Convenient synthesis of glycosylated hydroxylysine derivatives for use in solid-phase peptide synthesis

Article abstract of DOI:10.1016/S0040-4039(99)02281-9

δ-O-Glycosylated 9-fluorenylmethoxycarbonyl-hydroxylysine (Fmoc-Hyl) derivatives have been conveniently prepared by introduction of a β-D- galactopyranosyl group to copper-complexed Hyl[ε-tert-butyloxycarbonyl (Boc)] or Hyl[ε-allyloxycarbonyl (Aloc)], followed by decomposition of the copper complex and addition of an Fmoc group to the α-amino group. Fmoc- Hyl[ε-Boc,O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)] was used in the solid-phase synthesis of a type IV collagen derived sequence. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(99)02281-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 1137–1140
Convenient synthesis of glycosylated hydroxylysine derivatives
for use in solid-phase peptide synthesis
Navdeep B. Malkar, Janelle L. Lauer-Fields and Gregg B. Fields ∗
Department of Chemistry & Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, USA
Received 27 September 1999; accepted 29 November 1999
Abstract
δ-O-Glycosylated 9-fluorenylmethoxycarbonyl-hydroxylysine (Fmoc-Hyl) derivatives have been conveniently
prepared by introduction of a β-D-galactopyranosyl group to copper-complexed Hyl[ε-tert-butyloxycarbonyl
(Boc)] or Hyl[ε-allyloxycarbonyl (Aloc)], followed by decomposition of the copper complex and addition of an
Fmoc group to the α-amino group. Fmoc-Hyl[ε-Boc,O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)] was used in
the solid-phase synthesis of a type IV collagen derived sequence. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: glycopeptides/glycoproteins; amino acids and derivatives; solid-phase synthesis.
Hydroxylysine (Hyl)1 is the major glycosylation site within collagens. The δ-hydroxyl group may be
posttranslationally modified by the monosaccharide galactose (β-D-galactopyranosyl) or the disaccha-
ride glucose-galactose [α-D-glucopyranosyl-(1→2)-β-D-galactopyranosyl].2 Interest in glycosylation of
collagen stems from the recent reports of T-cell recognition of a glycosylated sequence within type II
collagen,3 the identification of melanoma and breast carcinoma binding sites within type IV collagen
that contain glycosylated Hyl residues,4,5 and the activation of specific tyrosine receptor kinases by
glycosylated type I collagen.6 Preparation of Hyl derivatives for incorporation by peptide synthesis
has a long and troubled history. Initial attempts at synthesizing Hyl(ε-Cbz) resulted in severe solubility
problems.7 The successful solution phase synthesis of a tetrapeptide containing glycosylated Hyl8 did
not use a Hyl ‘building block’, but rather the lactone form of the amino acid. Use of the lactone is
detrimental for efficient solid-phase synthesis. The recent preparation of glycosylated Fmoc-Hyl(ε-Boc)
required seven steps, with the final derivative obtained in 21.4% yield.3
We sought a more direct and convenient method for the synthesis of glycosylated Fmoc-Hyl deri-
vatives. Our previous work had shown that either the Boc or Aloc group was suitable for side-chain
protection of Hyl, but Cbz was not.9 Thus, Hyl derivatives bearing either Boc or Aloc side-chain
protection were prepared (Scheme 1). L-Hyl (1; 0.50 g, 2.52 mmol) was dissolved in 3.75 ml H2O,
and basic cupric carbonate (1.62 g, 4.03 mmol) was added. The mixture was stirred and refluxed for 4
h. The resulting suspension was filtered and washed with hot H2O until the filtrate was colorless. The
∗
Corresponding author. Tel: 561-297-2093; fax: 561-297-2759; e-mail: fieldsg@fau.edu (G. B. Fields)
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(99)02281-9
tetl 16209

1138
Scheme 1. Synthesis of glycosylated Fmoc-Hyl derivatives
combined filtrates were cooled to 0°C, and 150 µl of 4N NaOH was added. A solution of di-tert-butyl
dicarbonate (0.83 g, 3.83 mmol) in 7 ml p-dioxane was added in four aliquots over a period of 1 h. Prior
