Assembly/disassembly of drug conjugates using imide ligation

Article abstract of DOI:10.1021/ol101049g

A strategy is described that allows the easy assembly and controlled disassembly of drug conjugates. Imide ligation, that is, the reaction of a peptide thioacid with an azidoformate, is used for conjugate assembly. The imide bond participates also with an endopeptidase-triggered cyclization-based disassembly mechanism.

Full text of DOI:10.1021/ol101049g

ORGANIC
LETTERS
2010
Vol. 12, No. 18
3982-3985
Assembly/Disassembly of Drug
Conjugates Using Imide Ligation
Reda Mhidia,† Nicolas Be´zie`re,† Annick Blanpain,† Nicole Pommery,‡ and
Oleg Melnyk*,†
UMR CNRS 8161, UniVersite´ de Lille Nord de France, IFR 142, Institut Pasteur de
Lille, 1 rue du Pr Calmette 59021 Lille, France, and UniVersite´ de Lille Nord de
France, 3 rue du Professeur Laguesse BP83, 59006 Lille Cedex, France
oleg.melnyk@ibl.fr
Received July 2, 2010
ABSTRACT
A strategy is described that allows the easy assembly and controlled disassembly of drug conjugates. Imide ligation, that is, the reaction of
a peptide thioacid with an azidoformate, is used for conjugate assembly. The imide bond participates also with an endopeptidase-triggered
cyclization-based disassembly mechanism.
In recent years, tremendous efforts have been focused on the
design of novel ways to target drugs to specific cells1 or organs.2
Very often, polypeptide conjugates are used for targeting drugs
to specific biomarkers.3 Formation of a covalent bond between
the peptide and the drug usually results in a biologically inactive
conjugate. Release of the active substance at the delivery site
can be triggered by a photochemical,4 chemical, or biochemical
event.3b In particular, the design of self-immolative linkers,
whose fragmentation is triggered by a specific enzyme, has
recently attracted utmost attention.3b,5 However, preparation
of such carrier-linked prodrugs requires multistep procedures
and complex protection schemes.
We disclose here a novel strategy allowing the facile
assembly/disassembly of peptide-drug conjugates 3 (Scheme
1). The first step involves an imide bond formation between
a peptide carrier and a drug by reaction of thioacid 1 with
azidoformate 2. This imide bond plays a key role in the
disassembly process leading to drug release. Indeed, enzyme-
catalyzed cleavage of the peptide bond between amino acids
n-1 and n unmasks the intermediate 5 amine group, which
cyclizes on the imide to liberate drug 7. Besides the efficiency
of the assembly/disassembly process, the strategy depicted
in Scheme 1 shows several other advantages: (1) the half-
life of intermediate 5 can be potentially tuned by varying
the last amino acid n; (2) peptide thioacids are easy to
synthesize using standard solid-phase methods6 or by hy-
drothiolysis of thioesters;7 (3) the cleavage occurs after
penultimate amino acid n-1, thus offering the possibility to
optimize the last residue according to P-1′ endopeptidase
specificity.
† UMR CNRS 8161, Universite´ de Lille Nord de France.
‡ Universite´ de Lille Nord de France.
(1) (a) Chapuis, J.-C.; Schmaltz, R. M.; Tsosie, K. S.; Belohlavek, M.;
Hecht, S. M. J. Am. Chem. Soc. 2009, 131, 2438. (b) Kang, J.-H.; Asai,
D.; Kim, J.-H.; Mori, T.; Toita, R.; Tomiyama, T.; Asami, Y.; Oishi, J.;
Sato, Y. T.; Niidome, T.; Jun, B.; Nakashima, H.; Katayama, Y. J. Am.
Chem. Soc. 2008, 130, 14906. (c) Zhou, X.; Chang, Y.-C.; Oyama, T.;
McGuire, M. J.; Brown, K. C. J. Am. Chem. Soc. 2004, 126, 15656.
(2) Boonyarattanakalin, S.; Hu, J.; Dykstra-Rummel, S. A.; August, A.;
Peterson, B. R. J. Am. Chem. Soc. 2006, 129, 268.
