Synthesis of oligomers of β- L-arabinofuranosides of (4 R)-4-hydroxy- l -proline relevant to the mugwort pollen allergen, art v 1

Article abstract of DOI:10.1021/jo501191b

An efficient, convergent solution phase synthesis of monomer, dimer, trimer and tetramer of the β-l-arabinofuranosylated hydroxyproline (β-l-Araf-Hyp) glycocluster is described. This motif constitutes the carbohydrate-specific epitope of Art v 1, the major allergen of mugwort pollen. While a single monomeric unit was proposed at the outset, poor yields for the seemingly trivial steps of end-capping to replace protecting groups with N-terminal acetamides and C-terminal methyl amides led to the introduction of N-terminal, central and C-terminal β-l-Araf-Hyp building blocks. Dimer 2 was obtained in 60% yield by coupling of two monomers, followed by hydrogenolysis of benzyl ether protecting groups. Trimer 3 was obtained in 35% yield via a [2 + 1] coupling and tetramer 4 in 15% yield via a [2 + 2] fragment condensation. Circular dichroism spectra show that monomer 1 displays no organized structure, whereas compounds 2-4 show a strong negative band at 200 nm and a weak positive band at ～220 mn, as is characteristic of the polyproline II helix.

Full text of DOI:10.1021/jo501191b

Article
pubs.acs.org/joc
Synthesis of Oligomers of β‑L‑Arabinofuranosides of (4R)‑4-
Hydroxy‑L‑proline Relevant to the Mugwort Pollen Allergen, Art v 1
Ning Xie† and Carol M. Taylor*
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States
S
* Supporting Information
ABSTRACT: An efficient, convergent solution phase syn-
thesis of monomer, dimer, trimer and tetramer of the β-L-
arabinofuranosylated hydroxyproline (β-L-Araf-Hyp) glyco-
cluster is described. This motif constitutes the carbohydrate-
specific epitope of Art v 1, the major allergen of mugwort
pollen. While a single monomeric unit was proposed at the
outset, poor yields for the seemingly trivial steps of end-
capping to replace protecting groups with N-terminal
acetamides and C-terminal methyl amides led to the
introduction of N-terminal, central and C-terminal β-L-Araf-
Hyp building blocks. Dimer 2 was obtained in 60% yield by
coupling of two monomers, followed by hydrogenolysis of benzyl ether protecting groups. Trimer 3 was obtained in 35% yield
via a [2 + 1] coupling and tetramer 4 in 15% yield via a [2 + 2] fragment condensation. Circular dichroism spectra show that
monomer 1 displays no organized structure, whereas compounds 2−4 show a strong negative band at 200 nm and a weak
positive band at ∼220 mn, as is characteristic of the polyproline II helix.
INTRODUCTION
■
Pollen from Artemisia vulgaris, mugwort, is a major contributor
to hay fever in Europe and North America. The major allergen
is a heterogeneous glycoprotein known as Art v 1.1,2 In a study
involving 100 pediatric mugwort-allergic patients, 79% of the
patients reacted with natural Art v 1, but only 39% showed
reactivity with a recombinant allergen, signifying the role of
post-translational modifications in allergenicity.3 Leonard et al.
identified a novel motif containing up to four contiguous, β-
linked arabinofuranosides of hydroxyproline (β-L-Araf-Hyp)
that showed significant binding to IgE from the serum of
allergic patients. Moreover, this prolyl domain facilitates protein
folding4 and influences the conformation of the globular
domain bearing other epitopes.5,6
There are 21 proline residues in the 53-residue C-terminal
domain of Art v 1. Over 75% of the proline residues are
hydroxylated, and 16−17 of these are adorned with β-L-
arabinofuranosides. The glycoprotein was isolated in limited
quantities. Alkaline hydrolysis of the protein leads to complex
mixtures of amino acids, including the β-L-Araf-Hyp residue.
The heterogeneous nature of this digest means it is unrealistic
to isolate even miniscule amounts of pure amino acids and
oligopeptides, thus providing an opportunity for chemical
synthesis. In order to determine the minimal allergenic binding
motif, and for potential downstream development of diagnostic
tools, we set ourselves the target molecules 1−4 (Figure 1),
representing monomer, dimer, trimer and tetramer of the β-L-
Figure 1. Relevant glycopeptide target molecules.
sequence or backbone conformation. The Art v 1 protein
presents an example of the former, viz. a contiguous
Araf-Hyp moiety.
We define a glycocluster as an array of carbohydrate groups
that are present in close proximity as a result of primary
Received: May 28, 2014
Published: July 22, 2014
© 2014 American Chemical Society
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glycocluster where two, three or four sequential β-L-
arabinosylated hydroxyproline residues occur. The chemical
synthesis of contiguous glycoclusters has been largely
concerned with mucin-type motifs,7 dystroglycan8 and tumor
antigens (e.g., 5 and 6).9,10 Most precedents for glycocluster
assembly therefore relate to glycosylated serine and threonine
residues, not hydroxyproline. Preformed glycosylated residues
are linked via peptide bond formation. For example, Kunz and
co-workers had used ethyldimethylaminopropyl carbodiimide
hydrochloride (EDC) with hydroxybenzotriazole (HOBt) in
their synthesis of MUC1 core glycopeptides.11 During the
assembly of the TN and TF tumor-associated antigens,
Danishefsky and co-workers employed 2-isobutoxy-1-isobutox-
ycarbonyl-1,2- dihydroquinoline (IIDQ) which worked well in
the case of the TN antigen, viz. compound 5.9
co-workers prepared dodecapeptide 11 among a series of
potential antifreeze peptides.21 Their Fmoc-[β-(1,4)-D-GalNAc-
(OAc)3]Hyp-OH building block was prepared in seven steps
and 25% overall yield from N-acetylgalactosamine. They used
only 1.2 equiv of this building block in on-resin couplings
mediated by HATU and diisopropylethylamine with extended
reaction times (20 h vs 1 h for regular residues). To their credit,
a 15% yield of glycopeptide 11 was isolated. On this
background we embarked on the synthesis of the Art v 1 β-L-
Araf-Hyp oligomers.
