Membrane-permeant derivatives of mannose-1-phosphate

Article abstract of DOI:10.1016/S0968-0896(02)00269-9

For treatment of congenital disorder of glycosylation type Ia (CDG-Ia) membrane-permeant derivatives of mannose-1-phosphate are required. Employing biologically cleavable phosphate protecting groups advantageous precursor derivatives could be synthesized

Full text of DOI:10.1016/S0968-0896(02)00269-9

Bioorganic & Medicinal Chemistry 10 (2002) 4043–4049
Membrane-Permeant Derivatives of Mannose-1-phosphate
Synke Rutschow,a Joachim Thiem,a,* Christian Kranzb and Thorsten Marquardtb
aInstitut fur Organische Chemie, Universitat Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
¨
¨
bKlinik und Poliklinik fur Kinderheilkunde, Albert-Schweitzer-Str. 33, D-48129 Munster, Germany
¨
¨
Received 9 January 2002; accepted 3 July 2002
Abstract—For treatment of congenital disorder of glycosylation type Ia (CDG-Ia) membrane-permeant derivatives of mannose-1-
phosphate are required. Employing biologically cleavable phosphate protecting groups advantageous precursor derivatives could be
synthesized following a facile approach. Their enzymatic cleavages using esterase from porcine liver (E.C. 3.1.1.1) were investigated.
# 2002 Published by Elsevier Science Ltd.
Introduction
to be the acetoxymethyl (AM) and the pivaloyloxy-
methyl (POM) esters. After penetration the acyloxy
phosphate ester is expected to be converted into the
parent compound by intracellular enzymatic cleavage
by carboxylate esterases.9,10 The acyloxy ester linkages
should be hydrolyzed to the hydroxymethyl analogues.
This intermediate in turn is chemically labile and looses
one mole of formaldehyde11,12 (Scheme 1). In order to
test this in vitro, initial enzymatic deprotection experi-
ments were conducted. For improving the lipophilicity
the hydroxy groups of the mannose moiety were masked
as different esters and carbonates.
Congenital disorders of glycosylation, formerly called
carbohydrate-deficient glycoprotein syndrome (CDG
syndrome), belong to a new group of genetic, multi-
systemic, metabolic disorders. These autosomal recessive
diseases were first described in 1980.1
The genetic defect influences the biosynthesis of N-
linked glycans. CDG could be classified into two types
based on the intracellular localization of the genetic
defect. In type I the defect could be found in the synth-
esis or transfer of oligosaccharides in the endoplasmic
reticulum (ER) whereas in type II the defect inhibits the
processing of the N-linked glycoproteins in the Golgi.
Results and Discussion
Synthesis
In CDG-Ia phosphomannomutase 2 (PMM 2) is deficient,
therefore the conversion of mannose-6-phosphate to
mannose-1-phosphate fails, and thus the supply of
GDP-mannose will be limited. Because GDP-mannose
is the decisive mannose donor the biosynthesis of glyco-
proteins is significantly disturbed. One way of dealing
with the PMM 2 deficiency would be treatment of patients
with mannose-1-phosphate from external sources.
Starting from mannose the benzyl mannopyranoside was
obtained by Fischer glycosylation with benzyl alcohol.13
This compound was converted into the appropriate sub-
stituted mannopyranosides using butyryl chloride, piva-
loyl chloride or iso-propyl chloroformiate.14,15 Subsequent
hydrogenolysis of the benzyl groups on Pd/C (10%)
yielded in the anomerically unblocked mannose deriva-
tives 1–3. Further reaction with dibenzyl di-iso-propyl-
phosphoramidite using 1H-tetrazole gave the phosphite
triesters which were oxidised in situ by meta-chlor-
operbenzoic acid (MCPBA) to the appropriate phosphate
derivatives16 4–6. Subsequently the benzyl groups were
removed by hydrogenolysis on Pd/C (10%). The result-
ing phosphates 7–9 were converted into their acetoxy-
methyl (AM) and pivaloyloxymethyl (POM) esters 10–15
employing bromomethylacetate or iodomethylpivaloate,
Due to the high polarity, mannose-1-phosphate is unable
to penetrate through cellular membranes.2,3 To overcome
this limitation biologically reversible protecting groups
previously used for carboxylic acids, phosphonates,
nucleotides and inositoles4ꢀ8 could be employed to form
neutral phosphotriester. Suitable for this purpose seem
*Corresponding author. Tel.: +49-40-42838-4241; fax: +49-40-
42838-4325; e-mail: thiem@chemie.uni-hamburg.de
0968-0896/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0968-0896(02)00269-9

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S. Rutschow et al. / Bioorg. Med. Chem. 10 (2002) 4043–4049
for the enzyme will be investigated and in vitro tests will
be done.
Conclusion
In this study the preparation of various potential mem-
brane-permeable derivatives of mannose-1-phosphate
could be demonstrated combining biologically reversible
phosphate and carbohydrate protecting groups. Enzy-
matic tests have revealed that AM-groups were cleaved
to restore the original compound. It is envisaged to
transfer this conception for membrane-permeant deriva-
tives including biologically reversible protecting groups
to other sugar phosphates and further biologically
important components.
Experimental
Scheme 1.
General methods
respectively, in the presence of N-ethyl-di-iso-propyla-
mine (DIPEA) (Scheme 2).
NMR spectra were recorded on Bruker AC-250, AMX-
400 and DRX-500. Chemical shifts of 1H NMR and 13
C
NMR are given relative to tetramethylsilane. Eighty-five
per cent phosphoric acid was used as an external stan-
dard for 31P NMR. Optical rotations were measured
with Perkin–Elmer polarimeter 341. Melting points
were determined with ST-apotec and are uncorrected.
Elemental analyses were performed by the micro-analy-
tical service of the Institute of Organic Chemistry of the
University of Hamburg. MALDI-TOF spectra were
recorded on Bruker Biflex III and ESI spectra on aHP
series 1100MSD. TLC were run on precoated plates, silica
gel 60GF 254 (Merck). Detection was effected by observa-
tion under UV light at 254 nm, and by spraying with 10%
ethanolic sulfuric acid and subsequent heating. Column
chromatography was performed by flash technique using
silica gel 60 (230–400 mesh, 0.040–0.063 mm, Merck).
