Proc. Natl. Acad. Sci. USA
`Vol. 95, pp. 14020-14024, November 1998
`Chemistry
`
`Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing
`N-peptidyl-0-hydroxylamine peptidomimetics
`
`JIAN LIN, PAUL J. ToscANo, AND JOHN T. WELCI-1*
`Department of Chemistry, University at Albany, Albany, NY 12222
`
`Communicated by George A. Ola/z, University of Southern California, Los Angeles, CA, August 10, 1998 (received for review April 8, 1998)
`
`Dipeptidyl peptidase IV (EC 3.4.14.5; DPP
`ABSTRACT
`IV), also known as the leukocyte differentiation antigen CD26
`when found as an extracellular membrane-bound proline
`specific serine protease, cleaves a dipeptide from the N
`terminus of a polypeptide chain containing a proline residue
`in the penultimate position. Here we report
`that known
`(Z) -Ala-1[1[CF=C]-Pro dipeptide isosteres 1 and 2, which
`contain 0-acylhydroxylamines, were isolated as diastereo-
`meric pairs u-1,1-1, and 1-2. The effect of each diastereomeric
`pair as an inhibitor of human placental dipeptidyl peptidase
`DPP IV has been examined. The inhibition of DPP IV by these
`compounds is rapid and efficient. The diastereomeric pair u-1
`exhibits very potent inhibitory activity with a K, of 188 nM.
`Fluoroolefin containing N-peptidyl-0-hydroxylamine pep-
`tidomimetics, by virtue of their inhibitory potency and sta-
`bility, are superior to N-peptidyl-0-hydroxylamine inhibitors
`derived from an Ala-Pro dipeptide.
`
`Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV; CD26), dis-
`covered in 1966 (1),
`is a transmembrane serine peptidase
`found in a variety of human tissues and organs (2-4). In
`particular, DPP IV, when expressed on the surface of CD4+ T
`cells,
`is identical with the leukocyte differentiation antigen
`CD26 and is considered to be a lymphocyte activation marker
`(5, 6). Although the involvement of DPP IV in the immune
`response and regulation of lymphocyte activation has been
`implicated, the mechanism of the involvement is not clear (7,
`8). Of the many functions that have been postulated (9-14), the
`most intriguing is the role of DPP IV in T-cell activation and
`in the regulation of T-cell proliferation (13, 15-18). Recog-
`nized as a cell surface activation marker of lymphocytes (19),
`the failure to observe CD26 implies a reduced immune re-
`sponse (20). The presence of DPP IV is associated with the
`capacity of cells to produce interleukin 2 and to proliferate
`strongly in response to mitogen stimulation (20, 21). Impor-
`tantly, binding of mAbs to CD26 suppresses interleukin 2
`production (21). CD26 modulation also can lead to enhanced
`cell proliferation preceded by an increase in Ca“ mobilization
`(22). CD26 is associated physically with CD45, which regulates
`T-cell activation pathways through protein tyrosine phospha-
`tase action. CD26 apparently modulates the activity of CD45
`by affecting the accessibility of critical substrates, with the
`result that the CD2 /CD3 path amplifies the immune response
`(23). DPP IV, known to be localized on the surface of T cells
`with adenosine deaminase, seems to form a complex with
`adenosine deaminase that is involved in an important immu-
`noregulatory mechanism involving T-cell proliferation (24,
`25). DPP IV appears to be not only up—regulated among
`proliferating thymocytes but also by those undergoing pro-
`grammed cell death (26). The involvement of CD26 in HIV
`infection has been the subject of investigation for some time,
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked “advertisement” in
`accordance with 18 U.S.C. §1734 solely to indicate this fact.
`© 1998 by The National Academy of Sciences 0027-8424/98/9514020—5$2.00/O
`PNAS is available online at www.pnas.org.
