Solid-phase synthesis of succinylhydroxamate peptides: Functionalized matrix metalloproteinase inhibitors

Article abstract of DOI:10.1021/ol060409e

A novel solid-phase synthesis strategy toward succinylhydroxamate peptides, using an appropriately protected hydroxamate building block, is described. Rapid and efficient access is gained to amine-functionalized peptides, which can be decorated with, for instance, a fluorescent label. In addition, we demonstrate an on-resin synthesis of a biotinylated photoactivatable hydroxamate peptide, which can be used as an activity-based probe for matrix metalloproteinases and ADAMs.

Full text of DOI:10.1021/ol060409e

ORGANIC
LETTERS
2006
Vol. 8, No. 8
1705-1708
Solid-Phase Synthesis of
Succinylhydroxamate Peptides:
Functionalized Matrix Metalloproteinase
Inhibitors
Michiel A. Leeuwenburgh,† Paul P. Geurink,† Theo Klein,‡ Henk F. Kauffman,§
Gijs A. van der Marel,† Rainer Bischoff,‡ and Herman S. Overkleeft*,†
Department of Bio-organic Synthesis, Leiden UniVersity, P.O. Box 9502,
2300 RA Leiden, The Netherlands, GUIDE Office,
UniVersity Medical Center Groningen, 9713 AV, Groningen, The Netherlands, and
Department of Analytical Biochemistry, Groningen UniVersity,
P.O. Box 196, 9700 AD Groningen, The Netherlands
h.s.oVerkleeft@chem.leidenuniV.nl
Received February 16, 2006
ABSTRACT
A novel solid-phase synthesis strategy toward succinylhydroxamate peptides, using an appropriately protected hydroxamate building block,
is described. Rapid and efficient access is gained to amine-functionalized peptides, which can be decorated with, for instance, a fluorescent
label. In addition, we demonstrate an on-resin synthesis of a biotinylated photoactivatable hydroxamate peptide, which can be used as an
activity-based probe for matrix metalloproteinases and ADAMs.
Matrix metalloproteinases (MMPs) play a central role in the
degradation of extracellular matrix proteins (gelatin, elastin,
and collagen). Disturbances in their physiological regulation
can contribute to a wide range of pathological states, such
as tumor invasion, atherosclerosis, and arthritis.1 ADAMs
are membrane-bound proteins containing both a cellular ad-
hesion and an MMP domain2 and are for instance involved
in cell migration, muscle development, and fertilization. Some
ADAMs display sheddase activity,3 as exemplified by ADAM-
17 (also known as the TNFR converting enzyme or TACE).
The biological importance of these two classes of enzymes,
together with the fact that their activities are highly regulated
in vivo at both the gene expression and protein levels, has
made the development of chemical tools for their study
(inhibitors and activity-based probes4) an important and
active field of research in recent years.
The most commonly used MMP and ADAM inhibitors fall
into the class of succinylhydroxamate-containing peptide ana-
logues.5 Notably, commercially available members of this type
are marimastat, batimastat, and TAPI-2 (see Figure 1), each
† Leiden University.
‡ Groningen University.
§ University Medical Center Groningen.
(1) Johnson, L. J.; Dyer, R.; Hupe, D. J. Curr. Opin. Chem. Biol. 1998,
2, 466-471.
(2) (a) Seals, D.; Courtneidge, S. A. Genes DeV. 2003, 17, 7-30. (b)
Black, R. A.; White, J. M. Curr. Opin. Cell Biol. 1998, 10, 654-659.
Figure 1. Structures of some commercially available MMP/ADAM
inhibitors.
10.1021/ol060409e CCC: $33.50
© 2006 American Chemical Society
Published on Web 03/14/2006

