Preparation of a diglycosylated hydroxylysine building block used in solid-phase synthesis of a glycopeptide from type II collagen

Full text of DOI:10.1021/jo990853d

8948
J . Org. Chem. 1999, 64, 8948-8953
or epimerization of peptide stereocenters, especially for
P r ep a r a tion of a Diglycosyla ted
the more persistent O-benzoates.5 Benzyl ethers have
also been used in solid-phase glycopetide synthesis.6
However, cysteine, methionine, tyrosine, and to some
extent the indole moiety of tryptophan are not compatible
with hydrogenolytic removal of benzyl groups.7 Moreover,
during acid-catalyzed cleavage from the solid support,
several partially de-O-benzylated glycopeptides are formed,
thereby complicating isolation of the target glycopeptides.6a
Hyd r oxylysin e Bu ild in g Block Used in
Solid -P h a se Syn th esis of a Glycop ep tid e
fr om Typ e II Colla gen
J ohan Broddefalk, Mattias Forsgren,
Ingmar Sethson, and J an Kihlberg*
Organic Chemistry, Umeå University,
SE-901 87 Umeå, Sweden
We have developed a different protective group strat-
egy based on the use of acid-labile protective groups for
the sugar moieties of glycopeptides. This allows an acid-
catalyzed, global deprotection step simultaneously with
cleavage of the glycopeptide from the solid support.
Furthermore, the acid-labile carbohydrate protective
groups should be stable both during preparation of the
glycosylated building block and during peptide synthesis.
Thereby, potentially problematic protective group ma-
nipulations can be avoided and the number of synthetic
steps required for preparation of complex glycosylated
amino acids is reduced. We now report on the use of this
strategy to prepare glycopeptide 1, which corresponds to
residues 256-270 of type II collagen.
Received May 25, 1999
Collagen is the most abundant protein in mammals.
In slightly different forms this fibrous protein is found
in nearly all organs, where it has structural functions.
In collagen, lysine residues located in the sequence Gly-
Xaa-Lys can become posttranslationally hydroxylated
and then glycosylated, either with a â-D-galactopyranosyl
or an R-D-glucopyranosyl-(1f2)-â-D-galactopyranosyl moi-
ety.1 Immunization of mice with type II collagen (CII),
the major protein of joint cartilage, leads to collagen-
induced arthritis,2a i.e., it induces symptoms identical to
those of patients suffering from rheumatoid arthritis.
Using this animal model it was recently demonstrated
that recognition of a peptide epitope found between
residues 256 and 270 of CII [CII(256-270)] by autoim-
mune helper T cells is a key step in eliciting disease.2b,c
Furthermore, glycosylation of hydroxylysine located at
position 264 of CII(256-270) with a galactose moiety was
found to be important for the autoimmune response.
Access to a hydroxylysine derivative that carries an R-D-
glucopyranosyl-(1f2)-â-D-galactopyranosyl moiety3 is re-
quired to fully characterize the immune response and in
efforts to induce tolerance, i.e., to cure the disease. Such
a glycosylated building block should also be useful in
studies of the function of collagen in other situations.
Stepwise assembly, using NR-fluoren-9-ylmethoxy-
carbonyl (Fmoc)-protected glycosylated amino acids as
building blocks, is the method of choice for efficient
preparation of O-linked glycopetides on solid support.4
Traditionally, the carbohydrate moieties of the glycosy-
lated building blocks have been protected with acetyl or
benzoyl groups. These groups enhance the stability of
glycosidic linkages toward acid, but base-mediated depro-
tection may lead to side reactions such as â-elimination
Resu lts a n d Discu ssion
We have recently reported the synthesis of â-D-galac-
tosylated 5-hydroxy-L-norvaline and (5R)-5-hydroxy-L-
lysine (cf. 2 and 3, Scheme 1) carrying acid-labile
protective groups for both the carbohydrate moiety and
the ꢀ-amino group of hydroxylysine.2c,8 Cleavage of the
benzyl ester transformed 2 and 3 into glycosylated
building blocks that were incorporated in glycopeptides
related to type II collagen. Furthermore, the hydroxynor-
valine derivative 2 could be glycosylated using donor 4
under promotion by N-iodosuccinimide and silver trif-
luoromethanesulfonate9 to give the desired R-glucoside
8 in a high yield.8 However, attempted attachment of an
R-D-glucopyranosyl unit to galactosylated hydroxylysine
3 using glucosyl donor 4 was not successful (Scheme 1).
* Corresponding author. Phone: +46-90-786 68 90. Fax: +46-90-
13 88 85. E-mail: jan.kihlberg@chem.umu.se.
(1) Spiro, R. G. J . Biol. Chem. 1967, 242, 4813-4823.
(2) (a) Holmdahl, R.; Andersson, M.; Goldschmidt, T. J .; Gustafsson,
K.; J ansson, L.; Mo, J . A. Immunol. Rev. 1990, 118, 193-232. (b)
Michae¨lsson, E.; Malmstro¨m, V.; Reis, S.; Engstro¨m, A° .; Burkhardt,
H.; Holmdahl, R. J . Exp. Med. 1994, 180, 745-749. (c) Broddefalk, J .;
Ba¨cklund, J .; Almqvist, F.; J ohansson, M.; Holmdahl, R.; Kihlberg, J .
J . Am. Chem. Soc. 1998, 120, 7676-7683.
(3) Short glycopeptides in which hydroxylysine carried an R-D-Glc-
(1f2)-â-D-Gal moiety have previously been prepared in solution
(Koeners, H. J .; Schattenkerk, C.; Verhoeven, J . J .; van Boom, J . H.
Tetrahedron 1981, 37, 1763-1771). However, this synthesis was less
flexible because it involved glycosylation of hydroxylysine incorporated
in a dipeptide. Moreover, the R- and ꢀ-amino groups of hydroxylysine
carried identical protective groups, thereby preventing extension of
the peptide at the N-terminus.
(4) Reviewed in (a) Meldal, M. In Neoglycoconjugates: Preparation
and applications; Lee, Y. C., Lee, R. T., Eds.; Academic Press: San
Diego, 1994; pp 145-198. (b) Arsequell, G.; Valencia, G. Tetrahedron:
Asymmetry 1997, 8, 2839-2876. (c) Kihlberg, J .; Elofsson, M. Curr.
