Selective Derivation of Protein Carboxylate Esters: Development of A

Selective Derivation of Protein Carboxylate Esters: Development of a

New Detection and Quantification Method

Laura A. Gillies

Department of Chemistry, Washington State University, Pullman, WA 99164

Spring 2004 Senior Thesis

Advisor: Zhouhui Sunny Zhou ---..... Honors Thesis ************************* PASS WITH DISTINCTION

\. TO THE UNIVERSITY HONORS COLLEGE:

As thesis advisor for Lo..u (~ G,'I/ies,

I have read this paper and find it satisfactory.

~jlj#--

/vf 4it .. (.'" 2. ~, 2. 00't Date Selective Derivation of Protein Carboxylate Esters: Development of a

New Detection and Quantification Method

Laura A. Gillies

Department of Chemistry, Washington State University, Pullman, WA 99164

Spring 2004 Senior Thesis

Advisor: Zhouhui Sunny Zhou Precis

Post-translational modifications of a protein can affect the structural stability or biological activity of a protein. One such modification is the esterification of protein carboxylic acids. Formation of protein carboxylate esters is involved in protein damage repair and signaling transduction. Current methods for detecting protein carboxylate esters are cumbersome and only semi-quantitative. The ability to detect and quantify these modifications using a proteomics approach is an important step to aid in further study of these modifications. Here we show a method being developed for facile detection and precise quantification of protein carboxylate esters. In the first step of the method, esters react with hydrazine to fonn a hydrazide. We utilized several UV active model esters to optimize the conditions for this reaction. These compounds allow for quick and facile screening of reaction conditions using UV-visible spectrometry or high performance liquid chromatography. Against common notions, we found this reaction gave high yields of the hydrazide. Furthermore, under these conditions proteins tested up to 147 IlM remained stable. From the studies of model carboxylic esters, a 2nd-order kinetic dependence on hydrazine concentration was found. The stability of several randomly selected proteins in

1.0 M hydrazine was investigated; all of these proteins remained intact even after prolonged incubation up to 10 days. The second step is to detect and quantify the hydrazide intermediate. For precise detection it is important only the hydrazide formed in step one is derivatized, and not other amines in the sample. Possible derivatizing agents include UV active or fluorescent compounds, or compounds which can be easily detected by mass spectrometry. First, we tried derivatizing the hydrazide with an aldehyde tag to form a hydrazone. Several aldehyde tags were used to successfully derivatize model hydrazides.

2 Using L-Iysine we were able to show selectivity of the aldehyde tag for reaction with hydrazide. However the hydrazones formed were found to be unstable under the acidic conditions commonly used for mass spectrometry analysis of peptides. For facile detection and application for protein research it is essential our method is suitable for analysis by mass spectrometry. Therefore, as an alternative derivatizing agent we are testing a dansyl containing tag. Derivation with the dansyl tag will allow mass spectrometry analysis under acidic conditions, and has been shown selective for hydrazide. We will next use ~-delta sleep inducing peptide to further optimize the method conditions. When complete, our method will allow for facile and precise detection and quantification of protein carboxylate

ester in biological samples. Future applications include further study of protein repair processes, signaling transduction, and discovery of new biological functions of protein carboxylate esters.

3 Table of Contents

Selective Derivation of Protein Carboxylate Esters: Development of a New Detection and Quantification Method

Precis 2

List of Figures and Schemes 5

Acknowledgements 6

Abbreviations 6

Introduction 7

Materials and Methods 10

Results and Discussion 14

Conclusion 27

References 28

4 List of Figures and Schemes

Scheme 1: PIMT repair cycle 8

Scheme 2: Proposed method 9

Scheme 3: Rxn of 4-nitrophenoxy acylal with hydrazine 14

Figure 1: UV spectra of4-nitrophenoxy acylal with hydrazine 16

Figure 2: Rate is dependent on hydrazine concentration 16

Scheme 4: Rxn of Ac-Tyr-OMe with hydrazine 17

Figure 3: Ester converts to hydrazide in 82% yield 18

Scheme 5: [3-DSIP reaction 19

Figure 4: Acetic hydrazide with 4-nitrobenzaldehyde fonn hydrazone 20

Scheme 6: Rxn of4-nitrobenzaldehyde with acetic hydrazide 21

Figure 5: Hydrazone fonnation rate is pH dependent 21

Scheme 7: Rxn of Ac-Tyr-Hyd with 3-hydroxy-4-nitrobenzaldehyde 23

Figure 6: Aldehyde and hydrazone peaks co-elute 23

Scheme 8: Rxn of Lysine with aldehyde and Ac-Tyr-Hyd 24

Scheme 9: Reduction of hydrazone using pyridine borane 26

Scheme 10: Hydrazide derivatization with dansyl chloride 26

5 Acknowledgements

Many people contributed to the completion of this project through their support, feedback, and helpful discussions. I would like to thank Joshua Alfaro, Katy Ritter, Shahrzad

Mansouri, and my other lab mates for their technical support and project feedback. I am thankful for the support and encouragement provided by Ralph Yount, my family and friends. Thank you to the College of Sciences Undergraduate Mini-Grant, College of

Phannacy Summer Undergraduate Research Fellowship, and Hennan Frasch Foundation

(541-HF02 to Z.S.Z.) for their financial support.