The poor nucleophilicity of the phosphate and the lack
of stability of the phosphate group obvious by the
anomerically free mannose byproduct could be the rea-
son for the relatively low yield in this step which
requires improvements in further studies to come.
Enzymatic tests
The AM-ester of mannose protected with butyryl
groups (10) was used as the model compound. First, a
standard reaction was carried out without any enzyme
to control the stability of the mannose phosphate under
the reaction conditions. The ester was incubated in
phosphate buffer at 37 ꢁC overnight and the course of
the reaction followed by TLC. Then MALDI-TOF
analysis was carried out to show the stability of the ester
under these conditions.
Reagents
Iodomethylpivaloate was synthesized following known
procedure. Esterase (porcine liver, crude and 3.2 M
(NH4)2SO4 solution, EC 3.1.1.1) was purchased from
Sigma. One unit (U) is defined as the hydrolysis of 1.0
mmol of ethyl butyrate to butyric acid and ethanol per
min at pH 8.0at 25 ꢁC. DIPEA is stored over 4 A
molecular sieves.
Subsequently, the ester was incubated in phosphate
buffer in the presence of esterase (porcine liver, crude,
EC 3.1.1.1) at 37 ꢁC for 3 h and the reaction was con-
trolled by TLC. Again MALDI-TOF analysis proved
that the AM-groups were cleaved sucessively and that
one butyryl group was removed. It may be presumed
that the butyryl group in question is in position 6, as
this would be the ester of a primary alcohol, and as such
the most reactive site in the molecule. To our surprise,
the remaining ester groups on the sugar moiety were not
removed, however, as a side-product the phosphorus
free mannose was detected. The same enzymatic test
was conducted with pure enzyme (porcine liver, 3.2 M
(NH4)2SO4 solution, pH 8, EC 3.1.1.1). An ESI analysis
was made, in addition to MALDI-TOF. It could thus
be shown that again both AM-groups had been cleaved.
Furthermore, the ESI spectrum demonstrated that all
four butyryl protecting groups had been removed. The
hydroxymethyl intermediates, produced during enzy-
matic cleavage, could also be detected. Further studies
will be conducted in this area. Other possible substrates
Protection of benzyl mannopyranoside and hydrogen-
ation14,15
Benzyl mannopyranoside was dissolved in dry pyridine
(0.1 M solution) at 0ꢁC. Butyryl chloride (3 equiv/OH),
pivaloyl chloride (3 equiv/OH) or iso-propylchlorformiate
(1.5 equiv/OH, 1 M toluene) were added dropwise. The
mixture was stirred overnight at room temperature.
Workup procedure for butyryl chloride and pivaloyl
chloride: The reaction was quenched with methanol, then
the solution concentrated and codistilled with toluene
under reduced pressure. The residue was dissolved in
dichloromethane, washed twice with saturated sodium

S. Rutschow et al. / Bioorg. Med. Chem. 10 (2002) 4043–4049
4045
Scheme 2.
hydrogen carbonate and once with water, then dried
over magnesium sulfate, filtrated, concentrated under
reduced pressure and again codistilled with toluene. The
crude residue was purified by column chromatography
with petroleum ether/ethyl acetate (1:1).
2,3,4,6-Tetra-O-butyryl-ꢀ-D-mannopyranose (1). Com-
pound 1 was synthesized in the manner described above.
Yield: 0.85 g (1.85 mmol, 52% with respect to mannose,
colourless sirup); Rf 0.49 in 1:1 petroleum ether/ethyl
acetate; 1H NMR (400 MHz, CDCl3) d 5.44 (dd, 1H, H-
3), 5.37 (ddꢂt, 1H, H-4), 5.31 (dd, 1H, H-2), 5.24 (s,
1H, H-1), 4.27–4.16 (m, 3H, H-5, H-6a, H-6b), 3.12 (bs,
1H, OH), 2.42–2.16 (m, 8H, 4x-CO–CH2–), 1.77–1.52 (m,
Workup procedure for iso-propyl chloroformiate: The
mixture was diluted with chloroform, washed twice
with 1 M hydrochloric acid and once with water,
then dried over magnesium sulfate, filtrated, con-
centrated under reduced pressure and codistilled with
toluene. The crude residue was purified by column
chromatography with petroleum ether/ethyl acetate
(1:1). Subsequently, hydrogenation was in dry
methanol (0.1 M solution) and Pd/C (10%) added
cautiously and the mixture stirred at room tempera-
ture under normal H2 pressure. After termination the
solution was filtrated over Celite, concentrated under
reduced pressure and purified by column chromato-
graphy with petroleum ether/ethyl acetate (3:1) to
give compounds 1, 2 or 3.
8H, 4x-CH2–CH3), 1.03–0.88 (m, 12H, 4x-CH3); J1,2
=
2.0, J2,3=3.1, J3,4=10.2, J4,5=9.7 Hz; 13C NMR (10 0 .62
MHz, CDCl3) d 173.4, 172.7, 172.5, 172.3 (C¼O), 92.4 (C-
1), 69.7 (C-2), 68.8, 68.6 (C-3, C-5), 65.7 (C-4), 62.2 (C-6),
36.1, 36.0, 35.9 (–CO–CH2–), 18.5, 18.3, 18.2 (–CH2–
CH3), 13.7, 13.6 (–CH3); C22H36O10 (460.52).
2,3,4,6-Tetra-O-pivaloyl-ꢀ-D-mannopyranose (2). Com-
pound 2 was synthesized in the manner described above.
