Tunable acid-sensitive ester protecting groups in oligosaccharide synthesis

Article abstract of DOI:10.1039/c3cc49205b

A series of acid-cleavable ester-type protecting groups, with acid-sensitivity profiles parallel to those of 2-naphthylmethyl (NAP) or p-methoxybenzyl (PMB) ether, were designed and TFA in toluene was identified as a technically simple and effective deblocking cocktail for their global removal in the context of oligosaccharide synthesis. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3cc49205b

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Tunable acid-sensitive ester protecting groups in
oligosaccharide synthesis†
Cite this: Chem. Commun., 2014,
50, 3155
Yao Li and Xinyu Liu*
Received 3rd December 2013,
Accepted 20th December 2013
DOI: 10.1039/c3cc49205b
www.rsc.org/chemcomm
A series of acid-cleavable ester-type protecting groups, with acid- are generally required to release the native carbohydrate mole-
sensitivity profiles parallel to those of 2-naphthylmethyl (NAP) or cules. This practice is cumbersome and requires considerable
p-methoxybenzyl (PMB) ether, were designed and TFA in toluene was synthetic skill to execute successfully.6 In contrast, standardized
identified as a technically simple and effective deblocking cocktail for single-step acid- or base-mediated global deprotection procedures
their global removal in the context of oligosaccharide synthesis.
are widely adopted in peptide and nucleic acid chemistry, which
take advantage of the identical acid- or base-sensitive profiles of
Chemical synthesis of complex carbohydrates constitutes a permanent protecting groups used therein.7 To address this
powerful means to provide homogeneous glycans for their practical efficiency gap, we envision the possibility of designing
functional studies in an age of glycomics.1 The recent emergence and implementing a defined set of acid-cleavable permanent
of new synthetic tactics and strategies has notably accelerated protecting groups by strategically leveraging the reactivity space
the preparation of carbohydrates in solution and on solid that exists when applying the acidic conditions used in hydro-
supports,2 however the overall efficiency associated with oligo- lysing oligosaccharides and chemical glycosylations. In this
saccharide assembly remains unmatched to the efficiencies of communication, we report the generation and application of
oligonucleotide and oligopeptide synthesis. The practical applic- a series of acid-cleavable ester-type protecting groups, of which
ability of automated nucleic acid and peptide synthesis is a the acid-sensitive profiles are tuned to be in sync with those of
combined result of the development of highly efficient coupling 2-naphthylmethyl (NAP) or p-methoxybenzyl (PMB) ether. The
reagents and robust protecting group chemistry.3 While there combination of these designer esters with NAP/PMB ethers
has been a collective effort to improve the efficiency of chemical allows for the establishment of a versatile permanent protect-
glycosylations,4 strategic development of protecting group ing group inventory. It also elevates the efficiency and practical
chemistry to parallel the efficiency in preparing oligonucleotides applicability of late stage protecting group manipulation for
and oligopeptides lags behind.
hexose-containing oligosaccharide assemblies to a level that
The protecting group strategy in modern oligosaccharide has been traditionally reserved for peptide and nucleic acid
synthesis is to rely on highly acid/base-stable benzyl ethers as synthesis.
the dominant protecting groups for hydroxyl functionalities.
Protic acids such as trifluoroacetic acid (TFA), commonly
A further set of orthogonal protecting groups are applied that used for global deblocking events in peptide chemistry,7b are
serve their capabilities, either temporarily or permanently, in rarely explored analogously in oligosaccharide chemistry. This
ensuring regioselectivity and stereoselectivity as well as tuning is in part due to the perception that an oligosaccharide mole-
the reactivity of glycosylating agents during chemical glyco- cule is prone to acid-mediated hydrolysis,8 but is also due to the
sylation events.5 Due to the introduction of a diverse set of fact that the inventory of practically useful acid-labile protecting
orthogonal protecting groups, the lack of efficiency in protecting groups in carbohydrate chemistry is restricted to acetals and silyl-
group manipulation is markedly notable at the late stage of any and PMB-type ethers that possess limited stability in Lewis acid-
given oligosaccharide synthesis where multistep transformations mediated chemical glycosylation.9,10 We recently discovered that
NAP ether is readily cleaved from an oligosaccharide backbone
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue,
with a 20 equivalent excess of HFÁPy in toluene under ambient
Pittsburgh, PA 15260, USA. E-mail: xinyuliu@pitt.edu; Fax: +1-412-624-8200;
conditions without compromising a glycosidic bond, while
Tel: +1-412-624-6932
remaining stable in the presence of a stoichiometric amount of
† Electronic supplementary information (ESI) available: Details of synthetic
strong Lewis acid (TMSOTf or BF3ÁOEt2).11 This unique reactivity
procedures and spectral data of newly synthesized compounds. See DOI:
10.1039/c3cc49205b
of NAP ether towards acids suggests the possibility of using it as
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Scheme 1 Efficient global removal of NAP ethers in dodecyl maltoside 1
by TFA in toluene.