Finally, I would like to thank my mentor Professor Sunny Zhou for his support, patience, and mentoring on this project. I have gained a priceless amount of knowledge about what it means to be a scientist, experimental methods, and technical writing. His drive and enthusiasm encourage and inspire me to do more than I often think is possible.

Abbreviations

Ac-Tyr-Hydrazide, N-acetyl-L-tyrosine hydrazide; Ac-Tyr-OMe, N-acetyl-L-tyrosine methyl ester; AdoMet, S-adenosyl-L-methionine; ~-DSIP, ~-delta sleep inducing peptide;

DMF, dimethyl fonnamide; DMSO, dimethyl sulfoxide; HEPES, N-(2 hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; HPLC, high perfonnance liquid chromatography; LuxS, S-ribosylhomocysteinease; PIMT, protein isoaspartyl methyltransferase; TFA, trifluoric acetic acid.

6 Introduction

Protein post translational modifications can affect the biological activity and structural stability of a protein. [1,2] One such modification is the esterification of protein carboxylic acid groups. For instance, aspartyl and asparginyl groups on aged proteins can deaminate, racemize, and isomerize.[3] This leads to fonnation of carboxylic acid containing D and L-isoaspartyl and D and L-isoasparginyl residues. These damaged proteins are repaired by the enzyme protein isoaspartyl methyldtransferase (PIMT, Ee

2.1.1.77). PIMT converts the damaged residue from a carboxylic acid to methyl ester through transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the damaged residue.[4,5] The newly fonned methyl ester can cyclize with the protein backbone to fonn a succinimide intennediate. Depending on which bond is broken, succinimide can hydrolyze back to the natural L-aspartyl residue. This cycle allows for repair of age damaged proteins (Scheme 1).

7 o o ~NH2 ~lOH r-""'" N~( NH""0, r-""'" N~( NH""o, H 0 H 0

L-asparagine L-aspartate ~ oY / N""""' r-"""'N ~" H 0 L-succinimide 0/ ~o ,JlNH""" ~NH""" PIMT r-""'" NAyo H AdoMet • r-""'" N~O'CH3 H 0 H 0 L-isoaspartate L-isoaspartate methyl ester

SCHEME 1: Repair cycle for age-damaged proteins. From the succinic intermediate damaged proteins can, depending on what bond is broken, return to a normal residue, or continue in the repair cycle. Formation of carboxylate ester occurs during the repair stage of the cycle.

Further study of protein carboxylate esters requires precise detection and quantification. Current methods for detection of these esters are cumbersome and often biologically incompatible.[6] One common method uses radio labeled AdoMet to transfer a radioactive methyl group to the protein carboxylic acid. Another current method for protein carboxylate ester quantification measures the methanol formed during the conversion of carboxylic acid to carboxylate ester. Other methods require lengthy and cumbersome high performance liquid chromatography (HPLC) teclmiques. Here we show a method for facile

8 detection and precise quantification of protein carboxylate esters under biologically compatible conditions (Scheme 2.)

First, as shown in Scheme 2, the reaction of hydrazine with esters results in formation of a hydrazide intennediate. This reaction has been suggested, but not reported in the literature.[7] Utilizing model compounds we optimized conditions of this reaction in aqueous hydrazine at pH 8.0. Second, the hydrazide intennediate is derivatized with a selective tag, thus allowing the original carboxylate ester to be precisely detected and quantified. Derivatizations of hydrazides with aldehydes have been previously reported. [8

lO] In addition, as an alternative derivatizing agent we will use dansyl chloride to derivatize hydrazide.

STEP 1 H ~r~CH3 + H2N-NH2 • ~r~NH2 Hydrazine Y Ester Hydrazide Acid

STEP 2 Hr§) H H o O~N /~ ~N'~ 2-a H N Hydrazone °r 'NH2 .-vvv"" 'VJVVV Hydrazide 2·b s~ o NONH 'N-S /; ~ /\ N/ H " os/; frVVVJ\ "'"""""" 0 CI-S \ r o ~ /; Dansyl Hydrazide

Dansyl Chloride

SCHEME 2: In Step I, esters react with hydrazine to fonn a hydrazide. In Step 2, hydrazide is derivatized with a selective tag. Possible tags include aldehyde compounds, resulting in fonnation of a hydrazone product (2-a), or dansyl chloride (2-b.)

