A facile silyl linker strategy for the solid-phase synthesis of protected glycopeptide: Synthesis of an N-terminal fragment of IL-2 (1-10)

Article abstract of DOI:10.1016/S0040-4020(00)00588-3

An N-terminal glycodecapeptide fragment of interleukin 2 (1) was synthesized by solid-phase method utilizing a new silyl linker. The O-silylated Fmoc-Thr-OAll was attached to the commercial HMP-resin and peptide chain elongation was performed by Fmoc protocol to produce a protected heptapeptide (3-10), which was cleaved from the resin by fluoridolysis and used as the amino component for further condensation on the solid support. On the other hand, 6-hydroxyl group of an Fmoc-Thr(GalNAc)-OAll derivative was silylated with the linker and attached to the resin. Deallylation, block condensation with the heptapeptide (3-10), and elongation at N-terminal with two amino acids were performed on the resin. Fluoride ion-mediated cleavage released the N- and C-protected glycopeptide from the solid support in good efficiency. Fully deprotected glycopeptide was also synthesized through on-resin deallylation and acidic cleavage of the silyl ether linkage. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00588-3

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 6235±6243
A Facile Silyl Linker Strategy for the Solid-Phase Synthesis
of Protected Glycopeptide: Synthesis of an N-Terminal
Fragment of IL-2 (1±10)
Akira Ishii,a,b Hironobu Hojo,a,b Aki Kobayashi,a,b Kazuhiko Nakamura,a,b Yuko Nakahara,b,c
Yukishige Itob,c and Yoshiaki Nakaharaa,b,c,
*
aDepartment of Industrial Chemistry, Tokai University, Kitakaname 1117, Hiratsuka-shi, Kanagawa 259-1292, Japan
bCREST, Japan Science and Technology Corporation (JST), Japan
cThe Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
Received 19 May 2000; accepted 30 June 2000
AbstractÐAn N-terminal glycodecapeptide fragment of interleukin 2 (1) was synthesized by solid-phase method utilizing a new silyl linker.
The O-silylated Fmoc±Thr±OAll was attached to the commercial HMP-resin and peptide chain elongation was performed by Fmoc protocol
to produce a protected heptapeptide (3±10), which was cleaved from the resin by ¯uoridolysis and used as the amino component for further
condensation on the solid support. On the other hand, 6-hydroxyl group of an Fmoc±Thr(GalNAc)±OAll derivative was silylated with the
linker and attached to the resin. Deallylation, block condensation with the heptapeptide (3±10), and elongation at N-terminal with two amino
acids were performed on the resin. Fluoride ion-mediated cleavage released the N- and C-protected glycopeptide from the solid support in
good ef®ciency. Fully deprotected glycopeptide was also synthesized through on-resin deallylation and acidic cleavage of the silyl ether
linkage. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
(glyco)peptides, which provide the useful synthetic
intermediates for further transformation into the large oligo-
peptides (Fig. 1).
Solid-phase synthesis has been the principal technology in
the peptide chemistry since the ®rst introduction by Merri-
®eld.1 In order to facilitate the multistep synthesis on resin
and isolation of the products from the solid supports, various
anchoring groups have been designed on the basis of peptide
chemistry used. For example, p-alkoxybenzyl ester type
linkers are widely used to pursue Fmoc chemistry.2 The
synthesized peptides are released under acidic conditions
which allow simultaneous deprotection of the most side
chain functional groups. On the other hand, the speci®c
linkers involving such a very acid-labile linkage as chloro-
trityl ester have been utilized to synthesize partially
protected peptides. Recently, we have reported a novel
silyl ether-type linker which binds a side chain hydroxyl
group of (glyco-)peptides to the solid support.3 This
approach has several advantages by combination with the
use of Fmoc and allyl ester protecting groups: (1) The
peptide chain assembly is possible at both the N- and
C-terminal after selective removal of the protecting groups.4
(2) The orthogonal conditions for Si±O bond cleavage by
¯uoridolysis allow to liberate the N- and/or C-protected
As an extension of this work, we planned to apply this
strategy to the synthesis of larger glycooligopeptides and
study the on-resin key reactions in detail. By selecting a
fragment of human interleukin 25 (1±10) 1 as a suitable
synthetic model, the solid-phase synthesis was undertaken.6
The results are described below. The synthetic route to 1
was designed so as to use this silyl linker protocol twice,
one for the synthesis of peptide block (4±10), another for
on-resin block coupling at Thr (3) and the subsequent
condensation with the N-terminal amino acid units. The
necessary glycosyl threonine 9 was prepared in six steps
from the known compound 2.7 Reduction of the azide
with Zn±AcOH was followed by acetylation with Ac2O in
MeOH to quantitatively give the corresponding acetamide
3, which on debenzylidenation with aq. CF3CO2H afforded a
sparingly soluble amide-triol 4 in 95% yield. Regioselective
silyl-etheri®cation of 4 with the silyl chloride 5 (1.5 equiv.)
was achieved to give 6 (67%), when the reaction was
conducted in the presence of NaI and N-methylmorpholine
in DMF.3 A trace amount of disilylated product was also
formed. To diminish the dif®culty in handling less soluble 6,
the vicinal diol was masked by isopropylidenation to give 7
(95%). Alternatively, the compound 2 was ®rst debenzyl-
idenated to the triol 8 and regioselectively silylated with
Keywords: glycopeptides; solid-phase synthesis; silicon halides.
* Corresponding author. Tel.: 181-463-50-2075; fax: 1810-463-50-2075;
e-mail: yonak@keyaki.cc.u-tokai.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00588-3

6236
A. Ishii et al. / Tetrahedron 56 (2000) 6235±6243
Figure 1.
t-butylchlorodimethylsilane to produce the 6-O-mono-silyl-
ated deivative 9 (97%).7 3,4-O-Isopropylidenation afforded
10 (84%). At this stage, the azide was transformed into
acetamide 11 (90%). Removal of the t-butyldimethylsilyl
group followed by re-silylation with 5 produced 7 in 89%
yield. Though involved two steps more than the former
route, the latter allowed the easier access to compound 7.
attached to the H±Gly±HMP resin, derived from the
commercial Fmoc±Gly±HMP-resin (0.78 mmol/g), in the
presence of DCC, HOBt, and iPr2NEt in NMP using vortex
tube mixer. The loading of 14 was ascertained by amino
acid analysis. Comparison of threonine content with glycine
in the cleavage mixture revealed that ef®ciency of the load-
ing was 36%. Repeating of the loading procedure (double
coupling) with another 1.2 equiv. of 14, however, gave rise
to only a slight increase in the total yield (41%). It is note-
worthy that the unreacted glycosyl threonine was readily
recovered from the reaction mixture. After concentration
of the separated liquid layer followed by gel ®ltration,
desilylation of the crude glycosyl threonine derivatives
The nitro group was reduced by treatment with Zn±AcOH
in THF (94%) to give an aniline derivative 13. Conversion
of 13 into succinanilic acid 14 was performed by the reac-
tion with succinic anhydride and N-methylmorpholine in
CH2Cl2 (82%). The carboxylic acid 14 (2.5 equiv.) was
Scheme 1.