RESULTS AND DISCUSSION
■
In 2010 we reported the construction of the β-glycosidic
linkage in monomer 14 and the formation of diglycodipeptide22
17 (Scheme 1).23 The synthesis of the cis-1,2-glycosidic linkage
The TF antigen presented more sterically demanding
couplings, requiring 1-[bis(dimethylamino)-methylene]-1H-
1,2,3-triazolo-[4,5b]pyridinium hexafluorophosphate 3-oxide
(HATU)12,13 and 1-hydroxy-7-azabenzotriazole (HOAt) to
produce 6.14 Live, Barany and co-workers used glycosylated
threonine building blocks in solid phase peptide synthesis
(SPPS), either activated as pentafluorophenyl esters (in
combination with HOBt and diisopropylethylamine in DMF)
or acids activated by 2-(6-chloro-1H-benzotriazole-1-yl)-
1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU),15
HOBt and diisopropylethylamine in N-methylpyrrolidone
(NMP).7,8
Scheme 1. Synthesis of the (β-Araf)Hyp Monomer and
Dimer23
Peptide coupling16−18 to proline residues is naturally slower
than to primary amino acids due to steric hindrance. The
pyrrolidine nitrogen of (4R)-hydroxyproline (Hyp) is less
nucleophilic than that of unsubstituted proline (Pro) due to the
electronegative oxygen. Because electron withdrawal and steric
hindrance are exacerbated upon glycosylation, forging the
prolyl peptide bonds in the Art v 1 oligomers is a considerable
challenge for synthesis. On the upside, proline carboxyl
components are not susceptible to Cα-epimerization during
peptide bond formation, widening the scope for selecting
coupling reagents.
The CLAVATA3 peptide 9 (Figure 1), regulator of stem cell
signaling in Arabidopsis, contains a single glycosylated Hyp
residue at the center of a 13-residue peptide. In their recent
SPPS of compounds 7−9, Kaeothip et al. performed all
couplings including, and following, the glycosylated Hyp
residue manually.19 While 1-[bis(dimethylamino)methylene]-
1H-benzotriazolium hexafluorophosphate 3-oxide (HBTU) was
used for earlier couplings, (1-cyano-2-ethoxy-2-oxo-ethylide-
neaminooxy)-dimethylaminomorpholinouronium hexafluoro-
phospate (COMU) was used for the glycosylated residue and
beyond. Coupling times varied depending on the steric
demands of the nucleophile: 2 h for regular amino acids, 4 h
for secondary amines and 16 h in the case of glycosylated
building blocks. They employed 3 equiv of their Fmoc-
protected glycosylated building block and isolated compounds
7−9 in 31−37% yield based on initial loading of the resin.
Two solid phase syntheses of oligomeric hydroxyproline
glycosides have been reported. Schweizer and co-workers
employed tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluor-
oborate (TBTU) for the assembly of a nonamer of (β-D-Gal)-
Hyp, compound 10.20 The Fmoc-[β-(1,4)-D-Gal(OAc)4]Hyp-
OH building block was prepared in one step from commercially
available D-galactose pentaacetate and Fmoc-Hyp-OH. The
synthesis was conducted on 30 μmol scale, using 3 equiv of
building block in each cycle; no yield was reported. Payne and
presented a considerable challenge, and ultimately conditions
were identified that yielded 14 in 60% yield with a 4:1 ratio of
anomers that could be separated by flash chromatography. In
the 2010 paper, other strategies were discussed to direct the
formation of the β-glycoside including conformationally
restricted glycosyl donors24,25 and intramolecular aglycone
delivery.26−29 An updated review and references to these
approaches was provided by Kaetothip et al.19
The longest linear sequence, from L-arabinose, to produce
monomer 14 is 7 steps and was achieved 28% overall yield. In
order to optimize peptide coupling conditions in solution it was
desirable to carefully monitor reactions and quantify and
characterize compounds at each step. Moreover, given the
number of residues relative to targets 10 and 11, solid phase
synthesis did not seem advantageous. Deprotection of the
amine and carboxyl functionalities of 14 in parallel and coupling
of the two resulting monomers afforded the dimer 17. In our
original communication the allyl ester was removed from 14 via
Pd0-mediated transfer to morpholine. In ongoing studies,
impurities from this reaction were found difficult to eliminate.
Alkaline hydrolysis of the allyl ester was more satisfactory
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(Scheme 1) and an updated procedure is provided for the
production of 17.
principle, three approaches were possible to the tetraglycote-
trapeptide: [3 + 1], [2 + 2] and [1 + 3]. However, the increased
value of the trimer relative to dimer made the [2 + 2] strategy
the obvious choice.
At the dimerization level, coupling conditions were
investigated thoroughly. Bromo-tris-pyrrolidino-phosphonium-
hexafluorphosphate (PyBrOP),30 tetramethylfluoroformamidi-
nium hexafluorophosphate (TFFH)31 and HATU were studied,
on the basis of the track record of these reagents in challenging
couplings.32 The first two of those reagents activate the
carboxylic acid as a putative acyl halide, a bromide in the case of
PyBrOP and a fluoride in the case of TFFH. Reaction mixtures
were complex and there was considerable unreacted starting
material, affording an 11% yield of 17 in the case of PyBrOP
and 18−26% yields in the case of TFFH. HATU was the clear
leader. Gentle heating of the reaction mixture was also
advantageous.
Glycopeptide assembly is generally conducted with protec-
tion of the carbohydrate hydroxyl groups as acetate esters.33−37
This is largely due to the minimal number of steps required to
produce these building blocks from peracetylated mono-
saccarides. Following glycopeptide assembly, the peracetylated
compound is purified, treated with NaOMe/MeOH to effect
cleavage of the acetate esters, and purified again. In the current
context, the benzyl ethers were required to afford good
stereoselectivity during glycosylation. The hydrolysis and
acetylation of compound 14 was considered, in order to follow
the general protocol. However, it turned out that the benzyl
ethers could be cleaved at an advanced stage (vide infra) and
that minimal purification was required at this final step. We
therefore decided not to invoke additional protecting group
manipulations in order to follow the norm.
The downstream utility of the oligoglycopeptides produced
in this study depend on the feasibility of ligating them to other
species, carrier proteins, fluorophores, etc. In the first instance,
we sought simply to replace the N- and C-terminal protecting
groups with amides to mimic the extended peptide backbone.
These end-capped oligomers will be used for initial structural
and biological studies. The seemingly trivial manipulations for
end-capping the monomer to produce 22 are shown in Scheme
3. The benzyl ethers were cleaved by standard hydrogenolysis
to give the monomer 1.
To prepare a triglycotripeptide, two convergent approaches
were possible: a [2 + 1] coupling or a [1 + 2] coupling, the
former placing the greater steric burden in the carboxyl
component and the latter in the amino component (Scheme 2).
