17O NMR

Chapter 6.5 170

Solid State NMR of Polymers, edited by I. Ando and T. Asakura Studies in Physical and Theoretical Chemistry, Vol. 84 9 Elsevier Science B.V. All rights reserved

NMR

S. Kuroki Department of Polymer Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo, Japan

6.5.1

Introduction

The oxygen atom is one of the most important atoms constituting hydrogenbonding structures in polymers such as peptides and polypeptides. Nevertheless, solid-state 170 NMR studies for polymers have not been carried out previously. This is due to the very weak sensitivity of solid-state 170 NMR measurements which comes from the two following reasons: One is that the 170 nucleus has a very low natural abundance, which is 0.037%. Another is that the 170 nuclear spin quantum number (I) is 5/2, which implies a quadrupolar nucleus, and so the 170 signal is broadened by nuclear quadrupolar effects in the solid. On the other hand, solution-state 170 NMR spectroscopy has been successfully employed to elucidate a number of structural problems in organic chemistry [1-4], because 170 signal becomes very sharp due to the removal of the quadrupolar interaction by isotropic fast reorientation in solution. For example, as the oxygen atom is directly associated with the formation of a hydrogen bond, hydrogen bonding for the carbonyl group in various compounds often results in large low frequency shifts of the carbonyl 170 NMR signal [5, 6]. From these results, solution-state 170 NMR has been established as a means for investigating structural characterizations. From such situations, it can be expected that solid-state '70 NMR provides a deep insight into understanding the hydrogen-bonding structures of solid polymers such as peptides and polypeptides. In this chapter [7], we show the ~70 NMR spectra of polyglycine form I (PG I: B-sheet structure), polyglycine form II (PG II: 3~-helix structure), glycylglycine (GlyGly) and glycylglycine nitrate (GlyGly.HNO3) in the solid state, which cover a wide range of hydrogen bond lengths, and three types of NMR parameters such as chemical shift, quadrupolar coupling constant (eZqQ/h) and asymmetric parameter (rio), and to understand the relationship between the hydrogen-bonding structure and these NMR parameters.

237

170 NMR

6.5.2

Static 170 CP spectra of ~70-1abeled peptides and polypeptides

Figure 6.5.1 shows the plots of the 170 CP NMR signal intensity for GlyGly against the contact time. Since the carbonyl oxygen atom is not bonded to a hydrogen atom, a long contact time is need. The appropriate contact time was 9 ms. From the above plots, the Toi-i and 1HTlo values were determined to be 2.5 and 30.0 ms, respectively. The Toi-i value of GlyGly is much longer than those of inorganic solids such as A 1 0 (17OH), whose Toi-i value is 0.018 ms [8]. Static 170 CP NMR spectra of GlyGly were observed at 36.6, 54.2 and 67.8 MHz as shown in Fig. 6.5.2. Each of the spectra at 36.6 and 54.2 MHz consists of two major split signals, but the spectrum at 67.8 MHz has only one major signal. Such splitting comes from the quadrupolar interaction. By computer simulation, the quadrupolar coupling constant e2qQ/h was determined to be 8.55 MHz. This value is much larger than that for the amide 14N nitrogen nuclei (1.11 MHz). It is seen that the NMR spectra strongly depend on the measurement frequency, because the quadrupolar effect depends on the measurement frequency, and the broadening of the 170 NMR signal is further increased by quadrupolar effects as the measurement frequency decreases. Therefore, it can be said that a high-frequency measurement is needed for quadrupolar nuclei such as 170. Figure 6.5.3 shows the 67.8 MHz static 170 CP NMR spectra of PG I,

1.0 0.8 0.6 "~

0.4

o.2 II 0.0 ~ 0.0

I 5.0

!

I

10.0 15.0 Contact time(ms)

! 20.0

25.0

Fig. 6.5.1. Plots of peak intensity versus cross-polarization contact time for the GlyGly.

