Second Generation of cycloSal-Pronucleotides with Esterase-Cleavable Sites: The "Lock-In"-Concept

Article abstract of DOI:10.1081/NCN-120027820

A conceptual extension of the cycloSal-pronucleotide approach is presented. The characteristic feature of the new cycloSal-derivatives of the anti-HIV active nucleoside analogue d4T 1 is the incorporation of an enzymatically cleavable carboxylic ester moiety with the intention to trap the triesters inside cells ("lock-in"-concept). CycloSal-triesters bearing different ester groups in the 3-or 5-position of the cycloSal-moiety are described. Surprisingly, only acetyl-and levulinyl esters are cleaved readily in CEM cell extracts while alkyl esters were found to be stable. Nevertheless, in in-vitro anti-HIV assays most of the compounds achieve the thymidine-kinase bypass, thus proving that they act at least as nucleotide delivery systems.

Full text of DOI:10.1081/NCN-120027820

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Second Generation of cycloSal‐Pronucleotides with
Esterase‐Cleavable Sites: The ”Lock‐In”‐Concept
a
Chris Meier a , Manuel F.H. Ruppel a , Dalibor Vukadinović & Jan Balzarini b
a Institute of Organic Chemistry, University of Hamburg, Martin‐Luther‐King‐Platz 6,
D‐20146, Hamburg, Germany
b Rega‐Institute for Medical Research, K.U. Leuven, Minderbroedersstraat 10, B‐3000,
Leuven, Belgium
Version of record first published: 01 Jun 2007.
To cite this article: Chris Meier , Manuel F.H. Ruppel , Dalibor Vukadinović & Jan Balzarini (2004): Second Generation
of cycloSal‐Pronucleotides with Esterase‐Cleavable Sites: The ”Lock‐In”‐Concept , Nucleosides, Nucleotides and Nucleic
Acids, 23:1-2, 89-115
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 1 & 2, pp. 89–115, 2004
Second Generation of cycloSal-Pronucleotides
with Esterase-Cleavable Sites:
The ‘‘Lock-In’’-Concepty
1
1
Chris Meier,1, Manuel F. H. Ruppel, Dalibor Vukadinovic,
*
´
and Jan Balzarini2
1Institute of Organic Chemistry, University of Hamburg, Hamburg, Germany
2Rega-Institute for Medical Research, K.U. Leuven, Leuven, Belgium
ABSTRACT
A conceptual extension of the cycloSal-pronucleotide approach is presented. The
characteristic feature of the new cycloSal-derivatives of the anti-HIV active
nucleoside analogue d4T 1 is the incorporation of an enzymatically cleavable
carboxylic ester moiety with the intention to trap the triesters inside cells (’’lock-in’’-
concept). CycloSal-triesters bearing different ester groups in the 3-or 5-position of the
cycloSal-moiety are described. Surprisingly, only acetyl-and levulinyl esters are
cleaved readily in CEM cell extracts while alkyl esters were found to be stable.
Nevertheless, in in-vitro anti-HIV assays most of the compounds achieve the
thymidine–kinase bypass, thus proving that they act at least as nucleotide deliv-
ery systems.
Key Words: Pronucleotides; Nucleoside analogue; Anti-HIV; CycloSal-triesters.
yIn honor and celebration of the 70th birthday of Professor Leroy B. Townsend.
*
Correspondence: Chris Meier, Institute of Organic Chemistry, University of Hamburg, Martin-
Luther-King-Platz 6, D-20146 Hamburg, Germany; Fax: + 49-40-42838-2495; E-mail: chris.
meier@chemie.uni-hamburg.de.
89
DOI: 10.1081/NCN-120027820
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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Meier et al.
INTRODUCTION
Recently we developed a new class of chemically cleavable prodrugs of the
corresponding nucleotide analogues of antivirally active nucleosides like 2’,3’-dideoxy-
2’,3’-didehydrothymidine (d4T) 1.[1] It was unambiguously shown that the so-called
cycloSal-pronucleotides (prototype A, Figure 1) deliver d4T monophosphate and other
nucleotides inside human cells.[2,3] Thymidine kinase (TK) is a salvage pathway
enzyme that is responsible for the conversion of a nucleoside analogue into its
nucleoside monophosphate (nucleotide).[4] This conversion is the first and often most
critical step in the bioactivation of nucleoside analogues into their ultimately bioactive
triphosphates.[5] This problem cannot be circumvented by the direct use of the
nucleotide because of its high polarity and its efficient catabolism in the blood stream
by unspecific nucleotidases. In contrast, nucleotide releasing lipophilic systems like
the cycloSal-pronucleotide approach can bypass these limitations by achieving the
thymidine-kinase bypass (TK-bypass).[6,7] The nucleotide delivery from cyclo-
Sal-triesters is a result of a chemically triggered releasing process that liberates
the nucleotide by a cascade reaction.[8] The cycloSal-approach has been applied
successfully to various nucleoside analogues, e.g. d4T 1,[9,10] 5-(E)-bromovinyl-2’-
deoxyuridine,[11,12] acyclovir,[13,14] 2’,3’-dideoxyadenosine[15] and 2’-ribo-fluoro-2’,3’-
dideoxyadenosine.[16] However, it cannot be excluded that a concentration equilibrium
through the membrane is formed due to the lipophilic nature of the cycloSal-triesters
(Scheme 1).
For an effective expression of the antiviral activity it is a prerequisite that high
concentrations of the pronucleotide must accumulate inside cells. This would then lead
also to high concentrations of the mononucleotide and consequently also of the
bioactive triphosphate. Therefore, we report here on an extension of the cycloSal-
phosphate triester approach. In order to trap the cycloSal-triester intracellularly, an
enzyme-cleavable site has been attached to the cycloSal-moiety. An enzymatic reaction,
that should occur predominately inside the cells, releases a highly or at least a more
polar cycloSal-derivative.[17] For this reason we introduced an ester group that should
be cleaved by (carboxy)esterases or lipases (’’lock-in’’ triesters B; Figure 1).
The intracellular concentrations of esterases are known to be higher as compared to
the extracellular medium. The use of esters/esterases in prodrug development has been
Figure 1. General structures of the prototype cycloSal-phosphate triesters A of 2’,3’-dideoxy-
2’,3’-didehydrothymidine (d4T 1) and the second generation derivatives B.

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91
Scheme 1. Formation of an equilibrium through the membrane and the general idea of the ’’lock-
in’’-concept.
Figure 2. Structures of the target new cycloSal-phosphate triesters of d4TMP bearing different
ester modifications.

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Meier et al.
widely applied.[6,7,18,19] However, in the case of the cycloSal-pronucleotides the ester
group attached to the cycloSal-moiety, represents an electron-withdrawing group that
should also decrease the stability of the phosphate ester. In order to avoid this, an
ethylene linker was introduced as a spacer unit between the ester group and the
cycloSal-moiety. Two types of ester groups can be attached that differ in the resulting
cycloSal-triesters. After enzymatic cleavage, a cycloSal-d4TMP acid 2 can be liberated
from cycloSal-d4TMP acid ester 3 or a cycloSal-d4TMP alcohol 4 can be released from
cycloSal-d4TMP alcohol ester 5 (Figure 2). In both cases, a more polar cycloSal-
derivative (Y-prodrug; Scheme 1) as compared to the parent triester (X-prodrug,
Scheme 1) is released that should accumulate inside the cell (’’lock-in’’-concept).
Here we report on the synthesis and the properties of these new cycloSal-d4TMP
derivatives 2–5 having alkyl ester groups attached via a C2-linker in the cycloSal-
residue. In this report, we focused our interest on the nucleoside analog d4T 1. The
ester-linker group has been attached either to the 3-or to the 5-position of the cycloSal-
aromatic ring. Previous cycloSal-derivatives bearing substituents in these positions
showed appropriate chemical stability, selective d4TMP delivery and strong bioactivity
in previous work.[1,9,10,15,16]
CHEMISTRY, RESULTS AND DISCUSSION
In order to use our previously reported synthetic approach towards the title
triesters,[1,9,10] the different salicyl alcohols 6 were prepared first from the cor-
responding phenols 7 (Figure 3). The methyl, ethyl and i-propyl 3-(2-hydroxyphenyl)-
propionates were prepared by transesterification of dihydrocoumarin 8 with methanol,
ethanol and 2-propanol in the presence of H2SO4 in 73%–96%. The benzyl ester
was prepared by alkylation of 3-(2-hydroxyphenyl)propionic acid 9 using benzyl
bromide in the presence of DBU in toluene (97% yield)[20] while both t-butyl ester
were obtained by esterification of the same starting material or 3-(4-hydroxyphenyl)-
propionic acid 10 with N,N-dimethylformamide-dineopentylacetale in toluene in 85%
and 73% yield, respectively.[21] The methyl- and the benzyl esters of 3-(4-hydroxy-
phenyl)propionic acid 10 were prepared in 93% and 73% yield by acid catalyzed
[22]
esterification (H2SO4 in the former and H3PO4
in the latter case). The 2-propyl ester
was obtained in 62% using 2-propanol and dry HCl-gas.[23] The acetyl esters of 2-(2-
hydroxyphenyl)ethanol 11 and 2-(4-hydroxyphenyl)ethanol 12 were also obtained by a
transesterification reaction. However, here the transesterification of the acetyl group
of ethylacetate was achieved in the presence of SiO2ꢀNaHSO4 as a catalyst in 87%
yield.[24] This procedure has also been used for the formation of the levulinyl ester
of 2-(2-hydroxyphenyl)ethanol 11 from ethyllevulinate. Finally, the pivaloyl esters
of 2-(2-hydroxyphenyl)ethanol 11 and 2-(4-hydroxyphenyl)ethanol 12 were synthe-
sized using the ’’twisted amides’’ as an activated pivaloyl-donor[25] in 80% and 51%
yield, respectively.
All phenols 7 were then transformed into the corresponding salicyl alcohols 6 by
reaction of p-formaldehyde and phenylboronic acid in the presence of a trace amount of
propionic acid.[26] The intermediate 2-phenyl-4H-benzo[1.3.2]-dioxaborinanes were
purified and subsequently treated with a 30% aqueous solution of H2O2 to give the
salicylalcohols. Yields for this two step procedure were found to be between 50–85%.

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93
Figure 3. Synthesis pathways towards the title compounds. Method A: alcohol, H2SO4, reflux,
5–8 h; method B: benzyl bromide, DBU, toluene, reflux, 7 h; method C: (CH3)2NCH(OCH2tBu)2,
toluene, reflux, 5 h; method D: methanol, CH2Cl2, H2SO4, reflux, 5 h; method E: benzyl alcohol,
toluene, H3PO4, reflux, 10 h; method F: 2-propanol, HCl-gas, rt, 16 h; method G: ethylacylate, n-
hexane, SiO2ꢀNaHSO4, 678C, 6-18 h; method H: 3-pivaloyl-1,3-thiazolidine-2-thion, toluene, 658C,
48 h; method I: i. phenylboronic acid, propionic acid (cat.), p-formaldehyde, toluene, reflux, 6–8 h;
ii) H2O2, THF, 08C, 30 min; method J: PCl3, pyridine, diethylether, 0–218C, 12 h; method K: i.
d4T 1, AcCN, DIPEA, 0–208C; ii. tBuOOH, AcCN, rt, 30 min; method L: hydrazineꢀhydrate,
pyridine/acetic acid 3:2, pyridine, 08C, 10 min; method M: TFA (10 equiv.), CH2Cl2, rt, 1h.
These diols were then reacted with PCl3 in the presence of pyridine to yield cyclic
chlorophosphanes 13 as described previously.[27] The chlorophosphanes 13 were
used as crude products (85–95% pure as judged from the 31P-NMR) for the next
reactions. Typically, the chlorophosphanes showed resonance signals at ꢁ 140.5 ppm in
the 31P-NMR. These were then reacted with the nucleoside analog d4T 1, and the
intermediately formed phosphite triesters were oxidized by t-butyl hydroperoxide in a

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Meier et al.

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95
one-pot reaction to give the title triesters 3,5 in 30–50% yield.[9,10] Moreover,
cycloSal-d4TMP acids 2 were prepared by cleavage of the t-butyl ester in 3e and 3i,
respectively, by TFA treatment (87% yield). Finally, cycloSal-d4TMP alcohol 4a was
obtained after delevulinylation of 5b with hydrazine-hydrate (25% yield).[28]
All new cycloSal-d4TMP derivatives 2–5 were studied regarding their stability in
aqueous phosphate buffer, pH 7.3. The half-lives are shown in Table 1.
First, all cycloSal-d4TMP esters bearing the modification in the 3-position will be
discussed. It was interesting to note that the stability of cycloSal-d4TMP acid esters
3a–e varied between 7.3 h and 13.5 h. However, this variation cannot be attributed to
changing lipophilicities because no correlation was found between the t1/2 and the
logPcalc values (Table 1). As expected, cycloSal-d4TMP alcohol esters 5a–c showed a
stability of 13–13.5 hours. These results prove that the ethylene-spacer is long enough
to separate the electron-withdrawing ester group from the cycloSal-ring system. This
can be concluded from the half-lives of two cycloSal-triesters published before: the
prototype cycloSal derivatives without any substituent showed a t1/2 of 4.4 h and for the
donor substituted 3-methyl-cycloSal-d4TMP a t1/2 value of 17.5 h was measured.[8]
Interestingly, the chemical stability of the cycloSal-d4TMP acid 2a was 1.8–3-fold
higher as those found for of the corresponding esters 3a–e. This increase in stability
may be attributed to the charged carboxylate at pH 7.3 of the buffer, with the result of
an overall negative charge on the triester molecule. Thus, a nucleophilic attack at the
phosphate group is less efficient. In contrast, cycloSal-d4TMP alcohol 4a showed the
same chemical stability as triesters 5a–c as expected.
Within the series of cycloSal-triesters 3f–i,5d,5e bearing the ester modification in
the 5-position, the half-lives were found to be between 6.1 and 7.3 h. Again, no
correlation with the lipophilicity was observed. As for the 3-acid derivative 2a, the
5-acid cycloSal-derivative 2b showed an 1.8-fold increase in stability. As compared to
the prototype 5-methyl-cycloSal-d4TMP (t1/2 = 8 h)8, triesters 3f–i,5d,5e showed com-
parable chemical stability thus, proving the efficiency of the ethylene linker again.
Finally, in all cases the product formed after the hydrolysis in aqueous phosphate
buffer, pH 7.3 was d4TMP and the corresponding diol without cleaving the different
ester groups.
In contrast, the cleavage of the ester groups attached to the 3-position of the
cycloSal-moiety was clearly observed in the case triesters 5a (acetyl-ester; t1/2 = 1.9 h
instead of 13.6 in the chemical hydrolysis) and 5c (Lev-ester; t1/2 = 2.0 h instead of
12.5 in the chemical hydrolysis) in hydrolysis studies using T-lymphocyte (CEM) cell
extracts (CE; Table 1). In HPLC-co-elution experiments, it was unambiguously shown
that triester alcohol 4a was formed which then hydrolyzed to yield d4TMP. As
expected, the Piv-triester 5c was more stable because (carboxy)esterases are not readily
’’working’’ on branched alkyl esters. Nevertheless, the stability decreased still 3-fold.
Thus, for the first time, a fast enzymatic cleavage led to a product that is considerably
more polar (logPcalc = ꢂ0.53 (4a) vs. 0.42 (5a) and 0.37 (5b)) as the precursor.
However, the 3-modified cycloSal-d4TMP acid esters 3a–e that should release the even
more polar carboxylate group were found to be inert against enzymatic cleavage: no
formation of the cycloSal-d4TMP acid 2a (logPcalc = ꢂ3.96) has been observed in the
HPLC-chromatograms. This selectivity of the involved esterases is surprising because
often carboxyl groups are bioreversibly protected by esterification. Moreover, the
inability to observe ester cleavage by the (carboxy)esterases in the extracts was

