Synthesis of the highly glycosylated hydrophilic motif of extensins

Article abstract of DOI:10.1002/anie.201404904

Extensin, the structural motif of plant extracellular matrix proteins, possesses a unique highly glycosylated, hydrophilic, and repeating Ser ₁Hyp₄ pentapeptide unit, and has been proposed to include post-translational hydroxylation at proline residue and subsequent oligo-L-arabinosylations at all of the resultant hydroxyprolines as well as galactosylation at serine residue. Reported herein is the stereoselective synthesis of one of the highly glycosylated motifs, Ser(Galp₁)- Hyp(Araf₄)-Hyp(Araf₄)-Hyp(Araf₃)-Hyp(Araf ₁). The synthesis has been completed by the application of 2-(naphthyl)methylether-mediated intramolecular aglycon delivery to the stereoselective construction of the Ser(Galp₁) and Hyp(Araf _n) fragments as the key step, as well as Fmoc solid-phase peptide synthesis for the backbone pentapeptide.

Full text of DOI:10.1002/anie.201404904

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DOI: 10.1002/anie.201404904
Glycopeptides
Synthesis of the Highly Glycosylated Hydrophilic Motif of Extensins**
Akihiro Ishiwata,* Sophon Kaeothip, Yoichi Takeda, and Yukishige Ito*
Abstract: Extensin, the structural motif of plant extracellular
matrix proteins, possesses a unique highly glycosylated, hydro-
philic, and repeating Ser1Hyp4 pentapeptide unit, and has been
proposed to include post-translational hydroxylation at proline
residue and subsequent oligo-l-arabinosylations at all of the
resultant hydroxyprolines as well as galactosylation at serine
residue. Reported herein is the stereoselective synthesis of one
of the highly glycosylated motifs, Ser(Galp1)-Hyp(Araf4)-
Hyp(Araf4)-Hyp(Araf3)-Hyp(Araf1). The synthesis has been
completed by the application of 2-(naphthyl)methylether-
mediated intramolecular aglycon delivery to the stereoselective
construction of the Ser(Galp1) and Hyp(Arafn) fragments as
the key step, as well as Fmoc solid-phase peptide synthesis for
the backbone pentapeptide.
of highly conserved Tyr-Xaa-Tyr motifs by oxidative coupling
of their Tyr residues confers insolubility on extensins.[10,11] In
contrast, the most common repeating motif of their hydro-
philic region is the Ser-Hyp-Hyp-Hyp-Hyp pentapeptide
modified by Ser a-d-galactopyranosylation [Ser(Galp1)] and
Hyp
oligo-l-arabinofuranosylation
[Hyp(Araf)1-4]
(Figure 1).[12,13] The hydrophilic motif of a HRGP related to
H
ydroxyproline-rich glycoproteins (HRGPs),[1] which are
major structural components of plant extracellular matrices,
are produced by extensive post-translational modifications of
proline residues. They are first hydroxylated by prolyl 4-
hydroxylases[2] and resultant hydroxyproline (Hyp) residues
are glycosylated by l-arabinofuranosyltransferases (AFT).[3]
These modifications are widespread in plants and essential for
their developmental processes such as root hair growth.[4]
Secreted peptide hormones of plant origin,[5] such as CLV3,[6]
are modified in a similar manner. In addition to arabinofur-
anosylation, glycosylation of serine (Ser) residues has been
found in extensins.[7] Glycosylation has been proposed to
enhance their conformational rigidity and are important for
molecular recognition[8] required for self-assembly of plant
cell walls.[9]
Figure 1. The hydrophilic repeating motif of the extensins 1.
Extensins are pivotal components of plant cell wall
architectures, and are required for their self-assembly. Exten-
sin monomers are bipartite in nature, thus consisting of
hydrophobic and hydrophilic repeating motifs. Crosslinking
root hair growth[12] was proposed to consist of, from the N-to-
C terminus, Ser(Galp1), [Hyp(Araf4)]2, Hyp(Araf3), and Hyp-
(Araf1) residues. In Hyp(Araf1-3), all glycosides have been
found to be b-linked, while Hyp(Araf4)[14,15] has an a-l-Araf
residue 1!3-linked to the Hyp(Araf3). In addition to their
structures and activities, biosynthetic as well as metabolic
processes have been subjects of recent studies. For example,
b-l-arabinofuranosidases (AFases)[16] and b-l-AFT[17] have
been identified recently. Interestingly, HypBA1, one of the
AFases, was indicated to be a cysteine glycosidase.[18]
The hydrophilic motif 1 is synthetically challenging
because they are extensively modified by oligosaccharides
consisting of consecutive b-Arafs. Since stereoselective con-
struction of b-Araf glycosides is difficult to achieve because of
its 1,2-cis nature, various approaches[19] based on direct[20] or
intramolecular[21,22] glycosylation strategies have been exam-
ined by targeting extensin structure motifs.[23,24] In contrast,
preparation of Ser(Galp1) is intrinsically more straightfor-
ward and has been carried out in a conventional manner.[25]
For the synthesis of 1,2-cis glycosides, approaches based
on intramolecular aglycon delivery (IAD)[26] have been
[*] Dr. A. Ishiwata, Dr. Y. Ito
Synthetic Cellular Chemistry Laboratory, RIKEN
2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)
E-mail: aishiwa@riken.jp
yukito@riken.jp
Dr. S. Kaeothip, Dr. Y. Takeda, Dr. Y. Ito
ERATO glycotrilogy project, JST (Japan)
[**] We thank Dr. Hiroyuki Koshino (RIKEN Global Research Cluster)
and his staff for technical help for technical help with ESI MS and
CD, and Dr. Fumiaki Hayashi and Dr. Hui-ping Zhang (RIKEN
Center for Life Science Technologies) for 900 MHz NMR measure-
ments and are grateful to the Support Unit for Biomaterial Analysis
(RIKEN Brain Science Institute) for tandem MS analysis. We also
thank Ms. Akemi Takahashi for her kind technical assistance. This
work was partly supported by Incentive Research Grant (2013) in
RIKEN.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201404904.
