Synthesis of thiourea-tethered C-glycosyl amino acids via isothiocyanate-amine coupling

Article abstract of DOI:10.1039/b908156a

A new class of C-glycosyl amino acids displaying a thiourea segment as a linker has been designed and synthesized by addition of peracetylated glycosylmethyl isothiocyanates to an amine-functionalized amino acid (N _α-Fmoc-β-amino-l-alanine). Th

Full text of DOI:10.1039/b908156a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of thiourea-tethered C-glycosyl amino acids via
isothiocyanate–amine coupling†
Reham F. Barghash, Alessandro Massi* and Alessandro Dondoni*
Received 24th April 2009, Accepted 27th May 2009
First published as an Advance Article on the web 2nd July 2009
DOI: 10.1039/b908156a
A new class of C-glycosyl amino acids displaying a thiourea segment as a linker has been designed and
synthesized by addition of peracetylated glycosylmethyl isothiocyanates to an amine-functionalized
amino acid (Na-Fmoc-b-amino-L-alanine). Three pairs of compounds with a- and b-galacto, a- and
b-gluco, and a- and b-manno configuration have been prepared with yields ranging between 70 and
75%. The orthogonal set of protective groups (O-acetyl in the carbohydrate moiety and N-Fmoc in the
amino acid residue) makes these compounds suitable substrates for the co-translational modification of
natural peptides. The couplings of model hydroxy-free and perbenzylated glycosylmethyl
isothiocyanates with the above Na-Fmoc-b-amino-L-alanine and the Na-Boc-protected analogue have
been carried out as well, thus broadening the scope of the coupling reaction. Nevertheless, there are
limitations of this isothiocyanate–amine coupling in complex systems, and these are briefly discussed.
more specific reason was similar to that which inspired our recent
Introduction
work on heterocycle-tethered glycosyl amino acid synthesis,3,6
namely the facile assembly of the sugar and glycinyl moieties on
a substrate which can serve not only as a robust linker but can
also participate (by hydrogen bonding and dipolar interactions)
in molecular recognition processes. Peptides constituted of such
amino acids, once introduced into proteins, could affect protein
folding, conformation, stability, and biological activity.7 Thiourea-
linked carbohydrates have been extensively investigated in the
context of their molecular recognition properties by virtue of the
strong hydrogen-bonding donor capability of the thiourea segment
toward anions.8 Recognition of nucleic acids by thiourea-linked
glycooligomers through bidentate hydrogen-bonding interaction
with phosphodiester groups has been also advanced.9
A research topic that is actively investigated in modern glyco-
chemistry involves the synthesis of oligosaccharide and glyco-
conjugate mimics in which robust linkers hold together the two
carbohydrate residues or the carbohydrate and aglycone. It is
in fact a well-established concept that the replacement of the
native O-glycosidic linkage by hydrolase-resistant linkers results
in products of great utility either as probes in biological studies or
as inhibitors of carbohydrate-based metabolic disorders. Linkers
ranging from simple fragments such as the methylene group or
sulfur atom to more complex residues such as acetylene, aromatic
and heteroaromatic rings have been employed.1 Accordingly, given
the fundamental role exerted by cell surface glycoproteins in cell–
cell recognition and interaction,2 a great deal of research has been
carried out on the preparation of non-natural glycopeptides for use
in the post-translational modification of proteins.3 In turn, non-
natural glycopeptides can be prepared from carbon-linked4 and
sulfur-linked5 glycosyl amino acids for which numerous synthetic
methods are available.
Results and discussion
A. General plan
To design thiourea-linked glycosyl amino acids as suitable building
blocks for non-natural glycopeptide synthesis, the following issues
were addressed. The first was the introduction of a spacer between
the glycosyl residue and the thiourea segment, because the direct
linking of these fragments through a carbon-nitrogen bond would
form a labile hemiaminal system. The second was the need for
a set of orthogonal protective groups compatible with peptide
synthesis. Based on this reasoning, the model glycosyl thioureido-
amino acids 7 and esters 8 were designed as suitable products
(Scheme 1). The main structural features of these compounds
consisted of a methylene spacer between the sugar and the thiourea
segment10 and the readily removable acetyl protecting groups in the
carbohydrate moiety, while the glycinyl group was orthogonally
protected as N-Fmoc (N-fluoren-9-ylmetoxycarbonyl) and tert-
butyl ester or N-Fmoc only. We envisaged the synthesis of a
collection of amino acids 7 and 8 by introducing sugar fragments
of the gluco, galacto, and manno series with a- and b-configuration
at the anomeric center.
Very recently, reports from our laboratory have dealt with the
preparation of a special class of glycosyl amino acids in which a
N-heterocycle (isoxazole, triazole, tetrazole, or pyridine) consti-
tuted the connecting group of the sugar and amino acid residues.3,6
We would like to report in this paper the synthesis of a new class
of non-natural C-glycosyl amino acids in which the two bioactive
fragments are connected through a thiourea segment. Access to
these compounds relies on a thiourea-forming reaction such as
isothiocyanate–amine coupling. There were two main reasons that
spurred us in this synthetic endeavor. One reason was to assess the
efficiency of the isothiocyanate–amine addition as a ligation tool of
complex and multifunctionalized chiral reagents. The second and
Dipartimento di Chimica, Laboratorio di Chimica Organica, Universita`
di Ferrara, Via L. Borsari 46, I-44100, Ferrara, Italy. E-mail: adn@
unife.it, msslsn@unife.it
† Electronic supplementary information (ESI) available: NMR spectra.
See DOI: 10.1039/b908156a
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Scheme 2 Abortive approaches towards the synthesis of tetra-O-acetyl
glycosylmethyl amines of type b-1a.
Scheme 1 Isothiocyanate–amine addition-based approach toward the
synthesis of C-glycosylmethylthioureido L-alanines.
derivatives 12 were considered (Scheme 3) because they were
easily accessible via direct cyanation of anomeric glycosyl acetates
with trimethylsilyl cyanide (TMSCN).6c,15 The known cyanides
a-12a and b-12a with the galacto configuration and compounds
a-12b and b-12b with the manno configuration were prepared
Two approaches were considered for the synthesis of 7 and
8, one involving the coupling of tetra-O-acetyl glycosylmethyl
amines 1 with Na-Fmoc-b-isothiocyano-L-alanine 2 or the tert-
butyl ester 3 (route A), the other engaging in a reverse way the
glycosylmethyl isothiocyanate 4 and Na-Fmoc-b-amino-L-alanine
5 or tert-butyl ester 6 (route B). Route A turned out to be
impractical because of the difficulty of accessing peracetylated
aminomethyl glycosides 1 by reduction of cyanoglycosides. In fact,
the reduction of tetra-O-acetyl-b-galactosyl cyanide 9 via catalytic
hydrogenation11 afforded the N-acetyl aminomethyl galactose
derivative 10 in excellent yield due to the acyl transfer from 2-
OAc to the initially formed amino group (Scheme 2, eq. a).12
To prevent this undesired reaction, the hydrogenation of 9 was
performed in the presence of di-tert-butyl-dicarbonate (Boc2O)
(Scheme 2, eq. b).13 Indeed, the amine was trapped as the
Boc-carbamate 11, which upon treatment with trifluoroacetic
acid (TFA) afforded the trifluoroacetic salt of b-1a in nearly
quantitative yield. Unfortunately, neutralization of the above salt
to free amine b-1a by Et3N or saturated NaHCO3 aqueous solution
afforded the amide 10. Moreover, the trifluoroacetic salt of b-1a
proved to be quite unstable under storage even at -20 ◦C. Hence,
the unsuccessful access to the starting material needed for route A
led us to explore the viability of route B toward the development
of our synthetic program.
B. Synthesis of glycosylmethyl isothiocyanates 4, 14, and 15
These carbohydrate-containing heterocumulenes appeared to be
unknown compounds to the best of our knowledge.14 We planned
their synthesis starting from sugar cyanides and glycosylmethyl
amines as intermediates. Of course, on the basis of the results
reported in section A (above), we avoided using peracetylated
glycosyl cyanides as starting materials. Instead, tetra-O-benzyl
Scheme 3 Synthesis of hydroxy-free, peracetylated, and perbenzylated
glycosylmethyl isothiocyanates.
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Table 1 Glycosylmethyl isothiocyanates prepareda
a
See Experimental section for details. b See Ref. 6c,15 for cyanide preparation. c Isolated yield of crude 14. d Determined by 1H NMR analysis. e Isolated
yield by chromatography. f Homogeneous by chromatography on a preparative scale. a/b ratio 1:2.
and isolated as single isomers, whereas the gluco derivative a,b-
12c was obtained as a mixture of inseparable a- and b-anomers
(Table 1). Reduction of these compounds with lithium aluminum
hydride (LiAlH4) furnished the corresponding perbenzylated
glycosylmethyl amines 13 (Scheme 3) which, as crude materials,
were elaborated in two different ways. Firstly, debenzylation by
catalytic hydrogenation and reaction with thiophosgene (CSCl2)
was carried out16 to give crude hydroxy-free glycosylmethyl
isothiocyanates 14a–c in good yields and purity (Table 1). In
turn, these products were readily acylated with acetic anhydride
in the presence of pyridine to give the target tetra-O-acetyl
derivatives 4a-c, all isolated as pure individual a- and b-anomers.