In practice, the two strategies both gave yields of ∼35%. In
Scheme 2. Trimer and Tetramer Assembly
Scheme 3. End-Capping of Monomer
The 1H and 13C NMR spectra of compound 1 (CD3OD, 400
MHz) were fully assigned on the basis of 2D experiments. On
the time scale of the 1H NMR acquisition, a 4:1 ratio of species
was observed, reflecting cis−trans isomerization about the prolyl
amide bond (Scheme 4). The NOESY spectrum showed a
correlation between the acetamide CH3 signal (δ 2.08 ppm)
Scheme 4. cis−trans Isomerism about the Prolyl Amide Bond
of 1 with NOE Correlations Illustrated by Double-Headed
Arrows
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and the Hδ signal (δ 3.73 ppm) of the major species. Thus, the
major species in solution adopts the trans conformation about
the central amide bond (Scheme 4). A correlation is observed
between signals corresponding to the acetamide CH3 (δ 1.93
ppm) and Hα (δ 4.52 ppm) of the minor cis conformation An
NOE was also observed between the signals corresponding to
H1 of the arabinose (δ 4.99 ppm) and Hγ of the Hyp (δ 4.46
ppm).
by four, in the assembly of each oligomer, as illustrated in
Scheme 5 for the dimer 23.
Assembly of the trimer and tetramer are illustrated in
Schemes 7 and 8 respectively. Since the functionality in each
Scheme 7. Convergent Trimer Assembly
In the case of end-capping the dimer, the formation of the
methyl amide proceeded in much lower yield using EDC/
HOBt; better results were obtained with HATU, again with an
overall yield of 42% for the end-capping manipulations to give
23 (Scheme 5). The end-capped trimer was obtained in only
Scheme 5. Linear and Convergent Approaches to End-
Capped Dimer
Scheme 8. Convergent Tetramer Assembly
18% yield and no end-capped tetramer was isolated. At this
juncture, our strategy was revised to include end-caps prior to
peptide coupling. Thus, two new building blocks were prepared
(Scheme 6). Methyl ester 26 and N-Boc-protected 27 were
fully characterized; each was converted to the requisite free acid
or amine respectively prior to peptide coupling. This approach
was more convergent and decreased the number of linear steps
Scheme 6. Three Building Blocks for Oligomer Assembly
1
building block is the same, no new signals appeared in the H
NMR spectrum to provide evidence for the elongation of the
peptide. Integration of the signals due to the C-terminal
NHCH3 (∼δ 2.75 ppm) group and H1, the anomeric protons
of the arabinose moieties (∼δ 5.00 ppm) gave a ratio that
showed congruence with the number of residues incorporated
(see Supporting Information). With the addition of each
residue, the number of conformational isomers possible (4 for
dimer 2, 8 for trimer 3 and 16 for tetramer 4) and the
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molecular mass of the peptides made it increasingly difficult to
acquire meaningful 13C NMR spectra.
band (λmin = 199 nm, [θ] = −10423 deg cm2 dmol−1) that is
characteristic of the PPII conformation.41 This is significant as
previous studies have shown that at least three Pro residues are
required for formation of the PPII helix.42 It is tempting to
suggest that the added bulk of the sugars might be contributing
to their heightened structure. The CD spectra of both trimer
and tetramer showed typical PPII-type helical structure. The
relative band strength (ρ) is the ratio of the maximum positive
ellipticity to the maximum negative ellipticity.43 Pysh attributed
the increase or decrease in ρ to conformational differences or
Global debenzylation of all end-capped compounds was
carried out under 1 atm of hydrogen in the presence of
palladium on carbon. For the larger oligomers, high catalyst
loadings were necessary. Trace impurities were removed by
reverse extraction: the highly hydrophilic peptides were
dissolved in water and washed with dichloromethane. The
aqueous layer was then lyophilized to afford glycopeptides 1−4.
1
Both H and 13C NMR spectra were simplified dramatically
changes to solvent and carbonyl backbone interactions.44
A
following debenzylation and so we elected to characterize the
final oligomers but not the perbenzylated intermediates that
adopted a multitude of conformations. Chemical shifts were in
agreement with those reported by Leonard et al. for the Art v 1
protein.2 For the full length heterogeneous protein, they
described an “average” structure that distinguished three sets of
decreasing ρ value corresponds to an increasing solvent-
carbonyl interaction. The relative band strength of our dimer,
trimer, and tetramer are 0.28, 0.30, and 0.27 respectively. The
variation is probably attributable to experimental error. These
ρ-values are also in concordance with the ρ-value of the
galactosylated hydroxyproline nonamer 5 reported by Owens
(ρ = 0.29).20
1
signals for β-L-Araf-Hyp in the H NMR spectrum but a single
1
13C NMR signal at δ 100.9 ppm for C1. Likewise, the H−13C
HSQC experiment for monomer 1 showed a correlation
between the H1 signal and a 13C signal at 101.2 ppm. The
anomeric carbon of β-arabinosides falls in the 100−105 ppm
range, whereas for α-arabinosides the signal is further
downfield.36
CONCLUSIONS
■
In summary, we have synthesized oligomers of the β-L-Araf-
Hyp motif that occurs in the mugwort pollen allergen, Art v 1.
This was extremely challenging and demonstrates that there are
still problems to be addressed in the synthesis of complex
peptides. NMR was used to characterize the monomer and
dimer, but has limitations in revealing secondary structure due
to the lack of amide protons along the backbone. Circular
dichroism revealed distinct PPII character, even at the dimer
level, indicating that glycosylation promotes and stabilizes helix
formation. Ongoing studies will investigate the interaction of
compounds 1−4 and derived compounds with biological
systems to probe the molecular basis for the interaction of
this glycocluster with relevant antibodies.
Natural hydroxyproline-rich glycoproteins have been shown
to adopt a polyproline II (PPII) conformation, e.g., the GP1
protein of Chlamydomonas reinhardtii38 and a cell wall protein
from carrot root.39 It was therefore not surprising that
compounds 10 and 11 gave rise to stable PPII structures.
Schweizer reported an increase in stability of 10 relative to its
nonglycosylated counterpart,20 and subsequent studies have
attributed this increase in stability to stabilizing interactions
between water and the carbohydrate backbone.40 On the other
hand, compound 11 did not show enhanced thermal stability
compared to the nonglycosylated oligomer.21 Interestingly, the
peptides containing contiguous GalNAc residues (e.g., 11)
demonstrated an additional positive signal in their CD spectra
below 200 nm.21
EXPERIMENTAL SECTION
■
General Note. The NMR spectra of these oligoprolines are
complex. Signals in square brackets, [], refer to resolved signals of
minor conformations arising from restricted rotation about the prolyl
peptide bonds. 13C NMR signals grouped together in braces, {}, are all
the resonances ascribed to a type of 13C nucleus, e.g., the Cβ of the
proline residues in their various conformations. Where no such
parentheses appear, this indicates either predominantly a single
conformation, or signals for different conformations that could not be
distinguished.
Acid 15. A 40% aqueous solution of tetrabutylammonium
hydroxide (2.2 mL, 876 mg, 3.3 mmol, 3.0 equiv) was added to a
solution of compound 1423 (766 mg, 1.1 mmol, 1.0 equiv) in THF (9
mL). The mixture was stirred at rt for 1.5 h. The solvent was
evaporated and the residue dissolved in EtOAc (45 mL) and washed
with 1 M HCl (50 mL). The aqueous layer was back extracted with
EtOAc (25 mL). The organic layers were combined, filtered through
MgSO4, and concentrated to give acid 15 (quantitative) that was used
in subsequent reactions without further purification: Rf 0.33(10:1
CH2Cl2−MeOH).
