Synthesis of a galactosylated 4-hydroxylysine building block and its incorporation into a collagen immunodominant glycopeptide

Article abstract of DOI:10.1002/ejoc.200700806

An analogue of the immunodominant glycopeptide from type II collagen encompassing residues 256-270 has been prepared by substituting β-D-galactopyranosyl-(2S,4R)-4-hydroxy-L-lysyl (Gal-4-Hyl) for β-D-galactopyranosyl-(2S,5R)-5-hydroxy-L-lysyl (Gal-5-Hyl) at position 264. The synthesis of the 4-hydroxylysine aglycon started from the known (2S,4S)-4-hydroxy-6-oxo-1,2-piperidinedicarboxylate (3) and involved selective ring-opening of 3, lactone formation, and N-acylation of the lactone with glycyl esters. The resulting N-Fmoc-protected 4-hydroxylysyl-glycine dipeptide derivative was galactosylated in high yield to give a building block suitable for solid-phase peptide synthesis. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200700806

FULL PAPER
DOI: 10.1002/ejoc.200700806
Synthesis of a Galactosylated 4-Hydroxylysine Building Block and Its
Incorporation into a Collagen Immunodominant Glycopeptide
Julien Marin,[a][‡] Jean-Paul Briand,[a] and Gilles Guichard*[a]
Keywords: Glycopeptides / Amino acids / Hydroxylysine / Solid-phase synthesis / Lactones / Rheumatoid arthritis
An analogue of the immunodominant glycopeptide from type
II collagen encompassing residues 256–270 has been pre-
volved selective ring-opening of 3, lactone formation, and N-
acylation of the lactone with glycyl esters. The resulting N-
pared by substituting β-
droxy- -lysyl (Gal-4-Hyl) for β-
hydroxy- -lysyl (Gal-5-Hyl) at position 264. The synthesis of
D-galactopyranosyl-(2S,4R)-4-hy- Fmoc-protected 4-hydroxylysyl-glycine dipeptide derivative
L
D-galactopyranosyl-(2S,5R)-5-
was galactosylated in high yield to give a building block suit-
able for solid-phase peptide synthesis.
L
the 4-hydroxylysine aglycon started from the known (2S,4S)-
4-hydroxy-6-oxo-1,2-piperidinedicarboxylate (3) and in-
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
Interestingly, synthetic CII peptides with Gal-5-Hyl at posi-
tion 264 {[Gal-5-Hyl264]CII(256–270) (1)} have been found
to exert a protective effect against the development of CIA
in neonatal treatment experiments.[6] Furthermore, [Gal-5-
Hyl264]CII(259–273) complexed to Aq molecules was re-
ported to prevent the development of CIA in mice while
the nonglycosylated analogue/Aq complex did not.[7] Taken
together, these findings are relevant to the development of
new treatment against autoimmune arthritis based on CII
glycopeptides. In particular, CII glycopeptide mimetics may
prove useful as altered peptide ligands (APL)[8] to interfere
with the T-cell response in CIA and RA.
Introduction
In collagen-induced arthritis (CIA), a mouse model for
rheumatoid arthritis (RA), autoreactive
T
cells of
transgenic mice expressing MHC class II Aq molecule (or
human DR4 and DR1 haplotypes) recognize a peptide en-
compassing residues 256–270 when it is post-translationally
modified at position 264 (hydroxylation and glycosylation
of the Lys side-chain).[1,2] The resulting β--galactopyrano-
syl-(2S,5R)-5-hydroxy--lysyl (Gal-5-Hyl) side-chain at po-
sition 264 serves as a critical T-cell receptor (TCR) contact
point. The development of efficient routes to Gal-5-Hyl
building blocks and synthetic CII-derived glycopeptides[3–5]
We recently described the design and synthesis of several
has been instrumental in studies aimed at understanding analogues of [Gal-5-Hyl264]CII(256–270) carrying diverse
the role of lysine hydroxylation and glycosylation in CIA. modifications at the critical hydroxylysine side-chain and
their use as probes to explore the fine specificity of CII-
[a] CNRS, Institut de Biologie Moléculaire et Cellulaire, Labora-
reactive T cells involved in the initiation and/or regulation
of CIA.[9] To further investigate the basis of epitope re-
cognition by CII-reactive T cells, we became interested in
synthesizing and evaluating the [Gal-5-Hyl264]CII(256–
270) variant 2 obtained by substituting a galactosylated 4-
hydroxylysyl residue for the galactosylated 5-hydroxylysyl
residue at position 264. We have shown previously that
toire d’Immunologie et Chimie Thérapeutiques,
15 rue René Descartes, 67000 Strasbourg, France
Fax: +33-3-88610680
E-mail: g.guichard@ibmc.u-strasbg.fr
[‡] Present address: Novalyst Discovery, BioParc,
Boulevard Sébastien Brant, B. P. 30170, 67405 Illkirch cedex,
France
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2008, 1005–1012
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1005

J. Marin, J.-P. Briand, G. Guichard
FULL PAPER
enantiopure 5-hydroxy- and 4-hydroxy-6-oxo-1,2-piperid-
inedicarboxylates are versatile building blocks for the syn-
thesis of 5- and 4-hydroxylysine derivatives, respectively.[5,10]
In continuation of this work, we now report the preparation
of a N-Fmoc-protected galactosylated (2S,4R)-4-hydroxyl-
ysine derivative and its incorporation at position 264 of the
CII(256–270) peptide.
Results and Discussion
Our general approach to 4-hydroxylysine derivatives suit-
ably protected for subsequent glycosylation and solid-phase
synthesis using Fmoc chemistry started with di-tert-butyl
(2S,4S)-4-hydroxy-6-oxo-1,2-piperidinedicarboxylate
(Scheme 1).
(3)
Scheme 2. Reagents and conditions: (a) TBAF, THF; (b) TBAF,
AcOH, THF.
preparation of 5-hydroxylysine derivatives [i.e., p-tolu-
enesulfonic acid (PTSA) in acetonitrile] resulted in quanti-
tative lactonization to give PTSA salt 9 (Scheme 3).
Scheme 1. Reagents and conditions: (a) TBDMSCl, imidazole,
CH2Cl2; (b) NaBH4, EtOH; (c) MsCl, DIPEA, CH2Cl2; (d) NaN3,
DMF.
Diastereomerically pure 3 was prepared in three steps
from Boc-Asp-OtBu in an overall yield of 63%, as de-
scribed previously.[10] Protection of the secondary alcohol
prior to ring-opening with NaBH4 in EtOH was mandatory
for a successful and high-yielding conversion into the corre-
Scheme 3. Reagents and conditions: (a) PTSA, CH3CN; (b)
FmocOSu, K2CO3, acetone/H2O.
Similarly, treatment of the O-protected derivative 6 under
sponding 1,3-diol. This is in contrast to (2S,5S)-5-hydroxy- the same conditions also gave 9 quantitatively. The N-
6-oxo-1,2-piperidinedicarboxylate (5-hydroxylysine precur- Fmoc-protected lactone 10 was easily obtained in 92% yield
sor) which was readily and selectively reduced to the corre- by reaction of 9 with FmocOSu in the presence of aqueous
sponding 1,2-diol under the same conditions.[5] The primary K2CO3 as base. We then envisioned that N-protected γ-lac-
alcohol 5 was then converted into azide 6 in 86% yield. tones 7 and 10, subject to selective ring-opening with a gly-
The azide moiety, which can be selectively reduced to the cyl ester derivative {Gly is the amino acid residue immedi-
corresponding primary amine on a solid-phase support ately following Gal-5-Hyl in the sequence of the [Gal-5-
prior to cleavage of the peptide from the resin, is used as a Hyl264]CII(256–270) glycopeptide}, could be of practical
permanent protection for the ε-amino group. Our first at- value to generate 4-hydroxylysylglycyl dipeptide agly-
tempt to remove the TBDMS protection by treatment with cons.[13] The N-acylation of various glycyl esters by lactones
TBAF[11] resulted in spontaneous and quantitative forma- 7 and 10 has been examined and the results are reported in
tion of γ-lactone 7 (Scheme 2).
