Modular design of synthetic protein mimics. Crystal structures

Dec 1, 1990 - Isabella L. Karle , R. Balaji Rao , Ramesh Kaul , Sudhanand Prasad , P. ... conformational properties of (R)- and (S)-2-methylaspartic a...
0 downloads 0 Views 857KB Size
Download PDF
J . Am. Chem. SOC.1990, 112, 9350-9356

9350

Modular Design of Synthetic Protein Mimics. Crystal Structures, Assembly, and Hydration of Two 15- and 16-Residue Apolar, Leucyl-Rich Helical Peptides Isabella L. Karle,**+Judith L. Flippen-Anderson,+ K. Uma,' M. Sukumar,* and P. Balaram' Contribution from the Laboratory for the Structure of Matter. Naval Research Laboratory, Washington, D.C. 20375-5000, and the Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 01 2, India. Received April 30, 1990

Abstract: Two IS-and 16-residue peptides containing a-aminoisobutyric acid (Aib) have been synthesized, as part of a strategy to construct stereochemically rigid peptide helices, in a modular approach to design of protein mimics. The peptides Boc(11) have been crystallized. (Val-Ala-Leu-Aib),-OMe ( I ) and Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-(Val-Ala-Leu-Aib)z-OMe Both crystals are stable only in the presence of mother liquor or water. The crystal data are as follows. I: C78H140N16019~2H20, P2,, a = 16.391 (3) A, b = 16.860 (3) A, c = 18.428 (3) A, p = 103.02 (I)O, Z = 2, R = 9.6% for 3445 data with lFol > 30(F), resolution 0.93 A. 11: C7,Hl,,N,S018.7.5H,0, C2221,a = 18.348 ( 5 ) A, b = 47.382 ( 1 1) A, c = 24.157 ( 5 ) A, Z = 8, R = l0,6%,for 3147 data with lFol > 3a(F), resolution 1.00 A. The 15-residue peptide (11) is entirely a helical, while the 16-residue peptide ( I ) has a short segment of 310 helix at the N terminus. The packing of the helices in the crystals is rather incfficicnt with no particular attractions between Leu-Leu side chains, or any other pair. Both crystals have fairly large voids, which are filled with water molecules in a disordered fashion. Water molecule sites near the polar head-to-tail regions are well detcrmined, those closer to the hydrophobic side chains less so and a number of possible water sites in the remaining "empty" space are not determined. No interdigitation of Leu side chains is observed in the crystal as is hypothesized in the "leucine

zipper" class of DNA binding proteins.

Several distinct approaches are being considered for the design and construction of synthetic polypeptides, which mimic the folding motifs observed in proteins.' In the first approach the 20 genetically codcd amino acids are used and a choice of sequence is based on a knowledge of the propensities of specific sequences to fold in a predictable manner., This method has been used most recently to construct synthetic four-helix motif^,^ which have been characterized by circular dichroism and hydrodynamic properties in ~ o I u t i o n . ~The ~ *use ~ ~ of ~ covalently linked templates for organizing thc spatial arrangement of synthetic peptides has also been e ~ p l o r e d . A ~ third approach, which is being developed in thcsc laboratories, is also a modular a p p r o a ~ hbased , ~ on the ability of specific non-protein amino acids to direct polypeptide chain folding, by virtue of their unique stereochemical properties. I n particular, the use of a-aminoisobutyric acid (Aib)6*7to construct stereochemically rigid helices is being systematically investigated.* Thc schcmc illustrated in Figure 1 envisages modular assembly of conformationally rigid helices into super-secondary structures like the CY,CY motif by means of intervening, flexible linker sequences. This approach may be divided into three distinct steps: (i) the synthetic construction and unambiguous structural characterization of the helical modules; (ii) the design and synthesis of appropriate linker peptides, a problem that is being approached by a computer-based analysis of linker sequences occurring in a,a motifs identified in crystalline proteins; (iii) the covalent assembly of the modules and the linker peptides, and introduction of features to promotc spccific hclix orientations. In this report we describe the characterization in crystals of two apolar hclical peptides of 15 and 16 residues in length: Boo( Val-Ala-Leu-Aib),-OMe (1) Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-( Val-Ala-Leu-Aib),-OMe (11) Thc scqucnccs have been chosen to maintain a relatively low Aib content (1. 25%; I f , 20%) and to be completely apolar to facilitate studies in organic solvents, in order to avoid the influence of hydrophobic interactions on the folding process, at this stage of synthetic design. The distribution of Leu residues in I 1 is reminiscent of the arrangements observed in the dimer-forming leucine 'Naval Rcscarch Laboratory. I Indian Institute of Scicncc.

0002-7863/90/ I5 12-9350$02.50/0

zipper region of a class of DNA binding protein^.^ The pairs Leu3/Leul0 and Leu7/LeuI4 are seven residues apart in the sequence in 11. In the leucine zipper proteins such heptad repeats occur in succession four or five times and the stabilization of dimers is expected to be due to interdigitation of leucine side chains in a helical coiled-coil m ~ t i f . ~ Peptides *'~ I and I1 provide an opportunity to examine attractive interactions between leucyl side chains in helical peptides. The crystal structures of I and I1 demonstrate stable helical conformations over the entire length of the molecules. No interdigitation or particular association between Leu side chains has been observed. Large voids occur ( I ) DeGrado, W. F. Adu. Protein Chem. 1988, 39, 51-124. (2) (a) DeGrado, W. F.; Wasserman, Z. R.; Lear, J. D. Science 1989,243, 622-628. (b) Richardson, J. S.; Richardson, D. C. In Protein Engineerins Oxender, D. L., Fox, C. F., Eds.; Alan R. L i s Inc.: New York. 1987; pp 149-163. (c) Urry, D. W. Res. Deu. 1988,30,72-76. (d) Moser, R.; Thomas, R. M.; Gutte, B. FEBS Lett. 1983, 157, 247-251. (e) Moser, R.; Frey, S.;

