An improved synthesis of a galactosylated hydroxylysine building block and its use in solid-phase glycopeptide synthesis

Article abstract of DOI:10.1016/S0040-4020(00)00061-2

Different protective groups for (5R)-5-hydroxy-L-lysine were investigated in silver silicate promoted glycosylations with aceto- bromogalactose as glycosyl donor. Best results were obtained with Fmoc- Hyl(Cbz)-OAll, which was glycosylated in 80% yield. Re

Full text of DOI:10.1016/S0040-4020(00)00061-2

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 1579–1586
An Improved Synthesis of a Galactosylated Hydroxylysine
Building Block and its use in Solid-Phase Glycopeptide Synthesis
*
¨
Bjorn Holm, Johan Broddefalk, Sara Flodell, Eric Wellner and Jan Kihlberg
˚
˚
Department of Chemistry, Organic Chemistry, Umea University, SE-901 87 Umea, Sweden
Received 18 November 1999; revised 23 December 1999; accepted 13 January 2000
Abstract—Different protective groups for (5R)-5-hydroxy-l -lysine were investigated in silver silicate promoted glycosylations with aceto-
bromogalactose as glycosyl donor. Best results were obtained with Fmoc-Hyl(Cbz)-OAll, which was glycosylated in 80% yield. Removal of
the allyl group gave a b-d-galactosylated building block which was used in solid-phase synthesis of a glycopeptide from type II collagen.
Such glycopeptides are required for studies of rheumatoid arthritis in a mouse that is transgenic for HLA-DR4, i.e. the class II MHC molecule
associated with rheumatism in humans. ᭧ 2000 Elsevier Science Ltd. All rights reserved.
Introduction
galactosylated hydroxylysine at position 264 was revealed
to form critical contacts with the T cell receptor, while Ile260
and Phe263 anchored the glycopeptide in the P1 and P4
pockets of the disease associated H-2Aq MHC molecule.5
Collagen induced arthritis is the most common animal
model for rheumatoid arthritis. In this model, symptoms
identical to those displayed by patients suffering from
rheumatoid arthritis are induced by immunization of mice
with type II collagen from rat cartilage.1–3 The rat type II
collagen is degraded in antigen presenting cells (APCs) in
the mouse to fragments presented as complexes with H-2Aq
class II MHC molecules on the surface of the APC. We have
recently shown that a glycopeptide fragment from type II
collagen, consisting of residues 256-270 (CII256-270),
stimulates the majority of the autoimmune helper T
cells obtained in the mice (Fig. 1).4 Furthermore, a b-d-
In humans, rheumatoid arthritis is associated with HLA-
DR4 class II MHC molecules.1,2 Interestingly, mice that
are transgenic for HLA-DR4 acquire rheumatoid arthritis
on immunization with type II collagen.6,7 In this case, the
immunodominant epitope on type II collagen has been
found to be located between residues 261 and 273
(CII261-273). The peptide is anchored into the P1 and
P4 pockets of HLA-DR4 by residues Phe263 and Glu266
,
respectively, while Lys264, and possibly also Lys270, makes
Figure 1. Immunodominant T cell epitopes on type II collagen are located between residues 256 and 273. Anchor positions to H-2Aq and HLA-DR4, as well as
the known T cell contact at position 264 are indicated. In collagen, lysines may be post-translationally hydroxylated and then glycosylated.
Keywords: glycosylation; glycopeptides; solid-phase synthesis.
* Corresponding author. Tel.: ϩ46-90-786-6890; fax: ϩ46-90-13-88-85; e-mail: jan.kihlberg@chem.umu.se
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00061-2

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B. Holm et al. / Tetrahedron 56 (2000) 1579–1586
important contacts with the T cell receptor. Consequently,
the epitope is shifted as compared to when CII256-270 is
bound by H-2Aq.
In efforts to develop an improved route to b-d-galacto-
sylated derivatives of hydroxylysine the benzyloxycarbonyl
group was chosen as protective group for the side-chain of
hydroxylysine, whereas benzyl, diphenylmethyl, and allyl
esters were evaluated for protection of the a-carboxyl group
(cf. 8a–c, Scheme 2). The Cbz-group is known to be more
stable than the Boc-group towards acids, but can still be
removed with TFA under conditions that mediate cleavage
from the solid phase and deprotection of peptides and glyco-
peptides prepared by the Fmoc-protocol.10,14 The three
esters were chosen since conditions for their removal
which are orthogonal to Fmoc, Cbz and O-acetyl groups
have been reported, i.e. benzyl esters can undergo selec-
tive hydrogenolysis using a palladium–ethylenediamine
complex deposited on activated charcoal [Pd/C(en)],15
diphenylmethyl esters can be hydrolysed selectively with
acid, and allyl esters may be cleaved by palladium catalyzed
allyl transfer.16 The three esters were prepared by con-
verting Na-(fluoren-9-ylmethoxycarbonyl)-N e-benzyloxy-
carbonyl-5-hydroxy-l -lysine10 (7) into a cesium salt which
was alkylated with benzyl, diphenylmethyl, and allyl
bromide, respectively, in dry DMF (Scheme 2). Spon-
taneous lactonization of hydroxylysine derivative 7 during
the esterifications complicated isolation of 8a–c and
accounts for the modest yields. Lactonization is more
pronounced for 8a and 8b since, in contrast to when using
allyl bromide, a large excess of benzyl or diphenylmethyl
bromide cannot be employed if repeated purification by
chromatography (which also results in some lactonization)
is to be avoided.
Facile access to synthetic glycopeptides from type II
collagen is required in studies of the role of glycosylation
for development of arthritis in HLA-DR4 transgenic mice.
We have therefore evaluated different protective group
patterns for (5R)-5-hydroxy-l -lysine and developed an
improved route for attachment of a b-d-galactopyranosyl
residue. The versatility of the glycosylated hydroxylysine
building blocks have been demonstrated by synthesis of two
glycopeptides from type II collagen using Fmoc solid-phase
synthesis.