Table 1.
This side-reaction was completely reversed to the benefit
The Boc-protected γ-lactone 7 reacted readily with H-
of the desired 4-hydroxylysine derivative 8 by simply per- Gly-OtBu and H-Gly-OAll in Et2O to give the correspond-
forming the reaction in a mixture of acetic acid and THF ing N-Boc-protected (2S,4R)-4-hydroxylysylglycyl esters 11
as solvent.[12] However, attempts to selectively remove the and 12 in 65 and 63% yields, respectively.[13,14] Alterna-
N-Boc protecting group of 8 in the presence of the tert- tively, THF but not MeCN could be employed. Ring-open-
butyl ester under conditions previously developed for the ing was accompanied by some epimerization (18–25%
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Eur. J. Org. Chem. 2008, 1005–1012

A Galactosylated 4-Hydroxylysine-Containing Glycopeptide
Table 1. N-Acylation of glycyl esters by lactones 7 and 10.
Entry Lactone
R1
Reagents
R2
Solvent
THF
Time [d]
3
Product
Epimer ratio[a] % Yield[b]
1
7
HCl·H-Gly-OtBu,
Et3N
Boc
tBu
11
70:30
34 (43)[c,d]
2
3
4
5
6
7
8
7
7
Boc
Boc
Boc
Boc
Boc
Fmoc
Fmoc
H-Gly-OtBu
H-Gly-OtBu
H-Gly-OtBu
H-Gly-OAll
tBu
tBu
tBu
All
All
tBu
tBu
THF
Et2O
CH3CN
Et2O
THF
THF
7
7
7
7
7
4
2
11
11
11
12
12
13
13
75:25
78:22
–
63
65
7
0 (100)[c]
63
7
7
10
10
80:20
82:18
H-Gly-OAll
H-Gly-OtBu
H-Gly-OtBu, AlMe3
48
[e]
–
23
THF
–
0[f]
[a] Epimer ratio determined by analytical C18 RP-HPLC of the crude product. [b] Refers to the major (2S,4R) diastereomer after
purification. [c] Starting material recovered is given as a percentage in parentheses. [d] The minor isomer was isolated in 12% yield. [e]
Only one product was detected and characterized. [f] The starting material decomposed.
based on HPLC analysis of the crude product), probably at
Glycosylation of 13 and 14 with tetrapivaloylated galac-
the α-carbon of the 4-Hyl residue. The epimers were easily tosyl bromide under conditions optimized previously for
separated from (2S,4R)-11 and (2S,4R)-12 by flash column the galactosylation of 5-Hyl mimetics afforded 15 and 16 in
chromatography on silica gel. The N-Fmoc-protected lac- 91 and 86% yields, respectively. Removal of the tert-butyl
tone 10 was a poorer substrate and the linear dipeptide 13 ester in 15 gave galactosylated N-Fmoc-protected 4-hydroxy-
was only recovered in 23% yield. An attempt to increase lysylglycine dipeptide 17 in 93% yield. Similarly, deal-
the yield by conducting the reaction with AlMe3 was not lylation of 16 by treatment with Pd(PPh3)4 gave 17 in 89%
successful. Selective removal of the Boc protecting group of yield. With an overall yield of 31%, the route involving
11 and 12 followed by Fmoc reprotection gave protected ring-opening of lactone 7 with H-Gly-OAll is clearly more
dipeptide aglycons 13 and 14 in 43 and 95% yields, respec- efficient.[15] The N-Fmoc-protected Gal-4-Hyl-Gly building
tively (Scheme 4).
block 17 was next used for the solid-phase synthesis of gly-
copeptide 2. Peptide assembly was performed on a polystyr-
ene Wang resin[16] (20 mmol scale) by using a home-made
peptide synthesizer.[17] Glycosylated dipeptide 17 was cou-
pled with BOP in the presence of HOBt and DIEA. After
elongation of the peptide, the azido function was reduced
on the resin by treatment with triphenylphosphane (PPh3)
in THF/H2O for 72 h. Cleavage from the resin was per-
formed with TFA containing water and scavengers. At this
stage of the synthesis, the purity of the precursor of 2 bear-
ing a protected galactosyl moiety (18, see formula in Sup-
porting Information) was 92.7% based on analytical C18
RP-HPLC (see the electronic supporting information). An
additional step was necessary for the removal of the piva-
loyl ester protecting groups. To avoid epimerization of the
α-stereogenic centers of the amino acids along the peptide
chain, a diluted solution of NaOMe in MeOH (40 m) was
used. Monitoring the reaction by C18 RP-HPLC indicated
complete cleavage of all four pivaloyl groups after 16 h
without significant formation of degradation byproducts.
The purity of crude glycopeptide 2 was 75%. Finally, CII-
derived glycopeptide 2 was purified by RP-HPLC and reco-
vered in 63% yield based on the resin loading. The homo-
geneity of glycopeptide 2 was assessed by C18 RP-HPLC
(Ͼ99%) and its structure was confirmed by electrospray-
Scheme 4. Reagents and conditions: (a) PTSA, CH3CN; (b) Fmoc-
OSu, K2CO3, acetone/H2O; (c) TFA, CH2Cl2; (d) FmocOSu,
NaHCO3, THF/H2O; (e) β--Gal(Piv)4Br, silver silicate, 4-Å mol.
sieves, CH2Cl2; (f) TFA, CH2Cl2; (g) Pd(PPh3)4, morpholine, THF.
Eur. J. Org. Chem. 2008, 1005–1012
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J. Marin, J.-P. Briand, G. Guichard
FULL PAPER
1
Table 2. H NMR chemical shifts of glycopeptide 2 in [D6]DMSO after RP-HPLC purification.[a,b]
Residue
δ [ppm]
NH
αCH
βCH
γCH
Others
Gly 256
Glu 257
Hyp 258
Gly 259
Ile 260
Ala 261
Gly 262
Phe 263
4-Hyl 264
Gly 265
Glu 266
Gln 267
Gly268
n.d.
8.52
3.57, 3.51
4.64
4.36
3.77, 3.66
4.19 (4.19)
4.25 (4.26)
3.73
4.45 (4.49)
4.31
3.69
4.27 (4.27)
4.30
3.91, 3.61 (4.00, 3.82)
4.37 (4.46)
4.11 (4.17)
–
–
2.31
4.36
–
1.07, 1.38
–
–
–
3.35
–
2.26 (2.24)
2.14
–
–
1.92, 1.69
2.02, 1.88
–
1.72 (1.69)
1.21 (1.21)
–
2.98, 2.87 (3.06, 2.92)
2.01
–
1.91, 1.80 (1.92, 1.82)
1.92, 1.75
–
2.02, 1.88 (2.20, 1.96)
1.72, 1.58 (1.75, 1.62)
δCH2 3.72, 3.55
8.25
–
γ
7.65 (7.84)
8.12 (8.22)
8.00
8.13 (8.07)
8.39
7.96
7.86 (7.88)
8.13
δCH3 0.77; CH3 0.79
–
–
arom. 7.27, 6.75
εNH 6.95; CH2 2.95; CH2 1.75, 1.66
ε
δ
–
–
–
–
7.90 (7.96)
–
Pro 269
Lys 270
1.88 (1.78) δCH2 3.50, 3.55 (3.40, 3.47)
ε
δ
7.97 (8.17)
1.35
εNH n.d.; CH2 2.78; CH2 1.53
[a] Signals for a minor isomer are indicated in parentheses. [b] Chemical shifts for the galactosyl moiety at position 4-Hyl 264 in 2 are:
δ = 3.99 (1-H), 3.62 (3-H), 3.60, 3.56, 3.38, (6-H, 5-H, 4-H), 3.29 (2-H) ppm.
allowed to reach room temperature and stirred overnight. After
evaporation of the solvent, the residue was dissolved in AcOEt.
