Stereoselective synthesis of Arabidopsis CLAVATA3 (CLV3) glycopeptide, unique protein post-translational modifications of secreted peptide hormone in plant

Article abstract of DOI:10.1039/c3ob41212a

The unique hydroxylproline (Hyp)-linked O-glycan modification is a common process in hydroxyproline-rich glycoproteins (HRGPs). The modification occurs through post-translational hydroxylation at 4-position of proline residues some of which are followed by O-glycosylation at the resulting Hyp which is also found in some secreted peptide hormones such as CLAVATA3 (CLV3) of Arabidopsis thaliana plants. An active mature CLV3 is a tridecapeptide linked to β-l-Araf-(1→2)-β-l-Araf-(1→2)-β-l-Araf at a Hyp residue in the center of the peptide sequence such as Arg-Thr-Val-Hyp-Ser-Gly-Hyp(l- Araf_n)-Asp-Pro-Leu-His-His-His (n = 3). We report here the synthesis of the secreted and modified CLV3 glycopeptide with all glycoforms (Araf _0-3CLV3) of A. thaliana plants. A highly stereoselective β-arabinofuranosylation of Hyp derivatives as the key step of the synthesis of CLV3 glycopeptide was achieved by NAP ether-mediated IAD, which was effectively applied to the synthesis of oligoarabinosylated hydroxylproline [Hyp(l-Araf_1-3)] derivatives. Fmoc-solid phase peptide synthesis was carried out using COMU as the coupling reagent for the introduction of [Hyp(l-Araf_0-3)] derivatives as well as further elongation to the CLV3 glycopeptides.

Full text of DOI:10.1039/c3ob41212a

Organic&
BiomolecularChemistry
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Volume 11 | Number 35 | 21 September 2013 | Pages 5737–5964
ISSN 1477-0520
PAPER
Akihiro Ishiwata, Yukishige Ito et al.
Stereoselective synthesis of Arabidopsis CLAVATA3 (CLV3) glycopeptide, unique
protein post-translational modifications of secreted peptide hormone in plant

Organic &
Biomolecular Chemistry
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Stereoselective synthesis of Arabidopsis CLAVATA3
(CLV3) glycopeptide, unique protein post-translational
Cite this: Org. Biomol. Chem., 2013, 11,
5892
modifications of secreted peptide hormone in plant†
Sophon Kaeothip,a Akihiro Ishiwata*b and Yukishige Ito*a,b
The unique hydroxylproline (Hyp)-linked O-glycan modification is a common process in hydroxyproline-
rich glycoproteins (HRGPs). The modification occurs through post-translational hydroxylation at 4-posi-
tion of proline residues some of which are followed by O-glycosylation at the resulting Hyp which is also
found in some secreted peptide hormones such as CLAVATA3 (CLV3) of Arabidopsis thaliana plants. An
active mature CLV3 is a tridecapeptide linked to β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Araf at a Hyp residue in
the center of the peptide sequence such as Arg-Thr-Val-Hyp-Ser-Gly-Hyp(L-Arafn)-Asp-Pro-Leu-His-His-His
(n = 3). We report here the synthesis of the secreted and modified CLV3 glycopeptide with all glycoforms
(Araf0–3CLV3) of A. thaliana plants. A highly stereoselective β-arabinofuranosylation of Hyp derivatives as
the key step of the synthesis of CLV3 glycopeptide was achieved by NAP ether-mediated IAD, which was
effectively applied to the synthesis of oligoarabinosylated hydroxylproline [Hyp(L-Araf1–3)] derivatives.
Fmoc-solid phase peptide synthesis was carried out using COMU as the coupling reagent for the intro-
duction of [Hyp(L-Araf0–3)] derivatives as well as further elongation to the CLV3 glycopeptides.
Received 12th June 2013,
Accepted 17th July 2013
DOI: 10.1039/c3ob41212a
www.rsc.org/obc
CLE22,5 and PSY1,6 have been identified from several plants
which carry Hyp modified by oligo-arabinofuranodides (Araf).
Introduction
Posttranslational modifications regulate the function of pro-
CLV3, initially formed as a preproprotein of 96 amino acids,
teins in various ways. One of the most prominent among them is secreted from stem cells and regulates cell homeostasis
is glycosylation,1 which occurs by the combined action of a in the shoot apical meristem of Arabidopsis.7 As the ligand of
variety of glycosyltransferases and glycosidases. Whereas the CLV1 (a leucine-rich repeat transmembrane receptor serine
structures of glycoprotein glycans are diverse, most of them threonine kinase8), CLV3 is responsible for regulating the
can be categorized into two major classes, N- and O-linked stem-cell signaling pathway, possibly by the formation of a
glycans. They comprise oligosaccharides linked to the side complex with CLV1 and CLV2 (a similar protein to CLV1
chain of asparagine (Asn) or serine (Ser)/threonine (Thr) lacking the kinase domain9).
residue, respectively. In the plant kingdom, a unique hydroxyl-
proline (Hyp)-linked O-glycan modification process has been CLV3 of Arabidopsis thaliana plants, which was shown to be
found. It occurs through posttranslational hydroxylation and 13 amino-acid oligoarabinofuranosylated glycopeptide
In 2009, Ohyama et al. reported the structure of mature
a
subsequent glycosylation at 4-position of specific proline resi- (Araf3CLV3), Arg-Thr-Val-Hyp-Ser-Gly-Hyp(L-Arafn)-Asp-Pro-Leu-
dues. This modification is widely distributed in hydroxypro- His-His-His (n = 3) (Fig. 1).10 The trisaccharide [β-L-Araf-(1→2)-
line-rich glycoproteins (HRGPs)2 such as extensins, proline- β-L-Araf-(1→2)-β-L-Araf-(1→4)-β-O-Hyp] linked to the Hyp con-
rich proteins and arabinogalactan proteins. More recently, sists of consecutive β-linked arabinofuranoside linkages.
secreted peptide hormones,3 including CLAVATA3 (CLV3),4 Because of its unique structure and interesting biological
activity, we deemed this glycopeptide to be an attractive target.
In order to synthesize CLV3, a problem associated with the
construction of β-L-arabinofuranoside (β-L-Araf) was foreseen.
The difficulty derives from its 1,2-cis, non-axial nature, preclud-
ing the use of conventional methodologies in O-glycoside
synthesis.
In the context of the construction of β-D-Araf linkages found
in mycobacterial arabinan, various concepts have been tested.
Most notably, Lowary11 and Kim12 reported elegant approaches
aERATO, Japan Science and Technology Agency (JST), Ito Glycotrilogy Project,
2-1 Hirosawa, Wako, Saitama 351-0198, Japan. E-mail: yukito@riken.jp;
Fax: +81-46-462-4680; Tel: +81-46-467-9430
bRIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. E-mail: aishiwa@riken.jp;
Fax: +81-46-462-4680; Tel: +81-46-467-9434
†Electronic supplementary information (ESI) available: NMR spectra of synthetic
compounds in the Experimental section and HPLC charts and the results of
tandem MS/MS analysis of 1–4. See DOI: 10.1039/c3ob41212a
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Fig. 1 Post-translationally modified CLV3 (glyco)peptide 1–4.
based on SN2-type displacement of α-triflates derived from 2,3- (Araf2Hyp) and β-L-Araf-(1→2)-β-L-Araf-(1→2)-β-L-Araf-(1→4)-β-O-
anhydro-11 or carboxybenzyl-substituted (CB-substituted)12 Hyp (Araf3Hyp). Finally, the synthesis of CLV3 (glyco)peptides
donors. Alternatively, certain cyclic protections have proven was achieved by Fmoc-based solid phase peptide synthesis
beneficial in enhancing the β-selectivity, possibly due to their using Araf0–3Hyp derivatives.26
property to bring conformational constraints to arabinofurano-
syl donors. For instance, 3,5-O-di-t-butylsilylene-protected
(DTBS-protected) donors, reported by Boons13 and others,14–18
were shown to give substantial β-selectivity, while our study
Results and discussion
indicated the suitability of 3,5-O-tetra-i-propyldisiloxanylidene-
In the beginning, we examined the suitability of TIPDS-con-
trolled β-L-Araf formation14 to achieve our goal. Thus, the reac-
tion of the donor 527 with N-benzyloxycarbonyl (Cbz)-protected
Hyp-OBn 628 was conducted under our standard conditions to
give the disaccharide 11, but in a stereorandom manner (α : β =
1 : 1.3, Table 1, entry 6).
Anomeric configuration of the α- and β-isomers was deter-
mined by JH1–H2 (1.5 and 4.0 Hz) and δ(C1) (105.1 and
98.4 ppm) values.29 Since the products were obtained as mix-
tures of rotamers, rigorous confirmation of the structure was
made after removal of the carbamate moiety.30,31
Given the limited success obtained by direct glycosylation,
we turned our attention to the IAD approach,24,25 which was
examined by using the same donor (5)–acceptor (6) combi-
nation. As depicted in Fig. 2, formation of the mixed acetal 9
proceeded cleanly (87%) in the presence of 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ), whereas subsequent IAD
(MeOTf–DTBMP32) at room temperature was slow giving β-L-
arabinofuranosylhydroxylproline 10 in 37% yield (Table 1,
entry 1), with recovery of Cbz-Hyp-OBn and hydrolysis product
corresponding to the glycosyl donor 5. The same IAD at a
protection (TIPDS-protection) for this purpose.14
Recently, an attempt to construct the hydroxylproline-
linked β-L-Araf structure of Art v 1, the major allergen of
mugwort pollen,19 by using donors protected by a 3,5-O-di-t-
butylsilylene (DTBS) group was reported by Xie and Taylor.20
However, both the yield and the selectivity were reported to be
low.
In order to realize the exclusive formation of β-Araf linkage,
approaches based on intramolecular aglycon delivery (IAD)
have been investigated.21 To this end, 2-O-p-methoxybenzyl
(PMB)22,23 or 2-O-(2-naphthyl)methyl (NAP)24 and 5-O-NAP25
equipped donors were examined, which were applied to the
synthesis of mycobacterial arabinans.22,25 Recently, Matsubaya-
shi et al. reported the stereoselective synthesis of
CLV3 glycopeptide using PMB-mediated IAD as the key
reaction.23
In this report, we describe our own effort to stereoselectively
synthesize β-L-Araf linked hydroxyproline through NAP-ether
mediated intramolecular aglycon delivery (IAD). Elongation of
the arabinan chain gave β-L-Araf-(1→2)-β-L-Araf-(1→4)-β-O-Hyp
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Table 1 Examination of β-L-arabinofuranosylation
Entry
Method
Acceptor
MA/%
Temp./time
Product
Yielda /%
α : β
1
2
3
4
5
6
A
A
A
A
A
B
6
6
6
7
8
6
87
—
—
84
92
—
r.t./120 h
10
10
10
12
13
11
37 (32)
81 (70)
67b (58)
67 (56)
79 (72)
76
β only
β only
β only
β only
β only
1 : 1.3
40 °C/48 h
40 °C/48 ha
40 °C/48 h
40 °C/48 h
−40 °C–r.t./15 min
a Two step yield in parentheses. b The reaction carried out in the presence of 5 equiv. of TTMSS. Method A: intramolecular aglycon delivery; B:
intermolecular glycosylation.
slightly elevated temperature (40 °C) pleasingly gave a satisfac- the glycosyl donor 5. Again, the IAD approach was successful,
tory yield (81%) of 10.
giving the disaccharide 13 in good yield (72%) with complete
In our previous study of 1,2-cis hexopyranoside synthesis, it β-stereoselectivity (entry 5).
was found that the naphthylmethyl group was preserved in
Accordingly, a further plan was made to apply the NAP-IAD
IAD, when an appropriate hydride donor, in particular tris(tri- for the construction of mono-, di- and tri-arabinosylated hydro-
methylsilyl)silane (TTMSS),24 was included. However, the con- xyproline 21, 23, and 25 (Scheme 1). Treatment of a mixture of
ditions that re-generate NAP ether were not found in the IAD the L-Araf donor 5 and Araf1Hyp derivative 10 with DDQ
of 9, solely giving 10, even in the presence of TTMSS (entry 3). afforded the corresponding mixed acetal 15 in 58% yield.
Various types of hydride transfer reagents such as dicyclohexyl- Interestingly, the formation of the same mixed acetal was
isobutylamine,33 cycloheptatriene,34 and poly(methyl-hydro- revealed to be much more facile when a combination of 2-O-
siloxane)35 were examined; however, the formation of NAP ether NAP ether β-1139,40 and the 2-O-unprotected donor 1427 was
product was not observed. N-tert-Butyloxycarbonyl-protected employed. This combination cleanly gave 15 (87%), which was
(Boc-protected) Hyp acceptor 736 also gave the desired subjected to IAD to give Araf2Hyp derivative 1630,31 in 73%
β-L-arabinofuranosides 12, but in somewhat lower yield, yield from 5.
because of partial cleavage of the Boc group during work-up
We speculate that the reactivity of the hydroxyl group of the
under acidic conditions (entry 4).
Araf2Hyp derivative 16 is attenuated due to the steric hin-
Before conducting elongation of β-D-Araf linkages, the for- drance caused by a cis-oriented bulky aglycon. On the other
mation of β-L-Araf-(1→2)-β-L-Araf was tested using methyl arabi- hand, the methylenic carbon of the NAP ether may well be
nofuranoside 838 as a model acceptor, which was reacted with more accessible, because it is two bonds apart from the
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furanoside ring. Further introduction of an Araf residue was
also conducted by using the 2-O-unprotected donor 14.