Results and Discussion
Glycopeptides having (5R)-5-hydroxy-l -lysine residues
glycosylated8 with b-d-galactopyranose moieties have
recently been synthesized by the Fmoc-protocol using
building block 3, which was prepared from hydroxylysine
19 and galactal 2 (Scheme 1).4 This method was developed
to provide, not only a 1,2-trans-glycosidic linkage, but also
a hydroxyl group on C-2 of the galactose residue (cf. 3) for
further attachment of an a-d-glucosyl moiety. Although
most efficient for this purpose,4,10 the procedure is less
suitable when a building block carrying only a single galac-
tose moiety is desired. The reason for this is that protected
glycosyl acceptor 1, which is prepared in three steps from
expensive hydroxylysine, must be used in excess (2 equiv.)
over the more readily available donor11 2 in order to prevent
further glycosylation of HO-2 in 3. Building block 3 is
therefore obtained in 19% yield based on 1 (37% based on
2), but some unreacted 1 can be recovered after removal of a
small amount of the a-anomer corresponding to 3.
Attempted improvement by glycosylation12 of 1 with
peracetylated galactosyl bromide 4 under promotion by
silver silicate13 did improve the stereoselectivity (no
a-anomer detected) but not the yield (19%, Scheme 1). In
this case, the low yield was explained by the inadequate
stability of the N e-Boc protective group of acceptor 1
under the conditions of the glycosylation.10
Silver silicate promoted glycosylation of hydroxylysine
benzyl ester 8a with galactosyl bromide 4 in dichloro-
methane at 0ЊC gave glycoside 9a in 70% yield without
any detectable formation of the corresponding a-anomer
or orthoester. Unfortunately, in our hands, selective hydro-
genolysis of the benzyl ester failed both for 9a and the
model compound Fmoc-Lys(Cbz)-OBzl. Use of different
amounts of the catalyst Pd/C(en)15 in different solvents
resulted either in removal of both the N e-Cbz group and
the benzyl ester, or in no reaction at all.17 Attempted hydro-
genolysis of 9a with 10% Pd/C in ethyl acetate was non-
selective, as expected.
˚
Scheme 1. (a) Dimethyldioxirane, CH2Cl2, 0ЊC then 1, ZnCl2, AW-300, Ϫ50ЊC!room temp (cf. reference 4); (b) Silver silicate, 3 A MS, CH2Cl2, 0ЊC; (c) H2,
Pd/C, EtOAc.

B. Holm et al. / Tetrahedron 56 (2000) 1579–1586
1581
˚
Scheme 2. (a) 30% CsCO3(aq), 80% EtOH(aq), BTB then BzlBr, Ph2CHBr or allyl bromide, DMF; (b) Silver silicate, 3 A MS, CH2Cl2, 0ЊC; (c) H2, Pd/C(en);
(d) TFA/H2O/CH2Cl2, 9:1:10, yield calculated from 8b; (e) (PPh3)4Pd(0), N-methyl-aniline, THF.
Glycosylation of diphenylmethyl ester 8b under the same
conditions as used for 8a gave a mixture of b-glycoside 9b
and the corresponding orthoester in a 4:1 ratio, as deter-
mined by 1H NMR spectroscopy. Since the two compounds
could not be separated even using normal-phase HPLC, the
mixture was treated with trifluoroacetic acid–water (9:1) in
dichloromethane at room temperature. This led both to
decomposition of the orthoester and removal of the diphenyl-
methyl ester, thereby affording building block 10 in 48%
overall yield from hydroxylysine 8b.
synthesizer18 or a mechanically agitated reactor. Synthesis
was performed on a polystyrene resin grafted with poly-
ethylene glycol spacers (Tentagel resin) according to the
௣
Fmoc strategy, under conditions identical to those reported
previously by us.4 Building blocks 6 and 10 were activated
as azabenzotriazolyl esters19 and coupled to the peptide–
resin in a minimal volume of dry DMF during 24 h. After
completion of the synthesis, treatment with a mixture of
trifluoroacetic acid, water, thioanisole, and ethanedithiol
(35:2:2:1) for 2–3 h liberated the glycopeptides from the
resin, with simultaneous removal of all protective groups
except the O-acetyl groups of the galactosyl moieties.
This is in agreement with previous observations10 regarding
the lability of the Cbz group under acidic conditions.
Deacetylation of the galactosyl moieties was performed
either by treatment with a 20 mM solution of sodium
methoxide in methanol, or using methanol saturated with
ammonia. Purification by reversed-phase HPLC then gave
glycopeptides 11 and 12 in 33 and 12% overall yields,
respectively.
Finally, silver silicate promoted glycosylation of allyl ester
8c with 4 gave b-glycoside 9c in 82% yield. Under these
conditions orthoester formation or lactonization of acceptor
8c, which may be a problem during glycosylations,10 was
negligible. Deallylation of 9c by N-methylaniline in THF
catalysed by (PPh3)4Pd(0)16 then gave the target glycosyl-
ated building block 10 in 83% yield (66% from hydroxyly-
sine 8c).
Glycosylated building blocks 6 and 10 were successfully
used in solid-phase peptide synthesis of glycopeptides 12
and 11 (Fig. 2), respectively, using an automatic peptide
In conclusion, we have established a protective group pattern
for hydroxylysine which allows efficient b-glycosylation of
Figure 2. Glycopeptides 11 and 12.

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B. Holm et al. / Tetrahedron 56 (2000) 1579–1586
the unprotected hydroxyl group. Silver silicate was found to
be a suitable promoter for glycosylation in combination with
acetobromogalactose as glycosyl donor. The resulting
mono-galactosylated hydroxylysine building blocks were
then successfully used in solid-phase synthesis of glycopep-
tides from type II collagen. These are now being used in
investigations of the response of helper T cells generated in
a mouse model for rheumatoid arthritis.
of glycopeptides 11 and 12. In the amino acid analysis
glutamine was determined as glutamic acid.
