2Icy),
4.43–4.65 (m, 10H, CH2Ph, ꢁ-CHPhe), 4.71–5.03 (m, 11H, CH2Ph, 1-H, 1-NH), 5.14 (d, 1H, J ¼ 9.0 Hz,
1-NH), 5.43 (m, 2H, NHIcy), 6.91 (d, 1H, J ¼ 7.2 Hz, NHPhe), 7.03 (d, 1H, J ¼ 6.6 Hz, NHPhe), 7.17–7.36
(m, 50H, Harom) ppm; 13C NMR (CDCl3, 75MHz, APT, HETCOR): ꢂ ¼ 28.06 (CH3tBu), 37.67, 37.84
(ꢀ-CH2Phe), 41.89, 42.76 (CHIcy), 44.85, 45.43 (CH2Icy), 54.37, 54.62 (ꢁ-CHPhe), 68.52, 68.65 (6-CH2),
73.49, 73.58, 74.77, 74.99, 75.82 (CH2Ph), 76.02 (5-CH), 77.92, 78.01 (4-CH), 80.86, 81.04 (2-CH),
81.40, 81.53 (1-CH), 82.75 (CtBu), 85.88, 85.93 (3-CH), 127.9–129.6, 136.1–138.5 (arom), 157.23,
170.56, 171.06, 171.16, 171.59 ppm; MS (FAB): m=z ¼ 890.3 [Mþ H]þ.
Synthesis of O-Glycosylated Isoserine Derivatives
Protocol 5: To a solution of the glycosyl donor (1.2 equiv) and the glycosyl acceptor (1.0 equiv) in dry
3
DCM (10 cm per mmol) 0.5g of activated molecular sieves (4A) were added. After stirring for 2 h,
˚
trimethylsilyl triflate (TMSOTf, 0.1 equiv) was added and stirring was continued until complete con-
sumption of the glycosyl donor was judged by TLC (1–2 h). The mixture was diluted with DCM
(40 cm3 per mmol) and extracted with sat. NaHCO3 solution (10 cm3 per mmol) and water (10 cm3 per
mmol). After drying with MgSO4 the organic solvent was removed in vacuo and the residue purified by
column chromatography.
Protocol 6: To a solution of the allyl ester (1.0 equiv) and (Ph3P)4Pd (0.1 equiv) in dry THF
(25 cm3 per mmol) N-methylaniline (10 equiv) was added within 3 min. The mixture was stirred until
complete consumption of the starting material (TLC, 30min). After addition of chloroform (50cm3 per
mmol) and extraction with 1.5M HCl the organic layer was dried and evaporated in vacuo. The
remaining residue was purified by column chromatography.
5-(5S)-[(9-Fluorenylmethoxycarbonyl)amino]methyl-2,2-bis(trifluoromethyl)-
1,3-dioxolan-4-one (16)
9-Fluorenylmethanol (2.47 g, 12.6mmol) and 4 (4.05 g, 14.5mmol) were heated in dry chloroform
(50 cm3) for 40 h under reflux. The reaction mixture was concentrated under reduced pressure to
10cm3. After addition of petroleum ether (90 cm3) the product crystallized to give colorless, fine
1
needles. Yield 4.95g (83%) 16, mp 116–117ꢁC; Rf ¼ 0.59 (ethyl actate:petroleum ether¼ 1:1); H
NMR (CDCl3, 300 MHz): ꢂ ¼ 3.71 (m, 1H, CH2Ise), 3.81 (m, 1H, CH2Ise), 4.26 (t, 1H, J ¼ 6.6 Hz,
CHFmoc), 4.45–4.55 (m, 2H, CH2Fmoc), 4.82 (m, 1H, CHIse), 5.14 (br.t, 1H, NHIse), 7.36 (t, 2H,
J ¼ 7.5 Hz), 7.45 (t, 2H, J ¼ 7.5 Hz), 7.61 (d, 2H, J ¼ 7.5 Hz), 7.81 (d, 2H, J ¼ 7.5 Hz); 19F NMR
(CDCl3, 282 MHz): ꢂ ¼ ꢃ3.0 (m, 3F, CF3), ꢃ2.80 (m, 3F, CF3); for further data see Ref. [31a].
