Efficient use of the Dmab protecting group: Applications for the solid-phase synthesis of N-linked glycopeptides

Article abstract of DOI:10.1039/b821051a

An efficient protocol for the chemoselective removal of Dmab esters on the solid phase is reported; this method has been successfully utilised for the convergent solid phase synthesis of N-linked glycopeptides. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b821051a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Efficient use of the Dmab protecting group: applications for the solid-phase
synthesis of N-linked glycopeptides†
Trent Conroy, Katrina A. Jolliffe and Richard J. Payne*
Received 25th November 2008, Accepted 18th March 2009
First published as an Advance Article on the web 26th March 2009
DOI: 10.1039/b821051a
An efficient protocol for the chemoselective removal of Dmab
esters on the solid phase is reported; this method has been
successfully utilised for the convergent solid phase synthesis
of N-linked glycopeptides.
protecting group in the solid phase synthesis of modified peptides,
cyclic peptides and glycopeptides. These include facile (hydrazine)
deprotection conditions, which negate the need for expensive
palladium catalysts. This is especially important in cases where
biological evaluation of the ensuing peptide derivatives may be
hampered by catalyst contamination. In addition, the Dmab
deprotection reaction generates a UV active indazole moiety,
thereby providing a convenient means by which to follow the
reaction progress spectrophotometrically (Scheme 1).3
To date, several peptides protected with a C-terminal Dmab
ester have been synthesised on the solid phase and converted to
cyclic peptides in an efficient manner.4–7 However, it is somewhat
surprising that similar approaches to the synthesis of modified
peptides involving Dmab-derivatised aspartate and glutamate side
chains have proved problematic.2,3,8,9 This can be attributed to
either the formation of undesired side products2,8–10 or, perhaps,
incomplete removal of the protecting group under standard
deprotection conditions.3 In the context of synthetic endeavours
within this laboratory directed towards the construction of N-
linked glycopeptides on the solid phase, we required a protecting
group orthogonal to the allyl ester. We were hopeful that the
development of improved conditions for the incorporation and
removal of the Dmab ester would facilitate this goal.
The 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-
butyl]-amino} benzyl (Dmab) ester has recently been introduced
as an orthogonal protecting group for the modification of peptides
on resin.1 Aspartic and glutamic acids bearing this group on their
side chains or C-termini are commercially available in their Fmoc
protected form ready for incorporation into solid phase peptide
synthesis (SPPS). The Dmab group contains a 1-(4,4-dimethyl-2,6-
dioxocyclohexylidene)-3-methylbutyl (ivDde) group which can be
conveniently removed by treatment with 2% hydrazine. Following
ivDde removal, the residual aminobenzyl moiety is reported
to spontaneously eliminate to afford the corresponding free
carboxylate (Scheme 1).2
To this end, synthetic efforts commenced with the preparation
of a model resin bound peptide bearing a Dmab protected aspartic
acid residue 1 (Scheme 2). A dimethoxybenzyl (Dmb)-protected
glycine residue was incorporated adjacent to the aspartic acid
residue to prevent aspartimide formation.8 So as to gauge the
efficiency of the Dmab deprotection and a subsequent asparty-
lation reaction, we prepared an analogous resin bound model
peptide 2 bearing an allyl protected aspartic acid residue, which
is known to be efficiently removed and derivatised on the solid
phase.11 Both resin bound peptides were synthesised on Wang
resin following the Fmoc-strategy, to initially afford resin bound
tripeptide 3 (see ESI†). Fmoc-Gly(Dmb)-OH was coupled as
the penultimate amino acid followed by Fmoc-Asp(ODmab)-
OH or Fmoc-Asp(OAllyl)-OH for the synthesis of 1 and 2
respectively. Yields of amino acid coupling steps were ascertained
to be quantitative in all cases, as determined by measuring the
piperidine-fulvene adduct at l = 301 nm. Finally, a capping step
was employed to acetylate the N-terminus of the resin bound
peptides to afford 1 and 2.
Scheme 1 Mechanism of Dmab deprotection with hydrazine.