2,3,4,6-Tetra-O-acetyl-b-d-galactopyranosyl bromide
4
was prepared from peracetylated galactose by treatment
with HBr in HOAc/Ac2O. (5R)-N a-(Fluoren-9-ylmethoxy-
carbonyl)-Ne-benzyloxycarbonyl-5-hydroxy-l -lysine was
prepared as described previously.10 Silver silicate was
prepared as follows: Silica–alumina (25 g, Sigma–Aldrich
35,335-4) was stirred with aqueous NaOH (1 M, 250 mL) at
90ЊC for 1 h. The solid was filtered off and washed with
water (6×100 mL). The remaining steps were carried out
in the absence of light. After stirring overnight at room
temperature with aqueous AgNO3 (0.2 M, 500 mL) the
solid was filtered off and washed with water (6×100 mL)
and finally with acetone (2×100 mL). Drying under high-
vacuum at 105ЊC for 48 h gave the silver silicate as a brown
powder.
Experimental
General methods and materials
TLC was performed on Silica Gel 60 F254 (Merck) with
detection by UV light, charring with 10% sulfuric acid or
by phosphomolybdic acid/ceric sulphate in 6% aqueous
sulfuric acid followed by careful heating. Flash column
chromatography was performed on silica gel (Matrex,
(5R)-Na-(Fluoren-9-ylmethoxycarbonyl)-Ne-(tert-butoxy-
carbonyl)-5-hydroxy-l -lysine benzyl ester (1). Com-
pound 1 was prepared as described previously4 from (5R)-
5-hydroxy-l -lysine dihydrochloride monohydrate (992 mg,
3.9 mmol) with the following modifications. After forma-
tion of the cupric chelate of hydroxylysine Boc-protection
of the e-amino group was found to give more reproducible
results by using the following procedure. Solid NaHCO3
(704 mg, 8.4 mmol) was added to the cupric chelate in
water (25 mL) at 20ЊC. A solution of di-tert-butyl dicarbo-
nate (1.23 g, 5.6 mmol) in dioxane (10 mL) was then added
over 1.5 h, and the reaction mixture was stirred at 20ЊC.
After 4 h a second portion of di-tert-butyl dicarbonate
(520 mg, 2.4 mmol) dissolved in dioxane (5 mL) was
added together with NaHCO3 (50 mg), and the reaction
mixture was then stirred overnight. The resulting suspension
was filtered and the precipitate was washed with small
portions of water. The combined filtrates were concentrated,
diluted with water/THF (10 mL, 1:1), diethyl ether (2 mL)
was added and the solution was cooled to 5ЊC. After 16 h
another portion of precipitate was filtered off and washed
with water. Once again, the combined filtrates were concen-
trated to dryness, the residue was dissolved in water/THF
(10 mL, 1:1) and diethyl ether (2 mL) was added after which
the solution was stored at 5ЊC overnight. Addition of a few
crystals of the precipitate to this cooled solution accelerates
the crystallization. This procedure was repeated until no
appreciable amount of precipitation occurred. The
combined precipitates were then converted to 1 according
to the previously reported procedure. In brief the cupric
chelate of N e-(tert-butoxycarbonyl)-5-hydroxy-l -lysine
was dissociated using Naϩ Chelex 100 which had been
converted to its Hϩ form. The a-amino group was protected
with an Fmoc group before formation of a cesium carboxy-
late salt which was finally transformed into a benzyl ester
with benzyl bromide. Use of bromothymol blue as indicator
during formation of the cesium carboxylate was found to
reduce the amount of undesired cleavage of the Fmoc group.
˚
60 A, 35–70 mm, Grace Amicon) with solvents of HPLC
grade, analytical grade or distilled technical grade. DMF
˚
was distilled and then dried over 3 A molecular sieves.
Dry THF was obtained by distilling from sodium–
potassium alloy and dry CH2Cl2 by distilling from CaH2.
1
The H and 13C NMR spectra of compounds 5–10 were
recorded on a Bruker DRX-400 spectrometer at 400 MHz
and 100 MHz, respectively. Chemical shifts are referenced
to residual CHCl3 (dHˆ7.27 ppm) and CDCl3 (dCˆ77.0) for
solutions in CDCl3 or to CD2HOD (dHˆ3.31 ppm) and
CD3OD (dCˆ49.0) for solutions in a 1:1 mixture of
CD3OD/CDCl3. Spectra for glycopeptides 11 and 12 were
recorded on Bruker DRX-600 and ARX-500 spectrometers
in 40 mM phosphate buffer containing 1.5 mM NaN3 and
10% D2O at 278 K and pH 6.2 with H2O as internal standard
1
(dHˆ4.95 ppm). First-order H and 13C chemical shifts, as
1
well as H coupling constants, were determined from one-
dimensional spectra and resonances were assigned from
COSY, TOCSY, 1H–13C–HSQC, 1H–15N–HSQC,
1H–13C-HSQC-TOCSY, DEPT and NOESY experiments.
Optical rotations were recorded on a Perkin–Elmer 343
polarimeter. Analytical reversed-phase HPLC was
performed on a Kromasil C-8 column (250×4.6 mm,
˚
5 mm, 100 A), eluted with a linear gradient of MeCN in
H2O containing 0.1% TFA (a flowrate of 1.5 mL was used
and detection was at 214 nm). Preparative reversed-phase
HPLC was performed on a Kromasil C-8 column
˚
(250×20 mm, 5 mm, 100 A), with the same eluent, a flow-
rate of 11 mL/min and detection at 214 nm. Analytical
normal-phase HPLC was performed on a Kromasil silica
˚
column (250×4.6 mm, 5 mm, 100 A), using a flowrate of
2 mL/min and with detection at 254 nm. Preparative
normal-phase HPLC was performed on a Kromasil silica
˚
column (250×20 mm, 5 mm, 100 A) using a flowrate of
15 mL/min and detection at 254 nm.