A. Ishii et al. / Tetrahedron 56 (2000) 6235±6243
6237
Scheme 2.
with n-Bu4NF±AcOH gave regenerated 12 in a reasonable
amount.
automated synthesizer under the ready-made program
(FastMoc). After N-deprotection, the heptapeptide was
detached from the resin by ¯uoridolysis with CsF±
AcOH in DMF. Fig. 2a shows a chromatograms of the
released product. The major component was the desired
peptide and the structure was con®rmed by mass spectro-
metry and amino acid analysis. Thus the side chain- and
C-protected peptide 21 was isolated in a good yield by
preparative HPLC (Fig. 2b) and used for the on-resin
block condensation.The crucial deallylation and block
condensation were next investigated. The glycosyl threo-
nine-bound resin 15 was treated overnight with Pd(PPh3)4
(0.5 equiv.) and N-methylaniline8 in THF using a vortex
mixer. Then the ef®ciency was monitored by HPLC and
MS analyses of the acidic cleavage mixture from the resin
sample. Since the incomplete conversion was evidenced
by the analysis, the resin was submitted again to the
On the other hand, the resin was treated with Ac2O to cap
the unreacted amine on the resin (Schemes 1 and 2).
In a similar manner, the Thr (10) residue was attached to the
resin for the synthesis of heptapeptide block.
Fmoc±threonine allyl ester was silylated,3 and the p-nitro-
benzylsilyl ether 16 was reduced to give aniline 17 quanti-
tatively. Treatment of 17 with succinic anhydride
exclusively gave another key intermediate 18. Installation
of 18 (2 equiv.) to the H±Gly±HMP resin proceeded in a
68% of ef®ciency on the basis of amino acid analysis. Peptide
synthesis was conducted with Fmoc±Lys(Boc)±OH, Fmoc±
Thr(tBu)±OH, and Fmoc±Ser(tBu)±OH utilizing an
Figure 2. HPL chromatogram of 21 on Mightysil RP-18 (4.6£150 mm2) with eluent A (distilled water containing 0.1% TFA) and B (acetonitrile containing
0.1% TFA): (a) the crude product released from the resin; (b) the puri®ed 21.

6238
A. Ishii et al. / Tetrahedron 56 (2000) 6235±6243
Figure 3. HPL chromatogram of the acid-cleavage products from 22: (a) the sample derived after ®rst run of on-resin deallylation; (b) the sample released after
three repetitive deallylation.
Figure 4. HPL chromatogram of the glycodecapeptides: (a) the crude product released from resin 25 by ¯uoridolysis; (b) isolated 26.
Scheme 3.

A. Ishii et al. / Tetrahedron 56 (2000) 6235±6243
6239
deallylation conditions. In order to complete the deallyl-
ation, another repetition of this procedure was necessary
as shown in Fig. 3.
Elmer Applied Biosystems, Div. HPLC was performed
using Mightysil RP-18 (4.6£150 mm2 for analysis and
10£250 mm2 for preparation, Kanto Chemical Co.).
Amino acids were analyzed on a Hitachi L-8500 amino
acid analyzer.
On-resin block condensation with the heptapeptide frag-
ment 21 (1.3 equiv.) was achieved in the presence of
i
HBTU, HOBt, and Pr2NEt in DMF/NMP, whereas the use
N-(9-Fluorenylmethoxycarbonyl)-O-(2-acetamido-4,6-O-
benzylidene-2-deoxy-a-d-galactopyranosyl)-l-threonine
allyl ester 3. A mixture of compound 2 (860 mg,
1.33 mmol), AcOH (10.7 ml, 3.30 mmol), powdered Zn
(870 mg, 13.3 mmol) in CH2Cl2 (12 ml) was stirred at
room temperature for 20 min and ®ltered through Celite.
The ®ltrate was concentrated with toluene. The residue
was dissolved in MeOH (5 ml) and stirred with Ac2O
(0.7 ml, 6.7 mmol) at room temperature for 30 min.
MeOH was evaporated with toluene and the product was
extracted with EtOAc. The extract was washed with sat.
NaHCO3 and brine, dried over Na2SO4, and concentrated
in vacuo. The residue was chromatographed on silica gel
with toluene±EtOAc (1:2) to give 3 (868 mg, quant.), Rf
0.44 (toluene±EtOAc, 1:2), [a]Dˆ170.28 (c 0.5). 1H
NMR: d 7.66 (d, 2H, Jˆ6.6 Hz, Ar), 7.50 (d, 2H, Jˆ
7.3 Hz, Ar), 7.39±7.14 (m, 9H, Ar), 6.11 (d, 1H, Jˆ
8.5 Hz, AcNH), 5.76 (m, 1H, ±CHvCH2), 5.54 (d, 1H.
Jˆ9.5 Hz, Thr±NH), 5.42 [s, 1H, PhCH(O)2], 5.20 (d, 1H,
Jˆ10.9 Hz, ±CHvCH2), 5.17 (d, 1H, Jˆ10.5 Hz,
±CHvCH2), 4.80 (d, 1H, Jˆ3.1 Hz, H-1), 4.51 (brt, 2H,
±CH2CHvCH2), 3.56 (brs, 1H, H-4), 3.35 (d, 1H, Jˆ
10.0 Hz, H-3), 1.95 (s, 3H, Ac), 1.16 (brs, 3H, Thr±gH).
MALDI TOF´MS m/z Calcd for C37H41N2O10 (M1H)1:
673.27, found: 673.62. Anal. Calcd for C37H40N2O10´H2O:
C, 64.62; H, 5.72; N, 4.07%. Found: C, 64.56; H, 5.99; N,
4.11%.
of DCC in place of HBTU was a little ef®cient for this
coupling reaction. HPLC analysis indicated not only
complete disappearance of the glycosyl threonine com-
ponent but also formation of a new glycooctapeptide. The
resin-bound peptide was then N-deprotected with 10%
piperidine in NMP and coupled with Fmoc±Pro±OH
using HBTU/ HOBt conditions. Similarly, the N-terminal
alanine residue was introduced using Boc±Ala±OH.
Finally, the glycopeptide thus synthesized was released
from the resin by ¯uoridolysis with CsF±AcOH (Fig. 4a).