The Dmab group possesses several notable advantages over
the allyl ester, which is the traditional choice of orthogonal
Deallylation of 2 was smoothly achieved on the solid phase by
treatment with Pd(PPh3)4 and phenylsilane.12 Subsequent asparty-
lation of amino-sugar 513 using PyBOP and NMM, followed by
acidolytic side chain deprotection and cleavage from the resin,
gave the desired glycopeptide 6 which was analysed by LC-MS
and purified by preparative HPLC. As expected, the desired
School of Chemistry, The University of Sydney, Sydney, Australia. E-mail:
payne@chem.usyd.edu.au; Fax: 0061 2 9351 3329; Tel: 0061 2 9351 5877
† Electronic supplementary information (ESI) available: Experimental
details of SPPS and aspartylation protocols and characterisation of
glycopeptides. See DOI: 10.1039/b821051a
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Table 1 Conditions (a–k) used for the elimination of the aminobenzyl
ester. The efficiency of elimination was determined from the yield of the
solid phase aspartylation of 5
Aspartylation
Entry Conditions (a–k)
Time (h) yield (%)a
1
2
3
4
5
6
7
a. 20% DIPEA in 9:1 v/v DMF/H2O 12
b. 20% DIPEA in 9:1 v/v DMF/H2O 48
50
90
20
15
20
25
30
c. 20% DIPEA in DMF
d. 20% DIPEA in DCM
e. 20% pyridine in DMF
f. 20% piperidine in DMF
g. 20% triethylamine in DMF
12
12
12
12
12
8
9
10
11
h. 10% K2CO3 in 1:1 v/v MeOH/H2O 12
65
75
90
100
i. 5 mM NaOH in 1:1 v/v MeOH/H2O
j. 5 mM NaOH in 1:1 v/v MeOH/H2O
k. 5 mM NaOH in 1:1 v/v MeOH/H2O
1
2
3
a Yields were determined by LC-MS at l = 280 nm.
It has been reported that sluggish elimination of the aminoben-
zyl moiety can be remedied by treatment with an aqueous solution
of N,N-diisopropylethylamine (DIPEA).3 In this case, treatment
of the resin bound peptide with hydrazine, 20% DIPEA in 9:1
v/v DMF/H2O for 12 hours, followed by aspartylation of 5,
provided the desired glycopeptide 6 in an improved yield of 50%
(entry 1, Table 1). Extending the base treatment to 48 h (entry 2,
Table 1) prior to aspartylation afforded 6 in 90% yield. Although a
vast improvement, incomplete removal of the aminobenzyl moiety
would complicate separation of the glycopeptide from the peptide
containing the free side chain carboxylate, especially significant
in the case of longer peptide sequences. Additionally, we were
concerned that prolonged exposure to aqueous basic conditions
may lead to epimerisation and aspartimide formation with delicate
amino acid sequences.
In an effort to optimise reaction conditions, we examined a
range of organic bases in the absence of water. These included
DIPEA (entries 3 and 4, Table 1), pyridine (entry 5, Table 1),
piperidine (entry 6, Table 1) and triethylamine (entry 7, Table 1).
Unfortunately, these conditions resulted in low yields of glycopep-
tide 6 (15–30%), suggesting water was required to facilitate the
deprotection. The next step was to employ an inorganic aqueous
base (entries 8–11, Table 1). Gratifyingly, treatment of the resin
with 5 mM sodium hydroxide in methanol and water led to rapid
elimination of the aminobenzyl ester. A time course experiment
revealed that a 3 hour treatment with 5 mM NaOH resulted
in complete deprotection (entry 11, Table 1). Additionally, all
protecting groups commonly employed in Fmoc-strategy SPPS,
as well as the ester linkage to Wang resin, are stable to this low
concentration of hydroxide (see ESI†). The exception is the Fmoc
protecting group which is partially cleaved under the conditions,
Scheme 2 Solid phase synthesis of N-linked glycopeptides.
N-linked glycopeptide was produced in high yield, without the
formation of aspartimides or truncated products. In contrast,
Dmab deprotection using the reported conditions (2% hydrazine),1
followed by aspartylation of 5 with PyBOP and NMM, returned
only 5% of glycopeptide 6.
Since the solid phase aspartylation of allyl protected peptide 2
proceeded in high yield, it was postulated that the Dmab ester was
not cleaved in an efficient manner under the reported conditions.
As the ivDde moiety was found to be completely removed upon
five treatments with 2% hydrazine solution, it was evident that
spontaneous elimination of the aminobenzyl ester was very slow
(Scheme 1).
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however, this has no bearing on the utility of the reaction since
peptides are fully assembled prior to Dmab deprotection (see
ESI†). In addition, no side products relating to epimerisation
or aspartimide formation were formed under these optimised
reaction conditions, even after prolonged treatments (see ESI†).
We have observed that the nature of the amino acid C-terminal
to the site of derivatisation has a dramatic effect on the efficiency
of solid phase reactions of aspartic acid and glutamic acid side
chains. As such, our next goal was to investigate the potential
scope of this improved solid phase aspartylation protocol, via
the incorporation of alternative amino acids C-terminal to the
Dmab-protected aspartic acid residue. Resin bound tripeptide 3
served as the starting point for the synthesis of three resin bound
peptides (Scheme 3). Peptides bearing penultimate glycine, proline
and valine residues were synthesised by SPPS. Glycine was incor-
porated in its Dmb-protected form (again to prevent aspartimide
formation). The inability of proline to form aspartimides, and the
sterically hindered nature of the valine side chain, meant that the
backbone amides were not protected with Dmb groups in these
cases. Treatment of the resin bound pentapeptides with hydrazine,
followed by 5 mM NaOH, gave the corresponding free acids 4,
7 and 8, which were subsequently reacted with 5. Side chain
deprotection and cleavage from the resin using an acidic cocktail
gave the desired glycopeptides 6, 9 and 10 in high yields (quant.,
95% and 96%, respectively) as determined by LC-MS analysis.