In a second instance, amines 13 were transformed into per-
benzylated glycosylmethyl isothiocyanates 15, thereby providing
an additional element of diversity in this new family of sugar
isothiocyanates. The NH2-to-NCS transformation was carried out
by a literature procedure17 that involved the one-pot coupling of
13 with carbon disulfide and triethylamine and treatment of the
unstable dithiocarbamide acid triethylamine salt intermediate with
2-chloro-N-methylpyridinium iodide to induce b-elimination to
give isothiocyanate.18
C. Synthesis of b-amino-L-alanines 5 and 6
The preparation of the Na-Fmoc-b-amino-L-alanine 5 (see
Scheme 1) was carried out by Hofmann rearrangement of
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N-Fmoc-L-asparagine induced by iodosobenzene diacetate as
described.19 Compound 5 prepared in this way showed m.p.
formed while the unreacted isothiocyanate a-4b was recovered
unaltered by chromatography of the reaction mixture. On the other
hand, the use of excess isothiocyanate a-4b (entry 4) gave a lower
yield of the coupling product a-7b. We noticed that under these
conditions the carbohydrate moiety of a-4b deteriorated by loss
of the acetyl protecting groups and transfer to the free amino
group of 5. Accordingly, products from partial deacylation of a-
4b together with the N-acetyl derivative of 5 were isolated by
chromatography of the crude reaction mixture. As examples of
isothiocyanate–amine coupling were reported to occur efficiently
in the presence of organic bases,8 DMF was replaced by pyridine as
the solvent (entry 5). This change was disadvantageous rather than
profitable, since the base favored the formation of decomposition
products due to the removal of the labile acetyl groups from the
carbohydrate residue ofa-4b. Consequently, in this run the product
a-7b was obtained in lower yield than in run 3. The addition of 0.5
equivalents of dimethylaminopyridine (DMAP) affected greatly
the kinetic of the process (entry 6), but unfortunately this base
promoted both the deacylation of a-4b and the removal of the
labile N-Fmoc protective group from the amino acid 5. Increasing
the temperature by heating at 50 ◦C with an oil bath reduced
the reaction time (entry 7) while inducing partial epimerization
(10%) at C(a) stereocenter of the product a-7b.22 Heating by
microwave irradiation (entry 8) reduced the reaction time to 1 hour
but produced also an even more substantial amount (ca. 30%) of
the epimer of a-7b. From these results we concluded that the
best conditions were those adopted in run 3. Next, a substrate
modification was introduced by using the tert-butyl ester 6 in the
coupling reaction with a-4b (entry 9). The corresponding product
a-8b was isolated in lower yield than a-7b. The intermolecular
acyl transfer from the sugar to the free amino group of 6 appeared
to be the main responsible for the low yield of a-8b. The same
undesired reaction occurred in runs carried out in DMF in the
presence of DMAP or in pyridine as a solvent (entries 10 and 11),
thus affording a-8b in modest isolated yields.
◦
1
(148 C) and H NMR spectral data consistent with those of
the literature.19 Also the tert-butyl ester 6 turned out to be a
known compound, as it had been employed in a synthesis of
C-glycopeptides possessing a reversed amide bond,20 but no details
on its preparation and characteristics were reported. Therefore we
synthesized this key reagent by Hofmann rearrangement of tert-
butyl N-Fmoc-L-asparagine 16 as well (Scheme 4). In this case,
however, the more common bis(trifluoroacetoxy)iodobenzene in
the presence of pyridine in water-acetonitrile at room temperature
was employed as selective and mild amide oxidant.20,21 Indeed,
under these conditions the target ester 6 was obtained in very
good isolated yield (86%).
Scheme 4 Synthesis of b-amino-L-alanine 6.
D. Synthesis of glycosylmethylthioureido L-alanines
In the search of optimized conditions, we set out to investigate the
coupling of the peracetylated mannosylmethyl isothiocyanate a-4b
with Na-Fmoc b-amino-L-alanine 5 and tert-butyl ester 6 (Table 2).
While we were pleased to observe that a-4b and 5 reacted smoothly
in DMF at room temperature over 12 hours to give the product
a-7b (entry 1), we found that a longer reaction time afforded
the same product in higher isolated yield (entry 2). Moreover,
changing the a-4b/5 ratio from 1:1 to 1:1.5 increased the yield
of a-7b of about 15% (entry 3). Notably, no side-products were
Table 2 Screening of reaction conditions for isothiocyanate–amine
couplinga
Having optimized the reaction conditions for the coupling of
a-4b with 5, we extended the reaction of the amino acid 5 to other
peracetylated isothiocyanates presented in Table 1. The products
a- and b-7a–c with the relevant yields are presented in Table 3.
Notably, yields of the formed amino acids were all in the range
70–75%. This demonstrated uniform behavior of the coupling
reaction under substrate variation. However, the incomplete
conversion of the reagents into the corresponding thioureido
derivative 7 or 8 remained a point to be clarified. We suspected
that the thiourea segment of the product exerted an inhibitory
effect on the reaction by complexation of the NH2 group of the
b-amino alanine via double hydrogen bonding. In support of this
hypothesis, we performed qualitative NMR experiments repro-
ducing the reaction composition of optimized isothiocyanate–
amine a-4b/5 coupling (entry 3, Table 2) before reaction workup
(75% conversion). Accordingly, the 1H NMR spectra of product-
amine a-7b/5 1:1 mixture and product–isothiocyanate a-7b/a-
4b 3:1 mixture were taken in DMF-d7. These spectra were then
compared with those of authentic a-4b, 5, and a-7b to evaluate
potential hydrogen-bond interactions between the product and
each reaction partner. Indeed, while no changes were observed
for the proton resonances of mixed a-7b and a-4b, the NH2
proton signals in the a-7b/5 1:1 mixture were split and significantly
downfield-shifted compared to 5 (Fig. 1). This result supported the
AA
Additive
Product
Entry (equiv.) (equiv.)
Solvent Time (h) Temp (◦C) (yield, %)b
1
2
3
4
5
6
7
8
9
5 (1.0)
5 (1.0)
5 (1.5)
5 (0.5)
5 (1.5)
—
—
—
—
—
DMF
DMF
DMF
DMF
12
24
24
24
25
25
25
25
25
25
50
a-7b (55)
a-7b (66)
a-7b (75)
a-7b (57)
a-7b (58)
a-7b (68)
a-7b (57)c
Pyridine 12
5 (1.5) DMAP (0.5) DMF
5
12
1
24
2
5 (1.5)
5 (1.5)
6 (1.5)
—
—
—
DMF
DMF
DMF
80 (MW)d a-7b (71)c
25
25
25
a-8b (65)
a-8b (45)
a-8b (48)
10
11
6 (1.5) DMAP (0.5) DMF
6 (1.5)
—
Pyridine 12
a All reactions were run with 0.40 mmol of a-4b. b Isolated yield by
chromatography. c Evidence of epimerization at the a-stereocenter in the
amino acid moiety. d Experiment run in Biotage Initiator (temperature was
measured externally by an IR sensor).
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Table 3 Glycosyl amino acids prepareda
Glycosyl amino acid
Yield (%)b
74
b-7a
a-7a
b-7b
a-7b
70
71
75
Fig. 1 1H NMR spectra of thiourea product (a) a-7b, (b) amine 5, and
(c) a-7b/5 1:1 mixture taken in DMF-d7. Downfield-shifting and splitting
(NHa and NHb) of the NH2 signal of 5 in the a-7b/5 spectrum can be seen.
the undesired protective group migration that occurred with the
O-acetyl derivatives 4. Moreover, in order to perform reactions
in basic media to disrupt the suspected thiourea–amine complex-
ation via hydrogen bonding, the known N-tert-butoxycarbonyl
(N-Boc) b-amino-L-alanine 17 was prepared,19 as the Boc protec-
tive group is well known to be compatible with basic conditions.
We were aware, however, that the glycosyl amino acids obtained
from isothiocyanates 15 were inappropriate as building blocks for
the co-translational modification of glycopeptides because of the
difficult removal of the benzyl groups by catalytic hydrogenation in
the presence of the thiourea fragment. Nevertheless, we envisaged
the C-glycosyl amino acids of type 18 (Table 4) as valuable
substrates for other synthetic programs.23 Hence, the coupling
of isothiocyanate a-15a with the amino acid 17 was carried out
under various conditions (Table 4). Low yields of the product a-
18a were obtained in DMF and pyridine as the solvents (entries 1
and 2) while the presence of DMAP (entry 3) gave a satisfactory
yield although the product was a mixture of C(a) epimers.
Very likely, the steric congestion induced by the N-Boc and
O-Bn groups was the cause of the lower efficiency of the coupling
of 17 with a-15a (15% yield; entry 1, Table 4) in respect to that of
N-Fmoc and O-Ac protected counterparts 5 and a-4a (70% yield;
entry 2, Table 3). In order to overcome this additional limitation to
the achievement of a quantitative isothiocyanate–amine coupling,
the reaction of Na-Fmoc-b-amino-L-alanine 5 with hydroxy-
free glycosylmethyl isothiocyanates was investigated. Hence, the
coupling of model b-14a with 5 under optimized conditions (DMF,
rt, 24 h) afforded the target thiourea b-19a in 70% conversion as
estimated by 1H NMR analysis (entry 4, Table 4).24 This outcome
was similar to that obtained with the peracetylated derivative b-4a
and 5 (74% yield; entry 1, Table 3). This coincidence supports the
hypothesis that also in the case of the reaction of b-14a with 5,
inhibition by the thiourea fragment in the product is taking place.
Unfortunately, the evaluation of b-14a/5 coupling efficiency in a
basic solvent such as pyridine (entry 5, Table 4) was precluded by
the nearly total conversion of b-14a into the bicyclic tetrahydro-
1¢,3¢-oxazine-2¢-thione 20. The formation of this product can be
b-7c
a-7c
73
70
a All reactions were run with 0.40 mmol of 4 and 0.60 mmol of 5 in DMF
at rt for 24 h. b Isolated yield by chromatography.
participation of the NH2 group of 5 in intermolecular bidentate
hydrogen bonding with the thiourea moiety of a-7b as the donor.
Additionally, the influence on this interaction of the carboxylic
acid group of 5 and a-7b was evaluated and excluded. In fact, a
very similar outcome to that depicted in Fig. 1 resulted from the
1
parallel H NMR investigation on a-8b/6 couple displaying the
t-butyl ester group in place of the free carboxylic acid functionality.
While the above results demonstrate that the main goal of our
research program, i.e. the preparation of orthogonally protected
thiourea-tethered peracetylated glycosyl N-Fmoc amino acids 7
in good yields, had been achieved, we wondered whether the
efficiency of the investigated isothiocyanate–amine coupling could
be improved. To this end, a different set of protective groups in
the reactants was considered in order to avoid the occurrence of
the side-reactions which were observed in the preparation of the
C-glycosyl amino acids 7. In particular, we envisaged the use of
perbenzylated glycosylmethyl isothiocyanates 15 in order to avoid
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Table 4 Scope and limitation of the investigated isothiocyanate–amine
Experimental section
couplinga
All moisture-sensitive reactions were performed under a nitrogen
atmosphere using oven-dried glassware. Solvents were dried over
standard drying agent and freshly distilled prior to use. Com-
˚
mercially available powdered 4 A molecular sieves (5 mm average
particle size) were used without further activation. Reactions
were monitored by TLC on silica gel 60 F254 with detection by
charring either with sulfuric acid or ninhydrin. Flash column
chromatography was performed on silica gel 60 (230–400 mesh.
