elucidation of the presence and location of

Elucidation of the Presence and Locationof t-Boc Protecting Groups in Aminesand Dipeptides Using On-ColumnH/D Exchange HPLC/ESI/MS

Christian Wolf and Cristina N. VillalobosChemistry Department, Georgetown University, Washington, District of Columbia, USA

Paul G. Cummings, Sonya Kennedy-Gabb, Mark A. Olsen,and Gudrun TrescherChemical Development, GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania, USA

High performance liquid chromatography/mass spectrometry (HPLC/MS) has become awidely used technique for routine analysis of pharmaceutical compounds. The constant searchfor new drugs requires the development of time-efficient methods that can be employed inhigh-throughput screening of combinatorial libraries of a variety of compounds, includingamines and peptides. Conventional HPLC/MS is a powerful technique that can easily beautomated and is suitable for comprehensive screening purposes. However, the unequivocaldetermination of the presence and location of important carbamoyl protecting groups ofamines is often elusive because of their inherent instability under MS conditions. In this study,the use of on-column H/D exchange HPLC/ESI/MS for structure elucidation of t-Bocprotecting groups which can often not be detected by MS because of facile McLaffertyrearrangement has been examined. We demonstrate that employing a deuterated mobile phasein HPLC/MS analysis provides a convenient tool for the determination of the absence orpresence of t-Boc protecting groups in amines and peptides. (J Am Soc Mass Spectrom 2005,16, 553–564) © 2005 American Society for Mass Spectrometry

Afast and unequivocal identification of the pres-ence and location of t-Boc protecting groupsplays an essential role in routine analysis and

high-throughput screening of combinatorial libraries ofcomplex amines or peptides. The widespread use of thet-Boc protecting group in organic synthesis and inparticular in peptide chemistry requires a reliablemethod for the fast determination of its absence orpresence in amines and amino acids. AlthoughHPLC/MS is a very powerful technique for the deter-mination of the structure of organic compounds, infor-mation about the labile t-Boc functionality is oftenelusive because of the fast McLafferty rearrangement ofthe tert-butyl carbamoyl group. For example, t-Bocprotected amino alcohol 1 readily undergoes McLaf-ferty rearrangement under standard MS conditions toform fragment 2 exhibiting m/z 152, Scheme 1 [1]. Thisfragmentation pathway may proceed via a labile inter-mediate carbamoyl acid intermediate 3, which easilyeliminates carbon dioxide and is therefore often not

observed. Because the unprotected amino alcohol 4affords fragment 2 after ionization in the MS detector,mass spectrometry does not provide a means to differen-tiate between 1 and 4, i.e., the determination of thepresence of the t-Boc group requires isolation and furtherspectroscopic analysis or the use of reference samples fora time-consuming comparison of HPLC retention timeswhich is unsuitable for high-throughput screening.Structure elucidation by mass spectrometry has be-

come increasingly successful through the introductionof chemical ionization with ND3, D2O, and CD3OD forexchange of hydrogen for deuterium of organic com-pounds in the gas phase [2]. This technique can beapplied to H/D exchange MS analysis of various classesof compounds including alcohols, carboxylic acids,amines, amides, and thiols [3]. It has also been shownthat hydrogens bonded to carbon atoms in aromaticcompounds can be replaced by deuterium in the gasphase [4]. The coupling of HPLC and mass spectrome-try combines a powerful separation technique with thesensitive and informative MS detection mode, whichopens a new venue for H/D exchange MS analysis ofcomplex mixtures containing polar compounds. Todate, various MS techniques including chemical ioniza-tion detection mass spectrometry (CI/MS) [5], fast atom

Published online February 23, 2005Address reprint requests to Professor C. Wolf, Department of Chemistry,Georgetown University, 37th and O Streets, Washington, DC 20057, USA.E-mail: [email protected]

