Tn Antigen Mimics by Ring-Opening of Chiral Cyclic Sulfamidates with Carbohydrate C1- S- and C1- O-Nucleophiles

Article abstract of DOI:10.1021/acs.joc.7b03225

Starting from commercially available (S)-isoserine and effectively accessible (S)-α-methylserine, enantiopure cyclic sulfamidates have been prepared as chiral building blocks for the synthesis of various S- and O-glycosylated amino acid derivatives, inclu

Full text of DOI:10.1021/acs.joc.7b03225

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Tn Antigen Mimics by Ring-Opening of Chiral Cyclic
Sulfamidates with Carbohydrate C1-S- and C1-O-Nucleophiles
Pablo Tovillas, Iván García, Paula Oroz, Nuria Mazo, Alberto Avenoza, Francisco
Corzana, Gonzalo Jiménez-Osés, Jesús H Busto, and Jesús M. Peregrina
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b03225 • Publication Date (Web): 11 Apr 2018
Downloaded from http://pubs.acs.org on April 11, 2018
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Tn Antigen Mimics by Ring-Opening of Chiral
Cyclic Sulfamidates with Carbohydrate C1-S-
and C1-O-Nucleophiles
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Pablo Tovillas, Iván García, Paula Oroz, Nuria Mazo, Alberto Avenoza, Francisco
Corzana, Gonzalo Jiménez-Osés, Jesús H. Busto* and Jesús M. Peregrina*
Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La
Rioja, 26006 Logroño, La Rioja, Spain
hector.busto@unirioja.es; jesusmanuel.peregrina@unirioja.es
ABSTRACT
Starting from commercially available (S)ꢀisoserine and effectively accessible (S)ꢀαꢀ
methylserine, enantiopure cyclic sulfamidates have been prepared as chiral building blocks
for the synthesis of various Sꢀ and Oꢀglycosylated amino acid derivatives, including
unnatural variants of the Tn antigen, through highly chemoꢀ, regioꢀ and stereoselective
nucleophilic ringꢀopening reactions with carbohydrate C1ꢀSꢀ and C1ꢀOꢀnucleophiles.
TOC
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INTRODUCTION
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MUC1 mucin is an Oꢀglycoprotein that plays a pivotal role in the renewal and
differentiation of the epithelium, cell adhesions, immune response and cell signalling.1 In
healthy cells, the mucin peptide is decorated with complex oligosaccharides; however,
when overexpressed in tumour cancer cells, the backbone appears with simple, truncated
carbohydrates. Incomplete glycosylation exposes glycopeptide epitopes, normally masked
in healthy cells, to the immune system. Such tumour associated carbohydrate antigens [e.g.,
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Tn antigen (αꢀDꢀGalpNAcꢀLꢀSer/Thr)] are thus attractive targets for the development of
therapeutic cancer vaccines. However, to date, none of these vaccines based on this antigen
have succeeded in clinical trials. The main drawback with therapeutic vaccines is that
cancer cells can generate immune escape mechanisms,2 which results in an increased
tolerance of the antigens by the immune system.
An attractive approach to tackle this issue may be the use of unnatural derivatives that
mimic the structure of the Tn antigen.3 Because of this, nowadays, there is an active field of
research focused on the design of the Tn antigen mimics acting as better candidates for
anticancer vaccine generation.4,5 Our group has contributed to this field by synthetizing and
evaluating a new cancer vaccine incorporating αꢀ
methylserine).6
DꢀGalpNAcꢀαꢀMeSer (αꢀMeSer = αꢀ
Linkage of αꢀGalNAc to Ser, Thr or, in general, hydroxyamino acids is a quite difficult
synthetic operation due to the presence of the C2ꢀacetamido group in 1,2ꢀcis disposition.
This group often directs glycosylation to form a βꢀlinkage due to neighboring effects. These
effects are particularly relevant when the carbohydrate acts as an electrophile, often through
a planar, prochiral oxocarbenium intermediate. In our case, the unnatural glycosyl amino
acid (αꢀ
D
ꢀGalpNAcꢀαꢀMeSer) was prepared by two different methodologies, both based on
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the nucleophilic attack of protected αꢀMeSer to carbohydrate electrophiles such as glycosyl
halides (KoenigsꢀKnorr)6 or 2ꢀnitrogalactal (OꢀMichael addition).5,7 These methodologies
give mixtures of both αꢀ and βꢀanomers, leading in the best conditions to ca. 25% of the
undesired βꢀderivative, therefore they have the drawback of needing tedious
chromatographic separation from the reaction mixture. One way of mitigating such
neighboring effects is by using nucleophilic carbohydrates in which the anomeric carbon
provides the atom defining the glycosydic bond (normally O or S), and maintains its
configuration upon glycosylation.
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Along these lines, several groups including ours have developed alternative
glycosylation methods using the wellꢀestablished sulfamidate chemistry.8 Hence, we
designed a versatile synthetic methodology based on the ringꢀopening of hindered cyclic
sulfamidates derived from αꢀmethylisoserine, with various 1ꢀthiocarbohydrates as
nucleophiles.9 We demonstrated that this reaction proceeds with total inversion of the
configuration at the quaternary electrophilic carbon, preserving the enantiomeric excess of
the starting material. The synthesis and subsequent ringꢀopening of cyclic sulfamidates
derived from Lꢀserine and Lꢀthreonine with some 1ꢀthiocarbohydrates have also been
reported.10 However, nucleophilic displacement reactions involving oxygenated
nucleophiles derived from carbohydrates, especially pyranose C1ꢀOꢀhemiacetals, are often
problematic due to their poorer nucleophilicity and higher basicity compared to 1ꢀ
thiocarbohydrates. To the best of our knowledge, this type of reaction has only been
achieved using aziridineꢀ2ꢀcarboxamides as electrophiles,11 leading to αꢀ and βꢀOꢀglycosyl
serine conjugates in a highly stereoselective manner.
RESULTS AND DISCUSSION
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Inspired by the results reported with aziridineꢀ2ꢀcarboxamides,11 and encouraged by the
good results obtained with 1ꢀthiocarbohydrates using different sulfamidates derived from
amino acids, we planned a new synthetic route to access mimics of the Tn antigen
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featuring αꢀmethylserine (αꢀ
DꢀGalpNAcꢀαꢀMeSer) and isoserine (αꢀDꢀGalpNAcꢀαꢀisoSer),
by combining both synthetic strategies.
First, enantiopure sulfamidate 1 was synthesized from protected (S)ꢀαꢀmethylserine, which
in turn can be accessed in a gram scale from Lꢀserine following our published protocol.12
Cyclic sulfamidate was formed by using the wellꢀestablished twoꢀstep protocol consisting
on treatment of protected amino acid with thionyl chloride in the presence of imidazole and
subsequent oxidation of the intermediate sulfamidites with RuCl3/NaIO4. As a proofꢀofꢀ
concept, the subsequent nucleophilic ringꢀopening with (AcO)3GalpNAcꢀαꢀOH as a
pyranose C1ꢀOꢀnucleophile,13 using sodium hydride as a base in DMF, led to the desired
protected αꢀDꢀGalpNAcꢀαꢀMeSer 2 in good yield and as a single α anomer without further
chromatographic purification. Importantly, competitive elimination or deprotection
reactions observed with natural analogues14 were not observed. (Scheme 1).
Scheme 1. Synthesis and ringꢀopening reaction of αꢀMeSerꢀsulfamidate 1 with
(AcO)3GalpNAcꢀαꢀOH to obtain the Tn antigen mimic 2.
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The scope of this new methodology was extended to other carbohydrates such as glucose.
Hence, we carried out the ringꢀopening reaction of sulfamidate 1 with a mixture of αꢀ and
βꢀanomers (α/β = 9:1) of tetraꢀOꢀacetylꢀDꢀglucopyranose hemiacetal, (AcO)4GlcpꢀOH,15
under the same conditions described above (Scheme 2). The corresponding protected αꢀOꢀ
glucosylꢀαꢀmethylserine 3 was efficiently obtained with the same high diastereomeric ratio
of the starting carbohydrate (3α/3β = 9:1).