9 Methods and Materials

Materials Acetyl tyrosine hydrazide and 4-nitrophenoxy acylal were synthesized by Joshua Alfaro in our laboratory. All commercially available reagents were of American Chemical Society (ACS) grade or finer and were used without further purification. N-Acetyl-L-tyrosine, hydrazine hydrate, 3-hydroxy-4-nitrobenzaldehyde, 4 nitrobenzaldehyde, 4-nitrobenzaldehyde, trifluoric acetic acid, acetic hydrazide, and pyridine-borane complex were purchased from Aldrich. N-Acetyl-L-tyrosine methyl ester was purchased from Bachem. L-Lysine, dimethyl sulfoxide, and p dimethylaminobenzaldehyde were purchased from Fischer. 2-Hydroxy-I-napthaldehyde was purchased from Fluka. Dimethyl forrnamide and HPLC-grade methanol were purchased from Baker.

General procedure for spectroscopic analysis All assays were performed on a

Cary Bio 100 UV-Vis spectrophotometer (Varian, Palo Alto, California) at 37°C maintained by a Peltier temperature controller. Under scan-kinetic mode, spectra from 240 nm to 500 nm plus the corresponding time courses at any given wavelength were collected.

Reactions were carried out in a I cm path length quartz cell in a typical total volume of 1.0 mL. For each reaction initial spectra of buffers, solvents, and starting materials were collected. Reactions were monitored until there was no further spectral change.

General procedure for HPLC HPLC analyses were performed on an Apollo C-18 reverse-phase column (4.6 mm x 250 mm, Alltech, Deerfield, Illinois) equipped with a 20

10 j..lL sample loop. Prior to each analysis the column was washed with a solution of methanol and water. Aqueous solvents were prepared in Milli-Q water. All solvents were filtered through a nylon membrane filter (pre-cut 0.45 j..lM, Alltech, Deerfield, Illinois) and degassed with helium. A flow rate of 1 mLimin was used in all analyses. Chromatograms were analyzed using Varian Star 6.0 software.

Reaction ofhydrazine and 4-nitrophenoxy acylal Analyses were performed following the general procedure for spectroscopic analysis. Final concentrations of 0.25 M,

0.50 M, 1.0 M, 1.5 M, and 2.0 M hydrazine at pH 8.0 were combined with 4-nitrophenoxy acylal (50 j..lM final concentration.) Methyl hydrazine (final concentration 100 mM) was combined with 50 j..lM 4-nitrophenoxy acylal in 100 mM, pH 8.0 N-(2 hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES.) Absorbance change at 300 nm associated with 4-nitrophenoxy acylal and absorbance change at 400 nm associated with

4-nitrophenol (I> ~ 15,760 M- 1 cm- 1 determined experimentally) were recorded continuously for each reaction.

Reaction ofN-acetyl-L-tyrosine methyl ester with hydrazine General procedures for

UV-Vis and HPLC analysis were followed. N-acetyl-L-tyrosine methyl ester (Ac-Tyr

OMe, 300 j..lM final concentration, Amax 277 nm) was added to 1.0 M and 100 mM, pH 8.0 hydrazine. Absorbance change at 277 nm associated with Ac-Tyr-OMe and absorbance change at 275 nm associated with N-acetyl-L-tyrosine hydrazide (Ac-Tyr-Hydrazide, Amax

275 nm) were continuously recorded. Samples were analyzed by HPLC at various times from 0 minutes to 5 hours, and after incubation at 37°C overnight. The column was

11 isocratically eluted with a mixture of methanol (40%) and water containing 0.1% TFA

(60%) while being monitored at 277 nm. Standard solutions of 1.0 roM each Ac-Tyr-OMe,

Ac-Tyr-Hydrazide, and N-acetyl-L-tyrosine (Ac-Tyr-Acid) final concentrations were co injected under the same conditions as reaction samples.

Protein stability in hydrazine Proteins LuxS C84E (final concentrations 3.676 /l-M and 14.08 /l-M), LuxS C84D (final concentration 36 /l-M), LuxS WT (final concentration 34

/l-M), LuxS C84A (final concentration 42.4 /l-M), LuxS E57Q (fmal concentration 147 /l-M), cytochrome P450 2E1 (final concentration 0.51 /l-M), and colicin B (final concentrations , 0.0055 /l-M and 0.0109 /l-M) were placed in 1.0 M hydrazine at pH 8.0. The proteins were incubated at room temperature in hydrazine for 10 days. Additionally, LuxS WT (l mg/mL) and lysozyme (25 mg/mL) were placed in 1.0 mL, 1.0 M hydrazine at pH 8.0 and incubated at 37°C for 48 hours. 20 /l-g each LuxS WT and lysozyme were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after incubation.

Standard solutions of 100 roM, pH 8.0 HEPES were also incubated 48 hours at 37°C and analyzed by SDS-PAGE for comparison.

Ac- Tyr-Hydrazide derivatization with aldehydes General procedure for spectroscopic and HPLC analyses were followed. Acetic acid/acetate (100 mM) was used for reactions between pH 4.0 and pH 5.0. Potassium phosphate (100 mM) was used for reactions between pH 5.0 and pH 6.0. The aldehydes p-dimethylaminobenzaldehyde, 2 hydroxy-I-napthaldehyde, 4-nitrobenzaldehyde, and 3-hydroxy-4-nitrobenzaldehyde were prepared at high concentration in DMF or DMSO, and then diluted into Milli-Q water.

