Total synthesis of novel subclass of glyco-amino acid structure motif: C2-α-L-C-mannosylpyranosyl-L-tryptophan [17]

Full text of DOI:10.1021/ja990926a

9754
J. Am. Chem. Soc. 1999, 121, 9754-9755
Total Synthesis of Novel Subclass of Glyco-amino
Acid Structure Motif:
C2-r-L-C-Mannosylpyranosyl-L-tryptophan
Shino Manabe and Yukishige Ito*
RIKEN (The Institute of Physical and Chemical Research)
and CREST, Japan Science and Technology Corporation
(JST), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
ReceiVed March 22, 1999
It is now widely recognized that attachment of carbohydrates
is one of the most important posttranslational protein modifications
affecting biological activities by way of controlling higher order
structure, stability, immunogenicity, and carbohydrate-protein
interaction.1 In most cases, protein glycosylation can be classified
into two major subtypes: O-glycosylation, where an N-acetyl-
galactosamine residue is linked to the hydroxyl group of either
serine or threonine, and N-glycosylation, where a glycan chain
is linked via a glycosylamido linkage to an asparagine residue.2
However, in 1994, a new class of glycoprotein structural motif 1
(Figure 1) was identified in human RNase, where a mannose
residue is connected to tryptophan via a C-glycosidic linkage.3a-e
Further investigations have revealed that the consensus recognition
site for C-glycosylation is Trp-Aaa-Aaa-Trp (glycosylation site
indicated as italic).3d More recently, the same structural motif was
found in recombinant human IL-12, implying that this posttrans-
lational modification might be more widespread in various
proteins containing the sequence Trp-Aaa-Aaa-Trp.3f Herein we
report the total stereocontrolled synthesis of this novel type of
glyco-amino acid, C2-R-L-C-mannosylpyranosyl-L-tryptophan 2,
and preliminary results on the synthesis of this glycopeptide,
which constitute the partial structure of human RNase.4
Figure 1.
Table 1. Stereochemistry of Epoxide Opening Reaction by
Lithiated Indole Derivatives
N-Arylsulfonated indoles have been known to be amenable
to direct metalation at the 2-position, which on subsequent
quenching with an electrophile provides an easy access to
2-substituted indole derivatives.5 We have recently reported the
direct incorporation of C-1 linked mannose onto the 2-position
of indole by using C-2 lithiated indole derivatives and 1,2-
anhydro-mannose6 3 in the presence of BF3‚OEt2.4a Further
systematic investigation revealed that the strereoselectivity of this
transformation strongly depends on the protecting group and
substituent on the indole. Representative results are shown in
Table 1. When sulfonamide was used as a protecting group for
the indole nitrogen, acyclic C-2 substituents larger than methyl
uniformly gave a higher degree of stereoselectivity in favor of
the R products (entries 5, 6, 8, and 9). Encouraged by these results,
we designed the indole derivative 4j as a latent tryptophan moiety
of the target molecule.
a Calculated based on isolated yield except entry 2 and 10, where
1
the ratios were determined by H NMR. b Taken from ref 4a
Scheme 1
(1) Kobata, A. Acc. Chem. Res. 1993, 26, 319. Varki, A. Glycobiology
1993, 3, 97. Giannis, A. Angew. Chem., Int. Ed. Engl. 1994, 33, 178. Dwek,
R. A. Chem. ReV. 1996, 96, 683.
(2) For recent synthetic studies on glycopeptides, see; Meinjohanns, E.;
Meldal, M.; Paulsen, H.; Dwek, R. A.; Bock, K. J. Chem. Soc., Perkin Trans.
1 1998, 549. Elofsson, M.; Salvador, L. A.; Kihlberg, J. Tetrahedron 1997,
53, 369. Taylor, C. M. Tetrahedron 1998, 54, 11317 and references therein.
(3) (a) Hofsteenge, J.; Mu¨ller, D. R.; Beer, T.; Lo¨ffler, A.; Richter, W. J.;
Vliegenthart, J. F. G. Biochemistry 1994, 33, 13524. (b) Beer, T.; Vliegenthart,
J. F. G.; Lo¨ffler, A.; Hofsteenge, J. Biochemistry 1995, 34, 11785. (c) Lo¨ffler,
A.; Doucey, M.-A.; Jansson, A. M.; Mu¨ller, D. R.; Beer, T.; Hess, D.; Meldal,
M.; Richter, W. J.; Vliegenthart, J. F. G.; Hofsteenge, J. Biochemistry 1996,
35, 12005. (d) Kreig, J.; Hartmann, S.; Vicentini, A.; Gla¨sner, W.; Hess, D.;
Hofsteenge, J. Mol. Biol. Cell. 1998, 9, 301. (e) Vliegenthart, J. F. G.; Casset,
F. Curr. Opin. Struct. Biol. 1998, 8, 565. (f) Doucey, M.-A.; Hess, D.;
Blommers, M. J. J.; Hofsteenge, J. Glycobiology 1999, 9, 435.
