Phosphorothioate anti-sense oligonucleotides: The kinetics and mechanism of the generation of the sulfurising agent from phenylacetyl disulfide (PADS)

Article abstract of DOI:10.1039/c6ob01531j

The synthesis of phosphorothioate oligonucleotides is often accomplished in the pharmaceutical industry by the sulfurisation of the nucleotide-phosphite using phenylacetyl disulfide (PADS) which has an optimal combination of properties. This is best achieved by an initial 'ageing' of PADS for 48 h in acetonitrile with 3-picoline to generate polysulfides. The initial base-catalysed degradation of PADS occurs by an E1cB-type elimination to generate a ketene and acyldisulfide anion. Proton abstraction to reversibly generate a carbanion is demonstrated by H/D exchange, the rate of which is greatly increased by electron-withdrawing substituents in the aromatic ring of PADS. The ketene can be trapped intramolecularly by an o-allyl group. The disulfide anion generated subsequently attacks unreacted PADS on sulfur to give polysulfides, the active sulfurising agent. The rate of degradation of PADS is decreased by less basic substituted pyridines and is only first order in PADS indicating that the rate-limiting step is formation of the disulfide anion from the carbanion.

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Phosphorothioate Anti-sense Oligonucleotides: The Kinetics and
Mechanism of the Generation of the Sulfurising Agent from
Phenylacetyl Disulfide (PADS)
Received 00th January 20xx,
Accepted 00th January 20xx
James Scotsona, Benjamin I. Andrewsb, Andrew P. Lawsa and Michael I. Pagea
DOI: 10.1039/x0xx00000x
www.rsc.org/
The synthesis of phosphorothioate oligonucleotides is often accomplished in the pharmaceutical
industry by the sulfurisation of the nucleotide-phosphite using phenylacetyl disulfide (PADS) which has
an optimal combination of properties. This is best achieved by an initial ‘ageing’ of PADS for 48 hrs in
acetonitrile with 3-picoline to generate polysulfides. The initial base-catalysed degradation of PADS
occurs by an E1cB-type elimination to generate a ketene and acyldisulfide anion. Proton abstraction to
reversibly generate a carbanion is demonstrated by H/D exchange, the rate of which is greatly
increased by electron-withdrawing substituents in the aromatic ring of PADS. The ketene can be
trapped intramolecularly by an o-allyl group. The disulfide anion generated subsequently attacks
unreacted PADS on sulfur to give polysulfides, the active sulfurising agent. The rate of degradation of PADS is
decreased by less basic substituted pyridines and is only first order in PADS indicating that the rate-limiting
step is formation of the disulfide anion from the carbanion.
numerous clinical trials of other oligonucleotides4 and, more
recently, Kynamro (mipomersen) to treat genetically inherited high
Introduction
Synthetic oligonucleotides have been used in many different ways
for several decades1, such as therapeutic agents and in diagnostics.
An anti-sense oligonucleotide (ASO) is usually a single-stranded
deoxy-ribonucleotide (usually 15-35 base pairs in length) which is
complementary to a target mRNA. Complex dimer formation
between the ASO and the target mRNA through normal Watson-
Crick base pairing can lead to inhibition of translation and so
prevent synthesis of an unwanted target protein. The formation of
cholesterol levels5. Other mechanisms of action of ASOs include
‘exon-skipping’ where, instead of binding to mRNA, the ASO binds
to longer pre-mRNA chains sterically blocking an unwanted
mutation and its surrounding sequence preventing the exon,
containing the mutation, being spliced into the mRNA. Antisense-
mediated exon skipping is
a
promising therapeutic for
neuromuscular diseases 6, although trials for drisapersen to treat
Duchenne Muscular Dystrophy have recently been stopped. ASOs
are also useful as valuable genetic diagnostic tools, for example,
HyBeacon probes are single-stranded oligonucleotides with one or
more internal bases labelled with a fluorescent dye so that when
duplex formation occurs with its target sequence there is an
increase in fluorescence7.
the ASO–mRNA hetero-duplex often activates RNase
H
endonuclease activity which then catalyses the hydrolysis of the
mRNA leaving the ASO intact2. The first approved anti-sense
oligonucleotide was Vitravene (formiversen, for treating the
blinding viral condition cytomegalovirus induced retinitis afflicting
some of those infected with HIV3), which was followed by
However, the use of phosphodiester anti-sense oligonucleotides is
limited because they are rapidly hydrolysed by intracellular
endonucleases and exonucleases8. Consequently, ASOs have been
modified to decrease their susceptibility to nuclease cleavage and
to increase their uptake into cells, binding efficiency, bio-stability
a Department of Chemistry, University of Huddersfield, Queensgate,
Huddersfield HD1 3DH, UK
b GSK Medicines Research Centre, Gunnels Wood Road, Stevenage,
Hertfordshire, SG1 2NY, UK.