The solution was washed with 1  KHSO4, brine, and water, dried
with Na2SO4, filtered, and concentrated in vacuo. The crude prod-
uct was purified by filtration through a plug of silica gel (AcOEt/
Hex, 2:8) to yield 4 (650 mg, 95%). HPLC: tR = 18.16 min (linear
gradient, 30–100% B, 20 min); colorless oil. [α]D = –29.4 (c = 1.1,
CHCl3). 1H NMR (300 MHz, CDCl3): δ = 4.48 (dd, J = 7.6,
6.7 Hz, 1 H, NCH), 4.12–4.03 (m, 1 H, CHO), 2.74 (ddd, J = 16.6,
5.3, 1.8 Hz, 1 H, CH2CO), 2.49 (dd, J = 16.6, 8.5 Hz, 1 H,
ionization time-of-flight (ESI-TOF) mass spectrometry (see
the electronic supporting information) and 1H NMR analy-
sis (see Table 2).
In previous work we identified CII-reactive T cells [three
hybridomas (A8E2, A2G10, and A9E5) and a recurrent
pathogenic CD4+ T cell clone (A9.2)] in DBA/1 (H-2q)
mice immunized with bovine CII (bCII) that participate in
CIA course and regulation.[18,19] We also showed previously
that the hybridomas and the T-cell clone expressed closely
related T-cell receptors (TCR) and recognized glycosylated CH2CO), 2.37–2.28 (m, 1 H, NCHCH2), 2.08–1.96 (m, 1 H,
NCHCH2), 1.50 [s, 9 H, C(CH3)3], 1.46 [s, 9 H, C(CH3)3], 0.88 [s,
9 H, SiC(CH3)3], 0.07 (s, 3 H, SiCH3), 0.05 (s, 3 H, SiCH3) ppm.
13C NMR (100 MHz, CDCl3): δ = 170.0 (C), 168.9 (C), 152.0 (C),
83.5 (C), 82.2 (C), 64.4 (CH), 56.6 (CH), 44.4 (CH2), 34.9 (CH2),
27.9 (3 CH3), 27.9 (3 CH3), 25.8 (3 CH3), 18.1 (C), –4.7 (CH3),
–4.8 (CH3) ppm. C21H39NO6Si (429.25): calcd. C 58.71, H 9.15, N
3.26; found C 59.01, H 9.14, N 3.46.
peptides comprising the immunodominant bCII (256–270)
epitope. The capacity of these cells to recognize glycopep-
tide 2 containing 4-Hyl instead of 5-Hyl substitution at po-
sition 264 will be reported in a forthcoming study.
Experimental Section
tert-Butyl (2S,4S)-2-[(tert-Butoxycarbonyl)amino]-4-{[tert-butyl(di-
methyl)silyl]oxy}-6-hydroxyhexanoate (5): Compound 4 (500 mg,
1.16 mmol) was dissolved in EtOH (10.0 mL) and cooled to 0 °C.
After portionwise addition of NaBH4 (220 mg, 5.82 mmol), the
mixture was allowed to reach room temperature and stirred over-
night. After being quenched by water, the mixture was stirred for
a further 10 min. Evaporation of the solvent gave an oil which was
dissolved in AcOEt. The solution was washed with water and dried
with Na2SO4. Evaporation of the filtrate afforded an oil which was
filtered through a silica pad (AcOEt/Hex, 1:1). Pure 5 was reco-
vered (440 mg, 87%). HPLC: tR = 16.12 min (linear gradient, 30–
100% B, 20 min); colorless oil. [α]D = –2.9 (c = 1.0, CHCl3). 1H
NMR (300 MHz, CDCl3): δ = 5.26 (br. d, J = 7.3 Hz, 1 H, NH),
4.16–4.08 (m, 1 H, CHO), 4.08–4.00 (m, 1 H, NHCH), 3.82–3.68
(m, 2 H, CH2OH), 2.22 (br. s, 1 H, OH), 1.98–1.63 [m, 4 H,
CH2CH(OH)CH2] 1.45 [s, 9 H, C(CH3)3], 1.42 [s, 9 H, C(CH3)3],
0.90 [s, 9 H, SiC(CH3)3], 0.09 (s, 6 H, SiCH3) ppm. 13C NMR
(100 MHz, CDCl3): δ = 172.0 (C), 155.3 (C), 81.6 (C), 79.5 (C),
68.5 (CH), 59.5 (CH2), 52.0 (CH), 38.8 (CH2), 38.4 (CH2), 28.3 (3
CH3), 27.9 (3 CH3), 25.8 (3 CH3), 17.9 (C), –4.5 (CH3), –4.8
(CH3) ppm. C21H43NO6Si (433.29): calcd. C 58.16, H 9.99, N 3.23;
found C 57.89, H 10.35, N 3.23.
General Methods: Amino acid derivatives were purchased from
NeoMPS (Strasbourg, France). THF was distilled from Na/benzo-
phenone. CH2Cl2 and cyclohexane were distilled from CaH2. Thin-
layer chromatography (TLC) was performed on silica gel 60 F254
(Merck) with detection by UV light and charring with 1% (w/w)
ninhydrin in ethanol followed by heating. Flash column
chromatography was carried out on silica gel (0.063–0.200 nm).
HPLC analysis was performed on a Nucleosil C18 column (5 µm,
3.9ϫ150 mm) by using a linear gradient of A (0.1% TFA in H2O)
and B (0.08% TFA in CH3CN) at a flow rate of 1.2 mLmin–1 with
UV detection at 214 nm. Optical rotations were recorded with a
Perkin–Elmer polarimeter. 1H and 13C NMR spectra were recorded
by using Bruker Advance DPX-300 and DPX-400 instruments.
Mass spectra were recorded by using an ESI-TOF spectrometer
(Bruker microTOF). Tetrapivaloylated galactosyl bromide was pre-
pared by a two-step procedure starting from commercial -galac-
tose.[20] Silver silicate was prepared according to the reported pro-
cedure[21] and dried at 110 °C under high-vacuum for 6 h before
use.
Di-tert-butyl (2S,4S)-4-{[tert-Butyl(dimethyl)silyl]oxy}-6-oxo-1,2-
piperidinedicarboxylate (4): Imidazole (430 mg, 6.32 mmol) and
TBDMSCl (476 mg, 3.16 mmol) were added to a solution of 3
(500 mg, 1.58 mmol) in CH2Cl2 (5.0 mL) at 0 °C. The mixture was
tert-Butyl (2S,4R)-6-Azido-2-[(tert-butoxycarbonyl)amino]-4-{[tert-
butyl(dimethyl)silyl]oxy}hexanoate (6): N,N-Diisopropylethylamine
1008
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Eur. J. Org. Chem. 2008, 1005–1012

A Galactosylated 4-Hydroxylysine-Containing Glycopeptide
(DIPEA) (194 µL, 1.14 mmol) was added to a solution of 5
(330 mg, 0.76 mmol) in CH2Cl2 (5.0 mL) at ambient temperature.
The solution was cooled to 0 °C and MsCl (88 µL, 1.14 mmol) was
added through a hypodermic syringe. The mixture was allowed to
reach room temperature and stirred for 3 h. The reaction was
quenched by water, CH2Cl2 was evaporated and replaced by Ac-
OEt. The organic phase was washed with 1  KHSO4, brine, satu-
rated NaHCO3, and water, dried with Na2SO4, filtered, and con-
centrated in vacuo. The residue was dissolved in DMF (10.0 mL).