Namely, compound 16 was once converted to the 2-O-NAP
Experimental section
General procedures
ether 17,39 treatment of which with the thioglycoside 14 and Column chromatography was performed on silica gel 60 (EM
DDQ gave the mixed acetal 18 in good yield. As we hoped, sub- Science, 40–100 mesh). Reactions were monitored by thin-layer
sequent IAD afforded a desired Araf3Hyp derivative 1930,31 in chromatography (TLC) using Kieselgel 60 F254 (EM Science),
77% yield, successfully completing the construction of all and compounds were detected by examination under UV light
three β-glycosidic linkages.
and by charring with 10% sulfuric acid in MeOH. Solvents
The thus obtained Araf1–3Hyp derivatives 10, 16, and 19 were removed under reduced pressure at <40 °C. CH2Cl2 was
were converted to per-O-acetylated derivatives 20, 22, and 24 by freshly obtained from the Glass Contour solvent dispensing
deprotection of the TIPDS group40 followed by acetylation. Sub- system. AgOTf was co-evaporated with toluene (2–3 times) and
sequent hydrogenolysis and Fmoc protection gave Fmoc-Hyp dried in vacuo for 2–3 h directly prior to application. Molecular
(per-O-acetyl-Araf1–3) 21, 23, and 25. With the oligoarabinosy- sieves (4 Å) were activated at 200 °C for 2–3 h under vacuum
lated hydroxylproline derivative in hand, synthesis of the glyco- prior to application. All reactions were carried out under an
peptides (Araf0–3CLV3) was planned and it was executed by the argon atmosphere. 1H NMR and 13C NMR spectra were
Fmoc-solid phase peptide synthesis strategy.26
recorded using a Bruker Advance 500 spectrometer with a
1
Namely, starting from the hexapeptide Fmoc-Asp(tBu)-Pro- cryoprobe. H NMR spectra were recorded in CDCl3 and refer-
Leu-His(Trt)-His(Trt)-His(Trt), immobilized as a trityl ester on enced to TMS at 0.00 ppm and H2O at 4.79 ppm, and 13C NMR
NovaSyn®TGT alcohol resin 27, which was prepared from the spectra were referenced to the central peak of CDCl3 at
monomer 26, the glycopeptides (Araf0–3CLV3) were synthesized 77.00 ppm. Assignments were made by standard gCOSY and
as shown in Scheme 2. Throughout the chain elongation, gHSQC. Optical rotations were measured with a Jasco DIP-370
(1-cyano-2-ethoxy-2-oxo-ethylideneaminooxy)-dimethylamino- polarimeter. MALDI-TOF mass spectra were recorded using a
morpholinouronium hexa-fluorophosphate (COMU),41 a non- Bruker Autoflex Speed spectrometer with 2-hydroxy-5-
explosive alternative to the classic benzotriazole coupling
reagents, was used as an activating agent, which was expected
to reduce the incidence of N-terminal masking during coup-
ling steps.
Introduction of Fmoc-Hyp 28 or arabinosylated Fmoc-Hyp
(21, 23, 25) to the hexapeptide on resin 27 was carried out suc-
cessfully to give (glyco)heptapeptides (29–32), monitoring of
which was done by MALDI TOF-MASS analysis after cleavage of
small aliquots of resins. A subsequent chain elongation was
conducted manually, giving the expected glycosylated trideca-
peptides (33–36). Acidic cleavage from resin and deacetylation
completed the synthesis of Araf0–3CLV3 (1–4). Spectral data of
synthetic CLV3s were all identical with those of reported
compounds.23
These glycopeptides were converted to resin-immobilized
forms,42 which were useful in identifying novel lectins that
recognize CLV3 or other HRGPs.
Conclusions
We report here the synthesis of the secreted and modified
CLV3 glycopeptide (Araf0–3CLV3, 1–4) of A. thaliana plants.
Highly stereoselective β-arabinofuranosylation of Hyp deriva-
tives (6, 7) as the key step of the synthesis of 1–3 was achieved
by NAP ether-mediated IAD, which was effectively applied to
the synthesis of oligoarabinosylated hydroxylproline deriva-
tives (21, 23, 25). Fmoc-solid phase peptide synthesis was
carried out using COMU as the coupling reagent for the intro-
duction of Fmoc-Hyp(per-O-acetyl-Araf1–3) (21, 23, 25) and
Fmoc-Hyp(tBu) as well as further elongation to the CLV3
Fig. 2 Fragment ions observed by tandem MS/MS analysis of synthetic [Arafn]-
CLV3 (n = 0–3) (1–4). Numbers on the upper and lower sides of 13 (glycosyl)-
(glyco)tridecapeptide derivatives (34–36). These compounds
amino acid sequences are b- and y-ions, respectively. Ions in italic letters were
will be useful in finding new CLV3 binding lectins.
also detected though at a small intensity. See also ESI.†
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Scheme 1 Synthesis of oligoarabinosylated hydroxylproline derivative. Reagents and conditions: (a) 5 (1.0 equiv.), DDQ (1.1–2.0 equiv.), MS4A, CH2Cl2, r.t., 87%
(9 for 6 h), 58% (15 for 24 h), trace (18 for 24 h); (b) MeOTf (3.5 equiv.), DTBMP (4.0 equiv.), MS4A, CH2Cl2, 40 °C, 48 h; (c) 10% TFA in CHCl3, 0 °C, 0.5 h, 81% for
(10 from 9), 84% for (16 from 15), 77% (19 from 18); (d) NAPBr, NaH, TBAI, DMF, −20 °C, 6 h, 82% (13), 79% (17); (e) 14 (1.0 equiv.), DDQ (1.1 equiv.) MS4A,
CH2Cl2, r.t., 16 h, 87% (15), 67% (18); (f ) TBAF, pyridine–THF, 0 °C, 1 h; (g) Ac2O, pyridine, r.t., 3 h, 89% (20 from 10), 81% (22 from 16), 87% (24 from 19); (h) H2,
Pd(OH)2, EtOAc–EtOH (2 : 1), r.t., 16 h; (i) FmocCl, DIPEA, CH2Cl2, r.t., 5 h, 81% (21 from 20), 74% (23 from 22), 81% (25 from 24).
methoxybenzoic acid as the matrix. HRMS determinations
4-Methylphenyl 3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-1-
were performed by the use of a JEOL AccuTOF JMS-T700LCK thio-α-L-arabinofuranoside (14). To a solution of 4-methyl-
mass spectrometer with CF3CO2Na as the internal standard. phenyl 1-thio-α-L-arabinofuranoside37 (3.20 g, 12.4 mmol) in
Tandem MS/MS was analyzed using the AB Sciex 4800 Plus pyridine (30 mL) at 0 °C was added 1,3-dichloro-1,1,3,3-tetra-
MALDI TOF/TOF mass spectrometer at the Support Unit for isopropyldisiloxane (4.39 mL, 13.7 mmol) dropwise. After stir-
Bio-material Analysis in Research Resources Center, RIKEN ring for 3 h at 0 °C, the reaction mixture was gradually warmed
Brain Science Institute. All other reagents were purchased up to room temperature and stirred for 2 h. Upon completion,
from Wako Pure Chemical Industries Ltd, Kanto Chemical Co., the mixture was quenched with saturated NaHCO3 and
Inc., Tokyo Chemical Industry Co., Ltd, and Sigma-Aldrich Co.
extracted with CHCl3. The combined organic phase was
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Scheme 2 Synthesis of CLV3 glycopeptide. Reagents and conditions: (a) see the experimental procedure; (b) i. piperidine, DMF, room temperature, 10 min; ii.
Fmoc-Hyp(L-Araf0–3) (3.0 equiv.), COMU (3.0 equiv.), DIPEA (6.0 equiv.), room temperature, 16 h, see also the Experimental section; (c) i. piperidine, DMF, room temp-
erature, 10 min; ii. Fmoc-AA (5.0 equiv.), COMU (5.0 equiv.), DIPEA (10 equiv.), room temperature, 2–4 h. The completion of two step reactions (Fmoc cleavage and
coupling) had been estimated by MALDI-TOF MASS of the sample after micro-cleavage. See also the Experimental section; (d) i. TFA–TIPS–H2O (190 : 5 : 5), 1 h; ii.
NeOMe, MeOH, room temperature, 2 h, 37% (3), 31% (2) 34% (1); (e) TFA–TIPS–H2O (190 : 5 : 5), 1 h, 30% (4). The yields were calculated based on the initial
loading capacity of the used NovaSyn TGT alcohol resin (0.02 mmol g−1).
®
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washed with saturated NaHCO3 and brine. The organic layer activated molecular sieves (4 Å, 10 g) in dry CH2Cl2 (110 mL)
was dried with MgSO4 and concentrated in vacuo. The residue was stirred under an argon atmosphere at room temperature
was purified by silica gel chromatography (EtOAc–hexane gra- for 30 min. MeOTf (0.87 mL, 7.76 mmol) was then added to
dient elution) to afford the title compound as a colorless syrup the mixture and the mixture was stirred for 48 h at 40 °C.
(4.48 g, 72%). Analytical data for 14: Rf = 0.50 (EtOAc–hexane, Upon completion the reaction mixture was cooled down,
1
1.5/8.5, v/v); [α]D27 −122.39° (c 1.0, CHCl3); H NMR (500 MHz, quenched with Et3N, diluted with CHCl3, and filtered through
CDCl3): δ 0.94–1.17 (m, 28H, TIPDS), 2.32 (s, 3H, CH3), 2.43 (d, a pad of Celite. The filtrate was washed with saturated
1H, J = 4.5 Hz, –OH), 3.92–3.95 (m, 1H, 4-H), 3.97–3.98 (m, 2H, NaHCO3 and brine. The organic layer was concentrated
5-Ha, 5-Hb), 4.16–4.21 (m, 2H, 2-H, 3-H), 5.24 (d, 1H, J1,2
=
in vacuo. The crude residue was dissolved in CHCl3 (30 mL)
5.0 Hz, 1-H), 7.09 (d, 2H, J = 8.5 Hz, aromatic), 7.39 (d, 2H, J = and cooled at 0 °C. TFA (3 mL) was added and stirred for
8.5 Hz, aromatic); 13C NMR (125 MHz, CDCl3): δ 12.7, 13.0, 30 min at the same temperature. The reaction mixture was
13.3, 13.7, 17.20, 17.26, 17.30, 17.5, 17.6, 21.3, 61.4, 76.4, 80.6, diluted with CHCl3 and washed successively with H2O, satu-
81.9, 91.3, 129.9, 130.9, 132.0, 137.6; ESI-TOF HRMS: [M + Na]+ rated NaHCO3 and brine. The organic layer was dried with
calcd for C24H42NaO5SSi2 521.2189, found 521.2204.
4-Methylphenyl 2-O-(2-naphthyl)methyl-3,5-O-(tetraisopro- silica gel chromatography (EtOAc–hexane gradient elution) to
pyldisiloxane-1,3-diyl)-1-thio-α-L-arabinofuranoside (5). To afford the title compound as a colorless syrup (1.40 g, 81%,
MgSO4 and concentrated in vacuo. The residue was purified by
a
solution of 14 (2.50 g, 4.8 mmol) in DMF (20 mL) at 0 °C β-only as the rotamer mixture). Analytical data for 10: Rf = 0.48
was added 2-(bromomethyl)naphthalene (NAPBr) (1.27 g, (EtOAc–hexane, 4.0/6.0, v/v); [α]2D6 47.59° (c 1.0, CHCl3);
5.7 mmol) and NaH (60% dispersion in oil, 0.23 g, 5.7 mmol) 1H NMR (500 MHz, CDCl3): δ (separated chemical shifts for
portionwise. The reaction mixture was stirred at 0 °C for 5 h. minor rotamer in parentheses) 1.01–1.09 (m, 28H, TIPDS),
Upon completion, the reaction was diluted with EtOAc, poured 2.11–2.20 (m, 2H, –OH, Hyp-β-Ha), 2.48–2.53 (2.39–2.44) (m,
into ice-water, stirred until cessation of H2 evolution, and then 1H, Hyp-β-Hb), 3.66–3.81 (3.56) (m, 4H, 4-H, 5-Ha, Hyp-δ-H),
extracted with EtOAc. The combined organic phase was 3.90–3.96 (dd, 1H, J = 1.5, 9.5 Hz, 5-Hb), 4.06–4.12 (m, 1H,
washed with sat. NH4Cl, and dried with MgSO4 and concen- 2-H), 4.18 (dd, 1H, J = 6.5, 7.5 Hz, 3-H), 4.37–4.40 (m, 1H, Hyp-
trated in vacuo. The residue was purified by column chromato- γ-H), 4.46 (4.53) (t, 1H, J = 7.5 Hz, Hyp-α-H), 4.92 (4.88) (d, 1H,
graphy on silica gel (EtOAc–hexane gradient elution) to afford J = 5.0 Hz, 1-H), 4.98 (d, 1H, J = 12.5 Hz, CH2Ph), 5.02 (d, 1H,
the title compound as a colorless syrup (2.60 g, 84%). Analyti- J = 12.5 Hz, CH2Ph), 5.06 (d, H, J = 12.5 Hz, CH2Ph), 5.15
cal data for 2: Rf = 0.47 (EtOAc–hexane, 1.0/9.0, v/v); [α]2D7 (d, 1H, J = 12.5 Hz, CH2Ph), 7.15–7.40 (m, 10H, aromatic); 13C
−39.19° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ 0.94–1.14 NMR (125 MHz, CDCl3): δ (separated chemical shifts for minor
(m, 28H, TIPDS), 2.28 (s, 3H, CH3), 3.69–4.02 (m, 3H, 4-H, rotamer in parentheses) 12.6, 12.9, 13.4, 13.6, 17.1, 17.2, 17.50,
5-Ha, 5-Hb), 4.09 (dd, 1H, J2,3 = 4.5 Hz, 2-H), 4.36 (dd, 1H, J3,4
=
17.53, 17.57, 17.64, 36.2 (37.8), 52.4 (51.9), 58.4 (58.0), 65.0,
7.5 Hz, 3-H), 4.81 (d, 1H, J = 12.0 Hz, CH2Ar), 4.87 (d, 1H, J = 67.0 (67.2), 67.5, 75.6 (78.0), 77.9 (78.0), 78.2 (78.3), 81.95
12.0 Hz, CH2Ar), 5.44 (d, 1H, J1,2 = 4.5 Hz, 1-H), 7.04 (d, 2H, J = (81.99), 100.1 (1JC1–H1 = 172.1 Hz) (100.0), 128.02, 128.03,
8.0 Hz, aromatic), 7.35 (d, 2H, J = 8.0 Hz, aromatic), 7.44 (m, 128.18, 128.22, 128.24, 128.27, 128.52, 128.57, 128.66, 128.70,
3H, aromatic), 7.78 (m, 4H, aromatic); 13C NMR (125 MHz, 135.4 (135.7), 136.6 (136.5), 154.4 (154.9), 172.4 (172.2);
CDCl3): δ 12.7, 13.0, 13.3, 13.7, 17.2, 17.3, 17.5, 17.8, 21.2, ESI-TOF HRMS: [M
61.4, 73.0, 76.2, 77.5, 80.3, 89.3, 89.9, 126.0, 126.1, 126.2, 661.3888, found 661.3903.
