Application of a water-soluble pyridyl disulfide amine linker for use in Cu-free click bioconjugation

Article abstract of DOI:10.1016/j.tetlet.2011.06.042

Described herein is the design and synthesis of a discrete heterobifunctional PEG-based pyridyl disulfide/amine-containing linker that can be used in the Cu-free click preparation of bioconjugates. The title PEG-based pyridyl disulfide amine linker is a potentially useful reagent for preparing water-soluble disulfide-linked cargos, and that it may be particularly valuable in expanding the field of Cu-free click-based bioconjugations to include reductively labile antibody, polymer, or nanoparticle-based drug conjugates.

Full text of DOI:10.1016/j.tetlet.2011.06.042

Tetrahedron Letters 52 (2011) 4316–4319
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Application of a water-soluble pyridyl disulfide amine linker
for use in Cu-free click bioconjugation
⇑
Joshua D. Thomas , Terrence R. Burke Jr.
Chemical Biology Laboratory, Molecular Discovery Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Described herein is the design and synthesis of a discrete heterobifunctional PEG-based pyridyl disulfide/
amine-containing linker that can be used in the Cu-free click preparation of bioconjugates. The title
PEG-based pyridyl disulfide amine linker is a potentially useful reagent for preparing water-soluble disul-
fide-linked cargos, and that it may be particularly valuable in expanding the field of Cu-free click-based
bioconjugations to include reductively labile antibody, polymer, or nanoparticle-based drug conjugates.
Published by Elsevier Ltd.
Received 27 May 2011
Accepted 9 June 2011
Available online 17 June 2011
Keywords:
Bioconjugation
Cleavable linker
Cu-free click reaction
Pyridyl disulfide
In order to develop proteins that merge properties of antibodies
with biologically active small molecules,1,2 we have recently begun
modifying selenocysteine (Sec) antibody constructs (Fc-Sec) by
introducing click-compatible functionality.3 This potentially allows
the click-attachment of cytotoxic drugs, dyes, or other cargo to the
Fc portion. Clickable antibody conjugates may require biologically-
cleavable linkers that allow release of cargo once delivery to the
target has been achieved. However, traditional Huisgen azide–al-
kyne cycloaddition click reactions employing Cu-catalysis can be
incompatible with sensitive protein functionality, and this is one
reason why Cu-free click chemistries are gaining greater preva-
lence.4 Accordingly, versatile hetero-bifunctional linkers are
needed that are both compatible with multiple types of Cu-free
click reagents and incorporate biologically cleavable bonds.
One approach to introducing a point of bio-cleavage between
cargo and carrier is the inclusion of a disulfide bond within the
linking component.5–11 Disulfides are synthetically straight-for-
ward to manipulate, and although they are stable under a variety
of conditions, they can be efficiently cleaved by reducing agents.
Disulfides exhibit good stability in the circulation due to the low
reducing potential of blood. In contrast, intracellular concentra-
tions of reducing agents, such as glutathione, are typically 1000-
fold greater than in the blood,12 with the reductive potential within
cancer cells being even higher.13,14 For these reasons, disulfide-
containing constructs can afford effective vehicles for delivery to
the cellular targets, and subsequent reductive release of cargo.
Currently available Cu-free click reagents commonly exhibit
considerable hydrophobic character.4 Since cytotoxic drugs also
tend to be highly hydrophobic, overcoming poor water solubility
can be an important consideration in designing a Cu-free clickable
linker for use in Fc-Sec conjugates. The commercially available 2-
(pyridyldithio)-ethylamine (PDA)15 is a reagent used for the intro-
duction of short disulfide-containing linkages. Unfortunately, con-
jugates based on
a PDA linker could potentially exhibit
unacceptably low solubility in aqueous media. Alternatively, poly-
ethylene glycol (PEG) is a highly water-soluble construct that is
used in a range of hetero-bifunctional linkers. However, none of
the commercially-available PEG-based reagents contain the combi-
nation of an activated disulfide (e.g., pyridyl disulfide) together
with amine functionality that we desired in our current work.
Additionally, previously reported PEG-based pyridyl disulfide/
amine-containing linkers have been prepared by polymerization
reactions that yield heterogenous reactions products.16 Herein
we describe the design and synthesis of a discrete heterobifunc-
tional PEG-based pyridyl disulfide/amine-containing linker (1) that
can be used in the Cu-free click preparation of bioconjugates either
by initial coupling of a bioactive molecule through disulfide forma-
tion (Route A, Scheme 1) or by initial introduction of the click re-
agent by amide bond formation (Route B, Scheme 1).
Discrete, highly pure PEG linkers that range in length from 817
to 4818 ethylene glycol units can be prepared from tetraethylene
glycol precursors. Our route to 1 began by the monobenzylation
of tetraethylene glycol (Scheme 2). Subsequent tosylation of 2
and chain elongation with excess tetraethylene glycol gave 3 in
modest yield. Conversion of the primary alcohol to the mesylate
and reaction with potassium phthalimide provided the protected
amine 4, which was debenzylated, brominated, and then reacted
⇑
Corresponding author. Tel.: +1 340 846 5906; fax: +1 340 846 6033.
E-mail address: tburke@helix.nih.gov (T.R. Burke).
Current address: Mersana Therapeutics, Cambridge, MA 02139, USA.
0040-4039/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2011.06.042