Med. Chem. 1997, 4, 79-110.
(5) Sjo¨lin, P.; Elofsson, M.; Kihlberg, J . J . Org. Chem. 1996, 61, 560-
565.
(6) (a) Nakahara, Y.; Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1996,
292, 71-81. (b) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T.
Tetrahedron Lett. 1997, 38, 7211-7214. (c) Guo, Z.-W.; Nakahara, Y.;
Nakahara, Y.; Ogawa, T. Angew. Chem., Int. Ed. Engl. 1997, 36, 1464-
1466.
(7) (a) Bodanszky, M.; Martinez, J . Synthesis 1981, 333-356. (b)
Guo, Z.-W.; Nakahara, Y.; Nakahara, Y.; Ogawa, T. Carbohydr. Res.
1997, 303, 373-377.
(8) Broddefalk, J .; Bergquist, K.-E.; Kihlberg, J . Tetrahedron 1998,
54, 12047-12070.
(9) Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetrahedron
Lett. 1990, 31, 4313-4316.
10.1021/jo990853d CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/30/1999

Notes
J . Org. Chem., Vol. 64, No. 24, 1999 8949
Sch em e 1. Attem p ted Cou p lin g of a n
r-D-Glu cosyl Un it to th e Ga la ctosyla ted
Hyd r oxyn or va lin e 2 a n d Hyd r oxylysin e 3
Sch em e 2a
Promotion by various thiofilic reagents10 resulted in
formation of several byproducts,11 and the yield of dig-
lycosylated hydroxylysine 9 never exceeded ∼10%. Use
of glucoside donors 5-7, which had similar protective
groups as 4 but different leaving groups at the anomeric
center, were then investigated. Activation of sulfoxide 5
with trifluoromethanesulfonic anhydride12 in the pres-
ence of base or of the reactive thioethyl glucoside 6 with
various soft Lewis acids13 gave disappointing results, just
as did promotion of trichloroacetimidate 7 by a catalytic
amount of trimethylsilyl trifluoromethanesulfonate.14
Because the problems in glycosylation of 3 originated
in the lability of the N-tert-butoxycarbonyl (Boc) group
under the acidic conditions of glycosylations, a more
stable protective group had to be used for the ꢀ-amino
group of hydroxylysine. Preferably, such a protective
group should still be removed during acid-catalyzed
cleavage of the target glycopeptide from the solid phase
to maintain the overall requirement for one global
deprotection step. Acid-catalyzed cleavage of the N-
benzyloxycarbonyl (Cbz) group is usually performed with
strong acids such as HBr or sulfonic acids, which cause
degradation of glycosidic bonds. However, an early
investigation on the synthesis of Met-enkephalin in
solution revealed that the Cbz group could be cleaved by
trifluoroacetic acid, provided that thioanisole was in-
cluded as nucleophile.15 Because these conditions could
be compatible with the integrity of O-glycosidic bonds,
we decided to evaluate the use of the Cbz group in solid-
a
Reagents and conditions: (a) (i) CuCO3‚Cu(OH)2, CbzCl, H2O,
then Chelex 100 (H+-form); (ii) FmocCl, Na2CO3, H2O/dioxane (1:
1); (iii) Cs2CO3, allyl bromide, DMF; 37% overall. (b) ZnCl2, THF,
AW-300 MS, -50 °C f rt, 30%. (c) TIPSCl, imidazole, DMF, 96%.
(d) MpmCl, NaH, DMF, 74%. (e) NIS, AgOTf, CH2Cl2, 4 Å MS,
-45 °C, 47%. (f) (Ph3P)4Pd(0), PhNHMe, THF, 92%. (g) Solid-phase
peptide synthesis, 23%.
phase synthesis of glycopeptides. The Nꢀ-Cbz-protected
hydroxylysine derivative 11, having orthogonally cleav-
able allyl ester and NR-Fmoc protective groups, was
therefore prepared (Scheme 2). This was done essentially
as for the analogous Fmoc-Hyl(Boc)-OBzl,2c i.e., via
formation of a cupric chelate that allowed selective Nꢀ-
Cbz protection,16 followed by introduction of the Fmoc
group and formation of the allyl ester using a cesium
carboxylate.17 Lactonization was a significant problem in
the esterification even when using a large excess of allyl
bromide. This reduced the overall yield, as compared to
the analogous synthesis of Fmoc-Hyl(Boc)-OBzl.2c
(10) Reviewed in (a) Norberg, T. In Modern Methods in Carbohydrate
Synthesis; Kahn, S. H., O’Neill, R. A., Eds.; Harwood Academic
Publishers: The Netherlands, 1996; pp 82-106. (b) Garegg, P. J . Adv.
Carbohydr. Chem. Biochem. 1997, 52, 179.
(11) Promotion was attempted by (a) N-iodosuccinimide and silver
trifluoromethanesulfonate, (b) N-iodosuccinimide and trifluoromethane-
sulfonic acid, (c) N-iodosuccinimide and trimethylsilyl trifluoromethane-
sulfonate, (d) iodonium dicollidine trifluoromethanesulfonate, and (e)
methylsulfenyl bromide and silver trifluoromethanesulfonate in the
presence of 2,6-di-tert-butyl-4-methylpyridine.
(12) Kahne, D.; Walker, S.; Cheng, Y.; Van Engen, D. J . Am. Chem.
Soc. 1989, 111, 6881-6882.
(13) Promotion was attempted by (a) N-iodosuccinimide and silver
trifluoromethanesulfonate, and (b) N-iodosuccinimide and trifluo-
romethanesulfonic acid.
â-Galactosylation of protected hydroxylysine 11 was
performed in the same manner as reported recently in
the synthesis of 3.2c Treatment of the R-1,2-anhydrosugar
128 with 11 in the presence of zinc chloride18 gave an
(16) (a) Fones, W. S. J . Am. Chem. Soc. 1953, 75, 4865-4866. (b)
Scott, J . W.; Parker, D.; Parrish, D. R. Synth. Commun. 1981, 11, 303-
314.
(14) Wegmann, B.; Schmidt, R. R. J . Carbohydr. Chem. 1987, 6,
357-375.
(15) Kiso, Y.; Ukawa, K.; Akita, T. J . Chem Soc., Chem. Commun.
1980, 101-102.
