Hydrogen-bonded structure and NMR parameters of oxygen-17 labeled poly(L-alanine)s as studied by solid state oxygen-17 NMR spectroscopy

Journal of

MOLECULAR STRUCTURE ELSEVIER

Journal of Molecular Structure 442 (1998) 195-199

Hydrogen-bonded structure and NMR parameters of oxygen- 17 labeled poly(L-alanine)s as studied by solid state oxygen-17 NMR spectroscopy Akihiro Takahashi a, Shigeki Kuroki a, Isao Ando a'*, Takuo Ozaki b, Akira Shoji b aDepartment of Polymer Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo, Japan bDepartment of Biological Sciences, Gunma University, Tenjin-cho, Kiryu, Gunma, Japan Received 11 July 1997; accepted 20 August 1997

Abstract ~vO-labeled poly(L-alanine)s with different degrees of polymerization were synthesized. These poly(L-alanine)s take an a-helix form and a/3-sheet form. Solid state 170 NMR spectra of these polymers were measured at different frequencies. By comparing the obtained spectrum and theoretical simulation, the NMR parameters, such as the quadrupolar coupling constant and chemical shift tensor components, were determined. The relationship between the hydrogen-bonded structure and the NMR parameters was elucidated in relation to the main-chain conformation. In addition, these results were compared with those of polyglycine as reported previously, and the feature of ~70 NMR behavior of these polypeptides, associated with the hydrogen-bonded structure, was discussed. © 1998 Elsevier Science B.V.

Keywords: NMR spectroscopy; Solid state ~70 hydrogen bonding; Conformation; Polypeptides; Poly(L-alanine)

I. Introduction W e have successfully observed solid state 170 N M R spectra of polyglycines ((Gly)n) I and II and glycylglycine peptides with different hydrogen bonded lengths RN...o [1,2]. By computer simulations of these spectra, the quadrupolar coupling constant (e2qQ/h), and the chemical shift, were determined. From these results, it has been shown that as the hydrogen-bond length decreases, the e2qQ/h value decreases, and the isotropic oxygen-17 chemical * Corresponding author. Tel: 81357342139; fax: 81357342889; e-mail: [email protected]

shift and its tensor components move upfield. These results have been reasonably justified by the quantum chemical method [3]. From such situations, in addition to the work on glycine-containing peptides and polypeptides, the present purpose of this work is to measure 170 N M R chemical shift and quadrupolar coupling constant of poly(L-alanine)s with the s - h e l i x and/3-sheet forms in the solid state, and to clarify the relationship between the hydrogen-bond structure and these N M R parameters. This will lead to further accumulation for understanding through the relationship between the hydrogen-bonded structure and N M R parameters [1-91.

0022-28601981519.00 © 1998 Elsevier Science B.V, All rights reserved PII S0022-2860(97)0033 3-5

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A. Takahashi et al./Journal ~f Molecular Structure 442 (1998) 195 199

2. Experimental

2.3. Solid state 170 NMR spectral analyses

2.1. Sample preparations

The 170 nucleus has a spin number of 5/2 and so is a quadrupolar nucleus. Therefore, the static CP spectrum contains quadrupolar interaction and chemical shift tensor. The theoretical simulation leads to the separation of the quadrupolar coupling constant (e2qQ/h), the asymmetry parameter 07Q), the chemical shift tensor components (611, 622 and 633) and the orientation of its components in the quadrupolar frame of reference as defined by the three Euler angles (or, /3 and 3') from the observed spectrum. The computer simulations, as reported previously [2], were carried out repeatedly by superimposing the theoretical line shape onto the experimental spectrum until the satisfied agreement was reached with any specified allowance. A Sun 4 SPARC workstation was for calculations. It took about 1 min to obtain a simulated spectrum.

