Improving glycopeptide synthesis: A convenient protocol for the preparation of β-glycosylamines and the synthesis of glycopeptides

Article abstract of DOI:10.1021/jo047801v

(Chemical Equation Presented) Herein we apply a recently introduced protocol using ammonium carbamate in methanol to the amination of crude chitobiose leading to 1,β-aminochitobiose. This simple, one-step procedure allows a facile preparation of unstable glycosylamines in contrast to the commonly implemented ammonium bicarbonate based amination of water-soluble carbohydrates. The new amination protocol leads to an improved synthesis of the key chitobiosyl-asparagine building block for the SPPS of glycopeptides. The utility of the method is demonstrated with the synthesis of a 39-amino acid glycoprotein.

Full text of DOI:10.1021/jo047801v

Improving Glycopeptide Synthesis: A Convenient Protocol for the
Preparation of â-Glycosylamines and the Synthesis of
Glycopeptides
Christian P. R. Hackenberger, Mary K. O’Reilly, and Barbara Imperiali*
Department of Chemistry and Department of Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
imper@mit.edu
Received December 16, 2004
Herein we apply a recently introduced protocol using ammonium carbamate in methanol to the
amination of crude chitobiose leading to 1,â-aminochitobiose. This simple, one-step procedure allows
a facile preparation of unstable glycosylamines in contrast to the commonly implemented ammonium
bicarbonate based amination of water-soluble carbohydrates. The new amination protocol leads to
an improved synthesis of the key chitobiosyl-asparagine building block for the SPPS of glycopeptides.
The utility of the method is demonstrated with the synthesis of a 39-amino acid glycoprotein.
Significant effort has been devoted toward understand-
ing the various roles of protein glycosylation, which is
now known to modulate protein structure and to influ-
ence numerous biochemical and medicinally relevant
processes.1 Research in this area is, however, limited by
the accessibility of homogeneous glycoproteins for study
since the glycoconjugates isolated from natural sources
commonly display considerable heterogeneity in the
saccharide portion.2 Therefore, the development of new
synthetic routes toward glycoproteins, and glycoconju-
gates in general, continues to attract much attention.3
Carbohydrates in natural glycoproteins are attached
to peptides through the oxygen of serine or threonine
residues in O-linked glycoproteins or through the car-
boxamide nitrogen of asparagine in N-linked glycopro-
teins.4 To access N-linked glycoproteins via chemical
synthesis, particular emphasis has been placed on the
preparation of â-glycosylamines, as these intermediates
can be coupled to activated aspartic acid derivatives or
peptides to establish a native N-linked glycopeptide
linkage.4b,5 Unfortunately, glycosylamines are unstable
and prone to dimerization, hydrolysis, and isomerization.6
These side reactions make the purification of glycosyl-
amines extremely difficult and in general, separation
steps must be carried out later in the synthesis on more
complex intermediates.7
As part of the continuing effort to access quantities of
glycopeptides and glycoproteins for biochemical and
biophysical investigations, we now report the first ex-
ample of a 1,â-aminochitobiose (1) synthesis directly from
a crude chitobiose mixture derived from the enzymatic
digestion of chitin with Chitinase (Scheme 1).8 This
(5) For other examples to construct the carbohydrate N-linkage
see: (a) Gu¨nther, W.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1990,
29, 1050-1051. (b) Handlon, A. L.; Fraser-Reid, B. J. Am. Chem. Soc.
1993, 115, 3796-3797. (c) Damkaci, F.; DeShong, P. J. Am. Chem.
Soc. 2003, 125, 4408-4409. (d) Maunier, V.; Boullanger, P.; Lafont,
D. J. Carbohydr. Chem. 1997, 16, 231-235. (e) Mizuno, M.; Muramoto,
I.; Kobayashi, K.; Yaginuma, H.; Inazu, T. Synthesis 1999, 162-165.
(f) He, Y.; Hinklin, R. J.; Chang, J. Y.; Kiessling, L. L. Org. Lett. 2004,
6, 4479-4482.
(6) Likhoshertov, L. M.; Novikova, V. A.; Dervitskaja, V. A.; Kochet-
kov, N. K. Carbohydr. Res. 1986, 46, C1-C5.
(7) Takeda, T.; Utsuno, A.; Okamoto, N.; Ogihara, Y.; Shibata, S.