(5) (a) Shamis, M.; Lode, H. N.; Shabat, D. J. Am. Chem. Soc. 2004,
126, 1726. (b) Gomes, P.; Vale, N.; Moreira, R. Molecules 2007, 12, 2484.
(6) (a) Baca, M.; Kent, S. B. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
11638. (b) Schnolzer, M.; Kent, S. B. Science 1992, 256, 221. (c) Crich,
D.; Sharma, I. Angew. Chem., Int. Ed. 2009, 48, 2355.
(7) Tan, X. H.; Zhang, X.; Yang, R.; Liu, C. F. ChemBioChem 2008, 9,
1052.
(3) (a) El Alaoui, A.; Schmidt, F.; Amessou, M.; Sarr, M.; Decaudin, D.;
Florent, J. C.; Johannes, L. Angew. Chem., Int. Ed. 2007, 46, 6469. (b) Kratz,
F.; Muller, I. A.; Ryppa, C.; Warnecke, A. ChemMedChem 2008, 3, 20.
(4) (a) Banerjee, S. S.; Chen, D. H. Nanotechnology 2009, 20, 185103.
(b) Wecksler, S. R.; Mikhailovsky, A.; Korystov, D.; Ford, P. C. J. Am.
Chem. Soc. 2006, 128, 3831.
10.1021/ol101049g  2010 American Chemical Society
Published on Web 08/20/2010

imides toward nucleophiles11 suggested that the imide group
could react with an internal amine, unmasked in a preceding
step by a specific biochemical event (Scheme 1).
Examination of the literature revealed that a unique example
of a carbonyl carbamate bond formation by reaction of
thiobenzoic or thioacetic acid with azido benzylformate was
reported by the group of Williams.8g The feasibility of reacting
chemoselectively azidoformates 2 with polyfunctional thioacids
such as peptide thioacids 1 is to be established.
Scheme 1. Assembly of Conjugate 3 Using Imide Ligation:
Disassembly Is Triggered by an Endopeptidase
In this proof-of-concept study, cyclooxygenase (COX)
inhibitor 7a (Scheme 2) was used as the drug model (see
Scheme 2
.
Imide Ligation and Rearrangement Using Model
Thioacids 1a-c
The reaction of thioacids with azides has been studied by
several groups.8 The reaction with electron-rich azides such as
alkyl azides leads to the corresponding amides8g,i,j and can be
promoted by Ru(III).8h The reaction is particularly effective
with electron-poor azides such as sulfonyl azides8b-g,k,l and
has been used by Liskamp et al.8f,k and others8b,l for peptide
or protein conjugate synthesis. This chemistry is of great
interest for linking molecules to peptide carriers, but the
N-acyl sulfonamide bond formed in this reaction is stable
toward nucleophilic attack and thus cannot be easily cleaved
in a subsequent step as needed for our strategy (Scheme 1).
Activation of the N-acyl sulfonamide toward nucleophiles
can be achieved by alkylation9 but requires an additional
step and a protected peptide chain.10 Alternately, we envis-
aged the chemoselective formation of an imide bond by
reaction of peptide thioacids 1 with azidocarbonyl derivatives
2. Indeed, besides the potential interest of this reaction for
peptide-drug conjugate assembly, the known reactivity of
Supporting Information). COXs are involved in a number
of diseases.12 In particular, COX-2 is overexpressed in
prostate cancer13 and considered as a molecular target in this
disease.14 7a features an alcohol group, which was used for
attaching the azidocarbonyl moiety.
In a preliminary approach, we have examined the reaction
between model amino thioacids 1a-c and azidocarbonyl
derivative 2a. Reaction in N,N-dimethylformamide in the
presence of triethylamine successfully afforded imides 3a-c
in excellent yield, which were subsequently used to explore
the cyclization-based disassembly step (Scheme 2). To this end,
the amino group of 3a,c was deprotected in TFA to provide
(8) (a) Kolakowski, R. V.; Shangguan, N.; Sauers, R. R.; Williams, L. J.
