13C NMR chemical shift and conformation of β-benzyl l -aspartate residues in copolymers of β-benzyl l -aspartate and l -alanine in solution

Journal of Molecular Structure, 238 (1990) 347-355 Elsevler Science Publishers B V , Amsterdam

347

13C NMR CHEMICAL SHIFT AND CONFORMATION OF &BENZYL L-ASPARTATE RESIDUES IN COPOLYMERS OF j?-BENZYL L-ASPARTATE AND L-ALANINE IN SOLUTION

SATORU TUZI and ISA0 AND0 Department of Polymer Chemistry, Tokyo Znstctute of Technology, Ookayama, Meguro-ku, Tokyo (Japan) AKIRA SHOJI and TAKUO OZAKI Department of Zndustrlal Chemutry, College of Technology, Gunma Unwerslty, TenJu-cho, Kuyu-shl, Gunma (Japan) (Received 4 December

1989)

ABSTRACT High resolution 13C NMR spectra of copolymers of/?-benzyl L-aspartate and L-alanme residues have been recorded, m order to obtain detailed mformatlon about the conformatlonal behavlour of the main-chain and side-chains of the /I-benzyl L-aspartate residues m solution The observed results m solution show that the main-chain takes both the right- and left-handed a-helical forms, which 1s identical with the cy-helix conformation m the sohd state The inner side-chains m the right-handed a-helical form have different characterlstlc conformations m solution and m the solid state Such chemical shift behaviour of the side-chains suggests that inter-side-chain mteractions make the inner side-chains take a conformation with higher energy than the isolated sldechain Such mteractlons may account for the fact that m solution the mam-chain takes the lefthanded a-helix rather than the right-handed one

INTRODUCTION

It has been shown that the a-hehces of poly L- (ammo acld)s generally have the right--handed hehx sense m the sohd state and m solution However, poly (j?benzyl L-aspartate) ( [~-Asp ( OBzl) ] ,) exceptionally takes the left-handed LYhelix (cu,-hehx) form m solution, and takes the right-handed a-hehx ( aRhelix), the aL-helix, the left-handed o-hehx and thep-sheet forms m the solid state under specified condltlons [ 1,2]. This behavlour of [L-ASP ( OBzl) ] n may be caused by mteractlons between its long side-chains It 1s known that the cu,-helix of [L-ASP (OBzl) 1, 1s less stable than the other homopolypeptldes [3-51 There are two explanations for the mechanism by which the crR-helix form becomes unstable and the cuL-hehx form 1s stablhzed m solution One 1s that the sterlc hmdrances m the side-chains induced by mterslde-chain mter-

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actions lead to mstabihty of the an-helix form [ 31. The other is that the competition between the main-chain N-H. * * main-chain C=O (amide C=O) hydrogen bond and main-chain N-H*** side-chain C=O (ester C=O) hydrogen bond makes the an-hehx form of [L-ASP (OBzl) ] n unstable [6] In this work, in order to obtain detailed mformation about the contributions of the mam chain and side-chains to the conformational stability of [LAsp (OBzl) ] n m solution, we have measured 13CNMR spectra for copolymers of L-alanme (L-Ala) and L-ASP(OBzl) as a function of ~-Asp (OBzl) content m solution and, on the basis of these results, we discuss the conformational stabihty of the ~-Asp (OBzl) residues. Since poly (L-alanme) ( [L-Ala] ,) takes the cu,-helix form in solution, some of the L-ASP(OBzl) residues m a copolymer chain may be forced to change conformation from the al-helix to a,-hehx form For this, it can be expected that through the behavlour of the main-chain and side-chains of the L-ASP(OBzl) residues m the copolymer, some factors which make [L-ASP (OBzl) ] n take the more stable cul-hehx form compared to the an-helix form m solution will be clarified The conformation-dependent 13Cchemical shift values for [L-Ala] n and [L-ASP (OBzl) ] n m the solid state reported previously are used to analyze the main-chain and side-chains of the copolymer [ 7-101. EXPERIMENTAL Materials Samples of [~-Asp (OBzl) ,L-Ala] n were prepared according to the followmg procedure L-Asp(OBz1) was prepared from L-aspartic acid by esterlficatlon with benzyl alcohol. L-Asp(OBz1) and L-Ala were reacted with tnchloromethylchloroformate m tetrahydrofuran to prepare their N-carboxyanhydndes (NCA). The copolymers were prepared by heterogeneous polymerization of a mixture of ~-Asp (OBzl) and [L-Ala] n NCAs m acetonitrile or dloxane at room temperature (about 25 oC ) by using n-butylamme or triethylamme as the mltiator The mole ratio of NCA (A) and nntiator (I) (A/I) was chosen as 100 The reaction yields of the polymerization were about 90% Conditions of preparation for [L-ASP (OBzl) ,L-Ala] n samples are summarized m Table 1 L-Ala contents m samples were estimated from ‘H and 13CNMR spectra L-Ala contents of CP-1 and CP-2 were estimated by comparison of the mtensities of the L-Ala Cp and L-ASP(OBzl) C/3 peaks m ‘H NMR spectra L-Ala contents of other samples were estimated by comparison of the intensities of the L-Ala C=O and ~-Asp (OBzl) C1 peaks m 13CNMR spectra. The composition of each of the samples considered here is summarized m Table 1