Yield: 1.38 g (2.63 mmol, 34% with respect to mannose,
white crystals); mp 175.8 ꢁC; Rf 0.23 in 3:1 petroleum
1
ether/ethyl acetate; H NMR (400 MHz, CDCl3) d 5.53
(ddꢂt, 1H, H-4), 5.46 (dd, 1H, H-3), 5.28 (dd, 1H,

4046
S. Rutschow et al. / Bioorg. Med. Chem. 10 (2002) 4043–4049
H-2), 5.19 (bs, 1H, H-1), 4.29 (ddd, 1H, H-5), 4.22–4.13
(m, 2H, H-6a, H-6b), 3.17 (d, 1H, OH), 1.27, 1.24, 1.16,
g (1.84 mmol, 70%, sirup); [a]D +22.1 (c 0.7, CHCl3);
Rf 0.52 in 1:1 petroleum ether/ethyl acetate; H NMR
1
1.12 (4xs, 36H, 4x-C(CH3)3); J1,2=1.8, J2,3=3.1, J3,4
=
(400 MHz, CDCl3) d 7.38–7.33 (m, 10H, Ph), 5.58 (dd,
1H, H-1), 5.52 (ddꢂt, 1H, H-4), 5.31 (dd, 1H, H-3),
5.24 (ddꢂt, 1H, H-2), 5.16–5.08 (m, 4H, 2x-CH2–Ph),
4.07–3.99 (m, 2H, H-5, H-6a), 3.89 (dd, 1H, H-6b), 1.25,
10.2, J4,5=10.2; 13C NMR (100.62 MHz, CDCl3) d 178.3,
177.3, 176.7, 172.0(C ¼O), 92.5 (C-1), 69.9 (C-2), 69.1 (C-
3), 68.9 (C-5), 65.2 (C-4), 61.9 (C-6), 38.9, 38.8 (Cq,–C
(CH3)3), 27.2, 27.2, 27.1 (–C(CH3)3); C26H44O10 (516.63).
1.21, 1.14, 1.12 (4xs, 36H, 4x-C(CH3)3); J1,2=1.9, J2,3
3.2, J3,4=10.4, J4,5=10.1, J5,6a=2.8, J5,6b=1.3, J6,6
=
=
2,3,4,6-Tetra-O-iso-propylcarbonate-ꢀ-D-mannopyranose
(3). Compound 3 was synthesized in the manner descri-
bed above. Yield: 0.73 g (1.39 mmol, 73% with respect
to mannose, colourless sirup); Rf 0.34 in 3:1 petroleum
12.6, JH-1,P=6.3 Hz; 13C NMR (100.62 MHz, CDCl3) d
178.0, 176.6, 176.4, 172.0 (C¼O), 133.7–127.5 (Carom.),
95.6 (d, C-1), 70.6 (C-5), 70.1 (d,–CH2–Ph), 69.9 (d,–CH2–
Ph), 68.7 (C-3), 68.6 (d, C-2), 64.2 (C-4), 61.0(C-6), 38.9,
1
2
ether/ethyl acetate; H NMR (400 MHz, CDCl3) d 5.34
38.8 (Cq,–C(CH3)3), 27.2, 27.1 (–C(CH3)3); JC-1,P=5.6,
2x 2JCH2,P=5.6, 3JC-2,P=11.7 Hz; 31P NMR (101.26MHz,
(s, 1H, H-1), 5.26–5.22 (m, 2H, H-2, H-3), 5.09 (ddꢂt,
1H, H-4), 4.93–4.80(m, 4H, 4x-C H(CH3)2), 4.32–4.28
(dd, 3H, H-5, H-6a, H-6b), 1.34–1.26 (m, 24H, 8x-
CH(CH3)2); J4,5=9.7 Hz; 13C NMR (100.62 MHz,
CDCl3) d 154.3, 153.9, 153.6, 153.4 (C¼O), 92.1 (C-1),
73.0, 72.9, 72.7, 72.4 (–CH(CH3)2), 72.5, 72.1 (C-2, C-3),
70.0 (C-4), 68.5 (C-5), 66.1 (C-6), 21.7, 21.7, 21.6
(–CH(CH3)2; C22H36O14 (524.52).
CDCl3)
[M+Na]+, 815.46 [M+K]+. Anal. calcd for C40H57O13P
(776.86): C 61.84, H 7.40; Found: C 61.11, H 7.35.
d
ꢀ1.76; MALDI-TOF-MS: m/z 799.53
Dibenzyl-(2,3,4,6-tetra-O-iso-propylcarbonate-ꢀ-D-man-
nopyranosyl)-phosphate (6). Compound 3 (1.19 g, 2.27
mmol) was reacted in the manner described above. 1.54
g (1.96 mmol, 87%, sirup); [a]D +6.7 (c 0.5, CHCl3); Rf
0.40 in 1:1 petroleum ether/ethyl acetate; 1H NMR
(400 MHz, CDCl3) d 7.37–7.32 (m, 10H, Ph), 5.75 (dd,
1H, H-1), 5.23 (ddꢂt, 1H, H-2), 5.14–5.07 (m, 6H, H-3,
H-4, 2x-CH2–Ph), 4.86 (m, 4H, 4x-CH(CH3)2), 4.26 (dd,
1H, H-6a), 4.18–4.11 (m, 2H, H-5, H-6b), 1.33–1.23 (m,
Phosphorylation16
Under argon atmosphere 1H-tetrazole (5 equiv) was
suspended in dry dichloromethane (20mL). After addi-
tion of dibenzyl di-iso-propylphosphoramidite (2.5
equiv) the mixture was stirred at room temperature for
15 min in order to form the tetrazolide intermediate.
Then a solution of mannose derivatives 1, 2 or 3 in dry
dichloromethane (20mL) was added and the mixture
was stirred for further 3 h at room temperature before
being cooled to 0 ꢁC. MCPBA (3 equiv) was added and
stirring was continued for 1 h. The solvents were
removed under reduced pressure. Purification was by
column chromatography with petroleum ether/ethyl
acetate (3:1, 2:1) to give compounds 4, 5 or 6.
24H, 8x-CH(CH3)2); J1,2=1.6, J2,3=2.2, J5,6a=5.7, J6,6
=
11.7, JH-1,P=6.6 Hz; 13C NMR (100.62 MHz, CDCl3) d
154.3, 153.5, 153.4 (C¼O), 128.7–128.1 (Carom.), 94.9 (d,
C-1), 73.3, 73.1, 72.8, 72.3 (–CH(CH3)2), 71.7 (C-3),
71.5 (d, C-2), 70.2 (C-5), 70.0 (d,–CH2–Ph), 69.8 (d,–
CH2–Ph), 69.1 (C-4), 65.3 (C-6), 21.7–21.6 (–
2
2
3
CH(CH3)2); JC-1,P= 5.6, 2x JCH2,P=5.6, JC-2,P=11.7
Hz; 31P NMR (101.26 MHz, CDCl3) d ꢀ1.90; MALDI-
TOF-MS: m/z 807.44 [M+Na]+, 823.39 [M+K]+.
