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Conformational Analysis of Model Complexes for Methyl-Coenzyme-M Reductase from Methanogenic Bacteria A Comparison of Crystal and Solution Structures.

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Conformational Analysis of Model Complexes
for Methyl-Coenzyme-M Reductase from
Methanogenic Bacteria: A Comparison of Crystal
and Solution Structures**
6 are derivatives of Nil'-salen, in the sense that one C-N
double bond has been modified into a C-N single bond.". l o ]
The corresponding carbon atom now bears a thioether-containing side chain; the nitrogen atom is bound to a hydrogen
atom in rue-4 and to a methyl group in ruc-5.6. The flexibil-
By Albrccht Berkessel." Michuel Bolte, Christiun
Griesingcr." Gottfried Huttner, Thomus Neurnnnn,
Eerlhctld Schiemc~nz,Hurald Scht~vrrlbe,
and Thornus Schwenkreis
In the last step of the archaebacterial methanogenesis,
the methylthioether methyl-coenzyme-M 1 reacts with
the thiol N-7-mercaptoheptanoyl-0-phospho-L-threonine
("HS-HTP", 2) to yield methane and the mixed disulfide 3.[']
This unusual reaction is brought about by the enzyme
methyl-coenzyme-M reductase, which contains the nickel
tetrahydrocorphinoid "factor 430" (F430)as cofactor.[21In
general, metalloenzyme-mediated catalysis depends on the
specific interaction between substrate(s) and metal ion(s).
Also in the case of methyl-coenzyme-M reductase it is assumed that the nickel ion of F430participates in the catalytic
R' = Ph, R2 = H
5: R ' = P h , R 2 = C H 3
6 : R' = CH2Ph, R' = CH,
ity of the side chain enables the coordination of its heteroatom to the metal ion. Through rotation around the C - C
single bond marked by an arrow, however, other local conformations are possible. in which the sulfur atom is turned
away from the metal ion. Figure 1 shows the Newman projections of the three staggered conformations about this CC single bond.
HO,C"'* H..,,
. N- Ni
- N /
Transition metal complexes of multidentate ligands have
been used successfully as models for the study of metal-substrate interactions in metal lo enzyme^.[^^ For methyl-coenzyme-M reductase, nickel chelates such as rue-4 have been
used to simulate the interaction of the organosulfur substrates 1 and 2 with cofactor F430:the comparison of the
redox properties of model complexes made it possible to
develop a mechanistic hypothesis for the catalytic cycle of
methyl-coenzyme-M r e d ~ c t a s e .The
~ ~ ]Ni" complexes ruc-4[*] Prof. Dr. A. Berkessel. DipLChem. T. Schwenkreis
0rg;inisch-chemisches Institut der Universitgt
Irn Neuenheimer Feld 270, D-69120 Heidelberg (FRG)
Teiefax: Int. code +(6221)56-4205
Prof. Dr. C . Griesinger. Dr. M . Bolte, DipLChem. H. Schwalbe
lnstitut fur Organische Chemie der Universitit
Marie-Curie-Strasse 11. D-60439 Frankfurt am Main
Prof. Dr. G. Huttner. DipLChem. B. Schiemenz
Aiiorganisch-chemisches lnstitut der Universitit Heidelberg
DipLChem. T. Neumann
lnstitut fur Organische Chemie der Universitit Gottingen
Fig. 1. Newman projections for the staggered conformations of the side chains
ofthe nickel chelates roc-4- 6; antiperiplanar arrangement of H, and S, H,-H,), .
and H,-Hp,.
This work was supported by the Bundesministerium f i r Forschung und
Ttchnologie (Projekt 031 8801). the Deutsche Forschungsgerneinschaft
(Projekt Be 998;2-1.2). and the Fonds der Chemischen Industrie.
Angeii.. Chrin. I n / . Ed. EngI. 1993, 32, No. 12
In order to find out whether model complexes of the type
rac-4-6 can be used to demonstrate an attractive interaction
between the sulfur atom in the side chain (representing the
sulfur-containing substrates of methyl-coenyzme-M reductase) and the nickel ion (representing the nickel ion in F430).
it appeared promising to determine the preferred conformation of the thioether side chain in ruc-4-6. Herein we report
the conformational analysis of the nickel complexes rac-4-6
in solution by N M R spectroscopy and compare the preferred conformations thus found with the side chain conformations in the crystals of the respective compounds.
