Chemical synthesis of α-D-mannosylphosphate serine derivatives: A new class of synthetic glycopeptides

Article abstract of DOI:10.1055/s-2003-40344

α-D-Mannosylphosphate serine derivatives were conveniently synthesized by reaction of benzyl or cyanoethyl phosphochloroamidites with 2,3,4,6-tetra-O-acetyl-D-mannose to give intermediate α-mannosyl phosphoramidites, which were successfully reacted with p

Full text of DOI:10.1055/s-2003-40344

LETTER
1373
Chemical Synthesis of a-D-Mannosylphosphate Serine Derivatives:
A New Class of Synthetic Glycopeptides
Synthesis of
a
-
D
-M
a
annosylph
l
osphate
a
Serine
D
e
l
rivatives A. Elsayed, Geert-Jan Boons*
Complex Carbohydrate Research Center, The University of Georgia, 220 Riverbend Road, Athens, GA 30602, USA
E-mail: gjboons@ccrc.uga.edu
Received 31 March 2003
Dedicated to the memory of Professor Raymond U. Lemieux.
OAc
Abstract: a-D-Mannosylphosphate serine derivatives were conve-
O
AcO
AcO
niently synthesized by reaction of benzyl or cyanoethyl phospho-
chloroamidites with 2,3,4,6-tetra-O-acetyl-D-mannose to give
intermediate a-mannosyl phosphoramidites, which were success-
fully reacted with properly protected serine derivatives in the pres-
ence of 1H-tetrazole to give phosphite triesters which could be
oxidized to phosphotriesters using t-BuOOH.
OH
AcO
1
i, i-Pr2NPCl(OR) 2a) R = Bn
2b) R = (CH2)2CN
AcO
AcO
OAc
O
Key words: carbohydrates, glycopeptides, glycosyl phosphates,
phosphoglycosylation, phosphochloroamidites
NHFmoc
OAll
+
AcO
HO
OR
O
O
P
3a,b
4a
N(i-Pr)2
Most eukaryotic cellular proteins with the exception of
some hormones and enzymes are reliant on sugar units
covalently attached to them to confer a broad range of im-
portant biological functions such as immunogenicity, sol-
ubility, cell-cell communication, protection from
proteolytic attack, and induction and maintenance of the
protein conformation in a biologically active forms.1–6
The vast majority of glycoproteins can be divided into two
principal groups: the N-linked glycoproteins having an N-
glycosidic linkage to the side chain of L-asparagine and
the more diverse O-linked group, bearing an O-glycosidic
linkage to L-serine, L-threonine, 4-hydroxy-L-proline, or
-L-tyrosine.
ii
AcO
AcO
OAc
O
AcO
NHFmoc
OAll
O
O
O
P
5a,b
O
OR
Scheme 1 Reagents and conditions: i) DIPEA, CH2Cl2; ii) 1H-tetra-
zole, CH3CN followed by addition of t-BuOOH at –40 °C.
6Galb(1-4)Man and PO4-6[Glcb(1-3)]Galb(1-4)Man re-
peating units capped with a neutral mannosyl oligosac-
charide.
Recently, several new classes of glycoproteins have been
identified and an intriguing group contains oligosaccha-
rides linked to serine or threonine moieties via a phos-
phodiester linkage and this type of protein modification
has been referred to as protein phosphoglycosylation.7
The first reported example of a protein modified by a
phosphoglycoside was an endopeptidase known as pro-
teinase I, isolated from the slime mold Dicyostelium di-
coideum.8 It was shown that this glycoprotein contains a
serine moiety modified by a phosphodiester linked to N-
acetyl glucosamine (a-D-GlcNAc-1-PO4-Ser). Two other
cysteine proteinases isolated from D. discoideum have
also been shown to carry GlcNAc-1-PO4 modifications.9
Phosphoglycosylation of serine was also observed in a se-
creted acid phosphatase of Leishmania mexicana.10–12 In
this case, serine is modified by a-mannosidic phosphodi-
ester linkages which can either be monomeric or consist
of a series of neutral a(1-2)-linked oligomannanes or
phosphorylated oligosaccharides composed of PO4-
In order to study in detail the biological significance of
protein phosphoglycosylation, reasonable quantities of
well-defined compounds are required. We report here, for
the first time, a convenient synthetic approach for the
preparation of properly protected a-D-mannosylphos-
phate serine derivatives that can be used for the synthesis
of glycosylphosphopeptides.