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Meier et al.
surprising because prior to the cycloSal-triesters we incubated 3-propionylsalicylalcohol
methyl ester with pig liver esterase (PLE; E.C. 3.1.1.1.) in phosphate buffer and an
extremely rapid deesterification was observed. In contrast, 3-or 5-modified cycloSal-
d4TMP triesters were not cleaved by PLE! Thus, the attached nucleoside significantly
impacts the ability of these compounds to serve as PLE substrates.
In the series of the 5-modified cycloSal-d4TMP triesters 3f–i,5d,5e the situation
was comparable: only the acetyl ester 5d was cleaved although the half-life dropped
only 2.4-fold and the appearance of a reaction intermediate was observed in the HPLC.
Chemical hydrolysis was only observed for all other derivatives. Again, a selective
delivery of d4TMP was observed.
Finally, triesters 2–5 were tested for their anti-HIV activity in CEM/O and CEM/
TKꢂ cells. All triesters showed the TK-bypass effect (antiviral activity in the wild-type
CEM cells and in the CEM/TKꢂ cells) except triesters 2. The non-TK-bypass of
cycloSal-d4TMP acids 2 is most probably due to an inability of membrane penetration
due to the charged carboxylate moiety. In contrast, cycloSal-d4TMP alcohol 4a showed
antiviral activity in the TK-competent and the TK-deficient cells. Although the
lipophilicity is considerably different among the esters, this result is suggestive of a
membrane penetration even if a free hydroxy group is present in the molecule. It is
interesting to note that the triesters 5a,b that were enzymatically cleaved in the cell
extracts showed the best antiviral activity in the CEM/TKꢂ cells.
The promising results of the enzymatic cleavage of the cycloSal-d4TMP alcohol
esters 5a,b,d at the introduced ester site provides supporting evidence for the feasibility
of the concept. Further experiments will be conducted in our laboratories in order to
assess the role of ester lability on antiviral activity and enzymatic stability.
EXPERIMENTAL SECTION
All experiments involving water-sensitive compounds were conducted under
scrupulously dry conditions (argon or nitrogen atmosphere). Solvents: Anhydrous
methylene chloride (CH2Cl2) and anhydrous acetonitrile (CH3CN) were obtained in a
˚
Sure/Seal bottle from Fluka and stored over 4 A molecular sieves. Ethyldiisopropyl-
amine (DIPEA) was distilled from Na prior to use. The solvents for HPLC were
obtained from Merck (acetonitrile, HPLC grade). Ion pairing buffer solution was
prepared by mixing 6.6 mL tetrabutylammonium hydroxide with 1000 mL water. The
pH-value was adjusted to 3.8 by adding concentrated phosphoric acid (buffer I). To
60 mL of buffer I-solution, 1000 mL water were added (buffer II). Chromatography:
Chromatotron (Harrison Research 7924), silica gel 60Pf (Merck, ‘‘gipshaltig’’); UV
detection at 254 nm. TLC: analytical thin layer chromatography was performed on
Merck precoated aluminium plates 60 F254 with a 0.2-mm layer of silica gel containing
a fluorescence indicator; sugar-containing compounds were visualized with the sugar
spray reagent (0.5 mL of 4-methoxybenzaldehyde, 9 mL of ethanol, 0.5 mL of
concentrated sulfuric acid, and 0.1 mL of glacial acectic acid) by heating with a fan or
a hot plate. HPLC: (Merck-Hitachi) analytical HPLC, LiChroCART 250-3 with
LiChrospher 100 RP-18 endcapped (5 mm), gradient I 5–100% CH3CN (0–20 min),
5% CH3CN (20–35 min), flow 0.5 mL, UV detection at 265 nm; gradient II 8–100%
CH3CN (0–22 min), 100% CH3CN (22–27 min), 8% CH3CN (27–33 min), flow
0.6 mL, UV detection at 256 nm (in gradient II tetrabutylammonium phosphate buffer

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97
was used instead of water as in gradient I). NMR spectra were recorded using (1H
NMR) Bruker AC 250 at 250 MHz, Bruker AMX 400 at 400 MHz or Bruker DMX 500
at 500 MHz; (13C NMR) Bruker WM 400 at 101 MHz, Bruker AMX 400 at 101 MHz
or Bruker DMX 500 at 123 MHz (Calibration was done in both cases with the solvent);
(31P NMR) Bruker AMX 400 at 162 MHz or Bruker DMX 500 at 202 MHz (H3PO4 as
1
external standard). All H and 13C NMR chemical shifts (d) are quoted in parts per
million (ppm) downfield from tetramethylsilane, (CD3)(CD2H)SO being set at dH 2.49
as a reference. UV spectra were taken with a Varian Cary 1E UV/Vis spectrometer.
Infrared spectra were recorded with a Perkin Elmer 1600 Series FT-IR or a ATI
Mattson Genesis Series FT-IR spectrometer in KBr pellets. Mass spectra were obtained
with a Finnigan electrospray MAT 95 Trap XL (ESI) or a VG Analytical VG/70–250 F
spectrometer (FAB, matrix was m-nitrobenzylalcohol). The test compounds were
isolated as mixtures of diastereomers arising from the mixed stereochemistry at the
phosphate center. From our experience, elemental analyses are difficult to obtain from
phosphorus and nucleoside containing compounds. However, the obtained lyophilized
triesters were found to be pure by HPLC analysis, high-field multinuclear NMR
spectroscopy and mass spectrometry.
Preparation of the Phenols 7
Methyl-3-(2-hydroxyphenyl)propionate 7a: 10.4 g (70.0 mmol) Dihydrocu-
marin 45 have been added to 60 ml dry methanol. 100 ml H2SO2 were added and the
mixture was heated under reflux for 5 h. Then, the methanol was distilled off and the
residue was dissolved in diethylether and the organic phase was extracted with sodium
bicarbonate solution. The organic phase was dried with sodium sulfate, the solvent was
evaporated and the residue was subjected to a chromatography on silica gel (CH2Cl2-
hexane gradient 50 to 0 %). The product was isolated as colorless needles. Yield: 96%;
1
m.p.: 41.58C; TLC: Rf (CH2Cl2): 0.56; H-NMR (250 MHz, CDCl3) d: 7.33 (s, 1H,
aryl-OH); 7.16–7.10 (m, 1H, H5-aryl); 7.12 (d, JH–H = 7.3 Hz, 1H, H3-aryl); 6.91–
3
3
6.85 (m, 2H, H4-aryl + H6-aryl); 3.71 (s, 3H, H10); 2.96 (t, JH–H = 6.7 Hz, 2H, H8);
3
2.75 (t, JH–H = 6.7 Hz, 2H, H7); 13C-NMR (63 MHz, CDCl3) d: 175.7 (C9); 154.1
(C1); 130.3 (C3); 127.8 (C2); 127.0 (C5); 120.6 (C4); 116.5 (C6); 52.0 (C10); 34.6
(C7); 24.9 (C8); UV (CH3CN) lmax: 298, 272 nm; IR (KBr) nꢂ1: 3457 (OH), 3038,
1719 (CO), 1594, 1506, 1456, 1421, 1102, 989, 845, 786, 751, 707.
Ethyl-3-(2-hydroxyphenyl)propionate 7b: Phenol 7b was prepared as 7a. The
product was isolated as colorless needles. Yield: 81%; m.p. 34–358C; TLC: Rf
1
(CH2Cl2/MeOH 9:1 v/v): 0.66; H-NMR (250 MHz, CDCl3) d: 7.42 (s, 1H, aryl-OH);
3
4
3
7.13 (ddd, 2ꢀ JH–H = 6.7 Hz, JH–H = 1.5 Hz, 1H, H5-aryl); 7.11 (d, JH–H = 7.3 Hz,
3
3
1H, H3-aryl); 6.90 (d, JH–H = 7.9 Hz, 1H, H6-aryl); 6.88 (ddd, 2ꢀ JH–H = 7.6 Hz,
4JH–H = 1.2 Hz, 1H, H4-aryl); 4.17 (q, JH–H = 7.0 Hz, 2H, H10); 2.93 (t, JH–H = 6.7
3
3
Hz, 2H, H8); 2.74 (t, JH–H = 6.7 Hz, 2H, H7); 1.26 (t, JH–H = 7.0 Hz, 3H, H11); 13C-
3
3
NMR (63 MHz, CDCl3) d: 175.4 (C9); 154.2 (C1); 130.3 (C3); 127.7 (C5); 127.1 (C2);
120.5 (C4); 116.6 (C6); 61.1 (C10); 34.9 (C8); 24.9 (C7); 14.0 (C11); IR (KBr) nꢂ1
3500 (OH), 1705 (CO).
:
2-Propyl-3-(2-hydroxyphenyl)propionate 7c: Phenol 7c was prepared as 7a.
The product was isolated as a colorless oil. Yield: 73%; TLC: Rf (CH2Cl2/MeOH 9:1

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Meier et al.
v/v): 0.68; 1H-NMR (400 MHz, CDCl3) d: 7.40 (s(br), 1H, aryl-OH); 7.13 (ddd,
3
4
3
2ꢀ JH – H = 7.6 Hz, JH – H = 1.8 Hz, 1H, H5-aryl); 7.09 (dd, JH – H = 7.5 Hz,
4JH–H = 1.7 Hz, 1H, H3-aryl); 6.92–6.85 (m, 2H, H4-aryl + H6-aryl); 5.02 (sept,
3JH–H = 6.2 Hz, 1H, H10); 2.90 (t, JH–H = 6.2 Hz, 2H, H7); 2.69 (dd, JH–H = 6.5 Hz,
3
3
3JH–H = 6.2 Hz, 2H, H8); 1.21 (d, JH–H = 6.2 Hz, 6H, H11a,b); 13C-NMR (101 MHz,
3
CDCl3) d: 175.0 (C9); 154.3 (C1); 130.4 (C3); 127.7 (C5); 127.2 (C2); 120.5 (C4);
116.6 (C6); 68.7 (C10); 35.2 (C8); 24.9 (C7); 21.6 (C11a,b); UV (CH3CN) lmax: 272,
214, 202 nm; IR (film): nꢂ1: 3398 (OH), 3038, 1702 (CO), 1609, 1595, 1506, 1491,
1458, 1375, 1236, 1105, 754.
Benzyl-3-(2-hydroxyphenyl)propionate 7d: To a solution of 2.08 g (12.5 mmol)
3-(2-hydroxyphenyl)propionic acid 47 in 30 ml dry toluene, 1.87 ml DBU and 1.48 ml
(12.5 mmol) benzylbromide was added. The mixture was heated for 7 h under reflux.
The organic phase was washed with water, sodium hydrogencarbonate and sodium
chloride solutions. After drying, the solvent was evaporated and the residue was
purified by chromatography on silica gel (n-hexane - ethylacetate-gradient 10–50 %).
The product was isolated as a yellowish oil. Yield: 97%; TLC: Rf (n-hexan/ethylacetate
1
3:2 v/v): 0.48; Rf (CH2Cl2/MeOH 9:1 v/v): 0.71; H-NMR (400 MHz, DMSO-d6) d:
9.36 (s, 1H, aryl-OH); 7.37–7.29 (m, 5H, Ph); 7.02 (dd, JH–H = 7.3 Hz, 1H, H3-
3
3
4
aryl); 7.00 (ddd, 2 ꢀ JH–H = 7.6 Hz, JH–H = 1.8 Hz, 1H, H5-aryl); 6.77 (dd,
4
3
3JH–H = 7.9 Hz, JH–H = 1.1 Hz, 1H, H6-aryl); 6.67 (ddd, 2ꢀ JH–H = 7.4 Hz,
4JH–H = 1.2 Hz, 1H, H4-aryl); 5.06 (s, 2H, H10); 2.79 (t, JH–H = 7.7 Hz, 2H, H7);
3
2.61 (dd, JH–H = 8.1 Hz, JH–H = 7.2 Hz, 2H, H8); 13C-NMR (101 MHz, DMSO-d6)
d: 172.6 (C9); 155.3 (C1); 136.4 (C11); 129.9 (C3); 128.6 (2 ꢃ Cm-Ph); 128.1
(Cp-Ph); 128.1 (2 ꢃ Co-Ph); 127.4 (C5); 126.5 (C2); 119.1 (C4); 115.0 (C6); 65.5
(C10); 33.7 (C8); 25.7 (C7).
3
3
tert-Butyl-3-(2-hydroxyphenyl)propionate 7e: A solution of 1.50 g (9.03 mmol)
3-(2-hydroxyphenyl)propionic acid 47 and 8.70 g (117 mmol) tert-butanol was refluxed
in 22 ml toluene. To this solution, 6.27 g (27.1 mmol) N,N-DMF-dineopentylacetal was
added dropwise during 30 min and stirring was continued for further 5 h. The organic
phase was washed with sodium bicarbonate solution and water. After drying the
organic phase, the solvent was removed and the residue was purified by
chromatography (CH2Cl2- methanol-gradient 0–20 %). The product was isolated as a
1
colorless oil. Yield: 81%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.61; H-NMR (400 MHz,
3
4
CDCl3) d: 8.06 (s, 1H, aryl-OH); 7.13 (ddd, 2 ꢃ JH–H = 7.6 Hz, JH–H = 1.5 Hz, 1H,
3
4
H5-aryl); 7.08 (dd, JH–H = 7.5 Hz, JH–H = 1.6 Hz, 1H, H3-aryl); 6.90 (dd,
4
3
3JH–H = 8.1 Hz, JH–H = 1.0 Hz, 1H, H6-aryl); 6.86 (ddd, 2 ꢃ JH–H = 7.4 Hz,
4JH–H = 1.1 Hz, 1H, H4-aryl); 2.86 (t, JH–H = 6.2 Hz, 2H, H7); 2.65 (t, JH–H = 6.2
Hz, 2H, H8); 1.43 (s, 9H, H11a–c); 13C-NMR (101 MHz, CDCl3) d: 174.5 (C9); 153.8
(C1); 129.9 (C3); 127.3 (C5); 126.7 (C2); 120.0 (C4); 116.2 (C6); 73.5 (C10); 35.8
(C8); 26.8 (C11); 25.6 (C7).
3
3
Methyl-3-(4-hydroxyphenyl)propionate 7f: Phenol 7f was prepared as 7a. The
product was isolated as a colorless solid. Yield: 93%; TLC: Rf (CH2Cl2/MeOH 9:1
3
v/v): 0.70; 1H-NMR (400 MHz, CDCl3) d: 7.07 (d, JH–H = 8.3 Hz, 2H, H3-
aryl + H5-aryl); 6.76 (d, JH–H = 8.4 Hz, 2H, H4-aryl + H6-aryl); 3.68 (s, 3H, H10);
3

