Synthesis of aminooxy and N-alkylaminooxy amines for use in bioconjugation

Article abstract of DOI:10.1021/jo101066c

Five Boc-protected aminooxy and N-alkylaminooxy amines have been synthesized in 60-95% overall yield using a common synthetic strategy from readily available two- and three-carbon Cbz-protected amino alcohols. The amines can be linked to biomolecules via

Full text of DOI:10.1021/jo101066c

pubs.acs.org/joc
Synthesis of Aminooxy and N-Alkylaminooxy
Amines for Use in Bioconjugation
Michael R. Carrasco,* Carolina I. Alvarado,
Scott T. Dashner, Amanda J. Wong, and Michael A. Wong
Department of Chemistry and Biochemistry,
Santa Clara University, 500 El Camino Real, Santa Clara,
California 95053-0270
FIGURE 1. Aminooxy and N-alkylaminooxy strategies for bio-
conjugation chemistry. In mildly acidic aqueous buffers, aminooxy
groups react with aldehydes and ketones to form oximes and
N-alkylaminooxy groups react with reducing sugars to form glyco-
conjugates.
mcarrasco@scu.edu
Received June 2, 2010
proteins for imaging applications. N-Alkylaminooxy groups
have specific utility for glycosylation chemistry because they
react chemoselectively with native reducing sugars to form
glycoconjugates where the attached sugars maintain the
biologically relevant cyclic conformations.3 N-Alkylami-
nooxy strategies have been used for the glycosylation of
peptides,3,4 peptoids,5 proteins,6 carbohydrates,7 micro-
arrays,8 and small molecules of pharmaceutical interest.9
To effect these conjugation strategies, a wide array of special
amino acids, sugars, and linkers have been reported. Rather
than make different compounds for each context, however,
it would be valuable to have a small set of derivatives that
could be used to incorporate aminooxy and N-alkylami-
nooxy functionality into a wide array of desired molecules.
Because our own interest was in the synthesis of modified
peptoid oligomers, we envisioned making the amines 1-5.
These amines could be used in the synthesis of peptoids by
the submonomer method,10 and they could also be used in
the established bioconjugation methods for modification of
carboxyl groups in molecules such as peptides and proteins.
Removal of the Boc protection would then reveal the desired
aminooxy (from 1 and 2) or N-alkylaminooxy functiona-
lity (from 3-5). As a result, 1-5 would allow for easy
Five Boc-protected aminooxy and N-alkylaminooxy
amines have been synthesized in 60-95% overall yield
using a common synthetic strategy from readily available
two- and three-carbon Cbz-protected amino alcohols.
The amines can be linked to biomolecules via amide
formation and incorporated directly into peptoids via
submonomer synthesis. Subsequent deprotection of the
aminooxy and N-alkylaminooxy groups enables conjuga-
tion with desired target molecules via established chemo-
selective ligation methods. The range of derivatives
synthesized allows different distances to be established
between the conjugated molecules.
The reactions of aminooxy and N-alkylaminooxy groups
have proven exceedingly useful for bioconjugate chemistry.1
Aminooxy groups react chemoselectively with aldehydes and
ketones to form oximes in mild aqueous solutions, and with
appropriate additives the reactions can be performed at very
low concentrations (Figure 1).2 Oxime formation has been
the basis of conjugation chemistry as varied as dendrimer
synthesis; formation of peptide and protein assemblies;
glycosylation of peptides, proteins, and cells; and labeling
(3) Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54, 12269.
(4) (a) Carrasco, M. R.; Nguyen, M. J.; Burnell, D. R.; MacLaren, M. D.;
Hengel, S. M. Tetrahedron Lett. 2002, 43, 5727. (b) Carrasco, M. R.; Brown,
R. T. J. Org. Chem. 2003, 68, 8853.
(5) Seo, J.; Michaelian, N.; Owens, S. C.; Dashner, S. T.; Wong, A. J.;
Barron, A. E.; Carrasco, M. R. Org. Lett. 2009, 11, 5210.
(6) (a) Matsubara, N.; Oiwa, K.; Hohsaka, T.; Sadamoto, R.; Niikura,
K.; Fukuhara, N.; Takimoto, A.; Kondo, H.; Nishimura, S.-I. Chem.;Eur.
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2002, 1504. (b) Peri, F.; Jimenez-Barbero, J.; Garcıa-Aparicio, V.; Tvaroska,
I.; Nicotra, F. Chem.;Eur. J. 2004, 10, 1433.
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(1) (a) Peri, F.; Nicotra, F. Chem. Commun. 2004, 623. (b) Zatsepin, T. S.;
Stetsenko, D. A.; Gait, M. J.; Oretskaya, T. S. Bioconjugate Chem. 2005, 16,
471. (c) Langenhan, J. M.; Griffith, B. R.; Thorson, J. S. J. Nat. Prod. 2005,
68, 1696. (d) Langenhan, J. M.; Thorson, J. S. Curr. Org. Synth. 2005, 2, 59.
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DOI: 10.1021/jo101066c
r
Published on Web 07/15/2010
J. Org. Chem. 2010, 75, 5757–5759 5757
2010 American Chemical Society