12 Concentrations of aldehyde stock solutions in organic solvent were between 1.0 M and 200 mM. Final concentrations were determined by UV-Vis absorption. In all reactions DMF or

DMSO comprised no more than 5% of the total volume composition. Ac-Tyr-Hydrazide (l mM to 10 f.LM final concentration) and aldehyde (1.0 mM to 10 f.LM final concentration) were combined in buffer. Samples were analyzed by HPLC from 0 minutes to 5 hours at various times. Samples were also analyzed after incubation overnight at 37°C. Standard solutions of aldehyde and Ac-Tyr-Hydrazide were each analyzed by HPLC under the same conditions as reaction samples.

Acetic hydrazide derivatization with aldehydes Analyses followed procedure stated in Ac-Tyr-Hydrazide derivatization with aldehydes. Various concentrations of acetic hydrazide from 10 f.LM to 1.0 mM were combined with 1.0 mM to 10 f.LM final concentration aldehyde in 100 mM buffer. Reactions were run at pH 4.0 and pH 5.0 in 100

!TIM acetic acid/acetate, and at pH 6.0 in 100 mM potassium phosphate.

Reaction ofL-Lysine, Ac-Tyr-Hydrazide, and 2-hydroxy-I-napthaldehyde

Following general procedures, 2-hydroxy-l-napthaldehyde (l0 f.LM final concentration) was combined with L-lysine (l00 JlM final concentration) in 100 mM at pH

6.0 potassium phosphate. When the initial reaction was complete Ac-Tyr-Hydrazide (l00

JlM final concentration) was added to the mixture. This reaction was also run with aldehyde:lysine:hydrazide concentration ratios of 1: 1000: 100 following the same procedure. Absorbance was continually monitored through out the reaction.

13 Reduction ofhydrazone by pyridine-borane Hydrazone was first formed by mixing acetic hydrazide (200 Il-M final concentration) and 3-hydroxy-4-nitrobenzaldehyde (100 Il-M final concentration) in 100 mM, pH 5.0 acetic acid/acetate. To this two separate aliquots of pyridine-borane were added. The first aliquot of pyridine borane (831.6 Il-M final concentration) was added immediately after hydrazone fonnation was complete. The second aliquot of pyridine borane was added approximately 800 minutes after the first to a final concentration 1.40 mM. The reaction was followed using the general procedure for spectroscopic analysis.

Results and Discussion

Step One: Conversion ofester to hydrazide Previously, reactions of esters with hydrazine in aqueous solution have been suggested, but not yet reported.[7] Utilizing the chromogenic model esters 4-nitrophenoxy acylal and UV active Ac-Tyr-OMe we were able to prove the viability of reactions of ester and hydrazine in aqueous solution. The optimized conditions reported here will be used to convert protein carboxylate esters to protein hyrazides, which can then be derivatized. Our conditions allow for protein stability at 37°C and are within physiological pH range.

02N~ N o 0 H2N-NH2 ~ °2 + HO/'-OH + H2N'N~ ~O/'-O~ hydrazine ~OH H

4-nitrophenoxyacylal 4-n itrophenol methanediol acetic hydrazide

Scheme 3: 4-Nitrophenoxy acylal and hydrazine fonn acetic hydrazide, and side products 4-nitrophenol and fonnaldehyde.

14 To study the feasibility and kinetics of the reaction of esters with hydrazine in aqueous solution the model compound 4-nitrophenoxy acylal (ester) was used (Scheme 3.)

We needed a model compound with leaving group's pKa near the pKa of methanol (16.3.)

The pKa of 4-nitrophenoxy(methanol), the leaving group displaced by hydrazine during the reaction) is ~ 11.[11] The eventual formation of 4-nitrophenol from this leaving group allows both the absorbance decrease at 300 nm associated with 4-nitrophenoxy acylal, and absorbance increase at 400 nm associated with 4-nitrophenol to be monitored during the reaction (Figure 1.) This easy detection ofreaction progress by UV absorbance allows for quick screening and optimization ofthis reaction. Using this model system we detennined the reaction rate of4-nitrophenoxy acylal (ester) with hydrazine at pH 8.0. When 1.0 M hydrazine at pH 8.0 is reacted with 50 flM 4-nitrophenoxy acylal the reaction rate is 0.67 minot and was complete in less than 10 minutes. Using various hydrazine concentrations we found a 2nd-order rate dependence ofthe reaction on hydrazine concentration (Figure

2.)

15 1.0 0.811 l' ~ 0.6 en .Q « 0.4

0.2

0.0 250 300 350 400 450 500 Wavelength (nm)

Figure 1: Absorbance change associated with the reaction of 4-nitrophenoxy acylal with hydrazine. This assay contains 50 flM 4-nitrophenoxy acylal and 250 mM hydrazine at pH 8.0. Note the non-zero absorbance at 300 nm associated with initial 4-nitrophenoxy acylal absorbance.

