Synthesis of nicotinamide adenine dinucleotide (NAD) analogues with a sugar modified nicotinamide moiety

Article abstract of DOI:10.1002/hlca.200790127

The synthesis of nicotinamide adenine dinucleotide (NAD) analogues in which the ribose unit of the nicotinamide moiety is replaced by a hexitol, altritol, and cyclohexenyl sugar mimic is described.

Full text of DOI:10.1002/hlca.200790127

1266
Helvetica Chimica Acta – Vol. 90 (2007)
Synthesis of Nicotinamide Adenine Dinucleotide (NAD) Analogues with a
Sugar Modified Nicotinamide Moiety
by Natasha Goulioukinaa), Johny Wehbea), Damien Marchanda), Roger Bussona),
Eveline Lescriniera), Dieter Heindlb), and Piet Herdewijn*a)
a) Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Minderbroedersstraat 10,
B-3000 Leuven
(phone: þ 3216337387; fax: þ 3216337340; e-mail: Piet.Herdewijn@rega.kuleuven.be)
b) Roche Diagnostics GmbH, Nonnenwald 2, D-82372 Penzberg
The synthesis of nicotinamide adenine dinucleotide (NAD) analogues in which the ribose unit of the
nicotinamide moiety is replaced by a hexitol, altritol, and cyclohexenyl sugar mimic is described.
Introduction. – Nicotinamide adenine dinucleotide (NADþ) is a cofactor in more
than 400 biological redox reactions and requires the presence of enzymes such as
dehydrogenases [1]. As the most important role in these reactions is played by the
nicotinamide moiety, this fragment of the molecule was intensively studied. NADþ,
however, is a labile molecule, and the nicotinamide glycosyl bond is easily cleaved, both
chemically and enzymatically (by ADP ribosylating enzymes). Therefore, it would be
desirable to have a ꢀsugar-modifiedꢁ NADþ analogue available with a more stable
nicotinamide glycoside.
In solution, NADþ occurs as a mixture of folded and unfolded forms with the
aromate rings in close proximity in the folded forms [2]. In solid state (X-ray) and when
bound to enzymes, NADþ adopts an extended conformation [3][4]. The ribose ring of
the nicotinamide moiety adapt the exo-C(3’) conformation as well in solution (85% S
conformation) as in solid phase [2]. Interestingly, the S conformation is also the most
abundant (90% S) for the ribose ring when the nicotinamide moiety is reduced (i.e., in
NADPHþ) [5].
To find a sugar surrogate that can replace the ribose moiety of the nicotinamide part
of NADþ (so that a more stable compound is obtained while keeping the catalytic
activity), we should address questions about the importance of sugar conformation and
flexibility of NADþ during the catalytic process, and the relative importance of the
presence of an anomeric center for catalysis. Such questions can be approached by
making sugar-modified analogues of NADþ available.
In this area of research, our interest is mainly focused on the study of catalytic
activity of dehydrogenases. All protein structures containing NADþ (810) were
retrieved from the Protein Data Bank [6]. Analysis of these complexes revealed that
the ribose part of the nicotinamide moiety in NADþ predominantly adopts a
conformation in the southern half of the pseudorotation wheel (89% ¼ S-type). In
all structures of NADþ co-crystallized with malate dehydrogenase (e.g., 4MDH),
ꢂ 2007 Verlag Helvetica Chimica Acta AG, Zürich

Helvetica Chimica Acta – Vol. 90 (2007)
1267
glucose-6-phosphate dehydrogenase (e.g., 1 H9A) and histidynol dehydrogenase (e.g.,
1KAE), the studied ribose is in an S-type conformation (Fig.).
Figure. The ribose part of the nicotinamide moiety in NADþ predominantly adopts an S-type
conformation in dehydrogenases
It is clear that the chemical and enzymatic lability of NADþ is due to the presence of
an anomeric center and thus can be overcome by synthesizing carbocyclic or hexitol-
like analogues. The importance of having two free OH groups on the ꢀsugarꢁ ring can be
approached by synthesizing altritol analogues. However, these six-membered ana-
logues are rigid mimics of a nucleoside in the N-type conformation, while, in the crystal
structures with dehydrogenases, the ribose part of the nicotinamide moiety adopts an S-
type conformation. Therefore, we also synthesized the cyclohexenyl analogue as a
congener with more conformational diversity.
Results. – The NADþ analogue with a hexitol moiety was synthesized from 1,5-
anhydro-4,6-O-benzylidene-3-deoxy-d-glucitol (Scheme 1) [7][8]. The N3 group in the
2-position (i.e., 3) was introduced by mesylation of 1 (!2), followed by nucleophilic
substitution with NaN3 with inversion of configuration. The benzylidene protecting
group was removed with acid (!4), and the primary OH group was phosphorylated
using POCl3 in trimethyl phosphate (! 5). Then the N3 group at C(2) was reduced to an
amino group (!6), which was used as nucleophile in the Zincke reaction. The
nicotinamide nucleotide 7 was obtained by reaction of 6 with 1-(2,4-dinitrophenyl)-3-
carbamoylpyridinium chloride (NDC) (or tetrafluoroborate) and purification over 2-

1268
Helvetica Chimica Acta – Vol. 90 (2007)
Scheme 1
a) MsCl, 4-(dimethylamino)pyridine (DMAP), pyridine, r.t., 14 h. b) NaN3, DMF, 808, 31 h. c) 80%
AcOH, 958, 1 h. d) 1. POCl3, (MeO)3PO, 08, 3 h; 2. H2O, NH4OH. e) 1. H2 (30 psi), PtO2 · H2O, H2O/
MeOH, r.t., 2 h; 2. Dowex 50WX4-400 (Naþ). f) H2O/MeOH, Et3NHþHCO3ꢀ, 408, 4 d. g) 1. AgNO3,
DMF, pyridine, r.t., 40 h; 2. LiOH.
(diethylamino)ethyl (DEAE)-cellulose. The hexitol nicotinamide adenine dinucleo-
tide (hNADþ) 8 was obtained from the nucleotide 7 and adenosine 5’-(phosphoric
dibutylphosphinothioic anhydride) using AgNO3 as activating agent.
The synthesis of the altritol nicotinamide adenine dinucleotide (aNADþ; 15)
started from the epoxide of 4,6-benzylidene-1,5-anhydro-d-allitol 9 (Scheme 2) [9].
The epoxide was opened using NaN3 in methoxyethanol (!10), and the benzylidene
protecting group was removed using AcOH (!11). After phosphorylation of the
primary OH group (!12), the N3 group was reduced (!13). The amino group was
then reacted with NDC to obtain the nicotinamide nucleotide 14. Reaction of 14 with

Helvetica Chimica Acta – Vol. 90 (2007)
1269
the activated adenosine monophosphate (AMP) yielded altritol nicotinamide adenine
dinucleotide (aNADþ; 15).
Scheme 2
a) NaN3, NH4Cl, MeOCH2OH/H2O, 1008, 18 h. b) 80% AcOH, 958, 2 h. c) 1. POCl3, (MeO)3PO, 08, 3 h;
2. H2O, Et3N. d) 1. H2 (30 psi), PtO2 · H2O, H2O/MeOH, r.t., 85 h; 2. Dowex 50X8-200 (Naþ). e) 1-(2,4-
dinitrophenyl)-3-carbamoylpyridinium chloride (NDC), H2O/MeOH, Et3NHþHCO3ꢀ, 408, 6 d. f) 1.
AgNO3, DMF, pyridine, r.t., 65 h; 2. LiOH.
The synthesis of the cyclohexenyl congener 22 was somewhat more complex. It
started from the chiral cyclohexenyl alcohol precursor 16 which was described before
[10][11] (Scheme 3). The OH function was converted into an amino group with
inversion of the configuration at the C-atom via the phtalimides 17 and 18 as
intermediates. The primary alcohol 18 was first phosphorylated (!19) before the
phtalimide group was removed using NH2NH2 (!20). Condensation reaction with
NDC (!21) and with activated AMP was accomplished in a similar way as described
before to obtain 22. The obtained compounds 8, 15, and 22 are currently under