(17) Chang, C.-D.; Waki, M.; Ahmad, M.; Meienhofer, J .; Lundell,
E. O.; Haug, J . D. Int. J . Pept. Protein Res. 1980, 15, 59-66.
(18) Halcomb, R. L.; Danishefsky, S. J . J . Am. Chem. Soc. 1989,
111, 6661-6666.

8950 J . Org. Chem., Vol. 64, No. 24, 1999
Notes
anomeric mixture (â/R ) 3.8:1 in 51% yield), from which
the desired â-glycoside 13 could be isolated in 30% yield.
This reaction was also accompanied by lactonization of
hydroxylysine 11, which led to release of allyl alcohol that
in turn gave â-allyl galactoside 14 as a byproduct. In
addition, small quantities of less polar compounds were
obtained and identified as higher order glycosides of 13
and 14 by FABMS. Use of the softer Lewis acid silver
trifluoromethanesulfonate19 in an attempt to reduce
lactonization20 during opening of epoxide 12 gave poor
results. The anomeric selectivity shifted in favor of the
undesired R-glycoside,21 while the yield was unchanged
(R/â ) 1.6:1, 46%).
Coupling of an R-linked glucose unit to galactosylated
hydoxylysine 13 was then attempted with the reactive
thioethyl glucoside 6 (Scheme 2). This donor was pre-
pared from ethyl 1-thio-â-D-glucopyranoside 1522 in two
steps. Treatment of 15 with triisopropylsilyl chloride and
imidazole gave exclusive silylation of the primary hy-
droxyl group (f 16) and was followed by protection of
the secondary hydroxyls using 4-methoxybenzyl chloride
and sodium hydride to give 6 in 71% overall yield.
Activation of 6 with N-iodosuccinimide and silver trif-
luoromethanesulfonate9 in dichloromethane allowed cou-
pling to HO-2 of 13. An anomeric mixture of glycosides
(R/â ) 3.3:1, 80% yield) was obtained from which pure
17 could be isolated in 47% yield. Attempts to improve
the R-selectivity by using a mixture of toluene and diethyl
ether as solvent for this glycosylation23 were not success-
ful (R/â ) 2.6:1, 66% yield). Conversion of thioglycoside
6 into a chloro sugar by treatment with iodine monochlo-
ride24 and use of this for glycosylation of 13 promoted by
silver trifluoromethanesulfonate in the presence of 2,4,6-
trimethylpyridine also affected the preparation of 17 in
a negative manner (R/â ) 2.1:1, 68% yield). The lower
R-selectivity obtained in formation of hydroxylysine de-
rivate 17, as compared to hydroxynorvaline derivate 8
(R/â ) 8:1, 89% yield), indicates a steric influence from
the Cbz-protected ꢀ-amino group. Finally, deprotection
of the allyl ester in 17 using N-methylaniline and a
catalytic amount of tetrakis(triphenylphosphine) pal-
ladium(0)25 gave diglycosylated hydroxylysine building
block 18 (92% yield).
different protective groups used for the peptide part and
the disaccharide moiety. As revealed by analytical re-
versed-phase HPLC, this was achieved without affecting
the glycosidic bonds, which would have been degraded
using the strong acids that are usually employed for
removal of the Cbz group. Purification by reversed-phase
HPLC then gave glycopeptide 1 in 23% overall yield
based on the resin capacity.
Glycopeptide 1 was used to evaluate the specificity of
a panel of 29 T cell hydridomas obtained previously by
immunization of mice with type II collagen.2b It was found
that two of the hybridomas recognized 1 when presented
by antigen-presenting cells. Previously, 20 of the hydri-
domas have been shown to recognize the glycopeptide
analogue of 1 that lacks the R-D-glucose moiety.2c The
remaining seven hybridomas responded to peptides that
had either lysine or hydroxylysine at position 264.
Consequently, the type II collagen fragment CII(256-
270) carrying either a galactosyl or a glucosyl-galactosyl
residue on hydroxylysine 264 appears to play a crucial
role for disease development in the mouse model for
rheumatoid arthritis.
In conclusion, use of a Cbz group for protection of the
ꢀ-amino group of hydroxylysine was essential for the
successful synthesis of building block 18 having an R-D-
glucopyranosyl-(1f2)-â-D-galactopyranosyl moiety linked
to hydroxylysine. This building block for the first time
allows incorporation of diglycosylated hydroxylysine into
glycopeptides, such as the type II collagen fragment 1,
by Fmoc solid-phase synthesis. The protective groups of
18, including the Nꢀ-Cbz group, were all removed during
TFA-induced cleavage from the solid phase without
degradation of the glycosidic bonds. Access to synthetic
glycopeptide fragments from collagen is important in
research aimed at understanding how rheumatoid ar-
thritis occurs and in efforts to develop cures for this
disease.
Exp er im en ta l Section
Gen er a l Meth od s a n d Ma ter ia ls. All reactions were carried
out under an inert atmosphere with dry solvents under anhy-
drous conditions, unless otherwise stated. CH2Cl2 and THF were
distilled from calcium hydride and sodium-potassium/ben-
zophenone, respectively. DMF was distilled and then dried over
3 Å molecular sieves. TLC was performed on silica gel 60 F254
(Merck) with detection by UV light and charring with aqueous
sulfuric acid or phosphomolybdic acid/ceric sulfate/aqueous
sulfuric acid. Flash column chromatography was performed on
silica gel (Matrex, 60 Å, 35-70 µm, Grace Amicon) with solvents
of HPLC grade or distilled technical grade. Organic solutions
were dried over Na2SO4 before being concentrated.