NaN3 (148 mg, 2.28 mmol) was added to the solution which was
heated at 80 °C for 8 h. After the solution had cooled to room
temperature, water was added, and the solution was extracted twice
with AcOEt. The combined organic layers were washed with water,
dried with Na2SO4, filtered, and the solvents evaporated in vacuo.
The crude product was purified by flash column chromatography
(AcOEt/Hex, 1:9) to yield pure 6 (298 mg, 86% for two steps).
HPLC: tR 16.80 (linear gradient, 50–100% B, 20 min); colorless oil.
[α]D = +1.0 (c = 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ =
5.22 (br. d, J = 7.1 Hz, 1 H, NH), 4.18–4.12 (m, 1 H, NHCH),
3.97–3.89 (m, 1 H, CHO), 3.36 (m, 2 H, CH2N3), 1.94–1.83 (m, 1
H, NHCHCH2), 1.81–1.68 (m, 3 H, NHCHCH2, CH2CH2N3);
1.46 [s, 9 H, C(CH3)3], 1.43 [s, 9 H, C(CH3)3], 0.90 [s, 9 H,
SiC(CH3)3], 0.09 (s, 3 H, SiCH3), 0.07 (s, 3 H, SiCH3) ppm. 13C
NMR (100 MHz, CDCl3): δ = 171.8 (C), 155.3 (C), 81.7 (C), 79.5
(C), 67.0 (CH), 51.9 (CH), 47.5 (CH2), 39.1 (CH2), 35.8 (CH2),
28.3 (3 CH3), 27.9 (3 CH3), 25.8 (3 CH3), 17.9 (C), –4.5 (CH3),
–4.8 (CH3) ppm. C21H42N4O5Si (458.29): calcd. C 54.99, H 9.23,
N 12.22; found C 55.23, H 9.30, N 12.44.
13C NMR (100 MHz, CDCl3): δ = 171.7 (C), 157.0 (C), 82.5 (C),
80.6 (C), 64.1 (CH), 51.0 (CH), 48.3 (CH2), 42.2 (CH2), 35.6 (CH2),
28.2 (3 CH3), 27.9 (3 CH3) ppm. C15H28N4O5 (344.21): calcd. C
52.31, H 8.19, N 16.27; found C 52.59, H 8.14, N 16.28.
(3S,5R)-5-(Azidoethyl)-2-oxotetrahydro-3-furanaminium 4-Methyl-
1-benzenesulfonate (9): p-Toluenesulfonic acid (55 mg, 0.289 mmol)
was added to a solution of 8 (50 mg, 0.145 mmol) in CH3CN
(2.0 mL) at 0 °C. The mixture was stirred for 6 h (until complete
consumption of 8). A large volume of cold Et2O was added and
the suspension was filtered. The precipitate was washed with
CH2Cl2/Et2O and dried under high vacuum to afford pure 9
(50 mg, 100%): TLC: Rf = 0.45 (AcOEt/pyridine/acetic acid/water,
8:2:0.5:1); white solid. [α]D = +25.4 (c = 1.0, MeOH); m.p. 234–
1
240 °C (decomposition). H NMR (300 MHz, CD3OD): δ = 7.70
(d, J = 8.0 Hz, 2 H, arom. H), 7.23 (d, J = 7.9 Hz, 2 H, arom. H),
4.72–4.63 (m, 1 H, CHO), 4.40 (dd, J = 8.7, 12.0 Hz, 1 H, CHCO),
3.55–3.45 (m, 1 H, CH2N3), 2.87–2.78 (m, 1 H, CHCH2CH), 2.37
(s, 3 H, CH3Ph), 2.03–1.91 (m, 3 H, CHCH2CH, CH2CH2N3) ppm.
13C NMR (100 MHz, CDCl3): δ = 170.0 (C), 138.6 (C), 126.8 (2
CH), 124.0 (2 CH), 74.4 (CH), 47.6 (CH), 32.2 (CH2), 31.4 (CH2),
18.3 (CH3) ppm. C13H18N4O5S (342.10): calcd. C 45.61, H 5.30, N
16.36; found C 45.76, H 5.31, N 16.19.
[(9H-Fluoren-9-yl)methyl] N-[(3S,5R)-5-(Azidoethyl)-2-oxotetrahy-
dro-3-furanyl]carbamate (10): Compound 9 (75 mg, 0.219 mmol)
was dissolved in H2O (1.0 mL). Solid K2CO3 (90 mg, 0.651 mmol)
and FmocOSu (110 mg, 0.326 mmol) dissolved in acetone (1.0 mL)
were added to the solution stirred at ambient temperature. After
3 h, acetone was evaporated and replaced by AcOEt. The solution
was washed with 1  KHSO4, dried with Na2SO4, and concentrated
in vacuo. Purification of the crude product by flash column
chromatography (AcOEt/Hex, 1:1) gave pure 10 (79 mg, 92%).
HPLC: tR = 12.47 min (linear gradient, 30–100% B, 20 min); white
tert-Butyl
N-[(3S,5R)-5-(Azidoethyl)-2-oxotetrahydro-3-furanyl]-
carbamate (7): Tetrabutylammonium fluoride (543 mg, 2.08 mmol)
was added to a solution of 6 (250 mg, 0.429 mmol) in THF
(20.0 mL) at 0 °C. The mixture was allowed to reach room temp.
and stirred for 2 h. The reaction was quenched by adding a satu-
rated solution of NH4Cl. THF was evaporated and replaced by
AcOEt. The organic layer was washed with 1  KHSO4, brine, and
saturated NaHCO3, dried with Na2SO4, filtered, and concentrated
in vacuo. Crude 7 was filtered through a short plug of silica gel
1
solid. [α]D = +48.0 (c = 1.0, CHCl3); m.p. 132–134 °C. H NMR
(300 MHz, CDCl3): δ = 7.76 (d, J = 7.4 Hz, 2 H, arom. H), 7.58
(d, J = 7.4 Hz, 2 H, arom. H), 7.40 (t, J = 7.4 Hz, 2 H, arom. H),
7.31 (t, J = 7.4 Hz, 2 H, arom. H), 5.48 (br. d, J = 4.8 Hz, 1 H,
NH), 4.51–4.43 (m, 2 H, NHCH, CHO), 4.42 (d, J = 6.6 Hz, 2 H,
CH2OCO), 4.21 (t, J = 6.6 Hz, 1 H, CHCH2OCO), 3.51–3.47 (m,
2 H, CH2N3), 2.84 (m, 1 H, NHCHCH2), 2.04–1.83 (m, 1 H,
NHCHCH2), 1.80–1.59 (m, 2 H, CH2CH2N3)2 ppm. 13C NMR
(100 MHz, CDCl3): δ = 174.0 (C), 155.9 (C), 143.6 (2 C), 141.3 (2
C), 127.8 (2 CH), 127.1 (2 CH), 125.0 (2 CH), 120.0 (2 CH), 74.9
(CH), 67.3 (CH2), 51.6 (CH), 47.4 (CH2), 47.0 (CH), 36.1 (CH2),
34.5 (CH2) ppm. HRMS (ESI): calcd. for C21H21N4O4 [M + H]+
393.1557; found: 393.1549.
