Synthesis and evaluation of chromogenic and fluorogenic analogs of glycerol for enzyme assays

Article abstract of DOI:10.1002/hlca.200390199

The branched glycerol analogs 1 and 2 were prepared. Mono-ester derivatives of these triols undergo a chromogenic or fluorogenic reaction in the presence of NaIO₄. In contrast, both the diesters and the triols are themselves not chromogenic or fluorogenic. Diester derivatives of these triols can be used as probes for lipases. The tris-phosphate derivative of 1 is a fluorogenic substrate for various phosphatases.

Full text of DOI:10.1002/hlca.200390199

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Helvetica Chimica Acta Vol. 86 (2003)
Synthesis and Evaluation of Chromogenic and Fluorogenic Analogs of
Glycerol for Enzyme Assays
by Eva Maria Gonza¬lez-GarcÌa, Johann Grognux, Denis Wahler, and Jean-Louis Reymond*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern
(fax: ‡41316318057; e-mail:jean-louis.reymond@ioc.unibe.ch)
The branched glycerol analogs 1 and 2 were prepared. Mono-ester derivatives of these triols undergo a
chromogenic or fluorogenic reaction in the presence of NaIO4. In contrast, both the diesters and the triols are
themselves not chromogenic or fluorogenic. Diester derivatives of these triols can be used as probes for lipases.
The tris-phosphate derivative of 1 is a fluorogenic substrate for various phosphatases.
Introduction. Enzyme assays are indispensable tools in enzymology, where they
are used to identify enzymes and to evaluate their purity and activity [1]. When an
enzyme is discovered, one important question is always the identity of its natural
substrate, which is presumably also its best substrate in terms of kinetics behavior. In
the context of enzyme assays, the related problem is to find the optimal assay method
for any given enzyme, in particular the most-sensitive method. Simple assays giving
readily recordable spectroscopic signals are preferred in the context of high-throughput
screening for new enzymes by biodiversity mining or directed evolution. Herein, we
report the synthesis and evaluation of the branched triols 1 and 2 as building blocks for
enzyme substrates. Triols 1 and 2 are close analogs of glycerol, and their ester
derivatives can be used to prepare chromogenic and fluorogenic analogs of glycerides,
the natural substrates of lipases. The synthesis of these triols and their derivatives and
their applications in enzyme assays are presented.
HO
HO
HO
HO
NO2
O
O
O
O
OH
OH
1
2
Results and Discussion. We recently reported a general assay for hydrolytic
enzymes based on substrates such as 3, which release a 1,2-diol such as 4 upon reaction
with the enzymes (Scheme 1) [2]. The 1,2-diol product then reacts with NaIO4 to
generate a labile carbonyl product, e.g., 5, which subsequently undergoes a b-
elimination to give a fluorescent or colored phenol, e.g., umbelliferone 6, under
catalysis by bovine serum albumin (BSA) [3]. The indirect release strategy for the
colored/fluorescent products makes these precursors particularly resistant to non-
specific hydrolysis. This property can be ascribed to the aliphatic nature of the alcohol
leaving group in the enzyme-labile functional group, and provides a key advantage over

Helvetica Chimica Acta Vol. 86 (2003)
2459
the highly labile esters of 4-nitrophenol or umbelliferone (ˆ 7-hydroxy-2H-1-
benzopyran-2-one), which tend to hydrolyze spontaneously. Although these substrates
showed satisfactory reactivity with lipases and esterases, we were intrigued by the
possibility to improve their specific reactivities by designing a structure that would be a
close analog of glycerol, which is the core element of the natural triglycerides. Triols 1
and 2, which contain glycerol as part of their branched structures, were, therefore,
investigated as building blocks for specific lipase substrates.
Scheme 1. Principle of NaIO4 Coupled Enzyme Assay for Hydrolytic Enzymes
Coum ˆ 2-Oxo-2H-1-benzopyran-7-yl
Retrosynthetic analysis suggested to prepare triols 1 and 2 in a divergent synthesis
by alkylation of the phenols with the versatile precursor 7 (Scheme 2). The branched C-
framework in 7 would itself be assembled by addition of a carbon nucleophile to the
CˆO group of the protected dihydroxyacetone building block 8 to form the tertiary
alcohol. This strategy offered a significant simplification over a previously described
preparation of intermediate 7 starting with the alkylation of malonate [4].
Scheme 2. Retrosynthetic Analysis for the Synthesis of Glycerol Analogs
Me
Me
HO
HO
SN2
OX
O
O
Me
Me
O
O
OTos
O
OH
OH
C2-equivalent
8
1 X = Coum
2 X = PNP
7
Protected dihydroxyacetone 8 was obtained from 2-amino-2-(hydroxymethyl)pro-
pane-1,3-diol (9) by acetonide formation to 10 and oxidation with NaIO4 (Scheme 3)
[5]. Reaction with allylmagnesium bromide gave the tertiary alcohol 11 in 50% yield.
The yield could not be improved despite many modifications of the procedure,

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Helvetica Chimica Acta Vol. 86 (2003)
probably due to a competing deprotonation of the ketone by the Grignard reagent.
While reductive ozonolysis of 11 (O3, then; NaBH4, or Me2S and NaBH4, or (EtO)3P
and NaBH4) did not give isolable products, a three-step sequence involving OsO4-
catalyzed dihydroxylation to form diol 12, oxidative cleavage by NaIO4, and reduction
of the resulting aldehyde with NaBH4, gave the desired diol 13. Selective tosylation of
the primary alcohol was achieved with TsCl in pyridine to give the key intermediate 7.
Careful control of the reaction was necessary, as small amounts of the corresponding
bis[toluene-4-sulfonate] were, quite surprisingly, also formed. The toluene-4-sulfonate
was then substituted with either 4-nitrophenol or umbelliferone to give 1 and 2,
respectively.
Scheme 3. Synthesis of Triols 1 and 2
1. (MeO)2CMe2, DMF,
HO
HO
Me
O
TsOH (62 %)
Me
OH
O
Y
NH2
X
9
10 X = NH2, Y = CH2OH
8 X, Y = O
2. NaIO4 (86 %)
Me
Me
4. NMO, OsO4
O
3. AllylMgBr
(50 %)
O
OH
Me
Me
(47 %)
O
O
OH
OH
OH
11
12
9. H2O, H+
(100 %)
Me
5. NaIO4, then
NaBH4 (91 %)
HO
HO
O
Me
O
OX
OX
OH
OH
1 X = Coum
2 X = PNP
13 X = OH
6. TsCl, py (62 %)
7 X = OTs
7. KOCoum, DMF (87 %)
8. KOPNP, DMF (54 %)
14 X = OCoum
15 X = OPNP
NMO ˆ 4-Methylmorpholine 4-oxide, PNP ˆ 4-nitrophenyl, Coum ˆ 2-oxo-2H-benzopyran-7-yl
These triols were then converted to potential probes for enzyme activities
(Scheme 4). Acetylation gave the diacetates 16 and 17 quantitatively. Reaction with
decanoyl chloride or dodecanoyl chloride gave the corresponding diesters 18 21,