Munger. K.; Hehlgans, T.; Klauser, S.; Langen, H.; Winnacker, E.; Metz, R.; Gutte. B. Profein Eng. 1987, I , 339-343. (3) (a) Regan, L.; DeGrado, W. F. Science 1988, 241,976-978. (b) Ho, S. P.; DeGrado, W. F. J. Am. Chem. SOC.1987, 109, 6151-6758. (c) DeGrado, w . F.; Regan, L.; Ho, S. P. Cold Spring Harbor Symp. Quant. Biol. 1987,52,521-526. (d) Hecht, M.H.; Richardson, D. C.; Richardson, J. S.; Ogden, R. J. Cell. Biochem. 1989, 13A. 86. (e) Kaumaya, P. T. P.; Berndt, K. D.; Heidorn, D. B.; Trewhella, J.; Kezdy, F. J.; Goldberg, E. Biochemistry 1990, 29, 13-23. (4) Mutter, M.; Vuilleumier, S.Angew. Chem., Int. Ed. Engl. 1989, 28, 535-554. (5) Karle, I . L.; Flippen-Anderson, J. L.; Uma, K.; Balaram, P. Biochemisfry 1989, 28, 6696-6701. (6) (a) Abbreviations used: Aib, a-aminoisobutyric acid; Boc, (tert-bu-

tyloxyNarbonyl. (b) For definitions of torsional angles, see: IUPAC-IUB Commission on Biochemical Nomenclature. Biochemistry 1970, 9, 3471-3479. (c) All chiral amino acids are of the L configuration. (7) (a) Prasad, B. V. V.; Balaram. P. CRC Crit. Reo. Biochem. 1984, 16, 307-347. (b) Toniolo, C.; Benedetti, E. IS/ Aflas Sci.: Biochem. 1988, I , 225-230. ( 8 ) (a) Karle, 1. L.; Flippen-Anderson, J . L.; Uma, K.; Balaram, P. Biopolymers 1990, 29, 1835-1845. (b) Karle, I. L.; Flippen-Anderson. J. L.; Uma, K.; Balaram, P. Proteins: Sfrucr. Funct. Genet. 1990, 7 , 62-73. (c) Karle, 1. L.; Flippen-Anderson,J. L.; Uma, K.; Balaram, H.; Balaram, P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 765-769. (9) (a) Landschulz, W. H.; Johnson, P. F.; McKnight, S. L. Science 1988, 240, 1759-1 764. (b) OShea, E. K.;Rutkowski, R.; Kim, P. S. Science 1989, 243, 538-542. (c) Kouzarides, T.; Ziff, E. Nature 1988, 336, 646-651. (IO) Vinson, C. R.; Sigler, P. B.; McKnight, S. L. Science 1989, 246, 91 1-916.

0 i 990 American Chemical Society

J . Am. Chem. Soc.. Vol. 112, No. 25, 1990 9351

Modular Design of Synthetic Protein Mimics

Table 1. Diffraction Data for Boc-(Val-Ala-Leu-Aib),-OMe.2H20 (I) and Boc-Val-Ala-Leu-Aib-Val~Ala-Leu-(Val-Ala-Le~-Aib)~-OM~ 7.5H,O (11) I I1 empirical formula color/ habit crystal size, inm space group cell parameters Figure 1. Schematic illustration of the modular or 'Meccano set" approach to construction of an a,a motif.

betwccn helices, which are filled by water, providing insights into hydration and the occurrence of water in hydrophobic cavities.

Experimental Procedures Both peptides I and I I were synthesized by conventional solution-phase procedures by a fragment condensation approach using dicyclohexylcarbodiimide and I -hydroxybenzotriazole in dichloromethane or dimethylformamide." The final peptides were purified by high-performance liquid chromatography on a reverse-phase Lichrosorb RP- I8 column (8 mm X 250 mm; particle size, IO r m ) using isocratic elution (95% McOH in H,O: flow rate, 1.5 mL min-I). Repetitive injections of - 5 mg pcr run were carried out. Crystals were grown by slow evaporation from a CH,OH/H,O solution. Thc crystals of both peptides were stable only in contact with mother liquor, and cach was scaled in a thin-walled capillary for X-ray data collections. Crystals of peptide I I were particularly fragile and had to be handled with extreme care. A crystal of I I needed an unusually large amount of mother liquor sealed with it in a capillary to maintain its viability. To prevent the crystal from sliding inside the capillary, the temperature was lowered to 2 OC with a cold nitrogen stream for the data collect ion. For each crystal, X-ray diffraction data were measured on an automated four-circle diffractometer with a graphite monochromator. Three reflections used as standards, monitored after every 97 measurements, remained constant to within 3% in the two data sets. Pertinent diffraction data arc listcd in Tablc I . Both structurcs wcrc solvcd by a vector search procedure in the Patsee computcr programI2 contained in the SHELX84 package of programs (MicroVAX version of SHELXTL system of programs, Siemens instrumcnts. Madison. WI). Thc modcl used for the vector search consisted of 43 backbone atoms from the Boc-Aib-(Val-Ala-Le~-Aib)~-OMe s t r ~ c t u r e .After ~ rotation and translation to the correct position in the cell, the remainder of the atoms in both molecules were found with the partial structure procedure." Full-matrix, anisotropic Icast-squarcs refinement was performed on thc C, N , and 0 atoms after which hydrogen atoms were placed in idcalizcd position, with C-H of 0.96 A, and allowed to ride with the C or N atom to which each was bonded for the final cycles of refinement. The thermal factor for the hydrogcn atoms was fixed at Ui, = 0.125. Four water sites, each at 0.5 occupancy were found in crystal I. The sites for W ( 1 A ) and W( IB) are mutually exclusive as well as the sites for W(2A) and W(2B). I n crystal II, 13 water sites were identified in difference maps and remained stable in the least-squares refinement, 3 sites with full occupancy and IO sites with occupancies 0.4-0.6. Among thc IO sitcs with partial occupancy, only 5 are occupicd in any one asyrnmctric unit bccausc of mutual exclusivity. There may be additional disordered sitcs present in a few remaining voids in the cell of crystal I I , but this cannot bc substantiated bccausc of weak and diffusc clcctron density distributions i n the difference maps. Fractional coordinatcs for peptides I and 11 arc listed in Tables I I and I l l , respectively. Conformational angles arc listcd in Table I V .

