A preparative synthesis of human chitinase fluorogenic substrate (4' -deoxychitobiosyl)-4-methylumbelliferone

Article abstract of DOI:10.1002/ejoc.201000080

To meet the increasing clinical demand for the diagnostic agent (4′-deoxychitobiosyl)-4-methylumbelliferone, a flexible and scalable route of synthesis is needed. In this paper such a route is presented. The key to the route is the use of a partially protected thiophenyl glucosamine as starting material for the preparation of both the reducing and nonreducing end building blocks of the 4′-deoxychitobiose disaccharide.

Full text of DOI:10.1002/ejoc.201000080

FULL PAPER
DOI: 10.1002/ejoc.201000080
A Preparative Synthesis of Human Chitinase Fluorogenic Substrate
(4Ј-Deoxychitobiosyl)-4-methylumbelliferone
Jasper Dinkelaar,[a][‡] Boudewijn A. Duivenvoorden,[a][‡] Tom Wennekes,[a]
Herman S. Overkleeft,[a] Rolf G. Boot,[b] Johannes M. F. G. Aerts,[b] Jeroen D. C. Codée,*[a]
and Gijs A. van der Marel*[a]
Keywords: Glycosylation / Chromophores / Fluorescence / Diagnostic tools
To meet the increasing clinical demand for the diagnostic
agent (4Ј-deoxychitobiosyl)-4-methylumbelliferone, a flexi-
ble and scalable route of synthesis is needed. In this paper
of a partially protected thiophenyl glucosamine as starting
material for the preparation of both the reducing and nonre-
ducing end building blocks of the 4Ј-deoxychitobiose disac-
such a route is presented. The key to the route is the use charide.
zyme replacement therapy and substrate reduction ther-
Introduction
apy.[19–24] Both therapies are expensive and therefore moni-
toring their effect (or optimal dosage and treatment regi-
men) through measuring serum chitotriosidase activity
levels has considerable clinical value.
The existence of endogenous chitinases in mammals was
only discovered a decade ago.[1] Firstly identified was the
enzyme chitotriosidase (CHIT1), a chitinase that is strongly
expressed and secreted by lipid-laden tissue macrophages
that are found in patients suffering from the glycolipid stor-
age disorder Gaucher disease.[2–4] Relatively more modest
elevations in plasma chitotriosidase have subsequently been
detected in other disease conditions involving macrophages,
including several lysosomal storage disorders,[5–8] fungal
and parasite infections like malaria and visceral Leishmani-
asis,[2,9,10] thalassemia,[11] sarcoidosis,[12] and atherosclero-
sis.[13] The existence of a second mammalian chitinase,
named AMCase (acidic mammalian chitinase), has been
recognized recently,[14,15] and its role in the etiology of
asthma has recently been proposed.[16]
Measurement of plasma chitinase activity in man is now
widely applied for clinical purposes. The most important
application is in the monitoring of severity of disease in
Gaucher patients.[2,17] Chitotriosidase activity levels in
plasma of Gaucher patients correlate to the progression of
the disease and the effect of therapeutic intervention. The
enzyme is thus an ideal marker through which Gaucher
patients are identified and their reaction towards thera-
peutic agents is monitored.[18] Currently, two therapies for
the treatment of Gaucher patients are applied, namely, en-
In the years immediately following our discovery of chi-
totriosidase levels as Gaucher marker we made use of the
umbelliferyl chitobioside fluorogenic substrate 1 (Fig-
ure 1).[2] However, we soon found out that human chito-
triosidase possesses intrinsic transglycosylase activity,[25] in
that chitobiose or higher oligomers are formed through hy-
drolysis of 1 and connected to the nonreducing 4Ј-hydroxy
group of another substrate to form higher oligomers. This
side reaction complicates interpretation of the kinetics of
the enzyme-mediated generation of the fluorescent umbelli-
feronate anion and in fact also renders the efficiency of the
fluorogenic substrate suboptimal. To circumvent this prob-
lem we reported the development of fluorogenic substrate
2 (Figure 1), in which the 4Ј-OH group is removed, pre-
venting chitotriosidase-mediated transglycosylation.[25] De-
oxychitobiosyl umbelliferone 2 is a superior chitotriosidase
[a] Leiden Institute of Chemistry, Leiden University,
Gorlaeus Laboratories,
Einsteinweg 55, 2300 RA Leiden, The Netherlands
Fax: +31-71-527-4307
E-mail: marel_g@chem.leidenuniv.nl
jcodee@chem.leidenuniv.nl
[b] Department of Medical Biochemistry,
Academic Medical Center, University of Amsterdam
Amsterdam, The Netherlands
[‡] Both authors contributed equally to this work
Figure 1. Umbelliferyl chitobioside fluorogenic substrate 1 and 2.
