Synthesis and antibody binding of highly fluorinated amphiphilic MUC1 glycopeptide antigens

Article abstract of DOI:10.1002/ejoc.201100648

The analysis of humoral immune responses is of great importance for basic and clinical research. Mapping the structural requirements of epitope recognition with modified tumor-associated carbohydrate antigens allows both the development of biomarkers and the design of synthetic anticancer vaccines. For this purpose, double-tailed hydrocarbon/fluorocarbon membrane anchors have been prepared and conjugated to a T_N dipeptide. Furthermore, a novel hydrophobized MUC1 tandem repeat glycopeptide antigen was fully assembled on a solid support and its specific binding to different mouse anti-MUC1 antibodies was demonstrated through ELISA, QCM, and SPR measurements. Such functional fluorous MUC1 antigens are of great interest for specific glycan (micro-)array formats and allow a detailed analyses of serum antibodies obtained from immunization studies. In addition, the intriguing characteristics of fluorous surfactants, for example, their strong self-association tendency, might stimulate the use of novel fluorous-tagged antigen conjugates in the development of multivalent micellar glycopeptide vaccines. Copyright

Full text of DOI:10.1002/ejoc.201100648

FULL PAPER
DOI: 10.1002/ejoc.201100648
Synthesis and Antibody Binding of Highly Fluorinated Amphiphilic MUC1
Glycopeptide Antigens
Tobias Platen,[a] Timo Schüler,[b] Wolfgang Tremel,[b] and Anja Hoffmann-Röder*[a]
Keywords: Amphiphiles / Glycopeptides / Antigens / Antibodies / Fluorine
The analysis of humoral immune responses is of great impor-
tance for basic and clinical research. Mapping the structural
requirements of epitope recognition with modified tumor-as-
sociated carbohydrate antigens allows both the development
of biomarkers and the design of synthetic anticancer vac-
cines. For this purpose, double-tailed hydrocarbon/fluorocar-
bon membrane anchors have been prepared and conjugated
to a TN dipeptide. Furthermore, a novel hydrophobized
MUC1 tandem repeat glycopeptide antigen was fully as-
sembled on a solid support and its specific binding to dif-
ferent mouse anti-MUC1 antibodies was demonstrated
through ELISA, QCM, and SPR measurements. Such func-
tional fluorous MUC1 antigens are of great interest for spe-
cific glycan (micro-)array formats and allow a detailed analy-
ses of serum antibodies obtained from immunization studies.
In addition, the intriguing characteristics of fluorous surfac-
tants, for example, their strong self-association tendency,
might stimulate the use of novel fluorous-tagged antigen
conjugates in the development of multivalent micellar glyco-
peptide vaccines.
based on fluorinated analogues of amphiphilic compounds
have recently been developed.[7]
Introduction
Glycoproteins are fundamental for a variety of biological
events including fertilization, neuronal development, im-
mune surveillance, and inflammatory responses.[1] As a con-
sequence, glycoconjugates have become highly attractive
targets for medical applications and important tools for
chemical glycobiology. Alterations in cell surface carbo-
hydrate structures have been found to affect normal cellular
interactions and contribute to the pathogenesis and pro-
gression of neoplasia.[2] For example, most carcinoma ex-
press abnormal forms of the transmembrane glycoprotein
mucin-1 (MUC1) characterized by the exposure of
immunogenic peptide epitopes and truncated glycan
chains.[3] Hence, mucin-type glycopeptides decorated with
tumor-associated saccharide antigens have been shown to
be important tools for the development of cancer immuno-
therapy and diagnostics.[4] With regard to vaccine design,
the traditional approach of coupling glycopeptide antigens
to carrier proteins[5] has been expanded by attaching mul-
tiple copies of MUC1 epitopes to peptide templates, dendri-
mers, nanoparticles, and liposomes.[6] To enhance the sta-
bility of liposomal vaccine formulations and to limit unde-
sired biological side-effects, self-assembled delivery systems
Fluorocarbon moieties are characterized by very strong
intramolecular bonds and very weak intermolecular inter-
actions. In addition, perfluoroalkyl chains are larger and
more rigid than their hydrocarbon counterparts and confer
on fluorinated surfactants a strong tendency to collect at
interfaces and self-associate into supramolecular as-
semblies.[8] Because fluorocarbon amphiphiles are at the
same time hydrophobic, lipophobic, and fluorophilic, they
do not accumulate in lipid membranes and hence are less
haemolytic and less detergent than hydrocarbon amphi-
philes.[9] Moreover, partially fluorinated surfactants show
intriguing characteristics with regard to miscibility, phase
separation, and compartmentalization that might stimulate
novel applications in materials science and the field of medi-
cine.[10]
In addition, the temporary attachment of fluorocarbon
linkers and fluorous tags is an attractive strategy for facili-
tating solution-phase organic synthesis by fluorous separa-
tion and purification techniques.[11] The use of fluorocar-
bon tags for specific immobilization within fluorous
(micro-)array formats has also expanded their applica-
tion.[12] For example, fluorous-tagged small molecules,[13]
carbohydrates,[14] and glycosphingolipids[15] have been used
in this context. Recently, the first examples of fluorous-
tagged tumor-associated MUC1 glycopeptide antigens and
their successful recognition by specifically induced anti-
MUC1 antibodies were described.[16] These hydrophobized
glycoconjugates were based on tris(hydroxymethyl)amino-
methane onto which three perfluoroundecyl chains were
crafted. In this study, however, a modular approach towards
[a] Institut für Organische Chemie, Johannes Gutenberg-
Universität,
Duesbergweg 10–14, 55128 Mainz, Germany
Fax: +49-6131-3926006
E-mail: hroeder@uni-mainz.de
[b] Institut für Anorganische Chemie und Analytische Chemie,
Johannes Gutenberg-Universität,
Duesbergweg 10–14, 55128 Mainz, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100648.
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Amphiphilic MUC1 Glycopeptide Antigens
the preparation of novel MUC1 glycopeptide conjugates is responding acid with SOCl2/pyridine.[21] Hence, coupling of
presented based on a trifunctional chiral lysine core[17] with 4 to the deprotected amines 3a,b was accomplished in the
single- or double-chain fluorinated tails that could serve as presence of DIPEA in CH2Cl2,[22] furnishing the desired
potential building blocks in cancer immunotherapy and di- Cbz-protected derivatives 5a and 5b in 83 and 69% yields,
agnostics.
respectively, after column chromatography or recrystalli-
zation. The Cbz groups were then removed by hydro-
genolysis with Pd/C in EtOH to yield amphiphiles 6a and
6b in nearly quantitative yields. Notably, MeOH is not a
suitable solvent for this reaction because it provides N-
methylated products almost exclusively.[23]
Results and Discussion
Synthesis of Lysine-Based Fluorinated Amphiphiles
The synthetic route to the target double-tailed surfac-
To install the tumor-associated carbohydrate antigen
tants is depicted in Scheme 1 and starts from commercially (TACA) head group, the hydrophilic N-protected ω-spacer
available Nα-Boc-Nε-Cbz-l-lysine (1), which was coupled to amino acid 7a[24] was attached to amines 6a,b by using
hydrocarbon and fluorocarbon amines 2a and 2b. Although HCTU/HOBt[25] in the presence of N-methylmorpholine
the coupling reaction to 3a has already been performed (NMM) in CH2Cl2 under sonication. Column chromatog-
with dicyclohexylcarbodiimide (DCC),[18] in our hands, the raphy or fluorous solid-phase extraction[26] (F-SPE) in the
best results were obtained by using TBTU/HOBt[19] and di- case of the corresponding double-tailed fluorocarbon deriv-
isopropylethylamine (DIPEA) in CH2Cl2 at 40 °C. Subse- ative provided the desired surfactants in good yields. Subse-
quent Boc deprotection with trifluoroacetic acid (TFA) and quent N-terminal deprotection of the corresponding Fmoc-
anisole[20] then set the stage for further attachment of the functionalized conjugate was attempted by treatment with
perfluoroalkylated fatty acid. Because of the low solubility either 20% piperidine in CH2Cl2 or tetrabutylammonium
of the reactants and the reduced reactivity of the l-lysine α- fluoride (TBAF) in iPrOH/DMF. Although TLC and
amino group, this step required the use of the more reactive HPLC indicated complete conversion in both cases, the low
perfluoro acyl chloride 4, accessible by treatment of the cor- solubility and high aggregation tendency of the deprotected
Scheme 1. Synthesis of the double-tailed fluorocarbon and hydrocarbon/fluorocarbon membrane anchors and their incorporation into a
TN antigen-Thr-Val dipeptide. TBTU = O-(benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium tetrafluoroborate, HOBt = 1-hydroxybenzo-
triazole, DIPEA = diisopropylethylamine, HCTU = O-(6-chlorobenzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate,
NMM = N-methylmorpholine, TFA = trifluoroacetic acid.
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surfactants always impeded the isolation of significant with NMM in CH2Cl2 at 40 °C, providing the desired con-
amounts of pure material. Fortunately though, after attach- jugates 16a,b together with unreacted starting material that
ment of the corresponding Cbz-protected spacer 7b to 6a,b could not be completely removed by RP-HPLC at this
and subsequent hydrogenolysis, the desired products 9a,b stage. However, separation of the latter was possible after
were accessible in good chemical yields and sufficient puri- O-deacetylation of the carbohydrate moiety. Thus, after
ties upon simple filtration.
careful Zemplén transesterification with NaOMe in MeOH
at pH 9.5[28] and semipreparative RP-HPLC, glycolipid an-
alogues 17a,b were isolated in 15% yield over the two steps.
