Christopher J. McGee, Gary L. Juskowiak, John Greaves, and David L. Van Vranken*

Department of Chemistry, University of California, Irvine, CA 92697 email: dlvanvra@uci.edu

SYNTHESIS, SCREENING, AND SEQUENCING OF CYSTEINE-RICH ONE-BEAD ONE-COMPOUND PEPTIDE LIBRARIES

1. Abstract 2. Cysteine-rich Peptides Are of Significant Interest

5. Synthesis of a Heavily Cysteine-Biased OBOC Library 6. Capping Cysteine Leads to Clean Cleavage Products

9. Sequencing Cysteine-rich Peptides is a Success 10. Screening: Discovery of Optimized FlAsH Binders 11. Conclusion 12. References

3. OBOC Libraries Can Complement Other Screening Methods 4. Cysteine Causes Problems In Synthesis, Screens, & Sequencing

7. Rudimentary Libraries Have Been the Limit for MS/MS 8. De Novo Interpretation of MS/MS Specta

Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. In doing so, we have also successfully sequenced the longest and most diverse OBOC derived peptides to date. These methods were used to identify new cysteine-rich motifs for binding the biarsenical FlAsH. Overall, the results enable the use of OBOC libraries in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals.

■ FlAsH-EDT2 is highly selective small molecule biarsenical probe reagent that becomes fluorescent only after binding to a tetracysteine motif ( CCXXCC ) 2

Live SH-SY5Y cells expressing α-synuclein-C4, labeled with a mixture of FlAsH and ReAsH. α-Synuclein is a component of intraneuronal protein aggregates that are characteristic of Parkinson’s disease3

■ Additionally, cysteine-rich peptides bind toxic metals and can be used in biological and phytological enviornmental remediation4

-Cys-Cys-Pro-Gly-Cys-Cys-

O OO

COO-

As AsSS SS

FlAsH-EDT2

HSHS SHSH

OAs

OOSS

As SS

Cys

Cys

Cys

CysPro Gly

--

- --

- -... ...

-

COO-

Surprisingly, after two decades of research and over 1,000 references to OBOC peptide libraries, free cysteines have been included and identified in OBOC libraries in only a handful of instances

■ Finally, free cysteine thiols can undergo side reactions during Edman degradation and during cyanogen bromide cleavage — traditional methods of direct sequencing of OBOC-derived peptides

■ Additionally, thiols will form disulfides under ambient conditions, so screening and sequencing must be performed under reducing conditions

■ Synthetic problems with cysteine-containing peptides stem from protecting groups5

H2N

S

OOH

H2N

S

OOH

NH

O H2N

S

OOHS

Requires DeprotectionWith Toxic Metals

Requires AdditionalDeprotection Step

Forms Aggregates Which Hinder Coupling

D I V I D E C O U P L E R E C O M B I N E

T r p T y r S e r A s p L e u H i s G l y

L y s P r o Cys

~6.5 million beads (5 g)

(50% of resin)

■ A cysteine-rich library (Ac-X-X-X-X-X-X-X-X-K-M-TentaGel) with potential diversity of 188 (all AA’s except Met and Ile) was synthesized on 5 g of 130 µm TentaGel

5 g = 6.5 Million Beads 300 pmol peptide/bead

Potential Diversity = 188

■ Using NMP as the solvent for all couplings during cysteine-rich library synthesis allowed for use of the preferred trityl-protected cysteine with efficient couplings

Cys-rich Library Ac-X8KM-TentaGel

(50% incorporation of Cys @ each position)

H2N K MREPEAT 7

■ LC-MS analysis of the crude products of cyanogen bromide cleavage of Ac-WDCCPGCCKM (with free cysteines) from Tentagel

■ LC-MS analysis of the crude products of CNBr cleavage of Ac-WDC*C*PGC*C*KM from Tentagel (C* = cysteine alkylated with iodoacetamide)

HN

ONH

SO

HN

O O

N HN

O O

OCNBr1N HCl H2O

HPeptide Peptide

Without Capping Cysteine

Capping Cysteine

Peptide Cleavage

Cys Cap

HNO

SH

HNO

S

NH2

O

i) DTT

IO

NH2

ii)