The crude product was puri®ed by preparative HPLC. The
isopropylidene group on GalNAc residue was cleaved
during concentration of the TFA-containing eluate of the
product to give the N-, C- and peptide side chain-protected
fragment 26 in 86% yield from 15 (Fig. 4b). On the other
hand, the fully deprotected glycopeptide 27 was obtained
from 25 by on-resin deallylation and acidic cleavage. The
structures of the puri®ed products were established by NMR
and mass spectra. The physical data of 27 was in good
accordance with those reported (Scheme 3).6
In conclusion, we have demonstrated the potential of the
newly developed synthetic strategy to the protected glyco-
peptide. Human interleukin 2 fragment (1±10) was chosen
as the model compound and sucessfully synthesized through
both C- and N-terminal peptide coupling on the resin. The
key deallylation on the resin and block condensation were
ef®ciently monitored by the use of HPLC and MS analyses.
We could also demonstrate here that the new silyl linker is
compatible with the automated procedure of Fmoc peptide
synthesis.
N-(9-Fluorenylmethoxycarbonyl)-O-(2-acetamido-2-deoxy-
a-d-galactopyranosyl)-l-threonine allyl ester 4. A solu-
tion of 3 (197 mg, 0.92 mmol) in 80% aq TFA (4 ml) was
stirred at 08C for 3 h. The reaction mixture was concentrated
with toluene and water. The residue was chromatographed
on silica gel with CHCl3±EtOH 19:1 ±CHCl3±EtOH±
AcOH 19:1:0.1) to afford 4 (141 mg, 83%), Rf 0.52
1
(CHCl3±EtOH, 1:1), [a]Dˆ145.58 (c 0.3). H NMR: d
Experimental
7.76 (brd, 2H, Jˆ7.4 Hz, Ar), 7.71 (brd, 2H, Jˆ7.1 Hz,
Ar), 7.39 (brd, 2H, Jˆ7.0 Hz, Ar), 7.31 (brd, 2H, Jˆ
7.1 Hz, Ar), 6.75 (brs, 1H, AcNH), 5.91±5.81 (m, 2H,
Thr±NH, ±CHvCH2), 5.31 (d, 1H, Jˆ16.3 Hz,
±CHvCH2), 5.26 (d, 1H, Jˆ10.3 Hz, ±CHvCH2), 4.85
(brs, 1H, H-1), 4.69±4.56 (m, 3H, ±CH2CHvCH2,
Ar2CH-), 4.48 (brs, 1H, Thr±aH), 4.42 (d, 2H, Jˆ6.4 Hz,
Ar2CHCH2±), 4.23 (m, 3H, H-2, H-6, Thr±bH), 4.15 (brs,
1H, H-4), 3.89 (m, 3H, H-3, H-5, H-6), 2.10 (s, 3H, Ac),
1.28 (d, 3H, Jˆ6.6 Hz, Thr±gH). MALDI TOF´MS m/z
Calcd for C30H36N2O10Na (M1Na)1: 607.22, found:
607.73.
Optical rotations were determined with a Jasco DIP-370
polarimeter for solutions in CHCl3, unless noted otherwise.
Column chromatography was performed on Silica Gel-60
(E. Merck 70±230 mesh or 230±400 mesh). TLC and
HPTLC were performed on Silica Gel 60 F254 (E. Merck).
1H and 13C NMR spectra were recorded with a JEOL AL400
[1H (400 MHz), 13C (100 MHz)] spectrometer. Chemical
shifts are expressed in ppm down®eld from the signal for
internal Me4Si for solutions in CDCl3. MALDI´TOF mass
spectra were obtained with a PerSeptive Voyager-DE PRO
spectrometer (2,5-dihydroxybenzoic acid was used as a
matrix). High resolution Fab mass spectra were measured
with JEOL JMS HX-110 spectrometer (2,5-dihydroxy-
benzoic acid was used as a matrix). Peptide synthesis for
the fragment (4±10) was run with an Applied Biosystems
Model 433A peptide synthesizer. All other solid-phase
reactions were performed at room temperature in the capped
polypropylene test tubes with stirring on a vortex tube-
mixer. Fmoc±Gly±preloaded HMP resin and the reagents
for the peptide synthesis were purchased from Perkin±
N-(9-Fluorenylmethoxycarbonyl)-O-[2-acetamido-2-deoxy-
6-O-(a,a-dimethyl-4-nitrobenzyl)dimethylsilyl-a-d-galac-
topyranosyl]-l-threonine allyl ester 6. A mixture of 4
(30 mg, 0.05 mmol),
5
(15.5 mg, 0.06 mmol), NaI
(22.5 mg, 0.15 mmol), and 4-methylmorpholine (11 ml,
0.10 mmol) in dry DMF (2 ml) was stirred at 2208C
under Ar for 2 h. The mixture was diluted with EtOAc,
washed with water and brine, dried over Na2SO4, and

6240
A. Ishii et al. / Tetrahedron 56 (2000) 6235±6243
concentrated in vacuo. The crude product was puri®ed by
preparative t.l.c. with CHCl3±EtOH (9:1) to give 6 (28 mg,
68%), Rf 0.31 (CHCl3±EtOH, 9:1), [a]Dˆ128.68 (c 0.5). 1H
NMR: d 8.09 (m, 2H, Ar), 7.74 (m, 2H, Ar), 7.59±7.24 (m,
8H, Ar), 6.55 (d, 1H, Jˆ8.1 Hz, AcNH), 5.84 (m, 1H,
±CHvCH2), 5.75 (d, 1H, Jˆ9.5 Hz, Thr±NH), 5.30 (d,
1H, Jˆ17.3 Hz, ±CHvCH2), 5.25 (d, 1H, Jˆ10.3 Hz,
±CHvCH2), 4.78 (d, 1H. Jˆ3.4 Hz, H-1), 4.15 (m, 1H,
Thr±bH), 3.90 (brs, 1H, H-4), 3.48 (brs, 1H, OH), 2.07 (s,
3H, Ac), 1.39 (s, 6H, ±CMe2Ar), 1.28 (m, 3H, Thr±gH),
0.01 (s, 6H, ±SiMe2±). MALDI TOF´MS m/z Calcd for
C41H51N3O12SiNa (M1Na)1: 828.31, found: 828.70. Anal.