It was envisaged that the preparation of a-D-manno-
sylphosphate serine derivatives would be complicated by
the inherent acid lability of anomeric phosphates and the
possibility of base-mediated b-elimination of the serine
phosphotri(di)ester linkage. Furthermore, due to unfavor-
able hydrogen bonding, the side-chain hydroxyl of car-
bamate-protected serine and threonine derivatives are
generally of low nucleophilicity13–15 complicating the
phosphorylation. In addition, anomeric phosphorylations
are challenging due to the fact that these reactions need to
be performed with control of anomeric configuration.
A number of strategies have been reported for the pre-
paration of inter-glycosidic phosphodiesters and the
most successful approaches involve the use of H-
Synlett 2003, No. 9, Print: 11 07 2003.
Art Id.1437-2096,E;2003,0,09,1373,1375,ftx,en;S04603ST.pdf.
© Georg Thieme Verlag Stuttgart · New York

1374
G. A. Elsayed, G.-J. Boons
LETTER
phosphonates16,17 and phosphoamidites.13,18,19 In first in-
stance, 2,3,4,6-tetra-O-acetyl-D-mannose was reacted
with salicylchlorophosphite and although after hydrolysis
the expected H-phosphonate was obtained, further activa-
tion with pivaloyl chloride (PivCl) and reaction with
serine derivative 4a followed by oxidation with t-BuOOH
did not provide the expected phosphodiester. Also an in-
verse procedure, in which serine was first phosphonylated
followed by reaction with 2,3,4,6-tetra-O-acetyl-D-man-
nose, did also not lead to the expected compound. Fortu-
nately, reaction of 1 with benzyl or cyanoethyl
phosphochloroamidites 2 and 3 in the presence of diiso-
propylethylamine (DIPEA) gave the expected phosphoa-
midites 3a,b, respectively. These derivatives could be
further activated by 1H-tetrazole and reaction with serine
derivative 4a gave the expected intermediate phosphites,
which could be oxidized to phosphotriesters 5a and 5b, re-
spectively using t-BuOOH. After purification, by silica
gel column chromatography, 5a and 5b were obtained in
AcO
AcO
OAc
O
NHR
O
i
AcO
O
P
HO
OR'
NHR
O
O
OR'
NC(CH2)2O
O
4a-c
5c) R = Boc, R' = A ll (53%)
5d) R = Boc, R' = Bn (48%)
5e) R = Boc, R' = Nbz) (53%)
5f) R = Cbz, R' = All (52%)
AcO
OAc
N=CPh2
OBn
O
i
AcO
HO
HO
AcO
O
P
N=CPh2
(63%)
O
O
O
OBn
O
6
7 NC(CH2)2O
O
AcO
AcO
N3
OAc
O
i
OAll
AcO
O
P
N3
(71%)
O
O
OAll
1
yields of 50% and 53%, respectively. H NMR of 5a,b
8
9 NC(CH2)2O
O
showed H-1 at ca 5.8 ppm (JH1,P = 6.7 Hz) and deshielding
of H-3 and H-5 by ca 0.2 ppm compared to the same pro-
tons in hemiacetal 1 confirming the a-configuration of the
phosphites. Encouraged by these results, a range of Boc
and Cbz protected serine derivatives were prepared that
have allyl-, benzyl- or Nbz protected carboxylic acids.
As can be seen in Scheme 2, the coupling of these deriva-
tives with phosphoramidite 3b followed by oxidation with
t-BuOOH, gave the expected phosphotriesters 5c–5f in
acceptable yields.