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J. D. C. Codée, G. A. van der Marel et al.
FULL PAPER
substrate compared to 1. The same holds for the enzyme
AMCase.[17] Given the rapidly growing interest to monitor
plasma chitotriosidase also in other disease conditions, and
given the present interest in AMCase in relation to asthma,
deoxychitobiosyl umbelliferone 2 has become a very desired
fluorogenic substrate and we foresee that larger quantities
will be needed on an annual basis in the near future. We
were thus in need of a route for the synthesis of compound
2 that is more efficient and reliable than the original route
we reported in our transglycosylase activity studies.
In our original work[25] we started from the disaccharide
chitobiose, which we converted in nine steps into the target
compound. Although sufficiently effective for the prepara-
tion of several milligrams, the route falls short when aiming
for larger quantities. The nine-step sequence is quite inef-
ficient (3% overall yield), and furthermore, the starting di-
saccharide, chitobiose, is rather expensive. We thus devised
a new route of synthesis, the details of which we disclose
here.
Results and Discussion
Scheme 1. Synthetic outline for the large-scale synthesis of umbelli-
feryl chitobioside fluorogenic substrate 2.
Our synthetic plan is outlined in Scheme 1. We envisaged
that the formation of the glycosidic linkage between the chi-
tobiose and the umbelliferyl chromophore could best be
achieved by SN2 displacement of an anomeric chloride by purification was required in this sequence of reactions. Re-
the umbelliferyl phenolate anion, as the poor nucleophilic- ductive opening of the benzylidene acetal in the next step
ity of the protonated phenol precludes the use of Lewis was affected by treatment of 7 with trifluoroacetic acid
acidic glycosylation methods. For the construction of the (TFA) and triethylsilane (TES) to selectively provide key
chitobiose core we selected thiophenyl glycoside building thioglycoside 6 in 85% yield.[28] The formation of the re-
blocks, because the anomeric thiophenyl group can be eas- gioisomeric C-4 benzyl ether was not observed. To provide
ily introduced early in the synthesis; it is also stable to the the nonreducing end glucosamine building block, alcohol 6
reaction conditions employed throughout the synthesis of was treated with NaH and CS2 followed by MeI to provide
the synthons and can be selectively activated with a variety the methyl dithiocarbonate. Radical fragmentation with the
of soft nucleophiles to provide a glycosylating species.
use of Bu3SnH and AIBN as initiator in refluxing toluene
Furthermore, thiophenyl glycosides are shelf stable and then led to deoxygenated glucosamine 4 in 87% yield.[29]
often crystalline, which for the large-scale preparation of For the construction of the reducing end glucosamine
the building blocks is a valuable asset. To maximize the building block 5, partly protected thioglycoside 6 was con-
efficiency in the construction of deoxychitobiosyl umbelli- densed with benzyl alcohol employing N-iodosuccinimide
ferone 2 a route was designed, in which a single thioglyco- (NIS) and a catalytic amount of trimethylsilyl trifluoro-
side (i.e., 6) serves as an advanced precursor for both the methanesulfonate (TMSOTf) as activator.[30] The use of a
nonreducing and reducing end glucosamine building large excess of BnOH (5 equiv.), and the high nucleo-
blocks. We selected the phthaloyl group to protect the glu- philicity of this alcohol as compared to the glucosamine C-
cosamine amino function because it is cheap, robust under 4 hydroxy, completely prevented self-condensation of 6, and
both basic and acidic conditions, and can be readily intro- glucosamine 5 was obtained in 75% yield. In the ensuing
duced onto the glucosamine substrate on a large scale by NIS-mediated glycosylation, deoxyglucosamine 4 and benz-
using well-established chemistry. The phthalimide group re- yl glucosamine 5 were reacted in a 1:1 ratio to provide
liably provides anchimeric assistance in the coupling of the chitobioside derivative 8 in excellent yield.
two glucosamines to give the 1,2-trans glycosidic bond and
To introduce the umbelliferyl chromophore, disaccharide
does not give rise to oxazoline side products. Benzyl ethers 8 was transformed into disaccharide chloride 3. To this end,
will mask all hydroxy groups during the assembly of the both N-phthaloyl groups in 8 were removed by transamid-
chitobiose disaccharide.
