Replacing the pyrophosphate group of HMB-PP by a diphosphonate function abrogates its potential to activate human γδ T cells but does not lead to competitive antagonism

Article abstract of DOI:10.1016/S0960-894X(03)00138-0

The immunological characterization of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), and its methylenediphosphonate analogue, HMB-PCP, is described. With an EC₅₀ of 0.1-0.2 nM, HMB-PP is significantly more potent in stimulating human Vγ9/Vδ2 T cells than any other compound described so far. However, replacing the pyrophosphate by a P-CH₂-P function abrogates the bioactivity drastically, with HMB-PCP having a EC₅₀ of only 5.3 μM.

Full text of DOI:10.1016/S0960-894X(03)00138-0

Bioorganic & Medicinal ChemistryLetters 13 (2003) 1257–1260
Replacing the Pyrophosphate Group of HMB-PP by a
Diphosphonate Function Abrogates Its Potential to Activate
Human ꢀꢁ T Cells but does not Lead to Competitive
Antagonism
a
a
a
a
a
Armin Reichenberg, Martin Hintz, Yvonne Kletschek, Tanja Kuhl, Christian Haug,
b
b
c
a,b
b,
Rosel Engel, Jens Moll, DmitryN. Ostrovsk y, Hassan Jomaa and Matthias Eberl *
aJomaa Pharmaka GmbH, Frankfurter Str. 50, D-35392 Giessen, Germany
bBiochemisches Institut, Justus-Liebig-Universita¨t Giessen, Friedrichstr. 24, D-35392 Giessen, Germany
cBakh Institute of Biochemistry, Leninsky prospect 33, 119071 Moscow, Russia
Received 2 December 2002; accepted 24 January2003
Abstract—The immunological characterization of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), and its methyl-
enediphosphonate analogue, HMB-PCP, is described. With an EC50 of 0.1–0.2 nM, HMB-PP is significantlymore potent in sti-
mulating human Vg9/Vd2 T cells than anyother compound described so far. However, replacing the p yr ophosphate bya P–CH 2–P
function abrogates the bioactivitydrasticall y, with HMB-PCP having a EC 50 of only5.3 mM.
#
2003 Elsevier Science Ltd. All rights reserved.
Introduction
and pyrophosphorylated low-molecular weight antigens
seems to reside, at least in part, in the chemical nature
of the molecule. Thus, hydrolysis of the pyrophosphate
moietywas proposed to contribute criticallyto bio-
activity, with dephosphorylation-resistant diphos-
phonate analogues having drasticallyincreased EC
Recognition of low-molecular weight non-peptide anti-
gens byhuman gd T cells is in striking opposition to the
conventional recognition of small antigenic peptides by
ab T cells. Although the reactivityof gd T cells towards
these natural and synthetic metabolites depends on the
5
0
5
expression of a functional Vg9/Vd2 T cell receptor
values.
1
(
TCR) on the surface, the exact molecular mechanism
involved still remains to be elucidated, as it does not
require classical antigen presentation in the context of
the major histocompatibilitycomplex. Yet, more than
Here, the chemical synthesis and immunological char-
acterization of HMB-PCP, a methylenediphosphonate
analogue of HMB-PP, is presented.
7
0 compounds have alreadybeen shown to stimulate
2
Vg9/Vd2 T cells, the most potent of which is (E)-4-
hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-
PP), a recentlyidentified intermediate of the bacterial
Chemical Synthesis
3
non-mevalonate pathwayof isoprenoid bios yn thesis.
HMB-PCP was synthesized from tert-butyldimethyl-
6
silyloxypropanone 1, as depicted in Figure 1. A Wittig–
While direct contact of an antigen with the TCR has not
been demonstrated so far, the crystal structure of the
Vg9/Vd2 TCR insinuates a putative antigen-binding
motif. However, in contrast to immunogenic epitopes
of protein antigens, the bioactivityof phosphor yl ated
Horner reaction of 1 and P,P-diethylphosphonoacetate
yielded a mixture of the ethylesters (E)- and (Z)-2. After
separation of the isomers, reduction of pure (E)-2 led to
(E)-4-(tert-butyldimethylsilyl)oxy-3-methyl-but-2-en-1-ol
3. The alcohol 3 was then transformed into the corre-
4
sponding bromide 4, and the P–CH –P moietywas
2
added byreaction with Tris(tetra- n-butylammonium)
7
hydrogen methylenediphosphonate. Finally, the pro-
tecting group of 5 was set free bytetra- n-butylammo-
*
9
Corresponding author. Tel.: +49-641-9947442; fax: +49-641-
947499; e-mail: matthias.eberl@biochemie.med.uni-giessen.de
0
960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00138-0

1258
A. Reichenberg et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1257–1260
Figure 1. Chemical synthesis of HMB-PCP.