J. D. Thomas, T. R. Burke Jr. / Tetrahedron Letters 52 (2011) 4316–4319
4317
Route A
Route B
HO2C
S
NH2
S
O
N
7
SH
S
1
S
NH2
H
N
O
S
7
S
O
N
7
O
SH
HO2C
H
N
S
O
S
7
O
Bioconjugate
= Bioactive Molecule
= Click Reagent
Scheme 1. Structure of PEG pyridyl disulfide amine linker 1 and its use in the construction of bioconjugates by two routes.
O
a
c
HO
OH
BnO
b
OH
BnO
N
O
O
3
n
O
7
O
2 n = 3
3 n = 7
4
O
O
f
d
g
S
X
N
N
S
O
O
N
7
7
O
O
7
5 X = Br
e
6 X = TrtS
h
1
S
S
NHMtt
O
N
7
8
Scheme 2. Reagents and conditions: (a) BnBr, NaH, THF (74%); (b) TsCl, Et3N, DMAP, CH2Cl2 (93%); (c) (i) MsCl, Et3N, CH2Cl2; (ii) potassium phthalimide, DMF (60% over two
steps); (d) (i) PdÁC, H2, EtOH (78%); (ii) Ph3P, CBr4, CH2Cl2 (70%); (e) TrtSH, NaH, DMF (88%); (f) (i) TFA/CH2Cl2/(i-Pr)3SH; (ii) 2-mercaptopyridine, SOCl2, CH2Cl2, (51% over two
steps); (g) NH2NH2, (71%); (h) MttCl, DIEA, DMF (40%).
with triphenylmethanethiol to yield the doubly protected amino
thiol 6. An activated disulfide was introduced in two steps by
removing the S-trityl group in 6 and then reacting the free thiol
with pyridylsulfenyl chloride to afford 7 (Scheme 2).
performing the reaction at ambient temperature decreased
side reactions and resulted in an improved yield (71%). To
assure stability during storage, the free amine group in 1 was
protected with the acid-labile 4-methyltrityl (Mtt) group (com-
pound 8 Scheme 2). The mild conditions required to remove the
Mtt group make it compatible with a broad range of disulfide-
linked cargo.
To demonstrate the utility of 8, a cytotoxic derivative of
cemadotin22 was appended to one end of the linker via a disulfide
bond, and a tetrazine construct (9)23 was introduced to the other
end. The tetrazine serves as a diene that undergoes irreversible
Cu-free click reactions through very efficient [2+4] inverse-elec-
tron-demand Diels–Alder cycloadditions with strained alkenes.4
In order to avoid potential side reactions with free thiols,24 9 was
introduced following disulfide exchange with the thiol-containing
bioactive cemadotin analog 1025 (Scheme 3). Cemadotin deriva-
Given the tendency of unsymmetrical disulfides to undergo
base and temperature-induced degradation,19 cleavage of the
phthalimide group in 7 to yield the free amine 1 could be problem-
atic with commonly used conditions (hydrazine in refluxing
ethanol).20 However, the stability of a 2-pyridyldithio-based amino
protecting group under piperidine-mediated Fmoc-deblock condi-
tions,21 indicated that the 2-pyridyl group in 7 could potentially
stabilize the disulfide bond during treatment with hydrazine.
None-the-less, our initial efforts to remove the phthalimide group
in 7 using standard hydrazine conditions gave significant disulfide
disproportionation (as determined by ESI-MS) that resulted in a
relatively low yield of the desired product 1 (48%). However,