Results The Helix. Peptides I ( 16 residues) and I I ( 15 residues) differ only by the prcscncc of Aib8 in I and its removal in I I . The stereodiaprams in Figure 2a and b show the helical structures of both peptides. I n I. where the Leu residue occurs in every fourth ( I I ) Balaram, H.; Sukumar, M . ; Balaram, P. Biopolymers 1986, 25. 2209-2223. (12) Egcrt. E.: Shcldrick. G. M. Acta Crysrallogr. 1985, A41, 262-268. ( 13) Karle. J . Acta C'rystullogr. 1968, 824. 182-1 86. (14) Karlc, I. L.; Flippen-Anderson. J . L.;Sukumar, M.; Balaram, P. Int. J. Pept. Protein Res. 1988, 3 / , 567-516. (IS) Karlc, I. L.: Flippn-Anderson, J . L.: Sukumar, M.: Balaram. P. Int, J. Pept. Prorein Res. 1990. 35, 518-526.

a. A b. A c,

A

P, deg VOI, A' Z mol wt density, g/cm F(OO0)

radiation temp, OC mounted in capillary 28 range, deg resolution, A scan type scan speed, deg/min index ranges h k I

independent reflcns obsd reflcns [IFoI > 3dF)I final R indices (obsd data), % goodness of fit data to parameter ratio largest diff ratio. e A-' largest diff hole,'e A-'

S018'7.5H20 colorless/plate 0.65 X 0.30 X 0.15 c222,

C78H laN,6°19*2H20

C74H 133Nl

16.391 (3) 16.860 (3) 18.428 (3) 103.02 ( I ) 4962.4 (1.4) 2 1606.09 + 36.03 1.099 1784 Cu K a ambient Yes 1-1 12 0.93 8-28 7-1 3 (variable)

18.348 (5) 47.382 (1 I ) 24.157 (5)

-18 to 18 0-19 0-20 6762 3445

0-19 0-48 0-24 5909 3147

R 9.6

R 10.6

R , 9.2

R , 10.7

I .5 7.2: I 0.29 -0.33

12.2 6.7:l 0.36 -0.31

colorless/fragment 0.25 X 0.50 X 0.15 p2 I

21001 (9) 8 1520.98 + 135.12 I .048 7208 Cu K a , X = 1.5418 A 2 Yes 1-100 1 .oo

8-26 29'

position, the side chains of Leu', Leu1),and LeuI5 are extended to the left side of Leu3 of the helix whereas the side chain of Leu3 (near the top) projects forward, as viewed in Figure 2a. The removal of Aib8 from I, to yield peptide 11, results in the extension to the left of the side chains of all four Leu residues, Le., Leu3, Leu', Leulo, and LeuI4 (Figure 2b). Furthermore, the side chains of Vali, Val5, Val8, and Valfzare all extended to the right side of the helix. The helical structure of the backbone is quite similar in the two pcptides. A least-squares fit of backbone atoms N(3)-0( 14) in I with N(2)-0( 13) of I I yields a root mean square deviation of 0.28 A. Peptide I I is completely a-helical with 12 5 4 1 hydrogen bonds (Table V), while peptide I has 1 1 5-1 hydrogen bonds and 2 4-1 hydrogen bonds (310helix) near the N terminus (Table V). A partial transition from a to 310helix, particularly at the N terminus, is not unusual, even for the same peptide in different polymorphsI6 or for multiple independent molecules in the same The conformations obtained earlierEbfor the hepta- and octapeptide fragments, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe and Boc-(Val-Ala-Lcu-Aib)2-OMe, are largely similar to that obscrvcd for the corresponding segments in the longer peptides 1 and 1 1 . Head-to-Tail Hydrogen Bonding. Apolar helical peptides utilize head-to-tail hydrogen bonding to form continuous colu m n ~ . ~ . ~Crystal . ~ ~ -structure ~ ~ . ~ information ~ for helical peptides with polar side chains is not extensive; therefore, a general statement about head-to-tail hydrogen bonding cannot be made at this time. For the apolar helices, the head-to-tail hydrogen bonding motifs vary from three direct N H d = C bonds between thc first thrcc N H moicties in one helix and the last three C=O moieties from another helix with the two helices in perfect register with cach othcrgcto hydrogen bonding where the N H and O I C moictics from the scparatc helices are mediated by water or alcohol (16) Karle, I. L.; Flippen-Anderson, J . L.: Uma, K.; Balaram, P. Bio-

pol,vmers. in prcss.

9352 J. Am. Chem. Soc., Vol. 112, No. 25, 1990 0

A

U

Figure 3. Stereodiagram depicting the head-to-tail region of peptide I1 with 12 of the water sites shown as black dots with W(n) labels. Only the bottom portion of the top peptide helix and the top portion of the bottom peptide helix are shown. Hydrogen bonds are indicated by dashed lines. Water molecules W(nA) and W(nB) occur only at -50% occupancy.

I d

Figure 4. Water molecules [W(n)] in the head-to-tail region of peptide I. Fragments of three neighboring peptide molecules are shown. Hydrogen bonds are indicated by dashed lines. Each of the water sites has -50% occupancy.

peptide. Water molecules W(l), W(2), and W(7) [where W(7) lies on a symmetry element] occur at full occupancy, whereas the remaining water sites are occupied only half the time. Those water sitcs labeled with the same number, such as W(6A) and W(6B), are mutually exclusive; that is, if one site is occupied, the other Figure 2. (a) Stereodiagram of Boc-(Val-Ala-Leu-Aib),-OMe (peptide site must be empty. Some of the water molecules, especially I ) . (b) Stereodiagram of Boc-(Val-Ala-Leu-Aib)-Val-Ala-Leu-(ValW(3A) and W(3B), occur in voids between the peptides and are Ala-Leu-Aib)?-OMe (peptide 11). I n each diagram, the C" atoms are not close enough for any hydrogen bonding. There undoubtedly numbered. The helices are oriented so that the Leu side chains are are other water sites that are occupied to a lesser extent but could extended to the left and the Val side chains are extended to the right. not be definitely substantiated in our experiment. molecules.Is The latter motif is present to an extreme in peptide I n the crystal of peptide I, the water structure is much simpler, as shown in Figure 4, where fragments of three surrounding 11. peptide molecules are shown. Water sites W( I A ) and W( I B), The stereodiagram in Figure 3 depicts the lower portion of one helical molecule I 1 placed over an upper portion of another helical each with -50% occupancy on the average throughout the crystal, have full occupancy for one or the other with one site unoccupied molecule I 1 with numerous water molecule sites (labeled W) near in any particular region of the crystal lattice. The same pertains the head-to-tail region. The N ( I)H-.O( 13) hydrogen bond is the only direct hydrogen bond between the two separate helices. The to water sites W(2A) and W(2B). There is only one direct hydrogen bond, head-to-tail, between peptide molecules, N ( 1 )HN(2)H moiety forms a hydrogen bond with W(2), which in turn SO(15). The N(2)H moiety does not participate in any hydrogen participates as a donor in hydrogen bonds with O( 13) and O( 15) bonding and N(3)H forms an internal N(3)H-.0(0) bond of the of the upper peptide and as a receptor in a hydrogen bond with 3,0 helix type. W( IA) or W( 1 B) is a donor in a hydrogen bond W( I ) . The N(3)H moiety forms a hydrogen bond with W( I ) , which in turn is a donor to W(2) and W(8A). Water molecule with O( 14), and W(2B) is a donor to a hydrogen bond with O(16). W(8A) forms another hydrogen bond with W(5A) which, in W(2A) is at least 3.9 A from any other C or 0 atom. Obviously, succession, is a donor to a hydrogen bond with O( 12) of the upper the underutilized hydrogen-bonding potential of the water mol-

