Synthesis of photoaffinity derivatives of adenophostin A

Article abstract of DOI:10.1002/1099-0690(200009)2000:17<3085::AID-EJOC3085>3.0.CO;2-A

Photoaffinity derivatives 7 and 8 of adenophostin A, modified at the 5'- and 1'-positions, were prepared by a chemoselective reaction of the aminophostins 15 and 30 with N-succini-midyl p-benzoyl-2,3-dihydrocinnamate (p-benzoyldihydrocinnamoyl-N-hydroxysu

Full text of DOI:10.1002/1099-0690(200009)2000:17<3085::AID-EJOC3085>3.0.CO;2-A

FULL PAPER
Synthesis of Photoaffinity Derivatives of Adenophostin A
Martin de Kort,[a] Jaco Luijendijk,[a] Gijs A. van der Marel,[a] and Jacques H. van Boom*[a]
Keywords: Adenophostin A / Glycosylations / Nucleosides / Photoaffinity labeling
Photoaffinity derivatives 7 and 8 of adenophostin A, modified
innamoyl-N-hydroxysuccinimide; BZDC-NHS, 21). The lat-
at the 5Ј- and 1Ј-positions, were prepared by a chemoselec- ter compound was prepared by Heck coupling of 4-iodoben-
tive reaction of the aminophostins 15 and 30 with N-succini-
midyl p-benzoyl-2,3-dihydrocinnamate (p-benzoyldihydroc-
zophenone (16) with methyl acrylate.
Introduction
affinity label at either the 5Ј, the 6ЈЈ, or the 1Ј-position with-
out substantially impairing the biological activity.
Adenophostin A (1) and B (2) (see Scheme 1) have at-
tracted considerable interest as full agonists of the -myo-
inositol 1,4,5-trisphosphate receptor (IP3R), exhibiting
binding affinities and Ca2ϩ-mobilizing potencies 10Ϫ100
times higher than those of the natural ligand IP3 (3).[1] Ini-
tially, it was proposed that 1 and 2 interact with a regu-
latory ATP binding site of IP3R. However, recent findings[2]
confirmed that the binding site of both compounds is the
same as for IP3 (3). Furthermore, the Ca2ϩ signaling in-
duced by the metabolically resistant adenophostins is, in
contrast with that of IP3, prolonged and spatially re-
stricted.[3] These favorable properties render both com-
pounds useful pharmacological tools[4] in studying in more
detail the activation of IP3Rs at specific locations in the
cell.[5] The latter aspect was nicely illustrated by the recent
discovery[6] of a small sub-region in the endoplasmic reticu-
lum of hepatocytes that may be responsible for IP3R-in-
duced Ca2ϩ entry.[7] It was envisaged that the availability of
photoaffinity derivatives of adenophostin A (1) would be
of general value in unraveling the complex Ca2ϩ signaling
pathways. In order to attain this goal, it is essential that the
presence of a photolabel does not impair the binding affin-
ity for the receptor. It is well established that the high activ-
ity of adenophostin A (1) is mainly dictated by the phos-
phate triad,[1a,8] the adenine residue,[9] and[10] HO-2ЈЈ, while
the contributions of HO-5Ј (cf. 4)[9a,9b] and HO-6" (cf. 2)
are less important.[11,12] In addition, analogs of 1 in which
the adenine is replaced[13] by an alkyl group, as in ribophos-
tin 5,[14] still exert IP3-like potency. The same holds for the
corresponding phenyl derivative 6,[14] which exhibits a
slightly higher potency than 5. It is also of interest to note
that adenophostin analogs with surrogate bases[15] are more
active than IP3 (3). This scrutinizing of the structure-activ-
ity profile for 1 suggests a strategy of introducing a photo-
We report here the synthesis of the two photoaffinity de-
rivatives 7 and 8 (see Scheme 1) based on adenophostin A
(1). In compound 7, the chemically stable p-benzoyldihyd-
rocinnamoyl (BZDC) photoprobe[16] is joined directly by an
amide bond to the 5Ј-position, while in 8 it is tethered to
an aminopropyl spacer at the anomeric center of the ri-
bosyl moiety.
[a]
Leiden Institute of Chemistry, Gorlaeus Laboratories,
University of Leiden,
Results and Discussion
P. O. Box 9502, 2300 RA Leiden, The Netherlands
Fax: (internat.) ϩ 31-71/527-4307
E-mail: j.boom@chem.leidenuniv.nl
In the first instance, attention was focused on the syn-
thesis of a photoaffinity derivative in which either the 5Ј-
Eur. J. Org. Chem. 2000, 3085Ϫ3092
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/0917Ϫ3085 $ 17.50ϩ.50/0
3085

M. de Kort, J. Luijendijk, G. A. van der Marel, J. H. van Boom
FULL PAPER
or the 6ЈЈ-position was replaced by a BZDC moiety. Previ- Naϩ ion-exchange chromatography, the homogeneous 5Ј-
ous work from our laboratory[14,17] had revealed that the amino derivative of adenophostin A (15, Naϩ salt). The
silyl-protected disaccharide 9 (see Scheme 2) is a valuable observed low yield (27%) of intermediate 14 may be due to
building block in the construction of adenophostin analogs. the occurrence of an undesired Staudinger reaction[22] of
Retrosynthetic analysis shows that the same dimer is also the 5Ј-azide functionality with the phosphoramidite re-
an ideal starting compound for the synthesis of key 5Ј-ami- agent. The low recovery of 14 could in principle be over-
nophostin 15, which can readily be converted into the 5Ј- come by prior conversion of the azide into a protected
BZDC derivative 7 by reaction with N-hydroxysuccinimide amine function (cf. phosphorylation of 28 in Scheme 3).
21. Desilylation of 9 with n-tetrabutylammonium fluoride
(TBAF) and subjection of the resulting primary hydroxy
function to diphenylphosphoryl azide and diethyl azodicar-
boxylate[18] gave the 5-azido derivative 10. Subsequently,
concomitant removal of the 3,4-butane diacetal (BDA) and
isopropylidene protecting groups in 10, followed by
acetylation, yielded tetraacetate 11. Vorbrüggen-type con-
densation of 11 with silylated N6-benzoyladenine in the
presence of catalytic TMSOTf gave the expected[19] glucosyl
adenosine derivative 12. The introduction of the three phos-
phate groups was effected by the following well-estab-
lished[19] three-step sequence of reactions. Selective de-
acetylation of 12 by short treatment with potassium tert-
butoxide in MeOH resulted in 13. Phosphitylation of the
alcohol functions in 13 with N,N-diisopropylbis[2-(methyl-
sulfonyl)ethyl] (MSE) phosphoramidite, assisted by 1H-
tetrazole,[20] and in situ oxidation of the intermediate phos-
phite triesters with tert-butyl hydroperoxide, afforded tris-
phosphate 14. Deprotection of 14 was accomplished by re-
moval of the base-labile groups with Tesser’s base,[21] fol-
lowed by hydrogenolysis of the remaining benzyl ethers to
Scheme 3. Reagents and conditions: (i) see Table 1, 15 min; (ii) a.
80% HOAc, reflux, 1 h; b. Ac2O/pyridine, 30 min., 92%; (iii) a.
HOAc/H2O/(HOCH2)2, 14:6:3, v/v/v, reflux, 1 h; b. Ac2O/pyridine,
4 h, 94% (2 steps); (iv) SnCl4, HO(CH2)3NHZ, (CH2Cl)2, mol.
sieves 4 A, 16 h, 92%; (v) a. NaOMe, MeOH, then Dowex H ; b.
