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Determination of the HNЦH Coupling Constant in Large Isotopically Enriched Proteins.

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The parameters of the two isomers differ most dramatically in large variations between equivalent angles at the metal
atom (for example the angle C12-Mo-PI has the nearly linear
value of 177.8(1)” in l b but is reduced to 162.4(1)” in la),
and these distortions are reflected in significant differences in
many of the metal-ligand bond lengths. The Mo=O length
of 1.663(2) 8, observed in the high frequency from 1a is
significantly shorter than 1.675(3) 8,, the length in 1 b,[I5] in
line with the 12 cm-’ difference in their (Mo=O) stretching
frequency. This difference in Mo=O length together with the
differences in other bonds to the metal are undoubtedly related to the distortions of the coordination polyhedron and
Chatt’s original description of distortional isomers would
Fable 1 Selected bond lengths
0-Mo-X( 1)
[A] and dngles [ ] of 1 ( X
= Cl)
and 2 (X
la [a]
I h lbl
1.663(2) [1.676(7)]
2.538(1) [2.551(3)]
2.454(1) [2.464(3)]
2.491(1) [2.500(3)]
2.542(1) [2.541(3)]
2.539(1) [2.558(3)]
169.3(1) [168.8(3)]
162.4(1) [162.4(1)]
171.2(1) [171.2(1)]
1.682(7) [1.675(3)]
2.523(1) [2.528(3)]
2.478(3) [2.482(1)]
2.484(3) [2.489(1)]
2.519(3) [2.529(1)]
2.530(3) [2.533(1)]
170.0(3) [169.8(1)]
177.8(1) [177.6( 111
160.4(1) [160.5(1)]
[a] Vdues for “Chatt blue” in parentheses [7]. [b] Values for “Enemdrk/Parkin
blue” in pnrentheses [I 51.
still seem quite appropriate.“6] The phenomenon is not
simple polymorphism because it has been clearly established
that the differences are not merely a result of two different
crystal packings; the two blue forms contain different molecules readily distinguished by their characteristic (Mo=O)
stretching frequencies and symmetry.“ ’I Confirmation that
the (Mo=O) stretching frequencies are molecular properties
of the two isomers, not the result of a particular crystalline
arrangement. is provided by the stretching frequency
F(Mo=O) at 943 cm-’ exhibited by the low symmetry isomer in both the pure orthorhombic crystalline modification
and in the contaminated monoclinic polymorph studied by
Enemark[’21and Parkin.[l’J
This study demonstrates the importance of distinguishing
between the phenomena of distortional isomerism and bond
stretch isomerism. It should be noted that different ligand
conformations cannot explain the differences in IR spectra
reported for systems where the two “bond stretch isomers”
are isostructural and give isomorphous crystals.[’8]
Received: April 15. 1992
Revised version: September X. 1992 [Z 5309 IE]
German version: Angrit.. Chwi. 1992. 104, 1664
CAS Registry numbers:
1, 30134-06-6; 2 , 30134-05-5
[l] J. M. Mayer, Angrii.. Chem. 1992, 104, 293; Angew. Chem. In/. Ed. Engl.
1992. 31, 286; G . Parkin. Arc. Chem. Res. 1992. 25, 455.
[2] V. C . Gibson, M. McPartlin, J. Cliem. SOC.
Dalton Trans. 1992. 947.
[3] R. Baum. Chem. Eng. N e n s 1991,6Y, (9) 20; I. Amato, Science 1991,254,
[4] L. Milgrom. New Scr. 1991. 131 (1788) 22.
[5] J. Chatt. Lj. Manojlovic-Muir. K . W. Muir, Chem. Comrnun. 1971. 655; LJ.
Manojlovic-Muir. K. W. Muir. J. Chem. SOC.Dalton Trans. 1972, 686.
[6] A. V. Butcher. J. Chatt, J. Cliem. Soc. A 1970, 2652.
[7] LJ. Mano~lovic-Muir.Chem. Commun. 1971, 147; J. Chem. Soc. A 1971.