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Scheme 2. Ringꢀopening reaction of αꢀMeSer sulfamidate 1 with (AcO)4GlcpꢀOH.
We next extended this synthetic protocol to access new unnatural Tn antigen mimics,
namely glycosyl β2ꢀamino acids. The proteolytic stability of glycosylated βꢀpeptides toward
glycosidases has been studied, showing that glycoꢀβꢀpeptides are stable to degradation by
proteolytic enzymes.16 Additionally, the synthesis of new conjugates composed of βꢀ or α,βꢀ
peptides functionalized with biologically active carbohydrate residue constitutes an
important platform for dual structure–activity studies.17 These results emphasize the
potential use of βꢀpeptides functionalized with carbohydrates for biological and biomedical
investigations. Therefore, development of robust methodologies towards suitably protected,
enantiopure αꢀ or βꢀglycosylꢀβꢀamino acids building blocks is synthetically valuable.
Starting from commercially available (S)ꢀisoserine 4, we first prepared several enantiopure
isoserine derivatives 5ꢀ9, which were used as starting material to synthesize enantiopure
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cyclic sulfamidates 10ꢀ12 following standard methodologies (Scheme 3 and Supporting
Information). Different protecting schemes were selected for these new sulfamidates in
order to test the tolerance of our ringꢀopening methodology to different functional groups.
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Scheme 3. Synthesis of cyclic sulfamidates derived from (S)ꢀisoserine 4.
Before synthesizing the required glycoconjugates, we examined the reactivity of this new
isoserineꢀderived sulfamidates towards ringꢀopening reactions with sodium azide and
cesium paranitrobenzoate as probe nucleophiles. The stereochemical outcome of the ringꢀ
opening reaction was confirmed to proceed with complete inversion of configuration at the
Cα of isoserine sulfamidate, by derivatizing the corresponding adducts to known
compounds 2,3ꢀdiaminopropionic acid 13 (βꢀaminoalanine, Scheme 4) and NꢀBocꢀ(R)ꢀ
isoserine methyl ester 14 (Scheme 5) with matching optical properties.
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Scheme 4. Ringꢀopening reaction of isoserine sulfamidate 10 with sodium azide and
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synthesis of βꢀaminoalanine.
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Scheme 5. Ringꢀopening reaction of isoserine sulfamidate 10 with a Oꢀnucleophile and
synthesis of a protected (R)ꢀisoserine derivative.
The reactivity of these electrophiles towards glycosylation was first assayed with
configurationally stable αꢀ and βꢀ1ꢀthiocarbohydrates. Due to the growing importance of Sꢀ
glycopeptides18 as mimics of Oꢀglycopeptides with singular properties, cysteine Sꢀ
glycosylation, such as that found in glycopeptide bacteriocins,19 has emerged as a powerful
and synthetically accessible postꢀtranslational modification. Reaction of sulfamidates 10-12
with tetraꢀOꢀacetylꢀβꢀ1ꢀthioꢀDꢀglucopyranose as a nucleophile using DBU as a base in
DMF, led to protected β-Sꢀglycosylꢀβ2ꢀamino acids 15-17 in good yields (panel A, Scheme
6). Analogous treatment of sulfamidates 11 and 12 with triꢀOꢀacetylꢀαꢀ1ꢀthioꢀNꢀ
acetylglucosamine produced α-Sꢀglycosylꢀβ2ꢀamino acids 18 and 19, respectively (panel B,
Scheme 6). In both cases, the configuration of the glycosidic bond was fully maintained
with respect to the anomeric configuration of the starting carbohydrates, as observed in the
1H NMR spectra.
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Scheme 6. Ringꢀopening reactions of isoserine sulfamidates with Sꢀ and Oꢀcarbohydrates
as nucleophiles.
Finally, we assayed the ringꢀopening of sulfamidates 11 and 12 with tetraꢀOꢀacetylꢀαꢀDꢀ
glucopyranose,
(AcO)4GlcpꢀαꢀOH,15
and
triꢀOꢀacetylꢀαꢀDꢀNꢀacetylgalactosamine,
(AcO)3GalpNAcꢀαꢀOH,13 respectively, as pyranose C1ꢀOꢀnucleophiles using sodium
hydride as a base in DMF (panel C, Scheme 6). As a result, αꢀOꢀglucosylꢀβ2ꢀamino acid 20
was obtained with high anomeric selectivity (20α/20β = 9:1, the same ratio as the starting
material). Most importantly, αꢀOꢀGalpNAcꢀβ2ꢀamino acid 21 was obtained in good yield
and as single α anomer. In both cases, nucleophilic attack occurred with complete inversion
of configuration at the reacting center, and competitive elimination or deprotection
reactions were not observed in any case.
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αꢀAnomer 21 is particularly attractive, since it is a protected structural analogue of the Tn
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antigen,20 in which the αꢀ
DꢀGalpNAc is Oꢀlinked to isoserine (αꢀDꢀGalpNAcꢀisoSer).
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Once the protected Tn antigen mimics 2 and 21 were obtained and considering the novelty
of these structures, in order to demonstrate that the carbohydrate and amino acid moieties
can be liberated without breaking the glycosidic bond, removal of the protecting groups
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were carried out in two steps to achieve the corresponding free Tn antigen mimics αꢀ
Dꢀ
GalpNAcꢀαꢀMeSer (22) and αꢀ ꢀGalpNAcꢀαꢀisoSer (23), respectively. First, protected
D
glycosyl amino acids were treated with lithium hydroxide monohydrate (LiOH·H2O) at
room temperature, using a mixture of methanol/water (2:1) as a solvent. The basic
1
hydrolysis reaction was followed by TLC until starting material disappeared and by H
t
NMR to test the hydrolysis of ester groups (AcOꢀ of carbohydrate moiety and ꢀCO2 Bu or ꢀ
CO2Me of amino acid moiety). The basic solution was neutralized with ion exchanger
Dowex® 50WꢀX8, filtered, and evaporated to give the glycosyl amino acid whose amino
group is already protected as Boc carbamate. Transformation of Boc to amino group was
carried out by treatment with trifluoracetic acid (TFA) in dichloromethane at room
temperature. Hence, the corresponding unprotected glycosyl amino acids 22 (87%) and 23
(84%) were obtained without further purification (Scheme 7).
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Scheme 7. Deprotection of glycosyl amino acids 2 and 21 to obtain the free Tn antigen
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mimics αꢀDꢀGalpNAcꢀαꢀMeSer (22) and αꢀDꢀGalpNAcꢀαꢀisoSer (23), respectively.
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CONCLUSION
In summary, a new family of differently protected, enantiopure cyclic sulfamidates have
been prepared from effectively accessible (S)ꢀαꢀmethylserine and commercially available
(S)ꢀisoserine. Such sulfamidates have been used as efficient electrophiles for glycosylation
with C1ꢀSꢀ and C1ꢀOꢀcarbohydrates via highly chemoꢀ, regioꢀ and stereoselective ringꢀ
opening reaction in very mild conditions and with complete functional group tolerance. Of
note, the special architecture of this unnatural amino acid derivatives precludes the
undesired elimination reactions commonly observed on their natural analogues derived
from
LꢀSer and LꢀThr with basic nucleophiles. This methodology allows Sꢀ and Oꢀ
glycosylation in an efficient and highly stereocontrolled manner, even with challenging
carbohydrates such as αꢀGalpNAc. As a result, analogues of the Tn antigen derived from αꢀ
methylated and β2ꢀamino acids are now accessible.
