Synthesis of a heterobifunctional PEG spacer terminated with aminooxy and bromide functionality

Article abstract of DOI:10.1055/s-2006-950246

A simple and efficient synthesis of a novel heterobifunctional polyethylene glycol (PEG) spacer is described. The PEG spacer reagent is terminated with aminooxy and bromide functionality for ease of conjugation to a variety of electrophiles and/or nucleop

Full text of DOI:10.1055/s-2006-950246

LETTER
2821
Synthesis of a Heterobifunctional PEG Spacer Terminated with Aminooxy
and Bromide Functionality
H
eterobifunction
h
P
EGSpac
r
er istopher W. Dicus, Michael H. Nantz1
Department of Chemistry, University of California Davis, Davis, CA 95616, USA
Fax +1(502)8527214; E-mail: michael.nantz@louisville.edu
Received 12 June 2006
higher gene delivery efficiencies compared to experi-
ments using unmodified PEI.8
Abstract: A simple and efficient synthesis of a novel heterobifunc-
tional polyethylene glycol (PEG) spacer is described. The PEG
spacer reagent is terminated with aminooxy and bromide function-
ality for ease of conjugation to a variety of electrophiles and/or nu-
cleophiles.
Despite the importance of heterobifunctional PEG, the
availability of PEG reagents having differentiated termi-
nal functionality is limited.9 Therefore, we pursued the
synthesis of a heterobifunctional PEG spacer that would
enable straightforward, chemoselective ligation of either
end to corresponding electrophilic or nucleophilic func-
tionality. We report herein a synthesis of PEG spacer re-
agents 1a and 1b (Figure 1). The PEG composition in
these spacer reagents differs by one ethylene oxide unit
and their use in tethering applications would provide a hy-
drophilic span of 10–13 atoms, a range deemed suitable
for molecular presentation by a previous study on PEG
linkers.10
Key words: heterobifunctional PEG, aminooxy, bioconjugation,
molecular recognition, OEG, spacer
The modification of macromolecules by the covalent at-
tachment of polyethylene glycol (PEG) often improves
their transport to target tissues and, consequently, is a use-
ful strategy in many biomedical applications. PEGylation
of exogenous peptides or proteins provides hydrophilic
bulk that reduces renal excretion, and the high mobility of
PEG chains also suppress antigenic and immunogenic
factors as well as the action of proteolytic enzymes.2 Such
single-chain, site-specific applications obviate the need
for bifunctional PEG elements. Indeed, unreactive methyl
ether groups generally terminate PEG chains opposite
their reactive end.3 A more recent area of interest exam-
ines the utility of PEG as a tether or spacer unit between a
bioactive ligand and an anchor, such as a polymer.4 In
these applications, the PEG chain serves to improve mo-
lecular recognition between the ligand and its biological
receptor by enhancing ligand presentation.5 To facilitate
the covalent attachment of PEG chains to unique function-
al groups present on either ligands or anchors, the avail-
ability of PEG reagents having chemoselectively
differentiated termini becomes necessary.
H
N
O
Br
O
O
O
O
Boc
Boc
1a
1b
H
N
O
O
Br
Figure 1 a-Aminooxy-w-bromo PEG derivatives 1a and 1b
The alkyl bromide terminus was selected for its ability to
facilitate simple alkylation of amines (e.g., as in PEI alky-
lation; path a, Figure 2), to make possible PEG dimeriza-
tion or other PEG polymerization strategies,11 and to
permit end-group modification for attachment of other
targeting functional groups. For example, chemoselective
targeting of cysteine residues could be accomplished by
conversion of the bromide to either a thiol reactive male-
imide,12 vinyl sulfone,13 or an orthopyridyl disulfide14
moiety (paths b–d, Figure 2).