ORDER
REPRINTS
cycloSal-Pronucleotides
99
3
3
2.89 (t, JH–H = 7.7 Hz, 2H, H7); 2.61 (t, JH–H = 7.7 Hz, 2H, H8); 13C-NMR (101
MHz, CDCl3) d: 173.6 (C9); 154.0 (C1); 132.6 (C4); 129.4 (C5 + C3); 115.3
(C2 + C6); 51.6 (C10); 36.0 (C8); 30.1 (C7).
2-Propyl-3-(4-hydroxyphenyl)propionate 7g: To a stirred solution of 3.34 g
(20.1 mmol) 3-(4-hydroxyphenyl)propionic acid 51 in 150 ml (1.95 mol) 2-propanol
HCl-gas was blubbed through. The methanol solution started to boil. After 1 h the
addition of HCl was stopped and the reaction was kept at room temperature was further
16 h. The solvent was removed and the remaining residue was chromatographed
(CH2Cl2/MeOH-gradient 0 to 20 %). The product was isolated as a colorless solid.
1
Yield: 62%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.71; H-NMR (400 MHz, DMSO-d6)
3
d: 9.12 (s, 1H, aryl-OH); 6.98 (d, JH–H = 8.5 Hz, 2H, H3-aryl + H5-aryl); 6.64 (d,
3
3JH–H = 8.5 Hz, 2H, H4-aryl + H6-aryl); 4.84 (sept, JH–H = 6.3 Hz, 1H, H10); 2.70 (t,
3
3
3JH–H = 7.5 Hz, 2H, H7); 2.47 (t, JH–H = 7.5 Hz, 2H, H8); 1.12 (d, JH–H = 6.3 Hz,
6H, H11a,b); 13C-NMR (101 MHz, CDCl3) d: 171.9 (C9); 155.7 (C1); 132.6 (C4);
129.2 (C5 + C3); 115.2 (C2 + C6); 67.1 (C10); 36.0 (C8); 29.7 (C7); 21.7 (C11a,b); IR
(NaCl) nꢂ1: 3412, 3024, 1712 (CO), 1613, 1595, 1519, 1449, 1377, 1298, 1266, 1149,
1108, 904, 837, 820, 609.
Benzyl-3-(4-hydroxyphenyl)propionate 7h: 7.00 g (42.1 mmol) 3-(4-Hydro-
xyphenyl)propionic acid 51, 13 ml benzyl alcohol and 3 drops of 85 % phosphoric acid
in 80 ml toluene were heated under reflux. The formed water during the esterification
was continuously destilled off in a Dean-Stark-apparatus at 145–1508C for 10h. Then
the excess of benzyl alcohol was evaporated in a vacuo. The residue was taken up in
diethylether and washed with a saturated sodium bicarbonate solution and water and
was subsequently dried with sodium sulfate. The crude reaction product was purified by
distillation in a vacuo at 185–1908C. The product was isolated as a yellowish oil.
1
Yield: 73%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.85; H-NMR (400 MHz, DMSO-d6)
3
d: 9.15 (s, 1H, aryl-OH); 7.36–7.27 (m, 5H, Ph); 6.98 (d, JH–H = 8.4 Hz, 2H, H3-
3
aryl + H5-aryl); 6.64 (d, JH–H = 8.5 Hz, 1H, H4-aryl + H6-aryl); 5.06 (s, 2H, H10);
3
3
2.74 (t, JH–H = 7.5 Hz, 2H, H7); 2.60 (t, JH–H = 7.4 Hz, 2H, H8); 13C-NMR (101
MHz, DMSO-d6) d: 172.4 (C9); 155.8 (C1); 136.4 (C11); 130.6 (C4); 129.3 (C5 + C3);
128.6 (2 ꢀ Cm-Ph); 128.1 (Cp-Ph); 128.0 (2 ꢀ Co-Ph); 115.3 (C2 + C6); 65.5 (C10);
35.7 (C8); 29.7 (C7).
t-Butyl-3-(4-hydroxyphenyl)propionat 7i: Phenol 7i was prepared as 7e. The
product was isolated as a colorless solid. Yield: 73%; TLC: Rf (CH2Cl2/MeOH 9:1
1
3
v/v): 0.79; H-NMR (400 MHz, CDCl3) d: 7.05 (d, JH–H = 8.5 Hz, 2H, H3-aryl + H5-
aryl); 6.75 (d, JH–H = 8.5 Hz, 2H, H4-aryl + H6-aryl); 2.84 (t, JH–H = 7.7 Hz, 2H,
3
3
3
H7); 2.51 (t, JH–H = 7.7 Hz, 2H, H8); 1.42 (s, 9H; H-tBu); 13C-NMR (101 MHz,
CDCl3) d: 172.7 (C9); 154.3 (C1); 132.4 (C4); 129.3 (C5 + C3); 115.2 (C2 + C6); 73.5
(C10); 37.4 (C8); 30.3 (C7); 28.0 (C11).
2-(2-Hydroxyphenyl)ethylacetate 7j: To 1 mmol of the phenol 11 in 15 ml of a
30 % solution of ethylacetate in n-hexane 100 mg of the NaHSO4ꢀ SiO2-catalyst was
added. The reaction temperature was increased to 678C. When the starting material was
comsumed (18 h) completely the reaction was stopped. The catalyst was filtered of and

ORDER
REPRINTS
100
Meier et al.
washed with CH2Cl2. The organic phases were evaporated and the oily crude product
was chromatographed (CH2Cl2-methanol-gradient 0–20 %). The product was isolated
as a colorless, crystalline solid. Yield: 87%; TLC: Rf (n-hexane/ethylacetate 3:2 v/v):
1
3
0.44; H-NMR (400 MHz, CDCl3) d: 8.08 (s, 1H, aryl-OH); 7.13 (ddd, 2ꢃ JH–H = 7.7
4
3
4
Hz, JH–H = 1.6 Hz, 1H, H5-aryl); 7.12 (dd, JH–H = 7.5 Hz, JH–H = 1.6 Hz, 1H, H3-
3
aryl); 6.88 (dd, JH–H = 7.7 Hz, JH–H = 1.3 Hz, 1H, H4-aryl); 6.84 (dd, JH–H = 7.7
4
3
4
3
Hz, JH–H = 1.0 Hz, 1H, H6-aryl); 4.29 (t, JH–H = 7.0 Hz, 2H, H8); 2.97 (t,
3JH–H = 7.0 Hz, 2H, H7); 2.09 (s, 3H, H10); 13C-NMR (101 MHz, CDCl3) d: 172.0
(C9); 154.8 (C1); 130.9 (C3); 128.3 (C5); 123.6 (C2); 120.7 (C4); 115.9 (C6); 64.5
(C8); 30.1 (C7); 21.0 (C10).
2-(2-Hydroxyphenyl)ethyllevulinate 7k: Phenol 7k was prepared as described
for 7j. The reaction time here was 72 h. The product was isolated as a colorless oil.
1
Yield: 82%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.63; H-NMR (400 MHz, DMSO-d6)
3
3
d: 2.08 (s, 3H, H13), 2.43 (t, 2H, JHH = 6.5 Hz, H10), 2.68 (t, 2H, JHH = 6.5 Hz,
H11) 2.81 (t, 2H, JHH = 7.1 Hz, H7), 4.15 (t, 2H, JHH = 7.1 Hz, H8), 6.72 (ddd, 1H,
3
3
4
3
4
3JHH = 7.8 Hz, JHH = 1.2 Hz, H4), 6.79 (dd, 1H, JHH = 7.8 Hz, JHH = 1.2 Hz, H6),
3
4
3
7.03 (ddd, 1H, JHH = 7.8 Hz, JHH = 1.7 Hz, H5), 7.07 (dd, 1H, JHH = 7.8 Hz,
4JHH = 1.7 Hz, H3), 9.38 (s, 1H, aryl-OH); 13C-NMR (100 MHz, DMSO-d6) d: 28.02
(C10), 29.61 (C7), 29.88 (C13), 37.75 (C11), 63.57 (C8), 115.28 (C6), 119.27 (C4),
124.07 (C2), 127.92 (C5), 130.90 (C3), 155.71. (C1), 172.54 (C9), 207.14 (C12).
2-(2-Hydroxyphenyl)ethylpivalate 7l: A mixture of 1.0 mmol of the phenol and
3-pivaloyl-1,3-thiazolidine-2-thion (‘‘twisted-amide’’) was stirred for 48 h at 658C in
20 ml of dry toluene. After completion of the reaction, the solvent was evaporated and
the residue was chromatographed twice on a chromatotron on silica gel (1. eluent: n-
hexane/ethylacetate 3:1 v/v; 2. eluent: n-hexane/diethylether 7:3 v/v). The product was
isolated as a colorless oil. Yield: 80%; TLC: Rf (n-hexane/Et2O 7:3 v/v): 0.32;
3
1H-NMR (400 MHz, CDCl3) d: 8.01 (s, 1H, aryl-OH); 7.12 (ddd, 2ꢃ JH–H = 7.7 Hz,
3
4
4JH–H = 1.8 Hz, 1H, H5-aryl); 7.10 (dd, JH–H = 7.5 Hz, JH–H = 1.8 Hz, 1H, H3-
3
4
aryl); 6.85 (ddd, 2ꢃ JH–H = 7.4 Hz, JH–H = 1.2 Hz, 1H, H4-aryl); 6.83 (dd,
3JH–H = 7.9 Hz, JH–H = 1.1 Hz, 1H, H6-aryl); 4.27 (t, JH–H = 7.1 Hz, 2H, H8);
4
3
2.95 (t, JH–H = 7.1 Hz, 2H, H7); 1.19 (s, 9H, H-tBu); 13C-NMR (101 MHz, CDCl3) d:
3
176.8 (C9); 154.5 (C1); 130.9 (C3); 128.2 (C5); 123.5 (C2); 120.5 (C4); 115.8 (C6);
64.3 (C8); 38.8 (C10); 30.1 (C7); 27.1 (C11a–c); IR (film) nꢂ1: 3418, 1704 (CO),
1481, 1458, 1292, 1233, 1170, 753.
2-(4-Hydroxyphenyl)ethylacetate 7m: Phenol 7m was prepared as 7j. The
reaction time here was 18 h. The product was isolated as a colorless, crystalline solid.
Yield: 87%; TLC: Rf (n-hexane/ethylacetate 3:2 v/v): 0.44; 1H-NMR (400 MHz, CDCl3)
3
4
d: 8.08 (s, 1H, aryl-OH); 7.13 (ddd, 2ꢃ JH–H = 7.7 Hz, JH–H = 1.6 Hz, 1H, H5-aryl);
3
7.12 (dd, JH–H = 7.5 Hz, JH–H = 1.6 Hz, 1H, H3-aryl); 6.88 (dd, JH–H = 7.6 Hz,
4
3
3
4
4JH–H = 1.3 Hz, 1H, H4-aryl); 6.84 (dd, JH–H = 7.7 Hz, JH–H = 1.0 Hz, 1H, H6-aryl);
3
3
4.29 (t, JH–H = 7.0 Hz, 2H, H8); 2.97 (t, JH–H = 7.0 Hz, 2H, H7); 2.09 (s, 3H, H10);
13C-NMR (101 MHz, CDCl3) d: 172.0 (C9); 154.8 (C1); 130.9 (C3); 128.3 (C5); 123.6
(C2); 120.7 (C4); 115.9 (C6); 64.5 (C8); 30.1 (C7); 21.0 (C10).

ORDER
REPRINTS
cycloSal-Pronucleotides
101
2-(4-Hydroxyphenyl)ethylpivalate 7n: Phenol 7n was prepared as 7l. The
product was isolated as a colorless oil. Yield: 51%; TLC: Rf (n-hexane/ethylacetate 3:1
1
3
v/v): 0.81; H-NMR (400 MHz, CDCl3) d: 7.09 (d, JH–H = 8.6 Hz, 2H, H3-aryl + H5-
aryl); 6.78 (d, JH–H = 8.4 Hz, 2H, H4-aryl + H6-aryl); 4.24 (t, JH–H = 6.9 Hz, 2H,
3
3
3
H8); 2.87 (t, JH–H = 6.9 Hz, 2H, H7); 1.17 (s, 9H, H-tBu); 13C-NMR (101 MHz,
CDCl3) d: 177.2 (C9); 154.4 (C1); 130.0 (C3 + C5); 129.8 (C4); 115.3 (C2 + C6); 65.2
(C8); 38.7 (C10); 34.2 (C7); 27.1 (C-tBu); IR (film) nꢂ1: 3403, 2973, 1704, 1517,
1292, 1225, 1169.
General Procedure for the Preparation of the Salicylalcolhol 6: A mixture of
the phenol 7 (1 equiv.), phenylboronic acid (1.2 equiv.), p-formaldehyde (2 equiv.) and
propionic acid (0.5 equiv.) was heated in dry toluene under reflux. The formed water
was destilled of in a Dean-Stark-apparatus. After 2 h further 2 equiv. of p-formal-
dehyde were added in portions. Often this procedure was repeated two to three times.
After complete comsumption of the phenol, the solvent was removed by evaporation
and the remaining material was dissolved in CH2Cl2. The organic phase was washed
with 10% sodium carbonate solution and with water. After evaporation of the organic
solvent, the product was obtained as yellowish oil that crystallizes upon cooling. This
material was generally pure enough to continue immediately with the oxidation/
hydrolysis procedure. Then, the 2-phenyl-4H-benzo[1.3.2]-dioxaborine derivative was
stirred with 30% H2O2-solution in THF for 30 min. The reaction mixture was added to
water and this mixture was extracted several times with diethylether. The collected
ether phases were washed with a sodium hydrogensulfite solution and then dried by
addition of sodium sulfate. The ether was removed and the residue was
chromatographed using n-hexane-diethylether gradients of 10–60%.
Methyl-3-(2-hydroxy-3-hydroxymethyl-phenyl)propionate 6a: The product
was isolated as a colorless oil. Yield: 77%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.67;
3
1H-NMR (250 MHz, CDCl3) d: 8.11 (s, 1H, aryl-OH); 7.05 (d, JH–H = 7.6 Hz, 1H,
3
3
H4-aryl); 6.95 (d, JH–H = 7.6 Hz, 1H, H6-aryl); 6.79 (dd, 2ꢃ JH–H = 7.3 Hz, 1H, H5-
3
aryl); 4.79 (s, 2H, H7); 3.68 (s, 3H, H11); 2.96 (t, JH–H = 7.0 Hz, 2H, H8); 2.63 (t,
3JH–H = 7.0 Hz; 2H, H9); 13C-NMR (63 MHz, CDCl3) d: 175.2 (C10); 153.9 (C2);
130.0 (C4); 127.7 (C1); 126.4 (C6); 125.8 (C3); 119.9 (C5); 64.2 (C7); 51.9 (C11);
34.4 (C9); 25.0 (C8); IR (film) nꢂ1: 3358 (OH), 1712 (C = O), 1595, 1465, 1369, 1227,
1083, 1006, 845, 779, 750; UV (CH3CN): lmax: 275, 208 nm; MS(ESIꢂ, m/z): calc.:
210.2 (M), found: 209.4 (M–H+).
Ethyl-3-(2-hydroxy-3-hydroxymethyl-phenyl)propionate 6b: The product was
1
isolated as a colorless oil. Yield: 88%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.63; H-
3
NMR (400 MHz, DMSO-d6) d: 8.51 (s(br), 1H, aryl-OH); 7.06 (dd, JH–H = 7.5 Hz,
3
4
4JH–H = 1.6 Hz, 1H, H4-aryl); 6.96 (dd, JH–H = 7.5 Hz, JH–H = 1.6 Hz, 1H, H6-
3
aryl); 6.72 (dd, 2ꢃ JH–H = 7.6 Hz, 1H, H5-aryl); 5.38 (s(br), 1H, Bn-OH); 4.56 (s, 2H,
3
H7); 4.03 (q, JH–H = 7.1 Hz, 2H, H11); 2.80 (t, JH–H = 7.7 Hz; 2H, H8); 2.52 (t,
3
3JH–H = 7.7 Hz, 2H, H9); 1.15 (t, JH–H = 7.1 Hz, 3H, H12); 13C-NMR (101 MHz,
3
CDCl3) d: 172.7 (C10); 152.6 (C2); 128.4 (C4); 128.3 (C1); 127.4 (C3); 125.8 (C6);
119.4 (C5); 60.2 (C7); 60.0 (C11); 33.9 (C9); 25.5 (C8); 14.3 (C12).