Angew. Clwni. I n / , Ed, Engl. 1992. 31. N o . 12
[8] B. L. Haymore, W. A. Goddard 111, J. N. Allison, Proc. /nr. Con/: Coord.
Cheni. 23rd 1984, 535.
[9] Y Jean, A. Liedos. J. K. Burdett, R . Hoffmann. J. Am. Cliem. Sw. 1988.
110, 4506.
1101 W.-D. Stohrer. R. Hoffmann. J. Am. Chem. Soc. 1972. 94. 1661.
[ l l ] K. Yoon,G. Parkin. A. L. Rheingo1d.J. Am. Chem. SOC.1991. 113, 1437.
1121 P. J. Desrochers, K. W. Nebesny, M. J. LaBarre, S . E. Lincoln. T. M.
Loehr, J. H . Enemark. J. Am. Chem. Soc. 1991, 113. 9193.
[I31 W. Wardlaw. H. W. Webb, J. Clwm. Soc. 1930. 2100.
[14] Crystal data: All crystals were orthorhombic, space group Phcu, Z = 8.
l a : [C,,H,,CI,MoOP,],
u =16.922(3), h = 16.446(3), c = 19.262(4) A,
M = 597.3, V = 5360.6 A’. Q . ” , . ~ = 1.480 gcm-’, F(O00) = 2448, R =
0.038 for 4265 reflections. p(Mo,,) = 8.4 cm- blue crystal. 0.21 x 0.1 8 x
u =11.300(2), h = 17.636(3). c =
0.16 mm’. I b : [C,,H,,CI,MoOP,],
28.277(6) A, M = 597.3, Y = 5635.2 A’. Q ~ = 1.408
, gcm-’.
~ F(000) =
2448, R = 0.0539 for 1914 reflections, p(MoKJ = 7.6 cm-’, blue crystal.
a = 17.020(4), h =
0.33 x 0.32 x 0.15 mm’. Za: [C,,H,,Br,MoOP,],
16.797(4). < =19.348(5) A, M = 686.2.
V = 5531.3 A’.
Q , , , ~ ~=
1.648 gem-’,
F(O00) = 2736,
R = 0.0768 for 1869 reflections,
p(Mo,,) = 34.9cm-’,
blue crystal, 0.31 x0.24x0.18 mm’.
u = 11.439(2). b = 17.660(3), c = 28.283(6) A,
M = 686.2, V = 5713.5 A’. Q ~ = 1.595
, gcm-’,
~ F(000) = 2736. R =
0.0632 fur 1953 reflections, p(Mo,,) = 33.2 cm-’. blue crystal.
0.41 x 0.38 x 0.22 mm’. Diffrdctometer data with I / o ( / )2 3.0 were used in
all determinations, collected in the H-ranges 3.-30‘ (la) and 3 ‘ 2 5 (Ih, 2a,
and 2b).
[15] K. Yoon, G . Parkin, A. L. Rheingold. J. A m . CIietn. SO<.1992, 114. 2210.
[16] Different orientations of bulky organophosphane ligands in metal complexes were shown previously to cause significant differences in bond
lengths and angles but such a distinct and marked effect on the IR spectrum has not been noted before. For example ciss-[PtCI,(PMePh,),l crystallizes in two forms, differing markedly in phosphane orientation [Ho
K.-C., G . M. McLaughlin, M. McPartlin, G . B. Robertson, AC/U
Cryst. B38, 1982, 421 -4251. Very accurate X-ray structure analyses
showed small but significant differences in the Pt-Cl lengths related to
marked differences in the angles at the Pt atom.
[I71 The concepts invol\,ed in this type of isomerism in some ways parallel
those invoked for isomerism in the tetrahedral cluster systems
[(Ph,P),N][H,M,(CO),,] (M = Ru and 0 s ) . Tautomeric anions of C,, or
Cz symmetry equilibrate rapidly in solution and can only be separated in
the solid state [J. W. Koepke, J. R. Johnson. S. A. R. Knox, H. D. Kaesz,
J. Am. SOC.1975. Y7. 3947; B. F. G . Johnson. J. Lewis, P. R . Raithby.
G . M. Sheldrick. K. Wong. M. McPartlin. J. Cliern. SOC. Dal/on Trans.