2.5 I I i .!: -S Ql (ij n:: c o ~ 1.5 E <5 ~ (5 C Ql J::. ~ 0.5

Z --v = 064629' )("(1.7744) R'= 0.99957 .,;. o I J.,.. r I o 0.5 1 15 Hydrazlne Cone. (M)

Figure 2: 2nd -order rate dependence on hydrazine concentration for the reaction of 4 nitrophenoxy acylal and hydrazine. These reactions contain 50 flM 5-nitrophenoxy acylal each and various hydrazine concentrations.

16 We further investigated reactions of esters with hydrazine in aqueous solution using as a carboxylate ester model N-acetyl-L-tyrosine methyl ester (Ac-Tyr-OMe.) Ac-Tyr-OMe contains a methyl ester group and is thus a better model for protein carboxylate esters than the ester-like model compound 4-nitrophenoxy acylal. When reacted with hydrazine in aqueous solution Ac-Tyr-OMe is converted into the hydrazide, N-acetyl-L-tyrosine hydrazide (Ac-Tyr-Hydrazide), and N-acetyl-L-tyrosine (Ac-Tyr-Acid) as shown in Scheme

4. Each component, of this model system can be separated by HPLC and monitored by UV absorption. We found when 1.0 M hydrazine at pH 8.0 and 300 ~M Ac-Tyr-OMe react, there is a 4: I ratio of hydrazide to acid formed from the ester (Figure 3.) The half-life of this reaction is approximately 7 hours. In comparison, when 100 mM hydrazine at pH 8.0 and 300 ~M Ac-Tyr-OMe react we found a 1:4 ratio of hydrazide to acid formed from the ester, with a half-life of approximately 45 hours, or roughly six-times that of the reaction using 1.0 M hydrazine.

HO HO -:? :::::-..1 H2N-NH2 H hydrazine 1r-N~O" N'NH 1r-~ 2 o H 0 o o" N-acetyl-L-tyrosine N-acetyl-L-tyrosine methyl ester hydrazide

Scheme 4: Reaction ofN-acetyl-L-tyrosine methyl ester results in formation of N-acetyl-L tyrosine hydrazide. This reaction contained 333 llM Ac-Tyr-OMe in 1.0 M hydrazine at pH 8.0.

17 E".c ~ "

Acid

Hydrazide

25 Minutes 5.0 7.5

Figure 3: Chromatogram of reaction of Ac-Tyr-OMe with 1.0 M and 100 mM hydrazine. These reactions contain 333 mM Ac-Tyr-OMe each, and 1.0 M or 100 mM hydrazine at pH 8.0. Note the relative ratio of hydrazide and acid products formed for each reaction.

Protein stability in concentrated hydrazine has been questioned due to the corrosive

nature of hydrazine vapor and its use as a means to denature proteins. Using concentrated

hydrazine in aqueous solution we were able to demonstrate the stability of several randomly

selected proteins. Proteins tested include several concentrations of LuxS, cytochrome P450

2El, colicin B, and lysozyme. Proteins were incubated for up to 10 days at room

temperature in 1.0 M hydrazine at pH 8.0. The most concentrated protein incubated for 10

days was LuxS E57Q at 147 ).!M final concentration in hydrazine. Additionally, LuxS WT and lysozyme were incubated at 37°C for 48 hours, and then analyzed using SDS-PAGE.

We did not observe protein precipitation during the incubation periods. SDS-PAGE analysis of LuxS WT and lysozyme show a single band, indicating the protein was not denatured by hydrazine prior to analysis.

18 The reaction of esters with hydrazine in aqueous solution has shown sufficient yield and rates using simple model esters. We are now using the model peptide ~-delta sleep inducing peptide W-DSIP) to further optimize conditions in step one (Scheme 5.) ~-DSIP is a known methyltransferase accepting peptide. The isoaspartyl residue on ~-DSIP will be converted to a methyl ester using PIMT as previously described.[12] The methyl ester will then be converted to hydrazide using hydrazine and conditions optimized using model esters. HPLC and mass spectrometry will be used to detect the conversion of ~-DSIP from acid, to methyl ester, to hydrazide.

O O O ....,N~O PIMT H2N-NH2 H H ""'NTIHN ""'NFHN H HN ~~~~~'"" ( \' ""'NE + H o OH '"" o 0 '"" o NH '"" o OH AdoMe! AdoHey I I NH2

Scheme 5: ~-DSIP will be used to further optimize conditions for the conversion ofesters to hydrazides using hydrazine.

Step Two: Derivatization ofHydrazide In step two the hydrazide formed in step one is derivatized. For precise quantification the derivatizing agent must selectively react with the hydrazide only, and not other amines which may be present in the sample.

