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HETLOC an Efficient Method for Determining Heteronuclear Long-Range Couplings with Heteronuclei in Natural Abundance.

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that of the C-glucoside derivatives not substituted at the
benzyl group.
The direct C1 lithiation of the 2-phenylsulfinyl substituted
glycals (2, readily obtained from D-glUCal,'8' Scheme 2) was
used to synthesize the C-glucosides. Thus the C1-lithiated
species was formed from the S isomer, (S)-2, with lithium
diisopropylamide (LDA) in THFiHMPT at - 100°C and
subsequent reaction with benzaldehyde afforded an easily
separable mixture of diastereomers, (S)-3a and (S)-3 b (ca.
4: 1 , 86%) (Table 1). Treatment of the major product (S)-3a
Table 2. Type of inhibition and inhibition constants ( K , ) [a].
compound
pH
T["C]
inhibition type K , [MI
1-deoxynojirimycin
la
6-gluconolactone
6.8
6.8
6.2
6.8
6.8
6.8
6.2
6.2
27
30
27
30
30
30
27
27
competitive
competitive
competitive
competitive
mixed
competitive
competitive
(substrate)
5
8
lb
D-glucal
cellobiose
1.8 x 10-'[b]
7.0~10-~[c]
2.0 x 10-4[b]
2.6 x
6.1 x 10-3
7.6 10-3
2 . 0 10-'[b]
~
6.3 x 10- I [b]
[a] For method see [Ill. (b] See (51. [c] Measurement range up to an inhibitor
concentration of I = 1.0mM.
Table 1. Selected physical data of the compounds 4a, 6, 9, and 1 a, b [a]
4a: [?ID+ 22.5 (C = 1.0, CHCl,); 6 , = 3.28 (dd, J5,6
= J6,,= 9.0 Hz; H-6),
3.77 (dd. J2, = J 3 , & = 9.5 Hz; H-3), 3.86 (dd, J4, = 9.0 Hz; H-4). 3.92 (dd,
however, has an enzyme affinity two orders of magnitude
J 4 , , = J 5 , , = 9 . 0 H z ; H - 5 ) , 4 . 7 9 ( d , J,,,=9.0Hz;H-7).
smaller.
This observation gives an indication of the relative
6:[al,+ 1 0 . l ( c = 1.0,CHCI,);6,=2.72(dd,J6,,. = 6 , J ,.,. = 13.8Hz;H-7'),
position of the acid groups (AH) of the active site of the
= 13.8 Hz; H-7), 3.56 (ddd, Jl,z= 5.2, J,.,, = 2.3,
2.98 (dd, J6,,= 6, J7,7.
JZ,3=9.6Hz; H-2). 3.68 (dddd, J 5 s , 6 = I l . 4 ,J,,,,=1.9, J 6 , 7 = 6 , J6, =
enzyme which participate in the glycoside cleavage. The Ki
6H~;H-6);4.07(dd,J,,,.=12.1,J,.,,=2.3Hz;H-l'),4.26(dd,J,,,.=
12.1,
values of the deoxy compounds 5 and 8 lie in the same region
J,,' = 5.2 Hz; H-I), 4.90-4.98 (m; H-3,4).
as that of 1 b.
9: [alD- 4.6 (c = 1.0, CHCI,); 6, = 3.59 (ddd, J,,z= 5.2. J,,2= 2.4, Jz,3 =
Received: June 4, 1991 [Z4673 IE]
9.9 HZ; H-2). 3.66 (ddd, J5.6 = 9.8, J6,, = 6.5, J 6 , , . = 5.6 Hz; H-6), 4.95 (dd,
German version: Angew. Chem. 103 (1991) 1348
J+5
J 5 . 6 = 9.4 Hz; H-5). 5.04 (dd, Jz,3
= J3,4
= 9.4 Hz; H-3), 5.18 (dd,
J3, = J4. = 9.4 Hz; H-4).