EXPERIMENTAL SECTION
General and Experimental Methods. Commercial reagents were used without further
purification. Analytical thin layer chromatography (TLC) was performed on Machereyꢀ
Nagel precoated aluminium sheets with a 0.20 mm thickness of silica gel 60 with
fluorescent indicator UV254. TLC plates were visualized with UV light and by staining with
phosphomolybdic acid (PMA) solution (5 g of PMA in 100 mL of absolute ethanol) or
sulfuric acidꢀethanol solution. Column chromatography was performed on silica gel
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(230−400 mesh). H and C NMR spectra were measured with a 400 MHz spectrometer
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with TMS as the internal standard. Multiplicities are quoted as singlet (s), broad singlet (br
s), doublet (d), doublet of doublets (dd), triplet (t), or multiplet (m). Spectra were assigned
using COSY and HSQC experiments. All NMR chemical shifts (δ) were recorded in ppm
and coupling constants (J) were reported in Hz. The results of these experiments were
processed with MestreNova software. High resolution electrospray mass (ESI) spectra were
recorded on a microTOF spectrometer; accurate mass measurements were achieved by
using sodium formate as an external reference.
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General Procedure for the Synthesis of Chiral Cyclic Sulfamidates. To a solution of
imidazole (1.06 g, 15.7 mmol) in dichloromethane (15 mL), another solution of thionyl
chloride (0.33 mL, 4.7 mmol) in dichloromethane (5 mL) was slowly added, at 0ꢀ5 °C for
15 min. The reaction mixture was then stirred at room temperature for 1 h and then cooled
o
to ꢀ10 C. To the resulting suspension, a solution of the corresponding NꢀBocꢀprotected
hydroxyamino ester (2.6 mmol), in dichloromethane (8 mL) was added over 30 min at ꢀ10o
C and the mixture was then stirred at room temperature for 2 h. To the resulting suspension,
water (30 mL) was added and the mixture was stirred at room temperature for 10 min. The
organic phase was washed with 10% aqueous citric acid (25 mL) and brine (25 mL) and
dried over sodium sulphate. The solids were removed by filtration and washed with
dichloromethane. The combined filtrates were mixed with a 10% aqueous sodium periodate
o
solution (25 mL) and cooled to 0 C. To the well stirred mixture, ruthenium (III) chloride
o
hydrate (6 mg, 0.26 mmol) was added and the reaction was vigorously stirred at 0 C for 2
h and additional 2 h at room temperature. The organic phase was washed with 10% aqueous
sodium ascorbate solution (8 mL) and filtered over silica gel. After evaporation of the
volatiles, the product was chromatographed to give the corresponding sulfamidate.
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3-(tert-Butyl) 4-methyl (S)-4-methyl-1,2,3-oxathiazolidine-3,4-dicarboxylate 2,2-dioxide
(1). Following the general procedure described above for the synthesis of chiral cyclic
sulfamidates and starting from (S)ꢀNꢀBocꢀαꢀMeSerꢀOMe (606 mg, 2.6 mmol), sulfamidate
1 was obtained as a white solid in a 90% yield (690 mg), after purification by a column
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chromatography with hexane/ethyl acetate (3:2). Physical data: mp 113ꢀ115 °C. [α]D (c
1.00, CHCl3): ꢀ10.7. HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for C10H17NO7SNa+
1
318.0618; Found 318.0627. H NMR (400 MHz, CDCl3)
δ (ppm): 1.56 (s, 9H, C(CH3)3),
1.81 (s, 3H, CαCH3), 3.85 (s, 3H, CO2CH3), 4.32 (d, 1H, J = 9.3 Hz CH2β), 4.64 (d, 1H, J
= 9.3 Hz, CH2β).13C NMR (100 MHz, CDCl3)
δ
(ppm): 20.6 (CαCH3), 27.9 (C(CH3)3),
53.7 (CO2CH3), 65.5 (CHα), 72.7 (CH2β), 86.1 (C(CH3)3), 147.9, 169.2 (CO).
(S)-3-tert-Butyl-5-methyl-1,2,3-oxathiazolidine-3,5-dicarboxylate
2,2-dioxide
(10).
Following the general procedure described above for the synthesis of sulfamidates, and
starting from NꢀBocꢀ(S)ꢀisoserine methyl ester (7) (1.705 g, 7.8 mmol), sulfamidate 10 was
obtained as a white solid in an 80% yield (1.80 g), after purification by a column
chromatography with hexane/ethyl acetate (2:1). Physical data: mp 75ꢀ77 °C. [α]D20 (c 1.00,
CHCl3): +0.9. HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for C9H15NO7SNa+ 304.0467;
Found 304.0481. 1H NMR (400 MHz, CDCl3)
δ (ppm): 1.55 (s, 9H, C(CH3)3), 3.90 (s, 3H,
CO2CH3), 4.17 (dd, 1H, J = 10.1, 7.4 Hz CH2β), 4.26 (dd, 1H, J = 10.1, 7.4 Hz, CH2β),
5.17 (t, 1H, J = 7.2 Hz, CHα). 13C NMR (100 MHz, CDCl3)
δ
(ppm): 28.0 (C(CH3)3), 47.1
(CH2β), 53.8 (CO2CH3), 72.5 (CHα), 86.3(C(CH3)3), 148.3, 165.6 (CO).
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(S)-5-Benzyl-3-tert-Butyl
1,2,3-oxathiazolidine-3,5-dicarboxylate
2,2-dioxide
(11).
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8
Following the general procedure described above for the synthesis of sulfamidates, and
starting from hydroxyamino ester 8 (767 mg, 2.6 mmol), sulfamidate 11 (650 mg, 70%)
was obtained as a white solid, after column chromatography in hexane/ethyl acetate (1:1).
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Physical data: mp 65ꢀ67 °C. [α]D (c 1.00, CHCl3): +3.1. HRMS (ESIꢀTOF) m/z: [M +
Na]+ Calcd for C15H19NO7SNa+ 380.0780; Found 380.0801. 1H NMR (400 MHz, CDCl3)
δ
(ppm): 1.54 (s, 9H, C(CH3)3), 4.14 (dd, 1H, J = 10.2, 7.4 Hz, CH2β), 4.24 (dd, 1H, J =
10.2, 7.3 Hz, CH2β), 5.16 (t, 1H, J = 7.2 Hz, CHα), 5.30 (s, 2H, CH2Ph), 7.38 (s, 5H,
arom). 13C NMR (100 MHz, CDCl3)
δ (ppm): 28.0 (C(CH3)3), 47.1 (CH2β), 68.9 (CH2Ph),
72.5 (CHα), 86.3 (C(CH3)3), 128.8, 129.0, 129.2, 134.1 (arom), 148.3, 164.9 (CO).
(S)-di-tert-Butyl 1,2,3-oxathiazolidine-3,5-dicarboxylate 2,2-dioxide (12). Following the
general procedure described above for the synthesis of sulfamidates, and starting from
hydroxyamino ester 9 (679 mg, 2.6 mmol), sulfamidate 12 (647 mg, 77%) was obtained as
a white solid, after column chromatography in hexane/ethyl acetate (4:1). Physical data: mp
93ꢀ95 °C. [α]D (c 1.00, CHCl3): +2.1. HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for
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C12H21NO7SNa+ 346.0936; Found 346.0899. 1H NMR (400 MHz, CDCl3)
δ (ppm): 1.53 (s,
9H, C(CH3)3), 1.55 (s, 9H, C(CH3)3), 4.10 (dd, 1H, J = 10.2, 7.6 Hz CH2β), 4.21 (dd, 1H, J
= 10.2, 7.2 Hz, CH2β), 5.04 (t, 1H, J = 7.3 Hz, CHα). 13C NMR (100 MHz, CDCl3)
δ
(ppm): 28.0 (2C(CH3)3), 47.4 (CH2β), 72.9 (CHα), 85.4 (C(CH3)3), 86.0 (C(CH3)3), 148.4,
163.8 (CO).