ORDER
REPRINTS
102
Meier et al.
Isopropyl-3-(2-hydroxy-3-hydroxymethyl-phenyl)propionate 6c: The product
1
was isolated as a yellow oil. Yield: 49%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.72; H-
3
NMR (400 MHz, DMSO-d6) d: 8.50 (s(br), 1H, aryl-OH); 7.06 (dd, JH–H = 7.5 Hz,
4JH–H = 1.7 Hz, 1H, H4-aryl); 6.96 (dd, JH–H = 7.5 Hz, JH–H = 1.7 Hz, 1H, H6-
3
4
3
aryl); 6.72 (dd, JH–H = 7.5 Hz, 1H, H5-aryl); 5.38 (s(br), 1H, Bn-OH); 4.86 (sept,
3JH–H = 6.3 Hz, 1H, H11); 4.56 (s, 2H, H7); 2.79 (t, JH–H = 7.7 Hz, 2H, H8); 2.49 (t,
3
3JH–H = 7.7 Hz, 2H, H9); 1.14 (d, JH–H = 6.3 Hz, 6H, H12a,b); 13C-NMR (125 MHz,
3
DMSO-d6) d: 172.2 (C10); 152.6 (C2); 128.4 (C4); 128.2 (C1); 127.3 (C3); 125.8 (C6);
119.3 (C5); 67.2 (C11); 60.2 (C7); 34.2 (C9); 25.5 (C8); 21.8 (C12a,b); UV (CH3CN)
l
max: 275, 211 nm; IR (film) nꢂ1 [cmꢂ1]: 3352 (OH), 1705 (C = O), 1596, 1467, 1375,
1268, 1228, 1107, 1083, 1009, 940, 836, 778, 749.
Benzyl-3-(2-hydroxy-3-hydroxymethyl-phenyl)propionate 6d: The product was
isolated as a yellow solid. Yield: 86%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.56; Rf (n-
1
hexan/diethylether 1:1 v/v): 0.31; H-NMR (400 MHz, DMSO-d6) d: 8.49 (s(br), 1H,
aryl-OH); 7.05 (dd, JH–H = 7.8 Hz, JH–H = 2.2 Hz, 1H, H4-aryl); 6.96 (dd,
3
4
4
3
3JH–H = 7.6 Hz, JH–H = 2.0 Hz, 1H, H6-aryl); 6.72 (dd, JH–H = 7.5 Hz, 1H, H5-
3
aryl); 5.37 (s(br), 1H, Bn-OH); 4.55 (s, 2H, H7); 2.76 (t, JH–H = 7.8 Hz, 2H, H8); 2.43
(t, JH–H = 7.7 Hz, 2H, H9); 13C-NMR (101 MHz, DMSO-d6) d: 172.1 (C10); 152.6
(C2); 128.4 (C4); 128.2 (C1); 127.5 (C3); 125.8 (C6); 119.3 (C5); 79.8 (C11); 60.3
(C7); 35.1 (C9); 28.0 (C12a–c); 25.6 (C8).
3
t-Butyl-3-(2-hydroxy-3-hydroxymethyl-phenyl)propionate 6e: The product was
1
isolated as a yellowish oil. Yield: 33%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.77; H-
NMR (400 MHz, DMSO-d6) d: 8.52 (s(br), 1H, aryl-OH); 7.38–7.29 (m, 5H, H-
pharyl); 7.07 (dd, JH–H = 7.5 Hz, JH–H = 1.8 Hz, 1H, H4-aryl); 6.96 (dd, JH–H = 7.5
3
4
3
4
Hz, JH–H = 1.9 Hz, 1H, H6-aryl); 6.72 (dd, JH–H = 7.5 Hz, 1H, H5-aryl); 5.37 (t(br),
3
3
3JH–H = 4.2 Hz, 1H, Bn-OH); 5.07 (s, 2H, H11); 4.55 (d, JH–H = 4.2 Hz, 2H, H7);
3
3
3
2.83 (t, JH–H = 7.7 Hz, 2H, H8); 2.61 (dd, JH–H = 8.1 Hz, JH–H = 7.1 Hz, 2H, H9);
13C-NMR (101 MHz, DMSO-d6) d: 172.5 (C10); 152.6 (C2); 136.4 (C12); 128.6
(2 ꢃ Cm-Ph); 128.4 (C4); 128.3 (C1); 128.1 (2 ꢃ Co-Ph); 127.2 (C3); 125.8 (C6);
119.4 (C5); 65.5 (C11); 60.2 (C7); 33.8 (C9); 25.4 (C8).
Methyl-3-(3-hydroxymethyl-4-hydroxyphenyl)propionate 6f: The product was
1
isolated as a colorless solid. Yield: 80%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.53; H-
4
NMR (400 MHz, DMSO-d6) d: 9.09 (s(br), 1H, aryl-OH); 7.10 (d, JH–H = 2.4 Hz, 1H,
H6-aryl); 6.85 (dd, JH–H = 8.1 Hz, JH–H = 2.4 Hz, 1H, H4-aryl); 6.64 (d, JH–H = 8.3
3
4
3
3
Hz, 1H, H3-aryl); 4.88 (t, JH–H = 5.0 Hz, 1H, Bn-OH); 4.43 (d, JH–H = 4.8 Hz, 2H,
3
3
3
H7); 3.56 (s, 3H, H11); 2.73 (t, JH–H = 7.7 Hz, 2H, H8); 2.53 (t, JH–H = 7.6 Hz, 2H,
H9); 13C-NMR (101 MHz, DMSO-d6) d: 172.9 (C10); 152.6 (C2); 130.5 (C5); 128.2
(C1); 127.3 (C4); 127.0 (C6); 114.6 (C3); 58.4 (C7); 51.4 (C11); 35.6 (C9); 29.9 (C8).
2-Propyl-3-(3-hydroxymethyl-4-hydroxyphenyl)propionate 6g: The product
was isolated as a colorless oil. Yield: 93%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.61;
4
1H-NMR (400 MHz, DMSO-d6) d: 9.08 (s(br), 1H, aryl-OH); 7.10 (d, JH–H = 2.3 Hz,
1H, H6-aryl); 6.85 (dd, JH–H = 8.1 Hz, JH–H = 2.4 Hz, 1H, H4-aryl); 6.64 (d,
3
4
3
3JH–H = 8.1 Hz, 1H, H3-aryl); 4.85 (sept, JH–H = 6.3 Hz, 2H, H11 + Bn-OH); 4.43 (d,

ORDER
REPRINTS
cycloSal-Pronucleotides
103
3
3
3JH–H = 3.4 Hz, 2H, H7); 2.72 (t, JH–H = 7.5 Hz, 2H, H8); 2.47 (t, JH–H = 7.5 Hz,
2H, H9); 1.14 (d, JH–H = 6.3 Hz, 6H, H-i-Pr); 13C-NMR (101 MHz, DMSO-d6) d:
172.0 (C10); 152.6 (C2); 130.5 (C5); 128.4 (C1); 127.4 (C4); 127.1 (C6); 114.6 (C3);
67.1 (C11); 58.4 (C7); 36.2 (C9); 30.0 (C8); 21.8 (C-i-Pr).
3
Benzyl-3-(3-hydroxymethyl-4-hydroxyphenyl)propionate 6h: The product was
1
isolated as a yellowish solid. Yield: 87%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.60; H-
4
NMR (400 MHz, DMSO-d6) d: 9.10 (s(br), 1H, aryl-OH); 7.10 (d, JH–H = 2.3 Hz, 1H,
H6-aryl); 6.85 (dd, JH–H = 8.2 Hz, JH–H = 2.3 Hz, 1H, H4-aryl); 6.63 (d, JH–H = 8.1
3
4
3
3
Hz, 1H, H3-aryl); 4.88 (t, JH–H = 4.9 Hz, 1H, Bn-OH); 4.42 (d, JH–H = 4.9 Hz, 2H,
3
3
3
H7); 2.68 (t, JH–H = 7.5 Hz, 2H, H8); 2.41 (t, JH–H = 7.5 Hz, 2H, H9); 1.35 (s, 9H,
H12a–c); 13C-NMR (101 MHz, DMSO-d6) d: 173.7 (C10); 152.5 (C2); 130.6 (C5);
128.4 (C1); 127.4 (C4); 127.1 (C6); 114.5 (C3); 79.7 (C11); 58.4 (C7); 37.1 (C9); 30.2
(C8); 27.9 (C12a–c).
t-Butyl-3-(3-hydroxymethyl-4-hydroxyphenyl)propionate 6i: The product was
1
isolated as a yellowish solid. Yield: 87%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.60; H-
4
NMR (400 MHz, DMSO-d6) d: 9.10 (s(br), 1H, aryl-OH); 7.10 (d, JH–H = 2.3 Hz, 1H,
H6-aryl); 6.85 (dd, JH–H = 8.2 Hz, JH–H = 2.3 Hz, 1H, H4-aryl); 6.63 (d, JH–H = 8.1
3
4
3
3
Hz, 1H, H3-aryl); 4.88 (t, JH–H = 4.9 Hz, 1H, Bn-OH); 4.42 (d, JH–H = 4.9 Hz, 2H,
3
3
3
H7); 2.68 (t, JH–H = 7.5 Hz, 2H, H8); 2.41 (t, JH–H = 7.5 Hz, 2H, H9); 1.35 (s, 9H,
H-tBu); 13C-NMR (101 MHz, DMSO-d6) d: 173.7 (C10); 152.5 (C2); 130.6 (C5); 128.4
(C1); 127.4 (C4); 127.1 (C6); 114.5 (C3); 79.7 (C11); 58.4 (C7); 37.1 (C9); 30.2 (C8);
27.9 (C-tBu).
2-(2-Hydroxy-3-hydroxymethylphenyl)ethylacetate 6j: The product was isolat-
1
ed as a colorless oil. Yield: 81%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.51; H-NMR
3
4
(400 MHz, CDCl3) d: 7.73 (s(br), 1H, aryl-OH); 7.09 (dd, JH–H = 7.5 Hz, JH–H = 1.4
Hz, 1H, H4-aryl); 6.95 (dd, JH–H = 7.5 Hz, JH–H = 1.4 Hz, 1H, H6-aryl); 6.80 (dd,
3
4
3
3
2ꢀ JH–H = 7.5 Hz, 1H, H5-aryl); 4.86 (s, 2H, H7); 4.31 (t, JH–H = 7.1 Hz, 2H, H9);
2.98 (t, JH–H = 7.1 Hz, 2H, H8); 2.05 (s, 3H, H11); 13C-NMR (101 MHz, DMSO-d6)
3
d: 170.6 (C10); 153.0 (C2); 129.8 (C4); 128.0 (C6); 125.6 (C1); 125.3 (C3); 119.7
(C5); 63.5 (C9); 60.3 (C7); 29.6 (C8); 21.1 (C11).
2-(2-Hydroxy-3-hydroxymethylphenyl)ethyllevulinate 6k: The product was
1
isolated as a yellowish oil. Yield: 14%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.53; H-
NMR (400 MHz, DMSO-d6) d: 2.03 (s, 3H, H14), 2.43 (t, 2H, 3JHH = 6.5 Hz, H12), 2.68
(t, 2H, 3JHH = 6.5 Hz, H11), 2.86 (t, 2H, 3JHH = 7.0 Hz, H8), 4.15 (t, 2H, 3JHH = 7.0 Hz,
3
H9), 4.59 (s, 2H, H7), 5.30–5.70 (br, 1H, benzyl-OH), 6.76 (dd, 1H, JHH = 7.4 Hz,
3
4
4JHH = 1.3 Hz, H4), 6.77 (dd, 1H, JHH = 7.5 Hz, JHH = 1.3 Hz, H6), 6.99–7.11 (m,
2H, H4, H6), 8.40–8.80 (br, 1H, aryl-OH); 13C-NMR (100 MHz, DMSO-d6) d: 28.01
(C11), 29.47 (C8), 29.89 (C14), 37.75 (C12), 60.37 (C7), 63.72 (C9), 119.58 (C5),
122.54 (C1)124.77 (C3), 126.31(C6), 129.26 (C4), 153.02. (C2), 172.55 (C10), 207.14
(C13); MS (ESI+, m/z): calc.: 266.12 (M), found: 289.15 (M + Na+).
2-(2-Hydroxy-3-hydroxymethylphenyl)ethylpivalate 6l: The product was iso-
1
lated as a yellowish oil. Yield: 86%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.70; H-NMR

