Identification of self-assembly products fromN-phosphoamino acids by electrospray ionizationmass spectrometry

Yang Jiang1, Hua Fu1, Li Xu 2, Qiang Lu2, Jing-Zun Wang2 and Yu-Fen Zhao1*1Bioorganic Phosphorus Chemistry Laboratory, Department of Chemistry, School of Life Sciences and Engineering, TsinghuaUniversity, Beijing 100084, P. R. China2Beijing Institute of Microchemistry, Beijing 100091, P. R. China

The self-assembly products of N-(O,O'-diisopropyl)-L-a-amino acids in aqueous media,N-(O,O'-diisopropyl)phosphoryl dipeptides and tripeptides, were identified by electrospray ionization massspectrometry. A stepwise fragmentation of the [M� H]� ions from the C-terminus of N-phosphopeptideswas observed. Copyright# 2000 John Wiley & Sons, Ltd.

Received 19 May 2000; Revised 23 June 2000; Accepted 24 June 2000

N-Phospho-a-amino acids are a group of molecules withparticular properties, such as peptide formation, esterformation and ester exchange on phosphorus. It has beenproposed that they may activate themselves by forming anactive intermediate, a phosphoric-carboxylic mixed anhy-dride.1–3 The autocatalytic system ofN-phospho-a-aminoacids can, not only self-assemble into oligo-peptides, butalso phosphorylate nucleosides into nucleotides and oligo-nucleotides in aqueous media, and, for this reason, theywere proposed as a model for the co-evolution of proteinsand nucleic acids.4,5 In the reaction system ofN-phospho-a-amino acids in aqueous media, the reaction mixtures arecomplicated since many kinds of compounds are producedfrom the different reaction pathways theN-phosphoaminoacids undergo.N-Phosphodipeptides were one of theimportant products to be identified in the reaction mixtures,together with dialkyl phosphates, monoalkyl phosphates,and free amino acids. In this paper, four differentN-phosphodipeptides containing alanine and phenylalanine,and the tripeptideN-phospho-trialanine, were identified byelectrospray ionization mass spectrometry, and the stepwisefragmentations of their [M� H]� ions were studied.

EXPERIMENTAL

Preparation of samples

N-(O,O'-Diisopropyl)phosphoryl-L-a-alanine (DIPP-Ala)and N-(O,O'-diisopropyl)phosphoryl-L-a-phenylalanine(DIPP-Phe) were prepared by the method reported pre-viously.1 In the experiments involving preparation ofN-phosphoalanylalanine orN-phosphophenylalanylphenyl-alanine, 0.8 mmol of DIPP-Ala or DIPP-Phe was dissolvedin 5 mL of water and incubated at 40°C for 12 h, until the

starting material was almost consumed, as shown by31PNMR. In the experiments involvingN-phosphoalanyl-phenylalanine orN-phosphophenylalanylalanine, in addi-tion to 0.8mmol of one of theN-phosphoamino acids,0.8 mmol of another amino acid was added into the reactionsystem under the same reaction conditions. Followingreaction the mixture was freeze-dried and separated by aSephadex gel LH20 column (1� 25 cm), using methanol aseluant. The components with larger molecular weightwere eluted first, and were collected for analysis by massspectrometry.

Mass spectrometric conditions

The mass spectra were obtained using a Bruker Esquire-LCion-trap mass spectrometer. The sample dissolved inmethanol was ionized by electrospray ionization. The scanrange wasm/z50–600 in positive ion mode. The [M� H]�

ions of theN-phosphodipeptides were analyzed by multi-stage mass spectrometry.

RESULTS AND DISCUSSION

N-(O,O'-Diisopropyl)phosphoryl-a-amino acids, e.g. ala-nine, when incubated in water, can autocatalyze to formN-(O,O'-diisopropyl)phosphorylalanylalanine (DIPP-Ala2)via an intermediate consisting of a penta-coordinated

Scheme 1.Formation ofN-phosphodipeptide via an intramolecularphosphoric-carboxylic anhydride.

*Correspondence to: Y.-F. Zhao, Bioorganic Phosphorus ChemistryLaboratory, Department of Chemistry, School of Life Sciences andEngineering, Tsinghua University, Beijing 100084, P. R. China.Contract/grant sponsor: National Natural Science Foundation of China.Contract/grant sponsor: National Ministry of Science and Technologyof China.Contract/grant sponsor: National Ministry of Education of China.Contract/grant sponsor: Tsinghua University, China.