© 2005 American Society for Mass Spectrometry. Published by Elsevier Inc. Received November 25, 20041044-0305/05/$30.00 Revised January 14, 2005doi:10.1016/j.jasms.2005.01.008 Accepted January 14, 2005

bombardment mass spectrometry (FAB/MS) [6], liquidsecondary ion mass spectrometry (LSI/MS) [7], electro-spray mass spectrometry (ESI/MS) [8], and thermos-pray mass spectrometry (TSP/MS) [9] utilizing a deu-terated mobile phase or matrix have been reported.Deuterated mobile phase HPLC has also successfullybeen combined with NMR [10] and IR spectroscopy[11].We report herein the use of deuterated mobile phase

HPLC/MS for the fast elucidation of the absence orpresence of t-Boc protection groups in amines andpeptides. Employing on-line H/D exchange HPLC/ESI/MS in the analysis of aminopyridines and 16dipeptides, both t-Boc protected and unprotected, wefound that the presence and position of this labileprotecting group as well as the amino sequence canreadily be identified.

Experimental

All dipeptides studied herein were synthesized fromcommercially available t-Boc protected amino acids and

amino acid methyl esters using PyBop (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophos-phate) as the coupling agent [12]. Hydrolysis undermild acidic conditions allowed selective cleavage of thet-Boc group to afford the corresponding unprotecteddipeptides. All samples were analyzed using an Agilent(Palo Alto, CA) or HP/Bruker (Billerica, MA) ion trapmass spectrometer equipped with Agilent 1100 HPLCsystems. The replacement of a nondeuterated with adeuterated mobile phase can cause minor peak shifts. Itshould be noted that differences in elution times ex-ceeding 0.1 min. observed with some of the samplesstudied are not a consequence of employing a deuter-ated mobile phase. In these cases, the sample wasanalyzed on two instruments with different voids, i.e.,the change in retention time is not inherent to the HPLCmethod used but due to different instrumental setups.The samples were evaluated by flow injection analysis(FIA) and HPLC/ESI/MS. The MS data were collectedusing in-source CID (collisionally-induced dissocia-tion), which may be enhanced by varying the potentialbetween the skimmer and the capillary exit to increase

NN OH

O

O

NHN OD

m/z=155

NN OD

O

HO

1

Ionization and McLafferty rearrangement

NHN OH NDN OD

m/z=156

Ionization

4

d-3 d-2

d2-2

NN OD

O

Od-1

NDN ODd2-4

deuterated mobilephase HPLC

deuterated mobilephase HPLC

+ D + D

+ D

CO2

Scheme 2. McLafferty rearrangement of 1 and ionization of 4 using deuterated mobile phaseHPLC/MS.

NN OH

O

O NHN OH

m/z=152

H

NN OH

O

HO

O C O

NN OH

O

O

H

NHN OH

m/z=152

O C O

1

1

McLafferty

McLafferty

NHN OH NHN OH

m/z=152

Ionization

4

3 2

2

2

Scheme 1. McLafferty rearrangement of 1 and ionization of 4 under MS conditions.

554 WOLF ET AL. J Am Soc Mass Spectrom 2005, 16, 553–564

or decrease the level of fragmentation. The scan rangewas 50–850 Da. The capillary exit voltage was set at4000 V and the fragmentor voltage was 80 V. Thedrying gas temperature was 350 °C and the flow was 12L/min. Nebulizer pressure was set at 60 psi. HPLCseparation of dipeptides 5–20 was performed using aWaters YMC ODS AM column (4.6 � 250 mm, 5 mm)and a standard linear gradient over 40 min startingfrom 95%/5%/0.1% to 5%/95%/0.1% water/acetoni-trile/TFA at a flow rate of 1 mL/min at ambienttemperature. The deuterium exchange experimentswere performed using identical conditions replacingwater and TFA with deuterium oxide and d-TFA,respectively,°as°has°previously°been°described°[7].