௣
A TentalGel resin (Rapp Polymere, Germany) function-
(5R)-Na-(Fluoren-9-ylmethoxycarbonyl)-Ne-tert-butoxy-
carbonyl-5-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
nosyl)-5-hydroxy-l -lysine benzyl ester (5). Aceto-
bromogalactose (4, 206 mg, 0.50 mmol) was added to a
suspension of 1 (175 mg, 0.30 mmol), silver silicate
alized with a triphenylmethyl linker carrying an Na-Fmoc-
protected proline residue (TentaGel–S–Trt–Pro–Fmoc)
and N a-Fmoc-protected amino acids carrying triphenyl-
methyl (Gln), tert-butyl (Glu, Thr) and tert-butoxycarbonyl
(Lys) side chain protective groups were used in the synthesis

B. Holm et al. / Tetrahedron 56 (2000) 1579–1586
1583
˚
(500 mg) and crushed molecular sieves (3 A, 360 mg) in
(Na2SO4) and concentrated. Flash column chromatography
CH2Cl2 (10 mL) at 0ЊC in the absence of light. After stirring
for 2 h, Et3N (0.15 mL) was added and the suspension was
stirred for 20 min. The solid material was filtered off (Hyflo
Supercel), washed with CH2Cl2 (35 mL) and the combined
filtrates were concentrated. Flash column chromatography
of the residue (Toluene/MeCN, 9:2) gave 5 (54 mg, 19%) as
of the residue (heptane/EtOAc, 1:1) gave 8a (93 mg, 38%)
1
as a white amorphous solid: [a]2d0ˆϪ5 (c 1.0, CHCl3); H
NMR d 7.76 (d, Jˆ7.5 Hz, 2H, Fmoc), 7.59 (d, Jˆ7.2 Hz,
2H, Fmoc), 7.39 (t, Jˆ7.4 Hz, 2H, Fmoc), 7.36–7.28
(m, 14H, Cbz, Bzl, Fmoc), 5.63 (d, Jˆ7.4 Hz, 1H, NH-e),
5.24–5.08 (m, 5H, NeCO2CH2Ph, CO2CH2Ph, NH-a),
4.50–4.34 (m, 3H, H-a, Fmoc, Fmoc), 4.21 (t, Jˆ6.7 Hz,
1H, Fmoc), 3.73–3.62 (m, 1H, H-d), 3.33–3.20 and 3.07–
2.94 (2 m, each 1H, H-e), 2.83–2.72 (m, 1H, OH), 2.07–
1.96 and 1.82–1.70 (2 m, each 1H, H-b), 1.50–1.38 (m, 2H,
H-g,g0); 13C NMR d 172.1, 157.2, 156.2, 143.8, 143.7,
141.3, 136.3, 135.2, 128.6, 128.6, 128.5, 128.4, 128.2,
128.1, 127.7, 127.1, 125.1, 120.0, 70.8, 67.3, 67.1, 66.9,
53.5, 47.1, 46.9, 29.9, 29.2; HRMS (FAB): calcd for
C36H37N2O7 (MϩHϩ) 609.2601, found 609.2626.
1
a white amorphous solid: [a]2d0ˆϪ5 (c 1.0, CHCl3); H
NMR (CDCl3) d 7.75 (d, Jˆ7.6 Hz, 2H, Fmoc), 7.56 (d,
Jˆ7.3 Hz, 2H, Fmoc), 7.39 (t, Jˆ7.5 Hz, 2H, Fmoc),
7.35–7.24 (m, 9H, Fmoc, OCH2Ph), 5.43–5.34 (m, 2H,
NH-e, H-4), 5.28 (m, 1H, NH-a), 5.20–5.11 (m, 2H,
OCH2Ph, H-2), 4.98 (dd, Jˆ10.5, 3.4 Hz, 1H, H-3), 4.43
(m, 2H, H-1, Fmoc), 4.35 (m, 2H, H-a, Fmoc), 4.23–4.08
(m, 3H, H-6,6, Fmoc), 3.88 (t, Jˆ6.9 Hz, 1H, H-5), 3.62–
3.51 (m, 1H, H-d), 3.35–3.23 and 3.14–3.01 (2 m, each 1H,
H-e), 2.15 and 2.04 (2 s, each 3H, COCH3), 1.97–1.94 (m,
7H, H-b, 2 COCH3), 1.72–1.59 (m, 2H, H-b, H-g), 1.58–
1.47 (m, 1H, H-g), 1.44 (s, 9H, C(CH3)3); 13C NMR d 171.9,
170.3, 170.2, 170.1, 169.4, 156.2, 156.0, 143.7, 143.6,
141.2, 135.0, 128.7, 128.6, 128.4, 127.7, 127.1, 127.0,
125.1, 120.0, 101.3, 81.3, 79.4, 70.7, 70.7, 68.7, 67.4,
67.1, 66.8, 61.3, 53.7, 47.0, 44.1, 28.5, 28.4, 28.3, 20.7,
20.6, 20.6; HRMS (FAB): calcd for C47H56N2O16Na
(MϩNaϩ) 927.3528, found 927.3521.
(5R)-N a-(Fluoren-9-ylmethoxycarbonyl)-N e-benzyloxy-
carbonyl-5-hydroxy-l -lysine diphenyl-methyl ester (8b).
Compound 7 (104 mg, 0.2 mmol) was converted into the
cesium salt of 5 as described for 8a. The solid was dissolved
in dry DMF (0.5 mL) at 0ЊC and diphenylmethyl bromide
(250 mg, 1 mmol) dissolved in dry DMF (0.5 mL) was
added. After stirring for 4 h at room temperature the solu-
tion was diluted with H2O (10 mL) and extracted with
diethyl ether (3×25 mL). The combined organic layers
were dried (Na2SO4) and concentrated. Flash column
chromatography of the residue (heptane/EtOAc, 1:1) gave
8b contaminated by a small amount of lactone. Further
purification by normal-phase HPLC (0!40% EtOH in
hexane over 180 min) gave pure 8b (48 mg, 35%) as a
white amorphous solid: [a]2d0ˆϪ12 (c 1.0, CHCl3); 1H
NMR (CDCl3) d 7.75 (d, Jˆ7.6 Hz, 2H, Fmoc), 7.58 (d,
Jˆ7.3 Hz, 2 H, Fmoc), 7.39–7.27 (m, 19H, Fmoc, Cbz,
CHPh2), 6.91 (s, 1 H, CHPh2), 5.59 (bd, Jˆ6.9 Hz, 1H,
NH-a), 5.13–5.06 (m, 3H, NeCO2CH2Ph, NH-e), 4.60–
4.52 (m, 1H, H-a), 4.40 (ABdd, Jˆ10.6, 7.4 Hz, 1H,
Fmoc), 4.38 (ABdd, Jˆ10.6, 6.9 Hz, 1H, Fmoc), 3.71–
3.61 (m, 1H, H-d), 3.30–3.21 and 3.02–2.91 (2 m, each
1H, H-e), 2.68 (bs, 1H, OH), 2.11–2.00 and 1.85–1.74 (2
m, each 1H, H-b(b0), 1.44–1.34 (m, 2H, H-g,g0); 13C NMR
(CDCl3) d 171.3, 157.1, 156.1, 143.8, 143.6, 141.2, 139.5,
139.2, 136.3, 128.6. 128.5, 128.5, 128.2, 128.1, 128.1,
127.7, 127.2, 127.0, 126.9, 125.0, 78.2, 70.7, 67.1, 66.9,
47.1, 46.9, 29.7, 28.9; HRMS (FAB): calcd for
C42H41N2O7 (MϩHϩ) 685.2914, found 685.2930.