1270
Helvetica Chimica Acta – Vol. 90 (2007)
Scheme 3
a) Diisopropyl azodicarboxylate (DIAD), Ph3P, phtalimide, THF, 1 h. b) 80% AcOH, 958, 1 h. c) 1.
POCl3, (MeO)3PO, 08, 5 h; 2. triethylammonium bicarbonate (TEAB), 30 min. d) 1. NH2NH2, EtOH,
958, 16 h; 2. Dowex 400X. e) 1. iPr2EtN, MeOH, 558 for 4 h, and 16 h at r.t.; 2. TEAB; 3. Dowex 50X8-
200. f) 1. AgNO3, DMF, pyridine, r.t., 16 h; 2. H2S; 3. Sephadex A25.
evaluation as NADþ analogues in order to study the structural requirements for activity
as cofactors for dehydrogenases.
Experimental Part
General. All solvents used for reactions are anal. grade or freshly distilled. Anh. pyridine was
obtained by reflux on KOH and distilled. DMF was stored over Linde 4-Š molecular sieves, followed by
distillation under reduced pressure. Precoated aluminum sheets (Fluka silica gel/TLC cards, 254 nm)
were used for TLC, and spots were visualized with UV and anisaldehyde – sulfuric acid spray. Column
chromatography (CC) was performed on silica gel (0.060 – 0.200 nm). UV: UVIKON 940 apparatus; lmax

Helvetica Chimica Acta – Vol. 90 (2007)
1271
in nm. NMR Spectra: 200- or 500-MHz Varian apparatus with TMS as internal standard for 1H-NMR and
(D6)DMSO (39.6 ppm) for 13C-NMR. 31P-NMR Spectra: were obtained using 85% H3PO4 as external
standard. Exact mass measurements were performed on a quadruple time-of-flight mass spectrometer
(Q-TOF-2, Micromass), equipped with a standard electrospray-ionization (ESI) interface; in m/z.
1,5-Anhydro-4,6-O-benzylidene-3-deoxy-2-O-(methylsulfonyl)-d-ribo-hexitol (2). To a soln. of 1,5-
anhydro-4,6-O-benzylidene-3-deoxy-d-glucitol [8] (1; 5.000 g, 21.16 mmol) and a cat. amount of 4-
(dimethylamino)pyridine (DMAP) in 40 ml of pyridine at 08, MsCl (3 ml, 38 mmol) was added. The
mixture was stirred at r.t. for 14 h. After evaporation and co-evaporation with toluene, the residue was
extracted three times with warm CHCl3, and the combined extracts were filtered over a short path of
silica gel. Evaporation of the solvent gave the crude product, which was washed with Et2O and
precipitated from MeOH to afford 2 (5.103 g, 77%). Oil. Rf (hexane/AcOEt 1:2) 0.8, Rf (hexane/AcOEt
2 :1) 0.5. 1H-NMR (200 MHz, CDCl3): 1.92 (ddd, 2J(3ax,3eq) ¼ 12, 3J(3ax,2) ¼ 11.5, 3J(3ax,4) ¼ 11,
HaxꢀC(3)); 2.68 (ddd, 2J(3eq,3ax) ¼ 12, 3J(3eq,2) ¼ 5.5, 3J(3eq,4) ¼ 4, HeqꢀC(3)); 3.06 (s, Me); 3.32
(ddd, 3J(5,6ax) ¼ 10, 3J(5,4) ¼ 8, 3J(5,6eq) ¼ 5, HꢀC(5)); 3.41 (dd, 2J(1ax,1eq) ¼ 3J(1ax,2) ¼ 11, HaxꢀC(1));
3.59 (ddd, 3J(4,3ax) ¼ 11, 3J(4,5) ¼ 8, 3J(4,3eq) ¼ 4, HꢀC(4)); 3.68 (dd, 2J(6ax ,6eq) ¼ 3J(6ax,5) ¼ 10,
2
3
4
2
HaxꢀC(6)); 4.21 (ddd, J(1eq,1ax) ¼ 11, J(1eq,2) ¼ 5.5, J(1eq,3eq) ¼ 2, HeqꢀC(1)); 4.33 (dd, J(6eq,6ax) ¼
10, 3J(6eq,5) ¼ 5, HeqꢀC(6)); 4.72 – 4.90 (m, HꢀC(2)); 5.53 (s, PhCH), 7.33 – 7.51 (m, 5 arom. H).
13C-NMR: see Table 1.
Table 1. 13C-NMR Chemical Shifts [ppm] andAssignments for Compounds 1 – 7. J in Hz.
1
2
3
4
5
6
7
(CDCl3) (CDCl3) (CDCl3)
(CDCl3) (D2O)
(D2O)
(D2O)a)
C(1)
C(2)
C(3)
C(4)
C(5) (3J(C,P))
C(6) (3J(C,P))
Me
72.4
65.6
38.4
73.1
76.4
69.2
69.4
72.3
35.8
73.2
75.7
69.3
57.4
32.8
68.8
57.2
35.8
70.7
59.4
36.5
63.8
69.5
50.0
35.0
62.2
69.2 or 69.0
69.0 or 69.2
37.7
62.2
83.2 (6.1)
65.3 (3.1)
74.0 or 74.2 63.0
74.2 or 74.0 81.7
68.9
83.3 (7.6) 83.7 (6.1)
66.4 (4.6) 65.7 (4.6)
68.9
38.6
62.8
PhC
Cipso
Co
Cm
101.7
137.4
126.2
128.4
129.2
101.8
137.1
126.1
128.4
129.3
102.0
137.4
126.1
128.4
129.2
Cp
C(2), C(4) (py)
C(3) (py)
C(5) (py)
C(6) (py)
C¼O
146.3, 146.6
136.3
130.9
148.2
168.2
a) Position numbers of the glucitol part are primed in the text.
1,5-Anhydro-2-azido-4,6-O-benzylidene-2,3-dideoxy-d-arabino-hexitol (3). To a soln. of 2 (5.000 g,
15.91 mmol) in 120 ml of DMF, NaN3 (2.1 g, 32 mmol) was added, and the mixture was heated at 808
under N2 with stirring. After 31 h, the mixture was cooled to r.t., and the solvent was evaporated in vacuo.
The residue was triturated with warm CHCl3, and the extract was filtered over a short path of silica gel.
The solvent was removed, and the resultant solid was precipitated from hexane to yield 3 (4.073 g, 98%).
White powder. Rf (hexane/AcOEt 2 :1) 0.9. 1H-NMR (200 MHz, CDCl3): 1.91 (ddd, 2J(3ax,3eq) ¼ 13,
3J(3ax,4) ¼ 12, 3J(3ax,2) ¼ 4, HaxꢀC(3)); 2.26 – 2.40 (dm, 2J(3eq,3ax) ¼ 13, HeqꢀC(3)); 3.39 (dd, 3J(5,6ax) ¼
10, 3J(5,6eq) ¼ 5, HꢀC(5)); 3.69 (dd, 2J(1ax ,1eq) ¼ 12, 3J(1ax ,2) ¼ 2, HaxꢀC(1)); 3.77 (dd,