Scheme 3 Efficient single-step preparation of PMB/NAP-modified acetic
and benzoic acid homologues (3a–b and 4a–b respectively).
an acid-cleavable global protecting group in oligosaccharide
synthesis. To test the feasibility of this approach, we screened a
panel of protic acid conditions which were devoid of HF. This effort
led us to identify TFA in toluene (10 : 1, v/v) as an effective cocktail
to unmask all NAP ethers in an oligosaccharide, as demonstrated
by their quantitative removal under ambient conditions12 from a
per-NAP protected dodecyl maltose 1 (Scheme 1).
steric elements17 that allow for the generation of analogous acetate
and benzoate esters with reversed cleavage kinetics, assisted by
acid rather than base (vide infra).
To validate our design, we first set out to establish a
practical route to generate the acylating agents used for acces-
sing the target designer esters (Scheme 3). We identified that
alkylative opening of readily available18 g-butyrolactone 3 and
phthalide 4 with PMBCl or NAPBr and KOH in toluene con-
stitutes a highly efficient single-step method to prepare PMB-
and NAP-capped 4-hydroxybutanoic acids (3a and b) as well as
2-(hydroxymethyl)benzoic acid analogues (4a and b). All target
carboxylic acids can be obtained in pure forms and good yields
after a simple extraction-based workup protocol (ESI† method).
Carboxylic acids 3a/3b and 4a/4b were abbreviated as
PMBAcOH/NAPAcOH and PMBBzOH/NAPBzOH respectively to
highlight their structural characteristics, including their steric
similarities to acetic and benzoic acid, as well as the identity of
the acid-labile trigger groups. Installation of 3a, 3b, 4a and 4b
into a carbohydrate backbone was readily achieved using DCC
mediated esterification to afford a series of 6-O-designer-ester
capped 1-O-methyl 2,3,4-tri-O-benzoyl glucosides 6 in excellent
yields (Table 1, 5 - 6).
Acid-assisted cleavage of the designer esters 6 was sub-
sequently evaluated with low and high concentrations of TFA
in toluene (1 : 10 and 10 : 1, v/v respectively) under ambient
conditions. Consistent with our designs, a marked difference in
the sensitivity towards TFA was observed between the PMBAc
and NAPAc groups. At a low concentration of TFA, PMBAc was
readily removed within 1 h (Table 1, entry 1) and NAPAc
remained completely stable (Table 1, entry 3a). Clean removal
of NAPAc from 6c required 8 hours exposure to a high concen-
tration of TFA (Table 1, entry 3b), analogous to that used for
NAP ether. Neither low nor high concentrations of TFA affect
the stability of classical benzoyl esters embedded in 6, or cause
acyl migrations. Cleavage of the PMBBz and NAPBz groups was
accelerated 12- and 5-fold (Table 1, entries 2 and 4) respectively,
compared with their Ac analogues under otherwise identical
conditions. This observation is in agreement with our expecta-
tion that increased steric constraint poised by the phenyl ring
in 2-(hydroxymethyl)benzoyl ester promotes lactone formation.
With the acid-sensitivity profiles of designer esters elucidated,
we proceeded to examine in depth their compatibility with other
known esters in addition to benzoate (Scheme 4). Differentially
protected glucosides 7 and 8 were prepared from 1-O-methyl
2,3-di-O-benzoyl glucoside (ESI† method). Orthogonality between
PMBBz and levulinate (Lev) was demonstrated using glucoside 7,
where regiospecific removal of Lev (7 - 7a) and PMBBz (7 - 7b)
The acidic strength of TFA in toluene (10 : 1, v/v) resides
distinctly between the strengths of acids required for hexose-
containing oligosaccharide hydrolysis13 and acids for chemical
glycosylations, implicating its potential as a practically useful
global deblocking cocktail in oligosaccharide synthesis, reminiscent
of those used in peptide chemistry.7b To generalize this approach,
it is necessary to have a collection of readily accessible protecting
groups that share analogous acid-sensitivities to NAP-ethers.
