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Ion-Spray Mass Spectrometry and High-Performance Liquid ChromatographyЧMass Spectrometry of Synthetic Peptide Libraries.

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This new reaction should be applicable to other iodoalkynes. In this context, we have found that 1-(1-cyclohexeny1)2-iodoethyne (1e) can also be dimerized by reaction with
Ipy2BF4/HBF4in CH,CI, (Scheme 3). Running the reaction
Ion-Spray Mass Spectrometry and HighPerformance Liquid Chromatography-Mass
Spectrometry of Synthetic Peptide Libraries**
By Jorg W. Metzger, Karl-Heinz Wiesrniiller, Volker Gnau,
Jente Briinjes, and Giinther Jung*
n
Dedicated to Professor Theodor Wieland
on the occasion qf his 80th birthday
Q
C
Scheme 3. Synthesis of a cross-conjugated (dieny1)enyne by the head-to-tail
coupling of an iodoenyne.
at -50°C for 24 h, and using a 2 x
M solution of
Ipy,BF4 and a 3:l ratio of iodoalkyne to Ipy,BF4 gave a
32% yield of pure 2e. Although these results are preliminary, this conversion of an iodoalkenyne suggests the feasibility of extending this reaction to other type of substrates.
Studies on the optimization and further applications are at
present in progress.
In short, a new catalytic protocol for dimerizing l-iodoalkynes has been established. The strategy is based on the
efectrophilic character of iodine in Ipy,BF,/HBF4 solutions.
The reaction affords head-to-tail dimers with no evidence for
the significant formation of other oligomers. The final products have an attractive functionality and could nicely compete with those derived from the transition metal coupling of
alkynes.[’]
Received: December 21, 1992 (Z 5766 IE]
German version: Angew. Chem. 1993. 105, 928
[I] M. J. Winter in The Chemistry of the Metal-Curbon Bond, Vol. 3 (Eds.: F R.
Hartley, S. Patai), Wiley, New York, 1985, p. 259.
[2] P. A. Chaloner, Handbook of Coordination Cutu1.vsis in Organic Chemistrj,
Butterworths, London 1986, p. 812.
[3] See for example: a ) M . E. Thompson, S. M. Baxter, A. R. Bulls, B. J.
Burger, M. C. Nolan, B. D. Santarsiero, W. P. Schaefer, J. E. Bercaw. J Am.
Chem. Soc. 1987, 109, 203-219; h) B. M. Trost, C. Chan, G. Ruhter, ihid.
1987, 109, 3486-3487; c) W. T. Boese. A. S. Goldman, Orgunomelallics
1991,10,782-786; d) H. J. Heeres, J. H. Teuhen, ibid. 1991,10,1980-1986;
e) A. D. Horton, J Chem. Soc. Chem. Commun. 1992, 185-187.
[4] a) J. Barluenga, J. M. Gonzilez, M. A. Rodriguez. P. J. Campos, G. Asensio, Synthesis 1987, 661 -662; b) A. Ricci, M. Taddei, P. Demhech. A.
Guerrini, G. Seconi, ibid. 1989, 461 -463.
[5] a) For a study on the reactivity of Ipy,BF, towards alkynes in the presence
of nucleophiles see: J. Barluenga, M. A. Rodriguez, P. J. Campos, J. Org.
Chem. 1990,55,3104-3106; h) A recent report: J. Barluenga, J. M. Gonzalez, M. A. Garcia-Martin, P. J. Campos, G. Asensio, J Chrm. SOC.Chem.
Commun. 1992, 1016-1017.
[6] J. Bartuengd, J. M. Gonzalez, P. J. Campos, G. Asensio, Angew. Chem.
1988, 100, 1604-1605, Angew. Chem. Inr. Ed. Engl. 1988,27, 1546-1547.
171 HBF, was used as a 54% ethereal solution, which is commercially available
from Merck. The acid protonates the two pyridine units, liberating “electrophilic iodine”. Nucleophilic incorporation of pyridine into the substrate
is thus avoided.
[8] Mixtures of the ketones ArCOCHI, and ArCOCH,I (Ar = aryl) are obtained. The longer the delay in performing the analysis, the higher the
proportion of the latter ketone. Among other spectroscopic data, a,%diiodo- and a-iodoketones have distinctive features in their ‘H NMR spectra. Monoiodo derivatives (CHJ) show a singlet at 6 % 4.3, the corresponding diiodo derivatives (CHI,) at 6 % 6.5.