Optical rotations were measured at 20 2 ◦C in the stated solvent;
1
[a]D values are given in 10-1 deg cm2 g-1. H (300 MHz) and 13C
(75 MHz) NMR spectra were recorded for CDCl3 solutions at
room temperature unless otherwise specified. MALDI-TOF mass
spectra were acquired using a-cyano-4-hydroxycinnamic acid as
the matrix. ESI MS analyses were performed in positive-ion mode
with samples dissolved in a 10 mM solution of HCO2NH4 in
1:1 MeCN/H2O. Microwave-assisted reactions were carried out
using a single-mode cavity dedicated reactor (Biotage InitiatorTM).
Reactions were performed with temperature-controlled programs
in glass vials (0.5–2 mL or 2–5 mL depending on the scale) sealed
with a Teflon septum. The temperature was measured externally
by an IR sensor. The reaction time was counted starting from the
point at which the reaction mixture reached the stated temperature.
Pressure was measured by a non-invasive sensor integrated into
the cavity lid.
Entry
Isothiocyanate
Amine
Solvent
Product (yield, %)b
1
2
3
4
5
a-15a
a-15a
a-15a
b-14a
b-14a
17
17
17
5
DMF
a-18a (15)c
a-18a (48)c
a-18a (62)c,e
b-19a (70)f
20 (90)
Cyanides 12,6c,15 amines a-13a,6c b-13a,25 b-13b,25 b-13c,25
carbamate 11,13 amino acids 519 and 1719 are known compounds
and their spectroscopic data were identical to those reported.
Acetamide 10,12 and derivatives 620 and 1620 are described in the
literature without spectroscopic and analytical data.
Pyridine
DMFd
DMF
5
Pyridine
a All reactions were run with 0.40 mmol of isothiocyanate, 0.60 mmol of
amine at rt for 24 h. b Isolated yield by chromatography. c Product isolated
as methyl ester (see Experimental section). d Reaction performed in the
presence of DMAP (0.20 mmol) as additive. e Evidence of epimerization
at a-stereocenter in the amino acid moiety. f Estimated conversion by
1H NMR analysis; product isolated as peracetylated derivative b-7a (see
Experimental section).
(3,4,6-Tri-O-acetyl-b-D-galactopyranosyl)methyl acetamide (10)
A vigorously stirred mixture of cyanide 9 (357 mg, 1.00 mmol),
20% palladium hydroxide on carbon (180 mg), and THF-EtOH
(9 mL- 1 mL) was degassed under vacuum and saturated with
hydrogen (by a H2-filled balloon) three times. The suspension was
stirred at room temperature for 12 h under a positive pressure
of hydrogen (8 bars), filtered through a plug of cotton, and
formulated to occur via intramolecular nucleophilic attack of the
hydroxy group at C2 of the sugar moiety to the NCS group.16
concentrated to give crude amine 10 (343 mg, 95%) as a white
1
foam. H NMR: d = 6.02 (dd, 1 H, J1¢a,NH = 6.0 Hz, J1¢b,NH
=
9.0 Hz, NH), 5.41 (dd, 1 H, J3,4 = 3.5 Hz, J4,5 = 0.5 Hz, H-4), 4.96
(dd, 1 H, J2,3¢ = 9.5 Hz, H-3), 4.12 (dd, 1 H, J5,6a = 7.5 Hz, J6a,6b
=
Conclusion
11.5 Hz, H-6a), 4.04 (ddd, 1 H, J1,1¢a = 2.5 Hz, J1¢a,1¢b = 15.0 Hz,
H-1¢a), 4.02 (dd, 1 H, J5,6b = 6.5 Hz, H-6b), 3.88 (ddd, 1 H, H-5),
3.61 (dd, 1 H, J1,2 = 9.5 Hz, H-2), 3.34 (ddd, 1 H, J1,1¢b = 3.0 Hz,
H-1¢b), 2.08, 2.07, 2.04, and 2.03 (4 s, 12 H, CH3). 13C NMR: d =
172.5, 170.5, 170.4, 170.0, 79.7, 74.6, 73.0, 68.0, 64.9, 61.9, 40.2,
23.0, 20.8, 20.7, 20.6. MALDI-TOF MS: 384.4 (M+ + Na). Anal.
Calcd for C15H23NO9 (361.14): C, 49.86; N, 3.88; H, 6.42. Found:
C, 49.99; N, 3.66; H, 6.55.
We have established a viable route to a new family of non-
natural glycosyl amino acids via coupling of sugar isothiocyanates
with a b-aminated a-amino acid. The main feature of these
products is a thiourea tether holding together the two bioactive
moieties. Reaction partners have been designed in order to obtain
suitably protected products for N-Fmoc-based C-glycopeptide
synthesis. It is expected that the thiourea fragment in synthesized
glycopeptides might affect their secondary structure via hydrogen
bonding and provide increasing ability in molecular recognition
processes. It has been shown, however, that the isothiocyanate–
amine coupling of multifunctionalized substrates, required for the
synthesis of designed complex products, might have limitations
due to restricted reaction conditions and the need for a set of
orthogonal protective groups.
N-tert-Butoxycarbonyl-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)methyl amine (11)
A vigorously stirred mixture of cyanide 9 (357 mg, 1.00 mmol),
20% palladium hydroxide on carbon (180 mg), triethylamine
(1.39 mL, 10.0 mmol), di-tert-butyl-dicarbonate (655 mg,
3.00 mmol), and THF-EtOH (5 mL-5 mL) was degassed under
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vacuum and saturated with hydrogen (by a H2-filled balloon)
three times. The suspension was stirred at room temperature for
12 h under a positive pressure of hydrogen (8 bars), filtered through
a plug of cotton, concentrated, and eluted from a column of silica
gel with 1:1 cyclohexane-AcOEt to afford 1111 (384 mg, 83%) as a
white foam.
stirred at room temperature for 12 h under a positive pressure
of hydrogen (8 bars), filtered through a plug of cotton, and
concentrated to give the corresponding unprotected amine, which
was used in the next step without any purification.
To a stirred mixture of the above crude amine (185–195 mg,
~1.00 mmol), CaCO3 (300 mg, 3.00 mmol), and H2O-acetone
(3 mL - 2 mL) thiophosgene (114 mL, 1.50 mmol) was slowly added.
The mixture was vigorously stirred at room temperature for 2 h,
then filtered through a glass-sintered filter, and concentrated. The
resulting residue was eluted from a column of Sephadex LH-20
(2 ¥ 60 cm) with 2:1 MeOH-CH2Cl2 to give the corresponding
glycosylmethyl isothiocyanate 14 at least 80% pure as established
by 1H NMR analysis.
(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)methyl amine (b-1a)
To a cooled (0 ◦C), stirred solution of 11 (231 mg, 0.50 mmol)
in CH2Cl2 (1 mL) was slowly added a solution of TFA-CH2Cl2
(1 mL-3 mL). Stirring was continued at 0 ◦C for an additional
30 min then warmed to room temperature. After 30 min at room
temperature the solution was concentrated to give the TFA salt of
amine b-1a (225 mg, 95%). 1H NMR: d = 7.80 (bs, 2 H, NH2), 6.80
(bs, 1 H, NH), 5.49 (dd, 1 H, J3,4 = 3.5 Hz, J4,5 = 0.5 Hz, H-4), 5.13
(dd, 1 H, J2,3 = 9.5 Hz, H-3), 5.05 (dd, 1 H, J1,2 = 9.5 Hz, H-2),
4.24–4.10 (m, 1 H, H-1), 4.08–3.90 (m, 2 H, 2H-6), 3.85–3.72 (m,
1 H, H-5), 3.38–3.22 and 3.20–3.08 (2 m, 2 H, 2H-1¢), 2.17, 2.11,
2.07, and 2.01 (4 s, 12 H, CH3).
(b-D-Galactopyranosyl)methyl isothiocyanate (b-14a). 1H
NMR (D2O): d = 3.84 (dd, 1 H, J3,4 = 3.5 Hz, J4,5 = 0.5 Hz, H-4),
3.82 (dd, 1 H, J2,3 = 9.5 Hz, H-3), 3.74 (dd, 1 H, J1,1¢a = 5.5 Hz,
J1¢a,1¢b = 15.0 Hz, H-1¢a), 3.64–3.52 (m, 4 H, H-5, 2H-6, H-1¢b),
3.48 (dd, 1 H, J1,2 = 9.5 Hz, H-2), 3.37 (ddd, 1 H, J1,1¢b = 6.0 Hz,
H-1). 13C NMR (D2O): d = 130.9, 78.9, 77.7, 73.8, 69.3, 68.2,
61.5, 46.3.
The TFA salt of b-1a is a highly unstable species and it cannot be
stored even at low temperature (-20 ◦C). Moreover, neutralization
of the above salt by Et3N or saturated NaHCO3 aqueous solution
resulted in the formation of the by-product 10.
(a-D-Galactopyranosyl)methyl isothiocyanate (a-14a). 1H
NMR (D2O): d = 4.21 (ddd, 1 H, J4,5 = 1.5 Hz, J5,6a = 4.5 Hz,
J5,6b = 7.5 Hz, H-5), 3.95 (dd, 1 H, J1,1¢a = 9.5 Hz, J1¢a,1¢b = 15.0 Hz,
H-1¢a), 3.84 (dd, 1 H, J3,4 = 3.5 Hz, H-4), 3.72–3.50 (m, 6 H, H-1,
H-2, H-3, 2H-6, H-1¢b).). 13C NMR (D2O): d = 131.3, 73.3, 72.8,
69.8, 68.9, 67.4, 61.0, 41.7.
General procedure for the synthesis of benzylated amines 13
To a stirred suspension of LiAlH4 (304 mg, 8.00 mmol) in
anhydrous THF (10 mL) a solution of cyanide 12 (1.10 g,
2.00 mmol) in anhydrous THF (4 mL) was slowly added. The
resulting mixture was stirred under reflux until disappearance of
starting cyanide (TLC analysis, typically 1 h), cooled to room
temperature, and then diluted with 28% aqueous NH4OH (2 mL).