1978, 6731. The two ruthenium isomers have been structurally characterized in the solid state, but in the case of osmium, the two different crystalline forms proved to be polymorphs each containing the same monoanion isomer of C , symmetry [M. McPnrtlin, W. H. Nelson. J. Chcni. SO(.
Dallon Truns. 1986, 15571.
[18] A. Bashall, V. C. Gibson, T. P. Kee. M. McPartlin, 0. B. Robinson,
Angebv. Cheni. 1991,103.1021 ; Angew. Chrm. I n / . Ed. Engl. 1991.30.980;
K. Wieghardt, G. Backes-Dahmann. B. Nuber. J. Weiss, ihirl. 1985, Y7. 773
bzw. 1985. 24, 777.
Determination of the HN-Hu Coupling Constant
in Large Isotopically Enriched Proteins**
By Stephan Seip, Jochen Balbach, and Horst KessIer*
New multidimensional pulse techniques in NMR spectroscopy in combination with isotopic enrichment of the
important heteronuclei 5 N and 13C allow the structure determination of larger proteins up to a molecular weight of
> 30 kDa.[1-31The information obtained from the coupling
constants becomes more and more important in conformational analysis and molecular dynamics calculation^.[^^
The HN-Hu coupling constant plays an important role in the
[*] Prof. Dr. H. Kessler, Dr. S. Seip, DipLChem. J. Balbdch
Organisch Chemiscbes Institut der Technischen Universitiit Miinchen
Lichtenbergstrasse 4. W-8046 Garching (FRG)
[**I This work was supported by the Deutscbe Forschungsgemeinscbaft and
the Fonds der Chemischen Industrie. The authors thank Prof. Dr. B. Erni
and K. Fliikiger for the preparation of the sample of the uniformly labelled
P I 3 domain Stephan Seip thanks the Fonds der Chemischen lndustrie for
a grant.
@?? VCH ~ r l r i ~ s g e s e l l . ~ r l mhH.
i u f t W-6940 Weinheim, 1992
$ 3.50f .25iO
determination of the backbone conformation, because it restricts the range of values of the angle @] (Fig. 1). It can be
obtained from the splitting of the HN or H" signal in the
one-dimensional spectra of small peptides. For larger pep-
Fig. 1. Part of the polypeptide
chain with definition of the angle
@. The range of 0 can be restricted
with the aid of the H"- H" coupling
I1 T' I
1 x 1 1
,I T 2 '
f 2 (
tides (up to medium-sized proteins), methods are available
that allow the determination of the desired coupling from
antiphase crosspeaks in COSY spectra.",
This requires
sufficient magnetization transfer by homonuclear J-couC'
pling. No homonuclear transfer is possible, however, for
larger proteins (> 30 kDa). Therefore, a method must be
Fig. 2. Pulse sequences for the determination of the Hv-H" coupling constants
in large proteins. Both sequences correlate "N with I3C" and H N .The rffield
chosen that combines high sensitivity and large spectral disstrength from the X channel is reduced to 4 kHz for the selective excitation of
persion of the heteronuclear couplings to reduce overlap of
the c" carbon nuclei without affecting the C' nuclei, by using the first zero
resonances. In addition, it is desirable to determine the coucrossing of the excitation function. Phase-modulated pulses [2,3] are used for
pling constant independent of the width of the signal.
selective inversion of the C' spins, without switching of the transmitter frequenA number of pulse sequences were previously described
cy. a) Coupled HNCA (correlates H, N, and a-C) with "constant-time" evolution of nitrogen chemical shifts and jump-return pulse for selective excitation
that allow for the measurement of the HN-H" coupling conoftheHqresonances.~=2.25,A=4.5.r'=13,~,=10,6=7.75,7,=2.25ms.
