Design of peptide hydroxamate-based photoreactive activity-based probes of zinc-dependent metalloproteases

Article abstract of DOI:10.1002/ejoc.200901385

Metalloproteases (ADAMs, MMPs) are multidomain proteins that play key roles in extracellular matrix remodelling and degradation, in cell-cell and cell-matrix interactions and in the proteolytic liberation of membrane-anchored proforms of cytokines and gro

Full text of DOI:10.1002/ejoc.200901385

FULL PAPER
DOI: 10.1002/ejoc.200901385
Design of Peptide Hydroxamate-Based Photoreactive Activity-Based Probes of
Zinc-Dependent Metalloproteases
Paul P. Geurink,[a][‡] Theo Klein,[b][‡] Laurette Prèly,[b][‡] Krisztina Paal,[b]
Michiel A. Leeuwenburgh,[a] Gijs A. van der Marel,[a] Henk F. Kauffman,[c]
Herman S. Overkleeft,*[a] and Rainer Bischoff*[b]
Keywords: Metalloenzymes / Activity-based profiling / Photoaffinity labeling / Diazirine / Alkylation / Diastereoselectivity
Metalloproteases (ADAMs, MMPs) are multidomain proteins
that play key roles in extracellular matrix remodelling and
degradation, in cell–cell and cell–matrix interactions and in
the proteolytic liberation of membrane-anchored proforms of
cytokines and growth factors, the so-called ectodomain
shedding. In this work we describe the development of
photoactivatable activity-based probes with which active
metalloproteases can be visualised. Our probes are based on
the succinyl hydroxamate motif and differ in the positioning
of the trifluoromethylphenyldiazirine photoreactive group.
We demonstrate that directing the photoactivatable group
towards the S1Ј pocket yields activity-based probes more ef-
fective than the corresponding probe with the photoactivat-
able group directed towards the S2Ј pocket.
cursor protein (APP) processing[10] as well as the convertase
activity of the same enzyme in the Notch/delta signaling
pathway.[11] Deregulation of metalloprotease catalytic ac-
tivity has been linked to inflammatory processes such as
arthritis,[12] inflammatory bowel disorder,[7] Alzheimer’s dis-
ease,[13] cancer[14,15] and cardiac hypertrophy.[16]
Introduction
Matrix metalloproteases (MMPs) and A Disintegrin And
Metalloproteases (ADAMs) are catalytically active Zn2+
-
dependent proteins that belong to the metzincin super-
family. Members of the MMP and ADAM families con-
tain the consensus zinc-binding catalytic sequence
HEXGHXXGXXHD in their metalloprotease domain.[1]
MMPs have important physiological functions related to
the degradation and turnover of extracellular matrix com-
ponents, such as collagens and fibronectin.[2–5] ADAMs are
membrane-anchored metalloproteases that display adhesive
properties through their integrin-binding disintegrin do-
main and interaction of the cysteine-rich region with glyco-
proteins[6] and extracellular matrix proteins (for instance,
fibronectin).[7] Their proteolytic activity is involved in so-
called ectodomain shedding and is exemplified by the re-
lease of soluble TNFα from the cell membrane by ADAM-
17 (TACE, TNF-alpha converting enzyme),[8] the release of
heparin-binding epidermal growth factor by ADAM-9[9]
and the α-secretase function of ADAM-10 in amyloid pre-
Conventional proteomics approaches to determine the
relation of metalloproteases to disease states are limited by
the fact that they take the total protein amount into ac-
count, whereas in many cases the functionality, that is the
catalytic activity, is the relevant parameter. Several elegant
methods to determine proteolytic activity in biological sam-
ples have been developed, such as zymography and activity-
based ELISA.[17,18] Although these approaches visualise
and quantify active proteases, application to a family-wide
proteomics approach is difficult. Substrate specificity in
zymography (for instance, gelatinases in gelatinzymogra-
phy) and antibody specificity in ELISA make that both
techniques are inherently limited to specific enzymes. Due
to these limitations, there is a growing interest in the devel-
opment of family-wide functional proteomics probes. Con-
siderable progress have been made in the development and
[a] Leiden Institute of Chemistry and the Netherlands Proteomics application of activity-based probes targeting cysteine pro-
teases,[19–22] serine hydrolases[23–26] and proteasome sub-
Center, Leiden University,
Einsteinweg 55, 2300 RA Leiden, The Netherlands
units.[27–29] In these proteases a side-chain residue (serine,
E-mail: h.s.overkleeft@chem.leidenuniv.nl
[b] Department of Analytical Biochemistry, University Centre for
Pharmacy, University of Groningen,
cysteine, threonine) acts as the nucleophilic species involved
in amide-bond cleavage that is amenable to covalent and
irreversible modification by installment of an appropriate
electrophilic trap in the activity-based probes. MMPs and
ADAMs employ a water molecule as the nucleophile in
their active site, which precludes the use of such an electro-
Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
E-mail: r.p.h.bischoff@rug.nl
[c] Laboratory of Allergology and Pulmonology, University Medi-
cal Center Groningen,
9713 GZ Groningen, The Netherlands
[‡] These authors contributed equally to this work.
2100
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Photoreactive Activity-Based Probes of Metalloproteases
Figure 1. A) Generic binding mode of N-terminal peptide hydroxamates to metalloprotease active sites. B) Peptide hydroxamate-based
activity-based metalloprotease probe from our previous studies with the photoactivatable group at P2Ј.[32] C) Compounds targeted in this
study and featuring the photoactivatable group at the P1Ј site.
philic trap. Photoaffinity labeling represents an alternative
Results and Discussion
way to introduce tags into the active site of metalloprote-
ases.[30,31] In this context, we recently reported[32] the syn-
In our previous work,[32] on the synthesis of ABP 1, we
thesis of peptide hydroxamate 1 featuring both a biotin and employed a chiral, -leucine mimetic succinyl hydroxamate
a trifluoromethyldiazirine moiety (Figure 1, B). Upon incu- in which the hydroxamic acid moiety was protected with
bation of purified recombinant ADAM-10 and subsequent acid-labile protective groups [O(TBS)-N(Boc)-R] and the
irradiation with UV light (366 nm) the metalloprotease was carboxylate activated as the pentafluorophenyl ester. This
covalently and irreversibly modified, as was evidenced by building block can be readily incorporated in Fmoc-based
SDS PAGE of the denatured protein followed by streptavi- solid-phase peptide synthesis protocols as the final building
din blotting. The efficiency of the photoaffinity labeling block after which the immobilized peptide hydroxamate is
however proved rather modest. This raised the question in one step cleaved from the resin and concomitantly depro-
whether the photoactivatable group would be better di- tected. Our strategy appeared of general use as we demon-
rected towards the S1Ј pocket, rather than the S2Ј pocket strated by the construction of a peptide hydroxamate li-
(see part A of Figure 1 for a general picture of the binding brary[37] and we therefore decided to apply a similar strat-
mode of N-terminal peptide hydroxamate-based metallo- egy for the construction of our target compounds. This re-
protease inhibitors). Examination of the available 3-dimen- quired the synthesis of trifluoromethylphenyldiazirine func-
sional structures of metalloprotease-inhibitor complexes in- tionalised, pentafluorophenyl-activated ester 24 (Scheme 1),
dicates that the S1Ј pocket in general should be able to ac- which we prepared as follows. Reduction of 4-bromophen-
commodate rather bulky hydrophobic groups at this posi- ylpropionic acid (3) (LiAlH4) provided alcohol 4 that was
tion.[33–36] We decided to address this issue by the synthesis transformed into TBS ether 5. At this stage, the trifluo-
of peptide hydroxamate 2a (Figure 1, C) with the photoac- roacetyl moiety was introduced by first lithiation and subse-
tivatable group at P1Ј, and compare its MMP/ADAM label- quent addition of 1-trifluoroacetylpiperidine. Refluxing the
ing efficiency to that of probe 1 having the photoactivatable resulting ketone 6 and hydroxylamine in pyridine afforded
group at P2Ј. We here report an efficient synthesis of the oxime 7 as an E/Z mixture. The hydroxy group in 7 was
required diazirine-modified succinyl hydroxamate building converted into the tosylate 8 after which reaction with li-
block and its application in the synthesis of activity-based quid ammonia under 8 bar at room temperature led to the
probe 2a along with a pair of fluorescent analogues 2b and formation of diaziridine 9. Oxidation of the diaziridine
2c. We demonstrate in a head-to-head comparison that 2a using iodine provided diazirine 10. Acidolysis of the TBS
indeed is the more efficient photoactivatable MMP/ADAM ether followed by biphasic TEMPO/BAIB oxidation of the
activity-based probe towards a range of recombinant, puri- resulting alcohol 11[38] provided carboxylic acid 12 in 57%
fied metalloproteases.
overall yield over the nine steps.
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Scheme 1. Reagents and conditions: (i) LiAlH4, Et2O, 0 °C, quant.; (ii) TBSCl, imidazole, DMF, quant.; (iii) nBuLi, 1-trifluoroacetylpiper-
idine, Et2O, –78 °CǞr.t., 76%; (iv) HONH2·HCl, pyridine, ∆, 93%; (v) TsCl, Et3N, DMAP, DCM, 97%; (vi) NH3, Et2O, 8 bar, 95%;
(vii) I2, Et3N, MeOH, 94%; (viii) HCl, H2O, MeOH, 97%; (ix) TEMPO, BAIB, DCM, H2O, 96%; (x) (a) (ClCO)2, DMF, DCM; (b) 13,
nBuLi, THF, 0 °C, 82%; (xi) LiHMDS, tert-butyl bromoacetate, THF, –78 °C, 70%; (xii) BnOH, nBuLi, THF, 0 °C, 82%; (xiii) TFA,
DCM, 97%; (xiv) (a) (ClCO)2, DMF, DCM; (b) TBSONHBoc, nBuLi, THF, 27% for 18 and 77% for 22; (xv) several conditions, no
yield; (xvi) PFPOH, EDCI, DCM, 88% over 2 steps; (xvii) allyl alcohol, nBuLi, THF, 0 °C, 65%; (xviii) 23, Pd(PPh)4, THF.
The route of synthesis continued by condensation of 12, ing for a protocol in which the diaziridine is oxidised back
via its acyl chloride, with the lithium salt of chiral auxiliary to the diazirine after hydrogenation, we opted to adapt our
13.[39] Deprotonation of the resulting intermediate 14 fol- protective group scheme, as follows. Reaction of compound
lowed by enantioselective alkylation with tert-butyl bromo- 15 with the lithium salt of allyl alcohol afforded allyl ester
acetate afforded succinate 15 as the single observed dia- 20.[41] Partial deprotection and condensation with the pro-
stereomer in 57% yield over the two steps. In line with our tected hydroxylamine as described before gave succinyl hy-
previous studies[32] we substituted the Evans template with droxamate 22. Now, the allyl ester was removed by reaction
lithium benzyl alcoholate to give diester 16. Selective acidic with tetrakis(triphenylphosphane)palladium in the presence
removal of the tert-butyl group gave carboxylic acid 17 of N,NЈ-dimethylbarbaturic acid (23) as the allyl scavenger,
which was transformed into fully protected succinyl hydro- providing carboxylate 19. The latter was converted into the
xamate 18 by first transformation into the corresponding key intermediate, pentafluorophenyl ester 24, in 88% yield
acyl chloride and next reaction with the lithiate of N-Boc- over the last two steps. The choice for chiral alkylation by
O-TBS-hydroxylamine.[40] We previously found[32] that con- means of Evans template chemistry was based on our pre-
densation of N-Boc-O-TBS-hydroxylamine with related vious work in which we showed that alkylation and substi-
acyl chlorides proceeded well under the agency of two tution of the Evans template, both of which entail strong
equivalents of 4-dimethylaminopyridine (DMAP) as the basic conditions, gave optically pure products.[32] Indeed, in
base. However, this procedure proved less efficient in the the here presented synthesis we did not observe any kind of
transformation aimed for here, and optimal results were ob- epimerization. The chiral outcome of these steps however
tained by adding dropwise the lithiate of N-Boc-O-TBS-hy- can vary with the type of side chain used and the use of a
droxylamine to a THF solution of the acyl chloride. Unfor- different kind of chiral auxiliary, for example the thiazolid-
tunately deprotection of the benzyl ester to carboxylic acid inethione derivatives, which are known to display a better
19 failed under the conditions attempted [Pd/C, H2 or leaving group ability,[42] may then prove necessary.