Helvetica Chimica Acta Vol. 86 (2003)
2461
which might serve as chromogenic or fluorogenic analogs of glycerides. The mono-
dodecanoyl esters 22 and 23 were also prepared. Finally, the tris-phosphate derivative
24 was obtained by reaction with dibenzyl N,N-diisopropyl phosphoramidite in the
presence of 1H-tetrazole, followed by oxidation with m-chloroperbenzoic acid (m-
CPBA). Hydrogenolysis effected clean and quantitative removal of the Bn protecting
groups to give tris-phosphate 25 as a substrate for phosphatases. It should be noted that
the functionalization of the sparingly soluble triols 1 and 2 gave generally low yields
despite careful control of the reaction conditions, in particular, drying of the starting
triols. Similarly, no products were formed in a series of attempts at monophosphor-
ylation of triols 1 or 2, or monoesters 22 and 23 according to the published
phosphorylation protocols [6] to produce monophosphate or phosphocholine ester
derivatives (for the assembly of a phospholipase substrates).
Scheme 4. Functionalization of Triols 1 and 2 into Enzyme Substrates
HO
RCOCl or Ac2O,
HO
py (25 - 100%)
OX
R
O
O
OH
O
1 X = Coum
2 X = PNP
R
OX
OH
O
(BnO)2PN(i-Pr)2, tetrazole,
C9H19
HO
O
R
X
then m-CPBA (27 %)
16
17
18
19
20
21
Me
Me
Coum
PNP
O
OX
OR
C9H19
C9H19
C11H23
C11H23
Coum
PNP
OH
RO
P O
O
Coum
PNP
X
O
O
Coum
PNP
22
23
P
RO OR
OCoum
O
OR
P
RO
O
24 R = Bn
25 R = H
H2, Pd/C (100 %)
Coum ˆ 2-Oxo-2H-1-benzopyran-7-yl, PNP ˆ 4-nitrophenyl
First, we investigated the reactivity of the triols in aqueous buffer in the presence of
NaIO4 and BSA. There was only a very small release of fluorescence or color upon
treatment of 0.1 mm aqueous solutions of the triols under our standard conditions
(1 mm NaIO4, 2 mg/ml BSA, 20 mm aq. borate pH 8.8 or phosphate pH 7.0). The
absence of a fluorogenic/chromogenic reaction with excess NaIO4 can be interpreted in
terms of an overoxidation of the intermediate hydroxy ketone 27 by a second C,C-bond
cleavage to form acid 28 and a second equiv. of formaldehyde, this overoxidation taking

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Helvetica Chimica Acta Vol. 86 (2003)
place faster than the b-elimination induced by BSA (Scheme 5). The formation of acid
28 via an unstable intermediate was confirmed by analyzing the reaction of triol 1 by
HPLC (Table 1) and 1H-NMR (see Exper. Part). Intrigued by this observation, we also
investigated the reactivity of diesters 16 21, mono-esters 22 and 23, and tris-phosphate
25 in the presence of NaIO4 and BSA. There was no reaction with diesters or
triphosphate as expected from the fact that these substrates do not have any
unprotected 1,2-diol functionalities. Mono-esters 22 and 23, which do have the critical
1,2-diol functional groups, reacted with NaIO4 and BSA to give the colored/fluorescent
products 4-nitrophenol or umbelliferone. The oxidationÀb-elimination sequence
proceeded with a half-life of t1/2 ˆ 15 min, which is comparable to that of simple diols
such as 4.
Table 1. Reaction of Triol 1 with NaIO4 Analyzed by HPLCa)
tR 4.8 min (1 ‡ 8)
tR 7.8 min (27)
tR 12.5 min (28)
A, t ˆ 2 min
A, t ˆ 1 h
A, t ˆ 5 h
A, t ˆ 24 h
B, t ˆ 2 min
B, t ˆ 1 h
63
49
90
96
34
30
25
14
37
47
7
0
4
3
4
0
27
58
82
0
66
43
17
4
B, t ˆ 6 h
B, t ˆ 24 h
a) The peak area observed are given in % for the reaction of 100 mm triol 1 in aq. 20 mm borate pH 8.8, 2 mg ¥
mlÀ1 BSA, 258, and A: 100 mm NaIO4, B: 1 mm NaIO4. Isocratic elution at 1.5 ml ¥ minÀ1 of 88% H2O, 12%
MeCN, 0.1% CF3COOH; detection by UV at 325 nm; analytical column: Chromolit Performance RP-18e
(Merck), 100 Â 4.6 mm (1.02129.0001, Charge No. OB 14306)
We next investigated the mono- and diester derivatives for use as lipase probes. Due
to the overoxidation chemistry observed with the triols, one could expect that a
chromogenic or fluorescent signal would be obtained only for lipases converting
selectively one of the diesters 16 21 to a mono-ester. Formation of the mono-ester
would be immediately followed by oxidation by NaIO4 to give an acyloxy ketone of
type 26 as for the reaction of the mono-esters with NaIO4. At that stage, the b-
elimination leading to the spectroscopic signal would take place only when the BSA-
catalyzed reaction was faster than a possible further hydrolysis of 26 by the lipase,
which would lead to hydroxy ketone 27 and, ultimately, to acid 28. This reactivity
pattern was indeed observed. Three commercial lipases, Rhizopusarrizus lipase
(RAL), Pseudomonas fluorescens lipase (PFL), and Pseudomonas species type B
lipoprotein lipase (PSBL), were chosen as test enzymes (Fig.). The reactions at the
lower concentration of enzyme gave generally stronger signals, with the bis-decanoates
18 and 19 giving the best ratios of catalytic rate over background (Vrel, in parentheses in
Table 2). This can be interpreted in terms of the chemistry discussed above, with the
enzyme reacting rapidly with the diester substrates, and much more slowly with the
acyloxy-ketone intermediate 26, such that the BSA-catalyzed b-elimination could take
place. Excess enzyme induces hydrolysis of 26, which prevents the formation of
umbelliferone or 4-nitrophenol by b-elimination. Any reaction that does not give a

Helvetica Chimica Acta Vol. 86 (2003)
2463
Scheme 5. Reaction Pathwaysfor the Reaction of Triol 1 and 2, and Their Ester Derivatives 16 23 in Aqueous
BuffersContaining NaIO 4, BSA, and Lipases
R
O
R
O
O
lipase
O
R
O
HO
OX
OX
OH
OH
O
22, 23, ...
16-21
lipase
NaIO4
R
O
BSA
HO
HO
O
O
XO
OX
OX
OH
X = Coum, PNP
26
1, 2
BSA
lipase
NaIO4
HO
O
O
NaIO4
O
OX
OX
27
28
significant spectroscopic signal might, therefore, signal either lack of reactivity of the
lipase, or very high reactivity leading to the rapid formation of 28.
The nonchromogenic and nonfluorogenic oxidation of triols 1 and 2, respectively,
with NaIO4 offered, in principle, the opportunity to assay esterification reactions of
these substrates according to an endpoint oxidation protocol. The triols would be
subjected to an esterification reaction, and the formation of a mono-esterified product
would be revealed after some time by oxidation with NaIO4. Various attempts to
subject these triols sequentially to a lipase/esterase-catalyzed esterification with vinyl
acetate in aqueous or organic phase, followed by reaction with periodate and BSA to
reveal the formation of a mono-esterification product, gave no results.
We next investigated the use of tris-phosphate 25 as a fluorogenic probe for
phosphatases. This substrate seemed promising as a possible chemoselective probe for
phytases against alkaline phosphatase, and as a tool to facilitate the discovery of novel
phytases by biodiversity mining. Phytases hydrolyze phytate, which is hexaphosphoryl-
ated inositol, and are used to enhance the nutritional value of animal feed formulations