(BnO)2PN(iPr)2, 1H-tetrazole, (CH2Cl)2/CH3CN, 3:1, v/v, 30 min,
then tBuOOH, 0°C, 1 h, 77%; (vi) Pd/C, H2 (1 atm), NaOAc, 1,4-
dioxane/iPrOH/H2O, 4:2:1, v/v/v, 16 h, 82%; (vii) 21, DMF, 0.2 ,
ϩ
˚
give, after purification by HW-40 gel filtration and Dowex Et3NHCO3, pH ϭ 8.5, 16 h, 95%
Scheme 2. Reagents and conditions: (i) a. TBAF (1.0  in THF)/1,4-dioxane, 1:4, v/v, 50°C, 8 h; b. Ph3P, DEAD, (PhO)2P(O)N3, 16 h,
60%, (2 steps); (ii) a. HOAc/H2O/(HOCH2)2, 14:6:3, v/v/v, reflux, 1 h; b. Ac2O/pyridine, 4 h, 79% (2 steps); (iii) TMS-ABz,TMS, TMSOTf,
(CH2Cl)2, reflux, 16 h, 68%; (iv) tBuOK (1  in MeOH), 1 min, 88%; (v) (MSEO)2PN(iPr)2, 1H-tetrazole, 30 min, then tBuOOH, 0 °C,
30 min, 27%; (vi) a. NaOH (4 )/1,4-dioxane/MeOH, 1:14:5, v/v/v, 16 h; b. H2, Pd black, 16 h, 62% (2 steps); (vii) methyl propiolate,
PdCl2(PPh3)2, CuI, K2CO3, THF, 65 °C, 6 h, 56%; (viii) methyl acrylate, Pd(OAc)2, Bu4NCl, NaHCO3, DMF, 3 h, 85%; (ix) H2, PtO2,
EtOAc, 5 min, 95%; (x) 0.5  NaOH, 1,4-dioxane/H2O (2:1, v/v), 2 h, quant.; (xi) NHS, DCC, CH2Cl2; (xii) DMF, 0.2 , Et3NHCO3
H, pH ϭ 8.5, 20 h, 95%, see ref.[28]
3086
Eur. J. Org. Chem. 2000, 3085Ϫ3092

Synthesis of Photoaffinity Derivatives of Adenophostin A
FULL PAPER
phane,[34] followed by in situ oxidation of the phosphite
The BZDC succinimyl reagent 21, required for the intro- triesters with tert-butyl hydroperoxide, gave triphosphate 29
duction of the photoaffinity probe, was prepared according in 77% yield. One-step removal of the benzyloxycarbonyl
to the sequence of reactions presented in Scheme 2. Sono- (Z) and benzyl groups in 29 by hydrogenolysis afforded de-
gashira coupling[23] of 4-iodobenzophenone (16)[24] with rivative 30, containing the amino spacer. Treatment of 30
methyl propiolate gave the acetylene derivative 17 in a mod- with BZDC-NHS (21) as described for the preparation of
erate yield. Alternatively, Heck coupling[25] of iodide 16 7 gave, after purification by ion-exchange chromatography,
with methyl acrylate[26] proceeded smoothly to provide the 1Ј-BZDC-aminopropyl derivative 8 (Naϩ salt) in 95%
methyl (E)-p-benzoylcinnamate (18). Short treatment[27] yield. The identity of 8 was unambiguously corroborated
1
(5 min) of either 17 or 18 with H2/PtO2 furnished the dihy- by mass spectrometry and 31P, 13C, and H NMR spectro-
drocinnamoyl derivative 19, which after saponification of scopy.
the methyl ester afforded the free acid 20. Condensation
of 20 with N-hydroxysuccinimide (NHS) assisted by N,NЈ-
Table 1. NIS-mediated glucosylation of 23 with 22 and 25
dicyclohexylcarbodiimide (DCC) led to the activated ester
21, which was in all aspects identical to a sample prepared
previously.[28] LC-MS analysis showed that the condensa-
tion of amino derivative 15 with 21 proceeded, as ex-
pected,[29] in a chemoselective fashion to give 7. Purification
of the crude product by ion-exchange chromatography pro-
vided the homogeneous 5Ј-BZDC photoaffinity probe 7
(Naϩ salt) in 78% yield. The identity of 7 was unambigu-
ously corroborated by mass spectrometry, 31P and 1H
NMR spectroscopy.
Entry Donor Product Conditions[a] α/β ratio[b] Yield (%)
1
2
3
4
22
22
22
25
24
24
24
26
A
B
C
C
2.2:1
3.0:1
3.5:1
11.8:1
62
72
75
89
[a]
˚
Conditions: mol. sieves 4 A, room temp.; A: NIS, TfOH, Et2O;
B: NIS, TfOH, toluene/1,4-dioxane, 1:3, v/v; C: NIS, TMSOTf,
toluene/1,4-dioxane, 1:3, v/v. Ϫ [b] Determined by integration of the
1
1Ј-H signals in the H NMR spectra of the crude products.
It is evident that the production of photoaffinity analog
8, containing the BZDC group tethered via an aminopropyl
spacer to the 1Ј-position, could also be effected starting
from the disaccharide 9 mentioned earlier. Nonetheless, it
was decided to replace the 5Ј-O-tert-butyldiphenylsilyl
group in 9 by a benzyl group, as in the disaccharide 24.
This change in protecting group strategy would facilitate
the deprotection in a later stage of the synthesis (vide infra).
The construction of target compound 8 is presented in
Scheme 3 and commences with the preparation of dimer 24.
Condensation of the readily accessible[14,17] BDA-protected
donor 22 with the known[30] acceptor 5-O-benzyl-1,2-O-iso-
propylidene-α--ribose (23), mediated by iodonium ion
(NIS) and catalytic amounts of triflic acid (TfOH), gave
dimer 24 as a mixture of anomers (see Entry 1, Table 1).
The low stereoselectivity is in contrast with the exclusive
formation of the α anomer in the coupling of 22 with the
corresponding 5-O-tert-butyldiphenylsilyl-1,2-O-isopropyli-
dene-α--ribofuranoside.[17] Executing the glycosylation of
23 with 22 in a mixture of 1,4-dioxane and toluene (3:1,
v/v)[31] led to a slight improvement in the α/β ratio and yield
(Entry 2). A similar result was obtained using trimethylsilyl
trifluoromethanesulfonate (TMSOTf) as the catalyst (Entry
3). Interestingly, the coupling of 23 with the less reactive
Conclusion
This paper describes the first synthesis of photoaffinity
derivatives of adenophostin A (1) Ϫ i.e. the benzophenone
ligands 7 and 8 Ϫ by chemoselective functionalization of
the respective aminophostins 15 and 30. Preliminary biolo-
gical assay indicated that both ligands 7 and 8 are full agon-
ists of IP3R. A full account on the binding affinity, Ca2ϩ
-
releasing properties, and the potential of these novel ligands
to induce IP3R-mediated Ca2ϩ entry is in progress.
Experimental Section
General Procedures: 1H NMR, 13C NMR and 31P NMR spectra
were recorded with a Jeol JNM-FX-200 (200/50.1/80.7 MHz), a
Bruker WM-300 (300/75.1/121 MHz), or a Bruker DMX-600 spec-
trometer (600/150/242 MHz). H and 13C chemical shifts are given
1
in ppm (δ) relative to tetramethylsilane as internal standard, and
31P chemical shifts relative to 85% H3PO4 as external standard. Ϫ
Mass spectra were recorded with a Finnigan MAT TSQ70 triple
quadrupole or a PE-SCIEX API 165 mass spectrometer equipped
with a custom-made electrospray (ES) interface. Ϫ Optical rota-
tions were determined with a Propol automatic polarimeter. Ϫ
3,4-di-O-acetylated donor 25, prepared by demasking of the Melting points were determined with a Büchi (Flawil, Switzerland)
melting point apparatus. Ϫ Toluene, CH2Cl2, and pyridine were
BDA function in 22 and acetylation of the resulting diol,
proceeded with an acceptable degree of α selectivity, to af-
ford dimer 26 in good yield (Entry 4). Removal of the ace-
tonide group in α anomer 26, followed by acetylation of the
hydroxy functions, afforded the valuable[15,17,32] tetraacetate
27. Glycosidation of 27 with 3-(benzyloxycarbonylamino)-
1-propanol[33] under the influence of SnCl4 led to the ex-
clusive formation of the β-aminopropyl derivative 28. De-
acetylation of 28 and subsequent phosphitylation of the
boiled under reflux for 3 h with P2O5, distilled, and stored over
˚
molecular sieves (4 A). Et2O was freshly distilled from LiAlH4. 1,2-
Dichloroethane (Biosolve, HPLC grade), 2-propanol, DMF, and
˚
1,4-dioxane (Baker, p.a.) were stored over molecular sieves (4 A).
MeOH (Rathburn, HPLC grade) was stored over molecular sieves
˚
(3 A). Column chromatography was performed on Baker silica gel
(0.063Ϫ0.200 mm) and TLC analysis on ‘‘DC-Fertigfolien’’ (Schle-
icher & Schüll F1500, LS 254) with detection by UV absorption
(254 nm) and spraying with 20% H2SO4 in EtOH, or ammonium
triol
with
dibenzyloxy(N,N-diisopropylamino)phos- molybdate (25 gLϪ1) and ceric ammonium sulfate (10 gLϪ1) in 10%
Eur. J. Org. Chem. 2000, 3085Ϫ3092
3087

M. de Kort, J. Luijendijk, G. A. van der Marel, J. H. van Boom
FULL PAPER
H2SO4, followed by charring at 140 °C. Reactions were carried out
at ambient temperature, unless otherwise stated. Prior to reactions
that required anhydrous conditions, traces of H2O were removed
by repeated concentration with toluene, pyridine, or 1,4-dioxane.