1272
Helvetica Chimica Acta – Vol. 90 (2007)
2J(6ax ,6eq) ¼ 3J(6ax,5) ¼ 10, HaxꢀC(6)); 3.55 – 4.06 (m, HeqꢀC(1), HꢀC(2), HꢀC(4)); 4.28 (dd,
2J(6eq,6ax) ¼ 10, 3J(6eq,5) ¼ 5, HeqꢀC(6)); 5.60 (s, PhCH), 7.34 – 7.52 (m, 5 arom. H). 13C-NMR: see
Table 1.
1,5-Anhydro-2-azido-2,3-dideoxy-d-arabino-hexitol (4). A soln. of 3 (4.000 g, 15,31 mmol) in 180 ml
of 80% AcOH was heated at 958 for 1 h. After evaporation, and co-evaporation with H2O and then with
toluene, the residue was purified by silica gel CC (0 – 10% AcOEt in hexane) and dried in vacuo over
1
P4O10 to afford 4 (2.174 g, 82%). Oil. Rf (hexane/AcOEt 1:2) 0.25. H-NMR (200 MHz, CDCl3): 1.72
(ddd, 2J(3ax ,3eq) ¼ 14, 3J(3ax ,4) ¼ 11, 3J(3ax,2) ¼ 4, HaxꢀC(3)); 2.25 – 2.40 (dm, 2J(3eq,3ax) ¼ 14,
HeqꢀC(3)); 2.67 (br. s, OH); 3.05 (br. s, OH); 3.12 – 3.28 (dm, 3J(5,4) ¼ 9, HꢀC(5)); 3.59 (dd,
2J(1ax,1eq) ¼ 12, 3J(1ax,2) ¼ 2, HaxꢀC(1)); 3.74 – 4.04 (m, HeqꢀC(1), HꢀC(2), HꢀC(4), HꢀC(6a),
HꢀC(6b)). 13C-NMR: see Table 1.
1,5-Anhydro-2-azido-2,3-dideoxy-6-O-phosphono-d-arabino-hexitol Diammonium Salt (5). The
azide 4 (1.004 g, 5.80 mmol) was dissolved under N2 in 6 ml of (MeO)3PO (freshly distilled in vacuo
over BaO), cooled in an ice bath, and then triturated with 1.7 ml of a 1:1 (v/v) mixture of POCl3 (distilled
immediately prior to use) and (MeO)3PO. The POCl3 soln. was prepared at 08 under N2, stirred for
15 min, and added to the soln. of 4 in a single portion. After 3 h of stirring at 08, the reaction was
quenched by addition of 6 ml of ice water and 9 ml of cold Et3N. The resulting mixture was evaporated to
dryness in vacuo, the residue was washed with (i-Pr)2O and purified by silica-gel CC (0 – 35% NH4OH
(20% soln. in H2O) in i-PrOH) to give 5 (0.677 g, 43%). Pale yellow oil. Mixed fractions were combined
and repurified to give an additional portion of 5 (0.159 g, 10%). The total yield was 53%. Rf (i-PrOH/
25% NH4OHaq/H2O 6 :3 :1) 0.35. 1H-NMR (200 MHz, D2O): 1.80 (ddd, 2J(3ax,3eq) ¼ 13.5, 3J(3ax,4) ¼ 11,
3J(3ax,2) ¼ 3.5, HaxꢀC(3)); 2.26 – 2.42 (dm, 2J(3eq,3ax) ¼ 13.5, HeqꢀC(3)); 3.43 (ddd, 3J(5,4) ¼ 8.5,
3
2
3
3J(5,6a) ¼ 5.5, J(5,6b) ¼ 2, HꢀC(5)); 3.68 (dd, J(1ax,1eq) ¼ 12.5, J(1ax,2) ¼ 1.5, HaxꢀC(1)); 3.77 – 4.24
(m, HeqꢀC(1), HꢀC(2), HꢀC(4), HaꢀC(6), HbꢀC(6)). 13C-NMR: see Table 1.
2-Amino-1,5-anhydro-2,3-dideoxy-6-O-phosphono-d-arabino-hexitol Sodium Salt (6). The azide 5
(0.677 g, 2.51 mmol) was dissolved in 75 ml of H2O and 25 ml of MeOH. Adams catalyst (PtO2 · H2O,
0.068 g, 0.28 mmol) was added, and the mixture was shaken at r.t. for 2 h in a Parr hydrogenation
apparatus (30 psi). After removal of the catalyst, the filtrate was concentrated to dryness, dissolved in
H2O, and applied to a column of Dowex 50WX4-400 (Naþ) ion-exchange resin with H2O as the eluent.
Evaporation and drying the residue in a vacuum dessicator over P4O10 provided 6 (0.666 g, 98%). Pale
yellow glass. Rf (i-PrOH/25% NH4OHaq/H2O 6 :3 :1) 0.2. 1H-NMR (500 MHz, D2O): 1.93 (ddd,
2J(3ax,3eq) ¼ 14.4, 3J(3ax,4) ¼ 12.1, 3J(3ax,2) ¼ 4.1, HaxꢀC(3)); 2.34 (dddd, 2J(3eq,3ax) ¼ 14.4, 3J(3eq,4) ¼
5.1, 4J(3eq,1eq) ¼ 3J(3eq,2) ¼ 2.6, HeqꢀC(3)); 3.41 (dddd, 3J(5,4) ¼ 9.6, 3J(5,6a) ¼ 4.1, 3J(5,6b) ¼ 2.2,
4J(5,P) ¼ 0.5, HꢀC(5)); 3.73 (dddd, 3J(2,3ax) ¼ 4.1, 3J(2,3eq) ¼ 2.6, 3J(2,1ax) ¼ 2.0, 3J(2,1eq) ¼ 1.7,
HꢀC(2)); 3.78 (dd, 2J(1ax ,1eq) ¼ 13.2, 3J(1ax ,2) ¼ 2.0, HaxꢀC(1)); 3.97 (ddd, 2J(1eq,1ax) ¼ 13.2,
4J(1eq ,3eq) ¼ 2.6, 3J(1eq ,2) ¼ 1.7, HeqꢀC(1)); 3.98 (ddd, 3J(4,3ax) ¼ 12.1, 3J(4,5) ¼ 9.6, 3J(4,3eq) ¼ 5.1,
HꢀC(4)); 4.01 (ddd, 2J(6b,6a) ¼ 11.8, 3J(6b,P) ¼ 5.4, 3J(6b,5) ¼ 2.2, HbꢀC(6)); 4.05 (ddd, 2J(6a,6b) ¼
3
3
11.8, J(6a,P) ¼ 6.9, J(6a,5) ¼ 4.1, HaꢀC(6)). 13C-NMR: see Table 1. 31P-NMR (202 MHz, D2O): 3.51.
Anal. calc. for C6H13O6NNa (218.162): C 33.03, H 6.01, N 6.42; found: C 33.20, H 5.79, N 6.22.
1-(2,4-Dinitrophenyl)-3-carbamoylpyridinium tetrafluoroborate was prepared according to the
combination of protocols reported by Slama et al. [12] and Walt et al. [13]. Nicotinamide (5.0 g,
40.9 mmol) and 1-chloro-2,4-dinitrobenzene (20.0 g, 98.7 mmol) were placed in 1-l round-bottom flask
equipped with a magnetic stirring bar. The mixture was melted and heated to 1058 with stirring under an
inert atmosphere for 1 h to form a dark red solid. After cooling to around 30 – 408, the solid was dissolved
in 250 ml of reagent-grade MeOH, followed by addition of 190 ml of Et2O. The liquid was decanted from
the oily precipitate, and the procedure was repeated two times. The residue was dissolved in 125 ml of
H2O, extracted with CHCl3 (8 ꢁ 20 ml) and treated with activated charcoal (1 g).
After filtration through Celite, the soln. was concentrated in vacuo at 308 to 30 ml and mixed with a
soln. of NaBF4 (9.0 g, 82.0 mmol) in 20 ml of H2O. An oily material was formed, which was redissolved in
350 ml of H2O by heating on a steam bath and cooled to give yellow crystals. After two recrystallizations
from MeOH, the fine pale yellow crystals were collected, washed with Et2O, and dried in vacuo. M.p. 1668
([12]: 167 – 1698). Rf (BuOH/AcOH/H2O 5 :2 :3) 0.6. The yield of 1-(2,4-dinitrophenyl)-3-carbamoyl-
pyridinium tetrafluoroborate was 8.8 g (57%).