CAS Registry numbers:
l a : [?ID- 9.4 (c = 1.0, H,O); 6, = 3.27-3.31 (m, 4H; H-2,3,4,5), 3.56-3.68
l a base, 136034-25-8; l a salt, 136034-26-9; l b base, 136087-43-9; l b salt,
(m, 2 H; H-1'.6)? 3.74 (d, J,, = 12.0 Hz; H-1). 4.49 (d, J 6 , , = 4.5 Hz; H-7),
136171-90-9; 2 S isomer, 118916-46-4; 2 R isomer, 118798-67-7; 3a S isomer,
7.27-7.33 (m. 5 H ; C,H,).
136034-18-9;3a R isomer, 136087-42-8; 3 b S isomer, 136087-41-7; 3 b R iso1b: [ ~ I +
D 20.8 ( r = 1.0, HZO); 6 , = 2.63 (dd, J4,5
= J5,6
= 9.5 Hz; H-5), 2.88
mer, 136087-44-0; 4a, 136034-19-0; 5, 136034-20-3; 6, 136034-21-4; 7a.
(dd. J z , 3 = J,.4 = 9.5 Hz; H-3), 3.24-3.32 (m. 2H; H-2, 4), 3.48 (dd, J,,,. =
12.1, J,.z=7.5H~;H-l),3.64(dd,J,,~=9.5,J,.,=3.2Hz;H-6),3.76(dd, 136034-22-5; 8, 136034-23-6; 9, 136034-24-7; benzaldehyde, 100-52-7; B-glucosidase, 9001-22-3.
J,.,.= 12.1, J ,. , , = 1.9H z; H-l' ), 4. 56(d, J6,,=3.1Hz;H-7),7.24-7.34(m,
5 H ; C,H,).
[l] R.R. Schmidt, Angew. Chem. 98(1986) 213; Angew. Chem. Inr. Ed. Engl.
[a] Optical rotations recorded at 2 0 T , 'H NMR spectra at 250 MHzin CDCI,
25(1986)212; Picre Appl. Chem. 61 (1989) 1257; H. Paulsen, Angew. Chem.
f02 (1990) 851; Angew. Chem. Int. Ed. Engl. 29 (1990) 823, and references
(4a, 6. 9) or D20( 1 a, b).
cited therein.
[2] S. Hakomori. J. Biol. Chem. 265 (1990) 18 713. and referencescited therein.
[3] E. Truscheit, W Frommer, B. Junge, L. Muller, D. D. Schmidt. W. Winwith Raney nickel W I1 and thereafter with hydrogen in the
gender, Angew. Chem. 93 (1981) 738; Angew. Chem. Int. Ed. Engl. 20
(1981) 741.
presence of PdjC as catalyst yielded directly, through loss of
[4] T. Feizi, M . Larkin, Glycobiology l(1990) 17.
the 0-benzyl protecting groups and the benzylic hydroxy
[5] M. P. Dale, Biochemistry 24 (1985) 3530; R. Saul, R. J. Mulyneux, A. D.
group, the dideoxy derivative 5, whose structure was ascerElbein, Arch. Biochem. Biophys. 230 (1984) 668.
tained by 0-acetylation to form 6. If borane addition and
[6] H. Mehnert, Arineimitteltherapie 8 (1990) 378.
[7] L. M. Sinnott in M. 1. Page, A. Williams (Eds.): Enzyme Mechanisms, The
alkaline oxidation with hydrogen peroxide followed the
Royal Society of Chemistry, London 1987, p. 259; P. Lalegerie. G. Legler,
Raney nickel treatment, which causes the cleavage of the
J. M. You, Biochimie 64 (1982) 977.
phenylsulfinyl group, a 5,7-dihydroxy derivative was ob[S] R. PreuR, R. R. Schmidt, Liebigs Ann. Chem. 1989, 429; S . Maier, R.
tained stereoselectively. Its structure was determined by 'H
PreuD, R. R. Schmidt, ibid. 1990,483; R. R. Schmidt, R. PreuR, Tetrahedron Leu. 30 (1989) 3409.
NMR spectroscopy after transformation into the benzyli[9] The configuration for ( R ) - 2 was established by X-ray structure analysis:
dene derivative 4a (J4. = J5, = J6., = 9 Hz. The reducH. Dietrich, Diplomarbeit, Universitat Konstanz, 1990.
tive opening of the benzylidene group with LiAIH,/AlCl,
[lo] Obtained from Boehringer Mannheim GmbH.
proceeded chemoselectively and afforded the 1,3,4,5-tetra[Ill J. Lehmann, L. Ziser, Carbohvdr. Res. 188 (1989) 45; we thank Prof.