238

s. KUROKI 1

(e) 6

(b) 54.2 MHz

(a) 36.6 MHz

PPM i500

iO00

500

0

-500

-iO00

-1500

Fig. 6.5.2. (a) 36.6, (b) 54.2 and (c) 67.8 MHz 170 CP static spectra of GlyGly in the solid state, respectively.

PG II, GlyGly and GlyGly.HNO3. To determine the 170 NMR parameters unequivocally, the computer simulations for different frequencies such as 36.6, 54.2 and 67.8 MHz were carried out. Figure 6.5.4 shows 67.8, 54.2 and 36.6 MHz static 170 CP NMR spectra of GlyGly together with theoreticallycalculated spectra. Next, Fig. 6.5.5 shows the 67.8 MHz static 170 CP NMR spectra of PG I, PG II, GlyGly and GlyGly.HNO3 together with theoreticallycalculated spectra, respectively. The 170 NMR parameters of PG I, PG II,

170 NMR

239

(b) GlyGly

(a) GIyGIy.HNOs

PPM 800

700

600

500

400

300

200

tO0

0

-100

-200

-300

Fig. 6.5.3. (a) 67.8 MHz 170 CP static NMR spectra of GlyGly.HNO3, (b) GlyGly, (c) PG I and (d) PG II, respectively.

GlyGly and GlyGly.HNO3 obtained by computer simulations are shown in Table 6.5.1. From these results, the ~11, ~22 and 633 values of the samples change from 546 to 574 ppm, 382 to 425 ppm and -132 to -101 ppm, respectively. The magnitude of the change in 170 chemical shift is much larger than those of the carbonyl 13C and amide 15N chemical shifts. Every A6 defined by A6 = ~ 3 3 - ~iso, is about 400 ppm, which is much larger than those of carbonyl 13C and amide lSN chemical shifts, which are about 100

240

s. KUROKI

c) 67.8 MHz

. 9"

:.

600

~,:

400

M

200

b) 54.2MHz

I

I

l

600

ppm

0

!

"400

|

200

0

-200 -400

ppm

a) 36.6 MHz 9

9 ~.,,. g

..

1000

500"

0

-SO0 -1000

ppm

Fig. 6.5.4. (a) 36.6, (b) 54.2 and (c) 67.8 MHz 170 CP static spectra of GlyGly together with theoretically-simulated spectra, respectively.

ppm, respectively. Every r/value defined by r/= ( 8 1 1 - 622)1A6 is about 0.4. These values are common to the carbonyl oxygen in peptides. Though the chemical shift tensor is axially symmetry (611 = 622) for the case of the carbonyl oxygen of c-alanine amino acid as reported by Fiat et al. [9, 10], the chemical shift tensor of the carbonyl oxygen in peptides is not axially symmetric from these results. The eZqQ/h values change from 8.30 to 8.75 MHz. These values are larger than the eZqQ/h value of c-leucine which is 8.0 MHz at 190 K [10]. The rtQ values of polypeptides, such as PG I and II are 0.26 and 0.29, and the r/o values of oligopeptides such as GlyGly and GlyGly-HNO3 are 0.45, 0.47, respectively. This difference may come from large difference in molecular packing between them. Furthermore, it is found

170

C) PG I

NMR

" 2 . .~.

9

d)

241

PG

II

..:~. :.

9 -.." ~ .

.v',

,~.-'~'~" . ~ F'---

l

600

200

400

a) GIyGIy.HNO3

O'

ppm

,. "k"

600

, 400

, 200

,I 0

b)GlyGly

~."

'7 9 "~" : ~;.."

9 9 ... ~o

/

\

:~,~,,Z~

".

:

"-

: "~-"

~

"

,,.

...

:.