displaying sub- to low-nanomolar, broad MMP/ADAM
inhibitory activity.
The synthesis of 6 starts with the known compound 1.10a,11
Removal of the chiral auxiliary using lithium benzyl alco-
holate gave benzyl ester 2. Partial deprotection led to
monoester 3, the acyl chloride derivative of which was
reacted with N-Boc-O-TBS-hydroxylamine.12 Next, the ben-
zyl ester was removed by catalytic hydrogenation to obtain
free acid 5. It was soon discovered that 5 is not only labile
during storage (even at -20 °C) but also extremely base
sensitive. Attempts to precipitate it as several different
alkylammonium salts led to complete degradation. Also,
standard peptide coupling conditions (HCTU/iPr2EtN) led
to complicated reaction mixtures, presumably due to cy-
clization of 5 to the anhydride. Therefore, to minimize the
amount of base encountered by compound 5, it was decided
to prepare an active ester derivative, which can, in theory,
be coupled without additional base. The pentafluorophenyl
(PFP) ester 6, obtained by reaction of 5 with pentafluo-
rophenol under the influence of N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide (EDC), proved to be far more stable
during storage than acid 5.
Because these compounds are peptide-like structures, it
would be highly convenient to be able to synthesize
functionalized derivatives using standard solid-phase peptide
synthesis (SPPS) procedures. Although many SPPS proce-
dures for C-terminal hydroxamic acids have been reported,6
there are very few SPPS methods for N-terminal succinyl-
hydroxamate peptides7,8 which obviate the handling of
advanced hydroxamate precursors such as carboxylic acids9
or esters.10 Therefore, we devised a building block that can
be used in a linear SPPS strategy, immediately leading to
products with R1 ) H. In this paper, we describe the novel
building block 6 (see Scheme 1) and its use in the synthesis
Scheme 1. Synthesis of Building Block 6
Next, the potential of PFP ester 6 in SPPS was evaluated
(see Scheme 2). Dipeptide 7a was synthesized on Rink amide
Scheme 2. Solid-Phase Synthesis of Succinylhydroxamate
Peptides Using 6
of inhibitors and functionalized probes for the study of MMP
and ADAM activities.
(3) Huovila, A.-P. J.; Turner, A. J.; Pelto-Huikko, M.; Ka¨rkka¨inen, I.;
Ortiz, R. M. Trends Biochem. Sci. 2005, 30, 413-422.
(4) For a recent overview, see: Verhelst, S. H. L.; Bogyo, M. QSAR
Comb. Sci. 2005, 24, 261-269.
resin using standard protocols. After removal of the Fmoc
group, several conditions for the coupling of 6 were
investigated.13 The optimal conditions proved to be shaking
the resin for 2 h with 5 equiv of 6 and 2 equiv of iPr2EtN
relative to the resin-bound peptide in NMP. The resulting
product was cleaved from the resin and concomitantly
deprotected using 95% aqueous TFA, cleanly yielding
peptide 8a in 64% yield after HPLC purification. In the same
fashion, the analogous peptides in which phenylalanine is
replaced by leucine (8b, 56% yield), tryptophan (8c, 42%
yield), and tyrosine (8d, 71% yield) were synthesized.
Enzyme inhibition tests using a fluorogenic substrate (see
Supporting Information) revealed IC50 values in the low
(5) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. H. Chem.
ReV. 1999, 99, 2735-2776.
(6) See, for instance: (a) Mellor, S. L.; McGuire, C.; Chan, W. C.
Tetrahedron Lett. 1997, 38, 3311-3314. (b) Sasubilli, R.; Gutheil, W. G.
J. Comb. Chem. 2004, 6, 911-915. (c) Gazal, S.; Masterson, L. R.; Barany,
G. J. Pept. Res. 2005, 66, 324-332. (d) Yin, Z.; Low, K. S.; Lye, P. L.
Synth. Commun. 2005, 35, 2945-2950.
(7) Solid-phase strategies of marimastat analogues have been reported:
(a) Jenssen, K.; Sewald, K.; Sewald, N. Bioconjugate Chem. 2004, 15, 594-
600. (b) Barlaam, B.; Koza, P.; Berriot, J. Tetrahedron 1999, 55, 7221-
7232.
(8) During the preparation of this manuscript, a paper appeared reporting
the synthesis of a library of diastereomeric inhibitors with R ) H by using
an alternative, racemic building block with O-trityl protection: Wang, J.;
Uttamchandani, M.; Sun, L. P.; Yao, S. Q. Chem. Commun. 2006, 717-
719.
(9) For selected examples of hydroxamate solution syntheses from
carboxylic acids, see: (a) Reddy, A. S.; Kumar, M. S.; Reddy, G. R.
Tetrahedron Lett. 2000, 41, 6285-6288. (b) Giacomelli, G.; Porcheddu,
A.; Salaris, M. Org. Lett. 2003, 5, 2715-2717.
(10) For selected examples of hydroxamate solution syntheses from esters,
see: (a) Levy, D. E.; Lapierre, F.; Liang, W.; Ye, W.; Lange, C. W.; Li,
X.; Grobelny, D.; Casabonne, M.; Tyrrell, D.; Holme, K.; Nadzan, A.;
Galardy, R. E. J. Med. Chem. 1998, 41, 199-223. (b) Ho, C. Y.; Strobel,
E.; Ralbovsky, J.; Galemmo, R. A., Jr. J. Org. Chem. 2005, 70, 4873-
4875.
(11) Auge´, F.; Hornebeck, W.; Decarme, M.; Laronze, J.-Y. Bioorg. Med.
Chem. Lett. 2003, 13, 1783-1786.
(12) Altenburger, J. M.; Mioskowski, C.; d’Orchymont, H.; Schirlin, D.;
Schalk, C.; Tarnus, C. Tetrahedron Lett. 1992, 33, 5055-5058. Instead of
Et3N and DMAP for the coupling of the hydroxylamine to the acyl chloride,
we used 2 equiv of DMAP without Et3N because the acyl chloride of 3
appeared to be highly sensitive to tertiary amine bases.
(13) Five equivalents of 6 and no additional base gave incomplete, though
clean, coupling, and adding five equivalents of iPr2EtN resulted in complete
consumption of 7a but also substantial side reactions.
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Org. Lett., Vol. 8, No. 8, 2006