Calcd for C41H51N3O12Si´1/2H2O: C, 60.42; H, 6.43; N,
5.16%. Found: C, 60.37; H, 6.39; N, 4.86%.
allyl ester 12. To a stirred mixture of 11 (330 mg,
0.45 mmol) and AcOH (0.24 ml, 4.55 mmol) in dry THF
(10 ml) was added 1 M Bu4NF/THF (1.82 ml, 1.82 mmol)
at 08C under Ar. After stirring for 4 h at 08C±room
temperature, the mixture was concentrated to the half
volume and extracted with CHCl3. The extract was washed
with sat. NaHCO3 and brine, dried over Na2SO4, and
concentrated in vacuo. The product was chromatographed
on silica gel with CHCl3±MeOH (97:3) to give 12 (257 mg,
92%), Rf 0.50 (CHCl3±EtOH 9:1), [a]Dˆ174.08 (c 0.2). 1H
NMR: d 7.78 (d, 2H, Jˆ7.6 Hz, Ar), 7.61 (d, 2H, Jˆ7.1 Hz,
Ar), 7.43±7.30 (m, 4H, Ar), 5.91±5.82 (m, 2H, ±CHvCH2,
AcNH), 5.48 (d, 1H. Jˆ9.5 Hz, Thr±NH), 5.32 (brd, 2H,
±CHvCH2), 4.77 (d, 1H, Jˆ3.4 Hz, H-1), 4.67 (dd, 1H,
Jˆ5.9, 12.9 Hz, ±CH2CHvCH2), 4.58 (brd, 1H, Jˆ6.1,
12.9 Hz, ±CH2CHvCH2), 4.50 (brd, 2H, ±COOCH2
CHAr2), 4.39 (d, 1H, Jˆ9.5 Hz, Thr±aH), 4.17 (dd, 1H,
Jˆ2.2, 4.9 Hz, H-2), 4.10 (m, 1H, H-5), 4.03 (dd, 1H,
Jˆ4.9, 9.0 Hz, H-3), 3.94 (brd, 1H, H-6), 3.83 (brd, 1H,
H-6'), 2.03 (s, 3H, Ac), 1.74 (brs, 1H, OH), 1.57 [s, 3H,
±(O)2CMe2], 1.34 [s, 3H, ±(O)2CMe2], 1.30 (s, 3H, Thr±
gH). MALDI TOF´MS m/z Calcd for C33H40N2O10Na
(M1Na)1: 647.25, found: 647.71. Anal. Calcd for
C33H40N2O10´1/2H2O: C, 62.54; H, 6.52; N, 4.42%. Found:
C, 62.86; H, 6.51; N, 4.89%.
N-(9-Fluorenylmethoxycarbonyl)-O-(2-azido-2-deoxy-
3,4-O-isopropylidene-6-O-tert-butyldimethylsilyl-a-d-
galactopyranosyl)-l-threonine allyl ester 10. A mixture of
9 (340 mg, 0.50 mmol), 2,2-dimethoxypropane (1.22 ml,
9.96 mmol), and camphorsulfonic acid (57.0 mg, 0.25
mmol) in dry CH3CN (6 ml) was stirred at room temperature
for 5 min. The mixture was diluted with EtOAc, washed
with sat. NaHCO3 and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was chromatographed
on silica gel with toluene±EtOAc (7:3) to give 10 (365 mg,
quant.). Rf 0.64 (toluene±EtOAc, 1:1), [a]Dˆ 173.98 (c
1
0.7). H NMR: d 7.75 (d, 2H, Jˆ7.3 Hz, Ar), 7.61 (brd,
N-(9-Fluorenylmethoxycarbonyl)-O-[2-acetamido-2-deoxy-
3,4-O-isopropylidene-6-O-(a,a-dimethyl-4-nitrobenzyl)-
dimethylsilyl-a-d-galactopyranosyl]-l-threonine allyl
ester 7. Procedure A (from 6). The compound 6 (30 mg,
0.05 mmol) was isopropylidenated with, 2,2-dimethoxy-
propane (0.1 ml, 0.82 mmol), and camphorsulfonic acid
(5.0 mg, 0.02 mmol) in dry CH3CN (1 ml) as described
for 10. The product was puri®ed by preparative t.l.c. with
toluene±EtOH (1:1) to give 7 (31 mg, 91%), Rf 0.29
(toluene±EtOH, 1:1), [a]Dˆ160.38 (c 1). 1H NMR: d 8.09
(d, 2H, Jˆ8.8 Hz, Ar), 7.76 (d, 2H, Jˆ7.3 Hz, Ar), 7.60 (m,
2H, Ar), 7.39±7.30 (m, 4H, Ar), 5.85 (m, 1H, ±CHvCH2),
5.74 (d, 1H, Jˆ7.3 Hz, AcNH), 5.40 (d, 1H, Jˆ9.5 Hz, Thr±
NH), 5.31 (m, 2H, ±CHvCH2), 4.69 (d, 1H. Jˆ3.4 Hz, H-
1), 4.07 [brs, 1H, H-4), 4.02 (brs, 1H, H-5), 3.96 (dd, 1H,
Jˆ4.9, 9.0 Hz, H-3), 2.01 (s, 3H, Ac), 1.61 [s, 3H,
±(O)2CMe2], 1.41 (s, 6H, ±CMe2Ar), 1.40 [m, 6H,
±(O)2CMe2 and Thr±gH], 0.01 (s, 6H, ±SiMe2±). MALDI
TOF´MS m/z Calcd for C44H55N3O12SiNa (M1Na)1:
868.34, found: 868.63. Anal. Calcd for C44H55N3O12Si: C,
62.47; H, 6.55; N, 4.97%. Found: C, 62.26; H, 6.58; N,
4.79%.
2H, Ar), 7.40±7.28 (m, 4H, Ar), 5.93 (m, 1H,
±CHvCH2), 5.61 (d, 1H. Jˆ9.5 Hz, Thr±NH), 5.35 (d,
1H, Jˆ15.9 Hz, ±CHvCH2), 5.25 (d, 1H, Jˆ10.3 Hz,
±CHvCH2), 4.87 (d, 1H, Jˆ3.7 Hz, H-1), 4.68 (d, 2H,
Jˆ5.9 Hz, ±CH2CHvCH2), 4.11 (ddd, 1H, Jˆ2.4, 6.4,
8.8 Hz, H-5), 3.35 (dd, 1H, Jˆ3.7, 8.3 Hz, H-2), 1.50 [s,
3H, ±(O)2CMe2], 1.35 [br, 6H, ±(O)2CMe2 and Thr±gH],
0.89 (s, 9H, t-Bu), 0.07 (s, 6H, ±SiMe2). MALDI TOF´MS
m/z Calcd for C37H50N4O9SiNa (M1Na)1: 745.32, found:
745.71. Anal. Calcd for C37H50N4O9Si: C, 61.47; H, 6.97; N,
7.75%. Found: C, 61.58; H, 7.02; N, 7.28%.