Allyl N-(9-Fluorenylmethoxycarbonyl)isoserinate (17, C21H21NO5)
A solution of 16 (1.19 g, 2.50 mmol) and allyl alcohol (7.5cm3) in chloroform (15 cm3) was heated
under reflux for 2 d. The volatiles were distilled off under reduced pressure and the residue was purified
by column chromatography (ethyl acetate:petroleuum ether¼ 1:1, Rf ¼ 0.34). Yield 779 mg (85%) 17,
crystalline solid, mp 119–121ꢁC; [ꢁ]D ¼ þ3ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.6, CHCl3); 1H NMR (CDCl3,
300 MHz): ꢂ ¼ 3.57–3.71 (m, 2H, CH2Ise), 4.26 (t, 1H, J ¼ 6.9 Hz, CHFmoc), 4.32 (m, 1H, CHIse),
4.38 (d, 2H, J ¼ 6.9 Hz, CH2Fmoc), 4.68 (d, 2 H, J ¼ 4.5 Hz, OCH2All), 5.22 (br.t, 1H, NHIse), 5.26 (m,
1H, 3Jcis ¼ 10.5 Hz, ¼CH2 ), 5.34 (m, 1H, 3Jtrans ¼ 17.1Hz, ¼CH2All), 5.91 (m, 1H, ¼CH-All), 7.32
All
(t, 2H, J ¼ 7.5 Hz), 7.40 (m, 2H), 7.58 (d, 2H, J ¼ 7.5 Hz), 7.76 (d, 2H, J ¼ 7.5 Hz) ppm; 13C NMR
(CDCl3, 75 MHz): ꢂ ¼ 44.33 (CH2Ise), 47.25 (CHFmoc), 66.74, 67.14 (OCH2All, CH2Fmoc), 70.20
(CHIse), 119.47 (¼CH2All), 120.05, 125.10, 127.11, 127.76, 131.22 (¼CH-All), 141.33, 143.84,
156.66, 172.66 ppm; IR (KBr): ꢄꢂ¼ 3400, 1740, 1697, 1541 cm; MS (FAB): m=z ¼ 368.2 [Mþ H]þ.

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Allyl 3-[(9-Fluorenylmethoxycarbonyl)amino]-2-(2S)-[(2,3,4,6-tetra-O-
acetyl-ꢀ-D-glucopyranosyl)oxy]propionate (18a-b, C35H39NO14)
17 (250 mg, 0.68mmol) and 2,3,4,6-tetra-O-acetyl-ꢁ-D-glucopyranosyl trichloroacetimidate (402 mg,
0.82mmol) were reacted due to protocol 5. Purification by flash chromatography (CHCl3:
acetone ¼ 10:1, Rf (ꢁ) ¼ 0.26, Rf (ꢀ) ¼ 0.22). Yield 62mg (13%) of a 1:1 mixture of 18a-a and 18a-b
and 310 mg (65%) 18a-b, foam; [ꢁ]D ¼ ꢃ21ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 0.5, CHCl3); 1H NMR (CDCl3,
300 MHz, COSY): ꢂ ¼ 2.05 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.12 (s, 3H) (OAc), 3.54–3.66 (m,
2H, CH2Ise, 5-H), 3.73 (m, 1H, CH2Ise), 4.11 (dd, 1H, J ¼ 1.8, 12.0Hz, 6-Ha), 4.17 (dd, 1H, J ¼ 4.8,
12.0Hz, 6-Hb), 4.24 (t, 1H, J ¼ 6.9 Hz, CHFmoc), 4.38–4.48 (m, 3H, CH2Fmoc, CHIse), 4.56 (d, 1H,
J ¼ 8.1 Hz, 1-H), 4.67 (d, 2H, J ¼ 5.7 Hz, OCH2All), 5.07 (dd, 1H, J ¼ 8.1, 9.6 Hz, 2-H), 5.08 (dd, 1H,