Carrasco et al.
JOCNote
incorporation of aminooxy and N-alkylaminooxy moieties
into a wide range of desired substrates, and their variation
allows for four-, five-, or six-atom distances to be established
between the conjugated molecules. This distance variation is
particularly important in the glycopeptoid context, where it
allows control of the distances between attached sugars and
the peptoid backbone.
SCHEME 1. Synthesis of 1-5
Perusal of the literature revealed that syntheses of 111 and
212 as free amines had been reported, and we recently
disclosed a synthesis of the free amine of 4.5 However, to
enable their practical and general use, especially by practi-
tioners interested in bioconjugation but only minimally
trained in organic synthesis, it would be desirable to have a
common route to 1-5. Moreover, that route would comprise
few steps, be high-yielding, require no advanced techniques,
and be amenable to large scale synthesis. The published
syntheses lack common strategies and intermediates, and
several suffer from low overall yields and troublesome
purifications.
We now report a general, practical synthesis of 1-5 in
three steps and 60-95% overall yield. All reactions proceed
at room temperature and without particular sensitivity to
moisture or air. Further, the majority of the purifications
may be accomplished solely with extractive workup proce-
dures or triturations.
Our sequence is indicated in Scheme 1. Although bromides
8 and 9 are known,13 we found it most convenient to prepare
them from the corresponding, readily available Cbz-pro-
tected amino alcohols 6 and 7. Under a slight modification of
the conditions of Albrecht et al.,14 reaction of 8 or 9 with N-Boc-
hydroxylamine, 10, or N-methyl-N-Boc-hydroxylamine, 11,15
yielded the four bis-protected amino hydroxylamines 13-16;
reaction of 8 with N-Boc-O-methylhydroxylamine, 12,16 yielded
17. Notably, the alkylation reactions with 11 required solely
extractive workup procedures to provide excellent yields of
analytically pure 15 and 16; column chromatography was
required for the purification of 13, 14, and 17, but each was still
obtained in good yield. Use of the bromides 8 and 9 rather than
the corresponding mesylates of 6 and 7 resulted in higher yields
for the alkylation reactions as well as easier reaction monitoring
and purifications because the mesylates had very similar Rf’s by
TLC to the desired products.
Removal of the Cbz protecting group from 13-17 was
accomplished using the method of Mandal and McMurray,17
and the addition of chloroform to the reactions allowed 1-5 to
be isolated directly as their HCl salts in excellent yields. In the
case of 1, 3, and 5, simple trituration of the solids with hexane
was sufficient to obtain analytically pure material in nearly
quantitative yield; the HCl salts of 2 and 4 were not as crystal-
line, but both could be purified with a simple filtration through
a short plug of silica gel. We found the nature of the Pd/C
catalyst and the amount of chloroform added to be very
important for successful deprotection. Less active catalysts,
lesser amounts of catalyst, and greater amounts of chloroform
led to sluggish reactions where additional portions of Pd/C and
triethylsilane had to be added over time to effect complete
conversion. In the end, we settled on using a relatively large
amount (40 wt %) of Degussa type E101 NE/W 10% Pd/C and
only 125 mol % of chloroform as conditions that gave com-
plete reaction in short (<20 min) reaction times. Importantly,
there was no evidence of cleavage of the hydroxylamine N-O
bond or loss of the Boc protecting group, which could have
been a concern with the generation of HCl during the reaction.
In the end, we established a highly efficient synthesis of a
suite of aminooxy and N-alkylaminooxy amines. We are
currently using them for the synthesis of arrays of glycopep-
toids, and we expect that they will find broad application in
bioconjugation strategies.
(11) Gaertner, H.; Offord, R.; Botti, P.; Kuenzi, G.; Hartley, O. Biocon-
jugate Chem. 2008, 19, 480.
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42, 6067. (c) Gao, F.; Yan, X.; Shakya, T.; Baettig, O. M.; Ait-Mohand-
Brunet, S.; Berghuis, A. M.; Wright, G. D.; Auclair, K. J. Med. Chem. 2006,
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195.
Experimental Section
N-(2-Bromoethyl)carbamic Acid, Phenylmethyl Ester (8). A
flask was charged with benzyl N-(2-hydroxyethyl)carbamate, 6
(9.76 g, 50.0 mmol, 100 mol %), methanesulfonyl chloride
(4.65 mL, 60.0 mmol, 120 mol %), and CH2Cl2 (150 mL). To
this stirring solution was added NEt3 (9.01 mL, 65.0 mmol,
130 mol %). Stirring was continued for 45 min, and then LiBr
(43.5 g, 500 mmol, 1000 mol %) and acetone (150 mL) were
added. The reaction mixture was stirred for an additional 21.5 h,
(16) Kawase, M.; Kitamura, T.; Kikugawa, Y. J. Org. Chem. 1989, 54,
3394.
(17) Mandal, P. K.; McMurray, J. S. J. Org. Chem. 2007, 72, 6599.
5758 J. Org. Chem. Vol. 75, No. 16, 2010