J. Am. Chem. Soc. 2006, 128, 5695. (b) Zhang, X.; Li, F.; Lu, X. W.; Liu,
C. F. Bioconjugate Chem. 2009, 20, 197. (c) Merkx, R.; van Haren, M. J.;
Rijkers, D. T.; Liskamp, R. M. J. Org. Chem. 2007, 72, 4574. (d)
Kolakowski, R. V.; Shangguan, N.; Williams, L. J. Tetrahedron Lett. 2006,
47, 1163. (e) Barlett, K. N.; Kolakowski, R. V.; Katukojvala, S.; Williams,
L. J. Org. Lett. 2006, 8, 823. (f) Merkx, R.; Brouwer, A. J.; Rijkers, D. T.;
Liskamp, R. M. Org. Lett. 2005, 7, 1125. (g) Shangguan, N.; Katukojvala,
S.; Greenberg, R.; Williams, L. J. J. Am. Chem. Soc. 2003, 125, 7754. (h)
Fazio, F.; Wong, C.-H. Tetrahedron Lett. 2003, 44, 9083. (i) Paulsen, H.;
Peters, S.; Bielfeldt, T.; Meldal, M.; Bock, K. Carbohydr. Res. 1995, 268,
17. (j) Rosen, T.; Lico, I. M.; Chu, D. T. W. J. Org. Chem. 1988, 53, 1580.
(k) Rijkers, D. T.; Merkx, R.; Yim, C. B.; Brouwer, A. J.; Liskamp, R. M.
J. Pept. Sci. 2010, 16, 1. (l) Zhang, X.; Lu, X. W.; Liu, C. F. Tetrahedron
Lett. 2008, 49, 6122.
(10) (a) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem.
Soc. 1999, 121, 11369. (b) Shin, Y.; Winans, K.; Backes, B. J.; Kent,
S. B. H.; Ellman, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684.
(11) (a) Evans, D. A.; Ennis, M. D.; Mathre, D. J. J. Am. Chem. Soc.
1982, 104, 1737. (b) Tsunoda, T.; Sasaki, O.; Takeuchi, O.; Itoˆ, S.
Tetrahedron Lett. 1991, 47, 3925. (c) Hogberg, H.-E. Houben-Weyl, Methods
of Organic Chemistry: StereoselectiVe Synthesis. Thieme: Stuttgart, 1996;
Vol. 2, p 883.
(12) Dubois, R. N.; Abramson, S. B.; Crofford, L.; Gupta, R. A.; Simon,
L. S.; Van De Putte, L. B.; Lipsky, P. E. FASEB J. 1998, 12, 1063.
(13) Gupta, S.; Srivastava, M.; Ahmad, N.; Bostwick, D. G.; Mukhtar,
H. Prostate 2000, 42, 73.
(14) (a) Aparicio Gallego, G.; Diaz Prado, S.; Jimenez Fonseca, P.;
Garcia Campelo, R.; Cassinello Espinosa, J.; Anton Aparicio, L. M. Clin.
Transl. Oncol. 2007, 9, 694. (b) Liao, Z.; Mason, K. A.; Milas, L. Drugs
2007, 67, 821.
(9) (a) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem.
Soc., Chem. Commun. 1971, 118, 3055. (b) Heidler, P.; Link, A. Bioorg.
Med. Chem. 2005, 13, 585.
Org. Lett., Vol. 12, No. 18, 2010
3983

5a,c quantitatively. Then, 5a,c were dissolved in aqueous buffer
at pH 2.5 or 7.4 and analyzed by RP-HPLC (Figure 1). Both
Figure 1. Rearrangement of imides 5a (A,B) or 5c (C-F). RP-
HPLC analysis of the reaction mixture (detection at 215 nm, C18
Nucleosil column).
