Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition

Article abstract of DOI:10.1039/b917797c

A strained aza-dibenzocyclooctyne was prepared via a high-yielding synthetic route. Copper-free, strain-promoted click reaction with azides showed excellent kinetics, and a functionalised aza-cyclooctyne was applied in fast and efficient PEGylation of enzymes.

Full text of DOI:10.1039/b917797c

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Aza-dibenzocyclooctynes for fast and efficient enzyme
PEGylation via copper-free (3+2) cycloadditionw
Marjoke F. Debets,z Sander S. van Berkel,z Sanne Schoffelen, Floris P. J. T. Rutjes,
Jan C. M. van Hest* and Floris L. van Delft*
Received (in Cambridge, UK) 1st September 2009, Accepted 22nd October 2009
First published as an Advance Article on the web 6th November 2009
DOI: 10.1039/b917797c
A
strained aza-dibenzocyclooctyne was prepared via
a
the best of our knowledge, these systems have not yet been
employed in the PEGylation of proteins.
high-yielding synthetic route. Copper-free, strain-promoted
click reaction with azides showed excellent kinetics, and a
functionalised aza-cyclooctyne was applied in fast and efficient
PEGylation of enzymes.
Inspired by the dibenzocyclooctyne derivative (DIBC,
structure D in Fig. 1) developed by Boons et al.13 and
aza-dimethoxycyclooctyne (DIMAC, structure C in Fig. 1)
synthesised by Bertozzi et al.,17 we set out to develop the
hybrid structure aza-dibenzocyclooctyne (DIBAC, structure E
in Fig. 1). The aza-dibenzocyclooctyne was designed to
combine the favourable kinetics of DIBC (D)13 with the
increased hydrophilicity of DIMAC (C).17 With respect to
the latter, the nitrogen atom in our designed analogue should
allow straightforward functionalisation of the aniline moiety
and modification of the system, e.g. by sulfonation. At the
same time, we set ourselves the goal to develop a straight-
forward, high-yielding and fast synthetic route. Herein we
report the facile synthesis of aza-dibenzocyclooctyne and deri-
vatives thereof for the quantitative PEGylation of proteins.
The synthetic route towards key-intermediate dihydro-
dibenzo-azocine (5) is shown in Scheme 1, and is based on a
Sonogashira cross-coupling reaction and a reductive amina-
tion ring-closing step. Whereas the Sonogashira reaction
under standard inert atmosphere initially resulted in Glaser
coupling, performing the reaction under an N2/H2-atmosphere18
effectively nihilated Glaser coupling and led to the
Sonogashira coupling product 1 in quantitative yield. Next,
Boc-protection, generating compound 2, was found to be
essential, in order to make partial hydrogenation of the
acetylene to the Z-alkene possible, yielding 3 (95%). Next,
Dess–Martin oxidation led to aldehyde 4, the precursor for the
reductive amination. Boc-deprotection under acidic conditions
resulted in immediate and exclusive formation of a cyclic
imine, which was not isolated but subjected to NaBH4
reduction, generating the free secondary amine (5) in
quantitative yield over the two steps. Via this high-yielding
The process of PEG conjugation to either peptides or proteins,
known as PEGylation, is a procedure of growing interest in
both biotechnology and therapeutical science.1 PEGylation of
proteins improves their in vivo applicability by reducing
aggregation, shielding critical protein binding sites and
improving the water solubility.2 As a result, a large variety
of methods for the conjugation of PEG to either peptides or
proteins is currently available.3 In most cases, however, a large
excess of PEG and prolonged reaction times are required, and
nevertheless generally result in suboptimal conversions.4 With
the recent discovery of the Cu(I)-catalysed Huisgen cyclo-
addition reaction,5 a powerful new methodology was added
to the field of bioconjugation6 with the particular benefits
that the reaction is selective, fast and high-yielding. The
Cu(I)-catalysed 1,3-dipolar cycloaddition reaction has become
of great use in labelling studies, the development of new
therapeutics and in protein modification.7 In addition, the
Cu(I)-catalysed 1,3-dipolar cycloaddition reaction has also
been applied in the synthesis of PEG–protein bioconjugates.8
A disadvantage of this reaction in PEGylation is the potential
presence of residual copper in the product. Not only is Cu(I)
toxic to cells, it can also bind to the active site of enzymes,
thereby blocking or reducing biological activity.9,10 Further-
more, Cu(I) is easily disproportionated in an aqueous environ-
ment, thus reducing the rate of the reaction. Therefore, interest
is growing in methods involving Cu-free 1,3-dipolar cyclo-
addition reactions.11 Several strain-promoted systems, such as
oxanorbornadienes,12 cyclooctynes,10 and dibenzocyclooctynes13
(Fig. 1) have recently been developed for the fast and selective
reaction with azide-containing biomolecules and have found
application in e.g. tumour imaging,14 glycan labelling,10,13
in vivo imaging,15 and surface modification.16 However, to
Institute for Molecules and Materials, Radboud University Nijmegen,
Heyendaalseweg 135, NL-6525 AJ Nijmegen, The Netherlands.