nium fluoride. However, apart from the tetra-n-butyl
ammonium salt of HMB-PCP, the crude product
obtained also contained considerable amounts of
measuring the phosphorus content of the pure product,
in addition to the gross weight of the lyophilized mate-
rial), and thus a reliable determination of its bioactivity.
In the presence of HMB-PP, proliferation of human
PBMC was restricted to Vg9/Vd2 T cells, leading to a
significant expansion of this subpopulation. Impor-
tantly, synthetic HMB-PP and the natural compound
isolated from Escherichia coli ꢀlytB mutants displayed
identical activities in stimulating Vg9/Vd2 T cells, at
concentrations in the nM range and below (Table 1).
Compared with the bioactivities of the direct precursor
and successor of HMB-PP within its biosynthesis path-
way, 2-C-methyl-d-erythritol 2,4-cyclopyrophosphate
ꢀ
ꢀ
Br
and
F
ions, as well as an excess of
methylenediphosphonate. For subsequent purification,
1
1
the tetra-n-butylammonium ions were substituted by
+
NH4 prior to extraction, as carried out in analogous
8
approaches. However, we rather chose to perform
the final chromatographystep using an anion
exchange material instead of cellulose. After lyophili-
zation of the product, the purityof the end product
was established on a Dionex HPLC anion exchange
system.
(
MEcPP) and isopentenyl pyrophosphate (IPP), respec-
3
In order to compare the Vg9/Vd2 T cell stimulating
properties of HMB-PCP with that of synthetic HMB-
tively, HMB-PP was 3–4 magnitudes more potent in
stimulating Vg9/Vd2 T cells than IPP, and 6 magnitudes
more potent than MEcPP. With an EC50 value of
approx. 0.1 nM, HMB-PP is considerablymore active
than the strongest compounds described so far, and is
the onlymolecule known possessing a bioactivityof
significantlyless than 1 nM.
9
PP, a procedure described previouslywas emplo ye d,
and subjected to the above purification protocol.
2
Biological Results and Discussion
In contrast to a number of recent protocols on its che-
8
Strikingly, replacing the pyrophosphate by a P–CH –P
2
function abrogated the bioactivityof HMB-PP by4–5
magnitudes (yet leaving a residual activity still compar-
,10
mical synthesis, our approach allows an absolute
quantitation of the salt-free HMB-PP recovery(by
Table 1. Biological activities of selected low molecular weight antigens
Compd
EC50 range (mM)
other studies (ref 2)
EC50 range (mM)
this study
Mean EC50 (mM)
this study( n=5–13)
Activityagainst IPP
(-fold) this study
Phosphoribosyl pyrophosphate
d-ribose-1-phosphate
MEcPP
HMB-PCP
Pamidronate
IPP
Alendronate
Bromohydrin pyrophosphate
Iodohydrin pyrophosphate
HMB-PP (E. coli ꢀlytB)
HMB-PP (synthetic)
—
1000–10,000
—
>10,000
—
10–200
1–10
0.07–10
0.06–4.0
0.1–1.0
—
>10,000
—
114
5.30
2.21
1.02
0.75
—
—
<0.00005
—
0.0004–0.01
0.04–0.2
0.6–1.0
1
0.7–1.5
—
—
1200–21,500
3600–19,700
—
4–8
1–10
0.9
0.005–0.02
0.0005–0.003
—
—
0.000004–0.001
0.00001–0.0003
0.000181
0.000084
—

A. Reichenberg et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1257–1260
1259
able to that of the commonlyused ligands, IPP and
pamidronate), suggesting that the extensive bioactivity
of HMB-PP resides mainlyin the p yr ophosphate moi-
ety. This is in agreement with comparable data on
methylenediphosphonate analogues of a synthetic gd T
cell activator, bromohydrin pyrophosphate (EC =10
4. (a) Allison, T. J.; Winter, C. C.; Fournie
´
, J. J.; Bonneville,
M.; Garboczi, D. N. Nature 2001, 411, 820. (b) Morita, C. T.;
Lee, H. K.; Wang, H.; Li, H.; Mariuzza, R. A.; Tanaka, Y. J.