""' __.__J~'~" ~ 9

,,

,

600

400

200

,

0

ppm

!

ppm

/-/--I 600

9

"

I

400

200

0

ppm

Fig. 6.5.5. (a) 67.8 MHz 170 CP static NMR spectra of GlyGly.HNO3; (b) GlyGly; (c) PG I; and (d) PG II, together with theoretically-simulated spectra, respectively.

that the principal axis of the quadrupolar tensor and the principal axis of the 170 chemical shielding tensor for the carbonyl oxygens of peptides and polypeptides are not coincident with each other. The relationship between these two principal axes is shown in Fig. 6.5.6 for the situation of PG II. Each of the above N M R parameters is influenced strongly by the electronic structures of the molecules. This seems to reflect a slight difference in their affects by the characteristic electronic distributions of the carbonyl oxygen of peptides and polypeptides.

6.5.3 The direction of the principal axes of the electric field gradient tensor and the chemical shielding tensor of the carboyl oxygen

No studies have ever tried to determine the direction of the principal axes of the electric field gradient tensor and the chemical shielding of the carbonyl

t,~ 4~ t,,9

Table 6.5.1. Determined Sample

1 7 0 N M R parameters of solid peptides containing Gly residue

e2qQ/h (MHz)

Polypeptides PG II PG I Oligopeptides GlyGly GlyGly.HNO3

Angle ma (deg)

Chemical shift tensor (ppm) "I~Q

611

(~22

633

6iso

A6b

?c

a

/~

8.30 8.55

0.29 0.26

562 574

410 425

- 108 - 101

288 299

396 400

0.38 0.37

92 100

89 91

-81 -79

8.55 8.75

0.45 0.47

546 559

382 408

- 132 - 127

265 280

397 407

0.41 0.37

94 94

90 89

-87 -81

0

a Angle A indicates the Euler angles between the principal axes of the quadrupolar tensor and the chemical shift tensor.

b A6 = 6iso- 633. c T~-- ( 6 1 1 - t~-22)/A6,

170 N M R

243

V33 (5'11

~=8 . ~..........."'" o~=92 *

...-"

0'33.~ . . . . . . . . . . . .

> Vll

.-"

0"22 ~

v22 Fig. 6.5.6. The relationship between the principal axes of the quadrupolar tensor on the

carbonyl oxygen for the situation of PG II.

oxygen from experiment. The determined direction of the principal axes of the chemical shielding of the carbonyl oxygen is shown in Fig. 6.5.7 as determined by F P T - M N D O - P M 3 method. The 0"22 component lies approximately along the C = O bond, the cr11 component is aligned in the direction perpendicular to the C - - O bond in the peptide plane and, the 0"33 which is

io'33

\

~2

.--

.-''""

C0~

Fig. 6.5. 7. The direction of the principal axes of the chemical shielding tensor of the carbonyl

oxygen employing FPT-MNDO-PM3 method.

244

s. KUROKI

V33 "~

.

.

.

.

.

.

...'"co\

V,, Fig. 6.5.8. The direction of the principal axes of the electric field gradient tensor of the carbonyl oxygen employing FPT-MNDO-PM3 method.

the most shielded component, is aligned in the direction perpendicular to the peptide plane. It is a very interesting result that the most shielded component, 0"33, is not aligned along the direction of the C = O bond, or the direction of lone pair electron which are aligned 120~ or -120 ~ from the C---O bond direction on the peptide plane. It can be said that the sp 2 hybrid property of the carbonyl bond is removed due to the double bonding property of the peptide bond. On the other hand, the direction of the principal axes of the electric field gradient tensor of the carbonyl oxygen is shown in Fig. 6.5.8 as determined by the FPT-MNDO-PM3 method. The V22 component lies approximately along the ~ O bond, the V33 component is aligned in the direction perpendicular to the C = O bond on the peptide plane and the V i i component is aligned in the direction perpendicular to the peptide plane. It can be said that the largest component, V33 , of the electric field gradient lies along the molecular chain direction. The relationship between the electric field gradient tensor and the chemical shielding employing this calculation agree with the experimental results in Fig. 6.5.6.