stant in 1sN/'3C enriched proteins.['-''] They all exploit the
T , is decremented with increasing I,. The WALTZ decoupling is performed with
"E. COSY" pattern in the Fl/F3planes of a 3D spectrum for
an 8 kHz field. The water resonance is irradiated during the relaxation delay.
b) Coupled HNCA with "constant-time" evolution of the nitrogen chemical
extraction of the coupling constant: The large coupling of
shifts. The proton frequency is set on the water resonance. The jump-return
the directly bound proton ( ' J C , J is used to separate the two
pulse leads to a sinusoidal offset-dependent excitation In this way excitation of
multiplet components of the C" signal (H" in SI and in p state)
the H" protons is avoided (prerequisite for the "E. COSY" pattern) and the
in F1, where C' is allowed to evolve (that means no proton
water resonance is suppressed. Sequence b) is superior to sequence a) when
presaturation of the water resonance may lead to saturation transfer of Hq
decoupling is used in that dimension). This separation preprotons. Parameters are the same as in a) except for: r2 = 10, T , = 2.25 ms.
vents mutual interference between the two components,
: = 60 psec (at 600 MHz). T ; is decremented with increasing 2 , . Phase cycling
which may lead to a wrong coupling constant. The 3JHN,H, ifor
= x, x, --s.
-x. $ 3 = x.
both experiments is $, = x . x , -x, -1,
coupling leads to a displacement of the two multiplet compo$4 = X(x), 8(
.x), 45 = Ib(x), 16( .x), @6 = a. --1. q57 = - I, x,receiver = x,
.x, Y, 5 , 1.I,-1.2( - x, I,x, - x , x, s,- .x, x), .Y, -I, -,I,
nents in the directly detected dimension and is therefore
I, .Y. - x. All other pulses are applied from the x axis.
extracted in F3 (the dimension with the best digital resolution).
We propose two pulse sequences that utilize the same principle, but are optimized for sensitivity. The determination of
the 3JHN.H.
coupling constant with high sensitivity, even for
L25, 6(l5Nj = 118.43
proteins that are larger than 30 kDa, can be achieved with
these sequences. The pulse sequences correlate C", N, and HN
through large one-bond couplings
(ca. 90 Hz) and
(ca. 11 Hz)).
The pulse sequences shown in Figure 2 excite the Ca nucleus (via the NH proton and nitrogen), which evolves in F1
as antiphase magnetization with respect to I5N during t ,
without proton decoupling. After the constant-time evolution of I5N in t,, the magnetization is then transferred back
to protons and detected during I,. The "E.COSY" pattern of
the C"(F1)-HN(F3) crosspeaks is achieved by selective excita1560
tion of the protons bound to nitrogen, thereby avoiding the
F3 [ppml
mixing of the H" spin states.['*] In practice, this is achieved
by a "jump-return" sequence at the end of the pulse train,
Fig. 3. Application of the pulse sequence shown in Figure 2 a on a 1 mM solution of uniformly ' 5N,"C labeled P I 3 domain of mannose permease of E. c d r .
which acts as a selective 90" pulse for HN.[l3l
The experiment was performed on a Bruker AMX 600 spectrometer equipped
The application to larger proteins requires careful optiwith a multichannel interface and a triple-resonance probe. 128 x 54 x 512 real
mization of all pulsewidths and delays. In particular, the
points were taken with 32 scans each. Total measuring time was 74 hours.