1H and 13C NMR spectra were recorded at 400 and 100 MHz,
respectively, in CDCl3 [residual CHCl3 (δH 7.27 ppm) or CDCl3
(δC 77.0 ppm) as internal standard] or in a 1:1 mixture of CD3-
OD and CDCl3 [residual CD2HOD (δH 3.31 ppm) or CD3OD (δC
49.0 ppm) as internal standard] at 300 K. The 1H NMR spectrum
of glycopeptide 1 was recorded at 600 MHz in a 9:1 mixture of
H2O and D2O [H2O (δH 4.95 ppm) as internal standard] at 278
K. First-order chemical shifts and coupling constants were
obtained from one-dimensional spectra, and proton resonances
were assigned from COSY,27a TOCSY,27b and NOESY27c experi-
ments. Resonances for aromatic protons and resonances that
could not be assigned are not reported. Ions for positive fast atom
bombardment mass spectra were produced by a beam of Xenon
The target type II collagen-derived glycopeptide 1 was
prepared from 18 on a polystyrene resin grafted with
poly(ethylene glycol) spacers (TentaGel resin). The syn-
thesis was performed according to the Fmoc strategy
under standard conditions,2c,8 employing only a slight
excess of diglycosylated building block 18 (1.2 equiv),
activated as an azabenzotriazolyl ester.26 When solid-
phase synthesis had been completed, the resin was
treated with trifluoroacetic acid containing water, thio-
anisole, and ethanedithiol as scavengers during 3 h to
liberate the glycopeptide from the solid support. Impor-
tantly, these conditions were also found to remove the
Nꢀ-Cbz group of hydroxylysine, as well as the eight
(19) Du, Y.; Kong, F. J . Carbohydr. Chem. 1995, 14, 341-352.
(20) Szabo´, L.; Polt, R. Carbohydr. Res. 1994, 258, 293-297.
(21) Liu, K. K.-C.; Danishefsky, S. J . J . Org. Chem. 1994, 59, 1895-
1897.
(22) Schneider, W.; Sepp, J .; Stiehler, O. Ber. Dtsch. Chem. Ges.
1918, 51, 220-234.
(23) Demchenko, A.; Stauch, T.; Boons, G.-J . Synlett 1997, 818-
820.
(24) Kartha, K. P. R.; Field, R. A. Tetrahedron Lett. 1997, 38, 8233-
8236.
(25) Ciommer, M.; Kunz, H. Synlett 1991, 593-595.
(26) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397-4398.
(27) (a) Derome, A. E.; Williamson, M. P. J . Magn. Reson. 1990, 88,
177-185. (b) Bax, A.; Davis, D. G. J . Magn. Reson. 1985, 65, 355-
360. (c) Kumar, A.; Ernst, R. R.; Wu¨thrich, K. Biochem. Biophys. Res.
Comm. 1980, 95, 1-6.

Notes
J . Org. Chem., Vol. 64, No. 24, 1999 8951
atoms (6 keV) from a matrix of glycerol and thioglycerol. In the
amino acid analysis glutamine was determined as glutamic acid.
Analytical normal-phase HPLC was performed on a Kromasil
silica column (100 Å, 5 µm, 4.6 mm × 250 mm) with a flowrate
of 2 mL/min and detection at 254 nm. Preparative purifications
were performed on a Kromasil silica column (100 Å, 5 µm, 20
mm × 250 mm) with a flowrate of 20 mL/min. Glycopeptide 1
was analyzed on reversed phase using a Kromasil C-8 column
(100 Å, 5 µm, 4.6 mm × 250 mm) and a linear gradient of 0% f
100% of B in A over 60 min with a flow rate of 1.5 mL/min and
detection at 214 nm (solvent systems: A, 0.1% aqueous trifluo-
roacetic acid; B, 0.1% trifluoroacetic acid in CH3CN). Purification
of crude 1 was performed on a Kromasil C-8 column (100 Å, 5
µm, 20 mm × 250 mm) using the same eluant and a flow rate of
11 mL/min.
28.2, 28.1, 28.0, 26.7, 26.2, 19.1; HRMS (FAB) calcd for
C
57H66N2O12SiNa 1021.4283 (M + Na+), found 1021.4305.
Eth yl 6-O-Tr iisop r op ylsilyl-1-th io-â-D-glu cop yr a n osid e
(16). Triisopropylsilyl chloride (1.14 mL, 5.35 mmol) was added
to a solution of ethyl 1-thio-â-D-glucopyranoside22 (15, 1.00 g,
4.45 mmol) and imidazole (759 mg, 11.2 mmol) in DMF (10 mL)
at 0 °C. After 5 h the mixture was partitioned between EtOAc
and saturated aqueous NH4Cl. The aqueous phase was extracted
with, and the combined organic phases were dried and concen-
trated. Flash column chromatography (toluene/EtOH, 10:1) of
the residue gave 16 (1.63 g, 96%): [R]20 -49° (c 1.0, CHCl3);
D
1H NMR (CDCl3) δ 4.35 (d, 1 H, J ) 9.7 Hz, H-1), 4.05 (dd, 1 H,
J ) 4.9, 10.0 Hz, H-6), 3.89 (dd, 1 H, J ) 7.0, 10.0 Hz, H-6),
3.71 (s, 1 H, OH), 3.64 (t, 1 H, J ) 8.8 Hz, H-4), 3.61 (t, 1 H, J
) 8.7 Hz, H-3), 3.44 (ddd, 1 H, J ) 4.9, 7.0, 8.8 Hz, H-5), 3.41
(bt, 1 H, J ) 8.9 Hz, H-2), 3.08 (s, 1 H, OH), 2.74 (dq, 1 H, J )
7.5, 12.6 Hz, SCH2CH3), 2.70 (dq, 1 H, J ) 7.4, 12.7 Hz, SCH2-