349 TABLE 1 Preparation condltlons and L-Ala content of [~-Asp (OBzl),L-Ala],

samples

Polymerlzatlon condltlons

CP-1 CP-2 CP-3 CP-4 CP-5 CP-6 CP-7 CP-8 CP-9

Initiator

Solvent

L-Ala content (% )

n-Butylamme n-Butylamme Tnethylamme Trlethylamme Trlethylamme Trlethylamme Tnethylamme Trlethylamme Trlethylamme

Acetomtrlle Acetomtrde Dloxane Dloxane Dloxane Acetomtrlle Acetomtrlle Acetomtnle Acetomtrlle

0 51 0 85 44 12 17 19 40 55 75

NMR measurement High resolution 13CNMR spectra were recorded at 125 7 MHz with a JNM GX-500 NMR spectrometer at room temperature. Spectral width and data points were 27 kHz and 32 k, respectively. Repetition time was 5 s, and spectra were accumulated about 1000 times to achieve a reasonable signal-to-noise ratio. The 13Cchemical shifts were calibrated through external tetramethylesilane [ (CH,)&] . The samples were prepared to 2% solution with CDC13and contained m sample tubes of 10 mm mternal diameter RESULTS AND DISCUSSION

Figure 1 shows typical 13CNMR spectra of [~-Asp ( OBzl) ,L-Ala] n in CDCl, solution. All peaks appearing m the spectra can be easily assigned to carbons of the L-Ala and ~-Asp (OBzl) residues as given m Fig 1 with reference data to the 13C chemical shift data of the respective residues in [L-ASP (OBzl) ] n and [L-Ala] n reported previously (Table 2) [ 7-101 Before discussmg 13C NMR spectra of the copolymers, it is necessary to know whether the copolymers considered here are random copolymers or not In 13CNMR spectra of the copolymers with low L-Ala contents, we can observe the two Ccwpeaks of L-Asp (OBzl) which come from the (XR-and cxL-helixforms, and so can estimate the ratio of the (XR-and al-helix forms m [~-Asp (OBzl) ] n using the intensities of these peaks (Fig 1). Figure 2 shows the percentage of ~-Asp (OBzl) residues taking the cuL-hehx form as a function of the L-Ala content of the copolymers With increase of L-Ala content, the content of cu,-hehx form decreases, however the rate of its decrease is not fixed but dlmmlshes as L-Ala content increases. On the assumption that the samples are random co-

350

Phenyl

CDCl3

L-Ala amide

C=O ester

c=o Cl

a,cvxp. \ \

A

\ 8’ _’

B

, 200

I

I,

I

180

160

I

I

140

I,

11

120

I

100

I

80

I

I

60

I

I

40

I

I

20

I

f

0

b /PPM

Fig 1 Typlcal 13C NMR spectra of [L-Asp(OBzl),L-Ala]. m CDCl, solution A CP-1 (L-Ala content 0 51%), B CP-6 (L-Ala content 19%) (Ye and CX~ mdlcate the peaks assigned to the ffRhelix and cYL-hehx,respectwely

polymers, and that the existence of L-Ala residues makes a regular number of nelghbourmg L-ASP(OBzl) residues change from the cuL-helixto cx,-hehx form lrrespectlve of the L-Ala sequence length, rt can be expected that the ar,-hehx content will decrease in proportron to L-Ala content. However, the expenmental feature IS mconslstent with expectation This suggests that the copolymer 1s not a random one, but a block one With a block copolymer, the L-Ala sequence length increases with increasing L-Ala content Where the L-Ala content 1s less than about 12%, three types of structure are considered m the copolymers, the L-Ala an-helix, L-Asp(OBz1) an-helix and L-Asp(OBz1) cyLhelix forms The cw,-helix sequence length for ~-Asp (OBzl) residues decreases with increase of L-Ala content When the L-Ala content exceeds about 12%, the al-helix structure does not exrst and the a,-hehx sequence length for LAsp (OBzl) residues decreases with mcreasmg L-Ala content Therefore, It should be possible to drscuss the nature of the an-helix or crL-hehx structure of ~-Asp (OBzl) residues with different sequence length through observmg the “Y! NMR spectra of the copolymers