`
`with the initial report (27) that CD26 was a cofactor facilitating
`HIV entry in CD4+ cells refuted (28-30). Reports that DPP
`IV enzymatic activity may decrease the efficiency of HIV
`infection (31) may be related to the binding of HIV glyco-
`proteins gp120 and gp41, which have been shown to be
`responsible for cell killing by apoptosis in CD4+ cells (32). The
`Tat protein of HIV-1, known to be capable of suppressing CD3
`activation of T cells, also has been shown to bind to DPP IV
`with effects on cytokine production and DNA synthesis,
`implying that the DPP IV plays a role in Tat immunosuppres-
`sion (33, 34).
`DPP IV will cleave the dipeptides Xaa—Pro from the N
`terminus of a polypeptide while recognizing several key struc-
`tural features in substrate proteins or peptides. It has been
`postulated (35) that DPP IV substrates require the presence of
`a proline at the P1 position as well as a protonated free N
`terminus (36, 37). It also has been proposed that DPP IV
`possesses a high conformational specificity for a trans amide
`bond between the P1 and N-terminal P2 residues (38). There
`is the additional requirement for the L configuration of the
`amino acid residue, both in the penultimate and the N-terminal
`position (39, 40).
`Obviously, inhibition of CD26 may critically affect T-cell
`activation and function and may potentially have therapeutic
`utility in the modulation of the immune response. Relatively
`few of the compounds reported thus far are effective inhibitors
`of DPP IV, with most inhibitors suffering from either insta-
`bility or low reactivity. N-Peptidyl-0-acylhydroxylamines irre-
`versibly inhibit DPP IV, but most of the inhibitor is enzyme-
`hydrolyzed during the inactivation process (41, 42). The bo-
`ronic acids Ala-boroPro, Pro-b0r0Pro, and Val-b0r0Pro are
`potent and specific reversible inhibitors of DPP IV with K,
`values in the nanomolar range. However, these compounds
`lose their inhibitory activity in aqueous solution at neutral pH
`because of the formation of cyclic species in which the
`N-terminal amine nitrogen coordinates to the boron atom (37,
`43-45). Peptidyl (waminoalkyl) phosphonate esters (46) and
`diphenyl phosphonate esters (47) are moderate and specific
`DPP IV inhibitors. These compounds are quite stable because
`phosphonate esters are relatively unreactive with nitrogen
`nucleophiles or N-terminal amines. Aminoacylpyrrolidine-2-
`nitriles (48) and 4-cyanothiazolidides (49, 50) recently were
`reported as very potent and rather stable inhibitors of DPP IV.
`They were found to have K, values in the nanomolar to low
`submicromolar range and half-lives between 27 and 72 h.
`Many of the problems associated with inefficient inactiva-
`tion of DPP IV are a consequence of the importance of the
`trans conformation of the P1-P2 amide bond and the require-
`ment for a protonated free N terminus. The cyclization
`
`Abbreviation: DPP IV, dipeptidyl peptidase IV.
`Data deposition: The atomic coordinates and structure factors have
`been deposited in the Cambridge Crystallographic Data Centre, 12
`Union Road, Cambridge, CB2 IEZ, United Kingdom (reference
`103375).
`*To whom reprint requests should be addressed at: Department of
`Chemistry, University at Albany, 1400 Washington Avenue, Albany,
`NY 12222. e—mail: JTW06@cnsvax.albany.edu.
`AURO - EXHlB|T 1015
`
`14020
`
`

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`Chemistry: Lin et al.