CH3), 2.65 (s, 1 H, OH), 1.30 (t, 3H, J ) 7.4 Hz, SCH2CH3), 1.17-
1.04 (m, 21 H, iPr); 13C NMR (CDCl3) δ 85.7, 77.7, 77.5, 73.7,
72.1, 65.8, 24.3, 17.9, 15.4, 11.8; HRMS (FAB) calcd for C17H36O5-
SSiNa 403.1950 (M + Na+), found 403.1965. Anal. Calcd for
(5R)-Nr-(F lu or en -9-ylm et h oxyca r b on yl)-NE-b en zyloxy-
ca r bon yl-5-h yd r oxy-L-lysin e Allyl Ester (11). Compound 11
was prepared from (5R)-5-hydroxy-L-lysine dihydrochloride mono-
hydrate (10), benzyl chloroformate (1.6 equiv), 9-fluorenylmethyl
chloroformate (1 equiv), and allyl bromide (5 equiv) as described
for (5R)-NR-(fluoren-9-ylmethoxycarbonyl)-5-hydroxy-L-lysine ben-
zyl ester.2c After purification by flash column chromatography
(heptane/tert-butyl methyl ether/EtOH, 7:3:1) 11 was obtained
in 37% overall yield: [R]20D -2° (c 2.0, CHCl3); 1H NMR (CDCl3)
δ 5.90 (ddt, 1 H, J ) 5.8, 10.7, 17.0 Hz, OCH2CHCH2), 5.59 (d,
1 H, J ) 7.1 Hz, NHR), 5.33 (bd, 1 H, J ) 17.2 Hz, OCH2CHCH2),
5.19 (bs, 1 H, NHꢀ), 5.26 (dd, 1 H, J ) 1.0, 10.2 Hz, OCH2-
CHCH2), 5.11 (ABd, 1 H, J ) 12.8 Hz, PhCH2O), 5.08 (ABd, 1
H, J ) 12.8 Hz, PhCH2O), 4.64 (d, 2 H, J ) 5.5 Hz, OCH2-
CHCH2), 4.46-4.39 (m, 1 H, HR), 4.44 (ABdd, 1 H, J ) 7.1, 10.0
Hz, FmocCH2), 4.38 (ABdd, 1 H, J ) 6.8, 10.3 Hz, FmocCH2),
4.21 (t, J ) 6.9 Hz, 1 H, FmocCH), 3.75 (bs, 1 H, Hδ), 3.39-3.31
(m, 1 H, Hꢀ), 3.13-3.05 (m, 1 H, Hꢀ), 2.84 (bs, 1 H, OH), 2.09-
2.00 (m, 1 H, Hâ), 1.87-1.73 (m, 1 H, Hâ), 1.59-1.47 (m, 2 H,
Hγ), 1.44 (s, 9 H, tBu); 13C NMR (CDCl3) δ 172.0, 157.2, 156.2,
143.8, 143.7, 141.3, 136.3, 131.4, 128.5, 128.2, 128.1, 127.7, 127.1,
125.0, 120.0, 120.0, 119.2, 70.8, 67.1, 67.0, 66.1, 53.5, 47.1, 46.9,
30.0, 29.2; HRMS (FAB) calcd for C32H35N2O7 559.2444 (M +
H+), found 559.2421.
C
17H36O5SSi: C, 53.6; H, 9.5. Found: C, 53.4; H, 9.6.
Eth yl 6-O-Tr iisop r op ylsilyl-2,3,4-tr i-O-(4-m eth oxyben -
zyl)-1-th io-â-D-glu cop yr a n osid e (6). Sodium hydride (473 mg,
60% in mineral oil, 11.8 mmol) was added to a solution of 16
(1.00 g, 2.63 mmol) and 4-methoxybenzyl chloride (2.14 mL, 15.8
mmol) in DMF (10 mL) at 0 °C. The mixture was stirred at 0 °C
for 15 min and then at room temperature for a further 32 h.
MeOH (5 mL) was then added, and the mixture was partitioned
between toluene and saturated aqueous NH4Cl. The organic
phase was washed with saturated aqueous NaCl and water,
dried, and concentrated. Flash column chromatography (toluene/
EtOAc, 30:1 and heptane/EtOAc, 3:1) of the residue gave 6 (1.45
g, 74%): [R]20 4° (c 4.0, CHCl3); 1H NMR (CDCl3) δ 4.85 (ABd,
D
1 H, J ) 10.4 Hz, MeOPhCH2O), 4.83 (d, 1 H, J ) 9.5 Hz,
MeOPhCH2O), 4.80 (ABd, 1 H, J ) 10.9 Hz, MeOPhCH2O), 4.78
(d, 1 H, J ) 10.5 Hz, MeOPhCH2O), 4.68 (d, 1 H, J ) 9.9 Hz,
MeOPhCH2O), 4.63 (d, 1 H, J ) 10.5 Hz, MeOPhCH2O), 4.43
(d, 1 H, J ) 9.7 Hz, H-1), 3.94 (dd, 1 H, J ) 1.7, 11.2 Hz, H-6),
3.85 (dd, 1 H, J ) 4.5, 11.2 Hz, H-6), 3.81, 3.80, and 3.80 (3 s,
each 3 H, OCH3), 3.64 (t, 1 H, J ) 9.1 Hz, H-3), 3.60 (t, 1 H, J
) 9.1 Hz, H-4), 3.38 (dd, 1 H, J ) 8.7, 9.7 Hz, H-2), 3.27 (ddd,
1 H, J ) 1.7, 4.4, 9.3 Hz, H-5), 2.78 (dq, 1 H, J ) 7.4, 12.5 Hz,
SCH2CH3), 2.68 (dq, 1 H, J ) 7.5, 12.5 Hz, SCH2CH3), 1.29 (t, 3
H, J ) 7.4 Hz, SCH2CH3), 1.15-1.04 (m, 21 H, iPr); 13C NMR
(CDCl3) δ 159.3, 159.3, 159.2, 130.8, 130.5, 130.4, 129.9, 129.6,
129.5, 113.9, 113.8, 86.5, 84.3, 81.7, 80.3, 77.3, 75.5, 75.1, 74.6,
62.6, 55.3, 55.2, 24.1, 18.0, 18.0, 15.0, 12.0; HRMS (FAB) calcd
for C41H60O8SSiNa 763.3676 (M + Na+), found 763.3658.
(5R)-Nr-(F lu or en -9-ylm et h oxyca r b on yl)-NE-b en zyloxy-
ca r bon yl-5-O-{6-O-ter t-bu tyld ip h en ylsilyl-3,4-O-isop r op yl-
id en e-2-O-[6-O-tr iisop r op ylsilyl-2,3,4-tr i-O-(4-m eth oxyben -
zyl)-r-D-glu cop yr a n osyl]-â-D-ga la ctop yr a n osyl}-5-h yd r oxy-
L-lysin e Allyl Ester (17). A mixture of 6 (52 mg, 70 µmol), 13
(58 mg, 58 µmol), and powdered molecular sieves (4 Å, 150 mg)
in CH2Cl2 (2 mL) was stirred at room temperature for 15 min.
N-Iodosuccinimide (18 mg, 70 µmol) was added, and the mixture
was cooled to -45 °C and protected from light. Silver trifluo-
romethanesulfonate (7 mg, 28 µmol) was added, the reaction
mixture was stirred for 80 min at -45 °C, and the reaction was
then quenched by addition of triethylamine (41 µL, 0.29 mmol).
