Cyclization of S-(p-bromophenacyl)-l -cysteine as observed by nuclear magnetic resonance spectroscopy

ARCHIVES

OF

BIWHEMISTRY

Cyclization

AXD

115,

(1966)

237-z%

of S-(p-Bromophenacyl)-L-Cysteine clear

Departments

BIOPHIYWS

of Biochemistry

Magnetic

Resonance

as Observed Spectroscopy’

and Jlicrobiolog~, College of Physiciuru Xeto York, New York Received

January

by Nu-

und Slcrgeons,

Columbin

ikicersily,

25, 1966

Dissolution of S-(p-bromophenacyl)-L-cysteine in trifluoroacetic acid or methanol resulted in an intramolecular interaction of the curbonyl and amino groups followed, in trifluoroacetic acid, by dehydration. The nature of the carbonyl-amino group interaction and the course of the dehydration were studied by means of proton magnetic resonance spectroscopy. A first-order reaction rate constant for the dehydration step of li = 0.023 min+ was derived from the time-dependent spectra. Use of deuterated trifluoroacetic acid made possible the study of exchange phenomena during the coIuse of the reaction. The direct observation of the steps of the cyclization reaction established the validity of conclllsions drawn by previous workers from kinetic or spectrophotometric studies of similar carbouyl-nitrogen addition reactions. TOP resldts are part,iculsrly interesting in view of the possibility of cleavage of proteins at the site of active cysteinyl residues.

In t,he course of an examination of the propert’ies of p-bromophenacyl derivat’ives of amino acids (l), it was observed that the ultraviolet spect’rum of S-(p-bromophenacyl)-L-cysteine, (I), depended strongly on

spect.ral changes is the cyclization

(1) -Br

There have been frequent reports on int,ramoleculnr interact,ions of a carbonyl function wit,h oxygen, nitrogen, sulfur, Or phosphorus properly bonded and favorabIy located. Thus, in protein chemistry, there are the observations of Edman (5), Levy (6), and Holley and Holley (7) regarding the amide carbonyl in interaction with nitrogen or with sulfur. In cyclol (I) chemistry, the amide group has been repeatedly observed in a variety of interactions lvith hydroxylic oxygen, ester carbonyl, and amino nitrogen (9). Leonard and his collaborators have elegantly investigat’ed hhe ketone rarbonylnitrogen (10, II), -sulfur (l?), and phosphorous interactions (13). Their spcckophotomc~tric studies of t’he carbonyl-nitrogen

(I) the pH of the solution. The nat,ure of the spectral changes suggested the possibility of an intramolecular interaction involving the carbonyl and amino groups. Since (I) is a &aminoketone, a likely cause of the 1 Supported in part by grants from the National Science Foundation (NSF-GB-1788) and from the National Institute of Health (GM-11805.08). While this paper was in process, A. M. Wenthe and 14. H. Cordes published their interesting work on the mechanism of acid-catalyzed hydrolysis of ket,als, ortho esters and orthocarbonat.es based on prot.on magnetic resonance spectroseop,v [J. Am. Chem. Sot. 87, 3173 (1965)]. 237 Copyright

@ 196F by Academic

I’ress

Inc.

process

(2-4).

23s

GLASKL

interwtion wrretl to strengthen our conc*lusion wnwrning the cyclization of (I). Thus, w decided t)o invest)igate the: fnt,c of (I) using troscxopy.

proton

magnetic.

resonance

spec-

Fortunately, the nmr2 spectra involved wrc Al first order and the rate of t,he dehy&xtion step Ivas slovv enough lo be followed by the time sequence m&hod. Hence this system was ideal for the direct; elucidat,ion of t,he reaction mechanism by nmr spectroscopy, a type of st,udy which, to our knowledge, has not, as yet been reported. EXPERIMEENTAL

