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Iowa State University From the SelectedWorks of Mark S. Gordon October, 1992 Uncatalyzed Peptide Bond Formation In the Gas Phase Jan H. Jensen Kim K. Balgridge Mark S. Gordon Available at: https://works.bepress.com/mark_gordon/118/ 8340 J. Phys. Chern. 1992, 96, 834Q-8351 Uncatalyzed Peptide Bond Formation In the Gas Phase Jan H. Jensen,t Kim K. Baldridge,t and MarkS. Gordon•·t Department of Chemistry, North Dakota State University, Fargo, North Dakota 58105-5516, and San Diego Supercomputer Center, San Diego, California 92186-9784 (Received: May 26, 1992) Several levels of electronic structure theory are used to analyze the formation of a peptide bond between two glycine molecules. Both a stepwise and concerted mechanism were considered. The energetic requirements for the stepwise and concerted mechanisms are essentially the same within the expected accuracy of the methods used. A simpler model system comprised of formic acid and ammonia is found to provide a good representation of the essential features of dipeptide formation. Total electron densities and localized molecular orbitals are used to interpret the mechanisms. I. Introduction the formation of the amide bond, could not be probed due to the The peptide bond is of central importance to protein chemistry lower barrier. One way to address this problem is to consider a in particular and biological chemistry in general. It provides the related reaction, namely the hydrolysis of the amide bond, which link between amino acid subunits of proteins and imposes an mechanistically is related to amide bond formation since it is the important conformational restriction on the main chain. While reverse of ( 1) for R' = H. Thus, step 3 in reaction 1 corresponds Nature has crafted a very complicated machinery for the making to the initial hydration of the amide bond, to be followed by and breaking of peptide bonds, 1 chemists have succeeded in this complete hydrolysis through additional steps. 6 regard as well, 2 starting with Fisher's first peptide bond synthesis The hydration of amides is a special case of the general nu­ in 1903.3 Controlled hydrolysis of the peptide bond is central to cleophilic attack on carbonyl centers for which three competing 7 the field of protein sequencing, initiated by Sanger's determination mechanisms exist (2). Depending on the nucleophile and pH, of the amino acid sequences of insulin. 4 Therefore, analysis of • I the details of the mechanism leading to the formation of a peptide Nuc -c-o- + HA bond warrants investigation. Important questions to be answered in this regard are as follows: (1) Does this bond formation occur le in a concerted or stepwise manner? (2) What is the molecular and electronic structure of each transition state? (3) What are the associated barrier heights? (4) What are the effects of entropy on the details of the mechanism? (5) What is the nature of solvent " / I effects on the apparent mechanism? (6) How do enzymes and Nuc: + C =0 + H-A ; Nuc: · ·C ":":0 • ·H ··A other catalysts aid peptide bond formation/breaking? The first four of these questions will be addressed in this paper. / \ I The more general case of amide bond formation has been (2) extensively studied. The mechanism of amide bond formation 5 . I was studied experimentally by Jencks and co-workers for the Nuc: + "'-c=o·-H + A- acid/base catalyzed aminolysis of alkyl esters in aqueous solutions. / Evidence was presented to support a preference for a stepwise mechanism, based on pH-dependence studies using various esters. Q and S may either represent intermediates or mechanistically extreme representations The mechanism shown in (1) was proposed. 5° The rate-deter- of the transition state for the concerted path. In acidic solution the nucleophile is weak (Nuc: = H20) 0 OH and the mechanism is "S-Iike." For amides, this is facilitated II .H I 8 RNH2 + C-OR' R- N-C-0-R' through the delocalization of positive charge on the nitrogen I H I r• OH •oH/f I OH 1: -c --c l- -C• + 2a[HA]~ ~2a' ~[A"] [HA [ ' ,, '\\ \ NHR' •NHR' NHR' o- OH 1 .H I --;::::::::::::==~2 I R- N-C-0-R' - R-N-C-0-R' (1) Additional steps lead to complete hydrolysis8 H H I r± I T' I I • I H2o• -C-OH ; HO-C-OH; HO=C -OH + R'NH 2 - ~[8] [BH•Jf I I 2b[BH+]~ _/(8]2b' NHR' •NH2R' o- o ~ -C02H + R'NH3 • (3) I R II In basic solution the nucleophile is strong (Nuc: =OH-) and Q · R-N-C-0-R' -- 'N-C + "OR'(+H+) is regarded as an intermediate that is broken down by the following H I r / "-. steps8 mining step is the proton transfer (step 2), rather than the amine I Hz() I attack (step 1), mediated by an acid (step 2a) or base (step 2b) Ho-c-o- HO-c-o- (4) 1 ·oH I catalyst (through either r+ or 1, respectively) or the solvent. The NHR' +NH R' third step, i.e. the breakdown of the tetrahedral intermediate and 2 No experimental evidence exists for a concerted