E-mail: J.vanHest@science.ru.nl, F.vanDelft@science.ru.nl;
Fax: (+31) 24 3653393;
Tel: (+31) 24 3653204; (+31) 24 3652373
w Electronic supplementary information (ESI) available: Synthetic
procedures for compounds 1–16, kinetic experiments and plots,
enzyme functionalisation. See DOI: 10.1039/b917797c
z These authors contributed equally to this work.
Fig. 1 Strain-promoted systems for Cu-free click reactions.
Chem. Commun., 2010, 46, 97–99 | 97
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Table 1 Rate constants of the different cyclooctyne systems
Entry
Compound
Solvent
k (Mꢁ1 sꢁ1
)
1
2
3
DIBAC (10)
DIBAC (12)
DIBAC (12)
CD3OD
CD3OD
D2O
0.29 ꢃ 0.01
0.31 ꢃ 0.02
0.36a
a
Reaction was performed under basic conditions with 1.1 equiv. of 12.
respectively) with benzyl azide were determined in CD3OD,
giving rate constants of 0.29 Mꢁ1 sꢁ1 and 0.31 Mꢁ1 sꢁ1
,
respectively. Cycloaddition of DIBAC 12 and 2-azidopropanoic
acid was also investigated by performing the reaction in basic
D2O, since addition of a small amount of 2 M NaOH was
necessary to dissolve 12. In D2O cycloaddition was calculated
to proceed with a rate constant of 0.36 Mꢁ1 sꢁ1, slightly faster
than in CD3OD. The results of the kinetic experiments are
summarised in Table 1. Much to our satisfaction, the kinetic
parameters of our new system proved slightly better than
DIBC (k = 0.17 Mꢁ1 sꢁ1 13 and DIFO (k = 0.076 Mꢁ1 sꢁ1 10
)
)
Scheme 1 Reagents and conditions: (a) PdCl2(PPh3)2, CuI, Et3N,
THF, N2/H2-atmosphere, r.t., 4 h (99%); (b) Boc2O, THF, 70 1C,
2 d (83%); (c) 10% Pd/BaSO4, quinoline, H2, MeOH, r.t., 1.5 h (95%);
(d) Dess–Martin periodinane, NaHCO3, CH2Cl2, r.t., 40 min (90%);
(e) (1) 2 M HCl in EtOAc, r.t., 1 h; (2) NaBH4, H2O, r.t., o.n. (100%);
(f) CbzCl, Na2CO3, H2O, CH2Cl2, r.t., 3 h (86%); (g) ClCOC3H6-
CO2Me, Et3N, CH2Cl2, r.t., 1.5 h (94%); (h) Br2, CH2Cl2, 0 1C, 2 h
(8: 67%, 9: 81%); (i) KOtBu, THF, 0 1C - r.t., o.n. (10), ꢁ40 1C, 2 h
(11) (10: 87%, 11: 84%); (j) LiOH, THF, H2O, r.t., 3 h (92%).
and it reacted approximately 100-fold faster than the
hydrophilic DIMAC-system (k = 3ꢂ10ꢁ3 Mꢁ1
s
ꢁ1).17
To investigate the applicability of the Cu-free 1,3-dipolar
cycloaddition in the conjugation of polyethylene glycol to
proteins, the aza-dibenzocyclooctyne analogue 12 was
functionalised with H2N-PEG2000-OMe via an EDC-coupling,
yielding DIBAC-mPEG2000 (15, Scheme 2). For comparison,
DIBC-mPEG2000 (16) was also prepared via conversion of the
alcohol into a 4-nitrophenyl carbonate13 followed by substitu-
tion with H2N-PEG2000-OMe. Initially, azide-containing CalB
(AHA-CalB)19 was used for the conjugation studies. This
modified enzyme, obtained by recombinant expression in
auxotrophic E. coli, contains five azidohomoalanine residues,
four of which are concealed inside the protein. Consequently,
only one azido residue is exposed on the exterior of the
enzyme, readily available for ligation. We have previously
reported that this enzyme underwent PEGylation selectively
with the most accessible residue by applying the Cu(I)-catalysed
(3+2) cycloaddition reaction. However, full conversion could
never be reached, despite the use of 20 equivalents of
acetylene-functionalised acetylene-PEG5000 and stirring for
1-3 days.19 Now, PEGylation of AHA-CalB was pursued by
mixing AHA-CalB (1 mg/mL, corresponding to B30 mM) with
DIBAC-mPEG2000 15 or DIBC-mPEG2000 16 (one, two and
five equiv). After three hours the reaction was quenched with
benzyl azide and analysed by SDS-PAGE (Fig. 2). As becomes
clear, with five equivalents of DIBAC-mPEG2000 (15) full
conversion was observed within three hours (Fig. 2, lane 2).
