A new and efficient method for synthesis of 5′-conjugates of oligonucleotides through amide-bond formation on solid phase

Article abstract of DOI:10.1002/1522-2675(200208)85:8<2409::AID-HLCA2409>3.0.CO;2-P

An efficient method for synthesis of oligonucleotide 5′-conjugates through amide-bond formation on solid phase is described. Protected oligonucleotides containing a 5′-carboxylic acid function were obtained by use of a novel non-nucleosidic phosphoramidite building block, where the carboxylic acid moiety was protected by a 2-chlorotrityl group. The protecting group is stable to the phosphoramidite coupling conditions used in solid-phase oligonucleotide assembly, but is easily deprotected by mild acidic treatment. The protecting group may be removed also by ammonolysis. 5′-Carboxylate-modified oligonucleotides were efficiently conjugated on solid support under normal peptide-coupling conditions to various amines or to the N-termini of small peptides to yield products of high purity. The method is well-suited in principle for the synthesis of peptide-oligonucleotide conjugates containing an amide linkage between the 5′-end of an oligonucleotide and the N-terminus of a peptide.

Full text of DOI:10.1002/1522-2675(200208)85:8<2409::AID-HLCA2409>3.0.CO;2-P

Helvetica Chimica Acta Vol. 85 (2002)
2409
A New and Efficient Method for Synthesis of 5'-Conjugates of
Oligonucleotides through Amide-Bond Formation on Solid Phase
by Anna V. Kachalovaa), Dmitry A. Stetsenkob), Elena A. Romanovaa), Vadim N. Tashlitskya),
Michael J. Gait*b), and Tatiana S. Oretskaya*a)
a) Chemistry Department and A. N. Belozersky Institute of Physico-Chemical Biology, M. V. Lomonosov
Moscow State University, Moscow, 119992, Russia
(tel.: ‡7-095-939-5411; fax: ‡7-095-939-3181; e-mail: oretskaya@belozersky.msu.ru)
b) Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
(tel.: ‡44-1223-248011; fax: ‡44-1223-402070; e-mail: mgait@mrc-lmb.cam.ac.uk)
Dedicated to Prof. Dr. Wolfgang Pfleiderer on the occasion of his 75th birthday
An efficient method for synthesis of oligonucleotide 5'-conjugates through amide-bond formation on solid
phase is described. Protected oligonucleotides containing a 5'-carboxylic acid function were obtained by use of a
novel non-nucleosidic phosphoramidite buildingblock, where the carboxylic acid moiety was protected by a 2-
chlorotrityl group. The protecting group is stable to the phosphoramidite coupling conditions used in solid-phase
oligonucleotide assembly, but is easily deprotected by mild acidic treatment. The protecting group may be
removed also by ammonolysis. 5'-Carboxylate-modified oligonucleotides were efficiently conjugated on solid
support under normal peptide-couplingconditions to various amines or to the N-termini of small peptides to
yield products of high purity. The method is well-suited in principle for the synthesis of peptide-oligonucleotide
conjugates containing an amide linkage between the 5'-end of an oligonucleotide and the N-terminus of a
peptide.
1. Introduction. Conjugation of antisense oligonucleotides to peptides and basic
polymers (e.g., polylysine) is aimed at enhancement of their cellular permeability, their
targeting to specific tissues, or alteration of their intracellular locations (for reviews, see
[1 4]). Conjugation of oligonucleotides to small molecules may also alter such
parameters but is often used to attach specific labels (e.g., fluorescent) or effector
molecules, such as biotin (reviewed in [5][6]). In these ways, chemical functionaliza-
tion of synthetic oligonucleotides has found wide application with the rapid growth of
molecular biology and biotechnology.