Pd(OAc)2, Et3SiH, Et3N], either because the hydrogenation
The construction of peptide hydroxamates incorporating
of the benzyl proceeded sluggishly or the diazirine was re- the chiral succinic acid derivative is depicted in Scheme 2.
duced concomitantly to the diaziridine. Rather than search- In the first instance we reacted pentafluorophenyl ester 24
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Photoreactive Activity-Based Probes of Metalloproteases
Scheme 2. Reagents and conditions: (i) DMF, 45%; (ii) TFA/H2O, 95:5 (v:v), 67%; (iii) (a) 20% piperidine/DMF; (b) Fmoc-Ahx-OH,
HCTU, DIEA, NMP; (c) 20% piperidine/DMF; (d) Fmoc-Phe-OH, HCTU, DIEA, NMP; (iv) (a) 20% piperidine/DMF; (b) 24, DIEA,
NMP; (c) TFA/H2O/TIS, 95:2.5:2.5 (v:v:v), 30% from 28; (v) Bodipy(Tmr)-OSu or Bodipy(FL)-OSu, DIEA, DMF, 31% and 55%,
respectively.
with -phenylalanine methylamide (25)[43] in DMF to give are some differences in potency. The values differ especially
bisamide 26, which was finally deprotected [TFA/H2O, 95:5 for ADAM-17, for which hydroxamate 1 appeared about 25
(v:v)] to the free hydroxamic acid 27 in 30% yield over the fold more potent compared to 2a. Interestingly, both com-
two steps.[44] This experiment at once established that build- pounds appear equally efficient in labeling ADAM-17, as is
ing block 24 is compatible with the peptide coupling/global evidenced from Figure 2. In this experiment, recombinant
deprotection conditions envisioned and delivered a tag-free and purified ADAM-17 was exposed to either 1 or 2a and
analogue of our target compounds for control experiments UV light prior to denaturation, SDS PAGE and streptavi-
(see below). The target compound 2a was prepared by din blotting. It seems that inhibitory efficiency is not di-
Fmoc-based solid-phase peptide synthesis starting with rectly correlated to photoaffinity labeling and we argue that
RINK amide-bound Fmoc-biocytin 28. Standard Fmoc- the mechanism by which the trifluoromethyldiazirine disso-
based solid-phase peptide synthesis afforded immobilized ciates and reacts upon irradiation is behind this observa-
tripeptide 29, which was transformed in three steps (first tion. Upon photoexcitation nitrogen is expulsed with con-
Fmoc removal, then condensation with pentafluorophenyl comitant formation of a highly reactive carbene that will
ester 24 and finally removal from the solid support with insert in the first available X–H bond.[47] In case an (active
concomitant global deprotection) into peptide hydroxamate site) amino acid is nearby effective photoaffinity labeling is
2a in 30% overall yield after RP-HPLC purification and the expected result, whereas poor labeling will occur in case
based on 28. In a similar fashion, but employing FmocLys- the photoreactive group is solvent (water) exposed. We ten-
(Boc) instead of Fmoc-biocytin, peptide hydroxamate 30 tatively conclude that the diazirine moiety in 2a is bound
was prepared. Treatment of 30 with either Bodipy(TMR)- more tightly to ADAM-17 than the one in 1, even though
OSu[45] or Bodipy(FL)-OSu[46] gave compounds 2b and 2c, the latter compound is the more potent inhibitor. Perusal
respectively, in 31% and 55% yield.
of a panel of ten recombinant and purified MMPs and
We next set out to compare the labeling efficiency of three more ADAMs reveals that, in general, peptide hydro-
probes 1 and 2a against a panel of MMPs and ADAMs. In xamate 2a is the more effective affinity label (Figure 2). In
a first experiment we assessed the inhibitory potency of the each case compound 2a is at least as effective (compare for
two probes against MMP-9, MMP-12, ADAM-10 and instance the data obtained for ADAM-9 and ADAM-10)
ADAM-17 (Table 1). Although the four enzymes are in- and often in fact provides a signal where compound 1 does
hibited in the nanomolar range by both compounds, there not (see for instance the results obtained for MMP-13 and
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H. S. Overkleeft, R. Bischoff et al.
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Figure 2. Photoaffinity labeling of MMPs (A) and ADAMs (B) using probe 1 (upper panels) and probe 2a (lower panels) at 1 µ. The
modified proteins were visualized by anti-biotin Western blots with streptavidin-alkaline phosphatase. Ten recombinant MMP and four
recombinant ADAM proteases (4 pmol each; numbers above the lanes correspond to the respective protease) were studied. Multiple
biotinylated bands in the recombinant MMPs indicate auto-degradation. M: molecular weight marker.
ADAM-8). Interestingly, multiple bands appear for some of were detectably labeled. Preincubation of MMP-9 or MMP-
the MMP labeling experiments (see for instance, MMP-1 12 with twofold molar excess, relative to 2a, of the non-
and MMP-8 treated with 2a), with the band corresponding biotinylated inhibitor 27 also effectively abolished labeling.
to the highest molecular weight in each case corresponding Taken together, these data provide strong evidence that
to the molecular weight of the full-length MMP at hand. photoaffinity labeling is activity-dependent and that label-
These bands in all likelihood are the result of auto-degrada- ing occurs most likely in the active site of the enzymes.
tion, in which unmodified MMP processes its photoaffinity
labeled counterpart. From these experiments we conclude
Conclusions
that positioning the photoactivatable group at P1Ј as in 2a
indeed gives a comparatively more potent MMP/ADAM
photoactivatable activity-based probe.
In summary, we have described the development of an
efficient photoactivatable activity-based probe with which a
broad panel of MMPs and ADAMs can be covalently and
irreversibly modified in an activity-dependent fashion. Our
work demonstrates that the enantioselective synthesis strat-
egy we previously reported[32,37] for the preparation of an
enantiomerically pure, alkylated succinyl hydroxamate is
also effective, in adapted form, for the synthesis of the func-
tionally more challenging key building block 24. Further,
our hypothesis that placing the photoactivatable trifluoro-
methyldiazirine in the P1Ј position would lead to more ef-
fective activity-based probes proved valid. We are now
faced with the challenge to detect MMPs and ADAMs in
their natural environment and at natural abundance levels
in a photoactivatable activity-based proteomics profiling
experimental setting.[30] The ability to prepare, with relative
ease, peptide hydroxamates analoguous to compounds 1
and 2a such as Bodipy derivatives 2b,c may well be indis-
pensable in reaching this research objective, on which we
will report in the future.
Table 1. IC50 values (in n) of compounds 1 and 2a.
MMP-9
MMP-12
ADAM-10
ADAM-17
20.6[a]
490
1
2a
25.1
24.2
3.60[a]
12.5
114[a]
54.1
[a] From reference.[32]
In order to prove that labeling is activity-dependent, ali-
quots of MMP-9 and MMP-12 were incubated with either
the natural inhibitor, TIMP-1[48] or the non-biotinylated in-
hibitor 27 (Figure 3). In the presence of equimolar amounts
(relative to 2a) of TIMP-1 neither MMP-9 nor MMP-12
Experimental Section
Syntheses
General Methods: Tetrahydrofuran was distilled from LiAlH4 prior
to use. Acetonitrile (ACN), dichloromethane (DCM), dimethyl-
formamide (DMF), N-methyl-2-pyrrolidone (NMP), methanol
(MeOH), piperidine, diisopropylethylamine (DIEA) and trifluoro-
acetic acid (TFA) were of peptide synthesis grade, purchased at
Biosolve, and used as received. All general chemicals (Fluka, Acros,
Merck, Aldrich, Sigma) were used as received. Rink amide MBHA
resin (0.64 mmol/g) was purchased at Novabiochem, as well as all
Figure 3. Activity-dependence of photoaffinity labeling of MMP-9
and MMP-12 (4 pmol each) with probe 2a (200 n final concentra-
tion) as shown by competition with (A) an equimolar amount of
the endogenous MMP inhibitor TIMP-1 (200 n final concentra-
tion) and with (B) a twofold molar excess of compound 27 (400 n
final concentration).
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Photoreactive Activity-Based Probes of Metalloproteases
appropriately protected amino acids [Fmoc-Phe-OH, Fmoc-
Ahx-OH, Fmoc-Lys(Mtt)-OH]. O-(6-Chloro-1H-benzotriazolyl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) was
purchased at Iris Biotech (Marktrewitz, Germany). Traces of water
were removed from reagents used in reactions that require an-
hydrous conditions by co-evaporation with toluene. Solvents that
were used in reactions were stored over 4 Å molecular sieves, except
for methanol and acetonitrile, which were stored over 3 Å molecu-
lar sieves. Molecular sieves were flame-dried before use. Unless
noted otherwise all reactions were performed under argon. Column
chromatography was performed on silica gel (purchased from
Screening Devices b.v.; particle size 40–63 µm, pore diameter of
60 Å). The eluents toluene, ethyl acetate and petroleum ether (40–
60 °C boiling range) were distilled prior to use. TLC analysis was
conducted on Merck aluminium sheets (Silica gel 60 F254). Com-
pounds were visualized by UV absorption (254 nm), by spraying
with a solution of (NH4)6Mo7O24·4H2O (25 g/L) and (NH4)4Ce-
(SO4)4·2H2O (10 g/L) in 10% sulfuric acid, a solution of KMnO4
(20 g/L) and K2CO3 (10 g/L) in water, or ninhydrin (0.75 g/L) and
acetic acid (12.5 mL/L) in ethanol, where appropriate, followed by
charring at ca. 150 °C. 1H- and 13C-NMR spectra were recorded
with a Bruker AV-400 (400 MHz), a Bruker AV-500 (500 MHz) or
a Bruker DMX-600 (600 MHz) spectrometer. 19F NMR spectra
were recorded with a Bruker AV-200 (200 MHz) or a Bruker DMX-
400 (400 MHz). Chemical shifts are given in ppm (δ) relative to
tetramethylsilane, CD3OD, [D6]DMSO, CDCl3 or CFCl3 as in-
ternal standard. High-resolution mass spectra were recorded by di-
rect injection [2 µL of a 2 µ solution in water/acetonitrile, 1:1 (v/
v) and 0.1% formic acid] on a mass spectrometer (Thermo Finni-
gan LTQ Orbitrap) equipped with an electrospray ion source in
positive mode (source voltage 3.5 kV, sheath gas flow 10, capillary
temperature 250 °C) with resolution R = 60000 at m/z 400 (mass
range m/z = 150–2000) and dioctyl phthalate (m/z = 391.28428) as a
“lock mass”. The high-resolution mass spectrometer was calibrated
prior to measurements with a calibration mixture (Thermo Finni-
gan). Optical rotations [α]2D3 were recorded with a Propol automatic
polarimeter.