`
`Proc. Natl. Acad. Sci. USA 95 (1998)
`
`14021
`
`reaction of the free N-terminal amino group with the reactive
`site of the inhibitor does, however, require the molecule to
`assume the cis conformation, the conformation previously
`proposed to be unreactive with DPP IV (38). To obviate this
`mode of inactivation and to rigorously examine the cis–trans
`selectivity of DPP IV, we have prepared a series of confor-
`mationally constrained fluoroolefin dipeptide isosteres. The
`fluoroolefin dipeptide isostere was proposed as early as 1984
`(51) as a superior isoelectronic and isosteric replacement for
`the amide bond. Theoretical studies strongly have supported
`the original hypothesis behind introduction of the fluoroolefin
`amide surrogate (52–54). The syntheses of fluoroolefin dipep-
`tide surrogates, Ala-c[CF5C]-Pro containing N,O-diacylhy-
`droxamic acid type protease inhibitors 1 and 2, were reported
`recently by our laboratory (55).
`
`SCHEME 1.
`
`MATERIALS AND METHODS
`Materials. Human placenta dipeptidyl peptidase IV (EC
`3.4.14.5) was purchased from Calbiochem–Novabiochem (La
`Jolla, CA). The specific activity is 8,333 milliunits per milli-
`gram of protein. One milliunit, specified by Calbiochem–
`Novabiochem, is defined as the amount of enzyme that will
`hydrolyze 1.0 mM of Ala-Pro-7-amino-4-trifluoromethyl cou-
`marin per minute at 30°C, pH 7.8. The DPP IV substrate
`Gly-Pro-p-nitroanilide was obtained from Sigma. The phos-
`phate buffer (KH2PO4-NaHPO4, 90 mM, pH 7.6) and TriszHCl
`buffer (20 mM, pH 7.8) were prepared in our laboratory.
`1H, 13C, 19NMR spectra were recorded on a Gemini-300
`NMR spectrometer (Varian) with CD3OD as solvent and
`residual methanol or CFCl3 as the internal standard. Thin layer
`chromatography was performed with F254 (Merck) silica gel as
`the adsorbent on 0.2-mm thick, plastic-backed plates. The
`chromatograms were visualized under UV (254 nm) and by
`spraying with a 95:5 mixture of 0.2% ninhydrin in n-butanol
`and 10% aqueous acetic acid followed by heating. The UV-
`visible spectra were determined by using a Shimadzu UV-
`visible recording spectrophotometer (UV-160).
`Syntheses of Inhibitors 1–2. The general procedure for
`amine deprotection is shown in Scheme 3. Compound l-4 (14.7
`mg, 0.025 mmol), prepared as described (55), was treated with
`1 M HCl in acetic acid (1 ml), stirring at room temperature for
`1–2 h. The solvent was removed under high vacuum. Diethyl
`ether (2 ml) was added to the residue. This mixture was stored
`at 24°C overnight. After the supernatant was decanted, the
`resultant white solid was washed with ether, and dried, to yield
`the sufficiently pure hydrochloride salts (Z)-(R, R), (Z)-(S,
`S)-1-[(19-f luoro-29-amino)propylidene]-2-cyclopentane-O-
`benzoyl hydroxamate hydrochloride (l-1) (10.7 mg, 86%). Data
`for l-1: 19NMR (CD3OD) d-124.37 (d, J 5 27.1 Hz); 1H NMR
`(CD3OD) d 8.07 (d, 2H, J 5 7.3 Hz), 7.69 (t, 1H, J 5 7.4 Hz),
`7.36 (t, 2H, J 5 7.8 Hz), 4.29 (dq, 1H, J 5 27.2, 6.9 Hz),
`3.66–3.58 (m, 1H), 2.55–2.45 (m, 2H), 2.39–1.96 (m, 3H),
`
`1.84–1.72 (m, 1H), 1.47 (d, 3H, J 5 6.8 Hz); 13C NMR
`(CH3OD) d 171.81, 164.08, 148.52 (d, J 5 249.7 Hz), 133.67,
`129.14, 128.29, 126.74, 123.34 (d, J 5 13.6 Hz), 44.71, 43.23 (d,
`J 5 27.6 Hz), 31.76, 28.03 (d, J 5 1.8 Hz), 25.26, 14.46.
