Encouraging progress in the ω-aspartylation of complex oligosaccharides as a general route to β-N-linked glycopolypeptides

Article abstract of DOI:10.1021/ja110115a

Described herein is a method for the joining of complex peptides to complex oligosaccharides via an N-linkage. The ω-aspartylation is conducted by coupling fully deprotected glycosylamine with a peptide containing a unique thioacid at the ω-aspartate carb

Full text of DOI:10.1021/ja110115a

ARTICLE
pubs.acs.org/JACS
Encouraging Progress in the ω-Aspartylation of Complex
Oligosaccharides as a General Route to β-N-Linked Glycopolypeptides
Ping Wang,† Xuechen Li,† Jianglong Zhu,† Jin Chen,† Yu Yuan,† Xiangyang Wu,† and Samuel
J. Danishefsky*,†,‡
†Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York,
New York 10065, United States
‡Department of Chemistry, Columbia University, Havemeyer Hall, 3000 Broadway, New York, New York 10027, United States
S Supporting Information
b
ABSTRACT: Described herein is a method for the joining of
complex peptides to complex oligosaccharides via an N-link-
age. The ω-aspartylation is conducted by coupling fully
deprotected glycosylamine with a peptide containing a unique
thioacid at the ω-aspartate carboxyl. In the presence of HOBT,
under conditions that, in principle, allow for oxidation, com-
plex components are combined in encouraging yields to produce structurally and stereochemically defined N-linked glycopolypep-
tides wherein the carbohydrate domain can be quite complex. Various mechanisms for oxidative coupling are proposed.
’ INTRODUCTION
carbohydrate synthesis.6 This method provides access to very
complex, highly branched oligosaccharides with generally excel-
lent levels of stereocontrol, while easing the complexities of
protecting group management. Additionally, significant efforts in
our laboratory have focused on the development of a broad menu
of useful methods for the ligation of peptide and glycopeptide
fragments.7-9 However, at the outset of the program we describe
herein, solutions to the second challenge stated above, that is, the
actual construction of the β-N-linkage required to connect the
oligosaccharide and peptidic domains in nature’s way, remained
problematic.
The current, state-of-the-art method for merging glycans to
peptides through a natural N-linkage10 involves appendage of a
fully deprotected anomeric β-glycosylamine of a complex oligo-
saccharide, to a properly protected and differentiated, ω-aspar-
toyl acyl donor in the setting of a peptide, that is, the Lansbury
reaction (Figure 1).11 This approach must overcome several
limitations. First, the anomeric glycan amine is relatively weakly
nucleophilic and is also vulnerable to hydrolytic conversion to
the free reducing sugar. The ω-aspartylation reaction can be
seriously compromised by a competing side reaction, wherein, in
the presence of a requisite base (such as diisopropylethylamine
[DIPEA]), the activated ω-aspartyl donor undergoes cyclization
with the NH of the adjacent amino acid. Depending on the
nature of the peptide and the amount of base used, the levels of
aspartimide formation may be prohibitive.12 For instance, when
the next amino acid located at the C-terminus of the aspartate
residue is of the glycine, serine, or alanine type, aspartimide
formation is particularly pronounced. Because the weak nucleo-
Glycoproteins play pivotal roles in an array of biological
processes, from intercellular communication to neuronal devel-
opment and fertilization.1-3 For instance, protein glycosylation
is understood to be essential in promoting proper protein fold-
ing, thereby governing proteolytic stability. Unfortunately, rig-
orous investigations as to the relationship between the structure
and function of glycoproteins have been complicated by the often
prohibitive difficulties in obtaining homogeneous samples from
natural sources. This situation arises from the fact that glycopro-
teins, biosynthesized through nonspecific post-translational gly-
cosylation, are typically isolated as horrific mixtures of difficult to
separate glycoforms.
Our laboratory,4 and others,5 have sought to respond to this
challenge through the development of general strategies for the
de novo chemical synthesis of homogeneous glycoproteins.