(5R)-Na-(Fluoren-9-ylmethoxycarbonyl)-Ne-tert-butoxy-
carbonyl-5-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
nosyl)-5-hydroxy-l -lysine (6). Compound
5 (49 mg,
54 mmol) in EtOAc (3.6 mL) was hydrogenated over Pd/C
(49 mg) for 3 h at atmospheric pressure. The mixture was
filtered (Hyflo supercel) and the filtrate concentrated. Flash
column chromatography of the residue (CHCl3/MeOH,
20:1!5:1) gave 6 (32 mg, 72%) as a white amorphous
solid: [a]2d0ˆϪ11 (c 0.4, CHCl3); 1H NMR (CDCl3/
CD3OD, 1:1) d 7.74 (d, Jˆ7.2 Hz, 2H, Fmoc), 7.58 (m,
2H, Fmoc), 7.38 (t, Jˆ7.3 Hz, 2H, Fmoc), 7.30 (m, 2H,
Fmoc), 5.75 (m, 1H, NH-e), 5.75 (m, 1H, NH-a), 5.38 (s,
2H, H-4), 5.17 (t, Jˆ11.0 Hz, 1H, H-2), 5.01 (bd,
Jˆ10.2 Hz, 1H, H-3), 4.50 (d, Jˆ7.5 Hz, 1H, H-1), 4.35
(m, 2H, H-a, Fmoc), 4.17 (m, 4H, H-6,6, Fmoc, Fmoc),
3.90 (m, 1H, H-5), 3.63 (m, 1H, H-d), 3.35 and 3.15 (2 m,
each 1H, H-e), 2.15, 2.04, 2.00 and 1.97 (4 s, each 3H,
COCH3), 1.75 (m, 1 H,H-b), 1.60 (m, 2H, H-g,g0), 1.44
(s, 9H, C(CH3)3); 13C NMR (CDCl3/CD3OD, 1:1) d 170.9,
170.7, 170.4, 170.4, 156.8, 156.7, 144.0, 143.9, 141.3,
127.7, 127.1, 125.0, 119.9, 101.2, 81.2, 79.5, 71.1, 70.5,
69.1, 67.3, 66.8, 61.3, 55.2, 47.1, 44.4, 29.6, 29.0, 28.5,
28.1, 20.3, 20.2, 20.2; HRMS (FAB): calcd for
C40H50N2O16Na (MϩNaϩ) 837.3058, found 837.3050.
(5R)-N a-(Fluoren-9-ylmethoxycarbnoyl)-N e-benzyloxy-
carbonyl-5-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
nosyl)-5-hydroxy-l -lysine benzyl ester (9a). A solution of
4 (69 mg, 0.16 mmol) in CH2Cl2 (1 mL) was added to a
suspension of 8a (63 mg, 0.10 mmol), silver silicate
(5R)-Na-(Fluoren-9-ylmethoxycarbonyl)-Ne-benzyloxy-
carbonyl-5-hydroxy-l -lysine benzyl ester (8a). A solution
of crude 710 (210 mg, 0.4 mmol) and bromothymol blue
(BTB, Ͻ1 mg) in 80% aqueous EtOH (3 mL) at 0ЊC was
adjusted to pH 7 with 30% Cs2CO3 (aq) and then concen-
trated. Concentration twice from 99.5% EtOH followed by
drying under high vacuum overnight gave the cesium salt of
7 as a dry solid. The solid was dissolved in dry DMF (2 mL)
at 0ЊC and benzyl bromide (72 mL, 0.6 mmol) was added.