The resulting mixture was stirred at room temperature until the
formation of a white precipitate was observed (typically 30 min).
The mixture was then filtered through a pad of Celite and washed
thoroughly with AcOEt. The combined filtrates were concentrated
to give the corresponding crude amine 13 in almost quantitative
yield. Each amine 13 was used in the next step without any
purification.
(b-D-Mannopyranosyl)methyl isothiocyanate (b-14b). 1H
NMR (D2O): d = 3.81 (dd, 1 H, J1,2 = 1.0 Hz, J2,3 = 3.5 Hz, H-2),
3.75 (dd, 1 H, J1,1¢a = 4.0 Hz, J1¢a,1¢b = 14.5 Hz, H-1¢a), 3.63 (dd,
1 H, J1,1¢b = 5.5 Hz, H-1¢b), 3.58–3.48 (m, 3 H, H-1, H-3, H-6a),
3.42 (dd, 1 H, J3,4 = 9.0 Hz, J4,5 = 9.0 Hz, H-4), 3.40 (dd, 1 H,
J5,6a = 3.0 Hz, J6a,6b = 10.5 Hz, H-6b), 3.25 (ddd, 1 H, H-5). 13C
NMR (D2O): d = 130.7, 80.0, 76.1, 73.9, 69.8, 67.3, 61.7, 45.8.
(a-D-Mannopyranosyl)methyl isothiocyanate (a-14b). 1H
NMR (D2O): d = 4.08 (ddd, 1 H, J1,2 = 3.5 Hz, J1,1¢a = 8.5 Hz,
J1,1¢b = 5.5 Hz, H-1), 3.94 (dd, 1 H, J1¢a,1¢b = 15.0 Hz, H-1¢a), 3.83
(dd, 1 H, J2,3 = 3.5 Hz, H-2), 3.75–3.55 (m, 5 H, H-3, H-4, 2H-6,
H-1¢b), 3.51 (ddd, 1 H, J4,5 = 8.0 Hz, J5,6a = 5.0 Hz, J5,6b = 5.5 Hz,
H-5). 13C NMR (D2O): d = 131.8, 75.2, 75.1, 70.7, 68.4, 67.4,
60.7, 44.0.
(2,3,4,6-Tetra-O-benzyl-a-D-mannopyranosyl)methyl amine (a-
13b). 1H NMR: d = 7.40–7.20 (m, 20 H, Ph), 4.70–4.50 (m,
10 H, PhCH2), 4.04–3.98 (m, 1 H, NH), 3.94–3.80 (m, 4 H), 3.76
(dd, 1 H, J = 5.0 Hz, J = 10.5 Hz), 3.72 (dd, 1 H, J = 1.5 Hz, J =
7.0 Hz), 2.89 (d, 2 H, J = 6.5 Hz), 1.60 (bs, 2 H).
(2,3,4,6-Tetra-O-benzyl-b-D-glucopyranosyl)methyl amine (b-
13c) and (2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl)methyl amine
(a-13c). 1H NMR selected data for 2:1 b/a diastereomeric
mixture: d = 7.40–7.10 (m, 20 H, Ph), 3.09 (dd, 0.66 H, J =
4.5 Hz, J = 14.0 Hz), 2.98 (dd, 0.33 H, J = 4.5 Hz, J = 14.0 Hz),
2.78 (dd, 1 H, J = 7.5 Hz, J = 14.0 Hz), 1.90 (bs, 2 H).
(b-D-Glucopyranosyl)methyl isothiocyanate (b-14c) and (a-D-
glucopyranosyl)methyl isothiocyanate (a-14c). 1H NMR (D2O)
selected data: d = 4.26–4.16 (m, 0.66 H, H-5b), 3.98 (dd, 0.66
H, J1,1¢a = 10.5 Hz, J1¢a,1¢b = 15.0 Hz, H-1¢ab). 13C NMR (D2O)
selected data: d = 131.6 (NCSb), 131.2 (NCSa).
General procedure for the synthesis of acetylated glycosylmethyl
isothiocyanates 4
General procedure for the synthesis of unprotected glycosylmethyl
isothiocyanates 14
A mixture of crude 14 (225–235 mg, ~1.00 mmol), pyridine
(5.0 mL), and Ac2O (5.0 mL) was stirred at room temperature
for 12 h, then concentrated, and coevaporated with toluene
three times. The resulting residue was eluted from a column of
silica gel with the suitable elution system to give the corresponding
peracetylated glycosylmethyl isothiocyanate 4.
A vigorously stirred mixture of benzylated amine 13 (540–553 mg,
~1.00 mmol), 20% palladium hydroxide on carbon (227 mg), and
AcOH (8 mL) was degassed under vacuum and saturated with
hydrogen (by a H2-filled balloon) three times. The suspension was
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(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)methyl
cyanate (b-4a). Column chromatography with
isothio-
1.5:1
a-4c (93 mg, 23%) and then b-4c (181 mg, 45%). b-4c: [a]D= -21.4
(c 1.0, CHCl3). 1H NMR: d = 5.20 (dd, 1 H, J2,3 = 9.0 Hz, J3,4
=
cyclohexane-AcOEt (containing 10% of CH2Cl2) afforded
b-4a (282 mg, 70%) as a white foam; [a]D= -11.4 (c 0.6, CHCl3).
1H NMR: d = 5.43 (dd, 1 H, J3,4 = 3.5 Hz, J4,5 = 0.5 Hz, H-4),
5.17 (dd, 1 H, J1,2 = 9.5 Hz, J2,3 = 9.5 Hz, H-2), 5.05 (dd, 1 H,
H-3), 4.14 (dd, 1 H, J5,6a = 7.0 Hz, J6a,6b = 11.5 Hz, H-6a), 4.10
(dd, 1 H, J5,6b = 6.0 Hz, H-6b), 3.96 (ddd, 1 H, H-5), 3.70–3.60 (m,
3 H, H-1, 2H-1¢), 2.20, 2.10, and 1.98 (3 s, 12 H, CH3). 13C NMR:
d = 170.3, 170.08, 169.9, 169.5, 134.2, 76.2, 74.2, 71.5, 67.3, 66.9,
61.6, 46.1, 20.6 (2C), 20.4 (2C). MALDI-TOF MS: 326.9 (M+ +
Na). Anal. Calcd. for C16H21NO9S (403.09): C, 47.64; N, 3.47; H,
5.25; S, 7.95. Found: C, 47.45; N, 3.58; H, 5.04; S, 7.73.
9.0 Hz, H-3), 5.04 (dd, 1 H, J4,5 = 9.0 Hz, H-4), 4.94 (dd, 1 H,
J1,2 = 9.0 Hz, H-2), 4.22 (dd, 1 H, J5,6a = 5.5 Hz, J6a,6b = 12.0 Hz,
H-6a), 4.12 (dd, 1 H, J5,6b = 3.0 Hz, H-6b), 3.72 (ddd, 1 H, H-5),
3.66 (ddd, 1 H, J1,1¢a = 4.0 Hz, J1,1¢b = 4.5 Hz, H-1), 3.62–3.58 (m,
2 H, 2H-1¢), 2.08, 2.06, 2.02, and 1.98 (4 s, 12 H CH3). 13C NMR:
d = 170.6, 170.2, 169.5, 169.3, 135.0, 75.9, 75.8, 73.6, 69.6, 68.1,
61.9, 46.1, 20.7 (2 C), 20.2 (2 C). MALDI-TOF MS: 404.3 (M+ +
H). Anal. Calcd. for C16H21NO9S (403.09): C, 47.64; N, 3.47; H,
5.25; S, 7.95. Found: C, 47.49; N, 3.61; H, 5.42; S, 8.19.
(2,3,4,6-Tetra-O-acetyl-a-D-glucopyranosyl)methyl isothiocy-
anate (a-4c). Column chromatography (from 4:1 to 1:1
cyclohexane-AcOEt containing 10% of CH2Cl2) afforded first
a-4c (93 mg, 23%) and then b-4c (181 mg, 45%). a-4c: [a]D= 96.0
(2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl)methyl isothiocy-
anate (a-4a). Column chromatography with 3:1 cyclohexane-
AcOEt (containing 10% of CH2Cl2) afforded a-4a (274 mg, 68%)
as a white foam; [a]D= 94.2 (c 1.2, CHCl3).). 1H NMR: d = 5.42(dd,
1 H, J3,4 = 3.5 Hz, J4,5 = 3.5 Hz, H-4), 5.28 (dd, 1 H, J1,2 = 9.0 Hz,
(c 1.2, CHCl3). 1H NMR: d = 5.25 (dd, 1 H, J2,3 = 9.0 Hz, J3,4
=
8.5 Hz, H-3), 5.17 (dd, 1 H, J1,2 = 5.5 Hz, H-2), 5.05 (dd, 1 H,
J4,5 = 9.0 Hz, H-4), 4.44 (ddd, 1 H, J1,1¢a = 10.0 Hz, J1,1¢b = 4.5 Hz,
H-1), 4.37 (dd, 1 H, J5,6a = 5.5 Hz, J6a,6b = 11.5 Hz, H-6a), 4.16
(dd, 1 H, J5,6b = 3.0 Hz, H-6b), 3.98 (dd, 1 H, J1¢a,1¢b = 15.0 Hz,
H-1¢a), 3.97 (ddd, 1 H, H-5), 3.64 (dd, 1 H, H-1¢b), 2.16, 2.14,
2.08, and 2.02 (4 s, 12 H, CH3). 13C NMR: d = 170.7, 169.7, 169.3,
169.2, 135.3, 70.6, 70.3, 69.5, 68.6, 67.8, 61.5, 42.5, 20.8 (2 C),
20.6 (2 C). MALDI-TOF MS: 426.3 (M+ + Na). Anal. Calcd. for
C16H21NO9S (403.09): C, 47.64; N, 3.47; H, 5.25; S, 7.95. Found:
C, 47.93; N, 3.29; H, 5.02; S, 7.79.
