An advance in the chemical synthesis of homogeneous N-linked glycopolypeptides by convergent aspartylation

Article abstract of DOI:10.1002/anie.201205038

Like a Pro: A one-flask aspartylation/deprotection method, wherein long peptide fragments, bearing proximal pseudoproline functionality, are merged with complex glycan domains has been developed. Following aspartylation, acid-mediated global deprotection reveals the elaborated glycopeptide. The temporary pseudoproline functionality serves to suppress formation of aspartimide side products during solid-phase peptide synthesis and aspartylation. Copyright

Full text of DOI:10.1002/anie.201205038

Angewandte
Chemie
DOI: 10.1002/anie.201205038
Glycoprotein Synthesis
An Advance in the Chemical Synthesis of Homogeneous N-Linked
Glycopolypeptides by Convergent Aspartylation**
Ping Wang, Baptiste Aussedat, Yusufbhai Vohra, and Samuel J. Danishefsky*
Our research group has a longstanding interest in the
development of improved methods for the chemical synthesis
of structurally complex proteins and glycoproteins.[1] Our
efforts started with the study of new departures in oligo-
saccharide synthesis,[2] as well as in polypeptide synthesis.[1a]
Building from the foundations of these key constituents, we
have recently turned our attention to merging such subunits
to produce glycoproteins.[3] Toward these pursuits, the com-
plex, multiply glycosylated protein, erythropoietin (EPO),[4]
has served as a defining synthetic target.[5] Our focusing goal,
that is, a total synthesis of homogeneous EPO, has served to
prompt a variety of ventures directed to enabling advances in
glycoprotein synthesis. We disclose herein a valuable new
aspartylation technology, developed in the context of our
EPO program, which allows for the highly convergent
synthesis of complex glycopeptides from fully elaborated
peptide and carbohydrate fragments.
Several general strategies are commonly employed for the
assembly of N-linked glycoprotein and glycopeptide targets,[6]
though each approach suffers from significant limitations in
scope or efficiency. According to the stepwise glycosylamino
acid approach (Scheme 1a),[7] the carbohydrate domain is
first appended to an 9-fluorenylmethyloxycarbonyl- or tert-
butoxycarbonyl-protected Asp residue. The resultant glyco-
sylamino acid is subsequently used directly in solid-phase
peptide synthesis (SPPS). When the carbohydrate component
Scheme 1. Strategies for aspartylation.
is complex, the overall efficiency of this linear strategy is
significantly compromised by low reaction yields obtained
during the glycosylamino acid coupling step and the subse-
quent elongation of the next amino acid. Moreover, any sialic
acid motifs in the glycan must be protected during SPPS.
Glycopeptides of up to 20 amino acids in length may be
generated through this method.[8]
A second SPPS-based approach offers enhanced conver-
gence. According to the on-resin a-linked glycopeptide
strategy[9] (Scheme 1b), the resin-bound peptide, assembled
through SPPS, is selectively deprotected to reveal the Asp
residue. Coupling of the glycan domain, followed by TFA-
mediated cleavage from the resin, delivers the glycopeptide
fragment. Two factors serve to mitigate the broad utility of
this approach. In certain peptide sequences, particularly those
incorporating aggregation-prone sectors, the SPPS-derived
resin-bound peptide can suffer from significant impurities.
Appendage of a high-value glycan domain to impure peptide
substrate thus results in significant loss of precious material
and formation of difficultly separable mixtures of glycopep-
tide product. Moreover, release from the resin delivers
glycopeptide presenting a C-terminal carboxylic acid, which
must then be converted into an activated thioester function-
ality prior to subsequent native chemical ligation[10] with
a peptide or glycopeptide coupling partner.