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employed successfully. Among a number of variants reported,
2-(naphthyl)methyl (NAP) ether mediated IAD has been
shown to be most versatile,[27,28] and applied to the synthesis of
CLV3[29] and p-nitrophenyl b-l-Araf,[30] a valuable substrate
for HypBA1. Herein we report the first synthesis of a hydro-
philic repeating motif typical of extensins, Ser(Galp1)-Hyp-
(Araf4)-Hyp(Araf4)-Hyp(Araf3)-Hyp(Araf1) (1). It features
the extensive use of NAP-IAD for all 1,2-cis glycosides,
including Ser(Galp1) and Hyp(Arafn) (n = 1, 3, 4). Subsequent
Fmoc solid-phase peptide synthesis (Fmoc-SPPS) was carried
out with suppression of diketopiperazine (DKP) formation.
Imaginary disconnection of the target structure led us to
design fragments corresponding to Ser(Galp1) (5) and Hyp-
(Arafn) (n = 1, 3, 4; 2–4; Figure 2). Among them, 2 and 3 were
prepared as previously reported[29] (Scheme 1). Namely,
starting from 10, oxidative mixed acetal (MA) formation
with the donor 11 in the presence of 2,3-dichloro-5,6-
dicyanobenzoquinone (DDQ) was followed by IAD medi-
Scheme 1. Stereoselective synthesis of Hyp(Araf1-4) derivatives.
a) DDQ, 4ꢀ M.S., DCE, RT; b) MeOTf, DTBMP, 4ꢀ M.S., DCE, 408C,
48 h; c) TFA, CHCl3, 08C, 2 h, 70% (12 from 10), 73% (16 from 13),
51% (19 from 17); d) NAPBr, NaH, TBAI, DMF, ꢀ208C, 6 h, 82%
(13), 79% (17); e) TBAF, pyr/THF, 08C, 1 h; f) Ac2O, py, RT, 3 h;
g) H2, Pd(OH)2, EtOAc/EtOH (2:1), RT, 8–16 h; h) FmocCl, DIPEA,
CH2Cl2, RT, 5 h, 72% (2 from 12), 70% (3 from 19), 70% (4 from 26);
i) TBAF, THF, 08C, 1 h; j) Ac2O, py, 89% from 17; k) DDQ, 4ꢀ M.S.,
DCE, RT, 74%; l) MeOTf, DTBMP, 4ꢀ M.S., DCE, 408C, 48 h; m) TFA,
CHCl3, 08C, 0.5 h; n) Ac2O, py, 84% from 20; o) 0.1m NaOH, MeOH,
08C, 4 h; p) H2, Pd(OH)2, MeOH/H2O/HOAc (30:10:1), RT, 15 h, 70%
from 22. M.S.=molecular seives, DCE=(CH2Cl)2, DDQ=2,3-dichloro-
5,6-dicyano-1,4-benzoquinone, DIPEA=(iPr)2NEt, DTBMP=2,6-di-tert-
butyl-4-methylpyridine, Naph=2-naphthyl, py=pyridine, TBAF=
(nBu)4NF, TBAI=(nBu)4NI, Tf=trifluoromethanesulfonyl, TFA=
trifluoroacetic acid, TIPDS=tetraisopropyldisiloxanylidene.
Figure 2. Synthetic plan for (oligo)saccharyl amino acid fragments of
1. Cbz=benzyloxycarbonyl, Fmoc=9-fluorenylmethyloxycarbonyl,
Tol =4-methylphenyl.
ated by MeOTf and 2,6-di-tert-butyl-4-methylpyridine
(DTBMP) to afford the Hyp(Araf1) derivative 12 in a fully
stereoselective manner. Then 12 was converted into 2 in
a conventional manner.
linkages. Accordingly, 12 was converted into the correspond-
ing NAP ether 13, which was coupled with the donor 14
through the MA 15 under standard NAP-IAD conditions.
After conversion of 16 into 17, chain elongation was carried
out in a similar manner to give 3 from 17 through 19.