J2,3 = 5.5 Hz, H-2), 5.16 (dd, 1 H, H-3), 4.44 (ddd, 1 H, J1,1¢a
9.0 Hz, J1,1¢b = 5.0 Hz, H-1), 4.42 (dd, 1 H, J5,6a = 7.5 Hz, J6a,6b
=
=
11.5 Hz, H-6a), 4.18 (ddd, 1 H, J5,6b = 4.5 Hz, H-5), 4.09 (dd, 1
H, H-6b), 3.83 (dd, 1 H, J1¢a,1¢b = 15.0 Hz, H-1¢a), 3.59 (dd, 1 H,
H-1¢b), 2.10, 2.09, 2.08, and 2.02 (4 s, 12 H, CH3). 13C NMR: d =
170.8, 169.8, 169.6, 169.5, 134.7, 69.9, 69.8, 67.5, 67.3, 66.7, 60.6,
43.0, 20.8, 20.6 (2 C), 20.8. MALDI-TOF MS: 404.5 (M+ + H).
Anal. Calcd. for C16H21NO9S (403.09): C, 47.64; N, 3.47; H, 5.25;
S, 7.95. Found: C, 47.83; N, 3.61; H, 5.40; S, 8.18.
(2,3,4,6-Tetra-O-acetyl-b-D-mannopyranosyl)methyl isothiocy-
anate (b-4b). Column chromatography with 4:1 cyclohexane-
AcOEt (containing 10% of CH2Cl2) afforded b-4b (290 mg, 72%)
slightly contaminated by uncharacterized byproducts.1H NMR:
d = 5.41 (dd, 1 H, J1,2 = 1.0 Hz, J2,3 = 3.5 Hz, H-2), 5.21 (dd, 1 H,
J3,4 = 9.5 Hz, J4,5 = 9.5 Hz, H-4), 5.05 (dd, 1 H, H-3), 4.27 (dd,
General procedure for the synthesis of benzylated glycosylmethyl
isothiocyanates 15
To a stirred solution of crude amine 13 (540–553 mg, ~1.00 mmol)
in CH2Cl2 (20 mL) were added CS2 (120 mL, 2.00 mmol), and
Et3N (0.21 mL, 1.50 mmol). The mixture was stirred at room
temperature for 3 h, then 2-chloro-1-methylpyridinium iodide
(383 mg, 1.50 mmol) and Et3N (0.21 mL, 1.50 mmol) were
sequentially added. The resulting mixture was stirred at room
temperature for an additional 2 h, then diluted with AcOEt
(80 mL), and washed with brine (2 ¥ 25 mL). The aqueous layer was
extracted with AcOEt (2 ¥ 50 mL).The combined organic phases
were dried (Na2SO4), concentrated, and eluted from a column of
silica gel with the suitable elution system to give the corresponding
benzylated glycosylmethyl isothiocyanate 15.
1 H, J5,6a = 6.0 Hz, J6a,6 = 12.0 Hz, H-6a), 4.12 (dd, 1 H, J5,6b
=
2.5 Hz, H-6b), 3.88 (ddd, 1 H, J1,1¢a = 7.0 Hz, J1,1¢b = 5.0 Hz, H-1),
3.72 (dd, 1 H, J1¢a,1¢b = 14.5 Hz, H-1¢a), 3.71 (ddd, 1 H, H-5), 3.50
(dd, 1 H, H-1¢b), 2.22, 2.14, 2.07, and 2.00 (4 s, 12 H, CH3). 13C
NMR: d = 170.7, 170.1, 170.0, 169.6, 135.1, 77.3, 75.0, 71.8, 67.9,
65.7, 62.6, 45.5, 20.8, 20.6 (2 C), 20.5. MALDI-TOF MS: 426.4
(M+ + Na).
(2,3,4,6-Tetra-O-acetyl-a-D-mannopyranosyl)methyl isothiocy-
anate (a-4b). Column chromatography with 3:1 cyclohexane-
AcOEt (containing 10% of CH2Cl2) afforded a-4b (282 mg, 70%)
as a white foam; [a]D= 21.2 (c 1.0, CHCl3). 1H NMR: d = 5.29 (dd,
1 H, J2,3 = 3.5 Hz, J3,4 = 5.5 Hz, H-3), 5.11 (dd, 1 H, J1,2 = 7.5 Hz,
(2,3,4,6-Tetra-O-benzyl-b-D-galactopyranosyl)methyl isothiocy-
anate (b-15a). Column chromatography with 6:1 cyclohexane-
AcOEt afforded b-15a (506 mg, 85%) as a yellow syrup; [a]D=
-34.0 (c 0.9, CHCl3). 1H NMR: d = 7.40–7.20 (m, 20 H, Ph), 4.98
and 4.62 (2 d, 2 H, J = 11.0 Hz, PhCH2), 4.97 and 4.61 (2 d, 2
H, J = 11.5 Hz, PhCH2), 4.78 and 4.68 (2 d, 2 H, J = 11.8 Hz,
PhCH2), 4.51 and 4.44 (2 d, 2 H, J = 12.0 Hz, PhCH2), 4.02 (dd,
1 H, J3,4 = 3.5 Hz, J4,5 = 0.5 Hz, H-4), 3.84 (dd, 1 H, J1,2 = 9.5 Hz,
J2,3 = 9.5 Hz, H-2), 3.74(dd, 1 H, J1,1¢a = 3.0 Hz, J1¢a,1¢b = 14.5 Hz,
H-1¢a), 3.66–3.58 (m, 5 H, H-3, H-5, 2H-6, H-1¢b), 3.43 (ddd, 1 H,
J1,1¢b = 6.0 Hz, H-1¢b). 13C NMR: d = 138.5, 137.9, 137.7, 137.6,
132.7, 128.5–127.4 (20 C), 84.4, 77.4, 77.1, 75.3, 75.2, 74.4, 73.5,
73.3, 72.0, 68.7, 46.5. MALDI-TOF MS: 634.7 (M+ + K). Anal.
Calcd. for C36H37NO5S (595.24): C, 72.58; N, 2.35; H, 6.26; S, 5.38.
Found: C, 72.36; N, 2.22; H, 6.02; S, 5.22.
H-2), 4.98 (dd, 1 H, J4,5 = 5.5 Hz, H-4), 4.71 (dd, 1 H, J5,6a
=
8.0 Hz, J6a,6b = 11.5 Hz, H-6a), 4.20 (ddd, 1 H, J1,1¢a = 7.0 Hz,
J1,1¢b = 3.5 Hz, H-1¢a), 4.10 (ddd, 1 H, J5,6b = 3.5 Hz, H-6b), 4.05
(dd, 1 H, H-6b), 3.72 (dd, 1 H, J1¢a,1¢b = 15.0 Hz, H-1¢a), 3.63 (dd,
1 H, H-1¢b), 2.12, 2.11, 2.08, and 2.02 (4 s, 12 H, CH3). 13C NMR:
d = 170.7, 169.4 (2 C), 169.2, 134.7, 73.3, 68.7, 67.6, 67.1, 66.6,
60.3, 45.3, 20.7 (2 C), 20.6 (2 C). MALDI-TOF MS: 442.5 (M+ +
K). Anal. Calcd. for C16H21NO9S (403.09): C, 47.64; N, 3.47; H,
5.25; S, 7.95. Found: C, 47.81; N, 3.65; H, 5.08; S, 7.79.
(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)methyl isothiocy-
anate (b-4c). Column chromatography (from 4:1 to 1:1
cyclohexane-AcOEt containing 10% of CH2Cl2) afforded first
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(2,3,4,6-Tetra-O-benzyl-a-D-galactopyranosyl)methyl isothiocy-
anate (a-15a). Column chromatography with 7:1 cyclohexane-
AcOEt afforded a-15a (524 mg, 88%) as a yellow syrup; [a]D=
15.8 (c 1.3, CHCl3). 1H NMR: d = 7.40–7.20 (m, 20 H, Ph), 4.68
and 4.47 (2 d, 2 H, J = 12.0 Hz, PhCH2), 4.64 and 4.54 (2 d, 2
H, J = 11.8 Hz, PhCH2), 4.58 and 4.51 (2 d, 2 H, J = 11.0 Hz,
PhCH2), 4.57 (s, 2 H, PhCH2), 4.14–4.06 (m, 2 H, H-1, H-5),
4.00 (dd, 1 H, J3,4 = 4.0 Hz, J4,5 = 2.5 Hz, H-4), 3.91 (dd, 1 H,
J5,6a = 7.5 Hz, J6a,6b = 11.0 Hz, H-6a), 3.78–3.64 (m, 4 H, H-2,
H-3, H-6b, H-1¢a), 3.60 (dd, 1 H, J1,1¢b = 6.5 Hz, J1¢a,1¢b = 15 Hz,
H-1¢b). 13C NMR: d = 138.2, 138.0, 137.5, 137.4, 132.6, 128.5–
127.6 (20 C), 75.5, 74.9, 73.6, 73.4, 73.3, 73.2, 73.1, 72.9, 69.2,
66.3, 43.7. MALDI-TOF MS: 618.8 (M+ + Na). Anal. Calcd. for
C36H37NO5S (595.24): C, 72.58; N, 2.35; H, 6.26; S, 5.38. Found:
C, 72.39; N, 2.50; H, 6.02; S, 5.54.
3.10 (dd, 1 H, J1,1¢a = 3.0 Hz, J1¢a,1¢b = 15.0 Hz, H-1¢a), 2.97 (dd,
1 H, J1,1¢b = 5.0 Hz, H-1¢b), 2.87 (ddd, 1 H, H-1). 13C NMR: d =
138.2, 138.0, 137.8, 137.5, 133.0, 128.6–127.6 (20 C), 86.8, 79.2,
78.1, 77.3, 76.8, 75.6, 75.2, 75.1, 73.6, 68.8, 46.1. MALDI-TOF
MS: 634.9 (M+ + K). Anal. Calcd. for C36H37NO5S (595.24): C,
72.58; N, 2.35; H, 6.26; S, 5.38. Found: C, 72.37; N, 2.54; H, 6.42;
S, 5.09.
