Synthesis of novel DOXYL labelling reagents with electrophilic groups

Article abstract of DOI:10.1016/j.tetlet.2003.10.089

The oxalic acid salt of 2-aminomethyl-3-oxyl-2,4,4-trimethyl-1,3- oxazolidine has been synthesised from 1-chloroacetone. Being water soluble, the salt is a promising candidate for varied applications. It has been functionalised with electrophilic groups generating novel spin labelling reagents.

Full text of DOI:10.1016/j.tetlet.2003.10.089

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 179–182
Synthesis of novel DOXYL labelling reagents with electrophilic
groups
Jitendra R. Harjani, Susheel J. Nara, Prashant U. Naik and Manikrao M. Salunkhe*
Department of Chemistry, The Institute of Science, 15-Madam Cama Road, Mumbai 400 032, India
Received 1 September 2003; revised 6 October 2003; accepted 17 October 2003
Abstract—The oxalic acid salt of 2-aminomethyl-3-oxyl-2,4,4-trimethyl-1,3-oxazolidine has been synthesised from 1-chloroacetone.
Being water soluble, the salt is a promising candidate for varied applications. It has been functionalised with electrophilic groups
generating novel spin labelling reagents.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Over the last 50 years, nitroxide free radicals have found
applications as diverse as redox reagents,1 SOD mimics,2
MRI contrast agents3 and above all as reporter groups4
(either spin label or spin trap) for probing biological
systems, making them a strong and versatile tool
accessible to chemists and biophysicists. Despite the vast
developments witnessed in the area of spin labels,5 the
syntheses of spin labels possessing the desired blend of
properties6 has always been a challenge to synthetic
chemists. In this regard the newer applications of nitr-
oxides, in particular their utility in oxymetry7 and pH
determination8 of biological systems, has intensified
efforts towards the design of novel and more suitable
probes possessing properties suitable for these applica-
tions.9
Despite these advantageous properties and the proce-
dural simplicity with which they can be installed at a
ketonic site,10b DOXYLs have attracted only limited
attention from synthetic chemists, probably because the
formation of 1,3-oxazolidine rings either suffers from the
disadvantage of poor yields for some ketones or is not
possible with others.12 In addition, the oxidation of
oxazolidines with m-CPBA affords the corresponding
DOXYL typically in only about 30% yield (based on the
starting oxazolidine).5a The literature documents only a
few successful preparations of DOXYL based13 spin
labelling reagents. An alternative method of generation
of DOXYL from a carboxylic acid, which circumvents
the use of a ketone, is also well known.14;10c;10d The
disadvantage of this method is the relatively greater
number of steps required.
Spin labelling is rarely achieved by direct construction of
a nitroxide group on the biomolecule.10 The most
common methodology for spin labelling involves cova-
lent anchoring of a nitroxide by employing spin labelling
reagents,5a;11 which are functionalised with electrophilic
groups or with groups that easily condense with the
groups present on biomolecules. The DOXYL (4,4-di-
methyl-oxazolidine-N-oxyl) spin labels are the proto-
typical spin labels possessing distinct conformational
rigidity, which facilitates the interpretation of rotational
correlation times in terms of their local environment.
With the intention of developing DOXYL based spin
labelling reagents, we report herein the synthesis of a
novel amino DOXYL spin label, which was conve-
niently functionalised with electrophilic groups to
facilitate the nucleophilic labelling of biomolecules. We
sought to prepare an –NH2 group appended to a
DOXYL spin label, 1 (Scheme 1), as functionalisation of
spin labels with electrophilic groups via reactions with a
–NH2 group are well documented.5a;11a The disconnec-
tion for the amino DOXYL demands that the –NH2
group be blocked, so that the secondary amino group of
the 1,3-oxazolidine can be preferentially oxidised over
the –NH2 group. The most obvious precursors for the
amino blocked 1,3-oxazolidine 2 would be N-blocked
aminal 3 and a-N-blocked amino ketone 4, as ketones
can be conveniently functionalised at an a-position.
Keywords: 4,4-Dimethyl-oxazolidine-N-oxyls; Nitroxide; Synthesis;
Electrophilic groups; Spin labels.
* Corresponding author. Tel./fax: +91-22-22816750; e-mail:
mmsalunkhe@hotmail.com
0040-4039/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.089