TIS, 95:2.5:2.5 (v/v/v). The resulting filtrate was concentrated un-
der reduced pressure and the product was purified by RP-HPLC.
LC-MS Analysis and HPLC Purifications. System A: LC-MS
analysis was performed with a Finnigan Surveyor HPLC system
with a Gemini C18 50ϫ4.60 mm column (detection at 200–
600 nm), coupled to a Finnigan LCQ Advantage Max mass spec-
trometer with ESI. The applied buffers were H2O, ACN and 1.0 %
aq. TFA.
System B: HPLC purifications were performed with a Gilson
HPLC system coupled to
a
Phenomenex Gemini 5 µm
250ϫ10 mm column and a GX281 fraction collector. The applied
buffers were: 0.1% aq. TFA and ACN. Appropriate fractions were
pooled, and concentrated in a Christ rotary vacuum concentrator
overnight at room temperature at 0.1 mbar.
Compounds
3-(4-Bromophenyl)propanol (4): Commercially available 3-(4-bro-
mophenyl)propanoic acid (3, 10.8 g, 46.22 mmol) was dissolved in
Et2O (250 mL) and cooled to 0 °C. To this solution was carefully
added LiAlH4 (1.3 equiv., 60 mmol, 2.28 g) in portions. The reac-
tion was slowly warmed to room temperature in 1 h after which
TLC analysis indicated a completed reaction. 1  aq. HCl (200 mL)
was slowly added and the layers were separated. The organic layer
was extracted with 1  aq. HCl, saturated aq. NaHCO3 (twice) and
brine (200 mL, each), dried with MgSO4 and concentrated under
reduced pressure. The product was obtained as a colourless oil
(yield: 9.9 g, 46.2 mmol, quant.). The spectroscopic data corre-
spond with those reported in literature.[49]
[3-(4-Bromophenyl)propoxy](tert-butyl)dimethylsilane (5): To a solu-
tion of alcohol 4 (9.9 g, 46.2 mmol) in DMF (80 mL) were added
imidazole (4.77 g, 69.3 mmol) and tert-butylchlorodimethylsilane
(TBS-Cl) (7.82 g, 50.8 mmol). The reaction was stirred for 2 h after
which TLC analysis indicated complete conversion. Deionised H2O
(300 mL) was added and the mixture was extracted 3 times with
petroleum ether (200 mL). The combined organic layers were ex-
tracted with deionised H2O (4 times) and brine (200 mL, each),
dried with MgSO4 and concentrated under reduced pressure. The
product was obtained as a colourless oil (yield: 15.2 g, 46.2 mmol,
quant.) and subjected to the next step without further purification.
The spectroscopic data correspond with those reported in litera-
ture.[50]
General Procedure for Solid-Phase Peptide Synthesis: Fmoc Rink
Amide MBHA resin (0.64 mmol/g) was used as received. Prior to
first use, it was washed twice with DMF, twice with methanol and
twice with DCM. Fmoc deprotection was performed by shaking
the resin in a 20% piperidine/DMF (v/v) stock solution for 20 min.
The resin was washed twice with DMF and twice with DCM after
every coupling and deprotection step. The first amino acid was
loaded by reacting the resin with 4 equiv. of HCTU, 4 equiv. of
amino acid and 8 equiv. DIEA (0.45  stock solution in NMP).
The amino acid was pre-activated in solution (5 min) before adding
it to the resin and shaking the resin for 1 h (standard coupling
protocol). Loading was determined by UV spectroscopy at 300 nm
of a freshly prepared Fmoc-deprotected resin sample. A capping
step was performed by shaking the resin for 10 min with 0.45 
acetic anhydride and 0.45  DIEA/NMP solution. Mtt deprotec-
tion was done by shaking repeatedly in a 1% TFA/DCM solution
until the characteristic yellow colour of the Mtt cation did no
longer appear (7–12 times, 2 min each). After Mtt cleavage, imme-
diately before the following coupling step, the resin was washed
with a 0.45  DIEA stock solution in NMP. The coupling and
deprotection reactions were checked on the presence of free amines
by performing a Kaiser test. Before cleaving the peptide from the
1-{4-[3-(tert-Butyldimethylsilyloxy)propyl]phenyl}-2,2,2-trifluoroeth-
anone (6): Bromide 5 (7.03 g, 21.36 mmol) was dissolved in Et2O
(100 mL) and cooled to –78 °C. nBuLi (26.7 mmol, 16.7 mL, 1.6 
in THF) was added dropwise and the solution was slowly warmed
to room temperature at which it was stirred for 1 h. Then the mix-
ture was cooled again to –78 °C and a solution of 1-trifluoroacetyl-
piperidine (4.2 g, 23.2 mmol) in Et2O (5 mL) was added dropwise.
The solution was slowly warmed to 0 °C in 2 h after which TLC
analysis indicated a complete conversion. The reaction was
quenched with saturated aq. NH4Cl (100 mL) and the layers were
separated. The organic layer was extracted with saturated aq.
NH4Cl, deionised water and brine, dried with MgSO4 and concen-
trated under reduced pressure. The crude material was purified by
column chromatography (10 % Ǟ 30% toluene/petroleum ether)
and the product was obtained as a colourless oil (yield: 5.63 g,
1
16.3 mmol, 76%). H NMR (400 MHz, CDCl3): δ = 8.00 (d, J =
8.0 Hz, 2 H, Ph), 7.37 (d, J = 8.0 Hz, 2 H, Ph), 3.64 (t, J = 6.4 Hz,
2 H, OCH2), 2.79 (t, J = 7.6 Hz, 2 H, PhCH2), 1.89–1.84 (m, 2 H,
resin, it was washed 5 times alternatingly with DCM and MeOH. CH2), 0.91 (s, 9 H, 3ϫ CH3), 0.06 (s, 6 H, 2ϫ SiCH3) ppm. 13C
Cleavage from the solid support was done by shaking the resin
in a TFA/H2O/TIS, 95:2.5:2.5 (v/v/v) solution for 2 h, followed by
filtration and rinsing the resin with a small portion of TFA/H2O/
NMR (100 MHz, CDCl3): δ = 151.3, 130.2, 129.3, 127.7, 116.8 (q,
J = 290 Hz, CF3), 61.4, 33.9, 32.4, 25.9, 18.2, –5.4 ppm. HRMS:
calcd. for C17H25F3O2Si [M + H]+ 347.16487; found 347.16505.
Eur. J. Org. Chem. 2010, 2100–2112
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2105

H. S. Overkleeft, R. Bischoff et al.
FULL PAPER
1-{4-[3-(tert-Butyldimethylsilyloxy)propyl]phenyl}-2,2,2-trifluoroeth-
anone Oxime (7): Ketone 6 (9.43 g, 27.2 mmol) was dissolved in
0.90 (s, 9 H, 3ϫ CH3), 0.05 (s, 6 H, 2ϫ SiCH3) ppm. 13C NMR
(100 MHz, CDCl3): δ = 144.3, 129.1, 128.8, 127.9, 123.6 (q, J =
pyridine (30 mL) and hydroxylamine hydrochloride (5.68 mmol, 277 Hz, CF3), 61.9, 57.7 (q, J = 35.7 Hz, CNHNH), 34.0, 31.7,
81.7 mmol) was added. The mixture was stirred at reflux for 2 h
after which TLC analysis indicated a complete conversion. The
mixture was concentrated under reduced pressure and dissolved in
EtOAc (100 mL) and 0.2  aq. citric acid (100 mL). The layers were
separated and the organic layer was extracted with 100 mL of 0.2 
aq. citric acid, deionised water and brine, dried with MgSO4 and
concentrated under reduced pressure. The crude product was ob-
tained as a colourless oil (yield: 9.13 g, 25.3 mmol, 93%) as a Z/E
mixture. A small amount was purified by column chromatography
(5 % Ǟ 10 % EtOAc/petroleum ether) for characterization. 1H
NMR (400 MHz, CDCl3): δ = 8.51 (br. s, 1 H, OH), 8.25 (br. s, 1
H, OH), 7.44–7.39 (m, 2 H, Ph), 7.31–7.23 (m, 2 H, Ph), 3.67–3.62
(m, 2 H, OCH2), 2.74–2.69 (m, 2 H, PhCH2), 1.89–1.82 (m, 2 H,
CH2), 0.91 (s, 9 H, 3ϫ CH3), 0.06 (s, 6 H, 2ϫ SiCH3) ppm. 13C
NMR (100 MHz, CDCl3): δ = 147.4–146.5 (m, C=N), 144.9, 144.5,
129.2, 128.9, 128.9, 128.7, 128.6, 128.6, 128.4, 128.3, 128.2, 127.9,
123.7, 120.9 (q, J = 273 Hz, CF3), 118.6 (q, J = 281 Hz, CF3), 63.0,
62.8, 34.0, 33.9, 32.0, 31.9, 25.9, 18.4, –5.3, –5.5 ppm. HRMS:
calcd. for C17H26F3NO2Si [M + H]+ 362.17577; found 362.17583.
25.7, 18.1, –5.6 ppm. HRMS: calcd. for C17H27F3N2OSi [M +
H]+ 361.19175; found 361.19183.
3-{4-[3-(tert-Butyldimethylsilyloxy)propyl]phenyl}-3-(trifluorometh-
yl)-3H-diazirine (10): Et3N (46.2 mmol, 6.4 mL) was added to a
solution of diaziridine 8 (8.32 g, 23.1 mmol) in MeOH (25 mL).
Then I2 (23.1 mmol, 5.86 g) was added in portions, letting the mix-
ture decolorize after every portion. Eventually the reaction mixture
did not decolorize anymore and the mixture was stirred for another
30 min. TLC analysis indicated a completed reaction and a 10%
w/w aq. citric acid solution (100 mL) was added. The mixture was
extracted twice with Et2O (150 mL) and the combined organic lay-
ers were extracted with 10% w/w aq. citric acid (100 mL), a satu-
rated aq. NaHSO3 solution (100 mL), deionised water (100 mL)
and brine (100 mL), dried with MgSO4 and concentrated under
reduced pressure. The crude diazirine was obtained as a colourless
oil (yield: 7.77 g, 21.7 mmol, 94%). A small amount was purified
by column chromatography (1% Ǟ 2.5% EtOAc/petroleum ether)
1
for characterization. H NMR (600 MHz, CDCl3): δ = 7.22 (d, J
= 8.4 Hz, 2 H, Ph), 7.10 (d, J = 7.8 Hz, 2 H, Ph), 3.61 (t, J =
6.3 Hz, 2 H, OCH2), 2.69 (t, J = 7.5 Hz, 2 H, PhCH2), 1.82–1.79
(m, 2 H, CH2), 0.90 (s, 9 H, 3ϫ CH3), 0.05 (s, 6 H, 2ϫ SiCH3)
ppm. 13C NMR (150 MHz, CDCl3): δ = 144.2, 129.0, 126.6, 126.5,
122.4 (q, J = 273 Hz, CF3), 61.9, 34.2, 31.8, 28.4 (q, J = 40 Hz,
CNN), 25.9, 18.3, –5.5 ppm. HRMS: calcd. for C17H25F3N2OSi [M
+ H]+ 359.17610; found 359.17616.
