July 20, 1939
COMMUNICXTIOKS TO THE EDITOR
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trast, when dimethylamine-borine is heated, the properties of trimeric dimethylaminoborine were nearly identical to those of the reported “ [(CHa)2principal product is dimethylaminoborine dimer However the preparation of trimeric dimethyl- NIaB3H4,” and the preparation process was identical aminoborine, [ (CH3)2NBH2]3, has been accom- to that described by Burg, it appears likely that ished by heating the dimer with higher boron trimeric dimethylaminoborine and “ [(CHa)pN]r&Lrides. Both pentaborane(9) and the yellow BaHo”are the same compound. The generous support of this work by the Maboron hydride solids produced by the pyrolysis of terials Laboratory, Wright Air Development diborane have been effective in this conversion. A sample of [(CH&NBH2I3, prepared by heat- Center, U.S.A.F. (Contract No. AF 33(616)ing the dimer a t 100-llOo with pentaborane(9), 5931) is gratefully acknowledged. and purified on the vacuum line,a melted a t 97.0- U. S. BORAXRESEARCH CORPORATION GEORGE W. CAMPBELL 97.8’. The molecular weight (cryoscopic in ben- ANAHEIM,CALIFORXIA VARIAN ASSOCIATES LEROYJOHNSON zene) was 165.3 (calcd. 170.7). I n two experiments the acid methanolysis (16 hours a t S5”) PALOALTO,CALIFORNIA RECEIVED APRIL11, 1959 showed 3.48 and 3.50y0 active hydrogen, or an average of 5.91 moles of hydrogen produced per mole of [ ( C H ~ ) Z N B H (calcd. ~ ] ~ 6.00). ELECTRON TRANSFER FROM THE INDOLE NUCLEUS TO THE PYRIDINE COENZYMES Anal. Calcd.: C, 42.25; H, 14.17; N, 24.62; B, 19.01. Found: C, 42.24, 42.14; H I 14.10, 14.21; Sir: N, 24.44, 24.33; B, 19.02 and 19.28. We report the formation of charge transfer The proton magnetic resonance spectrum of [(CH&NBH2]3 consisted of four signals of equal complexes among biologically active compounds intensity with spacing of approximately 2 p.p.m., and a concomitant strong support for the views of with a very strong superimposed single peak. Mulliken’ and Kosower2 on the importance of An integration showed that the intensity of the charge transfer complexes in biochemical systems. Addition of DPN3or T P N or of their model comlarge single peak was three times the sum of intensities of the four smaller peaks, corrected for pound 1-benzyl-3-carboxamide pyridinium chloride 19% B lo concentration. The interpretation was to an aqueous solution of any of the indole derivathat the large single peak was due to the C-H tives available t o us (these in Table I and yohimhydrogen, and that the N(CH& protons are bine) developed instantaneously a faint yellow color. magnetically equivalent, whereas the smaller Spectroscopic examination indicates the appearmultiplet was due to the hydrogen atoms attached ance of a new, quite difuse band as a long tail to the to B”. The relative intensities, therefore, indi- longer wave length side of the indole nucleus abcate that there are three hydrogen atoms bonded sorption. to carbon for each B-H hydrogen] which is in TABLEI agreement with the formula [ (CH&NBH2]3. This DATAFOR CHARGETRANSFER COMPLEXES OF BENZYLinterpretation was substantiated by the B1’ 3-CARBOXAMIDE PVriIDINIUM CHLORIDE WITH INDOLE AND spectrum, run a t 16.2 mc. in a field of 11,900 gauss. DERIVATIVES’ A simple triplet was observed, with a 1-2-1 AssociaMolar tiou extinction intensity ratio, which strongly indicates that all constant coefficient boron atoms are magnetically equivalent and that 1. mol.-’ e mp each has two covalently bonded hydrogen atoms. Indole Water 2.5 540 370 The dimethylaminoborine trimer has a camphor- L-Tryptophan Water 2.2 860 370 like odor, and is quite unreactive in moist air and Glycyl-L-tryptophan Water 2.9 500 400 even when dissolved in wet acetone it does not Indole-3-acetic acid 1.7 X M hydrolyze measurably a t room temperature. I n phosphate this respect i t is quite comparable to the methylPH 6.7 4.1 1220 370 aminoborine trimer.’ In view of the nuclear Serotoninb PH 6.6 1 . 