Fluorinated glycosyl amino acids for mucin-like glycopeptide antigen analogues

Article abstract of DOI:10.1002/chem.200903294

The aberrant glycosylation profiles of mucin glycoproteins on epithelial tumour cells represent attractive target structures for the development of immunotherapy against cancer. Mucin-type glycopeptides have been successfully investigated as molecularly defined vaccine prototypes for triggering humoral immunity but are susceptible to rapid in vivo degradation. As a potential means to enhance the bioavailabilities of the antigenic structures, hydrolysis-resistant carbohydrate analogues with fluorine substituents at positions C6, C2′ and C6′ were synthesised and incorporated into the tandem repeat sequence of the mucin MUC1. The resulting pseudo-glycopeptides can be used to elucidate the effects of chemically modified antibody determinants on metabolic and immunological properties.

Full text of DOI:10.1002/chem.200903294

FULL PAPER
DOI: 10.1002/chem.200903294
Fluorinated Glycosyl Amino Acids for Mucin-Like Glycopeptide
Antigen Analogues
Sarah Wagner, Christian Mersch, and Anja Hoffmann-Rçder*[a]
Abstract: The aberrant glycosylation
ble to rapid in vivo degradation. As a
potential means to enhance the bio-
AHCTUNGTRENNUNG
logues with fluorine substituents at po-
profiles of mucin glycoproteins on epi-
thelial tumour cells represent attractive
target structures for the development
of immunotherapy against cancer.
Mucin-type glycopeptides have been
successfully investigated as molecularly
defined vaccine prototypes for trigger-
ing humoral immunity but are suscepti-
sitions C6, C2’ and C6’ were synthes-
ised and incorporated into the tandem
repeat sequence of the mucin MUC1.
The resulting pseudo-glycopeptides can
be used to elucidate the effects of
chemically modified antibody determi-
nants on metabolic and immunological
properties.
Keywords: antigens
glycopeptides
solid-phase synthesis
·
fluorine
mucin-1
·
·
·
Introduction
hydrolysis-resistant TACA mimics (e.g., C-glycosides, S-gly-
cosides) have been incorporated into carbohydrate-based
vaccines.[5] Deoxyfluoro carbohydrate analogues, however,
have been less widely investigated as antigen mimetics.[6]
The substitution of a sugar hydroxy group by a fluorine
atom is an established method for systematic examination of
the specificity of carbohydrate binding to a variety of pro-
tein binding sites with minimal steric perturbation.[6b,7] The
Pauling electronegativity and the van der Waals radius of
fluorine (4.0, 1.47 ꢀ) compare favourably with those of
Malignancies are frequently associated with the over-expres-
sion of various cell-surface glycoproteins and glycosphingoli-
pids.[1] In many carcinomas, the transmembrane glycoprotein
mucin-1 (MUC1) is strongly over-expressed and character-
ised by the exposure of immunogenic peptide epitopes and
truncated glycans, as a result of the down-regulation of cer-
tain glycosyltransferases.[2] Expression of various tumour-as-
sociated carbohydrate antigens (TACAs) has been found to
correlate with cancer progression and metastasis,[3] and so
these antigens are of particular interest for the development
of cancer diagnostics and immunotherapy.[4] A severe draw-
back in the development of efficient carbohydrate-based
vaccines is the low metabolic stabilities of their glycosidic
bonds, which are easily cleaved by endogenous glycosidases,
leading to reduced bioavailability of the antigens to the
immune system. Moreover, loss of essential saccharide rec-
ognition elements would be expected to affect antigen pre-
sentation and specificity of the immune response. In at-
tempts to circumvent hydrolytic degradation, a number of
ꢀ
oxygen (3.5, 1.52 ꢀ), allowing a C F moiety to serve as an
[8]
ꢀ
effective C OH mimic. Moreover, the electron-withdraw-
ing nature of fluorine affects the reactivity of an adjacent
glycosidic bond, enabling the use of fluorinated substrate
analogues as mechanistic probes for glycosyl-processing en-
zymes and metabolic studies.[9]
The strong interest in fluorinated mono- and oligosacchar-
ides has resulted in a number of methods for their synthe-
sis,[10] including the syntheses of mucin-like core struc-
tures.[11] In contrast, only a few pseudo-glycopeptides con-
taining fluorinated glycosyl amino acids are available from
the literature.[6c,e] We have recently described the first exam-
ples of TACA-threonine conjugates containing one or two
fluorine substituents in their glycan components, together
with their incorporation into short mucin-type model glyco-
peptides by solid-phase peptide synthesis (SPPS).[12] Here
we report the detailed syntheses of orthogonally protected
deoxyfluoro analogues of the TN, TF and 2,6-sialyl-TF anti-
gens, as well as the assembly of pseudo-glycopeptides each
containing a complete MUC1 tandem repeat sequence in-
[a] Dipl.-Chem. S. Wagner, Dipl.-Chem. C. Mersch,
Prof. Dr. A. Hoffmann-Rçder
Institut fꢁr Organische Chemie
Johannes Gutenberg-Universitꢂt Mainz
Duesbergweg 10–14, 55128 Mainz (Germany)
Fax : (+49)6131-39-24786
E-mail: hroeder@uni-mainz.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200903294.
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corporating a 2’-deoxy-2’-fluoro-TF or a 6,6’-dideoxy-6,6’-di-
fluoro-TF antigen analogue. The resulting modified glyco-
peptide antigens might prove to be valuable cancer vaccine
building blocks with enhanced immunogenicities and meta-
bolic stabilities.
presence of 2,4,6-collidine.[17] Subsequent acidolysis, acetyla-
tion and treatment with HBr in glacial acetic acid afforded
the galactosyl donor 10 in 70% yield over three steps after
column chromatography. Stereoselective coupling of 10 with
Fmoc-Thr(a-4,6-O-Bzn-GalNAc)-OtBu (7) was achieved
most successfully by the Helferich method[18] with micro-
wave assistance. Thus, on irradiation of the acceptor 7 with
6-deoxy-6-fluorogalactosyl bromide (10) and Hg(CN)2 in
MeNO2/CH2Cl2 3:2 at 100 W (808C) a clean conversion into
the b-configured disaccharide 17 (98%) was observed with-
out significant formation of orthoester or a-configured by-
products. Remarkably, conventional heating of the reaction
mixture only led to diminished yields of conjugate 17, due
to decomposition of the donor, although the corresponding
galactosylation with the non-fluorinated donor Ac4GalBr[19]
had proceeded smoothly even at room temperature without
microwave support.[20] Iodine-promoted cleavage[21] of the
benzylidene acetal of 17 at 608C finally provided the 6’-
deoxy-6’-fluoro-TF antigen-threonine analogue 2 (80%),
which was further converted into 1, the first known deoxy-
fluoro analogue of the 2,6-sialyl-TF antigen conjugate. The
natural 2,6-sialyl-TF antigen, together with its regioisomeric
2,3-sialylated congener, represents an important TACA for
breast cancer and leukaemia.[22]
Results and Discussion
From the point of view of the desired chemical and meta-
bolic stabilisation, TACA analogues with fluorine substitu-
ents at position C2 and C6 in the non-reducing glycan termi-
nus are particularly interesting because exo-glycosidases op-
erate by cleavage of these residues. In addition, the induc-
tive destabilising effect on a transition state resembling an
oxocarbenium ion should be at its maximum in close prox-
imity to C1. To limit the number of synthetic precursors and
protecting group manipulations, we decided to assemble the
corresponding antigen analogues by stepwise extension of
the saccharide chains on preformed GalNAc-threonine
building blocks, rather than coupling the deoxyfluoro sac-
charides individually to suitably protected threonine deriva-
tives. Our strategy for the synthesis of the fully protected
TACA analogues 1–6 (Scheme 1) was therefore based on
the TN antigen derivatives 7[13] and 8, the fluorinated galac-
tosyl donors 10,[6a] 13[14] and 14 and the sialyl donor 11.[15]
Compound 1 was directly obtained from 2 through
chemo- and stereoselective glycosylation at 6-OH with the
sialyl xanthate 11[15] as the donor (Scheme 2). Protection of
the carboxy function of 11 as a benzyl ester has proven ad-
vantageous in this reaction, because this ester can readily be
removed from the glycopeptide at a later stage under neu-
tral conditions, avoiding epimerisation of the amino acids or
b-elimination of the glycan.[20] Chemoselective sialylation of
the disaccharide conjugate 2 was performed at ꢀ408C in a
mixture of MeCN and CH2Cl2 as the solvents. Activation of
the xanthate 11 was promoted with methylsulfenyl triflate
generated in situ from methylsulfenyl bromide and silver
AHCTUNGTRENNUNG
triflate.[23] Under these conditions, formation of the a-sialo-
side is favoured and the desired conjugate 1 was obtained in
a yield of 46% after flash chromatographic removal of con-
comitant sialic acid glycal and minor amounts of the corre-
sponding b-anomer. In view of the planned glycopeptide
synthesis we abstained from acetylation of the remaining
axial 4-OH, because deacetylation of such sterically hin-
dered O-acetyl groups has been found to be difficult.[24]
Therefore, liberation of the carboxy terminus of 1 immedi-
ately provided a glycosyl amino acid building block suitable
for SPPS.
The TF antigen analogues 3 and 4, with fluorine substitu-
ents in their GalNAc moieties, were also accessible from the
galactosyl bromide 10 (Scheme 2). In an approach similar to
that used in the synthesis of the GalNAc-threonine conju-
gate 7,[13] the building block 10 was transformed into the 6-
deoxy-6-fluoro-galactal 18 by zinc-mediated reductive elimi-
nation of the 1-bromo and 2-acetoxy groups (89%). Subse-
quent azidonitration[25] and installation of the anomeric bro-
mide furnished the azido bromide 19 (31%, two steps),
which was used as the galactosyl donor in the Ag2CO3/
Scheme 1. Retrosynthetic plan for targeting the fluorinated TACA ana-
logues 1–6.
Synthesis of the 6-deoxy-6-fluoro-TACA analogues 1–4: The
6-deoxy-6-fluoro-glycosyl amino acid syntheses commenced
with the preparation of the fluorinated precursor 16
(Scheme 2),[16] easily available through microwave-assisted
deoxyfluorination of 1,2:3,4-di-O-isopropylidene galactose
(15) with N,N-diethylaminosulfur trifluoride (DAST) in the
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Fluorinated Glycosyl Amino Acids
FULL PAPER
Scheme 2. a) DAST, 2,6-collidine, microwave irradiation 100 W, 808C, 1 h, 84%; b) i) AcOH, reflux, then Ac2O/pyridine 1:2, 99%; ii) HBr/AcOH,
CH2Cl2, 08C to RT, 71%; c) 7, Hg(CN)2, MeNO2/CH2Cl2 3:2, MS (4 ꢀ), microwave irradiation 100 W, 808C, 1 h, 98% (b-17); d) I2, MeOH, reflux, 80%;
e) 11, MeSBr, AgOTf, MS (4 ꢀ), MeCN/CH2Cl2 2:1, ꢀ408C to RT, 46% (a-anomer); f) Zn, N-methylimidazole, EtOAc, reflux, 89%; g) i) CAN, NaN3,
MeCN, ꢀ188C, 47%; ii) LiBr, MeCN, 67%; h) 9, Ag2CO3, AgClO4, MS (4 ꢀ), toluene/CH2Cl2 1:1, 08C to RT, 45% (a-20); j) Zn, THF/Ac2O/AcOH
3:2:1, 75%; k) NaOMe (cat.), MeOH, pH 8.5, 78%; l) 10, Hg(CN)2, MeNO2/CH2Cl2 3:2, MS (4 ꢀ), microwave irradiation 100 W, 808C, 4 h, 73% (b-3);
m) TFA/anisole 10:1, 87%; n) 12, TMSOTf (cat.), MS (4 ꢀ), CH2Cl2, ꢀ208C to RT, 54% (b-4).
AgClO4-promoted coupling to Fmoc-Thr-OtBu (9).[26] The
glycosylation proceeded stereoselectively at 08C in a mix-
ture of toluene and CH2Cl2 as the solvents and led to the
formation of the desired a-configured conjugate 20 in 45%
yield. Concomitant traces of the corresponding b-anomeric
glycosyl amino acid were removed by flash column chroma-
tography and the conjugate 20 was transformed into the
acetamide 21 upon reductive acetylation (75%). De-O-ace-
tylation of 21 under Zemplꢄn conditions at pH 8.5[27] provid-
ed the hitherto unknown 6-deoxy-6-fluoro-TN antigen ana-
logue 8 in 78% yield, and this was regio- and stereoselec-
tively b-galactosylated at 3-OH with the 6-deoxy-6-fluoro-
galactosyl bromide 10 to yield the 6,6’-difluoro-conjugate 3.
As had also been the case for compound 17, formation of
the disaccharide 3 required microwave-assisted activation of
the galactosyl donor with Hg(CN)2 in a MeNO2/CH2Cl2 3:2
solvent mixture. Under these conditions, a clean conversion
into b-3 (73%) was again observed, with the less reactive
axial 4-hydroxy group being left untouched. Because protec-
tion of this free 4-OH had been found not to be essential
for SPPS (vide infra), the target glycopeptide building block
22 was obtained through acidolysis of the tert-butyl ester
(87%).
Ac4GalBr to yield compound 4 was found not to proceed
satisfactorily either at ambient temperature or under micro-
wave irradiation conditions. Instead, the use of the more re-
active Ac4Gal trichloroacetimidate 12[28] was required. Thus,
in the presence of catalytic amounts of trimethylsilyl triflate
in CH2Cl2 the desired 6-deoxy-6-fluoro-TF conjugate 4 was
isolated as the only glycosylated product in a moderate
54% yield. It is interesting to note that no formation of the
corresponding orthoester was observed, in contrast with the
related glycosylation of the nonfluorinated TN acceptor 7.[13]
However, the inherent low reactivity of the accepting 3-OH
group resulting from negative inductive effects and/or the
engagement of its lone pairs in an intramolecular hydrogen
bond to the 2-acetamido group seemed to impede the com-
plete conversion of acceptor 8, which was always recovered
from the reaction mixture to some extent.
Synthesis of the 2’-deoxy-2’-fluoro-TF analogues 5 and 6: In
view of the microwave-supported activation of the 6-deoxy-
6-fluoro-galactosyl donor 10, which had proved to be essen-
tial for the successful glycosylation of the TN acceptors 7
and 8, we also expected the corresponding 2-deoxy-2-fluoro-
galactosylations to be difficult, because anomeric reactivity
is greatly suppressed by the inductive effect of a 2-fluoro
group. Indeed, microwave-supported galactosylation of 7
Interestingly, the corresponding Hg(CN)2-promoted 3-b-
galactosylation of the monofluorinated TN derivative 8 with
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A. Hoffmann-Rçder et al.
with 2F-Ac3GalBr[29] proved unsuccessful for formation of
the desired 2’-deoxy-2’-fluoro-TF antigen analogue 5. We
therefore turned our attention to the use of the more reac-
tive 2-deoxy-2-fluoro trichloroacetimidate donor 13
(Scheme 3),[30] which can be conveniently prepared by Se-
non-participating character of the fluorine substituent, we
were not able to exceed a b/a ratio of 1:2, even after exten-
sive optimisation studies with use of various promoters and
solvents. Flash chromatographic separation of the anomeric
mixture proved difficult at this stage, but was conveniently
achieved after removal of the benzylidene acetal (26, 77%).