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Helvetica Chimica Acta Vol. 86 (2003)
Figure. Fluorogenic reaction of umbelliferyl ethers 22 (mC10) and 18 (diC10) with Rhizopus arrizus lipase
(Fluka No. 62305) at 100 mg ¥ mlÀ1 (100), 10 mg ¥ mlÀ1 (10), or no enzyme (noE). Conditions:100 mm substrate in
aq. 20 mm borate buffer pH 8.8, 2 mg ¥ mlÀ1 BSA, 1 mm NaIO4, 308. 0.1-ml Assays were carried out in round-
bottom polypropylene 96 well-plates (Costar) and recorded with a Cytofluor II Fluorescence Plate Reader
(Perseptive Biosystems; filters lex ˆ 360 Æ 20, lem ˆ 460 Æ 20 nm).
Table 2. Initial Rates of Formation of Umbelliferone or 4-Nitrophenol from Lipase Substratesa)
No.
X
Ester
No Enz. RALb)
100
RALb)
10
PFLc)
100
PFLc)
10
PSBLd)
100
PSBLd)
10
16
17
18
19
20
21
22
23
Coum di-C2
PNP di-C2
Coum di-C10
PNP di-C10
Coum di-C12
PNP di-C12
0.38
1.3
0.09
0.58
0.10
0.11
0.33
1.0
0.47
1.1
0.53
1.4
0.55
1.3
9.0 (99)
1.2
1.3 (12)
1.7
45
5.3 (13) 1.5 (3)
19 (14)
0.11
5.3 (3)
0.15
6.0 (66)
5.2 (8)
0.90 (8)
0.18 (84)
25
11
13 (143)
2.8 (4)
2.2 (21)
9.3 (4)
50
3.2 (35)
4.7 (7)
1.2 (11)
0.59 (14)
12
0.59
1.2
0.49 (4) 0.48 (4)
0.26
0.22
0.57
0.32
1.8
0.60
Coum mono-C10 53
PNP mono-C10 24
27
2.0
4.1
a) Apparent rate of formation of umbelliferone (6) or 4-nitrophenol, in nm ¥ sÀ1. The number in parentheses is
the relative reaction rate Vrel ˆ (Vobs/Vno enz.) À 1. Vrel is given only when Vrel > 3, which indicates the detection
limit for catalysis, and these numbers are marked in italics. Conditions:100 mm substrate in 20 mm aq. borate
buffer pH 8.8, 308, 2 mg ¥ mlÀ1 BSA, 1 mm NaIO4, and a lipase at the indicated concentration in mg ¥ mlÀ1
.
b) Rhizopusarrizus lipase (RAL), Fluka No. 62305. c) Pseudomonas fluorescens lipase (PFL), Fluka
No. 62321. d) Pseudomonas species type B lipoprotein lipase (PSBL), Fluka No. F 62336.
by promoting the release of phosphate from phytate, which is abundant in plants.
Phytase treatment makes phosphate bio-assimilable by the animal and, thus, prevents
the release of the polluting phytate into soil [7]. Due to its polyanionic nature
reminiscent of phytate, we expected that tris-phosphate 25 might be selectively cleaved

Helvetica Chimica Acta Vol. 86 (2003)
2465
by phytases only, and might not be recognized by alkaline phosphatase. Tris-phosphate
25 was tested with four different phosphatases at various pH values according to an
endpoint protocol, leading, in certain cases, to a fluorogenic reaction (Table 3). The
occurrence of a fluorogenic reaction implied that two of the three phosphate groups
were cleaved upon incubation with the various phosphatases to form a primary mono-
phosphate, which then underwent the fluorogenic oxidation/b-elimination sequence
upon treatment with NaIO4 and BSA during the endpoint treatment. Two of the three
phytases tested showed significant reactivity with 25 at pH 2.5, under which conditions
the reaction with alkaline phosphatase (CIAP) was very slow. However, alkaline
phosphatase also reacted with the substrate at pH 5.5, indicating that the observed
selectivity at low pH might be more related to the intrinsic pH optima of the different
enzymes than to actual substrate selectivity.
a
Table 3. Reaction of Triphosphate 25 with VariousPhosphatases
)
Enzyme
pH 2.5
pH 4.5
pH 5.5
pH 6.5
CIAPb)
1.3
58.0
3.6
2.2
67.0
5.4
15.2
79.5
2.7
1.3
0.6
0.7
0.9
A.f. phytasec)
Novo phytase
Natuphos phytase
26.8
8.0
4.5
a) Amount of umbelliferone (6) formed given in percent. Conditions:0.1 mg ¥ ml À1 enzyme, 100 mm substrate 25,
10 mm aq. citrate, acetate, or phosphate with 1 mm CaCl2, 558. Enzymes and substrates are incubated for 1 h at
the desired pH and 558, the pH is then adjusted to 7.2 with 0.5m aq. dibasic phosphate. NaIO4 (final conc. 1 mm)
and BSA (final conc. 2 mg ¥ mlÀ1) are added, and the reactions incubated for a further 60 min before
fluorescence reading. Fluorescence was converted to umbelliferone (6) concentration with a calibration curve.
b) CIAP:calf intestinal alkaline phosphatase. c) A. f. phytase: Aspergillus ficuum phytase.
The conversion of tris-phosphate 25 to monophosphate 31 probably takes place by
hydrolysis of a primary phosphate group, migration of the phosphate group to the
primary OH group thus liberated, and further hydrolysis of a primary phosphate group
to give a mono-phosphate intermediate capable of undergoing a fluorogenic oxidation/
b-elimination sequence (Scheme 6). This process could not be investigated in detail due
to the limited amount of trisphosphate available.
The favorable reactivity of our tris-phosphate 25 with enzymes, in particular the
suitable processing of monophosphate 31 to a fluorescent product, indicated that the
triols 1 and 2 might serve as useful probes for kinase reactions. The reaction with
glycerol kinase, which phosphorylates glycerol to glycerol 1-phosphate with adenosine
triphosphate (ATP), was investigated [8]. Unfortunately, we could not detect any sign
of phosphorylation by endpoint treatment with NaIO4 and BSA. The lack of reactivity
of glycerol kinase with triols 1 or 2 can be explained by the fact that this enzyme is
highly selective for glycerol.
Conclusions. In summary, glycerol analogs 1 and 2 were prepared by an efficient
synthesis starting with 2-amino-2-(hydroxymethyl)propane-1,3-diol (9). Bis-dec-
anoates 18 and 19 were found to be reactive probes for lipases under dilute enzyme
conditions, and the tris-phosphate 25 operates as a fluorogenic phosphatase substrate,
with selectivity for phytases at low pH values.