PKOCEDURE

Methods. Vltraviolet spectra wcrc obtained with a Gary model 11, and infrared spectxa with a Perkin-Elmer model 21 recording spectrophotometer. Nuclear magnetic resonance spectra were obtained on a \-arian Associates D-4-60 spect,rometer; tetramethylsilane was used as internal reference. The field lock and sweep of this instnlmcnt permits the use of precalibrated spectral chart,s. The resolution xas adjusted to 0.3 cps. The inprobe temperature was 26 f I”, according to the ethylene glycol method of determination (14). Precision 5.mm nmr tubes were used without, degassing the samples. At “zero seconds” approximatcly 1.5 ml of solvent was added to a known amount of material in a 2-ml volumetric flask; dissolution n;as effected by suction-expulsion with a glass dropper, and the solution was brought to volume, mixed. and added to the sample t,ube. The time in seconds at which the tetramethylsilane signal appeared was taken as the t.ime of each spectral run. In all spectra t.he sweep rate was 2 cps per second. Melting points were obtained with a Rershberg (capillary) melting point apparatlts, and are reported uncorrected. Elemental analyses were performed by J. F. Alicino, Metuchen, New Jersey. Mnleriais. p-Bromoacetophenone (Distillation Products, Inc.), was reagent grade, m.p. 50-51”. Trifluoroacetic acid (Dist,illation Products, Inc.), reagent grade, was glass-distilled from PzOj. The nIlclear magnetic resonance spectrum of this solvent, before and after distillation showed a small peak at 6 = 4.17. Trifluoroacetic acid-d1 was prepared by mixing trifluoroacetic anhydride (Distillation Products, Inc., reagent grade) with D20 (99.7’;;b isotope purity) in slight excess, and glassdistilling the mixture from PzOj. This solvent also used: e Abbreviations acid; T&IS, tetramethylsilanc; netic resonance.

TFA, triflaoroacetic nmr, nIlclear mag-

ET AL. showed a small peak ill its nrtclear magtletic resoIlance spectrtim at, 6 = 4.17. S-(p-nromophenncyZ)-r,-cys~e~ne. To 150 ml of isopropyl alcohol, at 55”, 6.15 gm (0.022 mole) of p-t)romophenac~-1 1)romide (I)ist illat icjll l’rodrlcts, twice-recryst,allizetl from alcohol) was added while stirring miLglu3t ically and brd~hlil~g fritrogetl through t.he solvcn~. Addition of 3.5 gm (0.020 mole) of L-cysteine HCl hydrate (Mann Itesearch Labs.) followed and result,ed in a light. yellow clear solution to which 10 ml of aq. s NaOH was added within 1 minllte. The mixture was stirred for 15 minutes at 55”, allowed lo stand for 15 minutes at room temperature, and then poured with stirring into a mixt,ure of 350 ml of water and 200 ml of diethyl ether. The insoluble product was collected by filtration, washed with distilled water, diethyl ether, and dried in t:ac~o. A white powder (3.78 gm, 60’$$ yield) was obtained; m.p. 119-120” (decomp.). And. Calcd. for C1lCH1&NO$: C, 41%; K, 3.80; N, 4.40; S, 10.05. Found: C, 41.67; II, 3.84; N, 4.49; s, 9.97. p-Bromophenacyl ethyl sulfide. To a stirred solution of 2.8 gm (0.01 mole) of p-bromophenacyl bromide in 100 ml of isopropyl alcohol, at AO”, 1.2 gm (5.0 ml, 0.007 mole) of et,hanethiol was added under the stirface over a period of 5 minutes, followed by 10 ml of N KaOH. The reaction mixture was stirred for 1 hour at 38-40” and for 15 minutes at 55-60”; it. was then evaporated to dryness in zlacuo at 30”. The residue was extracted twice with loo-ml portions of diet.hyl ether, the extract was filtered, and the ether was evaporated. The crystalline residue was recrystallized from 10 ml of isopropyl alcohol. Yield: 2.1 gm (81’3;,) of light ycllor crystals, m.p. 35.5-37.2”. Snal. Calcd. for CloFIllBrOS: C, 46.34; II, 4.28; S, 12.37. Found: C, 46.41; H, 1.55; S, 12.02. RESULTS