ONH

O (Gly)4 X X X X H

Amino Acids Used (X)

Potential Diversity Library

Members

A,F,G,H,L, P,Q,R,V, K, H, L, W, S

6,561

~ 1,000,000 ~ 24,500

32

Despite advantages such as compatibility with capped N-termini and nonribosomal amino acids, there are surprisingly few examples of MS/MS sequencing applied to peptides obtained from single beads 6

Best examples of MS/MS on

single-bead OBOC derived

peptides

2003 2005 2007

G, A, T, V, I, D, K, F, Y, W

1,000

~ 1,000

■ In no case has single-bead MS/MS analyses ever been applied to oligomers containing cysteine.

1336.7

3 letter0 0

Gly 57.02146 1279.68Ala 71.03711 1265.66Ser 87.03203 1249.67Pro 97.05276 1239.65Val 99.06841 1237.63Thr 101.04768 1235.65Leu/Ile 113.08406 1223.62Asn 114.04293 1222.66Asp 115.02694 1221.67Gln 128.05858 1208.64Lys 128.09496 1208.61Glu 129.04259 1207.66His 137.05891 1199.64Phe 147.06841 1189.63Arg 156.10111 1180.60Cys* 160.03070 1176.67Tyr 163.06333 1173.64Trp 186.07931 1150.62

y ions high to low monoisotopic

mass

[M+H]+ or charged fragment

Spectra of Ac-CYCFCRCCK-Hsl (Nanoflow LC-MS/MS)

Charged Fragments Occur When Amide Bond Cleaves

Entering the largest y-ion mass (1336.7) into a spreadsheet generates masses corresponding to all possible adjacent residues - in this case 1173.6 corresponds to

Tyrosine

y2" y3" y4" y5" y6" y7" y8" y9"Cys* Cys* Arg Cys* Phe Cys* TyrLys-Hsl Ac-Cys*

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

%

10

20

30

40

50

60

230.2 390.2 550.3 706.4 866.5 1013.5 1173.6 1336.7

b1 doubly charged molecular ion

NH

HN N

H

HN N

H

HN N

H

HN N

H

HN

S

NH2O

O

O

O

O

O

O

O

O

S

NH2O

NH

O

O

O

NH3

OS

y7" y4"y6" y5" y3"y9" y8" y2"

S

OH

S

NH2O

OH2N

NH2O

NH2

O

Cys01-09: Unambiguous sequences

Cys06: Truncated likely a problematic coupling

Cys10: Did not yield a complete set of y’’ ions

Four cleaved products were devoid of significant doubly charged molecular ion required for good MS/MS

Proteomic de novo MS/MS sequencing7 (all 20 AA’s) yields complete sequences in: ~ 30% of cases

Beads from a comparable cysteine-free library yielded complete sequences in: 6 out of 8 cases (75%)

This challenging class of cysteine-rich peptides yielded complete sequences in: 9 out 14 cases (60%)

BeadCys01 Ac-CYCFCRCCKM– Cys02 Ac-CCVCCYGYKM–Cys03 Ac-CVWKTCWCKM–Cys04 Ac-CFCCCCCCKM–Cys05 Ac-CCCACCHCKM–Cys06 Ac-DYCWCRCKM–Cys07 Ac-CCKQCFGFKM–Cys08 Ac-FLAKHGCCKM–Cys09 Ac-CLWFFKPAKM–Cys10 Ac-XXDQRCLAKM–

Sequence

Name Rel. Fluor. Sequence FB1 + + + AcCCCCKCCCKM FB2 + + + AcCCPCCCYNKM FB3 + + - FB4 + + - FB5 + + - FB6 + AcWDCCPGCCKM

■ We successfully identified sequences containing both known FlAsH tag motifs: CCPGCC & CCKXCC 8

■ CCPCCC is an novel motif that was also discovered

To identify improved tags for FlAsH, 430,000 beads from our cys-rich library were screened in Petri dishes with .005 mmol FlAsH over six hours. This smaller library was doped with 5 beads containing the optimized WDCCPGCCK sequence to ensure presence an optimized binder. Identification of the known sequence as a hit would validate our screening and sequencing protocols

Photographs of green fluorescent beads FB1 to FB6 (left to right)

