Journal of Biochemical ond Riophysical Methvds, 1 (: ?,7~) 135--143 © ElsevierlNorth-.~':cllandBic~ed:eal Preas

C. N I U

{, R.D. B E R T R A N D

2, H. S H ! N D O

135

: and J.S. CO}':EN

I Developmental Pharmacology Branch, National Instiiute of Child tleatth and Human Development, Notional !nslitutesof Hea~th, Bethesda, ~CD 20014, ond 2 Deportment of Naturol Sciences, Unit,ersity o f ~ichingan-'r--,earborn, Dearborn, M] 48128, L:S.A. (Re:eived 4 I)e~emher 1978; accei:ted £ 5 ~'.arch 1979)

Comparative IaC--~SN coupling constants are repcrtgd for the linear dipeptide tBoc~.%~-'3~'~AIa-~sN)G:yCMe and the corresponding cyei:.~ d".ketopiperazine, both "n dimethyisulfoxidc ( D M S C ~ and, upon tern.ova: of the tBoc group, :n water solut:ons. Spectra were obtained by ~aC N M R and by the firstapplication of J cross-~o:arization (JCP) :SN N M R , wh:ch graat:y reduces the time requ".red to accumulate XSN N M R spectra. :n D M S O there was evidence ~or the ,~cnr,at~or,of compiexed species which were not present in water. _~he values obtained for *.he cross-peptide bond coupling constant zJ~3c~t -'sl~-were consLstently "ass (by 2.2 Hz in D M S O , 4.3 Hz in water) for the cyc'lic than ~or the llnamr pep.tide, which r=.ay be r~!ated to the c:oss-pept:de bond =onformat:on. The ~sN resonance for the cycP.c peptide w ~ shifted on:y 2 p p m downfield from the t~.nearpeptide chemical sh'ftvalue in botch solvents. :~eywords: 13C IWMR; '-sN N M R ; l:~ptide bond; coup~:ng ~onstant.

".NTRODUCT-ON

The structural dependence of two-bond ~3C--:5N coupling constants has been descMbed for a seriesof amides Ill ar.d amines [2~. By contrast, threebond couplings were e'th~r not observed cr *.co sm,.!l to be useful in conformat:one! studies on pe~tides ~3~. (For a recent review of ~3C--'SN coup~ng constants, see Wasyl'.shen . : . ~ We wished to test the utility of cross-peptide bond :3C--]SN coupling ~onstants as monitors of confor.~-ationa~ effects. W~ used the simple s~atagem c f joining a ~3C-iabe1~ed a~.ino acid with an ~'~N~abellad amino acid by peptide synthesis. For con:.~pariscn o~ two extreme cases we con-..pared the Scme of this work was presented at the American Society cf Bio:ogical Chemists' meeting, A1tante, June, 1978. Abbreviation.s: DMSO, d.~methylsulphoxide; tBcc~ tbutyicxycarbor~yl; JCP, J crosspoladzat'on; F~D, free £nduetlc:n ~ecay; T'~C, thin-layer chromato~aphy; _~T, Four:er transfcrrr..

136 linear {blocked) peptide which is in the trans conformation with the corresponding cyclic diketopiperazine which is in the cis form. In addition to our use of the ~3C Fourier transform (FT) NMR technique, we also report the first application of the J cross-polarization (JCP) 'SN NMR technique [5]. This consists of the application of the double-resonance cross-polarization technique developed for solids, but applied in this case to liquids [6]. In liquids, spin--spin coupling (J) is responsible for the magnetization transfer which provides the name for the technique. Since the effective signal enhancement by this m e t h o d is determined by the ratio of the gyromagnetic ratios of the nuclei irradiated (in this c a s e ~ H / ~ / N • 9.9), and since no negative NOE factors are produced in the method, this technique is especially appropriate for observation of ~SN NMR spectra. With the capability of avoiding the long repetition times normally associated with ~SN T~ values, enhancements of up to 350 in time can be expected by JCP 'SN NMR over the standard technique [5]. With the application of both ~3C and ~SN NMR observation, consistent values of ~3C--ISN coupling constants were obtained. EXPERIMENTAL

Materials [lSN]Glycine (60%) was purchased from KOR Isotope, and L[Vfl3C]alanine (90%) was obtained from the Los Alamos Scientific Laboratory.