For the preparation of the Hyp(Araf4) component 4,
a fragment coupling between the donor 6[31] and the acceptor
7 was planned (Figure 2). The acceptor 7 was prepared from
17 through desilylation and acetylation of the resultant 18
From the preceding study,[29] we learned that in order for
the IAD to be most efficient, proper combination of donor
and acceptor is critical. Specifically, the use of an NAP-
masked acceptor in combination with a 2-O-unprotected
donor is optimal for the construction of consecutive b-Araf
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(Scheme 1). Coupling of 6 with 7 stereoselectively afforded 21
from the MA 20, which was converted into 22. Since, judging
from NMR spectra, 22 was a mixture of rotamers, its
homogeneity was confirmed after being converted into the
free Hyp(Araf4) 24 from 23. The compound 22 was then
converted into 4 by way of 25.
The Fmoc-Ser(a-Galp1) derivative 5 was synthesized from
the NAP-protected donor 8[31] (Figure 2, Scheme 2), although
5 was also obtained predominantly by direct intermolecular
glycosylation.[31] The donor 8 was treated with the Fmoc-Ser-
O-tBu 9 under oxidative mixed-acetal-formation conditions
to give the MA 26, which was converted into the expected a-
galactosylserine derivative 29 in a highly stereoselective
manner after mild acidic work-up (10% TFA in CHCl3 at
08C) of 27 and O-acetylation of the resultant triol 28. Further
acidic removal of the tert-butylester of 29 gave the desired
Fmoc-Ser(Galp1) derivative 5.[25]
We initially planned to conduct solution-phase peptide
synthesis using 1-[(1-(cyano-2-ethoxy-2-oxo-ethylidene-
aminooxy)-dimethylaminomorpholino)]uronium hexafluoro-
phosphate (COMU)[32] as a coupling reagent[29] (Scheme 3).
However, not unexpectedly,[33] treatment of the dipeptide
derivative 32[31] with piperidine to remove the Fmoc group
Scheme 2. Stereoselective synthesis of 5. a) DDQ, 4ꢀ M.S., DCE, RT,
98%; b) MeOTf, DTBMP, 4ꢀ M.S., DCE, 408C, 48 h; c) 10% TFA,
CHCl3, 08C, 2 h; d) Ac2O, py, 56% from 30; e) 20% TFA, CHCl3, RT,
2 h, 84%.
resulted in formation of a significant amount of the DKP 34
(MALDI-TOF MS: [M+Na]+ calc for C50H66N2Na1O30,
1197.35, found 1197.43). This problem was circumvented by
Fmoc-SPPS using the chlorotrityl (ClTr) resin 30.[34] Namely,
after introducing 2, the resin 31 was subjected to Fmoc
removal and COMU coupling with 3 to give the resin-bound
dipeptide 33. Subsequent Fmoc removal was carried out with
piperidine in the presence of 1-O-hydroxybenzotriazole
Scheme 3. Solid-phase glycopeptide synthesis toward 1. a) DIPEA, RT, 16 h; b) pip, DMF, RT, 10 min; c) COMU, DIPEA, RT, 16 h; d) pip, DMF,
RT, 84%; e) pip, HOBt, DMF, RT, 10 min; f) DIC, HOBt, RT, 16 h; g) TFA/(iPr)3SiH/H2O (190:5:5), 1 h; h) NaOH, MeOH, 08C, 21% based on
initial loading to ClTr resin. pip=piperidine.
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(HOBt)[35] to trap free amino groups, and DIC–HOBt
coupling[36] with 4 afforded the tripeptide 35 with suppression
of the DKP formation. The entire sequence of 1 was
successfully constructed by iterative chain elongation using
4 and 5 to give 37 via 36. Finally, the resultant pentapetide was
cleaved from the resin and was deacetylated at 08C,[37] thus
completing the synthesis of 1. High-resolution ESI-TOF mass
data ([M+H]+ calcd for C89H142N5O64, 2304.8011, found
2304.7968) provided evidence to support the target structure.
For structural analysis, we opted to use the CID method
for tandem MS-MS analysis, although glycosidic linkages
would be cleaved to a larger extent than in ETD/ECD
methods. The fragmentations of all sugar moieties one by one
was observed to support the presence of all required sugar
residues.[31] The structure of 1 was also analyzed by two-
dimensional NMR techniques in detail. The corresponding 13
glycosidic linkages were identified in HSQC spectra
(Figure 3), and full assignment of the peaks was made at
900 MHz.[31]
glycopeptide into a left-handed polyPro II helixlike struc-
ture[38] (Figure 4), as has been found in nonglycosylated as
well as in glycosylated Hyp derivatives.[39,31]
In summary, stereoselective synthesis of Ser(Galp1)-Hyp-
(Araf4)-Hyp(Araf4)-Hyp(Araf3)-Hyp(Araf1) (1) was realized
by NAP-IAD for stereoselective constructions of all 1,2-cis
glycosides containing the fragments Ser(Galp1) and Hyp-
(Arafn) (n = 1, 3, 4). Assembly of the highly glycosylated
pentapeptide unit was achieved by Fmoc solid-phase peptide
synthesis.
Received: May 2, 2014
Published online: July 15, 2014
Keywords: glycopeptides · proteins · solid-phase synthesis ·
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synthetic methods
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