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General Procedure for the Ring-opening Reactions of Sulfamidates with
Carbohydrate C1-O-Nucleophiles. Sodium hydride 60% dispersion in mineral oil (12 mg,
0.30 mmol) was added to a solution of the corresponding protected carbohydrate C1ꢀOꢀ
nucleophile (0.30 mmol) in dry DMF (2 mL) in a nitrogen atmosphere using standard
schlenk techniques. The resulting mixture was stirred at room temperature for 5 min and it
was then added by cannula to a solution of the corresponding sulfamidate (0.30 mmol) in
dry DMF (1 mL). The reaction mixture was kept at room temperature and stirred for 20
min. After that time, the volatiles were removed, and the residue dissolved in a mixture of
20% aqueous H2SO4/dichloromethane (1:1), which was stirred for 30 min at room
temperature to hydrolyze the sulfamic acid intermediate. The reaction crude was isolated
after extraction with dichloromethane (2 x 5 mL), dried over anhydrous Na2SO4, and
concentrated. That crude was purified by silica gel column chromatography and the
corresponding protected glycosyl amino acid was obtained.
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(2S)-O-((3',4',6')-Tri-O-acetyl-2’-acetamido-2’deoxy-α-D-galactopyranosyl)-N-(tert-
butoxycarbonyl)-2-methylserine methyl ester (2). Following the general procedure
described above for the ringꢀopening reactions of sulfamidates with carbohydrate C1ꢀOꢀ
nucleophiles, and
starting from 3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀαꢀDꢀ
galactopyranose (105 mg, 0.30 mmol) and sulfamidate 1 (88 mg, 0.30 mmol), protected
glycosyl amino acid 2 (119 mg, 71%) was obtained as an oil, after column chromatography
20
in hexane/ethyl acetate (1:1). Physical data: [α]D (c 1.00, CHCl3): +61.3. HRMS (ESIꢀ
1
TOF) m/z: [M + Na]+ Calcd for C24H38N2O13Na+ 585.2272; Found 585.2270. H NMR
(400 MHz, CDCl3) δ (ppm): 1.47 (s, 9H, C(CH3)3), 1.49 (s, 3H, CαCH3), 1.99 (s, 3H,
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NHCOCH3), 2.00 (s, 3H, CH3CO), 2.07 (s, 3H, CH3CO), 2.17 (s, 3H, CH3CO), 3.77 (s, 3H,
CO2CH3), 3.79ꢀ3.84 (d, 1H , J = 10.4 Hz, CH2β), 4.01ꢀ4.19 (m, 4H, 1CH2β, 2H6s, H5s), 4.57
(ddd, 1H, J = 11.3, 9.6, 3.7 Hz, H2s), 4.93 (d, J = 3.7 Hz, H1s), 5.08 (dd, J = 11.3, 3.3 Hz,
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H3s), 5.32 (s, 1H, NHBoc), 5.38 (d, J = 3.2 Hz, H4s), 6.15 (d, 1H, J = 9.6 Hz, NHAc). C
NMR (100 MHz, CDCl3) δ (ppm): 20.7, 20.8, 20.9 (3CH3CO), 23.3 (NHCOCH3), 28.1
(NCO2C(CH3)3), 28.5 (CαCH3), 42.9 (CH2β), 51.6 (C2s), 62.0 (C6s), 68.3 (C4s), 68.8 (C5s),
71.5 (C3s), 76.4 (CHα), 80.2 (C(CH3)3), 83.5 (CO2C(CH3)3), 98.5 (C1s), 155.7, 169.4, 170.8,
171.2 (CO).
(2S)-O-((2',3',4',6')-Tetra-O-acetyl-D-glucopyranosyl)-N-(-tert-butoxycarbonyl)-2-
methylserine methyl ester (3). Following the general procedure described above for the
ringꢀopening reactions of sulfamidates with carbohydrate C1ꢀOꢀnucleophiles, and starting
from 2,3,4,6ꢀtetraꢀOꢀacetylꢀαꢀDꢀglucopyranose (100 mg, 0.29 mmol) and sulfamidate 1 (50
mg, 0.17 mmol), protected glycosyl amino acid 3 (53 mg, 55%) was obtained as an oil in a
9:1 ratio in favor of the αꢀanomeric isomer, after column chromatography in hexane/ethyl
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acetate (1:1). Physical data: [α]D (c 1.00, CHCl3): +40.5. HRMS (ESIꢀTOF) m/z: [M +
1
Na]+ Calcd for C24H37NO14Na+ 586.2106; Found 586.2113. H NMR (300 MHz, CDCl3) δ
(ppm): 1.44 (s, 9H NCO2C(CH3)3), 1.52 (s, 3H, CαCH3), 2.01 (s, 3H, COCH3), 2.02 (s, 3H,
COCH3), 2.05 (s, 3H, COCH3), 2.10 (s, 3H, COCH3), 3.70ꢀ3.75 (m, 1H, CH2β), 3.75 (s,
3H, CO2CH3), 3.95ꢀ4.02 (m, 1H, H5s), 4.06ꢀ4.13 (m, 2H, 1 CH2β, 1 H6s), 4.23ꢀ4.30 (m, 1H,
1 H6s), 4.83 (dd, 1H, J = 10.2, 3.7 Hz, H2s), 5.01ꢀ5.07 (m, 2H, H1s, H4s), 5.37ꢀ5.45 (m, 2H,
13
H3s, NHBoc). C NMR (75 MHz, CDCl3) δ (ppm): 20.7 (4CH3CO), 20.9 (CαCH3), 28.4
(NCO2C(CH3)3), 52.9 (CO2CH3), 59.8 (Cα), 61.8 (C6s), 67.6 (C5s), 68.4 (C4s), 70.3 (C3s),
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70.3 (C2s), 70.9 (Cβ), 96.4 (C1s), 154.5 (NCO2C(CH3)3), 169.7, 170.2, 170.3, 170.3, 170.8,
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173.0 (CO).
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(2R)-O-((2',3',4',6')-Tetra-O-acetyl-D-glucopyranosyl)-N-(tert-butoxycarbonyl)isoserine
benzyl ester (20). Following the general procedure described above for the ringꢀopening
reactions of sulfamidates with carbohydrate C1ꢀOꢀnucleophiles, and starting from 2,3,4,6ꢀ
tetraꢀOꢀacetylꢀαꢀDꢀglucopyranose (70 mg, 0.20 mmol) and sulfamidate 11 (72 mg, 0.20
mmol), protected glycosyl amino acid 20 (89 mg, 71%) was isolated as a colorless oil in a
9:1 ratio in favor of αꢀanomeric isomer, after column chromatography in hexane/ethyl
acetate (5.5:4.5). Physical data: [α]D20 (c 1.00, CHCl3): +54.3. HRMS (ESIꢀTOF) m/z: [M +
1
Na]+ Calcd for C29H39NO14Na+ 648.2268; Found 648.2271. H NMR (400 MHz, CDCl3)
δ
(ppm): 1.44 (s, 9H, C(CH3)3), 2.00ꢀ2.06 (m, 9H, Ac), 2.09 (s, 3H, Ac), 3.54ꢀ3.62 (m, 2H,
CH2β), 4.03ꢀ4.15 (m, 2H, H6s, H5s), 4.27 (dd, 1H, J = 12.3, 4.0 Hz, H6s), 4.38 (t, 1H, J =5.0
Hz, CHα), 4.87 (dd, 1H, J= 12.4, 4.3 Hz, H3s), 4.91ꢀ4.99 (m, 1H, NHBoc), 5.06 (t, 1H, J =
9.9 Hz, H4s), 5.12ꢀ5.18 (m, 2H, CH2Ph), 5.25 (d, 1H, J = 3.3 Hz, H1s), 5.51 (t, 1H, J = 9.8
13
Hz, H3s), 7.29ꢀ7.41 (m, 5H, arom). C NMR (100 MHz, CDCl3)
δ (ppm): 20.8 (CH3CO),
28.5 (C(CH3)3), 42.7 (CH2β), 61.9 (C6s), 67.5 (CH2Ph), 68.2 (C5s), 68.7 (C4s), 70.0 (C3s),
70.5 (C2s), 74.2 (CHα), 80.1 (C(CH3)3), 95.0 (C1s), 128.2, 128.8, 128.9, 135.3 (arom),
155.4, 169.3, 169.7, 170.6, 170.9, 171.1, 171.6 (CO).