(AcOEt/Hex, 1:1) to afford pure 7 (281 mg, 75%). HPLC: tR
7.56 min (linear gradient, 30–100% B, 20 min); colorless oil. [α]D
=
=
+57.9 (c = 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ = 5.12 (br.
s, 1 H, NH), 4.56–4.46 (m, 1 H, CHO), 4.42–4.35 (m, 1 H, NHCH),
3.53–3.48 (m, 2 H, CH2N3), 2.89–2.79 (m, 1 H, NHCHCH2),
2.02–1.81 (m, 3 H, NHCHCH2, CH2CH2N3), 1.44 [s, 9 H, C-
(CH3)3] ppm. 13C NMR (100 MHz, CDCl3): δ = 174.3 (C), 155.3
(C), 80.6 (C), 74.8 (CH), 51.3 (CH), 47.4 (CH2), 36.5 (CH2), 34.6
(CH2), 28.2 (3 CH3) ppm. C11H18N4O4 (270.13): calcd. C 48.88, H
6.71, N 20.73; found C 49.15, H 6.72, N 20.66.
tert-Butyl
2-({(2S,4R)-6-Azido-2-[(tert-butoxycarbonyl)amino]-4-
tert-Butyl
(2S,4R)-6-Azido-2-[(tert-butoxycarbonyl)amino]-4-hy-
hydroxyhexanoyl}amino)acetate [(2S,4R)-11]: Lactone 7 (245 mg,
0.906 mmol) was placed in a 50 mL round-bottomed flask and dis-
solved in Et2O (1.0 mL). HCl·H-Gly-OtBu (761 mg, 4.54 mmol)
was washed with a 1  NH4OH solution (50.0 mL) and extracted
twice with CH2Cl2 (25.0 mL). The resulting residue was dissolved
in Et2O (3.0 mL) and added to the previous solution through a
hypodermic syringe. The solution was stirred at room temperature
and the reaction was monitored by TLC (AcOEt/Hex, 1:1). After
1 week, 7 had completely disappeared. The remaining solvent was
evaporated and replaced by AcOEt. The resulting solution was
washed with 1  KHSO4, brine, and water and the solvents evapo-
rated in vacuo to give crude 11. The crude product was purified by
flash column chromatography (AcOEt/Hex, 1:1) to yield (2S,4R)-
droxyhexanoate (8): Acetic acid (58 µL, 1.00 mmol) was added to a
solution of 6 (92 mg, 0.200 mmol) in THF (2.0 mL) at 0 °C. TBAF
(262 mg, 1.00 mmol) was added and the solution was stirred for
96 h at room temp. The reaction was quenched by adding a satu-
rated solution of NH4Cl. THF was evaporated and replaced by
AcOEt. The organic layer was washed with 1  KHSO4, brine, and
saturated NaHCO3, dried with Na2SO4, filtered, and concentrated
in vacuo. Purification of the crude product by flash column
chromatography (AcOEt/Hex, 2:8) gave pure 8 (66 mg, 96%).
HPLC: tR = 10.93 min (linear gradient, 30–100% B, 20 min); color-
less oil. [α]D = +15.1 (c = 1.1, CHCl3). 1H NMR (300 MHz,
CDCl3): δ = 5.39 (br. d, J = 7.7 Hz, 1 H, NH), 4.41–4.33 (m, 2 H,
NHCH, OH), 3.70 (br. t, J = 9.7 Hz, 1 H, CHOH), 3.50–3.37 (m, 11 (237 mg, 65%). HPLC: tR = 8.77 min (linear gradient, 30–100%
1
2 H, CH2N3), 1.90–1.80 (m, 1 H, NHCHCH2), 1.76–1.57 (m, 3 H, B, 20 min); colorless oil. [α]D = +14.5 (c = 1.0, CHCl3). H NMR
CH2CH2N3), 1.44 [s, 9 H, C(CH3)3], 1.43 [s, 9 H, C(CH3)3] ppm. (300 MHz, CDCl3): δ = 6.88 (m, 1 H, NHCH2), 5.70 (d, J = 7.9 Hz,
Eur. J. Org. Chem. 2008, 1005–1012
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1009

J. Marin, J.-P. Briand, G. Guichard
FULL PAPER
1 H, NHCHCO), 4.46–4.41 (m, 1 H, NHCHCO), 3.92 (d, J = 1.62 (m, 2 H, CH2CH2N3), 1.41 [s, 9 H, C(CH3)3] ppm. 13C NMR
5.1 Hz, 2 H, NHCH2CO), 3.81 (m, 1 H, CHOH), 3.51–3.37 (m, 2 (100 MHz, CDCl3): δ = 172.6 (C), 169.5 (C), 155.7 (C), 131.4 (CH),
H, CH2N3), 1.87–1.64 (m, 4 H, NHCHCH2, CH2CH2N3), 1.45 [s, 119.0 (CH2), 80.6 (C), 66.1 (CH2), 65.3 (CH), 51.5 (CH), 48.3
9 H, C(CH3)3], 1.43 [s, 9 H, C(CH3)3] ppm. 13C NMR (100 MHz, (CH2), 41.9 (CH2), 41.2 (CH2), 36.4 (CH2), 29.7 (CH2), 28.3 (3
CDCl3): δ = 171.9 (C), 168.9 (C), 156.7 (C), 82.5 (C), 80.5 (C), 65.1
CH3) ppm. HRMS (ESI): calcd. for C16H28N5O6 [M + H]+
(CH), 51.5 (CH), 48.4 (CH2), 42.0 (CH2), 41.9 (CH2), 35.9 (CH2), 386.2034; found 386.2034.
28.2 (3 CH3), 28.0 (3 CH3) ppm. HRMS (ESI): calcd. for
tert-Butyl 2-{[(2S,4R)-6-Azido-2-{[(9H-fluoren-9-ylmethoxy)-
C17H32N5O6 [M + H]+ 402.2347; found 402.2338.
carbonyl]amino}-4-hydroxyhexanoyl]amino}acetate (13): p-Tolu-
enesulfonic acid (95 mg, 0.499 mmol) was added to a solution of
(2S,4R)-11 (100 mg, 0.249 mmol) in CH3CN (2.0 mL) at 0 °C. The
mixture was stirred for 7 h [until complete consumption of (2S,4R)-
11]. The reaction was quenched by the addition of aqueous 1 
NH4OH (20.0 mL). The solution was extracted with CH2Cl2
(20.0 mL, 2ϫ10.0 mL) and the combined organic layers were dried
with Na2SO4, filtered, and concentrated in vacuo. The residue was
dissolved in acetone (3.0 mL) and the same volume of water was
added to the solution. Solid K2CO3 (103 mg, 0.745 mmol) and a
solution of FmocOSu (126 mg, 0.373 mmol) in acetone (1.0 mL)
were added to the solution which was stirred at room temperature
for 3 h. Acetone was evaporated and replaced by AcOEt. The solu-
tion was washed with a saturated NaHCO3 solution, brine, and 1 
KHSO4, dried with Na2SO4, and concentrated in vacuo. The crude
product was purified by flash column chromatography (AcOEt/
Hex, 6:4) to yield pure 13 (56 mg, 43% for two steps). HPLC: tR
= 12.71 min (linear gradient, 30–100% B, 20 min); colorless oil.