Results and Discussion

We assumed that on-line H/D exchange HPLC/ESI/MS would allow differentiation between analytes 1and 4 and derivatives thereof. Employing a deuteratedmobile phase in HPLC should afford complete deute-rium exchange for molecules containing unprotectedamino functions such as 4, whereas protected aminessuch as 1 can not undergo proton/deuterium exchange,Scheme 2. During fragmentation amines carrying at-Boc group will undergo a McLafferty rearrangementtransferring a hydrogen from a methyl group of thet-Boc moiety to the amino function or to the carbonyloxygen forming an intermediate carbamoyl acid. Theprotection of 1 thus prevents incorporation of deute-rium into the amino group during chromatography. Asa result, using a deuterated mobile phase in LC/MSanalysis affords distinguishable MS fragments d-2 andd2-2 corresponding to 1 and 4, respectively.Our on-column H/D exchange HPLC/ESI/MS strat-

egy was first utilized for the analysis of a three compo-nent°mixture°containing°unknown°aminopyridines,°Fig-ure°1.°Comparison°of°the°mass°spectra°obtained°by°MSfollowing nondeuterated and deuterated mobile phaseHPLC gave invaluable information for structure eluci-dation°of°each°peak.°(Figure°2)

Peak A does not carry any protecting groups on itsamino or alcohol functions and affords a (M � H)� ionsignal at m/z 153 and an ion fragment at m/z 135, whichcan be attributed to elimination of water. The deute-rium exchange data show the corresponding signals atm/z 136 and 156 and thus prove the presence of twoexchangeable protons, which is consistent with theproposed structure. Since H/D back-exchange is com-mon with amines, a signal at m/z 136 (M � D � D2O)

�

and m/z 137 (M � D � HOD)� is observed. The massspectrum obtained for Peak B by conventionalHPLC/MS is misleading because the data suggest amolecular weight of 166 Da according to the (M � H)�

ion°signal°at°m/z 167,°Figure°3.°However,°the°deuteratedmobile phase experiment reveals a (M � D)� ion signalat m/z 169 indicating the presence of only one exchange-able hydrogen which can be attributed to the carboxylicacid function. [On-column H/D exchange HPLC/ESI/MS analysis using a sample treated with dia-zomethane for selective methylation of the free carbox-ylic acid group provided further evidence for thisasumption.] By contrast, the secondary amino functiondoes not undergo H/D exchange because it carries at-Boc group. The hydrogen attached to the amino group

0 5 10 15 20 25 Time [min]0.0

0.5

1.0

1.5

7x10Intens.

AB

C

Figure 1. HPLC separation of a mixture of 3 unknown amino-pyridines.

0

20

40

60

80

100

Abund.

60 80 100 120 140 160 180 m/z

121.1

136.1

156.0

0

20

40

60

80

100

Abund.

60 80 100 120 140 160 180 m/z

120.2

135.1

144.0

153.1

(M + H)+

(M + H - H2O)+

(M + D)+

(M + D - D 2O)+

NND

OD

C8H10D2N2O MW 154

NNH

OH

C8H12N2O MW 152

Peak A

Peak ADeuterium Exchange

Figure 2. ESI mass spectrum of Peak A. HPLC was performed ona YMC ODS AM column using CH3CN/H2O/CH3CO2H (top)and CH3CN/D2O/CH3CO2D (bottom) as the mobile phase.

555J Am Soc Mass Spectrom 2005, 16, 553–564 ELUCIDATION OF t-Boc GROUPS USING H/D EXCHANGE

in the major fragment is a consequence of intramolecu-lar hydrogen transfer through McLafferty rearrange-ment. Due to H/D back-exchange, a signal at m/z 150(M�D�HOD)� is observed. The deuterium exchangeMS data clearly reveal the presence and position of thet-Boc group even though the molecular ion or thecorresponding carbamoyl acid fragment, which wouldbe indicative of a McLafferty rearrangement, can not beobserved.Routine analysis of the mass spectrum obtained for

Peak C obtained with a nondeuterated mobile phaseindicates the presence of a carboxylic acid with amolecular weight of 166 Da, which would be in accor-dance with a (M � H)� molecular ion signal at m/z 167,Figure°4°(top°panel).°[The°shown°carbamoyl°acid°exhib-iting a molecular weight of 166 Da would not be stableduring HPLC analysis. Accordingly, one would predictthe presence of a carboxylic acid isomer, e.g., 2-(6-methylamino-2-pyridyl)acetic acid.] However, the cor-responding ion signal following H/D exchange is ob-served at m/z 168 and suggests that exchangeablehydrogens are not present in this molecule. The de-tected fragment must therefore be a consequence of aMcLafferty rearrangement of a t-Boc protected tertiaryamine, which does not undergo H/D exchange. Assum-ing an intramolecular hydrogen transfer from a t-Boc