126.8, 127.9, 128.1, 128.2, 129.78, 129.81, 131.4, 131.7, 133.2, N-Benzyloxycarbonyl-4R-[3,5-O-(tetraisopropyldisiloxane-1,3-
133.4, 135.3, 137.4; ESI-TOF HRMS: [M + Na]+ calcd for diyl)-2-O-(2-naphthyl)methyl-L-arabinofuranosyl]oxy-L-proline
C35H50NaO5SSi2 661.2815, found 661.2795. benzyl ester (11). A mixture of a thioglycoside (5) (250.0 mg,
+
Na]+ calcd for C48H63NNaO10SSi2
N-Benzyloxycarbonyl-4R-[3,5-O-(tetraisopropyldisiloxane-1,3- 0.39 mmol), Z-Hyp-OBn (6) (115.0 mg, 0.32 mmol) and freshly
diyl)-β-L-arabinofuranosyl]oxy-L-proline benzyl ester (10). DDQ activated molecular sieves (4 Å, 750 mg) in CH2Cl2 (10.0 mL)
(1.84 g, 8.10 mmol) was added to a mixture of Z-Hyp-OBn28 (6) was stirred under argon at room temperature for 30 min. After
(2.0 g, 5.63 mmol), glycosyl donor (5) (4.31 g, 6.76 mmol), and the mixture was cooled to −40 °C, N-iodosuccinimide (NIS)
freshly activated molecular sieves (4 Å, 12 g) in dry CH2Cl2 (132.0 mg, 0.58 mmol) followed by silver trifluoromethanesul-
(30 mL) at room temperature. The reaction mixture was stirred fonate (AgOTf) (30.0 mg, 0.11 mmol) were added. The reaction
for 16 h under an argon atmosphere. The reaction mixture was mixture was warmed slowly to room temperature and stirred
quenched with aqueous ascorbate buffer, and filtered through for 15 min. The reaction was quenched by the addition of
a pad of Celite. The filtrate was extracted with CHCl3 and the Et3N. The suspension was diluted with CHCl3 and filtered
organic layer was washed with saturated NaHCO3 and brine. through a pad of Celite; the filtrate was washed successively
The organic layer was dried with MgSO4 and concentrated with 10% Na2S2SO3 and brine. The organic layer was dried
in vacuo. The residue was purified by silica gel chromatography with MgSO4 and concentrated in vacuo to give a residue. The
(EtOAc–hexane gradient elution) to afford mixed acetal (4.86 g, residue was purified by silica gel chromatography (EtOAc–
87%); MALDI-TOF MS: [M + Na]+ calcd for C55H69NNaO10SSi2 hexane gradient elution) to afford the corresponding oligosac-
1014.4079, found 1014.6565. A mixture of the mixed acetal charide (265 mg, 76%, α/β ratio 1.3 : 1). Analytical data for the
(2.2 g, 2.21 mmol), DTBMP (1.81 g, 8.87 nmol) and freshly rotamer mixture of β-11: Rf = 0.36 (EtOAc–hexane, 3.0/7.0, v/v);
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[α]D26 64.79° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ (separ- mixed acetal (1.25 g, 84%); MALDI-TOF MS: [M + Na]+ calcd
ated chemical shifts for minor rotamer in parentheses) for C52H71NNaO10SSi2 980.4235, found 980.5001. A mixture of
0.92–1.11 (m, 28H, TIPDS), 2.08–2.12 (m, 1H, J = 5.5 Hz, Hyp- the mixed acetal (0.64 g, 0.67 mmol), DTBMP (0.54 g,
β-Ha), 2.42–2.53 (m, 1H, Hyp-β-Hb), 3.62–3.68 (dd, 2H, J = 3.5, 2.67 mmol) and freshly activated molecular sieves (4 Å, 2 g) in
11.5 Hz, Hyp-δ-H2), 3.73–3.82 (m, 2H, 5-Ha, 5Hb), 3.88–3.94 dry CH2Cl2 (33 mL) was stirred under an argon atmosphere at
(m, 1H, 4-H), 3.97 (dd, 1H, J = 4.5, 7.5 Hz, 2-H), 4.22–4.27 (m, room temperature for 30 min. MeOTf (0.26 mL, 2.34 mmol)
1H, Hyp-γ-H), 4.52 (dd, 1H, J = 6.0, 8.0 Hz, 3-H), 4.64–4.71 (t, was then added to the mixture and the mixture was stirred for
1H, J = 7.0 Hz, Hyp-α-H), 4.79 (4.73), (d, 1H, J = 4.0 Hz, 1-H), 48 h at 40 °C. Upon completion the reaction mixture was
4.89 (d, 1H, J = 12.5 Hz, CH2Ph), 4.98 (s, 2H, CH2Ph), 5.02 (d, cooled down, quenched with Et3N, diluted with CHCl3, and fil-
1H, J = 12.5 Hz, CH2Ph), 5.14 (d, 1H, J = 12.5 Hz, CH2Ph), 5.20 tered through a pad of Celite. The filtrate was washed with
(d, 1H, J = 12.5 Hz, CH2Ph), 7.16–7.80 (m, 17H, aromatic); 13C saturated NaHCO3 and brine. The organic layer was concen-
NMR (125 MHz, CDCl3): δ (separated chemical shifts for minor trated in vacuo. The crude residue was dissolved in CHCl3
rotamer in parentheses) 12.6, 13.0, 13.4, 13.6, 17.17, 17.19, (10 mL) and cooled at 0 °C. TFA (1 mL) was added and stirred
17.22, 17.29, 17.56, 17.60, 17.72, 37.7 (36.7)*, 51.7 (51.4), 58.4 for 30 min at the same temperature. The reaction mixture was
(58.1), 66.3, 67.0 (67.1), 67.35 (67.43), 72.6 (72.9), 73.9, 75.0, diluted with CHCl3 and washed successively with H2O, satu-
77.4, 82.0 (81.9), 84.25 (84.28), 98.4 (1JC1–H1 = 169.7 Hz) (98.9), rated NaHCO3 and brine. The organic layer was dried with
125.9, 126.0, 126.1, 126.2, 126.28, 126.34, 126.7, 126.9, 127.85, MgSO4 and concentrated in vacuo. The residue was purified by
127.87, 127.90, 128.00, 128.04, 128.06, 128.09, 128.19, 128.22, silica gel chromatography (EtOAc–hexane gradient elution) to
128.24, 128.32, 128.35, 128.41, 128.48, 128.51, 128.65, 128.69, afford the title compound as a colorless syrup (0.32 g, 67%,
133.2, 133.33, 135.34, 135.45, 135.51, 135.7, 136.4, 136.6, 154.5 β-only as the rotamer mixture). Analytical data for 12: Rf = 0.48
(154.6), 172.6 (172.4); ESI-TOF HRMS: [M + Na]+ calcd for (EtOAc–hexane, 4.0/6.0, v/v); [α]D24 52.60° (c 1.0, CHCl3); 1H
C48H63NNaO10Si2 892.3888, found 892.3903.
NMR (500 MHz, CDCl3): δ (separated chemical shifts for minor
Rotamer mixture of α-11: Rf = 0.51 (EtOAc–hexane, 3.0/7.0, rotamer in parentheses) 0.91–1.09 (m, 28H, TIPDS), 1.34 (1.45)
1
v/v); [α]2D6 −39.19° (c 1.0, CHCl3); H NMR (500 MHz, CDCl3): δ (s, 9H, C(CH3)3), 2.10 (m, 1H, Hyp-β-Ha), 2.28 (2.24) (d, 1H, J =
(separated chemical shifts for minor rotamer in parentheses) 10.0 Hz, –OH), 2.46–2.51 (2.34–2.38) (m, 1H, Hyp-β-Hb),
0.89–1.12 (m, 28H, TIPDS), 1.89–1.98 (m, 1H, Hyp-β-Ha), 3.59–3.66 (3.45) (m, 2H, Hyp-δ-H), 3.75–3.83 (m, 2H, 4-H,
2.11–2.18 (m, 1H, Hyp-β-Hb), 3.65–3.89 (m, 4H, 5-Ha, 5-Hb, 5-Ha), 3.92–3.95 (m, 1H, J = 8.0 Hz, 5-Hb), 4.10 (dd, 1H, J = 4.5,
Hyp-δ-H), 3.73–3.82 (m, 2H, 5-Ha, 5-Hb), 3.93–3.95 (m, 1H, 7.5 Hz, 2-H), 4.19 (dd, 1H, J = 6.0, 7.5 Hz, 3-H), 4.34–4.37 (m,
4-H), 3.98 (dd, 1H, J = 1.5, 7.0 Hz, 2-H), 4.19–4.31 (m, 2H, 3-H, 2H, Hyp-α-H, Hyp-γ-H), 4.93 (4.89) (d, 1H, J = 5.0 Hz, 1-H), 5.13
Hyp-γ-H), 4.46 (4.36) (t, 1H, J = 7.5 Hz, Hyp-α-H), 4.71 (d, 1H, (d, 1H, J = 12.5 Hz, CH2Ph), 5.18 (d, 1H, J = 12.5 Hz, CH2Ph),
J = 12.0 Hz, CH2Ph), 4.79 (d, 1H, J = 12.0 Hz, CH2Ph), 4.97 7.35 (m, 5H, aromatic); 13C NMR (125 MHz, CDCl3): δ (separ-
(4.94) (d, 1H, J = 1.5 Hz, 1-H), 4.98–5.03 (m, 2H, CH2Ph), 5.14 ated chemical shifts for minor rotamer in parentheses) 12.6,
(d, 1H, J = 12.5 Hz, CH2Ph), 5.20 (d, 1H, J = 12.5 Hz, CH2Ph), 12.9, 13.4, 13.6, 28.3, 28.5, 36.8 (37.7), 51.8 (51.9), 65.2 (66.9),
7.19–7.79 (m, 17H, aromatic); 13C NMR (125 MHz, CDCl3): 75.7 (75.9), 77.9 (78.0), 78.4, 80.56 (80.56), 81.9 (82.0), 100.1
δ (separated chemical shifts for minor rotamer in parentheses) (100.0), 128.1 (128.3), 128.4 (128.5), 128.7 (128.6), 135.5
12.7, 13.0, 13.3, 13.72, 13.74, 17.17, 17.24, 17.3, 17.5, 17.6, 35.5 (135.75), 153.83 (154.4), 172.8 (172.4); ESI-TOF HRMS:
(36.3), 53.1 (53.6), 57.8 (58.1), 61.2, 67.1 (67.0), 67.30 (67.28), [M + Na]+ calcd for C34H57NNaO10Si2 718.3419, found 718.3408.