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Helvetica Chimica Acta Vol. 86 (2003)
Scheme 6. Hypothetical Mechanism for the Dephosphorylation of Triphosphate 25 Leading to the Fluorescent
Umbelliferone (6). Treatment with NaIO4 and BSA is carried out after incubation with phosphatase enzymes.
¬
¬
This work was supported by the Swiss National Science Foundation, the Swiss Office Federal de l×Education
¬
et de la Science, and ProteusSA , NÓmes, France.
Experimental Part
General. All reactions were monitored by TLC on Alugram SIL G/UV254 silica-gel sheets (Macherey-Nagel)
with detection by UV or with 0.5% phosphomolybdic acid soln. in 95% EtOH. Silica gel 60 (Macherey-Nagel
230 400 mesh) was used for flash chromatography (FC). M.p.: Kofler or B¸chi 510 apparatus; uncorrected. IR
Spectra: Perkin-Elmer Spectrum One series FTIR apparatus. 1H- and 13C-NMR spectra: Bruker AC-300
spectrometer.
7-[3,4-Dihydroxy-3-(hydroxymethyl)butoxy]-2H-1-benzopyran-2-one (1).
A soln. of compound 14
(420 mg, 1.30 mmol) in THF (8 ml) and 1n HCl (8 ml) was stirred for 1 h until TLC indicated disappearance
of starting material. The mixture was extracted with AcOEt (10 Â 200 ml), dried (Na2SO4), filtered, and the
solvent was evaporated in vacuo to yield 1 (362 mg, 100%). Pale-yellow solid. M.p. 124 1268. TLC (AcOEt):
Rf 0.67. IR (KBr):3437 s (br.), 1703s, 1622s, 1034m, 847s. 1H-NMR (CD3OD, 300 MHz):7.86 ( d, J ˆ 8.6, 1 H);
7.52 (d, J ˆ 8.6, 1 H); 6.95 6.92 (m, 2 H); 6.22 (d, J ˆ 9.6, 1 H); 4.27 (t, J ˆ 7.0, 2 H); 3.55 (s, 4 H); 2.04 (t, J ˆ 7.0,
2 H). 13C-NMR (CD3OD, 75 MHz):163.9; 163.4; 157.1; 145.7; 130.4; 114.2; 114.0; 113.3; 102.3; 74.8; 66.4 (2 C);
‡
65.9; 34.1. EI-MS:279 (6, [ M À H] ), 149 (63), 134 (21), 84 (76), 66 (100).
2-(Hydroxymethyl)-4-[(4-nitrophenyl)oxy]butane-1,2-diol (2).
A soln. of compound 15 (330 mg,
1.12 mmol) in THF (4 ml) and 1n HCl (4 ml) was stirred for 30 min at r.t. The mixture was extracted with
AcOEt (10 Â 40 ml). The org. phases were dried (Na2SO4), and the solvent was evaporated to provide 2
(288 mg) in a quant. yield. Colorless solid. M.p. 69 718. TLC (AcOEt): Rf 0.59. IR (KBr):3393 s (br.), 1594s,
1506s, 1343s, 1264s, 1112m. 1H-NMR (CD3OD, 300 MHz):8.20 ( d, J ˆ 9.2, 2 H); 7.08 (d, J ˆ 9.2, 2 H); 4.31
(t, J ˆ 7.0, 2 H); 3.55 (s, 4 H); 2.04 (t, J ˆ 7.0, 2 H). 13C-NMR (CD3OD, 75 MHz):165.4; 142.4; 126.7 (2 C); 115.6
‡
(2 C); 74.7; 66.3 (2 C); 65.9; 34.0. EI-MS:257 (5, M ), 226 (67), 208 (59), 152 (77), 140 (83), 101 (96), 71 (100),
‡
‡
‡
43 (82). HR-MS (ESI -TOF-MS):258.0979 (C 11H16NO6 , [M ‡ H] ; calc. 258.0977).
2-(5-Hydroxy-2,2-dimethyl-1,3-dioxan-5-yl)ethyl 4-Methylbenzenesulfonate (7). TsCl (554 mg, 2.90 mmol)
was added to a soln. of diol 13 (465 mg, 2.64 mmol) in anh. pyridine (10 ml) at 08. The mixture was stirred at 58
for 3 d. The mixture was quenched with H2O (5 ml) and extracted with AcOEt (3 Â 50 ml). The org. phases
were dried (Na2SO4), and the solvents were evaporated in vacuo. The crude product was chromatographed
(silica gel; hexane/AcOEt 1:1) to yield 7 (538 mg, 62%). Pale-yellow oil. TLC (hexane/AcOEt 1:1): Rf 0.33. IR
1
(film):3374 m, 1216s, 1187s, 1038m, 815m. H-NMR (CDCl3, 300 MHz):7.78 ( d, J ˆ 8.2, 2 H); 7.35 (d, J ˆ 8.2,