[α]D = +6.8 (c = 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ =
7.76 (d, J = 7.5 Hz, 2 H, arom. H), 7.58 (m, 2 H, arom. H), 7.39
(t, J = 7.5 Hz, 2 H, arom. H), 7.30 (t, J = 7.5 Hz, 2 H, arom. H),
6.95 (br. t, J = 5.0 Hz, 1 H, NHCH2CO), 6.08 (br. d, J = 7.9 Hz,
1 H, NHCHCO), 4.56–4.48 (m, 1 H, NHCHCO), 4.46–4.35 (m, 2
H, CH2OCO), 4.20 (t, J = 6.9 Hz, 1 H, CHCH2OCO), 3.93 (d, J
= 4.9 Hz, 2 H, NHCH2CO), 3.86–3.80 (m, 1 H, CHOH), 3.46–3.41
(m, 2 H, CH2N3), 1.86–1.79 (m, 2 H, NHCHCH2), 1.74–1.63 (m, 2
H, CH2CH2N3), 1.45 [s, 9 H, C(CH3)3] ppm. 13C NMR (100 MHz,
CDCl3): δ = 171.7 (C), 168.9 (C), 157.2 (C), 143.7 (C), 143.6 (C),
141.3 (2 C), 82.6 (C), 67.4 (CH2), 65.5 (CH), 52.1 (CH), 48.3 (CH2),
47.1 (CH), 42.0 (CH2), 41.7 (CH2), 36.0 (CH2), 28.0 (3 CH3) ppm.
tert-Butyl 2-({(2R,4R)-6-Azido-2-[(tert-butoxycarbonyl)amino]-4-
hydroxyhexanoyl}amino)acetate (epi-11): The diastereomer epi-11
was obtained from crude 11 after purification by flash column
chromatography (yield: 12%). HPLC: tR = 8.37 min (linear gradi-
ent, 30–100 % B, 20 min); colorless oil. 1H NMR (300 MHz,
CDCl3): δ = 6.99 (m, 1 H, NHCH2), 5.44 (d, J = 7.7 Hz, 1 H, Boc-
NH), 4.38 (m, 1 H, NHCHCO), 4.01 (dd, J = 5.7, 18.1 Hz, 1 H,
NHCH2CO), 3.90–3.87 (m, 1 H, CHOH), 3.86 (dd, J = 4.9,
18.1 Hz, 1 H, NHCH2CO), 3.46 (t, J = 6.4 Hz, 2 H, CH2N3), 1.
92–1.85 (m, 2 H, NHCHCH2); 1.75–1.67 (m, 2 H, CH2CH2N3),
1.45 [s, 9 H, C(CH3)3], 1.43 [s, 9 H, C(CH3)3] ppm. 13C NMR
(100 MHz, CDCl3): δ = 172.4 (C), 168.85 (C), 155.6 (C), 82.5 (C),
80.4 (C), 66.2 (CH), 52.0 (CH), 48.4 (CH2), 41.9 (CH2), 39.87
(CH2), 36.3 (CH2), 28.3 (3 CH3), 28.0 (3 CH3) ppm. HRMS (ESI):
calcd. for C17H32N5O6 [M + H]+ 402.2347; found 402.2347.
Allyl 2-({(2S,4R)-6-Azido-2-[(tert-butoxycarbonyl)amino]-4-hy-
droxyhexanoyl}amino)acetate [(2S,4R)-12]: Lactone 7 (187 mg,
0.692 mmol) was placed in a 50 mL round-bottomed flask and dis-
solved in THF (1.0 mL). TFA·H-Gly-OAll (793 mg, 3.46 mmol)
was washed with a 1  NH4OH solution (50.0 mL) and extracted
twice with CH2Cl2 (25.0 mL). After evaporation to dryness, the
resulting residue was dissolved in THF (2.0 mL) and added to the
previous solution through a hypodermic syringe. The white suspen-
sion was stirred at room temperature and the reaction was moni-
tored by TLC (AcOEt/Hex, 1:1). After 1 week, 7 had completely
disappeared. THF was evaporated and replaced by AcOEt. The
solution was washed with 1  KHSO4, brine, and water and the
solvents evaporated in vacuo. The crude product was purified by
flash column chromatography (AcOEt/Hex, 1:1) to yield (2S,4R)-
12 (128 mg, 48%). HPLC: tR = 7.57 min (linear gradient, 30–100%
Allyl 2-{[(2S,4R)-6-Azido-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]-
amino}-4-hydroxyhexanoyl]amino}acetate (14): The dipeptide
(2S,4R)-12 was dissolved in CH2Cl2 (1.0 mL) and TFA (2.0 mL)
was added to the resulting solution. The mixture was stirred at
room temperature for 2 h. The solvents were evaporated in vacuo
and coevaporated with hexane and the residue was dried under
high vacuum. The residue was dissolved in THF (1.0 mL) and the
same volume of water was added to the solution. Solid NaHCO3
(39 mg, 0.464 mmol) and a solution of FmocOSu (79 mg,
0.234 mmol) in THF (1.0 mL) was added to the solution which
was stirred at room temperature for 3 h. THF was evaporated and
replaced by AcOEt. The solution was washed with a saturated
NaHCO3 solution, brine, and 1  KHSO4, dried with Na2SO4, and
concentrated in vacuo. The crude product was purified by flash
column chromatography (AcOEt/Hex, 6:4) to yield pure 14 (75 mg,
1
B, 20 min); colorless oil. [α]D = +11.7 (c = 1.1, CHCl3). H NMR
(300 MHz, CDCl3): δ = 7.23 (br. t, J = 5.3 Hz, 1 H, NHCH2),
5.96–5.83 (m, 1 H, CH=CH2), 5.71 (br. d, J = 7.7 Hz, 1 H,
NHCH), 5.36–5.24 (m, 2 H, CH=CH2), 4.64 (dt, J = 5.8, 1.3 Hz,
2 H, CH2CH=CH2), 4.48–4.43 (m, 1 H, NHCHCO), 4.13–3.95 (m,
2 H, NHCH2CO), 3.80 (m, 1 H, CHOH), 3.50–3.35 (m, 2 H,
CH2N3), 1.88–1.79 (m, 1 H, NHCHCH2), 1.76–1.64 (m, 3 H,
CH2CH2N3), 1.43 [s, 9 H, C(CH3)3] ppm. 13C NMR (100 MHz,
CDCl3): δ = 172.2 (C), 169.5 (C), 156.8 (C), 131.3 (CH), 119.1
(CH2), 80.7 (C), 66.2 (CH2), 65.2 (CH), 51.5 (CH), 48.3 (CH2),
41.9 (CH2), 41.2 (CH2), 35.9 (CH2), 29.7 (CH2), 28.2 (3 CH3) ppm.
HRMS (ESI): calcd. for C16H28N5O6 [M + H]+ 386.2034; found
386.2027.