Scheme 2 Reagents and conditions: i) compound 1, 1H-tetrazole,
CH3CN followed by the addition of t-BuOOH at –40 °C.
phosphotriesters undergo piperidine mediated b-elimina-
tion to dehydroalanine and a-amino-butenoate. However,
it has been found that phosphodiesters are perfectly stable
under these conditions and furthermore, it has been shown
that phosphodiesters are compatible with stepwise phos-
phopeptide synthesis.22,23 Thus, the allyl ester of 5a was
cleaved by treatment with (Ph3P)4Pd and Bu3SnH and the
resulting compound was converted into phosphodiester
10 (Scheme 3)by removal of the benzyl protecting group
of phosphate using NaI in refluxing acetonitrile. After pu-
rification by size exclusion column chromatography using
Sephadex LH-20, compound 10 was isolated in an overall
yield of 53%. It is to be expected that 10 will be a suitable
building block for the stepwise assembly of glycosylphos-
phopeptides.
In order to increase the yields of the phosphorylations,
serine derivatives 6 was employed which is protected by
a benzophenone Schiff base. This compound could easily
be prepared in good yield (75%) by reaction of the benzyl
ester of serine with benzophenone imine.20 It was expect-
ed that the hydroxyl of 6 would be more nucleophilic than
a hydroxyl of similar serine derivatives that are protected
by carbamates. In the latter compounds, unfavorable hy-
drogen bonding between the lone pair of the serine hy-
droxyl and the amide moiety of the protecting group
reduces the electron density and hence nucleophilicity of
the hydroxyl. On the other hand, it has been shown that re-
placement of carbamates (H-bond donor) with an imine
type protecting group (H-bond acceptor) provides favor-
able hydrogen bonding that increases the electron density
of the serine hydroxyl.14 This finding has been elegantly
exploited in high yielding glycosylations of benzophe-
none Schiff base protected serine. As expected, coupling
of 1 with 6 followed by oxidation with t-BuOOH gave 7
in an improved yield of 63%. Even more gratifying was
the finding that the use of azido-modified serine 8, which
was obtained by an azido transfer reaction using TfN3,21
gave even a higher yield (71%) of the coupling product 9.
AcO
OAc
O
AcO
AcO
5a
NHFmoc
OH
O
P
O
O
10
O–Na+
O
Scheme 3 Reagents and conditions: i) Pd(Ph3P)4, BuSnH, AcOH,
THF; ii) NaI, CH3CN.
In conclusion, it has been demonstrated that benzyl and
cyanoethyl phosphochloroamidites are convenient re-
agents for the preparation of a wide range of a-D-manno-
sylphosphate serine derivatives. The best results were
obtained with serine derivatives that were modified by
amino protecting or masking groups that provide favor-
able hydrogen bonding. It is to be expected24 that the
methodology presented here can be employed for the glo-
Having established a convenient approach for the phos-
phoglycosylation of serine derivatives, attention was fo-
cused on partial deprotection to obtain a building block
that can be used in stepwise glycosylphosphopeptide as-
sembly. It is well know that serine and threonine protected
Synlett 2003, No. 9, 1373–1375 ISSN 1234-567-89 © Thieme Stuttgart · New York

LETTER
Synthesis of a-D-Mannosylphosphate Serine Derivatives
1375
bal glycosylphosphorylation of pre-assembled peptides.
Alternatively, building blocks such as 10 should be useful
for the stepwise synthesis of glycosylphosphopeptides.
Acknowledgement
We thank the Egyptian Cultural and Educational Bureau and the Of-
fice of the Vice President for Research, The University of Georgia,
Athens for financial support.