delays must be optimized under consideration of the relaxMultiplication with a 70' shifted sin' function was applied in all dimensions to
avoid truncation artifacts. The final spectrum consisted of 128 x 64 x 512 real
ation behavior of the different nuclei. Equation (a) is found
points. Left. Section of the 2D plane with the crosspeak of Leu2' at a "N
for 2 z, Equation (b) for z1 z2 + 6; the latter should be
47') =
sin(n'JN,C2T') cos (n2JN,c2~')
exp (27'/T2,
A ( 7 , . ~ ~ ,=
6 )sin(x'J,,,(s,
+ T~ 6))
C O S ( ~ * J ~ . ~+( 7Z
T , + 6)) exp ((zl + 7 > + J)/T2,NH)
resonance frequency of 6 = 11 8.43. The "E.COSY" pattern is evident. For the
determination of the coupling constants two rows which crossed the maxima of
the two peaks along the F3 dimension were extracted from the spectrum. The
displacement of the two maxima was measured after inverse Fourier transformation. zero-filling to 8192 points, and retransformation. The coupling constant is 8.0 Hz (the error is estimated at f 1 Hz).
shorter than 2t', because of the strong dipolar relaxation of
the proton, N,.,H, antiphase magnetization decays faster
than ' 5 N in-phase magnetization which is present during
The T, times can be extracted from Heteronuclear
Single Quantum Correlation (HSQC) and refocused HSQC
spectra." '1
The evaluation of the 3D spectrum is shown for the C"-HN
crosspeak of Leuz5 in the fully 1sN/13Clabeled P I 3 domain
of mannose permease (31 kDa)["I (Fig. 3). Because of the
low digitization in the 3 D spectrum, the coupling constant is
best determined from selected rows, in analogy to the
DISCO proced~re."'~The pulse sequence from Figure 2 b
yields an identical spectrum. Evaluation is performed in the
Rjn planes.
The experiments described here allow for the determinacoupling constant even in
tion of the homonuclear 3JHN.Hx
very large (by N M R standards) proteins, which d o not show
any correlations in COSY o r TOCSY spectra. Coupling constants provide structural information, which, together with
distances determined from NOESY spectra, significantly enhance the quality of protein structures obtained by N M R
Received. August 12. 1992 [Z 5514 IE]
German version: An,qew. Chem. 1992, 104, 1656
CAS Registry number:
Mannose permease. 99442-22-5.
[l] M. Ikura. L. E. Kay. A. Bax, Biorlienlisrry 1990, 29, 4659-4667.
[ 2 ] G . M Clore. A. M. Gronenborn. Prog. N u d . Mugn. Reson. SprcIrosc.
1991. 23. 43-92.
[3] L. E. Kay. M. Ikura, R. Tschudin. A. Bax, J Mugn. Rrson. 1991, 91.
422 428.
[4] Y. Kim, J. H. Prestegdrd, Prorein5 Strircr. Funcr. Gener. 1990, 8. 377-382.
[S] D. M . Mierke, H . Kessler, Biupo!l;mers, 1992. 32, 1277-1282.
[6] M . Kur7. P. Schmieder. H . Kessler, Anxeir.. Chem. 1991, f03. 1341-1342;
A n p i . Chmi. In/.Ed. EnRI. 1991, 30, 1329-1331.
17) H. Kcssler. M. Gehrke, C. Griesinger. Anjiew. Chem. 1988, 100, 507-554;
Angcii. Chcm. h i / . Ed. EnRI. 1988. 27, 490-536.
[XI Y Kim, J. H. Prestegdrd. L. Magn. Reson. 1989, K4, 9-13.
[9] G. T. Montelione. G. Wagner, J. Am. Cheni. Soc. 1989, l i i 3 5474-5475.
[lo] G. T. Montelione. G . Wagner, J. M q n . Reson. 1990. 87. 183-188; G.
Wagner. P. Schmieder. V. Thanabal, ;bid. 1991, 93. 436-440.
1111 0. W. Sorensen. J Mujin. Reson. 1990.90.433-438.
[I21 C. Griesinger. 0. W. Smensen. R. R. Emst, J. Mugn. Reson. 1987, 75,
1131 L. Mueller. J. Am. Clien?. Soc. 1979, 1 0 1 , 4481-4484.
1141 S. Campbell-Burk, P. Domaille, L. Mueller. J. Magn. Reson. 1991, 93,
171 - 176.