ORDER
REPRINTS
104
Meier et al.
3
4
(400 MHz, DMSO-d6) d: 7.08 (dd, JH–H = 7.5 Hz, JH–H = 1.8 Hz, 1H, H4-aryl); 6.98
3
4
(dd, JH–H = 7.5 Hz, JH–H = 1.8 Hz, 1H, H6-aryl); 6.74 (dd, 23JH–H = 7.5 Hz, 1H,
3
3
H5-aryl); 4.56 (s, 2H, H7); 4.15 (t, JH–H = 6.9 Hz, 2H, H9); 2.86 (t, JH–H = 6.9 Hz,
2H, H8); 1.08 (s, 9H, H12a–c); 13C-NMR (101 MHz, DMSO-d6) d: 177.6 (C10); 152.8
(C2); 129.4 (C4); 128.3 (C1); 126.1 (C6); 124.7 (C3); 119.3 (C5); 63.5 (C9); 60.2 (C7);
38.3 (C11); 29.3 (C8); 27.1 (C12a–c).
2-(3-Hydroxymethyl-4-hydroxyphenyl)ethylacetate 6m: The product was iso-
1
lated as a colorless solid. Yield: 95%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.51; H-
4
NMR (400 MHz, DMSO-d6) d: 9.16 (s, 1H, aryl-OH); 7.13 (d, JH–H = 2.1 Hz, 1H,
H6-aryl); 6.89 (dd, JH–H = 8.1 Hz, JH–H = 2.3 Hz, 1H, H4-aryl); 6.67 (d, JH–H = 8.1
3
4
3
3
Hz, 1H, H3-aryl); 4.92 (t, JH–H = 5.5 Hz, 1H, Bn-OH); 4.44 (d, JH–H = 5.4 Hz, 2H,
3
3
3
H7); 4.11 (t, JH–H = 7.1 Hz, 2H, H9); 2.75 (t, JH–H = 7.1 Hz, 2H, H8); 1.97 (s, 3H,
H11); 13C-NMR (101 MHz, DMSO-d6) d: 170.5 (C10); 152.8 (C2); 128.6 (C5); 127.9
(C1); 127.8 (C4); 127.7 (C6); 114.7 (C3); 65.0 (C9); 58.4 (C7); 34.0 (C8); 20.9 (C11).
2-(3-Hydroxymethyl-4-hydroxyphenyl)ethylacetate 6n: The product was iso-
1
lated as a yellowish solid. Yield: 72%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.61; H-
4
NMR (400 MHz, DMSO-d6) d: 9.14 (s, 1H, aryl-OH); 7.14 (d, JH–H = 2.1 Hz, 1H,
H6-aryl); 6.88 (dd, JH–H = 8.0 Hz, JH–H = 2.3 Hz, 1H, H4-aryl); 6.66 (d, JH–H = 8.1
3
4
3
3
3
Hz, 1H, H3-aryl); 4.90 (t, JH–H = 5.5 Hz, 1H, Bn-OH); 4.43 (d, JH–H = 5.5 Hz, 2H,
3
3
H7); 4.12 (t, JH–H = 6.9 Hz, 2H, H9); 2.75 (t, JH–H = 6.7 Hz, 2H, H8); 1.09 (s, 9H,
H12a–c); 13C-NMR (101 MHz, DMSO-d6) d: 177.5 (C10); 152.8 (C2); 130.6 (C5);
128.0 (C4); 128.0 (C1); 127.8 (C6); 114.5 (C3); 65.1 (C9); 58.4 (C7); 34.0 (C8); 30.9
(C11); 27.1 (C12a–c).
General procedure for the synthesis of the cyclic chlorophosphanes 13: The
reactions have been carried out as described previously in references 7 and 23. In all
cases, diethylether has been used as solvent. After evaporation of the solvent, the
chlorophosphanes were obtained as slightly yellow oils. 31P-NMR charcterisation
showed in all cases a resonance signal at ꢁ140 ppm. The crude reaction products have
been use directly for the synthesis of the triesters.
General procedure for the synthesis of the cycloSal-triesters 2–5: The
reactions have been carried out as described previously in reference 7. In all cases,
acetonitrile has been used as solvent. All reactions have been carried out in an inert
atmosphere at –208C. Two equivalents of the chlorophosphites 13 have been used.
Prior to the reaction, d4T 1 has been coevaporated twice with pyridine. Purification has
been done by chromatography on a Chromatotron on silica gel with EtOAc/MeOH and
CH2Cl2/MeOH gradients.
3-Methylpropionyl-cycloSal-d4TMP 3a: The product was isolated as a colorless
1
foam. Yield: 32%; TLC: Rf (CH2Cl2/MeOH) 9 : 1 v/v) 0.57; H-NMR (400 MHz,
DMSO-d6) d: 11.33, 11.32 (2s, 1H, NH), 7.26 (dddd, 1H, H4-aryl), 7.20 (q 1H1.D
H6), 7.18 (q, 1H2.D, H6), 7.16–7.08 (m, 2H, H6-aryl + H5-aryl), 6.79 (ddd, 1H1.D
,
,
H1’), 6.79 (ddd, 1H2.D, H1’), 6.40, 6.36 (2 ꢃ ddd 1H, H3’), 6.02 (ddd, 1H1.D, H2’), 6.00
(ddd, 1H2.D, H2’), 5.48 (dd, 1H1.D, H7-benzyl), 5.44 (dd, 1H1.D, H7-benzyl), 5.38 (dd

ORDER
REPRINTS
cycloSal-Pronucleotides
105
1H2.D, H7-benzyl), 5.35 (dd, 1H2.D, H7-benzyl), 4.98–4.93 (m, 1H, H4’), 4.34–4.24
(m, 2H, H5’), 3.57, 3.56 (2s, 3H, H11), 2.90–2.75 (m, 2H, H8), 2.59 (dd, 2H1.D, H9),
2.57 (dd, 2H2.D, H9), 1.63 (d, 3H1.D, H7), 1.59 (d, 3H2.D, H7); 13C-NMR (101 MHz,
DMSO-d6) d 172.5 (2 ꢃ C10), 163.9 (C4), 150.9, 150.8 (2 ꢃ C2), 148.1 (d,
C21.D-aryl), 147.9 (d, C22.D-aryl), 135.8, 135.7 (2 ꢃ C6), 133.0, 132.9 (2 ꢃ C3’),
130.3, 130.2 (2 ꢃ C4-aryl), 129.6, 129.5 (2 ꢃ C1-aryl), 127.6, 127.5 (2 ꢃ C2’), 124.5
(2 ꢃ C6-aryl), 124.3 (C5-aryl), 121.7, 121.6 (2 ꢃ C3-aryl), 109.9, 109.8 (2 ꢃ C5),
89.4, 89.3 (2 ꢃ C1’), 84.3 (2 ꢃ C4’), 68.8 (d, C5’1.D), 68.8 (d, C5’2.D), 68.4 (2d C7-
benzyl), 51.5 (2 ꢃ C11), 33.3, 33.2 (2 ꢃ C9), 24.3, 24.2 (2 ꢃ C8), 12.0, 11.9
(2 ꢃ C7); 31P-NMR (202 MHz, DMSO-d6) d ꢂ 7.53,–7.60; HPLC: tR = 11.3, 11.5
min (method I); tR = 10.4 min (method II); MS (ESI+, m/z): calc. 478.2 (M), found
478.9 (M + H+).
3-EtPr-cycloSal-d4TMP 3b: The product was isolated as a colorless foam.
1
Yield: 35%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.58; H-NMR: (500 MHz, DMSO-d6)
3
4
d: 11.32, 11.31 (2s, 1H, NH); 7.26 (dddd, JH–H = 7.1 Hz, 3ꢃ JH–H = 2.0 Hz, 1H, H4-
4
aryl); 7.20 (q, JH–H = 1.2 Hz, 1H1.D, H6); 7.18 (q, JH–H = 1.3 Hz, 1H2.D, H6); 7.15–
4
7.08 (m, 2H, H6-aryl + H5-aryl); 6.80–6.78 (m, 1H, H1’); 6.40, 6.36 (2ddd,
3
4
3
3JH–H = 6.0 Hz, JH–H = 1.8 Hz, JH–H = 1.8 Hz, 1H, H3’); 6.02 (ddd, JH–H = 6.0
3
4
3
3
Hz, JH–H = 2.5 Hz, JH–H = 1.3 Hz, 1H1.D, H2’); 6.00 (ddd, JH–H = 5.9 Hz, JH–
4
2
3
= 2.4 Hz, JH–H = 1.3 Hz, 1H2.D, H2’); 5.48 (dd, JH–H = 14.2 Hz, JH–P = 5.6 Hz,
1H1.D, H7-benzyl); 5.44 (dd, JH–H = 14.2 Hz, JH–P = 5.3 Hz, 1H1.D, H7-benzyl); 5.38
H
2
3
2
(dd, JH–H = 14.2 Hz, JH–P = 4.8 Hz, 1H2.D, H7-benzyl); 5.34 (dd, JH–H = 14.2 Hz,
3
2
3JH–P = 5.2 Hz, 1H2.D, H7-benzyl); 4.97–4.93 (m, 1H, H4’); 4.33–4.25 (m, 2H, H5’);
4.03, 4.02 (2q, JH–H = 7.1 Hz, 2H, H11); 2.88–2.77 (m, 2H, H8); 2.57 (dd,
3
3
4
2 ꢃ JH–H = 7.4 Hz, 2H, H9); 1.63, 1.58 (2d, JH–H = 1.3 Hz, 3H, H7); 1.14, 1.13 (2dd,
3JH–H = 7.1 Hz, 3H, H12); 13C-NMR (101 MHz, DMSO-d6) d: 173.1 (2 ꢃ C10); 163.9
2
(C4); 150.9, 150.8 (2 ꢃ C2); 148.1, 147.9 (2d, JC–P = 7.1 Hz, C2-aryl); 135.8, 135.7
(2 ꢃ C6); 133.0, 132.9 (2 ꢃ C3’); 130.3 (2 ꢃ C4-aryl); 129.7, 129.6 (2 ꢃ C1-aryl);
127.6, 127.5 (2 ꢃ C2’); 124.5 (2 ꢃ C6-Aryl); 124.3 (C5-aryl); 121.7, 121.6 (2 ꢃ C3-
aryl); 109.9, 109.8 (2 ꢃ C5); 89.4, 89.3 (2 ꢃ C1’); 84.3 (2 ꢃ C4’); 68.8 (2d,
2JC–P = 6.1 Hz, C5’); 68.4 (2d, JC–P = 6.6 Hz, C7-benzyl); 60.1 (C11); 33.5, 33.4
2
(2 ꢃ C9); 24.3, 24.2 (2 ꢃ C8); 14.2 (C12); 12.0, 11.9 (2 ꢃ C7); 31P-NMR (202 MHz,
DMSO-d6) d: ꢂ 7.52, –7.57; HPLC: tR = 12.8 min (method I); tR = 11.3 min (method
II); ); MS (ESI+, m/z): calc. 492.1298 (M), found 493.3 (M + H+).
3-i-PrPr-cycloSal-d4TMP 3c: The product was isolated as a colorless foam.
1
Yield: 54%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.60; H-NMR (500 MHz, DMSO-d6)
4
d: 11.35, 11.34 (2s, 1H, NH); 7.27–7.23 (m, 1H, H4-aryl); 7.20, 7.18 (2q, JH–H = 1.1
Hz, 1H, H6); 7.15–7.08 (m, 2H, H5-, H6-aryl); 6.80–6.78 (m, 1H, H1’); 6.40 (ddd,
3
4
3
3JH–H = 6.0 Hz, JH–H = 1.8 Hz, JH–H = 1.8 Hz, 1H1.D, H3’); 6.36 (ddd, JH–H = 5.9
3
4
3
Hz, JH–H = 1.8 Hz, JH–H = 1.8 Hz, 1H2.D, H3’); 6.02 (ddd, JH–H = 6.1 Hz,
4
3
3
3JH–H = 2.3 Hz, JH–H = 1.3 Hz, 1H1.D, H2’); 6.00 (ddd, JH–H = 6.0 Hz, JH–H = 2.5
4
2
3
Hz, JH–H = 1.3 Hz, 1H2.D, H2’); 5.48 (dd, JH–H = 14.1 Hz, JH–P = 4.8 Hz, 1H1.D
,
2
H7-benzyl); 5.44 (dd, JH–H = 14.2 Hz, JH–P = 4.3 Hz, 1H1.D, H7-benzyl); 5.38 (dd,
3
2JH–H = 12.4 Hz, JH–P = 4.8 Hz, 1H2.D, H7-benzyl); 5.34 (dd, JH–H = 12.6 Hz,
3
2
3JH–P = 4.5 Hz, 1H2.D, H7-benzyl); 4.97–4.93 (m, 1H, H4’); 4.85, 4.84 (2sept,

ORDER
REPRINTS
106
Meier et al.
3JH–H = 6.3 Hz, 1H, H11); 4.32–4.25 (m, 2H, H5’); 2.87–2.77 (m, 2H, H8); 2.54 (2dd,
4
3JH–H = 7.3 Hz, 2H, H9); 1.63, 1.57 (2d, JH–H = 1.1 Hz, 3H, H7); 1.14–1.11 (m, 6H,
H12a,b); 13C-NMR (101 MHz, DMSO-d6) d: 171.6, 171,5 (2 ꢃ C10); 163.9 (C4);
2
2
150.9, 150.8 (2 ꢃ C2); 148.0 (d, JC–P = 7.6 Hz, C2-aryl); 147.9 (d, JC–P = 8.1 Hz,
C2-aryl); 135.8 (2 ꢃ C6); 133.0 132.9 (2 ꢃ C3’); 130.3 (2 ꢃ C4-aryl); 129.6 (2 ꢃ C1-
aryl); 127.6, 127.5 (2 ꢃ C2’); 124.5 (2 ꢃ C6-aryl); 124.3 (C5-aryl); 121.7 (C3-aryl);
109.9, 109.8 (2 ꢃ C5); 89.4, 89.3 (2 ꢃ C1’); 84.4, 84.3 (2 ꢃ C4’); 68.8, 68.7
2
2
(2 ꢃ C5’); 68.4 (d, JC–P = 7.6 Hz, C7-benzyl); 68.4 (d, JC–P = 6.6 Hz, C7-benzyl);
67.5 (C11); 33.7 (2 ꢃ C9); 24.3 (2 ꢃ C8); 21.7 (C12a,b); 12.0, 11.9 (2 ꢃ C7); 31P-NMR
(202 MHz, DMSO-d6) d: ꢂ7.50, –7.53; HPLC: tR = 13.7 min (method I) tR = 12.3 min
(method II); ); MS (ESI+, m/z): calc. 506.1454 (M), found 507.2 (M + H+).
3-Benzylpropionyl-cycloSal-d4TMP 3d: The product was isolated as a
1
colorless foam. Yield 43%; H-NMR (500 MHz, DMSO-d6) d 11.33, 11.31 (2s, 1H,
NH), 7.36–7.28 (m, 5H, Ph) 7.26–7.23 (m, 1H, H4-aryl), 7.18 (q, 1H1.D, H6), 7.17 (q,
1H2.D, H6), 7.15–7.12 (m, 2H, H6-aryl), 7.09, 7.08 (2dd 1H, H5-aryl) 6.79–6.77 (m,
1H, H1’), 6.37 (ddd, 1H1.D, H3’), 6.32 (ddd, 1H2.D, H3’), 5.99 (ddd, 1H1.D, H2’), 5.97
(ddd, 1H2.D, H2’), 5.47 (dd, 1H1.D, H7-benzyl), 5.44 (dd, 1H1.D, H7-benzyl), 5.37 (dd,
1H2.D, H7-benzyl), 5.34 (dd, 1H2.D, H7-benzyl), 5.07, 5.06 (2s, 2H, H11) 4.94–4.90
(m, 1H, H4’), 4.32–4.22 (m, 2H, H5’), 2.92–2.80 (m, 2H, H8), 2.68–2.62 (m, 2H, H9),
1.64, 1.58 (2 ꢃ d, 3H, H7); 13C-NMR (101 MHz, DMSO-d6) d 172.4, 172,3
(2 ꢃ C10), 163.9 (C4), 150.8, 150.7 (2 ꢃ C2), 148.0 (2d, C2-aryl), 135.8, 135.7
(2 ꢃ C6), 132.9 (2 ꢃ C3’), 131.2 (C12), 130.3 (2 ꢃ C4-aryl), 129.5 (2 ꢃ C1-aryl),
128.6 (2 ꢃ Cm-Ph), 128.1 (2 ꢃ Co-Ph), 128.1 (Cp-Ph), 127.5 (2 ꢃ C2’), 124.5
(2 ꢃ C6-aryl), 124.3 (C5-aryl), 121.7 (C3-aryl), 109.9, 109.8 (2 ꢃ C5), 89.4, 89.3
(2 ꢃ C1’), 84.3, 84.2 (2 ꢃ C4’), 68.7 (2 ꢃ C5’), 68.4 (d, C7-benzyl), 68.4 (d,
C7-benzyl), 65.7 (C11), 33.4 (2 ꢃ C9), 24.3, 24.2 (2 ꢃ C8), 12.0, 11.9 (2 ꢃ C7);
31P-NMR (202 MHz, DMSO-d6) d ꢂ 7.57 (only 1 peak); Rf 0.64 (CH2Cl2/MeOH) 9 : 1
v/v); HPLC: tR = 15.0 min (method I); tR = 13.6 min (method II); MS (FAB, m/z):
calc. 554.15 (M), found 555.2 (M + H+), 555.1532 (M + H+, FAB-HR).
3-t-Butylpropionyl-cycloSal-d4TMP 3e: The product was isolated as a colorless
1
foam. Yield 73%; H-NMR (500 MHz, DMSO-d6) d 11.32, 11.31 (2s, 1H, NH), 7.27–
7.24 (m, 1H, H4-aryl), 7.20, 7.18 (2q, 1H, H6), 7.15–7.08 (m, 2H, H6-aryl + H5-aryl),
6.80–6.78 (m, 1H, H1’), 6.40, 6.36 (2ddd, 1H H3’), 6.02 (ddd, 1H1.D, H2’), 6.00 (ddd,
1H2.D, H2’), 5.47 (dd, 1H1.D, H7-benzyl), 5.44 (dd, 1H1.D, H7-benzyl), 5.37 (dd, 1H2.D
,
H7-benzyl), 5.35 (dd, 1H2.D, H7-benzyl), 4.97–4.93 (m, 1H, H4’), 4.34–4.25 (m, 2H,
H5’), 2.87–2.73 (m, 2H, H8), 2.47 (dd, 2H1.D, H9), 2.46 (dd, 2H2.D, H9), 1.64, 1.57 (2d,
3H, H7), 1.35, 1.34 (2s, 9H, H12a–c); 13C-NMR (101 MHz, DMSO-d6) d 171.4, 171,3
(2 ꢃ C10), 163.9 (C4), 150.8 (2 ꢃ C2), 148.0 (d, C2-aryl), 148.0 (d, C2-aryl), 135.8,
135.7 (2 ꢃ C6), 133.0 132.9 (2 ꢃ C3’), 130.3 (2 ꢃ C4-aryl), 129.7, 129.6 (2 ꢃ C1-
aryl), 127.5 (2 ꢃ C2’), 124.4 (2 ꢃ C6-aryl), 124.2 (C5-aryl), 121.7, 121.6 (C3-aryl),
109.8 (2 ꢃ C5), 89.3 (2 ꢃ C1’), 84.3 (2 ꢃ C4’), 80.0 (C11), 68.8 (d, C5’), 68.7 (d, C5’),
68.4 (d, C7-benzyl), 68.4 (d, C7-benzyl), 34.6, 34.5 (2 ꢃ C9), 27.9 (12a–c), 24.4
(2 ꢃ C8), 12.0, 11.9 (2 ꢃ C7); 31P-NMR (202 MHz, DMSO-d6) d ꢂ7.50, ꢂ7.53; Rf
0.46 (CH2Cl2/MeOH) 9 : 1 v/v); HPLC: tR = 14.7 min (method I); tR = 13.2 min
(method II); MS (FAB, m/z): calc. 520.1611 (M), found 521.2 (M + H+).