(2R)-O-((3',4',6')-Tri-O-acetyl-2’-acetamido-2’-deoxy-α-D-galactopyranosyl)-N-(tert-
butoxycarbonyl)isoserine tert-butyl ester (21). Following the general procedure described
above for the ringꢀopening reactions of sulfamidates with carbohydrate C1ꢀOꢀnucleophiles,
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and starting from 3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀαꢀDꢀgalactopyranose (70 mg,
0.20 mmol) and sulfamidate 12 (69 mg, 0.20 mmol), protected glycosyl amino acid 21 (42
mg, 68%) was obtained as an oil, after column chromatography in hexane/ethyl acetate
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(1:1). Physical data: [α]D (c 1.00, CHCl3): +41.5. HRMS (ESIꢀTOF) m/z: [M + Na]+
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Calcd for C26H42N2O13Na+ 613.2585; Found 613.2557. H NMR (400 MHz, CDCl3) δ
(ppm): 1.41ꢀ1.50 (m, 18H NCO2C(CH3)3, C(CH3)3), 2.01 (s, 3H, NHCOCH3), 2.01ꢀ2.05
(m, 6H, 2Ac), 2.10 (s, 3H, Ac), 3.54 (t, 2H, J = 4.8, CH2β), 4.03ꢀ4.16 (m, 2H, H5s, H6s),
4.21ꢀ4.28 (m, 2H, CHα, H6s), 4.37 (ddd, 1H, J = 11.0, 8.4, 5.4 Hz, H2s), 4.82 (d, 1H, J = 3.6
Hz, H1s), 4.91 (br s, 1H, NHBoc), 5.15 (t, 1H, J = 9.8 Hz, H4s), 5.26 (dd, 1H, J = 20.1, 10.4,
13
H3s) 6.60 (d, 1H, J = 8.9 Hz, NHAc). C NMR (100 MHz, CDCl3) δ (ppm): 20.7, 20.8,
20.9 (CH3CO), 23.3 (NHCOCH3), 28.1 (NCO2C(CH3)3), 28.5 (C(CH3)3), 42.9 (CH2β), 51.6
(C2s), 62.0 (C6s), 68.3 (C4s), 68.8 (C5s), 71.5 (C3s), 76.4 (CHα), 80.2 (C(CH3)3), 83.5
(CO2C(CH3)3), 98.5 (C1s), 155.7, 169.4, 170.8, 171.2 (CO).
General Procedure for the Ring-opening Reactions of Sulfamidates with
Carbohydrate C1-S-Nucleophiles. DBU (19 ꢀL, 0.13 mmol) was added to a solution of
the corresponding thiocarbohydrate (0.12 mmol) in dry DMF (1 mL) in a nitrogen
atmosphere using standard schlenk techniques. The resulting mixture was stirred at room
temperature for 5 min and it was then added by cannula to a solution of the corresponding
sulfamidate (0.12 mmol) in dry DMF (1 mL). The reaction mixture was kept at room
temperature and stirred for 30 min. After that time, the volatiles were removed, and the
residue dissolved in a mixture of 20% aqueous H2SO4/dichloromethane (1:1), which was
stirred for 30 min at room temperature to hydrolyze the sulfamic acid intermediate. The
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reaction crude was isolated after extraction with dichloromethane (2 x 5 mL), dried over
anhydrous Na2SO4, and concentrated. That crude was purified by silica gel column
chromatography to give the corresponding protected Sꢀglycosyl amino acid.
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(2R)-2-((2',3',4',6')-Tetra-O-acetyl-β-D-glucopyranosylthio)-3-tert-butoxycarbonylamino
propionic acid methyl ester (15). Following the general procedure described above for the
ringꢀopening reactions of sulfamidates with carbohydrate C1ꢀSꢀnucleophiles, and starting
from 2,3,4,6ꢀtetraꢀOꢀacetylꢀ1ꢀthioꢀβꢀDꢀglucopyranose (44 mg, 0.12 mmol) and sulfamidate
10 (34 mg, 0.12 mmol), protected Sꢀglycosyl amino acid 15 (49 mg, 72%) was obtained as
a colorless oil, after column chromatography in hexane/ethyl acetate (6.5:3.5). Physical
data: [α]D (c 1.00, CHCl3): +196.3. HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for
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C23H35NO13SNa+ 588.1727; Found 588.1707. H NMR (400 MHz, CDCl3)
δ (ppm): 1.44
(s, 9H, C(CH3)3), 2.00 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.09 (s, 3H, Ac), 3.30ꢀ
3.45 (m, 1H, CH2β), 3.58ꢀ3.69 (m, 1H, CH2β), 3.71ꢀ3.80 (m, 5H, CHα, H5s, CO2CH3),
4.11ꢀ4.22 (m, 2H, H6s), 4.82 (d, 1H, J = 10.2, H1s), 4.97 (t, 1H, J = 9.7, H2s), 5.04 (t, 1H, J =
9.7, H4s), 5.14 (t, 1H, J = 5.6 Hz, NHBoc), 5.23 (t, 1H, J = 9.3, H3s). 13C NMR (100 MHz,
CDCl3)
δ (ppm): 20.7 (4CH3CO), 28.5 (C(CH3)3), 41.7 (CH2β), 45.8 (CHα), 52.8
(CO2CH3), 62.3 (C6s), 68.4 (C4s), 69.9 (C2s), 73.8 (C3s), 76.0 (C5s), 80.0 (C(CH3)3), 82.2
(C1s), 155,9, 169.4, 169.6, 170.2, 170.7, 171.2 (CO).
(2R)-2-((2',3',4',6')-Tetra-O-acetyl-β-D-glucopyranosylthio)-3-tert-butoxycarbonylamino
propionic acid benzyl ester (16). Following the general procedure described above for the
ringꢀopening reactions of sulfamidates with carbohydrate C1ꢀSꢀnucleophiles, and starting
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from 2,3,4,6ꢀtetraꢀOꢀacetylꢀ1ꢀthioꢀβꢀDꢀglucopyranose (66 mg, 0.18 mmol) and sulfamidate
11 (64 mg, 0.18 mmol), protected Sꢀglycosyl amino acid 16 (89 mg, 77%) was obtained as
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an oil, after column chromatography in hexane/ethyl acetate (6:4). Physical data: [α]D (c
9
1.00, CHCl3): +8.3. HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for C29H39NO13SNa+
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664.2040; Found 664.2151. H NMR (400 MHz, CDCl3)
δ (ppm): 1.43 (s, 9H, C(CH3)3),
1.95 (s, 3H, Ac), 2.00 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.07 (s, 3H, Ac), 3.39 (ddd, 1H, J =
13.9, 7.6, 5.9 Hz, CH2β), 3.58ꢀ3.72 (m, 2H, CH2β, H5s), 3.77 (t, 1H, J = 7.2, CHα), 4.06ꢀ
4.21 (m, 2H, H6s), 4.74 (d, 1H, J = 10.3, H1s), 4.88ꢀ5.06 (m, 2H, H2s, H4s), 5.07ꢀ5.26 (m,
4H, NHBoc, H3s, CH2Ph), 7.33ꢀ7.41 (m, 5H, arom). 13C NMR (100 MHz, CDCl3)
δ (ppm):
20.7 (CH3CO), 28.5 (C(CH3)3), 41.8 (CH2β), 45.9 (CHα), 62.3 (C6s), 67.5 (CH2Ph) 68.4
(C4s), 69.8 (C2s), 73.8 (C3s), 75.9 (C5s), 80.0 (C(CH3)3), 82.2 (C1s), 128.4, 128.8, 128.9,
135.4 (arom), 155,9, 169.4, 169.5, 170.2, 170.4, 170.7 (CO).