73.13 (73.06), 74.4, 75.4, 75.8, 80.60 (80.63), 89.6 (89.7), 105.1
(1JC1–H1 = 170.0 Hz) (105.0), 125.9, 126.1, 126.3, 127.8, 128.0, furanosyl-(1→2)-3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-α-L-
128.11, 128.13, 128.28, 128.34, 128.45, 128.50, 128.54, 128.56, arabinofuranoside (13). 2,3-Dichloro-5,6-dicyano-1,4-benzo-
Methyl 3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-β-L-arabino-
128.61, 128.7, 133.4 (133.2), 135.3 (135.4), 135.5 (135.8), 136.7 quinone (DDQ) (80.5 mg, 0.35 mmol) was added to a mixture
(136.6), 155.1 (154.3), 172.5 (172.3); ESI-TOF HRMS: [M + Na]+ of glycosyl acceptor38 (8) (100.0 mg, 0.24 mmol), glycosyl
calcd for C48H63NNaO10Si2 892.3888, found 892.3900.
donor (5) (188.5 mg, 0.28 mmol), and freshly activated mole-
N-tert-Butyloxycarbonyl-4R-[3,5-O-(tetraisopropyldisiloxane- cular sieves (4 Å, 300 mg) in dry CH2Cl2 (5 mL) at room temp-
1,3-diyl)-β-L-arabinofuranosyl]oxy-L-proline benzyl ester (12). erature. The reaction mixture was stirred for 16 h under an
DDQ (0.51 g, 2.24 mmol) was added to a mixture of Boc-Hyp- argon atmosphere. The reaction mixture was quenched with
OBn (7)36 (0.50, 1.55 mmol), glycosyl donor (5) (1.19 g, aqueous ascorbate buffer, and filtered through a pad of Celite.
1.86 mmol), and freshly activated molecular sieves (4 Å, 3.5 g) The filtrate was extracted with CHCl3 and the organic layer was
in dry CH2Cl2 (10 mL) at room temperature. The reaction washed with saturated NaHCO3 and brine. The organic layer
mixture was stirred for 16 h under an argon atmosphere. The was dried with MgSO4 and concentrated in vacuo. The residue
reaction mixture was quenched with aqueous ascorbate buffer, was purified by silica gel chromatography (EtOAc–hexane gra-
and filtered through a pad of Celite. The filtrate was extracted dient elution) to afford corresponding mixed acetal (230.0 g,
with CHCl3 and the organic layer was washed with saturated 92%); MALDI-TOF MS: [M + Na]+ calcd for C53H86NaO11SSi4
NaHCO3 and brine. The organic layer was dried with MgSO4 1065.4866, found 1065.5035. A mixture of the mixed acetal
and concentrated in vacuo. The residue was purified by silica (150 mg, 0.14 mmol), 2,6-di-tert-butyl-4-methylpyridine
gel chromatography (EtOAc–hexane gradient elution) to afford (DTBMP) (118.0 mg, 0.57 mmol) and freshly activated
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molecular sieves (4 Å, 750 mg) in dry CH2Cl2 (7.5 mL) was CHCl3 (20 mL) and cooled at 0 °C. TFA (2 mL) was added and
stirred under an argon atmosphere at room temperature for kept stirred in vacuo for 30 min at the same temperature. The
30 min. Methyl trifluoromethanesulfonate (MeOTf) (57 μL, reaction mixture was diluted with CHCl3 washed successively
0.50 mmol) was then added to the mixture and the mixture with H2O, saturated NaHCO3 and brine. The organic layer was
was stirred for 48 h at 40 °C. Upon completion the reaction dried with MgSO4 and concentrated in vacuo. The residue was
mixture was cooled down, quenched with triethylamine (Et3N), purified by silica gel chromatography (EtOAc–hexane gradient
diluted with CHCl3, and filtered through a pad of Celite. The elution) to afford the title compound as a colorless syrup
filtrate was washed with saturated NaHCO3 and brine. The (1.23 g, 84%, β-only as the rotamer mixture). Analytical data
organic layer was concentrated in vacuo. The crude residue was for 16: Rf = 0.48 (EtOAc–hexane, 3.0/7.0, v/v); [α]2D6 73.60° (c 1.0,
dissolved in CHCl3 (2 mL) and cooled at 0 °C. Trifluoroacetic CHCl3); 1H NMR (500 MHz, CDCl3): δ (separated chemical
acid (TFA) (0.2 mL) was added and stirred for 30 min at the shifts for minor rotamer in parentheses) 0.93–1.11 (m, 56H,
same temperature. The reaction mixture was diluted with 2 × TIPDS), 2.09–2.14 (m, 1H, Hyp-β-Ha), 2.37–2.42 (m, 1H,
CHCl3 and washed successively with H2O, saturated NaHCO3 Hyp-β-Hb), 2.47 (2.50) (d, 1H, J = 10.0 Hz, –OH), 3.51–3.59 (m,
and brine. The organic layer was dried with MgSO4 and con- 1H, J = 12.0 Hz, Hyp-δ-H), 3.66–3.99 (m, 7H, 4-H, 5-Ha, 5-Hb,
centrated in vacuo. The residue was purified by silica gel 4′-H, 5′-a, 5′-Hb, Hyp-δ-H), 4.01–4.06 (m, 1H, 2′-H), 4.18 (dd,
chromatography (EtOAc–hexane gradient elution) to afford the 1H, J = 6.5, 7.0 Hz, 3′-H), 4.23 (dd, 1H, J = 4.5, 8.0 Hz, 2-H),
title compound as a colorless syrup (88.7 mg, 79%, β-only). 4.31–4.35 (m, 1H, Hyp-γ-H), 4.44 (dd, 1H, J = 6.5, 7.5 Hz, 3-H),
Analytical data for 13: Rf = 0.50 (EtOAc–hexane, 2.0/8.0, v/v); 4.52 (t, 1H, J = 7.5 Hz, Hyp-α-H), 4.74 (4.71) (d, 1H, J = 5.0 Hz,
[α]2D7 197.99° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ 1′-H), 4.98 (4.93) (d, 1H, J = 4.5 Hz, 1-H), 4.98–5.24 (m, 4H,
1.01–1.09 (m, 28H, TIPDS), 2.18 (d, 1H, J = 10.5 Hz, –OH), 3.37 2 × CH2Ph), 7.15–7.39 (m, 10H, aromatic); 13C NMR (125 MHz,
(s, 3H, –OCH3), 3.77 (dd, 1H, J = 10.0, 10.5 Hz, 5′-Ha), CDCl3): δ (separated chemical shifts for minor rotamer in par-
3.87–3.93 (m, 3H, 4-H, 4′-H, 5′-Hb), 3.97–4.02 (m, 2H, 5-Ha, entheses) 12.6, 12.98, 13.01, 13.4, 13.5, 16.98, 17.06, 17.18,
5-Hb), 4.11–4.16 (m, 1H, 2′-H), 4.20 (dd, 1H, J = 6.0, 7.0 Hz, 17.23, 17.33, 17.44, 17.49, 17.53, 17.65, 17.71, 37.7 (36.7), 51.3
3′-H), 4.24 (dd, 1H, J = 2.0, 6.0 Hz, 2-H), 4.28 (dd, 1H, J = 6.5, (51.2), 58.0 (58.2), 65.9 (65.8), 66.2 (66.1), 67.0 (67.1), 67.5,
7.0 Hz, 3-H), 4.80 (d, 1H, J = 2.0 Hz, 1-H), 5.02 (d, 1H, J = 73.8, 75.3, 76.7 (76.5), 79.04 (78.97), 80.1 (79.9), 80.7 (80.9),
5.0 Hz, 1′-H); 13C NMR (125 MHz, CDCl3): δ 12.7, 12.8, 13.1, 82.1 (82.0), 82.72 (82.67), 97.0 (1JC1–H1 = 172.2 Hz) (98.0), 99.5
13.2, 13.4, 13.5, 13.6, 17.09, 17.16, 17.31, 17.34, 17.41, 17.53, (1JC1–H1 = 170.4 Hz) (99.7), 127.97 (128.00), 128.2, 128.3, 128.5,
17.57, 17.61, 17.69, 17.76, 30.5, 55.37, 61.9, 66.1, 75.7, 79.4, 128.7 (128.6), 135.5 (135.7), 136.5 (136.6), 154.7 (154.9), 172.6
80.1, 81.3, 82.7, 86.0, 99.3, 106.2; ESI-TOF HRMS: [M + Na]+ (172.3); ESI-TOF HRMS: [M + Na]+ calcd for C54H89NNaO15SSi4
calcd for C35H72NaO11Si4 803.4049, found 803.4015.
1126.5207; found 1126.5173.
N-Benzyloxycarbonyl-4R-[3,5-O-(tetraisopropyldisiloxane-1,3-
N-Benzyloxycarbonyl-4R-[3,5-O-(tetraisopropyldisiloxane-1,3-
diyl)-β-L-arabinofuranosyl-(1→2)-3,5-O-(tetraisopropyldisiloxane- diyl)-2-O-(2-naphthyl)methyl-β-L-arabinofuranosyl-(1→2)-3,5-O-
1,3-diyl)-β-L-arabinofuranosyl]oxy-L-proline benzyl ester (16). (tetraisopropyldisiloxane-1,3-diyl)-β-L-arabinofuranosyl]oxy-L-
DDQ (0.41 g, 1.82 mmol) was added to a mixture of glycosyl proline benzyl ester (17). To a stirred solution of compound
acceptor 10 (0.91 g, 1.82 mmol), glycosyl donor 5 (1.27 g, 16 (1.60 g, 1.44 mmol) in dry DMF (20 mL) at −20 °C were
1.52 mmol), and freshly activated molecular sieves (4 Å, 3.5 g) added tetrabutylammonium iodide (TBAI) (80 mg,
in dry CH2Cl2 (20 mL) at room temperature. The reaction 0.22 mmol), NaH (60% dispersion in mineral oil, 70 mg,
mixture was stirred for 16 h under an argon atmosphere. The 1.74 mmol), and NAPBr (0.38 g, 1.74 mmol). The reaction
reaction mixture was quenched with aqueous ascorbate buffer, mixture was stirred for 16 h at the same temperature. Upon
and filtered through a pad of Celite. The filtrate was extracted completion, the reaction was diluted with EtOAc, poured into
with CHCl3 and the organic layer was washed with saturated ice-water, stirred until cessation of H2 evolution, and then
NaHCO3 and brine. The organic layer was dried with MgSO4 extracted with EtOAc. The combined organic phase was
and concentrated in vacuo. The residue was purified by silica washed with sat. NH4Cl, and dried with MgSO4 and concen-
gel chromatography (EtOAc–hexane gradient elution) to afford trated in vacuo. The residue was purified by column chromato-
mixed acetal 15 (1.81 g, 87%). 15: MALDI-TOF MS: [M + Na]+ graphy on silica gel (EtOAc–hexane gradient elution) to afford
calcd for C72H103NNaO15SSi4 1388.6023, found 1388.8651. A the title compound as a colorless syrup (1.42 g, 79%, rotamer
mixture of the mixed acetal 15 (1.81 g, 1.32 mmol), DTBMP mixture). Analytical data for rotamer mixture of 17: Rf = 0.54
(1.08 g, 5.30 mmol) and freshly activated molecular sieves (4 Å, (EtOAc–hexane, 3.0/7.0, v/v); [α]2D6 76.20° (c 1.0, CHCl3); 1H
10 g) in dry CH2Cl2 (67 mL) was stirred under an argon atmos- NMR (500 MHz, CDCl3): δ (major rotamer) 0.87–1.11 (m, 56H,
phere at room temperature for 30 min. MeOTf (0.52 mL, 2 × TIPDS), 2.07 (m, 1H, Hyp-β-Ha), 2.17–2.29 (m, 1H, Hyp-
4.63 mmol) was then added to the mixture and the mixture β-Hb), 3.54–4.02 (m, 9H, 2-H, 4-H, 5-Ha, 5-Hb, 4′-H, 5′-Ha,
was stirred for 48 h at 40 °C. Upon completion the reaction 5′-Hb, Hyp-δ-H), 4.21 (dd, 1H, J = 4.0, 8.0 Hz, 2′-H), 4.38 (t, 1H,
mixture was cooled down, quenched with Et3N, diluted with J = 7.5 Hz, Hyp-γ-H), 4.55 (m, 2H, 3-H, 3′-H), 4.75 (t, J = 6.0 Hz,
CHCl3, and filtered through a pad of Celite. The filtrate was Hyp-α-H), 4.84–5.14 (m, 8H, 1-H, 1′-H, 3 × CH2Ph), 7.06–7.77
washed with saturated NaHCO3 and brine. The organic layer (m, 17H, aromatic); 13C NMR (125 MHz, CDCl3): δ (rotamer
was concentrated in vacuo. The crude residue was dissolved in mixture) 12.56, 12.59, 12.9, 13.0, 13.4, 13.5, 13.6, 17.01, 17.07,
5900 | Org. Biomol. Chem., 2013, 11, 5892–5907
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17.09, 17.13, 17.19, 17.23, 17.29, 17.44, 17.48, 17.52, 17.59, 13.4, 13.5, 13.6, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.5, 17.6,
17.65, 17.76, 36.7, 37.7, 51.1, 51.8, 57.9, 58.2, 65.7, 66.5, 66.6, 17.7, 17.8, 37.66, 37.75, 50.88, 51.5, 57.9, 58.2, 65.76, 65.79,
66.8, 67.0, 67.4, 71.1, 71.2, 73.6, 74.3, 76.3, 76.4, 77.4, 78.2, 66.4, 66.5, 66.6, 67.0, 67.2, 67.4, 67.5, 73.5, 74.6, 76.5, 77.9,
78.3, 78.37, 78.40, 81.8, 81.9, 82.9, 85.2, 85.4, 96.7, 96.8, 97.3, 78.0, 78.1, 78.2, 79.1, 79.2, 80.4, 80.5, 81.9, 81.95, 81.96, 82.00,
97.4, 125.49, 125.57, 125.62, 125.8, 125.9, 126.0, 127.65, 82.5, 82. 6, 83.06, 83.08, 95.9, 96.0, 965, 97.5, 99.1, 99.3, 128.0,
127.71, 128.0, 128.1, 128.16, 128.18, 128.28, 128.31, 128.34, 128.1, 128.19, 128.24, 128.3, 128.45, 128.51, 128.6, 128.7,
128.4, 128.5, 128.6, 132.9, 133.0, 133.37, 133.40, 135.44, 135.7, 135.6, 135.7, 136.4, 136.7, 154.4, 154.8, 172.4, 172.7; ESI-TOF
136.0, 136.1, 138.5, 154.2, 154.7, 172.2, 172.3 ppm; ESI-TOF HRMS: [M + Na]+ calcd for C71H123NNaO20Si6 1500.7152,
HRMS: [M + Na]+ calcd for C65H97NNaO15Si4 1266.5833, found found 1500.7155.
