Facile synthesis of N-Fmoc-serine-S-GlcNAc: A potential molecular probe for the functional study of O-GlcNAc

Article abstract of DOI:10.1016/S0960-894X(00)00223-7

A methabolically stable β-N-acetylglucosaminyl-l-thio-N-Fmoc-serine (S-GlcNAc-Ser) derivative was synthesized in two procedures: one involving a coupling of a readily obtainable l-psuedo-thiourea of GlcNAc (S-GlcNAc) and iodo-N-Boc-L-alanine benzyl ester, and the other utilizing a modified Mitsunobu reaction of GlcNAc-SH and a serine derivative. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00223-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1289±1291
Facile Synthesis of N-Fmoc-Serine-S-GlcNAc: A Potential
Molecular Probe for the Functional Study of O-GlcNAc
Yuki Ohnishi, Mie Ichikawa and Yoshitaka Ichikawa*
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Received 9 March 2000; accepted 4 April 2000
AbstractÐA methabolically stable b-N-acetylglucosaminyl-1-thio-N-Fmoc-serine (S-GlcNAc-Ser) derivative was synthesized in two
procedures: one involving a coupling of a readily obtainable 1-pseudo-thiourea of GlcNAc (S-GlcNAc) and iodo-N-Boc-l-alanine
benzyl ester, and the other utilizing a modi®ed Mitsunobu reaction of GlcNAc-SH and a serine derivative. # 2000 Elsevier Science
Ltd. All rights reserved.
O-Linked N-acetylglucosamine (O-GlcNAc) is an
abundant and ubiquitous post-translational modi®ca-
tion of Ser/Thr residues in both nuclear and cytoplasmic
proteins, and its addition to proteins is thought to be
dynamically regulated as is protein phosphorylation.1 5
Accumulating evidence has suggested a putative functional
role for O-GlcNAc1,3 in a variety of diseases, including
cancer, diabetes, and Alzheimer's disease; however, due
to the lack of a suitable molecular probe (inhibitor), the
role of O-GlcNAc had not yet been fully understood at
the molecular level. Unfortunately, site-directed muta-
genesis of the Ser/Thr residues which are substituted with
GlcNAc does not o€er a promising approach to studying
this glycosylation, because these residues are also poten-
tial phosphorylation sites, as is the case for c-myc.6,7
an O-GlcNAc'ase-resistant analogue of O-GlcNAc
because thio-glycosides are known to be glycosidase
resistant.9 Although several synthetic procedures have
been reported for the preparation of neutral sugars thio-
linked to amino acids,10 14 there is still, to our knowl-
edge, none which describes the synthesis of thio-linked
GlcNAc to serine and S-GlcNAc-Ser-incorporated pep-
tides. We have examined several synthetic approaches
and report herein a facile synthesis of S-GlcNAc-N-
Fmoc-Ser, which serves as a building block for the pre-
paration of a peptide bearing S-GlcNAc.