Interestingly, not only was the enzyme fully functionalised, but
it appears that one of the less accessible azides also reacted,
generating di-PEGylated CalB, confirming the excellent
reactivity of aza-dibenzocyclooctyne towards azides. The
higher reactivity of the DIBAC system is further demonstrated
by the fact that a higher degree of PEGylation is observed than
with DIBC-mPEG2000. In both cases, the small amount of
‘unreacted’ CalB observed is not caused by failure to react, but
can be ascribed to the fact that AHA-CalB always contains a
small amount of non-modified enzyme.19 Performing the
PEGylation with either one or two equivalents of DIBAC
15, approximately 50% conversion to the single PEGylated
protocol the key aza-cyclooctene intermediate 5 was synthesised
in an excellent yield of 70% over five steps.
Unfortunately, direct bromination of dihydrodibenzo-
azocine 5 resulted in the intramolecular substitution of a
bromide, leading to an indoline, thus requiring protection of
the amine. Consequently,
a Cbz-group was introduced
generating compound 6 in good yield (86%). The alkene
moiety could then be smoothly converted into an alkyne via
successive bromination (67%) and elimination (87%).
Elimination was performed using KOtBu since other bases
such as LDA, n-BuLi, NaOH (6 or 12 M) all failed to give the
desired aza-dibenzocyclooctyne 10. Compound 10, although
fulfilling our aim to prepare an aza-cyclooctyne, is clearly
lacking a handle for further functionalisation. Unfortunately,
deprotection of the Cbz-group, using either acidic or basic
conditions, did not yield the free amine (i.e. E, R = H, Fig. 1).
Instead, 6H-isoindolo[2,1-a]indole (13) was formed, presumably
via an 5-endo-dig cyclisation, thereby relieving ring-strain with
formation of the indole. Consequently, N-functionalisation
was required prior to alkyne formation. To this end, 5
was equipped with a small functionalised linker, leading to
compound 7 in 94% yield (Scheme 1). Subsequent bromina-
tion (81%) and elimination with KOtBu (84%) cleanly
resulted in alkyne 11. A 1 M solution of KOtBu in THF was
used since the use of solid KOtBu resulted in hydrolysis of the
methyl ester, which gave rise to undesired side-reactions.
Finally, a functionalizable probe (12) was prepared via hydrolysis
of the methyl ester. The above reported reaction sequence
yielded the desired strained aza-cyclooctyne probe, with a
functional handle, in a good overall yield of 41% over 9 steps.
Next, the kinetics in the 1,3-dipolar cycloaddition of the
Cbz-protected and acid-functionalised DIBACs (10 and 12,
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was underlined by the effective PEGylation of enzymes.
The functionalisation, further derivatisation and application
of aza-dibenzocyclooctynes in bioconjugation are topics
currently under investigation in our laboratories.
Notes and references
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Scheme 2 Reagents and conditions: (a) H2N-PEG2000-OMe, EDC,
DMAP, CH2Cl2, 2 d, r.t.; (b) (1) (4-NO2C6H4)OCOCl, pyridine,
CH2Cl2,
4 h, r.t.; (2) H2N-PEG2000-OMe, CH2Cl2, 1 d, r.t.;
(c) PBS-buffer (pH 8.5, 30 mM), 3 h, r.t.
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product was observed (see lanes 3 and 4). Using DIBC 16
under the same conditions resulted in somewhat lower
conversions (see lanes 6 and 7).
The fast and quantitative ligation of both reagents to
AHA-CalB shows the high potential of these systems. To
investigate the reactivity and efficiency of these reagents
towards other enzymes, HRP was modified via diazotransfer
as recently developed in our group.20 The resulting HRP-N3
was then reacted with either 15 or 16 following the same
procedure as applied to AHA-CalB, giving almost identical
results (See ESI, Fig. S1w). The aza-dibenzocyclooctyne is
in both cases more reactive than the dibenzocyclooctyne,
especially when only one or two equivalents were used.
Nevertheless, in comparison to the Cu(I)-catalysed cyclo-
addition reaction, both DIBCs show fast and efficient
modification of both enzymes, clearly showing the power
and applicability of these copper-free systems.
In conclusion, we have developed a new, highly efficient
reagent for copper-free (3+2) cycloaddition reactions. The
aza-dibenzocyclooctyne is easily accessible via a versatile and
high-yielding synthetic route, and allows straightforward
functionalisation via the nitrogen atom. In addition, the
outstanding reaction rate constant (k = 0.3 Mꢁ1 ꢁ1) marks
s
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Fig. 2 SDS-PAGE gel of PEGylated AHA-CalB (1 mg/mL) using
various equivalents of cyclooctyne 15 or 16.
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