group to a carbonyl oxygen, the signals exhibiting m/z167 and 168, respectively, can be assigned to a car-bamoyl acid fragment generated under MS conditionsafter chromatographic separation.We decided to prepare sixteen representative dipep-

tides to further study the use of our on-column H/Dexchange HPLC/ESI/MS method for structure elucida-tion of N-terminal t-Boc-protected dipeptides and theirunprotected°analogs,°Figure°5.Employing a deuterated mobile phase in the chro-

matographic separation of dipeptides 5–20 should re-sult in complete proton/deuterium exchange for allprotons attached to a heteroatom. At the N-terminus,the unprotected dipeptides 5, 7, 9, 13, 15, 17, and 19 canexchange two protons (but only one proton in dipeptide11), whereas the t-Boc protected derivatives 6, 8, 10, 14,16, 18, and 20 can only exchange one proton fordeuterium (none in 12). In addition, the amide protonsand the proton of the Z-carbamate group of the orni-thine derivatives 15 and 16 will also be exchanged. Inaccordance with the results obtained with aminopyri-dines, we expected that comparison of mass spectraldata obtained in conjunction with nondeuterated anddeuterated HPLC would provide clear evidence of thepresence and position of t-Boc groups.Comparison of the HPLC/CID/MS spectral data

0

20

40

60

80

100

Abund.

50 75 100 125 150 175 200 225 250 275 m/z

106.3 134.0

149.1

169.0

0

20

40

60

80

100

Abund.

50 75 100 125 150 175 200 225 250 275 m/z

106.3

149.0

167.0

(m/z 167 - H2O)+

(m/z 169 - D2O)+

Peak B

Peak BDeuterium Exchange

NNO

O

OD

O

C13 H17 DN2 O4MW 267

NNO

O

OH

O

C13 H18 N2 O4 MW 266

NHN OH

O

+ H

+

NHN OD

O

+ D

+

Figure 3. ESI mass spectrum of Peak B. HPLC was performed ona YMC ODS AM column using CH3CN/H2O/CH3CO2H (top)and CH3CN/D2O/CH3CO2D (bottom) as the mobile phase.

0

20

40

60

80

100

Abund.

50 75 100 125 150 175 200 225 250 275 m/z

65.292.2

110.1

124.2

149.0

168.0

0

20

40

60

80

100

Abund.

50 75 100 125 150 175 200 225 250 275 m/z

65.2

92.2

110.2

123.2

149.0

167.0

NNO

O

C12 H18 N2 O2 MW 222

NNO

O

C12 H18 N2 O2 MW 222

NNHO

O

+ H

+

NNHO

O

+ D

+

(m/z 168 - CO 2)+

(m/z 168 - HDO)+

(m/z 167 - CO 2)+

(m/z 167 - H2O)+

N

N

Peak C

Peak CDeuterium Exchange

Figure 4. ESI mass spectrum of Peak C. HPLC was performed ona YMC ODS AM column using CH3CN/H2O/CH3CO2H (top)and CH3CN/D2O/CH3CO2D (bottom) as the mobile phase.


obtained for dipeptide 5 with and without a deuteratedmobile phase reveals the presence of three exchange-able°protons,°Figures°6°and°7.°The°molecular° ion° iseasily detected by conventional MS analysis as its (M �H)� or (M � Na)� signal respectively at m/z 203 and

225. Deuterated mobile phase HPLC/MS affords thecorresponding strong signals at m/z 207 (d3-M � D)�

and 228 (d3-M � Na)�. Because of the low stability oft-Boc groups discussed above, on-line H/D exchangeMS data are necessary to clearly prove the absence of a

Figure 5. Structure of dipeptides 5–20.