Subsequent O-acetylation proceeded smoothly (89%) and
afforded the pure conjugate b-5 in 89%. Acidolytic cleavage
of the tert-butyl ester finally provided the 2’-deoxy-2’-fluoro-
TF-glycopeptide building block 27 (89%), which was direct-
ly subjected to the solid-phase glycopeptide synthesis.
It is generally accepted that the natures of the carbohy-
drate protecting groups can modulate the efficiency of gly-
cosylation by influencing both the reactivities of the cou-
pling partners and the reaction stereoselectivity. Woerpel
and co-workers,[32] for instance, have shown that the pres-
ence of electronegative substituents, in particular at C3 and
C4 as in the galactosyl donor 13, favours the pseudoaxial
conformation of the intermediate oxocarbenium ion, which
then leads to a 1,4-trans-selective glycosylation reaction. In
contrast to this, the corresponding but more reactive benzy-
lated trichloroacetimidate donor 14 should react preferably
through the pseudoequatorially substituted oxocarbenium
ion, thus favouring b-anomeric selectivity. 3,4,6-Tri-O-
benzyl-2-deoxy-2-fluorogalactosyl trichloroacetimidate (14)
was obtained from the starting 3,4,6-tri-O-benzylgalactal
(28) over two steps and in 55% yield through Selectfluor-
mediated fluorination by a literature protocol[6d] (29, 66%),
followed by treatment with trichloroacetonitrile in the pres-
ence of DBU in CH2Cl2 (86%).
To our delight, the subsequent galactosylation of the ac-
ceptor 7 with the donor 14 in the presence of TMSOTf pro-
ceeded in an excellent yield of 86% and with a significantly
increased b-selectivity of 10:1 (b/a). Again, as with donor
13, elevated temperatures and long reaction times were re-
quired for the glycosylation to go to completion. Minor
amounts of the corresponding a-anomer were separated
from b-30 by flash chromatography, and the hydroxy pro-
tecting groups of b-30 were removed by hydrogenolysis in
the presence of palladium on activated charcoal in EtOH.
Unfortunately, this step was found not to be reliable under a
wide range of reaction conditions, including the use of
Scheme 3. a) R=Ac: Selectfluor, MeNO2/H2O 4:1, microwave irradiation
100 W, 1088C, 2 min, 66% (24), R=Bn: Selectfluor, MeNO2/H2O 4:1,
reflux, 66% (29); b) CCl3CN, DBU, CH2Cl2, 71% (13), 84% (14); c) 7,
TMSOTf (cat.), MS (4 ꢀ), CH2Cl2, 08C to RT, 12 h, 70% (25, a/b 2:1),
86% (30, a/b 1:10); d) R=Ac: NaHSO4/SiO2, CH2Cl2/MeOH 4:1, 77%;
R=Bn: i) H2, Pd/C, EtOH, 12 h; ii) FmocOSu, DIPEA, CH2Cl2;
e) Ac2O/pyridine 1:2, 89% from 26 and 30–90% from 30 over three
steps; f) TFA/anisole 10:1, CH2Cl2, 89%; g) NaHSO4/SiO2, CH2Cl2/
MeOH 4:1, 85%; h) Ac2O/pyridine 1:2, 88%; j) TFA/anisole 10:1,
CH2Cl2, 70%.
lectfluor-mediated electrophilic fluorination of 3,4,6-tri-O-
acetylgalactal (23).[29–31] However, in our synthesis of the di-
ACHTUNGTRENNUNGsaccharide 5, a slightly modified microwave-assisted proto-
col for the preparation of 24 was used. Treatment of 23 with
Selectfluor in MeCN/H2O 4:1 under microwave irradiation
conditions (100 W, 1088C) provided 3,4,6-tri-O-acetyl-2-
deoxy-2-fluorogalactose (24) in 66% yield within minutes,
in contrast with a 63% yield after 6.5 h at reflux without mi-
crowave support. Compound 24 was then transformed into
the trichloroacetimidate 13 by treatment with DBU in
CH2Cl2 (71%). The subsequent glycosylation of the acceptor
7 with the donor 13 afforded the desired disaccharide 25 in
good yields (70–77%) only at temperatures above 08C.
Under these conditions, however, poor stereoselectivity of
the glycosylation reaction was observed, with predominant
formation of the thermodynamically favoured a-glycoside of
25. Because the formation of the desired b-anomeric prod-
uct of 25 was impeded both by electronic effects and by the
PdACTHNGUTREN(UNG OAc)2 and MeOH/AcOH as the solvents. Besides the
sluggish course of the debenzylation reaction, partial loss of
the Fmoc protecting group was observed; it therefore had to
be reinstalled prior to O-acetyl protection, furnishing the 2’-
deoxy-2’-fluoro-TF building block 5 in variable yields of 30–
80% over three steps. In view of this tedious and inefficient
protecting group transformation, we decided to refrain from
global O-acetylation. Encouraged by literature precedence
with regard to the use of benzylated glycosyl amino acids
for SPPS[33] and in spite of the expected increase in acid-sen-
sitivity of the resulting conjugate 6, we exchanged only the
labile benzylidene acetal for acetyl groups. Thus, upon selec-
tive removal[34] of the benzylidene acetal (31, 77%), fol-
lowed by O-acetylation (6, 88%) and careful acidolysis of
the tert-butyl ester, the alternative 2’-deoxy-2’-fluoro-TF-gly-
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Fluorinated Glycosyl Amino Acids
FULL PAPER
copeptide building block 32 was obtained in 47% yield over
three steps.
(NMP) to remove the temporary Fmoc protecting group,
followed by coupling of excess (10 equiv) Fmoc-amino acid
activated by HBTU/HOBt[39] and diisopropylethylamine
(DIPEA) in DMF. Unreacted amino acids were capped
after each cycle with Ac2O in the presence of DIPEA and
catalytic amounts of HOBt in NMP. To allow for the steri-
cally demanding natures of their carbohydrate moieties, the
threonine conjugates 22 (1.3 equiv), 27 (2.0 equiv) and 32
(2.5 equiv) were each coupled with an extended reaction
time of 8 h and with use of the more reactive reagents
HATU/HOAt[40] with N-methylmorpholine (NMM) in NMP
for activation. Subsequent Fmoc removal from the glycosy-
lated peptides was again carried out with piperidine in
NMP.[13,20] After the final five Fmoc-amino acids of the TR
sequence had been coupled by the standard protocol, the
triethylene glycol spacer 34 (10 equiv) was attached to each
peptide, again by the standard coupling procedure. Simulta-
neous detachment of the glycopeptides from the resin and
cleavage of the acid-labile amino acid side chain protecting
Solid-phase synthesis of MUC1 glycopeptide antigen ana-
logues: After liberation of their C termini, the glycosyl
amino acids 22, 27 and 32 were directly used in the sequen-
tial solid-phase synthesis of two glycopeptide antigen ana-
logues, each containing a full tandem repeat (TR) sequence
of the epithelial mucin MUC1 and an N-terminal triethylene
glycol spacer. These can then be used for conjugation to im-
munostimulants such as BSA[35] or tetanus toxoid[36] and for
immobilisation onto microarray platforms[37] in functional
immunological studies. The MUC1 pseudo-glycopeptides
were assembled with an automated synthesiser by the Fmoc
strategy on a Tentagel S resin (33) modified with a bulky
trityl linker[38] to avoid diketopiperazine formation and pre-
loaded with Fmoc-proline (Scheme 4). The first 13 amino
acids of the MUC1 sequence were coupled under standard
conditions with use of piperidine in N-methylpyrrolidone
Scheme 4. Solid-phase synthesis of the tumour-associated MUC1 tandem repeat glycopeptide analogues 38 and 39 with fluorinated TF antigen glycan
chains at Thr15. NMP=N-methylpyrrolidone, HBTU=O-(1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate, HOBt=N-hy-
droxybenzotriazole, DIPEA=diisopropylethylamine, HATU=O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate, HOAt=
N-hydroxy-7-azabenzotriazole, NMM=N-methylmorpholine, TIS=triisopropylsilane.
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A. Hoffmann-Rçder et al.
chased from Orpegen Pharma. For solid-phase syntheses, pre-loaded Ten-
tagel S resins (Rapp Polymere) were employed. Reactions were moni-
groups was achieved by treatment with a mixture of TFA,
triisopropylsilane and water. The resulting partially de-
blocked glycopeptides 35 and 36 were isolated after purifica-
tion by semipreparative RP-HPLC in yields of 29 and 21%,
respectively (based on the loaded resin 33). The final de-O-
acetylation of the saccharide moiety was accomplished by
prolonged treatment either with aqueous NaOH (5 mm) so-
lution (38, 55%) or with catalytic amounts of NaOMe in
methanol (39, 60%) at pH 10–10.5. In each case, careful
monitoring of the reaction by analytical RP-HPLC was nec-
essary, in order to avoid epimerisation of amino acids and/or
b-elimination of the glycan. Deprotection of the glycan part
in the MUC1 pseudo-glycopeptide 37 was directly per-
formed on the crude product in a two-step sequence after
release from the resin. At first, debenzylation was achieved
tored by TLC with pre-coated silica gel (60 F254
) aluminium plates
(Merck KGaA, Darmstadt). Flash column chromatography was per-
formed with silica gel (230–400 mesh) from Merck. RP-HPLC analyses
were performed with
a JASCO-HPLC system and PerfectSil C18(2)
(250ꢅ4.6 mm, 5 mm), Phenomenex Luna C18(2) (250ꢅ4.6 mm, 10 mm) or
Phenomenex Jupiter C18(2) (250ꢅ4.6 mm, 10 mm) columns at a flow rate
of 1 mLminꢀ1. Preparative HPLC separations were carried out with a
JASCO-HPLC System and PerfectSil C18(2) (250ꢅ20 mm, 5 mm), Phe-
nomenex Luna C18(2) (250ꢅ30 mm, 10 mm) or Phenomenex Jupi-
ter C18(2) (250ꢅ30 mm, 10 mm) columns at a flow rate of 20 mLminꢀ1 or
10 mLminꢀ1. H2O/MeCN and H2O/MeOH mixtures were used as sol-
1
vents; if required TFA (0.1%) was added. H, 13C, 19F and 2D NMR spec-
tra were recorded with a Bruker AC 300 or a Bruker AM 400 spectrome-
ter. The chemical shifts are reported in ppm relative to the signal of the
deuterated solvent. Multiplicities are given as: s (singlet), brs (broad sin-
glet), d (doublet), t (triplet) and m (multiplet). Assignment of proton and
carbon signals was achieved by means of additional COSY, TOCSY,
HMQC and HMBC experiments when noted. The signals of the saccha-
ride portions were denoted as follows: N-acetyl-d-galactosamine (no
prime), d-galactose (’) and N-acetyl-neuraminic acid (’’). In the glycopep-
tide spectra, the glycosylated amino acid threonine is characterised by a
star. ESI and HR-ESI mass spectra were recorded with a Micromass
Q TOF Ultima 3 spectrometer, whereas MALDI-TOF mass spectra were
acquired with a Micromass Tofspec E spectrometer and use of 2,5-dihy-
droxybenzoic acid as the matrix. Optical rotations were measured at
546 nm and 578 nm with a Perkin–Elmer polarimeter 241.
by hydrogenolysis in the presence of Pd
lyst, followed by de-O-acetylation through careful trans-
ACHTUNGTRENNUNGesterification with NaOMe in MeOH at pH 9–10. Purifica-
ACHTUNGRTEN(NGNU OAc)2 as the cata-
tion by RP-HPLC finally furnished the desired glycopeptide
39 with an improved overall chemical yield of 34% based
on the loaded resin 33.
Conclusion
Detailed experimental procedures and characterisation data for com-
pounds 5, 10, 13, 14, 16, 18, 19, 24–26 and 29 are provided in the Support-
ing Information (see footnote at the first page of this article).
A set of orthogonally protected fluorinated analogues of the
tumour-associated carbohydrate antigens TN, TF and 2,6-
sialyl-TF with fluorine substituents at positions C6, C2’ and
C6’ has been successfully prepared. In general, the regio-
and stereoselective glycosylation reactions of fluorinated
galactosyl donors were characterised by markedly reduced
reactivities, in the case of glycosyl bromide 10 even requir-
ing microwave-supported activation to provide the mono-
and difluorinated TF antigen-threonine conjugates 17 and 3
in good chemical yields. In addition, use of the benzyl-pro-
tected 2-fluoro-substituted trichloroacetimidate donor 14 al-
lowed b-selective formation of the 2’-deoxy-2’-fluoro-TF an-
tigen analogue 5 despite the non-participating character of
its 2-fluoro substituent. The orthogonally protected 2’-fluori-
nated TF antigen building blocks 27 and 32, as well as the
6,6’-difluorinated derivative 22, were incorporated into
MUC1 glycopeptide sequences, each containing a non-im-
munogenic linker, for further conjugation to carrier proteins
or microarray platforms. The presented antigen analogues
are potentially interesting tools for the development of
novel glycoconjugate mimics with increased biological half-
lives and enhanced immunogenicities.