Of specific interest to us is the use of PEG to tether the
gene delivery polymer polyethylenimine (PEI) to ligands
that are capable of enhancing gene transport in mammali-
an cells.6 The efficacy of this technique has been demon-
strated by a recent study wherein TAT, a membrane
translocation peptide, was attached to PEI using a PEG
spacer.7 The administration of a derived DNA·PEI-
[PEG]n-TAT complex to lung tissue in vivo showed a sig-
nificant increase in delivery efficiency when compared to
either the corresponding DNA·PEI complex or the
DNA·PEI-TAT complex. Similarly, the use of PEG to
tether PEI to an integrin-targeted peptide also resulted in
We selected the aminooxy functionality for the other ter-
minus because of its ability to rapidly condense with alde-
hydes and ketones under a variety of conditions. The use
of aminooxy groups in coupling reactions has emerged as
a powerful tool for the labeling of modified proteins,15 cell
surfaces,16 and carbohydrates.17 These condensations of-
ten proceed in near quantitative yields under aqueous or
organic conditions, providing a robust oxime ether link-
age that is stable at physiological pH. The ease of oxime
ether formation provides a superior method for targeting
aldehyde and ketones compared to a reductive-amination
approach, which is often fraught with difficulties.18
SYNLETT 2006, No. 17, pp 2821–2823
1
7
.1
0
.2
0
0
6
Advanced online publication: 09.10.2006
DOI: 10.1055/s-2006-950246; Art ID: S12706ST
© Georg Thieme Verlag Stuttgart · New York

2822
C. W. Dicus, M. H. Nantz
LETTER
tography to remove small amounts of N,N-
bis(Boc)aminoxy products. Introduction of the bromide
terminus commenced by TBAF-mediated desilylation fol-
lowed by the alcohol-to-bromide transformation using the
Appel procedure.21 Carbon tetrabromide was slowly add-
ed to a mixture of triphenylphosphine and 7a or 7b in
CH2Cl2 at 0 °C to furnish a-(Boc)aminooxy-w-bromo
compounds 1a and 1b in 71% and 82% yield, respective-
ly.22
HN
x
NH2
NH2
(OCH2CH2)n
N
HN
y
O
a
(OCH2CH2)n
(OCH2CH2)n
N
b
O
(OCH2CH2)n Br
c
A Boc-protected aminooxy moiety can be deprotected by
stirring in trifluoroacetic acid (TFA), and the resultant,
crude trifluoroacetoxy salt generally can be reacted with
an aldehyde or ketone carbonyl group to obtain the oxime
ether conjugate in a good yield.23 To demonstrate this ap-
plication using the title PEG compounds, and, more im-
portantly, to illustrate that the presence of the primary
bromide does not react with the aminooxy group as it
deprotects, we linked spacer reagent 1a to benzaldehyde
as a representative coupling reaction (Scheme 2). The
aminiumoxy trifluoroacetoxy salt 8 was isolated by sim-
ple vacuum removal of TFA after 1a was stirred in TFA
at room temperature. Dissolution of the crude salt in a so-
lution of benzaldehyde in pyridine (ca. 0.2 M) followed
by aqueous work-up after 2 hours gave oxime ether con-
jugate 9 in 90% yield.24
O
d
S
O
(OCH2CH2)n
S
S
N
Figure 2 Reactions of PEG-bromide: (a) PEI alkylation; and trans-
formations to thiol-reactive (b) maleimide, (c) vinyl sulfone, or (d) or-
thopyridyl disulfide groups.
Our synthesis of a-aminooxy-w-bromo PEG spacers 1a
and 1b is summarized in Scheme 1.19 Desymmetrization
of commercially available tri- or tetra(ethylene glycol)
(2a or 2b) was accomplished by monosilylation of the
glycol using tert-butyldiphenylsilyl chloride (BPSCl).
The monosilyl ethers 3a and 3b were easily separated
from unreacted PEG by simple aqueous washing, and
were isolated from the corresponding bissilyl ether prod-
ucts by column chromatography (SiO2). Incorporation of
the aminooxy moiety followed a literature protocol20
wherein 3a or 3b was treated with Ph3P, N-hydroxyph-
thalimide and diisopropyl azodicarboxylate (DIAD) to af-
ford phthalimido ethers 4a and 4b. Hydrazinolysis of the
phthalimide group using hydrazine monohydrate in etha-
nol proceeded smoothly to give aminooxy compounds 5a
and 5b in 88% and 86% overall yield from 3, respectively.
At this stage we felt it prudent to protect the aminooxy
moiety to prevent PEG polymerization subsequent to bro-
mide installation. Protection of compounds 5 as their tert-
butyl carbamates was effected by reaction with di-tert-bu-
tyl dicarbonate (Boc2O) and triethylamine in refluxing
CH2Cl2. Boc-protected compounds 6a and 6b were ob-
tained in 76% and 84% yields, respectively, after chroma-
a
b
CF3CO2 H3N
O
1a
O
Br
2
8
Ph
N
O
O
Br
2
9
Scheme 2 Reagents and conditions: (a) CF3CO2H, r.t., 0.5 h; (b)
PhCHO, pyridine, r.t., 2 h.