6.5.4 Nuclear quadrupolar coupling constant of peptides and polypeptides

(e2qQIh) of carbonyl oxygen

The geometrical parameters and hydrogen-bonding geometrical parameters [11-14] of these peptides and polypeptides are shown in Table 6.5.2. Some

Table 6.5.2. The geometrical parameters and hydrogen-bonding geometrical parameters of peptides containing Gly residue Sample

P G II PG I GlyGly GlyGly'HNO3

Geometrical parameters

Hydrogen-bonding geometrical parameters

Dihedral angle (deg)

HB length (/k)

HB angle (deg)

Ref. HB dihedral angle (deg)

~b

~p

N... O

H... O

L C--~O. 99N

L N ~ H . 99O

N~C---~O. 99H

C z O - 99H ~ N

-78.0 -149.9 157.1 165.6

145.8 146,5 151.0 148.9

2.73 2.95 2.94 3.12

1.84 2.16 1.97 2.38

159 149 157 162

146 133 157 165

-47 68 -161 3

157 -173 -145 -156

9 Z 11 12 13 14

t,~ 4~ t.~

246

s. KUROKI 9.0

I

I

I

I

8.8

8.6 i.=,.,

8.4

8.2

I

8.0

2.7

I

I

I

2.8 2.9 3.0 3.1 Hydrogen bond length RN...o(,~)

3.2

Fig. 6.5.9. Plots of the e2qQ/hagainst the hydrogen bond length. of the geometrical parameters were calculated by using the unit cell parameters and fractional coordinates as given in the literature [11-14]. Figure 6.5.9 shows the plot of the observed e2qQ/h values against the hydrogen bond length. The e2qQ/h values decrease linearly with a decrease of the hydrogen bond length (RN... o). This relationship is expressed by

e2qO/h (MHz) = 5.15 + 1.16RN... o (/k).

(6.5.1)

This change comes from a change of the q values which are the largest component of electric gradient tensor (V33). This experimental result shows that a decrease in thehydrogen bond length leads to a decrease of the electric gradient. The q value seems to be very sensitive to the change in hydrogenbonding length.

6.5.5 170 NMR chemical shifts of carbonyl oxygen of peptides and polypeptides From Table 6.5.1, there is a large difference in the chemical shift value between peptides and polypeptides. Figure 6.5.10 shows the plot of the

~70 NMR

250

I

247

I

~" 260

I

I

"-.GlyGly

O

"0.

~

~ 27o "'O.

o 280

GIyGIy.HNO~

P G II

"0

o 290

e~

o "Q)..

o 300 o tt~ la,,u

PG I'" I

310 2.7

2.8

!

I

!

2.9 3.0 3.i Hydrogen bond length RN...o(/~)

3.2

Fig. 6.5.10. Plots of the observed isotropic 170 chemical shift against the hydrogen bond length. observed isotropic 170 chemical shifts (~iso) against the hydrogen bond length (RN... o). The 6iso values in both peptides and polypeptides move to low frequency with the decrease in the hydrogen bond length (RN... o). Figure 6.5.11(a-c) show the plot of the observed principal values of the 170 chemical shifts against the hydrogen bond length (RN... o). Every principal value in both peptides and polypeptides moves to low frequency with a decrease in the hydrogen bond length (RN... o).

6.5.6

Polyalanines

Figure 6.5.12 shows static 170 CP NMR spectra of solid (Ala),,[A/I = 100] with an c~-helix form at (a) 67.8 MHz and (b) 36.6 MHz together with theoretically-simulated spectra. The spectrum at 36.6 MHz consists of two major split signals, but that at 67.8 MHz has one major signal overlapping with two other signals. Such a large variation comes from the quadrupolar interaction because the appearance of the spectrum depends on the NMR frequency. If the NMR frequency is extremely high, the influence by quadrupolar interactions may be neglected in the spectrum. By computer simulation, the obtained e2qQ/h value and chemical shift tensor components for the a-

t,J

530

'

,

a)

'

,

,

380

,

I~

I

'

-140

I

i

GlyGly

b)

C)

a ~

l

l

GlyGly

t t

540 -

-130 -

390 GlyGiy "'O

~,GlyGly'HNO 3

%

.