After 5 min, the mixture was diluted with CH2Cl2, filtered (Hyflo
Supercel), and washed with 10% aqueous Na2S2O3 and saturated
aqueous NaHCO3. The organic layer was dried, concentrated,
and subjected to flash column chromatography (toluene/EtOAc,
8:1) to give 17 and the corresponding â-isomer (78 mg, R/â )
3.3:1, ∼80%). Purification of the mixture by normal-phase HPLC
(linear gradient 0% f 8% tert-butyl methyl ether in CH2Cl2
(5R)-Nr-(F lu or en -9-ylm et h oxyca r b on yl)-NE-b en zyloxy-
ca r bon yl-5-O-(6-O-ter t-bu tyld ip h en ylsilyl-3,4-O-isop r op yl-
id en e-â-D-ga la ctop yr a n osyl)-5-h yd r oxy-L-lysin e Allyl Ester
(13). A solution of 11 (305 mg, 0.546 mmol) in THF (3 mL) and
crushed molecular sieves (AW-300, 200 mg) was added to freshly
prepared 1,2-anhydro-6-O-tert-butyldiphenylsilyl-3,4-O-isopro-
pylidene-R-D-galactopyranoside8 (12, 0.273 mmol), and the mix-
ture was stirred for 20 min at room temperature and then cooled
to -50 °C. Zinc chloride (300 µL, 1.0 M in Et2O, 0.300 mmol)
was added, and the reaction mixture was allowed to attain room
temperature over 20 h. The mixture was then diluted with
EtOAc, filtered (Hyflo-Supercel), and washed with water. The
aqueous phase was extracted twice with EtOAc, and the
combined organic phases were dried and concentrated. Flash
column chromatography (toluene/EtOAc, 7:3 f 1:1) followed by
purification by normal-phase HPLC (linear gradient 0% f 20%
tert-butyl methyl ether in CH2Cl2 during 160 min) gave 13 (81
mg, 30%), the corresponding R-anomer (30 mg, 7%), and unre-
acted acceptor 11 contaminated with the corresponding lactone
(182 mg). Da ta for 13: [R]20 +9° (c 1.0, CHCl3); 1H NMR
D
(CDCl3) δ 5.91 (ddt, 1 H, J ) 5.7, 10.6, 17.2 Hz, OCH2CHCH2),
5.67 (bt, 1 H, J ) 5.2 Hz, NHꢀ), 5.62 (d, J ) 7.5 Hz, 1 H, NHR),
5.34 (d, 1 H, J ) 17.2 Hz, OCH2CHCH2), 5.27 (d, 1 H, J ) 10.3
Hz, OCH2CHCH2), 5.00 (ABd, 1 H, J ) 12.3 Hz, PhCH2O), 4.87
(ABd, 1 H, J ) 12.2 Hz, PhCH2O), 4.65 (bd, 2 H, J ) 5.6 Hz,
OCH2CHCH2), 4.44 (ABdd, 1 H, J ) 7.0, 10.6 Hz, FmocCH2),
4.43-4.37 (m, 1 H, HR), 4.37 (ABdd, 1 H, J ) 6.9, 10.4 Hz,
FmocCH2), 4.21 (t, 1 H, J ) 7.1 Hz, FmocCH), 4.20 (bd, 1 H, J
) 5.5 Hz, H-4), 4.18 (d, 1 H, J ) 8.3 Hz, H-1), 4.02 (dd, 1 H, J
) 5.6, 7.0 Hz, H-3), 3.94-3.91 (m, 2 H, H-6), 3.89-3.84 (m, 1 H,
H-5), 3.70 (bs, 1 H, Hδ), 3.52 (dt, 1 H, J ) 3.0, 7.6 Hz, H-2),
3.42-3.36 (m, 1 H, Hꢀ), 3.31 (d, 1 H, J ) 2.9 Hz, OH), 3.22 (dt,
1 H, J ) 5.6, 14.3 Hz, Hꢀ), 2.03-1.84 (m, 2 H, Hâ), 1.71-1.55
(m, 2 H, Hγ), 1.48 and 1.32 (2 s, each 3 H, CH3), 1.32 (s, 9 H,
tBu); 13C NMR (CDCl3) δ 171.8, 156.8, 156.0, 143.8, 143.7, 141.2,
136.6, 135.6, 135.5, 133.1, 133.0, 131.4, 129.8, 128.4, 128.0, 127.9,
127.8, 127.7, 127.7, 127.1, 125.1, 120.0, 120.0, 119.3, 110.1, 102.9,
80.0, 78.9, 73.6, 73.5, 73.0, 67.0, 66.4, 66.2, 62.6, 53.5, 47.1, 44.8,
during 160 min) gave 17 (46 mg, 47%): [R]20 +28° (c 1.0,
D
CHCl3); 1H NMR (CDCl3) δ 5.85 (ddt, 1 H, J ) 5.5, 10.7, 17.2
Hz, OCH2CHCH2), 5.65 (bs, 1 H, NHꢀ), 5.64 (d, 1 H, J ) 7.2 Hz,
NHR), 5.28 (bd, 1 H, J ) 17.2 Hz, OCH2CHCH2), 5.22 (d, 1 H, J
) 3.5 Hz, H-1′), 5.20 (dd, 1 H, J ) 1.0, 10.5 Hz, OCH2CHCH2),
4.98 (bs, 2 H, PhCH2O), 4.81 (d, 2 H, J ) 9.9 Hz, MeOPhCH2O),
4.76 (ABd, 1 H, J ) 10.5 Hz, MeOPhCH2O), 4.68 (d, 1 H, J )
10.2 Hz, MeOPhCH2O), 4.67 (bs, 2 H, MeOPhCH2O), 4.57 (bd,
2 H, J ) 5.3 Hz, OCH2CHCH2), 4.46 (d, 1 H, J ) 7.8 Hz, H-1),

8952 J . Org. Chem., Vol. 64, No. 24, 1999
Ta ble 1. 1H NMR Da ta (δ, p p m ) for Glycop ep tid e 1 in Wa ter Con ta in in g 10% D2Oa
Notes
residue
NH
H-R
H-â
H-γ
H-δ
others
Gly256
Glu257
Hyp258
Gly259
Ile260
3.76, 3.61
4.60
4.45
3.90, 3.84
4.12
8.70
1.97, 1.77
2.27, 1.01
2.22b
4.56
3.85, 3.79
0.77
8.67
8.05
8.62
8.43
8.10
8.46
7.87
8.46
8.63
8.40
1.81
1.31
1.35, 1.11
0.85 (â-CH3)
Ala261
Gly262,c
Phe263
Hyl264
Gly265,c
Glu266
Gln267
Gly268
Pro269
Lys270
4.21
3.83b
4.60
3.01b
1.87, 1.63
7.27, 7.17 (arom.)
