Proc. Nat. Acad. Sci. USAVol. 69, No. 8, pp. 2212-2215, August 1972

Stereochemistry of a Curare Alkaloid: O,O',N-Trimethyl-d-Tubocurarine(x-ray crystallography/neuromuscular blocking agent)

HENRY M. SOBELL*, T. D. SAKORE*T, S. S. TAVALE*§, F. G. CANEPAtW, PETER PAULINGt,AND TREVOR J. PETCHERt* Department of Chemistry, The University of Rochester, Rochester, New York 14627; * Department of Radiation Biologyand Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14620; and t Departmentof Chemistry, University College London, Gower Street, London W.C. 1

Communicated by Linus Pauling, June 12, 1972

ABSTRACT The three-dimensional structure of 0,0',-N-trimethyl-d-tubocurarine, a neuromuscular blockingagent, has been determined by x-ray crystallography. Thismay help to provide insight into its pharmacologic action.

d-Tubocurarine is a curare alkaloid that has been used forcenturies by South American Indians to prepare poison ar-rows for hunting wild animals for food. Death results fromrespiratory paralysis and subsequent asphyxiation. Its majoraction is the interruption of transmission of a nerve im-pulse at the neuromuscular junction. This is thought to re-flect complex formation between the drug and cholinergicreceptors located at the postjunctional membrane, com-petitively blocking the transmitter action of acetylcholine(1). The bis-quaternary ammonium structure of d-tubocur-arine and its more potent derivative, O,O',N-trimethyl-d-tubocurarine (see Fig. 1), suggests that coulombic inter-action occurs between the cationic centers of the drug andcertain anionic groups of the receptor site, and that these,as well as stereo-specific van der Waals interactions, prob-ably account for its binding specificity and biological activity.We report here the crystal and molecular structure of 0,0',-

N-trimethyl-d-tubocurarine di-iodide. Several interestingaspects of the conformation of the molecule may help toexplain its pharmacological action.

MATERIAL AND METHODS

Crystals of 0,0',N-trimethyl-d-tubocurarine di-iodide weresupplied to us independantly by Professors Edward Reichand S. Wilkinson. These are monoclinic, space group P21,with cell constants a = 15.22 A, b = 18.36 i, c = 15.44 A,X = 91.20. The asymmetric unit consists of two d-tubocur-arine molecules and four iodide anions, a total of 100 atoms,excluding hydrogens. Three-dimensional diffraction datawere collected at room temperature (22-24°) with a Pickerautomatic diffractometer by the theta-two theta scan tech-nique. 5675 Reflections were measured out to a maximum two-theta of 90°, this representing about half the data theoreticallyaccessible in the copper sphere. Of these, 4884 were recordedto be significantly above background. The positions of the

four iodide ions were determined unambiguously from anexamination of the Patterson function, which simultaneouslyconfirmed the space group assignment. The structure wasthen developed by successive Fourier synthesis, startingwith the heavy atom phases and extending phase informationas recognizable chemical groups appeared. Block diagonalisotropic least squares led to rapid convergence, leaving a

TABLE 1. Coordinates and temperature factors forOO',N-trimethyl-d-turbocurarine di-iodide crystal structure

after full matrix least squares

Atom

N1C2C3C4C5C6C7C8C9C10Cl1C12013C14015C16C17C18C19C20C21022C23C24C25C26C27C28C29C30031C32C33C34C35C36C37C38C39N40C41C42C43C44045C46047C48

x/a y/b

0.4257 0.77220.4929 0.80290.5817 0.78620.6207 0.79350.5490 0.79130.4547 0.80270.6994 0.79540.7297 0.79650.6588 0.79470.5770 0.79310.4390 0.69230.3292 0.78820.8095 0.79740.8712 0.80140.6948 0.79210.6278 0.77150.5939 0.81540.5431 0.78860.5373 0.71170.5674 0.66200.6219 0.68410.6683 0.64340.6464 0.56960.4224 0.88460.4446 0.91850.3872 0.89940.4214 0.92410.4858 0.97090.5338 0.99150.5136 0.95730.5160 0.99150.4561 1.01380.3962 1.06110.3444 1.08550.3573 1.05420.4226 1.00330.4779 0.97120.4639 0.97300.5049 0.90990.5739 0.90240.5385 0.90980.4836 0.85220.6400 0.82170.6168 0.97900.3909 1.10070.3099 1.09410.2829 1.14410.2594 1.1760

z/c

0.69750.77410.75360.65930.58730.60380.63230.55190.48220.50300.69810.72970. 52400.57920.40000.34390.27910.21800.20490.26380.32900.39100.39490.59500. 52050.45090.36560.34850.42120.50370.27050.21340.22310.13490.06130.04670.1269