Ϫ Propargyl alcohol, diethyl azodicarboxylate, trifluoromethanes-
1,2-Di-O-acetyl-3-O-(3Ј,4Ј-di-O-acetyl-2Ј,6Ј-di-O-benzyl-α-
D-
glucopyranosyl)-5-azido-5-deoxy-α- -ribofuranoside (11): Com-
D
pound 10 (1.17 g, 1.75 mmol) was heated to reflux in a mixture of
AcOH/H2O/(CH2OH)2 (20 mL, 14:6:3, v/v/v). After 1 h, the reac-
tion mixture was cooled (0°C) and quenched with sat. aq. NaHCO3
ulfonic
PdCl2(PPh3)2, methyl propiolate, copper(I) iodide, tetrabutylam-
monium chloride (Acros), ethylene glycol, tBuOK, tin(IV) chloride, dried (MgSO4), and concentrated. The intermediate tetraol (Rf ϭ
acid,
trimethylsilyl
trifluoromethanesulfonate, (30 mL). The resulting suspension was extracted with EtOAc (3 ϫ
25 mL). The organic layer was washed with H2O (3 ϫ 10 mL),
methyl acrylate, K2CO3, N-hydroxysuccinimide (NHS), dicyclohex-
ylcarbodiimide (DCC), tBuOOH (80% in di-tert-butyl peroxide)
(Merck), platinum(IV) oxide, 1H-tetrazole, palladium(II) acetate
(Aldrich), and acetic anhydride (Baker) were all used as received.
0.29, MeOH/EtOAc, 5:95, v/v) was dissolved in a mixture of acetic
anhydride/pyridine (25 mL, 3:7, v/v) and stirred for 4 h. The mix-
ture was diluted with toluene (10 mL) and concentrated under re-
duced pressure (3 ϫ). The oily product was subjected to column
Imino diacetate resin (Chelex , Naϩ salt) was purchased from chromatography by elution with Et2O/light petroleum ether
Sigma. Purification of the target compounds was performed by gel
filtration with a Fractogel column [HW 40(s), 26/60] with triethyl-
(1:9Ǟ1:1, v/v). Concentration of the appropriate fractions afforded
tetraacetate 11 as a white foam. Yield 0.95 g (1.38 mmol, 79%, 2
ammonium bicarbonate buffer (0.15 ) as eluent (1.5 mLminϪ1) or steps); α/β ഠ 1:7. Ϫ 13C{1H} NMR (CDCl3): δ ϭ 169.9, 169.6,
FPLC chromatography (Pharmacia) with ammonium bicarbonate
169.4, 168.7 (4 CϭO Ac), 137.6, 137,4 (2 Cq Bn), 128.2Ϫ127.5 (CH
arom), 97.9 (C-1ЈЈ), 96.1 (C-1Ј), 80.4, 76.4, 73.0, 72.7, 71.5, 69.0,
68.8 (C-2Ј, C-3Ј, C-4Ј, C-2ЈЈ, C-3ЈЈ, C-4ЈЈ, C-5ЈЈ), 73.3 (2 CH2 Bn),
67.8 (C-6ЈЈ), 50.8 (C-5Ј), 20.7, 20.5, 20.4, 20.1 (4 CH3 Ac). Ϫ ES-
MS; m/z: 687 [M ϩ H]ϩ.
(0.15 ) as eluent. Analytical anion exchange HPLC was performed
on a Mono Q HR 5/5 column (Pharmacia), flow rate 2.0 mLminϪ1
.
Elution was effected at pH ϭ 12.0 with a mixture of buffer A (0.01
 NaOH) and buffer B (0.01  NaOH ϩ 1.2  NaCl) with the
following gradient: t ϭ 0 to t ϭ 2.5 min 0% B, t ϭ 2.5 to t ϭ
12.5 min 0Ϫ40% B.
2Ј-O-Acetyl-3Ј-O-(3ЈЈ,4ЈЈ-di-O-acetyl-2ЈЈ,6ЈЈ-di-O-benzyl-α-D-
glucopyranosyl)-5Ј-azido-N6-benzoyl-5Ј-deoxyadenosine (12): A sus-
pension of 6-N-benzoyladenine (0.91 g, 3.79 mmol) in 1,1,1,3,3,3-
hexamethyldisilazane (6.8 mL) and pyridine (2.4 mL) was refluxed
for 7 h. The reaction mixture was cooled, diluted with toluene
(5 mL), and concentrated in vacuo. The residual oil was repeatedly
diluted with toluene (3 ϫ 5 mL) and concentrated in vacuo to re-
move excess 1,1,1,3,3,3-hexamethyldisilazane. Disaccharide 11
(0.95 g, 1.38 mmol) in 1,2-dichloroethane (18 mL), and a catalytic
amount of TMSOTf (61 µL, 0.36 mmol) were added to the silylated
N6-benzoyladenine. After stirring for 16 h at reflux temperature,
TLC analysis showed conversion of the starting tetraacetate into a
slower running product. The reaction mixture was quenched with
Et3N (1.5 mL), diluted with CH2Cl2 (30 mL), and poured into sat.
aq. NaHCO3 (15 mL). The organic phase was washed with H2O
(10 mL), dried (MgSO4), filtered, and concentrated under reduced
pressure. The crude product was purified by column chromato-
graphy (CH2Cl2/MeOH, 1:0 Ǟ95:5, v/v) to give 12 as a yellowish
foam. Yield 0.80 g (0.93 mmol, 68%). Ϫ [α]D ϭ ϩ74.0 (c ϭ1.0
5-Azido-5-deoxy-3-O-{2,6-di-O-benzyl-3,4-di-O-[(2ЈS,3ЈS)-2Ј,3Ј-
dimethoxybutane-2Ј,3Ј-diyl]-α-
D-glucopyranosyl}-1,2-O-
isopropylidene-α-
D
-ribofuranoside (10): Compound 9[17] (3.00 g,
3.40 mmol) was stirred at 50 °C in a mixture of 1,4-dioxane
(20 mL) and TBAF (1.0  in THF, 5.24 mL). After 8 h, TLC ana-
lysis revealed the reaction to be complete and the mixture was con-
centrated under reduced pressure. The residue was dissolved in
EtOAc (75 mL), washed with brine (2 ϫ 15 mL) and H2O (10 mL),
and dried (MgSO4). Purification by column chromatography
(Et2O/light petroleum ether, 1:1Ǟ1:0, v/v) gave the desilylated dis-
accharide in a yield of 2.02 g. Rf ϭ 0.47 (Et2O). The alcohol
(1.98 g, 3.06 mmol) was stirred in a mixture containing triphenyl-
phosphane (1.06 g, 4.10 mmol) and diethyl azodicarboxylate
(0.31 g, 4.10 mmol) in dry THF (15 mL). Diphenylphosphoryl az-
ide (0.88 mL, 4.10 mmol) was added over a period of 15 min. The
reaction mixture was stirred for 16 h, after which TLC analysis
showed conversion of the starting alcohol into a faster running
product. The solvent was removed under reduced pressure and the
product was purified by column chromatography (eluent: EtOAc/