Immediately upon dissolutjion of X-(pbromophenacyl)-L-cysteine (I) in trifluoroacet’ic acid or methanol, a clear pale yellow solution was obtained which developed an orange-red color upon standing. Figure 1 shows the ultraviolet absorption specatra of (I) in methanolic, aqueous acidic, and aqucous alkaline solut,ions. There is a strong absorption with a maximum in the region of 260 rnp under ayucous acidic conditions. This spect,ral feature contrasts st*rikingly with t,he absorption of (I) in methanol or in an aqueous alkaline environment. in which the 260 rnp band is absent, and is replaced by another band with a maximum in the

I

AQUEOUS

H+

2 METHANOL 3 AQUEOUS

WAVELENGTH

OH‘

(mu)

1. Ultraviolet absorption spectra of (I). Aqlleolls TIC: G.67 X loo-” BI in 0.1 N HCl. Aqueous OII-: Ci.G7 X 10-S M in 0.1 \L borate buffer, pH 7.X, 0.05 M in CuClz. ?IIrthsnol: 6.67 X 10e5 N in methanol. FIG.

I

I

,

I

I

1

I

12,600sec.

I L

7,912 sec.

4,220

sec.

3,145 set

2,230sec

780 sec.

5.0

4.0

3.0

, 2.0

226s+---d 1.0

L 0.0

PPM (6)

FIG. 2. Nmr spectra of (I) in trifluoroacetic acid: Concentration, 70 mg./ml. Refer to text and Table I for assignment of numbered peaks. (X) indicates solvent impurity present in blank. 239

240

GLASEL ET AL. I

I

8

I

I

I

I

I

1

FIG. 3. T\;mr spectra of: (A), r)-cysteine in trifluoroacetic acid, 7% solution; (B), p-bromophenacyl ethyl sulfide in trifluoroacetic acid, 7y0 solution; (C), aromatic port,ion of spectrum from (I) in trifluoroacetic acid-d1 170 seconds after dissolution; compare with the aromatic portion of (B).

region of 230 mp and a minor broad absorption between 290 and 350 n+. Figure 2 illustrates the changes in the nuclear magnetic resonance of t,he protons of (I) dissolved in TFA, at) selected times after complete dissolution. For comparison, the nmr spectra of the model compounds, p-bromophenacyl et’hyl sulfide and I,cyst,eine, in Tl:=i solution appear in Fig. 3. On the basis of t.he spcct.ra of these model compounds, the basic features of the spectra shown in Fig. 2 can be interpret’ed easily. Generally, with the exception of the group of lines at 6 = i.88, the spectra are clearly first-order spectra. Description of the protons as cy, 0, etc. will bc made with rcference to S-(p-bromophenacyl)-L-cysteinc (I). In sequence of increasing chemica1 shift, t,he spectral features are as follows: (a) Region cl/ 6 = 3.5: These absorptions are due to the mct,hylene protons (pmethylenc) of the cystcine moiety of (I) which is represented in cysteine by a

doublet of doublets (due to coupling with both alpha and sulfhydryl protons; Fig. 3). Accordingly, in the region of 6 = 3.5 we observe changes in the st’atus of the beta methylene of (I). A doublet! exist,ing at t,he beginning disappears with time, while another doublet develops at] a slightly lower field. Thus, there is an overlap of one component from each doublet, and the doublets can be identified as lines 1, 2 and 2, :J. (b) Reyion of 6 = 4.4: Inspection of the spectra of the model compounds leads to the expectation that the &methylenc protons of (I) (the methylene of the p-bromophenacyl moiety) will resonate in this region as a singlet. The a-proton of (I) is cxxpcctcd in the same region as a broad ~ripltt. Thcrefore, lines 4 and 5 in Fig. 2 arc associated wit,h the &methylcne of (I). Line 4 decreases in intensit’y, with time, and t,hcre is t’he concomitant rise of the line 5 sligh;htly downfield. Soon after dissolut,ion of (I) in TFA, it is possible to observe the broad