Figure 6. HPLC/CID/MS data of dipeptide 5 using a nondeuterated mobile phase.


t-Boc group in 5 and also allow one to exclude possiblycoeluting t-Boc-protected impurities.The MS data of Dipeptide 6 show that one t-Boc

group°is°incorporated°into°the°N-terminus,°Figures°8and°9.°This°dipeptide°affords°two°exchangeable°pro-tons, one on the t-Boc protected amino function and oneattached to the peptide bond. As the molecule frag-ments, the t-Boc protected amine acquires an additional

hydrogen via a McLafferty rearrangement. While con-ventional HPLC/MS data would also indicate the pres-ence of one t-Boc group because of the (M � Na)� ionsignal at m/z 325, the deuterated mobile phaseHPLC/MS approach allows one to exclude the presenceof additional carbamate functions. It should be notedthat the MS data of 6 obtained using a nondeuteratedmobile phase in the preceding HPLC separation contain

88.4

149.3

174.3

207.3

228.1

244.3

All, 7.7-7.9min (#832-#858), Background Subtracted, Background Subtracted

0.0

0.5

1.0

1.5

2.0

2.5

5x10Intens.

60 80 100 120 140 160 180 200 220 240 m/z

D2N

DN

O

O

O

C9H15D3N2O3 MW 205

Deuterium Exchange

( M + Na )+

m/z 228

( M + K )+

m/z 244

( M + D )+

m/z 207

D2NO

O

+ D+

D+DN

+

D2N

DN

O

O

D2N

DN

O

m/z 146

Figure 7. HPLC/CID/MS data of dipeptide 5 using a deuterated mobile phase.

86.4

146.2

171.1

203.2

225.2

269.1

325.2


0

1

2

3

6x10Intens.

50 100 150 200 250 300 m/z

H2NO

O

+ H+

O NH

HN

O

O

O

O

C14H26N2O5 MW 302

H2N

HN

O

O

O

+ H+

H2N

HN

O

O

O

+ Na+

( M + Na )+

m/z 325

HN+ H

+

H2N

HN

O

O

H2N

HN

O

m/z 143OH N

H

NH

O

O

O

O + Na

+



the same signals as observed with the deprotecteddipeptide 5, i.e., fragments at m/z 203 and 225. It is verywell known that the t-Boc protection group is readilycleaved under mild acidic conditions. This often com-plicates the analysis of such carbamates because unde-sirable cleavage can occur during isolation and purifi-cation steps of t-Boc protected compounds or duringchromatographic analysis. The coexistence of 5 and 6

could also be a consequence of incomplete derivatiza-tion°of°the°unprotected°dipeptide.°However,°Figure°9shows the prevalence of fragments at m/z 206 and 227observed in the mass spectra after H/D exchange andthe low intensity of fragments exhibiting a m/z 207 and228, which may be attributed to H/D back exchangeand 13C content of the sample, provides evidence thatdipeptide°6 is°not°contaminated°with°5, Figure°9.

160.2205.2 221.1 243.2

286.2

333.3

350.3

394.3

416.2

432.2


0

2

4

6

8

5x10Intens.

200 250 300 350 400 m/z

C20H31N3O5 MW 393

H2N

HN

O

O

HN O

O

O

( M + Na )+

m/z 416

( M + H )+

m/z 394

( M + K )+

m/z 432H2N

HN

O

O

HN

O

O

H2N

HN

O

O

HN

O

+ H

+

HN

HN O

O+ H

+

(m/z 350 - NH3 )+

m/z 333

H2N

HN

O


88.4

148.2

173.1

189.1

206.3

227.1

243.2250.1

271.1

327.2

343.2


0.00

0.25

0.50

0.75

1.00

1.25

1.50

6x10Intens.

100 150 200 250 300 m/z

O ND

DN

O

O

O

O

C14H24D2N2O5 MW 304

( M + Na )+

m/z 327

( M + K )+

m/z 343

N

DN

O

O

O

+ D

+D

H

NO

O

+ D+

H

D

( m/z 204 + K)+

m/z 243

Deuterium Exchange

DN + D+

N

DN

O

D

H

m/z 145

N

DN

O

OH

D

N

DN

O

O

O

+ Na

+D

H

OH NDND

O

O

O

O + Na

+



Employing the on-column H/D exchangeHPLC/MS methodology to Compound 15 carrying abenzyloxycarbonyl carbamate reveals the presence offour hydrogens which readily exchange in the deuter-ated°mobile°phase,°Figures°10°and°11.°The°ions°at°m/z394, 399, 416, and 420 corresponding, respectively, to (M� H)�, (d4-M � D)�, (M � Na)�, and (d4-M � Na)�

refer to the molecular ion of 15. Again, comparison of

the MS data clearly proves the absence of a t-Boc groupand coeluting carbamoyl impurities.HPLC/MS analysis of the N-t-Boc protected dipep-

tide 16 shows three exchangeable hydrogens, one on thet-Boc protected amino function, one on the Z-carbamateand° one° on° the° peptide° bond,° Figures° 12° and° 13.Deuterated mobile phase HPLC followed by MS frag-mentation of 16 results in incorporation of only one