Anal. calcd for C36H49O17P (784.76): C 55.10, H 6.29;
Found: C 55.23, H 6.45.
Dibenzyl-(2,3,4,6-tetra-O-butyryl-ꢀ-D-mannopyranosyl)-
phosphate (4). Compound 1 (1.80g, 3.92 mmol) was
reacted in the manner described above. Yield: 2.51 g
(3.48 mmol, 89%, sirup); [a]D +13.7 (c 0.4, CHCl3); Rf
0.45 in 1:1 petroleum ether/ethyl acetate; 1H NMR
(400 MHz, CDCl3) d 7.40–7.30 (m, 10H, Ph), 5.62 (dd,
1H, H-1), 5.36 (ddꢂt, 1H, H-4), 5.31 (dd, 1H, H-3), 5.26
(ddꢂt, 1H, H-2), 5.12–5.09 (m, 4H, 2x-CH2–Ph), 4.14
(dd, 1H, H-6a), 4.03 (ddd, 1H, H-5), 3.95 (dd, 1H, H-6b),
2.40–2.19 (m, 8H, 4x-CO–CH2–), 1.75–1.53 (m, 8H, 4x-
Hydrogenation
Pd/C (10%) was given cautiously to a solution of man-
nopyranosyl phosphate derivatives 4, 5 or 6 in ethyl
acetate/methanol/water (1:2:1). The mixture was stirred
at room temperature under H2 atmosphere (50bar).
After termination the solution was filtrated over Celite
and concentrated under reduced pressure. The residue was
purified by column chromatography with chloroform/
methanol/water (6:3.5:0.5) to give products 7, 8 or 9.
CH2–CH3), 1.01–0.88 (m, 12H, 4x-CH3); J1,2=1.5, J2,3
=
3.1, J3,4=10.2, J4,5=9.7, J5,6a=4.1, J5,6b=2.0, J6,6=12.2,
JH-1,P=6.1 Hz; 13C NMR (100.62 MHz, CDCl3) d 173.1,
172.3, 172.1, 172.0(C ¼O), 130.2, 129.8 (Cq), 128.8–128.0
(Carom.), 95.3 (d, C-1), 70.5 (C-5), 70.0 (d,–CH2–Ph),
69.9 (d,–CH2–Ph), 68.6 (d, C-2), 68.2 (C-3), 64.8 (C-4),
61.4 (C-6), 36.0, 35.9, 35.8 (–CO–CH2–), 18.4, 18.3,
18.2, 18.1 (–CH2–CH3), 13.7, 13.6 (–CH3); 2JC-1,P=4.8, 2x
2,3,4,6-Tetra-O-butyryl-ꢀ-D-mannopyranosyl phosphate
(7). Compound 4 (2.43 g, 3.37 mmol) was reacted in
the manner described above in 40mL solvent for 5 h.
Yield: 1.35 g (2.50mmol, 74%, yellow sirup); [ a]D
+37.3 (c 1.0, CHCl3); Rf 0.27 in 6:3.5:0.5 chloroform/
methanol/water; 1H NMR (400 MHz, CDCl3) d 5.60
(bs, 1H, H-1), 5.40(dd, 1H, H-3), 5.36 (bs, 1H, H-2),
5.17 (ddꢂt, 1H, H-4), 4.27–4.13 (m, 1H, H-5), 3.79–
3.62 (m, 2H, H6a, H-6b), 2.41–2.24 (m, 8H, 4x-CO–
CH2–), 1.71–1.53 (m, 8H, 4x-CH2–CH3), 1.0–0.87 (m,
12H, 4x-CH3); J2,3=3.6, J3,4=9.7, J4,5=10.2 Hz; 13C
NMR (100.62 MHz, CDCl3) d 174.9–172.5 (C¼O), 94.9
3
2JCH2,P=6.1, JC-2,P=10.9 Hz; 31P NMR (101.26 MHz,
CDCl3) d ꢀ1.97. Anal. calcd for C36H49O13P (720.76): C
59.99, H 6.85; Found: C 60.01, H 6.74.
Dibenzyl-(2,3,4,6-tetra- O-pivaloyl-ꢀ-D-mannopyrano-
syl)-phosphate (5). Compound 2 (1.38 g, 2.63 mmol)
was reacted in the manner described above. Yield: 1.43

S. Rutschow et al. / Bioorg. Med. Chem. 10 (2002) 4043–4049
4047
(bs, C-1), 72.2 (C-5), 69.1 (C-2), 68.5 (C-3), 65.8 (C-4),
61.6 (C-6), 36.0, 35.9 (–CO–CH2–), 18.4, 18.3, 18.1
mixture was stirred at room temperature overnight, the
solvents evaporated and the residue purified by column
chromatography with petroleum ether/ethyl acetate
(1:1) to give 10 as a colourless sirup (68 mg, 0.1 mmol)
in 41% yield; [a]D +7.5 (c 0.5, CHCl3); Rf 0.29 in 1:1
petroleum ether/ethyl acetate; 1H NMR (400 MHz,
CDCl3) d 5.73–5.64 (m, 5H, H-1, 2x-CH2–, AM), 5.41
(ddꢂt, 1H, H-4), 5.38 (ddꢂt, 1H, H-2), 5.35 (dd, 1H,
H-3), 4.28–4.15 (m, 3H, H-5, H-6a, H-6b), 2.42–2.19
(m, 8H, 4x-CO–CH2–), 2.17, 2.16 (2xs, 6H,–CH3, AM),
1.75–1.52 (m, 8H, 4x-CH2–CH3), 1.04–0.87 (m, 12H,
3
(–CH2–CH3), 13.5 (–CH3); JC-2,P=9.2 Hz; 31P NMR
(101.26 MHz, CDCl3) d ꢀ1.71; MALDI-TOF-MS: m/z
+
563.61 [M+Na]+, 579.50[M+K]
, 585.49 [M–
H+Na+Na]+, 601.41 [M–H+Na+K]+. Anal. calcd
for C22H37O13P (540.50): C 48.89, H 6.90; Found: C
48.90, H 6.60.
2,3,4,6-Tetra-O-pivaloyl-ꢀ-D-mannopyranosyl phosphate
(8). Compound 5 (1.30g, 1.67 mmol) was reacted in the
manner described above in 32 mL solvent for 6 h.