Helvetica Chimica Acta – Vol. 90 (2007)
1273
For the preparation of 1-(2,4-dinitrophenyl)-3-carbamoylpyridinium chloride (NDC), the soln.
obtained after filtration over charcoal was concentrated to a viscous oil, which was mixed while still warm
with 30 ml of BuOH. The flask was stopped with rubber septum, flushed with N2 and kept at 58 for 2 d.
The pale yellow crystals of NDC were isolated by removal of the mother liquor using a syringe, followed
by washing with dry Et2O. Traces of solvent were removed under vacuum. This procedure was required in
order to minimize exposure of NDC to moisture.
1,5-Anhydro-2-(3-carbamoylpyridinium)-2,3-dideoxy-6-O-phosphono-d-glucitol (7). To a stirred
soln. of 6 (0.100 g, 0.40 mmol) in 6 ml of H2O and 4 ml of MeOH a soln. of NDC (0.148 g, 0.46 mmol) in
1.5 ml of H2O was added dropwise over several hours. After the addition was complete, the reaction was
stirred for 4 d at 408, 0.3 ml of 0.5m aq. triethylammonium bicarbonate (TEAB) being added over this
period. The reaction was monitored by TLC. When quant. conversion of starting amine 6 was achieved,
the mixture was triturated with TEAB soln. and cooled to 08. The colored precipitate was separated by
centrifugation. The soln. was concentrated in vacuo and passed through a column (21 mm ꢁ 14 cm)
packed with 2-(diethylamino)ethyl (DEAE) Whatman DE-52 cellulose for crude purification. The
column was washed with 0.01m TEAB. Pure nucleotide 7 was isolated by HPLC on DEAE Sephadex A-
25 cellulose (column dimensions 18 mm ꢁ 24 cm), pre-equilibrated with 0.01m TEAB, eluting with
TEAB soln. (0.01 ! 0.5m). The desired fractions were collected and concentrated to dryness. TEAB was
removed by repeated co-evaporation with H2O, and the residue was dried in vacuo over P4O10 to yield 7
1
(0.083 g, 59%). Yellow glass. Rf (i-PrOH/25% NH4OHaq/H2O 6 :4 :1) 0.15. H-NMR (200 MHz, D2O):
2
2
2.40 (ddd, J(3ax,3eq) ¼ 15, 3J(3’ ,4’) ¼ 12, 3J(3a’x,2’) ¼ 5, HaxꢀC(3’)); 2.58 – 2.73 (dm, J(3eq,3ax,) ¼ 15,
’
’
’
’
ax
3
3
3
3
HeqꢀC(3’)); 3.59 – 3.72 (dm, J(5’,4’) ¼ 9, HꢀC(5’)); 3.95 (ddd, J(4’,3’ ) ¼ 12, J(4’,5’) ¼ 9, J(4’,3’ ) ¼ 5,
ax
eq
HꢀC(4’)); 4.06 – 4.26 (m, CH2(6’)); 4.26 (dd, J(1ax,1eq) ¼ 14, 3J(1a’x,2’) ¼ 3, HaxꢀC(1’)); 4.63 (br. d,
2
’
’
J(1eq,1ax) ¼ 14, HeqꢀC(1’)); 5.26 (m, HꢀC(2’)); 8.27 (dd, 3J(5,4) ¼ 8, 3J(5,6) ¼ 6, HꢀC(5)); 8.94 (d,
2
’
’
3J(4,5) ¼ 8, HꢀC(4)); 9.39 (d, J(6,5) ¼ 6, HꢀC(6)); 9.49 (s, HꢀC(2)). 13C-NMR: see Table 1. ESI-MS
3
(pos.): 333.1 (Mþ). ESI-MS (neg.): 331.4 ([M ꢀ 2 H]ꢀ).
Adenosine 5’-(Phosphoric dibutylphosphinothioic anhydride) was synthesized by the reaction of
adenosine monophosphate with dibutylphosphinothioyl bromide and isolated as a white solid (90%
yield) according to the procedure of Slama et al. [14]. Rf (i-PrOH/25% NH4OHaq/H2O 6 :3 :1) 0.9. For
potassium salt: 1H-NMR (500 MHz, D2O): 0.76 – 0.80 (m, 2 Me); 1.24 – 1.30 (m, 2 CH2 (Bu)); 1.43 – 1.48
(m, 2 CH2 (Bu)); 2.00 – 2.07 (m, 2 CH2 (Bu)); 4.15 – 4.19 (m, 1 H); 4.34 – 4.39 (m, 1 H); 4.54 (dd, J ¼ 5.1,
3.7, 1 H); 4.80 – 4.85 (m, 1 H); 6.12 (d, J ¼ 5.9, HꢀC(1’)); 8.24 (s, HꢀC(2)); 8.46 (s, HꢀC(8)). 13C-NMR
1
(125 MHz, D2O): 12.9 (Me); 12.9 (Me); 23.0 (Me CH2); 24.2 (EtCH2); 33.8 (d, J(C,P) ¼ 68.4, CꢀP);
33.8 (d, 1J(C,P) ¼ 68.4, CꢀP); 65.7 (d, 2J(C,P) ¼ 5.8, C(5’)); 70.7 (C(3’)); 74.1 (C(2’)); 83.9 (d, 3J(C,P) ¼
9.8, C(4’)); 87.1 (C(1’)); 118.8 (C(5)); 140.1 (C(8)); 149.4 (C(4)); 155.9 (C(2)); 160.4 (C(6)). 31P-NMR
(202 MHz, D2O): ꢀ 9.2 (d, J(P, P ) ¼ 34.2, P¼O); 103.9 (d, 2J(P, P ) ¼ 34.2, P¼S).
2
Dibutylphosphinothioyl bromide was prepared by bromination of tetrabutyldiphosphine disulfide
according to the procedure of Furusawa et al. [15] (see also [14]). The product was isolated as a pale
yellow liquid. B.p. 968/0.1 mm Hg [15][16].
Tetrabutyldiphosphine disulfide was prepared in 42% yield by the reaction of PSCl3 with BuMgBr
according to the procedure of Furusawa et al. [15] with slight modification: 2.5m soln. of BuMgBr was
used.
Hexitol Nicotinamide Adenine Dinucleotide Lithium Salt 8. Nucleotide 7 (0.067 g, 0.2 mmol) and
adenosine 5’-(phosphoric dibutylphosphinothioic anhydride) (0.211 g, 0.4 mmol) were dissolved in 1 ml
of dry, amine free DMF and 3 ml of pyridine (distilled over BaO immediately prior to use). The solvents
were evaporated in vacuo to remove traces of moisture, and the procedure was repeated three times. The
final residue was dissolved in 2.4 ml of DMFand 3 ml of pyridine. The flask was wrapped in aluminum foil
for protection from light, and AgNO3 (0.274 g., 1.61 mmol) was added in one portion. The flask was
immediately capped with a septum, charged with N2, and the mixture was stirred at r.t. After 40 h, 15 ml
of H2O was added, and H2S was bubbled into the mixture. The Ag2S precipitate was removed by filtration
through Celite. The filtrate was extracted by CHCl3 (3 ꢁ 5 ml). Combined org. extracts were washed with
15 ml of H2O. The combined aq. extracts were concentrated in vacuo at r.t. The product 8 was isolated by
HPLC in three steps. First on DEAE Sephadex A-25 cellulose (column dimensions 18 mm ꢁ 24 cm, pre-
equilibrated with 0.01m TEAB, eluting with TEAB soln. (0.01 ! 0.5m). The desired fractions were