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and potency9. Examples of these modifications are the inclusion of P=S to P=O ratio. The sulfurisation of phosphorus(III)
phosphorothioate backbones (1)10 and changes to the 2′ position of compounds has been achieved with a number of reagents such
the ribose sugar (e.g. 2′-O-methoxyethyl) or the sugar-phosphate as: phenylacetyl disulfide (PADS) (2),15 3H-1,2-benzodithiol-3-
(e.g. morpholino and locked nucleic acid, (S)-constrained-2′-O-ethyl one-1,1-dioxide (Beaucage reagent),16 tetraethylthiuram
- bicyclic constrained riboses)11. These changes do not disrupt H- disulfide
(TETD),17
dibenzoyl
tetrasulfide18
,
bis-(O,O-
bonding between the nucleobases.
diisopropoxyphosphinothioyl)
disulfide
(S-Tetra),19
benzyltriethylammonium tetrathiomolybdate (BTTM)20, bis-(p-
toluenesulfonyl) disulfide,21 3-ethoxy-1,2,4-dithiazoline-5-one
(EDITH)
and
1,2,4-dithiazolidine-3,5-dione
(DTSNH),22
bis(ethoxythiocarbonyl)tetrasulfide,23
3-methyl-1,2,4-
dithiazolin-5-one (MEDITH)24 and 3-amino-1,2,4-dithiazole-5-
thione (ADTT, xanthane hydride).25
Although PADS (2) has an optimal combination of properties and
is often used as the sulfurising reagent in the pharmaceutical
industry26,27, there have been very few mechanistic studies of
the sulfurisation reactions of phosphorus (III) analogues using
PADS. In particular, PADS must be ‘aged’ in a basic acetonitrile
solution to obtain optimal sulfurisation activity28 and the
reasons for this are not understood. Herein we report the
results of our kinetic and mechanistic studies which identify the
actual sulfurising agent and suggest an unexpected pathway for
its generation. Elsewhere we describe the kinetics and
mechanism of the sulfurisation step of substituted triphenyl
Phosphorothioates (1) are the most widely investigated
oligonucleotides because of their relative nuclease stability and
ease of synthesis and a naturally occurring phosphorothioate has
even been found in bacterial DNA12. The replacement of one of the
phosphate non-bridging oxygens by sulfur introduces chirality at
phosphorus and it is only the (S)-P phosphorothioate diastereomer
phosphites and trialkyl phosphites using PADS29
.
that is nuclease resistant13
.
However, phosphorothioate
Results and Discussion
oligonucleotides can reduce the affinity of the ASO for its mRNA
target as shown by the melting temperature of the ASO-mRNA
hetero-duplex which is decreased by approximately 0.5oC per
nucleotide14. Conversely, the introduction of the hydrophobic sulfur
increases cell uptake compared with the wild-type phosphodiester.
Phosphorothioate oligonucleotides remain highly water soluble, still
bind well to mRNA and activate RNase H making them the most
common modification of ASOs undergoing clinical trials.
It is known that a freshly made solution of PADS (2) is not an
optimal sulfurising agent26. Solutions of PADS usually consist of 50%
v/v 3-picoline in acetonitrile or other bases such as pyridine or
collidine but their efficiency improves greatly upon ‘ageing’ for
about 2 days26. PADS degrades completely over this period of time
as shown byHPLC, 1H NMR and MS and the colour of the solution
changes dramatically from pale yellow through green/blue to
brown/black over about
5 days. The pseudo first-order rate
The synthesis of phosphorothioate oligonucleotides is often
accomplished by the sulfurisation of the nucleotide-phosphite
through reaction of the corresponding P(III) analogue, usually
attached to a solid support, with an organic sulfurising agent
which is present in an organic solvent. As with all the steps
involved in the synthesis of oligonucleotide based
phosphorthioates, the sulfurisation step of the synthesis should
be be rapid, have a near quantitative yield and give a maximal
constants for the sulfurisation of 0.05 M triphenyl phosphite in
acetonitrile by 0.5 M PADS with 1.0 M 3-picoline increase with the
length of ‘ageing’ (Fig. 1), such that PADS ‘aged’ for 48 hrs. is 13-
fold more active than a freshly prepared solution of PADS. It is
apparent therefore that the degradation product(s) of PADS are
more efficient sulfurising agents, but their identity is not known.
