Synthesis of fluorinated analogues of tumor-associated carbohydrate and glycopeptide antigens

Article abstract of DOI:10.1055/s-0029-1217566

Partial structures of tumor-associated mucin glycoproteins are interesting target structures for the development of selective anticancer vaccines. To probe the effect of fluorination on the immunological and metabolic properties of mucin glycopeptides, six novel fluorinated glycosyl-threonine conjugates have been synthesized. The synthesis of the orthogonally protected glycosyl amino acids was achieved using microwave irradiation in key fluorination and glycosylation steps. The 2′-deoxy-2′-fluoro- and 6′-deoxy-6′-fluoro-T antigen building blocks were applied to the synthesis of analogues of MUC1 tandem repeat-glycopeptide antigens via SPPS. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0029-1217566

LETTER
2167
Synthesis of Fluorinated Analogues of Tumor-Associated Carbohydrate and
Glycopeptide Antigens
F
luorinated Anal
h
T
um
r
or-Asso
i
ciated
s
A
ntigens tian Mersch, Sarah Wagner, Anja Hoffmann-Röder*
Institut für Organische Chemie, Johannes Gutenberg-Universität Mainz, Duesbergweg 10-14, 55128 Mainz, Germany
Fax +49(6131)3924786; E-mail: hroeder@uni-mainz.de
Received 20 April 2009
We here describe the synthesis of a first series of deoxy-
Abstract: Partial structures of tumor-associated mucin glycopro-
teins are interesting target structures for the development of selec-
tive anticancer vaccines. To probe the effect of fluorination on the
immunological and metabolic properties of mucin glycopeptides,
fluoro analogues of the tumor-associated TN, T, and 2,6-
sialyl-T antigens. The assembly of the fluorinated glyco-
syl amino acids was accomplished by stepwise extension
six novel fluorinated glycosyl–threonine conjugates have been syn- of the saccharide chain.10 The synthesis of the 6-deoxy-6-
thesized. The synthesis of the orthogonally protected glycosyl ami-
no acids was achieved using microwave irradiation in key
fluoro-TN antigen analogue 6 started from the known pre-
cursor 211 for which a convenient and scalable micro-
fluorination and glycosylation steps. The 2¢-deoxy-2¢-fluoro- and
wave-assisted fluorination procedure was developed
6¢-deoxy-6¢-fluoro-T antigen building blocks were applied to the
(Scheme 1). Thus, reacting 1,2:3,4-di-O-isopropylidene
synthesis of analogues of MUC1 tandem repeat-glycopeptide anti-
galactose (1) with (diethylamino)sulfur trifluoride
(DAST) in the presence of 2,6-collidine11a under micro-
wave irradiation (100 W12) led to the desired product 2 in
84% yield within one hour. By contrast, the corresponding
thermal reaction of 1 with DAST only furnished 2 in a
markedly reduced yield (45%, 2.5 h). Conversion of 2 into
galactosyl bromide 35b,13 and subsequent reductive elimi-
nation of the 1-bromo and 2-acetoxy groups afforded the
6-deoxy-6F-galactal (4, 63% over 4 steps). Azido-
nitration14 and installation of the anomeric bromide fur-
nished galactosyl donor 5 for Ag2CO3/AgClO4-promoted
conjugation15 to Fmoc-Thr-Ot-Bu.16 Under these condi-
tions predominant formation of the desired a-configured
conjugate was observed. Finally, reductive acetylation
and de-O-acetylation under Zemplén conditions at pH
8.517 yielded TN analogue 7 in 35% over three steps.
gens via SPPS.
Key words: fluorinated carbohydrates, glycopeptide antigens,
MUC1, solid-phase synthesis, microwave-assisted glycosylations
Cell-surface glycoconjugates have received much atten-
tion in recent years owing to their crucial role in mediating
important cellular recognition processes.1 For example,
the potential of mucin glycopeptide antigens, resulting
from aberrant glycosylation of tumor cells,2 as selective
anticancer vaccines has been demonstrated in mouse
models.3 However, the immunogenicity of the tumor-as-
sociated carbohydrate antigens is low, and these structures
are readily metabolized by glycosidases. Both factors op-
pose the development of efficient synthetic anticancer
vaccines. An interesting approach to overcome these lim-
itations is the use of hydrolytically stable glycopeptide
mimics for synthetic tumor-associated antigen analogues.