J . Am. Chem. SOC.,Vol. 112, No. 25, 1990 9353

Modular Design of Synthetic Protein Mimics

Table 11. Atomic Coordinates ( X IO') and Equivalent Isotropic Displacement Coefficients (A2X IO3) X V z Ued" X Y 1498 (6) 3411 (6) 5986 (8) 1165 (7) 4 2 6 0 (8) 1787 (7) 3579 (8) 926 (8) 3328 (7) 6156 (8) 1310 (7) 4265 (7) 6277 (8) 590 (7) 4718 ( s i 1092 (5) 4220 (6) 1897 (8) 6413 (8) 4614 (9) 2721 (7) 3729 (8) 5145 (5) 1246 (5) 4438 (5) 3212 (8) 3017 (9) 1711 (6) 4680 (8) 4130 (7) 3933 (9) 3078 (8) 4918 (6) 2076 (5) 3629 (5) 1210 (6) 4889 (6) 3945 (6) 4448 (6) 1746 (6) 5588 (7) 780 (8) 3336 (8) 21 12 (7) 4103 (8) 5384 (7) -I20 (7) 3508 (8) 4177 (8) 2941 (8) -373 (6) 5661 (6) 3260 (6) 3298 (6) 3686 (6) 721 (8) 6246 (9) 2575 (8) 1989 (8) 4540 (8) -550 (6) 4924 (6) 2438 (9) 1853 (9) 4295 (IO) -1383 (7) 4716 (8) 1213 (9) 2341 (9) 4333 (9) 4124 (7) -1434 (7) 3267 (5) 4861 (6) 3913 (7) -1975 (5) 4229 (6) 5034 (9) 41 I9 (IO) 4039 (7) -1910 (8) 4398 (8) 4505 (IO) 4231 (7) 4307 (8) 4954 (9) -2070 (8) 4432 (7) 4883 (5) 4194 (6) -2538 (IO) 5660 (IO) 4338 (1 3) 4378 (8) 5763 (9) 4555 (9) -2577 (IO) 4988 (7) 3768 (5) 3775 (7) -857 (6) 3573 (6) 3966 (7) 5388 (8) 3103 (7) -787 (7) 3011 (8) 4076 (6) 4832 (9) 2420 (7) -726 (7) 3484 (9) 4535 (4) 5012 (6) 2027 (5) -1 117 (5) 3333 (6) 3426 (6) 6079 (8) 2740 (8) 2503 (8) 3436 (8) 6791 (9) 3266 (8) -6 (7) -1550 (7) 2468 (8) 4179 (9) 7181 (IO) 3430 ( 1 1 ) -180 (5) 4093 (6) 2818 (9) 2863 (1 0) 7343 (9) 4577 (7) -48 (6) 3661 (5) 2290 (6) 4191 (7) -811 (9) 5081 (8) 1697 (9) 3623 (9) 3734 (7) -1088 (6) 5103 (7) 1878 (9) 3324 (8) 4569 (8) 750 (8) 4926 (5) 5104 (9) 1312 (6) 3208 (6) 1500 (7) 4579 (9) 3238 (7) 2900 (8) 1733 (9) 837 (8) 5685 (IO) 787 (8) 3548 (7) 3953 (8) -I I78 (6) 5472 (6) 2662 (7) 4843 (5) 3155 (7) -1886 (8) 5985 (8) 3033 (9) 5609 (7) 2837 (8) -2664 (8) 5529 (9) 2939 (8) 6201 (8) 3444 (9) -3151 (6) 5860 (7) 2638 (7) 6733 (5) 3303 (7) -2080 (8) 6438 (8) 3904 ( 1 0) 5725 (8) 2539 (9) 3853 (IO) -2809 (6) 4813 (7) 5177 (9) 1867 (IO) 4280 (9) 2296 ( 1 1 ) -3524 (7) 4339 (7) 6417 (8) -3475 (9) 3851 (8) 4184 (8) 3188 (7) 6068 (5) 4809 (9) -4079 (6) 3538 (6) 6603 (8) 31 16 (8) -3778 (8) 3844 (8) 4943 (8) 2215 (8) 6604 (8) -4040 (8) 5108 (7) 2081 (5) 4334 (8) 7258 (5) -4762 (9) 5489 (9) 3494 (9) 4869 (9) 6382 (8) 4941 (8) 1606 (7) -4182 (9) 3785 (9) 5998 (6) 767 (8) 4984 (8) -2667 (7) 3778 (7) 6059 (7) 465 (8) 4374 (8) -2498 (IO) 3347 (IO) 6500 (7) 2549 ( I I ) -2882 (IO) 4579 (6) 6945 (5) 5 (6) -3221 (8) 2240 (9) 223 (7) 5062 ( I O ) 5264 (7) -702 ( 1 1 ) 5165 (IO) -I 554 (9) 3207 (IO) 5191 (8) -1245 (IO) 5015 ( 1 1 ) 3797 (IO) -2905 (1 0) 4408 (8) -900 ( 1 0) 5989 ( I I ) -2825 (7) 2166 (7) 5429 (9) 676 (7) 3609 (7) -3237 (IO) 1373 (IO) 6402 (6) 2651 (13) 973 ( I 2) 438 (8) 2987 (8) 6856 (8) 3626 (12) 407 (12) 705 (8) 3136 (7) 7695 (7) 8491 (12) 2007 (12) 213 (5) 3022 (6) 8069 (5) 7400 (12) 2491 (12) 889 (9) 2258 (9) 6619 (8) Cfl(8) -525 (8) 2824 (9) 6596 (7) a Equivalcni isotropic U dcrincd as one-third of the tracc of orghogonalizcd U,, tensor. bOccupancy -0.5.