1-{4-[3-(tert-Butyldimethylsilyloxy)propyl]phenyl}-2,2,2-trifluoroeth-
anone O-Tosyl Oxime (8): Oxime 7 (9.13 g, 25.3 mmol) was dis-
solved in DCM (20 mL). To this solution were added Et3N
(5.26 mL, 37.95 mmol) and DMAP (60 mg, 0.5 mmol) after which
TsCl (4.84 g, 25.3 mmol) in DCM (20 mL) was added dropwise
over 1 h. After stirring the mixture at room temperature for 30 min
TLC analysis indicated a complete conversion. 0.2  aq. citric acid
(100 mL) was added and the layers were separated. The organic
layer was extracted with 0.2  aq. citric acid (100 mL) and brine,
dried with MgSO4 and concentrated under reduced pressure yield-
ing the crude product (Z/E mixture) as a colourless oil (yield:
12.59 g, 24.4 mmol, 97%). A small amount was purified by column
chromatography (1.5% Ǟ 7.5% EtOAc/petroleum ether) for char-
3-{4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]phenyl}propan-1-ol (11):
Compound 10 (7.78 g, 21.7 mmol) was dissolved in MeOH (50 mL)
and concentrated HCl [37% (w/v), 3 mL] was added. The reaction
mixture was stirred at room temperature until TLC analysis re-
vealed a completed reaction after 2 h. The solvent was evaporated
under reduced pressure and the residue was dissolved in Et2O
(200 mL). The resulting solution was extracted twice with a satu-
rated aq. NaHCO3 solution (150 mL) and brine (100 mL), dried
with MgSO4 and concentrated under reduced pressure. The crude
mixture was purified by column chromatography (10% Ǟ 60%
EtOAc/petroleum ether) and the pure alcohol 11 was obtained as a
colourless oil (yield: 5.13 g, 21.0 mmol, 97%). 1H NMR (400 MHz,
CDCl3): δ = 7.25 (d, J = 8.0 Hz, 2 H, Ph), 7.12 (d, J = 8.0 Hz, 2
H, Ph), 3.66 (t, J = 6.4 Hz, 2 H, OCH2), 2.72 (d, J = 7.8 Hz, 2 H,
PhCH2), 1.90–1.83 (m, 2 H, CH2), 1.43 (br. s, 1 H, OH) ppm. 13C
NMR (100 MHz, CDCl3): δ = 143.7, 129.1, 126.5, 126.4, 122.2 (q,
J = 263 Hz, CF3), 61.4, 33.7, 31.6, 28.2 (q, J = 40.2 Hz, CNN)
ppm. HRMS: calcd. for C11H11F3N2O [M + H]+ 245.08962; found
245.08978.
1
acterization. H NMR (400 MHz, CDCl3): δ = 7.91–7.88 (m, 2 H,
Ph), 7.39–7.25 (m, 6 H, Ph), 3.66–3.62 (m, 2 H, OCH2), 2.73 (t, J
= 7.8 Hz, 2 H, PhCH2), 2.48 (s, 3 H, PhCH3), 2.46 (s, 3 H, PhCH3),
1.88–1.83 (m, 2 H, CH2), 0.91 (s, 9 H, 3ϫ CH3), 0.90 (s, 9 H, 3ϫ
CH3), 0.06 (s, 6 H, 2ϫ SiCH3), 0.05 (s, 6 H, 2ϫ SiCH3) ppm. 13C
NMR (100 MHz, CDCl3): δ = 153.8 (q, J = 34.4 Hz, C=N), 146.8,
146.6, 146.0, 145.9, 131.5, 131.2, 129.7, 129.1, 128.9, 129.8, 128.7,
128.4, 121.8, 119.7 (q, J = 276 Hz, CF3), 61.9, 61.8, 33.8, 33.8,
32.0, 31.9, 25.8, 21.5, 18.1, –5.5 ppm. HRMS:: calcd. for
C
24H32F3NO4SSiH+ 516.18462; found 516.18443, calcd. for
C24H32F3NO4SSi [M + Na]+ 538.16656; found 538.16655.
3-{4-[3-(tert-Butyldimethylsilyloxy)propyl]phenyl}-3-(trifluorometh-
yl)diaziridine (9): Tosylate 7 (12.6 g, 24.4 mmol) was dissolved in
Et2O (30 mL) in an autoclave and cooled to –78 °C. Freshly con-
densed ammonia (≈ 5 mL) was added and the autoclave was closed
and warmed to room temperature. The pressure inside increased to
8 bar. After 5 h the autoclave was cooled to –78 °C and opened.
Deionised water (40 mL) was carefully added and the mixture was
warmed to room temperature. The layers were separated and the
organic layer was extracted with deionised water (3ϫ 50 mL) and
brine, dried with MgSO4 and concentrated under reduced pressure.
The crude diaziridine was obtained as a colourless oil (yield: 8.32 g,
23.1 mmol, 95 %). A small amount was purified by column
chromatography (2% Ǟ 11% EtOAc/petroleum ether) for charac-
3-{4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]phenyl}propanoic
Acid
(12): Alcohol 11 (2.1 g, 8.6 mmol) was dissolved in a mixture of
DCM (25 mL) and deionised H2O (12.5 mL). To this mixture were
added 2,2,6,6-tetramethylpiperidinooxy (TEMPO) (0.1 equiv.,
0.86 mmol, 134 mg) and [bis(acetoxy)iodo]benzene (BAIB)
(2.5 equiv., 21.5 mmol, 6.92 g). The reaction was stirred overnight
at room temperature after which TLC analysis revealed a com-
pleted reaction. A saturated aq. Na2S2O3 solution (100 mL) was
added and the mixture was vigorously stirred for 5 min. EtOAc
(150 mL) was added and the layers were separated. The organic
layer was extracted with 1  aq. HCl and brine (100 mL), dried
with MgSO4 and concentrated under reduced pressure. The crude
material was purified by column chromatography (10% Ǟ 30%
EtOAc/petroleum ether) and the product was obtained as a colour-
1
terization. H NMR (400 MHz, CDCl3): δ = 7.51 (d, J = 8.0 Hz,
2 H, Ph), 7.24 (d, J = 8.0 Hz, 2 H, Ph), 3.63 (t, J = 6.2 Hz, 2 H,
OCH2), 2.77 (d, J = 8.8 Hz, 1 H, NH), 2.71 (t, J = 7.8 Hz, 2 H,
PhCH2), 2.20 (d, J = 8.8 Hz, 1 H, NH), 1.86–1.81 (m, 2 H, CH2), less solid (yield: 2.13 g, 8.23 mmol, 96%). 1H NMR (600 MHz,
2106
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 2100–2112

Photoreactive Activity-Based Probes of Metalloproteases
CDCl3): δ = 11.81 (br. s, 1 H, CO2H),7.22 (d, J = 9.0 Hz, 2 H, Ph),
7.11 (d, J = 9.6 Hz, 2 H, Ph), 2.94 (t, J = 8.1 Hz, 2 H, CH2), 2.65
(t, J = 8.1 Hz, 2 H, CH2) ppm. 13C NMR (150 MHz, CDCl3): δ =
179.4, 142.0, 128.8, 127.2, 126.6, 122.2 (q, J = 273 Hz, CF3), 35.2,
30.1, 28.4 (q, J = 40.5 Hz, CNN) ppm. HRMS: calcd. for
C11H9F3N2O2 [M + H]+ 259.06889; found 259.06909.
16.8, J2 = 4.2 Hz, 1 H, 0.5ϫ CH2,B), 1.34 (s, 9 H, 3ϫ CH3) ppm.
13C NMR (150 MHz, CDCl3): δ = 174.5, 170.8, 152.8, 139.9, 135.3,
129.6, 129.3, 128.7, 127.3, 127.1, 126.4, 121.9 (q, J = 273 Hz, CF3),
80.6, 65.7, 55.2, 41.0, 37.3, 36.2, 28.1 (q, J = 39.8 Hz, CNN),
27.2 ppm. [α]2D3 = +72.8 (c = 1, CHCl3). HRMS: calcd. for
C27H28F3N3O5 [M + H]+ 532.20538; found 532.20512.
(S)-4-Benzyl-3-(3-{4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl}-
propanoyl)oxazolidin-2-one (14): Carboxylic acid 12 (1.04 g,
4.03 mmol) was dissolved in DCM (25 mL) and DMF (1 drop) was
added. The mixture was cooled to 0 °C and oxalyl chloride
(1.7 mL, 20 mmol) was added dropwise. The reaction was warmed
to room temperature and stirred for 30 min after which gas forma-
tion ceased. Toluene was added and the mixture was concentrated
in vacuo followed by coevaporation with toluene (twice). The crude
product was subjected to the next step without further purification.
nBuLi (2.75 mL of a 1.6  solution in THF, 4.4 mmol) was put into
a flask under an argon atmosphere and cooled to 0 °C. To this was
added dropwise a solution of (S)-4-benzyloxazolidin-2-one[39] (13,
0.78 g, 4.43 mmol) in THF (15 mL) and the mixture was stirred at
0 °C for 15 min, thereby forming a white precipitant. Then the
freshly prepared acyl chloride in THF (10 mL) was added to the
reaction mixture dropwise and the mixture was slowly warmed to
room temperature. After 2 h at room temperature, TLC analysis
showed complete consumption of the starting material. To the mix-
ture was added deionised water (50 mL) and it was extracted twice
with EtOAc (50 mL). The combined organic layers were extracted
with a saturated aq. solution of NaHCO3 (2ϫ50 mL) and brine
(50 mL), dried with MgSO4 and concentrated under reduced pres-
sure. The obtained crude material was purified by column
chromatography (10 % Ǟ 30 % EtOAc/petroleum ether) and the
product was obtained as a colourless solid (yield: 1.38 g,
3.31 mmol, 82%). 1H NMR (600 MHz, CDCl3): δ = 7.32–7.25 (m,
5 H, Ph), 7.16 (d, J = 7.2 Hz, 2 H, Ph), 7.13 (d, J = 8.4 Hz, 2 H,
Ph), 4.67–4.63 (m, 1 H, NCH), 4.18–4.14 (m, 2 H, OCH2), 3.32–
3.19 (m, 3 H, CH2 + 0.5ϫ PhCH2), 3.04–3.01 (m, 2 H, CH2), 2.75
(dd, J1 = 13.8, J2 = 9.6 Hz, 1 H, 0.5ϫ PhCH2) ppm. 13C NMR
(150 MHz, CDCl3): δ = 171.9, 153.4, 142.4, 135.0, 129.3, 129.1,
128.9, 127.3, 127.0, 126.6, 122.1 (q, J = 273 Hz, CF3), 66.2, 55.0,
37.7, 36.7, 29.7, 28.3 (q, J = 40.0 Hz, CNN) ppm. [α]2D3 = +48.5 (c
= 1, CHCl3). HRMS: calcd. for C21H18F3N3O3 [M + H]+
418.13730; found 418.13743.