Fmoc-Thr(b-6F-Ac3Gal-(1–3)-a-4,6-O-Bzn-GalNAc)-OtBu (17): A solu-
tion of Fmoc-Thr-(a-4,6-O-Bzn-GalNAc)-OtBu (7, 812 mg, 1.18 mmol) in
dry MeNO2/CH2Cl2 3:2 (12.5 mL) was treated with activated powdered
molecular sieves (4 ꢀ, 800 mg) and Hg(CN)2 (596 mg, 2.36 mmol) and
was stirred for 30 min at room temperature under argon. The glycosyl
donor 10 (876 mg, 2.36 mmol), dissolved in dry MeNO2/CH2Cl2 3:2
(12.5 mL), was added and the reaction mixture was irradiated in a CEM
Discover microwave reactor at 100 W (808C) for 1 h. The reaction mix-
ture was diluted with CH2Cl2 (20 mL) and filtered through Hyflo Super-
cel into saturated aqueous NaHCO3 (80 mL), and the aqueous phase was
extracted with CH2Cl2 (2ꢅ80 mL). The combined organic phases were
washed with saturated aqueous NaHCO3 (40 mL) and brine (40 mL),
dried (MgSO4) and concentrated in vacuo. Flash chromatography on
silica gel (cHex/EtOAc, 2:3) afforded 17 as a colourless amorphous solid
(1.13 g, 1.15 mmol, 98%); Rf =0.29 (cHex/EtOAc 2:3); analytical RP-
HPLC (Luna, H2O/MeCN 25:75!0:100, 30 min): tR =6.89 min; [a]D23
=
64.7 (c=1.0 in CHCl3); 1H NMR (400 MHz, CDCl3, COSY): d=7.79 (d,
A
3J
H,2-H)=3J
Har-Bzn), 7.45–7.31 (m, 7H; 2-H-, 3-H-, 6-H-, 7-H-Fmoc, Har-Bzn), 5.85
(d, 3J(NH,2-H)=8.8 Hz, 1H; NH-Ac), 5.56 (s, 1H; CH-Bzn), 5.44 (d, 3J-
(NH,Ta)=9.0 Hz, 1H; NH-urethane), 5.40–5.37 (m, 1H; 4’-H), 5.21 (dd,
(5-H,6-H)=7.5 Hz, 2H; 4-H-, 5-H-Fmoc), 7.63 (d, 3J
ACHUTGTNRENNUG ACHTUNGTRENNUNG(1-
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
3J(2’-H,1’-H)=7.4 Hz, 3J(2’-H,3’-H)=9.8 Hz, 1H; 2’-H), 5.00–4.94 (m,
2H; 3’-H {4.99}, 1-H {4.94}), 4.76 (d, 3J(1’-H,2’-H)=7.2 Hz, 1H; 1’-H),
4.67–4.61 (m, 1H; 2-H), 4.50–4.37 (m, 3H; CH2-Fmoc {4.50}, 6a’-H
{4.48}), 4.37–4.33 (m, 1H; 4-H), 4.30–4.21 (m, 4H; 6b’-H {4.30}, 9-H-
Fmoc {4.25}, Ta {4.23}, 6a-H {4.21}, Tb {4.18}), 4.14–4.05 (m, 2H; 6b-H
{4.10}, 5’-H {4.06}), 3.88 (d, 3J
1H; 5-H), 2.15, 2.09, 2.00, 1.92 (4 ꢅ s, 12H; CH3-Ac), 1.46 (s, 9H; tBu),
1.25 ppm (d, 3J(Tg,Tb)=6.3 Hz, 3H; Tg); 13C NMR (100 MHz, CDCl3,
ACHTUNGTRENN(UNG 3-H,2-H)=8.7 Hz, 1H; 3-H), 3.70 (brs,
Experimental Section
AHCTUNGTRENNUNG
DEPT, HMQC): d=170.7, 170.2, 170.0, 169.9 (C=O), 156.4 (C=O-ure-
thane), 143.7, 143.6 (C-1a-, C-8a-Fmoc), 141.3 (C-4a-, C-5a-Fmoc), 137.4
(Cq-Bzn), 128.8, 128.2, 128.1 (Car-Bzn), 127.8, 127.7 (C-3-, C-6-Fmoc),
127.1 (C-2-, C-7-Fmoc), 126.2 (Car-Bzn), 124.9 (C-1-, C-8-Fmoc), 120.1
General remarks: Solvents for moisture-sensitive reactions (toluene,
MeCN, CH2Cl2, MeNO2) were distilled and dried by standard procedures.
Glycosylations were performed in flame-dried glassware under argon.
DMF (amine-free, for peptide synthesis) and NMP were purchased from
Roth, and Ac2O and pyridine in p.a. quality from Acros. Reagents were
purchased in the highest available commercial quality and were used as
supplied except where noted. Fmoc-protected amino acids were pur-
(C-4-, C-5-Fmoc), 101.4 (C-1’), 100.6 (CH-Bzn), 100.2 (C-1), 83.2 (Cq-
1
tBu), 81.3 (d, J
71.4 (d, J
(F,C-6’)=171.5 Hz; C-6’), 76.1 (Tb), 75.2 (C-4), 75.1 (C-3),
2
ACHTUNGERTN(NUNG F,C-5’)=21.3 Hz; C-5’), 70.3 (C-3’), 69.3 (C-6), 68.7 (C-2’), 67.6
(d, 3J(F,C-4’)=5.3 Hz; C-4’), 66.8 (CH2-Fmoc), 63.7 (C-5), 58.9 (Ta), 47.9
AHCTUNGTRENNUNG
7324
www.chemeurj.org
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 7319 – 7330

Fluorinated Glycosyl Amino Acids
FULL PAPER
(C-2), 47.3 (9-H-Fmoc), 28.0 (CH3-tBu), 20.8, 20.7, 20.6, 20.1 (CH3-Ac),
ACTHNGUTERNNUG
3J(NH,5-H)=8.7 Hz, 1H; NH-NeuNAc), 4.98 (dd, 3J(3’-H,4’-H)=2.7 Hz,
18.9 ppm (Tg); 19F NMR (376 MHz, CDCl3), d=ꢀ230.1 (dt, 3J
A
3J(3’-H,2’-H)=10.5 Hz, 1H; 3’-H), 4.87–4.80 (m, 1H; 4’’-H), 4.73 (d,
2
3
2
3
13.3 Hz, J
(F,6’-H)=46.7 Hz), ꢀ232.2 ppm (dt, J
(F,5-H)=13.8 Hz, J
H)=46.2 Hz, rotamer); HRMS (ESI-TOF): m/z: calcd for
3J
N
4.55–4.50 (m, 3H; CH2-Fmoc {4.53}, 2-H {4.52}), 4.46–4.43 (m, 1H; 6a’-
C50H59FN2O17Na: 1001.3698 [M+Na]+; found: 1001.3681.
H), 4.36–4.25 (m, 3H; 6b’-H {4.33}, 9a’’-H {4.30, dd, 3J(9a’’-H,8’-H’)=
2
3
2.1 Hz, J(9a’’-H,9b’’-H)=12.1 Hz}, 9H-Fmoc {4.25}), 4.16 (d, JACHTUNGTRENNUNG(Ta,Tb)=
Fmoc-Thr(b-6F-Ac3Gal-(1–3)-a-GalNAc)-OtBu (2): Iodine (a few milli-
grams) and water (a few drops) were added to a solution of the disac-
charide 17 (847 mg, 0.87 mmol) in methanol (90 mL). The reaction mix-
ture was heated at reflux for 5 h and diluted with CH2Cl2 (100 mL). The
organic layer was washed with saturated aqueous NaHCO3 (60 mL) and
brine (60 mL), dried (MgSO4) and concentrated in vacuo. Flash chroma-
tography on silica gel (cHex/EtOAc 2:3) afforded 2 as a colourless amor-
phous solid (617 mg, 0.69 mmol, 80%); Rf =0.13 (cHex/EtOAc 1:5); ana-
lytical RP-HPLC (Luna, H2O/MeCN 50:50!15:85 (40 min)): tR =
13.13 min; [a]2D3 =43.2 (c=1.0 in CHCl3); 1H NMR (300 MHz, CDCl3):
10.0 Hz, 1H; Ta), 4.11–4.01 (m, 4H; Tb {4.11}, 9a’’-H {4.08}, 5’’-H, 6’’-H
{4.05}), 3.98–3.89 (m, 3H; 5’-H {3.95}, 4-H {3.93}, 6a-H {3.89}), 3.81 (brs,
ACTHNUTRGNEUNG
1H; 5-H), 3.55 (dd, 3J(6b-H,5-H)=5.1 Hz, 2J(6-Ha,6b-H)=10.0 Hz, 1H;
6b-H), 2.61 (dd, 3J(3-Heq,4-H)=4.4 Hz, 2J(3-Heq,3-Hax)=12.7 Hz, 1H; 3’’-
eq), 2.48 (s, 1H; OH), 2.15, 2.11, 2.09, 2.00, 1.98, 1.95, 1.86 (7 ꢅ s, 27H;
9 ꢅ CH3-Ac), 1.94–1.90 (m, 1H; 3’’-Hax), 1.45 (s, 9H; tBu), 1.24 ppm (d,
3J(Tg,Tb)=6.2 Hz, 3H; Tg); 13C NMR (100 MHz, CDCl3, DEPT,
H
AHCTUNGTRENNUNG
HMQC): d=170.3, 170.3, 170.2, 169.9, 169.8, 167.5 (C=O), 156.6 (C=O-
urethane), 143.9, 143.8 (C-1a-, C-8a-Fmoc), 141.5 (C-4a-, C-5a-Fmoc),
135.0 (Cq-Bzn), 128.9, 128.6 (Car-Bzn), 128.5 (C-3-, C-6-Fmoc), 128.0 (C-
2-, C-7-Fmoc), 127.3 (Car-Bzn), 125.0 (C-1-, C-8-Fmoc), 120.2 (C-4-, C-5-
Fmoc), 101.8 (C-1’), 100.4 (C-1), 98.9 (C-2’’), 83.2 (Cq-tBu), 81.1 (d,
d=7.79 (d, 3J
7.62 (d, 3J(1-H,2-H)=3J
(3-H,4-H)=3J
(6-H,5-H)=7.4 Hz, 2H; 3-H-, 6-H-Fmoc), 7.32 (t,
(2-H,1-H)=3J
(7-H,8-H)=7.3 Hz, 2H; 2-H-, 7-H-Fmoc), 5.93 (d,
(NH,2-H)=9.5 Hz, 1H; NH-Ac), 5.44 (d, 3J
(NH,Ta)=9.3 Hz, 1H;
A
ACHTUNGTRENN(UNG 5-H,6-H)=7.4 Hz, 2H; 4-H-, 5-H-Fmoc),
A
ACHTUNGTRENNUNG
(t, 3J
G
A
ACHTUNGTRENNUNG
1J
2J
ACHTUNGTRENNUNG
3J
3J
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
C
ACHTUNGTRENNUNG
68.6 (C-2’), 67.6 (C-4), 67.5 (CH2-Bn), 67.1 (d, 3J
N
NH-urethane), 5.40 (d, 3J(4’-H,3’-H)=3.0 Hz, 1H; 4’-H), 5.21 (dd,
3J(2’-H,1’-H)=8.0 Hz, 3J(2’-H,3’-H)=10.3 Hz, 1H; 2’-H), 4.97 (dd,
3J(3’-H,4’-H)=3.1 Hz, 3J(3’-H,2’-H)=11.2 Hz, 1H; 3’-H), 4.83 (m, 1H;
1-H), 4.65 (d, 3J(1’-H,2’-H)=7.8 Hz, 1H; 1’-H), 4.57–4.45 (m, 4H; 2-H,
CH2-Fmoc, 6a’-H), 4.37–4.33 (m, 1H; 4-H), 4.27–4.20 (m, 2H; 6b’-H, 9H-
Fmoc), 4.17–4.09 (m, 2H; Ta, Tb), 3.97–3.92 (m, 2H; 6a/b-H), 3.86–3.81
(m, 2H; 5’-H, 3-H), 3.72–3.69 (m, 1H; 5-H), 2.87 (brs, 1H; OH), 2.60
(brs, 1H; OH), 2.16, 2.09, 2.01, 1.94 (4 ꢅ s, 12H; 4 ꢅ CH3-Ac), 1.45 (s,
19F NMR (376 MHz, CDCl3): d=ꢀ230.4 ppm (dt, 3J
2J
ACTHNURTGENUNG(F,6’-H)=46.2 Hz); HRMS (ESI-TOF): m/z: calcd for C69H86FN3O29Na:
1462.5228 [M+Na]+; found: 1462.5206.
Fmoc-Thr(a-6F-Ac2GalN3)-OtBu (20): Fmoc-Thr-OtBu (9, 283 mg,
0.71 mmol) was dissolved in a mixture of dry toluene and dry CH2Cl2 1:1
(6 mL) and stirred under argon with activated powdered molecular sieves
(4 ꢀ, 0.5 g) at room temperature for 45 min. The suspension was cooled
to 08C, after which silver carbonate (206 mg, 0.75 mmol) and silver per-
chlorate (28.1 mg, 0.14 mmol), dissolved in anhydrous toluene (1 mL),
were added. The glycosyl donor 19 (250 mg, 0.68 mmol), dissolved in a
mixture of dry toluene and dry CH2Cl2 1:1 (6 mL), was added dropwise
at 08C over a period of 25 min. The reaction mixture was stirred at 108C
for 16 h, diluted with CH2Cl2 (50 mL), and filtered through Hyflo Super-
cel. The organic layer was washed with saturated aqueous NaHCO3
(30 mL) and brine, (30 mL), dried (MgSO4) and concentrated in vacuo.
Flash chromatography on silica gel (cHex/EtOAc 3:1) afforded 20 as a
colourless amorphous solid (212 mg, 0.32 mmol, 45%); Rf =0.27 (cHex/
EtOAc 4:1); analytical RP-HPLC (Luna, H2O/MeCN 50:50!25:75
(40 min)!0:100, 10 min): tR =39.97 min; [a]2D3 =59.8 (c=1.0 in CHCl3);
ACTHNUTRGNEUNG
9H; tBu), 1.25 ppm (d, 3J(Tg,Tb)=6.6 Hz, 3H; Tg); 13C NMR (75 MHz,
CDCl3): d=170.2, 170.1, 170.0, 169.8 (C=O), 156.3 (C=O-urethane),
143.7 (C-1a-, C-8a-Fmoc), 141.3 (C-4a-, C-5a-Fmoc), 127.8 (C-3-, C-6-
Fmoc), 127.1 (C-2-, C-7-Fmoc), 124.8 (C-1-, C-8-Fmoc), 120.0 (C-4-, C-5-
Fmoc), 101.5 (C-1’), 100.1 (C-1), 83.1 (Cq-tBu), 80.9 (d, 1J
ACHTUNGTRENNUNG
172.6 Hz; C-6’), 77.3 (C-3), 76.2 (Tb), 71.7 (d, 2J
ACHTUNGTRENNUNG
C-5’), 70.5 (C-3’), 69.9 (C-4), 69.6 (C-6), 68.4 (C-2’), 66.9 (d, 3J
ACHTUNGTRENNUNG
6.5 Hz; C-4’), 66.7 (CH2-Fmoc), 62.6 (C-5), 59.0 (Ta), 47.6 (C-2), 47.3
(C-9-Fmoc), 28.0 (CH3-tBu), 20.7, 20.5, 20.41 (CH3-Ac), 18.7 ppm
(Tg); 19F NMR (376 MHz, CDCl3): d=ꢀ230.7 ppm (dt, 3J
ACHTUNGTRENN(UNG F,5’-H)=
12.4 Hz, 2J
ACHTUNGTRENNUNG(F,6’-H)=46.1 Hz); HRMS (ESI-TOF): m/z: calcd for
C43H55FN2O17Na: 913.3385 [M+Na]+; found: 913.3383.
Fmoc-Thr(b-6F-Ac3Gal-(1–3)-[a-Ac4NeuNAcCO2Bn-(2–6)]-a-GalNAc)-
OtBu (1): A solution of Ac4NeuNAcCO2Bn-Xan (11, 566 mg, 0.84 mmol)
in dry CH2Cl2 (3 mL) and dry MeCN (6 mL) was stirred with activated
powdered molecular sieves (4 ꢀ, 1 g) at room temperature for 16 h under
argon. The glycosyl acceptor 2 (300 mg, 0.34 mmol), dissolved in dry
CH2Cl2 (4 mL) and dry MeCN (7 mL), was added and the reaction mix-
ture was stirred at room temperature for 1 h with protection from light.