ORDER
REPRINTS
cycloSal-Pronucleotides
107
5-Methylpropionyl-cycloSal-d4TMP 3f: The product was isolated as a colorless
foam. Yield: 39%; TLC: Rf (CH2Cl2/MeOH 9 : 1 v/v) 0.52; 1H-NMR (500 MHz,
DMSO-d6) d 11.33, 11.32 (2s, 1H, NH), 7.23–7.18 (m, 1H, H4-aryl), 7.18, 7.15 (2q,
1H, H6), 7.13–7.11 (m, 1H, H6-aryl), 7.03, 7.00 (2d, 1H, H3-aryl), 6.79, 6.78 (2ddd,
1H, H1’), 6.40 (ddd, 1H1.D, H3’), 6.34 (ddd, 1H2.D, H3’), 6.01 (ddd, 1H1.D, H2’), 5.99
(ddd, 1H2.D, H2’), 5.45 (dd, 1H1.D, H7-benzyl), 5.41 (dd, 1H1.D, H7-benzyl), 5.35
(d(br), 1H2.D, H7-benzyl), 5.33 (d(br), 1H2.D, H7-benzyl), 4.96–4.91 (m, 1H, H4’),
4.34–4.22 (m, 2H, H5’), 3.57 (s, 3H, H11), 2.81 (dd, 2H, H8), 2.60 (dd, 2H, H9), 1.66,
1.59 (2d, 3H, H7); 13C-NMR (101 MHz, DMSO-d6) d 172.6 (2 ꢃ C10), 163.9
(2 ꢃ C4), 150.8 (C2), 148.0 (2d, C2-aryl), 137.0 (C5-aryl), 135.8 (2 ꢃ C6), 133.0,
132.9 (2 ꢃ C3’), 129.8, 129.7 (2 ꢃ C4-aryl), 129.0, 128.9 (2 ꢃ C1-aryl), 127.5, 127.4
(2 ꢃ C2’), 125.9 (2 ꢃ C6-aryl), 118.2, 118.1 (2 ꢃ C3-aryl), 109.8 (2 ꢃ C5), 89.3
(C1’), 84.3, 84.2 (2 ꢃ C4’), 68.5 (d, C5’1.D), 68.5 (d, C5’2.D), 68.4 (2d, C7-benzyl), 51.5
(C11), 34.8 (C9), 29.5 (C8), 12.1, 12.0 (2 ꢃ C7); 31P-NMR (202 MHz, DMSO-d6) d
ꢂ7.98, ꢂ8.04; HPLC: tR = 11.9, 12.0 min (method I); tR = 10.6 min (method II); MS
(FAB, m/z): calc. 478.1141 (M), found 479.4 (M + H+).
5-i-PrPr-cycloSal-d4TMP 3g: The product was isolated as a colorless foam.
1
Yield: 33%; TLC Rf (CH2Cl2/MeOH 9:1 v/v): 0.63; H-NMR (500 MHz, DMSO-d6) d:
11.32, 11.31 (2s, 1H, NH); 7.24–7.18 (m, 1H, H4-Aryl); 7.18, 7.15 (2q, 4JH–H = 1.1 Hz,
1H, H6); 7.13–7.11 (m, 1H, H6-aryl); 7.03 (d, 3JH–H = 8.6 Hz, 1H1.D, H3-aryl); 7.00 (d,
3
4
3JH–H = 8.4 Hz, 1H2.D, H3-aryl); 6.79 (ddd, JH–H = 3.6 Hz, 2ꢃ JH–H = 1.8 Hz, 1H1.D
,
H1’); 6.77 (ddd, 3JH–H = 3.7 Hz, 2ꢃ JH–H = 1.9 Hz, 1H2.D, H1’); 6.40 (ddd, 3JH–H = 6.0
4
3
4
3
3
Hz, JH–H = 1.8 Hz, JH–H = 1.8 Hz, 1H1.D, H3’); 6.33 (ddd, JH–H = 5.9 Hz, JH–H
=
=
1.7 Hz, 4JH–H = 1.7 Hz, 1H2.D, H3’); 6.00 (ddd, 3JH–H = 5.6 Hz, 3JH–H = 2.0 Hz, 4JH–H
2.0 Hz, 1H1.D, H2’); 5.99 (ddd, 3JH–H = 6.0 Hz, 3JH–H = 2.3 Hz, 4JH–H = 1.5 Hz, 1H2.D
,
=
2
3
2
H2’); 5.45 (dd, JH–H = 14.4 Hz, JH–P = 4.6 Hz, 1H1.D, H7-benzyl); 5.40 (dd, JH–H
14.2 Hz, JH–P = 4.1 Hz, 1H1.D, H7-benzyl); 5.35 (d(br), JH–H = 14.5 Hz, 1H2.D, H7-
3
2
2
benzyl); 5.32 (d(br), JH–H = 14.1 Hz, 1H2.D, H7-benzyl); 4.96–4.92 (m, 1H, H4’); 4.85
(2sept, 3JH–H = 6.2 Hz, 1H, H11); 4.34–4.21 (m, 2H, H5’); 2.80 (dd, 2ꢃ JH–H = 7.4 Hz,
3
2H, H8); 2.55 (dd, 2ꢃ JH–H = 7.5 Hz, 2H, H9); 1.66, 1.60 (2d, 4JH–H = 1.1 Hz, 3H, H7);
3
1.13, 1.12 (2d, JH–H = 6.2 Hz, 6H, H12a,b); 13C-NMR (101 MHz, DMSO-d6) d 172.6
3
(2 ꢃ C10); 163.9 (2 ꢃ C4); 150.8 (C2); 148.0 (2d, 2JC–P = 7.1 Hz, C2-aryl); 137.0 (C5-
aryl); 135.8 (2 ꢃ C6); 133.0, 132.9 (2 ꢃ C3’); 129.8, 129.7 (2 ꢃ C4-aryl); 129.0, 128.9
(2 ꢃ C1-aryl); 127.5, 127.4 (2 ꢃ C2’); 125.9 (2 ꢃ C6-aryl); 118.2, 118.1 (2 ꢃ C3-aryl);
2
109.8 (2 ꢃ C5); 89.3 (C1’); 84.3, 84.2 (2 ꢃ C4’); 68.5 (d, JC–P = 7.6 Hz, C5’1.D); 68.5
(d, 2JC–P = 6.1 Hz, C5’2.D); 68.4 (2d, 2JC–P = 6.6 Hz, C7-benzyl); 51.5 (C11); 34.8 (C9);
29.5 (C8); 12.1, 12.0 (2 ꢃ C7); 31P-NMR (202 MHz, DMSO-d6) d: ꢂ 8.00, ꢂ 8.02;
HPLC tR = 13.9 min (method I) tR = 12.4 min (method II); MS (FAB, m/z): calc.
506.1454 (M), found 507.4 (M + H+).
5-Benzylpropionyl-cycloSal-d4TMP 3h: The product was isolated as a color-
1
less foam. Yield: 59%; TLC: Rf (CH2Cl2/MeOH 9 : 1 v/v) 0.63; H-NMR (500 MHz,
DMSO-d6) d 11.33, 11.32 (2s, 1H, NH), 7.36–7.26 (m, 5H, Ph), 7.23–7.18 (m, 1H,
H4-aryl), 7.18, 7.15 (2q, 1H, H6), 7.11–7.09 (m, 1H, H6-aryl), 7.01, 6.99 (2d, 1H, H3-
aryl), 6.79 (ddd, 1H1.D, H1’), 6.77 (ddd, 1H2.D, H1’), 6.40, 6.34 (2ddd, 1H, H3’), 6.01
(ddd, 1H1.D, H2’), 5.99 (ddd, 1H2.D, H2’), 5.41 (dd, 1H1.D, H7-benzyl), 5.38 (dd, 1H1.D
,

ORDER
REPRINTS
108
Meier et al.
H7-benzyl), 5.33 (d(br), 1H2.D, H7-benzyl), 5.30 (d(br), 1H2.D, H7-benzyl), 5.06 (s, 2H,
H11), 4.96–4.92 (m, 1H, H4’), 4.34–4.22 (m, 2H, H5’), 2.83 (dd, 2H, H8), 2.67 (dd,
2H, H9), 1.66, 1.60 (2d, 3H, H7); 13C-NMR (101 MHz, DMSO-d6) d 172.1, 171.9
(2 ꢃ C10), 163.9 (2 ꢃ C4), 150.9 (2 ꢃ C2), 148.1 (C2-aryl), 136.9 (C12), 136.3 (C5-
aryl), 135.9, 135.8 (2 ꢃ C6), 133.0 (2 ꢃ C3’), 129.9, 129.8 (2 ꢃ C4-aryl), 128.6
(2 ꢃ Cm-Ph), 128.5 (C1-aryl), 128.2 (Cp-Ph), 128.1 (2 ꢃ Co-Ph), 127.5 (2 ꢃ C2’),
126.0, 125.9 (2 ꢃ C6-aryl), 118.2, 118.1 (2 ꢃ C3-aryl), 109.8 (2 ꢃ C5), 89.3 (C1’),
84.3, 84.2 (2 ꢃ C4’), 68.5, 68.4 (2d, C5’), 68.4 (d, C71.D-benzyl), 68.3 (d, C72.D
-
benzyl), 65.6 (C11), 35.0 (C9), 29.6 (C8), 12.1, 12.0 (2 ꢃ C7); 31P-NMR (202 MHz,
DMSO-d6) d ꢂ7.98, ꢂ8.05; HPLC tR = 15.3 min (method I) tR = 13.7 min (method
II); MS (FAB, m/z): calc. 554.1454 (M), found 555.2 (M + H+).
5-t-Butylpropionyl-cycloSal-d4TMP 3i: The product was isolated as a colorless
foam. Yield: 44%; TLC: Rf (CH2Cl2/MeOH 9 : 1 v/v) 0.49; 1H-NMR (500 MHz,
DMSO-d6) d 11.33, 11.32 (2s, 1H, NH), 7.23–7.18 (m, 1H, H4-aryl), 7.18, 7.15 (2q,
1H, H6), 7.12 (d, 1H, H6-aryl), 7.03 (d, 1H1.D, H3-aryl), 7.01 (d, 1H2.D, H3-aryl), 6.79
(ddd, 1H1.D, H1’), 6.77 (ddd, 1H2.D, H1’), 6.40 (ddd, 1H1.D, H3’), 6.34 (ddd, 1H2.D
,
H3’), 6.00 (ddd, 1H1.D, H2’), 5.99 (ddd, 1H2.D, H2’), 5.44, 5.41 (2dd, 1H1.D, H7-
benzyl), 5.35 (dd 1H2.D, H7-benzyl), 5.32 (dd, 1H2.D, H7-benzyl), 4.96–4.92 (m, 1H,
H4’), 4.33–4.22 (m, 2H, H5’), 2.77 (dd, 2H, H8), 2.48 (dd, 2H, H9), 1.67, 1.60 (2d, 3H,
H7), 1.34, 1.33 (2s, 9H, H-tBu); 13C-NMR (101 MHz, DMSO-d6) d 171.5 (C10), 163.9,
163.8 (2 ꢃ C4), 150.8 (2 ꢃ C2), 148.0 (C2-aryl), 137.1 (C5-aryl), 135.8 (2 ꢃ C6),
133.0, 132.9 (2 ꢃ C3’), 132.2 (C1-aryl), 129.8 (C4-aryl), 127.5, 127.4 (2 ꢃ C2’),
126.0, 125.9 (2 ꢃ C6-aryl), 118.1, 118.0 (2 ꢃ C3-aryl), 109.8 (2 ꢃ C5), 89.3 (C1’),
84.3, 84.2 (2 ꢃ C4’), 79.9 (C11), 68.5–68.3 (C5’ + C7-benzyl), 36.2 (C9), 29.8 (C8),
27.9 (2 ꢃ C-tBu), 12.1, 12.0 (2 ꢃ C7); 31P-NMR (202 MHz, DMSO-d6) d: ꢂ8.02
(only 1 Peak); HPLC tR = 14.6 min (method I) tR = 13.4 min (method II); MS (FAB,
m/z): calc. 520.1611 (M), found 521.3 (M + H+).
3-AcEt-cycloSal-d4TMP 5a: The product was isolated as a colorless foam.
1
Yield 31%; TLC Rf (CH2Cl2/MeOH 9:1 v/v): 0.61; H-NMR (500 MHz, DMSO-d6) d:
3
4
11.33, 11.31 (2s, 1H, NH); 7.29 (dddd, JH–H = 7.3 Hz, 3ꢃ JH–H = 1.7 Hz, 1H, H4-
4
aryl); 7.19, 7.18 (2q, JH–H = 1.3 Hz, 1H, H6); 7.16–7.10 (m, 2H, H5-aryl + H6-aryl);
3
3
4
6.80–6.78 (m, 1H, H1’); 6.40 (ddd, JH–H = 6.1 Hz, JH–H = 1.7 Hz, JH–H = 1.7 Hz,
3
3
4
1H1.D, H3’); 6.36 (ddd, JH–H = 6.0 Hz, JH–H = 1.8 Hz, JH–H = 1.8 Hz, 1H2.D, H3’);
3
3
4
6.02 (ddd, JH–H = 6.0 Hz, JH–H = 2.3 Hz, JH–H = 1.5 Hz, 1H1.D, H2’); 6.00 (ddd,
3JH–H = 6.0 Hz, JH–H = 2.2 Hz, JH–H = 1.7 Hz, 1H2.D, H2’); 5.48 (dd, JH–H = 14.3
Hz, JH–P = 7.7 Hz, 1H1.D, H7-benzyl); 5.45 (dd, JH–H = 14.3 Hz, JH–P = 7.3 Hz,
3
4
2
3
2
3
2
1H1.D, H7-benzyl); 5.38 (dd, JH–H = 14.0 Hz, JH–P = 7.4 Hz, 1H2.D, H7-benzyl); 5.35
3
2
3
(dd, JH–H = 14.2 Hz, JH–P = 8.0 Hz, 1H2.D, H7-benzyl); 4.96–4.93 (m, 1H, H4’);
4.32–4.26 (m, 2H, H5’); 4.22–4.12 (m, 2H, H9); 2.97–2.82 (m, 2H, H8); 1.96, 1.95
(2s, 3H, H11); 1.63, 1.59 (2d, JH–H = 1.3 Hz, 3H, H7); 13C-NMR (101 MHz, DMSO-
4
d6) d: 173.3, 173.0 (2 ꢃ C10); 163.9 (C4); 150.9, 150.8 (2 ꢃ C2); 148.3, 148.2
(2 ꢃ C2-aryl); 135.8 (2 ꢃ C6); 133.0 132.9 (2 ꢃ C3’); 131.1, 131.0 (2 ꢃ C4-aryl);
130.3, 130.2 (2 ꢃ C1-aryl); 127.6, 127.5 (2 ꢃ C2’); 124.8 (2 ꢃ C6-aryl); 124.3 (C5-
aryl); 121.7, 121.6 (2 ꢃ C3-aryl); 109.9, 109.8 (2 ꢃ C5); 89.4, 89.3 (2 ꢃ C1’); 84.3,
2
2
84.2 (2 ꢃ C4’); 68.8 (d, JC–P = 5.1 Hz, C5’1.D); 68.8 (d, JC–P = 6.1 Hz, C5’2.D); 68.4