ecules, as well as the free N(2)H moiety, indicates that additional water sites with low occupancy can be present in the head-to-tail region. Helix Aggregation. Completely parallel packing of apolar peptidcs with 8-16 rcsidues has been established for a number of p ~ p t i d c s . ' ~Some ' ~ of the peptides that aggregate in a parallel fashion in one polymorph aggregate also in an antiparallel fashion (17) Tcrwilligcr, T. C.; Eiscnberg, D. J . B i d . Chcm. 1982, 257, 6016-6022. (18) Karle, 1. L.;Flippen-Anderson. J. L.;Sukumar, M.;Balaram. p. floc. Narl. Arad. Sri. U S A . 1987.84, 5087-5091. (19) Karle, 1. L.; Flippen.An&rmn, J. L.; Nail. Acad. S r i . U.S.A. 1988. 85, 299-303.

uma,K,;Balaram.

p, proc,

z 7884 (6) 8690 (7) 8955 (7) 9532 (5) 8837 (7) 8684 ( I ) 9666 (7) 8511 (6) 8729 (7) 8824 (7) 9326 (5) 8205 (8) 8278 ( 5 ) 8340 (5) 8950 (6) 9297 (5) 7591 (7) 6938 (8) 7060 (8) 6236 (7) 9073 (5) 9646 (7) 10369 (8) 10881 (5) 9756 (7) 9535 (7) 10508 ( 5 ) 1 1 194 (7) 11248 (8) 11814 (5) 11228 (7) 11372 (7) 11869 (7) 10598 (6) 10581 (8) 10754 (8) 1 IO55 (6) 9863 (8) 10429 (6) 10461 (8) I 1 I45 (8) 11288 (6) 9765 (9) 9014 (8) 8959 (8) 8344 (8) 1 I575 (6) 12307 (9) 12140 (IO) 12646 (7) 12519 (8) 12881 (9) 11546 (7) I1413 (9) 7268 (11) 7402 ( I 0) 4573 ( 1 1 ) 4271 ( 1 1 )

U(eq)"

in a polymorph grown from a different s o l ~ e n t . ~The ~ J common ~ motif in the aggregation of apolar helical peptides in crystals is the formation of continuous columns by head-to-tail hydrogen bonding and the assembly of the columns into sheets with all helix axes in the parallel mode. The differences in packing occur in the mode of assembly of the sheets: parallel, antiparallel, or the sheets of parallel s k e ~ e d . ~ "In~ crystal ~ ~ ' I (space group R,), helices are related by the 2-fold screw axis perpendicular to the helix axes, resulting in an antiparallel assembly as shown in Figure 5. In Figure 6 (space group C222,), a sheet of parallel helices with all N termini at the top is shown for crystal 11. The sheets of hcliccs above and below the one shown are rotated by 1800, thus making an antiparallel a r r a n g m " in the direction into the Page.

Karle et al.

9354 J . Am. Chem. SOC.,Vol. 112, No. 25, 1990 Table Ill. Atomic Coordinates ( X IO') and Equivalent Isotropic Displacement Coefficients (A2 X IO3) X

I'

Z

Uea)'

X

Y

2

Uea)"

2109 (8) 2483 (IO) 2221 (9) 2283 (7) 3033 (9) 1979 (8) 1734 (IO) 1255 ( 1 1 ) 1235 (6) 1556 ( 1 1 ) 1304 (14) 1704 (12) 1088 (16) 853 (8) 384 (8) 569 ( 1 1 ) 345 (7) 99 (11) -25 (1 1) I010 (8) 1 I90 (1 I ) 1488 (IO) 1412 (7) 1524 (17) 1149 (13) 1784 (17) 1772 (8) 2023 (11) 1605 (9) 1705 (6) 2404 (IO) 1 I49 (8) 721 (9) 310 (IO) 43 (7) 434 (IO) 737 (16) 1094 (16) 450 ( I 3) 277 (7) -154 (IO) -67 (1 3) -140 (12) 283 (6) -730 ( I 3) -650 (6) -662 ( 1 1 ) 2765 (8) 3617 (7) 346 (26) -122 (25) -39 ( 1 9) 1262 (19) 1686 (26) I550 (26) 27 (20) 0 (73) 1057 (23) 2345 (48) 2373 (42)