Helvetica Chimica Acta Vol. 86 (2003)
2467
2 H); 4.22 (t, J ˆ 5.9, 2 H); 3.80 (d, 2J ˆ 12.0, 2 H); 3.50 (d, 2J ˆ 12.0, 2 H); 3.22 (br. s, 1 H); 2.46 (s, 3 H); 1.73
(t, J ˆ 5.9, 2 H); 1.42 (s, 6 H). 13C-NMR (CDCl3, 75 MHz):145.6; 133.5; 130.6 (2 C); 128.6 (2 C); 99.1; 69.1
‡
‡
(2 C); 66.7; 66.3; 33.8; 29.1; 22.3; 19.0. EI-MS:315 ([ M À Me] ), 172 (71), 91 (100), 41 (74). HR-MS (ESI -
‡
‡
TOF-MS):331.120 (C 15H23O6S , [M ‡ H] ; calc. 331.1215).
2,2-Dimethyl-1,3-dioxan-5-one (8). A soln. of NaIO4 (8.5 g, 39.9 mmol) in H2O (110 ml) was added
dropwise at 08, over a period of 3 h, to a slurry of 10 (6.4 g, 39.9 mmol) and KH2PO4 (5.4 g, 39.9 mmol) also in
H2O (130 ml). The resulting mixture was stirred at 58 for 1 h and then 5 h at r.t., Na2S2O3 (9.9 g, 39.9 mmol) was
added, and the resulting soln. was stirred for a further 15 min. Then, the mixture was extracted with CH2Cl2
(10 Â 50 ml). The combined org. phases were dried (Na2SO4), filtered, concentrated in vacuo, and purified by
bulb-to-bulb distillation (958) to afford 8 (4.47 g, 86%). Colorless volatile oil. TLC (hexane/AcOEt 1 :1):
Rf 0.46. EI-MS:130 (12, M ), 115 (27), 100 (23), 72 (44), 43 (100). IR (film):1752. 1H-NMR (CDCl3,
300 MHz):4.1 ( s, 4 H); 1.41 (s, 6 H). 13C-NMR (CDCl3, 75 MHz):208.7; 100.7; 67.4 (2 C); 24.1 (2 C). EI-HR-
‡
‡
MS:130.062960 (C 9H10O3 ; calc. 130.062994).
5-Amino-2,2-dimethyl-1,3-dioxane-5-methanol (10). TsOH (603 mg, 3.17 mmol) was added to a soln. of 2-
amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride (10 g, 63.4 mmol) in dry DMF (70 ml) at r.t., followed
by the addition of 2,2-dimethoxypropane (8.55 ml, 69.7 mmol) in one portion. The resulting white suspension
became clear and colorless after 30 min. After stirring 12 h, Et3N (0.5 ml, 36 mmol) was added, and the mixture
was stirred for an additional 10 min. The mixture was next concentrated in vacuo and treated with Et3N (7 ml)
and AcOEt (250 ml). The white precipitate formed upon addition of the base was collected by filtration. The
crude product was recrystallized in Et2O (7 ml) to afford 10 (6.40 g, 62%). Colorless solid. M.p. 67 698. TLC
‡
(CH2Cl2/MeOH 9 :1): Rf 0.31. EI-MS:146 (80, [ M À Me] ), 130 (85), 73 (100), 42 (73). IR (KBr):3266.0 s,
1
1603.7s, 1372.4s, 1059.8s, 830.0s. H-NMR (CDCl3, 300 MHz):3.80 ( d, 2J ˆ 12.0, 2 H); 3.54 (d, 2J ˆ 12.0, 2 H);
3.50 (s, 2 H); 1.98 (br. s, 3 H); 1.46 (s, 3 H); 1.42 (s, 3 H). 13C-NMR (CDCl3, 75 MHz):99.1; 67.9 (2 C); 65.6;
50.9; 25.6; 22.8.
2,2-Dimethyl-5-(prop-2-enyl)-1,3-dioxan-5-ol (11). Allylmagnesium bromide (ca. 1m in hexane, 14.1 ml,
14.15 mmol) was added to a soln. of 8 (920 mg, 7.08 mmol) in dry THF (40 ml) at 08 during 5 min. The mixture
was stirred for 2 h at r.t., then the reaction was quenched with 5n NH4Cl (5 ml), the mixture was extracted with
AcOEt (3 Â 50 ml) and washed with brine (2 Â 50 ml). The org. phases were dried (Na2SO4) and concentrated
in vacuo. The crude product was chromatographed (silica gel; hexane/AcOEt 7:3) to afford 11 (610 mg, 50%).
Colorless oil. TLC (hexane/AcOEt 7 :3): Rf 0.6. IR (film):3448 m, 2994m, 1641w, 1374s, 1200s, 830s. 1H-NMR
(CDCl3, 300 MHz):5.92 5.78 ( m, 1 H); 5.14 5.07 (m, 2 H); 3.78 (d, 2J ˆ 11.8, 2 H); 3.53 (d, 2J ˆ 11.8, 2 H);
3.17 (br. s, 1 H); 2.16 (d, J ˆ 7.3, 2 H); 1.43 (s, 3 H); 1.41 (s, 3 H). 13C-NMR (CDCl3, 75 MHz):132.3; 119.5; 89.9;
‡
69.0 (2 C); 67.2; 39.5; 28.5; 19.6. EI-MS:171 ([ M À H] ), 157 (32), 141 (29), 83 (30), 59 (47), 43 (100). HR-MS
‡
‡
(ESI -TOF-MS):173.1179 (C 9H17O3, [M ‡ H] ; calc. 173.1177).
3-(2,2-Dimethyl-5-hydroxy-1,3-dioxan-5-yl)propane-1,2-diol (12). 4-Methylmorpholine 4-oxide (1.54 g,
11.36 mmol) and catalytic OsO4 (0.05 mmol) were added at r.t. to a soln. of 11 (490 mg, 2.84 mmol) in acetone
(15 ml) and H2O (15 ml). The mixture was stirred for 5 h, the reaction was quenched with an aq. soln. of 1n
Na2SO3 (5 ml), and the mixture was extracted with AcOEt (10 Â 100 ml). The combined org. extracts were
dried (Na2SO4), filtered, and concentrated in vacuo, to produce a white precipitate, which was collected by
filtration and washed with hexane/AcOEt 1:1, affording 12 (278 mg, 47%). Colorless solid. M.p. 139 1418.
TLC (AcOEt): Rf 0.38. IR (KBr):3393 w, 2996m, 2521s, 1730s, 1292s, 1074s. 1H-NMR (CD3OD, 300 MHz):
3.97 3.90 (m, 1 H); 3.83 (dd, J ˆ 2.2, 11.9, 2 H); 3.64 (2dd, J ˆ 2.2, 11.9, 2 H); 3.44 (d, J ˆ 5.5, 2 H); 1.67 (dd, J ˆ
2.6, 14.7, 1 H); 1.49 (dd, J ˆ 9.6, 14.7, 1 H); 1.40 (s, 6 H). 13C-NMR (CD3OD, 75 MHz):99.4; 69.6; 69.4; 69.3;
67.9; 66.4; 38.9; 25.7; 22.0. Anal. calc. for C9H18O5 (206.24):C 52.41, H 8.80; found:C 52.33, H 8.77.
5-(2-Hydroxyethyl)-2,2-dimethyl-1,3-dioxan-5-ol (13). NaIO4 (303 mg, 1.42 mmol) was added to a soln. of
12 (278 mg, 1.35 mmol) in H2O (10 ml) at 08, the mixture was stirred at this temp. for 30 min until the TLC
showed that the starting material had disappeared. NaBH4 (64 mg, 1.69 mmol) was added directly, and the
mixture was stirred for further 45 min, extracted with AcOEt (5 Â 25 ml), dried (Na2SO4), filtered, and
concentrated in vacuo. The product was chromatographed (silica gel; AcOEt/hexane 4 :1) to afford 13 (270 mg,
1
91%). Pale-yellow oil. TLC (AcOEt): Rf 0.53. IR (film):3415 s, 2922s, 1376m, 1200m, 1077s, 829m. H-NMR
(CD3OD, 300 MHz):3.80 ( d, 2J ˆ 11.8, 2 H); 3.74 (t, J ˆ 6.6, 2 H); 3.60 (d, 2J ˆ 11.8, 2 H); 3.50 (br. s, 2 H); 1.69
(t, J ˆ 6.6, 2 H); 1.40 (s, 6 H). 13C-NMR (CD3OD, 75 MHz):99.3; 69.3 (2 C); 67.8; 58.1; 38.2; 25.6; 22.0. EI-MS:
‡
174 ([M À 2 H] ), 145 (34), 72 (76), 70 (46), 59 (100).
7-[2-(5-Hydroxy-2,2-dimethyl-1,3-dioxan-5-yl)ethoxy]-2H-1-benzopyran-2-one (14). To a suspension of 7-
hydroxy-2H-1-benzopyran-2-one (6; 97.5 mg, 0.6 mmol) in DMF (2 ml), oven-dried K2CO3 (167 mg,
1.21 mmol), and 18-crown-6 ether as catalyst, previously stirred for 1 h at r.t., was added a solution of 7