Notably, the relatively low yield of 17a was not a result of
Synthesis of F-Amphiphilic TACA Conjugates
With regard to the low solubility of compounds 9a,b, the significant β-elimination of the glycan but mainly due to
initial synthetic strategy for their incorporation into tumor- solubility problems.
associated MUC1 glycopeptide conjugates was slightly
For potential immunotherapeutic and diagnostic applica-
modified and TN antigen building block 10[27] was first cou- tions, that is, to map antibody specificities of humoral im-
pled to the spacer amino acid tert-butyl ester 11[24] by using mune responses and elucidate the corresponding binding
HCTU/HOBt with NMM in CH2Cl2. To enhance the epitopes, it would be desirable to attach larger tumor-asso-
chemical stability of the antigen derivative, conjugate 12 ciated antigen structures to the fluorous anchors. Towards
was then further condensed to Fmoc-protected l-valine un- this end, a mixed hydrocarbon/fluorocarbon double-tailed
der similar coupling conditions (76% over two steps). The glycolipopeptide carrying a complete MUC1 tandem repeat
labile Fmoc protecting group was exchanged for a perma- domain with a TN determinant at Thr6 as TACA was pre-
nent acetyl protecting group and the tert-butyl ester of the pared by solid-phase glycopeptide synthesis (SPPS). Thus,
resulting dipeptide 14 was cleaved under acidic conditions with the aid of automated solid-phase peptide chemistry,
(TFA, H2O) to afford building block 15 in 64% yield over the MUC1 glycopeptide 19, equipped with a hydrophilic
three steps. Final attachment of 15 to the surfactants 6a,b triethylene glycol spacer and a fluorous anchor, was as-
was again accomplished in the presence of HCTU/HOBt sembled on a Fmoc-Pro-trityl-TentaGel resin (Scheme 2).
Scheme 2. Solid-phase synthesis of the double-tailed fluorocarbon/hydrocarbon MUC1 glycopeptide antigen 19. A) Fmoc removal: 20%
piperidine in NMP; B) coupling: Fmoc-aa-OH, HBTU, HOBt, DIPEA, DMF; C) capping: cat. HOBt, Ac2O, DIPEA, NMP; D) coupling:
10 or 18, HATU, HOAt, NMM, NMP, 8 h; E) cleavage: TFA, TIS, H2O (10:1:1); F) deprotection: NaOMe, MeOH, pH 9.5. HBTU =
O-(1H-benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate, NMP = N-methylpyrrolidone, HATU = O-(7-azabenzo-
triazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate, HOAt = N-hydroxy-7-azabenzotriazole, TIS = triisopropylsilane.
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Amphiphilic MUC1 Glycopeptide Antigens
The first 13 amino acids of the tandem repeat sequence its use as an antibody profiling tool was conducted. Because
were attached under standard conditions (Fmoc protocol) antibodies possess multiple binding sites, the presentation,
with piperidine in N-methylpyrrolidone (NMP) for Fmoc orientation, and multivalent architecture of epitopes all
deprotection and HBTU, HOBt, and DIPEA[29] in DMF contribute to the recognition event. Hence, upon careful
to couple the amino acids (10 equiv. each). To minimize the analysis of humoral responses, different antibodies or sub-
amount of byproducts, unreacted amino groups were also populations of antibodies recognizing variations in binding
capped with Ac2O, HOBt, and DIPEA in NMP after each epitopes and/or their spatial orientations might be distin-
cycle. In the case of the sterically hindered glycosylated guished to help fine-tune vaccine design and biomarker de-
threonine derivative 10 (2.5 equiv.), successful attachment velopment. Towards this end, conjugate 19 was coated onto
was ensured by using extended reaction times (8 h) and the polystyrene microtiter plates and incubated with serum
more reactive reagents HATU/HOAt[30] with N-methyl- anti-MUC1 antibodies raised after immunization of mice
morpholine (NMM) in NMP for activation. After coupling with a structurally related glyco-6Thr(TF)-MUC1-TTox
the final five Fmoc-amino acids of the tandem repeat se- vaccine.[32] Although this glycopeptide vaccine does not
quence and the hydrophilic spacer 7a according to the stan- contain the exact TACA of the amphiphile 19, the induced
dard protocol, the fluorinated amphiphile 18[31] (2.0 equiv.) antibodies show relatively broad specificities and bind to
was attached over 8 h by using the reagent cocktail of various MUC1 peptide antigens. Hence, not unexpectedly,
HATU, HOAt, and NMM in NMP. Simultaneous detach- specific binding of these serum antibodies to the immobi-
ment of the glycopeptide from the resin and cleavage of lized fluorous amphiphile 19 was also proven in a corre-
the acid-labile amino acid side-chain protective groups was sponding ELISA test (see Figure 1).
achieved upon treatment with a mixture of TFA, triisopro-
pylsilane (TIS), and water. The subsequent de-O-acetyl-
ation of the saccharide moiety under Zemplén conditions
at pH 9.5 was directly performed on the crude product and
required stringent pH control to avoid epimerization of
amino acids and/or β-elimination of the glycan. Purification
of the resulting hydrophobized glycoconjugate by RP-
HLPC was impossible due to its limited solubility. Fortu-
nately though, precipitation from Et2O provided 19 (36%
based on the loaded resin) without concomitant deletion
sequences, as indicated by analytical RP-HPLC and
MALDI-TOF mass spectrometry.
The binding of the commercially available monoclonal
anti-MUC1 antibody SM3[33] to 19 was confirmed by using
a quartz crystal microbalance[34] (QCM) and surface plas-
mon resonance (SPR) spectroscopy. The SM3 antibody was
immobilized onto freshly cleaned SPR glass slides success-
ively sputtered with 1.5 nm chromium and 55 nm gold
films. Similarly, QCM measurements were performed by
using commercially available quartz crystals coated with
100 nm gold layers. Because all cysteine residues are en-
gaged in disulfide bridges inside the protein, binding of the
antibody to the gold surface is assumed to take place
through peripheral methionine residues, for example, Met18
and Met85.[35] Both residues are readily accessible and
should not interfere with the antigen binding-site (see the
Supporting Information). Thus, after addition of the anti-
Antibody Binding of Glycolipopeptide Analogue 19
With the mixed hydrocarbon/fluorocarbon glycolipo- genic amphiphile 19 to the QCM chamber, a typical Lang-
peptide conjugate 19 in hand, a preliminary study towards muir adsorption isotherm was obtained indicating a mass
Figure 1. ELISA of the anti-serum induced by a structurally related glyco-6Thr(TF)-MUC1-TTox vaccine demonstrates specific binding
of amphiphile 19 to the mouse serum antibodies. Optical density at λ = 410 nm.
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T. Platen, T. Schüler, W. Tremel, A. Hoffmann-Röder
FULL PAPER
Figure 2. Specific binding of the MUC1 amphiphile 19 to the commercially available anti-MUC1 antibody SM3 shown by QCM and
SPR spectroscopy. Left: QCM frequency shift vs. time for the binding of 19 to the preadsorbed antibody. Right: Coupling angle vs. time
in the SPR experiment. Before the injection of the antigen, the SM3-coated surface was washed three times with PBS buffer.
300 or AM-400 spectrometer. The chemical shifts are reported in
ppm relative to the signal of the deuteriated solvent. Multiplicities
are given as s (singlet), br. s (broad singlet), d (doublet), t (triplet),
and m (multiplet). The proton and carbon signals were assigned by
additional COSY, HSQC, and HMBC experiments as noted. The
signals of molecule fragments are denoted as follows: amino acids
(Greek indices), octyl fragment (O), decyl (F17) fragment (D) and
oligo(ethylene glycol) fragment (OEG). ESI-MS and ESI-HRMS
were recorded with a Micromass Q TOF Ultima 3 spectrometer
and MALDI-TOF MS were acquired with a Micromass Tofspec E
spectrometer using 2,5-dihydroxybenzoic acid as the matrix. Op-
tical rotations were measured at 546 and 578 nm with a Perkin–
Elmer 241 polarimeter.
increase upon antigen binding (Figure 2, left). Similarly, the
corresponding SPR experiment reflects the recognition of
the pre-adsorbed SM3 antibody through an increase of the
layer thickness (Figure 2, right).
Conclusions
Fluorous double-tailed surfactants carrying one or two
perfluoroalkyl chains and based on a lysine core have been
assembled. Attachment of these amphiphiles to TN antigen-
Thr-Val dipeptides provided novel hydrophobized TACAs
17a and 17b that are of interest as building blocks for fluo-
rous microarrays and/or multivalent cancer vaccines. With
regard to the latter, the first hydrocarbon/fluorocarbon
double-tailed MUC1 glycolipopeptide 19 was prepared by
solid-phase glycopeptide chemistry and its specific recogni-
tion by different anti-MUC1 mouse antibodies was demon-
strated through ELISA, SPR, and QCM measurements.
Owing to the strong hydrophobizing influence of the fluo-
rous tag, it is anticipated that such amphiphilic glycoconju-
gates might represent a novel potent type of self-aggregated
multivalent cancer vaccine for future immunizations.