1-Benzyl 4-tert-Butyl (2R)-2-{4-[3-(Trifluoromethyl)-3H-diazirin-3-
yl]benzyl}succinate (16): Benzyl alcohol (350 µL, 3.40 mmol) was
dissolved in THF (8 mL) and cooled to 0 °C. To this mixture was
added nBuLi (1.10 mL of a 1.6  solution, 1.76 mmol) and the re-
action was stirred at 0 °C for 30 min. Then a solution of compound
15 (784 mg, 1.47 mmol) in THF (4 mL) was added and the reaction
mixture was stirred for 1 h at 0 °C and then warmed to room tem-
perature. After 30 min TLC analysis indicated complete conversion
of the starting material. A saturated aq. NH4Cl solution (20 mL)
was added and the mixture was extracted twice with EtOAc
(20 mL). The combined organic layers were dried with MgSO4 and
concentrated under reduced pressure. After purification of the
crude mixture by column chromatography (3% Ǟ 10% EtOAc/
petroleum ether) the product was obtained as a colourless oil (yield:
1
558 mg, 1.21 mmol, 82%). H NMR (600 MHz, CDCl3): δ = 7.37
(t, J = 7.2 Hz, 1 H, Ph), 7.33 (d, J = 4.8 Hz, 2 H, Ph), 7.22 (t, J =
3.6 Hz, 2 H, Ph), 7.14 (d, J = 7.8 Hz, 2 H, Ph), 7.05 (d, J = 7.8 Hz,
2 H, Ph), 5.06 (dd, J1 = 36.6, J2 = 12.0 Hz, 2 H, PhCH2O), 3.13–
3.10 (m, 1 H, CHα), 2.99 (dd, J1 = 13.2, J2 = 7.2 Hz, 1 H, 0.5ϫ
PhCH2), 2.82 (dd, J1 = 13.8, J2 = 7.8 Hz, 1 H, 0.5ϫ PhCH2), 2.60
(dd, J1 = 16.2, J2 = 8.4 Hz, 1 H, 0.5ϫ CH2,α), 2.34 (dd, J1 = 16.8,
J2 = 5.4 Hz, 1 H, 0.5ϫ CH2,α), 1.40 (s, 9 H, 3ϫ CH3) ppm. 13C
NMR (150 MHz, CDCl3): δ = 173.4, 170.3, 140.0, 135.5, 129.3,
128.1, 128.0, 127.1, 126.4, 126.3, 122.0 (q, J = 273 Hz, CF3), 80.7,
66.3, 42.8, 37.1, 36.4, 28.1 (q, J = 40.5 Hz, CNN), 27.70 ppm.
[α]2D3 = +3.6 (c = 1, CHCl3).
(R)-4-(Benzyloxy)-4-oxo-3-{4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-
benzyl}butanoic Acid (17): tert-Butyl ester 16 (550 mg, 1.19 mmol)
was dissolved in DCM (4 mL). To this was added TFA (3 mL) and
the reaction was stirred at room temperature. After 1 h TLC analy-
sis indicated a completed reaction. Toluene was added and the mix-
ture was concentrated under reduced pressure. Co-evaporating the
mixture twice with toluene resulted in a yellowish oil. This crude
product was purified by column chromatography (10 % Ǟ 60%
EtOAc/petroleum ether) and the free carboxylic acid was obtained
as a colourless solid (yield: 1.09 g, 3.07 mmol, 97 %). 1H NMR
(600 MHz, CDCl3): δ = 11.15 (br. s, 1 H, CO2H), 7.35 (t, J =
7.2 Hz, 1 H, Ph), 7.32 (d, J = 4.8 Hz, 2 H, Ph), 7.21 (t, J = 3.6 Hz,
2 H, Ph), 7.11 (d, J = 7.8 Hz, 2 H, Ph), 7.04 (d, J = 7.8 Hz, 2 H,
Ph), 5.06 (dd, J1 = 36.6, J2 = 12.0 Hz, 2 H, PhCH2O), 3.17–3.12
(m, 1 H, CHα), 3.00 (dd, J1 = 13.8, J2 = 7.2 Hz, 1 H, 0.5ϫ PhCH2),
2.81 (dd, J1 = 13.8, J2 = 7.8 Hz, 1 H, 0.5ϫ PhCH2), 2.74 (dd, J1
= 17.4, J2 = 9.0 Hz, 1 H, 0.5ϫ CH2,α), 2.43 (dd, J1 = 17.4, J2 =
5.4 Hz, 1 H, 0.5ϫ CH2,α) ppm. 13C NMR (150 MHz, CDCl3): δ =
177.7, 173.4, 139.6, 135.3, 129.4, 128.5, 128.5, 128.4, 127.5, 126.5,
122.0 (q, J = 273 Hz, CF3), 66.7, 42.4, 37.0, 34.9, 28.2 (q, J =
39 Hz, CNN) ppm.
tert-Butyl (3R)-4-[(4S)-4-Benzyl-2-oxooxazolidin-3-yl]-4-oxo-3-{4-
[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}butanoate (15):
LiHMDS (5.5 mL of a 1  solution, 5.5 mmol) was put into a flask
under an argon atmosphere and cooled to –78 °C. To this was
added a solution of compound 14 (2.0 g, 5.0 mmol) in THF
(25 mL) over 15 min. The reaction was stirred for 1 h at –78 °C
after which tert-butyl bromoacetate (2.2 mL, 15 mmol) was added.
Then the mixture was slowly warmed to –10 °C in 4 h after which
TLC analysis showed a completed reaction. A saturated aq. NH4Cl
solution (50 mL) was added and the mixture was extracted with
EtOAc (3 ϫ 30 mL). The combined organic layers were dried with
MgSO4 and concentrated under reduced pressure. The resulting
crude mixture was purified by column chromatography (10% Ǟ
20% EtOAc/petroleum ether) and the product was obtained as a
Benzyl (2R)-4-[tert-Butoxycarbonyl(tert-butyldimethylsilyloxy)-
colourless solid (yield: 1.86 g, 3.50 mmol, 70 %). 1H NMR amino]-4-oxo-2-{4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}-
(600 MHz, CDCl3): δ = 7.30–7.25 (m, 4 H, Ph), 7.21–7.18 (m, 3 H,
Ph), 7.05 (d, J = 9.0 Hz, 2 H, Ph), 4.55–4.52 (m, 1 H, NCH), 4.42–
4.38 (m, 1 H, CHα), 4.05 (dd, J1 = 9.0, J2 = 1.8 Hz, 1 H, 0.5ϫ
butanoate (18): Carboxylic acid 17 (71 mg, 0.18 mmol) was dis-
solved in DCM (1.5 mL) and DMF (1 drop) was added. This mix-
ture was cooled to 0 °C and oxalyl chloride (4 equiv., 0.70 mmol,
OCH2), 3.92 (t, J = 8.4 Hz, 1 H, 0.5ϫ OCH2), 3.23 (dd, J1 = 13.5, 60 µL) was added dropwise. The reaction was warmed to room
J2 = 2.7 Hz, 1 H, 0.5ϫ PhCH2), 3.00 (dd, J1 = 13.2, J2 = 6.0 Hz, temperature and stirred for 30 min after which gas formation ce-
1 H, 0.5ϫ CH2,A), 2.79–2.70 (m, 2 H, 0.5ϫ PhCH2 + 0.5ϫ CH2,B),
2.57 (dd, J1 = 13.2, J2 = 9.6 Hz, 1 H, 0.5ϫ CH2,A), 2.25 (dd, J1 =
ased. Toluene was added and the mixture was concentrated in
vacuo followed by coevaporation with toluene (2ϫ).
Eur. J. Org. Chem. 2010, 2100–2112
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2107

H. S. Overkleeft, R. Bischoff et al.
FULL PAPER
O(TBS)-N(Boc)-protected hydroxylamine[44] (1.2 equiv., 0.19
mmol, 48 mg) was dissolved in THF (2 mL) and the solution was
cooled to 0 °C. nBuLi (1.05 equiv., 0.185 mmol, 0.11 mL of a 1.6 
solution in hexane) was added dropwise and the reaction mixture
was stirred at 0 °C for 30 min. In a separate flask the freshly pre-
pared crude acyl chloride was dissolved in THF (2 mL) and cooled
to 0 °C. To this the lithiated hydroxylamine mixture was added
dropwise and the reaction was stirred at 0 °C for 2.5 h after which
TLC analysis indicated complete conversion of compound 17. A
0.1  aq. HCl solution (10 mL) was added and the mixture was
extracted twice with EtOAc (10 mL). The combined organic layers
were extracted with 0.1  aq. HCl (10 mL) and brine, dried with
MgSO4 and concentrated under reduced pressure. The crude mix-
ture was purified by column chromatography (toluene Ǟ 3 %
EtOAc/toluene) and the product was obtained as a colourless oil
(yield: 30 mg, 47 µmol, 27%). 1H NMR (600 MHz, CDCl3): δ =
7.34–7.31 (m, 3 H, Ph), 7.20 (t, J = 3.6 Hz, 2 H, Ph), 7.15 (d, J =
8.4 Hz, 2 H, Ph), 7.04 (d, J = 7.8 Hz, 2 H, Ph), 5.06–5.01 (m, 2 H,
PhCH2O), 3.27–3.21 (m, 2 H, CHα + 0.5ϫ PhCH2), 2.98 (dd, J1
= 13.2, J2 = 6.6 Hz, 1 H, 0.5ϫ PhCH2), 2.90–2.84 (m, 2 H, CH2,α),
1.53 (s, 9 H, 3ϫ CH3), 0.98 (s, 9 H, 3ϫ CH3), 0.12 (s, 3 H, SiCH3),
0.085 (s, 3 H, SiCH3) ppm. 13C NMR (150 MHz, CDCl3): δ =
174.1, 170.3, 151.8, 140.0, 135.5, 129.4, 128.4, 128.2, 128.1, 127.3,
126.4, 122.1 (q, J = 273 Hz, CF3), 84.6, 66.4, 42.7, 38.8, 37.4, 28.2
(q, J = 40 Hz, CNN), 27.9, 25.6, 18.0, –5.1, –5.1 ppm.
8.4 Hz, 2 H, Ph), 7.12 (d, J = 8.4 Hz, 2 H, Ph), 5.82–5.76 (m, 1 H,
CH sp2), 5.22 (dd, J1 = 15.6, J2 = 1.8 Hz, 1 H, 0.5ϫ CH2 sp2),
5.19 (dd, J1 = 10.8, J2 = 1.2 Hz, 1 H, 0.5ϫ CH2 sp2), 4.57–4.52
(m, 2 H, OCH2), 3.15–3.10 (m, 1 H, CHα), 3.05 (dd, J1 = 13.8, J2
= 7.2 Hz, 1 H, 0.5ϫ PhCH2), 2.83 (dd, J1 = 13.8, J2 = 7.8 Hz, 1
H, 0.5ϫ PhCH2), 2.73 (dd, J1 = 17.4, J2 = 9.0 Hz, 1 H, 0.5ϫ
CH2,α), 2.44 (dd, J1 = 17.4, J2 = 5.4 Hz, 1 H, 0.5ϫ CH2,α) ppm.
13C NMR (150 MHz, CDCl3): δ = 177.7, 173.3, 139.8, 131.5, 129.5,
127.6, 126.6, 122.0 (q, J = 273 Hz, CF3), 118.5, 65.5, 42.5, 37.1,
34.8, 28.2 (q, J = 40.0 Hz, CNN) ppm. [α]2D3 = +13.2 (c = 1,
CHCl3). HRMS: calcd. for C16H15F3N2O4 [M + H]+ 357.10567;
found 357.10576.