to the addition of the second, third, and fourth aliquots, 450 µl of 4N NaOH was added to maintain
a pH>8. Once di-tert-butyl dicarbonate addition was complete the reaction proceeded overnight. The
resulting precipitate was filtered, washed three times each with 3.0 ml H2O and 3.0 ml CH3OH, and
then dried under vacuum to give copper-complexed Hyl(ε-Boc) (2; 1.10 g, 75% yield). The product
was taken in dry CH3CN (10 ml) under dry nitrogen atmosphere and NaH (0.2 g, 4.60 mmol) was
added. The reaction mixture was refluxed for 2 h and then cooled to room temperature. 2,3,4,6-Tetra-
O-acetyl-α-D-galactopyranosyl bromide10 (1.88 g, 4.60 mmol) in dry CH3CN (5 ml) was added and the
reaction mixture was stirred overnight. CH3CN was distilled off completely and the product was dried
under vacuum to give copper-complexed Hyl(ε-Boc,O-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl) (3;
1.80 g, 77% yield). The product was dissolved in 10 ml CH3OH, stirred overnight at 20°C, and diluted
with 12 ml H2O. Na+ Chelex 100 resin (10 g) was washed six times with 6 ml of 1N acetic acid and
15 times with 6 ml H2O, then added to the CH3OH–H2O solution containing 3 and stirred for 3 h at
20°C. The resin was filtered and washed five times each with 4 ml of CH3OH–H2O (1:1) and 6 ml
H2O. The combined filtrates were evaporated under high vacuum at 25–30°C to provide Hyl(ε-Boc,O-
2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl). A portion of the product (0.98 g, 1.66 mmol) and NaHCO3
(0.159 g, 1.90 mmol) were dissolved in 10 ml H2O, and a solution of 9-fluorenylmethoxycarbonyl-N-
hydroxysuccinimide ester (FmocOSu) (0.635 g, 1.88 mmol) in 5 ml acetone was added. An additional
5 ml acetone was added, and the reaction proceeded overnight at 30°C. The solution was acidified to
pH∼2 with dilute HCl and the acetone was removed under high vacuum at 25–30°C. The aqueous
suspension was extracted three times with 20 ml CHCl3. The CHCl3 layer was washed three times
with 20 ml H2O, dried over anhydrous sodium sulfate, and evaporated under high pressure at 25–30°C

1139
to provide Fmoc-Hyl(ε-Boc,O-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl) (4; 0.68 g, 50% yield, 29%
overall yield).11 For the synthesis of Fmoc-Hyl(ε-Aloc,O-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl),
allyl chloroformate (0.568 g, 4.71 mmol) in 7 ml p-dioxane was used instead of di-tert-butyl dicarbonate;
product yield was 29%.12
Derivative 4 was used for the solid-phase synthesis of the α1(IV)1263–1277 sequence from type IV
collagen (Fig. 1). This sequence promotes tumor cell binding and signal transduction,4 and contains
a glycosylated Hyl residue in position 1265.13 α1(IV)1266–1277 was assembled on a PE-Biosystems
433A Peptide Synthesizer using Fmoc-based chemistry as described.14 The H2N-peptidyl-resin was
then removed from the instrument, and the last three amino acids were coupled manually in an orbital
shaker. Glycosylated derivative 4 was coupled using threefold molar excesses of Fmoc-amino acid and
HOAt, a 2.7-fold molar excess of HATU, and a sixfold molar excess of DIEA in 10 ml DMF for
18 h. Fmoc-Val and Fmoc-Gly were coupled using fourfold molar excesses of Fmoc-amino acid and
HOBt, a 3.6-fold excess of HBTU, and an eightfold molar excess of DIEA in 10 ml DMF for 1 h.
Fmoc groups were removed with 20% piperidine in DMF for 1 h. Analysis of the peptide-resin by
Edman degradation chemistry15 revealed the desired sequence.16 Peptide-resin cleavage and side-chain
deprotection proceeded with H2O–TFA (1:19) for 1.5 h.17 The peptide was purified by preparative RP-
HPLC and deacetylated with methanolic sodium methoxide18 (2 M) for 1 h at 20°C. The product was
homogeneous by analytical RP-HPLC, and MALDI-MS analysis gave the desired mass ([M+Na]+ 1638.5
Da; theoretical 1637.77 Da). Treatment of the peptide with carbazole–sulfuric acid reagent19 was positive
for carbohydrate.