Boc-Lys(Z)-O-octyl
(3a):
Diisopropylethylamine
(DIPEA;
3.45 mL, 20.87 mmol) was added to a solution of Nα-Boc-Nε-Cbz-
l-lysine (1; 3.80 g, 9.98 mmol), O-(benzotriazol-1-yl)-N,N,NЈ,NЈ-
tetramethyluronium
tetrafluoroborate
(TBTU;
3.50 g,
10.89 mmol), and 1-hydroxybenzotriazole (HOBt; 1.47 g,
10.89 mmol) in dry CH2Cl2 (30 mL). The reaction mixture was
stirred for 60 min at room temperature under argon before oc-
tylamine (1.5 mL, 9.08 mmol) was added. The solution was heated
at reflux for 6 h and then stirred for a further 13 h at room tem-
perature. After dilution with CH2Cl2 (120 mL), the organic phase
was washed with 1 m aq. HCl (2ϫ 150 mL), dried (MgSO4), and
concentrated in vacuo. Flash chromatography (SiO2, cHex/EtOAc,
3:1Ǟ1:1) afforded 3a as a colorless amorphous solid (4.42 g,
8.99 mmol, 99%). Rf = 0.10 (cHex/EtOAc, 3:1). Analytical RP-
Experimental Section
HPLC (Luna-PFP, MeOH/H2O, 80:20Ǟ100:0, 20 min): Rt
=
8.7 min. [α]2D3 = –9.5 (c = 1.00, CHCl3). 1H NMR (400 MHz,
CDCl3, COSY): δ = 7.32–7.27 (m, 5 H, Har), 6.49 (br. s, 1 H,
NHamide), 5.35 (d, JNH,Kα = 7.6 Hz, 1 H, NHBoc), 5.10 (br. s, 1 H,
NHcarbamate), 5.06 (s, 2 H, CH2-ar), 4.07–3.97 (m, 1 H, Kα), 3.20–
3.13 (m, 4 H, Kε, 1O-H), 1.81–1.73 (m, 1 H, Kaβ), 1.63–1.54 (m, 1
H, Kbβ), 1.51–1.41 (m, 4 H, Kδ, 2O-H), 1.40 [s, 9 H, C(CH3)3],
1.35–1.33 (m, 2 H, Kγ), 1.23 (br. s, 10 H, 3O–7O-H), 0.85 (t, 3 H,
J8O-H,7O-H = 6.9 Hz, 8O-H) ppm. 13C NMR (100.6 MHz, CDCl3,
HSQC): δ = 172.1 (C=Oamide), 156.7, 155.9 (C=Ocarbamate), 136.7
(Cq-ar), 128.5, 128.1 (5ϫ CHar), 79.9 (Cq-tBu), 66.6 (CH2-ar), 54.4
(Kα), 40.5, 39.5 (Kε, C-1O), 32.1 (Kβ), 31.8 (C-6O), 29.6, 29.5 (Kδ,
C-2O), 29.3 (C-4O, C-5O), 28.4 [3ϫ C(CH3)3], 26.9 (C-3O), 22.7,
22.6 (Kγ, C-7O) 14.2 (C-8O) ppm. HRMS (ESI-TOF): calcd. for
C27H45N3O5Na [M + Na]+ 514.3257; found 514.3234.
General: Reagents were purchased in the highest available commer-
cial quality and used as supplied, except where noted. DMF
(amine-free, for peptide synthesis) and NMP were purchased from
Roth. Fmoc-protected amino acids were purchased from Orpegen
Pharma. For solid-phase syntheses, preloaded TentaGel S resin
(Rapp polymere) was employed. Reactions were monitored by TLC
using precoated silica gel 60 F254 aluminium plates (Merck KGaA,
Darmstadt). Flash column chromatography was performed with
silica gel (230–400 mesh) from Merck. RP-HPLC analyses were
performed with a Jasco HPLC system with Phenomenex Luna-
PFP(2) (250ϫ4.6 mm, 5 μm) and Phenomenex Jupiter C18(2)
(250ϫ4.6 mm, 5 μm) columns at flow rates of 1 mLmin–1. Prepar-
ative HPLC separations were carried out with a Jasco HPLC sys-
tem with Phenomenex Luna PFP(2) (250ϫ30 mm, 5 μm) columns
at a flow rate of 20 mLmin–1. Mixtures of MeCN/H2O and MeOH/
RF-Lys(Z)-O-octyl (5a): A solution of 3a (1.74 g, 3.54 mmol) and
anisole (0.5 mL) in CH2Cl2 (30 mL) was treated with trifluoroacetic
acid (TFA, 5.0 mL) and stirred for 15 h at room temperature. The
1
H2O were used as solvents. If required, 0.1% TFA was added. H,
13C, 19F, and 2D NMR spectra were recorded with a Bruker AC-
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Amphiphilic MUC1 Glycopeptide Antigens
reaction mixture was concentrated in vacuo and co-evaporated with
toluene (2ϫ 20 mL). The crude product was cooled in an ice bath,
treated with aq. NH4OH (50 mL), and extracted with Et2O/CH2Cl2
(10:1, 3ϫ 30 mL). The combined organic phases were washed with
H2O (40 mL) and brine (40 mL), dried (MgSO4), and concentrated
in vacuo to yield the free amine H2N-Lys(Z)-O-octyl as a colorless
amorphous solid (1.37 g, 3.50 mmol, 99%). Rf = 0.37 (CH2Cl2/
MeOH/NEt3, 19:1:0.1). Analytical RP-HPLC (Luna-PFP, MeOH/
H2O, 80:20Ǟ100:0, 20 min): Rt = 3.4 min. [α]2D3 = –10.0 (c = 1.00,
CHCl3). 1H NMR (400 MHz, CDCl3, COSY): δ = 7.35–7.28 (m, 5
H, Har), 7.26 (br. s, 1 H, NHamide), 5.08 (s, 2 H, CH2-ar), 4.92–4.84
(m, 1 H, NHcarbamate), 3.31 (dd, JKα,Kaβ = 7.9, JKα,Kbβ = 4.3 Hz, 1
H, Kα), 3.25–3.14 (m, 4 H, Kε, 1O-H), 1.87 (br. s, 2 H, NH2), 1.89–
1.76 (m, 1 H, Kaβ), 1.57–1.45 (m, 5 H, Kbβ, Kδ, 2O-H), 1.45–1.33
C=ORF], 53.8 (Kα), 41.4, 39.9 (Kε, C-1O), 32.3 (Kδ), 32.0, 31.9 (Kβ,
C-6O), 29.5 (C-2O), 29.3 (C-4O, C-5O), 27.0 (C-3O), 22.7, 22.3 (Kγ,
C-7O) 14.2 (C-8O) ppm. 19F NMR (376.4 MHz, CDCl3), δ = –81.20
(t, JF,F = 9.7 Hz, 3 F, CF3), –119.20 (m, 2 F), –120.06 (br. s, 2 F),
–121.91 (br. s, 2 F), –122.23 (br. s, 2 F), –122.76 (br. s, 2 F), –123.09
(br. s, 2 F), –126.50 (m, 2 F, CF2CF3) ppm. HRMS (ESI-TOF):
calcd. for C23H30F17N3O2H [M + H]+ 704.2145; found 704.2134.
RF-Lys(OEG-Cbz)-O-octyl (8a): N-Methylmorpholine (NMM;
30 μL, 0.273 mmol) was added to a solution of CbzHN-OEG-
CO2H (7b;[24] 56 mg, 0.156 mmol), O-(6-chlorobenzotriazol-1-yl)-
N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate (HCTU;
71 mg, 0.171 mmol), and HOBt (26 mg, 0.171 mmol) in dry
CH2Cl2 (5 mL). The reaction mixture was stirred for 60 min at
room temperature under argon, before 6a (100 mg, 0.142 mmol),
dissolved in dry CH2Cl2/DMF (1:1, 10 mL), was added. The reac-
tion mixture was stirred at 40 °C for 15 h, diluted with CH2Cl2
(20 mL), washed with 1 m aq. HCl (2ϫ 15 mL), dried (MgSO4),
and concentrated in vacuo. Purification by RP-HPLC (Luna-PFP,
MeOH/H2O, 80:20Ǟ100:0, 20 min) afforded 8a as a colorless lyo-
philisate (103 mg, 0.099 mmol, 70%). Rf = 0.66 (EtOAc). Analyti-
cal RP-HPLC (Luna-PFP, MeOH/H2O, 80:20Ǟ100:0, 20 min): Rt
(m, 2 H, Kγ), 1.33–1.20 (m, 10 H, 3O–7O-H), 0.87 (t, J8O-H,7O-H
=
6.9 Hz, 3 H, 8O-H) ppm. 13C NMR (100.6 MHz, CDCl3, HSQC):
δ = 174.9 (C=Oamide), 156.6 (C=Ocarbamate), 136.8 (Cq-ar), 128.6,
128.2 (5ϫ CHar), 68.7 (CH2-ar), 55.2 (Kα), 40.8, 39.2 (Kε, C-1O),
34.8 (Kβ), 31.9 (C-6O), 29.9, 29.8 (Kδ, C-2O), 29.4, 29.3 (C-4O, C-
5O), 27.1 (C-3O), 23.0, 22.8 (Kγ, C-7O) 14.2 (C-8O) ppm. HRMS
(ESI-TOF): calcd. for C22H37N3O3H [M + H]+ 392.2913; found
392.2925.