Compound 7 was then subjected to a selective 3-b-galac-
tosylation with known aAc4Gal-trichloroacetimidate
donor18 to provide the 6-deoxy-6-fluoro-T antigen ana-
logue 8 in 42%. The corresponding 6,6¢-difluoro-ana-
logue 9 was also obtained from acceptor 7. In this case,
however, best results of the desired disaccharide were ob-
tained using 6¢-fluoro-galactosyl donor 3 and Hg(CN)2 as
Fluorinated carbohydrate derivatives have emerged as
powerful tools for providing insight into the specificity of
carbohydrate binding to a variety of proteins.4 Fluorosug-
ars often retain much of the reactivity of natural saccha-
rides, with particular reference to polarity and structural
integrity. However, the presence of fluorine atoms close
to the anomeric center modulates the reactivity of the gly-
cosidic bond and stabilizes it against hydrolysis.5 Despite
examples of antibody–antigen complexes with fluorinated
saccharides,6 the use of such compounds in the develop-
ment of carbohydrate-based vaccines has not been stud-
ied. Thus, besides a number of methods for the synthesis
of fluorinated mono- and oligosaccharides,6,7 including
mucin-like core structures,8 only few examples of fluori-
nated glycosyl amino acids can be found in the litera-
ture.5c,9
promoter in
a
microwave-assisted glycosylation
reaction19 (Scheme 1). Thus, under microwave irradiation
(80 °C, 4 h, 100 W) clean conversion to the b-configured
disaccharide 9 (73%, b-anomer) was observed, while con-
ventional heating mainly led to decomposition of the
donor. It is remarkable, that the corresponding galacto-
sylation using the nonfluorinated galactosyl bromide
proceeded smoothly at room temperature10 without micro-
wave support.
In a similar manner, the 6¢-deoxy-6¢-fluoro-T antigen an-
alogue 11 was prepared in an excellent yield by micro-
wave-assisted 3-b-galactosylation of Fmoc-Thr(a4,6-O-
Bzn-GalNAc)-Ot-Bu (10)20 with 3 (Scheme 2). Again, the
desired b-anomer was obtained without significant forma-
tion of orthoester or a-configured byproducts. Subsequent
SYNLETT 2009, No. 13, pp 2167–2171
Advanced online publication: 15.07.2009
DOI: 10.1055/s-0029-1217566; Art ID: G13909ST
x
x
.x
x
.2
0
0
9
© Georg Thieme Verlag Stuttgart · New York

2168
C. Mersch et al.
LETTER
R1
AcO
AcO
AcO
AcO
O
F
F
b
c
O
O
O
O
AcO
O
Br
O
d
3
4
AcO
F
R1 = OH
1
O
a
2 R1 = F
AcO
N3
Br
5
e
R2O
R3
HO
O
AcO
AcO
F
F
O
O
O
R2O
g
AcHN
AcHN
AcO
O
O
R2 = Ac
Ot-Bu
R3 = OAc
6
8
9
Ot-Bu
f
FmocHN
FmocHN
7 R2 = H
R
3 = F
O
O
Scheme 1 Reagents and conditions: (a) i. DAST, 2,6-collidine, MW 100 W, 80 °C, 1 h, 84%; b) i. AcOH, reflux, then Ac2O–pyridine (1:2),
99%; ii. HBr/AcOH, CH2Cl2, 0 °C to r.t., 71%; (c) Zn, N-methylimidazole, EtOAc, reflux, 89%; (d) i. CAN, NaN3, MeCN, –18 °C, 47%; ii.