(2R)-2-((2',3',4',6')-Tetra-O-acetyl-β-D-glucopyranosylthio)-3-tert-butoxycarbonylamino
propionic acid tert-butyl ester (17). Following the general procedure described above for
the ringꢀopening reactions of sulfamidates with carbohydrate C1ꢀSꢀnucleophiles, and
starting from 2,3,4,6ꢀtetraꢀOꢀacetylꢀ1ꢀthioꢀβꢀDꢀglucopyranose (36 mg, 0.10 mmol) and
sulfamidate 12 (32 mg, 0.10 mmol), protected Sꢀglycosyl amino acid 17 (49 mg, 81%) was
obtained as a colorless oil, after column chromatography in hexane/ethyl acetate (6:4).
Physical data: [α]D (c 1.00, CHCl3): +30.7. HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for
20
C26H41NO13SNa+ 630.2196; Found 630.2203. H NMR (400 MHz, CDCl3)
δ (ppm): 1.44
1
(s, 9H, NCO2C(CH3)3), 1.47 (s, 9H, C(CH3)3), 2.00 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.04 (s,
3H, Ac), 2.08 (s, 3H, Ac), 3.13ꢀ3.39 (m, 1H, CH2β), 3.56 ( t, 1H, J = 11.2, CHα), 3.63ꢀ3.83
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(m, 2H, CH2β, H5s), 4.07ꢀ4.23 (m, 2H, H6s), 4.86ꢀ5.10 (m, 3H, H1s, H2s, H4s), 5.13ꢀ5.28 ( m,
5
6
13
2H, NHBoc, H3s). C NMR (100 MHz, CDCl3)
δ (ppm): 20.7 (CH3CO), 28.0 (C(CH3)3),
7
8
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28.5 (NCO2C(CH3)3), 41.3 (CH2β), 46.7 (CHα), 62.4 (C6s), 68.5 (C4s), 69.7 (C2s), 73.9
(C3s), 75.8 (C5s), 79.8 (NCO2C(CH3)3), 81.8 (C1s), 82.4 (C(CH3)3), 155,9, 169.6, 169.7,
170.2, 170.2, 170.7 (CO).
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(2R)-2-((3',4',6')-Tri-O-acetyl-2’-acetamido-2’-deoxy-α-D-glucopyranosylthio)-3-tert-
butoxycarbonylamino propionic acid benzyl ester (18). Following the general procedure
described above for the ringꢀopening reactions of sulfamidates with carbohydrate C1ꢀSꢀ
nucleophiles, and
starting from 3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀ1ꢀthioꢀαꢀDꢀ
glucopyranose (36 mg, 0.10 mmol) and sulfamidate 11 (32 mg, 0.10 mmol), protected Sꢀ
glycosyl amino acid 18 (48 mg, 75%) was obtained as a colorless oil, after column
chromatography in hexane/ethyl acetate (1:1). Physical data: [α]D20 (c 0.50, CHCl3): +42.8.
HRMS (ESIꢀTOF) m/z: [M + Na]+; Calcd for C29H40N2O12SNa+ 663.2200; Found
1
663.2194. H NMR (400 MHz, CDCl3) δ (ppm): 1.42 (s, 9H, C(CH3)3), 1.90 (s, 3H,
NHCOCH3), 2.03 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.08 (s, 3H, Ac), 3.43ꢀ3.60 (m, 2H, CH2β),
3.72 ( t, 1H, J = 8.4 Hz, CHα), 4.03ꢀ4.12 (m, 1H, H6s), 4.24ꢀ4.37 (m, 2H, H6s, H5s), 4.51
(ddd, 1H, J = 11.0, 8.5, 5.4 Hz, H2s), 4.94ꢀ5.08 (m, 2H, NHBoc, H3s), 5.12 (d, 1H, J = 9.6
Hz, H4s), 5.16ꢀ5.20 (m, 2H, CH2Ph), 5.66ꢀ5.75 (m, 2H, H1s, NHAc), 7.28ꢀ7.44 (m, 5H,
arom). 13C NMR (100 MHz, CDCl3) δ (ppm): 20.8 (CH3CO), 23.3 (NHCOCH3), 28.5
(C(CH3)3), 41.9 (CH2β), 45.4 (CHα), 52.5 (C2s), 62.0 (C6s), 67.8 (CH2Ph), 68.0 (C4s), 69.2
(C5s), 71.3 (C3s), 80.1 (C(CH3)3), 83.9 (C1s), 128.4, 128.7, 128.9, 135.2 (arom), 155.7,
169.4, 170.3, 170.9, 171.0, 171.8 (CO).
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(2R)-2-((3',4',6')-Tri-O-acetyl-2’-acetamido-2’-deoxy-α-D-glucopyranosylthio)-3-tert-
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8
9
butoxycarbonylamino propionic acid tert-butyl ester (19). Following the general procedure
described above for the ringꢀopening reactions of sulfamidates with carbohydrate C1ꢀSꢀ
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nucleophiles, and
starting from 3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀ1ꢀthioꢀαꢀDꢀ
glucopyranose (36 mg, 0.10 mmol) and sulfamidate 12 (32 mg, 0.10 mmol), protected Sꢀ
glycosyl amino acid 19 (42 mg, 68%) was obtained as a colorless oil, after column
chromatography in hexane/ethyl acetate (1:1). Physical data: [α]D20 (c 0.50, CHCl3): +29.3.
HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for C26H42N2O12SNa+ 629.2356; Found 629.2303.
1H NMR (400 MHz, CDCl3)
δ (ppm): 1.44 (s, 9H NCO2C(CH3)3), 1.47 (s, 9H, C(CH3)3),
1.95 (s, 3H, NHCOCH3), 2.04 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.11 (s, 3H, Ac), 3.42ꢀ3.58 (m,
3H, CH2β, CHα), 4.04ꢀ4.17 (m, 1H, H6s), 4.26ꢀ4.42 (m, 2H, H6s, H5s), 4.52 (ddd, 1H, J =
11.0, 8.4, 5.4 Hz, H2s), 4.99ꢀ5.10 (m, 2H, NHBoc, H3s), 5.16 (t, 1H, J = 12.6 Hz, H4s), 5.70ꢀ
5.82 ( m, 2H, H1s, NHAc). 13C NMR (100 MHz, CDCl3)
δ (ppm): 20.8 (CH3CO), 23.3
(NHCOCH3), 28.0 (C(CH3)3), 28.5 (NCO2C(CH3)3), 41.7 (CH2β), 45.9 (CHα), 52.7 (C2s),
62.0 (C6s), 68.0 (C4s), 69.1 (C5s), 71.2 (C3s), 82.9 (C(CH3)3), 84.1 (C1s), 155,8, 169.4, 170.0,
170.5, 170.9, 171.9 (CO).
Methyl (S)-3-amino-2-hydroxypropanoate hydrochloride (5). Acetyl chloride (2 mL) was
added dropwise at 0 °C in about 20 min to absolute methanol (12 mL) through a dropping
funnel. After complete addition, the ice bath was removed and (S)ꢀisoserine 4 (1.0 g, 9.52
mmol) was added in one portion and the solution was heated to reflux for 2 h. After that,
the reaction mixture was allowed to cool to room temperature and the volatiles were
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removed under reduced pressure to give the methyl ester hydrochloride 5 (1.11 g, 98%) as a
white solid, which was used without further purification in the next step. The spectroscopic
data are consistent with those described in the literature.21
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(S)-3-((tert-Butoxycarbonyl)amino)-2-hydroxypropanoic acid (6). (S)ꢀIsoserine 4 (1.04 g,
9.8 mmol) was dissolved in 1 M aqueous NaOH (20 mL) and dioxane (10 mL) at 0° C and
treated with diꢀtertꢀbutyl dicarbonate (2.57 g, 11.8 mmol) and the mixture was allowed to
warm to room temperature and stirred for 24 h. The dioxane was then evaporated and the
aqueous layer was washed with diethyl ether (30 mL) to remove diꢀtertꢀbutyl dicarbonate.
Ethyl acetate (50 mL) was then added to the aqueous layer and the mixture was stirred
while a 20% aqueous H2SO4 solution was added to give pH 2ꢀ3. The organic layer was
separated. The aqueous layer was saturated with NaCl and extracted with ethyl acetate (4 x
50 mL). The combined organic layers were dried and filtered and the volatiles were
removed to give compound 6 (1.83 g, 91%), as a white solid. Physical data: mp 85ꢀ88 °C.