The suspension was cooled to ꢀ408C, and silver triflate (130 mg,
0.84 mmol) and a pre-cooled (08C) solution of methylsulfenyl bromide in
dry 1,2-dichloroethane [527 mL of a 1.6m solution in 1,2-dichloroethane;
the methylsulfenyl bromide solution was prepared by addition of bro-
mine (205 mL, 3.99 mmol) to a solution of dimethyl disulfide (354 mL,
3.99 mmol) in dry 1,2-dichloroethane (5 mL) and stirring at room temper-
ature with exclusion of light for 13 h] were added slowly. The reaction
mixture was stirred for 24 h at ꢀ45 to ꢀ308C under argon with exclusion
of light. It was then neutralised with DIPEA and allowed to warm to
room temperature. The suspension was diluted with CH2Cl2 (20 mL) and
filtered through Hyflo Supercel, and the solvents were removed in vacuo.
Purification by flash chromatography on silica gel (cHex/EtOAc 5:1!
1H NMR (300 MHz, CDCl3): d=7.77 (d, 3J
G
ACHTUNGTRENUN(NG 5-H,6-H)=
3
7.0 Hz, 2H; 4-H-, 5-H-Fmoc), 7.63 (d, J
N
R
(3-H,4-H)=3J
ACHUTGTNRENNUG CAHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
A
U
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
3
AHCTUNGTRENNUNG
(Tg,Tb)=6.3 Hz, Tg);
13C NMR (75 MHz, CDCl3): d=170.1, 170.0, 169.5 (C=O), 157.0 (C=O-
urethane), 144.1, 144.0 (C-1a-, C-8a-Fmoc), 141.5 (C-4a-, C-5a-Fmoc),
127.9, 127.3 (C-3-, C-6-Fmoc), 127.3 (C-2-, C-7-Fmoc), 125.5, 125.3 (C-1-,
C-8-Fmoc), 120.2, 120.1 (C-4-, C-5-Fmoc), 99.3 (C-1), 83.0 (Cq-tBu), 81.6
1
2
(d, J
G
ACHTUNGERTN(NUNG F,C-5)=26.7 Hz; C-5),
68.0 (C-3), 67.7 (d, 3J
AHCTUNGTRENNUNG
58.0 (C-2), 47.4 (C-9-Fmoc), 28.2 (CH3-tBu), 20.9, 20.8 (CH3-Ac),
EtOAc) afforded a-1 as
a colourless amorphous solid (216 mg,
19.2 ppm (Tg); 19F NMR (376 MHz, CDCl3): d=ꢀ231.5 ppm (dt, 3J
ACHTUNGTRENNUNG(F,5-
0.15 mmol, 46%); Rf =0.12 (cHex/EtOAc 1:5); analytical RP-HPLC
(Luna, H2O/MeCN 70:30!23:77 (25 min)!0:100, 30 min): tR =
27.79 min; [a]2D3 =15.9 (c=1.0 in CHCl3); 1H NMR (600 MHz, CDCl3,
H)=14.8 Hz, 2J
ACTHNUGRTENUNG(F,6-H)=46.5 Hz); HRMS (ESI): m/z: calcd for
C33H39FN4O10Na: 693.2550 [M+Na]+; found: 693.2455.
COSY): d=7.78 (d, 3J
Fmoc), 7.62 (d, 3J(1-H,2-H)=3J
Fmoc), 7.43–7.32 (m, 9H; 2-H-, 3-H-, 6-H-, 7-H-Fmoc, Har-Bn), 5.89 (d,
3J(NH,2-H)=9.8 Hz, 1H; NH-Ac), 5.43 (d, 3J
(NH,Ta)=9.1 Hz, 1H;
A
U
Fmoc-Thr(a-6F-Ac2GalNAc)-OtBu (21): Activated zinc dust [783 mg,
11.3 mmol; activation was achieved by suspension in aqueous CuSO4 so-
lution (2%), followed by subsequent washings with water, EtOAc and
Et2O] was added to a stirred solution of 20 (760 mg, 1.13 mmol) in a
THF/AcO2/AcOH mixture 3:2:1 (48 mL). The reaction mixture was
stirred for 18 h at room temperature, diluted with THF (30 mL) and fil-
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
NH-urethane), 5.43–5.41 (m, 1H; 4’-H), 5.33–5.28 (m, 2H; 8’’-H {5.33},
7’’-H {5.30}), 5.23–5.18 (m, 3H; 2’-H {5.22}, CH2-Bn {5.20}), 5.12 (d,
Chem. Eur. J. 2010, 16, 7319 – 7330
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7325

A. Hoffmann-Rçder et al.
tered through Hyflo Supercel. The filtrate was concentrated in vacuo and
co-evaporated with toluene (5ꢅ20 mL) and CH2Cl2 (20 mL). The residue
was dissolved in CH2Cl2 (40 mL), washed with saturated aqueous
NaHCO3 (3ꢅ20 mL) and brine (20 mL), dried (MgSO4) and concentrat-
ed in vacuo. Flash chromatography on silica gel (cHex/EtOAc 1:1) af-
forded 21 as a colourless amorphous solid (603 mg, 0.87 mmol, 75%);
Rf =0.16 (cHex/EtOAc 1:1); analytical RP-HPLC (Luna, H2O/MeCN
70:30!25:75 (25 min)!0:100, 30 min): tR =30.21 min; [a]2D3 =35.6 (c=1.0
ed aqueous NaHCO3 (20 mL), and the aqueous phase was extracted with
CH2Cl2 (2ꢅ30 mL). The combined organic phases were washed with sa-
turated aqueous NaHCO3 (20 mL) and brine (20 mL), dried (MgSO4)
and concentrated in vacuo. Flash chromatography on silica gel (cHex/
EtOAc 1:5) afforded
3 as a colourless amorphous solid (162 mg,
0.18 mmol, 73%); Rf =0.53 (cHex/EtOAc 1:5); analytical RP-HPLC
(Luna, H2O/MeCN 70:30!23:77 (15 min)!0:100, 35 min): tR =
27.16 min; [a]2D3 =59.8 (c=1.0 in CHCl3); 1H NMR (400 MHz, CDCl3,
in CHCl3); 1H NMR (300 MHz, CDCl3): d=7.78 (d, 3J
G
(5-
COSY): d=7.78 (d, 3J
G
ACHTUNGTRENNUNG
H,6-H)=7.0 Hz, 2H; 4-H-, 5-H-Fmoc), 7.64 (d, 3J
H)=6.6 Hz, 2H; 1-H-, 8-H-Fmoc), 7.40 (t, 3J
7.4 Hz, 2H; 3-H-, 6-H-Fmoc), 7.32 (t, 3J
2H; 2-H-, 7-H-Fmoc), 6.06 (d, 3J
(d, 3J
G
R
Fmoc), 7.61 (d, 3J
A
ACHTUNGTRENNUNG
3
G
R
Fmoc), 7.42 (t, J
7.32 (t, J
N
N
3
G
N
(2-H,3-H)=3J
ACHUTGTNRENNUG CAHTUNGTRENNUNG
G
3J(NH,2-H)=8.8 Hz, 1H; NH-Ac), 5.43 (d, 3J
E
ACHTUNGTRENNUNG
U
NH-urethane), 5.40 (d, 3J(4’-H,3’-H)=2.7 Hz, 1H; 4’-H), 5.20 (dd,
3J(2’-H,1’-H)=8.0 Hz, 3J(2’-H,3’-H)=10.3 Hz, 1H; 2’-H), 4.97 (dd,
3J(3’-H,4’-H)=3.0 Hz, 3J(3’-H,2’-H)=10.5 Hz, 1H; 3’-H), 4.80 (d,
A
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
H)=9.9 Hz, 1H; 2-H), 4.53–4.39 (m, 3H; 5-H, 6a/b-H), 4.34–4.21 (m,
3J
ACHTUNGTRENNUNG
5H; 9-H-Fmoc, CH2-Fmoc, Ta, Tb), 2.15, 2.00 (2 ꢅ s, 6H; 2 ꢅ CH3-Ac),
2.15 (s, 3H; CH3-NHAc), 1.46 (s, 9H; tBu), 1.25 ppm (d, 3J
U
4.48–4.43 (m, 2H; 6a’-HACTHNGUTERNNU{G 4.47}, CH2-Fmoc {4.42}), 4.41–4.29 (m, 2H; 6b’-
H {4.34}, 5-H {4.30}), 4.25–4.17 (m, 3H; 9-H-Fmoc {4.23}, Tb {4.19}, Ta
3
{4.14}), 4.06 (s, 1H; 4-H), 4.00–3.92 (m, 1H; 5’-H), 3.71 (d, J
N
10.3 Hz, 1H; 3-H), 2.15, 2.08, 1.97 (3 ꢅ s, 9H; 3 ꢅ CH3), 2.00 (s, 3H; CH3-
ACTHNUTRGNEUNG
NHAc), 1.45 (s, 9H; tBu), 1.25 ppm (d, 3J(Tg,Tb)=6.3 Hz, 3H; Tg);
100.0 (C-1), 83.4 (Cq-tBu), 81.8 (d, 1J
G
13C NMR (100 MHz, CDCl3, DEPT, HMQC): d=170.2, 170.1, 170.1,
170.0 (C=O), 156.3 (C=O-urethane), 143.7, 143.6 (C-1a-, C-8a-Fmoc),
141.3 (C-4a-, C-5a-Fmoc), 127.8, 127.7 (C-3-, C-6-Fmoc), 127.0 (C-2-, C-
7-Fmoc), 124.9, 124.8 (C-1-, C-8-Fmoc), 120.1, 120.0 (C-4-, C-5-Fmoc),
68.8 (C-3), 68.5 (d, 2J(F,C-5)=22.5 Hz; C-5), 67.5 (d, 3J
ACHTUNGTRENNUNG ACHTUTGNREN(NUGN F,C-4)=6.7 Hz;
C-4), 67.4 (CH2-Fmoc), 59.1 (Ta), 52.1 (C-2), 47.4 (C-9-Fmoc), 28.3 (CH3-
tBu), 23.5 (CH3-NHAc), 20.9, 20.8 (CH3-Ac), 18.8 ppm (Tg); 19F NMR
(376 MHz, CDCl3): d=ꢀ231.1 ppm (dt, 3J
101.7 (C-1’), 100.0 (C-1), 83.4 (d, 1J
tBu), 81.0 (d, 1J
2J(F,C-5’)=22.2 Hz; C-5’), 70.5 (C-3’), 69.2 (d, 2J
68.3 (d, 3J(F,C-4)=7.2 Hz; C-4), 68.2 (C-2’), 66.9 (d, 3J
ACTHNUTRGENNUG CAHTUNGTRENNUNG
ACHTUNGTRENNUNG
45.1 Hz); HRMS (ESI-TOF): m/z: calcd for C35H43FN2O11Na: 709.2751
ACHTUNGTRENNUNG
[M+Na]+; found: 709.2744.
A
ACHTUNGTRENNUNG
Fmoc-Thr(a-6F-GalNAc)-OtBu (8): A solution of NaOMe in MeOH
(1%) was added dropwise to a solution of Fmoc-Thr(a-6F-Ac2GalNAc)-
OtBu (21, 573 mg, 0.83 mmol) in MeOH (10 mL) until pH 8.5 was
reached. The reaction mixture was stirred for 4 d, during which the pH
was carefully monitored and re-adjusted when necessary. After neutrali-
sation with acidic cation exchanger (Amberlyst IR 120) and filtration, the
solvent was removed at reduced pressure. Flash chromatography on silica
gel (EtOAc/MeOH 20:1) afforded 8 as a colourless amorphous solid
(394 mg, 0.66 mmol, 78%); Rf =0.10 (EtOAc/MeOH 20:1); analytical
RP-HPLC (Luna, H2O/MeCN 70:30!23:77 (25 min): tR =21.71 min;
[a]2D3 =26.5 (c=1.0 in CHCl3); 1H NMR (300 MHz, CDCl3): d=7.77 (d,
C-4’), 66.8 (CH2-Fmoc), 59.0 (Ta), 47.5 (C-2), 47.2 (C-9-Fmoc), 28.0
(CH3-tBu), 23.2 (CH3-NHAc), 20.9, 20.8, 20.6 (CH3-Ac), 18.7 ppm
(Tg); 19F NMR (376 MHz, CDCl3): d=ꢀ229.5 (dt, 3J
ACHTUNGTRENNUNG
2J
(F,6-H)=46.7 Hz), ꢀ230.6 ppm (dt, 3J
(F,5-H)=12.4 Hz, 2J
ACHUTGTNRENNUG ACHTUNGTRENNUNG
46.3 Hz); HRMS (ESI-TOF): m/z: calcd for C43H54F2N2O16Na [M+Na]+:
915.3338; found: 915.3334.
Fmoc-Thr(b-6F-Ac3Gal-(1–3)-a-6F-GalNAc)-OH (22): The disaccharide
3 (130 mg, 0.15 mmol) was treated with anisole (0.4 mL, 3.66 mmol) and
TFA (4 mL, 22.8 mmol). The reaction mixture was stirred for 4 h at room
temperature and was subsequently co-evaporated with toluene (5ꢅ
30 mL). The residue was purified by flash chromatography on silica gel
(EtOAc/MeOH/AcOH 4:1:0.05) to afford 22 as a colourless amorphous
solid (106 mg, 0.13 mmol, 87%), which was subjected to SPPS without
further characterisation; Rf =0.43 (EtOAc/MeOH/AcOH 4:1:0.05); ana-
lytical RP-HPLC (Luna, H2O/MeCN +0.1% TFA, 70:30!10:90,
25 min): tR =20.18 min; HRMS (ESI-TOF): m/z: calcd for
C39H46F2N2O16Na: 859.2715 [M+Na]+; found: 859.2726.
3J
3J
3J
3J
3J
N
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
Fmoc-Thr(b-Ac4Gal-ACTHNUTRGNE(UNG 1–3)-a-6F-GalNAc)-OtBu (4): A solution of the
ACHTUNGTRENNUNG
glycosyl acceptor 8 (200 mg, 0.33 mmol) and the donor 12 (145 mg,
0.29 mmol) in dry CH2Cl2 (8 mL) was stirred under argon with activated,
powdered molecular sieves (4 ꢀ, 260 mg) for 30 min at room tempera-
ture. The suspension was cooled to ꢀ208C and TMSOTf (16.3 mL,
0.09 mmol) in dry CH2Cl2 (1 mL) was added. After stirring for 25 h, the
reaction mixture was neutralised with solid NaHCO3 and filtered through
Hyflo Supercel. The organic phase was washed with saturated aqueous
NaHCO3 (10 mL) and brine (10 mL) and dried (MgSO4), and the solvent
was removed in vacuo. Purification by flash chromatography on silica gel
ACHTUNGTRENNUNG
CDCl3): d=174.2, 171.5 (C=O), 156.6 (C=O-urethane), 144.0, 143.8 (C-
1a-, C-8a-Fmoc), 141.5 (C-4a-, C-5a-Fmoc), 128.1, 128.0 (C-3-, C-6-
Fmoc), 127.3 (C-2-, C-7-Fmoc), 125.2, 125.1 (C-1-, C-8-Fmoc), 120.3,
120.2 (C-4-, C-5-Fmoc), 99.7 (C-1), 83.7 (Cq-tBu), 83.6 (d, 1J
ACHTUNGTRENNUNG
167.8 Hz; C-6), 76.8 (Tb), 71.4 (C-3), 69.7 (d, 2J
ACHTUNGTRENNUNG
68.3 (d, 3J(F,C-4)=7.2 Hz; C-4), 67.3 (CH2-Fmoc), 59.1 (Ta), 51.3 (C-2),
ACHTUNGTRENNUNG
47.4 (C-9-Fmoc), 28.3 (CH3-tBu), 23.0 (CH3-NHAc), 18.9 ppm (Tg);
19F NMR (376 MHz, CDCl3): d=ꢀ230.68 ppm (m); HRMS (ESI-TOF):
m/z: calcd for C31H39FN2O9Na: 625.2540 [M+Na]+; found: 625.2521.