Circular dichroism spectra of the synthetic glycopeptides 1−
4 were recorded in the far-ultraviolet region of the spectrum
(190−240 nm) (Figure 2). As expected, the monomer is largely
unordered. However, the diglycodipeptide Ac-[[β-L-Araf-
Hyp)]2-NHMe (2) displayed both a positive band (λmax
=
220 nm, [θ] = 2905 deg cm2 dmol−1) and a strong negative
Amine 16. Trifluoroacetic acid (3.3 mL) was added to a solution of
compound 14 (789 mg, 1.2 mmol) in dry CH2Cl2 (10 mL) at 0 °C.
The mixture was stirred at 0 °C for 3 h and concentrated. The residue
was purified by flash column chromatography, eluting with EtOAc and
then flushing with 4:1 CH2Cl2−MeOH to give 16 as a light brown oil
(581 mg, 72%): Rf 0.40 (2:1 EtOAc−hexanes).
Diglycodipeptide 17. Acid 15 (489 mg, 0.77 mmol, 1.2 equiv)
and amine 16 (443 mg, 0.64 mmol, 1.0 equiv) were suspended in dry
CH2Cl2 (14 mL) and the mixture was cooled to 0 °C.
Diisopropylethylamine (405 μL, 305 mg, 2.3 mmol, 3.7 equiv) and
HATU (380 mg, 1.0 mmol, 1.5 equiv) were added successively. The
reaction was heated to 30 °C while stirring under N2 overnight. The
Figure 2. CD spectra of compounds 1 (monomer), 2 (dimer), 3
(trimer) and 4 (tetramer).
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mixture was diluted with CH2Cl2 to a total volume of 70 mL, washed
with 1 M HCl (2 × 40 mL), sat’d aq. NaHCO3 (40 mL), and brine
(40 mL). The organic layer was filtered through MgSO4 and
concentrated. The residue was purified by flash column chromatog-
raphy, eluting with 1.5:1 hexanes−EtOAc → 1.5:1 EtOAc−hexanes to
give compound 17 as a light oil (431 mg, 56%). 1H and 13C NMR data
were in agreement with those reported previously.23
74.0, 74.1, 74.3 (Pro Cγ)}, {79.8, 79.9, 80.3, 80.4, 80.5, 80.6, 82.9,
83.1, 83.4, 83.7, 84.1, 84.5 (Araf C2,3,4; Boc 4 °C)}, {98.0, 98.1, 98.3,
98.6, 98.9, 99.2, 99.4, 100.8 (Araf C1)}, 118.5 [118.6], {127.9, 128.2,
128.3, 128.4, 128.6, 128.7, 128.9 (Ar CH)}, 131.8, {137.8, 138.0,
138.1, 138.3, 138.5 (Ar 4 °C)}, 154.2 [153.9], 170.4, 170.5, 170.7,
170.9, 171.4; HRMS (ESI) calcd for C131H146N4O27Na (M + Na)+
2242.0072, obsd 2241.9979.
Acid 18. A 40% aq. solution of tetrabutylammonium hydroxide
(296 μL, 160 mg, 0.45 mmol, 3.0 equiv) was added to a solution of
compound 17 (180 mg, 0.15 mmol, 1.0 equiv) in THF (3 mL). The
mixture was stirred at rt under N2 for 2 h. The solvent was evaporated,
the residue dissolved in EtOAc (20 mL) and washed with 1 M HCl
(15 mL). The aqueous layer was back-extracted with EtOAc (3 × 10
mL). The organic layers were combined, filtered through MgSO4 and
concentrated. The crude acid 18 was obtained in quantitative yield and
submitted to the subsequent reactions without further purification: Rf
0.31 (10:1 CH2Cl2/MeOH).
Ac-Hyp-OMe. Dicyclohexylcarbodiimide (119 mg, 0.58 mmol, 1.0
equiv) and DMAP (18 mg, 0.15 mmol, 0.25 equiv) were added
sequentially to a suspension of Ac-Hyp-OH (100 mg, 0.58 mmol, 1.0
equiv) in dry MeOH (2 mL) and CH2Cl2 (2 mL). The mixture was
stirred overnight at rt under N2. The solvent was evaporated and the
residue triturated with CH2Cl2 and filtered to remove dicyclohexylur-
ea. The filtrate was concentrated and purified by flash column
chromatography, eluting with CH2Cl2−MeOH (14:1 → 10:1) to give
Ac-Hyp-OMe as an amorphous solid (71 mg, 66%): Rf 0.33 (10:1
CH2Cl2/MeOH); [α]D25 −89.9 (c 1.0, CH2Cl2); 1H NMR (400 MHz,
CDCl3) δ 2.03−2.10 [2.15−2.23] (m, 1H), 2.07 [1.96] (s, 3H), 2.26−
2.32 [2.41−2.47] (m, 1H), 3.51 (d, J = 11.2 Hz, 1H), 3.72 [3.77] (s,
3H), 3.74−3.79 (m, 1H), 4.52−4.57 [4.44−4.47] (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 22.2 [21.6], 38.0 [39.7], 52.3 [52.7], 55.9 [54.5],
57.5 [58.8], 70.1 [68.5], 170.0 [170.7], 173.0 [172.7]; HRMS (ESI)
calcd for C8H14NO4 (M + H)+ 188.0917, obsd 188.0919.
Triglycotripeptide 20. Acid 18 (81 mg, 0.07 mmol, 1.0 equiv)
and amine 16 (48 mg, 0.07 mmol, 1.0 equiv) and were suspended in
dry CH2Cl2 (3 mL). Diisopropylethylamine (37 μL, 30 mg, 0.21
mmol, 3.0 equiv) and HATU (40 mg, 0.1 mmol, 1.5 equiv) were
added successively. The mixture was stirred for 21 h at rt under N2.
The mixture was diluted with CH2Cl2 to a total volume of 25 mL,
washed with 1 M HCl (2 × 20 mL), sat’d aq. NaHCO3 (20 mL), and
brine (20 mL). The organic layer was filtered through MgSO4,
concentrated and the residue purified by flash column chromatog-
raphy, eluting with 1.5:1.0 hexanes−EtOAc → 1.5:1 EtOAc−hexanes
→2:1 EtOAc−hexanes to give compound 20 as a light oil (42 mg,
Compound 26. A solution of glycosyl donor 1323 (342 mg, 0.65
mmol, 1.0 equiv) and Ac-Hyp-OMe (124 mg, 0.66 mmol, 1.0 equiv) in
dry CH2Cl2 (40 mL) was stirred with activated, powdered 4 Å
molecular sieves (1.0 g) under N2 for ∼30 min at rt. The suspension
was cooled to −78 °C (acetone/dry ice) and then NIS (231 mg, 1.0
mmol, 1.5 equiv) and AgOTf (83 mg, 0.32 mmol, 0.5 equiv) were
added. The reaction was gradually warmed to 0 °C over 1.5 h. The
reaction was quenched by the addition of Et3N (2 mL) and filtered.