LiBr, MeCN, 67%; (e) i. Fmoc-Thr-Ot-Bu, Ag2CO3/AgClO4, MS 4 Å, toluene–CH2Cl2 (1:1), 0 °C to r.t., 59% (a-anomer); ii. Zn, THF–Ac2O–
AcOH (3:2:1), 75%; (f) NaOMe (cat.), MeOH, pH 8.5, 78%; (g) aAc4Gal-trichloroacetimidate, TMSOTf (cat.), MS 4 Å, CH2Cl2, –20 °C to r.t.,
54% (8, b-anomer); 3, Hg(CN)2, MeNO2–CH2Cl2 (3:2), MS 4 Å, MW 100 W, 80 °C, 4 h, 73% (9, b-anomer).
cleavage of the benzylidene acetal furnished disaccharide building block 13 after acetylation and liberation of the
11 (72% over 2 steps), which after regio- and stereoselec- carboxy group (79%, 2 steps).
tive sialylation reaction at 6-OH with sialyl xanthate 1421
furnished fluorinated a2,6-sialyl-T antigen analogue 15 in
42% yield. Besides, 11 was converted into glycopeptide
Antigen analogues with fluoro substituents in 2-position
of the galactose moiety are particularly interesting, as the
AcO
CO2Bn
AcO
OAc
AcO
CO2Bn
AcO
O
OAc
O
OEt
S
AcHN
AcO
O
AcO
S
R1O
O
HO
AcHN
OR1
O
AcO
AcO
F
F
AcO
14
O
O
O
AcO
O
d
AcHN
AcHN
AcO
AcO
O
O
15
OR2
Ot-Bu
a
Ph
FmocHN
FmocHN
O
O
O
1 = H, R2 = t-Bu
1 = Ac, R2 = t-Bu
O
11
R
R
b
c
O
12
HO
AcHN
13 R1 = Ac, R2 = H
O
10
Ot-Bu
Ph
FmocHN
O
g
O
AcO
O
AcO
AcO
AcO
OAc
OAc
O
OAc
O
h
RO
RO
OAc
O
O
O
AcO
O
O
AcHN
R
AcHN
F
F
O
O
F
e
19
AcO
AcO
OAc
O
R = OH
17
Ot-Bu
f
OR
FmocHN
R =
t-Bu
20
FmocHN
18 R = OC(NH)CCl3
i
21 R = H
O
O
16
Scheme 2 Reagents and conditions: (a) i. 3, Hg(CN)2, MeNO2–CH2Cl2 (3:2), MS 4 Å, MW 100 W, 80 °C, 1 h, 98% (b-anomer); ii. I2, MeOH,
reflux, 73%; (b) Ac2O–pyridine (1:2), 60 °C, 81%; (c) TFA–anisole (10:1), CH2Cl2, 97%; (d) MeSBr, AgOTf, MS 4 Å, MeCN–CH2Cl2 (2:1),
–40 °C to r.t., 43% (a-anomer); (e) Selectfluor, MeNO2–H2O (4:1), MW 100 W, 108 °C, 2 min, 66%; (f) CCl3CN, DBU, CH2Cl2, 71%; (g)
TMSOTf, MS 4 Å, CH2Cl2, 70% (a/b = 2:1); (h) i. I2, MeOH, 60 °C; ii. Ac2O–pyridine (1:2), 70% over 2 steps; (i) TFA–anisole (10:1), CH2Cl2,
88%.
Synlett 2009, No. 13, 2167–2171 © Thieme Stuttgart · New York

LETTER
Fluorinated Analogues of Tumor-Associated Antigens
2169
R2
AcO
AcO
AcO
O
OAc
O
O
O
Cl
O
Fmoc
R1
AcHN
N
O
13 R1 = OAc, R2 = F
21 R1 = F, R2 = OAc
3x
a–c
4x
FmocHN
COOH
a, d, c
a–c
R2
HO
HO
HO
OH
e–g
22 R1 = OH, R2 = F
23 R1 = F, R2 = OH
O
O
O
R1
AcHN
O
O
O
O
O
H
H
N
N
N
OH
AcHN
N
N
N
N
N
H
H
H
O
O
O
O
O
OH
COOH
NH
HN
NH2
Scheme 3 Reagents and conditions: (a) piperidine–NMP (20%); (b) Fmoc-AA-OH (10 equiv), HBTU, HOBt, DIPEA, NMP; (c) Ac2O–
DIPEA–HOBt (4:1:0.12); (d) 13/21, HATU, HOAt, NMM, NMP, 9 h; (e) AcOH–CF3CH2OH–H2O (1:1:8), r.t., 1 h; (f) TFA–i-Pr3SiH–H2O
(15:0.9:0.9), r.t., 2 h; (g) NaOMe, MeOH, 0 °C to r.t., 24 h, 41% (22) and 18% (23).