First, several aldehyde tags were tested as derivatizing agents. The reaction of aldehyde with hydrazide results in formation of a hydrazone. Derivatizations of hydrazides with aldehydes have been previously reported.[8-10] Alternatively, dansyl chloride can also be used to derivatize hydrazides.[13,14]

We tested many aldehyde tags, including p-dimethylaminobenzaldehyde, 2 hydroxy-1-napthaldehyde, 3-hydroxy-4-nitrobenzaldehyde, and 4-nitrobenzaldehyde.

19 Initial screening of the aldehydes when reacted with acetic hydrazide showed p dimethylaminobenzaldehyde yielded the slowest reaction rate. In addition, p dimethylaminobenzaldehyde was difficult to work with due to its low solubility in water.

The aldehydes 4-nitrobenzaldehyde and 3-hydroxy-4-nitrobenzaldehyde yielded the fastest reaction rates with acetic hydrazide. Figure 4 shows by HPLC the formation of hydrazone product when 4-nitrobenzaldehyde and acetic hydrazide react. Structural considerations when selecting an aldehyde for hydrazone formation have been previously reported.[15]

Our findings follow those previously reported, with benzaldehyde tags containing electron- withdrawing groups in para position relative to the aldehyde yielding the fastest reaction rate.

1200 I I I I I I 600 .6 •...•..• 'LI. ~ 1000 .. 1500 Q) "0 >. .c 800 .~ 400 Q) I "0 '< ro 6. Q. N 600 300 iii c N Q) a .0 ::J a CD .~ 400 200 c I "'1" 200 i:i ~ '=2 I J:'" 50 100 150 200 250 Minutes

Figure 4: 4-Nitrobenzaldehyde (solid line, 0) and acetic hydrazide (dashed line, L) form a hydrazone product. Note the reaction is complete in -200 minutes.

Also suggested during the reaction of aldehyde with hydrazides are pH considerations.[ 16] Jencks found an approximate ten-fold dependence of reaction rate on each pH unit decrease during the reaction of benzaldehydes with hydrazides. We similarly

20 found a non-linear dependence of reaction rate on pH during the reaction of 4 nitrobenzaldehyde (l00 J.lM final concentration) and Ac-Tyr-Hydrazide (l mM final concentration) (Scheme 6 and Figure 5.) Reactions were carried out at pH 4.0 and pH 5.0 in 100 mM acetic acid/acetate and at pH 6.0 in 100 mM potassium phosphate. We found for each pH unit decrease the reaction rate increased by approximately 10-fold (Figure 5.)

N'~~°

I~ x + H2N'N~° ~ y H Q N02 N02 4-nitrobenzaldehyde acetic hydrazide 4-nitrophenyl acetic hydrazone

Scheme 6: 4-Nitrobenzaldehyde and acetic hydrazide react to form 4-nitrophenyl acetic hydrazone.

~ 0100 .~ S C .2 ro 0.010 § 0 U. Ql 0.001 C 0 N III U > 0.000 I 3.5 4 4.5 5 5.5 6 6.5 pH

Figure 5: pH profile for 4-nitrophenyl acetic hydrazone formation. This reaction contains 100 J.lM 4-nitrobenzaldehyde and I mM Ac-Tyr-Hydrazide in 100 mM acetic acid/acetate at pH 4.0 and 5.0, and 100 mM potassium phosphate at pH 6.0.

The model hydrazide, N-acetyl-L-tyrosine hydrazide (I mM final concentration) was combined with 3-hydroxy-4-nitrobenzaldehyde (100 J.lM final concentration) in 100 mM, pH 4.0 acetic acid/acetate (Scheme 7.) During HPLC analysis of this reaction several

21 problems were encountered. Measuring UV absorbance change, the conversion of Ac-Tyr

Hydrazide to hydrazone and depletion of 3-hydroxy-4-nitrobenzaldehyde could be easily seen; however HPLC only produced the Ac-Tyr-Hydrazide and 3-hydroxy-4 nitrobenzaldehyde peaks (Figure 6.) The expected hydrazone product peak was missing.

HPLC solvent pH and percents were changed to try and fmd the hydrazone peak without success. Finally, we looked at the log of partition coefficients (IogP) for each of the expected peaks. The 10gP of 3-hydroxy-4-nitrobenzaldehyde is 1.30 and the 10gP of the hydrazone product is 1.23. With 10gP values so close to each other we thought the aldehyde and hydrazone peaks may be co-eluting. The missing hydrazone peak was found by injecting samples immediately following one another, instead of allowing a time interval between each injection. The closely eluting 3-hydroxy-4-nitrobenzaldehyde and hydrazone peaks were seen as a double peak. The reaction at pH 4.0 was so fast (half-life less than 45 minutes) that between the first and second injections most of the 3-hydroxy-4 nitrobenzaldehyde had converted to hydrazone product, which eluted nearly on top of the original aldehyde peak. We had mistaken the hydrazone product peak for the 3-hydroxy-4 nitrobenzaldehyde peak, thus missing the formation of hydrazone product.