Most conjugation strategies involve prior attachment of a tether bearing a
nucleophilic group to a modified nucleoside or to a special linker, which is then
incorporated into DNA by synthesis of an appropriate phosphoramidite and
subsequent oligonucleotide synthesis under standard conditions. Commonly used
nucleophilic groups include primary amines and thiols [7 11]. For example, we
reported a few years ago the solid-phase conjugation of small peptides to oligonucleo-
tides that had been functionalized with an amino linker [12][13]. Electrophilic groups
have been introduced into synthetic oligonucleotides relatively rarely, for example, at
the 2'-position [14][15]. Modification of the 5'-terminus with an aldehyde or carboxylic
acid has been reported previously by a number of authors [16 20]. In each case, the
modifications were introduced into oligonucleotide chains after solid-phase oligonu-

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Helvetica Chimica Acta Vol. 85 (2002)
cleotide assembly, and the aldehyde or carboxylic acid function liberated either before
or simultaneously with the final deblockingand release of the oligonucleotide from the
support with ammonia. Thus, subsequent transformations could be achieved only by a
solution-phase method. As a result, these methods are frequently plagued by low yields,
longreaction times, the need for large excesses of reactant(s), and the formation of by-
products that are difficult to separate. Hovinen et al. described reaction of a solid-
supported oligonucleotide modified by a 5'-glycolic acid ester with several aliphatic
diamines over 6 12 h [18]. Very recently, a method was described for synthesis of 5'-
aldehydes via couplingof a protected diol-containinglinker phosphoramidite in solid-
phase oligonucleotide synthesis, where the acetal protecting group could be removed
under mild acidic conditions [21]. We wish to report here the synthesis of a new type of
modified oligonucleotide via couplingof a phosphoramidite linker reagent containinga
5'-carboxylic acid function carryinga 2-chlorotrityl ( ˆ(2-chlorophenyl)diphenylmeth-
yl) protectinggroup, which may be removed under mild acidic conditions. Subsequent
conjugation of the 5'-carboxylic acid function to a range of primary and secondary
aliphatic amines is achieved through amide-bond formation on solid support. We show
examples of synthesis of such carboxylate-modified oligonucleotides conjugated to
various reporter groups (e.g., fluorescent labels, biotin) or to small peptides.
2. Results and Discussion. To obtain oligonucleotides modified at the 5'-end with a
carboxylic group, we synthesized two novel non-nucleosidic phosphoramidite building-
block linkers 5 and 6 (Scheme). For the selection of a phosphoramidite useful in the
final coupling step in solid-phase oligonucleotide assembly, we investigated the utility
of the trityl (ˆtriphenylmethyl) family of protectinggroups for the carboxylic acid
moiety, since a 2-chlorotrityl ester had been found to be useful previously as a
carboxylic acid protecting group in fragment peptide coupling [22]. Furthermore, 2-
chlorotrityl ether has been used as a linker for polymer-supported peptide-fragment
synthesis [23], which gained further popularity when it was found to be cleaved under
extremely mild acidic conditions, for example, (CF3)2CHOH/CH2Cl2 1:4 [24]. In the
case of polymer-bound 2-chlorotrityl chloride, the Et(i-Pr)2NH salt of an N-a-Fmoc-
protected amino acid is usually used for coupling[23]. In our case, levulinic acid ( ˆ4-
oxopentanoic acid) was treated either with Ph3CCl or with 2-chlorotrityl chloride in the
presence of Et3N in dry CH2Cl2 to give high yields of crystalline trityl or 2-chlorotrityl
levulinate 1 and 2, respectively. Each compound was easily reduced by NaBH4 in EtOH
to afford the correspondinghydroxy esters 3 and 4, respectively, in good yield, but some
decomposition of the unsubstituted trityl compound was observed duringsilica-gel
column chromatography. The relative stabilities of the protecting groups under
oligonucleotide synthesis conditions were then checked by TLC (Table 1). Whilst the
trityl ester was only moderately stable to 0.5m 1H-tetrazole solution in MeCN, and
noticeable cleavage was seen even after 15 min, the corresponding 2-chlorotrityl
derivative remained unchanged after 1-h treatment. Both compounds were easily
deprotected by 80% aq. AcOH, again the trityl removal being faster. Cleavage of both
esters was almost instantaneous upon treatment with 2.5% (v/v) Cl2CCOOH in
CH2Cl2, a commonly used detritylating reagent in oligonucleotide synthesis.