`(Z)-(R, S), (Z)-(S, R)-1-[(19-fluoro-29-amino)propylidene]-
`2-cyclopentane-O-benzoyl hydroxamate hydrochloride (u-1)
`was prepared in the same manner from u-4 in 51% yield. Data
`for u-1: 19NMR (CD3OD) d-124.42 (d, J 5 24.4 Hz); 1H NMR
`(CD3OD) d 8.07 (d, 2H, J 5 8.5 Hz), 7.69 (t, 1H, J 5 7.5 Hz),
`7.53 (t, 2H, J 5 7.3 Hz), 4.29 (dq, 1H, J 5 26.8, 6.9 Hz),
`3.68–3.59 (m, 1H), 2.59–2.39 (m, 2H), 2.26–1.93 (m, 4H),
`1.84–1.68 (m, 1H), 1.49 (d, 3H, J 5 6.9 Hz); 13C NMR
`(CD3OD) d 172.84, 165.57, 149.98 (d, J 5 249.7 Hz),135.13,
`130.67, 129.78, 128.30, 124.69 (d, J 5 13.7 Hz), 47.35 (d, J 5
`27.5 Hz), 45.30, 33.20, 29.67 (d, J 5 3.2 Hz), 26.99, 16.32.
`(Z)-(R, R), (Z)-(S, S)-1-[(19-fluoro-29-amino)propylidene]-
`2-cyclopentane-(4-nitro)-O-benzoyl hydroxamate hydrochlo-
`ride (l-2) was prepared in the same manner from l-5 in 63%
`yield. Data for l-2: 19NMR (CD3OD) d-124.33 (d, J 5 27.1 Hz);
`1H NMR (CD3OD) d8.39 (d, 2H, J 5 9.1 Hz), 8.29 (d, 2H, J 5
`9.0 Hz), 4.29 (dq, 1H, J 5 27.1, 6.9 Hz), 3.67–3.59 (m, 1H),
`2.62–2.40 (m, 2H), 2.26–2.11 (m, 1H), 2.10–1.93 (m, 2H),
`1.87–1.70 (m, 1H), 1.48 (d, 3H, J 5 6.9 Hz); 13C NMR
`(CD3OD) d 173.22, 164.03, 152.51, 150.00 (d, J 5 249.7 Hz),
`133.66, 132.03, 124.84 (d, J 5 13.7 Hz), 124.83, 47.47 (d, J 5
`27.1 Hz), 45.07, 33.43, 29.70 (d, J 5 2.8 Hz), 26.93, 16.16.
`Crystal Structure Determination of l-3. Diastereomeric pair
`l-3 prepared as described (55) was recrystallized from a
`mixture of hexanes and ethyl acetate (1:1). Crystal data:
`C14H21FNO3, M 5 287.3, monoclinic, space group P21yn, a 5
`9.607 (4) Å, b 5 9.300 (3) Å, c 5 17.204 (6) Å, b 5 95.66 (3)
`°, V 5 1529.7 (9) Å3, Z 5 4, Dc 5 1.248 g cm21, m5 0.98 cm21,
`l(MoKa) 5 0.71073 Å, F(000) 5 616, T 5 298 K. Nicolet R3
`myV diffractometer was used to collect 2,038 reflections (3° ,
`2u , 45°) on a colorless crystal 0.15 3 0.15 3 0.40 mm3. Of
`these, 1,961 were unique and 1,193 were observed ( Fo . 6s
`Fo ). Lorentz and polarization corrections were applied to the
`data. The non-hydrogen atoms were located by direct methods.
`R 5 0.070, Rw 5 0.068, GOF 5 2.14.