22 HO~~I HO~~I ~OH ~'~UOH -_I ')-N N ')-N ~'NH2 + H QNO, H o 0 h- N0 o H 0 2

N-acetyl-L-tyrosine 3-hydroxy-4 N-acetyl-L-tyrosine hydrazide nitrobenzaldehyde hydrazone

Scheme 7: Ac-Tyr-Hydrazide and 3-hydroxy-4-nitrobenzaldehyde fonn the UV active hydrazone. . 3-hydroxy-4-nitrobenzladehyde

Ac-Tyr-Hydrazide

- ...---..;:; ~/':'-/----;>..L-----:;>L-~L------;7'~------:7""'~~:":""'-~:":""'----,..L //

Figure 6: Chromatograms showing separation of co-eluting aldehyde and hydrazone peaks during the reaction of3-hydroxy-4-nitrobenzaldehyde with Ac-Tyr-Hydrazide.

Using 2-hydroxy-l-napthaldehyde the selectivity of aldehyde to hydrazide was determined. 2-Hydroxy-l-napthaldehyde (l0 11M final concentration) was combined with the amine L-lysine (l00 11M final concentration) in 100 roM potassium phosphate at pH 6.0

23 (Scheme 8.) The reaction of aldehydes with amines results in Schiff base formation. We found the initial rate of Schiff base formation was greater than the rate of hydrazone formation when 2-hydroxy-I-napthaldehyde and Ac-Tyr-Hydrazide were reacted. After

Schiff base formation reached equilibrium, Ac-Tyr-Hydrazide (100 I-lM final concentration) was added to the mixture. We observed the equilibrium shift from Schiff base formation, to hydrazone formation after addition of Ac-Tyr-Hydrazide. This shift can be attributed to the higher stability of the hydrazone product versus the Schiff base product. Despite a faster initial formation rate of Schiff base, over time the dominant product present will be hydrazone. In the future, lysine may be used as a catalyst in this reaction.

o H 0 N~OH HO '-'::: '-'::: HzN~ H N 0 HO '-'::: '-'::: NHz + z,r---4I 15QI ~ ~ \-I 'oH mI ~ ~ 2-hydroxy-l-napthaldehyde L-lysine 2-hydroxy-1-napthlyene (lysine Schiff base)

HN~O~~ NH z I '-':::

~ OH

N-acetyl-L-tyrosine hydrazide

o NH 0 Hr i -\ ~N ~

2-hydroxy-1-napthlyene acetyl tyrosine hydrazone

Scheme 8: 2-Hydroxy-I-napthaldehyde will form the Schiff base with L-lysine, however with addition of Ac-Tyr-Hydrazine to the solution hydrazone product formation is favored.

24 We have used several aldehydes to derivatize model hydrazides. Using the aldehyde 2-hydroxy-I-napthaldehyde we showed aldehydes will preferentially form hydrazone product, rather than react with other amines present in solution to form a Schiff base product. These reactions were monitored by UV absorption, however for application of this derivatization technique to protein research it is essential mass spectrometry analysis can also be performed. Mass spectrometry is the preferred method for protein analysis and commonly occurs with samples in acidic conditions. After converting acetic hydrazide to hydrazone using 3-hydroxy-4-nitrobenzaldehyde, we attempted to analyze this sample using mass spectrometry. For analysis our sample was placed in 0.1% TFA solution commonly used for mass spectrometry. Unfortunately, we were unable to detect the hydrazone product using mass spectrometry, despite easy detection by UV absorption. The hydrazone was unstable under the acidic conditions needed for mass spectrometric analysis.

Several attempts were made to reduce the hydrazone-tagged product to a hydrazide-tagged product stable in acidic conditions following literature procedures (Scheme 9.)[17,18] We were unable to reduce our hydrazone product. There is some evidence that hydrazone reduction may be competing with reduction of excess aldehyde in the solution.

Consequently, derivatization of hydrazides with aldehydes is successful, but not well suited quantification by mass spectrometry. As an alternative derivatizing agent we will next try dansyl chloride (Scheme 10.) Derivatizations of hydrazides with dansyl chloride in the presence of other amines, and under acidic conditions have been previously reported.[13,14] We will modify these previous procedures to ensure derivatization is

25 selective for our hydrazide intermediate and maintains conditions suitable for mass spectrometry. )l~'N~H X + ~N/NH2° H I'" l(lOH // OH N02 acetic hydrazide N02 3-hydroxy-4-nitrobenzaldehyde (Z)-N-(3-hydroxy-4-nitrobenzylidene)acetohydrazide (hydrazone product)

pyridine-borane complex

)l~'~~H I'" // OH

N02 N -(3-hydroxy-4-n itrobenzyl)acetohydrazide (hydrazide product)

Scheme 9: Attempted reduction of hydrazone product to a hydrazide product.

\-S~ H H N ~ /; N ° I N If ~ °S-N/N ° 2 I\13 S-CI + ' ~ II H .-vvv,A 'VJVVV NH /; 0 °t ¥VVVJ'\ 'V.JVVV - 0 r Hydrazide Dansyl Chloride Dansyl Hydrazide

Scheme 10: Hydrazide is derivatized with dansyl chloride to form dansyl hydrazide product. Dansyl hydrazide will allow for precise quantification and detection using mass spectrometry, as well as spectrometry.