In summary, we have developed a reliable synthesis of a
heterobifunctional PEG spacer that features aminooxy
functionality at one terminus. The high reactivity of the
aminooxy moiety toward aldehydes and ketones allows
for chemoselective attachment of PEG to a wide range of
compounds. The synthetic route is amenable to other PEG
lengths and thus should find utility in bioconjugation ap-
plications.
O
a
b
c
O
O
O
N
O
n OBPS
HO
nOH
HO
n OBPS
O
2a: n = 2
2b: n = 3
3a (84%), 3b (80%)
4a (93%), 4b (91%)
H
d
f
H2N
O
O
N
O
1a (71%), 1b (82%)
n OBPS
Boc
O
n OR
5a (95%), 5b (95%)
6a (76%), 6b (84%): R = BPS
7a (94%), 7b (92%): R = H
e
Scheme 1 Reagents and conditions: (a) BPS-Cl (0.30 equiv), imidazole (2.0 equiv), DMAP (cat.), CH2Cl2, r.t., 4 h; (b) DIAD (1.1 equiv),
Ph3P (1.1 equiv), N-hydroxyphthalimide (1.1 equiv), CH2Cl2, –40 °C to r.t., 4 h; (c) H2NNH2·H2O (2.0 equiv), EtOH, r.t., 18 h; (d) Boc2O (1.0
equiv), Et3N (2.0 equiv), CH2Cl2, reflux, 18 h; (e) TBAF (1.2 equiv), THF, r.t., 3 h; (f) CBr4 (2.0 equiv), Ph3P (1.2 equiv), CH2Cl2, 0 °C, 30 min.
Synlett 2006, No. 17, 2821–2823 © Thieme Stuttgart · New York

LETTER
Heterobifunctional PEG Spacer
2823
Hz, 2 H), 1.48 (s, 9 H). 13C NMR: d = 156.7, 81.7, 75.4, 71.2,
70.5, 70.4, 69.4, 30.2, 28.2.
Acknowledgment
Compound 1b: 1H NMR: d = 7.59 (br s, 1 H), 4.03 (m, 2 H),
3.82 (t, J = 6.3 Hz, 2 H), 3.70–3.66 (m, 8 H), 3.48 (t, J = 6.3
Hz, 2 H), 1.48 (s, 9 H). 13C NMR: d = 156.7, 81.5, 75.3, 71.2,
70.6 (3 C), 70.5, 69.3, 30.2, 28.2.
This work was supported by the National Institutes of Health (NS-
046591).
References and Notes
Compound 3a: 1H NMR: d = 7.70–7.67 (m, 4 H), 7.44–7.35
(m, 6 H), 3.82 (t, J = 5.4 Hz, 2 H), 3.73–3.70 (m, 2 H), 3.66
(app. s, 4 H), 3.63 (m, 4 H), 2.17 (br s, 1 H), 1.05 (s. 9 H).
13C NMR: d = 135.6, 133.6, 129.6, 127.6, 72.5, 70.8, 70.5,
63.4, 61.8, 26.8, 19.2.
(1) Present address: Department of Chemistry, University of
Louisville, Louisville, KY 40292, USA
(2) (a) Bailon, P.; Berthold, W. Pharm. Sci. Technol. Today
1998, 352. (b) Veronese, F. M. Biomaterials 2001, 405.
(c) Pasut, G.; Veronese, F. M. Adv. Polym. Sci. 2006, 95.
(3) Dreborg, S.; Akerblom, E. B. Crit. Rev. Ther. Drug Carrier
Syst. 1990, 315.
(4) Smaller PEG units (MW <500), also referred to as
oligoethylene glycol (OEG), often are employed as spacers;
see, for example: Engel, A.; Chatterjee, S. K.; Al-Arifi, A.;
Riemann, D.; Langner, J.; Nuhn, P. Pharm. Res. 2003, 51.
(5) For recent examples, see: (a) Chen, H.; Chen, Y.;
Sheardown, H.; Brook, M. A. Biomaterials 2005, 7418.
(b) Otsuka, H.; Nagasaki, Y.; Kataoka, A. Langmuir 2004,
11285. (c) Manta, C.; Ferraz, N.; Betancor, L.; Antunes, G.;
Batista-Viera, F.; Carlsson, J.; Caldwell, K. Enzyme Microb.
Technol. 2003, 890.
(6) For recent reviews, see: (a) Kichler, A. J. Gene Med. 2004,
S3. (b) Kircheis, R.; Wightman, L.; Wagner, E. Adv. Drug
Delivery Rev. 2001, 341.