ation with ethylenediamine in refluxing n-butanol. Subse-
Scheme 2 depicts the synthesis of deoxychitobiosyl um- quent acetylation of the resulting free amines then provided
belliferone 2, which started with the synthesis of thioglyco- crystalline dimer 9. Removal of all benzyl groups from this
side 6. 4,6-Benzylidene-N-phthaloyl thioglucosamine (7) disaccharide proved to be more troublesome than expected
was obtained from -glucosamine in 40% yield over eight because of the low solubility of the partly benzylated-N-
steps on a 147-g scale.[26,27] Only a single chromatographic acetyled chitobiosides. The best results were obtained when
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A Preparative Synthesis of a Human Chitinase Fluorogenic Substrate
Scheme 2. Reagents and conditions: (a) DCM, BnOH, NIS, 0 °C, TMSOTf (75%); (b) i. THF, imidazole, CS2, 0 °C, NaH, 1 h, then r.t.,
MeI (93%); ii. Tol, Bn3SnH, AIBN, ∆ (87%); (c) DCM, NIS, 0 °C, TMSOTf (86%); (d) i. nBuOH, ethylenediamine, ∆; ii. MeOH, Ac2O,
Et3N (82% over two steps); (e) i. THF, MeOH, AcOH, Pd(OH)2, H2; ii. pyridine, Ac2O (65% over two steps); (f) AcOH, Ac2O, HCl
gas, 0 to 5 °C (74%); (g) CHCl3, H2O, NaHCO3, umbelliferone sodium salt, TBAHS (62%); (h) MeOH, NaOMe (28% after HPLC
purification).
disaccharide 9 was treated under 5 bar hydrogen pressure amorphous solid. Saponification of the acetyl esters with
with Pearlman’s catalyst (5 mol-%) in a THF/MeOH (1:1) NaOMe and HPLC purification completed the synthesis of
solvent mixture in the presence of AcOH (5 equiv.). The target compound 2.
fully deprotected 4Ј-deoxychitobioside was then acetylated
to give penta-O-acetate 10 in 65% yield as an amorphous
Conclusions
white solid over the two steps. The final stages of the syn-
thesis followed procedures slightly adapted from litera-
ture.[31] Chlorination of the reducing end glucosamine de-
rivative required careful tuning of the reaction conditions.
Anomeric acetate 10 was treated with dry HCl in a mixture
of AcOH and Ac2O at 5 °C for 42 h to afford 4Ј-deoxychi-
tobiosyl chloride 3 in 74%. We observed that shorter reac-
tion times led to incomplete chlorination and that at higher
reaction temperatures the GlcNAc–GlcNAc interglycosidic
bond was cleaved. Previously, it has been reported that the
anomeric chlorination of chitobiosyl acetate can be readily
accomplished at room temperature. Presumably the 4-deoxy
nature of the nonreducing end GlcNAc residue in 10 makes
the glycosidic linkage more labile towards acidic cleavage.
Introduction of the umbelliferyl chromophore was ac-
complished by SN2 displacement of the anomeric α-chloride
by the tetrabutylammonium salt of 4-methylumbelliferone,
generated under phase-transfer conditions.[32] The protected
In conclusion we have developed an efficient, reliable,
and scalable route for the synthesis of 4Ј-deoxychitobiosyl
umbelliferone 2. The synthesis is based on the use of a par-
tially protected thiophenyl glucosamide, which is readily
transformed into both the reducing and nonreducing end
building blocks for the construction of 4Ј-deoxychitobiose.
Experimental Section
General: Dichloromethane was heated at reflux with P2O5 and dis-
tilled before use. All other chemicals (Acros, Fluka, Merck,
Schleicher & Schuell) were used as received. Column chromatog-
raphy was performed on Merck silica gel 60 (0.040–0.063 mm).
TLC analysis was conducted on HPTLC aluminum sheets (Merck,
silica gel 60, F245). Compounds were visualized by UV absorption
(245 nm), by spraying with 20% H2SO4 in ethanol or with a solu-
tion of (NH4)6Mo7O24·4H2O (25 g/L), (NH4)4Ce(SO4)4·2H2O
1
umbelliferyl derivative was obtained in 62% yield as a white (10 g/L), 10% H2SO4 in H2O followed by charring at ≈140 °C. H
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J. D. C. Codée, G. A. van der Marel et al.