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Helvetica Chimica Acta Vol. 86 (2003)
(200 mg, 0.6 mmol) in DMF (2 ml). The slurry was stirred for 12 h at 958. After quenching with H2O (5 ml), the
mixture was extracted with AcOEt (3 Â 20 ml), washed with aq. NaOH (3 Â 20 ml) and brine (3 Â 20 ml). The
org. phases were dried (Na2SO4), filtered, concentrated in vacuo, and purified by chromatography to afford 14
(167 mg, 87%). Pale-yellow oil. TLC (hexane/AcOEt 1:1): Rf 0.22. IR (film):3452 m, 1729s, 1614s, 1230m,
1
1199m, 1126m. H-NMR (CDCl3, 300 MHz):7.63 ( d, J ˆ 9.2, 1 H); 7.36 (d, J ˆ 9.2, 1 H); 6.83 6.80 (m, 2 H);
6.24 (d, J ˆ 9.6, 1 H); 4.24 (t, J ˆ 5.9, 2 H); 3.90 (d, 2J ˆ 11.9, 2 H); 3.65 (d, 2J ˆ 11.9, 2 H); 3.39 (br. s, 1 H); 1.88
(t, J ˆ 5.9, 2 H); 1.46 (s, 3 H); 1.45 (s, 3 H). 13C-NMR (CDCl3, 75 MHz):162.3; 162.0; 156.6; 143.9; 129.5; 114.0;
‡
113.5; 113.4; 102.3; 99.1; 69.4 (2 C); 67.1; 64.3; 33.8; 28.9; 19.4. EI-MS:321 (9, [ M À Me] ), 320 (33, [M À
‡
H2O] ), 175 (81), 162 (100), 134 (60), 43 (73).
2,2-Dimethyl-5-[2-(4-nitrophenyloxy)ethyl]-1,3-dioxan-5-ol (15). A soln. of K2CO3 (234 mg, 1.7 mmol), 4-
nitrophenol (142 mg, 1.02 mmol), and 18-crown-6 ether (as catalyst, 0.34 mmol) in acetone (6 ml) was stirred for
40 min ar r.t. Compound 7 (280 mg, 0.85 mmol) was added, and the mixture was refluxed overnight. After
quenching with H2O (10 ml) and extraction with AcOEt (3 Â 30 ml), the org. phases were dried (Na2SO4), and
the solvents were evaporated in vacuo. The crude product was chromatographed (silica gel; hexane/AcOEt 7:3)
to afford 15 (137 mg, 54%). Yellow solid. M.p. 95 978. TLC (hexane/AcOEt 1:1): Rf 0.59. IR (KBr):3343 m,
1606m, 1594s, 1508s, 1341s, 1259s. 1H-NMR (CDCl3, 300 MHz):8.20 ( d, J ˆ 9.2, 2 H); 6.95 (d, J ˆ 9.2, 2 H); 4.29
(t, J ˆ 5.9, 2 H); 3.91 (d, 2J ˆ 11.7, 2 H); 3.65 (d, 2J ˆ 11.7, 2 H); 3.35 (br. s, 1 H); 1.89 (t, J ˆ 5.9, 2 H); 1.48
(s, 3 H); 1.46 (s, 3 H). 13C-NMR (CDCl3, 75 MHz):164.0; 142.2; 126.5 (2 C); 115.0 (2 C); 90.0; 69.2 (2 C); 66.9;
‡
‡
64.4; 33.7; 28.8; 19.2. EI-MS:298 (48, [ M ‡ 1] ), 282 (48, [M À Me] ), 240 (100), 152 (35), 119 (35). HR-MS
‡
‡
‡
(ESI -TOF-MS):298.1299 (C 14H20NO6 , [M ‡ H] ; calc. 298.1290).
2-(Acetoxymethyl)-2-hydroxy-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butyl Acetate (16). Ac2O (1 ml) was
added to a soln. of 1 (20 mg, 0.08 mol) in dry pyridine (1 ml), the mixture was stirred overnight at r.t. Then the
solvent was evaporated to give 16 in quant. yield. Pale-yellow oil. TLC (hexane/AcOEt 1:1): Rf 0.24. EI-MS:
‡
365 (100, [M ‡ 1] ), 305 (17), 245 (33), 155 (37), 135 (27), 119 (80). IR (film):3453 w, 1733s, 1515s, 1232s,
1127m, 1046m, 838w. 1H-NMR (CD3OD, 300 MHz):7.64 ( d, J ˆ 9.5, 1 H); 7.37 (d, J ˆ 9.4, 1 H); 6.84 6.82
(m, 2 H); 6.25 (d, J ˆ 9.4, 1 H); 4.26 (t, J ˆ 5.9, 2 H); 4.21 4.12 (m, 4 H); 2.90 (br. s, 1 H); 2.14 2.06 (m, 2 H);
2.11 (s, 6 H). 13C-NMR (CD3OD, 75 MHz):171.5 (2 C); 162.2; 161.7; 156.5; 143.9; 129.5; 114.0; 113.6; 113.5;
‡
‡
‡
102.1; 72.6; 67.5 (2 C); 64.5; 34.2; 21.5 (2 C). HR-MS (ESI -TOF-MS):365.1267 (C 18H21NO8 , [M ‡ H] ;
365.136).
2-(Acetoxymethyl)-2-hydroxy-4-[(4-nitrophenyl)oxy]butyl Acetate (17). Ac2O (1 ml) was added at 08 to a
soln. of 2 (20 mg, 0.08 mmol) in dry pyridine (1 ml), and the mixture was stirred overnight at r.t. The solvent was
evaporated to give 17 in quant. yield. Pale-yellow oil. TLC (hexane/AcOEt 1:1): Rf 0.42. IR (film):3483 w,
1
1740s, 1594s, 1515s, 1342s, 1259s, 1112m. H-NMR (CD3OD, 300 MHz):8.20 ( d, J ˆ 9.2, 2 H); 6.95 (d, J ˆ 9.2,
2 H); 4.29 (t, J ˆ 6.2, 2 H); 4.21 4.12 (m, 4 H); 2.14 2.04 (m, 2 H); 2.10 (s, 6 H). 13C-NMR (CD3OD, 75 MHz):
‡
171.5 (2 C); 163.9; 142.4; 126.6 (2 C); 115.1 (2 C); 72.5; 67.4 (2 C); 64.7; 34.1; 21.5 (2 C). EI-MS:341 (15, M ),
‡
‡
324 (96), 282 (25), 222 (42), 155 (40), 135 (47), 119 (100). HR-MS (ESI -TOF-MS):341.11110 (C 15H19NO8 ,
[M ‡ H] ; calc. 341.11106).
‡
2-[(Decanoyloxy)methyl]-2-hydroxy-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butyl Decanoate (18). Decano-
yl chloride (182 ml, 0.89 mmol) was added to a soln. of 1 (100 mg, 0.36 mmol) in dry pyridine (6.6 ml) at 08, and
the mixture was stirred at 58 overnight. The solvent was evaporated, and the crude product was extracted with
AcOEt (3 Â 25 ml), washed with aq. NaHCO3 (3 Â 20 ml), and dried (Na2SO4). The yellow oil obtained after
evaporation of the solvents was chromatographed (silica gel; hexane/AcOEt 4 :1) to yield 18 (96 mg, 43%).
Colorless crystals. M.p. 64 668. TLC (hexane/AcOEt 3 :2): Rf 0.45. IR (KBr):3410 m, 2925s, 2855s, 1733s,
1
1615m, 1470m. H-NMR (CDCl3, 300 MHz):7.64 ( d, J ˆ 9.3, 1 H); 7.38 (d, J ˆ 9.3, 1 H); 6.95 6.93 (m, 2 H);
6.27 (d, J ˆ 9.3, 1 H); 4.26 (t, J ˆ 6.5, 2 H); 4.21 4.11 (m, 4 H); 2.82 (br. s, 1 H); 2.35 (t, J ˆ 7.7, 4 H); 2.11 (t, J ˆ
6.5, 2 H); 1.66 1.61 (m, 4 H); 1.39 1.20 (m, 24 H); 0.88 (t, J ˆ 6.9, 6 H). 13C-NMR (CDCl3, 75 MHz):174.3
(2 C); 162.2; 161.7; 156.5; 143.9; 129.5; 114.0; 113.9; 113.5; 102.1; 72.7; 67.3 (2 C); 64.6; 34.8 (2 C); 34.2; 32.5
(2 C); 30.2 (2 C); 30.1 (2 C); 30.0 (2 C); 29.9 (2 C); 29.8 (4 C); 25.6 (2 C); 23.3 (2 C); 14.7 (2 C). FAB-MS:571
(45), 435 (94), 417 (33), 281 (100), 263 (63), 163 (76). Anal. calc. for C34H52O8 (588.77):C 69.36, H 8.90; found:
C 69.25, H 8.91.
2-[(Decanoyloxy)methyl]-2-hydroxy-4-[(4-nitrophenyl)oxy]butyl Decanoate (19). Decanoyl chloride
(157 ml, 0.77 mmol) was added to a soln. of 2 (67 mg, 0.29 mmol) in dry pyridine (4 ml) at 08, and the mixture
was stirred at 58 overnight. The solvent was evaporated, and the crude product was extracted with AcOEt (3 Â
15 ml), washed with aq. sat. NaHCO3 (3 Â 10 ml), and dried (Na2SO4). The crude product obtained after
evaporation of the solvents was chromatographed (silica gel; hexane/AcOEt 85 :15) to give 19 (63 mg, 38%).
Colorless oil. TLC (hexane/AcOEt 7:3): Rf 0.64. IR (film):3448 w, 2927s, 2856s, 2856s, 1742s, 1594m, 1516m,