Carbohydr. Res. 1990, 207, 71-79.
(1) (a) Varki, A. Glycobiology 1993, 3, 97-130. (b) Dwek, R. A. Chem.
Rev. 1996, 3, 683-720. (c) Bertozzi, C. R.; Kiessling, L. L. Science 2002,
291, 2357-2364. (d) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I.
A.; Dwek, R. A. Science 2001, 291, 2370-2376.
(2) Helenius, A.; Aebi, M. Science 2001, 291, 2364-2369.
(3) (a) Grogan, M. J.; Pratt, M. R.; Marcaurelle, L. A.; Bertozzi, C.
R. Annu. Rev. Biochem. 2002, 71, 593-634. (b) Arsequell, G.; Valencia,
G. Tetrahedron: Asymmetry 1999, 10, 3045-3094.
(4) Bura, P.; Aebi, M. Biochim. Biophys. Acta 1999, 1426, 239-257.
10.1021/jo047801v CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/31/2005
3574
J. Org. Chem. 2005, 70, 3574-3578

Improving Glycopeptide Synthesis
SCHEME 1. Route for the Synthesis of Glycopeptides Starting from Chitin
SCHEME 2. Proposed Mechanism of the
conversion allows a convenient six-step synthesis of the
acid labile Fmoc-protected chitobiose amino acid 2 in
gram quantities including two purification steps by
column chromatography.9 The building block 2 is then
used directly in the solid-phase peptide synthesis (SPPS)
of an N-linked glycopeptide modified with chitobiose 3.
In principle, two different synthetic routes to glycos-
ylamines have been explored recently. The carbohydrate
amine functionality can be introduced after the hydroxyl
group protection, which requires the use of organic
solvents as reaction media. Examples of this approach
include the conversion of glycosyl halides with am-
monia,10 azide11 or the Burgess reagent,12 and synthetic
schemes based on glycals13 or R-hydroxy nitriles.14 The
second route employs unprotected water-soluble carbo-
hydrates and in this case the amination procedure is
commonly carried out by the Kochetkov method, with use
of a saturated solution of ammonium bicarbonate in
water.5,15 Applications of this strategy include complex
carbohydrate syntheses pioneered by Danishefsky16 as
well as the previous route to building block 2; the latter
method implemented pure chitobiose as starting material
for the synthesis of 1,â-aminochitobiose (1) before cou-
pling with a preactivated aspartic acid derivative in an
overall yield of 12%.3b,9
Carbamate-Mediated Amination of Sugar
Derivatives
erstov, which used ammonium carbamate in methanol
as the reactant and showed the general applicability for
a series of mono- and disaccharide derivatives.17 The
major advantage of this procedure is the ease of isolation
of the reaction product. The glycosylamine, formed by the
reaction with ammonia that has been generated by
dissociation of ammonium carbamate, is precipitated as
a carbamic acid salt under the reaction conditions
(Scheme 2). This salt formation prevents the hydrolysis
and glycosylamine-dimer formation, as long as the amount
of water is kept to a minimum. Finally, the free amine
is generated by base treatment or under high vacuum.
We envisioned that this protocol could also allow the
purification of glycosylamines starting from a crude
carbohydrate preparation.
Recently, a new protocol for the selective amination of
unprotected sugar derivatives was introduced by Likhosh-
(8) Terayama, H.; Takahashi, S.; Kuzuhara, H. J. Carbohydr. Chem.
1993, 12, 81-93.
(9) For a previous example of the synthesis of building block 2 see:
Bosques, C. J.; Tai, V. W. F.; Imperiali, B. Tetrahedron Lett. 2001, 42,
7207-7210.
Results and Discussion
(10) Michael, F.; Frier, R.; Plate, E.; Hiller, A. Chem. Ber. 1952, 85,
1092-1096.
The investigations were initiated by evaluating ami-
nation of pure chitobiose (4) (Scheme 3), which was
obtained from chitin by enzymatic digestion with Chiti-
nase at pH 6.2 and subsequent recrystallization of the
acetyl-protected carbohydrate.18 Amination was carried
out with ammonium carbamate for 24 h at 37 °C in
methanol. The chitobiose amine-salt precipitated as a
(11) Gyo¨rdea´k, Z.; Szilagyi, L.; Paulsen, H. J. Carbohydr. Chem.