The thioether side chain in the crystal structure of the Ni"
complex rue-4 has the local conformation I ; the sulfur atom
is coordinated to the nickel ion, the dihedral angles H,-C-CH,, and H,-C-C-H,, are -59.2" and 59.7<, respectively
(Fig. 1, conformation I and Fig. 2a). A centrosymmetric
dimer is formed from one molecule each of 4 and cwt-4. This
dimer is stabilized by two N H . ' 0 hydrogen bonds. In this
arrangement the sixth coordination site of the nickel ion of
one molecule is filled by the phenolate oxygen atom of the
other molecule in the dimer, resulting in a distorted octahedral coordination geometry at Ni. The distances from the
nickel ions in ruc-4,5, and 6 to the N, 0, and S atoms of the
ligands are listed in Table 1 .
mhH. 0-69451 Weinlwin?, 1993
057o-OX33!93/1212-1777 3 lO.OO+ 25:O
Table 1. Distances [A] between the nickel ion and the heteroatoms in the Iigands in complexes uuc-4[7]. ruc-5[9]. and 611 1.121 (from crystal structures).
Table 2. Vicinal coupling constants[a] and distances determined by N O € experiments[a] on the nickel chelates ruc-4-6.
1 .834(3)
1 .X64(3)
I .843(73
Vicinal coupling
constants. distance
The crystal structure of the nickel complex rue-5 shows it
to be monomeric, and the sulfur atom of the thioether side
chain is not bound to the Ni" ion (Fig. 2b).[7.91Instead, the
side chain exhibits conformation I1 (Fig. 1). Hence, the Ni"
ion in rue-5 is four-coordinate with a square-planar coordination geometry. The exchange of the phenylthio moiety in
the side chain with a benzylthio group led to the Nil' complex
rue-6, which formed high-quality crystals readily. Its crystal
structurer7. 1 2 ] shows that 6 is monomeric in the crystalline state and is present in conformation I1 (Fig. 1).
The three Ni" chelates rac-4-6 are diamagnetic in solution
('H NMR); for ruc-4 in CDCI,, however, slightly broadened
signals were observed for H,,BI,,z.The addition of [D,jdimethylsulfoxide ([DJDMSO) led to "normal" line widths.
In spite of the completely different side chain conformations
found in the crystal structures, rac-4-6 exhibit similar vicinal coupling constants 3JsIT-H,,,
(Table 2). These
N M R data can be interpreted with three models: 1 ) one
staggered conformation (I or I1 or I11 in Fig. 11, 2) one nonstaggered conformation (x=
60,l180, - 60 "). and 3) a Pachler e q ~ i l i b r i u m [ ' of
~ ' the three staggered conformations 1I11 (Fig. 1 ) . Model 1 : The "large-small" coupling pattern
observed for all three complexes cannot be explained by the
exclusive presence of the staggered conformation I, which
was found in the crystal structure of rcrc-4 (Fig. 2a). The
Nickel chelate
[a] For experimental details see ref.[13]. [b] Calibrations based on NOE cross
peak intensities of the two geminal protons H,,,, Hd2; d(H,, H,J =
178 pm (standard bond lengths and angles; error i 10 pm). [c] pro-S-H of the
rnethylene group adjacent to the amine nitrogen atom. labeled H,
in Figures 2a-c.
approximately equal dihedral angles H,-C-C-H,, and Ha-CC-H,, ( x = i 60') should lead to roughly equal coupling
constants 3JHr.Hpl
and 3JHz_HB,.
The presence of exclusive/y
conformations I1 and 111 is contradicted by the value of
(Table 2. line 3), which is too small for a purely trans
coupling. Model 2: The attempt to interpret the experimental data with a nonstaggered conformation leads to high
standard deviations and incompatible results from the 3J
and NOE analyses. Model 3: Table 3 lists the results of the
Pachler analysis['41for the complexes rac-4-6. For all three
complexes an approximately 1 : I distribution of a transgauche (I1 or 111, Fig. 1 ) and the gauche-gcruclae conforma-
Fig. 2. a) Crystal structure of roc4. Top: only 4 is shown; bottom:
centrosymmetric dimer of 4 and
ent-4. The hydrogen bonds between N1 and 02' as well as between N1* and 0 2 are depicted by
dashed lines. the intermolecular
Ni -0 coordination IS indicated by
thin lines. b) Crystal structure of
rur-5 (only one enantiomer is
shown). c) Crystal structure of 6.