J ¼ 9.6, 9.6 Hz, 4-H), 5.25–5.40 (m, 4H, 3-H, NHIse, ¼CH2All), 5.95 (m, 1H, ¼CH-All), 7.33–7.48 (m,
4H), 7.62–7.66 (m, 2H), 7.78–7.81 (m, 2H) ppm; 13C NMR (CDCl3, 75 MHz, HETCOR): ꢂ ¼ 20.89
(2C), 21.02 (2C) (OAc), 43.25 (CH2Ise), 47.51 (CHFmoc), 61.98 (6-CH2), 66.42 (OCH2All), 66.97
(CH2Fmoc), 68.60, 71.27 (4=2-CH), 72.18 (5-CH), 72.49 (3-CH), 76.23 (CHIse), 100.40 (1-CH),
119.53 (¼CH2All), 120.25, 125.36, 127.36, 128.01, 131.62 (¼CH-All), 141.56, 144.14, 156.57,
168.88, 169.89, 170.13, 170.42, 170.82ppm; IR (KBr): ꢄꢂ¼ 1753, 1634, 1523 cmꢃ1; MS (FAB):
m=z ¼ 736.2 [M þ K]þ, 720.2 [Mþ Na]þ, 698.2 [Mþ H]þ.
Allyl 3-[(9-Fluorenylmethoxycarbonyl)amino]-2-(2S)-[(2,3,4,6-tetra-O-
acetyl-ꢀ-D-galactopyranosyl)oxy]propionate (18b-b, C35H39NO14)
17 (231 mg, 0.63 mmol) and 2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosyl trichloroacetimidate
(373 mg, 0.76 mmol) were reacted due to protocol 5. Purification by flash chromatography
(CHCl3:acetone ¼ 9:1, Rf(ꢁ) ¼ 0.32, Rf(ꢀ) ¼ 0.28). Yield 43 mg (9%) of a 1:1 mixture of 18b-a
and 18b-b and 312 mg (71%) 18b-b, foam; [ꢁ]D ¼ ꢃ12ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.1, CHCl3); 1H NMR
(CDCl3, 300 MHz, COSY): ꢂ ¼ 1.97 (s, 3H), 2.05 (s, 3H), 2.14 (s, 3H), 2.18 (s, 3H) (OAc),
3.57 (m, 1H, CH2Ise), 3.68–3.78 (m, 3H, CH2Ise, 5-H), 4.05–4.09 (m, 2H, 6-H2), 4.25 (t, 1H,
J ¼ 6.6 Hz, CHFmoc), 4.42 (m, 1H, CHIse), 4.46–4.55 (m, 3H, 1-H, CH2Fmoc), 4.65–4.70 (m, 2H,
OCH2All), 5.07 (dd, 1H, J ¼ 3.3, 10.2 Hz, 3-H), 5.25 (dd, 1H, J ¼ 7.8, 10.2 Hz, 2-H), 5.29–5.41
(m, 3H, 4-H, ¼CH2All), 5.45 (br.t, 1H, NHIse), 7.32–7.46 (m, 4H), 7.64–7.67 (m, 2H), 7.81 (d,
2H, J ¼ 7.5 Hz) ppm; 13C NMR (CDCl3, 75 MHz): ꢂ ¼ 20.76, 20.87, 20.91, 21.15 (OAc), 43.36
(CH2Ise), 47.51 (CHFmoc), 61.58 (6-CH2), 66.43 (OCH2All), 66.78 (CH2Fmoc), 67.13 (4-CH), 68.83
(2-CH), 70.68 (3-CH), 71.08 (5-CH), 76.42 (CHIse), 101.00 (1-CH), 119.56 (¼CH2All), 120.27,
125.29, 127.39, 128.02, 131.61 (¼CH-All), 141.63, 144.15, 156.63, 168.88, 170.40 (2C), 170.49,
170.62 ppm; IR (KBr): ꢄꢂ¼ 1745–1717, 1523 cmꢃ1; MS (FAB): m=z ¼ 720.2 [M þ Na]þ, 698.2
[M þ H]þ.