N-Acetylglucosamine was converted to the known 1-
isothiourea derivative 115,16 (Scheme 1), which was pre-
cipitated during the reaction and readily isolated (about
60% overall yield from GlcNAc) by simple ®ltration
after the reaction mixture was cooled. We con®rmed the
b-stereochemistry of the 1-thio linkage of 1 by 1H NMR
spectral analysis with a large J1,2 coupling (10.8 Hz) and
did not detect any a-isomer.17 The following coupling
reaction between the 1-thio-GlcNAc derivative 2 and
the iodo alanine derivative 318 was troublesome because
of the competing side reactions under various condi-
tions (Table 1). In a reaction in acetone±water at room
temperature,12 some of the iodo derivative 3 seemed to
undergo a b-elimination, giving an a,b-unsaturated ester
5 (evidenced by TLC), which then presumably served as
a Michael acceptor to produce a mixture of 4 (l and d,
Fig. 2). In a separate experiment, we indeed found that
2 reacted with the unsaturated ester 5 to give a mixture
of 4 under the same reaction conditions. Coupling of 2
and 3 in DMSO±H2O at 0 ꢀC (Monsigny's conditions)11
gave an almost pure product of 4. In addition, a bi-
phase reaction using CH2Cl2±H2O in the presence of
The proteins exist in three di€erent isoforms: the naked
(the Ser/Thr-free form), O-glycosylated (O-GlcNAc
form), and O-phosphate forms (Fig. 1), and the mechan-
ism of their interconversions is dynamically regulated by
corresponding enzymes. The key enzyme in this process is
an O-GlcNAc-speci®c N-acetylglucosaminidase (O-
GlcNAc'ase), which cleaves O-GlcNAc residues to liber-
ate the OH group of Ser/Thr,8 which can then be phos-
phorylated by a kinase(s). We thought that it would be
very valuable to prepare a peptide containing an O-
GlcNAc equivalent that is resistant to O-GlcNAc'ase
digestion, so that we could modulate such dynamic
transformations of nuclear protein structures. We
chose a 1-thio-b-N-acetyl-glucosamine (S-GlcNAc) as
*Corresponding author. Tel.: +1-410-614-2892; fax: +1-410-955-
3023; e-mail: ichikawa@jhmi.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00223-7

1290
Y. Ohnishi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1289±1291
Figure 1. Schematic presentation of the putative role of O-GlcNAc versus O-phosphorylation and the designed O-Glc-NAc'ase resistant S-GlcNAc-Ser.
Scheme 1. Synthesis of N-Boc-S-GlcNAc 4 by alkylation.
Table 1. Coupling reaction of 1 and 3
the product 4 in 53% yield after silica gel chromato-
graphy (toluene:EtOAc, 1:1) without epimerization.22
Entry
Condition
Ratio (l:d)a Yield (%)
Finally, the Boc group of 4 was removed with 50%
CF3CO2H±CHCl3 and the resulting amino compound
was treated with Fmoc-Cl to give the N-Fmoc derivative,
which was subsequently hydrogenated over Pd(OH)2/C
and puri®ed on SiO2 chromatography23 to a€ord
building block 824 (Scheme 3).
1
2
3
K2CO3/Na2S2O5/acetone/H2O
K2CO3/Na2S2O5/DMSO/H2O
K2CO3/Na2S2O5/Bu4NI/CH2Cl2/H2O
5:1
>70:1
100:0
81
58
34
aThe ratio was determined by 1H NMR analysis.
Bu4NI as a phase transfer catalyst also gave 4 without
epimerization. Such a coupling reaction of the 1-thio-
GlcNAc 2 and the iodo alanine derivative 3 may be
suitable for large-scale preparation but is rather labor-
ious; therefore, we searched for a more convenient syn-
thetic route for 4.19
In summary, we have established a facile synthetic route
to an O-GlcNAc'ase resistant S-GlcNAc-Ser as a build-
ing block for a solid-phase synthesis of S-GlcNAc-con-
taining peptides for use in studying the role of O-GlcNAc
in O-GlcNAc-bearing nuclear proteins. We are currently
preparing a peptide with Ser-S-GlcNAc from 8 using the
solid-phase FMOC chemistry and the biological evalua-
tion of such glycopeptide will be published elsewhere.
The Toth group has reported a modifed Mitsunobu
reaction (Me3P and 1,10-(azodicarbonyl)-dipiperidine,
ADDP) for the coupling of 1-thio-sugars and alco-
hols;20 however, it did not include the coupling reaction
of 1-thio-GlcNAc and an amino acid derivative. We
therefore examined a direct coupling reaction of the 1-
thio-GlcNAc 6 and a serine derivative 7 (Scheme 2). We
chose another modi®ed Mitsunobu condition21 utilizing
ADDP and Bu3P (due to the cost: Me3P, $62 for 0.1
mol; Bu3P: $3 for 0.1 mol from Aldrich), and found that
the coupling of 6 and 7 proceeded smoothly to a€ord
Scheme 3.