Allyl (2R)-4-[tert-Butoxycarbonyl(tert-butyldimethylsilyloxy)-
amino]-4-oxo-2-{4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}-
butanoate (22): Carboxylic acid 21 (1.22 g, 3.42 mmol) was dis-
solved in DCM (15 mL) and DMF (2 drops) were added. This
mixture was cooled to 0 °C and oxalyl chloride (4 equiv.,
13.68 mmol, 1.20 mL) was added dropwise. The reaction was
warmed to room temperature and stirred for 30 min after which
gas formation ceased. Toluene was added and the mixture was con-
centrated in vacuo followed by coevaporation with toluene (2ϫ).
The crude product was subjected to the next step without further
purification. O(TBS)-N(Boc)-protected hydroxylamine[44]
(1.2 equiv., 4.1 mmol, 1.01 g) was dissolved in THF (10 mL) and
the solution was cooled to 0 °C. nBuLi (1.05 equiv., 3.59 mmol,
1-Allyl 4-tert-Butyl (2R)-2-{4-[3-(Trifluoromethyl)-3H-diazirin-3- 2.25 mL of a 1.6  solution in hexane) was added dropwise and
yl]benzyl}succinate (20): Allyl alcohol (190 µL, 2.75 mmol) was dis-
solved in THF (5 mL) and cooled to 0 °C. To this mixture was
added nBuLi (812 µL of a 1.6  solution, 1.3 mmol) and the reac-
tion was stirred at 0 °C for 30 min. Then a solution of compound
15 (586 mg, 1.10 mmol) in THF (4 mL) was added and the reaction
mixture was stirred for 1 h at 0 °C and then warmed to room tem-
perature. After 2.5 h TLC analysis indicated complete conversion
of the starting material. A saturated aq. NH4Cl solution (20 mL)
was added and the mixture was extracted twice with EtOAc
(20 mL). The combined organic layers were dried with MgSO4 and
concentrated under reduced pressure. After purification of the
crude mixture by column chromatography (2.5% Ǟ 5% EtOAc/
the reaction mixture was stirred at 0 °C for 30 min. In a separate
flask the freshly prepared crude acyl chloride was dissolved in THF
(15 mL) and cooled to 0 °C. To this the lithiated hydroxylamine
mixture was added dropwise and the reaction was stirred at 0 °C
for 2.5 h after which TLC analysis indicated complete conversion
of compound 21. A 0.1  aq. HCl solution (30 mL) was added and
the mixture was extracted twice with EtOAc (30 mL). The com-
bined organic layers were extracted with 0.1  aq. HCl (30 mL)
and brine, dried with MgSO4 and concentrated under reduced pres-
sure. The crude mixture was purified by column chromatography
(100% toluene Ǟ 2.5% EtOAc/toluene) and the product was ob-
1
tained as a yellowish oil (yield: 1.54 g, 2.63 mmol, 77%). H NMR
petroleum ether) the product was obtained as a colourless oil (yield: (600 MHz, CDCl3): δ = 7.22 (d, J = 8.4 Hz, 2 H, Ph), 7.11 (d, J =
1
293 mg, 0.71 mmol, 65%). H NMR (600 MHz, CDCl3): δ = 7.21 8.4 Hz, 2 H, Ph), 5.80–5.75 (m, 1 H, CH sp2), 5.21 (d, J = 17.4 Hz,
(d, J = 8.4 Hz, 2 H, Ph), 7.11 (d, J = 7.8 Hz, 2 H, Ph), 5.83–5.77
1 H, 0.5ϫ CH2 sp2), 5.16 (d, J = 10.2 Hz, 1 H, 0.5ϫ CH2 sp2),
4.51 (d, J = 6.0 Hz, 2 H, OCH2), 3.24–3.20 (m, 2 H, CHα + 0.5ϫ
(m, 1 H, CH sp2), 5.22 (d, J = 16.8 Hz, 1 H, 0.5ϫ CH2 sp2), 5.18
(d, J = 10.8 Hz, 1 H, 0.5ϫ CH2 sp2), 4.57–4.50 (m, 2 H, OCH2), PhCH2), 3.03 (dd, J1 = 13.8, J2 = 6.6 Hz, 1 H, 0.5ϫ PhCH2), 2.89–
3.11–3.07 (m, 1 H, CHα), 3.02 (dd, J1 = 13.8, J2 = 7.2 Hz, 1 H, 2.85 (m, 2 H, CH2,α), 1.53 (s, 9 H, 3ϫ CH3), 0.99 (s, 9 H, 3ϫ
0.5ϫ PhCH2), 2.82 (dd, J1 = 13.8, J2 = 7.8 Hz, 1 H, 0.5ϫ PhCH2),
2.59 (dd, J1 = 16.2, J2 = 8.4 Hz, 1 H, 0.5ϫ CH2,α), 2.33 (dd, J1 =
CH3), 0.13 (s, 3 H, SiCH3), 0.10 (s, 3 H, SiCH3) ppm. 13C NMR
(150 MHz, CDCl3): δ = 173.7, 170.2, 151.8, 140.3, 131.8, 129.4,
16.8, J2 = 5.4 Hz, 1 H, 0.5ϫ CH2,α), 1.42 (s, 9 H, 3ϫ CH3) ppm. 127.3, 126.5, 122.0 (q, J = 273 Hz, CF3), 118.1, 84.5, 65.2, 42.7,
13C NMR (150 MHz, CDCl3): δ = 173.5, 170.6, 140.2, 131.7, 129.5, 38.6, 37.3, 28.2 (q, J = 40.5 Hz, CNN), 27.8, 25.6, 18.0, –5.2 ppm.
127.4, 126.5, 122.1 (q, J = 273 Hz, CF3), 118.3, 80.9, 65.3, 43.0,
37.2, 36.4, 28.2 (q, J = 40.5 Hz, CNN), 27.9 ppm. [α]2D3 = +9.9 (c
= 1, CHCl3). HRMS: calcd. for C20H23F3N2O4 [M + H]+
413.16827; found 413.16826.
[α]2D3 = +7.91 (c = 1, CHCl3). HRMS: calcd. for C27H38F3N3O6Si
[M + H]+ 586.25547; found 586.25562.
Pentafluorophenyl (2R)-4-[tert-Butoxycarbonyl(tert-butyldimethyl-
silyloxy)amino]-4-oxo-2-{4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-
benzyl}butanoate (24): Allyl ester 22 (1.53 g, 2.63 mmol) was dis-
solved in THF (15 mL). To this solution were added N,NЈ-dimeth-
ylbarbaturic acid (23, 0.5 equiv., 1.32 mmol, 210 mg) and tetrakis-
(triphenylphosphane)palladium (cat.). The reaction was stirred for
1 h at room temp. after which TLC analysis indicated complete
consumption of the starting compound. The mixture was concen-
trated under reduced pressure and dissolved again in DCM
(2R)-4-(Allyloxy)-4-oxo-3-{4-[3-(trifluoromethyl)-3H-diazirin-3-
yl]benzyl}butanoic Acid (21): tert-Butyl ester 20 (1.31 g, 3.17 mmol)
was dissolved in DCM (10 mL). To this was added TFA (10 mL)
and the reaction was stirred at room temperature. After 1 h TLC
analysis indicated a completed reaction. Toluene was added and
the mixture was concentrated under reduced pressure. Co-evaporat-
ing the mixture twice with toluene resulted in yellowish oil. This
crude product was purified by column chromatography (10% Ǟ (15 mL) without further purification (19). To this were added
50% EtOAc/petroleum ether) and the free acid was obtained as a pentafluorophenol (2 equiv., 5.26 mmol, 556 µL) and EDCI
colourless solid (yield: 1.09 g, 3.07 mmol, 97 %). 1H NMR (2 equiv., 5.26 mmol, 1.00 g) and the reaction was stirred for 12 h
(600 MHz, CDCl3): δ = 10.80 (br. s, 1 H, CO2H), 7.20 (d, J = at room temp. Et2O (50 mL) was added and the mixture was ex-
2108
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 2100–2112

Photoreactive Activity-Based Probes of Metalloproteases
tracted twice with 0.1  aq. HCl (50 mL) and brine, dried with
MgSO4 and concentrated under reduced pressure. The resulting
mixture was purified by column chromatography (1.5% EtOAc/
petroleum ether) and the product was obtained as a yellow solid
(yield: 1.65 g, 2.31 mmol, 88%). 1H NMR (500 MHz, CDCl3): δ =
7.30 (d, J = 8.5 Hz, 2 H, Ph), 7.16 (d, J = 8.0 Hz, 2 H, Ph), 3.62–
3.56 (m, 1 H, CHα), 3.33 (dd, J1 = 18.5, J2 = 9.5 Hz, 1 H, 0.5ϫ
PhCH2), 2.23 (dd, J1 = 13.5, J2 = 6.5 Hz, 1 H, 0.5ϫ PhCH2), 3.07–
4.38–4.36 (m, 1 H, NCHα), 3.00 (dd, J1 = 13.6, J2 = 5.2 Hz, 1 H,
0.5ϫ PhCH2), 2.98–2.92 (m, 1 H, CHα), 2.82–2.75 (m, 2 H, 2ϫ
0.5ϫ PhCH2), 2.62 (dd, J1 = 13.6, J2 = 5.6 Hz, 1 H, 0.5ϫ PhCH2),
2.57 (d, 3 H, NCH3), 2.11 (dd, J1 = 14.8, J2 = 7.2 Hz, 1 H, 0.5ϫ
CH2,α), 1.94 (dd, J1 = 14.6, J2 = 7.4 Hz, 1 H, 0.5ϫ CH2,α) ppm.
13C NMR (100 MHz, [D6]DMSO): δ = 173.7, 172.0, 168.3, 142.8,
139.0, 130.8, 130.0, 129.0, 127.1, 127.0, 126.2, 55.0, 44.1, 38.2, 37.8,
35.1, 26.4 ppm. [α]2D3 = –10.8 (c = 1, DMSO). LC-MS (system A):
2.99 (m, 2 H, CH2,α), 1.54 (s, 9 H, 3ϫ CH3), 0.99 (s, 9 H, 3ϫ gradient 10% Ǟ 90% ACN/(0.1% TFA/H2O): Rt (min): 7.20 [ESI-
CH3), 0.14 (s, 3 H, SiCH3), 0.12 (s, 3 H, SiCH3) ppm. 13C NMR MS: m/z = 492.07 [M + H+]]. HRMS: calcd. for C23H24F3N5O4
(125 MHz, CDCl3): δ = 170.5, 169.6, 151.9, 141.1 (dd, J1 = 250 H,
J2 = 9.0 Hz, o-CF PFP), 139.5 (dt, J1 = 264, J2 = 13.5 Hz, p-CF3
PFP), 139.3, 137.8 (dt, J1 = 249, J2 = 13.5 Hz, m-CF3 PFP), 129.5,
127.9, 126.8, 125.0 (t, J2 = 12.8 Hz, C-O PFP), 122.1 (q, J =
273 Hz, CF3), 84.9, 42.4, 38.7, 37.1, 28.3 (q, J = 40.1 Hz, CNN),
27.8, 25.6, 18.1, –5.2, –5.3 ppm. 19F NMR (188 MHz, CDCl3): δ =
–65.8 (s, 3 F), –152.4 (d, J = 18.6 Hz, 2 F), –158.6 (t, J = 21.1 Hz,
1 F), –163.0 (t, J = 21.4 Hz, 2 F) ppm. [α]2D3 = +8.8 (c = 1, CHCl3).
HRMS: calcd. for C30H33F8N3O6Si [M + Na]+ 734.19031; found
734.19061.