(EtOAc/MeOH 20:1) afforded
4 as a colourless amorphous solid
(170 mg, 0.18 mmol, 54%). Rf =0.49 (EtOAc/MeOH 20:1); analytical
RP-HPLC (Luna, H2O/MeCN 50:50!15:85, 40 min): tR =18.79 min;
[a]2D3 =59.8 (c=1.0 in CHCl3); 1H NMR (400 MHz, [D6]DMSO, COSY):
Fmoc-Thr(b-6F-Ac3Gal-(1–3)-a-6F-GalNAc)-OtBu (3): A solution of the
glycosyl acceptor 8 (150 mg, 0.25 mmol) in a mixture of dry MeNO2 and
dry CH2Cl2 3:2 (4 mL) was stirred under argon with activated, powdered
molecular sieves (4 ꢀ, 200 mg) and Hg(CN)2 (125 mg, 0.49 mmol) for
30 min at room temperature. The glycosyl donor 10 (185 mg, 0.49 mmol),
dissolved in a mixture of dry MeNO2 and dry CH2Cl2 3:2 (4 mL), was
added, and the reaction mixture was irradiated in a CEM Discoverꢀ mi-
crowave reactor for 4 h at 100 W (808C). The reaction mixture was dilut-
ed with CH2Cl2 (20 mL) and filtered through Hyflo Supercel into saturat-
d=7.89 (d, 3J
7.61 (m, 2H; 1-H-, 8-H-Fmoc), 7.53 (d, 3J
Ac), 7.51 (d, 3J
(NH,Ta)=9.5 Hz, 1H; NH-urethane), 7.44–7.39 (m, 2H;
(4-H,3-H)=3J
ACHUTGTNRENNUG CAHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
3
3-H-, 6-H-Fmoc), 7.34–7.27 (m, 2H; 2-H-, 7-H-Fmoc), 5.27 (d, J(3’-H,4’-
H)=3.5 Hz 1H; 4’-H), 5.02 (dd, 3J(3’-H,4’-H)=3.5 Hz, 3J(3’-H,2’-H)=
10.4 Hz, 1H; 3’-H), 4.99–4.94 (m, 1H; 2’-H), 4.73 (d, 3J(1’-H,2’-H)=
3
7.7 Hz, 1H; 1’-H,), 4.62 (d, J
U
7326
www.chemeurj.org
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Fluorinated Glycosyl Amino Acids
FULL PAPER
4H; 6a/b-H {4.53, 4.39}, CH2-Fmoc {4.48, 4.38}), 4.32–4.29 (m, 1H; 9-H-
0.177 mmol, 89%); analytical RP-HPLC (Luna, H2O/MeCN 50:50!
Fmoc), 4.26–4.20 (m, 2H; 2-H {4.21}, Tb {4.19}), 4.15–4.11 (m, 2H; 6a’-H-
10:90, 30 min): tR =10.91 min; Rf =0.77 (CH2Cl2/MeOH 5:0.3); [a]D23
=
3
3
G
E
N
55.7 (c=1.0 in CHCl3); 1H NMR (400 MHz, [D6]DMSO, COSY): d=
12.97 (brs, 1H; OH), 7.91 (d, 3J(4-H,3-H)=3J
(5-H,6-H)=7.6 Hz, 2H; 4-
H-, 5-H-Fmoc), 7.74 (dd, 3J(1-H,2-H)=3J(8-H,7-H)=7.4 Hz, 3J
(1-H,3-
(8-H,6-H)=3.7 Hz, 2H; 1-H-, 8-H-Fmoc), 7.47 (d, 3J
(NH,2-H)=
(NH,Ta)=9.7 Hz, 2H; NH-Fmoc, NH-Ac), 7.43 (t, 3J
(3-H,4-H)=
(6-H,5-H)=7.5 Hz, 2H; 3-H-, 6-H-Fmoc), 7.33 (t, 3J(2-H,1-H)=3J
(7-
(4-H,3-H)=3.1 Hz,
1H; 4-H), 5.25 (s, 1H; 4’-H), 5.21–5.18 (m, 1H; 3’-H), 4.87 (dd,
3J(1’-H,2’-H)=7.6 Hz, 3J(1’-H,F)=3.6 Hz, 1H; 1’-H), 4.72 (d, 3J
(1-H,2-
E
A
ACHTUNGTRENNUNG
A
U
A
R
ACHTUNGTRENNUNG
H)=3J
G
G
ACHTUNGTRENNUNG
3
CH3-NHAc), 1.34 (s, 9H; tBu), 1.12 ppm (d, J
N
3J
3J
ACHTUNGTRENNUNG
13C NMR (100 MHz, [D6]DMSO, DEPT, HMQC): d=170.1, 170.0, 169.8,
169.2, 169.1 (C=O), 156.9 (C=O-urethane), 143.8, 143.7 (C-1a-, C-8a-
Fmoc), 140.9 (C-4a-, C-5a-Fmoc), 127.9, 127.8 (C-3-, C-6-Fmoc), 127.2
(C-2-, C-7-Fmoc), 125.4, 125.3 (C-1-, C-8-Fmoc), 120.3 (C-4-, C-5-Fmoc),
C
E
ACHTUNGTRENNUNG
H,8-H)=6.8 Hz, 2H; 2-H-, 7-H-Fmoc), 5.35 (d, 3J
R
A
ACHTUNGTRENNUNG
101.5 (C-1’), 99.4 (C-1), 83.7 (d, 1J
N
H)=3.9 Hz, 1H; 1-H), 4.58–4.39 (m, 2H; CH2-Fmoc), 4.37–4.07 (m, 8H;
76.9 (C-3), 74.4 (Tb), 70.6 (C-3’), 69.7 (C-5’), 69.8 (d, 2J
AHCTUNGTRENNUNG
2’-H {4.34, 4.21}, 9-H-Fmoc {4.31}, Tb {4.25}, 2-H {4.20}, 5-H {4.18}, 5’-H
C-5), 68.5 (C-2’), 67.5 (C-4’), 67.3 (C-4), 65.7 (CH2-Fmoc), 61.2 (C-6’),
59.4 (Ta), 46.9 (C-2), 46.8 (C-9-Fmoc), 27.7 (CH3-tBu), 22.9 (CH3-
NHAc), 20.6, 20.5, 20.4 (CH3-Ac), 19.0 ppm (Tg); 19F NMR (376 MHz,
ACTHNGUTRENNUG ACHTUNGTRENNUNG(6a-
{4.14}, Ta {4.12}, 6a’-H{4.11}), 4.01 (dd, 2J(6a-H,6b-H)=11.3 Hz, 3J
H,5-H)=6.2 Hz, 1H; 6a-H), 3.96–3.79 (m, 2H; 6b-H {3.88}, 6b’-H {3.83}),
2.10, 2.06, 2.00, 1.99, 1.98 (5 ꢅ s, 15H; 5 ꢅ CH3-Ac), 1.80 (s, 3H; CH3-
CDCl3): d=ꢀ229.8 ppm (dt, 3J
G
G
ACTHNGUTERNNUG
NHAc), 1.12 ppm (d, 3J(Tg,Tb)=6.4 Hz, 3H; Tg); 13C NMR (100 MHz,
HRMS (ESI-TOF): m/z: calcd for C45H57FN2O18Na [M+Na]+: 955.3490;
[D6]DMSO, HMQC): d=171.7, 170.1, 170.0, 169.9, 169.8, 169.6, 169.2
(C=O), 156.9 (C=O-urethane), 143.8, 143.8 (C-1a-, C-8a-Fmoc), 140.9,
140.8 (C-4a-, C-5a-Fmoc), 127.7, 127.7 (C-3-, C-6-Fmoc), 127.1 (C-2-,
C-7-Fmoc), 125.2, 125.1 (C-1-, C-8-Fmoc), 120.3, 120.2 (C-4-, C-5-Fmoc),
found: 955.3470.
Fmoc-Thr(b-2F-Bn3Gal-(1–3)-a-4,6-O-Bzn-GalNAc)-OtBu (30): A solu-
tion of the glycosyl acceptor 7 (1.40 g, 2.35 mmol) and the donor 14
(1.02 g, 1.45 mmol) in dry CH2Cl2 (40 mL) was stirred under argon with
activated, powdered molecular sieves (4 ꢀ, 1.34 g) for 30 min at room
temperature. The suspension was cooled to 08C and TMSOTf (50 mL,
0.277 mmol) in dry CH2Cl2 (1 mL) was added. After having been stirred
for 20 h, the reaction mixture was diluted with CH2Cl2 (50 mL), neutral-
ised with solid NaHCO3 and filtered through Hyflo Supercel. The organic
phase was washed with saturated aqueous NaHCO3 (3ꢅ100 mL) and
brine (2ꢅ100 mL), dried (MgSO4) and concentrated in vacuo. Flash chro-
matography on silica gel (cHex/EtOAc 1:1) afforded 30 as a colourless
amorphous solid (1.51 g, 1.35 mmol, 92%); analytical RP-HPLC (Per-
fectSil, H2O/MeCN 25:75!0:100, 30 min): tR =16.64 min (b-anomer),
tR =19.57 min (a-anomer); Rf =0.33 (cHex/EtOAc 1:2), [a]2D3 =62.8 (c=
100.3 (d, 2J
185.2 Hz; C-2’), 75.4 (C-3), 75.1 (Tb), 70.6 (d, 2J
AHCTUNGTRENNUNG
N
ACHTUNGTRENNUNG(C-2’,F)=
(CH2-Fmoc), 63.2 (C-6’), 60.7 (C-6), 58.5 (Ta), 47.5 (C-2), 46.9 (C-9-
Fmoc), 22.7 (CH3-NHAc), 20.8, 20.5, 20.5, 20.4, 20.3 (CH3-Ac), 19.4 ppm
(Tg); 19F NMR (376 MHz, CDCl3): d=ꢀ206.7 ppm (d, 2J
ACHTUNGTRENNUNG(F,2-H)=
49.4 Hz); HRMS (ESI-TOF): m/z: calcd for C43H51FN2O19K: 957.2707
[M+K]+; found: 957.2675.
Fmoc-Thr(b-2F-Bn3Gal-(1–3)-a-GalNAc)-OtBu
(31):
NaHSO4/SiO2
(760 mg) was added as the de-O-acetylation catalyst to a solution of the
disaccharide 30 (991 mg, 0.883 mmol) in a mixture of CH2Cl2 and MeOH
4:1 (50 mL) and the suspension was stirred for 18 h at room temperature.