15 9

128 4

128 1

135 9

33 9 1686 170 7 66 4

1717

512 174 1

15 7

176 5

128 1 128 6

135 8

135 9

15 8

176 5

128 2 128 6

1686

34 8

66 4

53 5 513 1742

53 6 513 174 2 1716 34 8 34 0 168 6 170 7 66 4

176 6 16 0

128 4

135 9

66 6

168 7

35 0

174 3

53 5

CP-5

15 7

176 5

128 6

128 3

135 9

66 7

168 7

35 0

174 3

53 5

CP-6

1765 16 1

128 5

127 8

135 8

66 7

1690

34 9

1740

53 3

CP-7

CP-8

CP-9

16 0

1765

128 6

128 0

135 8

66 7

169 1

e

e

53 0

16 1

1769

128 6

128 0

136 0

66 6

169 3

e

e

52 9

128 4(aL)

128 Z(a,)

135 9(&L)

66 3(01L)

170 7

33 8

1717

513

128 O((YR) 128 6(aL)

66 3(&L) 135 7((YR) 134 7(a,)

65 l (aR)

168 6 169 4

33 6 34 0

1713

174 5

53 4 512

Sohd”

14 9

52 4 176 4

Sohdd

[L-Ala],

‘C1 carbon of phenyl group bChemlcal shift values m CDCl, solution “Chemical shift values m the solid state The values are calibrated through external benzene dFrom Ref 8 Chemical shift values m the solid state The values are calibrated through external benzene ‘Peak ISvery broad and so accurate chemical shift value cannot be obtained

CP

15 8

128 1 128 4

Phenyl (C,-C,)

L-Ala Ca Amide C=O

135 9

66 2

33 8 168 6 170 7

51 3 1742 171 7

CP-4 Solutionb

CP-3

CP-1

CP-2

[L-Asp(OBz1) In

[L-Asp (OBzl) &-Ala] n copolymer samples

Cla

OCH,

Ester C=O

CP

Amide C=O

~-Asp (OBzl) Ca

Helix sense

Observed 13Cchemical shifts of [L-ASP( OBzl) ,L-Ala], m solution (ppm from (CH,)&)

TABLE 2

352

10

5 L-Ala

content

15 A,*

content for L-Asp( OBzl) residues m [L-ASP(OBzl),L-Ala],

Fig 2 The plot of the al-helix L-Ala content

against

13Cchemzcal shafts of [L-Asp(OBzl), ~-Ala], m sol&on Figure 3 shows the chemical shift change in solution of the L-Asp(OBz1) residues m the copolymer with different L-Ala content. The chemical shift values of the main-chain carbons (Ccw and amide C=O) are almost the same as those of the a-helical [~-Asp ( OBzl) ] n and [L-Ala] n m the solid state. Furthermore, the chemical shift values for amide C=O carbon of L-ASP(OBzl) residues m the cm-helix and L-Ala residues are almost constant regardless of L-Ala content. Though the chemical shift values of Ccr carbon for the o!nhelical [L-ASP (OBzl) ,L-Ala] n change with mcreasmg L-Ala content, this change seems to be caused by the overlapping of the peaks of L-Ala Ccuand L-

50

0 L-Ala

Fig 3 I

100 content

/%

353

i-_so

‘a3

100

0

L-Ala

Flg3

content

/%

11

1

0

50 L-Ala content

100 1%

3 111The plot of 13C chemical shift values of [~-Asp (OBzl) ,L-Ala] n m solution against L-Ala content 0, L-Asp(OBz1) m the a,-helix, 0, L-Asp(OBz1) m the q,-helix, 0, L-Asp(OBz1) m the cya- and al-helix (overlapped), and 9, L-Ala The chemical shift values shown by triangles are the values of [L-Asp(OBzl)], and [L-Ala], m the sohd state A, L-Asp(OBz1) m the (~ahelix, A, L-ASP (OBzl) m the cY,-hehx, and A, L-Ala

Asp(OBzl) Ccu carbons These results suggest that the conformations of the mam-cham m solution and m the solid state are slmllar to each other. The peak of side-chain carbons of L-Ala residues (L-Ala C/I) appears at 15 7 N 16 0 ppm This peak appears at lower field by 0.8 N 1 ppm than the peak