1266.5866.
N-Benzyloxycarbonyl-4R-[2,3,5-tri-O-acetyl-β-L-arabinofurano-
N-Benzyloxycarbonyl-4R-[3,5-O-(tetraisopropyldisiloxane-1,3- syl]oxy-L-proline benzyl ester (20). To a solution of 10 (0.65 g,
diyl)-β-L-arabinofuranosyl-(1→2)-3,5-O-(tetraisopropyldisiloxane- 0.89 mmol) in pyridine–THF (1 : 4, 5 mL) at 0 °C was added
1,3-diyl)-β-L-arabinofuranosyl-(1→2)-3,5-O-(tetraisopropyldisil-
a solution of tetrabutylammonium fluoride (1.78 mL,
oxane-1,3-diyl)-β-L-arabinofuranosyl]oxy-L-proline benzyl ester 1.78 mmol, 1 M in THF). After being stirred for 1 h at 0 °C,
(19). DDQ (114 mg, 0.50 mmol) was added to a mixture of gly- CaCO3 (0.74 g), DOWEX® 50WX2 (2.20 g, used as supplied),
cosyl acceptor 17 (250 mg, 0.50 mmol), glycosyl donor 14 and MeOH (5.2 mL) were added.40 The suspension was stirred
(520 mg, 0.42 mmol), and freshly activated molecular sieves at room temperature for 1 h. All insoluble materials were
(4 Å, 450 mg) in dry CH2Cl2 (7 mL) at room temperature. The removed by filtration through a pad of Celite, and then filter
reaction mixture was stirred for 16 h under an argon atmos- Celite was washed with MeOH thoroughly. The combined fil-
phere. The reaction mixture was quenched with aqueous ascor- trates were concentrated in vacuo to give the crude product.
bate buffer, and filtered through a pad of Celite. The filtrate The crude product was subjected to the next step without
was extracted with CHCl3, and the organic layer was washed further purification. The crude product was dissolved in pyri-
with saturated NaHCO3 and brine. The organic layer was dried dine (8 mL), cooled to 0 °C, and treated with acetic anhydride
with MgSO4 and concentrated in vacuo. The residue was puri- (4 mL). The reaction mixture was stirred overnight at room
fied by silica gel chromatography (EtOAc–hexane gradient temperature. Upon completion, the reaction was diluted with
elution) to afford mixed acetal 18 (487 mg, 67%). 18: MALDI- CHCl3 and washed thoroughly with H2O, 1 N HCl, saturated
TOF MS: [M + Na]+ calcd for C89H137NNaO20SSi6 1764.6025, NaHCO3 and brine. The organic phase was dried with MgSO4
found 1764.8685. A mixture of the mixed acetal 18 (450 mg, and concentrated in vacuo. The residue was purified by silica
0.25 mmol), DTBMP (209 mg, 1.02 mmol) and freshly activated gel chromatography (EtOAc–hexane gradient elution) to afford
molecular sieves (4 Å, 1.5 g) in dry CH2Cl2 (15 mL) was stirred the title compound as a colorless syrup (0.48 g, 89%, rotamer
under an argon atmosphere at room temperature for 30 min. mixture). Analytical data for 20: Rf = 0.75 (EtOAc–CHCl3, 6.0/
MeOTf (101 μL, 0.89 mmol) was then added to the mixture 4.0, v/v); [α]2D6 37.19° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3):
and the mixture was stirred for 48 h at 40 °C. Upon completion δ (separated chemical shifts for minor rotamer in parentheses)
the reaction mixture was cooled down, quenched with Et3N, 1.94 (2.06) (s, 6H, 2 × OAc), 1.98 (2.01) (s, 3H, OAc), 2.09–2.12
diluted with CHCl3, and filtered through a pad of Celite. The (m, 1H, Hyp-β-Ha), 2.47–2.53 (2.42–2.45) (m, 1H, Hyp-β-Hb),
filtrate was washed with saturated NaHCO3 and brine. The 3.53–3.61 (m, 2H, Hyp-δ-H), 4.06–4.10 (m, 1H, 4-H), 4.20 (4.23)
organic layer was concentrated in vacuo. The crude residue was (dd, 1H, J = 5.5, 11.5 Hz, 5-Ha), 4.31–4.35 (m, 2H, 5-Hb, Hyp-
dissolved in CHCl3 (10.0 mL) and cooled at 0 °C. TFA (1.0 mL) γ-H), 4.49 (4.44) (t, 1H, J = 8.0 Hz, Hyp-α-H), 4.94 (4.90) (dd,
was added and stirred for 30 min at the same temperature. 1H, J = 4.5, 6.5 Hz, 2-H), 514 (4.99) (s, 2H, CH2Ph), 5.15 (5.01)
The reaction mixture was diluted with CHCl3 washed succes- (d, 1H, J = 12.0 Hz, CH2Ph), 5.21 (5.05) (d, 1H, J = 12.0 Hz,
sively with H2O, saturated NaHCO3 and brine. The organic CH2Ph), 5.28 (dd, 1H, J = 6.5, 7.5 Hz, 3-H), 5.32 (5.31) (d, 1H,
layer was dried with MgSO4 and concentrated in vacuo. The J = 4.5 Hz, 1-H), 7.19–7.38 (m, 10H, aromatic); 13C NMR
residue was purified by silica gel chromatography (EtOAc– (125 MHz, CDCl3): δ (separated chemical shifts for minor
hexane gradient elution) to afford the title compound as a col- rotamer in parentheses) 20.09 (20.06), 20.6 (20.5), 36.4 (37.3),
orless syrup (290 mg, 77%, β-only as the rotamer mixture). 51.1 (51.9), 57.7 (58.0), 65.13 (65.05), 66.8 (66.7), 67.2 (67.0),
Analytical data for 19: Rf = 0.54 (EtOAc–hexane, 3.0/7.0, v/v); 75.47 (75.39), 75.58 (75.54), 76.7, 76.9, 78.7 (78.8), 99.4 (98.7),
[α]2D7 90.00° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ (major 127.76, 127.87, 127.94, 128.0, 128.1, 128.2, 128.29, 128.34,
rotamer) 0.91–1.10 (m, 84H, 3 × TIPDS), 2.11–2.15 (m, 1H, 128.4, 135.2 (135.5), 136.2 (136.3), 154.8 (154.0), 169.96
Hyp-β-Ha), 2.37–2.42 (m, 2H, –OH, Hyp-β-Hb), 3.62–4.08 (m, (169.90), 170.03 (170.00), 170.3, 172.2 (171.9); ESI-TOF HRMS:
14H, 4-H, 5-Ha, 5-Hb, 2′-H, 3′-H, 4′-H, 5′-H, 5b′-H, 2′′-H, 4′′-H, [M + Na]+ calcd for C31H35NaO12 636.2057, found 636.2083.
5a′′-H, 5b′′-H, Hyp-δ-H), 4.18 (m, 2H, 2-H, 3′′-H), 4.33–4.36 (m,
N-9-Fluorenylmethyloxycarbonyl-4R-[2,3,5-tri-O-acetyl-β-L-
1H, Hyp-γ-H), 4.42–4.54 (m, 2H, 3-H, Hyp-α-H), 4.78 (d, 1H, arabinofuranosyl]oxy-L-proline (21). Compound 20 (0.48 g,
J1′′,2′′ = 4.00 Hz, 1′′-H), 4.88 (d, 1H, J1′,2′ = 4.5 Hz, 1′-H), 0.78 mmol) was dissolved in a mixture of EtOAc–MeOH (1 : 1,
4.95–5.01 (m, 3H, 1-H, CH2Ph), 5.10–5.23 (m, 2H, CH2Ph), 8 mL) and 20% Pd(OH)2 (0.48 g) was added. The reaction
7.19–7.36 (m, 10H, aromatic); 13C NMR (125 MHz, CDCl3): δ mixture was stirred under an atmosphere of H2 for 8 h. Upon
(rotamer mixture) 12.6, 12.67, 12.72, 12.9, 13.0, 13.1, 13.3, completion, the catalyst was filtered through a pad of Celite,
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Organic & Biomolecular Chemistry
and the filtrate was concentrated under reduced pressure to
N-Benzyloxycarbonyl-4R-[2,3,5-tri-O-acetyl-β-L-arabinofurano-
give a crude amine product. The crude product was then sub- syl-(1→2)-2,3,5-tri-O-acetyl-β-L-arabinofuranosyl]oxy-L-proline
jected to the next step without further purification. 9-Fluore- benzyl ester (22). To a solution of 16 (0.69 g, 0.62 mmol) in
nylmethyloxycarbonyl chloride (FmocCl) (0.23 g, 0.86 mmol) pyridine–THF (1 : 4, 10 mL) at 0 °C was added a solution of
was added to a stirred solution of the crude amine and N,N-di- tetrabutylammonium fluoride (2.5 mL, 2.50 mmol, 1 M in
isopropylethylamine (DIPEA) (150 μL, 0.86 mmol) in dry THF). After stirring for 3 h at 0 °C, CaCO3 (0.51 g), DOWEX
CH2Cl2 (5 mL) at 0 °C. The reaction mixture was stirred at 50WX2 (1.54 g, used as supplied), and MeOH (4 mL) were
room temperature for 5 h, after which it was diluted with added. The suspension was stirred at room temperature for
CHCl3 and washed with brine. The organic phase was dried 1 h. All insoluble materials were removed by filtration through
with MgSO4 and concentrated in vacuo. The residue was puri- a pad of Celite, and then filter Celite was washed with MeOH
fied by silica gel chromatography (EtOAc–hexane gradient thoroughly. The combined filtrates were concentrated in vacuo
elution) to afford the title compound as a colorless syrup to give the crude product. The crude product was subjected to
(0.34 g, 71%, rotamer mixture). Analytical data for 21: Rf = 0.62 the next step without further purification. The crude product
(MeOH–CHCl3, 1.0/9.0, v/v); [α]2D6 21.60° (c 1.0, CHCl3); was dissolved in pyridine (10 mL), cooled to 0 °C, and treated
1H NMR (500 MHz, CDCl3): δ (separated chemical shifts for with acetic anhydride (5 mL). The reaction mixture was
minor rotamer in parentheses) 1.94 (2.31) (s, 3H, OAc), 2.09 (s, allowed to stir overnight at room temperature. Upon com-
6H, 2 × OAc), 2.25–2.30 (2.16–2.20) (m, 1H, Hyp-β-Ha), pletion, the reaction was diluted with CHCl3 and washed
2.45–2.56 (m, 1H, Hyp-β-Hb), 3.55–3.62 (m, 2H, Hyp-δ-H), thoroughly with H2O, 1 N HCl, saturated NaHCO3 and brine.
4.08–4.15 (m, 1H, 4-H), 4.21–4.40 (m, 7H, 5-Ha, 5-Hb, Hyp-α-H, The organic phase was dried with MgSO4 and concentrated
Hyp-γ-H, CHFmoc, OCH2Fmoc), 4.94 (dd, 1H, J = 5.5, 10.0 Hz, in vacuo. The residue was purified by silica gel chromatography
2-H), 5.26–5.31 (m, 1H, 3-H), 5.34 (d, 1H, J = 4.5 Hz, 1-H), (EtOAc–CHCl3 gradient elution) to afford the title compound
7.24–7.75 (m, 8H, aromatic); 13C NMR (125 MHz, CDCl3): δ as a colorless syrup (0.42 g, 81%, rotamer mixture). Analytical
(separated chemical shifts for minor rotamer in parentheses) data for 22: Rf = 0.61 (EtOAc–CHCl3, 6.0/4.0, v/v); [α]2D6 59.40°
1
14.4, 20.6 (20.5), 21.01 (20.96), 36.2 (37.7), 47.3, 51.4 (52.2), (c 1.0, CHCl3); H NMR (500 MHz, CDCl3): δ (major rotamer)
58.3, 60.7, 65.3 (65.5), 68.3 (67.9), 75.74 (75.78), 75.85 (75.80), 2.05 (s, 3H, OAc), 2.07 (s, 9H, 3 × OAc), 2.10 (s, 3H, OAc), 2.19
77.4, 79.1 (79.0), 99.3 (99.9), 120.2, 125.3, 127.3, 128.0, (m, 1H, Hyp-β-Ha), 2.53 (m, 1H, Hyp-β-Hb), 3.65–3.70 (m, 2H,
141.5, 144.0 (143.9), 144.2, 156.1 (154.5), 170.36, 170.40, Hyp-δ-H2), 4.02–4.06 (m, 1H, 2-H), 4.10–4.20 (m, 3H, 4-H,
170.45, 170.9, 171.0; ESI-TOF HRMS: [M + Na]+ calcd for 5-Ha, 5′-Ha), 4.34–4.04 (m, 4H, 4′-H, 5-Hb, 5′-Hb, Hyp-γ-H),
C31H33NNaO12 634.1900, found 634.1917.