1274
Helvetica Chimica Acta – Vol. 90 (2007)
collected and evaporated in vacuo at r.t. TEAB was removed by repeated co-evaporation with H2O. The
second column with DEAE Whatman DE-52 cellulose (18 mm ꢁ 25 cm) was pre-equilibrated with H2O,
eluting with aq. HCOOH (0 ! 0.3m). The desired fractions were collected and lyophilized twice. The
residue was dissolved in H2O. The soln. was adjusted to pH 6 with 0.1m LiOH and applied to a column
with Sephadex LH-20 cellulose (18 mm ꢁ 31 cm) with H2O as eluent. Two lyophilizations yielded 8
(0.026 g, 19%). Fine white powder. Rf (i-PrOH/25% NH4OHaq/H2O 6 :4 :1) 0.5. UV (H2O): 259.
1
2
3
3
’
’
H-NMR (500 MHz, D2O): 2.37 (ddd, J(3ax,3eq) ¼ 15.0, J(3a’x,4’) ¼ 11.5, J(3a’x,2’) ¼ 5.2, HaxꢀC(N3’));
2.62 (ddd, J(3eq,3ax) ¼ 15.0, 2J(3’ ,4’) ¼ 4.8, 3J(3’ ,2’) ¼ 2.8, J(3eq,1eq) ¼ 2.8, HeqꢀC(N3’)); 3.66 (dt,
2
4
’
’
’
’
eq
eq
3J(5’,4’) ¼ 9.2, 3J(5’,6’) ¼ 3.1, HꢀC(N5’)); 3.95 (ddd, 3J(4’,3’ ) ¼ 11.5, 3J(4’,5’) ¼ 9.2, 3J(4’,3’ ) ¼ 4.8,
ax
eq
HꢀC(N4’)); 4.22 (dd, J(1ax,1eq) ¼ 14.4, 3J(1’ ,2’) ¼ 3.1, HaxꢀC(N1’)); 4.24 (dd, 2J(5’a,5’b) ¼ 12.1,
2
’
’
ax
3J(5’a,4’) ¼ 3.1, HaꢀC(A5’)); 4.24 – 4.30 (m, CH2(N6’)); 4.28 (dd, 2J(5’b,5’a) ¼ 12.1, 3J(5’b,4’) ¼ 3.1,
HbꢀC(A5’)); 4.40 (ddd, 3J(4’,3’) ¼ 3J(4’,5’a) ¼ 3J(4’,5’b) ¼ 3.1, 4J(4’,P) ¼ 1.9, HꢀC(A4’)); 4.53 (dd,
3J(3’,2’) ¼ 4.7, 3J(3’,4’) ¼ 3.1, HꢀC(A3’)); 4.57 (ddd, J(1eq,1ax) ¼ 14.4, J(1eq,3eq) ¼ 2.8, 3J(1’eq,2’) ¼ 2.6,
2
4
’
’
’
’
3
3
3
3
HeqꢀC(N1’)); 4.74 (dd, J(2’,1’) ¼ 5.4, J(2’,3’) ¼ 4.7, HꢀC(A2’)); 5.20 (dddd, J(2’,3’ax) ¼ 5.1, J(2’,1’ ) ¼
ax
3
3
3
3
3.1, J(2’,3’ ) ¼ 2.8, J(2’,1e’q) ¼ 2.6, HꢀC(N2’)); 6.14 (d, J(1’,2’) ¼ 5.4, HꢀC(A1’)); 8.25 (dd, J(5,4) ¼
eq
3
3
4
8.3, J(5,6) ¼ 6.1, HꢀC(N5)); 8.40 (s, HꢀC(A2)); 8.60 (s, HꢀC(A8)); 8.89 (dd, J(4,5) ¼ 8.3, J(4,2) ¼
1.5, HꢀC(N4)); 9.37 (d, J(6,5) ¼ 6.1, J(6,2) ¼ 1.5, HꢀC(N6)); 9.45 (t, J(2,4) ¼ 4J(2,6) ¼ 1.5, HꢀC(N-
2)). 13C-NMR (125 MHz, D2O): 38.4 (NC(3’)); 62.6 (NC(4’)); 67.5 (dd, 2J(6’,P) ¼ 4.9, 4J(6’,P) ¼ 2.8 ,
NC(6’)); 67.9 (dd, 2J(5’,P) ¼ 3.7, 4J(5’,P) ¼ 2.8, AC(5’); 69.6 (NC(2’)); 69.8 (NC(1’)); 73.0 (AC(3’)); 77.4
3
4
4
3
3
(AC(2’)); 83.2 (d, J(5’,P) ¼ 7.9, NC(5’)); 86.9 (d, J(4’,P) ¼ 8.7, AC(4’)); 90.5 (AC(1’)); 121.20 (AC(5));
131.5 (NC(5)); 136.8 (NC(3)); 144.7 (AC(8)); 146.7 (NC(2); 147.0 (NC(4)); 148.7 (NC(6)); 149.2
(AC(4)); 151.1 (AC(2)); 153.4 (AC(6)); 168.5 (CONH2). 31P-NMR (202 MHz, D2O): ꢀ 10.7 (d,
2J(P, P ) ¼ 20.5, AꢀP); ꢀ 10.3 (d, 2J(P, P ) ¼ 20.5, NꢀP). ESI-Q-TOF-MS: 660.1229 ([ M ꢀ 2 H]þ,
C22H28N7O13Pþ2 ; calc. 660.1220).
1,5-Anhydro-2-azido-4,6-O-benzylidene-2-deoxy-d-altritol (10). To a soln. of 1,5 :2,3-anhydro-4,6-O-
benzylidene-d-allitol [9] (9; 1.000 g, 4.27 mmol) in a mixture of 2-methoxyethanol/H2O 5 :1 (240 ml)
were added NaN3 (1.500 g, 23.07 mmol) and NH4Cl (1.500 g, 28.04 mmol), and the mixture was stirred at
1008 for 18 h under N2. After evaporation, the residue was treated with warm CHCl3 (100 ml) and
CH2Cl2 (100 ml), and extracts were filtered over a short path of silica gel to remove inorganic salts. The
solvents were removed in vacuo to yield pure 10 (1.126 g, 95%). Pale yellow oil. Rf (CH2Cl2/MeOH 98 :2)
0.7. 1H-NMR (200 MHz, CDCl3): 2.46 (d, 3J ¼ 1.5, OH); 3.68 – 3.94 (m, 5 H); 4.05 (dd, J ¼ 13, 2, HꢀC);
4.13 (br. s, HꢀC); 4.28 – 4.36 (m, HꢀC); 5.65 (s, PhCH); 7.35 – 7.55 (m, 5 arom. H). 13C-NMR: see
Table 2. ESI-MS (pos.): 278.0 ([ M þ H]þ), 300.0 ([M þ Na]þ).
1,5-Anhydro-2-azido-2-deoxy-d-altritol (11). A soln. of 10 (1.116 g, 4.02 mmol) in 80% AcOH
(60 ml) was heated at 958 for 2 h under N2. After evaporation and co-evaporation with H2O, then with
toluene, the residue was purified by silica gel CC (30 – 90% AcOEt in hexane). The obtained colorless
solid was washed with CHCl3 and dried in vacuo over P4O10: 11 (0.505 g, 66%). Rf (hexane/AcOEt 1:2)
0.1. 1H-NMR (500 MHz, (D6)DMSO): 3.38 – 3.45 (m, HꢀC(4), HꢀC(5), HaꢀC(6)); 3.61 – 3.65 (m,
HbꢀC(6), HeqꢀC(2)); 3.66 (d, 2J(1ax,1eq) ¼ 12.2, HaxꢀC(1)); 3.72 (br. s, Dn1/2 ¼ 10, HeqꢀC(3)); 3.75 (dd,
2J(1eq,1ax) ¼ 12.2, 3J(1eq,2) ¼ 1.5, HeqꢀC(1)); 4.46 (br. t, HOꢀC(6)); 4.69 (d, J(4,OH) ¼ 4.6, HOꢀC(4));
5.21 (d, J(3,OH) ¼ 4.1, HOꢀC(3)). 13C-NMR (125 MHz, (D6)DMSO): 61.2 (C(2)); 61.5 (C(6)); 63.1
(C(1)); 65.8 (C(4)); 68.2 (C(3)); 77.2 (C(5)); for other solvents, see also Table 2.
1,5-Anhydro-2-azido-2-deoxy-6-O-phosphono-d-altritol Diammonium Salt (12). Compound 12 was
obtained in an analogous way as phosphate 5. Azide 11 (0.503 g, 2.66 mmol) was dissolved under N2 in
3 ml of (MeO)3PO, cooled in an ice bath, and triturated with 1.7 ml of a 1:1 (v/v) mixture of POCl3 and
(MeO)3PO. After 3 h of stirring at 08, the reaction was quenched by addition of 3 ml of ice-water and
4 ml of cold Et3N. The resulting mixture was evaporated in vacuo to dryness, and the residue was washed
with (i-Pr)2O and purified by silica gel CC (0.35% NH4OH (20% soln. in H2O) in i-PrOH) to give 12
(0.353 g, 46%). Pale yellow oil. Rf (i-PrOH/25% NH4OHaq/H2O 6 :4 :1) 0.4. This compound was used
directly in the next step. 13C-NMR: see Table 2.
1,5-Anhydro-2-amino-2-deoxy-6-O-phosphono-d-altritol Sodium Salt (13). Azide 12 (0.353 g,
1.31 mmol) was dissolved in 10 ml of H2O and 25 ml of MeOH. Adams catalyst (PtO2 · H2O, 0.069 g,
0.28 mmol) was added, and the mixture was shaken for 85 h in a Parr hydrogenation apparatus (30 psi).