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The exponential rate of degradation of 3.3
ARTICLE
M
PADS (2) in ‘ageing’ of PADS by substituted pyridines (Table 1) generate a
acetonitrile 25oC was followed by HPLC and the associated first-
order rate constants depend on the concentration of 3-picoline (Fig.
2). This increase in rate is unlikely to be a consequence of a change
Bronsted β-value of 0.37 based on the estimated pKa in
acetonitrile30. This is compatible with the pyridine acting as either
a base or as a nucleophile with little charge development on the
basic nitrogen. The rate of degradation of PADS in acetonitrile is
faster with a more basic amine, e.g. with 1.0 M triethylamine it is
complete within 5 mins.
12
10
8
6
Table 1 The second-order rate constants for the decomposition
of PADS (2) in acetonitrile (ACN) as a function of the basicity of
substituted pyridines in ACN at 25oC.
4
2
Pyridine
substituent
4-MeO
3-Me
pKa (ACN) conjugate
0
kcat (M-1s-1)
0
10
20
30
40
50
acid
14.73
13.70
12.60
12.45
8.50
Time of ageing (hrs)
7.76 x 10-6
3.34 x 10-6
1.63 x 10-6
3.79 x 10-7
3.76 x 10-8
4.44 x 10-6
Figure 1 The pseudo first-order rate constants for the
sulfurisation of triphenyl phosphite (0.05 M) to (PhO)3P=S
by (0.5 M) PADS (2) as a function of the time that PADS has
been ‘aged’ in acetonitrile with (1.0 M) 3-picoline at 25oC
H
3-MeO
4-CN
2,6-diMe2
14.72
in the dielectric constant of the solution as, for example, PADS is
stable in a solution of acetonitrile / dimethylsulfoxide 90/10% v/v
over 24 hrs. Furthermore, the rate of degradation of PADS in
acetonitrile increases with the basicity of the pyridine base. The
slope of the dependence of the observed pseudo first-order rate
constants for the decomposition of PADS (2) (Fig. 2) gives the
associated second-order rate constant. These rate constants for the
The products of the ‘ageing’ of PADS are
a mixture of
diacylpolysulfides and acylpolysulfide anions. HPLC-mass
spectroscopy data performed on aged PADS solutions showed the
presence of m/z values corresponding to
a
variety of
diacylpolysulfides, in particular the tri-sulfide (3) at m/z = 357 (Na+
adduct) and the penta-sulfide (4) at m/z = 437 (K+ adduct). After
several days elemental sulfur crystallised from the solution. The
diphenylacetyl polysulfides are presumably formed from
nucleophilic attack of the disulfide anion on PADS at sulfur. It is
known that disulfide anions are better nucleophiles than the
corresponding thiolate31. The formation of phenylacetyl disulfide
anion (5) could result from either nucleophilic attack on PADS or a
general-base catalysed elimination reaction of PADS by the picoline
base. It is likely that there is an equilibrium set up between the
8.0
6.0
4.0
2.0
0.0
diacylpolysulfides and acylpolysulfide anions and sulfur32
.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
O
O
Concentration of 3-picoline (M)
(S)n
S
S
Figure 2 The dependence of the pseudo first-order rate
constants for the decomposition of PADS (2) in acetonitrile on
3 n = 1
4 n = 3
the concentration of 3-picoline at 25oC
.
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assumed to be
~
1010 M-1s-1. The pKa value for PADS in
acetonitrile/water (90/10% v/v) can then be calculated to be 26.0
4.0
Nucleophilic attack on PADS (2) by picoline would generate an
acyl-picolinium ion intermediate (6) but no direct experimental
evidence for this could be found nor could its presence be
3.0
2.0
1.0
0.0
inferred from trapping experiments with
a
variety of
nucleophiles. However, the general-base catalysed formation of
the disulfide anion by an E1cB - type mechanism (Scheme 1) is
indicated by both D-exchange and trapping of the ketene
intermediate.