light petroleum ether, 0:1Ǟ1:9, v/v) to afford azide 10. Yield 1.22 g
(1.82 mmol, 60%, 2 steps). Ϫ Rf ϭ 0.81 (EtOAc/light petroleum
ether, 1:9, v/v). Ϫ 1H NMR (CDCl3): δ ϭ 7.42Ϫ7.15 (m, 10 H, CH
arom), 5.78 (d, 1 H, 1Ј-H, J1Ј,2Ј ϭ 3.6 Hz), 5.14 (d, 1 H, 1ЈЈ-H,
J1ЈЈ,2ЈЈ ϭ 4.0 Hz), 4.76 (AB, 2 H, CH2 Bn, J ϭ Ϫ11.9 Hz), 4.70 (t,
1 H, 2Ј-H), 4.56 (AB, CH2 Bn, J ϭ Ϫ11.8 Hz ), 4.32 (m, 1 H, 4Ј-
H), 4.10 (t, 1 H, 3ЈЈ-H, J2ЈЈ,3ЈЈ ϭ J3ЈЈ,4ЈЈ ϭ 9.6 Hz), 4.04 (m, 1 H, 3Ј-
H), 3.87 (m, 1 H, 5ЈЈ-H), 3.78 (t, 1 H, 4ЈЈ-H), 3.73Ϫ3.66 (m, 3 H,
1
CHCl3). Ϫ H NMR (CDCl3): δ ϭ 9.15 (s, 1 H, NH), 8.81 (s, 1 H,
2-H), 8.23 (s, 1 H, 8-H), 8.02 (d, 2 H, CH arom Bz), 7.63Ϫ7.44 (m,
3 H, CH arom Bz), 7.37Ϫ7.23 (m, 10 H, CH arom Bn), 6.25 (d, 1
H, 1Ј-H, J1Ј,2Ј ϭ 5.1 Hz), 5.86 (t, 1 H, 2Ј-H, J2Ј,3Ј ϭ 5.2 Hz), 5.44
(t, 1 H, 3ЈЈ-H, J3ЈЈ,4ЈЈ ϭ 9.6 Hz), 5.00 (t, 1 H, 4ЈЈ-H, J4ЈЈ,5ЈЈ
ϭ
9.8 Hz), 4.98 (d, 1 H, 1ЈЈ-H, J1ЈЈ,2ЈЈ ϭ 3.6 Hz), 4.73 (t, 1 H, 3Ј-H,
J3Ј,4Ј ϭ 5.2 Hz), 4.64Ϫ4.43 (m, 4 H, 2 CH2 Bn), 4.45 (m, 1 H, 4Ј-H),
3.98 (m, 1 H, 5ЈЈ-H), 3.61 (AB, 2 H, 5Ј-H, J4Ј,5Ј 6.3 Hz, J5aЈ,5bЈ
ϭ
Ϫ10.2 Hz), 3.57 (dd, 1 H, 2ЈЈ-H, J2ЈЈ,3ЈЈ 10.1 Hz), 3.51Ϫ3.49 (m, 2
H, 6ЈЈ-H), 1.98, 1.95, 1.90 (3 s, 9 H, 3 CH3 Ac). Ϫ 13C{1H} NMR
(CDCl3): δ ϭ 169.7, 169.6 (3 CϭO Ac), 164.8 (CϭO Bz), 152.3 (C-
2), 151.2 (C-4), 149.5 (C-6), 140.3 (C-8), 137.1, 137.0 (2 Cq Bn),
133.0 (Cq Bz), 132.2 (CH Bz), 128.2Ϫ127.3 (CH arom), 123.5 (C-
5), 97.8 (C-1ЈЈ), 86.4 (C-1Ј), 81.3, 76.1, 75.7, 72.7, 71.1, 69.1, 68.7
(C-2Ј, C-3Ј, C-4Ј, C-2ЈЈ, C-3ЈЈ, C-4ЈЈ, C-5ЈЈ), 73.2, 72.9 (2 CH2 Bn),
6ЈЈ-H, 5aЈ-H), 3.64 (dd, 1 H, 2ЈЈ-H), 3.33 (dd, 1 H, 5bЈ-H, J5aЈ,5bЈ
ϭ
Ϫ9.7 Hz, J4Ј,5aЈ 5.8 Hz), 3.31, 3.19 (2 s, 6 H, CH3 OMe), 1,54, 1.35
(2 s, 6 H, CH3 BDA), 1.35, 1.30 (2 s, 6 H, CH3 isoprop). Ϫ 13C{1H}
NMR (CDCl3): δ ϭ 138.4, 137.6 (2 Cq Bn), 127.9Ϫ126.9 (CH
arom), 112.7 (Cq isoprop), 103.6 (C-1ЈЈ), 99.2, 99.1 (2 Cq BDA),
95.5 (C-1Ј), 76.7, 76.2, 75.6, 73.7, 69.0, 65.7 (C-2Ј, C-3Ј, C-4Ј, C-
2ЈЈ, C-3ЈЈ, C-4ЈЈ, C-5ЈЈ), 73.0, 71.7 (2 CH2 Bn), 67.6 (C-6ЈЈ), 50.1
(C-5Ј), 47.7, 47.5 (2 CH3 OMe), 26.3, 26.2 (2 CH3 BDA), 17.5, 17.3
(2 CH3 isoprop). Ϫ C34H45N3O11 (671.7 ((AUTHOR: We added
67.8 (C-6ЈЈ), 50.9 (C-5Ј), 20.4, 20.1, 19.8 (3 CH3 Ac).
Ϫ
C43H44N8O12 (864.9): calcd. C 59.72, H 5.13, N 12.96; found C
59.89, H 5.19, N 13.05. Ϫ ES-MS; m/z: 866 [M ϩ H]ϩ, 889 [M
ϩ Na]ϩ.
mol. masses, please check them all!))): calcd. C 60.79, H 6.75, N 5Ј-Azido-N6-benzoyl-3Ј-O-(2ЈЈ,6ЈЈ-di-O-benzyl-α-
D-glucopyranosyl)-
6.26; found C 60.60, H 6.84, N 6.21. Ϫ ES-MS; m/z: 694 [M ϩ
5Ј-deoxyadenosine (13): To a vigorously stirred solution of glucopy-
ranosyl adenosine 12 (0.73 g, 0.85 mmol) in 1,4-dioxane (25 mL)
Na]ϩ.
3088
Eur. J. Org. Chem. 2000, 3085Ϫ3092

Synthesis of Photoaffinity Derivatives of Adenophostin A
FULL PAPER
was added a solution of tBuOK in MeOH (1.0 , 35 mL). After
stirring for 1 min, the reaction mixture was neutralized upon the
addition of AcOH (2.0 mL, 35 mmol). The solution was poured
Methyl p-Benzoyl-2,3-didehydrocinnamate (17): 4-Iodobenzo-
phenone (16, 0.32 g, 1.04 mmol) and methyl propiolate (0.35 g,
0.37 mL, 4.16 mmol) were dissolved in THF (5 mL) and the solu-
into sat. aq. NaHCO3 (25 mL), and the solution was extracted with tion was degassed. PdCl2(PPh3)2 (15 mg, 0.02 mmol), copper(I)
CH2Cl2 (2 ϫ 25 mL). The organic phase was washed with H2O
(15 mL), dried (MgSO4), filtered, and concentrated in vacuo. Puri-
fication by column chromatography (eluent: CH2Cl2/MeOH, 1:0 to
iodide (8 mg, 0.05 mmol), and K2CO3 (0.29 g, 2.1 mmol) were ad-
ded and the mixture was refluxed for 6 h under argon. The dark
brown solution was concentrated and dissolved in Et2O (50 mL).
The organic phase was washed with sat. aq. NaHCO3 (25 mL) and
95:5, v/v) afforded triol 13 as
a white foam. Yield 0.55 g
(0.75 mmol, 88%). [α]D ϭ ϩ57.8 (c ϭ1.0 CHCl3). Ϫ Rf ϭ 0.45 H2O (25 mL), dried (MgSO4), and concentrated. The dark brown
(MeOH/EtOAc, 5:95, v/v). Ϫ 1H NMR (CDCl3): δ ϭ 9.41 (s, 1 H,
residue was applied to a column of silica gel, which was eluted
NH), 8.73 (s, 1 H, 2-H), 8.20 (s, 1 H, 8-H), 8.02 (d, 2 H, CH Bz), with EtOAc/light petroleum ether (1:9, v/v) to give 17. Yield 0.15 g
7.61Ϫ7.46 (m, 3 H, CH arom Bz), 7.36Ϫ7.16 (m, 10 H, CH arom (0.58 mmol, 56%). Ϫ Rf ϭ 0.36 (EtOAc/light petroleum ether, 1:9,
1
Bn), 6.07 (d, 1 H, 1Ј-H, J1Ј,2Ј ϭ 5.3 Hz), 4.89 (d, 1 H, 1ЈЈ-H, v/v). Ϫ H NMR (CDCl3): δ ϭ 7.85Ϫ7.75, 7.72Ϫ7.57, 7.54Ϫ7.44
J1ЈЈ,2ЈЈ ϭ 3.6 Hz), 4.72 (m, 1 H, 2Ј-H), 4.71 (AB, 2 H, CH2 Bn, J ϭ (m, 9 H, H arom), 3.87 (s, 3 H, OMe). Ϫ 13C{1H} NMR (CDCl3):
Ϫ11.7 Hz), 4.53 (AB, 2 H, CH2 Bn, J ϭ Ϫ12.2 Hz), 4.28Ϫ4.24 (m, δ ϭ 200.7 (CϭO benzophenone), 153.9 (CϭO), 137.8, 135.3, 123.2
2 H, 3Ј-H, 4Ј-H), 4.03 (t, 1 H, 3ЈЈ-H, J3ЈЈ,4ЈЈ ϭ J2ЈЈ,3ЈЈ ϭ 9.5 Hz), (Cq arom), 132.7, 132.6, 129.8, 128.3 (CH arom), 86.9 (C-2), 52.8
3.84 (m, 1 H, 5ЈЈ-H), 3.77 (dd, 1 H, 6aЈЈ-H, J5ЈЈ,6aЈЈ ϭ 6.9 Hz), (OMe). Ϫ ES-MS; m/z: 265 [M ϩ H]ϩ.