1993, 12, 139-163.
(12) Nicolaou, K. C.; Snyder, S. A.; Nalbandian, A. Z.; Longbottom,
L. A. J. Am. Chem. Soc. 2004, 126, 6234-6235.
(13) Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem., Int. Ed. Engl.
1996, 35, 1380-1419.
(14) Dorsey, A. D.; Barbarow, J. E.; Trauner, D. Org. Lett. 2003, 5,
3237-3239.
(15) Lubineau, A.; Auge, J.; Drouillat, B. Carbohydr. Res. 1995, 266,
211-219.
(16) (a) Miller, J. S.; Dudkin, V. Y.; Lyon, G. J.; Muir, T. W.;
Danishefsky, S. J. Angew. Chem., Int. Ed. 2003, 42, 431-434. (b) Wang,
Z.-G.; Zhang, X.; Live, D.; Danishefsky, S. J. Angew. Chem., Int. Ed.
2000, 39, 3652-3656.
(17) (a) Likhosherstov, L. M.; Novikova, O. S.; Shibaev, V. N. Dokl.
Chem. 2002, 383, 89-92. (b) Likhosherstov, L. M.; Novikova, O. S.;
Shibaev, V. N. Dokl. Chem. 2003, 389, 73-76. (c) Likhosherstov, L.
M.; Novikova, O. S.; Zheltova, A. O.; Shibaev, V. N. Russ. Chem. Bull.,
Int. Ed. 2004, 53, 709-713.
J. Org. Chem, Vol. 70, No. 9, 2005 3575

Hackenberger et al.
SCHEME 3. General Route for the Amination of
Chitobiosea
a Reagents and conditions: (a) HCl, sonication; (b) Chitinase,
pH 6.2, phosphate buffer; (c) Ac2O, NaOAc; (d) NaOMe, MeOH.
white solid, which was transformed into the correspond-
ing â-chitobioseamine (1) after brief drying under high
vacuum in 76% yield and high purity (see the Experi-
mental Section for details). Under these conditions, none
of the corresponding R-anomer was detected. Longer
reaction times did not improve the yield, whereas shorter
reaction reduced the yield significantly.
Next, amination of the crude chitobiose enzymatic
digestion product, instead of pure 4, was investigated to
evaluate whether this method would afford pure â-chi-
tobioseamine, since soluble byproducts could be removed
by simple filtration (Scheme 3). To test this hypothesis,
we directly compared ammonium carbamate and am-
monium bicarbonate as reagents for the amination of
crude chitobiose. We found that the ammonium carbam-
ate protocol leads to the formation of pure 1,â-aminochi-
tobiose (1), which was confirmed by 1H NMR analysis of
the anomeric R-hydrogens and the amide methyl groups
(Figure 1, bottom). In contrast, treatment of crude
chitobiose with ammonium bicarbonate and concentra-
tion of the aqueous reaction mixture results in an
inseparable mixture of compounds that was not further
characterized (Figure 1, top). Further optimization stud-
ies into the carbamate amination of crude chitobiose
revealed that reaction at 37 °C for 16 h with 4 equiv of
ammonium carbamate are the optimal reaction condi-
tions, with a yield of 69%.
After the successful optimization of the glycosylamine
synthesis, we concentrated on improving the preparation
of the Fmoc-amino acid building block 2 for the SPPS of
glycopeptides. This building block includes the fully
TBDMS-protected chitobiose attached to aspartic acid via
a â-glycosyl amide linkage.9,19 The acid-labile building
block 2 has proven to be advantageous for glycopeptide
SPPS in comparison to the acetyl protected analogue, as
the protecting groups can be removed during the resin
FIGURE 1. 1H NMR spectra in D2O of 1,â-aminochitobiose
(1) obtained from ammonium bicarbonate amination (top) and
ammonium carbamate amination (bottom). Spectra to the left
display the anomeric hydrogens, while spectra to the right
represent the acetamide-methyl group hydrogens.
cleavage in a single synthetic step by using standard
TFA-based cleavage cocktails.19d,e Additionally, aggrega-
tion, which occurs frequently upon treatment of unpro-
tected peptides with harsh basic conditions, is minimized
in the final acidic treatment.9
The final optimized sequence is summarized in Scheme
4. Coupling of the chitobioseamine 1 was performed
under HBTU/HOBt conditions in DMF with use of 2
equiv of chitobioseamine and 1 equiv of Fmoc-Asp-OAll.