354

of L-Ala C/3 m the solid state (15 9 ppm) This may be due to the fact that m the solid state inter-chain mteractlons between the ar-hehcal chains exist and m solution mteractlons between an a-helix chain and solvent molecules exist Side-chain carbons m L-ASP (OBzl) residues show characterlstlc chemrcal shift changes correspondmg to the change of L-Ala content of the copolymers The chemical shift values of the outer carbons of side-chains (OCH,, C, and other phenyl carbons) are almost constant (Fig 3), but there 1s no obvious chemical shift drfference between the aa- and cul-helix forms, and then chemical shift values disagree with those m the solid state. Furthermore, the sharp peaks of these carbons show that they have high mobrhty regardless of L-Ala content This suggests that the outer parts of side-chains move freely, and then averaged conformation 1s identical to the side-chain conformatron for the (Ynand cu,-hehx m the solid state The high mobility for the terminal carbons of srde-chams may arise from high degree of freedom of rotatron around the bonds (C=O) -0 and 0-CH 2 m side-chains The chemrcal shifts of the inner carbons of side-chams (Cp and ester C=O ) are character&c values correspondmg to the (xn- and cr,-hehx forms (Fig. 3) The chemical shaft of Cj? carbon m the al-helix form 1s the same as that m the solid state On the other hand, the chemical shift values of the ester C=O carbon m the al-helix form are dlsplaced to lower field by 1.2 N 13 ppm compared with those m the solid state. Since the solid cuL-hehx sample of [~-Asp (OBzl) ] n 1s prepared by preclpltatlon of [L-ASP (OBzl) In from chloroform solution, the conformation 1s expected to be almost the same m solution and m the solid state. The agreement between the chemical shift values of L-ASP (OBzl) C/I carbon m solution and m the solid state supports this explanation Therefore, the chemical shift displacement of ester C=O carbon seems to arise from mteractlons between the C=O carbon and solvent molecules rather than change of side-chain conformation. The 13C peak of C=O carbon which contributes to a hydrogen bond shifts lower field than the peak of Isolated C=O carbon From this, it can be said that the downfield shaft of ester C=O 1s due to a large hydrogen bondmg contrrbutlon between ester C=O and solvent molecules (CDCl,) Thus suggests that the ester C=O m the al-helix form 1s exposed to solvent molecules The Cp peak m the cuR-helix form appears at lower field by 1 1~ 14 ppm than that m the solid state, regardless of L-Ala content Furthermore, the Cppeak m the crR-hehx form appears at lower field than that in the al-hehx form in solutron, but m the sohd state the correspondmgpeak m cxR-helix form appears at hrgher field than that m the cwL-helix form (Frg. 3) This suggests that the conformation of the inner side-chain m the cx,-helix m solution 1s different from that m the solid state, regardless of the sequence length of L-ASP (OBzl) residues Such a drfference between the side-chain conformations m solution and m the solid state seems to result from the difference of the conformation of the outer side-chain or the difference m the mteractions between the side-chains and solvent molecules From these results, it IS obvious that the chemical shift

values of side-chains m the a,-hehx m solution cannot be compared directly with those in the solid state The chemical shift values of the ester C=O carbon in the cm-hehx shift to low field with decrease of the sequence length of LAsp (OBzl) residues This chemical shift change suggests that the rapid exchange among different conformations of ester C=O occurs. From the chemical shift change through the change of the sequence length of L-ASP (OBzl) resldues, it can be said that the stable side-chain conformation under mter-sldechain mteractions is different from the stable one of isolated side-chain not under inter-side-chain mteractlons Therefore, it seems that the inner sidechains m the ~-Asp (OBzl) residues under the mter-side-cham Interaction take the conformation with higher energy than the inner side-chain m the isolated residue This suggests that high conformational energy of the inner side-chain under the mfluence of inter-side-chain mteractlons is one of the factors which leads to the unstable cun-hehx of [L-ASP (OBzl) ] n m solution CONCLUSIONS As mentioned above, the factor which destabilizes the ar,-helix form of [LAsp ( OBzl) ] n m solution is attributed to the sterlc hindrance of side-chains m the cr,-helix form or the competition between amide C=O***N-H hydrogen bond and ester C=O.**N-H hydrogen bonding m the a,-hehx as stated m previous work From the characteristic chemical shift change of ester C=O m the aR-hehx, which depends on the change of L-Ala content, it can be concluded that the mner side-chains m the ~-Asp (OBzl) residues under the mfluence of inter-side-cham interaction take the conformation with higher energy than the inner side-chain m the isolated residue This high conformational energy of the inner side-chain must be one of the factors which leads to the unstable cxn-hehx and makes [L-ASP (OBzl) ] n form the cu,-hehx m solution. However the hydrogen-bond between ester C=O and solvent molecules (CDCl,) m solution seems to be one of the factors stabihzmg the cxL-hehx

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