Lehmann for support during the introduction of this method.
0-benzyl derivative 7a. Here the hydrogenolytic O-debenzy-
lation yielded the deoxy compound 8 directly and the derivative 9 on 0-acetylation. The target molecule l a was
obtained in good yield from 7 a by 7-O-mesylation, introduction of azide with tetrabutylammonium azide, release of the
amino group by reduction with LiAIH,, introduction of a
tert-butoxycarbonyl(Boc) protecting group, hydrogenolytic
0-debenzylation, and acid-catalyzed cleavage of the Boc protecting group. Compound 1 a was isolated as trifluoroacetate. The diastereoisomer 1 b was synthesized analogously
from (R)-2 via (R)-3b (the transformation with benzaldehyde afforded (R)-3a and (R)-3b in a 1 :4 ratio and 94 YO
yield) and the intermediates 4b and 7 b (diastereomeric to 4a
and 7a).
For the inhibition studies, the cleavage of O-nitrophenylP-D-glucoside with 8-glucosidase (cellobiase) from sweet almonds[''] was employed.['l] The resulting types of inhibition and the inhibition constants ( K i )are listed in Table 2. It
is remarkable that the amino compound 1 a yields a Ki value
similar to that of 1 -deoxynojirimycin. The diastereomer 1 b,
Angew. Chem. Inr. Ed. Engl. 30 (1991) No. 10
0 VCH
HETLOC, an Efficient Method for Determining
Heteronuclear Long-Range Couplings with
Heteronuclei in Natural Abundance **
By Michael Kurz. Peter Schmieder, and Horst Kessler *
Heteronuclear long-range couplings such as ,J(C, H) and
3J(N,H) are among the most important NMR parameters
[*] Prof. Dr. H. Kessler, Dip1.-Chem. M. Kurz, Dip1.-Chem. P. Schmieder
Organisch-Chemisches lnstitut der Technischen Universitat M iincben
Lichtenbergstrasse 4, W-8046 Garching (FRG)
[**I This work was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie. Peter Schmieder thanks the Fonds
der Chemischen Industrie for a fellowship.
Verlagsgesellschaft mbH. W-6940 Weinherm, 1991
0570-0833~9ljlOlO-1329$3.50+ ,2510
1329
for determining conformation and configuration.['] However, their intensive use has been hindered by the difficulty in
measuring them. Either the sensitivity of the existing methods of determination['] is too low or the methods are limited
to well-resolved proton spectra. Here we propose a procedure that can be used to determine the couplings to protonbearing heteroatoms without the disadvantages mentioned
above and without the necessity of a cumbersome enrichment of the heteronucleus (13C, "N).
An efficient method for determining these long-range couplings should meet the following requirements : highest sensitivity, applicability to overlapped proton and carbon signals,
and measurement of the coupling constants within the detection time (dimension with the better resolution). All of these
conditions are fulfilled by the method of Montelione et al.J3I
in which an E. COSY patternf4]is obtained in the conventional TOCSY or NOESY spectra of isotopically enriched
proteins. In the incremented dimension (F1 in 2 D spectra,
F 2 in 3D spectra), the cross peaks show the direct, large
coupling to the heteronucleus ('J(C,H), 'J(N, H)) and, in
the detected dimension (F2 in 2 D spectra, F 3 in 3 D spectra),
they show the desired heteronuclear long-range coupling to
the same heteronucleus through the shift of the multiplet. In
this way, coupling constants smaller than the line widths
may be measured.
In order to determine couplings to nuclei in natural abundance, it is necessary to suppress the signals of protons that
are not bonded to a magnetically active heteronucleus. We
suggest the use of a w , hetero half-filter,[51which selects for
such nuclei in F1 via the phase cycle. The pulse sequences are
shown in Figure 1 . BIRD presaturationt6l of the protons
bonded to "C allows a rapid pulse sequence['' (ca. two scans
per second). The resulting 2 D spectra are TOCSY spectra in
which the cross peaks show the desired "E. COSY" pattern.