stabilizing influence of the fluorine atom on the rate of (Scheme 3). The fluorinated T antigen–threonine building
hydrolysis will be maximal. As a consequence, 2-deoxy- blocks 13 and 21 were coupled over a period of 9 hours
2-fluoroglycosides represent powerful mechanism-based using HATU/HOAt31 and NMM for activation. After
inhibitors for retaining b-glycosidases.22 Following pub- Fmoc deprotection of the N-terminal Ser residue, the non-
lished literature reports on 2-deoxy-2-fluoro-glycosyla- apeptides were acetylated on-bead and detached from the
tions,9a,b,23 we relied upon the use of known resin by treatment with HOAc/CF3CH2OH. Complete
trichloroacetimidate derivative 189b for the preparation of deprotection using a sequence of acidolysis and de-O-
the 2¢-deoxy-2¢-fluoro-T antigen derivative 20 acetylation furnished the desired glycopeptides 2232 and
(Scheme 2). The latter can be conveniently prepared by 23 in overall yields of 41% and 18% (based on the loaded
electrophilic fluorination of 3,4,6-tri-O-acetylgalactal 16 resin) after HPLC purification.
with Selectfluor.24 Again, microwave irradiation proved
In summary, we have prepared a series of novel orthogo-
beneficial with regard to reaction time. Hence, 3,4,6-tri-
nally protected glycosyl amino acids with one or two flu-
O-acetyl-2-deoxy-2-fluorogalactose (17) was obtained in
oro substituents within the glycan portion for solid-phase
66% yield within minutes at 108 °C (100 W) as compared
glycopeptide synthesis. By incorporating these molecules
to 63% yield after 6.5 hours of reflux without microwave
into mucin peptide sequences, as shown for compounds
support. Compound 17 was then transformed into 18 us-
13 and 21, tumor-associated glycopeptide antigen ana-
ing DBU in CH2Cl2 (71%). In the subsequent glycosyla-
logues were obtained. These compounds are now avail-
tion, formation of the a-anomer of 19 was favored (a/b,
able for investigations of their metabolic stability and
2:1), due to electronic effects and the nonparticipating
their immunological properties.
character of the fluorine substituent. However, chromato-
graphic separation of the desired b-anomer was possible
Acknowledgment
after exchange of the benzylidene acetal with acetyl
groups (b-20,25 16% over 3 steps). Acidolytic cleavage of
the tert-butyl ester finally provided analogue 21 (88%) for
glycopeptide synthesis.
Financial support from the Deutsche Forschungsgemeinschaft
(Emmy Noether Fellowship to A.H.-R.) and the Fonds der Chemi-
schen Industrie (Liebig Fellowship to A.H.-R.) is gratefully ack-
nowledged.
Both T-antigen analogues 13 and 21 were incorporated
into a MUC1 tandem repeat-peptide sequence comprising
the immunodominant PDTRP epitope26 (SAPDTRPAP) References and Notes
by automated solid-phase peptide synthesis27 (SPPS) ac-
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was achieved by HBTU/HOBt30 and DIPEA in NMP
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Synlett 2009, No. 13, 2167–2171 © Thieme Stuttgart · New York

2170
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LETTER
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(MgSO4), and concentrated in vacuo. Flash chromatography
on silica gel (cyclohexane–EtOAc, 5:1) afforded 9 as a
colorless, amorphous solid (162 mg, 73%); Rf = 0.53
(cyclohexane–EtOAc, 5:1); [a]D23 39.42 (c 1, CHCl3).
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4150.