4x-CH3); J1,2=1.9, J2,3=3.2, J3,4=9.9, J4,5=9.9, J5,6a
=
3.8, J5,6b=1.5, J6,6=11.7, JH-1,P=7.7 Hz; 13C NMR
(100.62 MHz, CDCl3) d 173.1, 172.2, 172.1 (C¼O),
169.3, 169.2 (C¼O, AM), 95.9 (d, C-1), 82.7
(ddꢂt,–CH2–, AM), 70.8 (C-5), 68.3 (d, C-2), 68.1 (C-
3), 64.7 (C-4), 61.4 (C-6), 35.9, 35.8 (–CO–CH2–), 20.6
(–CH3, AM), 18.4, 18.3, 18.1 (–CH2–CH3), 13.7, 13.6,
13.5 (–CH3); JC-1,P=6.1, JCH2,P=6.1, JC-2,P=12.2
Hz; 31P NMR (101.26 MHz, CDCl3) d ꢀ5.05; MALDI-
TOF-MS: m/z 707.29 [M+Na]+, 723.19 [M+K]+.
Anal. calcd for C28H45O17P (684.51): C 49.12, H 6.63;
Found: C 49.60, H 6.79.
Yield: 0.82 g (1.37 mmol, 82%, white solid); [a]D +27.0
(c 1.0, CHCl3); mp ꢂ245 ꢁC decomposition; Rf 0.34 in
6:3.5:0.5 chloroform/methanol/water; 1H NMR (400
MHz, methanol-d4) d 5.58 (ddꢂt, 1H, H-4), 5.48
(ddꢂt, 2H, H-1, H-3), 5.32 (bs, 1H, H-2), 4.48–4.15 (m,
3H, H-5, H-6a, H-6b), 1.28, 1.23, 1.15, 1.10(4xs, 36H,
4x-C(CH3)3); J3,4=10.4, J4,5=10.1 Hz; 13C NMR
(100.62 MHz, methanol-d4) d 179.8, 179.3, 178.8 (C¼O),
95.2 (bs, C-1), 71.7 (C-2), 71.4 (C-3), 70.8 (C-5), 66.5 (C-
4), 62.9 (C-6), 40.3, 40.2, 40.1, 40.0 (Cq,–C(CH3)3), 28.0,
2
2
3
3
27.9, 27.8 (–C(CH3)3; JC-2,P=12.7 Hz; 31P NMR
(101.26 MHz, methanol-d4) d ꢀ1.75; MALDI-TOF-MS:
m/z 619.42 [M+Na]+, 635.35 [M+K]+, 641.40[M ꢀ
H+Na+Na]+, 657.33 [MꢀH+Na+K]+, 673.29
[MꢀH+K+K]+. Anal. calcd for C26H45O13P (596.61):
C 52.34, H 7.60; Found: C 45.28, H 6.61 (Material
hygroscopic).
bis-Pivaloyloxymethyl-(2,3,4,6-tetra-O-butyryl-ꢀ-D-man-
nopyranosyl)-phosphate (11). Mannopyranosyl-1-phos-
phate 7 (111 mg, 0.21 mmol) was suspended in dry
acetonitrile (1 mL). DIPEA (0.11 mL, 0.62 mmol) and
iodomethylpivaloate (0.15 g, 0.62 mmol) were added.
The mixture was stirred at room temperature overnight,
then the solvent removed and the residue dissolved in
ethyl acetate. The mixture was washed twice with satu-
rated brine, dried over sodium sulfate, filtrated and
concentrated under reduced pressure. Purification of the
crude was followed by column chromatography (petro-
leum ether/ethyl acetate+1% Et3N, 1:1) to give com-
pound 11 (39 mg, 0.05 mmol, sirup) in 24% yield; [a]D
+4.1 (c 0.4, CHCl3); Rf 0.88 in 1:1 petroleum ether/
2,3,4,6-Tetra-O-iso-propylcarbonate-ꢀ-D-mannopyranosyl
phosphate (9). Compound 6 (0.45 g, 0.57 mmol) was
reacted in the manner described above in 8 mL solvent
overnight.
Yield: 0.26 g (0.43 mmol, 75%, solid); [a]D +23.4 (c 1.0,
CHCl3); mp 184.1 ꢁC; Rf 0.30 in 6:3.5:0.5 chloroform/
methanol/water; 1H NMR (400 MHz, methanol-d4) d
5.58 (d, 1H, H-1), 5.26 (bs, 1H, H-2), 5.21 (dd, 1H, H-
3), 5.12 (ddꢂt, 1H, H-4), 4.90–4.79 (m, 4H, 4x-
CH(CH3)2), 4.36–4.21 (m, 3H, H-5, H-6a, H-6b), 1.23–
1.20(m, 24H, 8x-CH(C H3)2); J2,3=3.1, J3,4=10.2,
J4,5=9.9, JH-1,P=7.1 Hz; 13C NMR (100.62 MHz,
methanol-d4) d 156.2, 155.5, 155.1 (C¼O), 95.1 (d, C-1),
74.4, 74.2, 73.8, (–CH(CH3)2), 74.3 (C-3), 74.3 (d, C-2),
70.9 (C-4), 70.4 (C-5), 66.5 (C-6), 22.3, 22.2 (–
1
ethyl acetate; H NMR (400 MHz, CDCl3) d 5.67–5.58
(m, 5H, H-1, 2x-CH2–, POM), 5.34 (ddꢂt, 1H, H-4),
5.31 (bs, 1H, H-2), 5.29 (dd, 1H, H-3), 4.22–4.07 (m,
3H, H-5, H-6a, H-6b), 2.33 (dt, 2H, –CO–CH2–), 2.27
(t, 2H, –CO–CH2–), 2.19 (dt, 2H, –CO–CH2–), 2.12 (dt,
2H, –CO–CH2), 1.68–1.46 (m, 8H, 4x-CH2–CH3), 1.18 (s,
18H, –C(CH3)3, POM), 0.96–0.81 (m, 12H, 4x-CH3);
J1,2=1.5, J2,3=3.1, J3,4=9.2, J4,5=9.7, J5,6a=4.1, J6,6
=
12.2 Hz; 13C NMR (100.62 MHz, CDCl3) d 173.5, 172.6,
172.5, 172.4 (C¼O), 96.4 (d, C-1), 83.0(d, –CH 2–, POM),
82.8 (d, –CH2–, POM), 70.7 (C-5), 68.3 (d, C-2), 68.1 (C-
3), 64.7 (C-4), 61.4 (C-6), 39.1 (Cq, –C(CH3)3, POM),
36.4, 36.2 (–CO–CH2–), 27.2 (–C(CH3)3, POM), 18.8,
2
3
CH(CH3)2); JC-1,P=3.6, JC-2,P=9.7 Hz; 31P NMR
(101.26 MHz, methanol-d4) d ꢀ1.02; MALDI-TOF-MS:
m/z 627.35 [M+Na]+, 643.29 [M+K]+, 649.33
[MꢀH+Na+Na]+, 665.28 [MꢀH+Na+K]+. Anal.