191 Concerning the functionality in the final products, two synthetic aspects
deserve special attention. First, their potential applicability to the preparation of radialenes; see: H. Hopf, G. Mads, Angew. Chem. 1992. 104,953977; Angew. Chem. Int. Ed. Engl. 1992.31, 931 -954. Second, their usefulness in the preparation of unsaturated compounds having a high carbon
content and a low hydrogen content; see, for instance, F. Diederich, Y
Rubin, Anger+,.Chem. 1992, 104, 1123-1 145; Angen. Chem. Int. Ed. Engl.
1992.31, 1101-1123.A. M. Boldi, J. Anthony, C. B. Knobler, F. Diederich,
ihid. 1992, 104, 1270-1273 and 1992, 31. 1240-1242.
894
(4
VCH Verlugsgesellschuft mbH. W-6940 Weinheim. 1993
Synthetic peptide libraries are composed of equimolar
mixtures of immobilized or free peptides with defined length
and sequence motifs which are gaining increasing interest for
the screening for new lead structures (e.g. for antibiotics and
enzyme inhibitors) and the investigation of ligand-receptor
interactions.“] Novel analytical tools are required for optimization of the synthesis and characterization of such complex mixtures.[21In order to evaluate the potency of ionfor this purpose, we
spray (IS) mass ~pectrometry‘~]
investigated a representative peptide library of 100 synthetic
48-component mixtures of octapeptides 1 (0defined, X un-
defined positions, see Experimental Procedure) exhibiting
the murine major histocompatibility complex (MHC) class
I H2-Kb restricted sequence motif.[41In addition, a second
more complex library consisting of 24 576 nonapeptides 2
X I -x,ox,x,
2
representing the binding motif of the nonclassical murine
MHC class I complex Qa-2a[s1 was analyzed. Such synthetic
peptide mixtures resemble the isolated natural mixtures with
defined length and “anchor amino acids” in defined posit i o n ~ .-61
[ ~ These natural peptides originate from proteins
processed within the cell and are presented in the MHC
groove to cytotoxic T lymphocytes (CTL).[4*’I Virus-specific
CTL kill cells after recognition of MHC-bound foreign peptides processed, for example, from viral proteins.‘’]
Peptides 1 and 2 were synthesized by solid-phase peptide
synthesis on a multiple peptide synthesizer using Fmoc
chemistry.[’*61The peptide mixtures 1 and 2 were obtained
by treatment of the respective resins with trifluoroacetic acid
followed by precipitation from diethyl ether. The monoisotopic relative molecular masses (RMM) of the 48 peptides 1
range between 101I .6 (octapeptide LNYRFSNV) and
1099.5 (octapeptide LNYRFEKM). Only 17 different mass
values were obtained for these 48 peptides, since several peptides of the mixture are isobaric, that is, they have an identical molecular mass (20.1 u). As many as eight peptides
(LNYRFTNL, LNYRFTNI, LNYRFSKL, LNYRFSQI,
LNYRFSKI, LNYRFTQV, LNYRFTKV and LNYRFSQL) of this mixture, for example, have a RMM of 1039.6.
The IS mass spectrum of 1 shows two groups of peaks with
rnjz values that correspond to the singly protonated molecular ions [ M + HIf (m/z 1012.6-1 100.5) and doubly proton[*] Prof. Dr. G. Jung, Dr. J. W. Metzger, Dr. K.-H. Wiesmiiller, Dip1.-Chem.
V. Gnau, Dip].-Biol. J. Briinjes
Institut fur Organische Chemie der Universitat
Auf der Morgenstelle 18, D-W-7400 Tubingen (FRG)
[**I
This work was supported by the Deutsche Forschungsgemeinschaft (SFB
323, Teilprojekt C-3 Metzger and Landesforschungsschwerpunkt Teilprojekt Jung).
0570-0833/93/0606-0894 $10.00+ ,2510
Angew. Chem. Int. Ed. Engl. 1993, 32, No. 6
ated molecular ions [M + 2Hj2+ (m/z 506.8-550.8) of the
individual peptides (the region of the singly charged ions is
depicted in Fig. 1). Multiple charges result from the protona8x
1040.6
t II [min]
-
1000
two or more peptides eluting at the same time. We used this
on-line technique to confirm the presence of isobaric peptides in 1. Mixture 1 was subjected to narrow-bore reversedphase HPLC-IS mass spectrometry. After the gradient was
optimized, the 48 peptides could be partially separated (see
reconstructed total ion chromatogram; Fig. 2a). The ion
chromatogram of m/z 1040.6 corresponding to the monoisotopic [ M + HI' ions of eight different peptides shows five
major peaks and indicates that three peptides coelute with
other peptides with RMM 1039.6 (Fig. 2b). The mass spectra in the maxima of the five major peaks in the ion chromatogram of mjz 1040.6 show not only this ion but also
further ions revealing other coeluting peptides (Fig. 3). Se-
1050
mlz
1100
A
+
+
a) m h 800-1200
4 1001
b) mlz 1040.6
A 1001
0.0
lf.1
5.0
10.0
t [min] --+
15.0
Fig. 2. HPLC-IS mass spectrum of 1. a) Reconstructed total ion chromatogram
800 1200) and b) ion chrornatogram of m/r 1040.6 corresponding to the
monoisotopic [ M + HI+ ions of eight isobaric peptides of the mixture (TIC:
total ion current; XIC: extracted ion current).