Copyright# 2000 John Wiley & Sons, Ltd.

RAPID COMMUNICATIONS IN MASS SPECTROMETRYRapid Commun. Mass Spectrom.14, 1491–1493 (2000)

phosphoric-carboxylic mixed anhydride, in which thecarbonyl group is activated towards attack by the freeamino group from an amino acid (Scheme 1).1 Smallamounts of longerN-phosphopeptides can form in this

system via the same mechanism, as the intermediate(Scheme 1) can also be attacked by a peptide. In thisexperiment the formation ofN-phosphodipeptides andtripeptides was elucidated.

Table 1. Multi-stage mass spectra of protonatedN-phosphodipeptides

Compounds Precursor ions Fragment ions and relative intensity percentage (in parentheses)

DIPP-Ala-Ala (MW = 324) 325 307(48)a, 283(100)b, 279(15), 265(30), 241(35)b, 237(6), 236(10), 223(20), 195(5)307 279(95)c, 265(12)b, 237(8), 236(100), 208(15), 194(12), 166(6), 152(2), 124(7)279 236(100)d, 208(5), 194(3), 164(2)236 208(100)a, 194(60)b, 166(15), 152(8)b, 124(3), 98(15)208 166(50)b, 124(12)b, 87(6), 81(10)283 265(70), 241(100), 237(18), 223(60), 195(16)265 237(100)237 195(100), 194(25)

DIPP-Phe-Phe (MW = 476) 477 459(100)a, 435(97)b, 431(95), 417(50), 393(30)b, 389(50), 375(28), 347(33), 312(25), 284(22)435 417(100)c, 393(40)b, 389(37), 375(25)417 389(100)c, 347(8)

DIPP-Phe-Ala (MW = 400) 401 383(70)a, 359(100)b, 355(30), 341(75), 317(68)b, 313(35),312(42), 299(80), 284(33), 271(40), 200(18)355 312(80)d, 127(100)

DIPP-Ala-Phe (MW = 400) 401 383(45)a, 359(100)b, 355(70), 341(43), 317(80)b, 313(92), 299(63), 271(77),236(32), 231(40), 208(22), 194(18)355 313(100)b, 271(40)b, 253(39),236(98)d, 219(87), 208(23), 194(26), 152(11), 120(30)

a Expulsion of H2O.b Expulsion of one or two propylene moieties.c Expulsion of CO.d Expulsion of substituted imine.

Scheme 2.Fragmentation pathway of protonatedN-(O,O'-diisopropyl)phosphoryl-dipeptide.

Rapid Commun. Mass Spectrom.14, 1491–1493 (2000) Copyright# 2000 John Wiley & Sons, Ltd.

1492 ES-MS/MS ANALYSIS OF SELF-ASSEMBLY PRODUCTS OFN-PHOSPHOAMINO ACIDS

On the basis of the multi-stage mass spectra of the ion atm/z325, interpreted as protonated DIPP-Ala2, and of the ionat m/z477, identified as protonated DIPP-Phe2 (Table 1), amechanism for the fragmentation of protonatedN-phospho-dipeptides was proposed (Scheme 2). There are two routesfor the protonatedN-phosphodipeptide to dissociate, oneinvolving the loss of small molecules from the C-terminus,and the other the loss of one or two molecules of propylenefrom the phosphoryl group. The protonated DIPP-Ala2

could thus yield ions atm/z307, 279, 236 by stepwise loss ofwater, carbon monoxide and ethylidenimine, which togethercorrespond to the loss of the alanine residue from the C-terminus. For the precursor ion and daughter ions at everystage, propylene was easily expelled from the phosphorylgroup. For example, in the spectrum of the [M� H]� ion atm/z325, the ion [M� Hÿ C3H6]

� at m/z283 was the basepeak. Protonated DIPP-Phe2 at m/z477 followed the samefragmentation pattern of stepwise expulsion of water,carbon monoxide and phenylethylidenimine from the C-terminal residue, and the loss of propylene from thephosphoryl group.