4.19
1.58b
4.00
3.09, 2.91 (Hꢀ), 7.61 (ꢀ-NH2), GlcGald
3.81b
4.22
1.99, 1.85
2.07, 1.91
2.22b
2.32b
4.27
4.09, 3.90
4.34
7.58, 6.90 (CONH2)
2.20, 1.90
1.75, 1.63
1.93b
1.34b
3.53b
1.59b
8.14
4.08
2.90b(Hꢀ), 7.50 (ꢀ-NH2)
a
b
Obtained at 600 MHz, 278 K, and pH ) 5.4 with H2O as internal standard (δH 4.95 ppm). Degeneracy has been assumed. c The
assignment of Gly262 and Gly265 was exchanged by mistake in refs 2c and 8. Chemical shifts (δ, ppm) for the disaccharide moiety: Gal
d
4.53 (H-1), 3.85 (H-4), 3.71 (H-6,6), 3.62 (H-5), 3.65 (H-3), 3.58 (H-2); Glc 5.31 (H-1′), 3.99 (H-5′), 3.76 (H-6′), 3.70 (H-6′), 3.68 (H-3′), 3.41
(H-2′), 3.37 (H-4′).
4.45-4.38 (m, 1 H, FmocCH2), 4.21-4.10 (m, 3 H, HR, FmocCH,
FmocCH2), 4.12 (dd, 1 H, J ) 5.5, 7.1 Hz, H-3), 4.09 (dd, 1 H, J
) 1.6, 5.8 Hz, H-4), 4.05 (dd, 1 H, J ) 1.8, 11.1 Hz, H-6), 3.97-
3.92 (m, 1 H, H-4′), 3.96 (t, 1 H, J ) 9.4 Hz, H-3′), 3.90 (dd, 1 H,
J ) 3.0, 10.3 Hz, H-6′), 3.90-3.86 (m, 1 H, H-6), 3.85-3.68 (m,
4 H, H-5, H-5′, H-6′, Hδ), 3.79, 3.79, and 3.74 (3 s, each 3 H,
OCH3), 3.63 (t, 1 H, J ) 7.1 Hz, H-2), 3.49 (dd, 1 H, J ) 3.6, 9.7
Hz, H-2′), 3.40-3.30 (m, 2 H, Hꢀ), 1.80-1.58 (m, 4 H, Hâ, Hγ),
1.44 and 1.23 (2 s, each 3 H, CH3), 1.14-1.03 (m, 21 H, iPr),
1.01 (s, 9 H, tBu); 13C NMR (CDCl3) δ 171.8, 159.2, 159.1, 157.2,
156.2, 143.9, 143.8, 141.2, 136.4, 135.6, 135.5, 133.0, 133.0, 131.6,
131.1, 131.0, 130.4, 129.8, 129.5, 128.8, 128.4, 128.0, 127.9, 127.8,
127.7, 127.6, 127.1, 127.1, 125.2, 125.2, 119.9, 118.8, 113.8, 113.8,
109.7, 101.7, 96.7, 81.8, 79.8, 78.7, 78.0, 77.2, 76.2, 75.3, 74.7,
73.5, 73.1, 72.6, 71.4, 67.0, 66.7, 65.9, 63.0, 61.9, 55.3, 55.2, 53.8,
47.1, 43.0, 28.0, 27.5, 26.7, 26.1, 19.1, 18.1, 18.0, 12.0, 11.9;
HRMS (FAB) calcd for C96H120N2O20Si2Na 1699.7871 (M + Na+),
found 1699.7887. Anal. Calcd for C96H120N2O20Si2: C, 68.7; H,
7.2; N, 1.7. Found: C, 68.5; H, 7.3; N, 1.8.
C
93H115N2O20Si2Na2 1681.7377 (M - H + 2Na+), found 1681.7383.
Anal. Calcd for C93H116N2O20Si2: C, 68.2; H, 7.1; N, 1.7. Found:
C, 68.2; H, 7.2; N, 1.7.
Glycyl-L-glu ta m -1-yl-tr a n s-4-h yd r oxy-L-p r olyl-glycyl-L-
isoleu cyl-L-a la n yl-glycyl-L-p h en yla la n yl-[5-O-(2-O-r-D-glu -
cop yr a n osyl-â-D-ga la ctop yr a n osyl)-5-h yd r oxy-L-lysyl]-gly-
cyl-L-glu ta m -1-yl-L-glu ta m in yl-glycyl-L-p r olyl-L-lysin e (1).
Glycopeptide 1 was synthesized in a mechanically agitated
reactor on a resin consisting of a cross-linked polystyrene
backbone grafted with poly(ethylene glycol) chains. The resin
carried the C-terminal lysine on a p-hydroxymethylphenoxy
linker (TentaGel S PHB, Rapp Polymere, Germany). Reagent
solutions and DMF for washing were added manually to the
reactor. NR-Fmoc-amino acids (Bachem, Switzerland) with the
following protective groups were used: triphenylmethyl (Trt) for
glutamine; tert-butyl for glutamic acid and hydroxyproline; and
tert-butoxycarbonyl (Boc) for lysine.
The NR-Fmoc-amino acids were activated as 1-benzotriazolyl
esters28 and then added to the resin (15 µmol). Activation was
performed by reaction of the appropriate NR-Fmoc-amino acid
(60 µmol), 1-hydroxybenzotriazole (HOBt, 14 mg, 90 µmol), and
1,3-diisopropylcarbodiimide (58.5 µmol, 0.15 mL of a 0.39 mM
solution in DMF) in DMF (0.1 mL) for 30 min. The acylation
was performed during 1-15 h and was monitored by addition
of bromophenol blue (11 nmol, 7.5 µL of a 0.15 mM solution in
DMF). NR-Fmoc deprotection of the peptide resin was effected
by treatment with 20% piperidine in DMF during 10-20 min.