Modified phosphoramidites were obtained by phosphitylation of the corresponding
alcohols by (2-cyanoethoxy)bis(diisopropylamino)phosphine in the presence of

Helvetica Chimica Acta Vol. 85 (2002)
2411
Scheme
O
HO
O
O
Ph
Ph
Ph
NaBH4
O
Ph
O
Ph
O
Ph
Cl
EtOH
Et3N, CH2Cl2
R
O
R
OH
R
1 R = H
2 R = Cl
3 R = H
4 R = Cl
(iPr2N)2POCH2CH2CN,
diisopropylammonium
1H-tetrazolide
1. Solid-phase coupling
O
R
Ph
2. 2% (w/v) CCl3CO2H in CH2Cl2
Ph
O
CH2Cl2
O
O
P
CN
NiPr2
5 R = H
6 R = Cl
O
O
R3
N
1. HBTU, HOBt, DMF
HO
O
P
H
O
2. Excess of amine or peptide
3. 25% aq. NH3, 55o
O
O
O
O
P
CN
O
B
O
B
O
O
R2O
R1O
R1 ˆSupport-bound protected oligonucleotide; R2 ˆunprotected oligonucleotide; for the range of R3 ˆsee Table 2.
Table 1. Acid Stabilitya) of Trityl and 2-Chlorotrityl Esters
Reagent
Trityl ester 3
2-Chlorotrityl ester 4
0.5m 1H-Tetrazole in MeCN
AcOH/H2O 4 : 1 (v/v)
2.5% Cl2CCOOH in CH2Cl2
30 min
< 5 min
< 1 min
> 1 h
15 min
< 1 min
a) Time needed for ca. 50% removal of the protectinggroup (TLC).
diisopropylammonium tetrazolide [25]. The compounds were purified by silica-gel
column chromatography, the trityl phosphoramidite being obtained in lower yield and
purity. However, both amidites were used successfully in machine-assisted solid-phase
synthesis, demonstratingcomparable couplingefficiencies ( > 96%). However, we
decided to use the more stable and easily obtainable phosphoramidite 6 for conjugation
studies. This was utilized in the synthesis of a series of modified oligonucleotides
(Table 2). Oligodeoxyribonucleotide III was synthesized as a model to select and

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No.
Helvetica Chimica Acta Vol. 85 (2002)
Table 2. Properties of 5'-Carboxylated Oligonucleotides and Their Conjugates
Oligonucleotide sequence/
conjugated moleculea)
MALDI-TOF-MS,
calc./foundb)
Retention time Yield HPLC Purity
[min]b)
[%]c) [%]d)
I
II
III
I.7
I.8
I.9
5'-d(GCT CCC AGG CTC AAA)-3' 3502.8/3501.2
5'-d(AGC TCC CAG GCT CAA)-3' 4725.0/4724.9
13.1
12.8
13.0
12.8
57
67
5'-d(TTT TTT TTT T)-3'
3502.8/3501.2
4789.2/4787.1
4785.3/4783.1, 4784.2 13.8, 14.6
4805.0/4801.6, 4803.2 18.7, 19.4
4814.2/4812.2, 4811.7 14.7, 15.1
4848.2/4847.8, 4848.7 15.4, 16.1
2-(2-Hydroxyethoxy)ethylamine (7)
Tetrahydrofurfurylamine (8)
2-Phenylethylamine (9)
99
99
99
98
98
88
90
89
75
71
67
70
I.10 H-Leu-NH2 (10)
I.11 H-Phe-NH2 (11)
II.12 Spermine (12)
II.13 Octadecylamine (13)
II.14 Histamine (14)
II.15 Dansylcadaverine (15)
II.16 Biotin-PEG-amine (16)
II.17 3-Aminopropane-1,2-diol (17)
II.18 1-Pyrenemethylamine (18)
I.19 H-Gly-Phe-NH2 (19)
I.20 H-Gly-Leu-Met-NH2 (20)
I.21 H-Pro-Leu-Gly-NH2 (21)
II.22 H-Gly-Gly-Gly-Gly-Gly-NH2 (22)
4887.4/4888.3
4976.5/4974.1
4818.2/4818.1
5042.5/5043.4
5081.5/5079.1
4773.9/4771.6
4916.3/4919.6
4905.3/4904.7
5003.5/5006.5
4969.4/4973.0
4984.9/4981.2
11.4
e
)
11.8, 12.0
81
96
60
66
e
)
)
e
12.1
f
)
17.4
19.3
15.5
14.0
90
90
80
75
61
64
66
53
a) Amines 7 11 and peptides 19 21 were conjugated to oligonucleotide I; amines 12 18 and peptide 22 were
conjugated to oligonucleotide II. b) Values separated by commas correspond to individual diastereoisomers.
