Synthesis of neoglycopeptides by chemoselective reaction of carbohydrates with peptides containing a novel N′-methyl-aminooxy amino acid

Article abstract of DOI:10.1016/S0040-4039(02)01212-1

A novel N′-methyl-aminooxy amino acid has been designed, synthesized, and successfully incorporated into peptides using standard solid-phase peptide synthesis procedures. Reaction of these peptides with native reducing sugars yields neoglycopeptides via a

Full text of DOI:10.1016/S0040-4039(02)01212-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 5727–5729
Synthesis of neoglycopeptides by chemoselective reaction of
carbohydrates with peptides containing a novel
N%-methyl-aminooxy amino acid
Michael R. Carrasco,* Michael J. Nguyen, Dawn R. Burnell, Michael D. MacLaren and
Shawna M. Hengel
Department of Chemistry, Santa Clara University, Santa Clara, CA 95053-0270, USA
Received 15 May 2002; revised 19 June 2002; accepted 21 June 2002
Abstract—A novel N%-methyl-aminooxy amino acid has been designed, synthesized, and successfully incorporated into peptides
using standard solid-phase peptide synthesis procedures. Reaction of these peptides with native reducing sugars yields neogly-
copeptides via a chemoselective reaction with the aminooxy side chains. The key feature of the new amino acid is that it maintains
attached sugars in their cyclic conformations and close to the peptide backbone. © 2002 Elsevier Science Ltd. All rights reserved.
Glycopeptides are ubiquitous in nature and play a
variety of biological roles.1 Deciphering these roles in
controlled studies requires convenient synthetic access
to large numbers of structures. Although many impres-
sive glycopeptide syntheses have been reported, they
generally involve complicated techniques and the han-
dling of many sensitive compounds. To greatly reduce
the synthetic burden, several groups have turned to
chemoselective reactions between completely unpro-
tected peptides and carbohydrates.2–8 Although the
resulting linkages do not precisely replicate natural
connections, the simplified procedures allow rapid, con-
venient access to a wide array of neoglycopeptides,
which have been shown by some researchers to main-
tain their native biological activity.3
The most attractive of these chemoselective reaction
strategies involves using aminooxy derivatized peptides
with natural, unmodified reducing sugars (Fig. 1).
However, most published examples have suffered from
one of two problems that limit the biological relevance
of the product neoglycopeptides: either the aminooxy
side chains are extremely long or the attached sugars do
not maintain their cyclic conformations. Ideally, neo-
glycopeptides would have cyclic sugars attached as
close as possible to the peptide backbone to allow the
sugars to significantly modulate peptide structure and
function.
Recently, several research groups have introduced
amino acid derivatives that either have short aminooxy
Figure 1. General reaction of aminooxy-derivatized peptides with reducing sugars to form neoglycopeptides. Sugars react
chemoselectively with the aminooxy side chains in the presence of a wide variety of other functional groups. When R=alkyl,
cyclic sugar conformations are observed, but when R=H, only open-chain oxime-linked sugars are seen.
* Corresponding author. Tel.: +1-408-551-1878; fax: +1-408-554-7811; e-mail: mcarrasco@scu.edu
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01212-1