`Inactivation Assays. Method A (inactivation in the absence
`of substrate): The inhibitory activity of the compounds, u-1,
`l-1, and l-2, was estimated from the residual activity of DPP IV
`in a solution of the substrate Gly-Pro-p-nitroanilide. An
`aliquot of inhibitor (20 ml, from 50 mM stock solution in water)
`was added to 80 ml of a buffered enzyme solution [0.2 milliunit
`in TriszHCl buffer (pH 7.6)] to initiate the inactivation reac-
`tion. The concentration of inhibitor in the incubation mixture
`(total volume 100 ml) was 10 mM. After the enzyme and
`inhibitor were incubated for either 2 or 30 min at 30°C, the
`incubation mixture was added to a 1-ml cuvette containing 900
`ml of substrate Gly-Pro-p-nitroanilide (0.1 mM) in 45 mM
`phosphate buffer (pH 7.6, m5 0.123). The measuring cell had
`been equilibrated thermally in the spectrophotometer for 2
`min before enzyme-inhibitor preincubation solution was
`added. The rate of change in UV absorbance at 385 nm, with
`respect to a cuvette containing only 0.1 mM substrate in 45 mM
`buffer, gave a straight line with the slope proportional to the
`enzyme activity. The residual enzyme activity is expressed
`relative to a DPP IV control, which was prepared by adding
`only enzyme to the substrate solution. The percentage inhi-
`bition (% I) was calculated as % I 5 [(1 2 viyvo)] 3 100%,
`where vi and vo are the rate of change in absorbance at 385 nm,
`with and without inhibitor, respectively. The percentage inhi-
`bition (% I) at other inhibition concentrations was measured
`by the same method.
`Method B (inactivation in the presence of substrate): To a
`cuvette containing 5 to 20 ml of appropriate concentration of
`inhibitor, 20 ml of 5 mM substrate Gly-Pro-p-nitroanilide, 500
`ml of 90 mM phosphate buffer (pH 7.6), and enough water to
`bring the final volume to 1 ml was added 20 ml of enzyme
`
`

`
`14022
`
`Chemistry: Lin et al.
`
`Proc. Natl. Acad. Sci. USA 95 (1998)
`
`pair (l-3). Obviously, compound 399, as ‘‘unlike’’ diastereo-
`meric pair (u-3), contains the S, R isomer corresponding to the
`natural amino acid L-(S)-Ala-L-(S)-Pro and would be predicted
`to have higher biological activity.
`The two diastereomeric pairs were converted independently
`to l-4 and u-4 after three step transformation (55). The analog
`l-5 was prepared in the same manner by using 4-nitrobenzoyl
`chloride instead of benzoyl chloride, as shown in Scheme 2.
`Removal of the Boc-groups was accomplished by using 1 M
`HCl in AcOH to give compounds 1 and 2 (Scheme 3).
`
`FIG. 1. ORTEP drawing of the x-ray structure of (Z)-l-Boc-Ala-
`c[CF5C]-Pro (l-3).
`
`solution (0.2 milliunit) in pH 7.6 Tris buffer. The rate of
`change in the absorbance at 385 nm, with respect to a cuvette
`containing the same amount of inhibitor and substrate in
`buffer, gave the inactivation progress curves. All inhibition
`experiments were monitored by using a Shimadzu UV-160 at
`385 nm and 30 6 0.1°C.
`Determination of Ki Values. For inhibitors u-1, the data for
`two Dixon plots (1yV vs. [I]) were obtained by repeating
`method B at two concentrations of substrate (0.2 mM and 0.4
`mM) and six to seven different inhibitor concentrations (0,
`0.25, 0.5, 0.75, 1.0, 5.0, and 10.0 mM). The correlation coeffi-
`cients of both lines were .0.994. The Ki value of u-1 was
`calculated according to the method of Dixon (56). The Ki value
`for compound l-1 was determined by the method described
`above.
`Determination of the Half-Life of Inhibitor u-1. An aliquot
`(40 ml) of inhibitor u-1 from 5 mM stock solution in H2O was
`added to a 1-ml cuvette containing 500 ml of 90 mM phosphate
`buffer solution (pH 7.6) and 460 ml of water at 30°C, such that
`the final inhibitor concentration was 0.2 mM. Spontaneous
`decomposition was monitored by following the decrease in
`absorbance at 229 nm at various time intervals. The absor-
`bance data points at 229 nm were recorded and plotted as
`function of time, which gave a spontaneous degradation curve.