c) Conversion of oligonucleotide peak to the conjugate peak(s), calculated from HPLC trace. d) As integrated
from HPLC traces. e) Purified by PAGE. f) Not purified.
optimize the specific conditions for 5'-deblockingand purification. Initially, we
envisaged that the 2-chlorotrityl protecting group could be used in an analogous way to
the 5'-dimethoxytrityl group as a purification handle on a reversed-phase (RP) HPLC
column. However, the 2-chlorotrityl ester was cleaved after treatment with 25%
t
aqueous NH3 or BuNH2/MeOH/H2O 1:2 :1 (v/v/v) at room temperature for ca.
30 min. Therefore, the 5'-carboxylic group was routinely deprotected on column under
standard detritylatingconditions followingoliognucleotide assembly (2% (
Cl3CCOOH in CH2Cl2).
w/v)
Then, we investigated the H2O-soluble carbodiimide-mediated reaction of 5'-
modified fully deprotected oligonucleotide with aqueous ethylenediamine dihydro-
chloride. The yield of the reaction product was found to be almost quantitative, and
product integrity was confirmed by RP-HPLC and by MALDI-TOF mass analysis.
Further conjugations of 5'-carboxylic acid-modified oligonucleotides I and II were
carried out on solid phase in organic solvent. The choice of amine was influenced by the
expected application for the conjugate; for example, affinity labelling (biotin-PEG-
amine (16)), fluorescent labelling(dansylcadaverine ( 15) and 1-pyrenemethylamine
(18)), introduction of positive charge or nucleophile (spermine (12) and histamine
(14)), addition of hydrophobicity (octadecylamine (13)) or chemical ligation [15][16]
(3-aminopropane-1,2-diol (17)). To obtain a high yield of structurally diverse types of
conjugates (Table 2), we used pre-activation of polymer-bound carboxylic oligonu-
cleotide with HBTU/HOBT 1:1 at 358 in dry DMF for 35 min, followed by addition of

Helvetica Chimica Acta Vol. 85 (2002)
2413
the amine or short peptide solution in DMF and incubation for a further 1 h. An
equimolar amount of Et3N was included also if the amine was present as its salt. After
completion of the reaction, the polymer-bound oligonucleotide was cleaved from the
support and deprotected by standard ammonia treatment [26], and the reaction
mixtures were analyzed by RP-HPLC in ion-pair mode (Fig.). In some cases, HPLC
traces showed the formation of two products, which were separated and found to have
identical molecular masses by MALDI-TOF MS analysis (Table 2). Such twin products
were expected and may be assigned to the formation of a mixture of diastereoisomeric
conjugates that are resultant from the asymmetric C-atom coming from the alkyl side-
chain of phosphoramidite 6 (see Scheme).
In conclusion, we have presented a new method for the synthesis of oligodeoxy-
ribonucleotides containinga 5 '-carboxylic acid derivative via couplingof a novel
phosphoramidite derivative that contains a 2-chlorotrityl protectinggroup for the
Figure. HPLC Traces of crude reaction mixtures analyzed in ion-pair mode: a) 5'-carboxy group-modified
oligonucleotide II; b) d) 5'-conjugates with 2-phenylethylamine I.9 (b) (mixed diastereoisomers); spermine
II.12 (c); and tripeptide H-Pro-Leu-Gly-NH2 I.21 (d)

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Helvetica Chimica Acta Vol. 85 (2002)
carboxylic acid moiety 6. After deprotection under acidic conditions, such substituted
oligonucleotides could be conjugated, whilst still attached to the solid support, with a
range of small molecule amines or to the N-termini of short peptides. The methods
described could prove useful in the synthesis of small molecule libraries of conjugates
with oligonucleotides for diagnostic or therapeutic evaluation. In addition, the
conjugation method can be explored as a useful route for attachment of the N-termini
of cell penetratingpeptides to the 5 '-ends of oligonucleotides and their analogues for
cell uptake studies.