(2R)-N4-(tert-Butoxycarbonyl)-N4-(tert-butyldimethylsilyloxy)-N1-
[(2S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl]-2-{4-[3-(trifluoro-
methyl)-3H-diaziren-3-yl]benzyl}succinamide (26): -phenylala-
ninemethylamide[43] (25, 21 mg, 120 µmol) was added to a solution
of compound 24 (85 mg, 120 µmol) in DMF (2 mL). The reaction
was stirred at room temp. for 24 h after which no more starting
material was consumed (followed by LC-MS analysis). Et2O
(10 mL) and 0.1  aq. HCl solution (10 mL) were added and the
layers were separated. The aq. layer was extracted with Et2O
(10 mL) and the combined organic layers were extracted with a
[M + H]+ 492.18532; found 492.18494.
HA-succ(tmd)-Phe-Ahx-Lys(Biotin)-NH2 (2a): This compound was
synthesized on solid support on 20 µmol scale [based on the load-
ing of Fmoc-Lys(Biotin)] following the general procedure for solid-
phase synthesis. The final coupling step involved the addition of
compound 24 (70 µmol, 50 mg) and DIEA (40 µmol, 90 µL 0.45 
in NMP) in NMP (0.40 mL) to the resin and shaking for 2 h. The
compound was purified by HPLC [system B, gradient 10% Ǟ 90%
ACN/(0.1 % TFA/H2O)] and was obtained as a colourless solid
(yield: 4.3 mg, 6.0 µmol, 30% after 3 coupling steps). 1H NMR
(400 MHz, [D6]DMSO): δ = 10.38 (s, 1 H, NHOH), 8.25 (br. s, 1
H, NHOH), 8.13 (t, J = 8.72, 8.72 Hz, 1 H, NH-Phe), 7.80–7.72
(m, 4 H, NH-Ahx + α-NH-Lys), 7.65 (t, J = 5.58 Hz, 1 H, ε-NH-
Lys), 7.37–7.13 (m, 8 H, Ph), 7.10 (t, J = 7.74 Hz, 2 H, Ph), 6.94
(br. s, 1 H, 0.5ϫ NH2), 6.42 (br. s, 1 H, NH-Biotin), 6.36 (br. s, 1
H, NH-Biotin), 4.35–4.32 (m, 2 H, CHα-Phe + NCH-Biotin), 4.18–
4.09 (m, 2 H, CHα-Lys + NCH-Biotin), 3.09 (dd, J1 = 12.83, J2 =
6.04 Hz, 1 H, SCH), 3.03–2.87 (m, 6 H, 0.5ϫ CHα + 0.5ϫ PhCH2
+ NCH2-Ahx + NCH2-Lys), 2.82 (dd, J1 = 12.40, J2 = 5.07 Hz, 1
H, 0.5ϫ CH2S), 2.78–2.72 (m, 1 H, 0.5ϫ CHα), 2.68–2.66 (m, 1
0.1  aq. HCl solution (2ϫ10 mL) and brine, dried with MgSO4 H, 0.5ϫ PhCH2), 2.64–2.60 (m, 1 H, 0.5ϫ PhCH2), 2.60 (d, J =
and concentrated under reduced pressure. The resulting mixture
was purified by column chromatography (10% Ǟ 50% EtOAc/pe-
troleum ether) and the product was obtained as a colourless oil
12.4 Hz, 1 H, 0.5ϫ CH2S), 2.13 (t, J = 7.35 Hz, 2 H, CH2,α-Ahx),
2.07 (t, J = 7.60 Hz, 2 H, CH2,α-Biotin), 2.02–1.96 (m, 1 H, 0.5ϫ
CH2,α) 1.91 (dd, J1 = 13.98, J2 = 7.50 Hz, 1 H, 0.5ϫ CH2,α), 1.65–
(yield: 38 mg, 54 µmol, 45%). 1H NMR (400 MHz, CDCl3): δ = 1.56 (m, 1 H, 0.5ϫ CH2-Lys), 1.52–1.40 (m, 9 H, 0.5ϫ CH2-Lys
7.29–7.17 (m, 7 H, Ph), 7.06 (d, J = 8.0 Hz, 2 H, Ph), 6.14 (d, J =
+ 2ϫ CH2-Ahx+ 2ϫ CH2-Biotin), 1.40–1.22 (m, 6 H, 2ϫ CH2-
8.0 Hz, 1 H, NH), 5.29–5.28 (m, 1 H, NH), 4.50 (q, J = 6.9 Hz, 1 Lys, CH2-Biotin), 1.20–1.12 (m, 2 H, CH2-Ahx) ppm. LC-MS (sys-
H, NCHα), 3.21–3.11 (m, 2 H, CHα + 0.5ϫ PhCH2), 2.97–2.85 (m, tem A): gradient 10% Ǟ 90% ACN/(0.1% TFA/H2O): Rt (min):
4 H, PhCH2 + 0.5ϫ PhCH2 + 0.5ϫ CH2,α), 2.77–2.69 (m, 1 H, 6.45 (ESI-MS: m/z = 945.53 [M + H+]). HRMS: calcd. for
0.5ϫ CH2,α), 2.57 (d, J = 4.8 Hz, 3 H, NCH3), 1.54 (s, 9 H, 3ϫ C44H59F3N10O8S [M + H]+ 945.42629; found 945.42682.
CH3), 0.99 (s, 9 H, 3ϫ CH3), 0.14 (s, 3 H, SiCH3), 0.11 (s, 3 H,
(2R)-N1-[(2S)-1-{6-[(S)-1,6-Diamino-1-oxohexan-2-ylamino]-6-
SiCH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 173.6, 170.8,
oxohexylamino}-1-oxo-3-phenylpropan-2-yl]-N4-hydroxy-2-{4-[3-(tri-
170.7, 151.8, 140.7, 136.7, 129.5, 128.6, 127.5, 126.9, 126.5, 122.1
fluoromethyl)-3H-diazirin-3-yl]benzyl}succinamide or HA-succ-
(tmd)-Phe-Ahx-Lys-NH2 (30): This compound was synthesized on
(q, J = 273 Hz, CF3), 84.8, 54.4, 44.5, 39.9, 37.9, 37.6, 29.7, 28.3
(q, J = 40.1 Hz, CNN), 28.0, 26.0, 25.7, 18.1, –4.9 ppm. [α]2D3
=
solid support on 175 µmol scale [based on the loading of Fmoc-
Lys(Boc)] following the general procedure for solid-phase synthesis.
The final coupling step involved the addition of compound 24
(1.12 mmol, 800 mg) and DIEA (750 µmol, 124 µL 0.45  in NMP)
in NMP (10 mL) to the resin and shaking for 2 h. The compound
was purified by HPLC [system B, gradient 10% Ǟ 90% ACN/
(0.1% TFA/H2O)] and was obtained as a colourless solid (yield:
+5.6 (c = 1, CHCl3). LC-MS (system A): = gradient 50 Ǟ 90 ACN/
(0.1 TFA/H2O): Rt (min): 9.81 [ESI-MS: m/z = 705.87 [M + H+]].
HRMS: calcd. for C34H46F3N5O6Si [M + H]+ 706.32422; found
706.32419.
(2R)-N4-Hydroxy-N1-[(2S)-1-(methylamino)-1-oxo-3-phenylpropan-
2-yl]-2-{4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}succinamide
(27): TFA (1 mL) and deionised H2O (50 µL) were added to a solu-
tion of compound 26 (21 mg, 30 µmol) in DCM (1 mL). After 1 h
of stirring LC-MS and TLC analysis indicated complete conversion
of the starting material. Toluene was added and the mixture was
concentrated under reduced pressure. In order to remove excess
TFA the mixture was co-evaporated twice with toluene. The re-
sulting crude mixture was purified by HPLC [system B, gradient
1
45 mg, 54 µmol, 30% after 3 coupling steps). H NMR (400 MHz,
CD3OD): δ = 7.69 (t, J = 5.62 Hz, 1 H, NH), 7.38–7.34 (m, 1 H,
NH), 7.29–7.16 (m, 9 H, Ph + 2ϫ NH), 7.10 (d, J = 8.03 Hz, 2 H,
Ph), 4.43 (t, J = 7.49 Hz, 1 H, NCHα-Phe), 4.35 (dd, J1 = 8.89, J2 =
5.32 Hz, 1 H, NCHα-Lys), 3.15–3.05 (m, 3 H, NCH2-Ahx + 0.5ϫ
PhCH2-Phe), 3.04–2.99 (m, 1 H, CHα), 2.98–2.83 (m, 4 H, NCH2-
Lys + 0.5ϫ PhCH2-Phe + 0.5ϫ PhCH2), 2.70 (dd, J1 = 13.62, J2
40% Ǟ 65% ACN/(0.1% TFA/H2O)]. The title compound was ob- = 6.17 Hz, 1 H, 0.5ϫ PhCH2), 2.33–2.24 (m, 3 H, CH2,α-Ahx +
tained as a colourless solid (yield: 10.2 mg, 21 µmol, 69%). 1H
NMR (400 MHz, [D6]DMSO): δ = 10.44 (s, 1 H, NHOH), 8.78
0.5ϫ CH2,α), 2.11 (dd, J1 = 14.82, J2 = 6.37 Hz, 1 H, 0.5ϫ CH2,α),
1.87 (ddd, J1 = 14.60, J2 = 7.64, J3 = 4.70 Hz, 1 H, 0.5ϫ CH2-
(br. s, 1 H, NHOH), 8.17 (d, J = 8.0 Hz, 1 H, NH), 7.65–7.58 (m, Lys), 1.78–1.68 (m, 3 H, 0.5ϫ CH2-Lys + CH2-Lys), 1.57 (dd, J1
1 H, NH), 7.30–7.21 (m, 7 H, Ph), 7.14 (d, J = 8.0 Hz, 2 H, Ph), = 14.95, J2 = 7.45 Hz, 2 H, CH2-Ahx), 1.55–1.45 (m, 2 H, CH2-
Eur. J. Org. Chem. 2010, 2100–2112
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2109

H. S. Overkleeft, R. Bischoff et al.
FULL PAPER
Lys), 1.44–1.36 (m, 2 H, CH2-Ahx), 1.30–1.18 (m, 2 H, CH2-Ahx)
ppm. 13C NMR (100 MHz, CD3OD): δ = 177.0, 176.5, 173.8,
173.7, 171.2, 143.4, 139.5, 131.8, 131.3, 130.3, 128.9, 128.6, 128.4,
57.2, 54.9, 46.5, 41.4, 41.0, 39.4, 39.4, 37.4, 36.5, 33.4, 30.7, 28.9,
28.2, 27.2, 24.7 ppm. LC-MS (system A): gradient 10% Ǟ 90%
ACN/(0.1% TFA/H2O): Rt (min): 5.92 (ESI-MS: m/z = 719.27 [M
+ H+]). HRMS: calcd. for C34H45F3N8O6 [M + H]+ 719.34869;
found 719.34848.
HA-succ(tmd)-Phe-Ahx-Lys(Bodipy-Tmr)-NH2 (2b): Compound 30 Asp697, Murine myeloma cell line, NS0 derived), ADAM-10 [Cata-
(9.1 mg, 11 µmol) and Bodipy-Tmr-OSu[47] (4.6 mg, 12 µmol) were log Number 936-AD, catalytic domain of human ADAM-10
dissolved in DMF (0.5 mL). DIEA (4.5 µL, 27 µmol) was added
and the reaction was stirred for 24 h after which the solvent was
evaporated under reduced pressure. The resulting mixture was puri-
33.2 Hz, 2 F) ppm. LC-MS (system A): gradient 10% Ǟ 90%
ACN/(0.1% TFA/H2O): Rt (min): 8.13 (ESI-MS: m/z = 993.33 [M
+ H+]). HRMS: calcd. for C48H58BF5N10O7 [M + H]+ 993.45759;
found 993.45864.