Among the most widely utilized protecting groups for the hydroxyl
functionality in carbohydrate chemistry are esters, including
acetate, benzoate and pivaloate, which are immune to TFA in
toluene (vide infra). As esters play indispensable roles beyond
simple ‘‘protections’’ by balancing the reactivity of chemical
glycosylating agents, as well as serving as effective stereo-
directing groups for 1,2-trans glycosidic bond formation,14 we
set out to develop a series of designer esters that retain their
essential properties as acetates and benzoates, yet can be
readily released by exposure to TFA in toluene.
Our design principle (Scheme 2) for engineering acid-labile
esters was inspired by early works relating to pilocarpine
prodrugs, in which intramolecular cyclization of pilocarpic acid
esters to form pilocarpine occurs rapidly over a broad range
of pHs in aqueous media.15 Although relayed deprotection of
modified esters has been explored in synthetic carbohydrate
chemistry, precedent studies have extensively focused on their
development in the context of temporary protection and lack
modularity and practical efficiency in their preparation.16 We
rationalize that acid-triggered 5-membered lactone formation
is a versatile route for modular design of tunable acid-sensitive
esters where, the acid-sensitivity difference between PMB and
NAP ethers that masks the latent hydroxyl nucleophile can be
strategically explored for creating two sets of otherwise-identical
esters with different profiles towards acid (Scheme 2). In addition,
the rate of lactone formation can be readily tuned by incorporating
Scheme 2 Design principle for tunable acid-sensitive esters.
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Table 1 Preparation of designer ester modified glucosides 6 and their cleavage by TFA in toluene
Entry
5 - 6 yield
6 - 5 conditions, yield
1
2
3
6a (Protecting group, PG = PMBAc), 93%
6b (PG = PMBBz), 97%
6c (PG = NAPAc), 95%
TFA/PhMe (1 : 10, v/v), rt, 1 h, 98%
TFA/PhMe (1 : 10, v/v), rt, 5 min, 98%
(a) TFA/PhMe (1 : 10, v/v), rt, 1 h, N.R.
(b) TFA/PhMe (10 : 1, v/v), rt, 8 h, 94%
TFA/PhMe (10 : 1, v/v), rt, 1.5 h, 96%
4
6d (PG = NAPBz), 97%
Scheme 5 Application of NAPBz designer esters and NAP ethers in the
stereoselective preparation of a(1–6) di- and trimannosides and their
single-step global deprotection by TFA in toluene (10 : 1, v/v).
Scheme 4 Evaluation of the orthogonality between designer esters
(PMBBz & NAPBz) and classical esters (Ac, Lev and Bz).
was readily accessible from a mannosyl tricyclic orthoester
(ESI† method), along with a reducing-end mannoside 9a.
were readily achieved using hydrazine and a low concentration Glycosylation of 9a with 10 provided dimannoside 11a in
of TFA in toluene, respectively. Furthermore, the hydrolysis of excellent yield and the subsequent exposure of the primary
6-O-acetate in 8 was quantitatively achieved using dilute methanolic triisopropylsilyl (TIPS) ether group in 11a to methanolic HCl
HCl in the presence of NAPBz (8 - 8a, Scheme 4), demonstrating (ESI† method) quickly afforded dimannoside 9b with the NAP-
that NAPBz retains the essential character of benzoate, which in derived protecting groups intact. Combination of 9b with 10
comparison to acetate, is more resistant to acid- or base-catalyzed gave trimannoside 11b in 94% yield. Both 11a and 11b are
hydrolysis in water or methanol. Cleavage of 4-O-NAPBz in 8 by a representative end products of a chemical glycosylation assembly
high concentration of TFA in toluene was also equally effective, line, where three layers of apparent orthogonality exist for hydroxyl
leaving 6-O-acetate intact (8 - 8b, Scheme 4).
protecting groups. This would traditionally have required three
These experiments collectively demonstrate the versatile nature distinct chemical transformations including fluoride-mediated
of acid-sensitive designer esters that are robustly orthogonal to desilylation, Zemplen-type deacylation and hydrogenolytic
classical esters while retaining their essential characters, and set debenzylation to release the native oligomannosides. By
the stage for exploring them in complex carbohydrate assemblies. utilizing the uniform TFA-sensitive properties of NAP ether
We use the preparation of linear a(1–6) mannan as a model system and NAPBz ester, as well as the innate acid-sensitive nature of
to illustrate the enabling features of NAPBz ester and NAP ether silyl ether, conversions of 11a/11b to the corresponding azide-
(Scheme 5), including their interchangeability with the classical tagged 12a/12b were effectively achieved using TFA in toluene
benzoate and benzyl ether respectively and their global removal by (10 : 1, v/v) in 4 hours under ambient conditions, with minimal
TFA in toluene.
