Synthesis of phosphonoglycine backbone units for the development of phosphono peptide nucleic acids

Article abstract of DOI:10.1002/ejoc.201300523

A series of phosphono-modified backbone mimics based on achiral and chiral N-(dihydroxypropyl)glycine units were obtained by sequential addition of phosphonate and nucleobase moieties to suitably protected dihydroxypropylamines. Simple synthetic strategies enabled the preparation of various target derivatives that will be useful as building blocks for the preparation of new synthetic polymers containing a phosphonate internucleotide linkage in place of the standard phosphodiester bond. Copyright

Full text of DOI:10.1002/ejoc.201300523

FULL PAPER
DOI: 10.1002/ejoc.201300523
Synthesis of Phosphonoglycine Backbone Units for the Development of
Phosphono Peptide Nucleic Acids
Bogdan Doboszewski,[a,b] Elisabetta Groaz,[b] and Piet Herdewijn*[b]
Keywords: Medicinal chemistry / Asymmetric synthesis / Nucleotides / Peptide nucleic acids / Phosphonates
A series of phosphono-modified backbone mimics based on
ous target derivatives that will be useful as building blocks
achiral and chiral N-(dihydroxypropyl)glycine units were ob- for the preparation of new synthetic polymers containing a
tained by sequential addition of phosphonate and nucleo-
base moieties to suitably protected dihydroxypropylamines.
Simple synthetic strategies enabled the preparation of vari-
phosphonate internucleotide linkage in place of the standard
phosphodiester bond.
Crick base pairing can be supported by chemically simpli-
fied backbone structures.[2–4] Among such modifications,
one relevant class of DNA mimics consists of peptide nu-
cleic acids (PNAs),[5] which are linear pseudopeptide back-
bones connected to nucleobases through methylene carb-
onyl linkages. Many structural variants, closely related to
N-(2-aminoethyl)glycine PNA,[6] have been synthesized and
are capable of stable antiparallel homoduplex and triple he-
lix formation (Figure 1).[5] PNA strongly hybridizes with
natural complements in a sequence-specific manner to form
PNA·DNA and PNA·RNA binary complexes with in-
creased specificity and thermal stability compared to un-
Introduction
Genetic information in all living organisms is coded in se-
quences of pyrimidines and purines connected to a back-
bone of phosphorylated 2-deoxy-d-ribofuranoses (in DNA)
or d-ribofuranoses (in RNA). The development of unnatu-
ral nucleic acids, especially those with sugar mimics in their
backbone to replace the natural furanosyl units, for the for-
mation of new oligomeric pairing systems represents an im-
portant avenue of research in synthetic biology and in the
search for the origin of life.[1] Studies to define an etiology
of nucleic acids have demonstrated that canonical Watson–
Figure 1. Repeating monomeric units of DNA, PNA, and its closely related DNA mimics (selected examples).
[a] Departamento de Química, Universidade Federal Rural de
modified homoduplexes owing to its nonionic nature.[7] For
Pernambuco,
52171-900 Recife, PE, Brasil
in vivo applications, a significant drawback of PNAs as bio-
molecular tools is their inadequate membrane permeability.
An effective solution to the above critical problem re-
quires the development of structurally distinct oligomers.
To date, few independent literature reports have described
autonomous pairing systems built upon N-(2-hydroxyeth-
[b] Laboratory for Medicinal Chemistry, Rega Institute for
Medical Research,
KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
E-mail: Piet.Herdewijn@rega.kuleuven.be
Homepage: http://medchem.rega.kuleuven.ac.be/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300523.
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Figure 2. General chemical structures of planned monomeric targets.
yl)phosphonoglycine (O-pPNA) or N-(2-aminoethyl)phos- lowed by tritylation of 4, generated secondary alcohol 5,
phonoglycine (N-pPNA) backbones (Figure 1).[8–12] Al- which, upon harsh basic hydrolysis, afforded 3-amino-1-O-
though such mimic families benefit from better metabolic trityl-2-propanol (6) in good overall yield.
stability owing to the insertion of a phosphono ester unit
in their backbones, they display considerably weaker hy-
bridization affinity for cognate DNA and RNA than PNAs
and natural oligonucleotide counterparts. A certain im-
proved ability for heteroduplex formation resulted from
chimeric sequences with alternating pPNA and PNA/PNA-
like monomers.[13–15]
Given our long-standing interest in the search for new
artificial oligonucleotides, we set out to design and synthe-
size modified phosphono building blocks 1a–1c and 2 (Fig-
ure 2) as precursors to a new generation of polymers. We
are investigating the possibility that a supplementary hy-
droxy group in the backbone of pPNA might facilitate
Scheme 1. Reagents and conditions: (i) BzCl, K2CO3, H2O. (ii) Tri-
tyl chloride (TrCl), pyridine (Py). (iii) 4 n aq. NaOH, EtOH, Δ.
groove solvation and, thus, prove to be advantageous upon
Next, to obtain both enantiomeric forms of the above
binding with DNA and/or RNA targets. In the proposed
amino alcohol, different routes were explored. The known
structures, the internucleotide linkers contain seven or six
(S)-1-O-trityl-2,3-propanediol (7), previously obtained from
bonds dependent on which hydroxy group is used for ester
5-O-trityl-d-arabinofuranose,[19] was our initially chosen
formation, and both are compatible with hybridization
substrate. However, conventional tosylation of 7 proceeded
properties.[16] Here, we present our synthetic efforts for the
with surprisingly little selectivity, and a mixture of the de-
preparation of the achiral and chiral phosphonoglycol
sired 3-O-tosylate 8 (54%) and 2,3-bis-O-tosylate 9 (23%)
monomers depicted in Figure 2.
was formed (Scheme 2). More importantly, 8 and 9 were
difficult to separate by chromatography owing to their very
similar Rf values. In view of those difficulties, we resorted
Results and Discussion
to tin methodology.[20] Treatment of diol 7 with Bu2SnO
From a strategic point of view, it was first decided that followed by TsCl permitted the isolation of the required
the target building blocks 1a–1c and 2 should be assembled monotosylate 8 in 97% yield over two steps.
by stepwise addition of phosphonate and nucleobase moie-
Subsequent substitution by using NaN3 furnished the
ties to appropriately protected amino dihydroxy synthons. chiral azide (S)-11 in 70% yield, whereas the methodology
Preliminary efforts were directed towards the preparation described by Kutney et al.[21] via transient cyclic carbonate
of the key racemic glycol derivative 6, which would serve as 12 and the Mitsunobu reaction[22] with 7 and diisopropyl
precursor for 1a (Scheme 1). Thus, selective N-benz- azodicarboxylate/Ph3P/HN3 were both unsuccessful. Both
oylation[17,18] of 3-amino-1,2-dihydroxypropane (3), fol- 6 and 11 were abandoned later because of difficulties during
Scheme 2. Reagents and conditions: (i) TsCl, Py. (ii) Bu2SnO, MeOH. (iii) TsCl, tetrahydrofuran (THF). (iv) NaN3, N,N-dimethylform-
amide (DMF).
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a reduction of the azide function, during a phosphonylation give 21 by a Staudinger reaction[30] (Ph3P, wet THF) re-
step, and subsequent coupling with thymin-1-yl acetic acid sulted in a mixture of products, presumably as a result of
(see below).
migration of the acetyl group and possibly of the silyl group
As an alternative route, we decided to turn to commer- as well. Consequently, compound 20 as a precursor of 1b
cially available (R)-glycidol (13) as a precursor to our de- was abandoned. Evidently, a nonmigrating hydroxy-pro-
sired chiral synthon. After silylation of 13 at the primary tecting group such as a benzyl group must be used in 15 as
position,[23] the resulting protected epoxide derivative 14 well as in 11. This remains to be investigated. In light of
(which can also be obtained by kinetic resolution[24] or by these results, we decided to use (S)-azide 18 directly instead
asymmetric epoxidation)[25] underwent noticeable decom- of transforming it into hydrophobic compound 20. Treat-
position during its ring opening to azido alcohol 15 ment of 18 with either Na2S·9H2O[31] or more conveniently
(Scheme 3), and only a marginal quantity of the expected under Staudinger reaction conditions[30] accomplished the
compound (ca 3%) could be isolated. Pleasingly, the re- reduction of the azido function to afford the corresponding
placement of 13 with (S)-1,2-isopropylideneglycerol [(S)- amine (S)-22[32,33] in 70% isolated yield in both cases. The
Solketal, 16] as starting material led to much improved re- amine 22 is very polar and inconvenient to handle on a
sults in terms of isolated yields. Compound 16 and its en- small scale. However, the advantage of its use is that both
antiomer are produced in ton scale by desymmetrization of hydroxy groups can be liberated in a single deprotection
1,2-isopropylideneglycerol hydrogen phthalate with chiral step (see below).
1-phenylethylamine.[26]
Although the corresponding (R)-3-amino-1,2-O-isoprop-
Following tosylation of the hydroxy group on 16[27] and ylidenoxypropane could be obtained by repeating the same
substitution by using NaN3,[28] hydrolysis of the isoprop- synthetic sequence shown in Scheme 3, starting from readily
ylidene protection upon treatment with pTSA·H2O,[28,29] available enantiomeric (R)-Solketal,[26] we devised a dif-
and selective silylation at the primary hydroxy group gener- ferent approach based on d-galactose (Scheme 4). d-Galac-
ated the desired (S)-3-azido-1-tert-butyldiphenylsilyloxy-2- tose is the only easily available sugar that can be converted
propanol (15) in good yield (82%). This material was iden- in one step into its derivative 23, which exposes a primary
tical to that prepared through the previous approach. Acet- hydroxy group. We reasoned that this compound could be
ylation of 15 proceeded quantitatively, but attempted con- transformed into a 6-amino-6-deoxy-d-galactopyranose de-
version of the azide into the required amino function to rivative 28, which in turn could be degraded by NaIO4 to
Scheme 3. Reagents and conditions: (i) tert-butyl(chloro)diphenylsilane (TBDPSCl), Et3N, CH2Cl2. (ii) NaN3, DMF, 78 °C. (iii) TsCl, Py.
(iv) NaN3, DMF. (v) p-Toluenesulfonic acid monohydrate (pTsA·H2O), MeOH. (vi) TBDPSCl, imidazole (Im), DMF. (vii) Ac2O, Py.
(viii) Ph3P, H2O. (ix) Ph3P, THO, H2O or Na2S·9H2O.