72 (4) 58 (4) 62 (4) 100 (4) 109 (4) 58 (4) 63 (4) 68 (4) 59 (3) 76 (4) I12 (4) 136 (4) 177 (4) 59 (4) 45 (4) 77 (4) 69 (3) 85 (4) 67 (4) 63 (4) 75 (4) 53 (4) 83 (4) 165 (4) 129 (4) 324 (4) 67 (4) 81 (4) 47 (4) 72 (3) 67 (4) 54 (4) 67 (4) 50 (4) 80 (4) 75 (4) 152 (4) 186 (4) 131 (4) 53 (4) 74 (4) I l l (4) 65 (4) 96 (4) 132 (4) 85 (4) I I8 (4) 135 (4) 89 (4) 187 (7) 173 (7) 152 (7) 172 (7) 216 (7) 201 (7) 128 (7) 331 (7) 136 (7) 494 (7) 381 (7)

~~~

1352 ( 1 1 ) 8060 ( 1 1 ) 3200 (4) 6012 (7) 7937 ( 1 3) 2951 (4) 933 ( I t ) 5947 (7) 7362 (13) 1360 (15) 2757 (6) 6339 (6) 7346 ( 1 2) 1309 (15) 2508 (3) 5872 (8) 7720 ( I 8) 1927 (7) 3082 (5) 5980 (3) 6743 (9) 2100 ( 1 1 ) 2889 (4) 5738 (5) 1878 (7) 6203 ( 1 3) 2720 (5) 5504 (4) 6457 (14) 2553 (6) 2587 (7) 5772 (3) 6362 (7) 2292 (3) 2869 (8) 5535 (4) 2984 (8) 5545 (13) 2927 (5) 5277 (4) 4896 ( I 6) 2751 (7) 2844 (6) 5040 (3) 4505 (1 3) 2572 (8) 3385 (9) 5631 (4) 4392 (18) 2970 (8) 3626 ( 1 I ) 5380 (5) 2692 (4) 3288 ( I 1) 6875 (IO) 5880 (5) 7215 ( 1 1 ) 2536 (5) 3317 (7) 5347 (3) 7654 ( I 6) 2286 (5) 3530 (9) 51 I7 (5) 7562 (9) 2050 (3) 4947 (5) 3081 (9) 2743 (5) 7711 (14) 3094 (6) 4683 (3) 6541 (12) 2454 (5) 3957 (IO) 5208 (5) 8124 ( 1 I ) 2328 (4) 2654 (7) 5096 (3) 8581 (14) 2190 (9) 4927 (4) 2099 (5) 8177 (13) 1869 (4) 1873 (8) 4764 (5) 8334 (IO) 1755 (6) 1622 (3) 4512 (3) 9234 ( 1 8) 1831 (8) 2235 (7) 5146 (4) 1337 (IO) 9699 (18) 2405 (6) 4999 (6) 9595 (25) 1995 (12) 903 ( 1 5) 5239 (7) 1481 (IO) 7592 (1 I ) 1950 (4) 4807 (5) 1721 (5) 1751 (7) 7150 (15) 4886 (3) 6752 ( 1 1 ) 1532 (5) 1469 (9) 4726 (5) 6652 (IO) 1280 (3) 1737 (IO) 4458 (4) 1870 (4) 6587 ( 1 3) 1507 (5) 4227 (3) 6517 (9) 1661 (4) 1463 (9) 4906 (4) 1495 (5) 6057 (14) 865 ( I O ) 4669 (6) 6623 ( 1 2) 1349 (5) 2311 (7) 4473 (3) 6371 (9) 1147 (3) 2628 (9) 4229 (5) 5588 (13) 1684 (6) 2629 (9) 4019 (5) 5010 (19) 1814 (7) 2553 (7) 3750 (3) 4580 ( I 9) 1631 (8) 3223 (8) 4323 (5) 3571 ( 1 1 ) 4588 ( I 5) 2005 (7) 4075 (6) 7291 (IO) 1451 (4) 3221 (12) 4491 (5) 1356 (6) 7748 ( 1 5) 2714 (7) 41 14 (4) 8532 (15) 1478 (6) 2710 (8) 3907 (5) 1017 (4) 7843 ( 1 3) 2194 (7) 3742 (4) 901 (3) 7936 (12) 2221 (6) 3496 (3) 1475 (6) 7513 (18) 2874 ( 1 1 ) 4083 (5) 7931 ( 1 1 ) 914 (4) 1721 (7) 3898 (3) 8007 ( I 9) 1192 ( 1 1 ) 612 (4) 3751 (6) 6310 (14) 5697 (4) I055 (IO) 3545 (5) 859 (7) 7240 ( 1 0) 5908 (4) 3313 (3) 891 (33) 1644 (13) 720 (9) 3970 (5) 5771 (33) 695 ( I 3) 3248 (13) 4143 (6) 869 (24) 4396 (9) 4367 (6) 221 (IO) 9704 (24) 1310 (IO) 675 ( 1 3) 3988 (6) 2198 (14) 1769 (32) 1237 (7) 3605 (4) 1213 (12) 3401 (5) 2234 ( I 5) 2289 (33) 1562 (IO) 58 (27) 4318 (IO) 3144 (4) 6512 (32) 1437 (7) 2892 (3) 0 (73) 1056 (13) 9656 (29) 1292 ( I I ) 3536 (6) 832 ( 1 5) 157 (60) I059 (20) 3744 (6) 1345 (14) 879 (49) 1380 (21) 3328 (6) Equivalcnt isotropic U dcfincd as one-third of the trace of the orthogonalizcd U,,tensor. bOccupancy -0.5. 7589 (15) 8168 (17) 7406 (22) 6895 ( I 9) 7902 ( I 0) 8104 (16) 8032 ( I O ) 8424 (IO) 8777 ( 1 1 ) 8370 ( 1 2) 8512 (7) 9259 ( I 3) 9674 ( 1 4) 9819 ( 1 I ) 7791 (IO) 7399 ( I 3) 7009 ( 1 2) 6950 (8) 6851 (14) 6702 (9) 6392 ( 1 2) 6891 (12) 6749 (8) 5976 ( I 2) 5570 (14) 5323 (17) 4956 ( I 4) 7549 (9) 8109 ( 1 1 ) 8259 ( 1 0) 8282 (7) 8752 (IO) 7891 (14) 8385 (9) 8551 (13) 7981 (12) 8083 (9) 8754 ( I 3) 8781 (17) 9486 ( 1 3) 7272 ( I O ) 6680 ( 1 2) 6570 (IO) 6414 (8) 5972 ( 1 7) 6661 (IO) 6585 ( 1 3) 7134 (13) 7022 (IO) 6523 (14) 5830 ( I 3) 5885 (20) 5144 (13) 7818 (IO) 8390 (14) 8300 ( I 2) 8392 (3) 9162 (16) 9357 ( 14)

The packing of the hydrocarbon side chains in crystals of both peptidcs (Figurcs 5 and 6) is quite inefficient with considerable spacc around thcm. Ncarcst approaches between C-C in neighboring molecules are rarely closer than 3.9 A and generally considerably larger than 4.1 A. There is no particular attraction betwccn spccific types of side chains. The voids between helices arc cspccinlly apparent in the crystal of peptide I 1 (Figure 6). The largc amount of watcr in peptidc I I is ordered [ W ( 1) and W(2)] in thc hcad-to-tail rcgion where each water molecule participates in scvcral hydrogcn bonds with thc N H and C=O moictics of thc pcptidcs and bccomcs less ordered ((W(4), W(5). W(6)] when only onc W-.O hydrogcn bond or a W-W hydrogcn bond is formcd. Scvcrnl npparcnt watcr sitcs [W(3A), W(3B). W(8B)]

are not within any hydrogen bond distance or van der Waals distance of any other atom, an indication that there probably are other water sites filling the hydrophobic holes.

Discussion The helical conformations determined for I and I 1 suggest that long stable helices can be observed in peptides with relatively low Aib content. I!, in fact, contains a central stretch of six contiguous non-Aib residues. The results underscore the dominant role of Aib rcsidues in nucleating and stabilizing helical folding patterns in peptides. Both helices are straight, cylindrical structures, in contrast to the curved helix observed in a peptide of similar length containing internal Pro residues.I8 The observed structures

J . A m . Chem. SOC.,Vol. 112. No. 25, 1990 9355

Modular Design of Synthetic Protein Mimics

Table VI. Hvdroaen Bonds in PeDtide I€'

Table IV. Torsion Angles@(deg) residue