0.0
0.2
0.4
0.6
0.8
1.0
Concentration of 3-picoline (M)
Figure 3 The dependence of the pseudo observed first-order
rate constants for the H/D - exchange of PADS on the
concentration of 3-picoline in acetonitrile at 25oC.
by using this estimated diffusion-controlled rate constant, the
measured rate of D-exchange and the pKa of 13.7 for 3-picoline in
acetonitrile34. There are no pKa data for thioesters in acetonitrile
but that for ethyl thioacetate in water is 2135 and, based on
substituent and solvent effects30, that for ethyl thiophenylacetate
PhCH2COSEt in acetonitrile is ~ 31, so the estimated value of 26.0
for PADS is reasonable given the additional electron-withdrawing
SSCOCH2Ph residue.
The rate of H/D exchange was measured by 1H NMR using a
solution of 0.17M PADS (2) in d3-acetonitrile containing various
concentrations of 3-picoline and 0.6M D2O at 25oC. The CH2 singlet
at δ 4.15 ppm disappeared with a first-order decay generating a
triplet for CHD at δ 4.13 ppm which reached a maximum intensity
followed by its disappearance to give CD2. The pseudo first-order
rate constants for D-exchange showed a first-order dependence on
the concentration of 3-picoline (Fig. 3) which gives a second-order
rate constant kpic = 4.72 x 10-3 M-1s-1.
The mechanism of D-exchange presumably occurs through the
formation of the intermediate carbanion. The relatively low
dielectric constant of the solvent may mean that the ionization of
this carbon acid may give rise to ion-pairs in equilibrium with the
dissociated species (Scheme 2), where the product KiKd corresponds
to the normal ionization constant Ka. However, it is unlikely that
exchange can occur from the ion-pair and so if isotopic exchange
occurs with the dissociated carbanion, then the rate of protonation
The rate of degradation of PADS (2) is ca. 500-fold slower than the
rate of D-exchange under the same conditions, indicating that
carbanion formation is relevant to the formation of the degradation
products of PADS. Indeed, removing the dissociable protons in
PADS by α,α dimethylation prevents the degradation of the
tetramethyl derivative 7 which remains unchanged after 1 week in
acetonitrile with 5 equivalents of picoline at 25oC. Similarly, bis-
of the carbanion k-2 can be assumed to be diffusion-controlled33
,
which, based on a viscosity of 0.35cP for acetonitrile can be
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benzoyl disulfide is stable in acetonitrile with 5 equivalents of All of the above observations suggest that a polar mechanism is
picoline at 25oC for 1 week, confirming the elimination mechanism taking place, which is also supported by the rate of degradation of
for the degradation of PADS (2).
PADS (2) being unaffected by light or dark or the addition of the
radical scavenger 2,6-di-tert-butyl-4-methylphenol (BHT).
In summary, the degradation of PADS (2) in acetonitrile catalysed by
pyridine bases involves reversible carbanion formation, which
expels acyl disulfide anion to generate a ketene intermediate. The
disulfide anion then attacks unreacted PADS at sulfur to give
polysulfides (Scheme 3). Other possibilities are feasible, including
polysulfide anion attack on PADS (2) and diacylated polysulfides. It
is interesting that the rate of degradation is only first order in PADS
(2) indicating that the rate-limiting step is k2 formation of the
disulfide anion from the carbanion, rather than its subsequent
reaction with a second mole of PADS.
The proposed mechanism for the degradation of PADS (Scheme 1)
involves the generation of the ketene intermediate. The reaction of
o-allyl PADS (8) in 50% v/v acetonitrile/3-picoline yields the bicyclic
ketone (9) identified by MS and 1H NMR, consistent with trapping
the intermediate ketene by a [2+2] cycloaddition. With PADS itself,
the ketene forms the 3-hydroxycyclobutenone dimer, (10) and the
pyrononone trimer (11) as observed with the base-catalysed
reaction of phenylacetyl chloride36
.
O
Ph
O
Ph
Ph
HO
Ph
PhCH2
O
OH
10
11
The relative effect of substituents in the aromatic ring of PADS (2)
on the rate constants for H/D-exchange and the degradation
process are shown in Table 2.
Conclusion
The activation of PADS by its ‘ageing’ for 48 hrs in acetonitrile
with 3-picoline involves the generation of polysulfides which are
the actual nucleotide-phosphite sulfurising agents. The initial
base-catalysed degradation of PADS occurs by an E1cB-type
Table 2 The dependence of the second-order rate constants
for H/D-exchange and for the degradation of substituted
PADS (2) in acetonitrile at 250C
elimination to generate
a ketene and acyldisulfide anion.