The glycosylated amino acid 18 (27 mg, 16.5 µmol) was activated
as described above using 1,3-diisopropylcarbodiimide (16.5 µmol)
and 1-hydroxy-7-azabenzotriazole26 (HOAt, 6.7 mg, 50 µmol).
Activated 1 was then coupled during 24 h, and the coupling was
monitored by bromophenol blue as described above.
(5R)-Nr-(F lu or en -9-ylm et h oxyca r b on yl)-NE-b en zyloxy-
ca r bon yl-5-O-{6-O-ter t-bu tyld ip h en ylsilyl-3,4-O-isop r op yl-
id en e-2-O-[6-O-tr iisop r op ylsilyl-2,3,4-tr i-O-(4-m eth oxyben -
zyl)-r-D-glu cop yr a n osyl]-â-D-ga la ctop yr a n osyl}-5-h yd r oxy-
L-lysin e (18). N-Methylaniline (7.6 µL, 70 µmol) and tetrakis(tri-
phenylphosphine) palladium(0) (3 mg, 2 µmol) were added to a
solution of 17 (39 mg, 23 µmol) in THF (1 mL) at room
temperature. The reaction mixture was protected from light and
stirred for 40 min. The mixture was then diluted with EtOAc
and washed with saturated aqueous NH4Cl. The organic layer
was dried, concentrated, and subjected to flash column chroma-
tography (CH2Cl2/MeOH, 50:1 f 10:1) to give 18 (35 mg, 92%):
[R]20 +28° (c 1.0, CHCl3); 1H NMR (CDCl3/CD3OD) δ 5.23 (d, 1
D
H, J ) 3.6 Hz, H-1′), 4.93 (ABd, 1 H, J ) 12.4 Hz, PhCH2O),
4.89 (ABd, 1 H, J ) 12.3 Hz, PhCH2O), 4.78 (d, 1 H, J ) 10.5
Hz, MeOPhCH2O), 4.75 (d, 1 H, J ) 10.4 Hz, MeOPhCH2O),
4.66 (ABd, 1 H, J ) 10.3 Hz, MeOPhCH2O), 4.64 (d, 1 H, J )
10.4 Hz, MeOPhCH2O), 4.58 (ABd, 1 H, J ) 10.5 Hz, MeOPh-
CH2O), 4.49 (d, 1 H, J ) 7.8 Hz, H-1), 4.27 (dd, 1 H, J ) 6.4, 9.2
Hz, FmocCH2), 4.19-4.06 (m, 5 H, H-3, H-4, HR, FmocCH,
FmocCH2), 3.99 (dd, 1 H, J ) 1.8, 11.0 Hz, H-6), 3.92 (bt, 1 H,
J ) 10.2 Hz, H-4′), 3.90 (t, 1 H, J ) 9.3 Hz, H-3′), 3.91-3.82 (m,
4 H, H-5, H-6, H-6′, H-6′), 3.75, 3.74, and 3.67 (3 s, each 3 H,
OCH3), 3.75-3.64 (m, 2 H, H-5′, Hδ), 3.59 (dd, 1 H, J ) 6.1, 7.6
Hz, H-2), 3.44 (dd, 1 H, J ) 3.6, 9.7 Hz, H-2′), 3.34-3.31 (m, 1
H, Hꢀ), 3.23 (dd, 1 H, J ) 5.6, 14.5 Hz, Hꢀ), 1.87-1.78 (m, 1 H,
Hâ), 1.73-1.58 (m, 3 H, Hâ, Hγ, Hγ), 1.44 and 1.23 (2 s, each 3
H, CH3), 1.14-1.03 (m, 21 H, iPr), 1.01 (s, 9 H, tBu); 13C NMR
(CDCl3/CD3OD) δ 174.8, 159.9, 159.9, 159.8, 158.2, 157.6, 144.5,
141.9, 141.9, 137.1, 136.2, 136.1, 133.7, 133.6, 133.2, 133.1, 132.6,
132.5, 131.6, 131.5, 130.8, 130.4, 130.1, 130.1, 129.9, 129.4, 129.3,
128.9, 128.5, 128.4, 128.3, 127.7, 127.6, 125.7, 120.4, 114.4, 114.3,
110.3, 101.7, 97.2, 82.3, 80.2, 78.7, 77.8, 77.1, 75.9, 75.3, 73.9,
73.2, 72.0, 67.5, 67.2, 63.4, 62.6, 55.6, 55.5, 54.4, 47.7, 44.1, 28.4,
27.1, 26.4, 19.6, 18.4, 18.4, 12.6; HRMS (FAB) calcd for
After completion of the synthesis, the resin carrying protected
peptide 1 was washed with CH2Cl2 and dried under vacuum.
The glycopeptide-resin was cleaved, the amino acid side chains
were deprotected, and acid-labile carbohydrate protective groups
were removed by treatment with trifluoroacetic acid/water/
thioanisole/ethanedithiol (87.5:5:5:2.5, 14 mL) for 3 h followed
by filtration. Acetic acid was added to the filtrate, the solution
was concentrated, and acetic acid was added twice more followed
by concentration after each addition. The residue was triturated
with diethyl ether, which gave a solid, crude glycopeptide that
was dissolved in a mixture of acetic acid and water and freeze-
dried. Purification by preparative reversed-phase HPLC (linear
gradient 0% f 100% B in A during 60 min) gave 1 (9 mg, 71%
peptide content, 23% overall yield): 1H NMR data are given in
Table 1; MS (FAB) calcd 1828 (M + H+), found 1827. Amino
acid analysis: Ala 0.98 (1), Glu 3.03 (3), Gly 4.90 (5), Hyl 1.02
(1), Hyp 1.01 (1), Ile 1.02 (1), Lys 1.03 (1), Phe 1.01 (1), Pro 1.01
(1).
(28) Ko¨nig, W.; Geiger, R. Chem. Ber. 1970, 103, 788-798.

Notes
Ack n ow led gm en t. This work was funded by grants
from the Swedish Natural Science Research Council and
the Swedish Research Council for Engineering Sciences.
J . Org. Chem., Vol. 64, No. 24, 1999 8953
spectra for compounds 6, 11, and 13. This material is available
free of charge via the Internet at http://pubs.acs.org.
Su p p or tin g In for m a tion Ava ila ble: The procedure used
for synthesis of compound 11, as well as 1H and 13C NMR
J O990853D