Scheme 4. Reagents and conditions: (i) TsCl, Py. (ii) NaN3, DMF, 120 °C. (iii) Ph3P, THF, H2O.
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Phosphono Peptide Nucleic Acids
form (R)-amine 29, in which the stereogenic C-2 atom of 2-amino-1,3-propanediol (Scheme 5). An insignificant
would be the C-5 atom in its predecessor 27. Galactose does amount of monosilylated compound 32 was also formed
not seem to be much exploited in this sense.[34]
together with the desired 2-amino-1,3-bis-tert-butydiphen-
The logic of this procedure in terms of “economy of chi- ylsilyloxypropane (31).
rality” can be questioned as only one stereogenic center
present in the substrate 27 is used and three others are lost.
Nevertheless, the ease of accessibility to 1,2;3,4-di-O-iso-
propylidene-d-galactopyranose (23)[35] prompted us to use
it as described above. Commercially available 23 was con-
verted into its 6-O-tosylate 24,[35] which after treatment
Scheme 5. Reagents and conditions: (i) TBDPSCl, Im, DMF.
with an excess of NaN3 furnished azide 25.[36] This appar-
ently simple substitution at the primary position of 24 re-
Having successfully obtained the desired protected
quires extended reaction times, elevated temperatures, and amines 6, 22, 26, and 31 in workable quantities, we achieved
excess nucleophile. The use of a triflate derived from 23 the subsequent transformation to install the phosphonate
rather than tosylate 24 makes substitutions much eas- group under standard alkylation conditions, as illustrated
ier.[37–39] The azide 25 was reduced to the amine 26 in nearly in Table 1.
quantitative yield by using Staudinger conditions. The same
The required alkylating reagents 33a[41] (RЈ
=
compound was previously obtained by using catalytic hy- SO2C6H4CH3) and 33b[42] (RЈ = SO2CF3) were prepared
drogenation[36] of 25 or lithium aluminum hydride re- according to literature procedures. The reaction of 33a with
duction of the corresponding 6-oxime.[40]
primary amines 22 and 26 gave phosphonomethylamines 34
The last amine necessary for the synthesis of achiral and 35 in moderate yields (ca. 20–30%), whereas the amine
building block 2 was easily obtained by standard silylation 31 underwent alkylation with more reactive triflate 33b in
Table 1. Synthesis of phosphonomethylamines 34–38, 40.
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good yield (ca. 56%). Phosphonylation of racemic amine 6
with 33b was possible, but this reaction was complicated
by facile double alkylation at NH2, which led to mono-N-
alkylated derivative 37 (18%) together with 22% of doubly
phosphonomethylated product 38. Consequently, it proved
necessary to turn to another substrate, such as commer-
cially available 3-amino-1,2-isopropylidenoxypropane (39),
which gave 30% yield of alkylated product. Attempts to
increase yields by using [(diethylphosphono)methyl]sulfono-
imidazole in place of 33a or 33b were uniformly unsuccess-
ful. The sulfonoimidazole moiety was claimed to be a better
leaving group than a tosylate/mesylate, but inferior to tri-
flate,[43] and can be obtained in situ by reacting (EtO)2P-
(O)CH2OH with sulfuryl diimidazole[44] and NaH or with
sulfuryl chloride and imidazole.[43] Unexpectedly, the subse-
quent addition of amine produced no traces of the desired
products.
In the following step, phosphonomethylamines 34, 35,
36, and 40 were reacted in the presence of dicyclohexyl-
carbodiimide (DCC) and hydroxybenzotriazole (HOBt)
with 41 (Scheme 6), which was obtained by alkylation of
thymine with chloroacetic acid in basic medium.[45] The
corresponding amides were generally produced in excellent
yields; the only exception to this trend was the amide de-
rived from 36, most probably because of the steric hin-
drance of the bulky silyl groups.
Scheme 6. Reagents and conditions: (i) DCC, HOBt, DMF.
concluded our synthesis was a dealkylation with trimethyl-
silyl iodide (TMSI) and furnished the (R) monomer 50, a
precursor of 1c.
The reaction of TMSI in turn with 42 and 45 allowed
removal of the isopropylidene acetal and cleavage of the
diethyloxy ether groups in a single synthetic operation, as
depicted in Scheme 8. Purification on silica gel, followed by
anion-exchange chromatography completed access to phos-
phonates 51 and 52, precursors of 1b and 1a, respectively.
At this point, 43 was subjected to further manipulation
by the deprotection–degradation sequence discussed earlier
(Scheme 7). First, the acetonide protecting groups were re-
moved by exposure to aqueous trifluoroacetic acid (TFA),
which led to tetraol 46 in 86% yield. Next, this compound
was treated with four equivalents of sodium periodate un-
Scheme 8. Reagents and conditions: (i) TMSI, Et3N, DMF; ion-ex-
change chromatography (1 m TEAB/H2O).
Bis-silylated phosphonamido-thymine 44 was initially
der carefully controlled conditions. We recognized that the treated with tetra-n-butylammonium fluoride (TBAF) to ef-
resulting transient aldehyde would be prone to fast decom- fect liberation of the hydroxy functionalities. Unfortunately,
position; therefore, it was immediately reduced upon treat- this approach invariably provided a complex mixture of in-
ment with sodium borohydride. Gratifyingly, the desired separable products, most likely owing to the sensitivity of
diol 47 could be isolated in reasonable yield (52%), together the substrate to a basic media. The same outcome was ob-
with minor amounts of two degradation fragments, presum- tained with HF/pyridine complex. After those unsuccessful
ably formed in the initial oxidation step. The reaction that attempts, we found that the use of a milder reagent such as
Scheme 7. Reagents and conditions: (i) 80 % aq. TFA. (ii) NaIO4, EtOH, H2O. (iii) NaBH4, EtOH. (iv) TMSI, DMF; ion-exchange
chromatography (1 m TEAB/H2O).
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Phosphono Peptide Nucleic Acids
Scheme 9. Reagents and conditions: (i) TAS-F, DMF, 0 °C to room temp. (ii) TMSI, Et3N, DMF; ion-exchange chromatography (1 m
TEAB/H2O).
benzoyl chloride (13.5 mL, 16.3 g, 116 mmol) dropwise. The mix-
ture was then magnetically stirred overnight at 40 °C. The product
4 has Rf = 0.36 in CH2Cl2/MeOH (10:0.7, stained with ninhydrin).
The reaction mixture was quenched by addition of 1 m HCl and
subsequently evaporated to dryness. The resulting residue was re-
suspended in ethanol, the mixture was then filtered, and the filtrate
was evaporated under reduced pressure. Chromatography with
CH2Cl2/MeOH (gradient 10:0.7Ǟ10:1) furnished 4 (19.4 g, quanti-
tative) as a colorless syrup.[17,18] The NMR spectroscopic data
match those published previously.[18] This compound (20.0 g,
102 mmol) in dry CH2Cl2 (100 mL) and pyridine (35 mL) was co-
oled in an ice-water bath under an argon atmosphere, and trityl
chloride (TrCl, 32.0 g, 115 mmol) was added in one portion with
magnetic stirring. The reaction mixture was left overnight at room
temp. and then gently boiled for 48 h. TLC showed the product 5
(Rf = 0.55 in hexane/EtOAc 1:1) as yellow with CrO3/H2SO4 or
pink with ninhydrin. Extraction (CH2Cl2/H2O), drying, evapora-
tion, and coevaporation with xylenes resulted in spontaneous
crystallization. The crystals were collected by filtration, washed
with hexane, and dried to yield 5 (31.5 g, 70%), m.p. 170–174 °C.
1H NMR (300 MHz, [D6]DMSO): δ = 8.33 (t, J = 5.5 Hz, ex-
changeable, 1 H), 7.29 (d, J = 6.9 Hz, 2 H), 7.77–7.21 (m, aromatic,
18 H), 5.12 (d, J = 5.3 Hz, exchangeable, 1 H), 3.92 (sextet, J =
5.6 Hz, 1 H), 3.53–3.45 and 3.29–3.20 (two m, 1 H each), 3.01 (dd,
J = 5.5, 9.5 Hz, 1 H), 2.97 (dd, J = 5.0, 9.0 Hz, 1 H) ppm. After
D2O exchange: δ = 3.91 (quintet, J = 5.7 Hz), 3.37 (dd, J = 5.3,
13.4 Hz), 3.25 (dd, J = 7.1, 13.3 Hz) ppm. 13C NMR (75 MHz,
[D6]DMSO): δ = 166.4, 143.9, 134.5, 131.0, 128.3, 128.1, 127.8,
127.2, 126.9, 85.8, 68.5, 66.2, 45.4 ppm. HRMS (ESI+): calcd. for
C29H27NNaO3 [M + Na]+ 460.1883; found 460.1876.
tris(dimethylamino)sulfonium
difluorotrimethylsilicate
(TAS-F)[46] was critical to avoid the formation of undesired
byproducts, mainly arising from preferential formation of
cyclic phosphonates. Finally, the phosphono functionality
of 53 was deprotected by the action of TMSI (Scheme 9) to
give monomer 54.
Conclusions
In summary, synthetic routes have been developed for the
preparation of new backbone motifs comprising N-alkyl-
ated phosphonoglycine moieties. These monomers will be
used to construct new unnatural nucleic acids with a phos-
phonate internucleotide linkage.
Experimental Section
General Methods: NMR spectra were recorded with Bruker Avance
300 (1H 300 MHz, 13C 75 MHz, 31P 121 MHz) or Avance II 500
(1H 500 MHz, 13C 125 MHz, 31P 202 MHz) spectrometers. Spectra
were referenced to TMS, and chemical-shift (δ) values are expressed
in parts per million (ppm) downfield of tetramethylsilane (TMS).
The multiplicity of signals is reported as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), broad (br), or a combination
of any of these. Coupling constants are expressed in Hertz. For
1
final products, H and 13C resonance assignments were made with
the aid of 1H–1H and 1H–13C correlation experiments. High resolu-
tion mass spectra (HRMS) were acquired with a quadrupole or-
thogonal acceleration time-of-flight mass spectrometer (Synapt G2
HDMS, Waters, Milford, MA) with electrospray ionization (ESI).