I~-120

E 400

~CI, I'G H

_

GlyGly-HNO;

\

~550 -

~560

""0-. ,,

t

q.

PG II

e,I

oo410

PG II

~-110

GlyGly.HNO 3

C

% %

"~-~

%

PGI

0

%

570 -

" ",

%

420 -

PG I ~)-..

~

,9

-100 -

PG I

"O.

_

%

580

I

t

,

t

I

430

2.7 2.8 2.9 3.0 3.1 3.2 H y d r o g e n b o n d l e g t h (,~)

Fig. 6.5.11. Plots of the observed principal values of bond length R N . . . o.

,

2.7

I

I

2.8

2.9

Hydrogen 170

"l

I

3.0

3.1

-90

3.2

2.7

bond length(~)

chemical shift tensor (a)

t~11 ,

(b)

~

I

I

2.8

2.9

3.0

Hydrogen t~22

and (c)

633 ,

bond

,

,,

3.1

3.2

length(~)

respectively, against the hydrogen

NMR

170

(a) 6 7 . 8 M H z

249

A

wi

'..y

9.

I

,

1000

,

9

800

t

.

600

.,.

'...

~

~

~

v

~

400

200

0

-200

-400

(b) 36.6MHz

9

~ (ppm)

-600

t-. !;

t:"..

oI

I

'i

,%;

+'.,, . . , ;

,,,.

(ppm)

1 SO0

1000

SO0

0

-SO0

-1000

-1500

Fig. 6.5.12.

170 CP static N M R spectra of (L-Ala),,[A/I = 100] together with theoreticallysimulated spectra, respectively.

helix are shown in Table 6.5.3. Similarly, the static 170 NMR spectral analyses of solid (Ala),,[A/I = 5] with a /3-sheet form as the major components (Fig. 6.5.13) were carried out. The obtained NMR parameters for the /3sheet form are shown in Table 6.5.3, together with the NMR parameters obtained from PG I (/3-sheet form) and II (31-helix form). The e2qQ/hvalues for poly (L-alanine) and polyglycine with the same/3sheet form are very close to each other. This can be understood from the experimental results that the hydrogen bond lengths for both of the/3-sheet forms are very close to each other as shown from the determination of the hydrogen bond length (RN... o = 3.02 A) by the observation of the amide carbonyl 13C chemical shift [15]. The isotropic 170 chemical shift for (Ala)n appears at lower frequency than that for polyglycine. It comes from the large low frequency shift of (~11, the direction of which is perpendicular to the C ~ O bond in the amide plane. Therefore, the hydrogen bond angle for both of the/3-sheet forms may be different from each other. The a-helix form for (Ala),, has different e2qQ/h value from that of the /3-sheet form. The former is larger than the latter. Also, the chemical shift tensor components are very different from each other. Such differences come from the different hydrogen bond lengths and angles.

tO O

Table 6.5.3. Determined 170 N M R parameters of (Ala)n together with those of PGs Sample

(MHz) (Ala)n a-helix /3-sheet (Gly). PG II PG I

Angle ma (deg)

Chemical shift tensor (ppm)

e2qQ/h 70

~11

i~22

~33

~iso

a

j~

9.28 8.65

0.38 O.41

595 514

435 390

- 121 - 265

303 265

100 83

76 106

-80 -85

8.30 8.55

0.29 0.26

562 574

410 425

-108 -101

288 299

92 100

89 91

-81 -79

a Angle A indicates the Euler angles between the principal axes of the quadrupolar tensor and the chemical shift tensor.

0

170

NMR

(a) 67.8MHz

251

I

t'[

:

" ,v

,

~.

l v./]A' t ~t ....

t...,...1..,

1000

800

t...