The catalyst was filtered off and the filtrate was washed with saturated
aqueous NaHCO3 (3ꢅ70 mL) and brine (50 mL), dried (MgSO4) and
concentrated in vacuo. Flash chromatography on silica gel (cHex/EtOAc
1:10) afforded 31 as a colourless amorphous solid (775 mg, 0.75 mmol,
85); analytical RP-HPLC (Jupiter, H2O/MeOH 20:80!0:100, 30 min):
tR =13.25 min; Rf =0.53 (cHex/EtOAc 1:10); [a]2D3 =33.4 (c=1.0 in
1.0 in CHCl3); 1H NMR (400 MHz, CDCl3, COSY): d=7.81–7.75 (m,
3
2H; 4-H-, 5-H-Fmoc), 7.65 (d, J
G
E
H-, 8-H-Fmoc), 7.55 (d, 3J(3-H,4-H)=3J
ACHTNUTRGENNUG HCATUNGTRENN(GUN 6-H,5-H)=7.3 Hz, 2H; 3-H-, 6-
H-Fmoc), 7.44–7.22 (m, 22H; 2-H-, 7-H-Fmoc, Har), 5.75 (d, 3J
H)=9.0 Hz, 1H; NH-Ac), 5.57 (d, J
5.51 (s, 1H; CH-Bzn), 5.03 (d, 3J
2J
(H,H)=11.6 Hz, 1H; CH2-Bn), 4.89–4.70 (m, 1H; 2’-H), 4.81–4.70 (m,
2H; CH2-Bn, 2-H {4.75}), 4.67 (d, 2J
(H,H)=12.1 Hz, 1H; CH2-Bn), 4.58
(d, 2J
(H,H)=11.6 Hz, 1H; CH2-Bn), 4.57 (s, 1H; 1’-H), 4.54–4.45 (m,
A
CHCl3); 1H NMR (400 MHz, CDCl3, COSY): d=7.77 (d, 3J
3J(5-H,6-H)=7.5 Hz, 2H; 4-H-, 5-H-Fmoc), 7.63 (d, 3J(1-H,2-H)=3J
H,7-H)=6.7 Hz, 2H; 1-H-, 8-H-Fmoc), 7.39 (t, 3J(3-H,4-H)=3J
(6-H,5-
H)=7.3 Hz, 2H; 3-H-, 6-H-Fmoc), 7.37–7.23 (m, 17H; 2-H-, 7-H-Fmoc,
Har), 5.77 (d, 3J(NH,2-H)=9.5 Hz, 1H; NH-Ac), 6.14 (d, 3J
(NH,Ta)=
9.3 Hz, 1H; NH-Fmoc), 4.90 (d, J(H,H)=11.2 Hz, 1H; CH2-Bn, 4.90 (d,
3J
(1-H,2-H)=4.0 Hz, 1H; 1-H), 4.77 (d, J
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
A
ACHTUNGTRENNUNG
2H; CH2-Fmoc), 4.44–4.40 (m, 2H; CH2-Bn), 4.37–4.14 (m, 5H; 4-H
{4.34}, Ta {4.23}, Tb {4.22}, 9-H-Fmoc {4.22}, 6a’/b’-H {4.18, d, 2J(6a-H,6b-
H)=12.4 Hz}), 3.98–3.84 (m, 3H; 4’-H {3.89}, 3-H {3.88}, 6a’/b’-H {3.94, d,
2J(6a’-H,6b’-H)=12.1 Hz}), 3.65–3.50 (m, 5H; 5’-H {3.63}, 6a/b-H {3.62,
3.55}, 5-H {3.61}, 3’-H {3.58}), 2.00 (s, 3H; CH3-NHAc), 1.46 (s, 9H; tBu),
A
ACHTUNGTRENNUNG
2
AHCTUNGTRENNUNG
2
G
ACHTUNGTRENNUNG
4.67 (d, 2J
ACHTUNGTRENNUNG
1.28 ppm (d, 3J
N
(d, 2J
A
3
DEPT, HMQC): d=170.3, 169.9 (C=O), 156.5 (C=O-urethane), 143.8,
143.7 (C-1a-, C-8a-Fmoc), 141.3 (C-4a-, C-5a-Fmoc), 138.2, 137.9, 137.7,
137.6 (Cq-Bzn, Cq-Bn), 128.7, 128.4, 128.4, 128.2, 128.0, 127.7, 127.7 (Car-
Bzn, Car-Bn), 127.6, 127.5 (C-2-, C-7-Fmoc), 127.2, 127.0 (Car-Bzn, Car-
Bn), 126.4 (C-3-, C-6-Fmoc), 125.0 (C-1-, C-8-Fmoc), 120.0, 120.0 (C-4-,
(t, J
N
{4.18}, 4-H {4.15}), 3.92–3.85 (m, 2H; 6a/b-H {3.90}, 3.84–3.79 (m, 1H; 5-
H), 3.76–3.65 (m, 2H; 3-H {3.68}, 6a/b-H {3.72}), 3.61–3.52 (m, 3H; 3’-H
{3.58}, 6a’/b’-H {3.56}, 5’-H {3.59}), 3.51–3.44 (m, 1H; 6a’/b’-H {3.49}), 2.01
(s, 3H; CH3-Ac), 1.45 (s, 9H; tBu), 1.28 ppm (d, 3J
ACTHNUTRGNEUNG(Tg,Tb)=6.2 Hz, 3H;
C-5-Fmoc), 102.8 (d, 2J
(C-1), 91.2 (d, 1J
(C-2’,F)=183.4 Hz; C-2’), 83.0 (Cq-tBu), 80.1 (d,
2J(C-3’,F)=15.8 Hz; C-3’), 76.6 (C-3), 76.4 (Tb), 75.8 (C-4), 74.6 (CH2-
Bn), 74.1 (d, 3J
(C-1’,F)=23.3 Hz; C-1’), 100.7 (CH-Bzn), 100.6
Tg); 13C NMR (100 MHz, CDCl3, DEPT, HMQC): d=170.4, 170.1 (C=
O), 156.4 (C=O-urethane), 143.8, 143.7 (C-1a-, C-8a-Fmoc), 141.3 (C-4a-,
C-5a-Fmoc), 137.9, 137.8, 137.6 (Cq-Bn), 128.5, 128.5, 128.3, 128.0, 127.8,
127.8, 127.6, 127.1 (Car-Bn, C-3-, C-6-Fmoc, C-2-, C-7-Fmoc), 125.0, 125.0
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG(C-4’,F)=8.8 Hz; C-4’), 73.7 (C-5), 73.5, 72.6 (CH2-Bn),
69.0 (C-6’), 68.7 (C-6), 67.0 (CH2-Fmoc), 63.7 (C-5’), 59.2 (Ta), 48.0 (C-
(C-1-, C-8-Fmoc), 120.0 (C-4-, C-5-Fmoc), 102.5 (d, 2J
N
2), 47.2 (C-9-Fmoc), 28.1 (CH3-tBu), 23.3 (CH3-NHAc), 19.1 ppm (Tg);
C-1’), 100.3 (C-1), 91.4 (d, J
A
1
19F NMR (376 MHz, CDCl3): d=ꢀ204.4 ppm (dd, 2J
U
(C-3), 74.0 (d, 3J(C-4’,F)=9.5 Hz; C-4’), 74.8 (CH2-Bn), 74.0 (d, 2J
AHTCUNGTRENNUGN ACHTUNGTRENNUNG
3J
A
3’,F)=14.4 Hz; C-3’), 73.9 (C-5’), 73.6, 72.7 (CH2-Bn), 69.7 (C-5), 69.6
(C-4), 69.4 (Tb), 68.8 (C-6’), 67.0 (CH2-Fmoc), 63.1 (C-6), 59.0 (Ta), 47.5
(C-2), 47.2 (C-9-Fmoc), 28.1 (CH3-tBu), 23.3 (CH3-NHAc), 18.9 ppm
[M+Na]+: 1145.4787; found: 1147.4796.
Fmoc-Thr(b-2F-Ac3Gal-(1–3)-a-Ac2GalNAc)-OH (27): Anisole (0.4 mL,
3.66 mmol) and TFA (4.0 mL, 22.8 mmol) were added to a solution of
the disaccharide 5 (194 mg, 0.20 mmol) in dry CH2Cl2 (20 mL) and the
reaction mixture was stirred for 4 h at room temperature. The mixture
was co-evaporated with toluene (5ꢅ30 mL) and concentrated in vacuo.
The residue was purified by flash chromatography on silica gel (CH2Cl2/
MeOH 5:0.3) to afford 27 as a colourless amorphous solid (163 mg,
(Tg); 19F NMR (376 MHz, CDCl3): d=ꢀ205.1 ppm (dd, 2J
ACHTUNGTRENNUNG(F,2-H)=
51.6 Hz, 3J
ACHTNUTRGNE(NUG F,3-H)=12.2 Hz); HRMS (ESI-TOF): m/z: calcd for
C58H67FN2O14Na: 1057.4474 [M+Na]+; found: 1057.4476.
Fmoc-Thr(b-2F-Bn3Gal-(1–3)-a-Ac2GalNAc)-OtBu (6): The disaccharide
31 (235 mg, 0.28 mmol), dissolved in a mixture of pyridine and Ac2O
(9 mL, 2:1), was stirred under argon for 20 h at room temperature. The
Chem. Eur. J. 2010, 16, 7319 – 7330
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7327

A. Hoffmann-Rçder et al.
reaction mixture was poured into ice/water and was extracted with
CH2Cl2 (5ꢅ50 mL). The combined organic phases were washed with sa-
turated aqueous NaHCO3 (2ꢅ70 mL) and brine (2ꢅ50 mL), dried
(MgSO4) and concentrated in vacuo. Flash chromatography on silica gel
(cHex/EtOAc 1:1) afforded 6 as a colourless amorphous solid (199 mg,
0.18 mmol, 78%). Analytical RP-HPLC (Jupiter, H2O/MeOH 30:70!
0:100, 40 min): tR =21.00 min; Rf =0.37 (cHex/EtOAc 1:1); [a]2D3 =50.9
(c=1.0 in CHCl3); 1H NMR (400 MHz, CDCl3, COSY): d=7.77 (d,
65.4 (CH2-Fmoc), 63.1 (C-6), 58.3 (Ta), 47.1 (C-2), 46.8 (C-9-Fmoc), 22.7
(CH3-NHAc), 20.7, 20.5 (CH3-Ac), 18.5 ppm (Tg); 19F NMR (376 MHz,
CDCl3): d=ꢀ204.8 ppm (d, 2J
ACHTUNGTRNE(NUNG F,2-H)=44.4 Hz); HRMS (ESI-TOF):
m/z: calcd for C58H63FN2O16Na: 1085.4060 [M+Na]+; found: 1085.4034.
General procedure for the automated solid-phase glycopeptide synthesis:
Peptide syntheses were carried out with an Applied Biosystems
ABI 433 A peptide synthesiser (standard programme Fastmoc 0.1 mmol)
with use of pre-loaded Fmoc-Pro-Trt-Tentagel S resin (0.10 mmol, load-
ing: 0.21–0.23). For the coupling reactions, the amino acids Fmoc-Ala-
3J
3J
3J
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
OH, Fmoc-Arg
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
17H; 2-H-, 7-H-Fmoc, HAr), 5.83 (d, 3J
N
Fmoc-Val-OH were employed. In every coupling cycle, the N-terminal
Fmoc group was removed by treatment of the resin with a solution of pi-
peridine (20%) in NMP for at least 3ꢅ2.5 min. The coupling of the
amino acids (1 mmol or 10 equiv based on the loaded resin) was carried
out with HBTU (1 mmol), HOBt (1 mmol) and DIPEA (2 mmol) in
DMF (20–30 min vortex). After every coupling step, unreacted amino
5.51 (d, 3J
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
(d, 2J(H,H)=11.6 Hz, 1H; CH2-Bn), 4.27 (t, 3J
ACHTUNGTRENNUNG ACHTUNGTNER(NUGN 9-H,10-H)=6.7 Hz, 1H;
groups were capped by treatment with
a mixture of Ac2O (0.5m),
9-H-Fmoc), 4.24–4.15 (m, 3H; Ta {4.23}, 6a/b-H {4.19}, Tb {4.19}, 4.12–
DIPEA (0.125m) and HOBt (0.015m) in NMP (10 min vortex). Coupling
of the glycosylated threonine building blocks was performed with the use
of HATU (1.1 equiv with respect to the glycosyl amino acid), HOAt
(1.1 equiv) and NMM (2.2 equiv) for activation (8 h vortex). After cou-
pling of the remaining five amino acids by the standard procedure, the
triethylene glycol spacer (1 mmol, 10 equiv based on the loaded resin)
was coupled by use of HBTU (1 mmol), HOBt (1 mmol) and DIPEA
(2 mmol) in DMF (20–30 min vortex) and the N-terminal Fmoc group
was removed by treatment with piperidine (20%) in NMP. Detachment
from the resins and simultaneous removal of all side chain protecting
groups was performed by shaking with TFA (10 mL), TIS (1.0 mL) and
H2O (1.0 mL) in a Merrifield glass reactor for 3 h. The solutions were fil-
tered, the resins were washed with TFA (3ꢅ5 mL) and CH2Cl2 (3ꢅ
5 mL), and the combined TFA solutions were concentrated in vacuo to a
volume of 0.5 mL. After co-evaporation with toluene (5ꢅ20 mL), the
crude products were dissolved in H2O and subjected to lyophilisation.
4.06 (m, 1H; 5-H), 3.94 (dd, 2J(6a-H,6b-H)=11.1 Hz, 3J(6a/b-H,5-H)=
8.0 Hz, 1H; 6a/b-H), 3.90 (t, 3J(4’-H,3’-H)=4J
ACHTUNGTRENNUNG
H), 3.80 (dd, 3J(3-H,2-H)=11.1 Hz, 3J
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
3.60–3.52 (m, 4H; 6a’/b’-H {3.55}, 3’-H {3.54}, 4-H {3.53}), 2.10, 2.03 (2 ꢅ
s, 6 H; CH3-Ac), 1.92 (s, 3H; CH3-NHAc), 1.46 (s, 9H; tBu), 1.30 ppm (d,
ACHTUNGTRENNUNG
3H; 3J(Tg,Tb)=6.2 Hz, Tg); 13C NMR (100 MHz, CDCl3, DEPT,
HMQC): d=170.5, 170.4, 170.2, 169.9 (C=O), 156.5 (C=O-urethane),
143.7, 143.7 (C-1a-, C-8a-Fmoc), 141.3 (C-4a-, C-5a-Fmoc), 138.4, 137.9,
137.8 (Cq-Bn), 128.4, 128.2, 127.9, 127.9, 127.8, 127.7, 127.5, 127.1 (Car-Bn,
C-3-, C-6-Fmoc, C-2-, C-7-Fmoc), 125.0, 125.0 (C-1-, C-8-Fmoc), 120.0
(C-4-, C-5-Fmoc), 102.2 (d, 2J
(d, 1J(C-2’,F)=182.3 Hz; C-2’), 83.1 (Cq-tBu), 79.8 (d, 2J
15.5 Hz; C-3’), 77.0 (Tb), 76.2 (C-3), 74.6 (CH2-Bn), 74.0 (d, 3J
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
8.7 Hz; C-4’), 73.6 (CH2-Bn, C-4), 72.4 (CH2-Bn), 69.4 (C-5’), 68.2 (C-6’),
68.2 (C-5), 67.1 (CH2-Fmoc), 63.4 (C-6), 59.1 (Ta), 48.4 (C-2), 47.2 (C-9-
Fmoc), 28.1 (CH3-tBu), 23.3 (CH3-NHAc), 20.9, 20.7 (CH3-Ac), 18.7 ppm
Glycopeptide 38: The MUC1 glycopeptide analogue 38 was prepared by
the general procedure from Fmoc-Pro-Trt-Tentagel S resin (435 mg,
0.10 mmol; loading: 0.23 mmolgꢀ1) and the glycosylated threonine build-
ing block 22 (106.2 mg, 0.127 mmol). The crude product was purified by
RP-HPLC (Luna, MeOH/H2O +0.1% TFA, 10:90!100:0, 40 min, tR =
30.1 min) to afford the partially protected glycopeptide 35 as a colourless
amorphous solid (77 mg, 0.03 mmol, 29%). Glycopeptide 35 (66 mg,
0.026 mmol) was dissolved in aqueous NaOH (5 mL, pH 10.5) and the so-
lution was being stirred for 2 days at room temperature. The solution was
neutralised by addition of AcOH (3 drops), concentrated in vacuo and
co-evaporated with toluene (3ꢅ10 mL). Purification by RP-HPLC (Jupi-
ter, MeOH/H2O +0.1% TFA, 10:90!100:0, 30 min, tR =26.6 min) and
lyophilisation afforded the glycopeptide 37 as a colourless amorphous
solid (39 mg, 0.016 mmol, 55%); analytical RP-HPLC (Luna, MeOH/
H2O +0.1% TFA, 10:90!100:0, 40 min): tR =18.0 min; [a]2D3 =ꢀ166.6
(c=1.0 in H2O); 1H NMR (400 MHz, D2O, COSY): d=8.62 (d,
(Tg); 19F NMR (376 MHz, CDCl3): d=ꢀ204.4 ppm (dd, 2J
ACHTUNGTRENN(UNG F,2-H)=
51.6 Hz, 3J
ACHTUNGTRENNUNG(F,3-H)=12.2 Hz); HRMS (ESI-TOF): m/z: calcd for
C62H71FN2O16Na: 1141.4685 [M+Na]+; found: 1141.4691.
Fmoc-Thr(b-2F-Bn3Gal-(1–3)-a-Ac2GalNAc)-OH (32): Anisole (0.8 mL,
7.32 mmol) and TFA (8.0 mL, 45.6 mmol) were added to a solution of
disaccharide 6 (282 mg, 0.25 mmol) in dry CH2Cl2 (20 mL) and the reac-
tion mixture was stirred for 2 h at room temperature. The mixture was
co-evaporated with toluene (5ꢅ30 mL) and concentrated in vacuo. The
residue was purified by flash chromatography on silica gel (CH2Cl2/
MeOH 5:0.3) to afford 27 as a colourless amorphous solid (188 mg,
0.18 mmol, 70%); analytical RP-HPLC (Luna, H2O/MeCN 50:50!