Carbanion formation from PADS is reversible, demonstrated by
H/D exchange, the mechanism of which occurs by base catalysis
and is greatly increased by electron-withdrawing substituents in
the aromatic ring of PADS. The ketene can be trapped
intramolecularly by an o-allyl group. The disulfide anion
generated subsequently attacks unreacted PADS on sulfur to give
polysulfides, the active sulfurising agent. The rate of degradation of
PADS is decreased by less basic substituted pyridines and is only
PADS substituent
kex (M-1s-1)
1.38 x 10-3
5.16 x 10-3
2.20 x 10-2
1.09 x 10-1
σ
4-MeO
H
-0.27
0
4-Cl
0.23
0.66
4-CN
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first order in PADS indicating that the rate-limiting step is formation 0.55 mmol) were dissolved in THF (250 ml), stirred for 30 min at
room temperature. A solution of allylboronic acid pinacol ester (3.3
of the disulfide anion from the carbanion.
ml, 21.8 mmol) in THF (50 mL) was added and then heated under
reflux for 24 hr. The reaction mixture was diluted with petroleum
ether (bpt. 60–80°C, 300mL), extracted with water (2 x 200ml) and
Experimental
the combined aqueous layers washed with petroleum ether (bpt
NMR experiments were performed using a 400MHz Bruker Avance
60-80oC, 100ml). The combined organic layers were washed with
DP X400 NMR spectrometer. HPLC traces were acquired using a
water (100 mL), brine (100 mL), dried over MgSO4. The solvent was
Shimadzu SIL 50AH instrument with a Luna 5μ C18 4.67x250mm
removed under vacuum to give an orange oil product (2.5 g) which
column. The gradient used for kinetic measurements was 30-95%
was purified using a Biotage SP4 chromatography system fitted with
acetonitrile in water over 10 minutes followed by isocratic elution
a Biotage Snap 10 column eluting with a gradient of 0-25% ethyl
for a further 10 minutes.
acetate in hexane over 15 column volumes to give the product ester
H/D exchange of PADS To a solution of phenylacetyl disulfide in
as a yellow oil (1.6 g, 77.2% yield). HPLC retention time 5.630 min;
deuterated acetonitrile (0.2M, 400 µl) and D2O (50 µl) was added 3-
1H NMR (CDCl3, 400MHz) δ 3.39 (dt, 2H, J 6.29Hz, CH2), 3.61 (s, 2H,
1
picoline (15.5 µl, 16.2 mg, 0.17mmol). The H NMR of this mixture
CH2), 3.65 (s, 3H, CH3), 4.94-5.06 (m, 2H, CH2), 5.87-5.97 (m, 1H,
CH), 7.14-7.23 (m, 4H, ArH) ppm; 13C NMR (CDCl3, 100MHz) δ 37.35
was run every 2 minutes until exchange was complete.
General degradation kinetics of PADS A solution of PADS
(CH2), 38.45 (CH2), 51.94 (CH3), 115.96 (CH2), 126.65 (CH), 127.55
(1000ppm, 3.3mM) in HPLC-grade acetonitrile containing the
(CH), 129.91 (CH), 130.65 (CH), 132.66 (Cq), 136.57 (CH2), 138.35
(Cq), 171.95 (CO) ppm; IR (film) 1734.52 cm-1.
appropriate concentration of 3-picoline was divided between 2
HPLC vials and chromatograms were taken every 30 minutes for 48
2′-Allyl 2-phenylacetic acid: to methyl-2′-allyl 2-phenylacetate (1.7
hours.
g, 8.9 mmol) in THF (20ml) was added an aqueous lithium hydroxide
General sulfurisation kinetics with ‘aged’ PADS A solution of
solution (0.4M, 20ml) and the solution stirred at room temperature
PADS (1M) 50/50 v/v acetonitrile/3-picoline was left to age for 48
for 3 hr after which the mixture was diluted with water (40ml) and
hours. At T=n hrs a 1ml sample of this was removed and quenched
extracted with DCM (2 x 20ml). The pH of the aqueous layer was
with dilute hydrochloric acid (10ml, 2M) to remove the picoline as
reduced to pH 1 with HCl, extracted with DCM (2 x 20ml), dried over
the protonated pyridinium ion. This was then washed with DCM (2 x
MgSO4 and the solvent removed under vacuum giving the brown oil
10ml) the combined organic layers dried and the solvent removed
min; 1H NMR (CDCl3, 400MHz) δ 3.38 (dt, 2H, J 6.37 Hz, CH2), 3.65 (s,
under vacuum. The resulting oil was then made up to 1ml using
product (1.7 g, 9.7 mmol, 82.9% yield). HPLC retention time 4.634
deuterated acetonitrile. To an NMR tube containing triphenyl
(m, 4H, ArH), 10.76 (broad, s, 1H, OH) ppm; 13C NMR (CDCl3,
phosphite in deuterated acetonitrile (0.1M, 200 μL) and 3-picoline
2H, CH2), 4.94-5.06 (m, 2H, CH2), 5.86-5.96 (m, 1H, CH), 7.14-7.23
in deuterated acetonitrile (2M, 200 μL) was added ‘aged’ PADS
100MHz) δ 37.5 (CH2), 38.45 (CH2), 116.22 (CH2), 126.79 (CH),
solution (200 μL, 1M) and 31P NMR spectrum recorded every 90
127.96 (CH), 130.08 (CH), 130.88 (CH), 132.05 (Cq), 136.52 (CH),
138.55 (Cq), 178.41 (CO) ppm; IR (film) 1702.49cm-1.