1
= 18.0 min. [α]2D3 = –7.2 (c = 1.00, MeOH). H NMR (400 MHz,
Acyl chloride 4 (2.06 g, 4.22 mmol) was prepared according to a
known procedure[21] and added dropwise to H2N-Lys(Z)-O-octyl
(1.00 g, 2.55 mmol) and DIPEA (0.52 mL, 3.06 mmol) in dry
CD3OD, COSY): δ = 7.35–7.28 (m, 5 H, Har), 5.07 (s, 2 H, CH2-
ar), 4.37 (dd, JKα,Kaβ = 8.8, JKα,Kbβ = 6.1 Hz, 1 H, Kα), 3.69 (t,
JH3,H2 = 6.2 Hz, 2 H, 3OEG-H), 3.61–3.56 (m, 8 H, 5,6,8,9OEG-H),
CH2Cl2 (30 mL) at 0 °C. After stirring at room temperature for 3.53 (t, JH11,H12 = 5.5 Hz, 2 H, 11OEG-H), 3.30–3.28 (m, 2 H,
15 h, the solvent was removed in vacuo and the residue was purified
by flash chromatography (SiO2, cHex/EtOAc, 2:1) to provide 5a as
a colorless amorphous solid (1.80 g, 2.14 mmol, 84%). Rf = 0.71
(cHex/EtOAc, 1:1). Analytical RP-HPLC (Luna-PFP, MeOH/H2O,
80:20Ǟ100:0, 20 min): Rt = 18.7 min. [α]2D3 = –1.3 (c = 1.00,
12OEG-H), 3.24–3.10 (m, 4 H, 1O-H, Kε), 2.40 (t, JH2,H3 = 6.2 Hz,
2 H, 2OEG-H), 1.88–1.71 (m, 2 H, Kβ), 1.56–1.48 (m, 4 H, Kδ, 2O-
H), 1.40–1.33 (m, 2 H, Kγ), 1.32–1.29 (m, 10 H, 3O–7O-H), 0.89
(t, J8O-H,7O-H = 6.9 Hz, 3 H, 8O-H) ppm. 13C NMR (100.6 MHz,
CD3OD, HSQC):
δ = 173.9, 172.6 (2ϫ C=Oamide), 159.1
CHCl3). 1H NMR (300 MHz, CDCl3): δ = 7.46 [d, JNH,Kα
=
(C=Oamide(F)), 158.9 (C=Ocarbamate), 138.4 (Cq-ar), 129.5, 129.0,
7.4 Hz, 1 H, NHamide(F)], 7.39–7.29 (m, 5 H, Har), 6.13 (br. s, 1 H, 128.9 (5ϫ CHar), 71.6, 71.5, 71.3 (C-5,6,8,9OEG), 70.9 (C-11OEG),
NHamide), 5.08 (s, 2 H, CH2-ar), 4.88 (t, JNH,Kε = 5.9 Hz, 1 H, 68.3 (C-3OEG), 67.4 (CH2-ar), 55.5 (Kα), 41.8 (C-12OEG), 40.5, 40.0
NHcarbamate), 4.40 (dd, JKα,Kaβ = 12.7, JKα,Kbβ = 7.2 Hz, 1 H, Kα), (Kε, C-1O), 37.7 (C-2OEG), 33.0 (C-6O), 32.2 (Kβ), 30.4, 30.3, 29.9
3.31–3.11 (m, 4 H, Kε, 1O-H), 1.96–1.87 (m, 1 H, Kaβ), 1.81–1.71
(Kδ, C-2O, C-4O, C-5O), 27.9 (C-3O), 24.2 (Kγ), 23.7 (C-7O), 14.4
(m, 1 H, Kbβ), 1.57–1.47 (m, 4 H, Kδ, 2O-H), 1.38–1.30 (m, 2 H, (C-8O) ppm. 19F NMR (376.4 MHz, CD3OD): δ = –83.78 (t, JF,F
Kγ), 1.30–1.26 (m, 10 H, 3O–7O-H), 0.87 (t, J8O-H,7O-H = 6.6 Hz, 3
H, 8O-H) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 169.7 (C=O-
= 10.1 Hz, 3 F, CF3), –121.99 (t, JF,F = 12.8 Hz, 2 F), –123.87 (br.
s, 2 F), –124.25 (br. s, 4 F), –124.89 (br. s, 2 F), –125.14 (br. s, 2
amide), 156.9 (C=Ocarbamate), 136.6 (Cq-ar), 128.7, 128.3, 128.1 (5ϫ F), –128.64 to –128.73 (m, 2 F, CF2CF3) ppm. HRMS (ESI-TOF):
CHar), 66.9 (CH2-ar), 53.7 (Kα), 40.2, 40.0 (Kε, C-1O), 32.0, 31.9 calcd. for 1063.3490 C40H53F17N4O8Na [M
(Kβ, C-6O), 29.5 (Kδ, C-2O), 29.3 (C-4O, C-5O), 27.0 (C-3O), 22.7, 1063.3468.
22.1 (Kγ, C-7O) 14.2 (C-8O) ppm. 19F NMR (376.4 MHz, CDCl3),
δ = –81.12 (t, JF,F = 9.8 Hz, 3 F, CF3), –119.92 (m, 2 F), –121.86
(br. s, 2 F), –122.22 (br. s, 4 F), –122.69 (br. s, 2 F), –123.05 (br. s,
2 F), –126.45 (m, 2 F, CF2CF3) ppm. HRMS (ESI-TOF): calcd.
for C31H36F17N3O4H [M + H]+ 838.2513; found 838.2523.
+
Na]+; found
RF-Lys(OEG-NH2)-O-octyl (9a): Pd/C (10%, 10 mg) in a flask was
activated through three cycles of a vacuum/H2 flush. A solution of
8a (50 mg, 0.05 mmol) in iPrOH (10 mL) was then added and the
reaction mixture was stirred under H2 for 12 h. The catalyst was
removed by filtration through Hyflo Super Cel® and washed with
iPrOH (50 mL). The filtrate was concentrated in vacuo to give 9a
as a colorless amorphous solid (43 mg, 0.048 mmol, 99%). Rf =
0.05 (CH2Cl2/MeOH/NEt3, 19:1:0.1). [α]2D3 = –8.1 (c = 1.00,
RF-Lys-O-octyl (6a): Pd/C (10%, 200 mg) in a flask was activated
through three cycles of a vacuum/H2 flush. A solution of 5a (1.50 g,
1.79 mmol) in EtOH (25 mL) was then added and the reaction mix-
ture was stirred under H2 for 12 h. The catalyst was removed by
filtration through a Hyflo Super Cel® and washed with EtOH (4ϫ
50 mL). The filtrate was concentrated in vacuo to give 6a as a col-
orless viscous oil (1.25 g, 1.77 mmol, 99%). Rf = 0.06 (CH2Cl2/
MeOH). 1H NMR (300 MHz, CD3OD): δ = 4.37 (dd, JKα,Kaβ
8.8, JKα,Kbβ = 6.0 Hz, 1 H, Kα), 3.72 (t, JH3,H2 = 6.2 Hz, 2 H, 3OEG
H), 3.64–3.59 (m, 8 H, 5,6,8,9OEG-H), 3.56 (t, JH11,H12 = 5.2 Hz, 2
H, 11OEG-H), 3.22–3.12 (m, 4 H, 1O-H, Kε), 2.87 (t, JH12,H11
=
-
=
MeOH/NEt3, 19:1:0.1). Analytical RP-HPLC (Luna-PFP, MeCN/ 5.2 Hz, 2 H, 12OEG-H), 2.43 (t, JH2,H3 = 6.2 Hz, 2 H, 2OEG-H),
H2O, 20:80Ǟ100:0, 60 min): Rt = 25.3 min. [α]2D3 = –10.3 (c = 1.00,
MeOH). 1H NMR (300 MHz, CDCl3): δ = 6.70 (t, JNH,1OH
1.89–1.73 (m, 2 H, Kβ), 1.55–1.48 (m, 4 H, Kδ, 2O-H), 1.42–1.29
=
(m, 12 H, Kγ, 3O–7O-H), 0.90 (t, J8O-H,7O-H = 6.9 Hz, 3 H, 8O-
5.4 Hz, 1 H, NHamide), 4.46 (t, JKα,Ka/bβ = 6.7 Hz, 1 H, Kα), 3.30– H) ppm. 13C NMR (75.5 MHz, CD3OD): δ = 173.9, 172.6 (2ϫ
3.15 (m, 2 H, 1O-H), 2.81–2.63 (m, 2 H, Kε), 1.96–1.82 (m, 1 H, C=Oamide), 159.1 (C=Oamide(F)), 72.0 (C-11OEG), 71.6, 71.5, 71.3
Kaβ), 1.79–1.69 (m, 1 H, Kbβ), 1.57–1.44 (m, 4 H, Kδ, 2O-H), 1.44– (C-5,6,8,9OEG), 68.3 (C-3OEG), 55.5 (Kα), 41.7 (C-12OEG), 40.5, 40.0
1.35 (m, 2 H, Kγ), 1.30–1.23 (m, 10 H, 3O–7O-H), 0.86 (t, (Kε, C-1O), 37.6 (C-2OEG), 33.0 (C-6O), 32.2 (Kβ), 30.4, 30.3, 29.9
J8O-H,7O-H = 6.6 Hz, 3 H, 8O-H) ppm. 13C NMR (75.5 MHz, (Kδ, C-2O, C-4O, C-5O), 27.9 (C-3O), 24.2 (Kγ), 23.7 (C-7O) 14.4
CDCl3): δ = 169.8 (C=Oamide), 157.6 [t, 2J(C,F) = 26.5 Hz,
(C-8O) ppm. 19F NMR (376.4 MHz, CD3OD), δ = –81.15 (t, JF,F
Eur. J. Org. Chem. 2011, 3878–3887
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3883

T. Platen, T. Schüler, W. Tremel, A. Hoffmann-Röder
FULL PAPER
= 10.0 Hz, 3 F, CF3), –119.74 to –119.82 (m, 2 F), –121.90 (br. s,
2 F), –122.24 (br. s, 4 F), –122.62 (br. s, 2 F), –123.09 (br. s, 2
F), –126.49 (m, 2 F, CF2CF3) ppm. HRMS (ESI-TOF): calcd. for
C32H47F17N4O86H [M + H]+ 907.3302; found 907.3338.