Helvetica Chimica Acta – Vol. 90 (2007)
1275
Table 2. 13C-NMR Chemical Shifts [ppm] andAssignments for Compounds 10 – 14
10
(CDCl3)
11
(CDCl3)
11
(D2O)
12
(D2O)
13
(D2O)
14
(D2O)a)
C(1)
C(2)
C(3)
C(4)
C(5) (3J(C,P))
C(6) (2J(C,P))
PhC
64.9
60.6
66.9 or 66.7
76.9
66.9 or 66.7
68.9
63.9
60.6
68.6
66.0
74.8
63.3
65.7
63.0
70.1
66.9
78.4
63.2
65.9
63.0
70.1
66.4
77.5 (7.6)
66.1 (4.6)
65.0
53.6
68.6
65.4
78.2 (7.6)
65.6 (3.5)
66
72.7
71.6
67.8
79.7 (7.6)
65.3 (3.0)
102.1
Cipso
137.1
Co
126.2
Cm
128.4
Cp
129.4
C(2) (py)
C(3) (py)
C(4) (py)
C(5) (py)
C(6) (py)
C ¼ O
146.4
136.4
147.3
131.2
148.6
168.0
a) Position numbers of the altritol part are primed in the text.
After removal of the catalyst, the filtrate was concentrated to dryness, dissolved in H2O, and applied to a
column of Dowex 50X8-200 (Naþ) ion-exchange resin with H2O as the eluent. Evaporation and keeping
the residue in a vacuum dessicator over P4O10 gave 13 (0.342 g, 91%). Pale yellow amorphous powder. Rf
(i-PrOH/25% NH4OHaq/H2O 6 :4 :1) 0.25. 1H-NMR (500 MHz, D2O): 3.47 – 3.51 (m, HꢀC(2)); 3.72 (dt,
3J(5,4) ¼ 9.1, 3J(5,6) ¼ 3.4, HꢀC(5)); 3.82 (d, 2J(1ax,1eq) ¼ 13.2, HaxꢀC(1)); 3.86 (dd, 3J(4,5) ¼ 9.1,
3J(4,3) ¼ 3.2, HꢀC(4)); 3.94 (dd, J(6,5) ¼ 3.4, J(6,P) ¼ 6.0, CH2(6)); 3.97 (dd, 2J(1eq,1ax) ¼ 13.2,
3
3J(1eq,2) ¼ 1.7, HeqꢀC(1)); 4.20 (dd, J(3,2) ¼ 3J(3,4) ¼ 3.2, HꢀC(3)). 13C-NMR (125 MHz, D2O): 53.6
(CH, C(2)); 65.0 (CH2, C(1)); 65.4 (CH, C(4)); 65.6 (CH2, C(6)); 68.6 (CH, C(3)); 78.2 (d, 3J(5,P) ¼ 7.6,
CH, C(5)); see also Table 2. 31P-NMR (202 MHz, D2O): 3.92. Anal. calc. for C6H13O7NNa (234.162): C
30.78, H 5.60, N 5.98; found: C 30.93, H 5.41, N 5.74.
1,5-Anhydro-2-(3-carbamoylpyridinium)-2-deoxy-6-O-phosphono-d-altritol (14) was prepared in
42% yield analogously to 7 from 13 (0.100 g, 0.35 mmol, soln. in 4 ml of MeOH and 5 ml of H2O), NDC
(0.201 g, 0.62 mmol, soln. in 2.1 ml of H2O) and 0.6 ml of 0.5m aq. TEAB. Rf (i-PrOH/25% NH4OHaq/
H2O 6 :4 :1) 0.15. 1H-NMR (200 MHz, D2O): 4.02 – 4.23 (m, 4 H); 4.40 (dd, 2J(1’ax,1e’ q) ¼ 13, 3J(1a’ x,2’) ¼ 4,
HaxꢀC(1’)); 4.54 (dd, 2J(1a’ x,1e’ q) ¼ 13, 3J(1e’ q,2’) ¼ 4, HeqꢀC(1’)); 4.60 (dd, 3J(2’,3’) ¼ 6.5, 3J(3’,4’) ¼ 2,
HꢀC(3’)); 5.04 (ddd, 3J(2’,3’) ¼ 6.5, 3J(1’ax,2’) ¼ 3J(1e’ q,2’) ¼ 4, HꢀC(2’)); 8.30 (dd, 3J(4,5) ¼ 8, 3J(5,6) ¼ 7,
HꢀC(5)); 8.99 (d, 3J(4,5) ¼ 8, HꢀC(4)); 9.37 (d, 3J(5,6) ¼ 7, H ꢀC(6)); 9.51 (s, HꢀC(2)). 13C-NMR: see
Table 2.
Altritol Nicotinamide Adenine Dinucleotide Lithium Salt 15. Nucleotide 14 (0.051 g, 0.15 mmol) was
dissolved in 2.5 ml of formamide (p.a. grade). Pyridine (1 ml; distilled over BaO immediately prior to
use) was added and evaporated in vacuo to remove the traces of moisture. The procedure was repeated
three times. Adenosine 5’-(phosphoric dibutylphosphinothioic anhydride) (0.155 g, 0.30 mmol) was
added, and the resulting mixture was co-evaporated with pyridine three times. The final residue was
dissolved in 2 ml of pyridine. The flask was wrapped in aluminum foil for protection from light, and
AgNO3 (0.200 g, 1.18 mmol, dried at 1208 for 2 h) was added in one portion. The flask was immediately
capped with a septum, charged with N2, and the mixture was stirred at r.t. for 65 h. Separation of the
dinucleotide was performed as described for compound 8. Lithium salt 15 (0.039 g, 39%) was obtained as
1
fine white powder. Rf (i-PrOH/25% NH4OHaq/H2O 6 :4 :1) 0.4. UV (H2O): 259. H-NMR (500 MHz,

1276
Helvetica Chimica Acta – Vol. 90 (2007)
D2O): 4.06 – 4.13 (m, HꢀC(N5’)); 4.09 – 4.11 (m, HꢀC(N4’)); 4.24 (dd, 2J(5’a,5’b) ¼ 11.7, 3J(5’a,4’) ¼ 3.3,
HaꢀC(A5’)); 4.27 (dd, 2J(5’b,5’a) ¼ 11.7, 3J(5’b,4’) ¼ 2.6, HbꢀC(A5’)); 4.27 – 4.31 (m, CH2(N6’)); 4.36
2
3
2
’
’
’
’
(dd, J(1ax,1eq) ¼ 13.2, J(1a’x,2’) ¼ 4.1, HaxꢀC(N1’)); 4.39 – 4.43 (m, HꢀC(A4’)); 4.46 (dd, J(1eq,1ax) ¼
13.2, 3J(1e’q,2’) ¼ 4.6, HeqꢀC(N1’)); 4.53 (dd, 3J(3’,4’) ¼ 5.8, 3J(3’,2’) ¼ 2.7, HꢀC(N3’)); 4.53 (dd,
3J(3’,2’) ¼ 5.1, J(3’,4’) ¼ 3.2, HꢀC(A3’)); 4.77 (dd, J(2’,1’) ¼ 5.7, J(2’,3’) ¼ 5.1 , HꢀC(A2’)); 5.02 (ddd,
3
3
3
3J(2’,1e’q) ¼ 4.6, 3J(2’,1a’x) ¼ 4.1, 3J(2’,3’) ¼ 2.7, HꢀC(N2’)); 6.13 (d, 3J(1’,2’) ¼ 5.7, HꢀC(A1’)); 8.25 (dd,
3J(5,4) ¼ 8.3, J(5,6) ¼ 6.0, HꢀC(N5)); 8.31 (s, HꢀC(A2)); 8.53 (s, HꢀC(A8)); 8.91 (dd, J(4,5) ¼ 8.3,
3
3
4J(4,2) ¼ 1.5, HꢀC(N4)); 9.30 (dd, 3J(6,5) ¼ 6.0, 4J(6,2) ¼ 1.5, HꢀC(N6)); 9.44 (t, J(2,4) ¼ 4J(2,6) ¼ 1.5,
4
HꢀC(N2)). 13C-NMR (125 MHz, D2O): 66.4 (NC(1’)); 67.7 (d, 2J(6’,P) ¼ 5.0, NC(6’)); 67.9 (d, 2J(5’,P) ¼
6.4, AC(5’)); 68.0 (NC(4’)); 72.1 (NC(3’)); 73.1 (NC(2’)); 73.2 (AC(3’)); 77.1 (AC(2’)); 79.4 (d, 3J(5’,P) ¼
6.9, NC(5’)); 86.7 (d, 3J(4’,P) ¼ 7.5, AC(4’)); 89.9 (AC(1’)); 121.2 (AC(5)); 131.6 (NC(5)); 136.8 (NC(3));
144.3 (AC(8)); 146.8 (NC(2)); 147.7 (NC(4)); 148.9 (NC(6)); 151.2 (AC(4)); 151.5 (AC(2)); 153.4
(AC(6)); 165.7 (CONH2). 31P-NMR (202 MHz, D2O): ꢀ 10.68. ESI-MS (neg.): 676 ([M ꢀ 2 H]ꢀ), 698
([M ꢀ 3 H þ Na]ꢀ). ESI-MS (pos.): 678 (Mþ), 700 ([M ꢀ H þ Na]þ). ESI-Q-TOF-MS: 676.1147 ([M ꢀ
2 H]þ, C22H28N7O14Pþ2 ; calc. 676.1169).
N-[(4aR,7S,8a,S)-4a,7,8,8a-Tetrahydro-2-phenyl-4H-1,3-benzodioxin-7-yl]phtalimide (17). A soln.
of diisopropyl azodicarboxylate (DIAD; 5.3 ml, 25.52 ml) in dry THF (50 ml), was slowly added to a
stirred suspension of Ph3P (7 g, 26.68 mmol) [10 – 11], 16 (4.2 g, 18.08 mmol) and phtalimide (4 g,
27.18 mmol) in dry THF (170 ml) at r.t. under N2. After 1 h, the solvent was removed under reduced
pressure, and the crude material was purified by CC using a gradient of AcOEt/hexane (4 :6, 5 :5, 6 :4) to
1
afford 17 (5.23 g, 80%). White solid. H-NMR (500 MHz, CDCl3): 2.14 – 2.18 (m, 1 H, CH2(8)); 2.26 –
2.32 (m, 1 H, CH2(8)); 2.50 – 2.53 (m, HꢀC(4a)); 3.86 (d, 2J ¼ 11.1, 1 H, CH2(4)); 4.36 (dd, J ¼ 10.7,
4.4, 1 H, CH2(4)); 4.44 – 4.49 (m, HꢀC(8a)); 5.08 – 5.12 (m, HꢀC(7)); 5.65 – 5.70 (m, HꢀC(5), HꢀC(6),
PhCH); 7.34 – 7.83 (m, 9 arom. H). 13C-NMR: see Table 3. ESI-MS: 362 ([M þ H]þ), 384 ([M þ Na]þ).
Table 3. 13C-NMR Chemical Shifts [ppm] andAssignments for Compounds 17 – 21
17a)
(CDCl3)
18
20
(D2O)
21
(DMSO)
(D2O)b)
C(1)
C(2)
C(3)
C(4) (3 J(C,P))
C(5)
45.0
126.1
134.1
39.1
45.3
127.5
134.4
44.0
48.1
126.4
135.5
46.2 (7.5)
67.5
69.8
125.2
139.6
46.2 (7.5)
66.3
75.9
64.6
C(6)
C(7) (2 J(C,P))
PhC
34.4
70.6
102.1
30.4
62.8
34.2
66.6 (3.4)
38.6
67.4 (3.2)
arom. C (phth)
123.2, 126.4, 127.0, 128.2, 128.8,
131.7, 138.5
122.9, 128.1, 131.6
C(2) (py)
C(3) (py)
C(4) (py)
C(5) (py)
C(6) (py)
C ¼ O
146.2
136.5
147.3
131.3
148.2
164.0
168.0
167.7
a) Arbitrary numbering. b) Position numbers of the 4-(phosphonomethyl)cyclohex-2-enyl part are
primed in the text.
N-[(1S,4R,5S)-5-Hydroxy-4-(hydroxymethyl)cyclohex-2-en-1-yl]phtalimide (18). A suspension of
17 in an 80% soln. of AcOH was heated at 958. When the entire solid disappeared (no more starting
material on TLC), the volatiles were removed and co-evaporated with H2O to give 18, after