ester aminoly­ sis/amide hydrolysis mechanism. t North Dakota State University. New address: Department of Chemistry, Gilman Hall, Iowa State University, Ames, lA 50011-311. There have been several previous theoretical studies on pro­ I San Diego Supercomputer Center. totypical reactions which are intended to mimic peptide bond 0022-3654/92/2096-8340$03.00/0 © 1992 American Chemical Society Uncatalyzed Peptide Bond Formation in the Gas Phase The Journal of Physical Chemistry, Vol. 96, No. 21, 1992 8341 SCHEME 1: Stepwise Mechanism for Peptide Bond Formation II. Computational Approach v An important aspect of this study is to determine levels of H./~ ... ··? theoretical treatments of large molecules which are both efficient l \ and reliable. Therefore, several levels of theory will be discussed. :..... ~·~""OIR The molecular structures of all stationary points have been R"' \. 14 1 TSI Q-OH determined with both the semiempirical AM1 method and the minimal ST0-3G13 basis set, at the self-consistent field (SCF) level of theory. For the model system, stationary points were identified with the 6-31G(d) 11 basis set as well, at both the SCF and MP215 levels of theory. Geometry optimizations were per­ formed with the aid of analytically determined gradients and the search algorithms contained in MOPAC (version 5.0)16 (AM1; 17 18 ...................~~;.t ... t.~~L ................... A'h. the "NOMM " option was used where applicable}, GAMESS, R' 9 INT2 INTI GAUSSIAN86,19 and GAUSSIAN8820 (ab initio). The nature 'NH + R.t.oH of each SCF stationary point was established by calculating H/ R~ ?" N-C·R (analytically for ab initio wavefunctions, numerically for semi­ / I H OH empirical methods) and diagonalizing the matrix of energy second derivatives (hessian) to determine the number of imaginary fre­ SCHEME II: Concerted Mechanism for Peptide Bond Formation quencies (zero for a local minimum, one for a transition state). The MP2 and SCF geometries are sufficiently similar so that the .ov considerable computational expense of MP2 hessians was con­ II ---- : sidered unnecessary. The two mechanisms (stepwise and concerted) for the model 11 system were initially explored with the semiempirical methods. R'llt··~~~~; 1R The three transition states (TSI, TS2, and TS3; see Schemes I and II) were located and identified by following the gradient downhill in both directions using the gradient following routine implemented in MOPA C. The resulting structures were optimized and verified as minima by calculating the hessian. These geom­ etries were used as initial guesses for subsequent ab initio cal­ R' •••• .................. ~E 7 (&E 1 ror =·~~~;~~20a~~ 111 culations. 9 Several computational problems arise in the study of systems 'NH + a.t.ou R~ Y / N·C·R + H20 as complex as those in reaction 7. One is how to select the H H/ lowest-energy conformation for each structure from the many conformational isomers that exist in a system this large. Another formation, most notably the landmark series of papers by Oie et is how to efficiently optimize structures this complex. The ap­ al.9 This group91 initially studied the reaction of ammonia with proach taken in this study is the following. First we define that formic acid to form formamide and water part of the structure that glycylglycine and the model system have 0 in common as the "model system part" of each structure. Those II atoms which are directly involved in a transition state are col­ H3N + HCOOH - HCNH2 + H20 (5) lectively referred to as the "TS part." The AMI geometry for using both semiempirical and ab initio wavefunctions. Both a the model system part of the three transition states was taken from stepwise process and a concerted mechanism (Schemes I and II, the model system calculations. Then (N-)H and (C-)H were respectively; R R' H) were considered. The latter proceeds replaced with CH2COOH and CH2NH2, respectively, each ar­ = = ranged so as to most closely resemble the global minimum con­ through the four-center transition state (TS3}, while the former 21 involves the formation of the intermediate (INT2} as a result of formation of gas-phase glycine. These two parts were subse­ the addition of an ammonia N-H bond across the carboxyl double quently energy minimized while the geometry of the TS part was kept frozen. Then all geometrical parameters were relaxed and bond of the acid. It was found that at the highest level of theory 22 considered [fourth-order perturbation theory (MP410) with the the TS was optimized using the NLLSQ option in MOPAC. 6-31G(d,p) basis set11 at the 3-21G12 geometry, denoted MP4/ Again, the gradient was followed downhill in both directions 6-31G(d,p)/ /RHF/3-21G], the two mechanisms are energetically starting at each TS, the resulting structures optimized and verified competitive with each other.

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