Figure 2. Unmasking of the drug using an endopeptidase (PBS
compounds 5a and 5c were stable in water at pH 2.5 (5a, Figure
1A; 5c, Figure 1C) due to protonation of the amine. Importantly,
at pH 7.4 used hereinafter for endopeptidase-triggered disas-
sembly of conjugates 3e,f (Scheme 3, Figure 2), rearrangement
pH 7.4, 37 °C). Conjugate 3e (40 µM) was incubated with trypsin
(A,B) (40 ng/mL) or chymotrypsin (C,D) (40 ng/mL). Conjugate
3f (40 µM) was incubated with (E-H) (10 µg/mL) or without (I)
PSA. RP-HPLC analysis of the reaction mixtures on a C18
Nucleosil column (215 nm).
Imide 5b derived from alanine rearranged as rapidly as
the glycine analogue 5a (see Supporting Information).
Careful LC-MS analysis of the reaction mixture showed the
exclusive formation of hydantoin 6b and 7a. The potential
hydrolysis products of imide 5b, such as alanine or alanine
amide, were not observed.15 Moreover, thorough NMR
analysis of the same reaction mixture showed the formation
of hydantoin 6b16 and of 7a in a 1/1 molar ratio. Taken
together, these data support the cyclization mechanism
depicted in Scheme 1. These results also show that the nature
of the amino acid involved in the imide bond influences the
rate of cyclization and thus of drug liberation.
Scheme 3. Synthesis of Peptide Imides 3d-f
We next explored the usefulness of imide ligation for the
chemoselective and racemization-free modification of un-
protected peptides (Scheme 3). Azidoformate 2a can poten-
tially react with R or ε-amino groups within peptides to give
carbamate derivatives. One way to avoid this side reaction
is to work at acidic pH, thereby allowing amine protection
by protonation. In this context, peptide thioacid 1d featuring
a free N-terminal amino group and a Lys residue was reacted
of both imides 5a,c induced release of 7a. For 5a, rearrangement
was complete after a few seconds only (Figure 1B), whereas
about 3 h was necessary for imide 5c to rearrange (Figure
1D-F).
(15) Kahns, A. H.; Bundgaard, H. Int. J. Pharm. 1991, 71, 31.
(16) Huang, Z.; Ough, C. S. J. Agric. Food Chem. 1991, 39, 2218.
3984
Org. Lett., Vol. 12, No. 18, 2010

with compound 2a in water at different acidic pH values.
Interestingly, the reaction proceeded efficiently and chemose-
lectively at pH 2.5 within 24 h and permitted isolation of
conjugate 3d in good yield. These experimental conditions
appeared optimal since the reaction rate and purity of the
crude product diminished by increasing the reaction mixture
pH (see Supporting Information). Importantly, the ligation
proceeded without racemization of the C-terminal Ala residue
(0.28% D-Ala by GC-MS).
The data presented here show that imide ligation is
chemoselective, racemization-free, gives good yields using
simple reaction conditions in water, and requires only readily
available starting materials. Moreover, dinitrogen sulfide,
formed as a byproduct of this reaction,8a,d,g is highly unstable
and decomposes into molecular nitrogen and elemental
sulfur,17 which are nontoxic. Besides its utility for the facile
assembly/disassembly of conjugates as shown here, we
believe that imide ligation is a valuable chemical tool that
will enrich the ligation repertoire available for assembling
complex peptide scaffolds.18
cancer cells. This enzyme has been used by several groups
for vectorization of anticancer drugs because PSA is active
only in the prostate cells’ microenvironment.20
The sequence of peptide imide 3f (Figure 2) was chosen
according to the known substrate specificity of PSA.21
Incubation of imide 3f (40 µM) with PSA (10 µg/mL) in
PBS buffer at 37 °C led to successful unmasking of 7a (t1/2
∼ 40 min) and to concomitant formation of peptide Ac-
GISSGY-OH, in a similar manner as in the experiment using
trypsin and peptide 3e. The rate of hydrolysis in the absence
of PSA was 22 times lower (t1/2 ∼ 15 h), in accord with the
relative stability of acyclic imides in water at neutral pH.15
In summary, the data presented in this communication
show that the reaction of peptide thioacids with azidoformates
(i.e., imide ligation) allows the chemoselective and racem-
ization-free assembly of the peptide-drug conjugates. More-
over, we show that the disassembly of the conjugate can be
triggered specifically by an endopeptidase, which generates
an internal amine able to cyclize on the imide through a five-
membered ring intermediate. The amino acid residue engaged
in the imide bond has been shown to influence the rate of
cyclization and thus of liberation. The assembly/disassembly
strategy or imide ligation chemistry reported in this study
should find many interests in different fields of research and,
in particular, in the design of biologically relevant conjugates.