The filtrate was diluted with EtOAc (50 mL) and washed with 10%
aqueous Na2S2O3 (50 mL) and brine (50 mL). The organic layer was
filtered through MgSO4 and concentrated. The residue, determined to
be a 3:1 β:α ratio by NMR, was purified by column chromatography,
eluting with 3:1 hexanes−EtOAc to afford 26 as a mixture of anomers
25
1
35%): Rf 0.62 (2:1 EtOAc/Hex); [α]D +42.6 (c 1.0, CH2Cl2); H
NMR (400 MHz, CDCl3) δ 1.34 [1.33] (s, 9H), 1.75−1.80 (m, 1H),
1.90−2.00 (m, 1H), 2.04−2.34 (m, 4H), 3.06−3.67 (m, 8H), 3.84−
4.13 (m, 12H), 4.38−4.72 (m, 27H), 4.84 (d, J = 4.2 Hz, 0.5H), 4.87
(d, J = 4.1 Hz, 0.5H), 4.92 (d, J = 3.8, 0.5H), 5.09 (d, J = 2.8 Hz,
0.5H), 5.15 (d, J = 2.1 Hz, 0.5H), 5.16 (d, J = 4.0 Hz, 0.5H), 5.19−
5.30 (m, 2H), 5.81−5.91 (m, 1H), 7.28−7.33 (m, 45H); 13C NMR
(100 MHz, CDCl3) δ 28.5, {34.9, 35.1, 35.5, 35.7, 36.0, 36.3 (Pro
Cβ)}, {50.0, 50.1, 50.5, 50.7, 51.6, 51.9 (Pro Cδ)}, {56.6, 56.8, 56.9,
57.0, 57.9, 58.1 (Pro Cα)}, 65.7, {72.0, 72.2, 72.3, 73.1, 73.3 (Araf
C5)}, {73.6, 73.7, 74.5 (Pro Cγ)}, {79.6, 79.7, 79.9, 80.3, 82.6, 82.8,
83.1, 83.3, 83.8, 84.1 (Araf C2,C3,C4; Boc 4 °C)}, {98.2, 98.6, 98.9,
101.0 (Araf C1)}, {118.4, 127.8, 128.0, 128.1, 128.3, 128.4 (Ar CH)},
131.8, {137.5, 137.9, 138.0, 138.2 (Ar 4 °C)}, 154.2 [153.8], 170.8,
170.8, 171.0, 171.1, 171.4, 171.5; HRMS (ESI) calcd for
C101H112N3O21Na (M + Na)+ 1726.7759, obsd 1726.7750.
25
(317 mg, 83%): Rf 0.34 (8:1 EtOAc/Hex); [α]D +39.2 (c 0.5,
CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 2.02−2.08 [2.10−2.17] (m,
1H), 2.03 [1.84] (s, 3H), 2.31−2.40 (m, 1H), 3.41 (dd, J = 10.6, 3.6
Hz, 1H), 3.49−3.52 (m, 2H), 3.71 [3.75] (s, 3H), 3.72−3.74 (m, 1H),
4.07−4.14 (m, 3H), 4.29−4.43 (m, 1H), 4.48−4.73 (m, 7H), 4.90
[4.98] (d, J = 3.6 [4.0] Hz, 1H), 7.27−7.36 (m, 15H); 13C NMR (100
MHz, CDCl3) δ 22.3 [21.5], 36.0 [38.0], 52.3 [52.7], 52.8 [50.6], 57.5
[58.8], 71.9 [72.2], 72.5 [72.4], 72.7 [73.1], 73.4 [73.3], 76.3, 80.1,
82.4, 84.2 [83.9], 100.5 [99.1], 127.8, 127.9, 128.0, 128.1 (2C), 128.4,
128.5, 128.6, 137.6, 137.8, 137.9, 138.1 (2C), 169.3, 172.7 [172.6];
HRMS (ESI) calcd for C34H40NO8 (M + H)+ 590.2748, obsd
590.2758.
Amine 19. Trifluoroacetic acid (1.5 mL) was added to a solution of
diglycodipeptide 17 (168 mg, 0.14 mmol, 1 equiv) in dry CH2Cl2 (3
mL) at 0 °C. The mixture was stirred at 0 °C for 3 h and concentrated
to give compound 19 in quantitative yield, which was used in
subsequent steps without further purification: Rf 0.59 (9:1 CH2Cl2/
MeOH).
Acid 24. A 40% aq. solution of tetrabutylammonium hydroxide
(401 μL, 160 mg, 0.62 mmol, 3.0 equiv) was added to a solution of
compound 26 (121 mg, 0.21 mmol, 1.0 equiv) in dry THF (4 mL)
was and stirred at rt under N2 for 1.5 h. The solvent was evaporated
and the residue dissolved in EtOAc (25 mL), washed with 1 M HCl
(25 mL). The aqueous layer was back-extracted with EtOAc (10 mL).
The organic layers were combined, filtered through MgSO4 and
concentrated. The acid 24 was obtained in quantitative yield and used
in subsequent steps without further purification: Rf 0.19 (9:1 CH2Cl2/
MeOH).