Samples were infused at 3 μL/min, and spectra were obtained in
positive (or negative) ionization mode with a resolution of 15000
[full width at half-maximum (FWHM)]; leucine enkephalin was
used as lock mass. Chromatographic separations were performed
with silica gel (100–200 or 230–400 mesh) and analytical thin-layer
chromatography was performed on precoated aluminum backed
sheets (254 nm) supplied by Fluka. For all reactions, analytical
grade solvents were used. Anhydrous THF was refluxed over so-
dium benzophenone ketyl and distilled. Dry dichloromethane was
obtained by distillation over CaH2, and dry pyridine was refluxed
and stored over KOH. Dry dimethylformamide was supplied by
Aldrich. All final compounds were purified by anion-exchange
chromatography (Source 15Q) with H2O and 1 m tetraethylammo-
nium bromide (TEAB) as eluents. Optical rotations were recorded
with a Perkin–Elmer 341 polarimeter in a thermostatted 1 dm cell
at 20 °C by using the sodium D line, and a suitable solvent is re-
ported along with the concentration (c in g/100 mL).
(rac)-3-Amino-1-(triphenylmethyloxy)-2-propanol (6): Compound 5
(2.0 g, 4.5 mmol) in EtOH (40 mL) and 4 n NaOH (30 mL) was
stirred under reflux for 48 h. Water (60 mL) was added, and the
resulting heterogeneous mixture was kept in ice-water for 1 h. The
precipitated colorless material was collected by filtration, washed
with ice-water, dried under vacuum, and solubilized in EtOH
(50 mL) with warming. Some solid material was removed by fil-
tration, and the volatiles were evaporated. TLC showed a spot of
the product (Rf = 0.40, CH2Cl2/MeOH/25% NH4OH 20:2:0.1) as
purple with ninhydrin or yellow with Hanessian’s stain.
Chromatography in the same system furnished 6 (1.1 g, 72%) as a
glassy material. The addition of Et2O resulted in the formation of
1
a colorless powder; m.p. ca. 77–115 °C. H NMR (300 MHz, [D6]-
DMSO): δ = 7.45–7.22 (m, aromatic, 15 H), 3.67–3.59 (m, 1 H),
3.01 (dd, J = 5.5, 9.0 Hz, 1 H), 2.90 (dd, J = 6.0, 9.0 Hz, 1 H), 2.71
(dd, J = 4.1, 12.7 Hz, 1 H), 2.51 (dd, J = 7.1, 12.6 Hz, 1 H) ppm.
13C NMR (75 MHz, [D6]DMSO): δ = 143.9, 128.3, 127.8, 126.9,
85.7, 71.1, 65.9, 45.3 ppm. HRMS (ESI+): calcd. for C22H24NO3
[M + H]+ 334.1801; found 334.1819.
(R)-1-Tosyloxy-3-(triphenylmethyloxy)-2-propanol (8) and (R)-1,2-
(rac)-3-N-Benzamido-1-(triphenylmethyloxy)-2-propanol (5): To a Bis(tosyloxy)-3-(triphenylmethyloxy)propane (9): (S)-Diol 7[19]
cold (ice-water bath) solution of 3-amino-1,2-propane diol (9.1 g,
100 mmol) in H2O (70 mL) was added K2CO3 (30 g) followed by
(0.25 g, 0.75 mmol) in dry CH2Cl2 (15 mL) and Et3N (0.3 mL) was
treated with TsCl (0.19 g, 1 mmol) for 18 h under a protective at-
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mosphere (argon). TLC showed two closely positioned compounds
(Rf of mid-point = 0.38 in hexane/EtOAc 140:55). Extraction
(CH2Cl2/H2O), drying (MgSO4), evaporation, and flash
chromatography yielded 9 (0.11 g, 23%) and 8 (0.197 g, 54%). Data
for 9: foam, [α]2D0 = –2.1° (c 3.2, dioxane). 1H NMR (300 MHz,
CDCl3): δ = 7.70–7.60 (m, aromatic, 4 H), 7.25 (m, aromatic, 15
J = 5.2 Hz, 1 H), 3.71–3.62 (m, 2 H), 3.56 (apparent d, J = 5.5 Hz,
2 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 135.7, 133.0, 130.2,
128.1, 71.1, 65.2, 53.4, 27.0, 19.4 ppm. HRMS (ESI+): calcd. for
C19H25KN3O2Si [M + K]+ 394.1353; found 394.1347. Method (b):
The diol 19 (2.55 g, 21.8 mmol) and imidazole (3.2 g, 48 mmol) in
dry DMF (30 mL) was treated with tBuPh2SiCl (6.6 g, 6.2 mL,
H), 4.55 (quintet, J = 4.9 Hz, 1 H), 4.15–4.13 (m, 2 H), 3.28 (dd, 14 mmol), which was added dropwise by syringe over ca. 5 min; the
J = 5.2, 10.0 Hz, 1 H), 3.23 (dd, J = 4.9, 10.0 Hz,1 H), 2.43 (s, 6 mixture was cooled in an ice bath under an argon atmosphere. Af-
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 145.3, 145.2, 143.3,
133.3, 132.4, 130.1, 130.0, 128.7, 128.2, 128.2, 128.1, 127.4, 87.4,
77.5, 67.8, 62.1, 21.9 ppm. HRMS (ESI+): calcd. for
C36H34NaO7S2 [M + Na]+ 665.1638; found 665.1634. Data for 8:
foam, [α]2D0 = –11.3° (c 1.8, dioxane). 1H NMR (300 MHz, CDCl3):
δ = 7.76 (d, J = 8.2 Hz, 2 H), 7.37–7.22 (m, 17 H), 4.15 (dd, J =
4.4, 10.2 Hz, 1 H), 4.06 (dd, J = 6.0, 10.1 Hz, 1 H), 3.92 (sextet, J
= 5.2 Hz, 1 H), 3.21 (dd, J = 5.3, 9.7 Hz, 1 H), 3.16 (dd, J = 5.5,
9.8 Hz, 1 H), 2.42 (s, 3 H), 2.35 (d, J = 5.6 Hz, 1 H) ppm. 13C
NMR (75 MHz, CDCl3): δ = 145.2, 143.6, 132.8, 130.1, 128.7,
128.2, 128.1, 127.4, 87.1, 71.2, 68.9, 63.8, 21.8 ppm. HRMS (ESI+):
calcd. for C29H28NaO5S [M + Na]+ 511.1549; found 511.1554.
Compound 8 was also prepared by tin oxide methodology:[20] Diol
7 (0.24 g, 0.71 mmol) in dry toluene (20 mL) and dibutyltin oxide
(0.23 g, 0.92 mmol) were magnetically stirred at gentle reflux under
argon. The mixture became homogeneous after ca. 20 min. After a
total of 80 min, the mixture was cooled, the solvent was evapo-
rated, and the solid was dried under vacuum. The vacuum was
broken by using dry argon in a balloon. To product 10 thus ob-
tained was added dry THF (10 mL) followed by TsCl (0.15 g,
0.8 mmol), and this mixture was left overnight at room temp. TLC
showed the monotosylate 8 as the only product. Evaporation of
the solvent and flash chromatography in hexane/EtOAc (7:3) fur-
nished 8 (0.25 g, 97%).
ter addition was complete, the reaction proceeded at room temp.
for 6 h. TLC showed a spot with Rf = 0.33 (hexane/Et2O 4:1). Ex-
traction (CH2Cl2/H2O), drying, evaporation, and chromatography
with hexane/Et2O (3:1) furnished 15 (6.4 g, 82%) as an oil.
(S)-3-Azido-1,2-propanediol (19): Commercial (S)-2,3-isopropylid-
eneglycerol (16, Fluka, 15.4 g, 116.3 mmol) was dissolved in
CH2Cl2 (150 mL), and Et3N (25 mL) and TsCl (16.5 g, 139 mmol)
were then added. The mixture was stirred for 8 h. Isolation after
conventional extraction and chromatography in hexane/EtOAc
(4:1) yielded 17; Rf = 0.45, (31.6 g, 95%), [α]2D0 = –3.6° (c 6, EtOH;
ref.[27] –4.5°, c 1, EtOH). Compound 17 (10.0 g, 35 mmol) was then
reacted with NaN3 (11.3 g, 175 mmol) in dry DMF (120 mL) at
65 °C for 20 h. After extraction and chromatography with hexane/
Et2O (4:1, Rf = 0.43), azide 18 was isolated (5.3 g, 96.5%), [α]2D0
=
–52.2° (c 5.6, CHCl3; ref.[28] αD not given). This azide (5.1 g,
32.5 mmol) was dissolved in MeOH (360 mL), and pTSA monohy-
drate (0.32 g, 1.7 mmol) was added, the mixture was kept at room
temp. for 25 h.[28] TLC showed a more polar product (Rf = 0.40,
hexane/EtOAc 1:3), which shows with Hanessian’s stain. Neutral-
ization with solid NaHCO3 (5.3 g over 45 min), filtration, evapora-
tion, and chromatography with hexane/EtOAc (1:3) furnished 19
as an oil (2.8 g, 57%); [α]2D0 = +2.8° (c 4.7, CHCl3), [α]2D0 = –16.2°
(c 3.6, MeOH; ref.[29] –10.8°, c 0.98, MeOH). 1H NMR (300 MHz,
[D6]DMSO): δ = 5.09 (d, J = 5.1 Hz, exchangeable, 1 H), 4.67 (t,
J = 5.6 Hz, exchangeable, 1 H), 3.68–3.59 (15-line m, 1 H), 3.41–
3.14 (16-line m, 4 H) ppm. After D2O exchange: δ = 3.66–3.59 (12-
line m, 1 H), 3.36 (dd, J = 5.4, 10.9 Hz, 1 H), 3.28 (dd, J = 6.6,
10.6 Hz, 1 H), 3.26 (dd, J = 3.9, 12.7 Hz, 1 H), 3.16 (dd, J = 6.7,
12.7 Hz, 1 H) ppm. 13C NMR (75 MHz, [D6]DMSO): δ = 74.0,
63.2, 53.5 ppm.
(S)-1-Azido-3-(triphenylmethyloxy)-2-propanol (11): Tosylate
8
(0.13 g, 0.26 mmol) and NaN3 (0.65 g, 10 mmol) in dry DMF
(10 mL) were stirred under argon at 88 °C for 2.5 h. TLC showed
a new intense spot (Hanessian’s stain) less polar than the substrate
(Rf = 0.64, hexane/EtOAc 7:3; substrate Rf = 0.40). Extraction
(CH2Cl2/H2O), drying (MgSO4), filtration, evaporation, and purifi-
cation by preparative TLC with hexane/EtOAc (4:1) furnished a
glassy product (0.067 g, 70%). [α]2D0 = –8.4° (c 3.4, CHCl3). 1H
NMR (300 MHz, CDCl3): δ = 7.42–7.23 (m, aromatic, 15 H), 3.88
(t, J = 5.3 Hz, 1 H), 3.35 (d, J = 5.3 Hz, 2 H), 3.20 (d, J = 5.3 Hz,
2 H), 2.39 (br. s, 1 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 143.5,
128.5, 127.9, 127.2, 86.9, 70.1, 64.7, 53.8 ppm. HRMS (ESI+):
calcd. for C22H21N3NaO2 [M + Na]+ 382.1526; found 382.1532.