4 5 I; broad triplet i triplet 8

centered

4.33 4.48 4.68 5.32 7.88

nenr

ii 1,-I3rC~TI,--C-CII,~SCilri -

5

s-(211, of s-CI-I, of d.2I-T of WC11 of Aromatic n11tl (III)

(II) (III) (II) (III) system

of (II)

4.09

NHai I HS-CH,--C-COOTI -H a 7’;1 (w/v) solrltions 0 Refer to discllssion. c Numbering system

3.46

iu triflr1oroacetic indicated

acid.

011 Figs. 2 and 4

triplet absorption of the wproton of (I), close to line 4. This proton is spin coupled (kiplet) to the fl-methylene prot’ons. The broadening of its resonance is due to the coupling with the exchanging protons on the nitrogen. The cu-prot.on band disappears gradually, and simultaneously a new greatly deshielded triplet appears at 6 = 5X. (c) Region of 6 = 5.32: The new triplet emerging in this area has the same coupling consmnt as the new doublet at 6 = 3.57 (lines 2, 3), as does the old triplet 6 = 3.GS and the old doublet (lines 1, 2). The triplet developing at 6 = ~5.X2must represent the wproton in a new strongly dcshielding environment. (d) Region 01 6 = 7.88: The sharp feuturcs of the group in the region 6 = 7.SS are due to the aromatic prot,ons of (I). The nmr spectra of the model compounds pbromophenacyl ethyl sulfide and p-bromophenacyl bromide support this interpretn-

tion. The four aromatic protons give rise to an A2B2 spectrum that (‘an approximate the spectrum of an A B, :I’ H’ system, neglecting the effects of the long range couplings. In t,he course of time, the original pattern of t,hc aromatic spectrum degenerates to a triplet with an extremely intense central line. In addition to this gradual change, it should be noted and emphasized that even at the carlkst time, the para’meters of this aromatic proton resonance arc not, the same as those observed with p-bromophcnacyl et,hyl sulfide (l’ig. 15). Underlying t,he sharp lines, them is a broad absorption that is disappearing wit,h time. On the basis of the model compounds and previous work on the nmr spect,ra of amino acids in TFA (15), this broad absorption is attributed to protons on the nitrogen. Table I lists chemical shifts, assignments, and coupling constants derived from a firstorder analysis (except for t.hc A2B2 system)

GLASEL

ET AL.

3,815 set

A

3,190 set ‘/i

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0

PPM(@ FIG. 4. Nmr sprc~ra ing as in Fig. 2.

of (I) in trifiuoroacetic

of the data in Big. 2 and from spectra of model compounds in TFA solution. Dcuterated TF’A was used as a solvent of (I) to obtain the time-dependent nmr spcctra shown in Fig. 4. The spect,ra display the expected clarity specifically, sharpness of the triplets at 6 = 4.68 and 5.32 and lack of the broad background in the 6 = 7.SS region. Rather unexpectedly, though, the single peak at 6 = 4.33 is falling without the appearance of an equivalent peak, in contrast to the data in Fig. 2. Since the time dependence of t,hc nmr spectra of (I) dissolved in TFA or deuterated TVA indicated a reactlion in progress, the rate of this reaction was observed by following the fall of height of peaks 1 and 4 (Figs. 2 and 3). Figure 5 presents plots of the logarithms of t,he peaks’ intensities versus time.

acid-d,.