3-[9-Fluorenylmethoxycarbonyl)amino]-2-(2S)-[(2,3,4,6-tetra-O-
acetyl-ꢀ-D-glucopyranosyl)oxy]propionic acid (19a, C32H35NO14)
18a-b (140 mg, 0.20mmol) was deprotected due to protocol 6. Purification by flash chromatography
(eluent 1: CHCl3:MeOH:AcOH¼ 100:5:1, Rf ¼ 0.25; eluent 2: CHCl3:acetone:AcOH ¼ 20:20:1, Rf ¼
0.34). Yield 88 mg (67%), foamy solid; [ꢁ]D ¼ ꢃ14ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 0.6, CHCl3); 1H NMR
Ise
(CDCl3, 300 MHz): ꢂ ¼ 1.89 (s, 3H), 2.02 (s, 6H), 2.06 (s, 3H) (OAc), 3.52–3.68 (m, 3H, CH2
,
5-H), 4.04 (dd, 1H, J ¼ 1.8, 12.0 Hz, 6-Ha), 4.11 (dd, 1H, J ¼ 4.5, 12.0 Hz, 6-Hb), 4.18 (t, 1H,
J ¼ 6.6 Hz, CHFmoc), 4.39 (d, 2H, J ¼ 6.6 Hz, CH2Fmoc), 4.50 (d, 1H, J ¼ 7.8 Hz, 1-H), 4.98–5.05
(m, 2H, 2,4-H), 5.25 (dd, 1H, J ¼ 9.6, 9.6Hz, 3-H), 5.47 (t, J ¼ 5.5 Hz, NHIse), 7.28–7.42 (m, 4H),
7.58–7.61 (m, 2H), 7.74–7.77 (m, 2H) ppm; 13C NMR (CDCl3, 75MHz): ꢂ ¼ 20.70 (2C), 20.73, 20.81
(OAc), 43.17 (CH2Ise), 47.27 (CHFmoc), 61.79 (6-CH2), 66.91 (CH2Fmoc), 68.38, 71.21, 71.89, 72.29
(2,3,4,5-CH), 76.21 (CHIse), 100.31 (1-CH), 120.10, 125.24, 127.22, 127.89, 141.38, 143.89, 156.82,

Hexafluoroacetone as a Protecting and Activating Reagent
593
169.56, 170.25, 170.34, 170.73, 171.69ppm; IR (KBr): ꢄꢂ¼ 1751, 1630, 1522 cmꢃ1; MS (FAB):
m=z ¼ 696.2 [M þ K]þ, 680.2 [Mþ Na]þ, 658.2 [Mþ H]þ.