After stirring for 3.5 h at room temperature the solution was
diluted with H2O (10 mL) and extracted with diethyl ether
(3×25 mL). The combined organic layers were dried
˚
(155 mg) and crushed molecular sieves (3 A, 50 mg) in
CH2Cl2 (2 mL) at 0ЊC. After stirring for 2 h Et3N (25 mL)
was added and the suspension was stirred for 20 min. The
solid material was filtered off (Hyflo Supercel), washed with
CH2Cl2 (20 mL) and the combined filtrates were concen-
trated. Flash column chromatography of the residue
(Toluene/MeCN, 8:1) gave 9a (68 mg, 70%) as a white
amorphous solid: [a]2d0ˆ1 (c 1.0, CHCl3); 1H NMR
(CDCl3) d 7.76 (d, Jˆ7.5 Hz, 2H, Fmoc), 7.59 (d,
Jˆ7.5 Hz, 2H, Fmoc), 7.40–7.28 (m, 12H, Fmoc,
CO2CH2Ph, NeCO2CH2Ph), 5.54 (t, Jˆ5.5 Hz, 1H, NH-e),

1584
B. Holm et al. / Tetrahedron 56 (2000) 1579–1586
˚
5.44 (d, Jˆ7.9 Hz, 1H, NH-a), 5.35 (dd, Jˆ3.4, 1 Hz, 1H,
H-4), 5.18 (s, 2H, CO2CH2Ph), 5.15 (dd, Jˆ10.5, 8.0 Hz,
1H, H-2), 5.12 and 5.09 (2 ABd, Jˆ12.2 Hz, each 1H,
NeCO2CH2Ph), 4.96 (dd, Jˆ10.5, 3.4 Hz, 1H, H-3), 4.47–
4.30 (m, 4H, Fmoc, Fmoc, H-1, H-a), 4.20 (t, Jˆ7.0 Hz, 1H,
Fmoc), 4.12 (ABdd, Jˆ11.4, 6.2 Hz, 1H, H-6), 4.05 (ABdd,
Jˆ11.2, 6.9 Hz, 1H, H-6), 3.82 (t, Jˆ6.6 Hz, 1H, H-5),
3.66–3.58 (m, 1H, H-d), 3.42–3.33 and 3.19–3.10 (2 m,
each 1H, H-e), 2.14 (s, 3H, COCH3), 2.08–2.02 (m, 1H,
H-b), 1.98, 1.98 and 1.95 (3 s, each 3H, 3 COCH3), 1.74–
1.43 (m, 3H, H-b, H-g,g0); 13C NMR (CDCl3) d 171.9,
170.2, 170.1, 170.0, 169.3, 156.7, 155.9, 143.8, 143.7,
141.3, 136.5, 135.0, 129.0, 128.7, 128.6, 128.5, 128.4,
128.2, 128.2, 127.7, 127.1, 125.3, 125.0, 120.0, 101.3,
80.9, 70.8, 70.7, 68.8, 67.4, 67.1, 67.0, 66.8, 61.4, 53.7,
47.1, 44.7, 28.52, 28.3, 20.6, 20.5, 20.5; HRMS (FAB):
calcd for C50H54N2NaO16 (MϩNaϩ) 961.3371, found
961.3391.
sieves (3 A, 30 mg) in CH2Cl2 (1 mL) at Ϫ10ЊC in the
absence of light. After stirring for 17 h Et3N (25 mL) was
added and the suspension was stirred for 20 min. The solid
material was filtered off (Hyflo supercel), washed with
CH2Cl2 and the combined filtrates were concentrated.
Flash column chromatography of the residue (Toluene/
MeCN, 8:1) gave a solid which was dissolved in TFA/
H2O/CH2Cl2 (1 mL, 9:1:10) at 0ЊC. After stirring for
40 min at room temperature the solvent was removed in
vacuo, toluene was added and the resulting solution was
concentrated. Flash column chromatography of the residue
(Toluene/EtOH, 20:1!4:1) gave 10 (24 mg, 48%) as a
white amorphous solid. Compound 10 had: [a]2d0ˆ0 (c
2.0, CHCl3); 1H NMR (CDCl3/CD3OD 1:1) d 7.74 (d,
Jˆ7.5 Hz, 2H, Fmoc), 7.62 (t, Jˆ6.3 Hz, 2H, Fmoc), 7.36
(t, Jˆ7.5 Hz, 2H, Fmoc), 7.37–7.25 (m, 7H, Fmoc, Cbz),
5.33 (d, Jˆ2.8 Hz, 1H, H-4), 5.10 (ABdd, Jˆ10.5, 7.9 Hz,
1H, H-2), 5.05 (s, 2H, OCH2Ph), 5.01 (dd, Jˆ10.5, 3.3 Hz,
1H, H-3), 4.40–4.30 (m, 2H, Fmoc), 4.55 (d, Jˆ7.5 Hz, 1H,
H-1), 4.20 (t, Jˆ6.8 Hz, 1H, Fmoc), 4.17–4.15 (m, 1H,
H-a), 4.12 (dd, Jˆ12.2, 7.1 Hz, 1H, H-6), 4.04 (dd,
Jˆ11.8, 7.5 Hz, 1H, H-6), 3.71–3.61 (m, 1H, H-d), 3.37–
3.27 and 3.22–3.14 (2 m, each 1H, 2H-e), 2.11 and 2.00 (2 s,
each 3H, 2 COCH3), 1.97–1.93 (m, 7H, H-b, 2 COCH3),
1.70–1.61 (m, 1H, H-b), 1.61–152 (m, 2 H, H-g,g0); 13C
NMR (CD3OD/CDCl3, 1:1) d 171.5, 171.3, 171.0, 170.8,
157.8, 157.6, 144.5, 144.4, 141.9, 137.1, 129.4, 129.3,
129.0, 128.7, 128.3, 127.6, 125.6, 120.4, 101.8, 81.4, 71.6,
71.2, 69.7, 67.9, 67.4, 67.3, 62.0, 54.4, 54.0, 47.7, 45.4,
29.7, 28.5, 20.8, 20.7, 20.7; HRMS (FAB): calcd for
C43H48N2NaO16 (MϩNaϩ) 871.2902, found 871.2897.