nanomolar range for the four compounds against both MMP-
12 (catalytic domain) and ADAM-17 (ectodomain), as shown
in Table 1, entries 1-4. The observation that the aromatic
Scheme 4. Linear, On-Resin Synthesis of Biotinylated,
Photo-Cross-Linker-Containing Inhibitor 12a
Table 1. Inhibitory Activities of Newly Synthesized
Compounds (IC50 Values in nM)
entry
compound
MMP-12
ADAM-17
1
2
3
4
5
6
7
8a
8b
8c
8d
9
5.3
41.8
7.8
4.8
4.1
15.1
58.2
26.1
10.7
23.6
42.7
20.6
10
11
13.1
3.6
a Mtt ) 4-methyltrityl.
amino-acid-containing compounds 8a,c,d are more potent
inhibitors than the aliphatic 8b corroborates earlier findings.5
Functionalization of the free amine in the prepared
inhibitors with a fluorescent label can readily be ac-
complished, as demonstrated in Scheme 3. Compound 9, a
of the tagged enzymes, as well as for visualization by
streptavidin after blotting.
Rink amide-bound Fmoc-Lys(Mtt) (11) was side-chain
deprotected and then coupled to biotin. Next, the 6-amino-
hexanoic (Ahx) spacer and the photo-cross-linker amino acid
FmocPhe(tmd) were added. Finally, after removal of the
Fmoc group and coupling of 6, the entire construct was
removed from the resin and concomitantly deblocked, to give
12 in an overall yield of 26% after HPLC purification.
Scheme 3. Fluorescent Labeling of a Succinylhydroxamate
Peptide
Testing of compound 12 on MMP-12 and ADAM-17
revealed that it is a highly potent inhibitor of both enzymes
(see Table 1, entry 7), as well as ADAM-10 (IC50 ) 114
nM). To validate it as a potential activity-based probe, 11
was incubated with recombinant ADAM-10, followed by
irradiation at 366 nm, SDS-PAGE, and detection with
streptavidin-alkaline phosphatase. The results of this experi-
ment are shown in Figure 2.
modified version of 8a with an additional spacer, was
obtained via the presented method in 53% overall isolated
yield. It was established that this spacer does not significantly
influence the inhibitory activity (see entry 5, Table 1).
Reaction of 9 in DMF in the presence of 1 equiv of
BODIPY-TMR-OSu and iPr2EtN cleanly furnished labeled
compound 10 in 77% isolated yield. Incorporation of the
fluorescent label led only to a slight drop in inhibitory
potency (see Table 1, entry 6).
The usefulness of building block 6 is further demonstrated
in the on-resin synthesis of the biotinylated inhibitor 12
(Scheme 4), containing a photoactivatable group. This
compound is designed to bind to the active site of MMPs
and ADAMs, after which it can be covalently locked by
irradiating the photo-cross-linker at 366 nm.14 The biotin
moiety can then be used as a handle for affinity purification
Figure 2. Blot of recombinant ADAM-10 (100 ng) incubated with
photoactivatable inhibitor 12 at 2.5 µM (except lane 2). The
mixtures were irradiated with UV light (366 nm) for different times,
followed by 10% SDS-PAGE and staining with streptavidin-AP.
Lane 1: denatured ADAM-10 (preheated in SDS) and 12. Lane 2:
ADAM-10 + 500 nM of 12. Lane 3: ADAM-10 + 12 + TAPI-2
(100 µM). Lanes 4-8: ADAM-10 + 12 with decreasing irradiation
times of 60, 30, 15, 5, and 0 min, respectively.
(14) Two examples of similar compounds have recently been reported:
(a) Saghatelian, A.; Jessani, N.; Joseph, A.; Humphrey, M.; Cravatt, B. F.
Proc. Natl. Acad. Sci. 2004, 101, 10000-10005. (b) Chan, E. W. S.;
Chattopadhaya, S.; Panicker, R. C.; Huang, X.; Yao, S. Q. J. Am. Chem.
Soc. 2004, 126, 14435-14446.
Clearly, compound 12 is able to irreversibly bind ADAM-
Org. Lett., Vol. 8, No. 8, 2006
1707