Immunol. 2001, 167, 36.
5
. Belmant, C.; Espinosa, E.; Halary, F.; Tang, Y.; Peyrat,
M.; Sicard, H.; Kozikowski, A.; Buelow, R.; Poupot, R.;
Bonneville, M.; Fournie, J. J. FASEB J. 2000, 14, 1669.
. NMR-spectra were recorded on Bruker DRX 500 and AM
5
0
´
nM), with the PCP derivative having a bioactivityof
5
6
4
>> 12 mM. Even more, in the same publication
1
13
00 spectrometers. H- and C NMR spectra in D O were
2
dephosphorylation-resistant analogues of synthetic
activators were described as competitive antagonists,
with IC50 values ranging from 10 mM to 1 mM. How-
ever, the IC50 values of these compounds were 3–4
magnitudes higher than the EC50 values of the corre-
sponding agonists, thus raising doubts about the inhib-
itoryspecificityof the antagonism observed. Indeed, in
our hands the putative antagonist HMB-PCP showed
no inhibitoryactivityat all, whether titrated in the pre-
sence of a given concentration of the agonist HMB-PP, or
in the reciprocal experiment, when added at a fixed con-
centration to a serial dilution of HMB-PP (not shown).
referenced to internal 3-trimethylsilyl propionic acid sodium
salt (0.0 ppm and 1.7 ppm, respectively), or tetramethylsilane
in CDCl , proton-decoupled P NMR to external phosphoric
3
acid (0.0 ppm). (E)-4-(tert-Butyldimethylsilyl)oxy-3-methyl-
but-2-enoic acid ethylester, (E)-2. To a suspension of 2.40 g (60
mmol) NaH in 40 mL of dryTHF under argon was dropped a
3
1
solution of 8.97
phonoacetate in 15 mL of dryTHF at 0 C. After 1 h at 0 C,
.53 g (40 mmol) of tert-butyldimethylsilyloxy-propanone 1
Choo, H. Y.; Peak, K. H.; Park, J.; Kim, D. H.; Chung, H. S.
g (40 mmol) ethyl P,P-diethylphos-
ꢁ
ꢁ
7
(
Eur. J. Med. Chem. 2000, 35, 643) in 15 mL of dryTHF were
ꢁ
added, and stirring was continued for 15 min at 0 C and 2 h
at rt. Workup was carried out byslow addition of 50 mL of
4
saturated NH Cl solution, followed by10 mL of water and 30
Taken together, while for a number of synthetic com-
pounds the gd T cell stimulatorycapacitywas attributed
to their strong chemical reactivity, this seems not the
mL of ether. The aqueous layer was separated and extracted
with a further 30 mL of ether. The combined organic layers
were washed with two 30 mL portions of saturated NaCl
5
2 4
solution, dried with Na SO , and released from the solvent by
case for the comparablyinert natural activator, HMB-
PP. Consistently, quantitative structure–activity rela-
tionship analyses rather identified the presence of two
negative ionizable groups, an H-bond donor, and a
hydrophobic group as the most important features to
rotaryevaporation under reduced pressure to yi eld a crude E/
Z-mixture [(E)-2:(Z)-2=80:20; GC analysis], which was sepa-
rated byflash chromatographyon silica gel with n-hexane/tert-
butylmethylether (90:10) as eluent to yield 7.03 g (61%) of the
1
pure desired E-isomer (E)-2: H NMR (CDCl /TMS), d: 0.09
3
2
b
make a good gd T cell activator. Yet, this model still
does not consider the striking difference in the bioactiv-
ity between bromohydrin pyrophosphate and its imido-
(s, 6H, Si(CH ) ); 0.93 (s, 9H, SiC(CH ) ); 1.29 (t, 3H, J=7.1
3
2
3 3
Hz, CH
2H, CH
2
CH
3
); 2.05 (s (broad), 3H, C¼CCH
3
); 4.11 (s (broad),
CH ); 5.99 (s
C NMR (CDCl /TMS), d:ꢀ5.4
OSi); 4.17 (q, 2H, J=7.1 Hz, CH
2
2
3
1
3
5
(broad), 1H, C¼CH).