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and 13C NMR spectra were recorded with a Bruker AV 400. NMR
spectra were recorded in CDCl3 with chemical shifts (δ) relative
to tetramethylsilane unless stated otherwise. Optical rotations were
measured with a Propol automatic polarimeter. High-resolution
mass spectra were recorded with a LTQ-orbitrap (thermoelectron).
IR spectra were recorded with a Shimadzu FTIR-8300.
H, CH2 Bn), 5.15–5.17 (m, 1 H, 1-H), 6.89–6.93 (m, 3 H, H arom.),
7.02–7.07 (m, 7 H, H arom.), 7.28–7.36 (m, 5 H, H arom.), 7.53
(br. s, 1 H, H arom.), 7.62–7.63 (m, 2 H, H arom.), 7.76 (br. s, 1
H, H arom.) ppm. 13C NMR (100 MHz, CDCl3): δ = 55.3 (C-2),
70.3 (C-6), 70.6 (CH2 Bn), 73.5 (CH2 Bn), 73.7 (C-5), 73.8 (C-4),
74.1 (CH2 Bn), 78.4 (C-3), 97.2 (C-1), 123 (CH arom.), 127.2–128.3
(CH arom.), 131.4 (Cq arom.), 133.5 (CH arom.), 136.9, 137.6,
138.0 (Cq arom.), 168.0 (C=O Phth), 168.1 (C=O Phth) ppm.
HRMS: calcd. for C35H33NO7 + Na+ 602.21492; found 602.21471.
Phenyl 3,6-Di-O-benzyl-2,4-dideoxy-2-phthalimido-1-thio-β-D-gluco-
pyranoside (4): Glycoside 6 (25.4 g, 43.8 mmol) was coevaporated
three times with dioxane, then taken up in THF (220 mL). Imid-
azole (0.298 g, 4.38 mmol) and CS2 (7.9 mL, 131 mmol) were Phenyl 3,6-Di-O-benzyl-2-deoxy-2-phthalimido-4-O-(3,6-di-O-benz-
added, after which the mixture was cooled to 0 °C. NaH (60%
dispersion in oil, 2.63 g, 65.7 mmol) was added, and the reaction
mixture was kept at 0 °C for 1 h and then warmed to room tem-
yl-2,4-dideoxy-2-phthalimido-β-D-glucopyranosyl)-β-D-glucopyrano-
side (8): A mixture of donor 4 (20.1 g, 35.4 mmol) and acceptor 5
(20.6 g, 35.4 mmol) were coevaporated three times with toluene.
perature. At room temperature, MeI (4.82 mL, 77.5 mmol) was DCM (350 mL) and NIS (9.56 g, 42.5 mmol) were added, and the
added. After 30 min the mixture was quenched by the addition of
AcOH and subsequently diluted with EtOAc (250 mL). The mix-
ture was then washed with NaHCO3 (aq.). The layers were sepa-
mixture was stirred over activated 3 Å molecular sieves for 30 min.
The mixture was cooled to 0 °C before a catalytic amount of
TMSOTf (0.32 mL, 1.77 mmol) was added. After TLC analysis
rated, and the organic layer was dried with MgSO4 and concen- showed complete consumption of the starting material (3 h) at
trated in vacuo. Purification by column chromatography (toluene/
ethyl acetate, 100:0 Ǟ 95:5) yielded the thiocarbamate intermediate
0 °C, the reaction was quenched with Et3N (5.0 mL, 35 mmol). The
reaction mixture was diluted with DCM and washed with Na2S2O3
(aq.). The water layer was extracted twice with DCM, and the col-
lected organic layer was dried with MgSO4 and concentrated in
vacuo. Purification by column chromatography (petroleum ether/
as
a yellow oil (27.4 g, 93%). The thiocarbonate (27.4 g,
40.8 mmol) was coevaporated three times with toluene, dissolved
in toluene (800 mL), and degassed with sonication under argon
flow for 5 min. Bu3SnH (16.4 mL, 61.2 mmol) and AIBN (0.33 g, ethyl acetate, 100:0 Ǟ 70:30) yielded 8 (31.7 g, 86%) as a colorless
1
2.04 mmol) were added, and the mixture was warmed to 120 °C. oil. H NMR (400 MHz, CDCl3): δ = 1.52 (q, J = 12.0 Hz, 1 H,
After 1 h when TLC analysis showed complete consumption of the
starting material, the reaction was cooled to room temperature and
concentrated in vacuo. The residue was taken up in acetonitrile and
washed two times with hexane; the acetonitrile layer was concen-
trated in vacuo. Column chromatography (petroleum ether/ethyl
acetate, 100:0 Ǟ 70:30) afforded 4 (20.1 g, 87%) as an oil. 1H NMR
(400 MHz, CDCl3): δ = 1.59 (q, J = 11.6 Hz, 1 H, 4-H), 2.31 (dd,
J = 3.6, 12.8 Hz, 1 H), 3.54–3.58 (m, 1 H, C-6), 3.65–3.69 (m, 1 H,
4Ј-H), 2.28 (dd, J = 4.8, 12.8 Hz, 1 H, 4Ј-H), 3.34–3.58 (m, 6 H),
4.11–4.39 (m, 7 H), 4.44–4.58 (m, 6 H, CH2 Bn), 4.68 (d, J =
12.4 Hz, 1 H, CH2 Bn), 4.84 (d, J = 12.4 Hz, 1 H, CH2 Bn), 4.98
(d, J = 6.4 Hz, 1 H, 1-H), 5.29 (d, J = 8.0 Hz, 1 H, 1Ј-H), 6.82 (br.