(tn/:
Anxew. Clwtn. I n r .
ELI. EngI 1993, 32, No. 6
-
' g 7 7 h 1 1~ 0 9 1101
25.401
1041 1059 1083
Ml,
Fig. 1. IS mass spectrum of the 48-component peptide mixture 1 showing the
region of the singly charged quasi-molecular ions (the m / i values of the smallest
and larpest peptides of the mixture are indicated by vertical dashed lines; peaks
are labeled with the mi; value and the number isobaric peptides corresponding
to this mass)
tion of the basic centers of the peptides.". l o ] All peptides 1
contain at least two basic centers, the free N-terminus and
7
2
_
.
.
the arginyl residue at position 4. Sixteen peptides of 1 have
an additional positive charge located at the lysyl residue at
position 7. Triply charged ions IM 3HI3' of these peptides are observed between m/z 338.2 and 367.5. All m/z
values expected for the [ M HI+ ions of 1 can be found in
the mass spectrum (Fig. 1). The absence of additional peaks
in the mass spectrum is a strong hint that the mixture is free
of possible by-products such as deletion peptides (masses
lower than expected) and peptides containing noncleaved
protecting groups (masses higher than expected).
Despite possible MS-related discrimination effects (e.g.
due to the different basicity and hydrophilicity of the peptides) and the distribution of ion intensities to several charge
states, the intensity distribution of the [ M + HI' ions seems
to reflect the number of isobaric peptides (Fig. 1) in the
mixture.
Side products in peptide synthesis are easily detected by IS
mass spectrometry and high-performance liquid chromatography combined with IS (HPLC-IS) mass spectrometry." l 2 ] In contrast to HPLC-UV detection, HPLC-IS
mass spectrometry is capable of revealing the presence of
69.580
1041
.
14.5
59.596
1041
16.4
1
950
lo00
-
1050
mlz
1033
1100
Y
1150
Fig. 3. IS mass spectra obtained in the peak maxima of the major peaks of the
ion chromatogram of m / z 1040.6 (see Fig. 2 b ; I , : retention time).
quence information on single peptides of 1 was obtained by
tandem mass spectrometry (low-energy collison-induced dissociation)." 31 When other isobaric peptides were present.
on-line HPLC-MS/MS was used (data not shown).
Not only small peptide libraries can be analyzed by IS
mass spectrometry. The IS mass spectrum of the
24576 nonapeptides of 2 (Fig. 4a) is very similar to the spectrum of the 48-component mixture 1 (cf. Fig. 1). Distinct
peak groups corresponding to the singly protonated molecular ions are evident. The dashed vertical lines in the spectrum
represent the m/z values expected for [ M HIi of the
smallest (m/z977.6, AITPVIHNI and corresponding isobaric peptides) and largest peptides ( m / z 1179.5, EMNEIMHEF
and corresponding isobaric peptides) of this mixture.
The number of isobaric peptides of 2 was calculated with
a computer program and plotted versus the monoisotopic
masses calculated for {A4+ HI+ of these peptides (Fig. 4b).
This representation is very similar to the IS mass spectrum of
2 (Fig. 4a). As in the case of the 48-component mixture, the
observed intensity distribution of [ M + HI+ roughly reflects
the number of isobaric peptides (see above) and therefore
gives further information on the constituents of this mixture.
A more detailed investigation of these quantitative aspects
with defined mixtures of single purified peptides is in progress.
In summary, IS mass spectrometry alone or in combination with HPLC is a valuable tool for estimating the identity
(for example the differentiation between hexa- and octapeptide libraries), composition, and purity of small and larger
peptide mixtures. More detailed characterization of single
peptides (i.e. sequence information) is possible only with
mixtures consisting of a small number of peptides. According to our limited and preliminary experience with peptide
VCH V e r l u ~ . ~ ~ e . ~ e l mhH,
l . ~ ~ h W-6940
u~
Weinherm. 1993
+
OS70-0R33~93/060S-OS95
$ IO.00 i
.2SP
895
a)
1001
For on-line HPLC-MS an Applied Biosystems ABI 140A HPLC system and a
narrow-bore column (Nucleosil C-18, 2 x 100 mm, 5 pm; Grom, Herrenberg.