:r? VCH VerlugsgeseNrcluffi mbH, 0.69451 Weinheim. 1993
0570-0833/931I212-177RB I0.00+ ,2510
Angew. Chem. Inf. Ed. EngI. 1993, 32. No. 12
Table 3. Model 3: Calculated conformer populations (1. 11. 111) for the complexes ru( -4 6 (in "4).
Conformation ./ [a]
NOE [h] J [a]
NOE [h]
J [a]
H5 H6
N O € [h]
[a] The linrplu?, equation was parameterized according to McLaughlin et al.
[15c], 'JJ(HZ.H/I)(O)= 11.1 cos2 H - 1.6 cosM. [h] The conformer distribution
was calculated assuming an
averaging of the NOE. [c] Based on the
homonuclear 'J(Hr.H/i) coupling data and the NOES between these nuclei. it
15 impos\ihle to distinguish between equilibria of I1 and 1, and 111 and 1.
tion (1. Fig. 1) is found. The determination of the preferred
trcirzs-gctuchc conformation and the assignment of the diastereotopic protons H,, and H,, was possible by measuring
the NOE between the pro-S methylene proton adjacent to
N 2 in me-6 (labeled He. i'Hc,hylene"
in Fig. 2a-c) and H,,
and H,?.
respectively. If one assumes the conformation for
the ethylene group that was found in the crystal structure,r161
the local conformations I. 11, and I11 of the thioether side
chain lead to the distances between H y l lH,,, and Heihylene
that are summarized in Figure 3. By usrng the Pachler equi-
6.0 5.5
v (15N) [Hzl
6 ('H)
Fig. 4. 'H-I5N HMBC N M R spectrum of the complex ~ - 5 .
Fig. 3. Distances from the H, atom ("He,,,,,,,". pro-S-H) to the three possible
position\ of H,,, and H Y I .
librium. (derived from analysis of the coupling constants) for
complex rue-6 (conformation I, 49 YO;11, 10%; 111, 41 YO)
(Table 3). the protons H,, and H,, are calculated to be on
average 227 and 269 pm from Hethylene.
This is in agreement
with the experimentally found data (Table 2, lines 7,8). The
stereochemical assignment of H,, and H,, follows from this
equilibrium to be pro-S and pro-R, respectively. The reverse
population of conformers I1 and 111 (I, 49%; 11, 41 Y O ;111,
10 %) leads to roughly equal average distances between
Hsl.? and Hethylene
(225 and 221 pm), which is in contrast to
the NOE-derived distances of 213 and 286 pm (Table 2, lines
7.8) and is therefore ruled out. In all three complexes (ruc4 - 6 ) one observes average distances between HB1,*and
that are different in magnitude. Hence, it appears
justified to assume an approximately 1 : 1 equilibrium of the
conformers I and 111 for all three complexes in solution. The
equilibria found for the three complexes are in agreement
with the nearly equal average coupling constants 3JN-H,lI
and 3Jh-HllZ
for rue-5 of 1.4 and 1.8 Hz, respectively. These
couplings were determined according to the method of Keeler and Neuhausr'81from a 2D heteronuclear multiple bond
correlation (HMBC) N M R spectrum of the complex which
was "N labeled at N 2 (Fig. 4).
In summary, the following conclusions appear justified: 1)
There is no dominant, attractive Ni-S interaction in the
monomeric. neutral Nil1-dihydrosalen complexes rue-4-6 in
solution. Presumably the conformer equilibrium is regulated
in all three cases to about the same extent by nonbonding,
intramolecular interactions. 2) The formation of two
NH . . .O hydrogen bonds favors the dimerization of complex vuc-4 in the crystal["1 and leads to a higher coordination number for the nickel ion, initially by the axial coordination of a phenolate oxygen atom. The coordination of the
thioether sulfur atom then completes the pseudooctahedral
ligand sphere.