Same concentration

:LIICI rei’erenc-

Having observed that’ the d-methylene hydrogens of (I) are exchnngenblc in deutcrated TE’A solution (ITig. A), it was of interest to examine the exchange of the analogous methylene protons in the simpler compound, p-bromophenacyl ethyl sulfide. This exchange was clearly observed in the time-dependent nmr spectra of this model compound dissolved in dcuteratcd TFA. The rate of the exchange n-as observed as rate of disappearance of the methylene singlet at 6 = 4.09, and the kinet,ics of the react.ion are also included in Fig. 3. The numbers of hydrogen5 represented by areas under peaks in the rum spectra were determined by cutting the peaks out and weighing the excised papers. -%bsolute values for t,he number of hydrogens were obtained on the basis of the sound assumption that the system of lines in the region of

C~ltra~~ioletspcctrosropt~. ‘Phc react ion bethr cwh~~lyl ;uitI aniilw groups of (I) was first, indicated to 11sby the data from ultra\-iolct spwtroscopy appearing in l:ig. 1. The X0 1~1~absorption in aqueous acidic solution c~rrcspontls to the first primary band of bcnzcw at 230 1~1~(16, 17) associated with :t (]I,;, f-f ‘A) transition (Is) and shifted a5 cxpwtctl (16) hwausc of lcarbonyl, -l-txomo disubsl it ution of the benzene ring. This shiftrd high-intensit,y cited also as a charge absorption has hcctl trnnsfcr band (19, 20) involving an elect,ron t,ransfer hctwen the aromatic ring and the carbonyl group. Thrrcforc, a 260 mp absorption requires I he prescnc~~ of the Carbony1 group and its co-planarity nit,11 the ring. 1)-Bromopllctl:~.t:yl bromide and I)bromophenncyl ethyl sulfide both absforb at about X0 ink, t,he positions and intensit,& of the maxima hcing practically irisensitive to pH or solvent. However, the carbonyl struckwe in (I) is apparently lost in aqueous all~alinc or mcthanolic media; in&cad, a maximum appcws at about 230 mp. It. is possible to interpret the 230 111/l absorption as an c>xcit,ation of the chromophore t wc’n

EXCH/\NGE --------IO 20 30 4050 60 7080 90IO0 110 120 130 TIME

(MIN)

FIG. 5. Pemilogarithmic plot of intensity of indicated peaks (in arbitrary units; numbering refers to Figs. 2 and 4) vs. time. “Exchange” refers to intensity of exchangeable methylene peak in p-bromophenacyl ethyl sulfide dissolved in TF4-tll as a function of time. TABLE PROTON

COUNTS

SEQUEWE AMPLES

UNDER

SPECTIL.4,

system

226

TIME EX-

IK FICUIUCS 2 AXL) 4

is the same as shown

Proton cwnt -. ~. ~__

Lines 2) (Fig.

IS Nblli

~El’RESENTATIVE

A~rr;.\e

OF WHICH

Numbering these figures.

II PE.GS

1 1162

at indicated _-.__1 2560

times

in

(in secondsj

3630 / 1931

12,600

8

I 2.2 i 1.8 / 1.9 1.8 1.9 1.6 ~ 6.2 I 4.8 4.0 3.9 ~3.8 ~ 4.0

Lines (Fig Ji

j--

4 + 5

!Proton I xl 1+2+3

8

count

II

~Averape

value

~

~

4.0

at indiratcd

times

(in

seconds) 6365 -

-’ of 2 measurrments

!

= 2.11 *

10%

4.1

i / --~x-c-or-r I I on the basis of t,he spectra of aza cyclic kc tones, which undergo earbonyl-amino nitrogen transannular interact!ions (21). This would bc consistent with what is believed to be the mechanism of react’ions betwcen carbonyl and amino groups, namely that t,he first, step is an addition reaction (22). Methanol as n solvent appears t,o favor the carhonyl-amino group interaction. Although it,s polarity would be expected to stabilize the separation of charges in the form

244

I

GLASEL ET AL.