Tetraglycotetrapeptide 21. Acid 18 (145 mg, 0.13 mmol, 1
equiv) and amine 19 (170 mg, 0.14 mmol, 1.1 equiv) were suspended
in dry CH2Cl2 (5 mL). HATU (50 mg, 0.13 mmol, 1.0 equiv) and
iPr2NEt (110 μL, 82 mg, 0.63 mmol, 5.0 equiv) were added
successively. The mixture was stirred for 18 h under N2. Upon
completion, the solvent was evaporated and the residue diluted with
EtOAc (30 mL), washed with 1 M HCl (30 mL), sat’d aq. NaHCO3
(30 mL), and brine (30 mL). The organic layer was filtered through
MgSO4 and concentrated. The residue was purified by flash
chromatography, eluting with 1.5:1 hexanes−EtOAc → 1:1
hexanes−EtOAc → 1:1.5 hexanes−EtOAc → 1:2 hexanes−EtOAc to
give compound 21 as a light oil (138 mg, 49%): Rf 0.80 (2:1 EtOAc/
Compound 27. HATU (50 mg, 0.13 mmol, 1.5 equiv) and
triethylamine (62 μL, 45 mg, 0.45 mmol, 5 equiv) were added to a
solution of acid 15 (56 mg, 0.09 mmol, 1 equiv) and methylamine
hydrochloride (12 mg, 0.18 mmol, 2 equiv) in acetonitrile under an
atmosphere of N2. The mixture was stirred for 18 h and the solvent
evaporated. The residue was diluted with EtOAc (30 mL), washed
with 1 M HCl (30 mL) and aq. NaHCO3 (30 mL), filtered through
MgSO4, and concentrated. The residue was purified by flash column
chromatography, eluting with 8:1 EtOAc/Hex to give compound 27 as
25
1
Hex); [α]D +42.2 (c 1.0, CH2Cl2); H NMR (400 MHz, CDCl3) δ
1.30 [1.26] (s, 9H), 1.70−2.30 (m, 8H), 3.08−3.66 (m, 16H), 3.76−
4.20 (m, 15H), 4.23−4.67 (m, 31H), 4.76−5.10 (m, 4H), 5.18 (d, J =
10.4 Hz, 1H), 5.27 (d, J = 17.3 Hz, 1H), 5.78−5.88 (m, 1H), 7.26−
7.31 (m, 60H); 13C NMR (100 MHz, CDCl3) δ 28.5 [29.7], {34.8,
35.0, 35.1, 35.3, 35.4, 35.9, 36.2 (Pro Cβ)}, [50.0, 50.1, 50.2, 50.4 50.5,
50.8, 51.6, 52.1 (Pro Cδ)}, {56.7, 56.8, 57.0, 57.1, 57.2, 57.9 (Pro
Cα)}, 65.8, {72.4, 72.5, 72.6, 72.8, 72.9. 73.4, 73.6 (Araf C5)}, {73.8,
25
a light oil (40 mg, 70%): Rf 0.32 (8:1 EtOAc/Hex); [α]D +19.6 (c
1
1.0, CH2Cl2); H NMR (400 MHz, CDCl3) δ 1.44 [1.38] (s, 9H),
2.08 (br s, 1H), 2.43 [2.32] (br s, 1H), 2.78 [2.77] (s, 3H), 3.43−3.47
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[3.75−3.78] (m, 2H), 3..52 (app d, J = 3.3 Hz, 2H), 4.06−4.31 (m,
5H), 4.50−4.70 (m, 6H), 4.97 (s, 1H), 6.57 [5.74] (s, 1H), 7.26−7.36
(m, 15H); 13C NMR (100 MHz) δ 26.3 [26.2], 28.5 [28.4], 35.1
[37.8], 51.6 [51.3], 58.9 [60.1], 72.3, 72.4, 73.3, 75.4 [73.7], 80.1, 80.6,
83.0 [82.9], 84.0 [83.8], 99.7 [98.8], 127.7, 127.8, 127.9, 128.1, 128.2,
128.4, 128.5, 128.6, 137.6, 138.0, 138.2, 155.6 [154.5], 172.2 [173.0];
HRMS (ESI) calcd for C37H46N2O8 (M + H)+ 647.3327, obsd
647.3323.
2.49 (m, 1H), 2.58−2.63 (m, 1H), 2.73 (s, 3H), 3.55−3.61 (m, 2H),
3.69−3.78 (m, 7H), 3.89−3.99 (m, 4H), 4.12 (d, J = 11.1 Hz, 1H),
4.46 (t, J = 8.1 Hz, 1H), 4.52 (br s, 1H), 4.76 (t, J = 8.1 Hz, 1H), 5.01
(d, J = 4.2 Hz, 1H), 5.04 (d, J = 4.4 Hz, 1H); 13C NMR (100 MHz,
CD3OD) δ 20.8, 25.0, 35.6, 36.1, 52.4, 53.5, 56.9, 59.4, 74.9, 75.2, 76.2,
76.7, 77.3, 77.4, 83.0, 83.1, 100.9, 101.0, 170.7, 171.9, 173.4; HRMS
(ESI+) calcd for C23H38N3O13 (M + H)+ 564.2399, obsd 564.2390.
Triglycotripeptide 3. Acid 24 (157 mg, 0.27 mmol, 1 equiv) and
amine 16 (184 mg, 0.27 mmol, 1 equiv) were dissolved in dry CH2Cl2
(7 mL) and stirred under N2. HATU (156 mg, 0..41 mmol, 1.5 equiv)
Amine 25. Trifluoroacetic acid (1.6 mL) was added to a solution of
compound 27 (199 mg, 0.31 mmol, 1 equiv) in dry CH2Cl2 (5 mL) at
0 °C under an atmosphere of N2. The mixture was stirred for 3 h at 0
°C and concentrated. The free amine 25 was submitted to subsequent
reactions without further purification: Rf 0.42 (9:1 CH2Cl2−MeOH).
End-Capped Perbenzylated Monomer 22. Diisopropylethyl-
amine (19 μL, 14 mg, 0.11 mmol, 1.1 equiv), EDC (21 mg, 0.11
mmol, 1.1 equiv) and HOBt (17 mg, 0.13 mmol, 1.3 equiv) were
added sequentially to a suspension of acid 24 (56 mg, 0.10 mmol, 1.0
equiv) and methylamine hydrochloride (8 mg, 0.10 mmol, 1.0 equiv)
in dry CH2Cl2 (3 mL) at 0 °C under N2. The ice bath was removed
and the mixture left to stir overnight, diluted with CH2Cl2 (25 mL)
and washed with 1 M HCl (25 mL), sat’d aq. NaHCO3 (25 mL), and
brine (25 mL). The organic layer was filtered through MgSO4 and
concentrated. The residue was purified by flash chromatography,
eluting with 19:1 CH2Cl2−MeOH to give compound 22 (35 mg,
i
was added and the reaction stirred for 15 min, after which Pr2NEt
(174 mg, 233 μL, 1.35 mmol, 5 equiv) was added to the mixture. The
mixture was stirred for 19 h. The mixture was diluted with EtOAc (40
mL), washed with 1 M HCl (25 mL) and brine (25 mL), filtered
through MgSO4, and concentrated. The residue was purified by flash
chromatography, eluting with 14:1 CH2Cl2−MeOH to give the dimer
28 as a cloudy oil (176 mg, 58%): Rf 0.37 (4:1 EtOAc−hexanes).
A 40% aq. solution of tetrabutylammonium hydroxide (305 μL, 121
mg, 0.47 mmol, 3 equiv) was added dropwise to a suspension of
compound 28 (176 mg, 0.16 mmol, 1 equiv) in THF (5 mL) at 0 °C.
The reaction was stirred, warming to rt over 1.5 h. The solvent was
evaporated and the residue diluted with EtOAc (30 mL) and washed
with 1 M HCl (30 mL). The aqueous layer was back-extracted with
EtOAc (2 × 15 mL). The combined organic layers were filtered
through MgSO4 and concentrated to give the free acid 29 in
quantitative yield: Rf 0.46 (10:1 CH2Cl2−MeOH).