8 1410 380 magnetic resonance analysis, it appears that this Acetyltryptophan pH 6.5 4.0 510 400 compound has a cyclic structure, Comparable to As creatinine sulfate. Room temperature (25 =k 2’). the phosphinoborine trimer^.^ Under comparable conditions, no other amino It was reported by Burg6 that the reaction of pentaborane(9) with dimethylaminoborine (present acid is able to replace tryptophan in this kind of in excess) produced the compound “ [ ( C H S ) ~ N ] ~interaction. Chymotrypsinogen after preincubation with B3H4.” No compound of this composition was obtained from this system. Since the physical urea for a few hours also develops the charge transfer band by addition of DPN or its model, (2) A. B Burg and C. L. Randolph, TEIS JOURNAL, 73, 953 (1951). even at low PH’s where reactivity of other amino (3) The last traces of [(CHr)zN]2B&s were removed from the trimeric dimethylaminoborine by slow distillation at 0 to 5’. The infraacids is out of q ~ e s t i o n . ~ red spectrum of [(CHd)nS]zB4Heshowed strong absorption at 4 08 #, Application of the equation of Foster,6 et al., with a shoulder at 3 0 p , whereas [(CHa)zNBHz)a had low absorption Z ,
at these wave lengths, but absorbed strongly at 4.14, 4.25 and 4.42 p. Other absorption bands for [(CHs)zSIz13rH1,which were useful in detecting its presence in trimeric dimethylaminoborine, were found a l 8 65 p and 10.35 p. The complete absence of absorption at these wave lengths was prerequisite to further work with trimeric dime thy lam in^ borine. (41 W C Hamilton, Acta Cvysl , 8 , 199 (1955). ( 5 ) A . B. Burg. THISJOURNAL, 79, 2129 (1957).
(1) R. S. Mulliken, THISJOURNAL, 74, 811 (1952). (2) E. M Kosower, {bid., 78, 3497 (1956). (3) These abbreviations are used: DPN, diphosphopyridine nucleotide; TPN, triphosphopyridine nucleotide; GPD, glycerddehyde phosphate dehydrogenase; APDPN, the acetyl analogue of DPN. (4) See J. van Eys, J . Biol. Chem., 983, 1203 (1958) ( 5 ) R,Foster, D. L1. Hammick and A. A. Wardley J Chdm. SOC.. 3817: 19.53,
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COMMUNICATIONS TO
indicates that the complexes are formed in a 1:1 ratio. Values of the association constant, K , and of the molar extinction coefficient e are reported in Table I. The spectra of these change transfer complexes are similar in both contour and intensity to the spectrum of GPD in presence of DPN.6-9 This suggests that in the GPD-DPN complex the pyridinium moiety of the coenzyme molecule interacts with indole side chains of the enzymelo; this interaction should almost certainly occur in the case of GPD-APDPN complex, since iodoacetate only slightly reduces the absorption in the 360 mp r e g i ~ n . ~ Complexing said to be associated with electron transfer from the indole nucleus to flavines and pteridines has been reported recently.I1 Acknowledgment.--We are indebted to the Rockefeller Foundation and to the Brazilian “Conselho Nacional de Pesquisas” for grants, and to the latter also for a fellowship granted to P. G. (6) E. Racker a n d I. K. Krimsky, J . B i d . Chem., 198, 731 (1952). (7) S. F. Velick, ibid., 203, 563 (1953); in “Mechanism of Enzyme Action,” W. D. McElroy and B . Glass, eds., T h e John Hopkins Press, Baltimore, hfd., 1954, p. 500-505. (8) J. B. Fox, Jr., and W. B. Dandliker, J . Biol. Chem., 221, 1005 (1956). (9) N. 0. Kaplan, M. M. Ciotti and F. E. Stolzenbach, Archiu. Biochem. B i o g h y s . , 69, 441 (19573. (10) It is true t h a t -SI3 reagents abolish t h e 305 m).i interaction in the D P D - D P N complex (ref, 6-8 and also B. Chance in ”Mechanism of Enzyme Action,” W. D . McElroy and B. Glass, eds., T h e Johns Hopkins Press, Baltimore, Md., 1954, p. 434-438), b u t as pointed o u t by Chance and by Velick (ref. 7) this does not necessarily indicate a direct binding of coenzyme t o sulfur. (11) H . A. Harbury and K. X.Foley, Pvoc. S n l l . Acnd. S c i . , 4 4 , 662 (1958); I. Isenberg and A. Szent-Gyorgl-i, i b i d . , 44, 857 (1958); E. Fujimori, i b i d . , 45, 133 (1959); R . Pullman and A. Pullman, ibid.,44, 1197 (1958); 45, 136 (1959).