I

I

I

I

2

3

4

5

I

6

7

I

8 9 IO Wavelength (mccrons)

II

I2

13

14

15

FIG. 6. Solid state (KBr pellet) infrared spectra of: (1) X-(p-bromophenac!-l)-L-cysteine HCl, and (2) L-cysteine.

polarity cannot be the only reason for promotion of the interaction, since water, which is more polar than methanol, fails to induce the effect at acid pH. ,4 possible reason for the difference between t,he behavior in methanol and wat,er might be t,hat the ratio of uncharged to dipolar species can be 100 times higher in the former solvent (23). In TFA4 solution (see below), the interaction may be promoted by protonation of t,he carbony oxygen, a process that would increase the clcctrophilic charnct,er of t,he carbon at’om. InJra~ed and nrny’ spectroscopy. The solid state infrared spect,rum of (I) (Fig. 6), is in accord with that of a ketone in which an electronegative clement is bonded to n carbon alpha to the carbonyl. Its absorption at 1695 cm-’ is shifted with respect to the carbonyl absorption of p-bromoawtophenone (1680 cm-l) but, agrees wit’h the carbony1 frequency of p-bromophenacyl ethyl sulfide (1695 cm-l). Thus, we can conclude that, in the solid stat)e, the cnrbonyl function of (I) is intact. Evidence for a rapid alterat,ion of (I) upon dissolution in TFA is giron in the nmr spectra. Even in t#he earliest spectra (Figs. 2 and 4) the mcthylcne group at F = 4.3~3and the oc-proton in the same region have already shifted from their positions in the model compounds. More significantly, the parameters of the aromatic resonance,

6 = 7.88, have changed in comparison with t.hose of p-hromophenacyl
Br

(II)

may be the reason for the observation of only six protons in the region 6 = S a few minutes after dissolution of (I). The fast initial reaction yielding the cyclol (II) is followed by a second slower reaction as shown in Figs. 2 and 4. The second reaction results in more advanced paramagr&c shifts of the resonance of t,he high and low field methylenc protons and the ol-prot,on of (I). Since the rffcct on the wproton is enormous, one has to think of a product with a strongly deshiclding feature adjacent to the a-proton. This would he in agreement with a dehydration (22) process yielding

(III)

In form (III) the a-proton is strongly deshielded because of its allylic posit’ion with respect to the C=N bond which is conjugated with the aromatic ring. The postulated dehydration followed first-order kinetics (Fig. Z) with a reaction rate constant 16= 0.023 min--’ when either TFA or deuterated TFA is used as a solvent,. The reaction is 90% complete within 90 minutes (tlie = 30 minutes). It might be argued that there is an alternative possibility regarding the fate of (II), that in a conformer of (I) a transannulnr lnctonizat~ion may take place. This is not likely because this reaction would not result in a strongly dcshicldcd cr-proton. Parallel with the dehydration, a deukrium exchange on the &methylene of (I) was observed (Fig. 4). The rate of t,his phenomenon is not comparable with that associated with lreto-enol t.automerism in p-bromophenacyl ethyl sulfide (Fig. 5, 16= 0.042 min-‘, tljr = 10.5 minutes). The exchange in the former case can be undcrstood in terms of the tautomerism,

So \Gylic protons are observed in the nmr spectra, implying an quilibrium greatly in favor of form (III), which is required also for justification of the p:wamagrrclic~ shift of the cr-proton rwonanw. The predominant, cxistcnce of (III) is widewed by the i’wt that as tllc dehydration takw plaw, the proton count for thr pwks centcrcd at 6 = 7.U rcac+iw tlic \-aluc of 4 (aromatic: proton resonanw only). A plausible cxplarrzltion is that the I)roton on the nitrogen in the group -C=XH+-resonates now at :L much lower frequency owing IO the C==S bond c*onjugat,ctl to t.hc :trom& ring, and t.lrc charge on lhe nitrogen. WC inspwkl the spcct’ral region downfield from 6 = s without, obscwing t,hc hydrogen in question. No c~onclusion could bc drawn siIlcc the solvent absorption wntewtl nt 6 = 11.4 obscured the pict,urc. An nltcrnativc cxplanation for the failure to observe the proton signal from the group C=X+H is t,hc lower basicity of lhc nitrogen in (III), which would affect’ the rate of exchange with protons of IIK solvent to an extent not permitting a discreet signal from this proton. E~~itlcncc regarding the rapidity of hydrogen shift in the scheme (III) $ (IV) WLS obtnincd :LY follows: Compound (I) was allowed to stand in TICA solut,ion for 24 hours and then the solvent, was evaporated in ZQCUO.The residual powder presumably form (III) was dissolved in TE’12-tl, and the nmr spectrum was taken immediately (170 seconds). The spectrum displayed the ftat,ures of form (III) wit,11 the protons of 6-nict.hylenc (6 = 4.48) already cxchanged, and the fcaturcs of form (II) in lower conccntrat,ion. The latter is not surprising since, toward t,hc end of the c>liminat,ion of the TFA, some water of dehydration would have acted upon form (III) to convert it to form (I) so t,hat, upon addition of TI”A-dl, some form (II) w’:~s rapidly pro-