4318
J. D. Thomas, T. R. Burke Jr. / Tetrahedron Letters 52 (2011) 4316–4319
O
H
N
H
N
N
N
N
N
O
O
O
O
SH
10
a
O
H
N
H
N
N
N
N
N
S
O
O
NH2
O
O
S
O
7
11
N
N
b
O
N
OH
N
N
N
N
N
9
O
O
H
N
H
N
N
N
N
N
S
O
O
O
O
O
O
O
S
O
O
N
H
N
N
O
N
O
N
N
O
N
N
N
O
12
Scheme 3. Reagents and conditions: (a) (i) 8, MeOH (74%); (ii) TFA/CH2Cl2/(i-Pr)3SH (39% for two steps); (b) HOAt, DIC, DIEA, DMF (31%).
S
S
a
H
N
H
N
S
NH2
HN
HN
S
O
STrt
7
NH
NH
O
O
O
O
14
13
c
S
b
H
N
H
S
N
HN
O
N3
S
7
NH
HN
O
O
O
16
O
N
O
S
H
N
N
H
N
N
N
S
N
S
O
7
N
N
NH
O
O
N
15
Scheme 4. Reagents and conditions: a) (i) TFA/CH2Cl2/(i-Pr)3SH; (ii) 8, MeOH (74%); (iii) TFA/CH2Cl2/(i-Pr)3SH; (b) 9, HOAt, DIEA, DMSO (15% overall from 13); (c) 1-
[(azidoacetyl)oxy]pyrrolidine-2,5-dione, MeCN/H2O (6% overall from 13).