(4) Previous synthetic studies: (a) Manabe, S.; Ito, Y.; Ogawa, T. Chem.
Lett. 1998, 919. (b) Nishikawa, T.; Ishikawa, M.; Isobe, M. SYNLETT 1999,
123 (the synthesis of proctected 2 as a diastereomeric mixture is reported).
(c) Hassan, H. H. A. M.; Bredenkamp, M. W. XIXth International Carbohy-
drate Symposium, San Diego, August, 1998, Abstract, BP 295.
(5) Sundberg, R. J.; Russell. H. F. J. Org. Chem. 1973, 38, 3324.
(6) Du, Y.; Kong, F. J. Carbohydr. Chem. 1995, 14, 341.
The synthesis of 4j was straightforward and high yielding as
depicted in Scheme 1. Commercially available (L)-(-)-trypto-
phanol 7 was converted to the corresponding azide by treatment
of TfN3,7 and the hydroxyl group was protected as a tert-butyl-
dimethylsilyl (TBS) ether. The indole ring was further protected
as a benzenesulfonamide by the action of benzenesulfonyl chloride
and n-BuLi to give 4j in 61% overall yield from 7. Subsequent
coupling with 1,2-anhydro-mannose 3 proceeded in a satisfactory
manner, in terms of both selectively (95:5) and reaction efficiency
(7) Vasella, A.; Witzig, C.; Chiara, J.-L.; Martin-Lomas, M. HelV. Chim.
Acta. 1991, 74, 2073.
10.1021/ja990926a CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/04/1999

Communications to the Editor
J. Am. Chem. Soc., Vol. 121, No. 41, 1999 9755
(63% yield) to afford 5j together with a small amount of the
stereoisomer 6j (entry 10). On the other hand, the masked
tryptophan derivatives 4k8 and 4l9 also gave corresponding
coupled products, which can be seen as properly functionalized
precursors of the target molecule. However, in these cases, both
yields and stereoselectivities were far from satisfactory (entries
11, and 12).
Scheme 3
Compound 5j was transformed into 8, which was oxidized to
carboxylic acid 9 by TEMPO-iodosobenzene diacetate combina-
tion10,11 in an excellent yield (Scheme 2). Protection of the
Scheme 2
chair conformation.3a-c With rigorously defined synthetic 2 in
1
hand, H NMR analysis clearly revealed that the mannosylated
tryptophan adopts the 1C4-like conformation with an equatorially
oriented tryptophan moiety (3J1,2 ) 8.1 Hz), presumably because
of its bulkiness as well as the absence of the anomeric effect.
With the use of azide acids in “racemization free” peptide
synthesis in mind,14 peptide elongation was commenced from 9
as follows. Removal of the indole ring protection of compound
9 was achieved with 10% NaOH aqueous in EtOH to give
compound 11 in 72% yield (Scheme 3). The coupling reaction
of acid 11 and tripeptide 1215 proceeded in 90% yield in the
presence of tetramethylfluoroformidium hexafluorophosphate
(TFFH).16 Thus obtained, 13 showed no indication of epimer-
carboxylic acid as a methyl ester was followed by phosphine-
mediated azide reduction to afford the amine that was isolated as
Boc-protected 10, following the protocol of Ariza et al.12
Debenzylation was accomplished under standard conditions,
followed by acidic cleavage of the Boc group. Final deprotection
of the methyl ester and sulfonamide groups was performed under
alkaline hydrolytic conditions. After purification by gel filtration,
the desired product 2 was obtained as a sodium salt in 87% yield.
1
Compound 2 thus obtained gave fully assignable H NMR, and
13C NMR spectra that compared well with those reported for the
hexapeptide 1.13
1
ization within the detection limit of 500 MHz H NMR. After
reduction of the azide group in the mannose-linked tetrapeptide
by PMe3, the coupling reaction with Fmoc-Thr(t-Bu)-OH was
successfully performed to afford 14 in good yield. Adequacy of
the present strategy for further peptide elongation both in solution
phase and solid phase is quite obvious.17
In summary, a total synthesis of C-linked amino acid 2 was
achieved in a concise manner. Our strategy may well be readily
applicable to the incorporation of 13C- or 15N- labels using
appropriately labeled tryptophan precursors. Furthermore, the
pentapeptide sequence 14 was prepared using 11 as a glyco-amino
acid unit. Investigation on further elongation of the peptide chain
for conformational analysis of peptides containing 2 is underway.