10 in an activity-based manner, as evidenced by the weak
staining after preheating the enzyme (lane 1) in comparison
with active ADAM-10 (lane 2). The binding efficiency of
12 is shown in lane 3, where a 40-fold excess of the generic
MMP/ADAM inhibitor TAPI-2 only slightly decreases the
observed staining. Figure 2 also shows that the covalent
labeling is light dependent because no labeling is observed
without irradiation (lane 8) and the maximum labeling occurs
after approximately 30 min of irradiation. These facts make
compound 12 a promising candidate for activity-based
profiling of MMPs and ADAMs.
In conclusion, we have shown a straightforward solid-
phase synthesis of succinylhydroxamate peptides using the
readily accessible building block 6. Potent, free-amine-
containing MMP and ADAM inhibitors were conveniently
synthesized and could be functionalized with a fluorescent
label. Photoactivatable inhibitor 12 was efficiently synthe-
sized entirely on the solid phase and shown to be suited for
activity-based covalent labeling of a model ADAM. This
synthetic strategy is a convenient alternative for the recently
reported methodology8 and can be highly useful for the rapid,
efficient synthesis of libraries of hydroxamate peptides. When
conjugated to a solid support via their free amines, com-
pounds 8a-d can be used in activity-based solid-phase
extraction of MMPs or ADAMs.15 Compound 10 may be
used for fluorescent staining of active MMPs and ADAMs.
Compound 12 nicely complements recently disclosed activ-
ity-based probes for MMPs.14 The use of these compounds
as tools in activity-based profiling is currently ongoing.
Acknowledgment. Financial support for this work was
provided by the Technology Foundation STW (project GPC
6150). We thank Fons Lefeber and Leendert van den Bos
for NMR assistance, Rian van den Nieuwendijk for chiral
HPLC, and Hans van den Elst and Niels Martha for mass
spectrometry measurements.
Supporting Information Available: Detailed experi-
mental procedures and full analytical data for all new
compounds and details of the biochemical evaluations. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL060409E
(15) Freije, J. R.; Bischoff, R. J. Chromatogr., A 2003, 1009, 155-169.
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Org. Lett., Vol. 8, No. 8, 2006