4.50 (t, 1H, J = 8.0 Hz, Hyp-α-H), 4.93–5.30 (m, 9H, 1-H, 2-H,
4R-β-L-Arabinofuranosyloxy-L-proline (deprotected derivative 3-H, 1′-H, 3′-H, 2 × CH2Ph), 7.18–7.37 (m, 10H, aromatic); 13C
of NAP-IAD product 10). To a solution of 20 (40.0 mg, NMR (125 MHz, CDCl3): δ (rotamer mixture) 20.3, 20.4, 20.6,
65.2 μmol) in methanol (2 mL) at 0 °C was added a 0.1 M 20.65, 20.68, 20.69, 36.1, 37.1, 51.1, 57.7, 58.0, 65.5, 65.69,
NaOH solution (2 mL). The reaction mixture was stirred for 4 h 65.73, 66.9, 67.1, 67.2, 74.7, 75.6, 75.7, 75.8, 76.3, 76.4, 76.7,
at the same temperature and then neutralized with Dowex 76.8, 77.4, 77.6, 77.7, 79.1, 79.2, 79.3, 79.5, 98.6, 98.7, 98.8,
(H+), filtered, and concentrated in vacuo. The crude residue 99.2, 127.78, 127.85, 127.95, 127.97, 128.04, 128.08, 128.20,
was dissolved in MeOH–H2O–HOAc (3 mL/1 mL/0.1 mL) and 128.25, 128.31, 128.41, 128.47, 128.49, 135.3, 135.5, 136.3,
20% Pd(OH)2 (15 mg) was added. The reaction mixture was 136.4, 154.2, 154.6, 169.91, 169.95, 170.03, 170.06, 170.33,
stirred under an atmosphere of H2 for 15 h. The catalyst was 170.38, 170.42, 170.44, 172.1, 172.4; ESI-TOF HRMS: [M + Na]+
then filtered-off, washed with methanol and H2O, and the fil- calcd for C40H47NNaO18 852.2691, found 852.2667.
trate was concentrated under reduced pressure. The residue
N-9-Fluorenylmethyloxycarbonyl-4R-[2,3,5-tri-O-acetyl-β-L-
was purified by Sep-pek C-18 cartridge (H2O–methanol gradi- arabinofuranosyl-(1→2)-2,3,5-tri-O-acetyl-β-L-arabinofuranosyl]-
ent elution). Fractions containing the product were collected oxy-L-proline (23). Compound 22 (0.67 g, 0.80 mmol) was dis-
and concentrated in vacuo to afford the title compound (13. solved in a mixture of EtOAc–MeOH (1 : 1, 10 mL) and 20%
5 mg, 79% in two steps). Analytical data for the title com- Pd(OH)2 (0.50 g) was added. The reaction mixture was stirred
pound: 1H NMR (500 MHz, D2O): δ 2.22–2.33 (m, 1H, Hyp- under an atmosphere of H2 for 16 h. Upon completion, the
β-Ha), 2.69–2.74 (m, 1H, Hyp-β-Hb), 3.50 (dd, 1H, J = 4.0, 12.5 catalyst was filtered through a pad of Celite and the filtrate was
Hz, Hyp-δ-Ha), 3.58 (d, 1H, J = 12.5 Hz, Hyp-δ-Hb), 3.66 (dd, concentrated under reduced pressure to give a crude amine
1H, J = 6.5, 12.0 Hz, 5-Ha), 3.81 (dd, 1H, J = 5.0, 12.5 Hz, 5-Hb), product. The crude product was then subjected to the next
3.87–3.91 (m, 1H, 4-H), 4.04 (dd, 1H, J = 8.0 Hz, 3-H), 4.14 (dd, step without further purification. FmocCl (0.32 g, 1.21 mmol)
1H, J = 4.5, 8.0 Hz, 2-H), 4.55 (dd, 1H, J = 8.5 Hz, Hyp-α-H), was added to a stirred solution of the crude amine and DIPEA
4.70 (m, 1H, Hyp-γ-H), 5.13 (d, 1H, J = 4.5 Hz, 1-H); 13C (215 μL, 1.21 mmol) in dry CH2CL2 (10 mL) at 0 °C. The reac-
NMR (75 MHz, D2O): δ 36.41, 51.63, 59.55, 63.36, 74.62, tion mixture was stirred at room temperature for 7 h, after
76.66, 76.70, 82.47, 100.48, 172.97; ESI-TOF MS: [M + Na − H]+ which it was diluted with CHCl3 and washed with brine. The
calc for C10H17NNaO7 286.09, found 286.14; ESI-TOF HRMS: organic phase was dried with MgSO4 and concentrated
[M + Na − H]+ calc for C10H17NNaO7 286.0903, found in vacuo. The residue was purified by silica gel chromatography
286.0908.
(MeOH–CHCl3 gradient elution) to afford the title compound
5902 | Org. Biomol. Chem., 2013, 11, 5892–5907
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Paper
as a colorless syrup (0.49 g, 74%, rotamer mixture). Analytical Celite, and then filter Celite was washed with MeOH
data for 23: Rf = 0.68 (MeOH–CHCl3, 1.0/9.0, v/v); [α]2D6 63.20° thoroughly. The combined filtrates were concentrated in vacuo
(c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ (separated chemi- to give the crude product. The crude product was subjected to
cal shifts for minor rotamer in parentheses) major 2.03 (s, 3H, the next step without further purification. The crude product
OAc), 2.06 (s, 9H, 3 × OAc), 2.08 (s, 3H, OAc), 2.29 (m, 1H, Hyp- was dissolved in pyridine (10 mL), cooled to 0 °C, and treated
β-Ha), 2.43–2.58 (m, 1H, Hyp-β-Hb), 3.64–3.71 (m, 2H, Hyp- with acetic anhydride (5 mL). The reaction mixture was
δ-H2), 4.01–4.07 (m, 1H, H-4), 4.08–4.43 (m, 10H, 2-H, 5-Ha, allowed to stir overnight at room temperature. Upon com-
5-Hb, 4′-H, 5′-Ha, 5′-Hb, Hyp-γ-H, CHFmoc, OCH2Fmoc), 4.55 pletion, the reaction was diluted with CHCl3 and washed
(t, 1H, J = 7.5 Hz, Hyp-α-H), 4.94 (dd, 1H, J = 5.5, 11.0 Hz, thoroughly with H2O, 1 N HCl, saturated NaHCO3 and brine.
2′-H), 5.08 (d, 1H, J = 4.5 Hz, 1-H), 5.12 (dd, 1H, J = 5.5, 6.0 Hz, The organic phase was dried with MgSO4 and concentrated
3-H), 5.24 (d, 1H, J = 4.5 Hz, 1′-H), 5.29 (dd, 1H, J = 6.5 Hz, in vacuo. The residue was purified by silica gel chromatography
H-3′), 7.14–7.75 (m, 8H, aromatic); 13C NMR (125 MHz, (EtOAc–CHCl3 gradient elution) to afford the title compound
CDCl3): δ 20.5, 20.66, 20.70, 20.74, 20.77, 36.0, 37.4, 47.10, as a colorless syrup (532 mg, 87%, rotamer mixture). Analytical
47.11, 51.1, 51.5, 57.4, 57.9, 65.6, 65.7, 65.8, 67.8, 68.0, 74.7, data for 24: Rf = 0.50 (EtOAc–CHCl3, 6.0/4.0, v/v); [α]2D7 95.79°
75.7, 75.9, 76.4, 76.5, 76.8, 77.4, 77.7, 79.2, 79.3, 79.4, 98.58, (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ major isomer
98.64, 99.1, 99.3, 119.9, 120.0, 125.1, 125.2, 125.3, 127.0, 127.1, 2.02–2.12 (s, 21H, 7 × OAc), 2.22 (m, 1H, Hyp-β-Ha), 2.55–2.70
127.6, 127.7, 128.1, 129.0, 141.2, 141.3, 143.88, 143.91, 144.0, (m, 1H, Hyp-β-Hb), 3.51–3.72 (m, 2H, Hyp-δ-H2), 3.95–4.13 (m,
154.7, 155.2, 170.0, 170.1, 170.2, 170.48, 170.53, 170.66, 5H, 2H, 5a-H, 5b-H, 5a′-H, 5b′-H), 4.20–4.45 (m, 7H, 2′-H, 4′-H,
170.70, 170.8, 175.3, 176.4; ESI-TOF HRMS: [M + Na]+ calcd for 4′′-H, 5a′′-H, 5b′′-H), 4.48 (m, 1H, Hyp-γ-H), 4.60 (t, 1H, J = 7.5
C40H45NNaO18 850.2534, found 850.2509.
Hz, Hyp-α-H), 4.97–5.30 (m, 12H, 1′-H, 1′′-H, 2′′-H, 3-H, 3′-H,
4R-[β-L-Arabinofuranosyl-(1→2)-β-L-arabinofuranosyl]oxy-L- 3′′-H, 2 × CH2Ph), 5.32 (d, 1H, J = 4.5 Hz, 1-H), 7.19–7.36 (m,
proline (deprotected compound of NAP-IAD product 16). To a 10H, aromatic); 13C NMR (125 MHz, CDCl3): δ (rotamer
solution of 22 (40.0 mg, 48.2 μmol) in MeOH (2.0 mL) at 0 °C mixture) 20.55, 20.57, 20.85, 20.89, 20.92, 20.95, 20.97, 21.01,
was added a 0.1 M NaOH solution (2.0 mL). The reaction 36.37, 37.40, 51.3, 51.6, 58.0, 58.2, 65.5, 65.6, 66.2, 66.3, 66.41,
mixture was stirred for 3 h at the same temperature and then 66.43, 67.0, 67.2, 67.36, 67.42, 74.3, 74.9, 75.8, 76.65, 76.68,
neutralized with Dowex (H+), filtered, and concentrated 76.8, 77.3, 77.4, 77.7, 77.8, 79.2, 79.3, 79.9, 80.55, 80.58, 97.5,
in vacuo. The crude residue was dissolved in MeOH–H2O– 97.6, 97.7, 97.8, 98.4, 98.5, 127.7, 128.0, 128.25, 128.27,
HOAc (3 mL/1 mL/0.1 mL) and 20% Pd(OH)2 (15 mg) was 128.30, 128.4, 128.5, 128.6, 128.70, 128.74, 135.6, 135.8, 136.6,
added. The reaction mixture was stirred under an atmosphere 136.7, 154.5, 154.8, 169.9, 170.4, 170.5, 170.6, 170.7, 170.76,
of H2 for 15 h. The catalyst was then filtered-off, washed with 170.82, 170.9, 172.7; ESI-TOF HRMS: [M + Na]+ calcd for
MeOH and H2O, and the filtrate was concentrated under C49H59NNaO24 1068.3324, found 1068.3352.
reduced pressure. The residue was purified by Sep-Pak C-18
N-9-Fluorenylmethyloxycarbonyl-4R-[2,3,5-tri-O-acetyl-β-L-
cartridge (H2O–MeOH gradient elution). Fractions containing arabinofuranosyl-(1→2)-2,3,5-tri-O-acetyl-β-L-arabinofuranosyl-
the product were collected and concentrated in vacuo to afford (1→2)-2,3,5-tri-O-acetyl-β-L-arabinofuranosyl]oxy-L-proline (25).
the fully deprotected disaccharide-linked Hyp (14.1 mg, 74% Compound 24 (575 mg, 0.55 mmol) was dissolved in a mixture
in two steps). Analytical data for the title compound: [α]2D4 of EtOAc–MeOH (1 : 1, 5 mL) and 20% Pd(OH)2 (350 mg) was
1
47.59° (c 1.0, H2O); H NMR (500 MHz, D2O): δ 1.95–2.01 (m, added. The reaction mixture was stirred under an atmosphere
1H, Hyp-β-Ha), 2.43–2.48 (m, 1H, Hyp-β-Hb), 3.21–3.31 (m, 2H, of H2 for 8 h. Upon completion, the catalyst was filtered
Hyp-δ-H2), 3.41–3.48 (m, 2H, 5-Ha, 5′-Ha), 3.56–3.58 (m, 2H, through a pad of Celite and the filtrate was concentrated
5-Hb, 5′-Hb), 3.65–3.70 (m, 2H, 4-H, 4′-H), 3.87–3.95 (m, 3H, under reduced pressure to give a crude amine product. The
3-H, 2-H, 3′-H), 4.05 (dd, 1H, J = 4.5, 8.5 Hz, 2′-H), 4.19 (t, 1H, crude product was then subjected to the next step without
J = 8.5 Hz, Hyp-α-H), 4.44 (m, 1H, Hyp-γ-H), 4.84 (d, 1H, J = further purification. FmocCl (259 mg, mmol) was added to a
4.5 Hz, 1-H), 5.05 (d, 1H, J = 4.5 Hz, 1′-H); 13C NMR (125 MHz, stirred solution of the crude amine and DIPEA (190 μL,
D2O): δ 36.7, 51.7, 60.2, 63.3 (×2), 73.0, 74.3, 76.9, 77.3, 80.9, 0.66 mmol) in dry CH2Cl2 (7 mL) at 0 °C. The reaction mixture
82.1, 82.8, 99.2, 100.8, 174.0; ESI-TOF HRMS: [M + Na]+ calcd was stirred at room temperature for 5 h, after which it was
for C15H25NNaO11 418.1325, found 418.1310.