-0.0398-0.03520.03760.12670.15910.0259

-0.00440.28890.32970.14710.0640

B

3.803.961.781.912.514.302.032.185.611.145.455.153.885.732.864.222.303.854.783.226.174.754.313.601.222.473.523.505.582.863.143.794.263.753.004.454.205.628.813.332.473.694.706.383.546.237.014.30

Atom x/a

N1' -0.0727C2' -0.0154C3' 0.0727C4' 0.1047C5' 0.0461C6' -0.0533C7' 0.2065C8' 0.2227C9' 0.1700C10' 0.0707Cll' -0.0767C12' -0.1747013' 0.3110C14' 0.3787015' 0.1966C16' 0.1450C17' 0.1027C18' 0.0504C19' 0.0453C20' 0.0809C21' 0.1290022' 0.1793C23' 0.1739C24' -0.0832C25' -0.0596C26' -0.1028C27' -0.0920C28' -0.0179C29' 0.0392C30' 0.0187031' 0.0172C32' -0.0357C33' -0.1034C34' -0.1410C35' -0.1416C36' -0.0694C37' -0.0177C38' -0.0378C39' 0.0157N40' 0.0805C41' 0.0421C42' -0.0009C43' 0.1510C44' 0.1456045' -0.1057C46' -0.1807047' -0.2052C48' -0.2411

y/b0.73110.70220.71700. 70570.70620.69520.71260.70830.71810.70370.81480.71620.70890.70140.71250.73820.69410.72440.80540.84290.82180.85530.92820.62140.58640.60020.57520.53980.51940.54090.51590.50180.44830.43390.46840.52700.53920.56100.62020.62080.60210.66980.68080.55460.40640.39350.37160.3590

z/c B

0.2750 4.900.2123 1.490.2187 2.740.3181 2.500.3840 1.680.3710 0.980.3408 3.500.4170 1.030.4826 3.980.4699 2.820.2831 6.730.2375 5.190.4426 4.420.3912 4.130.5696 3.640.6240 2.640.6881 2.770.7504 1.940.7633 3.600.7030 2.670.6368 1.980.5845 2.550.5836 6.900.3727 3.440.4640 2.650.5325 4.590.6206 1.230.6262 1.480.5594 3.210.4769 2.470.7044 2.800.7716 2.230.7808 0.740.8479 1.130.9286 4.800.9302 4.000.8481 1.891.0105 3.430.9923 3.800.9347 3.980.8454 2.900.8020 1.960.9391 3.800.9607 5.090.6971 4.640.6525 7.500.8501 6.360.9172 5.64

t Department of Chemistry, Indian Institute of Technology,Bombay, India.§ National Chemical Laboratories, Poona, India.¶T Department of Physics, North of London Polytechnic, LondonN.7.

2212

Atom x/a y/b z/c

II 0.7731 0.5018 0.1759

I2 0.5907 0.1869 0.0412

13 0.3745 0.4955 0.2424

14 0.9080 0.8309 0.0128

11

0. 0063

0.0058

0.0068

0.0071

622 B 33 B 12 113 p23

0.0063 0.0085 -0.0001 -0.0035 0.0001

0.0032 0.0067 0.0014 -0.0014 -0.0015

0.0037 0.0110 -0.0003 -0.0032 -0.0015

0.0034 0.0104 0.0002 -0.0042 0.0012

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Proc. Nat. Acad. Sci. USA 69 (1972)

OCH3

CH30 2I-

FIG. 1. Structural formula for O,O',N-trimethyl-d-tubocura-rine di-iodide.

residual (unweighted) of 17.5%. Next, anisotropic temper-ature parameters were introduced for the iodide atoms andseveral cycles of full matrix least squares were performedin which all coordinates were varied simultaneously withanisotropic temperature factors for the iodide atoms and iso-tropic temperature factors for the light atoms. The final re-sidual after full matrix least squares is 11.9%, weightedusing a weighting scheme based on counting statistics (2).