Helvetica Chimica Acta – Vol. 90 (2007)
1277
1
chromatography. H-NMR (500 MHz, (D6)DMSO): 1.74 – 1.78 (m, HꢀC(4)); 2.14 – 2.18 (m, CH2(6));
3.39 – 3.43 (m, CH2(7)); 4.02 – 4.06 (m, HꢀC(5)); 4.69 (br. s, HOꢀC(7)); 4.76 (d, J ¼ 3.5, HOꢀC(5));
4.93 – 4.96 (m, HꢀC(1)); 5.61 – 5.65 (m, HꢀC(3)); 5.65 – 5.69 (m, HꢀC(2)); 7.80 – 7.86 (m, 4 arom. H).
13C-NMR: see Table 3. ESI-MS (C15H15NO4): 274 ([M þ H]þ), 296 ([M þ Na]þ).
(1S,4R,5S)-1-Amino-5-hydroxy-4-O-(phosphonomethyl)cyclohexene Ammonium Salt (20). To a
suspension of 18 (3 g, 10.98 mmol) in 25 ml of (MeO) 3PO at 08 was added POCl3 (0.8 ml) in one portion.
The mixture was stirred for 5 h at 08 until the mixture became homogenous. The reaction was quenched
with 250 ml of cold TEAB and stirred for 30 min. The volatiles were removed under reduced pressure,
and the crude mixture was adsorbed on a silica column and eluted using a gradient of CH2Cl2/MeOH
(100 :0, 95 :5, 90 :10, 80 :20 and 50 :50). The desired fractions were evaporated to yield the crude
phosphate 19 as a white solid (2.8 g, 60%).
The crude 19 (0.34 g, 0.6 mmol) was dissolved in 22 ml of EtOH and NH2NH2 (0.06 ml, 1.23 mmol)
was added. The mixture was heated at 958 overnight. EtOH was evaporated, and the residue was
dissolved in H2O and washed with AcOEt. The aq. layers were collected and evaporated under reduce
pressure. The residue was purified using an ion exchange-resin (Dowex 400X (Naþ)) CC packed with i-
PrOH and eluted with i-PrOH/NH4OH/H2O ( 9 :1:0! 6 :3 :1). Evaporation gave 20 (0.07 g, 43%).
White solid. 1H-NMR (500 MHz, D2O): 2.03 (ddd, J(6a,6b) ¼ 13.9, J(6a,5) ¼ 6.6, J(6a,1) ¼ 3.2,
HaꢀC(6)); 2.15 (ddd, J(6b,6a) ¼ 13.9, J(6b,5) ¼ 8.1, J(6b,1) ¼ 5.8, HbꢀC(6)); 2.41 – 2.45 (m, HꢀC(4));
3.86 (ddd, J(7a,7b) ¼ 10.2, J(7a,5) ¼ J(7a,P) ¼ 5.6, HaꢀC(7)); 3.98 (ddd, J(7b,7a) ¼ 10.2, J(7b,5) ¼
J(7b,P) ¼ 5.1, HbꢀC(7)); 4.01 – 4.05 (m, HꢀC(1)); 4.15 (ddd, J(5,6b) ¼ 8.2, J(5,6a) ¼ 6.6, J(5,4) ¼ 5.1,
HꢀC(5)); 5.86 (ddd, J(3,2) ¼ 10.2, J(3,4) ¼ J(3,1) ¼ 2.5, HꢀC(3)); 6.00 (dm, J(2,3) ¼ 10.2, HꢀC(2)).
13C-NMR: see Table 3. 31P-NMR (202 MHz, D2O): 1.95 (s). ESI-Q-TOF-MS: 222.0523 (C7H13NO5Pþ;
calc. 222.1477).
(1S,4R,5S)-1-(Carbamoylpyridinium)-5-hydroxy-4-(phosphonomethyl)cyclohex-2-ene Triethylam-
monium Salt (21). Compound 20 (0.07 g, 0.26 mmol) was dissolved in a mixture of MeOH (4.5 ml)
and dry EtN(i-Pr)2 (0.09 ml, 0.52 mmol). The mixture was stirred for 30 min. at r.t. under N2. The Zincke
salt (0.1 g, 0.28 mmol) was added in one portion. A deep red color was formed immediately. The mixture
was then heated at 558 for 4 h and overnight at r.t. After one night, the starting material was not totally
disappeared, and another 0.03 g of the tetrafluoroborate salt was added. The mixture was stirred for an
additional 2 h. The reaction was quenched with 50 ml of 1m TEAB soln., and the volatiles were removed.
The crude material was purified over Dowex 50X8-200 ion-exchange column (Naþ) and eluted with H2O.
After evaporation of the desired fractions, the residue was dissolved in H2O and purified with HPLC
using a column of DEAE Sephadex A-25 and an eluting system of 0.01m TEAB ! 0.5m TEAB.
1
Evaporation of the pure fractions gave 21 (0.09 g, 92%). White powder. H-NMR (500 MHz, D2O):
2.31 – 2.35 (ddd, J(6’a,6’b) ¼ 13.8, J(6’a,5’) ¼ 6.6, J(6’a,1’) ¼ 3.1, HaꢀC(6’)); 2.48 – 2.54 (dm, J(6’b,6’a) ¼
13.8, HbꢀC(6’)); 2.57 – 2.61 (m, HꢀC(4’)); 4.05 – 4.11 (m, CH2(7’), HꢀC(5’)); 5.65 – 5.69 (m,
HꢀC(1’)); 6.03 – 6.07 (m, HꢀC(3’)); 6.41 (dm, J(2’,3’) ¼ 11.5, HꢀC(2’)); 8.22 (dd, J(5,4) ¼ 8, J(5,6) ¼
6, HꢀC(5)); 8.92 (d, J(4,5) ¼ 8, HꢀC(4)); 9.12 (d, J(6,5) ¼ 6.1, HꢀC(6)); 9.35 (s, HꢀC(2)).
13C-NMR: see Table 3. 31P-NMR (202 MHz, D2O): 1.02 (s). ESI-Q-TOF-MS: 327.0739 (C13H16N2O6Pþ;
calc. 327.2514).
Cyclohexenyl Nicotinamide Adenine Dinucleotide Triethylammonium Salt 22. To a mixture of 21
(0.1 g, 0.23 mmol) and adenosine 5’-(phosphoric dibutylphosphinothioic anhydride) (0.24 g, 0.4 mmol)
were added 2 ml of DMF and 4 ml of dry pyridine, and the solvents were removed under reduced
pressure. This procedure was repeated two times. The residue was dried on an oil pump and then
dissolved under N2 in a mixture of DMF/pyridine 1:1. The mixture was stirred for 20 min at r.t. The flask
was wrapped with an aluminium foil to protect from light. AgNO3 (0.32 g, 1.87 mmol) was added, and the
mixture was stirred overnight at r.t. 30 ml of H2O was added, and H2S was bubbled into the mixture for
20 min. The black precipitate was filtered, and the filtrate was extracted 3 times with CHCl3. Combined
org. extracts were washed with H2O, and the aq. layers were concentrated under reduced pressure
(T(bath) < 258). The product was purified by HPLC on Sephadex-A25 cellulose using 0.01m TEAB !
0.5m TEAB in 280 min. The desired fractions were collected and lyophilized to give 22 as a solid TEA
salt (0.018 g, 12%). 1H-NMR (500 MHz, D2O): 2.24 (ddd, J(N6’a,N6’b) ¼ 14, J(N6’a,N5’) ¼ 8.3,
J(N6’a,N1’) ¼ 6, HaꢀC(N6’)); 2.4 (ddd, J(N6’b,N6’a) ¼ 14.2, J(N6’b,N1’) ¼ 6.2, J(N6’b,N5’) ¼ 3.1,