Fig. 1. Structure of the α1(IV)1266–1277 sequence
We have reported a convenient method for the preparation of glycosylated Hyl derivatives for use in
Fmoc solid-phase synthesis. The procedure described herein requires only four distinct steps, as opposed
to previous procedures of seven or more steps.3,8 The preparation of glycosylated derivatives using
amino acid copper complexes may also prove to be generally applicable for trifunctional compounds.
The incorporation of glycosylated Hyl into peptides will allow for the further study of the effects of
glycosylation on cell recognition and signalling.
Acknowledgements
We thank Katarzyna Pisarewicz for technical assistance and Degussa-Hüls AG for supplying L-Hyl,
and acknowledge the support of NIH (CA77402 and HL62427).

1140
References
1. Abbreviations used are: Aloc, allyloxycarbonyl; Boc, tertiary-butyloxycarbonyl; Cbz, benzyloxycarbonyl; DIEA, N,N-di-
isopropylethylamine; DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; HATU, O-(7-azabenzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, 1-hydroxybenzotriazole; Hyl, δ-hydroxylysine; MALDI-
MS, matrix-assisted laser desorption/ionization mass spectrometry; RP-HPLC, reversed-phase high-performance liquid
chromatography; TFA, trifluoroacetic acid.
2. (a) Butler, W. T.; Cunningham, L. W. J. Biol. Chem. 1966, 241, 3882–3888. (b) Spiro, R. G. J. Biol. Chem. 1969, 244,
602–612.
3. Broddefalk, J.; Bäcklund, J.; Almqvist, F.; Johansson, M.; Kihlberg, J. J. Am. Chem. Soc. 1998, 120, 7676–7683.
4. (a) Chelberg, M. K.; McCarthy, J. B.; Skubitz, A. P. N.; Furcht, L. T.; Tsilibary, E. C. J. Cell Biol. 1990, 111, 261–270. (b)
Fields, C. G.; Mickelson, D. J.; Drake, S. L.; McCarthy, J. B.; Fields, G. B. J. Biol. Chem. 1993, 268, 14153–14160. (c)
Fields, G. B.; Lauer, J. L.; Dori, Y.; Forns, P.; Yu, Y.-C.; Tirrell, M. Biopolymers 1998, 47, 143–151.
5. (a) Miles, A. J.; Skubitz, A. P. N.; Furcht, L. T.; Fields, G. B. J. Biol. Chem. 1994, 269, 30939–30945. (b) Miles, A. J.;
Knutson, J. R.; Skubitz, A. P. N.; Furcht, L. T.; McCarthy, J. B.; Fields, G. B. J. Biol. Chem. 1995, 270, 29047–29050. (c)
Li, C.; McCarthy, J. B.; Furcht, L. T.; Fields, G. B. Biochemistry 1997, 36, 15404–15410. (d) Lauer, J. L.; Gendron, C. M.;
Fields, G. B. Biochemistry 1998, 37, 5279–5287.
6. Vogel, W.; Gish, G. D.; Alves, F.; Pawson, T. Mol. Cell 1997, 1, 13–23.
7. (a) Zahn, H.; Umalauf, E. Hoppe-Seyler’s Z. Physiol. Chem. 1954, 297, 127–145. (b) Zahn, H.; Zuern, L. Liebigs Ann.
Chem. 1958, 613, 76–83.
8. (a) Koeners, H. J.; Schattenkerk, C.; Verhoeven, J. J.; van Boom, J. H. Tetrahedron Lett. 1980, 21, 2372–2376. (b) Koeners,
H. J.; Schattenkerk, C.; Verhoeven, J. J.; van Boom, J. H. Tetrahedron 1981, 37, 1763–1771.
9. Remmer, H. A.; Fields, G. B. In Peptides: Frontiers of Peptide Science; Tam, J. P.; Kaumaya, P. T. P., Eds.; Kluwer Academic
Publishers: Dordrecht, The Netherlands, 1999; pp. 287–288.