ORDER
REPRINTS
cycloSal-Pronucleotides
109
2
2
(d, JC–P = 6.6 Hz, C7-benzyl); 68.4 (d, JC–P = 7.6 Hz, C7-benzyl); 63.0, 62.9
(2 ꢃ C9); 28.3 (2 ꢃ C8); 20.8 (2 ꢃ C11); 12.0, 11.9 (2 ꢃ C7); 31P-NMR (202 MHz,
DMSO-d6) d: ꢂ7.50, ꢂ7.64; HPLC: tR = 10.9 min (method I); tR = 10.3 min (method
II); MS (FAB, m/z): calc. 478.1141 (M), found 479.2 (M + H+).
3-LevEt-cycloSal d4TMP 5b: The product was isolated as a colorless foam.
1
Yield: 30%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v) 0.42; H-NMR (500 MHz, DMSO-d6) d:
1.61 (d, 3H, JHH = 1.3 Hz, thymine-H7), 1.66 (d, 3H, JHH = 1.1 Hz, thymine-H7),
4
4
3
3
2.08 (s, 2 ꢃ 3H, 2 ꢃ H14), 2.42 (t, 2H, JHH = 6.5 Hz, H12), 2.43 (t, 2H, JHH = 6.7
3
3
Hz, H12), 2.67 (t, 2H, JHH = 6.7 Hz, H11), 2.68 (t, 2H, JHH = 6.5 Hz, H11), 2.83–
2.96 (m, 2 ꢃ 2H, 2 ꢃ H8), 4.13–4.23 (m,2 ꢃ 2H, 2 ꢃ H9) 4.26–4.35 (m, 2 ꢃ 2H,
2
3
2 ꢃ H5’), 4.95–4.99 (m, 2 ꢃ 1H, 2 ꢃ H4’) 5.37 (dd, 1H, JHH = 12.5 Hz, JHP = 7.0
2
Hz, H7), 5.40 (dd, 1H, JHH = 12.5 Hz, JHP = 7.0 Hz, H7), 5.47 (dd, 1H, JHH = 14.2
3
2
3
Hz, JHP = 7.2 Hz, H7), 5.51 (dd, 1H, JHH = 14.2 Hz, JHP = 7.2 Hz, H7), 6.02 (ddd,
2
3
3
3
4
3
1H, JHH = 6.0 Hz, JHH = 1.6 Hz, JHH = 2.4 Hz, H2’), 6.04 (ddd, 1H, JHH = 6.0 Hz,
3JHH = 1.6 Hz, JHH = 2.4 Hz, H2’), 6.38 (ddd, 1H, JHH = 6.0 Hz, JHH = 1.6 Hz,
4
3
3
4JHH = 1.8 Hz, H3’), 6.41 (ddd, 1H, JHH = 6.0 Hz, JHH = 1.6 Hz, JHH = 1.8 Hz,
H3’), 6.80–6.83(m, 2 ꢃ 1H, 2 ꢃ H1’), 7.13–7.21 (m, 2 ꢃ 3H, thymine-H6, H4, H5),
7.29–7.33 (m, 2 ꢃ 1H, H6), 11.33 (s, 1 ꢃ 1H, 1xNH), 11.35 (s, 1 ꢃ 1H, 1 ꢃ NH);
13C-NMR (100 MHz, DMSO-d6) d: 11.71, 11.82 (2x thymine-C7), 27.53, 27.55
(2 ꢃ C12), 28.07 (2 ꢃ C8), 29.47 (2 ꢃ C14), 37.34 (2 ꢃ C11), 62.85, 62.89 (2 ꢃ C9),
3
3
4
3
3
68.17 (d, JCP = 4.4 Hz, 1 ꢃ C7), 68.23 (d, JCP = 4.6 Hz, 1 ꢃ C7), 68.57 (d,
3JCP = 2.0 Hz, 1 ꢃ C5’), 68.63 (d, JCP = 2.5 Hz, 1 ꢃ C5’) 84.07, 84.13 (2 ꢃ C4’),
3
89.12, 89.22 (2 ꢃ C1’), 109.65, 109.70 (2 ꢃ thymine-C5), 120.36 (2 ꢃ C1) 124.15
(2 ꢃ C4), 124.62, 124.65 (2 ꢃ C5), 126.98, 127.05 (2 ꢃ C3), 127.33, 127.36
(2 ꢃ C2’), 130.84, 130.93 (2 ꢃ C6) 132.71, 132.78 (2 ꢃ C3’), 135.60, 135.66
(2 ꢃ thymine-C6) 150.67 (2 ꢃ thymine-C2), 155.71 (2 ꢃ C2), 163.69 (2 ꢃ thymine-
C4), 172.54 (2 ꢃ C10), 207.14 (2 ꢃ C13); 31P-NMR (202 MHz, DMSO-d6) d: ꢂ7.60,
ꢂ7.52; HPLC: tR = 12.22 min (method I); tR = 11..23, 11.41 min (method II); MS
(ESI+, m/z): calc. 534.14 (M), found 557.16 (M + Na+).
3-PivEt-cycloSal-d4TMP 5c: The product was isolated as a colorless foam.
Yield: 52%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.60; 1H-NMR (400 MHz, DMSO-d6) d:
11.33, 11.31 (2s, 1H, NH); 7.30–7.26 (m, 1H, H4-aryl); 7.19, 7.18 (2q, JH–H = 1.2 Hz,
4
1H, H6); 7.17–7.10 (m, 2H, H5-aryl + H6-aryl); 6.81–6.78 (m, 1H, H1’); 6.40 (ddd,
3
4
3
3JH–H = 6.0 Hz, JH–H = 1.7 Hz, JH–H = 1.7 Hz, 1H1.D, H3’); 6.36 (ddd, JH–H = 6.0
3
4
3
Hz, JH–H = 1.8 Hz, JH–H = 1.8 Hz, 1H2.D, H3’); 6.02 (ddd, JH–H = 6.2 Hz,
4
3
3
3JH–H = 2.4 Hz, JH–H = 1.2 Hz, 1H1.D, H2’); 6.00 (ddd, JH–H = 5.9 Hz, JH–H = 2.4
Hz, 4JH–H = 1.4 Hz, 1H2.D, H2’); 5.48 (dd, 2JH–H = 14.3 Hz, 3JH–P = 5.1 Hz, 1H1.D, H7-
benzyl); 5.44 (dd, JH–H = 14.3 Hz, JH–P = 4.8 Hz, 1H1.D, H7-benzyl); 5.38 (dd,
2
3
2JH–H = 14.2 Hz, JH–P = 4.8 Hz, 1H2.D, H7-benzyl); 5.35 (dd, JH–H = 14.2 Hz, JH–
3
2
3
= 5.3 Hz, 1H2.D, H7-benzyl); 4.97–4.92 (m, 1H, H4’); 4.35–4.24 (m, 2H, H5’); 4.22–
P
4.16 (m, 2H, H9); 2.99–2.82 (m, 2H, H8); 1.64, 1.59 (2d, 4JH–H = 1.2 Hz, 3H, H7); 1.05
(2s, 9H, H12a–c); 13C-NMR (101 MHz, DMSO-d6) d: 177.4 (2 ꢃ C10); 163.9 (C4);
2
2
150.8 (2 ꢃ C2); 148.2 (d, JH–H = 7.1 Hz, C21.D-aryl); 148.1 (d, JH–H = 7.6 Hz,
C22.D-aryl); 135.8 (2 ꢃ C6); 132.9 (2 ꢃ C3’); 131.2 (2 ꢃ C4-aryl); 127.5 (2 ꢃ C2’);
127.3, 127.2 (2 ꢃ C1-aryl); 124.8 (2 ꢃ C6-aryl); 124.2, 124.1 (2 ꢃ C5-aryl); 121.7,

ORDER
REPRINTS
110
Meier et al.
121.6 (2 ꢃ C3-aryl); 109.9, 109.8 (2 ꢃ C5); 89.4, 89.3 (2 ꢃ C1’); 84.3 (2 ꢃ C4’); 68.8
2
2
2
(d, JC–P = 5.1 Hz, C5’1.D); 68.7 (d, JC–P = 6.1 Hz, C5’2.D); 68.4 (d, JC–P = 7.1 Hz,
2
C7-benzyl); 68.4 (d, JC–P = 6.1 Hz, C7-benzyl); 62.9 (2 ꢃ C9); 30.8 (C11); 28.3
(2 ꢃ C8); 26.9 (C12a–c); 12.0, 11.9 (2 ꢃ C7); 31P-NMR (202 MHz, DMSO-d6)
d: ꢂ7.51, ꢂ7.58; HPLC: tR = 13.9 min (method I), tR = 13.3 min (method II); MS (FAB,
m/z): calc. 520.1611 (M), found 521.2 (M + H+), 521.1689 (M + H+, FAB-HR).
5-AcEt-cycloSal-d4TMP 5d: The product was isolated as a colorless foam. Yield:
1
41%; TLC Rf (CH2Cl2/MeOH 9:1 v/v): 0.65; H-NMR (500 MHz, DMSO-d6) d: 11.33,
11.32 (2s, 1H, NH); 7.26–7.21 (m, 1H, H4-aryl); 7.18 (q, JH–H = 1.3 Hz, 1H1.D, H6);
4
4
7.15 (q, JH–H = 1.1 Hz, 1H2.D, H6); 7.16–7.14 (m, 1H, H6-aryl); 7.05, 7.03 (2d,
3
4
3JH–H = 8.4 Hz, 1H, H3-aryl); 6.79 (ddd, JH–H = 3.5 Hz, 2ꢃ JH–H = 1.7 Hz, 1H1.D
,
H1’); 6.77 (ddd, 3JH–H = 3.7 Hz, 2ꢃ JH–H = 1.8 Hz, 1H2.D, H1’); 6.41 (ddd, 3JH–H = 6.0
4
3
4
3
Hz, JH–H = 1.8 Hz, JH–H = 1.8 Hz, 1H1.D, H3’); 6.34 (ddd, JH–H = 6.0 Hz,
3JH–H = 1.7 Hz, JH–H = 1.7 Hz, 1H2.D, H3’); 6.01 (ddd, JH–H = 6.1 Hz, JH–H = 2.4
4
3
3
4
3
3
Hz, JH–H = 1.3 Hz, 1H1.D, H2’); 5.99 (ddd, JH–H = 6.0 Hz, JH–H = 2.4 Hz,
4JH–H = 1.4 Hz, 1H2.D, H2’); 5.47 (dd, JH–H = 14.5 Hz, JH–P = 6.1 Hz, 1H1.D, H7-
benzyl); 5.43 (dd, JH–H = 14.4 Hz, JH–P = 5.9 Hz, 1H1.D, H7-benzyl); 5.37 (d(br),
2
3
2
3
2JH–H = 14.3 Hz, 1H2.D, H7-benzyl); 5.35 (d(br), JH–H = 14.2 Hz, 1H2.D, H7-benzyl);
2
3
4.96–4.92 (m, 1H, H4’); 4.34–4.23 (m, 2H, H5’); 4.17 (dd, 2 ꢃ JH–H = 6.9 Hz, 2H, H9);
2.85 (dd, 2ꢃ JH–H = 6.8 Hz, 2H, H8); 1.97 (s, 3H, H11); 1.66 (d, 4JH–H = 1.1 Hz, 3H1.D
,
3
H7); 1.59 (d, JH–H = 1.3 Hz, 3H2.D, H7); 13C-NMR (101 MHz, DMSO-d6) d: 170.4
4
2
(C10); 163.9 (2 ꢃ C4); 150.9, 150.8 (2 ꢃ C2); 148.3, 148.2 (2d, JC–P = 7.1 Hz, C2-
aryl); 135.8 (2 ꢃ C6); 134.5 (C5-aryl); 133.0, 132.9 (2 ꢃ C3’); 130.4, 130.3 (2 ꢃ C4-
aryl); 129.2 (C1-aryl); 127.5 (2 ꢃ C2’); 126.5, 126.4 (2 ꢃ C6-aryl); 118.2, 118.1
2
(2 ꢃ C3-aryl); 109.8 (2 ꢃ C5); 89.3 (C1’); 84.3, 84.2 (2 ꢃ C4’); 68.5 (d, JC–P = 8.1
2
2
Hz, C5’1.D); 68.4 (d, JC–P = 6.1 Hz, C5’2.D); 68.3 (d, JC–P = 7.1 Hz, C7-benzyl); 64.2
(C9); 33.6 (C11); 20.8 (C8); 12.1, 12.0 (2 ꢃ C7); 31P-NMR (202 MHz, DMSO-d6)
d: ꢂ 7.99, ꢂ 8.04; HPLC tR = 11.6 min (method I), tR = 10.4 min (method II); MS
(FAB, m/z): calc. 478.1141 (M), found 479.4 (M + H+), 479.1232 (M + H+, FAB-HR).
5-PivEt-cycloSal-d4TMP 5e: The product was isolated as a colorless foam.
Yield: 39%; TLC: Rf (CH2Cl2/MeOH 9:1 v/v): 0.56; 1H-NMR (500 MHz, DMSO-d6) d:
11.33, 11.32 (2s, 1H, NH); 7.26–7.20 (m, 1H, H4-aryl); 7.17, 7.15 (2q, JH–H = 1.1 Hz,
4
3
1H, H6); 7.15–7.13 (m, 1H, H6-aryl); 7.06, 7.04 (2d, JH–H = 8.4 Hz, 1H, H3-aryl);
4
6.79 (ddd, 3JH–H = 3.5 Hz, 2 ꢃ JH–H = 1.7 Hz, 1H1.D, H1’); 6.77 (ddd, 3JH–H = 3.7 Hz,
4
3
3
2ꢃ JH – H = 1.8 Hz, 1H2.D, H1’); 6.40 (ddd, JH – H = 6.0 Hz, JH – H = 1.7 Hz,
4JH–H = 1.8 Hz, 1H1.D, H3’); 6.34 (ddd, JH–H = 6.0 Hz, JH–H = 1.7 Hz, JH–H = 1.7
3
3
4
3
3
4
Hz, 1H2.D, H3’); 6.00 (ddd, JH–H = 5.9 Hz, JH–H = 2.3 Hz, JH–H = 1.3 Hz, 1H1.D
,
H2’); 5.99 (ddd, 3JH–H = 5.8 Hz, 3JH–H = 2.5 Hz, 4JH–H = 2.5 Hz, 1H2.D, H2’); 5.45 (dd,
2JH–H = 14.5 Hz, JH–P = 6.0 Hz, 1H1.D, H7-benzyl); 5.42 (dd, JH–H = 14.4 Hz,
3
2
3JH–P = 5.9 Hz, 1H1.D, H7-benzyl); 5.36 (dd, JH–H = 14.3 Hz, JH–P = 3.7 Hz, 1H2.D
,
H7-benzyl); 5.33 (dd, JH–H = 14.4 Hz, JH–P = 3.7 Hz, 1H2.D, H7-benzyl); 4.95–4.92
2
3
2
3
3
(m, 1H, H4’); 4.34–4.22 (m, 2H, H5’); 4.18 (dd, 2ꢃ JH–H = 6.6 Hz, 2H, H9); 2.85
3
4
(dd(br), 2ꢃ JH–H = 6.5 Hz, 2H, H8); 1.66, 1.60 (2d, JH–H = 1.1 Hz, 3H, H7); 1.06 (2s,
9H, H12a–c); 13C-NMR (101 MHz, DMSO-d6) d: 177.4 (C10); 163.9, 163.8 (2 ꢃ C4);