`The half-life was obtained from first-order plot of ln(AyA0) vs.
`time, where A is the absorbance of the mixture at time t, and
`A0 is the absorbance at initial time (t 5 0).
`
`RESULTS AND DISCUSSION
`Chemistry. (Z)-N-tert-Butyloxycarbonyl-1-[(19-fluoro-29-
`amino)propylidene]-2-cyclopentane carboxylic acid 3 was syn-
`thesized and isolated as two diastereomeric pairs 39 and 399, as
`described in a previous report (55). The relative stereochem-
`istry of these diastereomeric pairs was determined by single
`crystal x-ray diffraction studies. The structure of compound 39,
`crystallized from a mixture of hexanes and ethyl acetate (1:1),
`is shown in Fig. 1. The absolute configurations at C1 and C8
`of 39 were confirmed as R and R or S and S, respectively.
`Therefore, 39 can be designated as the ‘‘like’’ diastereomeric
`
`SCHEME 2. 1. Im2CO; 2. NH2OHzHCl; 3. BzCl or (4-NO2)BzCl, Py,
`0°C.
`
`SCHEME 3.
`
`Inhibition of DPP IV. The results of initial inhibition studies of
`DPP IV by diastereomeric pairs u-1, l-1, and l-2 are shown in
`Table 1. At the same inhibitor concentration with 2-min incuba-
`tion time or 30-min incubation time, the percentages of inhibition
`of DPP IV by u-1, l-1, and l-2 unexpectedly remained the same
`or changed only slightly with an increase in incubation time.
`The results presented in Table 1 revealed the following
`phenomena: (i) Inactivation of DPP IV by u-1 did not follow
`pseudo-first order reaction kinetics. The inactivation process
`was principally dependent on inhibitor concentration and
`independent of incubation time. At a concentration of 0.01
`mM, inhibitor u-1 showed nearly the same percentages of
`inhibition, 42% and 39% inhibition, at both 2 min and 30 min
`incubation time, respectively. At 0.25 mM, after both 2 min and
`30 min incubation time, 100% inhibition of the activity of DPP
`IV was observed. For inhibitors l-1 and l-2, the percentages of
`inhibition (% I) increased or remained the same within
`experimental error with increasing incubation time at the same
`inhibitor concentrations (shown in Table 1). (ii) Inhibitory
`potency of u-1 was much greater than that of the other
`diastereomeric pair l-1. At a concentration of 0.01 mM and a
`2-min incubation time, compound u-1 inhibited 42% of the
`enzymatic activity of DPP IV; however, the isomer l-1 was
`nearly ineffective (4% inhibition) under the same conditions.
`As mentioned above, DPP IV has an absolute requirement for
`the L configuration of the amino acid residue, both in penul-
`timate and N-terminal positions. Because the pair u-1 contains
`the compounds with the desired configuration (L, L), it was
`more reactive with DPP IV. (iii) Replacement of the benzoyl
`group (l-1) by a para-nitrobenzoyl group (l-2) enhanced the
`inhibitory activity slightly. This may be because the electron
`withdrawing group (4-NO2) facilitates the rate-determining
`N-O fission. (iv) Inhibitors u-1,
`l-1, and l-2 all exhibited
`
`

`
`Chemistry: Lin et al.
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`Proc. Natl. Acad. Sci. USA 95 (1998)
`
`14023
`
`Inhibition of DPP IV by fluoroolefin-containing
`Table 1.