Isolation of these glycopeptides by preparative HPLC provided 6,
9 and 10 in moderate to high yields.
Scheme 4 Solid phase synthesis of N-linked glycopeptide 11.
strates the orthogonal nature of the deprotection/aspartylation
conditions to a large range of protecting groups commonly
employed in solid-phase chemistry including Boc, tBu ethers, tBu
esters and trityl (Trt) groups and illustrates the utility of the solid
phase aspartylation method for the synthesis of more complex
glycopeptides.
In summary, improved conditions for the removal of Dmab
esters from the side chain of aspartic acid residues on the solid
phase have been developed and implemented in a novel solid-
phase strategy for the simple, rapid and efficient construction of
N-linked glycopeptides in a convergent manner. Applications of
this procedure are ongoing in this laboratory for the synthesis
of N-linked glycopeptide fragments which can be used for the
ligatory assembly of therapeutic glycopeptides and glycoproteins.
In addition, it is anticipated that the Dmab deprotection method
will find application in a host of synthetic endeavours, including
the synthesis of cyclic peptides.
Acknowledgements
We would like to thank Dr. Kelvin Picker for his assistance with
HPLC and LC-MS analysis.
Notes and references
1 W. C. Chan, B. W. Bycroft, D. J. Evans and P. D. White, J. Chem. Soc.,
Chem. Commun., 1995, 2209–2210.
2 T. Johnson, M. Liley, T. J. Cheeseright and F. Begum, J. Chem. Soc.,
Perkin Trans. 1, 2000, 2811–2820.
Scheme 3 Solid phase synthesis of N-linked glycopeptides 6, 9 and 10.
3 M. Valldosera, M. Monso, C. Xavier, P. Raposinho, J. D. G. Correia, I.
Santos and P. Gomes, Int. J. Pept. Res. Ther., 2008, 14, 273–281.
4 T. Berthelot, M. Goncalves, G. Lain, K. Estieu-Gionnet and G. Deleris,
Tetrahedron, 2006, 62, 1124–1130.
5 M. Goncalves, K. Estieu-Gionnet, G. Lain, M. Bayle, N. Betz and G.
Deleris, Tetrahedron, 2005, 61, 7789–7795.
6 J. P. Malkinson, M. Zloh, M. Kadom, R. Errington, P. J. Smith and M.
Searcey, Org. Lett., 2003, 5, 5051–5054.
7 M. Cudic, J. D. Wade and L. Otvos, Tetrahedron Lett., 2000, 41, 4527–
4531.
Finally, to demonstrate the applicability of our solid-phase
aspartylation method for the preparation of longer glycopeptides
bearing an internal glycan, we embarked on the synthesis of
11 (Scheme 4). Synthesis of fully protected peptide 12 was
achieved from 3 using standard Fmoc-SPPS followed by Dmab-
deprotection of the internal aspartic acid residue (see ESI†). Solid
phase aspartylation of 5 and acidolytic deprotection and cleavage
from the resin gave the desired glycopeptide 11 in 61% isolated
yield based on the Fmoc loading of 3. This example clearly demon-
8 J. Ruczynski, B. Lewandowska, P. Mucha and P. Rekowski, J. Pept.
Sci., 2008, 14, 335–341.
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9 D. R. Coleman, Z. Y. Ren, P. K. Mandal, A. G. Cameron, G. A. Dyer,
S. Muranjan, M. Campbell, X. M. Chen and J. S. McMurray, J. Med.
Chem., 2005, 48, 6661–6670.
10 K. F. Medzihradszky, N. P. Ambulos, A. Khatri, G. Osapay, H. A.
Remmer, A. Somogyi and S. A. Kates, Lett. Pept. Sci., 2001, 8,
1–12.
11 J. Offer, M. Quibell and T. Johnson, J. Chem. Soc., Perkin Trans. 1,
1996, 175–182.
12 S. Ficht, R. J. Payne, R. T. Guy and C. H. Wong, Chem.–Eur. J., 2008,
14, 3620–3629.
13 L. M. Likhosherstov, O. S. Novikova, V. A. Derevitskaja and N. K.
Kochetkov, Carbohydrate Res., 1986, 146, C1–C5.
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