[*] Dr. P. Wang, Dr. B. Aussedat, Y. Vohra, Prof. S. J. Danishefsky
Laboratory for Bioorganic Chemistry
Our research group has been employing[1] the convergent
aspartylation approach pioneered by Lansbury and co-work-
ers[11] (Scheme 1c). In this protocol, a moderately sized,
partially protected peptide, bearing the free aspartyl residue,
is merged with the glycosyl amine to generate a glycopeptide
fragment. While useful for producing short peptide fragments,
this method is often compromised when long sequences are
being joined with glycosylamine. Coupling yields may be
badly undermined by peptide decomposition pathways, pre-
Sloan-Kettering Institute for Cancer Research
1275 York Avenue, New York, NY 10065 (USA)
E-mail: s-danishefsky@ski.mskcc.org
Prof. S. J. Danishefsky
Department of Chemistry, Columbia University
3000 Broadway, New York, NY 10027 (USA)
[**] This research was supported by NIH grant HL25848 (S.J.D.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201205038.
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dominantly by aspartimide formation (Scheme 1d).[12]
Accordingly, our general approach to glycoprotein synthesis
has involved assembly of short glycopeptide fragments
through Lansbury aspartylation, followed by ligation with
a long peptide domain, which may itself contain a glycosyla-
tion site. Although not maximally convergent, this strategy is
apt to offer ease of use and a high measure of control over
glycopeptide purity.
In the context of our ongoing EPO synthesis, we sought to
apply this less convergent strategy to the assembly of the
EPO(29–78) glycopeptide fragment. Thus, glycopeptide 1,
encompassing the EPO(29–40) peptide sector bearing hex-
asaccharide 4,[13] was prepared through Lansbury aspartyla-
tion. However, all efforts to couple 1 with peptide 2 [EPO(41–
78)] delivered prohibitively low yields of 3 (Scheme 2). We
aspartimide avoidance in the Lansbury coupling. Clearly,
placement of a pseudoproline motif, derived from Ser or Thr,
at the (n + 1) position necessarily blocks aspartimide forma-
tion at the n position.[15] Driven by curiosity, rather than by
a convincing rationale, we wondered whether temporary
installation of the pseudoproline motif at the (n + 2) position
might also somehow serve to suppress the undesired asparti-
mide formation (Scheme 3). Because a Ser or Thr residue is
universally located at the (n + 2) position (relative to Asp) in
native protein sequences, success in this regard could bring
with it major enabling progress in the chemical synthesis of
homogeneous complex glycopolypeptides.
Scheme 3. Pseudoproline dipeptide.
We examined this possibility in the context of the
EPO(29–78) segment. Thus, through recourse to SPPS, we
prepared partially protected peptide 5, incorporating several
pseudoproline dipeptides[16] (highlighted in blue, Scheme 4),
including one at the (n + 2) position relative to Asp. In the
crucial transformation, peptide 5 readily underwent coupling
with chitobiose (7) under Lansbury conditions. Subsequent
addition of TFA cocktail (TFA/phenol/water/TIS; 88:5:5:2)
served to unmask the pseudoproline motifs and remove the
peptide protecting groups, thereby delivering the target
glycopeptide, 6a, with quantitative conversion and 53%
yield upon isolation. Peptide 5 also underwent one-flask
aspartylation/deprotection with the more complex hexasac-
Scheme 2. Attempted synthesis of EPO fragment 3. Reaction condi-
tions: Gn·HCl (6m), Na2HPO4 (0.1m), TCEP·HCl (50 mm), pH 6.8.
Gn=guanidine, NCL=native chemical ligation, TCEP=tris(2-carbox-
yethyl)phosphine.
attribute the failure of this attempted aspartylation to the low
solubility of peptide 2, which contains a large hydrophobic
domain. Even appendage of a C-terminal PEGylated thio-
ester did not improve the solubility of the peptide to an
acceptable level.[14]
An alternative approach, which would allow us to
circumvent issues of peptide solubility and would provide
maximum efficiency, envisions merger of the full peptide
sequence with the glycan through aspartylation. The success-
ful implementation of this strategy would require conditions
under which the problematic aspartimide formation (see
Scheme 1d) could be suppressed or even eliminated.