Notwithstanding substantial advances in this regard, the total
synthesis of significantly sized N-linked glycopolypeptides, let
alone N-linked glycoproteins, remains a daunting task. The chal-
lenges can be encompassed into four broad categories: (i) assembly
of complex oligosaccharide fragments; (ii) actual merger of the
oligosaccharide and peptide domains; (iii) ligation of component
glycopeptide and peptide fragments to provide the fully elabo-
rated glycoprotein structure; and (iv) deprotection, if necessary.
In the past two decades, our laboratory has attempted to
advance the field of glycopeptide and glycoprotein synthesis
through the development of enabling methodologies, both in
constructing oligosaccharides and in combining polypeptides,
including glycopolypeptides. Recent years have brought forth
major advances in the ability to reach single glycoforms of even
highly complex oligosaccharide targets. One of the initiatives from
our laboratory involved the development of “glycal assembly” for
Received: November 10, 2010
Published: January 5, 2011
r
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Figure 3. Thioacid peptides 7-13.
Figure 1. Merger of glycan and peptide domains through aspartylation;
undesired base-induced aspartimide formation.
Figure 2. Proposed base-free amide β-asparagine linkage through
interception of a thio-FCMA (formimidate carboxylate mixed anhydride)
intermediate.
philicity and the lability of the anomeric glycosylamine are given,
we focused on enhancing the intermolecular acyl donor capacity
of the ω-aspartoyl function. It was reasoned that if the need for
external base in the Lansbury method could be avoided, compe-
tition from aspartimide formation could well be attenuated.12 It
was with this view that we began.
Along these lines, we recently disclosed the development of an
efficient, base-free method for the generation of complex amide
bonds. Our line of discovery arose from revisitation of long
known, but under-appreciated functional groups, that is, iso-
nitriles.13,14 The presumed mechanism of our amidation reaction
(Figure 2) starts with addition of a thio acid (1) to an isonitrile
(2), thereby generating a thio formimidate-carboxylate mixed
anhydride (termed a thio-FCMA, i.e., intermediate 3). In the
absence of external nucleophile, 3 undergoes 1,3-SfN acyl
migration to generate 6 via what we have referred to13 as a
two-component coupling (2CC). Alternatively, the intermediate
thio-FCMA 3, being a powerful acyl donor, can be intercepted by
an external amine nucleophile (cf., 4), thus leading to the
formation of amide product (5).15 This reaction proceeds readily
at room temperature, thereby providing even quite hindered
amides in high yields. Notably, this new amide forming reaction
does not require the presence of an added base. It was in this
context that we theorized as to whether this newly developed
thio-FCMA amide coupling method could be applied to the
challenge of “ω-aspartylation” as required for the synthesis of
N-linked glycopeptides (vide supra). More specifically, we envi-
sioned a setting wherein thioacid 1 would represent the aspartate
residue of a peptide fragment, and structure 2 would correspond
to a “throwaway” isonitrile,16 which might otherwise revert to its
corresponding thioformamide, 6. Hopefully, in the presence of
trapping agent 4, thio-FCMA 3 would be interdicted to produce
Figure 4. Oligosaccharides 14-17.
5. To reach our objective, wherein 5 corresponds to N-linked
glycopeptides, the nucleophile must be the fully deprotected
β-anomeric glycosylamine of a suitably complex oligosaccharide.
’ RESULTS AND DISCUSSION
As shown in Figure 3, a range of peptides containing ω-Asp
thioacids was prepared for evaluation. The general synthetic
procedure for 9-13 is outlined in Figure 3 (thioacids 7 and 8
were prepared from previously described procedures;14b details
may be found in the Supporting Information). The requirement
of emplacement of a thioacid function at the ω-aspartyl terminus
does not constitute a significant complication to the method.
Solid-phase peptide synthesis (SPPS) served well to provide the
protected peptide substrates.17a,b Following conversion of the
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C-terminal carboxylic acids to their methyl ester functions, and
subsequent deprotection of the ω-aspartate esters, the ω-aspartyl
carboxylate moieties were converted to thioesters. Upon treat-
ment with cocktail TFA18 and purification by RP-HPLC, the
ω-aspartyl thioacid peptide substrates (9-13) were in hand.