diphosphate analogue (EC =12 mM), or between
3
5
0
(
Si(CH
(SiC(CH
3
)
2
); 14.4 (CH
2
CH
3
); 15.4 (C¼CCH
3
); 18.4
OSi);
HMB-PP and HMB-PCP (Table 1). While the proposed
5
3
)
3
); 25.9 (SiC(CH
3
)
3
); 59.6 (CH
2
CH ) 67.1 (CH
3
2
need for a hydrolysable pyrophosphate moiety may
1
1
13.4 (C¼CH) 157.1 (C¼CH); 167.1 (C¼O). (Z)-2 isomer: H
NMR (CDCl /TMS), d: 0.08 (s, 6H, Si(CH ); 0.91 (s, 9H,
SiC(CH ); 1.27 (t, 3H, J=7.1 Hz, CH CH ); 1.97 (s (broad),
H, C¼CCH ); 4.13 (q, 2H, J=7.1 Hz, CH CH ); 4.79 (s
explain the relativelylow bioactivities of the aminobi-
sphosphonates, alendronate and pamidronate, our own
data indicate that the bridging oxygen atom between the
two negative ionizable groups is clearlyimportant for
maximum activity.
3
3 2
)
3
)
3
2
3
3
3
2
3
13
(
broad), 2H, CH OSi); 5.67 (s (broad), 1H, C¼CH).
C
2
NMR (CDCl /TMS), d: ꢀ5.4 (Si(CH ) ); 14.3 (CH CH ); 18.3
3
3 2
2
3
(
SiC(CH
3
)
3
); 21.4 (C=CCH
3
); 25.9 (SiC(CH
3
)
3
); 59.7
(
CH CH ) 62.7 (CH OSi); 115.0 (C=CH) 160.8 (C=CH);
2 3 2
1
66.1 (C¼O). (E)-4-(tert-Butyldimethylsilyl)oxy-3-methyl-but-
Acknowledgements
2-en-1-ol, 3. In a 50 mL flask were placed 0.891 g (3.45 mmol)
of (E)-2 in 10 mL of drytoluene under argon and cooled to
¨
This studywas supported bythe Bundesministerium fu r
ꢁ
ꢀ
78 C. After dropping 8.6 mL (8.6 mmol, 1 M in hexane) of
ꢁ
Bildung und Forschung (BioChance 0312588). We
gratefullyacknowledge the help of Ute Bahr, Ewald
Beck, Ruth Gschwind, Irina Steinbrecher, Stefanie
Wagner and Jochen Wiesner.
DIBAH into this solution, the temperature was kept at ꢀ78 C
ꢁ
ꢁ
for 2 h, then raised to 0 C during 1 h, and held at 0 C for 30
min. Addition of 1 mL of methanol and 10 mL of saturated
sodium potassium tartrate solution formed a white gelatinous
precipitate, which was stirred for 1 h at rt prior to adding 10 mL
of water and 30 mL of ether. The aqueous layer was separated
and extracted twice more, each with 30 mL of ether. The com-
References and Notes
4
bined organic layers were dried with MgSO , filtered and the
1
. Bukowski, J. F.; Morita, C. T.; Tanaka, Y.; Bloom, B. R.;
Brenner, M. B.; Band, H. J. Immunol. 1995, 154, 998.
. (a) Espinosa, E.; Belmant, C.; Sicard, H.; Poupot, R.;
Bonneville, M.; Fournie, J. J. Microbes Infect. 2001, 3, 645. (b)
Gossman, W.; Oldfield, E. J. Med. Chem. 2002, 45, 4868.