Helvetica Chimica Acta Vol. 86 (2003)
2469
1342s, 1262s, 1111m, 847w. 1H-NMR (CDCl3, 300 MHz):8.19 ( d, J ˆ 9.2, 2 H); 6.95 (d, J ˆ 9.2, 2 H); 4.28 (t, J ˆ
6.5, 2 H); 4.21 4.10 (m, 4 H); 2.85 (br. s, 1 H); 2.34 (t, J ˆ 7.3, 4 H); 2.10 (t, J ˆ 6.5, 2 H); 1.64 1.60 (m, 4 H);
1.38 1.28 (m, 24 H); 0.87 (t, J ˆ 6.9, 6 H). 13C-NMR (CDCl3, 75 MHz):174.4 (2 C); 164.0; 142.4; 126.6 (2 C);
115.1 (2 C); 72.7; 67.3 (2 C); 64.7; 34.8 (2 C); 34.2; 32.5 (2 C); 30.0 (2 C); 29.9 (4 C); 29.8 (2 C); 25.5 (2 C); 23.3
‡
‡
(2 C); 14.7 (2 C). EI-MS:565 (6, M ), 535 (63), 380 (85), 155 (100), 83 (67). HR-MS (ESI -TOF-MS):548.3576
‡
‡
(C31H50NO7 , [M À H2 ‡ H] ; 548.3587).
2-[(Dodecanoyloxy)methyl]-2-hydroxy-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butyl Dodecanoate (20).
Dodecanoyl chloride (211 ml, 0.89 mmol) was added to a soln. of 2 (100 mg, 0.36 mmol) in dry pyridine
(6.6 ml) at 08, and the mixture was stirred at 58 overnight. The solvents were evaporated, and the crude product
was extracted with AcOEt (3 Â 25 ml), washed with aq. NaHCO3 (3 Â 20 ml), and dried (Na2SO4). The yellow
oil obtained was chromatographed (silica gel; hexane/AcOEt 4 :1). The crude product obtained was further
purified by recrystallization from hexane to afford 20 (55 mg, 25%). Colorless crystals. M.p. 69 718. TLC
(hexane/AcOEt 3 :2): Rf 0.45. IR (KBr):2958 m, 2922m, 2853m, 1738s, 1621m, 1470m. 1H-NMR (CDCl3,
300 MHz):7.63 ( d, J ˆ 9.4, 1 H); 7.36 (d, J ˆ 9.4, 1 H); 6.83 6.81 (m, 2 H); 6.25 (d, J ˆ 9.5, 1 H); 4.26 (t, J ˆ 5.9,
2 H); 4.21 4.11 (m, 4 H); 2.91 (br. s, 1 H); 2.34 (t, J ˆ 7.4, 4 H); 2.10 (t, J ˆ 5.9, 2 H); 1.64 1.59 (m, 4 H); 1.39
1.23 (m, 32 H); 0.87 (t, J ˆ 6.6, 6 H). 13C-NMR (CDCl3, 75 MHz):174.3 (2 C); 162.2; 161.7; 156.5; 143.9; 129.5;
114.0; 113.5; 113.4; 102.1; 72.7; 67.3 (2 C); 64.6; 34.8 (2 C); 34.2; 32.5 (2 C); 30.2 (2 C); 30.1 (2 C); 30.0 (2 C);
29.9 (2 C); 29.8 (4 C); 25.6 (2 C); 23.3 (2 C); 14.7 (2 C). Anal. calc. for C38H60O8 (644.88):C 70.77, H 9.38;
found:C 70.68, H 9.37.
2-[(Dodecanoyloxy)methyl]-2-hydroxy-4-[(4-nitrophenyl)oxy]butyl Dodecanoate (21). Dodecanoyl chlor-
ide (161 ml, 0.68 mmol) was added to a soln. of 2 (67 mg, 0.26 mmol) in dry pyridine (4 ml) at 08, and the mixture
was stirred at 58 overnight. The solvent was evaporated, and the crude product was extracted with AcOEt (3 Â
15 ml), washed with aq. sat. NaHCO3 (3 Â 10 ml), and dried (Na2SO4). The crude residue obtained after
evaporation of the solvent was chromatographed (silica gel, hexane/AcOEt 85 :15) to provide 21 (67 mg, 41%).
Colorless oil. TLC (hexane/AcOEt 7 :3): Rf 0.79. IR (film):3381 w, 2956s, 2921s, 2852s, 1711s, 1593m, 1343s,
1265.5s, 1111m, 849w. 1H-NMR (CDCl3, 300 MHz):8.20 ( d, J ˆ 9.2, 2 H); 6.95 (d, J ˆ 9.2, 2 H); 4.29 (t, J ˆ 6.0,
2 H); 4.21 4.11 (m, 4 H); 2.81 (br. s, 1 H); 2.35 (t, J ˆ 7.4, 4 H); 2.10 (t, J ˆ 6.0, 2 H); 1.65 1.60 (t, J ˆ 7.0, 4 H);
1.38 1.28 (m, 32 H); 0.88 (t, J ˆ 6.2, 6 H). 13C-NMR (CDCl3, 75 MHz):174.4 (2 C); 164.0; 142.4; 126.6 (2 C);
115.1 (2 C); 72.7; 67.3 (2 C); 64.7; 34.8 (2 C); 34.2; 32.6 (2 C); 30.2 (4 C); 30.1 (2 C); 30.0 (2 C); 29.9 (2 C); 29.8
‡
(2 C); 25.6 (2 C); 23.2 (2 C); 14.7 (2 C). EI-MS:621 (7, M ), 603 (63), 408 (11), 183 (79), 83 (100), 57 (59).
Anal. calc. for C35H59NO8 (621.84):C 67.60, H 9.56; found:C 67.72, H 9.55.
2-Hydroxy-2-(hydroxymethyl)-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butyl Decanoate (22). Decanoyl
chloride (17.5 ml, 0.085 mmol) was added to a soln. of 1 (20 mg, 0.071 mmol) in dry pyridine (2 ml) at 08, and
the mixture was stirred overnight at 58. The solvent was evaporated, and the crude product was extracted with
AcOEt (3 Â 15 ml), washed with aq. sat. NaHCO3 (3 Â 10 ml), and dried (Na2SO4). The crude residue obtained
after evaporation of the solvents was chromatographed (silica gel; hexane/AcOEt 7 :3) to yield 22 (15 mg,
49%). Pale-yellow oil. TLC (hexane/AcOEt 1:1): Rf 0.15. IR (film):3447.4 s, 2927.3s, 2856.6s, 1733.5s, 1615.8s,
1282.3s. 1H-NMR (CD3OD, 300 MHz):7.64 ( d, J ˆ 9.4, 1 H); 7.38 (d, J ˆ 9.4, 1 H); 6.85 6.83 (m, 2 H); 6.26
(d, J ˆ 9.4, 1 H); 4.27 (t, J ˆ 5.9, 2 H); 4.23 4.16 (m, 2 H); 3.60 3.52 (m, 2 H); 2.86 (br. s, 1 H); 2.36 (t, J ˆ 7.3,
2 H); 2.07 (t, J ˆ 5.9, 2 H); 1.65 1.61 (m, 2 H); 1.37 1.27 (m, 12 H); 0.88 (t, J ˆ 7.0, 3 H). 13C-NMR (CD3OD,
75 MHz):175.3; 163.7; 163.3; 157.1; 145.8; 130.4; 114.2; 114.0; 113.3; 102.3; 73.6; 67.6; 66.4; 65.4; 35.0; 34.4; 33.0;
‡
‡
30.6; 30.4; 30.3; 30.2; 26.0; 23.7; 14.4. EI-MS:435 (57, M ); 281 (100); 163 (71). HR-MS (ESI -TOF-MS):
435.2372 (C4H35O7 , M ‡ H] ; calc. 435.2382.
‡
‡
2-Hydroxy-2-(hydroxymethyl)-4-[(4-nitrophenyl)oxy]butyl Decanoate (23). Decanoyl chloride (91 ml,
0.44 mmol) was added to a soln. of 2 (105 mg, 0.37 mmol) in dry pyridine (10 ml) at 08, and the mixture was
stirred at 58 overnight. The solvent was evaporated, and the crude product was extracted with AcOEt (3 Â
15 ml), washed with aq. sat. NaHCO3 (3 Â 10 ml), and dried (Na2SO4). The crude residue obtained after
evaporation of the solvents in vacuo was chromatographed (silica gel; hexane/AcOEt 7:3) to give 23 (64 mg,
42%). Pale-yellow oil. TLC (hexane/AcOEt 1:1): Rf 0.41. IR (film):3449 m, 2927s, 2856s, 1733s, 1515s, 1262s,
1111s. 1H-NMR (CD3OD, 300 MHz):8.20 ( d, J ˆ 9.2, 2 H); 7.07 (d, J ˆ 9.2, 2 H); 4.30 (t, J ˆ 6.6, 2 H); 4.12
(s, 2 H); 3.54 (s, 2 H); 2.33 (t, J ˆ 7.7, 2 H); 2.07 (t, J ˆ 6.6, 2 H); 1.59 1.57 (m, 2 H); 1.38 1.28 (m, 12 H); 0.88
(t, J ˆ 6.9, 3 H). 13C-NMR (CD3OD, 75 MHz):175.3; 165.4; 142.8; 126.8 (2 C); 115.8 (2 C); 73.6; 67.7; 66.4; 65.7;
35.0; 34.4; 33.0; 30.6; 30.4 (2 C); 30.3; 30.2; 26.0; 23.7; 14.4. Anal. calc. for C21H33NO7 (411.49):C 61.30, H 8.08;
found:C 61.36, H 8.10.
2-[(Dibenzyloxyphosphoryloxy)methyl]-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butane-1,2-diyl Bis(diben-
zylphosphate) (24). A soln. of 1 (0.050 g, 0.18 mmol) and 1H-tetrazole (0.113 g, 9 equiv.) in anh. CH2Cl2