160.1 208.3224.2

246.3

290.3

335.4

355.4

399.3

420.4

436.4


0

1

2

3

4

5

5x10Intens.

150 200 250 300 350 400 450m/z

C20H27D4N3O5 MW 397

D2N

DN

O

O

DN O

O

O

Deuterium Exchange

( M + D )+

m/z 399

( M + Na )+

m/z 420

( M + K )+

m/z 436

D2N

DN

O

O

DN

O

OD2N

DN

O

O

DN

O

+ D

+

(m/z 355 - ND3 )+

m/z 335

DN

DN O

O+ D

+

D2N

DN

O


160.2205.2 221.1 243.2

286.2

333.3

350.3

394.3

416.3438.3 460.2

516.3

532.2


0.00

0.25

0.50

0.75

1.00

1.25

1.50

7x10Intens.

150 200 250 300 350 400 450 500 m/z

H2N

HN

O

O

HN

O

O

( M + Na )+

m/z 516

( M + K )+

m/z 532

H2N

HN

O

O

HN O

O

O+ H

+

( m/z 393 + Na )+

m/z 416

M + H -

m/z 438

+

HN

HN O

O+ H

+

H2N

HN

HN

O

O

O + H

+

(m/z 350 - NH3 )+

m/z 333

H2N

HN

O

O NH

OHN

O

O

HN O

O

O

C25H39N3O7 MW 493

M + Na -

m/z 460

+



hydrogen into the �-amino group of the ornithinemoiety. As expected, the amide bond and the Z-pro-tected �-amino group of the ornithine moiety showexchange of one proton for deuterium. Comparison ofthe fragment with m/z 394 and its C-13 isotope patternto the signal of the corresponding fragment obtained byHPLC/MS using a deuterated mobile phase exhibitingm/z 398 proves that 16 carries one t-Boc group and also

allows exclusion of the presence of impurities of 15 inthis sample. Deuterated mobile phase HPLC/MS datathus afford clear information about the purity and thepresence and location of different carbamoyl protectiongroups of complex molecules.Dipeptide 19 carrying a benzyl protecting group and

20,which also affords a t-Boc group, were also analyzedby on-column H/D exchange HPLC/MS. The spectral

226.2

269.1

286.2

297.1

357.2

379.2

All, 9.3-9.5min (#883-#896),Background Subtracted, Background Subtracted

0.0

0.5

1.0

1.5

2.0

2.5

3.0

6x10Intens.

150 175 200 225 250 275 300 325 350 375 400 m/z

C20H24N2O4 MW 356

H2N

HN

O

O

O

O

( M + H )+

m/z 357

( M + Na )+

m/z 379

H2N

O

+ H

+

( M + H - CH3CO2H )+

m/z 297

H2NO

O

O

+ H

+

(m/z 286 - NH3 )+

m/z 269


207.2 223.2

289.3

335.3

354.3

398.4

419.4442.4

463.3

519.5

535.4


0.0

0.2

0.4

0.6

0.8

1.0

1.2

7x10Intens.