Attempts to obtain the azide 11 via a cyclic carbonate 12 by anal-
ogy to the other substrates[21] (diol 7 and carbonyldiimidazole, fol-
lowed by NaN3) or by a Mitsunobu reaction[22] (7, PPh3, HN3,
iPrO2CNNCO2iPr) failed.
(S)-2-Acetoxy-3-azido-1-(tert-butyldiphenylsilyloxy)propane
(20):
Conventional acetylation of 15 (4.71 g, 13.3 mmol) in pyridine
(30 mL) and Ac2O (15 mL) overnight, followed by extractive
workup, coevaporation with xylenes, and exhaustive drying under
vacuum furnished 20 as a syrup (5.3 g, quantitative); Rf = 0.63 in
hexane/Et2O (4:1), [α]2D0 = –14.6° (c 2.8, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 7.68–7.35 (m, two groups of signals, 10 H),
5.06 (quintet, J = 5.2 Hz, 1 H), 3.79 (dd, J = 5.0, 10.9 Hz, 1 H),
3.74 (dd, J = 5.5, 10.9 Hz, 1 H), 3.53 (apparent d, J = 5.2 Hz, 2
H), 2.04 (s, 3 H), 1.06 (s, 9 H) ppm. 13C NMR (75 MHz, CDCl3):
δ = 170.3, 135.8, 135.7, 133.1, 130.1, 128.0, 127.9, 72.8, 62.6, 50.9,
26.9, 21.1, 19.4 ppm. HRMS (ESI+): calcd. for C21H27N3NaO3Si
[M + Na]+ 420.1714; found 420.1740. Attempted conversion of the
azide 20 to amine 21 by a Staudinger reaction[30] resulted in a com-
plex reaction mixture, which was not investigated.
(S)-3-Azido-1-(tert-butyldiphenylsilyloxy)-2-propanol (15): Method
(a) from (R)-glycidol: (R)-glycidol (Sigma–Aldrich, 13) was conven-
tionally silylated[23] to furnish 14 as an oil in ca. 90% yield after
chromatography (gradient, hexane/Et2O 10:0.5Ǟ10:1.5), Rf = 0.47
in hexane/Et2O (10:1), [α]2D0 = –1.9° (c 21.6, CHCl3; ref.[24] –1.4°, c
1, CHCl3; ref.[25] –2.3°, c 9.07, CHCl3; optical rotation is not pre-
sented in ref.[13]). This compound (1.21 g, 3.9 mmol) in dry DMF
(24 mL) and NaN3 (2.0 g, 30 mmol) was stirred under argon at
78 °C for 8 h. The mixture turned brown. TLC showed the product
15 (Rf = 0.34 in hexane/Et2O 4:1) along with decomposition prod-
ucts. Extraction (CH2Cl2/water), drying, evaporation of the solvent,
(S)-3-Amino-1,2-(isopropylidenoxy)propane (22): This compound
was obtained by reduction of the azide 18 by using either a Staud-
inger reaction[30] or Na2S·9H2O by analogy to the published pro-
cedure.[31] Staudinger reaction: To a magnetically stirred solution
of 18 (1.6 g, 10 mmol) in THF (20 mL) and water (four drops) was
added Ph3P (3.1 g, 12 mmol). Evolution of nitrogen started within
ca. 30 s. After overnight reaction, the product showed a TLC spot
and chromatography (gradient, hexane/Et2O 20:4Ǟ20:6) furnished
1
15 (0.049 g, ca. 3%) as an oil. H NMR (300 MHz, CDCl3): δ = visualized with ninhydrin Rf
=
0.50 in CH2Cl2/MeOH/25%
7.66–7.35 (m, two groups of signals, aromatic, 10 H), 3.86 (sextet,
NH4OH 20:2:1). Evaporation of the volatiles and chromatography
4810
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 4804–4815

Phosphono Peptide Nucleic Acids
in the abovementioned system furnished 22 as yellowish oil (1.06 g,
70%). Na2S·9H2O: To a solution of azide 18 (3.35 g, 21.3 mmol)
in MeOH (10 mL) was added a solution of Na2S·9H2O (6.4 g) in
H2O (10 mL). Stirring was continued for 16 h at 55 °C. The reac-
tion mixture was transferred to a separatory funnel charged with
Et2O and H2O. After exhaustive extraction with Et2O (six times),
the organic phase was dried, and the solvent was evaporated to
furnish an yellowish oil. Crude 22 thus obtained was used for the
subsequent transformation. [α]2D0 = +1.1° (c 14, CHCl3; ref.[33]
+0.9°, c 1, CHCl3; ref.[32] +1.48°, neat). 1H NMR (300 MHz,
CDCl3): δ = 4.13 (ddd, J = 4.3, 6.3, 12.6 Hz, 1 H), 4.04 (dd, J =
6.4, 7.9 Hz, 1 H), 3.52 (dd, J = 6.4, 7.9 Hz, 1 H), 2.85 (dd, J = 4.4,
13.1 Hz, 1 H), 2.78 (dd, J = 7.1, 13.1 Hz, 1 H), 1.43 (s, 3 H), 1.36
(s, 3 H), 1.26 (br. s, 2 H) ppm. 13C NMR (75 MHz, CDCl3): δ =
109.2, 77.5, 67.0, 44.8, 26.9, 25.4 ppm.
27.1, 19.5 ppm. HRMS (ESI+): calcd. for C35H46NO2Si2 [M +
1
H]+ 568.3061; found 568.3057. Data for 32: H NMR (300 MHz,
[D6]DMSO): δ = 7.64–7.41 (m, aromatic, 10 H), 3.59 (dd, J = 6.9,
9.7 Hz, 1 H), 3.51 (dd, J = 5.8, 9.6 Hz, 1 H), 3.45 (dd, J = 5.3,
10.4 Hz, 1 H), 3.33 (dd, J = 6.0, 10.4 Hz, 1 H), 3.23 (br. s, 2 H),
2.84 (quintet, J = 5.7 Hz, 1 H) ppm. 13C NMR (75 MHz, [D6]-
DMSO): δ = 135.1, 133.2, 129.8, 127.9, 66.1, 63.2, 54.3, 26.7,
18.9 ppm. HRMS (ESI+): calcd. for C19H28NO2Si [M + H]+
330.1884; found 330.1894.
Diethoxyphosphonylmethyl Tosylate (33a): Diethylhydroxymethyl
phosphonate (Aldrich, 13.5 g, 78 mmol) was added to pyridine
(30 mL) and CH2Cl2 (70 mL), and the resulting solution was co-
oled in an ice bath under an argon atmosphere. Tosyl chloride
(14.9 g, 78 mmol) was added, and the mixture was allowed to reach
room temp. over 5 h and left at room temp. for 20 h. The mixture
was partitioned between CH2Cl2 and water. The organic phase was
washed with saturated NaHCO3 followed by water, dried, and fil-
tered, and the volatiles were evaporated. Xylenes were added and
evaporated to remove any residual pyridine. Final drying under
vacuum yielded crude product (23.4 g, 91%), which was used with-
out any further purification. The yield obtained was higher than
that previously reported.[41] 1H NMR (300 MHz, CDCl3): δ = 7.80
(d, J = 8.3 Hz, 2 H), 7.37 (d, J = 8.0 Hz, 2 H), 4.23–4.09 (m, 6 H),
2.46 (s, 3 H), 1.31 (t, J = 7.1 Hz, 6 H) ppm. 13C NMR (75 MHz):
δ = 145.6, 131.8, 130.1, 128.3, 63.5 (d, JC,P = 6.4 Hz), 61.4 (d, JC,P
= 167.9 Hz), 21.7, 16.4 (d, JC,P = 5.7 Hz) ppm. 31P NMR
(121 MHz, CDCl3): δ = 15.2 ppm. Attempts to obtain [(diethyl-
phosphono)methyloxy]sulfonoimidazole (to be used instead of
triflate 33b or tosylate 33a) by direct reaction of diethyl (hy-
droxymethyl)phosphonate with sulfuryldiimidazole[44] and NaH, or
with sulfuryl chloride and imidazole,[43] followed by addition of
the amine 39 furnished no traces of the expected product 40 (see
below).
6-Amino-6-deoxy-1,2;3,4-di-O-isopropylidene-α-D-galactopyranose
(26): Conventional tosylation of commercial 1,2;3,4-di-O-isoprop-
ylidene-α-d-galactopyranose (23, Acros, [α]2D0 = –56.8°, c 5.9,
CHCl3,) in CH2Cl2, pyridine, or Et3N and TsCl (1.1 equiv.) over-
night, followed by extractive workup (CH2Cl2/H2O) and
chromatography in hexane/EtOAc (3:1, Rf = 0.41) furnished 24[35]
(89%); [α]2D0 = –62.4° (c 1.7, CHCl3; ref.[35] –63°, CHCl3, c not
given). No attempt was made to crystallize this compound. Tosyl-
ate 24 was converted into the corresponding 6-azide 25 by analogy
to the procedure for l-galactose[36] (5 equiv NaN3, DMF, ca.