3-[9-Fluorenylmethoxycarbonyl)amino]-2-(2S)-[(2,3,4,6-tetra-O-
acetyl-ꢀ-D-galactopyranosyl)oxy]propionic acid (19b, C32H35NO14)
18b-b (140mg, 0.20mmol) was deprotected due to protocol 6. Purification by flash chromatography
(CHCl3:MeOH:AcOH¼ 100:5:1, Rf ¼ 0.26). Yield 99mg (75%), foamy solid; [ꢁ]D ¼ ꢃ10ꢁ cm3
1
g
ꢃ1 dmꢃ1 (c ¼ 0.6, CHCl3); H NMR (CDCl3, 300 MHz): ꢂ ¼ 1.89 (s, 3H), 2.00 (s, 3H), 2.04 (s,
3H), 2.13 (s, 3H) (OAc), 3.52–3.68 (m, 3H, CH2Ise, 5-H), 3.96–4.00 (m, 2H, 6-H2), 4.18 (t, 1H,
J ¼ 6.6 Hz, CHFmoc), 4.29 (m, 1H, CHIse), 4.37 (d, 1H, J ¼ 7.8Hz, 1-H), 4.39–4.48 (m, 2H, CH2Fmoc),
5.01 (dd, 1H, J ¼ 3.3, 10.2Hz, 3-H), 5.17 (dd, 1H, J ¼ 7.8, 10.2 Hz, 2-H), 5.32 (d, 1H, J ¼ 3.3Hz, 4-H),
5.57 (br.t, 1H, NHIse), 7.28–7.42 (m, 4H), 7.58–7.61 (m, 2H), 7.75 (d, 2H, J ¼ 7.5 Hz) ppm; 13C NMR
(CDCl3, 75MHz): ꢂ ¼ 20.52, 20.70 (2C), 20.92 (OAc), 43.38 (CH2Ise), 47.24 (CHFmoc), 61.46 (6-CH2),
66.74, 66.97, 68.85, 70.36, 70.81 (CH2Fmoc, 2,3,4,5-CH); 76.61 (CHIse), 100.89 (1-CH), 120.11,
125.20, 127.25, 127.89, 141.38, 143.85, 156.94, 170.25, 170.34, 170.49, 170.59, 172.34 ppm; IR
(KBr): ꢄꢂ¼ 1747, 1628, 1531 cmꢃ1; MS (FAB): m=z ¼ 696.1 [Mþ K]þ, 680.2 [Mþ Na]þ, 658.2
[Mþ H]þ.
Allyl 2-(2S)-Hydroxy-3-[3-(2,3,4,6-tetra-O-acetyl-
ꢀ-D-glucopyranosyl)ureido]propanoate (20, C21H30N2O13)
A mixture of 6a-b (507 mg, 0.81mmol) and allyl alcohol (3cm3) in dry chloroform (3cm3) was heated
under reflux for 2 d. After removal of the volatiles under reduced pressure, the residue was purified
by flash chromatography (ethyl acetate, Rf ¼ 0.36). Yield 306 mg (73%), colorless foam, which was
contaminated by 5–10% of the ꢁ-anomer; [ꢁ]D ¼ ꢃ4ꢁ cm3 gꢃ1 dmꢃ1 (c ¼ 1.0, CHCl3); 1H NMR
(CDCl3, 300 MHz): ꢂ ¼ 2.00 (s, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.07 (s, 3H) (OAc), 3.52–3.66 (m,
2H, CH2Ise), 3.82 (ddd, 1H, J ¼ 2.1, 4.8, 9.9 Hz, 5-H), 4.08 (dd, 1H, J ¼ 1.8, 12.3Hz, 6-Ha), 4.30 (dd,
1H, J ¼ 4.8, 12.3 Hz, 6-Hb), 4.33 (m, 1H, CHIse), 4.68 (d, 2H, J ¼ 5.7 Hz, OCH2All), 4.91 (dd, 1H,
J ¼ 9.6, 9.6 Hz), 5.05 (dd, 1H, J ¼ 9.9, 9.3 Hz), 5.13 (dd, 1H, J ¼ 9.3, 9.6 Hz) (4,2,1-H), 5.25–5.37 (m,
3H, ¼CH2All, 3-H), 5.43 (t, 1H, J ¼ 6.0 Hz, NHIse), 5.78 (d, 1H, J ¼ 9.3 Hz, 1-NH), 5.91 (m, 1H,
¼CH-All) ppm; 13C NMR (CDCl3, 75 MHz, APT): ꢂ ¼ 20.83, 20.86, 21.00 (2C) (OAc), 43.88 (CH2Ise),
62.18 (6-CH2), 66.71 (OCH2All), 68.57, 70.79, 70.90, 73.34 (2C) (2,3,4,5-CH, CHIse), 80.13 (1-CH),
119.49 (¼CH2All), 131.64 (¼CH-All), 157.57, 169.98, 170.22, 171.05, 171.32, 173.10ppm; IR (KBr):
ꢄꢂ¼ 3400, 1700, 1684, 1635, 1542 cmꢃ1; MS (FAB): m=z ¼ 519.2 [Mþ H]þ.