q5

Val(l) Ala(2) Leu(3) Aib(4) Val(5) Ala(6) Leu(7) Aib(8) Val(9) Ala(l0) Leu(l I ) Aib(l2) Val( 13) Ala(14) Leu(l5) Aib(l6)

-61.7b -50.9 -68.4 -55.5 -64.3 -64.5 -57.2 -53.1 -66.0 -58.2 -71.8 -54.5 -67.1 -61.3 -82.3 +54.4

it

X2

XI

W

Boc-(Val-Ala-Leu-Aib),-OMe -33.0 -179.5 173.7, -66.9 -34.5 -176.7 -32.8 177.6 -69.8 -62.4. 172.9 -46.7 -178.2 -48.8 179.6 -61.5, 175.1 -39.8 172.9 -44.4 176.2 -176.6 -164.1, 73.1 -43.7 179.0 -45.1 -178.3 -63.4, 178.5 -44.0 179.8 -35.4 176.1 -62.3 -61.4, 178.6 -48.3 -178.2 -44.9 -1 78.2 -69.4, 172.6 -41.4 174.2 -14.9 176.6 -61.2 -60.6, 173.2 +40.7' -179.1d

Boc-Val-Ala-Leu-Aib-VaLAla-Leu-( Val-Ala-Leu-Aib)2-OMe Val( I ) -55.5b -60.2 -1 71.6 - I 74.2, -49.6 Ala(2) -65.5 -38.5 173.7 -64.8 -40.3 175.0 -176.4 -166.1, 66.5 Leu(3) Aib(4) -51.9 -50.0 -178.9 Val(5) -61.8 -45.8 179.2 168.8, -67.2 -60.3 -39.5 -178.2 Ala(6) Leu(7) -67.7 -34.0 -169.4 -69.6 176.0, -54.8 Val(8) -64.5 -45.1 178.4 -63.0, 172.6 180.0 Ala(9) -58.9 -42.7 Leu(l0) -62.4 -46.8 174.1 -177.0 69.0, -1 72.0 -46.0 -177.4 Aib(lI) -51.6 -30.6 174.3 -66.4, 169.4 Val(l2) -72.5 -34.4 -176.5 Ala(13) -67.8 Leu(l4) -87.3 -20.0 -169.1 -66.3 -46.7, 176.5 Aib(l5) -57.5 148.2' -179.0" "The torsion angles for rotation about bonds of the peptide backbone (I$, it, and W ) and about bonds of the amino acid side chains ( X I and x z ) arc dcscribcd in rcf 6b. csd's are 1 . 5 O for peptide I and ~ 2 . 5 ~ for pcptidc II. bC'(0), N ( I ) , Cu(I), C'(1). 'NV), Cmy). C'y), 0(OMc) whcrc f = final residue. dCuy), C'y), O(OMe), C ( 0 M e ) w h c r c j final residue.

,.

tvDe

head-to-tail water .peptide-to. 5+ 1

water-to-peptide or water

donor N(I) N(2) N(3) N(4) N(5) N(6) N(7) N(8) N(9) N(10) N(11) N(I2) N(13) N(14) N(15) W(1)

angle N** *O,Ha -0,' C d . * *N, acceDtor A A dea 2.95 0(13)c 2.02 155 1.99 W(2) 2.94 W(I) 2.95 2.03 O(0) 3.07 2.14 158 O(l) 2.99 2.04 161 O(2) 2.91 1.97 154 O(3) 2.91 1.97 165 O(4) 3.13 2.20 157 O(5) 2.82 1.88 163 O(6) 2.99 2.07 151 O(7) 2.95 2.00 157 O(8) 2.90 1.96 161 O(9) 2.95 2.07 146 O(l0) 3.01 2.15 166 O(ll) 2.89 2.25 158 W(8A)C 2.90

-

1

W(2) 2.85 W(1) O(13)' 2.80 W(2) O(lS)e 2.68 W(2) 2.73 W(4A)' O(14) 3.27 W(4By O(14) 2.94 W(5A)c O(12) 2.99 W(5A)c W(8A)' 3.32 W(6A)< O(9) 3.18 W(6B)c O(9) 3.10 W(7) W(4A)' "The H atoms were placed in idealized positions with the N-H distance equal to 0.96 A. bSymmetry equivalent I + x , y. -1 + z to coordinates listed in Table 11. eOccupancy -50%. A pair of disordered water oxygens [W(nA) and W(nB)] cannot coexist. dSymmetryequivalent -x, +y, 2 - z to coordinates listed in Table 11. 'Symmetry equivalent 1 - x , + y, - z to coordinates listed in Table 111. /Water oxygens W(3A). W(38). and W(8B) occur in large holes without any hydrogen bond distances.

-

Table V. Hydrogen Bonds in Peptide I donor acceptor A N(I) O(15)b 2.817 N(2) 4-+ I N(3) O(0) 3.076 N(4) O(I) 2.973 N(5) 5- I N(6) O(2) 3.047 N(7) O(3) 2.931 N(8) O(4) 3.197 N(9) O(5) 3.133 N(I0) O(6) 2.999 N(11) O(7) 2.865 N(12) 0(8) 2.971 N(13) O(9) 3.052 O(10) 2.954 N(14) O(11) 2.909 N(I5) 3.181 O(12) N(16) water-to-peptide W( IA)' 0(14)d 3.02 W(IB)' 0(14)d 3.20 W(2A)C W(2B)c 0(16)b 2.97 "dSee corresponding footnotes in Table VI. tYPC

head-to-tail

A 1.87

deg 140

2.17 2.27

131 125

2.11 1.98 2.27 2.20 2.06 1.92 2.07 2.13 2.04 2.00 2.49

156 155

146 161 154 162 152 155

I50 I60 146

comprise approximatcly four helical turns in each case and permit segrcgation of Lcu rcsiducs on onc face of thc hcliccs. Thcsc structurcs a r e therefore of some interest in evaluating t h e role of projccting sidc chains in mcdiating helix association. This is a problem of interest in t h c casc of t h c lcucine zippcr motif seen in a class of DNA binding protein^.^*'^ Peptide I I indeed has two pairs of Leu rcsiducs spaced scvcn positions a p a r t a n d may bc, in principle, il niodcl for t h c lcucine zippcr structure. However, sincc Lcu3, Leu', Lcu'O. a n d LcuI4 all project from one side of

Figure 5. Antipnrallel packing of helices in crystal I . Head-to-tail hydrogcn bonding is indicated by a dashcd linc. The disordered water molecules in the head-to-tail region arc omitted (see Figure 4). The molcculcs in thc shccts (or laycrs) pcrpcndicular to the page aggregate with all thc hcliccs in a parallcl modc. The b axis (2-fold screw axis) is horizontal.

the helix in I I , they cannot interdigitate with those from a second similar helix in the same manner as proposed for the leucine zipper. T h c obscrvcd packing arrangement in I I does not lead to any intcrdigitation of the Leu side chains. Instead, the hydrocarbon side chains are loosely packed, with large voids developing between hcliccs. In a morc direct comparison with 11, interdigitation of lcucyl sidc chains had been established in t h e crystal structure of Boc-(Aib-Ala-Leu)3-AibOMe, a hydrophobic decapeptide that

J . Am. Chem. SOC.1990, 112, 9356-9364

9356

b

Figure 6 . A sheet of parallel helices in crystal I I with all N termini pointing upward (six molecules are drawn). Sheets of helices above and below the one shown are rotated by 180’ to give an antiparallel arrangement. Water molecules in the head-to-tail region are omitted (see Figurc 3). Axial dircctions of the cell arc a-, b t , and c u p from the page.