The pulse sequence b in Figure 1 is the three-dimensional
variant[*' in which the chemical shift is allowed to evolve
further. This technique is especially applicable for overlapping signals. However, the method proposed here is restricted to systems in which the heteronucleus is bonded to a
proton belonging to the same spin system as the "J(H,X)coupled proton. If this is not the case, then the NOE cross
peaks obtained with the pulse sequence shown in Figure 1
can be used: for example, between the amide proton of an
amino acid and H" of the preceding amino acid. For small
molecules, however, the sensitivity is limited by the efficiency
of the NOE mixing process.
TOCSY
c
Fig. 2. Schematic representation of the determination of 'J(H", CB) in a
peptide. HNis linked to HP through the TOCSY transfer. If 'T-bonded Ha is
selected for in the experiment, the H" show long-range coupling to Co.
The method to obtain "JAX
is illustrated here to determine
the @ angle in peptides, the dihedral angle between the two
carbonyl C atoms along the peptide chain. Figure 2 shows
that the 3J(HN,CP)coupling, just like the homonuclear
3J(HN,Ha) coupling, is correlated with the angle @ through
a Karplus relation. The relevant Karplus-Bystrov relations
a)
BIRD,
111
90°
?
I
1800
A
18Do
1
A,
A,
900
I
I
A
j
tl
1 MLEVIyJ
900
I v2
v1
I
-180
.
.
-120
.
.
-60
.
.
0
.
.
60
-
.
.
120
.
.
180
#I0]
bl
BIRD,
90°
111
I
1800
I
18Do
t1/2
tl/2
A
9%
1
A
j
t2
Fig. 3. Graphical representation of the Karplus-Bystrov relations (a) and (b).
The curves shown pertain to the middle of an allowed range.
1 MLEVIyJ
g0OY2
are given in Equations (a) and (b).l9I A graphical representation is shown in Figure 3.
C)
+ 0.4
(a)
+ 60) - 0.2
(b)
3J(HN,Ha)= 9.4 cos'(4 - 60) - 1.1 cos(4 - 60)
1800
I
3J(HN,CB)= 4.7 COS'(~+ 60) - 1.5 COS($
9".;
Fig. 1. Pulse sequences of the NMR experiments discussed in the text. a) w ,
hetero half-filtered TOCSY without heteronuclear decoupling (HETLOC, determination of heteronuclear long-range couplings). b) 3 D HMQC-TOCSY
without heteronuclear decoupling. c) w,-filtered NOESY without heteronuclear decoupling. In all sequences, the BIRD pulse serves to suppress the signals
of C'*-bonded protons and makes it possible to perform the experiment rapidly
with two repetitions per second. The delay times are A = 3.57 ms, A1 = 3 ps; z
has to be optimized for each compound. The phase cycle is
= x, -x,x, -.x,
@* = x,x, - x , -x, receiver = + ,-,-, + . In (c), the phase cycle corresponds to
the phases 6,and
in the usual NOESY experiment. In the case of TOCSY
transfer, MLEV-17 was used as mixing sequence.
1330
0 VCH Verlagsgesellschafi mbH, W-6940 Weinheim, 1991
This approach will be exemplified with the cyclic pentapeptide cycZo(-A1a'-A1a'-Ala3-Pro4-Pros-),which exists in
two conformations (53 and 47 %). Figure 4 a shows the HN,
HP region of the w , hetero half-filtered TOCSY (HETLOC)
spectrum. The cross peak of A1a'-HN in Figure 4a clearly
reveals the E. COSY pattern.[41Coupling constants can be
determined either directly from the cross peak or, analogously to the DISCO procedure,["] from slices through the spectrum (Fig. 4c).
0570-0833/91/1010-1330 $3.50+.25/0
Angew. Chem. Ini. Ed. Engl. 30 (1991) No. i0
1I
to be determined only qualitatively from a selective HMBC
experiment.["] The possible angles thus determined are given in boldface in Table I ; they are in good agreement with
those obtained from MD simulations.