1-H-, 8-H-Fmoc), 7.41–7.36 (m, 2 H, 3-H-, 6-H-Fmoc), 7.32–7.27
(m, 2 H, 2-H-, 7-H-Fmoc), 7.08–7.00 (m, 2 H, NHT, NHOEG), 6.87
(d, JNH,H2 = 8.7 Hz, 1 H, NHGalAc), 5.61 (d, JNH,Vα = 7.9 Hz, 1 H,
NHV), 5.33 (d, JH4,H5 = 3.0 Hz, 1 H, 4-H), 5.07 (dd, JH3,H2 = 10.9,
JH3,H4 = 3.1 Hz, 1 H, 3-H), 4.93 (d, JH1,H2 = 3.4 Hz, 1 H, 1-H),
4.57–4.49 (m, 2 H, 2-H, Tα), 4.46–4.35 (m, 2 H, CH2-Fmoc), 4.29–
4.20 (m, 3 H, 5-H, Tβ, 9-H-Fmoc), 4.07–3.98 (m, 3 H, Vα, 6a/b-
H), 3.70 (t, JCH2,CH2 = 6.5 Hz, 2 H, 3OEG-H), 3.60–3.56 (m, 8 H,
5,6,8,9OEG-H), 3.53–3.49 (m, 2 H, 11OEG-H), 3.47–3.38 (m, 2 H,
12OEG-H), 2.49 (t, JCH2,CH2 = 6.4 Hz, 2 H, 2OEG-H), 2.25–2.16 (m,
1 H, Vβ), 2.13 [s, 3 H, CH3(Ac)], 2.01 [s, 3 H, CH3(Ac)], 1.93 [s, 3
H, CH3(Ac)], 1.91 [s, 3 H, CH3(Ac)], 1.43 [s, 9 H, C(CH3)3], 1.24
(d, JTγ,Tβ = 6.2 Hz, 3 H, Tγ), 0.99 (d, JVγ,Vβ = 6.8 Hz, 3 H, Vγ), 0.97
(d, JVγ,Vβ = 6.8 Hz, 3 H, Vγ) ppm. 13C NMR (75.5 MHz, CDCl3): δ
= 172.0, 171.1, 170.8, 170.5, 169.9 (7ϫ C=O), 156.4 (C=Ocarbamate),
144.0 (C-1a-, C-8a-Fmoc), 141.4 (C-4a-, C-5a-Fmoc), 127.9 (C-3-,
C-6-Fmoc), 127.2 (C-2-, C-7-Fmoc), 125.3, 125.2 (C-1-, C-8-
Fmoc), 120.1 (C-4-, C-5-Fmoc), 99.9 (C-1), 80.8 (Cq-tBu), 77.3
(Tβ), 70.6, 70.5, 70.4, 70.3 (C-5,6,8,9OEG), 69.4 (C-11OEG), 68.9 (C-
3), 67.5, 67.3, 67.2 (C-4, C-5, CH2-Fmoc), 67.0 (C-3OEG), 62.2 (C-
6), 61.1 (Vα), 56.7 (Tα), 47.8, 47.3 (C-2, C-9-Fmoc), 39.6 (C-12OEG),
36.3 (2OEG-H), 30.7 (Vβ), 28.2 [C(CH3)3], 23.1, 20.9, 20.7 [4ϫ
CH3(Ac)], 19.5 (2ϫ Vγ), 18.1 (Tγ) ppm. HRMS (ESI-TOF): calcd.
for C51H72N4O18Na [M + Na]+ 1051.4739; found 1051.4749.
Fmoc-Thr(αAc3GalNAc)-OEG-OtBu (12): The active ester was pre-
pared by adding NMM (0.8 mL, 7.26 mmol) to a solution of
Fmoc-Thr(αAc3GalNAc)-OH (10; 2.44 g, 3.63 mmol), HCTU
(1.64 g, 3.96 mmol), and HOBt (0.61 g, 3.96 mmol) in dry CH2Cl2
(35 mL). The reaction mixture was stirred at room temperature un-
der argon for 60 min and H2N-OEG-CO2tBu (11; 0.916 g,
3.30 mmol), dissolved in dry CH2Cl2 (15 mL), was added. The re-
action mixture was stirred at room temperature for 15 h, diluted
with CH2Cl2 (50 mL), washed with 1 m aq. HCl (2ϫ 50 mL), dried
(MgSO4), and concentrated in vacuo. Flash chromatography (SiO2,
CH2Cl2/MeOH, 39:1) afforded 12 as
a yellow oil (2.96 g,
3.18 mmol, 96%). Rf = 0.39 (CH2Cl2/MeOH, 39:1). Analytical RP-
HPLC (Jupiter C18, CH3CN/H2O, 50:50, 5 min, then
50:50Ǟ100:0, 25 min): Rt = 22.9 min. [α]2D3 = 36.5 (c = 1.00,
1
CHCl3). H NMR (300 MHz, CDCl3): δ = 7.75 (d, JH4,H3(Fmoc)
=
JH5,H6(Fmoc) = 7.6 Hz, 2 H, 4-H-, 5-H-Fmoc), 7.63 (d, JH1,H2(Fmoc)
= JH8,H7(Fmoc) = 7.4 Hz, 2 H, 1-H-, 8-H-Fmoc), 7.41–7.36 (m, 2 H,
3-H-, 6-H-Fmoc), 7.34–7.27 (m, 2 H, 2-H-, 7-H-Fmoc), 7.12 (t,
JNH,CH2 = 5.0 Hz, 1 H, NHOEG), 6.62 (d, JNH,H2 = 9.4 Hz, 1 H,
NHGalNAc), 5.90 (d, JNH,Tα = 9.1 Hz, 1 H, NHT), 5.39 (d, JH4,H5
=
2.2 Hz, 1 H, 4-H), 5.06 (dd, JH3,H2 = 11.4, JH3,H4 = 3.0 Hz, 1 H,
3-H), 4.91 (d, JH1,H2 = 3.4 Hz, 1 H, 1-H), 4.60–4.53 (m, 1 H, 2-H),
Ac-Val-Thr(αAc3GalNAc)-OEG-OtBu (14): Piperidine (1.35 mL,
13.6 mmol) was added to a solution of 13 (2.00 g, 1.94 mmol) in
4.46–4.38 (m, 2 H, CH2-Fmoc), 4.35–4.16 (m, 4 H, 5-H, Tα, Tβ, 9- CH2Cl2 (30 mL) and the solution was stirred at room temperature
H-Fmoc), 4.10–4.01 (m, 2 H, 6a/b-H), 3.67 (t, JCH2,CH2 = 6.4 Hz,
for 5 h, concentrated in vacuo, and co-evaporated with toluene (3ϫ
2 H, 3OEG-H), 3.60–3.57 (m, 8 H, 5,6,8,9OEG-H), 3.55–3.51 (m, 2 20 mL). The crude product was dissolved in dry CH2Cl2 (30 mL)
H, 11OEG-H), 3.47–3.42 (m, 2 H, 12OEG-H), 2.47 (t, JCH2,CH2
6.4 Hz, 2 H, 2OEG-H), 2.15 [s, 3 H, CH3(Ac)], 2.01 [s, 6 H, 2ϫ
CH3(Ac)], 1.97 [s, 3 H, CH3(Ac)], 1.41 [s, 9 H, C(CH3)3], 1.29 (d,
=
and successively treated with HOBt (89 mg, 0.583 mmol), DIPEA
(0.64 mL, 3.89 mmol), and Ac2O (1.29 mL, 13.60 mmol). After
stirring at room temperature for 15 h, the organic phase was
JTγ,Tβ = 6.2 Hz, 3 H, Tγ) ppm. 13C NMR (75.5 MHz, CDCl3): δ = washed with 1 m aq. HCl (2ϫ 15 mL), dried (MgSO4), and concen-
171.2, 171.1, 170.9, 170.5, 170.4, 170.3 (6ϫ C=O), 156.8 trated in vacuo. The residue was purified by flash chromatography
(C=Ocarbamate), 143.8 (C-1a-, C-8a-Fmoc), 141.4 (C-4a-, C-5a- (SiO2, CH2Cl2/MeOH, 39:1Ǟ19:1) to give 14 as an yellow
Fmoc), 127.9 (C-3-, C-6-Fmoc), 127.2 (C-2-, C-7-Fmoc), 125.3 (C-
amorphous solid (1.10 g, 1.30 mmol, 67%). Rf = 0.33 (CH2Cl2/
1-, C-8-Fmoc), 120.1 (C-4-, C-5-Fmoc), 100.2 (C-1), 80.8 (Cq-tBu), MeOH, 19:1). Analytical RP-HPLC (Jupiter C18, CH3CN/H2O,
78.1 (Tβ), 70.5, 70.4, 70.3, 70.2 (C-5,6,8,9OEG), 69.4 (C-11OEG), 69.0
(C-3), 67.5, 67.4, 67.3 (C-4, C-5, CH2-Fmoc), 66.9 (C-3OEG), 62.2
50:50, 5 min, then 50:50Ǟ100:0, 25 min): Rt = 12.4 min. [α]2D3
38.1 (c = 1.00, CHCl3). H NMR (400 MHz, CDCl3, COSY): δ =
=
1
(C-6), 58.6 (Tα), 47.6, 47.3 (C-2, C-9-Fmoc), 39.6 (C-12OEG), 36.3 7.27 (d, JNH,Tα = 8.0 Hz, 1 H, NHT), 7.13 (t, JNH,CH2 = 5.0 Hz, 1
(C-2OEG), 28.2 [C(CH3)3], 23.1, 20.9, 20.8, 20.7 [4ϫ CH3(Ac)], 18.0 H, NHOEG), 6.87 (d, JNH,H2 = 9.2 Hz, 1 H, NHGalNAc), 6.60 (d,
(Tγ) ppm. HRMS (ESI-TOF): calcd. for C46H63N3O17Na
[M + Na]+ 952.4055; found 952.4088.