Allyl 2-(2S)-[(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl)oxy]-3-
[3-(2,3,4,6-tetra-O-acetyl-ꢀ-D-glucopyranosyl)ureido]propanoate (21, C35H48N2O22)
20 (160 mg, 0.31mmol) and 2,3,4,6-tetra-O-acetyl-ꢁ-D-glucopyranosyl trichloroacetimidate (183 mg,
0.37mmol) were reacted due to protocol 5. Purification by flash chromatography (gradient elution,
CHCl3:acetone¼ 5:1 ! 3:1, Rf ¼ 0.28). Yield 130 mg (49%), foamy solid, [ꢁ]D ¼ þ1ꢁ cm3 gꢃ1 dmꢃ1
1
(c ¼ 1.0, DCM); H NMR(CDCl3, 300 MHz, COSY): ꢂ ¼ 1.99–2.14 (several s, OAc), 3.46–3.54 (m,
1H, CH2Ise), 3.64–3.74 (m, 2H, CH2 , 5- or 50-H), 3.81 (ddd, 1H, J ¼ 2.1, 4.2, 10.2Hz, 5- or 50-H),
Ise
4.07 (dd, 2H, J ¼ 2.1, 12.3Hz, 6-Ha, 60-Hb), 4.23–4.31 (m, 2H, CHIse, 6-Hb or 60-Hb), 4.52 (d, 1H,
J ¼ 8.1 Hz, 10-H), 4.50 (dd, 1H, J ¼ 2.1, 12.6Hz, 6-Hb or 60-Hb), 4.52 (d, 1H, J ¼ 8.1 Hz, 10-H), 4.60 (d,
All
2H, J ¼ 5.7 Hz, OCH2 ), 5.90 (dd, 1H, J ¼ 9.3, 9.6 Hz, 2-H), 4.98 (dd, 1H, J ¼ 8.1, 9.3 Hz, 20-H),
5.02–5.31 (m, 8H, 4,40,1,3,30-H, NHIse, ¼CH2All), 5.88 (m, 1H, ¼CH-All), 6.03 (d, 1H, J ¼ 9.6 Hz, 1-
NH) ppm; 13C NMR (CDCl3, 75 MHz, APT, HECTCOR): ꢂ ¼ 20.86 (4C), 20,99 (2C), 21.09, 21.15
Ise
All
(OAc), 42.32 (CH2 ), 61.34, 62.12 (6,60-CH2), 66.43 (OCH2 ), 68.22, 68.49 (4,40-CH), 70.79, 71.18

€
C. Bottcher et al.
594
(2-,20-H), 72.35 (3- or 30-H), 77.47 (CH2 ), 72.44, 73.19 (5, 50-H), 73.52 (3-, 30-H), 77.47 (CHIse),
80.17 (1-CH), 100.89 (10-H), 119.49 (¼CH2All), 131.61 (¼CH-All), 156.70, 168.82, 169.60, 169.88,
170.22, 170.40, 170.45, 170.60. 170.91, 171.73ppm; IR (KBr): ꢄꢂ¼ 1751, 1697–1633, 1558 cmꢃ1; MS
(FAB): m=z ¼ 871.3 [M þ Na]þ, 849.3 [Mþ H]þ.
Ise
Acknowledgements
The authors are grateful to DFG and VW-Stiftung, Hannover, for financial support.
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