HCl-glycine methyl ester 0.5 g ['SN]glycine was suspended in dry methanol, and the solution was cooled to 0°C in an ice-water bath for 15 min. After dry hydrogen chloride had been bubbled into the solution for 10 rain at 0°C, the methanol solution was stirred at room temperature for additional 60 min. After evaporating the solvent, crystals were formed, and dried with a vacuum pump overnight. However, TLC showed that the product was contaminated with approx. 10% unreacted glycine.

tBoe[U -13C]A laOH This was synthesized by the m e t h o d of Schnabel [7]. The yield was 95%.

tBoc [U -13C]A la-[ ~SN] Gly OMe This was prepared by the mixed anhydride method. The c o m p o u n d could not be cyrstallized; nonetheless, TLC showed that a single spot on silica gel plate with CHC13/CH3OH (93 : 7). The yield was 85%. To remove the tBoc group from this product it was dissolved in ethyl acetate and dry HC1 was bubbled for 10 min at 0°C, then stirred at room temperature for ! 5 min and the solvent was evaporated.

137

Cyclo(L-fU-~3C] A la-[' SN] Gly ) This was prepared by the method of Nitecki et al. [8]. The cyclic peptide was ninhydrin-negative but gave a positive result with chlorine reagent; TLC showed a single spot with CHC13/CH3OH/acetic acid ( 8 5 : 1 0 : 5 ) and ethanol/water/aqueous ammonia (7 : 3 : 1) as solvents. The yield was 40%. NMR methods "~N NMR spectra were obtained using the JCP pulse technique [6] as well as by the conventional pulse FT technique. This JCP method involves applying a 7r/2 pulse at the proton frequency followed by a 90 ° phase-shifted spin-locking pulse. RF radiation is simultaneously applied to the 'SN spins during the spin-locking pulse. The magnitudes of the RF field strengths are adjusted so that 3"B',sN =~/B', H. During observation of the FID's, noise decoupling was applied to the protons. The spectrometer used in these experiments operated at 2.3 tesla, with an observing 'SN frequency of 10.13 MHz 'with 3'B, = 3 kHz. Samples of approx. 80 mg in 0.5 ml were contained in 5-mm NMR tubes. Optimum or near-optimum cross-polarization times were used in obtaining the "~N NMR spectra. These were determined from equations given earlier [6] and measured 'SN--'H one-bond J couplings. Chemical shifts were determined relative to an external sample of a saturated solution of 'SN-enriched ammonium chloride in acidified water, lSN NMR spectra were also obtained at 27.3 MHz by the conventional probe FT NMR method with a partially home-built spectrometer equipped with a Bruker magnet operating at 6.4 tesla and a Nicolet 1180 computer. Proton noise decoupling was used and 10 000--14 000 pulses were accumulated with a 3 s recycle time. '3C NMR spectra were also obtained using the same superconductingmagnet spectrometer at 67.9 MHz. Resolution was 32 768 real points in 15 kHz, with a recycle-time of 2.16 s and a 7r pulse of 20 ~s. Either 500 or 1000 transients were collected with no exponential filtering. Samples from the JCP experiments were diluted to 1.3 ml and placed in 10-mm NMR tubes, containing an anti-vortex device. RESULTS AND DISCUSSION Several attempts have been made to determine the potential utility of "~N--'3C coupling constants for conformational studies of peptides [1--4]. In all cases the ~SN and '3C nuclei considered were within the same amino acid residue. We have measured cross-peptide bond coupling constants by using '3C- and 'SN-enriched amino acids coupled by standard peptide synthesis methods. In order to ascertain if conformational effects could cause changes in the values of the cross-peptide 'SN--'3C coupling constants, we compared the linear dipeptide and cyclic diketopiperazines in two solvents. The values of the coupling constants determined are shown in Table 1. Only one- and two-bond couplings were observed, no three-bond couplings could be resolved. The 'SN NMR spectra obtained by the JCP m e t h o d for

H20/2H20 ( 4 : I) DMSO

tBoc-Ala-GlyOMe 13C

J C P 15 N 13C

J C P 1s N 13C 13C

NMR method

9.3

9.0 8.9

6.8 6.8 5.0

2J13c _ I S N

J13C0--13C ~ -51.0 51.3 -53.0 52.5

JI3c0--15N 15.0 14.9 16.4 14.4 ld.2 16.6

C o u p l i n g c o n s t a n t (Hz) b

a Ala was u n i f o r m l y labelled a p p r o x . 90% 13C. Gly was a p p r o x . 60% lSN. b Errors are < ± 0 . 2 .