25
61%) as an oil: Rf 0.49 (10:1 CH2Cl2/MeOH); [α]D 20.6 (c 1.0,
1
CH2Cl2); H NMR (400 MHz, CDCl3) δ 2.02 [1.84] (s, 3H), 2.04−
2.07 (m, 1H), 2.53 (dt, J = 13.0, 5.1 Hz, 1H), 2.72 (2.77) (d, J = 4.8
Hz, 3H), 3.36 (3.43) (dd, J = 11.6 (12.7), 4.6 (4.1) Hz, 1H), 3.53−
3.56 (m, 2H), 3.61 (dd, J = 10.7, 5.8 Hz, 1H), 4.07−4.11 (m, 3H),
4.39−4.44 (app. p, J = 5.4 Hz, 1H), 4.48−4.73 (m, 7H), 4.94 (4.99)
(d, J = 3.8 Hz, 1H), 7.26−7.35 (m, 15H); 13C NMR (100 MHz,
CDCl3) δ 22.5, 26.2, 34.2, 52.9, 58.37, 72.1, 72.5, 72.7, 73.3, 76.3, 80.1,
82.7, 84.2, 100.3, 127.7, 127.9, 128.0, 128.1, 128.4, 128.5, 128.5, 137.6,
137.9, 138.1, 170.5, 171.4; HRMS (ESI) calcd for C34H41N2O7 (M +
H)+ 589.2908, obsd 589.2913.
The dipeptide acid 29 (156 mg, 0.14 mmol, 1.0 equiv) and amine
25 (94 mg, 0.14 mmol, 1.0 equiv) were dissolved in dry CH2Cl2 (5
mL) and stirred under N2. HATU (82 mg, 0.22 mmol, 1.5 equiv) was
added and the mixture stirred for 15 min, after which iPr2NEt (124 μL,
92 mg, 0.71 mmol, 5.0 equiv) was added. The mixture was stirred at rt
for 21 h, diluted with EtOAc (25 mL), washed with 1 M HCl (25 mL),
sat’d NaHCO3 (25 mL) and brine (25 mL), filtered through MgSO4
and concentrated. The residue was purified by flash column
chromatography, eluting with 19:1 CH2Cl2/MeOH to give the
protected trimer 30 as a light oil (81 mg, 35%): Rf 0.50 (10:1
CH2Cl2−MeOH).
End-Capped Monomer 1. Palladium on carbon (10% w/w, 45
mg) was added to a solution of compound 22 (35 mg, 0.06 mmol) in
MeOH (2 mL). The suspension was stirred under an atmosphere of
H2 gas for 18 h. The mixture was filtered through Celite and
concentrated to give the compound 1 as an amorphous solid (19 mg,
Palladium on carbon (100 mg, 10% w/w) was added to a
compound 30 (11 mg, 6.8 μmol) in MeOH (1.5 mL). The suspension
was stirred under an atmosphere of H2 for 24 h, filtered through
Celite, washing well with MeOH, and concentrated. The residue was
dissolved in H2O (10 mL) and washed with CH2Cl2 (3 × 10 mL) to
remove organic impurities. The aqueous layer was lyophilized to give
the fully deprotected trimer 3 (5.5 mg, quantitative) as an amorphous
25
quantitative): [α]D +25.6 (c 0.5, MeOH); 1H NMR (400 MHz,
CD3OD) δ 2.08 [1.93] (s, 3H), 2.03−2.10 [2.13−2.19] (m, 1H),
2.47−2.53 [2.59−2.64] (m, 1H), 2.73 [2.77] (s, 3H), 3.34 (s, 1H),
3.56 (dd, J = 11.6, 7.1 Hz, 1H), 3.68−3.78 (m, 3H), 3.85−3.91 (m,
1H), 3.96 (dd, J = 7.8, 4.6 Hz, 1H), 4.41 [4.51] (t, J = 8.0 [7.7] Hz,
1H), 4.45−4.47 (m, 1H), 4.99 [4.95] (d, J = 4.6 [4.5] Hz, 1H); 13C
NMR (100 MHz, CD3OD) δ 21.0 [20.2], 25.0 [25.1], 36.5 [38.4],
53.5 [51.7], 59.0 [60.1], 63.9 [63.8], 75.0 [74.4], 76.4, 77.2, 83.0,
101.2 [100.7], 171.2 [171.6], 173.7 [173.5]; HRMS (ESI) calcd for
C13H23N2O7 (M + H)+ 319.1500, obsd 319.1486.
End-Capped Diglycodipeptide 2. Acid 24 (94 mg, 0.14 mmol, 1
equiv) and amine 25 (104 mg, 0.14 mmol, 1 equiv) were dissolved in
dry CH2Cl2 and stirred under N2. HATU (93 mg, 0.21 mmol, 1.5
equiv) was added and the reaction stirred for 15 min, after which
iPr2NEt (137 μL, 102 mg, 0.79 mmol, 5.5 equiv) was added to the
mixture. The reaction was warmed to 30 °C and stirred for 18 h. The
mixture was diluted with CH2Cl2 (25 mL), washed with 1 M HCl (25
mL) and brine (25 mL), filtered through MgSO4, and concentrated.
The residue was purified by flash chromatography, eluting with 14:1
CH2Cl2−MeOH to give the dimer 23 as a cloudy oil (86 mg, 55%): Rf
0.49 (9:1 CH2Cl2/MeOH).
Palladium on carbon (20 mg, 10% w/w) was added to a portion of
compound 23 (19 mg, 0.018 mmol) in MeOH (1 mL). The
suspension was stirred under an atmosphere of H2 gas for 18 h, then
the mixture was filtered through Celite washing well with MeOH, and
concentrated. The residue was dissolved in H2O (10 mL) and washed
with CH2Cl2 (3 × 10 mL) to remove organic impurities. The aqueous
layer was lyophilized to give the fully deprotected dimer 2 (10 mg,
quantitative) as an amorphous solid: [α]D25 +30.8 (c 0.5, MeOH); 1H
NMR (400 MHz, CD3OD) δ 2.00−2.11 (m, 2H), 2.06 (s, 3H), 2.44−
solid: [α]D25 −17.4 (c 0.1, MeOH); H NMR (400 MHz, CD3OD) δ
1
2.01−2.17 (m, 3H), 2.11 (s, 3H), 2.47−2.66 (m, 3H), 2.75 (s, 3H),
3.58−3.66 (m, 3H), 3.69−3.78 (m, 10H), 3.86−4.01 (m, 6H), 4.15 (d,
J = 11.5 Hz, 1H), 4.26 (d, J = 11.1 Hz, 1H), 4.48 (t, J = 8.5 Hz, 2H),
4.53 (br s, 2H), 4.60 (br s, 1H), 4.79 (t, J = 8.3 Hz, 1H), 5.02 (d, J =
4.4 Hz, 1H), 5.04 (app. t, J = 4.8 Hz, 2H); 13C NMR (100 MHz,
CD3OD) δ 24.1, 28.6, 37.5, 37.7, 38.5, 55.4, 55.5, 56.4, 59.6, 60.2, 62.4,
63.2, 65.9, 66.0, 72.5, 74.4, 77.1, 77.2, 78.8, 78.9, 79.0, 79.3, 84.6, 84.7,
102.7, 102.8, 102.9, 173.8, 174.6, 174.8, 175.5, 176.5; HRMS (ESI+)
calcd for C33H52N4O19 (M + H)+ 809.3299, obsd 809.3314.