10% 170-labeled L-alanine methyl ester was placed in Nal7OH/methanol solution (where NaITOH was prepared by reaction of 20% 170-labeled water which was purchased from Cambridge Isotope Laboratories with Na metal). Further, L-Ala N-carboxyanhydride (NCA) was prepared using 10% J70-L-alanine. 10% 170-labeled poly(L-alanine) ((Ala),) was prepared by heterogeneous polymerization of 10% 170-labeled NCA in acetonitrile by using n-butylamine as the initiator. The mole ratio of NCA to initiator (A/I) was chosen as 100 and 5. The conformational characterizations of these polymers were made on the basis of ~3C cross polarization/ magic angle spinning (CP/MAS) by using reference data of J3C chemical shift values for poly(L-alanine), which are associated with the main-chain conformation [10,11].

2.2. Solid state 170 and 13C NMR measurements Static 170 CP NMR spectra were recorded with a JEOL GSX-500 spectrometer and a JEOL GSX-270 spectrometer operating at 67.8 and 36.6 MHz, respectively, with a CP/MAS accessory at room temperature. In the CP static methods, Mg(17OH)2 was used for ~H-170 CP matching (THBH = 3")'oBo). The IH ~r/2 pulse length was 5/~s and the IVO 7r/2 pulse length was 5 #s for a solid sample (which corresponded to 15/~s for a solid sample). The IH decoupling field strength was 50 kHz and the repetition time was 5 s. According to the previous work on 170 CP NMR experiments for polyglycines and its peptides, 9 ms was used as the appropriate contact time. The ~70 chemical shifts were calibrated through external liquid water (6 = 0 ppm). ~3C CP/MAS NMR spectra were recorded with a JEOL GSX-270 spectrometer operating at 67.8 MHz with a CP/MAS accessory. The IH decoupling field strength was 50 kHz, the contact time was 2 ms, the repetition time was 5 s, and the spectral width was 27 kHz. The 13C chemical shifts were calibrated indirectly through the adamantane peak observed upfield (29.5 ppm relative to tetramethylsilane).

3. Results and discussion 3.1. 1-~CCP/MAS CP NMR spectra of (Ala)n samples and the conformational characterization Fig. 1 shows the 13C CP/MAS NMR spectra of (Ala)n with A/I = 100 (a) and 5 (b). The carbonyl (C=O), Ca and C3 carbons appear at ca. 171-176 ppm, ca. 48-52 ppm and ca. 15-19 ppm, respectively. The reference data of (Ala)n (as reported previously) show that the carbonyl carbons for the o~-helix and 3-sheet forms appear at ca. 176 and 172 ppm, respectively, the Co~ carbons for the s-helix and 3-sheet forms at ca. 52 and 48 ppm, respectively, and the C3 carbons for the s-helix and 3-sheet forms at ca. 15 and 19 ppm, respectively. From these assignments, we can characterize the main-chain conformation for the above samples [10,11]. The conformation of (Ala)n withA/l= 100 and 5 is the o~-helix form and a mixture of the o~-helix and 3-sheet forms, of which the fractions are 0.2 and 0.8, where the fraction was estimated from the peak intensity.

3.2. The NMR parameters and structure of (Ala)n samples as obtained from static 170 NMR spectra Static 170 CP NMR spectra of (Ala)n with A/I = 5 (3-sheet form) and 100 (or-helix form) at 36.6 and

A. Takahashi et al./Journal of Molecular Structure 442 (1998) 195-199

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Fig. I. UC CP/MAS NMR spectra of (Ala)n with the c~-helix form (A/l= 100) (A) and the/3-sheet form (A/I = 5) (B).