3.68Ϫ3.62 (m, 2 H, 6bЈЈ-H, 5aЈ-H), 3.61Ϫ3.51 (m, 2 H, 4ЈЈ-H, 5bЈ-
(E)-Methyl p-Benzoylcinnamate (18): 4-Iodobenzophenone (16,
H), 3.44 (dd, 1 H, 2ЈЈ-H). Ϫ 13C{1H} NMR (MeOD ): δ ϭ 167.6
0.31 g, 1.00 mmol) was dissolved in DMF (4 mL) and the solution
was degassed. Pd(OAc)2 (11 mg, 0.05 mmol), methyl acrylate
(0.17 g, 0.18 mL, 2.0 mmol), tetrabutylammonium chloride (0.28 g,
1.0 mmol), and NaHCO3 (0.21 g, 0.25 mmol) were added, and the
solution was degassed again. The reaction mixture was stirred for
3 h under argon, after which it was concentrated to 1 mL, diluted
with Et2O (20 mL), and rinsed with H2O (10 mL), sat. aq.
(CϭO Bz), 153.1 (C-2), 152.9 (C-4), 150.8 (C-6), 144.2 (C-8), 139.3,
139.0 (2 Cq Bn), 134.6 (Cq Bz), 133.7 (CH Bz), 129.5Ϫ128.5 (CH
arom), 124.8 (C-5), 98.8 (C-1ЈЈ), 90.0 (C-1Ј), 82.9, 80.4, 77.8, 73.2,
71.7 (C-2Ј, C-3Ј, C-4Ј, C-2ЈЈ, C-3ЈЈ, C-4ЈЈ, C-5ЈЈ), 74.2, 74.1 (2 CH2
Bn), 70.8 (C-6ЈЈ), 52.8 (C-5Ј). Ϫ ES-MS: 740 [M ϩ H]ϩ, 762 [M
ϩ Na]ϩ.
NaHCO3 (10 mL), and H2O (10 mL). Purification was effected by
column chromatography (EtOAc/light petroleum ether, 0:1Ǟ2:8, v/
v) to give cinnamic acid derivative 18 in pure form. Yield 0.23 g
(0.85 mmol, 85%). Ϫ Rf ϭ 0.31 (EtOAc/light petroleum ether, 1:9,
5Ј-Amino-5Ј-deoxy-3ЈO-α-D-glucopyranosyl-3ЈЈ,4ЈЈ-bisphosphate)-
adenosine 2Ј-Monophosphate 15, Na؉ Salt: To a mixture of triol 13
(0.11 g, 0.14 mmol) and N,N-diisopropylbis[2-(methylsulfonyl)-
ethyl] phosphoramidite[19,20] (0.30 g, 0.81 mmol) in CH2Cl2 (4 mL)
was added a solution of 1H-tetrazole (75 mg, 1.1 mmol) in CH3CN
(4 mL). After stirring for 30 min, TLC analysis (MeOH/CH2Cl2,
15:85, v/v) showed complete conversion of starting triol into several
faster running products (major product: Rf ϭ 0.57). The reaction
mixture was cooled, tBuOOH (0.17 mL) was added, and stirring
was continued for 30 min, after which TLC analysis revealed com-
plete conversion of the intermediate phosphite triester into slower
running products. The phosphate triester 14 was obtained after
purification by column chromatography (eluent: CH2Cl2/MeOH,
1:0 to 9:2, v/v). Yield 55 mg (38 µmol, 27%). Ϫ Rf ϭ 0.41 (CH2Cl2/
MeOH, 90:8, v/v). Ϫ 31P NMR (CDCl3): δ ϭ Ϫ2.0, Ϫ1.9, Ϫ1.8.
Ϫ Compound 14 (33 mg, 23 µmol) was dissolved in a mixture of
NaOH (4 )/1,4-dioxane/MeOH (1:14:5, v/v/v, 10 mL) and stirred
for 16 h. The mixture was neutralized with AcOH (0.12 mL) and
concentrated. Purification of the trisphosphate was accomplished
by gel filtration on an HW-40 column eluted with triethyl ammo-
nium bicarbonate buffer (0.15 ). Complete deprotection of the
purified 2ЈЈ,6ЈЈ-di-O-benzyl-5Ј-azidophostin intermediate was ef-
fected by hydrogenation in MeOH/H2O and AcOH (3 drops) in the
presence of Pd black. After 16 h, the mixture was neutralized with
Et3N and filtered through glass fiber (Whatman GF/A) and con-
centrated under reduced pressure. Ion-exchange chromatography
by Dowex 50Wx4 (Naϩ salt) and treatment with imino diacetate
resin (Chelex , Naϩ salt), followed by lyophilization, gave pure 5Ј-
1
v/v). Ϫ H NMR (CDCl3): δ ϭ 7.85Ϫ7.44 (m, 10 H, H arom, 3-
H), 6.54 (d, 1 H, 2-H, Jtrans ϭ 16 Hz), 3.84 (s, 3 H, OMe). Ϫ
13C{1H} NMR (CDCl3): δ ϭ 195.6 (CϭO benzophenone), 166.8
(CϭO), 143.2 (C-3), 138.6, 137.9, 137.1 (Cq arom), 132.5, 131.3,
130.4, 129.8, 128.2, 127.7 (CH arom, C-3), 120.0 (C-2), 51.7 (OMe).
Ϫ C17H14O3: calcd. C 76.68, H 5.30; found C 76.61, H 5.25. Ϫ ES-
MS; m/z: 267 [M ϩ H]ϩ, 289 [M ϩ Na]ϩ, 555 [2 M ϩ Na]ϩ.
Methyl p-Benzoyl-2,3-dihydrocinnamate (19): Cinnamic ester deriv-
ative 17 (0.13 g, 0.50 mmol) or 18 (0.13 g, 0.50 mmol) was dissolved
in EtOAc (5 mL). Platinum(IV) oxide (7 mg, 0.03 mmol) was added
and the solution was degassed. The reaction mixture was vigor-
ously stirred under H2. After 5 min, the solution was degassed,
immediately filtered through glass fiber (GF/2A, Whatman ) and
concentrated. Purification was effected by column chromatography
(EtOAc/light petroleum ether, 1:9Ǟ3:7, v/v) to give compound 19
in pure form. Yield 0.12 g (0.45 mmol, 95%). Ϫ Rf ϭ 0.30 (EtOAc/
light petroleum ether, 2:8, v/v). Ϫ 1H NMR (CDCl3): δ ϭ
7.84Ϫ7.20 (m, 9 H, H arom), 3.69 (s, 3 H, CH3), 3.04 (t, 2 H, 3-
H, J2,3 ϭ 7.3 Hz), 2.69 (t, 2 H, CH2). Ϫ 13C{1H} NMR (CDCl3):
δ ϭ 178.2 (CϭO), 144.8, 137.9, 135.1 (Cq arom), 132.0, 130.6,
129.1, 128.4 (CH arom), 34.1, 29.2 (C-2, C-3). Ϫ C17H16O3 (268.3):
calcd. C 76.10, H 6.01; found C 75.99, H 6.03. Ϫ ES-MS; m/z: 269
[M ϩ H]ϩ, 291 [M ϩ Na]ϩ.