In contrast to the usually observed 4C1 conformation, the
1
mannose moiety in peptide 1 was reported to adopt a C4-like
(8) 4k was prepared from (S)-(-)-(Z)-Trp in five steps: (i) 3-methyl-3-
oxetanmethanol, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide‚HCl, DMAP,
DMF, quant., (ii) H2, 10% Pd-C, MeOH, (iii) TfN3, DMAP, CH3CN, 79%
in 2 steps, (iv) BF3‚OEt2 (0.25 equiv), CH2Cl2, 34% (starting material was
recovered in 18% yield.), (v) PhSO2Cl, n-BuLi, THF, 90%.
Acknowledgment. This work was supported by the Science and
Technology Agency of the Japanese Government through Special
Researcher’s Basic Science Program at RIKEN (S.M.). We thank Ms.
A. Takahashi for technical assistance. We thank Dr. J. Uzawa for
recording and measuring the NMR spectra. We thank Dr. Chihara and
his staff for elemental analysis.
For rearrangement of oxetan ester to 2,6,7-trioxabicyclo[2, 2, 2]octane ortho
ester, see; Corey, E. J.; Raju, N. Tetrahedon Lett. 1983, 24, 5571.
(9) Liu, R.; Zhang, P.; Gan, T.; Cook, J. M. J. Org. Chem. 1997, 62, 7447.
Scho¨llkopf, U.; Groth, U.; Deng, C. Angew. Chem., Int. Ed. Engl. 1981, 20,
798.
(10) Epp, J. B.; Widlanski, T. S. J. Org. Chem. 1999, 64, 293.
(11) The compound 5k was converted to the corresponding carboxylic acid
9 quantitatively in two steps ((i) 1 M HCl, THF, (ii) 1 M LiOH, aqueous
MeOH,) and was identical with 9 derived from alcohol 5j.
(12) Ariza, X.; Urp´ı, F.; Viladomat, C.; Vilarrasa, J. Tetrahedron Lett. 1998,
39, 9101.
Supporting Information Available: Experimental details and 1H
NMR spectra of compound 4j, 5j, 8, 9, 10, 11, 12, 13, and 14 (PDF).
This material is available free of charge via the Internet at http://pubs.acs.org.
JA990926A
(13) 1H NMR (600 MHz, D2O, t-BuOH as an internal standard; t-Bu was
adjusted 1.23 ppm) δ 7.73 (d, J ) 7.7 Hz, 1H, H-4), 7.52 (d, J ) 7.7 Hz, 1H,
H-7), 7.30 (t, J ) 7.3 Hz, 1H, H-6), 7.20 (t, J ) 7.3 Hz, 1H, H-5), 5.16 (d,
J ) 8.1 Hz, 1H, H-1′), 4.42 (dd, J ) 8.1, 3.3 Hz, 1H, H-2′) 4.25 (dd, J )
12.5, 8.8 Hz, 1H, H-6′), 4.11 (dd, J ) 3.3, 3.3 Hz, 1H, H-3′), 4.01 (dd, J )
9.2, 5.1 Hz, 1H, H-R), 3.94 (dd, J ) 4.0, 3.3 Hz, 1H, H-4′), 3.88 (ddd, J )
3.3, 3.3, 8.8 Hz, 1H, H-5′) 3.72 (dd, J ) 3.3, 12.5 Hz, 1H, H-6′), 3.55 (dd,
J ) 5.1, 15.3 Hz, 1H, H-â), 3.35 (dd, J ) 15.3, 9.2 Hz, 1H, H-â); 13C NMR
(150 MHz, D2O) δ 175.9 (C), 137.5 (C), 134.8 (C), 128.5 (C), 124.4 (CH,
C-6), 121.3 (CH, C-5), 120.2 (CH, C-4), 113.3 (CH, C-7), 109.8 (C), 80.5
(CH, C-5′), 71.9 (CH, C-3′), 70.4 (CH, C-4′), 69.1 (CH, C-2′), 67.4 (CH,
C-1′), 60.4 (CH2, C-6′), 56.6 (CH, C-R), 27.3 (CH2, C-â).
(14) Peptide synthesis using azido acids on solid phase has been reported
to proceed with a negligible degree of racemization: Meldal, M.; Juliano, M.
A.; Jansson, A. M. Tetrahedron Lett. 1997, 38, 2531.
(15) For preparation of 12, see Supporting Information.
(16) Carpino, L. A.; E-Faham, A. J. Am. Chem. Soc. 1995, 117, 5401.
(17) Further incorporation of a Phe residue was successfully performed to
give hexapeptide corresponding to 1 in a fully protected form. However, the
final deprotection turned out to be problematic, presumably due to the
instability of mannosylated tryptophan portion under acidic conditions required
for the complete removal of t-Bu and Boc groups. This problem would be
solved by making slight modification of amino acid protection strategy (Fmoc/
benzyl), which is currently under investigation.