JNH,Vα = 7.9 Hz, 1 H, NHV), 5.35 (d, JH4,H5 = 2.3 Hz, 1 H, 4-H),
5.08 (dd, JH3,H2 = 11.4, JH3,H4 = 3.2 Hz, 1 H, 3-H), 4.94 (d, JH1,H2
= 3.6 Hz, 1 H, 1-H), 4.55–4.48 (m, 2 H, 2-H {4.53}, Tα {4.50}),
4.31–4.25 (m, 2 H, 5-H {4.30}, Tβ {4.26}), 4.22 (pseudo-t, JVα,NH
= JVα,Vβ = 7.7 Hz, 1 H, Vα), 4.09–4.00 (m, 2 H, 6a/b-H), 3.69 (t,
Fmoc-Val-Thr(αAc3GalNAc)-OEG-OtBu (13): A solution of 12
(5.62 g, 6.04 mmol) in CH2Cl2 (80 mL) was treated with piperidine
(4.19 mL, 42.3 mmol) and stirred at room temperature for 14 h.
The solution was diluted with toluene (80 mL) and concentrated in
vacuo. The residue was purified by flash chromatography [SiO2,
CH2Cl2/MeOH/NEt3, 19:1:0.1. Rf = 0.36 (CH2Cl2/MeOH/NEt3,
19:1:0.1)], dissolved in dry CH2Cl2 (25 mL), and added to the
freshly prepared active ester of Fmoc-Val [obtained by treatment
of Fmoc-Val (2.25 g, 6.64 mmol) with HCTU (3.00 g, 7.25 mmol),
HOBt (1.11 g, 7.25 mmol), and NMM (1.46 mL, 13.29 mmol) in
dry CH2Cl2 (100 mL)]. The resulting reaction mixture was stirred
at room temperature for 15 h, diluted with CH2Cl2 (100 mL),
washed with 1 m aq. HCl (2ϫ 100 mL), dried (MgSO4), and con-
centrated in vacuo. Flash chromatography (SiO2, CH2Cl2/MeOH,
39:1) yielded 13 as a brown amorphous solid (4.72 g, 4.59 mmol,
76%). Rf = 0.11 (CH2Cl2/MeOH, 39:1). Analytical RP-HPLC (Jup-
iter C18, CH3CN/H2O, 50:50, 5 min, then 50:50Ǟ100:0, 25 min):
Rt = 23.5 min. [α]2D3 = 28.3 (c = 1.00, CHCl3). 1H NMR (300 MHz,
JCH2,CH2 = 6.4 Hz, 2 H, 3OEG-H), 3.60–3.56 (m, 8 H, 5,6,8,9OEG
-
H), 3.55–3.47 (m, 2 H, 11OEG-H), 3.46–3.38 (m, 2 H, 12OEG-H),
2.49 (t, JCH2,CH2 = 6.4 Hz, 2 H, 2OEG-H), 2.20–2.14 (m, 1 H, Vβ),
2.13 [s, 3 H, CH3(Ac)], 2.03 [s, 3 H, CH3(Ac)], 2.00 [s, 6 H, 2ϫ
CH3(Ac)], 1.95 [s, 3 H, CH3(Ac)], 1.42 [s, 9 H, C(CH3)3], 1.22 (d,
JTγ,Tβ = 6.4 Hz, 3 H, Tγ), 0.97 (d, JVγ,Vβ = 6.4 Hz, 3 H, Vγ), 0.95
(d, JVγ,Vβ = 6.4 Hz, 3 H, Vγ) ppm. 13C NMR (100.6 MHz, CDCl3,
HSQC): δ = 174.2, 172.1, 171.3, 171.2, 170.8, 170.7, 170.5,
169.9 (8ϫ C=O), 99.6 (C-1), 80.9 (Cq-tBu), 76.6 (Tβ), 70.5, 70.4,
70.3, (C-5,6,8,9OEG), 69.5 (C-11OEG), 68.7 (C-3), 67.5 (C-4), 67.2
(C-5), 66.4 (C-3OEG), 62.3 (C-6), 59.6 (Vα), 56.9 (Tα), 47.8 (C-2),
39.6 (C-12OEG), 36.3 (C-2OEG), 30.1 (Vβ), 28.2 [C(CH3)3], 23.1,
20.9, 20.8 [5ϫ CH3(Ac)], 19.5 (Vγ), 18.4 (Vγ), 18.2 (Tγ) ppm.
HRMS (ESI-TOF): calcd. for C38H64N4O17Na [M
871.4164; found 871.4149.
+
Na]+
CDCl3): δ = 7.75 (d, JH4,H3(Fmoc) = JH5,H6(Fmoc) = 7.5 Hz, 2 H, 4- Ac-Val-Thr(αAc3GalNAc)-OEG-OH (15): TFA (10.0 mL) was
H-, 5-H-Fmoc), 7.60 (d, JH1,H2(Fmoc) = JH8,H7(Fmoc) = 7.4 Hz, 2 H, added to a solution of 14 (1.00 g, 1.18 mmol) and anisole (1.0 mL)
3884
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3878–3887

Amphiphilic MUC1 Glycopeptide Antigens
in CH2Cl2 (30 mL) and the solution was stirred at room tempera-
ture for 15 h. The solvent was removed in vacuo and the residue
was co-evaporated with toluene (2ϫ 20 mL). Purification by flash
chromatography (SiO2, CH2Cl2/MeOH/HOAc, 19:1:0.1) yielded 15
(Vα), 57.5 (Tα), 54.8 (Kα), 48.2 (C-2), 40.1 (C-12OEG), 40.0 (C-1O),
39.2 (Kε), 37.5 (C-2OEG), 32.5 (C-6O), 32.2 (Kβ), 31.1 (Vβ), 27.9 (C-
3O), 23.4 (Kγ), 23.3 (C-7O), 22.9, 22.7 [2ϫ CH3(NHAc)], 20.7, 20.6,
20.5 [3ϫ CH3(Ac)], 19.9 (Vγ), 19.0 (Tγ), 18.6 (Vγ), 14.3 (C-
as a brown oil (889 mg, 1.12 mmol, 95%). Rf = 0.07 (CH2Cl2/ 8O) ppm. 19F NMR [376.4 MHz, (CD3)2O]: δ = –81.96 (t, JF,F
=
MeOH, 19:1). Analytical RP-HPLC (Jupiter C18, CH3CN/H2O, 9.8 Hz, 3 F, CF3), –119.90 to –120.20 (m, 2 F), –122.35 (br. s, 2 F),
50:50, 5 min, then 50:50Ǟ100:0, 25 min): Rt = 4.5 min. [α]2D3 = 36.0
(c = 1.00, MeOH). 1H NMR (400 MHz, CDCl3, COSY): δ = 7.72–
–122.70 (br. s, 4 F), –123.22 (br. s, 2 F), –123.57 (br. s, 2 F), –126.99
to –127.09 (m, 2 F, CF2CF3) ppm. HRMS (ESI-TOF): calcd. for
7.68 (m, 1 H, NHOEG), 7.54 (d, JNH,Tα = 9.0 Hz, 1 H, NHT), 7.07 C57H84F17N7O18Na [M + Na]+ 1500.5499; found 1500.5535.
(d, JNH,Vα = 9.1 Hz, 1 H, NHGalNAc), 6.56 (d, JNH,H2 = 8.3 Hz, 1
Ac-Val-Thr(αGalNAc)-OEG-RF-Lys-O-octyl (17a): NaOMe (2.5%
H, NHV), 5.35 (d, JH4,H5 = 2.5 Hz, 1 H, 4-H), 5.10 (dd, JH3,H2
=
in MeOH) was added dropwise to a solution of 16a (64 mg,
0.043 mmol) in MeOH (HPLC grade, 20 mL) until a pH of 9.5 was
reached. After stirring for 18 h at room temperature, the solution
was neutralized with HOAc and concentrated in vacuo. Purifica-
tion by RP-HPLC (Luna-PFP, MeOH/H2O, 80:20Ǟ100:0, 20 min)
afforded 17a as a colorless lyophilisate (23 mg, 0.017 mmol, 39%).
Analytical RP-HPLC (Luna-PFP, MeOH/H2O, 80:20Ǟ100:0,
11.4, JH3,H4 = 3.2 Hz, 1 H, 3-H), 4.92 (d, JH1,H2 = 3.5 Hz, 1 H, 1-
H), 4.66 (dd, JTα,NH = 8.9, JTα,Tβ = 1.9 Hz, 1 H, Tα), 4.57–4.50 (m,
1 H, 2-H), 4.41 (pt, JVα,NH = JVα,Vγ = 8.0 Hz, 1 H, Vα), 4.31–4.27
(m, 2 H, Tβ {4.31}, 5-H {4.29}), 4.06–4.04 (m, 2 H, 6a/b-H), 3.76–
3.71 (m, 2 H, 3OEG-H), 3.62–3.56 (m, 8 H, 5,6,8,9OEG-H), 3.50–
3.43 (m, 4 H, 11OEG-H {3.49}, 12OEG-H {3.44}), 2.62 (t, JCH2,CH2
= 5.4 Hz, 2 H, 2OEG-H), 2.14 [s, 3 H, CH3(Ac)], 2.12–2.08 (m, 1
H, Vβ), 2.05 [s, 3 H, CH3(Ac)], 2.03 [s, 3 H, CH3(Ac)], 2.01 [s, 3
H, CH3(Ac)], 1.95 [s, 3 H, CH3(Ac)], 1.26 (d, JTγ,Tβ = 6.4 Hz, 3 H,
Tγ), 0.97 (d, JVγ,Vβ = 6.8 Hz, 3 H, Vγ), 0.95 (d, JVγ,Vβ = 6.8 Hz, 3
H, Vγ) ppm. 13C NMR (100.6 MHz, CDCl3, HSQC): δ = 175.1,
172.5, 171.4, 171.3, 170.8, 170.7, 170.6, 169.9 (8ϫ C=O), 99.8 (C-
1), 77.0 (Tβ), 70.7, 70.6, 70.2 (C-5,6,8,9OEG), 69.7 (C-11OEG), 68.8
(C-3), 67.7 (C-4), 67.2 (C-5), 66.7 (C-3OEG), 62.3 (C-6), 59.2 (Vα),
57.4 (Tα), 47.8 (C-2), 39.7 (C-12OEG), 35.0 (C-2OEG), 30.8 (Vβ),
23.2, 23.1, 20.9, 20.8 [5ϫ CH3(Ac)], 19.5 (Vγ), 18.4 (Vγ), 18.2
(Tγ) ppm. HRMS (ESI-TOF): calcd. for C34H56N4O17Na
[M + Na]+ 815.3538; found 815.3541.