1278
Helvetica Chimica Acta – Vol. 90 (2007)
HbꢀC(N6’)); 2.47 (ddddd, J(N4’,N5’) ¼ 5.5, J(N4’,N7’a) ¼ 4.5, J(N4’,N7b’) ¼ 3.8, J(N4’,N3’) ¼ 3.1,
J(N4’,N2’) ¼ 2.8, HꢀC(N4’)); 4.04 (ddd, J(N5’,N6’a) ¼ 8.3, J(N5’,N4’) ¼ 5.5, J(N5’,N6’b) ¼ 3.1,
HꢀC(N5’)); 4.09 – 4.16 (dAB, J(N7’a,N7’b) ¼ 10.6, J(N7’a,N4’) ¼ 4.5, J(N7’b,N4’) ¼ 3.8, HaꢀC(N7’),
HbꢀC(N7’)); 4.20 – 4.23 (dAB, J(A5’a,A5’b) ¼ 11.9, J(A5’a,A4’) ¼ 3.1, J(A5’b,A4’) ¼ 3.5, HaꢀC(A5’),
HbꢀC(A5’)); 4.36 (ddd, J(A4’,A3’) ¼ 3.2, J(A4’,A5’a) ¼ 3.1, J(A4’,A5’b) ¼ 3.5, HꢀC(A4’)); 4.49 (dd,
J(A3’,A2’) ¼ 5.6, J(A3’,A4’) ¼ 3.2, HꢀC(A3’)); 4.75 (dd, J(A2’,A1’) ¼ 5.9, J(A2’,A3’) ¼ 5.6, HꢀC(A2’));
5.55 (dddd, J(N1’,N6’b) ¼ 6.2, J(N1’,N6’a) ¼ 6.0, J(N1’,N2’) ¼ 2.8, J(N1’,N3’) ¼ 1.6, HꢀC(N1’)); 5.89
(ddd, J(N2’,N3’) ¼ 10.2, J(N2’,N4’) ¼ J(N2’,N1’) ¼ 2.8 , HꢀC(N2’)); 6.05 (d, J(A1’,A2’) ¼ 5.9,
HꢀC(A1’)); 6.28 (ddd, J(N3’,N2’) ¼ 10.2, J(N3’,N4’) ¼ 3.1, J(N3’,N1’) ¼ 1.6, HꢀC(N3’); 8.14 (dd,
J(N5,N4) ¼ 8.1, J(N5,N6) ¼ 6.2, HꢀC(N5)); 8.15 (s, HꢀC(A2)); 8.43 (s, HꢀC(A8)); 8.78 (dd,
J(N4,N5) ¼ 8.1, J(N4,N2) ¼ 1.5, HꢀC(N4)); 8.99 (dd, J(N6,N5) ¼ 6.2, J(N6,N2) ¼ 1.5, HꢀC(N6));
9.26 (dd, J(N2,N4) ¼ 1.5, J(N2,N6) ¼ 1.5, HꢀC(N2)). 13C-NMR (125 MHz, D2O): 38.5 (NC(6’)); 46.2 (d,
J(NC4’,NP) ¼ 8.8, NC(4’)); 66.0 (NC(5’)); 68.0 (d, J(NC7’,NP) ¼ 5.8, NC(7’)); 68.2 (d, J(AC5’,AP) ¼ 4.8,
AC(5’)); 69.8 (NC(1’)); 73.1 (AC(3’)); 76.7 (AC(2’)); 86.5 (d, J(AC4’,AP) ¼ 8.7, AC(4’)); 89.4 (AC(1’));
121.1 (AC(5)); 125.0 (NC(2’)); 131.2 (NC(5)); 136.4 (NC(3)); 139.6 (NC(3 ’)); 142.4 (AC(8)); 146.0
(NC(2)); 147.1 (NC(4)); 148.0 (NC(6)); 151.7 (AC(4)); 155.6 (AC(2)); 158.1 (AC(6)); 168.7 (CONH2).
31P-NMR (202 MHz, D2O): ꢀ 10.9 (d, J(NP,AP) ¼ 21.5, NP); ꢀ 10.6 (d, J(NP,AP) ¼ 21.5, AP). ESI-Q-
TOF-MS: 657.1313 (C23H29N7O12Pþ2 ; calc. 657.471).
Authors are indebted to Roche Diagnostics GmbH, D-Penzberg, for financial support.
REFERENCES
[1] J. Gebicki, A. Marcinek, J. Zielonka, Acc. Chem. Res. 2004, 37, 379.
[2] P. E. Smith, J. J. Tanner, J. Am. Chem. Soc. 1999, 121, 8637.
[3] C. E. Bell, T. O. Yeates, D. Eisenberg, Protein Sci. 1997, 6, 2084.
[4] B. S. Reddy, W. Saenger, K. Mühlegger, G. Weimann, J. Am. Chem. Soc. 1981, 103, 907.
[5] E. Ragg, L. Scaglioni, R. Mondelli, I. Carelli, A. Casini, S. Totorella, Biochim. Biophys. Acta 1991,
1076, 49.
[6] H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E.
Bourne, Nucleic Acids Res. 2000, 28, 235; http://www.rcsb.org/pdb/.
[7] I. Verheggen, A. Van Aerschot, S. Toppet, R. Snoeck, G. Janssen, J. Balzarini, E. De Clercq, P.
Herdewijn, J. Med. Chem. 1993, 36, 2033.
[8] M. W. Andersen, S. M. Daluge, L. Kerremans, P. Herdewijn, Tetrahedron Lett. 1996, 37, 8147.
[9] B. Allart, K. Khan, H. Rosemeyer, G. Schepers, C. Hendrix, K. Rothenbacher, F. Seela, A. Van
Aerschot, P. Herdewijn, Chem.–Eur. J. 1999, 5, 2424; B. Allart, R. Busson, J. Rozenski, A. Van
Aerschot, P. Herdewijn, Tetrahedron 1999, 55, 6527.
[10] J. Wang, J. Morral, C. Hendrix, P. Herdewijn, J. Org. Chem. 2001, 66, 8478.
[11] P. Gu, C. Griebel, A. Van Aerschot, J. Rozenski, R. Busson, H.-J. Gais, P. Herdewijn, Tetrahedron
2004, 60, 2111.
[12] J. T. Slama, C. Radziejewski, S. Oruganti, E. T. Kaiser, J. Am. Chem. Soc. 1984, 106, 6778.
[13] D. R. Walt, M. A. Findeis, V. M. Rios-Mercadillo, J. Auge, G. M. Whitesides, J. Am. Chem. Soc. 1984,
106, 234.
[14] J. T. Slama, A. M. Simmons, Biochemistry 1988, 27, 183.
[15] K. Furusawa, M. Sekine, T. Hata, J. Chem. Soc., Perkin Trans. 1 1976, 1711.
[16] W. Kuchen, H. Buchwald, K. Strolenberg u. J. Metten, Liebigs Ann. Chem. 1962, 652, 28.
ReceivedMarch 2, 2007