seconds.
2′,2′-Diallyl 2,2-phenylacetyl disulfide (8)38: sodium hydrosulfide
Trapping the ketene intermediate A mixture of 2,2’-allyl-2,2’-
phenylacetyl disulfide (330mg, 0.852mmol) and 3-picoline (165.6 µl,
1.7mmol) in acetonitrile (10ml) was refluxed for 2 hr. The reaction
was followed by HPLC and was complete after 2 hrs. The mass spec
and NMR analysis showed evidence of the product 7,7a-dihydro-1H-
cyclobuta[a]inden-2(2aH)-one as described below.
(798 mg, 14.25 mmol) was dissolved in ethanol (5 ml) and chilled
over ice. The flask was fitted with an exhaust line allowing any
gasses generated to be scrubbed by sodium hypochlorite in a well-
ventilated fume hood. In a separate flask, to 2′-allyl 2-phenylacetic
acid (1.0 g, 5.7 mmol) dissolved in DCM (10 ml) was added Ghosez
reagent (1.04 g, 1.03 ml, 8.5 mmol) then stirred at room
Synthesis
Methyl 2′-iodo-2-phenylacetate: 2′-iodo-phenylacetic acid (5.01 g, temperature for 30 min. and the solvent removed under vacuum to
19.1 mmol) and H2SO4 (98%, 1.25 ml) were dissolved in methanol yield an orange oil. This oil was added to the ethanolic sodium
(7.5 ml). The reaction mixture was stirred for 2.5 hr at 65oC, then
hydrosulfide solution which instantly turned yellow and the
diluted with 250 ml dichloromethane (DCM), extracted with water precipitate of sodium chloride removed under vacuum filtration and
(2 x 100 ml) and brine (50 ml). The organic solution was dried over washed with ice cold ethanol (1.5ml). To the solution, stirred over
ice, iodine was slowly added until the colour of the suspension
MgSO4, and gravity filtered. DCM was removed under vacuum to
give a pale yellow oil (4.88 g, 92.6% yield). HPLC retention time
changed from white to pale brown (approximately 1 g, 7.8 mmol).
5.397 min; 1H NMR (CDCl3, 400MHz) δ 3.69 (s, 3H, CH3), 3.79 (s, 2H, The resulting mixture was diluted with DCM (10 ml), washed twice
CH2), 6.92-6.96 (m, 1H, ArH), 7.22-7.32 (m, 2H, ArH), 7.83 (d, 1H, J with saturated sodium thiosulfate solution (2 x 15 ml) and the
7.91Hz, ArH) ppm; 13C NMR (CDCl3, 100MHz) δ 46.12 (CH3), 52.22
organic layer concentrated under vacuum to yield the brown oil
(CH2), 101.07 (CI), 128.48 (CH), 128.94 (CH), 130.68 (CH), 137.72 product which was purified using a Biotage SP4 chromatography
(Cq), 139.54 (CH), 170.95 (CO) ppm; IR (film) 1732.81 cm-1.