20 min): Rt = 14.4 min. [α]2D3 = 24.2 (c = 1.00, MeOH). H NMR
1
(400 MHz, CD3OD, COSY): δ = 4.88 (s, 10 H, all OH and NH),
4.84 (d, JH1,H2 = 3.8 Hz, 1 H, 1-H), 4.52 (d, JTα,Tβ = 2.3 Hz, 1 H,
Tα), 4.37 (dd, JKα,Kaβ = 8.8, JKα,Kbβ = 6.1 Hz, 1 H, Kα), 4.26–4.22
(m, 2 H, Vα {4.24}, 2-H {4.24}), 4.20–4.14 (m, 1 H, Tβ), 3.91–3.89
(m, 2 H, 5-H {3.90}, 4-H {3.90}), 3.79 (dd, JH3,H2 = 10.9, JH3,H4
= 3.0 Hz, 1 H, 3-H), 3.74–3.70 (m, 4 H, 3OEG-H {3.73}, 6a/b-H
{3.71}), 3.64–3.57 (m, 8 H, 5,6,8,9OEG-H), 3.55–3.51 (m, 2 H,
11OEG-H), 3.31 (m, 2 H, 12OEG-H), 3.20–3.13 (m, 4 H, 1O-H, Kε),
2.43 (t, JCH2,CH2
= 6.3 Hz, 2 H, 2OEG-H), 2.08 [s, 3 H,
CH3(NHAc)], 2.12–2.07 (m, 1 H, Vβ), 2.01 [s, 3 H, CH3(NHAc)],
1.87–1.72 (m, 2 H, Kβ), 1.57–1.41 (m, 4 H, 2O-H, Kδ), 1.38–1.35
(m, 2 H, Kγ), 1.30 (m, 10 H, 3O–7O-H), 1.26 (d, JTγ,Tβ = 6.4 Hz, 3
H, Tγ), 1.00 (d, JVγ,Vβ = 6.8 Hz, 3 H, Vγ), 0.99 (d, JVγ,Vβ = 6.8 Hz,
3 H, Vγ), 0.90 (t, JCH3,CH2 = 6.9 Hz, 3 H, 8O-H) ppm. 13C NMR
(100.6 MHz, CD3OD, HSQC): δ = 174.3, 174.1, 173.9, 173.5,
172.6, 171.9 (6ϫ C=O), 100.9 (C-1), 78.2 (Tβ), 72.9 (C-5 or C-4),
71.5, 71.4, 71.3, 71.2 (C-5,6,8,9OEG), 70.4 (C-3, C-11OEG), 70.3 (C-
4 or C-5), 68.3 (C-3OEG), 62.7 (C-6), 60.7 (Vα), 58.0 (Tα), 55.5 (Kα),
51.5 (C-2), 40.5, 40.5, 40.0 (C-12OEG, C-1O, Kε), 37.7 (C-2OEG),
33.0 (C-6O), 32.2 (Kβ), 31.3 (Vβ), 30.4, 30.4, 30.3, 29.9 (C-2O, Kδ,
C-4O, C-5O), 27.4 (C-3O), 24.2 (Kγ), 23.7 (C-7O), 23.2, 22.4 [2ϫ
CH3(NHAc)], 19.9 (Vγ), 19.3 (Tγ), 19.0 (Vγ), 14.4 (C-8O) ppm. 19F
NMR (376.4 MHz, CD3OD): δ = –82.69 (t, JF,F = 9.8 Hz, 3 F,
CF3), –120.88 (t, JF,F = 12.6 Hz, 2 F), –122.78 (br. s, 2 F), –123.18
(br. s, 4 F), –123.80 (br. s, 2 F), –124.06 (br. s, 2 F), –127.55 to
–127.63 (m, 2 F, CF2CF3) ppm. HRMS (ESI-TOF): calcd. for
C51H78F17N7O15Na [M + Na]+ 1374.55182; found 1374.5153.
Ac-Val-Thr(αAc3GalNAc)-OEG-RF-Lys-O-octyl (16a): A solution
of 15 (100 mg, 0.126 mmol), HCTU (57 mg, 0.138 mmol), and
HOBt (221 mg, 0.138 mmol) in dry CH2Cl2 (5 mL) was treated
with NMM (30 μL, 0.273 mmol) and stirred for 60 min at room
temperature under argon. Compound 6a (81 mg, 0.115 mmol), dis-
solved in dry CH2Cl2/DMF (1:1, 10 mL), was added to this solu-
tion and stirring was continued at 40 °C for 15 h. The reaction
mixture was diluted with CH2Cl2 (20 mL), washed with 1 m aq.
HCl (2ϫ 15 mL), dried (MgSO4), and concentrated in vacuo. Puri-
fication by RP-HPLC (Luna-PFP, MeOH/H2O, 80:20Ǟ100:0,
20 min) afforded 16a as a colorless lyophilisate (64 mg, 0.043 mmol,
38%).
Analytical
RP-HPLC
(Luna-PFP,
MeOH/H2O,
80:20Ǟ100:0, 20 min): Rt = 16.9 min. [α]2D3 = 10.5 (c = 1.00,
MeOH). 1H NMR [400 MHz, (CD3)2O, COSY]: δ = 8.71 (d,
JNH,CH = 7.8 Hz, 1 H, NHK), 7.74–7.69 (m, 2 H, NHOEG, NHT),
7.61–7.56 (m, 1 H, NHV), 7.52 (t, JNH,CH2 = 5.0 Hz, 1 H, NHO),
7.34 (t, JNH,CH2 = 5.3 Hz, 1 H, NHK(ε)), 7.00 (d, JNH,H2 = 9.4 Hz,
1 H, NHGalNAc), 5.36 (d, JH4,H5 = 2.2 Hz, 1 H, 4-H), 5.04 (dd,
JH3,H2 = 11.5, JH3,H4 = 3.2 Hz, 1 H, 3-H), 4.99 (d, JH1,H2 = 3.6 Hz,
Glycolipopeptide 19: Starting from Fmoc-Pro-Trt-TentaGel S resin
(455 mg, 0.22 mmolg–1, 0.10 mmol), the solid phase glycopeptide
synthesis was conducted on a Perkin–Elmer ABI 433A peptide syn-
1 H, 1-H), 4.60 (dd, JTα,NH = 9.0, JTα,Tβ = 2.1 Hz, 1 H, Tα), 4.50– thesizer according to the Fastmoc protocol. In every coupling cycle,
4.29 (m, 5 H, Kα {4.45}, 2-H {4.43}, Tβ {4.39}, 5-H {4.36}, Vα
{4.30}), 4.13–4.03 (m, 2 H, 6a/b-H), 3.73 (t, JCH2,CH2 = 6.1 Hz, 2 with a solution of piperidine (20%) in NMP for at least 3ϫ
H, 3OEG-H), 3.60–3.57 (m, 8 H, 5,6,8,9OEG-H), 3.55–3.49 (m, 3 H, 2.5 min. The coupling of the amino acids (1 mmol or 10 equiv.
11OEG-H, 12OEG-H), 3.30–3.13 (m, 5 H, 1O-H, Kε, 12OEG-H), 2.41– based on the loaded resin) was carried out with O-(1H-benzotria-
2.39 (m, 2 H, 2OEG-H), 2.14–2.09 (m, 1 H, Vβ), 2.11 [s, 3 H, zol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium
hexafluorophosphate
CH3(NHAc)], 1.98 [s, 6 H, CH3(NHAc), CH3(Ac)], 1.90–1.80 (m, (HBTU, 1 mmol), HOBt (1 mmol), and DIPEA (2 mmol) in DMF
2 H, Kβ), 1.90, 1.85 (s, 6 H, OAc), 1.53–1.41 (m, 4 H, Kδ, 2O-H),
under 20–30 min of vortex. After every coupling step, unreacted
1.45–1.36 (m, 2 H, Kγ), 1.32–1.25 (m, 13 H, 3O–7O-H, Tγ), 0.96 (d, amino groups were capped by treatment with a mixture of Ac2O
the N-terminal Fmoc group was removed by treatment of the resin
JVγ,Vβ = 6.9 Hz, 3 H, Vγ), 0.94 (d, JVγ,Vβ = 6.9 Hz, 3 H, Vγ), 0.87
(t, JCH3,CH2 = 6.0 Hz, 3 H, 8O-H) ppm. 13C NMR [100.6 MHz,
(CD3)2O, HSQC]: δ = 172.8, 171.7, 171.1, 170.9, 170.8, 170.7,
170.6, 170.3 (9ϫ C=O), 157.7 (t, 2J = 26.1 Hz, C=Oamide(F)), 100.3
(C-1), 77.6 (Tβ), 71.2, 71.1, 70.9 (C-5,6,8,9OEG), 70.1 (C-11OEG),
69.5 (C-3), 68.4 (C-4), 68.0 (C-3OEG), 67.9 (C-5), 62.9 (C-6), 59.3
(0.5 m), DIPEA (0.125 m), and HOBt (0.015 m) in NMP (10 min
vortex). Coupling of the glycosylated threonine building block 10
(168 mg, 0.25 mmol) was performed using O-(7-azabenzotriazol-1-
yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate (HATU;
105 mg, 0.275 mmol), N-hydroxy-7-azabenzotriazole (HOAt,
36 mg, 0.275 mmol), and NMM (45 μL, 0.44 mmol) in N-methyl-
Eur. J. Org. Chem. 2011, 3878–3887
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3885

T. Platen, T. Schüler, W. Tremel, A. Hoffmann-Röder
FULL PAPER
pyrrolidone (NMP, 2.0 mL) for activation (8 h vortex). After at-
tachment of the remaining five amino acids and the spacer 7a
(1 mmol, 10 equiv. based on the loaded resin) using the standard
coupling procedure, again, the N-terminal Fmoc group was re-
moved by piperidine (20%) in NMP. The coupling of amphiphile
18 (164 mg, 0.20 mmol) required the use of HATU (84 mg,
0.22 mmol), HOAt (30 mg, 0.22 mmol), and NMM (45 μL,
0.44 mmol) in NMP (2.0 mL) under extended coupling times of
8 h. Detachment from the resin and simultaneous removal of all
side-chain protecting groups was performed in a Merrifield glass
reactor by shaking with TFA (10 mL), triisopropylsilane (TIS,
1.0 mL), and H2O (1.0 mL) for 3 h. The solution was filtered and
the resin was washed with TFA (3ϫ5 mL) and CH2Cl2 (3ϫ5 mL).