180
J. R. Harjani et al. / Tetrahedron Letters 45 (2004) 179–182
N
E
disconnection
.
N
R
O
O
.
.
N
R
O
O
N
R
O
O
R'
R'
R'
N
E
N
1
NH2
2
H
O
N
R
H
O
R'
R
+
R'
HO
NH2
N
N
3
4
Scheme 1. Strategic disconnection for DOXYL derivatised with electrophilic groups.
O
O
O
O
O
O
(ii)
(i)
N
H
N
NK
O
N
O
5
6
(iii)
O
O
Cl
.
N
O
(v)
O
N
1/2(COO-)2
H
(iv)
.
N
+
.
N
O
NH3
O
O
O
N
9
O
O
8
7
(vi)
.
O
N
O
O
N
H
10
Scheme 2. Synthesis of DOXYLs with electrophilic groups. Reagents and conditions: (i) 1-chloroacetone (1.1 equiv), DMF, 0 ꢁC, then rt, 1 h, reflux
under nitrogen, 2 h; (ii) 2-amino-2-methyl-1-propanol (3.3 equiv), toluene, TsOHÆH2O (cat.), reflux, Dean–Stark water separator, 24 h; (iii) m-CPBA
(1.2equiv), CH 2Cl2, Et2O, 0 ꢁC, then rt, 3 h; (iv) aq MeNH2 (37%, 5 equiv), Et2O, rt, 30 min, then aq (COOH)2Æ2H2O (0.5 equiv) after removal of
MeNH2; (v) Et3N (1.2equiv), chloroacetyl chloride (1.1 equiv), 0 ꢁC, then rt, 30 min; (vi) aq 20% NaOH (5 equiv), ClCOOEt (1.1 equiv), 0 ꢁC, then rt,
30 min.
A nucleophilic displacement reaction involving 1-chlo-
roacetone and potassium phthalimide in DMF as per
the reported procedure,15 resulted in the formation of
ketone 516 in 92% yield (Scheme 2). The ketone 5 upon
condensation with 2-amino-2-methyl-1-propanol with
azeotropic removal of water generated oxazolidine 617 in
85% yield. The oxazolidine 6 on oxidation with m-
CPBA generated phthaloyl DOXYL 718 almost quanti-
tatively (94%). The overall yield of phthaloyl DOXYL 7
from ketone 5 is 73%. To our knowledge, this is the best
reported yield for a DOXYL starting from the parent
ketone. A biphasic dephthaloylation of 7 in Et2O with
aq MeNH2 followed by subsequent removal of MeNH2
(at 400 mm pressure for 1 h and N2 bubbling for 3 h)
from the aqueous phase and neutralisation of the amino
DOXYL with aq oxalic acid generated the oxalate salt
of amino DOXYL 8.19 With 8 in hand, easy electrophilic
functionalisation was illustrated by the formation of N-
chloroacetyl 920 and N-ethoxycarbonyl 1021 derivatives
in 46% and 55% overall yields, respectively.
search for newer water soluble nitroxides,22 which are
needed for MRI,23 protection from oxidative stress and
radiative damage24 and nitroxide mediated controlled
free radical polymerisation reactions (NMCPR).25 Such
nitroxides are generally carboxylate26 or ammonium27 or
sulfonium22b salts and are hence highly hydrophilic in
nature. We believe that the oxalic acid salt of 2-amino-
methyl-3-oxyl-2,4,4-trimethyl-1,3-oxazolidine 8, will be
a valuable addition to the list of known water soluble
nitroxide radicals, especially in the light of the search for
nitroxides with the charged group separated from the
aminoxyl function by a shorter distance.22b This is
because such nitroxides are expected to be most effective
for the NMCPR reactions of hydrophilic monomers in
water.
To summarise, we have succeeded in the synthesis of the
oxalic acid salt of a novel amino DOXYL spin label,
2-aminomethyl-3-oxyl-2,4,4-trimethyl-1,3-oxazolidine,
which could be used for generating potential spin
labelling reagents with electrophilic groups, thus allow-
ing nucleophilic labelling of biomolecules with DOXYL
spin labels. In addition, it could also be applied to those
The limited solubility of piperidine and pyrrolidine
nitroxides has greatly intensified efforts towards the