600

400

!...

200

I.

. l...

0

-200

l...l(ppm

-400

)

-600

(b) 36.6MHz

9t

I

i

t .... 1 SO0

, .... 1000 .

, .... SO0

i .... 0

! ..... -SO0

I ..... -1000

~ (ppm) -1 SO0

Fig. 6.5.13. 170 CP static NMR spectra of (L-Ala),[A/I = 5] together with theoretically-simulated spectra, respectively. 6.5.6

Conclusion

From the observed carbonyl oxygen 1 7 0 N M R spectra of PG I, PG II, GlyGly and GlyGly.HNO3 in the solid state, it is found that the e2qQ/h values decrease linearly with a decrease in the hydrogen bond length. This indicates that it is possible to determine the hydrogen bond length through the observation of e2qQ/h values. It was found that there is a difference in r/o between the polypeptides and oligopeptides. This may come from large difference in the molecular packing between them. The chemical shift values in both peptides and polypeptides move to low frequency with a decrease in the hydrogen bond length. However, there is a difference in the chemical shift values between peptides and polypeptides. From these experimental findings, it is demonstrated that 1 7 0 N M R spectroscopy becomes a useful means for elucidating the hydrogen-bonding structure in solid peptides and polypeptides. Most recently, the multiple quantum MAS N M R technique was applied to obtain a high resolution solid-state 170 N M R spectrum [16-17], but it seems to need much more time before this technique becomes useful for solid-state 1 7 0 NMR.

252

s. KUROKI

References

.

.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

D.W. Boykinm (ed), 170 NMR Spectroscopy in Organic Chemistry. CRC Press, Boca Raton, FL, 1991. A.L. Baumstark and D.W. Boykin, 170 NMR spectroscopy: applications to structural problems in organic chemistry, in A.L. Baumstark (ed), Advances in Oxygenated Processes, vol. III. JAI Press, Greenwich, CT, 1991, 141. W.G. Klemperer, in J.B. Lambert and F.G. Riddell (eds), The Multinuclear Approach to NMR Spectroscopy. Kluwer, Dordrecht, 1983, 245. J.P. Kintzinger, in P. Laszlo (ed), Newly Accessible Nuclei, vol. 2. Academic Press, New York, 1983, 79. D.W. Boykin and A.L. Baumstark, New Journal of Chemistry 16 (1992) 357. D.W. Boykin and A. Kumar, J. Heterocyclic Chem. 29 (1992) 1. S. Kuroki, A. Takahashi, I. Ando, A. Shoji and T. Ozaki, J. Mol. Struct. 323 (1994) 197. T.H. Walter, G.L. Turner and E. Oldfield, J. Magn. Reson. 76 (1988) 106. R. Goc, E. Ponnusomy, J. Tritt-Goc and D. Fiat, Int. J. Pept. Protein Res. 31 (1988) 130. R. Goc, J. Tritt-Goc and D. Fiat, Bull. Magn. Reson. 11 (1989) 238. F.H.C. Crich and A. Rich, Nature 176 (1955) 780. W.T. Astbury, C.H. Dalgleish, S.E. Darmon and G.B.B.M. Sutherland, Nature 69 (1948), 596. A. Kevick, A.R. AI-Karaghouli and T.F. Koetzle, Acta. Cryst. B33 (1977) 3796. S.N. Rao and R. Parthasarathy, Acta Cryst. B29 (1973) 2379. K. Tsuchiya, A. Takahashi, N. Takeda, N. Asakawa, S. Kuroki, I. Ando, A. Shoji and T. Ozaki, J. Mol. Struct. 350 (1995) 233. G. Wu, D. Rovnyank, B. Sun and R.G. Griffin, Chem. Phys. Lett. 249 (1995) 210. P.J. Dirken, S.C. Kohn, M.E. Smith and E.R.H. van Eck, Chem. Phys. Lett. 266 (1997) 568.