(7) Kleemann, E.; Neu, M.; Jekel, N.; Fink, L.; Schmehl, T.;
Gessler, T.; Seeger, W.; Kissel, T. J. Controlled Release
2005, 299.
(8) Kim, W. J.; Yockman, J. W.; Lee, M.; Jeong, L. H.; Kim, Y.-
H.; Kim, S. W. J. Controlled Release 2005, 224.
(9) Roberts, M. J.; Bentley, M. D.; Harris, J. M. Adv. Drug
Delivery Rev. 2002, 459.
(10) Engel, A.; Chatterjee, S. K.; Al-Arifi, A.; Nuhn, P. J. Pharm.
Sci. 2003, 2229.
(11) Loiseau, F. A.; Hii, K. K.; Hill, A. M. J. Org. Chem. 2004,
639.
(12) Goodson, R. J.; Katre, N. V. Biotechnology (N.Y.) 1990, 8,
343.
Compound 3b: 1H NMR: d = 7.70–7.67 (m, 4 H), 7.44–7.35
(m, 6 H), 3.82 (t, J = 5.4 Hz, 2 H), 3.73–3.70 (m, 2 H), 3.66–
3.58 (m, 12 H), 2.25 (br s, 1 H), 1.05 (s, 9 H). 13C NMR:
d = 135.6, 133.6, 129.6, 127.6, 72.5, 72.4, 70.7, 70.7, 70.4,
63.4, 61.8, 26.8, 19.2.
Compound 4a: 1H NMR: d = 7.83–7.80 (m, 2 H), 7.72–7.70
(m, 2 H), 7.70–7.65 (m, 4 H), 7.44–7.35 (m, 6 H), 4.36 (m,
2 H), 3.86 (m, 2 H), 3.74 (t, J = 5.4 Hz, 2 H), 3.65–3.62 (m,
2 H), 3.58–3.56 (m, 2 H), 3.52 (t, J = 5.4 Hz, 2 H), 1.03 (s, 9
H). 13C NMR: d = 163.4, 135.6, 134.4, 133.7, 129.6, 129.0,
127.6, 123.4, 77.2, 72.4, 70.8, 70.7, 69.4, 63.3, 26.8, 19.2.
Compound 4b: 1H NMR: d = 7.84–7.81 (m, 2 H), 7.74–7.71
(m, 2 H), 7.70–7.66 (m, 4 H), 7.44–7.34 (m, 6 H), 4.36 (m,
2 H), 3.85 (m, 2 H), 3.79 (t, J = 5.4 Hz, 2 H), 3.67–3.63 (m,
2 H), 3.61–3.52 (m, 8 H), 1.04 (s, 9 H). 13C NMR: d = 163.4,
135.6, 134.4, 133.7, 129.6, 129.0, 127.6, 77.2, 72.4, 70.8,
70.7, 70.6, 70.5, 69.3, 63.4, 26.8, 19.2.
Compound 5a: 1H NMR: d = 7.70–7.67 (m, 4 H), 7.44–7.35
(m, 6 H), 3.84 (m, 2 H), 3.81 (t, J = 5.4 Hz, 2 H), 3.69–3.59
(m, 8 H), 1.05 (s, 9 H). 13C NMR: d = 135.6, 133.6, 129.6,
127.6, 74.7, 72.4, 70.7, 70.6, 69.7, 63.4, 26.8, 19.2.
Compound 5b: 1H NMR: d = 7.70–7.67 (m, 4 H), 7.44–7.35
(m, 6 H), 3.85 (m, 2 H), 3.81 (t, J = 5.4 Hz, 2 H), 3.69–3.63
(m, 10 H), 3.60 (t, J = 5.4 Hz, 2 H), 1.05 (s, 9 H). 13C NMR:
d = 135.6, 133.6, 129.6, 127.6, 74.6, 72.4, 70.7, 70.6, 70.5,
70.5, 69.7, 63.4, 26.8, 19.2.
Compound 6a: 1H NMR: d = 7.70–7.67 (m, 4 H), 7.51 (br s,
1 H), 7.44–7.34 (m, 6 H), 4.01 (m, 2 H), 3.82 (t, J = 5.4 Hz,
2 H), 3.72 (m, 2 H), 3.68–3.62 (m, 4 H), 3.61 (t, J = 5.4 Hz,
2 Hz), 1.46 (s, 9 H), 1.05 (s, 9 H). 13C NMR: d = 156.7,
135.6, 133.6, 129.6, 127.6, 81.5, 75.3, 72.4, 70.7, 70.6, 69.4,
63.4, 28.2, 26.8, 19.2.