s, 3 H, H arom.), 6.96–7.02 (m, 12 H, H arom.), 7.20–7.37 (m, 10
H, H arom.), 7.58–7.59 (m, 2 H, H arom.), 7.67–7.71 (m, 4 H, H
arom.), 7.88–7.89 (m, 2 H, H arom.) ppm. 13C NMR (100 MHz,
CDCl3): δ = 34.2 (C-4Ј), 55.6 (C-2), 57.7 (C-2Ј), 68.1, 70.3, 70.6,
6-H), 3.84 (m, 1 H, 5-H), 4.19–4.34 (m, 3 H, 2-H, 3-H, CH2 Bn), 71.1, 71.9, 72.4, 72.5, 73.2, 74.0, 74.5, 75.5, 76.5, 97.0 (C-1), 97.2
4.55–4.57 (m, 3 H, CH2 Bn), 5.57 (d, J = 10.4 Hz, 1 H, 1-H), 6.98– (C-1Ј), 122.9–123.5 (CH arom.), 126.7–128.3 (CH arom.), 131.5 (Cq
7.02 (m, 5 H, H arom.), 7.16–7.17 (m, 3 H, H arom.), 7.28–7.39 arom.), 133.4–133.7 (CH arom.), 136.9–138.6 (Cq arom.), 167.5–
(m, 7 H, H arom.), 7.66–7.83 (m, 4 H, H arom.) ppm. 13C NMR 168.1 (C=O Phth) ppm. HRMS: calcd. for C63H58N2O12 + Na+
(100 MHz, CDCl3): δ = 34.0 (C-4), 55.5 (C-2), 70.6 (CH2 Bn), 72.3 1057.38820; found 1057.38876.
(C-6), 73.3 (CH2 Bn), 73.5 (C-3), 75.4 (C-5), 83.6 (C-1), 123.1,
123.3, 127.3–128.6 (CH arom.), 131.5 (Cq arom.), 132.0 (CH
arom.), 132.5 (Cq arom.), 133.7 (CH arom.), 137.7, 138.0 (Cq
Phenyl 3,6-Di-O-benzyl-2-deoxy-2-acetamido-4-O-(3,6-di-O-benzyl-
2,4-dideoxy-2-acetamido-β-D-glucopyranosyl)-β-D-glucopyranoside
(9): Disaccharide 8 (31.7 g, 30.6 mmol) was dissolved in nBuOH
(275 mL) and ethylene diamine (30 mL). This mixture was heated
arom.), 167.7, 168.0 (C=O Phth) ppm. HRMS: calcd. for
C34H31NO5S + Na+ 588.18151; found 588.18115.