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Vincent, S. P.; Wong, C.-H. Angew. Chem. Int. Ed. 2005, 44,
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(25) Compound b-20
[a]D23 = 55.74 (c 1, CHCl3); tR = 14.6 min [Perfectsil C18,
grad.: MeCN–H2O, (50:50) → (90:10), 30 min → (100:0),
10 min]. 1H NMR (400 MHz, CDCl3, COSY): d = 5.43 (d, 1
H, 4-H, J3,4 = 2.0 Hz), 5.36–5.32 (m, 1 H, 4¢-H), 5.10–5.00
(m, 1 H, 3¢-H), 4.87 (d, 1 H, 1-H, J1,2 = 3.5 Hz), 4.65 (dt, 1
H, 2-H, J2,1 = 3.6 Hz, J2,3 = 10.6 Hz), 4.60–4.37 (m, 4 H, 1¢-
H {4.57}, 2¢-H, CH2 (Fmoc) {4.52}), 4.29–4.08 (m, 6 H, Ta
{4.23}, Tb {4.16}, 6a-H {4.11}, 5¢-H {4.13}, 6a¢-H {4.21},
9-H (Fmoc) {4.22}), 4.03–3.84 (m, 3 H, 5-H, 6b-H, 6b¢-H),
4.03–3.94 (m, 1 H, 3¢-H), 3.79 (dd, 1 H, 3-H, J3,4 = 3.2 Hz,
J2,3 = 11.0 Hz), 2.12 [s, 3 H, CH3(Ac)], 2.10 [s, 3 H,
CH3(Ac)], 2.03 [s, 9 H, 3 × CH3(Ac)], 1.97 [s, 3 H,
CH3(NHAc)], 1.44 [s, 9 H, CH3(t-Bu)], 1.28 (d, 3 H, Tg,
J
Tg,Tb = 6.1 Hz) ppm. 13C NMR (100.6 MHz, CDCl3, BB,
(10) (a) Brocke, C.; Kunz, H. Synlett 2003, 2052. (b) Brocke, C.;
HMQC): d = 101.9 (d, C1¢, JC1¢,F = 23.5 Hz), 100.3 (C1),
87.8 (d, C2¢, JC2¢,F = 186.2 Hz), 83.2 [Cq(t-Bu)], 77.5 (C3),
77.2 (Tb), 70.8 (d, C3¢, JC3¢,F = 19.1 Hz), 70.5 (C5), 69.0
(C4), 68.1 (C5¢), 67.2 (d, C4¢, JC4¢,F = 8.0 Hz,), 66.8 (CH2-
Fmoc), 63.2 (C6¢), 60.8 (C6) 59.0 (Ta), 48.0 (C2), 47.2 (CH-
Fmoc), 28.0 [CH3(t-Bu)], 23.0 [CH3(NHAc)], 20.8, 20.7,
20.6, 20.5, 20.5 [CH3 (Ac)], 18.5 (Tg) ppm. ESI-MS (pos. ion
mode): m/z calcd for C47H59FN2NaO19: 997.36; found:
997.34 [M + Na]+.
Kunz, H. Synthesis 2004, 525.
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(19) Typical Experimental Procedure
(32) Compound 22
[a]D25 = –97.61 (c 1, CHCl3); tR = 21.2 min [Luna C18,
grad.: MeCN–H2O + 0.1% TFA, (5:95) → (50:50), 80 min
→ (100:0), 20 min]. 1H NMR (400 MHz, DMSO-d6,
COSY): d = 8.25 (d, 1 H, RNH, JNH,a = 7.2 Hz), 8.21 (d, 1 H,
DNH, JNH,a = 8.7 Hz), 8.10 (d, 1 H, ANH, JNH,a = 7.3 Hz) 8.00
(d, 1 H, ANH, JNH,a = 7.3 Hz), 7.92 (d, 1 H, SNH, JNH,a = 8.0
Hz), 7.61–7.53 (m, 2 H, TNH {7.56}, RGua {7.58}), 6.93 (d, 1
H, NH-GalNAc, JNH,2 = 8.7 Hz), 4.69–4.62 (m, 2 H, 1-H
{4.67}, Da {4.65}), 4.58 (m, 1 H, 6¢a-H), 4.54-4.43 (m, 3 H,
Aa {4.51}, Ra {4.47}, 6¢b-H {4.47}), 4.43–4.36 (m, 2 H, Aa
A solution of acceptor 7 (150 mg, 0.25 mmol) in anhyd
MeNO2–CH2Cl2 (3:2, 4 mL) was stirred with Hg(CN)2 (125
mg, 0.49 mmol) and activated pulverized MS 4 Å (200 mg)
for 30 min under argon. A solution of donor 3 (185 mg, 0.49
mmol) in anhyd MeNO2–CH2Cl2 (3:2, 4 mL) was added, and
the reaction mixture was irradiated in a microwave reactor
for 5 h (80 °C, 100 W), diluted with CH2Cl2 (20 mL), and
filtered through Hyflo Supercel. The filtrate was washed
with sat. aq NaHCO3 (10 mL) and brine (10 mL), dried
Synlett 2009, No. 13, 2167–2171 © Thieme Stuttgart · New York