The experiment presented here provides a simple method
for determining heteronuclear coupling constants. For the
determination of structures in solution by NMR spectroscopy, these coupling constants are a valuable adjunct to the
distances obtained from NOESY spectra.
a)
i
!
9.5
8.5
9.0
8.0
7.5
7.0
6.5
6.0
-6
Received: June 7, 1991 [Z 46821E3
German version: Angew. Chem. 103 (1991) 1341
CAS Registry number:
cJ'clo(-Ala-Ala-Ala-Pro-Pro-),135866-42-1.
LO
-
30
C _ _ _ . . . . .
20
10
0
LO
30
.
20
J. L. Marshall: Carbon-Carbon and Carbon-Proton N M R couplings, Verlag
Chemie International, Deerfield Beach, FL. USA 1983; A. Demarco.
M. Llinas, K. Wuthrich, Biopolymers 1 7 (1978) 2727-2742.
A. Bax, R. Freeman, J. Am. Chem. SOC.104 (1982) 1099-1100; M. Ochs,
S . Berger, Magn. Reson. Chem. 28 (1990) 994-997; R. C. Crouch,
G. E. Martin,J. Mugn. Reson. 92(1991) 189-194; H. Kessler. M. Gehrke,
C. Griesinger, Angew. Chem. 100 (1988) 507-554; Angew. Chem. Inr. Ed.
Engl. 27 (1988) 490-536, and references cited therein.
G. T. Montelione, M. E. Winkler, P. Rauenbuehler, G. Wagner, J. Mugn.
Reson. 8 2 (1989) 198-204.
C. Griesinger, 0. W. Sorensen, R. R. Ernst, J. Magn. Reson. 7.5 (1987)
474-492.
G. Otting, H. Senn, G. Wagner, K. Wuthrich, J. Mugn. Reson. 70 (1986)
500- 505.
J. R. Garbow, D. P. Weitkamp, A. Pines, Chem. Phys. Letr. 93 (1982)504509.
A. Bax, S . Subramanian, J. Magn. Reson. 67 (1986) 565-569.
G. Wider, D. Neri, G. Otting, K. Wuthrich, J Magn. Reson. 8.5 (1989)
426-431 ; A. S . Edison, W. M. Westler. J. L. Markley, ibid. 92 (1991)434438; P. Schmieder, M. Kurz, H. Kessler, J. Biomol. N M R , unpublished.
V. F. Bystrov, Prog. Nucl. Magn. Reson. Spectrosc. 10 (1976) 41 -81.
H. Kessler, A. Miiller, H. Oschkinat, Magn. Reson. Chem. 23 (1985) 844852.
W. Bermel, K. Wagner, C. Griesinger, J. Magn. Reson. 83 (1989) 223-232;
H. Kessler, P. Schmieder, M. Kock, M. Kurz. ibid. 88 (1990) 615-618.
W. E van Gunsteren, H. J. C. Berendsen, Angew. Chem. 102 (1990) 10201055; Angew. Chem. Int. Ed. Engl. 29 (1990) 992-1017.
-
-6[Hzl
6[HZl
Fig. 4. Application of the pulse sequence shown in Figure 1a to the cyclic
The sample contains 10 mg
pentapeptide c~~clo(-Ala'-Ala'-Ala3-Pro4-Pros-).
of peptide in 0.5 mL of [DJDMSO (ca. 25 mM per conformation); the measurements were carried out at 300 K with a Bruker AMX-500 spectrometer.
512 1, increments with 128 repetitions each were carried out (T = 198 ms.
0, = x); the total measuring time was 9.5 hours. 4096 points were recorded in
the acquisition dimension; the spectrum was apodized in both dimensions with
a 90"-shifted quadratic sine function and, after the calculation, had a size of
8192 x 1024 real points. a) Region of the HN,HP cross peaks of the I3C a,-filtered TOCSY spectrum. b) Cross peaks of Ala2; the E. COSY pattern is clearly
evident. c) Two slices through the cross peak of (b); the slices were inverse
Fourier transformed, zero-filled, and Fourier transformed once again. The shift
of the signals with respect to one another gives the long-range coupling.