We thank Dr. Evgueni M. Zubin for his helpful suggestions and Donna Williams for some initial
oligonucleotide syntheses. This work was supported by Wellcome Trust grant No. 057361, and partially by
Universities of Russia Fundamental Research grant No. 05.03.010 and Leading Scientific Schools grant No. 00-
15-97944.
Experimental Part
General. Materials were obtained from commercial suppliers and used without further purification unless
otherwise noted. CH2Cl2 (BDH) was refluxed for 24 h over CaH2, and then distilled. DMF was distilled in vacuo
and used fresh. Et3N, 1-hydroxybenzotriazole (HOBt), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU), dansylcadaverine, and DMF were from Fluka. N'-[3-(dimethylamino)propyl]-
N'-ethylcarbodiimide, ethylenediamine dihydrochloride, LiClO4, and 2-(2-hydroxyethoxy)ethylamine were
from Sigma. Levulinic acid, Ph3CCl, 4-(dimethylamino)pyridine (DMAP), spermine, histamine, 1-pyreneme-
thylamine hydrochloride, 3-aminopropane-1,2-diol and NaBH4 were from Aldrich. Tetrahydrofurfurylamine
and 2-phenylethylamine were from Reakhim (USSR). Octadecylamine was from Lancaster. (‡)-d-Biotin 8-
amino-3,6-dioxaoctylamide (biotin-PEG-amine) was obtained from Pierce. 2-Chlorotrityl chloride was
purchased from Novabiochem, amino acids and peptides were obtained from Bachem. TLC Plates (Kieselgel
60 F254, Merck) were developed in the appropriate system: AcOEt/hexane 2 :3 (v/v) ‡ 1% Et3N (A); AcOEt/
hexane 3 :7 (v/v) ‡ 1% Et3N (B); AcOEt/hexane 1:9 ‡ 1% Et3N (C). Compounds were visualized by UV
shadowing(254 nm) or by CF 3COOH vapor staining(yellow color).
Oligonucleotide Synthesis. Oligodeoxyribonucleotides were assembled on a ABI 380B DNA Synthesizer by
the cyanoethyl phosphoramidite method [26] accordingto the manufacturer×s recommendations. Protected
2'-deoxyribonucleoside phosphoramidites and S-ethylthiotetrazole were purchased from Glen Research (via
Cambio). Prepacked 0.4-mmol functionalized columns of controlled-pore glass (Glen) were used throughout.
For couplings with modified phosphoramidite 6, a 0.15m concentration in anh. MeCN was used, and the coupling
time was increased to 15 min. Oligonucleotides were cleaved from the support and deprotected with
concentrated aq. NH3 overnight at 558.