150 200 250 300 350 400 450 500 m/z

( M + K )+

m/z 535

( M + Na )+

m/z 519

+ D

+

N

DN

DN O

O

O

D

H

O

O

( m/z 396 + Na )+

m/z 419N

DN

O

O

DN

O

O

D

H

Deuterium Exchange

N

DN

O

O

DN

O

+ D

+

H

D

(m/z 354 - NHD2 )+

m/z 335

N

DN

O

H

D

M + H -

m/z 442

+

M + Na -

m/z 463

+

O ND

ODN

O

O

DN O

O

O

C25H36D3N3O7 MW 496

+ D

+

HN

DN O

O



MS data observed for 19 reveal three exchangeableprotons and confirm the presence of the benzyl group,Figures°14°and°15.°The°location°of°the°benzyl°groupbecomes evident from the fragmentation pattern of 19and d3-19. The CID/MS of 19 shows a fragment referingto a benzylated methyl tyrosine ester (nondeuteratedionm/z 286, deuterated ionm/z 289) that eliminates NH3or ND3 to give a fragment at m/z 269. The presence of

this fragment ion and the H/D exchange MS datapattern reveal that the benzyl group must be attached tothe phenol moiety of tyrosine but not to its aminofunction.The analysis of Dipeptide 20 provides complete

structural information about the presence and locationof° the° protecting° groups,° Figures° 16° and° 17.° Twoexchangeable protons were detected and assigned to




protons attached to the peptide bond and one carbam-ate function. Again, MS data of nondeuterated 20 couldbe misinterpretated because of the intensive fragment atm/z 357 suggesting the presence of 19 as an impurity.This can be ruled out by comparison to the correspond-ing fragment obtained with deuterated 20. The frag-ment ion exhibiting m/z 360 carries a hydrogen attachedto a nitrogen and therefore must be assigned to aMcLafferty rearrangement fragment of 20. The ionsignals at m/z 286 and 357 (nondeuterated sample) andm/z 288 and 360 (deuterated sample) in conjunctionwith the fragment exhibiting m/z 269 reveal that thet-Boc group is attached to the alanine moiety at theN-terminus. H/D Exchange MS data thus also providesa tool for differentiating between N-t-Boc-(S)-Ala-O-Bn-(S)-Tyr-OMe, 20, and N-t-Boc-O-Bn-(S)-Tyr-(S)-Ala-OMe, i.e., applying this method to structure elucidationof 5–20 affords invaluable information about the aminoacid sequence in addition to the presence and locationof t-Boc protecting groups attached to these dipeptides.On-column H/D exchange HPLC combined with

mass spectrometry was also utilized for the analysis ofDipeptides 7–14, 17, and 18. In all cases, we found thatthis methodology greatly facilitates structure elucida-tion, i.e., determination of position and number of t-Bocprotecting groups and information about the primarypeptide structure. It also allows one to examine thepresence or absence of coeluting impurities that exhibitthe same MS spectrum using conventional HPLC/MS.

Conclusions

The usefulness of deuterated mobile phase HPLC/MSfor a fast determination of the absence or presence of

t-Boc protection group in various amines and peptideshas been demonstrated. This technique also allows forthe determination of the presence of unprotected impu-rities that may result from undesirable carbamate cleav-age under mild acidic conditions or incomplete deriva-tization. Employing this strategy in mass spectralstructural elucidation is expected to be invaluable forroutine MS analysis of complex molecules and for fastscreening of combinatorial libraries that require a rapidand automated analysis of the presence and location oft-Boc protecting groups.

Uncited References

Per journal style, the footnote type references whichyou have listed in the reference list have been moved tothe body text and enclosed in square brackets. They are13, 14 as explained in your author query. Any refer-ences not dealt with will be retained in this section.

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5. (a) Henion, J. D. J. Chromatogr. Sci. 1981, 19, 57–64. (b) Cairns,T.; Siegmund, E. G. Anal. Chem. 1982, 54, 2456–2461.

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91.3 135.2

228.1

269.1

288.1

343.2

360.2

381.2

404.3425.2

481.2


0

1

2

3

4

5

6x10Intens.

50 100 150 200 250 300 350 400 450 m/z

C25H30D2N2O6 MW 458

ND

DN

O

O

O

O

O

O

Deuterium Exchange

+ D

+N

O

O

O

D

H

( M + Na )+

m/z 381

( M + K )+

m/z 497(m/z 288 - NHD2 )+

m/z 269

(m/z 358 + Na )+

m/z 381

+ D

+N

DN

O

O

O

O

D

H

( m/z 360 - CH3CO2D )+

m/z 299

M + Na -

m/z 425

+DN

O

DN

O

+ D

+



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