(2,3,4,6-Tetra-O-benzyl-a-D-glucopyranosyl)methyl isothiocy-
anate (a-15c). Column chromatography (from 9:1 to 5:1
cyclohexane-AcOEt) afforded first a-15c (149 mg, 25%) and then
b-15c (298 mg, 50%). a-15c: [a]D= 59.5 (c 0.8, CHCl3). 1H NMR
(C6D6): d = 7.40–7.00 (m, 20 H, Ph), 4.78 and 4.59 (2 d, 2 H,
J = 11.5 Hz, PhCH2), 4.71 (s, 2 H, PhCH2), 4.45 and 4.30 (2 d, 2
H, J = 12.0 Hz, PhCH2), 4.20 and 4.12 (2 d, 2 H, J = 11.8 Hz,
PhCH2), 3.84 (ddd, 1 H, J1,2 = 5.5 Hz, J1,1¢a = 10.5 Hz, J1,1¢b
=
4.0 Hz, H-1), 3.74 (dd, 1 H, J3,4 = 8.5 Hz, J4,5 = 9.5 Hz, H-4), 3.70
(dd, 1 H, J5,6a = 2.5 Hz, J6a,6b = 10.5 Hz, H-6a), 3.67 (dd, 1 H,
J5,6b = 3.0 Hz, H-6b), 3,43 (dd, 1 H, J2,3 = 9.0 Hz, H-2), 3.38 (dd,
1 H, H-3), 3.32 (ddd, 1 H, H-5), 3.20 (dd, 1 H, J1¢a,1¢b = 15.0 Hz,
H-1¢a), 3.00 (dd, 1 H, H-1¢b). 13C NMR: d = 138.2, 137.9, 137.8,
137.4, 134.0, 128.6–127.7 (20 C), 81.6, 78.1, 77.3, 75.2, 74.8, 73.7,
73.6, 72.6, 72.3, 68.2, 42.0. MALDI-TOF MS: 618.8 (M+ + Na).
Anal. Calcd. for C36H37NO5S (595.24): C, 72.58; N, 2.35; H, 6.26;
S, 5.38. Found: C, 72.39; N, 2.17; H, 6.41; S, 5.54.
(2,3,4,6-Tetra-O-benzyl-b-D-mannopyranosyl)methyl isothiocy-
anate (b-15b). Column chromatography with 6:1 cyclohexane-
AcOEt afforded b-15b (482 mg, 81%) as a yellow syrup; [a]D=
-36.0 (c 1.0, CHCl3). 1H NMR: d = 7.50–7.10 (m, 20 H, Ph), 5.10
and 4.68 (2 d, 2 H, J = 11.5 Hz, PhCH2), 4.91 and 4.48 (2 d, 2
H, J = 11.0 Hz, PhCH2), 4.84 and 4.79 (2 d, 2 H, J = 11.8 Hz,
PhCH2), 4.65 and 4.56 (2 d, 2 H, J = 12.0 Hz, PhCH2), 3.98 (dd,
1 H, J3,4 = 9.0 Hz, J4,5 = 9.0 Hz, H-4), 3.92 (dd, 1 H, J1,2 = 1.0 Hz,
J2,3 = 3.5 Hz, H-2), 3.80–3.70 (m, 3 H, H-1, 2 H-6), 3.68 (dd, 1 H,
H-3), 3.55 (d, 2 H, J1¢a,1¢b = 12.5 Hz, 2H-1¢), 3.50 (ddd, 1 H, J5,6a
=
Na-(9-Fluorenylmethoxycarbonyloxy)-b-amino-L-alanine tert-
2.5 Hz, J5,6b = 5.5 Hz, H-5). 13C NMR: d = 138.0, 137.9, 137.8,
137.6, 132.0, 128.5–127.5 (20 C), 84.4, 79.8, 76.0, 75.2, 74.8, 74.5,
73.4, 73.0, 72.7, 69.1, 45.0. MALDI-TOF MS: 618.3 (M+ + Na).
Anal. Calcd. for C36H37NO5S (595.24): C, 72.58; N, 2.35; H, 6.26;
S, 5.38. Found: C, 72.40; N, 2.49; H, 6.52; S, 5.12.
butyl ester (6). To
a
cooled (0 ◦C), stirred mixture
of
ester
commercially
hydrochloride
available
(800
L-asparagine
mg, 3.56 mmol),
tert-butyl
N-(9-
fluorenylmethoxycarbonyloxy)succinimide (1.80 g, 5.34 mmol),
and THF-H2O (20 mL-12 mL) saturated NaHCO3 aqueous
solution was added till basic pH was reached. The resulting
mixture was warmed to room temperature, stirred for 14 h, cooled
to 0 ◦C, neutralized with saturated NH4Cl aqueous solution,
diluted with AcOEt (100 mL), and washed with brine (2 ¥ 20 mL).
The organic phase was dried (Na2SO4), concentrated, and
eluted from a column of silica gel with 1:2 cyclohexane-AcOEt
(containing 10% of CH2Cl2) to give Fmoc-Asn-Ot-Bu 1620 (1.31 g,
(2,3,4,6-Tetra-O-benzyl-a-D-mannopyranosyl)methyl isothiocy-
anate (a-15b). Column chromatography with 5:1 cyclohexane-
AcOEt afforded a-15b (494 mg, 83%) as a yellow syrup; [a]D=
29.7 (c 1.0, CHCl3). 1H NMR: d = 7.40–7.10 (m, 20 H, Ph), 4.58
and 4.42 (2 d, 2 H, J = 12.0 Hz, PhCH2), 4.53 and 4.48 (2 d, 2
H, J = 11.0 Hz, PhCH2), 4.50 and 4.46 (2 d, 2 H, J = 11.5 Hz,
PhCH2), 4.44 and 4.38 (2 d, 2 H, J = 11.8 Hz, PhCH2), 4.15 (ddd,
1 H, J4,5 = 8.0 Hz, J5,6a = 3.0 Hz, J5,6b = 5.5 Hz, H-5), 4.02 (ddd, 1
H, J1,2 = 3.0 Hz, J1,1¢a = 5.5 Hz, J1,1¢b = 9.0 Hz, H-1), 3.83 (dd, 1 H,
J6a,6b = 10.5 Hz, H-6a), 3.82 (dd, 1 H, J3,4 = 8.0 Hz, H-4), 3.78–3.70
(m, 4 H, H-2, H-3, 2H-1¢), 3.68 (dd, 1 H, H-6b). 13C NMR: d =
138.1, 137.9, 137.7, 137.4, 132.4, 128.5–127.6 (20 C), 74.6, 73.9,
73.4, 73.3, 73.0, 72.6, 71.8, 71.2, 68.6, 67.8, 46.6. MALDI-TOF
MS: 596.5 (M+ + H). Anal. Calcd. for C36H37NO5S (595.24): C,
72.58; N, 2.35; H, 6.26; S, 5.38. Found: C, 72.79; N, 2.59; H, 6.45;
S, 5.22.
◦
1
90%) as a white solid (mp 136–138 C, from MeCN). H NMR:
d = 7.81–7.78 (m, 2 H, Ar), 7.68–7.60 (m, 2 H, Ar), 7.45–7.20
(m, 4 H, Ar), 6.00 (d, 1 H, J2,NH = 8.5 Hz, NaH), 5.69 (bs, 1 H,
NH), 5.40 (bs, 1 H, NH), 4.50 (ddd, 1 H, J2,3a = 5.5 Hz, J2,3b
=
4.5 Hz, H-2), 4.42–4.32 (m, 2 H, FmocCH2), 4.30.4.20 (m, 1 H,
FmocCH), 2.96 (dd, 1 H, J3a,3b = 16.0 Hz, H-3a), 2.78 (dd, 1 H,
H-3b), 1.42 (s, 9 H, t-Bu).
To a stirred solution of 16 (500 mg, 1.22 mmol) in H2O-MeCN
(6 mL-6 mL) were added I,I-bis(trifluoroacetoxy)iodobenzene
(682 mg, 1.59 mmol) and pyridine (197 mL, 2.44 mmol). The
resulting mixture was stirred at room temperature for 2.5 h, then
diluted with AcOEt (80 mL), and washed with brine (2 ¥ 10 mL).
The organic phase was dried (Na2SO4), concentrated, and eluted
from a column of silica gel with 19:1 CH2Cl2-MeOH to afford 6
(2,3,4,6-Tetra-O-benzyl-b-D-glucopyranosyl)methyl isothiocy-
anate (b-15c). Column chromatography (from 9:1 to 5:1
cyclohexane-AcOEt) afforded first a-15c (149 mg, 25%) and then
b-15c (298 mg, 50%). b-15c: [a]D= -46.1 (c 1.1, CHCl3). 1H NMR
(C6D6): d = 7.40–7.00 (m, 20 H, Ph), 4.82 and 4.72 (2 d, 2 H, J =
11.5 Hz, PhCH2), 4.76 and 4.55 (2 d, 2 H, J = 10.5 Hz, PhCH2),
4.72 and 4.33 (2 d, 2 H, J = 11.0 Hz, PhCH2), 4.48 and 4.37 (2 d,
1
(401 mg, 86%) as a white foam; [a]D= -19.9 (c 1.0, MeOH). H
NMR: d = 7.80–7.70 (m, 2 H, Ar), 7.65–7.55 (m, 2 H, Ar), 7.42–
7.25 (m, 4 H, Ar), 5.58 (d, 1 H, J2,NH = 8.5 Hz, NaH), 4.41 (d, 2
2 H, J = 12.0 Hz, PhCH2), 3.64 (dd, 1 H, J5,6a = 4.5 Hz, J6a,6b
=
H, J = 8.0 Hz, FmocCH2), 4.28 (bddd, 1 H, J2,3a = 4.5 Hz, J2,3b =
9.5 Hz, H-6a), 3.62 (dd, 1 H, J3,4 = 9.0 Hz, J4,5 = 8.5 Hz, H-4),
3.59 (dd, 1 H, J5,6b¢ = 2.5 Hz, H-6b), 3.47 (dd, 1 H, J2,3 = 9.0 Hz,
H-3), 3.26 (dd, 1 H, J1,2 = 9.5 Hz, H-2), 3.18 (ddd, 1 H, H-5),
5.5 Hz, H-2), 4.22 (t, 1 H, J = 8.0 Hz, FmocCH), 3.10 (dd, 1 H,
J3a,3b = 12.5 Hz, H-3a), 3.04 (dd, 1 H, H-3b), 1.90 (bs, 2 H, NH2),
1.42 (s, 9 H, t-Bu). 13C NMR: d = 169.9, 156.3, 143.7 (2 C), 141.2
This journal is
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Org. Biomol. Chem., 2009, 7, 3319–3330 | 3327
©

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(2 C), 127.6 (2 C), 127.0 (2 C), 125.0 (2 C), 119.9 (2 C), 82.6, 67.0,
56.1, 47.0, 43.5, 27.8 (3 C). MALDI-TOF MS: 383.4 (M+ + H).