system fitted with a Biotage Snap 10 column eluting with 25% ethyl
Methyl 2′-allyl-2-phenylacetate37: methyl 2′-iodo 2-phenylacetate
acetate in hexane (Rf= 0.41) to give the product as a clear oil (384
(3.0 g, 10.9 mmol), CsF (6.6 g, 43.6 mmol), and Pd(PPh3)4 (0.63 g, mg, 1 mmol, 14.1% yield). HPLC retention time 7.6 min; 1H NMR
6 | J. Name., 2012, 00, 1-3
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(CDCl3, 400MHz) δ 3.41 (d, 4H, J= 6.3 Hz, CH2), 4.00 (s, 4H, CH2), cyanophenylacetic acid (2.753 g, 17.1 mmol) in DCM (20 ml);
4.96-5.60 (m, 4H, CH2), 5.88-5.98 (m, 2H, CH), 7.17-7.30 (m, 8H, Ghosez reagent (3.432 g, 3.120 ml, 25.7 mmol); iodine (0.5 g, 3.9
ArH) ppm; 13C NMR (CDCl3, 100MHz) δ 37.51 (CH2), 46.68 (CH2), mmol) to give the product as a oragne/yellow solid (1.6 g, 4.6
116.63 (CH2), 126.95 (CH), 128.58 (CH), 130.22 (CH), 130.88 (Cq), mmol, 53.8 % yield). HPLC retention time 6.0 min; 1H NMR (CDCl3,
131.53 (CH), 136.12 (CH), 139.07 (Cq), 191.81 (CO) ppm.
400MHz) δ 4.08 (s, 4H), 7.53 (d,d, 8H, J1 8.06 Hz, J2 92.53 Hz, ArH)
7,7a-dihydro-1H-cyclobuta[a]inden-2(2aH)-one (9): A mixture of 2′- ppm; 13C NMR (CDCl3, 100MHz) δ 48.82 (CH2), 112.06 (CN), 118.41
allyl 2-phenylacetylchloride (100 mg, 0.514 mmol) and 1-chloro-
N,N,2-trimethyl-1-propenylamine (74.7 µl, 0.56 mmol) in DCM (10
ml) was stirred for 30 min at room temperature, triethylamine (78
µl, 0.56 mmol) in DCM (4 ml) was added and refluxed for 2 hr. The
reaction mixture was cooled and extracted with 2M hydrochloric
acid (20 ml) to remove the 3-picoline. The crude bright orange oil
product mixture was purified using a Gilson prep. HPLC system
equipped with a phenomenex luna C18, 5µ 250 x 20 mm column.
The mobile phase consisted of a gradient of 50-100% acetonitrile
(0.05% TFA) in water (0.05% TFA) over 15minutes then isocratic
elution at 50% acetonitrile (0.05% TFA) in water (0.05% TFA) for a
further 3 minutes. The product eluted at 7.5 min. All fractions were
combined and the solvent removed under vacuum to give the
product as a white, crystalline solid (24 mg, 41.8% yield). HPLC
retention time 4.775 min; 1H NMR (d3ACN, 400MHz) δ 2 2.75-2.87
(m, 1H, CH2), 3.05 (d, 1H, J 16.73, CH2), 3.11-3.18 (m, 1H, CH), 3.30-
3.42 (m, 1H, CH2), 3.38-3.45 (m, 1H, CH2), 4.71 (broad, s, 1H), 7.23-
7.36 (m, 4H, ArH); 13C NMR (ACN-d3, 100MHz) δ 26.18 (CH), 38.82
(CH2), 52.32 (CH2), 71.81 (CH), 124.45 (CH), 125.50 (CH), 126.82
(CH), 127.52 (CH), 137.6 (Cq), 143.38 (Cq), 206.13 (CO), IR (film)
1771.39 cm-1.
(Cq), 130.47 (CH), 132.65 (CH), 137.28 (Cq), 189.76 (CO) ppm. HRMS
(m/z): [M+NH4]+ for C18H12N2O2S2, calculated 352.0340, measured
352.0352; IR: 1715.6 cm-1 (film).
2,2′-(4-methoxyphenyl)acetyl disulfide: as above using sodium
hydrosulfide (4.26 g, 75.3 mmol) in ethanol (20 ml); 4-
methoxyphenylacetic acid (5 g, 30.1 mmol) in DCM (20 ml); Ghosez
reagent (8.03g, 7.96 ml, 60.2 mmol); iodine (1 g, 7.8 mmol) to give
product (1.19 g, 3.28 mmol, 10.9 % yield). 1H NMR (CDCl3, 400MHz)
δ 3.79 (s, 6H, CH3), 3.92 (s, 4H, CH2), 7.04 (d,d, 8H, J1 8.75 Hz, J2
114.31, ArH) ppm; 13C NMR (CDCl3, 100MHz) δ 48.6 (CH3), 55.2
(CH2), 114.7 (CH), 124.5 (Cq), 131.1 (CH), 159.5 (Cq), 192.5 (CO)
ppm; HRMS (m/z): [M+NH4]+ for C18H18O4S2, calculated 362.0647,
measured 362.0634.