Preparation compound 3a. To a stirred solution of 1 (0.55 g, 1.43
mmol) and DIPEA (0.52 mL, 2.86 mmol) in CH2Cl2 (5 mL) was
added benzyl N,N-diisopropylchlorophosphoramidite 2a (0.3 g, 1.4
mmol). When TLC analysis (ethyl acetate/hexane 2/1) showed
completion of the reaction, the reaction mixture was diluted with
CH2Cl2 (20 mL) and successively washed with ice-cold 10% aque-
ous NaHCO3 (10 mL), brine (10 mL) and water (15 mL), followed
by drying over MgSO4. After evaporation of the solvent, compound
3a was obtained as a colorless oil (0.5g, 59%). 31P NMR d: 14.9,
References
(1) Lis, H.; Sharon, N. Eur. J. Biochem. 1993, 218, 1.
(2) Arsequell, G.; Valencia, G. Tetrahedron: Asymmetry 1997,
8, 2839.
(3) Varki, A. Glycobiology 1993, 3, 97.
(4) Dwek, R. A. Biochem. Soc. Trans. 1995, 23, 1.
(5) Dwek, R. A. Chem. Rev. 1996, 96, 683.
(6) Hayes, B. K.; Hart, G. W. Curr. Opin. Struct. Biol. 1994, 4,
692.
1
15.2 (2 × s, 2 diastereoisomers). H NMR (300 MHz, CDCl3): d:
7.22–7.16 (m, 5 H, Ar-H), 5.84 (dd, 1 H, H-1, J1,2 = 1.6 Hz, J1,P
=
8.0 Hz), 5.61 (s, 2 H, OCH2Ph), 5.38 (dd, 1 H, H-3, J2,3 = 3.6 Hz,
3,4 = 9.5 Hz). 5.11 (t, 1 H, H-4, J4,5 = 10 Hz), 5.09 (dd, 1 H, H-2),
(7) Haynes, P. A. Glycobiology 1998, 8, 1.
(8) Souza, G. M.; Hirai, J.; Mehta, D. P.; Freeze, H. H. J. Biol.
Chem. 1995, 270, 28938.
(9) Ord, T.; Adessi, C.; Wang, L.; Freeze, H. H. Glycobiology
1996, 6, 313.
(10) Ilg, T.; Overath, P.; Ferguson, M. A. J.; Rutherford, T.;
Campbell, D. G.; McConville, M. J. J. Biol. Chem. 1994,
269, 24073.
(11) McConville, M. J.; Thomasoates, J. E.; Ferguson, M. A. J.;
Homans, S. W. J. Biol. Chem. 1990, 265, 19611.
(12) Ilg, T.; Stierhof, Y. D.; Craik, D.; Simpson, R.; Handman,
E.; Bacic, A. J. Biol. Chem. 1996, 271, 21583.
(13) Westerduin, P.; Veeneman, G. H.; Marugg, J. E.; Van der
Marel, G. A.; Van Boom, J. H. Tetrahedron Lett. 1986, 27,
1211.
(14) Szabo, L.; Li, Y. S.; Polt, R. Tetrahedron Lett. 1991, 32, 585.
(15) Polt, R.; Szabo, L.; Treiberg, J.; Li, Y. S.; Hruby, V. J. J. Am.
Chem. Soc. 1992, 114, 10249.
(16) Westerduin, P.; Veeneman, G. H.; Van der Marel, G. A.;
Van Boom, J. H. Tetrahedron Lett. 1986, 27, 6271.
(17) Nikolaev, A. V.; Ivanova, I. A.; Shibaev, V. N.; Kochetkov,
N. K. Carbohydr. Res. 1990, 204, 65.
(18) Sinha, N. D.; Biernat, J.; McManus, J.; Koster, H. Nucl. Acid
Res. 1984, 12, 4539.
(19) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981,
22, 1859.