Anal. Calcd. for C22H26N2O4 (382.19): C, 69.09; N, 7.32; H, 6.85.
Found: C, 69.28; N, 7.15; H, 7.01.
FmocCH2, FmocCH, 2H-1¢¢), 2.08, 2.00, 1.95, and 1.90 (4 s, 12
H, CH3). 13C NMR (acetone-d6): d = 184.2, 172.0, 171.5, 171.3,
169.5, 169.0, 156.5, 144.4 (2 C), 141.4 (2 C), 127.9 (2 C), 127.4
(2 C), 125.7 (2 C), 120.2 (2 C), 76.6, 74.3, 72.1, 68.6, 64.5, 64.2,
62.3, 56.4, 47.6, 45.9, 44.5, 20.8 (2 C), 20.0 (2 C). m/z (ESI+) 731.0
(MH+, 100%). Anal. Calcd. for C34H39N3O13S (729.22): C, 55.96;
N, 5.76; H, 5.39; S, 4.39. Found: C, 56.20; N, 5.95; H, 5.54; S, 4.22.
General procedure for the synthesis of glycosyl amino acids 7a-c
A
solution of glycosylmethyl isothiocyanate 4 (161 mg,
0.40 mmol), Na-Fmoc-b-amino-L-alanine 519 (196 mg, 0.60 mmol)
in anhydrous DMF (2 mL) was stirred at room temperature for
24 h and then concentrated. The resulting residue was eluted from
a column of silica gel with the suitable elution system to give the
corresponding glycosyl amino acid 7.
(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(2¢,3¢,4¢,6¢-
tetra-O-acetyl-a-D-mannopyranosylmethyl-thioureido)propionic
acid (a-7b). Column chromatography with AcOEt first and then
20:1 AcOEt-AcOH afforded a-7b (219 mg, 75%) as a white foam;
1
[a]D= 7.6 (c 1.0, CHCl3). H NMR (acetone-d6): d = 7.90–7.80
1
Partial decomposition of derivatives 7 was observed when H
(m, 2 H, Ar), 7.70–7.60 (m, 2 H, Ar), 7.40–7.10 (m, 4 H, Ar), 5.28
NMR analysis was performed at high temperatures (DMSO-d6,
T > 100) as confirmed by the presence of fulvene signals in the
spectra.
(dd, 1 H, J2¢,3¢ = 3.5 Hz, J3¢,4¢ = 8.5 Hz, H-3¢), 5.20 (dd, 1 H, J1¢,2¢
=
3.0 Hz, H-2¢), 5.14 (dd, 1 H, J4¢,5¢ = 8.5 Hz, H-4¢), 4.54.4.48 (m, 1
H, H-2), 4.40–4.00 (m, 10 H, H-1¢, H-5¢, 2H-6¢, H-3a, FmocCH2,
FmocCH, 2H-1¢¢), 3.98–3.90 (m, 1 H, H-3b), 2.08, 2.04, 1.98, and
1.96 (4 s, 12 H, CH3). 1H NMR (DMF-d7) selected data: d = 8.19
(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(2¢,3¢,4¢,6¢-
tetra-O-acetyl-b-D-galactopyranosylmethyl-thioureido)propionic
acid (b-7a). Column chromatography with AcOEt first and then
20:1 AcOEt-AcOH afforded b-7a (216 mg, 74%) as a white foam;
[a]D= -21.0 (c 1.0, CHCl3). 1H NMR (acetone-d6): d = 7.90–7.80
(m, 2 H, Ar), 7.70–7.60 (m, 2 H, Ar), 7.50–7.20 (m, 4 H, Ar), 5.39
(bs, 2 H, NHC(S)NH), 7.90 (d, 1 H, J = 8.0 Hz, NHFmoc). 13
C
NMR (acetone-d6): d = 184.6, 171.9, 170.4, 169.7, 169.5 (2C),
156.4, 144.4 (2 C), 141.4 (2 C), 129.1 (2 C), 128.4 (2 C), 125.5 (2
C), 120.1 (2 C), 72.4, 72.1, 68.6, 68.3, 67.4, 66.8, 61.8, 54.6, 47.3,
45.1, 43.2, 20.2 (2 C), 20.1, 20.0. m/z (ESI+) 730.9 (MH+, 100%).
Anal. Calcd. for C34H39N3O13S (729.22): C, 55.96; N, 5.76; H, 5.39;
S, 4.39. Found: C, 55.77; N, 5.93; H, 5.21; S, 4.19.
(dd, 1 H, J3¢,4¢ = 3.5 Hz, J4¢,5¢ = 0.5 Hz, H-4¢), 5.12 (dd, 1 H, J2¢,3¢
=
9.5 Hz, H-3¢), 5.06 (dd, 1 H, J1¢,2¢ = 9.5 Hz, H-2¢), 4.52–4.48 (m, 1 H,
H-2), 4.34–4.00 (m, 8 H, H-3a, H-5¢, 2H-6¢, FmocCH2, FmocCH,
H-1¢¢a), 3.98–3.90 (m, 1 H, H-3b), 3.78 (ddd, 1 H, J1¢,1¢¢a = 2.5 Hz,
J1¢,1¢¢b = 8.0 Hz H-1¢), 3.36 (dd, 1 H, J1¢¢a,1¢¢b = 14.5 Hz, H-1¢¢b), 2.12,
2.08, 1.95, and 1.90 (4 s, 12 H, CH3). 13C NMR (acetone-d6): d =
184.3, 172.0, 170.1, 170.0, 169.9, 169.6, 156.4, 144.4 (2 C), 141.4
(2 C), 127. 9 (2 C), 127.4 (2 C), 125.7 (2 C), 120.2 (2 C), 77.2, 74.0,
71.9, 68.0, 67.5, 66.8, 61.5, 54.7, 47.3, 45.7, 45.2, 20.2, 20.0 (3 C).
m/z (ESI+) 730.9 (MH+, 100%). Anal. Calcd. for C34H39N3O13S
(729.22): C, 55.96; N, 5.76; H, 5.39; S, 4.39. Found: C, 56.17; N,
5.61; H, 5.18; S, 4.23.
(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(2¢,3¢,4¢,6¢-
tetra - O - acetyl - b - D - glucopyranosylmethyl - thioureido)propionic
acid (b-7c). Column chromatography with AcOEt first and then
20:1 AcOEt-AcOH afforded b-7c (213 mg, 73%) as a white foam;
[a]D= -23.3 (c 1.0, CHCl3). 1H NMR (acetone-d6): d = 7.90–7.80
(m, 2 H, Ar), 7.70–7.60 (m, 2 H, Ar), 7.50–7.20 (m, 4 H, Ar), 7.05
(d, 1 H, J = 8.5 Hz, NH), 5.22 (dd, 1 H, J2¢,3¢ = 9.5 Hz, J3¢,4¢
=
9.5 Hz, H-3¢), 4.99 (dd, 1 H, J4¢,5¢ = 9.5 Hz, H-4¢), 4.88 (dd, 1 H,
J1¢,2¢ = 9.5 Hz, H-2¢), 4.58–4.50 (m, 1 H, H-2), 4.36–3.90 (m, 8
H, 2H-6¢, 2H-3, FmocCH2, FmocCH, H-1¢¢a), 3.84–3.76 (m, 2 H,
H-1¢, H-5¢), 3.70–3.60 (m, 1 H, H-1¢¢b), 2.08, 2.04, 1.96, and 1.90
(4 s, 12 H, CH3). 13C NMR (acetone-d6): d = 184.7, 172.4, 170.2,
169.7, 169.6, 169.4, 156.5, 144.4 (2 C), 141.4 (2 C), 127.9 (2 C),
127.4 (2 C), 125.7 (2 C), 120.1 (2 C), 76.8, 75.4, 74.0, 69.9, 68.8,
66.8, 62.3, 54.8, 47.3, 45.4 (2 C), 20.2, 20.0, 19.9, 19.8. m/z (ESI+)
731.0 (MH+, 100%). Anal. Calcd. for C34H39N3O13S (729.22): C,
55.96; N, 5.76; H, 5.39; S, 4.39. Found: C, 55.95; N, 5.59; H, 5.59;
S, 4.13.
(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(2¢,3¢,4¢,6¢-
tetra-O-acetyl-a-D-galactopyranosylmethyl-thioureido)propionic
acid (a-7a). Column chromatography with AcOEt first and then
20:1 AcOEt-AcOH afforded a-7a (204 mg, 70%) as a white foam;
1
[a]D= 23.2 (c 1.0, CHCl3). H NMR (acetone-d6): d = 7.90–7.85
(m, 2 H, Ar), 7.80–7.70 (m, 2 H, Ar), 7.50–7.30 (m, 4 H, Ar), 5.20
(bs, 1 H, NH), 5.30–5.20 (m, 2 H), 4.60–3.80 (m, 12 H), 2.15, 2.12,
2.12, and 2.10 (4 s, 12 H, CH3). 13C NMR (acetone-d6): d = 184.1,
172.3, 170.2, 169.9, 169.7, 169.5, 156.5, 144.4 (2 C), 141.4 (2 C),
127.9 (2 C), 127.4 (2 C), 125.7 (2 C), 120.2 (2 C), 71.6, 69.0, 67.8,
67.7, 67.6, 66.8, 61.0, 54.8, 47.3, 45.2, 41.6, 20.2, 20.1, 20.0, 19.9.
m/z (ESI+) 731.0 (MH+, 100%). Anal. Calcd. for C34H39N3O13S
(729.22): C, 55.96; N, 5.76; H, 5.39; S, 4.39. Found: C, 55.71; N,
5.93; H, 5.19; S, 4.20.