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111
2
150.9, 150.8 (2 ꢃ C2); 148.0 (2d, JC–P = 7.1 Hz, C2-aryl); 135.8 (2 ꢃ C6); 134.6
(C5-aryl); 133.0, 132.9 (2 ꢃ C3’); 130.5, 130.4 (C4-aryl); 129.7, 129.6 (2 ꢃ C1-aryl);
127.5 (2 ꢃ C2’); 126.6, 126.5 (2 ꢃ C6-aryl); 118.1 (2 ꢃ C3-aryl); 109.8 (2 ꢃ C5); 89.3
2
2
(C1’); 84.3, 84.2 (2 ꢃ C4’); 68.5 (d, JC–P = 6.6 Hz, C5’1.D); 68.4 (d, JC–P = 8.1 Hz,
2
2
C5’2.D); 68.4 (d, JC–P = 6.1 Hz, C71.D-benzyl); 68.3 (d, JC–P = 7.1 Hz, C72.D-benzyl);
64.3 (C9); 38.3 (C11); 33.6 (C8); 27.0 (C12a–c); 12.1, 12.0 (2 ꢃ C7); 31P-NMR (202
MHz, DMSO-d6) d: ꢂ8.03, ꢂ8.06; HPLC: tR = 15.0 min (method I), tR = 13.4 min
(method II); MS (FAB, m/z): calc. 520.1611 (M), found 521.2 (M + H+), 520.1532
(M + H+, FAB-HR).
3-(2-Carboxyethyl)-cycloSal-d4TMP 2a: The product was isolated as a
1
colorless foam. Yield: 71%; TLC: Rf (CH2Cl2/MeOH 9 : 1 v/v) 0.16; H-NMR (500
MHz, DMSO-d6) d 12.17 (s(br), 1H, COOH), 11.31 (2s, 1H, NH), 7.27–7.25 (m, 1H,
H4-aryl), 7.19 (2q, 1H, H6), 7.14–7.09 (m, 2H, H5-aryl + H6-aryl), 6.80–6.78 (m, 1H,
H1’), 6.40 (ddd, 1H1.D, H3’), 6.35 (ddd, 1H2.D, H3’), 6.02, 6.00 (2ddd, 1H, H2’), 5.47
(dd, 1H1.D, H7-benzyl), 5.44 (dd, 1H1.D, H7-benzyl), 5.37 (dd, 1H2.D, H7-benzyl), 5.34
(dd, 1H2.D, H7-benzyl), 4.96–4.92 (m, 1H, H4’), 4.34–4.24 (m, 2H, H5’), 2.85–2.75
(m, 2H, H8), 2.51, 2.50 (2dd, 2H, H9), 1.65, 1.58 (2d, 3H, H7); 13C-NMR (101 MHz,
DMSO-d6) d 173.6 (2 ꢃ C10), 163.9 (C4), 150.9, 150.8 (2 ꢃ C2), 147.9 (d, C2-aryl),
147.9 (d, C2-aryl), 135.8, 135.7 (2 ꢃ C6), 133.0 132.9 (2 ꢃ C3’), 130.2 (2 ꢃ C4-aryl),
130.0, 129.9 (2 ꢃ C1-aryl), 127.5 (2 ꢃ C2’), 124.4 (2 ꢃ C6-aryl), 124.3 (C5-aryl),
121.7, 121.6 (C3-aryl), 109.9, 109.8 (2 ꢃ C5), 89.4, 89.3 (2 ꢃ C1’), 84.3 (2 ꢃ C4’),
68.7 (d, C5’), 68.7 (d, C5’), 68.4 (d, C7-benzyl), 68.4 (d, C7-benzyl), 33.5 (2 ꢃ C9),
24.2 (2 ꢃ C8), 12.0, 11.9 (2 ꢃ C7); 31P-NMR (202 MHz, DMSO-d6) d ꢂ7.51, ꢂ7.56;
HPLC: tR = 9.2, 9.5 min (method I), tR = 9.7 min (method II); MS (FAB, m/z): calc.
464.0985 (M + H+), found 464.2 (M + H+).
5-(2-Carboxyethyl)-cycloSal-d4TMP 2b: The product was isolated as a
1
colorless foam. Yield: 93%; TLC: Rf (CH2Cl2/MeOH 9 : 1 v/v) 0.16; H-NMR (500
MHz, DMSO-d6) d 12.08 (s(br), 1H, COOH), 11.33, 11.32 (2s, 1H, NH), 7.24–7.19
(m, 1H, H4-aryl), 7.18, 7.15 (2q, 1H, H6), 7.13–7.11 (m, 2H, H6-aryl), 7.03 (d, 1H1.D
,
H3-aryl), 7.01 (d, 1H2.D, H3-aryl), 6.79, 6.77 (2ddd, 1H, H1’), 6.40, 6.33 (2ddd, 1H,
H3’), 6.01 (ddd, 1H1.D, H2’), 5.99 (ddd, 1H2.D, H2’), 5.45 (dd, 1H1.D, H7-benzyl), 5.41
(dd, 1H1.D, H7-benzyl), 5.36 (d(br), 1H2.D, H7-benzyl), 5.33 (dd, 1H2.D, H7-benzyl),
4.96–4.92 (m, 1H, H4’), 4.33–4.22 (m, 2H, H5’), 2.78 (dd, 2H, H8), 2.51 (dd, 2H, H9),
1.67, 1.61 (2d, 3H, H7); 13C-NMR (101 MHz, DMSO-d6) d 173.7 (2 ꢃ C10), 163.9
(C4), 150.9, 150.8 (2 ꢃ C2), 146.5, 146.2 (d, C2-aryl), 137.4 (C5-aryl), 135.8
(2 ꢃ C6), 133.0 132.9 (2 ꢃ C3’), 129.8, 129.7 (2 ꢃ C4-aryl), 128.7 (C1-aryl), 127.5,
127.4 (2 ꢃ C2’), 125.9, 125.8 (2 ꢃ C6-aryl), 118.1 (2 ꢃ C3-aryl), 109.8 (2 ꢃ C5),
89.3 (C1’), 84.3, 84.2 (2 ꢃ C4’), 68.5 (d, C5’), 68.5 (d, C5’), 68.4 (2d, C7-benzyl), 35.1
(C9), 29.6 (C8), 12.1, 12.0 (2 ꢃ C7); 31P-NMR (202 MHz, DMSO-d6) dꢂ8.01, ꢂ8.06;
HPLC: tR = 9.5 min (method I), tR = 9.6 min (method II)MS (FAB, m/z): calc.
464.0985 (M + H+), found 464.3 (M + H+).
3-(2-Hydroxyethyl)-cycloSal-d4TMP 4a: 65 mg (0.12 mmol) 3-LevEt
cycloSal-d4TMP 5b were dissolved in 5 ml pyridine. To this solution, a mixture of

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112
Meier et al.
1 ml (0.24 mmol) hydrazine-hydrate, 3.1 ml (0.48 mmol) pyridine and 2 ml (0.36
mmol) acetic acid was added at 08C. After 10 min water and ethylacetate was added
and the separated organic phase was washed with a 5% sodium bicarbonate solution.
After drying with sodium sulfate, the organic solvent was removed and the crude
product was purified by chromatography (CH2Cl2-MeOH gradient 0–4%). The product
was isolated as a colorless foam. Yield: 25 %; TLC: Rf (CH2Cl2/MeOH 9:1 v/v) 0.32;
4
1H-NMR (500 MHz, DMSO-d6) d: 1.62 (d, 3H, JHH = 1.3 Hz, thymine-H7), 1.66 (d,
4
3H, JHH = 1.3 Hz, thymine-H7), 2.69–2.80 (m, 2 ꢃ 2H, 2 ꢃ H8), 4.26–4.35 (m,
3
3
2 ꢃ 2H, 2 ꢃ H5’), 4.68 (t, 1H, JHH = 5.2 Hz, -OH), 4.72 (t, 1H, JHH = 5.2 Hz, -OH),
2
3
4.92–4.98 (m, 2 ꢃ 1H, 2 ꢃ H4’) 5.36 (dd, 1H, JHH = 12.5 Hz, JHP = 5.4 Hz, H7),
2
3
5.38 (dd, 1H, JHH = 12.5 Hz, JHP = 5.4 Hz, H7), 5.45 (dd, 1H, JHH = 14.5 Hz,
2
3JHP = 8.0 Hz, H7), 5.54 (dd, 1H, JHH = 14.5 Hz, JHP = 8.0 Hz, H7), 6.02 (ddd, 1H,
2
3
3JHH = 6.0 Hz, JHH = 1.5 Hz, JHH = 2.5 Hz, H2’), 6.04 (ddd, 1H, JHH = 6.0 Hz,
3
4
3
3JHH = 1.3 Hz, JHH = 2.4 Hz, H2’), 6.36 (ddd, 1H, JHH = 6.0 Hz, JHH = 1.3 Hz,
4
3
3
3
3
4
4JHH = 1.8 Hz, H3’), 6.42 (ddd, 1H, JHH = 6.0 Hz, JHH = 1.5 Hz, JHH = 1.8 Hz,
H3’), 6.78–6.83(m, 2 ꢃ 1H, 2 ꢃ H1’), 7.13–7.21 (m, 2 ꢃ 3H, thymine-H6, H4, H5),
7.29–7.33 (m, 2 ꢃ 1H,2 ꢃ H6), 11.32–11.41 (br, 2 ꢃ 1H, 2 ꢃ NH); 13C-NMR (100
MHz, DMSO, d6) d: 11.74, 11.83 (2 ꢃ thymine-C7), 32.41 (2 ꢃ C8), 60.38 (2 ꢃ C9),
68.62–68.91 (m, 2 ꢃ C5’, 2 ꢃ C7) 84.04, (2 ꢃ C4’), 89.11, 89.19 (2 ꢃ C1’), 120.20
(2 ꢃ thymine-C5), 120.36 (2 ꢃ C1) 124.01 (2 ꢃ C4), 124.62, 124.65 (2 ꢃ C5),
126.98, 127.05 (2 ꢃ C3), 127.33, 127.36 (2 ꢃ C2’), 130.84, 130.93 (2 ꢃ C6)
132.71, 132.78 (2 ꢃ C3´), 135.60, 135.66 (2 ꢃ thymine-C6) 150.67 (2 ꢃ thymine-
C2), 155.71 (2 ꢃ C2), 163.69 (2 ꢃ thymine-C4), 172.54 (2 ꢃ C10), 207.14
(2 ꢃ C13); 31P-NMR (202 MHz, DMSO-d6) d: ꢂ7.48, ꢂ7.43; HPLC: tR = 10.27
min (method I); tR = 9.07, 9.36 min (method II); MS (ESI+, m/z): calc.: 436.10 (M),
found: 459.13 (M + Na+).
Kinetic Data. (a) Aqueous Buffers: 12 mL of DMSO stock solutions (50 mM)
of the triesters were diluted in 300 mL water or water/DMSO (c = 2.0 mM). 0.3 mL of
this solution were added to 0.3 mL of aqueous buffer (50 mM phosphate buffer, pH 7.3
or 50 mM phosphate buffer, pH 6.8) containing 5 mL of an aqueous AZT solution
(AZT as internal standard) at 378C. The final concentrations were 0.96 mM for the
triesters and 25 mM for the aqueous buffer. Aliquots of 60 mL of the hydrolysis
mixture were taken and the hydrolysis was stopped by addition of 5 mL glacial acetic
acid and frozen in liquid air. After thawing, samples were analyzed by analytical HPLC
(Merck LiChroCART column, LiChrospher 100 reversed-phase silica gel RP-18
endcapped (5 mm); UV detection at 250 nm). The hydrolysis of the compounds 2–4
was followed by integration of the peak areas in the HPLC chromatograms. The rate
constants k were determined from slope of the logarithmic degradation curve. The half-
lives (t1/2) were calculated by using the rate constants k.
(b) CEM Cell Extract: 3.0 mM stock solution of the triesters in DMSO were
prepared. 20 mL of this stock solution was mixed with 100 mL cell extract and 20 mL of
a 70 mM magnesium chloride solution. The hydrolysis process was stopped after
8 hours by addition of 300 mL acidic methanol and storage for 5 min at 08C. The
mixtures were centrifuged by 13000 rpm for 10 min and the supernatant was analyzed
as mentioned above.

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113
HIV-Assay: The anti-HIV evaluation has been carried out as described
previously.[8]
ACKNOWLEDGMENTS
Financial support by the Deutsche Forschungsgemeinschaft, Germany, the Fonds
der Chemischen Industrie, Germany and the Rene´ Descartes Prize 2001 of the
European Commission is gratefully acknowledged.
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Received July 31, 2003
Accepted September 19, 2003

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