`N-peptidyl-O-hydroxylamines
`
`Inhibitors
`I-1 (Z)-Ala-c[CF¢C]-Pro-NHO-Bz
`I-2 (Z)-Ala-c[CF¢C]-Pro-NHO-Bz(4-NO2)
`u-1 (Z)-Ala-c[CF¢C]-Pro-NHO-Bz
`
`%
`Inhibition*
`2
`30
`[I],
`min
`min
`mM
`4
`1
`0.01
`17
`25
`0.50
`22
`34
`0.50
`42
`39
`0.01
`100
`100
`0.25
`29†
`60
`1.10
`Ala-Pro-NHO-Bz(4-NO2)
`pPercentage inhibition was measured after 2- or 30-min incubation in
`45 mM phosphate buffer, pH 7.6, at 30°C. Gly-Pro-p-nitroanilide was
`used as substrate.
`†Incubation time was 10 min (41).
`
`inhibitory activity superior to the previously prepared Ala-
`Pro-NHO-Bz(4-NO2) compounds (41, 42) It has been pro-
`posed that the trans P2-Pro peptide bonds of the substrates are
`essential to the reactivity of enzyme DPP IV. The enhance-
`ment in inhibitory potency of our fluoroolefin containing
`dipeptide isosteres can be attributed to the efficient mimicking
`of the trans P1-P2 amide bonds of the original dipeptides by the
`(Z) fluoroolefin double bond conformation.
`The effect of inhibitor u-1 on the hydrolysis of substrate (S)
`by DPP IV (E) was demonstrated in two different ways:
`enzyme-initiated assay, (S 1 I) 1 E, and substrate initiated
`assay, (E 1 I) 1 S (Fig. 2). The results were unexpected. In
`both cases (curves B and C), the rates of hydrolysis of substrate
`by DPP IV increased linearly over 50 min and were nearly
`identical (curves B and C overlapped). The initial rate (u) of
`hydrolysis of substrate in an enzyme initiated assay is often
`larger than that found in a substrate initiated assay (57). The
`curves B and C shown in Fig. 1 indicate that inhibitor u-1 very
`rapidly inactivates DPP IV in a process much faster than the
`rate of hydrolysis of the substrate by DPP IV; thus, the
`presence of competing substrate did not slow down the inac-
`tivation process.
`To determine the values of the inhibition constant Ki for
`both compound u-1 and l-1, the rates of DPP IV-catalyzed
`hydrolysis of Gly-Pro-p-nitroanilide substrate were estimated
`at six to seven different concentrations for each inhibitor (0.25
`to 10 mM) in a competitive hydrolysis fashion. The Ki values
`reported in Table 3 for compounds u-1 and l-1 were obtained
`
`FIG. 2. Hydrolysis of substrate Gly-Pro-4-nitroanilide was monitored
`by the change in absorbance at 385 nm with time. In all cases, the final
`concentrations S, E, and I were 0.1 mM, 0.2 milliunit, and 1 mM,
`respectively.
`
`Table 2.
`
`Inhibition constants of inhibitors of DPP IV, I-1, and u-1
`Inhibitors
`Ki, nM
`I-1 (Z)-Ala-c[CF¢C]-Pro-NHO-Bz
`14,400
`u-1 (Z)-Ala-c[CF¢C]-Pro-NHO-Bz
`188
`Ala-Pro-NHO-Bz(4-NO2)
`30,000 (58)
`
`from the plots 1yv versus [I] according to the method of Dixon
`(56). The Dixon plot revealed that compounds u-1 and l-1 act
`as competitive inhibitors.
`As shown in Table 2, the diastereomeric pair u-1 (containing
`the L, L isomer) exhibited very potent inhibitory activity with
`a small Ki value in the nanomolar range (0.19 mM). The affinity
`of this isomer for DPP IV is two orders of magnitude greater
`than the other diastereomeric pair l-1 (Ki 5 14.4 mM). The
`Demuth’s inhibitor, Ala-Pro-NHO-Bz(4-NO2), also has a
`larger Ki value (30 mM) (58) and is a poorer inhibitor.