aminophostin 15. Yield 9.3 mg (14 µmol, 62%). Ϫ 1H NMR (D2O, N-Succinimidyl p-Benzoyl-2,3-dihydrocinnamate (21): Compound
600 MHz, HH-COSY): δ ϭ 8.22 (s, 1 H, 2-H), 8.18 (s, 1 H, 8-H),
6.27 (d, 1 H, 1Ј-H, J1Ј,2Ј ϭ 5.1 Hz), 5.37 (d, 1 H, 1ЈЈ-H, J1ЈЈ,2ЈЈ
3.9 Hz), 5.08 (ddd, 1 H, 2Ј-H, J2Ј,3Ј ϭ 2.9 Hz, J2Ј,P ϭ 8.4 Hz), 4.65
(dd, 1 H, 3Ј-H, J3Ј,4Ј ϭ 4.8 Hz), 4.52 (q, 1 H, 3ЈЈ-H, J3ЈЈ,4ЈЈ
19 (0.10 g, 0.37 mmol) was dissolved in a 0.5  solution of NaOH
in 1,4-dioxane/H2O (2:1, v/v, 5 mL). After stirring for 2 h, the mix-
ture was acidified with AcOH (pH ϭ 5). The solution was diluted
with EtOAc (15 mL), washed with H2O (2 ϫ 5 mL), dried
ϭ
ϭ
J2ЈЈ,3ЈЈ ϭ J3ЈЈ,P ϭ 8.4 Hz), 4.32 (m, 1 H, 4Ј-H), 3.91 (m, 2 H, 4ЈЈ-H, (MgSO4), and concentrated. The product 20 was used in the next
6aЈЈ-H), 3.73 (m, 3 H, 5ЈЈ-H, 6bЈЈ-H, 2ЈЈ-H), 3.32 (dAB, 2 H, 5Ј-
reaction without purification. Yield 95 mg (quant.). Ϫ Rf ϭ 0.23
1
H, J4Ј,5Ј ϭ 2.9 Hz, J5aЈ,5bЈ ϭ Ϫ10.7 Hz). Ϫ 31P NMR (D2O): δ ϭ (EtOAc/light petroleum ether 1:1, v/v). Ϫ H NMR (CDCl3): δ ϭ
4.25, 3.59, 3.08. Ϫ HR-MS: C16H26N6O17P3 [M Ϫ H]Ϫ calcd. 7.85Ϫ7.25 (m, 9 H, H arom), 3.05 (t, 2 H, 3-H, J2,3 ϭ 7.3 Hz), 2.74
667.0566; found 667.0573. Ϫ ES-MS; m/z: 669 [M ϩ H]ϩ.
(t, 2 H, 2-H, CH2). Ϫ 13C{1H} NMR (CDCl3): δ ϭ 178.4 (CϭO),
Eur. J. Org. Chem. 2000, 3085Ϫ3092
3089

M. de Kort, J. Luijendijk, G. A. van der Marel, J. H. van Boom
FULL PAPER
145.2, 137.6, 135.7 (Cq arom), 132.3, 130.5, 129.9, 128.2 (CH
arom), 35.0, 30.4 (C-2, C-3). Ϫ ES-MS; m/z: 255 [M ϩ H]ϩ, 277
[M ϩ Na]ϩ, 531 [2 M ϩ Na]ϩ. Ϫ Dihydrocinnamic acid 20 was
converted into N-succinimidyl cinnamate 21 as described by
Prestwich et al.[28]
(t, 1 H, 2-H, J2,3 ϭ 9.5 Hz), 2.83Ϫ2.72 (m, 2 H, CH2 SEt), 1.33 (t,
3 H, CH3 SEt, J ϭ 7.4 Hz). Ϫ ES-MS; m/z: 489 [M ϩ H]ϩ.
3-O-(3Ј,4Ј-Di-O-acetyl-2Ј,6Ј-di-O-benzyl-α-
D-glucopyranosyl)-5-O-
benzyl-1,2-O-isopropylidene-α- -ribofuranoside (26): Acceptor 23
D
(0.54 g, 1.94 mmol) and donor 25 (1.04 g, 2.13 mmol) were dis-
solved in a mixture of toluene and 1,4-dioxane (1:3, v/v, 20 mL)
and were coupled as described for 24. The α and β anomers were
separated by column chromatography (eluent: toluene/EtOAc
9:1Ǟ8:2, v/v), to give disaccharide 26 in pure form. Yield 1.31 g
(1.86 mmol, 89%). Ϫ Rf ϭ 0.64 (toluene/EtOAc/MeOH, 90:25:2.5,
5Ј-(p-Benzoyl-2,3-dihydrocinnamido)-5Ј-deoxy-3Ј-O-(α-D-
glucopyranosyl 3ЈЈ,4ЈЈ-bisphosphate)-adenosine 2Ј-Mono-
phosphate (7), Na؉ Salt: Aminophostin 15 (Naϩ salt, 5.0 mg, 7.5
µmol) was subjected to the activated ester derivative 21 (8.0 mg, 23
µmol) as described by Prestwich et al.[29] The reaction was mon-
itored by LC/MS analysis, which indicated complete conversion of
the starting material after 20 h. No side products arising from reac-
tion with the adenine base were detected. The product was purified
by Q-Sepharose column chromatography (Pharmacia). After ly-
ophilization of the appropriate fractions, photoaffinity derivative 7
was converted into the Naϩ salt by sequential treatment with
Dowex 50Wx4 (Naϩ salt) and imino diacetate resin (Chelex ,
Naϩ salt), followed by lyophilization. Yield 5.1 mg (5.5 µmol, 78%).
1
v/v/v). Ϫ [α]2D0 ϭ ϩ103.5 (c ϭ 2.0 CHCl3). Ϫ H NMR (CDCl3):
δ ϭ 7.36Ϫ7.22 (m, 15 H, H arom), 5.83 (d, 1 H, 1-H, J1,2
ϭ
4.4 Hz), 5.38 (t, 1 H, 3Ј-H, J2Ј,3Ј, J3,4 ϭ 9.5 Hz), 5.21 (d, 1 H, 1Ј-
H, J1Ј,2Ј ϭ 3.7 Hz), 5.09 (t, 1 H, 4Ј-H, J4Ј,5Ј ϭ 9.5 Hz), 4.72Ϫ4.47
(m, 7 H, 2 CH2 Bn, 2-H, 3-H, 4-H), 4.30 (d, 2 H, CH2 Bn), 4.16
(dd, 1 H, 5Ј-H, J5Ј,6Ј ϭ 4.4 Hz), 3.81 (dd, 1 H, 5a-H, J4,5a ϭ 2.2 Hz,
J5a,5b ϭ 11.7 Hz), 3.71 (dd, 1 H, 5b-H, J4,5b ϭ 3.0 Hz), 3.62 (dd, 1
H, 2Ј-H), 3.32 (m, 2 H, 6-H), 2.03, 1.88 (s, 6 H, 2 CH3 Ac), 1.53,
1.37 (s, 6 H, 2 CH3 isoprop). Ϫ 13C{1H} NMR (CDCl3): δ ϭ 168.8,
169.3 (2 CϭO Ac), 137.6, 137.3 (3 Cq Bn), 128.0Ϫ127.2 (CH
arom), 112.6 (Cq isoprop), 104.0 (C-1), 94.0 (C-1Ј), 77.3, 75.9, 75.5,
72.5, 71.7, 68.6, 68.2 (C-2, C-3, C-4, C-2Ј, C-3Ј, C-4Ј, C-5Ј), 73.2,
73.0 (3 CH2 Bn), 67.6, 67.1 (C-5, C-6Ј), 26.4 (2 CH3 Ac), 20.5, 20.3
(2 CH3 isoprop). Ϫ C39H46N8O12 (818.8): calcd. C 66.28, H 6.56;
found C 66.37, H 6.53. Ϫ ES-MS; m/z: 706 [M ϩ H]ϩ.
1
Ϫ H NMR (D2O, 600 MHz, HH-COSY): δ ϭ 8.24 (s, 1 H, 2-H),
8.13 (s, 1 H, 8-H), 7.82 (t, 1 H, J ϭ 7.5 Hz), 7.65 (m, 4 H), 7.51
(d, 2 H, J ϭ 7.9 Hz), 7.28 (d, 2 H, J ϭ 8.2 Hz), 6.28 (d, 1 H, 1Ј-
H, J1Ј,2Ј ϭ 1.1 Hz), 5.33 (d, 1 H, 1ЈЈ-H, J1ЈЈ,2ЈЈ ϭ 3.4 Hz), 4.52 (dd,
1 H, 2Ј-H, J2Ј,P ϭ 7.3 Hz), 4.27 (q, 1 H, 3ЈЈ-H, J ϭ 8.1 Hz), 4.22
(m, 2 H, 3Ј-H, 4Ј-H), 3.86 (dd, 1 H, 6aЈЈ-H, J6aЈЈ,6bЈЈ ϭ Ϫ9.6 Hz,
J5ЈЈ,6aЈЈ ϭ 3.4 Hz), 3.81 (m, 2 H, 5ЈЈ-H, 4ЈЈ-H), 3.68 (m, 2 H, 2ЈЈ-
H, 6bЈЈ-H), 3.10Ϫ3.01 (m, 2 H, 5Ј-H), 2.75 (m, 1 H, CH2 BZDC),
2.57 (m, 3 H, CH2 BZDC). Ϫ 31P NMR (D2O, 242 MHz): δ ϭ
2.86 (P-4Ј), 2.27 (P-3ЈЈ), 1.43 (P-2Ј). Ϫ HR-MS: C32H38N6O19P3
[M Ϫ H]Ϫ calcd. 903.1403; found 903.1398. Ϫ ES-MS; m/z: 905
[M ϩ H]ϩ.