at reflux overnight and subsequently concentrated in vacuo. The
Benzyl 3,6-Di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside
(5): Glycoside 6 (23.6 g, 40.5 mmol) was coevaporated three times
with toluene. DCM (810 mL), BnOH (21 mL, 202 mmol), and NIS
(10.9 g, 48.6 mmol) were added. The mixture was stirred over acti-
vated 3 Å molecular sieves for 30 min. After cooling to 0 °C, a cata-
lytic amount of TMSOTf (0.81 mL, 4.5 mmol) was added. After
1 h, the mixture was warmed to room temperature when TLC
analysis showed complete consumption of the starting material,
and the reaction was quenched by the addition of Et3N (5.6 mL,
40.5 mmol). The reaction mixture was diluted with DCM and
washed with Na2S2O3 (aq.). The water layer was extracted twice
with DCM, and the collected organic layer was dried with MgSO4
and concentrated in vacuo. Purification by column chromatog-
raphy (petroleum ether/ethyl acetate, 100:0 Ǟ 60:20) yielded 5
reaction was then coevaporated three times with toluene and taken
up in MeOH (300 mL). At 0 °C, Ac2O (30 mL, 300 mmol) and
Et3N (8.5 mL, 61.2 mmol) were added, and the mixture was
warmed to room temperature. The resulting mixture was concen-
trated in vacuo and taken up in CHCl3 and washed with H2O. The
collected organic layer was stirred over activated carbon and fil-
tered through hyflo-gel concentrated in vacuo. Crystallization (pe-
troleum ether/ethyl acetate) yielded 9 (26.6 g, 82%) as slightly yel-
low crystals. 1H NMR (400 MHz, CDCl3/CD3OD, 1:1): δ = 1.45
(q, J = 12.0 Hz, 1 H, 4Ј-H), 1.94 (s, 6 H, CH3 NHAc), 2.20 (dd, J
= 4.8, 12.8 Hz, 1 H, 4Ј-H), 3.37–3.38 (m, 1 H), 3.44–3.51 (m, 3 H),
3.63–3.79 (m, 5 H), 3.97 (t, J = 6.4 Hz, 1 H), 4.12 (t, J = 6.8 Hz,
1 H) 4.38–4.49 (m, 5 H, CH2 Bn, H1), 4.54–4.69 (m, 5 H, CH2 Bn,
H1Ј), 4.75 (d, J = 11.6 Hz, 1 H, CH2 Bn), 4.86 (d, J = 12.4 Hz, 1 H,
CH2 Bn), 7.21–7.35 (m, 25 H, H arom.) ppm. 13C NMR (100 MHz,
CDCl3/CD3OD, 1:1): δ = 22.2, 22.5 (CH3 NHAc), 33.1 (C-4Ј), 51.8
1
(17.6 g, 75%) as a colorless oil. H NMR (400 MHz, CDCl3): δ =
3.20 (d, J = 2.4 Hz, 1 H, OH), 3.62–3.65 (m, 1 H, 5-H), 3.82 (m,
3 H, 4-H, 6-H, 6-H), 4.23–4.25 (m, 2 H, 2-H, 3-H), 4.47 (d, J = (C-2), 55.5 (C-2Ј), 68.9, 69.8, 70.2, 70.5, 71.8, 72.3, 73.0, 73.1, 74.1,
12.4 Hz, 1 H, CH2 Bn), 4.51 (d, J = 12.4 Hz, 1 H, CH2 Bn), 4.58 74.4, 75.4, 78.4, 99.6, 100.2 (C-1, C-1Ј), 126.8–128.0 (CH arom.),
(d, J = 12.0 Hz, 1 H, CH2 Bn), 4.64 (d, J = 12.0 Hz, 1 H, CH2 137.1–138.3 (Cq arom.), 170.8, 171.4 (Cq NHAc) ppm. HRMS:
Bn), 4.74 (d, J = 12.4 Hz, 1 H, CH2 Bn), 4.78 (d, J = 12.4 Hz, 1
calcd. for C51H58N2O10 + Na+ 881.39837; found 881.39865.
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A Preparative Synthesis of a Human Chitinase Fluorogenic Substrate
1,3,6-Tri-O-acetyl-2-deoxy-2-acetamido-4-O-(3,6-di-O-acetyl-2,4-di- 72.1, 98.7 (C-1), 102.9 (C-1Ј), 103.5 (CH arom.), 112.4, 133.3 (CH
deoxy-2-acetamido-β-D-glucopyranosyl)-D-glucopyranoside (10): Di-
arom.), 114.7 (Cq arom.), 125.5 (CH arom.), 153.3, 154.4, 159.9,
saccharide 9 (22.9 g, 26.6 mmol) was dissolved in THF (250 mL) 160.1 (Cq arom.), 171.9–172.4 (Cq Ac) ppm. HRMS: calcd. for
and then MeOH (250 mL), AcOH (9 mL, 106 mmol), and
Pd(OH)2 (20% on activated carbon, 1 g, 1.33 mmol) were added.
