The first synthesis of N-Man-Trp: Alternative mannosylation modification of protein

Article abstract of DOI:10.1055/s-2008-1032099

A novel protein modification, N-mannosyl tryptophan (N-Man-Trp) was synthesized in a stereoselective manner. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2008-1032099

880
LETTER
The First Synthesis of N-Man-Trp: Alternative Mannosylation Modification of
Protein
F
irst Synthesis
h
of
N
-
Man-T
i
r
p
no Manabe,* Yukishige Ito*
RIKEN (The Institute of Physical and Chemical Research), Hirosawa, Wako, Saitama, 351-0198 Japan
Fax +81(48)4624680; E-mail: smanabe@riken.jp
Received 7 December 2007
O
Abstract: A novel protein modification, N-mannosyl tryptophan
NH2
(N-Man-Trp) was synthesized in a stereoselective manner.
HO
O
Key words: carbohydrate, glycopeptides, glycoamino acid, man-
nosyl tryptophan, total synthesis
OH
N
H
HO
OH
Glycosylation is the most widely recognized protein post-
translational modification and it affects various func-
tions.1 There are two major classifications based on the
structure of the linkage between the sugar and protein
parts; O-linked oligosaccharides and N-linked oligosac-
charides.2 In O-glycosylation, the sugar is attached to the
hydroxy group of serine or threonine, while in N-glycosy-
lation, sugar is attached to the protein via the side-chain
amide group of aspargine in the recognition sequence of
Asn-X-Ser/Thr, where X can be any amino acid except
proline.
OH
α-C-Man-Trp
(1)
CO2H
CO2H
NH2
OH
OH
O
N
O
N
NH2
HO
OH
HO
OH
OH
OH
α-N-Man-Trp (2a)
β-N-Man-Trp (2b)
However, a novel type of protein glycosylation pattern in-
volving mannosylation was recently reported. The C-
mannosylation was first identified as a mannosyl modifi-
cation in RNase.3 The mannose residue was connected to
the indole of tryptophan via carbon–carbon linkage
(Figure 1). C-Mannosyl tryptophan 1 (C-Man-Trp) has
been found in a number of mammalian proteins, including
interleukin-12 expressed in Chinese hamster ovary cells,
complements, properdin, thrombospondin, mucins, and a
bovine lens protein, as well as in marine ascidians and the
Ebola virus.4–6 The sequence Trp-X-X-Trp (Italic Trp is
mannosylated) appears to be the recognition motif, but
some exceptions have also been identified.7
Figure 1 The structure of mannosylated tryptophan as protein post-
translational modification
In 2005, Li et al. identified another mannosylation modi-
fication, N-linked mannosylation 2, in Asdes aegypti
chorion peroxidase.13 The structure was identified by
electrospray ionization/tandem mass spectrometry and
peptide-N4(N-acetyl-b-D-glucosaminyl) asparagine ami-
dase (PNGase) digestion. On the mass spectra, a 162-Da
subsistent was seen in each Trp residue, and abundant
fragmentation at m/z = 163 without the loss of 120 Da was
observed. Because of limitations in sample quantity, the
stereochemistry at the anomeric center was not deter-
mined rigorously. PNGase was reported able to hydrolyze
the C–N linkage between indole and mannose. But, since
PNGase hydrolyzes the b-amide bond between the as-
pargine side chain and sugar moiety, and it has very high
substrate specificity,14,15 the ability to hydrolyze the in-
dole sugar is critical to the biochemical mechanisms
(Scheme 1).
We previously succeeded in the first total synthesis of C-
mannosylated tryptophan and related peptides.8,9 Using an
antibody against C-Man-Trp and derivatives prepared
from chemically synthesized compounds, we were able to
investigate the relationship between diabetes and C-man-
nosylation.10 We also found that C-Man-Trp-containing
peptides enhance cytotoxicity together with lipopolysac-
charide via the upregulation of tumor necrosis factor-a.11
In addition, Pallavicini suggested a relationship between
breast cancer and C-mannosylation using high throughput
mass spectrometry techniques.12
In order to clarify this question, we planned to synthesize
the N-linked mannosyl tryptophan in a stereoselective
manner. We report here the first total synthesis of a-N-
linked mannosyl tryptophan 2a. The indole glycosylation
reaction is known to be difficult.16 Indole is not stable un-
der Lewis acidic conditions, and the glycosylation reac-
tion causes numerous side reactions including orthoester-
type 1,2-O-(indol-1-yl)ethylidene compound formation.