diluted with CHCl3 and washed with brine. The organic phase
N-Benzyloxycarbonyl-trans-4-hydroxy-4-O-[2,3,5-tri-O-acteyl- was dried with MgSO4 and concentrated in vacuo. The residue
β-L-arabinofuranosyl-(1→2)-2,3,5-tri-O-acetyl-β-L-arabinofurano- was purified by silica gel chromatography (MeOH–CHCl3 gradi-
syl-(1→2)-2,3,5-tri-O-acetyl-β-L-arabinofuranosyl]-L-proline benzyl ent elution) to afford the title compound as a colorless syrup
ester (24). To a solution of 19 (865 mg, 0.58 mmol) in pyri- (465 mg, 81%, rotamer mixture). Analytical data for 25: Rf =
dine–THF (1 : 4, 20 mL) at 0 °C was added a solution of tetra- 0.75 (MeOH–CHCl3, 1.0/9.0, v/v); [α]2D6 75.20° (c 1.0, CHCl3); 1H
butylammonium fluoride (3.48 mL, 3.48 mmol, 1 M in THF). NMR (500 MHz, CDCl3): δ major isomer 1.78–2.11 (m, 21H,
After stirring for 3 h at 0 °C, CaCO3 (725 mg), DOWEX 50WX2 7 × OAc), 2.30 (m, 1H, Hyp-β-Ha), 2.61–2.85 (m, 1H, Hyp-β-Hb),
(2.17 g, used as supplied), and MeOH (8 mL) were added. The 3.67–3.72 (m, 2H, Hyp-δ-H2), 3.94–4.14 (m, 6H, 2H, 5a-H, 5b-
suspension was stirred at room temperature for 1 h. All insolu- H, 5a′-H, 5b′-H, OCHFmoc), 4.21–4.53 (m, 13H, 2′-H, 4-H, 4′-H,
ble materials were removed by filtration through a pad of 4′′-H, 5a′′-H, 5b′′-H, OCH2Ph, Hyp-γ-H), OCH2Fmoc, 4.67
This journal is © The Royal Society of Chemistry 2013
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(t, 1H, J = 7.5 Hz, Hyp-α-H), 4.93 (dd, 1H, J = 4.0, 7.0 Hz, 2′′-H), (Fmoc-Asp(tBu)-Pro-Val-His(Trt)-His(Trt)-His(Trt)-Novasyn-TGT
5.00–5.06 (m, 2H, 1′-H, 3-H), 5.12–5.20 (m, 3H, 1′′-H, 3′-H, 3′′- alcohol resin, 100.0 mg, 0.02 mmol) was placed in an SPPS
H), 5.34 (d, 1H, J = 4.5 Hz, 1-H), 7.13–7.76 (m, 7H, aromatic); reaction vessel. Fmoc removal was achieved with 20% piper-
13C NMR (125 MHz, CDCl3): δ (rotamer mixture) 20.45, 20.49, idine in DMF (0.5 mL) for 10 min. The resin was washed with
20.6, 20.71, 20.73, 20.9, 21.5, 36.3, 37.6, 47.18, 47.21, 51.4, DMF (2 × 3 mL), CH2Cl2 (2 × 1 mL) and then DMF (2 × 1 mL).
51.8, 57.5, 58.0, 65.4, 65.5, 66.2, 66.3, 67.9, 68.0, 74.4, 75.2, Fmoc-amino acid (5.0 equiv., 0.1 mmol) or Fmoc-glycoamino
75.3, 75.5, 76.4, 76.5, 76.7, 77.1, 77.4, 78.9, 79.2, 79.5, 79.7, acid (3.0 equiv., 0.06 mmol), 1-[(1-(cyano-2-ethoxy-2-oxo-ethyl-
80.4, 80.5, 97.4, 97.6, 97.7, 97.8, 98.6, 119.9, 120.0, 120.1, ideneaminooxy)dimethylaminomorpholino)]uronium
hexa-
125.07, 125.11, 125.2, 125.38, 127.12, 127.17, 127.21, 127.6, fluorophosphate (COMU) (1 equiv. to amino acid) and N,N-
127.75, 127.84, 128.3, 129.1, 137.9, 141.2, 141.26, 141.31, diisopropylethylamine (DIPEA) (2 equiv. to amino acid) was
141.4, 143.6, 143.9, 144.1, 144.4, 154.7, 155.2, 169.88, 169.92, mixed in DMF (0.5 mL) and activated for 1–2 min before being
170.3, 170.4, 170.6, 170.7, 170.78, 170.83, 170.9, 171.0, 175.9, added to the resin. The reaction mixture was then agitated
177.0; ESI-TOF HRMS: [M + Na]+ calcd for C49H57NNaO24 slowly for 1 min and allowed to couple for 2 h (4 h for second-
1066.3168, found 1066.3140.
ary amine and 16 h for glycoamino acids 1–3). The reaction
4R-[β-L-Arabinofuranosyl-(1→2)-β-L-arabinofuranosyl-(1→2)- was monitored by a microcleavage method and then analyzed
β-L-arabinofuranosyl]oxy-L-proline (deprotected compound of by MALDI-TOF MS. Upon completion, the resin was washed
NAP-IAD product 19). To a solution of 24 (30.0 mg, 28.7 μmol) with DMF and deprotected by 20% piperidine in DMF for
in MeOH (2.0 mL) at 0 °C was added 0.1 M NaOH solution 10 min. It was then washed again with DMF, CH2Cl2 and DMF,
(2.0 mL). The reaction mixture was stirred for 3 h at the same and then coupled with the next amino acid. Coupling
temperature, and then neutralized with Dowex (H+), filtered, and deprotection were repeated until the desired tridecapep-
and concentrated in vacuo. The crude residue was dissolved in tide (Fmoc-Arg(Pbf)-Thr(tBu)-Hyp(tBu)-Val-Ser(tBu)-Gly-Hyp-
MeOH–H2O–HOAc (3 mL/1 mL/0.1 mL) and 20% Pd(OH)2 (L-Araf0–3)-Asp(tBu)-Pro-Val-His(Trt)-His(Trt)-His(Trt)-Novasyn-
(15 mg) was added. The reaction mixture was stirred under an TGT alcohol resin) was obtained. Peptide was cleaved from the
atmosphere of H2 for 15 h. The catalyst was then filtered-off, resin by treatment with a cocktail of 95% TFA, 2.5% water and
washed with MeOH and H2O, and the filtrate was concentrated 2.5% TIPS (1 mL) for 1 h. Each cleaved peptide resin was then
under reduced pressure. The residue was purified by Sep-Pak washed extensively with a fresh cleavage cocktail (2 × 1 mL).
C-18 cartridge (H2O–MeOH gradient elution). Fractions con- The peptide was precipitated by the addition of cold diethyl
taining the product were collected and concentrated in vacuo ether at least 10 times the initial cocktail volume. The precipi-
to afford the fully deprotected trisaccharide-linked Hyp tate was filtered and washed with cold diethyl ether. Precipi-
(10.7 mg, 71% in two steps). Analytical data for deprotected tated peptide was dissolved in 0.1% TFA in water and then
1
compound of 24: [α]2D4 77.79° (c 1.0, H2O); H NMR (500 MHz, lyophilized. The lyophilized O-acetylated glycopeptides were
D2O): δ 2.17–2.33 (m, 1H, Hyp-β-Ha), 2.67–2.72 (m, 1H, Hyp- then dissolved in MeOH for removal of O-acetyl groups. The
β-Hb), 3.48–3.53 (m, 2H, Hyp-δ-H2), 3.68–3.75 (m, 3H, 5-Ha, pH of the MeOH solution was adjusted to ∼10 (as detected by
5′-Ha, 5′′-Ha), 3.81–3.84 (m, 3H, 5-Hb, 5′-Hb, 5′′-Hb), 3.91–3.99 wet litmus paper) by adding 0.1 M NaOMe in MeOH. The reac-
(m, 3H, 4-H, 4′-H, 4′′-H), 4.14–4.20 (m, 3H, 2′′-H, 3′-H, 3′′-H), tion was monitored by MALDI-TOF mass spectrometry and
4.26–4.33 (m, 4H, 2-H, 3-H, 2′-H, Hyp-α-H), 4.66 (s, 1H, Hyp- found to be complete after 2 h. Powder CO2 (dry ice) was then
γ-H), 5.13 (d, 1H, J = 4.0 Hz, 1′′-H), 5.27 (d, 1H, J = 4.0 Hz, 1-H), added carefully to reach a pH of 6. The crude product was puri-
5.29 (d, 1H, J = 4.0 Hz, 1′-H); 13C NMR (75 MHz, D2O): δ 36.2, fied by semi-preparative reverse phase HPLC followed by lyo-
51.1, 60.1, 62.3, 62.6, 63.0, 72.5, 72.6, 74.1, 76.7, 77.1, 80.6, philization of the appropriate fractions. The purity of
1
80.7, 81.7, 82.0, 82.3, 98.0, 99.0, 100.1, 174.2; ESI-TOF HRMS: Araf0–3CLV3 (1–4) was shown by H NMR spectra as well as a
[M + Na]+ calcd for C20H33NNaO15 550.1748, found 550.1762.