The resulting unprotected amino acid derivative was
precipitated from ethyl acetate, isolated by filtration, and
directly used without further purification in the next
protection step. TBDMS-protection to yield the protected
amino acid 5 was performed with 16 equiv of TBDMS-
OTf, which were added over 24 h in three portions. It
was essential to implement the silyl triflate because the
less reactive silyl-based electrophiles, such as TBDMS-
chloride or bromide, did not lead to complete protection
of the building block. Final deprotection of the allyl ester
and purification by size exclusion chromatography yielded
building block 2 in 56% overall yield based on the amount
of Fmoc-amino acid used in this transformation.
(18) Crude chitobiose contains a mixture of R- and â-anomers of
chitobiose as well as amounts of GlcNAc monosaccharides as analyzed
by 1H NMR spectroscopy and HPLC of fluorescently labeled substrates
(see the Supporting Information for details).
(19) For examples of SPPS of glycopeptides using the building block
approach see: (a) Jobron, L.; Hummel, G. Angew. Chem., Int. Ed. 2000,
39, 1621-1624. (b) Christiansen-Brams, I.; Meldal, M.; Bock, K. J.
Chem. Soc., Perkin Trans. 1 1993, 1461-1471. (c) Meinjohanns, E.;
Meldal, M.; Paulsen, H.; Dwek, R. A.; Bock, K. J. Chem. Soc., Perkin
Trans. 1 1998, 549-560. (d) Holm, B.; Linse, S.; Kihlberg, J. Tetra-
hedron 1998, 54, 11995-12006. (e) Broddefalk, J.; Bergquist, K.;
Kihlberg, J. Tetrahedron 1998, 54, 12047-12070.
3576 J. Org. Chem., Vol. 70, No. 9, 2005

Improving Glycopeptide Synthesis
SCHEME 4. Optimized Synthesis of the Building Block 2a
a Reagents and conditions: (a) NH4CO2NH2, MeOH; then high vacuum; (b) Fmoc-Asp-OAll, HBTU, HOBt, DIPEA, DMF; (c) TBDMS-
OTf, DMAP, pyridine; (d) Pd(PPh3)4, PhSiH3, CH2Cl2.
SCHEME 5. SPPS of the Glycosylated C-Peptide
Analogue G1-G39a
a Reagents and conditions: (a) 2, PyAOP, collidine, CH2Cl2; (b)
Fmoc-Xaa-OH, HBTU, HOBt, DIPEA, DMF; (c) Cleavage cocktail
K (TFA, phenol, EDT, PhSMe, TIS, H2O).
Having established a convenient route toward the acid
labile building block 2, this derivative was used in the
preparation of a complex N-linked glycopeptide. We chose
a glycosylated C-peptide analogue as our synthetic target,
which, in the unglycosylated form, plays an important
FIGURE 2. Synthesis of the glycosylated C-peptide G1-G39
role in the inhibition of the HIV membrane fusion process
(indicated by arrow): (a) HPLC spectrum of crude SPPS
by binding tightly to the viral envelope glycoprotein
product after resin cleavage (constant flow: 5 min at 7% CH3-
gp41.20 The C-peptide consists of 39 amino acids and
CN, 15 min at 20% CH3CN; gradient 1: 45% to 53% CH3CN
includes a natural glycosylation sequon (NYT) from
positions 14 to 16.21 The C-peptide sequence is displayed
in Scheme 5 with the glycosylation site as indicated.
The peptide Y15-G39 was synthesized on a Tenta-Gel
resin, using standard Fmoc SPPS conditions with HOBt/
HBTU activation.22 Next, 2 equiv of the building block 2
were coupled manually under PyAOP/collidine activation
in CH2Cl2. PyAOP/collidine was also used for the coupling
of the next two amino acids before the conditions were
reverted to HOBt/HBTU activation for the remaining 11
amino acid couplings. The N-terminal deprotected pep-
tide G1-G39 was capped with an acetyl group and cleaved
over 20 min; gradient 2: 53% to 95% over 5 min); (b) HPLC
spectrum of purified glycopeptide (gradient: 7% to 95% CH3-
CN over 35 min); and (c) MALDI analysis (inset).
from the resin with the cleavage cocktail K.23 HPLC
analysis of the crude peptide product reveals the suc-
cessful utilization of the new chitobiose building block
in the glycopeptide synthesis. The crude peptide was
purified by preparative HPLC, and analyzed by ESI-MS
and MALDI (Figure 2).