(5R)-N a-(Fluoren-9-ylmethoxycarbonyl)-N e-benzyloxy-
carbonyl-5-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
nosyl)-5-hydroxy-l -lysine allyl ester (9c). A solution of 4
(247 mg, 0.60 mmol) in CH2Cl2 (1 mL) was added to a
suspension of 8c (223 mg, 0.4 mmol), silver silicate
˚
(600 mg) and crushed molecular sieves (3 A, 50 mg) in
CH2Cl2 (2 mL) at 0ЊC in the absence of light. Workup as
described for 9a followed by flash column chromatography
of the residue (Toluene/MeCN, 8:1) gave 9c (290 mg, 82%)
1
as a white amorphous solid: [a]2d0ˆ0 (c 3.0, CHCl3); H
NMR (CDCl3) d 7.76 (d, Jˆ7.5 Hz, 2H, Fmoc), 7.60 (d,
Jˆ7.3 Hz, 2 H, Fmoc), 7.40 (t, Jˆ7.4 Hz, 2H, Fmoc),
7.36–7.29 (m, 7H, Fmoc, Cbz), 5.96–5.85 (m, 1H,
OCH2CHCH2), 5.59 (m, 1H, NH-e), 5.45 (d, Jˆ8.2 Hz,
1H, NH-a), 5.36–5.25 (m, 3 H, H-4, OCH2CHvCH2),
5.16 (dd, Jˆ10.5, 8.0 Hz, 1H, H-2), 4.96 (dd, Jˆ10.5,
3.36 Hz, 1H, H-3), 4.64 (m, 2H, OCH2CHvCH2), 4.48–
4.43 (m, 2H, H-1, Fmoc), 4.38–4.30 (m, 2H, H-a, Fmoc),
4.22 (t, Jˆ6.9 Hz, 1H, Fmoc), 4.12 (dd, Jˆ11.4, 6.2 Hz, 1H,
H-6), 4.05 (dd, Jˆ11.2, 6.9 Hz, 1H, H-6), 3.83 (t, Jˆ6.6 Hz,
1H, H-5), 3.70–3.62 (m, 1H, H-d), 3.46–3.38 and 3.23–
3.14 (2 m, each 1H, H-e), 2.15 (s, 3 H, COCH3), 2.05–
1.93 (m, 10H, H-b, 3 COCH3), 1.75–1.50 (m, 3H, H-b,
H-g,g0); 13C NMR (CDCl3) d 171.7, 170.3, 170.1, 170.0,
169.3, 156.7, 156.0, 143.7, 143.6, 141.2, 136.4, 131.3,
128.5, 128.4, 128.3, 128.2, 127.7, 127.1, 127.0, 125.0,
120.0, 119.3, 101.3, 80.9, 70.8, 70.7, 68.8, 67.0, 66.9,
66.7, 66.2, 61.5. 53.6, 53.6, 47.0, 44.8, 28.6, 28.3, 20.6,
20.5; HRMS (FAB): calcd for C46H52N2NaO16 (MϩNaϩ)
911.3215, found 911.3221.
l -Alanylglycyl-l -phenylalanyl-l -lysylglycyl-l -glutam-1-
yl-l -glutaminylglycyl-l -prolyl-(5R)-5-O-(b-d-galacto-
pyranosyl)-5-hydroxy-l -lysylglycyl-l -glutam-1-yl-l -
threonylglycyl-l -prolyl-l -alanylglycyl-l -proline
(11).
Glycopeptide 11 (Table 1) was prepared under conditions
identical to those described recently.4,10 A custom-made,
fully automatic, continuous flow peptide synthesizer18 and
a TentaGel–S–Trt–Pro–Fmoc resin (480 mg, capacity
0.21 mmol/g, 0.100 mmol) was used for the synthesis. In
brief, the Fmoc-amino acids (4 equiv.) were activated as
benzotriazolyl esters20 using 1,3-diisopropylcarbodiimide
(DIC, 3.9 equiv.) and 1-hydroxybenzotriazole (HOBt,
6 equiv.) in DMF. The acylations were monitored21 using
the absorbance of bromophenol blue at 600 nm. Na-Fmoc
deprotections were performed by a flow of 20% piperidine
in DMF using the absorbance of the dibenzofulvene–piper-
idine adduct at 350 nm for monitoring.22 After coupling of
Gly271 and removal of the Fmoc group, half of the resin
(50 mmol) was allowed to swell in a minimal amount of
DMF. Glycosylated hydroxylysine 10 (1.5 equiv.) was
activated separately using DIC (1.5 equiv.) and 1-hydroxy-
7-azabenzotriazole (3 equiv.) in dry DMF for 45 min before
coupling to the peptide resin. The coupling was allowed to
proceed for 24 h, then the resin was washed six times with
DMF and transferred back to the peptide synthesizer where
the remaining couplings were performed. After completion
of the synthesis the resin was washed six times with DMF
and 10 times with CH2Cl2 and dried under vacuum over-
night giving 224 mg of resin. Cleavage and deprotection of
the peptide moiety was performed with a mixture of TFA/
H2O/thioanisole/ethanedithiol (35:2:2:1, 10 mL) as described
(5R)-N a-(Fluoren-9-ylmethoxycarbonyl)-N e-benzyloxy-
carbonyl-5-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyra-
nosyl)-5-hydroxy-l -lysine (10). From 9c: (PPh3)4Pd(0)
(29 mg, 25 mmol) was added to a solution of 9c (223 mg,
0.25 mmol) and N-methylaniline (82 ml, 0.75 mmol) in dry
THF in the absence of light. After stirring for 1.5 h at room
temperature the solution was diluted with EtOAc (50 mL)
and washed with saturated NH4Cl (aq), dried, filtered and
concentrated. Flash column chromatography of the residue
(CH2Cl2!CH2Cl2/MeOH, 10:1) gave 10 (182 mg, 86%).
From 8b: A solution of 4 (37 mg, 87 mmol) in CH2Cl2
(1.5 mL) was added to a suspension of 8b (40 mg,
58 mmol), silver silicate (87 mg) and crushed molecular

B. Holm et al. / Tetrahedron 56 (2000) 1579–1586
Table 1. H NMR data for glycopeptide 11, in water containing 10% D2O
1585
1
Residue
NH
a
b
g
Others
Ala261
Gly262
Phe263
Lys264
Gly265
Glu266
Gln267
Gly268
Pro269,c
Hyl270,b
Gly271
Glu272
Thr273
Gly274
Pro275,c
Ala276
Gly277
Pro278
4.03
1.44
8.64
8.41
8.52
7.95
8.38
8.60
8.42
3.89a
4.56
4.19
3.85, 3.80
4.21
4.31
4.10, 3.90
4.29
4.28
4.09, 3.89
4.32
4.32
4.12, 3.99
4.29
4.29
4.08, 3.92
4.34
3.00a
1.72, 1.62
7.27, 7.24 and 7.20 (arom.)