F R G ) equipped with a precolumn ( 2 x 10 mm; same filling) at a flow rate of
200 pLmin- were used. A linear gradient of 5 t o 20% solvent Bin 40 min was
used (solvent A . 0.1 % aqueous trifluoroacetic acid: solvent B: 0.1 "/o trifluoroacetic acid in acetonitrile). The flow was split so that 40 pLmin- I entered the
I S interface A solution of 500 pmol of 1 in 5 pL of water was injected.
1094
I
'c
1000
1050
mlz
1100
Conipurer progrum A program for calculation of the molecular masses of the
peptides and the number of isobaric peptides in libraries was designed. which
generated all possible permutations within the peptide sequences as defined by
the variable positions. The mass distribution was approximated by counting the
number of different peptides within one atomic mass unit. In order to assist
assignment of additional mass peaks in the IS mass spectriim corresponding to
by-products, the expected masses for uncleaved protecting groups or deletion
peptides can he calculated and displayed. too.
1150
--+
Received: September 17,1992
Revised version: January 26.1993 [Z 5723 IE]
German version: Angew. Chem. 1993. 105, 901
1000
1050
1100
1150
M Fig. 4. a) IS mass spectrum of the synthetic nonapeptide library 2 consisting of
24575 components (region of the singly charged quasi-molecular ions; vertical
dashed lines indicate calculated masses for the smallest and largest peptides of
the mixture). b) Calculated number of isobaric peptides n of 2 vs. the monoisotopic molecular mass M of [ M HI' ions.
+
libraries in bio-assays, however, the construction of huge
libraries seems to have restricted application.
Experimental Procedure
Pepride svnrhesis. The synthesis of 100 mixtures of octapeptides 1 (defined positions 0 , : R.I,L,S.A; 0,: N; 0,: Y,P; 0,: R,D,E.K,T; 0,: F,Y; undefined
positions X,: T,I,E,S; X,: N,Q,K and X,: L.M.1.V) was performed by using
premixed Fmoc-X,-Wang resins @-benzyloxybenzylalcohol-polystyrene crosslinked with 1 % divinylbenzene) and the FmoclrBu strategy. The moist resins
were divided into equivalent amounts and coupled with amino acids X,. The
Fmoc protecting groups were removed and the resin-bound amino acids
coupled with amino acids X,. The amino acids were used in threefold excess;
TBTU (O-benzotriazol-l-yl-N,N,N',N'-tetramethyluronium
tetrafluoroborate) was used for activation. Aliquots of the Finoc-X,X,X, resin mixture were
elongated to octapeptide mixtures on a multiple peptide synthesizer (SMPS 350.
Zinsser Analytic. Frankfurt, FRG) (see ref. [l]). After cleavage of the peptide
mixtures from the resins (trifluoroacetic acid/thioanisole/thiocresol190:5 : 5 ,
v:v:v) the synthetic 48-component mixture LNYRFX,X,X, was selected for
mass spectrometric analysis.
Identical procedures were used for the synthesis and cleavage of the nonapeptide library 2 (defined position 0: H: undefined positions X , : A,E,K.Q; X,:
M.I,L,Q;X,:L,I.N,T:X,:D.E.K,P;X,:V.I;X,:M.I,L.K;X,:
E,Q,N,K;X,:
L,I,F.
IS mu.ss sprctromerry: Samples of the synthetic peptide mixtures (ca. 100 pg)
were dissolved in methaiiol/l % formic acid (1 :1. 1 mL). IS mass spectra were
recorded on a triple-quadrupole mass spectrometer API Ill equipped with an
ion-spray (nebulizer-assisted electrospray) source (Sciex. Thornhill, Ontario,
Canada). The solutions were continuously infused with a medical infusion
pump (Model 22, Harvard Apparatus, South Natick. USA) at a flow rate of
5 pLmin-'. Cesium iodide was used to calibrate quadrupoles Q , and Q,. IS
mass spectra were acquired at unit resolution by scanning from mi- 800 to 1200
with a step size of 0.1 Da and a dwell time of 10 ms. Five to eight spectra were
summed The potential of the spray needle was held at +4.9 kV. Spectra containing singly. doubly. and triply charged quasi-molecular ions were recorded
at an orifice voltage of +60 V. A higher orifice voltage of 120 V was chosen
to enhance the intensity of[M + HI' and to suppress the formation of multiply
charged ions. The mass values above the (not centroided) mass peaks in the
mass spectra (Figs. 1-4) correspond to the observed peak maxima. The observed intensities for the monoisotopic [M + HI+ ions (calculated with
C = 12.000) are higher than for the isotopic "C ions. which is in agreement with
the natural isotope distribution calculated for the molecular peaks of the investigated peptides. Accuracy of mass assignment was f O . 1 u. Tandem mass
spectrometry (collision-induced dissociation) was performed with argon as collision gas.