The consensus sequence for N-glycosylation is Asn-Xaa-
Ser/Thr. With this generic structural pattern in mind, we
evaluated a potential general solution to the challenge of
Scheme 4. Synthesis of glycopeptides 6a and 6b. Pseudoproline
dipeptides are depicted in blue. Amino acids protected with acid-labile
protecting groups are shown in bold. Amino acid protecting groups
are: E(tBu), H(Trt), S(tBu), N(Trt), K(Boc), Y(tBu), W(Boc), R(Pbf),
Q(Trt). Boc=tert-butyloxycarbonyl, DIPEA=diisopropylethylamine,
DMSO=dimethylsulfoxide, HATU=O-(7-azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate, TFA=trifluoro-
acetic acid, TIS=triisopropylsilane, Trt=trityl.
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charide, 4, to generate 6b with 75% conversion, albeit in
somewhat lower yield upon isolation (38%).
In a further demonstration of the potential utility of this
transformation, we accomplished the aspartylation of the
modified EPO(30–78) peptide, 8, both with chitobiose and
with the challenging dodecasaccharide, 9.[17] As outlined in
Scheme 5, under our one-flask aspartylation/deprotection
Scheme 6. Attempted aspartylation of 12. Reaction conditions:
a) TMS-diazomethane CH2Cl2/MeOH; b) TFA/PhOH/H2O/TIS, 12%
over 3 steps; c) glycosylamine 7, HATU, DIPEA, DMSO. Pseudoproline
dipeptides are depicted in blue. Amino acids protected with acid-labile
protecting groups are shown in bold. Amino acid protecting groups
are: E(allyl), H(Trt), S(tBu), N(Dmcp), K(Alloc), Y(tBu), R(Pbf),
Q(Dmcp). Acm=acetamidomethyl, Alloc=allyloxycarbonyl,
Dmcp=dimethylcyclopropyl, Pbf=2,2,4,6,7-pentamethyldihydrobenzo-
furan-5-sulfonyl.
Scheme 5. Synthesis of glycopeptides 10a and 10b. Pseudoproline
dipeptides are depicted in blue. Amino acids protected with acid-labile
protecting groups are shown in bold. Amino acid protecting groups
are: E(tBu), H(Trt), S(tBu), N(Trt), K(Boc), Y(tBu), W(Boc), R(Pbf),
Q(Trt).
exposure of peptide 12 to chitobiose under Lansbury con-
ditions resulted in little or no glycopeptide formation. The
only observed products were those incorporating aspartimide
(13) and Asn (14) in place of the Asp residue. The latter
product presumably arises from addition of trace ammonia[18]
to the activated aspartate.
In contrast, the fully protected peptide 15, incorporating
the (n + 2) pseudoproline, was prepared through SPPS
(Scheme 7). C-Terminal methyl ester formation, followed by
palladium-mediated removal of the allyl group, yielded
peptide 16. The latter readily underwent one-flask aspartyla-
tion with deprotection to deliver the desired chitobiose-
containing glycopeptide 17 in 45% overall yield (starting
from SPPS). The results of this comparison study (Scheme 6
vs. 7) clearly reveal the capacity for incorporation of
a pseudoproline motif in the (n + 2) position to mitigate
aspartimide formation at the n position.
In addition to the examples provided above, this protocol
enabled the efficient and convergent syntheses of two key
glycopeptide fragments en route to homogeneous EPO,
namely, EPO(79–124) and EPO(1–28) (Scheme 8).[19]
Indeed, in the absence of the pseudoproline functionalities,
the precursor peptide domains were not amenable to
preparation through SPPS, presumably as a result of the
propensity of the peptides to undergo aspartimide formation.
In summary, through incorporation of a pseudoproline
motif at the (n + 2) Ser or Thr residue, it proved possible to
suppress otherwise competitive aspartimide-based peptide
conditions, glycopeptide 10a was obtained in 54% yield and
glycopeptide 10b was isolated in 32% yield. Notably, the
fucose and sialic acid motifs of the dodecasaccharide glycan
survived under these conditions, despite the potential sensi-
tivity of these functionalities to acid-mediated decomposition.