Purifications and Characterizations. HPLC Analysis and purification of deprotected oligonucleotides and
conjugates were carried out on a Tracor instrument 4 Â 250 mm DIAKS-130-CETYL column; buffer A: 0.1m
ammonium acetate (pH 7); buffer B: 0.1m ammonium acetate, 40% MeCN (pH 7); gradient of B from 0 to
100% in 80 min; flow rate 1 ml/min at 458. Oligonucleotides and conjugates were analyzed by a RP-HPLC (ion
pair mode; Waters) on a DIAKS-130-CETYL column (3 Â 250 mm) with a logarithmic gradient: 0 42.4% B
(1 min); 42.4 45.7% B (1 min); 45.7 50.1% B (3 min); 50.1 53.5% B (5 min); 53.5 56.8% B (10 min); 56.8
58.7% B (10 min); 58.7 60.1% B (10 min). Separation of oligomers with a retention time step of 0.5 min/
residue was carried out; mobile phase A: H2O/MeCN 95 :5 (v/v), 2 mm tetrabutylammonium dihydrogen
phosphate, 48 mm KH2PO4, pH 7; mobile phase B: H2O/MeCN 60 :40 (v/v), 2 mm tetrabutylammonium
dihydrogen phosphate, 48 mm KH2PO4, pH 7; flow rate 0.6 ml/min, and temp. 458. MALDI-TOF-MS: on a
Voyager DE workstation (PE Biosystems) in a freshly prepared 1 :1 (v/v) mixture of 2,6-dihydroxyacetophe-
none (40 mg/ml in MeOH), and aq. diammonium hydrogen citrate (80 mg/ml) as a matrix. Compounds 1
4
were prepared in 2,4,6-trihydroxyacetophenone (10 mg/ml in 50% aq. MeOH). Denaturing gel electrophoresis
of oligonucleotides was carried out on 15% PAGE containing 2m urea in Tris-borate buffer (50 mm Tris ¥ HCl,
50 mm boric acid, 1 mm EDTA, pH 7.5).
Reaction of the Modified Oligonucleotide I with Ethylenediamine Dihydrochloride. 5'-Carboxylic group-
modified oligonucleotide I (0.4 A260 units) was dissolved in 40 ml of deionized H2O, ethylenediamine
dihydrochloride (5.3 mg) and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride (3 mg) were

Helvetica Chimica Acta Vol. 85 (2002)
2415
added, and the mixture was incubated for 3 h at r.t. The oligonucleotide was then precipitated by addition of
100 ml of 2m LiClO4 and 1 ml of acetone, the pellet washed with 300 ml of acetone, redissolved in 100 ml of 2m
LiClO4 and precipitated again. Analysis of the products was carried out by RP-HPLC under conditions
described above. Yield of ethylenediamine-modified oligonucleotide was 98% (HPLC).
Conjugate Synthesis. After completion of the oligonucleotide assembly and 5'-carboxylic group
deprotection, a soln. of HBTU (100 equiv.) and HOBt (100 equiv.) in 150 ml of dry DMF was added to the
support. The slurry was incubated at 358 for 35 min with occasional swirling, then an amine or peptide soln. was
added (100 equiv.), with additional Et3N (100 equiv.), if needed. The reaction was carried out for 1 h at 358 and
shaking, the supernatant discarded, and glass beads washed successively with DMF (2 Â 200 ml), EtOH (2 Â
200 ml), and water (2 Â 200 ml). Conjugate cleavage from the support and deprotection of phosphate and
nucleobase residues were performed as usual. Reaction mixtures were analyzed by ion-pair RP-HPLC,
conjugates were purified by RP-HPLC under conditions described above for 5'-carboxylic oligonucleotides, or
by a denaturingPAGE. Their respective molecular masses were checked by MALDI-TOF-MS analysis.
Triphenylmethyl 4-Oxopentanoate (1). To a stirred soln. of 4-oxopentanoic acid (2.25 ml, 22 mmol) in 35 ml
i
of CH2Cl2, containingEtN ( Pr)2 (5.14 ml, 30 mmol) and DMAP (122 mg, 1 mmol), a soln. of Ph3CCl (5.58 g,
20 mmol) in 15 ml of CH2Cl2 was added dropwise over a period of 20 min. The mixture was stirred at 258 for 2 h.
After TLC (system A, Rf 6.6) showed the completion of the reaction, the soln. was evaporated, dissolved in
50 ml of AcOEt, washed with 5% aq. NaHCO3 soln. (2 Â 30 ml) and brine (25 ml), dried (anh. Na2SO4), and
evaporated to a small volume. Petroleum ether was then added, and the soln. was kept in a fridge overnight. The
crystalline precipitate was collected by filtration, washed with AcOEt/petroleum ether 1:2 (v/v), and dried in
vacuo: 6.68 g(93.1%) of 1. White crystals. 1H-NMR ((D6)DMSO): 2.06 (s, Me); 2.65 (q, J ˆ 5.1, 2 CH2); 7.23,
‡
7.29 (2m, 15 arom. H). MALDI-TOF-MS: 364.1 ([M ‡ H] ; calc. 359.4).