. (a) Hintz, M.; Reichenberg, A.; Altincicek, B.; Bahr, U.;
solvent was removed under reduced pressure to yield the crude
product quantitatively, which was used in the next step without
1
2
further purification. H NMR (CDCl /TMS), d: 0.07 (s, 6H,
3
´
Si(CH
C¼CCH
Hz, CH
)
); 0.92 (s, 9H, SiC(CH
), 4.03 (s (broad), 2H, CH
OH); 5.66 (dq, 1H, J =6.9 Hz, J
)
); 1.64 (s (broad), 3H,
3
2
3
3
3
2
OSi); 4.18 (d, 2H, J=6.9
3
2
1
2
=1.4 Hz, C¼CH).
1
3
Gschwind, R. M.; Kollas, A. K.; Beck, E.; Wiesner, J.; Eberl,
M.; Jomaa, H. FEBS Lett. 2001, 509, 317. (b) Eberl, M.;
Altincicek, B.; Kollas, A. K.; Sanderbrand, S.; Bahr, U.;
Reichenberg, A.; Beck, E.; Wiesner, J.; Hintz, M.; Jomaa, H.
Immunology 2002, 106, 200.
C NMR (CDCl /TMS), d: ꢀ5.6 (Si(CH ) ); 13.5 (C¼CCH );
3
3 2
3
18.4 (SiC(CH
(CH
) ); 25.7 (SiC(CH ) ); 58.9 (CH OH); 67.8
3 3 3 3 2
2
OSi); 123.0 (C¼CH); 137.9 (C¼CH). (E)-4-Hydroxy-3-
methyl-but-2-enyl methylenediphosphonate, HMB-PCP. To a
solution of 0.430 g (1.987 mmol) 3 in 10 mL of dryCH Cl at
2
2

1260
A. Reichenberg et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1257–1260
ꢁ
0 C under argon were dropped in succession 0.625 g (2.384
mmol) PPh and 0.791 g (2.384 mmol) CBr , each in 3 mL of
4.03 (s, 2H, CH
5.64 (t, J=6.7 Hz, 1H, C=CH). C NMR (D O), d: 17.7
2 1 2 2
OH); 4.49 (dd, J =J =7.2 Hz, 2H, CH OP);
1
3
3
4
2
ꢁ
dryCH
2
Cl
2
. After 1 h at 0 C and 4 h at rt, the reaction mix-
(CH
J=5.0 Hz; CH
); 32.8 (dd, J =124.8 Hz, PCH
3 1
=121.0 Hz, J
2
2
P); 65.3 (d,
ture was filtered through a short column of silica gel. The sol-
vent was removed byrotaryevaporation under reduced
pressure and the residue treated for 1 h in vacuo. The crude
product was triturated 3 times with n-pentane and filtered. The
combined filtrates were evaporated to leave 0.265 g of the
bromide 4 as a yellow oil, which was dissolved immediately in
.5 mL of dryacetonitril and dropped to (1.282 g, 1.424 mmol,
.5 equiv) freshlyprepared tris(tetra- n-butylammonium)
hydrogen methylenediphosphonate suspended in 3 mL of dry
acetonitril under argon. After stirring the product 5 overnight
2
OP); 71.1 (CH OH); 125.8 (d, J=6.5 Hz,
2
3
1
C=CH); 143.9 (C=CH). P NMR (D O), d: 13.76 (d, J=8.9
2
ꢀ
Hz); 20.95 (d, J=8.9 Hz). ESI–MS(mꢀH) , calcd 259.0137,
found 259.0147.
7. Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stranler,
K. E.; Muehlbacher, M.; Poulter, C. D. J. Org. Chem. 1986,
51, 4768.
8. Wolff, M.; Seemann, M.; Grosdemange-Billiard, C.;
´
´
Tritsch, D.; Campos, N.; Rodrıguez-Concepcion, M.; Bor-
0
1
onat, A.; Rohmer, M. Tetrahedron Lett. 2002, 43, 2555.