J. D. Thomas, T. R. Burke Jr. / Tetrahedron Letters 52 (2011) 4316–4319
4319
tives represent attractive drug cargos for validating the synthetic
Supplementary data
strategy outlined in Scheme 3, because they contain N-alkylated
tripeptide sequences that are prone to acid-catalyzed degrada-
tion.26 Disulfide exchange between 8 and 10 and evaporation of
solvent gave a crude reaction mixture, which was subjected to
Mtt removal (1% TFA in CH2Cl2) to provide 11 in modest yield
(Scheme 3). Importantly, no degradation of 10 was observed
during under these conditions. Coupling of 11 to 9 (HOAt, diisopro-
pylcarbodiimide and diisopropylethylamine in DMF) gave the fully
elaborated construct 12.
Supplementary data (experimental procedures and spectro-
scopic data for all new compounds) associated with this article
can be found, in the online version, at doi:10.1016/j.tetlet.
2011.06.042.
References and notes
1. Hofer, T.; Thomas, J. D.; Burke, T. R., Jr.; Rader, C. Proc. Natl. Acad. Sci. U.S.A. 2008,
105, 12451.
Linker 8 was also used to prepare clickable biotin-containing
derivatives 15 and 16 (Scheme 4). Biotin-(STrt)cystamine 13 was
converted in three steps to disulfide-containing amine 14 (Scheme
4). HATU-mediated coupling with 9 yielded biotin-containing
tetrazine 15, while reaction of 14 with OSu-activated azido acetic
acid yielded the corresponding azide 16. Since classical click
reactions3 require a reducing agent in order to maintain copper
in the +1 oxidation state, disulfides such as 16 are potentially
better suited for Cu-free reactions that utilize highly strained
cyclooctynes.4
In conclusion, we have shown that the PEG-based pyridyl
disulfide amine linker 1 is a useful reagent for preparing water-sol-
uble disulfide-linked cargos. It may be particularly valuable in
expanding the field of Cu-free click-based bioconjugations, where
applications have traditionally been related to imaging agents.
Reagents such as 1 may extend the use of Cu-free click reagents
to include reductively labile antibody, polymer, or nanoparticle-
based drug conjugates.
2. Hofer, T.; Skeffington, L. R.; Chapman, C. M.; Rader, C. Biochemistry 2009, 48,
12047.
3. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004.
4. Jewett, J. C.; Bertozzi, C. R. Chem. Soc. Rev. 2010, 39, 1272.
5. Saito, G.; Swanson, J. A.; Lee, K.-D. Adv. Drug Delivery Rev. 2003, 55, 199.
6. Ojima, I. Acc. Chem. Res. 2008, 41, 108.
7. Carter, P. J.; Senter, P. D. Cancer J. 2008, 14, 154.
8. Low, P. S.; Kularatne, S. A. Curr. Opin. Chem. Biol. 2009, 13, 256.
9. van der Vlies, A. J.; O’Neil, C. P.; Hasegawa, U.; Hammond, N.; Hubbell, J. A.
Bioconjugate Chem. 2010, 21, 653.
10. Leamon, C. P.; Low, P. S. J. Biol. Chem. 1992, 267, 24966.
11. Zhu, L.; Mahato, R. I. Bioconjugate Chem. 2010, 21, 2119.
12. Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52, 711.
13. Howie, A. F.; Forrester, L. M.; Glancey, M. J.; Schlager, J. J.; Powis, G.; Beckett, G.
J.; Hayes, J. D.; Wolf, C. R. Carcinogenesis 1990, 11, 451.
14. Balendiran, G. K.; Dabur, R.; Fraser, D. Cell Biochem. Funct. 2004, 22, 343.
15. Zugates, G. T.; Anderson, D. G.; Little, S. R.; Lawhorn, I. E. B.; Langer, R. J. Am.
Chem. Soc. 2006, 128, 12726.
16. Breitenkamp, K.; Sill, Kevin, N.; Skaff, H. In Patent Cooperation Treaty (PCT);
Organization, Publication WO 2007/127473 A2, November 8, 2007.
17. Pilkington-Miksa, M. A.; Sarkar, S.; Writer, M. J.; Barker, S. E.; Shamlou, P. A.;
Hart, S. L.; Hailes, H. C.; Tabor, A. B. Eur. J. Org. Chem. 2008, 2900.
18. French, A. C.; Thompson, A. L.; Davis, B. G. Angew. Chem. Int. Ed. 2009, 48, 1248.
19. Field, L.; Parsons, T. F.; Pearson, D. E. J. Org. Chem. 1966, 31, 3550.
20. Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in Organic Synthesis, 4th
ed.; Wiley-Interscience: Hoboken, NJ 07030-5774, 2007.
Acknowledgments
21. Lapeyre, M.; Leprince, J. M. M.; Oulyadi, H.; Renard, P. Y.; Romieu, A.; Turcatti,
G.; Vaudry, H. Chem. Eur. J. 2006, 12, 3655.
22. de Arruda, M.; Cocchiaro, C. A.; Nelson, C. M.; Grinnell, C. M.; Janssen, B.; Haupt,
A.; Barlozzari, T. Cancer Res. 1995, 55, 3085.
23. Pipkorn, R.; Waldeck, W.; Didinger, B.; Koch, M.; Mueller, G.; Wiessler, M.;
Braun, K. J. Pept. Sci. 2009, 15, 235.
24. Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008, 130, 13518.
25. Thomas Joshua, D.; Rader, C.; Burke, T. R., Jr. Unpublished results 2011.
26. Anteunis, M. J. O.; van der Auwera, C. Int. J. Pept. Protein Res. 1988, 31, 301.
This work was supported in part by the Intramural Research
Program of the NIH, Center for Cancer Research, NCI-Frederick
and the National Cancer Institute, National Institutes of Health.
The content of this publication does not necessarily reflect the
views or policies of the Department of Health and Human Services,
nor does mention of trade names, commercial products, or organi-
zations imply endorsement by the US Government.