Figure 2. Side reaction observed during the alkylation.
Scheme 2. Synthesis of 4 by a Mistunobu reaction.

Y. Ohnishi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1289±1291
1291
Acknowledgements
tion mixture was stirred for 1.5h at 0 ꢀC, and then poured into
ice-water. The products were extracted with CHCl3, and the
organic layer was washed with 0.5 M KHSO4, H2O, and brine.
The combined organic layer was dried over MgSO4, and con-
centrated in vacuo. The residue was puri®ed by SiO2 column
chromatography (toluene:EtOAc, 5:1 to 1:2) to a€ord 4
(252.5 mg, 58%). 1H NMR (CDCl3, 300 MHz): d 7.37±7.35
(5H, m, aromatic), 6.13 (1H, d, J=9.0 Hz,-NH), 5.89 (1H, d,
J=7.5 Hz,-NH), 5.24±5.03 (4H, m), 4.64±4.55 (2H, m), 4.22±
4.09 (5H, m), 3.73 (1H, m, H-5), 3.31 (1H, dd, J=14.5,
3.3 Hz,-CHa), 2.95 (1H, dd, J=14.5, 7.5 Hz,-CHb), 2.05 (3H,
s,-OAc), 2.02 (6H, s,-OAcÂ2), 1.94 (3H, s, NAc), 1.45 (9H, s,
tert-Bu); 13C NMR (CDCl3, 75 MHz) d 170.8, 170.6, 170.5,
170.2, 169.1, 135.1, 128.6, 128.5, 128.4, 128.1, 83.6, 80.1, 77.2,
75.8, 73.6, 68.3, 67.2, 62.1, 53.4, 52.5, 28.2, 23.0, 20.5.
The NMR studies were performed in the Biochemistry
NMR Facility at Johns Hopkins University, which was
established by a grant from the National Institutes of
Health (GM 27512) and a Biomedical Shared Instru-
mentation Grant (1S10-RR06262-0). This research was
partly supported by the National Institutes of Health
(GM 52324).
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22. Procedure for coupling of 2 and 6 by a Mitsunobu reac-
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K2CO3 (1.52 g, 11.0 mmol) and Na2S2O5 (1.74 g, 9.2 mmol) in
CH2Cl2:H2O (1:1, 80 mL) was added 1 (4.06 g, 9.2 mmol). The
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pound 6 (ca. 2.95 g, 8.1 mmol) in dry THF (30 mL) were added to
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concentrated in vacuo. The residue was further precipitated
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diluted with CH2Cl2, washed with H2O, brine, and dried over
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17. Spectral data for 1: 1H NMR (300 MHz, D2O): d 5.42 (1H,
d, J=10.8 Hz, H-1), 5.53 (1H, dd, J=9.8, 9.6 Hz, H-3), 5.15
(1H, dd, J=9.8, 9.6 Hz, H-4), 4.40±4.15 (4H, m, H-2,5,6a, and
6b), 2.10, 2.07, 2.04 (3H each, s, OAc), 1.97 (3H, s, NAc); 13C
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J=9.8, 9.6 Hz, H-30), 4.68 (1H, dd, J=9.8, 9.6 Hz, H-40), 4.77
(1H, d, J=10.5 Hz, H-10), 4.29±3.85 (8H, m), 3.14 (1H, dd,
J=13.8, 4.2 Hz, H-3a), 2.81 (1H, dd, J=13.8, 9.6 Hz, H-3b),
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68.5, 66.3, 65.7, 62.0, 54.2, 52.0, 46.5, 30.7, 28.9, 22.5, 20.3;
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673.2067, found 673.2065.
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H2O: To a cooled (0 ꢀC) and a stirred solution of K2CO3
(93.7 mg, 0.68 mmol) and Na2S2O5 (104.0 mg, 0.55 mmol) in
H2O (3mL) was added 1 (299.5 mg, 0.68mmol) in DMSO
(3mL) and 3 (274.8mg, 0.68 mmol) in DMSO (5mL). The reac-