THE
EDITOR
Vol. 81
S
/ \
c=o
II?C
I
H r C-CH
+ H2S-protc.iri -+
1
I
~HCOCH~ HS-CHaCH&HCO--NH--protein
AHCOCH~
(2)
Although direct reaction is not especially useful, with Agf as adjuvant3 thiolation of gelatins has been effected rapidly a t p H 7.5. However, the presence of silver, and subsequently 1 M thiourea for its removal, presents problems particularly with disulfide-containing proteins. These problems are avoided by a suitable extension of the reaction of acid anhydrides with proteins6 For example with S-acetylmercaptosuccinic anhydride 0
CH ,CO-S-C€I-C/
CH?CO-S-CIICO-SH-prot
ein
I
(IT)
+
CHjCOOH ~~S-C€€CO-~I~-I)r~)teiii (3) (111) &H.COOH
Solid anhydride6 is added to protein solution at, for example, fiH 7, over from 0.25-1 hour, depending on the amount of reagent. The p H is maintained by adding sodium hydroxide. Air is excluded throughout with nitrogen. (Coupling can be performed over a range of p H and temperature.) Hydrolyzed anhydride (I) is removed with an anion exchanger, salts with a mixed-bed exchanger DEPARTMENT OF CHEMISTRY or by dialysis. The mercaptosuccinylated proDE FILOSOFIA, CIENCIAS E LETRAS FACULDADE DE SXo PACLO GIUSEPPECILENTO tein is isolated by lyophilization. UNIVERSIDADE S.Xo PAULO,BRASIL PAOLAGICSTI Typical results at room temperature are sum RECEIVED ?IIIAY (5, 1959 inarized in Table I. TABLE I MERCAPTOSUCCINYL PROTEINS
A NEW METHOD FOR THE INTRODUCTION OF THIOL GROUPS INTO PROTEINS
hloles (I)
Iaaea _____
Sir: For fundamental and technical reasons many attempts have been made to introduce sulfur de novo into proteins. Using thioglycolides, Schoberl’ prepared highly thiolated casein and ovalbumin (-sCHYCO-sCH2CO-),
+ HzN>protein H2N I-IS-CHzCO-NH HS-CH3CO-NH
-+-
)protein
(1)
Since Schoberl’s reagent is difficult to characterize, the recent method of Benesch and Benesch2J using N-acetylhomocysteine thiolactone is more attractive (1) A. Schoberl, Atzgew, Chem., 60, 7 (1948). (2) R . Benesch and R. E . Benesch, TITISJ O U R N A L , 78, 1597 (l!X6). (3) R. Benesch and R. E . Benesch, P i q c .Vall, A r a d . Sci.. 44, 848
(1958).
Protein
per 106 grams of protein
Gelatin Gelatin Gelatin Bovine serum albumin Bovine serum albumin Ribonuclease
30 120 360 45 360 140
Moles introduced per 10s grams of protein Acetyl\fermer~... captocaptop H of succinyl succinyl reactlon (111) (11) ~~~
7
6
8 8
12
8 8 7
14 5 8 2
12 17 23 21
54 6
A reaction analogous to ( 3 ) giving mercaptosuccinylated esters has been achieved with polyhydroxylic molecules (e.g., dextran, polyvinyl alcohol). (4) It has been successfully applied a t high $11 (10.7) b y S. J . Singer, J. E . Fothergill and J. R. Shainoff, THISJ O U R N A L , 81, 2277 (1969). (5) H. Frankel-Conrat, R . S . Bean and H. Lineweaver, J . Biol. Chem., 177, 385 (1949); P . H. Maurer and H . I,ebovitz, J . Immuizol., 76, 335 (1956); A. F. S. A. Habeeb, H . G. Cassidy and S. J. Singer, Biochim. Biophys. Acta, 29, 687 (1958). (6) B. Holmberg and E. SchjBnberg, Arkiv Kemi Mineual. Geol., 1 4 8 , No. 7 (1940).