We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. A diverse, heavily-biased cysteine-rich library Ac(Xxx)8LysMet (Xxx = 50% Cys ≠ Met, Ile) was synthesized on Tentagel resin with Fmoc-tritylcysteine, using NMP to avoid tritylcysteine aggregation. Conditions were developed for iodoacetamide alkylation and cyanogen bromide cleavage of single beads and the samples were analyzed by tandem mass spectrometry. Over half of the peptide samples cleaved from beads gave unambiguous sequences. Cysteine-free peptides yielded slightly more unambiguous sequences than the cysteine-rich sequences. The method was validated by synthesizing and screening a cysteine-rich library against the pro-fluorescent reagent FlAsH. Using this method, we successfully identified sequences containing two of the known short biarsenical tag motifs: CCKXCC and CCPGCC. Our synthesis and sequencing methodology expands the power of OBOC libraries to include short, cysteine-rich peptides for applications such as biarsenical binding tags, metal-chelating species, and cysteine-rich pharmaceuticals.

1.Juskowiak, G.L.; McGee, C.M.; Greaves, J.; Van Vranken, D.L.; J. Comb Chem. 2008. 10, 726–731. 2. Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269–72. 3. Roberti, M.J.; Bertoncini,C.W.; Klement, R.; Jares-Erijam, E.A.; Jovin,K.M. Nature Methods. 2007, 4, 345–351. 4. (a) Rauser, W. E. Methods Enzymol. 1991, 205, 319–333.5. (b) McIntyre, T. Adv. Biochem. Eng. Biotechnol. 2003, 78, 97–123. (c) Meagher, R. B. Curr. Opin. Plant Bio. 2000, 3, 153–162. 5. (a) Kaiser, T.; Nicholson, G. J.; Kohlbau, H. J.; Voelter, W. Tetrahedron Lett. 1996, 37, 1187–119 (b)Lukszo, J.; Patterson, D.; Albericio, F.; Kates, S. A. Lett. Peptide Sci. 1996, 3, 157–166 (c) Spain, S. M.; Rabenstein, D. L. Anal. Chem. 2003, 75, 3712–3719. 6. (a) Redman, J. E.; Wilcoxen, K. M.; Ghadiri, M. R. J. Comb. Chem. 2003, 5, 33–40. (b) Kritzer, J. A.; Luedtke, N. W.; Harker, E. A.; Schepartz, A. J. Am. Chem. Soc. 2005, 127, 14584–14585. (c) Marani, M.M.; Oliveira, E.; Cote, S.; Camperi, S.A.; Albericio,. F.; Cascone, O. Anal. Biochem. 2007, 370,

215–222. 7. Frank, A. M.; Savitski, M. M.; Nielsen, M. L.; Zubarev, R. A.; Pevzner, P. A. J. Proteome Res. 2007, 6, 114–123. 8. Chen, B.; Cao, H.; Yan, P.; Mayer, M. U.; Squier, T. C. Bioconjugate Chem. 2007, 18, 1259–1265.

GAANN Fellowship

NIH R33-CA91216

13. Acknowledgements

Typical Capabilities of Chemical and

Biological Libraries

Compatible with Heavy Metals

Facile Incorporation Of

Nonribosmal Amino Acids

Capable of Large Size and Diversity

Members Screened Against

Native Form of Target

Cell Based Libraries

Phage Display Libraries

Spatial Adress Libraries

Bead Based Libraries

■ One-bead one-compound (OBOC) peptide libraries are powerful tools to simultaneously screen millions of peptides for exceptional reactivity and selectivity. They can incorporate both ribosomal and nonribosomal amino acids and can be screened under conditions toxic to cells.

NH

HN N

HN N

H

HN N

H

HN N

H

HN

S

NH2

OO

O

O

O

O

O

O

OS

S

NH2

O

S

NH2

O

O

O

S

NH2

O

NH2

ONH2

O O H2NO

y7" y4"y6" y5" y3"y9" y8" y2"Ac-X-X-X-X-X-X-X-X-K-M-

I NH2

Oi )ii ) CNBr

Cysteine-rich peptide from a one-bead one-compound library

OH

iii ) MS/MS