120 °C, 18 h under argon) to furnish 25 in 91% yield after extrac-
tive workup (CH2Cl2/H2O) and chromatography in hexane/EtOAc
(5:1, Rf = 0.39); [α]2D0 = –98° (c 2.4, CHCl3). To a magnetically
stirred solution of this azide (3.3 g, 11.6 mmol) in THF (50 mL)
was added H2O (0.3 mL) followed by Ph3P (3.7 g, 14 mmol) by
analogy to a similar reduction.[30] The evolution of nitrogen started
in ca. 30 s. TLC performed the following day, showed that all sub-
strate reacted to form the amine 26 (Rf = 0.61, CH2Cl2/MeOH
20:3). The solvent was evaporated, and the residue was purified by
chromatography (gradient CH2Cl2/MeOH 20:2Ǟ20:3) to furnish
26 as an oil (2.94 g, 98%). Alternatively, 25 was subjected to cata-
lytic hydrogenation (10% Pd/C, EtOH, 50 psi in a Parr apparatus,
6 h) to furnish amine 26 in nearly quantitative yield. [α]2D0 = –42.1°
(c 1.4, MeOH; ref.[40] –53.1°, c 1, CHCl3; +48.6°, c 1.4, MeOH for
the l form).[36] 1H NMR (300 MHz, CDCl3): δ = 5.56 (d, J =
5.0 Hz, 1 H), 4.60 (dd, J = 2.3, 7.9 Hz, 1 H), 4.32 (dd, J = 2.4,
5.0 Hz, 1 H), 4.23 (dd, J = 1.9, 7.9 Hz, 1 H), 3.84 (ddd, J = 1.7,
4.6, 7.2 Hz, 1 H), 3.54 (br. s, 2 H, in the other samples this signal
appeared at δ = 2.43 and 2.22 ppm), 3.04 (dd, J = 8.0, 13.2 Hz, 1
H), 2.96 (dd, J = 4.1, 13.2 Hz, 1 H), 1.57 (s, 3 H), 1.45 (s, 3 H),
1.33 (s, 6 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 109.5, 108.9,
96.5, 71.8, 71.0, 70.7, 68.3, 41.9, 26.3, 26.2, 25.1, 24.5 ppm.
Diethoxyphosphonylmethyl Triflate (33b): Compound 33b was pre-
pared from diethyl (hydroxymethyl)phosphonate, trifluorometh-
anesulfonic anhydride, and 2,6-lutidine as previously reported[42]
(an alternative is to use CF3SO2Cl and NaH[47]) and was used as
a crude product without any further purification (such as distil-
lation).[42] This material was most probably contaminated with
bis(diethylphosphonomethyl) ether.[47] A solution of diethyl (hy-
droxymethyl)phosphonate (5.2 g, 4.5 mL, 30.6 mmol) in dry
CH2Cl2 (50 mL, freshly distilled from CaH2) and 2,6-lutidine (dried
by storage over KOH, 4.3 mL, 37.6 mmol) was cooled to ca. –50 °C
(external temp.) under argon. To this solution was slowly added
trifluoromethanesulfonic anhydride (10.1 g, 6.0 mL, 35.6 mmol) by
syringe over ca. 10 min with magnetic stirring. After addition was
complete, the temperature was increased slowly to ca. 10 °C over
90 min, whereupon extraction was performed (CH2Cl2/dil. HCl).
The organic phase was washed once with ice-cold H2O, dried
(MgSO4), filtered, evaporated, and finally dried under vacuum to
furnish 9.5 g of red oil (theoretical yield is 9.2 g). This material was
used as such immediately after preparation.
2-Amino-1,3-bis(tert-butyldiphenylsilyloxy)propane (31) and (rac)-2-
Amino-1-(tert-butyldiphenylsilyloxy)-3-propanol (32): 2-Amino-1,3-
propane diol (Acros or Aldrich, 3.85 g, 42.8 mmol) was dissolved
in dry DMF (36 mL) under argon. To this solution was added
imidazole (12.6 g, 185.1 mmol) followed by tBuPh2SiCl (24.7 g,
23.3 mL, 90.1 mmol) by syringe with stirring. After overnight reac-
tion, TLC showed a UV-absorbing compound (Rf = 0.52, hexane/
EtOAc 2:1), which stains with ninhydrin upon heating. Extraction
(CH2Cl2/dil. NaHCO3), drying (MgSO4), evaporation, and
chromatography (gradient hexane/EtOAc, 3:1Ǟ2:1) gave 31 as col-
orless oil (20.3 g, 85%). Elution with CH2Cl2/MeOH (10:1) fur-
nished monosilylated product 32 (0.12 g, 0.9%). Data for 31: 1H
NMR (300 MHz, CDCl3): δ = 7.65–7.32 (m, aromatic, 20 H), 3.71
(dd, J = 5.2, 9.8 Hz, 2 H), 3.62 (dd, J = 5.8, 9.8 Hz, 2 H), 3.00
(Aminomethyl)phosphonate 34: (S)-Amine 22 (1.06 g, 8.0 mmol),
tosylate 33a (3.7 g, 11.5 mmol), and powdered molecular sieves 4 Å
(2 g) in dry DMF (25 mL) were stirred under argon at 80 °C for
20 h. TLC showed a spot for the product 34 (Rf = 0.28 CH2Cl2/
MeOH 20:1, revealed with ninhydrin). Unreacted amine remained
at the application point. The solid material was removed by fil-
tration, and extraction was performed (CH2Cl2/H2O). The organic
phase was dried (MgSO4), and the solvent was evaporated.
(quintet, J = 5.6 Hz, 1 H), 1.51 (s, 2 H), 1.03 (s, 18 H) ppm. 13C Chromatography with CH2Cl2/MeOH (10:1) furnished 34 (0.49 g,
NMR (75 MHz, CDCl3): δ = 135.8, 133.7, 129.8, 127.9, 65.9, 54.6, 21.5%) as a colorless oil. For the NMR spectroscopic data, see
Eur. J. Org. Chem. 2013, 4804–4815
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4811

B. Doboszewski, E. Groaz, P. Herdewijn
FULL PAPER
compound 40. Prolongation of the reaction time and/or higher
temperature did not improve the yield. [α]2D0 = +2.4° (c 3.7, CHCl3).
(rac)-3-[(Diethylphosphonomethyl)amino]-1-(triphenylmethyloxy)-2-
propanol (37) and (rac)-3-[Bis(diethylphosphonomethyl)amino]-1-O-
HRMS (ESI+): calcd. for C11H25NO5P [M + H]+ 282.1465; found (triphenylmethyloxy)-2-propanol (38): From 33b: Triflate 33b (0.5 g,
282.1478.
ca. 1.6 mmol) was added to a solution of 6 in THF (15 mL) under
an argon atmosphere. The mixture warmed up and became blue.
After 8 h at room temp., TLC showed the bis-phosphonate 38 (Rf =
0.55) and monophosphonate 37 (Rf = 0.47, CH2Cl2/MeOH 20:1.3).
Extractive workup (CH2Cl2/H2O) followed by chromatography in
CH2Cl2/MeOH (20:1.1) furnished 38 (0.23 g, 22%) and 37 (0.14 g,
18 %). From 33a: To a DMF (10 mL) solution of 6 (0.81 g,
2.4 mmol) was added 33a (0.84 g, 2.6 mmol), and this mixture was
incubated at 100 °C under argon for 9 h. The TLC profile was sim-
ilar to that described above, but additional minor byproducts were
present and made the purification difficult. Extractive workup and
chromatography furnished 37 as a syrup (0.34 g, 27%). The doubly
phosphonylated compound 38 was not isolated. Data for 38: 1H
NMR (300 MHz, CDCl3): δ = 7.45–7.18 (m, two groups of signals,
15 H), 4.11 (sextet, J = 7.4 Hz, 8 H), 3.87–3.80 (nonet, 1 H), 3.30–
3.15 (m, 5 H), 3.09–3.03 (m, 2 H), 5.89 (dd, J = 8.8, 13.5 Hz, 1 H),
1.30 (dt, J = 3.7, 7.0 Hz, 12 H) ppm. 13C NMR (75 MHz, CDCl3):
δ = 144.1, 128.8, 127.9, 127.1, 86.7, 68.8, 65.7, 62.3, 62.2, 62.2,
62.1, 62.1, 61.1, 61.1, 61.0, 52.1 (d, JC,P = 154.6 Hz), 16.7, 16.6,
16.6 ppm. 31P NMR (121 MHz, CDCl3): δ = 25.5 ppm. HRMS
(ESI+): calcd. for C32H45NNaO8P2 [M + Na]+ 656.2513; found
656.2504. Data for 37: 1H NMR (300 MHz, CDCl3): δ = 7.44–7.19
(m, two groups of signals, 15 H), 4.10 (quintet, J = 7.3 Hz, 4 H),
3.86 (unresolved quintet, 1 H), 3.17 (dd, J = 5.8, 9.4 Hz, 1 H), 3.12
(dd, J = 5.1, 9.5 Hz, 1 H), 2.97 (apparent dd, J = 2.5, 12.3 Hz, 2
H), 2.90–2.70 (m, 3 H), 1.29 (t, J = 7.1 Hz, 6 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 144.0, 128.8, 128.0, 127.2, 86.8, 69.3, 66.1,
(Aminomethyl)phosphonate 35: A mixture of tosylate 33a (0.88 g,
2.78 mmol), galactosylamine 26 (0.72 g, 2.78 mmol), and powdered
4 Å molecular sieves (3 g) in dry DMF (10 mL) was stirred under
argon at 80 °C for 20 h. TLC showed a new product (Rf = 0.40,
CH2Cl2/MeOH 20:0.8). Unreacted 33a and 26 were still present.
The solid material was removed by filtration with a sintered glass
filter, and the filtrate was extracted (CH2Cl2/H2O). After usual
workup, the residue was purified by chromatography with CH2Cl2/
MeOH (20:0.8) to furnish 35 as an oil (0.42 g, 37%). 1H NMR
(300 MHz, CDCl3): δ = 5.52 (d, J = 5.0 Hz, 1 H), 4.59 (dd, J =
2.3, 7.9 Hz, 1 H), 4.31 (dd, J = 2.4, 5.1 Hz, 1 H), 4.20–4.10 (m, 5
H), 3.89 (ddd, J = 1.7, 4.4, 8.1 Hz, 1 H), 3.03 (dd, J = 2.6, 12.5 Hz,
2 H), 2.97 (dd, J = 4.3, 7.6 Hz, 1 H), 2.85 (dd, J = 4.4, 12.7 Hz, 1
H), 1.94 (br. s, exchangeable, 1 H), 1.52 (s, 3 H), 1.44 (s, 3 H), 1.36
(s, 3 H), 1.34 (s, 3 H), 1.33 (s, 3 H), 1.31 (s, 3 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 109.4, 108.6, 96.5, 72.0, 71.0, 70.7, 66.7,
62.2 (d, JC,P = 6.6 Hz), 50.8 (d, JC,P = 14.9 Hz), 45.1 (d, JC,P
=
155.4 Hz), 26.2, 26.1, 25.1, 24.5, 16.6 (d, JC,P = 5.7 Hz) ppm. 31P
NMR (121 MHz, CDCl3): δ = 25.9 ppm. HRMS (ESI+): calcd. for
C17H33NO8P [M + H]+ 410.1938; found 410.1935.