calcd for C22H37O17P (604.51): C 43.71, H 6.17; Found:
C 44.32, H 6.06.
2
18.7, 18.5 (–CH2–CH3), 14.1, 14.0, 13.9 (–CH3); JC-
2
3
1,P=6.1, 2x JCH2,P=6.1, JC-2,P=12.2 Hz; 31P NMR
(101.26 MHz, CDCl3) d ꢀ4.92; MALDI-TOF-MS: m/z
791.32 [M+Na]+, 807.29 [M+K]+. Anal. calcd for
C34H57O17P (768.81): C 53.12, H 7.47; Found: C 53.75,
H 7.56.
bis-Acetoxymethyl-(2,3,4,6-tetra-O-butyryl-ꢀ-D-manno-
pyranosyl)-phosphate (10). A solution of mannopyr-
anosyl-1-phosphate 7 (131 mg, 0.24 mmol) in dry
acetonitrile (3 mL) was evaporated to dryness. DIPEA
(0.2 mL, 1.2 mmol) and dry acetonitrile (3 mL) were
added and the solution was evaporated again and then
in high vacuum. Subsequently, dry acetonitrile (3 mL),
DIPEA (0.41 mL, 2.4 mmol) and bromomethylacetate
(0.59 mL, 6.1 mmol) were added under argon. The
bis-Acetoxymethyl-(2,3,4,6-tetra-O-pivaloyl-ꢀ-D-manno-
pyranosyl)-phosphate (12). Mannopyranosyl-1-phos-
phate 8 (269 mg, 0.45 mmol) was suspended in dry
acetonitrile (3 mL) and dry toluene (0.5 mL). DIPEA
(0.22 mL, 1.35 mmol) and bromomethylacetae (0.13

4048
S. Rutschow et al. / Bioorg. Med. Chem. 10 (2002) 4043–4049
mL, 1.35 mmol) were added. The mixture was stirred
overnight at room temperature, the reaction being
monitored by TLC (petroleum ether/ethyl acetate, 1:1).
After further addition of bromomethylacetate (0.1 mL,
1.02 mmol) and DIPEA (0.1 mL, 0.59 mmol) the sus-
pension was stirred again at room temperature for 2
days. The solvents were removed, the residue dissolved
in ethyl acetate (5 mL) and dichloromethane (5 mL).
The mixture was washed twice with saturated brine,
dried over sodium sulfate, filtrated and concentrated
under reduced pressure. The crude residue was purified
by column chromatography (petroleum ether/ethyl ace-
tate, 2:1) to yield 12 (104 mg, 0.14 mmol) as yellowish
solid in 31%; [a]D +12.0(c 0.5, CHCl 3); mp 88.5 ꢁC; Rf
0.31 in 1:1 petroleum ether/ethyl acetate; 1H NMR
(400 MHz, CDCl3) d 5.74–5.63 (m, 5H, H-1, 2x-CH2–,
AM), 5.58 (ddꢂt, 1H, H-4), 5.39–5.35 (m, 2H, H-2, H-
3), 4.29–4.16 (m, 3H, H-5, H-6a, H-6b), 2.17, 2.16 (2xs,
6H, –CH3, AM), 1.28, 1.24, 11.16, 1.12 (4xs, 36H, 4x-
C(CH3)3); J1,2=1.8, J3,4=9.4, J4,5=10.2 Hz; 13C NMR
(100.62 MHz, CDCl3) d 178.0, 176.4 (C¼O), 171.2,
169.2, 169.1 (C¼O, AM), 96.2 (d, C-1), 82.7 (d,–CH2–,
AM), 82.6 (d, –CH2–, AM), 71.0(C-5), 68.6 (C-3), 68.4
(d, C-2), 64.1 (C-4), 61.1 (C-6), 38.9, 38.8, 38.7 (Cq,
–C(CH3)3), 27.1, 27.0(–C( CH3)3), 21.1, 20.6, 20.5 (–
mmol), bromomethylacetate (96 mL, 0.98 mmol), stirred
for 3d, column chromatography with petroleum ether/
ethyl acetate (1:1)] as compound 10. Product 14 (5.4 mg,
7.2 mmol) was obtained as a colourless sirup in 17%
yield.%; [a]D ꢀ1.6 (c 0.6, CHCl3); Rf 0.22 in 1:1 petro-
1
leum ether/ethyl acetate; H NMR (400 MHz, CDCl3) d
5.79 (dd, 1H, H-1), 5.73–5.64 (m, 4H, 2x-CH2–, AM),
5.51 (ddꢂt, 1H, H-2), 5.16–5.13 (m, 2H, H-3, H-4),
4.92–4.81 (m, 4H, 4x-CH(CH3)2), 4.33 (dd, 1H, H-6a),
4.28–4.22 (m, 2H, H-5, H-6b), 2.18, 2.16 (2xs, 6H,
–CH3, AM), 1.34–1.25 (m, 24H, 4x-CH(CH3)2);
J1,2=1.5, J2,3=2.0, J5,6a=6.4, J6,6=12.2 Hz; 13C NMR
(100.62 MHz, CDCl3) d 168.3, 168.2 (C¼O, AM), 154.2,
153.5, 153.4, 153.3 (C¼O), 95.5 (d, C-1), 82.8 (d, –CH2–
, AM), 82.7 (d,–CH2–, AM), 73.5, 73.2, 72.9, 72.4
(–CH(CH3)2), 71.5, 68.9 (C-3, C-4), 71.2 (d, C-2), 70.5
(C-5), 65.2 (C-6), 21.7, 21.6, 21.5 (–CH(CH3)2), 20.6,
2
2
3
20.5 (–CH3, AM); JC-1,P=5.1, 2x JCH2,P=5.1, JC-
2,P=12.2 Hz; 31P NMR (101.26 MHz, CDCl3) d ꢀ5.30;
MALDI-TOF-MS: m/z 771.08 [M+Na]+, 787.03
[M+K]+. Anal. calcd for C28H45O21P (748.51): C
44.92, H 6.06; Found: C 45.09, H 6.20.