[α]D (c 1.00, MeOH): +6.7. HRMS (ESIꢀTOF) m/z: [M + H]+; Calcd for C8H15NO5H+
20
1
206.1028; Found 206.1019. H NMR (400 MHz, CDCl3)
δ (ppm): 1.38 (s, 9H, C(CH3)3),
3.24 (dd, 1H, J = 13.9, 6.6 Hz, CH2β), 3.39 (dd, 1H, J = 13.8, 4.0 Hz, CH2β), 4.07ꢀ4.18 (m,
1H, CHα). 13C NMR (100 MHz, CDCl3)
δ (ppm): 28.7 (C(CH3)3), 45.1 (CH2β), 71.2
(CHα), 80.3(C(CH3)3), 158.5, 175.9 (CO).
Methyl (S)-3-((tert-butoxycarbonyl)amino)-2-hydroxypropanoate (7). Compound 5 (1.11 g,
9.32 mmol) was suspended in THF (24 mL) and triethylamine (2.84 mL, 20.5 mmol) was
added and the mixture cooled down to 0 °C. Diꢀtertꢀbutyl dicarbonate (2.06 g, 9.44 mmol),
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dissolved in THF (8 mL), was added slowly under nitrogen atmosphere over 1 h using a
dropping funnel. The resulting mixture was left stirring for 14 h at room temperature and
afterward was stirred at 50 °C for additional 3 h. The volatiles were then removed and the
crude residue was partitioned between ethyl ether (400 mL) and saturated NaHCO3 solution
(20 mL). The aqueous phase was extracted with ethyl ether three times. The combined
organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give 7 (1.705
g, 82%) as a colorless oil, which was used as such for the next step. [α]D20 (c 1.00, MeOH):
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+
+38.2. HRMS (ESIꢀTOF) m/z: [M + H]+ Calcd for C9H18NO5 220.1185; Found 220.1177.
The spectroscopic data are consistent with those described in the literature.22
Benzyl (S)-3-((tert-butoxycarbonyl)amino)-2-hydroxypropanoate (8). To a stirring solution
of NꢀBocꢀ(S)ꢀisoserine (6) (517 mg, 2.52 mmol) in DMF (100 mL) was added Cs2CO3 (822
mg, 2.52 mmol) and the stirring was continued 30 min. Benzyl bromide (300 ꢀL, 2.52
mmol) was then added and the resulting solution was stirred for 18 h. The reaction mixture
was then diluted with ethyl acetate (25 mL), washed with lithium bromide (3 x 15 mL),
NaHCO3 (2 x 15 mL), and brine (2 x 15 mL). The organic layer was dried over sodium
sulfate. The volatiles were then removed under reduced pressure and the resulting tan oil
was purified by flash chromatography, using a mixture of hexane/ethyl acetate (7:3), to
20
afford the product 8 (45%) as a colorless oil. Physical data: [α]D (c 1.00, CHCl3): ꢀ3.7.
HRMS (ESIꢀTOF) m/z: [M + Na]+ Calcd for C15H21NO5Na+ 318.1317; Found 318.1327. 1H
NMR (400 MHz, CDCl3)
δ (ppm): 1.43 (s, 9H, C(CH3)3), 3.34ꢀ3.56 (m, 2H, CH2β), 4.30
(dd, 1H, J = 9.3, 4.9 Hz, CHα), 4.93 (s, 1H, NHBoc), 5.15ꢀ5.26 (m, 2H, CH2Ph), 7.29ꢀ7.42
13
(m, 5H, arom). C NMR (100 MHz, CDCl3)
δ (ppm): 28.3 (C(CH3)3), 44.0 (CH2β), 67.7
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(CH2Ph), 70.4 (CHα), 79.9(C(CH3)3), 128.5, 128.6, 128.7, 135.0 (arom), 156.2, 173.0
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6
(CO).
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tert-Butyl (S)-3-((tert-butoxycarbonyl)amino)-2-hydroxypropanoate (9). tertꢀButanol (1.60
g, 21.6 mmol), DCC (3.50 g, 10.5 mmol) and CuCl (38 mg, 0.38 mmol) were stirred under
exclusion of light for 3 days. The mixture was diluted with dry dichloromethane (10 mL)
and a solution of the compound 6 (1.07 g, 5.2 mmol) in dry dichloromethane (15 mL) was
added at room temperature. After stirring for 3 h, N,N’ꢀdicyclohexylurea was filtered off
and, then, the solvent removed in vacuo. The remaining powder was subjected to flash
chromatography (hexane/ethyl acetate, 7:3) to give protected amino acid 9 (1.11 g, 4.26
20
mmol, 82%) as a white solid. Physical data: mp 83ꢀ86 °C. [α]D (c 1.00, CHCl3): +11.8.
1
HRMS (ESIꢀTOF) m/z: [M + H]+ Calcd for C12H23NO5H+ 262.1654; Found 262.1642. H
NMR (400 MHz, CDCl3)
δ
(ppm): 1.37 (s, 9H, C(CH3)3), 1.42 (s, 9H, NCO2C(CH3)3), 3.42
13
(d, 2H, J = 4.5 Hz, CH2β), 4.09 (t, 1H, J = 4.3 Hz, CHα). C NMR (100 MHz, CDCl3)
δ
(ppm): 28.3 (C(CH3)3), 28.7 (C(CH3)3), 44.1 (CH2β), 70.7 (CHα), 79.8, 83.4 (C(CH3)3),
156.3, 172.7 (CO).
Methyl (R)-2-azido-3-((tert-butoxycarbonyl)amino)propanoate (10-N3). To a well stirred
solution of sulfamidate 10 (33 mg, 0.12 mmol) in DMF (1 mL), at room temperature,
sodium azide (31 mg, 0.47 mmol) was added in one portion. The reaction was stirred for 30
min and the remaining DMF was evaporated under vacuum conditions. The crude was
dissolved in dichloromethane (5 mL) and an aqueous solution 20% H2SO4 (5 mL) was
added. The reaction mixture was stirred at room temperature for 30 min and the solvent
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mixture was extracted with dichloromethane. The combined organic phase was dried with
Na2SO4, concentred under vacuum and purified by column chromatography, using
hexane/ethyl acetate (6.5:3.5) as an eluent, to give the required product 10-N3 (25 mg, 85%)
as a colorless oil. Physical data: [α]D20 (c 1.01, CHCl3): +100.5. HRMS (ESIꢀTOF) m/z: [M
10
11
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+ Na]+ Calcd for C9H16N4O4Na+ 267.1069; Found 267.1097. 1H NMR (400 MHz, CDCl3)
δ
(ppm): 1.45 (s, 9H, C(CH3)3), 3.35ꢀ3.48 (m, 1H, CH2β), 3.51ꢀ3.67 (m, 1H, CH2β), 3.82 (s,
3H, CO2CH3), 4.15 (t, 1H, J = 5.7 Hz, CHα), 4.92 (br s, 1H, NHBoc). 13C NMR (100 MHz,
CDCl3)
δ (ppm): 28.4 (C(CH3)3), 41.8 (CH2β), 53.0 (CO2CH3), 61.7 (CHα), 80.2
(C(CH3)3), 155.7, 169.4 (CO).
(R)-2,3-Diaminopropanoic acid hydrochloride (13). The catalyst (Pd/C, 2.5 mg, 10% mass)
was suspended in methanol (3 mL) into a schlenk reactor and prehydrogenated for 10 min.
Then, we added, in one portion, compound 10-N3 (25 mg, 0.1 mmol) dissolved in methanol
(3 mL) and the reaction mixture was vigorously stirred at room temperature for 16 h. The
reaction mixture was filtered through diatomaceous earth and concentrated in vacuo.