Unverzagt reported a b-Glc-Trp synthesis using a Lewis
SYNLETT 2008, No. 6, pp 0880–0882
x
x.
x
x.
2
0
0
8
Advanced online publication: 26.02.2008
DOI: 10.1055/s-2008-1032099; Art ID: U12307ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
First Synthesis of N-Man-Trp
881
O
a)
H
N
HO
N
H
O
H
O
HO
N
NHAc
O
O
b)
HO
HO
HO
OH
O
PNGase
N
H
N
NH
PNGase
OH
?
PNGase
catalytic triad
O
HO
HO
HO
OH
O
NH
S Cys
O
N
N
His
H
O
+
Asp
HO
HN
O
H
O
H
NH
O
HO
N
N
NHAc
H
NH
then H2O
O
O
H
N
HO
N
H
+
O
HO
NH2
HO
NHAc
O
Scheme 1 a) Proposed mechanism of hydrolysis of amide bond by PNGase; b) hydrolysis of anomeric carbon–indole bond.
OBn
O
O
BnO
N
N
OMe
N
N
OMe
Br
OBn
i
6
+
MeO
MeO
N
N
iii
SO2Ph
R
3
4
5a R = SO2Ph
5b R = H
ii
NH2
OBn
N
OMe
OBn
v, vi
iv
O
N
2a
O
N
MeO
O
MeO
N
BnO
OH
BnO
OH
OBn
8
OBn
7
Scheme 2 Reagents and conditions; i) BuLi, THF, 64%; ii) Mg, EtOH, 98%; iii) KHMDS, THF, 49%, iv) HCl, dioxane, H2O, 71%; v) H2,
Pd(OH)2/C, AcOH, H2O; vi) LiOH·H2O, H2O, 75%.
acidic glycosylation reaction, but the yield was not suffi- then removed by Mg in EtOH without racemization at the
ciently high and a bulky pivaloyl group was necessary at chiral centers of bislactim moiety in 98% yield.19 The in-
the 2-position to prevent side reactions.17 To avoid Lewis dole anion generated from 5b attacked the anomeric posi-
acidic conditions, we planned to make the anomeric car- tion of the epoxide20 6 in SN2 fashion to give adduct 7 in
bon–indole nitrogen connection via nucleophilic attack 49% yield. After cleaving the bislactim group under acid-
under basic conditions.
ic conditions, the benzyl group was removed under typical
hydrogenolysis conditions using Pd(OH)2/C as a catalyst.
After hydrolysis of the methyl ester by LiOH, a-N-Man-
Trp (2a) was obtained in 75% yield (two steps) after puri-
fication by reverse-phase silica gel column chromatogra-
phy. The 1H NMR coupling constant of anomeric position
is 4.5 Hz.21 This large coupling constant at anomeric po-
sition suggests that the pyranose ring conformation is dis-
The amino acid moiety of the tryptophan was therefore
masked as a bislactim structure, which is stable under ba-
sic conditions. The tryptophan equivalent bislactim struc-
ture was prepared from indolyl bromide 3 and Schöllkopf
chiral auxiliary 418 (Scheme 2). The newly formed chiral
center was stereochemically pure based on 400 MHz H
NMR analysis. The sulfonamide group at indole 5a was
1
Synlett 2008, No. 6, 880–882 © Thieme Stuttgart · New York

882
S. Manabe, Y. Ito
LETTER
(8) (a) Manabe, S.; Ito, Y. J. Am. Chem. Soc. 1999, 121, 9754.
(b) Manabe, S.; Marui, Y.; Ito, Y. Chem. Eur. J. 2003, 9,
1435.