Table 1 compares the angles obtained from both couplings. Combination of the two relations allows the number
of possible angles to be reduced to one or two. The final
determination can be made by using two additional longwhich need
range couplings, 3J(HN,C;i,)
and 3J(Ha,Cii-
Table 1. Coupling constants [Hz] and corresponding angles ["I for the three alanines
for the major and minor conformers (Ala and
in r.~rlo(-Ala'-Ala2-Ala3-Pro4-Pros-)
ala, respectively).
A New Macrobicyclic Tris-bipyridine Ligand and
Its Cu: and Agi Complexes**
By Javier de Mendoza,* Esther Mesa,
Juan-Carlos Rodriguez- Ubis, Purijicacidn Vazquez,
Fritz Vogtle,* Paul-Michael Windscheif, Kari Rissanen,
Jean-Marie Lehn,* Daniel Lilienbaum, and Raymond Ziessel
The remarkable complexation properties of tris(2-aminoethyl)amine (Tren) 1"' towards transition-metal cations
~~
3J(H".H") -angle
Ala' 8.0
Ala'
6.7
AIa3 6.6
ala'
9.6
ala2 5.6
ala3 10.0
-153. -87, 44, 76
-160. -80, 32, 88
-161, -79. 31, 89
-141, -99 [d]
-167. -73, 24, 96
-137. -103 [d]
3J(HN,C0)-+a ngle
2.0
3.0
2.5
0.2
2.6
0.4
-90, -30,63, 177 [b]
-60, 73, 167 [c]
-81, -39, 68, 172 [c]
- 160, - 120, 0.40
-79, -41, 69, 171 [c],
-163, -117, -3.43
MD
angle [a]
63
-99
-67
- 95
-51
-100
[a] From restrained M D simulation with GROMOS [12] over 100 ps in vacuum.
Eighteen and seventeen distances were used for the major and minor conformer,
respectively. [b] Because of a very intense cross peak between the carbonyl C atom
of the preceding amino acid and Ala' Ha, the alternative (- 87"/90") can be ruled
out. [c] Owing to the weak or absent cross peak between the carbonyl C atom of the
preceding amino acid and HEof the amino acid being considered, the alternative
with positive @angle(88"/73", 89"/68", or 96"/69") can be ruled out. [d] Both angles
determined from the 'J(HN, H") coupling constants are consistent with the angles in
boldface determined from the 3J(HN,Cb)relation. A more precise distinction is not
possible, not even by using the other two long-range couplings. 3J(HN.C;i,) and
3J(Hc. Cij ,A
Angew. Chem. lnr. Ed. Engl. 30 (1991) No. 10
["I
0 VCH Verlagsgesellsehafl mbH.
[**I
Prof. Dr. J. de Mendoza, E. Mesa, Dr. I-C. Rodriguez-Ubis.
Dr. P. Vizquez
Departamento de Quimica, Universidad Autonoma de Madrid
Cantoblanco, E-28049 Madrid (Spain)
Prof. Dr. F. Vogtle, DipLChem. P.-M. Windscheif
Institut fur Organische Chemie und Biochemie der Universitat
Gerhard-Domagk-Strasse 1, W-5300 Bonn 1 (FRG)
Priz-Doz. Dr. K. Rissanen
Department of Chemistry, University of Jyvaskyla
Kyllikinatu 1-3, SF-40 100 JyvaskylP (Finland)
Prof. Dr. J.-M. Lehn, D. Lilienbaum, Dr. R. Ziessel
Institut Le Bel, Universite Louis Pasteur
4, rue Blaise Pascal, F-67000 Strasbourg (France)
This work was supported by Comisi6n Interministerial de Ciencia y Tecnologia (CICYT PB87-0109), by the Bundesministerium fur Forschung
und Technologie (BMFT-O329120A), by the CNRS (URA422) and by
the Academy of Finland (No. 1031 002). Since our three groups in Madrid,
Bonn, and Strasbourg, supplemented by a Finnish group, had been working independently on this same topic, we decided to publish our results
jointly, in a European spirit!
W-6940 Weinheim, 1991
0.570-0833/91j1010-1331~
3..50+.2.5/0
1331
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