(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(2¢,3¢,4¢,6¢-
tetra-O -acetyl- a- D -glucopyranosylmethyl-thioureido)propionic
acid (a-7c). Column chromatography with AcOEt first and then
20:1 AcOEt-AcOH afforded a-7c (204 mg, 70%) as a white foam;
1
[a]D= 28.3 (c 0.8, CHCl3). H NMR (acetone-d6): d = 7.90–7.80
(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(2¢,3¢,4¢,6¢-
tetra-O-acetyl-b-D-mannopyranosylmethyl-thioureido)propionic
acid (b-7b). Column chromatography with AcOEt first and then
20:1 AcOEt-AcOH afforded b-7b (207 mg, 71%) as a white foam;
[a]D= -8.6 (c 1.0, CHCl3). 1H NMR: d = 7.80–7.70 (m, 2 H, Ar),
7.60–7.50 (m, 2 H, Ar), 7.40–7.20 (m, 4 H, Ar), 7.10 (bs, 1 H,
(m, 2 H, Ar), 7.70–7.60 (m, 2 H, Ar), 7.50–7.20 (m, 4 H, Ar), 5.34
(dd, 1 H, J2¢,3¢ = 9.5 Hz, J3¢,4¢ = 9.5 Hz, H-3¢), 5.07 (dd, 1 H, J1¢,2¢
=
5.5 Hz, H-2¢), 4.99 (dd, 1 H, J4¢,5¢ = 9.0 Hz, H-4¢), 4.50–3.60 (m, 12
H, H-1¢, H-5¢, 2H-6¢, H-2, 2H-3, FmocCH2, FmocCH, 2H-1¢¢),
2.08, 2.00, 1.98, and 1.95 (4 s, 12 H, CH3). 13C NMR (acetone-d6):
d = 185.0, 171.8, 170.9, 170.1, 170.0, 189.9, 157.0, 145.0 (2 C),
142.0 (2 C), 128,5 (2 C), 127.0 (2 C), 126.2 (2 C), 120.7 (2 C),
72.1, 70.6, 70.2, 69.2, 67.4, 62.6, 62.5, 55.6, 47.9, 46.1, 41.0, 20.7
(2 C), 20.6 (2 C). m/z (ESI+) 730.8 (MH+, 100%). Anal. Calcd. for
NH), 6.80 (bs, 1 H, NH), 6.60 (bs, 1 H, NH), 5.40 (dd, 1 H, J1¢,2¢
0.5 Hz, J2¢,3¢ = 3.0 Hz, H-2¢), 5.20 (dd, 1 H, J3¢,4¢ = 9.0 Hz, J4¢,5¢
=
=
9.0 Hz, H-4¢), 4.50–3.20 (m, 12 H, H-1¢, H-5¢, 2H-6¢, H-2, 2H-3,
3328 | Org. Biomol. Chem., 2009, 7, 3319–3330
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©

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C34H39N3O13S (729.22): C, 55.96; N, 5.76; H, 5.39; S, 4.39. Found:
C, 55.80; N, 5.51; H, 5.17; S, 4.56.
Acknowledgements
We thank M. Passaponti (Universita` di Firenze, Italy) for the
elemental analyses and P. Formaglio (Universita` di Ferrara, Italy)
for NMR experiments. R. F. B. is grateful for a scholarship
(Channel Scholarship Program) from Chemical Industries Divi-
sion (National Research Center, Dokki, Cairo, Egypt) to spend
two years at the University of Ferrara. A. D. and A. M. greatly
acknowledge the University of Ferrara for financial support.
(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(2¢,3¢,4¢,6¢-
tetra-O-acetyl-a-D-mannopyranosylmethyl-thioureido)propionic
acid tert-butyl ester (a-8b). A solution of glycosylmethyl isothio-
cyanate a-4b (161 mg, 0.40 mmol), Na-Fmoc-b-amino-L-alanine
tert-butyl ester 6 (229 mg, 0.60 mmol) in anhydrous DMF (2 mL)
was stirred at room temperature for 24 h and then concentrated.
The resulting residue was eluted from a column of silica gel with
1:1 cyclohexane-AcOEt to give a-8b (204 mg, 65%) as a white
foam; [a]D= 9.3 (c 1.4, CHCl3). 1H NMR: d = 7.90.7.75 (m, 2 H,
Ar), 7.65–7.55 (m, 2 H, Ar), 7.50–7.30 (m, 4 H, Ar), 6.60 (bs, 2
References and notes
1 M. H. D. Postema, C-Glycoside Synthesis, CRC Press, Boca Raton,
1995.
H, NH), 5.92 (d, 1 H, J2,NH = 8.0 Hz, NH), 5.30 (dd, 1 H, J2¢,3¢
=
2 (a) A. Varki, Essentials of Glycobiology, ed. R. D. Cummings, J. D. Esko,
H. H. Freeze, G. W. Hart and J. Marth, Cold Spring Harbor Laboratory
Press, New York, 1999; (b) O. Seitz, ChemBioChem, 2000, 1, 214;
(c) H. C. Hang and C. R. Bertozzi, Acc. Chem. Res., 2001, 34, 727–736.
3 For a brief introduction to this topic with leading references, see: A.
Dondoni, and A. Massi, in Current Frontiers in Asymmetric Synthesis
and Application of alpha-Amino Acids, ed. V. A. Soloshonok and
K. Izawa, ACS Symposium Series, Oxford University Press, 2009, p.
13–30.
3.5 Hz, J3¢,4¢ = 7.5 Hz, H-3¢), 5.14 (dd, 1 H, J1¢,2¢ = 6.5 Hz, H-2¢),
5.06 (dd, 1 H, J4¢,5¢ = 7.5 Hz, H-4¢), 4.70–4.58 (m, 1 H), 4.404–3.60
(m, 11 H), 2.10, 2.08, 2.06, and 2.05 (4 s, 12 H, CH3), 1.42 (2 9
H, t-Bu). 13C NMR: d = 183.2, 171.1, 169.9, 169.5, 169.4, 169.1,
143.6 (2 C), 143.5 (2 C), 127.8 (2 C), 127.1 (2 C), 125.0 (2 C), 120.0
(2 C), 83.6, 72.9, 70.7, 67.8, 67.4, 67.3, 61.2, 60.3, 56.5, 47.0, 46.9,
44.0, 27.9, 20.9 (2 C), 20.7, 20.6. m/z (ESI+) 786.8 (MH+, 100%).
Anal. Calcd. for C34H39N3O13S (785.28): C, 58.08; N, 5.35; H, 6.03;
S, 4.04. Found: C, 58.32; N, 5.19; H, 6.19; S, 4.20.
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(2S)-2-(tert-Butoxycarbonylamino)-3-(2¢,3¢,4¢,6¢-tetra-O-ben-
zyl-a-D-galactopyranosylmethyl-thioureido)propionic acid (a-18a).
A solution of glycosylmethyl isothiocyanate a-15a (238 mg,
0.40 mmol), Na-Boc-b-amino-L-alanine 17 (122 mg, 0.60 mmol)
in anhydrous pyridine (2 mL) was stirred at room temperature for
24h andthenconcentrated. The resulting residue was elutedfrom a
column of silica gel with 20:1 AcOEt-AcOH to give a-18a (384 mg,
48%) slightly contaminated by uncharacterized byproducts. This
derivative was characterized as methyl ester by diazomethane
esterification. a-18a Me ester; 1H NMR (selected data): d = 7.40–
7.20 (m, 20 H, Ph), 6.38 (bs, 1 H), 5.78 (bs, 1 H), 3.75 (s, 3 H,
OCH3), 1.42 (s, 9 H, t-Bu). m/z (ESI+) 815.0 (MH+, 100%).
(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(b-D-gala-
ctopyranosylmethyl-thioureido)propionic acid (b-19a). A solution
of glycosylmethyl isothiocyanate b-14a (100 mg, ~0.40 mmol), Na-
Fmoc-b-amino-L-alanine 5 (197 mg, 0.60 mmol) in anhydrous
DMF (2 mL) was stirred at room temperature for 24 h and
then concentrated to give crude b-19a in 70% estimated (1H
NMR analysis). The resulting residue was diluted with pyridine
(0.5 mL) and Ac2O (0.5 mL), stirred at room temperature for 4 h,
concentrated, and eluted from a column of silica gel with AcOEt
first and then 20:1 AcOEt-AcOH to afford b-7a (145 mg, 50%) as
a white foam.
The same coupling performed in pyridine (2 mL) produced a
residue that was eluted from a column of silica gel with 5:1 AcOEt-
MeOH to give 20 (84 mg, 90%) as a white foam. 1H NMR (D2O):
d = 4.07 (dd, 1 H, J1,2 = 9.5 Hz, J2,3 = 9.5 Hz, H-2), 3.91 (dd, 1 H,
J3,4 = 3.5 Hz, J4,5 = 0.5 Hz, H-4), 3.82 (dd, 1 H, H-4), 3.70–3.62
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(m, 2 H, H-1, H-5), 3.61–3.55 (m, 2 H, 2H-6), 3.48 (dd, 1 H, J1,1¢a
=
6.0 Hz, J1¢a,1¢b = 12.0 Hz, H-1¢a), 3.12 (dd, 1 H, J1,1¢b = 10.0 Hz,
H-1¢b). 13C NMR (D2O): d = 186.2, 79.7, 78.8, 70.5, 69.5, 67.4,
61.3, 44.3. MALDI-TOF MS: 274.8 (M+ + K).
7 For examples of biological and biotechnological applications of
synthetic glycopeptides/glycoproteins, see: (a) M. Aebi and T. Hennet,
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22 A pure sample of this product was not obtained by chromatography
of the reaction mixture. Evidence for its formation relies on the signal
splitting of the 1H NMR spectrum of isolated a-7b.
23 The evaluation of the organocatalytic activity of stereodiversified and
polyfunctional thioureido-amino acids 7, 18, 19 and their derivatives is
currently under investigation in our laboratory.
24 Compound b-20a was isolated as peracetylated derivative b-7a after
acetylation (Ac2O, pyridine) of the crude reaction mixture (see Experi-
mental section).
25 A. Dondoni, A. Massi, S. Sabbatini and V. Bertolasi, J. Org. Chem.,
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3330 | Org. Biomol. Chem., 2009, 7, 3319–3330
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The Royal Society of Chemistry 2009
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