(2-Chlorophenyl)diphenylmethyl 4-Oxopentanoate (2). To a stirred soln. of 4-oxopentanoic acid (3.07 ml,
i
30 mmol) in 50 ml of CH2Cl2 containingEtN( Pr)2 (8.56 ml, 50 mmol), a soln. of (2-chlorophenyl)diphenyl-
methyl chloride (7.86 g, 25 mmol) in 25 ml of CH2Cl2 was added dropwise over a period of 15 min. The mixture
was stirred at 258 for 3 h. After TLC (system B, Rf 0.45) showed the completion of the reaction, the soln. was
evaporated, dissolved in 60 ml of AcOEt, washed with 5% aq. NaHCO3 soln. (2 Â 35 ml) and brine (30 ml),
dried (anh. Na2SO4), and evaporated to a small volume. Petroleum ether was then added, and the soln. was kept
in a fridge overnight. The crystalline precipitate was collected by filtration, washed with AcOEt/petroleum ether
1:2 (v/v), and dried in vacuo: 9.60 g(97.5%) of 2. White crystals. 1H-NMR ((D6)DMSO): 2.09 (s, CH2COMe);
‡
2.71 (s, Me); 3.31 (s, CH2CO2); 7.19, 7.30 (2m, 14 arom. H). MALDI-TOF-MS: 393.5 ([M ‡ H] ; calc. 393.9).
Triphenylmethyl 4-Hydroxypentanoate (3). To a stirred soln. of 1 (1.08 g, 3 mmol) in 10 ml of EtOH, solid
NaBH4 (0.17 g, 4.5 mmol) was added in small portions over 10 min. The mixture was stirred for another 1 h.
After TLC (system B, Rf 0.29) showed disappearance of the startingmaterial, the mixture was evaporated,
dissolved in 20 ml of CHCl3, washed by 5% aq. NaHCO3 soln. (2 Â 15 ml) and brine (10 ml), dried (anh.
Na2SO4), and evaporated in vacuo. The residue was redissolved in CHCl3 and chromatographed on a silica-gel
column with a gradient of 1 to 5% MeOH in CHCl3 (v/v) containing0.5% Et 3N as an eluent. The appropriate
fractions were pooled, evaporated to dryness, and dried in high vacuum: 0.77 g (71.5%) of 3. Colorless oil.
Triphenylmethanol (74 mg, 6.8%) was also isolated as a minor by-product. 1H-NMR ((D6)DMSO): 1.00 (d, J ˆ
6.1, Me); 1.55 (q, J ˆ 7.5, CH2CHOH); 2.49 (m, CH2CO); 3.31 (d, J ˆ 7.3, OH); 3.52 (m, CHOH); 7.28 (m, 15
‡
arom. H). MALDI-TOF-MS: 361.7 ([M ‡ H] ; calc. 360.5).
(2-Chlorophenyl)diphenylmethyl 4-Hydroxypentanoate (4). To a stirred soln. of 2 (3.94 g, 10 mmol) in
25 ml of EtOH, solid NaBH4 (0.57 g, 15 mmol) was added in small portions over 10 min. The mixture was stirred
for another 1 h. After TLC (system B, Rf 0.26) showed disappearance of the startingmaterial, the mixture was
evaporated, dissolved in 25 ml of AcOEt, washed with 5% aq. NaHCO3 soln. (2 Â 15 ml) and brine (10 ml),
dried (anh. Na2SO4), and evaporated in vacuo. The residue was dissolved in CHCl3 and chromatographed on a
silica-gel column with a gradient of 1 to 5% MeOH in CHCl3 (v/v) containing0.5% Et 3N as an eluent. The ap-
propriate fractions were pooled, evaporated to dryness, and dried in high vacuum: 3.48 g (87.9%) of 4. Colorless
1
oil. H-NMR ((D6)DMSO): 1.03 (d, J ˆ 6.2, Me); 1.61 (q, J ˆ 7.3, CH2CHOH); 2.57 (q, J ˆ 7.8, CH2CO); 3.58
‡
(m, CHOH); 4.51 (d, J ˆ 4.8, OH); 7.29 (m, 14 arom. H). MALDI-TOF-MS: 393.4 ([M ‡ H] ; calc. 395.1).