Drawing encouragement from these early experiments,
we sought to further establish the role of the (n + 2) pseudo-
proline in minimizing nonproductive peptide aspartimide
formation. For these studies, we selected as a model peptide
scaffold the 34-mer, 12, incorporating the aspartimide-prone
Asp-Ala-Thr sequence. It is of note that the successful
preparation of peptide 12, itself, through SPPS necessitated
the installation of the Thr-based pseudoproline motif at the
(n + 2) position (see 11). In the absence of pseudoproline
protection, serious competition from aspartimide-containing
peptide resulted, even at the SPPS stage (see the Supporting
Information for details). Apparently, the (n + 2) pseudopro-
line functionality effectively suppresses formation of asparti-
mide in SPPS, particularly at the stage of 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU)/piperidine-mediated deprotection.
In our control experiment, we evaluated partially pro-
tected 12, wherein the amine and acid functionalities are
masked by allyl and Alloc groups and the (n + 2) residue is
not protected as a pseudoproline (Scheme 6). As anticipated,
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[1] For recent reviews from our research group, see: a) C. Kan, S. J.
Danishefsky, Tetrahedron 2009, 65, 9047; b) R. M. Wilson, J. L.
Stockdill, X. Wu, X. Li, P. A. Vadola, P. K. Park, P. Wang, S. J.
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Sane, P. Nagorny, S. J. Danishefsky, J. Am. Chem. Soc. 2012, 134,
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[4] a) A. J. Sytkowski, Erythropoietin, Wiley-VCH, Weinheim,
2004; b) P. H. Lai, R. Everett, F. F. Wang, T. Arakawa, E.
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Acad. Sci. USA 2012, 109, 7196.
[6] For recent reviews on glycoprotein synthesis, see: a) B. G. Davis,
Chem. Rev. 2002, 102, 579; b) D. Gamblin, E. M. Scanlan, B. G.
Davis, Chem. Rev. 2009, 109, 131; c) R. J. Payne, C. H. Wong,
Chem. Commun. 2010, 46, 21.
Scheme 7. Synthesis of glycopeptide 17. a) TMS-Diazomethane
CH2Cl2/MeOH; b) [Pd(PPh3)4], CH2Cl2, PhSiH3; c) glycosylamine 7,
HATU, DIPEA, DMSO; then TFA/PhOH/H2O/TIS, 45% over 4 steps.
Pseudoproline dipeptides are depicted in blue. Amino acids protected
with acid-labile protecting groups are shown in bold. Amino acid
protecting groups are: E(tBu), H(Trt), S(tBu), N(Dmcp), K(Boc),
Y(tBu), R(Pbf), Q(Dmcp).
[7] a) Y. Kajihara, A. Yoshihara, K. Hirano, N. Yamamoto, Carbo-
hydr. Res. 2006, 341, 1333; b) N. Yamamoto, Y. Tanabe, R.
Okamoto, P. Dawson, Y. Kajihara, J. Am. Chem. Soc. 2008, 130,
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Maeda, M. Ouchi, K. Yoshida, Y. Nambu, J. Igarashi, N. Hayashi,
T. Tsuji, Y. Kajihara, J. Am. Chem. Soc. 2012, 134, 5428; d) T.
Takemura, H. Hojo, Y. Nakahara, T. Ishimuzu, S. Hase, Org.
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1997, 38, 5627; f) M. Murakami, R. Okamoto, M. Izumi, Y.
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Guo, N. Shao, Med. Res. Rev. 2005, 25, 655; j) C. Piontek, D.
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1974; Angew. Chem. Int. Ed. 2009, 48, 1941.
Scheme 8. EPO(79–124) and EPO(1–28).
[8] For recent improvements in the efficiency of the SPPS approach
to glycopeptide synthesis, see: C. Heinlein, D. V. Silva, A.
Trçster, J. Schmidt, A. Gross, C. Unverzagt, Angew. Chem. 2011,
123, 6530; Angew. Chem. Int. Ed. 2011, 50, 6406.
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1994, 266, 776.
[11] a) S. T. Anisfeld, P. T. Lansbury, J. Org. Chem. 1990, 55, 5560;
b) S. T. Cohen-Anisfeld, P. T. Lansbury, J. Am. Chem. Soc. 1993,
115, 10531.
decomposition pathways. This strategy is also effective for
minimizing aspartimide formation during SPPS. Moreover,
the aspartylation approach has been successfully applied to
convergent syntheses of the EPO glycopeptide fragments.