Subsequently, the residue was dissolved in an aqueous solution of 6 M HCl (6 mL) and
kept stirring at 60 °C for 12 h. The aqueous phase was evaporated and the residue dissolved
in H2O (2 mL) and eluted through a reverseꢀphase Sepꢀpak C18 cartridge to obtain, after
evaporation, the corresponding compound 13 (9 mg, 60%) as a colorless oil. Physical data:
20
[α]D (c 0.50, 1 M HCl): ꢀ24.2. HRMS (ESIꢀTOF) m/z: [M + H]+ Calcd for C3H8N2O2H+
105.0664; Found 105.0672. The spectroscopic data are consistent with those described in
the literature.23
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(R)-3-((tert-Butoxycarbonyl)amino)-1-methoxy-1-oxopropan-2-yl 4-nitrobenzoate (10-
OBz). Sulfamidate 10 (225 mg, 0.80 mmol), CsF (134 mg, 0.88 mmol), and pꢀnitrobenzoic
acid (pꢀNO2ꢀBzOH) (147 mg, 0.88 mmol) were dissolved in DMF (5 mL) and the mixture
was heated at 50° C for 12 h, until the total disappearance of starting material monitored by
TLC. After the volatiles were evaporated, the residue was dissolved in a mixture of aqueous
20% H2SO4/CH2Cl2 (1:1, 10 mL) and it was stirred at room temperature for 30 min. The
aqueous phase was extracted with dichloromethane (3 x 15 mL), the combined organic
phases were dried (Na2SO4) and evaporated to give a residue, which was purified by silica
gel column chromatography (hexane/ethyl acetate, 6.5:3.5). In this way, compound 10-OBz
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(212 mg, 72%) was isolated as an oil. Physical data: [α]D (c 0.50, CHCl3): +0.8. HRMS
(ESIꢀTOF) m/z: [M + Na]+ Calcd for C16H20N2O8Na+ 391.1117; Found 391.1249. 1H NMR
(400 MHz, CDCl3)
δ (ppm): 1.43 (s, 9H, C(CH3)3), 3.75ꢀ3.80 (m, 2H, CH2β), 3.81 (s, 3H,
CO2CH3), 4.86 (br s, 1H, NH), 5.38 (t, 1H, J = 4.6 Hz, CHα), 8.20ꢀ8.34 (m, 4H, arom). 13C
NMR (100 MHz, CDCl3)
δ (ppm): 28.4 (C(CH3)3), 41.4 (CH2β), 53.0 (CO2CH3), 73.0
(CHα), 80.3 (C(CH3)3) 123.8, 131.3, 134.7, 151.0 (arom), 155.8, 164.1, 168.4 (CO).
Methyl (R)-3-((tert-butoxycarbonyl)amino)-2-hydroxypropanoate (14). A solution of pꢀ
nitrobenzoate protected compound 10-OBz (151 mg, 0.41 mmol) and sodium azide (80 mg,
o
1.22 mmol) in dry MeOH (10 mL) was warmed at 40 C for 14 h under nitrogen. The
solvent was removed on a rotary evaporator, and the hydroxyl free compound was purified
by column chromatography using hexane/ethyl acetate (7:3) to give 14 (59 mg, 65%) as an
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oil. Physical data: [α]D (c 1.00, MeOH): ꢀ39.1. HRMS (ESIꢀTOF) m/z: [M + H]+ Calcd
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for C9H18NO5 220.1185; Found 220.1174. The spectroscopic data are consistent with those
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described in the literature for its enantiomer.22
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General Procedure for the Synthesis of Free Tn Antigen Mimics. Deprotection of
hydroxyl, carboxylic acid and amino groups of protected glycosyl amino acids were carried
out in two steps following this protocol. Protected glycosyl amino acid (0.1 mmol) was
dissolved in a mixture of methanol/water (2:1, 3 mL) and after the addition of lithium
hydroxide monohydrate (LiOH·H2O, 1.0 mmol), the mixture was stirred at room
temperature until starting material was consumed by TLC monitoring (2 h). Additionally,
the reaction was monitored by 1H NMR to test the hydrolysis of all ester groups. The basic
solution was neutralized with ion exchanger Dowex® 50WꢀX8, filtered and evaporated to
give an oil that was treated with trifluoracetic acid (TFA, 1 mL) in dichloromethane (3 mL)
at room temperature. After stirring for 1 h, the volatiles were removed and the
corresponding unprotected glycosyl amino acid was obtained as a white solid without
further purification.
(2S)-O-(2’-Acetamido-2’-deoxy-α-D-galactopyranosyl)-2-methylserine (22). Following the
general procedure and starting from compound 2 (50 mg, 0.09 mmol), free glycosyl amino
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acid 23 was obtained without further purification (25 mg, 87%). Physical data: [α]D (c
+
1.00, H2O): +57.1. HRMS (ESIꢀTOF) m/z: [M + H]+ Calcd for C12H23N2O8 323.1449;
1
Found 323.1459. H NMR (300 MHz, D2O) δ (ppm): 1.53 (s, 3H, CCH3), 1.99 (s, 3H,
NHCOCH3), 3.69ꢀ3.76 (m, 2H, 2H6s), 3.84ꢀ3.89 (m, 2H, H5s, H3s), 3.90ꢀ3.93 (m, 2H,
CH2β), 3.94ꢀ3.96 (m, 1H, H4s), 4.10 (dd, 1H, J = 11.80, 3.80 Hz, H2s), 4.92 (d, 1H, J = 3.80
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Hz, H1s). C NMR (75 MHz, D2O) δ (ppm): 18.0 (CCH3), 21.8 (NHCOCH3), 49.6 (C2s),
60.2 (Cα), 61.2 (C6s), 67.2 (C5s), 68.2 (C4s), 70.2 (Cβ), 71.5 (C3s), 98.1 (C1s), 172.3
(NHCOCH3), 174.5 (CO2H).
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(2R)-O-(2’-Acetamido-2’-deoxy-α-D-galactopyranosyl)isoserine (23). Following the
general procedure and starting from compound 21 (50 mg, 0.08 mmol), free glycosyl amino
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acid 23 was obtained without further purification (22 mg, 84%). Physical data: [α]D (c
+
1.00, H2O): +51.3. HRMS (ESIꢀTOF) m/z: [M + H]+ Calcd for C11H21N2O8 309.1292;
1
Found 309.1290. H NMR (300 MHz, D2O) δ (ppm): 1.93 (s, 3H, NHCOCH3), 3.35 (dd,
2H, J = 5.02, 2.79 Hz, CH2β), 3.61ꢀ3.69 (m, 2H, 2H6s), 3.83ꢀ3.90 (m, 3H, H3s, H4s, H5s ),
4.10 (dd, 1H, J = 10.91, 3.88 Hz, H2s), 4.54 (t, 1H, J = 5.01, Hα), 5.04 (d, 1H, J = 3.89 Hz,
H1s). 13C NMR (75 MHz, D2O) δ (ppm): 21.9 (NHCOCH3), 40.6 (Cβ), 49.4 (C2s), 61.3
(C6s), 67.1 (C5s), 68.3 (C4s), 71.2 (Cα), 72.1 (C3s), 96.9 (C1s), 171.8 (NHCOCH3), 174.8
(CO2H).
ASSOCIATED CONTENT
Supporting Information. Additional experimental section and copies of NMR spectra for
all new compounds. The Supporting Information is available free of charge on the ACS
Publications website at DOI: xxxx
AUTHOR INFORMATION
Corresponding Authors
*J.M.P.: eꢀmail, jesusmanuel.peregrina@unirioja.es
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The Journal of Organic Chemistry
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ORCID J.M.P.: 0000ꢀ0003ꢀ3778ꢀ7065
ORCID J.H.B.: 0000ꢀ0003ꢀ4403ꢀ4790
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We thank the Ministerio de Economía y Competitividad (Project Nos. CTQ2015ꢀ67727ꢀR
and UNLR13ꢀ4Eꢀ1931 to J.M.P. and F.C., CTQ2015ꢀ70524ꢀR and RYCꢀ2013ꢀ14706 to
G.J.ꢀO., and FPI fellowship to P.T.). I.G. thanks Universidad de La Rioja for a grant.
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