Inhibition and Labeling Studies: Recombinant ADAM-8 (Catalog
Number 1031-AD, catalytic domain of human ADAM-8 Glu158
-
Pro497, Murine myeloma cell line, NS0 derived), ADAM-9 (Catalog
Number 939-AD, catalytic domain of human ADAM-9 Ala206
-
Thr214-Glu672, Spodoptera frugiperda, Sf 21 (baculovirus) derived]
and ADAM-17 [ectodomain, Catalog Number 930-ADB, catalytic
domain of human ADAM-17 Arg215-Asn671, Spodoptera frugip-
fied by HPLC [system B, gradient 10% Ǟ 90% ACN/(0.1% TFA/ erda, Sf 21 (baculovirus) derived] were purchased from R&D sys-
H2O)] and the title compound was obtained as a brown solid (yield: tems (Minneapolis, MN, USA). ADAM-8 was autocatalytically ac-
3.4 mg, 3.4 µmol, 31%). 1H NMR (400 MHz, [D6]DMSO): δ = tivated by incubation at 37 °C for 5 d according to the manufactur-
10.38 (s, 1 H, NHOH), 8.73 (s, 1 H, NHOH), 8.15 (d, J = 8.0 Hz, er’s instructions. Recombinant catalytic domains (CD) of human
1 H, NH-Phe), 7.86 (d, J = 8.87 Hz, 2 H, Ph-Bodipy), 7.85 (t, J =
5.20 Hz, 1 H, NH-Ahx), 7.81 (d, J = 8.40 Hz, 1 H, α-NH-Lys) 7.67
MMP-1 (Catalog Number BML-SE180–0010, catalytic domain of
human MMP-1 Phe100-Gln268), MMP-2 (Catalog Number BML-
(s, 1 H, CHar-Bodipy), 7.68 (t, J = 5.49 Hz, 1 H, ε-NH-Lys), 7.32 SE237–0010, catalytic domain of human MMP-2 Tyr110-Asp452),
(s, 1 H, 0.5ϫ NH2), 7.30–7.11 (m, 10 H, Ph + CHar-Bodipy), 7.02
(d, J = 8.91 Hz, 2 H, Ph-Bodipy), 6.93 (s, 1 H, 0.5ϫ NH2), 6.70
(d, J = 4.02 Hz, 1 H, CHar-Bodipy), 4.41–4.30 (m, 1 H, CHα-Phe),
4.13 (dt, J1 = 8.80, J2 = 5.79 Hz, 1 H, CHα-Lys), 3.82 (s, 3 H,
MMP-3 (Catalog Number BML-SE109–0010, catalytic domain of
human MMP-3 Phe100-Thr272), MMP-7 (Catalog Number BML-
SE181–0010, catalytic domain of human MMP-7 Tyr95-Lys267),
MMP-8 (Catalog Number BML-SE255–0010, catalytic domain of
OCH3), 3.04–2.83 (m, 6 H, NCH2-Ahx + CHα + NCH2-Lys + human MMP-8 Phe99-Gln269), MMP-10 (Catalog Number BML-
0.5ϫ PhCH2-Phe), 2.82–2.70 (m, 2 H, 0.5ϫ PhCH2-Phe + 0.5ϫ
PhCH2), 2.64–2.58 (m, 3 H, 0.5ϫ PhCH2 + PhCH2-Bodipy), 2.48 MMP-11 (Catalog Number BML-SE282–0010, catalytic domain of
SE329–0010, catalytic domain of human MMP-10 Phe99-Glu271),
(s, 3 H, CH3), 2.24 (s, 3 H, CH3), 2.23 (t, J = 6.8 Hz, 2 H,
CH2,α-Bodipy), 2.14–2.07 (m, 3 H, 0.5ϫ CH2,α + CH2,α-Ahx), 1.88
(dd, J1 = 15.01, J2 = 7.43 Hz, 1 H, 0.5ϫ CH2,α), 1.68–1.58 (m, 1
H, 0.5ϫ CH2-Lys), 1.54–1.42 (m, 3 H, 0.5ϫ CH2-Lys + CH2-Ahx),
human MMP-11 Phe98-Ser266) and MMP-13 (Catalog Number
BML-SE246–0010, catalytic domain of human MMP-13 Tyr104
-
Asn274) were from Biomol International (Butler Pike, PA, USA).
All hrMMPs were expressed in E. coli. Recombinant human
1.39–1.11 (m, 8 H, 2ϫ CH2-Ahx + 2ϫ CH2-Lys) ppm. 19F NMR MMP-12 CD and recombinant human MMP-9 CD without fib-
(376 MHz, [D6]DMSO): δ = –64.09 (s, 3 F), –136.82 (q, J =
32.8 Hz, 2 F) ppm. LC-MS (system A): gradient 10% Ǟ 90%
ACN/(0.1% TFA/H2O): Rt (min): 8.84 (ESI-MS: m/z = 1099.40 [M
ronectin type II inserts (expressed in E. coli as described[51,52]) were
a kind gift from AstraZeneca R&D (Lund & Moelndal, Sweden).
TIMP-1 from human neutrophil granulocytes was from Calbi-
+ H+]). HRMS: calcd. for C55H64BF5N10O8 [M + H]+ 1099.49946; ochem (La Jolla, CA, USA). Alkaline phosphatase conjugated
found 1099.50039.
streptavidin was from Sigma–Aldrich (Zwijndrecht, The Nether-
lands). 5-Bromo-4-chloro-3-indoyl phosphate (BCIP) and nitro
blue tetrazolium (NBT) were from Duchefa (Haarlem, The Nether-
lands). Unless mentioned otherwise all other biochemicals were
from Sigma–Aldrich.
HA-succ(tmd)-Phe-Ahx-Lys(Bodipy-FL)-NH2 (2c): Compound 30
(9.1 mg, 11 µmol) and Bodipy-FL-OSu[48] (4.6 mg, 12 µmol) were
dissolved in DMF (0.5 mL). DIEA (4.5 µL, 27 µmol) was added
and the reaction was stirred for 24 h after which the solvent was
evaporated under reduced pressure. The resulting mixture was puri-
fied by HPLC [system B, gradient 10% Ǟ 90% ACN/(0.1% TFA/
H2O)] and the title compound was obtained as an orange solid
(yield: 5.9 mg, 6.0 µmol, 55%). H NMR (400 MHz, [D6]DMSO):
δ = 10.38 (s, 1 H, NHOH), 8.73 (s, 1 H, NHOH), 8.12 (d, J =
8.23 Hz, 1 H, NH-Phe), 7.91 (t, J = 5.35 Hz, 1 H, NH-Ahx), 7.80
Determination of IC50 Values: The affinity of the photoactivatable
probes for ADAM and MMP proteases was determined in a com-
petitive enzyme activity assay monitoring conversion of the fluo-
rogenic substrate Mca-PLAQAV-Dpa-RSSSR-NH2 (R&D Sys-
tems) by recombinant ADAM-9, -10 and -17 in the presence of
increasing concentrations photoactivatable probe. For MMP-9 and
1
(d, J = 8.15 Hz, 1 H, α-NH-Lys), 7.68 (s, 1 H, CHar-Bodipy), 7.65 MMP-12 inhibition of the conversion of fluorogenic substrate
(t, J = 5.49 Hz, 1 H, ε-NH-Lys), 7.31 (s, 1 H, 0.5ϫ NH2), 7.27– Mca-PLGL-Dpa-AR-NH2 (Bachem, Bubendorf, Switserland) was
7.13 (m, 7 H, Ph), 7.11–7.07 (m, 3 H, CHar-Bodipy + Ph), 6.95 (s,
1 H, 0.5ϫ NH2), 6.34 (d, J = 3.99 Hz, 1 H, CHar-Bodipy), 6.29 (s,
determined. Measurements were performed in Costar White 96-
well plates (Corning, Schiphol-Rijk, The Netherlands), where each
1 H, CHar-Bodipy), 4.34 (dd, J1 = 14.65, J2 = 8.31 Hz, 1 H, CHα- well contained either 10 ng ADAM-17, 100 ng ADAM-10 or 200
Phe), 4.15 (dt, J1 = 8.29, J2 = 5.47 Hz, 1 H, CHα-Lys), 3.15–3.03
(m, 4 H, NCH2-Ahx + PhCH2-Bodipy), 3.01–2.90 (m, 4 H, CHα
ng ADAM-9 and a final concentration of 10 µ substrate in a final
volume of 100 µL ADAM assay buffer (25 m Tris pH 9.0, 2.5 µ
+ NCH2-Lys + 0.5ϫ PhCH2-Phe), 2.83–2.77 (m, 2 H, 0.5ϫ ZnCl2, 0.005% w/v Brij-35). Inhibition of MMP proteolytic activity
PhCH2-Phe + 0.5ϫ PhCH2), 2.62 (dd, J1 = 13.6, J2 = 6.0 Hz, 1 H,
+ 0.5ϫ PhCH2), 2.50 (s, 3 H, CH3), 2.49 (t, signal under DMSO
was determined with 10 ng of MMP-9 or MMP-12 per well with a
final concentration of 2 µ substrate in 100 µL MMP assay buffer
signal, 3 H, CH3-Bodipy) 2.29 (s, 3 H, CH3-Bodipy), 2.18–2.06 (m, [50 m Tris pH 7.4, 0.2  NaCl, 10 m CaCl2, 2.5 µ ZnCl2,
3 H, 0.5ϫ CH2,α + CH2,α-Ahx), 1.88 (dd, J1 = 15.00, J2 = 7.51 Hz,
1 H, 0.5ϫ CH2,α), 1.70–1.60 (m, 1 H, 0.5ϫ CH2-Ahx), 1.57–1.38
(m, 5 H, 0.5ϫ CH2-Ahx + 2ϫ CH2-Ahx), 1.37–1.23 (m, 4 H, 2ϫ
CH2-Lys), 1.22–1.11 (m,
(376 MHz, [D6]DMSO): δ = –64.09 (s, 3 F), –142.77 (q, J =
0.05% (v/v) Brij-35]. Proteolysis rates were followed by measuring
fluorescence (λex,em = 320, 440 nm) increase using a Fluostar Op-
tima plate reader (BMG Labtech, Offenburg, Germany) at 37 °C.
Six-point inhibition curves (0–10 µ) were plotted in Origin 7.0
(Micronal) and IC50 values were determined by sigmoidal fitting.
2
H, CH2-Ahx) ppm. 19F NMR
2110
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 2100–2112

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Acknowledgments
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Financial support for this work was provided by the Technology
Foundation STW (STW grants GPC 6150 and 08008), the Nether-
lands Organisation for Scientific Research (NWO) and the Nether-
lands Genomics Initiative (NGI). We thank Fons Lefeber and Kees
Erkelens for NMR assistance.
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2111

H. S. Overkleeft, R. Bischoff et al.
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Received: November 30, 2009
[44] Compound 27 is a close analogue of a reported potent and
Published Online: February 19, 2010
broad-spectrum metalloprotease inhibitor featuring the leucine
2112
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Eur. J. Org. Chem. 2010, 2100–2112