Notably, peptides 9-11 were well suited for a discriminating
evaluation because they comprise aspartimide-prone (Asp-Ser)
sequences.12
As shown in Figure 4, the glycosylamines selected for inclusion
in this study are chitobiose 14, hexasaccharide 15, dodecasac-
charide 16, and the particularly complex tridecasaccharide 17.
The syntheses of these putative γ-aspartyl acceptors have been
described.19
Table 1. Thio-FCMA-Mediated Aspartylationa
With the peptide thioacids and glycosylamine substrates in
place, we were in a position to attempt their merger, through a
natural ω-asparagine linkage. As shown in Table 1, entry 1, in the
presence of cyclohexylisonitrile, dipeptide thioacid 7 and glyco-
sylamine 14 successfully underwent internal base-free aspartyla-
tion to afford the glycopeptide adduct in 70% yield, along with
low levels of peptide carboxylic acid, aspartimide, and products
arising from apparent intramolecular SfN rearrangement of the
thio-FCMA (cf., Figure 2, compound 6, where R1 = C-terminal
peptide and R3 = cyclohexyl). Attempts to accomplish coupling
with more complex peptide and glycosylamine substrates
(entries 2-3) required the use of significant excesses of peptide
thioacid (3 equiv), for glycopeptide adducts to be obtained in
moderate to good yield. Coupling of thioacid peptide 13 with
glycosylamine 15 proceeded in only 19% yield, even when
4 equiv of peptide was used (entry 4). Nonetheless, the results
shown in Table 1 already seemed to comprise significant progress
in the convergent formation of N-linked glycopolypeptides.
In light of these encouraging proof of principle findings, we
sought to identify improved reaction conditions, which would
allow for the use of fewer equivalents of peptide thioacid and
would give rise to adducts with higher efficiency (still operating
in the mechanistic logic adumbrated in Figure 2). We also hoped
a DMSO, 4 Å MS, 2-8 equiv of cyclohexyl isonitrile.
Figure 5. Possible mechanisms for HOBT-mediated oxidation.
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to better evaluate the mechanism proposed for the results shown
in Table 1. It was in this context that we came upon a fortuitous
discovery. In conducting a necessary control experiment, which
we fully expected to be negative, it was observed that the reaction
of peptide 7 and chitobiose 14 reacted to form small amounts of
coupled product (∼20%) in the absence of cyclohexylisonitrile.
At the time, this was unsettling, because the isonitrile had been
assumed to be the defacto activating agent. However, on the basis
of the analogy with our recent discoveries in the field of classical
peptide bond formation,20 we soon postulated that coupling
might be proceeding through low levels of an adventitious
oxidation agent (air!). Interestingly, when 2 equiv of HOBT21
was added to the reaction, conducted under air, the yields of the
glycopeptides were enhanced to as high as 85%, accompanied by
small amounts of peptide carboxylic acid and very low levels of
aspartimide.
Table 2. HOBT-Mediated Aspartylation
It is well to emphasize that the result of the control experiment
does not invalidate the thio-FCMA mechanism proposed above.
Indeed, that pathway must be operative at some level, because
significant levels of the thioformamide 6 type products are
produced when isonitrile 2 was in fact employed. However,
there is clearly another, perhaps concurrent, operative pathway,
which does not involve the isonitrile-mediated thio-FCMA
pathway. By analogy to our recent findings resulting in a novel
route to peptide ligation,20 we now propose a mechanistic
framework that is not unlike that suggested by Liu and Orgel,22,23
to explain the oxidative acylation of thioacids as a route to the
formation of amides under prebiotic conditions, although with
two major caveats. First, our chemistry seems to operate via low
levels of oxidation (air) and does not require strong oxidizing
agents. Moreover, the method works even with the rather weakly
nucleophilic and quite labile β-anomeric glycosyl amine even
when the glycans are quite complex. As a mechanistic model, it is
proposed that perhaps, upon exposure to air, the peptide thioacid
(22) suffers low levels of oxidation to afford a strong acyl donor
intermediate (Figure 5, eq 1). For the sake of discussion rather
than on the basis of solid proof, we consider a structure of the
type 23 in an oxidation-mediated mechanistic format to rationa-
lize our results (vide infra). Conjecturing further, perhaps in the
presence of HOBT, 23 reacts to give rise to HOBT ester 24,
along with 25. The latter could rapidly react with HOBT, to also
yield 24 (eq 2), which is then activated for coupling with the
glycosylamine nucleophile (24f26, eq 4). Alternatively, 22 can
react with 25 to reconstitute 23 (eq 3), which re-enters the cycle
as shown in Figure 5.