N-(9-Fluorenylmethoxycarbonyl)-O-(2-acetamido-2-deoxy-
3,4-O-isopropylidene-6-O-tert-butyldimethylsilyl-a-d-galac-
topyranosyl)-l-threonine allyl ester 11. The azide 10
(365 mg, 0.51 mmol) was treated with Zn and AcOH in
CH2Cl2, and then acetylated as described for 3. The
acetamide 11 (336 mg, 90%) was isolated by column chro-
matography on silica gel with CHCl3±EtOH (19:1). Rf 0.29
(toluene±EtOH, 1:1), [a]Dˆ161.78 (c 0.5).1H NMR: d 7.75
(d, 2H, Jˆ7.6 Hz, Ar), 7.59 (d, 2H, Jˆ4.9 Hz, Ar), 7.40±
7.28 (m, 4H, Ar), 5.85 (m, 1H, ±CHvCH2), 5.74 (d, 1H.
Jˆ9.5 Hz, Thr±NH), 5.42 (d, 1H, Jˆ9.5 Hz, AcNH), 5.32
(d, 1H, Jˆ17.1 Hz, ±CHvCH2), 5.27 (d, 1H, Jˆ10.5 Hz,
±CHvCH2), 4.69 (d, 1H, Jˆ3.2 Hz, H-1), 4.65 (brd, 1H,
±CH2CHvCH2), 4.55 (brd, 1H, ±CH2CHvCH2), 4.46
(brd, 2H, ±COOCH2CHAr2), 4.36 (d, 1H, Jˆ9.8 Hz,
Thr±aH), 4.05 (brd, 1H, H-5), 2.01 (s, 3H, Ac), 1.55 [s,
3H, ±(O)2CMe2], 1.30 [br, 6H, ±(O)2CMe2 and Thr±gH],
0.88 (s, 9H, t-Bu), 0.06 (s, 6H, ±SiMe2). MALDI TOF´MS
m/z Calcd for C39H54N2O10SiNa (M1Na)1: 761.34, found:
761.75. Anal. Calcd for C39H54N2O10Si: C, 63.39; H, 7.37;
N, 3.79%. Found: C, 63.21; H, 7.34; N, 3.75%.
Procedure B (from 12). The compound 12 (187 mg,
0.30 mmol) was silylated with 5 (93 mg, 0.36 mmol) and
NaI (134 mg, 0.90 mmol) in DMF (1.5 ml) at 2208 C for
45 min. Work-up and puri®cation by preparative t.l.c
afforded 7 (235 mg, 97%).
N-(9-Fluorenylmethoxycarbonyl)-O-[2-acetamido-2-deoxy-
3,4-O-isopropylidene-6-O-(a,a-dimethyl-4-aminobenzyl)-
dimethylsilyl-a-d-galactopyranosyl]-l-threonine allyl
ester 13. A mixture of 7 (54 mg, 0.06 mmol), AcOH
(0.06 ml, 1.15 mmol), and powdered Zn (400 mg,
6.12 mmol) was stirred in THF (1 ml) at room temperature
for 45 min. The mixture was diluted with EtOAc, ®ltered
through Celite, washed with sat. NaHCO3 and brine, dried
N-(9-Fluorenylmethoxycarbonyl)-O-(2-acetamido-2-deoxy-
3,4-O-isopropylidene-a-d-galactopyranosyl)-l-threonine

A. Ishii et al. / Tetrahedron 56 (2000) 6235±6243
6241
over Na2SO4, and concentrated in vacuo. The product was
chromatographed on silica gel with toluene±EtOAc (1:9) to
give 13 (49 mg, 94%), Rf 0.49 (CHCl3±EtOH, 9:1),
resultant resin exhibited a little increase in the total loading
(41%). Then the unreacted amino group on the resin was
capped with Ac2O (0.1 ml) and pyridine (0.05 ml) in NMP
(0.85 ml). The resin-attached 14 was also con®rmed by the
HPLC analysis and TOFMS of the acidic cleavage sample
from resin 15.
1
[a]Dˆ145.68 (c 1.4). H NMR: d 7.75 (d, 2H, Jˆ7.3 Hz,
Ar), 7.60 (d, 2H, Jˆ7.6 Hz, Ar), 7.40±7.29 (m, 4H, Ar),
7.01 (brd, 2H, Ar), 6.56 (brd, 2H, Ar), 5.84 (m, 1H, ±
CHvCH2), 5.76 (d, 1H, Jˆ7.3 Hz, AcNH), 5.55 (d, 1H,
Jˆ9.5 Hz, Thr±NH), 5.29 (m, 2H, ±CHvCH2), 4.66 (d,
1H. Jˆ3.4 Hz, H-1), 4.34 (dd, 1H, Jˆ1.7, 9.5 Hz, Thr±
aH), 4.06 [brs, 1H, H-4), 2.00 (s, 3H, Ac), 1.54 [s, 3H, -
(O)2CMe2], 1.30±1.25 [m, 12H, ±CMe2Ar, ±(O)2CMe2 and
Thr±gH], 0.01 (s, 6H, ±SiMe2±). MALDI TOF´MS m/z
Calcd for C44H58N3O10Si (M1H)1: 816.38, found: 816.64.
Anal. Calcd for C44H57N3O10Si: C, 64.76; H,7.04; N, 5.15%.
Found: C, 64.60; H, 7.03; N, 4.91%.
The unreacted glycosyl threonine unit was recovered from
the ®rst coupling mixture as follows. The combined ®ltrate
and washings were concentrated in vacuo and the residue
was chromatographed on Bio-beads S£3 with toluene±
EtOAc (1:1). The fractions containing glycosyl threonine
derivatives were collected and treated with 1 M Bu4NF/
THF (1.1 ml) and AcOH (0.14 ml) in THF (1 ml) for 10 h.
The reaction was worked up as described for 12. Chromato-
graphic puri®cation afforded a reasonable amount (65 mg,
50%) of recovered 12.
N-(9-Fluorenylmethoxycarbonyl)-O-[2-acetamido-2-deoxy-
3,4-O-isopropylidene-6-O-(a,a-dimethyl-4-succin-mono-
amidobenzyl)dimethylsilyl-a-d-galactopyranosyl]-l-threo-
nine allyl ester 14. A mixture of 13 (49 mg, 0.06 mmol),
succinic anhydride (6.6 mg, 0.07 mmol), and 4-methyl-
morpholine (6.6 ml, 0.06 mmol) in CH2Cl2 (1 ml) was
stirred under Ar at room temperature for 3 h. The mixture
was concentrated in vacuo, before the residue was puri®ed
by gel permeation chromatography on Bio-beads S£3 with
toluene±EtOAc (1:1). The obtained 14 (46 mg, 82%) was
used without further puri®cation, Rf 0.27 (CHCl3±EtOH,
9:1). The NMR spectrum of 14 was not fully assigned due
to the presence of more than three conformational isomers.