10:90, 30 min): tR =26.07 min; [a]2D3 =120.4 (c=1.0 in CHCl3); Rf =0.09
(CH2Cl2/MeOH 5:0.3); 1H NMR (400 MHz, CDCl3, COSY): d=12.94 (s,
1H; COOH), 8.32 (s, 1H; OH), 7.90 (d, 3J
G
ACHTUNGTRENUN(NG 4-H,6-H)=
3
7.5 Hz, 2H; 4-H-, 5-H-Fmoc), 7.73 (d, J
G
U
3J
(d, 3J
{4.68}, 6a-H {4.67}, Ta* {4.66}, Ra {4.65}, A3 {4.64}), 4.61–4.51
{4.69}, 6a’-HAHCTUNGTRNEGNU
a a a
A
(Hd,He)=1.2 Hz, 1H; Hd), 5.01
ACHTUNGTRENNUNG
2H; 1-H-, 8-H-Fmoc), 7.48–7.38 (m, 4H; 3-H-, 6-H-Fmoc, NH-Fmoc
{7.45}, NH-Ac {7.40}), 7.38–7.25 (m, 17H; 2-H-, 7-H-Fmoc, Har), 5.31 (d,
AHCTUNGTRENNUNG
3J
ACHTUNGTRENNUNG(4-H,3-H)=3.0 Hz, 1H; 4-H), 4.79–4.68 (m, 3H; CH2-Bn {4.77}, CH2-
a
Bn {4.72}, 1-H {4.71}), 4.66–4.59 (m, 2H; 1’-H {4.71}, CH2-Bn {4.62}),
(m, 5H; A2 {4.59}, 6b’-H {4.58}, A4 {4.57}, 6b-H {4.56}, S1 {4.52}), 4.48–
a
a
4.19 (m, 17H; S2 {4.67}, 1’-H {4.47}, Va {4.42}, P1–5 {4.41, 4.39, 4.38, 4.35,
4.55–4.41 (m, 5H; CH2-Bn {4.53}, CH2-Fmoc {4.51}, CH2-Bn {4.52, 4.44,
ACHTUNGTRENNUNG
d, 2J(H,H)=11.9 Hz}), 4.33–4.22 (m, 2H; 9-H-Fmoc {4.31}, Tb {4.27}),
a
a
4.33}, T1–2 {4.36, 4.31}, Tb* (4.31}, 5-H {4.28}, 2-H {4.24}, A1 {4.23}, 4-H
4.30–4.13 (m, 1H; 2’-H {4.28}, {4.15}), 4.19–4.11 (m, 2H; 2-H {4.18}, Ta-
ACHTUNGTRENNUNG{4.14}), 4.07–3.99 (m, 3H; 5-H {4.04}, 4’-H {4.03}, 6a-H {4.02}), 3.91–3.78
b
{4.22}, T1–2 {4.21, 4.20}), 4.03 (d, 3J
(3-H,4-H)=4.8 Hz, 1H; 3-H), 4.00–
3.95 (m, 5H; Ga {3.99, 3.97}, 4’-H {3.95}), 3.93–3.74 (m 17H; 5’-H {3.91},
b
d
(m, 2H; 3-H {3.86}, 6b-H {3.81}), 3.76–3.67 (m, 2H; 5’-H {3.73}, 3’-H
{3.71}), 3.55–3.46 (m, 2H; 6a’/b’-H {3.51}), 2.02, 1.97, (2 ꢅ s, 6H; CH3-
S1,2 {3.87, 3.83}, P1–4 {3.85, 3.81, 3.78, 3.75}, CH2O-spacer {3.75, 3.74}),
dA
3.69–3.65 (m, 10H; P5 CTHNUGRTNE{NUG 3.69}, CH2O-spacer {3.69, 3.68, 3.67, 3.64}, 3.62
ACTHNUTRGNEUNG
Ac), 1.81 (s, 3H; CH3-NHAc), 1.12 ppm (d, 3J(Tg,Tb)=6.4 Hz, 3H; Tg);
(dd, 3J(3’-H,4’-H)=3.2 Hz, 3J(3’-H,2’-H)=10.1 Hz, 1H; 3’-H), 3.51 (dd,
13C NMR (100 MHz, CDCl3, DEPT, HMQC): d=171.7, 170.0, 169.8 (C=
O), 156.9 (C=O-urethane), 143.8, 143.7 (C-1a-, C-8a-Fmoc), 140.8, 140.8
(C-4a-, C-5a-Fmoc), 138.6, 138.3, 138.2 (CAr-Bn), 128.3, 128.2, 127.6,
127.5, 127.4, 127.1 (C-2-, C-7-, C-3-, C-6-Fmoc, CAr-Bn), 125.2, 125.1 (C-
3J(2’-H,1’-H)=7.8 Hz, 3J(2’-H,3’-H)=9.9 Hz, 1H; 2’-H), 3.31 (dd,
2
3J
(Hba,Ha)=5.5 Hz, J
(Hba,Hbb)=15.5 Hz, 1H; Hba), 3.21–3.16 (m 5H;
Rd {3.2}, Hb {3.19} CH2NH2-spacer {3.17}), 3.00–2.87 (m, 2H; Db), 2.79–
b
2.63 (m, 2H; CH2CO-spacer), 2.38–2.21 (m, 6H; P1–3 {2.34, 2.30, 2.24},
1-, C-8-Fmoc), 120.2, 120.2 (C-4-, C-5-Fmoc), 101.2 (d, 2J
23.2 Hz; C-1’), 99.3 (C-1), 90.8 (d, 1J
2J(C-3’,F)=12.1 Hz; C-3’), 75.1 (Tb), 75.0 (C-3), 74.1 (CH2-Bn), 73.8 (C-
5), 72.7 (CH2-Bn), 72.6 (C-5’), 71.0 (CH2-Bn), 67.9 (C-6’), 67.4 (C-4’),
T
2.12–1.84 (m, 16H; Vb {2.10}, P1–5 {2.08, 2.05, 2.00, 1.94, 1.93}, CH3-
g
b
N
NHAc {2.00}, P4,5 {1.92}, Rba {1.84}), 1.75–1.68 (m, 3H; Rbb {1.72}, Rg
b
T
{1.66}), 1.39–1.30 (m, 12H; A1–4 {1.38, 1.36, 1.34, 1.32}), 1.26 (d,
3J
(Tg,Tb)=6.3 Hz, 3H; Tg*), 1.20 (d, 3J
(Tg,Tb)=6.5 Hz, 3H; T1 ), 1.18
g
7328
www.chemeurj.org
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 7319 – 7330

Fluorinated Glycosyl Amino Acids
FULL PAPER
g
b
a
(d, 3J
G
E
{4.04}), 3.97–3.91 (m, 2H; T2 {3.93}, 4-H {3.92}), 3.89–3.30 (m, 34H; G1
a
0.96 ppm (d, 3J
(Vg,Vb)=4.8 Hz, 3 H Vg); 13C NMR (100 MHz, D2O,
{3.85}, G2 {3.83}, 5-H {3.70}, 4’-H {3.69}, 3-H {3.66}, 3’-H {3.51}, 6a/b-H
d
DEPT, HMQC): d=176.5, 175.8, 174.9, 174.5, 174.4, 174.0, 173.7, 173.6,
173.5, 173.1, 173.0, 172.7, 172.5, 172.4, 172.0, 171.9, 171.5, 171.3, 171.1,
170.9, 170.7 (C=O), 156.7 (C=NH), 133.4 (He), 128.4 (Hg), 117.3 (Hd),
{3.51}, P1–5 {3.62, 3.55, 3.50, 3.45}, CH2-spacer {3.60, 3.58}, CH2O-spacer
{3.54, 3.48}, 6a’/b’-H {3.45}), 3.18–2.92 (m, 6H; Hba {3.14, dd,
2J(Hba,Hbb)=15.9 Hz, 3J(Hba,Ha)=5.0 Hz}, Rd {3.09}, Hba {2.97},
ACTHNUTRGENNUG CAHTUNGTRENNUGN
104.5 (C-1’), 99.2 (C-1), 83.7 (d, 1J
1J
20.4 Hz; C-5’), 72.3 (C-3’), 70.4 (C-2’), 69.5 (C-5), 69.6, 69.5, 69.5, 69.4
(CH2O-spacer), 68.5 (d, 3J(F,C-4)=9.0 Hz; C-4), 68.1 (d, 3J
(F,C-4’)=
CH2NH2-spacer {2.97}), 2.73 (dd, 2J
(Dba,Dbb)=16.1 Hz, 3J
ACHUTGTNRNEUNG CAHUTNGTRENN(GUN Dba,Da)=
G
5.3 Hz, 1H; Dba), 2.62–2.52 (m, 3H; Dbb {2.53}, CH2CO-spacer {2.52}),
b
g
2.19–1.66 (m, 24H; P1–5 {2.13, 1.99}, P1–5 {1.92, 1.89, 1.86, 1.82}, CH3-
NHAc {1.79}, Rba {1.69}), 1.52 (brs, 1H; Rg), 1.21 (d, 3J
(A1b,A1a)=
7.2 Hz, 3H; A1b), 1.19 (d, J(A2–4b,A2–4a)=7.0 Hz, 9H; A(2–4)b), 1.13 (d,
3
7.1 Hz; C-4’), 67.0 (T1b), 66.3 (T2b), 66.0 (CH2-spacer), 61.4, 61.1 (S1,2b),
60.8, 60.5, 60.1, 60.0 (P1–5a), 59.4 (Va), 59.9, 58.8 (T1,2a), 57.0 (Ta*), 55.6,
55.0 (S1,2a), 52.3 (Ha), 51.1 (Ra), 50.1 (Da), 49.6 (C-2), 48.2, (Aa), 48.1,
47.9, 47.8, (P1–3d), 47.7, 47.6 (Aa), 47.4, 47.3 (P4,5d), 42.4, 42.3 (G1,2a), 40.5
(Rd), 39.1 (CH2NH2-spacer), 34.9 (Db), 34.0 (CH2CONH-spacer), 30.1
(Vb), 29.6, 29.3, 29.2, 28.7 (P1–4b), 27.5 (Rb), 26.2 (Hb), 24.7, 24.6, 24.5,
24.3 (P1–5g), 23.9 (Rg), 22.3 (CH3-NHAc), 18.8, 18.7 (Tg), 18.5 (Vg), 18.2
(Tg*), 17.8 (Vg), 16.3, 15.3, 15.2, 15.1 ppm (A1–4b); 19F NMR (376 MHz,
3J(Tg,Tb)=6.2 Hz, 3H; Tg*), 1.03 (d, J
ACTHUNTRGENNGU ACHTUGNRTENUN(GN T1g,T1b)=6.4 Hz, 3H; T1 ), 1.00
3
g
3
g
3
(d, J
N
ACHTUNGTRENNUNG
0.84 ppm (d, 3J
N
V
[D6]DMSO, HMQC): d=173.2, 172.5, 172.2, 172.0, 171.9, 171.9, 171.7,
171.3, 171.1, 170.9, 170.4, 170.3, 170.2, 170.0, 169.5, 169.4, 169.3, 168.8
(C=O), 156.7 (C=NH), 133.8 (He), 129.2 (Hg), 117.1 (Hd), 102.2 (C-1’),
98.6 (C-1), 91.6 (d, 1J
74.4 (Tb*), 71.6 (C-5), 71.5 (d, J
ACTHNUGTRNEUN(G C-3’,F)=15.1 Hz; C-3’), 68.7 (C-4’), 67.9
ACHTUNGTREN(NGNU C-2’,F)=175.3 Hz; C-2’), 77.9 (C-3), 75.2 (C-5’),
2
D2O): d=ꢀ229.4 ppm (m, 2F; {-229.4 (dt, 3J
(C-4), 67.0, 69.7, 69.6 (CH2O-spacer), 66.8 (T2b), 66.7, 66.3 (CH2-spacer),
66.2 (T1b), 61.8 (S1,2b), 60.8 (C-6’), 60.2 (C-6), 59.8, 59.5, 59.2, 59.1 (P1–4a),
59.3 (Ta*), 58.5 (P5a), 58.0 (T2a), 57.7 (T1a), 57.2 (Va), 54.9 (S1a), 54.7
(S2a), 51.4 (Ha), 50.1 (Da), 49.5 (Ra), 48.4 (A1a), 47.1 (C-2), 47.0, 46.8,
46.7, 46.5 (P1–5d), 46.3, 46.3 (A2–4a), 42.0 (G1,2a), 40.6 (Rd), 38.6 (CH2NH2-
spacer), 35.5 (Db), 34.3 (CH2-spacer), 31.0 (Vb), 29.3, 29.1, 29.1, 29.0, 28.6
(P1–5b), 28.4 (Rb), 26.9 (Hb), 24.6, 24.5, 24.4, 24.3 (P1–5g), 24.4 (Rg), 22.8
48.5 Hz}); MALDI-TOF-MS (dhb, positive ion mode), m/z: calcd for
C103H166F2N27O40: 2460.57 [M+H]+; found: 2460.90; HRMS (ESI-TOF),
m/z: calcd for C43H55FN2O17Na: 913.3385 [M+Na]+; found: 913.3383.
Glycopeptide 39: The MUC1 glycopeptide analogue 39 was prepared by
the general procedure from Fmoc-Pro-Trt-Tentagel S resin (476 mg,
0.10 mmol; loading: 0.21 mmolgꢀ1) and the glycosylated threonine build-
ing block 27 (225.3 mg, 0.245 mmol). The crude product was purified by
RP-HPLC (Jupiter, MeOH/H2O +0.1% TFA, 10:90!100:0, 40 min, tR =
24.4 min) to afford the partially protected glycopeptide 36 as a colourless
amorphous solid (55.9 mg, 21%); tR =21.3 min (Phenomenex Luna,
g
g
(CH3-NHAc), 19.8 (T1 ), 19.6 (T2 ), 19.2 (Vga), 18.1 (Vgb), 18.1 (Tg*), 17.4
(A1b), 16.9, 16.8, 16.6 ppm (A2–4b); 19F NMR (376 MHz, [D6]DMSO): d=
ꢀ205.4 ppm (dd, 2J
(F,2-H)=51.9 Hz, 3J
ACHTUGTNRENNUG CAHTUNGTRENN(UGN F2,3-H)=14.6 Hz); MALDI-
TOF-MS, (dhb, positive ion mode): m/z: calcd for C103H167FN27O41:
2458.58 [M+H]+; found: 2458.29; HRMS (ESI-TOF): m/z: calcd for
C103H168FN27O41 [M+2H]2+: 1229.0890; found: 1229.0933.
grad.: CH3CN/H2O +0.1% TFA (5:95)!
10 min). A solution of the partially protected glycopeptide 36 in dry
MeOH (20 mL) was carefully adjusted to pH 9.5 by addition of small
portions of NaOMe and was stirred for 16 h at room temperature. The
solution was neutralised by addition of AcOH (3 drops), concentrated in
vacuo and co-evaporated with toluene (3ꢅ10 mL). Purification by RP-
HPLC (Luna, MeCN/H2O +0.1% TFA, 5:95!40:60, 30 min!100:0,
10 min, tR =17.2 min) and lyophilisation afforded the glycopeptide 39 as
a colourless amorphous solid (31 mg, 0.012 mmol, 60%);
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft
(Emmy Noether Fellowship to A.H.R.) and the Fonds der Chemischen
Industrie (Liebig Fellowship to A.H.R. and doctoral fellowship to S.W.).