LETTER
{4.40}, Ta {4.39}), 4.36–4.29 (m, 2 H, 2 × Pa {4.33},
Fluorinated Analogues of Tumor-Associated Antigens
2171
3 H, Ab, Ja,b = 7.4 Hz), 1.10 (d, 3 H, Tg, Jb,g = 6.4 Hz) ppm.
13C NMR (100.6 MHz, CDCl3, BB, HMQC): d = 173.3,
171.9, 171.8, 171.0, 170.8, 170.5, 170.5, 169.7, 169.7,
169.6, 169.4 (12 × C=O), 156.8 (C=N), 104.8 (C1¢), 98.6
(C1), 83.3 (d, C6¢, JC6¢,F = 166.9 Hz), 78.8 (C3), 75.0 (Tb),
73.1 (d, C5¢, JC5¢,F = 19.1 Hz), 72.5 (C3¢), 71.3 (C5), 70.5
(C2¢), 68.0 (d, C4¢, JC4¢,F = 6.5 Hz), 67.8 (C4), 61.8 (Sb), 60.6
(C6), 59.5, 59.0, 58.5 (3 × Pa), 55.8 (Ta), 55.0 (Sa), 50.1 (Ra),
49.3 (Da), 48.0 (C2), 46.7, 46.5, 46.3, 46.3 (2 × Aa, 3 × Pd),
40.5 (Rd), 35.9 (Db), 29.1, 28.8, 28.6 (3 × Pb), 28.3 (Rb), 24.8
(Rg), 24.6, 24.4, 24.3 (3 × Pg), 23.1, 22.6 [2 × CH3(NHAc)],
18.3 (Tg) 17.1, 16.6 (2 × Ab) ppm. ESI-MS (pos. ion mode):
m/z calcd for C54H87FN13O24: 1320.59; found: 1320.60 [M +
H]+.
{4.31}), 4.28–4.23 (m, 2 H, Sa {4.26}, 1¢-H {4,24}), 4.20
(dd, 1 H, Pa, Ja,ba = 4.4 Hz, Ja,bb = 8.7 Hz), 4.17–4.12 (m, 1
H, 2-H), 4.11–4.08 (m, 1 H, Tb), 3.89–3.85 (m, 1 H, 4-H),
3.76–3.60 (m, 3 H, 5¢-H {3.69}, 5-H {3.65}, 4¢-H {3.63}),
3.61–3.38 (m, 11 H, Sb {3.52}, 3 × Pd {3.54}, 6a-H {3.43},
6b-H {3.41}, 3-H {3.57}), 3.35–3.28 (m, 2 H, 2¢-H {3.52},
4¢-H {3.30}), 3.15–3.01 (m, 2 H, Rd), 2.75 (dd, 1 H, Dba,
Jba,a = 6.4 Hz, Jba,bb = 16.5 Hz), 2.49 (m, 1 H, Dbb, under
DMSO-d6), 2.18–2.09 (m, 1 H, Pba), 2.07–1.97 (m, 2 H,
2 × Pba, {2.06}, {2.01}), 1.94–1.74 [m, 15 H, 6 × Pg {1.91},
{1.88}, {1.83}, 3 × Pbb {1.84}, {1.81}, {1.78}, 2 × CH3
(NHAc)], 1.73–1.65 (m, 1 H, Rba), 1.59–1.42 (m, 3 H, Rb,
{1.52}, Rbb {1.49}), 1.19 (d, 3 H, Ab, Ja,b = 7.4 Hz), 1.17 (d,
Synlett 2009, No. 13, 2167–2171 © Thieme Stuttgart · New York

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