The mixture was shaken overnight on a par apparatus under 5 bar
hydrogen pressure. The resulting mixture was filtered over What-
mann filter paper, concentrated in vacuo, and taken up in pyridine
(180 mL). At 0 °C, Ac2O (55 mL) was added, and after 1 h the
mixture was warmed to room temperature and stirred overnight.
The reaction was quenched by the addition of MeOH at 0 °C and
then concentrated in vacuo. The residue was taken up in CHCl3
and washed with 1  HCl (aq.)/NaHCO3 (aq.) and brine. The or-
ganic layer was dried with MgSO4 and concentrated in vacuo. Puri-
fication by column chromatography (DCM/MeOH, 100:0 Ǟ 97:3)
C34H42N2O16 + Na+ 757.24265; found 757.24278.
4-Methylumbelliferyl 2-Deoxy-2-acetamido-4-O-(2,4-dideoxy-2-
acetamido-β-D-glucopyranosyl)-β-D-glucopyranoside (2): To a sus-
pension of 11 (0.878 g, 1.195 mmol) in MeOH (60 mL) was added
NaOMe (30wt.-% in MeOH, 44 µL, 0.24 mmol). The reaction was
stirred under exclusion of light. When LC–MS (gradient 0 to 50%
MeOH) showed complete conversion to the product, the mixture
was quenched with AcOH (70 µL, 1.2 mmol). The reaction was
diluted with H2O (60 mL), the MeOH was evaporated in vacuo,
and the remaining H2O was lyophilized. Purification by HPLC
(gradient H2O/MeOH + 0.1% TFA 80:20 Ǟ 60:40), evaporation
of MeOH, and lyophilizing H2O yielded 2 (227 mg, 28%) as white
fluffy solid. 1H NMR (400 MHz, [D6]DMSO): δ = 1.21 (q, J =
11.6 Hz, 1 H, 4Ј-H), 1.80 (s, 3 H, CH3 NHAc), 1.84 (s, 4 H, CH3
NHAc, 4Ј-H), 2.39 (s, 3 H, CH3 4-methylumbelliferyl), 3.36–3.68
(m, 10 H), 3.78 (q, J = 9.2 Hz, 1 H, C-2 or C-2Ј), 4.30 (d, J =
8.4 Hz, 1 H, 1Ј-H), 4.69 (br. s, 1 H, OH), 4.84–4.90 (m, 3 H, OH),
5.17 (d, J = 8.4 Hz, 1 H, 1-H), 6.25 (s, 1 H, 4-methylumbelliferyl),
6.94 (d, J = 8.8 Hz, 1 H, 4-methylumbelliferyl), 7.02 (d, J = 1.6 Hz,
1 H), 7.67–7.71 (m, 2 H, 4-methylumbelliferyl, NH), 7.90 (d, J =
8.8 Hz, 1 H, NH) ppm. 13C NMR (100 MHz, [D6]DMSO): δ =
18.1 (CH3 4-methylumbelliferyl), 23.0 (CH3 NHAc), 23.1 (CH3
NHAc), 35.8 (C-4Ј), 54.4, 57.0 (C-2 and C-2Ј), 59.7, 63.5 (C-6 and
C-6Ј), 68.2, 72.3, 72.9, 75.1, 80.9 (C-3, C-4, C-5, C-3Ј, C-5Ј), 98.3
(C-1), 102.5 (C-1Ј), 103.2 (CH arom.) 111.9 (CH arom.), 113.5 (CH
arom.), 114.3 (Cq arom.), 126.5 (CH arom.), 153.3, 154.4, 159.9,
160.1 (Cq arom.), 169.2, 169.4 (C=O Ac) ppm. HRMS: calcd. for
C26H34N2O12 + Na+ 589.20040; found 589.20031.