(Aminomethyl)phosphonate 40: Tosylphosphonate 33a (3.7 g,
7.6 mmol) was added to a solution of (rac)-3-amino-1,2-isopropyl-
idenoxypropane 39 (1.0 g, 7.6 mmol) in dry DMF (12 mL), fol-
lowed by powdered 4 Å molecular sieves (2 g). The mixture was
stirred under argon for 12 h at 80 °C. TLC showed a spot for the
product 40 (Rf = 0.28, CH2Cl2/MeOH 20:1, revealed with ninhyd-
rin). Unreacted amine remained at the application point. The solid
material was removed by filtration, and extraction was performed
(CH2Cl2/H2O). The organic phase was dried (MgSO4), and the sol-
vent was evaporated. Chromatography with CH2Cl2/MeOH (10:1)
furnished 40 as a yellowish oil (0.86 g, 30.2%). Prolongation of the
reaction time and/or higher temperature did not improve the yield.
1H NMR (300 MHz, [D6]DMSO): δ = 4.12 (quintet, J = 6.0 Hz, 1
H), 4.02 (quintet, J = 7.3 Hz, 4 H), 3.96 (dd, J = 6.5, 8.0 Hz, 1 H),
3.60 (dd, J = 6.5, 8.0 Hz, 1 H), 3.31 (br. s, exchangeable, 1 H), 2.94
(d, J = 11.4 Hz, 2 H), 2.71 (d, J = 2.1 Hz, 1 H), 2.69 (d, J = 1.7 Hz,
1 H), 1.31 (s, 3 H), 1.26 (s, 3 H), 1.23 (t, J = 7.1 Hz, 6 H) ppm.
13C NMR (75 MHz, [D6]DMSO): δ = 108.1, 74.8, 66.9, 61.2 (d,
JC,P = 6.5 Hz), 52.7 (d, JC,P = 12.8 Hz), 44.3 (d, JC,P = 154.0 Hz),
26.7, 25.3, 16.3 (d, JC,P = 5.5 Hz) ppm. 31P NMR (121 MHz, [D6]-
DMSO): δ = 26.1, 24.4 (impurity, ca. 6%) ppm. HRMS (ESI+):
calcd. for C11H25NO5P [M + H]+ 282.1465; found 282.1462.
62.2 (d, JC,P = 6.6 Hz), 53.8 (d, JC,P = 13.6 Hz), 45.3 (d, JC,P
=
153.3 Hz), 16.6 (d, JC,P = 6.0 Hz) ppm. 31P NMR (121 MHz,
CDCl3): δ = 26.0 ppm. HRMS (ESI+): calcd. for C27H35NO5P [M
+ H]+ 484.2247; found 484.2247.
(Thymin-1-yl)acetic Acid (41): Compound 41 was obtained by di-
rect alkylation of thymine with chloroacetic acid in alkaline me-
dium in 46 % yield, as described.[45] 1H NMR (300 MHz, [D6]-
DMSO): δ = 13.1 (br. s, exchangeable, 1 H), 7.49 (unresolved quar-
tet, J = 1.1 Hz, 1 H), 4.37 (s, 2 H), 1.75 (d, J = 1.1 Hz, 3 H) ppm.
13C NMR (75 MHz): δ = 169.7, 164.34, 151.0, 141.8, 108.4, 48.4,
11.9 ppm.
(Acetamidomethyl)phosphonate 42: A coupling procedure as de-
scribed below was performed with amine 34 (0.42 g, 3.2 mmol),
thymine acetic acid 41 (1.8 g, 9.6 mmol), DCC (2.0 g, 9.6 mmol),
and HOBt (1.0 g, 7.0 mmol) in dry DMF (20 mL) for 24 h. Workup
and chromatography yielded 42 (0.55 g, 82%). [α]2D0 = –18.2° (c 3.1,
CHCl3). HRMS (ESI+): calcd. for C18H31N3O8P [M + H]+
448.1843; found 448.1846.
(Aminomethyl)phosphonate 36: Triflate 33b (4.2 g, ca. 14 mmol) was
added to a solution of amine 31 (7.9 g, 14 mmol) in dry THF
(30 mL) under argon. The reaction mixture became blue and
slightly warm. After 8 h at room temp., the newly formed com-
pound 36 was detected by TLC (Rf = 0.31, hexane/EtOAc 3:2).
Extractive workup (CH2Cl2/H2O) and chromatography with hex-
ane/EtOAc (3:2) furnished the product (5.65 g, 56.6%). When tos-
ylate 33a was used instead of 33b (in DMF at 100 °C overnight),
36 was isolated in 14% yield. 1H NMR (300 MHz, CDCl3): δ =
(Acetamidomethyl)phosphonate 43: To a magnetically stirred solu-
tion of 35 (0.41 g, 1 mmol) and 41 (0.368 g, 2 mmol) in dry DMF
(10 mL) was added DCC (0.412 g, 2 mmol) and HOBt (0.27 g,
2 mmol) under argon. The mixture became turbid in a few seconds.
The solution was stirred for 36 h, and the solids were removed by
filtration with a sintered glass filter. The filtrate was extracted with
7.64–7.32 (m, aromatic, 20 H), 4.10 (quintet, J = 7.3 Hz, 4 H), 3.69 CH2Cl2 and aq. NaHCO3. The organic phase was dried (MgSO4),
(apparent d, J = 5.7 Hz, 4 H), 2.91 (d, JH,P = 13.5 Hz, 2 H), 2.80 and the volatiles were evaporated. The product (Rf = 0.38 in
(quintet, J = 5.6 Hz, 1 H), 1.86 (br. s, 1 H), 1.28 (t, J = 7.1 Hz, 6 CH2Cl2/MeOH 20:1.4) was isolated by chromatography in the same
H), 1.02 (s, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 135.7,
133.6, 129.9, 127.9, 63.4, 62.4, 62.2 (d, JC,P = 6.8 Hz), 43.7 (d, JC,P
= 156.3 Hz), 27.0, 19.4, 16.6 (d, JC,P = 5.6 Hz) ppm. 31P NMR
(121 MHz, CDCl3): δ = 26.0 ppm. HRMS (ESI+): calcd. for
C40H57NO5PSi2 [M + H]+ 718.3507; found 718.3517.
system to furnish 43 (0.52 g, 92%). [α]2D0 = –29.3° (c 4.1, CHCl3).
The NMR spectra of 43 (like those of 43, 44, and 45) are very
complex owing to couplings to 31P nuclei and the presence of
syn/anti isomers around the amide bond. 1H NMR (500 MHz,
CDCl3): δ = 9.42 (major) and 9.36 (minor, 2ϫ br. s, ca. 6:4 pro-
4812
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 4804–4815

Phosphono Peptide Nucleic Acids
portion rotamers, exchangeable, 1 H), 7.01 (minor) and 6.93 (major,
2ϫ d, rotamers, J = 1.1 Hz, 1 H), 5.53 (major) and 5.47 (minor,
2ϫ d, rotamers, J = 4.9 Hz, 1 H), 4.95 (major) and 4.86 (minor,
2ϫ d, J = 16.6 Hz, 1 H), 4.68 (d, J = 16.5 Hz) and 4.65 (dd, J =
2.2, 8.0 Hz,1 H), 4.57 (dd, J = 2.2, 7.9 Hz) and 4.52 (d, J = 16.5 Hz,
1 H), 4.40 (t, J = 15.1 Hz, 0.5 H), 4.35 (major) and 4.28 (minor,
2ϫ dd, rotamers, J = 2.3, 4.9 Hz, 1 H), 4.23–3.99 (m, 6.5 H), 3.94–
3.83 (m, 1.5 H), 3.71 (dd, J = 3.2, 15.7 Hz, 0.5 H), 3.46–3.36 (m,
1 H), 1.91 (major) and 1.89 (minor, 2ϫ s, rotamers, 3 H), 1.52–
1.26 (m, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 167.7, 167.6,
167.6, 164.5, 151.2 (major) and 151.1 (minor), 141.3 (minor) and
141.2 (major), 110.6 (major) and 110.5 (minor), 109.7 (major) and
109.6 (minor), 109.1 (minor) and 109.0 (major), 96.4 (major) and
96.3 (minor), 71.6 (minor) and 71.4 (major), 70.9, 70.5, 64.7, 63.1,
63.0, 62.97, 62.93, 62.8, 62.7, 62.6, 48.8, 48.3, 48.1, 47.5, 45.1
(minor, d, JC,P = 156.9 Hz), 40.4 (major, d, JC,P = 155.8 Hz), 26.2,
26.1, 26.0, 25.1, 24.9, 24.5, 24.4, 16.7, 16.6, 16.5, 16.4, 16.3, 12.5
(major) and 12.4 (minor) ppm. 31P NMR (121 MHz, CDCl3): δ =
21.44 (major) and 21.13 (minor) ppm. HRMS (ESI+): calcd. for
C24H38N3NaO11P [M + Na]+ 598.2136; found 598.2143.
50.8 (minor) and 49.7 (major), 48.4 (major) and 48.1 (minor), 44.7
(major, d, JC,P = 157.2 Hz), 41.5 (minor, d, JC,P = 155.1 Hz), 26.8,
25.5 (major) and 25.3 (minor), 16.7 (major, d, JC,P = 5.4 Hz), 16.6
(minor, d, JC,P = 5.6 Hz), 12.5 (minor) and 12.4 (major) ppm. 31P
NMR (121 MHz, CDCl3): δ = 21.40 (minor) and 21.09 (major)
ppm. HRMS (ESI+): calcd. for C18H31N3O8P [M + H]+ 448.1843;
found 448.1855.