1.29a
1.58a (d), 2.88a (e)
2.20a
2.30a
1.90, 1.84
2.07, 1.92
7.57 and 6.89 (CONH2)
1.83, 1.76
1.99, 1.76
2.25, 2.03
1.76a
3.49 and 3.42 (d)
4.0 (d), 2.93 and 3.13 (e)
8.67
8.43
8.50
8.42
8.38
2.21a
4.18
2.01, 1.87
1.15
1.91, 1.86
1.34
2.13a
3.50a (d)
3.54a (d)
8.57
8.23
1.95, 1.88
2.22a
a Degeneracy has been assumed.
b Chemical shifts (d, ppm) for the galactose moiety: 4.40 (H-1), 3.84 (H-4), 3.69 (H-5), 3.62 (H-6,6), 3.58 (H-3), 3.46 (H-2).
c The assignments of Pro269 and Pro275 are uncertain.
previously.4,10 The crude product obtained after freeze-
drying was purified by reversed-phase HPLC (0!100%
MeCN in 40 min) to give 12 mg of the O-acetylated glyco-
peptide. Deacetylation was performed using methanolic
NaOMe (20 mM, 10 mL) at 0!20ЊC for 3 h (monitoring
by reversed-phase HPLC, 0!100% MeCN during 60 min)
and the solution was neutralized (pH paper) by addition of
10% HOAc in MeOH. Concentration, followed by purifi-
cation using reversed-phase HPLC (0!100% MeCN during
50 min) gave 11 (6.8 mg, 81% peptide content, 12% overall
l -Alanylglycyl-l -phenylalanyl-(5R)-5-O-(b-d-galacto-
pyranosyl)-5-hydroxy-l -lysylglycyl-l -glutam-1-yl-l -
glutaminylglycyl-l -prolyl-l -lysylglycyl-l -glutam-1-yl-l -
threonylglycyl-l -prolyl-l -alanylglycyl-l -proline (12).
Synthesis of glycopeptide 12 (Table 2) was performed in
a mechanically agitated reactor on TentaGel–S–Trt–Pro–
Fmoc resin (167 mg, 30 mmol) using the same conditions
for synthesis and cleavage as described for glycopeptide 11.
However, the glycosylated hydroxylysine at position 264
was incorporated using building block 6 (1.3 equiv.) acti-
vated as a 7-azabenzotriazole ester. After cleavage from the
solid phase, purification by reversed-phase HPLC (0!
100% MeCN during 60 min) gave O-acetylated glyco-
peptide (33.5 mg). Deacetylation was performed using
saturated methanolic ammonia (50 mL) at 0!20ЊC for
1
yield) as a white amorphous solid after freeze-drying: H
NMR data, cf. Table 1; MS (FAB) calcd. 1863 (MϩHϩ),
found 1862; amino acid analysis: Ala 2.03 (2), Glu 2.90
(3), Gly 6.01 (6), Hyl 1.00 (1), Lys 1.04 (1), Phe 1.01
(1), Pro 2.98 (3), Thr 1.01 (1).
1
Table 2. H NMR data for glycopeptide 12 in water containing 10% D2O
Residue
NH
a
b
g
Others
Ala261
Gly262
Phe263
Hyl264,b
Gly265
Glu266
Gln267
Gly268
Pro269,c
Lys270
Gly271
Glu272,d
Thr273
Gly274
Pro275,c
Ala276
Gly277
Pro278
4.01
1.42
8.63
8.40
8.53
7.98
8.39
8.61
8.39
3.37a
4.55
4.20
3.85, 3.78
4.20
4.29
2.98a
1.91, 1.66
7.27 and 7.18 (arom)
1.51a
3.90 (d), 2.88 and 3.09 (e)
7.56 and 6.88 (CONH2)
1.60a (d), 2.9a (e)
2.21a
2.29a
1.96, 1.81
2.05, 1.76
4.06, 3.90
8.61
8.42
8.39
8.39
8.38
4.23
4.05, 3.91
1.77, 1.68
4.17
1.36a
1.12
4.31
4.10, 3.98
8.54
8.21
4.23
4.05, 3.91
1.32
a Degeneracy has been assumed.
b Chemical shifts (d, ppm) for the galactose moiety: 4.38 (H-1), 3.86 (H-4), 3.70 (H-6,6), 3.64 (H-5), 3.59 (H-3), 3.46 (H-2).
c The assignments of Pro278, Pro275 and Pro269 are uncertain. Three sets of resonances for Pro residues exist but they cannot be unambiguously identified.
d The remaining resonances of Glu272 could not be unambiguously assigned and are therefore not reported.

1586
B. Holm et al. / Tetrahedron 56 (2000) 1579–1586
glycopeptides. However, as both the a- and e-amino groups of
hydroxylysine carry identical protective groups, selective exten-
sion of the peptide at the N-terminus cannot easily be achieved.
9. A more reproducible procedure for synthesis of 1 than reported
in Ref. 4 has now been developed and is included in the
experimental part.
8 h. Concentration, followed by purification using reversed-
phase HPLC (0!100% MeCN during 60 min) and freeze
drying gave 12 (25 mg, 73% peptide content, 33% overall
yield) as a white amorphous solid: 1H NMR data, cf. Table
2; MS (FAB) calcd. 1863 (MϩHϩ), found 1862; amino acid
analysis: Ala 2.03 (2), Glu 3.00 (3), Gly 5.99 (6), Hyl 1.02
(1), Lys 1.02 (1), Phe 1.01 (1), Pro 2.92 (3), Thr 1.01 (1).
10. Broddefalk, J.; Forsgren, M.; Sethson, I.; Kihlberg, J. J. Org.
Chem. 1999, 64, 8948–8953.
11. Alonso, R. A.; Vite, G. D.; McDevitt, R. E.; Fraser-Reid, B.
J. Org. Chem. 1992, 57, 573–584.
Acknowledgements
12. In a related study, various promoters and conditions were
evaluated for glycosylation of hydroxylysine with 2,3,4-tri-O-
acetyl-a-d-fucopyranosyl-bromide or thioglycoside derivatives.
With exception of when silver silicate was used as promoter
these attempts gave the target building block in low yields, often
due to formation of orthoester side-products that could not be
removed.
This project was supported by grants from the programme
‘Glycoconjugates in Biological Systems’ sponsored by the
Swedish Foundation for Strategic Research, the Swedish
Natural Science Research Council and the Swedish
Research Council for Engineering Science.
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¨
20. Konig, W.; Geiger, R. Chem. Ber. 1970, 103, 788–798.
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