J. R. Harjani et al. / Tetrahedron Letters 45 (2004) 179–182
181
14. (a) Keana, J. F. W.; Lee, T. D. J. Am. Chem. Soc. 1975,
97, 1273–1274; (b) Lee, T. D.; Keana, J. F. W. J. Org.
Chem. 1976, 41, 3237–3241.
reactions/processes in which water-soluble nitroxides are
required.
15. Wimalasena, K.; May, S. W. J. Am. Chem. Soc. 1987, 109,
4036–4046.
16. N-(2-Oxopropyl)-phthalimide 5: White crystalline solid,
mp 118–120 ꢁC from CHCl3–hexane. Anal. calcd for
C11H9NO3: C, 65.02; H, 4.43; N, 6.90. Found: C, 64.97;
H, 4.46; N, 6.86%. IR (KBr): mmax 2969, 1770, 1714, 1417,
1182, 1019, 710 cmꢀ1. 1H NMR (CDCl3, 300 MHz): d 2.27
(s, 3H, CH3), 4.50 (s, 2H, CH2), 7.71–7.74 (m, 2H, 2 · CH
arom.), 7.84–7.87 (m, 2H, 2 · CH arom.) ppm. 13C NMR
(CDCl3, 300 MHz): d 27.01 (CH3), 47.08 (CH2), 123.38
(2 · CH arom.), 131.82(2 · C arom.), 134.00 (2 · CH
arom.), 167.39 (2 · CO), 199.41 (CO) ppm.
Acknowledgements
The authors are grateful to the University of Mumbai,
Mumbai, for generously funding this work under the
University Research Project (APD/237/110 of 2003). The
authors are also thankful to Professor Girish K. Trivedi
for fruitful discussions.
17. 2-(N-Phthalimidomethyl)-2,4,4-trimethyl-1,3-oxazolidine
6: White amorphous powder, mp 119–121 ꢁC from
CHCl3–hexane. Anal. calcd for C15H18N2O3: C, 65.69;
H, 6.57; N, 10.22. Found C, 65.65; H, 6.53; N, 10.17%. IR
(KBr): mmax 3357, 2966, 2855, 1708, 1392, 1211, 1062,
References and Notes
726 cmꢀ1 1H NMR (CDCl3, 500 MHz): d 1.21 (s, 3H,
.
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D2O exchangeable, NH), 3.60 (s, 2H, CH2), 3.68–3.70 (d,
J ¼ 12Hz, 1H, HC H), 3.82–3.85 (d, J ¼ 12Hz, 1H,
HCH), 7.68 (br s, 2H, 2 · CH arom.), 7.80 (br s, 2H,
2 · CH arom.) ppm. 13C NMR (CDCl3, 300 MHz): d
26.36 (CH3), 27.56 (CH3), 28.57 (CH3), 45.56 (CH2), 59.33
(C), 77.24 (CH2, overlapped with CDCl3 signal), 96.26
(C), 123.38 (2 · CH arom.), 131.89 (2 · C arom.), 134.05
(2 · CH arom.), 168.70 (2 · CO) ppm.
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18. 3-Oxyl-2-(N-phthalimidomethyl)-2,4,4-trimethyl-1,3-oxa-
zolidine 7: Orange yellow powder, mp 85–87 ꢁC from
EtOH–H2O. Anal. calcd for C15H17N2O4: C, 62.28, H,
5.88; N, 9.69. Found C, 62.32; H, 5.83; N, 9.72%. UV
(CH3CN): kmax 220 nm (emax 40,240 dm3 molꢀ1 cmꢀ1). IR
(KBr): mmax 2974, 2928, 2869, 1722, 1389, 1247, 1067,
724 cmꢀ1
.
1H NMR (CDCl3, Ph–NH–NH2, D2O,
300 MHz): d 0.96 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.38
(s, 3H, CH3), 3.42–3.45 (d, J ¼ 8:1 Hz, 1H, HCH), 3.57–
3.60 (d, J ¼ 8:7 Hz, 1H, HCH), 3.74 (s, 2H, CH2), 7.55–
7.58 (m, 2H, 2 · CH arom.), 7.72–7.74 (m, 2H, 2 · CH
arom.) ppm. 13C NMR (CDCl3, Ph–NH–NH2, 300 MHz):
d 19.65 (CH3), 20.62 (CH3), 26.12 (CH3), 44.18 (CH2),
61.11 (C), 75.22 (CH2), 96.89 (C), 122.90 (2 · CH arom.),
131.68 (2 · C arom.), 133.57 (2 · CH arom.), 168.38
(2 · CO) ppm. ESR: 10ꢀ4 M solution in CHCl3, three
equidistant lines with aN ¼ 14:5 G.
6. (a) Ozhogina, O. A. Tetrahedron Lett. 2002, 43, 553–555;
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ꢀ
}
ꢀ
ꢀ
}
8125–8127; (d) Kulcsar, G.; Kalai, T.; Jeko, J.; Hideg, K.
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19. Oxalic acid salt of 2-aminomethyl-3-oxyl-2,4,4-trimethyl-
1,3-oxazolidine 8: Orange crystals, Anal. calcd for
C8H16N2O4: C, 47.06; H, 7.84; N, 13.73. Found C,
47.01; H, 7.82; N, 13.69%. UV (CH3CN): kmax 195 nm
(emax 29,268 dm3 molꢀ1 cmꢀ1). IR (KBr): mmax 3404, 2987,
1617, 1381, 1212 cmꢀ1 1H NMR (Ph–NH–NH2, D2O,
.
500 MHz): d 1.10 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.29 (s,
3H, CH3), 2.96–3.00 (d, J ¼ 22 Hz, 2H, CH2), 3.62(s, 1H,
HCH), 3.78 (s, 1H, HCH) ppm. 13C NMR (D2O, Ph–
NH–NH2, 500 MHz) d 19.09 (CH3), 20.85 (CH3), 25.18
(CH3), 44.86 (CH2), 61.99 (C), 75.78 (CH2), 94.97 (C),
173.26 (CO) ppm. ESR: 10ꢀ4 M solution in H2O, three
equidistant lines with aN ¼ 15:0 G.
20. 2-(N-Chloroacetylaminomethyl)-3-oxyl-2,4,4-trimethyl-1,
3-oxazolidine 9: Orange oil, Anal. calcd for
C9H16ClN2O3: C, 45.86; H, 6.79; Cl, 15.07; N, 11.89.
Found C, 45.82; H, 6.77; Cl, 15.04; N, 11.85%. UV
(CH3CN): kmax 240 nm (emax 14,461 dm3 molꢀ1 cmꢀ1). IR
1
(CHCl3 film): mmax 3282, 2975, 1675, 1387, 1215 cmꢀ1. H
NMR (CDCl3, Ph–NH–NH2, D2O, 500 MHz): d 1.18 (s,
3H, CH3), 1.25 (s, 3H, CH3), 1.35 (s, 3H, CH3), 3.31–3.34
(d, J ¼ 13:5 Hz, 1H, HCH), 3.44–3.47 (d, J ¼ 13:5 Hz,