1,2-Di-O-acetyl-3-O-(3Ј,4Ј-di-O-acetyl-2Ј,6Ј-di-O-benzyl-α-
D-gluco-
pyranosyl)-5-O-benzyl-α- -ribofuranoside (27): Compound 26
D
(2.26 g, 3.20 mmol) was heated to reflux in a mixture of AcOH/
H2O/(CH2OH)2 (35 mL, 14:6:3, v/v/v). After 1 h, the reaction mix-
ture was cooled (0 °C) and quenched with sat. aq. NaHCO3
(60 mL). The resulting suspension was extracted with EtOAc (3 ϫ
50 mL). The organic layer was washed with H2O (3 ϫ 20 mL),
5-O-Benzyl-3-O-{2Ј,6Ј-di-O-benzyl-3Ј,4Ј-di-O-[(2ЈЈS,3ЈЈS)-2ЈЈ,3ЈЈ-di-
methoxybutane-2ЈЈ,3ЈЈ-diyl]-α-
D-glucopyranosyl}-1,2-O-isopropy-
dried (MgSO4), and concentrated. The intermediate diol (Rf
ϭ
lidene-α- -ribofuranoside (24): Acceptor 23 (0.97 g, 3.49 mmol) and
D
0.42, MeOH/EtOAc, 4:96, v/v) was dissolved in a mixture of acetic
anhydride/pyridine (40 mL, 3:7, v/v) and stirred for 4 h. The mix-
ture was diluted with toluene (3 ϫ 10 mL) and concentrated under
reduced pressure. The oily product was subjected to column chro-
matography, eluting with Et2O/light petroleum ether (1:9Ǟ1:1, v/
v). Concentration of the appropriate fractions afforded tetraacetate
27 as a white foam. Yield 2.33 g (94%, 3.10 mmol, 2 steps). Ϫ Rf ϭ
0.56 (EtOAc/light petroleum ether, 1:1, v/v). Ϫ α/β ഠ 1:7. The ana-
lytical data are in all aspects identical to a previously prepared
sample.[14,15a,17]
donor 22 (1.98 g, 3.83 mmol) were repeatedly concentrated with
toluene (3 ϫ 5 mL) and dissolved in a mixture of toluene and 1,4-
˚
dioxane (1:3, v/v, 30 mL). Activated molecular sieves (4 A) were
added and the mixture was stirred under a stream of N2. NIS
(0.86 g, 3.83 mmol) and a catalytic amount of TMSOTf (0.07 mL,
0.43 mmol) were subsequently added. After stirring for 10 min,
TLC analysis showed complete disappearance of 23. The reaction
mixture was filtered through Hyflo and the filtrate was diluted
with EtOAc (25 mL), washed with aq. Na2S2O3 (15 mL) and aq.
NaHCO3 (1 , 15 mL), dried with MgSO4 and concentrated in
vacuo. The α and β anomers were separated by column chromato-
graphy (eluent: toluene/EtOAc, 19:1Ǟ8:2, v/v), to give disaccharide
24 in pure form. Yield 2.09 g (2.62 mmol, 75%) The analytical data
are in all aspects identical to a previously prepared sample.[14,17]
1-((3-Benzyloxycarbonylamino)-propyl)-2-O-acetyl-5-O-benzyl-3-O-
(3Ј,4Ј-di-O-acetyl-2Ј,6Ј-di-O-benzyl-α-D-glucopyranosyl)-β-D-ribo-
furanoside (28): Tetraacetate 27 (0.97 g, 1.29 mmol) and 3-
(benzyloxycarbonylamino)-1-propanol[33] (0.24 g, 1.94 mmol) were
dissolved in 1,2-dichloroethane (20 mL). The solution was stirred
˚
Ethyl 3,4-Di-O-acetyl-2,6-di-O-benzyl-1-thio-β-
D-glucopyranoside
with powdered molecular sieves (4 A) under a stream of argon.
(25): Compound 22 (7.96 g, 15.6 mmol) was dissolved in 80% aq.
AcOH (75 mL) and heated to reflux. After stirring for 1 h at that
temperature, the reaction mixture was concentrated in vacuo. The
oily residue was repeatedly concentrated with pyridine (3 ϫ 10 mL)
and dissolved in a mixture of pyridine/acetic anhydride (25 mL,
2:1, v/v). After 30 min, the mixture was concentrated and the res-
idue was concentrated with toluene (3 ϫ 20 mL). The product 25
SnCl4 (76 µL, 0.65 mmol) was added and the mixture was stirred
overnight. The mixture was neutralized with Et3N (0.2 mL), filtered
through Hyflo, and diluted with Et2O (20 mL). The solution was
washed with H2O (10 mL) and sat. aq. NaHCO3 (10 mL), dried
(MgSO4), and concentrated under reduced pressure. The residue
was purified by column chromatography (EtOAc/light petroleum
ether, 1:6Ǟ1:1, v/v) to give 28 as a single isomer. Yield 1.07 g
was isolated as white needles after crystallization from Et2O/light (1.19 mmol, 92%). Ϫ Rf ϭ 0.86 (toluene/EtOAc/MeOH, 90:25:2.5,
petroleum ether (5:2, v/v). Yield 7.00 g (14.4 mmol, 92%). Ϫ Rf ϭ
v/v/v). Ϫ 1H NMR (300 MHz, HH COSY, CDCl3): δ ϭ 7.36Ϫ7.21
(m, 20 H, H arom), 5.38 (t, 1 H, 3ЈЈ-H, J2ЈЈ,3ЈЈϭ J3ЈЈ,4ЈЈ ϭ 9.7 Hz),
0.81 (EtOAc). Ϫ M.p. 108 °C. Ϫ 1H NMR (CDCl3): δ ϭ 7.30Ϫ7.26
(m, 10 H, H arom), 5.19 (t, 1 H, 3-H, J3,4 ϭ 9.5 Hz), 4.90 (t, 1 H, 5.19 (dd, 1 H, 2Ј-H, J1Ј,2Ј ϭ 1.1 Hz, J2Ј,3Ј ϭ 4.7 Hz), 5.07 (AB, 2
4-H, J4,5 ϭ 9.5 Hz), 4.71 (AB, 2 H, CH2 Bn, J ϭ Ϫ12.1 Hz), 4.52
(m, 3 H, 1-H, CH2 Bn), 3.67Ϫ3.54 (m, 3 H, 5-H, 6a-H, 6b-H), 3.47
H, CH2 Bn, J ϭ Ϫ12.0 Hz), 5.03 (t, 1 H, 4ЈЈ-H, J4ЈЈ,5ЈЈ ϭ 9.5 Hz),
4.96 (m, 2 H, 1Ј-H, 1ЈЈ-H), 4.51Ϫ4.44 (m, 5 H, 2 CH2 Bn, 3Ј-H),
3090
Eur. J. Org. Chem. 2000, 3085Ϫ3092

Synthesis of Photoaffinity Derivatives of Adenophostin A
FULL PAPER
4.32 (m, 1 H, 4Ј-H), 3.86 (ddd, 1 H, 5ЈЈ-H, J4ЈЈ,5ЈЈ ϭ 10.7 Hz,
give compound 8 as the Naϩ salt. Yield 14 mg (Naϩ salt, 14 µmol,
J5ЈЈ,6ЈЈ ϭ 4.5 Hz), 3.75 (m, 1 H, 1a-H), 3.66 (dd, 1 H, 5aЈ-H, J4Ј,5Ј
ϭ
95%). Ϫ 1H NMR (600 MHz, HH-COSY, D2O): δ ϭ 7.76Ϫ7.72
5.8 Hz), 3.56Ϫ3.50 (m, 2 H, 5bЈ-H, 2ЈЈ-H), 3.45 (m, 1 H, 1bЈ-H), (m, 4 H, H arom), 7.69 (t, 1 H, H arom, J ϭ 7.4 Hz), 7.55 (t, 2 H,
3.37 (dd, 1 H, 6aЈЈ-H, J6aЈЈ,6bЈЈ ϭ Ϫ11.0 Hz, J5ЈЈ,6ЈЈ ϭ 2.6 Hz), 3.27 H arom, J ϭ 7.9 Hz), 7.37 (d, 2 H, H arom, J ϭ 8.1 Hz), 5.28 (s,
(dd, 1 H, 6bЈЈ-H, J5ЈЈ,6ЈЈ ϭ 3.9 Hz), 3.19 (m, 2 H, 2-H), 1.93, 1.87,
1 H, 1Ј-H), 5.15 (d, 1 H, 1ЈЈ-H, J1ЈЈ,2ЈЈ ϭ 3.8 Hz), 4.47 (dd, 1 H, 2Ј-
1.82 (s, 3 CH3 Ac), 1.70 (m, 2 H, 3-H). Ϫ 13C{1H} NMR (CDCl3): H, J2Ј,3Ј ϭ 4.5 Hz, J2Ј,P ϭ 7.8 Hz), 4.37 (q, 1 H, 3ЈЈ-H, J ϭ 7.5 Hz),
δ ϭ 170.3, 169.3 (3 CϭO Ac), 156.3 (CϭO Z), 137.9, 137.7, 137.5 4.18 (dd, 1 H, 3Ј-H, J3Ј,4Ј ϭ 7.4 Hz), 4.09 (m, 1 H, 4Ј-H), 3.97 (q,
(3 Cq Bn), 136.6 (Cq Z), 128.8Ϫ127.5 (CH arom), 105.2 (C-1ЈЈ), 1 H, 4ЈЈ-H, J ϭ 8.1 Hz), 3.78 (dd, 1 H, 6aЈЈ-H), 3.68Ϫ3.63Ϫ3.58
96.5 (C-1Ј), 80.1, 76.5, 75.0, 73.8, 71.9, 69.0, 68.8 (C-2Ј, C-3Ј, C-4, (m, 4 H, 5ЈЈ-H, 5bЈ-H, 6bЈЈ-H, 1a-H), 3.28 (dt, 1 H, 1b-H, J1,2
C-2ЈЈ, C-3ЈЈ, C-4ЈЈ, C-5ЈЈ), 73.4, 73.2 (2 CH2 Bn), 70.3, 67.6, 66.5, 6.2 Hz, J1a,b ϭ 10.0 Hz), 3.18 (dt, 2 H, 3-H, J ϭ 6.5 Hz, J ϭ
65.5 (CH2 Bn, C-5Ј, C-6ЈЈ, C-1), 38.2 (C-3), 29.3 (C-2), 20.8, 20.6 13.5 Hz), 3.06 (t, 2 H, 6-H, J ϭ 6.7 Hz), 2.63 (t, 2 H, 7-H, J ϭ
(CH3 Ac). Ϫ C49H57NO15 (899.9): calcd. C 65.39, H 6.38, N 1.56; 7.2 Hz), 1.63 (m, 2 H, 2-H). Ϫ 13C{1H} NMR (D2O): δ ϭ 134.2,
ϭ
found C 65.51, H 6.42, N 1.59. Ϫ ES-MS; m/z: 923 [M ϩ Na]ϩ.