HPLC chart obtained by using a semi-micro reverse-phase
HPLC system equipped with a photo diode array detector
(Waters Alliance e2695 and 2475 at 220 nm) with a gradient of
General procedure for (glyco)peptide synthesis
Peptide chain assembly was starting with Fmoc-His(Trt) carry- 5–5–50–50% acetonitrile (containing 0.1% TFA) for 0–5–
ing as a trityl ester on NovaSyn®TGT alcohol resin 26 (loading 30–35 min respectively, at a flow rate of 500 μL min−1 through
capacity 0.2 mmol g−1). The first five amino acids were intro- InertSustain® C18 column [3 μm (3.0 × 150), GL Science
duced in an automatic peptide synthesizer (Life Technologies Inc.].10
ABI433A Peptide Synthesizer) employing the Nα-Fmoc-based
H-Arg-Thr-Val-Hyp-Ser-Gly-Hyp-Asp-Pro-Leu-His-His-His-OH
approach by the research resources center in RIKEN Brain (4). Compound 4 was obtained by the general procedure for
Science Institute. 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyl- peptide synthesis in 30% yield (8.9 mg) as an amorphous
uronium hexafluorophosphate (HBTU) and 1-hydroxybenzo- white solid. Analytical data for peptide 4 was in good agree-
triazole (HOBt) were employed as coupling reagents in an ment with those reported previously.23 Analytical data for 4: 1H
automatic peptide synthesizer. To the hexapeptide Fmoc-Asp- NMR (500 MHz, D2O): δ 0.83 (d, 3H, J = 6.0 Hz, Leu-δ-H3), 0.90
(tBu)-Pro-Leu-His(Trt)-His(Trt)-His(Trt) carrying as
a trityl (d, 3H, J = 6.5 Hz, Leu-δ-H3), 0.93 (d, 3H, J = 6.0 Hz, Val-γ-H3),
ester on NovaSyn®TGT alcohol resin 27, the rest of the amino 0.97 (d, 3H, J = 6.0 Hz, Val-γ-H3), 1.21 (d, 3H, J = 6.0 Hz, Thr-
acids were added manually. The hexapeptide-bounded resin γ-H3), 1.39–1.42 (m, 1H, Leu-γ-H), 1.52–1.58 (m, 2H, Leu-β-H2),
5904 | Org. Biomol. Chem., 2013, 11, 5892–5907
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Table 2 Selected NMR chemical shifts and 3JH1–H2 spin-couplings of saccharide
signals of synthetic [Arafn]CLV3 (n = 1–3) (1–3)
1.63–1.68 (m, 2H, Arg-γ-H2), 1.88–2.13 (m, 8H Arg-β-H2, Val-
β-H, 2 × Hyp-β-Ha, Pro-β-Ha, Pro-γ-H2), 2.24–2.33 (m, 2H, Hyp-
β-Ha, Pro-β-Hb), 2.36–2.41 (m, 1H, Hyp-β-Hb), 2.77–2.81 (m,
1H, Asp-β-Ha), 2.91–2.96 (dd, 1H, J = 8.0, 17.0 Hz, Asp-β-Hb),
3.13–3.34 (m, 8H, Arg-δ-H2, 3 × His-β-H2), 3.63–3.95 (m, 8H,
2 × Hyp-δ-H2, Pro-δ-H2, Ser-β-H2), 4.04–4.24 (m, 3H, Arg-α-H,
Leu-α-H, Gly-α-Ha), 4.21–4.24 (m, 1H, Gly-α-Hb), 4.36–4.39 (m,
1H, Pro-α-H), 4.42–4.53 (m, 4H, Hyp-α-H, Ser-α-H, Thr-α-H,
Hyp-α-H), 4.58–4.73 (m, 4H, 3 × His-α-H, Hyp-α-H), 4.95 (t, 1H,
H, C/ppm (3J Hz)
[Araf1]CLV3 (3)
[Araf2]CLV3 (2)
[Araf3]CLV3 (1)
1-H (3JH1–H2
C1
2-H
)
5.14 (4.5)
100.4
4.16
5.31 (4.5)
98.5
4.29
5.33 (4.5)
98.4
4.34
3-H
4-H
4.05
3.90
4.15
3.92
4.15
3.92
5-Ha
5-Hb
3.66
3.81
3.63
3.79
3.72
3.84
J
= 6.5 Hz, Asp-α–H), 7.27–7.31 (m, 4H, His-aromatic),
8.62–8.65 (m, 6H, His-aromatic); MALDI-TOF MS: [M + H]+
calcd for C63H97N22O20 1481.7249, found 1481.8541. See also
Fig. 2 and ESI.†
1′-H (3JH1′–H2′
C1′
2′-H
)
5.01 (4.0)
100.1
4.13
5.13 (4.0)
98.0
4.29
3′-H
4.11
4.23
4′-H
5′-Ha
5′-Hb
3.90
3.69
3.80
4.01
3.74
3.85
H-Arg-Thr-Val-Hyp-Ser-Gly-Hyp[Araf]-Asp-Pro-Leu-His-His-His-
OH (3). Compound 3 was obtained by the general procedure
for the peptide synthesis in 37% yield (12.0 mg) as an amor-
phous white solid. Analytical data for 3: 1H NMR (500 MHz,
D2O): δ 0.83 (d, 3H, J = 6.0 Hz, Leu-δ-H3), 0.90 (d, 3H, J =
6.5 Hz, Leu-δ-H3), 0.93 (d, 3H, J = 6.0 Hz, Val-γ-H3), 0.98 (d, 3H,
J = 6.0 Hz, Val-γ-H3), 1.21 (d, 3H, J = 6.0 Hz, Thr-γ-H3),
1.38–1.42 (m, 1H, Leu-γ-H), 1.50–1.58 (m, 2H, Leu-β-H2),
1.60–1.68 (m, 2H, Arg-γ-H2), 1.88–2.12 (m, 8H, Arg-β-H2,
Val-β-H, 2 × Hyp-β-Ha, Pro-β-Ha, Pro-γ-H2), 2.25–2.28 (m, 1H,
Pro-β-Hb), 2.37–2.41 (m, 1H, Hyp-β-Ha), 2.36–2.41 (m, 1H,
Hyp-β-Hb), 2.76–2.79 (m, 1H, Asp-β-Ha), 2.91–2.96 (m, 1H,
Asp-β-Hb), 3.12–3.34 (m, 8H, Arg-δ-H2, 3 × His-β-H2), 3.62 (dd,
1H, J = 7.5, 12.0 Hz, 5a-H), 3.75–3.79 (m, 3H, 5b-H, Hyp-δ-H2),
3.86–3.91 (m, 7H, 4-H, Hyp-δ-H2, Pro-δ-H2, Ser-β-H2), 4.05 (dd,
1H, J = 6.5, 8.0 Hz, 3-H), 4.06–4.16 (m, 3H, 2-H, Arg-α-H,
Gly-α-Ha), 4.19–4.20 (m, 1H, Leu-α-H, Gly-α-Hb), 4.36–4.39 (m,
1H, Pro-α-H), 4.43–4.53 (m, 4H, Hyp-α-H, Ser-α-H, Thr-α-H,
Hyp-α-H), 4.59–4.71 (m, 4H, 3 × His-α-H, Hyp-α-H), 4.96 (t, 1H,
J = 6.5 Hz, Asp-α–H), 5.14 (d, 1H, J = 4.5 Hz, 1-H), 7.27–7.31 (m,
4H, His-aromatic), 8.62–8.65 (m, 6H, His-aromatic); MALDI-
TOF MS calcd for C68H105N22O24 [M + H]+: 1613.7672, found
1613.7910. See also Tables 2, Fig. 2 and ESI.†
H-Arg-Thr-Val-Hyp-Ser-Gly-Hyp[Araf2]-Asp-Pro-Leu-His-His-His-
OH (2). Compound 2 was obtained by the general procedure
for the peptide synthesis in 31% yield (10.8 mg) as an amor-
phous white solid. Analytical data for 2: 1H NMR (500 MHz,
D2O): δ 0.84 (d, 3H, J = 6.0 Hz, Leu-δ-H3), 0.90 (d, 3H, J =
6.5 Hz, Leu-δ-H3), 0.93 (d, 3H, J = 6.0 Hz, Val-γ-H3), 0.98 (d, 3H,
J = 6.0 Hz, Val-γ-H3), 1.21 (d, 3H, J = 6.0 Hz, Thr-γ-H3),
1.36–1.41 (m, 1H, Leu-γ-H), 1.50–1.58 (m, 2H, Leu-β-H2),
1.60–1.68 (m, 2H, Arg-γ-H2), 1.88–2.12 (m, 8H Arg-β-H2, Val-
β-H, 2 × Hyp-β-Ha, Pro-β-Ha, Pro-γ-H2), 2.24–2.30 (m, 1H, Pro-
β-Hb), 2.37–2.42 (m, 1H, Hyp-β-Ha), 2.48–2.49 (m, 1H, Hyp-
β-Hb), 2.73–2.77 (m, 1H, Asp-β-Ha), 2.88–2.94 (m, 1H, Asp-
β-Hb), 3.11–3.31 (m, 8H, Arg-δ-H2, 3 × His-β-H2), 3.63 (dd, 1H,
J = 7.0, 12.0 Hz, 5a-H), 3.69 (dd, 1H, J = 7.0, 12.0 Hz, 5a′-H),
3.75–3.86 (m, 4H, 5b-H, 5b′-H, Hyp-δ-H2), 3.86–3.93 (m, 8H,
4-H, 4′-H, Hyp-δ-H2, Pro-δ-H2, Ser-β-H2), 4.01–4.16 (m, 6H, 2′-
H, 3-H, 3′-H Arg-α-H, Gly-α-Ha), 4.20–4.23 (m, 2H, Leu-α-H,
Gly-α-Hb), 4.29 (dd, 1H, J = 4.5, 8.0 Hz, 2-H), 4.37–4.40 (m, 1H,
Pro-α-H), 4.42–4.53 (m, 4H, Hyp-α-H, Ser-α-H, Thr-α-H, Hyp-
α-H), 4.59–4.69 (m, 4H, 3 × His-α-H, Hyp-α-H), 4.94 (t, 1H, J =
1′′-H (3JH1′′–H2′′
C1′′
2′′-H
3′′-H
4′′-H
)
4.95 (4.5)
100.2
4.11
4.13
3.91
3.69
3.85
5′′-Ha
5′′-Hb
6.5 Hz, Asp-α–H), 5.01 (d, 1H, J = 4.5 Hz, 1′-H), 5.31 (d, 1H, J =
4.5 Hz, 1-H), 7.27–7.31 (m, 4H, His-aromatic), 8.62–8.65
(m, 6H, His-aromatic); MALDI-TOF MS: [M + H]+ calcd for
C73H113N22O28 1745.8095, found 1745.6676. See also Table 2,
Fig. 2 and ESI.†
H-Arg-Thr-Val-Hyp-Ser-Gly-Hyp[Araf3]-Asp-Pro-Leu-His-His-
His-OH (1). Compound 1 was obtained by the general pro-
cedure for the peptide synthesis in 34% yield (12.8 mg) as an
amorphous white solid. Analytical data for peptide 1 was in
good agreement with those reported previously.23 Analytical
1
data for 1: H NMR (500 MHz, D2O): δ 0.84 (d, 3H, J = 6.0 Hz,
Leu-δ-H3), 0.90 (d, 3H, J = 6.5 Hz, Leu-δ-H3), 0.93 (d, 3H, J =
6.0 Hz, Val-γ-H3), 0.98 (d, 3H, J = 6.0 Hz, Val-γ-H3), 1.21 (d, 3H,
J = 6.0 Hz, Thr-γ-H3), 1.35–1.41 (m, 1H, Leu-γ-H), 1.48–1.57 (m,
2H, Leu-β-H2), 1.60–1.67 (m, 2H, Arg-γ-H2), 1.88–2.13 (m, 8H,
Arg-β-H2, Val-β-H, 2 × Hyp-β-Ha, Pro-β-Ha, Pro-γ-H2), 2.23–2.29
(m, 1H, Pro-β-Hb), 2.38–2.41(m, 1H, Hyp-β-Ha), 2.45–2.49 (m,
1H, Hyp-β-Hb), 2.74–2.79 (m, 1H, Asp-β-Ha), 2.89–2.94 (m, 1H,
Asp-β-Hb), 3.11–3.31 (m, 8H, Arg-δ-H2, 3 × His-β-H2), 3.62 (dd,
1H, J = 7.0, 12.0 Hz, 5a-H), 3.69–3.74 (m, 2H, 5a′-H, 5a′′-H),
3.77–3.86 (m, 5H, 5b-H, 5b′-H, 5′′b-H, Hyp-δ-H2), 3.88–4.03 (m,
9H, 4-H, 4′-H, 4′′-H, Hyp-δ-H2, Pro-δ-H2, Ser-β-H2), 4.09–4.16
(m, 6H, 2′′-H, 3-H, 3′′-H, Arg-α-H, Gly-α-H2), 4.21–4.25 (m, 3H,
3′-H, Leu-α-H, Gly-α-Hb), 4.30 (dd, 1H, J = 4.5, 8.0 Hz, 2′-H),
4.35 (dd, 1H, J = 4.5, 8.0 Hz, 2-H), 4.36–4.39 (m, 1H, Pro-α-H),
4.42–4.53 (m, 5H, 2′′-H, Hyp-α-H, Ser-α-H, Thr-α-H, Hyp-α-H),
4.59–4.70 (m, 4H, 3 × His-α-H, Hyp-α-H), 4.94 (t, 1H, J = 6.5 Hz,
Asp-α–H), 4.98 (d, 1H, J = 4.5 Hz, 1′′-H), 5.14 (d, 1H, J = 4.5 Hz,
1′-H), 5.34 (d, 1H, J = 4.5 Hz, 1-H), 7.27–7.31 (m, 4H, His-
aromatic), 8.62–8.65 (m, 6H, His-aromatic); MALDI-TOF MS:
[M
1877.7329. See also Table 2, Fig. 2 and ESI.†
+
H]+ calcd for C78H121N22NaO32 1877.8517, found
This journal is © The Royal Society of Chemistry 2013
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10 K. Ohyama, H. Shinohara, M. Ogawa-Ohnishi and
Acknowledgements
Y. Matsubayashi, Nat. Chem. Biol., 2009, 5, 578–580.
We thank Dr Yoichi Takeda and Dr Masakazu Hachisu (JST, 11 C. S. Callam, R. R. Gadikota, D. M. Krein and T. L. Lowary,
ERATO Ito Glycotrilogy Project) for their valuable advice and J. Am. Chem. Soc., 2003, 125, 13112–13119.
help. We thank Dr Hiroyuki Koshino (Collaboration Promotion 12 Y. J. Lee, K. Lee, E. H. Jung, H. B. Jeon and K. S. Kim, Org.
Unit, RIKEN) and his staff for ESI HRMS measurements, and Lett., 2005, 7, 3263–3266.
are grateful to the support unit for Bio-material Analysis of the 13 X. Zhu, S. Kawatkar, Y. Rao and G.-J. Boons, J. Am. Chem.
research resources center in RIKEN Brain Science Institute for Soc., 2006, 128, 11948–11957.
technical help with hexapeptide synthesis and tandem MS/MS 14 A. Ishiwata, H. Akao and Y. Ito, Org. Lett., 2006, 8, 5525–
analysis. We also thank Ms Akemi Takahashi and Ms Satoko 5528.
Shirahata for their kind technical assistance. This work was 15 D. Crich, C. M. Pedersen, A. A. Bowers and D. J. Wink,
partly supported by a Grant-in-Aid for Scientific Research from J. Org. Chem., 2007, 72, 1553–1565.
the Ministry of Education, Culture, Sports, Science, and Tech- 16 Y. Wang, S. Maguire-Boyle, R. T. Dere and X. Zhu, Carbo-
nology (no. 22510243).
hydr. Res., 2008, 343, 3100–3106.
17 A. Imamura and T. L. Lowary, Org. Lett., 2010, 12, 3686–
3689.
18 K. G. Fedina, P. I. Abronina, N. M. Podvalnyy,
N. N. Kondakov, A. O. Chizhov, V. I. Torgov and
L. O. Kononov, Carbohydr. Res., 2012, 357, 62–67.
Notes and references
1 (a) R. A. Dwek, Chem. Rev., 1996, 96, 683–720; (b) Essentials
in Glyocbiology, ed. A. Varki, R. Cummings, J. Esko, 19 (a) M. Himly, B. Jahn Schmid, A. Dedic, P. Kelemen,
H. Freeze, G. Hart and J. Marth, Cold Spring Harbor Labo-
ratory Press, New York, 1999.
2 (a) D. T. A. Lamport, Nature, 1967, 216, 1322–1324;
(b) D. T. A. Lamport and L. Clark, Biochemistry, 1969, 8,
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