Final coordinates and temperature factors are shown inTable 1.

RESULTS

Fig. 2 shows the crystal structure of O,O',N-trimethyl-d-tubocurarine di-iodide as viewed along the b crystallographicaxis. Both d-tubocurarine molecules in the asymmetric unit,possess almost identical molecular conformations and arerelated to one another by approximate (noncrystallographic)screw symmetry along the a axis. This same approximatescrew symmetry relates six of eight iodide anions in the unitcell, and this pseudosymmetry had initially suggested theorthorhombic space group, P21212 (in fact, F. G. Canepa,P. Pauling, and T. J. Petcher initially solved the structurein this space group, but experienced difficulty in refinement.The structure had to be refined by the rigid body least squares,and apparent disorder in one of two iodide positions wasencountered. In spite of this, however, the rough conforma-tion of the molecule could be established). The iodide anionsform numerous salt linkages with the positively chargedquaternary ammonium groups on adjacent d-tubocurarine

FIG. 2. The crystal structure of O,O',AN-trimethyl-d-tubocurarine di-iodide as viewed down the b crystallographic direction. The asym-metric unit has been labeled, and consists of two d-tubocurarine molecules and four iodide anions, a total of 100 atoms.

d-Tubocurarine Structure 2213

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2214 Biochemistry: Sobell et al.

A

D

FIG. 3. OR TEP computer-drawn illustration and the space-filling CPK model of the O,O',N-trimethyl-d-tubocurarine molecu e viewedfrom opposite sides. The molecule is leaf-like and possesses convex and concave surfaces. The convex surface (shown in A and B) is hydro-philic, while the concave surface is almost entirely hydrophobic (shown in C and D). This demarcation is an unusual feature that mayrelate to its pharmacological activity.

molecules, and relevant distances are shown in Table 2. Nowater has been observed in the crystal lattice, and since thereare no hydrogen-bond donor groups available, hydrogenbonding is absent in this lattice. The structure is stabilizedby coulombic and van der Waals interactions.The conformation of the molecule is of particular interest.

It is leaf-like, possessing convex and concave surfaces. Thisis illustrated in Fig. 3, which shows a space-filling CPK modelof the molecule and an OR TEP computer-drawn representa-tion viewed from opposite directions. The convex surfaceof the molecule (shown in Fig. 3A and B) contains the sixether oxygen atoms that lie in a fold dividing both halves ofthe molecule. This side is distinctly hydrophilic. The otherside (shown in Fig. 3C and D) is concave and is almost en-tirely hydrophobic. This demarcation between hydrophilic

and hydrophobic sides of the molecule is an unusual featurethat may relate to its pharmacological activity (see discus-sion below). The molecule has a tight compact structurethat possesses little flexibility. The interquaternary nitrogendistance is therefore rigidly fixed at 10.7 i

DISCUSSION

Although it has not yet been possible to characterize thechemical basis underlying the interaction between acetyl-choline and the cholinergic receptor or to understand indetail the events in neuromuscular transmission that follow,the site and mechanism of action of d-tubocurarine and othercompetitive neuromuscular blocking agents have been rea-sonably well defined (3, 4). d-Tubocurarine is known to com-bine with cholinergic receptor sites at the postjunctional

Proc. Nat. Acad. Sci. USA 69 (1972)

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Proc. Nat. Acad. Sci. USA 69 (1972)

membrane in the motor end-plate and to competitively in-hibit the binding of acetylcholine. Wher the drug is directlyapplied to the end-plate of a single isolated muscle fiber, themuscle cell becomes insensitive to motor-nerve impulses orto externally applied acetylcholine, although the remainderof the muscle fiber membrane retains its sensitivity to potas-sium ions and to direct electrical stimulation. Recent pro-gress in the purification of cholinergic receptor protein fromvarious sources (5-7) indicates that it is a membrane-boundprotein having a molecular weight of about 80,000. The pro-tein binds the snake venom toxin a-bungarotoxin, a poly-peptide of molecular weight 8000 that specifically and ir-reversibly blocks the depolarizing action of acetylcholineat the motor end-plate (8, 9). d-Tubocurarine protects thecholinergic receptor from the action of this toxin and thissuggests that the pharmacological competition between theseagents reflects a common binding site on the receptor pro-tein (7).Although no structural information is yet available on the