67.8 MHz are shown in Fig. 2, together with the theoretical simulation. The spectra at lower frequency NMR (36.6 MHz) consist of two major signals and those at higher frequency NMR (67.8 MHz) become one major signal due to the overlap with the two major

signals. This indicates that the separated two signals come from quadrupolar interaction. The influence on the spectral pattern by quadrupolar interaction at higher frequency NMR becomes very small. By a comparison of the experimental spectrum and

A. Takahashi et al./Journal of Molecular Structure 442 (1998) 195-199

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Fig. 2. Static tTo CP NMR spectra of(Ala), with the c~-helix form (A/1 = 100) (A) and the t3-sheet form (B) at (a) 67.8 MHz and (b) 36.6 MHz. theoretical simulation at both frequencies, the N M R parameters such as the quadrupolar c o u p l i n g constant and c h e m i c a l shift tensor w e r e determined. This determ i n e d quadrupolar c o u p l i n g constant (eZqQ/h), its a s y m m e t r i c p a r a m e t e r (qQ), c h e m i c a l shift tensor c o m p o n e n t s (61~, 622 and 633), and its isotropic c h e m i cal shift (CSiso) for the a b o v e samples, are listed in Table 1. At first w e w e r e c o n c e r n e d with the quadrupolar c o u p l i n g constant e2qQ/h o f (Ala)n with the ~/-sheet form and a - h e l i x form. The e2qQ/h v a l u e s for the 13-sheet and c~-helix forms are 8.65 and 9.28 M H z , respectively. The /3-sheet f o r m is smaller than the co-helix form. This m a y c o m e f r o m the difference in h y d r o g e n - b o n d length RN_.O b e t w e e n the /3-sheet and e~-helix form (to be 2.83 and 2.87 A,, respectively, as determined by X-ray diffraction) [12,13]. In the previous w o r k on glycine-containing peptides and

polypeptides, w e have demonstrated that the quadrupolar coupling constant e2qQ/h is linearly increased with an increase in the hydrogen-bond length. Also, the a s y m m e t r y parameter ~70 for the B-sheet form is s o m e w h a t larger than that for the c~-helix. The theoretical calculation shows that the 70 value is decreased with an increase in RN...o. This prediction agrees with the experimental results. F r o m these experimental and

Table 1 Determined e2qQ/h, r/O and 170 chemical shift of(Ala)n with the c~helix form and the/3-sheet form Conformation

eeqQ/h rtQ (MHz)

s-helix ~Lsheet

9.28 8.65

0.38 0.41

~70 chemical shift (ppm) 6iso 6~1 622

633

303 265

- 121 - 110

595 514

435 390

A. Takahashi et al./Journal of Molecular Structure 442 (1998) 195-199

theoretical findings~ the experimental results on the e2qQ/h and ~Q values o f (Ala)n m a y be understood. As seen f r o m Table 1, it is s h o w n that the isotropic c h e m i c a l shift for the a - h e l i x f o r m appears at a l o w e r field by about 40 p p m than that for the fl-sheet. This c o m e s f r o m the fact that all o f the c h e m i c a l shift tensor c o m p o n e n t s (61j, 622 and t~33) for the o~-helix form appear at l o w e r fields than those for the fl-sheet form. The 6 i i c o m p o n e n t for the fl-sheet f o r m appears at a higher field by about 80 p p m c o m p a r e d with that for the a - h e l i x form. Such larger upfield shift m a y be due to the discrepancy in the h y d r o g e n - b o n d angles and the e n v i r o n m e n t around a poly(L-alanine) chain c o m p a r e d with the o~-helix form. The h y d r o g e n bonds for the or-helix f o r m are i n t r a m o l e c u l a r l y f o r m e d and those for the fl-sheet f o r m is i n t e r m o l e c u l a r l y formed. This m a y lead to the discrepancy in the h y d r o g e n b o n d e d structure. It is suggested that the ~70 c h e m i c a l shift is greatly influenced as in the case o f the nitrogen-15 c h e m i c a l shift of polypeptides as reported previously. Finally, it can be said that solid state 170 N M R p r o v i d e s useful information about the h y d r o g e n bonded structure o f poly(L-alanine)s with the c~-helix and fl-sheet forms.

199

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