Tetraglycotetrapeptide 4. Acid 15 (155 mg, 0.24 mmol, 1.0
equiv) and amine 25 (161 mg, 0.24 mmol, 1.0 equiv) were suspended
in dry CH2Cl2 (5 mL) and stirred under N2. HATU (112 mg, 0.29
i
mmol, 1.2 equiv) and Pr2NEt (213 μL, 158 mg, 1.2 mmol, 5 equiv)
were added sequentially. The mixture was stirred for 24 h. The mixture
was diluted with EtOAc (50 mL) and washed with 5% citric acid (50
mL) and brine (50 mL). The organic layer was filtered through
MgSO4 and concentrated. The residue was purified by flash
chromatography eluting with 28:1 CH2Cl2−MeOH to give compound
31 as a light oil (138 mg, 49%). Trifluoroacetic acid (0.75 mL) was
added to the residue suspended in CH2Cl2 (1.5 mL) at 0 °C. The
mixture was stirred for 3 h at 0 °C and the solvent was evaporated to
give the free amine 32 in quantitative yield: Rf 0.33 (10:1 CH2Cl2−
MeOH).
Acid 29 (47 mg, 0.04 mmol, 1.0 equiv) and amine 32 (71 mg, 0.06
mmol, 1.4 equiv) were dissolved in dry CH2Cl2 (1.75 mL) and stirred
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under N2. HATU (17 mg, 0.04 mmol, 1.0 equiv) was added and the
reaction stirred for 15 min, after which diisopropylethylamine (124 μL,
92 mg, 0.16 mmol, 4.0 equiv) was added. The mixture was stirred at rt
for 20 h, diluted with EtOAc (25 mL), washed with 1 M HCl (25 mL)
and brine (25 mL), filtered through MgSO4 and concentrated. The
residue was purified by flash column chromatography, eluting with
19:1 CH2Cl2−MeOH to give the protected tetramer 33 as a light oil
(14 mg, 15%): Rf 0.42 (9:1 CH2Cl2−MeOH).
REFERENCES
■
(1) Himly, M.; Jahn-Schmid, B.; Dedic, A.; Kelemen, P.; Wopfner,
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Palladium on carbon (25 mg, 10% w/w) was added to a solution of
compound 33 (3 mg, 1.4 μmol) in MeOH (0.6 mL). The suspension
was stirred under an atmosphere of H2 gas for 24 h. The mixture was
filtered through Celite, washing well with MeOH, and concentrated.
The residue was dissolved in H2O (10 mL) and washed with CH2Cl2
(3 × 10 mL) to remove organic impurities. The aqueous layer was
lyophilized to give the fully deprotected tetramer 4 (1,5 mg,
25
quantitative) as an amorphous solid: [α]D +60.8 (c 0.075,
1
MeOH); H NMR (400 MHz, CD3OD) δ 2.04−2.19 (m, 4H), 2.09
(s, 3H), 2.47−2.67 (m, 4H), 2.76 (s, 3H), 3.59−3.65 (m, 4H), 3.73−
3.79 (m, 12H), 3.86−4.01 (m, 8H), 4.15 (app d, J = 11.5 Hz, 3H),
4.50 (t, J = 8.0 Hz, 4H), 4.56 (br s, 4H), 5.04 (d, J = 4.5 Hz, 2H), 5.04
(d, J = 4.3 Hz, 4H); HRMS (ESI+) calcd for C43H67N5O25Na (M +
Na)+ 1076.4017, obsd 1076.3992.
CD Spectroscopy. Compounds 1−4 were each lyophilized for 24
h prior to dilution to a concentration of 0.4 mM with purified water.
The pH of the samples were measured at rt and found to be 6.80, 7.38,
8.79, and 9.48 for compounds 1, 2, 3, and 4 respectively. For analysis,
175 μL of the sample was loaded into a quartz cell with a path length
of 0.1 cm. The CD spectra were recorded at a scan rate of 20 nm per
min, data pitch of 1.0 nm, and bandwidth of 2.0 nm. The accumulation
of three scans was averaged for each sample, after which a blank of the
solvent was subtracted. The CD signal was converted to molar
ellipticity per mean residue ([θ]) and the data was smoothed by
Savitzky−Golay algorithm. For Ac-([β-L-Araf ]Hyp)2-NHMe (2),
positive band (λmax = 220 nm, [θ] = 2905 deg cm2 dmol−1) and a
negative band (λmin = 199 nm, [θ] = −10423 deg cm2 dmol−1); trimer
Ac-([β-L-Araf ]Hyp)3-NHMe (3), positive band at 222 nm ([θ] =
4207 deg cm2 dmol−1) and a negative maxima at 203 nm ([θ] =
−13352 deg cm2 dmol−1); and Ac-([β-L-Araf]Hyp)4-NHMe (4),
positive band at 220 nm ([θ] = 3704 deg cm2 dmol−1) and a negative
maxima at 200 nm ([θ] = −13816 deg cm2 dmol−1).
(8) Liu, M.; Borgert, A.; Barany, G.; Live, D. Biopolymers Pept. Sci.
2008, 90, 358−368.
(9) Sames, D.; Chen, X.-T.; Danishefsky, S. J. Nature 1997, 389,
587−591.
(10) Shao, N.; Guo, Z. Org. Lett. 2005, 7, 3589−3592.
(11) Braun, P.; Davies, G. M.; Price, M. R.; Williams, P. M.; Tendler,
S. J. B.; Kunz, H. Bioorg. Med. Chem. 1998, 6, 1531−1545.
(12) Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang,
C.; Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay, C.; Mugge, C.;
Wenschuh, H.; Klose, J.; Beyermann, M.; Bienert, M. Angew. Chem.,
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AUTHOR INFORMATION
Corresponding Author
*E-mail: cmtaylor@lsu.edu.
■
Present Address
†TumorEnd LLC, Baton Rouge, Louisiana 70808, United
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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Ted Gauthier at LSU for his assistance in the acquisition of CD
spectra.
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