In addition to theN-phospho-homodipeptides of Phe orAla, theN-phospho-hetero-dipeptides, formed by the cross-reaction of one phosphoamino acid with another amino acid,were also studied by this method. Both DIPP-Ala-Phe, theproduct from the reaction of DIPP-Ala and phenylalanine,and DIPP-Phe-Ala, the product from the reaction of DIPP-Phe and alanine, yielded [M� H]� ions atm/z401. Whenanalyzed by multi-stage mass spectrometry, the formeryielded a series of daughter ions atm/z383, 355 and 236,which were again produced by stepwise expulsion of water,carbon monoxide and phenylethylidenimine, while the latterproduced corresponding daughter ions atm/z383, 355 and312. Hence the two isomers DIPP-Ala-Phe and DIPP-Phe-

Ala could be distinguished easily from each other by the lossof different amino acid residues from the C-terminus. Sincesmall peptides can be phosphorylated conveniently inaqueous media,6 this provides a novel method to determinethe amino acid at the C-terminus of a peptide, which mightbe applicable to peptide sequencing.

The [M� H]� ion of N-(O,O'-diisopropyl)phosphoryl-Ala-Ala-Ala (DIPP-Ala3) at m/z396, whose intensity wasmuch lower than that of protonated DIPP-Ala2, was studiedby three-stage mass spectrometry and was found to have asimilar fragmentation pattern (Scheme 3).

The N-phosphodipeptides had been studied previously byfast atom bombardment mass spectrometry (FAB-MS).7–9

Similar fragment ions, [M� Hÿ C3H6]�, [M � Hÿ

2C3H6]� and [M� Hÿ HCOOH]�, were observed, but

the fragmentation mechanisms were not studied in detail.Some aspects of the mass spectrometric fragmentation ofoligopeptides and modified peptides have been reportedrecently.10–12In these reports, the most common fragmenta-tions occurred at the amide bonds to form b� and y� ions.However, in our experiments onN-phosphopeptides, onlythe b� ions were observed, and the fragmentation proceededstepwise from the C-terminus.

CONCLUSIONS

Using ESI with multi-stage mass spectrometry, theN-phosphopeptide products from the reaction system ofN-phosphoamino acids in aqueous media could be identified.The N-phosphopeptides showed characteristic fragmenta-tion patterns, one of which involved the sequential losses ofwater, carbon monoxide and substituted imines, i.e. of thecomplete amino acid residue from the C-terminus, whichmight be useful in the sequence determination of peptidesand proteins.

Acknowledgements

The authors thank the National Natural Science Foundation of China,the National Ministry of Science and Technology of China, theNational Ministry of Education of China and Tsinghua University forfinancial support.

REFERENCES

1. Li YM, Yin YW, Zhao YF. Int. J. Peptide Protein Res.1992;39:375.

2. Zhao YF, Ju Y, Li YM, Wang Q, Yin YW, Tan B.Int. J. PeptideProtein Res.1995;45: 514.

3. Zhou WH, Ju Y, Zhao YF, Wang QG, Luo GA.Origins Life Evol.Biosphere1996;26: 547.

4. Zhao YF, Cao PS.Phosphorus, Sulfur Silicon, 1999; 144–146:757.

5. Zhao YF, Cao PS.Pure Appl. Chem.1999;71(6), 1163.6. Yin YW, Zhao YF, Chen Y, Ma Y, Xin B, Wang GH.Science

China B1994;24: 904.7. Xue CB, Yin YW, Yu X, Zhao YF.Org. Mass. Spectrom.1989;24:

253.8. Zhao YF, Zhang DQ, Xue CB, Zeng JN, Ji GJ.Org. Mass

Spectrom.1991;26: 510.9. Chai Wen-Gang, Ye Mao-Chun, Zhao Lin Yu-Fen.J. Org. Chem.

1987;52: 1617.10. Vaisar T, Urban J.J Mass Spectrom.1998;33: 505.11. McCormack AL, Somogyi A´ , DongreAR, Wysocki VH. Anal.

Chem.1993;65: 2859.12. Pfeider T, Schierhorn A, Friedemann R, Jakob M, Frank R,

Schutkowski M, Fischer G.J. Mass Spectrom.1997;32: 1064.

Scheme 3.Fragmentation pathway of protonatedN-(O,O'-diisopro-pyl)-phosphoryl-Ala-Ala-Ala.

Copyright# 2000 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom.14, 1491–1493 (2000)

ES-MS/MS ANALYSIS OF SELF-ASSEMBLY PRODUCTS OFN-PHOSPHOAMINO ACIDS 1493