bis-Pivaloyloxy-(2,3,4,6-tetra-O-iso-propylcarbonate-ꢀ-
D-mannopyranosyl)-phosphate (15). Compound 9 (189
mg, 0.31 mmol) was suspended in dry acetonitrile (3
mL). DIPEA (0.16 mL, 0.94 mmol) and iodomethylpi-
valoate (0.23 g, 0.94 mmol) were added. The mixture
was stirred overnight at room temperature and the
course of the reaction followed by TLC (petroleum
ether/ethyl acetate, 1:1). The mixture was stirred for a
further 3 days after addition of DIPEA (0.16 mL) and
iodomethylpivaloate (0.23 g). The solvents were
removed, the residue was dissolved in ethyl acetate (5
mL) and dichloromethane (5 mL). The mixture was
washed twice with saturated brine, dried over sodium
sulfate, filtrated and concentrated under reduced pres-
sure. The crude residue was purified by column chro-
matography (petroleum ether/ethyl acetate+1% Et3N,
3:1) to yield 15 (9.3 mg, 0.01 mmol) as colourless sirup
in 4%; [a]D +2.3 (c 0.5, CHCl3); Rf 0.5 in 1:1 petroleum
2
2
3
CH3, AM); JC-1,P=5.1, 2x JCH2,P=5.1, JC-2,P=12.2
Hz; 31P NMR (101.26 MHz, CDCl3) d ꢀ5.47; MALDI-
TOF-MS: m/z 763.52 [M+Na]+, 779.46 [M+K]+.
Anal. calcd for C32H53O17P (740.74): C 51.89, H 7.21;
Found: C 51.19, H 7.32.
bis-Pivaloyloxymethyl-(2,3,4,6-tetra- O-pivaloyl-ꢀ -D -
mannopyranosyl)-phosphate (13). Compound 8 (232 mg,
0.39 mmol) was treated by the same procedure [dry
acetonitrile (3 mL), dry toluene (1 mL), iodomethylpi-
valoate (0.57 g, 2.34 mmol), DIPEA (0.40 mL, 2.34
mmol), stirred for 3d] as for compound 11. The
obtained residue was purified by column chromato-
graphy (petroleum ether/ethyl acetate+1% Et3N, 3:1)
to give 13 as a white solid (18 mg, 0.02 mmol) in 6%
yield; [a]D +6.1 (c 0.5, CHCl3); mp 86.7 ꢁC; Rf 0.62 in
1
1
1:1 petroleum ether/ethyl acetate; H NMR (400 MHz,
ether/ethyl acetate; H NMR (400 MHz, CDCl3) d 5.78
CDCl3) d 5.75–5.64 (m, 5H, H-1, 2x-CH2–POM), 5.58
(ddꢂt, 1H, H-4), 5.41–5.36 (m, 2H, H-2, H-3), 4.32–4.22
(m, 2H, H-5, H-6a), 4.13 (d, 1H, H-6b), 1.27, 1.23, 1.16,
1.11 (4xs, 36H, 4x-C(CH3)3), 1.24 (2xs, 18H, 2x-C(CH3)3,
POM); J1,2=1.5, J2,3=3.1, J3,4=9.9, J4,5=9.9, J5,6a=2.8,
(dd, 1H, H-1), 5.75–5.65 (m, 4H, 2x-CH2–, POM), 5.30
(ddꢂt, 1H, H-2), 5.16–5.12 (m, 2H, H-3, H-4), 4.91–
4.81 (m, 4H, 4x-CH(CH3)2), 4.33 (dd, 1H, H-6a), 4.27–
4.22 (m, 2H, H-5, H-6b), 1.32–1.26 (m, 24H, 4x-
CH(CH3)2), 1.24, 1.23 (2xs, 18H, 2x-C(CH3)3, POM);
J1,2=1.8, J2,3=4.9, J5,6a=6.4, J5,6b=2.5, J6,6=12.2,
3
J6,6=11.2 JH-1,P=5.6 Hz; 13C NMR (100.62 MHz,
3
CDCl3) d 177.9, 176.9, 176.7, 176.5 (C¼O), 96.2 (d, C-
JCH,CH3=6.4, JH-1,P=5.6 Hz; 13C NMR (100.62 MHz,
1), 83.0(d, –CH –, POM), 82.9 (d, –CH2–, POM), 70.9
2
CDCl3) d 95.5 (d, C-1), 83.0(d, –CH –, POM), 82.9 (d,
2
(C-5), 68.5 (d, C-2), 68.5 (C-3), 64.2 (C-4), 61.2 (C-6),
38.9, 38.8, 38.7 (Cq, –C(CH3)3, Piv, POM), 27.1, 27.0
–CH2–, POM), 73.4, 73.1, 72.9, 72.4 (–CH(CH3)2), 71.6
(C-3), 71.3 (d, C-2), 70.5 (C-5), 69.0 (C-4), 65.2 (C-6), 26.8
(–C(CH3)3, POM), 21.7, 21.6 (–CH(CH3)2); 3JC-1,P=5.1,
2x 2JCH2,P=5.1, 3JC-2,P=12.2 Hz; 31P NMR
(101.26 MHz, CDCl3) d ꢀ5.23; MALDI-TOF-MS: m/z
855.26 [M+Na]+, 871.22 [M+K]+. Anal. calcd for
C34H57O21P (8.32.79): C 49.04, H 6.90; Found: C 50.45,
H 7.34.