(Acetamidomethyl)phosphonate 46: Compound 43 (1.76 g,
3.06 mmol) was mixed with 80% aq. TFA (50 mL). After 1 h, TLC
showed consumption of all the starting material. After removal of
all the volatiles in vacuo and drying, water was added to the resid-
ual oil, followed by Amberlite IRA 400 OH– until neutrality. The
resin was filtered and washed with water. The combined filtrates
were evaporated and dried in vacuo to leave a crude residue, which
was then purified by column chromatography (CH2Cl2/MeOH
80:20, 1% EtN3) to give 46 as a white foam (1.31 g, 86%). The
NMR spectra of 46 is very complex owing to couplings to 31P
nuclei, the presence of syn/anti isomers around the amide bond and
α/β anomers at C-1Ј. 1H NMR (500 MHz, MeOD): δ = 7.34
(major), 7.32 (minor), 7.30 (major) and 7.27 (minor, 4ϫ d, J =
1.2 Hz, 1 H), 5.50 (major, d, J = 3.7 Hz) and 5.17 (minor, d, J =
2.4 Hz) and 5.14 (minor, d, J = 3.2 Hz, 1 H), 4.92–4.77 (m), 4.46
(d, J = 7.3 Hz, 0.5 H), 4.40–4.38 (minor) and 4.34–4.32 (major, 2ϫ
m, 1 H), 4.25–4.00 (m, 6.5 H), 3.94–3.92 (m, 1 H), 3.85–3.74 (m, 3
H), 3.64–3.58 (m, 1 H), 3.54–3.43 (m, 1.5 H), 3.31–3.29 (m, 0.5 H),
3.20 (q, J = 7.4 Hz, 2 H), 1.86 (minor) and 1.85 (major, 2ϫ d, J =
1.1 Hz, 3 H), 1.37 (minor) and 1.30 (major, 2ϫ t, J = 7.2 Hz, 6 H)
(Acetamidomethyl)phosphonate 44: Amine 36 (3.9 g, 5.4 mmol) and
41 (3.0 g, 16.2 mmol) were condensed by using DCC (3.3 g,
16.2 mmol) and HOBt (1.4 g, 10 mmol) in dry DMF (60 mL) for
48 h under argon as described above. TLC showed the product 44
(Rf = 0.32, hexane/EtOAc 1:4). After usual workup and chromatog-
raphy in hexane/EtOAc (1:4), 44 was obtained (1.16 g, 24%). 1H
NMR (300 MHz, CDCl3): δ = 8.97 (minor) and 8.88 (major, 1:2
proportion, 2ϫ br. s, rotamers, exchangeable, 1 H), 7.59–7.34 (m, ppm. 13C NMR (125 MHz, MeOD): δ = 170.0, 169.8, 167.0, 166.9,
aromatic, 20 H), 6.89 (minor) and 6.50 (major, 2ϫ unresolved q,
rotamers, J ≈ 1 Hz, 1 H), 4.68 (minor) and 4.29 (major, 2ϫ br. s,
rotamers, 2 H), 4.15–3.69 (m, 11 H), 1.88 (minor) and 1.79 (major,
2ϫ unresolved d, J ≈ 1 Hz, 3 H), 1.23 (minor) and 1.16 (major,
2ϫ t, rotamers, J = 7.1 Hz, 6 H), 1.03 (major) and 1.00 (minor,
2 ϫ s, rotamers, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ =
167.7, 164.5, 151.1 (minor) and 150.9 (major), 141.2 (minor) and
141.1 (major), 135.7, 135.6, 133.2, 133.1, 132.9, 132.8, 130.2, 130.1,
129.9, 128.1, 127.9, 110.5 (minor) and 110.3 (major), 63.0 (minor,
d, JC,P = 7.4 Hz), 62.5 (major, d, JC,P = 6.5 Hz), 62.4 (minor) and
153.1, 153.0, 143.8, 143.7, 110.9, 110.8, 98.8, 98.7, 94.2, 94.1, 74.8,
74.7, 74.1, 73.6, 72.8, 71.5, 71.3, 71.0, 70.9, 70.8, 70.6, 70.2, 69.3,
68.4, 64.5, 64.23, 64.17, 64.1, 64.0, 53.6, 49.9, 49.8, 49.7, 47.8, 42.9,
42. 3 (major, d, JC,P = 157.1 Hz), 42. 3 (major, d, JC,P = 157.1 Hz)
and 42.2 (minor, d, JC,P = 156.8 Hz), 16.83, 16.79, 16.75. 16.70,
16.6, 12.2, 12.1 ppm. 31P NMR (121 MHz, MeOD): δ = 22.9, 22.8,
2 2 . 6 ( m a j o r ) a nd 21 . 8 pp m. HR MS (p os. ) : c al cd . for
C18H30N3NaO11P [M + Na]+ 518.1510; found 518.1516.
(R)-(Acetamidomethyl)phosphonate 47: To a stirred solution of 46
(0.30 g, 0.61 mmol) in absolute ethanol (8 mL) at 0 °C was added
sodium periodate (0.52 g, 2.43 mmol) solubilized in a small volume
of water (2.5 mL). The mixture turned immediately opaque. The
reaction was stirred for 1 h, after which it was complete. The mix-
ture was then filtered through a pad of Celite, which was washed
with ethanol, and the filtrate was concentrated in vacuo at 25 °C.
The resulting white solid residue was then dried under reduced
pressure and used in the following step without further purifica-
62.2 (major), 60.2, 48.6 (minor) and 48.2 (major), 39.1 (d, JC,P
=
158.7 Hz), 27.0 (major) and 26.9 (minor), 19.3 (major) and 19.2
(minor), 16.6 (minor, d, JC,P = 5.3 Hz), 16.4 (major, d, JC,P
=
6.0 Hz), 12.4 ppm. 31P NMR (121 MHz, CDCl3): δ = 22.47 (major)
and 20.89 (minor) ppm. HRMS (ESI+): calcd. for C47H62N3Na-
O8PSi2 [M + Na]+ 906.370; found 906.3716.
(Acetamidomethyl)phosphonate 45: To a magnetically stirred solu-
tion of 40 (0.3 g, 1.1 mmol) and 41[45] (0.55 g, 3.0 mmol) in dry tion. The crude residue was suspended in absolute ethanol, and the
DMF (10 mL) was added DCC (0.62 g, 3.0 mmol) and HOBt mixture was cooled to 0 °C. Sodium borohydride (0.045 g,
(0.27 g, 2.0 mmol) under argon. The mixture became turbid in few
seconds. Stirring was continued overnight. TLC showed the prod-
uct (Rf = 0.38, CH2Cl2/MeOH 20:1.3). The solids were removed by
filtration with a sintered glass filter. The filtrate was transferred to
a separatory funnel charged with CH2Cl2 and aq. NaHCO3, and
extraction was performed. The organic phase was dried (MgSO4),
and the volatiles were evaporated. Chromatography of the residue
(gradient CH2Cl2/MeOH 20:1.2Ǟ20:1.4) furnished 45 as a foam
1.19 mmol) was then added, and the reaction mixture was warmed
to room temp. The mixture was stirred for 3 h. The reaction mix-
ture was then quenched with 10% HOAc to pH 7. All the volatiles
were removed in vacuo to leave a crude product, which was purified
by column chromatography (CH2Cl2/MeOH 9:1) to give 47 (0.
11 g, 52%) as a white solid. A degradation fragment 49 was also
isolated (0.016 g, 8%), and traces (Ͻ 1%) of fragment 48 were iden-
tified by mass spectroscopy [HRMS (ESI–): calcd. for
C14H23N3O7P [M – H]– 376.1279; found 376.1266], but the com-
pound was not fully characterized. Data for 47: 1H NMR
1
(0.43 g, 90.1%). H NMR (300 MHz, CDCl3): δ = 9.86 (br. s, ex-
changeable, 1 H), 7.04 (major) and 7.00 (minor, 2ϫ br. s, rotamers,
proportion 3:2, 1 H), 4.88–3.67 (m, 13 H), 1.90 (s, 3 H), 1.47–1.27 (500 MHz, D2O): δ = 7.38 (major) and 7.36 (minor, 2ϫ d, rota-
(m, 12 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 167.7 (major) mers, J = 1.1 Hz, 1 H), 4.88 (ddd, J = 1.7, 17.0, 35.6 Hz, 2 H),
and 167.3 (major), 164.5 (major) and 164.4 (minor), 151.4 (major)
and 151.3 (minor), 141.2 (major) and 141.1 (minor), 110.8 (major)
and 110.5 (minor), 109.7, 75.4 (major) and 73.8 (minor), 67.1
(major) and 67.0 (minor), 63.2, 63.1, 63.0, 62.9, 62.9, 62.8, 62.7,
4.30–4.14 (m, 5 H), 4.09–4.07 (major) and 4.03–3.98 (minor, 2ϫ
m, rotamers, 1 H), 3.91 (dd, J = 11.1, 16.0 Hz, 1 H), 3.70–3.51 (m,
4 H), 1.90 (s, 3 H), 1.38 (minor, t, J = 7.1 Hz) and 1.33 (major, dt,
J = 1.5, 7.5 Hz, rotamers, 6 H) ppm. 13C NMR (75 MHz, D2O): δ
Eur. J. Org. Chem. 2013, 4804–4815
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4813

B. Doboszewski, E. Groaz, P. Herdewijn
FULL PAPER
= 169.0 (minor) and 168.8 (major), 166.7, 151.9 (minor) and 151.8
(major), 142.8 (major) and 142.7 (minor), 110.6 (minor) and 110.5
was added triethylamine (1.24 mL, 8.89 mmol), followed by iodo-
trimethylsilane (0.76 mL, 5.35 mmol). The reaction mixture was
stirred overnight under an argon atmosphere. The reaction was
then quenched with a 1 m TEAB solution, and the volatiles were
(major), 69.1 (minor) and 68.5 (major), 64.3 (minor, d, JC,P
=
7.2 Hz) and 63.9 (major, d, JC,P = 6.7 Hz), 62.9 (minor) and 62.6
(major), 50.1 (major) and 50.0 (minor), 49.2 (minor) and 49.1 evaporated under reduced pressure. The crude residue was first
(major), 43.6 (minor, d, JC,P = 156.9 Hz) and 41.2 (major, d, JC,P purified by column chromatography (CH2Cl2/MeOH/1 m TEAB
= 155.7 Hz), 15.5 (minor, d, JC,P = 5.4 Hz) and 15.3 (major, d, JC,P
5:2.5:0.5) and then by HPLC by using a Source 15Q anion-ex-
= 5.7 Hz), 11.0 ppm. 31P NMR (121 MHz, D2O): δ = 24.0 (major) change column (gradient 1 m TEAB from 1 to 25% in 30 min) to
1
and 22.4 (minor) ppm. HRMS (ESI+): calcd. for C15H26N3NaO8P
afford 52 as a triethylammonium salt (90%). H NMR (300 MHz,
D2O): δ = 7.33 (major, d, J = 1.1 Hz) and 7.27 (minor, br. s, rota-
[M + Na]+ 430.1349; found 430.1339. Data for 49: 1H NMR
(MeOD, 300 MHz): δ = 7.35 (d, J = 1.2 Hz, 1 H), 4.42 (d, J = mers, 1 H), 4.83 (br. s, 2 H), 4.08–3.99 (minor) and 3.92–3.86
1.4 Hz, 2 H), 4.18–4.08 (m, 4 H), 3.72 (d, J = 11.7 Hz, 2 H), 3.20
(q, J = 7.3 Hz, 1 H), 1.86 (d, J = 1.1 Hz, 3 H), 1.33–1.27 (m, 6 H)
(major, 2ϫ m, 1 H), 3.66 (minor) and 3.61 (major, 2ϫ d, rotamers,
J = 4.3 Hz, 1 H), 3.54–3.38 (m, 5 H), 3.01 (q, J = 7.3 Hz, 12 H),
ppm. 13C NMR (MeOD, 75 MHz): δ = 169.8, 165.2, 151.2, 141.8, 1.78 (s, 3 H), 1.16 (t, J = 7.3 Hz, 18 H) ppm. 13C NMR (75 MHz,
109.3, 62.5 (d, JC,P = 6.6 Hz), 49.0, 33.9 (d, JC,P = 158.3 Hz), 14.9 D2O): δ = 169.5, 168.9, 154.2, 143.0, 110.4, 69.2 (major) and 68.4
(d, JC,P = 5.9 Hz), 10.5 ppm. 31P NMR (MeOD, 121 MHz): δ = (minor), 63.1 (major) and 62.9 (minor), 50.6, 49.5, 47.8 (d, JC,P
=
22.8 ppm. HRMS (ESI+): calcd. for C12H21N3O6P [M + H]+
334.1163; found 334.1163.