26 Conclusion

We have shown our methods development through the use of model compounds. The reaction of esters with hydrazine in aqueous solution was found to be kinetically dependent on hydrazine concentration. In the derivatization step we found reactions ofaldehydes with hydrazides are pH dependent. We also found an aldehyde tag is selective for hydrazine, even in the presence of other amines; however derivatizations with aldehydes do not produce a product suitable for analysis by mass spectrometry. We will try to alternatively use dansyl chloride to derivatize hydrazides. With the success of the reaction of esters with hydrazine in water we have overcome a major hurdle in our proposed method; however there is still much optimization to do before we can detect and quantify carboxylate esters on protein samples. Our next step is to continue optimizing and developing our method using model peptides. Peptides will better mimic the behavior of protein carboxylate esters, but still allow for simplified analysis. Upon completion, our method will allow for simple and precise detection and quantification of protein carboxylate esters. Further study of the roles these esters play in many important biological functions can be performed. In the future we would like to develop a method for not only detection and quantification of protein carboxylate esters, but for determination of the location on protein samples of carboxylate esters.

27 References

1. Mamula MJ, Gee RJ, Elliott JI, Sette A, Southwood S, Jones PJ, Blier PR: Isoaspartyl

post-translational modification triggers autoimmune responses to self-proteins.

Journal ofBiological Chemistly 1999,274:22321-22327.

2. Esposito L, Vitagliano L, Sica F, Sorrentino G, Zagari A, Mazzarella L: The ultrahigh

resolution crystal structure of ribonuclease A containing an isoaspartyl residue:

hydration and stereochemical analysis. Journal ofMolecular Biology 2000,297:713

732.

3. Geiger T, Clarke S: Deamidation, isomerization, and racemization at asparaginyl

and aspartyl residues in peptides. Succinimide-linked reactions the contribute to

protein degradation. Journal ofBiological Chemistry 1987, 262:785-794.

4. Clarke S: A protein carboxyl methyltransferase that recognizes and repairs age

damaged peptides and proteins and participates in their repair. In S

Adenosylmethionine-dependent Methyltransferases: Structures and Functions. Edited by

Cheng XB, R.M.: World Scientific Pub.; 1999:123-148.

5. Aswad DW, Paranandi MY, Schurter BT: Isoaspartate in peptides and proteins:

formation, significance, and analysis. Journal ofPharm. Biomed. Anal. 2000, 21: 1129

1136.

6. Fujii N, Takemoto LJ: Formation of four isomers at the Asp-lSI residue of aged

human aA-Crystallin by natural ageing. Biochemical and Biophysical Research

Communications 1999,265:746-751.

28 7. Blumenfeld 0, Gallop P: The participation of aspartyl residues in the

hydroxylamine- or hydrazine-sensitive bonds of collagen. Biochemistry 1962, 1:947

959.

8. Pino T, Cordes EH: Kinetics and mechanism for pyruvic acid semicarbazone

formation. Journal ofOrganic Chemistry 1970, 36: 1668-1670.

9. Nguyen R, Hue I: Optimizing the reversibility of hydrazone formation for dynamic

combinatorial chemistry. Chemical Communications 2003:942-943.

10. Streuli CA, Averell PR: The Analytical Chemistry ofNitrogen and its Compounds, vol

28 edn 1. Edited by Averell PR. New York: Wiley-Interscience

11. Kirby AJ, Marriott RE: Mechanism of RNA c1evage by imidazole catalysis vs

medium effects. Journal ofthe American Chemical Society 1995, 117:883-834.

12. Jolmson BA, Aswad DW: Optimal conditions for the use of protein L-isoaspartyl

methyltransferase in assessing the isoaspartate content of peptides and proteins.

Analytical Biochemistry 1991, 192:384-391.

13. Kreutzer M, Sclunidt M, Stadier E, Zeitler H-J: Purification and characterization of

animal porphobilinogen synthases. I. Bovine liver porphobilinogen synthase.

Hoppe-Seyler's Z. Physiol. Chem. 1977, 358: 1081-1091.

14. Scluner G, Kreil G: Micro method for detection of formyl and acetyl groups in

proteins. Analytical Biochemistry 1969,29: 186-192.

15. Anderson BM, Jencks WP: The effect of structure on reactivity in semicarbazone

formation. Journal ofthe American Chemical Society 1960,82: 1773-1777.

16. Jencks WP: Mechanism and catalysis of simple carbonyl group reactions. Progress

in Physical Chemistry 1964,2:63-129.

?q 17. Wong DWS, Osuga DT, Feeney RE: Pyridine borane as a reducing agent for

proteins. Analytical BiochemistlY 1984, 139:58-67.

18. CabacWlgan lC, Ahmed AI, Feeney RE: Amine boranes as alternative reducing

agents for reductive alkylatioin of proteins. Analytical Biochemistry 1982, 124:272

278.