The study of the complexes ruc-4-6 by NMR spectroscopy and X-ray crystal structure analysis shows once
againrzo1that the conformation that is "frozen out" in the
crystal is not necessarily the preferred conformation in solution and that the support o r hindrance (in the crystal or by
the solvent) of hydrogen-bond- induced self-association can
result in dramatic changes in the conformer distribution. For
the substrate-cofactor interaction in methyl-coenzyme-M
reductase, this leads to the interesting possibility that the
strength of the binding could be regulated by the absence or
presence of a sixth ligand on the nickel ion of Fa3(].
Received: June 24. 1993 [Z 6167 IE]
German version: Anycil-. C I i m . 1993. tfJ.5. 1716
[ I ] J. Ellermann. R. Hedderich, R. Biicher. R. K. Thauer. E M . J. B/uc/it,nl.
1988, 172. 669-677.
[2] a ) G. Firber, W. Keller. C. Kratky. B. Jaun. A. PfaltL. C. Spinner. A.
Kohelt. A. Eschenmoser. H d v . Chin. Arra 1991, 74. 697 716: h) A. Fiiss-
ler, A. Kohelt. A. Pfaltz. A. Eschenmoser. C. Bladon. A. R. Battershy.
R. K. Thauer. h i d . 1985, 6X, 2287-2298.
[ 3 ] a ) S. Rospert. M. Voges. A. Berkessel, S. P. J. Alhracht. R. K . Thauer. Eirr.
J Biorhrin. 1992,210.101 - 107; h)S. Rosperl. R. Bocher. S. P J. Alhracht.
R. K. Thauer. FEES Lrlr. 1991. 291. 371 375.
[4] a ) H. Dugas. Biooryunic Cheinktr!. 2nd ed.. Springer. Heidelherg. 1989;
h) W. Kaim, B. Schwederski. Bir)unor~u/zi.s~l?r
( % z i ? i ~ i ~Teuhner.
Berkessel, Bioorg. C/irrn. 1991, I Y , 101-115.
[6] a ) M. D. Hobday. T. D. Smith. C o r d . Cheii?.Reis. 1972:1973. 9. 31 1 - 337:
b) A. G . Maiifredotti. C. Guastini. Actu Crwu//t,,r. Sect. C 1983. 3Y.
[7] The preparation of complex 1 . ~ and
~ 4 its crystal structure analysis have
both been reported [S.Xl. The synthesis of the N-methylated nickel chelates
roc-5.6 will be reported at a later date. In contrast to ruc-4[8],rw-5,6 are
1101 oxygen-sensitive.
1x1 A . Berkessel, J. W. Bats. C. Schwarz, Angcw. Chcnl. 1990. 1112, 81 -84:
A n g ~ w C/wii.
I n / . E d EngI. 1990. 29. 106- 108.
[9] Crystals of rue-5 suitable for structure determination were obtained from
a solution of the complex in methanol by slow evaporation of the solvent
at room temperalure. r n . - 5 : CL,H,,N,O,SNi. monoclinic, space group
P2, I ? . II = 8.577(X). / J = 9.936(8). c = 24.66(3)A. /{ = 99.53(6) . V =
20?3(4) A,. Z = 4. pC.,,' = 1.484 gem-'. Siemens (Nicolet-Syntex) R3m:V
diffractometer. Mo, radiation. 20 range = 2 -50 5119 total measured
reflections. 3655 symmetry-independent reflections: no absorption correction: 2880 reflections with I > 2 d l ) were used for the structure solution
and refinement [SHELX 76 (G. Sheldrick. Cambridge University. 1976).
SHELXTL-PLUS (G Sheldrick. University of Gottingen. 1988)]:
K = 0.038. R, = 0.034: data:parameter ratio =11.9: 1. maximuin residual
electron density 0.46 e k 3 . All non-hydrogen atoms were relined anisotropically. The imine H atom was located and refined isotropically. The
positions of all other H atoms were calculated and the temperature factors
"ere refined. Ref. [12b].
[lo] Examples for Nil'-dihydrosalen complexes without side chains: A. Bottcher. H. Ehas. L. Miiller. H . Paulus. Angeir. Clrriii. 1992. 104. 635-637:
Angrw. Cltcrii. In!. Ed. EngI. 1992. 31. 623-625.