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M. R. Carrasco et al. / Tetrahedron Letters 43 (2002) 5727–5729
side chains6 or utilize N-alkyl substitution of the
aminooxy group to ensure cyclic sugar conformations.4
We have now constructed a single derivative that com-
bines both of these elements to allow the synthesis of
neoglycopeptides with biologically relevant structures.
with t-butyl dicarbonate in basic conditions followed
by alkylation of the carboxylate anion with allyl bro-
mide gives the Boc-protected ester 3 in 64% yield for
the one-pot, two-step procedure. Subsequent conver-
sion of the alcohol to its mesylate and reaction with
lithium bromide yields 88% of the bromide 4. Displace-
ment of the bromine with the oxy-anion of N-methyl-
N-(2-Cl-Cbz)-hydroxylamine 5 (made by the reaction
of N-methyl-hydroxylamine with N-(2-chlorobenzyl-
oxycarbonyloxy)succinimide) provides the aminooxy
ester 6 in 93% yield. Quantitative removal of the allyl
ester with catalytic Pd(Ph3)4 in the presence of pyrro-
lidine then gives the desired amino acid 7,9 suitably
protected for Boc-chemistry-based SPPS. The overall
Our efforts have focused on the two derivatives shown
below. These derivatives are the O-(N-methyl)-amino
analogs of serine (1) and homoserine (2). Both offer the
shortest possible amino acid side chains that incorpo-
rate the functionality needed for chemoselective attach-
ment of sugars in cyclic conformations.
yield of 7 from
L-homoserine is over 50%, and the
procedures are amenable to large-scale synthesis.
We have successfully incorporated 7 into several model
peptides (3–9 amino acids each)10 using standard Boc-
chemistry-based SPPS procedures.11 Each has been
deprotected and cleaved from its solid support (tri-
fluoroacetic acid, bromotrimethylsilane, thioanisole)12
to yield peptides containing the desired N-methyl-
aminooxy amino acid 2.13
Initially, we focused on the serine analog 1, which we
successfully synthesized as its N-a-Boc, N-g-(2-Cl-Cbz)
derivative and incorporated into peptides via standard
solid-phase peptide synthesis (SPPS) procedures.
Unfortunately, in reactions of these peptides with
native reducing sugars, we found that elimination of the
N-methyl-aminooxy moiety to give dehydroalanine was
competitive with the addition of any sugars. In fact, we
determined that this elimination occurred sponta-
neously in aqueous solutions as mild as 0.1 M sodium
acetate buffer at pH 5.1. Although our results con-
firmed the viability of the general strategy, the elimina-
tion reaction rendered the derivative impractical for
neoglycopeptide syntheses.
Glycosylation of these peptides proceeds smoothly and
chemoselectively in aqueous conditions (0.1 M NaOAc,
pH 4.0 or 5.1, 40–45°C) with both
D-glucose and
lactose. HPLC analysis of a representative glycosyla-
tion reaction is shown in Fig. 2. The peptide H2N–
FKAZSK–NH2, where Z is our aminooxy amino acid
2, was synthesized as described above, purified by high
performance liquid chromatography (HPLC), and char-
acterized by electrospray ionization mass spectrometry
(ESI-MS). The lyophilized peptide powder (0.1 mg) was
dissolved in 0.1 M NaOAc, pH 4.0 buffer (100 ml),
treated with glucose (1 mg), and heated at 40°C. After
26 h, the chromatogram and ESI-MS analysis of the
fractions corresponding to the peaks indicated 85%
conversion of the parent peptide (peak A; calcd for
(M+2H)+2, 355.4; found, 355.5) to a single, monoglyco-
sylated peptide (peak B; calcd for (M+2H)+2, 436.5;
We then turned to the homoserine analog 2, reasoning
that the greatly reduced acidity of the carbonyl b
protons (relative to the a proton) would restrain any
tendency of the aminooxy group to eliminate. This
amino acid was conveniently synthesized in its N-a-
Boc, N-d-(2-Cl-Cbz) protected form from
L
L
-homoserine
-homoserine
as detailed in Scheme 1.9 Reaction of
Scheme 1. Reagents and conditions: (a) (Boc)2O, NaOH, H2O, CH3CN; (b) allyl bromide, DMF, 64% from L-homoserine; (c)
methanesulfonyl chloride, NEt3, CH2Cl2, then LiBr, acetone, 88%; (d) 5 with NaH, THF, 0°C, then 4, 93%; (e) Pd(PPh3)4, PPh3,
pyrrolidine, CH2Cl2, 99%.