We thank Prof. Dr. H. Kunz for fruitful discussions and D. Ferenc for as-
sistance with the glycopeptide syntheses.
The MUC1 glycopeptide analogue 39 was also prepared by the general
procedure from Fmoc-Pro-Trt-Tentagel S resin (454 mg, 0.10 mmol; load-
ing: 0.22 mmolgꢀ1) and the glycosylated threonine building block 32
(208 mg, 0.195 mmol). Without further purification the crude product was
dissolved in degassed MeOH (30 mL) under argon and treated with a
catalytic amount of PdACHTUNGTRENNUNG(OAc)2. The reaction flask was purged with hydro-
gen and the reaction mixture was stirred under hydrogen for 12 h. The
mixture was filtered through Hyflo Supercel, which was afterwards
washed with MeOH (5ꢅ50 mL). The solvent was evaporated in vacuo,
the residue was again dissolved in dry MeOH (30 mL), and the pH of the
solution was adjusted to pH 9 with a freshly prepared NaOMe solution
(1%). After the mixture had been stirred for 18 h at room temperature,
the reaction was stopped by addition of AcOH (5 drops) and the solvents
were evaporated in vacuo. Purification by RP-HPLC (Luna, CH3CN/
H2O +0.1% TFA, 5:95!40:60, 30 min!100:0, 10 min, tR =17.2 min)
and lyophilisation afforded the glycopeptide 39 as a colourless amor-
phous solid (83.1 mg, 0.033 mmol, 34%); analytical RP-HPLC (Luna,
MeCN/H2O +0.1% TFA, 5:95!40:60, 30 min!100:0, 10 min): tR =
13.2 min; [a]2D3 =ꢀ89.4 (c=1.0 in H2O); 1H NMR (400 MHz, [D6]DMSO,
[1] a) S. Hakomori, Adv. Cancer Res. 1989, 52, 257–331; b) S. Hako-
mori, Y. Zhang, Chem. Biol. 1997, 4, 97–104.
[2] a) G. F. Springer, Science 1984, 224, 1198–1206; b) J. Taylor-Papadi-
mitriou, J. Burchell, D. W. Miles, M. Daziel, Biochim. Biophys. Acta
Mol. Basis Dis. 1999, 1455, 301–313; c) F. G. Hanisch, S. Mꢁller,
Glycobiology 2000, 10, 439–449; d) J. M. Burchell, A. Mungul, J.
Taylor-Papadimitriou, J. Mammary Gland Biol. Neoplasia 2001, 6,
355–364.
[3] a) M. F. Wolf, A. Ludwig, P. Fritz, K. Schumacher, Tumour Biol.
1988, 9, 190–194; b) H. Zhang, S. Zhang, N.-K. V. Cheung, G. Ragu-
pathi, P. O. Livingston, Cancer Res. 1998, 58, 2844–2849; c) J. Heim-
burg, J. Yan, S. Morey, O. V. Glinskii, V. H. Huxley, L. Wild, R.
Klick, R. Roy, V. V. Glinsky, K. Rittenhouse-Olson, Neoplasia 2006,
8, 939–948; d) L. Yu, Glycoconjugate J. 2007, 24, 411–420.
[4] a) J. Taylor-Papadimitriou, A. A. Epenetos, Trends Biotechnol. 1994,
12, 227–233; b) P. O. Livingston, S. Zhang, K. O. Lloyd, Cancer Im-
munol. Immunother. 1997, 45, 1–9; c) S. J. Danishefsky, J. R. Allen,
Angew. Chem. 2000, 112, 882–912; Angew. Chem. Int. Ed. 2000, 39,
836–863; d) S. S. Slovin, S. J. Keding, G. Ragupathi, Immunol. Cell
Biol. 2005, 83, 418–428; e) T. Becker, S. Dziadek, S. Wittrock, H.
Kunz, Curr. Cancer Drug Targets 2006, 6, 491–517; f) A. Liakatos,
H. Kunz, Curr. Opin. Mol. Ther. 2007, 9, 35–44; g) T. Buskas, P.
Thompson, G.-J. Boons, Chem. Commun. 2009, 5335–5349.
COSY, TOCSY, NOESY): d=12.44 (brs, 2H; COOH), 8.96 (s, 1H; Hd),
NH
8.34–8.18 (m, 4H; A1
3J
A(2,3)NH), 8.13 (d, 3J
7.2 Hz, 1H; HNH), 8.04–7.88 (m, 4H; TNH* {8.00}, RNH {7.97}, VNH {7.91,
ACTHNGUTERNNUG
{8.31, d, 3J(NH,Aa)=7.0 Hz}, DNH {8.25, d,
NH
G
{8.21}), 8.14 (d, 3J
ACHTUNGTRENNUNG
E
ACHTUNGTRENNUNG
NH
NH
d, 3J
{7.75, d, 3J
5.5 Hz, 1H; NHimidazole), 7.38 (s, 1H; RNH–guanidine), 7.34 (d, 3J
8.0 Hz, 1H; T2NH), 6.99 (d, 3J
(NH,2-H)=9.3 Hz, 1H; NH-Ac)), 4.72 (d,
ACHTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
3J
(1-H,2-H)=2.5 Hz, 1H; 1-H), 4.60–4.35 (m, 10H; 1’-H {4.56}, Ha {4.55},
a
a
a
a
b
A2,3 {4.51, 4.48}, Da {4.50}, Ra {4.49}, Va {4.48}, Ta* {4.41}, S1 {4.37}, S2
[5] C-Glycosides: a) D. Urban, T. Skrydstrup, J.-M. Beau, Chem.
Commun. 1998, 955–956; b) C. H. Rçhrig, M. Takhi, R. R. Schmidt,
Synlett 2001, 1170–1172; c) B. Kuberan, S. A. Sikkander, H. Tomiya-
a
{4.36}), 4.34–4.01 (m, 13H; 2-H {4.23}, 2’-H {4.20, 4.07}, T1 {4.18}, T2
a
a
a
{4.17}, Tb* {4.09}, A1 {4.24}, P(1–5) {4.27, 4.25, 4.21}, A4 {4.15}, T1
Chem. Eur. J. 2010, 16, 7319 – 7330
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7329

A. Hoffmann-Rçder et al.
ma, R. J. Linhardt, Angew. Chem. 2003, 115, 2119–2121; Angew.
Chem. Int. Ed. 2003, 42, 2073–2075; d) L. Cipolla, M. Rescigno, A.
Leone, F. Peri, B. La Ferla, F. Nicotra, Bioorg. Med. Chem. 2002, 10,
1639–1646. S-Glycosides: e) J. J. Rich, D. R. Bundle, Org. Lett.
2004, 6, 897–900; f) E. Bousquet, A. Spadaro, M. S. Pappalardo, R.
Bernardini, R. Romeo, L. Panza, G. Ronsisvalle, J. Carbohydr.
Chem. 2000, 19, 527–541; g) D. R. Bundle, J. J. Rich, S. Jacques,
H. N. Yu, M. Nitz, C.-C. Ling, Angew. Chem. 2005, 117, 7903–7907;
Angew. Chem. Int. Ed. 2005, 44, 7725–7729; h) J. J. Rich, W. W. Wa-
karchuk, D. R. Bundle, Chem. Eur. J. 2006, 12, 845–858; i) X. Wu,
T. Lipinski, E. Paszkiewicz, D. R. Bundle, Chem. Eur. J. 2008, 14,
6474–6482.
[21] W. A. Szarek, A. Zamojski, K. N. Tiwari, E. R. Ison, Tetrahedron
Lett. 1986, 27, 3827–3830.
[22] a) S. Mꢁller, F.-G. Hanisch, J. Biol. Chem. 2002, 277, 26103–26112;
b) M. Fukuda, S. R. Carlsson, J. C. Klock, A. Dell, J. Biol. Chem.
1986, 261, 12796–12806.
[23] F. Dasgupta, P. J. Garegg, Carbohydr. Res. 1988, 177, c13-c17.
[24] S. Dziadek, H. Kunz, Synlett 2003, 1623–1626.
[25] R. U. Lemieux, R. M. Ratcliffe, Can. J. Chem. 1979, 57, 1244–1251.
[26] a) H. Paulsen, K. Adermann, Liebigs Ann. Chem. 1989, 751–769;
b) H. Kunz in Preparative Carbohydrate Chemistry (Ed.: S. Hanessi-
an), Marcel Dekker, New York, 1997, pp. 265–281.
[27] a) B. Liebe, H. Kunz, Angew. Chem. 1997, 109, 629–631; Angew.
Chem. Int. Ed. Engl. 1997, 36, 618–621; b) B. Liebe, H. Kunz, Helv.
Chim. Acta 1997, 80, 1473–1482.
[6] a) R. U. Lemieux, R. Cromer, U. Spohr, Can. J. Chem. 1988, 66,
3083–3098; b) C. P. J. Glaudemans, Chem. Rev. 1991, 91, 25–33, and
cited ref.; c) B. Holm, S. M. Baquer, L. Holm, R. Holmdahl, J. Kihl-
berg, Bioorg. Med. Chem. 2003, 11, 3981–3987; d) L. Barbieri, V.
Costantino, E. Fattorusso, A. Mangoni, N. Basilico, M. Mondani, D.
Taramelli, Eur. J. Org. Chem. 2005, 3279–3285; e) J. Wang, H. Li, G.
Zhou, L.-X. Wang, Org. Biomol. Chem. 2007, 5, 1529–1540.
[7] R. U. Lemieux, Chem. Soc. Rev. 1989, 18, 347–374.
[8] D. OꢆHagan, Chem. Soc. Rev. 2008, 37, 308–319.
[9] R. Pongdee, H. Liu, Bioorg. Chem. 2004, 32, 393–437.
[10] a) P. J. Card, J. Carbohydr. Chem. 1985, 4, 451–487; b) T. Tsuchiya,
Adv. Carbohydr. Chem. Biochem. 1990, 48, 91–277; c) K. Dax, M.
Albert, J. Ortner, B. J. Paul, Carbohydr. Res. 2000, 327, 47–86; d) R.
Miethchen, J. Fluorine Chem. 2004, 125, 895–901.
[11] a) L. T. Rexford, A. A. Saeed, K. L. Matta, Carbohydr. Res. 1987,
165, C14–C16; b) J. Xia, J. L. Alderfer, C. F. Piskorz, R. D. Locke,
K. L. Matta, Synlett 2003, 1291–1294; c) J. Xia, J. Xue, R. D. Locke,
E. V. Chandrasekaran, T. Srikrishnan, K. L. Matta, J. Org. Chem.
2006, 71, 3696–3706; d) J. Xue, V. Kumar, S. D. Khaja, E. V. Chan-
drasekaran, R. D. Locke, K. L. Matta, Tetrahedron 2009, 65, 8325–
8335.
[28] R. R. Schmidt, M. Stumpp, Liebigs Ann. Chem. 1983, 1249–1256.
[29] M. Albert, K. Dax, J. Ortner, Tetrahedron 1998, 54, 4839–4848.
[30] M. Albert, B. J. Paul, K. Dax, Synlett 1999, 1483–1485.
[31] a) M. D. Burkart, Z. Zhang, S.-C. Hung, C.-H. Wong, J. Am. Chem.
Soc. 1997, 119, 11743–11746; b) S. P. Vincent, M. D. Burkart, C.-Y.
Tsai, Z. Zhang, C.-H. Wong, J. Org. Chem. 1999, 64, 5264–5279.
[32] L. Ayala, C. G. Lucero, J. A. C. Romero, S. A. Tabacco, K. A. Woer-
pel, J. Am. Chem. Soc. 2003, 125, 15521–15528.
[33] a) S. Ando, Y. Nakahara, Y. Ito, T. Ogawa, Y. Nakahara, Carbohydr.
Res. 2000, 329, 773–780; b) Y. Takano, N. Kojima, Y. Nakahara, H.
Hojo, Y. Nakahara, Tetrahedron 2003, 59, 8415–8427; c) Y. Naka-
hara, C. Ozawa, E. Tanaka, K. Ohtsuka, Y. Takano, H. Hojo, Y. Na-
kahara, Tetrahedron 2007, 63, 2161–2169; d) E. Tanaka, Y. Naka-
hara, Y. Kuroda, Y. Takano, H. Hojo, Y. Nakahara, Biosci. Biotech-
nol. Biochem. 2006, 70, 2515–2522.
[34] Y. Niu, N. Wang, X. Cao, X.-S. Ye, Synlett 2007, 2116–2120.
[35] S. Dziadek, D. Kowalczyk, H. Kunz, Angew. Chem. 2005, 117, 7798–
7803; Angew. Chem. Int. Ed. 2005, 44, 7624–7630.
[36] A. Kaiser, N. Gaidzik, U. Westerlind, D. Kowalczyk, A. Hobel, E.
Schmitt, H. Kunz, Angew. Chem. 2009, 121, 7688–7692; Angew.
Chem. Int. Ed. 2009, 48, 7551–7555.
[12] C. Mersch, S. Wagner, A. Hoffmann-Rçder, Synlett 2009, 2167–
2171.
[13] S. Dziadek, C. Brocke, H. Kunz, Chem. Eur. J. 2004, 10, 4150–4162.
[14] M. C. Kasuya, A. Ito, K. Hatanaka, J. Fluorine Chem. 2007, 128,
562–565.
[15] S. Keil, C. Claus, W. Dippold, H. Kunz, Angew. Chem. 2001, 113,
379–382; Angew. Chem. Int. Ed. 2001, 40, 366–369.
[37] U. Westerlind, H. Schrçder, A. Hobel, N. Gaidzik, A. Kaiser, C. M.
Niemeyer, E. Schmitt, H. Waldmann, H. Kunz, Angew. Chem. 2009,
121, 8413–8417; Angew. Chem. Int. Ed. 2009, 48, 8263–8267.
[38] J. M. J. Frꢄchet, K. E. Haque, Tetrahedron Lett. 1975, 16, 3055–3056.
[39] V. Dourtoglou, B. Gross, V. Lambropoulou, C. Zioudrou, Synthesis
1984, 572–574.
[16] S. Kobayashi, A. Yoneda, T. Fukuhara, S. Hara, Tetrahedron Lett.
2004, 45, 1287–1289.
[40] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397–4398.
[17] M. D. Burkart, S. P. Vincent, A. Dꢁffels, B. W. Murray, S. V. Ley, C.-
H. Wong, Bioorg. Med. Chem. 2000, 8, 1937–1946.
[18] B. Helferich, K. Weis, Chem. Ber. 1956, 89, 314–321.
[19] P. A. J. Gorin, Carbohydr. Res. 1982, 101, 13–20.
[20] a) C. Brocke, H. Kunz, Synlett 2003, 2052–2056; b) C. Brocke, H.
Kunz, Synthesis 2004, 525–542.
Received: December 1, 2009
Revised: March 5, 2010
Published online: May 12, 2010
7330
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ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 7319 – 7330