(9) Other examples of synthesis of C-Man-Trp: (a) Nishikawa,
T.; Ishikawa, M.; Isobe, M. Synlett 1999, 123.
torted, probably due to the lack of anomeric effects and
steric hindrance of the indole ring. An antibody against a-
N-Man-Trp is now being prepared using the chemically
synthesized a-N-Man-Trp derivatives, and biological
studies will be reported. The synthesis of the other ste-
reoisomer, b-N-Man-Trp, is also currently under way.
(b) Nishikawa, T.; Ishikawa, M.; Wada, K.; Isobe, M. Synlett
2001, 945. (c) Nishikawa, T.; Koide, Y.; Kajii, S.; Wada, K.;
Ishikawa, M.; Isobe, M. Org. Biomol. Chem. 2005, 3, 687.
(d) Kohno, H.; Okabe, K.; Yonekawa, O.; Fujise, H.;
Horiuchi, K.; Adachi, K.; Sano, H.; Suzuki, K. WO
9909411, 1999.
Acknowledgment
We thank Dr. Teiji Chihara and his staff for elemental analyses, Dr.
Hiroyuki Koshino for NMR measurement (1H, 13C, HMBC, and
NOESY) and Ms. Akemi Takahashi for her technical assistance.
We also thank to President’s Discretionary Fund from RIKEN.
(10) (a) Ihara, Y.; Manabe, S.; Kanda, M.; Kawano, H.;
Nakayama, T.; Sekine, I.; Kondo, T.; Ito, Y. Glycobiology
2005, 15, 383. (b) We also found the antibody against C-
mannosylated peptide shows cancer metastasis inhibition:
Ihara, Y.; Takahito, K.; Muroi, E.; Ito, Y.; Manabe, S. WO
2007074859, 2007.
(11) Muroi, E.; Manabe, S.; Ikezaki, M.; Urata, Y.; Sato, S.;
Kondo, T.; Ito, Y.; Ihara, Y. Glycobiology 2007, 17, 1015.
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References and Notes
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(21) 1H NMR (600 MHz, D2O, KH2PO4–Na2HPO4 buffer pH 7,
dioxane 3.75 ppm as a ref., 25 °C): d = 7.74 (d, J = 8.1 Hz, 1
H, C-5), 7.72 (d, J = 8.1 Hz, 1 H, C-8), 7.44 (s, 1 H, C-2),
7.36 (dd, J = 8.1, 7.1 Hz, 1 H, C-7), 7.27 (dd, J = 8.1, 7.1 Hz,
1 H, C-6), 6.03 (d, J = 4.5 Hz, 1 H, C¢-1), 4.68 (dd, J = 4.5,
3.5 Hz, 1 H, C¢-2), 4.21 (dd, J = 6.5, 3.5 Hz, 1 H, C¢-3), 4.07
(dd, J = 8.1, 5.0 Hz, 1 H, CH of Trp) 4.00 (dd, J = 12.6, 7.6
Hz, 1 H, C¢-6), 3.92 (dd, J = 6.5, 6.5 Hz, 1 H, C¢-4), 3.76 (dd,
J = 12.6, 3.0 Hz, 1 H, C¢-6), 3.55 (ddd, J = 7.6, 6.5, 3.0 Hz,
1 H, C¢-5), 3.47 (dd, J = 15.1, 5.0 Hz, 1 H, CH2 of Trp), 3.31
(dd, J = 15.1, 8.1 Hz, 1 H, CH2 of Trp). 13C NMR (125 MHz,
D2O, KH2PO4–Na2HPO4 buffer pH 7): d = 175.0 (C, CO2H),
137.5 (C, C-9), 128.5 (C, C-4), 123.5 (CH, C-7), 125.8 (CH,
C-2), 121.4 (CH, C-6), 119.6 (CH, C-5), 112.3 (CH, C-8),
110.2 (C, C-3), 82.4 (CH, C¢-1), 77.5 (CH, C¢-5), 71.8 (CH,
C¢-3), 68.6 (CH, C¢-4), 68.3 (CH, C¢-2), 60.5 (CH2, C¢-6),
55.4 (CH, CH of Trp), 26.86 (CH2, CH2 of Trp); C–N
linkage between anomeric carbon and indole nitrogen is
confirmed by HMBC analysis; [a]D24 –17 (c 0.1, H2O).
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Synlett 2008, No. 6, 880–882 © Thieme Stuttgart · New York