246

GLASEL ET AL.

duced. This reversibility, (III) H()q (I) Tz (II) was proved in an independent experiment in which water was added to a solut,ion of (I) in Tilt whic*h had been standing for 24 hours, and the mixture was evapomt.ed to dryness. -4ddition of TI;A to the residue gave a solution whose spectrum was time dependent, exactly as prcscntcd in Fig. 2. The tautomerism III $ IV was caonsisknt also with t,he proton count of the mrthylenc lines at 6 = 4.48. The number of protons was 1.8-1.9 (Table II). In addiCon, the conclusions are in accord with the obsrrved line widt,hs. The line widt,h is narrower for the Cmethylene of (II) (beginning of t,hc reaction, limited exchange) than for t.he product (III) in TFA. Exchange is known to cause broadening of the lines (24). Exposure of (I) to TE’A for 24 hours gives rise to another change which is revealed using TFA-dl. A peak starts rising from the middle of the methylcne doublet of 6 = 3.57 (Fig. 4). This is ckarly due to exchange of the a-hydrogen with deutcrium leaving the @-met.hylenc proton isolat,ed and resonsting as a singlet’. This exchange is very slow (11:2= 36 hours). Any transient forms required are apparently not favored at equilibrium, and their features, therefore, are not observable in the nmr specks. The scheme prssented for the intramolecular cyclization of S-(p-bromophenacyl)-~cysteine derived from nmr time sequence spectra thus agrees with the mechanism suggested by kinetic studies for the reaction of amines with carbonyl groups. However, in TI;‘~4, dehydration was the rate-determining step, contrary to ihe results found in aqueous acbidic solut’ions (25, 26). Finally, it is worth not,ing that t,he intramolecular cyclizat,ion of S - (p-bromophenacyl)-L-cysteine may be a useful reaction in prokin chemistry as a means of breaking a pept,ide bond involving the amino group of a cysteine residue. The feasibilit,y of this application is now being &died.

I?EFE 1: ENCES 1. I':ILI,.\NGEII, B. F.. \S~~.\~~.\~~~~, 8. M., WASSERM.lNN, xi., .\ND CoofElt, iz. (;., ~1. Bid. Chem. 240, PC3447 (1965). 2. Lrt~, A., (‘hem. Ilei~. 26, 2190 (1892). 7c GABRIEL, S., ('hew. I%. 42, 4059 (1900). 4. G.\BRIEL, S., Chest. Ber. 41, 2010 (1908). 5. E:IJM.\N, P., -lcln Chetn. Scnntl. 4, 283 (1950). 6. LEV\-, A. L., J. Chem. Sot. 404 (1950). 7. I~OLLEY, It. W., .\Kl) HOLLEY, A. I)., J. dV2. Chem. Sot. 74, 1110 (1952). 8. WRINCH, I)., :\-UiUT’e 199, 564 (1963). 9. Gr.ov~a, (2. I.. SMITH, I:. B., .\SD RAI~~P~RT, II., .J. .t rn. f’hetrr. Sot. 87, 2003 (1965). N. J., IVl
14. 15. 16. 17. 18. 10.

20.

21. 22. 23.

24.

25. 26.

JR.,

;tbslr.

Xcrtl.

Mreluq

lint.

Chem.

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