The combined TFA solutions were concentrated in vacuo and lyo-
philized. The crude product was dissolved in MeOH (HPLC-grade,
40 mL) and treated dropwise with NaOMe (2.5% in MeOH) until
a pH of 9.5 was reached. After stirring at room temperature for
18 h, the solution was neutralized with HOAc and concentrated in
vacuo. The crude product was precipitated twice from cold Et2O
(40 mL) to furnish 19 (110 mg, 0.036 mmol, 36%) as a colorless
foam after lyophilization. Analytical RP-HPLC (Luna-PFP,
MeOH/H2O, 70:30Ǟ100:0, 20 min): Rt = 15.4 min. [α]2D3 = –52.7
(m, 2 F, CF2CF3) ppm. MS (MALDI-TOF; DHB, positive): calcd.
for C124H189F17N30O42H [M + H]+ 3094.34; found 3094.08.
Surface Plasmon Resonance Spectroscopy: SPR was conducted on
a customized Kretschmann-type spectrometer. For this purpose,
glass slides were cleaned with piranha solution and water prior to
sequentially sputtering with 1.5 nm chromium and 55 nm gold
films. The slides were rinsed with doubly deionized H2O and PBS
buffer (pH 7.5), attached to the SPR spectrometer, and immersed
in PBS buffer. The kinetic measurements were started with injec-
tion of the SM3 antibody or amphiphilic MUC1 antigen into the
measurement chamber, both at a concentration of 2 μgmL–1 in
PBS. All measurements were repeated three times.
Quartz Crystal Microbalance: QCM measurements were performed
on a Qsense© D3000 microbalance. Quartz crystals with a reso-
nance frequency of 5 MHz and with a 100 nm thick gold layer were
used from Qsense. All measurements were made at T = 25 °C. The
crystal was attached to the measurement chamber and immersed
in PBS buffer. The respective antibody or antigen PBS solution
(2 μgmL–1) was injected upon a constant frequency shift, followed
by washing steps until the frequency shifts remained constant. All
measurements were repeated three times.
1
(c = 1.00, H2O + TFA). H NMR (400 MHz, CD3OD + [D1]TFA,
COSY): δ = 8.79 (d, JHε,Hδ = 1.3 Hz, 1 H, Hε), 7.43 (s, 1 H, Hδ),
5.02 (d, JH1,H2 = 3.6 Hz, 1 H, 1-H), 4.71–4.23 (m, 22 H, Rα {4.69},
Supporting Information (see footnote on the first page of this arti-
cle): Experimental procedures for compounds 3b, 5b, 6b–9b, 16b,
17b, and 18 as well as the 1H and 19F NMR spectra of all new
compounds.
α
α
α
α
Hα {4.67}, Dα {4.62}, A4 {4.61}, A(2,3) {4.57}, S1 {4.52}, S2
{4.47}, Kα {4.40}, TTnα, Vα {4.39}, P(1-5) {4.36}, T(1,2) {4.35,
4.32}, A1 {4.31}, TTn {4.30}, T2 {4.29}, T1 {4.25}), 4.18 (dd,
JH2,H3 = 3.6, JH2,H1 = 3.6 Hz, 1 H, 2-H), 4.09–4.04 (m, 5 H, G1
{4.08}, 2ϫ -CqCH2O- {4.05}), 3.98–3.56 (m, 34 H, G1αb {3.96}, 5-
α
α
α
β
β
β
αa
Acknowledgments
β
δ
This work was supported by the Deutsche Forschungsgemeinschaft
(DFG) (Emmy Noether Fellowship to A. H.-R.) and the Fonds der
Chemischen Industrie (Kekulé fellowship to T. P.). We thank Prof.
Dr. Horst Kunz (University of Mainz) for fruitful discussions and
generous support of the work.
H {3.94}, S2 {3.89}, 3-H {3.83}, 6a/b-H {3.81}, P(1-2) {3.80},
α
β
δ
G2 {3.79}, S1 {3.77}, 4-H {3.69}, 3OEG-H {3.68}, P(3-5) {3.65},
5,6,8,9OEG-H {3.62}, 11OEG-H {3.58}), 3.45 (t, JCH2,CH2 = 5.3 Hz,
2 H, 12OEG-H), 3.39 (d, JHβ,Hα = 5.1 Hz, 1 H, Hβa), 3.28–3.14 (m,
8 H, Kε {3.24}, Hβb {3.21}, Rδ {3.20}, 1O-H {3.19}), 2.95–2.92 (m,
3 H, Dβa {2.94}, 2OEG-H {2.93}), 2.72–2.68 (m, 1 H, Dβb), 2.29–
1.83 (m, 25 H, P(1-5) {2.25}, Vβ (2.13), CH3(NHAc) {2.08},
βa
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γ
βb
P(1-5) {2.03, 1.99}, P(1-5) {1.97}, Rβa {1.90}), 1.82–1.77 (m, 2 H,
Kβ), 1.74–1.68 (m, 3 H, Rβb {1.72}, Rγ {1.68}), 1.61–1.55 (m, 2 H,
Kδ), 1.53–1.45 (m, 2 H, 2O-H), 1.42 (d, JA2/3β,A2/3α = 6.9 Hz, 6 H,
A(2,3)β), 1.38–1.26 (m, 21 H, A(1,4) {1.37}, Kγ {1.35}, 3O–7O-H
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{1.29}, TTnγ {1.27}), 1.20 (d, JT1/2γ,T1/2β = 6.4 Hz, 6 H, T(1,2)γ), 1.00
(d, JVγa,Vβ = 6.5 Hz, 3 H, Vγa), 0.98 (d, JVγb,Vβ = 6.6 Hz, 3 H, Vγb),
0.89 (t, JCH3,CH2
= 6.9 Hz, 3
H, 8O-H) ppm. 13C NMR
(100.6 MHz, CD3OD + [D1]TFA, HSQC, HMBC): δ = 174.1,
174.0, 173.9, 173.2, 173.0, 172.9, 172.7, 172.4, 172.3, 172.2, 172.1,
171.7, 171.6, 171.4, 171.2, 171.0, 170.7, 170.6, 170.5, 170.2 (26ϫ
C=O), 157.1 (C=NH), 133.7 (Hε), 129.4 (Hγ), 117.5 (Hδ), 99.1 (C-
1), 76.4 (TTnβ), 71.4 (C-5), 70.0 (2ϫ -CqCH2O-), 69.9 (C-3), 69.8
(C-4), 69.6 (C-5,6,8,9OEG), 69.1 (C-11OEG), 69.0 (C-3OEG), 66.9,
66.4 (T(1,2)β), 61.9 (S1β), 61.7 (C-6), 61.6 (S2β), 61.6, 61.5, 61.4, 61.1,
60.9 (P(1-5)α), 59.4 (T2α), 59.1 (Vα), 59.0 (T1α), 58.8 (TTnα), 55.8
(S2α), 55.2 (S1α), 54.2 (Kα), 52.4 (Rα), 50.8 (Hα), 50.5 (Dα), 50.0 (C-
2), 42.4 (G2α), 42.4 (G1α), 40.7 (Kε), 39.1 (C-1O), 38.4 (C-12OEG),
34.4 (C-2OEG), 34.3 (Dβ), 31.5 (C-6O), 30.8 (Kβ), 30.0 (Vβ), 29.4,
29.2 (2ϫ P(1-5)β), 29.1, 29.0 (C-4O, C-5O), 28.9, 28.8 (3ϫ P(1-5)β),
28.7 (C-2O), 28.5 (Kδ), 27.8 (Rβ), 26.5 (C-3O), 26.4 (Hβ), 24.9, 24.7,
24.6, 24.5, 24.4 (P(1-5)γ), 24.3 (Rγ), 22.8 (Kγ), 22.3 (C-7O), 21.8
[CH3(NHAc)], 18.8 (T(1,2)γ), 18.4 (Vγa), 17.9 (TTnγ), 17.6 (Vγb),
15.5, 15.3, 15.1 (A(1-4)β), 12.9 (C-8O) ppm. 19F NMR (376.4 MHz,
CD3OD + [D1]TFA), δ = –84.26 (t, JF,F = 10.2 Hz, 3 F, CF3),
–122.43 (t, JF,F = 13.0 Hz, 2 F), –124.32 (br. s, 2 F), –124.71 (br. s,
4 F), –125.32 (br. s, 2 F), –125.60 (br. s, 2 F), –129.11 to –127.22
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3886
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3878–3887

Amphiphilic MUC1 Glycopeptide Antigens
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