(13) Morpurgo, M.; Veronese, F. M.; Kachensky, D.; Harris, J.
M. Bioconjugate Chem. 1996, 7, 363.
(14) Woghiren, C.; Sharma, B.; Stein, S. Bioconjugate Chem.
Compound 6b: 1H NMR: d = 7.70–7.67 (m, 4 H), 7.62 (br s,
1 H), 7.44–7.34 (m, 6 H), 4.01 (m, 2 H), 3.81 (t, J = 5.4 Hz,
2 H), 3.70 (m, 2 H), 3.67–3.62 (m, 8 H), 3.60 (t, J = 5.4 Hz,
2 H), 1.46 (s, 9 H), 1.05 (s, 9 H). 13C NMR: d = 156.7, 135.6,
133.6, 129.6, 127.6, 81.4, 75.3, 72.3, 70.7, 70.6, 70.5, 70.5,
69.2, 63.4, 28.2, 26.8, 19.1.
1993, 4, 314.
(15) Tumelty, D.; Carnevali, M.; Miranda, L. P. J. Am. Chem.
Soc. 2003, 14238.
(16) (a) Lemieux, G. A.; Yarema, K. J.; Jacobs, C. L.; Bertozzi,
C. R. J. Am. Chem. Soc. 1999, 4278. (b) Sadamoto, R.;
Niikura, K.; Ueda, T.; Monde, K.; Fukuhara, N.; Nishimura,
S.-I. J. Am. Chem. Soc. 2004, 3755.
Compound 7a: 1H NMR: d = 7.82 (br s, 1 H), 4.03 (m, 2 H),
3.76–3.72 (m, 4 H), 3.69 (s, 4 H), 3.64–3.61 (m, 4 H), 2.84
(br s, 1 H), 1.48 (s, 9 H). 13C NMR: d = 156.8, 81.6, 75.2,
72.6, 70.4, 70.2, 69.2, 61.6, 28.2.
(17) Perouzel, E.; Jorgensen, M. R.; Keller, M.; Miller, A. D.
Bioconjugate Chem. 2003, 14, 884.
(18) Bertozzi, C. R.; Bednarski, M. D. J. Org. Chem. 1991, 4326.
(19) For moisture-sensitive reactions, optimized yields were
obtained only after drying the PEG intermediate by toluene
azeotropic distillation immediately prior to use.
(20) (a) Grochowski, E.; Jurczak, J. Synthesis 1976, 682.
(b) Nicolaou, K. C.; Groneberg, R. D. J. Am. Chem. Soc.
1990, 4085. (c) Su, S.; Giguere, J. R.; Schaus, S. E. Jr.;
Porco, J. A. Jr. Tetrahedron 2004, 8645.
(21) Appel, R. Angew. Chem., Int. Ed. Engl. 1975, 14, 801.
(22) 1H NMR and 13C NMR data (300 MHz and 75 MHz,
respectively; in CDCl3) for all compounds: Compound 1a:
1H NMR: d = 7.23 (br s, 1 H), 4.03 (m, 2 H), 3.82 (t, J = 6.3
Hz, 2 H), 3.74 (m, 2 H), 3.69 (app. s, 4 H), 3.48 (t, J = 6.3
Compound 7b: 1H NMR: d = 8.08 (br s, 1 H), 4.02 (m, 2 H),
3.74–3.66 (m, 12 H), 3.63 (m, 2 H), 2.80 (br s, 1 H), 1.48 (s,
9 H). 13C NMR: d = 156.9, 81.4, 75.1, 72.6, 70.6, 70.4, 70.4,
70.3, 69.0, 61.7, 28.2.
(23) Gaertner, H. F.; Offord, R. E. Bioconjugate Chem. 1996, 7,
38.
(24) Compound 9 was obtained as a single isomer: 1H NMR (600
MHz, CDCl3): d = 8.13 (s, 1 H), 7.58–7.57 (m, 2 H), 7.38–
7.36 (m, 3 H), 4.34 (t, J = 4.6 Hz, 2 H), 3.83–3.81 (m, 4 H),
3.70 (app. s, 4 H), 3.46 (t, J = 6.0 Hz, 2 H). 13C NMR (75
MHz, CDCl3): d = 149.0, 132.2, 129.8, 128.7, 127.0, 73.5,
71.2, 70.7, 70.5, 69.7, 30.3.
Synlett 2006, No. 17, 2821–2823 © Thieme Stuttgart · New York