The successful chemoselective and racemization-free as-
sembly of conjugates 3 sets the stage for studying drug
release using endopeptidases. To this end, conjugate 3e
featuring an Arg residue in the penultimate position was
synthesized as described before and incubated with trypsin,
which cleaves after Arg/Lys residues (Figure 2A,B). Chy-
motrypsin, which cuts after aromatic amino acid residues,
was used in the control experiment (Figure 2C,D). Figure
2A shows that unmasking of 7a was almost complete after
a few seconds of trypsin digestion only. Unmasking of 7a
occurred in the mixture because rearrangement of intermedi-
ate 5b did not occur in the eluent used for RP-HPLC analysis
(pH 2). The early eluting peak corresponded, by LC-MS, to
peptide H-ANIQR-OH, showing the Arg-Ala peptide bond
cleavage by trypsin. Chymotrypsin was unable to induce
release of 7a using the same experimental conditions (Figure
2C). Longer incubation times led to release of a small
proportion of 7a and 9 due to slow hydrolysis or degradation
of the imide bond in water (Figure 2D, t1/2 ∼ 6 h).
Acknowledgment. We thank Re´gion Nord Pas-de-Calais
and from Cance´ropoˆle Nord-Ouest for financial support. We
thank Emmanuelle Boll (CNRS) for NMR experiments.
Supporting Information Available: Experimental pro-
cedures and characterization data for all compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL101049G
(19) Balk, S. P.; Ko, Y. J.; Bubley, G. J. J. Clin. Oncol. 2003, 21, 383.
(20) (a) Denmeade, S. R.; Nagy, A.; Gao, J.; Lilja, H.; Schally, A. V.;
Isaacs, J. T. Cancer Res. 1998, 58, 2537. (b) Brady, S. F.; Pawluczyk, J. M.;
Lumma, P. K.; Feng, D. M.; Wai, J. M.; Jones, R.; DeFeo-Jones, D.; Wong,
B. K.; Miller-Stein, C.; Lin, J. H.; Oliff, A.; Freidinger, R. M.; Garsky,
V. M. J. Med. Chem. 2002, 45, 4706. (c) DeFeo-Jones, D.; Brady, S. F.;
Feng, D. M.; Wong, B. K.; Bolyar, T.; Haskell, K.; Kiefer, D. M.; Leander,
K.; McAvoy, E.; Lumma, P.; Pawluczyk, J. M.; Wai, J.; Motzel, S. L.;
Keenan, K.; Van Zwieten, M.; Lin, J. H.; Garsky, V. M.; Freidinger, R.;
Oliff, A.; Jones, R. E. Mol. Cancer Ther. 2002, 1, 451. (d) Kumar, S. K.;
Williams, S. A.; Isaacs, J. T.; Denmeade, S. R.; Khan, S. R. Bioorg. Med.
Chem. 2007, 15, 4973.
To illustrate applicability of the strategy to an enzyme of
medical interest, we have used a peptide substrate of prostate
specific antigen (PSA),19 an endopeptidase which is relatively
specific to prostate tissue and overexpressed in prostate
(17) Wentrup, C.; Kambouris, P. Chem. ReV. 1991, 91, 363.
(18) Hackenberger, C. P.; Schwarzer, D. Angew. Chem., Int. Ed. 2008,
47, 10030.
(21) Coombs, G. S.; Bergstrom, R. C.; Pellequer, J. L.; Baker, S. I.;
Navre, M.; Smith, M. M.; Tainer, J. A.; Madison, E. L.; Corey, D. R. Chem.
Biol. 1998, 5, 475.
Org. Lett., Vol. 12, No. 18, 2010
3985