Though the reasons for this observed phenomenon are
presently matters of conjecture, its consequences on this
field of research are apt to be quite important. Indeed, if
generalizable, it could well have solved one of the most
serious problems in the assembly of complex glycopolypep-
tides by chemical means.[20]
[12] a) M. Bodanszky, J. C. Tolle, S. S. Deshmane, A. Bodanszky, Int.
J. Pept. Protein Res. 1978, 12, 57; b) M. Bodanszky, G. F. Sigler,
A. Bodanszky, J. Am. Chem. Soc. 1973, 95, 2352.
[13] P. Wang, X. Li, J. Zhu, J. Chen, Y. Yuan, X. Wu, S. J. Danishefsky,
J. Am. Chem. Soc. 2011, 133, 1597.
Received: June 27, 2012
Revised: July 17, 2012
Published online: && &&, &&&&
[14] Attempts to improve reactivity by increasing the solubility of
peptide 2 through the use of dodecylphosphocholine were
unsuccessful.
Keywords: aspartimide · aspartylation · glycopeptides ·
pseudoproline dipeptide · solid-phase synthesis
.
[15] T. Haack, M. Mutter, Tetrahedron Lett. 1992, 33, 1589.
4
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[16] Installation of the Leu-Ser pseudoproline dipeptide at the C-
terminus served to minimize peptide aggregation: F. Garcꢁa-
Martꢁn, P. White, R. Steinauer, S. Cꢂtꢃ, J. Tulla-Puche, F.
Albericio, Biopolymers 2006, 84, 566.
[17] P. Nagorny, B. Fasching, X. Li, G. Chen, B. Aussedat, S. J.
Danishefsky, J. Am. Chem. Soc. 2009, 131, 5792.
Albericio, Tetrahedron 2011, 67, 8595. Another line of conjecture
focuses on the possible inhibition of the amidic NH deprotona-
tion step necessary for aspartamide formation. Perhaps the
hydrogen of the aspartamide NH group is transferred to the
adjacent amide of the (n + 1) amino acid through a hydrogen
bond (to
N or O). The tertiary amide character of the
[18] Small amounts of ammonia are often present in the glycosyl-
amine component, even following multiple lyophilizations.
[19] P. Wang, S. Dong, J. A. Brailsford, S. D. Townsend, Q. Zhang, K.
Iyer, R. C. Hendrickson, J. H. Shieh, M. Moore, S. J. Danishef-
sky, Angew. Chem. 2012, DOI: 10.1002/ange.201206090; Angew.
Chem. Int. Ed. 2012, DOI: 10.1002/anie.201206090.
pseudoproline engagement, would impede such a transfer to
either the N or O atom. Clearly the question of the basis for the
pseudoproline effect will be pursued by concerned research
groups, including our own.
[20] The basis for the (n + 2)pseudoproline effect in suppressing
aspartimide formation both in peptide synthesis as well as in
asparagine glycosylation invites various interpretations. It has
been argued, with computational support, that type II’ b turn,
stabilized by an intramolecular hydrogen bond (see structure A)
of a type which cannot be present in the pseudoproline case (see
structure B). This effect could be operative at the kinetic level,
leading to suppression of imide formation relative to biomolec-
ular acylation. See: a) S. Capasso, L. Mazzarella, A. Zagari,
Chirality 1995, 7, 605; b) R. Subirꢀs-Funosas, A. El-Faham, F.
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Communications
Glycoprotein Synthesis
Like a Pro: A one-flask aspartylation/
deprotection method, wherein long pep-
tide fragments, bearing proximal pseu-
doproline functionality, are merged with
complex glycan domains has been devel-
oped. Following aspartylation, acid-
mediated global deprotection reveals the
elaborated glycopeptide. The temporary
pseudoproline functionality serves to
suppress formation of aspartimide side
products during solid-phase peptide syn-
thesis and aspartylation.
P. Wang, B. Aussedat, Y. Vohra,
S. J. Danishefsky*
&&&& — &&&&
An Advance in the Chemical Synthesis of
Homogeneous N-Linked
Glycopolypeptides by Convergent
Aspartylation
6
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
These are not the final page numbers!