cholinergic receptor protein, conformational studies of acetyl-choline and a wide range of nicotinic acting drugs has sug-gested common stereo-specific features between them thatmay help to explain their action (10). These include a posi-tively charged quaternary nitrogen atom capable of inter-acting with anionic groups on the receptor protein, and ahydrogen-bond acceptor located about 5.9 it away (as mea-sured from its van der Waals surface). These features areobserved in the d-tubocurarine molecule (relevant distance5.6 X), suggesting that the hydrophilic convex surface ofthe molecule interacts with the acetylcholine receptor pro-tein. Its leaf-like shape and sharp demarcation between hy-drophilic and hydrophobic sides suggests an insulating rolefor the molecule, the hydrophobic portion of the moleculeperhaps interacting with the membrane and effecting depolari-zation through allosteric changes. Direct information concern-ing the interaction could, in principle, be obtained throughsuccessful cocrystallization of these components. The powerof this technique has recently been demonstrated in elucida-tion of the stereochemistry of actinomycin-DNA bindingthrough the determination of an actinomycin-deoxyguanosinecrystalline complex (11, 12).The compound, O,O',N-trimethyl-d-tubocurarine has until

now been described as O,O'-dimethyl-d-tubocurarine, an errorarising from the incorrect formulation of d-tubocurarine,which has recently been shown to have one IN (CH3)2

group and one 7N+HCH3 group (13). The increased potencyof the molecule described here, previously ascribed to 0-methylation, is almost certainly due in part to simultaneous

TABLE 2. Distances between iodide anions and quaternaryammonium nitrogen atoms

Atom 1

I1(I)I1(I)I2(I)I2(I)I2(I)I2(I)I3(I)I3(I)I4(I)I4(I)I4(I)I4(I)

Atom 2

Nl'(I)N1(II)N40(I)N1(II)N40(II)N40'(II)N1(II)N40(II)N1'(I)N40(I)N40'(I)N40'(II)

Cell

1 0 01 -1 10 -1 01 -1 11 -1 01 -1 11 -1 11 -1 01 0 00 0 01 0 -11 0 1

Distance (&)5.065.555.234.334.845.165.194.714.455.274.825.39

Note: (I) = x, y, z(II) = -x, 1/2 + Y, -z

methylation of the \N+HCH3/x group during methylationof the two free hydroxy groups in d-tubocurarine.

This work was supported in part by grants from the NationalInstitutes of Health, the National Science Foundation, the Ameri-can Cancer Society, arid the Atomic Energy Commission (awardedto H.M.S.), and the Medical Research Council (awarded toP. P.). We thank E. Reich, R. Baker, and C. Chothia for discus-sion, and M. Dellow and S. C. Jain for computing assistance.

1. Koelle, G. B. (1970) in The Pharmacological Basis of Thera-peutics, eds. Goodman, L. S. & Gilman, A. (The MacMillanCo., New York), 4th Ed., pp. 601-619.

2. Stout, G. H. & Jensen, L. H. (1968) X-ray Structure Deter-mination, A Practical Guide (The MacMillan Co., NewYork).

3. Bernard, C. (1956) C.R. H. Acad. Sci. 43, 825-829.4. Castillo, J. del. & Katz, B. (1957) Proc. Roy. Soc. Ser. B 146,

339-356.5. Changeux, J. P., Kasai, M. & Lee, C. Y. (1970) Proc. Nat.

Acad. Sci. USA 67, 1241-1247.6. Miledi, R.* Molinoff, P. & Potter, L. T. (1971) Nature 229,

554-557.7. Miledi, R. & Potter, L. T. (1971) Nature 233, 599-603.8. Chang, C. C. & Lee, C. Y. (1963) Arch. Int. Pharmacodyn.

Ther. 144, 241.9. Lee, C. Y., Tseng, L. F. & Chiu, T. H. (1967) Nature 215,

1177-1178.10. Beers, W. H. & Reich, E. (1970) Nature 228, 917-922.11. Sobell, H. M., Jain, S. C., Sakore, T. D. & Nordman, C. E.

(1971) Nature 231, 200-205.12. Sobell, H. M., in Progress in Nucleic Acid Research and

Molecular Biology eds. Davidson, J. N. & Cohn, W. E. (Aca-demic Press, Inc. New York), Vol. 13.

13. Everett, A. J., Lowe, L. A. & Wilkinson, S. (1970) Chem.Commun. 16, 1020-1021.

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