3
(–C(CH3)3), 26.8 (–C(CH3)3, POM); JC-1,P=5.6, 2x
3
2JCH2,P=5.1 JC-2,P=12.7 Hz; 31P NMR (101.26 MHz,
CDCl3)
[M+Na]+, 863.30[M+K] +. Anal. calcd for C38H65O17P
(824.90): C 55.33, H 7.94; Found: C 56.10, H 7.99.
d
ꢀ5.38; MALDI-TOF-MS: m/z 847.33
bis-Acetoxymethyl-(2,3,4,6-tetra-O-iso-propylcarbonate-
ꢀ-D-mannopyranosyl)-phosphate (14). Mannopyranosyl-
1-phosphate 9 (25.6 mg, 0.04 mmol) was treated by the
same procedure [dry acetonitrile (1 mL+1 mL+0.5
mL), DIPEA (0.02 mL, 0.12 mmol+0.04 mL, 0.24
Enzymatic tests
Crude enzyme. Compound 10 (10.1 mg) was incubated
in phosphate buffer (pH 7.4, 0.05 M, 1 mL) with porcine

S. Rutschow et al. / Bioorg. Med. Chem. 10 (2002) 4043–4049
4049
liver esterase (crude, EC 3.1.1., 1 U) at 37 ꢁC for 3 h.
Acknowledgements
The reaction was monitored by TLC (petroleum ether/
ethyl acetate, 1:1). After termination by quenching with
methanol (2 mL) the residue was centrifuged and desal-
ted using ZipTipC18 for direct spotting on a MALDI-
TOF MS target. Methanol/water solution in various
ratio was used for elution; MALDI-TOF-MS: m/z 706.98
[M+Na]+, 722.92 [M+K]+, 635.21 [M–AM+Na]+,
651.19 [M–AM+K]+, 657.27 [M–AM–H+Na+Na]+,
673.24 [M–AM–H+Na+K]+, 563.23 [M–2AM+Na]+,
585.24 [M–2AM–H+Na+Na]+, 601.21 [M–2AM–
H+Na+K]+, 587.26 [M–AM–Bt–H+Na+Na]+,
515.31 [M–2AM–Bt-H+Na+Na]+, 483.40[M–
PO3AM2+Na]+/[M–2Bt–2Ac+2H+Na]+, 499.31 [M–
PO3AM2+K]+/[M–2Bt–2Ac+2H+K]+, 413.68 [M–
PO3AM2–Bt+Na]+, 429.63 [M–PO3AM2–Bt+K]+.
Support of this work by the Deutsche For-
schungsgemeinschaft (SFB 470) and the Fonds der
Chemischen Industrie is gratefully acknowledged.
References and Notes
1. Jaeken, J.; Vanderschueren-Lodeweyckx, M.; Casaer, P.;
Snoeck, L.; Corbeel, L.; Eggermont, E.; Edckels, R. Pediatr.
Res. 1980, 14, 179.
2. Liebermann, K. C.; Heidelberger, C. J. Biol. Chem. 1955,
316, 823.
3. Roll, P. M.; Weinfeld, H.; Carroll, E.; Brown, G. B. J. Biol.
Chem. 1956, 220, 439.
4. Jansen, A. B. A.; Russell, T. J. J. Chem. Soc 1965, 2127.
5. Iyer, R. P.; Phillips, L. R.; Biddle, J. A.; Thakker, D. R.; Egan,
W.; Aoki, S.; Mitsuya, H. Tetrahedron Lett. 1989, 30, 7141.
6. Freed, J. J.; Farquhar, D.; Hampton, A. Biochem. Phar-
macol. 1989, 38, 3193.
7. Li, W.; Schultz, C.; Llopis, J.; Tsien, R. Y. Tetrahedron
Lett. 1997, 53, 12017.
8. Khan, S. R.; Farquhar, D. Tetrahedron Lett. 1999, 40, 607.
9. Farquhar, D.; Srivastva, D. N.; Kuttesch, N. J.; Saunders,
P. P. J. Pharma. Sci. 1983, 72, 324.
10. Srivastva, D. N.; Farquhar, D. Bioorg. Chem. 1984, 12, 118.
11. Sastry, J. K.; Nehete, P. N.; Khan, S.; Nowak, B. J.;
Plunkett, W.; Arlinghaus, R. B.; Farquhar, D. Mol. Pharma-
col. 1992, 41, 441.
12. Farquhar, D.; Khan, S.; Srivastva, D. N.; Saunders, P. P.
J. Med. Chem. 1994, 37, 3902.
13. Dziewiszek, K.; Banaszek, A.; Zamojski, A. Tetrahedron
Lett. 1987, 28, 1569.
14. Ogilvie, K. K.; Letsinger, R. L. J. Org. Chem. 1967, 32,
2365.
Pure enzyme. Compound 10 (9.7 mg) was incubated in
phosphate buffer (pH 7.4, 0.05 M, 1 mL) with porcine
liver esterase (pure, EC 3.1.1.1, 1 U) at 37 ꢁC for 3 h.
The reaction was monitored by TLC (petroleum ether/
ethyl acetate, 1:1). After termination by quenching with
methanol (2 mL) the residue was centrifuged and desal-
ted using ZipTipC18 for direct spotting on a MALDI-
TOF MS target and ESI analysis. Methanol/water
solution in various ratio was used for elution; MALDI-
TOF-MS: m/z 707.19 [M+Na]+, 723.09 [M+K]+,
635.22 [M–AM+Na]+, 651.17 [M–AM+K]+, 657.25
[M–AM–H+Na+Na]+, 673.22 [M–AM–H+Na+
K]+, 611.19 [M–Bt–Ac+H+K]+, 623.23 [M–2Ac+
H+Na]+, 563.21 [M–2AM+Na]+, 585.22 [M–2AM–
H+Na+Na]+, 601.11 [M–2AM–H+Na+K]+, 483.44
[M–PO3AM2+Na]+/[M–2Bt–2Ac+2H+Na]+, 499.22
[M–PO3AM2+K]+/[M–2Bt–2Ac+2H+K]+; ESI-MS:
m/z 354 [M–AM–4Bt+Na]+, 353 [M–2AM–3Bt+Na]+,
283 [M–2AM–4Bt+Na].
15. Nicolaou, K. C.; Webber, S. E. Synthesis 1986, 453.
16. Mills, S. J.; Potter, B. V. L. J. Chem. Soc., Perkin Trans. 1
1997, 1279.