(15) A . Bax. M. Ikura, L. E. Kay. D. A. Torchia. R . Tschudin. J M q n . Reson.
1990, 86. 304-318.
1161 B. Erni. B. Zanolari, H. P. Kocher. J. B i d . Chem. 1987, 262, 5238-5247.
117) H. Kessler. A. Muller. H. Oschkinat. M u p . Reson. Chon. 1985. 23. 844852.
Butatrienes by Desulfurization of Cyclic
By Ruincr Hrrges* and Cliristoph Hoock
Most of the numerous butatriene syntheses". are based
on dehalogenation of 1,4-dichlor0-2-butynes.~~~~~
we reported a novel method for the synthesis of the parent
butatriene 3 (R = H) starting from 1,4-dihalo- o r 1,4-bistosyl-2-butyne 1 via 4-vinylidene-I ,3-dithiolane-2-thione 2.1'1
[*I Dr. R. Herges, Dipl.-Chem. C. Hoock
Institut fur Organische Chemie der Universitit Erlangen-Nurnberg
Henkestrasse 42, D-W-8520 Erlangen (FRG)
[**I This research was supported by the Deutsche Forschungsgemeinschaft.
The subsequent Corey-Winter desulfurization to butatriene 3 (R = H ) was accomplished with the phosphorous
base 1,3-dimethyl-2-phenyl-I ,3.2-diazaphospholidine at
0 "C with yields exceeding 90%.
X = CI,Br, OTs
The net reaction is homologous to a fragmentation reaction that we discovered recently using computer-aided reaction design,I6] namely the reaction of 1,4-dichloro-2-butene
with potassium trithiocarbonate and subsequent desulfurization to 1,3-butatriene. Compared with the conventional
method of Schubert et aLt3] and Brandsma et al.I4] for the
synthesis of butatriene, our method gives higher yields and a
very pure product. For the extension to alkyl-substituted
butatrienes, one could use the easily accessible bistosylates 1
(X = OTs) instead of the 1,4-dihalogen-2-butynes 1 (X = Br,
CI, R = alkyl), which are difficult to prepare. Unfortunately,
the reaction is restricted to the parent butatriene when the
conventional phosphorous bases for Corey-Winter reactions are used. All attempts to desulfurize alkyl-substituted
dithiolanes 2 with the phosphorous bases trimethyl phosphite, triphenyl phosphite, tris(dimethylamino)phosphane,
and diazaphospholidine to the corresponding cumulenes
failed. After testing a number of transition metals and transition metal complexes[71we found Raney nickel[*' (deactivated with ketones) to be an excellent desulfurization agent.
Alkylated 1,4-butynediols 1 (X = OH) were easily prepared from acetylene and aldehydes or ketones.['] The corresponding bistosylates 1 (X = 0%)were then synthesized in
almost quantitative yields according to a slightly modified
method of Brandsma['ol with tosyl chloride. Reaction with
anhydrous potassium trithiocarbonate was accomplished in
dichloromethane with 10 mol YOcrown ether and using ultrasound to giveexcellent yields of the dithiolanes 2 (Table
Dithiolanes 2 are yellow, light- and air-sensitive oils that can
be stored in solution under nitrogen at -30 "C for several
weeks. Characteristic spectroscopic features of 2 are the lowfield N M R shift of the trithiocarbonate carbon atom
(6 = 223-224),["] the large C-H coupling constant of the
terminal allene carbon atom (165-171 Hz), the strong
I R stretching vibration of the C = S bond at 10701060 cm-1,[121 and the strong UV absorption at 330338 nm (cmar = 4000-15000 Lmolcm- 1).1131 According to
the I3C and 'H N M R spectra, the two stereoisomeres synand anti-2 (R = Me, iPr) are formed in a 1 : 1 ratio (Fig. I).
For subsequent desulfurization the crude product can be
used without purification. A proper choice of the solvent was
crucial for the desulfurization. Dimethylformamide and other higher boiling formamides are most suitable. The sensitive
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