`Stability of Inhibitors. As shown in Table 3, inhibitor u-1
`was very stable in buffer (pH 7.6) with a decomposition rate
`constant kd of 1.1 3 1024 min21 and a half-life of 103 h. In
`contrast, the stability of inhibitor Ala-Pro-NHO-Bz(4-NO2)
`under assay conditions was limited because of a 10-fold higher
`decomposition rate constant kd (1.3 3 1023 min21), resulting
`in a shorter half-life of only 8.8 h (42).
`We believe that intramolecular cyclization is probably respon-
`sible for facile, spontaneous degradation of natural peptide-based
`hydroxamic acid inhibitors. In a manner similar to enzyme-
`induced N-O fission, the free amino group at the N terminus
`nucleophilically attacks the amide carbonyl carbon, thus forming
`a tetrahedral intermediate and thereby promoting N-O scission
`and subsequent generation of the reactive acylnitrene interme-
`diate. The six-membered cyclic intermediate is hydrolyzed further
`either to hydroxamic acid or diketopiperazine products (Scheme
`4). Clearly, the findings of our study support this postulate.
`Considerable improvement in stability of our compounds in
`buffer at neutral pH can be ascribed to the constrained double
`bond conformation of (Z)-fluoroolefin, excluding the possibility
`of intramolecular cyclization caused by the amide bond rotation
`in the dipeptides.
`l-1, and l-2,
`(Z)-Fluoroolefin-containing dipeptides u-1,
`designed as the mimics of N-peptidyl-O-acylhydroxylamines,
`have been synthesized and tested as inhibitors of dipeptidyl
`peptidase DPP IV. One diastereomeric pair u-1 exhibits very
`potent inhibitory activity with a Ki of 188 nM. The inhibitory
`potency of this isomer is ’70-fold higher than the other
`diastereomer l-1 (Ki 5 14, 400 nM). In comparison with the
`Ala-Pro-NHO-Bz(4-NO2) analog, the dipeptide isosteres 1
`and 2 are better inhibitors of DPP IV by virtue of their superior
`inhibitory potency and stability; presumably, the (Z) double
`bond conformation of the fluoroolefin dipeptide isosteres
`efficiently mimics the trans P2-Pro amide bonds of the original
`dipeptides. In addition, the rates of inactivation of DPP IV by
`compounds 1 and 2 appeared to be very fast. More detailed
`biological studies, kinetic analysis for inactivation rate con-
`stant kinact, and investigations of inhibition mechanism are in
`progress at present. The results of this study reveal that a series
`of known inhibitors of DPP IV such as dipeptide boronic acids
`(43–45), dipeptide phosphonates (46, 47), peptidyl nitriles
`(49–51), and others can be modified by replacing the amide
`bonds by fluoroolefin moieties. Because of the anticipated high
`affinity and stability, the fluoroolefin containing dipeptide
`peptiomimetics should prove to be very promising inhibitors of
`
`Table 3. Spontaneous degradation rate constants kd and
`half-life t1/2
`
`Inhibitors
`u-1 (Z)-Ala-c[CF¢C]-Pro-NHO-Bz
`Ala-Pro-NHO-Bz(4-NO2)
`
`kdz104, min21
`1.1
`13.0
`
`t1/2, h
`103 6 3
`8.8
`
`

`
`14024
`
`Chemistry: Lin et al.
`
`Proc. Natl. Acad. Sci. USA 95 (1998)
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`SCHEME 4.
`
`DPP IV and therefore helpful in elucidating the biological
`functions of DPP IV in T-cell activation. These agents may also
`be potential therapeutic agents useful in modifying and con-
`trolling the immune response.
`
`Dedicated to Professor Dieter Seebach on the occassion of his 60th
`birthday. Financial support of this work by the National Science
`Foundation Grant CHE 9413004 and National Institutes of Health
`Grant A133690 is gratefully acknowledged.
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