Allyl 2-({(2R,4R)-6-Azido-2-[(tert-butoxycarbonyl)amino]-4- 95% for two steps). HPLC: tR = 12.13 min (linear gradient, 30–
hydroxyhexanoyl}amino)acetate (epi-12): The diastereomer epi-12
was obtained from crude 12 after purification by flash column
chromatography. epi-12 could not be isolated in pure form and was
contaminated by ca. 15% (2S,4R)-12. HPLC: tR = 7.11 min (linear
gradient, 30–100% B, 20 min); colorless oil. H NMR (300 MHz,
CDCl3): δ = 7.36 (m, 1 H, NHCH2), 5.95–5.82 (m, 1 H, CH=CH2),
100% B, 20 min); colorless oil. [α]D = +4.9 (c = 0.9, CHCl3). 1H
NMR (300 MHz, CDCl3): δ = 7.75 (d, J = 7.5 Hz, 2 H, arom. H),
7.57 (m, 2 H, arom. H), 7.40 (t, J = 7.5 Hz, 2 H, arom. H), 7.36
(tt, J = 7.5, 1.0 Hz, 2 H, arom. H), 7.12 (m, 1 H, NHCH2CO),
6.08 (br. d, J = 7.7 Hz, 1 H, NHCHCO), 5.95–5.82 (m, 1 H,
CH=CH2), 5.35–5.22 (m, 2 H, CH=CH2), 4.62 (d, J = 5.8 Hz, 2
1
5.58 (m, 1 H, NHCH), 5.35–5.22 (m, 2 H, CH=CH2), 4.62 (dt, J H, CH2CH=CH2), 4.58–4.51 (m, 1 H, NHCHCO), 4.46–4.38 (m,
= 5.8, 1.4 Hz, 2 H, CH2CH=CH2), 4.44 (m, 1 H, NHCHCO), 4.17– 2 H, CH2OCO), 4.20 (t, J = 6.9 Hz, 1 H, CHCH2OCO), 4.07–4.03
3.93 (m, 2 H, NHCH2CO), 3.94–3.84 (m, 1 H, CHOH), 3.44 (t, J (m, 2 H, NHCH2CO), 3.88–3.78 (m, 1 H, CHOH), 3.46–3.41 (m,
= 6.7 Hz, 2 H, CH2N3), 1.96–1.78 (m, 2 H, NHCHCH2), 1.74– 2 H, CH2N3), 1.91–1.79 (m, 2 H, NHCHCH2), 1.74–1.63 (m, 2 H,
1010
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1005–1012

A Galactosylated 4-Hydroxylysine-Containing Glycopeptide
CH2CH2N3) ppm. 13C NMR (100 MHz, CDCl3): δ = 171.9 (C),
169.4 (C), 157.2 (C), 143.7 (C), 143.5 (C), 141.3 (2 C), 131.3 (CH),
C), 141.3 (2 C), 131.6 (CH), 127.7 (2 CH), 127.2 (2 CH), 125.3 (2
CH), 120.0 (2 CH), 118.9 (CH2), 99.7 (CH), 74.9 (CH), 71.3 (CH),
127.8 (2 CH), 127.1 (2 CH), 125.0 (2 CH), 120.0 (2 CH), 119.2 70.8 (CH), 68.9 (CH), 67.3 (CH2), 66.5 (CH2), 65.9 (CH), 60.6
(CH2), 67.4 (CH2), 66.2 (CH2), 65.7 (CH), 52.2 (CH), 48.4 (CH2), (CH2), 52.2 (CH), 48.4 (CH2), 47.1 (CH), 41.6 (CH2), 41.3 (CH2),
47.1 (CH), 41.6 (CH2), 41.3 (CH2), 35.9 (CH2), 29.7 (CH2) ppm. 39.0 (C), 38.9 (C), 38.8 (C), 38.7 (C), 33.8 (CH2), 30.3 (CH2), 27.2
HRMS (ESI): calcd. for C26H30N5O6 [M + H]+ 508.2191; found
508.2186.
(3 CH3), 27.1 (9 CH3) ppm. HRMS (ESI): calcd. for C52H72N5O15
[M + H]+ 1006.5019; found 1006.5011.
tert-Butyl 2-{[(2S,4R)-6-Azido-2-{[(9H-fluoren-9-ylmethoxy)carbon-
yl]amino}-4-O-(2,3,4,6-tetra-O-pivaloyl-β-D-galactopyranosyl)-
2-{[(2S,4R)-6-Azido-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-
4-O-(2,3,4,6-tetra-O-pivaloyl-β-D-galactopyranosyl)hexanoyl]-
hexanoyl]amino}acetate [Fmoc-(GalPiv4)(4R)Hyl-Gly-OtBu (15)]:
The N-Fmoc-protected dipeptide 13 (56 mg, 0.107 mmol) and tet-
rapivaloylated galactosyl bromide (93 mg, 0.160 mmol) were placed
in an argon-filled round-bottomed flask and dissolved in CH2Cl2
(3.0 mL). Powdered 4-Å mol. sieves were added and the suspension
was stirred for 30 min. The mixture was protected from light and
silver silicate (200 mg) was added. The reaction was stirred for 8 h
at room temperature. The dark-brown suspension was filtered
through a plug of Celite® and the solvent was evaporated to give
a brownish residue which was purified by flash column chromatog-
amino}acetic Acid [Fmoc-(GalPiv4)(4R)Hyl-Gly-OH (17)]: From
the galactosylated dipeptide 15: 15 (80 mg, 0.078 mmol) was dis-
solved in CH2Cl2 (1.0 mL) and TFA (2.0 mL) was added to the
solution. The mixture was stirred at room temperature for 2 h. The
solvents were evaporated in vacuo and co-evaporated with hexane.
Crude 17 was purified by filtration through a short plug of silica
gel (AcOEt/Hex/AcOH, 5:5:0.1) to give pure 17 (70 mg, 93%).
From the galactosylated dipeptide 16: 16 (50 mg, 0.050 mmol) was
dissolved in THF (1.0 mL) and morpholine (22 µL, 0.250 mmol)
was added to the solution. Pd(PPh3)4 (5.8 mg, 0.005 mmol) was
added to the mixture which was stirred at room temperature for
2 h. THF was evaporated and replaced by AcOEt. The solution
was washed with 1  KHSO4 and brine, dried with Na2SO4, and
concentrated in vacuo. Crude 17 was purified by flash column
chromatography on silica gel (AcOEt/Hex/AcOH, 5:5:0.1) to give
raphy (Et2O/Pent, 7:3) to give pure 15 (100 mg, 91%). HPLC: tR
=
13.29 min (linear gradient, 60–100% B, 20 min); colorless oil. [α]D
= –8.4 (c = 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): δ = 7.76 (d,
J = 7.5 Hz, 2 H), 7.62 (m, 2 H), 7.39 (t, J = 7.5 Hz, 2 H), 7.32 (m,
2 H), 6.98 (m, 1 H), 6.29 (br. d, J = 7.1 Hz, 1 H), 5.42 (d, J =
2.9 Hz, 1 H), 5.29–5.23 (m, 1 H), 5.15 (dd, J = 10.4, 2.9 Hz, 1 H),
4.66 (d, J = 7.7 Hz, 1 H), 4.52 (m, 1 H), 4.44–4.25 (m, 3 H), 4.19–
4.07 (m, 3 H), 4.04–4.00 (m, 1 H), 3.97–3.95 (m, 2 H), 3.36 (m, 2
H), 2.17–1.99 (m, 2 H), 1.91–1.80 (m, 1 H), 1.77–1.65 (m, 1 H),
1.44 (s, 9 H), 1.18 (s, 9 H), 1.17 (s, 9 H), 1.15 (s, 9 H), 1.11 (s, 9
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 177.8 (C), 177.4 (C),
176.9 (C), 176.8 (C), 172.8 (C), 168.3 (C), 156.5 (C), 143.8 (C),
143.7 (C), 141.2 (2 C), 127.7 (2 CH), 127.1 (2 CH), 125.3 (2 CH),
119.9 (2 CH), 99.8 (CH), 82.5 (C), 74.4 (CH), 71.3 (CH), 70.8
(CH), 69.0 (CH), 67.6 (CH2), 66.4 (CH), 60.6 (CH2), 52.5 (CH),
47.1 (CH2), 47.0 (CH), 42.1 (CH2), 39.0 (C), 38.8 (2 C), 38.7 (C),
33.7 (CH2), 30.3 (CH2), 28.0 (3 CH3), 27.2 (3 CH3), 27.1 (3 CH3),
27.0 (6 CH3) ppm. HRMS (ESI): calcd. for C53H76N5O15
[M + H]+ 1022.5332; found 1022.5358.