Carrasco et al.
JOCNote
and then the solvents were removed by rotary evaporation. The
contents were partitioned between Et2O (300 mL) and H2O (200
mL), and the Et2O layer was washed with 0.1 M KHSO4 (3 Â 100
mL) and brine (1Â), dried (Na2SO4), filtered, evaporated to
dryness, and placed under vacuum to yield 12.77 g (49.5 mmol,
99%) of 8 as a white solid. Mp: 47-48 °C. 1H NMR (400 MHz,
CDCl3): δ 7.35 (m, 5H), 5.27 (br s, 1H), 5.11 (s, 2H), 3.59 (q, J =
5.9 Hz, 2H), 3.45 (t, J = 5.8 Hz, 2H). 13C NMR (100 MHz,
CDCl3): δ 156.3, 136.3, 128.7, 128.4, 128.3, 67.1, 42.8, 32.6. Anal.
Calcd for C10H12BrNO2: C, 46.53; H, 4.69; N, 5.43. Found: C,
46.92; H, 4.62; N, 5.53.
6,9,9-Trimethyl-7-oxo-5,8-dioxa-2,6-diazadecanoic Acid, Phen-
ylmethyl Ester (15). A flask was charged with N-Boc-N-methylhy-
droxylamine, 11 (2.06 g, 14.0 mmol, 140 mol %), DBU (2.24 mL,
15.0 mmol, 150 mol %), and Et2O (8 mL). To this stirring solution
was added 8 (2.58 g, 10.0 mmol, 100 mol %), and the walls of the
flask were rinsed with additional Et2O (2 mL). Stirring was con-
tinued for 15 min, and then the Et2O was removed by rotary
evaporation. The concentrated mixture was stirred for an additional
13 h and then partitioned between EtOAc (200 mL) and water (150
mL). The EtOAc layer was washed with 0.1 M KHSO4 (3Â 50 mL),
0.1 M NaOH (5 Â 50 mL), and brine (1Â) and then dried (Na2SO4)
and filtered. Evaporation of the solvents and drying under vacuum
yielded 3.18 g (9.80 mmol, 98%) of 15 as a clear oil. 1H NMR (400
MHz, CDCl3): δ 7.33 (m, 5H), 5.83 (br s, 1H), 5.11 (s, 2H), 3.87 (t,
J = 4.8 Hz, 2H), 3.40 (m, 2H), 3.07 (s, 3H), 1.48 (s, 9H). 13C NMR
(100 MHz, CDCl3):δ157.5, 156.6, 136.7, 128.5, 128.10, 128.07, 82.0,
73.0, 66.7, 39.5, 36.7, 28.3. Anal. Calcd for C16H24N2O5:C,59.24;H,
7.46; N, 8.64. Found: C, 58.86; H, 7.72; N, 8.66.
N-(2-Aminoethoxy)-N-methylcarbamic Acid, 1,1-Dimethy-
lethyl Ester Hydrochloride (3). A flask was charged with 15
(0.352 g, 1.09 mmol, 100 mol %), CHCl3 (0.109 mL, 1.36 mmol,
125 mol %), and CH3OH (3 mL). The flask was alternately
evacuated and flushed with Ar (3Â), and then Degussa type
E101 NE/W 10% Pd/C (0.282 g, 40 wt % on dry basis) was
added. Triethylsilane (1.74 mL, 10.9 mmol, 1000 mol %) was
added dropwise, and the reaction mixture was stirred for 50 min.
The solution was filtered through Celite and then evaporated to
dryness. The solids were triturated with hexane, filtered, and
dissolved in CH2Cl2. Evaporation of the solvent and drying
under vacuum yielded 0.242 g (1.07 mmol, 98%) of 3 as a white
solid. Mp: 148-150 °C. 1H NMR (400 MHz, CDCl3): δ 8.66 (br
s, 3H), 4.20 (br s, 2H), 3.28 (br t, J = 4.4 Hz, 2H), 3.10 (s, 3H),
1.48 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 158.8, 83.4, 69.9,
38.2, 37.2, 28.1. Anal. Calcd for C8H19ClN2O3: C, 42.38; H,
8.45; N, 12.36. Found: C, 42.15; H, 8.69; N, 12.10.
Acknowledgment. We thank Brian J. McNelis for many
helpful discussions and Joseph M. Langenhan for acquiring
NMR spectra. We are grateful to the National Institutes of
Health (R15 GM081851-01) and the Camille and Henry
Dreyfus Foundation for their support of this work.
Supporting Information Available: Synthetic procedures for
1, 2, 4, 5, 9, 13, 14, 16, and 17 and 1H and 13C NMR spectra for
1-5, 8, 9, and 13-17. This material is available free of charge via
the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 16, 2010 5759