technicalities on work up (ESI† method), to afford analytically
Glycosyl phosphate was chosen as the glycosylating agent for pure compounds in quantitative yields.
preparing the target di- and trimannosides 11a/11b, as it requires
In summary, we described the practical and modular genera-
a stoichiometric amount of strong Lewis acid (TMSOTf) for tion of a series of tunable acid-sensitive ester protecting groups
activation19 that serves as a test ground to evaluate the stabilities that are effective surrogates of acetate and benzoate. The
of NAPBz ester and NAP ether during chemical glycosylations. synchronized acid-sensitive profiles of these designer esters with
The key glycosylation building block, glycosyl phosphate 10, PMB and NAP ethers enabled the application of a single-step
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with unsaturated lipids. Strict anhydrous conditions are required to
minimize acid-mediated cleavage of the protecting groups in these
studies, according to the corresponding experimental supporting
information. (a) B. M. Swarts and Z. Guo, J. Am. Chem. Soc., 2010,
132, 6648; (b) A. V. Nikolaev and N. Al-Maharik, Nat. Prod. Rep., 2011,
28, 970.
TFA-mediated global deprotection protocol at the final stage of
oligosaccharide assembly that emulates the synthetic efficiency
traditionally reserved for peptide and nucleic acid chemistry.
As glycosidic linkages between mammalian hexoses are very
tolerant of TFA in toluene (10 :1, v/v) under ambient conditions,13
we envision that NAP ethers and NAPBz esters can readily find
broad applications in complex carbohydrate assemblies by
directly replacing benzyl ethers and acetates/benzoates. Further
generalization of the single-step TFA-mediated global deprotec-
tion protocol in oligosaccharide chemistry would call for the
development of acid-sensitive nitrogen protecting groups that
are effective during chemical glycosylations. Extension of this
strategy to mammalian oligosaccharides with more acid-labile
glycosidic linkages (such as a-fucoside) requires the implemen-
tation of PMB ethers and PMBBz esters, which would necessi-
tate the development of novel glycosylating agents, activatable
under ‘‘acid-neutral’’ conditions.20 The details of this work will
be reported in due course.
11 Y. Li, B. Roy and X. Liu, Chem. Commun., 2011, 47, 8952.
12 Ambient conditions refer to those during the period of time when
this work was conducted on the 5th floor of the Chevron Science
Center, University of Pittsburgh, fumehood #0376, with an average
air temperature of 15–22 1C from July 2011 to January 2012 (see ESI†
spectra). All TFA-mediated deprotections described in this manu-
script were carried out in a 10–25 mL round bottom flask capped
with a rubber septum under normal atmosphere. No nitrogen or
argon gas was used. HPLC grade toluene (Sigma-Aldrich, cat# 34866)
and reagent grade TFA (Sigma-Aldrich, cat# T6508) were used as
received throughout this study.
13 To the best of our knowledge, all precedent studies related to the
acid-stability of glycosidic linkages in naturally occurring oligo-
saccharides were conducted in aqueous solutions, see early works
(a) W. G. Overend, J. S. Sequeira and C. W. Rees, J. Chem. Soc., 1962,
3429; (b) R. D. Marshall and A. Neuberger, in Glycoproteins: Their
Composition, Structure and Function, ed. A. Gottschalk, Elsevier,
New York, 1966, pp. 224–299. We have tested the sensitivity of a
series of monosaccharides and oligosaccharides, including methyl
a-glucoside/a-mannoside/b-galactoside, maltose, lactose and malto-
triose to TFA in toluene (10 : 1, v/v) and observed no product
degradation up to 8 h under the ambient conditions outlined in
ref. 12. According to ref. 13a and b, methyl b-galactoside is the
most acid-labile methyl glycoside among mammalian hexoses and
hexosamines.
This work is supported by a start-up fund from the Depart-
ment of Chemistry, University of Pittsburgh.
Notes and references
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3158 | Chem. Commun., 2014, 50, 3155--3158
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