1H NMR: d 5.80 (m, 1H, ±CHvCH2), 5.21 (dd, 1H, Jˆ1.5,
17.1 Hz, ±CHvCH2), 5.18 (dd, 1H, Jˆ1.3, 10.5 Hz,
±CHvCH2), 4.44 (d, 1H, Jˆ4.0 Hz, H-1), 4.59±4.40 (m,
4H, ±CH2CHvCH2, Ar2CHCH2), 4.38±4.15 (m, 2H, Thr±
aH, Ar2CH±), 2.94±2.40 (m, 4H, ±COCH2CH2CO±), 1.92
(s, 3H, Ac), 1.44, 1.39, and 1.29 (3brs, 12H, ±CMe2Ar and
±(O)2CMe2), 0.18 (s, 6H, ±SiMe2±). MALDI TOF´MS m/z
Calcd for C48H61N3O13Si´Na (M1Na)1: 938.39, found:
938.80.
N-(9-Fluorenylmethoxycarbonyl)-O-(a,a-dimethyl-4-
aminobenzyl)dimethylsilyl-l-threonine allyl ester 17.
Compound 16 (350 mg, 0.58 mmol) was reduced with
powdered Zn (1.5 g) and AcOH (0.2 ml) in THF (5 ml) by
the same procedure as described for 13. The product was
chromatographed on silica gel with hexane±EtOAc (1:1) to
give 17 (331 mg, 96%), Rf 0.40 (hexane±EtOAc, 1:1),
1
[a]Dˆ27.58 (c 0.9). H NMR: d 7.75 (brd, 2H, Jˆ7.3 Hz,
Ar), 7.64 (dd, 2H, Jˆ7.8, 9.5 Hz, Ar), 7.41±7.29 (m, 4H,
Ar), 7.00 (brd, 2H, Jˆ8.5 Hz, Ar), 6.59 (brd, 2H, Jˆ8.5 Hz,
Ar), 5.88 (m, 1H, ±CHvCH2), 5.42 (d, 1H, Jˆ9.5 Hz, Thr±
NH), 5.31 (brd, 1H, Jˆ17.3 Hz, ±CHvCH2), 5.23 (brd, 1H,
Jˆ10.5 Hz, ±CHvCH2), 4.64±4.54 (m, 2H), 4.48±4.35 (m,
3H), 4.31±4.24 (m, 2H), 3.45 (brs, 2H, NH2), 1.28 and 1.27
(2s, 6H, ±CMe2Ar), 1.11 (d, 3H, Jˆ6.3 Hz, Thr±gH),
20.04 and 20.08 (2s, 6H, ±SiMe2±). Anal. Calcd for
C33H40N2O5Si´H2O: C, 67.10; H,7.17; N, 4.74%. Found:
C,67.40; H, 7.05; N, 4.46%.
N-(9-Fluorenylmethoxycarbonyl)-O-(a,a-dimethyl-4-
succin-mono-amidobenzyl)dimethylsilyl-l-threonine allyl
ester 18. Compound 17 (633 mg, 1.11 mmol) was
converted to 18 with succinic anhydride (122 mg,
1.22 mmol) and 4-methylmorpholine (121 ml, 1.11 mmol)
by the same procedure as described for 14. Chromatography
on Bio-beads S£3 with toluene±EtOAc (1:1) afforded 18
(732 mg, 98%), Rf 0.28 (CHCl3±MeOH, 9:1), [a]Dˆ27.38
Attachment of 14 to resin (synthesis of 15). Commercial
Fmoc±Gly±HMP-resin (73 mg, 56 mmol; 0.78 mmol/g)
was stirred with 50% piperidine/NMP solution (1 ml) at
room temperature for 1.5 h. The mixture was ®ltered on a
sintered glass disk, washed successively with NMP, CH2Cl2
and ether. The resulting resin was dried in vacuo for a little
while and used for further solid-phase synthesis. A mixture
of 14 (129 mg, 0.14 mmol), 1 M HOBt/NMP (183 ml,
0.18 mmol), 1 M DCC/NMP (183 ml, 0.18 mmol) and 2 M
iPr2NEt/NMP (71 ml, 0.14 mmol) in NMP (0.6 ml) was
stirred at room temperature for 30 min. To the mixture
was added the above N-deprotected resin and stirring was
continued overnight. The mixture was ®ltered and the resin
was washed with NMP, CH2Cl2 and ether. A small portion
of resin (3 mg) was heated in a mixture of propionic acid
(100 ml) and conc HCl (100 ml) at 1508C in an evacuated
sealed tube. The mixture was concentrated in vacuo before
dilution with 0.2N HCl. After ®ltration, the amino acids in
the ®ltrate were analyzed. Since result of the analysis
indicated that the loading ef®ciency of 14 was 36%, the
resin was again exposed to the freshly prepared mixture of
14 (84 mg, 0.09 mmol), HOBt (0.12 mmol), DCC
1
(c 1). H NMR: d 7.76 (brd, 2H, Jˆ7.6 Hz, Ar), 7.61 (dd,
2H, Jˆ7.7, 10.4 Hz, Ar), 7.41±7.20 (m, 6H, Ar), 7.15 (brd,
2H, Jˆ8.8 Hz, Ar), 5.86 (m, 1H, ±CHvCH2), 5.37 (d, 1H,
Jˆ9.8 Hz, Thr±NH), 5.29 (dd, 1H, Jˆ1.2, 17.2 Hz,
±CHvCH2), 5.22 (dd, 1H, Jˆ1.2, 10.5 Hz, ±CHvCH2),
4.63±4.51 (m, 2H), 4.47±4.35 (m, 3H), 4.31±4.22 (m,
2H), 2.67 and 2.51 (2brt, 4H, ±COCH2CH2CO±), 1.30
and 1.29 (2s, 6H, ±CMe2Ar), 1.09 (d, 3H, Jˆ6.1 Hz, Thr±
gH), 20.02 and ±0.08 (2s, 6H, ±SiMe2±). Anal. Calcd for
C37H44N2O8Si´3/2H2O: C, 63.50; H,6.77; N, 4.00%. Found:
C, 63.76; H, 6.45; N, 3.57%.