182
J. R. Harjani et al. / Tetrahedron Letters 45 (2004) 179–182
1H, HCH), 3.62–3.64 (d, J ¼ 7:5 Hz, 1H, HCH), 3.72–
62.05 (CH2), 75.43 (CH2), 97.27 (C), 151.05 (CO) ppm.
ESR: 10ꢀ4 M solution in CHCl3, three equidistant lines
with aN ¼ 14:5 G.
3.73 (d, J ¼ 7:5 Hz, 1H, HCH) and 3.98–4.05 (dd, J ¼ 15,
3.5 Hz, 2H, CH2). 13C NMR (CDCl3, Ph–NH–NH2,
500 MHz): d 20.30 (CH3), 21.10 (CH3), 25.71 (CH3),
42.79 (CH2), 46.60 (CH2), 62.32 (C), 75.78 (CH2), 97.17
(C) and 166.74 (CO) ppm. ESR: 10ꢀ4 M solution in
CHCl3, three equidistant lines with aN ¼ 14:75 G.
22. (a) Marx, L.; Rassat, A. Tetrahedron Lett. 2002, 43, 2613–
2614; (b) Marx, L.; Rassat, A. Chem. Commun. 2002, 632–
633, and references cited therein.
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2420; (b) Keana, J. F. W.; Heo, G. S.; Gaughan, G. T.
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1908.
21. 2-(N-Ethoxycarbonylaminomethyl)-3-oxyl-2,4,4-trimethyl-
1,3-oxazolidine 10: Orange yellow oil. Anal. calcd for
C10H19N2O4: C, 51.95; H, 8.23; N, 12.12. Found C, 51.90;
H, 8.20; N, 12.11%. UV (CH3CN): kmax 223 nm (emax
7104 dm3 molꢀ1 cmꢀ1). IR (CHCl3 film): mmax 3455, 2997,
1
1721, 1519, 1370, 1213 cmꢀ1. H NMR (CDCl3, Ph–NH–
NH2, D2O, 300 MHz): d 1.14 (s, 3H, CH3), 1.22 (6H,
2 · CH3), 1.31 (s, 3H, CH3), 3.06–3.10 (d, J ¼ 14:7 Hz,
1H, HCH), 3.34–3.39 (d, J ¼ 13:5 Hz, 1H, HCH), 3.56–
3.59 (d, J ¼ 8:4 Hz, 1H, HCH), 3.67–3.70 (d, J ¼ 8:4 Hz,
1H, HCH), 4.03–4.10 (q, J ¼ 7:2Hz, 2H, C H2). 13C NMR
(CDCl3, Ph–NH–NH2, 300 MHz): d 14.57 (CH3), 19.81
(CH3), 20.85 (CH3), 25.50 (CH3), 47.94 (CH2), 60.71 (C),