[l I J ruc-6 crystallizes a s a conglomerate of 6 and en/-6.
1121 a ) Crystals of 6 suitable for structural determination were obtained
by slouly cooling a solutioii of the complex ra(.-6 in ethyl acetate.
6: C,,H,,N,O,SNi.
hexagonal. space group P6,. (I = 21.351(2),
<. = 10.124(1) A.
Y = 3996.9(7)A,'. Z = 6. p = 1 91 m m - l : pLd,L
1 . I 90 gcm ': Enraf-Nonius CAD4 diffractometer. Cu,, radiation. 21)
range = 2-120 .3138 total measured reflections, 2802 symmetry-independent reflections: empirical absorption correction based on 3 scans of
nine reflections with relative transmissions of 0.78 1 .00; 2621 reflections
with F >3o(F) were used for the structure solution (Patterson method)
and relineineiit (SHELXTL-PLUS program package). nonhydrogen
atoms were refined anisotropically, H atoms were located using difference
electron density maps and refined using a "riding" model. The absolute
configuration was determined ( q = l . l ( l ) ) . R = 0.051 ( R , = 0.065.
11 = u'(F)
0.0008F2):data,parameter ratio = 9.4: 1. maximuin residual
electron density 0 . 7 7 e k 3 . b) Further details of the crystal structure
investigiitions of rue-5 and 6 may be obtained from the Fachinformationszentrum Karlsruhe. Gesellschaft fur wissenschaftlich-technischeInformation mbH. D-76344 Eggenstein-Leopoldshafen. ( F R G ) on quoting the
depository numbers CSD-57467 o r CSD-56660. respectively. the names of
the authors. and the journal citation.
[13] Experimental details: ROESY of r u - 4 : delay 150 ms. spin lock 3 kHz.
1 kHz off resonance. operating frequency 600 MHz. NOESY of rue-5.6:
operating frequeny 400 and 600 MHz, delay 150 ms. 200 ms. integration
over the entire negative and positive region; 512 experiments with 16 scans
per increment and 1024 complex points in I,.zero filling in both dimensions. apodiration using cos'. An off-resonance correction was performed
for the integration ofthe ROE spectra. The HMBC spectrum was obtained
at 600 M H r with 128 experiments and 8 scans. The 'J(Hs-H/{1.2) coupling
constants were determined using resolution-enhanced 1 D spectra. All measurements were conducted at 300K. rue-4 in CDCI,. rue-5.6 in
[DJDMSO. Solutions of ruc-4 were prepared under the exclusion of oxygen.
1141 a ) K. G. R. Pachler. Spec1rochr117.Acru 1963. I Y , 2085- 2092: b) ibrd. 1964.
20. 581 -587.
1151 a) M. Karplus, J. Am. Ch~nr.Sor. 1963. 85. 2870 2x71: b) V. F. Bystrov.
Prog. N M R SpecIrosc. 1976. 10. 41-81; c) R. J. Abraham. K. A.
McLauchlan. J. Mol. P/iI,s. 1962. 5, 513-523.
1161 I n the case of rm-4. the crystal structure indicated the "inverted" conformation for the ethylene bridge with a pseudoequatorial Hc,,,,,,,,, (pro-S-H)
(Fig. ?a). The assumption of this conformation for the ethylene bridge
leads unambiguously to the same results with quantitatively slightly differing distance data.
1171 A. DeMarco, M. Llinas. K. Wiithrich. Biopolynwrs 1978. 17, 2727-2742
[ l X ] J. Keeler. D. Neuhaus. J. Titman, Chenr. PIiw. Lrri. 1988. 146. 545 -548
[19] The line broadening observed in the ' H N M R spectrum of the Nil' complex I-nc-4in CDCI, indicates the onset of association~octahedralcoordination even in this low-polarity solvent.
[20] a ) H. Kessler. G . Zimmermann. H . Forster. J. Engel. G. Oepen. W. S.
Sheldrick. A J I ~ ~C/iciri.
1981. Y3, 1085- 1086: Airgei!. C/ierir. 117r. Ed.
€nR/. 1981. 20. 1053-1054: b) H. Kessler. J. W. Bats, J. Lautz, A. Miiller.