131.6, 131.0, 129.5, 129.3 (CH arom), 106.6 (C-1ЈЈ), 96.4 (C-1Ј),
81.7, 77.9, 75.7, 75.5, 73.0, 72.5, 71.7 (C-2Ј, C-3Ј, C-4Ј, C-2ЈЈ, C-
3ЈЈ, C-4ЈЈ, C-5ЈЈ), 66.1 (C-1), 63.3, 60.9 (C-5Ј, C-6ЈЈ), 37.8, 36.8 (C-
6, C-7), 32.3 (C-3), 28.9 (C-2). Ϫ 31P NMR (D2O): 1.89, 0.61. Ϫ
1-(3-Aminopropyl)-3-O-(α-D-glucopyranosyl)-β- -ribofuranoside
D
2,3Ј,4Ј-Trisphosphate (30) (Et3NH؉ Salt): Triacetate 28 (0.39 g,
0.43 mmol) was dissolved in dry MeOH (10 mL) containing so-
dium methoxide (7 mg, 0.12 mmol) and was stirred for 1.5 h. When
TLC analysis (toluene/EtOAc/MeOH, 90:25:2.5, v/v/v) showed
complete conversion of the starting material into a slower running
product (Rf ϭ 0.59), the solution was neutralized with Dowex Hϩ,
filtered, and concentrated under reduced pressure. Ϫ ES-MS; m/z:
774 [M ϩ H]ϩ, 797 [M ϩ Na]ϩ. Ϫ The triol (0.12 g, 0.15 mmol)
was repeatedly concentrated with CH3CN (2 ϫ 5 mL) and dis-
solved in CH2Cl2 (5 mL). Dibenzyloxy(N,N-diisopropylamino)-
phosphane (0.20 mL, 0.60 mmol) and a solution of 1H-tetrazole
(84 mg, 1.20 mmol) in CH3CN (2 mL) were subsequently added.
The mixture was stirred under nitrogen for 30 min, after which
TLC analysis (toluene/EtOAc, 9:1, v/v) revealed complete conver-
sion into a faster running product (Rf ϭ 0.72). The mixture was
cooled (0 °C) and tBuOOH (0.25 mL, 2.2 mmol) was added. After
1 h, the solution was diluted with EtOAc (20 mL), washed with
H2O, dried (MgSO4) and concentrated. The product was purified
by column chromatography (EtOAc/light petroleum ether, 1:4Ǟ1:0,
v/v) to give 29 as a colorless oil. Yield 0.18 g (0.16 mmol, 77%, 2
steps). Ϫ 31P NMR (CDCl3): δ ϭ 0.86, 1.39, 2.00. Ϫ Compound
29 (0.14 g, 89 µmol) and NaOAc (87 mg, 0.11 mmol) were dissolved
in a mixture of dioxane/2-propanol/H2O (15 mL, 4:2:1, v/v/v) and
the solution was degassed. Palladium on carbon (10%, 75 mg) was
added and the reaction mixture was stirred under H2. After 16 h,
the catalyst was removed by filtration through glass fiber (GF/2A,
Whatman). The filtrate was concentrated under reduced pressure
and the product was purified by gel filtration on a Fractogel HW-
40 column as described for 15, to give aminopropyl derivative 30.
Yield 0.11 g (Et3NHϩ salt, 73 µmol, 82%). An analytical sample
was prepared by conversion of the trisphosphate into the Naϩ salt
as described for compound 15. Ϫ 1H NMR (300 MHz, HH-COSY,
D2O): δ ϭ 5.22 (s, 1 H, 1Ј-H), 5.17 (d, 1 H, 1ЈЈ-H, J1ЈЈ,2ЈЈ ϭ 3.8 Hz),
4.55 (dd, 1 H, 2Ј-H, J2Ј,3Ј ϭ 4.2 Hz, J2Ј,P ϭ 8.3 Hz), 4.32 (q, 1 H,
3ЈЈ-H, J3ЈЈ,4ЈЈϭ J3ЈЈ,4ЈЈϭ J3ЈЈ,P ϭ 9.5 Hz), 4.29Ϫ4.16 (m, 2 H, 3Ј-H,
4Ј-H), 3.93Ϫ3.87 (m, 2 H, 5ЈЈ-H, 1a-H), 3.82 (dd, 1 H, 5aЈ-H,
J4Ј,5aЈ ϭ 3.0 Hz, J5aЈ,bЈ ϭ Ϫ12.4 Hz), 3.75Ϫ3.60 (m, 7 H, 2ЈЈ-H, 4ЈЈ-
H, 1b-H, 5bЈ-H, 6ЈЈ-H), 3.11 (dt, 2 H, 3-H, J ϭ 1.9 Hz, J ϭ
6.7 Hz), 1.95 (m, 2 H, 2-H). Ϫ 13C{1H} NMR (D2O): δ ϭ 106.9
(C-1ЈЈ), 97.8 (C-1Ј), 82.0, 77.8, 75.5, 74.0, 72.6, 71.7 (C-2Ј, C-3Ј, C-
4Ј, C-2ЈЈ, C-3ЈЈ, C-4ЈЈ, C-5ЈЈ), 66.9 (C-1), 62.8, 60.9 (C-5Ј, C-6ЈЈ),
38.7 (C-3), 27.2 (C-2). Ϫ HR-MS: C14H30NO19P3 [M Ϫ H]Ϫ calcd.
608.0546; found 608.0541. Ϫ ES-MS; m/z: 608 [M Ϫ H]Ϫ.
HR-MS: C30H42NO21P3 [M
Ϫ
H]Ϫ calcd. 844.1383; found
844.1390. Ϫ ES-MS; m/z: 846 [M ϩ H]ϩ, 905 [M ϩ Na]ϩ.
Acknowledgments
These investigations were supported by the Netherlands Founda-
tion for Chemical Research (SON) with financial aid from the
Netherlands Organization for Scientific Research (NWO). We
thank Hans van den Elst and Nico Meeuwenoord for recording
the mass spectra and assistance in purifying the target compounds.
We thank Cees Erkelens and Fons Lefeber for recording NMR
spectra.
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