Triphenylmethyl 4-[(2-Cyanoethoxy)(diisopropylamino)phosphanyloxy]pentanoate (5). Compound
3
(0.75 g, 2.07 mmol), dried by repeated co-evaporations with dry MeCN, was dissolved in dry CH2Cl2 (10 ml),
and diisopropylammonium tetrazolide (0.53 g, 3.11 mmol) and (2-cyanoethoxy)bis(diisopropylamino)phos-
phane (0.86 ml, 2.69 mmol) were added, and the mixture was stirred at 258 for 5 h. After TLC (system C, Rf
0.25) showed the completion of the reaction, the mixture was evaporated, dissolved in AcOEt (10 ml), washed
with 5% aq. NaHCO3 soln. (2 Â 5 ml) and brine (5 ml), dried (anh. Na2SO4), and evaporated to dryness. The

2416
Helvetica Chimica Acta Vol. 85 (2002)
residue was dissolved in CH2Cl2/hexane 3 :1 (v/v) and applied on the top of silica-gel column eluted first by 20%,
then by 15%, and finally by 10% hexane in CH2Cl2 containing2% Et N (v/v). The appropriate fractions were
3
pooled, evaporated to dryness, and dried in high vacuum: 0.44 g (38.2%) of 5. Colorless oil. 1H-NMR (CD3CN):
1.20 (m, 5 Me); 1.79 (m, CH2CHO); 2.61 (m, 2 CH2); 3.71 (2m, 2 CHN, CH2O); 3.99 (m, CHO); 7.31 (m, 15
arom. H). 31P-NMR (CD3CN): 147.37, 148.38 (two sets of diastereoisomers in ca. 2.5 :1 ratio). MALDI-TOF-
‡
MS: 561.5 ([M ‡ H] ; calc. 561.3).
(2-Chlorophenyl)diphenylmethyl 4-[(2-Cyanoethoxy)(diisopropylamino)phosphanyloxy]pentanoate (6).
Compound 4 (0.47 g, 1.2 mmol), dried by repeated co-evaporations with dry MeCN, was dissolved in dry
CH2Cl2 (5 ml) and diisopropylammonium tetrazolide (0.31 g, 1.8 mmol) and (2-cyanoethoxy)bis(diisopropyl-
amino)phosphane (0.49 ml, 1.5 mmol) were added, and the mixture was stirred at 258 for 4 h. After TLC (system
B, Rf 0.71, 0.76, pair of diastereoisomers) showed the completion of the reaction, the mixture was evaporated,
dissolved in AcOEt (10 ml), washed with 5% aq. NaHCO3 soln. (2 Â 5 ml) and brine (5 ml), dried (anh.
Na2SO4), and evaporated to dryness. The residue was dissolved in CHCl3/hexane 3 :2 (v/v) and chromato-
graphed on a silica-gel column eluted first by 40%, then by 30%, and finally by 20% hexane in CHCl3 containing
2% Et3N (v/v). The appropriate fractions were pooled, evaporated to dryness, and dried in high vacuum: 0.69 g
(97.4%) of 6. Colorless oil. 1H-NMR ((D6)DMSO): 1.14 (m, 5 Me); 1.72 (m, CH2CHO); 2.65 (m, CH2CH2CN);
3.54, 3.69 (2m, 2 CHN, CH2O); 3.95 (m, CHO); 7.18, 7.32 (2m, 14 arom. H). 31P-NMR (CD3CN): 147.35, 146.28
‡
(two sets of diastereoisomers). MALDI-TOF-MS: 597.9 ([M ‡ H] ; calc. 596.1).
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Received May 6, 2002