1
yielded 10 (10.6 g, 65%) as a white amorphous solid. H NMR of
alpha acetate (400 MHz, CD3OD): δ = 1.51 (q, J = 11.6 Hz, 1 H,
4Ј-H), 1.86–2.14 (22 H, CH3 Ac, 4Ј-H), 3.61 (t, J = 9.2 Hz, 1 H),
3.79 (m, 1 H), 3.88 (t, J = 9.6 Hz, 1 H), 3.98 (m, 1 H), 4.04–4.12 (m,
2 H), 4.23 (dd, J = 5.6, 11.6 Hz, 1 H), 4.30 (dd, J = 3.6, 10.8 Hz, 1
H), 4.44 (d, J = 12.0 Hz, 1 H), 4.56 (d, J = 8.0 Hz, 1 H, 1Ј-H), 5.04
(dt, J = 5.2, 10.8 Hz, 1 H), 5.24 (t, J = 10.0 Hz, 1 H), 5.99 (d, J =
3.6 Hz, 1 H, 1-H) ppm. 13C NMR of alpha acetate (100 MHz,
CD3OD): δ = 20.8–23.0 (CH3 Ac), 33.8 (C-4Ј), 52.2, 56.4 (C-2, C-
2Ј), 63.5, 66.7 (C-6, C-6Ј), 70.7, 71.6, 72.0, 72.4, 76.9, 91.5 (C-1),
102.6 (C-1Ј), 171.9–172.4 (C=O Ac) ppm. HRMS: calcd. for
C26H38N2O15 + Na+ 641.21644; found 641.21643.
4-Methylumbelliferyl 1,3,6-Tri-O-acetyl-2-deoxy-2-acetamido-4-O-
(3,6-di-O-acetyl-2,4-dideoxy-2-acetamido-β-D-glucopyranosyl)-β-D-
glucopyranoside (11): Disaccharide 10 (1.61 g, 2.59 mmol) was dis-
solved in AcOH (13 mL) and Ac2O (3.2 mL). At 0 °C dry HCl (g)
was bubbled through (liberated under Kipp conditions) for 3 h. The
reaction mixture was then placed at 5 °C for 42 h, after which TLC
analyses (DCM/acetone, 60:40) showed complete consumption of
the starting material. The reaction mixture was diluted with CHCl3
(50 mL, 0 °C) and washed twice with H2O (25 mL, 0 °C) and twice
with NaHCO3 (aq.) (25 mL, 0 °C). The organic layer was dried
with MgSO4 and concentrated in vacuo to yield amorphous solid
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1
3 (1.14 g). Its purity was evaluated by H NMR spectroscopy [1H
NMR (400 MHz, CDCl3): δ = 1.55 (q, J = 11.6 Hz, 1 H, 4Ј-H),
1.86–2.14 (22 H, CH3 Ac, 4Ј-H), 3.73–3.83 (m, 4 H), 4.03 (dd, J =
4.0, 11.6 Hz, 1 H), 4.20–4.25 (m, 2 H), 4.36–4.54 (m, 3 H), 4.48 (d,
J = 8.0 Hz, 1 H, 1Ј-H), 5.02 (dt, J = 5.2, 11.2 Hz, 1 H), 5.30 (t, J
= 10.0 Hz, 1 H), 5.94 (d, J = 8.0 Hz, 2 H, NHAc), 5.96 (d, J =
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solution of H2O (76 mL), NaHCO3 (1.29 g, 15 mmol), 4-methyl-
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phases were separated, and the organic layer was washed two times
with NaHCO3 (0.2 ) and two times with H2O. The organic layer
was dried with MgSO4 and concentrated in vacuo. Purification by
column chromatography (CHCl3/MeOH, 100:0 Ǟ 97:3) yielded 11
(0.88 g, 46%) as a white amorphous solid. 1H NMR (400 MHz,
CDCl3/CD3OD, 1:1): δ = 1.71 (q, J = 12.4 Hz, 1 H, 4Ј-H), 1.86–
2.14 (19 H, CH3 Ac, 4Ј-H), 2.38 (s, 3 H, CH3 4-methylumbelliferyl),
4.04–4.29 (m, 7 H), 5.14 (t, J = 8.1 Hz, 1 H), 5.23–5.26 (m, 3 H),
5.46–5.49 (m, 2 H), 6.09 (s, 1 H) 6.54 (d, J = 9.2 Hz, 1 H, NHAc),
6.72 (d, 1 H, J = 9.2 Hz, NHAc). 6.86 (m, 2 H), 7.44 (d, 1 H, J =
10.4 Hz) ppm. 13C NMR of (100 MHz, CDCl3/CD3OD, 1:1):δ =
18.5 (CH3 4-methylumbelliferyl), 20.5–23.2 (CH3 Ac), 32.5 (C-4Ј),
54.2, 54.4 (C-2, C-2Ј), 62.0, 65.2 (C-6, C-6Ј), 68.5, 69.9, 70.0, 72.0,
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Published Online: March 19, 2010
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 2565–2570