had been rendered amphipathic by the insertion of water molecules into the helix.I9 In this case the leucyl side chains occur in every third position, therefore Leu3 and Leu6 are adjacent to each other on thc hclix. Since there is not sufficient space between adjacent leucyl side chains for others to penetrate in the same “plane”, the interdigitating leucyl side chains from the neighboring molecule are offset, pcrpcndicular to the view in Figure 3 of ref 19. Thc loosc, inefficient packing in the crystals of peptide 11 results in thc occurrcncc of several water molecules in completely hydrophobic cavities, with at least some appearing to be uninvolved in hydrogen bonding. Presumably, the presence of water in hydrophobic holes does lead to some enthalpic stabilization by means of dipole-induced dipole (London) interactions with neighboring alkyl side chains.*O These structures provide a high-resolution (20) Burley, S. K.; Petsko, G. A. Adu. Protein Chem. 1988.39, 125-189.

glimpse of water molecules in totally hydrophobic environments and may be of use in modeling trapped water in the apolar interior of globular proteins.21,22 A recent analysis of protein crystal structures reveals that water molecules in the vicinity of apolar side chains lie predominantly at van der Waals contact distances, but most of these have a primary, shorter contact with a neighboring atom.** The unusual amount of water associated with the apolar peptide I1 is reminiscent of the antifreeze polypeptides (AFP) found in the blood of arctic fishes, which often are characterized by a very high alanine content and a high a-helical structure.23 The first report on a crystal structure of an antifreeze polypeptide, at 2.5 A, shows that the 36-residue peptide forms a single helix.24 The water content and placement within the AFP crystal was not determined at this time. The structures of I and I1 in crystals demonstrate that the incorporation of a few Aib residues permits the synthetic construction of largely a-helical structures. The length of the helices compares well with that found for helical segments in globular proteins.z5 Future studies will focus on the covalent linking of such helical modules and in the development of cylindrical peptide helices as a scaffold for the controlled orientation of functional residues and binding cavities. Acknowledgment. This research was supported in part by National Institutes of Health Grant GM30902, by the Office of Naval Research, and by a grant from the Department of Science and Technology, India. K.U. is the recipient of a fellowship from the Council of Scientific and Industrial Research, India. Supplementary Material Available: Tables of atomic coordinates, bond lengths, bond angles, anisotropic displacement coefficients, and H atom coordinates (23 pages); observed and calculated structure factors for peptides I and I1 (47 pages). Ordering information is given on any current masthead page. (21) Matsumura, M.; Wozniak, J. A,; Dao-Pin, S . ; Matthews, B. W. J . Biol. Chem. 1989, 264, 16059-1 6066.

(22) Thanki, N.; Thornton, J. M.;Goodfellow, J. M. J . Mol. B i d . 1988, 202. 631-657. (23) Ananthanarayanan, V. S.;Hew, C. L. Biochem. Biophys. Res. Commun. 1977, 74,685-689. (24) Yang, D. S. C.; Sax, M.; Chakrabarty, A,; Hew, C. L. Naature 1988, 333. 232-237. (25) Barlow, D. J.; Thornton, J. M. J . Mol. Biol. 1988, 201, 601-619. ~

Unified Synthesis of Vinylsilanes and Silylated Butadienes. Nickel-Catalyzed Olefination and Silylolefination of Dit hioacetals Zhi-Jie Ni,” Pin-Fan Yang12”Dennis K. P. Ng,2a,b,3 Yih-Ling Tzeng,2band Tien-Yau Luh*v”’ Contribution from the Department of Chemistry, The Chinese University of Hong Kong, Shatin, N . T . , Hong Kong, and the Department of Chemistry, National Taiwan University, Taipei, Taiwan 10764, Republic of China. Receioed April 30. 1990 Abstract: A simple unified reaction has been developed in the syntheses of various vinylsilanes and silylated butadienes by

thc nickcl-catalyzed coupling of dithioacetals with appropriate Grignard reagents. Reactions of 2-aryl-2-(trimethylsilyl)dithianes with MeMgl or cyclopropyl Grignard reagent give in good yields 1 -aryl-l -(trimethylsilyl)styrenes and 1 -aryl-I-(trimethylsilyl)butadicncs,respectively. Reactions of these substrates with Me3SiCH2MgCIafford 1,2-bis(silyl)styrenes.Allylic dithioacetals rcact with Mc3SiCHzMgCIfurnishing the synthesis of I-(trimethylsily1)butadienes. Reactions of I-(trimethylsily1)-substituted allylic dithioacctals with MeMgl or Me3SiCHzMgCIgive internal (trimethylsily1)butadienes or bis(trimethylsilyl)butadienes in satisfactory yiclds. Trcatment of allylic orthothimter with Me,SiCH,MgCI under similar conditions provides a facile synthesis of compounds having both vinyl- and allylsilanc functionalities in onc stcp.

The synthetic utility of vinylsilanes is Various litcrature proccdurcs are known for the preparation of this f~nctionality,~-’ 0002-7863/90/ I5 I2-9356$02.50/0

but there seems to be no general method for the synthesis of different kinds of vinylsilanes and silylated butadienes. Occa-

63 I990 Amcrican Chemical Society