Lichrg\ Ann. Clwn. 1989. 913-928: c) N. B Perry, J. W. Blunt. M. H. G.
Munro. Mugn. Rrson. Chmii. 1989, 27. 624-627: d ) M. Billeter. A. D.
Kline, W. Braun. R. Huber, K. Wiithrich. J. Mol. Bid. 1989.206.677-687:
e) E. T. Baldwin, I T. Weber. R. S. Charles. J:C. Xuan. E. Appella, M.
Yamada. K. Matsushima, B. F. P. Edwards. G. M. Clore. A. M Gronenborn, A. Wlodawer. Proc.. Nut/. Acud. Sci. USA 1991. 88. 502 506.
Cation Inclusion within the Mixed-Valence
Polyanion Cluster
[ ( M O ~ ' O , ) ~ M O ~ , O , , ( O H ):, ,Syntheses
Structures of (NH4),[NaMo,,(OH),,040]-4H,0
and (Me,NH,),[H,Mo,,(OH),,O,ol **
By M . Ishaque Khan, Achim Miiller,* Stephan Dillinger,
Hartmut Bogge, Qin Chen, and Jon Zubieta*
The understanding of the driving force for the formation
of high-nuclearity clusters is still a challenge. This is especially valid for polyoxometalates having a tremendous variety of
quasi-spherical structures with highly symmetrical core assemblies of MO, units."] Though the famous x-Keggin
anion [ M O ~ , O , , P ] ~ - , which
[ ~ ~ has an extremely high formation tendency, was isolated nearly 200 years ago and numerous compounds based on this isomer have been reported, the
E isomer (formally obtained by rotation of all four Mo301,
groups by 60") seems to be rather unstable. No pure MolO
or WjO conipound with r:-Keggin structure has yet been isolated. The reason seems to be that the density of packing of the
oxygen atoms decreases gradually from the x to the 8
whereby 12-membered ring systems result. This
class of compounds is also of interest with regard to the new
area of inorganic host-guest chemistry.[4- 'I The polyvanadates, for instance, adopt basket, bowl, belt, barrel, and
spherical structures which can encapsulate neutral, anionic.
and cationic species, and even cation-anion aggregates.[' Likewise, cyclic arsenic(Ir1) polytungstates and phosphorus
(v) polytungstates have been shown to encapsulate a variety
of cations.['7, "I In contrast, oxomolybdate cages or clusters
accommodating guest species remain relatively rare, with the
notable exception of the recently described cluster species
report here on the synthesis of a mixed-valence MoV/MoV'
polyanion cluster which exhibits a central E-Keggin unit
and a cavity capable of accommodating protons or metal
cations, as demonstrated by clusters 1 and 2.
Both 1 and 2 were isolated in 20% yield from aqueous
solutions containing molybdate and both organic and inorganic templates. The red needles of 1 and red-orange cubes
of 2 are water soluble, and the latter rapidly become opaque
when removed from the mother liquor. X-ray structural
analyses[201of 1 and 2 reveal the presence of discrete, ordered [Mo,,(OH),,O,,]sanions (Fig. 1) that have a central cavity encapsulating protons o r a Na' ion. Both anions
contain four MoV' centers, twelve Mo" centers, forty 0x0
groups, and twelve hydroxy groups in full tetrahedral Td
The twelve Mo" centers in 1 and 2 form six binuclear units
with an Mo-Mo distance of 2.62(1)A (single bond). The
[*] Prof. Dr. A. Miiller. DipLChem. S. Dillinger, Dr. H. Bogge
Fakultit fur Chemie der Universitat
Postfach 100131. D-33501 BieIefeid 1 (FRG)
Telefax: Int. code+ (521) 106-6003
Prof. Dr. J. Zubieta. Dr. M. I. Khan, Dr. Q. Chen
Department of Chemistry
Syracuse University
Syracuse. NY 13244 (USA)
TekpdX: int. code + (315) 443--4070
This work was supported by the National Science Foundation, Grant No.
CHE9119910. and the Fondsder Chemischen Industrie. S. D.. H. B.. and
A. M. thank Frau A. Armatage for her assistance in the X-ray structure
analysis of I .
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crystals, mode, coenzyme, complexes, comparison, reductase, conformational, methanogenic, methyl, solutions, structure, analysis, bacterial
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