In summary, we have developed an efficient synthesis
of glycopeptides bearing the native amide linkage, start-
ing from simple and inexpensive starting materials. The
major advantage of this route is the straightforward
isolation and purification of pure glycosylamines, which
are finally converted into a building block for the SPPS
(20) (a) Root, M. J.; Kay, M. S.; Kim, P. S. Science 2001, 291, 884-
888. (b) Root, M. J.; Hamer, D. H. PNAS 2003, 100, 5016-5021.
(21) For the SPPS of a glycopeptide-thioester with a 26 amino acid
sequence see: (a) Hojo, H.; Watabe, J.; Nakahara, Y.; Ito, Y.; Na-
beshima, K.; Toole, B. P. Tetrahedron Lett. 2001, 42, 3001-3004. (b)
Hojo, H.; Haginoya, E.; Matsumoto, Y.; Nakahara, Y.; Nabeshima, K.;
Toole, B. P.; Watanabe, Y. Tetrahedron Lett. 2003, 44, 2961-2964.
(22) We acknowledge Professor Michael J. Root for the SPPS of the
peptide Y15-G39.
(23) (a) King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Peptide Protein
Res. 1990, 36, 255-266. (b) Huang, H.; Rabenstein, D. L. J. Pept. Res.
1999, 53, 548-553.
J. Org. Chem, Vol. 70, No. 9, 2005 3577

Hackenberger et al.
dried at high vacuum for no more than 10 s to avoid
decomposition of the aminosugar. The resulting white solid
was directly used for analysis or for further synthetic trans-
formations.
of glycopeptides. This synthetic route greatly increases
the accessibility of homogeneous N-linked glycopeptides
for biochemical and biophysical investigations. We are
currently using this methodology to access large peptide
fragments for the semi-synthesis of glycoproteins via
native chemical ligation for protein folding studies as well
as for other biochemical studies.
1H NMR (500 MHz, D2O) δ (ppm) 4.50 (1 H, d, J ) 8.5 Hz,
H-1′), 4.12 (1 H, d, J ) 8.5 Hz, H-1), 3.90-3.40 (13 H, m), 2.04
(3 H, s, NAc), 2.01 (3 H, s, NAc).
MS (ESMS, [MH+]) 424.1 (obsd), 424.4 (calcd).
Experimental Section
Acknowledgment. This work was funded by the
NIH (GM 39334). C.P.R.H. acknowledges a postdoctoral
fellowship from the German Academic Exchange Service
(DAAD). We thank Dr. Mark Nitz, Dr. Carlos Bosques,
and Dr. Eugenio Vazquez for valuable discussions and
advice and Professor Michael J. Root for the gift of the
resin bound peptide intermediate Y15-G39.
General. Solvent purification, suppliers, and instruments,
as well as full synthetic protocols for the synthesis of pure and
crude chitobiose and building block 2, are described in the
Supporting Information.
Synthesis of 1,â-Aminochitobiose (1) via Amination of
Crude Chitobiose with Ammonium Carbamate. In a flask
equipped with a reflux condenser, 1 equiv of the crude
chitobiose and 4 equiv of ammonium carbamate were sus-
pended in methanol (5 mL/mmol of chitobiose). The suspension
was stirred for 16 h at 37 °C until the precipitate became white
and fluffy and TLC analysis (silica, 4:3:2 ethyl acetate/MeOH/
water; CAM and 10% H2SO4 detection; Rf(unprotected chito-
biose) ) 0.73; Rf(1) ) 0.33) indicated no further amination
product. The mixture was stirred for 1 h at 0 °C, and the
precipitate was filtered, washed twice with cold methanol and
Supporting Information Available: 1H NMR spectra of
amination products, HPLC analysis of crude chitobiose, full
experimental details, and analysis for the building block 2
synthesis and the SPPS of the glycosylated C-peptide ana-
logue. This material is available free of charge via the Internet
at http://pubs.acs.org.
JO047801V
3578 J. Org. Chem., Vol. 70, No. 9, 2005