J
4.93 (dd, 1 H, H6a, J5,6a = 5.0 Hz, J6a,6b = 12.4 Hz), 4.27 (ddd, 1 H,
H-5, J5,6b = 2.1 Hz), 3.96 (dd, 1 H, H-6b), 2.97–2.53 [m, 2 H, 2 ×
CH(CH3)2], 2.11–1.65 (4 × s, 12 H, 4 × COCH3), 1.63–1.59 [d, 12
H, 2 × CH(CH3)2]. 13C NMR (125 MHz, CDCl3) d: 172.13, 171.54,
170.19, 169.23, 139.14, 127.23, 126.78, 125.11, 123.45, 101.23,
98.34, 87.23, 77.12, 76.82, 72.14, 69.14, 69.11, 67.56, 58.33, 53.61,
44.16, 42.65, 41.86, 29.87, 24.56, 23.11, 22.13, 20.93, 20.84, 20.67,
20.55, 20.33, 19.89. FAB-MS: m/z 608.26 [M + Na]+.
Preparation compound 5a. To a stirred mixture of phosphoramid-
ite 3a (100 mg, 0.17 mmol) and amino acid 4a (62.4 mg, 0.17
mmol) in acetonitrile (1.5 mL) was added 1H-tetrazole (0.67 mL of
3 wt.% solution in acetonitrile). When TLC analysis (ethyl acetate/
hexane, 2 /3) showed the consumption of 3a, the reaction mixture
was cooled (–40 °C) and a solution of t-BuOOH in decane (77 mL,
5.0–6.0 M) added. The reaction mixture was left stirring at –40 °C
until TLC analysis (ethyl acetate/hexane, 2 /1) showed complete
formation of triester 5a. The reaction mixture was concentrated to a
small volume in vacuo and applied to a column of Sephadex LH-20
(eluent: CH2Cl2/MeOH, 2/1, v/v) to afford 5a as a colorless syrup
(70 mg, 50%). 31P NMR d: –3.19, –3.29 (2 × s, 2 diastereoisomers).
1H NMR (300 MHz, CDCl3) d: 8.10 (dd, 1 H, NH, J = 9.7 Hz, J =
8.7 Hz), 7.87 (d, 2 H, Ar-H, J = 7.3 Hz), 7.71 (d, 2 H, Ar-H, J = 6.8
Hz), 7.40–7.30 (m, 9 H, Ar-H), 5.90–5.85 (m, 1 H, OCH2CH=CH2),
5.68 (dd, 1 H, H-1, J1,2 = 1.5 Hz, J1,P = 7.3 Hz), 5.32–5.04 (m, 7 H,
H-2, H-3, OCH2Ph, H-9, Fmoc, OCH2CH=CH2), 4.61–4.55 (m, 3
H, OCH2CH=CH2, a-CH), 4.35–3.94 (m, 8 H, H-4, H-5, H-6a,b, b-
CH2, CH2O, Fmoc), 2.10–1.94 (4 × s, 12 H, 4 × CH3CO). 13C NMR
(125 MHz, DMSO) d: 170.63, 170.15, 170.02, 169.56, 132.81,
118.69, 118.50, 95.55, 79.47, 70.60, 68.84, 68.69, 68.39, 67.45,
65.98, 65.26, 63.59, 62.12, 28.74, 21.19, 21.12, 21.05, 19.69,
19.59. FAB-MS: m/z 730.99 [M + Na]+.
(20) O’Donnell, M. J.; Polt, R. J. Org. Chem. 1982, 47, 2663.
(21) Manabe, S.; Sakamoto, K.; Nakahara, Y.; Sisido, M.;
Hohsaka, T.; Ito, Y. Bioorg. Med. Chem. 2002, 10, 573.
(22) Otvos, L.; Elekes, I.; Lee, V. M. Y. Intern. J. Pept. Prot. Res.
1989, 34, 129.
(23) Kitas, E. A.; Perich, J. W.; Tregear, G. W.; Johns, R. B. J.
Org. Chem. 1990, 55, 4181.
(24) McMurray, J. S.; Coleman, D. R.; Wang, W.; Campbell, M.
L. Biopolymers 2001, 60, 3.
Synlett 2003, No. 9, 1373–1375 ISSN 1234-567-89 © Thieme Stuttgart · New York