9. (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, HMB-
PP. (E)-4-hydroxy-3-methyl-but-2-enyl bromide (127 mg) was
obtained according to ref 8, dissolved in 0.5 mL dryaceto-
nitril, and dropped into (1.042 g, 1.155 mmol) freshlyprepared
Tris(tetra-n-butylammonium) hydrogen pyrophosphate, sus-
pended in 3 mL dryacetonitril under argon. After 16 h at rt,
the solvent was evaporated to leave a slightlybrownish oil,
which was dissolved in 1 mL of ion-exchange buffer. This
solution was subjected to the same extraction protocol as
described for HMB-PCP, yielding 36.2 mg of HMB-PP as a
white solid material, calculated as free acid.
10. (a) Giner, J. L. Tetrahedron Lett. 2002, 43, 5457. (b)
Ward, J. L.; Beale, M. H. J. Chem. Soc., Perkin Trans. 1 2002,
6, 710. (c) Fox, D. T.; Poulter, C. D. J. Org. Chem. 2002, 67,
5009. (d) Amslinger, S.; Kis, K.; Hecht, S.; Adam, P.; Roh-
dich, F.; Arigoni, D.; Bacher, A.; Eisenreich, W. J. Org. Chem.
2002, 67, 4590. (e) Gao, W.; Loeser, R.; Raschke, M.; Dessoy,
M. A.; Fulhorst, M.; Alpermann, H.; Wessjohann, L. A.;
Zenk, M. H. Angew. Chem., Int. Ed. Engl. 2002, 41, 2604.
11. Flow cytometrical analysis of human Vg9/Vd2 T cells was
performed according to ref 3b, and Eberl, M.; Engel, R.; Beck,
E.; Jomaa, H. Cell. Immunol. 2003, 218, 1. Samples were tested
at appropriate dilutions and calibrated using serial dilutions of
IPP (Sigma). Natural HMB-PP was purified from E. coli
ꢀlytB (ref 3a); MEcPP was isolated from benzylviologen
treated Corynebacterium ammoniagenes (Ostrovsky, D.;
Shashkov, A.; Sviridov, A. Biochem. J. 1993, 295, 901). Alen-
dronate was purchased from Merck, pamidronate from
Novartis. Phosphoribosyl pyrophosphate (Sigma) served as
negative control.
.
2
at RT, 0.449 g (1.424 mmol, 1.5 equiv) of TBAF 3H O dis-
solved in 2 mL of acetonitril was added dropwise, and stirring
was continued for a further 3 h. Subsequent filtering of the
reaction mixture and evaporation of the solvent left a slightly
brownish oil, which was dissolved in 1 mL of ion-exchange
buffer (25 mM NH
4 3
HCO /2% isopropanol). This solution was
subjected to chromatographyon a 2.6 Â40 cm column of
+
4
Dowex 50Wx8 (100–200 mesh, NH -form, elution at 2.5 mL/
min). Fractions containing the desired product, analyzed on a
Dionex HPLC system using high capacity anion-exchange
columns, conductivitydetection, and KOH gradient elution,
were combined and lyophilized. For removal of dipho-
sphonate, the residue was dissolved in 1 mL of buffer (50 mM
NH
was shaken vigorouslyon a vortex mixer. After centrifugation
2000 g, 5 min, rt), the supernatant was removed and the resi-
4 3
HCO ), 2 mL of isopropanol were added, and the mixture
(
due extracted a further two times with 3 mL of a 1:2 mixture
of buffer/isopropanol, with each extraction step being mon-
itored on the Dionex HPLC system. The combined super-
natants were loaded on a 2.6Â40 cm Source 15Q column
(
4 3
Pharmacia) and gradient-eluted (A, 25 mM NH HCO /acet-
onitril 10:1; B, 1 M NH HCO /acetonitril 10:1; equilibration,
4
3
1
1
h 100% A; gradient, 10 min 100% A, 120 min A!B, 10 min
00% B; flow rate 4 mL/min, each fraction 8 mL). Product
containing fractions were lyophilized to yield 126.1 mg (cor-
responding to an overall yield for the last three steps of 24%)
of HMB-PCP as a white solid material, using the method for
phosphorus determination in organic compounds described
1
before (ref. 3a), and calculated as free acid. H NMR (D O),
2
d: 1.72 (s, 3H, CH ); 2.09 (dd, J =J =19.6 Hz, 2H, PCH P);
3
1
2
2