O-(tert-Butyl)-l-seryl-O-(tert-butyl)-l-seryl-O-(tert-butyl)-
l-seryl-O-(tert-butyl)-l-threonyl-Ne-(tert-butoxycarbonyl)-
l-lysyl-Ne-(tert-butoxycarbonyl)-l-lysyl-l-threonine allyl
ester 21. As described for 15, the carboxylic acid 18
(305 mg, 0.45 mmol) was activated with 1 M DCC/NMP
(589 ml, 0.59 mmol), 1 M HOBt/NMP (589 ml, 0.59
i
(0.12 mmol), and 2 M Pr2NEt/NMP (45 ml, 0.09 mmol) in
NMP (0.7 ml). Amino acid analysis of the sample from the

6242
A. Ishii et al. / Tetrahedron 56 (2000) 6235±6243
mmol), and 2 M iPr2NEt/NMP (227 ml, 0.45 mmol) in NMP
(0.6 ml), and then stirred with H±Gly±HMP-resin (293 mg,
0.23 mmol) overnight. A part of the obtained resin (3 mg)
was hydrolyzed with HCl as described above. The hydro-
lysate was submitted to amino acid analysis, which showed
1:1.5 Thr/Gly ratio. The resin (385 mg) was treated with
Ac2O in pyridine and used for further solid-phase synthesis.
The peptide assembly was performed with Fmoc±
A mixture of the deallylated resin 22 (111 mg, 0.06 mmol),
heptapeptide 21 (92 mg, 0.08 mmol), 0.45 M HBTU/DMF
(214 ml, 0.10 mmol), 1 M HOBt/NMP (30 ml, 0.03 mmol)
and 2 M iPr2NEt/NMP (48 ml, 0.10 mmol) in NMP (0.5 ml)
was stirred overnight. The resin was collected by ®ltration
and washed successively with NMP, CH2Cl2 and ether.
Monitoring of the condensation was performed using a
small resin sample (4 mg). HPLC and MS analyses of the
hydrolyzed sample revealed the complete conversion of 22,
where generation of glycosyl octapeptide was detected as
MALDI TOF´MS m/z 1304.55 [Calcd for C59H90N11O22
(M1H)1, 1304.62]. The resin 23 thus obtained was stirred
with 10% piperidine/NMP (1 ml) for 1.5 h. After ®ltration
and washing with NMP, the resin was again treated with
10% piperidine/NMP (1 ml) for another 30 min. The resin
was washed with NMP, CH2Cl2 and ether. A mixture of
Fmoc±Pro±OH (24 mg, 0.07 mmol), 0.45 M HBTU/DMF
(0.2 ml, 0.09 mmol), 1 M HOBt/NMP (90 ml, 0.09 mmol)
and 2 M iPr2NEt/NMP (72 ml, mmol) was stirred for
30 min. The N-deprotected resin was added to the activated
proline mixture and stirring was continued overnight. The
coupling was readily monitored by HPLC and MS of the
hydrolyzed sample, MALDI TOF´MS m/z 1401.67 [Calcd
for C64H97N12O23 (M1H)1, 1401.67]. After washing, the
resin (24) was N-deprotected as described above. In a
similar manner, Boc±Ala±OH (14 mg, 0.07 mmol) was
activated and reacted with the resin to give 25 (131 mg).
Lys(Boc)±OH,
Fmoc±Thr(tBu)±OH,
and
Fmoc±
Ser(tBu)±OH on an automated synthesizer according to
FastMoc program, where HBTU/HOBt/iPr2NEt was the
condensing agent. N-Deprotection in each cycle was
effected with 20% piperidine/NMP. After six cycles of
amino acid coupling and N-deprotection, the hepta-
peptide-bound resin was stirred with CsF (152 mg,
0.20 mmol) and AcOH (0.3 ml, 1.0 mmol) in DMF (5 ml)
overnight. The mixture was ®ltered and the resin was
washed with DMF, CH2Cl2, and ether. The combined ®ltrate
and washings were concentrated in vacuo and the residue
was extracted with CHCl3. The extract was washed with
water and brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was puri®ed by preparative
HPLC on C18 silica gel (Mightysil, 10£250 mm2) with a
gradient elution of aq CH3CN containing 0.1% TFA (45±
1
75%/0±30 min, 2.5 ml/min) to give 21 (64 mg, 88%). H
NMR (CD3OD): d 5.85 (m, 1H, ±CHvCH2), 5.25 (dd,
1H, Jˆ1.5, 15.6 Hz, ±CHvCH2), 5.13 (dd, 1H, Jˆ1.5,
10.5 Hz, ±CHvCH2), 4.55 (brd, 2H, Jˆ5.6 Hz,
±CH2CHvCH2), 4.47 (brt, 1H, Jˆ6.4 Hz, Ser±aH), 4.38
(d, 1H, Jˆ3.2 Hz, Thr±aH), 4.36±4.28 (m, 3H, Ser±aH, 2
Lys±aH), 4.26 (d, 1H, Jˆ6.9 Hz, Thr±aH), 4.22 (dd, 1H,
Jˆ3.0, 6.4 Hz, Thr±bH), 4.09 (dd, 1H, Jˆ3.4, 6.3 Hz, Thr±
bH), 3.99 (dd, 1H, Jˆ3.0, 6.4 Hz, Ser±aH), 3.77 (dd, 1H,
Jˆ4.6, 10.0 Hz, Ser±bH), 3.65 (dd, 1H, Jˆ4.4, 9.3 Hz,
Ser±bH), 3.62±3.56 (m, 3H, 3 Ser±bH), 3.52 (dd, 1H,
Jˆ6.1, 12.7 Hz, Ser±bH), 2.92 (m, 4H, Lys±eH), 1.61
(m, 4H, Lys±bH), 1.34 (m, 4H, Lys±bH), 1.42±1.25 (m,
26H, 2 t-Bu, 4 Lys±gH, 4 Lys±dH), 1.16±1.13 (m, 36H, 4
t-Bu), 1.09 (d, 3H, Jˆ6.4 Hz, Thr±gH), 1.02 (d, 3H,
Jˆ6.3 Hz, Thr±gH). MALDI TOF´MS m/z Calcd for
C58H108N9O17Na (M1H)1: 1203.52, found: 1203.0.
The resin 25 (65 mg) was stirred with CsF (15 mg,
0.10 mmol) and AcOH (30 ml, 0.10 mmol) in DMF
(0.5 ml) for two days. The resin was ®ltered off and washed
with DMF, CH2Cl2 and ether. The combined ®ltrate and
washings were concentrated in vacuo and the residue was
extracted with CHCl3. The extract was washed with sat.
NaHCO3 and brine, dried over Na2SO4, and concentrated
in vacuo. The crude product was chromatographed by
preparative HPLC on C18 silica gel with a gradient elution
of aq. CH3CN containing 0.1% TFA (50±80%/0±30 min).