136.8 Hz), 46.2, 11.3, 7.95 ppm. 31P NMR (121 MHz, D2O): δ =
12.6 (minor) and 12.0 (major) ppm. HRMS (ESI–): calcd. for
C11H17N3O8P [M – H]– 350.0759; found 350.0762.
(R)-(Acetamidomethyl)phosphonate Triethylammonium Salt 50: To
a solution of 47 (0.11 g, 0.27 mmol) in dry CH2Cl2 (5 mL) at 0 °C
was added iodotrimethylsilane (0.15 mL, 1.08 mmol). The reaction
mixture was stirred overnight at room temp. under an argon atmo-
sphere. The reaction was then quenched with a 1 m TEAB solution,
and the volatiles were evaporated under reduced pressure. The
crude residue was first purified by column chromatography
(CH2Cl2/MeOH/1 m TEAB 5:2.5:0.5) and then by HPLC by using
a Source 15Q anion-exchange column (gradient 1 m TEAB from 1
to 25% in 30 min) to afford 50 as a triethylammonium salt (76%).
(Acetamidomethyl)phosphonate 53: To a solution of 44 (0.20 g,
0.23 mmol) in dry DMF (2 mL) at 0 °C was added a solution of
tris(dimethylamino)sulfonium difluorotrimethylsilicate (TAS-F,
0.15 g, 0.54 mmol) in dry DMF (2 mL). The reaction mixture was
stirred at 0 °C for 2 h and then warmed to room temp. The mixture
was then diluted with EtOAc and washed with pH 7 buffer. The
aqueous layer was extracted with EtOAc, and the combined or-
ganic layers were dried with Na2SO4, filtered, and concentrated in
vacuo. The resulting crude residue was purified by column
chromatography (CH2Cl2/MeOH 9:1) to give 53 (0.050 g, 54%) as
[α]2D0 = +19.1° (c 0.52, DMSO/PBS). H NMR (300 MHz, D2O): δ
1
1
= 7.41 (major, d, J = 1.1 Hz) and 7.35 (minor, br. s, rotamers, 1 H,
6-H), 4.91 (br. s, 2 H, COCH2), 4.16–4.08 (minor) and 4.02–3.95
(major, 2ϫ m, rotamers, 1 H), 3.74 (minor) and 3.69 (major, 2ϫ
d, rotamers, J = 4.2 Hz, 1 H), 3.67–3.47 (m, 5 H), 3.14 [q, J =
7.3 Hz, 12 H, +N(CH2CH3)3], 1.86 (s, 3 H, CH3), 1.25 [t, J =
7.3 Hz, 18 H, +N(CH2CH3)3] ppm. 13C NMR (75 MHz, D2O): δ =
169.6, 168.9, 154.3, 143.0, 110.4, 69.3 (major) and 68.4 (minor),
63.1 (major) and 63.0 (minor), 50.7, 49.5, 47.9 (d, JC,P = 136.9 Hz),
46.3, 11.3, 8.00 ppm. 31P NMR (121 MHz, D2O): δ = 12.6 (minor)
and 12.0 (major) ppm. HRMS (ESI+): calcd. for C11H17N3O8P
[M – H]– 350.0759; found 350.0765.
a white solid. H NMR (300 MHz, MeOD): δ = 7.27 (major, d, J
= 1.2 Hz) and 7.25 (minor, br. s, rotamers, 1 H), 4.80 (apparent d,
J = 1.6 Hz, 2 H), 4.26–4.18 (minor) and 4.17–4.08 (major, 2ϫ m,
rotamers, 4 H), 4.03–3.97 (m, 1 H), 3.87 (d, J = 12.0 Hz, 2 H),
3.70–3.68 (m, 2 H), 3.33–3.28 (m, 2 H), 1.86 (major) and 1.85
(minor, 2ϫ s, rotamers, 3 H), 1.38 and 1.30 (2ϫ t, rotamers, J =
7.1 Hz, 6 H) ppm. 13C NMR (75 MHz, MeOD): δ = 169.0, 165.3,
151.3, 142.0, 109.2, 62.5 (d, JC,P = 6.6 Hz), 61.1, 59.5 (minor) and
58.6 (major), 48.4, 37.7 (d, JC,P = 158.9 Hz), 14. 9 (d, JC,P
=
6.2 Hz), 10.5 ppm. 31P NMR (121 MHz, MeOD): δ = 25.6 (major)
and 22.0 (minor) ppm. HRMS (ESI+): calcd. for C15H26N3NaO8P
[M + Na]+ 430.1350; found 430.1350.
(S)-(Acetamidomethyl)phosphonate Triethylammonium Salt 51: To a
solution of 42 (0.10 g, 0.22 mmol) in dry CH2Cl2 (5 mL) at 0 °C
was added triethylamine (0.31 mL, 2.23 mmol), followed by iodo-
trimethylsilane (0.25 mL, 1.80 mmol). The reaction mixture was
stirred overnight under an argon atmosphere. The reaction was
then quenched with a 1 m TEAB solution, and the volatiles were
evaporated under reduced pressure. The crude residue was first
purified by column chromatography (CH2Cl2/MeOH/1 m TEAB
5:2.5:0.5) and then by HPLC by using a Source 15Q anion-ex-
change column (gradient 1 m TEAB from 1 to 25% in 30 min) to
afford 51 as a triethylammonium salt (78%). [α]2D0 = –9.8° (c 0.88,
DMSO/PBS). 1H NMR (300 MHz, D2O): δ = 7.41 (major) and
7.35 (minor, 2ϫ br. s, rotamers, 1 H), 4.91 (br. s, 2 H), 3.98–3.96
(m, 1 H), 3.74 (minor) and 3.69 (major, 2ϫ d, rotamers, J = 4.3 Hz,
1 H), 3.66–3.48 (m, 5 H), 3.14 (q, J = 7.4 Hz, 12 H), 1.86 (s, 3 H),
1.24 (t, J = 7.4 Hz, 18 H) ppm. 13C NMR (75 MHz, D2O): δ =
169.5, 169.2, 154.2, 143.4, 110.7, 69.6 (major) and 68.7 (minor),
63.4 (major) and 63.3 (minor), 51.0, 49.8, 48.1 (d, JC,P = 136.9 Hz),
46.6, 11.6, 8.31 ppm. 31P NMR (121 MHz, D2O): δ = 12.6 (minor)
and 12.0 (major) ppm. HRMS (ESI–): calcd. for C11H17N3O8P
[M – H]– 350.0759; found 350.0760.
(Acetamidomethyl)phosphonate Triethylammonium Salt 54: To a
solution of 53 (0.05 g, 0.12 mmol) in dry CH2Cl2 (3 mL) at 0 °C
was added triethylamine (0.17 mL, 1.18 mmol), followed by iodo-
trimethylsilane (0.07 mL, 0.49 mmol). The reaction mixture was
stirred overnight at room temp. under an argon atmosphere. The
reaction was then quenched with a 1 m TEAB solution, and the
volatiles were evaporated under reduced pressure. The crude resi-
due was first purified by column chromatography (CH2Cl2/MeOH/
1 m TEAB, 5:2.5:0.5) and then by HPLC by using a Source 15Q
anion-exchange column (gradient 1 m TEAB from 1 to 25% in
1
30 min) to afford 54 as a triethylammonium salt (82%). H NMR
(300 MHz, D2O): δ = 7.49 (major, d, J = 1.1 Hz) and 7.42 (minor,
br. s, 1 H, rotamers, 6-H), 5.06 (br. s, 2 H, COCH2), 4.45–4.40 (m,
1 H, CH), 3.81 (dd, J = 12.3, 8.6 Hz, 2 H, CH2), 3.67 (dd, J =
12.3, 4.7 Hz, 2 H, CH2), 3.46 (d, J = 10.3 Hz, 2 H, POCH2), 3.20
[q, J = 7.3 Hz, 12 H, +N(CH2CH3)3], 1.89 (minor) and 1.88 (major,
s, 3 H, CH3), 1.27 [t, J = 7.3 Hz, 18 H, +N(CH2CH3)3] ppm. 13C
NMR (75 MHz, D2O): δ = 169.8, 167.1, 152.3, 143.3, 110.4, 61.4,
58.6 (major) and 57.8 (minor), 49.9, 46.3, 44.7 (d, JC,P = 134.9 Hz),
11.0, 7.92 ppm. 31P NMR (121 MHz, D2O): δ = 13.7 (minor) and
12.3 (major) ppm. HRMS (ESI+): calcd. for C11H17N3O8P [M –
H]– 350.0759; found 350.0766.
(rac)-(Acetamidomethyl)phosphonate Triethylammonium Salt 52: To
a solution of 45 (0.40 g, 0.89 mmol) in dry CH2Cl2 (10 mL) at 0 °C
4814
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 4804–4815

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Published Online: June 25, 2013
Eur. J. Org. Chem. 2013, 4804–4815
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4815