+
896
:Ci VCH
~~riu~.s~rs~,Il.sehuJ(
mbH, W-6940 Weinhriwi. 1993
[ I ] G. Jung, A. G Beck-Sickinger, Angcir. Chem. 1992. 104,375- 391 ; Angeir.
Chm?. Inr. Ed. Eiigl. 1992, 31, 367 383.
[2] J. W. Metzger, K.-H. Wiesmiiller, S. Stevanovic. G . Jung in Peprides 1992
(Pro<. 22nd Eur. P e p . Symp. Inrerlukm, S c h w i r , 19921 (Eds : C .
Schneider, A. Eberle), Escom. Leiden, 1993.
[3] A. P. Bruins, T. R. Covey. J. D. Henion. A n d . Chem. 1987.59.2642-2646.
[41 K . Falk. 0. Rotzschke, S. Stevanovic. G . Jung. H.-G. Rammensee, Narurr
1991. 351, 290-296
[ 5 ] 0. Rotzschke. K . Falk, S. Stevanovit, 8.Grahovac, M. J. Soloski. G .
Jung. H:G. Rammensee, Nururr 1993. 361, 642-644
[6] S . Stevanovic, L H . Wiesmiiller. J. W Metrger. A. G. Beck-Sickinger, G .
Jung, Bioorg. M r d . Chern. L e u 1993, 3, 431 -436.
171 N. Zimmermaiin. 0. Rotzschke, K. Falk. D. Rognan. G. Folkers, H.-G.
Rammensee, G . Jung. Angen. Chem. 1992, 104,928-931 ; Angen,. Chem.
Int. Ed. Engl. 1992. 31. 886-890.
[8] A. Townsend, H. Bodmer. Annu. Re),. fmmunol. 1989,354, 601-624.
[9] T. R. Covey. R. F. Bonner, B. I. Shushan, J. Henion, Rupid Commun.
Muss. Spccrrom 1988, 2, 249-256.
[lo] R . D. Smith. J. A. Loo, C. G . Edmonds. C. J. Barinaga. H. R. Udseth,
And. Chem. 1990. 62.882-899.
Ill] A. Beck-Sickinger, G. Schnorrenberg, J. Metzger. G. Jung, Inr. J Pepride
Prorein Res. 1991, 38, 25-31.
[12] H. Durr, A. G. Beck-Sickinger, G . Schnorrenberg, W. Rapp. G. Jung. I n f .
J Peptide Protein Rex 1991, 38, 146-153.
[13] D. F. Hunt. 3. R. Yates. J. Shabanowitz. S. Winston, C. R. Hauer. Pror..
Nut/. Arud Sci. U S A 1986. 83. 6233-6237.
[P(MeNCH,CH,),N] as a Superior Catalyst for
the Conversion of Isocyanates to Isocyanurates
By Jian-Sheng Tang and John G. Verkade*
Triaryl isocyanurates are useful as activators for the continuous anionic polymerization and postpolymerization of
ecaprolactam to nylon-6 possessing a low unreacted
monomer content and a highly stable melt viscosity."' Recently the superior thermal properties and hydrolytic stability of isocyanurate-based foams and plastics have generated
considerable interest in the development of efficient isocyanate trimerization catalysts.['] A wide variety of catalysts
for the trimerization of aryl isocyanates to triaryl isocyanurates has been reported.", 31 Because impure triaryl isocyanurates are detrimental to the quality of nylon-6, much attention has been given to obtaining such activators in high
purity.""] However, purification often results in substantial
lowering of product yield, and attempts to increase the yield
of trimer frequently require large amounts of catalyst, ex-
3:s. Tang
Department of Chemistry, Iowa State University
Ames, IA 5001 I (USA)
Telefax: Int. code + (515)294-0105
This work was supported by the National Science Foundation
[*] Prof. J. G . Verkade.
I**]
0570-0~-~3:93/0606-0~96
3 10.00 + ,2510
Anfew. Chem. hi.Ed. Engl. 1993, 32. No. 6
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