The collected glycopeptide fractions were concentrated in
vacuo to give 26 (13 mg, 86%). The isopropylidene group
was readily removed during concentration of the acidic
1
solution. [a]Dˆ25.38 (c 0.2, MeOH). H NMR: d 7.65±
Deallylation of 15 (synthesis of 22). The above glycosyl
threonine-bound resin 15 (79 mg, 0.04 mmol) was stirred
with Pd(PPh3)4 (9 mg, 8 mmol) and N-methylaniline
(87 ml, 0.8 mmol) in THF (1 ml) under Ar overnight. The
mixture was ®ltered and the resin was washed successively
with THF, CH2Cl2 and ether. A small resin sample (2 mg)
was stirred with TFA (30 ml) and the cleavage product was
analyzed by HPLC on C18 silica gel and TOF mass spectra.
The presence of the unreacted allyl ester derivative (m/z
584.24) in the hydrolysate indicated 39% conversion. The
deallylation was completed by two more repetition of this
procedure to give 22. The deallylated product in the hydro-
lysate was con®rmed as Fmoc±Thr(GalNAc)±OH, MALDI
TOF´MS m/z 567.15 [Calcd for C27H32N2O10Na (M1Na)1,
567.19].
7.12 (m, 6H, 3 Ser±NH, 3 Thr±NH), 5.91 (m, 1H,
±CHvCH2), 5.32 (d, 1H, Jˆ17.3 Hz, ±CHvCH2), 5.22
(d, 1H, Jˆ10.5 Hz, ±CHvCH2), 4.98 (d, 1H, Jˆ3.7 Hz,
H-1), 4.65 (brd, 2H, Jˆ5.6 Hz, ±CH2CHvCH2), 4.49±
4.41 (m, 3H, 2 Ser±aH, Thr±aH), 4.37±4.30 (m, 3H,
Ser±aH, 2 Thr±bH), 4.20 (brd, 1H, Jˆ3.1 Hz, Thr±bH),
4.19±4.14 (m, 3H, H-2, 2 Thr±aH), 3.93 (brs, 1H, H-4),
3.91±3.81 (m, 3H, H-3, 2 Ser±bH), 3.73 (brd, 1H, Jˆ
4.9 Hz, Ser±bH), 3.12±3.02 (m, 4H, 2 Lys±eH), 1.22,
1.21, 1.95, and 1.18 (4s, 36H, 4 t-Bu), 1.17, 1.15, and
1.13 (3brs, 9H, 3 Thr±gH). HR FAB´MS m/z Calcd for
C83H148N13O28 (M1H)1: 1775.0557, found: 1775.0554.
l-Alanyl-l-prolyl-O-(2-acetamido-2-deoxy-a-d-galacto-
pyranosyl)-l-threonyl-l-seryl-l-seryl-l-seryl-l-threonyl-
l-lysyl-l-lysyl-l-threonine 27. The resin 25 (64 mg,
8.35 mmol) was deallylated by the same procedure as
described for the synthesis of 22 and the glycopeptide was
detached from the resin by treatment with TFA (1 ml). The
cleavage solution and aq. CH3CN washings were concen-
trated in vacuo. The crude product was puri®ed by reversed
N-(tert-Butoxycarbonyl)-l-alanyl-l-prolyl-O-(2-aceta-
mido-2-deoxy-a-d-galactopyranosyl)-l-threonyl-O-(tert-
butyl)-l-seryl-O-(tert-butyl)-l-seryl-O-(tert-butyl)-l-seryl-
O-(tert-butyl)-l-threonyl-Ne-(tert-butoxycarbonyl)-l-lysyl-
Ne-(tert-butoxycarbonyl)-l-lysyl-l-threonine allyl ester 26.

A. Ishii et al. / Tetrahedron 56 (2000) 6235±6243
6243
phase chromatography as described above. By the gradient
References
elution of aq. CH3CN containing 0.1% TFA (5±25%/0±20
min), was isolated pure 27 (8 mg, 79%). [a]Dˆ244.18 (c
0.3, H2O), lit.6 233.48 (c 1, H2O). 1H NMR (D2O, t-BuOH at
d 1.23): d 4.94 (brs, 1H, H-1), 4.68 (brt, 1H, Jˆ6.4 Hz, Pro±
aH), 3.98 (brs, 1H, H-4), 3.75 and 3.64 (2m, 2H, Pro±dH),
2.99 (m, 4H, Lys±eH), 2.40 (m, 1H, Pro±bH), 2.13±1.93
(m, 3H, Pro±bH, 2 Pro±gH), 2.04 (s, 3H, Ac), 1.92±1.70
(m, 4H, Lys±gH), 1.66 (m, 4H, Lys±dH), 1.53 (d, 3H,
Jˆ6.6 Hz, Ala±bH), 1.46 (m, 4H, Lys±bH), 1.33 (d, 3H,
Jˆ6.3 Hz, Thr±gH), 1.21 (d, 3H, Jˆ6.4 Hz, Thr±gH), 1.18
(d, 3H, Jˆ6.1 Hz, Thr±gH). HR FAB´MS m/z Calcd for
C49H88N13O22 (M1H)1: 1210.6167, found: 1210.6176.
1. Merri®eld, R. B. J. Am. Chem. Soc. 1963, 85, 2149±2154.
2. Reviews in Chan, W. C., White, P. D., Eds. Fmoc Solid Phase
Peptide Synthesis; Oxford University Press: New York, 2000.
3. (a) Nakamura, K.; Hanai, N.; Kanno, M.; Kobayashi, A.;
Ohnishi, Y.; Ito, Y., Nakahara, Y. Tetrahedron Lett. 1999, 40,
515±518. (b) Nakamura, K.; Ishii, A.; Ito, Y.; Nakahara, Y.
Tetrahedron 1999, 55, 11253±11266.
4. The strategically similar access to the oligopeptides has been
reported recently: Alsina, J.; Yokum, T. S.; Albericio, F.; Barany,
G. J. Org. Chem. 1999, 64, 9761±8769.
5. (a) Taniguchi, T.; Matsui, H.; Fujita, T.; Takaoka, C.; Kashima,
N.; Yoshimoto, R.; Hamuro, J. Nature 1983, 302, 305±310.
(b) Smith, K. A. Annu. Rev. Immun. 1984, 2, 319±333.
6. The synthesis of 1 in solution has been reported previously:
Paulsen, H.; Adermann, K. Liebigs Ann. Chem. 1989, 751±769.
7. Singh, L.; Nakahara, Yu.; Ito, Y.; Nakahara, Yo. Carbohydr.
Res. 2000, 325, 132±142.
Acknowledgements
We thank Dr T. Chihara and his staff for elemental analyses,
Mr M. Chijimatsu for amino acid analyses, and Ms A. Taka-
hashi for technical assistance.
8. Kunz, H.; Waldmann, H. Angew. Chem., Int. Ed. Engl. 1984,
23, 71±72.