H20/2H20 (4:])

DMSO

Cyelo(Ala-Gly)

Ala-GlyOMe

Solvent

Compound a

DIPEPTIDE 13C_l s N COUPLING C O N S T A N T S

TABLE 1

-35.5 33.9

-36.6 35A

J13c~--13C ~

139 t h e t w o p e p t i d e s s h o w n in Figs. 1 a n d 2 w e r e r e a d i l y a n a l y z e d t o give t w o c o u p l i n g c o n s t a n t s . T h e '3C s p e c t r a o f t h e s a m e s a m p l e s w e r e n a t u r a l l y m o r e c o m p l e x d u e t o t h e p r e s e n c e o f [U-~3C]Ala, b u t w e r e r e a d i l y a n a l y z e d f o r

the various 'SN--'3C and '3C--'3C coupling constants (Fig. 3). The consistency of the 'SN--'3C coupling constants obtained by both methods is quite striking, and helps to confirm the coupling patterns shown. While the one-bond J,3c0_,SN coupling constant showed little variation the two-bond 2J,3ca_,sN showed a significant change between the linear and cyclic dipeptides in both solvent systems. Although it is quite likely that these differences, of 2.2 Hz in DMSO and 4.3 Hz in water solutions, represent conformational effects, other considerations must be taken into account. Thus, it is clear by a comparison of the spectra of the peptides in DMSO and water that some form of complexation is occurring in DMSO giving rise to extra peaks and more complex spectra (Fig. 4). While these extra peaks remain sharp for the cyclic peptide they are broadened for the blocked linear peptides (Figs. 2 and 4). In addition, from the '3C chemical shifts in DMSO compared to those in water (Table 2) it is clear that different modes of complexation with DMSO are occurring in each case. Thus, the earbonyl-~3C resonance, relative to the same compound in water is shifted downfield by approx. 3 ppm for the cyclic peptide and upfield by approx. 1.5 ppm "in the linear case. It should be noted that in order to dissolve the linear peptide in aqueous solvent it was necessary to deblock the amino group by removing the tBoc blockinggroup. This too may have an effect on

~Co Gly N

-raN 's N

Ii i

! I i

I

yl

i

'

'~i"'

'"

Fig. 1. JCP 15N NMR spectrum of eyelo([U-t3C]Ala-[lSN]Gly) in DMSO. Shown in a 48.3 Hz portion of a 500 Hz spectrum obtained by Fourier transformation of 1412 signal-averaged FID's digitized into 8192 points. The FID's were produced using the JCP pulse sequence [6] with a 10 ms cross-polarization time, repeated every 9.19 s. Chemical shift values are given in Table 2.

140

GIyN ,

. ~

i i

i i

I~l

' ~

; t ' i

I

t

I '

i

;

"

i ,.

/.',

,

;

:.

,>

',/

~ .:,

d%c_,SN

, 4,'J~''~

V~, ; i , . , , I , Y'l ",' Ii~t

Fig. 2. JCP ISN N M R s p e c t r u m of t B o c - [ U - 1 3 C ] A l a - [ l S N J G l y O M e in DMSO, S h o w n is a 4 4 . 2 Hz p o r t i o n o f a 5 0 0 Hz s p e c t r u m o b t a i n e d by F o u r i e r t r a n s f o r m a t i o n of 256 signalaveraged F I I ) ' s digitized i n t o 8 1 9 2 p o i n t s . A 0.2 Hz e x p o n e n t i a l line b r o a d e n i n g was applied b e f o r e : t r a n s f o r m a t i o n . T h e F I D ' s were p r o d u c e d b y using the JCP pulse s e q u e n c e [.6 ] with a n 11 ms c r o s s - p o l a r i z a t i o n t i m e , r e p e a t e d every 9.19 s.

A l a Co

A l a C ;i

AIoCc

I

- -tJ

I

b j°

J 3~

(

i

ll! i! !

i

r

.

.

1','

,

..

,

I i I

h/.

i

,, i~ '! ,/

L

A.

175

.

.

.

l . _ _

I~

_ _

125

IO0

75

SO

2~

PPM

Fig. 3. 13C N M R s p e c t r u m o f [U -13C]Ala-[ l s N ] G l y O M e in water o b t a i n e d at 67.9 MHz. T h e insets are: e x p a n s i o n s o f t h e r e s o n a n c e s for each 13C-enriched a t o m s h o w i n g t h e splitting pattern.