10. (a) Redemann, C. E.; Niemann, C. Org. Synth. 1942, 3, 11–14. (b) Conchie, J.; Levvy, G. A. Meth. Carbohydr. Chem. 1963,
2, 335–337. (c) β-D-Galactose pentaacetate (2.0 g) was taken in 7 ml acetic acid. The reaction mixture was cooled to 0–5°C
while stirring. To this 2 ml of 33% HBr in acetic acid solution was added dropwise over a period of 15 min. The reaction
mixture was further stirred for 2 h at 0–5°C. Following work-up, 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide was
crystallized from diethyl ether.
1
11. MALDI-MS analysis indicated the desired mass (814 Da; theoretical 814.32 Da). H NMR analysis (500 MHz, CDCl3):
δ 7.84–7.28 (m, 8H, Fmoc-ArH), 4.70 (d, 2H, J=7.0, 6.9 Hz, Fmoc-CH2), 4.46 (d, 1H, J=6.8 Hz, Fmoc-CH), 5.43 (d, 1H,
J=7.3 Hz, NHα), 5.41 (d, 1H, NHε), 4.46 (dt, 1H, J=5.8, 14.2 Hz, Hα), 1.78 (m, 2H, Hβ), 1.42 (m, 2H, Hχ), 3.46 (m, 1H,
Hδ), 3.09 (m, 2H, Hε), 1.40 (s, 9H, Boc-CH3); chemical shifts for the galactose moiety were 5.54 (d, 1H, J=8.0 Hz, H1),
4.98 (m, 1H, J=9.3, 8.0 Hz, H2), 5.25 (m, 1H, J=9.3 Hz, H3), 4.65 (m, 1H, J=9.7 Hz, H4), 4.78 (d, 1H, J=6.0 Hz, H5), 4.21
21
(d, 2H, J=12.3, 2.4 Hz, H50), 2.01 (s, 12H, CH3COO-). [α]D 9.0° (c 1.0, CHCl3).
1
12. MALDI-MS gave the desired mass (798 Da; theoretical 798.28 Da). H NMR analysis (500 MHz, CDCl3): δ 7.84–7.28
(m, 8H, Fmoc-ArH), 4.70 (d, 2H, J=7.0, 6.9 Hz, Fmoc-CH2), 4.46 (d, 1H, J=6.8 Hz, Fmoc-CH), 5.43 (d, 1H, J=7.4 Hz,
NHα), 5.41 (d, 1H, NHε), 4.46 (dt, 1H, J=5.8, 14.2 Hz, Hα), 1.78 (m, 2H, Hβ), 1.42 (m, 2H, Hχ), 3.46 (m, 1H, Hδ), 3.09
(m, 2H, Hε), 4.75 (d, 2H, J=7.0, 10.6 Hz, Aloc-CH2), 5.89 (m, 1H, J=7.2, 10.7 Hz, Aloc-CH), 5.23–5.24 (d, 2H, J=7.4 Hz,
Aloc-CH2); chemical shifts for the galactose moiety were 5.54 (d, 1H, J=8.0 Hz, H1), 4.98 (m, 1H, J=9.3, 8.0 Hz, H2), 5.25
(m, 1H, J=9.3 Hz, H3), 4.65 (m, 1H, J=9.7 Hz, H4), 4.78 (d, 1H, J=6.0 Hz, H5), 4.21 (d, 2H, J=12.3, 2.4 Hz, H50), 2.01 (s,
12H, CH3COO-).
13. Babel, W.; Glanville, R. W. Eur. J. Biochem. 1984, 143, 545–556.
14. Lauer, J. L.; Fields, C. G.; Fields, G. B. Lett. Pept. Sci. 1995, 1, 197–205.
15. Sequencing was performed on an ABI 477A Protein Sequencer; see: Fields, C. G.; VanDrisse, V. L.; Fields, G. B. Pept. Res.
1993, 6, 39–47.
16. Sequence analysis showed a derivative in cycle 3, presumably Hyl(ε-Boc,O-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl),
eluting at a similar position as Lys.
17. Fields, C. G.; Fields, G. B. Tetrahedron Lett. 1993, 34, 6661–6664.
18. Paulsen, H.; Schultz, M.; Klamann, J. D.; Waller, B.; Pall, M. Liebigs Ann. Chem. 1985, 2028–2048.
19. Chrums, S. C. CRC Handbook of Chromatography; CRC Press: Boca Raton, FL, 1982; Vol. 1, p. 190.