Several findings support this oxidative mechanistic proposal.
Thus, when the reaction is performed under conditions designed
to suppress the oxidation, the resulting yields of N-linked glycan
are sharply attenuated. Furthermore, even when only 1 equiv of
peptide thioacid is used, in the presence of air, the glycopeptide
product yields are well over 50%, suggesting that oxidized inter-
mediates (perhaps 23) can ultimately give rise to two coupling
acyl donors (possibly another molecule of 23), thus allowing the
yields to rise to their observed levels.
While the data above, particularly in the context of the earlier
experiments, are strongly suggestive that the thioacid (22)
undergoes oxidative activation at substoichiometric levels, they
do not preclude the possibility of concurrent nonoxidative varia-
tions to account for the acyl donor quality of the thioacid. For
instance, nothing in our data package convincingly precludes the
possibility that a thioacid, such as 22, has some intrinsic acyl
donor capacity that can be tapped for nonoxidative amide bond
a Isolated yield. The ratio of products was determined by LC-MS of the
crude reaction mixture (product:acid:aspartimide). b 89:10:1. c 82:17:1.
d 59:37:4. e 52:44:2. f 60:35:5. g 63:34:3. h 62:34:4.
formation (eq 5). Alternatively, it is possible that two thioacid
molecules could give rise to a thio-anhydride, which, of course,
can function as an acyl donor (eq 6). Conceivably, even the per
thioacid (25, generated as shown in eq 1) has inherent acyl donor
capacity. Finally, we cannot rule out the possibility that the
thioacid reacts with HOBT to produce a thiol-BT acyl donor
(eq 7). Clearly, this cannot be the sole pathway, because the
asparagine linkage can be established in the absence of HOBT.
The precise delineation of the operative mechanism(s) could
well require extensive research. However, the data in hand point
to substoichiometric oxidation as an important pathway leading
to amide from thioacid and β-anomeric glycosyl amines.
As shown in Table 2, the HOBT-mediated protocol has been
extended to an impressive range of complex peptide thioacid and
oligosaccharide domains. Thus, the aspartimide-prone peptides,
10 and 11, were successfully merged with chitobiose 14 and
hexasaccharide 15 in good yields (entries 4-6, 8). In the
coupling of 11 and 15, an excess of peptide (1.5:1) was used,
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given the complexity of the hexasaccharide component. In this
case, a 75% yield of coupled product was obtained, and the excess
peptide thioacid had decomposed to carboxylic acid and
aspartimide (entry 8). Coupling of peptide 11 with dodeca-
saccharide 16 gave glycopeptide 33 in 49% yield (entry 9).
The aspartylation of peptide 11 with the complex tridecasac-
charide 17 proceeded in 39% yield (entry 10). By comparison,
aspartylation under standard conditions18 is accomplished in
only 20% yield following the use of larger excesses of peptide
thioacid acyl donors.
’ AUTHOR INFORMATION
Corresponding Author
s-danishefsky@ski.mskcc.org
’ ACKNOWLEDGMENT
Support was provided by the National Institutes of Health
(CA103823 to S.J.D.). We thank Dr. George Sukenick, Ms. Hui
Fang, and Ms. Sylvi Rusli of the Sloan-Kettering Institute’s NMR
core facility for mass spectral and NMR spectroscopic analysis,
and Ms. Rebecca Wilson and Dr. William Berkowitz for valuable
discussions.
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’ ASSOCIATED CONTENT
S
Supporting Information. General experimental proce-
b
dures, including spectroscopic and analytical data for new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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