141 C~

t2o

I

Ca

I

b i _°

',

,b ¢

11 I

I~ Ii*l I ,

!

j

1

~*~1 I

T ' I I

i l)i iI

Fig. 4. D i f f e r e n c e s in t h e 13C r e s o n a n c e s for t h e cyclic ( u p p e r ) a n d linear b l o c k e d (lower) p e p t i d e s in DMSO at 67.9 MHz. T h e s e s h o u l d also be c o m p a r e d to t h e a p p r o p r i a t e 13C r e s o n a n c e s in Fig. 3.

the various chemical shifts, although it is not likely to affect greatly the carbonyl -~3C chemical shift. It should be noted that the formation of hydrogenbonded peptide--DMSO complexes has been described previously [9]. Thus, the difference in the 2J13ca_lSN coupling constants might be ascribed to the apparent differences in complexation of DMSO between the cyclic and linear peptides. On the other hand, the results in aqueous solution, with no evidence of complexation or other species formed, give an even larger difference for t h e 2Jl3ca._ls N values. At the present time the precise origin of the electronic changes in the '3C--'SN bonds giving rise to these differences is not known. It should be noted, however, that the angle subtended by the N--C0--C a atoms is generally considered to be 120 ° [10]. The values of the

TABLE 2 CHEMICAL SHIFT VALUES OF SYNTHETIC PEPTIDES Compound

Cyclo(Ala-Gly) tBoc-Ala-GlyOMe Ala-GlyOMe

Solvent

DMSO H20 DMSO HEO

C h e m i c a l shift ( p p m ) a 13Co ~

I ~C~ a

1 3C o

15 N

48.46 49.5 48.33 48.36

17.51 17.73 17.02 15.64

167.73 !70.65 172.23 170.67

80.6 -78.5 --

a x3C c h e m i c a l shifts are in p p m d o w n f i e l d f r o m i n t e r n a l l a C H a C N , lSN c h e m i c a l shifts were o b t a i n e d using t h e J C P t e c h n i q u e a n d are in p p m d o w n f i e l d f r o m e x t e r n a l lSNH4C1.

142 '3C--'3C c o u p l i n g c o n s t a n t s o b t a i n e d were c o n s i s t e n t with t h o s e r e p o r t e d previously [ 11 ]. A p a r t f r o m the c o u p l i n g c o n s t a n t s described above 'SN c h e m i c a l shifts were also measured. T h e ~SN N M R spectra o b t a i n e d by the JCP m e t h o d o n the small peptides were a c c u m u l a t e d a p p r o x i m a t e l y an o r d e r o f m a g n i t u d e m o r e q u i c k l y than b y the c o n v e n t i o n a l pulse FT N M R m e t h o d , f o r approxim a t e l y the same S/N. H o w e v e r , the differences in field strength and sample size p r e c l u d e a m o r e e x a c t c o m p a r i s o n o f t o t a l a c c u m u l a t i o n time in each case. Similarly, a l t h o u g h absolute values o f chemical shifts c a n n o t be compared f r o m t h e results o f the t w o m e t h o d s , due to the d i f f e r e n t m a g n e t c o n f i g m ' a t i o n s , different bulk susceptibilities o f the samples a n d d i f f e r e n t standards used, differences in chemical shifts are r e a s o n a b l y c o m p a r a b l e . The "~N r e s o n a n c e o f t h e cyclic p e p t i d e was shifted o n l y 2.1 p p m d o w n f i e l d f r o m the p o s i t i o n o f the linear p e p t i d e r e s o n a n c e in DMSO (Table 2). At t h e higher f r e q u e n c y a c o n s i s t e n t d i f f e r e n c e o f 2.0 p p m was also f o u n d for the t w o peptides in w a t e r at p H 7.0, with the t B o c g r o u p r e m o v e d f r o m the linear p e p t i d e to c o n f e r solubility. This d i f f e r e n c e is q u i t e small c o m p a r e d to o t h e r 'SN chemical shift effects w h i c h have been r e p o r t e d [ 1 2 , 1 3 ] * SIMPLIFIED DESCRIPTION OF THE METHOD AND ITS APPLICATIONS The JCP lSN NMR technique enabled us to measure lSN spectra of small peptides very readily. Comparison of cross-peptide 2J13Ca--lSN for '3C- and I SN.enriched linear and cyclic dipeptides in DMSO and water revealed a small but consistent difference which may reflect the cross-peptide bond conformation. ACKNOWLEDGEMENTS T h e JCP ~SN N M R s p e c t r a were o b t a i n e d while one o f us (R.D.B.) was o n sabbatical leave at the Naval Research L a b o r a t o r y . The assistance o f W.B. M o n k , A.N. G a r r o w a y and G.C. Chingas is gratefully a c k n o w l e d g e d . REFERENCES 1 Lichter, R.L., Fehder, C.G., Patton, P.H. and Combes, J. (1974) Chem. Commun. 114--115

* The 15N resonance of the synthetic amino-terminal peptide of ribonuclease, containing 15 amino acids with lSN enrichment at Ala-6, in H20 at pH 2 was shifted downfield by approx. 15 ppm compared to the linear dipeptide and an lSN-enriched tripeptide. However, no significant difference (