Bis-benzoyl disulfide39: To sodium sulfide (1.84g, 23.6mmol) in
water (12ml) and heated to 50oC was added tetra-N-
butylammonium bromide (380mg, 1.18mmol) then stirred for 30
minutes. In a separate flask, (fitted with an exhaust line allowing
any gasses generated to be scrubbed by sodium hypochlorite in a
well-ventilated fume hood) benzoyl chloride (2ml, 1.65g, 11.8mmol)
2,2,2′,2′-Tetramethyl 2,2′phenylacetyl disulfide (7): Sodium
hydrosulfide (4.2 g, 75 mmol) was dissolved in ethanol (30 ml) and in toluene (12ml). This was chilled in an ice bath. After 30 minutes
chilled over ice. The flask was fitted with an exhaust line allowing
any gasses generated to be scrubbed by sodium hypochlorite in a
well-ventilated fume hood. In a separate flask, to 2-phenylisobutyric
acid (5 g, 30 mmol) dissolved in DCM (20 ml) was added Ghosez
reagent (6.16 g, 5.6 ml, 45 mmol) then stirred at room temperature
for 30 min. and the solvent removed under vacuum to yield an
orange oil. This oil was added to the ethanolic sodium hydrosulfide
solution which instantly turned yellow and the precipitate of
sodium chloride removed under vacuum filtration and washed with
ice cold ethanol (1.5ml). To the solution, stirred over ice, iodine (2
g, 15.6 mmol) was slowly added. The resulting precipitate was
collected by vacuum filtration and washed with ice-cold ethanol (5
ml) and water (10ml) to give a waxy yellow solid (2.2 g, 5.9 mmol,
39.3 % yield). HPLC retention time 7.6 min; 1H NMR (CDCl3,
400MHz) δ 1.68 (s, 12H), 7.34 (m, 10H) ppm; 13C NMR (CDCl3,
100MHz) δ 26.82, 54.21, 127.27, 127.86, 128.61, 142.14, 199.39
ppm. HRMS (m/z): [M+NH4]+ for C20H22O2S2, calculated 390.0782,
measured 390.0788; IR: 1707.3 cm-1 (film).
the sodium sulfide solution was cooled to room temperature and
slowly added to the rapidly stirring, chilled benzoyl chloride solution
under a nitrogen atmosphere creating a biphasic reaction mixture.
This was stirred for 24 hours at room temperature. The organic and
aqueous layers were separated, the aqueous layer washed with 2 x
5ml portions of toluene and the organic layers combined. The
toluene was then removed under vacuum giving the product as
white crystals (738mg, 45.7% yield) from which a single crystal X-ray
diffraction pattern was resolved. The aqueous layer contains
residual Na2S which will generate H2S if exposed to acid and was
1
disposed of carefully. H NMR (CDCl3, 400MHz) δ 7.43 (t, 4H, ArH),
7.57 (t, 2H, ArH), 7.89 (d, 4H, ArH)ppm; 13C NMR (CDCl3, 100MHz) δ
127.91 (CH), 128.79 (CH), 134.02 (CH), 136.61 (Cq), 190.37 (CO)
ppm.
2,2′-(4-chlorophenyl)acetyl disulfide: as above using sodium
hydrosulfide (1.6 g, 29.25 mmol) in ethanol; (15 ml) 4-
chlorophenylacetic acid (2 g, 11.7 mmol) in DCM (20 ml); Ghosez
reagent (1.72 g, 1.57 ml, 12.9 mmol); iodine (0.5 g, 3.9 mmol) to
give product (1.5 g, 4.1 mmol, 70.1 % yield). HPLC retention time
7.3 min; 1H NMR (CDCl3, 400MHz) δ 3.94 (s, 4H, CH2), 7.17-7.39 7.52
(d,d, 8H, J1 8.28 Hz, J2 92.54 Hz, ArH) ppm; 13C NMR (CDCl3,
100MHz) δ 48.37 (CH2), 129.12 (CH), 130.48 (Cq), 131.06 (CH),
134.12 (Cq), 190.83 (CO) ppm. HRMS (m/z): [M+NH4]+ for
C16H12Cl2O2S2, calculated 387.9994, measured 388.0000; IR:
1720.2cm-1 (film).
Acknowledgements
JS is grateful to GSK for the award of an BBSRC CASE
studentship
2,2′-(4-cyanophenyl)acetyl disulfide: as above using sodium
hydrosulfide (2.4 g, 43 mmol) in ethanol (20 ml); 4-
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Notes and references
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