1
pure 17 (52 mg, 89%); colorless oil. H NMR (300 MHz, CDCl3):
δ = 7.75 (d, J = 7.4 Hz, 2 H), 7.62 (m, 2 H), 7.38 (t, J = 7.4 Hz, 2
H), 7.35–7.27 (m, 2 H), 7.11 (m, 1 H), 6.22 (br. d, J = 6.9 Hz, 1
H), 5.41 (d, J = 3.1 Hz, 1 H), 5.25 (dd, J = 10.4, 7.6 Hz, 1 H), 5.15
(dd, J = 10.4, 2.8 Hz, 1 H), 4.64 (d, J = 7.5 Hz, 1 H), 4.52–4.46
(m, 1 H), 4.40–4.34 (m, 2 H), 4.30–4.23 (m, 1 H), 4.17–4.06 (m, 4
H), 4.02–3.98 (m, 2 H), 3.34 (m, 2 H), 2.07–1.79 (m, 3 H), 1.77–
1.48 (m, 1 H), 1.77–1.65 (m, 1 H), 1.17 (s, 9 H), 1.16 (s, 9 H), 1.15
(s, 9 H), 1.11 (s, 9 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 177.8
(C), 177.4 (C), 176.9 (C), 176.8 (C), 172.8 (C), 168.3 (C), 156.5 (C),
143.8 (C), 143.7 (C), 141.2 (2 C), 127.7 (2 CH), 127.1 (2 CH), 125.3
(2 CH), 119.9 (2 CH), 99.8 (CH), 82.5 (C), 74.4 (CH), 71.3 (CH),
70.8 (CH), 69.0 (CH), 67.6 (CH2), 66.4 (CH), 60.6 (CH2), 52.5
(CH), 47.1 (CH2), 47.0 (CH), 42.1 (CH2), 39.0 (C), 38.8 (2 C), 38.7
(C), 33.7 (CH2), 30.3 (CH2), 28.0 (3 CH3), 27.2 (3 CH3), 27.1 (3
CH3), 27.0 (6 CH3) ppm. HRMS (ESI): calcd. for C49H68N5O15 [M
+ H]+ 966.4706; found 966.4688.
Allyl 2-{[(2S,4R)-6-Azido-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]-
amino}-4-O-(2,3,4,6-tetra-O-pivaloyl-β-D-galactopyranosyl)-
hexanoyl]amino}acetate [Fmoc-(GalPiv4)(4R)Hyl-Gly-OAll (16)]:
The N-Fmoc-protected dipeptide 14 (54 mg, 0.106 mmol) and tet-
rapivaloylated galactosyl bromide (92 mg, 0.159 mmol) were placed
in an argon-filled round-bottomed flask and dissolved in CH2Cl2
(3.0 mL). Powdered 4-Å mol. sieves were added and the suspension
was stirred for 30 min. The mixture was protected from light and
silver silicate (150 mg) was added. The reaction was stirred for 8 h
at room temperature. The dark-brown suspension was filtered
through a plug of Celite® and the solvent was evaporated to give
a brownish residue which was purified by flash column chromatog-
Gly256-Glu-Hyp-Gly-Ile-Ala-Gly-Phe-(Gal)(4R)Hyl-Gly-Glu-Gln-
Gly-Pro-Lys270 (2): The glycopeptide 2 was synthesized on Wang
resin by using a homemade semiautomatic peptide synthesizer on
a 20 µmol scale. For each coupling step, the reactants were intro-
duced manually as a solution in DMF (2.0 mL). Nα-Fmoc amino
acids (5.0 equiv.) with standard side-chain protecting groups were
coupled twice by using BOP (5.0 equiv.), HOBt (5.0 equiv.), and
DIEA (10.0 equiv.) in DMF for 20 min. The galactosylated build-
ing block 17 (1.5 equiv.) was coupled twice by using BOP
(1.5 equiv.), HOBt (1.5 equiv.), and DIEA (3.0 equiv.) in DMF for
60 (1st coupling) and 20 min (2nd coupling). The washing of the
raphy (Et2O/Pent, 8:2) to give pure 16 (92 mg, 86%). HPLC: tR
=
11.78 min (linear gradient, 60–100% B, 20 min); colorless oil. [α]D
1
= –12.0 (c = 1.0, CHCl3). H NMR (300 MHz, CDCl3): δ = 7.76
(d, J = 7.4 Hz, 2 H), 7.61–7.64 (m, 2 H), 7.39 (t, J = 7.4 Hz, 2 H), resin and Fmoc deprotection (by using a freshly prepared solution
7.34–7.28 (m, 2 H), 7.06 (m, 1 H), 6.17 (br. d, J = 7.7 Hz, 1 H),
5.97–5.84 (m, 1 H), 5.41 (d, J = 3.1 Hz, 1 H), 5.32 (dq, J = 17.2,
of 20% piperidine in DMF) were performed automatically. The
coupling and deprotection steps were monitored by the Kaiser
1.4 Hz, 1 H), 5.26–5.22 (m, 2 H), 5.16 (dd, J = 10.4, 3.1 Hz, 1 H), test.[22] At the end of the elongation of the peptide chain, the resin
4.67 (d, J = 8.0 Hz, 1 H), 4.63 (d, J = 5.7 Hz, 2 H), 4.51–4.25 (m, was washed with CH2Cl2 and dried with Et2O. The resin was placed
4 H), 4.22–4.16 (m, 1 H), 4.14–4.06 (m, 2 H), 4.06–3.98 (m, 2 H),
3.36 (t, J = 6.8 Hz, 2 H), 2.15–2.04 (m, 2 H), 1.92–1.78 (m, 1 H),
1.77–1.68 (m, 1 H), 1.18 (s, 9 H), 1.16 (s, 9 H), 1.14 (s, 9 H), 1.11
(s, 9 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 177.7 (C), 177.2
in a syringe equipped with a frit and allowed to swell by addition
of THF (1.0 mL). A solution of PPh3 (10 equiv.) in a 3:1 mixture
of THF/H2O (4.0 mL) was added and the mixture was gently
shaken for 72 h. The resin was washed with THF and CH2Cl2
(C), 176.8 (C), 176.7 (C), 172.1 (C), 169.3 (C), 157.2 (C), 143.9 (2 and dried with Et2O.
A
mixture of TFA/H2O/TIPS/DTT
Eur. J. Org. Chem. 2008, 1005–1012
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1011

J. Marin, J.-P. Briand, G. Guichard
FULL PAPER
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(8.8:0.5:0.2:0.5, 10.0 mL) was added to the resin. The mixture was
gently shaken for 2.5 h and the resulting solution was flushed
through a frit with cold Et2O. The precipitate was recovered by
centrifugation, dissolved in a mixture of AcOH and H2O, and
freeze-dried. The resulting protected glycopeptide 18 (purity of
crude 18 estimated by RP-HPLC: 92.7%) was placed in a 50.0 mL
round-bottomed flask and dissolved in a freshly prepared 40 m
solution of NaOMe in MeOH (25.0 mL). The deprotection was
monitored by C18 RP-HPLC. After 16 h the solution was neutral-
ized by the dropwise addition of AcOH and MeOH was evaporated
in vacuo (purity of crude 2: 75%). Purification by semipreparative
C18 RP-HPLC gave the glycopeptide 2 (27.5 mg, 63%, Ͼ99% pu-
1
rity). HPLC: tR = 7.59 min (linear gradient, 5–65% B, 20 min). H
NMR (300 MHz, [D6]DMSO): see Table 2. (ESI-TOF): calcd. for
C71H113N18O28 [M + H]+ 1665.7966; found 1665.8012.
Supporting Information (see also the footnote on the first page of
this article): Formula of compound 18, C18 RP-HPLC profiles for
crude 18, crude 2, and purified 2; the high-resolution ESI-MS spec-
trum of pure 2, and 1H NMR spectra of compounds 7, 11, 12 and
17.
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[15] For early work on the use of allyl esters as protecting groups
in peptide and glycopeptide synthesis, see: H. Waldmann, H.
Kunz, Liebigs Ann. Chem. 1983, 1712–1725.
Acknowledgments
This research was supported in part by the Centre National de la
Recherche Scientifique (CNRS) and NeoMPS (PolyPeptide
Laboratories Group, Strasbourg). A BDI fellowship to J. M. from
NeoMPS and the CNRS is gratefully acknowledged. The authors
thank Roland Graff and Lionel Allouche for their assistance with
2D NMR measurements.
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Received: August 30, 2007
Published Online: January 2, 2007
1012
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1005–1012