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Helvetica Chimica Acta Vol. 86 (2003)
(5 ml) and DMF (2 ml) was treated at 258 under stirring with dibenzyl N,N-diisopropylphosphoramidite
(0.180 ml, 3 equiv.) and stirred at 258 for 3 h. The soln. was cooled to À788, m-CPBA (0.250 g, 6 equiv.) was
added, and stirring was prolonged for 1 h at 08. A soln. of sat. aq. NaHCO3 (1 ml) was added, and the mixture
was concentrated in vacuo. The residue was taken in AcOEt, washed (sat. aq. NaHCO3, H2O, and brine) and
purified by FC (CH2Cl2/MeCN 75 :25) to give pure 24 (0.052 g, 27%). Yellow syrup. TLC (CH2Cl2/MeCN
90 :10, UV 365 nm): Rf 0.43. IR (KBr):3447 s, 2092w, 1732s, 1615s, 1558w, 1509w, 1499w, 1457w, 1399w, 1279s,
1016s. 1H-NMR (300 MHz, CDCl3):7.60 ( d, J ˆ 9.6, 1 H); 7.35 7.23 (m, 31 H); 6.71 6.59 (m, 2 H); 6.25 (d, J ˆ
9.6, 1 H); 5.00 4.93 (m, 12 H); 4.30 (dd, J ˆ 4.5, 10.7, 2 H); 4.23 (dd, J ˆ 4.5, 10.7, 2 H); 3.94 (t, J ˆ 5.9, 2 H);
2.22 (t, J ˆ 5.9, 2 H). 13C-NMR (75 MHz, CDCl3):162.1; 161.7; 156.4; 143.9; 136.3; 136.2; 129.5; 129.4; 129.3;
128.9; 128.7; 128.6; 114.0; 113.5; 102.1; 83.9; 70.3; 70.2; 67.7; 63.7; 32.5. 31P-NMR (81 MHz, CDCl3): À0.4; À5.0.
2-[(Dihydroxyphosphoryloxy)methyl]-4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butane-1,2-diyl Bis(dihydro-
genphosphate) (25). A soln. of 24 (0.040 g, 0.038 mmol) in EtOH/H2O 4 :1 (10 ml) was treated with Pd on
charcoal (0.002 g, 0.1 equiv.) at 258 under 1 atm H2 and stirred vigorously for 3 h. Filtration over Celite and
concentration in vacuo gave pure 25 (0.019 g, quant.). Yellow syrup. IR (KBr):3438 s, 2929w, 2346w, 1702m,
1619s, 1557w, 1509w, 1385m, 1084s. 1H-NMR (300 MHz, D2O/CD3CN):7.85 ( d, J ˆ 9.4, 1 H); 7.50 (d, J ˆ 7.4,
1 H); 6.93 (m, 2 H); 6.21 (d, J ˆ 9.4, 1 H); 4.28 (m, 6 H); 2.25 2.15 (m, 2 H). 13C-NMR (75 MHz, D2O/
CD3CN):164.4; 162.9; 156.3; 146.3; 130.5; 114.2; 113.8; 112.9; 102.5; 82.6; 66.9; 65.5; 64.9; 32.7. 31P-NMR
(81 MHz, D2O/CD3CN):3.1; À1.4.
KineticsMeasurements . All substrates were diluted from stock solns. in 50% aq. MeCN and stored at ‡ 48.
Enzymes were diluted from 1 mg ¥ mlÀ1 stock solns. of the supplied solid in PBS (10 mm phosphate, 150 mm
NaCl, pH 7.4). Assays (0.1 ml) were followed in individual wells of round-bottom polypropylene 96-well-plates
(Costar) with a Cytofluor II Fluorescence Plate Reader (Perseptive Biosystems, filters lex ˆ 360 Æ 20, lem ˆ 460 Æ
20 nm), or of polystyrene 96-well-plates (Costar) with a Spectramax 250 Microplate Spectrophotometer
(Molecular Devices). Fluorescence data were converted to umbelliferone or nitrophenol concentration by
means of a calibration curve. The rates indicated in Table 2 are derived from the linear portion in each curve.
Oxidation of 1 with NaIO4. A soln. of triol 1 (8.2 mg, 29.3 mmol, 1 equiv.) in MeCN/H2O 1 :1 (35 ml) was
stirred at 258 with 10 equiv. of NaIO4 (62.6 mg, 292.6 mmol) for 24 h. The mixture was then lyophilized to give a
crude product containing acid 28 (87%) and umbelliferone 8 (13%) as analyzed by HPLC (for conditions, see
1
Table 1) and H-NMR integration.
Data of 28: 1H-NMR (300 MHz, (D6)DMSO):7.99 ( d, J ˆ 9.2, 1 H); 7.63 (d, J ˆ 8.8, 1 H); 7.00 (d, J ˆ 2.6,
1 H); 6.94 (dd, J ˆ 8.8, 2.6, 1 H); 6.29 (d, J ˆ 9.2, 1 H); 4.27 (t, J ˆ 5.9, 2 H); 2.72 (t, J ˆ 5.89, 2 H). 13C-NMR
(100 MHz, (D6)DMSO):172.10; 161.50; 160.30; 155.37 (2 C); 144.34; 129.55; 112.64; 112.57; 101.21; 64.45;
33.89. 13C-NMR (DEPT-135; 100 MHz, (D6)DMSO):144.09 (CH); 129.30 (CH); 112.39 (CH); 112.32 (CH);
‡
100.96 (CH); 64.20 (CH2); 33.63 (CH2). EI-MS:234 ( M ).
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Received February 10, 2003