M. R. Carrasco et al. / Tetrahedron Letters 43 (2002) 5727–5729
5729
to the peptide backbone, these neoglycopeptides should
serve as excellent mimics of natural glycopeptides. We
expect them to enable many new studies of how glyco-
sylation affects the structure and function of biological
peptides.
Acknowledgements
Acknowledgement is made to the Donors of The
Petroleum Research Fund, administered by the Ameri-
can Chemical Society, for partial support of this
research. We also gratefully acknowledge financial sup-
port from the Camille and Henry Dreyfus Faculty
Start-up Grant Program for Undergraduate Institutions
and the National Science Foundation (NSF-REU grant
CHE98-20382). This research was also supported by an
award to Santa Clara University under the Undergrad-
uate Biological Sciences Education Program of the
Howard Hughes Medical Institute.
Figure 2. Analytical HPLC chromatograms of the glycosyla-
tion reaction between H2N–FKAZSK–NH2 and -glucose in
D
0.1 M NaOAc, pH 4.0, at 40°C. Peak A is the non-glycosy-
lated peptide, and peak B is the glycosylated peptide. Samples
were run on a Microsorb-MV™ C18 column with a gradient
of 0–25% buffer B (CH3CN+0.08% TFA) in buffer A (H2O+
0.1% TFA) over 30 min and monitored at 214 nm.
found, 436.5). Importantly, no additional glycosylation
was observed at the N-terminus or lysine or serine side
chains,14 and the glycosylated and non-glycosylated
peptides could be separated cleanly by HPLC.
References
In our various trials, we have found 60–85% glycosyla-
tion yields (D-glucose or lactose) after 24–48 h. An
1. Varki, A. Glycobiology 1993, 3, 97–130.
2. Cervigni, S. E.; Dumy, P.; Mutter, M. Angew. Chem., Int.
Ed. Engl. 1996, 35, 1230–1232.
equilibrium mixture is reached quickly: the ratio of
glycosylated to non-glycosylated peptide does not
change significantly after 24 h. As would be expected
with an intermolecular process, conversion yields are
greatest when the concentration of sugar and peptide
are high. In cases of incomplete glycosylation, the
non-glycosylated peptide can be recovered, and no pep-
tide or neoglycopeptide decomposition or elimination
products have been observed under our reaction
conditions.
3. Marcaurelle, L. A.; Rodriguez, E. C.; Bertozzi, C. R. Tet-
rahedron Lett. 1998, 39, 8417–8420.
4. Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54,
12269–12278.
5. Lang, I.; Donze´, N.; Garrouste, P.; Dumy, P.; Mutter, M.
J. Peptide Sci. 1998, 4, 72–80.
6. Spetzler, J. C.; Hoeg-Jensen, T. J. Peptide Sci. 1999, 5,
582–592.
7. Zhao, Y.; Kent, S. B. H.; Chait, B. T. Proc. Natl. Acad.
Sci. USA 1997, 94, 1629–1633.
8. Peluso, S.; Imperiali, B. Tetrahedron Lett. 2001, 42, 2085–
2087.
9. All synthetic intermediates exhibited satisfactory H and
After isolation, the neoglycopeptides are stable in
aqueous solutions at neutral pH. However, at pH 2
they lose their sugars and revert to the parent peptides.
We expect that the ability to induce deglycosylation by
changing the pH will be useful for potential biological
and structural studies.
1
13C NMR and HRMS data. Characterization for 7: 1H
NMR (400 MHz, CDCl3, 55°C): l 8.60 (bs, 1H), 7.39 (m,
2H) 7.26 (m, 2H), 5.63 (bs, 1H), 5.29 (s, 2H), 4.43 (bs, 1H),
4.01 (m, 2H), 3.18 (s, 3H), 2.17 (m, 1H), 2.05 (m, 1H), 1.43
(s, 9H). 13C NMR (125 MHz, CDCl3, 55°C): l 175.7,
157.6, 156.2, 133.93, 133.90, 130.1, 129.82, 129.80, 127.2,
80.6, 71.0, 65.5, 51.8, 36.7, 30.7, 28.5. FAB-HRMS calcd
for C18H26ClN2O7 (M+H)+: 417.1429; found: 417.1421.
10. Peptide sequences synthesized: Ac–ZAF–NH2, H2N–
FKAZSK–NH2, and H2N–SEZYFLASK–NH2, where Z
is the N-methyl-aminooxy amino acid 2.
Our current focus is making neoglycopeptide libraries
by reacting series of N-methyl-aminooxy-containing
peptides combinatorially with a variety of sugars. These
libraries are designed to address hypothesized structural
and functional roles of glycosylation in biological pep-
tides and proteins. We are also undertaking NMR
studies of our neoglycopeptides to confirm cyclic con-
formations for sugars not previously investigated.4
Lastly, to extend the use of our strategy, we are synthe-
sizing 2 protected appropriately for Fmoc-based SPPS.
11. Schno¨lzer, M.; Alewood, P.; Jones, A.; Alewood, D.;
Kent, S. B. H. Int. J. Peptide Protein Res. 1992, 40, 180–
193.
12. Hughes, J. L.; Leopold, E. J. Tetrahedron Lett. 1993, 34,
7713–7716.
Our new N-methyl-aminooxy amino acid derivative 7
can be synthesized in large quantities in very good
overall yield. Its incorporation into peptides is accom-
plished with standard SPPS procedures, and the N-
methyl-aminooxy side chain provides a selective site for
glycosylation under mild conditions. Our methods
provide rapid and convenient access to large numbers
of neoglycopeptides. By containing cyclic sugars close
13. All peptides were purified by reversed-phase HPLC and
characterized
spectrometry.
by
electrospray
ionization
mass
14. We have conducted control reactions with H2N–FKASK–
NH2 under the same glycosylation conditions to verify
that no co-eluting mono-glycosylated peptides with a
sugar attached at another location were formed. No glyco-
sylation of any kind was observed in the control reactions.